Dysferlin regulation of acetylcholine signaling at the C. elegans NMJ

Dysferlin 对线虫 NMJ 乙酰胆碱信号传导的调节

• 批准号: 8085729
• 负责人: Jessica E Tanis
• 金额: $ 5.3万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-06-01 至 2012-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8085729
• 关键词: Acetylcholine; Affect; Animal Model; Animals; Behavioral Assay; Biological Models; Caenorhabditis elegans; Cells; Cholinergic Receptors; DYSF gene; Defect; Development; Event; Exhibits; Functional disorder; Genes; Genetic; Hereditary Disease; Lead; Levamisole; Limb-Girdle Muscular Dystrophies; Locomotion; Mediating; Membrane; Modeling; Molecular; Muscle; Muscle Weakness; Muscle function; Muscular Dystrophies; Mutation; Myopathy; Neuromuscular Junction; Neurons; Neurotransmitters; Orthologous Gene; Phenotype; Play; Proteins; RNA Interference; Regulation; Research; Resistance; Role; Signal Transduction; Site; Skeletal Muscle; Synapses; Synaptic Membranes; System; Testing; Therapeutic; United States; Vesicle; acetylcholine receptor agonist; base; cholinergic; in vivo; loss of function mutation; muscle degeneration; mutant; novel; promoter; repaired; research study; synaptic function

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.

描述（由申请人提供）：肌营养不良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，影响着美国约 250,000 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。 2B 型肢带型肌营养不良症 (LGMD2B) 是由 Dysferlin 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 的缺失究竟如何导致 LGMD2B 表型尚不清楚。线虫中令人兴奋的结果表明 Dysferlin 直系同源物 fer-1 在体壁肌肉中表达，通过调节神经肌肉接头 (NMJ) 处乙酰胆碱受体 (AChR) 的定位，在突触功能中发挥新作用。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 的缺失会导致 NMJ 处含有 AChR 的囊泡融合减少，从而导致突触功能缺陷，从而可能导致 LGMD2B 表型。 使用模型生物线虫，将使用三个独立的实验线来研究 FER-1 的这种新的突触作用，以及更广泛地研究突触后乙酰胆碱 (ACh) 信号传导的调节。线虫是用于分析肌肉功能的强大遗传系统，控制线虫运动的体壁肌肉在功能上与脊椎动物骨骼肌相当。尽管线虫 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 在何处发挥作用。将进行额外的拯救实验以确定线虫 fer-1 和哺乳动物 Dysferlin 在突触功能调节中是否具有功能直系同源。尽管 fer-1 突变体在药理学行为测定和突触后 AChR 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，并进一步确定秀丽隐杆线虫 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不完全清楚。通过对对 AChR 激动剂左旋咪唑有抗性的动物进行 RNA 干扰 (RNAi) 筛选，可以鉴定调节 ACh 信号传导所需的其他基因，例如 fer-1。总之，我将通过测试 FER-1/Dysferlin 在突触功能调节中的新作用并识别突触后 ACh 信号调节所需的新基因，进一步了解 NMJ 处 ACh 信号传导的分子机制。