Raghavan Madhavan

Tyrosine phosphatase regulation of MuSK-dependent acetylcholine receptor clustering

During vertebrate neuromuscular junction (NMJ) development, nerve-secreted agrin induces acetylcholine receptor (AChR) clustering in muscle by activating the muscle-specific tyrosine kinase MuSK. Recently, it has been recognized that MuSK activation-dependent AChR clustering occurs in embryonic muscle even in the absence of agrin, but how this process is regulated is poorly understood. We report that inhibition of tyrosine phosphatases in cultured C2 mouse myotubes using pervanadate enhanced MuSK auto-activation and agrin-independent AChR clustering. Moreover, phosphatase inhibition also enlarged the AChR clusters induced by agrin in these cells. Conversely, in situ activation of MuSK in cultured Xenopus embryonic muscle cells, either focally by anti-MuSK antibody-coated beads or globally by agrin, stimulated downstream tyrosine phosphatases, which could be blocked by pervanadate treatment. Immunoscreening identified Shp2 as a major tyrosine phosphatase in C2 myotubes and down-regulation of its expression by RNA interference alleviated tyrosine phosphatase suppression of MuSK activation. Significantly, depletion of Shp2 increased both agrin-independent and agrin-dependent AChR clustering in myotubes. Our results suggest that muscle tyrosine phosphatases tightly regulate MuSK activation and signaling and support a novel role of Shp2 in MuSK-dependent AChR clustering.

Molecular regulation of postsynaptic differentiation at the neuromuscular junction

The neuromuscular junction (NMJ) is a synapse that develops between a motor neuron and a muscle fiber. A defining feature of NMJ development in vertebrates is the re‐distribution of muscle acetylcholine (ACh) receptors (AChRs) following innervation, which generates high‐density AChR clusters at the postsynaptic membrane and disperses aneural AChR clusters formed in muscle before innervation. This process in vivo requires MuSK, a muscle‐specific receptor tyrosine kinase that triggers AChR re‐distribution when activated; rapsyn, a muscle protein that binds and clusters AChRs; agrin, a nerve‐secreted heparan‐sulfate proteoglycan that activates MuSK; and ACh, a neurotransmitter that stimulates muscle and also disperses aneural AChR clusters. Moreover, in cultured muscle cells, several additional muscle‐ and nerve‐derived molecules induce, mediate or participate in AChR clustering and dispersal. In this review we discuss how regulation of AChR re‐distribution by multiple factors ensures aggregation of AChRs exclusively at NMJs. IUBMB Life, 57: 719‐730, 2005

The function of cortactin in the clustering of acetylcholine receptors at the vertebrate neuromuscular junction.

Background Postsynaptic enrichment of acetylcholine receptors (AChRs) at the vertebrate neuromuscular junction (NMJ) depends on the activation of the muscle receptor tyrosine MuSK by neural agrin. Agrin-stimulation of MuSK is known to initiate an intracellular signaling cascade that leads to the clustering of AChRs in an actin polymerization-dependent manner, but the molecular steps which link MuSK activation to AChR aggregation remain incompletely defined. Methodology/Principal Findings In this study we used biochemical, cell biological and molecular assays to investigate a possible role in AChR clustering of cortactin, a protein which is a tyrosine kinase substrate and a regulator of F-actin assembly and which has also been previously localized at AChR clustering sites. We report that cortactin was co-enriched at AChR clusters in situ with its target the Arp2/3 complex, which is a key stimulator of actin polymerization in cells. Cortactin was further preferentially tyrosine phosphorylated at AChR clustering sites and treatment of myotubes with agrin significantly enhanced the tyrosine phosphorylation of cortactin. Importantly, forced expression in myotubes of a tyrosine phosphorylation-defective cortactin mutant (but not wild-type cortactin) suppressed agrin-dependent AChR clustering, as did the reduction of endogenous cortactin levels using RNA interference, and introduction of the mutant cortactin into muscle cells potently inhibited synaptic AChR aggregation in response to innervation. Conclusion Our results suggest a novel function of phosphorylation-dependent cortactin signaling downstream from agrin/MuSK in facilitating AChR clustering at the developing NMJ.

Axonal filopodial asymmetry induced by synaptic target

Early nerve–muscle interaction that leads to neuromuscular junction formation is mediated by axonal filopodia that are oriented preferentially toward the muscle as a result of target-derived basic fibroblast growth factor.

