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

项目摘要 在美国，烟草使用率，包括越来越多地使用电子烟，仍然保持在20%左右， 对个人、家庭和社会造成长期的健康和经济后果。许多个人 吸烟对大脑的潜在神经生物学影响 仍然知之甚少。最近的大规模全基因组关联研究（GWAS）提供了 超过2500个位点与吸烟行为相关的令人信服的证据。了解生物学 这些基因影响吸烟行为的机制可能为研究吸烟行为提供新的视角。 烟草制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，是造成吸烟的许多影响的原因，从最初的 从多方面奖励退出。其作用机制通常归因于其对烟碱的作用。 乙酰胆碱受体（nAChRs）。然而，星形胶质细胞也表达nAChRs， 对尼古丁的依赖越来越多的证据表明，星形胶质细胞积极参与行为 在动物模型和人类中对尼古丁和尼古丁戒断的反应。因此，一些基因 与人类尼古丁行为相关的基因可能通过改变星形胶质细胞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 R21期的体外功能测定，以在功能上评估和优先考虑可能 通过它们在星形胶质细胞中的作用来促进尼古丁的行为。感兴趣的基因将在 体内尼古丁暴露后作为该基因的初始GO NO-GO。条件性基因敲除（cKO）小鼠 然后，将为通过筛选确定的前两个基因建立模型，并且GO NO-GO决策将 基于从星形胶质细胞中去除的选择性和效率。R33阶段将使用cKO小鼠 以确认所选基因的功能性星形胶质细胞效应并评估特定尼古丁表型 通过在星形胶质细胞中选择性地靶向这些基因来影响。最后，从动物的大脑区域 测试将用于转录组研究，以确定新的基因和途径在星形胶质细胞的差异 受基因型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， 我们将是第一个测试星形胶质细胞培养可用于功能评估GWAS作用的想法的人- 在尼古丁相关的神经生物学中发现了基因。我们将利用新的基因试剂靶向GOI， 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以下问题： GOI在烟碱神经反应中的功能，特别是在星形胶质细胞中。所提出的研究是有意义的 因为它将提供对星形胶质细胞和GWAS鉴定的与尼古丁使用相关的基因机制的深入了解， 这对提高尼古丁神经生物学的理解和开发更多的 帮助减少尼古丁使用和戒断症状的有效疗法。

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.