Xiaoju Yang

The Mu-Opioid Receptor Polymorphism A118G Predicts Cortisol Responses to Naloxone and Stress

A polymorphism in the mu-opioid receptor (MOR) (A118G) has been shown to increase β-endorphin binding affinity, theoretically placing greater inhibitory tone on hypothalamic corticotropin-releasing hormone (CRH) neurons. We hypothesized that the minor allele (G) would predict cortisol responses to both pharmacological (naloxone) and psychological (stress) activation of the hypothalamic–pituitary–adrenal (HPA) axis. Healthy subjects (mean age 25.2 years, SD 9.2 years) completed a naloxone challenge (n=74) and/or the modified Trier Social Stress Test (TSST) (n=86). For the naloxone challenge, two baseline blood samples were obtained. Then, five increasing doses of i.v. naloxone were administered at 30-min intervals and 12 additional blood samples were collected at 15-min intervals. The TSST consisted of 5-min of public speaking and 5-min of mental arithmetic exercises. Three baseline and five post-TSST blood samples were drawn. Both the naloxone and TSST groups had significant adrenocorticotropin (ACTH) and cortisol responses to their respective challenges (P<0.001). There were no differences in baseline ACTH, baseline cortisol, or ACTH response by genotype in either the naloxone or the TSST group. Among subjects expressing a G allele, there was a higher cortisol response to naloxone (P=0.046), but a lower cortisol response to the TSST (P=0.044). In conclusion, the minor allele (G) was associated with a robust cortisol response to naloxone blockade, but a blunted response to psychosocial stress. We speculate that increased opioid avidity of the minor allele receptor contributes to the differential response to naloxone vs stress.

Glucocorticoid-induced loss of DNA methylation in non-neuronal cells and potential involvement of DNMT1 in epigenetic regulation of Fkbp5

Glucocorticoids may play a significant role in the etiology of neuropsychiatric illnesses. Abnormalities in plasma cortisol levels, glucocorticoid sensitivity, and HPA-axis function often accompany clinical symptoms of stress-related illnesses such as PTSD and depression. Of particular interest are genetic association studies that link single nucleotide polymorphisms of HPA-axis genes with illnesses only in the context of an early-life trauma exposure such as child abuse. These studies suggest that dysregulation of HPA-axis function can have lasting repercussions in shaping mood and anxiety, long after termination of the traumatic experience. As persistent glucocorticoid-induced loss of DNA methylation in FK506 binding protein 5 (Fkbp5) was previously observed in the hippocampus and blood and in the neuronal cell line HT-22, we asked whether these epigenetic alterations occur in non-neuronal, HPA-axis relevant cells. We used the pituitary adenoma cell line AtT-20 to demonstrate that the intronic enhancer region of Fkbp5 undergoes loss of DNA methylation in response to dexamethasone treatment in a dose-dependent manner. We also focused on the mouse hippocampal dentate gyrus to test whether these changes would be enriched in a region implicated in the HPA-axis stress response, neurogenesis, and synaptic plasticity. We observed an increase in enrichment of DNA methylation loss in the dentate gyrus, as compared to whole hippocampal tissues that were similarly treated with glucocorticoids. We then asked whether DNA methyltransferase 1 (Dnmt1), a methyltransferase enzyme involved in maintaining DNA methylation following cell division, is involved in the observed epigenetic alterations. We found a dose-dependent decrease of Dnmt1 expression in the AtT-20 cells following dexamethasone treatment, and a similar decrease in corticosterone-treated mouse hippocampus. Taken together, we provide evidence that these glucocorticoid-induced epigenetic alterations have a broader validity in non-neuronal cells and that they may involve the DNA methylation machinery.

Chronic ethanol administration decreases phosphorylation of cyclic AMP response element-binding protein in granule cells of rat cerebellum

Abstract: To help define the molecular basis of ethanols actions on the nervous system, we have in previous studies demonstrated that ethanol administration triggers a robust increase in cyclic AMP‐response element‐binding protein (CREB) phosphorylation in the cerebellum. The purpose of the present study was to compare the effects of acute and chronic ethanol exposure on the phosphorylation of CREB in rat cerebellum and to determine which cell types in the cerebellum display this response to ethanol. An acute ethanol challenge (3.0 g/kg of body weight) induced a rapid increase in content of the phosphorylated form of CREB, peaking at 30 min and declining to basal levels within 2 h. Immunocytochemical studies revealed prominent ethanol‐induced changes in phosphoCREB in the granule cell layer, with little phosphoCREB apparent in Purkinje cells. Following chronic ethanol exposure (5 weeks), induction of CREB phosphorylation by a subsequent acute ethanol challenge was markedly attenuated. The attenuation in CREB phosphorylation was associated with a significant reduction in the levels of the catalytic unit of protein kinase A and calcium/calmodulin‐dependent protein kinase IV. In summary, induction of CREB phosphorylation in cerebellum is most prominent in the granule cell layer. Neuroadaptation to chronic ethanol exposure includes a reduction in nuclear protein kinase A and calcium/calmodulin‐dependent protein kinase IV levels, an event associated with impaired CREB phosphorylation.

Influence of OPRM1 Asn40Asp variant (A118G) on [11C]carfentanil binding potential: preliminary findings in human subjects

The Asn40Asp variant (A118G) of the μ opioid receptor (OPRM1) gene is thought to contribute to the development and treatment of alcohol dependence. Employing positron emission tomography (PET), we first examined whether the single nucleotide polymorphism (SNP) modifies binding potential (BP(ND)) of the μ-selective ligand [(11)C]carfentanil in healthy control (Con) and 5-d abstinent alcohol-dependent (AD) subjects (unblocked basal scan). Second, we examined whether the allelic variants were associated with differences in OPRM1 occupancy by naltrexone (50 mg) in AD subjects. Con and AD carriers of the G allele (AG) had lower global BP(ND) at the basal scan than subjects homozygous for the A allele (AA). In AD subjects, naltrexone occupancy was slightly higher in AG subjects (98.9%) compared to AA subjects (93.1%), but this was not significant. We are the first to demonstrate using PET in healthy normal and AD subjects that the A118G SNP alters OPRM1 availability.

