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Modification of genes by homologous recombination

Posted in Genetics & Innovations in Treatment, Genetics & Pharmaceutical, Genome Biology, Neurological Diseases, Stem Cells for Regenerative Medicine, tagged , , on September 5, 2015| Leave a Comment »

Modification of genes by homologous recombination

Larry H. Bernstein, MD, FCAP, Curator

Leaders in Pharmaceutical Innovation

Series E: 2; 2.15

Mario Capecchi, Martin Evans, Oliver Smithies

2007 Nobel Prize for their work on targeted gene modification.

Born in Italy in 1937, scientist Mario R. Capecchi emigrated to the United States after World War II and later became a geneticist and professor. His groundbreaking work on targeted gene modification won him a Nobel Prize in 2007. He is Distinguished Professor of Human Genetics at the University of Utah School of Medicine. Mario Capecchi is interested in the molecular genetic analysis of mammalian development, with emphasis on neurogenesis, organogenesis, patterning of the vertebral column, and limb development. He also contributes to the modeling of human disease in the mouse, from cancer to neuropsychiatric disorders.

Capecchi MR. (2005). Gene targeting in mice: functional analysis of the mammalian genome for the twenty-first century. Nat Rev Genet, Jun;6(6):507-12. Review.

https://youtu.be/WQr6ZeNe-vE

 

Sir Martin John Evans FRS FMedSci (b. 1 January 1941, Stroud, Gloucestershire[1][5]) is a Welsh biologist who, with Matthew Kaufman, was the first to culture mice embryonic stem cells and cultivate them in a laboratory in 1981. He is also known, along with Mario Capecchi and Oliver Smithies, for his work in the development of the knockout mouse and the related technology of gene targeting, a method of using embryonic stem cells to create specific gene modifications in mice.[5][6] In 2007, the three shared the Nobel Prize in Physiology or Medicine in recognition of their discovery and contribution to the efforts to develop new treatments for illnesses in humans.[7][8][9][10][11]

He won a major scholarship to Christ’s College, Cambridge at a time when advances in genetics were occurring there and became interested in biology and biochemistry. He then went to University College London where he learned laboratory skills supervised by Elizabeth Deuchar. In 1978, he moved to the Department of Genetics, at the University of Cambridge, and in 1980 began his collaboration with Matthew Kaufman. They explored the method of using blastocysts for the isolation of embryonic stem cells. After Kaufman left, Evans continued his work, upgrading his laboratory skills to the newest technologies, isolated the embryonic stem cell of the early mouse embryo and established it in a cell culture. He genetically modified and implanted it into adult female mice with the intent of creating genetically modified offspring, work for which he was awarded the Nobel Prize in 2007.

In 1981, Evans and Kaufman published results for experiments in which they described how they isolated embryonic stem cells from mouse blastocysts and grew them in cell cultures.[23][24] This was also achieved by Gail R. Martin, independently, in the same year.[25] Eventually, Evans was able to isolate the embryonic stem cell of the early mouse embryo and establish it in a cell culture. He then genetically modified it and implanted it into adult female mice with the intent of creating genetically modified offspring, the forbearers of the laboratory mice that are considered so vital to medical research today.[23] The availability of these cultured stem cells eventually made possible the introduction of specific gene alterations into the germ line of mice and the creation of transgenic mice to use as experimental models for human illnesses.[23]

Evans and his collaborators showed that they could introduce a new gene into cultured embryonic stem cells and then use such genetically transformed cells to make chimeric embryos.[26] In some chimeric embryos, the genetically altered stem cells produced gametes, thus allowing transmission of the artificially induced mutation into future generations of mice.[27] In this way, transgenic mice with induced mutations in the enzyme Hypoxanthine-guanine phosphoribosyltransferase (HPRT) were created.[28] Today, genetically modified mice are considered vital for medical research.

In the 1990s, he was a fellow at St Edmund’s College, Cambridge. In 1999, he became Professor of Mammalian Genetics and Director of the School of Biosciences at Cardiff University,[5][17] where he worked until he retired at the end of 2007.[18] He became a Knight Bachelor in the 2004 New Year Honours in recognition of his work in stem cell research.[5][19] He received the accolade from Prince Charles at Buckingham Palace on 25 June 2004.[20] In 2007, he was awarded the Nobel Prize in Physiology or Medicine along with Mario Capecchi and Oliver Smithies for their work in discovering a method for introducing homologous recombination in mice employing embryonic stem cells.[7] Evans was appointed president of Cardiff University and was inaugurated into that position on 23 November 2009.[21] Subsequently Evans became Chancellor of Cardiff University in 2012. [22]

 

The Whole of a Scientific Career: An Interview with Oliver Smithies

Jane Gitschier*

PLoS Genet. 2015 May; 11(5): e1005224.

Published online 2015 May 28. doi:  10.1371/journal.pgen.1005224

Smithies, of course, is well worth any pilgrimage. Nearing 90 years of age, he still works at the bench, seven days a week. He is enthusiastic, curious, gentle, and fearless in attacking new problems, to which he applies his gifts both as a tinkerer and a thinker. He is generous with his ideas and advice and beloved by his colleagues, students, and postdocs, now numbering so many that he has lost count. His scientific journey began in the mid-late 1940s as an undergraduate at Balliol College, Oxford, where his tutor introduced him to a new field, now called “molecular biology.” Smithies embraced the young field, and after a brief postdoctoral stint at University of Wisconsin, took his first job in Toronto. There, in the early 1950s, he invented starch gel electrophoresis, which had the property of fractionating proteins on the basis of size and led him to discover inherited differences in haptoglobin, a serum protein that binds hemoglobin. One variant, the product of an abnormal genetic exchange, piqued his life-long interest in homologous recombination. Three decades later, after an arduous, three-year experiment, he was able to demonstrate homologous recombination between a plasmid and the human genome in the pursuit of correcting genetic defects, a discovery for which he, much later, won the Nobel Prize.

 

Genetic engineering, also called genetic modification, is the direct manipulation of an organism’s genome using biotechnology. It is therefore a set of technologies used to change the genetic makeup of cells, including the transfer of genes within and across species boundaries to produce improved or novel organisms. New DNA may be inserted in the host genome by first isolating and copying the genetic material of interest using molecular cloning methods to generate a DNA sequence, or by synthesizing the DNA, and then inserting this construct into the host organism. Genes may be removed, or “knocked out”, using a nuclease. Gene targeting is a different technique that uses homologous recombination to change an endogenous gene, and can be used to delete a gene, remove exons, add a gene, or introduce point mutations.

An organism that is generated through genetic engineering is considered to be a genetically modified organism (GMO). The first GMOs were bacteria generated in 1973 and GM mice in 1974. Insulin-producing bacteria were commercialized in 1982 and genetically modified food has been sold since 1994. Glofish, the first GMO designed as a pet, was first sold in the United States December in 2003.[1]

Genetic engineering techniques have been applied in numerous fields including research, agriculture, industrial biotechnology, and medicine. Enzymes used in laundry detergent and medicines such as insulin and human growth hormone are now manufactured in GM cells, experimental GM cell lines and GM animals such as mice or zebrafish are being used for research purposes, and genetically modified crops have been commercialized.

In 1972 Paul Berg created the first recombinant DNA molecules by combining DNA from the monkey virus SV40 with that of the lambda virus.[26] In 1973 Herbert Boyer andStanley Cohen created the first transgenic organism by inserting antibiotic resistance genes into the plasmid of an E. coli bacterium.[27][28] A year later Rudolf Jaenisch created a transgenic mouse by introducing foreign DNA into its embryo, making it the world’s first transgenic animal.[29] These achievements led to concerns in the scientific community about potential risks from genetic engineering, which were first discussed in depth at the Asilomar Conference in 1975. One of the main recommendations from this meeting was that government oversight of recombinant DNA research should be established until the technology was deemed safe.[30][31]

In 1976 Genentech, the first genetic engineering company, was founded by Herbert Boyer and Robert Swanson and a year later the company produced a human protein (somatostatin) in E.coli. Genentech announced the production of genetically engineered human insulin in 1978.[32] In 1980, the U.S. Supreme Court in the Diamond v. Chakrabarty case ruled that genetically altered life could be patented.[33] The insulin produced by bacteria, branded humulin, was approved for release by the Food and Drug Administration in 1982.[34]

The most common form of genetic engineering involves inserting new genetic material randomly within the host genome.[citation needed] Other techniques allow new genetic material to be inserted at a specific location in the host genome or generate mutations at desired genomic loci capable of knocking out endogenous genes. The technique of gene targeting uses homologous recombination to target desired changes to a specific endogenous gene. This tends to occur at a relatively low frequency in plants and animals and generally requires the use of selectable markers. The frequency of gene targeting can be greatly enhanced with the use of engineered nucleases such as zinc finger nucleases,[62][63] engineered homing endonucleases,[64][65] or nucleases created from TAL effectors.[66][67] In addition to enhancing gene targeting, engineered nucleases can also be used to introduce mutations at endogenous genes that generate a gene knockout.[68][69]

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Voluntary and Involuntary S-Insufficiency

Posted in Alzheimer's Disease, Amino acids, Best evidence, Biochemical pathways, Cell Biology, Chemical Biology and its relations to Metabolic Disease, Curation, Developmental biology, Disease Biology, Etiology, Metabolism, Metabolomics, Mutant Gene Expression, Neurological Diseases, Nutrition, Nutrition Disorders, Population Health Management, Proteins, Proteomics, Signaling & Cell Circuits, Stress Disorders, Systemic Inflammatory Response Related Disorders, Translational Research, Translational Science, tagged , , , , , , , , , , , , on March 7, 2015| Leave a Comment »

Voluntary and Involuntary S- Insufficiency

Writer and Curator: Larry H Bernstein, MD, FCAP 

Transthyretin and the Stressful Condition

Introduction

This article is written among a series of articles concerned with stress, obesity, diet and exercise, as well as altitude and deep water diving for extended periods, and their effects.  There is a reason that I focus on transthyretin (TTR), although much can be said about micronutients and vitamins, and fat soluble vitamins in particular, and iron intake during pregnancy.    While the importance of vitamins and iron are well accepted, the metabolic basis for their activities is not fully understood.  In the case of a single amino acid, methionine, it is hugely important because of the role it plays in sulfur metabolism, the sulfhydryl group being essential for coenzyme A, cytochrome c, and for disulfide bonds.  The distribution of sulfur, like the distribution of iodine, is not uniform across geographic regions.  In addition, the content of sulfur found in plant sources is not comparable to that in animal protein.  There have been previous articles at this site on TTR, amyloid and sepsis.

Transthyretin and Lean Body Mass in Stable and Stressed State

http://pharmaceuticalintelligence.com/2013/12/01/transthyretin-and-lean-body-mass-in-stable-and-stressed-state/

A Second Look at the Transthyretin Nutrition Inflammatory Conundrum

http://pharmaceuticalintelligence.com/2012/12/03/a-second-look-at-the-transthyretin-nutrition-inflammatory-conundrum/

Stabilizers that prevent transthyretin-mediated cardiomyocyte amyloidotic toxicity

http://pharmaceuticalintelligence.com/2013/12/02/stabilizers-that-prevent-transthyretin-mediated-cardiomyocyte-amyloidotic-toxicity/

Thyroid Function and Disorders

http://pharmaceuticalintelligence.com/2015/02/05/thyroid-function-and-disorders/

Proteomics, Metabolomics, Signaling Pathways, and Cell Regulation: a Compilation of Articles in the Journal http://pharmaceuticalintelligence.com

http://pharmaceuticalintelligence.com/2014/09/01/compilation-of-references-in-leaders-in-pharmaceutical-intelligence-about-proteomics-metabolomics-signaling-pathways-and-cell-regulation-2/

Malnutrition in India, high newborn death rate and stunting of children age under five years

http://pharmaceuticalintelligence.com/2014/07/15/malnutrition-in-india-high-newborn-death-rate-and-stunting-of-children-age-under-five-years/

Vegan Diet is Sulfur Deficient and Heart Unhealthy

http://pharmaceuticalintelligence.com/2013/11/17/vegan-diet-is-sulfur-deficient-and-heart-unhealthy/

How Methionine Imbalance with Sulfur-Insufficiency Leads to Hyperhomocysteinemia

http://pharmaceuticalintelligence.com/2013/04/04/sulfur-deficiency-leads_to_hyperhomocysteinemia/

Amyloidosis with Cardiomyopathy

http://pharmaceuticalintelligence.com/2013/03/31/amyloidosis-with-cardiomyopathy/

Advances in Separations Technology for the “OMICs” and Clarification of Therapeutic Targets

http://pharmaceuticalintelligence.com/2012/10/22/advances-in-separations-technology-for-the-omics-and-clarification-of-therapeutic-targets/

Sepsis, Multi-organ Dysfunction Syndrome, and Septic Shock: A Conundrum of Signaling Pathways Cascading Out of Control

http://pharmaceuticalintelligence.com/2012/10/13/sepsis-multi-organ-dysfunction-syndrome-and-septic-shock-a-conundrum-of-signaling-pathways-cascading-out-of-control/

Automated Inferential Diagnosis of SIRS, sepsis, septic shock

http://pharmaceuticalintelligence.com/2012/08/01/automated-inferential-diagnosis-of-sirs-sepsis-septic-shock/

Transthyretin and the Systemic Inflammatory Response 

Transthyretin has been widely used as a biomarker for identifying protein-energy malnutrition (PEM) and for monitoring the improvement of nutritional status after implementing a nutritional intervention by enteral feeding or by parenteral infusion. This has occurred because transthyretin (TTR) has a rapid removal from the circulation in 48 hours and it is readily measured by immunometric assay. Nevertheless, concerns have been raised about the use of TTR in the ICU setting, which prompts a review of the actual benefit of using this test in a number of settings. TTR is easily followed in the underweight and the high risk populations in an ambulatory setting, which has a significant background risk of chronic diseases.  It is sensitive to the systemic inflammatory response syndrom (SIRS), and needs to be understood in the context of acute illness to be used effectively. There are a number of physiologic changes associated with SIRS and the injury/repair process that will affect TTR and will be put in context in this review. The most important point is that in the context of an ICU setting, the contribution of TTR is significant in a complex milieu.  copyright @ Bentham Publishers Ltd. 2009.

Transthyretin as a marker to predict outcome in critically ill patients.
Arun Devakonda, Liziamma George, Suhail Raoof, Adebayo Esan, Anthony Saleh, Larry H. Bernstein.
Clin Biochem Oct 2008; 41(14-15): 1126-1130

A determination of TTR level is an objective method od measuring protein catabolic loss of severly ill patients and numerous studies show that TTR levels correlate with patient outcomes of non-critically ill patients. We evaluated whether TTR level correlates with the prevalence of PEM in the ICUand evaluated serum TTR level as an indicator of the effectiveness of nutrition support and the prognosis in critically ill patients.

TTR showed excellent concordance with patients classified with PEM or at high malnutrition risk, and followed for 7 days, it is a measure of the metabolic burden. TTR levels did not respond early to nutrition support because of the delayed return to anabolic status. It is particularly helpful in removing interpretation bias, and it is an excellent measure of the systemic inflammatory response concurrent with a preexisting state of chronic inanition.

 The Stressful Condition as a Nutritionally Dependent Adaptive Dichotomy

Yves Ingenbleek and Larry Bernstein
Nutrition 1999;15(4):305-320 PII S0899-9007(99)00009-X

The injured body manifests a cascade of cytokine-induced metabolic events aimed at developing defense mechanisms and tissue repair. Rising concentrations of counterregulatory hormones work in concert with cytokines to generate overall insulin and insulin-like growth factor 1 (IGF-1), postreceptor resistance and energy requirements grounded on lipid dependency. Dalient features are self-sustained hypercortisolemia persisting as long as cytokines are oversecreted and down-regulation of the hypothalamo-pituitary-thyroid axis stabilized at low basal levels. Inhibition of thyroxine 5’deiodinating activity (5’DA) accounts for the depressed T3 values associated with the sparing of both N and energy-consuming processes. Both the liver and damaged territories adapt to stressful signals along up-regulated pathways disconnected from the central and peripheral control systems. Cytokines stimulate 5’DA and suppress the synthesis of TTR, causing the drop of retinol-binding protein (RBP) and the leakage of increased amounts of T4 and retinol in free form. TTR and RBP thus work as prohormonal reservoirs of precursor molecules which need to be converted into bioactive derivatives (T3 and retinoic acids) to reach transcriptional efficiency. The converting steps (5’DA and cellular retinol-binding protein-1) are activated to T4 and retinol, themselves operating as limiting factors to positive feedback loops. …The suicidal behavior of TBG, CBG, and IGFBP-3 allows the occurrence of peak endocrine and mitogenic influences at the site of inflammation. The production rate of TTR by the liver is the main determinant of both the hepatic release and blood transport of holoRBP, which explains why poor nutritional status concomitantly impairs thyroid- and retinoid-dependent acute phase responses, hindering the stressed body to appropriately face the survival crisis.  …
abbreviations: TBG, thyroxine-binding globulain; CBG, cortisol-binding globulin; IGFBP-3, insulin growth factor binding protein-3; TTR, transthyretin; RBP, retionol-binding protein.

Why Should Plasma Transthyretin Become a Routine Screening Tool in Elderly Persons? 

Yves Ingenbleek.
J Nutrition, Health & Aging 2009.

The homotetrameric TTR molecule (55 kDa as MM) was first identified in cerebrospinal fluid (CSF).  The initial name of prealbumin (PA)  was assigned based on the electrophoretic migration anodal to albumin. PA was soon recognized as a specific binding protein for thyroid hormone. and also of plasma retinol through the mediation of the small retinol-binding protein (RBP, 21 kDa as MM), which has a circulating half-life half that of TTR (24 h vs 48 h).

There exist at least 3 goos reasons why TTR should become a routine medical screening test in elderly persons.  The first id grounded on the assessment of protein nutritional status that is frequently compromized and may become a life threatening condition.  TTR was proposed as a marker of protein-energy malnutrition (PEM) in 1972. As a result of protein and energy deprivation, TTR hepatic synthesis is suppressed whereas all plasma indispensable amino acids (IAAs) manifest declining trends with the sole exception of methionine (Met) whose concentration usually remains unmodified. By comparison with ALB and transferrin (TF) plasma values, TTR did reveal a much higher degree of reactivity to changes in protein status that has been attributed to its shorter biological half-life and to its unusual tryptophan richness. The predictive ability of outcome offered by TTR is independent of that provided by ALB and TF. Uncomplicated PEM primarily affects the size of body nitrogen (N) pools, allowing reduced protein syntheses to levels compatible with survival.  These adaptiver changes are faithfully identified by the serial measurement of TTR whose reliability has never been disputed in protein-depleted states. On the contrary, the nutritional relevance of TTR has been controverted in acute and chronic inflammatory conditions due to the cytokine-induced transcriptional blockade of liver synthesis which is an obligatory step occurring independently from the prevailing nutritional status. Although PEM and stress ful disorders refer to distinct pathogenic mechanisms, their combined inhibitory effects on TTR liber production fueled a long-lasting strife regarding a poor specificity.  Recent body compositional studies have contributed to disentagling these intermingled morbidities, showing that evolutionary patterns displayed by plasma TTR are closely correlated with the fluctuations of lean body mass (LBM).

The second reason follows from advances describing the unexpected relationship established between TTR and homocysteine (Hcy), a S-containing AA not found in customary diets but resulting from the endogenous transmethylation of dietary methionine.  Hcy may be recycled to Met along a remethylation pathway (RM) or irreversibly degraded throughout the transsulfuration (TS) cascade to relase sulfaturia as end-product. Hcy is thus situated at the crossrad of RM and TS pathways which are in equilibrium keeping plasma Met values unaltered.  Three dietary water soluble B viatamins are implicated in the regulation of the Hcy-Met cycle. Folates (vit B9) are the most powerful agent, working as a supplier of the methyl group required for the RM process whereas cobalamines (vit B12) and pyridoxine (vit B6) operate as cofactors of Met-synthase and cystathionine-β-synthase.  Met synthase promotes the RM pathway whereas the rate-limiting CβS governs the TS degradative cascade. Dietary deficiency in any of the 3 vitamins may upregulate Hcy plasma values, an acquied biochemiucal anomaly increasingly encountered in aged populations.

The third reason refers to recent and fascinating data recorded in neurobiology and emphasizing the specific properties of TTR in the prevention of brain deterioration. TTR participates directly in the maintenance of memory and normal cognitive processes during the aging process by acting on the retinoid signaling pathway.  Moreover, TTR may bind amyloid β peptide in vitro, preventing its transformation into toxic amyloid fibrils and amyloid plaques.  TTR works as a limiting factor for the plasma transport of retinoid, which in turn operates as a limiting determinant of both physiologically active retinoic acid (RA) derivatives, implying that any fluctuation in protein status might well entail corresponding  alterations in cellular bioavailability of retinoid compounds.  Under normal aging circumstances, the concentration of retinoid compounds declines in cerebral tissues together with the downregulation of RA receptor expression. In animal models, depletion of RAs causes the deposition of amyloid-β peptides, favoring the formation of amyloid plaques.

Prealbumin and Nutritional Evaluation

Larry Bernstein, Walter Pleban
Nutrition Apr 1996; 12(4):255-259.
http://nutritionjrnl.com/article/S0899-9007(96)90852-7

We compressed 16-test-pattern classes of albumin (ALB), cholesterol (CHOL), and total protein (TPR) in 545 chemistry profiles to 4 classes by conveerting decision values to a number code to separate malnourished (1 or 2) from nonmalnourished (NM)(0) patients using as cutoff values for NM (0), mild (1), and moderate (2): ALB 35, 27 g/L; TPR 63, 53 g/L; CHOL 3.9, 2.8 mmol/L; and BUN 9.3, 3.6 mmol/L. The BUN was found to have  to have too low an S-value to make a contribution to the compressed classification. The cutoff values for classifying the data were assigned prior to statistical analysis, after examining information in the structured data. The data was obtained by a natural experiment in which the test profiles routinely done by the laboratory were randomly extracted. The analysis identifies the values used that best classify the data and are not dependent on distributional assumptions. The data were converted to 0, 1, or 2 as outcomes, to create a ternary truth table (eaxch row in nnn, the n value is 0 to 2). This allows for 34 (81) possible patterns, without the inclusion of prealbumin (TTR). The emerging system has much fewer patterns in the information-rich truth table formed (a purposeful, far from random event). We added TTR, coded, and examined the data from 129 patients. The classes are a compressed truth table of n-coded patterns with outcomes of 0, 1, or 2 with protein-energy malnutrition (PEM) increasing from an all-0 to all-2 pattern.  Pattern class (F=154), PAB (F=35), ALB (F=56), and CHOL (F=18) were different across PEM class and predicted PEM class (R-sq. = 0.7864, F=119, p < E-5). Kruskall-Wallis analysis of class by ranks was significant for pattern class E-18), TTR (6.1E-15) ALB (E-16), CHOL (9E-10), and TPR (5E-13). The medians and standard error (SEM) for TTR, ALB, and CHOL of four TTR classes (NM, mild, mod, severe) are: TTR = 209, 8.7; 159, 9.3; 137, 10.4; 72, 11.1 mg/L. ALB – 36, 0.7; 30.5, 0.8; 25.0, 0.8; 24.5, 0.8 g/L. CHOL = 4.43, 0.17; 4.04, 0.20; 3.11, 0.21; 2.54, 0.22 mmol/L. TTR and CHOL values show the effect of nutrition support on TTR and CHOL in PEM. Moderately malnourished patients receiving nutrition support have TTR values in the normal range at 137 mg/L and at 159 mg/L when the ALB is at 25 g/L or at 30.5 g/L.

An Informational Approach to Likelihood of Malnutrition 

Larry Bernstein, Thomas Shaw-Stiffel, Lisa Zarney, Walter Pleban.
Nutrition Nov 1996;12(11):772-776.  PII: S0899-9007(96)00222-5.
http://dx.doi.org:/nutritionjrnl.com/article/S0899-9007(96)00222-5

Unidentified protein-energy malnutrition (PEM) is associated with comorbidities and increased hospital length of stay. We developed a model for identifying severe metabolic stress and likelihood of malnutrition using test patterns of albumin (ALB), cholesterol (CHOL), and total protein (TP) in 545 chemistry profiles…They were compressed to four pattern classes. ALB (F=170), CHOL (F = 21), and TP (F = 5.6) predicted PEM class (R-SQ = 0.806, F= 214; p < E^-6), but pattern class was the best predictor (R-SQ = 0.900, F= 1200, p< E^-10). Ktuskal-Wallis analysis of class by ranks was significant for pattern class (E^18), ALB (E^-18), CHOL (E^-14), TP (@E^-16). The means and SEM for tests in the three PEM classes (mild, mod, severe) were; ALB – 35.7, 0.8; 30.9, 0.5; 24.2, 0.5 g/L. CHOL – 3.93, 0.26; 3.98, 0.16; 3.03, 0.18 µmol/L, and TP – 68.8, 1.7; 60.0, 1.0; 50.6, 1.1 g/L. We classified patients at risk of malnutrition using truth table comprehension.

Downsizing of Lean Body Mass is a Key Determinant of Alzheimer’s Disease

Yves Ingenbleek, Larry Bernstein
J Alzheimer’s Dis 2015; 44: 745-754.
http://dx.doi.org:/10.3233/JAD-141950

Lean body mass (LBM) encompasses all metabolically active organs distributed into visceral and structural tissue compartments and collecting the bulk of N and K stores of the human body. Transthyretin (TTR)  is a plasma protein mainly secreted by the liver within a trimolecular TTR-RBP-retinol complex revealing from birth to old age strikingly similar evolutionary patterns with LBM in health and disease. TTR is also synthesized by the choroid plexus along distinct regulatory pathways. Chronic dietary methionine (Met) deprivation or cytokine-induced inflammatory disorders generates LBM downsizing following differentiated physiopathological processes. Met-restricted regimens downregulate the transsulfuration cascade causing upstream elevation of homocysteine (Hcy) safeguarding Met homeostasis and downstream drop of hydrogen sulfide (H2S) impairing anti-oxidative capacities. Elderly persons constitute a vulnerable population group exposed to increasing Hcy burden and declining H2S protection, notably in plant-eating communities or in the course of inflammatory illnesses. Appropriate correction of defective protein status and eradication of inflammatory processes may restore an appropriate LBM size allowing the hepatic production of the retinol circulating complex to resume, in contrast with the refractory choroidal TTR secretory process. As a result of improved health status, augmented concentrations of plasma-derived TTR and retinol may reach the cerebrospinal fluid and dismantle senile amyloid plaques, contributing to the prevention or the delay of the onset of neurodegenerative events in elderly subjects at risk of Alzheimer’s disease.

Amyloidogenic and non-amyloidogenic transthyretin variants interact differently with human cardiomyocytes: insights into early events of non-fibrillar tissue damage

Pallavi Manral and Natalia Reixach
Biosci.Rep.(2015)/35/art:e00172 http://dx.doi.org:/10.1042/BSR20140155

TTR (transthyretin) amyloidosis are diseases characterized by the aggregation and extracellular deposition of the normally soluble plasma protein TTR. Ex vivo and tissue culture studies suggest that tissue damage precedes TTR fibril deposition, indicating that early events in the amyloidogenic cascade have an impact on disease development. We used a human cardiomyocyte tissue culture model system to define these events. We previously described that the amyloidogenic V122I TTR variant is cytotoxic to human cardiac cells, whereas the naturally occurring, stable and non-amyloidogenic T119M TTR variant is not. We show that most of the V122I TTR interacting with the cells is extracellular and this interaction is mediated by a membraneprotein(s). In contrast, most of the non-amyloidogenic T119M TTR associated with the cells is intracellular where it undergoes lysosomal degradation. The TTR internalization process is highly dependent on membrane cholesterol content. Using a fluorescent labelled V122I TTR variant that has the same aggregation and cytotoxic potential as the native V122I TTR, we determined that its association with human cardiomyocytes is saturable with a KD near 650nM. Only amyloidogenic V122I TTR compete with fluorescent V122I force ll-binding sites. Finally, incubation of the human cardiomyocytes with V122I TTR but not with T119M TTR, generates superoxide species and activates caspase3/7. In summary, our results show that the interaction of the amyloidogenic V122I TTR is distinct from that of a non-amyloidogenic TTR variant and is characterized by its retention at the cell membrane, where it initiates the cytotoxic cascade.

Emerging roles for retinoids in regeneration and differentiation in normal and disease states

Lorraine J. Gudas
Biochimica et Biophysica Acta 1821 (2012) 213–221
http://dx.doi.org:/10.1016/j.bbalip.2011.08.002

The vitamin (retinol) metabolite, all-transretinoic acid (RA), is a signaling molecule that plays key roles in the development of the body plan and induces the differentiation of many types of cells. In this review the physiological and pathophysiological roles of retinoids (retinol and related metabolites) in mature animals are discussed. Both in the developing embryo and in the adult, RA signaling via combinatorial Hoxgene expression is important for cell positional memory. The genes that require RA for the maturation/differentiation of T cells are only beginning to be cataloged, but it is clear that retinoids play a major role in expression of key genes in the immune system. An exciting, recent publication in regeneration research shows that ALDH1a2(RALDH2), which is the rate-limiting enzyme in the production of RA from retinaldehyde, is highly induced shortly after amputation in the regenerating heart, adult fin, and larval fin in zebrafish. Thus, local generation of RA presumably plays a key role in fin formation during both embryogenesis and in fin regeneration. HIV transgenic mice and human patients with HIV-associated kidney disease exhibit a profound reduction in the level of RARβ protein in the glomeruli, and HIV transgenic mice show reduced retinol dehydrogenase levels, concomitant with a greater than 3-fold reduction in endogenous RA levels in the glomeruli. Levels of endogenous retinoids (those synthesized from retinol within cells) are altered in many different diseases in the lung, kidney, and central nervous system, contributing to pathophysiology.

The Membrane Receptor for Plasma Retinol-Binding Protein, A New Type of Cell-Surface Receptor

Hui Sun and Riki Kawaguchi
Intl Review Cell and Molec Biol, 2011; 288:Chap 1. Pp 1:34
http://dx.doi.org:/10.1016/B978-0-12-386041-5.00001-7

Vitamin A is essential for diverse aspects of life ranging from embryogenesis to the proper functioning of most adul torgans. Its derivatives (retinoids) have potent biological activities such as regulating cell growth and differentiation. Plasma retinol-binding protein (RBP) is the specific vitamin A carrier protein in the blood that binds to vitamin A with high affinity and delivers it to target organs. A large amount of evidence has accumulated over the past decades supporting the existence of a cell-surface receptor for RBP that mediates cellular vitamin A uptake. Using an unbiased strategy, this specific cell-surface RBP receptor has been identified as STRA6, a multi-transmembrane domain protein with previously unknown function. STRA6 is not homologous to any protein of known function and represents a new type of cell-surface receptor. Consistent with the diverse functions of vitamin A, STRA6 is widely expressed in embryonic development and in adult organ systems. Mutations in human STRA6 are associated with severe pathological phenotypes in many organs
such as the eye, brain, heart, and lung. STRA6 binds to RBP with high affinity and mediates vitamin A uptake into cells. This review summarizes the history of the RBP receptor research, its expression in the context of known functions of vitamin A in distinct human organs, structure/function analysis of this new type of membrane receptor, pertinent questions regarding its very existence, and its potential implication in treating human diseases.

Choroid plexus dysfunction impairs beta-amyloid clearance in a triple transgenic mouse model of Alzheimer’s disease

Ibrahim González-Marrero, Lydia Giménez-Llort, Conrad E. Johanson, et al.
Front Cell Neurosc  Feb2015; 9(17): 1-10
http://dx.doi.org:/10.3389/fncel.2015.00017

Compromised secretory function of choroid plexus (CP) and defective cerebrospinal fluid (CSF) production, along with accumulation of beta-amyloid (Aβ) peptides at the blood-CSF barrier (BCSFB), contribute to complications of Alzheimer’s disease (AD). The AD triple transgenic mouse model (3xTg-AD) at 16 month-old mimics critical hallmarks of the human disease: β-amyloid (Aβ) plaques and neurofibrillary tangles (NFT) with a temporal-and regional-specific profile. Currently, little is known about transport and metabolic responses by CP to the disrupted homeostasis of CNS Aβ in AD. This study analyzed the effects of highly-expressed AD-linked human transgenes (APP, PS1 and tau) on lateral ventricle CP function. Confocal imaging and immunohistochemistry revealed an increase only of Aβ42 isoform in epithelial cytosol and in stroma surrounding choroidal capillaries; this buildup may reflect insufficient clearance transport from CSF to blood. Still, there was increased expression, presumably compensatory, of the choroidal Aβ transporters: the low density lipoprotein receptor-related protein1 (LRP1) and the receptor for advanced glycation end product (RAGE). A thickening of the epithelial basal membrane and greater collagen-IV deposition occurred around capillaries in CP, probably curtailing solute exchanges. Moreover, there was attenuated expression of epithelial aquaporin-1 and transthyretin(TTR) protein compared to Non-Tg mice. Collectively these findings indicate CP dysfunction hypothetically linked to increasing Aβ burden resulting in less efficient ion transport, concurrently with reduced production of CSF (less sink action on brain Aβ) and diminished secretion of TTR (less neuroprotection against cortical Aβ toxicity). The putative effects of a disabled CP-CSF system on CNS functions are discussed in the context of AD.

Endoplasmic reticulum: The unfolded protein response is tangled In neurodegeneration

Jeroen J.M. Hoozemans, Wiep Scheper
Intl J Biochem & Cell Biology 44 (2012) 1295–1298
http://dx.doi.org/10.1016/j.biocel.2012.04.023

Organelle facts•The ER is involved in the folding and maturation ofmembrane-bound and secreted proteins.•The ER exerts protein quality control to ensure correct folding and to detect and remove misfolded proteins.•Disturbance of ER homeostasis leads to protein misfolding and induces the UPR.•Activation of the UPR is aimed to restore proteostasis via an intricate transcriptional and (post)translational signaling network.•In neurodegenerative diseases classified as tauopathies the activation of the UPR coincides with the pathogenic accumulation of the microtubule associated protein tau.•The involvement of the UPR in tauopathies makes it a potential therapeutic target.

The endoplasmic reticulum (ER) is involved in the folding and maturation of membrane-bound and secreted proteins. Disturbed homeostasis in the ER can lead to accumulation of misfolded proteins, which trigger a stress response called the unfolded protein response (UPR). In neurodegenerative diseases that are classified as tauopathies, activation of the UPR coincides with the pathogenic accumulation of the microtubule associated protein tau. Several lines of evidence indicate that UPR activation contributes to increased levels of phosphorylated tau, a prerequisite for the formation of tau aggregates. Increased understanding of the crosstalk between signaling pathways involved in protein quality control in the ERand tau phosphorylation will support the development of new therapeutic targets that promote neuronal survival.

Chemical and/or biological therapeutic strategies to ameliorate protein misfolding diseases

Derrick Sek Tong Ong and Jeffery W Kelly
Current Opin Cell Biol 2011; 23:231–238
http://dx.doi.org:/10.1016/j.ceb.2010.11.002

Inheriting a mutant misfolding-prone protein that cannot be efficiently folded in a given cell type(s) results in a spectrum of human loss-of-function misfolding diseases. The inability of the biological protein maturation pathways to adapt to a specific misfolding-prone protein also contributes to pathology. Chemical and biological therapeutic strategies are presented that restore protein homeostasis, or proteostasis, either by enhancing the biological capacity of the proteostasis network or through small molecule stabilization of a specific misfolding-prone protein. Herein, we review the recent literature on therapeutic strategies to ameliorate protein misfolding diseases that function through either of these mechanisms, or a combination thereof, and provide our perspective on the promise of alleviating protein misfolding diseases by taking advantage of proteostasis adaptation.

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Stress and Anxiety

Posted in Cell Biology, Signaling & Cell Circuits, Child and Adolescent Psychiatry, Cognition, Curation, Disease Biology, Epigenetics and Cardiovascular Risks, Gastroenterology, HTN, Indigent Nutrition, Innovations in Neurophysiology & Neuropsychology, Lipids, Liver & Digestive Diseases Research, Neurological Diseases, Nitric Oxide in Health and Disease, Nutrition and Phytochemistry, Nutrition Disorders, Origins of Cardiovascular Disease, Population Health Management, Population Health Management, Nutrition and Phytochemistry, Prescription Drugs Costs, Uninsured and Underinsured, tagged , , , , , , on March 4, 2015| Leave a Comment »

Stress and Anxiety

Writer and Curator: Larry H Bernstein, MD, FCAP

 

Introduction

This article follows immediately after two on diet and obesity and diet and exercise. The hypothalamus has been discussed in some detail, although There is more that needs to be said about glutamate receptors, which is a topic in itself. However, this material fits in place quite well.  There is a considerable amount of obesity, and exercise is limited by time and commitment.  The shrinking middle class and the working poor, and the unemployed poor as well, have a struggle to make ends meet, and with the divorce rates that we are seeing, it is stressful for a single mother to carry on a complete life as mother and caregiver, and it is not unusual to see one or both couples in a household, regardless of sex, to hold two jobs.  Students enter colleges for higher education and leave with significant debts.  Graduates with advanced degrees may have to compete with a crowd of qualified applicants for an academic position, or even for a job in technology.  In addition, there is an increase in stress related disorders in the   pre-school, elementary and middle school population.  We no longer have to read the front pages to learn that a violent act has been carried out somewhere, in some neighborhood in our great nation that has experienced a great civil war, two world wars, the Mc Carthy hearings, the Cold War, and Vietnam, and the Iraq War, all of which was accompanied by migrations, immigration, and outsourcing of jobs.  The following is another look at how we are adjusting.

 

Effectiveness of a meditation-based stress management program as an adjunct to pharmacotherapy in patients with anxiety disorder

Sang Hyuk Lee, Seung Chan Ahn, Yu Jin Lee, Tae Kyu Choi, et al.
J Psychosomatic Research 62 (2007) 189–195
http://dx.doi.org:/10.1016/j.jpsychores.2006.09.009

Objective: The objective of this study was to examine the effectiveness of a meditation-based stress management program in patients with anxiety disorder.
Methods: Patients with anxiety disorder were randomly assigned to an 8-week clinical trial of either a meditation-based stress management program or an anxiety disorder education program. The Hamilton Anxiety Rating Scale (HAM-A), the Hamilton Depression Rating Scale (HAM-D), the State–Trait Anxiety Inventory (STAI), the Beck Depression Inventory, and the Symptom Checklist- 90 — Revised (SCL-90-R) were used to measure outcome at 0, 2, 4, and 8 weeks of the program. Results: Compared to the education group, the meditation-based stress management group showed significant improvement in scores on all anxiety scales (HAM-A, P=.001; STAI state, P=.001; STAI trait, P=.001; anxiety subscale of SCL-90-R,P=.001) and in the SCL-90-R hostility subscale (P=.01). Findings on depression measures were inconsistent, with no significant improvement shown by subjects in the meditation-based stress management group compared to those in the education group. The meditation-based stress management group did not show significant improvement in somatization, obsessive–compulsive symptoms, and interpersonal sensitivity scores, or in the SCL-90-R phobic anxiety subscale compared to the education group. Conclusions: A meditation-based stress management program can be effective in relieving anxiety symptoms in patients with anxiety disorder. However, well-designed, randomized, and controlled trials are needed to scientifically prove the worth of this intervention prior to treatment.

 

Evidence and Potential Mechanisms for Mindfulness Practices and Energy Psychology for Obesity and Binge-Eating Disorder

Renee Sojcher, Susan Gould Fogerite, and Adam Perlman
Explore 2012; 8(5):271-276
http://dx.doi.org/10.1016/j.explore.2012.06.003

Obesity is a growing epidemic. Chronic stress produces endocrine and immune factors that are contributors to obesity’s etiology. These biochemical alsocan affect appetite and eating behaviors that can lead to binge-eating disorder. The inadequacies of standard care and the problem of patient noncompliance have inspired a search for alternative treatments. Proposals in the literature have called for combination therapies involving behavioral or new biological therapies. This manuscript suggests that mindbody interventions would be ideal for such combinations. Two mind body modalities, energy psychology and mindfulness meditation, are reviewed for their potential in treating weight loss, stress, and behavior modification related to binge-eating disorder.

Whereas mindfulness meditation and practices show more compelling evidence, energy psychology, in the infancy stages of elucidation, exhibits initially promising outcomes but requires further evidence-based trials. “Diets Don’t Work” has been a mantra repeated over and over in the media. In fact, in a 2006 study in which investigators compared several popular diets comprising either high carbohydrates, high protein, or high fat, they found a rapid regression of compliance after six months, to the extent that it did not matter which diet had initially been more effective. In another study, authors examined a combination of diet and exercise compared with diet alone and observed that 50% of their subjects in both groups regained the weight that they lost after one year, despite their having lost more weight with the combination therapy. Despite the failure of diet alone in most studies, strategies incorporating both diet and exercise can be effective: a Cochrane review on exercise for overweight or obesity concluded that exercise had a positive effect on body weight and cardiovascular risk factors and that this effect was enhanced by a combination of exercise with dietary interventions.

The authors of a more recent study found that the benefits of exercise in inducing weight loss may come through psychological pathways rather than through actual energy expenditure. These factors include self-regulation and self-efficacy, which may mediate the relationship between exercise and weight change. Psychological interventions, particularly behavioral therapy and CBT, have been shown to be effective, especially when combined with diet and exercise. However, these interventions are costly and require extensive clinical contact for long durations to achieve efficacy. The authors of a recent randomized controlled trial (RCT) with a three-year follow-up period looked at a new form of CBT that addresses patients’ overeating and low level of activity, as well as factors that impede weight maintenance, and found that this form of therapy did not result in improved weight maintenance. These authors concluded that CBT is not sufficiently effective in helping patients maintain their weight loss in the long term. Although 20% of people will not change their eating behaviors under stress, most do; approximately 40% will increase and 40% will decrease their eating.

The emotional eaters, who tend to increase food intake, are more likely to crave high-fat/sweet and rewarding comfort foods. The basis for this behavior is becoming understood to entail brain pathways that involve learning and memory of reward and pleasure. Habit formation and decreased cognitive control are also involved. These habits form the basis of BED. Binge eating occurs when a person eats larger amounts of food than normal in a short amount of time. It therefore involves a loss of control and is often precipitated by a range of negative emotions, such as anxiety, depression, anger, and loneliness. Overweight subjects may or may not be characterized as binge eaters.

The stress response, also known as the “fight or flight response,” involves the interaction of the autonomic nervous system, which includes the sympathetic and the parasympathetic nervous systems, the hypothalamic pituitary adrenal axis and endocrine secretion. Together, these systems comprise neuro-endocrine pathways that collaborate to maintain the body’s regulation of homeostasis. This mechanism is very effective when stress is acute, but in the case of chronic stress, the effect can be injurious to one’s physiological state. Over time, chronic exposure to stress hormones contributes to“ allostatic load.” The stress hormones released by the body, mostly cortisol, can alter the body’s fuel metabolism, especially by adipose tissue, leading to an increase in upper-body obesity. Furthermore, hormones such as leptin, ghrelin, and neuropeptide Y can affect appetite and cause changes in fat mass storage. This results in the linking of stress and obesity.

Given the limited success of conventional approaches and the new information about the psychological and physiological mechanisms underlying obesity, we propose that a specific sub-group of mind-body therapies, including energy psychology and mindfulness-based approaches, could add an important new dimension to the integrative treatment of eating disorders. Energy psychology refers to a family of therapies that are used for treating physical disorders and psychological symptoms, which includes Thought Field Therapy, Emotional Freedom Techniques (EFT), Eye Movement Desensitization and Reprocessing, and Tapas Acupressure Technique (TAT). These therapies incorporate concepts originating from non-Western healing and spiritual systems, including acupuncture, acupressure, yoga, meditation, and qigong, and they combine physical activity with mental activation on the basis of the premise that the body is composed of electrical signals or energy fields. Energy psychology has been quite controversial among psychotherapists and has been the subject of much heated debate in the literature. Nonetheless, the clinical application of these practices is growing and is beginning to be investigated for efficacy. Mindfulness-Based Eating Awareness Training (ie,MB-EAT) involves the cultivation of mindfulness, mindful eating, emotional balance, and self-acceptance.

A pilot trial of a six-week group curriculum for providing mindfulness training to obese individuals, called Mindful Eating and Living (ie,MEAL), showed significant increases in measures of mindfulness and cognitive restraint around eating and significant decreases in weight, eating disinhibition, bingeeating, depression, perceived stress, physical symptoms, negative affect ,and C-reactive protein. In a recent systematic review of eight studies, authors examined a variety of mindfulness techniques in treating eating disorders, including anorexia, bulimia, and BED. Because trial quality varied and sample sizes were small, the researchers concluded that mindfulness may be effective in treating eating disorders but that further research was needed. The authors noted, however, that all of the articles that met the study’s criterion reported positive outcomes for the mindfulness intervention. Two additional studies recently addressed the treatment of obesity with a combination of mindfulness strategies and ACT. Lillis et al. conducted a RCT on 87 subjects who had all completed at least a six-month weight loss program. Using a wait list control against treatment of the experimental group through a one-day workshop, the authors found that, compared with the control group, the experimental group showed greater improvements in obesity-related stigma, quality of life, psychological distress, and reduction of body mass in a three-month follow-up. Alberts et al. conducted an RCT on 19 participants in a 10-week dietary group treatment that examined the effect of mindfulness plus ACT on food cravings. Experimental subjects underwent an additional seven-week, manual-based mindfulness/acceptance training. The control group received information on healthy food choices. The experimental group showed significantly lower food cravings, a lower preoccupation with food in four subscales, less loss of control, and better positive outcome expectancy, as compared with the control group. There was no significant effect observed for emotional craving. The authors of both of these studies conclude that mindfulness strategies combined with acceptance are effective in reducing the behaviors that lead many obese patients to overeat. With regards to stress, mindfulness can reduce psychological factors that have been shown to contribute to obesity.

In a recent well conducted systematic review, Mars and Abbey examined 22 studies with conditions ranging from participants with Axis I disorders, various diagnosed medical disorders, and healthy subjects. Axis I disorders include a range of psychopathologies such as childhood developmental and adjustment abnormalities, adult anxiety, and mood, sleep, and sexual disorders. Subjects with BED are known to have greater comorbidity forAxis I disorders. The authors report that five studies examining Axis I disorders showed statistically significant results for an eight-week, two hours per week MBCT program in reducing psychological stress, recurring bouts of depression, and pain. They conclude that, despite some methodological difficulties in the trials, mindfulness therapy may have a positive impact on reducing stress and depression. Despite increasing public awareness of obesity’s detrimental effects on health, the conventional approaches to managing this condition have not been effective. The recommended standard care for overweight and obesity, namely diet and exercise, are for the most part ineffective in the long term. Behavioral therapy and CBT may have some effect but are costly and difficult to implement. Issues with bariatric surgery and pharmacological therapies attributable to cost and the potential for harm, as well as lack of long-term efficacy, have limited their utility.

The effectiveness of a stress coping program based on mindfulness meditation on the stress, anxiety, and depression experienced by nursing students in Korea

Yune Sik Kang, So Young Choi, Eunjung Ryu
Nurse Education Today 29 (2009) 538–543
http://dx.doi.org:/10.1016/j.nedt.2008.12.003

This study examined the effectiveness of a stress coping program based on mindfulness meditation on the stress, anxiety, and depression experienced by nursing students in Korea. A nonequivalent, control group, pre-posttest design was used. A convenience sample of 41 nursing students were randomly assigned to experimental (n=21) and control groups (n=20). Stress was measured with the PWI-SF(5-point) developed by Chang. Anxiety was measured with Spieberger’s state anxiety y inventory. Depression was measured with the Beck depression inventory. The experimental group attended 90-min sessions for eight weeks. No intervention was administered to the control group. Nine participants were excluded from the analysis because they did not complete the study due to personal circumstances, resulting in16 participants in each group for the final analysis. Results for the two groups showed

(1) a significant difference in stress scores (F=6.145,p=0.020),

(2) a significant difference in anxiety scores (F=6.985,p=0.013), and

(3) no significant difference in depression scores (t=1.986,p=0.056).

A stress coping program based on mindfulness meditation was an effective intervention for nursing students to decrease their stress and anxiety, and could be used to manage stress in student nurses. In the future, long-term studies should be pursued to standardize and detail the program, with particular emphasis on studies to confirm the effects of the program in patients with diseases, such as cancer.

 

 

Meditation and Anxiety Reduction: A Literature Review

M. M. Delmonte Clin
Psychol Rev 1985; 5: 91-102
Meditation is increasingly being practiced as a therapeutic technique. The effects of practice on psychometrically assessed anxiety levels has been extensively researched. Prospective meditators tend to report above average anxiety. In general, high anxiety levels predict a subsequent low frequency of practice. However, the evidence suggests that those who practice regularly tend to show significant decreases in anxiety. Meditation does not appear to be more effective than comparative interventions in reducing anxiety. There is evidence to suggest that hypnotizability and expectancy may both play a role in reported anxiety decrease. Certain individuals with a capacity to engage in autonomous self-absorbed relaxation, may benefit most from meditation.

 

Meta-analysis on the effectiveness of mindfulness-based stress reduction therapy on mental health of adults with a chronic disease: What should the reader not make of it?

Ernst Bohlmeijer, Rilana Prenger, ErikTaal
Letters to the Editor/J Psychosom Res 69 (2010) 613–615
http://dx.doi.org:/10.1016/j.jpsychores.2010.09.005

In a letter to the editor, Nyklíček et al. discuss the study of Bohlmeijer et al. [1]on the meta-analysis on the effectiveness of mindfulness-based stress reduction (MBSR) therapy on mental health of adults with a chronic disease. They claim that the effects of MBSR are underestimated in this meta-analysis due to the inclusion of a study using an active education support group as control group and to the omission of some subscales for which larger effect sizes have been found. We do not agree that the study using an active education support group as a control group should not have been included in the meta-analysis. It is a common procedure to include studies with various types of control groups, e.g., waiting-list, placebo, minimal interventions, or evidence-based treatment. Normally, subgroup analyses can be conducted, contrasting studies that use differen ttypes of control groups. As seven studies used a waiting-list control condition and only one study used an education support group, this subgroup comparison was not useful. However, when we conducted a meta-analysis of the seven RCTs using a waiting-list control group an overall effect size of 0.30 instead of 0.26 was found. In addition, it is often found in meta-analyses that the largest effect sizes are reported in studies that use waiting-list control groups, e.g. ,Refs.[2,3]. The fact that almost all studies included in our meta-analysis in fact used waiting-list control groups makes it unlikely that the effects of MBSR were underestimated. As to the second claim by Nyklíček e tal.that some outcomes were selectively omitted from the meta-analysis, we can state that the subscales of the POMS were included in the meta-analysis.The program that was used in our study, Comprehensive Meta-Analysis, combined the scales that measure the same outcome, e.g., anxiety in one study. So the larger effects sizes for the subscales of the POMS were included in the meta-analysis. Lastly, Nyklíčeketal. State that ‘decentering’ is not an exclusive process of MBCT but is a central feature of MBSR as well. MBCT was specifically developed for people with recurrent depression and on the basis of a thorough analysis of the role of specific cognitions in people with recurrent depression. In ouropinion, this may explain the large effect sizes that have been found in randomized controlled trials, e.g., [4]. In general, other studies have shown that integrating MBSR in behavioral therapy is a very promising strategy for enhancing the efficacy of treatments of psychological  distress[5,6]. However, more studies with different target groups are needed to answer the question as to which mindfulness-based intervention is most effective for which target group in which setting. Overall, in response to the letter to the editor by Nyklíček et al. we cannot corroborate their claim that the effects of MBSR were underestimated and have to stand with our conclusion that, on the basis of current RCTs, MBSR has small leffects on depression and anxiety in people with chronic medical diseases.

[1] BohlmeijerET, PrengerR, TaalE, CuijpersP.
The effects of mindfulness-based stress reduction therapy on the mental health of adults with a chronic medical disease: A meta-analysis.
JPsychosom Res 2010; 68:539–44.

[2]Powers MB, Zum Vörde Sive Vörding MB, Emmelkamp PMG.
Acceptance and commitment therapy: A meta-analytic review.
Psychoth Psychosom 2009; 78:73–80.

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Depth Underwater and Underground

Posted in Amino acids, Best evidence, Biochemical pathways, Biological Networks, Biological Networks, Gene Regulation and Evolution, Biomedical Measurement Science, Cell Biology, Cell Biology, Signaling & Cell Circuits, Curation, Curation methodology, Developmental biology, Diabetes Mellitus, Dialectic, Discovery process, Electrophysiology, Evolutionary cognition, Explanatory, Fatty acids, Gene Regulation, Gene Regulation and Evolution, Genome Biology, Hematopoiesis, Historical relevance, Innovations, Lipid metabolism, Metabolism, Mutant Gene Expression, Myocardial Ischemia, Myocardial Metabolism, Na-K transport, Na-K-ATPase, Neurological Diseases, Nutrition, Proteins, Proteomics, Signaling & Cell Circuits, Theoretic convergence, Translational Research, Translational Science, tagged , , , , , , , , , , , , , , , , on February 24, 2015| Leave a Comment »

Depth Underwater and Underground

Writer and Curator: Larry H. Bernstein, MD, FCAP 

 

Introduction

Deep diving for mammals is dangerous for humans and land based animals for too long, and it has dangerous consequences, most notable in nitrogen emboli  with very deep underwater diving. Other mammals live in water and have adapted to a water habitat.  This is another topic that needs further exploration.

Deep diving has different meanings depending on the context. Even in recreational diving the meaning may vary:

In recreational diving, a depth below about 30 metres (98 ft), where nitrogen narcosis becomes a significant hazard for most divers, may be considered a “deep dive”

In technical diving, a depth below about 60 metres (200 ft) where hypoxic breathing gas becomes necessary to avoid oxygen toxicity may be considered a “deep dive”.

Early experiments carried out by Comex S.A. (Compagnie maritime d’expertises) using hydrox and trimix attained far greater depths than any recreational technical diving. One example being the Comex Janus IV open-sea dive to 501 metres (1,644 ft) in 1977. The open-sea diving depth record was achieved in 1988 by a team of Comex divers who performed pipe line connection exercises at a depth of 534 metres (1,752 ft) in the Mediterranean Sea as part of the Hydra 8 program. These divers needed to breathe special gas mixtures because they were exposed to very high ambient pressure (more than 50 times atmospheric pressure).

Then there is the adaptation to the water habitat as a living environment. The two main types of aquatic ecosystems are marine ecosystems and freshwater ecosystems.

http://en.wikipedia.org/wiki/Deep_diving

Marine ecosystems are part of the earth’s aquatic ecosystem. The habitats that make up this vast system range from the productive nearshore regions to the barren ocean floor. The marine waters may be fully saline, brackish or nearly fresh. The saline waters have a salinity of 35-50 ppt (= parts per thousand). The freshwater has a salinity of less than 0.5 ppt. The brackish water lies in between these 2. Marine habitats are situated from the coasts, over the continental shelf to the open ocean and deep sea. The ecosystems are sometimes linked with each other and are sometimes replacing each other in other geographical regions. The reason why habitats differ from another is because of the physical factors that influence the functioning and diversity of the habitats. These factors are temperature, salinity, tides, currents, wind, wave action, light and substrate.

Marine ecosystems are home to a host of different species ranging from planktonic organisms that form the base of the marine food web to large marine mammals. Many species rely on marine ecosystems for both food and shelter from predators. They are very important to the overall health of both marine and terrestrial environments. Coastal habitats are those above the spring high tide limit or above the mean water level in non-tidal waters.  They are close to the sea and include habitats such as coastal dunes and sandy shores, beaches , cliffs and supralittoral habitats. Coastal habitats alone account for approximately 30% of all marine biological productivity.

http://www.marbef.org/wiki/marine_habitats_and_ecosystems

All plant and animal life forms are included from the microscopic picoplankton all the way to the majestic blue whale, the largest creature in the sea—and for that matter in the world. It wasn’t until the writings of Aristotle from 384-322 BC that specific references to marine life were recorded. Aristotle identified a variety of species including crustaceans, echinoderms, mollusks, and fish.
Today’s classification system was developed by Carl Linnaeus external link as an important tool for use in the study of biology and for use in the protection of biodiversity. Without very specific classification information and a naming system to identify species’ relationships, scientists would be limited in attempts to accurately describe the relationships among species. Understanding these relationships helps predict how ecosystems can be altered by human or natural factors.

Preserving biodiversity is facilitated by taxonomy. Species data can be better analyzed to determine the number of different species in a community and to determine how they might be affected by environmental stresses. Family, or phylogenetic, trees for species help predict environmental impacts on individual species and their relatives.

http://marinebio.org/oceans/marine-taxonomy/

For generations, whales and other marine mammals have intrigued humans. 2,400 years ago, Aristotle, a Greek scientist and philosopher, recognized that whales are mammals, not fish, because they nurse their young and breathe air like other mammals. There are numerous myths and legends surrounding marine mammals. The Greeks believed that killing a dolphin was as bad as murdering a human. An Amazon legend said that river dolphins came to shore dressed as men to woo pretty girls during fiestas. During the Middle Ages, there were numerous legends surrounding the narwhals’ amazing tusk, which was thought to have come from the unicorn.

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Marine mammals evolved from their land dwelling ancestors over time by developing adaptations to life in the water. To aid swimming, the body has become streamlined and the number of body projections has been reduced. The ears have shrunk to small holes in size and shape. Mammary glands and sex organs are not part of the external physiology, and posterior (hind) limbs are no longer present.

Mechanisms to prevent heat loss have also been developed. The cylindrical body shape with small appendages reduces the surface area to volume ratio of the body, which reduces heat loss. Marine mammals also have a counter current heat exchange mechanism created by convergent evolution external link where the heat from the arteries is transferred to the veins as they pass each other before getting to extremities, thus reducing heat loss. Some marine mammals also have a thick layer of fur with a water repellent undercoat and/or a thick layer of blubber that can’t be compressed. The blubber provides insulation, a food reserve, and aids with buoyancy. These heat loss adaptations can also lead to overheating for animals that spend time out of the water. To prevent overheating, seals or sea lions will swim close to the surface with their front flippers waving in the air. They also flick sand onto themselves to keep the sun from directly hitting their skin. Blood vessels can also be expanded to act as a sort of radiator.

One of the major behavioral adaptations of marine mammals is their ability to swim and dive. Pinnipeds swim by paddling their flippers while sirenians and cetaceans move their tails or flukes up and down.

Some marine mammals can swim at relatively high speeds. Sea lions swim up to 35 kph and orcas can reach 50 kph. The fastest marine mammal, however, is the common dolphin, which reaches speeds up to 64 kph. While swimming, these animals take very quick breaths. For example, fin whales can empty and refill their huge lungs in less than 2 seconds. Marine mammals’ larynx and esophagus close automatically when they open their mouths to catch prey during dives. Oxygen is stored in hemoglobin in the blood and in myoglobin in the muscles. The lungs are also collapsible so that air is pushed into the windpipe preventing excess nitrogen from being absorbed into the tissues. Decreasing pressure can cause excess nitrogen to expand in the tissues as animals ascend to shallower depths, which can lead to decompression sickness,  aka “the bends.” Bradycardia, the reduction of heart rate by 10 to 20%, also takes place to aid with slowing respiration during dives and the blood flow to non-essential body parts. These adaptations allow sea otters to stay submerged for 4 to 5 minutes and dive to depths up to 55 m. Pinnipeds can often stay down for 30 minutes and reach average depths of 150-250 m. One marine mammal with exceptional diving skills is the Weddell seal, which can stay submerged for at least 73 minutes at a time at depths up to 600 m. The length and depth of whale dives depends on the species. Baleen whales feed on plankton near the surface of the water and have no need to dive deeply so they are rarely seen diving deeper than 100 m external link. Toothed whales seek larger prey at deeper depths and some can stay down for hours at depths of up to 2,250 m external link.

http://marinebio.org/oceans/marine-mammals/

Human Experience

Albert Behnke: Nitrogen Narcosis

Casey A. Grover and David H. Grover
The Journal of Emergency Medicine, 2014; 46(2):225–227
http://dx.doi.org/10.1016/j.jemermed.2013.08.080

As early as 1826, divers diving to great depths noted that descent often resulted in a phenomenon of intoxication and euphoria. In 1935, Albert Behnke discovered nitrogen as the cause of this clinical syndrome, a condition now known as nitrogen narcosis. Nitrogen narcosis consists of the development of euphoria, a false sense of security, and impaired judgment upon underwater descent using compressed air below 34 atmospheres (99 to 132 feet). At greater depths, symptoms can progress to loss of consciousness. The syndrome remains relatively unchanged in modern diving when compressed air is used. Behnke’s use of non-nitrogencontaining gas mixtures subsequent to his discovery during the 1939 rescue of the wrecked submarine USS Squalus pioneered the use of non-nitrogencontaining gas mixtures, which are used by modern divers when working at great depth to avoid the effects of nitrogen narcosis.

Behnke’s first duty station as a licensed physician was as assistant medical officer for Submarine Division 20 in San Diego, which was then commanded by one of the Navy’s rising stars, Captain Chester W. Nimitz of World War II fame.
In this setting, Dr. Behnke spent his free time constructively by learning to dive, using the traditional ‘‘hard-hat’’ gear aboard the USS Ortalon, a submarine rescue vessel to which he also rotated. Diving was not a notable specialty of the Navy at the time, and the service was slow in developing the infrastructure for it. Dr. Behnke devoted his efforts to research on the topic of diving medicine, as well as developing a more sound understanding of the biophysics of diving. In 1932, he wrote a letter to the Surgeon General describing some of his observations on arterial gas embolism, which earned him some accolades from the Navy and resulted in his transfer to Harvard’s School of Public Health as a graduate fellow. After 2 years at Harvard, the Navy assigned duty to Dr. Behnke at the Navy’s submarine escape training tower at Pearl Harbor. He worked extensively here on developing techniques for rescuing personnel from disabled submarines on the sea floor. In 1937, he was one of three Navy physicians assigned to the Navy’s Experimental Diving Unit. This team worked on improving the rescue system, plus updating the diving recompression tables originally developed by the British in 1908.

The intoxicating effects of diving were first described by a French physician named Colladon in 1826, who reported that descent in a diving bell resulted in his feeling a ‘‘state of excitement as though I had drunk some alcoholic liquor’’.
The etiology of this phenomenon remained largely unknown until the 1930s, when the British military researcher Damant again highlighted the issue, and reported very unpredictable behavior in his divers during descents as deep as 320 feet during the British Admiralty Deep Sea diving trials. Two initial theories arose as to the etiology for this effect, the first being from psychological causes by Hill and Phillip in 1932, and the second being from oxygen toxicity by Haldane in 1935.

Dr. Behnke and his colleagues at the Harvard School of Public Health had another idea as to the etiology of this phenomenon. In 1935, based on observation of individuals in experiments with a pressure chamber, Dr. Behnke published an article in the American Journal of Physiology in which he posited that nitrogen was the etiology of the intoxicating effects of diving.

Nitrogen narcosis, described as ‘‘rapture of the deep’’ by Jacques Cousteau, still remains a relatively common occurrence in modern diving, despite major advances in diving technology since Behnke’s initial description of the pathophysiologic cause of the condition in 1935. The development of symptoms of this condition varies from diver to diver, but usually begins when a depth of 4 atmospheres (132 feet) is reached in divers using compressed air. More sensitive divers can develop symptoms at only 3 atmospheres (99 feet), and other divers may not be affected up to depths as high as 6 atmospheres (198 feet). Interestingly, tolerance to nitrogen narcosis can be developed by frequent diving and exposure to the effects of compressed air at depth.

  1. Acott C. A brief history of diving and decompression illness. SPUMS J 1999;29:98–109.
    2. Bornmann R. Dr. Behnke, founder of UHMS, dies. Pressure 1992; 21:14.
    3. Behnke AR, Thomson RM, Motley P. The psychologic effects from breathing air at 4 atmospheric pressures. Am J Physiol 1935; 112:554–8.
    4. Behnke AR, Johnson FS, Poppen JR, Motley P. The effect of oxygen on man at pressures from 1 to 4 atmospheres. AmJ Physiol 1934; 110:565–72.

Exhaled nitric oxide concentration and decompression-induced bubble formation: An index of decompression severity in humans?

J.-M. Pontier, Buzzacott, J. Nastorg, A.T. Dinh-Xuan, K. Lambrechts
Nitric Oxide 39 (2014) 29–34
http://dx.doi.org/10.1016/j.niox.2014.04.005

Introduction: Previous studies have highlighted a decreased exhaled nitric oxide concentration (FE NO) in divers after hyperbaric exposure in a dry chamber or following a wet dive. The underlying mechanisms of this decrease remain however unknown. The aim of this study was to quantify the separate effects of submersion, hyperbaric hyperoxia exposure and decompression-induced bubble formation on FE NO after a wet dive.
Methods: Healthy experienced divers (n = 31) were assigned to either

  • a group making a scuba-air dive (Air dive),
  • a group with a shallow oxygen dive protocol (Oxygen dive) or

a group making a deep dive breathing a trimix gas mixture (deep-dive).
Bubble signals were graded with the KISS score. Before and after each dive FE NO values were measured using a hand-held electrochemical analyzer.
Results: There was no change in post-dive values of FE NO values (expressed in ppb = parts per billion) in the Air dive group (15.1 ± 3.6 ppb vs. 14.3 ± 4.7 ppb, n = 9, p = 0.32). There was a significant decrease in post-dive values of FE NO in the Oxygen dive group (15.6 ± 6 ppb vs. 11.7 ± 4.7 ppb, n = 9, p = 0.009). There was an even more pronounced decrease in the deep dive group (16.4 ± 6.6 ppb vs. 9.4 ± 3.5 ppb, n = 13, p < 0.001) and a significant correlation between KISS bubble score >0 (n = 13) and percentage decrease in post-dive FE NO values (r = -0.53, p = 0.03). Discussion: Submersion and hyperbaric hyperoxia exposure cannot account entirely for these results suggesting the possibility that, in combination, one effect magnifies the other. A main finding of the present study is a significant relationship between reduction in exhaled NO concentration and dive-induced bubble formation. We postulate that exhaled NO concentration could be a useful index of decompression severity in healthy human divers.

Brain Damage in Commercial Breath-Hold Divers

Kiyotaka Kohshi, H Tamaki, F Lemaıtre, T Okudera, T Ishitake, PJ Denoble
PLoS ONE 9(8): e105006 http://dx.doi.org:/10.1371/journal.pone.0105006

Background: Acute decompression illness (DCI) involving the brain (Cerebral DCI) is one of the most serious forms of diving related injuries which may leave residual brain damage. Cerebral DCI occurs in compressed air and in breath-hold divers, likewise. We conducted this study to investigate whether long-term breath-hold divers who may be exposed to repeated symptomatic and asymptomatic brain injuries, show brain damage on magnetic resonance imaging (MRI).
Subjects and Methods: Our study subjects were 12 commercial breath-hold divers (Ama) with long histories of diving work in a district of Japan. We obtained information on their diving practices and the presence or absence of medical problems, especially DCI events. All participants were examined with MRI to determine the prevalence of brain lesions.
Results: Out of 12 Ama divers (mean age: 54.965.1 years), four had histories of cerebral DCI events, and 11 divers demonstrated ischemic lesions of the brain on MRI studies. The lesions were situated in the cortical and/or subcortical area (9 cases), white matters (4 cases), the basal ganglia (4 cases), and the thalamus (1 case). Subdural fluid collections were seen in 2 cases. Conclusion: These results suggest that commercial breath-hold divers are at a risk of clinical or subclinical brain injury which may affect the long-term neuropsychological health of divers.

Decompression illness

Richard D Vann, Frank K Butler, Simon J Mitchell, Richard E Moon
Lancet 2010; 377: 153–64

Decompression illness is caused by intravascular or extravascular bubbles that are formed as a result of reduction in environmental pressure (decompression). The term covers both arterial gas embolism, in which alveolar gas or venous gas emboli (via cardiac shunts or via pulmonary vessels) are introduced into the arterial circulation, and decompression sickness, which is caused by in-situ bubble formation from dissolved inert gas. Both syndromes can occur in divers, compressed air workers, aviators, and astronauts, but arterial gas embolism also arises from iatrogenic causes unrelated to decompression. Risk of decompression illness is
affected by immersion, exercise, and heat or cold. Manifestations range from itching and minor pain to neurological symptoms, cardiac collapse, and death. First aid treatment is 100% oxygen and definitive treatment is recompression to increased pressure, breathing 100% oxygen. Adjunctive treatment, including fluid administration and prophylaxis against venous thromboembolism in paralyzed patients, is also recommended. Treatment is, in most cases, effective although residual deficits can remain in serious cases, even after several recompressions.

Bubbles can have mechanical, embolic, and biochemical effects with manifestations ranging from trivial to fatal. Clinical manifestations can be caused by direct effects from extravascular (autochthonous) bubbles such as mechanical distortion of tissues causing pain, or vascular obstruction causing stroke-like signs and symptoms. Secondary effects can cause delayed symptom onset up to 24 h after surfacing. Endothelial damage by intravascular bubbles can cause capillary leak, extravasation of plasma, and haemoconcentration. Impaired endothelial function, as measured by decreased effects of vasoactive compounds, has been reported in animals and might occur in man. Hypotension can occur in severe cases. Other effects include platelet activation and deposition, leucocyte-endothelial adhesion, and possibly consequences of vascular occlusion believed to occur in thromboembolic stroke such as ischaemia-reperfusion injury, and apoptosis.

Classification of initial and of all eventual manifestations of decompression illness in 2346 recreational diving accidents reported to the Divers Alert Network from 1998 to 2004 For all instances of pain, 58% consisted of joint pain, 35% muscle pain, and 7% girdle pain. Girdle pain often portends spinal cord involvement. Constitutional symptoms included headache, lightheadedness, inappropriate fatigue, malaise, nausea or vomiting, and anorexia. Muscular discomfort included stiffness, pressure, cramps, and spasm but excluded pain. Pulmonary manifestations included dyspnoea and cough.

Other than depth and time, risk of decompression sickness is affected by other factors that affect inert gas exchange and bubble formation, such as immersion (vs dry hyperbaric chamber exposure), exercise, and temperature. Immersion decreases venous pooling and increases venous return and cardiac output. Warm environments improve peripheral perfusion by promoting vasodilation, whereas cool temperatures decrease perfusion through vasoconstriction. Exercise increases both peripheral perfusion and temperature. The effect of environmental conditions on risk of decompression sickness is dependent on the phase of the pressure exposure. Pressure, exercise, immersion, or a hot environment increase inert gas uptake and risk of decompression sickness. During decom-pression these factors increase inert gas elimination and therefore decrease the risk of decompression sickness. Conversely, uptake is reduced during rest or in a cold environment, hence a diver resting in a cold environment on the bottom has decreased risk of decompression sickness. Rest or low temperatures during decompression increase the risk. If exercise occurs after decompression when super-saturation is present, bubble formation increases and risk of decompression sickness rises.

Exercise at specific times before a dive can decrease the risk of serious decompression sickness in animals and incidence of venous gas emboli in both animals and man. The mechanisms of these effects are unknown but might involve modulation of nitric oxide production and effects on endothelium. Venous gas emboli and risk of decompression sickness increase slightly with age and body-mass index.

Arterial gas embolism should be suspected if a diver has a new onset of altered consciousness, confusion, focal cortical signs, or seizure during ascent or within a few minutes after surfacing from a compressed gas dive.

If the diver spends much time at depth and might have absorbed substantial inert gas before surfacing, arterial gas embolism and serious decompression sickness can coexist, and in such cases, spinal cord manifestations can predominate. Other organ systems, such as the heart, can also be affected, but the clinical diagnosis of gas embolism is not reliable without CNS manifestations. Arterial gas embolism is rare in altitude exposure; if cerebral symptoms occur after altitude exposure, the cause is usually decompression sickness.

Nondermatomal hypoaesthesia and truncal ataxia are common in neurological decompression sickness and can be missed by cursory examination. Pertinent information includes level of consciousness and mental status, cranial nerve function, and motor strength. Coordination can be affected disproportionately, and abnormalities can be detected by assessment of finger-nose movement, and, with eyes open and closed, ability to stand and walk and do heel-toe walking backwards and forwards. Many of these simple tests can be done on the scene by untrained companions.

Panel: Differential diagnosis of decompression illness
Inner-ear barotrauma
Middle-ear or maxillary sinus overinfl ation
Contaminated diving gas and oxygen toxic effects
Musculoskeletal strains or trauma sustained before, during, or after diving
Seafood toxin ingestion (ciguatera, pufferfish, paralytic shellfish poisoning)
Immersion pulmonary edema
Water aspiration
Decompression chamber

Decompression chamber

Decompression chamber. fluidic or pneumatic ventilator is shown at the left. The infusion pump is contained within a plastic cover, in which 100% nitrogen is used to decrease the fi re risk in the event of an electrical problem. The monitor screen is outside the chamber and can be seen through the viewing port. Photo from Duke University Medical Center, with permission.

Long-term outcomes of 69 divers with spinal cord decompressionsickness, by manifestation
n %
No residual symptoms 34 49·3
Any residual symptom 35 50·7
Mild paraesthesias, weakness, or pain 14 20·3
Some impairment of daily activities 21 30·4
Difficulty walking 11 15·9
Impaired micturition 13 18·8
Impaired defecation 15 21·7
Impaired sexual function 15 21·7

Decompression illness occurs in a small population but is an international problem that few physicians are trained to recognise or manage. Although its manifestations are often mild, the potential for permanent injury exists in severe cases, especially if unrecognised or inadequately treated. Emergency medical personnel should be aware of manifestations of decompression illness in the setting of a patient with a history of recent diving or other exposure to substantial pressure change, and should contact an appropriate consultation service for advice.

Diving Medicine: Contemporary Topics and Their Controversies

Michael B. Strauss and Robert C. Borer, Jr
Am J Emerg Med 2001; 19:232-238
http://dx.doi.org:/10.1053/ajem.2001.22654

SCUBA diving is a popular recreational sport. Although serious injuries occur infrequently, when they do knowledge of diving medicine and/or where to obtain appropriate consultation is essential. The emergency physician is likely to be the first physician contact the injured diver has. We discuss 8 subjects
in diving medicine which are contemporary, yet may have controversies associated with them. From this information the physician dealing primarily with the injured diver will have a basis for understanding and managing, as
well as where to find additional help, for his/her patients’ diving injuries.

Over the past 10 years, new knowledge and equipment improvements have made diving safer and more enjoyable. Estimates of actively participating sports divers show a striking increase over this time interval while the number of SCUBA diving deaths annually has remained nearly level at approximately 100. A further indicator of recreational diving safety is that reflected in the nearly constant number of diving injuries (1000 per annum) over the most recent 5 reported years, or approximately 0.53 to 3.4 incidents/10,000 dives.

Divers Alert Network.
The Divers Alert Network (DAN) is a nonprofit organization directed and staffed by experts in the specialty of diving medicine.6 DAN provides immediate consultation for both divers and physicians in the diagnosis and initial management of diving injuries. This 24-hour service is available free world-wide through a dedicated emergency telephone line: 1-919-684-4326. The DAN staff will also identify the nearest appropriate recompression treatment facility and knowledgeable physicians for an expedient referral. General diving medical inquiries can be answered during normal weekday hours either through an information telephone line: 1-919-684-2948 or through an interactive web site http://www.diversalertnetwork.org.

Use of 100% Oxygen for Initial, on the Scene, Management of Diving Accidents
The breathing of pure oxygen is crucial for the initial management of the diving related problems of arterial gas embolism (AGE), decompression sickness (DCS), pulmonary barotrauma (thoracic squeeze), aspiration pneumonitis, and hypoxic encephalopathy associated with near drowning. In 1985, Dick reported that in many cases the neurologic symptoms of AGE and DCS were resolved with the immediate breathing of pure oxygen on the surface. The breathing of pure oxygen reduces bubble size by increasing the differential pressure for the inert gas to diffuse out of the bubble and it also speeds the washout of inert gas from body tissues. The early elimination of the bubble prevents hypoxia and the interaction of the bubble with the blood vessel lining. This interaction leads to secondary problems of capillary leak, bleeding, inflammation, ischemia, and cell death. These secondary problems are the reasons not all DCS symptoms resolve with recompression chamber treatment. The immediate use of pure oxygen for the medical management of these diving problems is analogous to the use of cardiopulmonary resuscitation for the witnessed cardiac arrest; the sooner initiated the better the results.

Diving Education

Medical Fitness for Diving

Asthma has the potential risk for AGE. Neuman reviewed the subject of asthma and diving. He and his coauthors recommend that asthmatics who are asymptomatic, not on medications and have no exercised induced abnormality on pulmonary function studies be allowed to dive.

Conditions leading to loss of consciousness, such as insulin dependent diabetes and epilepsy, can result in drowning. Carefully controlled diving studies in diabetics, who are free from complications, are now defining the safe requirements for diving. Epilepsy remains as a disqualification except in individuals with a history of febrile seizures ending prior to 5 years of age.

Availability of Hyperbaric Oxygen Treatment Facilities

The availability of these chambers makes it possible for divers who become symptomatic after SCUBA diving to readily receive recompression treatment. This is important because the closer the initiation of recompression treatment to the onset of DCS (and AGE) signs and symptoms, the greater the likelihood of full recovery.

Improved Diving Equipment

Mixed and Rebreather Gas Diving
Mixed gas diving involves changing the breathing gas from air which has 20% oxygen to higher oxygen percentages (nitrox). As the amount of oxygen is increased in the gas mixture, the amount of the inert gas (nitrogen) is reduced. With oxygen enriched air there is less tissue deposition of inert gas per unit of time under water for any given depth. However, because of increased oxygen partial pressures, the seizure threshold for oxygen toxicity is lowered. For normal sports diving activities, oxygen toxicity with mixed gas diving is only a theoretical concern.

Decompression Illness is More Than Bubbles

When AGE occurs, DCS symptoms may be concurrent or appear during or after recompression treatment even though the decompression tables were not violated on the dive. When DCS occurs in this situation it appears resistant to recompression treatment (Neuman) perhaps because of the inflammatory reaction generated by the bubble-blood vessel interaction from the AGE. In cases of DCI where components of both DCS and AGE are suspected, the diver should be observed for a minimum of 24 hours after the recompression treatment is completed for the delayed onset of DCS.

No theory of DCS discounts the primary role of bubbles in this condition. However, new information suggests that there are precursors to bubble formation and post-bubbling events that occur as a consequence of the bubbles. As mentioned earlier, venous gas emboli are a common occurrence diving ascent and ordinarily are filtered out harmlessly by the lungs. Precursors to DCS include stasis, dehydration and too rapid of ascents. These conditions allow the ubiquitous VGE to enlarge, coalesce and occlude the venous side of the circulation. Massive venous bubbling to the lungs can cause pulmonary vessel obstruction described as the chokes. If right to left shunts occur in the heart, VGE can become AGE to the brain. If the arterial flow is slow enough and/or the gradients large enough, autochthonus (ie, spontaneous) bubbles can form in the arterial circulation and lead to any of the consequences of AGE. In such situations it could be difficult to determine whether the DCI event was from AGE or DCS even after careful analysis of the dive profile. Hollenbeck’s model for diving paraplegia includes the setting of venous stasis (Batson’s plexus of veins) in the spinal canal, bubble formation, bubble enlargement possibly from off gassing of the spinal cord, blood vessel occlusion, and venous side infarctions of the spinal cord.
Contemporary Management of DCS

Problem Intervention Effect
Bubble Recompression
with HBO		 Reduce bubble size
1. Washout inert gas.
2. Change bubble composition by diffusion.
Stasis and dehydration Hydration: oral fluids if alert, IV fluids otherwise. Improve blood flow.
InflammationCell Ischemia ? Anti-inflammatory medicationsHBO Reduce interaction between bubble and blood vessel endothelium.
Improve oxygen availability to hypoxic tissues, reduce edema and also reduces the interaction between bubble and blood vessel endothelium.

