Neurotransmitter acetylcholine negatively regulates neuromuscular synapse formation by a Cdk5-dependent mechanism

Synapse formation requires interactions between pre- and postsynaptic cells to establish the connection of a presynaptic nerve terminal with the neurotransmitter receptor-rich postsynaptic apparatus. At developing vertebrate neuromuscular junctions, acetylcholine receptor (AChR) clusters of nascent postsynaptic apparatus are not apposed by presynaptic nerve terminals. Two opposing activities subsequently promote the formation of synapses: positive signals stabilize the innervated AChR clusters, whereas negative signals disperse those that are not innervated. Although the nerve-derived protein agrin has been suggested to be a positive signal, the negative signals remain elusive. Here, we show that cyclin-dependent kinase 5 (Cdk5) is activated by ACh agonists and is required for the ACh agonist-induced dispersion of the AChR clusters that have not been stabilized by agrin. Genetic elimination of Cdk5 or blocking ACh production prevents the dispersion of AChR clusters in agrin mutants. Therefore, we propose that ACh negatively regulates neuromuscular synapse formation through a Cdk5-dependent mechanism.     

Agrin elicits membrane lipid condensation at sites of acetylcholine receptor clusters in C2C12 myotubes

The formation of the neuromuscular junction is characterized by the progressive accumulation of nicotinic acetylcholine receptors (AChRs) in the postsynaptic membrane facing the nerve terminal, induced predominantly through the agrin/muscle-specific kinase (MuSK) signaling cascade. However, the cellular mechanisms linking MuSK activation to AChR clustering are still poorly understood. Here, we investigate whether lipid rafts are involved in agrin-elicited AChR clustering in a mouse C2C12 cell line. We observed that in C2C12 myotubes, both AChR clustering and cluster stability were dependent on cholesterol, because depletion by methyl-beta-cyclodextrin inhibited cluster formation or dispersed established clusters. Importantly, AChR clusters resided in ordered membrane domains, a biophysical property of rafts, as probed by Laurdan two-photon fluorescence microscopy. We isolated detergent-resistant membranes (DRMs) by three different biochemical procedures, all of which generate membranes with similar cholesterol/GM1 ganglioside contents, and these were enriched in several postsynaptic components, notably AChR, syntrophin, and raft markers flotillin-2 and caveolin-3. Agrin did not recruit AChRs into DRMs, suggesting that they are present in rafts independently of agrin activation. Consequently, in C2C12 myotubes, agrin likely triggers AChR clustering or maintains clusters through the coalescence of lipid rafts. These data led us to propose a model in which lipid rafts play a pivotal role in the assembly of the postsynaptic membrane at the neuromuscular junction upon agrin signaling.     

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.     

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.     

Muscle-derived agrin in cultured myotubes: expression in the basal lamina and at induced acetylcholine receptor clusters.

The synaptic basal lamina (SBL) directs key aspects of the differentiation of regenerating neuromuscular junctions. A range of experiments indicate that agrin or a closely related molecule is stably associated with the SBL and participates in inducing the formation of the postsynaptic apparatus after damage to adult muscle. The selective concentration of agrin-related molecules in the SBL suggests that agrin is secreted locally by cellular components of the nerve-muscle synapse. In vivo studies on aneural embryonic muscle indicate that the muscle cell is one source of the agrin-like molecules in the SBL. Here we have used cultured chick muscle cells to study the expression of agrin-related molecules in the absence of innervation. Immunofluorescence and immunoelectron microscopy show that myogenic cells in culture express agrin-related molecules on their surfaces, and that at least a subset of these molecules are associated with the basal lamina. Moreover, in short term cultures agrin-like molecules accumulate on the surfaces of myogenic cells grown in unsupplemented basal media. We quantified the expression of agrin-like molecules on the cell surface using a solid-phase radioimmune assay. The expression of these molecules is relatively low during the first 6 days of culture and increases fourfold during the second week. The stimulation of the expression of agrin-related molecules in these long-term cultures requires the presence of chick embryo extract or fetal calf serum. We also characterized the expression of muscle-derived agrin-like molecules at clusters of AChR. These agrin-related molecules are not consistently colocalized at spontaneous AChR aggregates; however, they are selectively concentrated at greater than or equal to 90% of the AChR clusters that are induced by Torpedo agrin. These data, together with previous results from in vivo developmental experiments indicate that the agrin-like molecules in the synaptic basal lamina are derived at least in part from the muscle cell. In addition, the expression of agrin-like molecules can be regulated by soluble factors present in CEE and FBS. Finally, the selective localization of these molecules at induced AChR clusters, taken together with their localization in the basal lamina, suggests that agrin-like molecules secreted by the muscle cell play an important role in the formation and/or the stabilization of the postsynaptic apparatus.     

