Cooperative regulation by Rac and Rho of agrin-induced acetylcholine receptor clustering in muscle cells

A key aspect of neuromuscular synapse formation is the clustering of muscle acetylcholine receptors (AChR) at synaptic sites in response to neurally secreted agrin. Agrin-induced AChR clustering in cultured myotubes proceeds via the initial formation of small microclusters, which then aggregate to form AChR clusters. Here we show that the coupling of agrin signaling to AChR clustering is dependent on the coordinated activities of Rac and Rho GTPases. The addition of agrin induces the sequential activation of Rac and Rho in C2 muscle cells. The activation of Rac is rapid and transient and constitutes a prerequisite for the subsequent activation of Rho. This temporal pattern of agrin-induced Rac and Rho activation reflects their respective roles in AChR cluster formation. Whereas agrin-induced activation of Rac is necessary for the initial phase of AChR cluster formation, which involves the aggregation of diffuse AChR into microclusters, Rho activation is crucial for the subsequent condensation of these microclusters into full-size AChR clusters. Co-expression of constitutively active forms of Rac and Rho is sufficient to induce the formation of mature AChR clusters in the absence of agrin. These results establish that Rac and Rho play distinct but complementary roles in the mechanism of agrin-induced AChR clustering.     

Collagen synthesis inhibition reduces clustering of heparan sulfate proteoglycan and acetylcholine receptors but not agrin or p65, at neuromuscular contacts in vitro

We have studied presynaptic and postsynaptic differentiation at neuromuscular junctions in vitro by examining the localization of synapse-specific proteins. In nerve–muscle co-cultures, the synaptic vesicle protein synaptotagmin (p65) accumulated in the nerve terminal overlying myotubes in association with postsynaptic cluster of acetylcholine receptors (AChRs), heparan sulfate proteoglycan (HSPG), laminin, and agrin. Inhibition of collagen synthesis with cis-hydroxyproline decreased the nerve-induced clustering of AChRs in muscle cells as well as that caused by exogenous agrin in muscle-only cultures. Moreover, accumulation of HSPG at contacts was also inhibited in cis-hydroxyproline–treated cultures. However, accumulation of p65 in nerve fibers at sites of muscle contact, a sign of presynaptic differentiation, was unaffected by cis-hydroxyproline treatment. In addition, even in cis-hydroxyproline–inhibited cultures, agrin was evident at more than 90% of contacts showing accumulation of p65 in the nerve terminal. Therefore, a mechanism exists to maintain agrin concentrations at nerve–muscle contacts, even when at least some extracellular matrix (ECM) proteins are disrupted. Our results suggest that HSPG is not required for the induction of nerve terminal differentiation but are consistent with the idea that HSPG or other ECM proteins are important in both nerve-and agrin-induced AChR clustering. In particular, agrin accumulation at sites of nerve–muscle contact is not sufficient to induce AChR clusters when the ECM at these contacts is disrupted. © 1995 John Wiley & Sons, Inc.     

Nicotine decreases agrin signaling and acetylcholine receptor clustering in C2C12 myotube culture

The clustering of acetylcholine receptors (AChRs) in skeletal muscle fibers is a critical event in neuromuscular synaptogenesis. AChRs in concert with other molecules form postsynaptic scaffolds in response to agrin released from motor neurons as motor neurons near skeletal muscle fibers in development. Agrin drives an intracellular signaling pathway that precedes AChR clustering and includes the tyrosine phosphorylation of AChRs. In C2C12 myotube culture, agrin application stimulates the agrin signaling pathway and AChR clustering. Previous studies have determined that the frequency of spontaneous AChR clustering is decreased and AChRs are partially inactivated when bound by the acetylcholine agonist nicotine. We hypothesized that nicotine interferes with AChR clustering and consequent postsynaptic scaffold formation. In the present study, C2C12 myoblasts were cultured with growth medium to stimulate proliferation and then differentiation medium to stimulate fusion into myotubes. They were bathed in a physiologically relevant concentration of nicotine and then subject to agrin treatment after myotube formation. Our results demonstrate that nicotine decreases agrin-induced tyrosine phosphorylation of AChRs and decreases the frequency of spontaneous as well as agrin-induced AChR clustering. We conclude that nicotine interferes with postsynaptic scaffold formation by preventing the tyrosine phosphorylation of AChRs, an agrin signaling event that precedes AChR clustering.     

