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  • br Results br Discussion The

    2024-02-09


    Results
    Discussion The present study uncovers a role of calpain, a family of calcium-dependent protease, in regulating postsynaptic differentiation at the NMJ. Cholinergic activation stimulates calpain, whose inhibition stabilizes AChR clusters in cultured muscle Coenzyme Q10 and in agrin mutant mice. Interestingly, rapsyn interacts with calpain and inhibits calpain activity. Blockade of the rapsyn-calpain interaction enhances CCh-induced AChR cluster dispersal. Furthermore, agrin increases rapsyn interaction with calpain and inhibits CCh-induced calpain activation. One substrate of calpain may be p35, whose cleavage by calpain generates p25, a potent activator of Cdk5 (Ahlijanian et al., 2000, Patrick et al., 1999, Patzke et al., 2003). Cdk5 inhibition counteracts the effects of calpain on AChR cluster dispersal. Together, these results support a model depicted in Figure 8 supporting the following hypothesis: AChR clusters are dispersed in muscle cells by electric activity stimulated by cholinergic inputs; agrin stimulates the interaction of rapsyn, which is spatially localized in synapses, with calpain to inhibit its enzyme activity to allow for formation of new AChR clusters and/or maintenance of existing AChR clusters. Activation of AChRs leads to calcium influx via the ligand-gated ion channel (i.e., AChRs) or indirectly via sodium influx-stimulated calcium from intracellular stores (Allard et al., 1996, Cherednichenko et al., 2004). However, the role of calcium in AChR clustering is complex (Bloch, 1983, Megeath and Fallon, 1998, Tseng et al., 2003). How calcium acts to alter the stability of AChR clusters is yet to be investigated. Calpain is a family of proteases that are activated by calcium. Our findings in this paper suggest a role of calpain in regulating AChR clusters. By using a specific substrate or probing the levels of p25 to monitor calpain activity, we show that CCh causes calpain activation (Figure 1). Furthermore, inhibition of calpain stabilizes AChR clusters in vitro (Kim and Nelson, 2000) (Figure 2) and in vivo (Figure 6, Figure 7), whereas overexpression of calpain promotes cluster dispersal (Figure 5). These results indicate a negative role of calpain in AChR clustering. One possible target of calpain is Cdk5, which is activated by CCh in cultured muscle cells and whose ablation leads to enlarged AChR clusters (Fu et al., 2005, Lin et al., 2005). This notion is supported by the following observations. First, CCh-induced Cdk5 activation was dependent on calpain. Second, CCh treatment caused the accumulation of p25, a potent activator of Cdk5. Third, overexpression of calpain promoted CCh-induced AChR cluster dispersion, which was ameliorated by inhibition of Cdk5. In addition to p25, calpain may also have other substrates. Several molecules have been identified to function downstream of agrin/MuSK signaling to induce AChR clustering (Luo et al., 2002, Luo et al., 2003, Okada et al., 2006, Wang et al., 2003, Weston et al., 2000). It is possible that calpain may also cleave other substrates to destabilize AChR clusters. We note that Cdk5 activation happens shortly after “cholinergic” stimulation (Lin et al., 2005) (data not shown), whereas the production of p25 happens slowly (Figure 1). This suggests that other events, such as phosphorylation modifications (Hallows et al., 2003, Sharma et al., 1999), may also contribute to Cdk5 activation. Intracellular calcium elevation, elicited by synaptic activity, stimulates calpain, which has been implicated in regulating the integrity and localization of synaptic proteins, and synaptic plasticity (Lee et al., 2000, Lynch and Seubert, 1989, Patrick et al., 1999, Siman and Noszek, 1988). Calpain activity is regulated by multiple mechanisms including phosphorylation, autolysis, and interaction with phospholipids (Carafoli and Molinari, 1998, Glading et al., 2002). In addition, calpain activity is also regulated by interacting proteins, mainly calpastatin (Benetti et al., 2001, Carafoli and Molinari, 1998, Glading et al., 2002). Here we identified rapsyn as a regulator for calpain. Rapsyn is a peripheral membrane protein that is essential for AChR clustering and neuromuscular junction formation (Gautam et al., 1995, Lin et al., 2001, Phillips et al., 1991a, Phillips et al., 1991b, Qu et al., 1996). This protein is thought to function downstream of the agrin/MuSK signaling, leading to AChR clustering (Apel et al., 1997). The present study reveals an unexpected mechanism by which rapsyn regulates AChR clusters. Rapsyn interacts with calpain and inhibits calpain activity in vitro and in vivo. The region in calpain for rapsyn interaction is domain III, a region that is known to regulate calpain activity (Hosfield et al., 2001, Rizo and Sudhof, 1998, Tompa et al., 2001). Disruption of the rapsyn-calpain interaction activates calpain and promotes AChR cluster dispersal. Remarkably, p25 levels, which reflect calpain activity, at the synaptic region are lower than that of nonsynaptic region (Figure 4I), supporting the conclusion that calpain is spatially inhibited at the NMJ, most probably by agrin and rapsyn. In agreement with this notion, inhibition of calpain partially prevented the loss of AChR clusters in agrin mutant mice (Figure 6, Figure 7). Recently it has been shown that inhibition or ablation of Cdk5 or ablation of ChAT had a similar rescue effect (Lin et al., 2005, Misgeld et al., 2005). In addition to the rescue phenotypes, AGD/ChAT double mutants had increased presynaptic differentiation, and all AChR clusters were opposed by synaptic nerve terminals in AGD/ChAT or mice (Lin et al., 2005, Misgeld et al., 2005). However, this phenotype was not observed in mice, probably due to the muscle-specific expression of calpastatin. It is possible that the presynaptic phenotypes of ChAT or Cdk5 mutant mice were caused by the ablation of these genes in motor neurons.