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Decline in Sperm Count – Epigenetics, Well-being and the Significance for Population Evolution and Demography
Dr. Marc Feldman, Expert Opinion on the significance of Sperm Count Decline on the Future of Population Evolution and Demography
Dr. Sudipta Saha, Effects of Sperm Quality and Quantity on Human Reproduction
Dr. Aviva Lev-Ari, Psycho-Social Effects of Poverty, Unemployment and Epigenetics on Male Well-being, Physiological Conditions affecting Sperm Quality and Quantity
Updated on 10/6/2022
There Are Two Americas Now: One With a B.A. and One Without’
Carol Graham, a senior fellow at Brookings, described the erosion of economic and social status for whites without college degrees in a 2021 paper:
From 2005 to 2019, an average of 70,000 Americans died annually from deaths of despair (suicide, drug overdose, and alcohol poisoning). These deaths are concentrated among less than college educated middle-aged whites, with those out of the labor force disproportionately represented. Low-income minorities are significantly more optimistic than whites and much less likely to die of these deaths. This despair reflects the decline of the white working class. Counties with more respondents reporting lost hope in the years before 2016 were more likely to vote for Trump.
A 2010 Pew Research Center study that examined the effects of the Great Recession on Black and white Americans reported that Black Americans consistently suffered more in terms of unemployment, work cutbacks and other measures, but remained far more optimistic about the future than whites. Twice as many Black as white Americans were forced during the 2008 recession to work fewer hours, to take unpaid leave or switch to part-time, and Black unemployment rose from 8.9 to 15.5 percent from April 2007 to April 2009, compared with an increase from 3.7 to 8 percent for whites.
Despite experiencing more hardship, 81 percent of Black Americans agreed with the statement “America will always continue to be prosperous and make economic progress,” compared with 59 percent of whites; 45 percent of Black Americans said the country was still in recession compared with 57 percent of whites
In “Trends in Extreme Distress in the United States, 1993-2019,” David G. Blanchflower and Andrew J. Oswald, economists at Dartmouth and the University of Warwick in Britain, note that “the proportion of the U.S. population in extreme distress rose from 3.6 percent in 1993 to 6.4 percent in 2019. Among low-education midlife white persons, the percentage more than doubled, from 4.8 percent to 11.5 percent.”
In her 2020 paper, “Trends in U.S. Working-Age Non-Hispanic White Mortality: Rural-Urban and Within-Rural Differences,” Shannon M. Monnat, a professor of sociology at Syracuse University’s Maxwell School, explained that “between 1990-92 and 2016-18, the mortality rates among non-Hispanic whites increased by 9.6 deaths per 100,000 population among metro males and 30.5 among metro females but increased by 70.1 and 65.0 among nonmetro (rural and exurban) males and females, respectively.”
Three economists, David Autor, David Dorn and Gordon Hanson of M.I.T., the University of Zurich and Harvard, reported in their 2018 paper, “When Work Disappears: Manufacturing Decline and the Falling Marriage Market Value of Young Men,” on the debilitating consequences for working-class men of the “China shock”
There is some evidence that partisanship correlates with mortality rates.
Anne Case wrote in her email, that the United States is fast approaching a point where
Education divides everything, including connection to the labor market, marriage, connection to institutions (like organized religion), physical and mental health, and mortality. It does so for whites, Blacks and Hispanics. There has been a profound (not yet complete) convergence in life expectancy by education. There are two Americas now: one with a B.A. and one without.
Aside from the decline in sperm counts, growing numbers of sperm appear defective — there’s a boom in two-headed sperm — while others loll aimlessly in circles, rather than furiously swimming in pursuit of an egg. And infants who have had greater exposures to a kind of endocrine disruptor called phthalates have smaller penises, Swan found.
Recent studies concluded via rigorous and comprehensive analysis found that Sperm Count (SC) declined 52.4% between 1973 and 2011 among unselected men from western countries, with no evidence of a ‘leveling off’ in recent years. Declining mean SC implies that an increasing proportion of men have sperm counts below any given threshold for sub-fertility or infertility. The high proportion of men from western countries with concentration below 40 million/ml is particularly concerning given the evidence that SC below this threshold is associated with a decreased monthly probability of conception.
1.Temporal trends in sperm count: a systematic review and meta-regression analysis
Hagai Levine, Niels Jørgensen, Anderson Martino‐Andrade, Jaime Mendiola, Dan Weksler-Derri, Irina Mindlis, Rachel Pinotti, Shanna H Swan. Human Reproduction Update, July 25, 2017, doi:10.1093/humupd/dmx022.
4. Long, mysterious strips of RNA contribute to low sperm count – Long non-coding RNAs can be added to the group of possible non-structural effects, possibly epigenetic, that might regulate sperm counts.
Thus, we postulated that some lncRNAs may also impact mammalian spermatogenesis and fertility. In this study, we identified a dynamic expression pattern of lncRNAs during murine spermatogenesis. Importantly, we identified a subset of lncRNAs and very few mRNAs that appear to escape meiotic sex chromosome inactivation (MSCI), an epigenetic process that leads to the silencing of the X- and Y-chromosomes at the pachytene stage of meiosis. Further, some of these lncRNAs and mRNAs show strong testis expression pattern suggesting that they may play key roles in spermatogenesis. Lastly, we generated a mouse knock out of one X-linked lncRNA, Tslrn1 (testis-specific long non-coding RNA 1), and found that males carrying a Tslrn1 deletion displayed normal fertility but a significant reduction in spermatozoa. Our findings demonstrate that dysregulation of specific mammalian lncRNAs is a novel mechanism of low sperm count or infertility, thus potentially providing new biomarkers and therapeutic strategies.
This article presents two perspectives on the potential effects of Sperm Count decline.
One Perspective identifies Epigenetics and male well-being conditions
as a potential explanation to the Sperm Count decline, and
as evidence for decline in White male longevity in certain geographies in the US since the mid 80s.
The other Perspective, evaluates if Sperm Count Decline would have or would not have a significant long term effects on Population Evolution and Demography.
The Voice of Prof. Marc Feldman, Stanford University – Long term significance of Sperm Count Decline on Population Evolution and Demography
Poor sperm count appears to be associated with such demographic statistics as life expectancy (1), infertility (2), and morbidity (3,4). The meta-analysis by Levine et al. (5) focuses on the change in sperm count of men from North America, Europe, Australia, and New Zealand, and shows a more than 50% decline between 1973 and 2011. Although there is no analysis of potential environmental or lifestyle factors that could contribute to the estimated decline in sperm count, Levine et al. speculate that this decline could be a signal for other negative changes in men’s health.
Because this study focuses mainly on Western men, this remarkable decline in sperm count is difficult to associate with any change in actual fertility, that is, number of children born per woman. The total fertility rate in Europe, especially Italy, Spain, and Germany, has slowly declined, but age at first marriage has increased at the same time, and this increase may be more due to economic factors than physiological changes.
Included in Levine et al.’s analysis was a set of data from “Other” countries from South America, Asia, and Africa. Sperm count in men from these countries did not show significant trends, which is interesting because there have been strong fertility declines in Asia and Africa over the same period, with corresponding increases in life expectancy (once HIV is accounted for).
What can we say about the evolutionary consequences for humans of this decrease? The answer depends on the minimal number of sperm/ml/year that would be required to maintain fertility (per woman) at replacement level, say 2.1 children, over a woman’s lifetime. Given the smaller number of ova produced per woman, a change in the ovulation statistics of women would be likely to play a larger role in the total fertility rate than the number of sperm/ejaculate/man. In other words, sperm count alone, absent other effects on mortality during male reproductive years, is unlikely to tell us much about human evolution.
Further, the major declines in fertility over the 38-year period covered by Levine et al. occurred in China, India, and Japan. Chinese fertility has declined to less than 1.5 children per woman, and in Japan it has also been well below 1.5 for some time. These declines have been due to national policies and economic changes, and are therefore unlikely to signal genetic changes that would have evolutionary ramifications. It is more likely that cultural changes will continue to be the main drivers of fertility change.
The fastest growing human populations are in the Muslim world, where fertility control is not nearly as widely practiced as in the West or Asia. If this pattern were to continue for a few more generations, the cultural evolutionary impact would swamp any effects of potentially declining sperm count.
On the other hand, if the decline in sperm count were to be discovered to be associated with genetic and/or epigenetic phenotypic effects on fetuses, newborns, or pre-reproductive humans, for example, due to stress or obesity, then there would be cause to worry about long-term evolutionary problems. As Levine et al. remark, “decline in sperm count might be considered as a ‘canary in the coal mine’ for male health across the lifespan”. But to date, there is little evidence that the evolutionary trajectory of humans constitutes such a “coal mine”.
References
Jensen TK, Jacobsen R, Christensen K, Nielsen NC, Bostofte E. 2009. Good semen quality and life expectancy: a cohort study of 43,277 men. Am J Epidemiol 170: 559-565.
Eisenberg ML, Li S, Behr B, Cullen MR, Galusha D, Lamb DJ, Lipshultz LI. 2014. Semen quality, infertility and mortality in the USA. Hum Reprod 29: 1567-1574.
Eisenberg ML, Li S, Cullen MR, Baker LC. 2016. Increased risk of incident chronic medical conditions in infertile men: analysis of United States claims data. Fertil Steril 105: 629-636.
Latif T, Kold Jensen T, Mehlsen J, Holmboe SA, Brinth L, Pors K, Skouby SO, Jorgensen N, Lindahl-Jacobsen R. Semen quality is a predictor of subsequent morbidity. A Danish cohort study of 4,712 men with long-term follow-up. Am J Epidemiol. Doi: 10.1093/aje/kwx067. (Epub ahead of print]
Levine H, Jorgensen N, Martino-Andrade A, Mendiola J, Weksler-Derri D, Mindlis I, Pinotti R, Swan SH. 2017. Temporal trends in sperm count: a systematic review and meta-regression analysis. Hum Reprod Update pp. 1-14. Doi: 10.1093/humupd/dmx022.
