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impairment of cognitive function and neurogenesis

Posted in Biological Networks, Cell Biology, Signaling & Cell Circuits, Cerebrovascular and Neurodegenerative Diseases, Curation, Disease Biology, Immunology, Innovations in Neurophysiology & Neuropsychology, tagged Aging, blood brain barrier, Cognition, cytochrome c, β2-microglobulin, neurogenesis on October 31, 2015| Leave a Comment »

impairment of cognitive function and neurogenesis

Larry H. Bernstein, MD, FCAP, Curator

LPBI

β2-microglobulin is a systemic pro-aging factor that impairs cognitive function and neurogenesis

Lucas K Smith, Yingbo He, Jeong-Soo Park, Gregor Bieri, Cedric E Snethlage, Karin Lin, Geraldine Gontier, Rafael Wabl, et al.
Nature Medicine 21,932–937(2015) http://dx.doi.org:/10.1038/nm.3898

Aging drives cognitive and regenerative impairments in the adult brain, increasing susceptibility to neurodegenerative disorders in healthy individuals^{1, 2, 3, 4}. Experiments using heterochronic parabiosis, in which the circulatory systems of young and old animals are joined, indicate that circulating pro-aging factors in old blood drive aging phenotypes in the brain^{5, 6}. Here we identify β2-microglobulin (B2M), a component of major histocompatibility complex class 1 (MHC I) molecules, as a circulating factor that negatively regulates cognitive and regenerative function in the adult hippocampus in an age-dependent manner. B2M is elevated in the blood of aging humans and mice, and it is increased within the hippocampus of aged mice and young heterochronic parabionts. Exogenous B2M injected systemically, or locally in the hippocampus, impairs hippocampal-dependent cognitive function and neurogenesis in young mice. The negative effects of B2M and heterochronic parabiosis are, in part, mitigated in the hippocampus of young transporter associated with antigen processing 1 (Tap1)-deficient mice with reduced cell surface expression of MHC I. The absence of endogenous B2M expression abrogates age-related cognitive decline and enhances neurogenesis in aged mice. Our data indicate that systemic B2M accumulation in aging blood promotes age-related cognitive dysfunction and impairs neurogenesis, in part via MHC I, suggesting that B2M may be targeted therapeutically in old age.

Figure 1: Systemic B2M increases with age and impairs hippocampal-dependent cognitive function and neurogenesis

Systemic B2M increases with age and impairs hippocampal-dependent cognitive function and neurogenesis.

http://www.nature.com/nm/journal/v21/n8/carousel/nm.3898-F1.jpg

(a,b) Schematics of unpaired young versus aged mice (a), and young isochronic versus heterochronic parabionts (b). (a,b) Changes in plasma concentration of B2M with age at 3, 6, 12, 18 and 24 months (a) and between young isochronic and…

Figure 2: B2M expression increases in the aging hippocampus and impairs hippocampal-dependent cognitive function and neurogenesis.close

http://www.nature.com/nm/journal/v21/n8/carousel/nm.3898-F2.jpg

(a,b) Western blot and quantification of hippocampal lysates probed with B2M- and actin-specific antibodies from young (3 months) and aged (18 months) unpaired animals (a), or young isochronic and young heterochronic parabionts five wee…

Figure 3: Reducing MHC I surface expression mitigates the negative effects of heterochronic parabiosis on neurogenesis.close

http://www.nature.com/nm/journal/v21/n8/carousel/nm.3898-F3.jpg

(a) Schematic of young (3 months) WT and Tap1^−/− isochronic parabionts and young WT and Tap1^−/− heterochronic parabionts. (b,c) Representative (of six sections per mouse) images of the DG (b) and quantification of DCX immunostaining (c)…

Figure 4: Absence of B2M enhances hippocampal-dependent cognitive function and neurogenesis in aged animals.

Absence of B2M enhances hippocampal-dependent cognitive function and neurogenesis in aged animals.

http://www.nature.com/nm/journal/v21/n8/carousel/nm.3898-F4.jpg

(a–d) Learning and memory in young (3 months) and aged (17 months) WT and B2m-knockout (B2m^−/−) mice by RAWM (a,c) and contextual fear conditioning (b,d). Data are from 10 young WT, 10 young B2m^−/−, 8 aged WT, and 12 aged B2m^−/− mice. (…

Neuroscience. 2015 Nov 12;308:75-94. doi: 10.1016/j.neuroscience.2015.09.012. Epub 2015 Sep 10.

Synergistic neuroprotection by epicatechin and quercetin: Activation of convergent mitochondrial signaling pathways.

Nichols M¹, Zhang J², Polster BM³, Elustondo PA⁴, Thirumaran A⁵, Pavlov EV⁶, Robertson GS⁷. Author information

In view of evidence that increased consumption of epicatechin (E) and quercetin (Q) may reduce the risk of stroke, we have measured the effects of combining E and Q on mitochondrial function and neuronal survival following oxygen-glucose deprivation (OGD). Relative to mouse cortical neuron cultures pretreated (24h) with either E or Q (0.1-10μM), E+Q synergistically attenuated OGD-induced neuronal cell death. E, Q and E+Q (0.3μM) increased spare respiratory capacity but only E+Q (0.3μM) preserved this crucial parameter of neuronal mitochondrial function after OGD. These improvements were accompanied by corresponding increases in cyclic AMP response element binding protein (CREB) phosphorylation and the expression of CREB-target genes that promote neuronal survival (Bcl-2) and mitochondrial biogenesis (PGC-1α). Consistent with these findings, E+Q (0.1 and 1.0μM) elevated mitochondrial gene expression (MT-ND2 and MT-ATP6) to a greater extent than E or Q after OGD. Q (0.3-3.0μM), but not E (3.0μM), elevated cytosolic calcium (Ca(2+)) spikes and the mitochondrial membrane potential. Conversely, E and E+Q (0.1 and 0.3μM), but not Q (0.1 and 0.3μM), activated protein kinase B (Akt). Nitric oxide synthase (NOS) inhibition with L-N(G)-nitroarginine methyl ester (1.0μM) blocked neuroprotection by E (0.3μM) or Q (1.0μM). Oral administration of E+Q (75mg/kg; once daily for 5days) reduced hypoxic-ischemic brain injury. These findings suggest E and Q activate Akt- and Ca(2+)-mediated signaling pathways that converge on NOS and CREB resulting in synergistic improvements in neuronal mitochondrial performance which confer profound protection against ischemic injury.

MiR-34a regulates blood–brain barrier permeability and mitochondrial function by targeting cytochrome c

Mimi Bukeirat1, Saumyendra N Sarkar1, Heng Hu1,2, Dominic D Quintana1, James W Simpkins1,2, Xuefang Ren1,2 ⇑
J Cereb Blood Flow Metab Sep 30, 2015 0271678X15606147
http://jcb.sagepub.com/content/early/2015/09/15/0271678X15606147.abstract http://dx.doi.org:/10.1177/0271678X15606147

The blood–brain barrier is composed of cerebrovascular endothelial cells and tight junctions, and maintaining its integrity is crucial for the homeostasis of the neuronal environment. Recently, we discovered that mitochondria play a critical role in maintaining blood–brain barrier integrity. We report for the first time a novel mechanism underlying blood–brain barrier integrity: miR-34a mediated regulation of blood–brain barrier through a mitochondrial mechanism. Bioinformatics analysis suggests miR-34a targets several mitochondria-associated gene candidates. We demonstrated that miR-34a triggers the breakdown of blood–brain barrier in cerebrovascular endothelial cell monolayer in vitro, paralleled by reduction of mitochondrial oxidative phosphorylation and adenosine triphosphate production, and decreased cytochrome c levels.

The blood–brain barrier (BBB) is composed of highly specialized cerebrovascular endothelial cells (CECs), separates brain tissue from the circulating blood, and maintains homeostasis of the neuronal environment.¹ The CECs are interconnected by tight junctions including cytoplasmic zonula occludens (ZO) proteins, and various transmembrane proteins such as occludin and claudins.² Disruption of BBB tight junctions has been well documented in cerebrovascular diseases and neurodegenerative disorders and is considered to be a pathological condition of the diseases and plays a key role in disease progression as well.²

A recent study demonstrates that the mitochondrial mechanisms regulate BBB integrity and permeability using oxygen–glucose deprivation and reoxygenation (OGD-R), anin vitro model of ischemic reperfusion injury.³ Our work demonstrates that compromised mitochondria lead to the disruption of tight junctions, opening of the BBB, and exacerbation of stroke outcomes.⁴ As such, regulation of mitochondrial function may affect BBB openings and could be critical in limiting the pathological progression of cerebrovascular diseases and neurodegenerative disorders.

MicroRNAs (miRNAs) are short non-coding functional RNAs that target certain messenger RNAs (mRNAs) through complementary base-pairing between the miRNAs and its mRNA targets, resulting in the inhibition of mRNA translation or degradation of mRNA.⁵ It has been documented that miRNAs are involved in mitochondrial structure and function, such as miR-181c which regulates mitochondrial morphology,⁶ miR-1 which affects mitochondrial mRNA translation,⁷ and miR-378 which targets mitochondrial enzymes involved in oxidative energy metabolism.⁸ Additionally, several miRNAs have recently been found to regulate BBB permeability. MiR-155, miR-181c, and miR-29c negatively affect BBB function by targeting tight junction protein genes directly or affecting related signal pathways.^9⇓–11 The miR-34 family members were discovered computationally and later verified experimentally as a part of the p53 tumor suppressor network. Recent work demonstrates that miR-34a modulates the expression of synaptic targets and neuronal morphology and function.¹² However, little is known regarding the role of miR-34a in mitochondrial function and BBB permeability.

In the present study, we report that the overexpression of miR-34a breaks down the BBB through inhibition of mitochondrial function. Furthermore, cytochrome c (CYC) is experimentally verified as a target of miR-34a in vitro.

Overexpression of miR-34a affects BBB permeability and disrupts tight junctions in CECs

To determine whether miR-34a functionally affected the BBB, we transfected CECs with miR34a plasmid versus vector control in 24-well plates, cultured the cells for 48 h, conducted a BBB permeability assay in a CEC monolayer transwell system in vitro with an additional culture of 48 h, and measured the fluorescent dye FD-4 permeability of each well (Figure 1(a)). As shown in Figure 1(a), FD-4 permeability was significantly increased in wells containing miR-34a overexpression CEC monolayer. Papp, the permeability coefficient, was also significantly higher in CECs overexpressed with miR-34a in comparison to vector controls (Figure 1(a)). Furthermore, immunohis-tochemistry staining of tight junction-related proteins revealed that ZO-1 was continuously distributed in the control, but a discontinuous distribution of ZO-1 was observed in miR-34a overexpressed CEC monolayer (Figure 1(b)). Disruption of tight junctions was not associated with cell viability in CECs transfected with plasmids for 48 h or 96 h (Supplementary Figure 2). Altogether, these data suggest that overexpression of miR-34a increases BBB permeability and compromises BBB tight junctions.

View larger version:

Figure 1.

Overexpression of miR-34a increases BBB permeability in vitro. (a) A schematic protocol using fluorescein isothiocyanate–dextran-4 (FD-4) to detect BBB permeability in vitro. FD-4 permeability in CECs that overexpressed miR-34a plasmid (0.017 ng) versus control was presented as real-time rate of FD-4 mean fluorescent intensity (2-way ANOVA followed by post hoc Dunnett’s test; n = 3; **, P < 0.01; ****, P < 0.0001). Calculated apparent permeability coefficient P_app(Student’s t-test; ****, P < 0.0001) is expressed as mean ± SD. (b) Confocal fluorescence images of CECs confluent monolayers confirmed microscopically after transfection with miR-34a plasmid versus control. Fluorescent staining: tight junctions ZO-1 (red), cell nuclei (DAPI, blue). Overexpression of miR-34a apparently disrupted tight junctions and resulted in gaps between cells (white arrows). Results are representative of three independent experiments.

MiR-34a affects mitochondrial function by targeting CYC in CECs

Our recent work demonstrated that mitochondria play a pivotal role in the maintenance of BBB integrity. BBB tight junctions are rapidly disrupted if oxidative phosphorylation is reduced by mitochondrial inhibitors.⁴ To investigate whether the miR-34a regulates BBB openings via affecting mitochondrial function in CECs, we examined cellular energetic OCRs in CECs transfected with miR-34a plasmid versus vector control. Interestingly, overexpression of miR-34a significantly impaired mitochondrial function in CECs (Figure 2(a) and Supplementary Figure 3). Basal respiration, ATP production, maximal respiration, and spare capacity were all significantly reduced in CECs overexpressing miR-34a for 48 and 72 h (Figure 2(a)). ATP level was also substantially reduced in CECs following overexpression of miR-34a in a dose dependent manner at 72 h (Figure 2(b)).

View larger version:

Figure 2.

Overexpression of mir-34a reduces mitochondrial function and decreases CYC level in cerebrovascular endothelial cells. (a) Basal respiration, ATP production, maximal respiration, and spare capacity were calculated from the bioenergetics functional assay at post-transfection 48 and 72 h (raw data in Supplementary Figure 3). Data are expressed as mean ± SD (n = 5). 1-way ANOVA followed by post hoc Tukey’s test. (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001). (b) ATP level was measured at 72 h post-transfection. Data are expressed as mean ± SD (n = 5). 1-way ANOVA followed by post hoc Tukey’s test. (****, P < 0.0001). (c) Bioinfomatic analysis of miR-34a-targeting candidates related to mitochondria. (d) Flow cytometry analysis of mitochondrial specific proteins for complex I proteins (NDUFAF1, NDUFC2 and NDUFS2), complex II protein (SDHC), complex III protein (CYB), complex IV protein (CYC oxidase, Cox IV), cytochrome c (CYCS), pyruvate dehydrogenase kinase (PDK), and voltage-dependent anion channel protein (VDAC) at 72 h post-transfection. CYC level was significantly lower in the cells that were transfected with the miR-34a plasmid. Data are presented as mean ± SD (n = 3) and analyzed by Student’s t-test, *, P < 0.05; ***, P < 0.001; ****, P < 0.0001. Results are representative of three independent experiments.

To further determine miR-34a targets and uncover the mechanism that is used to affect mitochondria, we performed a bioinformatics analysis of the miR-34a database (miRbase and TargetScan). MiR-34a potentially targets several mitochondria-associated gene candidates including succinate dehydrogenase subunit c (SDHC), cytochrome B reductase 1 (CYBRD1), cytochrome B5 reductase 3 (CYBRD5), cytochrome c (CYCS), pyruvate dehydrogenase kinase isozyme 1 and 2 (PDK1 and PDK2) (Figure 2(c). However, CECs transfected with the miR-34a plasmid had robustly decreased CYCS levels measured by flow cytometry, suggesting that CYCS is one of the miR-34a targets among the potential candidates (Figure 2(d)). Moreover, overexpression of miR-34a slightly increased potential target SDHC but did not change the protein level of CYB and PKD (Figure 2(d)). Off-target genes, NDUFAF1, and VDAC showed no significant change in protein level, but NDUFC2, NDUFS2, and Cox IV were all increased in parallel with overexpression of miR-34a (Figure 2(d)). Taken together, these results experimentally verified CYCS as a miR-34a target, which is associated with the reduction of mitochondrial oxidative phosphorylation in CECs.

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Discussion

In the present study, we demonstrated that the overexpression of miR-34a results in an increased BBB permeability and the disruption of tight junctions ZO-1 in CECs. Consistently, overexpression of miR-34a impaired mitochondrial oxidative phosphorylation and reduced ATP production in CECs. Bioinformatics analysis revealed series of potential miR-34a-targeting candidates related to mitochondrial function. We elucidated that CYCS is a miR-34a target, and the overexpression of miR-34a inhibited the CYCS expression and increased with the expression of other mitochondria-associated genes.

The overexpression of miR-34a disrupted tight junction protein ZO-1 (Figure 1). However, bioinformatics analysis indicated that miR-34a did not target the ZO-1 gene or other tight junction related genes, which suggests that the increased BBB permeability is not directly caused by the targeting of tight junction protein genes. The compromised mitochondrial function by overexpression of miR-34a may influence cellular metabolism in a way that is critical to maintain BBB tight junctions. Among several potential mitochondria-associated gene targets (Figure 2(c)), miR-34a initiated the reduction of CYCS level. Interestingly, potential target SDHC and other off-target gene proteins (NDUFC2, NDUFS2, and Cox IV) were concurrently upregulated (Figure 2(d)), which might be due to the compensation for the reduced target gene protein CYCS, or the disturbance of the coordinated gene translation in mitochondria. We therefore concluded that CYCS is a miR-34a target and is responsible for the miR-34a-induced reduction of mitochondrial oxidative phosphorylation.

Protein kinase C (PKC) signaling has also been shown to affect BBB or other endothelial barriers in vitro and in vivo. A recent study reported that miR-34a regulated blood–tumor barrier by targeting PKCɛ using glioma endothelial cells.¹³ In this study, we did not assess the PKC pathways that could contain additional targets of miR-34a. However, our data do support that miR-34a affects BBB via a mitochondrial mechanism, which is novel and may lead a new direction for designing BBB-related therapeutics.

We have noted several limitations in our study. First, we did not examine the effects of knockdown or knockout miR-34a on BBB function, which might fully establish the role of miR-34a in the BBB and mitochondria. Second, this work was conducted in cell culture models, which adequately address the mechanism of effect that miR-34a exerts on the BBB and mitochondria but do not provide evidence of its involvement in cerebrovascular or neurodegenerative conditions. Further studies in relevant experimental models are warranted.

Mitochondria play a pivotal role in cellular bioenergetics and cell survival, participating in a variety of cellular processes, including the generation of ATP, and the regulation of apoptotic signaling and other signaling pathways.¹⁴ MiR-34a targets and represses multiple genes involved in cell proliferation, apoptosis, cell cycle, migration, etc.,¹⁵ but it is not known if these effects are modulated by the observed mitochondrial effects as well. The present study provides the first description of miR-34a affecting mitochondrial activity, which could lead to a revision of current miR-34a targets and may lead to discovery of new mechanisms. The elucidation of the miR-34a’s role in mitochondrial oxidative phosphorylation and the BBB integrity offers a novel therapeutic strategy for targeting miR-34a to treat cerebrovascular and neurodegenerative diseases such as stroke and Alzheimer’s disease. These neuropathological diseases are known to involve a host of conditions that lead to mitochondrial impairment and BBB disruption. Finally, transient opening of the BBB could prove to be useful for CNS drug delivery.

Long-term aerobic exercise prevents age-related brain deterioration
http://www.kurzweilai.net/long-term-aerobic-exercise-prevents-age-related-brain-deterioration

October 30, 2015

A study of the brains of mice shows that structural deterioration associated with old age can be prevented by long-term aerobic exercise starting in mid-life, according to the authors of an open-access paper in the journal PLOS Biologyyesterday (October 29).

Old age is the major risk factor for Alzheimer’s disease, like many other diseases, as the authors at The Jackson Laboratory in Bar Harbor, Maine, note. Age-related cognitive deficits are due partly to changes in neuronal function, but also correlate with deficiencies in the blood supply to the brain and with low-level inflammation.

“Collectively, our data suggests that normal aging causes significant dysfunction to the cortical neurovascular unit, including basement membrane reduction and pericyte (cells that wrap around blood capillaries) loss. These changes correlate strongly with an increase in microglia/monocytes in the aged cortex,” said Ileana Soto, lead author on the study.*

Benefits of aerobic exercise

However, the researchers found that if they let the mice run freely, the structural changes that make the blood-brain barrier leaky and result in inflammation of brain tissues in old mice can be mitigated. That suggests that there are also beneficial effects of exercise on dementia in humans.**

Further work will be required to establish the mechanism(s): what is the role of the complement-producing microglia/macrophages, how does Apoe decline contribute to age-related neurovascular decline, does the leaky blood-brain barrier allow the passage of damaging factors from the circulation into the brain?

This work was funded in part by The Jackson Laboratory Nathan Shock Center, the Fraternal Order of the Eagle, the Jane B Cook Foundation and NIH.

* The authors investigated the changes in the brains of normal young and aged laboratory mice by comparing by their gene expression profiles using a technique called RNA sequencing, and by comparing their structures at high-resolution by using fluorescence microscopy and electron microscopy. The gene expression analysis indicated age-related changes in the expression of genes relevant to vascular function (including focal adhesion, vascular smooth muscle and ECM-receptor interactions), and inflammation (especially related to the complement system, which clears foreign particles) in the brain cortex.

These changes were accompanied by a decline in the function of astrocytes (key support cells in the brain) and loss of pericytes (the contractile cells that surround small capillaries and venules and maintain the blood-brain barrier). There were also effects on the basement membrane, which forms an integral part of the blood-brain barrier, as well as an increase in the density and functional activation of the immune cells known as microglia/monocytes, which scavenge the brain for infectious agents and damaged cells.

** To investigate the impact of long-term physical exercise on the brain changes seen in the aging mice, the researchers provided the animals with a running wheel from 12 months old (equivalent to middle aged in humans) and assessed their brains at 18 months (equivalent to ~60yrs old in humans, when the risk of Alzheimer’s disease is greatly increased). Young and old mice alike ran about two miles per night, and this physical activity improved the ability and motivation of the old mice to engage in the typical spontaneous behaviors that seem to be affected by aging.

This exercise significantly reduced age-related pericyte loss in the brain cortex and improved other indicators of dysfunction of the vascular system and blood-brain barrier. Exercise also decreased the numbers of microglia/monocytes expressing a crucial initiating component of the complement pathway that others have shown previously to play are role in age-related cognitive decline. Interestingly, these beneficial effects of exercise were not seen in mice deficient in a gene called Apoe, variants of which are a major genetic risk factor for Alzheimer’s disease. The authors also report that Apoe expression in the brain cortex declines in aged mice and this decline can also be prevented by exercise.

Abstract of APOE Stabilization by Exercise Prevents Aging Neurovascular Dysfunction and Complement Induction

Aging is the major risk factor for neurodegenerative diseases such as Alzheimer’s disease, but little is known about the processes that lead to age-related decline of brain structures and function. Here we use RNA-seq in combination with high resolution histological analyses to show that aging leads to a significant deterioration of neurovascular structures including basement membrane reduction, pericyte loss, and astrocyte dysfunction. Neurovascular decline was sufficient to cause vascular leakage and correlated strongly with an increase in neuroinflammation including up-regulation of complement component C1QA in microglia/monocytes. Importantly, long-term aerobic exercise from midlife to old age prevented this age-related neurovascular decline, reduced C1QA+ microglia/monocytes, and increased synaptic plasticity and overall behavioral capabilities of aged mice. Concomitant with age-related neurovascular decline and complement activation, astrocytic Apoe dramatically decreased in aged mice, a decrease that was prevented by exercise. Given the role of APOE in maintaining the neurovascular unit and as an anti-inflammatory molecule, this suggests a possible link between astrocytic Apoe, age-related neurovascular dysfunction and microglia/monocyte activation. To test this, Apoe-deficient mice were exercised from midlife to old age and in contrast to wild-type (Apoe-sufficient) mice, exercise had little to no effect on age-related neurovascular decline or microglia/monocyte activation in the absence of APOE. Collectively, our data shows that neurovascular structures decline with age, a process that we propose to be intimately linked to complement activation in microglia/monocytes. Exercise prevents these changes, but not in the absence of APOE, opening up new avenues for understanding the complex interactions between neurovascular and neuroinflammatory responses in aging and neurodegenerative diseases such as Alzheimer’s disease.

references:

Ileana Soto, Leah C. Graham, Hannah J. Richter, Stephen N. Simeone, Jake E. Radell, Weronika Grabowska, W. Keith Funkhouser, Megan C. Howell, Gareth R. Howell. APOE Stabilization by Exercise Prevents Aging Neurovascular Dysfunction and Complement Induction. PLOS Biology, October 29, 2015; DOI: 10.1371/journal.pbio.1002279 (open access)

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Pyrroloquinoline quinone (PQQ) – an unproved supplement

Posted in Biological Networks, Cell Biology, Cerebrovascular and Neurodegenerative Diseases, Chemical Biology and its relations to Metabolic Disease, Clinical & Translational, tagged cognitive function, glutamine, Mitochondrial biogenesis, PQQ, Sulfur, vitamin-like agent on October 15, 2015| Leave a Comment »

Pyrroloquinoline quinone (PQQ) – an unproved supplement

Larry H. Bernstein, MD, FCAP, Curator

LPBI

Pyrroloquinoline quinone (PQQ)

Pyrroloquinoline quinone (henceforth PQQ) is a small quinone molecule which has the ability to be a REDOX agent, capable of reducing oxidants (an antioxidant effect) and then being recycled by glutathione back into an active form. It appears to be quite stable as it can undergo several thousand cycles before being used up, and it is novel since it associates with protein structures inside the cell (some antioxidants, mostly notably carotenoids like β-carotene and Astaxanthin, are located at specific areas of a cell where they exert proportionally more antioxidant effects due to proximity; PQQ seems to do this near proteins like carotenoids do so at the cell membrane).

The aforementioned REDOX functions can alter protein function and signaling pathways, and while there is a lot of promising in vitro (outside of a living model) research on what it could do there are only a few promising results of PQQ supplementation, mostly related to either altering some signaling pathways or via its benefits to mitochondria (producing more of them and increasing their efficiency).

It is a coenzyme in bacteria (so, to bacteria, this would be something like a B-vitamin) but this role does not appear to extend to humans. Since this does not extend to humans, the designation of PQQ as a vitamin compound has fallen through and it is only considered ‘vitamin-like’ at best.

PQQ seems to modify oxidation in a cell after binding to some proteins, and this modulatory role it plays can alter the signalling processes that go on in a cell. Due to PQQ being a REDOX agent (capable of both reducing and oxidizing) it is not a pure antioxidant, but it is involved in a cyclical antioxidative cycle with an antioxidant enzyme known as glutathione

For human evidence, the limited evidence we have right now suggests a possible neuroprotective role in the aged (no research in clinical situations of neurodegeneration nor in youth) and it may have an antiinflammatory role. This limited evidence also suggests that the main claim of PQQ, an enhancement of mitochondrial function, occurs in otherwise healthy humans given PQQ supplementation.

The animal evidence that might apply to humans (using oral supplementation at doses similar to what humans use) include a radioprotective effect, possible benefits to insulin resistance, and being a growth factor when PQQ is added to the diet over a long period of time. Higher than normal oral doses in rodents seem to also enhance peripheral neurogenesis (nerve growth outside of the brain) but not necessarily in the brain.

A large amount of the evidence for a direct antioxidant role or the neurological actions related to NMDA signalling of PQQ seem to use very high concentrations in cells, due to possible transportation issues to the brain and low concentrations of PQQ found in the blood following oral ingestion.

It holds a potential to modify signalling in humans, and although the oxidation in the blood (easiest thing to measure) in mostly unaffected it also retains the potential to act as an intracellular antioxidant. The enhancement of mitochondrial function may also occur, but beyond some alterations in signalling and the mitochondrial biogenesis most other properties of PQQ are unlikely to extend to humans.

Sources and Structure

1.1. Sources

Pyrroloquinoline quinone (PQQ) is a quinone molecule that was first identified as an enzymatic cofactor in bacteria, acting as a prosthetic group similar to how B-vitamins work in humans.^[1] It is doubtful that PQQ is an enzymatic cofactor in humans, although it still appears to have affinity to proteins in the human body and can bind to them to confer biolgical effects. The proteins that seem to bind to PQQ are called quinoproteins,^[2] and via modifying their actions in the body PQQ can exert biological activity.

PQQ was once thought to be a novel vitamin compound, although this view has since had doubts cast upon it and is no longer seen as accurate. Despite the lack of a vitamin role in mammals, it does appear to have growth promoting properties in rodents and may be active in humans following supplementation

PQQ naturally occurs in most foods (in miniscule amounts) although the highest levels can be found in:

Fermented Soybeans products such as Nattō (highest estimate of 61+/-31 ng/g wet weight,^[3] lower estimates in the range of 1.42 +/- 0.32ng/g^[4])
Green Soybeans (9.26+/-3.82ng/g wet weight)^[3]
Spinach (7.02 +/- 2.17ng/g fresh weight)^[4]
Rape blossoms (blossoms of the brassica napus plant at 5.44 +/- 0.8ng/g fresh weight)^[4]
Field Mustard (5.54 +/-1.50ng/g fresh weight)^[4]
Tofu (24.4+/-12.5ng/g wet weight)^[3]
Teas from Camellia Sinensis, aka Green Tea (around 30ng/g dry weight of leaves)^[3] with the lower range of estimates at 0.16 +/- 0.05^[4]
Green peppers, Parsely, and Kiwi fruits (around 30ng/g wet weight or so)^[3] although some estimates are lower (2.12 +/- 0.40ng/g for green peppers)^[4]
Human Breast milk at 140-180ng/mL (total PQQ and IPQ)^[5]

Overall content of PQQ in foods seems to range from 0.19-7.02ng/g fresh weight in one study^[4] up to 3.7-61ng/g in another,^[3] low numbers may not adequately reflect total content in foods due to excluding IPQ in the measurements whereas higher levels tend to include both PQQ and IPQ.^[5]

PQQ is present in a wide variety of foods, but currently the estimates of its contents are quite variable. This may be due to confusion as to whether solely PQQ should be counted or PQQ conjugates (it is not known if these confer dietary benefit). In general, the PQQ content of food products listed above is substantially lower than the content of supplemented PQQ (10-20mg) and food ingestion is unlikely to replicate the effects of supplementation due to the magnitude of difference

It should be noted that due to an affinity of PQQ to bind to amino acids and form imidazolopyrroloquinoline derivatives that the PQQ content of foods may not be the same as the total bioactive amounts of PQQ,^[6] probably due to rapid association with proteins forming amino acid conjugates (Imidazolopyrroloquinoline, or IPQ).^[7] Human milk, for example, contained 15% PQQ and 85% IPQ derivatives. That being said, no direct studies have been undertaken to see whether PQQ and IPQ have similar or different properties in vivo.

PQQ may form conjugates with dietary protein similar to how it is known to react with proteins in the body, but it is not known if this potential interaction with dietary protein is beneficial or negatively influences bioavailability

1.2. Structure and Properties

Pyrroloquinoline quinone is heat-stable and water soluble,^[1] and appears to be stable at ambient temperatures in the form of PQQ disodium salt either as trihydrate (12.7% water^[8]) or pentahydrate (22.9% water^[9]). It is thought to be a relatively stable REDOX factor in vivo, and is able to carry generally around 20,000 REDOX reactions before degradation,^[10]^[11] and when it carries out REDOX reactions by itself it gets converted into its reduced form known as pyrroloquinoline dihydroquinone (PQQH₂)^[12] and is replenished (back to the PQQ form) by glutathione.^[12]

PQQ binds to proteins via forming a schiff base, which is a spontaneous (no enzyme required) reaction to amino acids found in the protein structures such as lysine.^[13] The binding of PQQ to proteins uses the carbonyl groups (C=O),^[14]including the three carboxylic groups opposite of the two ketones used in REDOX reactions.

Pyrroloquinline quinone (PQQ) is a quinone structure with three carboxylic acid groups which are used to bind to proteins, and two ketone groups which are involved in the REDOX capacities of the molecule

In some in vitro studies, combining PQQ with reducing agents (SIN-1, sodium borohydride) can form a green precipitate^[15] and the reddish coloration of PQQ turns increasing brown when water content is removed.^[8]

PQQ (as a powder) appears to be able to change color depending on its hydration status and oxidation status

1.3. Biological Significance

PQQ was initially thought to be synthesized via the α-amino adipic acid-Δ-semialdehyde (AASDH; also known as U26) enzyme^[16] although this seems to be incorrect^[17]^[18] since despite this protein having many PQQ binding sites^[19] its mRNA levels are not negatively regulated by PQQ levels^[20] which would most likely occur if the enzyme synthesized PQQ. It is known to be synthesized (in bacteria) from the amino acids L-Tyrosine and glutamate^[21]^[22] in a process requiring a series of enzymes labelled PqqA-F where PqqA formed the peptide precursor and the other enzymes structurally modify it into active PQQ.^[23]

Although mammalian synthesis is not certain, PQQ does occur normally in the mammalian body^[24] and approxiamtely 100-400 nanograms of PQQ are thought to be made in humans each day;^[3]^[25] leading some authors to claim an estimated tissue concentration of approximately 0.8−5.9ng/g in humans.^[3]

Since complete deprivation from the diet of animals has been shown to hinder growth and reproductive performance,^[26]^[7] it was initially thought that this (paired with the initial guess of endogenous synthesis via AASDH) indicated a vitamin deficiency. However, due to the definition of vitamins being one that requires a disease state to occur during deficiency^[16] and no apparent dysfunction aside from impaired growth seen with PQQ deficeincy it was not classified as an essential vitamin;^[17] this claim of no vitamin-like property being supported by the idea that AASDH is not actually used for PQQ sythesis in humans.^[17]^[18]

Pyrroloquinoline quinone (PQQ) is known to occur in both the diet and in mammalian tissue, and appears to have biological activity in the body. It was initially thought to be a new vitamin, but this conclusion seems unlikely and it is more likely a bioactive non-vitamin compound.

PQQ has been investigated for being a growth factor in youth (since deprivation in rats impairs growth^[26]^[7]), secondary to its effects at improving mitochondrial biogenesis (making more mitochondria) at seemingly effective doses of 0.2-0.3mg/kg foodstuff (in mice),^[27] which is surprisingly close to the levels found in human breast milk.^[5] Preliminary evidence for mitochondrial efficacy has also been noted in adult humans given 0.075-0.3mg/kg daily,^[28] with the latter dose being close to the recommended 20mg serving for a 150lb adult.

PQQ is thought to be a non-vitamin growth factor, in part due to its naturally high levels in breast milk and reduced growth in rats without dietary PQQ. It may do so via beneficially influencing mitochondrial function

It is seen as a novel REDOX catalyzing agent due to its stability, which prevents most self-oxidation (seen in catechins) and polymerization (tannins).^[10] A case has been made that PQQs effects are constant between species and bacteria, which aims to validate extrapolation from one species to humans.^[10] The potency of PQQ and its quinoproteins in REDOX cycling appears to be approximately 100-fold greater than Vitamin C or other polyphenolic compounds, when in alkaline conditions.^[25]^[29]^[30]

PQQ, after associating with proteins (not in the role of a cofactor) appears to be capable of REDOX cycling suggesting that it can have conditional prooxidant and antioxidant roles. The association with proteins suggests that it can modify their structures either directly or via modifying the levels of oxidation at the level of the protein (similar to how carotenoids such as Astaxanthin are located at the cellular membrane which localizes their effects)

Molecular Targets

2.1. Enzymatic Cofactor

Pyrroloquinoline quinone (PQQ) was discovered in 1979 as an enymatic cofactor in bacteria;^[31] preliminary evidence in pig kidneys and adrenal glands suggested a similar role in mammals.^[32]^[33]^[34]^[35] Doubts were later cast upon the role of PQQ as a mammalian enzymatic cofactor,^[36]^[37]^[38] and currently the consensus is that PQQ is unlikely to be an enzymatic cofactor in humans as it is in bacteria and plants.

Pyrroloquinoline quinone (PQQ) was first discovered as a bacterial enzymatic cofactor (being required by bacterial enzymes to function properly) and preliminary evidence suggested it could play the same role in mammals, which would make PQQ a vitamin. But further study found no quality evidence supporting this role in mammals; it is currently believed that PQQ does not act as an enzymatic cofactor in humans

2.2. REDOX Signalling

REDOX (REDuction OXidation) signalling refers to stimulation or inhibition of cellular signalling systems by molecules that can switch from an oxidized state to a reduced state, such as the well-known REDOX-acting supplements Vitamin C andAlpha-Lipoic Acid.^[39] Pyrroloquinoline quinone (PQQ) may have this property as well, although its primary mode of action seems to be acting on known REDOX proteins in the cell; this is in line with its high binding affinity for some proteins, despite not acting as their coenzyme.^[40]^[21] For example, PQQ may function as a mammalian growth factor via signal transduction modification by both oxidation and redox cycling^[41], and has been shown to improve insulin signalling in mice by redox cycling.^[42]

PQQ may have an indirect influence on REDOX signalling in a cell by modifying the actions of proteins, which may underlie some antioxidative (and prooxidative) changes in a cell similar to any other REDOX agent

2.3. Thioredoxin Reductase 1

PQQ has been noted to partially inhibit thioredoxin reductase 1 (TrxR1), which is an enzyme in the cytosol that reducesthioredoxin.^[43] PQQ has low potency yet high affinity in binding to TrxR1 and seems to outcompete thioredoxin binding.^[44] When PQQ binds to TrxR1, the enzyme’s activity is modified so it acts more on an alternate substrate known as juglone.^[45] Overall, NADPH oxidase activity of TrxR1 (a measure of the activity of this enzyme) is increased in the presence of 10-50µM PQQ due to increased activity of the TrxR1-Juglone interaction.^[45]

Pyrroloquinoline quinone (PQQ) binds to an antioxidant enzyme (TrxR1) and alters its function, reducing its affinity towards its normal substrate and increasing its affinity towards an alternate substrate. Overall activity of this enzyme appears to be enhanced at high concentrations of PQQ, but the effect of more physiologically realistic (nanomolar) concentrations are not known

Inhibition of TrxR1 activity is known to cause an increase in the activity of the Nrf2 protein, which acts on the nucleus (via the antioxidant response element or ARE) to increase antioxidant gene expression.^[46]^[47]^[48] Since oral supplementation of PQQ appears to influence a large amount of genes under control of TrxR1-related transcripts^[49] it is thought that TrxR1 inhibition by PQQ occurs in vivo.^[49]

It is thought that PQQ inhibits thioredoxin reductase (TrxR1) when ingested orally, since genes that would normally be activated when TrxR1 is inhibited do seem to be activated with PQQ in rats

2.4. Glutathione Reductase

PQQ has also been shown to inhibit glutathione reductase, but despite a decreased K_M towards juglone (which would increase NAPDH oxidation and enzyme activity) the K_cat was also reduced and enzyme activity remains similar with or without PQQ.^[45] However, GSSG reduction with 5µM PQQ was reduced approximately 2-fold relative to control.^[45]

An inhibitory effect has been noted in regards to glutathione reductase as well, although the practical significance of this particular enzyme interaction is not known

2.5. Mitochondrial Biogenesis

In rats, PQQ depletion is known to influence genetic expression (238 out of 10,000 tested genes) and dietary repletion is known to influence 847 transcripts;^[49] of these, the major pathways affected include Thioredoxin and MAPK signalling but also PGC-1α, a positive regulator of mitochondrial biogenesis^[50]).^[49] PQQ activates PGC-1α via CREB phosphorylation^[51]and appears to positively regulate mitochondrial biogenesis in vivo. It also has other possible roles in blood pressure regulation, cellular cholesterol homeostasis, energy production, and protection of mitochondrial activity, all of which are beneficially associated with increased PGC-1α activity^[10]^[50]).

When studies are undertaken in rats comparing a PQQ deficient diet, in which the rats must rely solely on de novobiogenesis of PQQ) against PQQ sufficient diets, the PQQ supplemented diets tend to promote up to 20-30% more mitochondria in the liver (on a mass basis, as assessed by mtDNA) over the rats’ lifetime.^[27]^[26]^[10]^[52]^[7]^[49]^[51] Decreased permeability of the mitochondrial membrane has also been noted without alterations in functional capacity or mitochondrial size,^[26] along with the mitochondrial count per cell increasing 60% from 56.8+/-7.8 to 91+/-6.6 with 2mg/kg PQQ fed by gavage starting from 2 weeks of age in rats on a PQQ deficient diet.^[26]

Pyrroloquinoline quinone (PQQ) appears to be capable to increasing the activity of PGC-1α, which then promotes mitochondrial proliferation and membrane stabilization. This occurs in rats using oral doses similar to those in humans, and occurs secondary to CREB phosphoylation; this may suggest bioenergetic benefits of supplementation, but human evidence does not yet exist

When humans supplement PQQ (0.075-0.3mg/kg for one week at a time for each dose), urinary lactate decreased by 15% along with a reduction in urinary pyruvic acid.^[28] A minor reduction of fumarate was noted, but other Kreb’s cycle intermediates (Isoaconitate, Citric acid, 2-oxoglutarate, and succinate) were not altered in the urine.^[28] It was hypothesized, on the assumption that urinary metabolites reflect cellular energy status, that this indicated an increase in mitochondrial efficiency.^[53]^[54]

A nonsignificant decreasing trend in urinary 4-hydroxyphenylacetate was noted with PQQ;^[28] decreases in this and other urinary metabolites tend to suggest increased β-oxidation rates.^[55]

The currently lone human study using doses of PQQ commonly found in supplements suggest that supplementation may increase mitochondrial efficiency

2.6. PTP1B

Pyrroloquinoline quinone (PQQ) is known to enhance signalling of some MAPK proteins, most notably ERK1/2, to significant extents, rivalling its effects on thioredoxin and PGC-1α.^[49]^[56] This may be secondary to oxidative changes on the PTP1B protein; the changes occur when PQQ facilitates the production of hydrogen peroxide by associating with other proteins^[57]) within a cell via direct REDOX cycling.^[41] Hydrogen peroxide then modifies PTP1B on Cys-215.^[58] The change of Cys-215 from a sulfenic acid moiety (-SOH) into a more oxidized sulfinic acid (–SO2H) or sulfonic acid (–SO3H) causes reversible inhibition of PTP1B.^[59]^[60]

PTP1B is a negative regulator of the insulin receptor,^[61] and is also a negative regulator of the epidermal growth factor receptor (EGFR).^[58] By alleviating a negative inhibition, PQQ (via H₂O₂) can enhance signalling through the EGFR resulting in more ERK1/2 activation.

