An ancient polymorphic regulatory region within the BDNF gene associated with obesity modulates anxiety-like behaviour in mice and humans.

Obesity and anxiety are morbidities notable for their increased impact on society during the recent COVID-19 pandemic. Understanding the mechanisms governing susceptibility to these conditions will increase our quality of life and resilience to future pandemics. In the current study, we explored the function of a highly conserved regulatory region (BE5.1) within the BDNF gene that harbours a polymorphism strongly associated with obesity (rs10767664; p = 4.69 × 10-26). Analysis in primary cells suggested that the major T-allele of BE5.1 was an enhancer, whereas the obesity-associated A-allele was not. However, CRISPR/CAS9 deletion of BE5.1 from the mouse genome (BE5.1KO) produced no significant effect on the expression of BDNF transcripts in the hypothalamus, no change in weight gain after 28 days and only a marginally significant increase in food intake. Nevertheless, transcripts were significantly increased in the amygdala of female mice and elevated zero maze and marble-burying tests demonstrated a significant increase in anxiety-like behaviour that could be reversed by diazepam. Consistent with these observations, human GWAS cohort analysis demonstrated a significant association between rs10767664 and anxiousness in human populations. Intriguingly, interrogation of the human GTEx eQTL database demonstrated no effect on BDNF mRNA levels associated with rs10767664 but a highly significant effect on BDNF-antisense (BDNF-AS) gene expression and splicing. The subsequent observation that deletion of BE5.1 also significantly reduced BDNF-AS expression in mice suggests a novel mechanism in the regulation of BDNF expression common to mice and humans, which contributes to the modulation of mood and anxiety in both species.

PACAP Induces Light Aversion in Mice by an Inheritable Mechanism Independent of CGRP.

The neuropeptides CGRP (calcitonin gene-related peptide) and PACAP (pituitary adenylate cyclase-activating polypeptide) have emerged as mediators of migraine, yet the potential overlap of their mechanisms remains unknown. Infusion of PACAP, like CGRP, can cause migraine in people, and both peptides share similar vasodilatory and nociceptive functions. In this study, we have used light aversion in mice as a surrogate for migraine-like photophobia to compare CGRP and PACAP and ask whether CGRP or PACAP actions were dependent on each other. Similar to CGRP, PACAP induced light aversion in outbred CD-1 mice. The light aversion was accompanied by increased resting in the dark, but not anxiety in a light-independent open field assay. Unexpectedly, about one-third of the CD-1 mice did not respond to PACAP, which was not seen with CGRP. The responder and nonresponder phenotypes were stable, inheritable, and not sex linked, although there was a trend for greater responses among male mice. RNA-sequencing analysis of trigeminal ganglia yielded hierarchical clustering of responder and nonresponder mice and revealed a number of candidate genes, including greater expression of the Trpc5 and Kcnk12 ion channels and glycoprotein hormones and receptors in a subset of male responder mice. Importantly, an anti-PACAP monoclonal antibody could block PACAP-induced light aversion but not CGRP-induced light aversion. Conversely, an anti-CGRP antibody could not block PACAP-induced light aversion. Thus, we propose that CGRP and PACAP act by independent convergent pathways that cause a migraine-like symptom in mice.SIGNIFICANCE STATEMENT The relationship between the neuropeptides CGRP (calcitonin gene-related peptide) and PACAP (pituitary adenylate cyclase-activating polypeptide) in migraine is relevant given that both peptides can induce migraine in people, yet to date only drugs that target CGRP are available. Using an outbred strain of mice, we were able to show that most, but not all, mice respond to PACAP in a preclinical photophobia assay. Our finding that CGRP and PACAP monoclonal antibodies do not cross-inhibit the other peptide indicates that CGRP and PACAP actions are independent and suggests that PACAP-targeted drugs may be effective in patients who do not respond to CGRP-based therapeutics.

A narrative review of converging evidence addressing developmental toxicity of pyrethroid insecticides.

