Wnt/β-Catenin Signaling Suppresses Rapsyn Expression and Inhibits Acetylcholine Receptor Clustering at the Neuromuscular Junction

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 β-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 β-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/β-catenin signaling in dispersing AChR clusters was also investigated in vivo by electroporation of Wnt3a or β-catenin into mouse limb muscles, where ectopic Wnt3a or β-catenin caused disassembly of postsynaptic apparatus. Together, these results suggest that Wnt/β-catenin signaling plays a negative role for postsynaptic differentiation at the neuromuscular junction, probably by regulating the expression of synaptic proteins, such as Rapsyn. 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 β-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 β-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/β-catenin signaling in dispersing AChR clusters was also investigated in vivo by electroporation of Wnt3a or β-catenin into mouse limb muscles, where ectopic Wnt3a or β-catenin caused disassembly of postsynaptic apparatus. Together, these results suggest that Wnt/β-catenin signaling plays a negative role for postsynaptic differentiation at the neuromuscular junction, probably by regulating the expression of synaptic proteins, such as Rapsyn. The precisely patterned synaptic connections are crucial for normal information flow. At the vertebrate neuromuscular junction (NMJ), 2The abbreviations used are: NMJ, neuromuscular junction; GFP, green fluorescent protein; ACh, acetylcholine; AChR, AChR receptor; HA, hemagglutinin; R-BTX, tetramethylrhodamine-α-bangarotoxin; R5k-Luc, Rapsyn5k-firefly luciferase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; TCF, T-cell-specific transcription factor; DN-TCF, dominant negative TCF; ANOVA, analysis of variance; siRNA, small interfering RNA; CAG, cytomegalovirus early enhancer/chicken β-actin. 2The abbreviations used are: NMJ, neuromuscular junction; GFP, green fluorescent protein; ACh, acetylcholine; AChR, AChR receptor; HA, hemagglutinin; R-BTX, tetramethylrhodamine-α-bangarotoxin; R5k-Luc, Rapsyn5k-firefly luciferase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; TCF, T-cell-specific transcription factor; DN-TCF, dominant negative TCF; ANOVA, analysis of variance; siRNA, small interfering RNA; CAG, cytomegalovirus early enhancer/chicken β-actin. AChR clusters underneath nerve terminals are stabilized by Agrin, a motor neuron-derived glycoprotein, and the AChR-associated protein Rapsyn (1McMahan U.J. Cold Spring Harbor Symp. Quant. Biol. 1990; 55: 407-418Crossref PubMed Scopus (566) Google Scholar, 2Sanes J.R. Lichtman J.W. Annu. Rev. Neurosci. 1999; 22: 389-442Crossref PubMed Scopus (1209) Google Scholar, 3Sanes J.R. Lichtman J.W. Nat. Rev. Neurosci. 2001; 2: 791-805Crossref PubMed Scopus (793) Google Scholar). On the other hand, motor neurons release negative signals to disperse non-innervated clusters and refine clusters at the synapses (4Lin W. Dominguez B. Yang J. Aryal P. Brandon E.P. Gage F.H. Lee K.F. Neuron. 2005; 46: 569-579Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar, 5Misgeld T. Kummer T.T. Lichtman J.W. Sanes J.R. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 11088-11093Crossref PubMed Scopus (179) Google Scholar). The dynamic interactions between positive and negative signals are thought to be necessary for AChR cluster remodeling to better match nerve terminals and to form perforated or pretzel-like structures (2Sanes J.R. Lichtman J.W. Annu. Rev. Neurosci. 1999; 22: 389-442Crossref PubMed Scopus (1209) Google Scholar, 3Sanes J.R. Lichtman J.W. Nat. Rev. Neurosci. 2001; 2: 791-805Crossref PubMed Scopus (793) Google Scholar, 6Marques M.J. Conchello J.A. Lichtman J.W. J. Neurosci. 2000; 20: 3663-3675Crossref PubMed Google Scholar). Genetic studies suggest that ACh may serve as a negative signal, and Agrin acts by counteracting the inhibitory effect of ACh to stabilize AChR clusters (4Lin W. Dominguez B. Yang J. Aryal P. Brandon E.P. Gage F.H. Lee K.F. Neuron. 2005; 46: 569-579Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar, 7Misgeld T. Burgess R.W. Lewis R.M. Cunningham J.M. Lichtman J.W. Sanes J.R. 