Translational examination of alcohol-associated epigenetic signatures: from primates to rodents

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PROJECT ABSTRACT Although approximately 54% of Americans over the age of 18 consume alcohol, only 6.5% meet the criteria for an alcohol use disorder (AUD). To date, there are three FDA approved medications to treat AUD, each having varying efficacy on an individual basis. Understanding the molecular mechanisms underlying circuit-specific changes that lead to AUD is essential to developing novel, targeted, and effective treatments for AUD. In advancement of this goal, we have begun to identify genome-wide DNA methylation (DNAm) signals within the nucleus accumbens core (NAcC) that distinguish low and heavy ethanol drinking monkeys. A subset of these differential DNAm (D-DNAm) signals were associated with the expression of genes that play a role in modulating neurotransmission. In particular, we found D-DNAm signals in genes that are functionally associated with different compartments of the tetrapartite synapse that includes pre- and postsynaptic elements, astroglial processes, and the extracellular matrix. Glutamatergic inputs into the NAcC arise from brain regions outside of the NAcC, including Brodmann area 32 (A32, in primates; prelimbic cortex (PL) in rodents). By investigating A32 inputs into NAcC, we will begin to elucidate circuit-specific DNAm signals that distinguish low from heavy/very heavy ethanol drinkers. We will use to advantage the highly relevant and well- characterized nonhuman primate (NHP) alcohol drinking model in which rhesus macaques imbibe alcohol daily for over 12 months and self-select into low and heavy drinkers. Genome-wide DNAm sequencing (GW-DNAm) will be used to identify alcohol-dose associated differentially methylated cytosines (DMCs) and regions (DMRs) in the A32 and NAcC. Using a combination of a neuron specific antibody (NeuN) with a fluorescent tag and fluorescence-activated cell sorting, we will begin to elucidate cell specificity (i.e. neuron-specific) in the DNAm signals of the NHP A32. In parallel, we will examine the DNAm state of these same NHP A32 targets using amplicon bisulfite sequencing in PL in high-ethanol preference mice that have consumed alcohol for 3 months and self-select into low and heavy drinkers. These studies will yield gene/regulatory regions as targets that are highly correlated with ethanol dose, based on differences in consumption, conserved across species and play a role in the tetrapartite synapse. Using engineered viral vectors that alter the expression, function or methylation level of gene targets, we will perform a mouse functional assay that will test their role in ethanol drinking (self-administration of ethanol for 3 months) and in the PL-NAcC circuit (tested using ex vivo slice electrophysiology). The most efficacious targets from the mouse functional assay will be tested in ex vivo slices obtained from chronic ethanol drinking NHPs for their ability to alter A32 and NAcC circuitry. In total, this work will identify alcohol dose-dependent DNAm modifications that are specific to the A32/PL-NAcC circuit, and provide functional support for the design of promising new AUD treatments.