Psycho-Social Effects of Poverty, Unemployment and Epigenetics on Male Well-being, Physiological Conditions as POTENTIAL effects on Sperm Quality and Quantity and Evidence of its effects on Male Longevity
The IMPACT of Well-being, Stress induced by Worry, Pain, Perception of Hope related to Employment and Lack of employment on deterioration of Physiological Conditions as evidence by Decrease Longevity
In recent work based on our well-being metrics in the Gallup polls and on the mortality data from the Centers for Disease Control and Prevention, we find a robust association between lack of hope (and high levels of worry) among poor whites and the premature mortality rates, both at the individual and metropolitan statistical area (MSA) levels. Yet we also find important differences across places. Places come with different economic structures and identities, community traits, physical environments and much more. In the maps below, we provide a visual picture of the differences in in hope for the future, worry, and pain across race-income cohorts across U.S. states. We attempted to isolate the specific role of place, controlling for economic, socio-demographic, and other variables.
One surprise is the low level of optimism and high level of worry in the minority dense and generally “blue” state of California, and high levels of pain and worry in the equally minority dense and “blue” states of New York and Massachusetts. High levels of income inequality in these states may explain these patterns, as may the nature of jobs that poor minorities hold.
We cannot answer many questions at this point. What is it about the state of Washington, for example, that is so bad for minorities across the board? Why is Florida so much better for poor whites than it is for poor minorities? Why is Nevada “good” for poor white optimism but terrible for worry for the same group? One potential issue—which will enter into our future analysis—is racial segregation across places. We hope that the differences that we have found will provoke future exploration. Readers of this piece may have some contributions of their own as they click through the various maps, and we welcome their input. Better understanding the role of place in the “crisis” of despair facing our country is essential to finding viable solutions, as economic explanations, while important, alone are not enough.
There has been a genuine decline in semen quality over the past 50 years. There is lot of controversy about this as there are limitations in studies that have attempted to address it. Sperm count is of considerable public health importance for several reasons. First, sperm count is closely linked to male fecundity and is a crucial component of semen analysis, the first step to identify male factor infertility.
Reduced sperm count is associated with cryptorchidism, hypospadias and testicular cancer. It may be associated with multiple environmental influences, including endocrine disrupting chemicals, pesticides, heat and lifestyle factors, including diet, stress, smoking and BMI. Therefore, sperm count may sensitively reflect the impacts of the modern environment on male health throughout the life span.
This study provided a systematic review and meta-regression analysis of recent trends in sperm counts as measured by sperm concentration (SC) and total sperm count (TSC), and their modification by fertility and geographic group. Analyzing trends by birth cohorts instead of year of sample collection may aid in assessing the causes of the decline (prenatal or in adult life) but was not feasible owing to lack of information.
This rigorous and comprehensive analysis found that SC declined 52.4% between 1973 and 2011 among unselected men from western countries, with no evidence of a ‘leveling off’ in recent years. Declining mean SC implies that an increasing proportion of men have sperm counts below any given threshold for sub-fertility or infertility. The high proportion of men from western countries with concentration below 40 million/ml is particularly concerning given the evidence that SC below this threshold is associated with a decreased monthly probability of conception.
Declines in sperm count have implications beyond fertility and reproduction. The decline reported in this study is consistent with reported trends in other male reproductive health indicators, such as testicular germ cell tumors, cryptorchidism, onset of male puberty and total testosterone levels. The public health implications are even wider. Recent studies have shown that poor sperm count is associated with overall morbidity and mortality. While the current study is not designed to provide direct information on the causes of the observed declines, sperm count has been plausibly associated with multiple environmental (including unwanted chemical exposure in alarming levels) and lifestyle influences, both prenatally and in adult life. In particular, endocrine disruption from chemical exposures or maternal smoking during critical windows of male reproductive development may play a role in prenatal life, while lifestyle changes and exposure to pesticides may play a role in adult life.
These findings strongly suggest a significant decline in male reproductive health, which has serious implications beyond fertility concerns. Research on causes and implications of this decline is urgently needed.
REFERENCES
Temporal trends in sperm count: a systematic review and meta-regression analysis
Hagai Levine, Niels Jørgensen, Anderson Martino‐Andrade, Jaime Mendiola, Dan Weksler-Derri, Irina Mindlis, Rachel Pinotti, Shanna H Swan. Human Reproduction Update, July 25, 2017, doi:10.1093/humupd/dmx022.
Intake of Fruits and Vegetables with Low-to-Moderate Pesticide Residues Is Positively Associated with Semen-Quality Parameters among Young Healthy Men.
The pattern of electrical signals propagated through neuronal networks determines brain function. Adam Cohen examines the possibility of visualizing these signals inside an intact brain using fluorescent transmembrane proteins that are sensitive to voltage. Cohen discusses the barriers to this approach, something he predicts scientists from many disciplines will eventually overcome.
Adam Cohen is Professor in the Departments of Chemistry and Physics at Harvard University and Investigator of the Howard Hughes Medical Institute. He develops biological tools and analytical approaches to investigate the behaviors of molecules and cells in vitro and in vivo. His lab merges protein engineering, optics, and physics, among other disciplines, on a variety of projects. For example, they have developed a fluorescent transmembrane protein that detects membrane voltage, which is useful in visualizing electrical activity in cells, such as cultured neurons.
Evolution can be defined as a change in heritable characteristics. In her fist talk, Hale does a excellent job of explaining how these changes occur. She uses examples, such as the variable color of the pepper moth, to explain selection of characteristics and she describes how geographic isolation can lead to the evolution of new species. In her second lecture, Hale describes work from her lab on the startle response, a highly conserved behavior found in fish and other vertebrates. Comparisons of the neurons which control the startle response, across many species of fish, have allowed Hale and her colleagues to determine how this neuronal circuit, and this behavior, have evolved over hundreds of millions of years.
Part 1 is an outstanding video for high school or undergraduate educators looking for material to teach evolution.
This is an expression of all the genes of a neuron during the first hours after its birth. Each circle represents a development stage (6h, 12h, 24h), and the colored points within each circle represent the level of gene expression. (Credit: Jabaudon Lab/ UNIGE)
Our brain is home to different types of neurons, each with their own genetic signature that defines their function. These neurons are derived from progenitor cells, which are specialized stem cells that have the ability to divide to give rise to neurons. Neuroscientists from the Faculty of Medicine at the University of Geneva (UNIGE) shed light on the mechanisms that allow progenitors to generate neurons. By developing a novel technology called FlashTag that enables them to isolate and visualize neurons at the very moment they are born, they have deciphered the basic genetic code allowing the construction of a neuron. This discovery, which is published in Science, allows not only to understand how our brain develops, but also how to use this code to reconstruct neurons from stem cells. Researchers will now be able to better understand the mechanisms underlying neurological diseases such as autism and schizophrenia.
Directed by Denis Jabaudon, a neuroscientist and neuroscientist at the Department of Basic Neurosciences at UNIGE Faculty of Medicine and neurologist at the University Hospitals Geneva (HUG), the researchers developed a technology termed FlashTag, which visualizes neurons as they are being born. Using this approach, at the very moment where a progenitor divides, it is tagged with a fluorescent marker that persists in its progeny. Scientists can then visualize and isolate newborn neurons in order to dynamically observe which genes are expressed in the first few hours of their existence. Over time, they can then study their evolution and changes in gene expression. “Previously, we only had a few photos to reconstruct the history of neurons, which left a lot of room for speculation. Thanks to FlashTag, there is now a full genetic movie unfolding before our eyes. Every instant becomes visible from the very beginning, which allows us to understand the developmental scenario at play, identify the main characters, their interactions and their incentives”, notes Jabaudon. Working in the cerebral cortex of the mouse, the scientists have thus identified the key genesto neuronal development, and demonstrated that their expression dynamics is essential for the brain to develop normally.
A very precise primordial choreography
This discovery, by giving access to the primordial code of the formation of neurons, helps us to understand how neurons function in the adult brain. And it appears that several of these original genes are also involved in neurodevelopmental and neurodegenerative diseases, which can occur many years later. This suggests that a predisposition may be present from the very first moments in the existence of neurons, and that environmental factors can then impact on how diseases may develop later on. By understanding the genetic choreography of neurons, the researchers can therefore observe how these genes behave from the start, and identify potential anomalies predicting diseases.
After successfully reading this genetic code, the scientists we able to rewrite it in newborn neurons. By altering the expression of certain genes, they were able to accelerate neuronal growth, thus altering the developmental script. With FlashTag, it is now possible to isolate newborn neurons and recreate cerebral circuits in vitro, which enables scientists to test their function as well as to develop new treatments.
A website open to all
The UNIGE team posted a website where it is possible to enter the name of a gene and observe how it is expressed, and how it interacts with other genes. “Each research team can only focus on a handful of genes at a time, while our genome is made up of close to 20,000 genes. We therefore made our tool available for other researchers to use it, in a fully open way,” highlights Jabaudon.
A new study suggests that long-term stress can hurt short-term memory, in part due to inflammation brought on by an immune response.
Researchers from Ohio State University performed experiments where mice were exposed to repeated social defeat by exposure to an aggressive, larger, alpha mouse. The mice that were under chronic stress, had difficulty remembering where the escape hole was in a maze they had previously mastered before the stressful period.
The findings were published in The Journal of Neuroscience.
“The stressed mice didn’t recall it. The mice that weren’t stressed really remembered it,” lead researcher Johnathan Godbout, associate professor of neuroscience at Ohio State, said in statement.
The researchers noted that this kind of stress isn’t the once-in-a-while, acute stress someone might feel before a big meeting or presentation, but prolonged, continued stress.
The mice also displayed depressive-like behavior through social avoidance that continued after four weeks of observation.
