LRP4 signaling in neuromuscular junction formation

描述(由申请人提供)：多PI提案是为了研究LRP4如何调节神经肌肉接头(NMJ)的形成，NMJ是一种突触，对理解突触发生和神经肌肉疾病做出了巨大贡献。NMJ的形成需要运动神经元(MN)和肌肉纤维之间的精确相互作用。在MN到达之前，肌肉纤维预先形成了小的原始AChR簇，这被认为是NMJ形成的关键，尽管目前缺乏遗传证据。在已建立的途径中，MNS释放集聚蛋白来促进突触后发育。它与LRP4结合，LRP4在顺式作用下刺激受体酪氨酸激酶麝香；随后的信号事件导致AChR聚集。然而，关于信号是如何从agrin传递到马斯克的，人们知之甚少。在初步研究中，我们解决了集聚蛋白-LRP4复合体的第一个晶体结构，这为深入了解集聚蛋白信号级联的初始步骤提供了深入的了解。我们对细胞特异性基因敲除(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形成的最初阶段，肌肉LRP4可能是突触前分化的关键。另一方面，MN LRP4可能作为反式集聚蛋白的受体，诱导AChR簇。这些发现中的许多都是出乎意料的，并提出了一些关键问题。LRP4是如何将agrin的信号传递给马斯克的？MN LRP4是否足以诱导NMJ的形成？肌肉纤维前构型对NMJ的形成至关重要吗？突触生成活动对NMJ的形成至关重要吗？肌肉LRP4如何调节运动神经末梢分化？为了解决这些问题，我们将1)通过求解agrin-LRP4-Musk复合体的结构来了解LRP4如何将信号转导到Musk；2)确定MN LRP4是否足以诱导NMJ的形成以及NMJ的形成是否需要肌肉纤维的预构图；以及3)研究LRP4在肌肉细胞中控制突触前分化的机制。这项研究代表了一种协同战略，它利用两个实验室的互补专业知识、优势和现有资源。这些结果将为更好地理解哺乳动物NMJ形成的细胞和分子机制提供帮助。众所周知，神经肌肉疾病的发病机制涉及NMJ结构和功能的异常。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。来自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.