Epigenetic Mechanisms of Negative Affective State of AUD

项目摘要： 新出现的临床前和临床证据表明，与酒精相关的症状 戒断/负面影响是酒精使用障碍（AUD）复发和维持的主要风险因素。 解剖结构包括延伸的杏仁核，特别是杏仁核中央核（CeA）， 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起着重要作用。这 CARE的研究部分将研究慢性酒精诱导的表观遗传修饰 在全基因组水平上的暴露和戒断导致异常的基因网络通路调节 杏仁核中的各种生物过程，产生类似焦虑的行为和酒精的升级 喝酒我们提出以下具体目的：1）检查a）染色质可及性和基因座的状态， 在大鼠杏仁核中使用ATAC-seq和H3 K27 me 3 ChIP-seq的基因组表观遗传标记（雄性和雌性） 在慢性乙醇暴露后的乙醇戒断过程中。新出现的数据集将与现有的 RNA-seq和ChIP-seq（H3 K9/14 ac标记）鉴定差异基因的整合表观遗传调控 在杏仁核中的表达。B）双特异性磷酸酶6（Dusp 6，通过合并 H3 K9/14 ac ChIP-seq/RNA-seq）siRNA将通过激活CBP和增加 乙醇戒断过程中杏仁核基因的组蛋白乙酰化。2)检查a）G9 a siRNA输注 将减弱焦虑样行为，使表观遗传和基因表达变化正常化， 乙醇戒断期间大鼠杏仁核中突触和树突棘的缺陷; B）神经元 使用化学发生学（Gq偶联DREADD）方法刺激CeA将减弱焦虑样 通过恢复HDAC 2/CBP调节的适当平衡，从而导致组蛋白增加， 乙醇戒断期间杏仁核中靶基因的乙酰化和c）神经元特异性p300 HAT引导的 通过CRISPR-dCas 9输注到CeA中将增加靶基因处的组蛋白乙酰化，导致基因表达增加。 杏仁核中的表达，焦虑样行为的减弱和戒断期间饮酒量的增加。第三章 为了检查HDAC 2和G9 a siRNA的CeA输注是否会减弱乙醇依赖性诱导的细胞凋亡， 通过操作性乙醇自我给药测量，雄性和雌性大鼠饮酒量增加。4)到 酒精依赖大鼠死后杏仁核的表观遗传动力学和基因表达 杏仁核，并将其与饮酒数据相关联。完成拟议的研究将 提供了关于杏仁核中整个转录组的表观遗传调控的新信息， 从而鉴定用于开发AUD药物疗法的分子靶点。

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.