SHP2 inhibitor protects AChRs from effects of myasthenia gravis MuSK antibody

ObjectiveTo determine whether an SRC homology 2 domain-containing phosphotyrosine phosphatase 2 (SHP2) inhibitor would increase muscle-specific kinase (MuSK) phosphorylation and override the inhibitory effect of MuSK-antibodies (Abs).MethodsThe effect of the SHP2 inhibitor NSC-87877 on MuSK phosphorylation and AChR clustering was tested in C2C12 myotubes with 31 MuSK-myasthenia gravis (MG) sera and purified MuSK-MG IgG4 preparations.ResultsIn the absence of MuSK-MG Abs, NSC-87877 increased MuSK phosphorylation and the number of AChR clusters in C2C12 myotubes in vitro and in DOK7-overexpressing C2C12 myotubes that form spontaneous AChR clusters. In the presence of MuSK-MG sera, the AChR clusters were reduced, as expected, but NSC-87877 was able to protect or restore the clusters. Two purified MuSK-MG IgG4 preparations inhibited both MuSK phosphorylation and AChR cluster formation, and in both, clusters were restored with NSC-87877.ConclusionsStimulating the agrin-LRP4-MuSK-DOK7 AChR clustering pathway with NSC-87877, or other drugs, could represent a novel therapeutic approach for MuSK-MG and could potentially improve other NMJ disorders with reduced AChR numbers or disrupted NMJs.

An improved method for culturing myotubes on laminins for the robust clustering of postsynaptic machinery

Motor neurons form specialized synapses with skeletal muscle fibers, called neuromuscular junctions (NMJs). Cultured myotubes are used as a simplified in vitro system to study the postsynaptic specialization of muscles. The stimulation of myotubes with the glycoprotein agrin or laminin-111 induces the clustering of postsynaptic machinery that contains acetylcholine receptors (AChRs). When myotubes are grown on laminin-coated surfaces, AChR clusters undergo developmental remodeling to form topologically complex structures that resemble mature NMJs. Needing further exploration are the molecular processes that govern AChR cluster assembly and its developmental maturation. Here, we describe an improved protocol for culturing muscle cells to promote the formation of complex AChR clusters. We screened various laminin isoforms and showed that laminin-221 was the most potent for inducing AChR clusters, whereas laminin-121, laminin-211, and laminin-221 afforded the highest percentages of topologically complex assemblies. Human primary myotubes that were formed by myoblasts obtained from patient biopsies also assembled AChR clusters that underwent remodeling in vitro. Collectively, these results demonstrate an advancement of culturing myotubes that can facilitate high-throughput screening for potential therapeutic targets for neuromuscular disorders.

MuSK Kinase Activity is Modulated By A Serine Phosphorylation Site in The Kinase Loop

The neuromuscular junction (NMJ) forms when a motor neuron contacts a muscle fibre. A reciprocal exchange of signals initiates a cascade of signalling events that result in pre- and postsynaptic differentiation. At the centre of these signalling events stands muscle specific kinase (MuSK). MuSK activation, kinase activity and subsequent downstream signalling are crucial for NMJ formation as well as maintenance. Therefore MuSK kinase activity is tightly regulated to ensure proper NMJ development. We have identified a novel serine phosphorylation site at position 751 in MuSK that is increasingly phosphorylated upon agrin stimulation. S751 is also phosphorylated in muscle tissue and its phosphorylation depends on MuSK kinase activity. A phosphomimetic mutant of S751 increases MuSK kinase activity in response to non-saturating agrin concentrations . In addition, basal MuSK and AChR phosphorylation as well as AChR cluster size are increased. We believe that the phosphorylation of S751 provides a novel mechanism to relief the autoinhibition of the MuSK activation loop. Such a lower autoinhibition could foster or stabilize MuSK kinase activation, especially during stages when no or low level of agrin are present. Phosphorylation of S751 might therefore represent a novel mechanism to modulate MuSK kinase activity during prepatterning or NMJ maintenance.

Acetylcholine Receptor (AChR) Clustering Is Regulated Both by Glycogen Synthase Kinase 3β (GSK3β)-dependent Phosphorylation and the Level of CLIP-associated Protein 2 (CLASP2) Mediating the Capture of Microtubule Plus-ends

The postsynaptic apparatus of the neuromuscular junction (NMJ) traps and anchors acetylcholine receptors (AChRs) at high density at the synapse. We have previously shown that microtubule (MT) capture by CLASP2, a MT plus-end-tracking protein (+TIP), increases the size and receptor density of AChR clusters at the NMJ through the delivery of AChRs and that this is regulated by a pathway involving neuronal agrin and several postsynaptic kinases, including GSK3. Phosphorylation by GSK3 has been shown to cause CLASP2 dissociation from MT ends, and nine potential phosphorylation sites for GSK3 have been mapped on CLASP2. How CLASP2 phosphorylation regulates MT capture at the NMJ and how this controls the size of AChR clusters are not yet understood. To examine this, we used myotubes cultured on agrin patches that induce AChR clustering in a two-dimensional manner. We show that expression of a CLASP2 mutant, in which the nine GSK3 target serines are mutated to alanine (CLASP2–9XS/9XA) and are resistant to GSK3β-dependent phosphorylation, promotes MT capture at clusters and increases AChR cluster size, compared with myotubes that express similar levels of wild type CLASP2 or that are noninfected. Conversely, myotubes expressing a phosphomimetic form of CLASP2 (CLASP2–8XS/D) show enrichment of immobile mutant CLASP2 in clusters, but MT capture and AChR cluster size are reduced. Taken together, our data suggest that both GSK3β-dependent phosphorylation and the level of CLASP2 play a role in the maintenance of AChR cluster size through the regulated capture and release of MT plus-ends.

