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

Cyfip2 通过精确基因组工程解析成瘾相关信号整合

• 批准号: 10424633
• 负责人: VIVEK KUMAR
• 金额: $ 40.47万
• 依托单位: JACKSON LABORATORY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-07-15 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10424633
• 关键词: Dissection; Addiction; Relevant; Signal; Integration

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.

项目摘要 成瘾是一个巨大的经济，个人和社会负担，在美国每年花费超过6000亿美元。 了解成瘾的脆弱性，并开发有效的治疗方法，需要确定基因和 介导成瘾过程的途径。我们的长期目标是开发新的遗传模型， 成瘾相关的表型，并使用这些模型来表征成瘾的遗传机制。在这里， 我们建议扩展我们先前的工作，克隆一个调控成瘾的QTL， 随后在小鼠亚系中鉴定Cyfip 2作为可卡因急性和致敏调节剂 应答我们和其他人已经证明，这种突变调节食物奖励，尼古丁偏好， 酒精偏好（初步数据）。此外，我们已经表明，Cyfip 2调节自愿自我- 在IVSA分析中，可卡因的施用是成瘾领域的金标准。Cyfip 2是一个信号中枢 来自多个途径的整合，包括小GT3 RAC 1、WIRS结构域受体和脆性X 家庭信号我们假设Cyfip 2的这种信号整合对奖励行为至关重要。响应 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 Cyfip 2中的5种变体（各1-2个氨基酸取代），特异性干扰这些信号整合中的每一种 事件这些突变是使用已发表的生物化学数据设计的，并与我们的合作伙伴进行了磋商。 陈博士是Cyfip生物物理学和结构的领导者。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，通过CRISPR/Cas9产生一组5个Cyfip 2 信号突变体进展到R33期的具体里程碑是（i）突变体的活力，因为突变体的活力是由突变体的存活率决定的。 Cyfip 2的敲除是出生后致死的，和（ii）缺乏功能性脱靶编辑。然后我们将描述 这些突变体全面的可卡因和自然奖励行为（R33，目标2）。深入了解 这些行为背后的机制，我们将确定小鼠中每个突变体的生化相互作用组 大脑区域使用全面的质谱为基础的研究（R33，目标3）。圆满完成 该项目将为科学界提供5种成瘾过渡的临床前小鼠模型，以及 关于特定信号通路的信息，这些信号通路对成瘾的过渡至关重要，并且可以被靶向 治疗