Neurotransmitter Acetylcholine Negatively Regulates Neuromuscular Synapse Formation by a Cdk5-Dependent Mechanism

Abstract

Synapse formation requires interactions between pre- and postsynaptic cells to establish the connection of a presynaptic nerve terminal with the neurotransmitter receptor-rich postsynaptic apparatus. At developing vertebrate neuromuscular junctions, acetylcholine receptor (AChR) clusters of nascent postsynaptic apparatus are not apposed by presynaptic nerve terminals. Two opposing activities subsequently promote the formation of synapses: positive signals stabilize the innervated AChR clusters, whereas negative signals disperse those that are not innervated. Although the nerve-derived protein agrin has been suggested to be a positive signal, the negative signals remain elusive. Here, we show that cyclin-dependent kinase 5 (Cdk5) is activated by ACh agonists and is required for the ACh agonist-induced dispersion of the AChR clusters that have not been stabilized by agrin. Genetic elimination of Cdk5 or blocking ACh production prevents the dispersion of AChR clusters in agrin mutants. Therefore, we propose that ACh negatively regulates neuromuscular synapse formation through a Cdk5-dependent mechanism.

MuSK controls where motor axons grow and form synapses

“…Our studies show that MuSK overexpression alters this balancing act, since non-synaptic AChR clusters are not extinguished but are maintained in mice that lack Agrin and overexpress MuSK. Taken together with experiments showing that the AChR prepattern is maintained in agrin mutant mice that fail to synthesize ACh (Lin et al, 2005;Misgeld et al, 2005), these studies demonstrate a fine balancing act between the signaling pathways mediated by Agrin/MuSK, which stabilizes the muscle prepattern, and ACh/AChRs, which destabilizes the prepattern. Surprisingly, neuromuscular synapses form and function in mice that lack Agrin and express a low-or high-expressing MuSK transgene.…”

“…In ChAT mutant mice, which fail to synthesize ACh, non-synaptic AChR clusters are not dispersed, so non-synaptic, prepatterned AChR clusters persist while synaptic AChR clusters are stabilized. The mechanisms by which these two signaling pathways converge are poorly understood, but the combination of these two signals, one promoting and one extinguishing AChR clusters, leads to the selective stabilization of postsynaptic differentiation at nascent synapses (Lin et al, 2005;Misgeld et al, 2005). Our studies show that MuSK overexpression alters this balancing act, since non-synaptic AChR clusters are not extinguished but are maintained in mice that lack Agrin and overexpress MuSK.…”

“…Agrin stimulates MuSK and stabilizes postsynaptic differentiation, whereas ACh stimulates AChRs and destabilizes AChR clusters that are not apposed by nerve terminals (Lin et al, 2005;Misgeld et al, 2005). In agrin mutant mice, motor neurons fail to provide Agrin, so prepatterned AChR clusters are extinguished by ACh (Lin et al, 2005;Misgeld et al, 2005).…”

“…Subsequent studies, which examined the roles of Agrin and MuSK in nerveindependent muscle patterning, demonstrated that MuSK, but not Agrin is required to cluster AChRs in the central region of the muscle (Lin et al, 2001;Yang et al, 2001). This nerveindependent prepattern of AChR expression is then modified and sharpened by two neural signals: Agrin, which stabilizes AChR clusters, and acetylcholine (ACh), which extinguishes AChR clusters (Lin et al, 2005;Misgeld et al, 2005). As a consequence, AChR clusters are selectively maintained at nascent synapses and dispersed at non-synaptic sites.…”

Rapsyn carboxyl terminal domains mediate muscle specific kinase–induced phosphorylation of the muscle acetylcholine receptor

“…is prepatterned and AChR clusters occur in the central region of the muscle prior to and even in the absence of neural innervation (Lin et al, 2001, Yang et al, 2001). However, upon innervation, agrin is required for stable aggregation of AChR at nerve-muscle contacts, counteracting an acetylcholine-driven dispersal of AChR that eliminates aneural aggregates (Lin et al, 2005, Misgeld et al, 2005. Indeed, in agrin and MuSK knockout mice, AChR clusters are largely eliminated by birth and the mice die due to an inability to move and breath (DeChiara et al, 1996, Gautam et al, 1996.…”