LRP4 signaling in neuromuscular junction formation

描述（由申请人提供）：多PI提案是为了研究LRP 4如何调节神经肌肉接头（NMJ）的形成，NMJ是一种对理解突触发生和神经肌肉疾病有很大贡献的突触。NMJ的形成需要运动神经元（MN）和肌纤维之间的精确相互作用。在MN到来之前，肌纤维被预先形成小的原始AChR簇，这被认为是NMJ形成的关键，尽管目前缺乏遗传证据。在已建立的通路中，MN释放聚集蛋白以促进突触后发育。它与LRP 4结合，LRP 4顺式作用以刺激受体酪氨酸激酶MuSK;随后的信号传导事件导致AChR聚集。然而，很少有人知道信号是如何从聚集蛋白到MuSK转导。在初步研究中，我们解决了聚集蛋白-LRP 4复合物的第一个晶体结构，这提供了对聚集蛋白信号级联的初始步骤的深入了解。我们对细胞特异性敲除（KO）和双KO小鼠的研究揭示了LRP 4在NMJ形成中的新功能。例如，肌肉LRP 4可能是关键的突触前分化在NMJ形成的初始步骤。另一方面，MN LRP 4可能作为聚集蛋白的反式受体诱导AChR簇。这些发现中有许多是出乎意料的，并提出了关键问题。LRP 4如何将信号从agrin传递到MuSK？MN LRP 4是否足以诱导NMJ形成？肌纤维预形成对NMJ形成至关重要吗？突触发生活性对NMJ的形成至关重要吗？肌肉LRP 4如何调节运动神经终末分化？为了解决这些问题，我们将1）通过解决聚集蛋白-LRP 4-MuSK复合物的结构来理解信号如何从LRP 4转导到MuSK; 2）确定MN LRP 4是否足以诱导NMJ形成以及肌纤维预图案化是否是NMJ形成所必需的;以及3）研究肌细胞中的LRP 4控制突触前分化的机制。这项研究代表了一种协同战略，利用了两个实验室的互补专业知识、优势和现有资源。结果将提供一个更好的理解细胞以及哺乳动物NMJ形成的分子机制。已知神经肌肉疾病的发病机制涉及异常的NMJ结构和功能。事实上，聚集蛋白/LRP 4/MuSK信号蛋白的突变与先天性肌无力综合征（CMS）有关。包括我们在内的多个实验室的最新证据表明，重症肌无力（MG）患者会产生针对MuSK和LRP 4的抗体。因此，我们的研究将有助于更好地了解这些神经肌肉疾病的发病机制。

DESCRIPTION (provided by applicant): The multi-PI proposal is to investigate how LRP4 regulates the formation of the neuromuscular junction (NMJ), a synapse that has contributed greatly to understanding of synaptogenesis and of neuromuscular disorders. NMJ formation requires precise interaction between motoneurons (MNs) and muscle fibers. In advance of MN arrival, muscle fibers are prepatterned with small, primitive AChR clusters, which are believed to be critical for NMJ formation although genetic evidence is lacking at the moment. In an established pathway, MNs release agrin to promote postsynaptic development. It binds to LRP4 which acts in cis to stimulate the receptor tyrosine kinase MuSK; and ensuing signaling events lead to AChR clustering. However, little is known of how signals are transduced from agrin to MuSK. In preliminary studies, we solved the first crystal structure of an agrin-LRP4 complex, which provides insight into the initial step of the agrin signaling cascade. Our studies of cell-specific knockout (KO) and double KO mice revealed novel functions of LRP4 in NMJ formation. For example, muscle LRP4 may be critical for presynaptic differentiation during initial steps of NMJ formation. On the other hand, MN LRP4 may serve as agrin's receptor in trans to induce AChR clusters. Many of these findings are unexpected and raise critical questions. How does LRP4 relay the signal from agrin to MuSK? Is MN LRP4 sufficient to induce NMJ formation? Is muscle fiber prepatterning critical for NMJ formation? Is the synaptogenic activity critical for NMJ formation? How does muscle LRP4 regulate motor nerve terminal differentiation? To address these questions, we will 1) understand how signal is transduced from LRP4 to MuSK by solving the structure of the agrin- LRP4-MuSK complex; 2) determine if MN LRP4 is sufficient to induce NMJ formation and if muscle fiber prepatterning is necessary for NMJ formation; and 3) investigate mechanisms by which LRP4 in muscle cells controls presynaptic differentiation. The research represents a synergistic strategy that leverages complementary expertise, strengths and existing resources of the two labs. Results will provide a better understanding of cellular as well as molecular mechanisms of mammalian NMJ formation. Pathogenesis of neuromuscular disorders is known to involve abnormal NMJ structure and function. In fact, mutations of agrin/LRP4/MuSK signaling proteins have been implicated in congenital myasthenic syndrome (CMS). Recent evidence from various laboratories including ours indicates that patients with myasthenia gravis (MG) develop antibodies against MuSK and LRP4. Therefore, our research will contribute to a better understanding of pathogenic mechanisms of these neuromuscular disorders.