Glucocorticoid Receptor Gene Research Articles

From maternal glucocorticoid and thyroid hormones to epigenetic regulation of offspring gene expression: An experimental study in a wild bird species.

Offspring phenotype at birth is determined by its genotype and the prenatal environment including exposure to maternal hormones. Variation in both maternal glucocorticoids and thyroid hormones can affect offspring phenotype, but the underlying molecular mechanisms, especially those contributing to long-lasting effects, remain unclear. Epigenetic changes (such as DNA methylation) have been postulated as mediators of long-lasting effects of early-life environment. In this study, we determined the effects of elevated prenatal glucocorticoid and thyroid hormones on handling stress response (breath rate) as well as DNA methylation and gene expression of glucocorticoid receptor (GR) and thyroid hormone receptor (THR) in great tits (Parus major). Eggs were injected before incubation onset with corticosterone (the main avian glucocorticoid) and/or thyroid hormones (thyroxine and triiodothyronine) to simulate variation in maternal hormone deposition. Breath rate during handling and gene expression of GR and THR were evaluated 14 days after hatching. Methylation status of GR and THR genes was analyzed from the longitudinal blood cells sampled 7 and 14 days after hatching, as well as the following autumn. Elevated prenatal corticosterone level significantly increased the breath rate during handling, indicating an enhanced metabolic stress response. Prenatal corticosterone manipulation had CpG-site-specific effects on DNA methylation at the GR putative promoter region, while it did not significantly affect GR gene expression. GR expression was negatively associated with earlier hatching date and chick size. THR methylation or expression did not exhibit any significant relationship with the hormonal treatments or the examined covariates, suggesting that TH signaling may be more robust due to its crucial role in development. This study provides some support to the hypothesis suggesting that maternal corticosterone may influence offspring metabolic stress response via epigenetic alterations, yet their possible adaptive role in optimizing offspring phenotype to the prevailing conditions, context-dependency, and the underlying molecular interplay needs further research.

Glucocorticoid Receptor Gene (NR3C1) Polymorphisms and Metabolic Syndrome: Insights from the Mennonite Population.

The regulation of the hypothalamic-pituitary-adrenal (HPA) axis is associated with polymorphisms and the methylation degree of the glucocorticoid receptor gene (NR3C1) and is potentially involved in the development of metabolic syndrome (MetS). In order to evaluate the association between MetS with the polymorphisms, methylation, and gene expression of the NR3C1 in the genetically isolated Brazilian Mennonite population, we genotyped 20 NR3C1 polymorphisms in 74 affected (MetS) and 138 unaffected individuals without affected first-degree relatives (Co), using exome sequencing, as well as five variants from non-exonic regions, in 70 MetS and 166 Co, using mass spectrometry. The methylation levels of 11 1F CpG sites were quantified using pyrosequencing (66 MetS and 141 Co), and the NR3C1 expression was evaluated via RT-qPCR (14 MetS and 25 Co). Age, physical activity, and family environment during childhood were associated with MetS. Susceptibility to MetS, independent of these factors, was associated with homozygosity for rs10482605*C (OR = 4.74, pcorr = 0.024) and the haplotype containing TTCGTTGATT (rs3806855*T_ rs3806854*T_rs10482605*C_rs10482614*G_rs6188*T_rs258813*T_rs33944801*G_rs34176759*A_rs17209258*T_rs6196*T, OR = 4.74, pcorr = 0.048), as well as for the CCT haplotype (rs41423247*C_ rs6877893*C_rs258763*T), OR = 6.02, pcorr = 0.030), but not to the differences in methylation or gene expression. Thus, NR3C1 polymorphisms seem to modulate the susceptibility to MetS in Mennonites, independently of lifestyle and early childhood events, and their role seems to be unrelated to DNA methylation and gene expression.

Genetic glucocorticoid receptor variants differ between ethnic groups but do not explain variation in age of diabetes onset, metabolic and inflammation parameters in patients with type 2 diabetes.