Brain changes were also observed in the stressed mice, including inflammation associated with the presence of immune cells, known as macrophages, in the brain. The researchers also recorded shortfalls in the development of new neurons at 10 days and 28 days after the chronic stress ended.
John Sheridan, associate director of Ohio State’s Institute for Behavioral Medicine said in a statement that there might be ways to interrupt the inflammation that occurs in the brain.
When the mice were given a chemical that inhibited inflammation, both memory loss and the inflammatory macrophages disappeared, leading researchers to conclude that post-stress memory deficits is directly tied to inflammation and the immune system. The depressive symptoms and the brain-cell problem did not go away.
“Stress releases immune cells from the bone marrow and those cells can traffic to brain areas associated with neuronal activation in response to stress,” Sheridan said. “They’re being called to the brain, to the center of memory.”
The team aims to understand the underpinnings of stress and responses that could one day lead to treatments for people that suffer from anxiety, depression, or post-traumatic stress disorder.
New information from this study could lead to immune-based treatments, Godbout said.
Blood-brain barrier (BBB) models are often used to investigate BBB function and screen brain-penetrating therapeutics, but it has been difficult to construct a human model that possesses an optimal BBB phenotype and is readily scalable. To address this challenge, we developed a human in vitro BBB model comprising brain microvascular endothelial cells (BMECs), pericytes, astrocytes and neurons derived from renewable cell sources. First, retinoic acid (RA) was used to substantially enhance BBB phenotypes in human pluripotent stem cell (hPSC)-derived BMECs, particularly through adherens junction, tight junction, and multidrug resistance protein regulation. RA-treated hPSC-derived BMECs were subsequently co-cultured with primary human brain pericytes and human astrocytes and neurons derived from human neural progenitor cells (NPCs) to yield a fully human BBB model that possessed significant tightness as measured by transendothelial electrical resistance (~5,000 Ωxcm2). Overall, this scalable human BBB model may enable a wide range of neuroscience studies.
The blood-brain barrier (BBB) is composed of brain microvascular endothelial cells (BMECs) which line brain capillaries and control molecular and cellular trafficking between the bloodstream and neural tissue. These properties are tightly regulated by the surrounding neurovascular microenvironment throughout BBB development and into adulthood. While this barrier is essential for preserving healthy brain activity, its dysfunction has been implicated in a number of neurological diseases1. Moreover, an intact BBB serves as a major bottleneck for brain drug delivery2. Studies regarding BBB development and regulation can be difficult and time-consuming to conduct in vivo and testing brain penetration of therapeutics in vivo is a low throughput endeavor. As such, in vitro BBB models have been widely implemented to study interactions between BMECs and other cells of the neurovascular unit and to conduct screens for prospective BBB-permeant drugs.
In vitro BBB models are typically constructed using primary BMECs isolated from animal brain tissue, including bovine, porcine, rat, and mouse (reviewed extensively in ref. 3). These BMECs are then co-cultured with combinations of other cells of the neurovascular unit, such as neurons, pericytes, and astrocytes, to upregulate BBB properties4,5,6,7. Models derived from animal tissue have proven extremely useful in studying various aspects of the BBB, such as developmental and regulatory mechanisms8,9,10,11,12 and assaying drug permeability, but it is generally well-accepted that owing to species differences, a robust human BBB model is vital to achieve a detailed understanding of human developmental pathways and to conduct relevant drug discovery and design studies13. Human BMEC sources for BBB models have previously consisted of either primary biopsied brain tissue14,15 or immortalized cell lines16. Primary human BMECs typically possess moderate barrier properties but are of limited scale14,15, and immortalized BMECs are clonal and readily scalable but often suffer from suboptimal barrier properties16,17. From a co-culture perspective, human neurons, astrocytes, and pericytes can also be difficult to obtain from primary tissue sources in sufficient quantities for modeling purposes. These collective issues have hindered the development of in vitro human BBB models that are both high fidelity and scalable3.
We have recently demonstrated that stem cells may be attractive candidates to replace primary cells in human BBB models. Human pluripotent stem cells (hPSCs), including both human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs), can be differentiated into cells possessing both endothelial and BBB properties (coined hPSC-derived BMECs) via co-differentiation of neural and endothelial progenitors, followed by selection and subsequent culture of the endothelial cells18. The iPSC-derived BMECs co-cultured with rat astrocytes possessed reasonable barrier tightness as measured by TEER (860 ± 260 Ωxcm2)18, but the TEER remained below some primary bovine19 and porcine20,21 models (800–2,000 Ωxcm2) and substantially lower than in vivo TEER (measured up to 5,900 Ωxcm2)22. In searching for candidates to improve the BBB phenotype, we identified all-trans retinoic acid (RA). BMECs in vivo have been shown to express retinol-binding protein and its membrane receptor STRA623,STRA6 expression has been detected in brain endothelium but not peripheral endothelium in adult mice24, and STRA6 expression was increased during the differentiation of hPSC-derived BMECs in our previous work18. Moreover, RA has been shown to upregulate certain BBB properties in immortalized rodent25,26 and human27 BMEC lines. In this manuscript, we demonstrate maturation of hPSC-derived BMEC phenotypes following retinoic acid (RA) addition during the differentiation process, including enhanced adherens junction protein expression, barrier function, and multidrug resistance protein (MRP) efflux activity. We also demonstrated in previous work that primary human neural progenitor cells (NPCs) could be differentiated to a defined mixture of neurons and astrocytes capable of inducing BBB properties in rat BMECs in co-culture7. In this manuscript, it is shown that under optimized culture conditions, RA-treated hPSC-derived BMECs sequentially co-cultured with primary human brain pericytes and NPC-derived astrocytes and neurons can achieve physiologic TEER values, forming a scalable, fully human BBB model.
The purpose of this work was to construct a renewable, robust human BBB multicellular co-culture model employing hPSCs, NPCs, and pericytes. Using previous studies as guides25,26,31, RA was identified as a significant modulator of BMEC properties during hPSC differentiation that greatly enhanced physical barrier characteristics as demonstrated by elevated TEER in BMECs cultured alone or with neurovascular cell co-culture. In recent work, RA treatment on the hCMEC/D3 human brain endothelial cell line served to increase occludin and VE-cadherin expression, and the authors suggested that RA secreted by radial glia may be involved in BBB development27. In our study, when RA was added during the endothelial progenitor expansion phase of hPSC-derived BMEC differentiation, similar results were observed including an earlier onset of VE-cadherin expression and increased occludin expression. Moreover, the BMEC yield was increased 2-fold and the tightness of the hPSC-derived BMEC monolayers as measured by elevated TEER was significantly enhanced for three different hPSC lines. Somewhat unexpectedly, RA treatment resulted in decreased claudin-5 expression. However, the Western blotting analysis was conducted using whole-cell lysates and does not take into account the substantially improved intercellular claudin-5 junctional continuity upon RA treatment (Fig. 2C). We and others have previously observed a strong correlation between such junctional continuity and resultant barrier phenotype6,29,32. In addition, previous work has demonstrated claudin-5 expression is relatively constant across peripheral and BBB endothelium while occludin expression is increased at the BBB relative to other vascular beds31. Thus, a combination of claudin-5 localization and elevated occludin expression may be the key phenotypic indicators of increased barrier function31,33. RA treatment of hPSC-derived BMECs also selectively increased MRP efflux activity, which agrees with reports demonstrating that signaling via nuclear receptors can regulate efflux transporter expression and activity at the blood-brain barrier in vitro and in vivo34,35,36,37. RA influences many aspects of brain development, such as anterior/posterior axis patterning in the hindbrain and anterior spinal cord38,39,40 and regulation of neurogenesis41,42,43. During BMEC differentiation, RA could trigger several modes of action. RA may act directly on the developing endothelial cells to upregulate BBB properties, it could induce changes in the neural cells to indirectly promote BBB differentiation, or it could act by a combination of these mechanisms. Future work will be necessary to deconvolute the RA signaling mechanisms affecting the hPSC-derived BMEC differentiation scheme.
In Your Dreams
Understanding the sleeping brain may be the key to unlocking the secrets of the human mind.
Many scientists who study the mind live in fantasyland. They ought to move back to reality: neuroscientists, psychologists, computer scientists pursuing artificial intelligence, and the philosophers of mind who are, in many cases, the sharpest thinkers in the room.
The mind makes us rational. That mind is the one we choose to study. When we study sleep or dreaming, we isolate them first—as the specialized topics they are. But, as I argue in my new book The Tides of Mind, we will never reach a deep understanding of mind unless we start with an integrated view, stretching from rational, methodical thought to nightmares.
Integrating dreaming with the rest of mind is something like being asked to assemble a car from a large pile of metal, plastic, rubber, glass, and an ocelot. Dreaming is hallucination, centering on a radically different self from our waking selves, within unreal settings and stories. Dreams can please or scare us far more vividly than our ordinary thoughts. And they are so slippery, so hard to grasp, that we start losing them the moment we wake up.
But dreaming fits easily into the big picture of mind; and we will make no basic progress on understanding the mind until we see how. Dreaming is the endpoint of the spectrum of consciousness, the smooth progression from one type of consciousness to the next, that we each experience daily.
The simplest approach to the spectrum centers on mental focus. The quality of our attention goes from concentrated to diffuse over the course of a normal day; from a state in which we can concentrate—we can think and remember in a relatively disciplined way—to one in which, with our minds wandering and memory growing increasingly vibrant and distracting, we approach sleep. Then our thinking becomes hallucinatory (as we pass through “sleep-onset thought”); and finally, we are asleep and dreaming. Usually, we oscillate down and up more than once during the day. We move partway down, come partway back, then finally slide slowly to the bottom, when we sleep and dream.
We can also describe the spectrum as a steady shift from a mind dominated by action to one dominated by passive mental experience; from mental doing to mental being. In the upper spectrum, we tend to ignore emotion as we pursue some mental object by means of reasoning or analysis. But the daydreams and fantasies that occupy us as we move down-spectrum are often emotional. And in dreaming we encounter the most saturated emotions, good and bad, that the mind can generate.
