Wnt proteins regulate acetylcholine receptor clustering in muscle cells

Abstract

BackgroundThe neuromuscular junction (NMJ) is a cholinergic synapse that rapidly conveys signals from motoneurons to muscle cells and exhibits a high degree of subcellular specialization characteristic of chemical synapses. NMJ formation requires agrin and its coreceptors LRP4 and MuSK. Increasing evidence indicates that Wnt signaling regulates NMJ formation in Drosophila, C. elegans and zebrafish.ResultsIn the study we systematically studied the effect of all 19 different Wnts in mammals on acetylcholine receptor (AChR) cluster formation. We identified five Wnts (Wnt9a, Wnt9b, Wnt10b, Wnt11, and Wnt16) that are able to stimulate AChR clustering, of which Wnt9a and Wnt11 are expressed abundantly in developing muscles. Using Wnt9a and Wnt11 as example, we demonstrated that Wnt induction of AChR clusters was dose-dependent and non-additive to that of agrin, suggesting that Wnts may act via similar pathways to induce AChR clusters. We provide evidence that Wnt9a and Wnt11 bind directly to the extracellular domain of MuSK, to induce MuSK dimerization and subsequent tyrosine phosphorylation of the kinase. In addition, Wnt-induced AChR clustering requires LRP4.ConclusionsThese results identify Wnts as new players in AChR cluster formation, which act in a manner that requires both MuSK and LRP4, revealing a novel function of LRP4.

Schwann Cells in Neuromuscular Junction Formation and Maintenance

“…Glial-cell-derived TGF␤ was shown to positively regulate NMJ formation (Fuentes-Medel et al, 2012;Kerr et al, 2014), possibly by increasing Agrin gene expression in motoneurons via TGF␤ signaling at motoneuron terminals and somas (Krieglstein et al, 1998). Recently, Wnt ligands were shown to be involved in postsynaptic differentiation (Packard et al, 2002;Henriquez et al, 2008;Koles and Budnik, 2012;Strochlic et al, 2012;Zhang et al, 2012;Barik et al, 2014a;Messéant et al, 2015). Wnts are expressed in glia (Castelo-Branco et al, 2006;Kerr et al, 2014) and are important in clustering postsynaptic receptors on muscles in Drosophila (Kerr et al, 2014).…”

“…The aneural clusters are thought to contribute to forming larger, synaptic AChR aggregates, in response to innervation, and outline a region that attract nerve terminals (Flanagan-Steet et al, 2005;Jing et al, 2009). Nerve induction of AChR clustering requires agrin, an extracellular proteoglycan, which acts by stimulating the Lrp4-MuSK receptor complex (Godfrey et al, 1984;Nitkin et al, 1987;McMahan, 1990;Ruegg et al, 1992;Kim et al, 2008;Zhang et al, 2008;Zong et al, 2012). Primitive, extrasyn-aptic AChR clusters are dispersed by muscle activity, caused by AChR activation by ACh (Avila et al, 1989;Misgeld et al, 2002;Brandon et al, 2003).…”

Wnts contribute to neuromuscular junction formation through distinct signaling pathways

“…Growing evidence in several vertebrate species, using both in vitro and in vivo models, suggests that Wnt morphogens act as regulators of NMJ initiation and/or formation (Gordon et al, 2012;Henriquez et al, 2008;Jing et al, 2009;Messéant et al, 2015;Packard et al, 2002;Strochlic et al, 2012;Zhang et al, 2012). Yet, Wnt function and the molecular mechanisms through which Wnts collaborate at the mammalian NMJ remain elusive and controversial.…”

“…At the vertebrate NMJ, Wnt ligands transduce their signals through activation of the receptor complex formed by the muscle-specific tyrosine kinase MuSK and low-density lipoprotein receptor-related protein 4 (Lrp4) and through the activation of classical Frizzled (Fzd) receptors (Avilés et al, 2014;Zhang et al, 2012;Strochlic et al, 2012;Gordon et al, 2012;Messéant et al, 2015). The MuSK-Lrp4 complex constitutes the central scaffold for the formation of the neuromuscular synapse (DeChiara et al, 1996;Weatherbee et al, 2006;Zhang et al, 2008).…”

“…Among the 19 Wnts currently identified in mammals, Wnt2, 3a, 4, 6, 7b, 9a and 11 directly interact with MuSK but only Wnt4, 9a and 11 enhance AChR clustering in muscle cells (Barik et al, 2014;Strochlic et al, 2012;Zhang et al, 2012). In zebrafish, both Wnt4a and Wnt11r initiate muscle prepatterning, probably by stimulating PCP-dependent MuSK endocytosis in muscle cells (Gordon et al, 2012;Jing et al, 2009).…”