The Role of Ninein in Ethanol Anxiolysis

项目概要/摘要 酒精使用障碍 (AUD) 是一种复杂的疾病，受遗传变异和多种因素影响。 环境因素。在人群中，焦虑和压力被认为是酗酒的重要驱动因素 乙醇的抗焦虑特性被认为有助于酒精的逐渐增加 消耗和复发。虽然 AUD 和焦虑都有遗传基础 疾病，几乎没有证据来定义和支持驱动乙醇的机制中的遗传变异性 具有抗焦虑药的功效。 Miles 实验室发表了一项先前的研究，使用重组近交 BXD 小鼠 菌株，证明了急性乙醇抗焦虑作用的强大数量性状基因座（QTL）[3]。从这里 QTL，Ninein (Nin) 被认为是驱动抗焦虑变异的强候选数量性状基因。 就像焦虑的明暗转变模型中对乙醇的反应一样[7]。 Ninein 是一种微管相关蛋白 （MAP）位于中心体和细胞质中，锚定微管的负端，并且已被 与轴突生长和分支有关 [6] Ninein 已被证明与 Gsk3b（一种已知的乙醇）相互作用。 迈尔斯实验室发现了与突触可塑性和神经递质运输有关的反应基因 显示可以调节乙醇消耗[28]。选择性剪接变体和外显子特异性相关变化 神经元微管动力学研究表明，Nin 基因组序列的变异性可能驱动遗传 对焦虑和乙醇的行为反应的变化。该项目将研究 Nin 和 Nin 的作用 基础焦虑、乙醇诱导的抗焦虑样活性和乙醇消耗中的转录变异表达 使用受调控的 Nin 敲除模型、脑区域选择性病毒载体基因递送和 CRISPR/Cas9 外显子 跳过分析。这些目标将通过以下具体目标来实现： 1) 描述 使用他莫昔芬诱导的基因敲除，研究区域特异性 Nin 对 C57BL/6J (B6) 小鼠乙醇相关行为的影响。 2） 表征 Ninein 的选择性剪接对 C57BL/6J 的乙醇和焦虑相关行为的作用 (B6) 小鼠，使用 CRISPR/Cas9 病毒载体进行外显子特异性删除。

Project Summary/ Abstract Alcohol Use Disorder (AUD) is a complex disease influenced by both genetic variability and multiple environmental factors. In the population, anxiety and stress are thought to be important drivers of alcohol consumption, with ethanol’s anxiolytic properties suggested as contributing to progressive increases in alcohol consumption and relapse. While there are foundations for genetic underpinnings of both AUD and anxiety disorders, there is little evidence to define and support the genetic variability in the mechanisms driving ethanol’s efficacy as an anxiolytic. The Miles laboratory has published a prior study, using recombinant inbred BXD mouse strains, demonstrating a robust quantitative trait locus (QTL) underlying acute ethanol anxiolysis [3]. From this QTL, Ninein (Nin) was implicated as a strong candidate quantitative trait gene driving variation in the anxiolytic- like response to ethanol in the light-dark transition model of anxiety [7]. Ninein is a microtubule associated protein (MAP) located in the centrosome and cytoplasm that anchors the minus ends of microtubules and has been implicated in axonal growth and branching [6] Ninein has been shown to interact with Gsk3b, a known ethanol- responsive gene implicated in synaptic plasticity and neurotransmitter trafficking that the Miles laboratory has shown to modulate ethanol consumption [28]. Alternative splicing variants and exon-specific associated changes in microtubule dynamics in neurons implicate that variability in Nin genomic sequence could drive genetic variation in behavioral responses to anxiety and ethanol. This project will examine the role of Nin and Nin transcript variant expression in basal anxiety, ethanol-induced anxiolytic-like activity and ethanol consumption using a regulated Nin knockout model, brain region selective viral vector gene delivery and CRISPR/Cas9 exon- skiping analysis. These objectives will be approached via the following specific aims: 1) Characterize role of region-specific Nin on ethanol-related behaviors in C57BL/6J (B6) mice using a tamoxifen induced knockout. 2) Characterize the role of alternative splicing of Ninein on ethanol and anxiety-related behaviors of C57BL/6J (B6) mice, using CRISPR/Cas9 viral vectors in exon-specific deletions.