The spectrum clarifies important aspects of the mind. “Intentionality,” the quality of aboutness (“I believe that bird is a sparrow” is about “that bird”), is sometimes called “the mark of the mental”—the distinguishing attribute of mental states. But intentionality belongs strictly to the upper spectrum, and disappears gradually as we descend. At the bottom, our minds are dominated by experience, pure being. Happiness or pain or “the experience of seeing purple” are states that have causes but are about nothing.
Software simulations of the upper spectrum, of thinking-about, have grown steadily stronger over the years. That trend will continue. Being, however, is not computable. Software can no more reproduce “being happy” than it can reproduce “being rusty.” Such states depend on physical properties of particular objects. A digital computer resembles only the upper-spectrum mind. Software will never come close to reproducing the mind as a whole.
Leaving sleep outside our investigation is a good way not to see any of this. Arbitrarily hacking off one end of any natural spectrum is an invitation to conceptual chaos. There has been plenty of that in the science of mind. We must start by understanding sleep and dreaming, and go from there.
N.M. Murray et al., “Insomnia caused by serotonin depletion is due to hypothermia,” Sleep, 38:1985-93, 2015.
Sleepless nights
Early research into serotonin’s functions suggested that the neurotransmitter promotes sleep: lab animals deprived of the chemical often developed insomnia. More recent evidence indicated that serotonin plays a part in wakefulness instead, a theory that has gained significant traction. But explanations of the initial experimental data were scarce—so Nick Murray, then a research fellow at the University of Iowa Carver College of Medicine, started digging.
Faulty furnace?
“Over the past 5 or 10 years, we’ve found that serotonin is a key neurotransmitter for generating body heat,” says Murray. To investigate whether this role was related to serotonin’s impact on sleep, he and his colleagues injected para-chlorophenylalanine into mice to inhibit serotonin synthesis.
On ice
When kept at room temperature (20 °C), the mice with depleted serotonin slept less and developed a lower body temperature compared with their control counterparts. However, when housed at 33 °C—a thermoneutral temperature for mice—the sleep and body temperature of the treated mice stayed normal. “Serotonin isn’t a sleep-promoting neurotransmitter,” concludes Murray, now a resident at California Pacific Medical Center. He suggests that mice lacking serotonin had a tough time sleeping under early experimental conditions simply because the animals were cold, and that at higher temperatures other neurotransmitter systems in the brain would function to allow them a normal sleep-wake cycle.
Case closed
The study “solves a long-standing mystery” in the field, says Clifford Saper of Harvard University. “Not very many labs measure sleep and body temperature at the same time,” he adds. “It just basically escaped everybody’s notice for all these years.”
Green fluorescently-tagged nanospheres flow through a lymph vessel from an unstressed mouse (top) and a mouse that has been administered the stress hormone norepinephrine (bottom). Scale bar: 20 μmNATURE COMMUNICATIONS, LE ET AL.
Stress is implicated in increased tumor progression risk and poor survival in cancer patients. A number of recent studies have linked these effects to the promotion of tumor cell dissemination through the bloodstream via stress-induced pathways. Now, a mouse study led by researchers in Australia has revealed the mechanisms by which stress modulates cancer’s spread through another transport network open to tumor cells—the lymphatic system. The findings were published today (March 1) in Nature Communications.
Chronic stress in mice remodels lymph vasculature to promote tumour cell dissemination
Chronic stress induces signalling from the sympathetic nervous system (SNS) and drives cancer progression, although the pathways of tumour cell dissemination are unclear. Here we show that chronic stress restructures lymphatic networks within and around tumours to provide pathways for tumour cell escape. We show that VEGFC derived from tumour cells is required for stress to induce lymphatic remodelling and that this depends on COX2 inflammatory signalling from macrophages. Pharmacological inhibition of SNS signalling blocks the effect of chronic stress on lymphatic remodelling in vivo and reduces lymphatic metastasis in preclinical cancer models and in patients with breast cancer. These findings reveal unanticipated communication between stress-induced neural signalling and inflammation, which regulates tumour lymphatic architecture and lymphogenous tumour cell dissemination. These findings suggest that limiting the effects of SNS signalling to prevent tumour cell dissemination through lymphatic routes may provide a strategy to improve cancer outcomes.
In everyday life, we encounter stressful experiences that pose a threat to physiological homeostasis. These threats trigger stress responses, including activation of the sympathetic nervous system (SNS), which leads to elevated local and systemic levels of catecholaminergic neurotransmitters that signal to cells1. Stress-induced SNS signalling is important to enhance alertness and physiological functions for rapid reaction to threat2. However, chronic periods of stress can be detrimental to health by increasing inflammation and promoting the progression of diseases including cancer3, 4, 5, 6. Clinical studies have linked experience of stressful events to poor cancer survival7, 8. This is supported by preclinical studies that show chronic stress promotes cancer progression3, 4, 6. These studies found that stress recruits inflammatory cells to tumours and increases the formation of blood vessels3, 6, which may provide routes for tumour cell dissemination. In addition to dissemination through blood vessels, cancer cells also escape from tumours through lymphatic vasculature9, 10, 11.
The lymphatic system plays an important role in immune function and therefore can influence the trajectory of disease progression. Under normal physiological conditions, the lymphatic system maintains homeostasis by directing cells and solutes from the interstitial fluid of peripheral tissues through lymphatic vessels and into lymph nodes, where they undergo immune examination12, 13. In addition, the lymphatic system aids in the resolution of inflammation by transporting immune cells away from sites of infection14. In cancer, the lymphatic system contributes to disease progression by providing a pathway for tumour cell escape while also being a rich source of chemokines that can promote the invasive properties of tumour cells15. Furthermore, tumour-draining lymph nodes and associated lymphatic endothelium have been shown to develop an immunosuppressive environment, which promotes immune tolerance to the cancer and facilitates tumour growth and spread16, 17, 18. The importance of the lymphatic system in cancer progression is supported by vast clinical data that show tumour-associated lymphatic vessel density (LVD), tumour cell invasion into lymphatic vasculature and the presence of tumour cells in lymph nodes are each associated with increased clinical tumour stage and reduced disease-free survival19, 20,21.
The lymphatic system is innervated by fibres of the SNS22, and acute SNS activity has been shown to increase lymphatic vessel contraction23, 24 and lymphocyte output into lymphatic circulation25. However, little is known about whether stress-induced SNS signalling affects tumour lymphatic vasculature and the consequences this may have on cancer progression.
In this study, we show that chronic stress increases intratumoural LVD while also inducing dilation and increasing flow in lymphatic vessels that drain metastatic tumour cells into lymphatic circulation. Inhibition of COX2 activity blocked the effect of stress on lymphatic vascular remodelling, and showed a key role for macrophage-mediated inflammation in the effects of stress. In addition, we show a critical role for tumour cell-derived VEGFC in the effects of stress on lymphatic vasculature. In both clinical and preclinical studies we demonstrate that disrupting SNS regulation of lymphatics, by blocking β-adrenoceptor signalling, protects against lymphatic dissemination and cancer progression. These findings identify stress signalling as a regulator of lymphatic remodelling and provide evidence for the feasibility of clinically targeting SNS regulation of lymphatics to prevent tumour cell dissemination through lymphatic routes.
(a) Schematic representation of the chronic stress paradigm. (b) Quantification and representative images of tumour LVD (LYVE-1+, green; nuclear, blue) immunostaining of MDA-MB-231 orthotopic tumours. Scale bar, 200μm (n=5). (c) Quantification of MDA-MB-231 primary tumour size in control or stressed BALB/c nu/nu mice over time (n=5 at each time point). (d) Quantification and representative images of tumour-draining lymphatic vessel diameter (LV, blue) in mice with MDA-MB-231 tumours. Scale bar, 1mm (n7). (e) Left: skin flap preparation after injection of Patent Blue V dye into the primary tumour (PT) showing the dye taken up into the tumour-draining LV and into the tumour-draining axillary lymph node (AxLN). The LV is adjacent to a blood vessel (BV). Right top panel: epifluorescence image of mCherry-tagged MDA-MB-231 tumour cells (TCs, red) that had spontaneously disseminated from orthotopic PT and were present in the tumour-draining LV that contained microspheres (green) and was adjacent to an autofluorescent BV. Right lower panel: corresponding maximum projection of multiphoton image. Scale bar, 100μm (Supplementary Movie 1). (f) Representative in vivo bioluminescence image of orthotopic MDA-MB-231 breast cancer model showing PT, and spontaneous metastasis to draining lymph node (LN) and lung 21 days after tumour cell injection. (g) Representative images of LN and lung metastasis and quantification of metastasis by ex vivo bioluminescence (BLI) imaging in control versus stressed mice with MDA-MB-231 tumours (n=5). (h) Metastasis in vivo over time (n=5 at each time point). (i) LN metastasis in mice that were negative or positive for tumour cells in collecting lymphatic vessels (n13). (j) Ex vivo quantification of bioluminescence from LN at day 28 of 66cl4 tumour progression from control or stressed mice (n=5). (k) Area of lymph node metastasis when primary tumour diameter reached 12mm in control or stressed MMTV-PyMT mice (n8). Experiments were completed 2–4 times. All data represent mean±s.e. **P<0.01 and ***P<0.001 by Student’s t-test or Mann–Whitney U-test (post hocBonferroni correction).
Stress remodels lymphatic vasculature through a tumour neural-inflammatory axis to promote lymphogenous tumour cell dissemination and metastasis. Tumour cell-derived VEGFC is necessary for stress-enhanced lymphatic remodelling but is not directly activated by β-adrenoceptor signalling. Tumour-associated macrophages respond to β-adrenoceptor signalling to produce inflammatory molecules such as PGE2, which may then signal to tumour cells to produce VEGFC required for lymphatic remodelling. These effects may be clinically blocked using BBs, anti-VEGFC therapeutics (αVEGFC) or COX2 inhibitors (COX2i). E, epinephrine; NE, norepinephrine; β-AR, β-adrenoceptor.
