Genetic Analysis of Neuromuscular Junction Formation

神经肌肉接头形成的遗传分析

基本信息

批准号：
7789609
负责人：
JOHN Y KUWADA
金额：
$ 32.92万
依托单位：
UNIVERSITY OF MICHIGAN AT ANN ARBOR
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-04-01 至 2012-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7789609
关键词：
Accounting Affect Agrin Animal Model Antibodies Axon Behavior Binding Biochemical Biological Assay Biological Process Candidate Disease Gene Cells Cholinergic Receptors Chromosome Mapping Complex Congenital Myasthenic Syndromes Cytoplasmic Tail Development Disease Dystroglycan Embryo Etiology Exhibits Extracellular Domain Family Genes Genetic Genomics Human Immunoprecipitation Integral Membrane Protein Label Length Low Density Lipoprotein Receptor Mammals Mediating Molecular Motor Motor Neurons Mus Muscle Muscle Fibers Muscle Weakness Mutagenesis Mutation Myasthenia Gravis Nerve Neuromuscular Diseases Neuromuscular Junction Neuromuscular Junction Diseases Nonsense Mutation Organism Patients Pattern Phenotype Physiology Process Proteins RNA Splicing Role Siblings Signal Pathway Signal Transduction Staging Swimming Synapses Testing Therapeutic Agents Transcript Vertebrates Zebrafish aggregation factor agrin receptor base computerized data processing dosage gene function genetic analysis in vivo interest loss of function member mutant nerve supply null mutation protein protein interaction public health relevance receptor research study response tool

项目摘要

DESCRIPTION (provided by applicant): The molecular process regulating the localization of acetylcholine receptors (AChRs) at the neuromuscular junction (NMJ) requires agrin, a factor secreted by motor neurons. This process involves various other molecules, but the signaling process in muscles initiated by agrin is poorly understood. We are examining this process in zebrafish which are vertebrates amenable to genetic analysis for the identification of genes important for a biological process and analysis of the in vivo function of these genes. We generated the zebrafish ennui mutation in which AChRs are mislocalized and identified the ennui gene as one encoding for LRP4 that in mammals is required for proper clustering of AChRs. We propose to use the ennui mutants to better understand the in vivo role of LRP4 for the formation of the vertebrate NMJ. We isolated the viable ennui mutation that showed a decreased electrophysiological response at the NMJ. The reduced response was due to a dramatic decrease in synaptic AChRs and high levels of AChRs mislocalized to the ends of muscles. The mutant phenotype is cell autonomous, and exogenous agrin induced AChR clusters in wildtype muscles but not in ennui muscles. These results suggested that the ennui gene encoded for a muscle factor required for agrin-induced localization of AChRs to the NMJ. The ennui gene was identified as lrp4 by a combination of genetic mapping of the mutation and genomic analysis. LRP4 is a member of the low-density lipoprotein receptor family and is expressed by early stage muscles. Although lrp4 was recently found to be critical for proper localization of AChRs in mice, there is little known about how LRP4 may mediate agrin signaling and how it might interact with other well studied components of the agrin-initiated signaling pathway. We propose to explore these issues with experiments that utilize the advantages of zebrafish for examining in vivo gene function. Aim 1: We will examine how LRP4 is distributed in muscle by generating antibodies and/or expression of fluorescently labeled LRP4 and see if LRP4 co-localizes with other known NMJ components. Aim 2: We will analyze how LRP4 regulates aneural AChR clusters that form prior to innervation by a combination of antisense knockdowns and expression of specific forms of LRP4. Aim 3: We will establish whether LRP4 is an aggregation factor and see how LRP4 and MuSK, a component of the agrin receptor complex, are functionally related. Aim 4: We will assay how the interaction of LRP4 and MuSK affects the in vivo development of the NMJ. PUBLIC HEALTH RELEVANCE: Zebrafish mutants could serve as animal models for human neuromuscular disorders such as myasthenia gravis and congenital myasthenic syndromes. The phenotype of ennui mutants is reminiscent of the muscle weakness and AChR deficiencies seen in patients afflicted with these diseases. This makes lrp4 a candidate gene for neuromuscular junction disorders in patients of unknown etiology. The fact that ennui mutants are viable makes them more analogous to human disorders and useful for assaying therapeutic agents.

描述（由申请人提供）：调节神经肌肉接头（NMJ）乙酰胆碱受体（AChR）定位的分子过程需要集聚蛋白（agrin），这是一种由运动神经元分泌的因子。这一过程涉及各种其他分子，但人们对集聚蛋白引发的肌肉信号传导过程知之甚少。我们正在斑马鱼中研究这一过程，斑马鱼是适合进行遗传分析的脊椎动物，以鉴定对生物过程重要的基因并分析这些基因的体内功能。我们生成了斑马鱼 ennui 突变，其中 AChR 定位错误，并将 ennui 基因鉴定为编码 LRP4 的基因，在哺乳动物中，LRP4 是 AChR 正确聚集所必需的。我们建议使用 ennui 突变体来更好地了解 LRP4 在脊椎动物 NMJ 形成中的体内作用。我们分离出了可行的倦怠突变，该突变显示 NMJ 的电生理反应降低。反应降低是由于突触 AChR 急剧减少以及高水平的 AChR 错误定位到肌肉末端。突变表型是细胞自主的，外源集聚蛋白在野生型肌肉中诱导 AChR 簇，但在倦怠肌肉中则不然。这些结果表明 ennui 基因编码了集聚蛋白诱导的 AChR 定位至 NMJ 所需的肌肉因子。通过突变的遗传图谱和基因组分析相结合，ennui 基因被鉴定为 lrp4。 LRP4 是低密度脂蛋白受体家族的成员，由早期肌肉表达。尽管最近发现 lrp4 对于小鼠体内 AChR 的正确定位至关重要，但人们对 LRP4 如何介导集聚蛋白信号传导以及它如何与集聚蛋白启动的信号通路中其他经过充分研究的成分相互作用知之甚少。我们建议通过利用斑马鱼的优势来检查体内基因功能的实验来探索这些问题。目标 1：我们将通过生成抗体和/或荧光标记的 LRP4 表达来检查 LRP4 如何在肌肉中分布，并观察 LRP4 是否与其他已知的 NMJ 成分共定位。目标 2：我们将分析 LRP4 如何通过反义敲低和 LRP4 特定形式的表达相结合来调节神经支配之前形成的神经 AChR 簇。目标 3：我们将确定 LRP4 是否是一种聚集因子，并了解 LRP4 和 MuSK（集聚蛋白受体复合物的一个组成部分）如何在功能上相关。目标 4：我们将分析 LRP4 和 MuSK 的相互作用如何影响 NMJ 的体内发育。公共健康相关性：斑马鱼突变体可以作为人类神经肌肉疾病（例如重症肌无力和先天性肌无力综合征）的动物模型。 ennui 突变体的表型让人想起患有这些疾病的患者中出现的肌肉无力和 AChR 缺陷。这使得 lrp4 成为病因不明的患者神经肌肉接头疾病的候选基因。事实上，无聊突变体是可行的，这使得它们更类似于人类疾病，并且可用于分析治疗剂。