.

Conclusions

We anticipate that in the future there will be further improvements for the safety and enjoyment of the recreational SCUBA diver. For example, the dive computer of the future will be able to individualize dive profiles for different personal medical parameters such as age, body composition and fitness level. Diver locators could quickly target a missing diver and save time and gas consumption as well as prevent serious diving mishaps. Drugs may be developed that would minimize the effect of bubbles interacting with body tissues and prevent DCS and AGE.

Extracorporeal membrane oxygenation therapy for pulmonary decompression illness

Yutaka Kondo, Masataka Fukami and Ichiro Kukita
Kondo et al. Critical Care 2014; 18:438 http://ccforum.com/content/18/3/438/10.1186/cc13935

Pulmonary decompression illness is rarely observed in clinical settings, and most patients die prior to hospitalization. We administered ECMO therapy to rescue a patient, even though this therapy has rarely been reported with good outcome in patients with decompression illness. In addition, we had to select venovenous ECMO even with the patient showing right ventricular failure. A lot of physicians may select venoarterial ECMO if the patient shows right ventricular failure, but the important physiological mechanism of pulmonary decompression illness is massive air embolism in the pulmonary arteries, and the bubbles diminish within the first 24 hours. The management of decompression illness therefore differs substantially from the usual right-sided heart failure.

Extremes of barometric pressure

Jane E Risdall, David P Gradwell
Anaesthesia and Intensive Care Medicine 16:2
Ascent to elevated altitude, commonly achieved through flight, by climbing or by residence in highland regions, exposes the individual to reduced ambient pressure. Although there are physical manifestations of this exposure as a consequence of Boyle’s law, the primary physiological challenge is of hypobaric hypoxia. The acute physiological and longer-term adaptive responses of the cardiovascular, respiratory, hematological and neurological systems to altitude are described, together with an outline of the presentation and management of acute mountain sickness, high-altitude pulmonary edema and high-altitude cerebral edema. While many millions experience modest exposure to altitude as a result of flight in pressurized aircraft, fewer individuals are exposed to increased ambient pressure. The pressure changes during diving and hyperbaric exposures result in greater changes in gas load and gas toxicity. Physiological effects include the consequences of increased work of breathing and redistribution of circulating volume. Neurological manifestations may be the direct result of pressure or a consequence of gas toxicity at depth. Increased tissue gas loads may result in decompression illness on return to surface or subsequent ascent in flight.

  • understand the physical effects of changes in ambient pressure and the physiological consequences on the cardiovascular respiratory and neurological systems
  • gain an awareness that exposure to reduced ambient pressure produces both acute and more chronic effects, with differing signs, symptoms and time to onset at various altitudes
  • develop an awareness of the toxic effects of ‘inert’ gases at increased ambient pressures and the pathogenesis and management of decompression illness

Decompression illness According to Henry’s law, at a constant temperature the amount of gas which dissolves in a liquid is proportional to the pressure of that gas or its partial pressure, if it is part of a mixture of gases. Breathing gases at increased ambient pressure will increase the amount of each gas dissolved in the fluid phases of body tissues. On ascent this excess gas has to be given up. If the ascent is controlled at a sufficiently slow rate, elimination will be via the respiratory system. If the ascent is too fast, excess gas may come out of solution and form free bubbles in the tissues or circulation. Bubbles may contain any of the gases in the breathing mixture, but it is the presence of inert gas bubbles (nitrogen or helium) that are thought most likely to give rise to problems, since the elimination of excess oxygen is achieved by metabolism as well as ventilation. These bubbles may act as venous emboli or may trigger inflammatory tissue responses giving rise to symptoms of decompression illness (DCI). Signs and symptoms of DCI may appear up to 48 hours after exposure to increased ambient pressure and include joint pains, motor and sensory deficits, dyspnoea, cough and skin rashes.

Neurological effects of deep diving

Marit Grønning, Johan A. Aarli
Journal of the Neurological Sciences 304 (2011) 17–21
http://dx.doi.org:/10.1016/j.jns.2011.01.021

Deep diving is defined as diving to depths more than 50 m of seawater (msw), and is mainly used for occupational and military purposes. A deep dive is characterized by the compression phase, the bottom time and the decompression phase. Neurological and neurophysiologic effects are demonstrated in divers during the compression phase and the bottom time. Immediate and transient neurological effects after deep dives have been shown in some divers. However, the results from the epidemiological studies regarding long term neurological effects from deep diving are conflicting and still not conclusive.

Possible immediate neurological effects of deep diving
Syndrome Pressure
Hyperoxia/oxygen seizures >152 kPa (5 msw)
HypoxiaHypercapnia
Nitrogen narcosis >354 kPa (25 msw)
High pressure nervous syndrome >1.6 MPa (150 msw)
Neurological decompression sickness

Neurological effects have been demonstrated, both clinically and neurophysiologically in divers during the compression phase and the bottom time. Studies of divers before and after deep dives have shown immediate and transient neurological effects in some divers. However, the results from the epidemiological and clinical studies regarding long term neurological effects from deep diving are conflicting and still not conclusive. Prospective clinical studies with sufficient power and sensitivity are needed to solve this important issue.

Today deep diving to more than 100 msw is routinely performed globally in the oil- and gas industry. In the North Sea remote underwater intervention and maintenance is performed by the use of remotely operated vehicles (ROV), both in conjunction to and as an alternative to manned underwater operations. There will, however, always be a need for human divers in the technically more advanced underwater operations and for contingency repair operations.

P300 latency indexes nitrogen narcosis

Barry Fowler, Janice Pogue and Gerry Porlier
Electroencephalography, and clinical Neurophysiology, 1990, 75:221-229

This experiment investigated the effects of nitrogen narcosis on reaction time (RT) and P300 latency and amplitude, Ten subjects breathed either air or a non-narcotic 20% oxygen-80% helium (heliox) mixture in a hyperbaric chamber at 6.5, 8.3 and 10 atmospheres absolute (ATA), The subjects responded under controlled accuracy conditions to visually presented male or female names in an oddball paradigm. Single-trial analysis revealed a strong relationship between RT and P300 latency, both of which were slowed in a dose-related manner by hyperbaric air but not by heliox. A clear-cut dose-response relationship could not be established for P300 amplitude. These results indicate that P300 latency indexes nitrogen narcosis and are interpreted as support for the slowed processing model of inert gas narcosis.

Adaptation to Deep Water Habitat

Effects of hypoxia on ionic regulation, glycogen utilization and antioxidative ability in the gills and liver of the aquatic air-breathing fish Trichogaster microlepis

Chun-Yen Huang, Hui-Chen Lina, Cheng-Huang Lin
Comparative Biochemistry and Physiology, Part A 179 (2015) 25–34
http://dx.doi.org/10.1016/j.cbpa.2014.09.001

We examined the hypothesis that Trichogaster microlepis, a fish with an accessory air-breathing organ, uses a compensatory strategy involving changes in both behavior and protein levels to enhance its gas exchange ability. This compensatory strategy enables the gill ion-regulatory metabolism to maintain homeostasis during exposure to hypoxia. The present study aimed to determinewhether ionic regulation, glycogen utilization and antioxidant activity differ in terms of expression under hypoxic stresses; fish were sampled after being subjected to 3 or 12 h of hypoxia and 12 h of recovery under normoxia. The air-breathing behavior of the fish increased under hypoxia. No morphological modification of the gills was observed. The expression of carbonic anhydrase II did not vary among the treatments. The Na+/K+-ATPase enzyme activity did not decrease, but increases in Na+/K+-ATPase protein expression and ionocyte levels were observed. The glycogen utilization increased under hypoxia as measured by glycogen phosphorylase protein expression and blood glucose level, whereas the glycogen content decreased. The enzyme activity of several components of the antioxidant system in the gills, including catalase, glutathione peroxidase, and superoxidase dismutase, increased in enzyme activity. Based on the above data, we concluded that T. microlepis is a hypoxia-tolerant species that does not exhibit ion-regulatory suppression but uses glycogen to maintain energy utilization in the gills under hypoxic stress. Components of the antioxidant system showed increased expression under the applied experimental treatments.

Divergence date estimation and a comprehensive molecular tree of extant cetaceans

Michael R. McGowen , Michelle Spaulding, John Gatesy
Molecular Phylogenetics and Evolution 53 (2009) 891–906
http://dx.doi.org/10.1016/j.ympev.2009.08.018

Cetaceans are remarkable among mammals for their numerous adaptations to an entirely aquatic existence, yet many aspects of their phylogeny remain unresolved. Here we merged 37 new sequences from the nuclear genes RAG1 and PRM1 with most published molecular data for the group (45 nuclear loci, transposons, mitochondrial genomes), and generated a supermatrix consisting of 42,335 characters. The great majority of these data have never been combined. Model-based analyses of the supermatrix produced a solid, consistent phylogenetic hypothesis for 87 cetacean species. Bayesian analyses corroborated odontocete (toothed whale) monophyly, stabilized basal odontocete relationships, and completely resolved branching events within Mysticeti (baleen whales) as well as the problematic speciose clade Delphinidae (oceanic dolphins). Only limited conflicts relative to maximum likelihood results were recorded, and discrepancies found in parsimony trees were very weakly supported. We utilized the Bayesian supermatrix tree to estimate divergence dates among lineages using relaxed-clock methods. Divergence estimates revealed rapid branching of basal odontocete lineages near the Eocene–Oligocene boundary, the antiquity of river dolphin lineages, a Late Miocene radiation of balaenopteroid mysticetes, and a recent rapid radiation of Delphinidae beginning [1]10 million years ago. Our comprehensive,  time calibrated tree provides a powerful evolutionary tool for broad-scale comparative studies of Cetacea.

Mitogenomic analyses provide new insights into cetacean origin and evolution

Ulfur Arnason, Anette Gullberg, Axel Janke
Gene 333 (2004) 27–34
http://dx.doi.org:/10.1016/j.gene.2004.02.010

The evolution of the order Cetacea (whales, dolphins, porpoises) has, for a long time, attracted the attention of evolutionary biologists. Here we examine cetacean phylogenetic relationships on the basis of analyses of complete mitochondrial genomes that represent all extant cetacean families. The results suggest that the ancestors of recent cetaceans had an explosive evolutionary radiation 30–35 million years before present. During this period, extant cetaceans divided into the two primary groups, Mysticeti (baleen whales) and Odontoceti (toothed whales). Soon after this basal split, the Odontoceti diverged into the four extant lineages, sperm whales, beaked whales, Indian river dolphins and delphinoids (iniid river dolphins, narwhals/belugas, porpoises and true dolphins). The current data set has allowed test of two recent morphological hypotheses on cetacean origin. One of these hypotheses posits that Artiodactyla and Cetacea originated from the extinct group Mesonychia, and the other that Mesonychia/Cetacea constitutes a sister group to Artiodactyla. The current results are inconsistent with both these hypotheses. The findings suggest that the claimed morphological similarities between Mesonychia and Cetacea are the result of evolutionary convergence rather than common ancestry.

The order Cetacea traditionally includes three suborders: the extinct Archaeoceti and the recent Odontoceti and Mysticeti. It is commonly believed that the evolution of ancestral cetaceans from terrestrial to marine (aquatic) life was accompanied by a fast and radical morphological adaptation. Such a scenario may explain why it was, for a long time, difficult to morphologically establish the position of Cetacea in the mammalian tree and even to settle whether Cetacea constituted a monophyletic group.

Biochemical analyses in the 1950s  and 1960s had shown a closer relationship between cetaceans and artiodactyls (even-toed hoofed mammals) than between cetaceans and any other eutherian order and karyological studies in the late 1960s and early 1970s unequivocally supported cetacean monophyly (Arnason, 1969, 1974). The nature of the relationship between cetaceans and artiodactyls was resolved in phylogenetic studies of mitochondrial (mt) cytochrome b (cytb) genes (Irwin and Arnason, 1994; Arnason and Gullberg, 1996) that placed Cetacea within the order Artiodactyla itself as the sister group of the Hippopotamidae (see also Sarich, 1993). The Hippopotamidae/ Cetacea relationship was subsequently supported in studies of nuclear data (Gatesy et al., 1996; Gatesy, 1997) and statistically established in analysis of complete mt genomes (Ursing and Arnason, 1998). The relationship has also been confirmed in analyses of combined nuclear and mt sequences (Gatesy et al., 1999; Cassens et al., 2000) and in studies of short interspersed repetitive elements (SINEs). Artiodactyla and Cetacea are now commonly referred to as Cetartiodactyla.

Previous analyses of the complete cytb gene of more than 30 cetacean species (Arnason and Gullberg, 1996) identified five primary lineages of recent cetaceans, viz., Mysticeti and the four odontocete lineages Physeteridae (sperm whales), Platanistidae (Indian river dolphins), Ziphiidae (beaked whales) and Delphinoidea (iniid river dolphins, porpoises, narwhals and dolphins). However, these studies left unresolved the relationships of the five lineages as well as those between the three delphinoid families Monodontidae (narwhals, belugas), Phocoenidae (porpoises) and Delphinidae (dolphins). Similarly, the relationships between the four mysticete families Balaenidae (right whales), Neobalaenidae (pygmy right whales), Eschrichtiidae (gray whales) and Balaenopteridae (rorquals) were not conclusively resolved in analyses of cytb genes.

Fig. (not shown). Cetartiodactyl relationships and the estimated times of their divergences. The tree was established on the basis of maximum likelihood analysis of the concatenated amino acid (aa) sequences of 12 mt protein-coding genes. Length of alignment 3610 aa. Support values for branches A–H are shown in the insert.
Cetruminantia (branch A) receives moderate support and Cetancodonta (B) strong support. Cetacea (C) splits into monophyletic Mysticeti (baleen whales) and monophyletic Odontoceti (toothed whales). Odontoceti has four basal lineages, Physeteridae (sperm whales: represented by the sperm and pygmy sperm whales), Ziphiidae (beaked whales: bottlenose and Baird’s beaked whales), Platanistidae (Indian river dolphins: Indian river dolphin) and Delphinoidea. Delphinoidea encompasses the families Iniidae (iniid river dolphins: Amazon river dolphin, La Plata dolphin), Monodontidae (narwhals/belugas: narwhal), Phocoenidae (porpoises: harbour porpoise) and Delphinidae (dolphins: white-beaked dolphin). The common odontocete branch and the branches separating the four cetacean lineages are short. These relationships are therefore somewhat unstable (cf. Section 3.1 and Table 1). Iniid river dolphins (F) are solidly nested within the Delphinoidea (E). Thus, traditional river dolphins (Platanistidae + Iniidae) do not form a monophyletic unit. Molecular estimates of divergence times (Sanderson 2002) were based on two calibration points, A/C-60 and O/M-35 (cf. Section 3.2). Due to the short lengths of internal branches, some estimates for these divergences overlap. NJ: neighbor joining; MP: maximum parsimony; LBP: local bootstrap probability; QP: quartet puzzling. The bar shows the number of aa substitutions per site.

The limited molecular resolution among basal cetacean lineages has been known for some time. Studies of hemoglobin and myoglobin (Goodman, 1989; Czelusniak et al., 1990) have either joined Physeteridae and Mysticeti to the exclusion of Delphinoidea (myoglobin data) or Mysticeti and Delphinoidea to the exclusion of Physeteridae (hemoglobin data). Thus, neither of the data sets identified monophyletic Odontoceti by joining the two odontocete lineages (Physeteridae and Delphinoidea) to the exclusion of Mysticeti. A similar instability was recognized and cautioned against in analyses of some mt data, notably, sequences of rRNA genes (Arnason et al., 1993b). The suggestion (Milinkovitch et al., 1993) of a sister group relationship between Physeteridae and the mysticete family Balaenopteridae (rorquals) was based on a myoglobin data set (which joins Physeteridae and Mysticeti to the exclusion of Delphinoidea) that was complemented with partial data of the mt 16S rRNA gene.

The cetancodont divergence times calculated using A/C-60 and O/M-35 as references have been included in Fig. 1. As a result of the short branches separating several cetacean lineages, the estimates of these divergences overlap. The same observation has been made in calculations based on SINE flanking sequences (Nikaido et al., 2001). There is a general consistency between the current and the flanking sequence datings, except for those involving the Balaenopteridae, which are somewhat younger in our analysis than in the SINEs study. The currently estimated age of the divergence between Hippopotamus and Cetacea (c53.5 MYBP) is consistent with the age (>50 MY) of the oldest archaeocete fossils identified so far (Bajpai and Gingerich, 1998). This suggests that the ages allocated to the two references, A/C-60 (the divergence between ruminant artiodactyls and cetancodonts) and O/M-35 (the divergence between odontocetes and mysticetes) are reasonably accurate.

The dating of the divergence between the blue and fin whales is of interest regarding hybridization between closely related mammalian species. Previous molecular analyses (Arnason et al., 1991b; Spilliaert et al., 1991) demonstrated the occurrence of hybridization between these two species. These studies, which were based on three hybrids (one female and two males), showed that either species could be the mother or father in these hybridizations. The two male hybrids had rudimentary testes, whereas the female hybrid was in her second pregnancy. This suggests that the blue and fin whales may be close to the limit for permissible species hybridization among mammals.

The current data set has allowed examination of the coherence between the molecular results and two prevalent morphological hypotheses related to cetacean evolution. The first hypothesis, which in essence originates from Van Valen (1966, 1968), postulates that monophyletic Artiodactyla and monophyletic Cetacea evolved separately from the extinct Palaeocene group Mesonychia. This hypothesis was recently reinforced in a morphological study (Thewissen et al., 2001) that included mesonychians, two archaeocete taxa (Ambuloocetus and Pakicetus) and some extant and fossil artiodactyls. The study of Thewissen et al. (2001) showed a sister group relationship between monophyletic Artiodactyla and monophyletic Cetacea, with Mesonychia as the basal sister group of Artiodactyla/Cetacea, a conclusion consistent with the palaeontological age of Mesonychia relative to that of Artiodactyla and Cetacea. The second hypothesis favours a sister group relationship between Mesonychia and Cetacea with the Mesonychia/Cetacea clade as the sister group of monophyletic Artiodactyla (O’Leary and Geisler, 1999; see also Gatesy and O’Leary, 2001).

Although the position of Mesonychia differs in the two morphological hypotheses, both correspond to a sister group relationship between Cetacea and monophyletic Artiodactyla among extant cetartiodactyls. Thus, both hypotheses can be tested against the current data set. The result of such a test has been included in Table 1, topology (m)(not shown). As evident, both these morphological hypotheses are incongruent with the mitogenomic findings.

Morphological studies have not provided an answer to the question whether mysticetes and odontocetes had separate origins among the archaeocetes (Fordyce and de Muizon, 2001). However, the long common cetacean branch and the short branches separating the five extant cetacean lineages strongly suggest an origin of modern cetaceans from the same archaeocete group (probably the Dorudontidae).

The limbs of Ambulocetus constitute somewhat of an evolutionary enigma. As evident in Thewissen et al.’s (1994) paper, Ambulocetus has very large hind limbs compared to its forelimbs, a difference that is less pronounced in later silhouette drawings of the animal. It is nevertheless evident that evolution from the powerful hindlimbs of Ambulocetus to their rudimentation in archaeocetes constitutes a remarkable morphological reversal if Ambulocetus is connected to the cetacean branch after the separation of the hippopotamid and cetacean lineages.

For natural reasons, systematic schemes have traditionally been based on external morphological characteristics. The rates of morphological and molecular evolution are rarely (if ever) strictly correlated, however, and this may give rise to inconsistency between traditional systematics and molecular findings. The emerging consensus that the order Cetacea resides within another traditional order, Artiodactyla, makes apparent the incongruity in cetartiodactyl nomenclature (Graur and Higgins, 1994). In this instance, a possible solution for maintaining reasonable consistency between nomenclature and phylogeny would be to recognize Cetartiodactyla as an order with three suborders: Suina, Tylopoda and Cetruminantia. According to such a scheme, Cetacea would (together with the Hippopotamidae) constitute a parvorder within the infraorder Cetancodonta.

Cytochrome b and Bayesian inference of whale phylogeny

Laura May-Collado, Ingi Agnarsson
Molecular Phylogenetics and Evolution 38 (2006) 344–354
http://dx.doi.org//10.1016/j.ympev.2005.09.019

In the mid 1990s cytochrome b and other mitochondrial DNA data reinvigorated cetacean phylogenetics by proposing many novel

and provocative hypotheses of cetacean relationships. These results sparked a revision and reanalysis of morphological datasets, and the collection of new nuclear DNA data from numerous loci. Some of the most controversial mitochondrial hypotheses have now become benchmark clades, corroborated with nuclear DNA and morphological data; others have been resolved in favor of more traditional views. That major conflicts in cetacean phylogeny are disappearing is encouraging. However, most recent papers aim specifically to resolve higher-level conflicts by adding characters, at the cost of densely sampling taxa to resolve lower-level relationships. No molecular study to date has included more than 33 cetaceans. More detailed molecular phylogenies will provide better tools for evolutionary studies. Until more genes are available for a high number of taxa, can we rely on readily available single gene mitochondrial data? Here, we estimate the phylogeny of 66 cetacean taxa and 24 outgroups based on Cytb sequences. We judge the reliability of our phylogeny based on the recovery of several deep-level benchmark clades. A Bayesian phylogenetic analysis recovered all benchmark clades and for the Wrst time supported Odontoceti monophyly based exclusively on analysis of a single mitochondrial gene. The results recover the monophyly, with the exception of only one taxa within Cetacea, and the most recently proposed super- and subfamilies. In contrast, parsimony never recovered all benchmark clades and was sensitive to a priori weighting decisions. These results provide the most detailed phylogeny of Cetacea to date and highlight the utility of both Bayesian methodology in general, and of Cytb in cetacean phylogenetics. They furthermore suggest that dense taxon sampling, like dense character sampling, can overcome problems in phylogenetic reconstruction.

Some long standing debates are all but resolved: our understanding of deeper level cetacean phylogeny has grown strong. However, the strong focus of most recent studies, aiming specifically to resolve these higher level conflicts by adding mostly characters rather than taxa, has left our understanding of lower level relationships among whale species lagging behind. Mitogenomic data, for example, is available only for 16 cetacean species, and no molecular study to date has included more than 33 cetaceans. It seems timely to focus on more detailed (genus, and species level) molecular phylogenies. These will provide better tools for detailed evolutionary studies, and are necessary to test existing morphological phylogenetic hypotheses, and current cetacean classification.

We judge the reliability of our phylogeny based on the recovery of the previously mentioned benchmark clades, in addition to the less controversial clades Perissodactyla, Euungulata (sensu Waddell et al., 2001; Perissodactyla+ Cetartiodactyla), Cetacea, and Mysticeti. Because Cytb is thought to be most reliable at lower taxonomic levels (due to high substitution rates), recovering ‘known’ deeper clades gives credibility to these new findings which have not been addressed by studies using few taxa. We compare the performance of Bayesian analyses versus parsimony under four different models, and briefly examine the sensitivity of the results to taxon sampling. We use our results to discuss agreement and remaining conflict in cetacean phylogenetics, and provide comments on current classification.

The Bayesian analysis recovered all seven benchmark clades. Support for five of the benchmark clades is high (100 posterior probabilities) but rather low for Cetancodonta (79) and marginal for the monophyly of Odontoceti. The analysis also recovered all but one family level, and most sub- and superfamily level cetacean taxa. The results broadly corroborate current cetacean classiffcation, while also pointing to some lower-level groups that may need redefinition.

Many recent cetacean phylogenetic studies include relatively few taxa, in part due to a focus on generating more characters to resolve higher level phylogenetics. While addressing crucial questions and providing the backbone for lower level phylogenies, such studies have limited utility for classification, and for comparative evolutionary studies. In some cases sparse taxon sampling may also confound the results. Of course, taxon sampling is usually simply constrained by the availability of character data, but for some reason many studies have opted to include only one, or a few outgroup taxa, even if many are available.

We find that as long as outgroup taxon sampling was extensive, Bayesian analyses of Cytb recovered all the a priori identified benchmark clades. When only a few outgroups were chosen, however, the Bayesian analysis negated Odontoceti monophyly, as have many previous parsimony analyses of mitochondrial DNA. Furthermore, in almost every detailed comparison possible our results mirror the findings O’Leary et al. (2004), the most ‘character-complete’ (but including relatively few cetacean taxa) analysis to date (37,000 characters from morphology, SINE, and 51 gene fragments). This result gives credibility to our findings, including previously untested lower level clades.

  • Monophyly and placement of Mysticeti (baleen whales).
  • Monophyly of Odontoceti (toothed whales)
  • Delphinoids
  • River Dolphins
  • Beaked and sperm whales

A major goal of phylogenetics is a phylogeny of life (i.e., many taxa), based on multiple lines of evidence (many characters of many types). However, when phylogenies based on relatively few characters can be judged reliable based on external evidence (taxonomic congruence with other phylogenies using many characters, but few taxa), they seem like very promising and useful ‘first guess’ hypotheses. The evolution of sexual dimorphism, echolocation, social behavior, and whistles and other communicative signals, and major ecological shifts (e.g., transition to fresh water) are among the numerous interesting questions in cetacean biology that this phylogeny can help answer.

Deep-diving sea lions exhibit extreme bradycardia in long duration dives

Birgitte I. McDonald1, and Paul J. Ponganis
The Journal of Experimental Biology (2014) 217, 1525-1534 http://dx.doi.org:/10.1242/jeb.098558

Heart rate and peripheral blood flow distribution are the primary determinants of the rate and pattern of oxygen store utilization and ultimately breath-hold duration in marine endotherms. Despite this, little is known about how otariids (sea lions and fur seals) regulate heart rate (fH) while diving. We investigated dive fH in five adult female California sea lions (Zalophus californianus) during foraging trips by instrumenting them with digital electrocardiogram (ECG) loggers and time depth recorders. In all dives, dive fH (number of beats/duration; 50±9 beats min−1) decreased compared with surface rates (113±5 beats min−1), with all dives exhibiting an instantaneous fH below resting (<54 beats min−1) at some point during the dive. Both dive fH and minimum instantaneous fH significantly decreased with increasing dive duration. Typical instantaneous fH profiles of deep dives (>100 m) consisted of:

(1) an initial rapid decline in fH resulting in the lowest instantaneous fH of the dive at the end of descent, often below 10 beats min−1 in dives longer than 6 min in duration;
(2) a slight increase in fH to ~10–40 beats min−1 during the bottom portion of the dive; and
(3) a gradual increase in fH during ascent with a rapid increase prior to surfacing.

Thus, fH regulation in deep-diving sea lions is not simply a progressive bradycardia. Extreme bradycardia and the presumed associated reductions in pulmonary and peripheral blood flow during late descent of deep dives should

(a) contribute to preservation of the lung oxygen store,
(b) increase dependence of muscle on the myoglobin-bound oxygen store,
(c) conserve the blood oxygen store and
(d) help limit the absorption of nitrogen at depth.

This fH profile during deep dives of sea lions may be characteristic of deep-diving marine endotherms that dive on inspiration as similar fH profiles have been recently documented in the emperor penguin, another deep diver that dives on inspiration.

The resting ƒH measured in this study (54±6 beats min−1) was lower than predicted for an animal of similar size (~80 beats min−1 for an 80 kg mammal). In part, this may be due to the fact that the sea lions were probably sleeping. The resting ƒH in our study was also lower than previous measurements in captive juvenile California sea lions (87±17 beats min−1, average mass 30 kg)  and wild Antarctic fur seals (78±5 beats min−1, body mass 30–50 kg). However, we found a significant negative relationship between mass and resting ƒH even with our small sample size of five sea lions (resting ƒH = –0.58 Mb +100.26, r2=0.81, F1,3=12.37, P=0.039). For a 30 kg sea lion, this equation predicts a resting ƒH of 83 beats min−1, which is similar to what was measured previously in juvenile sea lions, suggesting this equation may be useful in estimating resting ƒH in sea lions.

The sea lions exhibited a distinct sinus arrhythmia fluctuating between a minimum of 42±9 and a maximum of 87±12 beats min−1, comparable to the sinus arrhythmias described in other diving birds and mammals, including sea lions. The minimum instantaneous ƒH during the sinus arrhythmia was similar to the mean minimum ƒH in dives less than 3 min (37±7 beats min−1), indicating that in dives less than 3 min (estimated cADL), ƒH only decreased to levels observed during exhalation at rest. This is consistent with observations in emperor penguins and elephant seals, where it was proposed that in dives shorter than the aerobic dive limit (ADL) the reduction in ƒH is regulated by a mechanism of cardiorespiratory control similar to that governing the respiratory sinus arrhythmia, with a further reduction only occurring in dives longer than the ADL.

Fig. 3. (not shown) Instantaneous fH and dive depth profiles of a California sea lion (CSL12_2). Data are from (A) a short, shallow dive (1.3 min, 45 m), (B) a mid-duration dive (4.8 min, 239 m) and (C) a long-duration dive (8.5 min, 305 m). Minimum instantaneous fH reached 37 beats min−1 in the short dive
(A) 19 beats min−1 in the mid-duration dive
(B) and 7 beats min−1 in the long duration dive
(C) Prominent features typical of mid- and long-duration dives include

  • a surface interval tachycardia (pre- and post-dive);
  • a steady rapid decrease in fH during initial descent;
  • a gradual decline in fH towards the end of descent with the lowest fH of the dive at the end of descent;
  • a slight increase and sometimes variable fH during the bottom portion of the dive; and
  • a slow increase in fH during ascent,
  • often ending in a rapid increase just before surfacing.

We obtained the first diving ƒH data from wild sea lions on natural foraging trips, demonstrating how they regulate ƒH over a range of dive durations. Sea lions always decreased dive ƒH from surface ƒH values; however, individual sea lions exhibited different dive ƒH, accounting for a significant amount of the variation in the relationship between dive duration and ƒH (intra-individual correlation: 75–81%)). The individual differences in dive ƒH exhibited in this study suggest that different dive capacities of individual sea lions may partially account for the range of dive strategies exhibited in a previous study (Villegas-Amtmann et al., 2011). Despite the individual differences in ƒH, the pattern of the dive ƒH response was similar in all the sea lions. As predicted, sea lions only consistently displayed a true bradycardia on mid- to long- duration dives (>4 min) (Fig. 5A). Additionally, as seen in freely diving phocids, dive ƒH and minimum ƒH were negatively related to dive duration, with the longest duration dives having the lowest dive ƒH and displaying the most intense bradycardia, often below 10 beats min−1 (Fig. 5A,B).

Profiles of mean fH at 10 s intervals of dives

Profiles of mean fH at 10 s intervals of dives

Fig 4.  Profiles of mean fH at 10 s intervals of dives for (A) six duration categories and (B) five depth categories. Standard error bars are shown. Data were pooled from 461 dives performed by five sea lions. The number of dives in each category and the number of sea lions performing the dives in each category are provided in the keys.

The mild bradycardia and the dive ƒH profiles observed in the shorter duration dives (<3 min) were similar to those observed in trained juvenile California sea lions and adult Stellar sea lions, but much more intense than ƒH observed in freely diving Antarctic fur seals. Surprisingly, although dive ƒH of trained Steller sea lions was similar, Steller sea lions regularly exhibited lower minimum ƒH, with minimum ƒH almost always less than 20 beats min−1 in dives less than 2 min in duration. In the wild, California sea lions rarely exhibited a minimum ƒH less than 20 beats min−1 in similar duration dives (Fig. 5B), suggesting greater blood oxygen transport during these natural short-duration dives.

Fig. 5. (not shown)  fH decreases with increasing dive duration. Dive duration versus (A) dive fH (total number of beats/dive duration), (B) minimum instantaneous fH and (C) bottom fH (total beats at bottom of dive/bottom time) for California sea lions (461 dives from five sea lions).

Although California sea lions are not usually considered exceptional divers, they exhibited extreme bradycardia, comparable to that of the best diving phocids, during their deep dives. In dives greater than 6 min in duration, minimum ƒH was usually less than 10 beats min−1 and sometimes as low as 6 beats mins−1 (Fig. 5B), which is similar to extreme divers such as emperor penguins (3 beats min−1), elephant seals (3 beats min−1), grey seals (2 beats min−1) and Weddell seals (<10 beats min−1), and even as low as what was observed in forced submersion studies. Thus, similar to phocids, the extreme bradycardia exhibited during forced submersions is also a routine component of the sea lion’s physiological repertoire, allowing them to perform long-duration dives.

While the degree of bradycardia observed in long dives of California sea lions was similar to the extreme bradycardia observed in phocids, the ƒH profiles were quite different. In general, phocid ƒH decreases abruptly upon submergence. The intensity of the initial phocid bradycardia either remains relatively stable or intensifies as the dive progresses, and does not start to increase until the seal begins its ascent. In contrast, the ƒH profiles of sea lions were more complex, showing a more gradual decrease during descent, with the minimum ƒH of the dive usually towards the end of descent (Figs 3, 6). There was often a slight increase in ƒH during the bottom portion of the dive, and as soon as the sea lions started to ascend, the ƒH slowly started to increase, often becoming irregular during the middle of ascent, before increasing rapidly as the sea lion approached the surface.

Fig. 6. (not shown) Instantaneous fH and dive depth profiles of the longest dive (10.0 min, 385 m) from a California sea lion (CSL12_1). During this dive, instantaneous fH reached 7 beats min−1 and was less than 20 beats min−1 for over 5.5 min. Post-dive fH was high in the first 0.5–1 min after surfacing, but then declined to ~100 beats min−1 towards the end of the surface interval.

Implications for pulmonary gas exchange

The moderate dive ƒH in short, shallow dives compared with the much slower ƒH of deep long-duration dives suggests more pulmonary blood flow and greater potential for reliance on lung O2. Most of these dives were to depths of less than 100 m (well below the estimated depth of lung collapse near 200 m), so maintenance of a moderate ƒH during these dives may allow sea lions to maximise use of the potentially significant lung O2 stores (~16% of total body O2 stores) throughout the dive. This is supported by venous blood O2 profiles, where, occasionally, there was no decrease in venous blood O2 between the beginning and end of the dive; this can only occur if pulmonary gas exchange continues throughout the dive. Greater utilization of the lung O2 store in sea lions is consistent with higher dive ƒH in other species that both dive on inspiration and typically perform shallow dives (dolphins, porpoises, some penguin species), and in deeper diving species when they perform shallow dives (emperor penguins).

In deeper dives of sea lions, although ƒH was lower and bradycardia more extreme, the diving ƒH profiles suggest that pulmonary gas exchange is also important. In long-duration dives, even though ƒH started to decrease upon or shortly after submergence, the decrease was not as abrupt as in phocids. Additionally, in long deep dives, despite having overall low dive ƒH, there were more heart beats before resting ƒH was reached compared with short, shallow dives. In dives less than 3 min in duration, there were ~10–15 beats until instantaneous ƒH reached resting values. In longer duration dives (>3 min), there were usually ~30–40 beats before instantaneous ƒH reached resting values. We suggest the greater number of heart beats early in these deeper dives enables more gas exchange and blood O2 uptake at shallow depths, thus allowing utilisation of the postulated larger respiratory O2 stores in deeper dives The less abrupt decline in ƒH we observed in sea lions is similar to the more gradual declines documented in emperor penguins and porpoises, where it has also been proposed that the gradual decrease in ƒH allows them to maximise pulmonary gas exchange at shallower depths. However, as sea lions swam deeper, ƒH decreased further (Figs 3, 6), and by 200 m depth (the approximate depth of lung collapse, instantaneous ƒH was 14 beats min−1. Such an extreme decline in ƒH in conjunction with increased pulmonary shunting due to lung compression at greater depths will result in minimization of both O2 and N2 uptake by blood, even before the depth of full lung collapse (100% pulmonary shunt) is reached.

Implications for blood flow

ƒH is often used as a proxy to estimate blood flow and perfusion during diving because of the relative ease of its measurement. This is based on the assumption that stroke volume does not change during diving in sea lions, and, hence, changes in ƒH directly reflect changes in cardiac output. As breath-hold divers maintain arterial pressure while diving, changes in cardiac output should be associated with changes in peripheral vascular resistance and changes in blood flow to tissues. In Weddell seals, a decrease in cardiac output of ~85% during forced submersions resulted in an 80–100% decrease in tissue perfusion in all tissues excluding the brain, adrenal glands and lung. Sea lions exhibited extremely low instantaneous ƒH values that often remained low for significant portions of the dive (Figs 4, 6), suggesting severe decreases in tissue perfusion in dives greater than 5 min in duration. In almost all dives greater than 6 min in duration, instantaneous ƒH reached 10 beats min−1, and stayed below 20 beats min−1 for more than a minute. At a ƒH of 20 beats min−1, cardiac output will be ~36% of resting cardiac output and only about 18% of average surface cardiac output. At these levels of cardiac suppression, most of this flow should be directed towards the brain and heart.

Conclusions

We successfully obtained diving ƒH profiles from a deep-diving otariid during natural foraging trips. We found that

(1) ƒH decreases during all dives, but true and more intense bradycardia only occurred in longer duration dives and
(2) in the longest duration dives, ƒH and presumed cardiac output were as low as 20% of resting values.

We conclude that, although initial high ƒH promotes gas exchange early in deep dives, the extremely low ƒH in late descent of deep dives (a) preserves lung O2, (b) conserves blood O2, (c) increases the dependence of muscle on myoglobin-bound O2 and (d) limits N2 absorption at depth. This ƒH profile, especially during the late descent/early bottom phase of deep dives is similar to that of deep-diving emperor penguins, and may be characteristic of deep diving endotherms that dive on inspiration.

Dive duration was the fixed effect in all models, and to account for the lack of independence caused by having many dives from the same individual, individual (sea lion ID) was included as a random effect. Covariance and random effect structures of the full models were evaluated using Akaike’s information criterion (AIC) and examination of residual plots. AICs from all the tested models are presented with the best model in bold.

Additionally, dives were classified as short-duration (less than 3 min, minimum cADL), mid-duration (3–5 min, range of cADLs) or long-duration (>5 min) dives. Differences in pre-dive ƒH, dive ƒH, minimum ƒH, post-dive ƒH, and heart beats to resting between the categories were investigated using mixed effects ANOVA, followed by post hoc Tukey tests. In all models, dive duration category was the fixed effect and individual (sea lion ID) was included as a random effect. Model fit was accessed by examination of the residuals. All means are expressed ±s.d. and results of the Tukey tests were considered significant at P<0.05. Statistical analysis was performed in R.

Investigating Annual Diving Behaviour by Hooded Seals (Cystophora cristata) within the Northwest Atlantic Ocean

Julie M. Andersen, Mette Skern-Mauritzen, Lars Boehme
PLoS ONE 8(11): e80438. http://dx.doi.org:/10.1371/journal.pone.0080438

With the exception of relatively brief periods when they reproduce and molt, hooded seals, Cystophora cristata, spend most of the year in the open ocean where they undergo feeding migrations to either recover or prepare for the next fasting period. Valuable insights into habitat use and diving behavior during these periods have been obtained by attaching Satellite Relay Data Loggers (SRDLs) to 51 Northwest (NW) Atlantic hooded seals (33 females and 18 males) during icebound fasting periods (200422008). Using General Additive Models (GAMs) we describe habitat use in terms of First Passage Time (FPT) and analyze how bathymetry, seasonality and FPT influence the hooded seals’ diving behavior described by maximum dive depth, dive duration and surface duration. Adult NW Atlantic hooded seals exhibit a change in diving activity in areas where they spend .20 h by increasing maximum dive depth, dive duration and surface duration, indicating a restricted search behavior. We found that male and female hooded seals are spatially segregated and that diving behavior varies between sexes in relation to habitat properties and seasonality. Migration periods are described by increased dive duration for both sexes with a peak in May, October and January. Males demonstrated an increase in dive depth and dive duration towards May (post-breeding/pre-molt) and August–October (post-molt/pre-breeding) but did not show any pronounced increase in surface duration. Females dived deepest and had the highest surface duration between December and January (post-molt/pre-breeding). Our results suggest that the smaller females may have a greater need to recover from dives than that of the larger males. Horizontal segregation could have evolved as a result of a resource partitioning strategy to avoid sexual competition or that the energy requirements of males and females are different due to different energy expenditure during fasting periods.

Novel locomotor muscle design in extreme deep-diving whales

P. Velten, R. M. Dillaman, S. T. Kinsey, W. A. McLellan and D. A. Pabst
The Journal of Experimental Biology 216, 1862-1871
http://dx.doi.org:/10.1242/jeb.081323

Most marine mammals are hypothesized to routinely dive within their aerobic dive limit (ADL). Mammals that regularly perform deep, long-duration dives have locomotor muscles with elevated myoglobin concentrations that are composed of predominantly large, slow-twitch (Type I) fibers with low mitochondrial volume densities (Vmt). These features contribute to extending ADL by increasing oxygen stores and decreasing metabolic rate. Recent tagging studies, however, have challenged the view that two groups of extreme deep-diving cetaceans dive within their ADLs. Beaked whales (including Ziphius cavirostris and Mesoplodon densirostris) routinely perform the deepest and longest average dives of any air-breathing vertebrate, and short-finned pilot whales (Globicephala macrorhynchus) perform high-speed sprints at depth. We investigated the locomotor muscle morphology and estimated total body oxygen stores of several species within these two groups of cetaceans to determine whether they

(1) shared muscle design features with other deep divers and
(2) performed dives within their calculated ADLs.

Muscle of both cetaceans displayed high myoglobin concentrations and large fibers, as predicted, but novel fiber profiles for diving mammals. Beaked whales possessed a sprinterʼs fiber-type profile, composed of ~80% fast-twitch (Type II) fibers with low Vmt. Approximately one-third of the muscle fibers of short-finned pilot whales were slow-twitch, oxidative, glycolytic fibers, a rare fiber type for any mammal. The muscle morphology of beaked whales likely decreases the energetic cost of diving, while that of short-finned pilot whales supports high activity events. Calculated ADLs indicate that, at low metabolic rates, both beaked and short-finned pilot whales carry sufficient onboard oxygen to aerobically support their dives.

Serial cross-sections of the m. longissimus dorsi of Mesoplodon densirostris

Serial cross-sections of the m. longissimus dorsi of Mesoplodon densirostris

Fig. Serial cross-sections of the m. longissimus dorsi of Mesoplodon densirostris (A–D) and Globicephala macrorhynchus (E–H). Scale bars, 50μm. Muscle sections stained for the alkaline (A,E) and acidic (B,F) preincubations of myosin ATPase were used to distinguish Type I and II fibers. Muscle sections stained for succinate dehydrogenase (C,G) and α-glycerophosphate dehydrogenase (D,H) were used to distinguish glycolytic (gl), oxidative (o) and intermediate (i) fibers.

Previous studies of the locomotor muscles of deep-diving marine mammals have demonstrated that these species share a suite of adaptations that increase onboard oxygen stores while slowing the rate at which these stores are utilized, thus extending ADL. Their locomotor muscles display elevated myoglobin concentrations and are composed predominantly of large Type I fibers. Vmt are also lower in deep divers than in shallow divers or athletic terrestrial species. The results of this study indicate that beaked whales and short-finned pilot whales do not uniformly display these characteristics and that each possesses a novel fiber profile compared with those of other deep divers.

The phylogeny of Cetartiodactyla: The importance of dense taxon sampling, missing data, and the remarkable promise of cytochrome b to provide reliable species-level phylogenies

Ingi Agnarsson, Laura J. May-Collado
Molecular Phylogenetics and Evolution 48 (2008) 964–985
http://dx.doi.org:/10.1016/j.ympev.2008.05.046

We perform Bayesian phylogenetic analyses on cytochrome b sequences from 264 of the 290 extant cetartiodactyl mammals (whales plus even-toed ungulates) and two recently extinct species, the ‘Mouse Goat’ and the ‘Irish Elk’. Previous primary analyses have included only a small portion of the species diversity within Cetartiodactyla, while a complete supertree analysis lacks resolution and branch lengths limiting its utility for comparative studies. The benefits of using a single-gene approach include rapid phylogenetic estimates for a large number of species. However, single-gene phylogenies often differ dramatically from studies involving multiple datasets suggesting that they often are unreliable. However, based on recovery of benchmark clades—clades supported in prior studies based on multiple independent datasets—and recovery of undisputed traditional taxonomic groups, Cytb performs extraordinarily well in resolving cetartiodactyl phylogeny when taxon sampling is dense. Missing data, however, (taxa with partial sequences) can compromise phylogenetic accuracy, suggesting a tradeoff between the benefits of adding taxa and introducing question marks. In the full data, a few species with a short sequences appear misplaced, however, sequence length alone seems a poor predictor of this phenomenon as other taxa.

The mammalian superorder Cetartiodactyla (whales and eventoed ungulates) contains nearly 300 species including many of immense commercial importance (cow, pig, and sheep) and of conservation interest and aesthetic value (antelopes, deer, giraffe, dolphins, and whales) (MacDonald, 2006). Certain members of this superorder count among the best studied organisms on earth, whether speaking morphologically, behaviorally, physiologically or genetically. Understanding the interrelationships among cetartiodactyl species, therefore, is of obvious importance with equally short sequences were not conspicuously misplaced. Although we recommend awaiting a better supported phylogeny based on more character data to reconsider classification and taxonomy within Cetartiodactyla, the new phylogenetic hypotheses provided here represent the currently best available tool for comparative species-level studies within this group. Cytb has been sequenced for a large percentage of mammals and appears to be a reliable phylogenetic marker as long as taxon sampling is dense. Therefore, an opportunity exists now to reconstruct detailed phylogenies of most of the major mammalian clades to rapidly provide much needed tools for species-level comparative studies.

Our results support the following relationship among the four major cetartiodactylan lineages (((Tylopoda ((Cetancodonta (Ruminantia + Suina))), with variable support. This arrangement has not been suggested previously, to our knowledge (see review in O’Leary and Gatesy, 2008 and discussion).

Relationships among clades within Cetancodonta are identical to those found by May-Collado and Agnarsson (2006).

Within Ruminantia all our analyzes suggest the following relationships among families: (((((Tragulidae((((Antilocapridae(((Giraffidae(( Cervidae(Moschidae + Bovidae))))) with relatively high support, supporting the subdivision of Ruminantia into Tragulina and Pecora.
In the rare cases where our results are inconsistent with benchmark clades, ad hoc explanations seem reasonable. The placement of M. meminna (Tragulidae) within Bovidae is likely an artifact of missing data, although remarkably it is the only conspicuous misplacement of a species across the whole phylogeny at the family level (while three species appear to be misplaced at the subfamily level within Cervidae in the full analysis, see Fig. 5a). This is supported by the fact that the placement of Moschiola receives low support, and the removal of Moschiola prior to analysis increases dramatically the support for clades close to where it nested (not shown, analysis available from authors), suggesting it had a tendency to ‘jump around’. Two other possibilities cannot be ruled out, however. One, that possibly the available sequence in Genbank may be mislabeled. And second, it should be kept in mind that the validity of Tragulidae has never been tested with molecular data including more than two species.

Oxygen and carbon dioxide fluctuations in burrows of subterranean blind mole rats indicate tolerance to hypoxic–hypercapnic stresses

Imad Shams, Aaron Avivi, Eviatar Nevo
Comparative Biochemistry and Physiology, Part A 142 (2005) 376 – 382
http://dx.doi.org:/10.1016/j.cbpa.2005.09.003

The composition of oxygen (O2), carbon dioxide (CO2), and soil humidity in the underground burrows from three species of the Israeli subterranean mole rat Spalax ehrenbergi superspecies were studied in their natural habitat. Two geographically close populations of each species from contrasting soil types were probed. Maximal CO2 levels (6.1%) and minimal O2 levels (7.2%) were recorded in northern Israel in the breeding mounds of S. carmeli in a flooded, poor drained field of heavy clay soil with very high volumetric water content. The patterns of gas fluctuations during the measurement period among the different Spalax species studied were similar. The more significant differentiation in gas levels was not among species, but between neighboring populations inhabiting heavy soils or light soils: O2 was lower and CO2 was higher in the heavy soils (clay and basaltic) compared to the relatively light soils (terra rossa and rendzina). The extreme values of gas concentration, which occurred during the rainy season, seemed to fluctuate with partial flooding of the tunnels, animal digging activity, and over-crowded breeding mounds inhabited by a nursing female and her offspring. The gas composition and soil water content in neighboring sites with different soil types indicated large differences in the levels of hypoxic–hypercapnic stress in different populations of the same species. A growing number of genes associated with hypoxic stress have been shown to exhibit structural and functional differences between the subterranean Spalax and the aboveground rat (Rattus norvegicus), probably reflecting the molecular adaptations that Spalax went through during 40 million years of evolution to survive efficiently in the severe fluctuations in gas composition in the underground habitat.

map of the studied sites

map of the studied sites

Schematic map of the studied sites: S. galili (2n =52): 1— Rehania (chalk); 2— Dalton (basaltic); S. golani (2n =54): 3— Majdal Shams (terra tossa); 4—Masa’ada (basaltic soils); S. carmeli (2n =58): 5— Al-Maker (heavy clay); 6— Muhraqa (terra rossa).

Comparison of gas composition (O2 and CO2) and water content between light and heavy soils inhabited by S. carmeli

Comparison of gas composition (O2 and CO2) and water content between light and heavy soils inhabited by S. carmeli

Comparison of gas composition (O2 and CO2) and water content between light and heavy soils inhabited by S. carmeli, Al-Maker (heavy soil) and Muhraqa (light soil). AverageTSD of measurements in the burrows of approximately 10 animals at a given date is presented. **p <0.01, T-test and Mann– Whitney test).

Subterranean mammals, which live in closed underground burrow systems, experience an atmosphere that is different from the atmosphere above-ground. Gas exchange between these two atmospheres depends on diffusion through the soil, which in turn, depends on soil particle size, water content, and burrow depth. Heavy soils (clay and basaltic), hold water and have little air space for gas diffusion. A large deviation from external gas composition is found in the burrows of Spalax living in these soil types. The maximal measured concentration of CO2 was 6.1% in Spalax breeding mounds, which is one of the highest concentrations among studied mammals in natural conditions. At the same time 7.2% O2 was measured in water saturated heavy clay soil

seasonal variation from August to March in mean O2, CO2, and soil water content

seasonal variation from August to March in mean O2, CO2, and soil water content

Example of seasonal variation from August to March in mean O2, CO2, and soil water content (VWC) in the Al-Maker population (2n =58, heavy soil). Values are presented as mean TSD.

In this study new data were presented for a wild mammal that survives in an extreme hypoxic–hypercapnic environment. Interestingly, the very low concentrations of O2 experienced by Spalax are correlated with the expression pattern of hypoxia related genes.  So far, we have shown higher and longer-term mRNA expression of erythropoietin, the main factor that regulates the level of circulating red blood cells, in subterranean Spalax compared to the above-ground rat in response to hypoxic stress, as well as differences in the response of erythropoietin to hypoxia in different populations of Spalax experiencing different hypoxic stress in nature. We also demonstrated that erythropoietin pattern of expression is different in Spalax than in Rattus throughout development, a pattern suggesting more efficient hypoxic tolerance in Spalax starting as early as in the embryonic stages. Furthermore, vascular endothelial growth factor (VEGF), which is a critical angiogenic factor that responds to hypoxia, is constitutively expressed at maximal levels in Spalax muscles, the most energy consuming tissue during digging. This level is 1.6-fold higher than in Rattus muscles and is correlated with significantly higher blood vessel concentration in the Spalax muscles compared to the Rattus muscles. Likewise, myoglobin the globin involved in oxygen homeostasis in skeletal muscles, exhibits different expression pattern under normoxia and in response to hypoxia in Spalax muscles compared to rat muscles as well as between different populations of Spalax exposed to different hypoxic stress in nature (unpublished results). Similarly, neuroglobin, a brain-specific globin involved in reversible oxygen binding, i.e., presumably in cellular homeostasis, is expressed differently in the Spalax brain compared to Rattus brain. Like erythropoietin and myoglobin also neuroglobin is expressed differently in Spalax populations experiencing different oxygen supply (unpublished results). Furthermore, Spalax p53 harbors two amino acid substitutions in its binding domain, which are identical to mutations found in p53 of human cancer cells. These substitutions endow Spalax p53 with several-fold higher activation of cell arrest and DNA repair genes compared to human p53 and favor activation of DNA repair genes over apoptotic genes. The study of specific tumoral variants indicates that such preference of growth arrest over apoptosis possibly results as a response to the hypoxic environmental stress known in tumors. Differences in the structure of other molecules related to homeostasis, namely, hemoglobin, haptoglobin (Nevo, 1999), and cytoglobin (unpublished) were also observed in Spalax.

Stress, adaptation, and speciation in the evolution of the blind mole rat, Spalax, in Israel

Eviatar Nevo
Molecular Phylogenetics and Evolution 66 (2013) 515–525
http://dx.doi.org/10.1016/j.ympev.2012.09.008

Environmental stress played a major role in the evolution of the blind mole rat superspecies Spalax ehrenbergi, affecting its adaptive evolution and ecological speciation underground. Spalax is safeguarded all of its life underground from aboveground climatic fluctuations and predators. However, it encounters multiple stresses in its underground burrows including darkness, energetics, hypoxia, hypercapnia, food scarcity, and pathogenicity. Consequently, it evolved adaptive genomic, proteomic, and phenomic complexes to cope with those stresses. Here I describe some of these adaptive complexes, and their theoretical and applied perspectives. Spalax mosaic molecular and organismal evolution involves reductions or regressions coupled with expansions or progressions caused by evolutionary tinkering and natural genetic engineering. Speciation of Spalax in Israel occurred in the Pleistocene, during the last 2.00–2.35 Mya, generating four species associated intimately with four climatic regimes with increasing aridity stress southwards and eastwards representing an ecological speciational adaptive trend: (Spalax golani, 2n = 54?S. galili, 2n = 52?S. carmeli, 2n = 58?S. judaei, 2n = 60). Darwinian ecological speciation occurred gradually with relatively little genetic change by Robertsonian chromosomal and genic mutations. Spalax genome sequencing has just been completed. It involves multiple adaptive complexes to life underground and is an evolutionary model to a few hundred underground mammals. It involves great promise in the future for medicine, space flight, and deep-sea diving.

Stress is a major driving force of evolution (Parsons, 2005; Nevo, 2011). Parsons defined stress as the ‘‘environmental factor causing potential injurious changes to biological systems with a potential for impacts on evolutionary processes’’. The global climatic transition from the middle Eocene to the early Oligocene (45–35 Ma = Million years ago) led to extensive convergent evolution underground of small subterranean mammals across the planet (Nevo, 1999; Lacey et al., 2000; Bennett and Faulkes, 2000; Begall et al., 2007). The subterranean ecotope provided small mammals with shelter from predators and extreme aboveground climatic stressful fluctuations of temperature and humidity. However, they had to evolve genomic adaptive complexes for the immense underground stresses of darkness, energy for burrowing in solid soil, low productivity and food scarcity, hypoxia, hypercapnia, and high infectivity. These stresses have been described in Nevo (1999, 2011) and Nevo et al. (2001); and Nevo list of Spalax publication at http://evolution.haifa.ac.il with many cited references relevant to these stresses).

blind subterranean mole rat of the Spalax ehrenbergi superspecies

blind subterranean mole rat of the Spalax ehrenbergi superspecies

The blind subterranean mole rat of the Spalax ehrenbergi superspecies in Israel. An extreme example of adaptation to life underground

Circadian rhythm and genes

adaptive circadian genes. We identified the circadian rhythm of Spalax
(Nevo et al., 1982) and described, cloned, sequenced, and expressed several circadian genes in Spalax. These include Clock, MOP3, three Period (Per), and cryptochromes (Avivi et al., 2001, 2002, 2003). The Spalax circadian genes are differentially conserved, yet characterized by a significant number of amino acid substitutions. The glutamine-rich area of Clock, which is assumed to function in circadian rhythmicity, is expanded in Spalax compared with that of mice and humans and is different in amino acid composition from that of rats. All three Per genes of Spalax oscillate with a periodicity of 24 h in the suprachaismatic nucleus, eye, and Harderian gland and are expressed in peripheral organs. Per genes are involved in clock resetting. Spalax Per 3 is unique in mammals though its function is still unresolved. The Spalax Per genes contribute to the unique adaptive circadian rhythm to life underground. The cryptochrome (Cry) genes, found in animals and plants, act both as photoreceptors and as ingredients of the negative feedback mechanism of the biological Clock. The CRY 1 protein is significantly closer to the human homolog than to that of mice, as was also shown in parts of the immunogenetic system. Both Cry 1 and Cry 2 mRNAs were found in the SCN, eye, harderian gland, and in peripheral tissues. Remarkably, the distinctly hypertrophied harderian gland is central in Spalax’s unique underground circadian rhythmicity (Pevet et al., 1984).

  • Spalax eye mosaic evolution
  • Gene expression in the eye of Spalax
  • Brain evolution in Spalax to underground stresses
  • Spalax: four species in Israel

The morphological, physiological, and behavioral Spalax eye patterns are underlain by gene expression representing regressive and progressive associated transcripts. Regressive transcripts involve B-2 microglobulin, transketolase, four keratins, alpha enolase, and different heat shock proteins. Several proteins may be involved in eye degeneration. These include heat shock protein 90alpha (hsp90alpha), found also in the blind fish Astyanax mexicanus, two transcripts of programmed cell death proteins, oculospanin, and peripherin 2, both belonging to the Tetraspanin family, in which 60 different mutations cause eye degeneration in humans. Several progressive transcripts in the Spalax eye are found in the retina of many mammals involving gluthatione, peroxidase 4, B spectrin, and Ankyrin; the last two characterize rod cells in the retina. Some transcripts are involved in metabolic processing of retinal, a vertebrate key component in phototransduction, and a relative of vitamin A.

cross section of the developing eye of the mole rat

cross section of the developing eye of the mole rat

Light micrographs showing cross section of the developing eye of the mole rat Spalax ehrenbergi. (A) Optic cup and lens vesicle initially develop normally (x100). (B) Eye at a later embryonic stage. Note appearance of iris-ciliary body rudiment (arrows), and development of the lens nucleus (L). ON, optic nerve (x100). (C) Eye at a still later fetal stage. Note massive growth of the iris-ciliary body complex colobomatous opening (arrow) (x100). (D) Early postnatal stage. The iris-ciliary body complex completely fills the chamber. The lens is vascularized and vacuolated (x100). (E) Adult eye. Eyelids are completely closed and pupil is absent. Note atrophic appearance of the optic disc region (arrow) (x65). (F) Higher magnification of the adult retina. The different retinal layers are retained: PE, pigment epithelium: RE, receptor layer; ON, outer nuclear layer: IN, inner nuclear layer; GC, ganglion cell layer (x500) (from Sanyal et al., 1990, Fig. 1).

The brains of subterranean mammals underwent dramatic evolution in accordance with underground stresses for digging and photoperiodic perception associated with vibrational, tactile, vocal, olfactory, and magnetic communication systems replacing sight, as is seen in Spalax. The brain of Spalax is twice as large as that of the laboratory rat of the same body size. The somatosensory region in the isocortex of Spalax is 1.7 times, the thalamic nuclei 1.3 times, and the motor cortex 3.1 times larger than in the sighted laboratory rat Rattus norvegicus matched to body size.

The ecological stress determinant in Spalax brain evolution is highlighted by the four species of the Spalax ehrenbergi superspecies in Israel. They differentiated chromosomally (by means of Robertsonian mutations and fission), allopatrically, and clinally southwards into four species associated with different climatic regimes, following the gradient of increasing aridity stress and decreasing predictability southwards towards the desert: Spalax galili (2n = 52) ->S. golani (2n = 54)->S. carmeli (2n = 58)->S. judaei (2n = 60), and eastwards S. galili ->S. golani (2n = 52–>54) (Fig. 2). This chromosomal speciation trend southwards is associated with the regional aridity stress southwards (and eastwards) in Israel, budding new species adapted genomically, proteomically, and phenomically (i.e., in morphology, physiology, and behavior) to increasing stresses of higher solar radiation, temperature, and drought southwards (Nevo, 1999; Nevo et al., 2001; Nevo
list of Spalax at http://evolution.haifa.ac.il). A uniquely recent discovery of incipient sympatric ecological speciation at a microscale in Spalax triggered by local stresses occurs within Spalax galili.

retinal input to primary visual structures in Spalax

retinal input to primary visual structures in Spalax

Relative degree of retinal input to primary visual structures in Spalax, hamster, rat, and Spalacopus cyanus (South American Octodontidae, ‘‘coruro’’). These rodents are of similar body size (120–140 g). B. Relative degree of change in the proportions of retinal input to different primary visual structures in Spalax compared with measures obtained in other rodents. A relative progressive development in Spalax is seen in structures involved in photoperiodic and neuroendocrine functions (SCN, BNST).The main regressive feature is the drastic relative reduction of retinal input to the superior colliculus. The main regressive feature is the drastic reduction of retinal input to the superior colliculus. The relative size of other visual structures in Spalax is modified compared to that of the other species. c. Comparison of the absolute size (volume, mm3 x 10-4) of visual structures in Spalax and other rodents. The size of the SCN is equivalent in all species. The vLGN and dLGN are reduced by 87–93% in Spalax. The retino-recipient layers of the superior colliculus are reduced by 97%. Abbreviations: SCN: suprachiasmatic nucleus; BNST: bed nucleus of the stria terminalis; dLGN: dorsal lateral geniculate nucleus; SC: superior colliculus [From Cooper et al., 1993 (Fig 3)].

Subterranean life has a high energetic cost if an animal has to burrow in order to obtain its food. For a 150 g Thomomys bottae, burrowing 1 m may be 360–3400 times more expensive energetically than moving the same distance on the surface (Vleck, 1979). Mean rates of oxygen consumption during burrowing at 22 oC are from 2.8 to 7.1 times the RMR. Vleck developed a model examining the energetics of foraging by burrowing and found that, in the desert, Thomomys adjusts the burrow segment length to minimize the cost of burrowing. Since burrowing becomes less economic as body size increases, Vleck (1981) predicted that the maximum possible body size that a subterranean mammal can attain depends on a balance between habitat productivity and the cost of burrowing in local soils. Vleck’s cost of burrowing hypothesis has been verified in multiple cases. Heth (1989) demonstrated longer burrows in the rendzina soil and shorter ones in the terra rossa soil, associating lower productivity in the former for Spalax.

Food is a limiting factor for subterranean mammals. The abundance and distribution of food explain some of the ecological, physiological, and behavioral characteristics of subterranean mammals. In a field test of Spalax foraging strategy, we concluded that Spalax was a generalist due to the constraints of the subterranean ecotope. Restricted foraging time primarily during the winter when soil is wet, and the high energetic investment of tunneling to get to food items is significantly reduced than in summertime.
We also identified a decrease in the basic metabolic rate towards the desert, i.e., economizing energetics. The maintenance of adequate O2 transport in a subterranean mammal confronting hypoxia requires adaptation along the O2 transport system, achieved by increasing the flow of O2 in the convection systems (ventilation and perfusion) and by reduction of oxygen pressure (PO2) gradients at the diffusion barriers (lung blood, blood-tissue (Arieli, 1990). The PO2 gradient between blood capillaries and respiring mitochondria capillaries is large, and any adaptation at this level could be significant for O2 transport. Reduction of diffusion distance in a muscle can be achieved, like in Spalax, by increasing the number of capillaries that surround muscle fiber or by reducing fiber areas.

Geographic distribution in Israel of the four chromosomal species belonging to the S. ehrenbergi superspecies

Geographic distribution in Israel of the four chromosomal species belonging to the S. ehrenbergi superspecies

Geographic distribution in Israel of the four chromosomal species belonging to the S. ehrenbergi superspecies that are separated by narrow hybrid zones (2n = 52, 54, 58, and 60, now named as S. galili, S. golani, S. carmeli, and S. judaei, respectively; see Nevo et al., 2001).

Spalacid evolution, based on mtDNA, is driven by climatic oscillations and stresses. The underground ecotope provided subterranean mammals with shelter from extreme climate (temperature and humidity) fluctuations, and predators. However, they had to extensively and intensively adapt to the multiple underground stresses (darkness, energetic, low productivity and
food scarcity, hypoxia, hypercapnia, and high infectivity). All subterranean mammals, including spalacids as an extreme case, share convergent molecular and organismal adaptations to their shared unique underground ecotope. Evolution underground, as exemplified here in spalacids, led to mosaic molecular and organismal evolutionary syndromes to cope with multiple stresses.

Speciation involves all rates – from gradual to rapid. Subterranean mammals, with the spalacid example discussed above, provide uniquely rich evolutionary global tests of speciation and adaptation, convergence, regression, progression, and mosaic evolutionary processes. Adaptation and speciation underground was one of the most dramatic natural experiments verifying Darwinian evolution.

The Spalax genome sequencing has just been completed. It is being analyzed and will soon be published in 2012. This will be a milestone in understanding how numerous mammals across the globe, who found underground shelter from climatic fluctuations and stresses above ground, cope with the new suite of stresses they encountered underground, demanding a new engineering overhaul on all organizational levels, selecting for adaptive complexes to cope with the new underground stresses. The main current and future challenges are to compare and contrast genome sequences and identify the genomic basis of adaptation and speciation.

This global Cenozoic experiment could answer the following open questions: How heterozygous is the whole genome? How prevalent are retrotransposons and what is their functional role? How many genes are involved in the Spalax genome and how are they regulated? What are the genic and regulatory networks resisting the multiple stresses underground? How much of the Spalax genome is conserved and how much is reorganized to cope with the underground stresses? How is the solitary blind mole rat, Spalax, different from the social naked mole rat Heterocephalus? How are the processes of reduction, expansion, and genetic tinkering and engineering reflected across the genome? How effective is copy number variation in regulation? Is there similarity in the transcriptomes of subterranean mammals? How could we harness the rich genome repertoire of Spalax to revolutionize medicine, especially in the realm of hypoxia tolerance and the related major diseases of the western world, e.g., cancer, stroke, and cardiovascular diseases? What is the phylogenetic origin of Spalax? How much of the Spalax genome represents its phylogenetic roots and how much of coding and noncoding genomic regions are shared with other subterranean mammals across the globe in adapting to life underground?

The Atmospheric Environment of the Fossorial Mole Rat (Spalax Ehrenbergi): Effects of Season, Soil Texture, Rain, Temperature and Activity

  1. Arieli
    Comp Biochen Physiol. 1978; 63A:569-5151. The fossorial mole rat (Spalax ehrenbergi) may inhabit heavy soil with low gas permeability.
  2. Air composition in burrows in heavy soil deviates from atmospheric air more than that of burrows in light soil.
  3. In winter and spring O2 and CO2 concentrations in breeding mounds were 16.5% O2 and 2.5-3x CO2 and the extreme values measured were 14.0% O2 and 4.8% Cot.
  4. Hypoxia and hypercapnia in the burrow develop shortly after rain and when ambient temperature drops.
  5. Composition of the burrows air is influenced by the solubility of CO2 in soil water and by faster penetration of oxygen than outflowing of CO2.

Hypo-osmotic stress-induced physiological and ion-osmoregulatory responses in European sea bass (Dicentrarchus labrax) are modulated differentially by nutritional status

Amit Kumar Sinha, AF Dasan, R Rasoloniriana, N Pipralia, R Blust, G De Boeck
Comparative Biochemistry and Physiology, Part A 181 (2015) 87–99
http://dx.doi.org/10.1016/j.cbpa.2014.11.024

We investigated the impact of nutritional status on the physiological, metabolic and ion-osmoregulatory performance of European sea bass (Dicentrarchus labrax)when acclimated to seawater (32 ppt), brackishwater (20 and 10 ppt) and hyposaline water (2.5 ppt) for 2 weeks. Following acclimation to different salinities, fish were either fed or fasted (unfed for 14 days). Plasma osmolality, [Na+], [Cl−] and muscle water contentwere severely altered in fasted fish acclimated to 10 and 2.5 ppt in comparison to normal seawater-acclimated fish, suggesting ion regulation and acid–base balance disturbances. In contrast to feed-deprived fish, fed fish were able to avoid osmotic perturbation more effectively. This was accompanied by an increase in Na+/K+-ATPase expression and activity, transitory activation of H+-ATPase (only at 2.5 ppt) and down-regulation of Na+/K+/2Cl− gene expression. Ammonia excretion rate was inhibited to a larger extent in fasted fish acclimated to low salinities while fed fish were able to excrete efficiently. Consequently, the build-up of ammonia in the plasma of fed fish was relatively lower. Energy stores, especially glycogen and lipid, dropped in the fasted fish at low salinities and progression towards the anaerobic metabolic pathway became evident by an increase in plasma lactate level. Overall, the results indicate no osmotic stress in both feeding treatments within the salinity range of 32 to 20 ppt. However, at lower salinities (10–2.5 ppt) feed deprivation tends to reduce physiological, metabolic, ion-osmo-regulatory and molecular compensatory mechanisms and thus limits the fish’s abilities to adapt to a hypo-osmotic environment.

The absence of ion-regulatory suppression in the gills of the aquatic air-breathing fish Trichogaster lalius during oxygen stress

Chun-Yen Huang, Hsueh-Hsi Lin, Cheng-Huang Lin, Hui-Chen Lin
Comparative Biochemistry and Physiology, Part A 179 (2015) 7–16
http://dx.doi.org/10.1016/j.cbpa.2014.08.017

The strategy for most teleost to survive in hypoxic or anoxic conditions is to conserve energy expenditure, which can be achieved by suppressing energy-consuming activities such as ion regulation. However, an air-breathing fish can cope with hypoxic stress using a similar adjustment or by enhancing gas exchange ability, both behaviorally and physiologically. This study examined Trichogaster lalius, an air-breathing fish without apparent gill modification, for their gill ion-regulatory abilities and glycogen utilization under a hypoxic  treatment. We recorded air-breathing frequency, branchial morphology, and the expression of ion-regulatory proteins (Na+/K+-ATPase and vacuolar-type H+-ATPase) in the 1st and 4th gills and labyrinth organ (LO), and the expression of glycogen utilization (GP, glycogen phosphorylase protein expression and glycogen content) and other protein responses (catalase, CAT; carbonic anhydrase II, CAII; heat shock protein 70, HSP70; hypoxia-inducible factor-1α, HIF-1α; proliferating cell nuclear antigen, PCNA; superoxidase dismutase, SOD) in the gills of T. lalius after 3 days in hypoxic and restricted conditions. No morphological modification of the 1st and 4th gills was observed. The air breathing behavior of the fish and CAII protein expression both increased under hypoxia. Ion-regulatory abilities were not suppressed in the hypoxic or restricted groups, but glycogen utilization was enhanced within the groups. The expression of HIF-1α, HSP70 and PCNA did not vary among the treatments. Regarding the antioxidant system, decreased CAT enzyme activity was observed among the groups. In conclusion, during hypoxic stress, T. lalius did not significantly reduce energy consumption but enhanced gas exchange ability and glycogen expenditure.

The combined effect of hypoxia and nutritional status on metabolic and ionoregulatory responses of common carp (Cyprinus carpio)

Sofie Moyson, HJ Liew, M Diricx, AK Sinha, R Blusta, G De Boeck
Comparative Biochemistry and Physiology, Part A 179 (2015) 133–143
http://dx.doi.org/10.1016/j.cbpa.2014.09.017

In the present study, the combined effects of hypoxia and nutritional status were examined in common carp (Cyprinus carpio), a relatively hypoxia tolerant cyprinid. Fish were either fed or fasted and were exposed to hypoxia (1.5–1.8mgO2 L−1) at or slightly above their critical oxygen concentration during 1, 3 or 7 days followed by a 7 day recovery period. Ventilation initially increased during hypoxia, but fasted fish had lower ventilation frequencies than fed fish. In fed fish, ventilation returned to control levels during hypoxia, while in fasted fish recovery only occurred after reoxygenation. Due to this, C. carpio managed, at least in part, to maintain aerobic metabolism during hypoxia: muscle and plasma lactate levels remained relatively stable although they tended to be higher in fed fish (despite higher ventilation rates). However, during recovery, compensatory responses differed greatly between both feeding regimes: plasma lactate in fed fish increased with a simultaneous breakdown of liver glycogen indicating increased energy use, while fasted fish seemed to economize energy and recycle decreasing plasma lactate levels into increasing liver glycogen levels. Protein was used under both feeding regimes during hypoxia and subsequent recovery: protein levels reduced mainly in liver for fed fish and in muscle for fasted fish. Overall, nutritional status had a greater impact on energy reserves than the lack of oxygen with a lower hepatosomatic index and lower glycogen stores in fasted fish. Fasted fish transiently increased Na+/K+-ATPase activity under hypoxia, but in general ionoregulatory balance proved to be only slightly disturbed, showing that sufficient energy was left for ion regulation.

The effect of temperature and body size on metabolic scope of activity in juvenile Atlantic cod Gadus morhua L.

Bjørn Tirsgaard, Jane W. Behrens, John F. Steffensen
Comparative Biochemistry and Physiology, Part A 179 (2015) 89–94
http://dx.doi.org/10.1016/j.cbpa.2014.09.033

Changes in ambient temperature affect the physiology and metabolism and thus the distribution of fish. In this study we used intermittent flow respirometry to determine the effect of temperature (2, 5, 10, 15 and 20 °C) and wet body mass (BM) (~30–460 g) on standard metabolic rate (SMR, mg O2 h−1), maximum metabolic rate (MMR, mg O2 h−1) and metabolic scope (MS, mg O2 h−1) of juvenile Atlantic cod. SMR increased with BM irrespectively of temperature, resulting in an average scaling exponent of 0.87 (0.82–0.92). Q10 values were 1.8–2.1 at temperatures between 5 and 15 °C but higher (2.6–4.3) between 2 and 5 °C and lower (1.6–1.4) between 15 and 20 °C in 200 and 450 g cod. MMR increased with temperature in the smallest cod (50 g) but in the larger cod MMR plateaued between 10, 15 and 20 °C. This resulted in a negative correlation between the optimal temperature for MS (Topt) and BM, Topt being respectively 14.5, 11.8 and 10.9 °C in a 50, 200 and 450 g cod. Irrespective of BM cold water temperatures resulted in a reduction (30–35%) of MS whereas the reduction of MS at warm temperatures was only evident for larger fish (200 and 450 g), caused by plateauing of MMR at 10 °C and above. Warm temperatures thus seem favorable for smaller (50 g) juvenile cod, but not for larger conspecifics (200 and 450 g).

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Neonatal Pathophysiology

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Neonatal Pathophysiology

Writer and Curator: Larry H. Bernstein, MD, FCAP 

 

Introduction

This curation deals with a large and specialized branch of medicine that grew since the mid 20th century in concert with the developments in genetics and as a result of a growing population, with large urban populations, increasing problems of premature deliveries.  The problems of prematurity grew very preterm to very low birth weight babies with special problems.  While there were nurseries, the need for intensive care nurseries became evident in the 1960s, and the need for perinatal care of pregnant mothers also grew as a result of metabolic problems of the mother, intrauterine positioning of the fetus, and increasing numbers of teen age pregnancies as well as nutritional problems of the mother.  There was also a period when the manufacturers of nutritional products displaced the customary use of breast feeding, which was consequential.  This discussion is quite comprehensive, as it involves a consideration of the heart, the lungs, the brain, and the liver, to a large extent, and also the kidneys and skeletal development.

It is possible to outline, with a proportionate emphasis based on frequency and severity, this as follows:

  1. Genetic and metabolic diseases
  2. Nervous system
  3. Cardiovascular
  4. Pulmonary
  5. Skeletal – bone and muscle
  6. Hematological
  7. Liver
  8. Esophagus, stomach, and intestines
  9. Kidneys
  10. Immune system

Fetal Development

Gestation is the period of time between conception and birth when a baby grows and develops inside the mother’s womb. Because it’s impossible to know exactly when conception occurs, gestational age is measured from the first day of the mother’s last menstrual cycle to the current date. It is measured in weeks. A normal gestation lasts anywhere from 37 to 41 weeks.

Week 5 is the start of the “embryonic period.” This is when all the baby’s major systems and structures develop. The embryo’s cells multiply and start to take on specific functions. This is called differentiation. Blood cells, kidney cells, and nerve cells all develop. The embryo grows rapidly, and the baby’s external features begin to form.

Week 6-9:   Brain forms into five different areas. Some cranial nerves are visible. Eyes and ears begin to form. Tissue grows that will the baby’s spine and other bones. Baby’s heart continues to grow and now beats at a regular rhythm. Blood pumps through the main vessels. Your baby’s brain continues to grow. The lungs start to form. Limbs look like paddles. Essential organs begin to grow.

Weeks 11-18: Limbs extended. Baby makes sucking motion. Movement of limbs. Liver and pancreas produce secretions. Muscle and bones developing.

Week 19-21: Baby can hear. Mom feels baby – and quickening.

http://www.nlm.nih.gov/medlineplus/ency/article/002398.htm

fetal-development

fetal-development

https://polination.files.wordpress.com/2014/02/abortion-new-research-into-fetal-development.jpg

Inherited Metabolic Disorders

The original cause of most genetic metabolic disorders is a gene mutation that occurred many, many generations ago. The gene mutation is passed along through the generations, ensuring its preservation.

Each inherited metabolic disorder is quite rare in the general population. Considered all together, inherited metabolic disorders may affect about 1 in 1,000 to 2,500 newborns. In certain ethnic populations, such as Ashkenazi Jews (Jews of central and eastern European ancestry), the rate of inherited metabolic disorders is higher.

Hundreds of inherited metabolic disorders have been identified, and new ones continue to be discovered. Some of the more common and important genetic metabolic disorders include:

Lysosomal storage disorders : Lysosomes are spaces inside cells that break down waste products of metabolism. Various enzyme deficiencies inside lysosomes can result in buildup of toxic substances, causing metabolic disorders including:

  • Hurler syndrome (abnormal bone structure and developmental delay)
  • Niemann-Pick disease (babies develop liver enlargement, difficulty feeding, and nerve damage)
  • Tay-Sachs disease (progressive weakness in a months-old child, progressing to severe nerve damage; the child usually lives only until age 4 or 5)
  • Gauchers disease and others

Galactosemia: Impaired breakdown of the sugar galactose leads to jaundice, vomiting, and liver enlargement after breast or formula feeding by a newborn.

Maple syrup urine disease: Deficiency of an enzyme called BCKD causes buildup of amino acids in the body. Nerve damage results, and the urine smells like syrup.

Phenylketonuria (PKU): Deficiency of the enzyme PAH results in high levels of phenylalanine in the blood. Mental retardation results if the condition is not recognized.

Glycogen storage diseases: Problems with sugar storage lead to low blood sugar levels, muscle pain, and weakness.

Metal metabolism disorders: Levels of trace metals in the blood are controlled by special proteins. Inherited metabolic disorders can result in protein malfunction and toxic accumulation of metal in the body:

Wilson disease (toxic copper levels accumulate in the liver, brain, and other organs)

Hemochromatosis (the intestines absorb excessive iron, which builds up in the liver, pancreas, joints, and heart, causing damage)

Organic acidemias: methylmalonic acidemia and propionic acidemia.

