The Role of lncRNA MALAT1 in Controlling Behavior and Neuroinflammation in Alcohol Use Disorder

项目摘要 酒精使用障碍（AUD）是美国一种普遍的精神疾病， 对社会的成本和每年造成数千人死亡。虽然许多行为改变 与疾病相关的潜在生理状况，如 神经炎症和转录组学改变，需要进一步研究。这一建议提出了一个新的 用于测试协调这些分子和行为变化的潜在机制的假设。 结合以往的文献，我推测长链非编码RNA（lncRNA）Malat 1可能介导了 神经炎症反应的乙醇和乙醇消费。 非编码RNA一直被认为是几种基本细胞生物学过程中不可或缺的参与者。 功能协调发展的Malat 1已被证明协调基因表达和炎症的转录调节 多项研究表明Malat 1和高迁移率族蛋白1之间存在联系 （Hmgb 1）/Toll样受体4（Tlr 4）神经炎症级联反应。在这个研究计划中，我想确定 如果Malat 1调节Hmgb 1/Tlr 4通路对乙醇的反应，特别是在星形胶质细胞中， 调节神经免疫反应并在控制行为方面发挥关键作用的中枢神经系统细胞类型。第一 该提案的目的是利用CRISPR/Cas9敲低原代星形胶质细胞培养物中的Malat 1表达 并评估乙醇诱导的神经免疫基因表达和细胞因子产生的变化。的 第二个目标是创造一种新的floxed Malat 1条件性敲除小鼠，将其培育成一个品系 在星形胶质细胞中选择性表达重组酶Cre。这种交配的后代将拥有 星形胶质细胞特异性敲除Malat 1。评估乙醇诱导的神经免疫应答和乙醇 这些动物的饮水行为将提供对Malat 1和星形胶质细胞在调节 AUD的潜在病理学。 该项目是了解lncRNA Malat 1在细胞内功能贡献的重要一步。 AUD以及该疾病的一些生理表型如何持续存在。很少有有效的 目前可用于患有AUD的个体的治疗，从拟议研究中获得的结果 可能指向新的预防策略和治疗目标。此外，该项目将大大提高 我的研究生培训，因为它将给我机会设计和执行假设驱动的研究， 令人兴奋的新研究领域和掌握创新技术。最终，这个露丝提供的训练 L. Kirschstein国家研究服务奖（F31）将有助于我作为研究生的科学训练， 以及我作为独立调查员的职业发展。

Project Summary Alcohol use disorder (AUD) is a prevalent psychiatric condition in the United States, imposing a huge cost on society and contributing to thousands of deaths each year. While many of the behavioral changes associated with the disorder have been characterized, the underlying physiological conditions, such as neuroinflammation and transcriptomic alterations, require further study. This proposal presents a novel hypothesis for testing the potential mechanism(s) coordinating these molecular and behavioral changes. Integrating previous literature, I hypothesize that the long non-coding RNA (lncRNA) Malat1 may mediate neuroinflammatory responses to ethanol and ethanol consumption. Non-coding RNAs are continually being recognized as integral players in several basic cellular functions. Malat1 has been shown to coordinate transcriptional regulation of gene expression and inflammatory processes, with multiple studies suggesting a link between Malat1 and the high mobility group box 1 (Hmgb1)/Toll-like receptor 4 (Tlr4) neuroinflammatory cascade. In this research proposal, I want to determine if Malat1 regulates expression of the Hmgb1/Tlr4 pathway in response to ethanol, particularly in astrocytes, a CNS cell-type that regulates neuroimmune responses and plays a pivotal role in controlling behavior. The first aim of this proposal will utilize CRISPR/Cas9 to knockdown Malat1 expression in primary astrocyte cultures and assess ethanol-induced changes in neuroimmune gene expression and the production of cytokines. The second aim will create a novel floxed Malat1 conditional knockout mouse which will be bred to a line of mice expressing the recombinase enzyme Cre selectively in astrocytes. Offspring of this mating will possess astrocyte-specific knockout of Malat1. Assessing the ethanol-induced neuroimmune response and ethanol drinking behavior of these animals will provide insight into the role of both Malat1 and astrocytes in regulating the underlying pathology of AUD. This project is an important step in understanding the functional contribution of the lncRNA Malat1 in AUD and how some of the physiological phenotypes of the disorder may be perpetuated. With few effective treatments currently available for individuals suffering from AUD, results obtained from the proposed studies may point to new prevention strategies and therapeutic targets. Additionally, this project will greatly enhance my graduate training, as it will give me the opportunity to design and perform hypothesis driven research in an exciting new area of research and master innovative techniques. Ultimately, the training provided by this Ruth L. Kirschstein National Research Service Award (F31) will aid in my scientific training as a graduate student, as well as in my professional development as an independent investigator.