The Role of Ninein in Ethanol Anxiolysis

项目总结/摘要 酒精使用障碍（AUD）是一种复杂的疾病，受遗传变异和多种遗传因素的影响。 环境因素在人群中，焦虑和压力被认为是酒精的重要驱动力 乙醇的抗焦虑特性被认为是导致酒精摄入量逐步增加的原因 消费和复发。虽然有基础的遗传基础的澳元和焦虑 尽管这些疾病中，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， 作为抗焦虑药的功效。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，证明了一个强大的数量性状基因座（QTL）潜在的急性乙醇抗焦虑[3]。从这个 QTL Ninein（Nin）被认为是一个强有力的候选数量性状基因，驱动抗焦虑基因的变异， 就像在焦虑的明暗过渡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，并已被 参与轴突生长和分支[6] Ninein已被证明与Gsk 3b相互作用，Gsk 3b是一种已知的乙醇- Miles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， 显示调节乙醇消耗[28]。可变剪接变异体和外显子特异性相关变化 在神经元微管动力学中，Nin基因组序列的变异性可以驱动遗传 对焦虑和酒精的行为反应的变化。这个项目将研究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体表达 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 区域特异性Nin对使用他莫昔芬诱导的敲除的C57 BL/6 J（B6）小鼠中乙醇相关行为的影响。（二） 表征Ninein的选择性剪接对C57 BL/6 J（B6）的乙醇和焦虑相关行为的作用 小鼠，使用CRISPR/Cas9病毒载体进行外显子特异性缺失。

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.