The involvement of calcineurin in acetylcholine receptor redistribution in muscle

In this study the intracellular signaling involved in acetylcholine receptor (AChR) redistribution in muscle was investigated. In cultured Xenopus embryonic muscle cells, both the dispersal of preexisting AChR clusters and the induction of new AChR clusters by growth factor-coated polystyrene beads were inhibited by two specific antagonists of the ser/thr phosphatase calcineurin (CaN), Cyclosporin A, (CsA) and FK-506, but not by KN-93 that blocks CaM kinases. Moreover, CaN inhibition decreased AChR clustering in Xenopus muscle cells by beads coated with antibodies that cross-link and activate the agrin receptor MuSK (muscle-specific kinase) and reduced the agrin-induced tyrosine phosphorylation of MuSK in cultured mouse (C2) myotubes. Last, the length and the number of AChR clusters generated by agrin in C2 myotubes were decreased by treatment with CsA, but not KN-93, and following the forced expression of a dominant negative CaN mutant in these cells, but not wild-type CaN or reporter green fluorescent protein. Collectively, our results support a role for CaN signaling in the redistribution of AChRs in muscle induced by synaptogenic signals.

A synaptic balancing act: Local and global signaling in the clustering of ACh receptors at vertebrate neuromuscular junctions

The clustering of acetylcholine receptors (AChRs) in muscle is a hallmark step in the development of vertebrate neuromuscular junctions (NMJs). It involves localized signaling, which is initiated by the activation of MuSK (muscle-specific kinase) and leads to the concentration and cytoskeletal anchoring of AChRs at the synapse, and global signaling, which traverses the muscle to disperse extra-synaptic AChR clusters. Here we review some of the recent findings that indicate important roles for the actin cytoskeleton and tyrosine phosphatases in mediating these processes.

Reciprocal Regulation of Axonal Filopodia and Outgrowth during Neuromuscular Junction Development

Background The assembly of the vertebrate neuromuscular junction (NMJ) is initiated when nerve and muscle first contact each other by filopodial processes which are thought to enable close interactions between the synaptic partners and facilitate synaptogenesis. We recently reported that embryonic Xenopus spinal neurons preferentially extended filopodia towards cocultured muscle cells and that basic fibroblast growth factor (bFGF) produced by muscle activated neuronal FGF receptor 1 (FGFR1) to induce filopodia and favor synaptogenesis. Intriguingly, in an earlier study we found that neurotrophins (NTs), a different set of target-derived factors that act through Trk receptor tyrosine kinases, promoted neuronal growth but hindered presynaptic differentiation and NMJ formation. Thus, here we investigated how bFGF- and NT-signals in neurons jointly elicit presynaptic changes during the earliest stages of NMJ development. Methodology/Principal Findings Whereas forced expression of wild-type TrkB in neurons reduced filopodial extension and triggered axonal outgrowth, expression of a mutant TrkB lacking the intracellular kinase domain enhanced filopodial growth and slowed axonal advance. Neurons overexpressing wild-type FGFR1 also displayed more filopodia than control neurons, in accord with our previous findings, and, notably, this elevation in filopodial density was suppressed when neurons were chronically treated from the beginning of the culture period with BDNF, the NT that specifically activates TrkB. Conversely, inhibition by BDNF of NMJ formation in nerve-muscle cocultures was partly reversed by the overexpression of bFGF in muscle. Conclusions Our results suggest that the balance between neuronal FGFR1- and TrkB-dependent filopodial assembly and axonal outgrowth regulates the establishment of incipient NMJs.

The function of p120 catenin in filopodial growth and synaptic vesicle clustering in neurons

The signaling by p120 catenin via its downstream effector RhoA is essential for filopodial growth and synaptic vesicle clustering along spinal axons and contributes to the formation of the neuromuscular junction.

Differential regulation of axonal growth and neuromuscular junction assembly by HGF/c-Met signaling.

Background: During vertebrate neuromuscular junction (NMJ) development, contact between motor axons and muscle fibers is followed by pre‐ and post‐synaptic specialization. Using Xenopus nerve‐muscle cocultures, we recently showed that spinal neurons initially contacted muscle cells by means of filopodial processes, and that muscle‐derived basic fibroblast growth factor induced axonal filopodia and slowed axonal advance to promote nerve‐muscle interaction and NMJ establishment. In contrast, neurotrophins enhanced axonal growth but suppressed the extension of axonal filopodia and blocked NMJ formation. Results: Here we report that hepatocyte growth factor (HGF), which also supports motor neuron survival, was expressed by Xenopus muscle cells, and that forced expression of HGF in Xenopus spinal neurons inhibited the extension of axonal filopodia. Overexpression of the HGF‐receptor c‐Met in neurons also blocked the formation of axonal filopodia and furthermore sped up axonal growth, but a kinase‐dead form of c‐Met was unable to effect these changes. Importantly, treatment of nerve‐muscle cocultures with recombinant HGF or the expression of HGF or active c‐Met in neurons, or that of excess HGF in muscle, inhibited nerve‐induced AChR clustering in muscle. Conclusions: Our results suggest that HGF/c‐Met signaling in neurons promotes axonal growth but suppresses filopodial assembly in neurons and hinders NMJ establishment. Developmental Dynamics 241:1562–1574, 2012.