Genetic modifiers of Chrna5 deletion in mice: role in nicotine behaviors modulated by the medial habenula-IPN pathway

项目概要/摘要 每10个试图戒烟的人中，只有不到1个人能坚持戒烟1年。这个糟糕的戒烟率是 部分原因是，目前用于帮助戒烟的药物仅为中等剂量， 有效的，最好的。因此，非常需要开发对吸烟更有效的新型药物 停止该项目的目标是使用一种新的遗传策略来确定新的生物靶点， 开发新型戒烟药物的潜力。遗传策略是基于识别 修饰基因，改变小鼠体内的尼古丁反应，这些小鼠在Chrna 5中存在无效突变， 烟碱受体α 5亚基的基因。实际上，修饰基因是有助于生理和/或生理功能的基因。 这些分子过程对感兴趣的行为很重要，但通常在生物体内未被检测到。 它们所修饰的基因中没有干扰。因为Chrna 5的变异改变了尼古丁的风险 人类的依赖性和啮齿动物的研究清楚地表明，Chrna 5对许多尼古丁至关重要， 相关行为，我们认为，确定基因修改的影响，Chrna 5删除尼古丁 行为将揭示与尼古丁依赖相关的新基因，这些基因可能作为新的靶点， 戒烟药物治疗。重要的是，我们计划筛选修饰符的行为不是 不仅依赖于Chrna 5，而且还依赖于内侧缰核-IPN通路， 被认为在尼古丁依赖中起着关键作用。为了确定遗传修饰剂的影响， Chrna5基因缺失对尼古丁行为的影响，我们提出了3个目标。在具体目标1中，我们将培育Chrna 5 null 突变到每种B6-ChrA/J染色体置换菌株（CSS）上，并鉴定 含有修饰基因，用于Chrna 5缺失对三种尼古丁行为的影响，口服尼古丁摄入， 尼古丁戒断的躯体症状和尼古丁条件性位置偏爱。具体目标2： 使用连续的同类菌株对那些携带修饰基因的染色体进行精细作图。通常，3 从CSS菌株开始的几代同源菌株提供了与 任何高分辨率绘图群体。最后，在具体目标3中，我们将使用RNA-seq来鉴定基因 其表达被鉴定的修饰基因改变。重要的是，我们将使用最先进的基因 策略，这将使我们能够检查神经细胞群体中的基因表达，这与神经细胞的功能高度相关。 行为：脚间核的Chrna 5表达细胞。通过将这一目标的结果与 通过目标1和目标2确定修饰基因位点，我们期望缩小潜在候选修饰基因的范围 并识别出受修饰基因特异性影响的途径。总之，我们认为，这一战略将 导致识别以前未知的基因和/或遗传途径，有助于 生理和/或分子过程对尼古丁的反应是重要的。这些基因和/或途径 可以作为新的药物治疗的发展，以帮助戒烟的新目标。

PROJECT SUMMARY/ABSTRACT Less than 1 in 10 individuals who attempt to quit smoking remain abstinent for 1 year. This poor quit rate is driven, in part, by the fact that currently available drugs used to aid in smoking cessation are only moderately effective, at best. Thus, there is a great need to develop novel drugs that are more effective for smoking cessation. It is the goal of this project to use a novel genetic strategy to identify new biological targets for the potential development of novel smoking cessation drugs. The genetic strategy is based upon identifying modifier genes that alter nicotine responses in mice that have a null mutation in Chrna5, the gene that codes for the nicotinic receptor α5 subunit. In effect, modifier genes are genes that contribute to physiological and/or molecular processes that are important for the behavior of interest but that generally go undetected in the absence of a perturbation in the gene that they modify. Because variants in Chrna5 alter risk for nicotine dependence in humans and studies in rodents clearly demonstrate that Chrna5 is critical for many nicotine- related behaviors, we believe that identifying genes that modify the effect of Chrna5 deletion on nicotine behaviors will uncover new genes relevant to nicotine dependence that may serve as novel targets for novel smoking cessation pharmacotherapies. Importantly, the behaviors that we plan to screen for modifiers are not only dependent upon Chrna5, but also dependent uponthe medial habenula-IPN pathway, a neural pathway that is thought to play a critical role in nicotine dependence. To identify genetic modifiers of the effect of Chrna5 deletion on nicotine behaviors, we propose 3 aims. In specific aim 1, we will breed the Chrna5 null mutation onto each of the B6-ChrA/J chromosome substitution strains (CSS) and identify chromosomes that harbor modifier genes for the effect of Chrna5 deletion on three nicotine behaviors, oral nicotine intake, somatic signs of nicotine withdrawal, and nicotine conditioned place preference. For specific aim 2, we will fine map those chromosomes that harbor modifier genes using sequential congenic strains. Typically, 3 generations of congenic strains starting from a CSS strain provides mapping resolution equivalent to that of any high resolution mapping population. Finally, in specific aim 3, we will use RNA-seq to identify genes whose expression is altered by the identified modifier genes. Importantly, we will use a state of the art genetic strategy that will allow us to examine gene expression in a neural cell population that is highly relevant to the behaviors: Chrna5 expressing cells of the interpeduncular nucleus. By combining the results of this aim with modifier loci identified through aims 1 and 2, we expect to narrow the list of potential candidate modifier genes and identify pathways specifically impacted by the modifier genes. In short, we believe that this strategy will lead to the identification of previously unknown genes and/or genetic pathways that contribute to the physiological and/or molecular processes important for the response to nicotine. These genes and/or pathways may serve as novel targets for the development of new pharmacotherapies to aid in smoking cessation.