Role of lipid rafts in agrin-elicited acetylcholine receptor clustering

Emerging concepts of membrane organization point to the compartmentalization of the plasma membrane into distinct lipid microdomains. This lateral segregation within cellular membranes is based on cholesterol-sphingolipid-enriched microdomains or lipid rafts which can move laterally and assemble into large-scale domains to create plasma membrane specialized cellular structures at specific cell locations. Such domains are likely involved in the genesis of the postsynaptic specialization at the neuromuscular junction, which requires the accumulation of acetylcholine receptors (AChRs), through activation of the muscle specific kinase MuSK by the neurotropic factor agrin and the reorganization of the actin cytoskeleton. We used C2C12 myotubes as a model system to investigate whether agrin-elicited AChR clustering correlated with lipid rafts. In a previous study, using two-photon Laurdan confocal imaging, we showed that agrin-induced AChR clusters corresponded to condensed membrane domains: the biophysical hallmark of lipid rafts [F. Stetzkowski-Marden, K. Gaus, M. Recouvreur, A. Cartaud, J. Cartaud, Agrin elicits membrane condensation at sites of acetylcholine receptor clusters in C2C12 myotubes, J. Lipid Res. 47 (2006) 2121-2133]. We further demonstrated that formation and stability of AChR clusters depend on cholesterol. We also reported that three different extraction procedures (Triton X-100, pH 11 or isotonic Ca++, Mg++ buffer) generated detergent resistant membranes (DRMs) with similar cholesterol/GM1 ganglioside content, which are enriched in several signalling postsynaptic components, notably AChR, the agrin receptor MuSK, rapsyn and syntrophin. Upon agrin engagement, actin and actin-nucleation factors such as Arp2/3 and N-WASP were transiently recovered within raft fractions suggesting that the activation by agrin can trigger actin polymerization. Taken together, the present data suggest that AChR clustering at the neuromuscular junction relies upon a mechanism of raft coalescence driven by agrin-elicited actin polymerization.     

Disruption of Trkb-mediated signaling induces disassembly of postsynaptic receptor clusters at neuromuscular junctions

Neurotrophins and tyrosine receptor kinase (Trk) receptors are expressed in skeletal muscle, but it is unclear what functional role Trk-mediated signaling plays during postnatal life. Full-length TrkB (trkB.FL) as well as truncated TrkB (trkB.t1) were found to be localized primarily to the postsynaptic acetylcholine receptor- (AChR-) rich membrane at neuromuscular junctions. In vivo, dominant-negative manipulation of TrkB signaling using adenovirus to overexpress trkB.t1 in mouse sternomastoid muscle fibers resulted in the disassembly of postsynaptic AChR clusters at neuromuscular junctions, similar to that observed in mutant trkB+/- mice. When TrkB-mediated signaling was disrupted in cultured myotubes in the absence of motor nerve terminals and Schwann cells, agrin-induced AChR clusters were also disassembled. These results demonstrate a novel role for neurotrophin signaling through TrkB receptors on muscle fibers in the ongoing maintenance of postsynaptic AChR regions.     

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.     

