Screening for drug targets in a Drosophila model of muscle degeneration

描述（由申请人提供）：肌肉萎缩症是指以一系列症状为典型的多种衰弱性遗传性肌肉疾病，包括进行性肌肉无力和肌肉萎缩。为了研究导致肌肉损失的分子机制，我们建立了果蝇肌肉退化的有效模型。果蝇幼虫体细胞肌肉中蜕皮激素受体（EcR）的组成性表达触发了它们的退化，从而作为一种模型来阐明遗传易感生物肌肉萎缩/退化的分子基础。利用果蝇系统，我们提出以下建议：建立分解代谢和合成代谢信号对ecr介导的肌肉退化的相对贡献：利用遗传和生化技术的结合，我们将研究通过蛋白酶体途径（即分解代谢信号）抑制细胞凋亡、自噬和降解以及胰岛素信号网络（合成代谢信号）的过度刺激对ecr介导的肌肉退化的影响；目标2。确定苍蝇肌肉变性的通用生物标志物，并为随后的遗传筛选生成“肌肉变性报告基因”：我们将比较ecr介导的肌肉变性的mRNA和蛋白质组学表达谱与其他三种已建立的肌肉变性模型（即肌营养不良蛋白模型、parkin/pinkl模型和散发性包涵体肌炎模型），以确定几种肌肉泛变性标志物。最后，我们将开发一种适合肌肉变性的检测方法，最有可能是基于泛变性标记之一的荧光素酶转录报告；和Aim 3。通过转基因RNAi筛选肌肉中的药物靶点，以鉴定EcR介导的肌肉退行性变的抑制因子：使用公开可用的RNAi转基因系，我们将在幼虫肌肉中进行全基因组的体内RNAi筛选（使用Aim 2中开发的基于荧光素酶的报告基因），以鉴定抑制可以改善EcR介导的肌肉退行性变的基因/途径。公共卫生相关性：考虑到果蝇和人类基因组之间的高度保守性，我们预计本研究确定的分子靶点将最终为解决人类肌肉萎缩/退化问题提供线索。综上所述，我们试图提供果蝇中触发或抑制蜕皮激素介导的肌肉萎缩的分子机制的全基因组图谱，最终目标是确定减轻人类肌肉退化的合适药物靶点。

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.