The role of lncRNA Gas5 in Glucocorticoid-Mediated Ethanol Dependence Phenotypes

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Project Summary Alcohol use disorder (AUD) is a chronic, relapsing brain disease that imposes a tremendous socioeconomic cost in the United States. Although the diagnostic criteria for AUD have been established, the underlying molecular pathogenesis is largely unknown. Prior AUD studies have demonstrated a critical role for dysregulation of glucocorticoid signaling throughout multiple brain-regions, including the medial prefrontal cortex (mPFC). Specific alterations in glucocorticoid receptor (GR) activity are important for the progression of alcohol dependence, a critical facet of AUD that contributes to escalation of alcohol consumption, withdrawal, and anxiety-like phenotypes. The overwhelming majority of GR studies for understanding AUD have focused on protein-coding genes despite less than 2% of the genome being protein-coding. Non-coding RNAs play vital roles in basic cellular functions in physiology and disease. This proposal will test a novel hypothesis that the long non-coding RNA (lncRNA) growth arrest specific 5 (Gas5) is a sex-dependent and [neuron]-specific modulator of alcohol responsive GR-dependent gene expression and behavior. Gas5 is a known repressor of GR activity and has previously been linked to AUD phenotypes in mice and humans. Our previous studies have shown that changes in the expression of Gas5, particularly in mPFC neurons, are related to chronic alcohol-induced withdrawal and escalated ethanol consumption in male mice. However, the biological mechanism(s) for these Gas5-associated changes in male animals are unknown. Additionally, no concurrent studies have been conducted in female animals to determine potential sex-dependent effects of Gas5 for chronic alcohol-induced behaviors. This project will directly study the role of mPFC neuronal Gas5 in the alcohol dependent transcriptome and behavioral phenotypes related to GR in both sexes. To accomplish this, the chronic intermittent ethanol vapor exposure (CIEV) model will be used in tandem with stereotaxic injections of an adeno-associated virus (AAV) to induce CRISPR/Cas9-mediated knockdown (KD) of Gas5 in mPFC neurons. The three aims of this study will determine the molecular and behavioral outcomes of mPFC neuron-specific Gas5 KD and CIEV. The first aim of this study uses RNA immunoprecipitation followed by real time, quantitative PCR (RIP-qPCR) to define changes in mPFC Gas5-GR binding. The second aim uses [next-generation RNA Sequencing (3’Tag- Seq) to determine neuron-specific] alterations in the GR-dependent transcriptome. The third aim employs a comprehensive battery to assay chronic ethanol exposure-related behavioral phenotypes. This project is a vital step in the functional characterization of Gas5 in AUD and how GR-dependent phenotypes emerge. The proposed studies may aid in rational development of more effective GR-mediated pharmacotherapies for AUD and co-morbid disorders (e.g., chronic stress and anxiety).