Dysferlin regulation of acetylcholine signaling at the C. elegans NMJ

描述（由申请人提供）：肌肉萎缩症是一种无法治愈的遗传性疾病，会导致进行性肌肉无力和退化，在美国约有25万人患有这种疾病。许多肌肉萎缩症的遗传原因是已知的，然而，进一步了解肌肉萎缩症的病理生理需要制定适当的治疗策略。肢体带状肌营养不良2B型（LGMD2B）是由调节囊泡融合事件以修复受损肌肉膜的Dysferlin功能突变缺失引起的。Dysferlin缺失究竟如何导致LGMD2B表型尚不清楚。在秀丽隐杆线虫中令人兴奋的结果表明，Dysferlin同源物fer-1在体壁肌肉中表达，并通过调节神经肌肉接点（NMJ）乙酰胆碱受体（achr）的定位在突触功能中发挥新的作用。将这些发现与先前的Dysferlin介导的囊泡融合模型相结合，表明FER-1/Dysferlin的缺失会导致NMJ中含有achr的囊泡融合减少，从而导致突触功能缺陷，这可能有助于LGMD2B表型。利用模式生物秀丽隐杆线虫，三个独立的实验线将用于研究FER-1在突触中的新作用，以及更广泛的突触后乙酰胆碱（ACh）信号的调节。秀丽隐杆线虫是一个强大的遗传系统，用于分析肌肉功能，控制秀丽隐杆线虫运动的体壁肌肉在功能上与脊椎动物骨骼肌相当。虽然秀丽隐杆线虫fer1在肌肉中表达，但其作用部位尚不清楚。细胞特异性启动子将用于在肌肉或神经元中表达fer-1，以确定fer-1在何处起作用。进一步的修复实验将确定秀丽隐杆线虫fer1和哺乳动物Dysferlin在调节突触功能方面是否在功能上同源。尽管fer-1突变体在药理学行为分析和突触后achr定位中表现出缺陷，但fer-1缺失对体壁肌肉活动的影响尚不清楚。因此，体内电生理方法将用于确定fe -1突变对乙酰胆碱诱发肌电流的影响，并进一步确定秀丽隐杆线虫fe -1在突触功能中的作用。最后，调节和维持突触后乙酰胆碱（ACh）信号的分子机制尚不完全清楚。通过对乙酰胆碱受体激动剂左旋咪唑耐药的动物进行RNA干扰（RNAi）筛选，可以确定调节乙酰胆碱信号所需的其他基因，如fe -1。总之，我将通过测试FER-1/Dysferlin在突触功能调控中的新作用，以及鉴定突触后乙酰胆碱信号调控所需的新基因，进一步了解NMJ中乙酰胆碱信号传导的分子机制。

DESCRIPTION (provided by applicant): The muscular dystrophies, incurable genetic disorders that result in progressive muscle weakness and degeneration, affect about 250,000 people in the United States. Genetic causes of many muscular dystrophies are known, however, further understanding of muscular dystrophy pathophysiology is required to develop appropriate therapeutic strategies. Limb Girdle Muscular Dystrophy type 2B (LGMD2B) is caused by loss of function mutations in Dysferlin, which regulates vesicle fusion events to repair damaged muscle membranes. Exactly how loss of Dysferlin lead to LGMD2B phenotypes is unknown. Exciting results in C. elegans show that the Dysferlin ortholog fer-1 is expressed in body-wall muscles where it plays a novel role in synaptic function by regulating the localization of acetylcholine receptors (AChRs) at the neuromuscular junction (NMJ). Integration of these findings with previous models of Dysferlin- mediated vesicle fusion, suggests that loss of FER-1/Dysferlin causes a reduction in AChR-containing vesicle fusion at the NMJ, leading to defects in synaptic function that may contribute to LGMD2B phenotypes. Using the model organism C. elegans, three independent lines of experimentation will be used to study this novel synaptic role of FER-1 and more broadly, the regulation of post-synaptic acetylcholine (ACh) signaling. C. elegans is a powerful genetic system used for analysis of muscle function, and the body-wall muscles which control C. elegans locomotion are functionally comparable to vertebrate skeletal muscle. Although C. elegans fer-1 is expressed in muscles, its site of action is not known. Cell-specific promoters will be used to express fer-1 in muscles or neurons in order to determine where FER-1 functions. Additional rescue experiments will be performed to determine if C. elegans fer-1 and mammalian Dysferlin are functionally orthologous in the regulation of synaptic function. Although fer-1 mutants exhibit defects in pharmacological behavioral assays and the localization of post-synaptic AChRs, the effect of loss of fer-1 on body-wall muscle activity is unknown. Thus, an in vivo electrophysiological approach will be used to determine the effect of fer-1 mutations on acetylcholine- evoked muscle currents and further define the role of C. elegans FER-1 in synaptic function. Finally, the molecular mechanisms that regulate and maintain proper post-synaptic acetylcholine (ACh) signaling are not fully understood. Additional genes that, like fer-1, are required for the modulation of ACh signaling will be identified by performing an RNA interference (RNAi) screen for animals resistant to the AChR agonist levamisole. In conclusion, I will achieve a further understanding of the molecular mechanisms underlying ACh signaling at the NMJ by testing a novel role for FER-1/Dysferlin in the regulation of synaptic function and identifying novel genes required for regulation of post-synaptic ACh signaling. PUBLIC HEALTH RELEVANCE: Limb Girdle Muscular Dystrophy type 2B (LGMD2B), which is caused by loss of function mutations in the gene Dysferlin, results in progressive muscle weakness. Recent results suggest that loss of Dysferlin causes defects in synaptic function and this may contribute to LGMD2B phenotypes. Our research using C. elegans to study this novel pathophysiological mechanism of LGMD2B may lead to a better understanding of the molecular mechanisms that cause progressive muscle weakness and could lead to the development of new therapies to treat this incurable muscle disease.