Pyrethroid insecticides are broadly used in agriculture and household products throughout the world. Exposure to this class of insecticides is widespread, and while generally believed to be safe for use, there is increasing concern regarding their effects on neurodevelopment. Due to the critical roles that molecular targets of pyrethroids play in the regulation of neurodevelopment, particular focus has been placed on evaluating the effects of in utero and childhood pyrethroid exposure on child cognition and behavior. As such, this narrative review synthesizes an assessment of converging study types; we review reports of neonatal pyrethroid levels together with current epidemiological literature that convergently address the risk for developmental toxicity linked to exposure to pyrethroid insecticides. We first address studies that assess the degree of direct fetal exposure to pyrethroids in utero through measurements in cord blood, meconium, and amniotic fluid. We then focus on the links between prenatal exposure to these insecticides and child neurodevelopment, fetal growth, and other adverse birth outcomes. Furthermore, we assess the effects of postnatal exposure on child neurodevelopment through a review of the data on pediatric exposures and child cognitive and behavioral outcomes. Study quality was evaluated individually, and the weight of evidence was assessed broadly to characterize these effects. Overall, while definitive conclusions cannot be reached from the currently available literature, the available data suggest that the potential links between pyrethroid exposure and child neurodevelopmental effects deserve further investigation.

Sex-specific maternal programming of corticosteroid-binding globulin by predator odour.

Predation is a key organizing force in ecosystems. The threat of predation may act to programme the endocrine hypothalamic-pituitary-adrenal axis during development to prepare offspring for the environment they are likely to encounter. Such effects are typically investigated through the measurement of corticosteroids (Cort). Corticosteroid-binding globulin (CBG) plays a key role in regulating the bioavailability of Cort, with only free unbound Cort being biologically active. We investigated the effects of prenatal predator odour exposure (POE) in mice on offspring CBG and its impact on Cort dynamics before, during and after restraint stress in adulthood. POE males, but not females, had significantly higher serum CBG at baseline and during restraint and lower circulating levels of Free Cort. Restraint stress was associated with reduced liver transcript abundance of SerpinA6 (CBG-encoding gene) only in control males. POE did not affect SerpinA6 promoter DNA methylation. Our results indicate that prenatal exposure to a natural stressor led to increased CBG levels, decreased per cent of Free Cort relative to total and inhibited restraint stress-induced downregulation of CBG transcription. These changes suggest an adaptive response to a high predator risk environment in males but not females that could buffer male offspring from chronic Cort exposure.

Genome-wide DNA methylation investigation of glucocorticoid exposure within buccal samples.

AIM: Glucocorticoids play a major role in regulating the stress response, and an imbalance of glucocorticoids has been implicated in stress-related disorders. Within mouse models, CpGs across the genome have been shown to be differentially methylated in response to glucocorticoid treatment, and using the Infinium 27K array, it was shown that humans given synthetic glucocorticoids had DNA methylation (DNAm) changes in blood. However, further investigation of the extent to which glucocorticoids affect DNAm across a larger proportion of the genome is needed. METHODS: Buccal samples were collected before and after synthetic glucocorticoid treatment in the context of a dental procedure. This included 30 tooth extraction surgery patients who received 10 mg of dexamethasone. Genome-wide DNAm was assessed with the Infinium HumanMethylationEPIC array. RESULTS: Five CpGs showed genome-wide significant DNAm changes that were >10%. These differentially methylated CpGs were in or nearest the following genes: ZNF438, KLHDC10, miR-544 or CRABP1, DPH5, and WDFY2. Using previously published datasets of human blood gene expression changes following dexamethasone exposure, a significant proportion of genes with false-discovery-rate-adjusted significant CpGs were also differentially expressed. A pathway analysis of the genes with false-discovery-rate-adjusted significant CpGs revealed significant enrichment of olfactory transduction, pentose and glucuronate interconversions, ascorbate and aldarate metabolism, and steroid hormone biosynthesis pathways. CONCLUSION: High-dose synthetic glucocorticoid administration in the setting of a dental procedure was significantly associated with DNAm changes within buccal samples. These findings are consistent with prior findings of an influence of glucocorticoids on DNAm in humans.

Epigenetic impacts of stress priming of the neuroinflammatory response to sarin surrogate in mice: a model of Gulf War illness.

