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

项目摘要 酒精使用障碍(AUD)是一种慢性、复发性脑部疾病，会造成巨大的社会经济损失 在美国。虽然AUD的诊断标准已经建立，但潜在的分子 发病机制很大程度上还不清楚。先前的AUD研究已经证明了调节失调的关键作用 糖皮质激素信号贯穿多个大脑区域，包括内侧前额叶皮质(MPFC)。 糖皮质激素受体(GR)活性的特异性改变对酒精的进展很重要 依赖，这是AUD的一个关键方面，导致酒精消费、戒断和 焦虑样的表型。绝大多数关于了解AUD的GR研究都集中在 蛋白质编码基因，尽管基因组中只有不到2%的基因是蛋白质编码基因。非编码RNA起着至关重要的作用 在生理和疾病中对基本细胞功能的作用。这一提议将检验一个新的假设，即长期 非编码RNA(LncRNA)生长抑制特异性5(Gas5)是一种性别依赖和[神经元]特异性的调节剂 酒精反应GR依赖的基因表达和行为。Gas5是一种已知的GR活性抑制因子 此前曾被认为与小鼠和人类的AUD表型有关。我们之前的研究表明， Gas5表达的变化，特别是在mPFC神经元中的表达变化与慢性酒精诱导有关 戒断和雄性小鼠酒精消耗量的增加。然而，造成这些的生物学机制(S) 雄性动物中与Gas5相关的变化尚不清楚。此外，还没有同时进行的研究 在雌性动物身上进行，以确定Gas5对慢性酒精诱导的潜在性别依赖效应 行为。本项目将直接研究mPFC神经元Gas5在酒精依赖转录组中的作用。 性别与GR相关的行为表型。要做到这一点，慢性间歇性乙醇 蒸汽暴露(CIEV)模型将与腺相关病毒的立体定向注射一起使用 (AAV)诱导CRISPR/Cas9介导的mPFC神经元Gas5基因敲除(KD)。这样做的三个目的 研究将确定mPFC神经元特异性Gas5KD和CIEV的分子和行为结果。这个 本研究的第一个目的是使用RNA免疫沉淀，然后实时定量聚合酶链式反应(RIP-qPCR) 定义mPFC Gas5-GR绑定中的更改。第二个目标是使用[下一代RNA测序(3‘标记- SEQ)以确定GR依赖的转录组中神经元特异性的]变化。第三个目标使用一个 综合电池检测慢性酒精暴露相关行为表型。这个项目是一个至关重要的项目 Gas5在AUD中的功能特征以及依赖GR的表型是如何出现的。这个 拟议的研究可能有助于合理开发更有效的GR介导的AUD药物疗法 以及共病障碍(例如，慢性压力和焦虑)。

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).