Abstract
The dynamic interaction between positive and negative signals is necessary for remodeling of postsynaptic structures at the neuromuscular junction. Here we report that Wnt3a negatively regulates acetylcholine receptor (AChR) clustering by repressing the expression of Rapsyn, an AChR-associated protein essential for AChR clustering. In cultured myotubes, treatment with Wnt3a or overexpression of beta-catenin, the condition mimicking the activation of the Wnt canonical pathway, inhibited Agrin-induced formation of AChR clusters. Moreover, Wnt3a treatment promoted dispersion of AChR clusters, and this effect was prevented by DKK1, an antagonist of the Wnt canonical pathway. Next, we investigated possible mechanisms underlying Wnt3a regulation of AChR clustering in cultured muscle cells. Interestingly, we found that Wnt3a treatment caused a decrease in the protein level of Rapsyn. In addition, Rapsyn promoter activity in cultured muscle cells was inhibited by the treatment with Wnt3a or beta-catenin overexpression. Forced expression of Rapsyn driven by a promoter that is not responsive to Wnt3a prevented the dispersing effect of Wnt3a on AChR clusters, suggesting that Wnt3a indeed acts to disperse AChR clusters by down-regulating the expression of Rapsyn. The role of Wnt/beta-catenin signaling in dispersing AChR clusters was also investigated in vivo by electroporation of Wnt3a or beta-catenin into mouse limb muscles, where ectopic Wnt3a or beta-catenin caused disassembly of postsynaptic apparatus. Together, these results suggest that Wnt/beta-catenin signaling plays a negative role for postsynaptic differentiation at the neuromuscular junction, probably by regulating the expression of synaptic proteins, such as Rapsyn.
Highlights
Wnt is a family of secreted proteins that are implicated in neural development [12, 13], neurite outgrowth, navigation, and synaptogenesis (14 –18)
Wnt3a Inhibits Formation of acetylcholine receptor (AChR) Clusters in Cultured Muscle Cells—To investigate the role of Wnt in postsynaptic differentiation, we examined the expression of different members of Wnt proteins in the mouse limb muscle
Wnt1, -4, -5a, -7a, -8a, and -11 were barely detectable, whereas Wnt3a, barely detectable at embryonic day 12 (E12), gradually increased in developing skeletal muscles and peaked at embryonic day 17 (E17) (Fig. 1A), a pattern coinciding with the period of postsynaptic differentiation at the NMJ in vivo [30]
Summary
Reagents—Antibodies against Rapsyn and MuSK were described in the previous studies [8, 25]. Vector-based siRNAs were encoded by pSUPER vectors, with the following sequences: -catenin, 1 (5Ј-ACCAC CCTGG TGCTG ACTA-3Ј), 2 (5ЈCATGC AGAAT ACAAA TGAT-3Ј), and 3 (5Ј-ACATA ATGAG GACCT ACAC-3Ј); Rapsyn, 233 (5Ј-ATGCT GACTT CCTGC TCGA-3Ј) and 616 (5Ј-GCCAT GAGCC AGTAC CACA-3Ј). Differentiated myotubes transfected with testing plasmids or treated with various reagents were subjected to immunoblotting, immunostaining, or luciferase assay. Quantitative Reverse Transcription-PCR—The mRNA samples from C2C12 myotubes treated with LiCl or NaCl were prepared using Trizol reagent (Invitrogen) and reverse transcribed into cDNA using oligo(dT) primers according to the manufacturer’s instructions. Immunohistochemistry—Mice muscles or muscle cells in culture were stained with the indicated antibodies, washed, and incubated with fluorescein isothiocyanate-conjugated goat anti-rabbit or anti-mouse antibody and together with R-BTX to label the AChR [29]. The muscles were dissected out 6 weeks after electroporation and analyzed for AChR clusters by whole mount staining with R-BTX (see Ref. 29)
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