The effect of excess glucocorticoid receptor (GR) stimulation through glucocorticoid medication or cortisol on glucose metabolism is well established. There are genetic GR variants that result in increased or decreased GR stimulation. We aimed to determine the prevalence of genetic GR variants in different ethnic groups in a cohort of patients with type 2 diabetes, and we aimed to determine their association with age of diabetes onset and metabolic and inflammation parameters. A cross-sectional analysis was performed in a multiethnic cohort (n = 602) of patients with established type 2 diabetes. Polymorphisms in the GR gene that have previously been associated with altered glucocorticoid sensitivity (TthIIII, ER22/23EK N363S, BclI and 9β) were determined and combined into 6 haplotypes. Associations with age of diabetes onset, HbA1c, hs-CRP and lipid values were evaluated in multivariate regression models. The prevalence of the SNPs of N363S and BclI was higher in Dutch than in non-Dutch patients. We observed a lower prevalence of the SNP 9β in Dutch, South(East) Asian and Black African patients versus Turkish and Moroccan patients. We did not detect an association between SNPs and diabetes age of onset or metabolic parameters. We only found a trend for lower age of onset and higher HbA1c in patients with 1 or 2 copies of haplotype 3 (TthIIII + 9β). The prevalence of genetic GR variants differs between patients of different ethnic origins. We did not find a clear association between genetic GR variants and age of diabetes onset or metabolic and inflammation parameters. This indicates that the clinical relevance of GR variants in patients with established type 2 diabetes is limited.

Polymorphism of glucocorticoid receptor gene (rs41423247) in functional seizures (psychogenic nonepileptic seizures/attacks).

We investigated the association between the glucocorticoid receptor (GR) gene, also known as the nuclear receptor subfamily 3, group C, member 1 (NR3C1), rs41423247 polymorphism, and functional seizures (psychogenic nonepileptic seizures/attacks) in a case-control study. We hypothesized that the tested polymorphism has significant associations with functional seizures (psychogenic nonepileptic seizures/attacks) independent from comorbid depression. Seventy patients with functional seizures (psychogenic nonepileptic seizures/attacks), 70 with major depressive disorder (MDD), and 70 healthy controls (HCs) were studied. Their DNAs were analyzed for NR3C1 rs41423247 polymorphism. Genotype and allele frequencies of rs41423247 were different between the three groups. G allele carriers were more frequent in patients with functional seizures (psychogenic nonepileptic seizures/attacks) and those with MDD compared to HCs (p = 0.0001). However no significant difference was observed with respect to allele distributions between functional seizures (psychogenic nonepileptic seizures/attacks) and MDD groups (p = 0.391). CC genotype was less often associated with functional seizures (psychogenic nonepileptic seizures/attacks) versus HC: Codominant model; p = 0.001, OR = 0.11, 95% CI = 0.05-0.24, and -2loglilkelihood = 231.7. In comparison between functional seizures (psychogenic nonepileptic seizures/attacks) group and other (MDD + HC) groups, we observed a significant association between CG genotype and functional seizures (psychogenic nonepileptic seizures/attacks) (Codominant model; p = 0.001, OR = 5.63, 95% CI = 2.60-12.40 and -2loglikelihood = 245.99). Patients with functional seizures (psychogenic nonepileptic seizures/attacks) and those with MDD were significantly more often G allele carriers in rs41423247 compared with HCs. We observed a significant association between CG genotype and functional seizures (psychogenic nonepileptic seizures/attacks). However, we could not exclude the possibility of confounding effects of depression. Future genetic studies of patients with functional seizures (psychogenic nonepileptic seizures/attacks) should include a comparison group with depression in addition to a comparison group of HCs.

Glucocorticoids and Their Receptor Isoforms: Roles in Female Reproduction, Pregnancy, and Foetal Development.