Urea cycle disorders: ornithine transcarbamylase deficiency and citrullinemia

Hemoglobinopathies – thalassemias, sickle cell disease

Red cell enzyme disorders – glucose-6-phosphate dehydrogenase, pyruvate kinase

This list is by no means complete.

http://www.webmd.com/a-to-z-guides/inherited-metabolic-disorder-types-and-treatments

New variations in the galactose-1-phosphate uridyltransferase (GALT) gene

Clinical and molecular spectra in galactosemic patients from neonatal screening in northeastern Italy: Structural and functional characterization of new variations in the galactose-1-phosphate uridyltransferase (GALT) gene

E Viggiano, A Marabotti, AP Burlina, C Cazzorla, MR D’Apice, et al.
Gene 559 (2015) 112–118
http://dx.doi.org/10.1016/j.gene.2015.01.013
Galactosemia (OMIM 230400) is a rare autosomal recessive inherited disorder caused by deficiency of galactose-1-phosphate uridyltransferase (GALT; OMIM 606999) activity. The incidence of galactosemia is 1 in 30,000–60,000, with a prevalence of 1 in 47,000 in the white population. Neonates with galactosemia can present acute symptoms, such as severe hepatic and renal failure, cataract and sepsis after milk introduction. Dietary restriction of galactose determines the clinical improvement in these patients. However, despite early diagnosis by neonatal screening and dietary treatment, a high percentage of patients develop long-term complications such as cognitive disability, speech problems, neurological and/or movement disorders and, in females, ovarian dysfunction.

With the benefit of early diagnosis by neonatal screening and early therapy, the acute presentation of classical galactosemia can be prevented. The objectives of the current study were to report our experience with a group of galactosemic patients identified through the neonatal screening programs in northeastern Italy during the last 30 years.

No neonatal deaths due to galactosemia complications occurred after the introduction of the neonatal screening program. However, despite the early diagnosis and dietary treatment, the patients with classical galactosemia showed one or more long-term complications.

A total of 18 different variations in the GALT gene were found in the patient cohort: 12 missense, 2 frameshift, 1 nonsense, 1 deletion, 1 silent variation, and 1 intronic. Six (p.R33P, p.G83V, p.P244S, p.L267R, p.L267V, p.E271D) were new variations. The most common variation was p.Q188R (12 alleles, 31.5%), followed by p.K285N (6 alleles, 15.7%) and p.N314D (6 alleles, 15.7%). The other variations comprised 1 or 2 alleles. In the patients carrying a new mutation, the biochemical analysis of GALT activity in erythrocytes showed an activity of < 1%. In silico analysis (SIFT, PolyPhen-2 and the computational analysis on the static protein structure) showed potentially damaging effects of the six new variations on the GALT protein, thus expanding the genetic spectrum of GALT variations in Italy. The study emphasizes the difficulty in establishing a genotype–phenotype correlation in classical galactosemia and underlines the importance of molecular diagnostic testing prior to making any treatment.

Diagnosis and Management of Hereditary Hemochromatosis

Reena J. Salgia, Kimberly Brown
Clin Liver Dis 19 (2015) 187–198
http://dx.doi.org/10.1016/j.cld.2014.09.011

Hereditary hemochromatosis (HH) is a diagnosis most commonly made in patients with elevated iron indices (transferrin saturation and ferritin), and HFE genetic mutation testing showing C282Y homozygosity.

The HFE mutation is believed to result in clinical iron overload through altering hepcidin levels resulting in increased iron absorption.

The most common clinical complications of HH include cirrhosis, diabetes, nonischemic cardiomyopathy, and hepatocellular carcinoma.

Liver biopsy should be performed in patients with HH if the liver enzymes are elevated or serum ferritin is greater than 1000 mg/L. This is useful to determine the degree of iron overload and stage the fibrosis.

Treatment of HH with clinical iron overload involves a combination of phlebotomy and/or chelation therapy. Liver transplantation should be considered for patients with HH-related decompensated cirrhosis.

Health economic evaluation of plasma oxysterol screening in the diagnosis of Niemann–Pick Type C disease among intellectually disabled using discrete event simulation

CDM van Karnebeek, Tima Mohammadi, Nicole Tsaod, Graham Sinclair, et al.
Molecular Genetics and Metabolism 114 (2015) 226–232
http://dx.doi.org/10.1016/j.ymgme.2014.07.004

Background: Recently a less invasive method of screening and diagnosing Niemann–Pick C (NP-C) disease has emerged. This approach involves the use of a metabolic screening test (oxysterol assay) instead of the current practice of clinical assessment of patients suspected of NP-C (review of medical history, family history and clinical examination for the signs and symptoms). Our objective is to compare costs and outcomes of plasma oxysterol screening versus current practice in diagnosis of NP-C disease among intellectually disabled (ID) patients using decision-analytic methods.
Methods: A discrete event simulation model was conducted to follow ID patients through the diagnosis and treatment of NP-C, forecast the costs and effectiveness for a cohort of ID patients and compare the outcomes and costs in two different arms of the model: plasma oxysterol screening and routine diagnosis procedure (anno 2013) over 5 years of follow up. Data from published sources and clinical trials were used in simulation model. Unit costs and quality-adjusted life-years (QALYs) were discounted at a 3% annual rate in the base case analysis. Deterministic and probabilistic sensitivity analyses were conducted.
Results: The outcomes of the base case model showed that using plasma oxysterol screening for diagnosis of NP-C disease among ID patients is a dominant strategy. It would result in lower total cost and would slightly improve patients’ quality of life. The average amount of cost saving was $3642 CAD and the incremental QALYs per each individual ID patient in oxysterol screening arm versus current practice of diagnosis NP-C was 0.0022 QALYs. Results of sensitivity analysis demonstrated robustness of the outcomes over the wide range of changes in model inputs.
Conclusion: Whilst acknowledging the limitations of this study, we conclude that screening ID children and adolescents with oxysterol tests compared to current practice for the diagnosis of NP-C is a dominant strategy with clinical and economic benefits. The less costly, more sensitive and specific oxysterol test has potential to save costs to the healthcare system while improving patients’ quality of life and may be considered as a routine tool in the NP-C diagnosis armamentarium for ID. Further research is needed to elucidate its effectiveness in patients presenting characteristics other than ID in childhood and adolescence.

Neurological and Behavioral Disorders

Estrogen receptor signaling during vertebrate development

Maria Bondesson, Ruixin Hao, Chin-Yo Lin, Cecilia Williams, Jan-Åke Gustafsson
Biochimica et Biophysica Acta 1849 (2015) 142–151
http://dx.doi.org/10.1016/j.bbagrm.2014.06.005

Estrogen receptors are expressed and their cognate ligands produced in all vertebrates, indicative of important and conserved functions. Through evolution estrogen has been involved in controlling reproduction, affectingboth the development of reproductive organs and reproductive behavior. This review broadly describes the synthesis of estrogens and the expression patterns of aromatase and the estrogen receptors, in relation to estrogen functions in the developing fetus and child. We focus on the role of estrogens for the development of reproductive tissues, as well as non-reproductive effects on the developing brain. We collate data from human, rodent, bird and fish studies and highlight common and species-specific effects of estrogen signaling on fetal development. Morphological malformations originating from perturbed estrogen signaling in estrogen receptor and aromatase knockout mice are discussed, as well as the clinical manifestations of rare estrogen receptor alpha and aromatase gene mutations in humans. This article is part of a Special Issue entitled: Nuclear receptors in animal development.

 

Memory function and hippocampal volumes in preterm born very-low-birth-weight (VLBW) young adults

Synne Aanes, Knut Jørgen Bjuland, Jon Skranes, Gro C.C. Løhaugen
NeuroImage 105 (2015) 76–83
http://dx.doi.org/10.1016/j.neuroimage.2014.10.023

The hippocampi are regarded as core structures for learning and memory functions, which is important for daily functioning and educational achievements. Previous studies have linked reduction in hippocampal volume to working memory problems in very low birth weight (VLBW; ≤1500 g) children and reduced general cognitive ability in VLBW adolescents. However, the relationship between memory function and hippocampal volume has not been described in VLBW subjects reaching adulthood. The aim of the study was to investigate memory function and hippocampal volume in VLBW young adults, both in relation to perinatal risk factors and compared to term born controls, and to look for structure–function relationships. Using Wechsler Memory Scale-III and MRI, we included 42 non-disabled VLBW and 61 control individuals at age 19–20 years, and related our findings to perinatal risk factors in the VLBW-group. The VLBW young adults achieved lower scores on several subtests of the Wechsler Memory Scale-III, resulting in lower results in the immediate memory indices (visual and auditory), the working memory index, and in the visual delayed and general memory delayed indices, but not in the auditory delayed and auditory recognition delayed indices. The VLBW group had smaller absolute and relative hippocampal volumes than the controls. In the VLBW group inferior memory function, especially for the working memory index, was related to smaller hippocampal volume, and both correlated with lower birth weight and more days in the neonatal intensive care unit (NICU). Our results may indicate a structural–functional relationship in the VLBW group due to aberrant hippocampal development and functioning after preterm birth.

The relation of infant attachment to attachment and cognitive and behavioural outcomes in early childhood

Yan-hua Ding, Xiu Xua, Zheng-yan Wang, Hui-rong Li, Wei-ping Wang
Early Human Development 90 (2014) 459–464
http://dx.doi.org/10.1016/j.earlhumdev.2014.06.004

Background: In China, research on the relation of mother–infant attachment to children’s development is scarce.
Aims: This study sought to investigate the relation of mother–infant attachment to attachment, cognitive and behavioral development in young children.                                                                                                                            Study design: This study used a longitudinal study design.
Subjects: The subjects included healthy infants (n=160) aged 12 to 18 months.
Outcome measures: Ainsworth’s “Strange Situation Procedure” was used to evaluate mother–infant attachment types. The attachment Q-set (AQS) was used to evaluate the attachment between young children and their mothers. The Bayley scale of infant development-second edition (BSID-II) was used to evaluate cognitive developmental level in early childhood. Achenbach’s child behavior checklist (CBCL) for 2- to 3-year-oldswas used to investigate behavioral problems.
Results: In total, 118 young children (73.8%) completed the follow-up; 89.7% of infants with secure attachment and 85.0% of infants with insecure attachment still demonstrated this type of attachment in early childhood (κ = 0.738, p b 0.05). Infants with insecure attachment collectively exhibited a significantly lower mental development index (MDI) in early childhood than did infants with secure attachment, especially the resistant type. In addition, resistant infants were reported to have greater social withdrawal, sleep problems and aggressive behavior in early childhood.
Conclusion: There is a high consistency in attachment development from infancy to early childhood. Secure mother–infant attachment predicts a better cognitive and behavioral outcome; whereas insecure attachment, especially the resistant attachment, may lead to a lower cognitive level and greater behavioral problems in early childhood.

representations of the HPA axis

representations of the HPA axis

representations of limbic stress-integrative pathways from the prefrontal cortex, amygdala and hippocampus

representations of limbic stress-integrative pathways from the prefrontal cortex, amygdala and hippocampus

Fetal programming of schizophrenia: Select mechanisms

Monojit Debnatha, Ganesan Venkatasubramanian, Michael Berk
Neuroscience and Biobehavioral Reviews 49 (2015) 90–104
http://dx.doi.org/10.1016/j.neubiorev.2014.12.003

Mounting evidence indicates that schizophrenia is associated with adverse intrauterine experiences. An adverse or suboptimal fetal environment can cause irreversible changes in brain that can subsequently exert long-lasting effects through resetting a diverse array of biological systems including endocrine, immune and nervous. It is evident from animal and imaging studies that subtle variations in the intrauterine environment can cause recognizable differences in brain structure and cognitive functions in the offspring. A wide variety of environmental factors may play a role in precipitating the emergent developmental dysregulation and the consequent evolution of psychiatric traits in early adulthood by inducing inflammatory, oxidative and nitrosative stress (IO&NS) pathways, mitochondrial dysfunction, apoptosis, and epigenetic dysregulation. However, the precise mechanisms behind such relationships and the specificity of the risk factors for schizophrenia remain exploratory. Considering the paucity of knowledge on fetal programming of schizophrenia, it is timely to consolidate the recent advances in the field and put forward an integrated overview of the mechanisms associated with fetal origin of schizophrenia.

NMDA receptor dysfunction in autism spectrum disorders

Eun-Jae Lee, Su Yeon Choi and Eunjoon Kim
Current Opinion in Pharmacology 2015, 20:8–13
http://dx.doi.org/10.1016/j.coph.2014.10.007

Autism spectrum disorders (ASDs) represent neurodevelopmental disorders characterized by two core symptoms;

(1)  impaired social interaction and communication, and
(2)  restricted and repetitive behaviors, interests, and activities.

ASDs affect ~ 1% of the population, and are considered to be highly genetic in nature. A large number (~600) of ASD-related genetic variations have been identified (sfari.org), and target gene functions are apparently quite diverse. However, some fall onto common pathways, including synaptic function and chromosome remodeling, suggesting that core mechanisms may exist.

Abnormalities and imbalances in neuronal excitatory and inhibitory synapses have been implicated in diverse neuropsychiatric disorders including autism spectrum disorders (ASDs). Increasing evidence indicates that dysfunction of NMDA receptors (NMDARs) at excitatory synapses is associated with ASDs. In support of this, human ASD-associated genetic variations are found in genes encoding NMDAR subunits. Pharmacological enhancement or suppression of NMDAR function ameliorates ASD symptoms in humans. Animal models of ASD display bidirectional NMDAR dysfunction, and correcting this deficit rescues ASD-like behaviors. These findings suggest that deviation of NMDAR function in either direction contributes to the development of ASDs, and that correcting NMDAR dysfunction has therapeutic potential for ASDs.

Among known synaptic proteins implicated in ASD are metabotropic glutamate receptors (mGluRs). Functional enhancement and suppression of mGluR5 are associated with fragile X syndrome and tuberous sclerosis, respectively, which share autism as a common phenotype. More recently, ionotropic glutamate receptors, namely NMDA receptors (NMDARs) and AMPA receptors (AMPARs), have also been implicated in ASDs. In this review, we will focus on NMDA receptors and summarize evidence supporting the hypothesis that NMDAR dysfunction contributes to ASDs, and, by extension, that correcting NMDAR dysfunction has therapeutic potential for ASDs. ASD-related human NMDAR genetic variants.

Chemokines roles within the hippocampus

Chemokines roles within the hippocampus

IL-1 mediates stress-induced activation of the HPA axis

IL-1 mediates stress-induced activation of the HPA axis

A systemic model of the beneficial role of immune processes in behavioral and neural plasticity

A systemic model of the beneficial role of immune processes in behavioral and neural plasticity

Three Classes of Glutamate Receptors

Three Classes of Glutamate Receptors

Clinical studies on ASDs have identified genetic variants of NMDAR subunit genes. Specifically, de novo mutations have been identified in the GRIN2B gene, encoding the GluN2B subunit. In addition, SNP analyses have linked both GRIN2A (GluN2A subunit) and GRIN2B with ASDs. Because assembled NMDARs contain four subunits, each with distinct properties, ASD-related GRIN2A/ GRIN2B variants likely alter the functional properties of NMDARs and/or NMDAR-dependent plasticity.

Pharmacological modulation of NMDAR function can improve ASD symptoms. D-cycloserine (DCS), an NMDAR agonist, significantly ameliorates social withdrawal and repetitive behavior in individuals with ASD. These results suggest that reduced NMDAR function may contribute to the development of ASDs in humans.

We can divide animal studies into two groups. The first group consists of animals in which NMDAR modulators were shown to normalize both NMDAR dysfunction and ASD-like behaviors, establishing strong association between NMDARs and ASD phenotypes (Fig.). In the second group, NMDAR modulators were shown to rescue ASD-like behaviors, but NMDAR dysfunction and its correction have not been demonstrated.

ASD models with data showing rescue of both NMDAR dysfunction and ASD like behaviors Mice lacking neuroligin-1, an excitatory postsynaptic adhesion molecule, show reduced NMDAR function in the hippocampus and striatum, as evidenced by a decrease in NMDA/AMPA ratio and long-term potentiation (LTP). Neuroligin-1 is thought to enhance synaptic NMDAR function, by directly interacting with and promoting synaptic localization of NMDARs.

Fig not shown.

Bidirectional NMDAR dysfunction in animal models of ASD. Animal models of ASD with bidirectional NMDAR dysfunction can be positioned on either side of an NMDAR function curve. Model animals were divided into two groups.

Group 1: NMDAR modulators normalize both NMDAR dysfunction and ASD-like behaviors (green).

Group 2: NMDAR modulators rescue ASD-like behaviors, but NMDAR dysfunction and its rescue have not been demonstrated (orange). Note that Group 2 animals are tentatively placed on the left-hand side of the slope based on the observed DCS rescue of their ASD-like phenotypes, but the directions of their NMDAR dysfunctions remain to be experimentally determined.

ASD models with data showing rescue of ASD-like behaviors but no demonstrated NMDAR dysfunction

Tbr1 is a transcriptional regulator, one of whose targets is the gene encoding the GluN2B subunit of NMDARs. Mice haploinsufficient for Tbr1 (Tbr1+/-) show structural abnormalities in the amygdala and limited GluN2B induction upon behavioral stimulation. Both systemic injection and local amygdalar infusion of DCS rescue social deficits and impaired associative memory in Tbr1+/- mice. However, reduced NMDAR function and its DCS-dependent correction have not been demonstrated.

Spatial working memory and attention skills are predicted by maternal stress during pregnancy

André Plamondon, Emis Akbari, Leslie Atkinson, Meir Steiner
Early Human Development 91 (2015) 23–29
http://dx.doi.org/10.1016/j.earlhumdev.2014.11.004

Introduction: Experimental evidence in rodents shows that maternal stress during pregnancy (MSDP) negatively impacts spatial learning and memory in the offspring. We aim to investigate the association between MSDP (i.e., life events) and spatial working memory, as well as attention skills (attention shifting and attention focusing), in humans. The moderating roles of child sex, maternal anxiety during pregnancy and postnatal care are also investigated.  Methods: Participants were 236mother–child dyads that were followed from the second trimester of pregnancy until 4 years postpartum. Measurements included questionnaires and independent observations.
Results: MSDP was negatively associated with attention shifting at 18monthswhen concurrent maternal anxiety was low. MSDP was associated with poorer spatial working memory at 4 years of age, but only for boys who experienced poorer postnatal care.
Conclusion: Consistent with results observed in rodents, MSDP was found to be associated with spatial working memory and attention skills. These results point to postnatal care and maternal anxiety during pregnancy as potential targets for interventions that aim to buffer children from the detrimental effects of MSDP.

Acute and massive bleeding from placenta previa and infants’ brain damage

Ken Furuta, Shuichi Tokunaga, Seishi Furukawa, Hiroshi Sameshima
Early Human Development 90 (2014) 455–458
http://dx.doi.org/10.1016/j.earlhumdev.2014.06.002

Background: Among the causes of third trimester bleeding, the impact of placenta previa on cerebral palsy is not well known.
Aims: To clarify the effect ofmaternal bleeding fromplacenta previa on cerebral palsy, and in particular when and how it occurs.
Study design: A descriptive study.
Subjects: Sixty infants born to mothers with placenta previa in our regional population-based study of 160,000 deliveries from 1998 to 2012. Premature deliveries occurring atb26 weeks of gestation and placenta accrete were excluded.
Outcome measures: Prevalence of cystic periventricular leukomalacia (PVL) and cerebral palsy (CP).
Results: Five infants had PVL and 4 of these infants developed CP (1/40,000 deliveries). Acute and massive bleeding (>500 g) within 8 h) occurred at around 30–31 weeks of gestation, and was severe enough to deliver the fetus. None of the 5 infants with PVL underwent antenatal corticosteroid treatment, and 1 infant had mild neonatal hypocapnia with a PaCO2 < 25 mm Hg. However, none of the 5 PVL infants showed umbilical arterial academia with pH < 7.2, an abnormal fetal heart rate monitoring pattern, or neonatal hypotension.
Conclusions: Our descriptive study showed that acute and massive bleeding from placenta previa at around 30 weeks of gestation may be a risk factor for CP, and requires careful neonatal follow-up. The underlying process connecting massive placental bleeding and PVL requires further investigation.

Impact of bilirubin-induced neurologic dysfunction on neurodevelopmental outcomes

Courtney J. Wusthoff, Irene M. Loe
Seminars in Fetal & Neonatal Medicine 20 (2015) 52e57
http://dx.doi.org/10.1016/j.siny.2014.12.003

Extreme neonatal hyperbilirubinemia has long been known to cause the clinical syndrome of kernicterus, or chronic bilirubin encephalopathy (CBE). Kernicterus most usually is characterized by choreoathetoid cerebral palsy (CP), impaired upward gaze, and sensorineural hearing loss, whereas cognition is relatively spared. The chronic condition of kernicterus may be, but is not always, preceded in the acute stage by acute bilirubin encephalopathy (ABE). This acute neonatal condition is also due to hyperbilirubinemia, and is characterized by lethargy and abnormal behavior, evolving to frank neonatal encephalopathy, opisthotonus, and seizures. Less completely defined is the syndrome of bilirubin-induced neurologic dysfunction (BIND).

Bilirubin-induced neurologic dysfunction (BIND) is the constellation of neurologic sequelae following milder degrees of neonatal hyperbilirubinemia than are associated with kernicterus. Clinically, BIND may manifest after the neonatal period as developmental delay, cognitive impairment, disordered executive function, and behavioral and psychiatric disorders. However, there is controversy regarding the relative contribution of neonatal hyperbilirubinemia versus other risk factors to the development of later neurodevelopmental disorders in children with BIND. In this review, we focus on the empiric data from the past 25 years regarding neurodevelopmental outcomes and BIND, including specific effects on developmental delay, cognition, speech and language development, executive function, and the neurobehavioral disorders, such as attention deficit/hyperactivity disorder and autism.

As noted in a technical report by the American Academy of Pediatrics Subcommittee on Hyperbilirubinemia, “it is apparent that the use of a single total serum bilirubin level to predict long-term outcomes is inadequate and will lead to conflicting results”. As described above, this has certainly been the case in research to date. To clarify how hyperbilirubinemia influences neurodevelopmental outcome, more sophisticated consideration is needed both of how to assess bilirubin exposure leading to neurotoxicity, and of those comorbid conditions which may lower the threshold for brain injury.

For example, premature infants are known to be especially susceptible to bilirubin neurotoxicity, with kernicterus reported following TB levels far lower than the threshold expected in term neonates. Similarly, among extremely preterm neonates, BBC is proportional to gestational age, meaning that the most premature infants have the highest UB, even for similar TB levels. Thus, future studies must be adequately powered to examine preterm infants separately from term infants, and should consider not just peak TB, but also BBC, as independent variables in neonates with hyperbilirubinemia. Similarly, an analysis by the NICHD NRN found that, among ELBW infants, higher UB levels were associated with a higher risk of death or NDI. However, increased TB levels were only associated with death or NDI in unstable infants. Again, UB or BBC appeared to be more useful than TB.

Are the neuromotor disabilities of bilirubin-induced neurologic dysfunction disorders related to the cerebellum and its connections?

Jon F. Watchko, Michael J. Painter, Ashok Panigrahy
Seminars in Fetal & Neonatal Medicine 20 (2015) 47e51
http://dx.doi.org/10.1016/j.siny.2014.12.004

Investigators have hypothesized a range of subcortical neuropathology in the genesis of bilirubin induced neurologic dysfunction (BIND). The current review builds on this speculation with a specific focus on the cerebellum and its connections in the development of the subtle neuromotor disabilities of BIND. The focus on the cerebellum derives from the following observations:
(i) the cerebellum is vulnerable to bilirubin-induced injury; perhaps the most vulnerable region within the central nervous system;
(ii) infants with cerebellar injury exhibit a neuromotor phenotype similar to BIND; and                                                       (iii) the cerebellum has extensive bidirectional circuitry projections to motor and non-motor regions of the brain-stem and cerebral cortex that impact a variety of neurobehaviors.
Future study using advanced magnetic resonance neuroimaging techniques have the potential to shed new insights into bilirubin’s effect on neural network topology via both structural and functional brain connectivity measurements.

Bilirubin-induced neurologic damage is most often thought of in terms of severe adverse neuromotor (dystonia with or without athetosis) and auditory (hearing impairment or deafness) sequelae. Observed together, they comprise the classic neurodevelopmental phenotype of chronic bilirubin encephalopathy or kernicterus, and may also be seen individually as motor or auditory predominant subtypes. These injuries reflect both a predilection of bilirubin toxicity for neurons (relative to glial cells) and the regional topography of bilirubin-induced neuronal damage characterized by prominent involvement of the globus pallidus, subthalamic nucleus, VIII cranial nerve, and cochlear nucleus.

It is also asserted that bilirubin neurotoxicity may be associated with other less severe neurodevelopmental disabilities, a condition termed “subtle kernicterus” or “bilirubin-induced neurologic dysfunction” (BIND). BIND is defined by a constellation of “subtle neurodevelopmental disabilities without the classical findings of kernicterus that, after careful evaluation and exclusion of other possible etiologies, appear to be due to bilirubin neurotoxicity”. These purportedly include:

(i) mild-to-moderate disorders of movement (e.g., incoordination, clumsiness, gait abnormalities, disturbances in static and dynamic balance, impaired fine motor skills, and ataxia);                                                                                             (ii) disturbances in muscle tone; and
(iii) altered sensorimotor integration. Isolated disturbances of central auditory processing are also included in the spectrum of BIND.

  • Cerebellar vulnerability to bilirubin-induced injury
  • Cerebellar injury phenotypes and BIND
  • Cerebellar projections
Transverse section of cerebellum and brainstem

Transverse section of cerebellum and brainstem

Transverse section of cerebellum and brain-stem from a 34 gestational-week premature kernicteric infant formalin-fixed for two weeks. Yellow staining is evident in the cerebellar dentate nuclei (upper arrow) and vestibular nuclei at the pontomedullary junction (lower arrowhead). Photo is courtesy of Mahmdouha Ahdab-Barmada and reprinted with permission from Taylor-Francis Group (Ahdab Barmada M. The neuropathology of kernicterus: definitions and debate. In: Maisel MJ, Watchko JF editors. Neonatal jaundice. Amsterdam: Harwood Academic Publishers; 2000. p. 75e88

Whether cerebellar injury is primal or an integral part of disturbed neural circuitry in bilirubin-induced CNS damage is unclear. Movement disorders, however, are increasingly recognized to arise from abnormalities of neuronal circuitry rather than localized, circumscribed lesions. The cerebellum has extensive bidirectional circuitry projections to an array of brainstem nuclei and the cerebral cortex that modulate and refine motor activities. In this regard, the cerebellum is characteristically subdivided into three lobes based on neuroanatomic and phylogenetic criteria as well as by their primary afferent and efferent connections. They include:
(i) flocculonodular lobe (archicerebellum);
(ii) anterior lobe (paleocerebellum); and
(iii) posterior lobe (neocerebellum).

The archicerebellum, the oldest division phylogenically, receives extensive input from the vestibular system and is therefore also known as the vestibulocerebellum and is important for equilibrium control. The paleocerebellum, also a primitive region, receives extensive somatosensory input from the spinal cord, including the anterior and posterior spinocerebellar pathways that convey unconscious proprioception, and is therefore also known as the spinocerebellum. The neocerebellum is the most recently evolved region, receives most of the input from the cerebral cortex, and is thus termed the cerebrocerebellum. This area has greatly expanded in association with the extensive development of the cerebral cortex in mammals and especially primates. To cause serious longstanding dysfunction, cerebellar injury must typically involve the deep cerebellar nuclei and their projections.

Schematic of the bidirectional connectivity between the cerebellum and other

Schematic of the bidirectional connectivity between the cerebellum and other

Schematic of the bidirectional connectivity between the cerebellum and other brain regions including the cerebral cortex. Most cerebro-cerebellar afferent projections pass through the basal (anterior or ventral) pontine nuclei and intermediate cerebellar peduncle, whereas most cerebello-cerebral efferent projections pass through the dentate and ventrolateral thalamic nuclei. DCN, deep cerebellar nuclei; RN, red nucleus; ATN, anterior thalamic nucleus; PFC, prefrontal cortex; MC, motor cortex; PC, parietal cortex; TC, temporal cortex; STN, subthalamic nucleus; APN, anterior pontine nuclei. Reprinted under the terms of the Creative Commons Attribution License from D’Angelo E, Casali S. Seeking a unified framework for cerebellar function and dysfunction: from circuit to cognition. Front Neural Circuits 2013; 6:116.

Given the vulnerability of the cerebellum to bilirubin-induced injury, cerebellar involvement should also be evident in classic kernicterus, contributing to neuromotor deficits observed therein. It is of interest, therefore, that cerebellar damage may play a role in the genesis of bilirubin-induced dystonia, a prominent neuromotor feature of chronic bilirubin encephalopathy in preterm and term neonates alike. This complex movement disorder is characterized by involuntary sustained muscle contractions that result in abnormal position and posture. Moreover, dystonia that is brief in duration results in chorea, and, if brief and repetitive, leads to athetosis ‒ conditions also classically observed in kernicterus. Recent evidence suggests that dystonic movements may depend on disruption of both basal ganglia and cerebellar neuronal networks, rather than isolated dysfunction of only one motor system.

Dystonia is also a prominent feature in Gunn rat pups and neonatal Ugt1‒/‒-deficient mice both robust models of kernicterus. The former is used as an experimental model of dystonia. Although these models show basal ganglia injury, the sine qua non of bilirubin-induced murine neuropathology is cerebellar damage and resultant cerebellar hypoplasia.

Studies are needed to define more precisely the motor network abnormalities in kernicterus and BIND. Magnetic resonance imaging (MRI) has been widely used in evaluating infants at risk for bilirubin-induced brain injury using conventional structural T1-and T2-weighted imaging. Infants with chronic bilirubin encephalopathy often demonstrate abnormal bilateral, symmetric, high-signal intensity on T2-weighted MRI of the globus pallidus and subthalamic nucleus, consistent with the neuropathology of kernicterus. Early postnatal MRI of at-risk infants, although frequently showing increased T1-signal in these regions, may give false-positive findings due to the presence of myelin in these structures.

Diffusion tensor imaging and tractography could be used to delineate long-term changes involving specific white matter pathways, further elucidating the neural basis of long-term disability in infants and children with chronic bilirubin encephalopathy and BIND. It will be equally valuable to use blood oxygen level-dependent (BOLD) “resting state” functional MRI to study intrinsic connectivity in order to identify vulnerable brain networks in neonates with kernicterus and BIND. Structural networks of the CNS (connectome) and functional network topology can be characterized in infants with kernicterus and BIND to determine disease-related pattern(s) with respect to both long- and short-range connectivity. These findings have the potential to shed novel insights into the pathogenesis of these disorders and their impact on complex anatomical connections and resultant functional deficits.

Audiologic impairment associated with bilirubin-induced neurologic damage

Cristen Olds, John S. Oghalai
Seminars in Fetal & Neonatal Medicine 20 (2015) 42e46
http://dx.doi.org/10.1016/j.siny.2014.12.006

Hyperbilirubinemia affects up to 84% of term and late preterm infants in the first week of life. The elevation of total serum/plasma bilirubin (TB) levels is generally mild, transitory, and, for most children, inconsequential. However, a subset of infants experiences lifelong neurological sequelae. Although the prevalence of classic kernicterus has fallen steadily in the USA in recent years, the incidence of jaundice in term and premature infants has increased, and kernicterus remains a significant problem in the global arena. Bilirubin-induced neurologic dysfunction (BIND) is a spectrum of neurological injury due to acute or sustained exposure of the central nervous system(CNS) to bilirubin. The BIND spectrum includes kernicterus, acute bilirubin encephalopathy, and isolated neural pathway dysfunction.

Animal studies have shown that unconjugated bilirubin passively diffuses across cell membranes and the blood‒brain barrier (BBB), and bilirubin not removed by organic anion efflux pumps accumulates within the cytoplasm and becomes toxic. Exposure of neurons to bilirubin results in increased oxidative stress and decreased neuronal proliferation and presynaptic neuro-degeneration at central glutaminergic synapses. Furthermore, bilirubin administration results in smaller spiral ganglion cell bodies, with decreased cellular density and selective loss of large cranial nerve VIII myelinated fibers. When exposed to bilirubin, neuronal supporting cells have been found to secrete inflammatory markers, which contribute to increased BBB permeability and bilirubin loading.

The jaundiced Gunn rat is the classic animal model of bilirubin toxicity. It is homozygous for a premature stop codon within the gene for UDP-glucuronosyltransferase family 1 (UGT1). The resultant gene product has reduced bilirubin-conjugating activity, leading to a state of hyperbilirubinemia. Studies with this rat model have led to the concept that impaired calcium homeostasis is an important mechanism of neuronal toxicity, with reduced expression of calcium-binding proteins in affected cells being a sensitive index of bilirubin-induced neurotoxicity. Similarly, application of bilirubin to cultured auditory neurons from brainstem cochlear nuclei results in hyperexcitability and excitotoxicity.

The auditory pathway and normal auditory brainstem response (ABR).

The auditory pathway and normal auditory brainstem response (ABR).

The auditory pathway and normal auditory brain-stem response (ABR). The ipsilateral (green) and contralateral (blue) auditory pathways are shown, with structures that are known to be affected by hyperbilirubinemia highlighted in red. Roman numerals in parentheses indicate corresponding waves in the normal human ABR (inset). Illustration adapted from the “Ear Anatomy” series by Robert Jackler and Christine Gralapp, with permission.

Bilirubin-induced neurologic dysfunction (BIND)

Vinod K. Bhutani, Ronald Wong
Seminars in Fetal & Neonatal Medicine 20 (2015) 1
http://dx.doi.org/10.1016/j.siny.2014.12.010

Beyond the traditional recognized areas of fulminant injury to the globus pallidus as seen in infants with kernicterus, other vulnerable areas include the cerebellum, hippocampus, and subthalamic nuclear bodies as well as certain cranial nerves. The hippocampus is a brain region that is particularly affected by age related morphological changes. It is generally assumed that a loss in hippocampal volume results in functional deficits that contribute to age-related cognitive deficits. Lower grey matter volumes within the limbic-striato-thalamic circuitry are common to other etiological mechanisms of subtle neurologic injury. Lower grey matter volumes in the amygdala, caudate, frontal and medial gyrus are found in schizophrenia and in the putamen in autism. Thus, in terms of brain volumetrics, schizophrenia and autism spectrum disorders have a clear degree of overlap that may reflect shared etiological mechanisms. Overlap with injuries observed in infants with BIND raises the question about how these lesions are arrived at in the context of the impact of common etiologies.

Stress-induced perinatal and transgenerational epigenetic programming of brain development and mental health

Olena Babenko, Igor Kovalchuk, Gerlinde A.S. Metz
Neuroscience and Biobehavioral Reviews 48 (2015) 70–91
http://dx.doi.org/10.1016/j.neubiorev.2014.11.013

Research efforts during the past decades have provided intriguing evidence suggesting that stressful experiences during pregnancy exert long-term consequences on the future mental wellbeing of both the mother and her baby. Recent human epidemiological and animal studies indicate that stressful experiences in utero or during early life may increase the risk of neurological and psychiatric disorders, arguably via altered epigenetic regulation. Epigenetic mechanisms, such as miRNA expression, DNA methylation, and histone modifications are prone to changes in response to stressful experiences and hostile environmental factors. Altered epigenetic regulation may potentially influence fetal endocrine programming and brain development across several generations. Only recently, however, more attention has been paid to possible transgenerational effects of stress. In this review we discuss the evidence of transgenerational epigenetic inheritance of stress exposure in human studies and animal models. We highlight the complex interplay between prenatal stress exposure, associated changes in miRNA expression and DNA methylation in placenta and brain and possible links to greater risks of schizophrenia, attention deficit hyperactivity disorder, autism, anxiety- or depression-related disorders later in life. Based on existing evidence, we propose that prenatal stress, through the generation of epigenetic alterations, becomes one of the most powerful influences on mental health in later life. The consideration of ancestral and prenatal stress effects on lifetime health trajectories is critical for improving strategies that support healthy development and successful aging.

Sensitive time-windows for susceptibility in neurodevelopmental disorders

Rhiannon M. Meredith, Julia Dawitz and Ioannis Kramvis
Trends in Neurosciences, June 2012; 35(6): 335-344
http://dx.doi.org:/10.1016/j.tins.2012.03.005

Many neurodevelopmental disorders (NDDs) are characterized by age-dependent symptom onset and regression, particularly during early postnatal periods of life. The neurobiological mechanisms preceding and underlying these developmental cognitive and behavioral impairments are, however, not clearly understood. Recent evidence using animal models for monogenic NDDs demonstrates the existence of time-regulated windows of neuronal and synaptic impairments. We propose that these developmentally-dependent impairments can be unified into a key concept: namely, time-restricted windows for impaired synaptic phenotypes exist in NDDs, akin to critical periods during normal sensory development in the brain. Existence of sensitive time-windows has significant implications for our understanding of early brain development underlying NDDs and may indicate vulnerable periods when the brain is more susceptible to current therapeutic treatments.

Fig (not shown)

Misregulated mechanisms underlying spine morphology in NDDs. Several proteins implicated in monogenic NDDs (highlighted in red) are linked to the regulation of the synaptic cytoskeleton via F-actin through different Rho-mediated signaling pathways (highlighted in green). Mutations in OPHN1, TSC1/2, FMRP, p21-activated kinase (PAK) are directly linked to human NDDs of intellectual disability. For instance, point mutations in OPHN1 and a PAK isoform are linked to non-syndromic mental retardation, whereas mutations or altered expression of TSC1/2 and FMRP are linked to TSC and FXS, respectively. Cytoplasmic interacting protein (CYFIP) and LIM-domain kinase 1 (LIMK1) are known to interact with FMRP and PAK, respectively [105]. LIMK1 is one of many dysregulated proteins contributing to the NDD Williams syndrome. Mouse models are available for all highlighted (red) proteins and reveal specific synaptic and behavioral deficits. Local protein synthesis in synapses, dendrites and glia is also regulated by proteins such as TSC1/2 and the FMRP/CYFIP complex. Abbreviations: 4EBP, 4E binding protein; eIF4E, eukaryotic translation initiation factor 4E.

Fig (not shown)

Sensitive time-windows, synaptic phenotypes and NDD gene targets. Sensitive time-windows exist in neural circuits, during which gene targets implicated in NDDs are normally expressed. Misregulation of these genes can affect multiple synaptic phenotypes during a restricted developmental period. The effect upon synaptic phenotypes is dependent upon the temporal expression of these NDD genes and their targets. (a) Expression outside a critical period of development will have no effect upon synaptic phenotypes. (b,c) A temporal expression pattern that overlaps with the onset (b) or closure (c) of a known critical period can alter the synaptic phenotype during that developmental time-window.

Outstanding questions

(1) Can treatment at early presymptomatic stages in animal models for NDDs prevent or ease the later synaptic, neuronal, and behavioral impairments?

(2) Are all sensory critical periods equally misregulated in mouse models for a specific NDD? Are there different susceptibilities for auditory, visual and somatosensory neurocircuits that reflect the degree of impairments observed in patients?

(3) If one critical period is missed or delayed during formation of a layer-specific connection in a network, does the network overcome this misregulated connectivity or plasticity window?

(4) In monogenic NDDs, does the severity of misregulating one particular time-window for synaptic establishment during development correlate with the importance of that gene for that synaptic circuit?

(5) Why do critical periods close in brain development?

(6) What underlies the regression of some altered synaptic phenotypes in Fmr1-KO mice?

(7) Can the concept of susceptible time-windows be applied to other NDDs, including schizophrenia and Tourette’s syndrome?

Cardiovascular

Cardiac output monitoring in newborns

Willem-Pieter de Boode
Early Human Development 86 (2010) 143–148
http://dx.doi.org:/10.1016/j.earlhumdev.2010.01.032

There is an increased interest in methods of objective cardiac output measurement in critically ill patients. Several techniques are available for measurement of cardiac output in children, although this remains very complex in newborns. Cardiac output monitoring could provide essential information to guide hemodynamic management. An overview is given of various methods of cardiac output monitoring with advantages and major limitations of each technology together with a short explanation of the basic principles.

Fick principle

According to the Fick principle the volume of blood flow in a given period equals the amount of substance entering the blood stream in the same period divided by the difference in concentrations of the substrate upstream respectively downstream to the point of entry in the circulation. This substance can be oxygen (O2-Fick) or carbon dioxide (CO2-FICK), so cardiac output can be calculated by dividing measured pulmonary oxygen uptake by the arteriovenous oxygen concentration difference. The direct O2-Fick method is regarded as gold standard in cardiac output monitoring in a research setting, despite its limitations. When the Fick principle is applied for carbon dioxide (CO2 Fick), the pulmonary carbon dioxide exchange is divided by the venoarterial CO2 concentration difference to calculate cardiac output.

In the modified CO2 Fick method pulmonary CO2 exchange is measured at the endotracheal tube. Measurement of total CO2 concentration in blood is more complex and simultaneous sampling of arterial and central venous blood is required. However, frequent blood sampling will result in an unacceptable blood loss in the neonatal population.

Blood flow can be calculated if the change in concentration of a known quantity of injected indicator is measured in time distal to the point of injection, so an indicator dilution curve can be obtained. Cardiac output can then be calculated with the use of the Stewart–Hamilton equation. Several indicators are used, such as indocyanine green, Evans blue and brilliant red in dye dilution, cold solutions in thermodilution, lithium in lithium dilution, and isotonic saline in ultrasound dilution.

Cardiovascular adaptation to extra uterine life

Alice Lawford, Robert MR Tulloh
Paediatrics And Child Health 2014; 25(1): 1-6.

The adaptation to extra uterine life is of interest because of its complexity and the ability to cause significant health concerns. In this article we describe the normal changes that occur and the commoner abnormalities that are due to failure of normal development and the effect of congenital cardiac disease. Abnormal development may occur as a result of problems with the mother, or with the fetus before birth. After birth it is essential to determine whether there is an underlying abnormality of the fetal pulmonary or cardiac development and to determine the best course of management of pulmonary hypertension or congenital cardiac disease. Causes of underdevelopment, maldevelopment and maladaptation are described as are the causes of critical congenital heart disease. The methods of diagnosis and management are described to allow the neonatologist to successfully manage such newborns.

Fetal vascular structures that exist to direct blood flow

Fetal structure Function
Arterial duct Connects pulmonary artery to the aorta and shunts blood right to left; diverting flow away from fetal lungs
Foramen ovale Opening between the two atria thatdirects blood flow returning to right

atrium through the septal wall into the left atrium bypassing lungs
Ductus venosus Receives oxygenated blood fromumbilical vein and directs it to the

inferior vena cava and right atrium
Umbilical arteries Carrying deoxygenated blood fromthe fetus to the placenta
Umbilical vein Carrying oxygenated blood from theplacenta to the fetus

Maternal causes of congenital heart disease

Maternal disorders rubella, SLE, diabetes mellitus
Maternal drug use Warfarin, alcohol
Chromosomal abnormality Down, Edward, Patau, Turner, William, Noonan

 

Fetal and Neonatal Circulation  The fetal circulation is specifically adapted to efficiently exchange gases, nutrients, and wastes through placental circulation. Upon birth, the shunts (foramen ovale, ductus arteriosus, and ductus venosus) close and the placental circulation is disrupted, producing the series circulation of blood through the lungs, left atrium, left ventricle, systemic circulation, right heart, and back to the lungs.

Clinical monitoring of systemic hemodynamics in critically ill newborns

Willem-Pieter de Boode
Early Human Development 86 (2010) 137–141
http://dx.doi.org:/10.1016/j.earlhumdev.2010.01.031

Circulatory failure is a major cause of mortality and morbidity in critically ill newborn infants. Since objective measurement of systemic blood flow remains very challenging, neonatal hemodynamics is usually assessed by the interpretation of various clinical and biochemical parameters. An overview is given about the predictive value of the most used indicators of circulatory failure, which are blood pressure, heart rate, urine output, capillary refill time, serum lactate concentration, central–peripheral temperature difference, pH, standard base excess, central venous oxygen saturation and color.

Key guidelines

➢ The clinical assessment of cardiac output by the interpretation of indirect parameters of systemic blood flow is inaccurate, irrespective of the level of experience of the clinician

➢ Using blood pressure to diagnose low systemic blood flow will consequently mean that too many patients will potentially be undertreated or overtreated, both with substantial risk of adverse effects and iatrogenic damage.

➢ Combining different clinical hemodynamic parameters enhances the predictive value in the detection of circulatory failure, although accuracy is still limited.

➢ Variation in time (trend monitoring) might possibly be more informative than individual, static values of clinical and biochemical parameters to evaluate the adequacy of neonatal circulation.

Monitoring oxygen saturation and heart rate in the early neonatal period

J.A. Dawson, C.J. Morley
Seminars in Fetal & Neonatal Medicine 15 (2010) 203e207
http://dx.doi.org:/10.1016/j.siny.2010.03.004

Pulse oximetry is commonly used to assist clinicians in assessment and management of newly born infants in the delivery room (DR). In many DRs, pulse oximetry is now the standard of care for managing high risk infants, enabling immediate and dynamic assessment of oxygenation and heart rate. However, there is little evidence that using pulse oximetry in the DR improves short and long term outcomes. We review the current literature on using pulse oximetry to measure oxygen saturation and heart rate and how to apply current evidence to management in the DR.

Practice points

  • Understand how SpO2 changes in the first minutes after birth.
  • Apply a sensor to an infant’s right wrist as soon as possible after birth.
  • Attach sensor to infant then to oximeter cable.
  • Use two second averaging and maximum sensitivity.

Using pulse oximetry assists clinicians:

  1. Assess changes in HR in real time during transition.
  2. Assess oxygenation and titrate the administration of oxygen to maintain oxygenation within the appropriate range for SpO2 during the first minutes after birth.

Research directions

  • What are the appropriate centiles to target during the minutes after birth to prevent hypoxia and hyperoxia: 25th to 75th, or 10th to 90th, or just the 50th (median)?
  • Can the inspired oxygen be titrated against the SpO2 to keep the SpO2 in the ‘normal range’?
  • Does the use of centile charts in the DR for HR and oxygen saturation reduce the rate of hyperoxia when infants are treated with oxygen.
  • Does the use of pulse oximetry immediately after birth improve short term outcomes, e.g. efficacy of immediate respiratory support, intubation rates in the DR, percentage of inspired oxygen, rate of use of adrenalin or chest compressions, duration of hypoxia/hyperoxia and bradycardia.
  • Does the use of pulse oximetry in the DR improve short term respiratory and long term neurodevelopmental outcomes for preterm infants, e.g. rate of intubation, use of surfactant, and duration of ventilation, continuous positive airway pressure, or supplemental oxygen?
  • Can all modern pulse oximeters be used effectively in the DR or do some have a longer delay before giving an accurate signal and more movement artefact?
  • Would a longer averaging time result in more stable data?

Peripheral haemodynamics in newborns: Best practice guidelines

Michael Weindling, Fauzia Paize
Early Human Development 86 (2010) 159–165
http://dx.doi.org:/10.1016/j.earlhumdev.2010.01.033

Peripheral hemodynamics refers to blood flow, which determines oxygen and nutrient delivery to the tissues. Peripheral blood flow is affected by vascular resistance and blood pressure, which in turn varies with cardiac function. Arterial oxygen content depends on the blood hemoglobin concentration (Hb) and arterial pO2; tissue oxygen delivery depends on the position of the oxygen-dissociation curve, which is determined by temperature and the amount of adult or fetal hemoglobin. Methods available to study tissue perfusion include near-infrared spectroscopy, Doppler flowmetry, orthogonal polarization spectral imaging and the peripheral perfusion index. Cardiac function, blood gases, Hb, and peripheral temperature all affect blood flow and oxygen extraction. Blood pressure appears to be less important. Other factors likely to play a role are the administration of vasoactive medications and ventilation strategies, which affect blood gases and cardiac output by changing the intrathoracic pressure.

graphic

NIRS with partial venous occlusion to measure venous oxygen saturation

NIRS with partial venous occlusion to measure venous oxygen saturation

NIRS with partial venous occlusion to measure venous oxygen saturation. Taken from Yoxall and Weindling

Schematic representation of the biphasic relationship between oxygen delivery and oxygen consumption in tissue

Schematic representation of the biphasic relationship between oxygen delivery and oxygen consumption in tissue

graphic

Schematic representation of the biphasic relationship between oxygen delivery and oxygen consumption in tissue.  (a) oxygen delivery (DO2). (b) As DO2 decreases, VO2 is dependent on DO2. The slope of the line indicates the FOE, which in this case is about 0.50. (c) The slope of the line indicates the FOE in the normal situation where oxygenation is DO2 independent, usually < 0.35

The oxygen-dissociation curve

The oxygen-dissociation curve

graphic

The oxygen-dissociation curve

Considerable information about the response of the peripheral circulation has been obtained using NIRS with venous occlusion. Although these measurements were validated against blood co-oximetry in human adults and infants, they can only be made intermittently by a trained operator and are thus not appropriate for general clinical use. Further research is needed to find other better measures of peripheral perfusion and oxygenation which may be easily and continuously monitored, and which could be useful in a clinical setting.

Peripheral oxygenation and management in the perinatal period

Michael Weindling
Seminars in Fetal & Neonatal Medicine 15 (2010) 208e215
http://dx.doi.org:/10.1016/j.siny.2010.03.005

The mechanisms for the adequate provision of oxygen to the peripheral tissues are complex. They involve control of the microcirculation and peripheral blood flow, the position of the oxygen dissociation curve including the proportion of fetal and adult hemoglobin, blood gases and viscosity. Systemic blood pressure appears to have little effect, at least in the non-shocked state. The adequate delivery of oxygen (DO2) depends on consumption (VO2), which is variable. The balance between VO2 and DO2 is given by fractional oxygen extraction (FOE ¼ VO2/DO2). FOE varies from organ to organ and with levels of activity. Measurements of FOE for the whole body produce a range of about 0.15-0.33, i.e. the body consumes 15-33% of oxygen transported.

Fig (not shown)

Biphasic relationship between oxygen delivery (DO2) and oxygen consumption (VO2) in tissue. Dotted lines show fractional oxygen extraction (FOE). ‘A’ indicates the normal situation when VO2 is independent ofDO2 and FOE is about 0.30. AsDO2 decreases in the direction of the arrow, VO2 remains independent of DO2 until the critical point is reached at ‘B’; in this illustration, FOE is about 0.50. The slope of the dotted line indicates the FOE (¼ VO2/DO2), which increases progressively as DO2 decreases.

Relationship between haemoglobin F fraction (HbF) and peripheral fractional oxygen extraction

Relationship between haemoglobin F fraction (HbF) and peripheral fractional oxygen extraction

Graphic
(A)Relationship between haemoglobin F fraction (HbF) and peripheral fractional oxygen extraction in anaemic and control infants. (From Wardle et al.)  (B) HbF synthesis and concentration. (From Bard and Widness.) (C) Oxygen dissociation curve.

Peripheral fractional oxygen extraction in babies

Peripheral fractional oxygen extraction in babies

graphic

Peripheral fractional oxygen extraction in babies with asymptomatic or symptomatic anemia compared to controls. Bars represent the median for each group. (From Wardle et al.)

Practice points

  • Peripheral tissue DO2 is complex: cardiac function, blood gases, Hb concentration and the proportion of HbF, and peripheral temperature all play a part in determining blood flow and oxygen extraction in the sick, preterm infant. Blood pressure appears to be less important.
  • Other factors likely to play a role are the administration of vasoactive medications and ventilation strategies, which affect blood gases and cardiac output by changing intrathoracic pressure.
  • Central blood pressure is a poor surrogate measurement for the adequacy of DO2 to the periphery. Direct measurement, using NIRS, laser Doppler flowmetry or other means, may give more useful information.
  • Reasons for total hemoglobin concentration (Hb) being a relatively poor indicator of the adequacy of the provision of oxygen to the tissues:
  1. Hb is only indirectly related to red blood cell volume, which may be a better indicator of the body’s oxygen delivering capacity.
  2. Hb-dependent oxygen availability depends on the position of the oxygen-hemoglobin dissociation curve.
  3. An individual’s oxygen requirements vary with time and from organ to organ. This means that DO2 also needs to vary.
  4. It is possible to compensate for a low Hb by increasing cardiac output and ventilation, and so the ability to compensate for anemia depends on an individual’s cardio-respiratory reserve as well as Hb.
  5. The normal decrease of Hb during the first few weeks of life in both full-term and preterm babies usually occurs without symptoms or signs of anemia or clinical consequences.

The relationship between VO2 and DO2 is complex and various factors need to be taken into account, including the position of the oxygen dissociation curve, determined by the proportion of HbA and HbF, temperature and pH. Furthermore, diffusion of oxygen from capillaries to the cell depends on the oxygen tension gradient between erythrocytes and the mitochondria, which depends on microcirculatory conditions, e.g. capillary PO2, distance of the cell from the capillary (characterized by intercapillary distances) and the surface area of open capillaries. The latter can change rapidly, for example, in septic shock where arteriovenous shunting occurs associated with tissue hypoxia in spite of high DO2 and a low FOE.

Changes in local temperature deserve particular consideration. When the blood pressure is low, there may be peripheral vasoconstriction with decreased local perfusion and DO2. However, the fall in local tissue temperature would also be expected to be associated with a decreased metabolic rate and a consequent decrease in VO2. Thus a decreased DO2 may still be appropriate for tissue needs.

Pulmonary

Accurate Measurements of Oxygen Saturation in Neonates: Paired Arterial and Venous Blood Analyses

Shyang-Yun Pamela K. Shiao
Newborn and Infant Nurs Rev,  2005; 5(4): 170–178
http://dx.doi.org:/10.1053/j.nainr.2005.09.001

Oxygen saturation (So2) measurements (functional measurement, So2; and fractional measurement, oxyhemoglobin [Hbo2]) and monitoring are commonly investigated as a method of assessing oxygenation in neonates. Differences exist between the So2 and Hbo2 when blood tests are performed, and clinical monitors indicate So2 values. Oxyhemoglobin will decrease with the increased levels of carbon monoxide hemoglobin (Hbco) and methemo-globin (MetHb), and it is the most accurate measurements of oxygen (O2) association of hemoglobin (Hb). Pulse oximeter (for pulse oximetry saturation [Spo2] measurement) is commonly used in neonates. However, it will not detect the changes of Hb variations in the blood for accurate So2 measurements. Thus, the measurements from clinical oximeters should be used with caution. In neonates, fetal hemoglobin (HbF) accounts for most of the circulating Hb in their blood. Fetal hemoglobin has a high O2 affinity, thus releases less O2 to the body tissues, presenting a left-shifted Hbo2 dissociation curve.5,6 To date, however, limited data are available with HbF correction, for accurate arterial and venous (AV) So2 measurements (arterial oxygen saturation [Sao2] and venous oxygen saturation [Svo2]) in neonates, using paired AV blood samples.

In a study of critically ill adult patients, increased pulmonary CO production and elevation in arterial Hbco but not venous Hbco were documented by inflammatory stimuli inducing pulmonary heme oxygenase–1. In normal adults, venous Hbco level might be slightly higher than or equal to arterial Hbco because of production of CO by enzyme heme oxygenase–2, which is predominantly produced in the liver and spleen. However, hypoxia or pulmonary inflammation could induce heme oxygenase–1 to increase endogenous CO, thus elevating pulmonary arterial and systemic arterial Hbco levels in adults. Both endogenous and exogenous CO can suppress proliferation of pulmonary smooth muscles, a significant consideration for the prevention of chronic lung diseases in newborns. Despite these considerations, a later study in healthy adults indicated that the AV differences in Hbco were from technical artifacts and perhaps from inadequate control of different instruments. Thus, further studies are needed to provide more definitive answers for the AV differences of Hbco for adults and neonates with acute and chronic lung diseases.

Methemoglobin is an indicator of Hb oxidation and is essential for accurate measurement of Hbo2, So2, and oxygenation status. No evidence exists to show the AV MetHb difference, although this difference was elucidated with the potential changes of MetHb with different O2 levels.  Methemoglobin can be increased with nitric oxide (NO) therapy, used in respiratory distress syndrome (RDS) to reduce pulmonary hypertension and during heart surgery. Nitric oxide, in vitro, is an oxidant of Hb, with increased O2 during ischemia reperfusion. In hypoxemic conditions in vivo, nitrohemoglobin is a product generated by vessel responsiveness to nitrovasodilators. Nitro-hemoglobin can be spontaneously reversible in vivo, requiring no chemical agents or reductase. However, when O2 levels were increased experimentally in vitro following acidic conditions (pH 6.5) to simulate reperfusion conditions, MetHb levels were increased for the hemolysates (broken red cells). Nitrite-induced oxidation of Hb was associated with an increase in red blood cell membrane rigidity, thus contributing to Hb breakdown. A newer in vitro study of whole blood cells, however, concluded that MetHb formation is not dependent on increased O2 levels. Additional studies are needed to examine in vivo reperfusion of O2 and MetHb effects.

Purpose: The aim of this study was to examine the accuracy of arterial oxygen saturation (Sao2) and venous oxygen saturation (Svo2) with paired arterial and venous (AV) blood in relation to pulse oximetry saturation (Spo2) and oxyhemoglobin (Hbo2) with fetal hemoglobin determination, and their Hbo2 dissociation curves. Method: Twelve preterm neonates with gestational ages ranging from 27 to 34 weeks at birth, who had umbilical AV lines inserted, were investigated. Analyses were performed with 37 pairs of AV blood samples by using a blood volume safety protocol. Results: The mean differences between Sao2 and Svo2, and AV Hbo2 were both 6 percent (F6.9 and F6.7 percent, respectively), with higher Svo2 than those reported for adults. Biases were 2.1 – 0.49 for Sao2, 2.0 – 0.44 for Svo2, and 3.1 – 0.45 for Spo2, compared against Hbo2. With left-shifted Hbo2 dissociation curves in neonates, for the critical values of oxygen tension values between 50 and 75 millimeters of mercury, Hbo2 ranged from 92 to 93.4 percent; Sao2 ranged from 94.5 to 95.7 percent; and Spo2 ranged from 93.7 to 96.3 percent (compared to 85–94 percent in healthy adults). Conclusions: In neonates, both left-shifted Hbo2 dissociation curve and lower AV differences of oxygen saturation measurements indicated low flow of oxygen to the body tissues. These findings demonstrate the importance of accurate assessment of oxygenation statues in neonates.

In these neonates, the mean AV blood differences for both So2 and Hbo2 were about 6 percent, which was much lower than those reported for healthy adults (23 percent) for O2 supply and demand. In addition, with very high levels of HbF releasing less O2 to the body tissue, the results of blood analyses are worrisome for these critically ill neonates for low systemic oxygen states.  O’Connor and Hall determined AV So2 in neonates without HbF determination. Much of the AV So2 difference is dependent on Svo2 measurement. The ranges of Svo2 spanned for 35 percent, and the ranges of Sao2 spanned 6 percent in these neonates. The greater intervals for Svo2 measurements contribute to greater sensitivity for the measurements (than Sao2 measurements) in responding to nursing care and changes of O2 demand. Thus, Svo2 measurement is essential for better assessment of oxygenation status in neonates.

The findings of this study on AV differences of So2 were limited with very small number of paired AV blood samples. However, critically ill neonates need accurate assessment of oxygenation status because of HbF, which releases less O2 to the tissues. Decreased differences of AV So2 measurements added further possibilities of lower flow of O2 to the body tissues and demonstrated the greater need to accurately assess the proper oxygenation in the neonates. The findings of this study continued to clarify the accuracy of So2 measurements for neonates. Additional studies are needed to examine So2 levels in neonates to further validate these findings by using larger sample sizes.

Neonatal ventilation strategies and long-term respiratory outcomes

Sandeep Shetty, Anne Greenough
Early Human Development 90 (2014) 735–739
http://dx.doi.org/10.1016/j.earlhumdev.2014.08.020

Long-term respiratory morbidity is common, particularly in those born very prematurely and who have developed bronchopulmonary dysplasia (BPD), but it does occur in those without BPD and in infants born at term. A variety of neonatal strategies have been developed, all with short-term advantages, but meta-analyses of randomized controlled trials (RCTs) have demonstrated that only volume-targeted ventilation and prophylactic high-frequency oscillatory ventilation (HFOV) may reduce BPD. Few RCTs have incorporated long-term follow-up, but one has demonstrated that prophylactic HFOV improves respiratory and functional outcomes at school age, despite not reducing BPD. Results from other neonatal interventions have demonstrated that any impact on BPD may not translate into changes in long-term outcomes. All future neonatal  ventilation RCTs should have long-term outcomes rather than BPD as their primary outcome if they are to impact on clinical practice.

A Model Analysis of Arterial Oxygen Desaturation during Apnea in Preterm Infants

Scott A. Sands, BA Edwards, VJ Kelly, MR Davidson, MH Wilkinson, PJ Berger
PLoS Comput Biol 5(12): e1000588
http://dx.doi.org:/10.1371/journal.pcbi.1000588

Rapid arterial O2 desaturation during apnea in the preterm infant has obvious clinical implications but to date no adequate explanation for why it exists. Understanding the factors influencing the rate of arterial O2 desaturation during apnea (_SSaO2 ) is complicated by the non-linear O2 dissociation curve, falling pulmonary O2 uptake, and by the fact that O2 desaturation is biphasic, exhibiting a rapid phase (stage 1) followed by a slower phase when severe desaturation develops (stage 2). Using a mathematical model incorporating pulmonary uptake dynamics, we found that elevated metabolic O2 consumption accelerates _SSaO2 throughout the entire desaturation process. By contrast, the remaining factors have a restricted temporal influence: low pre-apneic alveolar PO2 causes an early onset of desaturation, but thereafter has little impact; reduced lung volume, hemoglobin content or cardiac output, accelerates _SSaO2 during stage 1, and finally, total blood O2 capacity (blood volume and hemoglobin content) alone determines _SSaO2 during stage 2. Preterm infants with elevated metabolic rate, respiratory depression, low lung volume, impaired cardiac reserve, anemia, or hypovolemia, are at risk for rapid and profound apneic hypoxemia. Our insights provide a basic physiological framework that may guide clinical interpretation and design of interventions for preventing sudden apneic hypoxemia.

A novel approach to study oxidative stress in neonatal respiratory distress syndrome

Reena Negi, D Pande, K Karki, A Kumar, RS Khanna, HD Khanna
BBA Clinical 3 (2015) 65–69
http://dx.doi.org/10.1016/j.bbacli.2014.12.001

Oxidative stress is an imbalance between the systemic manifestation of reactive oxygen species and a biological system’s ability to readily detoxify the reactive intermediates or to repair the resulting damage. It is a physiological event in the fetal-to-neonatal transition, which is actually a great stress to the fetus. These physiological changes and processes greatly increase the production of free radicals, which must be controlled by the antioxidant defense system, the maturation of which follows the course of the gestation. This could lead to several functional alterations with important repercussions for the infants. Adequately mature and healthy infants are able to tolerate this drastic change in the oxygen concentration. A problem occurs when the intrauterine development is incomplete or abnormal. Preterm or intrauterine growth retarded (IUGR) and low birth weight neonates are typically of this kind. An oxidant/antioxidant imbalance in infants is implicated in the pathogenesis of the major complications of prematurity including respiratory distress syndrome (RDS), necrotizing enterocolitis (NEC), chronic lung disease, retinopathy of prematurity and intraventricular hemorrhage (IVH).

Background: Respiratory distress syndrome of the neonate (neonatal RDS) is still an important problem in treatment of preterm infants. It is accompanied by inflammatory processes with free radical generation and oxidative stress. The aim of study was to determine the role of oxidative stress in the development of neonatal RDS. Methods: Markers of oxidative stress and antioxidant activity in umbilical cord blood were studied in infants with neonatal respiratory distress syndrome with reference to healthy newborns. Results: Status of markers of oxidative stress (malondialdehyde, protein carbonyl and 8-hydroxy-2-deoxy guanosine) showed a significant increase with depleted levels of total antioxidant capacity in neonatal RDS when compared to healthy newborns. Conclusion: The study provides convincing evidence of oxidative damage and diminished antioxidant defenses in newborns with RDS. Neonatal RDS is characterized by damage of lipid, protein and DNA, which indicates the augmentation of oxidative stress. General significance: The identification of the potential biomarker of oxidative stress consists of a promising strategy to study the pathophysiology of neonatal RDS.

Neonatal respiratory distress syndrome represents the major lung complications of newborn babies. Preterm neonates suffer from respiratory distress syndrome (RDS) due to immature lungs and require assisted ventilation with high concentrations of oxygen. The pathogenesis of this disorder is based on the rapid formation of the oxygen reactive species, which surpasses the detoxification capacity of antioxidative defense system. The high chemical reactivity of free radical leads to damage to a variety of cellular macro molecules including proteins, lipids and nucleic acid. This results in cell injury and may induce respiratory cell death.

Malondialdehyde (MDA) is one of the final products of polyunsaturated fatty acids peroxidation. The present study showed increased concentration of MDA in neonates with respiratory disorders than that of control in consonance with the reported study.

Anemia, Apnea of Prematurity, and Blood Transfusions

Kelley Zagol, Douglas E. Lake, Brooke Vergales, Marion E. Moorman, et al
J Pediatr 2012;161:417-21
http://dx.doi.org:/10.1016/j.jpeds.2012.02.044

The etiology of apnea of prematurity is multifactorial; however, decreased oxygen carrying capacity may play a role. The respiratory neuronal network in neonates is immature, particularly in those born preterm, as demonstrated by their paradoxical response to hypoxemia. Although adults increase the minute ventilation in response to hypoxemia, newborns have a brief increase in ventilation followed by periodic breathing, respiratory depression, and occasionally cessation of respiratory effort. This phenomenon may be exacerbated by anemia in preterm newborns, where a decreased oxygen carrying capacity may result in decreased oxygen delivery to the central nervous system, a decreased efferent output of the respiratory neuronal network, and an increase in apnea.

Objective Compare the frequency and severity of apneic events in very low birth weight (VLBW) infants before and after blood transfusions using continuous electronic waveform analysis. Study design We continuously collected waveform, heart rate, and oxygen saturation data from patients in all 45 neonatal intensive care unit beds at the University of Virginia for 120 weeks. Central apneas were detected using continuous computer processing of chest impedance, electrocardiographic, and oximetry signals. Apnea was defined as respiratory pauses of >10, >20, and >30 seconds when accompanied by bradycardia (<100 beats per minute) and hypoxemia (<80% oxyhemoglobin saturation as detected by pulse oximetry). Times of packed red blood cell transfusions were determined from bedside charts. Two cohorts were analyzed. In the transfusion cohort, waveforms were analyzed for 3 days before and after the transfusion for all VLBW infants who received a blood transfusion while also breathing spontaneously. Mean apnea rates for the previous 12 hours were quantified and differences for 12 hours before and after transfusion were compared. In the hematocrit cohort, 1453 hematocrit values from all VLBW infants admitted and breathing spontaneously during the time period were retrieved, and the association of hematocrit and apnea in the next 12 hours was tested using logistic regression. Results Sixty-seven infants had 110 blood transfusions during times when complete monitoring data were available. Transfusion was associated with fewer computer-detected apneic events (P < .01). Probability of future apnea occurring within 12 hours increased with decreasing hematocrit values (P < .001). Conclusions Blood transfusions are associated with decreased apnea in VLBW infants, and apneas are less frequent at higher hematocrits.

Bronchopulmonary dysplasia: The earliest and perhaps the longest lasting obstructive lung disease in humans

Silvia Carraro, M Filippone, L Da Dalt, V Ferraro, M Maretti, S Bressan, et al.
Early Human Development 89 (2013) S3–S5
http://dx.doi.org/10.1016/j.earlhumdev.2013.07.015

Bronchopulmonary dysplasia (BPD) is one of the most important sequelae of premature birth and the most common form of chronic lung disease of infancy, an umbrella term for a number of different diseases that evolve as a consequence of a neonatal respiratory disorder. BPD is defined as the need for supplemental oxygen for at least 28 days after birth, and its severity is graded according to the respiratory support required at 36 post-menstrual weeks.

BPD was initially described as a chronic respiratory disease occurring in premature infants exposed to mechanical ventilation and oxygen supplementation. This respiratory disease (later named “old BPD”) occurred in relatively large premature newborn and, from a pathological standpoint, it was characterized by intense airway inflammation, disruption of normal pulmonary structures and lung fibrosis.

Bronchopulmonary dysplasia (BPD) is one of the most important sequelae of premature birth and the most common form of chronic lung disease of infancy. From a clinical standpoint BPD subjects are characterized by recurrent respiratory symptoms, which are very frequent during the first years of life and, although becoming less severe as children grow up, they remain more common than in term-born controls throughout childhood, adolescence and into adulthood. From a functional point of view BPD subjects show a significant airflow limitation that persists during adolescence and adulthood and they may experience an earlier and steeper decline in lung function during adulthood. Interestingly, patients born prematurely but not developing BPD usually fare better, but they too have airflow limitations during childhood and later on, suggesting that also prematurity per se has life-long detrimental effects on pulmonary function. For the time being, little is known about the presence and nature of pathological mechanisms underlying the clinical and functional picture presented by BPD survivors. Nonetheless, recent data suggest the presence of persistent neutrophilic airway inflammation and oxidative stress and it has been suggested that BPD may be sustained in the long term by inflammatory pathogenic mechanisms similar to those underlying COPD. This hypothesis is intriguing but more pathological data are needed.  A better understanding of these pathogenetic mechanisms, in fact, may be able to orient the development of novel targeted therapies or prevention strategies to improve the overall respiratory health of BPD patients.

We have a limited understanding of the presence and nature of pathological mechanisms in the lung of BPD survivors. The possible role of asthma-like inflammation has been investigated because BPD subjects often present with recurrent wheezing and other symptoms resembling asthma during their childhood and adolescence. But BPD subjects have normal or lower than normal exhaled nitric oxide levels and exhaled air temperatures, whereas they are higher than normal in asthmatic patients.

Of all obstructive lung diseases in humans, BPD has the earliest onset and is possibly the longest lasting. Given its frequent association with other conditions related to preterm birth (e.g. growth retardation, pulmonary hypertension, neurodevelopmental delay, hearing defects, and retinopathy of prematurity), it often warrants a multidisciplinary management.

Effects of Sustained Lung Inflation, a lung recruitment maneuver in primary acute respiratory distress syndrome, in respiratory and cerebral outcomes in preterm infants

Chiara Grasso, Pietro Sciacca, Valentina Giacchi, Caterina Carpinato, et al.
Early Human Development 91 (2015) 71–75
http://dx.doi.org/10.1016/j.earlhumdev.2014.12.002

Background: Sustained Lung Inflation (SLI) is a maneuver of lung recruitment in preterm newborns at birth that can facilitate the achieving of larger inflation volumes, leading to the clearance of lung fluid and formation of functional residual capacity (FRC). Aim: To investigate if Sustained Lung Inflation (SLI) reduces the need of invasive procedures and iatrogenic risks. Study design: 78 newborns (gestational age ≤ 34 weeks, weighing ≤ 2000 g) who didn’t breathe adequately at birth and needed to receive SLI in addition to other resuscitation maneuvers (2010 guidelines). Subjects: 78 preterm infants born one after the other in our department of Neonatology of Catania University from 2010 to 2012. Outcome measures: The need of intubation and surfactant, the ventilation required, radiological signs, the incidence of intraventricular hemorrhage (IVH), periventricular leukomalacia, retinopathy in prematurity from III to IV plus grades, bronchopulmonary dysplasia, patent ductus arteriosus, pneumothorax and necrotizing enterocolitis. Results: In the SLI group infants needed less intubation in the delivery room (6% vs 21%; p b 0.01), less invasive mechanical ventilation (14% vs 55%; p ≤ 0.001) and shorter duration of ventilation (9.1 days vs 13.8 days; p ≤ 0.001). There wasn’t any difference for nasal continuous positive airway pressure (82% vs 77%; p = 0.43); but there was less surfactant administration (54% vs 85%; p ≤ 0.001) and more infants received INSURE (40% vs 29%; p=0.17). We didn’t found any differences in the outcomes, except for more mild intraventricular hemorrhage in the SLI group (23% vs 14%; p = 0.15; OR= 1.83). Conclusion: SLI is easier to perform even with a single operator, it reduces the necessity of more complicated maneuvers and surfactant without statistically evident adverse effects.

Long-term respiratory consequences of premature birth at less than 32 weeks of gestation

Anne Greenough
Early Human Development 89 (2013) S25–S27
http://dx.doi.org/10.1016/j.earlhumdev.2013.07.004

Chronic respiratory morbidity is a common adverse outcome of very premature birth, particularly in infants who had developed bronchopulmonary dysplasia (BPD). Prematurely born infants who had BPD may require supplementary oxygen at home for many months and affected infants have increased healthcare utilization until school age. Chest radiograph abnormalities are common; computed tomography of the chest gives predictive information in children with ongoing respiratory problems. Readmission to hospital is common, particularly for those who have BPD and suffer respiratory syncytial virus lower respiratory infections (RSV LRTIs). Recurrent respiratory symptoms requiring treatment are common and are associated with evidence of airways obstruction and gas trapping. Pulmonary function improves with increasing age, but children with BPD may have ongoing airflow limitation. Lung function abnormalities may be more severe in those who had RSV LRTIs, although this may partly be explained by worse premorbid lung function. Worryingly, lung function may deteriorate during the first year. Longitudinal studies are required to determine if there is catch up growth.

Long-term pulmonary outcomes of patients with bronchopulmonary dysplasia

Anita Bhandari and Sharon McGrath-Morrow
Seminars in Perinatology 37 (2013)132–137
http://dx.doi.org/10.1053/j.semperi.2013.01.010

Bronchopulmonary dysplasia (BPD) is the commonest cause of chronic lung disease in infancy. The incidence of BPD has remained unchanged despite many advances in neonatal care. BPD starts in the neonatal period but its effects can persist long term. Premature infants with BPD have a greater incidence of hospitalization, and continue to have a greater respiratory morbidity and need for respiratory medications, compared to those without BPD. Lung function abnormalities, especially small airway abnormalities, often persist. Even in the absence of clinical symptoms, BPD survivors have persistent radiological abnormalities and presence of emphysema has been reported on chest computed tomography scans. Concern regarding their exercise tolerance remains. Long-term effects of BPD are still unknown, but given reports of a more rapid decline in lung function and their susceptibility to develop chronic obstructive pulmonary disease phenotype with aging, it is imperative that lung function of survivors of BPD be closely monitored.

Neonatal ventilation strategies and long-term respiratory outcomes

Sandeep Shetty, Anne Greenough
Early Human Development 90 (2014) 735–739
http://dx.doi.org/10.1016/j.earlhumdev.2014.08.020

Long-term respiratory morbidity is common, particularly in those born very prematurely and who have developed bronchopulmonary dysplasia (BPD), but it does occur in those without BPD and in infants born at term. A variety of neonatal strategies have been developed, all with short-term advantages, but meta-analyses of randomized controlled trials (RCTs) have demonstrated that only volume-targeted ventilation and prophylactic high-frequency oscillatory ventilation (HFOV) may reduce BPD. Few RCTs have incorporated long-term follow-up, but one has demonstrated that prophylactic HFOV improves respiratory and functional outcomes at school age, despite not reducing BPD. Results from other neonatal interventions have demonstrated that any impact on BPD may not translate into changes in long-term outcomes. All future neonatal ventilation RCTs should have long-term outcomes rather than BPD as their primary outcome if they are to impact on clinical practice.

Prediction of neonatal respiratory distress syndrome in term pregnancies by assessment of fetal lung volume and pulmonary artery resistance index

Mohamed Laban, GM Mansour, MSE Elsafty, AS Hassanin, SS EzzElarab
International Journal of Gynecology and Obstetrics 128 (2015) 246–250
http://dx.doi.org/10.1016/j.ijgo.2014.09.018

Objective: To develop reference cutoff values for mean fetal lung volume (FLV) and pulmonary artery resistance index (PA-RI) for prediction of neonatal respiratory distress syndrome (RDS) in low-risk term pregnancies. Methods: As part of a cross-sectional study, women aged 20–35 years were enrolled and admitted to a tertiary hospital in Cairo, Egypt, for elective repeat cesarean at 37–40 weeks of pregnancy between January 1, 2012, and July 31, 2013. FLV was calculated by virtual organ computer-aided analysis, and PA-RI was measured by Doppler ultrasonography before delivery. Results: A total of 80 women were enrolled. Neonatal RDS developed in 11 (13.8%) of the 80 newborns. Compared with neonates with RDS, healthy neonates had significantly higher FLVs (P b 0.001) and lower PA-RIs (P b 0.001). Neonatal RDS is less likely with FLV of at least 32 cm3 or PA-RI less than or equal to 0.74. Combining these two measures improved the accuracy of prediction. Conclusion: The use of either FLV or PA-RI predicted neonatal RDS. The predictive value increased when these two measures were combined

Pulmonary surfactant - a front line of lung host defense, 2003 JCI0318650.f2

Pulmonary surfactant – a front line of lung host defense, 2003 JCI0318650.f2

Pulmonary hypertension in bronchopulmonary dysplasia

Sara K.Berkelhamer, Karen K.Mestan, and Robin H. Steinhorn
Seminars In  Perinatology 37 (2013)124–131
http://dx.doi.org/10.1053/j.semperi.2013.01.009

Pulmonary hypertension (PH) is a common complication of neonatal respiratory diseases, including bronchopulmonary dysplasia (BPD), and recent studies have increased aware- ness that PH worsens the clinical course, morbidity and mortality of BPD. Recent evidence indicates that up to 18% of all extremely low-birth-weight infants will develop some degree of PH during their hospitalization, and the incidence rises to 25–40% of the infants with established BPD. Risk factors are not yet well understood, but new evidence shows that fetal growth restriction is a significant predictor of PH. Echocardiography remains the primary method for evaluation of BPD-associated PH, and the development of standardized screening timelines and techniques for identification of infants with BPD-associated PH remains an important ongoing topic of investigation. The use of pulmonary vasodilator medications, such as nitric oxide, sildenafil, and others, in the BPD population is steadily growing, but additional studies are needed regarding their long-term safety and efficacy.
An update on pharmacologic approaches to bronchopulmonary dysplasia

Sailaja Ghanta, Kristen Tropea Leeman, and Helen Christou
Seminars In Perinatology 37 (2013)115–123
http://dx.doi.org/10.1053/j.semperi.2013.01.008

Bronchopulmonary dysplasia (BPD) is the most prevalent long-term morbidity in surviving extremely preterm infants and is linked to increased risk of reactive airways disease, pulmonary hypertension, post-neonatal mortality, and adverse neurodevelopmental outcomes. BPD affects approximately 20% of premature newborns, and up to 60% of premature infants born before completing 26 weeks of gestation. It is characterized by the need for assisted ventilation and/or supplemental oxygen at 36 weeks postmenstrual age. Approaches to prevention and treatment of BPD have evolved with improved understanding of its pathogenesis. This review will focus on recent advancements and detail current research in pharmacotherapy for BPD. The evidence for both current and potential future experimental therapies will be reviewed in detail. As our understanding of the complex and multifactorial pathophysiology of BPD changes, research into these current and future approaches must continue to evolve.

