Abstract
At the neuromuscular junction (NMJ), acetylcholine receptor (AChR) clustering is mediated by spinal motor neuron (SMN)-derived agrin and its receptors on the muscle, the low-density lipoprotein receptor-related protein 4 (LRP4) and muscle-specific receptor tyrosine kinase (MuSK). Additionally, AChR clustering is mediated by the components of the Wnt pathway. Laser capture microdissection of SMNs revealed that a secreted activator of Wnt signaling, R-spondin 2 (Rspo2), is highly expressed in SMNs. We found that Rspo2 is enriched at the NMJ, and that Rspo2 induces MuSK phosphorylation and AChR clustering. Rspo2 requires Wnt ligands, but not agrin, for promoting AChR clustering in cultured myotubes. Leucine-rich repeat-containing G-protein coupled receptor 5 (Lgr5), an Rspo2 receptor, is also accumulated at the NMJ, and is associated with MuSK via LRP4. Lgr5 is required for Rspo2-mediated AChR clustering in myotubes. In Rspo2-knockout mice, the number and density of AChRs at the NMJ are reduced. The Rspo2-knockout diaphragm has an altered ultrastructure with widened synaptic clefts and sparse synaptic vesicles. Frequency of miniature endplate currents is markedly reduced in Rspo2-knockout mice. To conclude, we demonstrate that Rspo2 and its receptor Lgr5 are Wnt-dependent and agrin-independent regulators of AChR clustering at the NMJ.
Highlights
At the neuromuscular junction (NMJ), acetylcholine receptor (AChR) clustering is mediated by spinal motor neuron (SMN)-derived agrin and its receptors on the muscle, the low-density lipoprotein receptor-related protein 4 (LRP4) and muscle-specific receptor tyrosine kinase (MuSK)
Agrin binds to the low-density lipoprotein receptor-related protein 4 (LRP4) on the postsynaptic membrane and it phosphorylates the muscle-specific receptor tyrosine kinase (MuSK)[7,8], which leads to the facilitation of AChR clustering through a scaffold composed of the subsynaptic structural protein rapsyn[9,10]
In Leucine-rich repeat-containing G-protein coupled receptor 5 (Lgr5)-deficient C2C12 myotubes, agrin increased the length and number of AChR clusters, whereas Rspo[2] could not induce the formation of AChR clusters (Fig. 3G,H and Supplementary Fig. S2D). These results suggest that leucine-rich-repeat– containing G-protein coupled receptor (Lgr)[5] is a receptor for Rspo[2], and is required for phosphorylation of MuSK and AChR clustering mediated by Rspo[2], but not by agrin
Summary
At the neuromuscular junction (NMJ), acetylcholine receptor (AChR) clustering is mediated by spinal motor neuron (SMN)-derived agrin and its receptors on the muscle, the low-density lipoprotein receptor-related protein 4 (LRP4) and muscle-specific receptor tyrosine kinase (MuSK). Agrin binds to the low-density lipoprotein receptor-related protein 4 (LRP4) on the postsynaptic membrane and it phosphorylates the muscle-specific receptor tyrosine kinase (MuSK)[7,8], which leads to the facilitation of AChR clustering through a scaffold composed of the subsynaptic structural protein rapsyn[9,10]. We report that a secreted protein, Rspo[2], is highly expressed in the SMNs and binds to its receptor, Leucine-rich repeat-containing G-protein coupled receptor 5 (Lgr5), at the NMJ. The loss of Rspo[2] in mice compromises AChR clustering, the ultrastructure of the NMJ, and neuromuscular signal transduction
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