By acting as a direct REDOX couple, PQQ can inhibit PTP1B activity via hydrogen peroxide production within a cell. This inhibition of PTP1B enhances growth factor signalling (via EGFR signalling) and can enhance insulin sensitivity in a cell (by enhancing insulin receptor signalling)

Pharmacology

3.1. Absorption

PQQ is absorbed well in the intestines, but its absorption is highly variable; 62% of PQQ is absorbed on average in rats in a fed state, with a range from 19-89%.^[62]

3.2. Serum

A single dose (0.2mg/kg) of PQQ ingested by humans in a fruit-flavored drink has a t_max of about two hours and a C_max of approximately 9nM.^[28] Doubling PQQ dose from 0.075mg/kg PQQ daily for one week to 0.15mg/kg and then 0.3mg/kg in healthy subjects increased plasma PQQ levels in a linear manner. Fasting blood levels of PQQ ranged from 2 to 14nM when measurements were taken on day four of supplementation.^[28] These levels may be similar to the steady state values as they were measured after the fourth day of dosing on the morning after PQQ was ingested.^[28]

Daily supplementation of pyrroloquinoline quinone (PQQ) appears to increase plasma PQQ concentrations to a steady state level of around 10nM in humans

3.3. Distribution

PQQ appears to be eliminated from mice 24 hours after ingestion except in the skin and kidneys, which retain detectable levels of PQQ following oral ingestion.^[62] In the skin, it was noted that 0.3% of the ingested dose was detectable six hours following a dose and 1.3% of the oral dose was detected after 24 hours. Greater than 95% of the PQQ in the blood seems to be associated with the blood cell fraction, with less than 5% remaining in the plasma fraction.^[62]

3.4. Elimination

86% of an ingested dose of PQQ in mice appears to be eliminated via the kidneys within 24 hours of oral ingestion^[62] and is excreted in a manner directly correlated with serum levels in humans;^[28] in humans, less than 0.1% of the ingested dose is detected as unmodified PQQ, suggesting that PQQ is highly metabolized prior to elimination.^[28]

3.5. Mineral Bioaccumulation

Pyrroloquinoline quinone has been noted to bind directly to metals such as uranium. This explains the toxicity of uranium to bacteria, which depend on PQQ as a cofactor for enzymes;^[63] uranium displaces a calcium ion which is required to bind PQQ to certain enzymes in bacteria.^[64]^[65]

Pyrroloquinoline quinone has a known affinity for some minerals, but the role of PQQ in the human body in regards to minerals is not known. It is unlikely to play a role in heavy mineral elimination due to the very low serum concentrations of PQQ

Interactions with Neurology

4.1. Glutaminergic Neurotransmission

The NMDA receptor possesses a sulfhydryl REDOX modulatory site that is susceptible to oxidation^[66] where oxidation suppresses NMDA signalling and reduction enhances NMDA signalling.^[67]^[68] PQQ (50µM) does not affect basal currents through the receptor, but it can block reducing agents from enhancing signalling^[69]^[70] in the 5-200µM range. The reduction of signalling is thought to be due to acting on the REDOX site, since PQQ can reduce excitotoxicity but fails to protect from H₂O₂ (which causes toxicity independent of the NMDA receptor).^[71]

This mechanism is thought to underlie protective benefits of PQQ supplementation^[70] seen at low concentrations of 5µM (other mechanisms require PQQ concentrations of up to 50µM in order to become appreciable).^[71]

PQQ appears to have a regulatory effect on the glutamate receptor known as NMDA, by causing some oxidation of the REDOX site and preventing excess reduction from occurring it can suppress abnormal spikes in NMDA signalling; since an excess of NMDA signalling can be toxic, the result is a neuroprotective effect. This is thought to be applicable to oral supplementation due to a low concentration being required

4.2. Neuroprotection

100µM PQQ has been noted to protect cells from glutamate-induced cytotoxicity^[72]^[73] associated with an increase in antioxidant enzyme activity, as assessed by Nrf2 and HO-1.^[72] This is thought to be downstream of Akt/PI3K and GSK-3β activation,^[74] of which the former is known to occur with PQQ in the 50-100µM range in vitro.^[74]

PQQ also appears to prevent an increase in JNK signalling seen with NMDA-mediated toxicity, but it is not related to the protective effects on cellular survival^[74] and PI3K activation cannot fully predict the protective effects of PQQ.^[72]

PQQ appears to be related to an activation of PI3K/Akt signalling, which is known to cause an induction in antioxidant enzymes via Nrf2. This is thought to underlie some of the protective benefits of PQQ on cellular structure seen in vitro, but its significance to oral supplementation is not known

Protective effects against glutamate have been noted when PQQ is directly injected into the brain in a manner that is associated with the aforementioned antioxidant effects (PI3K activation and Nrf2/HO-1 induction).^[73]

Injections of PQQ into the brain are known to be neuroprotective, but it is not known if this applies to oral ingestion as well

4.3. Neurogenesis

In fibroblastic cells (L-M), incubation of PQQ disodium salt (approximately 100µg/mL) for 24 hours has resulted in a peak 40-fold increase in Nerve Growth Factor (NGF) synthesis, with minor (around 5 to 10-fold) increases at 10-20µg/mL^[75]^[76]in a manner dependent on COX2 induction^[77] and PI3K/Akt.^[78] Prostaglandins D2 and E2 (from Arachidonic acid) have been reported in vitro,^[77] and while they were not tested as a mandatory intermediate the former (and its metabolite prostaglandin J2) are known to promote NGF synthesis in the 6.3-25µg/mL range^[77] via CHRT2^[79] extending to a variety of cell lines.^[80]^[81]^[82]

This increase in NGF synthesis has also been noted in isolated mouse astrocytes exceeding Alpha-Lipoic Acid (ALA) in potency, but less than ALA in c/3T3 (embyotic fibroblast) cells.^[83]

When tested in vitro, PQQ appears to concentration-dependently increase NGF synthesis up to a peak efficacy at 100µg/mL. The increase noted in isolated cells appears to be quite large. Eicosanoid signalling appears to be involved in this phenomena, suggesting that PQQ works via manipulating the actions of eicosanoids

When fat-soluble derivatives were tested (PQQ trimethyl esters) at injections of 0.1-1mg/kg every other day, it was noted that peripheral sciatic nerves had enhanced regeneration;^[75] injections into the periphery failed to cause an increase in NGF in the neocortex, thought to be due to poor diffusion of PQQ across the blood brain barrier due to complexation with proteins in serum.^[75] A pharmaceutical modification of the PQQ enzyme (oxapyrroloquinoline; OPQ) was able to enhance brain NGF concentrations,^[75] and since OPQ is known to be metabolized into PQQ in bacteria (hypothesized to occur in rodents) and is fat soluble it was thought to act as a prodrug.

When tested later, PQQ added to silicon tubes confirmed an increase in the rate of physical recovery in a mouse model of physical nerve injury with benefits seen after four weeks extending to twelve weeks.^[84] This improvement was associated with an increase in well-myelinated neurons.^[84]

In a spinal cord injury model, 5mg/kg PQQ injected into the spine daily for a week after injury was able to suppress the expression of iNOS after one day (a biomarker for inflammation^[85]^[86]) and improved both locomotor performance and neuronal health (axonal density) in the area relative to control.^[87] Benefits to peripheral nerve function (in a rat model of sciatic nerve injury) have been noted orally; a low dose (20mg/kg) prevented hyperalgesia from the nerve injury while only the higher dose (40mg/kg) prevented muscular atrophy and lipid peroxidation.^[88]

The enhancement of neurogenesis has been noted in the periphery (tissue excluding the brain) with injections of low doses of PQQ, but an increase in neurogenesis in the brain has failed to be noted which is thought to be due to transportation issues to the brain. While there are no oral studies in rodents yet, PQQ has been noted to enhance peripheral neurogenesis following nerve injuries

4.4. Neurooxidation

As mentioned in the glutaminergic section, the oxidative effects of PQQ on the NMDA modulatory site^[69]^[70] can ultimately cause a reduction in NMDA-induced superoxide formation in the neuron^[71] at concentrations (5uM) that do not affect oxidation per se (no effect against hydrogen peroxide which circumvents the receptor).^[71]

The anti-glutaminergic effects that occur at lower concentrations may also ultimately cause anti-oxidative effects by suppressing NMDA signalling, despite this mechanism being reliant on the pro-oxidant effects of PQQ

PQQ does not appear to influence the toxicity of peroxynitrate (a combination of nitric oxide and the superoxide radical), despite inhibiting its formation.^[89] When using SIN-1 as a way to produce peroxynitrate and induce cell death in vitro, PQQ at 100uM abolished cell death prior to peroxynitrate formation with an EC₅₀ of 15+/-8.4uM, yet actually potentiated pre-existing peroxynitrate toxicity (also seen with superoxide dismutase, an anti-oxidant enzyme, when catalase was not present).^[15] The mechanism appears to be through sequestering the superoxide radical without significantly influencing nitric oxide, as PQQ does not appear to modify many parameters of nitric oxide or peroxynitrate per se yet potentiated a SIN-1 induction of cGMP and production of nitrate, theoretically caused by a backlog of nitric oxide that could not convert to peroxynitrate due to less free superoxide radicals.^[15] Interactions with PQQ and superoxide radicals has been noted previously.^[90]^[91]

Can prevent superoxide radical induced cell death, but does not significantly influence nitric oxide cell death per se

4.5. Epilepsy and Convulsions

NMDA receptors are involved in the pathology of seizures (as seizures are involved with excessive NMDA signalling^[92]^[93]) and the REDOX modulatory site that PQQ is known to interact with (suppressing high levels of activity) is further implicated^[94] since seizures are associated with a high level of reducing agents in the brain^[95]^[96] which can act upon that site to promote increased NDMA signalling;^[94] it is thought that PQQ could have a therapeutic role (seen with pharmaceutical NMDA antagonist^[97]^[98]) since by its oxidative role it hinders this particular site on NMDA receptors^[69]^[70]and PQQ is thought to not associated with side-effects from excess suppression due to only suppressing high levels of NMDA signalling but not basal levels.

When seizures occur, they are potentiated by excessive signalling through the NMDA receptors and due to this NMDA receptor antagonists (or anything that can suppress excess signalling) are thought to be therapeutic. Since PQQ has been implicated in suppressing excess NMDA signalling, it is being investigated for anti-epileptic effects

Application of 200µM PQQ to isolated neurons undergoing epileptic activity can fully abolish such activity if induced by reducing agents (no effect on epileptic activity induced by other means),^[94] supporting the role PQQ plays in epilepsy via NMDA antagonism which may occurs to limited levels at concentrations as low as 5µM.^[71]

In vitro evidence support a role for PQQ, but due to quite high concentrations being used (relative to what is seen in the blood) and a hypothesized low transportation to the brain it is not sure if this will occur in a living organism following oral ingestion

4.6. Hypoxia and Stroke

Pyrroloquinoline quinone (PQQ) appears to have protective effects against ischemia (assessed by infart size) when 10mg/kg is injected either 30 minutes prior to ischemia (reducing the infarct size from a 95+/-3.6% increase to 68.8+/-10.4%)^[99] and is slightly less effective when injected immediately after rather than preloaded (37.6% reduction seen previously reduced to 18.5%).^[99] This has been replicated elsewhere with 3-10mg/kg (70-81% protection) but not 1mg/kg was given an hour after MCAO injury.^[100]

Injections of PQQ have been noted to have protective effects in rats subject to stroke, but due to high injection doses being used and the low dose being ineffective preliminary evidence does not appear to look promising for oral supplementation of PQQ in this role; oral testing, however, has not yet been conducted

4.7. Brain Injury

Injections (intraperitoneal) of PQQ in the range of 5-10mg/kg to rats for three days prior to tramautic brain injury was able to dose-dependently protect the brain from injury with the highest dose appearing to confer absolute protection (assessed by histology and cognitive behaviour post-injury).^[101]

4.8. Memory and Learning

When injected into rats at 10mg/kg bodyweight, PQQ does not appear to cause overt behavioural changes in regards to sedation, activty, or heart rate^[99] with no alterations in EEG readings being observed.^[99]

Several morphological changes are associated with PQQ that may confer pro-cognitive effects, such as proliferation of Schwann cells secondary to PI3K/Akt activation,^[78] PQQ is also able to induce production of Nerve Growth Factor (NGF)^[76] secondary to COX induction;^[77] increases in NGF have been observed in vivo when using trimethylesters (for permeability into the brain) with a maximal increase of 1.7-fold over baseline associated with a PQQ metabolite named oxazopyrroloquinoline.^[75]

PQQ supplementation has also been associated with preventing stress-associated (oxidative stress mediated) declines in memory^[102] reducing damage done by methylmercury toxicity,^[103]^[104] and reducing memory impairment induced by a lack of oxygen;^[105] at 20mg/kg bodyweight PQQ has a potency nonsignificantly different than 200mg/kg Vitamin E (as R-R-R-Alpha tocopherol) in reversing age-related memory decline in rats.^[105] which, together with its neuroprotective status, assure it a position as a rehabilitative Nootropic.

Currently, one study has been conducted in humans using PQQ at 20mg daily or using PQQ at 20mg paired with 300mgCoQ10.^[106] This study used the supplements once-daily at breakfast for 12 weeks in persons aged 51.7-52.3yrs with the three tests being a Verbal Memory test (seven words read aloud and then asked to recite), the Stroop Test, and the CogHealth test. The results suggested a tendency towards improvement in the Verbal memory test (nonsignificant) a significant increase in performance in the Stroop test with PQQ+CoQ10 but not PQQ in isolation, and the choice reaction and simple reactions subsets of the CogHealth test showed statistically significant improvements with PQQ and PQQ+CoQ10 but the degree of improvement was not recorded.^[106]

General nootropic benefit for those with impaired cognitive function (due to age, neural damage, etc.) but does not have ample evidence to be claimed a cognition promoting nootropic in otherwise healthy. The one study conducted in humans does not claim a 50% or doubling of memory, and was not suited to answer this question

4.9. Sedation

One open-label human study conducted with 20mg PQQ for 8 weeks in 17 persons with fatigue or sleep impairing disorder noted that PQQ was able to significantly improve sleep quality, with improvements in sleep duration and quality appearing at the first testing period 4 weeks after usage while a decrease in sleep latency required 8 weeks to reach significance.^[107] This study also noted improved appetite, obsession, and pain ratings that may have been secondary to improved sleep; contentness with life trended toward significance over 8 weeks but did not reach.^[107]

Cardiovascular Health

5.1. Cardiac Tissue

Protective effects have been noted in cardiac myocytes subject to ischemia, secondary to scavenging of peroxynitrate radicals, at injectible doses of 15mg/kg bodyweight 30 minutes prior to ischemia.^[108]^[109] PQQ was studied alongside metprolol as a combiantion anti-oxidant/beta-blocker therapy, and 3mg/kg PQQ and 1mg/kg metprolol were both insignificantly different in reducing mortality (40% of control passed, 8% of PQQ and 14% of metprolol) while no deaths were recorded in combination therapy.^[110] Combination was also more effective in reducing infarct size relative to either therapy in isolation, and both groups using PQQ had a reduction of creatine kinase release that was insignificantly different between groups.^[110]

The combination therapy study noted increased cardiac mitochondrial respiration with PQQ but neither metprolol nor PQQ+metprolol, and respiration was further increased even in the contrl groups with no ischemia/reperfusion done.^[110]

Secondary to the pro-mitochondrial effects and anti-oxidative effects during ischemia/reperfusion, PQQ appears to be cardioprotective under certain contexts

5.2. Atherosclerosis

In otherwise healthy humans supplementing PQQ at 0.075-0.3mg/kg for three weeks (increasing the dose each week), supplementation was associated with a decrease in C-reactive protein concentrations in serum (45%).^[28] This study also noted that urinary trimethylamine-N-oxide (TMAO) was reduced^[28] and since both C-reactive protein (CRP)^[111] and TMAO^[112] are thought to be biomarkers for atherosclerosis PQQ is thought to have a role.

5.3. Triglycerides

In rats fed a PQQ deficient diet relative to the same diet fed with 2mg/kg PQQ, plasma diglycerides and triglycerides (DAG and TAG) were elevated 20-50% (higher value related to triglycerides) in the PQQ deficient diet relative to 2mg/kg with no significant difference in free fatty acids,^[27] which is similar to levels previously seen with this experimental protocol.^[26] The elevation of triglycerides in the deficient mice does not influence the n3/n6 omega fatty acid ratios.^[27]

The increase seen in triglycerides may be due to this study being conducted for a long period of time, where previous research has demonstrated that PQQ deficient diets reduce mitochondrial density by 20-30%^[26] and levels of mRNA for PPAR, Fatty Acid binding protein, and Acyl CoA oxidase being significantly reduced with PQQ deficiency.^[27] Additionally, higher levels of beta-hydroxybutryic acid (indicative of less beta-oxidation) were seen in PQQ deficient rats. Inducing PQQ deficiency from a sufficient state can also elevate triglyceride levels to almost two-fold the previous levels, with the trend being reversed upon acute administration of PQQ in pharmacological amounts (2mg/kg bodyweight).^[49]

Appears to reduce triglycerides very potently (to a greater extent than Fish Oil, empirically) in research animals relative to a PQQ deficient diet, and this is thought to be due to increased mitochondrial β-oxidation of fatty acids

The one human study to use supplemental PQQ (0.075-0.3mg/kg for three weeks in escalating doses) failed to find any significant influence on triglyceride concentrations in serum of otherwise healthy adults consuming a standard (but uncontrolled) diet.^[28] This study also noted alterations in urinary metabolites (4-Hydroxyphenylacetate and 4-Hydroxyphenylactate) suggestive of an increase in mitochondrial β-oxidation despite no apparent changes in triglycerides.^[28]

First study to assess the effects of PQQ on triglycerides has failed to find an influence in otherwise healthy humans

Interactions with Glucose Metabolism

6.1. Glucose Deposition

PQQ (500nM) has been noted to inhibit protein tyrosine phosphatase 1B (PTP1B) secondary to producing H₂O₂^[41] (H₂O₂is known to inactivate PTP1B in a reversible manner^[58]), and aside from PTP1B being a negative regulator of a growth factor receptor (EGFR^[58]) it also negatively influences insulin receptor signalling;^[61] inhibition of PTP1B, seen also withBerberine and Ursolic Acid (albeit by different mechanisms), tends to increase the activity of the insulin receptor.

Sequestering the hydrogen peroxide made from PQQ appears to block its inhibition on PTP1B.^[41]

Via prooxidative changes within a cell, PQQ can produce hydrogen peroxide which then impairs PTP1B function. Since PTP1B normally suppresses signalling via the insulin receptor, the result is a compensatory increase in insulin signalling

6.2. Serum Glucose

In young rats (before sexual maturation), PQQ either at 3mg/kg in the diet or having a PQQ deficient diet does not seem to significantly affect blood glucose or insulin levels.^[27] An increased glucose AUC was seen when PQQ deficient mice were subject to an oral glucose tolerance test, but no single time point was significnatly different.^[27] Injections of PQQ at 4.5mg/kg bodyweight also did not significantly influence blood sugar or insulin levels in healthy rats, but was able to significantly reduce glucose AUC (by 7%) and glucose disposition in diabetic rats fed glucose and injected with PQQ, with no effect of PQQ on fasting glucose levels in rats.^[27]

6.3. Insulin resistance

It has potential for alleviating fat-induced insulin resistance (characterized by a dysregulation in beta-oxidation of the TCA cycle) by increasing mitochondrial biogenesis in muscle cells, similar to exercise.^[113]

At this moment in time, nothing remarkable about PQQ and glucose metabolism

Interactions with Obesity

7.1. Metabolic Rate

When comparing a rat diet deemed sufficient in dietary pyrroloquinoline quinone (PQQ; 2mg/kg) to a diet deficient in one, the deficient diet appeared to have a decreased metabolic rate (reaching only 90% of the control rats)^[27] with the difference being more prominent during the fed rather than fasted state;^[27] it appears that this decreased metabolic rate did not influence the rats of lipolysis nor glycolysis as assessed by the respiratory quotient.^[27]

Depleting the rat diet of PQQ appears to reduce their metabolic rates relative to a diet with adequate levels of PQQ, but no studies have investigated whether an increase in metabolic rate occurs with extra supplemental PQQ

Bone and Joint Health

8.1. Osteoclasts

Pyrroloquinoline quinone (PQQ) has been noted to inhibit RANKL-induced osteclast formation in RAW 264.7 macrophage-like cells at a concentration of 10µM, which occurred at all stages of cell maturation.^[114]

RANKL normally signals through the transcription factor NFATc1^[115]^[116] via a particular AP-1 signalling protein that contains c-Fos and c-Jun.^[117]^[118] PQQ inhibited c-Fos induction from RANKL,^[114] but other RANKL-induced proteins (NF-kB and MAPKs) were unaffected suggesting that RANKL signalling overall was unaffected.^[114]

There is a negative regulatory pathway from RANKL, where RANKL increases IFN-β production which signals via its receptor (IFNAR^[119]) to activate STAT1 and JAK1 to suppress the actions of RANKL.^[120]^[121] IFN-β was not affected by PQQ, but the receptor expression (and its targets) appeared to be increased which were thought to underlie the observed inhibitory effects seen with PQQ.^[114]

PQQ appears to enhance the negative feedback mechanism controlling osteoclastogenesis (production of osteoclasts, which are negative regulators of bone mass) and via this enhancement overall osteoclast activity is hindered somewhat and this is thought to promote bone mass over time. Due to a higher than normal concentration being used, it is not sure if this occurs following oral supplementation

Skeletal Muscle and Physical Performance

9.1. Mechanisms

One study using 0.075-0.3mg/kg PQQ supplementation daily for three weeks (increasing with dose each week) in otherwise healthy adults has noted a decrease in overall urinary amino acid levels by approximately 15%,^[28] with the decrease in some (serine, asparangine, aspartic acid) being biomarkers for skeletal muscle consumption of nitrogen (via being converted into Glutamine and alanine^[28]^[122]).

Preliminary evidence suggests that oral PQQ supplementation can influence skeletal muscle metabolism in otherwise healthy humans with standard supplemental doses, but the practical significance of this is not yet known

Immunology and Inflammation

10.1. Mechanisms

PQQ appears to have some interactions with the immune system, as deprivation of PPQ from the diet (relative to a PQQ sufficient diet) appears to cause abnormal immune function in mice, with altered immune response after stressors.^[52]^[7]

A study on parental (intravenous) nutrition found that the addition of 3mcg PQQ to the parental nutrition in mice was able to increase the count of CD8+ cells and lymphocytes in intestinal Peyer’s Patches, although not to the level of oral control.^[123]

10.2. Macrophages

Application of PQQ to macrophages in vitro was able to prevent osteoclast differentiation at doses as low as 0.1uM (but more potency at 10uM) secondary to increasing IFN-β secretion; IFNβ is a negative regulator of osteoclast differentiation normally released after inflammation, and PQQ increases its release (and subsequent suppression), which is also demonstrated by increased levels of proteins induced by IFN-β (iNOS, STAT1, JAK1).^[114] PQQ was found to phosphorylate NF-kB, p38, and IKKβ in these cells which is a pro-inflammatory response in macrophages.^[114]

Practical relevance unknown

Interactions with Oxidation

11.1. Singlet Oxygen

The reduced form of pyrroloquinoline quinone (PQQ), known as pyrroloquinoline equinol or dihydroquinone pyrroloquinoline (PQQH₂) appears to be able to sequester singlet oxygen (¹O₂) with a potency 6.4-fold less than β-carotene as reference yet higher than that of Vitamin E (2.2-fold) and Vitamin C (6.3-fold).^[12]

PQQH₂ appears to be produced (via reduction) from PQQ when in a buffer in the presence of glutathione^[12] and this process is known to use the semiquinone (PQQH) as an intermediate;^[57] exposure to oxygen either by ambient atmosphere or by singlet oxygen readily oxidizes PQQH₂ back into PQQ.^[12] This suggests that glutathione is capable of recycling PQQ as an antioxidant.

PQQ and its reduced form PQQH₂ appear to form a cyclical relationship where PQQH₂ sequesters oxygen radicals, and glutathione reduces it back into PQQ so it may sequester more radicals; the potency of this reaction, on a molecular level, seems intermediate to β-carotene (PQQ is lesser) and Vitamin C/E (greater)

11.2. Reactive Nitrogen Species

One study assessing whether PQQ could directly sequester peroxynitrate (ONOO^–) failed to find such a property of PQQ, as despite protecting cells form the toxic effects of SIN-1 (produces nitric oxide and superoxide radicals,^[124] of which PQQ scavenged the superoxide radicals^[15]) the toxicity of peroxynitrate directly was not protected against (in fact, it appeared to be augmented at 100-300µM PQQ).^[15]

Pyrroloquinoline quinone (PQQ), even at impractically high concentrations, does not appear to direct sequester reactive nitrogen species (nitrogen based pro-oxidants) such as peroxynitrate

11.3. Lipid Peroxidation

One human study using supplemental pyrroloquinoline quinone (PQQ) and measuring serum antioxidant capacity via TBARS and TRAP values failed to find any significant influence on TRAP values but noted a decrease in TBARS (indicative of lipid peroxidation) to the degree of 0.2% when measured at peak serum PQQ values (6-12nM) seen with up to 300µg/kg supplementation;^[28] this decrease in TBARS was noted to be significantly less than other dietary supplements such as procyanidins from Cocoa Extract which (560mg) can reduce TBARS by 25-35%^[125] or sources of anthocyanins such as Aronia melanocarpa or Blueberry.

The decrease in serum biomarkers of lipid peroxidation that is known with PQQ supplementation is probably much too low to be indicative of anything significant

11.4. Radiation

Oral ingestion of 4mg/kg PQQ to mice (more effective than both 2mg/kg and 8mg/kg, as well as the reference drug of 10mg/kg nilestriol^[126]) appears to reduce death from gamma irradiation when given an hour before and again seven days after irradiation; damage to select cells tested (white blood cells, reticulocytes, bone marrow cells) was also reduced with 4mg/kg PQQ supplementation to mice.^[126]

Oral ingestion of PQQ (estimated human equivalent of 0.32mg/kg) appears to be able to protect mice from gamma irradiation to a respectable degree

Peripheral Organ Systems

12.1. Liver

An intraperitoneal injection of pyrroloquinoline quinone (PQQ) to rats at 5mg/kg twice before CCl₄ liver toxicity appeared to exert protective effects;^[127] when tested in vitro, PQQ showed protective effects in isolated liver cells with most potency at 3µM.^[127]

12.2. Intestines

Due to the involvement of pyrroloquinoline quinone (PQQ) in bacteria (from where it was discovered in 1979^[31]) and the involvement of quinoproteins in the fermentation process ^[128] (which PQQ associates with) and the above higher count of PQQ recorded in fermented foods; it is hypothesized that fermentation may increase PQQ content. Interestingly, common strains of bacteria in the human intestinal tract do not appear to synthesis much PQQ^[129]^[130] and in antibiotic fed mice (lacking intestinal microflora) it seems that dietary intake is the major determinent of bodily PQQ levels.^[130]

Pyrroloquinoline quinone was thought to be synthesized by intestinal bacteria due to its discovery being that of a bacterial cofactor, but preliminary evidence does not support the intestinal microflora as a major producer of PQQ in the body

12.3. Kidney

Pyrroloquinoline quinone (PQQ) was once implicated in being an enzymatic cofacter for diamine oxidase (pig kidney)^[32]^[33]and DOPA decarboxylase (pig kidney)^[34] (as well as dopamine β-hydroxylase, albeit in the renal medulla^[35]), although it is generally accepted to not be a significant component of eukaryotic enzymes in vivo (in the role of a cofactor) like it is in bacterial and plant enzymes.^[36]^[37]^[38] Still, it is detectable in the kidney after oral ingestion in the rat^[62] and elimination of PQQ is primarily via the urine^[62] suggesting it may still play a role independent of being an enzymatic cofactor.

PQQ is not thought to play a role as a cofactor of enzymes in the kidneys like initially thought, but due to being eliminated by the kidneys and accumulating in them following oral ingestion in the rat it is still thought to play a role (perhaps as a REDOX couplet, like other mechanisms)

Interactions with Cancer

13.1. Leukemia

PQQ has been shown to be cytotoxic to U937 leukemia cells, but not NIH3T3 nor L929 cells, in a dose-dependent manner.^[131] Catalase treatment neutralized these effects, as they appear to be secondary to hydrogen peroxide production in cells which PQQ has been repeatedly shown to induce.^[132] Superoxide dismutase had no effect on PQQ cytotoxicity, while glutathione or N-AcetylCysteine increased cytotoxicity 2-5fold without affecting the cells on their own (and thus working via PQQ by increasing H₂O₂ production form PQQ 1.5-2fold).^[131] PQQ by itself decreased intracellular glutathione levels, and when glutathione was depleted (via BSO, an inhibitor of γ-glutamylcysteine synthetase) the apoptosis of cells morphed into necrosis, and this necrosis was still mediated by H₂O₂ due to being inhibited by catalase.^[131]

Induces cell death via H₂O₂, and uses glutathoine to produce even more H₂O₂ to augment its efficacy. A depletion of glutathione induces necrosis

13.2. Melanoma

PQQ has been implicated in reducing melanogenic (melanin producing) protein expression in cultured B16 cells, where it can inhibit tyrosinase expression and reduce gene activity^[133] and can prevent stimulation of tryosinase mRNA by alpha-melanocyte stimulating hormone.^[134]

Interactions with Medical Conditions

14.1. Parkinson’s Disease

Parkinson’s disease is known to be associated with what are known as Lewy Bodies (irregular cytoplasmic inclusions^[135]^[136]) which are comprised of a molecule known as α-synuclein^[137] which is known to damange dopaminergic neurons and is involved in the pathology of Parkinson’s disease when it aggregates.^[138]^[139] It is involved in normal physiological function (as a chaperone) when unaggregated,^[140] so the process of α-synuclein aggregation itself is seen as pathological.

Pyrroloquinline quinone (PQQ) is known to bind to some of these α-synuclein peptides directly via forming a schiff basewith the lysine amino acids in the peptides^[13] similar to both EGCG (Green Tea Catechins) and baicalein (skullcap)^[13]although baicalein seems relatively more potent.^[141] This direct binding also reduces formation of truncated α-synuclein^[142] (which accelerate the formation of larger aggregates^[143]) and the larger protein aggregates themselves^[13] by around 14.8-50% at 280µM.^[142] This may indirectly reduce the cytotoxicity that is seen with large aggregates,^[13] although PQQ seems to be capable of reducing cytotoxicity from pre-formed aggregates independent of the aforementioned binding.^[142]

Protein aggregates tend to occur normally in the brain, and their aggregation is accelerated and seem to be central to the development of Parkinson’s Disease. PQQ appears to physically bind to these proteins in vitro to prevent the aggregation, but it occurs at a very high concentration and it does not seem likely to occur with respectable potency following oral supplementation

6-hydroxydopamine (6-OHDA), a metabolite of dopamine which is known to cause oxidative damage to dopaminergic neurons and detected at higher levels in persons with Parkinson’s,^[144] may have its toxicity attenuated with coincubation of PQQ.^[145] Oxidative neurotoxicity and DNA fragmentation induced by 6-hydroxydopamine was reduced in a concentration dependent manner with concentrations of 300nM showing efficacy, yet this protective effect was not seen with Vitamin C or Vitamin E, two other anti-oxidants tested at concentrations up to 100µM.^[145]

Elsewhere in isolated neurons, the protein DJ-1 (plays roles in oxidative protection^[146]^[147] and mutations in it underly some genetic cases of early onset Parkinson’s Disease^[148]) does not have its expression altered by PQQ^[149] but 15µM PQQ appeared to preserve cell survival in the presence of oxidants by preserving the actions of DJ-1;^[149] excessive oxidation of DJ-1 at C106 ablates its antioxidant potential^[150] and PQQ appears to prevent this from occurring despite no direct binding.^[149]

There may be some protective effects at the level of dopaminergic neurons with PQQ that is not related to preventing the formation of protein aggregation, and although this happens at a much more respectable (lower) concentration it is still uncertain if this applies to oral supplementation of PQQ

14.2. Alzheimer’s Disease

Pyrroloquinline quinone appears to inhibit the formation of amyloid fibrils (Aβ_1-42; full inhibition at 70μM PQQ^[151]), and although it can also bind to α-synuclein this binding does not indirectly inhibit Aβ_1-42 aggregation.^[13]

and to reduce the cytotoxicity of these fibrils on neuronal cells.^[152]

Nutrient-Nutrient Interactions

15.1. Glutathione

PQQ has been shown to be cytotoxic to U937 leukemia cells, but not NIH3T3 nor L929 cells (but was observed in EL-4), in a dose-dependent manner with most significance at 20-50uM.^[131] Catalase treatment neutralized these effects, as they appear to be secondary to hydrogen peroxide production in cells which PQQ has been repeatedly shown to induce.^[132]Superoxide dismutase had no effect on PQQ cytotoxicity, while glutathione or N-AcetylCysteine increased cytotoxicity 2-5fold without affecting the cells on their own (and thus working via PQQ by increasing H₂O₂ production form PQQ 1.5-2fold).^[131] PQQ by itself decreased intracellular glutathione levels, and when glutathione was depleted (via BSO, an inhibitor of γ-glutamylcysteine synthetase) the apoptosis of cells morphed into necrosis, and this necrosis was still mediated by H₂O₂ due to being inhibited by catalase.^[131]

Glutathione can be increased by cysteine containing supplements including N-AcetylCysteine or Whey Protein

In cancer cells susceptible to PQQ’s induction of H₂O₂, adding glutathione to the cell by consuming Cysteine-containing supplements can augment the efficacy of PQQ

Safety and Toxicology

16.1. General

PQQ has been associated with renal tubule inflammation at the dose of 11-12mg/kg bodyweight in rats after injections, and some symptoms of both renal and hepatic toxicity are seen with injections of 20mg/kg in rats.^[110]^[153] Acute death from PQQ injections between doses of 500-1000mg/kg bodyweight has been recorded in rats.^[10]^[153]

11-12mg/kg bodyweight, based on rudimentary body surface area conversions, is approximately 120-131mg/PQQ daily (although injections) if extrapolated to humans.

One human study using 20mg PQQ alone or in combination with 300mg CoQ10 noted that there were no toxicological signs or symptoms associated with treatment over a 12 week period,^[106] and consumption of up to 0.3mg/kg PQQ (around 20mg for a 150lb person) for one week has been noted to be safe.^[28]

Chronic toxicity to the kidneys and liver may be achieved at a relatively low dose, although acute death requires a very high and unpractical dose. Until more evidence surfaces, it would be prudent to avoid superloading

16.2. Genotoxicity

In an Ames test (TA1535, TA1537, TA98, and TA100 strains), 10-5000μg PQQ per plate (without metabolic activation) and 156-5000μg per plate (with activation) has failed to show appreciable genotoxic effects.^[154]

In lung fibroblasts derived from chinese hamsters, 12.5-400μg/mL (no metabolic activation) and 117.2-3750μg/mL (with activation; highest concentration being 10mM) and the latter concentration in isolated lymphocytes failed to exert appreciable genotoxic effects as assessed by structural abberations and polyploidy.^[154]

The aforementioned disodium salt of PQQ has failed to acutely exert genotoxic effects in mice (up to 2,000mg/kg) as assessed by a micronucleus assay and in bone marrow erythrocytes.^[154]

No genotoxiticity has been noted with the disodium salt of PQQ

Scientific Support & Reference Citations

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(Users who contributed to this page include shrillthrill, GregoryLopez, KurtisFrank, KamalPatel )

Pyrroloquinoline quinone

Pyrroloquinoline quinone (PQQ) was discovered by J.G. Hauge as the third redox cofactor after nicotinamide and flavin in bacteria (although he hypothesised that it was naphthoquinone).[1] Anthony and Zatman also found the unknown redox cofactor in alcohol dehydrogenase and named it methoxatin.[2] In 1979, Salisbury and colleagues[3] as well as Duine and colleagues[4] extracted this prosthetic group from methanol dehydrogenase of methylotrophs and identified its molecular structure. Adachi and colleagues identified that PQQ was also found in Acetobacter.[5]

These enzymes containing PQQ are called quinoproteins. Glucose dehydrogenase, one of the quinoproteins, is used as a glucose sensor. Subsequently, PQQ was found to stimulate growth in bacteria.[6] In addition, antioxidant and neuroprotective effects were also found.[7]

https://en.wikipedia.org/wiki/Pyrroloquinoline_quinone

Research in animals[edit]

Mitochondrial biogenesis in mice[edit]

In 2010, researchers at the University of California at Davis released a peer-reviewed publication showing that PQQ’s critical role in growth and development stems from its unique ability to activate cell signaling pathways directly involved in cellular energy metabolism, development, and function. The study demonstrated that PQQ not only protects mouse hepatocyte mitochondria from oxidative stress—it promotes the spontaneous generation of new mitochondria within aging cells, a process known asmitochondrial biogenesis.^[8]

The team of researchers at the University of California analyzed PQQ’s influence over cell signaling pathways involved in the generation of new mitochondria and found that there are three mouse proteins activated by PQQ that cause cells to undergo spontaneous mitochondrial biogenesis: peroxisome proliferator-activated receptor gamma coactivator 1-alpha, cAMP response element-binding protein, and the DJ-1 protein.^[8]

Cardioprotection in rat models[edit]

Damage from a heart attack, like a stroke, is inflicted via ischemic reperfusion injury. PQQ administration reduces the size of damaged areas in animal models of acute heart attack (myocardial infarction). Significantly, this occurs irrespective of whether the chemical is given before or after the ischemic event itself, suggesting that administration within the first hours of medical response may offer benefits to heart attack victims.^[9]

Researchers at the University of California at San Francisco investigated this potential, comparing PQQ with the beta blocker metoprolol—a standard post-MI clinical treatment. Independently, both treatments reduced the size of the damaged areas and protected against heart muscle dysfunction. When given together, the left ventricle’s pumping pressure was enhanced. The combination of PQQ with metoprolol also increased mitochondrial energy-producing functions—but the effect was modest compared with PQQ alone. Only PQQ favorably reduced lipid peroxidation. These results led the researchers to conclude that “PQQ is superior to metoprolol in protecting mitochondria from ischemia/reperfusion oxidative damage.” ^[10]

Subsequent research has also demonstrated that PQQ helps heart muscle cells resist acute oxidative stress by preserving and enhancing mitochondrial function.^[11]

Radiation poisoning in mice[edit]

In a study of gamma radiation poisoning in mice, 4mg/kg of PQQ improved 30-day survival from 2/20 to 12/20 at an 8 Gy dose.^[12]

Neuroprotection[edit]

PQQ is a neuroprotective compound that has been shown in a small number of preliminary studies to protect memory and cognition in aging animals and humans.^[13]^[14] It has been shown to reverse cognitive impairment caused by chronic oxidative stress in animal models and improve performance on memory tests.^[15] PQQ supplementation stimulates the production and release of nerve growth factors in cells that support neurons in the brain,^[16] a possible mechanism for the improvement of memory function it appears to produce in aging humans and rats.

PQQ has also been shown to safeguard against the self-oxidation of the DJ-1 protein, an early step in the onset of some forms of Parkinson’s disease.^[17]

PQQ protects brain cells against oxidative damage following ischemia-reperfusion injury—the inflammation and oxidative damage that result from the sudden return of blood and nutrients to tissues deprived of them by stroke.^[18] Reactive nitrogen species (RNS) arise spontaneously following stroke and spinal cord injuries and impose severe stresses on damaged neurons, contributing to subsequent long-term neurological damage.^[19] PQQ suppresses RNS in experimentally induced strokes,^[20] and provides additional protection following spinal cord injury by blocking inducible nitric oxide synthase (iNOS), a major source of RNS.^[21]

In animal models, administration of PQQ immediately prior to induction of stroke significantly reduces the size of the damaged brain area.^[22] These observations have been compounded by the observation in vivo that PQQ protects against the likelihood of severe stroke in an experimental animal model for stroke and brain hypoxia.^[18]

PQQ also affects some of the brain’s neurotransmitter systems. It protects neurons by modulating the properties of the N-methyl-D-aspartate (NMDA) receptor,^[23]^[24] and so reducing excitotoxicity—the damaging consequence of long-term overstimulation of neurons that is associated with many neurodegenerative diseases and seizures.^[25]^[26]^[27]^[28]

PQQ also protects the brain against neurotoxicity induced by other powerful toxins, including mercury^[29](a suspected factor in the development of Alzheimer’s disease^[30]) and oxidopamine^[31] (a potent neurotoxin used by scientists to induce Parkinsonism in laboratory animals by destroying dopaminergic and noradrenergic neurons.^[32])

PQQ prevents aggregation of alpha-synuclein, a protein associated with Parkinson’s disease.^[33] PQQ also protects nerve cells from the toxic effects of the amyloid-beta protein linked with Alzheimer’s disease,^[34]and reduces the formation of new amyloid beta aggregates.^[35]

Controversy[edit]

Although Nature Magazine published the 2003 paper by Kasahara and Kato which essentially stated that PQQ was a new vitamin, they also subsequently published, in 2005, an article by Chris Anthony and his colleague L.M. Fenton of the University of Southhampton which states that the 2003 Kasahara and Kato paper drew incorrect and unsubstantiated conclusions.^[36] On his website,^[37] Anthony discusses the Nature Magazine publications:

When I pointed out to the journal Nature that their high reputation was being used to justify investments of millions of dollars in the development of PQQ as a vitamin, they investigated the original paper, agreed with our objections and published our argument against it (Felton & Anthony, Nature Vol. 433, 2005). They also published (alongside ours) a paper by Rucker disagreeing with the conclusions of Kasahara and Kato on nutritional grounds, concluding “that insufficient information is available so far to state that PQQ uniquely performs an essential vitamin function in animals”.

