The Role and Mechanisms of Clic4 in Modifying Ethanol-Related Behaviors

项目总结 酒精使用障碍(AUD)有许多危险因素，既有环境因素，也有遗传因素，并且表现出复杂的 产生深刻的生物医学和社会后果的行为特征。识别产生AUD的基因 风险和对潜在神经生物学机制的贡献是 设计有效的治疗方法。已发表的和初步的研究表明，细胞内的氯 通道4(Clic4)的表达对酒精敏感性有重要影响，酒精敏感性是酒精的主要风险因素 人类的依赖性。最近未发表的数据也定义了Clic4在确定乙醇中的作用 偏好和摄入量。总之，这些研究强调了clic4在分子基础上的潜在影响。 并确认进一步的调查。在这里提出的工作中，将在#年单独删除CLIC4。 小鼠前额叶皮质神经元和少突胶质细胞及Clic4在脑内的细胞类型特异性作用 将对饮酒行为、酒精敏感性和焦虑样行为进行评估。这些删除将是 通过立体定向微量注射AAV-Cre到成年Clic4系小鼠体内。行为将是 使用3瓶可供选择的间歇性酒精通道、翻正反射丧失和明/暗盒子测试进行评估。 除了描述Clic4在酒精相关行为中的作用外，一个新的和有影响力的特征描述 它的机制将被实施。CLIC4已被证明移位到原子核并与之相互作用 转录因子，从而改变基因的表达。用操纵法研究乙醇-Clic4相互作用组 在乙醇存在的情况下，Clic4可以揭示其在乙醇相关的机制框架中的作用 行为。这一潜力将通过表征随后PFC中转录转录的变化来评估 神经元和少突胶质细胞中Clic4缺陷小鼠的酒精暴露。特定单元格类型的删除 CLIC4将在成年小鼠身上使用他莫昔芬诱导的Cre/loxP系统进行。RNAseq将提供 差异基因表达的鉴定和下游生物信息学分析将识别丰富的 生物途径和相关的基因网络。本提案中定义的研究将描述 Clic4修饰乙醇相关行为的程度并提供了理解的重要第一步 这个机制。

PROJECT SUMMARY Alcohol use disorder (AUD) has many risk factors, both environmental and genetic, and represents a complex behavioral trait producing profound biomedical and social consequences. Identifying genes that engender AUD risk and contribute to the underlying neurobiological mechanisms represents an important first step in designing effective treatments. Published and preliminary studies make it apparent that chloride intracellular channel 4 (Clic4) expression has a major influence on ethanol sensitivity, a major risk factor for alcohol dependence in humans. Recent unpublished data has also defined a role for Clic4 in determining ethanol preference and intake. Together, these studies underscore the potential impact of Clic4 on the molecular basis of AUD and validate further investigation. In the work proposed here, Clic4 will be deleted separately in neurons and oligodendrocytes in the prefrontal cortex (PFC) of mice and the cell type-specific roles of Clic4 in drinking behavior, ethanol sensitivity, and anxiety-like behavior will be evaluated. These deletions will be performed through stereotactic microinjection of AAV-Cre into adult Clic4-floxed mice. Behaviors will be evaluated using 3-bottle-choice intermittent ethanol access, loss of righting reflex, and the light/dark box test. In addition to describing the role of Clic4 in ethanol-related behavior, a novel and impactful characterization of its mechanisms will be conducted. CLIC4 has been shown to translocate to the nucleus and interact with transcription factors thereby altering gene expression. Studying the ethanol-Clic4 interactome by manipulating Clic4 in the presence of ethanol could reveal the mechanistic framework underlying its role in ethanol-related behavior. This potential will be evaluated by characterizing the transcriptomic changes in the PFC that follow ethanol exposure in mice deficient for Clic4 in neurons and oligodendrocytes. Cell type-specific deletions of Clic4 will be performed in adult mice using a tamoxifen-inducible Cre/loxP system. RNAseq will provide identification of differential gene expression and downstream bioinformatic analysis will identify enriched biological pathways and relevant gene networks. The studies defined in this proposal will characterize the extent which Clic4 modifies ethanol-related behavior and provide an important first step towards understanding the mechanism.