Epigenetic Mechanisms of Positive Affective State of AUD

项目摘要 作为酒精使用障碍(AUD)的标志，戒断综合征包括两种阴性症状，如 焦虑和动机症状，如对酒精的渴望，这会促进复发和酗酒。 了解戒酒如何改变大脑生理是发展的重要一步 减少酗酒的有效治疗方法。腹侧被盖区(VTA)是大脑的一个重要区域 投射到延伸的杏仁核的组成部分，包括伏隔核，前额叶皮质， 杏仁核和海马体。戒断引起的VTA神经元生理学改变可能是其发生机制之一 AUD期间的饮酒情况。VTA神经元对γ-氨基丁酸抑制的敏感性 (GABA)在酒精戒断过程中降低，但被组蛋白脱乙酰酶(HDAC)抑制剂正常化， 提示VTA撤退引起的表观遗传学改变可能是药物作用的结果。 操纵。该项目的全基因组测序和分子研究确定了一组基因 编码胆固醇合成途径酶，这些酶在酒精戒断过程中表达减少， 在功能上与戒断时VTA神经元GABA敏感性降低有关。建议数 该项目计划进一步表征特定的表观遗传修饰在戒断诱导调节中的作用 负责胆固醇合成的基因，以及这些变化对戒断诱导的GABA的影响 过敏和饮酒行为。通过使用电生理、行为和最先进的分子 通过采用化学遗传学和CRISPR技术等生物方法，我们预计将实现以下目标： 1)揭示与慢性酒精暴露相关的新的表观遗传标记和基因表达的变化 和停用全基因组方法，2)检查是否减少组蛋白乙酰化和 戒断时组蛋白甲基化增加降低VTA中胆固醇合成酶的表达 以细胞类型特异性的方式，探讨胆固醇合成酶基因在戒断相关过程中的作用 饮酒行为和GABA低敏感性，3)确定是否有针对性的表观遗传干预 通过使用CRISPR防止组蛋白乙酰化水平下降来对抗戒断诱导的表型 关键胆固醇合成酶基因的启动子，以及4)转化为死后的人酒精性VTA 已发现的与GABA低敏感性相关基因的表观遗传动力学和表达 大鼠VTA。最终，需要这些研究来理解vta神经元的表观遗传适应。 在戒酒过程中处于积极的情感状态，并与该中心的其他组成部分一起，将 提供了大量关于戒断诱导的脑变化的表观遗传机制的信息， 以及更有效治疗AUD的可能药理学方法。

Project Summary As a hallmark of alcohol use disorder (AUD), the withdrawal syndrome includes both negative symptoms like anxiety and motivational symptoms such as craving for alcohol, which promotes relapse and alcohol seeking. Understanding how alcohol withdrawal changes brain physiology is an important step towards developing effective treatments to reduce alcohol abuse. The ventral tegmental area (VTA) is an important brain area that projects to components of the extended amygdala, including the nucleus accumbens, prefrontal cortex, amygdala and hippocampus. Changes in the physiology of VTA neurons induced by withdrawal may underlie the alcohol-seeking during AUD. The sensitivity of neurons of the VTA to inhibition by gamma aminobutyric acid (GABA) is decreased during alcohol withdrawal but normalized by histone deacetylase (HDAC) inhibitors, indicating epigenetic changes induced by withdrawal in the VTA may be amenable to pharmacological manipulation. Whole genome sequencing and molecular studies of this project identified a cluster of genes that encode cholesterol synthesis pathway enzymes that showed decreased expression during alcohol withdrawal, and are functionally relevant to decreased GABA sensitivity in VTA neurons during withdrawal. The proposed project plans to further characterize the role of specific epigenetic modifications in withdrawal-induced regulation of genes responsible for cholesterol synthesis, and the effect of these changes in withdrawal-induced GABA hyposensitivity and drinking behaviors. By using electrophysiological, behavioral, and state of the art molecular biological methods such as chemogenetics and CRISPR technology, we anticipate achieving the following goals: 1) to reveal novel epigenetic marks and changes in gene expression associated with chronic alcohol exposure and withdrawal with whole genome approaches, 2) to examine whether decreased histone acetylation and increased histone methylation reduce expression of cholesterol synthesis enzymes in the VTA during withdrawal in a cell-type specific manner, and probe the role of cholesterol synthesis enzyme genes in withdrawal-related drinking behavior and GABA hyposensitivity, 3) to determine whether targeted epigenetic intervention counteracts withdrawal-induced phenotypes by using CRISPR to prevent decreases in histone acetylation on promoters of key cholesterol synthesis enzyme genes, and 4) to translate to post-mortem human alcoholic VTA the epigenetic dynamics and expression of genes related to GABA hyposensitivity that have been identified in the rat VTA. Ultimately, these studies are needed to understand epigenetic adaptation of VTA neurons involved in the positive affective state during alcohol withdrawal, and, with the other components of this Center, will provide a great deal of information on epigenetic mechanisms involved in withdrawal-induced brain changes, and possible pharmacological approaches toward more effective treatment of AUD.