Anthony further states on his website that “No mammalian PQQ-containing enzyme (quinoprotein) has been described” and that PQQ therefore cannot be called a “vitamin”. The latter statement is an exaggeration, since there is one mammalian enzyme which appears to use PQQ as a cofactor:^[38]

EC 1.2.1.31 – L-aminoadipate-semialdehyde dehydrogenase ^[39]^[40]

References[edit]

Jump up^ Hauge JG (1964). “Glucose dehydrogenase of bacterium anitratum: an enzyme with a novel prosthetic group”. J Biol Chem 239: 3630–9. PMID 14257587.
Jump up^ Anthony C, Zatman LJ (1967). “The microbial oxidation of methanol. The prosthetic group of the alcohol dehydrogenase of Pseudomonas sp. M27: a new oxidoreductase prosthetic group”. Biochem J 104 (3): 960–9. PMC 1271238. PMID 6049934.
Jump up^ Salisbury SA, Forrest HS, Cruse WB, Kennard O (1979). “A novel coenzyme from bacterial primary alcohol dehydrogenases”. Nature 280 (5725): 843–4. doi:10.1038/280843a0. PMID 471057.
Jump up^ Westerling J, Frank J, Duine JA (1979). “The prosthetic group of methanol dehydrogenase from Hyphomicrobium X: electron spin resonance evidence for a quinone structure”. Biochem Biophys Res Commun87 (3): 719–24. doi:10.1016/0006-291X(79)92018-7. PMID 222269.
Jump up^ Ameyama M, Matsushita K, Ohno Y, Shinagawa E, Adachi O (1981). “Existence of a novel prosthetic group, PQQ, in membrane-bound, electron transport chain-linked, primary dehydrogenases of oxidative bacteria”.FEBS Lett 130 (2): 179–83. doi:10.1016/0014-5793(81)81114-3. PMID 6793395.
Jump up^ Ameyama M, Matsushita K, Shinagawa E, Hayashi M, Adachi O (1988). “Pyrroloquinoline quinone: excretion by methylotrophs and growth stimulation for microorganisms”. BioFactors 1 (1): 51–3. PMID 2855583.
Jump up^ Rucker R, Chowanadisai W, Nakano M. (2009). “Potential physiological importance of pyrroloquinoline quinone”. Altern Med Rev. 14 (3): 179–83.
^ Jump up to:^a ^b Chowanadisai, W.; Bauerly, K. A.; Tchaparian, E.; Wong, A.; Cortopassi, G. A.; Rucker, R. B. (January 2010). “Pyrroloquinoline quinone stimulates mitochondrial biogenesis through cAMP response element-binding protein phosphorylation and increased PGC-1alpha expression”. Journal of Biological Chemistry 285 (1): 142–152. doi:10.1074/jbc.M109.030130. PMC 2804159. PMID 19861415.
Jump up^ Zhu, B. Q.; Zhou, H. Z.; Teerlink, J. R.; Karliner, J. S. (November 2004). “Pyrroloquinoline quinone (PQQ) decreases myocardial infarct size and improves cardiac function in rat models of ischemia and ischemia/reperfusion”. Cardiovascular Drugs and Therapy 18 (6): 421–431. doi:10.1007/s10557-004-6219-x.PMID 15770429.
Jump up^ Zhu, B. -Q.; Simonis, U.; Cecchini, G.; Zhou, H. -Z.; Li, L.; Teerlink, J. R.; Karliner, J. S. (June 2006). “Comparison of pyrroloquinoline quinone and/or metoprolol on myocardial infarct size and mitochondrial damage in a rat model of ischemia/reperfusion injury”. Journal of Cardiovascular Pharmacology and Therapeutics 11 (2): 119–128. doi:10.1177/1074248406288757. PMID 16891289.
Jump up^ Tao, R; Karliner, J; Simonis, U; Zheng, J; Zhang, J; Honbo, N; Alano, C (2007). “Pyrroloquinoline quinone preserves mitochondrial function and prevents oxidative injury in adult rat cardiac myocytes”. Biochemical and Biophysical Research Communications 363 (2): 257–62. doi:10.1016/j.bbrc.2007.08.041. PMC 2844438.PMID 17880922.
Jump up^ Xiong, X. H.; Zhao, Y; Ge, X; Yuan, S. J.; Wang, J. H.; Zhi, J. J.; Yang, Y. X.; Du, B. H.; Guo, W. J.; Wang, S. S.; Yang, D. X.; Zhang, W. C. (2011). “Production and radioprotective effects of pyrroloquinoline quinone”. International Journal of Molecular Sciences 12 (12): 8913–23. doi:10.3390/ijms12128913.PMC 3257108. PMID 22272111.
Jump up^ Takatsu, H; Owada, K; Abe, K; Nakano, M; Urano, S (2009). “Effect of vitamin E on learning and memory deficit in aged rats”. Journal of nutritional science and vitaminology 55 (5): 389–93. doi:10.3177/jnsv.55.389.PMID 19926923.
Jump up^ Nakano M, Ubukata K, Yamamoto T, Yamaguchi H. (2009). “Effect of pyrroloquinoline quinone (PQQ) on mental status of middle-aged and elderly persons”. Food Style 21 13 (7): 50–52.
Jump up^ Ohwada, K.; Takeda, H.; Yamazaki, M.; Isogai, H.; Nakano, M.; Shimomura, M.; Fukui, K.; Urano, S. (January 2008). “Pyrroloquinoline quinone (PQQ) prevents cognitive deficit caused by oxidative stress in rats”. Journal of Clinical Biochemistry and Nutrition 42 (1): 29–34. doi:10.3164/jcbn.2008005.PMC 2212345. PMID 18231627.
Jump up^ Murase, K; Hattori, A; Kohno, M; Hayashi, K (1993). “Stimulation of nerve growth factor synthesis/secretion in mouse astroglial cells by coenzymes”. Biochemistry and molecular biology international 30 (4): 615–21.PMID 8401318.
Jump up^ Nunome, K; Miyazaki, S; Nakano, M; Iguchi-Ariga, S; Ariga, H (2008). “Pyrroloquinoline quinone prevents oxidative stress-induced neuronal death probably through changes in oxidative status of DJ-1”. Biological & Pharmaceutical Bulletin 31 (7): 1321–6. doi:10.1248/bpb.31.1321. PMID 18591768.
^ Jump up to:^a ^b Jensen, FE; Gardner, GJ; Williams, AP; Gallop, PM; Aizenman, E; Rosenberg, PA (1994). “The putative essential nutrient pyrroloquinoline quinone is neuroprotective in a rodent model of hypoxic/ischemic brain injury”. Neuroscience 62 (2): 399–406. doi:10.1016/0306-4522(94)90375-1. PMID 7830887.
Jump up^ Ono, K.; Suzuki, H.; Sawada, M. (2010-10-05). “Delayed neural damage is induced by iNOS-expressing microglia in a brain injury model”. Neuroscience Letters 473 (2): 146–150. doi:10.1016/j.neulet.2010.02.041.PMID 20178828.
Jump up^ Zhang, Y; Rosenberg, PA (2002). “The essential nutrient pyrroloquinoline quinone may act as a neuroprotectant by suppressing peroxynitrite formation”. The European Journal of Neuroscience 16 (6): 1015–24. doi:10.1046/j.1460-9568.2002.02169.x. PMID 12383230.

PQQ and Statin Damage
By Dr. Duane Graveline MD, MPH

Those of you who have been following my research during the past two years will know that I consider mitochondrial DNA damage as the ultimate result for some of statin drug intake.

Through mevalonate blockade, statins directly inhibit CoQ10 synthesis making mitochondrial damage and mutation all but inevitable. Furthermore, the inhibitory effect of statins on dolichol synthesis makes repair of DNA damage all the more difficult because of dolichol’s vital role in glycoprotein (glycohydrolase) synthesis.

Recently I have learned of another biochemical substance that also is implicated in this process of mitochondrial maintenance. The name of this biochemical is pyrroloquinoline quinone with the shorthand version being PQQ.

This substance has been discovered only in the past decade with its vital role in mitochondrial support having been documented only in the past several years. From what I have read of this substance, trying to get beyond the hype, it is worth considering for those of us who have been damaged by statins, whether by cognitive dysfunction, permanent myopathy, ALS like symptoms, or peripheral neuropathy.

Dietary sources of PQQ include many fruits and vegetables and egg yolk. Natto ( fermented soybeans ) has the highest concentration but parsley, green peppers, papaya, kiwi fruit and spinach are all good sources. PQQ is also available as a dietary supplement. Human trials and studies will need to be performed to support any claims for the benefits of PQQ supplementation.

One promotion for PQQ begins with, “The more functional mitochondria you have in your cells, the greater your overall health and durability,” which is the premise of my new e-book, The Dark Side of Statins, so my interest in this substance is obvious.

The problem is that as we age, our mitochondria degrade and become dysfunctional. Compared with nuclear DNA, mitochondrial DNA is left almost entirely exposed to the ravages of free radicals. It attaches directly to the inner membrane where the mitochondria’s furnace rages continuously.

Statin drugs directly hasten this process of mitochondrial DNA degradation by direct inhibition of CoQ10 and dolichol synthesis. The ultimate cause of statin associated adverse reactions is this progressive deterioration of mitochondrial DNA. PQQ is being touted not only for its extra anti-oxidant protection in the fight against free radicals but also for its potential use for mitochondrial genesis

https://youtu.be/-PA-buwI3q4

https://youtu.be/-PA-buwI3q4?t=406

https://youtu.be/-PA-buwI3q4?t=455

https://youtu.be/j1FmK4582mA

This is part 1 (of nine parts) of the Preventing and Reversing Alzheimer’s Disease presentation, an earlier version of which was presented to the San Francisco bay area Smart Life Forum in January of 2009. This part covers the verbal introduction and the falling-dominoes illustration of the Alzheimer’s cascade

https://youtu.be/hQipKkFppzI

https://youtu.be/oX6RG6ky0yU

This is part three of the Prevention and Reversal of Alzheimer’s Disease presentation. This part covers the Alzheimer’s Map (schematic), mitochondria, and creatine kinase (the first domino in the Alzheimer’s disease cascade).

https://youtu.be/2dutY1zUM7k

https://youtu.be/kktDaCRwnFM

This is part six of the Prevention and Reversal of Alzheimer’s Disease presentation. This part covers the antioxidant defense system, glutathione (the “star of the movie”), and the brain’s phosphorylation cycle (the brains “biorhythm).

https://youtu.be/kktDaCRwnFM?t=81

https://youtu.be/kktDaCRwnFM?t=118

https://youtu.be/iC9GOU78OwU

Read Full Post »

Current Advances in Medical Technology

Posted in Bio Instrumentation in Experimental Life Sciences Research, BioIT: BioInformatics, Biological Networks, Cell Biology, Cerebrovascular and Neurodegenerative Diseases, Clinical & Translational, Clinical Diagnostics, Curation, Drug Development using MultiOrgan Chip, Gastroenterology, Genetics & Pharmaceutical, Genome Biology, Image Processing/Computing, Infectious Disease Immunodiagnostics, Innovations, Innovations in Neurophysiology & Neuropsychology, Organ-on-a-Chip, Programmable Sensors (Carbon Nano Tubes), tagged biomedical imaging, gene editing, Influenza virus typing, Medical technology, organ on a chip on October 13, 2015| Leave a Comment »

Current Advances in Medical Technology

Larry H. Bernstein, MD, FCAP, Curator

LPBI

Pumpkin-Shaped Molecule Enables 100-Fold Improved MRI Contrast

Tue, 10/13/2015 – 9:16amby Forschungsverbund Berlin e.V. (FVB)

http://www.mdtmag.com/news/2015/10/pumpkin-shaped-molecule-enables-100-fold-improved-mri-contrast

Assuming that we could visualize pathological processes such as cancer at a very early stage and additionally distinguish the various different cell types, this would represent a giant step for personalized medicine. Xenon magnetic resonance imaging has the potential to fulfil this promise – if suitable contrast media are found that react sensitively enough to the “exposure”. Researchers at the Leibniz-Institut für Molekulare Pharmakologie in Berlin have now found that a class of pumpkin-shaped molecules called cucurbiturils together with the inert gas xenon, enables particularly good image contrast – namely around 100 times better than has been possible up to now. This finding published in the November issue cover article of Chemical Science by the Royal Society of Chemistry points the way to the tailoring of new contrast agents to different cell types and has the potential to enable molecular diagnostics even without tissue samples in the future.

Personalized medicine instead of one treatment for all – especially in cancer medicine, this approach has led to a paradigm shift. Molecular diagnostics is the key that will give patients access to tailor-made therapy. However, if tumors are located in poorly accessible areas of the body or several tumor foci are already present, this often fails due to a lack of sufficient sensitivity of the diagnostic imaging. But such sensitivity is needed to determine the different cell types, which differ considerably even within a tumor. Although even the smallest of tumor foci and other pathological changes can be detected using the PET-CT, a differentiation according to cell type is usually not possible.

Scientists from the FMP are therefore focusing on xenon magnetic resonance imaging: The further development of standard magnetic resonance imaging makes use of the “illuminating power” of the inert gas xenon, which can provide a 10,000-fold enhanced signal in the MRI. To do this, it must be temporarily captured by so-called “cage molecules” in the diseased tissue. This has been more or less successful with the molecules used to date, but the experimental approach is still far from a medical application.

Cucurbituril Provides Stunning Image Contrasts
The research group led by Dr. Leif Schröder at the Leibniz-Institut für Molekulare Pharmakologie (FMP) has now discovered a molecule class for this purpose that eclipses all of the molecules used to date. Cucurbituril exchanges around 100 times more xenon per unit of time than its fellow molecules, which leads to a much better image contrast. “It very quickly became clear that cucurbituril might be suitable as a contrast medium,” reports Leif Schröder. “However, it was surprising that areas marked with it were imaged with a much better contrast than previously.” The explanation is to be found in the speed. Upon exposure, so to speak, cucurbituril generates contrast more rapidly than all molecules used to date, as it only binds the xenon very briefly and thus transmits the radio waves to detect the inert gas to very many xenon atoms within a fraction of a second. In this way, the inert gas is passed through the molecule much more efficiently.

In the study, which appeared in the specialist journal “Chemical Science”, the world’s first MRI images with cucurbituril have been achieved. With the aid of a powerful laser and a vaporized alkali metal, the researchers initially greatly strengthened the magnetic properties of normal xenon. The hyperpolarized gas was then introduced into a test solution with the cage molecules. A subsequent MRI image showed the distribution of the xenon in the object. In a second image, the curcurbituril together with radio waves destroyed the magnetization of the xenon, leading to dark spots on the images.

“Comparison of the two images demonstrates that only the xenon in the cages has the right resonance frequency to produce a dark area,” explains Schröder. “This blackening is possible to a much better degree with cucurbituril than with previous cage molecules, for it works like a very light-sensitive photographic paper. The contrast is around 100 times stronger.”

Time-of-Flight IC Revolutionizes Object Detection and Distance Measurement

Tue, 10/13/2015 – 9:07amby Intersil

New ISL29501 signal processing IC detects objects up to two meters

http://www.mdtmag.com/product-release/2015/10/time-flight-ic-revolutionizes-object-detection-and-distance-measurement
Intersil Corporation has introduced an innovative time-of-flight (ToF) signal processing IC that provides a complete object detection and distance measurement solution when combined with an external emitter (LED or laser) and photodiode. The ISL29501 ToF device offers one-of-a-kind functionality, including ultra-small size, low-power consumption and superior performance ideal for connected devices that make up the Internet of Things (IoT), as well as consumer mobile devices and the emerging commercial drone market.

The ISL29501 overcomes the shortcomings of traditional amplitude-based proximity sensors and other ToF solutions that perform poorly in lighting conditions above 2,000 lux, or cannot provide distance information unless the object is perpendicular to the sensor.

The ISL29501 applies Intersil’s power management expertise to save power and extend battery life through several innovations.

“Prior to Intersil’s time-of-flight technology breakthrough, there was no practical way to measure distance up to two meters in a small form factor,” said Andrew Cowell, senior vice president of Mobile Power Products at Intersil. “The innovative ISL29501 provides customers a cost-effective, small footprint solution that also gives them the flexibility to use multiple devices to increase the field of view to a full 360 degrees for enhanced object detection capabilities.”

Key Features and Specifications

On-chip DSP calculates ToF for accurate proximity detection and distance measurement up to two meters
Modulation frequency of 4.5MHz prevents interference with other consumer products such as IR TV remote controls that operate at 40kHzOn-chip emitter DAC with programmable current up to 255mA
Allows designers to choose the desired current level to optimize distance measurement and power budget
Operates in single shot mode for initial object detection and approximate distance measurement, while continuous mode improve distance accuracy
On-chip active ambient light rejection minimizes or eliminates the influence of ambient light during distance measurement
Programmable distance zones: allows the user to define three ToF distance zones for determining interrupt alerts
Interrupt controller generates interrupt alerts using distance measurements and user defined thresholds
Automatic gain control sets optimum analog signal levels to achieve best SNR response
Supply voltage range of 2.7V to 3.3V
I2C interface supports 1.8V and 3.3V bus

The ISL29501 can be combined with the ISL9120 buck-boost regulator to further reduce power consumption and extend battery life in consumer and home automation applications.

Optoelectronic Implantable Could Enable Two-Way Communication with Brain

Mon, 10/12/2015 – 4:04pmby Brown University

http://www.mdtmag.com/news/2015/10/optoelectronic-implantable-could-enable-two-way-communication-brain

Brown University researchers have created a new type of optoelectronic implantable device to access brain microcircuits, synergizing a technique that enables scientists to control the activity of brains cells using pulses of light. The invention, described in the journal Nature Methods, is a cortical microprobe that can stimulate multiple neuronal targets optically by specific patterns on micrometer scale while simultaneously recording the effects of that stimulation in the underlying neural microcircuits of interest with millisecond precision.

“We think this is a window-opener,” said Joonhee Lee, a senior research associate in Professor Arto Nurmikko’s lab in the School of Engineering at Brown and one of the lead authors of the new paper. “The ability to rapidly perturb neural circuits according specific spatial patterns and at the same time reconstruct how the circuits involved are perturbed, is in our view a substantial advance.”

First introduced around 2005, optogenetics has enriched ability of scientists seeking to understand brain function at the neuronal level. The technique involves genetically engineering neurons to express light-sensitive proteins on their membranes. With those proteins expressed, pulses of light can be used to either promote or suppress activity in those particular cells. The method gives researchers in principle unprecedented ability to control specific brain cells at specific times.

But until now, simultaneous optogenetic stimulation and recording of brain activity rapidly across multiple points within a brain microcircuit of interest has proven difficult. Doing it requires a device that can both generate a spatial pattern of light pulses and detect the dynamical patterns of electrical reverberations generated by excited cellular activity. Previous attempts to do this involved devices that cobbled together separate components for light emission and electrical sensing. Such probes were physically bulky, not ideal for insertion into a brain. And because the emitters and the sensors were necessarily a hundreds of micrometers apart, a sizable distance, the link between stimulation and recorded signal was ambiguous.

The new compact, integrated device developed by Nurmikko’s lab begins with the unique advantages endowed by a so-called wide bandgap semiconductor called zinc oxide. It is optically transparent yet able readily to conduct an electrical current.

“Very few materials have that pair of physical properties,” Lee said. “The combination makes it possible to both stimulate and detect with the same material.”

Joonhee Lee, with Assistant Research Professor Ilker Ozden and Professor Yoon-Kyu Song at Seoul National University in Korea, co-developed a novel microfabrication method with Nurmikko to shape the material into a monolithic chip just a few millimeters square with sixteen micrometer sized pin-like “optoelectrodes,” each capable of both delivering light pulses and sensing electrical current. The array of optoelectrodes enables the device to couple to neural microcircuits composed of many neurons rather than single neurons.

Such ability to stimulate and record at the network level on spatial and time scales at which they operate is key, Nurmikko says. Brain functions are driven by neural circuits rather than single neurons.

“For example, when I move my hand, that’s an example of action driven by specific network-level activity in the brain,” he said. “Our new device approach gives scientists and engineers a tool in applying the full power of optogenetics as a means of neural stimulation, while providing the means to read activity of perturbed networks at multiple points at high spatial precision and time resolution.”

Ozden led the initial testing of the device in rodent models. The researchers looked at the extent to which different light intensities could stimulate network activity. The tests showed that increasing optical power led to distinct recruitment of neuronal circuits revealing functional connectivity in the targeted network.

“We went over a range of optical power that was large–over three orders of magnitude–and in so doing we got a range of network-related responses, in particular we could replicate an activity pattern naturally occurring in the brain.” Ozden said. “It gave us a new insight into how optogenetics operates on the network level. This gives us encouragement to go ahead and extend the repertoire and application of the device technology.”

Nurmikko’s group together with the Song lab in Seoul plan to continue further development of the device, ultimately include an access via wireless means. Their next steps anticipate the use of the new device technology as chronic implant in non-human primates at potentially hundreds of points and, depending on progress in worldwide research on optogenetics ahead, perhaps even one day in humans.

“At least, the initial building blocks are here,” Nurmikko said, who conceived the idea with his Korean colleague Song.

Study Advances Possibility of Mind-Controlled Devices

Mon, 10/12/2015 – 10:50amby Ryan Bushey, Associate Editor, R&D

A study published in the journal Nature Medicine has shown a possible path to creating effective neural prosthetics.

http://www.mdtmag.com/blog/2015/10/study-advances-possibility-mind-controlled-devices

The study’s subjects, only listed as T6 and T7, have Amyotropic Lateral Sclerosis (ALS). The scientists performed surgery on them one year ago to place a “neural recording device” in the part of the brain in charge of controlling hand function, notes Bloomberg.

The test documented in the study required T6 and T7 to perform a variety of tasks, such as moving a cursor to hit different targets on a computer screen. The device receives electrical impulses from the brain and morphs them into a computer signal to operate the cursor.

Both test subjects had the highest published performance so far, and even doubled the results of the previous clinical trial participant, according to the study.

The hope is that these devices can improve quality of life for people suffering from paralysis.

You can watch how T6 performed in her test below.

https://youtu.be/9P-qsiIORVU

Removing 62 Barriers to Pig–to–Human Organ Transplant in One Fell Swoop

Mon, 10/12/2015 – 9:09amby Wyss Institute for Biologically Inspired Engineering

The largest number of simultaneous gene edits ever accomplished in the genome could help bridge the gap between organ transplant scarcity and the countless patients who need them

http://www.mdtmag.com/news/2015/10/removing-62-barriers-pig%E2%80%93%E2%80%93human-organ-transplant-one-fell-swoop

Never before have scientists been able to make scores of simultaneous genetic edits to an organism’s genome. But now in a landmark study by George Church, Ph.D., and his team at the Wyss Institute for Biologically Inspired Engineering at Harvard University and Harvard Medical School, the gene editing system known as “CRISPR–Cas9” has been used to genetically engineer pig DNA in 62 locations – an explosive leap forward in CRISPR’s capability when compared to its previous record maximum of just six simultaneous edits. The 62 edits were executed by the team to inactivate retroviruses found natively in the pig genome that have so far inhibited pig organs from being suitable for transplant in human patients. With the retroviruses safely removed via genetic engineering, however, the road is now open toward the possibility that humans could one day receive life–saving organ transplants from pigs.

Church is a Wyss Core Faculty member, the Robert Winthrop Professor of Genetics at Harvard Medical School (HMS) and Professor of Health Sciences and Technology at Harvard and MIT. The advance, reported by Church and his team including the study’s lead author Luhan Yang, Ph.D., a Postdoctoral Fellow at HMS and the Wyss Institute, was published in the October 11 issue of Science.

The concept of xenotransplantation, which is the transplant of an organ from one species to another, is nothing new. Researchers and clinicians have long hoped that one of the major challenges facing patients suffering from organ failure – which is the lack of available organs in the United States and worldwide – could be alleviated through the availability of suitable animal organs for transplant. Pigs in particular have been especially promising candidates due to their similar size and physiology to humans. In fact, pig heart valves are already commonly sterilized and de–cellularized for use repairing or replacing human heart valves.

This artistic rendering shows pig chromosomes (background) which reside in the nucleus of pig cells and contain a single strand of RNA, and the Cas9 protein targeting DNA (foreground). The CRISPR–Cas9 gene editing system works like molecular scissors to precisely edit genes of interest. A new advance reported in Science by Wyss Core Faculty member George Church and his team used Cas9 to make 62 edits to the pig genome to remove latent retroviruses, presenting a solution to one of the largest safety concerns that has so far blocked progress in making pig organs compatible for xenotransplant in humans. (Credit: Wyss Institute at Harvard University)

But the transplant of whole, functional organs comprised of living cells and tissue constructs has presented a unique set of challenges for scientists. One of the primary problems has been the fact that most mammals including pigs contain repetitive, latent retrovirus fragments in their genomes – present in all their living cells – that are harmless to their native hosts but can cause disease in other species.

“The presence of this type of virus found in pigs – known as porcine endogenous retroviruses or PERVs – brought over a billion of dollars of pharmaceutical industry investments into developing xenotransplant methods to a standstill by the early 2000s,” said Church. “PERVs and the lack of ability to remove them from pig DNA was a real showstopper on what had been a promising stage set for xenotransplantation.”

Now – using CRISPR–Cas9 like a pair of molecular scissors – Church and his team have inactivated all 62 repetitive genes containing a PERV in pig DNA, surpassing a significant obstacle on the path to bringing xenotransplantation to clinical reality. With more than 120,000 patients currently in the United States awaiting transplant and less than 30,000 transplants on average occurring annually, xenotransplantation could give patients and clinicians an alternative in the future.

“Pig kidneys can already function experimentally for months in baboons, but concern about the potential risks of PERVs has posed a problem for the field of xenotransplantation for many years,” said David H. Sachs, M.D., Director of the TBRC Laboratories at Massachusetts General Hospital, Paul S. Russell Professor of Surgery Emeritus at Harvard Medical School, and Professor of Surgical Sciences at Columbia University’s Center for Translational Immunology. Sachs has been developing special pigs for xenotransplantation for more than 30 years and is currently collaborating with Church on further genetic modifications of his pigs. “If Church and his team are able to produce pigs from genetically engineered embryos lacking PERVs by the use of CRISPR-Cas9, they would eliminate an important potential safety concern facing this field.”

Yang says the team hopes eventually they can completely eliminate the risk that PERVs could cause disease in clinical xenotransplantation by using modified pig cells to clone a line of pigs that would have their PERV genes inactivated.

“This advance overcomes a major hurdle that has until now halted the progress of xenotransplantation research and development,” said Wyss Institute Founding Director Donald Ingber, M.D., Ph.D., who is also the Judah Folkman Professor of Vascular Biology at Harvard Medical School and Professor of Bioengineering at the Harvard John A. Paulson School of Engineering and Applied Sciences. “The real value and potential impact is in the number of lives that could be saved if we can one day use xenotransplants to close the huge gap between the number of available functional organs and the number of people who desperately need them.”

The remarkable and newly demonstrated capability for CRISPR to edit tens of repetitive genes such as PERVs will also unlock new ways for scientists to study and understand repetitive regions in the genome, which has been estimated to comprise more than two–thirds of our own human genome.

Contributors to the work also included: co–lead authors Marc Güell of the Wyss Institute and Harvard Medical School Department of Genetics, Dong Niu of HMS Dept. of Genetics and Zhejiang University’s College of Animal Sciences, and Haydy George of HMS Dept. of Genetics; and co–authors Emal Lesha, Dennis Grishin, Jürgen Poci, Ellen Shrock, and Rebeca Cortazio of HMS Dept. of Genetics, Weihong Xu of Massachusetts General Hospital Department of Surgery, and Robert Wilkinson and Jay Fishman of MGH’s Transplant Infection Disease & Compromised Host Program.

Novel Gut-on-a-Chip Nearly Indistinguishable from Human GI Tract

Fri, 10/09/2015 – 1:17pmby University of North Carolina Healthcare

http://www.mdtmag.com/news/2015/10/novel-gut-chip-nearly-indistinguishable-human-gi-tract?et_cid=4876632&et_rid=535648082

A team of researchers from the University of North Carolina at Chapel Hill and NC State University has received a $5.3 million, five-year Transformative Research (R01) Award from the National Institutes of Health (NIH) to create fully functioning versions of the human gut that fit on a chip the size of a dime.

Such “organs-on-a-chip” have become vital for biomedical research, as researchers seek alternatives to animal models for drug discovery and testing. The new grant will fund a technology that represents a major step forward for the field, overcoming limitations that have mired other efforts.

The technology will use primary cells derived directly from human biopsies, which are known to provide more relevant results than the immortalized cell lines used in current approaches. In addition, the device will sculpt these cells into the sophisticated architecture of the gut, rather than the disorganized ball of cells that are created in other miniature organ systems.

This is a picture of a schematic of colonic epithelial tissue. Crypt units are pointed down, flat surface faces center of the gut tube. Stem cells are red, progenitor cells are pink, differentiated cells are grey, blue and green. Yellow cells are stem cell niche cells. Lumenal surface is above crypts. (Credit: Scott Magness, PhD, UNC School of Medicine)

“We are building a device that goes far beyond the organ-on-a-chip,” said Nancy L. Allbritton, MD, PhD, professor and chair of the UNC-NC State joint department of biomedical engineering and one of four principle investigators on the NIH grant. “We call it a ‘simulacrum,’ a term used in science fiction to describe a duplicate. The idea is to create something that is indistinguishable from your own gut.”

Allbritton is an expert at microfabrication and microengineering. Also on the team are intestinal stem cell expert Scott T. Magness, PhD, associate professor of medicine, biomedical engineering, and cell and molecular physiology in the UNC School of Medicine; microbiome expert Scott Bultman, PhD, associate professor of genetics in the UNC School of Medicine; and bioinformatics expert Shawn Gomez, associate professor of biomedical engineering at UNC-Chapel Hill and NC State.

The impetus for the “organ-on-chip” movement comes largely from the failings of the pharmaceutical industry. For just a single drug to go through the discovery, testing, and approval process can take as many as 15 years and as much as $5 billion dollars. Animal models are expensive to work with and often don’t respond to drugs and diseases the same way humans do. Human cells grown in flat sheets on Petri dishes are also a poor proxy. Three-dimensional “organoids” are an improvement, but these hollow balls are made of a mishmash of cells that doesn’t accurately mimic the structure and function of the real organ.

Basically, the human gut is a 30-foot long hollow tube made up of a continuous single-layer of specialized cells. Regenerative stem cells reside deep inside millions of small pits or “crypts” along the tube, and mature differentiated cells are linked to the pits and live further out toward the surface. The gut also contains trillions of microbes, which are estimated to outnumber human cells by ten to one. These diverse microbial communities — collectively known as the microbiota — process toxins and pharmaceuticals, stimulate immunity, and even release hormones to impact behavior.

These are fluorescent images of the side view of two synthetic crypts. Blue: nuclei of the cells. Red: proliferating stem cells in similar location to those in the human colon. (Credit: Scott Magness, PhD, UNC School of Medicine)

To create a dime-sized version of this complex microenvironment, the UNC-NC State team borrowed fabrication technologies from the electronics and microfluidics world. The device is composed of a polymer base containing an array of imprinted or shaped “hydrogels,” a mesh of molecules that can absorb water like a sponge. These hydrogels are specifically engineered to provide the structural support and biochemical cues for growing cells from the gut. Plugged into the device will be various kinds of plumbing that bring in chemicals, fluids, and gases to provide cues that tell the cells how and where to differentiate and grow. For example, the researchers will engineer a steep oxygen gradient into the device that will enable oxygen-loving human cells and anaerobic microbes to coexist in close proximity.

“The underlying concept — to simply grow a piece of human tissue in a dish — doesn’t seem that groundbreaking,” said Magness. “We have been doing that for a long time with cancer cells, but those efforts do not replicate human physiology. Using native stem cells from the small intestine or colon, we can now develop gut tissue layers in a dish that contains stem cells and all the differentiated cells of the gut. That is the thing stem cell biologists and engineers have been shooting for, to make real tissue behave properly in a dish to create better models for drug screening and cell-based therapies. With this work, we made a big leap toward that goal.”

Right now, the team has a working prototype that can physically and chemically guide mouse intestinal stem cells into the appropriate structure and function of the gut. For several years, Magness has been isolating and banking human stem cells from samples from patients undergoing routine colonoscopies at UNC Hospitals. As part of the grant, he will work with the rest of the team to apply these stem cells to the new device and create “simulacra” that are representative of each patient’s individual gut. The approach will enable researchers to explore in a personalized way how both the human and microbial cells of the gut behave during healthy and diseased states.

“Having a system like this will advance microbiota research tremendously,” said Bultman. “Right now microbiota studies involve taking samples, doing sequencing, and then compiling an inventory of all the microbes in the disease cases and healthy controls. These studies just draw associations, so it is difficult to glean cause and effect. This device will enable us to probe the microbiota, and gain a better understanding of whether changes in these microbial communities are the cause or the consequence of disease.”

On-Chip Optical Sensing Technique Detects Multiple Flu Strains

Tue, 10/06/2015 – 10:11amby University of California – Santa Cruz

http://www.mdtmag.com/news/2015/10/chip-optical-sensing-technique-detects-multiple-flu-strains?et_cid=4876632&et_rid=535648082

A schematic view shows the optical waveguide intersecting a fluidic microchannel containing target particles. Targets are optically excited as they flow past well-defined excitation spots created by multi-mode interference; fluorescence is collected by the liquid-core waveguide channel and routed into solid-core waveguides (red). (Credit: Ozcelik et al., PNAS 2015)

New chip-based optical sensing technologies developed by researchers at UC Santa Cruz and Brigham Young University enable the rapid detection and identification of multiple biomarkers. In a paper published October 5 in Proceedings of the National Academy of Sciences, researchers describe a novel method to perform diagnostic assays for multiple strains of flu virus on a small, dedicated chip.

“A standard flu test checks for about ten different flu strains, so it’s important to have an assay that can look at ten to 15 things at once. We showed a completely new way to do that on an optofluidic chip,” said senior author Holger Schmidt, the Kapany Professor of Optoelectronics in the Baskin School of Engineering at UC Santa Cruz.

Over the past decade, Schmidt and his collaborators at BYU have developed chip-based technology to optically detect single molecules without the need for high-end laboratory equipment. Diagnostic instruments based on their optofluidic chips could provide a rapid, low-cost, and portable option for identifying specific disease-related molecules or virus particles.

In the new study, Schmidt demonstrated a novel application of a principle called wavelength division multiplexing, which is widely used in fiber-optic communications. By superimposing multiple wavelengths of light in an optical waveguide on a chip, he was able to create wavelength-dependent spot patterns in an intersecting fluidic channel. Virus particles labeled with fluorescent markers give distinctive signals as they pass through the fluidic channel depending on which wavelength of light the markers absorb.

“Each color of light produces a different spot pattern in the channel, so if the virus particle is labeled to respond to blue light, for example, it will light up nine times as it goes through the channel, if it’s labeled for red it lights up seven times, and so on,” Schmidt explained.

The researchers tested the device using three different influenza subtypes labeled with different fluorescent markers. Initially, each strain of the virus was labeled with a single dye color, and three wavelengths of light were used to detect them in a mixed sample. In a second test, one strain was labeled with a combination of the colors used to label the other two strains. Again, the detector could distinguish among the viruses based on the distinctive signals from each combination of markers. This combinatorial approach is important because it increases the number of different targets that can be detected with a given number of wavelengths of light.

For these tests, each viral subtype was separately labeled with fluorescent dye. For an actual diagnostic assay, fluorescently labeled antibodies could be used to selectively attach distinctive fluorescent markers to different strains of the flu virus.

While previous studies have shown the sensitivity of Schmidt’s optofluidic chips for detection of single molecules or particles, the demonstration of multiplexing adds another important feature for on-chip bioanalysis. Compact instruments based on the chip could provide a versatile tool for diagnostic assays targeting a variety of biological particles and molecular markers.

The optofluidic chip was fabricated by Schmidt’s collaborators at Brigham Young University led by Aaron Hawkins. The joint first authors of the PNAS paper are Damla Ozcelik and Joshua Parks, both graduate students in Schmidt’s lab at UC Santa Cruz. Other coauthors include Hong Cai and Joseph Parks at UC Santa Cruz and Thomas Wall and Matthew Stott at BYU.

In another recent paper, published September 25 in Nature Scientific Reports, Schmidt’s team reported the development of a hybrid device that integrates an optofluidic chip for virus detection with a microfluidic chip for sample preparation.

“These two papers represent important milestones for us. Our goal has always been to use this technology to analyze clinically relevant samples, and now we are doing it,” Schmidt said.

Boom in Gene-Editing Studies amid Ethics Debate over Its Use

Mon, 10/12/2015 – 1:54pmby Lauran Neergaard, AP Medical Writer

http://www.mdtmag.com/news/2015/10/boom-gene-editing-studies-amid-ethics-debate-over-its-use-0

The hottest tool in biology has scientists using words like revolutionary as they describe the long-term potential: wiping out certain mosquitoes that carry malaria, treating genetic diseases like sickle cell, preventing babies from inheriting a life-threatening disorder.

It may sound like sci-fi, but research into genome editing is booming. So is a debate about its boundaries, what’s safe and what’s ethical to try in the quest to fight disease.

Does the promise warrant experimenting with human embryos? Researchers in China already have, and they’re poised to in Britain.

Should we change people’s genes in a way that passes traits to future generations? Beyond medicine, what about the environmental effects if, say, altered mosquitoes escape before we know how to use them?

“We need to try to get the balance right,” said Jennifer Doudna, a biochemist at the University of California, Berkeley. She helped develop new gene-editing technology and hears from desperate families, but urges caution in how it’s eventually used in people.

The U.S. National Academies of Science, Engineering and Medicine will bring international scientists, ethicists and regulators together in December to start determining that balance. The biggest debate is whether it ever will be appropriate to alter human heredity by editing an embryo’s genes.

“This isn’t a conversation on a cloud,” but something that families battling devastating rare diseases may want, Dr. George Daley of Boston Children’s Hospital told specialists meeting this week to plan the ethics summit. “There will be a drive to move this forward.”

Laboratories worldwide are embracing a technology to precisely edit genes inside living cells — turning them off or on, repairing or modifying them — like a biological version of cut-and-paste software. Researchers are building stronger immune cells, fighting muscular dystrophy in mice and growing human-like organs in pigs for possible transplant. Biotech companies have raised millions to develop therapies for sickle cell disease and other disorders.

The technique has a wonky name — CRISPR-Cas9 — and a humble beginning.

Doudna was studying how bacteria recognize and disable viral invaders, using a protein she calls “a genetic scalpel” to slice DNA. That system turned out to be programmable, she reported in 2012, letting scientists target virtually any gene in many species using a tailored CRISPR recipe.

There are older methods to edit genes, including one that led to an experimental treatment for the AIDS virus, but the CRISPR technique is faster and cheaper and allows altering of multiple genes simultaneously.

“It’s transforming almost every aspect of biology right now,” said National Institutes of Health genomics specialist Shawn Burgess.

In this photo provided by UC Berkeley Public Affairs, taken June 20, 2014 Jennifer Doudna, right, and her lab manager, Kai Hong, work in her laboratory in Berkeley, Calif. The hottest tool in biology has scientists using words like revolutionary as they describe the long-term potential: wiping out certain mosquitoes that carry malaria, treating genetic diseases like sickle-cell, preventing babies from inheriting a life-threatening disorder. “We need to try to get the balance right,” said Doudna. She helped develop new gene-editing technology and hears from desperate families, but urges caution in how it’s eventually used in people. (Cailey Cotner/UC Berkeley via AP)

CRISPR’s biggest use has nothing to do with human embryos. Scientists are engineering animals with human-like disorders more easily than ever before, to learn to fix genes gone awry and test potential drugs.

Engineering rodents to harbor autism-related genes once took a year. It takes weeks with CRISPR, said bioengineer Feng Zhang of the Broad Institute at MIT and Harvard, who also helped develop and patented the CRISPR technique. (Doudna’s university is challenging the patent.)

A peek inside an NIH lab shows how it works. Researchers inject a CRISPR-guided molecule into microscopic mouse embryos, to cause a gene mutation that a doctor suspects of causing a patient’s mysterious disorder. The embryos will be implanted into female mice that wake up from the procedure in warm blankets to a treat of fresh oranges. How the resulting mouse babies fare will help determine the gene defect’s role.

Experts predict the first attempt to treat people will be for blood-related diseases such as sickle cell, caused by a single gene defect that’s easy to reach. The idea is to use CRISPR in a way similar to a bone marrow transplant, but to correct someone’s own blood-producing cells rather than implanting donated ones.

“It’s like a race. Will the research provide a cure while we’re still alive?” asked Robert Rosen of Chicago, who has one of a group of rare bone marrow abnormalities that can lead to leukemia or other life-threatening conditions. He co-founded the MPN Research Foundation, which has begun funding some CRISPR-related studies.

So why the controversy? CRISPR made headlines last spring when Chinese scientists reported the first-known attempt to edit human embryos, working with unusable fertility clinic leftovers. They aimed to correct a deadly disease-causing gene but it worked in only a few embryos and others developed unintended mutations, raising fears of fixing one disease only to cause another.

If ever deemed safe enough to try in pregnancy, that type of gene change could be passed on to later generations. Then there are questions about designer babies, altered for other reasons than preventing disease.

In the U.S., the NIH has said it won’t fund such research in human embryos.

In Britain, regulators are considering researchers’ request to gene-edit human embryos — in lab dishes only — for a very different reason, to study early development.

Medicine aside, another issue is environmental: altering insects or plants in a way that ensures they pass genetic changes through wild populations as they reproduce. These engineered “gene drives” are in very early stage research, too, but one day might be used to eliminate invasive plants, make it harder for mosquitoes to carry malaria or even spread a defect that gradually kills off the main malaria-carrying species, said Kevin Esvelt of Harvard’s Wyss Institute for Biologically Inspired Engineering.

No one knows how that might also affect habitats, Esvelt said. His team is calling for the public to weigh in and for scientists to take special precautions. For example, Esvelt said colleagues are researching a tropical mosquito species unlikely to survive cold Boston even if one escaped locked labs.

“There is no societal precedent whatsoever for a widely accessible and inexpensive technology capable of altering the shared environment,” Esvelt told a recent National Academy of Sciences hearing.

Researchers Use ‘Avatar’ Experiments to Get Leg Up On Locomotion

Mon, 10/12/2015 – 5:09pmby North Carolina State University

North Carolina State University scientists take a giant leap closer to understanding locomotion from the leg up

http://www.mdtmag.com/news/2015/10/researchers-use-avatar-experiments-get-leg-locomotion

Simple mechanical descriptions of the way people and animals walk, run, jump and hop liken whole leg behavior to a spring or pogo stick.

But until now, no one has mapped the body’s complex physiology – which in locomotion includes multiple leg muscle-tendons crossing the hip, knee and ankle joints, the weight of a body, and control signals from the brain – with the rather simple physics of spring-like limb behavior.

Using an “Avatar”-like bio-robotic motor system that integrates a real muscle and tendon along with a computer controlled nerve stimulator acting as the avatar’s spinal cord, North Carolina State University researchers have taken a giant leap closer to understanding locomotion from the leg up. The findings could help create robotic devices that begin to merge human and machine in order to assist human locomotion.

Despite the complicated physiology involved, NC State biomedical engineer Greg Sawicki and Temple University post-doctoral researcher Ben Robertson show that if you know the mass, the stiffness and the leverage of the ankle’s primary muscle-tendon unit, you can predict neural control strategies that will result in spring-like behavior.

“We tried to build locomotion from the bottom up by starting with a single muscle-tendon unit, the basic power source for locomotion in all things that move,” said Greg Sawicki, associate professor in the NC State and UNC-Chapel Hill Joint Department of Biomedical Engineering and co-author of a paper published in Proceedings of the National Academy of Sciences that describes the work. “We connected that muscle-tendon unit to a motor inside a custom robotic interface designed to simulate what the muscle-tendon unit ‘feels’ inside the leg, and then electrically stimulated the muscle to get contractions going on the benchtop.”

The researchers showed that resonance tuning is a likely mechanism behind springy leg behavior during locomotion. That is, the electrical system – in this case the body’s nervous system – drives the mechanical system – the leg’s muscle-tendon unit – at a frequency which provides maximum ‘bang for the buck’ in terms of efficient power output.