These findings suggest that it may be important to identify stressed individuals who may be particularly susceptible to lymphogenous dissemination. One approach may be through transcriptional profiling using a stress signature55. Alternatively, as cancer is often a highly stressful experience, it is plausible that SNS intervention may be generally useful to improve cancer outcome. In support of that contention, we found here that clinical BB use was linked to a significant reduction in lymph node metastasis (and reduced distant metastasis) in a cancer cohort without prior evaluation of stress levels.
Stress regulation of lymphatic vasculature may have evolved to promote survival during times of threat. Co-ordinated regulation of the fight-or-flight stress response with increased lymphatic function may have provided an evolutionary advantage by enhancing immune surveillance and activating a rapid immune response to physical threat. However, the findings presented here demonstrate that SNS-regulated lymphatic function can have adverse effects in the context of chronic diseases such as cancer. Importantly, these findings identify multiple points of clinical intervention to limit these adverse effects of stress.
“Stress not only affects your well-being, but it also affects your biology,” said study coauthor Erica Sloan, a cancer researcher at Monash University in Melbourne. “Our study particularly highlights the early steps of tumor cell dissemination into the lymphatic system.”
“This is an excellent contribution,” said Kari Alitalo, a professor of translational cancer biology at the University of Helsinki, Finland, who was not involved in the study. “It’s certainly a very refreshing, novel aspect of biology that they explore in this paper.”
Chronic stress, mediated partly through the sympathetic nervous system, has been associated in cancer patients with a number of physiological changes that promote metastasis (the spread of cancer), including the promotion of blood vessel formation and the recruitment of inflammatory cells like macrophages.
To investigate whether stress could also induce changes in lymph vasculature, the researchers subjected various types of mammary tumor–bearing mice—including strains genetically engineered to develop tumors spontaneously, as well as animals given tumor transplants—to a paradigm designed to induce chronic stress: confinement in a tight space. Comparing stressed mice to controls that bore the same cancerous tumors but had been kept in normal cage conditions, the researchers found no difference in primary tumor growth, but significant differences in lymph vasculature architecture and the frequency of metastases.
“We found that stress helps to build new lymphatic freeways out of the tumor [and] modulates how quickly lymph flows through lymph vessels,” said Sloan, adding that “stress increases the speed limit on these little lymphatic highways and helps cells transit more quickly out of the tumor.”
Since tumor cell dissemination is a key step in cancer metastasis, the team wanted to test whether dissemination through the lymphatic system could be reduced by blocking stress signaling pathways. The researchers turned to beta-blockers—cheap, widely available drugs commonly used to treat hypertension—which inhibit signaling of norepinephrine (or noadrenaline), a stress hormone already implicated in cancer progression risk.
Administering beta-blockers to tumor-bearing mice, the researchers were able to minimize changes in the density of lymph vessels at the primary tumor site, and subsequently reduce metastasis to the lymph nodes. By contrast, artificially stimulating norepinephrine receptors increased both lymph vessel density and metastasis. Through a series of further experiments, the team demonstrated important roles for macrophages involved in inflammatory signaling and a set of tumor-secreted vascular endothelial growth factors (VEGFs) in the regulation of lymph vasculature remodeling and tumor cell dissemination.
“It’s an important step in understanding how stress pathways can influence metastasis,” said Anil Sood, a professor of translational research at MD Anderson Cancer Center in Houston, Texas, who was not involved in the research. “It really helps us to understand the possible mechanisms by which sympathetic nervous system pathways can affect how lymphatics may be remodeled.”
The study also included an analysis of observational data from a cohort of nearly 1,000 breast cancer patients in Milan, which corroborate the team’s findings in mice: patients taking beta-blockers showed a significantly lower incidence of lymph node and distant metastases, even once potentially confounding factors such as age and treatment type had been taken into account.
But Alitalo cautioned against drawing strong conclusions from these data. “Stress biology is complex,” he said. “In laboratory conditions with mice, it’s easier to define and measure stress. In real life, these things fluctuate a lot, especially in cancer patients.” He added that beta-blockers show “no specificity to the lymphatic system, so [their] effects as such could be transduced via a variety of pathways.”
Sloan and colleagues are now working to further resolve the molecular mechanisms involved in stress-induced remodeling of the tumor microenvironment in mice, and are investigating potential interactions between beta-blockers and standard cancer treatments, with a view to using the drugs to tackle stress-related metastasis risk in the clinic.
“This is something that, when we treat cancer, we should be considering,” Sloan said. “By actually addressing stress in the patient, we’re giving our cancer therapies a better chance to work.”
C.P. Le et al., “Chronic stress in mice remodels lymph vasculature to promote tumour cell dissemination,” Nature Communications,http://dx.doi.org:/10.1038/ncomms10634,2016.
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
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
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
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?
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.
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
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
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
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
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 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. 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. 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
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
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
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
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
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. 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. 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
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 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
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
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
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
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) 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
(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
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
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
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
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
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
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. 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 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
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. 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
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
T cell receptor (TCR) and costimulatory molecule mediated signaling
culminate in maximal cytokine mRNA production and stability.
The transcriptional responses to co-stimulatory T cell signaling involve calcineurin and NF-AT, which
can be antagonized by interference with the cis-trans peptidyl-prolyl isomerases (PPIase), cyclophilin A and FKBP.
Signaling molecules downstream of CD28
which are essential for the stabilization of cytokine mRNAs are largely unknown.
Pin1, a third member of the PPIase family
mediates the post-transcriptional regulation of Th1 cytokines by activated T cells.
Blockade of Pin1 by pharmacologic or genetic means
greatly attenuated IFN-γ, IL-2 and CXCL-10 mRNA
stability,
accumulation and
protein expression after cell activation.
In vivo, Pin1 blockade prevented
both the acute and chronic rejection of MHC mismatched, orthotopic rat lung transplants by
reducing the expression of IFN-γ and CXCL-10.
Combined transcriptional and post-transcriptional blockade with
cyclosporine A and the Pin1 inhibitor, juglone, was synergistic.
These data suggest Pin1 inhibitors should be explored for use as immunosuppressants and employed with available calcineurin inhibitors to reduce toxicity and enhance effectiveness.
Esnault S, Braun RK, Shen Z-J, Xiang Z, Heninger E, et al. (2007) Pin1 Modulates the Type 1 Immune Response. PLoS ONE 2(2): e226. http://dx. doi.org/10.1371/journal.pone.0000226
Mixed-lineage kinase 3 phosphorylates prolyl-isomerase Pin1 to regulate its nuclear translocation and cellular function
Velusamy Rangasamya,1, Rajakishore Mishraa,1, Gautam Sondarvaa, Subhasis Dasa, et al.
Nuclear protein peptidyl-prolyl isomerase Pin1-mediated prolyl isomerization is
an essential and novel regulatory mechanism for protein phosphorylation.
Therefore, tight regulation of Pin1 localization and catalytic activity is
crucial for its normal nuclear functions.
Pin1 is commonly dysregulated during oncogenesis and likely contributes to these pathologies; The mechanism by which Pin1 catalytic activity and nuclear localization are increased is unknown.
Here we demonstrate that
mixed-lineage kinase 3 (MLK3), a MAP3K family member,
phosphorylates Pin1 on a Ser138 site
to increase its catalytic activity and nuclear translocation.
This phosphorylation event
drives the cell cycle and
promotes cyclin D1 stability and centrosome amplification.
Pin1 pSer138 is significantly
up-regulated in breast tumors and
is localized in the nucleus.
These findings collectively suggest that the MLK3-Pin1 signaling cascade plays a critical role
in regulating the cell cycle,
centrosome numbers, and
oncogenesis. breast cancer
JNK Peptidyl-prolyl isomerase Pin1 plays a critical role in
regulating cellular homeostasis by
isomerizing the prolyl bond preceded by
a phosphorylated Ser or Thr residue (pSer/Thr-Pro) (1).
This isomerization by Pin1 regulates the biological function of several target proteins, including
cell-cycle regulators,
protooncogenes,
tumor suppressors, and
transcription factors (2).
Due to its role in controlling the cell cycle, apoptosis, growth, and stress responses, Pin1 has been linked to the pathogenesis of human diseases, including
cancer (3, 4),
asthma (5),
Alzheimer’s disease (AD) (6), and
Parkinson disease (PD) (7).
It is thus quite likely that tight regulation of Pin1 catalytic activity or expression is important for normal physiology. It is reported that Pin1 is
overexpressed in most types of cancer (8), whereas
its expression is diminished in AD brains (2).
Accumulating evidence suggests that Pin1 isomerase activity
and thus function are regulated by posttranslational modifications (2).
Pin1 function is also dependent on its
predominant nuclear localization (2),
consistent with its substrates being involved in transcription and cell-cycle progression.
It was recently reported that Pin1 nuclear import is regulated by a novel nuclear localization sequence in the PPIase domain, composed of basic amino acids (9). Nonetheless, the detailed mechanism that regulates Pin1 nuclear translocation is still not known. It also remains unknown whether any posttranslational modification of Pin1 can regulate its nuclear translocation or catalytic activity, and therefore directly affect its function.
Stereospecific gating of functional motions in Pin1 Andrew T. Namanjaa, Xiaodong J. Wangb, Bailing Xub, et al.
University of Notre Dame, Notre Dame, IN 46556; Virginia Tech, Blacksburg, VA 24061
Edited by Peter E. Wright, The Scripps Research Institute, La Jolla, CA, and approved June 2, 2011 Pin1 is a modular enzyme that
accelerates the cis-trans isomerization of phosphorylated-Ser/Thr-Pro (pS/T-P) motifs
found in numerous signaling proteins regulating cell growth and neuronal survival.