BACKGROUND: Gulf War illness (GWI) is an archetypal, medically unexplained, chronic condition characterised by persistent sickness behaviour and neuroimmune and neuroinflammatory components. An estimated 25-32% of the over 900,000 veterans of the 1991 Gulf War fulfil the requirements of a GWI diagnosis. It has been hypothesised that the high physical and psychological stress of combat may have increased vulnerability to irreversible acetylcholinesterase (AChE) inhibitors leading to a priming of the neuroimmune system. A number of studies have linked high levels of psychophysiological stress and toxicant exposures to epigenetic modifications that regulate gene expression. Recent research in a mouse model of GWI has shown that pre-exposure with the stress hormone corticosterone (CORT) causes an increase in expression of specific chemokines and cytokines in response to diisopropyl fluorophosphate (DFP), a sarin surrogate and irreversible AChE inhibitor. METHODS: C57BL/6J mice were exposed to CORT for 4 days, and exposed to DFP on day 5, before sacrifice 6 h later. The transcriptome was examined using RNA-seq, and the epigenome was examined using reduced representation bisulfite sequencing and H3K27ac ChIP-seq. RESULTS: We show transcriptional, histone modification (H3K27ac) and DNA methylation changes in genes related to the immune and neuronal system, potentially relevant to neuroinflammatory and cognitive symptoms of GWI. Further evidence suggests altered proportions of myelinating oligodendrocytes in the frontal cortex, perhaps connected to white matter deficits seen in GWI sufferers. CONCLUSIONS: Our findings may reflect the early changes which occurred in GWI veterans, and we observe alterations in several pathways altered in GWI sufferers. These close links to changes seen in veterans with GWI indicates that this model reflects the environmental exposures related to GWI and may provide a model for biomarker development and testing future treatments.

A comprehensive review of genetic and epigenetic mechanisms that regulate BDNF expression and function with relevance to major depressive disorder.

Major depressive disorder (MDD) is a mood disorder that affects behavior and impairs cognition. A gene potentially important to this disorder is the brain derived neurotrophic factor (BDNF) as it is involved in processes controlling neuroplasticity. Various mechanisms exist to regulate BDNF's expression level, subcellular localization, and sorting to appropriate secretory pathways. Alterations to these processes by genetic factors and negative stressors can dysregulate its expression, with possible implications for MDD. Here, we review the mechanisms governing the regulation of BDNF expression, and discuss how disease-associated single nucleotide polymorphisms (SNPs) can alter these mechanisms, and influence MDD. As negative stressors increase the likelihood of MDD, we will also discuss the impact of these stressors on BDNF expression, the cellular effect of such a change, and its impact on behavior in animal models of stress. We will also describe epigenetic processes that mediate this change in BDNF expression. Similarities in BDNF expression between animal models of stress and those in MDD will be highlighted. We will also contrast epigenetic patterns at the BDNF locus between animal models of stress, and MDD patients, and address limitations to current clinical studies. Future work should focus on validating current genetic and epigenetic findings in tightly controlled clinical studies. Regions outside of BDNF promoters should also be explored, as should other epigenetic marks, to improve identification of biomarkers for MDD.

Effects of negative stressors on DNA methylation in the brain: implications for mood and anxiety disorders.

Stress is a major contributor to anxiety and mood disorders. The recent discovery of epigenetic changes in the brain resulting from stress has enhanced our understanding of the mechanism by which stress is able to promote these disorders. Although epigenetics encompasses chemical modifications that occur at both DNA and histones, much attention has been focused on stress-induced DNA methylation changes on behavior. Here, we review the effect of stress-induced DNA methylation changes on physiological mechanisms that govern behavior and cognition, dysregulation of which can be harmful to mental health. A literature review was performed in the areas of DNA methylation, stress, and their impact on the brain and psychiatric illness. Key findings center on genes involved in the hypothalamic-pituitary-adrenal axis, neurotransmission and neuroplasticity. Using animal models of different stress paradigms and clinical studies, we detail how DNA methylation changes to these genes can alter physiological mechanisms that influence behavior. Appropriate levels of gene expression in the brain play an important role in mental health. This dynamic control can be disrupted by stress-induced changes to DNA methylation patterns. Advancement in other areas of epigenetics, such as histone modifications and the discovery of the novel DNA epigenetic mark, 5-hydroxymethylcytosine, could provide additional avenues to consider when determining the epigenetic effects of stress on the brain.