Methylxanthines
Diuretics and bronchodilators
Corticosteroids
Macrolide antibiotics
Recombinant human Clara cell 10-kilodalton protein(rhCC10)
Vitamin A
Surfactant
Leukotriene receptor antagonist
Pulmonary vasodilators

Skeletal and Muscle

Skeletal Stem Cells in Space and Time

Moustapha Kassem and Paolo Bianco
Cell  Jan 15, 2015; 160: 17-19
http://dx.doi.org/10.1016/j.cell.2014.12.034

The nature, biological characteristics, and contribution to organ physiology of skeletal stem cells are not completely determined. Chan et al. and Worthley et al. demonstrate that a stem cell for skeletal tissues, and a system of more restricted, downstream progenitors, can be identified in mice and demonstrate its role in skeletal tissue maintenance and regeneration.

The groundbreaking concept that bone, cartilage, marrow adipocytes, and hematopoiesis-supporting stroma could originate from a common progenitor and putative stem cell was surprising at the time when it was formulated (Owen and Friedenstein, 1988). The putative stem cell, nonhematopoietic in nature, would be found in the postnatal bone marrow stroma, generate tissues previously thought of as foreign to each other, and support the turnover of tissues and organs that self-renew at a much slower rate compared to other tissues associated with stem cells (blood, epithelia). This concept also connected bone and bone marrow as parts of a single-organ system, implying their functional interplay. For many years, the evidence underpinning the concept has been incomplete.

While multipotency of stromal progenitors has been demonstrated by in vivo transplantation experiments, self-renewal, the defining property of a stem cell, has not been easily demonstrated until recently in humans (Sacchetti et al., 2007) and mice (Mendez-Ferrer et al., 2010). Meanwhile, a confusing and plethoric terminology has been introduced into the literature, which diverted and confounded the search for a skeletal stem cell and its physiological significance (Bianco et al., 2013).

Two studies in this issue of Cell (Chan et al., 2015; Worthley et al., 2015), using a combination of rigorous single-cell analyses and lineage tracing technologies, mark significant steps toward rectifying the course of skeletal stem cell discovery by making several important points, within and beyond skeletal physiology.

First, a stem cell for skeletal tissues, and a system of more restricted, downstream progenitors can in fact be identified and linked to defined phenotype(s) in the mouse. The system is framed conceptually, and approached experimentally, similar to the hematopoietic system.

Second, based on its assayable functions and potential, the stem cell at the top of the hierarchy is defined as a skeletal stem cell (SSC). As noted earlier (Sacchetti et al., 2007) (Bianco et al., 2013), this term clarifies, well beyond semantics, that the range of tissues that the self-renewing stromal progenitor (originally referred to as an ‘‘osteogenic’’ or ‘‘stromal’’ stem cell) (Owen and Friedenstein, 1988) can actually generate in vivo, overlaps with the range of tissues that make up the skeleton.

Third, these cells are spatially restricted, local residents of the bone/bone marrow organ. The systemic circulation is not a sizable contributor to their recruitment to locally deployed functions.

Fourth, a native skeletogenic potential is inherent to the system of progenitor/ stem cells found in the skeleton, and internally regulated by bone morphogenetic protein (BMP) signaling. This is reflected in the expression of regulators and antagonists of BMP signaling within the system, highlighting potential feedback mechanisms modulating expansion or quiescence of specific cell compartments.

Fifth, in cells isolated from other tissues, an assayable skeletogenic potential is not inherent: it can only be induced de novo by BMP reprogramming. These two studies (Chan et al., 2015, Worthley et al., 2015) corroborate the classical concept of ‘‘determined’’ and ‘‘inducible’’ skeletal progenitors (Owen and Friedenstein, 1988): the former residing in the skeleton, the latter found in nonskeletal tissues; the former capable of generating skeletal tissues, in vivo and spontaneously, the latter requiring reprogramming signals in order to acquire a skeletogenic capacity; the former operating in physiological bone formation, the latter in unwanted, ectopic bone formation in diseases such as fibrodysplasia ossificans progressiva.

To optimize our ability to obtain specific skeletal tissues for medical application, the study by Chan et al. offers a glimpse of another facet of the biology of SSC lineages and progenitors. Chan et al. show that a homogeneous cell population inherently committed to chondrogenesis can alter its output to generate bone if cotransplanted with multipotent progenitors. Conversely, osteogenic cells can be shifted to a chondrogenic fate by blockade of vascular endothelial growth factor receptor, consistent with the avascular and hypoxic milieu of cartilage. This has two important implications:

  • commitment is flexible in the system;
  • the choir is as important as the soloist and can modulate the solo tune.

Reversibility and population behavior thus emerge as two features that may be characteristic, albeit not unique, of the stromal system, resonating with conceptually comparable evidence in the human system.

The two studies by Chan et al. and Worthely et al. emphasize the relevance not only of their new data, but also of a proper concept of a skeletal stem cell per se, for proper clinical use. Confusion arising from improper conceptualization of skeletal stem cells has markedly limited clinical development of skeletal stem cell biology.

Gremlin 1 Identifies a Skeletal Stem Cell with Bone, Cartilage, and Reticular Stromal Potential

Daniel L. Worthley, Michael Churchill, Jocelyn T. Compton, Yagnesh Tailor, et al.
Cell, Jan 15, 2015; 160: 269–284
http://dx.doi.org/10.1016/j.cell.2014.11.042

The stem cells that maintain and repair the postnatal skeleton remain undefined. One model suggests that perisinusoidal mesenchymal stem cells (MSCs) give rise to osteoblasts, chondrocytes, marrow stromal cells, and adipocytes, although the existence of these cells has not been proven through fate-mapping experiments. We demonstrate here that expression of the bone morphogenetic protein (BMP) antagonist gremlin 1 defines a population of osteochondroreticular (OCR) stem cells in the bone marrow. OCR stem cells self-renew and generate osteoblasts, chondrocytes, and reticular marrow stromal cells, but not adipocytes. OCR stem cells are concentrated within the metaphysis of long bones not in the perisinusoidal space and are needed for bone development, bone remodeling, and fracture repair. Grem1 expression also identifies intestinal reticular stem cells (iRSCs) that are cells of origin for the periepithelial intestinal mesenchymal sheath. Grem1 expression identifies distinct connective tissue stem cells in both the bone (OCR stem cells) and the intestine (iRSCs).

Identification and Specification of the Mouse Skeletal Stem Cell

Charles K.F. Chan, Eun Young Seo, James Y. Chen, David Lo, A McArdle, et al.
Cell, Jan 15, 2015; 160: 285–298
http://dx.doi.org/10.1016/j.cell.2014.12.002

How are skeletal tissues derived from skeletal stem cells? Here, we map bone, cartilage, and stromal development from a population of highly pure, postnatal skeletal stem cells (mouse skeletal stem cells, mSSCs) to their downstream progenitors of bone, cartilage, and stromal tissue. We then investigated the transcriptome of the stem/progenitor cells for unique gene-expression patterns that would indicate potential regulators of mSSC lineage commitment. We demonstrate that mSSC niche factors can be potent inducers of osteogenesis, and several specific combinations of recombinant mSSC niche factors can activate mSSC genetic programs in situ, even in nonskeletal tissues, resulting in de novo formation of cartilage or bone and bone marrow stroma. Inducing mSSC formation with soluble factors and subsequently regulating the mSSC niche to specify its differentiation toward bone, cartilage, or stromal cells could represent a paradigm shift in the therapeutic regeneration of skeletal tissues.

Bone mesenchymal development

Bone mesenchymal development

Bone mesenchymal development

The bone-remodeling cycle

The bone-remodeling cycle

Nuclear receptor modulation – Role of coregulators in selective estrogen receptor modulator (SERM) actions

Qin Feng, Bert W. O’Malley
Steroids 90 (2014) 39–43
http://dx.doi.org/10.1016/j.steroids.2014.06.008

Selective estrogen receptor modulators (SERMs) are a class of small-molecule chemical compounds that bind to estrogen receptor (ER) ligand binding domain (LBD) with high affinity and selectively modulate ER transcriptional activity in a cell- and tissue-dependent manner. The prototype of SERMs is tamoxifen, which has agonist activity in bone, but has antagonist activity in breast. Tamoxifen can reduce the risk of breast cancer and, at same time, prevent osteoporosis in postmenopausal women. Tamoxifen is widely prescribed for treatment and prevention of breast cancer. Mechanistically the activity of SERMs is determined by the selective recruitment of coactivators and corepressors in different cell types and tissues. Therefore, understanding the coregulator function is the key to understanding the tissue selective activity of SERMs.

Hematopoietic

Hematopoietic Stem Cell Arrival Triggers Dynamic Remodeling of the Perivascular Niche

Owen J. Tamplin, Ellen M. Durand, Logan A. Carr, Sarah J. Childs, et al.
Cell, Jan 15, 2015; 160: 241–252
http://dx.doi.org/10.1016/j.cell.2014.12.032

Hematopoietic stem and progenitor cells (HSPCs) can reconstitute and sustain the entire blood system. We generated a highly specific transgenic reporter of HSPCs in zebrafish. This allowed us to perform high resolution live imaging on endogenous HSPCs not currently possible in mammalian bone marrow. Using this system, we have uncovered distinct interactions between single HSPCs and their niche. When an HSPC arrives in the perivascular niche, a group of endothelial cells remodel to form a surrounding pocket. This structure appears conserved in mouse fetal liver. Correlative light and electron microscopy revealed that endothelial cells surround a single HSPC attached to a single mesenchymal stromal cell. Live imaging showed that mesenchymal stromal cells anchor HSPCs and orient their divisions. A chemical genetic screen found that the compound lycorine promotes HSPC-niche interactions during development and ultimately expands the stem cell pool into adulthood. Our studies provide evidence for dynamic niche interactions upon stem cell colonization.

Neonatal anemia

Sanjay Aher, Kedar Malwatkar, Sandeep Kadam
Seminars in Fetal & Neonatal Medicine (2008) 13, 239e247
http://dx.doi.org:/10.1016/j.siny.2008.02.009

Neonatal anemia and the need for red blood cell (RBC) transfusions are very common in neonatal intensive care units. Neonatal anemia can be due to blood loss, decreased RBC production, or increased destruction of erythrocytes. Physiologic anemia of the newborn and anemia of prematurity are the two most common causes of anemia in neonates. Phlebotomy losses result in much of the anemia seen in extremely low birthweight infants (ELBW). Accepting a lower threshold level for transfusion in ELBW infants can prevent these infants being exposed to multiple donors.

Management of anemia in the newborn

Naomi L.C. Luban
Early Human Development (2008) 84, 493–498
http://dx.doi.org:/10.1016/j.earlhumdev.2008.06.007

Red blood cell (RBC) transfusions are administered to neonates and premature infants using poorly defined indications that may result in unintentional adverse consequences. Blood products are often manipulated to limit potential adverse events, and meet the unique needs of neonates with specific diagnoses. Selection of RBCs for small volume (5–20 mL/kg) transfusions and for massive transfusion, defined as extracorporeal bypass and exchange transfusions, are of particular concern to neonatologists. Mechanisms and therapeutic treatments to avoid transfusion are another area of significant investigation. RBCs collected in anticoagulant additive solutions and administered in small aliquots to neonates over the shelf life of the product can decrease donor exposure and has supplanted the use of fresh RBCs where each transfusion resulted in a donor exposure. The safety of this practice has been documented and procedures established to aid transfusion services in ensuring that these products are available. Less well established are the indications for transfusion in this population; hemoglobin or hematocrit alone are insufficient indications unless clinical criteria (e.g. oxygen desaturation, apnea and bradycardia, poor weight gain) also augment the justification to transfuse. Comorbidities increase oxygen consumption demands in these infants and include bronchopulmonary dysplasia, rapid growth and cardiac dysfunction. Noninvasive methods or assays have been developed to measure tissue oxygenation; however, a true measure of peripheral oxygen offloading is needed to improve transfusion practice and determine the value of recombinant products that stimulate erythropoiesis. The development of such noninvasive methods is especially important since randomized, controlled clinical trials to support specific practices are often lacking, due at least in part, to the difficulty of performing such studies in tiny infants.
The Effect of Blood Transfusion on the Hemoglobin Oxygen Dissociation Curve of Very Early Preterm Infants During the First Week of Life

Virginie De HaUeux, Anita Truttmann, Carmen Gagnon, and Harry Bard
Seminars in Perinatology, 2002; 26(6): 411-415
http://dx.doi.org:/10.1053/sper.2002.37313

This study was conducted during the first week of life to determine the changes in Ps0 (PO2 required to achieve a saturation of 50% at pH 7.4 and 37~ and the proportions of fetal hemoglobin (I-IbF) and adult hemoglobin (HbA) prior to and after transfusion in very early preterm infants. Eleven infants with a gestational age <–27 weeks have been included in study. The hemoglobin dissociation curve and the Ps0 was determined by Hemox-analyser. Liquid chromatography was also performed to determine the proportions of HbF and HbA. The mean gestational age of the 11 infants was 25.1 weeks (-+1 weeks) and their mean birth weight was 736 g (-+125 g). They received 26.9 mL/kg of packed red cells. The mean Ps0 prior and after transfusion was 18.5 +- 0.8 and 21.0 + 1 mm Hg (P = .0003) while the mean percentage of HbF was 92.9 -+ 1.1 and 42.6 -+ 5.7%, respectively. The data of this study show a decrease of hemoglobin oxygen affinity as a result of blood transfusion in very early preterm infants prone to O 2 toxicity. The shift in HbO 2 curve after transfusion should be taken into consideration when oxygen therapy is being regulated for these infants.

Effect of neonatal hemoglobin concentration on long-term outcome of infants affected by fetomaternal hemorrhage

Mizuho Kadooka, H Katob, A Kato, S Ibara, H Minakami, Yuko Maruyama
Early Human Development 90 (2014) 431–434
http://dx.doi.org/10.1016/j.earlhumdev.2014.05.010

Background: Fetomaternal hemorrhage (FMH) can cause severe morbidity. However, perinatal risk factors for long-term poor outcome due to FMH have not been extensively studied.                                                                                 Aims: To determine which FMH infants are likely to have neurological sequelae.
Study design: A single-center retrospective observational study. Perinatal factors, including demographic characteristics, Kleihauer–Betke test, blood gas analysis, and neonatal blood hemoglobin concentration ([Hb]), were analyzed in association with long-term outcomes.
Subjects: All 18 neonates referred to a Neonatal Intensive Care Unit of Kagoshima City Hospital and diagnosed with FMH during a 15-year study period. All had a neonatal [Hb] b7.5 g/dL and 15 of 17 neonates tested had Kleihauer–Betke test result N4.0%.
Outcome measures: Poor long-term outcome was defined as any of the following determined at 12 month old or more: cerebral palsy, mental retardation, attention deficit/hyperactivity disorder, and epilepsy.
Results: Nine of the 18 neonates exhibited poor outcomes. Among demographic characteristics and blood variables compared between two groups with poor and favorable outcomes, significant differences were observed in [Hb] (3.6 ± 1.4 vs. 5.4 ± 1.1 g/dL, P = 0.01), pH (7.09 ± 0.11 vs. 7.25 ± 0.13, P = 0.02) and base deficits (17.5 ± 5.4 vs. 10.4 ± 6.0 mmol/L, P = 0.02) in neonatal blood, and a number of infants with [Hb] ≤ 4.5 g/dL (78%[7/9] vs. 22%[2/9], P= 0.03), respectively. The base deficit in neonatal arterial blood increased significantly with decreasing neonatal [Hb].
Conclusions: Severe anemia causing severe base deficit is associated with neurological sequelae in FMH infants

Clinical and hematological presentation among Indian patients with common hemoglobin variants

Khushnooma Italia, Dipti Upadhye, Pooja Dabke, Harshada Kangane, et al.
Clinica Chimica Acta 431 (2014) 46–51
http://dx.doi.org/10.1016/j.cca.2014.01.028

Background: Co-inheritance of structural hemoglobin variants like HbS, HbD Punjab and HbE can lead to a variable clinical presentation and only few cases have been described so far in the Indian population.
Methods: We present the varied clinical and hematological presentation of 22 cases (HbSD Punjab disease-15, HbSE disease-4, HbD Punjab E disease-3) referred to us for diagnosis.
Results: Two of the 15 HbSDPunjab disease patients had moderate crisis, one presented with mild hemolytic anemia; however, the other 12 patients had a severe clinical presentation with frequent blood transfusion requirements, vaso occlusive crisis, avascular necrosis of the femur and febrile illness. The 4 HbSE disease patients had a mild to moderate presentation. Two of the 3 HbD Punjab E patients were asymptomatic with one patient’s sibling having a mild presentation. The hemoglobin levels of the HbSD Punjab disease patients ranged from 2.3 to 8.5 g/dl and MCV from 76.3 to 111.6 fl. The hemoglobin levels of the HbD Punjab E and HbSE patients ranged from 10.8 to 11.9 and 9.8 to 10.0 g/dl whereas MCV ranged from 67.1 to 78.2 and 74.5 to 76.0 fl respectively.
Conclusions: HbSD Punjab disease patients should be identified during newborn screening programs and managed in a way similar to sickle cell disease. Couple at risk of having HbSD Punjab disease children may be given the option of prenatal diagnosis in subsequent pregnancies.

Sickle cell anemia is the most common hemoglobinopathy seen across the world. It is caused by a point mutation in the 6th codon of the beta (β) globin gene leading to the substitution of the amino acid glutamic acid to valine. The sickle gene is frequently seen in Africa, some Mediterranean countries, India, Middle East—Saudi Arabia and North America. In India the prevalence of hemoglobin S (HbS) carriers varies from 2 to 40% among different population groups and HbS is mainly seen among the scheduled tribe, scheduled caste and other backward class populations in the western, central and parts of eastern and southern India. Sickle cell anemia has a variable clinical presentation in India with the most severe clinical presentation seen in central India whereas patients in the western region show a mild to moderate clinical presentation.

Hemoglobin D Punjab (HbD Punjab) (also known as HbD Los-Angeles, HbD Portugal, HbD North Carolina, D Oak Ridge and D Chicago) is another hemoglobin variant due to a point mutation in codon 121 of the β globin gene resulting in the substitution of the amino acid glutamic acid to glycine. It is a widely distributed hemoglobin with a relatively low prevalence of 0.86% in the Indo-Pak subcontinent, 1–3% in north-western India, 1–3% in the Black population in the Caribbean and North America and has also been reported among the English. It accounts for 55.6% of all the Hb variants seen in the Xenjiang province of China.

Hemoglobin E (HbE) is the most common abnormal hemoglobin in Southeast Asia. In India, the frequency ranges from 4% to 51% in the north eastern region and 3% to 4% in West Bengal in the east. The HbE mutation (β26 GAG→AAG) creates an alternative splice site and the βE chain is insufficiently synthesized, hence the phenotype of this disorder is that of a mild form of β thalassemia.

Though these 3 structural variants are prevalent in different regions of India, their interaction is increasingly seen in all states of the country due to migration of people to different regions for a better livelihood. There are very few reports on interaction of these commonly seen Hb variants and the phenotypic–genotypic presentation of these cases is important for genetic counseling and management.

HbF of patients with HbSD Punjab disease with variable clinical severity. The HbF values of 4 patients are not included as they were post blood transfusion

The genotypes of the patients were confirmed by restriction enzyme digestion and ARMS (Fig). Patients 1 to 15 were characterized as compound heterozygous for HbS and HbD Punjab whereas patients 16 to 19 were characterized as compound heterozygous for HbS and HbE. Patient nos. 20 to 22 were characterized as compound heterozygous for HbE and HbD Punjab.

Molecular characterization of HbS and HbDPunjab by restriction enzyme digestion and of HbE by ARMS.

Molecular characterization of HbS and HbDPunjab by restriction enzyme digestion and of HbE by ARMS.

Molecular characterization of HbS and HbDPunjab by restriction enzyme digestion and of HbE by ARMS.

The 3 common β globin gene variants of hemoglobin, HbS, HbE and HbD Punjab are commonly seen in India, with HbS having a high prevalence in the central belt and some parts of western, eastern and southern India, HbE in the eastern and north eastern region whereas HbD is mostly seen in the north western part of India. These hemoglobin variants have been reported in different population groups. However, with migration and intermixing of the different populations from different geographic regions, occasional cases of HbSD Punjab and HbSE are being reported. There are several HbD variants like HbD Punjab, HbD Iran, HbD Ibadan. However, of these only HbD Punjab interacts with HbS to form a clinically significant condition as the glutamine residue facilitates polymerization of HbS. HbD Iran and HbD Ibadan are non-interacting and produce benign conditions like the sickle cell trait. The first case of HbSD Punjab disease was a brother and sister considered to have atypical sickle cell disease in 1934. This family was further reinvestigated and reported as the first case of HbD Los Angeles which has the same mutation as the HbD Punjab. Serjeant et al. reported HbD Punjab in an English parent in 6 out of 11 HbSD-Punjab disease cases. This has been suggested to be due to the stationing of nearly 50,000 British troops on the Indian continent for a period of 200 y and the introduction into Britain of their Anglo-Indian children.

HbSD Punjab disease shows a similar pattern to HbS homozygous on alkaline hemoglobin electrophoresis but can be differentiated on acid agar gel electrophoresis and on HPLC. In HbSD Punjab disease cases, the peripheral blood films show anisocytosis, poikilocytosis, target cells and irreversibly sickled cells. Values of HbF and HbA2 are similar to those in sickle homozygous cases. HbSD Punjab disease is characterized by a moderately severe hemolytic anemia.

Twenty-one cases of HbSDPunjab were reported by Serjeant of which 16 were reported by different workers among patients originating from Caucasian, Spanish, Australian, Irish, English, Portuguese, Black, American, Venezuelan, Caribbean, Mexican, Turkish and Jamaican backgrounds. Yavarian et al. 2009 reported a multi centric origin of HbD Punjab which in combination with HbS results in sickle cell disease. Patel et al. 2010 have also reported 12 cases of HbSD Punjab from the Orissa state of eastern India. Majority of these cases were symptomatic, presenting with chronic hemolytic anemia and frequent painful crises.

HbF levels >20% were seen in 4 out of our 11 clinically severe patients of HbSD-Punjab disease with the mean HbF levels of 16.8% in 8 clinically severe patients, while 3 clinically severe patients were post transfused. However, the 3 patients with a mild to moderate clinical presentation showed a mean HbF level of 8.6%. This is in contrast to the relatively milder clinical presentation associated with high HbF seen in patients with sickle cell anemia. This was also reported by Adekile et al. 2010 in 5 cases of HbS-DLos Angeles where high HbF did not ameliorate the severe clinical presentation seen in these patients.

These 15 cases of HbSDPunjab disease give us an overall idea of the severe clinical presentation of the disease in different regions of India. However the HbDPunjabE cases were milder or asymptomatic and the HbSE cases were moderately symptomatic. Since most of the cases of HbSDPunjab disease were clinically severe, it is important to pick up these cases during newborn screening and enroll them into a comprehensive care program with the other sickle cell disease patients with introduction of therapeutic interventions such as penicillin prophylaxis if required and pneumococcal immunization. In fact, 2 of our cases (No. 6 and 7) were identified during newborn screening for sickle cell disorders. The parents can be given information on home care and educated to detect symptoms that may lead to serious medical emergencies. The parents of these patients as well as the couples who are at risk of having a child with HbSDPunjab disease could also be counseled about the option of prenatal diagnosis in subsequent pregnancies. It is thus important to document the clinical and hematological presentation of compound heterozygotes with these common β globin chain variants.

Common Hematologic Problems in the Newborn Nursery

Jon F. Watchko
Pediatr Clin N Am – (2015) xxx-xxx
http://dx.doi.org/10.1016/j.pcl.2014.11.011

Common RBC disorders include hemolytic disease of the newborn, anemia, and polycythemia. Another clinically relevant hematologic issue in neonates to be covered herein is thrombocytopenia. Disorders of white blood cells will not be reviewed.

KEY POINTS

(1)               Early clinical jaundice or rapidly developing hyperbilirubinemia are often signs of hemolysis, the differential diagnosis of which commonly includes immune-mediated disorders, red-cell enzyme deficiencies, and red-cell membrane defects.

(2)             Knowledge of the maternal blood type and antibody screen is critical in identifying non-ABO alloantibodies in the maternal serum that may pose a risk for severe hemolytic disease in the newborn.

(3)             Moderate to severe thrombocytopenia in an otherwise well-appearing newborn strongly suggests immune-mediated (alloimmune or autoimmune) thrombocytopenia.

Hemolytic conditions in the neonate

1. Immune-mediated (positive direct Coombs test)  a. Rhesus blood group: Anti-D, -c, -C, -e, -E, CW, and several others

  b. Non-Rhesus blood groups: Kell, Duffy, Kidd, Xg, Lewis, MNS, and others

  c. ABO blood group: Anti-A, -B

2. Red blood cell (RBC) enzyme defects

  a. Glucose-6-phosphate dehydrogenase (G6PD) deficiency

  b. Pyruvate kinase deficiency

  c. Others

3. RBC membrane defects

  a. Hereditary spherocytosis

  b. Elliptocytosis

  c. Stomatocytosis

  d. Pyknocytosis

  e. Others

4. Hemoglobinopathies

  a. alpha-thalassemia

  b. gamma-thalassemia
Standard maternal antibody screeningAlloantibody                                 Blood Group

D, C, c, E, e, f, CW, V                     Rhesus

K, k, Kpa, Jsa                                  Kell

Fya, Fyb                                          Duffy

Jka, Jkb                                           Kidd

Xga                                                  Xg

Lea, Leb                                          Lewis

S, s, M, N                                        MNS

P1                                                    P

Lub                                                  Lutheran
Non-ABO alloantibodies reported to cause moderate to severe hemolytic disease of the newbornWithin Rh system: Anti-D, -c, -C, -Cw, -Cx, -e, -E, -Ew, -ce, -Ces, -Rh29, -Rh32, -Rh42, -f, -G, -Goa, -Bea, -Evans, -Rh17, -Hro, -Hr, -Tar, -Sec, -JAL, -STEM

Outside Rh system:  Anti-LW, -K, -k, -Kpa, -Kpb, -Jka, -Jsa, -Jsb, -Ku, -K11, -K22, -Fya, -M, -N, -S, -s, -U, -PP1 pk, -Dib, -Far, -MUT, -En3, -Hut, -Hil, -Vel, -MAM, -JONES, -HJK, -REIT

 

Red Blood Cell Enzymopathies

G6PD9 and pyruvate kinase (PK) deficiency are the 2 most common red-cell enzyme disorders associated with marked neonatal hyperbilirubinemia. Of these, G6PD deficiency is the more frequently encountered and it remains an important cause of kernicterus worldwide, including the United States, Canada, and the United Kingdom, the prevalence in Western countries a reflection in part of immigration patterns and intermarriage. The risk of kernicterus in G6PD deficiency also relates to the potential for unexpected rapidly developing extreme hyperbilirubinemia in this disorder associated with acute severe hemolysis.

Red Blood Cell Membrane Defects

Establishing a diagnosis of RBC membrane defects is classically based on the development of Coombs-negative hyperbilirubinemia, a positive family history, and abnormal RBC smear, albeit it is often difficult because newborns normally exhibit a marked variation in red-cell membrane size and shape. Spherocytes, however, are not often seen on RBC smears of hematologically normal newborns and this morphologic abnormality, when prominent, may yield a diagnosis of hereditary spherocytosis (HS) in the immediate neonatal period. Given that approximately 75% of families affected with hereditary spherocytosis manifest an autosomal dominant phenotype, a positive family history can often be elicited and provide further support for this diagnosis. More recently, Christensen and Henry highlighted the use of an elevated mean corpuscular hemoglobin concentration (MCHC) (>36.0 g/dL) and/or elevated ratio of MCHC to mean corpuscular volume, the latter they term the “neonatal HS index” (>0.36, likely >0.40) as screening tools for HS. An index of greater than 0.36 had 97% sensitivity, greater than 99% specificity, and greater than 99% negative predictive value for identifying HS in neonates. Christensen and colleagues also provided a concise update of morphologic RBC features that may be helpful in diagnosing this and other underlying hemolytic conditions in newborns.

The diagnosis of HS can be confirmed using the incubated osmotic fragility test when coupled with fetal red-cell controls or eosin-5-maleimide flow cytometry. One must rule out symptomatic ABO hemolytic disease by performing a direct Coombs test, as infants so affected also may manifest prominent micro-spherocytosis. Moreover, HS and symptomatic ABO hemolytic disease can occur in the same infant and result in severe hyperbilirubinemia and anemia.  Of other red-cell membrane defects, only hereditary elliptocytosis,  stomato-cytosis, and infantile pyknocytosis have been reported to exhibit significant hemolysis in the newborn period. Hereditary elliptocytosis and stomatocytosis are both rare. Infantile pyknocytosis, a transient red-cell membrane abnormality manifesting itself during the first few months of life, is more common.

Risk factors for bilirubin neurotoxicityIsoimmune hemolytic disease

G6PD deficiency

Asphyxia

Sepsis

Acidosis

Albumin less than 3.0 g/dL
Data from Maisels MJ, Bhutani VK, Bogen D, et al. Hyperbilirubinemia in the newborn infant > or 535 weeks’ gestation: an update with clarifications. Pediatrics 2009; 124:1193–8.

Polycythemia

Polycythemia (venous hematocrit 65%) in seen in infants across a range of conditions associated with active erythropoiesis or passive transfusion.76,77 They include, among others, placental insufficiency, the infant of a diabetic mother, recipient in twin-twin transfusion syndrome, and several aneuploidies, including trisomy. The clinical concern related to polycythemia is the risk for microcirculatory complications of hyperviscosity. However, determining which polycythemic infants are hyperviscous and when to intervene is a challenge.

 

 

Liver

Metabolic disorders presenting as liver disease

Germaine Pierre, Efstathia Chronopoulou
Paediatrics and Child Health 2013; 23(12): 509-514
The liver is a highly metabolically active organ and many inherited metabolic disorders have hepatic manifestations. The clinical presentation in these patients cannot usually be distinguished from liver disease due to acquired causes like infection, drugs or hematological disorders. Manifestations include acute and chronic liver failure, cholestasis and hepatomegaly. Metabolic causes of acute liver failure in childhood can be as high as 35%. Certain disorders like citrin deficiency and Niemann-Pick C disease may present in infancy with self-limiting cholestasis before presenting in later childhood or adulthood with irreversible disease. This article reviews important details from the history and clinical examination when evaluating the pediatric patient with suspected metabolic disease, the specialist and genetic tests when investigating, and also discusses specific disorders, their clinical course and treatment. The role of liver transplantation is also briefly discussed. Increased awareness of this group of disorders is important as in many cases, early diagnosis leads to early intervention with improved outcome. Diagnosis also allows genetic counselling and future family planning.

Adult liver disorders caused by inborn errors of metabolism: Review and update

Sirisak Chanprasert, Fernando Scaglia
Molecular Genetics and Metabolism 114 (2015) 1–10
http://dx.doi.org/10.1016/j.ymgme.2014.10.011

Inborn errors of metabolism (IEMs) are a group of genetic diseases that have protean clinical manifestations and can involve several organ systems. The age of onset is highly variable but IEMs afflict mostly the pediatric population. However, in the past decades, the advancement in management and new therapeutic approaches have led to the improvement in IEM patient care. As a result, many patients with IEMs are surviving into adulthood and developing their own set of complications. In addition, some IEMs will present in adulthood. It is important for internists to have the knowledge and be familiar with these conditions because it is predicted that more and more adult patients with IEMs will need continuity of care in the near future. The review will focus on Wilson disease, alpha-1 antitrypsin deficiency, citrin deficiency, and HFE-associated hemochromatosis which are typically found in the adult population. Clinical manifestations and pathophysiology, particularly those that relate to hepatic disease as well as diagnosis and management will be discussed in detail.

Inborn errors of metabolism (IEMs) are a group of genetic diseases characterized by abnormal processing of biochemical reactions, resulting in accumulation of toxic substances that could interfere with normal organ functions, and failure to synthesize essential compounds. IEMs are individually rare, but collectively numerous. The clinical presentations cover a broad spectrum and can involve almost any organ system. The age of onset is highly variable but IEMs afflict mostly the pediatric population.

Wilson disease is an autosomal recessive genetic disorder of copper metabolism. It is characterized by an abnormal accumulation of inorganic copper in various tissues, most notably in the liver and the brain, especially in the basal ganglia. The disease was first described in 1912 by Kinnier Wilson, and affects between 1 in 30,000 and 1 in 100,000 individuals. Clinical features are variable and depend on the extent  and the severity of copper deposition. Typically, patients tend to develop hepatic disease at a younger age than the neuropsychiatric manifestations. Individuals withWilson disease eventually succumb to complications of end stage liver disease or become debilitated from neurological problems, if they are left untreated.

The clinical presentations of Wilson disease are varied affecting many organ systems. However, the overwhelming majority of cases display hepatic and neurologic symptoms. In general, patients with hepatic disease present between the first and second decades of life although patients as young as 3 years old or over 50 years old have also been reported. The most common modes of presentations are acute self-limited hepatitis and chronic active hepatitis that are indistinguishable from other hepatic disorders although liver aminotransferases are generally much lower than in autoimmune or viral hepatitis. Acute fulminant hepatic failure is less common but is observed in approximately 3% of all cases of acute liver failure. Symptoms of acute liver failure include jaundice, coagulopathy, and hepatic encephalopathy. Cirrhosis can develop over time and may be clinically silent. Hepatocellular carcinoma (HCC) is rarely associated with Wilson disease, but may occur in the setting of cirrhosis and chronic inflammation.

Copper is an essential element, and is required for the proper functioning of various proteins and enzymes. The total body content of copper in a healthy adult individual is approximately 70–100 mg, while the daily requirements are estimated to be between 1 and 5 mg. Absorption occurs in the small intestine. Copper is taken up to the hepatocytes via the copper transporter hTR1. Once inside the cell, copper is bound to various proteins including metallothionein and glutathione, however, it is the metal chaperone, ATOX1 that helps direct copper to the ATP7B protein for intracellular transport and excretion. At the steady state, copper will be bound to ATP7B and is then incorporated to ceruloplasmin and secreted into the systemic circulation. When the cellular copper concentration arises, ATP7B protein will be redistributed from the trans-Golgi network to the prelysosomal vesicles facilitating copper excretion into the bile. The molecular defects in ATP7B lead to a reduction of copper excretion. Excess copper is accumulated in the liver causing tissue injury. The rate of accumulation of copper varies among individuals, and it may depend on other factors such as alcohol consumption, or viral hepatitis infections. If the liver damage is not severe, patients will accumulate copper in various tissues including the brain, the kidney, the eyes, and the musculoskeletal system leading to clinical disease. A failure of copper to incorporate into ceruloplasmin leads to secretion of the unsteady protein that has a shorter half-life, resulting in the reduced concentrations of ceruloplasmin seen in most patients with Wilson disease.

Wilson disease used to be a progressive fatal condition during the first half of the 20th century because there was no effective treatment available at that time. Penicillamine was the first pharmacologic agent introduced in 1956 for treating this condition. Penicillamine is a sulfhydryl-bearing amino acid cysteine doubly substituted with methyl groups. This drug acts as a chelating agent that promotes the urinary excretion of copper. It is rapidly absorbed in the gastrointestinal track, and over 80% of circulating penicillamine is excreted via the kidneys. Although it is very effective, approximately 10%–50% of Wilson disease patients with neuropsychiatric presentations may experience worsening of their symptoms, and often times the worsening symptoms may not be reversible.

Alpha1-antitrypsin deficiency

Alpha1-antitrypsin deficiency (AATD) is one of the most common genetic liver diseases in children and adults, affecting 1 in 2000 to 1 in 3000 live births worldwide. It is transmitted in an autosomal co-dominant fashion with variable expressivity. Alpha1 antitrypsin (A1AT) is a member of the serine protease inhibitor (SERPIN) family. Its function is to counteract the proteolytic effect of neutrophil elastase and other neutrophil proteases. Mutations in the SERPINA1, the gene encoding A1AT, result in changes in the protein structure with the PiZZ phenotype being the most common cause of liver and lung disease-associated AATDs. Although, it classically causes early onset chronic obstructive pulmonary disease (COPD) in adults, liver disease characterized by chronic inflammation, hepatic fibrosis, and cirrhosis is not uncommon in the adult population. Decreased plasma concentration of A1AT predisposes lung tissue to be more susceptible to injury from protease enzymes. However, the underlying mechanism of liver injury is different, and is believed to be caused by accumulation of polymerized mutant A1AT in the hepatocyte endoplasmic reticulum (ER). Currently, there is no specific treatment for liver disease-associated AATD, but A1AT augmentation therapy is available for patients affected with pulmonary involvement.

A1AT is a single-chain, 52-kDa polypeptide of approximately 394 amino acids [56]. It is synthesized in the liver, circulates in the plasma, and functions as an inhibitor of neutrophil elastase and other proteases such as cathepsin G, and proteinase 3. A1AT has a globular shape composed of two central β sheets surrounded by a small β sheet and nine α helices. The pathophysiology underlying liver disease is thought to be a toxic gain-of-function mutation associated with the PiZZ phenotypes. This hypothesis has been supported by the fact that null alleles which produce no detectable plasma A1AT, are not associated with liver disease. In addition, the transgenic mouse model of AATD PiZZ developed periodic acid-Schiff-positive diastase-resistant intrahepatic globule early in life similar to AATD patients. The PiZZ phenotype results in the blockade of the final processing of A1AT in the liver, as only 15% of the A1AT reaches the circulation whereas 85% of non-secreted protein is accumulated in the hepatocytes.

Citrin deficiency

Citrin deficiency is a relatively newly-defined autosomal recessive disease. It encompasses two different sub-groups of patients, neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD), and adult onset citrullinemia type 2 (CTLN 2).

AGC2 exports aspartate out of the mitochondrial matrix in exchange for glutamate and a proton. Thus, this protein has an important role in ureagenesis and gluconeogenesis. In CTLN2, a defect in this protein is believed to limit the supply of aspartate for the formation of argininosuccinate in the cytosol resulting in impairment of ureagenesis. Interestingly, the mouse model of citrin deficiency (Ctrn−/−) fails to develop symptoms of CTLN2 suggesting that the mitochondrial aspartate is not the only source of ureagenesis. However, it should be noted that the rodent liver expresses higher glycerol-phosphate shuttle activity than the human counterpart. With the intact glycerol-phosphate dehydrogenase, it can compensate for the deficiency of AGC2, as demonstrated by the AGC2 and glycerol-phosphate dehydrogenase double knock-out mice that exhibit similar features to those observed in human CTLN2.

HFE-associated hemochromatosis

HFE-associated hemochromatosis is an inborn error of iron metabolism characterized by excessive iron storage resulting in tissue and organ damage. It is the most common autosomal recessive disorder in the Caucasian population, affecting 0.3%–0.5% of individuals of Northern European descent. The term “hemochromatosis” was coined in 1889 by the German pathologist Friedrich Daniel Von Recklinghausen, who described it as bronze stain of organs caused by a blood borne pigment.

The classic clinical triad of cirrhosis, diabetes, and bronze skin pigmentation is rarely observed nowadays given the early recognition, diagnosis, and treatment of this condition. The most common presenting symptoms are nonspecific including weakness, lethargy, and arthralgia.

The liver is a major site of iron storage in healthy individuals and as such it is the organ that is universally affected in HFE-associated hemochromatosis. Elevation of liver aminotransferases indicative of hepatocyte injury is the most common mode of presentation and it can be indistinguishable from other causes of hepatitis. Approximately 15%–40% of patients with HFE-associated hemochromatosis have other liver conditions, including chronic viral hepatitis B or C infection, nonalcoholic fatty liver disease, and alcoholic liver disease.

 

The liver in haemochromatosis

Rune J. Ulvik
Journal of Trace Elements in Medicine and Biology xxx (2014) xxx–xxx
http://dx.doi.org/10.1016/j.jtemb.2014.08.005

The review deals with genetic, regulatory and clinical aspects of iron homeostasis and hereditary hemochromatosis. Hemochromatosis was first described in the second half of the 19th century as a clinical entity characterized by excessive iron overload in the liver. Later, increased absorption of iron from the diet was identified as the pathophysiological hallmark. In the 1970s genetic evidence emerged supporting the apparent inheritable feature of the disease. And finally in 1996 a new “hemochromato-sis gene” called HFE was described which was mutated in about 85% of the patients. From the year2000 onward remarkable progress was made in revealing the complex molecular regulation of iron trafficking in the human body and its disturbance in hemochromatosis. The discovery of hepcidin and ferroportin and their interaction in regulating the release of iron from enterocytes and macrophages to plasma were important milestones. The discovery of new, rare variants of non-HFE-hemochromatosis was explained by mutations in the multicomponent signal transduction pathway controlling hepcidin transcription. Inhibited transcription induced by the altered function of mutated gene products, results in low plasma levels of hepcidin which facilitate entry of iron from enterocytes into plasma. In time this leads to progressive accumulation of iron and subsequently development of disease in the liver and other parenchymatous organs. Being the major site of excess iron storage and hepcidin synthesis the liver is a cornerstone in maintaining normal systemic iron homeostasis. Its central pathophysiological role in HFE-hemochromatosis with downgraded hepcidin synthesis, was recently shown by the finding that liver transplantation normalized the hepcidin levels in plasma and there was no sign of iron accumulation in the new liver.

Gastrointestinal

Decoding the enigma of necrotizing enterocolitis in premature infants

Roberto Murgas Torrazza, Nan Li, Josef Neu
Pathophysiology 21 (2014) 21–27
http://dx.doi.org/10.1016/j.pathophys.2013.11.011

Necrotizing enterocolitis (NEC) is an enigmatic disease that affects primarily premature infants. It often occurs suddenly and when it occurs, treatment attempts at treatment often fail and results in death. If the infant survives, there is a significant risk of long term sequelae including neurodevelopmental delays. The pathophysiology of NEC is poorly understood and thus prevention has been difficult. In this review, we will provide an overview of why progress may be slow in our understanding of this disease, provide a brief review diagnosis, treatment and some of the current concepts about the pathophysiology of this disease.

Necrotizing enterocolitis (NEC) has been reported since special care units began to house preterm infants .With the advent of modern neonatal intensive care approximately 40 years ago, the occurrence and recognition of the disease markedly increased. It is currently the most common and deadly gastro-intestinal illness seen in preterm infants. Despite major efforts to better understand, treat and prevent this devastating disease, little if any progress has been made during these 4 decades. Underlying this lack of progress is the fact that what is termed “NEC” is likely more than one disease, or mimicked by other diseases, each with a different etiopathogenesis.

Human gut microbiome

Human gut microbiome

Term or near term infants with “NEC” when compared to matched controls usually have occurrence of their disease in the first week after birth, have a significantly higher frequency of prolonged rupture of membranes, chorio-amnionitis, Apgar score <7 at 1 and 5 min, respiratory problems, congenital heart disease, hypoglycemia, and exchange transfusions. When a “NEC” like illness presents in term or near term infants, it should be noted that these are likely to be distinct in pathogenesis than the most common form of NEC and should be differentiated as such.

The infants who suffer primary ischemic necrosis are term or near term infants (although this can occur in preterms) who have concomitant congenital heart disease, often related to poor left ventricular output or obstruction. Other factors that have been associated with primary ischemia are maternal cocaine use, hyperviscosity caused by polycythemia or a severe antecedent hypoxic–ischemic event. Whether the dis-ease entity that results from this should be termed NEC can be debated on historical grounds, but the etiology is clearly different from the NEC seen in most preterm infants.

The pathogenesis of NEC is uncertain, and the etiology seems to be multifactorial. The “classic” form of NEC is highly associated with prematurity; intestinal barrier immaturity, immature immune response, and an immature regulation of intestinal blood flow (Fig.). Although genetics appears to play a role, the environment, especially a dysbiotic intestinal microbiota acting in concert with host immaturities predisposes the preterm infant to disruption of the intestinal epithelia, increased permeability of tight junctions, and release of inflammatory mediators that leads to intestinal mucosa injury and therefore development of necrotizing enterocolitis.

NEC is a multifactorial disease

NEC is a multifactorial disease

What causes NEC? NEC is a multifactorial disease with an interaction of several etiophathologies

It is clear from this review that there are several entities that have been described as NEC. What is also clear is that despite having some overlap in the final parts of the pathophysiologic cascade that lead to necrosis, the disease that is most commonly seen in the preterm infant is likely to have an origin that differs markedly from that seen in term infants with congenital heart disease or severe hypoxic–ischemic injury. Thus, epidemiologic studies will need to differentiate these entities, if the aim is to dissect common features that are most highly associated with development of the disease. At this juncture, we areleft with more of a population based preventative approach, where the use of human milk, evidence based feeding guide-lines, considerations for microbial therapy once these are proved safe and effective and approved as such by regulatory authorities, and perhaps even measures that prevent prematurity will have a major impact on this devastating disease.

Influenced by the microbiota, intestinal epithelial cells (IECs) elaborate cytokines

Influenced by the microbiota, intestinal epithelial cells (IECs) elaborate cytokines

Influenced by the microbiota, intestinal epithelial cells (IECs) elaborate cytokines, including thymic stromal lymphoprotein (TSLP), transforming growthfactor (TGF), and interleukin-10 (IL-10), that can influence pro-inflammatory cytokine production by dendritic cells (DC) and macrophages present in the laminapropria (GALT) and Peyer’s patches. Signals from commensal organisms may influence tissue-specific functions, resulting in T-cell expansion and regulation of the numbers of Th-1,
Th-2, and Th-3 cells. Also modulated by the microbiota, other IEC derived factors, including APRIL (a proliferation-inducing ligand),B-cell activating factor (BAFF), secretory leukocyte peptidase inhibitor (SLPI), prostaglandin E2(PGE2), and other metabolites, directly regulate functions ofboth antigen presenting cells and lymphocytes in the intestinal ecosystem. NK: natural killer cell; LN: lymph node; DC: dendritic cells.Modified from R. Sharma, C. Young, M. Mshvildadze, J. Neu, Intestinal microbiota does it play a role in diseases of the neonate? NeoReviews 10 (4) (2009)e166, with permission

Cross-talk between monocyte.macrophage cells and T.NK lymphocytes

Cross-talk between monocyte.macrophage cells and T.NK lymphocytes

Current Issues in the Management of Necrotizing Enterocolitis

Marion C. W. Henry and R. Lawrence Moss
Seminars in Perinatology, 2004; 28(3): 221-233
http://dx.doi.org:/10.1053/j.semperi.2004.03.010

Necrotizing enterocolitis is almost exclusively a disease of prematurity, with 90% of all cases occurring in premature infants and 90% of those infants weighing less than 2000 g. Prematurity is the only risk factor for necrotizing enterocolitis consistently identified in case control studies and the disease is rare in countries where prematurity is uncommon such as Japan and Sweden. When necrotizing enterocolitis does occur in full-term infants, it appears to by a somewhat different disease, typically associated with some predisposing condition.

NEC occurs in one to three in 1,000 live births and most commonly affects babies born between 30-32 weeks. It is most often diagnosed during the second week of life and occurs more often in previously fed infants. The mortality from NEC has been cited as 10% to 50% of all NEC cases. Surgical mortality has decreased over the last several decades from 70% to between 20 and 50%. The incremental cost per case of acute hospital care is estimated at $74 to 186 thousand compared to age matched controls, not including additional costs of long term care for the infants’ with lifelong morbidity. Survivors may develop short bowel syndrome, recurrent bouts of catheter-related sepsis, malabsorption, malnutrition, and TPN induced liver failure.

Although extensive research concerning the pathophysiology of necrotizing enterocolitis has occurred, a complete understanding has not been fully elucidated. The classic histologic finding is coagulation necrosis; present in over 90% of specimens. This finding suggests the importance of ischemia in the pathogenesis of NEC. Inflammation and bacterial overgrowth also are present. These findings support the assumptions by Kosloske that NEC occurs by the interaction of 3 events:

  • intestinal ischemia,
  • colonization by pathogenic bacteria and
  • excess protein substrate in the intestinal lumen.

Additionally, the immunologic immaturity of the neonatal gut has been implicated in the development of NEC. Reparative tissue changes including epithelial regeneration, formation of granulation tissue and fibrosis, and mixed areas of acute and chronic inflammatory changes suggest that the pathogenesis of NEC may involve a chronic process of injury and repair.

Premature newborns born prior to the 32nd week of gestational age may have compromised intestinal peristalsis and decreased motility. These motility problems may lead to poor clearance of bacteria, and subsequent bacterial overgrowth. Premature infants also have an immature intestinal tract in terms of immunologic immunity.

There are fewer functional B lymphocytes present and the ability to produce sufficient secretory IgA is reduced. Pepsin, gastric acid and mucus are also not produced as well in prematurity. All of these factors may contribute to the limited proliferation of intestinal flora and the decreased binding of these flora to mucosal cells (Fig).

Role of nitric oxide in the pathogenesis of NEC

Role of nitric oxide in the pathogenesis of NEC

Role of nitric oxide in the pathogenesis of NEC.

Characteristics of the immature gut leading to increased risk of necrotizing enterocolitis

Characteristics of the immature gut leading to increased risk of necrotizing enterocolitis

Characteristics of the immature gut leading to increased risk of necrotizing enterocolitis.

As understanding of the pathophysiology of necrotizing enterocolitis continues to evolve, a unifying concept is emerging. Initially, there is likely a subclinical insult leading to NEC. This may arise from a brief episode of hypoxia or infection. With colonization of the intestines, bacteria bind to the injured mucosa eliciting an inflammatory response which leads to further inflammation.

Intestinal Microbiota Development in Preterm Neonates and Effect of Perinatal Antibiotics

Silvia Arboleya, Borja Sanchez,, Christian Milani, Sabrina Duranti, et al.
Pediatr 2014;-:—).  http://dx.doi.org/10.1016/j.jpeds.2014.09.041

Objectives Assess the establishment of the intestinal microbiota in very low birth-weight preterm infants and to evaluate the impact of perinatal factors, such as delivery mode and perinatal antibiotics.
Study design We used 16S ribosomal RNA gene sequence-based microbiota analysis and quantitative polymerase chain reaction to evaluate the establishment of the intestinal microbiota. We also evaluated factors affecting the microbiota, during the first 3 months of life in preterm infants (n = 27) compared with full-term babies (n = 13).
Results Immaturity affects the microbiota as indicated by a reduced percentage of the family Bacteroidaceae during the first months of life and by a higher initial percentage of Lactobacillaceae in preterm infants compared with full term infants. Perinatal antibiotics, including intrapartum antimicrobial prophylaxis, affects the gut microbiota, as indicated by increased Enterobacteriaceae family organisms in the infants.

Human gut microbiome

Human gut microbiome

Conclusions Prematurity and perinatal antibiotic administration strongly affect the initial establishment of microbiota with potential consequences for later health.

Ischemia and necrotizing enterocolitis: where, when, and how

Philip T. Nowicki
Seminars in Pediatric Surgery (2005) 14, 152-158
http://dx.doi.org:/10.1053/j.sempedsurg.2005.05.003

While it is accepted that ischemia contributes to the pathogenesis of necrotizing enterocolitis (NEC), three important questions regarding this role subsist. First, where within the intestinal circulation does the vascular pathophysiology occur? It is most likely that this event begins within the intramural microcirculation, particularly the small arteries that pierce the gut wall and the submucosal arteriolar plexus insofar as these represent the principal sites of resistance regulation in the gut. Mucosal damage might also disrupt the integrity or function of downstream villous arterioles leading to damage thereto; thereafter, noxious stimuli might ascend into the submucosal vessels via downstream venules and lymphatics. Second, when during the course of pathogenesis does ischemia occur? Ischemia is unlikely to the sole initiating factor of NEC; instead, it is more likely that ischemia is triggered by other events, such as inflammation at the mucosal surface. In this context, it is likely that ischemia plays a secondary, albeit critical role in disease extension. Third, how does the ischemia occur? Regulation of vascular resistance within newborn intestine is principally determined by a balance between the endothelial production of the vasoconstrictor peptide endothelin-1 (ET-1) and endothelial production of the vasodilator free radical nitric oxide (NO). Under normal conditions, the balance heavily favors NO-induced vasodilation, leading to a low resting resistance and high rate of flow. However, factors that disrupt endothelial cell function, eg, ischemia-reperfusion, sustained low-flow perfusion, or proinflammatory mediators, alter the ET-1:NO balance in favor of constriction. The unique ET-1–NO interaction thereafter might facilitate rapid extension of this constriction, generating a viscous cascade wherein ischemia rapidly extends into larger portions of the intestine.

Schematic representation of the intestinal microcirculation

Schematic representation of the intestinal microcirculation

Schematic representation of the intestinal microcirculation. Small mesenteric arteries pierce the muscularis layers and terminate in the submucosa where they give rise to 1A (1st order) arterioles. 2A (2nd order) arterioles arise from the 1A. Although not shown here, these 2A arterioles connect merge with several 1A arterioles, thus generating an arteriolar plexus, or manifold that serves to pressurize the terminal downstream microvasculature. 3A (3rd order) arterioles arise from the 2A and proceed to the mucosa, giving off a 4A branch just before descent into the mucosa. This 4A vessel travels to the muscularis layers. Each 3A vessel becomes the single arteriole perfusing each villus.

Collectively, these studies indicate that disruption of endothelial cell function has the potential to disrupt the normal balance between NO and ET-1 within the newborn intestinal circulation, and that such an event can generate significant ischemia. In this context, it is important to note that NO and ET-1 each regulate the expression and activity of the other. An increased [NO] within the microvascular environment reduces ET-1 expression and compromises ligand binding to the ETA receptor (thus decreasing its contractile efficacy), while ET-1 compromises eNOS expression. Thus, factors that upset the balance between NO and ET-1 will have an immediate and direct effect on vascular tone, but also exert an additional indirect effect by extenuating the disruption of balance between these two factors.

It is not difficult to construct a hypothesis that links the perturbations of I/R and sustained low-flow perfusion with an initial inflammatory insult. Initiation of an inflammatory process at the mucosal–luminal interface could have a direct impact on villus and mucosal 3A arterioles, damaging arteriolar integrity and disrupting villus hemodynamics. Ascent of proinflammatory mediators to the submucosal 1A–2A arteriolar plexus could occur via draining venules and lymphatics, generating damage to vascular effector systems therein; these mediators might include cytokines and platelet activating factor, as these elements have been recovered from human infants with NEC. This event, coupled with a generalized loss of 3A flow throughout a large portion of the mucosal surface, could compromise flow rate within the submucosal arteriolar plexus.

Necrotizing enterocolitis: An update

Loren Berman, R. Lawrence Moss
Seminars in Fetal & Neonatal Medicine 16 (2011) 145e150
http://dx.doi.org:/10.1016/j.siny.2011.02.002

Necrotizing enterocolitis (NEC) is a leading cause of death among patients in the neonatal intensive care unit, carrying a mortality rate of 15e30%. Its pathogenesis is multifactorial and involves an over reactive response of the immune system to an insult. This leads to increased intestinal permeability, bacterial translocation, and sepsis. There are many inflammatory mediators involved in this process, but thus far none has been shown to be a suitable target for preventive or therapeutic measures. NEC usually occurs in the second week of life after the initiation of enteral feeds, and the diagnosis is made based on physical examination findings, laboratory studies, and abdominal radiographs. Neonates with NEC are followed with serial abdominal examinations and radiographs, and may require surgery or primary peritoneal drainage for perforation or necrosis. Many survivors are plagued with long term complications including short bowel syndrome, abnormal growth, and neurodevelopmental delay. Several evidence-based strategies exist that may decrease the incidence of NEC including promotion of human breast milk feeding, careful feeding advancement, and prophylactic probiotic administration in at-risk patients. Prevention is likely to have the greatest impact on decreasing mortality and morbidity related to NEC, as little progress has been made with regard to improving outcomes for neonates once the disease process is underway.

Immune Deficiencies

Primary immunodeficiencies: A rapidly evolving story

Nima Parvaneh, Jean-Laurent Casanova,  LD Notarangelo, ME Conley
J Allergy Clin Immunol 2013;131:314-23.
http://dx.doi.org/10.1016/j.jaci.2012.11.051

The characterization of primary immunodeficiencies (PIDs) in human subjects is crucial for a better understanding of the biology of the immune response. New achievements in this field have been possible in light of collaborative studies; attention paid to new phenotypes, infectious and otherwise; improved immunologic techniques; and use of exome sequencing technology. The International Union of Immunological Societies Expert Committee on PIDs recently reported on the updated classification of PIDs. However, new PIDs are being discovered at an ever-increasing rate. A series of 19 novel primary defects of immunity that have been discovered after release of the International Union of Immunological Societies report are discussed here. These new findings highlight the molecular pathways that are associated with clinical phenotypes and suggest potential therapies for affected patients.

Combined Immunodeficiencies

  • T-cell receptor a gene mutation: T-cell receptor ab1 T-cell depletion

T cells comprise 2 distinct lineages that express either ab or gd T-cell receptor (TCR) complexes that perform different tasks in immune responses. During T-cell maturation, the precise order and efficacy of TCR gene rearrangements determine the fate of the cells. Productive β-chain gene rearrangement produces a pre-TCR on the cell surface in association with pre-Tα invariant peptide (β-selection). Pre-TCR signals promote α-chain recombination and transition to a double-positive stage (CD41CD81). This is the prerequisite for central tolerance achieved through positive and negative selection of thymocytes.

  • Ras homolog gene family member H deficiency: Loss of naive T cells and persistent human papilloma virus infections
  • MST1 deficiency: Loss of naive T cells

New insight into the role of MST1 as a critical regulator of T-cell homing and function was provided by the characterization of 8 patients from 4 unrelated families who had homozygous nonsense mutations in STK4, the gene encoding MST1. MST1 was originally identified as an ubiquitously expressed kinase with structural homology to yeast Ste. MST1 is the mammalian homolog of the Drosophila Hippo protein, controlling cell growth, apoptosis, and tumorigenesis. It has both proapoptotic and antiapoptotic functions.

  • Lymphocyte-specific protein tyrosine kinase deficiency: T-cell deficiency with CD41 lymphopenia

Defects in pre-TCR– and TCR-mediated signaling lead to aberrant T-cell development and function (Fig). One of the earliest biochemical events occurring after engagement of the (pre)-TCR is the activation of lymphocyte-specific protein tyrosine kinase (LCK), a member of the SRC family of protein tyrosine kinases. This kinase then phosphorylates immunoreceptor tyrosine-based activation motifs of intracellular domains of CD3 subunits. Phosphorylated immunoreceptor tyrosine-based activation motifs recruit z-chain associated protein kinase of 70 kDa, which, after being phosphorylated by LCK, is responsible for activation of critical downstream events. Major consequences include activation of the membrane-associated enzyme phospholipase Cg1, activation of the mitogen-activated protein kinase, nuclear translocation of nuclear factor kB (NFkB), and Ca21/Mg21 mobilization. Through these pathways, LCK controls T-cell development and activation. In mice lacking LCK, T-cell development in the thymus is profoundly blocked at an early double-negative stage.

TCR signaling

TCR signaling

TCR signaling. Multiple signal transduction pathways are stimulated through the TCR. These pathways collectively activate transcription factors that organize T-cell survival, proliferation, differentiation, homeostasis, and migration. Mutant molecules in patients with TCR-related defects are indicated in red.

  • Uncoordinated 119 deficiency: Idiopathic CD41 lymphopenia

Idiopathic CD41 lymphopenia (ICL) is a very heterogeneous clinical entity that is defined, by default, by persistent CD41 T-cell lymphopenia (<300 cells/mL or <20% of total T cells) in the absence of HIV infection or any other known cause of immunodeficiency.

Well-Defined Syndromes with Immunodeficiency

  • Wiskott-Aldrich syndrome protein–interacting protein deficiency: Wiskott-Aldrich syndrome-like phenotype

In hematopoietic cells Wiskott-Aldrich syndrome protein (WASP) is stabilized through forming a complex with WASP interacting protein (WIP).

  • Phospholipase Cg2 gain-of-function mutations: Cold urticaria, immunodeficiency, and autoimmunity/autoinflammatory

This is a unique phenotype, sharing features of antibody deficiency, autoinflammatory diseases, and immune dysregulatory disorders, making its classification difficult. Two recent studies validated the pleiotropy of genetic alterations in the same gene.

Predominantly Antibody Defects

  • Defect in the p85a subunit of phosphoinositide 3-kinase: Agammaglobulinemia and absent B cells
  • CD21 deficiency: Hypogammaglobulinemia
  • LPS-responsive beige-like anchor deficiency:
  • Hypogammaglobulinemia with autoimmunity and

early colitis

Defects Of Immune Dysregulation

  • Pallidin deficiency: Hermansky-Pudlak syndrome type 9
  • CD27 deficiency: Immune dysregulation and
  • persistent EBV infection

Congenital Defects Of Phagocyte Number, Function, Or Both

  • Interferon-stimulated gene 15 deficiency: Mendelian susceptibility to mycobacterial diseases

Defects In Innate Immunity

  • NKX2-5 deficiency: Isolated congenital asplenia
  • Toll/IL-1 receptor domain–containing adaptor inducing IFN-b and TANK-binding kinase 1 deficiencies: Herpes simplex encephalitis
  • Minichromosome maintenance complex component 4 deficiency: NK cell deficiency associated with growth retardation and adrenal insufficiency

Autoinflammatory Disorders

  • A disintegrin and metalloproteinase 17 deficiency: Inflammatory skin and bowel disease

 

Cross-talk between monocyte.macrophage cells and T.NK lymphocytes

Cross-talk between monocyte.macrophage cells and T.NK lymphocytes

Cross-talk between monocyte/macrophage cells and T/NK lymphocytes. Genes in the IL-12/IFN-g pathway are particularly important for protection against mycobacterial disease. IRF8 is an IFN-g–inducible transcription factor required for the induction of various target genes, including IL-12. The NF-kB essential modulator (NEMO) mutations in the LZ domain impair CD40-NEMO–dependent pathways. Some gp91phox mutations specifically abolish the respiratory burst in monocyte-derived macrophages. ISG15 is secreted by neutrophils and potentiates IFN-g production by NK/T cells. Genetic defects that preclude monocyte development (eg, GATA2) can also predispose to mycobacterial infections (not shown). Mutant molecules in patients with unusual susceptibility to infection are indicated in red.

The field of PIDs is advancing at full speed in 2 directions. New genetic causes of known PIDs are being discovered (eg, CD21 and TRIF). Moreover, new phenotypes qualify as PIDs with the identification of a first genetic cause (eg, generalized pustular psoriasis). Recent findings contribute fundamental knowledge about immune system biology and its perturbation in disease. They are also of considerable clinical benefit for the patients and their families. A priority is to further translate these new discoveries into improved diagnostic methods and more effective therapeutic strategies, promoting the well-being of patients with PIDs.

Primary immunodeficiencies

Luigi D. Notarangelo
J Allergy Clin Immunol 2010; 125(2): S182-194
http://dx.doi.org:/10.1016/j.jaci.2009.07.053

In the last years, advances in molecular genetics and immunology have resulted in the identification of a growing number of genes causing primary immunodeficiencies (PIDs) in human subjects and a better understanding of the pathophysiology of these disorders. Characterization of the molecular mechanisms of PIDs has also facilitated the development of novel diagnostic assays based on analysis of the expression of the protein encoded by the PID-specific gene. Pilot newborn screening programs for the identification of infants with severe combined immunodeficiency have been initiated. Finally, significant advances have been made in the treatment of PIDs based on the use of subcutaneous immunoglobulins, hematopoietic cell transplantation from unrelated donors and cord blood, and gene therapy. In this review we will discuss the pathogenesis, diagnosis, and treatment of PIDs, with special attention to recent advances in the field.

 

 

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Neural Activity Regulating Endocrine Response

Posted in Curation, Disease Biology, Genetics & Innovations in Treatment, Genome Biology, Human Sensation and Cellular Transduction: Physiology and Therapeutics, Innovations in Neurophysiology & Neuropsychology, Neurohumoral Transmission, Neurological Diseases, Pain: Etiology, Genetics & Innovations in Treatment, Pharmaceutical Analytics, Pharmaceutical Discovery, Schizophrenia, Severe Autism, Signaling & Cell Circuits, Small Molecules in Development of Therapeutic Drugs, Social Development, tagged , , , , on February 13, 2015| Leave a Comment »

Neural Activity Regulating Endocrine Response

Writer and Curator: Larry H. Bernstein, MD, FCAP

 

Defensive responses of Brandt’s voles (Lasiopodomys brandtii) to chronic predatory stress

Ibrahim M. Hegab, Guoshen Shang, Manhong Ye, Yajuan, et al.
Physiology & Behavior 126 (2014) 1–7
http://dx.doi.org/10.1016/j.physbeh.2013.12.001

Predator odors are non-intrusive natural stressors of high ethological relevance. The objective of this study was to investigate the processing of a chronic, life-threatening stimulus during repeated prolonged presentation to Brandt’s voles. One hundred and twenty voles were tested by repeated presentation of cat feces in a defensive withdrawal apparatus. Voles exposed to feces for short periods showed more avoidance, more concealment in the hide box, less contact time with the odor source, more freezing behavior, less grooming, more jumping, and more vigilant rearing than did non-exposed voles, and those exposed for longer periods. Serum levels of adrenocortico-tropic hormone and corticosterone increased significantly when animals were repeatedly exposed to cat feces for short periods. The behavioral and endocrine responses  habituated during prolonged presentation of cat feces.  ΔfosB mRNA expression level was highest in voles exposed to cat feces for 6 and 12 consecutive days, and subsequently declined in animals exposed to cat feces for 24 days. We therefore conclude that the behavioral and endocrine responses to repeated exposure to cat feces undergo a process of habituation, while ΔfosB changes in the medial hypothalamic region exhibit sensitization. We propose that habituation and sensitization are complementary rather than contradictory processes that occur in the same individual upon repeated presentation of the same stressor.

Neuroendocrine changes upon exposure to predator odors

Ibrahim M. Hegab, Wanhong Wei
Physiology & Behavior 131 (2014) 149–155
http://dx.doi.org/10.1016/j.physbeh.2014.04.041

Predator odors are non-intrusive and naturalistic stressors of high ethological relevance in animals. Upon exposure to a predator or its associated cues, robust physiological and molecular anti-predator defensive strategies are

elicited thereby allowing prey species to recognize, avoid and defend against a possible predation threat. In this review, we will discuss the nature of neuroendocrine stress responses upon exposure to predator odors. Predator odors can have a profound effect on the endocrine system, including activation of the hypothalamic–pituitary–adrenal axis, and induction of stress hormones such as corticosterone and adrenocorticotropic hormone. On a neural level, short-term exposure to predator odors leads to induction of the c-fos gene, while induction of ΔFosB in a different brain region is detected under chronic predation stress. Future research should aim to elucidate the relationships between neuroendocrine and behavioral outputs to gage the different levels of antipredator responses in prey species.

Involvement of NR1, NR2A different expression in brain regions in anxiety-like behavior of prenatally stressed offspring

Hongli Sun, Ning Jia, Lixia Guan, Qing Su, et al.
Behavioural Brain Research 257 (2013) 1– 7
http://dx.doi.org/10.1016/j.bbr.2013.08.044

Prenatal stress (PS) has been shown to be associated with anxiety. However, the underlying neurological mechanisms are not well understood. To determine the effects of PS on anxiety-like behavior in the adult offspring, we evaluated anxiety-like behavior using open field test (OFT) and elevated plus maze (EPM) in the 3-month offspring. Both male and female offspring showed a significant reduction of crossing counts in the center, total crossing counts, rearing counts and time spent in the center in the OFT, and only male offspring showed a decreased percentage of open-arm entries and open-arm time in open arms in the EPM. Additionally, expression of NR1 and NR2A subunit of N-methyl-d-aspartate receptor (NMDAR) in the hippocampus (HIP), prefrontal cortex (PFC) and striatum (STR) was studied. Our results showed that PS reduced NR1 and NR2A expression in the HIP, NR2A expression in the PFC and STR in the offspring. The altered NR1 and NR2A could have potential impact on anxiety-like behavior in the adult offspring exposed to PS.

Acute serotonergic treatment changes the relation between anxiety and HPA-axis functioning and periaqueductal gray activation

Dietmar Hestermann, Yasin Temel, Arjan Bloklan, Lee Wei Lim
http://dx.doi.org/10.1016/j.bbr.2014.07.003

Serotonergic (5-HT) drugs are widely used in the clinical management of mood and anxiety disorders. However, it is reported that acute 5-HT treatment elicits anxiogenic-like behavior. Interestingly, the periaqueductal gray (PAG), a midbrain structure which regulates anxiety behavior – has robust 5-HT fibers and reciprocal connections with the hypothalamic–pituitary–adrenal (HPA) axis. Although the HPA axis and the 5-HT system are well investigated, the relationship between the stress hormones induced by 5-HT drug treatment
and the PAG neural correlates of the behavior remain largely unknown. In
this study, the effects of acute and chronic treatments with buspirone (BUSP)
and escitalopram (ESCIT) on anxiety related behaviors were tested in an open-
field (OF). The treatment effects on PAG c-Fos immunoreactivity (c-Fos-ir) and corticosterone (CORT) concentration were measured in order to determine the neural endocrine correlates of anxiety-related behaviors and drug treatments. Our results demonstrate that acute BUSP and ESCIT treatments induced anxiogenic behaviors with elevation of CORT compared to the baseline. A decrease of c-Fos-ir was found in the dorsomedial PAG region of both the treatment groups. Correlation analysis showed that the CORT were not associated with the OF anxiogenic behavior and PAG c-Fos-ir. No significant differences were found in behaviors and CORT after chronic treatment.
In conclusion, acute BUSP and ESCIT treatments elicited anxiogenic response with activation of the HPA axis and reduction of c-Fos-ir in the dorsomedial PAG. Although no correlation was found between the stress hormone and
the PAG c-Fos-ir, this does not imply the lack of cause-and-effect relationship between neuroendocrine effects and PAG function in anxiety responses. These correlation studies suggest that the regulation of 5-HT system was probably disrupted by acute 5-HT treatment.

Neuroendocrine mechanisms for immune system regulation during stress in fish

Gino Nardocci,, Cristina Navarro, Paula P. Cortes, Monica Imarai
Fish & Shellfish Immunology 40 (2014) 531e538
http://dx.doi.org/10.1016/j.fsi.2014.08.001

In the last years, the aquaculture crops have experienced an explosive and intensive growth, because of the high demand for protein. This growth has increased fish susceptibility to diseases and subsequent death. The constant biotic and abiotic changes experienced by fish species in culture are challenges that induce physiological, endocrine and immunological responses. These changes mitigate stress effects at the cellular level to maintain homeostasis. The effects of stress on the immune system have been studied for many years. While acute stress can have beneficial effects, chronic stress inhibits the immune response in mammals and teleost fish. In response to stress, a signaling cascade is triggered by the activation of neural circuits in the central nervous system because the hypothalamus is the central modulator of stress. This leads to the production of catecholamines, corticosteroid-releasing hormone, adrenocorticotropic hormone and glucocorticoids, which are the essential neuroendocrine mediators for this activation. Because stress situations are energetically demanding, the neuroendocrine signals are involved in metabolic support and will suppress the “less important” immune function.  Understanding the cellular mechanisms of the neuroendocrine regulation of immunity in fish will allow the development of new pharmaceutical strategies and therapeutics for the prevention and treatment of diseases triggered by stress at all stages of fish cultures
for commercial production.

Stress and immune modulation in fish

Lluis Tort
Developmental and Comparative Immunology 35 (2011) 1366–1375
http://dx.doi.org:/10.1016/j.dci.2011.07.002

Stress is an event that most animals experience and that induces a number of responses involving all three regulatory systems, neural, endocrine and immune. When the stressor is acute and short-term, the response pattern is stimulatory and the fish immune response shows an activating phase that specially enhances innate responses. If the stressor is chronic the immune response shows suppressive effects and therefore the chances of an infection may be enhanced. In addition, coping with the stressor imposes an allostatic cost that may interfere with the needs of the immune response. In this paper the mechanisms behind these immunoregulatory changes are reviewed and the role of the main neuroendocrine mechanisms directly affecting the building of the immune response and their consequences are considered.

Stress is a general term proposed by Hans Selye in 1953 (Selye, 1953) applying to a situation in which a person or an animal is subjected to a challenge that may result in a real or symbolic danger for its integrity. The stress response applies to a wide range of physiological mechanisms, including gene and protein changes, metabolism, energetics, immune, endocrine, neural and even behavioral changes that will first try to overcome that situation and then compensate for the imbalances produced by either the stressor or the consequences generated by the first array of responses.

The stress response is a general and widespread reaction in animals and it
may be assumed that this response has common traits along the phylogenetic tree. Thus, responses such as the fight and flight reaction and therefore the repertoire of energetic arrangements to serve the surplus of activity are observed in all animals. For instance, in terms of molecular responses, the increase in heat shock proteins is observed from invertebrates to fish to humans; the induction of acute phase proteins is also a common trait.

Stress and immune response

Stress and immune response

Stress and immune response. Main events regarding the principal hormones and immune mechanisms involved in acute and chronic stress

A variety of immune changes have been described after applying different kinds of stressors in fish. Hence, both activating and suppressive processes have been described following stress episodes, although the majority of changes often result in deleterious effects. Immediate responses during the activation phase enhance innate humoral immunity such as increased levels of lysozyme and C3 proteins after acute stress or the increase of the number of myeloid-type leukocytes in the peritoneum after intraperitoneal bacterial injection. Moreover, glucocorticoid receptor sites increase in head kidney leukocytes after acute handling stress.

Longer term treatments normally show suppressive effects: Sea bass subjected to crowding stress show reduced immunocompetence, as shown by reduced rates of cytotoxicity and chemiluminescence. A decrease of complement activity, lysozyme levels, agglutination activity and antibody titers is observed after 3 days onwards after repeated stress in sea bream. Stress reduces the number of circulating B-lymphocytes, and decreases the antibody response after immunization in vivo.

Effects of cortisol on cell immune responses

Effects of cortisol on cell immune responses

Effects of cortisol on cell immune responses. The arrow indicates permissive and the cross indicates suppressive. Neuroendocrine response to stress after perception by the sensors of the nervous system involves the immediate secretion of corticosteroid releasing hormone (CRH) by the preoptic nucleus of the hypothalamus. The stimulated CRH receptors in the corticotropic cells of the pituitary gland induce release of adrenocorticotropic hormone (ACTH) into the circulation that subsequently stimulates release of cortisol by the head kidney interrenal cells. ACTH as well as melanocyte-stimulating hormone (α-MSH) are derived from cleavage of the pro-opiomelanocortin gene product. In most fishes this hormone releasing sequence is taking place in seconds for CRH, seconds to minutes for ACTH, and minutes for cortisol. Since the effect of corticosteroids is exerted in most tissues, a number of studies looking at the consequences of cortisol release on the immune system have been performed but less work has been done on its precursors.

It is assumed that the nervous system plays a principal role in stress episodes as the main center for sensing the challenge and developing fight-or-flight responses. At the same time, endocrine networks are responsible for a number of responses related to the subsequent reorganization of energetic resources and modification of metabolism. Finally, the immune system is not only activated very early in the time course response but it has been shown to appear as a main partner in the regulatory network that is able to modulate non-specific immediate responses and modify hormonal activity. Therefore, in summary

  • all three regulatory systems have a role in the building of a stress response
    (b) their interaction modulates and fine tunes the initial response to avoid excessive activation and adapting resources to the specific challenge.
    These interactions will not only serve for any particular stress episode but also for adapting and preparing the response for future challenges.

Neural Input Is Critical for Arcuate Hypothalamic Neurons to Mount Intracellular Signaling Responses to Systemic Insulin and Deoxyglucose Challenges in Male Rats: Implications for Communication Within Feeding and Metabolic Control Networks

Arshad M. Khan, Ellen M. Walker, Nicole Dominguez, and Alan G. Watts
Endocrinology 155: 405–416, 2014
http://dx.doi.org:/10.1210/en.2013-1480

The hypothalamic arcuate nucleus (ARH) controls rat feeding behavior in part through peptidergic

neurons projecting to the hypothalamic paraventricular nucleus (PVH). Hindbrain catecholaminergic

(CA) neurons innervate both the PVH and ARH, and ablation of CA afferents to PVH neuroendocrine

neurons prevents them from mounting cellular responses to systemic metabolic challenges such as insulin or 2-deoxy-D-glucose (2-DG). Here, we asked whether ablating CA afferents also limits their ARH responses to the same challenges or alters ARH connectivity with the PVH. We examined ARH neurons for three features:

(1) CA afferents, visualized by dopamine-β-hydroxylase (DBH)– immunoreactivity;

(2) activation by systemic metabolic challenge, as measured by increased numbers of neurons immunoreactive (ir) for phosphorylated ERK1/2 (pERK1/2);

(3) density of PVH-targeted axons immunoreactive for the feeding control peptides Agouti-related peptide and  α-melanocyte-stimulating hormone (αMSH).
Loss of PVH DBH immunoreactivity resulted in concomitant ARH reductions of DBH-ir and pERK1/2-ir neurons in the medial ARH, where AgRP neurons are enriched. In contrast, pERK1/2 immunoreactivity after systemic metabolic challenge was absent in αMSH-ir ARH neurons. Yet surprisingly, axonal αMSH immune-reactivity in the PVH was markedly increased in CA-ablated animals. These results indicate that

(1) intrinsic ARH activity is insufficient to recruit pERK1/2-ir ARH neurons during systemic metabolic challenges (rather, hindbrain-originating CA neurons are required); and

(2) rats may compensate for a loss of CA innervation to the ARH and PVH by increased expression of αMSH.
These findings highlight the existence of a hierarchical dependence for ARH responses to neural and humoral signals that influence feeding behavior and metabolism.

Acute hypernatremia dampens stress-induced enhancement of long-term potentiation in the dentate gyrus of rat hippocampus

Chiung-Chun Huang, Chiao-Yin Chu, Che-Ming Yeh , Kuei-Sen Hsu
Psychoneuroendocrinology (2014) 46, 129—140
http://dx.doi.org/10.1016/j.psyneuen.2014.04.016

Stress often occurs within the context of homeostatic threat, requiring integration of physiological and psychological demands to trigger appropriate behavioral, autonomic and endocrine responses. However, the neural mechanism underlying stress integration remains elusive. Using an acute hypernatremic challenge (2.0 M NaCl subcutaneous), we assessed whether physical state may affect subsequent responsiveness to psychogenic stressors. We found that experienced forced swimming (FS, 15 min in 25 8C), a model of psychogenic stress, enhanced long-term potentiation (LTP) induction in the dentate gyrus (DG) of the rat hippocampus ex vivo. The effect of FS on LTP was prevented when the animals were adrenalectomized or given mineralocorticoid receptor antagonist RU28318 before experiencing stress. Intriguingly, relative to normonatremic controls, hypernatremic challenge effectively elevated plasma sodium concentration and dampened FS-induced enhancement of LTP, which was prevented by adrenalectomy. In addition, acute hypernatremic challenge resulted in increased extracellular signal regulated kinase (ERK)1/2 phosphorylation in the DG and occluded the subsequent activation of ERK1/2 by FS. Moreover, stress response dampening effects by acute hypernatremic challenge remained intact in conditional oxytocin receptor knockout mice. These results suggest that acute hypernatremic challenge evokes a sustained increase in plasma corticosterone concentration,

Long-term dysregulation of brain corticotrophin and glucocorticoid receptors and stress reactivity by single early-life pain experience in male and female rats

Nicole C. Victoria, Kiyoshi Inoue, Larry J. Young, Anne Z. Murphy
Psychoneuroendocrinology (2013) 38, 3015—3028
http://dx.doi.org/10.1016/j.psyneuen.2013.08.013

Inflammatory pain experienced on the day of birth (postnatal day 0: PD0) significantly dampens behavioral responses to stress- and anxiety-provoking stimuli in adult rats. However, to date, the mechanisms by which early life pain permanently alters adult stress responses remain unknown. The present studies examined the impact of inflammatory pain, experienced on the day of birth, on adult expression of receptors or proteins implicated in the activation and termination of the stress response, including corticotrophin releasing factor receptors (CRFR1 and CRFR2) and glucocorticoid receptor (GR). Using competitive receptor autoradiography, we show that Sprague Dawley male and female rat pups administered 1% carrageenan into the intraplantar surface of the hindpaw on the day of birth have significantly decreased CRFR1 binding in the basolateral amygdala and midbrain periaqueductal gray in adulthood. In contrast, CRFR2 binding, which is associated with stress termination, was significantly increased in the lateral septum and cortical amygdala. GR expression, measured with in situ hybridization and immunohistochemistry, was significantly increased in the paraventricular nucleus of the hypothalamus and significantly decreased in the hippocampus of neonatally injured adults. In parallel, acute stress-induced corticosterone release was significantly attenuated and returned to baseline more rapidly in adults injured on PD0 in comparison to controls.
Collectively, these data show that early life pain alters neural circuits that regulate responses to and neuroendocrine recovery from stress, and suggest that pain experienced by infants in the Neonatal Intensive Care Unit may permanently alter future responses to anxiety- and stress provoking stimuli.