We have used NMR to investigate the interaction of Pin1 with three related ligands that include
a pS-P substrate peptide, and
two pS-P substrate analogue inhibitors
locked in the cis and trans conformations.
We compared the
ligand binding modes and
binding-induced changes
in Pin1 side-chain flexibility.
The cis and trans binding modes differ, and
produce different mobility in Pin1.
The cis-locked inhibitor and substrate produced a
loss of side-chain flexibility
along an internal conduit of conserved hydrophobic residues,
connecting the domain interface with the isomerase active site.
The trans-locked inhibitor
produces a weaker conduit response.
Thus, the conduit response is stereoselective. We further show
interactions between the peptidyl-prolyl isomerase and
Trp-Trp (WW) domains
amplify the conduit response, and
alter binding properties at the remote peptidyl-prolyl isomerase active site.
These results suggest that
specific input conformations can gate dynamic changes that support intraprotein communication.
Such gating may help control the propagation of chemical signals by Pin1, and other modular signaling proteins.
allostery ∣ protein dynamics ∣ ligand dynamics ∣ protein evolution Phospho-serine/threonine-proline (pS/T-P) motifs are
signaling motifs within
intrinsically disordered loops of cell cycle proteins (1).
The imide bond between the pS/T and P residues can adopt
either the cis or trans conformation.
These conformations differ
in their susceptibility to kinases and phosphatases
that propagate the chemical signals governing the cell cycle.
Accordingly, the cell must regulate the cis/trans populations of these pS/T-P motifs
to ensure proper signal routing.
In this context, the peptidyl-prolyl isomerase Pin1 has emerged as a critical regulator (2, 3). Pin1 is a reversible enzyme that
catalyzes the cis-trans isomerization of the pS/T-P imide linkages (2, 3) of other signaling proteins, such as
CDC25C,
p53,
c-Myc,
NF-kB,
cyclin D1, and
tau (3).
Pin1 engages when external events, such as
S/T (de)-phosphorylation, change the cis-trans equilibrium.
Pin1 then
catalyzes the cis-trans isomerization, thereby
accelerating the approach to the new equilibrium (1).
Pin1 is a modular protein of 163 residues consisting of a
catalysis is the sole province of the PPIase domain.
Earlier structural studies of Pin1 revealed
conformational changes upon substrate interaction, thus
motivating flexibility-function studies of Pin1 (4–6).
Peptidyl-prolyl Isomerase Pin1 Controls Down-regulation of Conventional Protein Kinase C Isozymes
JBC Papers in Press, Feb 8, 2012. http://dx.doi.org/10.1074/jbc.M112.349753
H Abrahamsen, AK O’Neill, N Kannan, N Kruse¶, et al.
From the University of California, San Diego, La Jolla, California 92093
Background: Conventional PKC isozymes have a putative Pin1
isomerization sequence at their turn motif phosphorylation site.
Results: Pin1binds conventional PKCs and
promotes their activation-induced down-regulation.
Conclusion: Pin1isomerizes the phosphorylated turn motif of conventional PKC isozymes,
priming them for subsequent down-regulation.
Significance: Pin1 provides a switch regulating the lifetime of conventional PKCs. The down-regulation or cellular depletion of protein kinase C (PKC)
attendant to prolonged activation by phorbol esters is a
widely described property of this key family of signaling enzymes.
However, neither the mechanism of down-regulation nor whether this mechanism occurs following stimulation by physiological agonists is known.
**the peptidylprolyl isomerase Pin1 provides a timer for the lifetime of conventional PKC isozymes,
converting the enzymes into a species that can be dephosphorylated and ubiquitinated
following activation induced by either phorbol esters or natural agonists.
The regulation by Pin1 requires both the catalytic activity of the isomerase and the presence of a Pro immediately following the phosphorylated Thr of
the turn motif phosphorylation site,
one of two C-terminal sites that is phosphorylated during the maturation of PKC isozymes.
the second C-terminal phosphorylation site, the hydrophobic motif, docks
Pin1 to PKC.
Our data are consistent with a model in which Pin1
binds the hydrophobic motif of conventional PKC isozymes to catalyze the isomerization of the phospho-Thr-Pro peptide bond at the turn motif, thus
converting these PKC isozymes into species that can be efficiently down-regulated following activation.
The peptidyl-prolyl cis-trans isomerase Pin1 is emerging as an important regulator of signal transduction pathways (1).
Pin1-catalyzed isomerization plays a key role in the control of normal cellular functions, most notably proliferation where
Pin1 is essential for cell cycle progression (2).
Pin1 belongs to the Parvulin family of peptidyl-prolyl cis-trans isomerases and is the only member that
the enzyme displays an 1000-fold selectivity for peptides phosphorylated on the
Ser/Thr preceding the Pro compared with unphosphorylated peptides (3).
Pin1–induced conformational changes in target proteins
affect a variety of protein properties from
folding to
regulation of activity and stability.
As a consequence, deregulation of phosphorylation steps and their attendant conformational changes often lead to disease (4). For example, Pin1 is
downregulated in degenerating neurons from Alzheimer disease patients, correlating with age-dependent neurodegeneration (5). Pin1 has also been implicated in cancer progression:
levels of this protein are increased in many cancers, including those of the
breast,
prostate,
brain,
lung, and
colon (6–9).
Thus, Pin1 has been proposed to function as a catalyst for oncogenic pathways (10). The molecular mechanisms that lead to disease progression
most likely involve postphosphorylation conformational changes
The human immunophilin protein FKBP12 colored by hydrophobicity (white = hydrophobic) with bound FK506, an immunosuppressant used in treating organ transplant patients to prevent rejection. FKBP also has unrelated prolyl isomerase activity. (Photo credit: Wikipedia)
The human immunophilin protein FKBP12 colored by secondary structure with bound FK506, an immunosuppressant used in treating organ transplant patients to prevent rejection. FKBP also has unrelated prolyl isomerase activity. (Photo credit: Wikipedia)
What is the Future for Genomics in Clinical Medicine?
Author and Curator: Larry H Bernstein, MD, FCAP
Introduction
This is the last in a series of articles looking at the past and future of the genome revolution. It is a revolution indeed that has had a beginning with the first phase discovery leading to the Watson-Crick model, the second phase leading to the completion of the Human Genome Project, a third phase in elaboration of ENCODE. But we are entering a fourth phase, not so designated, except that it leads to designing a path to the patient clinical experience.
What is most remarkable on this journey, which has little to show in treatment results at this time, is that the boundary between metabolism and genomics is breaking down. The reality is that we are a magnificent “magical” experience in evolutionary time, functioning in a bioenvironment, put rogether like a truly complex machine, and with interacting parts. What are those parts – organelles, a genetic message that may be constrained and it may be modified based on chemical structure, feedback, crosstalk, and signaling pathways. This brings in diet as a source of essential nutrients, exercise as a method for delay of structural loss (not in excess), stress oxidation, repair mechanisms, and an entirely unexpected impact of this knowledge on pharmacotherapy. I illustrate this with some very new observations.
Gutenberg Redone
The first is a recent talk on how genomic medicine has constructed a novel version of the “printing press”, that led us out of the dark ages.
In our series The Creative Destruction of Medicine, I’m trying to get into critical aspects of how we can Schumpeter or reboot the future of healthcare by leveraging the big innovations that are occurring in the digital world, including digital medicine.
We have this big thing about evidence-based medicine and, of course, the sanctimonious randomized, placebo-controlled clinical trial. Well, that’s great if one can do that, but often we’re talking about needing thousands, if not tens of thousands, of patients for these types of clinical trials. And things are changing so fast with respect to medicine and, for example, genomically guided interventions that it’s going to become increasingly difficult to justify these very large clinical trials.
For example, there was a drug trial for melanoma and the mutation of BRAF, which is the gene that is found in about 60% of people with malignant melanoma. When that trial was done, there was a placebo control, and there was a big ethical charge asking whether it is justifiable to have a body count. This was a matched drug for the biology underpinning metastatic melanoma, which is essentially a fatal condition within 1 year, and researchers were giving some individuals a placebo.
The next observation is a progression of what he have already learned. The genome has a role is cellular regulation that we could not have dreamed of 25 years ago, or less. The role is far more than just the translation of a message from DNA to RNA to construction of proteins, lipoproteins, cellular and organelle structures, and more than a regulation of glycosidic and glycolytic pathways, and under the influence of endocrine and apocrine interactions. Despite what we have learned, the strength of inter-molecular interactions, strong and weak chemical bonds, essential for 3-D folding, we know little about the importance of trace metals that have key roles in catalysis and because of their orbital structures, are essential for organic-inorganic interplay. This will not be coming soon because we know almost nothing about the intracellular, interstitial, and intrvesicular distributions and how they affect the metabolic – truly metabolic events.
I shall however, use some new information that gives real cause for joy.
Reprogramming Alters Cells’ Fate
Kathy Liszewski
Gordon Conference Report: June 21, 2012;32(11)
New and emerging strategies were showcased at Gordon Conference’s recent “Reprogramming Cell Fate” meeting. For example, cutting-edge studies described how only a handful of key transcription factors were needed to entirely reprogram cells.
M. Azim Surani, Ph.D., Marshall-Walton professor at the Gurdon Institute, University of Cambridge, U.K., is examining cellular reprogramming in a mouse model. Epiblast stem cells are derived from the early-stage embryonic stage after implantation of blastocysts, about six days into development, and retain the potential to undergo reversion to embryonic stem cells (ESCs) or to PGCs.” They report two critical steps both of which are needed for exploring epigenetic reprogramming. “Although there are two X chromosomes in females, the inactivation of one is necessary for cell differentiation. Only after epigenetic reprogramming of the X chromosome can pluripotency be acquired. Pluripotent stem cells can generate any fetal or adult cell type but are not capable of developing into a complete organism.”
The second read-out is the activation of Oct4, a key transcription factor involved in ESC development. The expression of Oct4 in epiSCs requires its proximal enhancer. Dr. Surani said that their cell-based system demonstrates how a systematic analysis can be performed to analyze how other key genes contribute to the many-faceted events involved in reprogramming the germline.
