Agrin and acetylcholine receptors are removed from abandoned synaptic sites at reinnervated frog neuromuscular junctions

Changes in the distribution of agrin and acetylcholine receptors (AChRs) were examined during reinnervation and following permanent denervation as a means of understanding mechanisms controlling the distribution of these molecules. Following nerve damage in the peripheral nervous system, regenerating nerve terminals preferentially return to previous synaptic sites leading to the restoration of synaptic activity. However, not all portions of original synaptic sites are reoccupied: Some of the synaptic sites are abandoned by both the nerve terminal and the Schwann cell. Abandoned synaptic sites contain agrin, AChRs, and acetylcholinesterase (AChE) without an overlying nerve terminal or Schwann cell providing a unique location to observe changes in the distribution of these synapse-specific molecules. The distribution of anti-agrin and AChR staining at abandoned synaptic sites was altered during the process of reinnervation, changing from a dense, wide distribution to a punctate, pale pattern, and finally becoming entirely absent. Agrin and AChRs were removed from abandoned synaptic sites in reinnervated frog neuromuscular junctions, while in contralateral muscles which were permanently denervated, anti-agrin and AChR staining remained at abandoned synaptic sites. Decreasing synaptic activity during reinnervation delayed the removal of agrin and AChRs from abandoned synaptic sites. Altogether, these results support the hypothesis that synaptic activity controls a cellular mechanism that directs the removal of agrin from synaptic basal lamina and the loss of agrin leads to the dispersal of AChRs. © 1997 John Wiley & Sons, Inc. J Neurobiol 33: 999–1018, 1997     

Agrin-like molecules at synaptic sites in normal, denervated, and damaged skeletal muscles

Several lines of evidence have led to the hypothesis that agrin, a protein extracted from the electric organ of Torpedo, is similar to the molecules in the synaptic cleft basal lamina at the neuromuscular junction that direct the formation of acetylcholine receptor and acetylcholinesterase aggregates on regenerating myofibers. One such finding is that monoclonal antibodies against agrin stain molecules concentrated in the synaptic cleft of neuromuscular junctions in rays. In the studies described here we made additional monoclonal antibodies against agrin and used them to extend our knowledge of agrin-like molecules at the neuromuscular junction. We found that anti-agrin antibodies intensely stained the synaptic cleft of frog and chicken as well as that of rays, that denervation of frog muscle resulted in a reduction in staining at the neuromuscular junction, and that the synaptic basal lamina in frog could be stained weeks after degeneration of all cellular components of the neuromuscular junction. We also describe anti-agrin staining in nonjunctional regions of muscle. We conclude the following: (a) agrin-like molecules are likely to be common to all vertebrate neuromuscular junctions; (b) the long-term maintenance of such molecules at the junction is nerve dependent; (c) the molecules are, indeed, a component of the synaptic basal lamina; and (d) they, like the molecules that direct the formation of receptor and esterase aggregates on regenerating myofibers, remain associated with the synaptic basal lamina after muscle damage.     

Activity dependent removal of agrin from synaptic basal lamina by matrix metalloproteinase 3

Agrin is a heparan sulfate proteoglycan, which plays an essential role in the development and maintenance of the neuromuscular junction. Agrin is a stable component of the synaptic basal lamina and strong evidence supports the hypothesis that agrin directs the formation of the postsynaptic apparatus, including aggregates of AChRs, and junctional folds. Changes in the distribution of agrin during synaptic remodeling, denervation and reinnervation reveal that agrin can be quickly and efficiently removed from the synaptic basal lamina in a regulated manner. In order to fully understand this mechanism we sought to identify those molecules that were responsible for the removal of agrin. Matrix Metalloproteinases (MMPs) were the most likely molecules since MMPs are involved in the regulation of the pericellular space, including the cleavage of matrix proteins. In particular, MMP3 has been shown to be effective in cleaving heparan sulfate proteoglycans. Antibodies to MMP3 recognize molecules concentrated in the extracellular matrix of perisynaptic Schwann cells. MMP3 specific phylogenic compounds reveal that active MMP3 is localized to the neuromuscular junction. Purified recombinant MMP3 can directly cleave agrin, and it can also remove agrin from synaptic basal lamina. MMP3 activity is itself regulated as activation of MMP3 is lost in denervated muscles. MMP3 null mutant mice have altered neuromuscular junction structure and function, with increased AChRs, junctional folds and agrin immunoreactivity. Altogether these results support the hypothesis that synaptic activity induces the activation of MMP3, and the activated MMP3 removes agrin from the synaptic basal lamina.     