The Impact of Ventral Noradrenergic Bundle Lesions on Increased IL-1 in the PVN and Hormonal Responses to Stress in Male Sprague Dawley Rats

Peter Blandino Jr, CM Hueston, CJ Barnum, C Bishop, and Terrence Deak
Endocrinology 154: 2489–2500, 2013
http://dx.doi.org:/10.1210/en.2013-1075

The impact of acute stress on inflammatory signaling within the central nervous system is of interest because these factors influence neuroendocrine function both directly and indirectly. Exposure to certain stressors increases expression of the proinflammatory cytokine, Il-1 in the hypothalamus. Increased IL-1 is reciprocally regulated by norepinephrine (stimulatory) and corticosterone (inhibitory), yet neural pathways underlying increased IL-1 have not been clarified.
These experiments explored the impact of bilateral lesions of the ventral noradrenergic bundle (VNAB) on IL-1 expression in the paraventricular nucleus of the hypothalamus (PVN) after foot shock. Adult male Sprague Dawley rats received bilateral 6-hydroxydopamine lesions of the VNAB (VNABx) and were exposed to intermittent foot shock. VNABx depleted approximately 64% of norepinephrine in the PVN and attenuated the IL-1 response produced by foot shock. However, characterization of the hypothalamic-pituitary-adrenal response, a crucial prerequisite for interpreting the effect of VNABx on IL-1 expression, revealed a profound dissociation between ACTH and corticosterone.

Specifically, VNABx blocked the intronic CRH response in the PVN and the increase in plasma ACTH, whereas corticosterone was unaffected at all time points examined. Additionally, foot shock led to a rapid and profound increase in cyclooxygenase-2 and IL-1 expression within the adrenal glands, whereas more subtle effects were observed in the pituitary gland.

Together the findings were

1) demonstration that exposure to acute stress increased expression of inflammatory factors more broadly throughout the hypothalamic-pituitary-adrenal axis;

2) implication of a modest role for norepinephrine-containing fibers of the VNAB as an upstream regulator of PVN IL-1; and

3) suggestion of an ACTH-independent mechanism controlling the release of corticosterone in VNABx rats.

Stress and trauma: BDNF control of dendritic-spine formation and regression

M.R. Bennett,  J. Lagopoulos
Progress in Neurobiology 112 (2014) 80–99
http://dx.doi.org/10.1016/j.pneurobio.2013.10.005

Chronic restraint stress leads to increases in brain derived neurotrophic factor (BDNF) mRNA and protein in some regions of the brain, e.g. the basal lateral amygdala (BLA) but decreases in other regions such as the CA3 region of the hippocampus and dendritic spine density increases or decreases in line with these changes in BDNF. Given the powerful influence that BDNF has on dendritic spine growth, these observations suggest that the fundamental reason for the direction and extent of changes in dendritic spine density in a particular region of the brain under stress is due to the changes in BDNF there. The most likely cause of these changes is provided by the stress initiated release of steroids, which readily enter neurons and alter gene expression, for example that of BDNF. Of particular interest is how glucocorticoids and mineralocorticoids tend to have opposite effects on BDNF gene expression offering the possibility that differences in the distribution of their receptors and of their downstream effects might provide a basis for the differential transcription of the BDNF genes. Alternatively, differences in the extent of methylation and acetylation in the epigenetic control of BDNF transcription
are possible in different parts of the brain following stress. Although present evidence points to changes in BDNF transcription being the major causal agent for the changes in spine density in different parts of the brain following stress, steroids have significant effects on downstream pathways from the TrkB receptor once it is acted upon by BDNF, including those that modulate the density of dendritic spines. Finally, although glucocorticoids play a canonical role in determining BDNF modulation of dendritic spines, recent studies have shown a role for corticotrophin releasing factor (CRF) in this regard. There is considerable improvement in the extent of changes in spine size and density in rodents with forebrain specific knockout of CRF receptor 1 (CRFR1) even when the glucocorticoid pathways are left intact. It seems then that CRF does have a role to play in determining BDNF control of dendritic spines.

Chronic restraint stress leads to increases in brain derived neurotrophic factor (BDNF) mRNA and protein in some regions of the brain, e.g. the basal lateral amygdala (BLA) but decreases in other regions such as the CA3 region of the hippocampus and dendritic spine density increases or decreases in line with these changes in BDNF. Given the powerful influence that BDNF has on dendritic spine growth, these observations suggest that the fundamental reason for the direction and extent of changes in dendritic spine density in a particular region of the brain under stress is due to the changes in BDNF
there. The most likely cause of these changes is provided by the stress initiated release of steroids, which readily enter neurons and alter gene expression, for example that of BDNF. Of particular interest is how glucocorticoids and mineralocorticoids tend to have opposite effects on BDNF gene expression offering the possibility that differences in the distribution of their receptors and of their downstream effects might provide a basis for the differential transcription of the BDNF genes. Alternatively, differences in the extent of methylation and acetylation in the epigenetic control of BDNF transcription are possible in different parts of the brain following stress.

Structure of the rodent BDNF gene

Structure of the rodent BDNF gene

Structure of the rodent BDNF gene. Exons are represented as boxes and the introns as lines. Numbers of the exons are indicated in Roman numerals. The coding exon (exon IX) contains two polyadenylation sites (poly A). The start codon (ATG) that marks the initiation of transcription is indicated. The red box shows the region of exon IX coding for the pro-BDNF protein. Some exons, like exon II and IX, contain different transcript variants with alternative splice-donor sites. Also shown is part of the BDNF exon IV sequence in adults with adverse infant experiences showing cytosine methylation (M) at three of the 12 CG dinucleotide sites (numbered with superscripts). See Boulle et al. (2012).

Epigenetic mechanism associated with repression and activation of BDNF exon IV transcription.

Epigenetic mechanism associated with repression and activation of BDNF exon IV transcription.

Epigenetic mechanism associated with repression and activation of BDNF exon IV transcription. The BDNF exon IV displays 12 distinct CpG sites, which can be methylated and interact selectively with MeCp2 to form complexes that repress gene transcription (see also Fig. 1). Histone methyltransferases (HMT) are responsible for adding methyl groups at histone tails (Panel A), whereas histone deacetylases (HDAC) remove acetylation at histone tails (Panel B), both processes that repress gene transcription. Moreover, low levels of nicotinamine adenine dinucleotide (NAD) promote DNA methylation at the BDNF locus. BDNF gene activation is associated with increased histone H3 and H4 acetylation, which is mediated by histone acetyl transferase (HAT) activity. DNA demethylation might be facilitated by growth arrest and DNA damage proteins such as Gadd45b. An increased binding of CREB to its specific binding protein, CREB binding protein (CBP), is also associated with an increase in BDNF gene transcription. See Boulle et al. (2012).

signaling and epigenetic pathways in granule neurons of the dentate gyrus

signaling and epigenetic pathways in granule neurons of the dentate gyrus

Schematic representation of the signaling and epigenetic pathways in granule neurons of the dentate gyrus thought to be involved in the consolidation process of memory formation after a psychologically stressful challenge. Activation of NMDAR results in stimulation of the MAPK/ERK signaling cascade, the AC /PKA cascade and the CaMKII cascade. In conjunction with activated GR these signaling cascades result in the activation of MSK and ERK leading to the formation of dual histone acetylation marks along the c-Fos promoter and subsequently induction of gene transcription. Signaling via CREB also leads to the same outcome. The induction of gene transcription is thought to be instrumental in the consolidation of memory formation in various stressful learning events. See Trollope et al. (2012).

Model for G9a-GLP complex transcriptional activity in the hippocampus

Model for G9a-GLP complex transcriptional activity in the hippocampus

Model for G9a/GLP complex transcriptional activity in the hippocampus during fear memory consolidation. Shown (panels A and B) is the role of G9a/GLP in the regulation of chromatin remodeling during long-term memory consolidation. Regulation of histone lysine methylation mediates active and repressive transcriptional regulation of genes in the hippocampus. The
changes in chromatin structure results in transcriptional gene silencing in the hippocampus. H3K9me2 dimethylation is associated with transcriptional silencing (not shown). The G9a/GLP complex methyltransferase is specific for producing this modification. Abbreviations: Ac, acetylation; M, methylation; MLLI, histone H3 lysine 4 methyltransferase (which regulates memory formation); H3K9me2, histone H3 lysine 9 dimethylation; HAT, histone acetyltransferase; G9a/GLP, G9a/G9a-like protein (GLP) complex methyltransferase.

Modification of serotonin reuptake transport, with inhibitors such as fluoxetine, augments BDNF exon I mRNA levels in the BLA as well as in the hippocampus. This augmentation is lost and replaced by a decrease in BDNF levels if the mice are homozygous for the BDNF Val66Met SNP. A better outcome is obtained for erasing fear memories in PTSD subjects than using D-cycloserine if a combination is used of extinction training with chronic fluoxetine treatment that augments BDNF exon I mRNA.

Conclusion

The following points are suggested by the present review on identifying the changes in dendritic spine synapses in neural networks under stress, the mechanisms that drive these, and how these networks can be reinstated to normality.

Dendritic spines and BDNF

Activation of BDNF leads to the sprouting of dendrites in many areas of the brain, such as CA1 in the hippocampus. As glucocorticoids decrease BDNF expression they decrease dendritic spine density in these areas . Thus activation of both GR and MR with corticosterone leads to an increase in dendritic spine turnover on pyramidal neurons in these areas. In other areas of the brain glucocorticoids do not have this.  Extinction of a fear memory, such as, of the negative effects of opiate withdrawal, involves increases of BDNF mRNA and protein in the ventromedial prefrontal cortex, through the action of CREB at histone H3 of the BDNF exon I transcript promoter with acetylation of the histone. This could be enhanced before extinction training with histone deacetylase inhibitors such as trichostatin A or inhibitors such as U0126 of ERK.
Major risk factors for PTSD are low levels of cortisol in the blood immediately after the trauma occasion; and before the trauma, in peripheral blood mononuclear cells, the presence of high GR numbers, low FKBP5 expression, and high levels of GILZ mRNA. All of these risk factors are involved in the action of cytoplasmic GR in modulating gene transduction, including most likely that for the BDNF gene, as well as regulating the capacity for BDNF itself to act. This emphasis on GR in PTSD is enforced by the observations that there is an association between two polymorphisms in the GR gene (N363S and Bcl1) and PTSD as there is between that of FKBP5 and GILZ on the one hand and the capacity of GR to modulate gene function on the other.

Brain-derived neurotrophic factor in the amygdala mediates susceptibility to fear conditioning

Dylan Chou, Chiung-Chun Huang, Kuei-Sen Hsu
Experimental Neurology 255 (2014) 19–29
http://dx.doi.org/10.1016/j.expneurol.2014.02.016

Fear conditioning in animals has been used extensively tomodel clinical anxiety disorders. While individual animals exhibit marked differences in their propensity to undergo fear conditioning, the physiologically relevant mediators have not yet been fully characterized. Here, we demonstrate that C57BL/6 inbred mouse strain subjected to a regimen of chronic social defeat stress (CSDS) can be separated into susceptible and resistant subpopulations that display different levels of fear responses in an auditory fear conditioning  paradigm. Susceptible mice had significantly more c-Fos protein expression
in neurons of the basolateral amygdala (BLA) following CSDS and showed exaggerated conditioned fear responses, while there were no significant differences between groups in innate anxiety- and depressive-like behaviors. Through the use of conditional brain-derived neurotrophic factor (BDNF) knockout strategies, we find that elevated BLA BDNF level following fear conditioning training is a key mediator contributing to determine the levels of conditioned fear responses. Our results also show that relative to susceptible mice, resistant mice had a much faster recovery from conditioned stimuli-induced cardiovascular and corticosterone responses. Systemic administration of norepinephrine reuptake inhibitor atomoxetine increased c-Fos protein expression in BLA neurons following fear conditioning training and promoted the expression of conditioned fear in resistant mice. Conversely, administration of β-adrenergic receptor antagonist propranolol reduced fear conditioning training-induced c-Fos protein expression in BLA neurons and reduced conditioned fear responses in susceptible mice. These findings reveal a novel role for the BDNF signaling within the BLA in mediating individual differences in autonomic, neuroendocrine and behavioral reactivity to fear conditioning.

Melanocortin-4 receptor in the medial amygdala regulates emotional stress-induced anxiety-like behavior, anorexia and corticosterone secretion

Jing Liu, Jacob C. Garza, Wei Li and Xin-Yun Lu
Intl J Neuropsychopharmacology (2013), 16, 105–120.
http://dx.doi.org:/10.1017/S146114571100174X

The central melanocortin system has been implicated in emotional stress-induced anxiety, anorexia and activation of the hypothalamo-pituitary-adrenal (HPA) axis. However, the underlying neural substrates have not been identified. The medial amygdala (MeA) is highly sensitive to emotional stress and expresses high levels of the melanocortin-4 receptor (MC4R). This study investigated the effects of activation and blockade of MC4R in the MeA
on anxiety-like behavior, food intake and corticosterone secretion. We demonstrate that MC4R-expressing neurons in the MeA were activated by acute restraint stress, as indicated by induction of c-fos mRNA expression. Infusion of a selective MC4R agonist into the MeA elicited anxiogenic-like effects in the elevated plus-maze test and decreased food intake. Local MeA infusion of SHU 9119, an MC4R antagonist, on the other hand, blocked restraint stress-induced anxiogenic and anorectic effects. Moreover, plasma corticosterone levels were increased by intra-MeA infusion of the MC4R agonist under non-stressed conditions and restraint stress-induced elevation of plasma corticosterone levels was attenuated by pretreatment with SHU 9119 in the MeA. Thus, stimulating MC4R in the MeA induces stress-like anxiogenic and anorectic effects as well as activation of the HPA axis, whereas antagonizing MC4R in this region blocks such effects induced by restraint stress. Together, our results implicate MC4R signaling in the MeA in behavioral and endocrine responses to stress.

The neuroendocrine functions of the parathyroid hormone 2 receptor

Arpád Dobolyi, Eugene Dimitrov, Miklós Palkovits and Ted B. Usdin
Front in Endocr Oct 2012 | Volume 3 | Article 121, 1-10
http://dx.doi.org:/10.3389/fendo.2012.00121

The G-protein coupled parathyroid hormone 2 receptor (PTH2R) is concentrated in endocrine and limbic regions in the forebrain. Its endogenous ligand, tuberoinfundibular peptide of 39 residues (TIP39), is synthesized in only two brain regions, within the posterior thalamus and the lateral pons.TIP39-expressing neurons have a widespread projection pattern, which matches the PTH2R distribution in the brain. Neuroendocrine centers including the preoptic area, the periventricular, paraventricular, and arcuate nuclei contain the highest density of PTH2R-positive networks. The administration of TIP39 and an antagonist of the PTH2R as well as the investigation of mice that lack functional TIP39 and PTH2R revealed the involvement of the PTH2R in a variety of neural and neuroendocrine functions. TIP39 acting via the PTH2R modulates several aspects of the stress response. It evokes corticosterone release by activating corticotropin-releasing hormone-containing neurons in the hypothalamic paraventricular nucleus. Block of TIP39 signaling elevates the anxiety state
of animals and their fear response, and increases stress-induced analgesia.

TIP39 has also been suggested to affect the release of additional pituitary hormones including arginine-vasopressin and growth hormone. A role of the TIP39-PTH2R system in thermoregulation was also identified. TIP39 may play
a role in maintaining body temperature in a cold environment via descending excitatory pathways from the preoptic area. Anatomical and functional studies also implicated the TIP39-PTH2R system in nociceptive information processing. Finally, TIP39 induced in postpartum dams may play a role in the release of prolactin during lactation. Potential mechanisms leading to the activation ofTIP39 neurons and how they influence the neuroendocrine system are also described. The unique TIP39-PTH2R neuromodulator system provides the possibility for developing drugs with a novel mechanism of action to control neuroendocrine disorders.

Interaction of the Serotonin Transporter-Linked Polymorphic Region and Environmental Adversity: Increased Amygdala-Hypothalamus Connectivity as a Potential Mechanism Linking Neural and Endocrine Hyperreactivity

Nina Alexander, T Klucken, G Koppe, R Osinsky, B Walter, et al.
Biol Psychiatry 2012;72:49–56
http://dx.doi.org:/10.1016/j.biopsych.2012.01.030

Background: Gene by environment (GE) interaction between genetic variation in the promoter region of the serotonin transporter gene (serotonin transporter-linked polymorphic region [5-HTTLPR]) and stressful life events (SLEs) has been extensively studied in the context of depression. Recent findings suggest increased neural and endocrine stress sensitivity as a possible mechanism conveying elevated vulnerability to psychopathology. Furthermore, these GE mediated alterations very likely reflect interrelated biological processes. Methods: In the present functional magnetic resonance imaging study, amygdala reactivity to fearful stimuli was assessed in healthy male adults (n[1]44),who were previously found to differ with regard to endocrine stress reactivity as a function of 5-HTTLPRSLEs. Furthermore, functional connectivity between the amygdala and the hypothalamus was measured as a potential mechanism linking elevated neural and endocrine responses during stressful/threatening situations. The study sample was carefully preselected regarding 5-HTTLPR genotype and SLEs. Results: We report significant GE interaction on neural response patterns and functional amygdala-hypothalamus connectivity. Homozygous carriers of the 5-HTTLPR S’ allele with a history of SLEs (S’S’/high SLEs group) displayed elevated bilateral amygdala activation in response to fearful faces. Within the same sample, a comparable GE interaction effect has previously been demonstrated regarding increased cortisol reactivity, indicating a cross-validation of heightened biological stress sensitivity. Furthermore, S’S’/high SLEs subjects were characterized by an increased functional coupling between the right amygdala and the hypothalamus, thus indicating a potential link between neural and endocrine hyperreactivity.

Amygdala reactivity to fearful faces as a function of the serotonin transporter-linked polymorphic region (5-HTTLPR)

Amygdala reactivity to fearful faces as a function of the serotonin transporter-linked polymorphic region (5-HTTLPR)

Amygdala reactivity to fearful faces as a function of the serotonin transporter-linked polymorphic region (5-HTTLPR) stressful life events (SLEs). The color bar depicts t values for the gene by environment interaction effect. For illustration reasons, the data were thresholded with a t value at 2.5 (see color bar for exact t values).

We report a significant 5-HTTLPRxSLEs interaction effect on bilateral amygdala reactivity to fearful faces in a sample of healthy male adults. As hypothesized, S’S’/high SLEs individuals appeared to be most reactive, which can be interpreted in terms of elevated amygdala reactivity to briefly presented (phasic) aversive stimuli. Interestingly, we have observed a similar response pattern regarding cortisol reactivity to acute stress within the same sample, indicating a cross-validation of neuroendocrine hyperreactivity to threatening/stressful stimuli as a function of 5-HTTLPRxSLEs.

Thus, our results are in line with findings from a small sample sized (n = 15) study reporting a positive association between amygdala reactivity to fearful faces and SLEs in S allele carriers during an unconscious fear processing condition. In contrast, a study using a comparable paradigm and sample size (n = 44) to our own found amygdala activity in the contrast neutral faces versus fixation to be negatively associated with SLEs in S allele carriers. The authors interpret the latter finding in support of a tonic model, by which SLEs interact with 5-HTTLPR on amygdala resting activation. Similar inconsistencies have been reported regarding the association of 5-HTTLPR and amygdala activation independent of environmental adversity, with studies supporting either a phasic or tonic model. Likewise, increased resting blood perfusion in S allele carriers has been reported in independent studies, whereas the largest study
to date could not replicate these findings.

Functional connectivity between the right amygdala as the seed region

Functional connectivity between the right amygdala as the seed region

  • Functional connectivity between the right amygdala as the seed region

(blue circle, right figure) and the hypothalamus (red circles). The middle figure depicts significant differences in activation patterns between the S’S’/high stressful life events (SLEs) and the L’/low SLEs groups and the left figure displays significant differences between S’S’/high SLEs and S’S’/high SLEs subjects. For illustration reasons, threshold was t =2.5 b (below).
(B) Surface plot of functional connectivity at the z-slice location of the peak coordinate. Voxel intensities are given in t values. 5-HTTLPR, serotonin-transporter-linked polymorphic region.

In conclusion, we report increased amygdala responsivity to aversive stimuli in healthy S’S’/high SLEs subjects who have previously been shown to display elevated cortisol secretion in response to psychosocial stress. Thus, our findings contribute to the current debate on potential mechanisms mediating susceptibility for the development of psychiatric disorders as a function of 5-HTTLPRxSLEs. Moreover, the present study extends previous findings by demonstrating altered functional coupling between the amygdala and the hypothalamus, thus indicating a potential link between threat/stress related neural and endocrine alterations associated with 5-HTTLPR x SLEs.

Identifying Molecular Substrates in a Mouse Model of the Serotonin Transporter Environment Risk Factor for Anxiety and Depression

 

Valeria Carola, Giovanni Frazzetto, Tiziana Pascucci, Enrica Audero, et al.
Biol Psychiatry 2008;63:840–846
http://dx.doi.org:/10.1016/j.biopsych.2007.08.013

Background: A polymorphism in the serotonin transporter (5-HTT) gene modulates the association between adverse early experiences and risk for major depression in adulthood. Although human imaging studies have begun to elucidate the neural circuits involved in the 5-HTT environment risk factor, a molecular understanding of this phenomenon is lacking. Such an understanding might help to identify novel targets for the diagnosis and therapy of mood disorders. To address this need, we developed a gene-environment screening paradigm in the mouse.

Methods: We established a mouse model in which a heterozygous null mutation in 5-HTT moderates the effects of poor maternal care on adult anxiety and depression-related behavior. Biochemical analysis of brains from these animals was performed to identify molecular substrates of the gene, environment, and gene environment effects.

Results: Mice experiencing low maternal care showed deficient ϒ-aminobutyric acid–A receptor binding in the amygdala and 5-HTT  heterozygous null mice showed decreased serotonin turnover in hippocampus and striatum. Strikingly, levels of brain-derived neurotrophic factor (BDNF) messenger RNA in hippocampus were elevated exclusively in 5-HTT heterozygous null mice experiencing poor maternal care, suggesting that developmental programming of hippocampal circuits might underlie the 5-HTT environment risk factor.

Conclusions: These findings demonstrate that serotonin plays a similar role in modifying the long-term behavioral effects of rearing environment in diverse mammalian species and identifies BDNF  as a molecular substrate of this risk factor. In summary, we have produced a mouse model of the 5-HTT environment risk factor for human depression and have used this model to identify molecular substrates underlying this risk factor.

Elevated GABA-A receptor expression in amygdala, decreased 5-HT turnover in hippocampus, and enhanced BDNF expression in hippocampus each correlated significantly with the behavioral phenotype seen in our mice. In particular, increased expression of BDNF in CA1 pyramidal neurons was found in mice with reduced 5-HTT function and exposed to low maternal care. This defect was accompanied by an increased bias in the response to threatening cues as assessed by ambiguous cue fear conditioning.

Our data suggest that alterations in hippocampal gene expression and function underlie at least part of the interaction between 5-HTT and rearing environment and point to a role for this structure in the increased anxiety and depression-related behavior that is a risk factor for major depression.

Gene—environment interactions predict cortisol responses after acute stress: Implications for the etiology of depression

Nina Alexander, Yvonne Kuepper, Anja Schmitz, Roman Osinsky, et al.
Psychoneuroendocrinology (2009) 34, 1294—1303
http://dx.doi.org:/10.1016/j.psyneuen.2009.03.017

Background: Growing evidence suggests that the serotonin transporter polymorphism (5-HTTLPR) interacts with adverse environmental influences to produce an increased risk for the development of depression while the underlying mechanisms of this association remain largely unexplored. As one potential intermediate phenotype, we investigated alterations of hypothalamic—pituitary—adrenal (HPA) axis responses to stress in individuals with no history of psychopathology depending on both 5-HTTLPR and stressful life events.

Methods: Healthy male adults (N = 100) were genotyped and completed a questionnaire on severe stressful life events (Life Events Checklist). To test for gene-by-environment interactions on endocrine stress reactivity, subjects were exposed to a standardized laboratory stress task (Public Speaking). Saliva cortisol levels were obtained at 6 time points prior to the stressor and during an extended recovery period.

Results: Subjects homozygous for the s-allele with a significant history of stressful life events exhibited markedly elevated cortisol secretions in response to the stressor compared to all other groups, indicating a significant gene-by-environment interaction on endocrine stress reactivity. No main effect of either 5-HTTLPR (biallelic and triallelic) or stressful life events on cortisol secretion patterns appeared.

Conclusion: This is the first study reporting that 5-HTTLPR and stressful life events interact to predict endocrine stress reactivity in a non-clinical sample. Our results underpin the potential moderating role of HPA-axis hyper-reactivity as a premorbid risk factor to increase the vulnerability for depression in subjects with low serotonin transporter efficiency and a history of severe life events.

The immune system and developmental programming of brain and behavior

Staci D. Bilbo, Jaclyn M. Schwarz
Frontiers in Neuroendocrinology 33 (2012) 267–286
http://dx.doi.org/10.1016/j.yfrne.2012.08.006

The brain, endocrine, and immune systems are inextricably linked. Immune molecules have a powerful impact on neuroendocrine function, including hormone–behavior interactions, during health as well as sickness. Similarly, alterations in hormones, such as during stress, can powerfully impact immune function or reactivity. These functional shifts are evolved, adaptive responses that organize changes in behavior and mobilize immune resources, but can also lead to pathology or exacerbate disease if prolonged or exaggerated. The developing brain in particular is exquisitely sensitive to both endogenous and exogenous signals, and increasing evidence suggests the immune system has a critical role in brain development and associated behavioral outcomes for the life of the individual. Indeed, there are associations between many neuropsychiatric disorders and immune dysfunction, with a distinct etiology in neurodevelopment. The goal of this review is to describe the important role of the immune system during brain development, and to discuss some of the many ways in which immune activation during early brain development can affect the later-life outcomes of neural function, immune function, mood and cognition.

Neuroplasticity signaling pathways linked to the pathophysiology of schizophrenia

Darrick T. Balua, Joseph T. Coyle
Neuroscience and Biobehavioral Reviews 35 (2011) 848–870
http://dx.doi.org:/10.1016/j.neubiorev.2010.10.005

Schizophrenia is a severe mental illness that afflicts nearly 1% of the world’s population. One of the cardinal pathological features of schizophrenia is perturbation in synaptic connectivity. Although the etiology of schizophrenia is unknown, it appears to be a developmental disorder involving the interaction of a potentially large number of risk genes, with no one gene producing a strong effect except rare, highly penetrant copy number variants. The purpose of this review is to detail how putative schizophrenia risk genes (DISC-1, neuregulin/ErbB4, dysbindin, Akt1, BDNF, and the NMDA receptor) are involved in regulating neuroplasticity and how alterations in their expression may contribute to the disconnectivity observed in schizophrenia. Moreover, this review highlights how many of these risk genes converge to regulate common neurotransmitter systems and signaling pathways. Future studies aimed at elucidating the functions of these risk genes will provide new insights into the pathophysiology of schizophrenia and will likely lead to the nomination of novel therapeutic targets for restoring proper synaptic connectivity in the brain in schizophrenia and related disorders.

Glutamate receptor composition of the post-synaptic density is altered in genetic mouse models of NMDA receptor hypo- and hyperfunction

Darrick T. Balu, Joseph T. Coyle
Brain Research 1392 (2011 ) 1–7
http://dx.doi.org:/10.1016/j.brainres.2011.03.051

The N-methyl-D-aspartate receptor (NMDAR) and α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate receptor (AMPAR) are ionotropic glutamate receptors responsible for excitatory neurotransmission in the brain. These excitatory synapses are found on dendritic spines, with the abundance of receptors concentrated at the postsynaptic density (PSD).
We utilized two genetic mouse models, the serine racemase knockout (SR−/−) and the glycine transporter subtype 1 heterozygote mutant (GlyT1+/−), to determine how constitutive NMDAR hypo- and hyperfunction, respectively, affect the glutamate receptor composition of the PSD in the hippocampus and prefrontal cortex (PFC).

Using cellular fractionation, we found that SR−/− mice had elevated protein levels of NR1 and NR2A NMDAR subunits specifically in the PSD-enriched fraction from the hippocampus, but not from the PFC. There were no changes in the amounts of AMPAR subunits (GluR1, GluR2), or PSD protein of 95 kDa (PSD95) in either brain region. GlyT1+/− mice also had elevated protein expression of NR1 and NR2A subunits in the PSD, as well as an increase in total protein. Moreover, GlyT1+/− mice had elevated amounts of GluR1 and GluR2 in the PSD, and higher total amounts of GluR1. Similar to SR−/− mice, there were no protein changes observed in the PFC. These findings illustrate the complexity of synaptic adaptation to altered NMDAR function.

Interleukin-1 (IL-1): A central regulator of stress responses

Inbal Goshen, Raz Yirmiya
Frontiers in Neuroendocrinology 30 (2009) 30–45
http://dx.doi.org:/10.1016/j.yfrne.2008.10.001

Ample evidence demonstrates that the pro-inflammatory cytokine interleukin-1 (IL-1), produced following exposure to immunological and psychological challenges, plays an important role in the neuroendocrine and behavioral stress responses. Specifically, production of brain IL-1 is an important link in stress induced activation of the hypothalamus-pituitary-adrenal axis and secretion of glucocorticoids, which
mediate the effects of stress on memory functioning and neural plasticity, exerting beneficial effects at low levels and detrimental effects at high levels. Furthermore, IL-1 signaling and the resultant glucocorticoid secretion mediate the development of depressive symptoms associated with exposure to acute and chronic stressors, at least partly via suppression of hippocampal neurogenesis. These findings indicate
that whereas under some physiological conditions low levels of IL-1 promote the adaptive stress responses necessary for efficient coping, under severe and chronic stress conditions blockade of IL-1 signaling can be used as a preventive and therapeutic procedure for alleviating stress-associated neuropathology
and psychopathology.

IL-1 mediates stress-induced activation of the HPA axis

IL-1 mediates stress-induced activation of the HPA axis

IL-1 mediates stress-induced activation of the HPA axis. Immunological and
psychological stressors increase the levels of IL-1 in various brain areas, including
several brain stem nuclei, the hypothalamus and the hippocampus. In turn, IL-1
induces the secretion of CRH from the hypothalamic paraventricular nucleus (PVN),
ACTH from the pituitary and glucocorticoids from the adrenal. Following immunological
stressors, peripheral IL-1 can directly influence brain stem nuclei, such as
the nucleus tractus solitarius (NTS) and ventrolateral medulla (VLM) as well as the
hypothalamus via penetration to adjacent circumventricular organs, (the area
postrema (AP) and the organum vasculosum of the lamina terminalis (OVLT),
respectively). Concomitantly, IL-1 in the periphery can activate vagal afferents,
which innervate and activate the NTS and VLM. These nuclei project to the
hypothalamus, in which the secretion of NE induces further elevation of IL-1 levels,
possibly by microglial activation. Psychological stressors can also activate the NTS
and VLM, either by intrinsic brain circuits or via vagal feedback from physiological
systems (e.g., the cardiovascular system) that are stimulated by the sympathetic
nervous system. Similarly to their role in immunological stress, the NTS and VLM
then elevate hypothalamic IL-1 levels, stimulating the CRH neurons.

The inverted U-shaped effect of IL-1 on memory and plasticity is mediated by glucocorticoids

The inverted U-shaped effect of IL-1 on memory and plasticity is mediated by glucocorticoids

The inverted U-shaped effect of IL-1 on memory and plasticity is mediated by glucocorticoids. The influence of IL-1 on memory and plasticity follows an inverted Ushape pattern, i.e., learning-associated increase in IL-1 levels is needed for memory formation (green), whereas any deviation from the physiological range, either by excess elevation in IL-1 levels or by blockade of IL-1 signaling, results in memory and plasticity impairment (red). Low dose GCs can also facilitate memory, whereas chronic or severe stressors, as well as high GC levels, can impair memory and neural plasticity. Studies on the implications of the interaction between stress, IL-1 and GCs on memory
and plasticity show that IL-1 mediates the detrimental effects of stress on memory, and that GCs are involved in both the detrimental and the beneficial effects of IL-1 on memory formation. Based on these studies, the following model is proposed: stressful stimuli induce an increase in brain IL-1 levels, which in turn contributes to the activation of the HPA axis. Subsequently, the secretion of GCs affects memory and plasticity processes in an inverted U-shaped pattern.

Immune modulation of learning, memory, neural plasticity and neurogenesis

Raz Yirmiya ⇑, Inbal Goshen
Brain, Behavior, and Immunity 25 (2011) 181–213
http://dx.doi.org:/10.1016/j.bbi.2010.10.015

Over the past two decades it became evident that the immune system plays a central role in modulating learning, memory and neural plasticity. Under normal quiescent conditions, immune mechanisms are activated by environmental/psychological stimuli and positively regulate the remodeling of neural circuits, promoting memory consolidation, hippocampal long-term potentiation (LTP) and neurogenesis.
These beneficial effects of the immune system are mediated by complex interactions among brain cells with immune functions (particularly microglia and astrocytes), peripheral immune cells (particularly T cells and macrophages), neurons, and neural precursor cells. These interactions involve the responsiveness of non-neuronal cells to classical neurotransmitters (e.g., glutamate and monoamines) and hormones
(e.g., glucocorticoids), as well as the secretion and responsiveness of neurons and glia to low levels of inflammatory cytokines, such as interleukin (IL)-1, IL-6, and TNFa, as well as other mediators, such as prostaglandins and neurotrophins. In conditions under which the immune system is strongly activated by infection or injury, as well as by severe or chronic stressful conditions, glia and other brain immune cells change their morphology and functioning and secrete high levels of pro-inflammatory
cytokines and prostaglandins. The production of these inflammatory mediators disrupts the delicate balance needed for the neurophysiological actions of immune processes and produces direct detrimental effects on memory, neural plasticity and neurogenesis. These effects are mediated by inflammation induced neuronal hyper-excitability and adrenocortical stimulation, followed by reduced production of neurotrophins and other plasticity-related molecules, facilitating many forms of neuropathology
associated with normal aging as well as neurodegenerative and neuropsychiatric diseases.

It is now firmly established that the immune system can modulate brain functioning and behavioral processes. This modulation is exerted by plasticity are among the most important aspects of brain functioning that are modulated by immune mechanisms. The aim of the present review is to present a comprehensive and integrative view of the complex dual role of the immune system in learning,memory, neural plasticity and neurogenesis. The first part of the review will focus on the physiological
beneficial effects of the immune system under normal, quiescent conditions. Under such conditions, immune mechanisms are activated by environmental/psychological stimuli and positively regulate neuroplasticity and neurogenesis, promoting learning, memory, and hippocampal long-term potentiation (LTP). The second part of the review will focus on the detrimental effects of inflammatory conditions induced by infections and injury as well as severe or chronic stress, demonstrating that under such
conditions the delicate physiological balance between immune and neural processes is disrupted, resulting in neuronal hyperexcitability, hormonal aberrant ions, reduced neurotrophic factors production and suppressed neurogenesis, leading to impairments in learning, memory and neuroplasticity.

A systemic model of the beneficial role of immune processes in behavioral and neural plasticity

A systemic model of the beneficial role of immune processes in behavioral and neural plasticity

A systemic model of the beneficial role of immune processes in behavioral and neural plasticity. Learning, memory and synaptic plasticity involve neural activation of hippocampal circuits by glutamatergic inputs that originate mainly in multiple cortical areas. Long-term memory consolidation also requires emotional (limbic) activation (particularly of the amygdala and hypothalamus), inducing a mild stressful condition, which in turn results in HPA axis and sympathetic nervous system (SNS) stimulation. The peripheral organs that are the targets of these systems (e.g., the adrenal glad, heart, blood vessels and gastrointestinal (GI) tract), in turn, send afferent inputs to the brain that culminate in stimulation of receptors for glucocorticoids, norepinephrine, dopamine and serotonin on hippocampal cells. These inputs are critical for memory consolidation, neural plasticity and neurogenesis. Furthermore, these inputs induce the production of IL-1, and possibly other cytokines, chemokines and immune mediators in the hippocampus, as well as in other brain areas (such as the hypothalamus and brain stem) that are critically important for neurobehavioral plasticity. Moreover, these cytokines, in turn further activate the HPA axis and SNS, thus participating in a brain-to-body-to-brain reverberating feedback loops.

Chemokines and the hippocampus: A new perspective on hippocampal plasticity and vulnerability

Lauren L. Williamson, Staci D. Bilbo
Brain, Behavior,and Immunity 30(2013)186–194
http://dx.doi.org/10.1016/j.bbi.2013.01.077

Chemokines roles within the hippocampus

Chemokines roles within the hippocampus

Chemokines have important roles within the hippocampus and may modulate plasticity and vulnerability within this unique structure. Neuroimmune signaling can occur across the blood-brain-barrier (BBB) via endothelial cells, astrocytes, and microglia within the BBB that recapitulate the immune signal from the periphery by secreting their own cohort of cytokines into the brain. Chemokines recruit cells to sites of injury as well . Microglia receive input from neurons via several membrane-bound and secreted factors, including neuronal CX3CL1 (fractalkine) and its receptor, CX3CR1, on microglia, which allow direct neuroimmune interaction. CXCL12 is released from vesicles concomitantly with GABA from basket cells onto immature neurons in the DG granule cell layer.  In the healthy brain, chemokines may modulate neuronal signaling during behavior, though this phenomenon remains to be explored. The spatial and temporal signaling and cellular sources of chemokines and their receptors are critical for understanding

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Endocrine Action on Midbrain

Posted in Biological Networks, Calcium Signaling, Cell Biology, Cerebrovascular and Neurodegenerative Diseases, Chemical Biology and its relations to Metabolic Disease, Curation, Cytoskeleton, Developmental biology, Diabetes Mellitus, Disease Biology, Embryology, Endocrine Diseases, Genomic Endocrinology, Innovations in Neurophysiology & Neuropsychology, Lipid metabolism, Metabolism, Metabolomics, Neurodegenerative Diseases, Neurological Diseases, Nutrition, Pharmaceutical Analytics, Pharmaceutical Discovery, Pharmacologic toxicities, Preimplantation Genetic Diagnosis and Reproductive Genomics, Proteomics, Reproductive Andrology, Embryology, Genomic Endocrinology, Preimplantation Genetic Diagnosis and Reproductive Genomics, Signaling, Signaling & Cell Circuits, Small Molecules in Development of Therapeutic Drugs, tagged , , , , , , , , , , , on February 12, 2015| Leave a Comment »

Endocrine Action on Midbrain

Writer and Curator: Larry H. Bernstein, MD, FCAP

 
  • Brain’s Role in Browning White Fat
  • Insulin and leptin act on specialized neurons in the mouse hypothalamus to promote conversion of white to beige fat.

By Anna Azvolinsky | January 15, 2015

JUSTIN HEWLETT, MNHS MULTIMEDIA, MONASH UNIVERSITY

Ever since energy-storing white fat has been shown to convert to metabolically active beige fat, through a process called browning, scientists have been trying to understand how this switch occurs. The immune system has been shown to contribute to activation of brown fat cells. Now, researchers from Monash University in Australia and their colleagues have shown that insulin and leptin—two hormones that regulate glucose metabolism and satiety and hunger cues—activate “satiety” neurons in the mouse hypothalamus to promote the conversion of white fat to beige. The results are published today (January 15) in Cell.

Hypothalamic appetite-suppressing proopiomelanocortin (POMC) neurons are known to relay the satiety signals in the bloodstream to other parts of the brain and other tissues to promote energy balance. “What is new here is that one way that these neurons promote calorie-burning is to stimulate the browning of white fat,” said Xiaoyong Yang, who studies the molecular mechanisms of metabolism at the Yale University School of Medicine, but was not involved in the work. “The study identifies how the brain communicates to fat tissue to promote energy dissipation.”

“The authors show that [insulin and leptin] directly interact in the brain to produce nervous-system signaling both to white and brown adipose tissue,” said Jan Nedergaard, a professor of physiology at Stockholm University who also was not involved in the study. “This is a nice demonstration of how the acute and chronic energy status talks to the thermogenic tissues.”

Although the differences between beige and brown fat are still being defined, the former is currently considered a metabolically active fat—which converts the energy of triglycerides into heat—nestled within white fat tissue. Because of their energy-burning properties, brown and beige fat are considered superior to white fat, so understanding how white fat can be browned is a key research question. Exposure to cold can promote the browning of white fat, but the ability of insulin and leptin to act in synergy to signal to the brain to promote browning was not known before this study, according the author Tony Tiganis, a biochemist at Monash.

White fat cells steadily produce leptin, while insulin is produced by cells of the pancreas in response to a surge of glucose into the blood. Both hormones are known to signal to the brain to regulate satiety and body weight. To explore the connection between this energy expenditure control system and fat tissue, Garron Dodd, a postdoctoral fellow in Tiganis’s laboratory, and his colleagues deleted one or both of two phosphatase enzymes in murine POMC neurons. These phosphatase enzymes were previously known to act in the hypothalamus to regulate both glucose metabolism and body weight, each regulating either leptin or insulin signaling. When both phosphatases were deleted, mice had less white fat tissue and increased insulin and leptin signaling.

“These [phosphatase enzymes] work in POMC neurons by acting as ‘dimmer switches,’ controlling the sensitivity of leptin and insulin receptors to their endogenous ligands,” Dodd told The Scientist in an e-mail. The double knockout mice also had an increase in beige fat and more active heat-generating brown fat. When fed a high-fat diet, unlike either the single knockout or wild-type mice, the double knockout mice did not gain weight, suggesting that leptin and insulin signaling to POMC neurons is important for controlling body weight and fat metabolism.

The researchers also infused leptin and insulin directly into the hypothalami of wild-type mice, which promoted the browning of white fat. But when these hormones were infused but the neuronal connections between the white fat and the brain were physically severed, browning was prevented. Moreover, hormone infusion and cutting the neuronal connection to only a single fat pad resulted in browning only in the fat pad that maintained signaling ties to the brain. “This really told us that direct innervation from the brain is necessary and that these hormones are acting together to regulate energy expenditure,” said Tiganis.

These results are “really exciting as, perhaps, resistance to the actions of leptin and insulin in POMC neurons is a key feature underlying obesity in people,” said Dodd.

Another set of neurons in the hypothalamus, the agouti-related protein expressing (AgRP) or “hunger” neurons, are activated by hunger signals and promote energy storage. Along with Tamas Horvath, Yale’s Yang recently showed that fasting activates AgRP neurons that then suppress the browning of white fat. “These two stories are complimentary, providing a bigger picture: that the hunger and satiety neurons control browning of fat depending on the body’s energy state,” said Yang. Activation of POMC neurons during caloric intake protects against diet-induced obesity while activation of AgRP neurons tells the body to store energy during fasting.

Whether these results hold up in humans has yet to be explored. Expression of the two phosphatases in the hypothalamus is known to be higher in obese people, but it is not clear whether this suppresses the browning of white fat.

“One of the next big questions is whether this increased expression and prevention of insulin plus leptin signaling, and conversion of white to brown fat perturbs energy balance and promotes obesity,” said Tiganis. Another, said Dodd, is whether other parts of the brain are involved in signaling to and from adipose tissue.

  1. Dodd et al., “Leptin and insulin act on POMC neurons to promote the browning of white fat,”

Cell, 2015.    http://dx.doi.org:/10.1016/j.cell.2014.12.022   http://medicine.yale.edu/lab/horvath/index.aspx

Our main interest is the neuroendocrine regulation of homeostasis with particular emphasis on metabolic disorders, such as obesity and diabetes, and the effect of metabolic signals on higher brain functions and neurodegeneration. We have active research programs to pursue the role of synaptic plasticity in the mediation of peripheral hormones’ effects on the central nervous system.

We also study the role of mitochondrial membrane potential in normal and pathological brain functions with particular emphasis on the acute effect of mitochondria in neuronal transmission and neuroprotection. We combine classical neurobiological approaches, including electrophysiology and neuroanatomy, with endocrine and genetic techniques to better understand biological events at the level of the organism.

Leptin and Insulin Act on POMC Neurons to Promote the Browning of White Fat

Garron T. Dodd, Stephanie Decherf, Kim Loh, Stephanie E. Simonds, Florian Wiede, Eglantine Balland, Troy L. Merry, et al.

http://dx.doi.org/10.1016/j.cell.2014.12.022

Highlights

  • Insulin and leptin act synergistically on POMC neurons to promote WAT browning
  • Increased POMC-mediated WAT browning prevents diet-induced obesity
  • PTP1B and TCPTP attenuate leptin and insulin signaling in POMC neurons
  • Combined PTP1B and TCPTP deficiency in POMC neurons promotes white fat browning

The primary task of white adipose tissue (WAT) is the storage of lipids. However, “beige” adipocytes also exist in WAT. Beige adipocytes burn fat and dissipate the energy as heat, but their abundance is diminished in obesity. Stimulating beige adipocyte development, or WAT browning, increases energy expenditure and holds potential for combating metabolic disease and obesity. Here, we report that insulin and leptin act together on hypothalamic neurons to promote WAT browning and weight loss. Deletion of the phosphatases PTP1B and TCPTP enhanced insulin and leptin signaling in proopiomelanocortin neurons and prevented diet-induced obesity by increasing WAT browning and energy expenditure. The coinfusion of insulin plus leptin into the CNS or the activation of proopiomelanocortin neurons also increased WAT browning and decreased adiposity. Our findings identify a homeostatic mechanism for coordinating the status of energy stores, as relayed by insulin and leptin, with the central control of WAT browning.  http://www.cell.com/cms/attachment/2023992410/2043906325/fx1.jpg

Light on the Brain

Researchers find that photoreceptors expressed in zebrafish hypothalamus contribute to light-dependent behavior.

By Sabrina Richards | September 20, 2012

A 21 day old zebrafish. Their optical clarity and relatively easy maintenance make them a favorite for geneticists and developmental biologists. In this fish, the muscles can be seen as chevron shapes in the tail, the swim bladder as a “bubble” just behind the head, and the food that the fish has been eating as a brown patch just below the swim bladder.

Juvenile zebrafish. Shawn Burgess, NHGRI

Zebrafish larvae without eyes or pineal glands can still respond to light using photopigments located deep within their brains.  Published today (September 20) in Current Biology, the findings are the first to link opsins, photoreceptors in the hypothalamus and other brain areas, to increased swimming in response to darkness, a behavior researchers hypothesize may help the fish move toward better-lit environments.

“[It’s a] strong demonstration that opsin-dependent photoreceptors in deep brain areas affect behaviors,” said Samer Hattar, who studies light reception in mammals at Johns Hopkins University but did not participate in the research.

Photoreceptors in eyes enable vision, and photoreceptors in the pineal gland, a small endocrine gland located in the center of the vertebrate brain, regulate circadian rhythms. But photoreceptors are also found in other brain areas of both invertebrates and vertebrate lineages. The function of these extraocular photoreceptors has been best studied in birds, where they regulate seasonal reproduction, explained Harold Burgess, a behavioral neurogeneticist at the Eunice Kennedy Shriver National Institute for Child Health and Human Development. Many opsins have been reported in the brains of tiny and transparent larval zebrafish, raising the possibility that light could be stimulating the photoreceptors even deep in the brain. To test for behaviors that may be regulated by deep brain photoreceptors, Burgess and his colleagues in Wolfgang Driever’s lab at the University of Freiburg removed the eyes of zebrafish larvae, and compared their behavior to larvae that retained their eyes. Although most light-dependent behavior required eyes, the eyeless larvae did respond when the lights were turned off, increasing their activity for a several minutes, though to a somewhat lesser extent than control larvae. But the fact that they responded at all suggests that non-retinal photoreceptors contributed to the behavior.

To confirm the role of the deep brain photoreceptors, the researchers also tested eyeless larvae that had been genetically modified to block expression of photoreceptors in the pineal gland. This fish still showed this jump in activity for several minutes after entering darkness.

Two different types of opsins—melanopsin and multiple tissue opsin—are expressed in the same type of neuron in zebrafish hypothalamus. Burgess and his colleagues looked at zebrafish missing the transcription factor Orthopedia, which is unique to these neurons, and found that the darkness-induced activity boost is nearly absent in these fish. To further narrow the search for the responsible photoreceptors, the researchers overexpressed melanopsin in hypothalamus neurons that co-express Orthopedia and melanopsin, and found that it increased the sensitivity of eyeless zebrafish to reductions in light. The results point to both melanopsin and Orthopedia as key players in modulating this behavior and pinpoint the location to neurons that coexpress these factors in the zebrafish hypothalamus.

Interestingly, the hypothalamus is one of the oldest parts of the vertebrate brain, said Detlev Arendt, a developmental biologist at the European Molecular Biology Laboratory in Heidelberg. “It’s very possible that this is one of the oldest functions”—one that evolved in “non-visual organisms” that had no eyes but still needed to sense light.

Although not as directed and efficient as eye-dependent behaviors that help fish swim toward light, Burgess speculates that deep brain opsins can still benefit zebrafish larvae. “You could imagine situation where it can’t see light, if a leaf falls on it and it doesn’t know where to swim. I think this behavior puts it in a hyperactive state where it swims wildly for several minutes until it reaches enough light for eyes to take over,” he explained, noting that such behavior is common in invertebrates.

It remains to be seen whether these deep brain opsins regulate other behaviors, perhaps in similar fashion to seasonal hormonal regulation in birds, but Hattar believes it is likely. “It’s beyond reasonable doubt there are many functions for these deep brain photoreceptors.”

Fernandes et al., “Deep brain photoreceptors control light-seeking behavior in zebrafish larvae,” Current Biology, 22:1-6, 2012.

Neuroendocrine basis of sexuality, mood, anxiety, social consciousness

Physiology, signaling, and pharmacology of galanin peptides and receptors: Three decades of emerging diversity

Lang, R., Gundlach, A.L., Holmes, F.E., (…), Hökfelt, T., Kofler, B.
Pharmacological Reviews 2015: 67 (1), pp. 118-175
http://dx.doi.org:/10.1124/pr.112.006536

Galanin was first identified 30 years ago as a “classic neuropeptide,” with actions primarily as a modulator of neurotransmission in the brain and peripheral nervous system. Other structurally-related peptides—galanin-like peptide and alarin—with diverse biologic actions in brain and other tissues have since been identified, although, unlike galanin, their cognate receptors are currently unknown. Over the last two decades, in addition to many neuronal actions, a number of nonneuronal actions of galanin and other galanin family peptides have been described. These include actions associated with neural stem cells, nonneuronal cells in the brain such as glia, endocrine functions, effects on metabolism, energy homeostasis, and paracrine effects in bone. Substantial new data also indicate an emerging role for galanin in innate immunity, inflammation, and cancer. Galanin has been shown to regulate its numerous physiologic and pathophysiological processes through interactions with three G protein–coupled receptors, GAL1, GAL2, and GAL3, and signaling via multiple transduction pathways, including inhibition of cAMP/PKA (GAL1, GAL3) and stimulation of phospholipase C (GAL2). In this review, we emphasize the importance of novel galanin receptor–specific agonists and antagonists. Also, other approaches, including new transgenic mouse lines (such as a recently characterized GAL3 knockout mouse) represent, in combination with viral-based techniques, critical tools required to better evaluate galanin system physiology. These in turn will help identify potential targets of the galanin/galanin-receptor systems in a diverse range of human diseases, including pain, mood disorders, epilepsy, neurodegenerative conditions, diabetes, and cancer.

Estradiol regulates responsiveness of the dorsal premammillary nucleus of the hypothalamus and affects fear- and anxiety-like behaviors in female rats

Litvin, Y., Cataldo, G., Pfaff, D.W., Kow, L.-M.
European Journal of Neuroscience 2014; 40 (2), pp. 2344-2351
10.1111/ejn.12608

Research suggests a causal link between estrogens and mood. Here, we began by examining the effects of estradiol (E2) on rat innate and conditioned defensive behaviors in response to cat odor. Second, we utilized whole-cell patch clamp electrophysiological techniques to assess noradrenergic effects on neurons within the dorsal premammillary nucleus of the hypothalamus (PMd), a nucleus implicated in fear reactivity, and their regulation by E2. Our results show that E2 increased general arousal and modified innate defensive reactivity to cat odor. When ovariectomized females treated with E2 as opposed to oil were exposed to cat odor, they showed elevations in risk assessment and reductions in freezing, indicating a shift from passive to active coping. In addition, animals previously exposed to cat odor showed clear cue + context conditioning 24 h later. However, although E2 persisted in its effects on general arousal in the conditioning task, its effects on fear disappeared. In the patch clamp experiments noradrenergic compounds that typically induce fear clearly excited PMd neurons, producing depolarizations and action potentials. E2 treatment shifted some excitatory effects of noradrenergic agonists to inhibitory, possibly by differentially affecting α- and β-adrenoreceptors. In summary, our results implicate E2 in general arousal and fear reactivity, and suggest these may be governed by changes in noradrenergic responsivity in the PMd. These effects of E2 may have ethological relevance, serving to promote mate seeking even in contexts of ambiguous threat and shed light on the involvement of estrogen in mood and its associated disorders.

Endogenous opiates and behavior: 2013

Richard J. Bodnar
Peptides 62 (2014) 67–136
http://dx.doi.org/10.1016/j.peptides.2014.09.013

This paper is the thirty-sixth consecutive installment of the annual review of research concerning the endogenous opioid system. It summarizes papers published during 2013 that studied the behavioral effects of molecular, pharmacological and genetic manipulation of opioid peptides, opioid receptors, opioid agonists and opioid antagonists. The particular topics that continue to be covered include the molecular-biochemical effects and neurochemical localization studies of endogenous opioids and their receptors related to behavior, and the roles of these opioid peptides and receptors in pain and analgesia; stress and social status; tolerance and dependence; learning and memory; eating and drinking; alcohol and drugs of abuse; sexual activity and hormones, pregnancy, development and endocrinology; mental illness and mood; seizures and neurologic disorders; electrical-related activity and neurophysiology; general activity and locomotion; gastrointestinal, renal and hepatic functions; cardiovascular responses; respiration and thermoregulation; and immunological responses.

Brain aromatase (cyp19a1b) and gonadotropin releasing hormone (gnrh2 and gnrh3) expression during reproductive development and sex change in black sea bass (Centropristis striata)

Timothy S Breton, Matthew A DiMaggio, Stacia A Sowe, David L Berlinsky, et al.
Comparative Biochemistry and Physiology, Part A 181 (2015) 45–53
http://dx.doi.org/10.1016/j.cbpa.2014.11.020

Teleost fish exhibit diverse reproductive strategies, and some species are capable of changing sex. The influence of many endocrine factors, such as gonadal steroids and neuropeptides, has been studied in relation to sex change, but comparatively less research has focused on gene expression changes within the brain in temperate grouper species with non-haremic social structures. The purpose of the present study was to investigate gonadotropin releasing hormone (GnRH) and brain aromatase (cyp19a1b) gene expression patterns during reproductive development and sex change in protogynous (female to male) black sea bass (Centropristis striata). Partial cDNA fragments for cyp19a1b and eef1a (a reference gene) were identified, and included with known gnrh2 and gnrh3 sequences in real time quantitative PCR. Elevated cyp19a1b expression was evident in the olfactory bulbs, telencephalon, optic tectum, and hypothalamus/
midbrain region during vitellogenic growth, which may indicate changes in the brain related to neurogenesis or sexual behavior. In contrast, gnrh2 and gnrh3 expression levels were largely similar among gonadal states, and all three genes exhibited stable expression during sex change. Although sex change in black sea bass is not associated with dramatic changes in GnRH or cyp19a1b gene expression among brain regions, these genes may mediate processes at other levels, such as within individual hypothalamic nuclei, or through changes in neuron size, that warrant further research.

Evaluation for roles of neurosteroids in modulating forebrain mechanisms controlling vasopressin secretion and related phenomena in conscious rats

Ken’ichi Yamaguchi
Neuroscience Research xxx (2015) xxx–xxx
http://dx.doi.org/10.1016/j.neures.2015.01.002

Anteroventral third ventricular region (AV3V) regulates autonomic functions through a GABAergic mechanism that possesses neuroactive steroid (NS)-synthesizing ability. Although NS can exert effects by acting on a certain type of GABAA-receptor (R), it is not clear whether NS may operate to modulateAV3V GABAergic activity for controlling autonomic functions. This study aimed to investigate the issue.AV3V infusion with a GABAA antagonist bicuculline increased plasma vasopressin (AVP), glucose, blood pressure (BP), and heart rate in rats. These events were abolished by preinjecting its agonist muscimol, whereas the infusion with allopregnanolone, a NS capable of potentiating GABAA-R function, affectednone of the variables in the absence or presence of such bicuculline actions. Similarly, AV3V infusion with pregnanolone sulfate, a NS capable of antagonizing GABAA-R, produced no effect on those variables.AV3V infusion with muscimol was effective in inhibiting the responses of plasma AVP or glucose, orBP to an osmotic loading or bleeding. However, AV3V infusion with aminoglutethimide, a NS synthesis inhibitor, did not affect any of the variables in the absence or presence of those stimuli. These results suggest that NS may not cause acute effects on the AV3V GABAergic mechanism involved in regulating AVP release and other autonomic function.

Novel receptor targets for production and action of allopregnanolone in the central nervous system: a focus on pregnane xenobiotic receptor

Cheryl A. Frye, Carolyn J. Koonce, and Alicia A. Walf
Front in Cell Neurosci Apr 2014; 8(106)
http://dx.doi.org:/10.3389/fncel.2014.00106

Neurosteroids are cholesterol-based hormones that can be produced in the brain, independent of secretion from peripheral endocrine glands, such as the gonads and adrenals. A focus in our laboratory for over 25 years has been how production of the pregnane neurosteroid, allopregnanolone, is regulated and the novel (i.e., non steroid receptor) targets for steroid action for behavior. One endpoint of interest has been lordosis, the mating posture of female rodents. Allopregnanolone is necessary and sufficient for lordosis, and the brain circuitry underlying it, such as actions in the midbrain ventral tegmental area (VTA), has been well-characterized. Published and recent findings supporting a dynamic role of allopregnanolone are included in this review. First, contributions of ovarian and adrenal sources of precursors of allopregnanolone, and the requisite enzymatic actions for de novo production in the central nervous system will be discussed.
Second, how allopregnanolone produced in the brain has actions on behavioral processes that are independent of binding to steroid receptors, but instead involve rapid modulatory actions via neurotransmitter targets (e.g., g-amino butyric acid-GABA, Nmethyl-D-aspartate- NMDA) will be reviewed.
Third, a recent focus on characterizing the role of a promiscuous nuclear receptor, pregnane xenobiotic receptor (PXR), involved in cholesterol metabolism and expressed in the VTA, as a target for allopregnanolone and how this relates to both actions and production of allopregnanolone will be addressed. For example, allopregnanolone can bind PXR and knocking down expression of PXR in the midbrain VTA attenuates actions of allopregnanolone via NMDA and/or GABAA for lordosis. Our understanding of allopregnanolone’s actions in the VTA for lordosis has been extended to reveal the role of allopregnanolone for broader, clinically-relevant questions, such as neurodevelopmental processes, neuropsychiatric disorders, epilepsy, and aging.

Long-term dysregulation of brain corticotrophin and glucocorticoid receptors and stress reactivity by single early-life pain experience in male and female rats

Nicole C. Victoria, Kiyoshi Inoue, Larry J. Young, Anne Z. Murphy
Psychoneuroendocrinology (2013) 38, 3015—3028
http://dx.doi.org/10.1016/j.psyneuen.2013.08.013

Inflammatory pain experienced on the day of birth (postnatal day 0: PD0) significantly dampens behavioral responses to stress- and anxiety-provoking stimuli in adult rats. However, to date, the mechanisms by which early life pain permanently alters adult stress responses remain unknown. The present studies examined the impact of inflammatory pain, experienced on the day of birth, on adult expression of receptors or proteins implicated in the activation and termination of the stress response, including corticotrophin releasing factor receptors (CRFR1 and CRFR2) and glucocorticoid receptor (GR). Using competitive receptor autoradiography, we show that Sprague Dawley male and female rat pups administered 1% carrageenan into the intraplantar surface of the hindpaw on the day of birth have significantly decreased CRFR1 binding in the basolateral amygdala and midbrain periaqueductal gray in adulthood. In contrast, CRFR2 binding, which is associated with stress termination, was significantly increased in the lateral septum and cortical amygdala. GR expression, measured with in situ hybridization and immunohistochemistry, was significantly increased in the paraventricular nucleus of the hypothalamus and significantly decreased in the hippocampus of neonatally injured adults. In parallel, acute stress-induced corticosterone release was significantly attenuated and returned to baseline more rapidly in adults injured on PD0 in comparison to controls. Collectively, these data show that early life pain alters neural circuits that regulate responses to and neuroendocrine recovery from stress, and suggest that pain experienced by infants in the Neonatal Intensive Care Unit may permanently alter future responses to anxiety- and stress provoking stimuli.

Dysruption of Corticotropin Releasing Factor in hypocampal region

Stress and trauma: BDNF control of dendritic-spine formation and regression

M.R. Bennett, J. Lagopoulos
Progress in Neurobiology 112 (2014) 80–99
http://dx.doi.org/10.1016/j.pneurobio.2013.10.005

Chronic restraint stress leads to increases in brain derived neurotrophic factor (BDNF) mRNA and protein in some regions of the brain, e.g. the basal lateral amygdala (BLA) but decreases in other regions such as the CA3 region of the hippocampus and dendritic spine density increases or decreases in line with these changes in BDNF. Given the powerful influence that BDNF has on dendritic spine growth, these observations suggest that the fundamental reason for the direction and extent of changes in dendritic spine density in a particular region of the brain under stress is due to the changes in BDNF there.
The most likely cause of these changes is provided by the stress initiated release of steroids, which readily enter neurons and alter gene expression, for example that of BDNF. Of particular interest is how glucocorticoids and mineralocorticoids tend to have opposite effects on BDNF gene expression offering the possibility that differences in the distribution of their receptors and of their downstream effects might provide a basis for the differential transcription of the BDNF genes. Alternatively, differences in the extent of methylation and acetylation in the epigenetic control of BDNF transcription are possible in different parts of the brain following stress.
Although present evidence points to changes in BDNF transcription being the major causal agent for the changes in spine density in different parts of the brain following stress, steroids have significant effects on downstream pathways from the TrkB receptor once it is acted upon by BDNF, including those that modulate the density of dendritic spines.
Finally, although glucocorticoids play a canonical role in determining BDNF modulation of dendritic spines, recent studies have shown a role for corticotrophin releasing factor (CRF) in this regard. There is considerable improvement in the extent of changes in spine size and density in rodents with forebrain specific knockout of CRF receptor 1 (CRFR1) even when the glucocorticoid pathways are left intact. It seems then that CRF does have a role to play in determining BDNF control of dendritic spines.

Central CRF system perturbation in an Alzheimer’s disease knockin mouse model

Qinxi Guo, Hui Zheng, Nicholas John Justice
Neurobiology of Aging 33 (2012) 2678–2691
http://dx.doi.org:/10.1016/j.neurobiolaging.2012.01.002

Alzheimer’s disease (AD) is often accompanied by changes in mood as well as increases in circulating cortisol levels, suggesting that regulation of the stress responsive hypothalamic-pituitary-adrenal (HPA) axis is disturbed. Here, we show that amyloid precursor protein (APP) is endogenously expressed in important limbic, hypothalamic, and midbrain nuclei that regulate hypothalamic-pituitary-adrenal axis activity. Furthermore, in a knockin mouse model of AD that expresses familial AD (FAD) mutations of both APP with humanized amyloid beta (hA), and presenilin 1 (PS1), in their endogenous patterns (APP/hA/PS1 animals), corticotropin releasing factor (CRF) levels are increased in key stress-related nuclei, resting corticosteroid levels are elevated, and animals display increased anxiety-related behavior. Endocrine and behavioral phenotypes can be normalized by loss of 1 copy of CRF receptor type-1 (Crfr1), consistent with a perturbation of central CRF signaling in APP/hA/PS1 animals. However, reductions in anxiety and corticosteroid levels conferred by heterozygosity of CRF receptor type-1 do not improve a deficit in working memory observed in APP/hA/PS1 mice, suggesting that perturbations of the CRF system are not the primary cause of decreased cognitive performance.

Alzheimer’s disease-like neuropathology of gene-targeted APP-SLxPS1mut mice expressing the amyloid precursor protein at endogenous levels

Christoph Kohler, Ulrich Ebert, Karlheinz Baumann, and Hannsjorg Schroeder
Neurobiology of Disease 20 (2005) 528 – 540
http://dx.doi.org:/10.1016/j.nbd.2005.04.009

Most transgenic mice used for preclinical evaluation of potential disease-modifying treatments of Alzheimer’s disease develop major histopathological features of this disease by several-fold overexpression of the human amyloid precursor protein. We studied the phenotype of three different strains of gene-targeted mice which express the amyloid precursor protein at endogenous levels. Only further crossing with transgenic mice overexpressing mutant human presenilin1 led to the deposition of extracellular amyloid, accompanied by the deposition of apolipoprotein E, an astrocyte and microglia reaction, and the occurrence of dilated cholinergic terminals in the cortex. Features of neurodegeneration, however, were absent. The pattern of plaque development and deposition in these mice was similar to that of amyloid precursor protein overproducing strains if crossed to presenilin1-transgenics. However, plaque development started much later and developed slowly until the age of 18 months but then increased more rapidly.

Central Cholinergic Functions In Human Amyloid Precursor Protein Knock-In/Presenilin-1 Transgenic Mice

Hartmann, C. Erb, U. Ebert, K. H. Baumann, A. Popp, G. Koenig, J. Klein
Neuroscience 125 (2004) 1009–1017
http://dx.doi.org:/10.1016/j.neuroscience.2004.02.038

Alzheimer’s disease is characterized by amyloid peptide formation and deposition, neurofibrillary tangles, central cholinergic dysfunction, and dementia; however, the relationship between these parameters is not well understood. We studied the effect of amyloid peptide formation and deposition on central cholinergic function in knock-in mice carrying the human amyloid precursor protein (APP) gene with the Swedish/London double mutation (APP-SL mice) which were crossbred with transgenic mice overexpressing normal (PS1wt) or mutated (M146L; PS1mut) human presenilin-1. APP-SLxPS1mut mice had increased levels of Aβ peptides at 10 months of age and amyloid plaques at 14 months of age while APP-SLPS1wt mice did not have increased peptide levels and did not develop amyloid plaques. We used microdialysis in 15–27 months old mice to compare hippocampal acetylcholine (ACh) levels in the two mouse lines and found that extracellular ACh levels were slightly but significantly reduced in the APP-SLPS1mut mice (-26%; P=0.044). Exploratory activity in the open field increased hippocampal ACh release by two-fold in both mouse lines; total and relative increases were not significantly different for the two strains under study. Similarly, infusion of scopolamine (1 µM) increased hippocampal ACh release to a similar extent (3–5-fold) in both groups. High-affinity choline uptake, a measure of the ACh turnover rate, was identical in both mouse lines. Neurons expressing choline acetyltransferase were increased in the septum of APP-SLPS1mut mice (26%; P =0.046). We conclude that amyloid peptide production causes a small decrease of extracellular ACh levels. The deposition of amyloid plaques, however, does not impair stimulated ACh release and proceeds without major changes of central cholinergic function.

Glutamate Neurotoxicity

Glutamate Neurotoxicity and Diseases of the Nervous System

Dennis W. Choi
Neuron. Oct, 1988; 1: 623-634

A growing number of studies now suggest that the cellular mechanisms which normally participate in signaling in the central nervous system (CNS) can be transformed by disease into instruments of neuronal cell destruction. Excitatory synaptic transmission in the mammalian CNS is principally mediated by L-glutamate. In fact, glutamate excites virtually all central neurons and is present in nerve terminals at millimolar levels (Curtis and Johnston, 1974). Normally, the extracellular levels of glutamate rise to high levels only in the brief and spatially localized fashion appropriate to synaptic transmission. This is fortunate, because as Lucas and Newhouse first showed in 1957, sustained exposure to glutamate can destroy retinal neurons. In a subsequent set of pioneering experiments, Olney (Olney and Sharpe, 1969; Olney et al., 1971) established that this toxicity, which he later called excitotoxicity, was not unique to glutamate or to retinal neurons, but was a feature common to the actions of all excitatory amino acids on central neurons. He postulated therefore that glutamate, or related compounds, might be the cause of the neuronal cell loss found in certain neurological diseases. In recent years, this hypothesis has gathered considerable support, fueled by new insights into glutamate receptor function and the development of effective glutamate antagonist drugs. The evidence is most convincing in diseases involving an acute insult to the brain, as occurs in a stroke, with abrupt deprivation of blood supply. But neurotoxicity due to excitatory amino acids may also be involved in slowly progressive degenerative diseases such as Huntington’s disease. Although the detailed molecular basis of glutamate neurotoxicity is not known, it appears that Ca2+ influx may play a critical role.
Glutamate interacts with at least three classes of membrane receptors, each commonly referred to by preferred pharmacological agonists: N-methyl-o-aspartate (NMDA), quisqualate, and kainate (Watkins and Olverman, 1987) (Figure I). These three classes are linked to membrane cation channels. A second type of quisqualate receptor has been additionally linked to a second messenger system (see below). It has been suggested that all three classes might actually be substates of a single molecular complex, but binding studies and newer physiological studies favor separate structures.

Quisqualate                         NMDA                       Kainate

Three Classes of Glutamate Receptors

Three Classes of Glutamate Receptors

Three Classes of Glutamate Receptors

One type of quisqualate receptor stimulates the formation of inositol 1,4,5-trisphosphate UPS) and diacylglycerol (DAG) from phosphatidylinositol-4,5-biphosphate (PIP,); the other is linked directly to a Na+ ionophore. Activation of the quisqualate receptor-ionophore complex can be potentiated by Zn2+. The NMDA receptor opens a channel permeable to Ca2+ as well as Na+; this receptor-channel complex has several modulatory sites discussed in the text. The kainate receptor opens an ionophore permeable to Na+.

Best defined is the NMDA receptor. This receptor opens a distinctive membrane channel characterized by high conductance (main state about 50 pS), voltage dependent Mgz+ blockade and permeability to both Ca2+ and Na+. The NMDA receptor can be selectively activated by several endogenous compounds, including L-aspartate, homocysteate, and quinolinate. Activation requires the coavailability of glycine in near micromolar concentrations. The action of glutamate at the NMDA receptor can be selectively antagonized: competitively by 2-amino-5-phosphonovalerate (APV) and 2-amino-5-phosphonoheptanoate (APH), or noncompetitively by drugs that bind to the phencyclidine site within the open channel (such as phencyclidine, MK-801, dextrorphan, or ketamine. The NMDA receptor-activated channel can also be blocked noncompetitively by Znz+, most likely at a site different from that which binds Mg2.
Although glutamate has high affinity for all three classes of postsynaptic receptors, it is not easy to demonstrate its neurotoxicity in vivo. Even when directly injected into brain, bypassing the blood-brain barrier, extremely high doses of glutamate are required to create lesions.  Mangano & Schwartz found that they could infuse 0.5 crl/hr of a 300 mM glutamate solution into the hippocampus of a rat for 2 weeks without producing neuronal injury. This apparent low in vivo neurotoxic potency of glutamate may represent one reason why Olney’s “glutamate hypothesis” of neurological disease did not initially achieve a more widespread following. However, in fact, glutamate is a potent and rapidly acting neurotoxin; its neurotoxicity in vivo is likely masked by the efficiency of normal cellular uptake mechanisms in removing glutamate from the extracellular space. Glutamate neurotoxicity can be most directly studied in cell culture where bath exposure is not limited by cellular uptake.
The toxic changes produced by glutamate or related excitatory amino acids in vivo are of two sorts:

  1. acute swelling of neuronal dendrites and cell bodies and a
  2. more slowly evolving neuronal degeneration (Olney, 1986).

Axons and glia are relatively spared, although high levels of excitatory amino acids can produce some swelling of glia. A hallmark of excitatory amino acid neurotoxicity is its cellular selectivity, with distinctive patterns of neuronal loss produced by different excitatory amino acids and different routes of administration. For example, Nadler and co-workers (1978) found that intraventricular kainate preferentially destroys hippocampal CA3 neurons but spares dentate granule neurons. Different neuronal subpopulations
may differ in their intrinsic vulnerability to damage.

Possible Mechanisms Involved in Glutamate Neurotoxicity

How Ca*+ may mediate glutamate-induced neuronal degeneration. Glutamate acts on NMDA, non-NMDA, and “metabotropic” receptors (the quisqualate receptor linked to a second messenger system) to produce an increase in cytosolic free Ca*+. This cytosolic Ca *+, in concert with diacylglycerol liberated by the quisqualate-triggered second messenger system, activates protein kinase C, which acts via a number of mechanisms (primarily by altering membrane ion channels) to increase neuronal excitability and further increase cytosolic Ca*+. Elevated cytosolic Ca2+ then activates several enzymes capable of either directly or indirectly (through free radical formation) destroying cellular structure. Glutamate released from synaptic terminals or leaking nonspecifically from ruptured neurons contributes to additional injury propagation.

Glutamate Neurotoxicity in Perspective

The hypothesis that excitatory amino acids may specifically mediate pathological neuronal injury gives new form to this age-old enemy and raises the tantalizing possibility that current molecular and cellular insights into excitatory amino acid transmitter systems might be harnessed to develop an efficacious clinical therapy. Some points of attack are already apparent; others will likely be defined as the biology of excitatory amino acids continues to be unraveled. An intriguing area for investigation is the relationship between excitatory amino acid neurotoxicity and normal neuronal processes such as maturation, neurite outgrowth, and synaptic plasticity.

Glutamate Toxicity in a Neuronal Cell line Involves Inhibition of Cystine Transport Leading to Oxidative Stress

Timothy H. Murphy, M Miyamoto, A Sastre, R Schnaar and JT Coyle
Neuron 1989: 2: 1547-88.

Glutamate binds to both excitatory neurotransmitter binding sites and a W-dependent, quisqualate- and cystine-inhibited transport site on brain neurons. The neuroblastoma-primary retina hybrid cells (NWRE-105) are susceptible to glutamate-induced cytotoxicity. The Cl–dependent transport site to which glutamate and quisqualate (but not kainate or NMDA) bind has a higher affinity for cystine than for glutamate. Towering cystine concentrations in the cell culture medium results in cytotoxicity similar to that induced by glutamate addition in its morphology, kinetics, and CaZ+ dependence. Glutamate-induced cytotoxicity is directly proportional to its ability to inhibit cystine uptake. Exposure to glutamate (or lowered cystine) causes a decrease in glutathione levels and an accumulation of intracellular peroxides. Like NW-RE-105 cells, primary rat hippocampal neurons (but not glia) in culture degenerate in medium with lowered cystine concentration. Thus, glutamate-induced cytotoxicity in N18-RE-105 cells is due to inhibition of cystine uptake, resulting in lowered glutathione levels leading to oxidative stress and cell death.

Mechanism of glutamate-induced neurotoxicity in HT22 mouse hippocampal cells

Masayuki Fukui, Ji-Hoon Song, Jinyoung Choi, Hye Joung Choi, Bao Ting Zhu
European Journal of Pharmacology 617 (2009) 1–11
http://dx.doi.org:/10.1016/j.ejphar.2009.06.059

Glutamate is an endogenous excitatory neurotransmitter. At high concentrations, it is neurotoxic and contributes to the development of certain neurodegenerative diseases. There is considerable controversy in the literature with regard to whether glutamate-induced cell death in cultured HT22 cells (an immortalized mouse hippocampal cell line) is apoptosis, necrosis, or a new form of cell death. The present study focused on investigating the mechanism of glutamate-induced cell death. We found that glutamate induced, in a time dependent manner, both necrosis and apoptosis in HT22 cells. At relatively early time points (8–12 h), glutamate induced mostly necrosis, whereas at late time points (16–24 h), it induced mainly apoptosis. Glutamate-induced mitochondrial oxidative stress and dysfunction were crucial early events required for the induction of apoptosis through the release of the mitochondrial apoptosis-inducing factor (AIF), which catalyzed DNA fragmentation (an ATP-independent process). Glutamate-induced cell death proceeded independently of the Bcl-2 family proteins and caspase activation. The lack of caspase activation likely resulted from the lack of intracellular ATP when the mitochondrial functions were rapidly disrupted by the mitochondrial oxidative stress. In addition, it was observed that activation of JNK, p38, and ERK signaling molecules was also involved in the induction of apoptosis by glutamate. In conclusion, glutamate-induced apoptosis is AIF-dependent but caspase-independent, and is accompanied by DNA ladder formation but not chromatin condensation.

Understanding Low Reliability of Memories for Neutral Information Encoded under Stress: Alterations in Memory-Related Activation in the Hippocampus and Midbrain

Shaozheng Qin, EJ Hermans, HJF van Marle, and G Fernandez, et al.
The Journal of Neuroscience, Mar 21, 2012; 32(12): 4032–4041
http://dx.doi.org:/10.1523/JNEUROSCI.3101-11.2012

Exposure to an acute stressor can lead to unreliable remembrance of intrinsically neutral information, as exemplified by low reliability of eyewitness memories, which stands in contrast with enhanced memory for the stressful incident itself. Stress-sensitive neuromodulators (e.g., catecholamines) are believed to cause this low reliability by altering neurocognitive processes underlying memory formation. Using event-related functional magnetic resonance imaging, we investigated neural activity during memory formation in 44 young, healthy human participants while incidentally encoding emotionally neutral, complex scenes embedded in either a stressful or neutral context.
We recorded event-related pupil dilation responses as an indirect index of phasic noradrenergic activity. Autonomic, endocrine, and psychological measures were acquired to validate stress manipulation. Acute stress during encoding led to a more liberal response bias (more hits and false alarms) when testing memory for the scenes 24 h later. The strength of this bias correlated negatively with pupil dilation responses and positively with stress-induced heart rate increases at encoding. Acute stress, moreover, reduced subsequent memory effects (SMEs; items later remembered vs forgotten) in hippocampus and midbrain, and in pupil dilation responses.
The diminished SMEs indicate reduced selectivity and specificity in mnemonic processing during memory formation. This is in line with a model in which stress-induced catecholaminergic hyperactivation alters phasic neuromodulatory signaling in memory-related circuits, resulting in generalized (gist-based) processing at the cost of specificity. Thus, one may speculate that loss of specificity may yield less discrete memory representations at time of encoding, thereby causing a more liberal response bias when probing these memories.