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In addition, treatment with or expression of in cultured muscle cells or mouse limb muscles, caused dispersion of AChR clusters. we investigated possible mechanisms underlying Wnt3a regulation of AChR clustering. Rapsyn and in the studies (8Chen F. Qian L. Yang Z.H. Huang Y. Ngo S.T. Ruan N.J. Wang J. Schneider C. Noakes P.G. Ding Y.Q. Mei L. Luo Z.G. Neuron. 2007; 55: 247-260Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, Z.G. Wang Q. Zhou J.Z. Wang J. Luo Z. Liu M. He X. Wynshaw-Boris A. Xiong W.C. Lu B. Mei L. Neuron. 2002; 35: 489-505Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar). and and and and was Agrin, mouse and the Rapsyn gene was the at and used as for the of the Rapsyn for the and The into to Rapsyn5k-firefly by with the β-catenin, and Rapsyn, and The that not gene was into as a into Rapsyn to the that to to as Wnt3a was into has fluorescent protein and X. J. Yang Wang Qian L. F. Y. Wang T. Luo Z.G. Nat. 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Wnt3a of AChR in we the effect of Wnt3a on the of AChR clusters. with Agrin for to the formation of AChR clusters and to or or of for the found that Wnt3a the of AChR clusters and and cluster in suggesting that Wnt3a treatment of The in AChR cluster and by Wnt3a was in cells with the effect of in a suggesting the of the Wnt3a Interestingly, the condition treatment caused a in the and of AChR clusters and suggesting a role of Wnt in AChR cluster are in with the that Wnt3a acts as a negative for the of AChR clusters. Wnt the of the that Wnt3a inhibits and AChR clusters, we investigated the of Wnt3a is that Wnt/β-catenin signaling regulates gene expression K. Nusse R. Curr. Dev. 1998; PubMed Scopus Google we that Wnt/β-catenin signaling may regulate AChR clustering by expression of synaptic at the this with or an of GSK3β, of activate Wnt canonical pathway and stabilize treatment with Wnt3a or caused an of β-catenin in muscle cells and Interestingly, these caused a of Rapsyn and the of other synaptic proteins, such as AChR or The effect of Wnt3a or on Rapsyn was not to the on muscle is a for myotubes, was not by Wnt3a or A and Rapsyn a by Agrin the formation of AChR clusters, as shown in a report J. 2003; PubMed Scopus Google Scholar). Together, these results suggest that Wnt/β-catenin signaling negatively regulates AChR probably by down-regulating the expression of Rapsyn. of the of AChR Wnt3a treatment in of β-catenin and inhibited AChR clustering and we investigated the role of β-catenin in AChR clustering. with stabilized β-catenin, a form of β-catenin with and to is to be to C. Y. M. C. Y. Z. X. He X. Cell. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar), and with Agrin to the formation of AChR clusters In with cells with and the cells β-catenin to Agrin the of AChR clusters or cluster was in these cells and Thus, of β-catenin inhibits AChR clustering. Rapsyn the that Wnt3a or treatment the protein level of Rapsyn in muscle cells we Wnt/β-catenin signaling transcription of Rapsyn by or promoter found that the level of Rapsyn was in with a that β-catenin shown in and is to AChR clustering J. 2003; PubMed Scopus Google Scholar). investigate the negative role of Wnt/β-catenin signaling in Rapsyn we a R5k-Luc, that the of the Rapsyn gene and the The activity of the by was in with and with for or for and for found that Wnt3a and an inhibitory effect on Rapsyn promoter activity and of cells with β-catenin also caused a in Rapsyn promoter with an effect in a In with β-catenin shown to the expression of β-catenin caused an in Rapsyn promoter activity β-catenin as a transcription for T-cell-specific transcription transcription V. N. R. B. Science. PubMed Scopus Google Scholar). of β-catenin caused a in the activity of an for Wnt canonical signaling V. N. R. B. Science. PubMed Scopus Google Scholar). of the dominant negative form of TCF, DN-TCF, to β-catenin, inhibited activity of to have effect on Rapsyn promoter activity and of effect on of Rapsyn promoter activity the effect of β-catenin is the in the Rapsyn we