Alterations in the hypothalamic-pituitary-adrenal (HPA) axis and associated changes in circulating levels of glucocorticoids are integral to an organism's response to stressful stimuli. Glucocorticoids acting via glucocorticoid receptors (GRs) play a role in fertility, reproduction, placental function, and foetal development. GRs are ubiquitously expressed throughout the female reproductive system and regulate normal reproductive function. Stress-induced glucocorticoids have been shown to inhibit reproduction and affect female gonadal function by suppressing the hypothalamic-pituitary-gonadal (HPG) axis at each level. Furthermore, during pregnancy, a mother's exposure to prenatal stress or external glucocorticoids can result in long-lasting alterations to the foetal HPA and neuroendocrine function. Several GR isoforms generated via alternative splicing or translation initiation from the GR gene have been identified in the mammalian ovary and uterus. The GR isoforms identified include the splice variants, GRα and GRβ, and GRγ and GR-P. Glucocorticoids can exert both stimulatory and inhibitory effects and both pro- and anti-inflammatory functions in the ovary, in vitro. In the placenta, thirteen GR isoforms have been identified in humans, guinea pigs, sheep, rats, and mice, indicating they are conserved across species and may be important in mediating a differential response to stress. Distinctive responses to glucocorticoids, differential birth outcomes in pregnancy complications, and sex-based variations in the response to stress could all potentially be dependent on a particular GR expression pattern. This comprehensive review provides an overview of the structure and function of the GR in relation to female fertility and reproduction and discusses the changes in the GR and glucocorticoid signalling during pregnancy. To generate this overview, an extensive non-systematic literature search was conducted across multiple databases, including PubMed, Web of Science, and Google Scholar, with a focus on original research articles, meta-analyses, and previous review papers addressing the subject. This review integrates the current understanding of GR variants and their roles in glucocorticoid signalling, reproduction, placental function, and foetal growth.

Glucocorticoid receptor expression in blood, but not across brain regions, reveals long-term effects of early life adversity in zebra finches

Early-life environment can affect organisms for life on many levels. The glucocorticoid receptor (GR) gene has a pivotal role mediating organismal physiological and behavioral responses to environmental change, and is sensitive to early-life environmental conditions and epigenetic programming. Longitudinal studies require non-lethal sampling of peripheral tissues (e.g. blood), but this approach is dependent on the extent to which GR expression in peripheral tissues covaries with GR expression in central tissues. To test for the long-term effects of early life adversity on GR expression across brain and peripheral tissues, we manipulated developmental conditions of captive zebra finches (n = 45), rearing them in either benign or harsh conditions through manipulation of parental foraging costs. We measured relative GR mRNA expression in blood and five brain regions in adulthood: hippocampus, hypothalamus, amygdala, ventral striatum, and the nidopallium caudolaterale (analogous to the mammalian prefrontal cortex), using qPCR. We further tested whether GR expression was modulated by natal brood size (which affected growth), age at sampling, and sex. GR expression correlations among tissues varied widely in magnitude and direction, ranging from -0.27 to +0.80, indicating that our understanding of developmental effects on GR expression and associated phenotypes needs to be region specific rather than organism wide. A more consistent pattern was that GR expression increased with age in blood, ventral striatum and hippocampus; GR expression was independent of age in other tissues. Developmental treatment did not affect GR expression in any of the tissues measured directly, but in blood and ventral striatum of adult females we found a positive correlation between nestling mass and GR expression. Thus, GR expression in blood was affected by early life conditions as reflected in growth in adult females, a pattern also found in one brain tissue, but not ubiquitous across brain regions. These results point at sex-dependent physiological constraints during development, shaping early life effects on GR expression in females only. Further study is required to investigate whether these tissue-dependent effects more generally reflect tissue-dependent long-term effects of early life adversity. This, together with investigating the physiological consequences of GR expression levels on individual performance and coping abilities, will be fundamental towards understanding the mechanisms mediating long-term impacts of early life, and the extent to which these can be quantified through non-lethal sampling.

Methylation of the Glucocorticoid Receptor Gene (NR3C1) in Adolescents with a History of Childhood Adversity Engaging in Non-Suicidal Self-Injury

Introduction: Non-suicidal self-injury (NSSI) is a large phenomenon among adolescents, and adverse childhood experiences (ACEs) are a major risk factor in its development. Malfunctioning of the hypothalamus-pituitary-adrenal (HPA) axis has been repeatedly reported for ACE as well as for NSSI. The glucocorticoid receptor (GR) is essential for the correct functioning of the HPA axis, thus alterations in the expression of the GR through altered methylation of the GR gene (NR3C1) (and more specifically exon 1F) might contribute to the development of NSSI in individuals with a history of ACEs, as has been reported for different other mental disorders. Methods: In this case-control study, we compared the methylation levels of exon 1F of the GR gene (NR3C1-1F) in adolescents with engagement in NSSI (n = 67) and a healthy control group (HC; n = 47). We preserved buccal swabs and used a mass spectrometry-based method called EpiTYPER for analyzing mean methylation of NR3C1-1F. Results: Adolescents in the NSSI group reported significantly more ACEs. The mean methylation level was about 3% in both groups with no significant group differences. Furthermore, no significant relation was found between ACE and methylation of NR3C1-1F, neither in the overall sample nor in the NSSI or HC group. Conclusion: Our results are contradictory to previous research showing an increased methylation in individuals with ACE. Regarding relations between methylation of NR3C1-1F and mental disorders, previous studies reported inconsistent findings. Our study points to NSSI being either unrelated to methylation of NR3C1-1F or to yet not identified moderators on relations between methylation of NR3C1-1F and engagement in NSSI during adolescence.

