Genetic Analysis of Neuromuscular Junction Formation

神经肌肉接头形成的遗传分析

• 批准号: 8204505
• 负责人: JOHN Y KUWADA
• 金额: $ 32.53万
• 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 2013-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8204505
• 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; 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

PROJECT SUMMARY 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.

项目概要 调节乙酰胆碱受体 (AChR) 定位的分子过程 神经肌肉接头 (NMJ) 需要集聚蛋白，这是运动神经元分泌的一种因子。这个过程涉及到 各种其他分子，但对集聚蛋白在肌肉中引发的信号传导过程知之甚少。我们 正在研究斑马鱼的这一过程，斑马鱼是适合进行遗传分析的脊椎动物 鉴定对生物过程重要的基因并分析这些基因的体内功能 基因。我们生成了斑马鱼倦怠突变，其中 AChR 定位错误，并确定了 ennui 基因作为 LRP4 的编码之一，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 在形成 脊椎动物 NMJ。 我们分离出了可行的倦怠突变，该突变在 国家医学杂志。反应降低是由于突触 AChR 急剧减少和高水平的 AChR 错误定位到肌肉末端。突变表型是细胞自主的，并且是外源的 agrin 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 编码肌肉因子的 ennui 基因是集聚蛋白诱导的 AChR 定位到 NMJ 所需的。 通过突变和基因图谱的结合，ennui 基因被鉴定为 lrp4。 基因组分析。 LRP4 是低密度脂蛋白受体家族的成员，由 早期肌肉。尽管最近发现 lrp4 对于 AChR 的正确定位至关重要 小鼠中，人们对 LRP4 如何介导 agrin 信号传导以及它如何与 集聚蛋白引发的信号通路的其他经过充分研究的成分。我们建议探索这些 利用斑马鱼的优势来检查体内基因功能的实验存在问题。 目标 1：我们将通过产生抗体和/或表达 LRP4 来研究 LRP4 如何在肌肉中分布。 荧光标记 LRP4，看看 LRP4 是否与其他已知的 NMJ 成分共定位。 目标 2：我们将分析 LRP4 如何调节在神经支配之前形成的神经 AChR 簇 反义敲低和 LRP4 特定形式表达的组合。 目标 3：我们将确定 LRP4 是否是聚合因子，并了解 LRP4 和 MuSK 如何作为聚合因子 集聚蛋白受体复合物的组成部分，在功能上是相关的。 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。

项目成果

Defective glycinergic synaptic transmission in zebrafish motility mutants.

• DOI: 10.3389/neuro.02.026.2009
• 发表时间: 2009
• 期刊: Frontiers in molecular neuroscience
• 影响因子: 4.8
• 作者: Hirata H;Carta E;Yamanaka I;Harvey RJ;Kuwada JY
• 通讯作者: Kuwada JY

RING finger protein 121 facilitates the degradation and membrane localization of voltage-gated sodium channels

• DOI: 10.1073/pnas.1414002112
• 发表时间: 2015-03-03
• 期刊: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
• 影响因子: 11.1
• 作者: Ogino, Kazutoyo;Low, Sean E.;Hirata, Hiromi
• 通讯作者: Hirata, Hiromi