DNA methylation in the medial prefrontal cortex regulates alcohol-induced behavior and plasticity

Abstract

Recent studies have suggested an association between alcoholism and DNA methylation, a mechanism that can mediate long-lasting changes in gene transcription. Here, we examined the contribution of DNA methylation to the long-term behavioral and molecular changes induced by a history of alcohol dependence. In search of mechanisms underlying persistent rather than acute dependence-induced neuroadaptations, we studied the role of DNA methylation regulating medial prefrontal cortex (mPFC) gene expression and alcohol-related behaviors in rats 3 weeks into abstinence following alcohol dependence. Postdependent rats showed escalated alcohol intake, which was associated with increased DNA methylation as well as decreased expression of genes encoding synaptic proteins involved in neurotransmitter release in the mPFC. Infusion of the DNA methyltransferase inhibitor RG108 prevented both escalation of alcohol consumption and dependence-induced downregulation of 4 of the 7 transcripts modified in postdependent rats. Specifically, RG108 treatment directly reversed both downregulation of synaptotagmin 2 (Syt2) gene expression and hypermethylation on CpG#5 of its first exon. Lentiviral inhibition of Syt2 expression in the mPFC increased aversion-resistant alcohol drinking, supporting a mechanistic role of Syt2 in compulsive-like behavior. Our findings identified a functional role of DNA methylation in alcohol dependence-like behavioral phenotypes and a candidate gene network that may mediate its effects. Together, these data provide novel evidence for DNA methyltransferases as potential therapeutic targets in alcoholism.

Keywords: DNA methylation; DNMT; alcoholism; epigenetics; neurotransmitter release; plasticity.

Figure 1.

Experimental timeline: Rats are exposed to alcohol vapor for 7 weeks (14 h per day). A, Amg, Hipp, NAc, and mPFC were collected 3 weeks after the end of alcohol exposure. B, Alcohol self-administration (SA) was measured 3 weeks after alcohol exposure. Once SA was stable (baseline), cannulae connected to an osmotic mini-pump containing either RG108 or vehicle were implanted into mPFC of PD and control rats. The rats were tested for SA after 1 week recovery.

Figure 2.

History of alcohol dependence increases DNMT1 expression in neuronal cells in mPFC. A, Western blot analysis shows no difference in DNMT1, DNMT3a, and DNMT3b protein expression. Black bars indicate mean values (± SEM) of control rats. Gray bars indicate mean values (± SEM) of PD rats. Protein expression is represented as DNMT/β-tubulin relative to control. B, C, Immunohistochemical detection of DNMT1 (green) and neuronal cells (red) in the mPFC of control (top) and PD rats (bottom). Right top and bottom, Merge DNMT1/NeuN. C, Average density of DNMT1 in neuronal cells. ^#p < 0.05, control versus PD rats.

Figure 3.

A, Immunohistochemical detection of 5MeC (red) and neuronal cells (green) in the mPFC of control (top) and PD (bottom). Right top and bottom, Merge 5MeC/NeuN. B, Average density of 5MeC in neuronal cells. ^#p < 0.05, control versus PD rats.

Figure 4.

Intracerebroventricular infusion of RG108 prevents escalation in alcohol intake in PD rats. Black bars indicate mean values (± SEM) of control rats. Gray bars indicate mean values (± SEM) of PD rats. PD rats show escalated alcohol consumption that is prevented by intracerebroventricular infusion of RG108. ^#p < 0.05, control versus PD rats. *p < 0.05, vehicle versus RG108.

Figure 5.

Infusion of RG108 directly into mPFC prevents escalation in alcohol intake in PD rats. A, Black bars indicate mean values (± SEM) of rats before drug injection. Gray bars indicate mean values (± SEM) of rats after drug injection. Ctl refers to Control rats. Infusion of RG108 into mPFC prevents the increased alcohol consumption observed in the PD compared with control rats. B, RG108 does not modify locomotor activity. White bars indicate mean values (± SEM) of rats treated with vehicle. Black bars indicate mean values (± SEM) of rats treated with RG108. ^#p < 0.05, control versus PD rats. *p < 0.05, vehicle versus RG108.

Figure 6.

RG108 prevents gene expression changes induced by a history of alcohol dependence. A, Figure from Ingenuity Pathway Analysis showing one of the top gene networks that is downregulated by chronic alcohol exposure. This network includes genes involved in calcium release and exocytosis, two processes critical for synaptic transmission. B, Table shows the fold change in gene expression that is obtained by RNA sequencing and qPCR validation. RG108 prevents alcohol-induced decrease expression of syt1 (C), syt2 (D), cacna1a (E), and wnk2 (F). White bars indicate mean values (± SEM) of rats treated with vehicle. Black bars indicate mean values (± SEM) of rats treated with RG108. ^#p < 0.05, control versus PD rats. *p < 0.05, vehicle versus RG108.

Figure 7.

History of alcohol dependence increases DNA methylation on exon 1 of syt2. A, Figure shows sequence of exon 1 of syt2 and the location of the 7 CpG sites. Bar graph represents DNA methylation level (%) at CpG#5 (B) and at CpG#6 (C) on exon 1. Black bars indicate mean values (± SEM) of control rats. Gray bars indicate mean values (± SEM) of PD rats. ^#p < 0.05, control versus PD rats. *p < 0.05 vehicle versus RG108.

Figure 8.

Syt2 inhibition increases tolerance to quinine adulteration. A, Immunohistochemical detection of syt2 (red) and cell infected by shRNA lentiviral vector specific to syt2. The figure shows that cells infected by lentivirus do not express SYT2 (green). B, Number of reward for alcohol in rats that received injection of shRNA lentiviral vector specific to Syt2 (square) and rats that received the scrambled lentiviral vector. C, Compulsive-like drinking (i.e., persistent alcohol drinking despite the aversive bitter taste of quinine added to the alcohol solution). The data represent the percentage change from baseline (i.e., lever presses for alcohol alone before adulteration with quinine). *p < 0.05; scramble vs. Syt2. D, Syt2 inhibition does not modify quinine consumption. Bar graph represents quinine consumption as measured by two-bottle free choice. E, Graph shows mapping of viral injection sites within the mPFC (•).