Stefan Bleich

DNA hypermethylation of the alpha synuclein promoter in patients with alcoholism

The aim of this study was to investigate whether the DNA methylation pattern within the alpha synuclein promoter region is altered in intoxicated and early abstinence patients with alcoholism undergoing alcohol withdrawal. We observed a significant increase of the alpha synuclein promoter DNA methylation in patients with alcoholism which was significantly associated with their elevated homocysteine levels. No significant differences of the promoter DNA methylation within a control gene (presenilin-1) in alcoholics and controls were found. The present results hint to a gene specific DNA promoter hypermethylation within the alpha synuclein gene. Since hypermethylation of DNA is an important epigenetic factor in the down regulation of gene expression and since alpha synuclein has been linked to craving these findings may explain the reduced value of craving under alcohol drinking conditions.

Homocysteine associated genomic DNA hypermethylation in patients with chronic alcoholism.

Summary.Higher plasma homocysteine concentrations can influence genomic DNA methylation in peripheral blood cells. In the present controlled study we observed a significant increase (10%) of genomic DNA methylation in patients with alcoholism (t = −3.16, df = 158, p = 0.002) which was significantly associated with their elevated homocysteine levels (multiple linear regression, p < 0.001). Since methylation of DNA is an important epigenetic factor in regulation of gene expression these findings may have important implications for a possible subsequent derangement of epigenetic control these patients.

Lowered DNA methyltransferase (DNMT-3b) mRNA expression is associated with genomic DNA hypermethylation in patients with chronic alcoholism

Summary.DNA methyltransferases (DNMTs) are involved within the epigenetic control of DNA methylation processes. Recently, it has been shown that the genomic DNA methylation in patients with alcoholism is increased. In the present controlled study we observed a significant decrease of mRNA expression of DNMT-3a and DNMT-3b when comparing alcoholic patients (n = 59) with healthy controls (n = 66): DNMT-3a (t = −2.38, p = 0.019), DNMT-3b (t = −2.65, p = 0.008). No significant differences were seen for DNMT-1 and Mbd-2 (Methyl-CpG-Binding-Domain protein 2) expression. Additionally, we observed a significant negative correlation between DNMT-3b expression and the blood alcohol concentration (r = −0.45, p = 0.003) which might explain the decrease of DNMT-3b mRNA expression in alcoholic patients. Using a multivariate model we observed that the increase (10%) of genomic DNA methylation in patients with alcoholism was significantly associated with their lowered DNMT-3b mRNA expression (multiple linear regression, p = 0.014). Since methylation of DNA is an important epigenetic factor in regulation of gene expression these findings may have important implications for a possible subsequent derangement of epigenetic control in these patients.

Epigenetic dysregulation of dopaminergic genes in eating disorders

OBJECTIVE The pathophysiology of eating disorders such as anorexia nervosa (AN) and bulimia nervosa (BN) has been linked to an impaired dopaminergic neurotransmission, still the origin of this disturbance remains unknown. The aim of the present study was, therefore, to evaluate whether the expression of dopaminergic genes is altered in the blood of patients suffering from eating disorders and if these alterations can be explained by changes in the promoter specific DNA methylation of the genes. METHOD We used quantitative real-time PCR to measure both the expression and the promoter specific DNA methylation of the dopamine transporter (DAT), and the D2 (DRD2) and D4 receptor (DRD4) gene in the blood of 46 patients (22 AN, 24 BN) and 30 healthy controls. RESULTS Patients showed an elevated expression of DAT mRNA when compared with the controls and a downregulation of the DRD₂ expression. The upregulation of the DAT gene was accompanied by a hypermethylation of the genes promoter in the AN and BN group while a significant hypermethylation of the DRD₂ promoter was only present in the AN group. No differences in expression or methylation were found for the other dopamine receptors investigated. DISCUSSION Our study shows a disturbed expression of dopaminergic genes that is accompanied by a dysregulation of the epigenetic DNA methylation. Further studies are necessary to provide more insight into the epigenetic dysregulation of the dopaminergic neurotransmission in the pathophysiology of eating disorders.