Sawicki likened resonance tuning to interacting with a slinky toy. “When you get it oscillating well, you hardly have to move your hand – it’s the timing of the interaction forces that matters.

“In locomotion, resonance comes from tuning the interaction between the nervous system and the leg so they work together,” Sawicki said. “It turns out that if I know the mass, leverage and stiffness of a muscle-tendon unit, I can tell you exactly how often I should stimulate it to get resonance in the form of spring-like, elastic behavior.”

The findings have design implications relevant to designing exoskeletons for able-bodied individuals, as well as exoskeleton or prosthetic systems for people with mobility impairments.

“In the end, we found that the same simple underlying principles that govern resonance in simple mechanical systems also apply to these extraordinarily complicated physiological systems,” said Robertson, the corresponding author of the paper.

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Outstanding Achievement in Pathology

Posted in BioIT: BioInformatics, Biological Networks, Biomarkers & Medical Diagnostics, Bone Disease and Musculoskeletal Disease, Cancer and Current Therapeutics, CANCER BIOLOGY & Innovations in Cancer Therapy, Cancer Screening, Cardiac and Cardiovascular Surgical Procedures, Cell Biology, Cerebrovascular and Neurodegenerative Diseases, Peripheral Arterial Disease & Peripheral Vascular Surgery, tagged biomarkers, cancer biomarkers, cardiac biomarkers, Clinical Pathology, Experimental Pathology, neuropathology, Pathology, surgical pathology on September 26, 2015| Leave a Comment »

Outstanding Achievement in Pathology

Curator: Larry H Bernstein, MD, FCAP

Olympus America Honors Outstanding Pathologists During First Annual “Unsung Heroes” Awards

Melville, Ny—Tracey Corey Handy, M.D., Chief Medical Examiner of Kentucky, and Matthew Zarka, M.D., affiliated with the University of Vermont and the Fletcher Allen Health Center, were recognized as the 1999 winners of the “Unsung Heroes” Awards. The awards, sponsored by Olympus America Inc., a world leading manufacturer of microscopes, in cooperation with the College of American Pathologists (CAP), were presented at a ceremony during the Fall CAP Conference in New Orleans.

The awards are the first in the on-going “Unsung Heroes” program sponsored by Olympus for the purpose of increasing public awareness of the vital and often invisible role pathologists have in saving lives. In addition to their expertise with a microscope, pathologists are the doctors who ensure that clinical laboratory testing is reliable and that diseases are accurately diagnosed. They are on the front lines whenever the public is threatened with disease. Their role in forensic science is crucial in helping prevent people from falling prey to abuse or avoidable illness. As Dan Biondi, Olympus Senior Vice President, points out, “Olympus is committed to supporting the work of the world’s pathologists and to advocating an educated patient population.”

Dr. Tracey Corey Handy is recognized as an “Unsung Hero” for her role in upgrading the well-being of children as Kentucky’s Chief Medical Examiner. Along with several colleagues, Dr. Handy founded the state’s “Living Forensics” team in 1991. Since its inception, the team has consulted on more than 700 cases of suspected child abuse. This effort has led to an increased conviction rate of abuse perpetrators and helps to reduce further cases of child abuse. In addition, Dr. Handy has initiated a program of routine screening for metabolic defects apparent in victims of Sudden Infant Death Syndrome (SIDS), which has resulted in the correct diagnosis of conditions that would have otherwise been attributed to SIDS. Dr. Handy has also chaired the state’s first child mortality review group that has resulted in the initiation of prevention programs, particularly in the event of accidental child death. A frequent speaker and contributor of her expertise to organizations throughout the country, she also teaches forensic pathology and has been published in more than a dozen peer-reviewed journals and books.

Dr. Matthew Zarka is recognized as an “Unsung Hero” for his efforts in aiding the extremely poor Mexican-Indian population in the remote mountain regions of Oaxaca, Mexico. Over the last two years Dr. Zarka has volunteered his time and services to bring much needed medical care to these impoverished communities. He and his OB/GYN team have been setting up the very first clinics throughout the area, enjoining the coffee companies of Mexico to spread word of the clinics to the local population and to help transport patients to the clinics. After each female patient underwent a gynecological examination, Dr. Zarka stained and read her Pap test. When needed, more extensive evaluations, biopsies, treatment and counsel were provided. Overwhelmingly successful, Dr. Zarka’s outreaching medical mission has grown to include additional professional staff. By volunteering his time and expertise, Dr. Zarka provides the only real access most people of the region have to modern medical care. His contribution has undoubtedly saved lives that might otherwise have been lost.

Stanford University

Benjamin Pinsky, MD, PhD, Assistant Professor of Pathology and Medicine (Infectious Diseases) is the recipient of the 2014 Siemens Healhcare Diagnostics Young Investigator Award. This award “honors outstanding laboratory research in clinical microbiology or antimicrobial agents and is intended to further the career development of a young clinical scientist and promote awareness of clinical microbiology as a career.”

Stephen J. Galli, MD, Chair of Pathology, Professor of Pathology and Microbiology and Immunology, and the Mary Hewitt Loveless, MD Professor, is the recipient of the 2014 ASIP (American Society of Investigative Pathology) Rouse Whipple Award. This award is presented to a senior scientist with a distinguished career in research who has advanced the understanding of disease and has continued productivity at the time of this award.

Dr. Raffick Bowen, Clinical Associate Professor and Associate Medical Director of SHC’s Clinical Chemistry and Immunology Laboratory is the recipient of the American Association of Clinical Chemistry’s Outstanding Speaker Award for 2013. This award recognizes his achievement in earning a speaker evaluation rating of 4.5 or higher during a 2013 continuing education activity accredited by AACC. The title of Dr. Bowen’s presentation is “Implementation of Autoverification in a Clinical Chemistry Laboratory: Theory to Practice”

Richard Kempson, MD,

Emeritus Professor of Pathology, is the recipient of the 2014 United States and Canadian Academy of Pathology (USCAP) President’s Award. The USCAP President’s Award is given annually to recognize an individual for outstanding service to the field of pathology.

Dr. Kempson is richly deserving of this award. Dr. Kempson has not only contributed substantially to the surgical pathology literature, particularly in gynecologic and soft tissue pathology but also, with Dr. Ronald Dorfman, he trained a substantial percentage of this and the next generation’s academic and community leaders in surgical pathology.

Dr. Kempson’s affiliation with Stanford University began in 1968 when he and Dr. Ronald Dorfman were recruited to Stanford to develop a program in surgical pathology. In short order, they established an internationally recognized residency and clinical fellowship program which went on to train more than 275 pathologists in the art and science of diagnostic surgical pathology. Dr. Kempson developed a distinctive teaching style that emphasized precise diagnostic criteria, approaching diagnosis with a broad morphologic differential diagnosis, and most importantly, always highlighting the relevance to patient management of the morphologic distinctions being made.

Prior to his recruitment to Stanford, Dr. Kempson was an Assistant Professor of Pathology and Surgical Pathology at Washington University. Dr. Kempson served as an Associate Professor of Pathology at Stanford from 1968 to 1974 and a Professor of Pathology from 1974 to 2001. In addition to his academic duties, he served as Co-Director of Surgical Pathology from 1968 until 2001. He also has served as President of the Association of Directors of Surgical Pathology (1993-1995), the United States and Canadian Academy of Pathology (1996) and the Arthur Purdy Stout Society (1996) and the California Society of Pathologists. The Richard Kempson, MD, Professorship in Surgical Pathology was established by the Department of Pathology in 2002 to honor him and his remarkable contributions to surgical pathology.

University of California, San Diego

A new era in diagnostics has emerged within the concept of Personalized Medicine. Imagine selecting cancer chemotherapy drugs based on knowledge of the precise mutations in a cancer. Can we predict who may have an adverse response to a medication based on that individual’s genetic blueprint? At UCSD, we are dedicated to making these resources available to our patients in the very near future. This is why we recently established the Pathology Center for Personalized Medicine. The goal of the Center is to conduct leading research necessary to form the foundation for advanced personalized medicine diagnostic testing and then to make this testing available in the CALM. For more information on the Center for Personalized Medicine, click here.

The research enterprise in Pathology at UCSD has grown dramatically in the past five years, and we are now amongst the top 15 programs in the country. Basic and translational research laboratories in the UCSD Pathology Department tackle important problems concerning cancer development and progression, angiogenesis, stem cell biology, neurodegenerative diseases, peripheral neuropathy, inflammation, infectious diseases, and wound healing. Our laboratories provide excellent environments for learning cell biology, molecular genetics, biochemistry, and animal physiology. Our faculty includes many active participants in the Biomedical Sciences (BMS) Graduate Program. For more information on this program, click here. We also have excellent opportunities for postdoctoral researchers. Please click here to visit our web page on summarizing the Pathology Department research enterprise. Then visit individual web pages for each of our faculty member to view specific research interests.

The Department of Pathology is home to both an outstanding Comparative Pathology and Medicine Program (for more information, click here) and the UCSD Research Ethics Program. We provide major educational support to the School of Medicine and the Skaggs School of Pharmacy and Pharmaceutical Sciences. For further information on these training opportunities, click here.

The La Jolla/San Diego community is a fertile environment for research and the pharmaceutical industry. The Sanford Burnham Medical Research Institute, the Scripps Research Institute, the Sidney Kimmel Cancer Center, the Salk Institute for Biological Studies, and the La Jolla Institute for Allergy and Immunology house exciting scientific programs and provide for numerous scientific collaborations. We also boast a plethora of biotechnology companies, located nearby on the La Jolla mesa.

The overall theme and focus of the Department of Pathology is to elucidate the molecular basis and pathology of human disease. The faculty is comprised of basic, translational and physician scientists that utilize the latest techniques in genomics, proteomics, cell biology, molecular biology and physiology to develop new diagnostic and therapeutic approaches for a wide range of diseases, including cancer, neurological disease, microbial infection, and inflammatory disease.

Steven L. Gonias, M.D., Ph.D.

Our laboratory is interested in identifying and characterizing novel pathways by which proteases and their cell-surface receptors regulate cell physiology. We are particularly interested in the function of proteases in cancer but also have active projects related to peripheral nerve injury, Alzheimer’s disease and cardiovascular biology. One focus involves urokinase-type plasminogen activator (uPA), a serine protease and plasminogen activator that binds with high affinity to a GPI-anchored receptor called uPAR. This event activates multiple cell-signaling pathways that affect cell migration, survival, and phenotype. We are actively working to elucidate mechanisms by which uPAR-initiated cell-signaling promotes cancer metastasis. We are particularly interested in breast cancer, but also work on prostate cancer and cancers of the central nervous system.

The complex of uPA with its inhibitor, PAI-1, is a ligand for a receptor called LRP-1. LRP-1 also is the receptor for other ligands, including extracellular matrix proteins, growth factors and foreign toxins. Our laboratory elucidated a pathway in which LRP-1 regulates cell-signaling indirectly, by regulating the cell-surface level of uPAR. However, recent studies suggest that LRP-1 also directly regulates cell-signaling by binding adaptor proteins, such as Shc and JIP. By this mechanism, LRP-1 regulates cell survival and gene transcription. Our current research is aimed at determining the role of LRP-1 in cancer and peripheral nerve injury, using in vitro and in vivomodel systems. Using proteomics approaches, we also are actively investigating the ability of LRP-1 to model the composition of the plasma membrane.

Our third area of focus concerns the plasma protease inhibitor, alpha2M. Our laboratory has demonstrated that this protein functions as a conformation-dependent carrier of growth factors. Alpha2M may also function in cell-signaling by binding to LRP-1. By site-directed mutagenesis, we have isolated and individually modified various functional sites in this multifunctional protein.

David Bailey, MD, PhD

David N. Bailey received his Bachelor of Science degree in Chemistry “with high distinction” from Indiana University and his Doctor of Medicine degree from Yale University. He completed a National Institutes of Health postdoctoral fellowship in Laboratory Medicine and a residency in Clinical Pathology, both at Yale, serving as Chief Resident in his final year. He is certified in Clinical Pathology and Chemical Pathology by the American Board of Pathology.

Dr. Bailey joined the University of California (UC) San Diego faculty in 1977 and served as Director of the Toxicology Laboratory of UC San Diego Medical Center (1977-2007), Head of the Division of Laboratory Medicine (1983-1989, 1994-1998), Acting Chair (1986-1988) and permanent Chair of the Department of Pathology (1988-2001), Director of the Pathology Residency Program (1986-1999), Director of Clinical Laboratories of UCSD Medical Center (1982-1999), Interim Vice Chancellor for Health Sciences and Dean of the UC San Diego School of Medicine (1999-2000 and 2006-2007), Deputy Vice Chancellor for Health Sciences (2001-2007), and Dean for Faculty & Student Matters in UC San Diego School of Medicine (2003-2007). From 2007 to 2009, he was Vice Chancellor for Health Affairs, Dean of the School of Medicine, and Professor of Pathology and Laboratory Medicine at the University of California, Irvine.

Dr. Bailey was recognized by the Institute of Scientific Information as one of the world’s ten most cited authors in forensic sciences (1981-93). He received the Gerald T. Evans Award from the Academy of Clinical Laboratory Physicians and Scientists in 1993 for his leadership and service to the Academy. Dr. Bailey has served as President of the California Association of Toxicologists (1981-1982), President of the Academy of Clinical Laboratory Physicians and Scientists (1988-89), and Secretary-Treasurer of the Association of Pathology Chairs (1996-99). He has also served on the Chemical Pathology Test Development and Advisory Committee of the American Board of Pathology; the Editorial Boards of Clinical Chemistry, the Journal of Analytical Toxicology, and the American Journal of Clinical Pathology; the Doris A. Howell Foundation for Women’s Health Research Board of Directors; the Board of Directors of the George G. Glenner Alzheimer’s Family Centers, Inc.; the Board of Directors of the Children’s Hospital of Orange County; the Board of Directors of Children’s Healthcare of California; the Board of Directors of the Rady Children’s Hospital of San Diego; the Board of Directors of the Veterans Medical Research Foundation (San Diego); and the Executive Committee and Governing Board of the California Institute of Telecommunications and Information Technology, among others.

David A. Herold, M.D., Ph.D.

My laboratory research interests are in the area of mass spectrometry application to clinical diagnostics. This includes prostaglandins, trace metal and steroids. Additionally, we has been involved in the development and validation of “classical” clinical chemistry diagnostic tests. The application of the mass spectrometry to determine the validity of endocrine tests, in particular testosterone, has been of particular interest. We have been using GC-MS, LC-MS, and MS-MS techniques for these investigations. At the present time, we are involved with the use of Accelerator Mass Spectrometry for the determination of calcium flux in serum and urine using 41Ca as a marker. The purpose of these studies is to better understand bone remodeling in normal and diseased patients. We have also investigated the use of microfluidics for the application to clinical diagnostics to measure selected proteins in a rapid and accurate manner.

David Cheresh, Ph.D.

Tumor growth, invasion, stem cells and drug resistance. Molecular regulation of tumor growth and angiogenesis. Drug development targeting molecular pathways involved in tumor growth metastasis and angiogenesis.

The Cheresh laboratory focuses on the discovery of molecular pathways involved in the progression of cancer. Cheresh’s earlier work identified integrin αυβ3 as a biomarker of tumor angiogenesis and tumor progression, and was involved in the discovery of a drug called cilentigide which targets integrins αυβ3 and αυβ5.

The Cheresh laboratory has identified a series of critical microRNAs that regulate the growth of blood vessels. These microRNAs control the angiogenic switch that occurs during the earliest stages of tumor growth and neovascularization in the retina. As such one of these microRNAs may have therapeutic application as it is capable of maintaining blood vessels in the quiescent state.

Cheresh and colleagues have identified integrin αυβ3 as a biomarker of tumor stem cells during intrinsic or acquired resistance of a wide range of tumors including: cancer of the lung, pancreas, breast, and colon. Cheresh and his lab discovered that αυβ3 expression is both necessary and sufficient to account for tumor stemness and drug resistance based on its ability to drive a molecular pathway regulating these processes. This has led to the development of new therapeutic strategies to resensitize patients to drugs such as erlotinib and lapatinib that target EGFR.

The Cheresh laboratory has identified RAF kinase as an important target involved in tumor growth and angiogenesis. They have developed a new drug design strategy to target RAF and other relevant kinases by designing allosteric inhibitors of these targets. This is based on the use of defined chemical scaffolds to dock into an allosteric pocket on these kinases to render them inactive. The combined use of in silico and biological screening has yielded drugs with nM anti-tumor activity that produce strong anti-tumor growth in mouse models following once a day oral dosing. This approach appears to yield drugs that target tumors that are resistant to ATP mimetic inhibitors of RAF, Kit or PDGFR

John Lowe

Senior Director, Pathology

I joined Genentech in 2008 as Senior Director of Pathology, after having spent more than 18 years as an HHMI Investigator at the University of Michigan and then 3 years as Chair of Pathology at Case Western Reserve University School of Medicine. The role of Senior Director of Pathology in Research at Genentech offered attractive opportunities to do research in an outstanding, disease-focused scientific environment, while also helping to lead the scientific and research support activities of the Pathology department. These latter efforts help Genentech continue to make a major positive difference to the health and well being of a large number of patients afflicted with cancer, autoimmune syndromes, neurodegenerative diseases and other illnesses for which therapies are unsatisfactory or nonexistent.

An exceptional team of pathologists, laboratory managers, scientific associates and administrative staff in the department collaborate with me in these efforts. Additional outstanding pathologists, scientists, and managers continue to be recruited to assist us in ensuring that the department performs at the highest level. Our task is made more straightforward by the environment at Genentech, which is characterized by exceptionally bright, motivated and collaborative colleagues at every level, spectacular facilities, and workplace philosophies that are conducive to the highest levels of achievement.

Postdoctoral Mentor

The opportunity to mentor postdoctoral fellows at Genentech has been a stimulating and gratifying experience for me. This derives in part from the freedom afforded by the program to pursue research directions that are deemed to be important and interesting, even if these have no immediate therapeutic relevance. The special mentoring experience also derives from extraordinary breadth and quality of the core laboratories at Genentech, and the spectacular intellectual environment. Together, these circumstances provide an unparalleled opportunity for postdoctoral fellows, and their mentors, to engage in biomedical discovery of the highest caliber.

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Outstanding Achievement in Anesthesiology

Posted in Academic Publishing, Cardiac and Cardiovascular Surgical Procedures, Cardiovascular and Vascular Systems, Cerebrovascular and Neurodegenerative Diseases, Clinical & Translational, tagged anesthesiology, Pain management, Pain Therapy, surgical procedures on September 26, 2015| Leave a Comment »

Outstanding Achievement in Anesthesiology

Curator: Larry H Bernstein, MD, FCAP

Thomas J. Rick, MD, for Outstanding Achievements in Anesthesiology and Pain Management

The International Association of HealthCare Professionals has carefully selected Thomas J. Rick, MD, to represent anesthesiology in their publication, The Leading Physicians of the World. Dr. Rick’s selection is a significant representation of his enduring passion and complete dedication for the field of anesthesiology and pain management. He is considered to be among the best throughout his 19 years in practice.

A well versed and respected anesthesiologist and pain management physician practicing in Phoenix, Arizona, Dr. Rick features a track record of achievements that have marked his 19-year professional journey in his challenging specialty. In a relaxed and friendly setting with accommodating support staff, Dr. Rick receives his at Thomas J. Rick, MD PC, his well equipped private office where he deals with anesthetic and pain management services for patients undergoing surgeries. While he primarily devotes his time to his office in Phoenix, he additionally provides anesthesiology-related services to patients of the St. Joseph’s Hospital and Medical Center and Banner Good Samaritan Medical Center, also in Phoenix, Arizona.

Dr. Rick embraced a career in medicine upon completing his medical degree in 1994 at Hahnemann University Hospital. His postgraduate training led him to the University of Arizona and his continuing learning enhanced his certification in anesthesiology by the American Board of Anesthesiology. As an affirmation of his commitment to education and his desire to advance by adapting his practice to the latest discoveries and technologies in his field, Dr. Rick joined the American Society of Anesthesiology and the Arizona Society of Anesthesiology. An active man in his free time, passionate by tennis, fitness, and drums, he attributes his exceptional success to his availability, as well affordability and accessibility of service.

Stanford Medical School

Myer “Mike” Rosenthal

Ellis N. Cohen Award for Outstanding Achievement in Anesthesiology, Stanford University Department of Anesthesia (1980)
Jack R. Collins Memorial Award for Outstanding Leadership in Anesthesia Education, Dannemiller Society (1990)
Kaiser Award for Clinical Teaching, Stanford University School of Medicine (1991, 2004)

Board of Directors (President and Chairman of Board – 2001-2004), Foundation for Anesthesia Education and Research (2000 – 2009)
Director (President – 1997-1998), American Board of Anesthesiology (1986 – 1998)
Medical Director of Intensive and Intermediate Intensive Care Units, Stanford University Hospital (1975 – 1997)

Arthur Bert, MD

Senior staff anesthesiologist, Rhode Island Hospital

Arthur Bert, MD, has served as director of cardiac anesthesia (1986-2002) at Rhode Island Hospital and as director of pediatric cardiac anesthesia (1996-2005) at Hasbro Children’s Hospital. Bert continues to pursue his interests in adult and pediatric cardiovascular and thoracic anesthesia as a senior staff anesthesiologist. He is a clinical professor of surgery (anesthesiology) at the the Warren Alpert Medical School at Brown University. He also holds the position of director of experimental cardiac surgery, anesthesiology and cardiac imaging at the cardiac surgery research laboratories of Children’s Mercy Hospital, in Kansas City, MO, where he is part of a funded research team that is growing tissue-engineered heart valves. He is a consultant anesthesiologist at Women & Infants Hospital for neonatal anesthesia.

Education

Bert graduated as president of Alpha Omega Alpha Medical Honor Society from Mount Sinai School of Medicine in New York City. He served as a resident in internal medicine at Beth Israel Deaconess Medical Center in Boston and was awarded the Dr. Nathan Sidel Prize for outstanding achievement. He completed his anesthesia residency and an adult cardiac anesthesia fellowship at Beth Israel Hospital in Boston, followed by a pediatric anesthesia fellowship at Children’s Hospital Boston.

Board Certification

Diplomate of the American Board of Anesthesiology (1985) and re-certified in 2008

Testamur of the National Board of Echocardiography in Perioperative Transesophageal Echocardiography (1998)

Diplomate (2006) and re-certified in 2007

Awards

Top Physicians, Rhode Island Monthly magazine (2000, 2002, 2004, 2006 and 2008)

Guide to America’s Top Physicians, Consumers’ Research Council of America, Washington, DC (2005, 2006)

Teaching Recognition Award, Brown Medical School (2005)

Dr. Charles A. Hill Award from the RI Medical Society (2006)

Interests

Applications of transesophageal echocardiography to intraoperative patient management

Techniques of reducing blood product transfusions during surgery

Cerebral function monitoring during general anesthesia

Research: Echocardiographic evaluation of tissue-engineered valve function

ASA Award for Excellence in Research

Henrik Kehlet, M.D., Ph.D.

The annual ASA Award for Excellence in Research recognizes an individual for outstanding achievement in research that has or is likely to have an important impact on the practice of anesthesiology.

The individual’s work must represent a body of original, mature and sustained contribution to the advancement of the science of anesthesiology. The nominee need not be a physician, an anesthesiologist or a member of ASA, but must be presently engaged in research related to anesthesiology, academically accomplished with peer-reviewed publications and funded research, and nominated in response to a call for nominations. The completed application must include the nominee’s current curriculum vitae, a letter of nomination and a seconding letter from two individuals with an understanding of the research contributions of the individual.

The 2014 Award for Excellence in Research was presented to Henrik Kehlet, M.D., Ph.D., at the ANESTHESIOLOGY™ 2014 annual meeting in New Orleans on Monday, October 13, 2014. Dr. Kehlet is a Professor at Rigshospitalet, Copenhagen University, Denmark.

Dr. Kehlet is known for his research and writing in surgical pathophysiology, surgical stress response and the transition from acute to chronic pain, among other topics.

Henrik Kehlet, M.D., Ph.D. is perhaps the most well-known surgeon among physician anesthesiologists around the world due to his substantial contributions toward the understanding of surgical pathophysiology. After Dr. Kehlet completed his medical studies and surgical residency at the University of Copenhagen, Denmark, he enrolled in a Ph.D. program within the same institution, authoring a thesis pertaining to the study of the hypothalamic-pituitary-adrenocortical function in glucocorticoid-treated surgical patients. Dr. Kehlet served as the Chief of Surgery and Professor of Surgery, Copenhagen University at Hvidovre University Hospital from 1989 to 2004. He was subsequently appointed as a Professor of Perioperative Therapy and Head of the Section for Surgical Pathophysiology at the Rigshospitalet in Copenhagen. Dr. Kehlet continues to be an extremely prolific writer, having authored more than 950 scientific articles covering topics of surgical pathophysiology, acute pain physiology and pharmacotherapy, surgical stress response, regional anesthesia and analgesia, perioperative immune function, fast-track surgery and the transition from acute to chronic pain.

Dr. Kehlet’s research led to the creation of the concept of fast-track surgery, or enhanced recovery after surgery (ERAS), with the aim of painless and safe surgeries. His work related to pain relief and surgical outcomes led to the multimodal analgesia approach of combining different analgesics for better pain control and fewer side effects that is widely used today. Dr. Kehlet also is credited with the concept of pre-emptive analgesia, or administering an analgesic prior to surgical injury in order to decrease the intensity and duration of postoperative pain. In addition to his many contributions to perioperative pain management, Dr. Kehlet is responsible for establishing the first nationwide hernia database in Denmark, with the purpose of optimizing outcomes and documenting different approaches to improve care.

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Proteins that control neurotransmitter release

Posted in Academic Publishing, Biological Networks, Cell Biology, Cerebrovascular and Neurodegenerative Diseases, Clinical & Translational, Clinical Genomics, Disease Biology, Gene Regulation, Genetics & Pharmaceutical, Genome Biology, Innovations in Neurophysiology & Neuropsychology, Intellectual Property, International Global Work in Pharmaceutical, Investment in Technological Breakthrough, Molecular Genetics & Pharmaceutical, Neurological Diseases, Personalized and Precision Medicine & Genomic Research, Pharmaceutical Analytics, Pharmaceutical Drug Discovery, Pharmaceutical Industry Competitive Intelligence, Pharmacogenomics, Population Health Management, Proteins, Proteomics, Regulated Clinical Trials: Design, Methods, Components and IRB related issues, Schizophrenia, Signaling & Cell Circuits, Small Molecules in Development of Therapeutic Drugs, tagged Genentech, Neuroscience, Richard H. Scheller, Roche Holding, Stanford University School of Medicine on September 6, 2015| Leave a Comment »

Proteins that control neurotransmitter release

Author & Curator: Larry H. Bernstein, MD, FCAP

Richard H. Scheller, PhD

The sec6/8 Complex Is Located at Neurite Outgrowth and Axonal Synapse-Assembly Domains

Christopher D. Hazuka, Davide L. Foletti, Shu-Chan Hsu, Yun Kee, F. Woodward Hopf, and Richard H. Scheller
Department of Molecular and Cellular Physiology, Howard Hughes Medical Institute, Stanford University School of Medicine, Stanford, California 94305-5428

The Journal of Neuroscience, February 15, 1999, 19(4):1324–1334 http://www.jneurosci.org/content/19/4/1324.full.pdf

The molecules that specify domains on the neuronal plasma membrane for the delivery and accumulation of vesicles during neurite outgrowth and synapse formation are unknown. We investigated the role of the sec6/8 complex, a set of proteins that specifies vesicle targeting sites in yeast and epithelial cells, in neuronal membrane trafficking. This complex was found in layers of developing rat brain undergoing synaptogenesis. In cultured hippocampal neurons, the sec6/8 complex was present in regions of ongoing membrane addition: the tips of growing neurites, filopodia, and growth cones. In young axons, the sec6/8 complex was also confined to periodic domains of the plasma membrane. The distribution of synaptotagmin, synapsin1, sec6, and FM1–43 labeling in cultured neurons suggested that the plasma membrane localization of the sec6/8 complex preceded the arrival of synaptic markers and was downregulated in mature synapses. We propose that the sec6/8 complex specifies sites for targeting vesicles at domains of neurite outgrowth and potential active zones during synaptogenesis. Key words: synaptogenesis; neurotransmission; secretion; exocytosis; synaptic vesicle; vesicle targeting

Targeting of vesicles to synaptic sites during development may use similar mechanisms as those involved in vesicle fusion underlying membrane outgrowth. Before contact with a postsynaptic target, axons possess mobile vesicle clusters bearing synaptotagmin, which fuse with the plasma membrane after stimulation (Matteoli et al., 1992; Kraszewski et al., 1995; Dai and Peng, 1996). Thus, growing axons must contain the molecular machinery required for constitutive exocytosis, endocytosis, and activitydependent vesicle release. However, it is unclear how vesicles become clustered at synapses. Although vesicle fusion in axons might occur anywhere along the plasma membrane, there must be membrane targets that signal the clustering of vesicles for synapse formation. Furthermore, it is unclear how sites of vesicle exocytosis are modified as the neuron forms stable contacts with postsynaptic partners.

Identification of a Novel Rab11/25 Binding Domain Present in Eferin and Rip Proteins

Rytis Prekeris*, Jason M. Davies*, and Richard H. Scheller#
JBC Papers in Press. Published on July 31, 2001 as Manuscript M106133200
http://www.jbc.org/content/early/2001/07/31/jbc.M106133200.full.pdf

Rab11, a low molecular weight GTP binding protein, has been shown to play a key role in a variety of cellular processes, including endosomal recycling, phagocytosis, and transport of secretory proteins from the TGN. In this study we describe a novel Rab11 effector, EF hands containing Rab11 interacting protein (eferin). In addition, we identify a 20 amino acid domain that is present at the C-terminus of eferin and other Rab11/25 interacting proteins, such as Rip11 and nRip11. Using biochemical techniques we demonstrate that this domain is necessary and sufficient for Rab11 binding in vitro and that it is required for localization of Rab11 effector proteins in vivo. The data suggest that various Rab effectors compete with each other for the binding to Rab11/25 possibly accounting for the diversity of Rab11 functions.

Members of the Rab/Ypt GTPase family have emerged as important regulators of vesicular trafficking (1). Rab proteins have been proposed to mediate a variety of functions, including vesicle translocation and docking at a specific fusion sites. Like all small GTPases, Rabs cycle between active (GTP bound) and inactive (GDP bound) conformations (2). In the GTP bound state, Rab proteins can bind a variety of downstream effector proteins, while GTP hydrolysis leads to a conformational change in the “switch” region that renders the Rab GTPase unrecognizable to its effector proteins (3,4). A key question in understanding the interactions between Rabs and their effectors concerns the mechanisms by which Rab GTPases specifically bind a diverse spectrum of effectors and how this is regulated by the common structural motif used as a GTP switch. Biochemical and genetic studies have identified several hypervariable regions that might be involved in determining Rab specificity, including N- and C-termini, as well as α3/β5 by guest on September 6, 2015 http://www.jbc.org/ Downloaded from loop (5,6). Indeed, the recently reported structure of Rab3a bound to a putative effector, rabphillin-3a, revealed that Rab3a/rabphillin-3a complex interacts through two main regions (7). The first consists of conformationally sensitive “switch” regions of Rab3a bound to the a1 helix and the C-terminal part of rabphillin-3a. The second involves the SGAWFF domain of rabphillin-3a which fits into a pocket formed by the three hypervariable complementary determining regions (CDRs) of Rab3a, corresponding to the N- and C-termini and α3/β5 loop. Thus, it appears that the hypervariable RabCDR are involved in determining the specificity of effector binding, while the conserved “switch” regions impart GTP dependency and binding. It remains to be determined, however, whether this paradigm also applies to other Rab/effector complexes. Rab11a, -11b, and -25 are closely related members of Rab GTPase family that have been implicated in regulating a variety of different post-Golgi trafficking pathways, such as protein recycling (8), phagocytosis (9), insulin-stimulated Glut4 insertion in the plasma membrane (10), and membrane trafficking from early endosomes to the transGolgi network (11). During the last few years several Rab11/25 interacting proteins have been identified, including Rab11BP/Rabphilin-11, Rip11, nRip11, and myosin Vb (12- 15). However, the mechanisms of their function, as well as molecular aspects of their interactions with Rab11, remain to be fully understood. In the present study, we report the identification of EF-hands containing Rab11/25 interacting protein (eferin). Furthermore, we characterized a Rab binding domain (RBD11) which is present at the Cterminus of eferin as well as other Rab11/25 binding proteins, such as Rip11 and nRip11. Using biochemical techniques we demonstrated that RBD11 is the region which encodes the specificity for Rab11/25, but is distinct from the region interacting with Rab “switch” domain, since its interactions with the Rab11/25 are not GTP-dependent.

The functional significance of the differences in Rip and eferin interactions with Rab11/25 remains to be determined. One possibility is that additional cellular factors can regulate the affinity of Rab11/25 binding to its effectors. Indeed, the recombinant full length Rip11 binds poorly to Rab11a in pull down and yeast-two hybrid assays as compared to full length endogenous Rip11 from cellular TX-100 extracts (data not shown). Furthermore, it has been previously shown that Rip11 can also interact with γSNAP and cytoskeleton (13,24). Thus, the interactions of Rips and eferin with different factors could be used as a means of differentially regulating Rab11/25 binding. Alternatively, the Rab11/25 binding motif in eferin and Rip11 might be conformationlly hidden and require activation before binding to Rab11/25. We have previously demonstrated that phosphorylation of Rip11 plays an important role in its trafficking (13). Thus, differential phosphorylation on Rab11/25 binding motifs could also play a role in regulating the binding of Rip11 and eferin to Rab GTPases. Despite to recent progress in understanding the roles of Rabs and their effectors in regulating membrane trafficking, we are only beginning to unravel the structural determinants of their function. Identification and characterization of the Rab11/25 binding regions in Rip and Eferin proteins will be of a crucial importance in understanding the molecular mechanisms involved in differential regulation of the variety of Rab11-dependent trafficking pathways.

Rapid identification of reactive cysteine residues for site-specific labeling of antibody-Fabs.

J. Immunol. Methods

J Immunol Methods 2008 Mar 14;332(1-2):41-52. Epub 2008 Jan 14.

Jagath R Junutula, Sunil Bhakta, Helga Raab, Karen E Ervin, Charles Eigenbrot, Richard Vandlen, Richard H Scheller,Henry B Lowman

Genentech Inc., 1DNA Way, South San Francisco, California, 94080, United States. jagath@gene.com

Cysteines with reactive thiol groups are attractive tools for site-specific labeling of proteins. Engineering a reactive cysteine residue into proteins with multiple disulfide bonds is often a challenging task as it may interfere with structural and functional properties of the protein. Here we developed a phage display-based biochemical assay, PHESELECTOR (Phage ELISA for Selection of Reactive Thiols) to rapidly screen reactive thiol groups on antibody fragments without interfering with their antigen binding, using trastuzumab-Fab (hu4D5Fab) as a model system

Site-specific conjugation of a cytotoxic drug to an antibody improves the therapeutic index.

Nat. Biotechnol.

Nat Biotechnol 2008 Aug 20;26(8):925-32. Epub 2008 Jul 20.

Genentech Inc, South San Francisco, California 94080, USA.

Antibody-drug conjugates enhance the antitumor effects of antibodies and reduce adverse systemic effects of potent cytotoxic drugs. However, conventional drug conjugation strategies yield heterogenous conjugates with relatively narrow therapeutic index (maximum tolerated dose/curative dose). Using leads from our previously described phage display-based method to predict suitable conjugation sites, we engineered cysteine substitutions at positions on light and heavy chains that provide reactive thiol groups and do not perturb immunoglobulin folding and assembly, or alter antigen binding.

Isolation and characterization of a Drosophila neuropeptide gene.

Neuron

Neuron 2008 Nov;60(3):400-1

Richard H Scheller, John R Nambu

Engineered thio-trastuzumab-DM1 conjugate with an improved therapeutic index to target human epidermal growth factor receptor 2-positive breast cancer.

Clin. Cancer Res.

Clin Cancer Res 2010 Oct 30;16(19):4769-78. Epub 2010 Aug 30.

Genentech, Inc., South San Francisco, California 94080, USA. jagath@gene.com

Antibody drug conjugates (ADCs) combine the ideal properties of both antibodies and cytotoxic drugs by targeting potent drugs to the antigen-expressing tumor cells, thereby enhancing their antitumor activity. Successful ADC development for a given target antigen depends on optimization of antibody selection, linker stability, cytotoxic drug potency, and mode of linker-drug conjugation to the antibody. Here, we systematically examined the in vitro potency as well as in vivo preclinical efficacy and safety profiles of a heterogeneous preparation of conventional trastuzumab-mcc-DM1 (TMAb-mcc-DM1) ADC with that of a homogeneous engineered thio-trastuzumab-mpeo-DM1 (thioTMAb-mpeo-DM1) conjugate.

Primary cilia membrane assembly is initiated by Rab11 and transport protein particle II (TRAPPII) complex-dependent trafficking of Rabin8 to the centrosome.

Proc. Natl. Acad. Sci. U.S.A.

Proc Natl Acad Sci U S A 2011 Feb 27;108(7):2759-64. Epub 2011 Jan 27.

Christopher J Westlake, Lisa M Baye, Maxence V Nachury, Kevin J Wright, Karen E Ervin, Lilian Phu, Cecile Chalouni,John S Beck, Donald S Kirkpatrick, Diane C Slusarski, Val C Sheffield, Richard H Scheller, Peter K Jackson

Genentech, South San Francisco, CA 94080, USA.

Sensory and signaling pathways are exquisitely organized in primary cilia. Bardet-Biedl syndrome (BBS) patients have compromised cilia and signaling. BBS proteins form the BBSome, which binds Rabin8, a guanine nucleotide exchange factor (GEF) activating the Rab8 GTPase, required for ciliary assembly.

Conjugation site modulates the in vivo stability and therapeutic activity of antibody-drug conjugates.

Nat. Biotechnol.

Nat Biotechnol 2012 Feb 22;30(2):184-9. Epub 2012 Jan 22.

Genentech Inc., 1 DNA Way, S. San Francisco, California, USA.

The reactive thiol in cysteine is used for coupling maleimide linkers in the generation of antibody conjugates. To assess the impact of the conjugation site, we engineered cysteines into a therapeutic HER2/neu antibody at three sites differing in solvent accessibility and local charge. The highly solvent-accessible site rapidly lost conjugated thiol-reactive linkers in plasma owing to maleimide exchange with reactive thiols in albumin, free cysteine or glutathione.

Rac and Rab GTPases dual effector Nischarin regulates vesicle maturation to facilitate survival of intracellular bacteria.

EMBO J.

EMBO J 2013 Mar 5;32(5):713-27. Epub 2013 Feb 5.

Coenraad Kuijl, Manohar Pilli, Suresh K Alahari, Hans Janssen, Poh-Sim Khoo, Karen E Ervin, Monica Calero,Sobhanaditya Jonnalagadda, Richard H Scheller, Jacques Neefjes, Jagath R Junutula

Genentech Inc., South San Francisco, CA 94080, USA. kuijlc@gene.com

The intracellular pathogenic bacterium Salmonella enterica serovar typhimurium (Salmonella) relies on acidification of the Salmonella-containing vacuole (SCV) for survival inside host cells. The transport and fusion of membrane-bound compartments in a cell is regulated by small GTPases, including Rac and members of the Rab GTPase family, and their effector proteins. However, the role of these components in survival of intracellular pathogens is not completely understood.

In search of the molecular mechanism of intracellular membrane fusion and neurotransmitter release.

Nat. Med.

Nat Med 2013 Oct;19(10):1232-5

Richard H Scheller

Genentech Research and Early Development, 1 DNA Way, San Francisco, California, USA.

An improved and robust DNA immunization method to develop antibodies against extracellular loops of multi-transmembrane proteins.

MAbs

MAbs 2014 Jan-Feb;6(1):95-107

Meredith Hazen, Sunil Bhakta, Rajesh Vij, Steven Randle, Dara Kallop, Vicki Chiang, Isidro Hötzel, Bijay S Jaiswal, Karen E Ervin, Bing Li, Robby M Weimer, Paul Polakis, Richard H Scheller, Jagath R Junutula, Jo-Anne S Hongo

Multi-transmembrane proteins are especially difficult targets for antibody generation largely due to the challenge of producing a protein that maintains its native conformation in the absence of a stabilizing membrane. Here, we describe an immunization strategy that successfully resulted in the identification of monoclonal antibodies that bind specifically to extracellular epitopes of a 12 transmembrane protein, multi-drug resistant protein 4 (MRP4). These monoclonal antibodies were developed following hydrodynamic tail vein immunization with a cytomegalovirus (CMV) promoter-based plasmid expressing MRP4 cDNA and were characterized by flow cytometry.

Site-specific trastuzumab maytansinoid antibody-drug conjugates with improved therapeutic activity through linker and antibody engineering.

J. Med. Chem.

J Med Chem 2014 Oct 18;57(19):7890-9. Epub 2014 Sep 18.

Genentech, Inc. , 1 DNA Way, South San Francisco, California 94080, United States.

Antibody-drug conjugates (ADCs) have a significant impact toward the treatment of cancer, as evidenced by the clinical activity of the recently approved ADCs, brentuximab vedotin for Hodgkin lymphoma and ado-trastuzumab emtansine (trastuzumab-MCC-DM1) for metastatic HER2+ breast cancer. DM1 is an analog of the natural product maytansine, a microtubule inhibitor that by itself has limited clinical activity and high systemic toxicity. However, by conjugation of DM1 to trastuzumab, the safety was improved and clinical activity was demonstrated.

Richard H Scheller, PhD

Published on 16 Sep 2014

The Keck School of Medicine of USC is the first medical school in the nation to host the Lasker Lectures, featuring recipients of the prestigious 2013 Albert Lasker Basic Medical Research Award. In this installment, Richard H. Scheller, PhD, executive vice president of Genentech research and early development, discusses breakthoughs in drug development that are turning the tide in the war against cancer.

https://www.youtube.com/watch?v=Fx54EVJMcxM

Kavli Prize 2015

Xenon Pharmaceuticals Appoints Dr. Richard H. Scheller to Its Board of Directors http://fb.me/78z2HoJKa

Biopharmaceutical company Xenon Pharmaceuticals (NasdaqGM:XENE) reported on Monday the addition of Richard H. Scheller, PhD to its board of directors.

Most recently, Dr Scheller has served as chief science officer and head of Therapeutics at 23andMe.