Perceived Parental Support and Psychological Control, DNA Methylation, and Loneliness: Longitudinal Associations Across Early Adolescence.

A broad range of factors have been associated with the development of adolescent loneliness. In the family context, a lack of parental support and high levels of parental psychological control have systematically been linked to loneliness. On the biological level, DNA methylation (which is an epigenetic process that suppresses gene expression) is believed to play a role in the development of loneliness. Specifically, high levels of DNA methylation in genes that play an important role in the functioning of the human stress response system are believed to elevate the risk of loneliness. Moreover, DNA methylation levels in these stress-related genes can be influenced by stressful environmental factors, suggesting a potential mediating role of DNA methylation in the association between parenting behaviors and loneliness. The current 3-year longitudinal study is the first study to examine the potential bidirectional longitudinal associations between loneliness, DNA methylation in stress-related genes, and both perceived parental support and psychological control. Furthermore, we explored the potential mediating role of DNA methylation in stress-related genes in the associations between perceived parenting and loneliness. The sample comprised 622 early adolescents (55% girls, Mage T1 = 10.77 years, SDage T1 = 0.48) who were followed from Grade 5 to 7. Parental support, psychological control, and loneliness were assessed annually by adolescent self-report questionnaires and DNA methylation was determined from saliva samples. Cross-Lagged Panel Models (CLPM) revealed that higher levels of loneliness predicted lower perceived parental support and higher perceived psychological control over time, as well as higher DNA methylation in some stress-related genes, that is, the glucocorticoid receptor gene (NR3C1) and the brain-derived neurotrophic factor (BDNF). In addition, higher NR3C1 methylation was predictive of lower perceived parental support and higher psychological control over time. No evidence was found for a mediating role of DNA methylation. Overall, our longitudinal findings challenge the current focus on DNA methylation and parenting behaviors as risk factors for adolescent loneliness. Instead, they suggest that the less considered direction of effects, which implies that loneliness predicts DNA methylation and aspects of parenting such as support and psychological control, should receive greater attention in future research.

The mediation effect of glucocorticoid receptor gene (NR3C1) methylation between childhood maltreatment and depressive symptoms in Chinese adolescents: A 2-year longitudinal study.

This three-wave longitudinal study examined whether methylation alterations in promoter exon 1F of a stress-related gene-NR3C1 (NR3C1-1F)-explained the longitudinal associations between childhood maltreatment and adolescent depressive symptoms. A total of 370 Han Chinese adolescents (Mage = 16.31 ± 1.28 years; 51.4% girls) recruited from Shandong, China were tracked from 2018 to 2020. The results showed that the severity of childhood maltreatment, especially that of emotional abuse and physical neglect, conferred risk for adolescent depressive symptoms via reducing NR3C1-1F methylation levels. These mediation effects of NR3C1-1F methylation did not vary between adolescent sex or NR3C1 BclI and Tth111I polymorphisms. The findings highlight how childhood maltreatment contributes to psychopathology development at a biological level.

