Recycling of acetylcholine receptors at ectopic postsynaptic clusters induced by exogenous agrin in living rats

During the development of the neuromuscular junction, motor axons induce the clustering of acetylcholine receptors (AChRs) and increase their metabolic stability in the muscle membrane. Here, we asked whether the synaptic organizer agrin might regulate the metabolic stability and density of AChRs by promoting the recycling of internalized AChRs, which would otherwise be destined for degradation, into synaptic sites. We show that at nerve-free AChR clusters induced by agrin in extrasynaptic membrane, internalized AChRs are driven back into the ectopic synaptic clusters where they intermingle with pre-existing and new receptors. The extent of AChR recycling depended on the strength of the agrin stimulus, but not on the development of junctional folds, another hallmark of mature postsynaptic membranes. In chronically denervated muscles, in which both AChR stability and recycling are significantly decreased by muscle inactivity, agrin maintained the amount of recycled AChRs at agrin-induced clusters at a level similar to that at denervated original endplates. In contrast, AChRs did not recycle at agrin-induced clusters in C2C12 or primary myotubes. Thus, in muscles in vivo, but not in cultured myotubes, neural agrin promotes the recycling of AChRs and thereby increases their metabolic stability.     

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.     

Guanylate cyclase and cyclic GMP-dependent protein kinase regulate agrin signaling at the developing neuromuscular junction

During formation of the neuromuscular junction (NMJ), agrin secreted by motor axons signals the embryonic muscle cells to organize a postsynaptic apparatus including a dense aggregate of acetylcholine receptors (AChRs). Agrin signaling at the embryonic NMJ requires the activity of nitric oxide synthase (NOS). Common downstream effectors of NOS are guanylate cyclase (GC), which synthesizes cyclic GMP, and cyclic GMP-dependent protein kinase (PKG). Here we show that GC and PKG are important for agrin signaling at the embryonic NMJ of the frog, Xenopus laevis. Inhibitors of both GC and PKG reduced endogenous AChR aggregation in embryonic muscles by 50-85%, and blocked agrin-induced AChR aggregation in cultured embryonic muscle cells. A cyclic GMP analog, 8-bromo-cyclic GMP, increased endogenous AChR aggregation in embryonic muscles to 3- to 4-fold control levels. Overexpression of either GC or PKG in embryos increased AChR aggregate area by 60-170%, whereas expression of a dominant negative form of GC inhibited endogenous aggregation by 50%. These results indicate that agrin signaling in embryonic muscle cells requires the activity of GC and PKG as well as NOS.     

Neuromuscular synapses can form in vivo by incorporation of initially aneural postsynaptic specializations

Synapse formation requires the coordination of pre- and postsynaptic differentiation. An unresolved question is which steps in the process require interactions between pre- and postsynaptic cells, and which proceed cell-autonomously. One current model is that factors released from presynaptic axons organize postsynaptic differentiation directly beneath the nerve terminal. Here, we used neuromuscular junctions (NMJs) of the zebrafish primary motor system to test this model. Clusters of neurotransmitter (acetylcholine) receptors (AChRs) formed in the central region of the myotome, destined to be synapse-rich, before axons extended and even when axon extension was prevented. Time-lapse imaging revealed that pre-existing clusters on early-born slow (adaxial) muscle fibers were incorporated into NMJs as axons advanced. Axons were, however, required for the subsequent remodeling and selective stabilization of synaptic clusters that precisely appose post- to presynaptic elements. Thus, motor axons are dispensable for the initial stages of postsynaptic differentiation but are required for later stages. Moreover, many AChR clusters on later-born fast muscle fibers formed at sites that had already been contacted by axons, suggesting heterogeneity in the signaling mechanisms leading to synapse formation by a single axon.     