Previously Dr Scheller was the executive vice president at Genentech Research and Early Development & a member of the Roche Corporate Executive Committee; chief scientific officer, executive vice president of Research and senior vice president of Research at Genentech; as well as a professor of Molecular and Cellular Physiology and of Biological Sciences at Stanford University Medical Center and an investigator of the Howard Hughes Medical Institute.

Dr Scheller is currently an adjunct professor in the Department of Biochemistry and Biophysics, School of Medicine at the University of California, San Francisco.

He has been a Director at Xenon Pharmaceuticals Inc. since March 16, 2015 and Medrio, Inc. since November 2012. He serves as a Member of the Medical and Scientific Review Board of Evotec (US), Inc. (Renovis Inc.). In 2014, he was named a trustee of Caltech. He served as a Member of Scientific Advisory Board of Intra-Cellular Therapies, Inc. and Rinat Neuroscience Corporation.

He served on numerous advisory boards including the National Advisory Mental Health Council of the National Institutes of Health. Dr. Scheller served as chairman of the Genentech Foundation’s board of directors. He is a globally recognized leader in biomedical research.

He has published over 200 papers in scientific journals, and worked in cell biology. He has received several additional awards for his work elucidating the molecular mechanisms governing neurotransmitter release, including the 2013 Albert Lasker Basic Medical Research Award, the 2014 California Institute of Technology’s Caltech Distinguished Alumni Award, the 2010 Kavli Prize in Neuroscience, and the 1997 U.S. National Academy of Sciences Award in Molecular Biology. He is a Fellow of the American Academy of Arts and Sciences. Dr. Scheller holds a Doctorate in Chemistry from the California Institute of Technology in 1980, where he was also a Postdoctoral Fellow, Division of Biology. He was also a Postdoctoral Fellow at Columbia University, College of Physicians & Surgeons. He has Bachelor’s Degree in Biochemistry in 1975 at the University of Wisconsin, Madison.

Education: 1971-1975 University of Wisconsin-Madison B.S. – Biochemistry with Honors 1975-1980 California Institute of Technology Ph.D. – Chemistry – Advisor: Eric H. Davidson 1980-1981 California Institute of Technology Postdoctoral Fellow-Division of Biology Advisor: Eric H. Davidson 1981-1982 Columbia University-College of Physicians & Surgeons Postdoctoral Fellow-Molecular Neurobiology Advisors: Richard Axel and Eric R. Kandel Industry Positions: 2001-2003 Senior Vice President – Research Genentech, Inc. 2003-2009 Executive Vice President – Research Genentech, Inc. 2008-2009 Chief Scientific Officer and Executive Vice President – Research Genentech, Inc. 2009- Executive Vice President – Genentech Research and Early Development (gRED) and Member of the Enlarged Roche Corporate Executive Committee Academic Appointments: 1982-1987 Assistant Professor, Department of Biological Sciences, Stanford University 1987-1990 Associate Professor, Department of Biological Sciences, Stanford University 1990-1993 Associate Professor, Department of Molecular and Cellular Physiology, Stanford University Associate Professor (by courtesy), Department of Biological Sciences, Stanford University

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Where is the most promising avenue to success in Pharmaceuticals with CRISPR-Cas9?

Posted in Advanced Drug Manufacturing Technology, Cancer and Current Therapeutics, Cerebrovascular and Neurodegenerative Diseases, Chemical Genetics, Clinical & Translational, CRISPR/Cas9 & Gene Editing, Curation, Disease Biology, Drug Delivery Platform Technology, Drug Toxicity, Embryology, Genetics & Innovations in Treatment, Genetics & Pharmaceutical, Genome Biology, Historical relevance, Technology Advance Assessment of, tagged CRISPR-Cas9, pharmaceutical application on September 1, 2015| Leave a Comment »

Where is the most promising avenue to success in Pharmaceuticals with CRISPR-Cas9?

Author: Larry H. Bernstein, MD, FCAP

2.1.2.3

Where is the most promising avenue to success in Pharmaceuticals with CRISPR-Cas9? Volume 2 (Volume Two: Latest in Genomics Methodologies for Therapeutics: Gene Editing, NGS and BioInformatics, Simulations and the Genome Ontology), Part 2: CRISPR for Gene Editing and DNA Repair

There has been a rapid development of methods for genetic engineering that is based on an initial work on bacterial resistance to viral invasion. The engineering called RNA inhibition (RNA_i) has gone through several stages leading to a more rapid and more specific application with minimal error.

It is a different issue to consider this application with respect to bacterial, viral, fungal, or parasitic invasion than it would be for complex human metabolic conditions and human cancer. The difference is that humans and multi-organ species are well differentiated systems with organ specific genome translation to function.

I would expect to see the use of genomic alteration as most promising in the near term for the enormous battle against antimicrobial, antifungal, and antiparasitic drug resistance. This could well be expected to be a long-term battle because of the invading organisms innate propensity to develop resistance.

A CRISPR/Cas system mediates bacterial innate immune evasion and virulence

Timothy R. Sampson, Sunil D. Saroj, Anna C. Llewellyn, Yih-Ling Tzeng & David S. Weiss

Affiliations, Contributions, Corresponding author

Nature 497, 254–257 (09 May 2013), http://dx.doi.org:/10.1038/nature12048

CRISPR/Cas (clustered regularly interspaced palindromic repeats/CRISPR-associated) systems are a bacterial defence against invading foreign nucleic acids derived from bacteriophages or exogenous plasmids¹^,²^,³^,⁴. These systems use an array of small CRISPR RNAs (crRNAs) consisting of repetitive sequences flanking unique spacers to recognize their targets, and conserved Cas proteins to mediate target degradation⁵^,⁶^,⁷^,⁸. Recent studies have suggested that these systems may have broader functions in bacterial physiology, and it is unknown if they regulate expression of endogenous genes⁹^,¹⁰. Here we demonstrate that the Cas protein Cas9 of Francisella novicida uses a unique, small, CRISPR/Cas-associated RNA (scaRNA) to repress an endogenous transcript encoding a bacterial lipoprotein. As bacterial lipoproteins trigger a proinflammatory innate immune response aimed at combating pathogens¹¹^,¹², CRISPR/Cas-mediated repression of bacterial lipoprotein expression is critical for F. novicida to dampen this host response and promote virulence. Because Cas9 proteins are highly enriched in pathogenic and commensal bacteria, our work indicates that CRISPR/Cas-mediated gene regulation may broadly contribute to the regulation of endogenous bacterial genes, particularly during the interaction of such bacteria with eukaryotic hosts.

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Zhang lab unlocks crystal structure of new CRISPR/Cas9 genome editing tool

Paul Goldsmith, 2015 Aug

In a paper published today in Cell researchers from the Broad Institute and University of Tokyo revealed the crystal structure of theStaphylococcus aureus Cas9 complex (SaCas9)—a highly efficient enzyme that overcomes one of the primary challenges to in vivo mammalian genome editing.

First identified as a potential genome-editing tool by Broad Institute core member Feng Zhang and his colleagues (and published by Zhang lab in April 2015), SaCas9 is expected to expand scientists’ ability to edit genomes in vivo. This new structural study will help researchers refine and further engineer this promising tool to accelerate genomic research and bring the technology closer to use in the treatment of human genetic disease.

“SaCas9 is the latest addition to our Cas9 toolbox, and the crystal shows us its blueprint,” said co-senior author Feng Zhang, who in addition to his Broad role, is also an investigator at the McGovern Institute for Brain Research, and an assistant professor at MIT.

The engineered CRISPR-Cas9 system adapts a naturally-occurring system that bacteria use as a defense mechanism against viral infection. The Zhang lab first harnessed this system as an effective genome-editing tool in mammalian cells using the Cas9 enzymes from Streptococcus thermophilus (StCas9) andStreptococcus pyogenes (SpCas9). Now, Zhang and colleagues have detailed the molecular structure of SaCas9, providing scientists with a high-resolution map of this enzyme. By comparing the crystal structure of SaCas9 to the crystal structure of the more commonly-used SpCas9 (published by the Zhang lab in February 2014), the team was able to focus on aspects important to Cas9 function— potentially paving the way to further develop the experimental and therapeutic potential of the CRISPR-Cas9 system.

Paper cited: Nishimasu H et al. “Crystal Structure of Staphylococcus aureus Cas9.” Cell, http://dx.doi.org:/10.1016/j.cell.2015.08.007

Advances in CRISPR-Cas9 genome engineering: lessons learned from RNA interference

Rodolphe Barrangou1,†, Amanda Birmingham2,†, Stefan Wiemann3, Roderick L. Beijersbergen4, Veit Hornung5 and Anja van Brabant Smith2
Nucleic Acids Research, 2015 Mar 23. http:dx.doi.org:/10.1093/nar/gkv226

RNAi and CRISPR-Cas9 have many clear similarities. Indeed, the mechanisms of both use small RNAs with an on-target specificity of ∼18–20 nt. Both methods have been extensively reviewed recently (3–5) so we only highlight their main features here. RNAi operates by piggybacking on the endogenous eukaryotic pathway for microRNA-based gene regulation (Figure 1A). microRNAs (miRNAs) are small, ∼22-nt-long molecules that cause cleavage, degradation and/or translational repression of RNAs with adequate complementarity to them(6).RNAi reagentsfor research aim to exploit the cleavage pathway using perfect complementarity to their targets to produce robust downregulation of only the intended target gene. The CRISPRCas9 system, on the other hand, originates from the bacterial CRISPR-Cas system, which provides adaptive immunity against invading genetic elements (7). Generally, CRISPR-Cas systems provide DNA-encoded (7), RNAmediated (8), DNA- (9) or RNA-targeting(10) sequencespecific targeting. Cas9 is the signature protein for Type II CRISPR-Cas systems (11

Figure 1. (not shown) The RNAi and CRISPR-Cas9 pathways in mammalian cells. (A) miRNA genes code for primary miRNAs that are processed by the Drosha/DGCR8 complex to generate pre-miRNAs with a hairpin structure. These molecules are exported from the nucleus to the cytoplasm, where they are further processed by Dicer to generate ∼22-nt-long double-stranded mature miRNAs. The RNA duplex associates with an Argonaute (Ago) protein and is then unwound; the strand with a more unstable 5 end (known as the guide strand) is loaded into Ago to create the RNA-induced silencing complex (RISC) while the unloaded strand is discarded. Depending on the degree of complementarity to their targets, miRNAs cause either transcript cleavage and/or translational repression and mRNA degradation. siRNAs directly mimic mature miRNA duplexes, while shRNAs enter the miRNA pathway at the pre-miRNA hairpin stage and are processed into such duplexes. (B) CRISPR-Cas9-mediated genome engineering in mammalian cells requires crRNA, tracrRNA and Cas9. crRNA and tracrRNA can be provided exogenously through a plasmid for expression of a sgRNA, or chemically synthesized crRNA and tracrRNA molecules can be transfected along with a Cas9 expression plasmid. The crRNA and tracrRNA are loaded into Cas9 to form an RNP complex which targets complementary DNA adjacent to the PAM. Using the RuvC and HNH nickases, Cas9 generates a double-stranded break (DSB) that can be either repaired precisely (resulting in no genetic change) or imperfectly repaired to create a mutation (indel) in the targeted gene. There are a myriad of mutations that can be generated; some mutations will have no effect on protein function while others will result in truncations or loss of protein function. Shown are mutations that will induce a frame shift in the coding region of the mRNA (indicated by red X’s), resulting in either a truncated, non-functional protein or loss of protein expression due to nonsense-mediated decay of the mRNA.

Both RNAi and CRISPR-Cas9 have experienced significant milestones in their technological development, as highlighted in Figure 2 (7–14,16–22,24–51) (highlighted topics have been detailed in recent reviews (2,4,52–58)). The CRISPR-Cas9 milestones to date have mimicked a compressed version of those for RNAi, underlining the practical benefit of leveraging similarities to this well-trodden research path. While RNAi has already influenced many advances in the CRISPR-Cas9 field, other applications of CRISPR-Cas9 have not yet been attained but will likely continue to be inspired by the corresponding advances in the RNAi field (Table 1). Of particular interest are the potential parallels in efficiency, specificity, screening and in vivo/therapeutic applications, which we discuss further below.

Figure2. Timeline of milestones for RNAi and CRISPR-Cas9. Milestones in the RNAi field are noted above the line and milestones in the CRISPR-Cas9 field are noted below the line. These milestones have been covered in depth in recent reviews (2,4,52–29).
Table 1. Summary of improvements in the CRISPR-Cas9 field that can be anticipated by corresponding RNAi advances

Work performed during the first few years of intensive RNAi investigations demonstrated that, when taking 70– 75% reduction in RNA levels as a heuristic threshold for efficiency (59), only a small majority of siRNAs and shRNAs function efficiently (24,60) when guide strand sequences are chosen randomly. This observation led to the development in 2004 of rational design algorithms for siRNA molecules (Figure2), followed later by similar algorithms for shRNAs. These methods have been able to achieve∼75% correlation and >80% positive predictive power in identifying functional siRNAs (61) but have been somewhat less effective for shRNAs (62) (perhaps because in most cases, shRNAs produce less knockdown than do siRNAs, likely due to a smaller number of active molecules in each cell). crRNAs also vary widely in efficiency: reports have demonstrated indel (insertion and deletion) creation rates between 5 and 65% (20,25), though the average appears to be between 10 and 40% in unenriched cell populations. Indeed, a growing amount of evidence suggests a wide range of crRNA efficiency between genes and even between exons of the same gene, yielding some ‘super’ crRNAs that are more functional(26,27).

Perhaps in no other area are the lessons of RNAi as obvious as in that of specificity. While RNAi was originally hailed as exquisitely specific (64), subsequent research has shown that in some circumstances it can trigger non-specific effects and/or sequence-specific off-target effects (65). Many non-specific effects seen with this approach are mediated by the inadvertent activation of pattern recognition receptors (PRRs) of the innate immune system that have evolved to sense the presence of nucleic acids in certain sub-cellular compartments. siRNA length, certain sequence motifs, the absence of 2-nt 3 overhangs and cell type are important factors for induction of the mammalian interferon response (66–68). Additionally, the general perturbation of cellular or tissue homeostasis by the delivery process itself can also trigger unwanted responses (most likely secondary to innate immune damage-sensing pathways) such as the wide-spread alteration of gene expression caused by cationic lipids, especially when used at high concentrations (69). Such nonspecific effects associated with delivery will still exist for CRISPR-Cas9 but can likely be overcome by minimizing lipid concentration as is now routinely done in RNAi studies. Similarly, the introduction of chemical modifications into the backbone of an siRNA duplex (e.g. 2-O-methyl ribosyl) can block the recognition of RNA molecules by PRRs (66,70–71),

RNAi can also produce sequence-specific off-target effects, which were initially described in early 2003 (31), but whose potential impact was not fully appreciated until well after the method had become a widely used research and screening technique (e.g. (74)). Cleavage-based off-targeting, which occurs when RISC encounters an unintended transcript target with perfect or near-perfect complementarity to its guide strand, can induce knockdownequivalenttothatofintendedtargetdown-regulation and was originally hypothesized to be the main cause of sequence-specific off-target effects. It took several years to determine that these effects were in fact primarily caused byRNAireagentsactingina‘miRNA-like’fashion,downregulating unintended targets by small (usually <2-fold) amounts primarily through seed-based interactions with the 3 UTR of those unintended targets. Because miRNAlike off-targeting is generally seed-based and all transcripts contain matches to a variety of 6–8-base motifs, such off targeting can affect tens to hundreds of transcripts. Furthermore, if the RNAi reagent contains a seed mimicking that of an endogenous miRNA, the off-targeting may affect the pathway or family of targets evolutionarily selected for regulation by that miRNA. It is not possible to design RNAi reagents that do not contain seed regions found in the transcriptome’s 3 UTRs and the non-seed factors that conclusively determine whether or not a seed-matched transcript is in fact off-targeted have not yet been identified. Both rational design and chemical modifications such as 2 O-methyl ribosyl substitutions can mitigate seed-based off-target effects (32), but without a full solution, specificity remains a well-known pain point for RNAi users.

Of particular importance is evaluating whether the lower efficiencies seen using CRISPR-Cas9 are sufficient to generate a desired phenotype in the screening assay––that is, determining whether the phenotype is detectable in the targeted cell population. In this regard, two factors are of special concern: the ploidy of the gene locus of interest (as tumor cell lines are often aneuploid) and the likelihood of disrupting the reading frame by the induced mutation (since +3 or−3 indels would not serve this purpose). Taking these factors into account, the chance of obtaining a high percentage of cells that have a functional knockout in a bulk cell culture is relatively low under typical screening conditions. Consequently, it is unlikely that traditional arrayed loss-of-signal screens such as those common in RNAi will be widely feasible in bulk-transfected cells using CRISPR-Cas9.

RNAi has demonstrated tremendous value as a functional genomics tool, especially with the technological advances described above that enhance efficiency and decrease offtarget effects (118). Likewise, CRISPR-Cas9 has already proven to be a valuable tool for functional genomics studies. Although we have highlighted many points on which the RNAi field can offer pertinent guidance for the effective development and exploitation of CRISPR-Cas9, it is important to remember the fundamental differences that underlie these techniques (Table 3). These contrasts must be considered when selecting the most appropriate method for studying a particular gene or genome.
Molecular consequences. One such fundamental difference between the two is the molecular consequences of their actions. RNAi results in knockdown at the RNA level while CRISPR-Cas9 causes a change in the DNA of the genome; as a corollary, RNAi happens predominantly in the cytoplasm, while CRISPRCas9 acts in the nucleus. These contrasts highlight the differing applicability of the techniques: for example, circRNAs (119,120) that differ from their linear counterparts by splice order in the final transcript can be interrogated by RNAi but not CRISPR-Cas9, while intron functionality can be investigated by CRISPR-Cas9 but not RNAi. For more prosaic targets of interest, in some cases the resulting phenotype associated with either knockdown or knockout may be similar but in others there may be significant differences that result from repression of gene expression compared to a complete null genotype.AlthoughCRISPRCas9-based approaches for drug target identification have been developed (121), repression of gene expression may better model a potential drug’s means of activity and thus be more relevant for drug discovery efforts.

Duration of effect. Because of differences in their mode of action, CRISPRCas9 and RNAi also differ in their duration of effect. siRNA knockdown is typically transient (lasting 2–7 days), while genome engineering with CRISPR-Cas9 induces a permanent effect that, if all alleles are affected, sustainably removes gene function and activity. shRNA knockdown can be either short- or long-term depending on whether the shRNA is continuously expressed, providing some middleground; shRNA activity can also be turned on and off with inducible vectors (122,123) although some leakage can occur even in the off state, depending on the inducible system. Inducible or transient systems will also likely be necessary for studying essential genes viaCRISPR-Cas9

Modulation of non-coding genes Most protein-coding genes will be easily down-modulated by either RNAi or CRISPR-Cas9. For permanent disruption of protein-coding genes using CRISPR-Cas9, frameshift mutations in a critical coding exon (i.e. an early protein-coding exon that is used by all relevant transcript variants) must occur, while RNAi reagents can be targeted essentially anywhere within the transcript.However,knockdown or knockout of non-coding RNAs is more nuanced. The study of small non-coding genes, particularly, is complicated for both RNAi and CRISPR-Cas9 by the limited design space for targeting the non-coding gene without affecting nearby genes.

The fact that CRISPR-Cas9 is not an endogenous mammalian system provides the opportunity for innovative protein evolution studies that are not possible with RNAi. Given this, we anticipate that the CRISPR-Cas9 field will expand beyond the canonical S. pyogenes SpyCas9 in combination with the NGG PAM that has been the focus of virtually all mammalian applications to date. Indeed, other Cas9 proteins are being increasingly characterized (145) with their respective PAMs (of various sizes and sequences) in order to expand targeting specificity.

The new frontier of genome engineering with CRISPR-Cas9
GENOME EDITING
Jennifer A. Doudna* and Emmanuelle Charpentier
Science 346, 1258096 (2014). http://dx.doi .org/10.1126/ science.125809

Fig. 1.Timeline of CRISPR-Cas and genome engineering research fields. Key developments in both fields are shown. These two fields merged in 2012 with the discovery that Cas9 is an RNA-programmable DNA endonuclease, leading to the explosion of papers beginning in 2013 in which Cas9 has been used to modify genes in human cells as well as many other cell types and organisms.

Functionality of CRISPR-Cas9 Bioinformatic analyses first identified Cas9 (formerly COG3513, Csx12, Cas5, or Csn1) as a large multifunctional protein (36) with two putative nuclease domains, HNH (38, 43, 44) and RuvC-like (44). Genetic studies showed that S. thermophilus Cas9 is essential for defense against viral invasion (45, 66), might be responsible for introducing DSBs into invading plasmids and phages (67), enables in vivo targeting of temperate phages and plasmids in bacteria (66, 68), and requires the HNH and RuvC domains to interfere with plasmid transformation efficiency (68). In 2011 (66), trans-activating crRNA (tracrRNA) —a small RNA that is trans-encoded upstream of the type II CRISPR-Cas locus in Streptococcus pyogenes—was reported to be essential for crRNA maturation by ribonuclease III and Cas9, and tracrRNA-mediated activation of crRNA maturation was found to confer sequence-specific immunity against parasite genomes. In 2012 (64), the S.pyogenes CRISPR-Cas9proteinwasshown tobeadual-RNA–guidedDNAendonucleasethat uses the tracrRNA:crRNA duplex (66) to direct DNA cleavage (64) (Fig. 2). Cas9 uses its HNH domain to cleave the DNA strand that is complementary to the 20-nucleotide sequence of the crRNA; the RuvC-like domain of Cas9 cleaves the DNA strand opposite the complementary strand (64, 65) (Fig. 2). Mutating either the HNH or the RuvC-like domain in Cas9 generates a variant protein with single-stranded DNA cleavage (nickase) activity, whereas mutating both domains (dCas9; Asp10 → Ala, His840 → Ala) results in an RNA guided DNA binding protein(64,65). DNA target recognition requires both base pairing to the crRNA sequence and the presence of a short sequence (PAM) adjacent to the targeted sequence in the DNA (64, 65) (Fig. 2). The dual tracrRNA:crRNA was then engineered as a single guide RNA (sgRNA) that retains two critical features: the 20-nucleotide sequence at the 5′end of the sgRNA that determines the DNA target site by Watson-Crick base pairing,and the double-stranded structure at the 3′ side of the guide sequence that binds to Cas9 (64) (Fig. 2). This created a simple two-component system in which changes to the guide sequence (20 nucleotides in the native RNA) of the sgRNA can be used to program CRISPR-Cas9 to target any DNA sequence of interest as long as it is adjacent to a PAM (64).

Fig. 2. Biology of the type II-A CRISPR-Cas system.The type II-A system from S. pyogenes is shown as an example. (A) The cas gene operon with tracrRNA and the CRISPR array. (B) The natural pathway of antiviral defense involves association of Cas9 with the antirepeat-repeat RNA (tracrRNA: crRNA) duplexes, RNA co-processing by ribonuclease III, further trimming, R-loop formation, and target DNA cleavage. (C) Details of the natural DNA cleavage with the duplex tracrRNA:crRNA

Mechanism of CRISPR-Cas9–mediated genome targeting. Structural analysis of S. pyogenes Cas9 has revealed additional insights into the mechanism of CRISPR-Cas9 (Fig. 3). Molecular structures of Cas9 determined by electron microscopy and x-ray crystallography show that the protein undergoes large conformational rearrangement upon binding to the guide RNA, with a further change upon association with a target doublestranded DNA (dsDNA). This change creates a channel, running between the two structural lobes of the protein, that binds to the RNA-DNA hybrid as well as to the coaxially stacked dualRNA structure of the guide corresponding to the crRNA repeat–tracrRNA antirepeat interaction (77, 78). An arginine-rich a helix (77–79) bridges the two structural lobes of Cas9 and appears to be the hinge between them.

Fig. 4. CRISPR-Cas9 as a genome engineering tool. (A) Different strategies for introducing blunt double-stranded DNA breaks into genomic loci, which become substrates for endogenous cellular DNA repair machinery that catalyze nonhomologous end joining (NHEJ) or homology-directed repair (HDR). (B) Cas9 can function as a nickase (nCas9) when engineered to contain an inactivating mutation in either the HNH domain or RuvC domain active sites. When nCas9 is used with two sgRNAs that recognize offset target sites in DNA, a staggered double-strand break is created. (C) Cas9 functions as an RNA-guided DNA binding protein when engineered to contain inactivating mutations in both of its active sites.This catalytically inactive or dead Cas9 (dCas9) can mediate transcriptional down-regulation or activation, particularly when fused to activator or repressor domains. In addition, dCas9 can be fused to fluorescent domains, such as green fluorescent protein (GFP), for live-cell imaging of chromosomal loci. Other dCas9 fusions, such as those including chromatin or DNA modification domains, may enable targeted epigenetic changes to genomic DNA.

The programmable binding capability of dCas9 can also be used for imaging of specific loci in live cells. An enhanced green fluorescent protein– tagged dCas9 protein and a structurally optimized sgRNA were shown to produce robust imaging of repetitiveand nonrepetitiveelementsin telomeres and coding genes in living cells (131). This CRISPR imaging tool has the potential to improve the current technologies for studying conformational dynamics of native chromosomes in living cells, particularlyifmulticolorimagingcanbedeveloped using multiple distinct Cas9 proteins. It may also be possible to couple fluorescent proteins or small molecules to the guide RNA, providing an orthogonal strategy for multicolor imaging using Cas9. Novel technologies aiming to disrupt proviruses may be an attractive approach to eliminating viral genomes from infected individuals and thus curing viral infections. An appeal of this strategy is that it takes advantage of the primary native functions of CRISPR-Cas systems as antiviral adaptive immune systems in bacteria. The targeted CRISPR-Cas9 technique was shown to efficiently cleave and mutate the long terminal repeat sites of HIV-1 and also to remove internal viral genes from the chromosome of infected cells (132, 133). CRISPR-Cas9 is also a promising technology in the field of engineering and synthetic biology. A multiplex CRISPR approach referred to as CRISPRm was developed to facilitate directed evolution of biomolecules (134). CRISPRm consists of the optimization of CRISPR-Cas9 to generate quantitative gene assembly and DNA library insertion into the fungal genomes, providing a strategy to improve the activity of biomolecules. In addition, it has been possible to induce Cas9 to bind single stranded RNA in a programmable fashion by using short DNA oligonucleotides containing PAM sequences (PAMmers) to activate the enzyme, suggesting new ways to target transcripts without prior affinity tagging (135). Several groups have developed algorithmic tools that predict the sequence of an optimal sgRNA with minimized off-target effects (for example, http://tools.genome-engineering.org, http://zifit.partners.org, and www.e-crisp.org) (141–145).

Our understanding of how genomes direct development, normal physiology, and disease in higher organisms has been hindered by a lack of suitable tools for precise and efficient gene engineering. The simple two-component CRISPRCas9system,usingWatson-Crickbasepairing by aguideRNAtoidentifytargetDNAsequences,is a versatile technology that has already stimulated innovative applications in biology. Understanding the CRISPR-Cas9 system at the biochemical and structural level allows the engineering of tailored Cas9 variants with smaller size and increased specificity. A crystal structure of the smaller Cas9 protein from Actinomyces, for example, showed how natural variation created a streamlined enzyme, setting the stage for future engineered Cas9 variants (77). A deeper analysis of the large panel of naturally evolving bacterial Cas9 enzymes may also reveal orthologs with distinct DNA binding specificity, will broaden the choice of PAMs, and will certainly reveal shorter variants more amenable for delivery in human cells.

Furthermore, specific methods for delivering Cas9 and its guide RNA to cells and tissues should benefit the field of human gene therapy. For example, recent experiments confirmed that the Cas9 protein-RNA complex can be introduced directly into cells using nucleofection or cell-penetrating peptides to enable rapid and timed editing (89,152), and transgenic organisms
that express Cas9 from inducible promoters are being tested. An exciting harbinger of future research in this area is the recent demonstration that Cas9–guide RNA complexes, when injected into adult mice, provided sufficient editing in the liver to alleviate a genetic disorder (153). Understanding the rates of homology-directed repair afterCas9-mediatedDNAcuttingwilladvancethe field by enabling efficient insertion of new or corrected sequences into cells and organisms. In addition, the rapid advance of the field has raised excitement about commercial applications of CRISPR-Cas9.

CRISPR Needle with DNA Nanoclews

GEN 2015 Aug

A team of researchers from North Carolina State University (NC State) and the University of North Carolina at Chapel Hill (UNC-CH) have created and utilized a nanoscale vehicle composed of DNA to deliver the CRISPR-Cas9 gene editing complex into cells both in vitro and in vivo.

When the nanoclew comes into contact with a cell, the cell absorbs the nanoclew completely—swallowing it and wrapping it an endosome. Nanoclews are coated with a positively charged polymer that breaks down the endosome, setting the nanoclew free inside the cell, thus allowing CRISPR-Cas9 to make its way to the nucleus. [North Carolina State University]

“Traditionally, researchers deliver DNA into a targeted cell to make the CRISPR RNA and Cas9 inside the cell itself—but that limits control over its dosage,” explained co-senior author Chase Beisel, Ph.D., assistant professor in the department of chemical and biomolecular engineering at NC State. “By directly delivering the Cas9 protein itself, instead of turning the cell into a Cas9 factory, we can ensure that the cell receives the active editing system and can reduce problems with unintended editing.”
The findings from this study were published recently in Angewandte Chemie through an article entitled “Self-Assembled DNA Nanoclews for the Efficient Delivery of CRISPR-Cas9 for Genome Editing.”

The nanoclews are made of a single, tightly-wound strand of DNA. The DNA is engineered to partially complement the relevant CRISPR RNA it will carry, allowing the CRISPR-Cas9 complex to loosely attach itself to the nanoclew. “Multiple CRISPR-Cas complexes can be attached to a single nanoclew,” noted lead author Wujin Sun, a Ph.D. student in Dr. Gu’s laboratory.
When the nanoclew comes into contact with a cell, the cell absorbs the nanoclew completely through typical endocytic mechanisms. The nanoclews are coated with a positively charged polymer, in order to break down the endosomal membrane and set the nanoclew free inside the cell. The CRISPR-Cas9 complexes will then free themselves from the nanoclew structure to make their way to the nucleus. Once the CRISPR-Cas9 complex reaches the nucleus than the gene editing can begin.
In order to test their delivery method, the investigators created fluorescently labeled cancer cells in culture and within mice. The CRISPR nanoclew was then designed to target the gene generating fluorescent protein in the cells—if the glowing stopped than the nanoclews worked. “And they did work. More than one-third of cancer cells stopped expressing the fluorescent protein,” Dr. Beisel stated.

Imitating Viruses to Deliver Drugs to Cells

2015 Aug – by CNRS (Délégation Paris Michel-Ange)

Figure (not shown). Assembly of the artificial virus and protein delivery: the virus consists of an initial polymer (pGi-Ni2+, left) on which the proteins to be delivered bind. It is encapsulated (right) by a second polymer (πPEI), which binds to the cell surface.

Viruses are able to redirect the functioning of cells in order to infect them. Inspired by their mode of action, scientists from the CNRS and Université de Strasbourg have designed a “chemical virus” that can cross the double lipid layer that surrounds cells, and then disintegrate in the intracellular medium in order to release active compounds. To achieve this, the team used two polymers they had designed, which notably can self-assemble or dissociate, depending on the conditions. This work, the result of collaborative efforts by chemists, biologists and biophysicists, is published in the 1st September issue of Angewandte Chemie International Edition.

Biotechnological advances have offered access to a wealth of compounds with therapeutic potential. Many of these compounds are only active inside human cells but remain unusable because the lipid membrane surrounding these cells is a barrier they cannot cross. The challenge is therefore to find transfer solutions that can cross this barrier.

By imitating the ability of viruses to penetrate into cells, chemists in the Laboratoire de Conception et Application de Molécules Bioactives (CNRS/Université de Strasbourg) sought to design particles capable of releasing macromolecules that are only active inside cells. To achieve this, these particles must comply with several, often contradictory, constraints. They must remain stable in the extracellular medium, they must be able to bind to the cells so that they be internalized, but they must be more fragile inside the cells so that they can release their content. Using two polymers designed by the team, the scientists succeeded in creating a “chemical virus” that meets the conditions necessary for the direct delivery of active proteins into cells.

In practice, the first polymer (pGi-Ni2+) serves as a substrate for the proteins that bind to it. The second, recently patented polymer (πPEI), encapsulates this assembly thanks to its positive charges, which bind to the negative charges of pGi-Ni2+. The particles obtained (30-40 nanometers in diameter) are able to recognize the cell membrane and bind to it. This binding activates a cellular response: the nanoparticle is surrounded by a membrane fragment and enters the intracellular compartment, called the endosome. Although they remain stable outside the cell, the assemblies are attacked by the acidity that prevails within this new environment. Furthermore, this drop in pH allows the πPEI to burst the endosome, releasing its content of active compounds.

Thanks to this assembly, the scientists were able to concentrate enough active proteins within the cells to achieve a notable biological effect. Thus by delivering a protein called caspase 3 into cancer cell lines, they succeeded in inducing 80% cell death.¹

The in vitro results are encouraging, particularly since this “chemical virus” only becomes toxic at a dose ten times higher than that used during the study. Furthermore, preliminary results in the mouse have not revealed any excess mortality. However, elimination by the body of the two polymers remains an open question. The next stage will consist in testing this method in-depth and in vivo, in animals. In the short term, this system will serve as a research tool to vectorize² recombinant and/or chemically modified proteins into cells. In the longer term, this work could make it possible to apply pharmaceutical proteins to intracellular targets and contribute to the development of innovative drugs.

This work was made possible by the collaboration of biophysicists and biologists. The skills in electron cryomicroscopy available at the Institut de Génétique et de Biologie Moléculaire et Cellulaire (CNRS/Université de Strasbourg/Inserm), and the expertise in atomic force microscopy of the Laboratoire de Biophotonique et Pharmacologie (CNRS/Université de Strasbourg) enabled highly precise characterization of the molecular assemblies. The Laboratoire Biotechnologie et Signalisation Cellulaire (CNRS/Université de Strasbourg) supplied the recombinant proteins encapsulated in the artificial virus.

A CRISPR view of development

Melissa M. Harrison,1 Brian V. Jenkins,2 Kate M. O’Connor-Giles,3,4 and Jill Wildonger2
1Department of Biomolecular Chemistry, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin 53706, USA; 2Biochemistry Department, University of Wisconsin-Madison, Madison, Wisconsin 53706, USA; 3Laboratory of Genetics, 4Laboratory of Cell and Molecular Biology, University of Wisconsin-Madison, Madison, Wisconsin 53706, USA
GENES & DEVELOPMENT 2015 Aug; 28:1859–1872
http://www.genesdev.org/cgi/doi/10.1101/gad.248252.114.

The CRISPR (clustered regularly interspaced short palindromic repeat)–Cas9 (CRISPR-associated nuclease 9) system is poised to transform developmental biology by providing a simple, efficient method to precisely manipulate the genome of virtually any developing organism. This RNA-guided nuclease (RGN)-based approach already has been effectively used to induce targeted mutations in multiple genes simultaneously, create conditional alleles, and generate endogenously tagged proteins. Illustrating the adaptability of RGNs, the genomes of >20 different plant and animal species as well as multiple cell lines and primary cells have been successfully modified. Here we review the current and potential uses of RGNs to investigate genome function during development.

Through the regulated process of development, a single cell divides and differentiates into the multitude of specialized cells that compose a mature organism. This process is controlled in large part by differential gene expression, which generates cells with distinct identities and phenotypes despite nearly identical genomes. Recent advances in genome engineering provide the opportunity to efficiently introduce almost any targeted modification in genomic DNA and, in so doing, the unprecedented ability to probe genome function during development in a diverse array of systems.

The CRISPR–Cas9 system has propelled genome editing from being a technical possibility to a practical reality for developmental biology studies due to the simplicity with which the Cas9 nuclease is recruited to a specific DNA sequence by a small, easily generated guide RNA (gRNA) that recognizes its genomic target via standard Watson-Crick base-pairing.

Cas9 enzymes from type II CRISPR–Cas systems are emerging as the sequence-specific nucleases of choice for genome engineering for several reasons. Most notably, as anRNA-guidednuclease(RGN),Cas9isguidedbyasingle gRNA that is readily engineered. In the case of the most commonly used Cas9, derived from Streptococcus pyogenes, the gRNA targeting sequence comprises 20 nucleotides (nt) that can be ordered as a pair of oligonucleotides and rapidly cloned. In contrast, generating an effective ZFN or TALEN is labor-intensive (see Box 1). ZFNs and TALENs are proteins that combine uniquely designed and generated DNA-binding sequences with the FokI nuclease cleavage domain. FokI is an obligate dimer, necessitating the generation of two novel proteins per editing experiment compared with a single gRNA for CRISPR–Cas9-mediated targeting.

Figure 1. (not shown) The flexibility and adaptability of the CRISPR–Cas9 system offers vast potential for genome manipulations. (A) Overview of the CRISPR–Cas9 system. At its simplest, the system consists of the chimeric gRNA (purple), which guides the Cas9 nuclease to the genomic target site (red). The genomic target site is composed of 20 base pairs (bp) of homology with the gRNA (red) and a PAM sequence (white). Cleavage (scissors) occurs 3 bp 59 of the PAM. (B) Components required for RGN-mediated genome editing. The CRISPR–Cas9 components can be delivered as DNA, RNA, or protein, as indicated, and introduced into the cell or embryo through injection, transfection, electroporation, or infection. Organisms and cells expressing transgenic Cas9 are available, and in Drosophila, both the transgenic Cas9-expressing strains and those expressing transgenic gRNA have been shown to increase targeting efficacy. To introduce designer mutations and/or exogenous sequence, a ssDNA or dsDNA donor template is included. (C) Genome engineering outcomes. Cas9-induced DSBs can be repaired by either NHEJ or HDR. (Top left) The DSB generated by a single gRNA can be repaired by NHEJ to generate indels. (Bottom left, dashed box) With the use of two gRNAs, NHEJ can result in larger deletions. If the gRNAs target sequences on different chromosomes, it is possible to generate chromosomal translocations and inversions. (Right) With the inclusion of a researcher-designed donor template, HDR makes it possible to generate conditional alleles (top), fluorescently or epitope tagged proteins (middle), specific mutations (bottom), or any combination thereof. The donor template can also be designed to correct a mutation in the organism or cell or replace a gene. (D) Catalytically inactive dCas9 provides a platform for probing genomic function. dCas9 can be fused to any number of different effectors to allow for the visualization of where specific DNA sequences localize, the repression or activation of transcription, or the immunoprecipitation of the bound chromatin.

Box: 1. A miniguide to genome engineering techniques

Zinc finger nucleases (ZFNs), transcriptional activator-like effector nucleases (TALENs), and CRISPR (clustered regularly interspaced short palindromic repeat)–Cas9 (CRISPR-associated nuclease 9) all function on a similar principle: A nuclease is guided to a specific sequence within the genome to induce a double strand DNA break (DSB). Once a DSB is generated, the cell’s intrinsic DNA repair machinery is set in motion, and it is during the repair of the DSB that the genome is modified. DSBs are typically repaired by either non-homologous end joining (NHEJ) or homology-directed repair (HDR) (Fig. 1C). In NHEJ, the two cleaved ends of the DSB are ligated together. During this process, DNA of varying sizes, generally on the order of a few base pairs, is occasionally inserted and/or deleted randomly. When a DSB is targeted to a coding exon, these insertions or deletions (indels) can result in a truncated gene product. If two DSBs are induced, NHEJ can generate deletions, eliminating an entire gene or region. HDR uses homologous sequence as a template to repair the DSB. Researchers can take advantage of this repair pathway to introduce designer mutations or exogenous sequence, such as genetically encoded tags, by supplying the cell with a donor DNA template that has homology with the sequence flanking the DSB. Note that cells can also use endogenous DNA as a template, in which case the DSB is repaired without incorporation of the donor-supplied edits. It is important to keep in mind that although the researcher directs where the DSB occurs in the genome, the cell is in control of how the DSB is repaired, which determines the ultimate outcome of a genome-editing experiment.

ZFNs

ZFNs are fusion proteins comprised of DNA-binding C2H2 zinc fingers fused to the nonspecific DNA cleavage domain of the nuclease Fok1 (for review, see Carroll 2011). Each zinc finger can be engineered to recognize a nucleotide triplet, and multiple (typically three to six) zinc fingers are joined in tandem to target specific genome sequences. Because the Fok1 cleavage domain must dimerize to be active, two ZFNs are required to create a DSB. This technique, which was first successfully used in fruit flies more than a decade ago (Bibikova et al. 2002), has since been used to modify the genomes of many different organisms, including those that had not previously been developed as genetic model systems.

TALENs

Similar to ZFNs, TALENs are chimeric proteins comprised of a programmable DNA-binding domain fused to the Fok1 nuclease domain (for review, see Joung and Sander 2013). TALEs are naturally occurring proteins that are secreted by the bacteria Xanthamonas and bind to sequences in the host plant genome, activating transcription. The TALE DNA binding domain is composed of multiple repeats, each of which are 33–35 amino acids long. Each repeat recognizes a single nucleotide in the target DNA sequence. Nucleotide specificity is conferred by a two-amino-acid hypervariable region present in each repeat. Sequence-specific TALENs are generated by modifying the two residues in the hypervariable region and concatenating multiple TALE repeats together. Because the TALE DNA-binding domain is fused to Fok1, TALENs, like ZFNs, must also be used as dimers to generate DSBs.

RGNs hold great potential for dissecting how the genome functions during development. Since the CRISPR–Cas9 system has been recently described in detail elsewhere (Hsu et al. 2014; Sander and Joung 2014), we provide just a brief overview of the system (Box1; Fig.1A–C) and focus here on a few practical considerations for using RGNs to edit the genome of a developing organism.

