Comparing DRD2 Promoter Methylation Between Blood and Brain in Alcohol Dependence.

AIMS: The dopamine receptor D2 (DRD2) is substantially involved in several forms of addiction. In addition to genetic polymorphisms, epigenetic mechanisms have emerged as an important means of regulation. Previously, DRD2 hypo- and hyper-methylation have been observed in alcohol use disorder (AUD). Blood samples are commonly used as a surrogate marker of epigenetic alterations in epigenetic research, but few specific comparisons between blood and brain tissue samples in AUD exist. METHODS: We used post-mortem brain tissue samples of 17 deceased patients with AUD and 31 deceased controls to investigate the relationship between blood and brain methylation of the DRD2 promoter. RESULTS: When investigating individual cytosine methylation sites (CpG), several significant differences were found in the nucleus accumbens and hippocampus in the study population. Investigating binding sites with significant differences in methylation levels revealed hypomethylated CpGs targeting mainly activating transcription factors. CONCLUSION: These findings support an altered transcription of the DRD2 gene in AUD specimens with a consecutively changed reward response in the brain. While methylation between specific brain regions and blood is comparable, our study further suggests that blood methylation cannot provide meaningful perspectives on DRD2 promoter methylation in the brain.

Epigenetic Regulation of Neural Transmission after Cerebellar Fastigial Nucleus Lesions in Juvenile Rats.

Structural and functional abnormalities in the cerebellar midline region, including the fastigial nucleus, have been reported in neuropsychiatric disorders, also comprising the cerebellar cognitive affecting syndrome. In rats, early fastigial lesions reduce social interaction during development and lead to cognitive and emotional deficits in adults, accompanied by compromised neuronal network activity. Since epigenetic mechanisms are implicated in the etiology of neuropsychiatric disorders, we investigated whether fastigial nucleus lesions in juvenile rats would impact epigenetic regulation of neural transmission. The fastigial nucleus was lesioned bilaterally in 23-day-old male rats. Sham-lesion and naïve rats served as controls. DNA methylation was investigated for target genes of the GABAergic, dopaminergic, glutamatergic and oxytocinergic systems in brain regions with anatomic connections to the fastigial nucleus, i.e., medial prefrontal cortex, nucleus accumbens, striatum, thalamus, and sensorimotor cortex. Protein expression was examined for the respective target genes in case of altered DNA methylation between lesion and control groups. Lesioning of the fastigial nucleus led to significant differences in the epigenetic regulation of glutamate decarboxylase 1 and the oxytocin receptor in the nucleus accumbens and the prefrontal cortex. No differences were found for the other target genes and brain regions. Our findings indicate that epigenetic dysregulation after lesioning of the fastigial nucleus may influence long-term recovery and the emergence of behavioral changes. Together with previous behavioral and electrophysiological investigations of this rat model, these observations can play a role in the cerebellar cognitive affective syndrome and other neuropsychiatric disorders.

Epigenetic Effects of Intravenous Diacetylmorphine on the Methylation of POMC and NR3C1.

The administration of diacetylmorphine (DAM) reduces the activation of the hypothalamic-pituitary-adrenal (HPA) axis in opioid-maintained patients. However, the epigenetic effects of DAM on addiction-related genes have not been investigated yet. In a randomized controlled study, we examined the immediate effects of intravenous DAM versus placebo on the promoter methylation of the POMC (pro- opiomelanocortin) and NR3C1 (glucocorticoid receptor 1) genes. Twenty-eight heroin-dependent patients on DAM-assisted treatment received either DAM or saline in a randomized crossover design and 17 healthy participants received saline only. EDTA blood samples were taken 25 min before and 10 min after the injection of DAM or saline. We found reciprocal regulation effects for DAM versus saline application regarding the methylation of POMC; while DAM injection significantly increased methylation, saline injection led to a significant decrease in methylation for patients as well as controls. NR3C1 data did not show significant changes in methylation. Injection of DAM blunted stress hormone levels and the POMC promoter methylation of heroin-dependent patients. These findings provide first preliminary insights into the epigenetic mechanisms underlying the emotional regulation effects of DAM-assisted treatment in severe heroin-dependent patients.

DNA methylation of the LEP gene is associated with craving during alcohol withdrawal.

Different studies have described evidence for an association between leptin serum levels and craving in alcohol dependent patients. As leptin expression is regulated by DNA methylation we investigated changes of DNA methylation of the LEP gene promoter region in alcohol dependent patients undergoing withdrawal. Results show that low methylation status is associated with increasing serum leptin levels and elevation of craving for alcohol in the referring patients group. These findings point towards a pathophysiological relevance of changes in DNA methylation of the LEP gene promoter region in alcohol dependence.

Alterations of homocysteine serum levels during alcohol withdrawal are influenced by folate and riboflavin: results from the German Investigation on Neurobiology in Alcoholism (GINA).

