Epigenetic Mechanisms of Neuroimmune Activation in AUD

项目摘要 表观遗传学酒精研究中心（CARE）的首要假设是， 慢性酒精暴露和戒断后， 神经适应，促进依赖诱导的行为，如焦虑，抑郁，和升级 乙醇摄入量CARE的研究项目3已经确定了先天性免疫基因的异常表达， 海马在从慢性饮酒戒断过程中。神经炎性反应增加， 在戒断过程中，海马体可能会导致抑郁相关的行为，促进酒精摄入量的增加。 初步数据表明，戒断过程中神经免疫基因的诱导可能是由以下因素驱动的： 增加组蛋白去甲基化酶KDM 6 B的表达，其去除转录抑制性甲基 组蛋白H3赖氨酸27（H3 K27 me 3）。慢性戒断过程中KDM 6 B的上调 预计酒精会降低特定基因启动子处的H3 K27 me 3并增加染色质可接近性， 导致基因表达增加。 第一组实验将使用无偏的方法来检查染色质可及性的变化 和H3 K27 me 3在戒断期间海马中基因组位点的水平。这将通过使用 H3 K27 me 3抗体检测转座酶可及染色质及染色质免疫沉淀 随后在已经被注射的大鼠的海马中进行DNA测序（分别为ATAC-Seq和ChIP-Seq）。 在依赖模型（Lieber DeCarli乙醇液体饮食）中用慢性乙醇处理并戒断。这些 实验将与CARE的表观遗传学和行为核心合作进行。第二 一组实验将检查KDM 6 B和H3 K27 me 3在特异性神经免疫球蛋白表达中的作用。 基因靶向，并使用病毒介导的RNA干扰或KDM 6 B的药理学抑制剂， KDM 6 B在戒断期间抑郁样行为中的功能作用。 在第三组实验中，酒精戒断过程中Kdm 6 b基因诱导的机制将被证实。 追究已知Kdm 6 b表达受STAT 3调节，STAT 3也被增加和激活 在海马体中的变化。以前的研究表明，STAT 3 可以与组蛋白去乙酰化酶HDAC 6相互作用以调节转录。我们会研究 通过STAT 3和HDAC 6的Kdm 6 b基因表达，并确定是否有药理学抑制或RNAi介导的 戒断过程中STAT 3或HDAC 6的下调影响抑郁样行为和乙醇浓度升高 在戒断期间摄入（与行为核心合作）。成功完成这些实验 将进一步实现CARE的两个目标，即描述酒精使用障碍的表观遗传机制 并确定新的表观遗传靶点，用于开发治疗酒精使用障碍的药物疗法。

Project Summary The overarching hypothesis of the Center for Alcohol Research in Epigenetics (CARE) is that epigenetic mechanisms drive alterations in the brain transcriptome after chronic alcohol exposure and withdrawal, leading to neuroadaptations that promote dependence-induced behaviors such as anxiety, depression, and escalated ethanol intake. Research project 3 of CARE has identified aberrant expression of innate immune genes in the hippocampus during withdrawal from chronic alcohol drinking. An increased neuroinflammatory response in the hippocampus during withdrawal may cause depression-related behaviors that promote escalated alcohol intake. Preliminary data suggests that the induction of neuroimmune genes during withdrawal may be driven by increased expression of the histone demethylase KDM6B, which removes transcriptionally-repressive methyl groups from histone H3 lysine 27 (H3K27me3). The upregulation of KDM6B during withdrawal from chronic alcohol is predicted to decrease H3K27me3 at specific gene promoters and increase chromatin accessibility, leading to increased gene expression. The first set of experiments will use unbiased methods to examine changes in chromatin accessibility and H3K27me3 levels at genomic loci in the hippocampus during withdrawal. This will be accomplished using the assay for transposase accessible chromatin and chromatin immunoprecipitation with H3K27me3 antibody followed by DNA sequencing (ATAC-Seq and ChIP-Seq, respectively) in the hippocampus of rats that have been treated with chronic ethanol and withdrawal in a dependence model (Lieber DeCarli ethanol liquid diet). These experiments will be performed in collaboration with the Epigenetics and Behavioral Cores of CARE. The second set of experiments will examine the role of KDM6B and H3K27me3 in the expression of specific neuroimmune gene targets and determine, using viral-mediated RNA interference or a pharmacological inhibitor of KDM6B, the functional role of KDM6B in depression-like behavior during withdrawal. In the third set of experiments, the mechanism of Kdm6b gene induction during alcohol withdrawal will be investigated. Kdm6b expression is known to be regulated by STAT3, which is also increased and activated in the hippocampus during withdrawal from chronic alcohol drinking. Previous studies have shown that STAT3 can interact with the histone deacetylase HDAC6 to regulate transcription. We will examine the regulation of Kdm6b gene expression by STAT3 and HDAC6, and determine if pharmacological inhibition or RNAi-mediated down-regulation of STAT3 or HDAC6 during withdrawal affects depression-like behavior and escalated ethanol intake (in collaboration with the Behavioral Core) during withdrawal. Successful completion of these experiments will further two goals of CARE, which are to delineate epigenetic mechanisms operative in alcohol use disorder and identify new epigenetic targets for development of pharmacotherapy to treat alcohol use disorder.