LRP4 signaling in neuromuscular junction formation

描述（由申请人提供）：多pi提案是研究LRP4如何调节神经肌肉连接（NMJ）的形成，NMJ是一种突触，对突触发生和神经肌肉疾病的理解有很大贡献。NMJ的形成需要运动神经元（MNs）和肌肉纤维之间的精确相互作用。在MN到来之前，肌肉纤维已经预先形成了小的、原始的AChR簇，这被认为是NMJ形成的关键，尽管目前缺乏遗传证据。在一个已建立的通路中，MNs释放agrin促进突触后发育。它与顺式作用的LRP4结合，刺激受体酪氨酸激酶MuSK；随后的信号事件导致AChR聚集。然而，人们对信号是如何从agrin转导到MuSK的知之甚少。在初步研究中，我们解决了agrin- lrp4复合物的第一个晶体结构，这为agrin信号级联的初始步骤提供了见解。我们对细胞特异性敲除（KO）和双KO小鼠的研究揭示了LRP4在NMJ形成中的新功能。例如，在NMJ形成的初始阶段，肌肉LRP4可能对突触前分化至关重要。另一方面，MN LRP4可能在反式中作为agrin的受体诱导AChR簇。这些发现中有许多是出乎意料的，并提出了关键的问题。LRP4是如何将信号从agrin传递给马斯克的？MN LRP4是否足以诱导NMJ的形成？肌纤维预模式化对NMJ的形成至关重要吗？突触发生活性对NMJ的形成至关重要吗？肌肉LRP4如何调控运动神经末梢分化？为了解决这些问题，我们将1)通过解决agrin- LRP4-MuSK复合物的结构，了解信号如何从LRP4转导到MuSK；2)确定MN LRP4是否足以诱导NMJ的形成，以及NMJ的形成是否需要肌纤维预图案化；3)研究肌肉细胞中LRP4调控突触前分化的机制。该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。研究结果将有助于更好地理解哺乳动物NMJ形成的细胞和分子机制。神经肌肉疾病的发病机制与NMJ结构和功能异常有关。事实上，agrin/LRP4/MuSK信号蛋白的突变与先天性肌无力综合征（CMS）有关。最近来自包括我们在内的多个实验室的证据表明，重症肌无力（MG）患者会产生针对MuSK和LRP4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.