Hyperhomocysteinemia as a new risk factor for brain shrinkage in patients with alcoholism.

Chronic alcohol consumption can induce brain atrophy, whereby the exact mechanism of brain damage in alcoholics remains unknown. There is evidence that chronic alcoholism is associated with hyperhomocysteinemia. Homocysteine is an excitatory amino acid which markedly enhances the vulnerability of neuronal cells to excitotoxic and oxidative injury in vitro and in vivo. The present volumetric magnetic resonance imaging study included 52 chronic alcoholics and 30 non-drinking healthy controls. Patients were active drinkers and had an established diagnosis of alcohol dependence. We investigated the influence of different variables on the hippocampal volume of patients suffering from chronic alcoholism. We observed that pathological raised levels of plasma homocysteine showed the most significant correlation to hippocampal volume reduction (P<0.001, multiple regression analysis). Raised plasma levels of homocysteine are associated with hippocampal (brain) atrophy in alcoholism.

Flupirtine shows functional NMDA receptor antagonism by enhancing Mg2+ block via activation of voltage independent potassium channels

Summary. The spectrum of action of flupirtine includes analgesia, muscle relaxation and neuroprotection. N-methyl-D-aspartate (NMDA) receptor antagonism has been discussed as a possible mechanism of action of this compound with little direct evidence. The objective of the present study was to develop a plausible model to explain flupirtines spectrum of action. A four-stage strategy was selected for this purpose: Firstly, the serum concentration of flupirtine under therapeutic conditions was determined on the basis of the current literature. The second stage involved assessing the known in-vitro effects in light of the therapeutic active concentration. Using whole cell patch clamp recordings from cultured rat superior colliculus neurones interactions between flupirtine and NMDA receptors were assessed. Only very high concentrations of flupirtine antagonized inward currents to NMDA (200 μM) at −70 mV with an lC50 against steady-state responses of 182.1 ± 12.1 μM. The effects of flupirtine were voltage-independent and not associated with receptor desensitization making actions within the NMDA receptor channel or at the glycine modulatory site unlikely. NMDA receptor antagonism probably has little relevance for the clinical efficacy of flupirtine as the concentrations needed were far higher than those achieved in clinical practice. However, the activation of a G-protein-regulated inwardly rectifying K+ channel was identified as an interesting molecular target site of flupirtine. In the next stage, the central nervous spectrum of action of experimental K+ channel openers (PCO) was considered. As far as they have been studied, experimental K+ channel openers display a spectrum of action comparable to that of flupirtine. In the final stage, a global model was developed in which flupirtine stabilizes the resting membrane potential by activating inwardly rectifying K+ channels, thus indirectly inhibiting the activation of NMDA receptors. The model presented here reconciles the known functional NMDA receptor antagonism of flupirtine with the activation of K+ channels that occurs at therapeutic concentrations, thus providing an understanding of flupirtines spectrum of action. This makes flupirtine the prototype of a clinically applicable substance group with analgesic, muscle-relaxant and neuroprotective properties.

Evidence of Increased Homocysteine Levels in Alcoholism: The Franconian Alcoholism Research Studies (FARS)

BACKGROUND A limited number of investigations have studied clearly defined patients with alcoholism and blood alcohol concentrations with their correlation to plasma homocysteine values and differentiated actively drinking patients from those with early abstinence. Therefore, this power analysis-based study was undertaken to determine whether plasma homocysteine levels are evidently altered in actively drinking alcoholic patients and patients with early abstinence. METHODS Two groups of patients with an established diagnosis of alcohol dependence. For both groups, a power of 90% (alpha = 0.05) was applied. Group A comprised 144 consecutively admitted actively drinking patients with alcoholism. Group B consisted of 56 patients with alcoholism who had abstained from alcohol for 24 to 72 hr before admission to the hospital. RESULTS Plasma homocysteine levels were significantly (t test: df = 198, t = -8.6, p < 0.0001) higher at admission when comparing group A with group B. The highly increased homocysteine levels in actively drinking patients with alcoholism were based on a strong significant positive correlation with the blood alcohol concentration (multiple regression analysis, p < 0.0001). CONCLUSIONS Plasma homocysteine levels are evidently altered in actively drinking patients with alcoholism. Even though it has been described, the authors found no evidence for an increase of homocysteine levels in alcoholic patients with early abstinence. The current results emphasize the proposed pathogenetic role of increased plasma homocysteine levels in alcohol-related disorders (i.e., brain atrophy, alcohol withdrawal seizures).