Sialic acid inhibits agrin signaling in C2 myotubes

Acetylcholine receptor (AChR) clustering is an early event in neuromuscular synapse formation that is commonly studied using muscle cell culture. Motor neuron-derived agrin induces the postsynaptic tyrosine phosphorylation of both a muscle-specific kinase (MuSK) and the AChR beta-subunit. These phosphorylation events are required for AChR clustering, suggesting an agrin-driven signaling pathway. Both the phosphorylation events and AChR clustering can also be induced by neuraminidase, an enzyme that cleaves sialic acid from glycoconjugates, suggesting that neuraminidase is able to activate the agrin signaling pathway. A postulated signal for postsynaptic differentiation at sites of nerve-muscle contact during vertebrate development is the enzymatic removal of basal lamina components. We show here that bath-applied sialic acid has an effect directly opposite that of agrin or neuraminidase. Sialic acid not only decreases AChR clustering but also diminishes the tyrosine phosphorylation of MuSK and the AChR beta-subunit signal-transduction events normally driven by agrin. However, sialic acid does not prevent agrin-binding molecules from colocalizing with the decreased number of AChR clusters that do form, suggesting that sialic acid is acting to inhibit the agrin signaling pathway downstream of agrin binding to the muscle cell membrane. We propose a regulatory role for sialic acid in the signal transduction events of neuromuscular synapse formation, in which agrin or neuraminidase can overcome this sialic acid repression, resulting in the clustering of AChRs and other postsynaptic molecules.     

Common Molecular Mechanisms in Field- and Agrin-Induced Acetylcholine Receptor Clustering

1. The aggregation of acetylcholine receptors at the developing neuromuscular junction is critical to the development and function of this synapse. In vitro studies have shown that receptor aggregation can be induced by the finding of agrin to the muscle cell surface and by the electric field-induced concentration of a (nonreceptor) molecule at the cathodal cell pole.2. We report here on the interaction between agrin binding and electric fields with respect to the distribution of receptors and agrin binding sites.3. (a) Pretreatment of cells with agrin completely blocks the development of field-induced receptor clusters. (b) Field-induced aggregation of receptors precedes the field-induced aggregation of agrin binding sites by approximately 30min. (c) Electric fields prevent agrin-induced receptor clustering despite the presence of agrin binding sites and freely diffusing receptors.4. These results indicate that another membrane component—but not the agrin binding site and not the receptor—is required for agrin-induced receptor clustering. They also suggest that electric fields and agrin cause receptor clustering via common molecular mechanisms.     

Phosphoinositide 3-kinase acts through RAC and Cdc42 during agrin-induced acetylcholine receptor clustering

The formation of the neuromuscular junction (NMJ) is regulated by the nerve-derived heparan sulfate proteoglycan agrin and the muscle-specific kinase MuSK. Agrin induces a signal transduction pathway via MuSK, which promotes the reorganization of the postsynaptic muscle membrane. Activation of MuSK leads to the phosphorylation and redistribution of acetylcholine receptors (AChRs) and other postsynaptic proteins to synaptic sites. The accumulation of high densities of AChRs at postsynaptic regions represents a hallmark of NMJ formation and is required for proper NMJ function. Here we show that phosphoinositide 3-kinase (PI3-K) represents a component of the agrin/MuSK signaling pathway. Muscle cells treated with specific PI3-K inhibitors are unable to form full-size AChR clusters in response to agrin and AChR phosphorylation is reduced. Moreover, agrin-induced activation of Rac and Cdc42 is impaired in the presence of PI3-K inhibitors. PI3-K is localized to the postsynaptic muscle membrane consistent with a role during agrin/MuSK signaling. These results put PI3-K downstream of MuSK as regulator of AChR phosphorylation and clustering. Its role during agrin-stimulated Rac and Cdc42 activation suggests a critical function during cytoskeletal reorganizations, which lead to the redistribution of actin-anchored AChRs.     

