Signaling Mechanisms Behind nAChR Clustering at the NMJ

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。作为这一组织方案的一部分，某些肌肉蛋白，如烟碱型乙酰胆碱受体(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。增强和维持nAChR的NMJ定位的一个主要因素是神经源性蛋白集聚蛋白。Agin通过包括肌肉特异性激酶(Musk)在内的受体复合体发挥作用。虽然穆斯克已经被很好地描述了，但它导致聚集素诱导的nAChR聚集的信号通路仍然知之甚少。阐明这一级联反应的成分可能为未来治疗各种神经和肌肉无力疾病提供见解，如重症肌无力和肌营养不良症。例如，可以设计治疗方法来绕过、增强或抑制级联反应中断的疾病中的特定途径步骤。此外，这一途径可能揭示了突触形成和/或维持的重要机制。为了揭示集聚蛋白-“麝香--nAChR信号通路的最初步骤，利用细菌双杂交实验鉴定了一种与麝香细胞质区域强相互作用的蛋白质--热休克蛋白40同源蛋白(Hsp40h)。在本提案中，假设Musk和hsp40h之间的相互作用是负责集聚蛋白介导的nAChR聚集的信号通路的关键组成部分。本研究旨在证实Musk与hsp40h之间的相互作用，确定Musk与hsp40h之间的相互作用如何影响集聚蛋白介导的nAChR聚集，并探讨hsp40h在NMJ的发育和维持中的作用。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介导的蛋白质敲除以及去神经研究来实现。这项提案中概述的实验很可能为神经肌肉连接的组织机制提供见解。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衰弱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.