Neuregulin-1 potentiates agrin-induced acetylcholine receptor clustering through muscle-specific kinase phosphorylation

At neuromuscular synapses, neural agrin (n-agrin) stabilizes embryonic postsynaptic acetylcholine receptor (AChR) clusters by signalling through the muscle-specific kinase (MuSK) complex. Live imaging of cultured myotubes showed that the formation and disassembly of primitive AChR clusters is a dynamic and reversible process favoured by n-agrin, and possibly other synaptic signals. Neuregulin-1 is a growth factor that can act through muscle ErbB receptor kinases to enhance synaptic gene transcription. Recent studies suggest that neuregulin-1–ErbB signalling can modulate n-agrin-induced AChR clustering independently of its effects on transcription. Here we report that neuregulin-1 increased the size of developing AChR clusters when injected into muscles of embryonic mice. We investigated this phenomenon using cultured myotubes, and found that in the ongoing presence of n-agrin, neuregulin-1 potentiates AChR clustering by increasing the tyrosine phosphorylation of MuSK. This potentiation could be blocked by inhibiting Shp2, a postsynaptic tyrosine phosphatase known to modulate the activity of MuSK. Our results provide new evidence that neuregulin-1 modulates the signaling activity of MuSK and hence might function as a second-order regulator of postsynaptic AChR clustering at the neuromuscular synapse. Thus two classic synaptic signalling systems (neuregulin-1 and n-agrin) converge upon MuSK to regulate postsynaptic differentiation

Molecular control of neuromuscular junction development

Skeletal muscle innervation is a multi-step process leading to the neuromuscular junction (NMJ) apparatus formation. The transmission of the signal from nerve to muscle occurs at the NMJ level. The molecular mechanism that orchestrates the organization and functioning of synapses is highly complex, and it has not been completely elucidated so far. Neuromuscular junctions are assembled on the muscle fibers at very precise locations called end plates (EP). Acetylcholine receptor (AChR) clusterization at the end plates is required for an accurate synaptic transmission. This review will focus on some mechanisms responsible for accomplishing the correct distribution of AChRs at the synapses. Recent evidences support the concept that a dual transcriptional control of AChR genes in subsynaptic and extrasynaptic nuclei is crucial for AChR clusterization. Moreover, new players have been discovered in the agrin–MuSK pathway, the master organizer of postsynaptical differentiation. Mutations in this pathway cause neuromuscular congenital disorders. Alterations of the postynaptic apparatus are also present in physiological conditions characterized by skeletal muscle wasting. Indeed, recent evidences demonstrate how NMJ misfunctioning has a crucial role at the onset of age-associated sarcopenia

Src, Fyn, and Yes are not required for neuromuscular synapse formation but are necessary for stabilization of agrin-induced clusters of acetylcholine receptors

Mice deficient in src and fyn or src and yes move and breathe poorly and die perinatally, consistent with defects in neuromuscular function. Src and Fyn are associated with acetylcholine receptors (AChRs) in muscle cells, and Src and Yes can act downstream of ErbB2, suggesting roles for Src family kinases in signaling pathways regulating neuromuscular synapse formation. We studied neuromuscular synapses in src(-/-); fyn(-/-) and src(-/-); yes(-/-) mutant mice and found that muscle development, motor axon pathfinding, clustering of postsynaptic proteins, and synapse-specific transcription are normal in these double mutants, showing that these pairs of kinases are not required for early steps in synapse formation. We generated muscle cell lines lacking src and fyn and found that neural agrin and laminin-1 induced normal clustering of AChRs and that agrin induced normal tyrosine phosphorylation of the AChR beta subunit in the absence of Src and Fyn. Another Src family member, most likely Yes, was associated with AChRs and phosphorylated by agrin in myotubes lacking Src and Fyn, indicating that Yes may compensate for the loss of Src and Fyn. Nevertheless, PP1 and PP2, inhibitors of Src-class kinases, did not inhibit agrin signaling, suggesting that Src class kinase activity is dispensable for agrin-induced clustering and tyrosine phosphorylation of AChRs. AChR clusters, however, were less stable in myotubes lacking Src and Fyn but not in PP1- or PP2-treated wild-type cells. These data show that the stabilization of agrin-induced AChR clusters requires Src and Fyn and suggest that the adaptor activities, rather than the kinase activities, of these kinases are essential for this stabilization

A single pulse of agrin triggers a pathway that acts to cluster acetylcholine receptors