HGF induction of postsynaptic specializations at the neuromuscular junction

A critical event in the formation of vertebrate neuromuscular junctions (NMJs) is the postsynaptic clustering of acetylcholine receptors (AChRs) in muscle. AChR clustering is triggered by the activation of MuSK, a muscle-specific tyrosine kinase that is part of the functional receptor for agrin, a nerve-derived heparan sulfate proteoglycan (HSPG). At the NMJ, heparan sulfate (HS)-binding growth factors and their receptors are also localized but their involvement in postsynaptic signaling is poorly understood. In this study we found that hepatocyte growth factor (HGF), an HS-binding growth factor, surrounded muscle fibers and was localized at NMJs in rat muscle sections. In cultured Xenopus muscle cells, HGF was enriched at spontaneously occurring AChR clusters (hot spots), where HSPGs were also concentrated, and, following stimulation of muscle cells by agrin or cocultured neurons, HGF associated with newly formed AChR clusters. HGF presented locally to cultured muscle cells by latex beads induced new AChR clusters and dispersed AChR hot spots, and HGF beads also clustered phosphotyrosine, activated c-Met, and proteins of dystrophin complex; clustering of AChRs and associated proteins by HGF beads required actin polymerization. Lastly, although bath-applied HGF alone did not induce new AChR clusters, addition of HGF potentiated agrin-dependent AChR clustering in muscle. Our findings suggest that HGF promotes AChR clustering and synaptogenic signaling in muscle during NMJ development.     

Overexpression of rapsyn inhibits agrin-induced acetylcholine receptor clustering in muscle cells

Rapsyn is a protein on the cytoplasmic face of the postsynaptic membrane of skeletal muscle that is essential for clustering acetylcholine receptors (AChR). Here we show that transfection of rapsyn cDNA can restore AChR clustering function to muscle cells cultured from rapsyn deficient (KORAP) mice. KORAP myotubes displayed no AChR aggregates before or after treatment with neural agrin. After transfection with rapsyn expression plasmid, some KORAP myotubes expressed rapsyn at physiological levels. These formed large AChR-rapsyn clusters in response to agrin, just like wild-type myotubes. KORAP myotubes that overexpressed rapsyn formed only scattered AChR-rapsyn microaggregates, irrespective of agrin treatment. KORAP cells were then transfected with mutant forms of rapsyn. A deletion mutant lacking residues 16–254 formed rapsyn microaggregates, but failed to aggregate AChRs. Substitution mutation to the C-terminal serine phosphorylation site of rapsyn (M43^D405,D406) did not impair the response to agrin, showing that differential phosphorylation of this site is unlikely to mediate agrin-induced clustering. The results indicate that rapsyn expression is essential for agrin-induced AChR clustering but that its overexpression inhibits this pathway. The approach of using rapsyn-deficient muscle cells opens the way for defining the role of rapsyn in agrin-induced AChR clustering.     

Local induction of acetylcholine receptor clustering in myotube cultures using microfluidic application of agrin

During neuromuscular synaptogenesis, the exchange of spatially localized signals between nerve and muscle initiates the coordinated focal accumulation of the acetylcholine (ACh) release machinery and the ACh receptors (AChRs). One of the key first steps is the release of the proteoglycan agrin focalized at the axon tip, which induces the clustering of AChRs on the postsynaptic membrane at the neuromuscular junction. The lack of a suitable method for focal application of agrin in myotube cultures has limited the majority of in vitro studies to the application of agrin baths. We used a microfluidic device and surface microengineering to focally stimulate muscle cells with agrin at a small portion of their membrane and at a time and position chosen by the user. The device is used to verify the hypothesis that focal application of agrin to the muscle cell membrane induces local aggregation of AChRs in differentiated C2C12 myotubes.     

What controls the position,number, size,and distribution of neuromuscular junctions on rat muscle fibers?