Glucocorticoid Receptor Polymorphism A3669G Is Associated with Airflow Obstruction in Mild-to-Severe Asthma

Background: Glucocorticoids (GCs) represent the mainstay therapy for asthmatics. A subset of severe asthmatics fails to respond to steroid-based therapies, leading to important healthcare costs. Single nucleotide polymorphisms (SNPs) of glucocorticoid receptor genes were associated with a response to GC. We evaluate the possible relation of BclI and A3669G SNPs to clinical, biological and functional characteristics of asthmatics. Methods: We recruited 172 mild-to-severe asthmatic outpatients referring to the Severe Asthma and Rare Lung Disease Unit at San Luigi University Hospital. Clinical data were obtained at recruitment when spirometry tests and peripheral blood sampling were performed. Patients were genotyped for BclI and A3669G through the pyrosequencing assay results. Results: Patients with the A3669G AG genotype were younger, allergic and had higher IgE levels compared to AA genotype (p < 0.05). Moreover, asthmatics with the AA genotype had a lower post-bronchodilator FEV1/FVC ratio than the GG genotype (p < 0.05), and a higher RV/TLC ratio than the AG genotype (p < 0.05). Conclusions: The A3669G AG genotype might be related to type-2 allergic asthma; in particular, allele A of A3669G SNP was associated with GC response in our asthmatics. In conclusion, this observational cross-sectional study suggests a possible role of A3669G SNP as a predictor of asthma severity and phenotype.

Differential effect of panic on the DNA methylation of the glucocorticoid receptor gene exon 1F in chronic subjective tinnitus with distress.

Tinnitus can be regarded as a chronic stressor, leading to dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis. There is important comorbidity with anxiety, particularly panic, potentially associated with differences in HPA axis functioning and methylation patterns of HPA axis-related genes. This study examines DNA methylation of the glucocorticoid receptor gene ( NR3C1 ) exon 1F in adults with chronic subjective tinnitus and the possible differential effect of panic. In a well characterized tinnitus sample ( n = 22, half of which had co-occurring panic attacks), and unaffected controls ( n = 31) methylation patterns of the CpG sites were determined using pyrosequencing and compared between groups through linear mixed models. Gene expression was determined using quantitative PCR on mRNA. Comparing the combined tinnitus groups to the control group, no DNA methylation differences were observed; however, the tinnitus group with panic attacks showed consistently higher mean methylation values across all CpGs compared to the tinnitus-only and the control group ( P = 0.03 following Tukey correction), which became even more pronounced when accounting for childhood trauma ( P = 0.012). Moreover, a significant positive correlation was found between methylation of the CpG7 site and the Beck Anxiety Inventory total score ( P = 0.001) in the total population. NR3C1 -1F expression was not significantly different between the three groups. Panic is associated with higher DNA methylation of the NR3C1 exon 1F in adults with chronic subjective tinnitus, consistent with the reduced negative glucocorticoid feedback and HPA axis hyperfunction observed in individuals with panic disorder.

Role of glucocorticoid receptor (GR) in white adipose tissue beiging

AimGlucocorticoids (GCs) play a crucial role in energy homeostasis including white adipose tissue function; however, chronic GC excess is detrimental to mammals' health. White hypertrophic adiposity is a main factor for neuroendocrine-metabolic dysfunctions in monosodium L-glutamate (MSG)-damaged hypercorticosteronemic rat. Nevertheless, little is known about the receptor path in endogenous GC impact on white adipose tissue-resident precursor cells to bring them into beige lineage. Thus, our aim was to explore whether transient/chronic endogenous hypercorticosteronemia affects browning capacity in white adipose tissue pads from MSG rats during development. Main methodsControl and MSG male rats aged 30 and 90 days were 7-day exposed to cold conditions in order to stimulate wet white epidydimal adipose tissue (wEAT) beiging capacity. This procedure was also replicated in adrenalectomized rats. Key findingsData indicated that whereas epidydimal white adipose tissue pads from prepubertal hypercorticosteronemic rats retained full expression of GR/MR genes resulting in a drastic reduction in wEAT beiging capacity, conversely, chronic hypercorticosteronemic adult MSG rats developed down-regulation of corticoid genes (and reduced GR cytosolic mediators) in wEAT pads and consequently partially restored local beiging capacity. Finally, wEAT pads from adrenalectomized rats revealed up-regulation of GR gene accompanied by full local beiging capacity. SignificanceThis study strongly supports a GR-dependent inhibitory effect of GC excess on white adipose tissue browning, an issue strongly supporting a key role of GR in the non-shivering thermogenic process. As a consequence, normalizing the GC milieu could be a relevant factor to handle dysmetabolism in white hyperadipose phenotypes.

Methylation of the glucocorticoid receptor gene (NR3C1) in dyads mother-child exposed to intimate partner violence in Cameroon: Association with anxiety symptoms.