Matrix metalloproteinase‐3 removes agrin from synaptic basal lamina

Agrin, a heparin sulfate proteoglycan, is an integral member of the synaptic basal lamina and plays a critical role in the formation and maintenance of the neuromuscular junction. The N‐terminal region of agrin binds tightly to basal lamina, while the C‐terminal region interacts with a muscle‐specific tyrosine kinase (MuSK) to induce the formation of the postsynaptic apparatus. Although the binding of agrin to basal lamina is tight, the binding of agrin to MuSK has yet to be shown; therefore, basal lamina binding is critical for maintaining the presentation of agrin to MuSK. Here we report evidence that supports our hypothesis that matrix metalloproteinase‐3 (MMP‐3) is responsible for the removal of agrin from synaptic basal lamina. Antibodies to the hinge region of human MMP‐3 recognize molecules concentrated at the frog neuromuscular junction in both cross sections and whole mounts. Electron microscopy of neuromuscular junctions stained with antibodies to MMP‐3 reveals that staining is found in the extracellular matrix surrounding the Schwann cell. Treatment of sections from frog anterior tibialis muscle with MMP‐3 results in a clear and reproducible removal of agrin immunoreactivity from synaptic basal lamina. The same MMP‐3 treatment does not alter anti‐laminin staining. These results support our hypothesis that synaptic activity results in the activation of MMP‐3 at the neuromuscular junction and that MMP‐3 specifically removes agrin from synaptic basal lamina. © 2000 John Wiley & Sons, Inc. J Neurobiol 43: 140–149, 2000     

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.     

Motor neurons contain agrin-like molecules

Molecules antigenically similar to agrin, a protein extracted from the electric organ of Torpedo californica, are highly concentrated in the synaptic basal lamina of neuromuscular junctions in vertebrate skeletal muscle. On the basis of several lines of evidence it has been proposed that agrin-like molecules mediate the nerve-induced formation of acetylcholine receptor (AChR) and acetylcholinesterase (AChE) aggregates on the surface of muscle fibers at developing and regenerating neuromuscular junctions and that they help maintain these postsynaptic specializations in the adult. Here we show that anti-agrin monoclonal antibodies selectively stain the cell bodies of motor neurons in embryos and adults, and that the stain is concentrated in the Golgi apparatus. We also present evidence that motor neurons in both embryos and adults contain molecules that cause the formation of AChR and AChE aggregates on cultured myotubes and that these AChR/AChE-aggregating molecules are antigenically similar to agrin. These findings are consistent with the hypothesis that agrin-like molecules are synthesized by motor neurons, and are released from their axon terminals to become incorporated into the synaptic basal lamina where they direct the formation of synapses during development and regeneration.     

Matrix metalloproteinase-3 removes agrin from synaptic basal lamina

Agrin, a heparin sulfate proteoglycan, is an integral member of the synaptic basal lamina and plays a critical role in the formation and maintenance of the neuromuscular junction. The N-terminal region of agrin binds tightly to basal lamina, while the C-terminal region interacts with a muscle-specific tyrosine kinase (MuSK) to induce the formation of the postsynaptic apparatus. Although the binding of agrin to basal lamina is tight, the binding of agrin to MuSK has yet to be shown; therefore, basal lamina binding is critical for maintaining the presentation of agrin to MuSK. Here we report evidence that supports our hypothesis that matrix metalloproteinase-3 (MMP-3) is responsible for the removal of agrin from synaptic basal lamina. Antibodies to the hinge region of human MMP-3 recognize molecules concentrated at the frog neuromuscular junction in both cross sections and whole mounts. Electron microscopy of neuromuscular junctions stained with antibodies to MMP-3 reveals that staining is found in the extracellular matrix surrounding the Schwann cell. Treatment of sections from frog anterior tibialis muscle with MMP-3 results in a clear and reproducible removal of agrin immunoreactivity from synaptic basal lamina. The same MMP-3 treatment does not alter anti-laminin staining. These results support our hypothesis that synaptic activity results in the activation of MMP-3 at the neuromuscular junction and that MMP-3 specifically removes agrin from synaptic basal lamina.     