The CRISPR–Cas9 system

The CRISPR–Cas9 genome-editing method is derived from a prokaryotic RNA-guided defense system (Gasiunas et al. 2012; Jinek et al. 2012, 2013; Cong et al. 2013; Mali et al. 2013c). CRISPR repeats were first discovered in the Escherichia coli genome as an unusual repeat locus (Ishino et al. 1987). The significance of this structure was appreciated later when investigators realized that phage and plasmid sequences are similar to the spacer sequences in CRISPR loci (Bolotin et al. 2005; Mojica et al. 2005; Pourcel et al. 2005). Soon afterward, it was shown that spacers are derived from viral genomic sequence (Barrangou et al. 2007). In the CRISPR–Cas system, short sequences (referred to as ‘‘protospacers’’) from an invading viral genome are copied as‘‘spacers’’ between repetitive sequences in the CRISPR locus of the host genome. The CRISPR locus is transcribed and processed into short CRISPR RNAs (crRNAs) that guide the Cas to the complementary genomic target sequence. There are at least eleven different CRISPR– Cas systems, which have been grouped into three major types (I–III). In the type I and II systems, nucleotides adjacent to the protospacer in the targeted genome comprise the protospacer adjacent motif (PAM). The PAM is essential for Cas to cleave its target DNA, enabling the CRISPR–Cas system to differentiate between the invading viral genome and the CRISPR locus in the host genome, which does not incorporate the PAM. For additional details on this fascinating prokaryotic adaptive immune response, see recent reviews (Sorek et al. 2013; Terns and Terns 2014). Type II CRISPR–Cas systems have been adapted as a genome-engineering tool. In this system, crRNA teams up with a second RNA, called trans-acting CRISPR RNA (tracrRNA), which is critical for crRNA maturation and recruiting the Cas9 nuclease to DNA (Deltcheva et al. 2011; Jinek et al. 2012). The RNA that guides Cas9 uses a short (;20-nt) sequence to identify its genomic target. This three-component system was simplified by fusing together crRNA and tracrRNA, creating a single chimeric ‘‘guide’’ RNA (abbreviated as sgRNA or simply gRNA) (Gasiunas et al. 2012; Jinek et al. 2012). While some early experiments indicated that a gRNA may not cleave a subset of targets as efficiently as a crRNA in combination with tracrRNA (Mali et al. 2013c), the ease of using a single RNA has led to the widespread adoption of gRNAs for genome engineering. A number of resources for designing experiments using the CRISPR–Cas9 system are freely available online. (A comprehensive list is available at http://www. geewisc.wisc.edu.)

The current methods of producing the CRISPR–Cas9 components provide great flexibility in terms of expression and delivery, and biologists can exploit these options to control when and where DSBs are generated in an organism. To introduce DSBs and generate modifications early in development, the CRISPR–Cas9 components can be injected as DNA, RNA, or protein into most developing organisms. This approach, which has been widely used, generates mosaic organisms for analysis. To gain control over which tissues are affected, a plasmid expressing Cas9 under the control of tissue-specific enhancers can be used. Since each cell has a choice of whether to repair a breakthrough NHEJ or HDR, a variety of different repair events will be present in the injected organism (and in individual cells). The frequency at which both alleles of a gene are affected has been reported to be high enough to visualize null phenotypes in developing mice and zebrafish (Jao et al. 2013; Wang et al. 2013a; Yasue et al. 2014; Yen et al. 2014).

Genome engineering with RGNs enables the direct manipulation of nearly any sequence in the genome to determine its role in development. The major limitation as to which genomic loci can be targeted is the requirement of a specific protospacer adjacent motif (PAM). The PAM is a short DNA motif adjacent to the Cas9 recognition sequence in the target DNA and is essential for cleavage. The most commonly used S. pyogenes Cas9 requires the PAM sequence 59-NGG (in cell lines, other PAMs are recognized, including 59-NAG, but at a lower frequency) (Jinek et al. 2012; Esvelt et al. 2013; Hsu et al. 2013; Jiang et al. 2013a; Zhang et al. 2014). The PAM is critical for cleavage and increases target specificity but, conversely, can also make some segments of the genome refractory to Cas9 cleavage. For example, AT-rich genomic sequences may contain fewer PAM sites that would be recognized and cleaved by S. pyogenes Cas9. Thus, some poly(dA-dT) tracts, which are implicated in nucleosome positioning (for review, see Struhl and Segal 2013), may be difficult to manipulate using S. pyogenes Cas9.

With RGNs, a variety of genomic manipulations are brought within reach of developmental biologists studying a diversity of organisms (Table 1 [nt shown]). This approach also makes it possible to readily generate mutations in different genetic strains, making it easier to control genetic background and eliminating the need to carry out multigenerational mating schemes to bring different mutations together in the same animal. While the CRISPR–Cas9 system has been widely used to introduce indels and deletions, HDR makes it possible to introduce more precise gene mutations, deletions, and exogenous sequences, such as loxP sites and green fluorescent protein (GFP).

Multiplexing advantages

Genes that have essential roles in development are often functionally redundant, and thus the effects of mutating a single gene can be masked by the presence of another gene. Due to the ease and efficiency with which gRNAs can be generated, multiple gRNAs can be used in a single experiment to simultaneously mutate multiple genes, overcoming issues of redundancy. Recent technical innovations now make it possible to express multiple gRNAs from a single transcript (Nissim et al. 2014; Tsai et al. 2014), making RGN multiplexing experiments even easier to carry out. Such multiplexing experiments will also facilitate multifaceted experiments, including epistasis tests and manipulating genes that are physically very close together in the genome. Multiplexing has already been used successfully to simultaneously disrupt both Tet1 and Tet2 in developing mice following injection into zygotes (Wang et al. 2013a). The CRISPR– Cas9 system has also been used to eliminate two genes in monkeys (Niu et al. 2014b).

Many gene products of interest to developmental biologists are essential early in development, and mutations in these genes are lethal to an animal before it reaches later developmental stages. Conditional alleles provide spatial and temporal control over gene inactivation and therefore have been invaluable tools for working with genes that cause early lethality. Conditional alleles have also been used to determine where and when a gene is acting during development. The utility of exerting conditional control over gene activity is widely recognized, and an international consortium is currently working to create a library of conditional alleles for ~ 20,000 genes in the mouse genome (Skarnes et al. 2011). Since the expression of the conditional allele reflects the expression pattern of the recombinase, it is advantageous to have a variety of lines that express recombinase in specific tissues or at discrete developmental stages. The CRISPR– Cas9 system was recently used to generate two different Cre recombinase-expressing lines in rats (Ma et al. 2014b). Thus, RGNs are being used to rapidly generate the tools necessary to probe gene function in a tissue- and time-dependent manner.

RGNs open the door to quickly and easily tagging endogenous genes for developmental studies. Furthermore, because the CRISPR–Cas9 system is amenable to multiplexing, tags could be added simultaneously to multiple genes or different splice isoforms of a single gene. There is an ever-growing number of genetically encoded molecular tags that can be used for functional analysis, protein purification, or protein and RNA localization studies.

One of the first reportsof the use of RGNs for genome engineering demonstrated success in induced pluripotent stem cells (iPSCs) with a frequency of between 2% and 4% when assayed by deep sequencing of bulk culture (Mali et al. 2013c). Recovery of engineered cells is increased when Cas9-expressing cells are marked with a fluorescent marker and selected by cell sorting (Ding et al. 2013). Using this strategy, it was reported that clones containing at least one mutant allele could be isolated at frequencies between 51% and 79%. In comparison, TALENs designed against the same set of genes resulted in between 0% and 34% of clones containing at least one mutant allele.

The relative ease of generating mutant animals will yield many additional animal models of disease and supply a means of testing whether specific polymorphisms are the proximal cause of disease in vivo. Additionally, the CRISPR–Cas9 system is amenable to application in organisms not widely used for genetic studies. Organisms that may be better suited to mimic human disease can now be more easily used to generate disease models. For example, mouse models of the bleeding disorder von Willebrand disease fail to fully recapitulate the human disease.

Apart from point mutations and gene deletions, large chromosomal rearrangements can drive specific cancers. By simultaneously introducing gRNAs targeting two different chromosomes or two widely separated regions of the same chromosome, RGNs have been used to introduce targeted inversions and translocations into otherwise wild-type human cells (Choi and Meyerson 2014; Torres et al. 2014). These engineered cells will ultimately allow for studies of the causative role of these gene fusions in cancer progression. Translocations that drive lung adenocarcinoma (Choi and Meyerson 2014), acute myeloid leukemia, and Ewing’s sarcoma (Torres et al. 2014) have been generated in both HEK293 cells and more physiologically relevant cell types (nontransformed immortalized lung epithelial cells and human mesenchymal stem cells). Additionally, cell lines harboring chromosomal inversions found in lung adenocarcinoma have also been created (Choi and Meyerson 2014).

The first RGN based genetic screens were recently carried out in cultured mammalian cells (Koike-Yusa et al. 2014; Shalem et al. 2014; Wang et al. 2014; Zhou et al. 2014). When carrying out such a screen, it is important to consider both the number of genes targeted by the library and the degree of coverage of each gene. The largest library reported to date is comprised of 90,000 gRNAs designed to target 19,000 genes, which equates to about four to five gRNAs per targeted gene (Koike-Yusa et al. 2014).The screens identified targets affecting the DNA mismatch repair pathway (Koike-Yusa et al. 2014; Wang et al. 2014), resistance to bacterial and chemical toxins (Koike-Yusa et al. 2014; Wang et al. 2014; Zhou et al. 2014), and cell survival and proliferation (Shalem et al. 2014; Wang et al. 2014). The Zheng group (Shalem et al. 2014) also compared the results of their screen for genes involved in resistance to a drug that inhibits B-Raf with a prior RNAi screen that used the same cell line and drug. This comparison revealed that gRNAs identified targets that could be validated more consistently and efficiently than shRNAs, pointing to the potential advantages of using gRNAs to knock out, rather than knock down, gene function in genetic screens.

The question remains whether similar screens can be performed in a developing organism. Excitingly, two recent proof-of-principle studies using worms and mice indicate that RGNs will likely be useful for in vivo genetic screens, including unbiased forward genetic screens (Liu et al. 2014a; Mashiko et al. 2014).

In regards to knocking down gene expression, it remains to be determined how effective CRISPRi and dCas9 chimeras are in comparison with RNAi. Notably, CRISPRi and the dCas9 chimeras designed to inhibit gene expression are reportedly less effective in cultured mammalian cells than in bacteria (Gilbert et al. 2013). Nonetheless, given the ease with which dCas9 and TALE platforms can be programmed and their versatility, the potential application of these approaches to investigating genome dynamics in vivo is enticing to consider.

The majority of RGN-editing experiments have taken advantage of NHEJ to create small indels and larger deletions, which are useful for disrupting gene expression. However, to introduce specific mutations or other tailored modifications (e.g., genetically encoded tags), the HDR pathway must be activated. In most eukaryotic cells, DSBs are repaired more frequently through NHEJ than HDR (for review, see Lieber et al. 2003; Carroll 2014).

Pharma IQ (PiQ), 2015 Sep 1

Pharma IQ spoke to Bhuvaneish, a Post Doctorate Fellow in neurodegenerative disorders.

Bhuvaneish T.S joined the Scottish Centre for Regenerative Medicine – University of Edinburgh, almost two years ago to establish and drive the use of CRISPR Cas9 within the University’s lab and apply it as a model for different disorders

Aim: To model motor neuron diseases using human pleuripotent stem cells

Bhuvaneish notes: “The disease modelling of neurodegenerative disorders, using human IPS (Induced Pluripotent Stem Cells), is quite challenging because of the technical variability in generating the IPS lines between different patient samples and also the varied genetic background between the donors. So this is a complex problem and leads to [difficulties when] interpreting the results and it’s also possible to generate erroneous results rather than proper scientific results because of the variations.

“One way to overcome this problem is using multiple lines for our study. So instead of using two or three patient donors, increasing their sample number to five or six, which is a tedious process.

“The other option, which [is] the ideal scenario, is to generate isogenic stem cells that differ only in the disease causing genetic variant. So that’s where the CRISPR Cas9 comes in and it’s a quite handy tool for us.

“In a nutshell what you could do is take patients’ stem cells and then perform a gene correction in CRISPR Cas9. So now we have two types of cell, one is the mutant and the other is the gene corrected. Both are pretty much identical apart from the disease variant. It could be either a point mutation, [or] an expansion repeat, etc. This allows us to nail different phenotypes for motor neurone disorders.

“So generally we generate motor neurones from these two lines and model the disease in a dish, which also helps us to understand the mechanism of the disease.”

Bhuvaneish’s lab also generates different knock outs, which is highly efficient with the CRISPR technique.

Challenges with CRISPR Cas9

With Bhuvaneish leading the use of this technique in the lab, he encountered various challenges regarding the delivery system into the stem cells.

These challenges include off target effects and the efficiency of CRISPR Cas9.

On the latter point, he explains: “Although people say that the efficiency of CRISPR is much better than other gene editing systems like TAL effectors or zinc fingers, it is still pretty low. I mean, the efficiencies you are talking about is 2%, so it is still low.

He continues: “These are the two challenges which we have and I think it’s a challenge the entire world has at the moment with this technology. And we’ve been trying to increase efficiencies with certain drugs, which has also been published recently. I haven’t got any data to back it up myself but looks promising, though.”

“So that itself is a really good thing because now I can dissect the disease causing phenotypes which we see in our culture and that has been reversed after gene correction. You can completely reverse the phenotype. So that itself is proof of concept that the disease causing the mutation is causing this phenotype.”

“In the research field it’s a really, really important tool but for gene therapy as a therapeutic we are still very behind because of the ethical issues. The big challenge is in how to deliver these Cas9 proteins and the guide RNAs to the required donor. It could be that the disease has affected only one particular organ rather than the whole body so you would try to target those particular organs. And it’s a challenge in delivering those Cas9 and the guide RNAs to the particular organ because it’s quite a huge protein compared to conventional proteins which have been used for gene therapy.

“Although it’s highly efficient when compared to the others, for therapeutics we need precise targeting with very, very minimal off target mutations. So that would be CRISPR’s bottleneck coming into the medicine field as a therapeutic.

“For the research it is great at the moment. It has enabled most of the researchers to do the genome editing in human stem cells, which was virtually impossible before.”

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News Picks | Hs-Troponin T, Silent Cerebral Ischemia and Complications in Afib

Posted in Acute Myocardial Infarction, Atrial Fibrilation (a-Fib), Cardiac Pacing and Arrhythmias, Cerebrovascular and Neurodegenerative Diseases, Coagulation Therapy and Internal Bleeding, Electrophysiology, Frontiers in Cardiology and Cardiovascular Disorders, Stroke on June 29, 2015| Leave a Comment »

News Picks | Hs-Troponin T, Silent Cerebral Ischemia and Complications in Afib

Reporter: Aviva Lev-Ari, PhD, RN

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https://www.youtube.com/v/W3X3bMT_F-M?fs=1&hl=fr_FR

High-sensitivity troponin T outperformed other assays in chest pain patients. Silent cerebral ischemia in Afib. Acute cardioversion.

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See on Scoop.it – Cardiovascular and vascular imaging

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Neonatal Pathophysiology

Posted in Amino acids, Anemia, Autism Spectrum Disorders, Behavioral Genetics, Best evidence, Biochemical pathways, Biological Networks, Bone Disease and Musculoskeletal Disease, Ca2+ triggered activation, Calcium Signaling, Cell Biology, Cerebrovascular and Neurodegenerative Diseases, Chemical Biology and its relations to Metabolic Disease, Child and Adolescent Psychiatry, Chronic Thromboembolic Pulmonary Hypertension (CTEPH) and Pulmonary Arterial Hypertension (PAH), Congenital Heart Disease, Curation, Cytoskeleton, Developmental biology, Diabetes Mellitus, Diagnostics and Lab Tests, Disease Biology, Embryology, Enzymes and isoenzymes, Explanatory, Fatty acids, Genetics & Pharmaceutical, Genome Biology, Health Economics and Outcomes Research, Hematology, Hematopoiesis, Historical relevance, Human Immune System in Health and in Disease, Inferential analysis, Innovations in Neurophysiology & Neuropsychology, Lipid metabolism, Liver & Digestive Diseases Research, Metabolism, Metabolomics, Microbiologial genetics, Molecular Genetics & Pharmaceutical, Mutant Gene Expression, Neurohumoral Transmission, Neurological Diseases, Neutropenia, Nitric Oxide in Health and Disease, Nutrition, Personalized and Precision Medicine & Genomic Research, Pharmacogenomics, Placenta, Proteins, Proteomics, Regenerative Biology and Medicine, Reproductive Andrology, Embryology, Genomic Endocrinology, Preimplantation Genetic Diagnosis and Reproductive Genomics, Schizophrenia, Severe Autism, Signaling, Signaling & Cell Circuits, Small Molecules in Development of Therapeutic Drugs, Social Development, Stem Cells for Regenerative Medicine, Synaptic vesicle, Systemic Inflammatory Response Related Disorders, Translational Effectiveness, Translational Research, Translational Science, tagged acute and chronic gastroenteritis, Alzheimers Disease, bilirubin toxicity, Congenital Heart Disease, fetal hemoglobin, genetics, hemochromatosis, hemoglobinopathies, hemolytic diseases, inborn errors of metabolism, intestinal microbiome, neonatal intensive care, oxygen dissociation curve, oxygen saturation, prematurity, pulmonary bronchodysplasia, thalassemia, very low birthweight on February 22, 2015| Leave a Comment »

Neonatal Pathophysiology

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

Introduction

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

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

Genetic and metabolic diseases
Nervous system
Cardiovascular
Pulmonary
Skeletal – bone and muscle
Hematological
Liver
Esophagus, stomach, and intestines
Kidneys
Immune system

Fetal Development

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

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

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

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

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

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

fetal-development

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

Inherited Metabolic Disorders

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

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

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

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

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

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

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

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

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

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

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

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

Organic acidemias: methylmalonic acidemia and propionic acidemia.

Urea cycle disorders: ornithine transcarbamylase deficiency and citrullinemia

Hemoglobinopathies – thalassemias, sickle cell disease

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

This list is by no means complete.

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

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

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

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

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

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

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

Diagnosis and Management of Hereditary Hemochromatosis

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

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

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

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

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

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

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

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

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

Neurological and Behavioral Disorders

Estrogen receptor signaling during vertebrate development

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

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

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

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

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

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

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

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

representations of the HPA axis

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

Fetal programming of schizophrenia: Select mechanisms

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

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

NMDA receptor dysfunction in autism spectrum disorders

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

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

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

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

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

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

Chemokines roles within the hippocampus

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

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

Three Classes of Glutamate Receptors

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

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

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

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

Fig not shown.

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

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

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

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

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

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

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

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

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

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

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

Impact of bilirubin-induced neurologic dysfunction on neurodevelopmental outcomes

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

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

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

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

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

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

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

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

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

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

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

Cerebellar vulnerability to bilirubin-induced injury
Cerebellar injury phenotypes and BIND
Cerebellar projections

Transverse section of cerebellum and brainstem

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

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

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

Schematic of the bidirectional connectivity between the cerebellum and other

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

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

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

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

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

Audiologic impairment associated with bilirubin-induced neurologic damage

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

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

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

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

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

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

Bilirubin-induced neurologic dysfunction (BIND)

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

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

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

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

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

Sensitive time-windows for susceptibility in neurodevelopmental disorders

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

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

Fig (not shown)

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

Fig (not shown)

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

Outstanding questions

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

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

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

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

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

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

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

Cardiovascular

Cardiac output monitoring in newborns

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

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

Fick principle

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

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

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

Cardiovascular adaptation to extra uterine life

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

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

Fetal vascular structures that exist to direct blood flow

Fetal structure	Function
Arterial duct	Connects pulmonary artery to the aorta and shunts blood right to left; diverting flow away from fetal lungs
Foramen ovale	Opening between the two atria thatdirects blood flow returning to right atrium through the septal wall into the left atrium bypassing lungs
Ductus venosus	Receives oxygenated blood fromumbilical vein and directs it to the inferior vena cava and right atrium
Umbilical arteries	Carrying deoxygenated blood fromthe fetus to the placenta
Umbilical vein	Carrying oxygenated blood from theplacenta to the fetus

Maternal causes of congenital heart disease

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

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

Clinical monitoring of systemic hemodynamics in critically ill newborns

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

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

Key guidelines

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

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

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

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

Monitoring oxygen saturation and heart rate in the early neonatal period

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

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

Practice points

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

Using pulse oximetry assists clinicians:

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

Research directions

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

Peripheral haemodynamics in newborns: Best practice guidelines

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

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

graphic

NIRS with partial venous occlusion to measure venous oxygen saturation

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

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

graphic

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

The oxygen-dissociation curve

graphic

The oxygen-dissociation curve

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

Peripheral oxygenation and management in the perinatal period

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

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

Fig (not shown)

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

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

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

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

$Peripheral fractional oxygen extraction in babies$

Peripheral fractional oxygen extraction in babies

graphic

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

Practice points

Peripheral tissue DO2 is complex: cardiac function, blood gases, Hb concentration and the proportion of HbF, and peripheral temperature all play a part in determining blood flow and oxygen extraction in the sick, preterm infant. Blood pressure appears to be less important.
Other factors likely to play a role are the administration of vasoactive medications and ventilation strategies, which affect blood gases and cardiac output by changing intrathoracic pressure.
Central blood pressure is a poor surrogate measurement for the adequacy of DO2 to the periphery. Direct measurement, using NIRS, laser Doppler flowmetry or other means, may give more useful information.
Reasons for total hemoglobin concentration (Hb) being a relatively poor indicator of the adequacy of the provision of oxygen to the tissues:

Hb is only indirectly related to red blood cell volume, which may be a better indicator of the body’s oxygen delivering capacity.
Hb-dependent oxygen availability depends on the position of the oxygen-hemoglobin dissociation curve.
An individual’s oxygen requirements vary with time and from organ to organ. This means that DO2 also needs to vary.
It is possible to compensate for a low Hb by increasing cardiac output and ventilation, and so the ability to compensate for anemia depends on an individual’s cardio-respiratory reserve as well as Hb.
The normal decrease of Hb during the first few weeks of life in both full-term and preterm babies usually occurs without symptoms or signs of anemia or clinical consequences.

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

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

Pulmonary

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

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

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

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

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

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

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

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

Neonatal ventilation strategies and long-term respiratory outcomes

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

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

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

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

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

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

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

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

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

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

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

Anemia, Apnea of Prematurity, and Blood Transfusions

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Long-term pulmonary outcomes of patients with bronchopulmonary dysplasia

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

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

Neonatal ventilation strategies and long-term respiratory outcomes

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

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

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

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

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

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

Pulmonary hypertension in bronchopulmonary dysplasia

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

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

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

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

Methylxanthines

Diuretics and bronchodilators

Corticosteroids

Macrolide antibiotics

Recombinant human Clara cell 10-kilodalton protein(rhCC10)

Vitamin A

Surfactant

Leukotriene receptor antagonist

Pulmonary vasodilators

Skeletal and Muscle

Skeletal Stem Cells in Space and Time

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Identification and Specification of the Mouse Skeletal Stem Cell

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

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

Bone mesenchymal development

The bone-remodeling cycle

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

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

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

Hematopoietic

Hematopoietic Stem Cell Arrival Triggers Dynamic Remodeling of the Perivascular Niche

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

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

Neonatal anemia

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

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

Management of anemia in the newborn

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

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

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

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

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

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

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

Clinical and hematological presentation among Indian patients with common hemoglobin variants

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Common Hematologic Problems in the Newborn Nursery

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

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

KEY POINTS

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

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

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

Hemolytic conditions in the neonate

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

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

c. ABO blood group: Anti-A, -B

2. Red blood cell (RBC) enzyme defects

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

b. Pyruvate kinase deficiency

c. Others

3. RBC membrane defects

a. Hereditary spherocytosis

b. Elliptocytosis

c. Stomatocytosis

d. Pyknocytosis

e. Others

4. Hemoglobinopathies

a. alpha-thalassemia

b. gamma-thalassemia

Standard maternal antibody screeningAlloantibody Blood Group

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

K, k, Kpa, Jsa Kell

Fya, Fyb Duffy

Jka, Jkb Kidd

Xga Xg

Lea, Leb Lewis

S, s, M, N MNS

P1 P

Lub Lutheran

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

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

Red Blood Cell Enzymopathies

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

Red Blood Cell Membrane Defects

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

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

Risk factors for bilirubin neurotoxicityIsoimmune hemolytic disease

G6PD deficiency

Asphyxia

Sepsis

Acidosis

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

Polycythemia

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

Liver

Metabolic disorders presenting as liver disease

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

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

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

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

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

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

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

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

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

Alpha1-antitrypsin deficiency

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

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

Citrin deficiency

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

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

HFE-associated hemochromatosis

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

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

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

The liver in haemochromatosis

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

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

Gastrointestinal

Decoding the enigma of necrotizing enterocolitis in premature infants

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

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

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

Human gut microbiome

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

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

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

NEC is a multifactorial disease

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

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

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

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

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

Current Issues in the Management of Necrotizing Enterocolitis

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

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

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

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

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

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

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

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

Role of nitric oxide in the pathogenesis of NEC

Role of nitric oxide in the pathogenesis of NEC.

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

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

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

Intestinal Microbiota Development in Preterm Neonates and Effect of Perinatal Antibiotics

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

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

Human gut microbiome

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

Ischemia and necrotizing enterocolitis: where, when, and how

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

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

Schematic representation of the intestinal microcirculation

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

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

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

Necrotizing enterocolitis: An update

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

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

Immune Deficiencies

Primary immunodeficiencies: A rapidly evolving story

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

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

Combined Immunodeficiencies

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

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

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

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

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

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

TCR signaling

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

Uncoordinated 119 deficiency: Idiopathic CD41 lymphopenia

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

Well-Defined Syndromes with Immunodeficiency

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

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

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

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

Predominantly Antibody Defects

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

early colitis

Defects Of Immune Dysregulation

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

Congenital Defects Of Phagocyte Number, Function, Or Both

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

Defects In Innate Immunity

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

Autoinflammatory Disorders

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

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

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

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

Primary immunodeficiencies

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

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

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Endocrine Action on Midbrain

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

Endocrine Action on Midbrain

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

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

By Anna Azvolinsky | January 15, 2015

JUSTIN HEWLETT, MNHS MULTIMEDIA, MONASH UNIVERSITY

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Highlights

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

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

Light on the Brain

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

By Sabrina Richards | September 20, 2012

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

Juvenile zebrafish. Shawn Burgess, NHGRI

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

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

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

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

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

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

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

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

Fernandes et al., “Deep brain photoreceptors control light-seeking behavior in zebrafish larvae,” Current Biology, 22:1-6, 2012.

Neuroendocrine basis of sexuality, mood, anxiety, social consciousness

Physiology, signaling, and pharmacology of galanin peptides and receptors: Three decades of emerging diversity

Lang, R., Gundlach, A.L., Holmes, F.E., (…), Hökfelt, T., Kofler, B.
Pharmacological Reviews 2015: 67 (1), pp. 118-175
http://dx.doi.org:/10.1124/pr.112.006536

Galanin was first identified 30 years ago as a “classic neuropeptide,” with actions primarily as a modulator of neurotransmission in the brain and peripheral nervous system. Other structurally-related peptides—galanin-like peptide and alarin—with diverse biologic actions in brain and other tissues have since been identified, although, unlike galanin, their cognate receptors are currently unknown. Over the last two decades, in addition to many neuronal actions, a number of nonneuronal actions of galanin and other galanin family peptides have been described. These include actions associated with neural stem cells, nonneuronal cells in the brain such as glia, endocrine functions, effects on metabolism, energy homeostasis, and paracrine effects in bone. Substantial new data also indicate an emerging role for galanin in innate immunity, inflammation, and cancer. Galanin has been shown to regulate its numerous physiologic and pathophysiological processes through interactions with three G protein–coupled receptors, GAL1, GAL2, and GAL3, and signaling via multiple transduction pathways, including inhibition of cAMP/PKA (GAL1, GAL3) and stimulation of phospholipase C (GAL2). In this review, we emphasize the importance of novel galanin receptor–specific agonists and antagonists. Also, other approaches, including new transgenic mouse lines (such as a recently characterized GAL3 knockout mouse) represent, in combination with viral-based techniques, critical tools required to better evaluate galanin system physiology. These in turn will help identify potential targets of the galanin/galanin-receptor systems in a diverse range of human diseases, including pain, mood disorders, epilepsy, neurodegenerative conditions, diabetes, and cancer.

Estradiol regulates responsiveness of the dorsal premammillary nucleus of the hypothalamus and affects fear- and anxiety-like behaviors in female rats

Litvin, Y., Cataldo, G., Pfaff, D.W., Kow, L.-M.
European Journal of Neuroscience 2014; 40 (2), pp. 2344-2351
10.1111/ejn.12608

Research suggests a causal link between estrogens and mood. Here, we began by examining the effects of estradiol (E2) on rat innate and conditioned defensive behaviors in response to cat odor. Second, we utilized whole-cell patch clamp electrophysiological techniques to assess noradrenergic effects on neurons within the dorsal premammillary nucleus of the hypothalamus (PMd), a nucleus implicated in fear reactivity, and their regulation by E2. Our results show that E2 increased general arousal and modified innate defensive reactivity to cat odor. When ovariectomized females treated with E2 as opposed to oil were exposed to cat odor, they showed elevations in risk assessment and reductions in freezing, indicating a shift from passive to active coping. In addition, animals previously exposed to cat odor showed clear cue + context conditioning 24 h later. However, although E2 persisted in its effects on general arousal in the conditioning task, its effects on fear disappeared. In the patch clamp experiments noradrenergic compounds that typically induce fear clearly excited PMd neurons, producing depolarizations and action potentials. E2 treatment shifted some excitatory effects of noradrenergic agonists to inhibitory, possibly by differentially affecting α- and β-adrenoreceptors. In summary, our results implicate E2 in general arousal and fear reactivity, and suggest these may be governed by changes in noradrenergic responsivity in the PMd. These effects of E2 may have ethological relevance, serving to promote mate seeking even in contexts of ambiguous threat and shed light on the involvement of estrogen in mood and its associated disorders.

Endogenous opiates and behavior: 2013

Richard J. Bodnar
Peptides 62 (2014) 67–136
http://dx.doi.org/10.1016/j.peptides.2014.09.013

This paper is the thirty-sixth consecutive installment of the annual review of research concerning the endogenous opioid system. It summarizes papers published during 2013 that studied the behavioral effects of molecular, pharmacological and genetic manipulation of opioid peptides, opioid receptors, opioid agonists and opioid antagonists. The particular topics that continue to be covered include the molecular-biochemical effects and neurochemical localization studies of endogenous opioids and their receptors related to behavior, and the roles of these opioid peptides and receptors in pain and analgesia; stress and social status; tolerance and dependence; learning and memory; eating and drinking; alcohol and drugs of abuse; sexual activity and hormones, pregnancy, development and endocrinology; mental illness and mood; seizures and neurologic disorders; electrical-related activity and neurophysiology; general activity and locomotion; gastrointestinal, renal and hepatic functions; cardiovascular responses; respiration and thermoregulation; and immunological responses.

Brain aromatase (cyp19a1b) and gonadotropin releasing hormone (gnrh2 and gnrh3) expression during reproductive development and sex change in black sea bass (Centropristis striata)

Timothy S Breton, Matthew A DiMaggio, Stacia A Sowe, David L Berlinsky, et al.
Comparative Biochemistry and Physiology, Part A 181 (2015) 45–53
http://dx.doi.org/10.1016/j.cbpa.2014.11.020

Teleost fish exhibit diverse reproductive strategies, and some species are capable of changing sex. The influence of many endocrine factors, such as gonadal steroids and neuropeptides, has been studied in relation to sex change, but comparatively less research has focused on gene expression changes within the brain in temperate grouper species with non-haremic social structures. The purpose of the present study was to investigate gonadotropin releasing hormone (GnRH) and brain aromatase (cyp19a1b) gene expression patterns during reproductive development and sex change in protogynous (female to male) black sea bass (Centropristis striata). Partial cDNA fragments for cyp19a1b and eef1a (a reference gene) were identified, and included with known gnrh2 and gnrh3 sequences in real time quantitative PCR. Elevated cyp19a1b expression was evident in the olfactory bulbs, telencephalon, optic tectum, and hypothalamus/
midbrain region during vitellogenic growth, which may indicate changes in the brain related to neurogenesis or sexual behavior. In contrast, gnrh2 and gnrh3 expression levels were largely similar among gonadal states, and all three genes exhibited stable expression during sex change. Although sex change in black sea bass is not associated with dramatic changes in GnRH or cyp19a1b gene expression among brain regions, these genes may mediate processes at other levels, such as within individual hypothalamic nuclei, or through changes in neuron size, that warrant further research.

Evaluation for roles of neurosteroids in modulating forebrain mechanisms controlling vasopressin secretion and related phenomena in conscious rats

Ken’ichi Yamaguchi
Neuroscience Research xxx (2015) xxx–xxx
http://dx.doi.org/10.1016/j.neures.2015.01.002

Anteroventral third ventricular region (AV3V) regulates autonomic functions through a GABAergic mechanism that possesses neuroactive steroid (NS)-synthesizing ability. Although NS can exert effects by acting on a certain type of GABAA-receptor (R), it is not clear whether NS may operate to modulateAV3V GABAergic activity for controlling autonomic functions. This study aimed to investigate the issue.AV3V infusion with a GABAA antagonist bicuculline increased plasma vasopressin (AVP), glucose, blood pressure (BP), and heart rate in rats. These events were abolished by preinjecting its agonist muscimol, whereas the infusion with allopregnanolone, a NS capable of potentiating GABAA-R function, affectednone of the variables in the absence or presence of such bicuculline actions. Similarly, AV3V infusion with pregnanolone sulfate, a NS capable of antagonizing GABAA-R, produced no effect on those variables.AV3V infusion with muscimol was effective in inhibiting the responses of plasma AVP or glucose, orBP to an osmotic loading or bleeding. However, AV3V infusion with aminoglutethimide, a NS synthesis inhibitor, did not affect any of the variables in the absence or presence of those stimuli. These results suggest that NS may not cause acute effects on the AV3V GABAergic mechanism involved in regulating AVP release and other autonomic function.

Novel receptor targets for production and action of allopregnanolone in the central nervous system: a focus on pregnane xenobiotic receptor

Cheryl A. Frye, Carolyn J. Koonce, and Alicia A. Walf
Front in Cell Neurosci Apr 2014; 8(106)
http://dx.doi.org:/10.3389/fncel.2014.00106

Neurosteroids are cholesterol-based hormones that can be produced in the brain, independent of secretion from peripheral endocrine glands, such as the gonads and adrenals. A focus in our laboratory for over 25 years has been how production of the pregnane neurosteroid, allopregnanolone, is regulated and the novel (i.e., non steroid receptor) targets for steroid action for behavior. One endpoint of interest has been lordosis, the mating posture of female rodents. Allopregnanolone is necessary and sufficient for lordosis, and the brain circuitry underlying it, such as actions in the midbrain ventral tegmental area (VTA), has been well-characterized. Published and recent findings supporting a dynamic role of allopregnanolone are included in this review. First, contributions of ovarian and adrenal sources of precursors of allopregnanolone, and the requisite enzymatic actions for de novo production in the central nervous system will be discussed.
Second, how allopregnanolone produced in the brain has actions on behavioral processes that are independent of binding to steroid receptors, but instead involve rapid modulatory actions via neurotransmitter targets (e.g., g-amino butyric acid-GABA, Nmethyl-D-aspartate- NMDA) will be reviewed.
Third, a recent focus on characterizing the role of a promiscuous nuclear receptor, pregnane xenobiotic receptor (PXR), involved in cholesterol metabolism and expressed in the VTA, as a target for allopregnanolone and how this relates to both actions and production of allopregnanolone will be addressed. For example, allopregnanolone can bind PXR and knocking down expression of PXR in the midbrain VTA attenuates actions of allopregnanolone via NMDA and/or GABAA for lordosis. Our understanding of allopregnanolone’s actions in the VTA for lordosis has been extended to reveal the role of allopregnanolone for broader, clinically-relevant questions, such as neurodevelopmental processes, neuropsychiatric disorders, epilepsy, and aging.

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

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

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

Dysruption of Corticotropin Releasing Factor in hypocampal region

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

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

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

Central CRF system perturbation in an Alzheimer’s disease knockin mouse model

Qinxi Guo, Hui Zheng, Nicholas John Justice
Neurobiology of Aging 33 (2012) 2678–2691
http://dx.doi.org:/10.1016/j.neurobiolaging.2012.01.002

Alzheimer’s disease (AD) is often accompanied by changes in mood as well as increases in circulating cortisol levels, suggesting that regulation of the stress responsive hypothalamic-pituitary-adrenal (HPA) axis is disturbed. Here, we show that amyloid precursor protein (APP) is endogenously expressed in important limbic, hypothalamic, and midbrain nuclei that regulate hypothalamic-pituitary-adrenal axis activity. Furthermore, in a knockin mouse model of AD that expresses familial AD (FAD) mutations of both APP with humanized amyloid beta (hA), and presenilin 1 (PS1), in their endogenous patterns (APP/hA/PS1 animals), corticotropin releasing factor (CRF) levels are increased in key stress-related nuclei, resting corticosteroid levels are elevated, and animals display increased anxiety-related behavior. Endocrine and behavioral phenotypes can be normalized by loss of 1 copy of CRF receptor type-1 (Crfr1), consistent with a perturbation of central CRF signaling in APP/hA/PS1 animals. However, reductions in anxiety and corticosteroid levels conferred by heterozygosity of CRF receptor type-1 do not improve a deficit in working memory observed in APP/hA/PS1 mice, suggesting that perturbations of the CRF system are not the primary cause of decreased cognitive performance.

Alzheimer’s disease-like neuropathology of gene-targeted APP-SLxPS1mut mice expressing the amyloid precursor protein at endogenous levels

Christoph Kohler, Ulrich Ebert, Karlheinz Baumann, and Hannsjorg Schroeder
Neurobiology of Disease 20 (2005) 528 – 540
http://dx.doi.org:/10.1016/j.nbd.2005.04.009

Most transgenic mice used for preclinical evaluation of potential disease-modifying treatments of Alzheimer’s disease develop major histopathological features of this disease by several-fold overexpression of the human amyloid precursor protein. We studied the phenotype of three different strains of gene-targeted mice which express the amyloid precursor protein at endogenous levels. Only further crossing with transgenic mice overexpressing mutant human presenilin1 led to the deposition of extracellular amyloid, accompanied by the deposition of apolipoprotein E, an astrocyte and microglia reaction, and the occurrence of dilated cholinergic terminals in the cortex. Features of neurodegeneration, however, were absent. The pattern of plaque development and deposition in these mice was similar to that of amyloid precursor protein overproducing strains if crossed to presenilin1-transgenics. However, plaque development started much later and developed slowly until the age of 18 months but then increased more rapidly.

Central Cholinergic Functions In Human Amyloid Precursor Protein Knock-In/Presenilin-1 Transgenic Mice

Hartmann, C. Erb, U. Ebert, K. H. Baumann, A. Popp, G. Koenig, J. Klein
Neuroscience 125 (2004) 1009–1017
http://dx.doi.org:/10.1016/j.neuroscience.2004.02.038

Alzheimer’s disease is characterized by amyloid peptide formation and deposition, neurofibrillary tangles, central cholinergic dysfunction, and dementia; however, the relationship between these parameters is not well understood. We studied the effect of amyloid peptide formation and deposition on central cholinergic function in knock-in mice carrying the human amyloid precursor protein (APP) gene with the Swedish/London double mutation (APP-SL mice) which were crossbred with transgenic mice overexpressing normal (PS1wt) or mutated (M146L; PS1mut) human presenilin-1. APP-SLxPS1mut mice had increased levels of Aβ peptides at 10 months of age and amyloid plaques at 14 months of age while APP-SLPS1wt mice did not have increased peptide levels and did not develop amyloid plaques. We used microdialysis in 15–27 months old mice to compare hippocampal acetylcholine (ACh) levels in the two mouse lines and found that extracellular ACh levels were slightly but significantly reduced in the APP-SLPS1mut mice (-26%; P=0.044). Exploratory activity in the open field increased hippocampal ACh release by two-fold in both mouse lines; total and relative increases were not significantly different for the two strains under study. Similarly, infusion of scopolamine (1 µM) increased hippocampal ACh release to a similar extent (3–5-fold) in both groups. High-affinity choline uptake, a measure of the ACh turnover rate, was identical in both mouse lines. Neurons expressing choline acetyltransferase were increased in the septum of APP-SLPS1mut mice (26%; P =0.046). We conclude that amyloid peptide production causes a small decrease of extracellular ACh levels. The deposition of amyloid plaques, however, does not impair stimulated ACh release and proceeds without major changes of central cholinergic function.

Glutamate Neurotoxicity

Glutamate Neurotoxicity and Diseases of the Nervous System

Dennis W. Choi
Neuron. Oct, 1988; 1: 623-634

A growing number of studies now suggest that the cellular mechanisms which normally participate in signaling in the central nervous system (CNS) can be transformed by disease into instruments of neuronal cell destruction. Excitatory synaptic transmission in the mammalian CNS is principally mediated by L-glutamate. In fact, glutamate excites virtually all central neurons and is present in nerve terminals at millimolar levels (Curtis and Johnston, 1974). Normally, the extracellular levels of glutamate rise to high levels only in the brief and spatially localized fashion appropriate to synaptic transmission. This is fortunate, because as Lucas and Newhouse first showed in 1957, sustained exposure to glutamate can destroy retinal neurons. In a subsequent set of pioneering experiments, Olney (Olney and Sharpe, 1969; Olney et al., 1971) established that this toxicity, which he later called excitotoxicity, was not unique to glutamate or to retinal neurons, but was a feature common to the actions of all excitatory amino acids on central neurons. He postulated therefore that glutamate, or related compounds, might be the cause of the neuronal cell loss found in certain neurological diseases. In recent years, this hypothesis has gathered considerable support, fueled by new insights into glutamate receptor function and the development of effective glutamate antagonist drugs. The evidence is most convincing in diseases involving an acute insult to the brain, as occurs in a stroke, with abrupt deprivation of blood supply. But neurotoxicity due to excitatory amino acids may also be involved in slowly progressive degenerative diseases such as Huntington’s disease. Although the detailed molecular basis of glutamate neurotoxicity is not known, it appears that Ca2+ influx may play a critical role.
Glutamate interacts with at least three classes of membrane receptors, each commonly referred to by preferred pharmacological agonists: N-methyl-o-aspartate (NMDA), quisqualate, and kainate (Watkins and Olverman, 1987) (Figure I). These three classes are linked to membrane cation channels. A second type of quisqualate receptor has been additionally linked to a second messenger system (see below). It has been suggested that all three classes might actually be substates of a single molecular complex, but binding studies and newer physiological studies favor separate structures.

Quisqualate NMDA Kainate

Three Classes of Glutamate Receptors

One type of quisqualate receptor stimulates the formation of inositol 1,4,5-trisphosphate UPS) and diacylglycerol (DAG) from phosphatidylinositol-4,5-biphosphate (PIP,); the other is linked directly to a Na+ ionophore. Activation of the quisqualate receptor-ionophore complex can be potentiated by Zn2+. The NMDA receptor opens a channel permeable to Ca2+ as well as Na+; this receptor-channel complex has several modulatory sites discussed in the text. The kainate receptor opens an ionophore permeable to Na+.

