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）：在美国，肌肉营养不良，无法治愈的遗传疾病，导致渐进的肌肉无力和退化，影响了美国约25万人。许多肌肉营养不良的遗传原因是已知的，但是，需要进一步了解肌肉营养不良的病理生理学才能开发出适当的治疗策略。肢体腰围肌肉营养不良2b型（LGMD2B）是由Dysferlin功能突变丧失引起的，该功能突变调节囊泡融合事件以修复受损的肌肉膜。 dysferlin的损失如何导致LGMD2B表型尚不清楚。秀丽隐杆线虫的令人兴奋的结果表明，Dysferlin ortholog Fer-1在身体壁肌肉中表达，在该肌肉中，它通过调节在神经肌肉连接（NMJ）上的乙酰胆碱受体（ACHR）的定位，在突触功能中起新颖的作用。这些发现与先前的dysferlin介导的囊泡融合模型的整合表明，FER-1/Dysferlin的损失会导致NMJ处含ACHR的囊泡融合的减少，从而导致突触功能的缺陷，这可能有助于LGMD2B表型。 使用模型有机体秀丽隐杆线虫，将使用三个独立的实验线来研究FER-1的新型突触作用，更广泛地调节突触后乙酰胆碱（ACH）信号传导。秀丽隐杆线虫是一种强大的遗传系统，用于分析肌肉功能，并且控制秀丽隐杆线虫运动的身体壁肌肉在功能上与脊椎动物骨骼肌相媲美。尽管秀丽隐杆线虫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。将进行其他救援实验，以确定秀丽隐杆线虫FER-1和哺乳动物dysferlin是否在突触功能的调节中是直系同源的。尽管FER-1突变体在药理学行为测定中表现出缺陷和突触后ACHR的定位，但FER-1损失对身体壁肌肉活性的影响尚不清楚。因此，将使用一种体内电生理方法来确定FER-1突变对乙酰胆碱诱发的肌肉电流的影响，并进一步定义了秀丽隐杆线虫FER-1在突触功能中的作用。最后，尚未完全了解调节和维持适当后突触后乙酰胆碱（ACH）信号的分子机制。通过对ACHR激动剂左旋硅烷抗性的动物进行RNA干扰（RNAI）筛查，将确定ACH信号传导调节所需的其他基因。总之，我将通过测试FER-1/Dysferlin在调节突触功能中的新作用并确定调节突触后ACH信号所需的新基因，进一步了解NMJ的分子机制。