The glucocorticoid receptor (GR), which is encoded by the NR3C1 (Nuclear Receptor Subfamily 3 Group C Member 1) gene plays an important role in the modulation of the hypothalamic-pituitary-adrenal (HPA) axis activity by providing feedback regulation which allows termination of the stress response. Little is known about epigenetic programming at the level of NGFI-A (nerve growth factor-inducible protein A) putative binding site (CpG) of the NR3C1 exon 1F in dyads mother-child exposed to intimate partner violence (IPV) more specifically in an unstudied region such as the sub-Saharan Africa where levels of violence are very high. Examine NR3C1 exon 1F methylation in response to IPV and possible association with cortisol concentration and mental health. We recruited 20 mother-child dyads exposed to IPV and a control group of 20 mother-child dyads not exposed to IPV. We administered self-reported questionnaires to measure mother's mental health and collected saliva samples for cortisol dosage and bisulfite sequencing of DNA methylation. Regarding the mothers, our results showed a significant difference in methylation level at CpG 16-21 sites of the NR3C1 exon 1F promoter region between the groups. In the exposed group as compared to the control group, there was a significant positive association between the level of methylation at CpG 16-21 sites and mother's mental health in particular anxiety symptoms. However, we did not find any significant correlation between methylation level and cortisol concentration. In children, we did not find any significant results. This study highlights a NGFI-A putative binding site (CpG 16-21) that is more methylated in mothers exposed to IPV and which may have the potential to confer vulnerability for psychopathologies.

Maternal sensitivity and child internalizing and externalizing behavior: a mediating role for glucocorticoid receptor gene (NR3C1) methylation?

The early caregiving environment can have lasting effects on child mental health. Animal models suggest that glucocorticoid receptor gene (NR3C1) DNA methylation plays a mediating role in linking more responsive caregiving to improved behavioral outcomes by its impact on the stress regulatory system. In this longitudinal study, we examined whether children's NR3C1 methylation levels mediate an effect of maternal sensitivity in infancy on levels of child internalizing and externalizing behavior in a community sample. Maternal sensitivity of 145 mothers was rated at infant age 5 weeks, 12 months, and 30 months by observing mother-infant interactions. Buccal DNA methylation was assessed in the same children at age 6 years and maternal-reported internalizing and externalizing behavior was assessed at age 6 and 10 years. Higher sensitivity at age 5 weeks significantly predicted lower DNA methylation levels at two NR3C1 CpG loci, although methylation levels at these loci did not mediate an effect of maternal sensitivity on levels of child internalizing and externalizing behavior. Overall, the study provides evidence that maternal sensitivity in early infancy is associated with DNA methylation levels at loci involved in stress regulation, but the significance of this finding for child mental health remains unclear.

Is chronic stress a causal mechanism for small mammal population cycles? Reconciling the evidence.

Chronic stress has long been hypothesized to play a role in driving population cycles. Christian (1950) hypothesized that high population density results in chronic stress and mass "die-offs" in small mammal populations. Updated variations of this hypothesis propose that chronic stress at high population density may reduce fitness, reproduction, or program aspects of phenotype, driving population declines. We tested the effect of density on the stress axis in meadow voles (Microtus pennsylvanicus) by manipulating population density in field enclosures over three years. Using fecal corticosterone metabolites as a non-invasive measure of glucocorticoid (GC) concentrations, we found that density alone was not associated with GC differences. However, we found that the seasonal relationship of GC levels differed by density treatment, with high-density populations having elevated GC levels early in the breeding season and decreasing towards late summer. We additionally tested hippocampal glucocorticoid receptor and mineralocorticoid receptor gene expression in juvenile voles born at different densities, with the hypothesis that high density may reduce receptor expression, altering negative feedback of the stress axis. We found that females had marginally higher glucocorticoid receptor expression at high density, no effect in males, and no detectable effect of density on mineralocorticoid receptor expression in either sex. Hence, we found no evidence that high density directly impairs negative feedback in the hippocampus, butrather female offspring may be better equipped for negative feedback. We compare our findings with prior studies to attempt to disentangle the complicated relationship between density, seasonality, sex, reproduction and the stress axis.