Distribution of alpha-dystroglycan during embryonic nerve-muscle synaptogenesis

The distribution of alpha-dystroglycan (alpha DG) relative to acetylcholine receptors (AChRs) and neural agrin was examined by immunofluorescent staining with mAb IIH6 in cultures of nerve and muscle cells derived from Xenopus embryos. In Western blots probed with mAb IIH6, alpha DG was evident in membrane extracts of Xenopus muscle but not brain. alpha DG immunofluorescence was present at virtually all synaptic clusters of AChRs and neural agrin. Even microclusters of AChRs and agrin at synapses no older than 1-2 h (the earliest examined) had alpha DG associated with them. alpha DG was also colocalized at the submicrometer level with AChRs at nonsynaptic clusters that have little or no agrin. The number of large (> 4 microns) nonsynaptic clusters of alpha DG, like the number of large nonsynaptic clusters of AChRs, was much lower on innervated than on noninnervated cells. When mAb IIH6 was included in the culture medium, the large nonsynaptic clusters appeared fragmented and less compact, but the accumulation of agrin and AChRs along nerve-muscle contacts was not prevented. It is concluded that during nerve-muscle synaptogenesis, alpha DG undergoes the same nerve- induced changes in distribution as AChRs. We propose a diffusion trap model in which the alpha DG-transmembrane complex participates in the anchoring and recruitment of AChRs and alpha DG during the formation of synaptic as well as nonsynaptic AChR clusters.     

Agrin Binds to the Nerve–Muscle Basal Lamina via Laminin

Agrin is a heparan sulfate proteoglycan that is required for the formation and maintenance of neuromuscular junctions. During development, agrin is secreted from motor neurons to trigger the local aggregation of acetylcholine receptors (AChRs) and other proteins in the muscle fiber, which together compose the postsynaptic apparatus. After release from the motor neuron, agrin binds to the developing muscle basal lamina and remains associated with the synaptic portion throughout adulthood. We have recently shown that full-length chick agrin binds to a basement membrane-like preparation called Matrigel™. The first 130 amino acids from the NH₂ terminus are necessary for the binding, and they are the reason why, on cultured chick myotubes, AChR clusters induced by full-length agrin are small. In the current report we show that an NH₂-terminal fragment of agrin containing these 130 amino acids is sufficient to bind to Matrigel™ and that the binding to this preparation is mediated by laminin-1. The fragment also binds to laminin-2 and -4, the predominant laminin isoforms of the muscle fiber basal lamina. On cultured myotubes, it colocalizes with laminin and is enriched in AChR aggregates. In addition, we show that the effect of full-length agrin on the size of AChR clusters is reversed in the presence of the NH₂-terminal agrin fragment. These data strongly suggest that binding of agrin to laminin provides the basis of its localization to synaptic basal lamina and other basement membranes.     

Agrin is required for posterior development and motor axon outgrowth and branching in embryonic zebrafish

Although recent studies have extended our understanding of agrin's function during development, its function in the central nervous system (CNS) is not clearly understood. To address this question, zebrafish agrin was identified and characterized. Zebrafish agrin is expressed in the developing CNS and in nonneural structures such as somites and notochord. In agrin morphant embryos, acetylcholine receptor (AChR) cluster number and size on muscle fibers at the choice point were unaffected, whereas AChR clusters on muscle fibers in the dorsal and ventral regions of the myotome were reduced or absent. Defects in the axon outgrowth by primary motor neurons, subpopulations of branchiomotor neurons, and Rohon-Beard sensory neurons were also observed, which included truncation of axons and increased branching of motor axons. Moreover, agrin morphants exhibit significantly inhibited tail development in a dose-dependent manner, as well as defects in the formation of the midbrain-hindbrain boundary and reduced size of eyes and otic vesicles. Together these results show that agrin plays an important role in both peripheral and CNS development and also modulates posterior development in zebrafish.     