Correlation of telomere length in brain tissue with peripheral tissues in living human subjects.

Telomeres are important to chromosomal stability, and changes in their length correlate with disease, potentially relevant to brain disorders. Assessing telomere length in human brain is invasive, but whether peripheral tissue telomere length correlates with that in brain is not known. Saliva, buccal, blood, and brain samples were collected at time points before, during, and after subjects undergoing neurosurgery (n = 35) for intractable epilepsy. DNA was isolated from samples and average telomere length assessed by qPCR. Correlations of telomere length between tissue samples were calculated across subjects. When data were stratified by sex, saliva telomere length correlated with brain telomere length in males only. Buccal telomere length correlated with brain telomere length when males and females were combined. These findings indicate that in living subjects, telomere length in peripheral tissues variably correlates with that in brain and may be dependent on sex. Peripheral tissue telomere length may provide insight into brain telomere length, relevant to assessment of brain disorder pathophysiology.

Transcriptomic Evaluation of a Stress Vulnerability Network Using Single-Cell RNA Sequencing in Mouse Prefrontal Cortex.

BACKGROUND: Increased vulnerability to stress is a major risk factor for several mood disorders, including major depressive disorder. Although cellular and molecular mechanisms associated with depressive behaviors following stress have been identified, little is known about the mechanisms that confer the vulnerability that predisposes individuals to future damage from chronic stress. METHODS: We used multisite in vivo neurophysiology in freely behaving male and female C57BL/6 mice (n = 12) to measure electrical brain network activity previously identified as indicating a latent stress vulnerability brain state. We combined this neurophysiological approach with single-cell RNA sequencing of the prefrontal cortex to identify distinct transcriptomic differences between groups of mice with inherent high and low stress vulnerability. RESULTS: We identified hundreds of differentially expressed genes (padjusted < .05) across 5 major cell types in animals with high and low stress vulnerability brain network activity. This unique analysis revealed that GABAergic (gamma-aminobutyric acidergic) neuron gene expression contributed most to the network activity of the stress vulnerability brain state. Upregulation of mitochondrial and metabolic pathways also distinguished high and low vulnerability brain states, especially in inhibitory neurons. Importantly, genes that were differentially regulated with vulnerability network activity significantly overlapped (above chance) with those identified by genome-wide association studies as having single nucleotide polymorphisms significantly associated with depression as well as genes more highly expressed in postmortem prefrontal cortex of patients with major depressive disorder. CONCLUSIONS: This is the first study to identify cell types and genes involved in a latent stress vulnerability state in the brain.

Single Cell Transcriptome of Stress Vulnerability Network in mouse Prefrontal Cortex.

Increased vulnerability to stress is a major risk factor for the manifestation of several mood disorders, including major depressive disorder (MDD). Despite the status of MDD as a significant donor to global disability, the complex integration of genetic and environmental factors that contribute to the behavioral display of such disorders has made a thorough understanding of related etiology elusive. Recent developments suggest that a brain-wide network approach is needed, taking into account the complex interplay of cell types spanning multiple brain regions. Single cell RNA-sequencing technologies can provide transcriptomic profiling at the single-cell level across heterogenous samples. Furthermore, we have previously used local field potential oscillations and machine learning to identify an electrical brain network that is indicative of a predisposed vulnerability state. Thus, this study combined single cell RNA-sequencing (scRNA-Seq) with electrical brain network measures of the stress-vulnerable state, providing a unique opportunity to access the relationship between stress network activity and transcriptomic changes within individual cell types. We found especially high numbers of differentially expressed genes between animals with high and low stress vulnerability brain network activity in astrocytes and glutamatergic neurons but we estimated that vulnerability network activity depends most on GABAergic neurons. High vulnerability network activity included upregulation of microglia and mitochondrial and metabolic pathways, while lower vulnerability involved synaptic regulation. Genes that were differentially regulated with vulnerability network activity significantly overlapped with genes identified as having significant SNPs by human GWAS for depression. Taken together, these data provide the gene expression architecture of a previously uncharacterized stress vulnerability brain state, enabling new understanding and intervention of predisposition to stress susceptibility.

