Signaling Mechanisms Behind nAChR Clustering at the NMJ

NMJ nAChR 聚类背后的信号机制

• 批准号: 7331196
• 负责人: JENNY J LINNOILA
• 金额: $ 4.47万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-07-01 至 2009-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7331196
• 关键词: Adult; Affect; Agrin; Autoantibodies; Bacteria; Binding; Biochemical; Biological Assay; Cells; Chromosome Pairing; Complex; Condition; Denervation; Development; Disease; Disruption; Drosophila genus; Electroporation; Embryo; Future; Goals; Homologous Gene; Human; Immunoblotting; Kinetics; Maintenance; Mediating; Morphology; Motor Neurons; Mus; Muscle; Muscle Fibers; Muscle Weakness; Muscle-Specific Kinase; Muscular Dystrophies; Myasthenia Gravis; N-terminal; Nerve; Neurologic; Neuromuscular Junction; Nicotinic Receptors; Numbers; Pathway interactions; Pattern; Personal Satisfaction; Process; Protein Overexpression; Proteins; RNA Interference; Rattus; Receptor Signaling; Research; Rodent; Role; Scheme; Signal Pathway; Signal Transduction; Staining method; Stains; Structure; Synapses; Techniques; Tumor Suppressor Proteins; Two-Hybrid System Techniques; Variant; Western Blotting; Yeasts; design; heat-shock proteins 40; imaginal disc; insight; knock-down; neurotransmission; postnatal; protein expression; receptor; relating to nervous system; research study; small hairpin RNA; transmission process; yeast two hybrid system

DESCRIPTION (provided by applicant): The neuromuscular junction (NMJ) has a structure that is optimized to relay signals from nerve to muscle. As part of this organizational scheme, certain muscular proteins, like the nicotinic acetylcholine receptor (nAChR), are clustered preferentially at the NMJ. Clustering of the nAChR at the NMJ is essential for efficient nerve and muscle transmission. A major factor that strengthens and sustains the NMJ localization of the nAChR is the neural-derived protein agrin. Agrin acts via a receptor complex that includes muscle-specific kinase (MuSK). Although MuSK has been well characterized, the signaling pathway by which it leads to agrin-induced clustering of the nAChR remains poorly understood. Elucidation of the components of this cascade may provide insights into future therapies for the treatment of a variety of neurological and muscle weakness disorders, such as myasthenia gravis and muscular dystrophy. For instance, treatments can be designed to circumvent, enhance, or inhibit specific steps of the pathway in diseases where the cascade is disrupted. In addition, this pathway may reveal mechanisms important for the formation and/or maintenance of synapses. In an effort to uncover the initial steps of the agrin -" MuSK -> nAChR signaling pathway, a bacterial two hybrid assay was used to identify a protein, a heat shock protein 40 homologue (hsp40h), that interacts strongly with the cytoplasmic region of MuSK. In the present proposal, it is hypothesized that interaction between MuSK and hsp40h is a key component of the signaling pathway responsible for agrin-mediated clustering of the nAChR. The proposed research aims to confirm the interaction between MuSK and hsp40h, to determine how interaction between MuSK and hsp40h affects agrin-mediated clustering of the nAChR, and to examine hsp40h's role in the development and maintenance of the NMJ. These goals will be accomplished by performing immunoblotting, coimmunoprecipitation, immunofluorescent staining, mutational analyses, RNAi-mediated protein knock-down, and denervation studies with cultured myotubes and rodent muscles. The experiments outlined in this proposal are likely to provide insights into the mechanisms responsible for the organization of the neuromuscular junction. Many diseases result from disruptions of the normal structures of nerves and muscles. Thus, studying the fundamental process of neuromuscular development is critical to understanding the causes of and to designing more effective treatments for these debilitating conditions.

描述（由申请人提供）： 神经肌肉接头 (NMJ) 的结构经过优化，可以将信号从神经传递到肌肉。作为该组织方案的一部分，某些肌肉蛋白，如烟碱乙酰胆碱受体 (nAChR)，优先聚集在 NMJ。 nAChR 在 NMJ 处的聚集对于有效的神经和肌肉传递至关重要。加强和维持 nAChR 的 NMJ 定位的一个主要因素是神经源蛋白 agrin。集聚蛋白通过包含肌肉特异性激酶 (MuSK) 的受体复合物发挥作用。尽管 MuSK 已得到很好的表征，但它导致集聚蛋白诱导的 nAChR 聚集的信号通路仍然知之甚少。阐明该级联的组成部分可能会为治疗各种神经和肌肉无力疾病（例如重症肌无力和肌营养不良症）的未来疗法提供见解。例如，可以设计治疗方法来规避、增强或抑制级联被破坏的疾病中途径的特定步骤。此外，该途径可能揭示对突触形成和/或维持重要的机制。为了揭示集聚蛋白 -” MuSK -> nAChR 信号通路的初始步骤，使用细菌二杂交测定来鉴定一种蛋白质，即热休克蛋白 40 同源物 (hsp40h)，它与细菌的细胞质区域强烈相互作用。 MuSK。在本提案中，假设 MuSK 和 hsp40h 之间的相互作用是负责 agrin 介导的 nAChR 聚类的信号通路的关键组成部分。目的是确认 MuSK 和 hsp40h 之间的相互作用，确定 MuSK 和 hsp40h 之间的相互作用如何影响 agrin 介导的 nAChR 聚类，并检查 hsp40h 在 NMJ 的发育和维持中的作用。这些目标将通过进行免疫印迹来实现。免疫共沉淀、免疫荧光染色、突变分析、RNAi 介导的蛋白质敲除以及培养肌管和去神经研究该提案中概述的实验可能会深入了解负责神经肌肉接头组织的机制。许多疾病是由神经和肌肉的正常结构破坏引起的。因此，研究神经肌肉发育的基本过程对于了解这些使人衰弱的疾病的原因和设计更有效的治疗方法至关重要。