Dysferlin regulation of acetylcholine signaling at the C. elegans NMJ

描述（由申请人提供）：肌营养不良症，导致进行性肌肉无力和退化的不可治愈的遗传性疾病，影响美国约25万人。许多肌营养不良症的遗传原因是已知的，然而，需要进一步了解肌营养不良症的病理生理学，以制定适当的治疗策略。肢带型肌营养不良症2B型（LGMD 2B）是由Dysferlin的功能缺失突变引起的，Dysferlin调节囊泡融合事件以修复受损的肌肉膜。Dysferlin的丢失如何导致LGMD 2B表型尚不清楚。令人兴奋的结果在C。elegans的研究表明Dysferlin直系同源物fer-1在体壁肌肉中表达，在那里它通过调节乙酰胆碱受体（AChR）在神经肌肉接头（NMJ）的定位而在突触功能中发挥新的作用。将这些发现与之前的Dysferlin介导的囊泡融合模型相结合，表明FER-1/Dysferlin的缺失导致NMJ处含AChR的囊泡融合减少，导致可能导致LGMD 2B表型的突触功能缺陷。 以模式生物C. elegans，三个独立的实验线将用于研究FER-1的这种新的突触作用，更广泛地说，突触后乙酰胆碱（ACh）信号的调节。C.秀丽隐杆线虫是一个强大的遗传系统，用于分析肌肉功能，控制秀丽隐杆线虫的体壁肌肉。秀丽线虫运动在功能上与脊椎动物骨骼肌相当。虽然C.秀丽线虫FER-1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的功能。将进行额外的拯救实验以确定C.秀丽线虫Fer-1和哺乳动物Dysferlin在突触功能的调节中是功能上正交的。尽管fer-1突变体在药理学行为测定和突触后AChRs定位方面表现出缺陷，但fer-1缺失对体壁肌肉活动的影响尚不清楚。因此，体内电生理学方法将用于确定fer-1突变对乙酰胆碱诱发的肌肉电流的影响，并进一步确定C。Elegans FER-1在突触功能中的作用。最后，调节和维持适当的突触后乙酰胆碱（ACh）信号传导的分子机制尚未完全了解。通过对AChR激动剂左旋咪唑耐药的动物进行RNA干扰（RNAi）筛选，将确定其他基因（如fer-1），这些基因是调节ACh信号所必需的。总之，我将通过测试FER-1/Dysferlin在突触功能调节中的新作用和鉴定突触后ACh信号调节所需的新基因，进一步了解NMJ ACh信号转导的分子机制。

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.