Dissection of Addiction Relevant Signal Integration by Cyfip2 through Precise Genome Engineering

项目总结 上瘾是一个巨大的经济、个人和社会负担，在美国每年造成超过6000亿美元的损失。 了解上瘾的易感性，并开发有效的治疗方法，需要识别基因和 调节成瘾过程的途径。我们的长期目标是开发新的遗传模型 与成瘾相关的表型，并使用这些模型来表征成瘾的遗传机制。这里, 我们建议扩展我们之前的工作，导致克隆一个调控成瘾和成瘾的QTL 随后在小鼠亚系中鉴定Cyfip2作为可卡因的急性和致敏调节因子 回应。自那以后，我们和其他人已经表明，这种突变调节食物奖励、尼古丁偏好和 饮酒偏好(初步数据)。此外，我们还表明，Cyfip2调节自愿的自我 在静脉注射可卡因测试中使用可卡因，这是成瘾领域的金标准。Cyfip2是信号的中枢 来自多条途径的整合，包括小GTP酶RAC1、WIRS结构域受体和脆性X 家庭信号。我们假设，Cyfip2的这种信号整合对奖励行为至关重要。作为回应 对于PAR-19-278，我们现在建议在小鼠中使用精确的基因组工程来产生和功能验证 Cyfip2的5个变异体(每个1-2个氨基酸替换)专门干扰这些信号整合 事件。这些突变是使用已发表的生化数据并与我们的合作伙伴协商设计的。 研究人员陈博士是青洽会生物物理和结构方面的领军人物。在R21阶段(目标1)，我们将 利用杰克逊实验室的小鼠遗传学专业知识，由CRISPR/Cas9产生一组5个Cyfip2 信号突变体。进展到R33阶段的具体里程碑是：(I)突变体的生存能力，因为 Cyfip2的敲除是出生后致命的，以及(Ii)缺乏功能性的非靶标编辑。然后我们将描述 这些突变体综合了可卡因和自然奖赏行为(R33，目标2)。深入了解 这些行为的潜在机制，我们将确定每个突变体在小鼠中的生化相互作用组 使用基于质谱学的综合研究(R33，Aim 3)研究大脑区域。圆满完成 将为科学界产生5个成瘾转化的临床前小鼠模型，以及 有关特定信号通路的信息，这些信号通路对转化成瘾至关重要，并且可以成为靶向 接受治疗。

PROJECT SUMMARY Addiction is an enormous economic, personal, and social burden, costing over $600 billion per year in the U.S. Understanding vulnerability to addiction, and developing effective therapies, requires identifying the genes and pathways that mediate the addiction process. Our long-term goal is to develop novel genetic models for addiction-relevant phenotypes, and use these models to characterize the genetic mechanisms of addiction. Here, we propose to extend our previous work that led to the cloning of a QTL that regulates addiction and the subsequent identification of Cyfip2 in mouse substrains as a regulator of cocaine acute and sensitized responses. We and others have since shown that this mutation regulates food reward, nicotine preference, and alcohol preference (preliminary data). In addition, we have shown that Cyfip2 regulates voluntary self- administration of cocaine in the IVSA assay, the gold standard in the addiction field. Cyfip2 is a hub for signal integration from multiple pathways, including the small GTPase RAC1, WIRS domain receptors, and Fragile X family signaling. We hypothesize that this signal integration by Cyfip2 is critical for reward behaviors. In response to PAR-19-278, we now propose to use precise genome engineering in mice to generate and functionally validate 5 variants in Cyfip2 (1-2 amino acid substitutions each) that specifically perturb each of these signal integration events. These mutations are designed using published biochemical data and in consultation with our Co- Investigator Dr. Chen, who is a leader in Cyfip biophysics and structure. In the R21 phase (Aim 1), we will leverage the mouse genetics expertise of the Jackson Laboratory to generate by CRISPR/Cas9 a set of 5 Cyfip2 signaling mutants. Specific milestones for progression to the R33 phase are (i) viability of the mutants, since the knockout of Cyfip2 is postnatal lethal, and (ii) the lack of functional off-target edits. We will then characterize these mutants comprehensively for cocaine and natural reward behavior (R33, Aim 2). To gain insight into mechanisms underlying these behaviors, we will determine the biochemical interactome of each mutant in mouse brain regions using a comprehensive mass spectrometry-based study (R33, Aim 3). The successful completion of this project will yield 5 preclinical mouse models of addiction transition for the scientific community, as well as information about specific signaling pathways that are critical for transition to addiction and that can be targeted for therapy.