Altered offspring neurodevelopment in an arginine vasopressin preeclampsia model.

Preeclampsia is a severe gestational hypertensive condition linked to child neuropsychiatric disorders, although underlying mechanisms are unclear. We used a recently developed, clinically relevant animal model of preeclampsia to assess offspring. C57BL/6J mouse dams were chronically infused with arginine vasopressin (AVP) or saline (24 ng/h) throughout pregnancy. Adult offspring were behaviorally tested (Y-maze, open field, rotarod, social approach, and elevated plus maze). Offspring brain was assessed histologically and by RNA sequencing. Preeclampsia-exposed adult males exhibited increased anxiety-like behavior and social approach while adult females exhibited impaired procedural learning. Adult AVP-exposed males had reduced total neocortical volume. Adult AVP-exposed females had increased caudate-putamen volume, increased caudate-putamen cell number, and decreased excitatory synapse density in hippocampal dentate gyrus (DG), CA1, and CA3. At postnatal day 7 (P7), AVP-exposed male and female offspring both had smaller neocortex. At P7, AVP-exposed males also had smaller caudate-putamen volume, while females had increased caudate-putamen volume relative to neocortical size. Similar to P7, E18 AVP-exposed offspring had smaller dorsal forebrain, mainly in reduced intermediate, subventricular, and ventricular zone volume, particularly in males. Decreased volume was not accounted for by cell size or cerebrovascular vessel diameter changes. E18 cortical RNAseq revealed 49 differentially-expressed genes in male AVP-exposed offspring, over-representing cytoplasmic translation processes. In females, 31 genes were differentially-expressed, over-representing collagen-related and epithelial regulation pathways. Gene expression changes in E18 AVP-exposed placenta indicated potential underlying mechanisms. Deficits in behavior and forebrain development in this AVP-based preeclampsia model were distinctly different in males and females, implicating different neurobiological bases.

Correction: Chronic maternal interleukin-17 and autism-related cortical gene expression, neurobiology, and behavior.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Chronic maternal interleukin-17 and autism-related cortical gene expression, neurobiology, and behavior.

Chronic inflammation during pregnancy (e.g., preeclampsia, diabetes) is linked to increased risk for offspring neurodevelopmental disorders such as autism spectrum disorder (ASD). However, mediators of such exposures that could be targeted with maternal intervention are unclear, as few chronic gestational inflammation models have been tested. One potential mediator is interleukin-17 (IL-17), a pro-inflammatory cytokine implicated in neurodevelopmental disorders and gestational disease. To test chronic maternal IL-17 impacts on offspring, C57BL/6J dams were administered IL-17A continuously throughout pregnancy. Offspring were assessed for body weight; cortical volume, gene expression, and cellular composition; and adult behavior. IL-17A-condition offspring exhibited decreased somatic and cortical size at embryonic day 18 (E18) and as adults. mRNA sequencing of E18 cortex revealed 320 differentially expressed genes in males, but none in females. These were significantly enriched for ASD (Simons Foundation Autism Research Initiative), synaptic, and cell cycle genes. By adulthood, neocortical glial cell density and gene expression were decreased, while GABAergic synaptic gene expression was increased in males. Furthermore, IL-17A-condition male but not female offspring exhibited reduced anxiety-like behavior. Social approach deficits in males were negatively correlated with neocortical GABAergic synaptic gene expression. Chronic gestational IL-17A was sufficient to cause ASD-like phenotypes early and persistently in male offspring. This echoes the male bias, altered cortical development, and behavioral findings in ASD, suggesting that chronic maternal IL-17 contributes to offspring ASD pathogenesis. Furthermore, the trajectory from embryonically dysregulated synaptic and cell cycle genes to disrupted adult glia, inhibitory synapses, and behavior suggests a mechanism for chronic maternal IL-17 effects on offspring.

CX3CR1-deficiency is associated with increased severity of disease in experimental autoimmune uveitis.

