Genetic Analysis of Neuromuscular Junction Formation

神经肌肉接头形成的遗传分析

• 批准号: 7992358
• 负责人: JOHN Y KUWADA
• 金额: $ 32.56万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 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/7992358
• 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; Health; 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; Receptor Aggregation; 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; receptor; research study; response; tool

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）：调节乙酰胆碱受体（ACHR）在神经肌肉结（NMJ）上的定位的分子过程需要Agrin，这是运动神经元分泌的因素。该过程涉及各种其他分子，但是对阿格林发起的肌肉的信号传导过程知之甚少。我们正在研究斑马鱼中的这一过程，斑马鱼是脊椎动物的遗传分析，以鉴定对这些基因的体内功能的生物学过程和分析重要的基因。我们生成了斑马鱼ENNUI突变，其中ACHR被错误定位并鉴定为ENNUI基因是一个编码LRP4的一种，即在哺乳动物中需要适当的ACHR聚类。我们建议使用ENNUI突变体更好地了解LRP4在脊椎动物NMJ形成中的体内作用。我们分离了可行的ENNUI突变，该突变显示NMJ处的电生理反应降低。响应的降低是由于突触ACHR的急剧下降以及高水平的ACHR降低，将其定位于肌肉的末端。突变表型是细胞自主的，外源Agrin诱导的ACHR簇在野生型肌肉中，但在ENNUI肌肉中却不是。这些结果表明，ENNUI基因编码为Agrin诱导的ACHR定位所需的肌肉因子。通过突变和基因组分析的遗传图的组合将ENNUI基因鉴定为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对于在小鼠中正确定位ACHR至关重要，但鲜为人知LRP4如何介导Agrin信号以及它如何与Agrin启动的信号通路的其他良好研究组件相互作用。我们建议通过实验探索这些问题，这些实验利用斑马鱼的优势来检查体内基因功能。 AIM 1：我们将通过产生抗体和/或表达荧光标记的LRP4的LRP4分布在肌肉中，并查看LRP4是否与其他已知的NMJ成分共定位。 AIM 2：我们将分析LRP4如何通过反义敲低的结合和特定形式的LRP4的表达来调节在神经支配之前形成的动脉ACHR簇。 AIM 3：我们将确定LRP4是否是一个聚集因子，并查看LRP4和MUSK是Agrin受体复合物的组成部分，在功能上是相关的。 AIM 4：我们将测定LRP4和MUSK的相互作用如何影响NMJ的体内发展。公共卫生相关性：斑马鱼突变体可以充当人类神经肌肉疾病的动物模型，例如肌无力重症肌无力和先天性肌无力综合征。 ENNUI突变体的表型让人联想到患有这些疾病的患者的肌肉无力和ACHR缺陷。这使LRP4成为病因未知患者神经肌肉连接疾病的候选基因。 ENNUI突变体可行的事实使其更类似于人类疾病，对于分析治疗剂有用。