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.     

Acetylcholinesterase from the motor nerve terminal accumulates on the synaptic basal lamina of the myofiber

Acetylcholinesterase (AChE) in skeletal muscle is concentrated at neuromuscular junctions, where it is found in the synaptic cleft between muscle and nerve, associated with the synaptic portion of the myofiber basal lamina. This raises the question of whether the synaptic enzyme is produced by muscle, nerve, or both. Studies on denervated and regenerating muscles have shown that myofibers can produce synaptic AChE, and that the motor nerve may play an indirect role, inducing myofibers to produce synaptic AChE. The aim of this study was to determine whether some of the AChE which is known to be made and transported by the motor nerve contributes directly to AChE in the synaptic cleft. Frog muscles were surgically damaged in a way that caused degeneration and permanent removal of all myofibers from their basal lamina sheaths. Concomitantly, AChE activity was irreversibly blocked. Motor axons remained intact, and their terminals persisted at almost all the synaptic sites on the basal lamina in the absence of myofibers. 1 mo after the operation, the innervated sheaths were stained for AChE activity. Despite the absence of myofibers, new AChE appeared in an arborized pattern, characteristic of neuromuscular junctions, and its reaction product was concentrated adjacent to the nerve terminals, obscuring synaptic basal lamina. AChE activity did not appear in the absence of nerve terminals. We concluded therefore, that the newly formed AChE at the synaptic sites had been produced by the persisting axon terminals, indicating that the motor nerve is capable of producing some of the synaptic AChE at neuromuscular junctions. The newly formed AChE remained adherent to basal lamina sheaths after degeneration of the terminals, and was solubilized by collagenase, indicating that the AChE provided by nerve had become incorporated into the basal lamina as at normal neuromuscular junctions.     

The signaling pathways mediated by P2Y nucleotide receptors in the formation and maintenance of the skeletal neuromuscular junction

The motor neuron, the Schwann cell and the muscle cell are highly specialized at the vertebrate skeletal neuromuscular junction (NMJ). The muscle cell surface contains a high local density of acetylcholine (ACh) receptors (AChRs), acetylcholinesterase (AChE) and their interacting macromolecules at the NMJ, forming the postsynaptic specializations. During the early stages of development, the incoming nerve terminal induces the formation of these postsynaptic specializations; the nerve secretes agrin and neuregulin (NRG), which are known to aggregate existing AChRs and to increase the expression of AChR at the synaptic region, respectively. In addition, adenosine 5'-triphosphate (ATP) is stored at the motor nerve terminals and is coreleased with ACh during muscle contraction. Recent evidence suggests that ATP can play a role in forming and maintaining the postsynaptic specializations by activating its corresponding receptors. In particular, one of the nucleotide receptor subtypes, the P2Y(1) receptor, is specifically localized at the NMJs. The gene expression of AChR and AChE is upregulated after the activation of P2Y(1) receptors. Thus, the synaptic ATP together with agrin and NRG can act as a synapse-organizing factor to induce the expression of postsynaptic functional effectors.     

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.     

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.     