Reprogramming Expressway
A number of other recent studies have shown the importance of Oct4 for self-renewal of undifferentiated ESCs. It is sufficient to induce pluripotency in neural tissues and somatic cells, among others. The expression of Oct4 must be tightly regulated to control cellular differentiation. But, Oct4 is much more than a simple regulator of pluripotency, according to Hans R. Schöler, Ph.D., professor in the department of cell and developmental biology at the Max Planck Institute for Molecular Biomedicine.
Oct4 has a critical role in committing pluripotent cells into the somatic cellular pathway. When embryonic stem cells overexpress Oct4, they undergo rapid differentiation and then lose their ability for pluripotency. Other studies have shown that Oct4 expression in somatic cells reprograms them for transformation into a particular germ cell layer and also gives rise to induced pluripotent stem cells (iPSCs) under specific culture conditions.
Oct4 is the gatekeeper into and out of the reprogramming expressway. By modifying experimental conditions, Oct4 plus additional factors can induce formation of iPSCs, epiblast stem cells, neural cells, or cardiac cells. Dr. Schöler suggests that Oct4 a potentially key factor not only for inducing iPSCs but also for transdifferention. “Therapeutic applications might eventually focus less on pluripotency and more on multipotency, especially if one can dedifferentiate cells within the same lineage. Although fibroblasts are from a different germ layer, we recently showed that adding a cocktail of transcription factors induces mouse fibroblasts to directly acquire a neural stem cell identity.
Stem cell diagram illustrates a human fetus stem cell and possible uses on the circulatory, nervous, and immune systems. (Photo credit: Wikipedia)
Transforming growth factor beta (TGF-β) is a secreted protein that controls proliferation, cellular differentiation, and other functions in most cells. http://en.wikipedia.org/wiki/TGFbeta (Photo credit: Wikipedia)
Pioneer Transcription Factors
Pioneer transcription factors take the lead in facilitating cellular reprogramming and responses to environmental cues. Multicellular organisms consist of functionally distinct cellular types produced by differential activation of gene expression. They seek out and bind specific regulatory sequences in DNA. Even though DNA is coated with and condensed into a thick fiber of chromatin. The pioneer factor, discovered by Prof. KS Zaret at UPenn SOM in 1996, he says, endows the competence for gene activity, being among the first transcription factors to engage and pry open the target sites in chromatin.
FoxA factors, expressed in the foregut endoderm of the mouse,are necessary for induction of the liver program. They found that nearly one-third of the DNA sites bound by FoxA in the adult liver occur near silent genes
A Nontranscriptional Role for HIF-1α as a Direct Inhibitor of DNA Replication
Many of the cellular responses to reduced O2 availability are mediated through the transcriptional activity of hypoxia-inducible factor 1 (HIF-1). We report a role for the isolated HIF-1α subunit as an inhibitor of DNA replication, and this role was independent of HIF-1β and transcriptional regulation. In response to hypoxia, HIF-1α bound to Cdc6, a protein that is essential for loading of the mini-chromosome maintenance (MCM) complex (which has DNA helicase activity) onto DNA, and promoted the interaction between Cdc6 and the MCM complex. The binding of HIF-1α to the complex decreased phosphorylation and activation of the MCM complex by the kinase Cdc7. As a result, HIF-1α inhibited firing of replication origins, decreased DNA replication, and induced cell cycle arrest in various cell types. To whom correspondence should be addressed. E-mail: gsemenza@jhmi.edu
Citation: M. E. Hubbi, Kshitiz, D. M. Gilkes, S. Rey, C. C. Wong, W. Luo, D.-H. Kim, C. V. Dang, A. Levchenko, G. L. Semenza, A Nontranscriptional Role for HIF-1α as a Direct Inhibitor of DNA Replication. Sci. Signal. 6, ra10 (2013).
Identification of a Candidate Therapeutic Autophagy-inducing Peptide
Beth Levine and colleagues have constructed a cell-permeable peptide derived from part of an autophagy protein called beclin 1. This peptide is a potent inducer of autophagy in mammalian cells and in vivo in mice and was effective in the clearance of several viruses including chikungunya virus, West Nile virus and HIV-1.
Could this small autophagy-inducing peptide may be effective in the prevention and treatment of human diseases?
PR-Set7 Is a Nucleosome-Specific Methyltransferase that Modifies Lysine 20 of
Histone H4 and Is Associated with Silent Chromatin
We have purified a human histone H4 lysine 20methyl-transferase and cloned the encoding gene, PR/SET07. A mutation in Drosophila pr-set7 is lethal: second in-star larval death coincides with the loss of H4 lysine 20 methylation, indicating a fundamental role for PR-Set7 in development. Transcriptionally competent regions lack H4 lysine 20 methylation, but the modification coincided with condensed chromosomal regions polytene chromosomes, including chromocenter euchromatic arms. The Drosophila male X chromosome, which is hyperacetylated at H4 lysine 16, has significantly decreased levels of lysine 20 methylation compared to that of females. In vitro, methylation of lysine 20 and acetylation of lysine 16 on the H4 tail are competitive. Taken together, these results support the hypothesis that methylation of H4 lysine 20 maintains silent chromatin, in part, by precluding neighboring acetylation on the H4 tail.
Next-Generation Sequencing vs. Microarrays
Shawn C. Baker, Ph.D., CSO of BlueSEQ
GEN Feb 2013
With recent advancements and a radical decline in sequencing costs, the popularity of next generation sequencing (NGS) has skyrocketed. As costs become less prohibitive and methods become simpler and more widespread, researchers are choosing NGS over microarrays for more of their genomic applications. The immense number of journal articles citing NGS technologies it looks like NGS is no longer just for the early adopters. Once thought of as cost prohibitive and technically out of reach, NGS has become a mainstream option for many laboratories, allowing researchers to generate more complete and scientifically accurate data than previously possible with microarrays.
Gene Expression
Researchers have been eager to use NGS for gene expression experiments for a detailed look at the transcriptome. Arrays suffer from fundamental ‘design bias’ —they only return results from those regions for which probes have been designed. The various RNA-Seq methods cover all aspects of the transcriptome without any a priori knowledge of it, allowing for the analysis of such things as novel transcripts, splice junctions and noncoding RNAs. Despite NGS advancements, expression arrays are still cheaper and easier when processing large numbers of samples (e.g., hundreds to thousands).
Methylation
While NGS unquestionably provides a more complete picture of the methylome, whole genome methods are still quite expensive. To reduce costs and increase throughput, some researchers are using targeted methods, which only look at a portion of the methylome. Because details of exactly how methylation impacts the genome and transcriptome are still being investigated, many researchers find a combination of NGS for discovery and microarrays for rapid profiling.
Diagnostics
They are interested in ease of use, consistent results, and regulatory approval, which microarrays offer. With NGS, there’s always the possibility of revealing something new and unexpected. Clinicians aren’t prepared for the extra information NGS offers. But the power and potential cost savings of NGS-based diagnostics is alluring, leading to their cautious adoption for certain tests such as non-invasive prenatal testing. Cytogenetics
Perhaps the application that has made the least progress in transitioning to NGS is cytogenetics. Researchers and clinicians, who are used to using older technologies such as karyotyping, are just now starting to embrace microarrays. NGS has the potential to offer even higher resolution and a more comprehensive view of the genome, but it currently comes at a substantially higher price due to the greater sequencing depth. While dropping prices and maturing technology are causing NGS to make headway in becoming the technology of choice for a wide range of applications, the transition away from microarrays is a long and varied one. Different applications have different requirements, so researchers need to carefully weigh their options when making the choice to switch to a new technology or platform. Regardless of which technology they choose, genomic researchers have never had more options.
Sequencing Hones In on Targets
Greg Crowther, Ph.D.
GEN Feb 2013
Cliff Han, PhD, team leader at the Joint Genome Institute in the Los Alamo National Lab, was one of a number of scientists who made presentations regarding target enrichment at the “Sequencing, Finishing, and Analysis in the Future” (SFAF) conference in Santa Fe, which was co-sponsored by the Los Alamos National Laboratory and DOE Joint Genome Institute. One of the main challenges is that of target enrichment: the selective sequencing of genomic or transcriptomic regions. The polymerase chain reaction (PCR) can be considered the original target-enrichment technique and continues to be useful in contexts such as genome finishing. “One target set is the unique gaps—the gaps in the unique sequence regions. Another is to enrich the repetitive sequences…ribosomal RNA regions, which together are about 5 kb or 6 kb.” The unique-sequence gaps targeted for PCR with 40-nucleotide primers complementary to sequences adjacent to the gaps, did not yield the several-hundred-fold enrichment expected based on previously published work. “We got a maximum of 70-fold enrichment and generally in the dozens of fold of enrichment,” noted Dr. Han.
“We enrich the genome, put the enriched fragments onto the Pacific Biosciences sequencer, and sequence the repeats,” continued Dr. Han. “In many parts of the sequence there will be a unique sequence anchored at one or both ends of it, and that will help us to link these scaffolds together.” This work, while promising, will remain unpublished for now, as the Joint Genome Institute has shifted its resources to other projects.
At the SFAF conference Dr. Jones focused on going beyond basic target enrichment and described new tools for more efficient NGS research. “Hybridization methods are flexible and have multiple stop-start sites, and you can capture very large sizes, but they require library prep,” said Jennifer Carter Jones, Ph.D., a genomics field applications scientist at Agilent. “With PCR-based methods, you have to design PCR primers and you’re doing multiplexed PCR, so it’s limited in the size that you can target. But the workflow is quick because there’s no library preparation; you’re just doing PCR.” She discussed Agilent’s recently acquired HaloPlex technology, a hybrid system that includes both a hybridization step and a PCR step. Because no library preparation is required, sequencing results can be obtained in about six hours, making it suitable for clinical uses. However, the hybridization step allows capture of targets of up to 5 megabases—longer than purely PCR-based methods can deliver. The Agilent talk also provided details on the applications of SureSelect, the company’s hybridization technology, to Methyl-Seq and RNA-Seq research. With this technology, 120-mer baits hybridize to targets, then are pulled down with streptavidin-coated magnetic beads.