This review focuses on mechanisms that determine the position, number, size, and distribution of neuromuscular junctions (NMJs) on skeletal muscle fibers. Most of the data reviewed derive from studies of ectopic NMJ formation on soleus (SOL) muscle fibers in adult rats, which recapitulates essential aspects of NMJ formation in normal development. Transplanted axons induce acetylcholine receptor (AChR) aggregates, which are multiple and irregularly distributed initially but subsequently undergo massive reorganization such that one or a few winners survive and reach a certain size while the rest are eliminated (the losers). Results obtained by blocking nerve activity early and stimulating the SOL electrically show that evoked muscle impulse activity is responsible for the growth of winners to a given size and the creation of refractory zones, about 0.75 long, on each side of the winners, in which the elimination of losers occurs. Consequently, when two or more aggregates or NMJs survive on one fiber, they are, on average, at least 1.5 mm apart. Locally applied neural agrin induces comparable aggregation of AChRs and other postsynaptic proteins on denervated SOL fibers and such aggregates undergo similar activity-dependent selection for survival or elimination in refractory zones. In a dose-dependent way, neural agrin alone also induces expression of ε-AChR subunits and stabilizes AChRs to a half-life of 10 days, as found at normal NMJs. It is argued that signs of prepatterning of innervation sites by intrinsic muscle mechanisms may refer to epiphenomena that play no important role in NMJ formation. The conclusion is that neural agrin initiates and then maintains NMJs where motor axons happen to contact receptive muscle fibers and that evoked muscle impulse activity then ensures that the NMJs reach their appropriate size, efficiency and spatial distribution along each fiber.     

Agrin-Deficient Myotube Retains Its Acetylcholine Receptor Aggregation Ability when Challenged with Agrin

Abstract: Agrin is a synapse-organizing molecule that mediates the nerve-induced aggregation of acetylcholine receptors (AChRs) and other postsynaptic components at the developing and regenerating vertebrate neuromuscular junctions. At the neuromuscular junction, three different cell types can express agrin, i.e., neuron, muscle, and Schwann cell. Several lines of evidence suggested that neuron-derived agrin is the AChR-aggregating factor, but the possible roles of muscle-derived agrin in the formation of AChR aggregate are not known. By using the recombinant DNA method, a clonal stable C2C12 cell line transfected with antisense agrin cDNA was created. RNA dot blot and western blot analysis indicated that the expression of agrin in the transfected cell was abolished by DNA transfection. When the agrin-deficient C2C12 cells were induced to form myotubes and subsequently cocultured with agrin cDNA transfected fibroblasts, AChR aggregates were formed in the cocultures. In addition, acetylcholinesterase (AChE) aggregates in agrin-deficient myotubes were also induced by exogenous agrin and the AChE aggregates were colocalized with the AChR aggregates. The agrin-deficient myotubes could also respond to neuron-induced AChR aggregation after coculturing with neuroblastoma cells. Thus, the agrin-deficient myotubes retain their ability to exhibit the agrin- or neuron-induced AChR aggregation. This result suggests that the formation of postsynaptic specializations during development and regeneration is mediated by neuron-derived agrin but not the agrin from muscle.     

Localized acetylcholine receptor clustering dynamics in response to microfluidic focal stimulation with agrin

Agrin is a proteoglycan secreted by the motor neuron's growing axon terminal upon contact with the muscle during embryonic development. It was long thought that agrin's role was to trigger the clustering of acetylcholine receptors (AChRs) to nascent synapse sites. However, agrin-predating, protosynaptic AChR clusters are present well before innervation in the embryo and in myotube cultures, yet no role has been conclusively ascribed to agrin. We used a microfluidic device to focally deliver agrin to protosynaptic AChR clusters in micropatterned myotube cultures. The distribution of AChRs labeled with fluorescent bungarotoxin was imaged at various time points over >24 h. We find that a 4-h focal application of agrin (100 nM) preferentially reduces AChR loss at agrin-exposed clusters by 17% relative to the agrin-deprived clusters on the same myotube. In addition, the focal application increases the addition of AChRs preferentially at the clusters by 10% relative to the agrin-exposed, noncluster areas. Taken together, these findings suggest that a focal agrin stimulus can play a key stabilizing role in the aggregation of AChRs at the early stages of synapse formation. This methodology is generally applicable to various developmental processes and cell types, including neurons and stem cells.     