Best defined is the NMDA receptor. This receptor opens a distinctive membrane channel characterized by high conductance (main state about 50 pS), voltage dependent Mgz+ blockade and permeability to both Ca2+ and Na+. The NMDA receptor can be selectively activated by several endogenous compounds, including L-aspartate, homocysteate, and quinolinate. Activation requires the coavailability of glycine in near micromolar concentrations. The action of glutamate at the NMDA receptor can be selectively antagonized: competitively by 2-amino-5-phosphonovalerate (APV) and 2-amino-5-phosphonoheptanoate (APH), or noncompetitively by drugs that bind to the phencyclidine site within the open channel (such as phencyclidine, MK-801, dextrorphan, or ketamine. The NMDA receptor-activated channel can also be blocked noncompetitively by Znz+, most likely at a site different from that which binds Mg2.
Although glutamate has high affinity for all three classes of postsynaptic receptors, it is not easy to demonstrate its neurotoxicity in vivo. Even when directly injected into brain, bypassing the blood-brain barrier, extremely high doses of glutamate are required to create lesions. Mangano & Schwartz found that they could infuse 0.5 crl/hr of a 300 mM glutamate solution into the hippocampus of a rat for 2 weeks without producing neuronal injury. This apparent low in vivo neurotoxic potency of glutamate may represent one reason why Olney’s “glutamate hypothesis” of neurological disease did not initially achieve a more widespread following. However, in fact, glutamate is a potent and rapidly acting neurotoxin; its neurotoxicity in vivo is likely masked by the efficiency of normal cellular uptake mechanisms in removing glutamate from the extracellular space. Glutamate neurotoxicity can be most directly studied in cell culture where bath exposure is not limited by cellular uptake.
The toxic changes produced by glutamate or related excitatory amino acids in vivo are of two sorts:

acute swelling of neuronal dendrites and cell bodies and a
more slowly evolving neuronal degeneration (Olney, 1986).

Axons and glia are relatively spared, although high levels of excitatory amino acids can produce some swelling of glia. A hallmark of excitatory amino acid neurotoxicity is its cellular selectivity, with distinctive patterns of neuronal loss produced by different excitatory amino acids and different routes of administration. For example, Nadler and co-workers (1978) found that intraventricular kainate preferentially destroys hippocampal CA3 neurons but spares dentate granule neurons. Different neuronal subpopulations
may differ in their intrinsic vulnerability to damage.

Possible Mechanisms Involved in Glutamate Neurotoxicity

How Ca*+ may mediate glutamate-induced neuronal degeneration. Glutamate acts on NMDA, non-NMDA, and “metabotropic” receptors (the quisqualate receptor linked to a second messenger system) to produce an increase in cytosolic free Ca*+. This cytosolic Ca *+, in concert with diacylglycerol liberated by the quisqualate-triggered second messenger system, activates protein kinase C, which acts via a number of mechanisms (primarily by altering membrane ion channels) to increase neuronal excitability and further increase cytosolic Ca*+. Elevated cytosolic Ca2+ then activates several enzymes capable of either directly or indirectly (through free radical formation) destroying cellular structure. Glutamate released from synaptic terminals or leaking nonspecifically from ruptured neurons contributes to additional injury propagation.

Glutamate Neurotoxicity in Perspective

The hypothesis that excitatory amino acids may specifically mediate pathological neuronal injury gives new form to this age-old enemy and raises the tantalizing possibility that current molecular and cellular insights into excitatory amino acid transmitter systems might be harnessed to develop an efficacious clinical therapy. Some points of attack are already apparent; others will likely be defined as the biology of excitatory amino acids continues to be unraveled. An intriguing area for investigation is the relationship between excitatory amino acid neurotoxicity and normal neuronal processes such as maturation, neurite outgrowth, and synaptic plasticity.

Glutamate Toxicity in a Neuronal Cell line Involves Inhibition of Cystine Transport Leading to Oxidative Stress

Timothy H. Murphy, M Miyamoto, A Sastre, R Schnaar and JT Coyle
Neuron 1989: 2: 1547-88.

Glutamate binds to both excitatory neurotransmitter binding sites and a W-dependent, quisqualate- and cystine-inhibited transport site on brain neurons. The neuroblastoma-primary retina hybrid cells (NWRE-105) are susceptible to glutamate-induced cytotoxicity. The Cl–dependent transport site to which glutamate and quisqualate (but not kainate or NMDA) bind has a higher affinity for cystine than for glutamate. Towering cystine concentrations in the cell culture medium results in cytotoxicity similar to that induced by glutamate addition in its morphology, kinetics, and CaZ+ dependence. Glutamate-induced cytotoxicity is directly proportional to its ability to inhibit cystine uptake. Exposure to glutamate (or lowered cystine) causes a decrease in glutathione levels and an accumulation of intracellular peroxides. Like NW-RE-105 cells, primary rat hippocampal neurons (but not glia) in culture degenerate in medium with lowered cystine concentration. Thus, glutamate-induced cytotoxicity in N18-RE-105 cells is due to inhibition of cystine uptake, resulting in lowered glutathione levels leading to oxidative stress and cell death.

Mechanism of glutamate-induced neurotoxicity in HT22 mouse hippocampal cells

Masayuki Fukui, Ji-Hoon Song, Jinyoung Choi, Hye Joung Choi, Bao Ting Zhu
European Journal of Pharmacology 617 (2009) 1–11
http://dx.doi.org:/10.1016/j.ejphar.2009.06.059

Glutamate is an endogenous excitatory neurotransmitter. At high concentrations, it is neurotoxic and contributes to the development of certain neurodegenerative diseases. There is considerable controversy in the literature with regard to whether glutamate-induced cell death in cultured HT22 cells (an immortalized mouse hippocampal cell line) is apoptosis, necrosis, or a new form of cell death. The present study focused on investigating the mechanism of glutamate-induced cell death. We found that glutamate induced, in a time dependent manner, both necrosis and apoptosis in HT22 cells. At relatively early time points (8–12 h), glutamate induced mostly necrosis, whereas at late time points (16–24 h), it induced mainly apoptosis. Glutamate-induced mitochondrial oxidative stress and dysfunction were crucial early events required for the induction of apoptosis through the release of the mitochondrial apoptosis-inducing factor (AIF), which catalyzed DNA fragmentation (an ATP-independent process). Glutamate-induced cell death proceeded independently of the Bcl-2 family proteins and caspase activation. The lack of caspase activation likely resulted from the lack of intracellular ATP when the mitochondrial functions were rapidly disrupted by the mitochondrial oxidative stress. In addition, it was observed that activation of JNK, p38, and ERK signaling molecules was also involved in the induction of apoptosis by glutamate. In conclusion, glutamate-induced apoptosis is AIF-dependent but caspase-independent, and is accompanied by DNA ladder formation but not chromatin condensation.

Understanding Low Reliability of Memories for Neutral Information Encoded under Stress: Alterations in Memory-Related Activation in the Hippocampus and Midbrain

Shaozheng Qin, EJ Hermans, HJF van Marle, and G Fernandez, et al.
The Journal of Neuroscience, Mar 21, 2012; 32(12): 4032–4041
http://dx.doi.org:/10.1523/JNEUROSCI.3101-11.2012

Exposure to an acute stressor can lead to unreliable remembrance of intrinsically neutral information, as exemplified by low reliability of eyewitness memories, which stands in contrast with enhanced memory for the stressful incident itself. Stress-sensitive neuromodulators (e.g., catecholamines) are believed to cause this low reliability by altering neurocognitive processes underlying memory formation. Using event-related functional magnetic resonance imaging, we investigated neural activity during memory formation in 44 young, healthy human participants while incidentally encoding emotionally neutral, complex scenes embedded in either a stressful or neutral context.
We recorded event-related pupil dilation responses as an indirect index of phasic noradrenergic activity. Autonomic, endocrine, and psychological measures were acquired to validate stress manipulation. Acute stress during encoding led to a more liberal response bias (more hits and false alarms) when testing memory for the scenes 24 h later. The strength of this bias correlated negatively with pupil dilation responses and positively with stress-induced heart rate increases at encoding. Acute stress, moreover, reduced subsequent memory effects (SMEs; items later remembered vs forgotten) in hippocampus and midbrain, and in pupil dilation responses.
The diminished SMEs indicate reduced selectivity and specificity in mnemonic processing during memory formation. This is in line with a model in which stress-induced catecholaminergic hyperactivation alters phasic neuromodulatory signaling in memory-related circuits, resulting in generalized (gist-based) processing at the cost of specificity. Thus, one may speculate that loss of specificity may yield less discrete memory representations at time of encoding, thereby causing a more liberal response bias when probing these memories.

Neuroendocrinology – Signaling, neuron plasticity and memory

Leptin Signaling Modulates the Activity of Urocortin 1 Neurons in the Mouse Nonpreganglionic Edinger-Westphal Nucleus

Lu Xu, Wim J. J. M. Scheenen, Rebecca L. Leshan, Christa M. Patterson, et al.
Endocrinology 152(3): 979–988, 2011
http://dx.doi.org:/10.1210/en.2010-1143

A recent study systematically characterized the distribution of the long form of the leptin receptor (LepRb) in the mouse brain and showed substantial LepRb mRNA expression in the nonpreganglionic Edinger-Westphal nucleus (npEW) in the rostroventral part of the midbrain. This nucleus hosts the majority of urocortin 1 (Ucn1) neurons in the rodent brain, and because Ucn1 is a potent satiety hormone and electrical lesioning of the npEW strongly decreases food intake, we have hypothesized a role of npEW-Ucn1 neurons in leptin-controlled food intake. Here, we show by immunohistochemistry that npEW-Ucn1 neurons in the mouse contain LepRb and respond to leptin administration with induction of the Janus kinase 2-signal transducer and activator of transcription 3 pathway, both in vivo and in vitro. Furthermore, systemic leptin administration increases the Ucn1 content of then pEW significantly, whereas in mice that lack LepRb (db/db mice), then pEW contains considerably reduced amount of Ucn1. Finally, we reveal by patch clamping of midbrain Ucn1 neurons that leptin administration reduces the electrical firing activity of the Ucn1 neurons. In conclusion, we provide ample evidence for leptin actions that go beyond leptin’s well-known targets in the hypothalamus and propose that leptin can directly influence the activity of the midbrain Ucn1 neurons.

Leptin regulation of hippocampal synaptic function in health and disease

Andrew J. Irving and Jenni Harvey
Trans. R. Soc. B 369: 20130155 http://dx.doi.org/10.1098/rstb.2013.0155

The endocrine hormone leptin plays a key role in regulating food intake and body weight via its actions in the hypothalamus. However, leptin receptors are highly expressed in many extra-hypothalamic brain regions and evidence is growing that leptin influences many central processes including cognition. Indeed, recent studies indicate that leptin is a potential cognitive enhancer as it markedly facilitates the cellular events underlying hippocampal-dependent learning and memory, including effects on glutamate receptor trafficking, neuronal morphology and activity-dependent synaptic plasticity. However, the ability of leptin to regulate hippocampal synaptic function markedly declines with age and aberrant leptin function has been linked to neurodegenerative disorders such as Alzheimer’s disease (AD). Here, we review the evidence supporting a cognitive enhancing role for the hormone leptin and discuss the therapeutic potential of using leptin-based agents to treat AD.

The Y2 receptor agonist PYY3–36 increases the behavioral response to novelty and acute dopaminergic drug challenge in mice

Ulrike Stadlbauer, Elisabeth Weber, Wolfgang Langhans and Urs Meyer
International Journal of Neuropsychopharmacology (2014), 17, 407–419
http://dx.doi.org:/10.1017/S1461145713001223

The gastrointestinal hormone PYY3–36 is a preferential Y2 neuropeptide Y (NPY) receptor agonist. Recent evidence indicates that PYY3–36 acts on central dopaminergic pathways, but its influence on dopamine-dependent behaviors remains largely unknown. We therefore explored the effects of peripheral PYY3–36 treatment on the behavioral responses to novelty and to dopamine-activating drugs in mice. In addition, we examined whether PYY3–36 administration may activate distinct dopamine and γ-aminobutyric acid (GABA) cell populations in the mesoaccumbal and nigrostriatal pathways. We found that i.p. PYY3–36 injection led to a dose-dependent increase in novel object exploration. The effective dose of PYY3–36 (1 μg/100 g body weight) also potentiated the locomotor reaction to the indirect dopamine receptor agonist amphetamine and increased stereotyped climbing/leaning responses following administration of the direct dopamine receptor agonist apomorphine. PYY3–36 administration did not affect activity of midbrain dopaminergic cells as evaluated by double immuno-enzyme staining of the neuronal early gene product c-Fos with tyrosine hydroxylase. PYY3–36 did, however, lead to a marked increase in the number of cells co-expressing c-Fos with glutamic acid decarboxylase in the nucleus accumbens and caudate putamen, indicating activation of GABAergic cells in dorsal and ventral striatal areas. Our results support the hypothesis that acute administration of the preferential Y2 receptor agonist PYY3–36 modulates dopamine-dependent behaviours. These effects do not seem to involve direct activation of midbrain dopamine cells but instead are associated with neuronal activation in the major input areas of the mesoaccumbal and nigrostriatal pathways.

Somatostatin and nociceptin inhibit neurons in the central nucleus of amygdala that project to the periaqueductal grey

Billy Chieng, MacDonald J. Christie
Neuropharmacology 59 (2010) 425e430
http://dx.doi.org:/10.1016/j.neuropharm.2010.06.001

The central nucleus of amygdala (CeA) plays an important role in modulation of the descending antinociceptive pathways. Using whole-cell patch clamp recordings from brain slices, we found that CeA neurons responded to the endogenous ligands somatostatin (SST) and nociceptin/orphanin FQ (OFQ) via an increased K-conductance. Co-application with selective antagonists suggested that SST and OFQ act on SSTR2 and ORL1 receptors, respectively. Taking account of anatomical localisation of recorded neurons, the present study showed that many responsive neurons were located within the medial subdivision of CeA and all CeA projection neurons to the midbrain periaqueductal grey invariably responded to these peptides. Randomly selected agonist-responsive neurons in CeA predominantly classified physiologically as low-threshold spiking neurons. The similarity of SST, OFQ and, as previously reported, opioid responsiveness in a sub-population of CeA neurons suggests converging roles of these peptides to inhibit the activity of projections from CeA to vlPAG, and potentially similar antinociceptive actions in this pathway.

In vitro identification and electrophysiological characterization of dopamine neurons in the ventral tegmental area

Tao A. Zhang, Andon N. Placzek, John A. Dani
Neuropharmacology 59 (2010) 431e436
http://dx.doi.org:/10.1016/j.neuropharm.2010.06.004

Dopamine (DA) neurons in the ventral tegmental area (VTA) have been implicated in brain mechanisms related to motivation, reward, and drug addiction. Successful identification of these neurons in vitro has historically depended upon the expression of a hyperpolarization-activated current (I_h) and immunohistochemical demonstration of the presence of tyrosine hydroxylase (TH), the rate-limiting enzyme for DA synthesis. Recent findings suggest that electrophysiological criteria may be insufficient for distinguishing DA neurons from non-DA neurons in the VTA. In this study, we sought to determine factors that could potentially account for the apparent discrepancies in the literature regarding DA neuron identification in the rodent brain slice preparation. We found that confirmed DA neurons from the lateral VTA generally displayed a larger amplitude I_h relative to DA neurons located in the medial VTA. Measurement of a large amplitude I_h (>100 pA) consistently indicated a dopaminergic phenotype, but non-dopamine neurons also can have I_h current. The data also showed that immunohistochemical TH labeling of DA neurons can render false negative results after relatively long duration (>15 min) wholecell patch clamp recordings. We conclude that whole-cell patch clamp recording in combination with immunohistochemical detection of TH expression can guarantee positive but not negative DA identification in the VTA.

Dopamine Enables In Vivo Synaptic Plasticity Associated with the Addictive Drug Nicotine

Jianrong Tang and John A. Dani
Neuron, Sept 10, 2009; 63, 673–682
http://dx.doi.org:/10.1016/j.neuron.2009.07.025

Addictive drugs induce a dopamine signal that contributes to the initiation of addiction, and the dopamine signal influences drug-associated memories that perpetuate drug use. The addiction process shares many commonalities with the synaptic plasticity mechanisms normally attributed to learning and memory. Environmental stimuli repeatedly linked to addictive drugs become learned associations, and those stimuli come to elicit memories or sensations that motivate continued drug use. Applying in vivo recording techniques to freely moving mice, we show that physiologically relevant concentrations of the addictive drug nicotine directly cause in vivo hippocampal synaptic potentiation of the kind that underlies learning and memory. The drug-induced long-term synaptic plasticity required a local hippocampal dopamine signal. Disrupting general dopamine signaling prevented the nicotine-induced synaptic plasticity and conditioned place preference. These results suggest that dopaminergic signaling serves as a functional label of salient events by enabling and scaling synaptic plasticity that underlies drug-induced associative memory.

NCS-1 in the Dentate Gyrus Promotes Exploration, Synaptic Plasticity, and Rapid Acquisition of Spatial Memory

Bechara J. Saab, John Georgiou, Arup Nath, Frank J.S. Lee, et al.
Neuron, Sept 10, 2009; 63, 643–656
http://dx.doi.org:/10.1016/j.neuron.2009.08.014

The molecular underpinnings of exploration and its link to learning and memory remain poorly understood. Here we show that inducible, modest overexpression of neuronal calcium sensor 1 (Ncs1) selectively in the adult murine dentate gyrus (DG) promotes a specific form of exploratory behavior. The mice also display a selective facilitation of longterm potentiation (LTP) in the medial perforant path and a selective enhancement in rapid-acquisition spatial memory, phenotypes that are reversed by direct application of a cell-permeant peptide (DNIP) designed to interfere with NCS-1 binding to the dopamine type-2 receptor (D2R). Moreover, the DNIP and the D2R-selective antagonist L-741,626 attenuated exploratory behavior, DG LTP, and spatial memory in control mice. These data demonstrate a role for NCS-1 and D2R in DG plasticity and provide insight for understanding how the DG contributes to the origin of exploration and spatial memory acquisition.

Neuroligin 2 Drives Postsynaptic Assembly at Perisomatic Inhibitory Synapses through Gephyrin and Collybistin

Alexandros Poulopoulos, Gayane Aramuni, Guido Meyer, Tolga Soykan, et al.
Neuron 63, 628–642, Sept 10, 2009
http://dx.doi.org:/10.1016/j.neuron.2009.08.023

In the mammalian CNS, each neuron typically receives thousands of synaptic inputs from diverse classes of neurons. Synaptic transmission to the postsynaptic neuron relies on localized and transmitter-specific differentiation of the plasma membrane with postsynaptic receptor, scaffolding, and adhesion proteins accumulating in precise apposition to presynaptic sites of transmitter release. We identified protein interactions of the synaptic adhesion molecule neuroligin 2 that drive postsynaptic differentiation at inhibitory synapses. Neuroligin 2 binds the scaffolding protein gephyrin through a conserved cytoplasmic motif and functions as a specific activator of collybistin, thus guiding membrane tethering of the inhibitory postsynaptic scaffold. Complexes of neuroligin 2, gephyrin and collybistin are sufficient for cell-autonomous clustering of inhibitory neurotransmitter receptors. Deletion of neuroligin 2 in mice perturbs GABAergic and glycinergic synaptic transmission and leads to a loss of postsynaptic specializations specifically at perisomatic inhibitory synapses.

A Subset of Ventral Tegmental Area Neurons is Inhibited by Dopamine, 5-Hydroxytryptamine and Opioids

L. Cameron, M. W. Wessendorf and J. T. Williams
Neuroscience 1997; 77(1), pp. 155–166 PII: S0306-4522(96)00444-7

Neurons originating in the ventral tegmental area are thought to play a key role in the formation of addictive behaviors, particularly in response to drugs such as cocaine and opioids. In this study we identified different populations of ventral tegmental area neurons by the pharmacology of their evoked synaptic potentials and their response to dopamine, 5-hydroxytryptamine and opioids. Intracellular recordings were made from ventral tegmental area neurons in horizontal slices of guinea-pig brain and electrical stimulation was used to evoke synaptic potentials. The majority of cells (61.3%) hyperpolarized in response to dopamine, depolarized to 5-hydroxytryptamine, failed to respond to [Met]5enkephalin and exhibited a slow GABAB-mediated inhibitory postsynaptic potential. A smaller proportion of cells (11.3%) hyperpolarized in response to [Met]5enkephalin, depolarized to 5-hydroxytryptamine, failed to respond to dopamine and did not exhibit a slow inhibitory postsynaptic potential. These two groups of cells corresponded to previously described ‘‘principal’’ and ‘‘secondary’’ cells, respectively. A further group of cells (27.4%) was identified that, like the principal cells, hyperpolarized to dopamine.

However, these ‘‘tertiary cells’’ also hyperpolarized to both 5-hydroxytryptamine and [Met]5enkephalin and exhibited a slow, cocaine-sensitive 5-hydroxytryptamine1A-mediated inhibitory postsynaptic potential. When principal and tertiary cells were investigated immuno-histochemically, 82% of the principal cells were positive for tyrosine hydroxylase compared
with only 29% of the tertiary cells. The 5-hydroxytryptamine innervation of both these cell types was investigated and a similar density of putative contacts was observed near the somata and dendrites in both groups. This latter finding suggests that the existence of a 5-hydroxytryptamine-mediated inhibitory postsynaptic potential in the tertiary cells may be determined by the selective expression of 5-hydroxytryptamine receptors, rather than the distribution or density of the 5-hydroxytryptamine innervation.
We conclude that tertiary cells are a distinct subset of ventral tegmental area neurons where cocaine and μ-opioids both mediate inhibition.

Dopamine reward circuitry: Two projection systems from the ventral midbrain to the nucleus accumbens–olfactory tubercle complex

Satoshi Ikemoto
Brain Research Reviews 56 (2007) 27–78
http://:dx.doi.org:/10.1016/j.brainresrev.2007.05.004

Anatomical and functional refinements of the meso-limbic dopamine system
of the rat are discussed. Present experiments suggest that dopaminergic neurons localized in the posteromedial ventral tegmental area (VTA) and central linear nucleus raphe selectively project to the ventromedial striatum (medial olfactory tubercle and medial nucleus accumbens shell), whereas
the anteromedial VTA has few if any projections to the ventral striatum,
and the lateral VTA largely projects to the ventrolateral striatum (accumbens
core, lateral shell and lateral tubercle). These findings complement the recent behavioral findings that cocaine and amphetamine are more rewarding when administered into the ventromedial striatum than into the ventrolateral striatum. Drugs such as nicotine and opiates are more rewarding when administered into the posterior VTA or the central linear nucleus than into
the anterior VTA. A review of the literature suggests that
(1) the midbrain has corresponding zones for the accumbens core and medial shell;
(2) the striatal portion of the olfactory tubercle is a ventral extension of the nucleus accumbens shell; and
(3) a model of two dopamine projection systems from the ventral midbrain to the ventral striatum is useful for understanding reward function.
The medial projection system is important in the regulation of arousal characterized by affect and drive and plays a different role in goal directed learning than the lateral projection system, as described in the variation–selection hypothesis of striatal functional organization.

Metabolic hormones, dopamine circuits, and feeding

Nandakumar S. Narayanan, Douglas J. Guarnieri, Ralph J. DiLeone
Frontiers in Neuroendocrinology 31 (2010) 104–112
http://dx.doi.org:/10.1016/j.yfrne.2009.10.004

Recent evidence has emerged demonstrating that metabolic hormones such as ghrelin and leptin can act on ventral tegmental area (VTA) midbrain dopamine neurons to influence feeding. The VTA is the origin of mesolimbic dopamine neurons that project to the nucleus accumbens (NAc) to influence behavior. While blockade of dopamine via systemic antagonists or targeted gene delete can impair food intake, local NAc dopamine manipulations have little effect on food intake. Notably, non-dopaminergic manipulations in the VTA and NAc produce more consistent effects on feeding and food choice. More recent genetic evidence supports a role for the substantia nigra-striatal dopamine pathways in food intake, while the VTA–NAc circuit is more likely involved in higher-order aspects of food acquisition, such as motivation and cue associations. This rich and complex literature should be considered in models of how peripheral hormones influence feeding behavior via action on the midbrain circuits.

Control of brain development and homeostasis by local and systemic insulin signaling

Liu, P. Speder & A. H. Brand
Diabetes, Obesity and Metabolism 16 (Suppl. 1): 16–20, 2014

Insulin and insulin-like growth factors (IGFs) are important regulators of growth and metabolism. In both vertebrates and invertebrates, insulin/IGFs are made available to various organs, including the brain, through two routes: the circulating systemic insulin/IGFs act on distant organs via endocrine signaling, whereas insulin/IGF ligands released by local tissues act in a paracrine or autocrine fashion. Although the mechanisms governing the secretion and action of systemic insulin/IGF have been the focus of extensive investigation, the significance of locally derived insulin/IGF has only more recently come to the fore. Local insulin/IGF signaling is particularly important for the development and homeostasis of the central nervous system, which is insulated from the systemic environment by the blood–brain barrier. Local insulin/IGF signaling from glial cells, the blood–brain barrier and the cerebrospinal fluid has emerged as a potent regulator of neurogenesis. This review will address the main sources of local insulin/IGF and how they affect neurogenesis during development. In addition, we describe how local insulin/IGF signaling couples neural stem cell proliferation with systemic energy state in Drosophila and in mammals.

Pharmacology, Physiology, and Mechanisms of Action of Dipeptidyl Peptidase-4 Inhibitors

Erin E. Mulvihill and Daniel J. Drucker
Endocrine Reviews 35: 992–1019, 2014
http://dx.doi.org/10.1210/er.2014-1035

Dipeptidyl peptidase-4 (DPP4) is a widely expressed enzyme transducing actions through an anchored transmembrane molecule and a soluble circulating protein. Both membrane-associated and soluble DPP4 exert
catalytic activity, cleaving proteins containing a position 2 alanine or proline. DPP4-mediated enzymatic cleavage alternatively inactivates peptides or generates new bioactive moieties that may exert competing or novel activities. The widespread use of selective DPP4 inhibitors for the treatment of type 2 diabetes has heightened interest in the molecular mechanisms through which DPP4 inhibitors exert their pleiotropic actions. Here we review the biology ofDPP4with a focus on:
1) identification of pharmacological vs physiological DPP4 substrates; and
2) elucidation of mechanisms of actions of DPP4 in studies employing genetic elimination or chemical reduction of DPP4 activity.
We review data identifying the roles of key DPP4 substrates in transducing the glucoregulatory, anti-inflammatory, and cardiometabolic actions of DPP4 inhibitors in both preclinical and clinical studies. Finally, we highlight experimental pitfalls and technical challenges encountered in studies designed to understand the mechanisms of action and downstream targets activated by inhibition of DPP4.
Dipeptidyl peptidase-4 (DPP4) is a multifunctional protein that exerts biological activity through pleiotropic actions including:

protease activity (1),
association with adenosine deaminase (ADA) (2),
interaction with the extracellular matrix (3),
cell surface coreceptor activity mediating viral entry (4), and
regulation of intracellular signal transduction coupled to control of cell migration and proliferation (5).

The complexity of DPP4 action is amplified by the panoply of bioactive DPP4 substrates, which in turn act as elegant biochemical messengers in multiple tissues, including the immune and neuroendocrine systems.

DPP4 transmits signals across cell membranes and interacts with other membrane proteins (Figure). Remarkably, most of the protein is extracellular, including the C-terminal catalytic domain, a cysteine-rich area, and a large glycosylated region linked by a flexible stalk to the transmembrane segment. Only six N-terminal amino acids are predicted to extend into the cytoplasm. The active site, Ser 630, is flanked by the classic serine peptidase motif Gly-Trp-Ser630-Tyr-Gly-Gly-Tyr-Val.

Membrane-bound DPP4

Membrane-bound DPP4 contains residues 1–766, whereas sDPP4 contains residues 39–766. sDPP4 is lacking the cytoplasmic domain [residues 1–6], transmembrane domain [residues 7–28], and the flexible stalk [residues 29–39]. Both membrane-bound and circulating sDPP4 share many domains including the glycosylated region [residues 101–535, specific residues 85,92, 150], ADA binding domain [340–343], fibronectin binding domain [468–479], cysteine-rich domain [351–506, disulfide bonds are formed from 385–394, 444–472, and 649–762], and the catalytic domain [507–766 including residues composing the catalytic active site 630, 708, and 740].

DPP4 activity is subject to regulation at many levels, including control of gene and protein expression, interaction with binding partners, and modulation of enzyme activity. The DPP4 gene does not contain conventional TATAA or CCAAT promoter sequences but is characterized by a cytosine/guanine-rich promoter region.
DPP4 contains eight to 11 potential N-glycosylation sites, which can contribute to its folding and stability. Although glycosylation may contribute approximately 18–25% of the total molecular weight, mutational analysis has determined that the glycosylation sites are not required for dimerization, catalytic activity, or ADA binding. However, N-terminal sialylation facilitates trafficking of DPP4 to the apical membrane. Interestingly, molecular analysis of DPP4 isoforms isolated from the rat kidney brush border membrane reveals extensive heterogeneity in the oligosaccharides of DPP4.DPP4 was first investigated for its role in hydrolysis of dietary prolyl peptides (58); subsequent studies using DPP4 isolated using immunoaffinity chromatography and ADA binding identified DPP4 as the primary enzyme responsible for the generation of Gly-Prop-nitroanilide substrates in human serum. It is now known that DPP4 can cleave dozens of peptides, including chemokines, neuropeptides, and regulatory peptides, most containing a proline or alanine residue at position 2 of the amino-terminal region. Despite the preference for a position 2 proline, alternate residues (hydroxyproline, dehydroproline > alanine >, glycine, threonine, valine, or leucine) at the penultimate position are also cleaved by DPP4, suggesting a required stereochemistry. The DPP4 cleavage at postproline peptide bonds inactivates peptides and/or generates new bioactive peptides (see Figure), thereby regulating diverse biological processes.

DPP4 cleavage regulates substrate-receptor interactions

DPP4 cleavage regulates substrate/receptor interactions. A, DPP4 cleaves NPY [1–36] and PYY [1–36]. The intact forms of these peptides signal through Y1R-Y5R. After DPP4 cleavage, NPY [3–36] and PYY [3–36] are generated and preferentially signal through the Y2R and Y5R. B, DPP4 cleaves SP [1–11], which signals through the NK1R receptor to generate SP [5–11], which can signal through (NK1R, -2R, -3R).

GHRH and IGF-1

GHRH [1–44] and [1–40] are produced in the arcuate nucleus of the hypothalamus and bind its receptor on the anterior pituitary to stimulate the release of GH, and in turn, GH stimulates hepatic IGF-1 release. GHRH was among the first peptides to be identified as a DPP4 substrate; it is rapidly degraded in rodent and human plasma to GHRH [3–44]/GHRH [3–40], and this cleavage was blocked upon incubation of human plasma with the DPP4 inhibitor, diprotin A (99).GHRH[1–44] or [1–40] exhibits a very short half-life (6 min) andDPP4 cleavage was initially perceived to be a critical regulator of GHRH bioactivity and, in turn, the GH-IGF-1 axis. IGF-1, the downstream effector of GHRH and GH, is a 105-amino acid protein produced mainly by the liver.
IGF-1 was identified as a pharmacological DPP4 substrate by matrix-assisted laser desorption/ionization-time of flight analysis of molecular forms of IGF-1 generated after incubation with DPP4 purified from baculovirus-infected insect cells. However, studies in pigs treated with sitagliptin at doses inhibiting 90% of DPP4 activity failed to demonstrate an increase in active intact IGF-1.
Clinically, treatment of healthy human male subjects with sitagliptin (25–600 mg) for 10 days did not produce increased concentrations of serum IGF-1 or IGF-binding protein 3 as measured by ELISA. Furthermore, Dpp4/ mice or rats do not exhibit increased organ growth or body size. Hence, the available data suggest that although DPP4 cleaves and inactivates both GHRH and IGF-1, enzymatic inactivation by DPP4 is not the major mechanism regulating the bioactivity of the GHRH-IGF-1 axis.

The role of acute cortisol and DHEAS in predicting acute and chronic PTSD symptoms

Joanne Mouthaan, Marit Sijbrandij, Jan S.K. Luitse
Psychoneuroendocrinology (2014) 45, 179—186
http://dx.doi.org/10.1016/j.psyneuen.2014.04.001

Background: Decreased activation of the hypothalamus—pituitary—adrenal (HPA) axis in response to stress is suspected to be a vulnerability factor for posttraumatic stress disorder (PTSD). Previous studies showed inconsistent findings regarding the role of cortisol in predicting PTSD. In addition, no prospective studies have examined the role of dehydroepiandrosterone (DHEA), or its sulfate form DHEAS, and the cortisol-to-DHEA(S) ratio in predicting PTSD. In this study, we tested whether acute plasma cortisol, DHEAS and the cortisol-to-DHEAS ratio predicted PTSD symptoms at 6 weeks and 6 months post-trauma. Methods: Blood samples of 397 adult level-1 trauma center patients, taken at the trauma resuscitation room within hours after the injury, were analyzed for cortisol and DHEAS levels. PTSD symptoms were assessed at 6 weeks and 6 months post-trauma with the Clinician Administered PTSD Scale. Results: Multivariate linear regression analyses showed that lower cortisol predicted PTSD symptoms at both 6 weeks and 6 months, controlling for age, gender, time of blood sampling, injury, trauma history, and admission to intensive care. Higher DHEAS and a smaller cortisol-to-DHEAS ratio predicted PTSD symptoms at 6 weeks, but not after controlling for the same variables, and not at 6 months. Conclusions: Our study provides important new evidence on the crucial role of the HPA-axis in response to trauma by showing that acute cortisol and DHEAS levels predict PTSD symptoms in survivors of recent trauma.
Neurobiology of DHEA and effects on sexuality, mood and cognition

Pluchino, P.Drakopoulos, F.Bianchi-Demicheli, J.M.Wenger
J Steroid Biochem & Molec Biol 145 (2015) 273–280
http://dx.doi.org/10.1016/j.jsbmb.2014.04.012

Dehydroepiandrosterone (DHEA) and its sulfate ester, DHEAS, are the most abundant steroid hormones in the humans. However, their physiological significance, their mechanisms of action and their possible roles as treatment are not fully clarified. Biological actions of DHEA(S) in the brain involve neuroprotection, neurite growth, neurogenesis and neuronal survival, apoptosis, catecholamine synthesis and secretion, as well as anti-oxidant, anti- inflammatory and antiglucocorticoid effects. In addition, DHEA affects neurosteroidogen is and endorphin synthesis/release. We also demonstrated in a model of ovariectomized rats that DHEA therapy increases proceptive behaviors, already after 1 week of treatment, affecting central function of sexual drive. In women, the analyses of clinical outcomes are far from being conclusive and many issues should still be addressed. Although DHEA preparations have been available in the market since the 1990s, there are very few definitive reports on the biological functions of this steroid. We demonstrate that 1 year DHEA administration at the dose of 10mg provided a significant improvement in comparison with vitamin D in sexual function
and in frequency of sexual intercourse in early postmenopausal women. Among symptomatic women, the spectrum of symptoms responding to DHEA requires further investigation, to define the type of sexual symptoms (e.g. decreased sexual function or hypoactive sexual desire disorder) and the degree of mood/cognitive symptoms that could be responsive to hormonal treatment.
In this regard, our findings are promising, although they need further exploration with a larger and more representative sample size.
Although adrenal cortex is considered to be the primary source of DHEAS in the brain, it was reported that DHEAS did not dis- appear or decrease in the brain 15 days neither after orchiectomy, adrenalectomy, or both, nor after the inhibition of adrenal secretion by dexamethasone. DHEA and DHEAS were among the first neurosteroids identified in rat brains. Cytochrome P450c17 was found in a subset of neurons of embryonic rodent brains. While P450c17 protein was readily detected in the brain, the abundance of P450c17 mRNA transcripts in the embryonic mouse brain or hippocampus of adult male rats was low, and was approximated to be 1/200^th of the expression in testis.
DHEAS may be synthesized in the brain from DHEA. Sulfation of DHEA has been observed in the brains of rhesus monkeys in vivo and in human fetal brain slices in vitro. DHEA sulfotransferase (HSTor SULT2A1) is an enzyme that sulfonates DHEA (in addition to pregnenolone).Western blotting and immune-histochemistry showed protein expression of an HST in adult Wistar rat brain. In addition SULT2A1 mRNA expression has been shown in rat brains. DHEAS is predominately transported out of the brain across the blood–brain barrier and DHEAS found in the brain is most likely due to local synthesis . DHEA(S) may mediate some of its actions through conversion into more potent sex steroids and activation of androgen or estrogen receptors in tissue.
According to existing assumption of the biology of depression, DHEA(S) ability to modulate many neurobiological actions could underlie relationships between endogenous and/or exogenously- supplemented DHEA(S) concentrations and depression. There is evidence that DHEAS concentrations are negatively correlated with ratings of depressed mood. However, the remaining literature examining plasma and serum DHEA(S) concentrations in depression is still inconsistent and other plasma indexes were studied in order to more accurately discriminate depressed from nondepressed individuals. Hypothalamic–pituitary–adrenal axis (HPA) hyperactivity has
been demonstrated in chronic diseases affecting nervous system disorders like depression. The end products of HPA axis, glucocorticoids (GCs), regulate many physiological functions and play an important role in affective regulation and dysregulation. Despite DHEAS levels which markedly decrease throughout adulthood, an increase in circulating cortisol with advanced age has been observed in human and nonhuman primates.
The most relevant aspect meriting attention is certainly the controversial finding among the studies that investigate the correlation of the endogenous DHEA sulfate (DHEAS) level, the aging process or organ illness with the results coming from studies focusing on the effects of exogenous DHEAS administration on brain function, sexuality, cardiovascular health and metabolic syndrome. Indeed, the marked age-related decline in serum DHEA and DHEAS has suggested that a deficiency of these steroids may be causally related to the development of a series of diseases that are generally associ- ated with aging. The postulated consequences of low DHEA levels include insulin resistance, obesity, cardiovascular disease, cancer, reduction of the immune defense system as well as psychosocial problems such as depression and a general deterioration in the sensation of well-being and cognitive function, DHEA replacement may seem an attractive treatment opportunity. Nevertheless, the analyses of clinical outcomes are far from being conclusive.

Dehydroepiandrosterone, its metabolites and ion channels

Hill, M. Dusková, L. Stárka
J Steroid Biochem & Molec Biology 145(2015)293–314
http://dx.doi.org/10.1016/j.jsbmb.2014.05.006

This review is focused on the physiological and pathophysiological relevance of steroids influencing the activities of the central and peripheral nervous systems with regard to their concentrations in body fluids and tissues in various stages of human life like the fetal development or pregnancy. The data summarized in this review shows that DHEA and its unconjugated and sulfated metabolites are physiologically and pathophysiologically relevant in modulating numerous ion channels and participate in vital functions of the human organism. DHEA and its unconjugated and sulfated metabolites including 5 _/ _-reduced androstane steroids participate in various physiological and pathophysiological processes like the management of GnRH cyclic release, regulation of glandular and neurotransmitter secretions, maintenance of glucose homeostasis on one hand and insulin insensitivity on the other hand, control of skeletalmuscle and smooth muscle activities including vasoregulation, promotion of tolerance to ischemia and other neuroprotective effects. In respect of prevalence of steroid sulfates over unconjugated steroids in the periphery and the opposite situation in the CNS, the sulfated androgens and androgen metabolites reach relevance in peripheral organs. The unconjugated androgens and estrogens are relevant in periphery and so much the more in the CNS due to higher concentrations of most unconjugated steroids in the CNS tissues than in circulation and peripheral organs.

Neurotrophins are proteins found within a broad range of cell types in the brain and periphery that facilitate neuronal growth, survival, and plasticity. The neurotrophin ‘‘superfamily’’ includes nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT3), neurotrophin-4/5 (NT4/5), and neurotrophin-6. Target tissues are hypothesized to regulate neuron survival by making neurotrophins available in limited amounts, resulting in selection of neurons with the best connectivity to the target tissue. NGF, in particular, is released by the target tissue and taken up in responsive neurons by receptor-mediated endocytosis. It is then transported retrogradedly into the cell where it exerts trophic effects. Lu et al. proposed a ‘‘Yin and Yang model,’’ whereby neurotrophic action is mediated by two principal classes of transmembrane receptor systems: the tyrosine kinase (Trk) receptors (including TrkA [selective for NGF], TrkB [selective for BDNF and NT4/5], and TrkC [selective for NT3]) and the neurotrophin receptor p75NTR. Each receptor type binds mature neurotrophins and/or neurotrophin precursors (proneurotrophins), creating a complex ‘‘balance’’ that then causes neuronal survival or death.
DHEA has been shown to evoke NGF mRNA expression in target cells. In a study of pregnant women, Schulte-Herbrüggen et al. showed no relationships between serum DHEAS and NGF. In contrast, we showed that DHEAS independently associated with salivary NGF (sNGF) in military men under baseline conditions, while DHEA did not. We now know that both DHEA(S) and NGF respond affirmatively to stressful insult, yet the association between these analytes during stress exposure is not understood. Characterization of this relationship has implications for prevention and treatment of traumatic stress and injury, degenerative disease management, and nerve repair. In this report, we extended our prior study of neuroprotective properties of DHEAS in men under baseline conditions to a prospective paradigm involving intense stress exposure in both men and women. We hypothesized that

(a) robust associations would prevail between total output of DHEAS and sNGF across the stress trajectory and at each time point,
(b) changes in DHEAS would predict corresponding changes in sNGF, and
(c) baseline DHEAS would positively predict total sNGF output across the stress trajectory.
We also explored the roles of testosterone and cortisol. In light of less definitive prior literature, directional hypotheses were not stated regarding these analytes.

In the first regression model, total hormone output (AUCG) of the independent variables (DHEAS, testosterone, and cortisol) combined to explain 63.7% of variance in sNGF output (F = 65.4, p < 0.001). Standardized beta coefficients revealed that testosterone exerted an independent effect (b = 0.80, p < 0.001), while the other predictors were not significant. In light of this unexpected finding, we then used regression-based causal steps modeling to evaluate whether testosterone mediated a hypothesized direct effect of DHEAS on sNGF. Following this approach, DHEAS predicted sNGF in an initial regression model (b = 0.45, p < 0.001). When testosterone was added, the direct effect of DHEAS (path c0) on sNGF was nearly eradicated and no longer significant (b = .04, p = .57), thus suggesting a mediated effect. An alternate statistical test (Sobel Test; 34) evaluating the hypothesized difference between the total effect (path c) and the direct effect (path c0) of DHEAS on sNGF produced a similar result (test statistic = 4.0, p < 0.001). Fig. 1 depicts positive association of DHEAS to sNGF, while Fig. 2 depicts Positive association of testosterone to sNGF.