A Single Pulse of Agrin Triggers a Pathway That Acts To Cluster Acetylcholine Receptors

ABSTRACT 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.

Laminin-1 redistributes postsynaptic proteins and requires rapsyn, tyrosine phosphorylation, and Src and Fyn to stably cluster acetylcholine receptors

Clustering of acetylcholine receptors (AChRs) is a critical step in neuromuscular synaptogenesis, and is induced by agrin and laminin which are thought to act through different signaling mechanisms. We addressed whether laminin redistributes postsynaptic proteins and requires key elements of the agrin signaling pathway to cause AChR aggregation. In myotubes, laminin-1 rearranged dystroglycans and syntrophins into a laminin-like network, whereas inducing AChR-containing clusters of dystrobrevin, utrophin, and, to a marginal degree, MuSK. Laminin-1 also caused extensive coclustering of rapsyn and phosphotyrosine with AChRs, but none of these clusters were observed in rapsyn −/− myotubes. In parallel with clustering, laminin-1 induced tyrosine phosphorylation of AChR β and δ subunits. Staurosporine and herbimycin, inhibitors of tyrosine kinases, prevented laminin-induced AChR phosphorylation and AChR and phosphotyrosine clustering, and caused rapid dispersal of clusters previously induced by laminin-1. Finally, laminin-1 caused normal aggregation of AChRs and phosphotyrosine in myotubes lacking both Src and Fyn kinases, but these clusters dispersed rapidly after laminin withdrawal. Thus, laminin-1 redistributes postsynaptic proteins and, like agrin, requires tyrosine kinases for AChR phosphorylation and clustering, and rapsyn for AChR cluster formation, whereas cluster stabilization depends on Src and Fyn. Therefore, the laminin and agrin signaling pathways overlap intracellularly, which may be important for neuromuscular synapse formation.

The Actin-Driven Movement and Formation of Acetylcholine Receptor Clusters

A new method was devised to visualize actin polymerization induced by postsynaptic differentiation signals in cultured muscle cells. This entails masking myofibrillar filamentous (F)-actin with jasplakinolide, a cell-permeant F-actin–binding toxin, before synaptogenic stimulation, and then probing new actin assembly with fluorescent phalloidin. With this procedure, actin polymerization associated with newly induced acetylcholine receptor (AChR) clustering by heparin-binding growth-associated molecule–coated beads and by agrin was observed. The beads induced local F-actin assembly that colocalized with AChR clusters at bead–muscle contacts, whereas both the actin cytoskeleton and AChR clusters induced by bath agrin application were diffuse. By expressing a green fluorescent protein–coupled version of cortactin, a protein that binds to active F-actin, the dynamic nature of the actin cytoskeleton associated with new AChR clusters was revealed. In fact, the motive force generated by actin polymerization propelled the entire bead-induced AChR cluster with its attached bead to move in the plane of the membrane. In addition, actin polymerization is also necessary for the formation of both bead and agrin-induced AChR clusters as well as phosphotyrosine accumulation, as shown by their blockage by latrunculin A, a toxin that sequesters globular (G)-actin and prevents F-actin assembly. These results show that actin polymerization induced by synaptogenic signals is necessary for the movement and formation of AChR clusters and implicate a role of F-actin as a postsynaptic scaffold for the assembly of structural and signaling molecules in neuromuscular junction formation.

A Role of Tyrosine Phosphatase in Acetylcholine Receptor Cluster Dispersal and Formation

Innervation of the skeletal muscle involves local signaling, leading to acetylcholine receptor (AChR) clustering, and global signaling, manifested by the dispersal of preexisting AChR clusters (hot spots). Receptor tyrosine kinase (RTK) activation has been shown to mediate AChR clustering. In this study, the role of tyrosine phosphatase (PTPase) in the dispersal of hot spots was examined. Hot spot dispersal in cultured Xenopus muscle cells was initiated immediately upon the presentation of growth factor–coated beads that induce both AChR cluster formation and dispersal. Whereas the density of AChRs decreased with time, the fine structure of the hot spot remained relatively constant. Although AChR, rapsyn, and phosphotyrosine disappeared, a large part of the original hot spot–associated cytoskeleton remained. This suggests that the dispersal involves the removal of a key linkage between the receptor and its cytoskeletal infrastructure. The rate of hot spot dispersal is inversely related to its distance from the site of synaptic stimulation, implicating the diffusible nature of the signal. PTPase inhibitors, such as pervanadate or phenylarsine oxide, inhibited hot spot dispersal. In addition, they also affected the formation of new clusters in such a way that AChR microclusters extended beyond the boundary set by the clustering stimuli. Furthermore, by introducing a constitutively active PTPase into cultured muscle cells, hot spots were dispersed in a stimulus- independent fashion. This effect of exogenous PTPase was also blocked by pervanadate. These results implicate a role of PTPase in AChR cluster dispersal and formation. In addition to RTK activation, synaptic stimulation may also activate PTPase which acts globally to destabilize preexisting AChR hot spots and locally to facilitate AChR clustering in a spatially discrete manner by countering the action of RTKs.