These are selections from the SFAF conference, which is expected to be a boost to work on the microbiome, and lead to infectious disease therapeutic approaches.
Summary
We have finished a breathtaking ride through the genomic universe in several sessions. This has been a thorough review of genomic structure and function in cellular regulation. The items that have been discussed and can be studied in detail include:
the classical model of the DNA structure
the role of ubiquitinylation in managing cellular function and in autophagy, mitophagy, macrophagy, and protein degradation
the nature of the tight folding of the chromatin in the nucleus
intramolecular bonds and short distance hydrophobic and hydrophilic interactions
trace metals in molecular structure
nuclear to membrane interactions
the importance of the Human Genome Project followed by Encode
the Fractal nature of chromosome structure
the oligomeric formation of short sequences and single nucletide polymorphisms (SNPs)and the potential to identify drug targets
Enzymatic components of gene regulation (ligase, kinases, phosphatases)
Methods of computational analysis in genomics
Methods of sequencing that have become more accurate and are dropping in cost
Chromatin remodeling
Triplex and quadruplex models not possible to construct at the time of Watson-Crick
sequencing errors
propagation of errors
oxidative stress and its expected and unintended effects
The hypothalamic-pituitary-adrenal (HPA) axis – which can be thought of as a series of closely linked endocrine structures in the brain – has a key role in triggering the body’s stress response through the secretion of cortisol. In explaining how the HPA axis is itself regulated, for example how its activity is increased in response to a perceived environmental threat, we can infer that the diverse brain areas with which it shares neural interconnections have a crucial role (for a review, see [1]). An equally important question relates to how the activity of the HPA axis is returned to normal when the stress response is no longer needed. To answer this, it is well known that the same “neurosteroid” hormones released by the HPA axis that trigger stress-related biological adaptations also serve to dampen its activity through a “negative feedback” mechanism. In re-defining the biological model of how neurosteroids control the HPA axis, a study led by Jamie Maguire, PhD at Tufts University (Boston, MA) provides some fascinating insights [2]. Moreover, this study has some extremely interesting and counter-intuitive implications for understanding the functions of the “inhibitory” brain chemical gamma-aminobutyric acid (GABA), which is best known for opposing the effects of “excitatory” brain chemicals in order to balance the flow of electrical activity in the brain.
To study how the HPA axis is regulated by neurosteroids, Maguire’s team performed investigations in mice using the neurosteroid tetrahydrodeoxycorticosterone (THDOC). The investigators found that THDOC, when applied to a discrete population of cells in the thalamus called the paraventricular nucleus (PVN), resulted in a decrease in blood levels of corticosterone (the mouse equivalent of the human stress hormone, cortisol). This finding highlights the importance of the PVN as a key anatomical locus in the brain where neurosteroids act, and is consistent with the traditional view of neurosteroids as “negative regulators” of the HPA axis. However, in mice that underwent a stressful “restraint” procedure, it was found that a prior treatment with THDOC (thirty minutes before the stressful experience) resulted in augmentation of corticosterone levels (i.e. relative to mice that underwent the stressful experience but did not receive prior THDOC treatment). In parallel, it was shown that while application of THDOC normally decreased the electrical activity of PVN cells, it actually led to increases in mice that had undergone restraint. Taken together, these findings provide evidence that neurosteroids can have opposite effects on the HPA axis depending on the “stressed” state of the organism.
Thinking about how a neurosteroid hormone can exert opposite effects on PVN cells in the thalamus may be confusing, but what may be more confusing is that these different actions depend on the same “inhibitory” brain chemical, GABA (a neurotransmitter), as well as the same molecular “machinery” (or receptors) with which GABA interacts. This was demonstrated by using mice in which a particular sub-component (or subunit) of the GABA receptor, the gamma subunit, had been genetically deleted; neurosteroids had absolutely no effect on the activity of the HPA axis (neither positive nor negative) in these gamma subunit-deficient mice.
How is it possible to explain the seemingly paradoxical finding that neurosteroids can exert opposite effects on the HPA axis through the same neurotransmitter system? In addressing this question, it is important to remember that although neurotransmitters may be thought of as excitatory or inhibitory, their ability to trigger these effects depends solely on the molecular and cellular apparatus with which they interact. Normally, the inhibitory actions of GABA upon the electrical activity of nerve cells depend on the maintenance of an “electrochemical” gradient by a “transporter” molecule called KCC2 (which transports chloride ions out of cells). Maguire’s team showed that “dephosphorylation” (i.e. the removal of a small chemical moiety – the phosphate group – which is covalently bound at a specific site on the molecule) of KCC2 resulted in lower detectable levels of this transporter in the PVN. Similarly to innumerable other examples in biology where dephosphorylation (or the reverse, phosphorylation) serves as an exquisite regulatory mechanism for controlling the activity of molecular networks, removal of the phosphate group from KCC2 acts as a molecular “switch” that causes the breakdown of the electrochemical gradient. The outcome is that GABA has an excitatory influence on neural activity instead of the inhibitory influence with which it is usually associated.
In common with many important contributions to scientific understanding, these findings should serve as a reminder that it is often necessary to challenge and question what is already “accepted” in our theoretical models, in the light of unexpected and sometimes counter-intuitive experimental results. Whatever the line of scientific inquiry may be, the reward for doing so will be a deeper and more comprehensive understanding of the natural phenomena being studied. The findings of Maguire and colleagues, published in the Journal of Neuroscience, have possible therapeutic implications for disorders associated with disrupted function of the HPA axis, including epilepsy and depression.
Though much attention is on radiolabeled markers, imaging βamyloid is problematic because many cognitively normal elderly have large amounts of β-amyloid in their brain, and appear as “positives” in the imaging tests.
At the same time therapeutic approaches for Alzheimer’s disease have not been focused much on the process of producing a neurofibrillary tangle composed on tau protein.
Various brain sections showing tau protein (Photo credit: WBUR)
Now the BUSM researchers identified a new group of proteins, termed RNA-binding proteins, which accumulate in the brains of patients with Alzheimer’s disease, and are present at much lower levels in subjects who are cognitively intact.
The researchers believe this work opens up novel approaches to diagnose and stage the likelihood of progression by quantifying the levels of these RNA-binding protein biomarkers that accumulate in the brains of Alzheimer patients.
The group found two different proteins, both of which show striking patterns of accumulation. “Proteins such as TIA-1 and TTP, accumulate in neurons that accumulate tau protein, and co-localize with neurofibrillary tangles. These proteins also bind to tau, and so might participate in the disease process,” explained senior author Benjamin Wolozin, MD, PhD, a professor in the departments of pharmacology and neurology at BUSM.
“A different RNA binding protein, G3BP, accumulates primarily in neurons that do not accumulate pathological tau protein.
This observation is striking because it shows that neurons lacking tau aggregates (and neurofibrillary tangles) are also affected by the disease process,” he added.
Wolozin’s group also pursued the observation that some of the RNA binding proteins bind to tau protein, and tested whether one of these proteins, TIA-1, might contribute to the disease process.
‘Stress’ induced aggregation of RNA-binding proteins
Previously, scientists like Tara Vanderweyde et. al., have demonstrated that TIA-1 spontaneously aggregates in response to stress as a normal part of the stress response. They examined the relationship between Stress Granules (SGs) and neuropathology in brain tissue from P301L Tau transgenic mice, as well as in cases of Alzheimer’s disease and FTDP-17.
Stress Granules (SGs) are ‘Stress’ induced aggregation of RNA-binding proteins.
The pattern of SG pathology differed dramatically based on the RNA-binding protein examined. SGs positive for T-cell intracellular antigen-1 (TIA-1) or tristetraprolin (TTP) initially did not co-localize with tau pathology, but then merge with tau inclusions as disease severity increases. In contrast, G3BP (ras GAP-binding protein) identifies a novel type of molecular pathology that shows increasing accumulation in neurons with increasing disease severity, but often is not associated with classic markers of tau pathology. TIA-1 and TTP both bind phospho-tau, and TIA-1 overexpression induces formation of inclusions containing phospho-tau. These data suggest that SG formation might stimulate tau pathophysiology.
Thus, study of RNA-binding proteins and SG biology highlights novel pathways interacting with the pathophysiology of AD.
With this understanding, Wolozin and his colleagues hypothesize that since TIA-1 binds tau, it might stimulate tau aggregation during the stress response. They introduced TIA-1 into neurons with tau protein, and subjected the neurons to stress. Consistent with their hypothesis, tau spontaneously aggregated in the presence of TIA-1, but not in the absence. Thus, the group has potentially identified an entirely novel mechanism to induce tau aggregates de novo.
In future work, the group hopes to use this novel finding to understand how neurofibrillary tangles for in Alzheimer’s disease and to screen for novel compounds that might inhibit the progression of Alzheimer’s disease.
They believe that it may open up novel approaches to diagnose and stage the progression likelihood of the disease in Alzheimer patients.
http://www.ibiology.org/ibiomagazine/adam-cohen-visualizing-activity-brain.html
7). (e) Left: skin flap preparation after injection of Patent Blue V dye into the primary tumour (PT) showing the dye taken up into the tumour-draining LV and into the tumour-draining axillary lymph node (AxLN). The LV is adjacent to a blood vessel (BV). Right top panel: epifluorescence image of mCherry-tagged MDA-MB-231 tumour cells (TCs, red) that had spontaneously disseminated from orthotopic PT and were present in the tumour-draining LV that contained microspheres (green) and was adjacent to an autofluorescent BV. Right lower panel: corresponding maximum projection of multiphoton image. Scale bar, 100
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