Alcohol use disorder (AUD) is highly prevalent among U.S. military veterans. Excessive alcohol consumption, defined as the acquisition of a blood alcohol concentration (BAC) ? 0.08 g/dl (binging) or ? 15 drinks/week for men, is an important risk factor for many serious medical and psychiatric conditions, including AUD. The medial prefrontal cortex (mPFC) is important for integrating various internal and external states in order to determine approach/avoidant behavior to rewarding or aversive stimuli. Prior studies demonstrated that Poly- ADP ribose Polymerase (PARP), through its ability to regulate synaptic plasticity gene expression, promotes cocaine addictive behaviors. Whether PARP enzymatic activity plays a similar role in the addictive properties of ethanol (EtOH) has yet to be studied. The hypothesis of this grant proposal is that EtOH increases PARP activity causing reduced expression of certain neuronal synaptic plasticity genes in excitatory mPFC neurons ultimately increasing alcohol drinking behavior. EtOH increases PARP enzymatic activity in cell culture, adult binging animals, and during fetal development. PARP can silence gene expression by catalyzing reactions adding PAR groups (PARylation) to downstream gene regulatory proteins, including the transcription factor peroxisome proliferator-activated receptor-? (PPAR?) and KDM4D, a demethylase of the transcriptionally repressive dimethylated lysine 9 of histone 3 (H3K9me2). Examples of genes implicated in AUD that are well- established to be regulated by H3K9me2 are Brain-derived Neurotrophic Factor (Bdnf) and Ppar?. BDNF and PPAR? are expressed by excitatory neurons in the cortex, where both have roles in synaptic plasticity and neuronal survival. Therefore, an alcohol induced suppression of BDNF and PPAR? expression would be expected to alter mPFC excitatory outputs, promoting continued alcohol drinking behavior. This is supported by reports indicating higher BDNF expression and PPAR? agonists reduce alcohol consumption. We reported that PARP activity is in part responsible for EtOH-induced decreases in Bdnf IV and IXa mRNA expression in primary cortical neuron cultures. We found several lines of evidence indicating that reduced PPAR? promoter binding may serve as an intermediary step between increased PARP activity and decreased Bdnf expression. We now propose to examine these same pathways in vivo. Our preliminary data indicate that mice that voluntarily consumed EtOH in the binge-like drinking-in-the-dark (DID) paradigm had increased PARP mRNA expression and enzymatic activity in the PFC. DID consumed EtOH reduced BDNF and PPAR? expression, effects that were reversed by PARP inhibitor treatment. DID EtOH consumption decreased PPAR? DNA binding ability generally and specifically at the Bdnf IXa promoter. Also, DID consumed EtOH increased global levels of H3K9me2, and PARP inhibition decreased H3K9me2 at Ppar? and Bdnf IXa gene promoters. Finally, we found that PARP inhibition reduced EtOH consumption in the DID paradigm. In the first aim, we propose dissecting the molecular mechanisms connecting PARP to changes in expression of genes involved in AUD in the mPFC following DID and 2-bottle free-choice drinking paradigms. In the second aim, we will examine the gene regulatory effects of EtOH-induced PARP in cortical excitatory neurons. This cell specific approach will utilize the Translating Ribosome Affinity Purification (TRAP) method for RNA isolation, and Fluorescence activated cell sorting (FACS) isolation of excitatory neuronal nuclei for KDM4D, H3K9me2, and PPAR? binding experiments. We will also perform data-driven experiments using RNA isolated from excitatory neurons in transcriptome-wide microarray analysis. In the third aim, we will study the role of PARP in EtOH drinking behavior, such as DID and 2-bottle free-choice. We will test whether these changes are reversible using PARP inhibitors and PPAR? agonists. Results of the proposed studies are expected to clarify PARP?s role in regulating gene expression in relation to AUD. Based on these data it is possible that PARP inhibitors may be useful pharmacological tools for treating AUD.
Alcohol use disorder (AUD) is highly prevalent among U.S. military veterans. The molecular mechanisms that underlie alcohol-seeking behavior have yet to be discovered. This project is highly innovative as it examines how the gene regulating enzyme, Poly ADP Ribose Polymerase (PARP), an enzyme induced by ethanol exposure, perturbs gene expression in the medial prefrontal cortex (mPFC). We hypothesize that activated PARP enzymatic activity promotes increased alcohol consumption through changes in mPFC excitatory neuronal gene expression. This study has important clinical implications as there exist FDA-approved drugs that target this pathway that may be able to reverse the underlying gene expression changes caused by ethanol-induced PARP.
| Gavin, David P; Hashimoto, Joel G; Lazar, Nathan H et al. (2018) Stable Histone Methylation Changes at Proteoglycan Network Genes Following Ethanol Exposure. Front Genet 9:346 |
