Role of glial expression in nicotine behaviors for genes identified through human GWAS

项目总结 烟草使用率，包括越来越多地使用电子烟，在美国保持在20%左右， 给个人、家庭和社会带来长期的健康和经济后果。很多人 谁想戒烟就停不下来，以及吸烟对大脑的潜在神经生物学影响 人们对此仍然知之甚少。最近的大规模全基因组关联研究(GWAS)提供了 2500多个基因座与吸烟行为相关的令人信服的证据。理解生物学 这些基因影响吸烟行为的机制可能会为我们提供对 烟草产品使用的潜在生物机制。众所周知，尼古丁是 烟草中的主要精神活性成分，从一开始就对吸烟的许多影响负责 奖励要从多方面撤资。其作用机制一般归因于对烟碱的作用。 神经元上的乙酰胆碱受体(NAChRs)。然而，星形胶质细胞也表达nAChRs，并可能发挥作用 尼古丁依赖。越来越多的证据表明，星形胶质细胞积极参与行为 动物模型和人类对尼古丁和尼古丁戒断的反应。因此，一些基因 与尼古丁相关的人类行为可能通过改变星形胶质细胞的功能来影响这些行为。 由于对星形胶质细胞在尼古丁行为中的作用知之甚少，该项目旨在使用星形胶质细胞 在R21期进行体外功能试验，从功能上评估和优先选择可能 通过它们在星形胶质细胞中的作用促进尼古丁的行为。感兴趣的基因将在 在尼古丁暴露后的体内，作为该基因的初始Go-no-go。条件性基因敲除(CKO)小鼠 然后将为通过屏幕识别的前两个基因制作模型，并且通过不通过的决定将 基于从星形胶质细胞中去除的选择性和效率。R33期将利用CKO小鼠 确认所选基因对星形胶质细胞的功能作用并评估特定的尼古丁表型 通过选择性地将这些基因定位于星形胶质细胞而受到影响。最后，来自动物的适当的大脑区域 测试将用于转录组研究，以区别地识别星形胶质细胞中的新基因和新途径 受基因或尼古丁暴露的影响。我们的方法在概念和技术上都是创新的，因为 我们将是第一个测试星形胶质细胞培养可以用来从功能上评估GWAS作用的想法- 确定了尼古丁相关神经生物学中的基因。我们将利用新的遗传试剂来靶向GOIS 特别是大脑中的星形胶质细胞。这项研究也是第一次使用转录组学来解决 GOI在尼古丁神经反应中发挥作用，特别是在星形胶质细胞中。这项拟议的研究具有重要意义 因为它将提供对星形胶质细胞和GWA识别的与尼古丁使用相关的基因机制的洞察， 这对于更好地理解尼古丁神经生物学和开发更多 有效的治疗方法有助于减少尼古丁的使用和戒断症状。

PROJECT SUMMARY Tobacco use rates, including the increasing use of electronic cigarettes, remain at ~20% in the United States, leading to long-term health and financial consequences for individuals, families, and society. Many individuals who desire to quit smoking cannot stop, and the underlying neurobiological effects of smoking on the brain remain poorly understood. Recent large-scale genome-wide association studies (GWAS) have provided convincing evidence for the association of over 2500 loci with smoking behaviors. Understanding the biological mechanisms through which these genes impact smoking behavior may provide novel insights into the underlying biological mechanisms responsible for the use of tobacco products. It is well known that nicotine is the main psychoactive component in tobacco and is responsible for many of the effects of smoking, from initial reward to many aspects of withdrawal. Its mechanism of action generally is attributed to its action on nicotinic acetylcholine receptors (nAChRs) on neurons. However, astrocytes also express nAChRs and may play a role in nicotine dependence. Accumulating evidence indicates that astrocytes actively participate in behavioral responses to nicotine and nicotine abstinence in both animal models and humans. Therefore, some genes associated with nicotine behaviors in humans may impact these behaviors by altering astrocyte function. Because little is known about the role of astrocytes in nicotine behaviors, this project aims to use an astrocyte in vitro functional assay in the R21 phase to functionally assess and prioritize nicotine GWAS genes that may contribute to nicotine behaviors through their actions in astrocytes. The genes of interest will be validated in vivo following nicotine exposure as an initial GO NO-GO for that gene. Conditional knock-out (cKO) mouse models will then be made for the top two genes identified through the screen, and the GO NO-GO decision will be based on the selectivity and efficiency of removal from astrocytes. The R33 phase will utilize the cKO mice to confirm functional astrocyte effects of the selected genes and assess the specific nicotine phenotypes impacted by targeting these genes selectively in astrocytes. Finally, appropriate brain regions from animals tested will be used for transcriptome studies to identify novel genes and pathways in astrocytes differentially affected by genotype or nicotine exposure. Our approach is conceptually and technically innovative because we will be the first to test the idea that astrocyte culture can be used to functionally assess the role of GWAS- identified genes in nicotine-related neurobiology. We will utilize novel genetic reagents to target GOIs in astrocytes in the brain specifically. This study is also the first to use transcriptomics to address questions of GOI function in nicotinic neural responses, specifically in astrocytes. The proposed research is significant because it will provide insight into astrocyte and GWAS identified gene mechanisms related to nicotine use, which has important implications for improved understanding of nicotine neurobiology and developing more effective therapies to help reduce nicotine use and withdrawal symptoms.