Discovering cardiomyopathy modifiers in TOR signaling via zebrafish genetics

描述（由申请人提供）：心肌病和相关的心力衰竭影响数百万美国人。因此，基于途径的疗法是非常期望的。该提案的目标是利用斑马鱼中独特的遗传工具来阐明雷帕霉素靶点（TOR）信号转导的心脏保护功能，并通过修饰剂筛选开发新型治疗化合物。有两个障碍，禁止修改器屏幕上进行成年斑马鱼：缺乏成人分析心肌病样表型，难以跟踪成年突变鱼。为了解决前一个挑战，我们建立了第一个成年鱼心肌病模型，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，便于通过RFP标签识别所有突变体。我们已经进行了基于表型的插入诱变筛选和化学筛选，并确定了依次影响DOX诱导的心肌病的TOR信号传导的基因修饰剂和化合物修饰剂。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。在这项提案中，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，这可以通过成年斑马鱼中的修饰筛选来利用，以确定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式生物的保护。在具体目标2中，我们将通过对两种差异影响TOR信号传导的修饰突变体进行表型分析，来辨别自噬和pS 6 K子通路在心肌病中的功能。在具体目标3中，我们计划发现新的TOR信号传导的化合物修饰剂，这些修饰剂可能对心肌病具有更大的治疗价值。在项目结束时，我们希望验证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，并鉴定特异性干扰TOR-自噬子途径的化合物，其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一个保守的动物模型，以确定新的心肌病修饰剂，以及一个完整的体内模型生物体，以促进药物发现。

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.