Agrin released by motor neurons induces the aggregation of acetylcholine receptors at neuromuscular junctions.

To test the hypothesis that agrin mediates motor neuron-induced aggregation of acetylcholine receptors (AChRs) in skeletal muscle fibers and to determine whether the agrin active in this process is released by motor neurons, we raised polyclonal antibodies to purified ray agrin that blocked its receptor aggregating activity. When the antibodies were applied to chick motor neuron--chick myotube cocultures, they inhibited the formation of AChR aggregates at and near neuromuscular contacts, demonstrating that agrin plays a role in the induction of the aggregates. Rat motor neurons, like chick motor neurons, induce AChR aggregates on chick myotubes. This effect was not inhibited by our antibodies, indicating that, although the antibodies inhibited the activity of chick agrin, they did not have a similar effect on rat agrin. We conclude that agrin released by rat motor neurons induced the chick myotubes to aggregate AChRs.     

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.     

Alpha3Na+/K+-ATPase is a neuronal receptor for agrin

Agrin, through its interaction with the receptor tyrosine kinase MuSK, mediates accumulation of acetylcholine receptors (AChR) at the developing neuromuscular junction. Agrin has also been implicated in several functions in brain. However, the mechanism by which agrin exerts its effects in neural tissue is unknown. Here we present biochemical evidence that agrin binds to the alpha3 subunit of the Na+/K+-ATPase (NKA) in CNS neurons. Colocalization with agrin binding sites at synapses supports the hypothesis that the alpha3NKA is a neuronal agrin receptor. Agrin inhibition of alpha3NKA activity results in membrane depolarization and increased action potential frequency in cortical neurons in culture and acute slice. An agrin fragment that acts as a competitive antagonist depresses action potential frequency, showing that endogenous agrin regulates native alpha3NKA function. These data demonstrate that, through its interaction with the alpha3NKA, agrin regulates activity-dependent processes in neurons, providing a molecular framework for agrin action in the CNS.     

The COOH-terminal domain of agrin signals via a synaptic receptor in central nervous system neurons

Agrin is a motor neuron-derived factor that directs formation of the postsynaptic apparatus of the neuromuscular junction. Agrin is also expressed in the brain, raising the possibility that it might serve a related function at neuron-neuron synapses. Previously, we identified an agrin signaling pathway in central nervous system (CNS) neurons, establishing the existence of a neural receptor that mediates responses to agrin. As a step toward identifying this agrin receptor, we have characterized the minimal domains in agrin that bind and activate it. Structures required for agrin signaling in CNS neurons are contained within a 20-kD COOH-terminal fragment of the protein. Agrin signaling is independent of alternative splicing at the z site, but requires sequences that flank it because their deletion results in a 15-kD fragment that acts as an agrin antagonist. Thus, distinct regions within agrin are responsible for receptor binding and activation. Using the minimal agrin fragments as affinity probes, we also studied the expression of the agrin receptor on CNS neurons. Our results show that both agrin and its receptor are concentrated at neuron-neuron synapses. These data support the hypothesis that agrin plays a role in formation and/or function of CNS synapses.     

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     

Formation of functional synaptic connections between cultured cortical neurons from agrin‐deficient mice

Numerous studies suggest that the extracellular matrix protein agrin directs the formation of the postsynaptic apparatus at the neuromuscular junction (NMJ). Strong support for this hypothesis comes from the observation that the high density of acetylcholine receptors (AChR) normally present at the neuromuscular junction fails to form in muscle of embryonic agrin mutant mice. Agrin is expressed by many populations of neurons in the central nervous system (CNS), suggesting that this molecule may also play a role in neuron–neuron synapse formation. To test this hypothesis, we examined synapse formation between cultured cortical neurons isolated from agrin‐deficient mouse embryos. Our data show that glutamate receptors accumulate at synaptic sites on agrin‐deficient neurons. Moreover, electrophysiological analysis demonstrates that functional glutamatergic and gamma‐aminobutyric acid (GABA)ergic synapses form between mutant neurons. The frequency and amplitude of miniature postsynaptic glutamatergic and GABAergic currents are similar in mutant and age‐matched wild‐type neurons during the first 3 weeks in culture. These results demonstrate that neuron‐specific agrin is not required for formation and early development of functional synaptic contacts between CNS neurons, and suggest that mechanisms of interneuronal synaptogenesis are distinct from those regulating synapse formation at the neuromuscular junction. © 1999 John Wiley & Sons, Inc. J Neurobiol 39: 547–557, 1999     

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.     