Laminin-induced Acetylcholine Receptor Clustering: An Alternative Pathway

The induction of acetylcholine receptor (AChR) clustering by neurally released agrin is a critical, early step in the formation of the neuromuscular junction. Laminin, a component of the muscle fiber basal lamina, also induces AChR clustering. We find that induction of AChR clustering in C2 myotubes is specific for laminin-1; neither laminin-2 (merosin) nor laminin-11 (a synapse-specific isoform) are active. Moreover, laminin-1 induces AChR clustering by a pathway that is independent of that used by neural agrin. The effects of laminin-1 and agrin are strictly additive and occur with different time courses. Most importantly, laminin- 1–induced clustering does not require MuSK, a receptor tyrosine kinase that is part of the receptor complex for agrin. Laminin-1 does not cause tyrosine phosphorylation of MuSK in C2 myotubes and induces AChR clustering in myotubes from MuSK−/− mice that do not respond to agrin. In contrast to agrin, laminin-1 also does not induce tyrosine phosphorylation of the AChR, demonstrating that AChR tyrosine phosphorylation is not required for clustering in myotubes. Laminin-1 thus acts by a mechanism that is independent of that used by agrin and may provide a supplemental pathway for AChR clustering during synaptogenesis.     

Isolated primary osteocytes express functional gap junctions in vitro

The differentiation of the neuromuscular junction is a multistep process requiring coordinated interactions between nerve terminals and muscle. Although innervation is not needed for muscle production, the formation of nerve-muscle contacts, intramuscular nerve branching, and neuronal survival require reciprocal signals from nerve and muscle to regulate the formation of synapses. Following the production of muscle fibers, clusters of acetylcholine receptors (AChRs) are concentrated in the central regions of the myofibers via a process termed “prepatterning”. The postsynaptic protein MuSK is essential for this process activating possibly its own expression, in addition to the expression of AChR. AChR complexes (aggregated and stabilized by rapsyn) are thus prepatterned independently of neuronal signals in developing myofibers. ACh released by branching motor nerves causes AChR-induced postsynaptic potentials and positively regulates the localization and stabilization of developing synaptic contacts. These “active” contact sites may prevent AChRs clustering in non-contacted regions and counteract the establishment of additional contacts. ACh-induced signals also cause the dispersion of non-synaptic AChR clusters and possibly the removal of excess AChR. A further neuronal factor, agrin, stabilizes the accumulation of AChR at synaptic sites. Agrin released from the branching motor nerve may form a structural link specifically to the ACh-activated endplates, thereby enhancing MuSK kinase activity and AChR accumulation and preventing dispersion of postsynaptic specializations. The successful stabilization of prepatterned AChR clusters by agrin and the generation of singly innervated myofibers appear to require AChR-mediated postsynaptic potentials indicating that the differentiation of the nerve terminal proceeds only after postsynaptic specializations have formed.     

Modulation of Agrin-induced Acetylcholine Receptor Clustering by Extracellular Signal-regulated Kinases 1 and 2 in Cultured Myotubes

Agrin released by motoneurons induces and/or maintains acetylcholine receptor (AChR) clustering and other aspects of postsynaptic differentiation at the vertebrate neuromuscular junction. Agrin acts by binding and activating a receptor complex containing LDL receptor protein 4 (Lrp4) and muscle-specific kinase (MuSK). Two critical downstream components of this signaling cascade, Dox-7 and rapsyn, have been identified. However, additional intracellular essential elements remain unknown. Prior observations by others and us suggested antagonistic interactions between agrin and neuregulin-1 (Nrg-1) signaling in cultured myotubes and developing muscle fibers in vivo. A hallmark of Nrg-1 signaling in skeletal muscle cells is the activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2). ERK1/2 are also activated in most cells by phorbol 12-myristate 13-acetate, a classical inhibitor of agrin-induced AChR clustering in myotubes. Here, it was investigated whether agrin activates ERK1/2 directly and whether such activation modulates agrin-induced AChR clustering. Agrin induced a rapid but transient activation of ERK1/2 in myotubes that was Lrp4/MuSK-dependent. However, blocking this ERK1/2 activation did not prevent but potentiated AChR clustering induced by agrin. ERK1/2 activation was dispensable for Nrg-1-mediated inhibition of the AChR clustering activity of agrin, but was indispensable for such activity by phorbol 12-myristate 13-acetate. Together, these results suggest agrin-induced activation of ERK1/2 is a negative modulator of agrin signaling in skeletal muscle cells.     