Promoter specific methylation of the dopamine transporter gene is altered in alcohol dependence and associated with craving

Dopaminergic neurotransmission plays a crucial role in the genesis and maintenance of alcohol dependence. Epigenetic regulation via promoter specific DNA methylation of the dopamine transporter gene (DAT) may influence altered dopaminergic neurotransmission in alcoholism. Aim of the present study was to investigate DNA promoter methylation of DAT in early alcohol withdrawal and in relation to alcohol craving. We analyzed blood samples of 76 patients admitted for detoxification treatment and compared them to 35 healthy controls. Methylation specific quantitative real-time PCR was used to measure the promoter specific DNA methylation of the dopamine transporter. We assessed the extent of alcohol craving using the obsessive compulsive drinking scale (OCDS). Compared to healthy controls we found a significant hypermethylation of the DAT-promoter (Mann-Whitney U-test: p=0.001). Ln-transformed methylation of the DAT-promoter was negatively associated with the OCDS (linear regression: Beta=-0.275, p=0.016), particularly with the obsessive subscale (Beta=-0.300, p=0.008). Findings of the present study show that the epigenetic regulation of the DAT-promoter is altered in patients undergoing alcohol withdrawal. Furthermore, hypermethylation of the DAT-promoter may play an important role in dopaminergic neurotransmission and is associated with decreased alcohol craving.

Homocysteine and risk of open-angle glaucoma.

Summary.Homocysteine levels and the frequency of heterozygous methylenetetrahydrofolate reductase (MTHFR) C677T mutation are increased in open-angle glaucoma. Since homocysteine can induce vascular injury, alterations in extracellular matrix remodelling, and neuronal cell death, these findings may have important implications for understanding glaucomatous optic neuropathy.

Homocysteine as a neurotoxin in chronic alcoholism.

There is evidence from in vitro and in vivo studies that homocysteine induces neuronal damage and cell loss by both excitotoxicity and different apoptotic processes. Clinical evidence suggest a strong relationship between higher plasma homocysteine levels and brain atrophy in healthy elderly subjects as well as in elderly at risk of and with Alzheimers disease. Chronic alcoholism leads to elevated plasma homocysteine levels, as shown by clinical investigations and animal experiments. In addition, an association between brain atrophy and increased levels of homocysteine in chronic alcoholism was shown. This may have important implications for the pathogenesis of alcoholism-associated brain atrophy. Furthermore, taking into account that high plasma homocysteine levels are helpful in the prediction of alcohol withdrawal seizures, early anticonvulsive therapy could prevent this severe complication. Homocysteine plays a role in a shared biochemical cascade involving overstimulation of N-methyl-D-aspartate (NMDA) receptors, oxidative stress, activation of caspases, DNA damage, endoplasmic reticulum and mitochondrial dysfunction. These mechanisms are believed to be important in the pathogenesis of both excitotoxicity and apoptotic neurotoxicity. Prospective intervention studies may show whether the incidence of complications of alcohol withdrawal or alcoholism-associated disorders can be reduced by therapeutic measures with early lowering of elevated homocysteine levels (e.g. folate administration). The most important pathophysiological and pathobiochemical features of glutamatergic neurotransmission and of ethanol-induced hyperhomocysteinaemia are reviewed in relation to their excitotoxic and apoptotic potential.