Signaling Mechanisms Behind nAChR Clustering at the NMJ

描述（由申请人提供）： 神经肌肉接头（NMJ）具有优化的结构，以将信号从神经传递到肌肉。作为这种组织结构的一部分，某些肌肉蛋白质，如烟碱乙酰胆碱受体（nAChR），优先聚集在NMJ。nAChR在NMJ的聚集对于有效的神经和肌肉传递是必不可少的。加强和维持nAChR的NMJ定位的主要因素是神经源性蛋白聚集蛋白。聚集蛋白通过包括肌肉特异性激酶（MuSK）的受体复合物起作用。虽然MuSK已被很好地表征，但其导致聚集蛋白诱导的nAChR聚集的信号通路仍然知之甚少。阐明这一级联反应的组成部分可能会为未来治疗各种神经和肌肉无力疾病（如重症肌无力和肌营养不良）的疗法提供见解。例如，可以设计治疗方法来规避、增强或抑制级联反应被破坏的疾病中通路的特定步骤。此外，该通路可能揭示了突触形成和/或维持的重要机制。为了揭示聚集蛋白-MuSK -nAChR信号传导途径的初始步骤，使用细菌双杂交测定来鉴定与MuSK的胞质区域强烈相互作用的蛋白质，热休克蛋白40同源物（hsp 40 h）。在本提案中，假设MuSK和hsp 40 h之间的相互作用是负责聚集蛋白介导的nAChR聚集的信号通路的关键组成部分。该研究旨在确认MuSK和hsp 40 h之间的相互作用，以确定MuSK和hsp 40 h之间的相互作用如何影响聚集蛋白介导的nAChR的聚集，并检查hsp 40 h在NMJ的发展和维持中的作用。这些目标将通过对培养的肌管和啮齿动物肌肉进行免疫印迹、免疫共沉淀、免疫荧光染色、突变分析、RNAi介导的蛋白质敲低和去神经研究来实现。本提案中概述的实验可能会为神经肌肉接头的组织机制提供见解。许多疾病是由于神经和肌肉的正常结构受到破坏而引起的。因此，研究神经肌肉发育的基本过程对于理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。

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.