Agrin isoforms and their role in synaptogenesis.

Agrin is thought to mediate the motor neuron-induced aggregation of synaptic proteins on the surface of muscle fibers at neuromuscular junctions. Recent experiments provide direct evidence in support of this hypothesis, reveal the nature of agrin immunoreactivity at sites other than neuromuscular junctions, and have resulted in findings that are consistent with the possibility that agrin plays a role in synaptogenesis throughout the nervous system.     

Formation of functional synaptic connections between cultured cortical neurons from agrin-deficient mice.

Numerous studies suggest that the extracellular matrix protein agrin directs the formation of the postsynaptic apparatus at the neuromuscular junction (NMJ). Strong support for this hypothesis comes from the observation that the high density of acetylcholine receptors (AChR) normally present at the neuromuscular junction fails to form in muscle of embryonic agrin mutant mice. Agrin is expressed by many populations of neurons in the central nervous system (CNS), suggesting that this molecule may also play a role in neuron-neuron synapse formation. To test this hypothesis, we examined synapse formation between cultured cortical neurons isolated from agrin-deficient mouse embryos. Our data show that glutamate receptors accumulate at synaptic sites on agrin-deficient neurons. Moreover, electrophysiological analysis demonstrates that functional glutamatergic and gamma-aminobutyric acid (GABA)ergic synapses form between mutant neurons. The frequency and amplitude of miniature postsynaptic glutamatergic and GABAergic currents are similar in mutant and age-matched wild-type neurons during the first 3 weeks in culture. These results demonstrate that neuron-specific agrin is not required for formation and early development of functional synaptic contacts between CNS neurons, and suggest that mechanisms of interneuronal synaptogenesis are distinct from those regulating synapse formation at the neuromuscular junction.     

Neural agrin controls maturation of the excitation-contraction coupling mechanism in human myotubes developing in vitro

The aim of this study was to elucidate the mechanisms responsible for the effects of innervation on the maturation of excitation-contraction coupling apparatus in human skeletal muscle. For this purpose, we compared the establishment of the excitation-contraction coupling mechanism in myotubes differentiated in four different experimental paradigms: 1) aneurally cultured, 2) cocultured with fetal rat spinal cord explants, 3) aneurally cultured in medium conditioned by cocultures, and 4) aneurally cultured in medium supplemented with purified recombinant chick neural agrin. Ca(2+) imaging indicated that coculturing human muscle cells with rat spinal cord explants increased the fraction of cells showing a functional excitation-contraction coupling mechanism. The effect of spinal cord explants was mimicked by treatment with medium conditioned by cocultures or by addition of 1 nM of recombinant neural agrin to the medium. The treatment with neural agrin increased the number of human muscle cells in which functional ryanodine receptors (RyRs) and dihydropyridine-sensitive L-type Ca(2+) channels were detectable. Our data are consistent with the hypothesis that agrin, released from neurons, controls the maturation of the excitation-contraction coupling mechanism and that this effect is due to modulation of both RyRs and L-type Ca(2+) channels. Thus, a novel role for neural agrin in skeletal muscle maturation is proposed.     

Neural Agrin Changes the Electrical Properties of Developing Human Skeletal Muscle Cells