The putative agrin receptor binds ligand in a calcium-dependent manner and aggregates during agrin-induced acetylcholine receptor clustering.

Agrin derived from Torpedo electric organ induces the clustering of acetylcholine receptors (AChRs) on cultured myotubes. As a first step toward characterizing the plasma membrane receptor for agrin, we have examined agrin binding to cultured myotubes. Agrin binding is saturable as measured by radioimmunoassay and, like agrin-induced AChR clustering, requires extracellular calcium. Immunofluorescence shows that on myotubes incubated with agrin at 4 degrees C, agrin binds in a uniform, finely punctate pattern that correlates poorly with the distribution of AChRs. Myotubes stimulated with agrin at 37 degrees C for greater than or equal to 2 hr show a coclustering of agrin binding sites and AChRs. By contrast, if anti-AChR antibodies are used either to cluster or to internalize AChRs, the distribution and number of agrin binding sites remain unchanged. The aggregation and calcium dependence of the putative agrin receptor may represent important control points in postsynaptic differentiation.     

Effects of purified recombinant neural and muscle agrin on skeletal muscle fibers in vivo.

Aggregation of acetylcholine receptors (AChRs) in muscle fibers by nerve-derived agrin plays a key role in the formation of neuromuscular junctions. So far, the effects of agrin on muscle fibers have been studied in culture systems, transgenic animals, and in animals injected with agrin--cDNA constructs. We have applied purified recombinant chick neural and muscle agrin to rat soleus muscle in vivo and obtained the following results. Both neural and muscle agrin bind uniformly to the surface of innervated and denervated muscle fibers along their entire length. Neural agrin causes a dose-dependent appearance of AChR aggregates, which persist > or = 7 wk after a single application. Muscle agrin does not cluster AChRs and at 10 times the concentration of neural agrin does not reduce binding or AChR-aggregating activity of neural agrin. Electrical muscle activity affects the stability of agrin binding and the number, size, and spatial distribution of the neural agrin--induced AChR aggregates. Injected agrin is recovered from the muscles together with laminin and both proteins coimmunoprecipitate, indicating that agrin binds to laminin in vivo. Thus, the present approach provides a novel, simple, and efficient method for studying the effects of agrin on muscle under controlled conditions in vivo.     

An amino-terminal extension is required for the secretion of chick agrin and its binding to extracellular matrix

Agrin is an extracellular matrix (ECM) protein with a calculated relative molecular mass of more than 200 kD that induces the aggregation of acetylcholine receptors (AChRs) at the neuromuscular junction. This activity has been mapped to its COOH terminus. In an attempt to identify the functions of the NH2-terminal end, we have now characterized full-length chick agrin. We show that chick agrin encoded by a previously described cDNA is not secreted from transfected cells. Secretion is achieved with a construct that includes an additional 350 bp derived from the 5' end of chick agrin mRNA. Recombinant agrin is a heparan sulfate proteoglycan (HSPG) of more than 400 kD with glycosaminoglycan side chains attached only to the NH2-terminal half. Endogenous agrin in tissue homogenates also has an apparent molecular mass of > 400 kD. While the amino acid sequence encoded by the 350-bp extension has no homology to published rat agrin, it includes a stretch of 15 amino acids that is 80% identical to a previously identified bovine HSPG. The extension is required for binding of agrin to ECM. AChR aggregates induced by recombinant agrin that includes the extension are considerably smaller than those induced by agrin fragments, suggesting that binding of agrin to ECM modulates the size of receptor clusters. In addition, we found a site encoding seven amino acids at the NH2-terminal end of agrin that is alternatively spliced. While motor neurons express the splice variant with the seven amino acid long insert, muscle cells mainly synthesize isoforms that lack this insert. In conclusion, the cDNAs described here code for chick agrin that has all the characteristics previously allocated to endogenous agrin.