Sex differences in hippocampal structural plasticity and glycosaminoglycan disaccharide levels after neonatal handling.

In this study we investigated the effects of a neonatal handling protocol that mimics the handling of sham control pups in protocols of neonatal exposure to brain insults on dendritic arborization and glycosaminoglycan (GAG) levels in the developing brain. GAGs are long, unbranched polysaccharides, consisting of repeating disaccharide units that can be modified by sulfation at specific sites and are involved in modulating neuronal plasticity during brain development. In this study, male and female Sprague-Dawley rats underwent neonatal handling daily between post-natal day (PD)4 and PD9, with brains analyzed on PD9. Neuronal morphology and morphometric analysis of the apical and basal dendritic trees of CA1 hippocampal pyramidal neurons were carried out by Golgi-Cox staining followed by neuron tracing and analysis with the software Neurolucida. Chondroitin sulfate (CS)-, Hyaluronic Acid (HA)-, and Heparan Sulfate (HS)-GAG disaccharide levels were quantified in the hippocampus by Liquid Chromatography/Mass Spectrometry analyses. We found sex by neonatal handling interactions on several parameters of CA1 pyramidal neuron morphology and in the levels of HS-GAGs, with females, but not males, showing an increase in both dendritic arborization and HS-GAG levels. We also observed increased expression of glucocorticoid receptor gene Nr3c1 in the hippocampus of both males and females following neonatal handling suggesting that both sexes experienced a similar stress during the handling procedure. This is the first study to show sex differences in two parameters of brain plasticity, CA1 neuron morphology and HS-GAG levels, following handling stress in neonatal rats.

Methylation and expression of glucocorticoid receptor exon-1 variants and FKBP5 in teenage suicide-completers

A dysregulated hypothalamic-pituitary-adrenal (HPA) axis has repeatedly been demonstrated to play a fundamental role in psychiatric disorders and suicide, yet the mechanisms underlying this dysregulation are not clear. Decreased expression of the glucocorticoid receptor (GR) gene, which is also susceptible to epigenetic modulation, is a strong indicator of impaired HPA axis control. In the context of teenage suicide-completers, we have systematically analyzed the 5’UTR of the GR gene to determine the expression levels of all GR exon-1 transcript variants and their epigenetic state. We also measured the expression and the epigenetic state of the FK506-binding protein 51 (FKBP5/FKBP51), an important modulator of GR activity. Furthermore, steady-state DNA methylation levels depend upon the interplay between enzymes that promote DNA methylation and demethylation activities, thus we analyzed DNA methyltransferases (DNMTs), ten-eleven translocation enzymes (TETs), and growth arrest- and DNA-damage-inducible proteins (GADD45). Focusing on both the prefrontal cortex (PFC) and hippocampus, our results show decreased expression in specific GR exon-1 variants and a strong correlation of DNA methylation changes with gene expression in the PFC. FKBP5 expression is also increased in both areas suggesting a decreased GR sensitivity to cortisol binding. We also identified aberrant expression of DNA methylating and demethylating enzymes in both brain regions. These findings enhance our understanding of the complex transcriptional regulation of GR, providing evidence of epigenetically mediated reprogramming of the GR gene, which could lead to possible epigenetic influences that result in lasting modifications underlying an individual’s overall HPA axis response and resilience to stress.

Pilot study suggests DNA methylation of the glucocorticoid receptor gene (NR3C1) is associated with MDMA-assisted therapy treatment response for severe PTSD.

Previous research has demonstrated that epigenetic changes in specific hypothalamic-pituitary-adrenal (HPA) genes may predict successful psychotherapy in post-traumatic stress disorder (PTSD). A recent Phase 3 clinical trial reported high efficacy of 3,4-methylenedioxymethamphetamine (MDMA)-assisted therapy for treating patients with severe PTSD compared to a therapy with placebo group (NCT03537014). This raises important questions regarding potential mechanisms of MDMA-assisted therapy. In the present study, we examined epigenetic changes in three key HPA axis genes before and after MDMA and placebo with therapy. As a pilot sub-study to the parent clinical trial, we assessed potential HPA epigenetic predictors for treatment response with genomic DNA derived from saliva (MDMA, n = 16; placebo, n = 7). Methylation levels at all 259 CpG sites annotated to three HPA genes (CRHR1, FKBP5, and NR3C1) were assessed in relation to treatment response as measured by the Clinician-Administered PTSD Scale (CAPS-5; Total Severity Score). Second, group (MDMA vs. placebo) differences in methylation change were assessed for sites that predicted treatment response. Methylation change across groups significantly predicted symptom reduction on 37 of 259 CpG sites tested, with two sites surviving false discovery rate (FDR) correction. Further, the MDMA-treatment group showed more methylation change compared to placebo on one site of the NR3C1 gene. The findings of this study suggest that therapy-related PTSD symptom improvements may be related to DNA methylation changes in HPA genes and such changes may be greater in those receiving MDMA-assisted therapy. These findings can be used to generate hypothesis driven analyses for future studies with larger cohorts.

