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

基本信息

批准号：
10450066
负责人：
VIVEK KUMAR
金额：
$ 38.28万
依托单位：
JACKSON LABORATORY
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-07-15 至 2025-06-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10450066
关键词：
Actins Acute Affect Alcohols Alleles Amino Acid Substitution Behavior Behavioral Binding Biochemical Biochemistry Biological Assay Biophysics Brain region CRISPR/Cas technology Cell Surface Receptors Cessation of life Cloning Cocaine Communities Complement Complex Consultations Critical Pathways Data Dideoxy Chain Termination DNA Sequencing Discipline Dissection Eating Economics Environment Event FMR1 Family Food Fragile X Syndrome Future Generations Genes Genetic Genetic Models Genome engineering Goals Gold Health Injury Intravenous Knock-out Knowledge Learning Mass Spectrum Analysis Measures Mediating Mediator of activation protein Modeling Molecular Monomeric GTP-Binding Proteins Mus Mutant Strains Mice Mutation Nicotine Palate Pathway interactions Phase Phenotype Play Process Proteins Proteomics Publishing Quantitative Trait Loci Research Research Personnel Rewards Role Series Signal Pathway Signal Transduction Signaling Protein Site Structure Sucrose Testing The Jackson Laboratory Therapeutic Intervention Translations Variant Work addiction base behavioral phenotyping chronic alcohol ingestion cocaine self-administration conditioned place preference cost design drug of abuse effective therapy genetic approach genome editing genome sequencing in vivo insight mesolimbic system mouse genetics mouse model multidisciplinary multidrug abuse mutant neurobiological mechanism new therapeutic target novel polymerization postnatal pre-clinical precise genome editing preference protein protein interaction public health relevance receptor recruit repaired response social targeted treatment therapeutically effective tool whole genome

项目摘要

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.

项目概要成瘾是一种巨大的经济、个人和社会负担，在美国每年造成的损失超过 6000 亿美元。了解成瘾的脆弱性并开发有效的治疗方法需要识别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。这里，我们建议扩展我们之前的工作，克隆出调节成瘾的 QTL 和随后在小鼠亚系中鉴定出 Cyfip2 作为可卡因急性和致敏的调节剂回应。我们和其他人后来证明，这种突变调节食物奖励、尼古丁偏好和酒精偏好（初步数据）。此外，我们还表明 Cyfip2 调节自愿的自我调节 IVSA 检测中可卡因的给药，这是成瘾领域的金标准。 Cyfip2 是信号集线器来自多种途径的整合，包括小 GTPase RAC1、WIRS 结构域受体和 Fragile X 家庭信号。我们假设 Cyfip2 的这种信号整合对于奖励行为至关重要。作为回应根据 PAR-19-278，我们现在建议在小鼠中使用精确的基因组工程来生成和功能验证 Cyfip2 中的 5 个变体（每个变体 1-2 个氨基酸取代）专门扰乱这些信号整合事件。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员陈博士，Cyfip生物物理和结构领域的领军人物。在 R21 阶段（目标 1），我们将利用 Jackson 实验室的小鼠遗传学专业知识，通过 CRISPR/Cas9 生成一组 5 个 Cyfip2 信号突变体。进展到 R33 阶段的具体里程碑是 (i) 突变体的活力，因为 Cyfip2 的敲除是产后致死的，并且 (ii) 缺乏功能性脱靶编辑。然后我们将表征这些突变体全面评估可卡因和自然奖赏行为（R33，目标 2）。为了深入了解为了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使用基于质谱的综合研究（R33，目标 3）对大脑区域进行研究。顺利完成该项目将为科学界提供 5 个成瘾转变的临床前小鼠模型，以及有关特定信号通路的信息，这些信号通路对于成瘾过渡至关重要并且可以作为目标用于治疗。