Neuroendocrinology – Signaling, neuron plasticity and memory

Leptin Signaling Modulates the Activity of Urocortin 1 Neurons in the Mouse Nonpreganglionic Edinger-Westphal Nucleus

Lu Xu, Wim J. J. M. Scheenen, Rebecca L. Leshan, Christa M. Patterson, et al.
Endocrinology 152(3): 979–988, 2011
http://dx.doi.org:/10.1210/en.2010-1143

A recent study systematically characterized the distribution of the long form of the leptin receptor (LepRb) in the mouse brain and showed substantial LepRb mRNA expression in the nonpreganglionic Edinger-Westphal nucleus (npEW) in the rostroventral part of the midbrain. This nucleus hosts the majority of urocortin 1 (Ucn1) neurons in the rodent brain, and because Ucn1 is a potent satiety hormone and electrical lesioning of the npEW strongly decreases food intake, we have hypothesized a role of npEW-Ucn1 neurons in leptin-controlled food intake. Here, we show by immunohistochemistry that npEW-Ucn1 neurons in the mouse contain LepRb and respond to leptin administration with induction of the Janus kinase 2-signal transducer and activator of transcription 3 pathway, both in vivo and in vitro. Furthermore, systemic leptin administration increases the Ucn1 content of then pEW significantly, whereas in mice that lack LepRb (db/db mice), then pEW contains considerably reduced amount of Ucn1. Finally, we reveal by patch clamping of midbrain Ucn1 neurons that leptin administration reduces the electrical firing activity of the Ucn1 neurons. In conclusion, we provide ample evidence for leptin actions that go beyond leptin’s well-known targets in the hypothalamus and propose that leptin can directly influence the activity of the midbrain Ucn1 neurons.

Leptin regulation of hippocampal synaptic function in health and disease

Andrew J. Irving and Jenni Harvey
Trans. R. Soc. B 369: 20130155 http://dx.doi.org/10.1098/rstb.2013.0155

The endocrine hormone leptin plays a key role in regulating food intake and body weight via its actions in the hypothalamus. However, leptin receptors are highly expressed in many extra-hypothalamic brain regions and evidence is growing that leptin influences many central processes including cognition. Indeed, recent studies indicate that leptin is a potential cognitive enhancer as it markedly facilitates the cellular events underlying hippocampal-dependent learning and memory, including effects on glutamate receptor trafficking, neuronal morphology and activity-dependent synaptic plasticity. However, the ability of leptin to regulate hippocampal synaptic function markedly declines with age and aberrant leptin function has been linked to neurodegenerative disorders such as Alzheimer’s disease (AD). Here, we review the evidence supporting a cognitive enhancing role for the hormone leptin and discuss the therapeutic potential of using leptin-based agents to treat AD.

The Y2 receptor agonist PYY3–36 increases the behavioral response to novelty and acute dopaminergic drug challenge in mice

Ulrike Stadlbauer, Elisabeth Weber, Wolfgang Langhans and Urs Meyer
International Journal of Neuropsychopharmacology (2014), 17, 407–419
http://dx.doi.org:/10.1017/S1461145713001223

The gastrointestinal hormone PYY3–36 is a preferential Y2 neuropeptide Y (NPY) receptor agonist. Recent evidence indicates that PYY3–36 acts on central dopaminergic pathways, but its influence on dopamine-dependent behaviors remains largely unknown. We therefore explored the effects of peripheral PYY3–36 treatment on the behavioral responses to novelty and to dopamine-activating drugs in mice. In addition, we examined whether PYY3–36 administration may activate distinct dopamine and γ-aminobutyric acid (GABA) cell populations in the mesoaccumbal and nigrostriatal pathways. We found that i.p. PYY3–36 injection led to a dose-dependent increase in novel object exploration. The effective dose of PYY3–36 (1 μg/100 g body weight) also potentiated the locomotor reaction to the indirect dopamine receptor agonist amphetamine and increased stereotyped climbing/leaning responses following administration of the direct dopamine receptor agonist apomorphine. PYY3–36 administration did not affect activity of midbrain dopaminergic cells as evaluated by double immuno-enzyme staining of the neuronal early gene product c-Fos with tyrosine hydroxylase. PYY3–36 did, however, lead to a marked increase in the number of cells co-expressing c-Fos with glutamic acid decarboxylase in the nucleus accumbens and caudate putamen, indicating activation of GABAergic cells in dorsal and ventral striatal areas. Our results support the hypothesis that acute administration of the preferential Y2 receptor agonist PYY3–36 modulates dopamine-dependent behaviours. These effects do not seem to involve direct activation of midbrain dopamine cells but instead are associated with neuronal activation in the major input areas of the mesoaccumbal and nigrostriatal pathways.

Somatostatin and nociceptin inhibit neurons in the central nucleus of amygdala that project to the periaqueductal grey

Billy Chieng, MacDonald J. Christie
Neuropharmacology 59 (2010) 425e430
http://dx.doi.org:/10.1016/j.neuropharm.2010.06.001

The central nucleus of amygdala (CeA) plays an important role in modulation of the descending antinociceptive pathways. Using whole-cell patch clamp recordings from brain slices, we found that CeA neurons responded to the endogenous ligands somatostatin (SST) and nociceptin/orphanin FQ (OFQ) via an increased K-conductance. Co-application with selective antagonists suggested that SST and OFQ act on SSTR2 and ORL1 receptors, respectively. Taking account of anatomical localisation of recorded neurons, the present study showed that many responsive neurons were located within the medial subdivision of CeA and all CeA projection neurons to the midbrain periaqueductal grey invariably responded to these peptides. Randomly selected agonist-responsive neurons in CeA predominantly classified physiologically as low-threshold spiking neurons. The similarity of SST, OFQ and, as previously reported, opioid responsiveness in a sub-population of CeA neurons suggests converging roles of these peptides to inhibit the activity of projections from CeA to vlPAG, and potentially similar antinociceptive actions in this pathway.

In vitro identification and electrophysiological characterization of dopamine neurons in the ventral tegmental area

Tao A. Zhang, Andon N. Placzek, John A. Dani
Neuropharmacology 59 (2010) 431e436
http://dx.doi.org:/10.1016/j.neuropharm.2010.06.004

Dopamine (DA) neurons in the ventral tegmental area (VTA) have been implicated in brain mechanisms related to motivation, reward, and drug addiction. Successful identification of these neurons in vitro has historically depended upon the expression of a hyperpolarization-activated current (Ih) and immunohistochemical demonstration of the presence of tyrosine hydroxylase (TH), the rate-limiting enzyme for DA synthesis. Recent findings suggest that electrophysiological criteria may be insufficient for distinguishing DA neurons from non-DA neurons in the VTA. In this study, we sought to determine factors that could potentially account for the apparent discrepancies in the literature regarding DA neuron identification in the rodent brain slice preparation. We found that confirmed DA neurons from the lateral VTA generally displayed a larger amplitude Ih relative to DA neurons located in the medial VTA. Measurement of a large amplitude Ih (>100 pA) consistently indicated a dopaminergic phenotype, but non-dopamine neurons also can have Ih current. The data also showed that immunohistochemical TH labeling of DA neurons can render false negative results after relatively long duration (>15 min) wholecell patch clamp recordings. We conclude that whole-cell patch clamp recording in combination with immunohistochemical detection of TH expression can guarantee positive but not negative DA identification in the VTA.

Dopamine Enables In Vivo Synaptic Plasticity Associated with the Addictive Drug Nicotine

Jianrong Tang and John A. Dani
Neuron, Sept 10, 2009; 63, 673–682
http://dx.doi.org:/10.1016/j.neuron.2009.07.025

Addictive drugs induce a dopamine signal that contributes to the initiation of addiction, and the dopamine signal influences drug-associated memories that perpetuate drug use. The addiction process shares many commonalities with the synaptic plasticity mechanisms normally attributed to learning and memory. Environmental stimuli repeatedly linked to addictive drugs become learned associations, and those stimuli come to elicit memories or sensations that motivate continued drug use. Applying in vivo recording techniques to freely moving mice, we show that physiologically relevant concentrations of the addictive drug nicotine directly cause in vivo hippocampal synaptic potentiation of the kind that underlies learning and memory. The drug-induced long-term synaptic plasticity required a local hippocampal dopamine signal. Disrupting general dopamine signaling prevented the nicotine-induced synaptic plasticity and conditioned place preference. These results suggest that dopaminergic signaling serves as a functional label of salient events by enabling and scaling synaptic plasticity that underlies drug-induced associative memory.

NCS-1 in the Dentate Gyrus Promotes Exploration, Synaptic Plasticity, and Rapid Acquisition of Spatial Memory

Bechara J. Saab, John Georgiou, Arup Nath, Frank J.S. Lee, et al.
Neuron, Sept 10, 2009; 63, 643–656
http://dx.doi.org:/10.1016/j.neuron.2009.08.014

The molecular underpinnings of exploration and its link to learning and memory remain poorly understood. Here we show that inducible, modest overexpression of neuronal calcium sensor 1 (Ncs1) selectively in the adult murine dentate gyrus (DG) promotes a specific form of exploratory behavior. The mice also display a selective facilitation of longterm potentiation (LTP) in the medial perforant path and a selective enhancement in rapid-acquisition spatial memory, phenotypes that are reversed by direct application of a cell-permeant peptide (DNIP) designed to interfere with NCS-1 binding to the dopamine type-2 receptor (D2R). Moreover, the DNIP and the D2R-selective antagonist L-741,626 attenuated exploratory behavior, DG LTP, and spatial memory in control mice. These data demonstrate a role for NCS-1 and D2R in DG plasticity and provide insight for understanding how the DG contributes to the origin of exploration and spatial memory acquisition.

Neuroligin 2 Drives Postsynaptic Assembly at Perisomatic Inhibitory Synapses through Gephyrin and Collybistin

Alexandros Poulopoulos, Gayane Aramuni, Guido Meyer, Tolga Soykan, et al.
Neuron 63, 628–642, Sept 10, 2009
http://dx.doi.org:/10.1016/j.neuron.2009.08.023

In the mammalian CNS, each neuron typically receives thousands of synaptic inputs from diverse classes of neurons. Synaptic transmission to the postsynaptic neuron relies on localized and transmitter-specific differentiation of the plasma membrane with postsynaptic receptor, scaffolding, and adhesion proteins accumulating in precise apposition to presynaptic sites of transmitter release. We identified protein interactions of the synaptic adhesion molecule neuroligin 2 that drive postsynaptic differentiation at inhibitory synapses. Neuroligin 2 binds the scaffolding protein gephyrin through a conserved cytoplasmic motif and functions as a specific activator of collybistin, thus guiding membrane tethering of the inhibitory postsynaptic scaffold. Complexes of neuroligin 2, gephyrin and collybistin are sufficient for cell-autonomous clustering of inhibitory neurotransmitter receptors. Deletion of neuroligin 2 in mice perturbs GABAergic and glycinergic synaptic transmission and leads to a loss of postsynaptic specializations specifically at perisomatic inhibitory synapses.

A Subset of Ventral Tegmental Area Neurons is Inhibited by Dopamine, 5-Hydroxytryptamine and Opioids

L. Cameron, M. W. Wessendorf and J. T. Williams
Neuroscience 1997; 77(1), pp. 155–166 PII: S0306-4522(96)00444-7

Neurons originating in the ventral tegmental area are thought to play a key role in the formation of addictive behaviors, particularly in response to drugs such as cocaine and opioids. In this study we identified different populations of ventral tegmental area neurons by the pharmacology of their evoked synaptic potentials and their response to dopamine, 5-hydroxytryptamine and opioids. Intracellular recordings were made from ventral tegmental area neurons in horizontal slices of guinea-pig brain and electrical stimulation was used to evoke synaptic potentials. The majority of cells (61.3%) hyperpolarized in response to dopamine, depolarized to 5-hydroxytryptamine, failed to respond to [Met]5enkephalin and exhibited a slow GABAB-mediated inhibitory postsynaptic potential. A smaller proportion of cells (11.3%) hyperpolarized in response to [Met]5enkephalin, depolarized to 5-hydroxytryptamine, failed to respond to dopamine and did not exhibit a slow inhibitory postsynaptic potential. These two groups of cells corresponded to previously described ‘‘principal’’ and ‘‘secondary’’ cells, respectively. A further group of cells (27.4%) was identified that, like the principal cells, hyperpolarized to dopamine.

However, these ‘‘tertiary cells’’ also hyperpolarized to both 5-hydroxytryptamine and [Met]5enkephalin and exhibited a slow, cocaine-sensitive 5-hydroxytryptamine1A-mediated inhibitory postsynaptic potential. When principal and tertiary cells were investigated immuno-histochemically, 82% of the principal cells were positive for tyrosine hydroxylase compared
with only 29% of the tertiary cells. The 5-hydroxytryptamine innervation of both these cell types was investigated and a similar density of putative contacts was observed near the somata and dendrites in both groups. This latter finding suggests that the existence of a 5-hydroxytryptamine-mediated inhibitory postsynaptic potential in the tertiary cells may be determined by the selective expression of 5-hydroxytryptamine receptors, rather than the distribution or density of the 5-hydroxytryptamine innervation.
We conclude that tertiary cells are a distinct subset of ventral tegmental area neurons where cocaine and μ-opioids both mediate inhibition.

Dopamine reward circuitry: Two projection systems from the ventral midbrain to the nucleus accumbens–olfactory tubercle complex

Satoshi Ikemoto
Brain Research Reviews 56 (2007) 27–78
http://:dx.doi.org:/10.1016/j.brainresrev.2007.05.004

Anatomical and functional refinements of the meso-limbic dopamine system
of the rat are discussed. Present experiments suggest that dopaminergic neurons localized in the posteromedial ventral tegmental area (VTA) and central linear nucleus raphe selectively project to the ventromedial striatum (medial olfactory tubercle and medial nucleus accumbens shell), whereas
the anteromedial VTA has few if any projections to the ventral striatum,
and the lateral VTA largely projects to the ventrolateral striatum (accumbens
core, lateral shell and lateral tubercle). These findings complement the recent behavioral findings that cocaine and amphetamine are more rewarding when administered into the ventromedial striatum than into the ventrolateral striatum. Drugs such as nicotine and opiates are more rewarding when administered into the posterior VTA or the central linear nucleus than into
the anterior VTA. A review of the literature suggests that
(1) the midbrain has corresponding zones for the accumbens core and medial shell;
(2) the striatal portion of the olfactory tubercle is a ventral extension of the nucleus accumbens shell; and
(3) a model of two dopamine projection systems from the ventral midbrain to the ventral striatum is useful for understanding reward function.
The medial projection system is important in the regulation of arousal characterized by affect and drive and plays a different role in goal directed learning than the lateral projection system, as described in the variation–selection hypothesis of striatal functional organization.

Metabolic hormones, dopamine circuits, and feeding

Nandakumar S. Narayanan, Douglas J. Guarnieri, Ralph J. DiLeone
Frontiers in Neuroendocrinology 31 (2010) 104–112
http://dx.doi.org:/10.1016/j.yfrne.2009.10.004

Recent evidence has emerged demonstrating that metabolic hormones such as ghrelin and leptin can act on ventral tegmental area (VTA) midbrain dopamine neurons to influence feeding. The VTA is the origin of mesolimbic dopamine neurons that project to the nucleus accumbens (NAc) to influence behavior. While blockade of dopamine via systemic antagonists or targeted gene delete can impair food intake, local NAc dopamine manipulations have little effect on food intake. Notably, non-dopaminergic manipulations in the VTA and NAc produce more consistent effects on feeding and food choice. More recent genetic evidence supports a role for the substantia nigra-striatal dopamine pathways in food intake, while the VTA–NAc circuit is more likely involved in higher-order aspects of food acquisition, such as motivation and cue associations. This rich and complex literature should be considered in models of how peripheral hormones influence feeding behavior via action on the midbrain circuits.

Control of brain development and homeostasis by local and systemic insulin signaling

Liu, P. Speder & A. H. Brand
Diabetes, Obesity and Metabolism 16 (Suppl. 1): 16–20, 2014

Insulin and insulin-like growth factors (IGFs) are important regulators of growth and metabolism. In both vertebrates and invertebrates, insulin/IGFs are made available to various organs, including the brain, through two routes: the circulating systemic insulin/IGFs act on distant organs via endocrine signaling, whereas insulin/IGF ligands released by local tissues act in a paracrine or autocrine fashion. Although the mechanisms governing the secretion and action of systemic insulin/IGF have been the focus of extensive investigation, the significance of locally derived insulin/IGF has only more recently come to the fore. Local insulin/IGF signaling is particularly important for the development and homeostasis of the central nervous system, which is insulated from the systemic environment by the blood–brain barrier. Local insulin/IGF signaling from glial cells, the blood–brain barrier and the cerebrospinal fluid has emerged as a potent regulator of neurogenesis. This review will address the main sources of local insulin/IGF and how they affect neurogenesis during development. In addition, we describe how local insulin/IGF signaling couples neural stem cell proliferation with systemic energy state in Drosophila and in mammals.

Pharmacology, Physiology, and Mechanisms of Action of Dipeptidyl Peptidase-4 Inhibitors

Erin E. Mulvihill and Daniel J. Drucker
Endocrine Reviews 35: 992–1019, 2014
http://dx.doi.org/10.1210/er.2014-1035

Dipeptidyl peptidase-4 (DPP4) is a widely expressed enzyme transducing actions through an anchored transmembrane molecule and a soluble circulating protein. Both membrane-associated and soluble DPP4 exert
catalytic activity, cleaving proteins containing a position 2 alanine or proline. DPP4-mediated enzymatic cleavage alternatively inactivates peptides or generates new bioactive moieties that may exert competing or novel activities. The widespread use of selective DPP4 inhibitors for the treatment of type 2 diabetes has heightened interest in the molecular mechanisms through which DPP4 inhibitors exert their pleiotropic actions. Here we review the biology ofDPP4with a focus on:
1) identification of pharmacological vs physiological DPP4 substrates; and
2) elucidation of mechanisms of actions of DPP4 in studies employing genetic elimination or chemical reduction of DPP4 activity.
We review data identifying the roles of key DPP4 substrates in transducing the glucoregulatory, anti-inflammatory, and cardiometabolic actions of DPP4  inhibitors in both preclinical and clinical studies. Finally, we highlight experimental pitfalls and technical challenges encountered in studies designed to understand the mechanisms of action and downstream targets activated by inhibition of DPP4.
Dipeptidyl peptidase-4 (DPP4) is a multifunctional protein that exerts biological activity through pleiotropic actions including:

  • protease activity (1),
  • association with adenosine deaminase (ADA) (2),
  • interaction with the extracellular matrix (3),
  • cell surface coreceptor activity mediating viral entry (4), and
  • regulation of intracellular signal transduction coupled to control of cell migration and proliferation (5).

The complexity of DPP4 action is amplified by the panoply of bioactive DPP4 substrates, which in turn act as elegant biochemical messengers in multiple tissues, including the immune and neuroendocrine systems.

DPP4 transmits signals across cell membranes and interacts with other membrane proteins (Figure). Remarkably, most of the protein is extracellular, including the C-terminal catalytic domain, a cysteine-rich area, and a large glycosylated region linked by a flexible stalk to the transmembrane segment. Only six N-terminal amino acids are predicted to extend into the cytoplasm. The active site, Ser 630, is flanked by the classic serine peptidase motif Gly-Trp-Ser630-Tyr-Gly-Gly-Tyr-Val.

Membrane-bound DPP4

Membrane-bound DPP4

Membrane-bound DPP4 contains residues 1–766, whereas sDPP4 contains residues 39–766. sDPP4 is lacking the cytoplasmic domain [residues 1–6], transmembrane domain [residues 7–28], and the flexible stalk [residues 29–39]. Both membrane-bound and circulating sDPP4 share many domains including the glycosylated region [residues 101–535, specific residues 85,92, 150], ADA binding domain [340–343], fibronectin binding domain [468–479], cysteine-rich domain [351–506, disulfide bonds are formed from 385–394, 444–472, and 649–762], and the catalytic domain [507–766 including residues composing the catalytic active site 630, 708, and 740].

DPP4 activity is subject to regulation at many levels, including control of gene and protein expression, interaction with binding partners, and modulation of enzyme activity. The DPP4 gene does not contain conventional TATAA or CCAAT promoter sequences but is characterized by a cytosine/guanine-rich promoter region.
DPP4 contains eight to 11 potential N-glycosylation sites, which can contribute to its folding and stability. Although glycosylation may contribute approximately 18–25% of the total molecular weight, mutational analysis has determined that the glycosylation sites are not required for dimerization, catalytic activity, or ADA binding. However, N-terminal sialylation facilitates trafficking of DPP4 to the apical membrane. Interestingly, molecular analysis of DPP4 isoforms isolated from the rat kidney brush border membrane reveals extensive heterogeneity in the oligosaccharides of DPP4.DPP4 was first investigated for its role in hydrolysis of dietary prolyl peptides (58); subsequent studies using DPP4 isolated using immunoaffinity chromatography and ADA binding identified DPP4 as the primary enzyme responsible for the generation of Gly-Prop-nitroanilide substrates in human serum. It is now known that DPP4 can cleave dozens of peptides, including chemokines, neuropeptides, and regulatory peptides, most containing a proline or alanine residue at position 2 of the amino-terminal region. Despite the preference for a position 2 proline, alternate residues (hydroxyproline, dehydroproline > alanine >,  glycine, threonine, valine, or leucine) at the penultimate position are also cleaved by DPP4, suggesting a required stereochemistry. The DPP4 cleavage at postproline peptide bonds inactivates peptides and/or generates new bioactive peptides (see Figure), thereby regulating diverse biological processes.

DPP4 cleavage regulates substrate-receptor interactions

DPP4 cleavage regulates substrate-receptor interactions

DPP4 cleavage regulates substrate/receptor interactions. A, DPP4 cleaves NPY [1–36] and PYY [1–36]. The intact forms of these peptides signal through Y1R-Y5R. After DPP4 cleavage, NPY [3–36] and PYY [3–36] are generated and preferentially signal through the Y2R and Y5R. B, DPP4 cleaves SP [1–11], which signals through the NK1R receptor to generate SP [5–11], which can signal through (NK1R, -2R, -3R).

GHRH and IGF-1

GHRH [1–44] and [1–40] are produced in the arcuate nucleus of the hypothalamus and bind its receptor on the anterior pituitary to stimulate the release of GH, and in turn, GH stimulates hepatic IGF-1 release. GHRH was among the first peptides to be identified as a DPP4 substrate; it is rapidly degraded in rodent and human plasma to GHRH [3–44]/GHRH [3–40], and this cleavage was blocked upon incubation of human plasma with the DPP4 inhibitor, diprotin A (99).GHRH[1–44] or [1–40] exhibits a very short half-life (6 min) andDPP4 cleavage was initially perceived to be a critical regulator of GHRH bioactivity and, in turn, the GH-IGF-1 axis. IGF-1, the downstream effector of GHRH and GH, is a 105-amino acid protein produced mainly by the liver.
IGF-1 was identified as a pharmacological DPP4 substrate by matrix-assisted laser desorption/ionization-time of flight analysis of molecular forms of IGF-1 generated after incubation with DPP4 purified from baculovirus-infected insect cells. However, studies in pigs treated with sitagliptin at doses inhibiting 90% of DPP4 activity failed to demonstrate an increase in active intact IGF-1.
Clinically, treatment of healthy human male subjects with sitagliptin (25–600 mg) for 10 days did not produce increased concentrations of serum IGF-1 or IGF-binding protein 3 as measured by ELISA. Furthermore, Dpp4/ mice or rats do not exhibit increased organ growth or body size. Hence, the available data suggest that although DPP4 cleaves and inactivates both GHRH and IGF-1, enzymatic inactivation by DPP4 is not the major mechanism regulating the bioactivity of the GHRH-IGF-1 axis.

The role of acute cortisol and DHEAS in predicting acute and chronic PTSD symptoms

Joanne Mouthaan, Marit Sijbrandij, Jan S.K. Luitse
Psychoneuroendocrinology (2014) 45, 179—186
http://dx.doi.org/10.1016/j.psyneuen.2014.04.001

Background: Decreased activation of the hypothalamus—pituitary—adrenal (HPA) axis in response to stress is suspected to be a vulnerability factor for posttraumatic stress disorder (PTSD). Previous studies showed inconsistent findings regarding the role of cortisol in predicting PTSD. In addition, no prospective studies have examined the role of dehydroepiandrosterone (DHEA), or its sulfate form DHEAS, and the cortisol-to-DHEA(S) ratio in predicting PTSD. In this study, we tested whether acute plasma cortisol, DHEAS and the cortisol-to-DHEAS ratio predicted PTSD symptoms at 6 weeks and 6 months post-trauma. Methods: Blood samples of 397 adult level-1 trauma center patients, taken at the trauma resuscitation room within hours after the injury, were analyzed for cortisol and DHEAS levels. PTSD symptoms were assessed at 6 weeks and 6 months post-trauma with the Clinician Administered PTSD Scale. Results: Multivariate linear regression analyses showed that lower cortisol predicted PTSD symptoms at both 6 weeks and 6 months, controlling for age, gender, time of blood sampling, injury, trauma history, and admission to intensive care. Higher DHEAS and a smaller cortisol-to-DHEAS ratio predicted PTSD symptoms at 6 weeks, but not after controlling for the same variables, and not at 6 months. Conclusions: Our study provides important new evidence on the crucial role of the HPA-axis in response to trauma by showing that acute cortisol and DHEAS levels predict PTSD symptoms in survivors of recent trauma.
Neurobiology of DHEA and effects on sexuality, mood and cognition

  1. Pluchino, P.Drakopoulos, F.Bianchi-Demicheli, J.M.Wenger
    J Steroid Biochem & Molec Biol 145 (2015) 273–280
    http://dx.doi.org/10.1016/j.jsbmb.2014.04.012

Dehydroepiandrosterone (DHEA) and its sulfate ester, DHEAS, are the most abundant steroid hormones in the humans. However, their physiological significance, their mechanisms of action and their possible roles as treatment are not fully clarified. Biological actions of DHEA(S) in the brain involve neuroprotection, neurite growth, neurogenesis and neuronal survival, apoptosis, catecholamine synthesis and secretion, as well as anti-oxidant, anti- inflammatory and antiglucocorticoid effects. In addition, DHEA affects neurosteroidogen is and endorphin synthesis/release. We also demonstrated in a model of ovariectomized rats that DHEA therapy increases proceptive behaviors, already after 1 week of treatment, affecting central function of sexual drive. In women, the analyses of clinical outcomes are far from being conclusive and many issues should still be addressed. Although DHEA preparations have been available in the market since the 1990s, there are very few definitive reports on the biological functions of this steroid. We demonstrate that 1 year DHEA administration at the dose of 10mg provided a significant improvement in comparison with vitamin D in sexual function
and in frequency of sexual intercourse in early postmenopausal women. Among symptomatic women, the spectrum of symptoms responding to DHEA requires further investigation, to define the type of sexual symptoms (e.g. decreased sexual function or hypoactive sexual desire disorder) and the degree of mood/cognitive symptoms that could be responsive to hormonal treatment.
In this regard, our findings are promising, although they need further exploration with a larger and more representative sample size.
Although adrenal cortex is considered to be the primary source of DHEAS in the brain, it was reported that DHEAS did not dis- appear or decrease in the brain 15 days neither after orchiectomy, adrenalectomy, or both, nor after the inhibition of adrenal secretion by dexamethasone. DHEA and DHEAS were among the first neurosteroids identified in rat brains. Cytochrome P450c17 was found in a subset of neurons of embryonic rodent brains. While P450c17 protein was readily detected in the brain, the abundance of P450c17 mRNA transcripts in the embryonic mouse brain or hippocampus of adult male rats was low, and was approximated to be 1/200th of the expression in testis.
DHEAS may be synthesized in the brain from DHEA. Sulfation of DHEA has been observed in the brains of rhesus monkeys in vivo and in human fetal brain slices in vitro. DHEA sulfotransferase (HSTor SULT2A1) is an enzyme that sulfonates DHEA (in addition to pregnenolone).Western blotting and immune-histochemistry showed protein expression of an HST in adult Wistar rat brain. In addition SULT2A1 mRNA expression has been shown in rat brains. DHEAS is predominately transported out of the brain across the blood–brain barrier and DHEAS found in the brain is most likely due to local synthesis . DHEA(S) may mediate some of its actions through conversion into more potent sex steroids and activation of androgen or estrogen receptors in tissue.
According to existing assumption of the biology of depression, DHEA(S) ability to modulate many neurobiological actions could underlie relationships between endogenous and/or exogenously- supplemented DHEA(S) concentrations and depression. There is evidence that DHEAS concentrations are negatively correlated with ratings of depressed mood. However, the remaining literature examining plasma and serum DHEA(S) concentrations in depression is still inconsistent and other plasma indexes were studied in order to more accurately discriminate depressed from nondepressed individuals. Hypothalamic–pituitary–adrenal axis (HPA) hyperactivity has
been demonstrated in chronic diseases affecting nervous system disorders like depression. The end products of HPA axis, glucocorticoids (GCs), regulate many physiological functions and play an important role in affective regulation and dysregulation. Despite DHEAS levels which markedly decrease throughout adulthood, an increase in circulating cortisol with advanced age has been observed in human and nonhuman primates.
The most relevant aspect meriting attention is certainly the controversial finding among the studies that investigate the correlation of the endogenous DHEA sulfate (DHEAS) level, the aging process or organ illness with the results coming from studies focusing on the effects of exogenous DHEAS administration on brain function, sexuality, cardiovascular health and metabolic syndrome. Indeed, the marked age-related decline in serum DHEA and DHEAS has suggested that a deficiency of these steroids may be causally related to the development of a series of diseases that are generally associ- ated with aging. The postulated consequences of low DHEA levels include insulin resistance, obesity, cardiovascular disease, cancer, reduction of the immune defense system as well as psychosocial problems such as depression and a general deterioration in the sensation of well-being and cognitive function, DHEA replacement may seem an attractive treatment opportunity. Nevertheless, the analyses of clinical outcomes are far from being conclusive.

Dehydroepiandrosterone, its metabolites and ion channels

Hill, M. Dusková, L. Stárka
J Steroid Biochem & Molec Biology 145(2015)293–314
http://dx.doi.org/10.1016/j.jsbmb.2014.05.006

This review is focused on the physiological and pathophysiological relevance of steroids influencing the activities of the central and peripheral nervous systems with regard to their concentrations in body fluids and tissues in various stages of human life like the fetal development or pregnancy. The data summarized in this review shows that DHEA and its unconjugated and sulfated metabolites are physiologically and pathophysiologically relevant in modulating numerous ion channels and participate in vital functions of the human organism. DHEA and its unconjugated and sulfated metabolites including 5 _/ _-reduced androstane steroids participate in various physiological and pathophysiological processes like the management of GnRH cyclic release, regulation of glandular and neurotransmitter secretions, maintenance of glucose homeostasis on one hand and insulin insensitivity on the other hand, control of skeletalmuscle and smooth muscle activities including vasoregulation, promotion of tolerance to ischemia and other neuroprotective effects. In respect of prevalence of steroid sulfates over unconjugated steroids in the periphery and the opposite situation in the CNS, the sulfated androgens and androgen metabolites reach relevance in peripheral organs. The unconjugated androgens and estrogens are relevant in periphery and so much the more in the CNS due to higher concentrations of most unconjugated steroids in the CNS tissues than in circulation and peripheral organs.

Neurotrophins are proteins found within a broad range of cell types in the brain and periphery that facilitate neuronal growth, survival, and plasticity. The neurotrophin ‘‘superfamily’’ includes nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT3), neurotrophin-4/5 (NT4/5), and neurotrophin-6. Target tissues are hypothesized to regulate neuron survival by making neurotrophins available in limited amounts, resulting in selection of neurons with the best connectivity to the target tissue. NGF, in particular, is released by the target tissue and taken up in responsive neurons by receptor-mediated endocytosis. It is then transported retrogradedly into the cell where it exerts trophic effects. Lu et al. proposed a ‘‘Yin and Yang model,’’ whereby neurotrophic action is mediated by two principal classes of transmembrane receptor systems: the tyrosine kinase (Trk) receptors (including TrkA [selective for NGF], TrkB [selective for BDNF and NT4/5], and TrkC [selective for NT3]) and the neurotrophin receptor p75NTR. Each receptor type binds mature neurotrophins and/or neurotrophin precursors (proneurotrophins), creating a complex ‘‘balance’’ that then causes neuronal survival or death.
DHEA has been shown to evoke NGF mRNA expression in target cells. In a study of pregnant women, Schulte-Herbrüggen et al. showed no relationships between serum DHEAS and NGF. In contrast, we showed that DHEAS independently associated with salivary NGF (sNGF) in military men under baseline conditions, while DHEA did not. We now know that both DHEA(S) and NGF respond affirmatively to stressful insult, yet the association between these analytes during stress exposure is not understood. Characterization of this relationship has implications for prevention and treatment of traumatic stress and injury, degenerative disease management, and nerve repair. In this report, we extended our prior study of neuroprotective properties of DHEAS in men under baseline conditions to a prospective paradigm involving intense stress exposure in both men and women. We hypothesized that

(a) robust associations would prevail between total output of DHEAS and sNGF across the stress trajectory and at each time point,
(b) changes in DHEAS would predict corresponding changes in sNGF, and
(c) baseline DHEAS would positively predict total sNGF output across the stress trajectory.
We also explored the roles of testosterone and cortisol. In light of less definitive prior literature, directional hypotheses were not stated regarding these analytes.

In the first regression model, total hormone output (AUCG) of the independent variables (DHEAS, testosterone, and cortisol) combined to explain 63.7% of variance in sNGF output (F = 65.4, p < 0.001). Standardized beta coefficients revealed that testosterone exerted an independent effect (b = 0.80, p < 0.001), while the other predictors were not significant. In light of this unexpected finding, we then used regression-based causal steps modeling to evaluate whether testosterone mediated a hypothesized direct effect of DHEAS on sNGF. Following this approach, DHEAS predicted sNGF in an initial regression model (b = 0.45, p < 0.001). When testosterone was added, the direct effect of DHEAS (path c0) on sNGF was nearly eradicated and no longer significant (b = .04, p = .57), thus suggesting a mediated effect. An alternate statistical test (Sobel Test; 34) evaluating the hypothesized difference between the total effect (path c) and the direct effect (path c0) of DHEAS on sNGF produced a similar result (test statistic = 4.0, p < 0.001). Fig. 1 depicts positive association of DHEAS to sNGF, while Fig. 2 depicts Positive association of testosterone to sNGF.

Positive association of DHEAS total output and sNGF total output

Positive association of DHEAS total output and sNGF total output

Positive association of DHEAS total output and sNGF total output

Positive association of testosterone total output and sNGF total output

Positive association of testosterone total output and sNGF total output

Positive association of testosterone total output and sNGF total output.
The models were then decomposed at each time point. At baseline, the independent variables (DHEAS, testosterone, and cortisol) combined to account for 10.2% of variance in sNGF (F = 5.3, p < 0.01). Standardized beta coefficients showed that DHEAS exerted an independent effect on sNGF (b = 0.39, p < 0.001), while the other predictors were not significant. During stress exposure, the independent variables combined to account for 28.0% of variance in NGF (F = 15.8, p < 0.001). Again, DHEAS exerted an independent effect (b = 0.56, p < 0.001) while the other predictors were not significant. During recovery, the predictor set accounted for 18.0% of variance in sNGF (F = 9.2, p < 0.001), and DHEAS exerted an independent effect (b = 0.47, p < 0.001) while the other predictors did not.
The models were then decomposed relative to each change index. In terms of reactivity, the independent variables (DHEAS, testosterone, and cortisol reactivity) and covariate (sex) combined to account for 20.3% of variance in sNGF reactivity (F = 8.2, p < 0.001). Standardized beta coefficients revealed that DHEAS reactivity exerted an independent effect (b = 0.39, p < 0.001), while the other predictors were not significant. In terms of recovery, the predictors combined to account for 28.2% of variance in sNGF recovery (F = 15.5, p < 0.001); DHEAS recovery exerted an independent effect (b = 0.52, p < 0.001), as did testosterone recovery (b = [1]0.27, p < 0.01). In terms of residual elevation/depression, the independent variables explained 12.4% of variance in sNGF residual elevation (F = 6.2, p < 0.001). DHEAS residual elevation exerted an independent effect (b = 0.35, p < 0.001), while the other predictors did not.

Endocrine-Disrupting Chemicals and Human Growth and Maturation: A Focus on Early Critical Windows of Exposure

Julie Fudvoye, Jean-Pierre Bourguignon, Anne-Simone Parent
Vitamins and Hormones, 2014; 94: Chapt 1. 1-25.
http://dx.doi.org/10.1016/B978-0-12-800095-3.00001-8

Endocrine-disrupting chemicals (EDCs) are exogenous substances that interfere with hormone synthesis, metabolism, or action. In addition, some of them could cause epigenetic alterations of DNA that can be transmitted to the following generations. Because the developing organism is highly dependent on sex steroids and thyroid hormones for its maturation, the fetus and the child are very sensitive to any alteration of their hormonal environment. An additional concern about that early period of life comes from the shaping of the homeostatic mechanisms that takes place also at that time with involvement of epigenetic mechanisms along with the concept of fetal origin of health and disease. In this chapter, we will review the studies reporting effects of EDCs on human development. Using a translational approach, we will review animal studies that can shed light on some mechanisms of action of EDCs on the developing organism. We will focus on the major hormone-dependent stages of development: fetal growth, sexual differentiation, puberty, brain development, and energy balance. We will also discuss the possible epigenetic effects of EDCs on human development.

Several studies have reported that prenatal or early postnatal exposure to some EDCs is associated with alterations of cognitive or motor functions in children. Knowing the fundamental role played by thyroid hormones and sex steroids in cortex development, one can hypothesize that disruption of those hormones could cause alteration of the development of the cerebral cortex and of its functions later in life. We will review here the human data suggesting a causal effect for endocrine disrupters on impairment of cortical functions and approach some EDC mechanisms of action using animal models.

Thyroid hormones are known to be essential for brain development. They regulate progenitor proliferation and differentiation, neuron migration, and dendrite outgrowth (Parent, Naveau, Gerard, Bourguignon, & Westbrook, 2011). Even mild thyroid hormone insufficiency in humans can produce measurable deficits in cognitive functions (Zoeller & Rovet, 2004). Thyroid hormone action is mediated by two classes of nuclear receptors (Forrest & Vennstro¨m, 2000) that exhibit differential spatial and temporal expressions in the brain, suggesting that thyroid hormones have variable functions during brain development. This differential expression of thyroid hormone receptors explains the critical period of thyroid hormone action on brain development as suggested by models of maternal hypothyroidism or congenital hypothyroidism.

Depending on the timing of onset of hypothyroidism, the offspring will display problems of visual attention, gross or fine motor skills, or language and memory skills. Similarly, one can hypothesize that disruption of thyroid function by EDCs will have different effects based on the timing of exposure. However, few studies focused on that aspect. Polychlorinated biphenyls (PCBs) form a group of widespread environmental contaminants composed of 209 different congeners used in a wide variety of applications. Their production was banned in the 1970s but PCBs are still present in the environment due to their high stability. PCBs were among the first EDCs identified as responsible for alterations of cognitive functions. Indeed, impaired memory and altered learning abilities have been associated with prenatal exposure to EDCs in humans and In animal models, perinatal exposure to PCBs has been consistently associated with a decrease of thyroid hormone concentration in maternal serum as well as pup serum. Some but not all epidemiological studies in human have found an association between PCB body burden and thyroid hormone levels. This disruption of thyroid function could explain some of the effects of PCBs on the developing brain. Indeed, animal models have shown that the ototoxic effects of PCBs could be partially ameliorated by thyroxin replacement and PCBs seem to alter some of the developmental processes in the cortex and the cerebellum that are dependent on thyroid hormones. However, recent publications raise important issues.

As it is the case for other EDCs, some windows of susceptibility have been identified during pre- and postnatal brain development. Recent studies have shown that exposure to PBDEs causes alteration of thyroid hormone levels in pregnant women and infants as it is the case in rodents. Only very few studies, however, have focused on the molecular or cellular effects of perinatal exposure to PBDEs in vivo. Viberg et al. have reported a decrease of cholinergic nicotinic receptors in the hippocampus after exposure to BDE-99 and BDE-153. However, the link between such a decrease and the behavioral effects of PBDEs is still unclear. Other teams have reported that exposure to PBDEs reduced hippocampal long term potentiation and decreased brain-derived neurotrophic factor expression in the brain. While several studies have reported negative effect of PBDEs on brain development and cognitive function in animals, there is relatively little information about adverse health effects of PBDEs in humans. Some very recent studies have identified a correlation between prenatal exposure to PBDEs and alteration of cognitive functions.

Endocrine-Disrupting Chemicals: Elucidating Our Understanding of Their Role in Sex and Gender-Relevant End Points

Cheryl A. Frye
Vitamins and Hormones, 2014; 94: 41-98
http://dx.doi.org/10.1016/B978-0-12-800095-3.00003-1

Endocrine-disrupting chemicals (EDCs) are diverse and pervasive and may have significant consequence for health, including reproductive development and expression of sex-/gender-sensitive parameters. This review chapter discusses what is known about common EDCs and their effects on reproductively relevant end points. It is proposed that one way that EDCs may exert such effects is by altering steroid levels (androgens or 17-estradiol, E2) and/or intracellular E2 receptors (ERs) in the hypothalamus and/or hippocampus. Basic research findings that demonstrate developmentally sensitive end points to androgens and E2 are provided. Furthermore, an approach is suggested to examine differences in EDCs that diverge in their actions at ERs to elucidate their role in sex-/gender-sensitive parameters.

Reproductive dysfunction among adults and emotional, attentional, and behavioral disorders among children are on the rise. Sperm counts and fertility have declined in the last 50 years . Incidence of attention-deficit hyperactivity disorder (ADHD) and autism has increased in the last 30 years. These increases in reproductive dysfunction and developmental disorders may be due to increased exposure to environmental contaminants, although there is controversy about the relationship between exposure and these effects.
Many contaminants in the environment, including polychlorinated biphenyls (PCBs), dioxins, and metals, accumulate in exposed individuals and may have adverse consequences due to effects as endocrine-disrupting chemicals (EDCs). EDCs may have effects by altering steroid levels (androgens or 17β-estradiol, E2) and/or intracellular E2 receptors (ERs) in the hypothalamus and/or hippocampus.
Steroid hormones, during critical periods of development, organize sexual dimorphisms in brain and behavior and give rise to sex differences in later responses to steroid hormones. EDCs can profoundly disrupt reproductive responses following adult exposure and result in pervasive effects that extend throughout the life of their offspring. Many nonreproductive behaviors, such
as spatial performance, activity, and arousal, are also sexually dimorphic and organized and activated by steroid hormones. Thus, EDCs may affect reproductive and the aforementioned nonreproductive parameters by altering E2 levels and/or ER binding in the hypothalamus and/or hippocampus.
Results from the literature and preliminary data will be presented that demonstrate our use of a whole-animal model to begin to investigate effects of exposure (in adulthood and/or development) to EDCs on steroid levels (androgens and E2), actions at ERs (in hypothalamus and hippocampus), and reproductive-sensitive measures (anogenital distance, accessory structure weight, onset of puberty and sexual maturity, and reproductive behavior) and nonreproductive behaviors (spatial performance, play behavior, and arousal) throughout development.

A common feature of many environmental contaminants is their estrogenic effects. Some contaminants can alter production of E2 and/or androgens or act as agonists or antagonists for intracellular or membrane ERs. Thus, the term “endocrine-disrupting chemicals” (EDCs) in this chapter is used to refer to contaminants with these effects. An important question considered here is the extent to which EDCs’ actions to alter E2 levels and/or ER binding in the hypothalamus or hippocampus mitigates effects on reproductive or nonreproductive processes. There are potential pervasive, negative effects of endocrine disrupters on steroid sensitive tissues, which may confer risk to disease states, such as cancer, heart disease, and neurodegenerative disorders. The following discussion provides evidence that exposure to EDCs during development may result in permanent, lifelong differences in sexual function and reproductive ability, as well as cognitive function and/or emotional reactivity/arousal. Gonad development, sex determination, and reproductive success of offspring are highly dependent on sex hormone systems. The developing organism is exquisitely sensitive to alterations in hormone function. In the early embryonic state, the gonads of human males and females are morphologically identical. Sexual differentiation begins under hormonal influence during the fifth and sixth weeks of fetal development, and thus, alterations in hormones during this highly sensitive period can have profound consequences. Disruption of the sex steroid system during fetal stages of life results in profound adverse developmental reproductive effects, as is well known from the effects of DES. The balance of estrogens and androgens is critical for normal development, growth, and functioning of the reproductive system. Although especially important during development, this balance is important throughout life for the preservation of normal feminine or masculine traits, as well as the expression of some sexually dimorphic behaviors (sex, spatial performance, and arousal).

Proposed negative effects of exposure to endocrine disrupters during development in people and in animals. The focus here is on vulnerability to sexually dimorphic processes that are estrogen-sensitive, such as reproductive, cognitive, and emotional development and associated behavioral processes

The existing data clearly indicate that developmental exposure to EDCs can adversely affect sexual development of people and animals; however, there are different effects depending upon the EDCs and when in development exposure occurs. Therefore, we consider the next effects of EDCs exposure at different point in development and the consequences for reproductive development and behavior, as well as E2 levels and hypothalamic ER binding.
Steroid hormones also play a critical role in neurodevelopment that influences not only reproductive but also nonreproductive behaviors that show sex differences. Specific behavioral differences in nonreproductive behaviors between males and females include differences in spatial learning, play, exploration, activity levels, novelty-seeking behavior, and emotional reactivity. These sex dimorphisms are thought to reflect adaptive differences for behavioral strategies in coping as a result of sexual selection. Moreover, these sexually dimorphic behaviors may be relevant for concerns regarding increased developmental, cognitive, or emotional disabilities over the past 30 years. Also, behaviors are particularly sensitive measures of effects of EDCs.
EDCs can alter cognitive development. Some, but not all, studies have shown a predictive relationship between prenatal PCB exposure and cognitive development in infancy through preschool years. EDCs have direct effects on nervous system function. Long-term potentiation (LTP), a form of synaptic plasticity used as a model system for study of cognitive potential, is altered by PCBs and lead. The protein kinase C (PKC)-signaling pathway is involved in the modulation of learning, memory, and motor behavior and may be a target of E2’s actions. PCBs also alter PKC signaling. Although findings provide evidence that EDCs can alter cognitive performance, these measures of cognition are neither sexually dimorphic nor E2- or ER-dependent.
There are sex-specific effects of perinatal PCB and dioxin exposure on spatial learning. Yu-Cheng boys that were prenatally exposed to high levels of PCBs and PCDFs when their mothers were accidentally exposed to these contaminants in rice oil show more disrupted cognitive development, mainly spatial function, than did exposed girls. In animal studies, spatial learning that favors males is mediated by perinatal exposure to androgens. Gestational and lactational exposure to ortho-substituted PCBs produces spatial deficits at adolescence in male mice or adulthood in male rats. The sparse data suggest that developmental exposure to EDCs disrupts spatial memory. Furthermore, Exposure during adulthood to EDCs can also have activational effects on spatial memory. Females exposed to a phytoestrogen-rich diet exhibit “masculinized” spatial performance in a radial arm maze, while males fed with a phytoestrogen-free diet show “feminized” performance.
An important question is what are the mechanisms by which developmental and/or adult exposure to EDCs alters spatial performance? There is evidence for sex differences in spatial performance and activational effects of E2 in adulthood to alter spatial performance of rats. Systemic or intrahippocampal administration of E2 improves spatial performance of female rats. Further, E2’s actions at intracellular ERs in the hippocampus of adults do not seem to be required to mediate these effects on spatial performance.
EDCs may have effects on E2 metabolism in a number of ways. First, some EDCs can alter serum lipid concentrations. Cholesterol is the precursor for the production of E2 and other steroid hormones (see Fig. 3.3). Second, there is also evidence that some EDCs can alter metabolism enzymes that are necessary for converting cholesterol to steroid hormones. Induction of CYP occurs when EDCs, such as TCDD, bind the aromatic hydrocarbon receptor (AhR). There is a firm link between PCBs, enzyme induction, and AhR. The binding of EDCs with AhR can result in antiestrogenic activity through increased metabolism and depletion of endogenous E2. Elevated levels of CYP enzymes, primarily expressed not only in the liver but also in the brain and other tissues, result in increased E2 metabolism and excretion. Alternatively, compounds that are metabolized by P450s may result in a net estrogenic effect if they inhibit endogenous estrogens from being metabolized.
Steroid hormones are lipid molecules with limited solubility in plasma and are accordingly carried through the plasma compartment to target cells by specific plasma transporter proteins. Each transporter protein has a specific ligand-binding domain for its associated hormone. It is generally accepted that the “free” formof the steroid hormone, and not the conjugate of the hormone with its plasma transport protein, enters target cells and binds with the appropriate receptor. Receptors for the steroid hormones are proteins located primarily in the cell nucleus or partitioned between the cytoplasm and the nucleus. The unoccupied steroid receptors may reside in the cell as heterodimeric complexes with the 90 kDa heat-shock protein, which prevents the receptor from binding with the DNA until the receptor has first bound with its steroid hormone. Once the hormone binds to the receptor, the hormone receptor complexes with the heterodimeric heat-shock protein and undergoes a conformational change and is activated. The activated receptor binds with DNA at a specific site, initiating gene transcription.

Traditional effects of steroid hormones at their cognate steroid receptors

Traditional effects of steroid hormones at their cognate steroid receptors

Traditional effects of steroid hormones at their cognate steroid receptors, which act as transcription factors. In this example, effects of steroid hormones, such as estradiol, to bind estrogen receptor (ER) subtypes, referred to as ERa and ERb, are shown.

Beyond traditional actions solely through intracellular cognate estrogen receptors (ERs; ERa and ERb), steroids, such as estradiol, and estradiol-mimetics (endocrine disrupters) may have novel actions involving membrane bound ERs, other neurotransmitter systems (e.g., NMDA receptor), and signal transduction cascades (e.g., growth factors, MAPK).

To date, there has been little investigation in a whole-animal model of the effects of EDCs on E2 levels and/or activity at intracellular ERs in the brain. Thus, changes in E2 levels and ER activity in the hypothalamus and hippocampus, concomitant with alterations in endocrine parameters and reproductive behavior and nonreproductive behavior, respectively, are
needed to elucidate tissue specificity of EDCs’ functions and mechanisms.

Low-Dose Effects of Hormones and Endocrine Disruptors

Laura N. Vandenberg
Vitamins and Hormones, 2014; 94: 129-165
http://dx.doi.org/10.1016/B978-0-12-800095-3.00005-5

Endogenous hormones have effects on tissue morphology, cell physiology, and behaviors at low doses. In fact, hormones are known to circulate in the part-per-trillion and part-per-billion concentrations, making them highly effective and potent signaling molecules.

Many endocrine-disrupting chemicals (EDCs) mimic hormones, yet there is strong debate over whether these chemicals can also have effects at low doses. In the 1990s, scientists proposed the “low-dose hypothesis,” which postulated that EDCs affect humans and animals at environmentally relevant doses. This chapter focuses on data that support and refute the low-dose hypothesis. A case study examining the highly controversial example of bisphenol A and its low-dose effects on the prostate is examined through the lens of endocrinology. Finally, the chapter concludes with a discussion of factors that can influence the ability of a study to detect and interpret low-dose effects appropriately.

Since EDCs began to be studied in depth in the 1990s, there has been intense debate over whether the public should be concerned about low level exposures to these chemicals. The low-dose hypothesis, proposed at that time, has steadily accumulated evidence that EDCs have actions at low doses, and these effects are not necessarily predicted from high-dose toxicology testing. In 2002, the NTP expert panel reported evidence for low-dose effects for a small number of EDCs and estradiol. In 2012, an updated approach identified several dozen additional EDCs with evidence for low-dose effects. Further, epidemiology studies continue to find relationships between EDC exposure levels and diseases in the general public, which has raised concerns because the general public is exposed to a large number of environmental chemicals at low doses. For decades, hormones have been known to produce striking changes in tissue morphology, physiology, and behaviors at exceedingly low doses.

A relatively large body of evidence suggests that EDCs, and in particular those environmental chemicals that mimic endogenous hormones, have similar effects at low doses. Although there is still no consensus about the universality of “low-dose effects” in the toxicology community, the Endocrine Society (Diamanti-Kandarakis et al., 2009; Zoeller et al., 2012) believes not only that there is sufficient evidence in support of this phenomenon but also that it is time for public health agencies to make changes to risk assessment paradigms and give greater consideration to studies that specifically identify low-dose effects when considering risks from chemical exposures.

Bisphenol A interferes with synaptic remodeling

Tibor Hajszan, Csaba Leranth
Frontiers in Neuroendocrinology 31 (2010) 519–530
http://dx.doi.org:/10.1016/j.yfrne.2010.06.004

The potential adverse effects of Bisphenol A (BPA), a synthetic xenoestrogen, have long been debated. Although standard toxicology tests have revealed no harmful effects, recent research highlighted what was missed so far: BPA-induced alterations in the nervous system. Since 2004, our laboratory has been investigating one of the central effects of BPA, which is interference with gonadal steroid-induced synaptogenesis and the resulting loss of spine synapses. We have shown in both rats and nonhuman primates that BPA completely negates the ~70–100% increase in the number of hippocampal and prefrontal spine synapses induced by both estrogens and androgens. Synaptic loss of this magnitude may have significant consequences, potentially causing cognitive decline, depression, and schizophrenia, to mention those that our laboratory has shown to be associated with synaptic loss. Finally, we discuss why children may particularly be vulnerable to BPA, which represents future direction of research in our laboratory.

Bisphenol-A rapidly promotes dynamic changes in hippocampal dendritic morphology through estrogen receptor-mediated pathway by concomitant phosphorylation of NMDA receptor subunit NR2B

Xiaohong Xu ⁎, Yinping Ye, Tao Li, Lei Chen, Dong Tian, Qingqing Luo, Mei Lu
Toxicology and Applied Pharmacology 249 (2010) 188–196
http://dx.doi.org:/10.1016/j.taap.2010.09.007

Bisphenol-A (BPA) is known to be a potent endocrine disrupter. Evidence is emerging that estrogen exerts a rapid influence on hippocampal synaptic plasticity and the dendritic spine density, which requires activation of NMDA receptors. In the present study, we investigated the effects of BPA (ranging from 1 to 1000 nM), focusing on the rapid dynamic changes in dendritic filopodia and the expressions of estrogen receptor (ER) β and NMDA receptor, as well as the phosphorylation of NMDA receptor subunit NR2B in the cultured hippocampal neurons. A specific ER antagonist ICI 182,780 was used to examine the potential involvement of ERs. The results demonstrated that exposure to BPA (ranging from 10 to 1000 nM) for 30 min rapidly enhanced the motility and the density of dendritic filopodia in the cultured hippocampal neurons, as well as the phosphorylation of NR2B (pNR2B), though the expressions of NMDA receptor subunits NR1, NR2B, and ERβ were not changed. The antagonist of ERs completely inhibited the BPA-induced increases in the filopodial motility and the number of filopodia extending from dendrites. The increased pNR2B induced by BPA (100 nM) was also completely eliminated. Furthermore, BPA attenuated the effects of 17β-estradiol (17β-E2) on the dendritic filopodia outgrowth and the expression of pNR2B when BPA was co-treated with 17β-E2. The present results suggest that BPA, like 17β-E2, rapidly results in the enhanced motility and density of dendritic filopodia in the cultured hippocampal neurons with the concomitant activation of NMDA receptor subunit NR2B via an ER-mediated signaling pathway. Meanwhile, BPA suppressed the enhancement effects of 17β-E2 when it coexists with 17β-E2. These results provided important evidence suggesting the neurotoxicity of the low levels of BPA during the early postnatal development of the brain.

Bisphenol-A rapidly enhanced passive avoidance memory and phosphorylation of NMDA receptor subunits in hippocampus of young rats

Xiaohong Xu⁎, Tao Li, Qingqing Luo, Xing Hong, Lingdan Xie, Dong Tian
Toxicology and Applied Pharmacology 255 (2011) 221–228
http://dx.doi.org:/10.1016/j.taap.2011.06.022

Bisphenol-A (BPA), an endocrine disruptor, is found to influence development of brain and behaviors in rodents. The previous study indicated that perinatal exposure to BPA impaired learning-memory and inhibited N-methyl-D-aspartate receptor (NMDAR) subunits expressions in hippocampus during the postnatal development in rats; and in cultured hippocampal neurons, BPA rapidly promotes dynamic changes in dendritic morphology through estrogen receptor-mediated pathway by concomitant phosphorylation of NMDAR subunit NR2B. In the present study, we examined the rapid effect of BPA on passive avoidance memory and NMDAR in the developing hippocampus of Sprague–Dawley rats at the age of postnatal day 18. The results showed that BPA or estradiol benzoate (EB) rapidly extended the latency to step down from the platform 1 h after foot shock and increased the phosphorylation levels of NR1, NR2B, and mitogen-activated extracellular signal-regulated kinase (ERK) in hippocampus within 1 h. While 24 h after BPA or EB treatment, the improved memory and the increased phosphorylation levels of NR1, NR2B, ERK disappeared. Furthermore, pre-treatment with an estrogen receptors (ERs) antagonist, ICI182, 780, or an ERK-activating kinase inhibitor, U0126, significantly attenuated EB- or BPA-induced phosphorylations of NR1, NR2B, and ERK within 1 h. These data suggest that BPA rapidly enhanced short-term passive avoidance memory in the developing rats. A non-genomic effect via ERs may mediate the modulation of the phosphorylation of NMDAR subunits NR1 and NR2B through ERK signaling pathway.

Bisphenol A promotes dendritic morphogenesis of hippocampal neurons through estrogen receptor-mediated ERK1/2 signal pathway

Xiaohong Xu, Yang Lu, Guangxia Zhang, Lei Chen, Dong Tian, et al.
Chemosphere 96 (2014) 129–137
http://dx.doi.org/10.1016/j.chemosphere.2013.09.063

Bisphenol A (BPA), an environmental endocrine disruptor, has attracted increasing attention to its adverse effects on brain developmental process. The previous study indicated that BPA rapidly increased motility and density of dendritic filopodia and enhanced the phosphorylation of N-methyl-D-aspartate (NMDA) receptor subunit NR2B in cultured hippocampal neurons within 30 min. The purpose of the present study was further to investigate the effects of BPA for 24 h on dendritic morphogenesis and the underlying mechanisms. After cultured for 5 d in vitro, the hippocampal neurons from 24 h-old rat were infected by AdV-EGFP to indicate time-lapse imaging of living neurons. The results demonstrated that the exposure of the cultured hippocampal neurons to BPA (10, 100 nM) or 17β-estradiol (17β-E2, 10 nM) for 24 h significantly promoted dendritic development, as evidenced by the increased total length of dendrite and the enhanced motility and density of dendritic filopodia. However, these changes were suppressed by an ERs antagonist, ICI182,780, a non-competitive NMDA receptor antagonist, MK-801, and a mitogen activated ERK1/2-activating kinase (MEK1/2) inhibitor, U0126. Meanwhile, the increased F-actin (filamentous actin) induced by BPA (100 nM) was also completely eliminated by these blockers. Furthermore, the result of western blot analyses showed that, the exposure of the cultures to BPA or 17β-E2 for 24 h promoted the expression of Rac1/Cdc42 but inhibited that of RhoA, suggesting Rac1 (Ras related C3 botulinum toxinsubstrate 1)/Cdc42 (cell divisioncycle 42) and RhoA (Ras homologous A), the Rho family of small GTPases, were involved in BPA- or 17β-E2-induced changes in the dendritic morphogenesis of neurons. These BPA- or 17b-E2-induced effects were completely blocked by ICI182,780, and were partially suppressed by U0126. These results reveal that, similar to 17β-E2, BPA exerts its effects on dendritic morphogenesis by eliciting both nuclear actions and extranuclear-initiated actions that are integrated to influence the development of dendrite in hippocampal neurons.

Tyreoliberin (Trh) – The Regulatory Neuropeptide Of Cns Homeostasis
Danuta Jantas
Advances In Cell Biology 2;(4)/2010 (139–154)
http://dx.doi.org:/10.2478/v10052-010-0008-4

The physiological role of thyreoliberin (TRH) is the preservation of homeostasis within four systems
(i) the hypothalamic-hypophsysiotropic neuroendocrine system,
(ii) the brain stem/midbrain/spinal cord system,
(iii) the limbic/cortical system, and
(iv) the chronobiological system.

Thus TRH, via various cellular mechanisms, regulates a wide range of biological processes (arousal, sleep, learning, locomotive activity, mood) and possesses the potential for unique and widespread applications for treatment of human illnesses. Since the therapeutic potential of TRH is limited by its pharmacological profile (enzymatic instability, short half-life, undesirable effects), several synthetic analogues of TRH were constructed and studied in mono- or adjunct therapy of central nervous system (CNS) disturbances. The present article summarizes the current state of understanding of the physiological role of TRH and describes its putative role in clinical indications in CNS maladies with a focus on the action of TRH analogues.

Breakthrough in neuroendocrinology by discovering novel neuropeptides and neurosteroids: 2. Discovery of neurosteroids and pineal neurosteroids

Kazuyoshi Tsutsui, Shogo Haraguchi
General and Comparative Endocrinology 205 (2014) 11–22
http://dx.doi.org/10.1016/j.ygcen.2014.03.008

Bargmann–Scharrer’s discovery of ‘‘neurosecretion’’ in the first half of the 20th century has since matured into the scientific discipline of neuroendocrinology. Identification of novel neurohormones, such as neuropeptides and neurosteroids, is essential for the progress of neuroendocrinology. Our studies over the past two decades have significantly broadened the horizons of this field of research by identifying novel neuropeptides and neurosteroids in vertebrates that have opened new lines of scientific investigation in neuroendocrinology. We have established de novo synthesis and functions of neurosteroids in the brain of various vertebrates. Recently, we discovered 7α-hydroxypregnenolone (7α-OH PREG), a novel bioactive neurosteroid that acts as a key regulator for inducing locomotor behavior by means of the dopaminergic system. We further discovered that the pineal gland, an endocrine organ located close to the brain, is an important site of production of neurosteroids de novo from cholesterol (CHOL). The pineal gland secretes 7α-OH PREG and 3α,5α-tetrahydroprogesterone (3α,5α-THP; allopregnanolone) that are involved in locomotor rhythms and neuronal survival, respectively. Subsequently, we have demonstrated their mode of action and functional significance. This review summarizes the discovery of these novel neurosteroids and its contribution to the progress of neuroendocrinology.

Mechanisms of crosstalk between endocrine systems: Regulation of sex steroid hormone synthesis and action by thyroid hormones

Paula Duarte-Guterman, Laia Navarro-Martín, Vance L. Trudeau
General and Comparative Endocrinology 203 (2014) 69–85
http://dx.doi.org/10.1016/j.ygcen.2014.03.015

Thyroid hormones (THs) are well-known regulators of development and metabolism in vertebrates. There is increasing evidence that THs are also involved in gonadal differentiation and reproductive function. Changes in TH status affect sex ratios in developing fish and frogs and reproduction (e.g., fertility), hormone levels, and gonad morphology in adults of species of different vertebrates. In this review, we have summarized and compared the evidence for cross-talk between the steroid hormone and thyroid axes and present a comparative model. We gave special attention to TH regulation of sex steroid synthesis and action in both the brain and gonad, since these are important for gonad development and brain sexual differentiation and have been studied in many species. We also reviewed research showing that there is a TH system, including receptors and enzymes, in the brains and gonads in developing and adult vertebrates. Our analysis shows that THs influences sex steroid hormone synthesis in vertebrates, ranging from fish to pigs. This concept of crosstalk and conserved hormone interaction has implications for our understanding of the role of THs in reproduction, and how these processes may be dysregulated by environmental endocrine disruptors.

Insights into the structure of class B GPCRs

Kaspar Hollenstein, Chris de Graaf, Andrea Bortolato, Ming-Wei Wang, et al.
Trends in Pharmacological Sciences, Jan 2014; 35(1)
http://dx.doi.org/10.1016/j.tips.2013.11.001

The secretin-like (class B) family of G protein-coupled receptors (GPCRs) are key players in hormonal homeostasis and are interesting drug targets for the treatment of several metabolic disorders (such as type 2 diabetes, osteoporosis, and obesity) and nervous system diseases (such as migraine, anxiety, and depression). The recently solved crystal structures of the transmembrane domains of the human glucagon receptor and human corticotropin-releasing factor receptor 1 have opened up new opportunities to study the structure and function of class B GPCRs. The current review shows how these structures offer more detailed explanations to previous biochemical and pharmacological studies of class B GPCRs, and provides new insights into their interactions with ligands.

Class B G protein-coupled receptors (GPCRs), also referred to as the secretin family of GPCRs, include receptors for 15 peptide hormones, which can be grouped into five subfamilies based on their physiological role (see Table 1 for an overview) [1]. These receptors are important drug targets in many human diseases, including diabetes, osteoporosis, obesity, cancer, neurodegeneration, cardiovascular disease, headache, and psychiatric disorders. However, the identification of small-molecule oral drugs for this family has proved extremely challenging.

(A,B) Crystal structures of the class B G protein-coupled receptors corticotropin-releasing factor receptor 1 (CRF1) [Protein Data Bank (PDB) identifier: 4K5Y] and glucagon receptor (PDB identifier: 4L6R) are shown in blue and orange ribbons, respectively, in two different views from within the membrane. Transmembrane (TM) helices and helix 8 are labelled. The disulfide bond tethering extracellular loop 2 (ECL2) to the tip of TM3 is shown as purple sticks. In CRF1 the small-molecule antagonist CP-376395 is shown in stick representation with carbon, nitrogen, and oxygen atoms colored magenta, blue, and red, respectively, and as skeletal formula in an inset. (C) Superposition of the two structures, with insets highlighting regions of particular interest. To highlight the structural differences in the extracellular halves of CRF1 and glucagon receptor, the distance of approximately 11 A° between the Ca-atoms of residues 7.33b at the N-terminal end of TM7 is indicated with a red arrow. The small molecule binding pocket is shown as a superposition of the two receptors around CP-376395, illustrating the antagonist binding mode and the substantial structural differences observed for TM6 of the two receptors.

  • Overview of NMR and crystal structures of class B G protein-coupled

receptor (GPCR) extracellular domains (ECDs; magenta) and their complexes with peptide ligands (different colors). A complete overview of corresponding Protein Data Bank identifiers is presented in Table 1 (not shown). (B) Structure-based sequence alignment of representative peptide ligands of class B GPCR, adopted from Parthier et al. [6]. The residues of the peptide ligands solved in ECD–ligand complex crystal structures are marked using the same colour as in Figure 2A. The residues that are boxed black are found in an α-helical conformation in the complex. Peptide ligand residues that covalently bind receptors in photo-crosslinking or cysteine-trapping studies are colored and boxed green, whereas peptide ligand residues that have been mutated and studied in combination with receptor mutants are colored and boxed red. Note that the first residue of glucagon-like peptide-1 (GLP-1) is His7. A complete overview of all ECD structures and important peptide ligands for all class B GPCRs is presented in Table 1. Putative helix-capping residues [6] are coloured blue and cysteines involved in a disulfide-bridge (calcitonin) are coloured orange. D-phenylalanine (f), and norleucine (m) residues are indicated in stressin and astressin. The last 41 and 99 residues of parathyroid hormone (PTH) and PTH-related protein.  (PTHrP), respectively, are not displayed. Abbreviations: CGRP, calcitonin gene-related peptide; CLR, calcitonin receptor-like receptor; CRF, corticotropin-releasing factor; CT, calcitonin; Ext-4, exendin-4; GHRHR, growth hormone releasing hormone receptor; GIP, glucose-dependent insulinotropic peptide; PAC, pituitary adenylate cyclase; PACAP, pituitary adenylate cyclase activating polypeptide; RAMP, receptor-activity modifying proteins; SCTR, secretin receptor; Ucn, urocortin; VPAC, vasoactive pituitary adenylate cyclase.

Figure 3. (not shown) (A) The spatial correspondence between residues in transmembrane (TM) helices of class A and class B G protein-coupled receptors (GPCRs) makes it possible to align the most conserved residues in class A (designated X.50, Ballesteros–Weinstein numbering) and class B (designated X.50b, Wootten numbering) for comparisons between GPCR classes (Box 1). (B) Structural alignment of corticotropin-releasing factor receptor 1 (CRF1; blue) and glucagon receptor (GCGR; orange) to two representative class A GPCRs, histamine H1 receptor (H1R; Protein Data Bank identifier: 3RZE) and CXC-chemokine receptor 4 (CXCR4; Protein Data Bank identifier: 3ODU/3OE0) (in grey). Helices are depicted as cylinders, and the ligands glucagon (for GCGR), CP-376395 (for CRF1), doxepin (for H1R), and IT1t and CVX15 (for CXCR4) are shown as sticks. The

location of the Ca-atoms of the most conserved residues of TM1–3 and TM5 among class A and class B GPCRs (Box 1) are indicated by spheres (TM4 is not depicted for clarity).

The GCGR and CRF1 crystal structures show distinct structural features and different binding pockets compared to class A GPCRs, and give new insights into the molecular details of peptide and small-molecule binding to class B GPCRs. The first two crystal structures of the TM domains of class B GPCRs provide a structural framework that will enable the design of biochemical and biophysical experiments detailing the complex structure of this class of receptors, and facilitate the design of stabilized constructs that might lead to the solution of full-length class B GPCR–ligand complexes. The structures furthermore present more reliable structural templates for the design of specific and potent small molecules for the treatment of type 2 diabetes (GCGR) and depression (CRF1) in particular, and open new avenues for structure-based small-molecule drug discovery for class B GPCRs as a whole.

Novel receptor targets for production and action of allopregnanolone in the central nervous system: a focus on pregnane xenobiotic receptor

Cheryl A. Frye, Carolyn J. Koonce and Alicia A. Walf
Front in Cell Neurosci  Apr 2014; 8(106): 1-13.
http://dx.doi.org:/10.3389/fncel.2014.00106

Neurosteroids are cholesterol-based hormones that can be produced in the brain,

independent of secretion from peripheral endocrine glands, such as the gonads and

adrenals. A focus in our laboratory for over 25 years has been how production of the

pregnane neurosteroid, allopregnanolone, is regulated and the novel (i.e., non steroid

receptor) targets for steroid action for behavior. One endpoint of interest has been lordosis, the mating posture of female rodents. Allopregnanolone is necessary and sufficient for lordosis, and the brain circuitry underlying it, such as actions in the midbrain ventral tegmental area (VTA), has been well-characterized. Published and recent findings supporting a dynamic role of allopregnanolone are included in this review.
First, contributions of ovarian and adrenal sources of precursors of allopregnanolone, and the requisite enzymatic actions for de novo production in the central nervous system will be discussed.
Second, how allopregnanolone produced in the brain has actions on behavioral processes that are independent of binding to steroid receptors, but instead involve rapid modulatory actions via neurotransmitter targets (e.g., g-amino butyric acid-GABA, N methyl-D-aspartate- NMDA) will be reviewed.
Third, a recent focus on characterizing the role of a promiscuous nuclear receptor, pregnane xenobiotic receptor (PXR), involved in cholesterol metabolism and expressed in the VTA, as a target for allopregnanolone and how this relates to both actions and production of allopregnanolone will be addressed. For example, allopregnanolone can bind PXR and knocking down expression of PXR in the midbrain VTA attenuates actions of allopregnanolone via NMDA and/or GABAA for lordosis. Our understanding of allopregnanolone’s actions in the VTA for lordosis has been extended to reveal the role of allopregnanolone for broader, clinically-relevant questions, such as neurodevelopmental processes, neuropsychiatric disorders, epilepsy, and aging.

Genetically Encoded Chemical Probes in Cells Reveal the Binding Path of Urocortin-I to CRF Class B GPCR

Irene Coin, Vsevolod Katritch, Tingting Sun, Zheng Xiang, Fai Yiu Siu
Cell  Dec 2013; 155, 1258–1269
http://dx.doi.org/10.1016/j.cell.2013.11.008

Molecular determinants regulating the activation of class B G-protein-coupled receptors (GPCRs) by native peptide agonists are largely unknown. We have investigated here the interaction between the corticotropin releasing factor receptor type 1 (CRF1R) and its native 40-mer peptide ligand Urocortin- I directly in mammalian cells. By incorporating unnatural amino acid photochemical and new click chemical probes into the intact receptor expressed in the native membrane of live cells, 44 intermolecular spatial constraints have been derived for the ligand-receptor interaction. The data were analyzed in the context of the recently resolved crystal structure of
CRF1R transmembrane domain and existing extracellular domain structures, yielding a complete conformational model for the peptide-receptor complex. Structural features of the receptor-ligand complex yield molecular insights
on the mechanism of receptor activation and the basis for discrimination between agonist and antagonist function.

Investigation of GPCR-Ligand Interactions under Native Conditions Using Genetically Encoded Chemical Probes GPCRs are integral membrane proteins containing multiple domains and various posttranslational modifications. To understand GPCR-ligand interactions by crystallography, receptors have to be extracted from the cell membrane and modified with a series of expedients such as deglycosylation, therm-stabilizing mutations, fusions with soluble proteins, or complexes with stabilizing nanobodies. We present here a method to investigate GPCR-ligand interactions at the intact fully posttranslationally modified receptor bound to its WT ligand on the membrane of the live cell, which mimics the native conditions for GPCR function. We first genetically incorporated into the receptor the photocrosslinking Uaa Azi, which served as
a proximity probe to provide an overall map of the ligand binding sites on the receptor. We then determined the relative position of the ligand in the binding pocket using a residue-specific chemical crosslinking reaction between Ffact genetically incorporated into the receptor and Cys introduced into the ligand. The derived intermolecular spatial constraints served eventually to build a detailed conformational model for the receptor-ligand complex.

Glutamate Neurons within the Midbrain Dopamine Regions

  1. Morales and D. H. Root
    Neuroscience 282 (2014) 60–68
    http://dx.doi.org/10.1016/j.neuroscience.2014.05.032

Midbrain dopamine (DA) neurons are hypothesized to play roles in reward-based behavior and addiction, reward prediction and learning by error detection, effort-based decision making, flexible reward-directed behaviors,

incentive salience, stimulus salience (e.g., prediction of rewarding and aversive events), aversion, depression, and fear. The extensive, divergent behavioral roles of midbrain dopamine neurons, predominantly from the ventral tegmental area (VTA), indicate that this system is highly heterogeneous.
This heterogeneity may be reflected in part by the diverse phenotypic characteristics among DAergic neurons and their interactive brain structures.

Midbrain dopamine systems play important roles in Parkinson’s disease, schizophrenia, addiction, and depression. The participation of midbrain dopamine systems in diverse clinical contexts suggests these systems are highly complex. Midbrain dopamine regions contain at least three neuronal phenotypes: dopaminergic, GABAergic, and glutamatergic. Here, we review the locations, subtypes, and functions of glutamatergic neurons within midbrain dopamine regions. Vesicular glutamate transporter 2 (VGluT2) mRNA-expressing neurons are observed within each midbrain dopamine system. Within rat retrorubral field (RRF), large populations of VGluT2 neurons are observed throughout its anteroposterior extent. Within rat substantia nigra pars compacta (SNC), VGluT2 neurons are observed centrally and caudally, and are most dense within the laterodorsal subdivision. RRF and SNC rat VGluT2 neurons lack tyrosine hydroxylase (TH), making them an entirely distinct population of neurons from dopaminergic neurons. The rat ventral tegmental area (VTA) contains the most heterogeneous populations of VGluT2 neurons. VGluT2 neurons are found in each VTA subnucleus but are most dense within the anterior midline subnuclei. Some subpopulations of rat VGluT2 neurons co-express TH or glutamic acid decarboxylase (GAD), but most of the VGluT2 neurons lack TH or GAD. Different subsets of rat VGluT2-TH neurons exist based on the presence or absence of vesicular monoamine transporter 2, dopamine transporter, or D2 dopamine receptor. Thus, the capacity by which VGluT2-TH neurons may release dopamine will differ based on their capacity to accumulate vesicular dopamine, uptake extracellular dopamine, or be autoregulated by dopamine. Rat VTA VGluT2 neurons exhibit intrinsic VTA projections and extrinsic projections to the accumbens and to the prefrontal cortex. Mouse VTA VGluT2 neurons project to accumbens shell, prefrontal cortex, ventral pallidum, amygdala, and lateral habenula. Given their molecular diversity and participation in circuits involved in addiction, we hypothesize that individual VGluT2 subpopulations of neurons play unique roles in addiction and other disorders. This article is part of a Special Issue entitled: Ventral Tegmentum & Dopamine. Published by Elsevier Ltd. On behalf of IBRO.

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Gastrointestinal Endocrinology

Posted in Cell Biology, Curation, Disease Biology, Endocrine Diseases, Genomic Endocrinology, Human Immune System in Health and in Disease, Imaging-based Cancer Patient Management, Innovations, Liver & Digestive Diseases Research, Microbiology, Neurological Diseases, Nutrition, Signaling & Cell Circuits, Small Molecules in Development of Therapeutic Drugs, tagged , , , on February 10, 2015| Leave a Comment »

Gastrointestinal Endocrinology

Writer and Curator: Larry H Bernstein, MD, FCAP

The Gut Microbial Endocrine Organ: Bacterially Derived Signals Driving Cardiometabolic DiseasesMark Brown and Stanley L. Hazen

Annual Review of Medicine Jan 2015; 66: 343-359
http://dx.doi.org:/10.1146/annurev-med-060513-093205

The human gastrointestinal tract is home to trillions of bacteria, which vastly outnumber host cells in the body. Although generally overlooked in the field of endocrinology, gut microbial symbionts organize to form a key endocrine organ that converts nutritional cues from the environment into hormone-like signals that impact both normal physiology and chronic disease in the human host. Recent evidence suggests that several gut microbial-derived products are sensed by dedicated host receptor systems to alter cardiovascular disease (CVD) progression. In fact, gut microbial metabolism of dietary components results in the production of proatherogenic circulating factors that act through a meta-organismal endocrine axis to impact CVD risk. Whether pharmacological interventions at the level of the gut microbial endocrine organ will reduce CVD risk is a key new question in the field of cardiovascular medicine. Here we discuss the opportunities and challenges that lie ahead in targeting meta-organismal endocrinology for CVD prevention.