on these are in the of the Rapsyn gene found that Rapsyn with the of or to β-catenin Together, these results suggest the activity of β-catenin in inhibiting Rapsyn gene In with this in effect on Agrin-induced formation of AChR clusters Wnt3a Rapsyn to AChR expression of Rapsyn is shown to be inhibited by Wnt3a and the dispersing effect of Wnt3a is indeed caused by the of Rapsyn we the expression of Rapsyn driven by a promoter that not to Wnt regulation in cells with Rapsyn of that has been shown to the expression of Rapsyn (8Chen F. Qian L. Yang Z.H. Huang Y. Ngo S.T. Ruan N.J. Wang J. Schneider C. Noakes P.G. Ding Y.Q. Mei L. Luo Z.G. Neuron. 2007; 55: 247-260Abstract Full Text Full Text PDF PubMed Scopus (79) Google of prevented Agrin the formation of AChR clusters the expression of Rapsyn in cells with Rapsyn we an a of Rapsyn that was to as of prevented the in the formation of AChR clusters caused by and the of Rapsyn to the of in down-regulating Rapsyn The in and of AChR clusters in cells with and with cells with and was probably to the between Rapsyn and is that the of AChR and Rapsyn in muscle cells is for AChR cluster formation J. Biol. Chem. Full Text PDF PubMed Google Scholar, J. Cell Biol. PubMed Scopus Google Scholar). and expression of Rapsyn have been shown to AChR clustering in J. Cell Biol. PubMed Scopus Google Scholar). The dispersing effect of Wnt3a was in these cells. shown in treatment with Wnt3a promoted dispersion of AChR clusters in muscle cells with of and Wnt3a and this effect was not in cells with Rapsyn of and Wnt3a and the effect of siRNA, we the of a and the Rapsyn Wnt3a caused dispersion of AChR clusters in muscle cells with siRNA, and the Rapsyn expression in Rapsyn cells prevented the dispersing effect of Wnt3a Thus, the expression of Rapsyn by Wnt3a is indeed for the dispersing effect of The of Wnt3a or on in the role of Wnt3a and β-catenin in development in we Wnt3a or β-catenin in the muscle of by the electroporation L. A. A. R. L. J.P. J. Proc. Natl. Acad. Sci. U. S. A. 2004; PubMed Scopus Google Scholar, P. 2004; PubMed Scopus Google Scholar, S. R.M. K. S. Neuron. 2007; 55: Full Text Full Text PDF PubMed Scopus Google Scholar). Wnt3a was in a that fluorescent protein and X. J. Yang Wang Qian L. F. Y. Wang T. Luo Z.G. Nat. Cell Biol. 2007; PubMed Scopus Google Scholar). 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A. 2005; 102: 11088-11093Crossref PubMed Scopus (179) Google Scholar, 8Chen F. Qian L. Yang Z.H. Huang Y. Ngo S.T. Ruan N.J. Wang J. Schneider C. Noakes P.G. Ding Y.Q. Mei L. Luo Z.G. Neuron. 2007; 55: 247-260Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). The decrease of release may the of AChR clusters. is also possible that there are or mechanisms in the Nevertheless, these suggest the of β-catenin at the The level and of β-catenin be crucial for the normal and of the is that β-catenin in the nucleus a with to regulate gene expression (19Woodgett J.R. Sci. STKE 2001. 2001; : RE12Google Scholar, 20Moon R.T. Bowerman B. Boutros M. Perrimon N. Science. 2002; 296: 1644-1646Crossref PubMed Scopus (874) Google Scholar). dominant negative of to have effect on AChR clustering and Rapsyn promoter suggesting that β-catenin in probably in a β-catenin is found to the expression of J. Wang W. Y. Zhou Y. Cell. 2002; 2: Full Text Full Text PDF PubMed Scopus Google Scholar). Interestingly, the Rapsyn promoter several and that is responsive to a of including and K. M. J.M. 2003; PubMed Scopus Google Scholar). in the Rapsyn promoter with K. M. J.M. 2003; PubMed Scopus Google Scholar). be to β-catenin regulates Rapsyn expression via these Wnt expression or release has been shown to be by activity in cultured neurons or S. T. V. Neuron. Full Text Full Text PDF PubMed Scopus Google Scholar, J. S.J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google it be of to Wnt expression or release in the muscle or motor is by is also possible that nerve terminals may release factors to Wnt/β-catenin to regulate Rapsyn In addition, other members of the Wnt family may also in development by distinct are to L. Mei for on the and C. X. and X. for valuable and Q. of the for with