Recent investigations suggest that the effects of neural agrin might not be limited to neuromuscular junction formation and maintenance and that other aspects of muscle development might be promoted by agrin. Here we tested the hypothesis that agrin induces a change in the excitability properties in primary cultures of non-innervated human myotubes. Electrical membrane properties of human myotubes were recorded using the whole-cell patch-clamp technique. Cell incubation with recombinant chick neural agrin (1 nM) led to a more negative membrane resting potential. Addition of strophanthidin, a blocker of the Na⁺/K⁺ ATPase, depolarized agrin-treated myotubes stronger than control, indicating, in the presence of agrin, a higher contribution of the Na⁺/K⁺ ATPase in establishing the resting membrane potential. Indeed, larger amounts of both the α1 and the α2 isoforms of the Na⁺/K⁺ ATPase protein were expressed in agrin-treated cells. A slight but significant down-regulation of functional apamin-sensitive K⁺ channels was observed after agrin treatment. These results indicate that neural agrin might act as a trophic factor promoting the maturation of membrane electrical properties during differentiation, confirming the role of agrin as a general promoter of muscle development. Tomaz Mars and Marina Sciancalepore contributed equally to this article. A preliminary account of our data has been presented in abstract form. Jurdana M, Mars T, Grubic Z, Sciancalepore M (2006) Agrin promotes the maturation of non-innervated human myotubes. Acta Physiol 188(Suppl 652):P6.     

Dystroglycan overexpression in vivo alters acetylcholine receptor aggregation at the neuromuscular junction

Dystroglycan is a member of the transmembrane dystrophin glycoprotein complex in muscle that binds to the synapse-organizing molecule agrin. Dystroglycan binding and AChR aggregation are mediated by two separate domains of agrin. To test whether dystroglycan plays a role in receptor aggregation at the neuromuscular junction, we overexpressed it by injecting rabbit dystroglycan RNA into one- or two-celled Xenopus embryos. We measured AChR aggregation in myotomes by labeling them with rhodamine-alpha-bungarotoxin followed by confocal microscopy and image analysis. Dystroglycan overexpression decreased AChR aggregation at the neuromuscular junction. This result is consistent with dystroglycan competition for agrin without signaling AChR aggregation. It also supports the hypothesis that dystroglycan is not the myotube-associated specificity component, (MASC) a putative coreceptor needed for agrin to activate muscle-specific kinase (MuSK) and signal AChR aggregation. Dystroglycan was distributed along the surface of muscle membranes, but was concentrated at the ends of myotomes, where AChRs normally aggregate at synapses. Overexpressed dystroglycan altered AChR aggregation in a rostral-caudal gradient, consistent with the sequential development of neuromuscular synapses along the embryo. Increasing concentrations of dystroglycan RNA did not further decrease AChR aggregation, but decreased embryo survival. Development often stopped during gastrulation, suggesting an essential, nonsynaptic role of dystroglycan during this early period of development.     

A neuronal inhibitory domain in the N-terminal half of agrin.

Agrin is required for appropriate pre- and postsynaptic differentiation of neuromuscular junctions. While agrin's ability to orchestrate postsynaptic differentiation is well documented, more recent experiments have suggested that agrin is also a "stop signal" for the presynaptic neuron, and that agrin has actions on neurons in the CNS. To elucidate the neuronal activities of agrin and to define the receptor(s) responsible for these functions, we have examined adhesions of neurons and their neurite-outgrowth responses to purified agrin in vitro. We find that both full-length agrin and the C-terminal 95 kDa of agrin (agrin c95), which is sufficient to induce postsynaptic differentiation, are adhesive for chick ciliary ganglion (CG) and forebrain neurons. Consistent with previous findings, our results show that N-CAM binds to full-length agrin, and suggest that alpha-dystroglycan is a neuronal receptor for agrin c95. In neurite outgrowth assays, full-length agrin inhibited both laminin- and N-cadherin-induced neurite growth from CG neurons. The N-terminal 150 kDa fragment of agrin, but not agrin c95, inhibited neurite outgrowth, indicating that domains in the N-terminal portion of agrin are sufficient for this function. Adhesion assays using protein-coated beads and agrin-expressing cells revealed differential interactions of agrin with members of the immunoglobulin superfamily of cell adhesion molecules. However, none of these, including N-CAM, appeared to be critical for neuronal adhesion. In summary, our results suggest that the N-terminal half of agrin is involved in agrin's ability to inhibit neurite outgrowth. Our results further suggest that neither alpha-dystroglycan nor N-CAM, two known binding proteins for agrin, mediate this effect.     

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.     