Agrin triggers signaling mechanisms of high temporal and spatial specificity to achieve phosphorylation, clustering, and stabilization of postsynaptic acetylcholine receptors (AChRs). Agrin transiently activates the kinase MuSK; MuSK activation has largely vanished when AChR clusters appear. Thus, a tyrosine kinase cascade acts downstream from MuSK, as illustrated by the agrin-evoked long-lasting activation of Src family kinases (SFKs) and their requirement for AChR cluster stabilization. We have investigated this cascade and report that pharmacological inhibition of SFKs reduces early but not later agrin-induced phosphorylation of MuSK and AChRs, while inhibition of Abl kinases reduces late phosphorylation. Interestingly, SFK inhibition applied selectively during agrin-induced AChR cluster formation caused rapid cluster dispersal later upon agrin withdrawal. We also report that a single 5-min agrin pulse, followed by extensive washing, triggered long-lasting MuSK and AChR phosphorylation and efficient AChR clustering. Following the pulse, MuSK phosphorylation increased and, beyond a certain level, caused maximal clustering. These data reveal novel temporal aspects of tyrosine kinase action in agrin signaling. First, during AChR cluster formation, SFKs initiate early phosphorylation and an AChR stabilization program that acts much later. Second, a kinase mechanism rapidly activated by agrin acts thereafter autonomously in agrin's absence to further increase MuSK phosphorylation and cluster AChRs

Acetylcholine receptors are required for agrin-induced clustering of postsynaptic proteins

We have investigated the role of acetylcholine receptors (AChRs) in an early step of postsynaptic assembly at the neuromuscular synapse, the clustering of postsynaptic proteins induced by nerve-released agrin. To achieve this, we used two variants of C2 myotubes virtually lacking AChRs and C2 cells in which surface AChRs were down-regulated by AChR antibodies. In all cases, agrin caused normal clustering of the agrin receptor component MuSK, alpha-dystrobrevin and utrophin, but failed to aggregate AChRs, alpha- and beta-dystroglycan, syntrophin isoforms and rapsyn, an AChR-anchoring protein necessary for postsynaptic assembly and AChR clustering. In C2 variants, the stability of rapsyn was decreased, whereas in antibody-treated cells, rapsyn efficiently co-localized with remaining AChRs in microaggregates. Upon ectopic injection into myofibers in vivo, rapsyn did not form clusters in the absence of AChRs. These results show that AChRs and rapsyn are interdependent components of a pre-assembled protein complex that is required for agrin-induced clustering of a full set of postsynaptic proteins, thus providing evidence for an active role of AChRs in postsynaptic assembly

Cholesterol and lipid microdomains stabilize the postsynapse at the neuromuscular junction

Stabilization and maturation of synapses are important for development and function of the nervous system. Previous studies have implicated cholesterol-rich lipid microdomains in synapse stabilization, but the underlying mechanisms remain unclear. We found that cholesterol stabilizes clusters of synaptic acetylcholine receptors (AChRs) in denervated muscle in vivo and in nerve–muscle explants. In paralyzed muscles, cholesterol triggered maturation of nerve sprout-induced AChR clusters into pretzel shape. Cholesterol treatment also rescued a specific defect in AChR cluster stability in cultured src(−/−);fyn(−/−) myotubes. Postsynaptic proteins including AChRs, rapsyn, MuSK and Src-family kinases were strongly enriched in lipid microdomains prepared from wild-type myotubes. Microdomain disruption by cholesterol-sequestering methyl-β-cyclodextrin disassembled AChR clusters and decreased AChR–rapsyn interaction and AChR phosphorylation. Amounts of microdomains and enrichment of postsynaptic proteins into microdomains were decreased in src(−/−);fyn(−/−) myotubes but rescued by cholesterol treatment. These data provide evidence that cholesterol-rich lipid microdomains and SFKs act in a dual mechanism in stabilizing the postsynapse: SFKs enhance microdomain-association of postsynaptic components, whereas microdomains provide the environment for SFKs to maintain interactions and phosphorylation of these components

Increased ratio of rapsyn to ACh receptor stabilizes postsynaptic receptors at the mouse neuromuscular synapse

The metabolic turnover of nicotinic ACh receptors (AChR) at the neuromuscular synapse is regulated over a tenfold range by innervation status, muscle electrical activity and neural agrin, but the downstream effector of such changes has not been defined. The AChR-associated protein rapsyn is essential for forming AChR clusters during development. Here, rapsyn was tagged with enhanced green fluorescent protein (EGFP) to begin to probe its influence at the adult synapse. In C2 myotubes, rapsyn–EGFP participated with AChR in agrin-induced AChR cluster formation. When electroporated into the tibialis anterior muscle of young adult mice, rapsyn–EGFP accumulated in discrete subcellular structures, many of which colocalized with Golgi markers, consistent with the idea that rapsyn assembles with AChR in the exocytic pathway. Rapsyn–EGFP also targeted directly to the postsynaptic membrane where it occupied previously vacant rapsyn binding sites, thereby increasing the rapsyn to AChR ratio. At endplates displaying rapsyn–EGFP, the metabolic turnover of AChR (labelled with rhodamine-α-bungarotoxin) was slowed. Thus, the metabolic half-life of receptors at the synapse may be modulated by local changes in the subsynaptic ratio of rapsyn to AChR