Mechanisms of unplugged/MuSK-mediated neuromuscular synapse formation

Formation of neuromuscular connectivity involves proper navigation of motor axons from the spinal cord to their muscle targets as well as precise alignment of these motor axons with acetylcholine receptors (AChRs) on the muscle surface. One key molecular player in this process is the mu scle specific kinase, unplugged/MuSK, which is expressed only in muscle cells. When activated by the nerve-derived ligand Agrin, Unplugged/MuSK triggers the accumulation of AChR clusters in the center of muscle fibers. Even before the arrival of motor axons, AChRs accumulate in the center of muscle fibers in an unplugged/MuSK-dependent manner and prefigure the sites of the first neuromuscular synapses. However, how Unplugged/MuSK activation translates into centralized AChR clustering is not well understood. The lab previously showed that Wnt11r acts through Unplugged/MuSK to restrict anueral AChR clusters and motor axons to the center of the muscle fiber. Despite a high efficiency of knockdown, the wnt11r knockdown phenotype was incompletely penetrant when compared to unplugged null mutants, which suggested that other Wnts might be contributing to unplugged/MuSK-mediated signaling. In this thesis work, I show that loss of two noncanonical Wnts, wnt11r and wnt4a, results in axon guidance errors as well as a complete loss of anueral AChR clusters, identical to the loss of the unplugged/MuSK receptor. In vivo, Wnt11r/4a initiate Unplugged/MuSK translocation from the muscle membrane to recycling endosomes, and this transition is critical for AChR localization to future synaptic sites. Moreover, I show that components of the planar cell polarity pathway colocalize to recycling endosomes, and that this localization is unplugged/MuSK dependent. Knockdown of several core components of the planar cell polarity pathway disrupts Unplugged/MuSK translocation to endosomes, AChR localization and axonal guidance. I propose that Wnt-induced trafficking of the Unplugged/MuSK receptor to endosomes initiates a signaling cascade to align pre- with postsynaptic elements. Collectively, these findings suggest a general mechanism by which Wnt signals shape synaptic connectivity through localized receptor endocytosis