Emerging Role of One-Carbon Metabolism and DNA Methylation Enrichment on δ-Containing GABAA Receptor Expression in the Cerebellum of Subjects with Alcohol Use Disorders (AUD)

Abstract

Background: Cerebellum is an area of the brain particularly sensitive to the effects of acute and chronic alcohol consumption. Alcohol exposure decreases cerebellar Purkinje cell output by increasing GABA release from Golgi cells onto extrasynaptic α6/δ-containing GABAA receptors located on glutamatergic granule cells. Here, we studied whether chronic alcohol consumption induces changes in GABAA receptor subunit expression and whether these changes are associated with alterations in epigenetic mechanisms via DNA methylation.

Methods: We used a cohort of postmortem cerebellum from control and chronic alcoholics, here defined as alcohol use disorders subjects (n=25/group). S-adenosyl-methionine/S-adenosyl-homocysteine were measured by high-performance liquid chromatography. mRNA levels of various genes were assessed by reverse transcriptase-quantitative polymerase chain reaction. Promoter methylation enrichment was assessed using methylated DNA immunoprecipitation and hydroxy-methylated DNA immunoprecipitation assays.

Results: mRNAs encoding key enzymes of 1-carbon metabolism that determine the S-adenosyl-methionine/S-adenosyl-homocysteine ratio were increased, indicating higher "methylation index" in alcohol use disorder subjects. We found that increased methylation of the promoter of the δ subunit GABAA receptor was associated with reduced mRNA and protein levels in the cerebellum of alcohol use disorder subjects. No changes were observed in α1- or α6-containing GABAA receptor subunits. The expression of DNA-methyltransferases (1, 3A, and 3B) was unaltered, whereas the mRNA level of TET1, which participates in the DNA demethylation pathway, was decreased. Hence, increased methylation of the δ subunit GABAA receptor promoter may result from alcohol-induced reduction of DNA demethylation.

Conclusion: Together, these results support the hypothesis that aberrant DNA methylation pathways may be involved in cerebellar pathophysiology of alcoholism. Furthermore, this work provides novel evidence for a central role of DNA methylation mechanisms in the alcohol-induced neuroadaptive changes of human cerebellar GABAA receptor function.

Keywords: GABAA receptor; alcohol; cerebellum; methylation; one-carbon metabolism.

Figure 1.

One-carbon metabolism, transmethylation reactions and DNA demethylation. DNA methylation on specific cytosine moieties is catalyzed by DNA-methyltransferases (DNMTs). This reaction is dependent on the levels of S-adenosyl-methionine (SAM), which is synthesized in the brain by the methionine-adenosyl-transferase (MAT) 2. After the DNA methyl transfer reaction, S-adenosyl-homocysteine (SAH) is formed as a by-product. SAH exerts a feedback inhibitory activity on DNMT and is used as a substrate for the adenosyl homocysteine hydrolase (AHCY) leading to the synthesis of homocysteine. The remethylation of homocysteine to form methionine is catalyzed via the folate cycle, that is, the conversion of folic acid into tetrahydrofolate (THF) and 5,10-methylenete-trahydrofolate (5,10-MTHF). The methylene tetrahydrofolate reductase (MTHFR) will produce 5-methyltetrahydrofolate (5-MTHF) that is used as a substrate by methionine synthase (MTR) to form methionine. The synthesis of methionine may also involve the activation of the betaine homocysteine methyltransferase (BHMT) pathway. DNA demethylation is initiated by ten-eleven-translocase (TET) enzymes that hydroxylate 5-methylcytosine (5mC) forming 5-hydroxymethylcytosine (5hmC). In the end, through a GADD45B coordinated process, 5hmC is deaminated to 5-hydroxyuracil (5hmU) by cytidine deaminases (i.e., AID/APOBECs). The base excision repair (BER) pathway removes 5hmU and substitutes it with cytosine.

Figure 2.

Location of human GABA_A receptor (A) δ (GABRD) and (B) α₆ (GABRA6) subunit promoter regions. CpG islands are illustrated as black squares and exons (grey rectangles) are in bold letters. Methylated DNA immunoprecipitation and hydroxymethylated DNA immunoprecipitation were performed using the primers underlined and italicized in the sequence.

Figure 3.

Chronic ethanol intake increases expression of key enzymes of the 1-carbon metabolism in cerebellum. mRNA levels of (A) methionine adenosyltransferase (MAT) 2B, (B) MAT2A, (C) methylenetetrahydrofolate reductase (MTHFR), (D) adenosyl homocysteine hydrolase (AHCY), (E) methionine synthase (MTR), and (F–H) DNA methyltransferase (DNMT) 1, 3A, and 3B, respectively, are expressed as fold change of controls. Values are mean ± SEM of 24 samples per group with exception of DNMT1, where only 21 samples per group were measured. *P < .05, Student’s t test vs controls.

Figure 4.

Chronic ethanol intake impairs demethylation pathways. mRNA levels of Ten-Eleven Translocase (TET) 1, 2, and 3, as well as Growth Arrest and DNA Damage Inducible Beta (GADD45B) and Apolipoprotein B mRNA editing enzyme catalytic subunit 3C (APOBEC 3C) are expressed as fold change of controls. Values are mean ± SEM of 24 samples per group. *P < .05, Student’s t test vs controls.

Figure 5.

Chronic ethanol intake changes the “methylation index” in cerebellum. (A) Amounts of S-adenosyl-methionine (SAM) and S-adenosyl-homocysteine (SAH) were measured (mol/mg tissue x10^-12) and (B) expressed as a ratio, indicating the methylation index. Global (C) methylation (5mC) and (D) hydroxymethylation (5hmC) were determined. Values are mean ± SEM of 18 samples per group. *P < .05, Student’s t test vs controls.

Figure 6.

Pearson’s correlation analysis of global methylation (5mC) and hydroxymethylation (5hmC) with S-adenosyl-methionine (SAM) and S-adenosyl-homocysteine (SAH). Correlations were performed in alcohol use disorders (AUD) subjects (A–D) and control subjects (E–H), n =18 samples per group.

Figure 7.

GABA_A receptor delta subunit expression is reduced in the cerebellum of alcohol use disorders subjects. (A) mRNA levels of GABA_A receptor δ (GABRD), α₆ (GABRA6), α₁ (GABRA1) subunits, and (B) 5-α-reductase 1 (SRD5A1) are expressed as fold change of controls. Methylated DNA immunoprecipitation (MeDIP) of the regulatory promoter regions of (C) GABRD and (D) GABRA6 are measured as percentage of input. Hydroxymethylated DNA immunoprecipitation (hMeDIP) of the regulatory promoter regions of (E) GABRD and (F) GABRA6. (G) Protein levels of GABRD measured as ratio of GABRD/GAPDH O.D. values are expressed as percentage of controls. Representative western blot is shown in the lower right-hand corner. Values are mean ± SEM of 24 samples per group for mRNA assays, 11 to 13 samples per group for the immunoprecipitation analysis; 25 samples per group were used for the western blot. *P < .05, Student’s t test vs controls.