Screening for drug targets in a Drosophila model of muscle degeneration

描述(申请人提供)：肌营养不良症是指一组不同的衰弱遗传性肌肉疾病，以一系列症状为典型，包括进行性肌肉无力和肌肉萎缩。为了研究导致肌肉丧失的分子机制，我们建立了一个果蝇肌肉退化的有效模型。蜕皮激素受体(ECR)在果蝇幼虫的躯体肌肉中的结构性表达触发了它们的退化，从而为阐明遗传上易驯化的生物体中肌肉萎缩/退化的分子基础提供了一个模型。利用这个果蝇系统，我们提出如下建议：目的1.建立分解代谢和合成代谢信号在ECR介导的肌肉萎缩中的相对贡献：我们将结合遗传和生化技术，研究通过蛋白酶体途径抑制细胞凋亡、自噬和降解(即分解代谢信号)以及过度刺激胰岛素信号网络(合成代谢信号)在ECR介导的肌肉退化中的作用；目的2.确定果蝇肌肉退变的通用生物标志物，并为后续的遗传筛查生成“肌肉退变报告者”：我们将比较ECR介导的肌肉退行性变与其他三种成熟的肌肉退行性变模型(即dystrophin模型、parkin/pinkl模型和散发性包涵体肌炎模型)的mRNA和蛋白质组表达谱，以确定几种肌肉泛退化标记。最后，我们将开发一种适合肌肉退化的测试方法，最有可能的是基于其中一种泛退化标记的荧光素酶转录报告；以及目标3.通过在肌肉中进行转基因RNAi筛选来确定药物靶点，以确定ECR介导的肌肉退化的抑制因子：使用公开可用的RNAi转基因株，我们将在幼虫肌肉中进行全基因组体内RNAi筛选(使用在目标2中开发的基于荧光素酶的报告)，以确定其抑制可以改善ECR介导的肌肉退化的基因/途径。公共卫生相关性：鉴于果蝇和人类基因组之间的高度保守，我们预计这项研究确定的分子靶标最终将为解决人类肌肉萎缩/退化问题提供线索。总之，我们试图提供一幅全基因组的图像，描述在果蝇中触发或抑制蜕皮激素介导的肌肉萎缩的分子机制，最终目标是确定合适的药物靶点来减轻人类肌肉退化。

DESCRIPTION (provided by applicant): Muscular dystrophy refers to a diverse group of debilitating hereditary muscle disorders typified by a host of symptoms, including progressive muscular weakness and muscle wasting. To examine the molecular mechanisms that contribute to muscle loss, we have established a potent model of muscle degeneration in Drosophila. Constitutive expression of the ecdysone receptor (EcR) in somatic muscles of Drosophila larvae triggers their degeneration, thereby serving as a model for elucidating the molecular underpinnings of muscle atrophy/degeneration in a genetically tractable organism. Using this Drosophila system, we propose the following: Aim 1. Establish the relative contribution of catabolic and anabolic signals to EcR-mediated muscle wasting: Using a combination of genetic and biochemical techniques, we will examine the effect of inhibiting apoptosis, autophagy and degradation via the proteasomal pathway (i.e catabolic signals) as well as hyperstimulation of the insulin signaling network (anabolic signals) on EcR-mediated muscle degeneration; Aim 2. Identify universal biomarkers of muscle degeneration in flies and generate a "muscle degeneration reporter" for subsequent genetic screens: We will compare the mRNA and proteomic expression profiles of EcR-mediated muscle degeneration with that of three other well-established fly models of muscle degeneration (i.e. the dystrophin model, parkin/pinkl model and a sporadic inclusion-body myositis model) to identify several muscle pan-degeneration markers. Finally, we will develop a suitable assay for muscle degeneration, most likely a Luciferase transcriptional reporter based on one of the pan-degeneration markers; and Aim 3. Identify drug targets via a transgenic RNAi screen in muscles to identify suppressors of EcR- mediated muscle degeneration: Using publicly available RNAi transgenic lines, we will carry out a genome- wide in vivo RNAi screen (using the Luciferase-based reporter developed in Aim 2) in larval muscles to identify genes/pathways whose inhibition can ameliorate EcR-mediated muscle degeneration. PUBLIC HEALTH RELEVANCE: Given the high degree of conservation between the Drosophila and human genomes, we anticipate that molecular targets identified by this study will eventually provide leads for addressing muscle wasting/degeneration in humans. In summary, we seek to provide a genome-wide portrait of the molecular mechanisms that either trigger or suppress ecdysone-mediated muscle wasting in Drosophila, with the ultimate goal of identifying suitable drug targets for attenuating muscle degeneration in humans.