The Role of Ninein in Ethanol Anxiolysis

项目摘要/摘要 酒精使用障碍(AUD)是一种受遗传变异和多种因素影响的复杂疾病 环境因素。在人群中，焦虑和压力被认为是酒精的重要驱动因素 消费，乙醇的抗焦虑特性被认为是酒精逐渐增加的原因 消费和旧病复发。虽然AUD和焦虑的遗传基础是有基础的 在疾病方面，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 作为一种抗焦虑药的功效。迈尔斯实验室已经发表了一项先前的研究，使用重组近交系BXD小鼠 菌株，显示了一个强大的数量性状基因座(QTL)的急性乙醇焦虑症[3]。由此 QTL，Ninein(Nin)是影响抗焦虑基因变异的一个重要候选数量性状基因。 就像在焦虑的明暗转换模型中对酒精的反应一样[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，一直是 与轴突生长和分支有关[6]Ninein已被证明与Gsk3b相互作用，Gsk3b是已知的乙醇- 迈尔斯实验室发现的与突触可塑性和神经递质运输有关的反应基因 显示可以调节乙醇的消耗[28]。选择性剪接变异体和外显子特异性相关改变 在神经元的微管动力学中，Nin基因组序列的可变性可以驱动基因 对焦虑和酒精的行为反应的变化。本项目将考察Nin和Nin的角色 基础焦虑、乙醇诱导的抗焦虑样活性和酒精消费中的转录变异表达 利用受调控的Nin基因敲除模型，脑区选择性病毒载体基因传递和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)确定 使用他莫昔芬诱导的基因敲除对C57BL/6J(B6)小鼠酒精相关行为的区域特异性NIN。2) Ninein选择性剪接对C57BL/6J(B6)细胞酒精和焦虑相关行为的影响 小鼠，使用外显子特异缺失的CRISPR/Cas9病毒载体。

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.