Exogenous glucagon-like peptide 1 reduces contractions in human colon circular muscle

Antonella Amato, Sara Baldassano, Rosa Liotta1, Rosa Serio and Flavia Mulè
J Endocrinol April 1, 2014 221 29-37
http://dx.doi.org:/10.1530/JOE-13-0525

Glucagon-like peptide 1 (GLP1) is a naturally occurring peptide secreted by intestinal L-cells. Though its primary function is to serve as an incretin, GLP1 reduces gastrointestinal motility. However, only a handful of animal studies have specifically evaluated the influence of GLP1 on colonic motility. Consequently, the aims of this study were to investigate the effects induced by exogenous GLP1, to analyze the mechanism of action, and to verify the presence of GLP1 receptors (GLP1Rs) in human colon circular muscular strips. Organ bath technique, RT-PCR, western blotting, and immunofluorescence were used. In human colon, exogenous GLP1 reduced, in a concentration-dependent manner, the amplitude of the spontaneous contractions without affecting the frequency and the resting basal tone. This inhibitory effect was significantly reduced by exendin (9–39), a GLP1R antagonist, which per se significantly increased the spontaneous mechanical activity. Moreover, it was abolished by tetrodotoxin, a neural blocker, or Nω-nitro-L-arginine – a blocker of neuronal nitric oxide synthase (nNOS). The biomolecular analysis revealed a genic and protein expression of the GLP1R in the human colon. The double-labeling experiments with anti-neurofilament or anti-nNOS showed, for the first time, that immunoreactivity for the GLP1R was expressed in nitrergic neurons of the myenteric plexus. In conclusion, the results of this study suggest that GLP1R is expressed in the human colon and, once activated by exogenous GLP1, mediates an inhibitory effect on large intestine motility through NO neural release.

The impact of dipeptidyl peptidase 4 inhibition on incretin effect, glucose tolerance, and gastrointestinal-mediated glucose disposal in healthy subjects

N A Rhee, S H Østoft, J J Holst, C F Deacon, T Vilsbøll and F K Knop
Eur J Endocrinol September 1, 2014 171 353-36
http://dx.doi.org:/10.1530/EJE-14-0314

Objective Inhibition of dipeptidyl peptidase 4 (DPP4) is thought to intensify the physiological effects of the incretin hormones. We investigated the effects of DPP4 inhibition on plasma levels of glucose-dependent insulinotropic polypeptide (GIP), glucagon-like peptide 1 (GLP1), incretin effect, glucose tolerance, gastrointestinal-mediated glucose disposal (GIGD) and gastric emptying in healthy subjects. Design A randomised, controlled and open-labelled study. Methods Ten healthy subjects (six women; age, 40±5 years (mean±S.E.M.); BMI, 24±3 kg/m2; fasting plasma glucose, 5.1±0.2 mmol/l and HbA1c, 34±1 mmol/mol (5.3±0.1%)) were randomised to two-paired study days comprising a 4-h 50 g oral glucose tolerance test (OGTT) with paracetamol (A) and an isoglycaemic intravenous (i.v.) glucose infusion (B), with (A1+B1) and without (A2+B2) preceding administration of the DPP4 inhibitor sitagliptin. Results Isoglycaemia was obtained in all subjects on the paired study days. Significant increases in fasting levels and OGTT-induced responses of active GLP1 and GIP were seen after DPP4 inhibition. No significant impact of DPP4 inhibition on fasting plasma glucose (5.1±0.1 vs 4.9±0.1 mmol/l, P=0.3), glucose tolerance (area under the curve (AUC) for plasma glucose, 151±35 vs 137±26 mmol/l×min, P=0.7) or peak plasma glucose during OGTT (8.5±0.4 vs 8.1±0.3 mmol/l, P=0.3) was observed. Neither incretin effect (40±9% (without DPP4 inhibitor) vs 40±7% (with DPP4 inhibitor), P=1.0), glucagon responses (1395±165 vs 1223±195 pmol/l×min, P=0.41), GIGD (52±4 vs 56±5%, P=0.40) nor gastric emptying (Tmax for plasma paracetamol: 86±9 vs 80±12 min, P=0.60) changed following DPP4 inhibition. Conclusions These results suggest that acute increases in active incretin hormone levels do not affect glucose tolerance, GIGD, incretin effect, glucagon responses or gastric emptying in healthy subjects.

Morphology and Tissue Distribution of Four Kinds of Endocrine Cells in the Digestive Tract of the Chinese Yellow Quail (Coturnix japonica)

He, M., Liang, X., Wang, K., (…), Li, X., Liu, L.
Analytical and Quantitative Cytology and Histology 2014; 36 (4), pp. 199-205

Objective: To describe the tissue distribution, density, and the morphological characteristics of 4 kinds of endocrine cells in the digestive tract of the Chinese yellow quail (Coturnix japonica). Study design: The streptavidin-biotin-peroxidase complex immunohistochemical method was used to identify the distribution of somatostatin (SS), serotonin (5-HT), gastrin and neuropeptide Y (NPY) in digestive tracts including proventriculus, duodenum, jejunum, ileum, and rectum. SPSS 19.0 software was used to perform biological statistical analysis. Results: The results showed that the SS and 5-HT secreting cells were mainly distributed in the proventriculus (19.2±6.9 and 16.1±3.4 cfu/mm2) and duodenum (2.9±2.0 and 1.9±0.6 cfu/mm2). Gastrin and NPY were not detected in each section of the digestive tract. Moreover, there was no significant difference in the quantitative distribution and morphological characteristics of SS and 5-HT secreting cells in the digestive tract between male and female quails. Conclusion: The distribution and morphological characteristics of endocrine cells were closely related to the physiological functions of different parts in the digestive tract. The preferential location of endocrine cells provides additional information for future studies on the physiological roles of gastrointestinal peptides in the gastrointestinal tract of the Chinese yellow quail

GEP-NETS update: Functional localisation and scintigraphy in neuroendocrine tumours of the gastrointestinal tract and pancreas (GEP-NETs)

Wouter W de Herder
Eur J Endocrinol May 1, 2014 170 R173-R183
http://dx.doi.org:/10.1530/EJE-14-0077

For patients with neuroendocrine tumours (NETs) of the gastrointestinal tract and pancreas (GEP) (GEP-NETs), excellent care should ideally be provided by a multidisciplinary team of skilled health care professionals. In these patients, a combination of nuclear medicine imaging and conventional radiological imaging techniques is usually mandatory for primary tumour visualisation, tumour staging and evaluation of treatment. In specific cases, as in patients with occult insulinomas, sampling procedures can provide a clue as to where to localise the insulin-hypersecreting pancreatic NETs. Recent developments in these fields have led to an increase in the detection rate of primary GEP-NETs and their metastatic deposits. Radiopharmaceuticals targeted at specific tumour cell properties and processes can be used to provide sensitive and specific whole-body imaging. Functional imaging also allows for patient selection for receptor-based therapies and prediction of the efficacy of such therapies. Positron emission tomography/computed tomography (CT) and single-photon emission CT/CT are used to map functional images with anatomical localisations. As a result, tumour imaging and tumour follow-up strategies can be optimised for every individual GEP-NET patient. In some cases, functional imaging might give indications with regard to future tumour behaviour and prognosis.

An immunohistochemical study on the distribution of endocrine cells in the digestive tract of gray goose (Anser anser)

Jun YANG1, Lei ZHANG,, Xin LI, , Leii ZHANG, , Xiangjiang LIU, , Kemei PENG

Turk. J. Vet. Anim. Sci. 2012; 36(4): 373-379
http://dx.doi.org:/10.3906/vet-1101-654

The objective of this study was to investigate the morphology and the distribution of 5-hydroxytryptamine (5-HT), somatostatin (SS), gastrin (Gas), glucagon (Glu), and substance P immunoreactive (IR) cells in the digestive tract of gray goose by the immunohistochemical streptavidin-peroxidase method.

The samples were taken from 10 healthy  adult gray geese. Th e results showed that 5 kinds of IR cells were mainly distributed between the mucous epithelium and intestinal gland. The number of 5-HT-IR cells was highest in the rectum and duodenum, but none were observed  in the pylorus. SS-IR cells appeared in great numbers in the pylorus, duodenum, and cecum; however, they were not found in esophagus. Gas-IR cells were mainly distributed in the glandular stomach and jejunum. Glu-IR cells appeared  in small numbers in the glandular stomach, duodenum, and jejunum, but were not detected in other tissues. Substance  P-IR cells were located in the jejunum, cecum, and rectum. Analysis of the present study showed that the distribution and morphological features of these 5 different endocrine cells were related to the feeding habits and metabolism in the digestive tract of the gray goose

Chapter 154 – Somatostatin

Mathias Guggera, Jean-Claude Meunierb

Handbook of Biologically Active Peptides 2006, Pages 1123–1130
http:/dx.doi.org:/10.1016/B978-012369442-3/50157-4

Somatostatin is abundant in the mucosa and in the enteric nervous system of the gastrointestinal tract and in the pancreas. In these tissues, it exerts a broad range of mainly inhibitory physiological actions in multiple targets, including endocrine glands, exocrine glands, smooth muscles, blood vessels, and immune cells, mediated by up to six somatostatin receptor subtypes. Several diseases of the gastrointestinal tract are characterized by disturbances in the somatostatin production or by overexpression of somatostatin receptors. In particular, somatostatin receptors have been found to be overexpressed in neuroendocrine gastroenteropancreatic tumors. These tumors can be diagnostically and therapeutically targeted with somatostatin analogs. In addition, various nonneoplastic diseases, including bleeding in the upper gastrointestinal tract, fistulas, and diarrhea can also be treated with somatostatin analogs.

Immunocytochemical study of the distribution of endocrine cells in the pancreas of the Brazilian sparrow species Zonotrichia Capensis Subtorquata (Swaison, 1837)

Nascimento, AA.*; Sales, A.; Cardoso, TRD.; Pinheiro, NL.; Mendes, RMM.
Braz. J. Biol. Nov. 2007; 67(4):  São Carlos

In the present study, we investigated types of pancreatic endocrine cells and its respective peptides in the Brazilian sparrow species using immunocytochemistry. The use of polyclonal specific antisera for somatostatin, glucagon, avian pancreatic polypeptide (APP), YY polypeptide (PYY) and insulin, revealed a diversified distribution in the pancreas. All these types of immunoreactive cells were observed in the pancreas with different amounts. Insulin- Immunoreactive cells to (B cells) were most numerous, preferably occupying the central place in the pancreatic islets. Somatostatin, PPA, PYY and glucagon immunoreactive cells occurred in a lower frequency in the periphery of pancreatic islets.

Immunolocalisation of the serotonin in the fundus ventriculi and duodenum of the Asia minor ground squirrel: (Spermophilus xanthoprymnus)

Timurkaan, S., Özkan, E., Ilgün, R., Gür, F.M
Veterinarski Arhiv 2009; 79 (1), pp. 69-76

Serotonin immunoreactive cells were located and distributed in the fundus and duodenum with variable frequencies. They were spherical or spindle-shaped and the highest frequency serotonin immunoreactive cells were detected in the whole fundic region. The regional distribution of the endocrine cells in the fundus and duodenum of the citellus resembled other mammalian species.

An Immunohistochemical Study of Gastrointestinal Endocrine Cells in the BALB/c Mouse

Ku, S.K., Lee, H.S., Lee, J.H.
J Vet Med Series C: Anatomia Histologia Embryologia 2004; 33 (1), pp. 42-48

The distributions and frequencies of some endocrine cells in the eight portions of the gastrointestinal tract (GIT) of BALB/c mouse were studied. Endocrine cells were stained using immunohistochemical method with seven types of anti-sera against bovine chromogranin (BCG), serotonin, gastrin, cholecystokinin (CCK)-8, somatostatin, glucagon and human pancreatic polypeptide (HPP), and the regional distributions and their relative frequencies were observed in the eight portions of the GIT of BALB/c mice. All seven types of immunoreactive (IR) cells were identified. Most of the IR cells in the intestinal portion were generally spherical or spindle in shape (open type cell) while round-shaped cells (closed type cell) were found in the intestinal gland and stomach regions occasionally. Their relative frequencies varied according to each portion of the GIT. BCG-IR cells were observed throughout the whole GIT except for the rectum and they were most predominant in the pylorus. Serotonin-IR cells were detected throughout the whole GIT and they showed the highest frequency in the fundus. Gastrin- and CCK-IR cells were restricted to the pylorus and duodenum with a majority in the pylorus and rare or a few frequencies in the duodenum. Compared with other mammals, somatostatin-IR cells were restricted to the fundus and pylorus with a few frequencies, respectively. In addition, glucagon- and HPP-IR cells were restricted to the fundus and duodenum, respectively, with relative low frequencies. Some species-dependent unique distributions and frequencies of endocrine cells were observed in the GIT of BALB/c mouse compared with other rodents.

Immunohistochemical study of the distribution of serotonin in the gastrointestinal tract of the porcupines (Hystrix cristata)

Timurkaan, S., Karan, M., Aydin, A.
Revue de Medecine Veterinaire 2005; 156 (11), pp. 533-536

Serotonin immunoreactive cells were located in the gastric glands and in the intestinal epithelium with variable frequencies. They were spherical or spindle-shaped. Serotonin immuno-reactive cells were detected in almost all regions of the gastrointestinal tract and they showed highest frequency in the stomach and colon.

Effects of carbachol on gastrin and somatostatin release in rat antral tissue culture

Wolfe, M.M., Jain, D.K., Reel, G.M., McGuigan, J.E.
Gastroenterology 1984; 87 (1), pp. 86-93

Recent studies have demonstrated that somatostatin-containing cells are in close anatomic proximity to gastrin-producing cells in antral mucosa, suggesting a potential local regulatory role for somatostatin. The purpose of this study was to examine further the relationships between gastrin and somatostatin and the effects of the cholinergic agonist carbachol on content and release of gastrin and somatostatin using rat antral mucosa in tissue culture. Antral mucosa was cultured at 37 °C in KrebsHenseleit buffer, pH 7.4, gassed with 95% O2-5% CO2. After 1 h, the culture medium was decanted and the tissue was boiled to extract mucosal gastrin and somatostatin. Inclusion of carbachol 2.5 × 10-6 M in the culture medium decreased medium somatostatin from 1.91 ± 0.28 (SEM) ng/mg tissue protein to 0.62 ± 0.12 ng/mg (p < 0.01), extracted mucosal somatostatin from 2.60 ± 0.30 to 1.52 ± 0.16 ng/mg (p < 0.001), and percentage of somatostatin released from 42% ± 2.6% to 27% ± 2.2% (p < 0.01). Carbachol also increased culture media gastrin from 14 ± 2.5 to 27 ± 3.0 ng/mg protein (p < 0.01). Tissue content and release of gastrin and somatostatin were also examined during culture of rat antral mucosa in culture media containing antibodies to somatostatin in the presence and in the absence of carbachol. Incubation with somatostatin antisera, both with and without carbachol, markedly increased culture media concentrations of somatostatin, all of which was effectively bound by antibodies present in the media. Antibody binding of somatostatin was accompanied by significant increases in culture media gastrin concentrations, both in the presence and in the absence of carbachol. Results of these studies support the hypothesis that antral somatostatin exerts a local regulatory effect on gastrin release and that cholinergic stimulation of gastrin release is mediated, at least in part, through inhibition of somatostatin synthesis and release.

Endogenous somatostatin-28 modulates postprandial insulin secretion. Immunoneutralization studies in baboons

J W Ensinck, R E Vogel, E C Laschansky, D J Koerker, et al.
J Clin Invest. 1997; 100(9):2295–2302.
http://dx.doi.org:/10.1172/JCI119767

Somatostatin-28 (S-28), secreted into the circulation from enterocytes after food, and S-14, released mainly from gastric and pancreatic D cells and enteric neurons, inhibit peripheral cellular functions. We hypothesized that S-28 is a humoral regulator of pancreatic B cell function during nutrient absorption. Consistent with this postulate, we observed in baboons a two to threefold increase in portal and peripheral levels of S-28 after meals, with minimal changes in S-14. We attempted to demonstrate a hormonal effect of these peptides by measuring their concentrations before and after infusing a somatostatin-specific monoclonal antibody (mAb) into baboons and comparing glucose, insulin, and glucagon-like peptide-1 levels before and for 4 h after intragastric nutrients during a control study and on 2 d after mAb administration (days 1 and 2). Basal growth hormone (GH) and glucagon levels and parameters of insulin and glucose kinetics were also measured. During immunoneutralization, we found that (a) postprandial insulin levels were elevated on days 1 and 2; (b) GH levels rose immediately and were sustained for 28 h, while glucagon fell; (c) basal insulin levels were unchanged on day 1 but were increased two to threefold on day 2, coincident with decreased insulin sensitivity; and (d) plasma glucose concentrations were similar to control values. We attribute the eventual rise in fasting levels of insulin to its enhanced secretion in compensation for the heightened insulin resistance from increased GH action. Based on the elevated postmeal insulin levels after mAb administration, we conclude that S-28 participates in the enteroinsular axis as a decretin to regulate postprandial insulin secretion.

The Therapeutic Value of Somatostatin and Its Analogues

Sadaf Farooqi, John S. Bevan, Michael C. Shepperd, John A. H. Wass
Pituitary June 1999; 2(1), pp 79-88
http:/dx.doi.org:/10.1023/A:1009978106476

In this review we discuss the physiological effects of somatostatin, which are mediated by specific receptor subtypes on different tissues. These observations have suggested new therapeutic possibilities for the use of the synthetic somatostatin analogues in the treatment of acromegaly as well as a number of other endocrine and non-endocrine disorders.

Somatostatin and Somatostatin Receptors

Ujendra Kumar, Michael Grant
Cellular Peptide Hormone Synthesis and Secretory Pathways
(Results and Problems in Cell Differentiation) 2010; 50: pp 97-120
http://dx.doi.org:/10.1007/400_2009_29

The biological effects of somatostatin (SST) were first encountered unexpectedly in the late 1960s in two unrelated studies, one by Krulich et al. (1968) who reported on a growth hormone (GH)-releasing inhibitory substance from hypothalamic extracts, and the other, by Hellman and Lernmark (1969), on the presence of a potent insulin inhibitory factor from the extracts of pigeon pancreatic islets. However, the inhibitory substance was not officially identified until 1973 by Guillemin’s group (Brazeau et al. 1973). In both synthetic and naturally occurring forms, this tetradecapeptide, originally coined as somatotropin release-inhibitory factor (SRIF, SST-14) was shown by Brazeau et al. to be the substance controlling hypothalamic GH release. This single achievement not only pioneered SST research but was also duly recognized, as Guillemin shared the 1977 Nobel Prize in Medicine. The following years bequeathed an exponential increase in SST-related studies. It soon became clear that SST-synthesis was not restricted to the hypothalamus. Its production is widely distributed throughout the central nervous system (CNS), peripheral neurons, the gastrointestinal tract, and the pancreatic islets of Langerhans (Luft et al. 1974; Arimura et al. 1975; Dubois 1975; Hokfelt et al. 1975; Orci et al. 1975; Pelletier et al. 1975; Polak et al. 1975; Patel and Reichlin 1978). In fact, SST-like immunoreactivity can be found throughout various tissues of vertebrates and invertebrates, including the plant kingdom (Patel 1992; Tostivint et al. 2004). Given its broad anatomical distribution, it is no wonder that SST produces a wide spectrum of biological effects. Generally regarded as an inhibitory factor, SST can function either locally on neighboring cells or distantly through the circulation, to regulate such physiological processes as glandular secretion, neurotransmission, smooth muscle contractility, nutrient absorption, and cell division (Reichlin 1983a, b; Patel 1992, 1999; Patel et al. 2001; Barnett 2003).

Receptor-Mediated Tumor Targeting with Radiopeptides. Part 1. General Concepts and Methods: Applications to Somatostatin Receptor-Expressing Tumors

Alex N. Eberle, Gabriele Mild, and Sylvie Froidevaux
Journal of Receptors and Signal Transduction  2004; 24(4) , Pages 319-455
http://dx.doi.org:/10.1081/RRS-200040939

Radiolabeled peptides have become important tools in nuclear oncology, both as diagnostics and more recently also as therapeutics. They represent a distinct sector of the molecular targeting approach, which in many areas of therapy will implement the old “magic bullet” concept by specifically directing the therapeutic agent to the site of action. In this three-part review, we present a comprehensive overview of the literature on receptor-mediated tumor targeting with the different radiopeptides currently studied. Part I summarizes the general concepts and methods of targeting, the selection of radioisotopes, chelators, and the criteria of peptide ligand development. Then, the >400 studies on the application to somatostatin/somatostatin-release inhibiting factor receptor-mediated tumor localization and treatment will be reviewed, demonstrating that peptide radiopharmaceuticals have gained an important position in clinical medicine.

The somatostatin neuroendocrine system: physiology and clinical relevance in gastrointestinal and pancreatic disorders

Malcolm J. Low
Best Practice & Res Clin Endocr & Metab, 2004; 18(4), pp. 607–622
http://dx.doi.org:/10.1016/j.beem.2004.08.005

The physiologic functions of hypothalamic somatostatin in the regulation of pituitary hormone secretion and the clinical use of somatostatin analogs for the treatment of pituitary adenomas have been reviewed. Similarly, the distribution, normal function and potential pathogenic roles of somatostatin in the central nervous system have been reported in detail. This review will focus exclusively on the physiologic actions of somatostatin and its receptors in the gastrointestinal tract, pancreas and immune system. Diagnostic and therapeutic roles of somatostatin analogs in a diverse catalog of neoplastic, inflammatory and autoimmune conditions affecting peripheral systems are outlined, with an emphasis on both well-established indications and current areas of exploration.

Somatostatin is produced in enteroendocrine D cells and intrinsic neurons of the stomach, intestines and pancreas. Its physiologic actions are mediated primarily by somatostatin receptors type 2 and 5, and include the inhibition of secretion of most endocrine and exocrine factors. Diseases directly attributable to somatostatin excess or deficiency are rare, although there is a complex pathogenic relationship between persistent Helicobacter pylori infection and reduced somatostatin in chronic gastritis. Abundant somatostatin receptors on many neoplastic and inflammatory cells are the basis for sensitive in vivo imaging with radiolabeled somatostatin analogs and provide a therapeutic target. Current indications for somatostatin therapy include hormone-expressing neuroendocrine tumors, intractable diarrhea and variceal bleeding secondary to portal hypertension. Exciting advances are being made in the development of high-affinity nonpeptide analogs with receptor-subtype selectivity and increased bioavailability. Somatostatin analogs coupled to high-energy radionuclides show promise as novel cytotoxic agents for certain metastatic tumors.

Evolution of the somatostatin gene family Both forms of mammalian somatostatin are derived post-translationally from a common pro-hormone by the action of specific pro-protein convertases (PCs). Genetic studies indicate a primary role for PC2 in the generation of SST147, which is the predominant form of somatostatin produced in the brain and most other tissues. SST28 is found in its highest concentrations in the gastrointestinal tract, especially the mucosal epithelial cells of the intestines.
A revised evolutionary concept of the somatostatin gene family is that a primordial gene underwent duplication during or before the advent of chordates and that the two resulting genes subsequently underwent differing rates of mutation to produce the distinct prepro-somatostatin and prepro-cortistatin genes in mammals. A second gene duplication event likely occurred in teleosts to generate PSS1 and PSS-II from the ancestral somatostatin gene.
It is possible that additional related genes have not yet been identified. Recent studies utilizing unique polyclonal antisera and a strain of somatostatin-deficient mouse have demonstrated the existence of a novel gastrointestinal peptide with homology to the amino acid sequence of SST28(1–13) that has been named thrittene.
Somatostatin gene organization and regulation The mammalian PPS1 (or SMST) gene has a relatively simple organization consisting of two coding exons separated by one intron. A single promoter directs transcription of the PPS1 gene in all tissues, and there are no known alternative mRNA splicing events. The molecular mechanisms underlying the developmental and hormonal regulation of somatostatin gene transcription have been most extensively studied in pancreatic islets and islet-derived cell lines. The proximal enhancer elements in the somatostatin gene promoter that bind complexes of homeodomain-containing transcription factors (PAX6, PBX, PREP1) to upregulate transcription in pancreatic islets may actually represent gene silencer elements in neurons (promoter elements TSEII and UE-A). Conversely, another related cis-element in the somatostatin gene (promoter element TSEI) apparently binds a homeodomain transcription factor PDX1 (also called STF1/ IDX1/IPF1) that is common to developing brain, pancreas and foregut, and regulates gene expression in both the CNS and gut.
Enteroendocrine cells of the gut mucosa differentiate from pluripotential stem cells in the crypts, share molecular phenotypes and retain close paracrine interactions among the daughter cells. Similarly, pancreatic islet cells share common precursors. Recent studies have demonstrated that bone marrow contains a stem cell population capable of producing islet-like cell clusters in vitro that contain somatostatin-positive cells together with the other cell types found in normally differentiated islets.
Somatostatin Receptors  There are five somatostatin receptor subtypes (SSTR1–5) encoded by separate genes located on different chromosomes. Alternative mRNA splicing generates SSTR2α and SSTR2β from heteronuclear RNA after transcription from the single SSTR2 gene. SSTRs are members of the rhodopsin-like G protein-coupled receptor superfamily and are most closely related structurally to the opioid receptors. The unique amino acid signature of SSTRs is contained in a seven-element fingerprint of peptide sequences located in conserved regions of the N and C termini, extra- and intra-cellular loops, and transmembrane domains. SSTRs are expressed in discrete or partially overlapping distributions in multiple target organs and differ in their coupling to second messenger signaling molecules, and therefore in their range and mechanism of intracellular actions. The subtypes also differ in their binding affinity to specific somatostatin-like ligands. Some of these differences have important implications for the use of somatostatin analogs in diagnostic imaging and in pharmacotherapy.
All SSTR subtypes are coupled to pertussis toxin-sensitive G proteins and bind SST14 and SST28 with high affinity in the low nanomolar range, although SST28 has a modestly higher affinity for SSTR5. All the subtypes are expressed in brain and pituitary to varying degrees with different distributions, but SSTR2 and SSTR5 are clearly the most abundant in peripheral tissues. These two subtypes are also the most physiologically important in pancreatic islets. SSTR5 is responsible for the inhibition of insulin secretion from b-cells, and SSTR2 is essential for the inhibition of glucagon from a-cells. SSTR1 is expressed at low levels in gastrointestinal structures. The binding of somatostatin to its receptors leads to the activation of one or more inhibitory G proteins (Gi/o), which in turn decrease adenylyl cyclase activity and the concentration of intracellular cAMP. Other G protein-mediated actions common to all The somatostatin neuroendocrine system 609 SSTRs are activation of a vanadate-sensitive phosphotyrosine phosphatase (PTP) and modulation of mitogen-activated protein kinases (MAPKs).
Inhibition of endocrine and exocrine secretion Somatostatin has diverse biologic activities in the gastrointestinal system. It is secreted from D cells into the extracellular space to act as a paracrine factor on nearby endocrine cells and as an autocrine factor to inhibit its own secretion. Most of the circulating hormonal somatostatin originates from the stomach and intestines. Basal plasma levels are in the range of 30–100 pg/ml and increase postprandially by as much as 100% over baseline for a duration up to 2 hours. The release of somatostatin from enteric D cells is regulated by a combination of nutritional, humoral, neural and paracrine signals.

The modulatory role of somatostatin in gastric acid secretion by parietal cells illustrates the typical complexity of hormonal, paracrine and neural integration within the gastrointestinal tract.
Somatostatin secreted from gastric D cells modulates the gastrin-enterochromaffin-like cell—parietal cell axis. Gastrin, secreted from G cells, stimulates the release of histamine from enterochromaffin-like cells (ECL), which is in turn a major secretagog of hydrochloric acid (HCl) from gastric parietal (P) cells. Somatostatin (SST14) inhibits secretion from each of these cell types, although the predominant actions are on the G and ECL cells. Food intake mediates gastric acid secretion by activating both vagal nerves and intrinsic gastric neurons. D cells are stimulated by the autocrine release of amylin, the paracrine release of bombesin and atrial natiuretic peptide (ANP), the enteric neuron release of pituitary adenylate cyclase-activating peptide (PACAP) and cholecystokinin (CCK), and the T lymphocyte release of interleukin-4 (IL-4).D cells are inhibited by histamine acting on H3 receptors in a negative paracrine feedback loop from ECL cells and by other factors, including gamma-aminobutyric acid (GABA) and opioid peptides. The pathways illustrated are not all-inclusive but represent many of the key regulatory steps.

Practice points

† long-acting somatostatin analogs are primary therapeutic tools for the symptomatic treatment of the excessive hormone and monoamine secretion from carcinoids and other neuroendocrine tumors

† somatostatin and long-acting somatostatin analogs are effective first-line
medical treatment for upper gastrointestinal bleeding from esophageal varices associated with hepatic cirrhosis and portal hypertension but are not indicatedfor the treatment of bleeding from gastric varices or duodenal ulcers

† radiolabeled somatostatin analogs provide a sensitive imaging technique for a wide range of neoplastic and inflammatory disorders, including neuroendocrine tumors, meningiomas and sarcoidosis because of their high level expression of somatostatin receptors.

The role(s) of somatostatin, structurally related peptides and somatostatin receptors in the gastrointestinal tract: a review

J Van Op den bosch, D Adriaensen, L Van Nassauw, Jean-Pierre Timmermans
Regulatory Peptides 156 (2009) 1–8
http://dx.doi.org:/10.1016/j.regpep.2009.04.003

Extensive functional and morphological research has demonstrated the pivotal role of somatostatin (SOM) in the regulation of a wide variety of gastrointestinal activities. In addition to its profound inhibitory effects on gastrointestinal motility and exocrine and endocrine secretion processes along the entire gastrointestinal tract, SOM modulates several organ-specific activities. In contrast to these well-known SOM-dependent effects, knowledge on the SOM receptors (SSTR) involved in these effects is much less conclusive. Experimental data on the identities of the SSTRs, although species- and tissue-dependent, point towards the involvement of multiple receptor subtypes in the vast majority of gastrointestinal SOM-mediated effects. Recent evidence demonstrating the role of SOM in intestinal pathologies has extended the interest of gastrointestinal research in this peptide even further. More specifically, SOM is supposed to suppress intestinal inflammatory responses by interfering with the extensive bidirectional communication between mucosal mast cells and neurons. This way, SOM not only acts as a powerful inhibitor of the inflammatory cascade at the site of inflammation, but exerts a profound anti-nociceptive effect through the modulation of extrinsic afferent nerve fibers. The combination of these physiological and pathological activities opens up new opportunities to explore the potential of stable SOM analogues in the treatment of GI inflammatory pathologies.

Schematic overview of the distribution of the SSTRs 1–5

Schematic overview of the distribution of the SSTRs 1–5

Schematic overview of the distribution of the SSTRs 1–5 in the murine small intestine under control conditions (left panel) and during intestinal schistosomiasis (right panel). In non-inflamed conditions, SSTR1, SSTR2A and SSTR4 are expressed in non-neuronal (glial cells, enterocytes…) and neuronal cells, both from intrinsic and extrinsic origin. SSTR3 and SSTR5 are undetectable. In response to intestinal schistosomiasis, profound sprouting of nerve fibres expressing SSTR1, SSTR3 and SSTR4 is observed, in addition to the expression of SSTR1 and SSTR3 in mucosal mast cells (MMC).

Somatostatin and Its Receptor Family

Yogesh C. Patel
Frontiers in Neuroendocrinology 1999; 20, 157–198 Article ID frne.1999.0183

Somatostatin (SST), a regulatory peptide, is produced by neuroendocrine, inflammatory, and immune cells in response to ions, nutrients, neuropeptides, neurotransmitters, thyroid and steroid hormones, growth factors, and cytokines. The peptide is released in large amounts from storage pools of secretory cells, or in small amounts from activated immune and inflammatory cells, and acts as an endogenous inhibitory regulator of the secretory and proliferative responses of target cells that are widely distributed in the brain and periphery. These actions are mediated by a family of seven  transmembrane (TM) domain G-protein-coupled receptors that comprise five distinct subtypes (termed SSTR1–5) that are endoded by separate genes segregated on different chromosomes. The five receptor subtypes bind the natural SST peptides, SST-14 and SST-28, with low nanomolar affinity. Short synthetic octapeptide and hexapeptide analogs bind well to only three of the subtypes, 2, 3, and 5. Selective nonpeptide agonists with nanomolar affinity have been developed for four of the subtypes (SSTR1, 2, 3, and 4) and putative peptide antagonists for SSTR2 and SSTR5 have been identified. The ligand binding domain for SST ligands is made up of residues in TMs III–VII with a potential contribution by the second extracellular loop. SSTRs are widely expressed in many tissues, frequently as multiple subtypes that coexist in the same cell. The five receptors share common signaling pathways such as the inhibition of adenylyl cyclase, activation of phosphotyrosine phosphatase (PTP), and modulation of mitogen-activated protein kinase (MAPK) through G-protein-dependent mechanisms.

Somatostatin receptors

Lars Neisig Møller, Carsten Enggaard Stidsen, Bolette Hartmann, Jens Juul Holst
Biochimica et Biophysica Acta 1616 (2003) 1 – 84
http://dx.doi.org:/10.1016/S0005-2736(03)00235-9

In 1972, Brazeau et al. isolated somatostatin (somatotropin release-inhibiting factor, SRIF), a cyclic polypeptide with two biologically active isoforms (SRIF-14 and SRIF-28). This event prompted the successful quest for SRIF receptors. Then, nearly a quarter of a century later, it was announced that a neuropeptide, to be named cortistatin (CST), had been cloned, bearing strong resemblance to SRIF. Evidence of special CST receptors never emerged, however. CST rather competed with both SRIF isoforms for specific receptor binding. And binding to the known subtypes with affinities in the nanomolar range, it has therefore been acknowledged to be a third endogenous ligand at SRIF receptors. This review goes through mechanisms of signal transduction, pharmacology, and anatomical distribution of SRIF receptors. Structurally, SRIF receptors belong to the superfamily of G protein-coupled (GPC) receptors, sharing the characteristic seven-transmembrane-segment (STMS) topography. Years of intensive research have resulted in cloning of five receptor subtypes (sst1-sst5), one of which is represented by two splice variants (sst2A and sst2B). The individual subtypes, functionally coupled to the effectors of signal transduction, are differentially expressed throughout the mammalian organism, with corresponding differences in physiological impact. It is evident that receptor function, from a physiological point of view, cannot simply be reduced to the accumulated operations of individual receptors. Far from being isolated functional units, receptors co-operate. The total receptor apparatus of individual cell types is composed of different-ligand receptors (e.g. SRIF and non-SRIF receptors) and co-expressed receptor subtypes (e.g. sst2 and sst5 receptors) in characteristic proportions. In other words, levels of individual receptor subtypes are highly cell-specific and vary with the co-expression of different-ligand receptors. However, the question is how to quantify the relative contributions of individual receptor subtypes to the integration of transduced signals, ultimately the result of collective receptor activity. The generation of knock-out (KO) mice, intended as a means to define the contributions made by individual receptor subtypes, necessarily marks but an approximation. Furthermore, we must now take into account the stunning complexity of receptor co-operation indicated by the observation of receptor homo- and heterodimerisation, let alone oligomerisation. Theoretically, this phenomenon adds a novel series of functional megareceptors/super-receptors, with varied pharmacological profiles, to the catalogue of monomeric receptor subtypes isolated and cloned in the past. SRIF analogues include both peptides and non-peptides, receptor agonists and antagonists. Relatively long half lives, as compared to those of the endogenous ligands, have been paramount from the outset. Motivated by theoretical puzzles or the shortcomings of present-day diagnostics and therapy, investigators have also aimed to produce subtype-selective analogues. Several have become available.

Somatostatin And Its Analogues In The Therapy Of Gastrointestinal Disease

Wynick, J. M. Polak And S. R. Bloom
Pharmac. Ther. 1989; 41, pp. 353-370

During the course of efforts to determine the distribution of growth hormone-releasing factor (GHRF) in rat hypothalamus a substance that inhibited growth hormone release was unexpectedly detected by Krulich et aL (1968). Their findings led them to hypothesize that the secretion of growth hormone from the pituitary was regulated by two different interacting neurohumoral factors–one stimulatory, the other inhibitory–each under the control of the nervous system. At about the same time Hellman and Lernmark (1969) found a factor in extracts of pigeon pancreatic islet-cells that inhibited insulin release in vivo from cultured pancreatic islet-cells. These two observations, seemingly unrelated, were ultimately to converge with the chemical identification of somatostatin, as an inhibitory peptide found in both the hypothalamus and pancreas.

Growth hormone-release inhibitory activity was re-discovered in 1972 by Brazeau et al. (1973). A concentrated effort to isolate and sequence the active principal was successful and it proved to be a cyclic peptide, to which the term ‘somatostatin’ (somatotrophin release inhibitory factor) was applied.               Subsequent work (Reichlin, 1982a,b, 1983a,b; Iverson, 1983; Guillemin, 1978a,b) has considerably expanded the initially simple concept of somatostatin as a 14 amino-acid containing peptide (tetradecapeptide), bridged by a sulphur-sulphur bond whose main function was the regulation of growth-hormone secretion (Bonfils, 1985). Somatostatin related peptides are now known to constitute a family that includes the original identified peptide (designated somatostatin 14), an N-terminal extended somatostatin (somatostatin 28), several species specific variants and larger prohormone forms.
The name somatostatin may now be considered to be inappropriate because this compound is distributed widely in cells that have nothing to do with growth-hormone regulation or release. Tissues where somatostatin may be found include the nervous system, the gut and endocrine glands.
Somatostatin is present in every vertebrate class and even in primitive invertebrates (Vale et al., 1976; Falkmer et al., 1978; Jackson, 1978). This would suggest that this molecule and its controlling gene or genes evolved before the appearance on earth of differentiated cell-cell and nerve-cell communication (Roth et al., 1982). The evolutionary paths of mammals and fish are thought to have diverged at least 400 million years ago. The fact that the phenotype of somatostatin 14 is so well conserved (as to a lesser degree is that of somatostatin 28) suggests that throughout evolutionary history the specific configuration of somatostatin 14 has endowed a selective advantage on the animal kingdom, and its absence is not compatible with life.
Though widely distributed in cells throughout the body of vertebrates somatostatin does not in Guillemin’s words (1978a), “inhibit secretion of everything and anything” (since, for example it has no effect on the release of LH and FSH). Despite this it has certainly earned itself the nickname ‘endocrine cyanide’ (Bloom and Polak, 1987). The peptide is found in most but not all organs and displays specific and selective functions depending on its location. Within the nervous system somatostatinergic neurons are found in the cortex, limbic system, anterior pituitary, brain stem and spinal cord.
The various biological effects of somatostatin seem to be mediated through its specific high affinity receptors found in the brain, pituitary, adrenal, pancreas and gastrointestinal tract. Not only normal target tissue, but also tumors from the same endocrine tissues i.e. human pituitary adenomas, human and hamster pancreatic insulinomas, glucagonomas and VIPomas all bear somatostatin receptors (Reubi et al., 1981, 1982a, 1984a, 1985a, 1987a,b). Interestingly, tumors from tissues which are not established targets for somatostatin also seem to bear somatostatin receptors (Goodman et al., 1982; Reubi et al., 1986). Reubi et al. (1987b) demonstrated that many endocrine tumors including meningiomas, breast, pancreatic and pituitary tumors all have somatostatin receptors however, they demonstrated no receptors in prostatic carcinomas, ovarian carcinomas, endometrial carcinomas, primary liver cell carcinomas, pheochromocytomas, aldosterone secreting tumors, medullary carcinoma of the thyroid and a number of pulmonary carcinoids. Somatostatin receptors were also found in benign or malignant tumors originating from tissues not primarily known as somatostatin target organs, the biological function of such receptors is therefore unknown though it may be that they mediate the anti-proliferative effect of somatostatin and may therefore potentially be of therapeutic interest (Blankenstein et al., 1983, 1984).

Review article: somatostatin analogues in the treatment of gastroenteropancreatic neuroendocrine (carcinoid) tumoursModlin,

M. Pavel[1], M. Kidd & B. I. Gustafsson
Aliment Pharmacol Ther 2009; 31, 169–188
http://dx.doi.org:/10.1111/j.1365-2036.2009.04174.x

Background

The discovery of somatostatin (SST) and the synthesis of a variety of analogues constituted a major therapeutic advance in the treatment of gastroenteropancreatic neuroendocrine (carcinoid) tumours (GEP-NETs). They currently provide the most efficient treatment to achieve symptomatic relief and have recently been demonstrated to inhibit tumour growth.

Aim To review 35 years of experience regarding the clinical application and

efficacy of SST analogues. Methods The PubMed database (1972–2009) was searched using somatostatin as a search term with combinations of terms including ‘treatment’; ‘neuroendocrine’; ‘carcinoid’; ‘tumor’; ‘octreotide’; ‘lanreotide’ and ‘pasireotide’. Results In a review of 15 studies including 481 patients, the slow-release formulations Sandostatin LAR and Somatuline SR⁄ Autogel achieved symptomatic relief in 74.2% (61.9–92.8%) and 67.5% (40.0–100%), biochemical response in 51.4% (31.5–100%) and 39.0% (17.9–58%), and tumor response in 69.8% (47.0–87.5%) and 64.4% (48.0–87.0%) respectively. New SST analogues like SOM230 (pasireotide) that exhibit pan SST receptor activity and analogues with high affinity to specific somatostatin receptor (sstr) subtypes show promise. Conclusion As more precise understanding of NET cell biology evolves and molecular biological tools advance, more accurate identification of individual tumours sstr profile will probably facilitate a more precise delineation of SST analogue treatment.

Novel Autonomic Neurotransmitters And Intestinal Function

S. Taylor and R. A. R. Bywater
Pharmac. Ther. 1989; 40, pp. 401 to 438

In this review we will discuss some of the difficulties encountered in ascribing a neurotransmitter function to the more recently discovered peptides and other substances within the intestine. We will also provide a brief (and of necessity incomplete) account of some of the properties of intestinal putative neurotransmitters, and their possible roles in the functions of the small and large intestine.
The Enteric Nervous System The diverse intestinal functions associated with transit, digestion and absorption rely upon an intact enteric nervous system. The enteric nervous system essentially consists of those neurons whose cell bodies lie within the walls of the gastrointestinal tract. In the small and large intestine the cell bodies lie within the myenteric and submucous plexuses; their processes ramify throughout the majority of the intestinal wall and in many areas give rise to additional plexuses (Furness and Costa, 1987; Gabella, 1987). Functionally, these neurons can be divided into sensory neurons, interneurons and motor neurons. Some enteric neurons receive projections from extrinsic neurons and/or send projections centrally; we will not consider these projections further here.
The early observations of the co-existence of peptides in the enteric nervous system (Schultzberg et al., 1980) have now been extended and these studies demonstrate that the co-existence of two or more peptides is the rule rather than the exception (H6kfelt et al., 1987). The mix of peptides within neurons does not appear to be random; rather, there appears to be a systematic grouping of peptides in neurons with particular projections. This has led to the concept of “chemical coding” of enteric neurons. According to this concept, particular combinations of peptides are associated with particular neural pathways and perhaps with particular functions. For example, in the guinea pig small intestine, two chemically coded groups of submucous neurons have projections of different lengths running to the mucosa. Cell bodies with longer projections show immunoreactivity for dynorphin (DYN) and VIP. The other group shows immunoreactivity for choline acetyltransferase (CHAT), cholecystokinin, (CCK), calcitonin gene-related peptide (CGRP), neuropeptide Y (NPY) and somatostatin (SOM) (Costa et al., 1986a; Furness et al., 1987a). More recently it has been demonstrated that both groups of neurons show immunoreactivity for galanin (GAL) (Furness et al., 1987a,b). As for the neurotransmitter roles in the gut, the key question then becomes; “How does the presence of specific combinations of chemical substances (including peptides) relate to neuronal function?” It has been known for several years that “classical” transmitter substances can coexist in combination with various peptides (H6kfelt et al., 1980; Gilbert and Emson, 1983).
The above commentary upon the possible co-existence of several putative transmitter substances highlights the complex neurochemistry of the enteric nervous system. A corresponding degree of complexity appears to exist for the neuronal circuitry that ultimately directs the differing, but highly organized, patterns of motility and the secretory/absorptive functions of the intestinal tract. In vitro electrophysiological studies of the myenteric and submucous plexuses have indicated that several different types of neurons are present, each with their own biophysical characteristics. Furthermore, neurotransmission through, and probably between, the plexuses involves synaptic potentials which have time courses ranging from several milliseconds up to several minutes, depending upon the characteristics (stimulus strength, frequency and train length, etc.) and location of the applied electrical stimulus (see Wood, 1987, for references). Intracellular recordings from smooth muscle cells have also shown that excitatory and inhibitory junction potentials (EJPs and IJPS) of varying time courses can be evoked at various locations along the intestine during transmural electrical stimulation in response to selective stimulus regimens (see, for instance, Bywater and Taylor, 1986).
A number of authors have proposed criteria which should be fulfilled in order that transmitter status can be bestowed upon a particular substance (see Furness and Costa, 1982, for references). These criteria were developed with reference to the classical transmitter substances such as ACh, using the paradigm of a single transmitter per neuron. Regardless of the coexistence of several putative transmitters, status can only be granted to those substances that are found to be released from that nerve terminal. In the enteric nervous system a particular putative transmitter may be contained in several different functional pathways. However, in general, the methods used for eliciting release of putative transmitter substances (e.g. transmural electrical stimulation) are not specific for particular projections. Thus, for any substance, the association of demonstrated release with a given transmitter role is not facile.

New roles of the multidimensional adipokine: Chemerin

Syeda Sadia Fatima, Rehana Rehman, Mukhtiar Baig, Taseer Ahmed Khan
Peptides 62 (2014) 15–20
http://dx.doi.org/10.1016/j.peptides.2014.09.019

The discovery of several adipokines with diverse activities and their involvement in regulation of various pathophysiological functions of human body has challenged the researchers. In the family of adipokine, chemerin is a novel and unique addition. Ever since the first report on chemerin as a chemo-attractant protein, there are numerous studies showing a multitasking capacity of chemerin in the maintenance of homeostasis, for the activation of natural killer cells, macrophages and dendritic cells in both innate and adaptive immunity. Its diversity ranges from generalized inflammatory cascades to being explicitly involved in the manifestation of arthritis, psoriasis and peritonitis. Its association with certain cancerous tissue may render it as a potential tumor marker. In present review, we aim to consolidate recent data of investigations on chemerin in context to functional characteristics with a special reference to its role as a metabolic signal in inflammation and non-metabolic syndromes.

Neuropeptide Y is expressed in subpopulations of insulin- and non-insulin-producing islet cells in the rat after dexamethasone treatment: a combined immunocytochemical and in situ hybridisation study

Myrs6n a, *, B. Ahr6n b, F. Sundler
Regulatory Peptides 1995; 60, 19-31

Neuropeptide Y (NPY) is known to occur in adrenergic and non-adrenergic nerves in rat pancreatic islets. Analysis of islet extracts has revealed local NPY synthesis after glucocorticoid treatment. The cellular localization of NPY expression in rat islets following dexamethasone treatment (2 mg/kg daily, for 12 days), was investigated by a combination of immunocytochemistry (ICC) and in situ hybridization (ISH). NPY-immunoreactive nerve fibers were seen in pancreatic islets of both control and dexamethasone-treated rats. In the controls weak NPY immunoreactivity but no NPY mRNA was observed in occasional i:dets. After dexamethasone treatment, clusters of islet cells distributed both centrally and peripherally displayed intense NPY immunoreactivity and NPY mRNA labelling. Immunocytochemical double staining and ISH combined with ICC for NPY and islet hormones revealed that most NPY expressing cells were identical with insulin cells; a few cells were identical[ with somatostatin or pancreatic polypeptide (PP) cells. In contrast, glucagon cells seemed to be devoid of NPY immunoreactivity and NPY mRNA labelling. Thus, in the rat, glucocorticoids cause a marked upregulation of NPY expression in islet cells, preferentially the insulin cells. The expression of NPY might represent an islet adaptation mechanism to the reduced peripheral insulin sensitivity.

Neuropeptide Y is expressed in islet somatostatin cells of the hamster pancreas: a combined immunocytochemical and in situ hybridization study

Ulrika Myrsrn, Frank Sundler
Regulatory Peptides 1995; 57, 65-76

Neuropeptide Y (NPY) is known to occur in the autonomic nervous system, including the pancreatic islet innervation. We now present evidence that NPY is also expressed in endocrine islet cells in hamster pancreas. Thus, NPY-immunoreactivity and gene expression were detected in peripheral islet cells, using immunocytochemistry (ICC), in situ hybridization (ISH), and a combination of these techniques. Double immunostaining for NPY and somatostatin enabled localization of NPY to the vast majority of the somatostatin cells. However, a few somatostatin cells were devoid of NPY immunoreactivity and an occasional NPY-immunoreactive cell was devoid of somatostatin. ISH with an NPY mRNA specific probe, showed labelling of cells in the islet periphery. Furthermore, combined ISH for NPY mRNA and ICC for somatostatin showed autoradiographic labelling of somatostatin cells to a varying degree. Both somatostatin and NPY are inhibitors of insulin and/or glucagon secretion. Thus, in the islets these two peptides may be coreleased and cooperate in the, regulation of islet hormone secretion. The role for NPY emanating from islet cells is probably paracrine rather than endocrine.

Neuropeptide Y and Peptide YY Immunoreactivities in the Pancreas of Various Vertebrates

Wei-Guang Ding, Hiroshi Kimura, Masaki Fujimura And Mineko Fujimiya
Peptides,  1997; 18(10), pp. 1523–1529   PII S0196-9781(97)00237-4

NPY-like immunoreactivity was observed in nerve fibers and endocrine cells
in pancreas of all species examined except the eel, which showed no NPY innervation. The density of NPY-positive nerve fibers was higher in mammals than in the lower vertebrates. These nerve fibers were distributed throughout the parenchyma, and were particularly associated with the pancreatic duct
and vascular walls. In addition, the density of NPY-positive endocrine cells was found to be higher in lower vertebrates than mammals; in descending order; eel 5 turtle 5 chicken . bullfrog . mouse 5 rat 5 human . guinea pig 5 dog. These NPY-positive cells in the eel and certain mammals tended to be localized throughout the islet region, whereas in the turtle and chicken they were mainly scattered in the exocrine region. PYY-immunoreactivity was only present in the pancreatic endocrine cells of all species studied, and localized similarly to NPY. Thus these two peptides may play endocrine or paracrine roles in the regulation of islet hormone secretion in various vertebrate species.

Inhibitory effect of somatostatin on inflammation and nociception

Erika Pintér, Zsuzsanna Helyes, János Szolcsányi
Pharmacology & Therapeutics 112 (2006) 440–456

Somatostatin is released from capsaicin-sensitive, peptidergic sensory nerve endings in response to noxious heat and chemical stimuli such as vanilloids, protons or lipoxygenase products. It reaches distant parts of the body via the circulation and exerts systemic anti-inflammatory and analgesic effects. Somatostatin binds to G-protein coupled membrane receptors (sst1–sst5) and diminishes neurogenic inflammation by prejunctional action on sensory-efferent nerve terminals, as well as by postjunctional mechanisms on target cells. It decreases the release of pro-inflammatory neuropeptides from sensory nerve endings and also acts on receptors of vascular endothelial, inflammatory and immune cells. Analgesic effect is mediated by an inhibitory action on peripheral terminals of nociceptive neurons, since circulating somatostatin cannot exert central action.
Somatostatin itself is not suitable for drug development because of its broad spectrum and short elimination half-life, stable, receptor-selective agonists have been synthesized and investigated. The present overview is aimed at summarizing the physiological importance of somatostatin and sst receptors, pharmacological significance of synthetic agonists and their potential in the development of novel anti-inflammatory and analgesic drugs. These compounds might provide novel perspectives in the pharmacotherapy of acute and chronic painful inflammatory diseases, as well as neuropathic conditions.

the sources, target cells and effects of somatostatin (SST) involved in inflammatory and nociceptive processes

the sources, target cells and effects of somatostatin (SST) involved in inflammatory and nociceptive processes

This schematic drawing demonstrates the sources, target cells and effects of somatostatin (SST) involved in inflammatory and nociceptive processes

Characterization, detection and regulation of somatostatin receptors

The physiological actions of SST are initiated by its binding to membrane receptors. Five human somatostatin receptors (sst), have been cloned and characterized and referred to as sst1-5 receptors using the nomenclature suggested by Hoyer et al. (1995). Structurally, sst receptors are 7 transmembrane domain glycoproteins, comprised of 7 membrane spanning α helical domains connected by short loops, an N-terminal extracellular domain and a C-terminal intracellular domain. On the basis of binding studies using synthetic somatostatin analogs, sst receptors can be divided into 2 different subgroups: SRIF1 group comprising sst2, sst3 and sst5 are able to bind octapeptide analogs, whereas SRIF2 group comprising sst1 and sst4 have negligible affinity for these compounds. Within sst2 receptors, sst2A and sst2B are encoded on the same chromosome 17 and generated through alternative splicing of sst2 mRNA (Patel et al., 1993). None of the peptide analogs bind exclusively to only one of the sst subtypes, although new approaches might yield subtype-selective agonists and antagonists (Hofland et al., 1995; Hoyer et al., 1995; Patel et al., 1995; Reisine & Bell, 1995; Florio & Schettini, 1996; Patel, 1997; Meyerhof, 1998; Janecka et al., 2001). Somatostatin receptors are linked to multiple cellular effector systems via G-proteins. They mediate the inhibition of adenylate cyclase activity (Jakobs et al., 1983; Patel et al., 1995), reduce the conductance of voltage-dependent Ca2+ channels (Schally, 1988; Patel et al., 1995) and activate K+ channels (Mihara et al., 1987; Moore et al., 1988; Wang et al., 1989). Somatostatin receptors also mediate the stimulation of tyrosine phosphatase activity, induce a reduction of cell proliferation and inhibit a Na+/H+ exchanger (NHE1) (Barber et al., 1989; Buscail et al., 1994; Patel et al., 1995). Sst receptors represent a major class of inhibitory receptors which play an important role in modulating higher brain functions, secretory processes, cell proliferation and apoptosis.
Endogenous Somatostatin-28 Modulates Postprandial Insulin Secretion Immunoneutralization Studies in Baboons

John W. Ensinck, Robin E. Vogel, Ellen C. Laschansky, Donna J. Koerker, et al.
J Clin Invest 1997. 100: 2295–2302.).  http://dx.doi.org/10.1172/JCI119767

Somatostatin-28 (S-28), secreted into the circulation from enterocytes after food, and S-14, released mainly from gastric and pancreatic δ cells and enteric neurons, inhibit peripheral cellular functions. We hypothesized that S-28 is a humoral regulator of pancreatic β cell function during nutrient absorption. Consistent with this postulate, we observed in baboons a two to threefold increase in portal and peripheral levels of S-28 after meals, with minimal changes in S-14. We attempted to demonstrate a hormonal effect of these peptides by measuring their concentrations before and after infusing a somatostatin-specific monoclonal antibody (mAb) into baboons and comparing glucose, insulin, and glucagon-like peptide-1 levels before and for 4 h after intragastric nutrients during a control study and on 2 d after mAb administration (days 1 and 2). Basal growth hormone (GH) and glucagon levels and parameters of insulin and glucose kinetics were also measured. During immunoneutralization, we found that
(a) postprandial insulin levels were elevated on days 1 and 2;
(b) GH levels rose immediately and were sustained for 28 h, while glucagon fell; (c) basal insulin levels were unchanged on day 1 but were increased two to threefold on day 2, coincident with decreased insulin sensitivity; and
(d) plasma glucose concentrations were similar to control values.
We attribute the eventual rise in fasting levels of insulin to its enhanced secretion in compensation for the heightened insulin resistance from increased GH action. Based on the elevated postmeal insulin levels after mAb administration, we conclude that S-28 participates in the enteroinsular axis as a decretin to regulate postprandial insulin secretion.

Effects of glucagon-like peptide 1 on appetite and body weight: focus on the CNS

L van Bloemendaal, J S ten Kulve, S E la Fleur, R G Ijzerman and M Diamant
Journal of Endocrinology 2014; 221, T1–T16
http://dx.doi.org:/10.1530/JOE-13-0414

The delivery of nutrients to the gastrointestinal tract after food ingestion activates the secretion of several gut-derived mediators, including the incretin hormone glucagon-like peptide 1 (GLP-1). GLP-1 receptor agonists (GLP-1RA), such as exenatide and liraglutide, are currently employed successfully in the treatment of patients with type 2 diabetes mellitus. GLP-1RA improve glycaemic control and stimulate satiety, leading to reductions in food intake and body weight. Besides gastric distension and peripheral vagal nerve activation, GLP-1RA induce satiety by influencing brain regions involved in the regulation of feeding, and several routes of action have been proposed. This review summarises the evidence for a physiological role of GLP-1 in the central regulation of feeding behavior and the different routes of action involved. Also, we provide an overview of presently available data on pharmacological stimulation of GLP-1 pathways leading to alterations in CNS activity, reductions in food intake and weight loss.

Critical role for peptide YY in protein-mediated satiation and body-weight regulation

Rachel L. Batterham, Helen Heffron, Saloni Kapoor, Joanna E. Chivers, et al.
Cell Metab 2006; 4, 223–233 http://dx.doi.org:/10.1016/j.cmet.2006.08.001

Dietary protein enhances satiety and promotes weight loss, but the mechanisms by which appetite is affected remain unclear. We investigated the role of gut hormones, key regulators of ingestive behavior, in mediating the satiating effects of different macronutrients. In normal-weight and obese human subjects, high-protein intake induced the greatest release of the anorectic hormone peptide YY (PYY) and the most pronounced satiety. Long-term augmentation of dietary protein in mice increased plasma PYY levels, decreased food intake, and reduced adiposity. To directly determine the role of PYY in mediating the satiating effects of protein, we generated PYY null mice, which were selectively resistant to the satiating and weight-reducing effects of protein and developed marked obesity that was reversed by exogenous PYY treatment. Our findings suggest that modulating the release of endogenous satiety factors, such as PYY, through alteration of specific diet constituents could provide a rational therapy for obesity.

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Thyroid Function and Disorders

Posted in Amino acids, Calcium Signaling, Clinical Diagnostics, Curation, Developmental biology, Disease Biology, Endocrine Diseases, Genomic Endocrinology, Metabolism, Neurological Diseases, Proteins, Translational Science, tagged , , , , , , , , , , on February 5, 2015| Leave a Comment »

Thyroid Function and Disorders

Writer and Curator: Larry H. Bernstein, MD, FCAP 

Normal thyroid function is maintained by endocrine interactions between the hypothalamus, anterior pituitary and thyroid gland [Matfin, 2009]. Iodide is transported across the basement membrane of the thyroid cells by an intrinsic membrane protein called the Na/I symporter (NIS). At the apical border, a second iodide transport protein called pendrin moves iodide into the colloid, where it is involved in hormono-genesis. Once inside the follicle, most of the iodide is oxidized by the enzyme thyroid peroxidase (TPO) in a reaction that facilitates combination with a tyrosine molecule to ultimately form thyroxine (T4) and triiodothyronine (T3). Thyroxine is the major thyroid hormone secreted into the circulation (90%, with T3 composing the other 10%). There is evidence that T3 is the active form of the hormone and that T4 is converted into T3 before it can act physiologically.

All of the major organs in the body are affected by altered levels of thyroid hormone. These actions are mainly mediated by T3. In the cell, T3 binds to a nuclear receptor, resulting in transcription of specific thyroid hormone response genes.

Maternal thyroid hormones are essential for neural development in zebrafish.

Marco A Campinho, João Saraiva, Claudia Florindo, Deborah M Power Molecular endocrinology (Baltimore, Md.) 05/2014;
http://dx.doi.org:/10.1210/me.2014-1032

ABSTRACT Teleost eggs contain an abundant store of maternal thyroid hormones (THs) and early in zebrafish embryonic development all the genes necessary for TH signalling are expressed. Nonetheless the function of THs in embryonic development remains elusive. To test the hypothesis that THs are fundamental for zebrafish embryonic development an MCT8 knockdown strategy was deployed to prevent maternal TH uptake. Absence of maternal THs did not affect early specification of the neural epithelia but profoundly modified later dorsal specification of the brain and spinal cord as well as specific neuron differentiation. Maternal THs acted upstream of pax2a, pax7 and pax8 genes but downstream of shha and fgf8a signalling. The lack of inhibitory spinal cord interneurons and increased motorneurons in the MCT8 morphants is consistent with their stiff axial body and impaired mobility. MCT8 mutations are associated with X-linked mental retardation in humans and the cellular and molecular consequences of MCT8 knockdown during embryonic development in zebrafish provides new insight into the potential role of THs in this condition.
Relationship between thyroid status and renal function in a general population of unselected outpatients

Giuseppe Lippi, Martina Montagnana, Giovanni Targher, Gian Luca Salvagno, Gian Cesare Guidi
Clin Biochem May 2008; 41(7–8): 625-627

When compared with euthyroid subjects, those with TSH < 0.2 mIU/L and > 2.5 mIU/L had increased and decreased estimated glomerular filtration rate (e-GFR), respectively. TSH levels were an independent predictor of e-GFR.

Serum Thyroid-Stimulating Hormone Measurement for Assessment of Thyroid Function and Disease

Douglas S. Ross
Endocr and Metab Clinics of N Am, Jun 2001; 30(2, 1): 245-264

Thyrotropin, or thyroid-stimulating hormone (TSH), is one of a family of glycoprotein hormones including luteinizing hormone (LH), follicle-stimulating hormone (FSH), and human chorionic gonadotropin (hCG) that share a common α-subunit and a unique β-subunit. Pituitary TSH regulates the secretion of the thyroid hormones T4 (thyroxine) and T3 (triiodothyronine). TSH secretion, in turn, is controlled through negative feedback by thyroid hormone on the pituitary thyrotrope. This relationship is negative log-linear. Small changes in serum free thyroid hormone concentrations result in large changes in serum TSH concentrations, and even subtle changes in thyroid hormone production are best assessed by measurement of serum TSH . Until the late 1980s, the detection limit of TSH assays was within the normal range, and these first-generation TSH assays were useful only for the detection of hypothyroidism. Free T4 measurements were primarily used for assessing thyroid function despite the technical difficulties in free thyroid hormone measurements owing to abnormal binding proteins, changes in binding protein concentrations, and the effects of drugs and illness on thyroid hormone binding. With the use of sensitive second- and third-generation TSH assays, TSH measurement has emerged as the single most useful test of thyroid function. It is widely and appropriately used as a screening test. Unfortunately, the trend has been to rely on TSH measurements alone for the assessment of complicated thyroid disease and patients undergoing treatment for thyroid dysfunction. This article focuses on the potential and real limitations of TSH measurement.
Correlation of creatinine with TSH levels in overt hypothyroidism — A requirement for monitoring of renal function in hypothyroid patients?

Vandana Saini, Amita Yadav, Megha Kataria Arora, Sarika Arora, Ritu Singh, Jayashree Bhattacharjee
Clin Biochem  Feb 2012; 45(3): 212-214

Highlights
► Increase serum creatinine levels in both subclinical and overt hypothyroidism. ► Creatinine levels progressively increase with increasing degree of hypothyroidism. ► Increase in creatinine correlated with TSH levels in overt hypothyroid subjects. ► Regular monitoring of renal function is required in hypothyroid patients.

Renal function is influenced by thyroid status. Therefore, this study was done to determine the relationship between renal function and different degrees of thyroid dysfunction.
Design and methods
Thyroid and kidney function tests were analyzed in 47 patients with overt (TSH ≥ 10.0 μIU/L) and 77 patients with subclinical hypothyroidism (TSH 6.0–9.9 μIU/L) in a cross-sectional study. These were compared with 120 age- and sex-matched euthyroid controls.
Results
Overt hypothyroid subjects showed significantly raised serum urea, creatinine and uric acid levels as compared to controls whereas subclinical hypothyroid patients showed significant increased levels of serum urea and creatinine levels. TSH showed significant positive correlation with creatinine and uric acid values and, fT4 had a negative correlation with uric acid in overt hypothyroidism.
Conclusion
Hypothyroid state is associated with significant derangement in biochemical parameters of renal function. Hence the renal function should be regularly monitored in hypothyroid patients.

  1. Ability of Serum Thyroid-Stimulating Hormone Levels to Reflect Peripheral and Central Thyroid Hormone Action Appropriately
  • Uncertainty Owing to Heterogeneity of T4 Deiodinases
  • Uncertainty Owing to Heterogeneity of T3 Receptors
  • Uncertainty Owing to Resetting of the Threshold for Negative Feedback
  1. Clinical Utility of Thyroid-Stimulating Hormone Measurement
  2. Screening for Thyroid Disease and Assessment of Patients Suspected of Having Thyroid Disease
  • Limitations of Thyroid-Stimulating Hormone Testing in Patients with Known Thyroid Disease Central Hypothyroidism
  • Thyrotoxicosis Owing to Inappropriate Thyroid-Stimulating Hormone Secretion
  • Monitoring Thyroid Hormone Therapy
  • Patients Treated for Hyperthyroidism
  1. The Pituitary-Thyroid Axis in Nonthyroidal Illness
  • Measurement of Thyroid-Stimulating Hormone
  • Drugs that Affect Serum Thyroid-Stimulating Hormone Concentrations

Investigations into the etiology of elevated serum T3 levels in protein-malnourished rats

Robert C. Smallridge, Allan R. Glass, Leonard Wartofsky, Keith R. Latham, Kenneth D. Burman
Metabolism, V June 1982; 31(6): 538-542

Thyroid function studies and the peripheral metabolism of thyroid hormone were examined in rats fed a low protein diet (9% casein) for 4–8 wk. Compared to animals fed a normal protein diet ad libitum, both the low protein rats and a pair-fed control group weighed less at the end of the study. However, serum total T3 levels were significantly higher only in the protein deficient rats. The elevated serum T3 was not explainable by enhanced peripheral T4 to T3 conversion, as there was no evidence of any change in hepatic or renal 5′-deiodinase activity when homogenates were examined for conversion of T4 to T3, reverse T3 to 3,3′-diiodothyronine, or 3′,5′-diiodothyronine to 3′-monoiodothyronine. Neither was there an effect on hepatic T3 receptor maximal binding capacity (204 ± 24 versus 168 ± 15 fmol/mg DNA control) or binding affinity (2.07 ± 0.38 versus 2.49 ± 0.24 × 10−10 M control). In two separate experiments the dialyzable fraction of T3 was significantly lower in the low protein group while free T3 concentrations were unchanged or reduced. In contrast, serum total and free T4 were either normal or reduced and dialyzable T4 was unaffected by protein deficiency. We conclude that while serum total T3 is elevated in rats chronically fed a low protein diet, this elevation is not due to enhanced T4 to T3 conversion. Rather, the increased T3 levels can be accounted for by a striking alteration in protein binding to T3. Moreover, the failure to demonstrate similar changes in serum total and dialyzable T4 suggests that in the rat, protein deficiency has different effects on binding to the two major thyroid hormones. Dietary induced changes in serum thyroid hormone binding must be kept in mind in nutrition studies in the rat.

Role of thyrotropin in metabolism of thyroid hormones in nonthyroidal tissues

Udaya M. Kabadi
Metabolism, Jun 2006; 55(6): 748-750

T4 conversion into T3 in peripheral tissues is the major source of circulating T3. However, the exact mechanism of this process is ill defined. Several in vitro studies have demonstrated that thyrotropin facilitates deiodination of T4 into T3 in liver and kidneys. However, there is a paucity of in vitro studies confirming this activity of thyrotropin. Therefore, this study was conducted to examine the influence of thyrotropin on thyroid hormone metabolism in nonthyroidal tissues. We assessed T4, T3, reverse T3 (rT3), and T3 resin uptake (T3RU) responses up to 12 hours at intervals of 4 hours in 6 thyroidectomized female mongrel dogs rendered euthyroid with LT4 replacement therapy before and after subcutaneous (SC) administration of bovine thyrotropin (5 U) on one day and normal saline (0.5 mL) on another in a randomized sequence between 08:00 and 09:00 am. Euthyroid state after LT4 replacement was confirmed before thyrotropin administration. Serum T4, T3, rT3, and T3RU all remained unaltered after SC administration of normal saline. No significant alteration was noted in serum T3RU values on SC administration of thyrotropin. However, serum T3 rose progressively reaching a peak at 12 hours with simultaneous declines being noted in both serum T4 and rT3 concentrations (P < .05 vs prethyrotropin values for all determinations). The changes after SC administration were significantly different (P < .001) in comparison to those noted on SC administration of normal saline. Thyrotropin may promote both the conversion of T4 to T3 and metabolism of rT3 into T2 in nonthyroidal tissues via enhancement of the same monodeionase.

Effects of growth hormone administration on fuel oxidation and thyroid function in normal man

Jens Møller, Jens O.L. Jørgensen, Niels Møller, Jens S. Christiansen, Jørgen Weeke
Metabolism, Jul 1992;  41(7): 728-731

In a randomized, double-blind, placebo-controlled, cross-over study, we examined the effects of 14 days of growth hormone (GH) administration (12 IU/d subcutaneously) on energy expenditure (EE), respiratory exchange ratio (RER), and thyroid function in 14 normal adults of normal weight (eight men and six women). EE (kcal/24 h) was significantly elevated after GH administration (2,073 ± 392, [GH], 1,900 ± 310, [placebo], P = .01). RER was significantly lowered during GH administration (0.73 ± 0.04 v 0.78 ± 0.06, P = .02), reflecting increased oxidation of lipids. Total triiodothyronine (TT3) (nmol/L) and free T3 (FT3) (pmol/L) increased significantly during GH (TT3: 1.73 ± 0.06 [GH], 1.48 ± 0.08 [placebo], P = .01; FT3: 6.19 ± 0.56 [GH], 5.49 ± 0.56 [placebo], P = .01). Concomitantly, an insignificant decrease in reverse T3 (rT3) (nmol/L) was observed (0.07 ± 0.01 [GH], 0.15 ± 0.01 [placebo], P = .08). GH caused a highly significant increase in T3/thyroxine (T4 (×100) ratio (1.84 ± 0.12 [GH], 1.37 ± 0.06 [placebo]). Serum thyrotropin (TSH) was not significantly changed by GH. No changes in total thyroxine (TT4) (nmol/L) (98 ± 6 [GH], 111 ± 8 [placebo], P = .40) and free thyroxine (FT4) (pmol/L) (17.4 ± 1.3 [GH], 18.6 ± 1.1 [placebo], P = .37) after 14 days of GH administration were observed. In conclusion, 2 weeks of GH administration increases EE and lipidoxidation. This finding may partly be mediated by an increase in peripheral T4 to T3 conversion.

Studies on the deiodination of thyroid hormones in Xenopus laevis tadpoles

Helen Robinson, Valerie Anne Galton
Gen Compar Endocr, Sept 1976; 30(1): 83-90

Liver and tail tissues from Xenopus laevis tadpoles possess deiodinating systems capable of degrading both thyroxine (T4) and 3,5,3′-triiodothyronine (T3). Deiodinating activity in liver remains at a constant level throughout late development and metamorphosis with the exception of a transient increase at stage 59, the onset of metamorphosis. Tail activity remains constant during development but rises sharply during metamorphosis when the tail is undergoing regression. In contrast to these findings on spontaneously metamorphosing tadpoles, tail tips induced to regress in vitro do not exhibit any rise in deiodinating activity, even when the tail tips are undergoing extensive autolysis. These results indicate that, while a rise in deiodinating activity may coincide temporarily with hormone action during metamorphosis, the two phenomena may be separated. The deiodinating activity present in tadpole tissues appears to be enzymic and possesses properties characteristic of peroxidase activity. The reaction catalyzed by this mechanism does not appear to involve monodeiodination and hence cannot be considered a mechanism for the peripheral conversion of T4 to T3.

Mechanisms governing the relative proportions of thyroxine and 3,5,3′-triiodothyronine in thyroid secretion

Peter Laurberg
Metabolism, Apr 1984; 33(4): 379-392

In subjects with normal thyroid function only a minor part of circulating 3,5,3′-triiodothyronine (T3) originates directly from the thyroid; the majority is produced in the peripheral tissues by deiodination of thyroxine (T4). However, T3 of thyroidal origin constitutes a relatively high fraction of the total T3 produced in many patients with thyroid hyperfunction or hypofunction. Such a relatively high T3 content in the secretion of the thyroid could be caused by a low T4T3 ratio in thyroglobulin. Severe iodine deficiency is a well-known inducer of a low T4T3 ratio, but a low T4T3 ratio can also be produced independent of the iodine content. This is seen in in vitro studies of thyroglobulin iodination when small amounts of DIT are added to the incubation mixture and in vivo in TSH-treated animals and in patients with Graves’ disease. Another mechanism for high thyroidal secretion of T3 could be an enhanced fractional deiodination of T4 to T3 in the thyroid. In vitro thyroid perfusion studies have shown that the T3 content of thyroid secretions is higher than would be expected from the T4T3 ratio of thyroid hydrolysate and that the major mechanism is deiodination of T4 to T3. Thyroxine deiodinases are also present in the human thyroid, and the amount of T4 deiodinase is enhanced in the thyroids from patients with medically treated Graves’ disease and in the hyperstimulated thyroids of rats. Other factors of possible importance for the mixture of T3 and T4 secreted by the thyroid are a relatively faster liberation of T3 than of T4 from thyroglobulin during partial hydrolysis (this faster release of T3 is probably the mechanism behind the more “rapid” secretion of T3 than of T4), or some kind of thyroid heterogeneity leading to pinocytosis and hydrolysis of thyroglobulin with a lower T4T3 ratio than that of average thyroglobulin.

Starvation-induced alterations of circulating thyroid hormone concentrations in man

Thomas J. Merimee, E.S. Fineberg
Metabolism Jan 1976; 25(1): 79-83

Serum concentrations of triiodothyronine (T3), thyroxine (T4), and TSH were examined in seven men and seven women of normal weight during a 60-hr fast. Similar studies were conducted in two women who received daily for 1 mo before and during a similar fast, 0.4 mg and 0.5 mg of l-thyroxine.
The serum concentrations of T3 decreased in each of the untreated normal subjects (sign test of significance, p < 0.001). The mean control concentration of T3 in women was 152 ± 9 ng100 ml (X ± SEM); after 24 hr of fasting, 131 ± 31 ng100 ml; and at the termination of the fast, 90 ± 15 ng100 ml. The latter value differed from the control value with a p value of < 0.01. Similar changes of T3 concentration occurred in men (mean basal T = 160 ± 11 ng100 ml; mean at termination of fast = 87 ± 16 ng100 ml). The range of decrease for T3 in all subjects varied from 24% to 55%.
The mean T4 concentration at the beginning of the fast was  6.9 ± 0.9, and at the termination of the fast, 7.5 ± 0.6 (p = NS). TSH concentrations remained unchanged (Control, 3.8 ± 0.45 μU/ml; at 60 hr, 4.0 ± 0.26 μU/ml, p = NS).
Studies in two women who received, before and during a fast, T4, indicate that a decreased peripheral conversion of T4 to T3 is the most likely mechanism responsible for this change.

Effect of estrogens on thyroid function. II. Alterations in plasma thyroid hormone levels and their metabolism

Ramesh C. Sawhney, Indra Rastogi, Gopal K. Rastogi
Metabolism Mar 1978; 27(3): 279-288

The circulating levels of total triiodothyronine (TT3), thyroxine (TT4, and T4-bbinding globulin (TBG) and the kinetics of T3 and T4 were studied in five menstruating rhesus monkeys before, during, and after prolonged treatment with estradiol monobenzoate (E2B, 50 μg/kg body weight/day subcutaneously). A significant increase over pretreatment (p < 0.01) plasma TT3, TT4, and TBG was recorded on day 6 of E2B therapy. A further significant stepwise increase in these parameters was noted up to day 19 of E2B, when the levels plateaued for the rest of the period of E2B treatment. Two weeks after discontinuation of E2B, plasma TT3, TT4, and TBG had returned to the pretreatment range and remained so up to 40 days of observation. Although the percent free T3 and percent free T4 were significantly decreased (p < 0.01) during E2B therapy, the absolute concentrations of free T3 and free T4 were not altered. After prolonged E2B treatment the metabolic clearance rate, distribution space, and production rate (PR) of both T3 and T4 were decreased (p < 0.01). The extrathyroidal T4 pool (ETT4P) was significantly increased (p < 0.01), whereas ETT3P did not show any significant alterations (p > 0.05). The decreased PR of T4 might have been due to a direct inhibitory effect of E2B on the thyroid, whereas the decrease in PR of T3 might have been due to either decreased conversion of T4 to T3, to decreased secretion by the thyroid, or both.
Zebrafish as a model to study peripheral thyroid hormone metabolism in vertebrate development

Marjolein Heijlen, Anne M. Houbrechts, Veerle M. Darras
Gen Compar Endocr 1 Jul 2013; 188: 289-296

To unravel the role of thyroid hormones (THs) in vertebrate development it is important to have suitable animal models to study the mechanisms regulating TH availability and activity. Zebrafish (Danio rerio), with its rapidly and externally developing transparent embryo has been a widely used model in developmental biology for some time. To date many of the components of the zebrafish thyroid axis have been identified, including the TH transporters MCT8, MCT10 and OATP1C1, the deiodinases D1, D2 and D3, and the receptors TRα and TRβ. Their structure and function closely resemble those of higher vertebrates. Interestingly, due to a whole genome duplication in the early evolution of ray-finned fishes, zebrafish possess two genes for D3 (dio3 and dio3a) and for TRα (thraa and thrab). Transcripts of all identified genes are present during embryonic development and several of them show dynamic spatio-temporal distribution patterns. Transient morpholino-knockdown of D2, D3 or MCT8 expression clearly disturbs embryonic development, confirming the importance of each of these regulators during early life stages. The recently available tools for targeted stable gene knockout will further increase the value of zebrafish to study the role of peripheral TH metabolism in pre- and post-hatch/post-natal vertebrate development.

The consequences of inappropriate treatment because of failure to recognize the syndrome of pituitary and peripheral tissue resistance to thyroid hormone

Samuel Refetoff, Angel Salazar, Terry J. Smith, Neal H. Scherberg
Metabolism  Aug 1983; 32(8); 822-834

Since the description of the syndrome of global (peripheral tissues and pituitary) resistance to thyroid hormone, new cases are being recognized with increasing frequency. The patient described herein had a markedly elevated serum TSH concentration of 260 μU/mL at the time of diagnosis. Studies suggest that elevations of serum TSH levels in this and other patients with the syndrome are most likely iatrogenic in origin. The patient was 312 years old when a goiter and a high serum T4 concentration were detected. Despite subtotal thyroidectomy, antithyroid drugs were required to maintain her T4 level in the normal range. She was referred at age 1112 years because of recurrent goiter. Her parents and five older siblings had normal thyroid function. Off therapy, her serum T4 level was 14.9 μg/dL, FT4I was 17.0, T3 was 362 ng/dL, TSH was 260 μU/mL, and antibodies were negative. There were no signs of thyrotoxicosis, her bone age was 7 years, her growth was stunted (third percentile), her intellectual quotient (IQ) was 67, and there was a 30–50 dB sensorineural hearing loss. The presence of a pituitary adenoma was ruled out. Her TSH had normal bioreactivity and rose to 540 μU/mL in response to TRH. Triiodothyronine was given in incremental doses of 50, 100, 200, and 400 μg/d over 28 days. The log concentrations of serum TSH showed an inverse linear correlation with serum T3. While receiving the highest dose of T3, on which the level of serum T3 ranged from 1400 to 2500 ng/dL, the TSH response to TRH normalized (basal 4.2 and peak 20 μU/mL), as did the high levels of serum cholesterol, carotene, and T4. Her BMR rose from +5 to +22%, her IQ rose to 77, and she gained weight without an increase in caloric intake. Only minimal changes were observed in levels of urinary cAMP, hydroxyproline, magnesium, and nitrogen. All values, with the exception of the weight gain, returned to baseline 2 months after T3 treatment was discontinued. The TSH level was suppressed by l-dopa and by prednisone. Long-term therapy with equivalent doses of T4 (from 300 to 1000 μg/d) produced a growth of 3 cm during the initial 6 weeks, 10.5 cm over the ensuring year (above the 10th percentile), and regression of goiter without thyrotoxicosis. The patient exhibited resistance to thyroid hormone in pituitary and peripheral tissues. The optimal dose of T4 replacement could be predicted by studying tissue responses to incremental doses of T3. The marked elevation in serum TSH concentration, stunted growth, and laboratory evidence of hypothyroidism were due to the limited thyroidal reserve caused by thyroidectomy. All patients with an impaired ability to compensate for the defect as a result of inappropriate treatment should be given thyroid hormone in amounts short of producing catabolic effects. Such a dose is expected to normalize the basal serum TSH concentration and its response to TRH.