A neuronal inhibitory domain in the N‐terminal half of agrin

Agrin is required for appropriate pre‐ and postsynaptic differentiation of neuromuscular junctions. While agrin's ability to orchestrate postsynaptic differentiation is well documented, more recent experiments have suggested that agrin is also a “stop signal” for the presynaptic neuron, and that agrin has actions on neurons in the CNS. To elucidate the neuronal activities of agrin and to define the receptor(s) responsible for these functions, we have examined adhesions of neurons and their neurite‐outgrowth responses to purified agrin in vitro. We find that both full‐length agrin and the C‐terminal 95 kDa of agrin (agrin c95), which is sufficient to induce postsynaptic differentiation, are adhesive for chick ciliary ganglion (CG) and forebrain neurons. Consistent with previous findings, our results show that N‐CAM binds to full‐length agrin, and suggest that α‐dystroglycan is a neuronal receptor for agrin c95. In neurite outgrowth assays, full‐length agrin inhibited both laminin‐ and N‐cadherin–induced neurite growth from CG neurons. The N‐terminal 150 kDa fragment of agrin, but not agrin c95, inhibited neurite outgrowth, indicating that domains in the N‐terminal portion of agrin are sufficient for this function. Adhesion assays using protein‐coated beads and agrin‐expressing cells revealed differential interactions of agrin with members of the immunoglobulin superfamily of cell adhesion molecules. However, none of these, including N‐CAM, appeared to be critical for neuronal adhesion. In summary, our results suggest that the N‐terminal half of agrin is involved in agrin's ability to inhibit neurite outgrowth. Our results further suggest that neither α‐dystroglycan nor N‐CAM, two known binding proteins for agrin, mediate this effect. © 2002 Wiley Periodicals, Inc. J Neurobiol 50: 164–179, 2002; DOI 10.1002/neu.10025     

Retina development in zebrafish requires the heparan sulfate proteoglycan agrin

Recent studies from our laboratory have begun to elucidate the role of agrin in zebrafish development. One agrin morphant phenotype that results from agrin knockdown is microphthalmia (reduced eye size). To begin to understand the mechanisms underlying the role of agrin in eye development, we have analyzed retina development in agrin morphants. Retinal differentiation is impaired in agrin morphants, with retinal lamination being disrupted following agrin morpholino treatment. Pax 6.1 and Mbx1 gene expression, markers of eye development, are markedly reduced in agrin morphants. Formation of the optic fiber layer of the zebrafish retina is also impaired, exhibited as both reduced size of the optic fiber layer, and disruption of retinal ganglion cell axon growth to the optic tectum. The retinotectal topographic projection to the optic tectum is perturbed in agrin morphants in association with a marked loss of heparan sulfate expression in the retinotectal pathway, with this phenotype resembling retinotectal phenotypes observed in mutant zebrafish lacking enzymes for heparan sulfate synthesis. Treatment of agrin morphants with a fibroblast growth factor (Fgf) receptor inhibitor, rescue of the retinal lamination phenotype by transplantation of Fgf8-coated beads, and disruption of both the expression of Fgf-dependent genes and activation of ERK in agrin morphants provides evidence that agrin modulation of Fgf function contributes to retina development. Collectively, these agrin morphant phenotypes provide support for a crucial role of agrin in retina development and formation of an ordered retinotectal topographic map in the optic tectum of zebrafish.     

LRP4 serves as a coreceptor of agrin

Neuromuscular junction (NMJ) formation requires agrin, a factor released from motoneurons, and MuSK, a transmembrane tyrosine kinase that is activated by agrin. However, how signal is transduced from agrin to MuSK remains unclear. We report that LRP4, a low-density lipoprotein receptor (LDLR)-related protein, is expressed specifically in myotubes and binds to neuronal agrin. Its expression enables agrin binding and MuSK signaling in cells that otherwise do not respond to agrin. Suppression of LRP4 expression in muscle cells attenuates agrin binding, agrin-induced MuSK tyrosine phosphorylation, and AChR clustering. LRP4 also forms a complex with MuSK in a manner that is stimulated by agrin. Finally, we showed that LRP4 becomes tyrosine-phosphorylated in agrin-stimulated muscle cells. These observations indicate that LRP4 is a coreceptor of agrin that is necessary for MuSK signaling and AChR clustering and identify a potential target protein whose mutation and/or autoimmunization may cause muscular dystrophies.