Epigenetic Mechanisms of Negative Affective State of AUD

项目总结： 新出现的临床前和临床证据表明，与酒精有关的症状 戒断/消极情绪是酒精使用障碍(AUD)复发和维持的主要危险因素。 构成延伸的杏仁核，特别是杏仁中央核(CEA)的解剖结构， 与焦虑和饮酒行为密切相关。表观遗传机制，如组蛋白和 DNA化学修饰在基因表达调控中发挥着重要作用。这 CARE的研究部分将检查慢性酒精引起的表观遗传修饰 全基因组水平的暴露和停药导致基因网络调节途径的异常 杏仁核中的各种生物过程，产生焦虑样行为和酒精升级 喝酒。我们提出以下具体目标：1)检查a)染色质可及性和基因座的状态 ATAC-seq和H3K27me3芯片-seq在大鼠杏仁核(雌雄)基因组表观遗传标记中的应用 在慢性酒精暴露后的酒精戒断期间。新兴数据集将与现有数据集合并 RNA-SEQ和CHIP-SEQ(H3K9/14ac标记)鉴定差异基因的整合表观遗传调控 杏仁核中的表达。B)双特异性磷酸酶6(Dusp6，通过合并确定的新基因 H3K9/14ac芯片-seq/rna-seq)siRNA将通过激活CBP和增加 酒精戒断过程中杏仁核基因的组蛋白乙酰化。2)检查a)G9a siRNA输注 在CEA中将减轻焦虑样行为，正常化表观遗传和基因表达变化以及 酒精戒断大鼠杏仁核内突触和树突棘的缺失；b)神经元 使用化学发生(GQ偶联DREADD)方法刺激CEA将减轻焦虑样症状 通过恢复HDAC2/CBP调节的适当平衡从而导致组蛋白增加的行为 酒精戒断时杏仁核靶基因的乙酰化及c)神经元特异性p300HAT的引导 通过CRISPR-dCas9注入CEA，会增加靶基因的组蛋白乙酰化，导致基因增加 在戒断过程中，杏仁核的表达、焦虑样行为的减弱以及饮酒增加。3) 检测CEA输注HDAC2和G9a siRNA是否能减轻酒精依赖 雄性和雌性大鼠饮酒的升级，通过可操作的乙醇自身给药来衡量。4)至 酒精依赖大鼠杏仁核表观遗传动力学和基因表达的死后转换 人类酗酒者的杏仁核，并将其与饮酒数据相关联。完成拟议的研究将 提供关于杏仁核整个转录组的表观遗传调控的新信息， 从而确定AUD药物治疗发展的分子靶点。

Project Summary: The emerging preclinical and clinical evidence suggests that symptoms associated with alcohol withdrawal/negative affect is a primary risk factor for relapse and maintenance of alcohol use disorder (AUD). Anatomical structures comprising the extended amygdala, particularly the central nucleus of amygdala (CeA), are strongly implicated in anxiety and alcohol-drinking behaviors. Epigenetic mechanisms such as histone and DNA chemical modifications, have been shown to play important roles in the regulation of gene expression. This research component of CARE will examine how epigenetic modifications induced by chronic alcohol exposure and withdrawal at genome-wide level lead to an aberrant gene network pathway regulating various biological processes in the amygdala, producing anxiety-like behavior and escalated alcohol drinking. We propose the following Specific Aims: 1) To examine a) status of chromatin accessibility and loci of genomic epigenetic marks using ATAC-seq and H3K27me3 ChIP-seq in the amygdala of rats (male & female) during ethanol withdrawal after chronic ethanol exposure. The emerging data set will be merged with existing RNA-seq and ChIP-seq (H3K9/14ac mark) to identify integrated epigenetic regulation of differential gene expression in the amygdala. b) Dual specificity phosphatase 6 (Dusp6, novel gene identified by merger of H3K9/14ac ChIP-seq/RNA-seq) siRNA will attenuate anxiety-like behaviors via activating CBP and increasing histone acetylation of genes in the amygdala during ethanol withdrawal. 2) To examine if a) G9a siRNA infusion in the CeA will attenuate anxiety-like behaviors, normalize epigenetic and gene expression changes as well as deficits in synapses and dendritic spines in the amygdala of rats during ethanol withdrawal; b) Neuronal stimulation in the CeA using a chemogenetic (Gq coupled DREADD) approach will attenuate anxiety-like behaviors via restoring the proper balance in HDAC2/CBP regulation thereby leading to increased histone acetylation of target genes in the amygdala during ethanol withdrawal and c) Neuron-specific p300HAT guided by CRISPR-dCas9 infusion into CeA will increase histone acetylation at target genes, leading to increased gene expression in the amygdala, attenuation of anxiety-like behaviors and escalated drinking during withdrawal. 3) To examine whether CeA infusion of a HDAC2 and G9a siRNA will attenuate ethanol dependence-induced escalation in drinking in male and female rats, as measured by operant ethanol-self administration. 4) To translate epigenetic dynamics and expression of genes in the amygdala of alcohol dependent rats to postmortem amygdala of human alcoholics and correlate them to drinking data. Completion of proposed studies will provide new information on the epigenetic regulation of the whole transcriptome in the amygdala, leading to identification of molecular targets for the development of pharmacotherapy of AUD.