Epigenetic modulation of amygdalar circuits that control alcohol compulsivity

PROJECT SUMMARY We propose to investigate the neurobiological basis of alcohol addiction focusing on epigenetic factors that underlie individual vulnerabilities. A key feature of alcohol addiction is alcohol drinking despite negative consequences or at the expense of other rewards. These clinically-significant “compulsive” behaviors occur in a minority of individuals that consume alcohol – a strong indication that individual vulnerabilities are at the core of disease. Our previous work has shown that behavioral changes associated with alcohol addiction manifest in response to persistent epigenetic reprogramming of transcription networks in specific brain regions and neuronal populations. Epigenetic mechanisms integrate genetic risk and environmental factors such as stress and alcohol itself. Therefore, elucidating the epigenetic networks associated with the addictive state is a major goal of the research community, but unfortunately current understanding of these networks and the downstream impact on neurocircuits involved in alcohol dependence remains limited. In preliminary studies, we investigated a vulnerable minority of laboratory animals that self-administer alcohol despite pairing with punishment (foot- shock) – a compulsion-like alcohol self-administration behavior. Using a combination of techniques to profile gene expression patterns in the amygdala of compulsive rats, we identified a critical role for Enhancer of Zeste 2 (Ezh2), the histone 3 lysine 27 (H3K27) methyltransferase and catalytic component of the polycomb repressive complex 2 (PRC2). Subsequently, we found that pharmacological inhibition of EZH2 attenuates compulsivity in rats. Therefore, we propose to clarify the role of EZH2 and H3K27 methylation in regulating transcription programs in the amygdala, a brain region linked to addiction that is critical for integrating response to adverse stimuli. Using a relatively novel and highly-efficient epigenetic profiling technology called CUT&RUN we will profile H3K27me3 specifically in amygdalar neurons and non-neuronal cells of compulsive rats. In parallel, the functional consequence of differential H3K27me3 will be defined using RNA sequencing. Additional preliminary data show that inhibition of GABAergic protein kinase C delta (PRKCD) neurons in the amygdala reduces compulsivity. To further investigate the role of PRKCD-neurons, we will selectively manipulate their activity using chemogenetics, and assess the effects on compulsivity. These experiments will utilize a recently validated transgenic rat model that we have created where expression of Cre-recombinase is driven by the endogenous Prkcd promoter. Using the PRKCD-Cre rat and a Cre-inducible nuclear tagging approach, we will define differential H3K27me3 levels and gene expression patterns specifically in PRKCD-neurons of compulsive rats. Finally, we will knock down EZH2 specifically in PRKCD-neurons to define its role in compulsivity with brain- region and cell-type specificity. Since epigenetic enzymes/effectors, including EZH2, have emerged as druggable therapeutic targets for complex psychiatric diseases such as alcohol addiction, our study could provide a new avenue for developing much-needed therapeutic strategies.