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

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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.