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.

项目摘要 调节乙酰胆碱受体（ACHR）在该分子过程 神经肌肉连接（NMJ）需要Agrin，这是运动神经元分泌的因素。这个过程涉及 其他各种分子，但是艾格林发起的肌肉中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，斑马鱼是可以接受遗传分析的脊椎动物 鉴定基因对生物学过程重要的基因和分析这些基因的体内功能 基因。我们产生了斑马鱼ENNUI突变，其中ACHR被错误定位并确定 ENNUI基因作为一种编码LRP4的编码，即在哺乳动物中适当聚类ACHR所必需。我们 建议使用ENNUI突变体更好地了解LRP4的体内作用 脊椎动物NMJ。 我们分离了活的ENNUI突变，该突变显示 NMJ。响应减少是由于突触ACHR的急剧下降和高水平 ACHR被错误地定位到肌肉的末端。突变表型是细胞自主和外源性的 Agrin诱导了野生型肌肉中的ACHR簇，但没有在Ennui肌肉中。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。 通过突变的遗传映射和 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对于正确定位ACHR至关重要 小鼠，关于LRP4如何介导Agrin信号及其如何与之相互作用的小鼠，鲜为人知 Agrin引起的信号通路的其他精心研究的组件。我们建议探索这些 利用斑马鱼在体内基因功能检查的实验问题。 目标1：我们将通过产生抗体和/或表达来检查LRP4如何在肌肉中分布 荧光标记LRP4，看看LRP4是否与其他已知NMJ组件共定位。 AIM 2：我们将分析LRP4如何调节在通过A神经支配之前形成的动脉ACHR簇 反义敲低和特定形式LRP4的表达的组合。 目标3：我们将确定LRP4是否是一个聚合因素，并查看LRP4和Musk如何 Agrin受体复合物的成分在功能上相关。 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。

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