Solving the mystery of iodine uptake

Valda Vinson
Science 20 Jun 2014; 344(6190), p. 1355
http://dx.doi.org:/10.1126/science.344.6190.1355-a

The thyroid gland produces iodine-containing hormones that regulate metabolism. The cell membrane protein NIS (sodium/iodine symporter) transports iodine into thyroid cells, but because iodine concentrations outside of the cell are so low, how it does so is a mystery. The key? Moving two sodium ions along with the iodine ion, Nicola et al found. NIS also does not bind sodium very tightly, but the high concentrations of sodium outside the cell allow one sodium ion to bind. This binding increases the affinity of NIS for a second sodium ion and also for iodine. With the three ions bound, NIS changes its conformation so that it opens to the inside of the cell, where the sodium concentration is low enough for NIS to release its sodium ions. When the sodium goes away, so does NIS’s affinity for iodine, leading NIS to release it.

Unliganded Thyroid Hormone Receptor α Regulates Developmental Timing via Gene Repression in Xenopus tropicalis

Jinyoung Choi, Ken-ichi T. Suzuki, Tetsushi Sakuma, Leena Shewade, Takashi Yamamoto, and Daniel R. Buchholz
Endocr Feb 2015; 156(2): 735–744 http://dx.doi.org:/10.1210/en.2014-1554

Thyroid hormone (TH) receptor (TR) expression begins early in development in all vertebrates when circulating TH levels are absent or minimal, yet few developmental roles for unliganded TRs have been established. Unliganded TRs are expected to repress TH-response genes, increase tissue responsivity to TH, and regulate the timing of developmental events. Here we examined the role of unliganded TRα in gene repression and development in Xenopus tropicalis. We used transcription activator-like effector nuclease gene disruption technology to generate founder animals with mutations in the TRα gene and bred them to produce F1 offspring with a normal phenotype and a mutant phenotype, characterized by precocious hind limb development. Offspring with a normal phenotype had zero or one disrupted TRα alleles , and tadpoles with the mutant hind limb phenotype had two truncated TRα alleles with frame shift mutations between the two zinc fingers followed by 40–50 mutant amino acids and then an out-of-frame stop codon. We examined TH-response gene expression and early larval development with and without exogenous TH in F1 offspring. As hypothesized, mutant phenotype tadpoles had increased expression of TH-response genes in the absence of TH and impaired induction of these same genes after exogenous TH treatment, compared with normal phenotype animals. Also, mutant hind limb phenotype animals had reduced hind limb and gill responsivity to exogenous TH. Similar results in methimazole-treated tadpoles showed that increased TH-response gene expression and precocious development were not due to early production of TH. These results indicate that unliganded TRα delays developmental progression by repressing TH-response genes.
The discovery of thyroid replacement therapy. Part 2: The critical 19th century
Conceptualizing the link between the thyroid and myxoedema

Stefan Slater
R Soc Med 2011; 104: 59–63. http://dx.doi.org:/10.1258/jrsm.2010.10k051

Sir William Withey Gull (1816–1890)

Frederik Ruysch, anatomist in Leyden around 1690, adopted, according to Albrecht von Haller in 1766, the opinion that a peculiar fluid was elaborated in the gland and poured into the veins’. The 19th century thus began with thyroidology at best in embryo; but during that century endocrinology was born and the thyroid was its standard bearer. In 1836, Thomas Wilkinson King of Guys Hospital, regarded by some as the ‘Father of Endocrinology’, anticipated on the basis of observation and experiment the internal secretion of the thyroid. In a meticulous paper on its anatomy: he wrote of the thyroid gland that ‘its absorbent vessels carry its peculiar secretion to the great veins of the body’. This language is almost identical to that of Ruysch and Haller more than a century earlier. The idea was prompted by the thyroid’s disproportionately large vascular supply in the absence of any evident mechanical or other local function and also at what he described as its ‘peculiar’ fluid. King notes that his view ‘has been indirectly surmised by Morgagni [probably in 1761] and others’.
In 1850, at a meeting of the Royal Medical and Chirurgical Society of London, chaired by Thomas Addison, Thomas Blizzard Curling, surgeon at the London Hospital, provided a clear clinicopathological correlate in a paper entitled ‘Two cases of absence of the thyroid body and symmetrical swellings of fat tissue at the sides of the neck, connected with defective cerebral development’.  Postmortem examination in each revealed no trace of thyroid tissue and that the swellings consisted only of fat.  Curling’s important observation was not pursued until 1871 when, at another meeting of the Society, Curling himself then in the chair, Charles Hilton Fagge, a physician at Guy’s Hospital, presented a paper on sporadic cretinism. He described four living cases and noted that none of them had a goiter and that one had been well up to the age of eight and, although now physically cretinous at age 16, she remained very intelligent. He referred to Curling’s paper and reached the same conclusion that the ‘healthy thyroid body is capable of exerting a counteracting influence [on cretinism]’.
Two years later, in 1873, Fagge’s senior colleague at Guy’s, Sir William Withey Gull, presented before the Clinical Society of London two of the five cases he had seen of what he called ‘A Cretinoid State supervening in Adult Life in Women’. He described their cretin-like appearance, drawing particular attention to the broad and thick tongue and the guttural voice and its pronunciation ‘as if the tongue were too large for the mouth’. He acknowledged his remarks were tentative, hence, he said, his use of the word ‘cretinoid’, but he had no doubt this was a ‘substantive’ condition and not one of cardiac or renal origin.
Gull was an interesting personality with apparently a remarkable presence, resembling Napoleon in face, form and manner (Figure). In the 1970s, 80 years after his death in 1890, he was the subject of a theory, quickly discredited, that he had been ‘Jack the Ripper’, the killer in the still unsolved murders and mutilations of at least five Whitechapel prostitutes in 1888. He figured in the 1988 TV film series, Jack the Ripper, starring Michael Caine as the detective. Gull is credited with the first description of hypothyroidism in adults and his paper was important in defining a recognizable clinical syndrome.
Then, in 1877, William Miller Ord, read his paper before the Royal Medical and Chirurgical Society of London and proposed the term ‘myxoedema’ for the adult condition. He described the non-pitting, ‘mucous edema’.   He also presented an engaging theory to explain the lethargy, inertia and slow responses associated with the disease. He suggested that these might result from the sheathing and insulation of the body in a ‘jelly-like’, mucin-laden integument that interfered with sensory perceptions and stimulation. Six years later, he chaired the committee set up by the Clinical Society of London to investigate the whole matter. He also later undertook some of the earliest metabolic studies of the effects of treating myxoedema with thyroid extract, showing the rapid weight loss and rise in temperature and in urinary volume and nitrogen excretion that occurred.
The key papers, which advanced these English authors observations, were those of the Swiss surgeons, Jaques-Louis Reverdin in Geneva and Emil Theodor Kocher in Bern, Kocher later receiving the Nobel Prize for his work on the thyroid. How fitting it is that it should be two Swiss doctors whose practices unlocked an understanding of the importance of the thyroid. For they each identified the late effects of total ablation (extirpation) of goiters. they

noted the great similarity of Gull’s and Ord’s myxoedema cases with their affected postoperative patients, referring to the comparison as a ‘rapprochement complet’, clearly making the connection. They acknowledged Gull’s primacy in describing the clinical manifestations and Ord’s ‘christening’ the condition ‘myxoedema’, and proposed that surgical cases be known as ‘myxoedème opératoire’. In light of his findings in 1882, Reverdin thereafter sought to conserve a part of the gland during thyroidectomy for goiter, speculating that its complete removal may have been responsible for these late effects. He had noticed that no such problems followed a just unilateral lobectomy. Kocher called the disease picture in his affected cases ‘cachexia strumipriva’ – literally, a bad condition due to the removal of a struma (goiter) without reference to the earlier work of Reverdin. Halsted noted in his monumental review of goiter surgery: ‘It is interesting to follow the argumentation of a mind so exceptionally keen and sane as Kocher’s in its futile efforts to explain insufficiently illuminated phenomena’. In reading Kocher’s 1909 Nobel Prize Lecture (in English translation), one gets the impression that Kocher was aware in 1883 of Gull’s and Ord’s reports, despite not referring to them, and he dismisses Reverdin’s contribution.
There ensued a competition over the contribution to the thyroid discovery.  When post-thyroidectomy myxedema wsas brought to the attention of Kocher, he agreed it was analogous to his cases of cachexia strumipriva. It is also obvious that Kocher, like many surgeons of the time, cannot have engaged in routine postoperative outpatient follow-up, for otherwise the ensuing problems in his goiter-operated patients would have been detected years earlier. In respect of this key moment in the history of the thyroid, Reverdin could be said to hold the intellectual property. The thought has been expressed that perhaps he should have shared the 1909 Nobel Prize with Kocher.
The Emerging Roles of Thyroglobulin

Yuqian Luo, Yuko Ishido, Naoki Hiroi, Norihisa Ishii, and Koichi Suzuki
Adv in Endocr 2014, Article ID 189194, 7 pp http://dx.doi.org/10.1155/2014/189194

Thyroglobulin (Tg), the most important and abundant protein in thyroid follicles, is well known for its essential role in thyroid hormone synthesis. In addition to its conventional role as the precursor of thyroid hormones, we have uncovered a novel function of Tg as an endogenous regulator of follicular function over the past decade. The newly discovered negative feedback effect of Tg on follicular function observed in the rat and human thyroid provides an alternative explanation for the observation of follicle heterogeneity. Given the essential role of the regulatory effects of Tg, we consider that dysregulation of normal Tg function is associated with multiple human thyroid diseases including autoimmune thyroid disease and thyroid cancer. Additionally, extrathyroid Tg may serve a regulatory function in other organs. Further exploration of Tg action, especially at the molecular level, is needed to obtain a better understanding of both the physiological and pathological roles of Tg.

The Surgical Management of Thyroid Cancer

Sara A. Morrison, Hyunsuk Suh, and Richard A. Hodin
Rambam Maimonides Med J 2014; 5(2):e0008. http://dx.doi.org:/10.5041/RMMJ.10142

There are approximately 63,000 reported cases of thyroid carcinoma annually in the United States, representing roughly 4% of all documented malignancies.1 Diagnosis typically stems from work-up of a thyroid nodule. Data from the Framingham study suggests that palpable thyroid nodules are present in 4% of the US population,2 but non-palpable nodules may exist in up to 67% of the population. Such nodules are often found incidentally secondary to the rising use of imaging modalities in medical settings. The large majority of thyroid nodules are benign, with an overall reported risk of malignancy from 5% to 15%.
Thyroid cancer has been increasing in incidence, with the number of reported cases in the US rising by 25% over the last 3 years. With growing technological advances in the field and improved contributions of diagnostics, surgical decision-making and operative planning have taken on new challenges. Herein, we review the current clinical practice recommendations and active areas of surgical controversy, reflective of the most recently published professional consensus guidelines and a systematic review of the literature.
The use of FNA in current clinical practice has resulted in post-surgical pathology findings of malignancy in over 50% of specimens.7 The Bethesda System for Reporting Thyroid Cytopathology (TBSRTC) was developed in order to allow pathologists among varying institutions to communicate results to clinical care-takers with widely under-stood descriptors. Results of FNA biopsies are broken down into the following categories with the corresponding risks of malignancy: non-diagnostic or unsatisfactory (1%–4%), benign (0%–3%), atypia of undetermined significance or follicular lesion of undetermined significance (AUS/FLUS; 5%–15%), follicular neoplasm or suspicious for a follicular neoplasm (FN/sFN; 15%–30%), suspicious for malignancy (60%–75%), and malignant (97%–99%).
Mutational Panels.
AsuragenmiR Inform (Austin, TX, USA) mutation analysis assay and Thyroid Cancer Mutation Panel by Quest Diagnostics (Madison, NJ, USA) are the two main commercially available mutational tests which test for known genetic alterations such as BRAF, RAS, RET/PTC, and PAX8/PPAR. These mutational panels are highly specific for malignancy; however, due to the low overall frequency of these mutations in thyroid cancers, negative results do not rule out cancer. Therefore, mutational panel tests are considered a “rule-in” test. If a preoperative mutational test is positive, the nodule should be considered malignant, and total thyroidectomy should be recommended.
Gene Expression Profiling.
The most widely known gene expression profiling test is Afirma Gene Expression Classifier (Veracyte, San Francisco, CA, USA), and, with its recent clinical validation by Alexander et al., Afirma is already being utilized in many clinical settings. The Afirma Gene Expression Classifier (GEC) is an RNA-based assay that utilizes FNA samples to evaluate 167 molecular genes associated with benign nodules based on their proprietary algorithm. Unlike the mutational panel testing, Afirma testing is considered a “rule-out” test since the test has a high negative predictive value in distinguishing benign nodules. However, a positive result reported as “suspicious” carries only 38% risk of malignancy.
In all, these molecular tests should be utilized judiciously and should be considered as a complementary diagnostic tool in the management of thyroid nodules. In the future, molecular testing could become more cost-effective and accurate as a diagnostic tool while providing prognostic and therapeutic information.
Papillary Thyroid Cancer.
Total thyroidectomy is the gold standard for patients with a preoperative diagnosis of papillary thyroid cancer when the nodule is greater than 1 cm in size. Completion thyroidectomy is indicated in patients who have undergone prior lobectomy and are found on final pathology to have papillary thyroid cancer that is larger than 1 cm. The completion thyroidectomy should generally be performed within 6 months of the original procedure in order to minimize the risk of lymph node metastasis.
Involvement of cervical lymph nodes in papillary thyroid cancer is frequent, reported to occur in up to 50% of patients. The role of neck dissection at the time of total thyroidectomy is somewhat controversial, however, since most of the nodal involvement is microscopic and does not affect overall survival. It is generally agreed upon that a therapeutic neck dissection should be pursued in the setting of well-differentiated thyroid cancer patients with clinically positive lymph nodes, whether in the central or lateral neck compartments. Prophylactic neck dissection is not done for follicular thyroid cancer, as the rates of lymph node metastasis are typically less than 10%.
Medullary thyroid cancer (MTC) comprises 4% of all thyroid malignancies. The majority of cases are sporadic in nature; approximately 20%–25% represent familiar/hereditary syndromes. Diagnosis is commonly made by FNA biopsy with specific staining for the presence of calcitonin in the tissue specimen. All patients with a diagnosis of medullary thyroid cancer must be evaluated for multiple endocrine neoplasia (MEN) 2 and be ruled out for the synchronous presence of pheochromocytoma prior to scheduling thyroid surgery.
Effects of Dose Level of Anti-thyroid Drug Carbimazole on Thermoregulation and Blood Constituents in Male Rabbits (Oryctolagus cuniculus)

Intisar H. Saeed, Abdalla M. Abdelatif and Mohamed E. Elnageeb
Adv in Research 2014; 2(3): 129-144. Article no. AIR.2014.002

Carbimazole (CBZ) is an anti-thyroid drug commonly used in the treatment of hyperthyroidism. The objective of this study was to evaluate the effects of dose level of CBZ on thermoregulation and blood constituents in mature male rabbits. Twenty animals were assigned to 4 groups (A, B, C, D) of 5 each. Group A served as control and treated animals in groups B,C,D, received daily orally CBZ doses of 10, 15 and 20 mg/animal for 3 weeks, respectively.
The values of rectal temperature (Tr,), respiration rate (RR) and heart rate (HR) decreased in treated rabbits and the mean values of HR decreased with increase in the dose level of CBZ. The packed cell volume (PCV),  Hb concentration and total leukocyte count (TLC) were lower in CBZ treated rabbits. Serum levels of total protein and globulins increased and serum albumin level decreased in treated groups of rabbits. Serum urea level was lower in CBZ treated groups and there was an increase in serum urea level with increase in CBZ dose level. Serum cholesterol level was higher in treated groups and there was an increase in serum cholesterol level with increase in CBZ dose level. Plasma glucose level decreased significantly in CBZ treated groups compared with the control and the mean values decreased with increase in the dose level of CBZ. The results indicate that the responses of basic physiological parameters were almost dose dependent in the range adopted in this study.
Phosphatase Inhibitor Calyculin A Activates TRPC2 Channels in Thyroid FRTL-5 Cells

Pramod Sukumaran, MY Asghar, C Löf, T Viitanen, and Kid Törnquist
Calcium Signaling Jun 2014; 1(2)  http://www.researchpub.org/journal/cs/cs.html

We have previously shown that rat thyroid FRTL-5 cells express a calcium entry pathway regulated by a phosphatase. The nature of the calcium entry pathway is presently unknown. We have also shown that FRTL-5 cells express only the TRPC2 channel of the TRPC family of cation channels. In the present investigation we show, using pharmacological inhibitors, the measurement of sodium and calcium entry, stable TRPC2 knock-down cells, and transfection with a non-conducting form of TRPC2, that the calcium entry pathway regulated by a phosphatase is, in fact, the TRPC2 channel. Our data thus point to a novel mechanism by which the TRPC2 channels can be regulated.

Thyroxine Uptake by Perfused Rat Liver
No Evidence for Facilitation by Five Different Thyroxine-binding Proteins

Carl M. Mendel and Richard A. Weisiger
J. Clin. Invest.  1990; 86: 1840-1847

For each of the five protein-hormone complexes studied, the rate of hepatic uptake of T4 (measured under conditions expected to result in dissociation-limited uptake) closely approximated the rate of spontaneous dissociation of the protein-hormone complex within the hepatic sinusoids. These findings indicate an absence of special cellular mechanisms that facilitate the hepatic uptake of T4 from its plasma binding proteins, and support the view that uptake occurs from the free T4 pool after spontaneous dissociation of T4 from its binding proteins.
Thyroxine Transport and Distribution in Nagase Analbuminemic Rats

Carl M. Mendel, RR Cavalieri, LA Gavin, T Pettersson, and M Inoue
J. Clin. Invest. 1989; 83: 143-148

The postulate that thyroxine (T4) in plasma enters tissues by protein-mediated transport or enhanced dissociation from plasma-binding proteins leads to the conclusion that almost all T4 uptake by tissues in the rat occurs via the pool of albumin bound T4 (Pardridge, W. M., B. N. Premachandra, and G. Fierer. 1985. Am. J. Physiol. 248:G545-G550).
To directly test this postulate, and to test more generally whether albumin might play a special role in T4 transport in the rat, we performed in vivo kinetics studies in six Nagase analbuminemic rats and in six control rats, all of whom had similar serum T4 concentrations and percent free T4 values.
Evaluation of the plasma disappearance curves of simultaneously injected 125I-T4 and I31I-albumin indicated that the flux of T4 from the extracellular compartment into the rapidly exchangeable intracellular compartment was similar in the analbuminemic rats (51±21 ng/min, mean±SD) and in the control rats (54±15 ng/min), as was the size of the rapidly exchangeable intracellular pool of T4 (1.13±0.53 vs. 1.22±036 Mg). This latter finding was confirmed by direct analysis of tissue samples (liver, kidney, and brain). We also performed in vitro kinetics studies using the isolated perfused rat liver. The single-pass fractional extraction by normal rat liver of T4 in pooled analbuminemic rat serum was indistinguishable from that of T4 in pooled control rat serum (10.9±3.3%, n = 3, vs. 11.4±3.4%). When > 98% of the albumin was removed from normal rat serum by chromatography with Affi-Gel blue, the single-pass fractional extraction of T4 (measured by a bolus injection method) did not change (16.3±2.1%, n = 5, vs. 15.2±2.5%). These data provide the first valid experimental test of the enhanced

dissociation hypothesis and indicate that there is no special, substantive role for albumin in T4 transport in the rat.
Influence of thyroid receptors on breast cancer cell proliferation

  1. Conde, R. Paniagua, J. Zamora, M. J. Blanquez, B. Fraile, A. Ruiz & M. I. Arenas
    Ann Oncol 2005; http://dx.doi.org:/10.1093/annonc/mdj040

Background: The involvement of thyroid hormones in the development and differentiation of normal breast tissue has been established. However, the association between breast cancer and these hormones is controversial. Therefore, the objective of the present study was to determine the protein expression pattern of thyroid hormone receptors in different human breast pathologies and to evaluate their possible relationship with cellular proliferation.
Patients and methods: The presence of thyroid hormone receptors was evaluated by immunohistochemistry and western blot analysis in 84 breast samples that included 12 cases of benign proliferative diseases, 20 carcinomas in situ and 52 infiltrative carcinomas.
Results: TR-α was detected in the nuclei of epithelial cells from normal breast ducts and acini, while in any pathological type this receptor was located in the cytoplasm. However, TR-b presented a nuclear location in benign proliferative diseases and carcinomas in situ and a cytoplasmatic location in normal breast and infiltrative carcinomas. The highest proliferation index was observed in carcinomas in situ, although in infiltrative carcinomas an inverse correlation between this index and the TR-α expression was encountered.
Conclusions: The results of this study reveal substantial changes in the expression profile of thyroid hormone.
Zebrafish as a model for monocarboxyl transporter 8-deficiency

GD Vatine, D Zada, T Lerer-Goldshtein, A Tovin, G Malkinson, K Yaniv and L Appelbaum
J Biol Chem Nov 2012; Manuscript M112.413831
http://dx.doi.org:/10.1074/jbc.M112.413831

Background: Mutations in the thyroid hormone transporter MCT8 are associated with psychomotor retardation AHDS.
Results: In zebrafish, as in humans, mct8 is expressed primarily in the nervous system. Elimination of MCT8 causes severe neural impairment.
Conclusion: MCT8 is a crucial regulator during zebrafish embryonic development. Significance: Establishment of the first vertebrate model for MCT8-deficiency, which exhibits a neurological phenotype.
Unusual Ratio between Free Thyroxine and Free Triiodothyronine in a Long-Lived Mole-Rat Species with Bimodal Ageing

Yoshiyuki Henning, Christiane Vole, Sabine Begall, Martin Bens, et al.
PlusOne Nov 2014; 9(11),e113698. http://dx.doi.org:/10.1371/journal.pone.0113698

Ansell’s mole-rats (Fukomys anselli) are subterranean, long-lived rodents, which live in eusocial families, where the maximum lifespan of breeders is twice as long as that of non-breeders. Their metabolic rate is significantly lower than expected based on allometry, and their retinae show a high density of S-cone opsins. Both features may indicate naturally low thyroid hormone levels.
In the present study, we sequenced several major components of the thyroid hormone pathways and analyzed free and total thyroxine and triiodothyronine in serum samples of breeding and non-breeding F. anselli to examine whether
a) their thyroid hormone system shows any peculiarities on the genetic level,
b) these animals have lower hormone levels compared to euthyroid rodents (rats and guinea pigs), and
c) reproductive status, lifespan and free hormone levels are correlated.
Genetic analyses confirmed that Ansell’s mole-rats have a conserved thyroid hormone system as known from other mammalian species. Interspecific comparisons revealed that free thyroxine levels of F. anselli were about ten times lower than of guinea pigs and rats, whereas the free triiodothyronine levels, the main biologically active form, did not differ significantly amongst species. The resulting fT4:fT3 ratio is unusual for a mammal and potentially represents a case of natural hypothyroxinemia.
Comparisons with total thyroxine levels suggest that mole-rats seem to possess two distinct mechanisms that work hand in hand to downregulate fT4 levels reliably. We could not find any correlation between free hormone levels and reproductive status, gender or weight. Free thyroxine may slightly increase with age, based on subsignificant evidence. Hence, thyroid hormones do not seem to explain the different ageing rates of breeders and nonbreeders. Further research is required to investigate the regulatory mechanisms responsible for the unusual proportion of free thyroxine and free triiodothyronine.
Transthyretin Regulates Thyroid Hormone Levels in the Choroid Plexus, But Not in  the Brain Parenchyma: Study in a Transthyretin-Null Mouse Model

JA Palha, R Fernandes, GM De Escobar, V Episkopou, M Gottesman, and MJ Saraiva
Endocr 2000; 141(9): 3267–3272.

Transthyretin (TTR) is the major T4-binding protein in rodents. Using a TTR-null mouse model we asked the following questions.
1) Do other T4 binding moieties replace TTR in the cerebrospinal fluid (CSF)?
2) Are the low whole brain total T4 levels found in this mouse model associated with hypothyroidism, e.g. increased 59-deiodinase type 2 (D2) activity and RC3-neurogranin messenger RNA levels?
3) Which brain regions account for the decreased total whole brain T4 levels?
4) Are there changes in T3 levels in the brain?
Our results show the following.
1) No other T4-binding protein replaces TTR in the CSF of the TTR-null mice.
2) D2 activity is normal in the cortex, cerebellum, and hippocampus, and total brain RC3-neurogranin messenger RNA levels are not altered.
3) T4 levels measured in the cortex, cerebellum, and hippocampus are normal. However T4 and T3 levels in the choroid plexus are only 14% and 48% of the normal values, respectively.
4) T3 levels are normal in the brain parenchyma.
The data presented here suggest that TTR influences thyroid hormone levels in the choroid plexus, but not in the brain. Interference with the blood-choroid-plexus-CSF-TTR-mediated route of T4 entry into the brain caused by the absence of TTR does not produce measurable features of hypothyroidism. It thus appears that TTR is not required for T4 entry or for maintenance of the euthyroid state in the mouse brain.
Identification of monocarboxylate transporter 8 as a specific thyroid hormone transporter

E.C.H. Friesema, S Ganguly, A. Abdalla, J.E.M. Fox, AP. Halestrap, and TJ. Visser
J Biol Chem 2003; Manuscript M300909200
http://dx.doi.org/10.1074/jbc.M300909200

Transport of thyroid hormone across the cell membrane is required for its action and

metabolism. Recently, a T-type amino acid transporter was cloned which transports aromatic amino acids but not iodothyronines. This transporter belongs to the monocarboxylate transporter (MCT) family, and is most homologous with MCT8 (SLC16A2). Therefore, we cloned rat MCT8, and tested it for thyroid hormone transport in Xenopus laevis oocytes. Oocytes were injected with rat MCT8 cRNA, and after 3 days immunofluorescence microscopy demonstrated expression of the protein at the plasma membrane. MCT8 cRNA induced a ~10-fold increase in uptake of 10 nM 125I-labeled thyroxine (T4), 3,3′,5-triiodothyronine (T3), 3,3′,5′-triiodothyronine (rT3) and 3,3′-diiodothyronine. Due to the rapid uptake of the ligands, transport was only linear with time for <4 min. MCT8 did not transport Leu, Phe, Trp or Tyr. [125I]T4 transport was strongly inhibited by L-T4, D-T4, L-T3, D-T3, 3,3’,5-triiodothyroacetic acid, N-bromoacetyl-T3, and bromosulfophthalein. T3 transport was less affected by these inhibitors. Iodothyronine uptake in uninjected oocytes was reduced by albumin but the stimulation induced by MCT8 was markedly increased. Saturation analysis provided apparent Km values of 2-5 μM for T4, T3 and rT3. Immunohistochemistry showed high expression in liver, kidney, brain and heart. In conclusion, we have identified MCT8 as a very active and specific thyroid hormone transporter.
Thyroid hormones,T3 andT4, in the brain
Amy C. Schroeder and Martin L. Privalsky
Front Endocr Mar 2014; 5 article 40.  http://dx.doi.org:/10.3389/fendo.2014.00040

Thyroid hormones (THs) are essential for fetal and post-natal nervous system development and also play an important role in the maintenance of adult brain function. Of the two major THs, T4 (3,5,30,50-tetraiodo-l-thyronine) is classically viewed as an pro-hormone that must be converted toT3 (3,5,30-tri-iodo-l-thyronine) via tissue-level deiodinases for biological activity. THs primarily mediate their effects by binding to thyroid hormone receptor (TR) isoforms, predominantly TRα1 and TRβ1, which are expressed in different tissues and exhibit distinctive roles in endocrinology. Notably, the ability to respond toT4 and toT3 differs for the two TR isoforms, with TRα1 generally more responsive to T4 than TRβ1. TRα1 is also the most abundantly expressed TR isoform in the brain, encompassing 70–80% of all TR expression in this tissue. Conversion of T4 into T3 via deiodinase 2 in astrocytes has been classically viewed as critical for generating local T3 for neurons. However, deiodinase-deficient mice do not exhibit obvious defectives in brain development or function. Considering that TRα1 is well-established as the predominant isoform in brain, and that TRα1 responds to both T3 and T4, we suggest T4 may play a more active role in brain physiology than has been previously accepted.
Thyroid hormone action: astrocyte–neuron communication

Beatriz Morte and Juan Bernal
Front Endocr May 2014; 5, Article 82 http://dx.doi.org:/10.3389/fendo.2014.00082

Thyroid hormone (TH) action is exerted mainly through regulation of gene expression by binding of T3 to the nuclear receptors.T4 plays an important role as a source of intracellular T3 in the central nervous system via the action of the type 2 deiodinase (D2), expressed in the astrocytes. A model of T3 availability to neural cells has been proposed and validated. The model contemplates that brain T3 has a double origin: a fraction is available directly from the circulation, and another is produced locally from T4 in the astrocytes by D2. The fetal brain depends almost entirely on theT3 generated locally. The contribution of systemic T3 increases subsequently during development to account for approximately 50% of total brain T3 in the late postnatal and adult stages. In this article, we review the experimental data in support of this model, and how the factors affectingT3 availability in the brain, such as deiodinases and transporters, play a decisive role in modulating local TH action during development.
The Significance of Thyroid Hormone Transporters in the Brain

Juan Bernal
Endocr Apr 2005; 146(4):1698–1700. http://dx.doi.org:/10.1210/en.2005-0134

The MCT family comprises up to 14 members, some of which are involved in the transport of important substrates for the brain such as lactate and pyruvate. MCT8 has been shown to act as a specific transporter for T4 and T3 and displays slightly higher affinity for T3. Heuer et al. have also studied the regional expression of MCT8 mRNA. In addition to high expression levels in the choroid plexus, they found that MCT8 is expressed in neurons of the neocortex, hippocampus, basal ganglia, amygdala, hypothalamus, and the Purkinje cells of the cerebellum, all regions known to be sensitive to thyroid hormones. Expression of MCT8 in neurons suggests that neuronal uptake of the T3 produced in astrocytes is facilitated by this transporter.
The physiological significance ofMCT8 as a transporter for thyroid hormone is supported by the finding of mutations in humans by Dumitrescu et al. and Friesema et al.  The syndrome affects children from an early age and consists of severe developmental delay and neurological damage together with an unusually altered pattern of thyroid hormone levels in blood. The patients presented low total and free T4, high total and free T3, and low rT3. TSH was moderately elevated in two of the patients and normal or slightly elevated in the other five. Inactivating mutations of the MCT8 transporter could result in the altered thyroid hormone levels. In vitro uptake of T4 and T3 by fibroblasts isolated from affected males was strongly reduced, and intracellular D2 was increased 6- to 8-fold. It is thus hypothesized that the resulting increase in intracellularly generated T3 accumulates in blood because of its poor reuptake into cells.
The second trimester is also the period when thyroid hormone receptors increase in concentration in the brain. If MCT8 is needed at this stage of development for T3 entry into neurons, mutations of the transporter could interfere with T3-dependent developmental processes. Knowledge of the ontogenetic patterns of MCT8 in the human fetal brain would certainly be helpful. On the other hand, there is also the possibility that MCT8 mutations interfere with transport of other substrates for brain metabolism that could be even more important than T3 in determining the severity and outcome of the syndrome. Other members of the family transport metabolic substrates such as pyruvate and lactate, but MCT8 so far appears to be specific for iodothyronines

Peripheral markers of thyroid function: The effect of T4 monotherapy versus T4/T3 combination therapy in hypothyroid subjects: A randomized cross-over study

Ulla Schmidt, B Nygaard, EW Jensen, J Kvetny, A Jarløv, and Jens Faber
Endocrine Connections Jan 10, 2013 http://dx.doi.org:/10.1530/EC-12-0

Background: A recent randomized controlled trial suggests that hypothyroid subjects may find L-T4 and L-T3 combination therapy to be

superior to L-T4 monotherapy in terms of quality of life, suggesting that the brain registered increased T3 availability during the

combination therapy.

Hypothesis: Peripheral tissue might also be stimulated during T4/T3 combination therapy compared to T4 monotherapy.
Methods: Serum levels of Sex Hormone-Binding Globulin (SHBG), pro-collagen-1-N-terminal peptide (PINP), and N-terminal pro-brain natriuretic peptide (NT-proBNP) (representing hepatocyte, osteoblast, and cardiomyocyte stimulation, respectively) were measured in 26 hypothyroid subjects in a double blind, randomized, cross-over trial, which compared the replacement therapy with T4/T3 in combination (50 Fg T4 was substituted with 20 Fg T3) to T4 alone (once daily regimens). This was performed to obtain unaltered serum thyroid stimulating hormone (TSH) levels during the trial and between the two treatment groups. Blood sampling was performed 24 hours after the last intake of thyroid hormone medication.
Results: TSH remained unaltered between the groups ((median) 0.83 vs. 1.18 mU/l in T4/T3 combination and T4 mono-therapy, respectively; p=0.534). SHBG increased from (median) 75 nmol/l at baseline to 83 nmol/l in the T4/T3 group (p=0.015), but remained unaltered in the T4 group (67 nmol/l); thus, it was higher in the T4/T3 vs. T4 group (p=0.041). PINP levels were higher in the T4/T3 therapy (48 vs. 40 Fg/l (p<0.001)). NT-proBNP did not differ between the groups. Conclusions: T4/T3 combination therapy in hypothyroidism seems to have more metabolic effects than the T4 monotherapy.
Stimulatory effects of thyroid hormone on brain angiogenesis in vivo and in vitro

Liqun Zhang, CM Cooper-Kuhn, U Nannmark, K Blomgren and HG Kuhn
J Cereb Blood Flow & Metab 2010; 30:323–335. http://dx.doi.org:/10.1038/jcbfm.2009.216

Thyroid hormone is critical for the proper development of the central nervous system. However, the specific role of thyroid hormone on brain angiogenesis remains poorly understood. Treatment of rats from birth to postnatal day 21 (P21) with propylthiouracil (PTU), a reversible blocker of triiodothyronine (T3) synthesis, resulted in decreased brain angiogenesis, as indicated by reduced complexity and density of microvessels. However, when PTU was withdrawn at P22, these parameters were fully recovered by P90. These changes were paralleled by an  altered expression of vascular endothelial growth factor A (Vegfa) and basic fibroblast growth factor (Fgf2). Physiologic concentrations of T3 and thyroxine (T4) stimulated proliferation and tubulogenesis of rat brain derived endothelial (RBE4) cells in vitro. Protein and mRNA levels of VEGF-A and FGF-2 increased after T3 stimulation of RBE4 cells. The thyroid hormone receptor blocker NH-3 abolished T3-induced Fgf2 and Vegfα upregulation, indicating a receptor-mediated effect. Thyroid hormone inhibited the apoptosis in RBE4 cells and altered mRNA levels of apoptosis-related genes, namely Bcl2 and Bad. The present results show that thyroid hormone has a substantial impact on vasculature development in the brain. Pathologically altered vascularization could, therefore, be a contributing factor to the neurologic deficits induced by thyroid hormone deficiency.

Molecules important for thyroid hormone

synthesis and action – known facts and future perspectives

Klaudia Brix, Dagmar Führer, Heike Biebermann
Thyroid Research 2011, 4(Suppl 1):S9 http://www.thyroidresearchjournal.com/content/4/S1/S9

Thyroid hormones are of crucial importance for the functioning of nearly every organ. Remarkably, disturbances of thyroid hormone synthesis and function are among the most common endocrine disorders affecting approximately one third of the working German population. Over the last ten years our understanding of biosynthesis and functioning of these hormones has increased tremendously. This includes the identification of proteins involved in thyroid hormone biosynthesis like Thox2 and Dehal where mutations in these genes are responsible for certain degrees of hypothyroidism. One of the most important findings was the identification of a specific transporter for triiodothyronine (T3), the monocarboxylate transporter 8 (MCT8) responsible for directed transport of T3 into target cells and for export of thyroid hormones out of thyroid epithelial cells. Genetic disturbances of MCT8 in patients result in a biochemical constellation of high T3 levels in combination with low or normal TSH and thyroxine levels leading to a new syndrome of severe X-linked mental retardation. Importantly mice lacking MCT8 presented only with a mild phenotype, indicating that compensatory mechanisms exist in mice. Moreover, it has become clear that not only genomic actions of T3 exist. T3 is also capable to activate adhesion receptors and it signals via activation of PI3K and MAPK pathways. Most recently, thyroid hormone derivatives were identified, the thyronamines which are decarboxylated thyroid hormones initiating physiological actions like lowering body temperature and heart rate, thereby acting in opposite direction to the classical thyroid hormones. So far it is believed that thyronamines function via the activation of a G-protein coupled receptor, TAAR1. The objective of this review is to summarize the recent findings in thyroid hormone synthesis and action and to discuss their implications for diagnosis of thyroid disease and for treatment of patients.

Retinoic Acid Induces Expression of the Thyroid Hormone Transporter, Monocarboxylate Transporter 8 (Mct8)

T Kogai, Yan-Yun Liu, LL Richter, K Mody, H Kagechika, and GA Brent
J Biol Chem Jun 2010. Manuscript M110.123158
http://www.jbc.org/cgi/doi/10.1074/jbc.M110.123158

Retinoic acid (RA) and thyroid hormone are critical for differentiation and organogenesis in the embryo. The monocarboxylate transporter-8 (Mct8), expressed predominantly in brain and placenta, mediates thyroid hormone uptake from the circulation and is required for normal neural development. RA induces differentiation of F9 mouse teratocarcinoma cells towards neurons as well as extraembryonal endoderm. We hypothesized that Mct8 is functionally expressed in F9 cells and induced by RA.  All trans RA (tRA), and other RA receptor (RAR) agonists, dramatically (> 300-fold) induced Mct8. tRA treatment significantly increased uptake of triiodothyronine and thyroxine (4.1 fold and 4.3 fold, respectively), which was abolished by a selective Mct8 inhibitor, bromosulfophthalein. Sequence inspection of the Mct8 promoter region and
5′-rapid amplification of cDNA ends (5’-RACE) PCR analysis in F9 cells identified
11 transcription start sites and a proximal Sp1 site, but no TATA-box.  tRA significantly enhanced Mct8 promoter activity through a consensus RA responsive element located 6.6 kilobases upstream of the coding region. Chromatin immunoprecipitation assay demonstrated binding of RAR and retinoid-X receptor (RXR) to the RA response element. The promotion of thyroid hormone uptake through the transcriptional up-regulation of Mct8 by RAR is likely to be important for extraembryonic endoderm development and neural differentiation. This finding demonstrates crosstalk between RA signaling and thyroid hormone signaling in early development at the level of the thyroid hormone transporter.
Abnormal thyroid hormone metabolism in mice lacking the monocarboxylate transporter 8

Marija Trajkovic, Theo J. Visser, Jens Mittag, Sigrun Horn, et al.
J. Clin. Invest.  2007; 117:627–635. http://dx.doi.org:/10.1172/JCI28253

In humans, inactivating mutations in the gene of the thyroid hormone transporter monocarboxylate transporter 8 (MCT8; SLC16A2) lead to severe forms of psychomotor retardation combined with imbalanced thyroid hormone serum levels. The MCT8-null mice described here, however, developed without overt deficits but also exhibited distorted 3,5,3′-triiodothyronine (T3) and thyroxine (T4) serum levels, resulting in increased hepatic activity of type 1 deiodinase (D1). In the mutants’ brains, entry of T4 was not affected, but uptake of T3 was diminished. Moreover, the T4 and T3 content in the brain of MCT8-null mice was decreased, the activity of D2 was increased, and D3 activity was decreased, indicating the hypothyroid state of this tissue. In the CNS, analysis of T3 target genes revealed that in the mutants, the neuronal T3 uptake was impaired in an area-specific manner, with strongly elevated thyrotropin-releasing hormone transcript levels in the hypothalamic paraventricular nucleus and slightly decreased RC3 mRNA expression in striatal neurons; however, cerebellar Purkinje cells appeared unaffected, since they did not exhibit dendritic outgrowth defects and responded normally to T3 treatment in vitro.
In conclusion, the circulating thyroid hormone levels of MCT8-null mice closely resemble those of humans with MCT8 mutations, yet in the mice, CNS development is only partially affected.
3-Monoiodothyronamine: the rationale for its action as an endogenous adrenergic-blocking neuromodulator

HS Gompf, JH Greenberg, G Aston-Jones, A Ianculescu, TS Scanlan, and MB Dratman
Brain Res. 2010 Sep 10; 1351: 130–140. http://dx.doi.org:/10.1016/j.brainres.2010.06.067

The investigations reported here were designed to gain insights into the role of
3-monoiodothyronamine (T1AM) in the brain, where the amine was originally identified and characterized.
Extensive deiodinase studies indicated that T1AM was derived from the T4 metabolite, reverse triiodothyronine (revT3), while functional studies provided well-confirmed evidence that T1AM has strong adrenergic blocking effects. Because a state of adrenergic overactivity prevails when triiodothyronine (T3) concentrations becomes excessive, the possibility that T3’s metabolic partner, revT3, might give rise to an antagonist of those T3 actions was thought to be reasonable.
All T1AM studies thus far have required use of pharmacological doses.
Therefore we considered that choosing a physiological site of action was a priority and focused on the locus coeruleus (LC), the major noradrenergic control center in the brain. Site-directed injections of T1AM into the LC elicited a significant, dose-dependent neuronal firing rate change in a subset of adrenergic neurons with an EC50=2.7 μM, a dose well within the physiological range. Further evidence for its physiological actions came from autoradiographic images obtained following intravenous carrier-free 125I-labeled T1AM injection. These showed that the amine bound with high affinity to the LC and to other selected brain nuclei, each of which is both an LC target and a known T3 binding site. This new evidence points to a physiological role for T1AM as an endogenous adrenergic-blocking neuromodulator in the central noradrenergic system.

Thyroid hormones are transported through the blood-brain barrier

Thyroid hormones are transported through the blood-brain barrier

Thyroid hormones are transported through the blood-brain barrier (OATP) or the blood-CSF barrier (OATP and MCT8). In the astrocytes and tanycytes T4 is converted to T3 which then enters the neurons through MCT8. In the neurons both T4 and T3 are degraded by D3. T3 from the tanycytes may reach the portal vessels in the median eminence. Other transporters may be present on the astrocyte or tanycyte membranes. In most cases the transport could be bidirectional, although only one direction is shown.
Juan Bernal – Instituto de Investigaciones Biomedicas – 28029 Madrid, Spain

the interactions of maternal, placental and fetal thyroid

the interactions of maternal, placental and fetal thyroid

Old and new concepts of thyroid hormone action.

A: Old concept of thyroid hormone action. In former times it was assumed that thyroid hormones are able to pass the plasma membrane by passive transport. Once in the cytosol T4 is deiodinated to T3 which exerts genomic effects by binding to the thyroid hormone receptor (TR). After hetero-dimerization with other nuclear receptors like retinoic X receptor (RXR), transcriptional regulation is initiated resulting in activation or inactivation of target genes.
B: New concepts of thyroid hormone action. Thyroid hormones enter a target cell via specific transporters, e.g. T3 uses the monocarboxylate transporter MCT8 while T4 entry is mediated by Lat2 or Oatp14. Moreover, T3 can interact with avb3 integrins to induce ERK1/2 signalling. Cytosolic T3 exerts genomic effects but can additionally also act by non-genomic means after TR binding and activation of down-stream PI-3 kinase. Likewise, the naturally occurring iodothyronine T2 is believed to stimulate metabolic rates via mitochondrial pathways, thereby bypassing genomic regulation. Besides thyroid hormones, derivatives like the thyronamines T1AM or T0AM, modulate the action of T3, e.g. counter-acting its effects in certain target cells. Thyronamines (TAMs) bind to and activate G-protein coupled receptors (GPCRs) of the trace amine associated receptor (TAAR) family. So far, it is only known that TAAR1 is activated by TAMs and signals via adenylylcyclase (AC) activation with subsequent rise of cAMP levels. However other GPCRs are likely targets for thyroid hormone derivatives

Brix et al.: Molecules important for thyroid hormone synthesis and action – known facts and future perspectives. Thyroid Research 2011 4(Suppl 1):S9.
http://dx.doi.org:/10.1186/1756-6614-4-S1-S9

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More Complexity in Protein Evolution

Posted in Alzheimer's Disease, Amino acids, Autism Spectrum Disorders, Biological Networks, Biomarkers & Medical Diagnostics, Ca2+ triggered activation, Cerebrovascular and Neurodegenerative Diseases, Chemical Biology and its relations to Metabolic Disease, Clinical Diagnostics, Curation, Disease Biology, Endocrine Diseases, Enzymes and isoenzymes, Etiology, Evolutionary cognition, Experimental validation, Explanatory, Gene Regulation and Evolution, Metabolism, Metabolomics, Mutant Gene Expression, Neurodegenerative Diseases, Neurohumoral Transmission, Neurological Diseases, Pharmaceutical Drug Discovery, Pharmacotherapy and Cell Activity, Proteins, Pyridine nucleotides, Signaling & Cell Circuits, Social Development, Translational Science, tagged , , , , , , , , on January 18, 2015| Leave a Comment »

More Complexity in Protein Evolution

Author and Curator: Larry H. Bernstein, MD, FCAP 

Lactate dehydrogenase like crystallin: a potentially protective shield for Indian spiny-tailed lizard (Uromastix ltardwickit) lens against environmental stress?
A Atta, A Ilyas, Z Hashim, A Ahmed and S Zarina
The Protein Journal 2014; 33(2), p. 128-34.
http://dx.doi.org/10.1007/s10930-014-9543-4

Taxon specific lens crystallins in ve1iebrates are either similar or identical with various metabolic enzymes. These bifunctional crystallins serve as structural protein in lens along with their catalytic role. In the present study, we have partially purified and characterized lens crystallin from Indian spiny-tailed lizard (Uroma stix hardwickii). We have found lactate dehydrogenase (LDH) activity in lens indicating presence of an enzyme crystallin with dual functions. Taxon specific lens crystallins are product of gene sharing or gene duplication phenomenon where a pre-existing enzyme is recruited as lens crystallin in addition to structural role. In lens, same gene adopts refractive role in lens without modification or loss of pre-existing function during gene sharing phenomenon. Apart from conventional role of structural protein, LDH activity containing crystallin in Uromastix hardwickii lens is likely to have adaptive characteristics to offer protection against toxic effects of oxidative stress and ultraviolet light, hence justifying its recruitment. Taxon specific crystallins may serve as good models to understand structure-function relationship of these proteins.

αB-Crystallin and 27-kd Heat Shock Protein Are Regulated by Stress Conditions in the Central Nervous System and Accumulate in Rosenthal Fibers
T Iwaki, A Iwaki, J Tateishi, Y Sakaki, and JE Goldmant
Ameri J Pathol  1993; 143(2):487-495.

To understand the significance of the accumulation of αB-crystallin in Rosenthal fibers within astrocytes, the expression and metabolism of αB-crystallin in glioma cell lines were examined under the conditions of heat and oxidative stress. αB-crystallin mRNA was increased after both stresses, and αB-crystallin protein moved from a detergent-soluble to a detergent-insoluble form. In addition, Western blotting of Alexander’s  disease brain homogenates revealed that the 27-kd heat shock protein (HSP27), which is related to αB-crystallin, accumulates along with αB-crystallin. The presence of HSP27 in Rosenthal fibers was directly demonstrated by immunohistochemistry. Our results suggest that astrocytes in Alexander’s disease may be involved in an as yet unknown kind of stress reaction that causes the accumulation of αB-ccystallin and HSP27 and results in Rosenthal fiber formation.

α-Crystallin can function as a molecular chaperone
Joseph Horwitz
Proc. Nadl. Acad. Sci. USA Nov 1992; 89: 10449-10453. Biochemistry

The α-crystallins (αA and αB) are major lens structural proteins of the vertebrate eye that are related to the small heat shock protein family. In addition, crystallins (especially αB) are found in many cells organs outside the lens, and aα is overexpressed in several neurological disorders and in cell lines under stress conditions. Here I show that α-crystallin can function as a molecular chaperone. Stoichiometric amounts of αA and αB suppress thermally induced aggregation of various enzymes. In particular, α-crystalln is very efficient in suppressing the thermally induced aggregation of β- and y-crystallins, the two other major mammalian stuctural lens proteins. α-Crystallin was also effective in preventing aggregation and in refolding guanidine hydrochloride-denatured y-crystallin, as judged by circular dichroism spectroscopy. My results thus indicate that α-crystallin refracts light and protects proteins from aggregation in the transparent eye lens and that in nonlens cells α-crystallin may have other functions in addition to its capacity to suppress aggregation of proteins.

Gene sharing by δ-crystallin and argininosuccinate Iyase
J Piatigorsky, WE O’Brient, BL Norman, K Kalumuckt, GJ Wistow, T Borras, et al.
Proc. Natl. Acad. Sci. USA  May 1988; 85: 3479-3483. Evolution.

The lens structural protein δ-crystallin and the metabolic enzyme argininosuccinate lyase (ASL; Largininosuccinate argine-lyase, EC 4.3.2.1) have striking sequence similarity. We have demonstrated that duck δ-crystallin has enormously high ASL activity, while chicken δ-crystallin has lower but significant activity. The lenses of these birds had much greater ASL activity than other tissues, suggesting that ASL is being expressed at unusually high levels as a structural component. In Southern blots of human genomic DNA, chicken δ1-crystallin cDNA hybridized only to the human ASL gene; moreover, the two chicken δ-crystallin genes accounted for all the sequences in the chicken genome able to cross-hybridize with a human ASL cDNA, with preferential hybridization to the δ2 gene. Correlations of enzymatic activity and recent data on mRNA levels in the chicken lens suggest that ASL activity depends on expression of the δ2-crystallin gene. The data indicate that the same gene, at least in ducks, encodes two different functions, an enzyme (ASL) and a structural protein (δ-crystallin), although in chickens specialization and separation of functions may have occurred.

Gecko i-crystallin: How cellular retinol-binding protein became an eye lens ultraviolet filter
PJ L Werten, Beate Roll, DMF van Aalten, and WW de Jong
PNAS Mar 2000; 97(7): 3282–3287 http://pnas.org/cgi/doi/10.1073ypnas.050500597

Eye lenses of various diurnal geckos contain up to 12% i-crystallin. This protein is related to cellular retinol-binding protein type I (CRBP I) but has 3,4-didehydroretinol, rather than retinol, as a ligand. The 3,4-didehydroretinol gives the lens a yellow color, thus protecting the retina by absorbing short-wave radiation. i-Crystallin could be either the gecko’s housekeeping CRBP I, recruited for an additional function in the lens, or the specialized product of a duplicated CRBP I gene. The finding of the same CRBP I-like sequence in lens and liver cDNA of the gecko Lygodactylus picturatus now supports the former option. Comparison with i-crystallin of a distantly related gecko, Gonatodes vittatus, and with mammalian CRBP I, suggests that acquiring the additional lens function is associated with increased amino acid changes. Compared with the rat CRBP I structure, the i-crystallin model shows reduced negative surface charge, which might facilitate the required tight protein packing in the lens. Other changes may provide increased stability, advantageous for a long-living lens protein, without frustrating its role as retinol transporter outside the lens. Despite a number of replacements in the ligand pocket, recombinant i-crystallin binds 3,4-didehydroretinol and retinol with similar and high affinity (1.6 nM). Availability of ligand thus determines whether it binds 3,4-didehydroretinol, as in the lens, or retinol, in other tissues. i-Crystallin presents a striking example of exploiting the potential of an existing gene without prior duplication.

Expression of βA3/A1-crystallin in the developing and adult rat eye
G Parthasarathy, Bo Ma, C Zhang, C Gongora, JS Zigler, MK Duncan, D Sinha
J Molec Histol 2011; 42(1): 59-69. http://dx.doi.org:/10.1007/s10735-010-9307-1

Crystallins are very abundant structural proteins of the lens and are also expressed in other tissues. We have previously reported a spontaneous mutation in the rat βA3/A1-crystallin gene, termed Nuc1, which has a novel, complex, ocular phenotype. The current study was undertaken to compare the expression pattern of this gene during eye development in wild type and Nuc1 rats by in situ hybridization (ISH) and immunohistochemistry (IHC).
βA3/A1-crystallin expression was first detected in the eyes of both wild type and Nuc1 rats at embryonic (E) day 12.5 in the posterior portion of the lens vesicle, and remained limited to the lens fibers throughout fetal life.
After birth, βA3/A1-crystallin expression was also detected in the neural retina (specifically in the astrocytes and ganglion cells) and in the retinal pigmented epithelium (RPE).
This suggested that βA3/A1-crystallin is not only a structural protein of the lens, but has cellular function(s) in other ocular tissues.
In summary, expression of βA3/A1-crystallin is controlled differentially in various eye tissues with lens being the site of greatest expression.
Similar staining patterns, detected by ISH and IHC, in wild type and Nuc1 animals suggest that functional differences in the protein, rather than changes in mRNA/protein level of expression likely account for developmental abnormalities in Nuc1.

βA3/A1Crystallin controls anoikis-mediated cell death in astrocytes by modulating PI3K/AKT/mTOR and ERK survival pathways through the PKD/Bit1-signaling axis
B Ma, T Sen, L Asnaghi, M Valapala, F Yang, S Hose, D S McLeod, Y Lu, et la.
Cell Death and Disease 2011; 2(10). http://dx.doi.org:/10.1038/cddis.2011.100

During eye development, apoptosis is vital to the maturation of highly specialized structures such as the lens and retina. Several forms of apoptosis have been described, including anoikis, a form of apoptosis triggered by inadequate or inappropriate cell–matrix contacts. The anoikis regulators, Bit1 (Bcl-2 inhibitor of transcription-1) and protein kinase-D (PKD), are expressed in developing lens when the organelles are present in lens fibers, but are downregulated as active denucleation is initiated.
We have previously shown that in rats with a spontaneous mutation in the Cryba1 gene, coding for βA3/A1-crystallin, normal denucleation of lens fibers is inhibited. In rats with this mutation (Nuc1), both Bit1 and PKD remain abnormally high in lens fiber cells. To determine whether βA3/A1-crystallin has a role in anoikis, we induced anoikis in vitro and conducted mechanistic studies on astrocytes, cells known to express βA3/A1-crystallin.
The expression pattern of Bit1 in retina correlates temporally with the development of astrocytes. Our data also indicate that loss of βA3/A1-crystallin in astrocytes results in a failure of Bit1 to be trafficked to the Golgi, thereby suppressing anoikis. This loss of βA3/A1-crystallin also induces insulin-like growth factor-II, which increases cell survival and growth by modulating the phosphatidylinositol-3-kinase (PI3K)/AKT/mTOR and extracellular signal-regulated kinase pathways. We propose that βA3/A1-crystallin is a novel regulator of both life and death decisions in ocular astrocytes.

βA3/A1-crystallin in astroglial cells regulates retinal vascular remodeling during development
D Sinha, A Klise, Y Sergeev, S Hose, IA Bhutto, L Hackler Jr., T Malpic-llanos, et al.
Molec Cell Neurosci 2008; 37(1): 85-95.

http://dx.doi.org:/10.1016/j.mcn.2007.08.016

Vascular remodeling is a complex process critical to development of the mature vascular system. Astrocytes are known to be indispensable for initial formation of the retinal vasculature; our studies with the Nuc1 rat provide novel evidence that these cells are also essential in the retinal vascular remodeling process.
Nuc1 is a spontaneous mutation in the Sprague–Dawley rat originally characterized by nuclear cataracts in the heterozygote and microphthalmia in the homozygote. We report here that the Nuc1 allele results from mutation of the βA3/A1-crystallin gene, which in the neural retina is expressed only in astrocytes. We demonstrate striking structural abnormalities in Nuc1 astrocytes with profound effects on the organization of intermediate filaments. While vessels form in the Nuc1 retina, the subsequent remodeling process required to provide a mature vascular network is deficient. Our data implicate βA3/A1-crystallin as an important regulatory factor mediating vascular patterning and remodeling in the retina.

A developmental defect in astrocytes inhibits programmed regression of the hyaloid vasculature in the mammalian eye
C Zhang, L Asnaghi, C Gongora, B Patek, S Hose, Bo Ma, MA Fard, L Brako, et al.
Eur J Cell Biol 2011; 90(5): 440-448.
http://dx.doi.org:/10.1016/j.ejcb.2011.01.003

Previously we reported the novel observation that astrocytes ensheath the persistent hyaloid artery, both in the Nuc1 spontaneous mutant rat, and in human PFV (persistent fetal vasculature) disease (Developmental Dynamics 234:36–47, 2005). We now show that astrocytes isolated from both the optic nerve and retina of Nuc1 rats migrate faster than wild type astrocytes. Aquaporin 4 (AQP4), the major water channel in astrocytes, has been shown to be important in astrocyte migration. We demonstrate that AQP4 expression is elevated in the astrocytes in PFV conditions, and we hypothesize that this causes the cells to migrate abnormally into the vitreous where they ensheath the hyaloid artery. This abnormal association of astrocytes with the hyaloid artery may impede the normal macrophage-mediated remodeling and regression of the hyaloid system.

βA3/A1-crystallin is required for proper astrocyte template formation and vascular remodeling in the retina.
D Sinha; WJ Stark; M Valapala; IA Bhutto; M Cano; S Hose; GA Lutty; et al.  Transgenic research 2012; 21(5):1033-42.

Nuc1 is a spontaneous rat mutant resulting from a mutation in the Cryba1 gene, coding for βA3/A1-crystallin. Our earlier studies with Nuc1 provided novel evidence that astrocytes, which express βA3/A1-crystallin, have a pivotal role in retinal remodeling. The role of astrocytes in the retina is only beginning to be explored. One of the limitations in the field is the lack of appropriate animal models to better investigate the function of astrocytes in retinal health and disease. We have now established transgenic mice that overexpress the Nuc1 mutant form of Cryba1, specifically in astrocytes. Astrocytes in wild type mice show normal compact stellate structure, producing a honeycomb-like network. In contrast, in transgenics over-expressing the mutant (Nuc1) Cryba1 in astrocytes, bundle-like structures with abnormal patterns and morphology were observed. In the nerve fiber layer of the transgenic mice, an additional layer of astrocytes adjacent to the vitreous is evident. This abnormal organization of astrocytes affects both the superficial and deep retinal vascular density and remodeling. Fluorescein angiography showed increased venous dilation and tortuosity of branches in the transgenic retina, as compared to wild type. Moreover, there appear to be fewer interactions between astrocytes and endothelial cells in the transgenic retina than in normal mouse retina. Further, astrocytes overexpressing the mutant βA3/A1-crystallin migrate into the vitreous, and ensheath the hyaloid artery, in a manner similar to that seen in the Nuc1 rat. Together, these data demonstrate that developmental abnormalities of astrocytes can affect the normal remodeling process of both fetal and retinal vessels of the eye and that βA3/A1-crystallin is essential for normal astrocyte function in the retina.

Ontogeny of oxytocin and vasopressin receptor binding in the lateral septum in prairie and montane voles
Z. Wang, L.J. Young
Developmental Brain Research 1997; 104:191–195.

Adult prairie (Microtus ochrogaster). and montane voles (M. montanus). differ in the distribution of oxytocin OT. and vasopressin AVP receptor binding in the brain. The present study examined the ontogenetic pattern of these receptor bindings in the lateral septum in both species to determine whether adult differences in the receptor binding are derived from a common pattern in development. In both species, OT and AVP receptor binding in the lateral septum were detected neonatally, increased during development, and reached the adult level at weaning third week. The progression of OT and AVP receptor differed, as OT receptor binding increased continually until weaning while AVP receptor binding did not change in the first week, increased rapidly in the second week, and was sustained thereafter. For both receptors, the binding increased more rapidly in montane than in prairie voles, resulting in species differences in receptor binding at weaning and in adulthood. Together, these data indicate that OT and AVP could affect the brain during development in a peptide- and species-specific manner in voles.

Evolution of the vasopressin/oxytocin superfamily: Characterization of a cDNA encoding a vasopressin-related precursor, preproconopressin, from the mollusc Lymnaea stagnalis
RE Van Kesteren, AB Smit, RW Dirksi, ND De With, WPM Geraerts, and J Joosse
Proc. Nadl. Acad. Sci. USA May 1992; 89: 4593-4597. Neurobiology

Although the nonapeptide hormones vasopressin, oxytocin, and related peptides from vertebrates and some nonapeptides from invertebrates share similarities in amino acid sequence, their evolutionary relationships are not dear. To investigate this issue, we doned a cDNA encoding a vasopressin-related peptide, Lys-conopressin, produced in the central nervous system of the gastropod mollusc Lymnaea stagnalis. The predicted preproconopressin has the overall architecture of vertebrate preprovasopressins, with a signal peptide, Lys-conopressin, that is flanked at the C terminus by an amidation signal and a pair of basic residues, followed by a neurophysin domain. The Lymnaea neurophysin and the vertebrate neurophysins share high sequence identity, which includes the conservation of all 14 cysteine residues. In addition, the Lymnaea neurophysin possesses unique structural characteristics. It contains a putative N-linked glycosylation site at a position in the vertebrate neurophysins where a strictly conserved tyrosine residue, which plays an essential role in binding of the nonapptide hormones, is found. The C-terminal copeptin homologous extension of the Lymnaea neurophysin has low sequence identity with the vertebrate counterparts and is probably not cleaved from the prohormone, as are the mammalin copeptins. The conopressin gene is expressed in only a few neurons in both pedal ganglia of the central nervous system. The conopressin transcript is present in two sizes, due to alternative use of polyadenylylation signals. The data presented here demonstrate that the typical organization of the prohormones of the vasopressin/oxytocin superfamily must have been present in the common ancestors of vertebrates and invertebrates.

A common allele in the oxytocin receptor gene (OXTR) impacts prosocial temperament and human hypothalamic-limbic structure and function
H Tosta, B Kolachanaa, S Hakimia, H Lemaitrea, BA Verchinskia, et al.
PNAS Aug 3, 2010; 107(31): 13936–13941
http://pnas.org/cgi/doi/10.1073/pnas.1003296107

The evolutionarily highly conserved neuropeptide oxytocin is a key mediator of social and emotional behavior in mammals, including humans. A common variant (rs53576) in the oxytocin receptor gene (OXTR) has been implicated in social-behavioral phenotypes, such as maternal sensitivity and empathy, and with neuropsychiatric disorders associated with social impairment, but the intermediate neural mechanisms are unknown. Here, we used multimodal neuroimaging in a large sample of healthy human subjects to identify structural and functional alterations in OXTR risk allele carriers and their link to temperament. Activation and interregional coupling of the amygdala during the processing of emotionally salient social cues was significantly affected by genotype. In addition, evidence for structural alterations in key oxytocinergic regions emerged, particularly in the hypothalamus. These neural characteristics predicted lower levels of reward dependence, specifically in male risk allele carriers. Our findings identify sex-dependent mechanisms impacting the structure and function of hypothalamic-limbic circuits that are of potential clinical and translational significance.
Test of Association Between 10 SNPs in the Oxytocin Receptor Gene and Conduct Disorder
JT Sakai, TJ Crowley, MC Stallings, M McQueen, JK Hewitt, C Hopfer, et al.
Psychiatr Genet. 2012 Apr; 22(2): 99–102. http://dx.doi.org:/10.1097/YPG.0b013e32834c0cb2

Animal and human studies have implicated oxytocin (OXT) in affiliative and prosocial behaviors. We tested whether genetic variation in the OXT receptor (OXTR) gene is associated with conduct disorder (CD).
Utilizing a family-based sample of adolescent probands recruited from an adolescent substance abuse treatment program, control probands and their families (total sample n=1,750), we conducted three tests of association with CD and 10 SNPs (single nucleotide polymorphisms) in the OXTR gene: (1) family-based comparison utilizing the entire sample; (2) within-Whites, case control comparison of adolescent patients with CD and controls without CD; and (3) within-Whites case-control comparison of parents of patients and parents of controls.
Family-based association tests failed to show significant results (no results p<0.05). While strictly correcting for the number of tests (α=0.002), adolescent patients with CD did not differ significantly from adolescent controls in genotype frequency for the OXTR SNPs tested; similarly, comparison of OXTR genotype frequencies for parents failed to differentiate patient and control family type, except a trend association for rs237889 (p=0.004). In this sample, 10 SNPs in the OXTR gene were not significantly associated with CD.

Leu55Pro transthyretin accelerates subunit exchange and leads to rapid formation of hybrid tetramers
CA Keetch, EHC Bromley, MG McCammon, N Wang, J Christodoulou, CV Robinson
JBC  Oct 11, 2005 M508753200. http://jbc.org/cgi/doi/10.1074/jbc.M508753200

Transthyretin is a tetrameric protein associated with the commonest form of

systemic amyloid disease. Using isotopically labeled proteins and mass spectrometry we compared subunit exchange in wild-type transthyretin with that of the variant associated with the most aggressive form of the disease, Leu55Pro. Wild-type subunit exchange occurs via both monomers and dimers , while exchange via dimers is the dominant mechanism for the Leu55Pro variant. Since patients with the Leu55Pro mutation are heterozygous, expressing both proteins simultaneously, we also analyzed the subunit exchange reaction between wild-type and Leu55Pro tetramers . We found that hybrid tetramers containing two or three Leu55Pro subunits dominate in the early stages of the reaction. Surprisingly we also found that in the presence of Leu55Pro transthyretin, the rate of dissociation of wild-type transthyretin is increased. This implies interactions between the two proteins that accelerate the formation of hybrid tetramers, a result with important implications for transthyretin amyloidos is.

Beyond Genetic Factors in Familial Amyloidotic Polyneuropathy: Protein Glycation and the Loss of Fibrinogen’s Chaperone Activity
G da Costa, RA Gomes, A Guerreiro, E Mateus, E Monteiro, et al.
PLoS ONE 2011; 6(10): e24850. http://dx.doi.org:/10.1371/journal.pone.0024850

Familial amyloidotic polyneuropathy (FAP) is a systemic conformational disease characterized by extracellular amyloid fibril formation from plasma transthyretin (TTR). This is a crippling, fatal disease for which liver transplantation is the only effective therapy. More than 80 TTR point mutations are associated with amyloidotic diseases and the most widely accepted disease model relates TTR tetramer instability with TTR point mutations. However, this model fails to explain two observations. First, native TTR also forms amyloid in systemic senile amyloidosis, a geriatric disease. Second, age at disease onset varies by decades for patients bearing the same mutation and some mutation carrier individuals are asymptomatic throughout their lives. Hence, mutations only accelerate the process and non-genetic factors must play a key role in the molecular mechanisms of disease. One of these factors is protein glycation, previously associated with conformational diseases like Alzheimer’s and Parkinson’s. The glycation hypothesis in FAP is supported by our previous discovery of methylglyoxal-derived glycation of amyloid fibrils in FAP patients. Here we show that plasma proteins are differentially glycated by methylglyoxal in FAP patients and that fibrinogen is the main glycation target. Moreover, we also found that fibrinogen interacts with TTR in plasma. Fibrinogen has chaperone activity which is compromised upon glycation by methylglyoxal. Hence, we propose that methylglyoxal glycation hampers the chaperone activity of fibrinogen, rendering TTR more prone to aggregation, amyloid formation and ultimately, disease.

Aromatic Sulfonyl Fluorides Covalently Kinetically Stabilize Transthyretin to Prevent Amyloidogenesis while Affording a Fluorescent Conjugate
NP Grimster, S Connelly, A Baranczak, J Dong, …, JW Kelly
J Am Chem Soc. 2013 Apr 17; 135(15): 5656–5668. http://dx.doi.org:/10.1021/ja311729d

Molecules that bind selectively to a given protein and then undergo a rapid chemoselective reaction to form a covalent conjugate have utility in drug development. Herein a library of 1,3,4-oxadiazoles substituted at the 2 position with an aryl sulfonyl fluoride and at the 5 position with a substituted aryl known to have high affinity for the inner thyroxine binding subsite of transthyretin (TTR) were conceived of by structure-based design principles and were chemically synthesized. When bound in the thyroxine binding site, most of the aryl sulfonyl fluorides react rapidly and chemoselectively with the pKa-perturbed K15 residue, kinetically stabilizing TTR and thus preventing amyloid fibril formation, known to cause polyneuropathy. Conjugation t50s range from 1 to 4 min, ~ 1400 times faster than the hydrolysis reaction outside the thyroxine binding site. Xray crystallography confirms the anticipated binding orientation and sheds light on the sulfonyl fluoride activation leading to the sulfonamide linkage to TTR. A few of the aryl sulfonyl fluorides efficiently form conjugates with TTR in plasma. A few of the TTR covalent kinetic stabilizers synthesized exhibit fluorescence upon conjugation and therefore could have imaging applications as a consequence of the environment sensitive fluorescence of the chromophore.

Identification of S-sulfonation and S-thiolation of a novel transthyretin Phe33Cys variant from a patient diagnosed with familial transthyretin amyloidosis
A Lim, T Prokaeva, ME Mccomb, LH Connors, M Skinner, and CE Costello
Protein Science 2003; 12:1775–1786.
http://proteinscience.org/cgi/doi/10.1110/ps.0349703.

Familial transthyretin amyloidosis (ATTR) is an autosomal dominant disorder associated with a variant form of the plasma carrier protein transthyretin (TTR). Amyloid fibrils consisting of variant TTR, wild-type TTR, and TTR fragments deposit in tissues and organs. The diagnosis of ATTR relies on the identification of pathologic TTR variants in plasma of symptomatic individuals who have biopsy proven amyloid disease. Previously, we have developed a mass spectrometry-based approach, in combination with direct DNA sequence analysis, to fully identify TTR variants. Our methodology uses immunoprecipitation to isolate TTR from serum, and electrospray ionization and matrix-assisted laser desorption/ionization mass spectrometry (MS) peptide mapping to identify TTR variants and posttranslational modifications. Unambiguous identification of the amino acid substitution is performed using tandem MS (MS/MS) analysis and confirmed by direct DNA sequence analysis. The MS and MS/MS analyses also yield information about posttranslational modifications. Using this approach, we have recently identified a novel pathologic TTR variant. This variant has an amino acid substitution (Phe — Cys) at position 33. In addition, like the Cys10 present in the wild type and in this variant, the Cys33 residue was both S-sulfonated and S-thiolated (conjugated to cysteine, cysteinylglycine, and glutathione). These adducts may play a role in the TTR fibrillogenesis.

Evolutionary relationships of lactate dehydrogenases (LDHs) from mammals, birds, an amphibian, fish, barley, and bacteria: LDH cDNA sequences from Xenopus, pig, and rat
S Tsuji, MA Qureshi, EW Hou, WM Fitch, and S S.-L. Li
Proc. Natl. Acad. Sci. USA Sep 1994; 91: 9392-9396. Evolution

The nucleotide sequences of the cDNAs encoding LDH (EC 1.1.1.27) subunits LDH-A (muscle), LDH-B (liver), and LDH-C (oocyte) from Xenopus laevis, LDH-A (muscle) and LDH-B (heart) from pig, and LDH-B (heart) and LDH-C (testis) from rat were determined. These seven newly deduced amino acid sequences and 22 other published LDH sequences, and three unpublished fish LDH-A sequences kindly provided by G. N. Somero and D. A. Powers, were used to construct the most parsimonious phylogenetic tree of these 32 LDH subunits from mammals, birds, an amphibian, fish, barley, and bacteria. There have been at least six LDH gene duplications among the vertebrates. The Xenopus LDH-A, LDH-B, and LDH-C subunits are most closely related to each other and then are more closely related to vertebrate LDH-B than LDH-A. Three fish LDH-As, as well as a single LDH of lamprey, also seem to be more related to vertebrate LDH-B than to land vertebrate LDH-A. The mammalian LDH-C (testis) subunit appears to have diverged very early, prior to the divergence of vertebrate LDH-A and LDH-B subunits, as reported previously.

Evidence for neutral and selective processes in the recruitment of enzyme-crystallins in avian lenses
Graeme Wistow, Andrea Anderson, and Joram Piatigorsky
Proc. Natl. Acad. Sci. USA Aug 1990; 87: 6277-6280, Evolution

In apparent contrast to most other tissues, the ocular lenses in vertebrates show striking differences in protein composition between taxa, most notably in the recruitment of different enzymes as major structural proteins. This variability appears to be the result of at least partially neutral evolutionary processes, although there is also evidence for selective modification in molecular structure. Here we describe a bird, the chimney swift (Chaetura pelagica), that lacks δ-crystallin/ argininosuccinate lyase, usually the major crystallin of avian lenses. Clearly, δ-crystallin is not specifically required for a functionally effective avian lens. Furthermore the lens composition of the swift is more similar to that of the related hummingbirds than to that of the barn swallow (Hirundo rustica), suggesting that phylogeny is more important than environmental selection in the recruitment of crystallins. However differences in ε-crystallin/lactate dehydrogenase-B sequence between swift and hummingbird and other avian and reptilian species suggest that selective pressures may also be working at the molecular level. These differences also confirm the close relationship between swifts and hummingbirds.

Enzyme/crystallins and extremely high pyridine nucleotide levels in the eye lens.
Zigler, J. S., Jr.; Rao, P. V.
FASEB J. 1991; 3: 223-225.

Taxon-specific crystallins are proteins present in high abundance in the lens of phylogenetically restricted groups of animals. Recently it has been found that these proteins are actually enzymes which the lens has apparently adopted to serve as structural proteins. Most of these proteins have been shown to be identical to, or related to, oxidoreductases. In guinea pig lens, which contains zeta-crystallin, a protein with an NADPH dependent oxidoreductase activity, the levels of both NADPH and NADP* are extremely high and correlate with the concentration of zeta-crystallin. We report here nucleotide assays on lenses from vertebrates containing other enzyme/crystallins. In each case where the enzyme/crystallin is a pyridine nucleotide-binding protein the level of that particular nucleotide is extremely high in the lens. The presence of an enzyme/crystallin does not affect the lenticular concentrations of those nucleotides which are not specifically bound. The possibility that nucleotide binding may be a factor in the selection of some enzymes to serve as enzyme/crystallins is considered.

Comparison of stability properties of lactate dehydrogenase B4/ε-crystallin from different species
CEM Voorter, LTM Wintjes, PWH Heinstra, H Bloemendal and WW De Jong
Eur. J. Biochem. 1993; 211: 643-648

ε-Crystallin occurs as an abundant lens protein in many birds and in crocodiles and has been identified as heart-type lactate dehydrogenase (LDH-B4). Lens proteins have, due to their longevity and environmental conditions, extraordinary requirements for structural stability. To study lens protein stability, we compared various parameters of LDH-B4/ε-crystallin from lens and/or heart of duck, which has abundant amounts of this enzyme in its lenses, and of chicken and pig, which have no λ-crystallin. Measuring the thermostability of LDH-B4 from the different sources, the t50 values (temperature at which 50% of the enzyme activity remains after a 20-min period) for LDH-B4 from duck heart, duck lens and chicken heart were all found to be around 76°C whereas pig heart LDHB4 was less thermostable, having a t50 value of 625°C. A similar tendency was found with urea inactivation studies. Plotting the first-order rate constants obtained from inactivation kinetic plots against urea concentration, it was clear that LDH-B4 from pig heart was less stable in urea than the homologous enzymes from duck heart, chicken heart and duck lens. The duck and chicken enzymes were also much more resistant against proteolysis than the porcine enzyme. Therefore, it is concluded that avian LDH-B4 is structurally more stable than the homologous enzyme in mammals. This greater stability might make it suitable to function as a ε-crystallin, as in duck, but is not necessarily associated with high lens expression, as in chicken.

Duck lens ε-crystallin and lactate dehydrogenase B4 are identical: A single-copy gene product with two distinct functions
W Hendriks, JWM Mulders, MA Bibby, C Slingsby, H Bloemendal, and WW De Jong
Proc. Natl. Acad. Sci. USA Oct 1988; 85: 7114-7118. Biochemistry

To investigate whether or not duck lens ε-crystaliin and duck heart lactate dehydrogenase (LDH) B4 are the product of the same gene, we have isolated and sequenced cDNA clones of duck ε-crystallin. By using these clones we demonstrate that there is a single-copy Ldh-B gene in duck and in chicken. In the duck lens this gene is overexpressed, and its product is subject to posttranslational modification. Reconstruction of the evolutionary history of the LDH protein family reveals that the mammalian Ldh-C gene most probably originated from an ancestral Ldh-A gene and that the amino acid replacement rate in LDH-C is approximately 4 times the rate in LDH-A. Molecular modeling of LDH-B sequences shows that the increased thermostability of the avian tetramer might be explained by mutations that increase the number of ion pairs. Furthermore, the replacement of bulky side chains by glycines on the corners of the duck protein suggests an adaptation to facilitate close packing in the lens.

Lactate Dehydrogenase A as a Highly Abundant Eye Lens Protein in Platypus (Ornithorhynchus anatinus): Upsilon (υ)-Crystallin
T van Rheede,  R Amons, N Stewart, and WW de Jong
Mol. Biol. Evol. 2003; 20(06):994–998. http://dx.doi.org:/10.1093/molbev/msg116

Vertebrate eye lenses mostly contain two abundant types of proteins, the α-crystallins and the β/λ-crystallins. In addition, certain housekeeping enzymes are highly expressed as crystallins in various taxa. We now observed an unusual approximately 41-kd protein that makes up 16% to 18% of the total protein in the platypus eye lens. Its cDNA sequence was determined, which identified the protein as muscle-type lactate dehydrogenase A (LDH-A). It is the first observation of LDH-A as a crystallin, and we designate it upsilon (υ)-crystallin. Interestingly, the related heart-type LDH-B occurs as an abundant lens protein, known as ε-crystallin, in many birds and crocodiles. Thus, two members of the ldh gene family have independently been recruited as crystallins in different higher vertebrate lineages, suggesting that they are particularly suited for this purpose in terms of gene regulatory or protein structural properties. To establish whether platypus LDH-A/υ-crystallin has been under different selective constraints as compared with other vertebrate LDH-A sequences, we reconstructed the vertebrate Ldh-A gene phylogeny. No conspicuous rate deviations or amino acid replacements were observed.

Isozymes, moonlighting proteins and promiscous enzymes
M Nath Gupta, M Kapoor, AB Majumder and V Singh
Current Science Apr 2011; 100(8): 1152-1162.

The structures of isoenzymes differ and yet these catalyse the same type of reaction. These structures evolved to suit the physiological needs and are located in different parts of cells or tissues. Moonlighting proteins represent the same structure performing very different biological functions. Biological promiscuity reveals that the same active sites can catalyse different types of reactions. These three different phenomena, all illustrate similar evolutionary strategies. Viewed together, it emerges that biologists need to take a hard look at the ‘structure–function’ paradigm as well as the notions of biological specificity. Meanwhile, biotechnologists  continue to exploit the opportunities which ‘nonspecificity’ offers.

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