The role of CX3CR1 in regulating the function of monocytes and microglia was examined in mice in which CX3CR1 had been replaced by green fluorescent protein (GFP). Induction of experimental autoimmune uveitis (EAU) in these mice resulted in increased disease severity at day 23 postimmunization with uveitogenic peptide when compared with CX3CR1-positive mice and increased apoptosis of neuronal cells in the inner nuclear layer. Resident microglia within the retina were activated equally as EAU developed in mice with or without CX3CR1, as determined by changes in morphology, suggesting that the microglial cell response did not account for the differences. Although the inflammatory infiltrate had increased in mice without CX3CR1 at day 23 postimmunization, the percentage of natural killer cells in the infiltrate was not changed in these mice. Similarly, increased disease severity at this stage was not associated with an overall increased percentage of macrophages in the retinal inflammatory infiltrate or in increased activation of these cells. The increased recruitment of monocytes to the retina in response to EAU induction in CX3CR1(GFP/GFP) mice compared with CX3CR1(GFP/+) mice was not reflected in increased migration away from vessels, leading to marked clustering of GFP(+) cells around veins and venules in these mice. It is possible that this monocyte/macrophage clustering leads to the increased severity of disease seen in the mice by focusing and so intensifying the inflammatory response.

Genome-wide DNA methylation comparison between live human brain and peripheral tissues within individuals.

Differential DNA methylation in the brain is associated with many psychiatric diseases, but access to brain tissues is essentially limited to postmortem samples. The use of surrogate tissues has become common in identifying methylation changes associated with psychiatric disease. In this study, we determined the extent to which peripheral tissues can be used as surrogates for DNA methylation in the brain. Blood, saliva, buccal, and live brain tissue samples from 27 patients with medically intractable epilepsy undergoing brain resection were collected (age range 5-61 years). Genome-wide methylation was assessed with the Infinium HumanMethylation450 (n = 12) and HumanMethylationEPIC BeadChip arrays (n = 21). For the EPIC methylation data averaged for each CpG across subjects, the saliva-brain correlation (r = 0.90) was higher than that for blood-brain (r = 0.86) and buccal-brain (r = 0.85) comparisons. However, within individual CpGs, blood had the highest proportion of CpGs correlated to brain at nominally significant levels (20.8%), as compared to buccal tissue (17.4%) and saliva (15.1%). For each CpG and each gene, levels of brain-peripheral tissue correlation varied widely. This indicates that to determine the most useful surrogate tissue for representing brain DNA methylation, the patterns specific to the genomic region of interest must be considered. To assist in that objective, we have developed a website, IMAGE-CpG, that allows researchers to interrogate DNA methylation levels and degree of cross-tissue correlation in user-defined locations across the genome.

Genome-wide association study of seasonal affective disorder.

Family and twin studies have shown a genetic component to seasonal affective disorder (SAD). A number of candidate gene studies have examined the role of variations within biologically relevant genes in SAD susceptibility, but few genome-wide association studies (GWAS) have been performed to date. The authors aimed to identify genetic risk variants for SAD through GWAS. The authors performed a GWAS for SAD in 1380 cases and 2937 controls of European-American (EA) origin, selected from samples for GWAS of major depressive disorder and of bipolar disorder. Further bioinformatic analyses were conducted to examine additional genomic and biological evidence associated with the top GWAS signals. No susceptibility loci for SAD were identified at a genome-wide significant level. The strongest association was at an intronic variant (rs139459337) within ZBTB20 (odds ratio (OR) = 1.63, p = 8.4 × 10-7), which encodes a transcriptional repressor that has roles in neurogenesis and in adult brain. Expression quantitative trait loci (eQTL) analysis showed that the risk allele "T" of rs139459337 is associated with reduced mRNA expression of ZBTB20 in human temporal cortex (p = 0.028). Zbtb20 is required for normal murine circadian rhythm and for entrainment to a shortened day. Of the 330 human orthologs of murine genes directly repressed by Zbtb20, there were 32 associated with SAD in our sample (at p < 0.05), representing a significant enrichment of ZBTB20 targets among our SAD genetic association signals (fold = 1.93, p = 0.001). ZBTB20 is a candidate susceptibility gene for SAD, based on a convergence of genetic, genomic, and biological evidence. Further studies are necessary to confirm its role in SAD.

Chronic social stress induces DNA methylation changes at an evolutionary conserved intergenic region in chromosome X.