AIMS: Various studies have shown that plasma homocysteine (HCY) serum levels are elevated in actively drinking alcohol-dependent patients a during alcohol withdrawal, while rapidly declining during abstinence. Hyperhomocysteinemia has been associated not only with blood alcohol concentration (BAC), but also with deficiency of different B-vitamins, particularly folate, pyridoxine and cobalamin. METHODS: Our study included 168 inpatients (110 men, 58 women) after admission for detoxification treatment. BAC, folate, cobalamin, pyridoxine, thiamine and riboflavin were obtained on admission (Day 1). HCY was assessed on Days 1, 7 and 11. RESULTS: HCY levels significantly declined during withdrawal. General linear models and linear regression analysis showed an influence of BAC, folate and riboflavin on the HCY levels on admission as well as on HCY changes occurring during alcohol withdrawal. No significant influence was found for thiamine, cobalamin and pyridoxine. CONCLUSIONS: These findings show that not only BAC and plasma folate levels, but also plasma levels of riboflavin influence HCY plasma levels in alcohol-dependent patients.

Promoter specific DNA methylation and gene expression of POMC in acutely underweight and recovered patients with anorexia nervosa.

Proopiomelanocortin (POMC) and its derived peptides, in particular alpha-MSH, have been shown to play a crucial role in the regulation of hunger, satiety and energy homeostasis. Studies in patients with anorexia nervosa (AN) suggest an abnormal expression of appetite-regulating hormones. Hormone expression levels may be modulated by epigenetic mechanisms, which were recently shown to be implicated in the pathophysiology of eating disorders. We hypothesised that POMC promoter specific DNA methylation and gene expression will be affected by malnutrition and therefore differ in AN patients at distinct stages of the disorder. Promoter specific DNA methylation of the POMC gene and expression of POMC mRNA variants were determined in peripheral blood mononuclear cells (PBMC) of 30 healthy control women (HCW), 31 underweight (acAN) and 30 weight-recovered patients with AN (recAN). Malnutrition was characterized by plasma leptin. Expression of the functionally relevant long POMC mRNA transcript was significantly correlated with leptin levels and higher in acAN compared to recAN and HCW. Expression of the truncated form and mean promoter DNA methylation was similar in all three subgroups. Methylation of single CpG residues in the E2F binding site was inversely related to POMC expression. Our preliminary data on pattern of POMC regulation suggests an association with the underweight state rather than with persisting trait markers of AN. In contrast to POMC expression in the central nervous system, peripheral POMC mRNA expression decreased with malnutrition and hypoleptinemia. This may represent a counterregulatory mechanism as part of the crosstalk between the immune and neuroendocrine systems.

Epigenetic regulation and gene expression of vasopressin and atrial natriuretic peptide in alcohol withdrawal.

Disturbances of volume regulating peptides like vasopressin and atrial natriuretic peptide (ANP) have been described in early abstinent patients. Aim of the present study was to evaluate possible alterations of the promoter-related DNA methylation of the ANP and vasopressin precursor genes and the related mRNA-expression of these genes in early alcohol withdrawal. We analyzed blood samples of 57 healthy controls and of 111 patients suffering from alcohol dependence that were admitted for detoxification treatment. Promoter-related DNA methylation and mRNA-expression of vasopressin and ANP genes were assessed using real-time PCR. Vasopressin mRNA-expression was not statistically different between patients and controls. However, we found a significantly elevated promoter-related DNA methylation of the vasopressin gene in patients with alcohol dependence (Mann-Whitney U-test: Z=-2.178, p=0.029). ANP mRNA-expression was significantly elevated in alcoholic patients (Z=-6.240, p<0.001) while promoter-related DNA methylation of ANP was significantly decreased (Z=-2.282, p=0.023). Furthermore, promoter-related DNA methylation of ANP was significantly correlated to the extent of craving measured with the OCDS (r=-0.197, p=0.040). The findings of the present study show significant alterations of the mRNA-expression and promoter-related DNA methylation of vasopressin and especially ANP precursor genes in patients with alcohol dependence. Further studies focusing on longitudinal changes of epigenetic regulation and gene expression of both peptides are needed to clarify the pathophysiological role of these findings.

Global DNA methylation is influenced by smoking behaviour.

The level of epigenetic DNA methylation is an important factor in the pathogenesis of various human diseases. As smoking may influence DNA methylation, we investigated the effect of smoking habits on global DNA methylation in 298 genomic DNA samples (73 fathers, 69 mothers and 156 offspring). We did not find a direct effect of smoking on global DNA methylation. However, there was an association of the offspring's DNA methylation with paternal DNA methylation that was strongest if both had never smoked (R2corr=0.41, Beta=0.68, p=0.02) and completely vanished if the offspring smoked or had ever smoked. These findings suggest an association between smoking behaviour and global DNA methylation, which may be of importance for a wide range of diseases.