Mechanistic Causes and Therapeutic Strategies for Dystroglycan-related Muscular

这项提案的总体目标是提高我们对肌营养不良蛋白聚糖如何结合的基本理解 层粘连蛋白，并探讨表达LARGE和应用几类药物的有效性， 治疗肌营养不良聚糖相关的肌营养不良症。特别是，一种新的翻译后 磷酸修饰：O-连接的甘露糖，是层粘连蛋白结合聚糖结构的一部分， 将研究α-肌营养不良聚糖在层粘连蛋白结合中的作用。同时，新的抗体只识别未成熟的 将开发具有末端磷酸修饰的α-DG，并用于评估翻译后 从患有各种类型的糖尿病的个体收集的组织或细胞中的α-DG的修饰状态 肌营养不良聚糖病（通过免疫荧光、免疫印迹和放射性同位素标记）。这些研究 将提高我们对肌营养不良蛋白聚糖作为层粘连蛋白受体所需的修饰的理解， 并通过改善生物标志物与临床严重程度的相关性来推进患者诊断。体外实验 与患者细胞的研究表明，糖基转移酶LARGE的过度表达可以绕过 α-肌营养不良聚糖糖基化缺陷在广泛的肌营养不良聚糖病中的应用。这一假设将是 通过评估在不同的肌营养不良聚糖病小鼠模型中预防疾病的能力在体内证实 通过Large转基因的系统性或肌肉特异性表达。这将提供更好的 了解广泛应用大规模治疗的可行性和成功的可能性， 遗传多样的一组营养不良聚糖病。此外，将对现有的临床药物进行测试， 改善营养不良聚糖病小鼠模型中疾病的潜力。待测试的药物组包括 过早终止密码子通读药物（PTC 124）以及类固醇（泼尼松）和PDES抑制剂 （西地那非）;后两种化合物已经显示出治疗非肌营养不良蛋白聚糖的前景 相关的肌肉萎缩症。这些治疗的有效性将通过以下方法进行探索： 细胞培养和体内小鼠研究以及测量肌营养不良蛋白聚糖功能、肌肉生理学和病理学。 拟议的研究将为新的治疗途径提供一个平台，其中一些将有可能 直接应用于营养不良症患者的护理

The overall objectives of this proposal are to improve our basic understanding of how dystroglycan binds laminin, and to explore the effectiveness of expressing LARGE and applying drugs of several classes in treating dystroglycan-related muscular dystrophies. In particular, the role of a novel post-translational phosphate modification¿an O-linked mannose that is part of the laminin-binding glycan structure of alpha-dystroglycan¿in laminin binding will be investigated. Also, new antibodies that recognize only immature alpha-DG with a terminal phosphate modification will be developed and used to evaluate the post-translational modification status of alpha-DG in tissues or cells collected from individuals with various types of dystroglycanopathies (by immunofluorescence, immunoblotting, and radioisotope-labeling). These studies will improve our understanding of the modifications required for dystroglycan to serve as a laminin receptor, and advance patient diagnosis by improving biomarker correlations with clinical severity. In vitro experiments with patient cells have suggested that overexpression of the glycosyltransferase LARGE can bypass alpha-dystroglycan glycosylation defects in a broad range of dystroglycanopathies. This hypothesis will be corroborated In vivo by assessing the ability to prevent disease in different dystroglycanopathy mouse models through either systemic or muscle-specific expression of the Large transgene. This will provide a better understanding of the feasibility and likelihood of success of broadly applying a LARGE-based therapy to the genetically diverse group of dystroglycanopathies. Furthermore, existing clinical drugs will be tested for their potential to ameliorate disease in dystroglycanopathy mouse models. The group of drugs to be tested includes a premature stop-codon readthrough drug (PTC124), as well as steroids (Prednisone) and PDES inhibitors (Sildenafil); the latter two compounds have already shown promise in the treatment of non-dystroglycan related muscular dystrophies. The effectiveness of these treatments will be explored using a combination of cell culture and in vivo mouse studies and measuring dystroglycan function, muscle physiology and pathology. The proposed research will provide a platform for new therapeutic avenues, some of which it will be possible to implement directly in the care of dystroglycanopathy patients