Discovering cardiomyopathy modifiers in TOR signaling via zebrafish genetics

通过斑马鱼遗传学发现 TOR 信号传导中的心肌病修饰因子

• 批准号: 8081575
• 负责人: Xiaolei Xu
• 金额: $ 35.15万
• 依托单位: MAYO CLINIC ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-04-01 至 2014-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8081575
• 关键词: Address; Adult; Affect; American; Animal Model; Attenuated; Autophagocytosis; Benchmarking; Biological Assay; Cardiac; Cardiac Myocytes; Cardiomyopathies; Chemicals; Development; Doxorubicin; Energy Metabolism; Experimental Designs; Fishes; Fluorescence; Gene Targeting; Genes; Genetic; Goals; Heart failure; Human Pathology; Insertional Mutagenesis; Label; Mitochondria; Modeling; Molecular Genetics; Mutagenesis; Pathogenesis; Pathway interactions; Phenotype; Phosphotransferases; Proteins; Screening procedure; Signal Pathway; Signal Transduction; Sirolimus; Stress; Testing; Therapeutic; Transgenic Organisms; Zebrafish; base; chemical genetics; drug discovery; gene cloning; genetic resource; in vivo Model; inhibition of autophagy; inhibitor/antagonist; mouse model; mutant; novel; novel therapeutics; response; small molecule; therapeutic gene; tool

DESCRIPTION (provided by applicant): Cardiomyopathy and related heart failure affect millions of Americans. Therefore, pathway-based therapies are highly desirable. The goal of this proposal is to leverage unique genetic tools in zebrafish to elucidate cardioprotective functions of target of rapamycin (TOR) signaling and develop novel therapeutic compounds via modifier screens. There are two obstacles prohibiting modifier screens from being conducted in adult zebrafish: the lack of adult assays to analyze cardiomyopathy-like phenotypes, and the difficulty to track adult mutant fish. To address the former challenge, we established and characterized first adult fish models of cardiomyopathy, including a model induced by Doxorubicin (DOX). To address the latter challenge, we adapted a transposon-based insertional mutagenesis strategy that facilitates the identification of all mutants by a RFP tag. We have conducted both phenotype-based insertional mutagenesis screens and chemical screens, and identified gene modifiers and compound modifiers of TOR signaling that sequentially affect DOX-induced cardiomyopathy. Preliminary studies using these genetic resources suggested that activated autophagy conveys the cardioprotective function of TOR inhibition. In this proposal, we will continue to leverage zebrafish genetics to test our central hypothesis that the cardioprotective functions of TOR inhibition are conferred by the activated autophagy, which can be harnessed by modifier screens in adult zebrafish to identify novel genes and therapeutics for treating cardiomyopathy. In Specific Aim 1, we will determine the conservation of adult zebrafish as a model organism for cardiomyopathy. In Specific Aim 2, we will discern functions of autophagy and pS6K subpathways in cardiomyopathy via phenotyping two modifier mutants that differentially affect TOR signaling. In Specific Aim 3, we plan to discover novel compound modifiers of TOR signaling that could be of greater therapeutic value for cardiomyopathy. At the end of the project, we expect to validate a cardioprotective function of TOR inhibition, to define autophagy as the major downstream signaling branch that confers this cardioprotective function, and to identify compounds that specifically interfere with the TOR-autophagy subpathway that might be of better therapeutic value than rapamycin. Our studies will establish adult zebrafish as a conservative animal model to identify novel modifiers of cardiomyopathy, as well as a complete in vivo model organism to facilitate drug discovery. PUBLIC HEALTH RELEVANCE: This proposal leverages the unique tools in zebrafish to seek TOR signaling-based therapies for cardiomyopathy and heart failure. We aim to use this unique animal model to identify gene modifiers of cardiomyopathy that will elucidate cardioprotective functions of TOR inhibition, and compounds in this pathway with therapeutic value for cardiomyopathy.

描述（由申请人提供）：心肌病和相关心力衰竭影响着数百万美国人。因此，基于途径的治疗非常可取。该提案的目标是利用斑马鱼独特的遗传工具来阐明雷帕霉素靶点（TOR）信号传导的心脏保护功能，并通过修饰筛选开发新型治疗化合物。有两个障碍阻碍在成年斑马鱼中进行修饰筛选：缺乏分析心肌病样表型的成体检测方法，以及追踪成年突变鱼的困难。为了解决前一个挑战，我们建立并表征了第一个成年鱼类心肌病模型，包括阿霉素（DOX）诱导的模型。为了解决后一个挑战，我们采用了基于转座子的插入诱变策略，该策略有助于通过 RFP 标签识别所有突变体。我们进行了基于表型的插入诱变筛选和化学筛选，并鉴定了 TOR 信号传导的基因修饰剂和复合修饰剂，这些修饰剂依次影响 DOX 诱导的心肌病。使用这些遗传资源的初步研究表明，激活的自噬传递了 TOR 抑制的心脏保护功能。在本提案中，我们将继续利用斑马鱼遗传学来测试我们的中心假设，即 TOR 抑制的心脏保护功能是由激活的自噬赋予的，可以通过成年斑马鱼的修饰筛选来利用它来识别治疗心肌病的新基因和疗法。在具体目标 1 中，我们将确定成年斑马鱼作为心肌病模型生物的保护。在具体目标 2 中，我们将通过对两种对 TOR 信号传导产生不同影响的修饰突变体进行表型分析，来辨别自噬和 pS6K 亚通路在心肌病中的功能。在具体目标 3 中，我们计划发现 TOR 信号传导的新型复合修饰剂，这可能对心肌病具有更大的治疗价值。在项目结束时，我们期望验证 TOR 抑制的心脏保护功能，将自噬定义为赋予这种心脏保护功能的主要下游信号分支，并鉴定出特异性干扰 TOR 自噬亚通路的化合物，这些化合物可能比雷帕霉素具有更好的治疗价值。我们的研究将建立成年斑马鱼作为保守的动物模型，以识别心肌病的新型修饰物，以及完整的体内模型生物体，以促进药物发现。 公共健康相关性：该提案利用斑马鱼的独特工具来寻求基于 TOR 信号传导的心肌病和心力衰竭疗法。我们的目标是利用这种独特的动物模型来鉴定心肌病的基因修饰剂，从而阐明 TOR 抑制的心脏保护功能，以及该途径中对心肌病具有治疗价值的化合物。