The influence of basal lamina on the accumulation of acetylcholine receptors at synaptic sites in regenerating muscle

If skeletal muscles are damaged in ways that spare the basal lamina sheaths of the muscle fibers, new myofibers develop within the sheaths and neuromuscular junctions form at the original synaptic sites on them. At the regenerated neuromuscular junctions, as at the original ones, the muscle fiber plasma membrane is characterized by infoldings and a high concentration of acetylcholine receptors (AChRs). The aim of this study was to determine whether or not the synaptic portion of the myofiber basal lamina sheath plays a direct role in the formation of the subsynaptic apparatus on regenerating myofibers, a question raised by the results of earlier experiments. The junctional region of the frog cutaneous pectoris muscle was crushed or frozen, which resulted in disintegration and phagocytosis of all cells at the synapse but left intact much of the myofiber basal lamina. Reinnervation was prevented. When new myofibers developed within the basal lamina sheaths, patches of AChRs and infoldings formed preferentially at sites where the myofiber membrane was apposed to the synaptic region of the sheaths. Processes from unidentified cells gradually came to lie on the presynaptic side of the basal lamina at a small fraction of the synaptic sites, but there was no discernible correlation between their presence and the effectiveness of synaptic sites in accumulating AChRs. We therefore conclude that molecules stably attached to the myofiber basal lamina at synaptic sites direct the formation of subsynaptic apparatus in regenerating myofibers. An analysis of the distribution of AChR clusters at synaptic sites indicated that they formed as a result of myofiber-basal lamina interactions that occurred at numerous places along the synaptic basal lamina, that their presence was not dependent on the formation of plasma membrane infoldings, and that the concentration of receptors within clusters could be as great as the AChR concentration at normal neuromuscular junctions.     

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.     

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.     

Regulation of agrin-induced acetylcholine receptor aggregation by Ca++ and phorbol ester

Agrin, a protein extracted from the electric organ of Torpedo californica, induces the formation of specializations on cultured chick myotubes that resemble the postsynaptic apparatus at the neuromuscular junction. The aim of the studies reported here was to characterize the effects of agrin on the distribution of acetylcholine receptors (AChRs) and cholinesterase as a step toward determining agrin's mechanism of action. When agrin was added to the medium bathing chick myotubes small (less than 4 micron 2) aggregates of AChRs began to appear within 2 h and increased rapidly in number until 4 h. Over the next 12-20 h the number of aggregates per myotube decreased as the mean size of each aggregate increased to approximately 15 micron 2. The accumulation of AChRs into agrin-induced aggregates occurred primarily by lateral migration of AChRs already in the myotube plasma membrane at the time agrin was added to the cultures. Aggregates of AChRs and cholinesterase remained as long as agrin was present in the medium; if agrin was removed the number of aggregates declined slowly. The formation and maintenance of agrin-induced AChR aggregates required Ca++, Co++ and Mn++ inhibited agrin-induced AChR aggregation and increased the rate of aggregate dispersal. Mg++ and Sr++ could not substitute for Ca++. Agrin-induced receptor aggregation also was inhibited by phorbol 12-myristate 13-acetate, an activator of protein kinase C, and by inhibitors of energy metabolism. The similarities between agrin's effects on cultured myotubes and events that occur during formation of neuromuscular junctions support the hypothesis that axon terminals release molecules similar to agrin that induce the differentiation of the postsynaptic apparatus.     

Acetylcholine receptors in regenerating muscle accumulate at original synaptic sites in the absence of the nerve

We examined the role of nerve terminals in organizing acetylcholine receptors on regenerating skeletal-muscle fibers. When muscle fibers are damaged, they degenerate and are phagocytized, but their basal lamina sheaths survive. New myofibers form within the original basal lamina sheaths, and they become innervated precisely at the original synaptic sites on the sheaths. After denervating and damaging muscle, we allowed myofibers to regenerate but deliberately prevented reinnervation. The distribution of acetylcholine receptors on regenerating myofibers was determined by histological methods, using [125I] alpha-bungarotoxin or horseradish peroxidase-alpha-bungarotoxin; original synaptic sites on the basal lamina sheaths were marked by cholinesterase stain. By one month after damage to the muscle, the new myofibers have accumulations of acetylcholine receptors that are selectively localized to the original synaptic sites. The density of the receptors at these sites is the same as at normal neuromuscular junctions. Folds in the myofiber surface resembling junctional folds at normal neuromuscular junctions also occur at original synaptic sites in the absence of nerve terminals. Our results demonstrate that the biochemical and structural organization of the subsynaptic membrane in regenerating muscle is directed by structures that remain at synaptic sites after removal of the nerve.