Chronic stress resulting from prolonged exposure to negative life events increases the risk of mood and anxiety disorders. Although chronic stress can change gene expression relevant for behavior, molecular regulators of this change have not been fully determined. One process that could play a role is DNA methylation, an epigenetic process whereby a methyl group is added onto nucleotides, predominantly cytosine in the CpG context, and which can be induced by chronic stress. It is unknown to what extent chronic social defeat, a model of human social stress, influences DNA methylation patterns across the genome. Our study addressed this question by using a targeted-capture approach called Methyl-Seq to investigate DNA methylation patterns of the dentate gyrus at putative regulatory regions across the mouse genome from mice exposed to 14 days of social defeat. Findings were replicated in independent cohorts by bisulfite-pyrosequencing. Two differentially methylated regions (DMRs) were identified. One DMR was located at intron 9 of Drosha, and it showed reduced methylation in stressed mice. This observation replicated in one of two independent cohorts. A second DMR was identified at an intergenic region of chromosome X, and methylation in this region was increased in stressed mice. This methylation difference replicated in two independent cohorts and in Major Depressive Disorder (MDD) postmortem brains. These results highlight a region not previously known to be differentially methylated by chronic social defeat stress and which may be involved in MDD.

Epigenetics of Delirium and Aging: Potential Role of DNA Methylation Change on Cytokine Genes in Glia and Blood Along With Aging.

Background: Delirium in elderly patients is common and dangerous. Major risk factors include aging and exogenous insults, such as infection or surgery. In animal models, aging enhances pro-inflammatory cytokine release from microglia in response to exogenous insults. The epigenetic mechanism DNA methylation (DNAm) regulates gene expression and changes with age. Older individuals may have methylation changes that influence the increased cytokine upon insult, but the degree to which aging affects DNAm of cytokine genes is not fully understood. Methods: The relationship between DNAm and aging of pro-inflammatory cytokine genes (TNF-alpha, IL1-beta, IL-6) was investigated using methylation array data in two cohorts. Brain and blood samples were collected from a neurosurgery cohort (NSG) of 21 subjects who underwent brain resection. A second cohort, the Grady Trauma Project (GTP), included blood samples from 265 subjects. Results: In the NSG cohort, a significant negative correlation between age and DNAm in brain was found at a CpG in IL-6. With the GTP dataset, significant negative correlations between age and DNAm were seen at most of the CpGs in TNF-alpha. Also, TNF-Alpha expression increases with age. These GTP DNAm correlations were also nominally significant in NSG blood samples. In neuronal negative NSG brain tissue, a similar negative trend was observed. Conclusions: With aging, a decrease in DNAm of cytokines gene CpGs in glia and blood was seen. As this can affect their expression, additional research is needed to fully elucidate the role of DNAm in aging and how it may influence the pathogenesis of delirium.

Adaptation of the targeted capture Methyl-Seq platform for the mouse genome identifies novel tissue-specific DNA methylation patterns of genes involved in neurodevelopment.

Methyl-Seq was recently developed as a targeted approach to assess DNA methylation (DNAm) at a genome-wide level in human. We adapted it for mouse and sought to examine DNAm differences across liver and 2 brain regions: cortex and hippocampus. A custom hybridization array was designed to isolate 99 Mb of CpG islands, shores, shelves, and regulatory elements in the mouse genome. This was followed by bisulfite conversion and sequencing on the Illumina HiSeq2000. The majority of differentially methylated cytosines (DMCs) were present at greater than expected frequency in introns, intergenic regions, near CpG islands, and transcriptional enhancers. Liver-specific enhancers were observed to be methylated in cortex, while cortex specific enhancers were methylated in the liver. Interestingly, commonly shared enhancers were differentially methylated between the liver and cortex. Gene ontology and pathway analysis showed that genes that were hypomethylated in the cortex and hippocampus were enriched for neuronal components and neuronal function. In contrast, genes that were hypomethylated in the liver were enriched for cellular components important for liver function. Bisulfite-pyrosequencing validation of 75 DMCs from 19 different loci showed a correlation of r = 0.87 with Methyl-Seq data. We also identified genes involved in neurodevelopment that were not previously reported to be differentially methylated across brain regions. This platform constitutes a valuable tool for future genome-wide studies involving mouse models of disease.