Mild daily stress, in interaction with NR3C1 DNA methylation levels, is linked to alterations in the HPA axis and ANS response to acute stress in early adolescents

BackgroundDaily Hassles (DH) or daily stress - is a mild type of stressor with unique contributions to psychological distress. Yet, most prior studies that investigate the effects of stressful life experiences focus on childhood trauma or on early life stress and little is known about the effects of DH on epigenetic changes in stress system related genes and on the physiological response to social stressors. MethodsIn the present study, conducted among 101 early adolescents (mean age = 11.61; SD = 0.64), we investigated whether Autonomic Nervous System (ANS) (namely heart rate and heart rate variability) and Hypothalamic-Pituitary-Adrenal (HPA) axis functioning (measured as cortisol stress reactivity and recovery) are associated with DNA methylation (DNAm) in the glucocorticoid receptor gene (NR3C1), the level of DH and their interaction. To assess the stress system functioning the TSST protocol was used. ResultsOur findings show that higher NR3C1 DNAm in interaction with higher levels of daily hassles, is associated with blunted HPA axis reactivity to psychosocial stress. In addition, higher levels of DH are associated with extended HPA axis stress recovery. In addition, participants with higher NR3C1 DNAm had lower ANS adaptability to stress, specifically lower parasympathetic withdrawal; for heart rate variability this effect was strongest for participants with higher level of DH. ConclusionsThe observation that interaction effects between NR3C1 DNAm levels and daily stress on the functioning of the stress-systems, are already detectable in young adolescents, highlights the importance of early interventions, not only in the case of trauma, but also daily stress. This might help to prevent stress-induced mental and physical disorders later in life.

Covariation between glucocorticoid levels and receptor expression modulates embryo development and postnatal phenotypes in gulls

The hypothalamic-pituitary-adrenocortical axis can translate, through glucocorticoid secretion, the prenatal environment to development to produce phenotypes that match prevailing environmental conditions. However, whether developmental plasticity is modulated by the interaction between circulating glucocorticoids and receptor expression remains unclear. Here, we tested whether covariation between plasma corticosterone (CORT) and glucocorticoid receptor gene (Nr3c1) expression in blood underlies embryonic developmental programming in yellow-legged gulls (Larus michahellis). We examined variations in circulating levels of CORT and the expression and DNA methylation patterns of Nr3c1 in response to two ecologically relevant prenatal factors: adult alarm calls (a cue of predator presence) and changes in prenatal light environment (a cue of competitive disadvantage). We then determined whether embryonic development and postnatal phenotypes were associated with CORT levels and Nr3c1 expression, and explored direct and indirect relationships between the prenatal environment, hormone-receptor covariation, and postnatal phenotypes. Prenatal exposure to alarm calls increased CORT levels and up-regulated Nr3c1 expression in gull chicks, while exposure to light cues reduced both hormone levels and receptor expression. Chicks prenatally exposed to alarm calls showed altered DNA methylation profiles in the Nr3c1 regulatory region, but patterns varied throughout the breeding season and between years. Moreover, our results suggest a negative relationship between DNA methylation and expression in Nr3c1 , at least at specific CpG sites. The interplay between circulating CORT and Nr3c1 expression affected embryo developmental timing and vocalizations, as well as hatchling mass and fitness-relevant behaviours. These findings provide a link between prenatal inputs, glucocorticoid function and phenotypic outcomes, suggesting that hormone-receptor interaction may underlie developmental programming in free-living animals.