Restraint Stress and Ethanol Consumption in Two Mouse Strains

BACKGROUND This study examined the interaction between restraint stress and ethanol drinking in mice that consume low and high amounts of ethanol. METHODS Two strains of mice (129SVEV and C57BL/6J) underwent 1 hour of restraint stress twice per day for 4 days in the presence of a CRF-1 receptor antagonist, a glucocorticoid receptor antagonist or vehicle. Ethanol preference and consumption were assessed using a two bottle choice design. In another study, mice were implanted with pellets containing corticosterone; ethanol preference and consumption were assessed using a two bottle choice design. RESULTS Restraint stress significantly increased ethanol preference and consumption in 129SVEV mice but not in C57BL/6J mice. Then 129SVEV mice underwent the identical stress procedure; however, mice received either the CRF-1 receptor antagonist, R121919 (15 or 20 mg/kg, ip) or vehicle 30 minutes prior to stress. R121919 did not block the stress-induced change in ethanol preference despite causing a significant blunting in the HPA axis. Negative results were also obtained using the CRF-1 receptor antagonist, Antalarmin (20 mg/kg, ip). In another study, 129SVEV mice were administered either the glucocorticoid receptor antagonist Mifepristone (25, 50 or 100 mug/kg, ip) or vehicle under the same procedure. Mifepristone did not alter ethanol preference. Moreover, the three receptor antagonist did not alter nonstress ethanol consumption either. In the last study, both mouse strains underwent active or sham adrenalectomy, then pellets containing corticosterone or placebo were implanted and preference for ethanol versus water was tested. Corticosterone administration decreased ethanol consumption in a strain-dependent manner. CONCLUSION These data show the restraint model for stress can modestly increase ethanol consumption in 129SVEV mice but not in C57BL/6J mice. Pharmacologic manipulation of CRF and corticosterone did not blunt baseline or stress-induced change in ethanol preference nor did administration of corticosterone mimic the effects of restraint stress on ethanol consumption. These findings suggest the mechanism responsible for increasing ethanol consumption in this model is independent of the HPA axis and extra-hypothalamic CRF.

Genome-wide Methyl-Seq analysis of blood-brain targets of glucocorticoid exposure

ABSTRACT Chronic exposure to glucocorticoids (GCs) can lead to psychiatric complications through epigenetic mechanisms such as DNA methylation (DNAm). We sought to determine whether epigenetic changes in a peripheral tissue can serve as a surrogate for those in a relatively inaccessible tissue such as the brain. DNA extracted from the hippocampus and blood of mice treated with GCs or vehicle solution was assayed using a genome-wide DNAm platform (Methyl-Seq) to identify differentially methylated regions (DMRs) induced by GC treatment. We observed that ∼70% of the DMRs in both tissues lost methylation following GC treatment. Of the 3,095 DMRs that mapped to the same genes in both tissues, 1,853 DMRs underwent DNAm changes in the same direction. Interestingly, only 209 DMRs (<7%) overlapped in genomic coordinates between the 2 tissues, suggesting tissue-specific differences in GC-targeted loci. Pathway analysis showed that the DMR-associated genes were members of pathways involved in metabolism, immune function, and neurodevelopment. Also, changes in cell type composition of blood and brain were examined by fluorescence-activated cell sorting. Separation of the cortex into neuronal and non-neuronal fractions and the leukocytes into T-cells, B-cells, and neutrophils showed that GC-induced methylation changes primarily occurred in neurons and T-cells, with the blood tissue also undergoing a shift in the proportion of constituent cell types while the proportion of neurons and glia in the brain remained stable. From the current pilot study, we found that despite tissue-specific epigenetic changes and cellular heterogeneity, blood can serve as a surrogate for GC-induced changes in the brain.

Independent and interactive effects of OPRM1 and DAT1 polymorphisms on alcohol consumption and subjective responses in social drinkers

BACKGROUND The current study examined independent and interactive effects of polymorphisms of the mu opioid receptor gene (OPRM1, A118G) and variable number tandem repeats of the dopamine transporter gene (DAT1, SLC6A3) on alcohol consumption and subjective responses to alcohol in 127 young, healthy, social drinkers. METHODS Participants completed an in-person assessment, which included self-reported alcohol drinking patterns and blood sampling for DNA, and in a second visit, a cumulative alcohol dosing procedure with subjective ratings across multiple time points and breath alcohol contents (0.03 to 0.1%). DNA was analyzed for OPRM1 AA versus AG/GG (*G) genotypes, DAT1 10-repeat allele (A10) versus 9 or lesser alleles (A9), and ancestral informative markers. RESULTS There were significant epistatic interactions between OPRM1 and DAT1 genotypes. Subjective High Assessment Scale scores after alcohol consumption were highest in *G and A9 carriers, and lowest in *G and A10 carriers. Negative subjective effects were also highest in *G and A9 carriers. Effects were similar in a sensitivity analysis limited to Caucasian subjects. There were independent and epistatic interactions on drinking. The OPRM1 *G allele was independently associated with fewer heavy drinking days. The A9 allele was associated with a greater number of drinking days, which was attenuated in carriers of the *G allele. CONCLUSIONS These findings highlight the biological importance of interactions between these 2 genes and interactions between brain opioid and dopamine systems.