The mammalian neuromuscular junction (NMJ) is a cholinergic synapse between motoneuron axons and skeletal muscles that is essential to control muscle contraction. The acetylcholine receptor (AChR), a ligand gated ion channel, is highly concentrated at the postsynaptic membrane to ensure fast and efficient neurotransmission. NMJ formation requires agrin, a factor utilized by motor neurons to direct postsynaptic differentiation, and MuSK, a receptor tyrosine kinase that is activated by agrin. Auto-activation of MuSK has been implicated in forming aneural AChR clusters (or prepatterning) in advance of the arrival of motor nerve terminals and in guiding motor axons whereas agrin-activation of MuSK is essential for nerved-induced AChR clustering. However, regulation of MuSK is not well understood. Although agrin is able to stimulate MuSK, the two proteins do not interact. Our preliminary studies indicate that MuSK is regulated by two novel proteins: LRP4 and Wnt11. LRP4 is a member of the low-density lipoprotein receptor (LDLR) family. It directly binds to agrin and MuSK, and thus transduces signal from agrin to MuSK. In addition, it is able to activate MuSK in the absence of agrin. On the other hand, soluble Wnt11 co-precipitates with MuSK, suggesting that Wnt11 may be a ligand for the receptor tyrosine kinase. Importantly, treatment of muscle cells with Wnt1 increases AChR clusters in the absence of agrin. These results suggest that MuSK activity is regulated by multiple mechanisms: LRP4 regulates both basal as well as agrin-induced activation of MuSK whereas Wnt11 stimulates MuSK to form AChR clusters in the absence of agrin. This proposal is to investigate how LRP4 and Wnt activate and regulate MuSK by a combination of in vitro and in vivo approaches. We will investigate how LRP4 regulates MuSK activation, how it transduces signal, mechanisms of Wnt regulation of mammalian NMJ formation. Results will provide a better understanding of cellular as well as molecular mechanisms of mammalian NMJ formation. They will also contribute to a better understanding of pathophysiology of muscular dystrophy and to developing strategies of gene therapy and of diagnostic tools.
The long-term goal of our laboratory is to understand molecular mechanisms underlying the formation and maintenance of synapses. This proposal is to investigate how MuSK is regulated by two newly identified proteins: LRP4 and Wnt11. A combination of in vitro and in vivo experiments will be performed. Results will provide a better understanding of cellular as well as molecular mechanisms of mammalian NMJ formation. They will also contribute to a better understanding of pathophysiology of muscular dystrophy and to developing strategies of gene therapy and of diagnostic tools.
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