Positive association of DHEAS total output and sNGF total output

Positive association of testosterone total output and sNGF total output

Positive association of testosterone total output and sNGF total output.
The models were then decomposed at each time point. At baseline, the independent variables (DHEAS, testosterone, and cortisol) combined to account for 10.2% of variance in sNGF (F = 5.3, p < 0.01). Standardized beta coefficients showed that DHEAS exerted an independent effect on sNGF (b = 0.39, p < 0.001), while the other predictors were not significant. During stress exposure, the independent variables combined to account for 28.0% of variance in NGF (F = 15.8, p < 0.001). Again, DHEAS exerted an independent effect (b = 0.56, p < 0.001) while the other predictors were not significant. During recovery, the predictor set accounted for 18.0% of variance in sNGF (F = 9.2, p < 0.001), and DHEAS exerted an independent effect (b = 0.47, p < 0.001) while the other predictors did not.
The models were then decomposed relative to each change index. In terms of reactivity, the independent variables (DHEAS, testosterone, and cortisol reactivity) and covariate (sex) combined to account for 20.3% of variance in sNGF reactivity (F = 8.2, p < 0.001). Standardized beta coefficients revealed that DHEAS reactivity exerted an independent effect (b = 0.39, p < 0.001), while the other predictors were not significant. In terms of recovery, the predictors combined to account for 28.2% of variance in sNGF recovery (F = 15.5, p < 0.001); DHEAS recovery exerted an independent effect (b = 0.52, p < 0.001), as did testosterone recovery (b = [1]0.27, p < 0.01). In terms of residual elevation/depression, the independent variables explained 12.4% of variance in sNGF residual elevation (F = 6.2, p < 0.001). DHEAS residual elevation exerted an independent effect (b = 0.35, p < 0.001), while the other predictors did not.

Endocrine-Disrupting Chemicals and Human Growth and Maturation: A Focus on Early Critical Windows of Exposure

Julie Fudvoye, Jean-Pierre Bourguignon, Anne-Simone Parent
Vitamins and Hormones, 2014; 94: Chapt 1. 1-25.
http://dx.doi.org/10.1016/B978-0-12-800095-3.00001-8

Endocrine-disrupting chemicals (EDCs) are exogenous substances that interfere with hormone synthesis, metabolism, or action. In addition, some of them could cause epigenetic alterations of DNA that can be transmitted to the following generations. Because the developing organism is highly dependent on sex steroids and thyroid hormones for its maturation, the fetus and the child are very sensitive to any alteration of their hormonal environment. An additional concern about that early period of life comes from the shaping of the homeostatic mechanisms that takes place also at that time with involvement of epigenetic mechanisms along with the concept of fetal origin of health and disease. In this chapter, we will review the studies reporting effects of EDCs on human development. Using a translational approach, we will review animal studies that can shed light on some mechanisms of action of EDCs on the developing organism. We will focus on the major hormone-dependent stages of development: fetal growth, sexual differentiation, puberty, brain development, and energy balance. We will also discuss the possible epigenetic effects of EDCs on human development.

Several studies have reported that prenatal or early postnatal exposure to some EDCs is associated with alterations of cognitive or motor functions in children. Knowing the fundamental role played by thyroid hormones and sex steroids in cortex development, one can hypothesize that disruption of those hormones could cause alteration of the development of the cerebral cortex and of its functions later in life. We will review here the human data suggesting a causal effect for endocrine disrupters on impairment of cortical functions and approach some EDC mechanisms of action using animal models.

Thyroid hormones are known to be essential for brain development. They regulate progenitor proliferation and differentiation, neuron migration, and dendrite outgrowth (Parent, Naveau, Gerard, Bourguignon, & Westbrook, 2011). Even mild thyroid hormone insufficiency in humans can produce measurable deficits in cognitive functions (Zoeller & Rovet, 2004). Thyroid hormone action is mediated by two classes of nuclear receptors (Forrest & Vennstro¨m, 2000) that exhibit differential spatial and temporal expressions in the brain, suggesting that thyroid hormones have variable functions during brain development. This differential expression of thyroid hormone receptors explains the critical period of thyroid hormone action on brain development as suggested by models of maternal hypothyroidism or congenital hypothyroidism.

Depending on the timing of onset of hypothyroidism, the offspring will display problems of visual attention, gross or fine motor skills, or language and memory skills. Similarly, one can hypothesize that disruption of thyroid function by EDCs will have different effects based on the timing of exposure. However, few studies focused on that aspect. Polychlorinated biphenyls (PCBs) form a group of widespread environmental contaminants composed of 209 different congeners used in a wide variety of applications. Their production was banned in the 1970s but PCBs are still present in the environment due to their high stability. PCBs were among the first EDCs identified as responsible for alterations of cognitive functions. Indeed, impaired memory and altered learning abilities have been associated with prenatal exposure to EDCs in humans and In animal models, perinatal exposure to PCBs has been consistently associated with a decrease of thyroid hormone concentration in maternal serum as well as pup serum. Some but not all epidemiological studies in human have found an association between PCB body burden and thyroid hormone levels. This disruption of thyroid function could explain some of the effects of PCBs on the developing brain. Indeed, animal models have shown that the ototoxic effects of PCBs could be partially ameliorated by thyroxin replacement and PCBs seem to alter some of the developmental processes in the cortex and the cerebellum that are dependent on thyroid hormones. However, recent publications raise important issues.

As it is the case for other EDCs, some windows of susceptibility have been identified during pre- and postnatal brain development. Recent studies have shown that exposure to PBDEs causes alteration of thyroid hormone levels in pregnant women and infants as it is the case in rodents. Only very few studies, however, have focused on the molecular or cellular effects of perinatal exposure to PBDEs in vivo. Viberg et al. have reported a decrease of cholinergic nicotinic receptors in the hippocampus after exposure to BDE-99 and BDE-153. However, the link between such a decrease and the behavioral effects of PBDEs is still unclear. Other teams have reported that exposure to PBDEs reduced hippocampal long term potentiation and decreased brain-derived neurotrophic factor expression in the brain. While several studies have reported negative effect of PBDEs on brain development and cognitive function in animals, there is relatively little information about adverse health effects of PBDEs in humans. Some very recent studies have identified a correlation between prenatal exposure to PBDEs and alteration of cognitive functions.

Endocrine-Disrupting Chemicals: Elucidating Our Understanding of Their Role in Sex and Gender-Relevant End Points

Cheryl A. Frye
Vitamins and Hormones, 2014; 94: 41-98
http://dx.doi.org/10.1016/B978-0-12-800095-3.00003-1

Endocrine-disrupting chemicals (EDCs) are diverse and pervasive and may have significant consequence for health, including reproductive development and expression of sex-/gender-sensitive parameters. This review chapter discusses what is known about common EDCs and their effects on reproductively relevant end points. It is proposed that one way that EDCs may exert such effects is by altering steroid levels (androgens or 17-estradiol, E2) and/or intracellular E2 receptors (ERs) in the hypothalamus and/or hippocampus. Basic research findings that demonstrate developmentally sensitive end points to androgens and E2 are provided. Furthermore, an approach is suggested to examine differences in EDCs that diverge in their actions at ERs to elucidate their role in sex-/gender-sensitive parameters.

Reproductive dysfunction among adults and emotional, attentional, and behavioral disorders among children are on the rise. Sperm counts and fertility have declined in the last 50 years . Incidence of attention-deficit hyperactivity disorder (ADHD) and autism has increased in the last 30 years. These increases in reproductive dysfunction and developmental disorders may be due to increased exposure to environmental contaminants, although there is controversy about the relationship between exposure and these effects.
Many contaminants in the environment, including polychlorinated biphenyls (PCBs), dioxins, and metals, accumulate in exposed individuals and may have adverse consequences due to effects as endocrine-disrupting chemicals (EDCs). EDCs may have effects by altering steroid levels (androgens or 17β-estradiol, E2) and/or intracellular E2 receptors (ERs) in the hypothalamus and/or hippocampus.
Steroid hormones, during critical periods of development, organize sexual dimorphisms in brain and behavior and give rise to sex differences in later responses to steroid hormones. EDCs can profoundly disrupt reproductive responses following adult exposure and result in pervasive effects that extend throughout the life of their offspring. Many nonreproductive behaviors, such
as spatial performance, activity, and arousal, are also sexually dimorphic and organized and activated by steroid hormones. Thus, EDCs may affect reproductive and the aforementioned nonreproductive parameters by altering E2 levels and/or ER binding in the hypothalamus and/or hippocampus.
Results from the literature and preliminary data will be presented that demonstrate our use of a whole-animal model to begin to investigate effects of exposure (in adulthood and/or development) to EDCs on steroid levels (androgens and E2), actions at ERs (in hypothalamus and hippocampus), and reproductive-sensitive measures (anogenital distance, accessory structure weight, onset of puberty and sexual maturity, and reproductive behavior) and nonreproductive behaviors (spatial performance, play behavior, and arousal) throughout development.

A common feature of many environmental contaminants is their estrogenic effects. Some contaminants can alter production of E2 and/or androgens or act as agonists or antagonists for intracellular or membrane ERs. Thus, the term “endocrine-disrupting chemicals” (EDCs) in this chapter is used to refer to contaminants with these effects. An important question considered here is the extent to which EDCs’ actions to alter E2 levels and/or ER binding in the hypothalamus or hippocampus mitigates effects on reproductive or nonreproductive processes. There are potential pervasive, negative effects of endocrine disrupters on steroid sensitive tissues, which may confer risk to disease states, such as cancer, heart disease, and neurodegenerative disorders. The following discussion provides evidence that exposure to EDCs during development may result in permanent, lifelong differences in sexual function and reproductive ability, as well as cognitive function and/or emotional reactivity/arousal. Gonad development, sex determination, and reproductive success of offspring are highly dependent on sex hormone systems. The developing organism is exquisitely sensitive to alterations in hormone function. In the early embryonic state, the gonads of human males and females are morphologically identical. Sexual differentiation begins under hormonal influence during the fifth and sixth weeks of fetal development, and thus, alterations in hormones during this highly sensitive period can have profound consequences. Disruption of the sex steroid system during fetal stages of life results in profound adverse developmental reproductive effects, as is well known from the effects of DES. The balance of estrogens and androgens is critical for normal development, growth, and functioning of the reproductive system. Although especially important during development, this balance is important throughout life for the preservation of normal feminine or masculine traits, as well as the expression of some sexually dimorphic behaviors (sex, spatial performance, and arousal).

Proposed negative effects of exposure to endocrine disrupters during development in people and in animals. The focus here is on vulnerability to sexually dimorphic processes that are estrogen-sensitive, such as reproductive, cognitive, and emotional development and associated behavioral processes

The existing data clearly indicate that developmental exposure to EDCs can adversely affect sexual development of people and animals; however, there are different effects depending upon the EDCs and when in development exposure occurs. Therefore, we consider the next effects of EDCs exposure at different point in development and the consequences for reproductive development and behavior, as well as E2 levels and hypothalamic ER binding.
Steroid hormones also play a critical role in neurodevelopment that influences not only reproductive but also nonreproductive behaviors that show sex differences. Specific behavioral differences in nonreproductive behaviors between males and females include differences in spatial learning, play, exploration, activity levels, novelty-seeking behavior, and emotional reactivity. These sex dimorphisms are thought to reflect adaptive differences for behavioral strategies in coping as a result of sexual selection. Moreover, these sexually dimorphic behaviors may be relevant for concerns regarding increased developmental, cognitive, or emotional disabilities over the past 30 years. Also, behaviors are particularly sensitive measures of effects of EDCs.
EDCs can alter cognitive development. Some, but not all, studies have shown a predictive relationship between prenatal PCB exposure and cognitive development in infancy through preschool years. EDCs have direct effects on nervous system function. Long-term potentiation (LTP), a form of synaptic plasticity used as a model system for study of cognitive potential, is altered by PCBs and lead. The protein kinase C (PKC)-signaling pathway is involved in the modulation of learning, memory, and motor behavior and may be a target of E2’s actions. PCBs also alter PKC signaling. Although findings provide evidence that EDCs can alter cognitive performance, these measures of cognition are neither sexually dimorphic nor E2- or ER-dependent.
There are sex-specific effects of perinatal PCB and dioxin exposure on spatial learning. Yu-Cheng boys that were prenatally exposed to high levels of PCBs and PCDFs when their mothers were accidentally exposed to these contaminants in rice oil show more disrupted cognitive development, mainly spatial function, than did exposed girls. In animal studies, spatial learning that favors males is mediated by perinatal exposure to androgens. Gestational and lactational exposure to ortho-substituted PCBs produces spatial deficits at adolescence in male mice or adulthood in male rats. The sparse data suggest that developmental exposure to EDCs disrupts spatial memory. Furthermore, Exposure during adulthood to EDCs can also have activational effects on spatial memory. Females exposed to a phytoestrogen-rich diet exhibit “masculinized” spatial performance in a radial arm maze, while males fed with a phytoestrogen-free diet show “feminized” performance.
An important question is what are the mechanisms by which developmental and/or adult exposure to EDCs alters spatial performance? There is evidence for sex differences in spatial performance and activational effects of E2 in adulthood to alter spatial performance of rats. Systemic or intrahippocampal administration of E2 improves spatial performance of female rats. Further, E2’s actions at intracellular ERs in the hippocampus of adults do not seem to be required to mediate these effects on spatial performance.
EDCs may have effects on E2 metabolism in a number of ways. First, some EDCs can alter serum lipid concentrations. Cholesterol is the precursor for the production of E2 and other steroid hormones (see Fig. 3.3). Second, there is also evidence that some EDCs can alter metabolism enzymes that are necessary for converting cholesterol to steroid hormones. Induction of CYP occurs when EDCs, such as TCDD, bind the aromatic hydrocarbon receptor (AhR). There is a firm link between PCBs, enzyme induction, and AhR. The binding of EDCs with AhR can result in antiestrogenic activity through increased metabolism and depletion of endogenous E2. Elevated levels of CYP enzymes, primarily expressed not only in the liver but also in the brain and other tissues, result in increased E2 metabolism and excretion. Alternatively, compounds that are metabolized by P450s may result in a net estrogenic effect if they inhibit endogenous estrogens from being metabolized.
Steroid hormones are lipid molecules with limited solubility in plasma and are accordingly carried through the plasma compartment to target cells by specific plasma transporter proteins. Each transporter protein has a specific ligand-binding domain for its associated hormone. It is generally accepted that the “free” formof the steroid hormone, and not the conjugate of the hormone with its plasma transport protein, enters target cells and binds with the appropriate receptor. Receptors for the steroid hormones are proteins located primarily in the cell nucleus or partitioned between the cytoplasm and the nucleus. The unoccupied steroid receptors may reside in the cell as heterodimeric complexes with the 90 kDa heat-shock protein, which prevents the receptor from binding with the DNA until the receptor has first bound with its steroid hormone. Once the hormone binds to the receptor, the hormone receptor complexes with the heterodimeric heat-shock protein and undergoes a conformational change and is activated. The activated receptor binds with DNA at a specific site, initiating gene transcription.

Traditional effects of steroid hormones at their cognate steroid receptors

Traditional effects of steroid hormones at their cognate steroid receptors, which act as transcription factors. In this example, effects of steroid hormones, such as estradiol, to bind estrogen receptor (ER) subtypes, referred to as ERa and ERb, are shown.

Beyond traditional actions solely through intracellular cognate estrogen receptors (ERs; ERa and ERb), steroids, such as estradiol, and estradiol-mimetics (endocrine disrupters) may have novel actions involving membrane bound ERs, other neurotransmitter systems (e.g., NMDA receptor), and signal transduction cascades (e.g., growth factors, MAPK).

To date, there has been little investigation in a whole-animal model of the effects of EDCs on E2 levels and/or activity at intracellular ERs in the brain. Thus, changes in E2 levels and ER activity in the hypothalamus and hippocampus, concomitant with alterations in endocrine parameters and reproductive behavior and nonreproductive behavior, respectively, are
needed to elucidate tissue specificity of EDCs’ functions and mechanisms.

Low-Dose Effects of Hormones and Endocrine Disruptors

Laura N. Vandenberg
Vitamins and Hormones, 2014; 94: 129-165
http://dx.doi.org/10.1016/B978-0-12-800095-3.00005-5

Endogenous hormones have effects on tissue morphology, cell physiology, and behaviors at low doses. In fact, hormones are known to circulate in the part-per-trillion and part-per-billion concentrations, making them highly effective and potent signaling molecules.

Many endocrine-disrupting chemicals (EDCs) mimic hormones, yet there is strong debate over whether these chemicals can also have effects at low doses. In the 1990s, scientists proposed the “low-dose hypothesis,” which postulated that EDCs affect humans and animals at environmentally relevant doses. This chapter focuses on data that support and refute the low-dose hypothesis. A case study examining the highly controversial example of bisphenol A and its low-dose effects on the prostate is examined through the lens of endocrinology. Finally, the chapter concludes with a discussion of factors that can influence the ability of a study to detect and interpret low-dose effects appropriately.

Since EDCs began to be studied in depth in the 1990s, there has been intense debate over whether the public should be concerned about low level exposures to these chemicals. The low-dose hypothesis, proposed at that time, has steadily accumulated evidence that EDCs have actions at low doses, and these effects are not necessarily predicted from high-dose toxicology testing. In 2002, the NTP expert panel reported evidence for low-dose effects for a small number of EDCs and estradiol. In 2012, an updated approach identified several dozen additional EDCs with evidence for low-dose effects. Further, epidemiology studies continue to find relationships between EDC exposure levels and diseases in the general public, which has raised concerns because the general public is exposed to a large number of environmental chemicals at low doses. For decades, hormones have been known to produce striking changes in tissue morphology, physiology, and behaviors at exceedingly low doses.

A relatively large body of evidence suggests that EDCs, and in particular those environmental chemicals that mimic endogenous hormones, have similar effects at low doses. Although there is still no consensus about the universality of “low-dose effects” in the toxicology community, the Endocrine Society (Diamanti-Kandarakis et al., 2009; Zoeller et al., 2012) believes not only that there is sufficient evidence in support of this phenomenon but also that it is time for public health agencies to make changes to risk assessment paradigms and give greater consideration to studies that specifically identify low-dose effects when considering risks from chemical exposures.

Bisphenol A interferes with synaptic remodeling

Tibor Hajszan, Csaba Leranth
Frontiers in Neuroendocrinology 31 (2010) 519–530
http://dx.doi.org:/10.1016/j.yfrne.2010.06.004

The potential adverse effects of Bisphenol A (BPA), a synthetic xenoestrogen, have long been debated. Although standard toxicology tests have revealed no harmful effects, recent research highlighted what was missed so far: BPA-induced alterations in the nervous system. Since 2004, our laboratory has been investigating one of the central effects of BPA, which is interference with gonadal steroid-induced synaptogenesis and the resulting loss of spine synapses. We have shown in both rats and nonhuman primates that BPA completely negates the ~70–100% increase in the number of hippocampal and prefrontal spine synapses induced by both estrogens and androgens. Synaptic loss of this magnitude may have significant consequences, potentially causing cognitive decline, depression, and schizophrenia, to mention those that our laboratory has shown to be associated with synaptic loss. Finally, we discuss why children may particularly be vulnerable to BPA, which represents future direction of research in our laboratory.

Bisphenol-A rapidly promotes dynamic changes in hippocampal dendritic morphology through estrogen receptor-mediated pathway by concomitant phosphorylation of NMDA receptor subunit NR2B

Xiaohong Xu ⁎, Yinping Ye, Tao Li, Lei Chen, Dong Tian, Qingqing Luo, Mei Lu
Toxicology and Applied Pharmacology 249 (2010) 188–196
http://dx.doi.org:/10.1016/j.taap.2010.09.007

Bisphenol-A (BPA) is known to be a potent endocrine disrupter. Evidence is emerging that estrogen exerts a rapid influence on hippocampal synaptic plasticity and the dendritic spine density, which requires activation of NMDA receptors. In the present study, we investigated the effects of BPA (ranging from 1 to 1000 nM), focusing on the rapid dynamic changes in dendritic filopodia and the expressions of estrogen receptor (ER) β and NMDA receptor, as well as the phosphorylation of NMDA receptor subunit NR2B in the cultured hippocampal neurons. A specific ER antagonist ICI 182,780 was used to examine the potential involvement of ERs. The results demonstrated that exposure to BPA (ranging from 10 to 1000 nM) for 30 min rapidly enhanced the motility and the density of dendritic filopodia in the cultured hippocampal neurons, as well as the phosphorylation of NR2B (pNR2B), though the expressions of NMDA receptor subunits NR1, NR2B, and ERβ were not changed. The antagonist of ERs completely inhibited the BPA-induced increases in the filopodial motility and the number of filopodia extending from dendrites. The increased pNR2B induced by BPA (100 nM) was also completely eliminated. Furthermore, BPA attenuated the effects of 17β-estradiol (17β-E2) on the dendritic filopodia outgrowth and the expression of pNR2B when BPA was co-treated with 17β-E2. The present results suggest that BPA, like 17β-E2, rapidly results in the enhanced motility and density of dendritic filopodia in the cultured hippocampal neurons with the concomitant activation of NMDA receptor subunit NR2B via an ER-mediated signaling pathway. Meanwhile, BPA suppressed the enhancement effects of 17β-E2 when it coexists with 17β-E2. These results provided important evidence suggesting the neurotoxicity of the low levels of BPA during the early postnatal development of the brain.

Bisphenol-A rapidly enhanced passive avoidance memory and phosphorylation of NMDA receptor subunits in hippocampus of young rats

Xiaohong Xu⁎, Tao Li, Qingqing Luo, Xing Hong, Lingdan Xie, Dong Tian
Toxicology and Applied Pharmacology 255 (2011) 221–228
http://dx.doi.org:/10.1016/j.taap.2011.06.022

Bisphenol-A (BPA), an endocrine disruptor, is found to influence development of brain and behaviors in rodents. The previous study indicated that perinatal exposure to BPA impaired learning-memory and inhibited N-methyl-D-aspartate receptor (NMDAR) subunits expressions in hippocampus during the postnatal development in rats; and in cultured hippocampal neurons, BPA rapidly promotes dynamic changes in dendritic morphology through estrogen receptor-mediated pathway by concomitant phosphorylation of NMDAR subunit NR2B. In the present study, we examined the rapid effect of BPA on passive avoidance memory and NMDAR in the developing hippocampus of Sprague–Dawley rats at the age of postnatal day 18. The results showed that BPA or estradiol benzoate (EB) rapidly extended the latency to step down from the platform 1 h after foot shock and increased the phosphorylation levels of NR1, NR2B, and mitogen-activated extracellular signal-regulated kinase (ERK) in hippocampus within 1 h. While 24 h after BPA or EB treatment, the improved memory and the increased phosphorylation levels of NR1, NR2B, ERK disappeared. Furthermore, pre-treatment with an estrogen receptors (ERs) antagonist, ICI182, 780, or an ERK-activating kinase inhibitor, U0126, significantly attenuated EB- or BPA-induced phosphorylations of NR1, NR2B, and ERK within 1 h. These data suggest that BPA rapidly enhanced short-term passive avoidance memory in the developing rats. A non-genomic effect via ERs may mediate the modulation of the phosphorylation of NMDAR subunits NR1 and NR2B through ERK signaling pathway.

Bisphenol A promotes dendritic morphogenesis of hippocampal neurons through estrogen receptor-mediated ERK1/2 signal pathway

Xiaohong Xu, Yang Lu, Guangxia Zhang, Lei Chen, Dong Tian, et al.
Chemosphere 96 (2014) 129–137
http://dx.doi.org/10.1016/j.chemosphere.2013.09.063

Bisphenol A (BPA), an environmental endocrine disruptor, has attracted increasing attention to its adverse effects on brain developmental process. The previous study indicated that BPA rapidly increased motility and density of dendritic filopodia and enhanced the phosphorylation of N-methyl-D-aspartate (NMDA) receptor subunit NR2B in cultured hippocampal neurons within 30 min. The purpose of the present study was further to investigate the effects of BPA for 24 h on dendritic morphogenesis and the underlying mechanisms. After cultured for 5 d in vitro, the hippocampal neurons from 24 h-old rat were infected by AdV-EGFP to indicate time-lapse imaging of living neurons. The results demonstrated that the exposure of the cultured hippocampal neurons to BPA (10, 100 nM) or 17β-estradiol (17β-E2, 10 nM) for 24 h significantly promoted dendritic development, as evidenced by the increased total length of dendrite and the enhanced motility and density of dendritic filopodia. However, these changes were suppressed by an ERs antagonist, ICI182,780, a non-competitive NMDA receptor antagonist, MK-801, and a mitogen activated ERK1/2-activating kinase (MEK1/2) inhibitor, U0126. Meanwhile, the increased F-actin (filamentous actin) induced by BPA (100 nM) was also completely eliminated by these blockers. Furthermore, the result of western blot analyses showed that, the exposure of the cultures to BPA or 17β-E2 for 24 h promoted the expression of Rac1/Cdc42 but inhibited that of RhoA, suggesting Rac1 (Ras related C3 botulinum toxinsubstrate 1)/Cdc42 (cell divisioncycle 42) and RhoA (Ras homologous A), the Rho family of small GTPases, were involved in BPA- or 17β-E2-induced changes in the dendritic morphogenesis of neurons. These BPA- or 17b-E2-induced effects were completely blocked by ICI182,780, and were partially suppressed by U0126. These results reveal that, similar to 17β-E2, BPA exerts its effects on dendritic morphogenesis by eliciting both nuclear actions and extranuclear-initiated actions that are integrated to influence the development of dendrite in hippocampal neurons.

Tyreoliberin (Trh) – The Regulatory Neuropeptide Of Cns Homeostasis
Danuta Jantas
Advances In Cell Biology 2;(4)/2010 (139–154)
http://dx.doi.org:/10.2478/v10052-010-0008-4

The physiological role of thyreoliberin (TRH) is the preservation of homeostasis within four systems
(i) the hypothalamic-hypophsysiotropic neuroendocrine system,
(ii) the brain stem/midbrain/spinal cord system,
(iii) the limbic/cortical system, and
(iv) the chronobiological system.

Thus TRH, via various cellular mechanisms, regulates a wide range of biological processes (arousal, sleep, learning, locomotive activity, mood) and possesses the potential for unique and widespread applications for treatment of human illnesses. Since the therapeutic potential of TRH is limited by its pharmacological profile (enzymatic instability, short half-life, undesirable effects), several synthetic analogues of TRH were constructed and studied in mono- or adjunct therapy of central nervous system (CNS) disturbances. The present article summarizes the current state of understanding of the physiological role of TRH and describes its putative role in clinical indications in CNS maladies with a focus on the action of TRH analogues.

Breakthrough in neuroendocrinology by discovering novel neuropeptides and neurosteroids: 2. Discovery of neurosteroids and pineal neurosteroids

Kazuyoshi Tsutsui, Shogo Haraguchi
General and Comparative Endocrinology 205 (2014) 11–22
http://dx.doi.org/10.1016/j.ygcen.2014.03.008

Bargmann–Scharrer’s discovery of ‘‘neurosecretion’’ in the first half of the 20th century has since matured into the scientific discipline of neuroendocrinology. Identification of novel neurohormones, such as neuropeptides and neurosteroids, is essential for the progress of neuroendocrinology. Our studies over the past two decades have significantly broadened the horizons of this field of research by identifying novel neuropeptides and neurosteroids in vertebrates that have opened new lines of scientific investigation in neuroendocrinology. We have established de novo synthesis and functions of neurosteroids in the brain of various vertebrates. Recently, we discovered 7α-hydroxypregnenolone (7α-OH PREG), a novel bioactive neurosteroid that acts as a key regulator for inducing locomotor behavior by means of the dopaminergic system. We further discovered that the pineal gland, an endocrine organ located close to the brain, is an important site of production of neurosteroids de novo from cholesterol (CHOL). The pineal gland secretes 7α-OH PREG and 3α,5α-tetrahydroprogesterone (3α,5α-THP; allopregnanolone) that are involved in locomotor rhythms and neuronal survival, respectively. Subsequently, we have demonstrated their mode of action and functional significance. This review summarizes the discovery of these novel neurosteroids and its contribution to the progress of neuroendocrinology.

Mechanisms of crosstalk between endocrine systems: Regulation of sex steroid hormone synthesis and action by thyroid hormones

Paula Duarte-Guterman, Laia Navarro-Martín, Vance L. Trudeau
General and Comparative Endocrinology 203 (2014) 69–85
http://dx.doi.org/10.1016/j.ygcen.2014.03.015

Thyroid hormones (THs) are well-known regulators of development and metabolism in vertebrates. There is increasing evidence that THs are also involved in gonadal differentiation and reproductive function. Changes in TH status affect sex ratios in developing fish and frogs and reproduction (e.g., fertility), hormone levels, and gonad morphology in adults of species of different vertebrates. In this review, we have summarized and compared the evidence for cross-talk between the steroid hormone and thyroid axes and present a comparative model. We gave special attention to TH regulation of sex steroid synthesis and action in both the brain and gonad, since these are important for gonad development and brain sexual differentiation and have been studied in many species. We also reviewed research showing that there is a TH system, including receptors and enzymes, in the brains and gonads in developing and adult vertebrates. Our analysis shows that THs influences sex steroid hormone synthesis in vertebrates, ranging from fish to pigs. This concept of crosstalk and conserved hormone interaction has implications for our understanding of the role of THs in reproduction, and how these processes may be dysregulated by environmental endocrine disruptors.

Insights into the structure of class B GPCRs

Kaspar Hollenstein, Chris de Graaf, Andrea Bortolato, Ming-Wei Wang, et al.
Trends in Pharmacological Sciences, Jan 2014; 35(1)
http://dx.doi.org/10.1016/j.tips.2013.11.001

The secretin-like (class B) family of G protein-coupled receptors (GPCRs) are key players in hormonal homeostasis and are interesting drug targets for the treatment of several metabolic disorders (such as type 2 diabetes, osteoporosis, and obesity) and nervous system diseases (such as migraine, anxiety, and depression). The recently solved crystal structures of the transmembrane domains of the human glucagon receptor and human corticotropin-releasing factor receptor 1 have opened up new opportunities to study the structure and function of class B GPCRs. The current review shows how these structures offer more detailed explanations to previous biochemical and pharmacological studies of class B GPCRs, and provides new insights into their interactions with ligands.

Class B G protein-coupled receptors (GPCRs), also referred to as the secretin family of GPCRs, include receptors for 15 peptide hormones, which can be grouped into five subfamilies based on their physiological role (see Table 1 for an overview) [1]. These receptors are important drug targets in many human diseases, including diabetes, osteoporosis, obesity, cancer, neurodegeneration, cardiovascular disease, headache, and psychiatric disorders. However, the identification of small-molecule oral drugs for this family has proved extremely challenging.

(A,B) Crystal structures of the class B G protein-coupled receptors corticotropin-releasing factor receptor 1 (CRF1) [Protein Data Bank (PDB) identifier: 4K5Y] and glucagon receptor (PDB identifier: 4L6R) are shown in blue and orange ribbons, respectively, in two different views from within the membrane. Transmembrane (TM) helices and helix 8 are labelled. The disulfide bond tethering extracellular loop 2 (ECL2) to the tip of TM3 is shown as purple sticks. In CRF1 the small-molecule antagonist CP-376395 is shown in stick representation with carbon, nitrogen, and oxygen atoms colored magenta, blue, and red, respectively, and as skeletal formula in an inset. (C) Superposition of the two structures, with insets highlighting regions of particular interest. To highlight the structural differences in the extracellular halves of CRF1 and glucagon receptor, the distance of approximately 11 A° between the Ca-atoms of residues 7.33b at the N-terminal end of TM7 is indicated with a red arrow. The small molecule binding pocket is shown as a superposition of the two receptors around CP-376395, illustrating the antagonist binding mode and the substantial structural differences observed for TM6 of the two receptors.

Overview of NMR and crystal structures of class B G protein-coupled

receptor (GPCR) extracellular domains (ECDs; magenta) and their complexes with peptide ligands (different colors). A complete overview of corresponding Protein Data Bank identifiers is presented in Table 1 (not shown). (B) Structure-based sequence alignment of representative peptide ligands of class B GPCR, adopted from Parthier et al. [6]. The residues of the peptide ligands solved in ECD–ligand complex crystal structures are marked using the same colour as in Figure 2A. The residues that are boxed black are found in an α-helical conformation in the complex. Peptide ligand residues that covalently bind receptors in photo-crosslinking or cysteine-trapping studies are colored and boxed green, whereas peptide ligand residues that have been mutated and studied in combination with receptor mutants are colored and boxed red. Note that the first residue of glucagon-like peptide-1 (GLP-1) is His7. A complete overview of all ECD structures and important peptide ligands for all class B GPCRs is presented in Table 1. Putative helix-capping residues [6] are coloured blue and cysteines involved in a disulfide-bridge (calcitonin) are coloured orange. D-phenylalanine (f), and norleucine (m) residues are indicated in stressin and astressin. The last 41 and 99 residues of parathyroid hormone (PTH) and PTH-related protein. (PTHrP), respectively, are not displayed. Abbreviations: CGRP, calcitonin gene-related peptide; CLR, calcitonin receptor-like receptor; CRF, corticotropin-releasing factor; CT, calcitonin; Ext-4, exendin-4; GHRHR, growth hormone releasing hormone receptor; GIP, glucose-dependent insulinotropic peptide; PAC, pituitary adenylate cyclase; PACAP, pituitary adenylate cyclase activating polypeptide; RAMP, receptor-activity modifying proteins; SCTR, secretin receptor; Ucn, urocortin; VPAC, vasoactive pituitary adenylate cyclase.

Figure 3. (not shown) (A) The spatial correspondence between residues in transmembrane (TM) helices of class A and class B G protein-coupled receptors (GPCRs) makes it possible to align the most conserved residues in class A (designated X.50, Ballesteros–Weinstein numbering) and class B (designated X.50b, Wootten numbering) for comparisons between GPCR classes (Box 1). (B) Structural alignment of corticotropin-releasing factor receptor 1 (CRF1; blue) and glucagon receptor (GCGR; orange) to two representative class A GPCRs, histamine H1 receptor (H1R; Protein Data Bank identifier: 3RZE) and CXC-chemokine receptor 4 (CXCR4; Protein Data Bank identifier: 3ODU/3OE0) (in grey). Helices are depicted as cylinders, and the ligands glucagon (for GCGR), CP-376395 (for CRF1), doxepin (for H1R), and IT1t and CVX15 (for CXCR4) are shown as sticks. The

location of the Ca-atoms of the most conserved residues of TM1–3 and TM5 among class A and class B GPCRs (Box 1) are indicated by spheres (TM4 is not depicted for clarity).

The GCGR and CRF1 crystal structures show distinct structural features and different binding pockets compared to class A GPCRs, and give new insights into the molecular details of peptide and small-molecule binding to class B GPCRs. The first two crystal structures of the TM domains of class B GPCRs provide a structural framework that will enable the design of biochemical and biophysical experiments detailing the complex structure of this class of receptors, and facilitate the design of stabilized constructs that might lead to the solution of full-length class B GPCR–ligand complexes. The structures furthermore present more reliable structural templates for the design of specific and potent small molecules for the treatment of type 2 diabetes (GCGR) and depression (CRF1) in particular, and open new avenues for structure-based small-molecule drug discovery for class B GPCRs as a whole.

Novel receptor targets for production and action of allopregnanolone in the central nervous system: a focus on pregnane xenobiotic receptor

Cheryl A. Frye, Carolyn J. Koonce and Alicia A. Walf
Front in Cell Neurosci Apr 2014; 8(106): 1-13.
http://dx.doi.org:/10.3389/fncel.2014.00106

Neurosteroids are cholesterol-based hormones that can be produced in the brain,

independent of secretion from peripheral endocrine glands, such as the gonads and

adrenals. A focus in our laboratory for over 25 years has been how production of the

pregnane neurosteroid, allopregnanolone, is regulated and the novel (i.e., non steroid

receptor) targets for steroid action for behavior. One endpoint of interest has been lordosis, the mating posture of female rodents. Allopregnanolone is necessary and sufficient for lordosis, and the brain circuitry underlying it, such as actions in the midbrain ventral tegmental area (VTA), has been well-characterized. Published and recent findings supporting a dynamic role of allopregnanolone are included in this review.
First, contributions of ovarian and adrenal sources of precursors of allopregnanolone, and the requisite enzymatic actions for de novo production in the central nervous system will be discussed.
Second, how allopregnanolone produced in the brain has actions on behavioral processes that are independent of binding to steroid receptors, but instead involve rapid modulatory actions via neurotransmitter targets (e.g., g-amino butyric acid-GABA, N methyl-D-aspartate- NMDA) will be reviewed.
Third, a recent focus on characterizing the role of a promiscuous nuclear receptor, pregnane xenobiotic receptor (PXR), involved in cholesterol metabolism and expressed in the VTA, as a target for allopregnanolone and how this relates to both actions and production of allopregnanolone will be addressed. For example, allopregnanolone can bind PXR and knocking down expression of PXR in the midbrain VTA attenuates actions of allopregnanolone via NMDA and/or GABAA for lordosis. Our understanding of allopregnanolone’s actions in the VTA for lordosis has been extended to reveal the role of allopregnanolone for broader, clinically-relevant questions, such as neurodevelopmental processes, neuropsychiatric disorders, epilepsy, and aging.

Genetically Encoded Chemical Probes in Cells Reveal the Binding Path of Urocortin-I to CRF Class B GPCR

Irene Coin, Vsevolod Katritch, Tingting Sun, Zheng Xiang, Fai Yiu Siu
Cell Dec 2013; 155, 1258–1269
http://dx.doi.org/10.1016/j.cell.2013.11.008

Molecular determinants regulating the activation of class B G-protein-coupled receptors (GPCRs) by native peptide agonists are largely unknown. We have investigated here the interaction between the corticotropin releasing factor receptor type 1 (CRF1R) and its native 40-mer peptide ligand Urocortin- I directly in mammalian cells. By incorporating unnatural amino acid photochemical and new click chemical probes into the intact receptor expressed in the native membrane of live cells, 44 intermolecular spatial constraints have been derived for the ligand-receptor interaction. The data were analyzed in the context of the recently resolved crystal structure of
CRF1R transmembrane domain and existing extracellular domain structures, yielding a complete conformational model for the peptide-receptor complex. Structural features of the receptor-ligand complex yield molecular insights
on the mechanism of receptor activation and the basis for discrimination between agonist and antagonist function.

Investigation of GPCR-Ligand Interactions under Native Conditions Using Genetically Encoded Chemical Probes GPCRs are integral membrane proteins containing multiple domains and various posttranslational modifications. To understand GPCR-ligand interactions by crystallography, receptors have to be extracted from the cell membrane and modified with a series of expedients such as deglycosylation, therm-stabilizing mutations, fusions with soluble proteins, or complexes with stabilizing nanobodies. We present here a method to investigate GPCR-ligand interactions at the intact fully posttranslationally modified receptor bound to its WT ligand on the membrane of the live cell, which mimics the native conditions for GPCR function. We first genetically incorporated into the receptor the photocrosslinking Uaa Azi, which served as
a proximity probe to provide an overall map of the ligand binding sites on the receptor. We then determined the relative position of the ligand in the binding pocket using a residue-specific chemical crosslinking reaction between Ffact genetically incorporated into the receptor and Cys introduced into the ligand. The derived intermolecular spatial constraints served eventually to build a detailed conformational model for the receptor-ligand complex.

Glutamate Neurons within the Midbrain Dopamine Regions

Morales and D. H. Root
Neuroscience 282 (2014) 60–68
http://dx.doi.org/10.1016/j.neuroscience.2014.05.032

Midbrain dopamine (DA) neurons are hypothesized to play roles in reward-based behavior and addiction, reward prediction and learning by error detection, effort-based decision making, flexible reward-directed behaviors,

incentive salience, stimulus salience (e.g., prediction of rewarding and aversive events), aversion, depression, and fear. The extensive, divergent behavioral roles of midbrain dopamine neurons, predominantly from the ventral tegmental area (VTA), indicate that this system is highly heterogeneous.
This heterogeneity may be reflected in part by the diverse phenotypic characteristics among DAergic neurons and their interactive brain structures.

Midbrain dopamine systems play important roles in Parkinson’s disease, schizophrenia, addiction, and depression. The participation of midbrain dopamine systems in diverse clinical contexts suggests these systems are highly complex. Midbrain dopamine regions contain at least three neuronal phenotypes: dopaminergic, GABAergic, and glutamatergic. Here, we review the locations, subtypes, and functions of glutamatergic neurons within midbrain dopamine regions. Vesicular glutamate transporter 2 (VGluT2) mRNA-expressing neurons are observed within each midbrain dopamine system. Within rat retrorubral field (RRF), large populations of VGluT2 neurons are observed throughout its anteroposterior extent. Within rat substantia nigra pars compacta (SNC), VGluT2 neurons are observed centrally and caudally, and are most dense within the laterodorsal subdivision. RRF and SNC rat VGluT2 neurons lack tyrosine hydroxylase (TH), making them an entirely distinct population of neurons from dopaminergic neurons. The rat ventral tegmental area (VTA) contains the most heterogeneous populations of VGluT2 neurons. VGluT2 neurons are found in each VTA subnucleus but are most dense within the anterior midline subnuclei. Some subpopulations of rat VGluT2 neurons co-express TH or glutamic acid decarboxylase (GAD), but most of the VGluT2 neurons lack TH or GAD. Different subsets of rat VGluT2-TH neurons exist based on the presence or absence of vesicular monoamine transporter 2, dopamine transporter, or D2 dopamine receptor. Thus, the capacity by which VGluT2-TH neurons may release dopamine will differ based on their capacity to accumulate vesicular dopamine, uptake extracellular dopamine, or be autoregulated by dopamine. Rat VTA VGluT2 neurons exhibit intrinsic VTA projections and extrinsic projections to the accumbens and to the prefrontal cortex. Mouse VTA VGluT2 neurons project to accumbens shell, prefrontal cortex, ventral pallidum, amygdala, and lateral habenula. Given their molecular diversity and participation in circuits involved in addiction, we hypothesize that individual VGluT2 subpopulations of neurons play unique roles in addiction and other disorders. This article is part of a Special Issue entitled: Ventral Tegmentum & Dopamine. Published by Elsevier Ltd. On behalf of IBRO.

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Long-term aerobic exercise prevents age-related brain deterioration
http://www.kurzweilai.net/long-term-aerobic-exercise-prevents-age-related-brain-deterioration