Involvement of p120 catenin in myopodial assembly and nerve-muscle synapse formation

At developing neuromuscular junctions (NMJs), muscles initially contact motor axons by microprocesses, or myopodia, which are induced by nerves and nerve-secreted agrin, but it is unclear how myopodia are assembled and how they influence synaptic differentiation at the NMJ. Here, we report that treatment of cultured muscle cells with agrin transiently depleted p120 catenin (p120ctn) from cadherin junctions in situ, and increased the tyrosine phosphorylation and decreased the cadherin-association of p120ctn in cell extracts. Whereas ectopic expression of wild-type p120ctn in muscle generated myopodia in the absence of agrin, expression of a specific dominant-negative mutant form of p120ctn, which blocks filopodial assembly in nonmuscle cells, suppressed nerve- and agrin-induction of myopodia. Significantly, approaching neurites triggered reduced acetylcholine receptor (AChR) clustering along the edges of muscle cells expressing mutant p120ctn than of control cells, although the ability of the mutant cells to cluster AChRs was itself normal. Our results indicate a novel role of p120ctn in agrin-induced myopodial assembly and suggest that myopodia increase muscle-nerve contacts and muscle's access to neural agrin to promote NMJ formation.     

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.     

Involvement of p120 catenin in myopodial assembly and nerve–muscle synapse formation

At developing neuromuscular junctions (NMJs), muscles initially contact motor axons by microprocesses, or myopodia, which are induced by nerves and nerve‐secreted agrin, but it is unclear how myopodia are assembled and how they influence synaptic differentiation at the NMJ. Here, we report that treatment of cultured muscle cells with agrin transiently depleted p120 catenin (p120ctn) from cadherin junctions in situ, and increased the tyrosine phosphorylation and decreased the cadherin‐association of p120ctn in cell extracts. Whereas ectopic expression of wild‐type p120ctn in muscle generated myopodia in the absence of agrin, expression of a specific dominant‐negative mutant form of p120ctn, which blocks filopodial assembly in nonmuscle cells, suppressed nerve‐ and agrin‐induction of myopodia. Significantly, approaching neurites triggered reduced acetylcholine receptor (AChR) clustering along the edges of muscle cells expressing mutant p120ctn than of control cells, although the ability of the mutant cells to cluster AChRs was itself normal. Our results indicate a novel role of p120ctn in agrin‐induced myopodial assembly and suggest that myopodia increase muscle–nerve contacts and muscle's access to neural agrin to promote NMJ formation. © 2006 Wiley Periodicals, Inc. J Neurobiol, 2006     

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.     

Agrin fragments differentially induce ectopic aggregation of acetylcholine receptors in myotomal muscles of Xenopus embryos

Agrin is an extracellular synaptic protein that organizes the postsynaptic apparatus, including acetylcholine receptors (AChRs), of the neuromuscular junction. The COOH-terminal portion of agrin has full AChR-aggregating activity in culture, and includes three globular domains, G1, G2, and G3. Portions of the agrin protein containing these domains bind to different cell surface proteins of muscle cells, including alpha-dystroglycan (G1-G2) and heparan sulfate proteoglycans (G2), whereas the G3 domain is sufficient to aggregate AChRs. We sought to determine whether the G1 and G2 domains of agrin potentiate agrin activity in vivo, as they do in culture. Fragments from the COOH-terminal of a neuronal agrin isoform (4,8) containing G3, both G2 and G3, or all three G domains were overexpressed in Xenopus embryos during neuromuscular synapse formation in myotomal muscles. RNA encoding these fragments of rat agrin was injected into one-cell embryos. All three fragments increased the ectopic aggregation of AChRs in noninnervated regions near the center of myotomes. Surprisingly, ectopic aggregation was more pronounced after overexpression of the smallest fragment, which lacks the heparin- and alpha-dystroglycan-binding domains. Synaptic AChR aggregation was decreased in embryos overexpressing the fragments, suggesting a competition between endogenous agrin secreted by nerve terminals and exogenous agrin fragments secreted by muscle cells. These results suggest that binding of the larger agrin fragments to alpha-dystroglycan and/or heparan sulfate proteoglycans may sequester the fragments and inhibit their activity in embryonic muscle. These intermolecular interactions may regulate agrin activity and differentiation of the neuromuscular junction in vivo.     

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.     

Effect of agrin on the distribution of acetylcholine receptors and sodium channels on adult skeletal muscle fibers in culture

We used the loose patch voltage clamp technique and rhodamine-conjugated alpha-bungarotoxin to study the regulation of Na channel (NaCh) and acetylcholine receptor (AChR) distribution on dissociated adult skeletal muscle fibers in culture. The aggregate of AChRs and NaChs normally found in the postsynaptic membrane of these cells gradually fragmented and dispersed from the synaptic region after several days in culture. This dispersal was the result of the collagenase treatment used to dissociate the cells, suggesting that a factor associated with the extracellular matrix was responsible for maintaining the high concentration of AchRs and NaChs at the neuromuscular junction. We tested whether the basal lamina protein agrin, which has been shown to induce the aggregation of AChRs on embryonic myotubes, could similarly influence the distribution of NaChs. By following identified fibers, we found that agrin accelerated both the fragmentation of the endplate AChR cluster into smaller patches as well as the appearance of new AChR clusters away from the endplate. AChR patches which were fragments of the original endplate retained a high density of NaChs, but no new NaCh hotspots were found elsewhere on the fiber, including sites of newly formed AChR clusters. The results are consistent with the hypothesis that extracellular signals regulate the distribution of AChRs and NaChs on skeletal muscle fibers. While agrin probably serves this function for the AChR, it does not appear to play a role in the regulation of the NaCh distribution.     

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.     

Nitric oxide synthase activity is required for postsynaptic differentiation of the embryonic neuromuscular junction

Agrin, a synapse-organizing protein externalized by motor axons at the neuromuscular junction (NMJ), initiates a signaling cascade in muscle cells leading to aggregation of postsynaptic proteins, including acetylcholine receptors (AChRs). We examined whether nitric oxide synthase (NOS) activity is required for agrin-induced aggregation of postsynaptic AChRs at the embryonic NMJ in vivo and in cultured muscle cells. Inhibition of NOS reduced AChR aggregation at embryonic Xenopus NMJs by 50-90%, whereas overexpression of NOS increased AChR aggregate area 2- to 3-fold at these synapses. NOS inhibitors completely blocked agrin-induced AChR aggregation in cultured embryonic muscle cells. Application of NO donors to muscle cells induced AChR clustering in the absence of agrin. Our results indicate that NOS activity is necessary for postsynaptic differentiation of embryonic NMJs and that NOS is a likely participant in the agrin-MuSK signaling pathway of skeletal muscle cells.     

CLASP2-dependent microtubule capture at the neuromuscular junction membrane requires LL5β and actin for focal delivery of acetylcholine receptor vesicles

A hallmark of the neuromuscular junction (NMJ) is the high density of acetylcholine receptors (AChRs) in the postsynaptic muscle membrane. The postsynaptic apparatus of the NMJ is organized by agrin secreted from motor neurons. The mechanisms that underlie the focal delivery of AChRs to the adult NMJ are not yet understood in detail. We previously showed that microtubule (MT) capture by the plus end–tracking protein CLASP2 regulates AChR density at agrin-induced AChR clusters in cultured myotubes via PI3 kinase acting through GSK3β. Here we show that knockdown of the CLASP2-interaction partner LL5β by RNAi and forced expression of a CLASP2 fragment blocking the CLASP2/LL5β interaction inhibit microtubule capture. The same treatments impair focal vesicle delivery to the clusters. Consistent with these findings, knockdown of LL5β at the NMJ in vivo reduces the density and insertion of AChRs into the postsynaptic membrane. MT capture and focal vesicle delivery to agrin-induced AChR clusters are also inhibited by microtubule- and actin-depolymerizing drugs, invoking both cytoskeletal systems in MT capture and in the fusion of AChR vesicles with the cluster membrane. Combined our data identify a transport system, organized by agrin through PI3 kinase, GSK3β, CLASP2, and LL5β, for precise delivery of AChR vesicles from the subsynaptic nuclei to the overlying synaptic membrane.     

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.