Clustering of nicotinic acetylcholine receptors: From the neuromuscular junction to interneuronal synapses

Fast and accurate synaptic transmission requires high-density accumulation of neurotransmitter receptors in the postsynaptic membrane. During development of the neuromuscular junction, clustering of acetylcholine receptors (AChR) is one of the first signs of postsynaptic specialization and is induced by nerve-released agrin. Recent studies have revealed that different mechanisms regulate assembly vs stabilization of AChR clusters and of the postsynaptic apparatus. MuSK, a receptor tyrosine kinase and component of the agrin receptor, and rapsyn, an AChR-associated anchoring protein, play crucial roles in the postsynaptic assembly. Once formed, AChR clusters and the postsynaptic membrane are stabilized by components of the dystrophin/utrophin glycoprotein complex, some of which also direct aspects of synaptic maturation such as formation of postjunctional folds. Nicotinic receptors are also expressed across the peripheral and central nervous system (PNS/CNS). These receptors are localized not only at the pre- but also at the postsynaptic sites where they carry out major synaptic transmission. In neurons, they are found as clusters at synaptic or extrasynaptic sites, suggesting that different mechanisms might underlie this specific localization of nicotinic receptors. This review summarizes the current knowledge about formation and stabilization of the postsynaptic apparatus at the neuromuscular junction and extends this to explore the synaptic structures of interneuronal cholinergic synapses.     

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.     

Immobilization of nicotinic acetylcholine receptors in mouse C2 myotubes by agrin-induced protein tyrosine phosphorylation

Agrin induces the formation of highly localized specializations on myotubes at which nicotinic acetylcholine receptors (AChRs) and many other components of the postsynaptic apparatus at the vertebrate skeletal neuromuscular junction accumulate. Agrin also induces AChR tyrosine phosphorylation. Treatments that inhibit tyrosine phosphorylation prevent AChR aggregation. To examine further the relationship between tyrosine phosphorylation and receptor aggregation, we have used the technique of fluorescence recovery after photobleaching to assess the lateral mobility of AChRs and other surface proteins in mouse C2 myotubes treated with agrin or with pervanadate, a protein tyrosine phosphatase inhibitor. Agrin induced the formation of patches in C2 myotubes that stained intensely with anti-phosphotyrosine antibodies and within which AChRs were relatively immobile. Pervanadate, on the other hand, increased protein tyrosine phosphorylation throughout the myotube and caused a reduction in the mobility of diffusely distributed AChRs, without affecting the mobility of other membrane proteins. Pervanadate, like agrin, caused an increase in AChR tyrosine phosphorylation and a decrease in the rate at which AChRs could be extracted from intact myotubes by mild detergent treatment, suggesting that immobilized receptors were phosphorylated and therefore less extractable. Indeed, phosphorylated receptors were extracted from agrin-treated myotubes more slowly than nonphosphorylated receptors. AChR aggregates at developing neuromuscular junctions in embryonic rat muscles also labeled with anti- phosphotyrosine antibodies, suggesting that tyrosine phosphorylation could mediate AChR aggregation in vivo as well. Thus, agrin appears to induce AChR aggregation by creating circumscribed domains of increased protein tyrosine phosphorylation within which receptors become phosphorylated and immobilized.     

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.     

Acetylcholine receptor aggregation parallels the deposition of a basal lamina proteoglycan during development of the neuromuscular junction

To determine the time course of synaptic differentiation, we made successive observations on identified, nerve-contacted muscle cells developing in culture. The cultures had either been stained with fluorescent alpha-bungarotoxin, or were maintained in the presence of a fluorescent monoclonal antibody. These probes are directed at acetylcholine receptors (AChR) and a basal lamina proteoglycan, substances that show nearly congruent surface organizations at the adult neuromuscular junction. In other experiments individual muscle cells developing in culture were selected at different stages of AChR accumulation and examined in the electron microscope after serial sectioning along the entire path of nerve-muscle contact. The results indicate that the nerve-induced formation of AChR aggregates and adjacent plaques of proteoglycan is closely coupled throughout early stages of synapse formation. Developing junctional accumulations of AChR and proteoglycan appeared and grew progressively, throughout a perineural zone that extended along the muscle surface for several micrometers on either side of the nerve process. Unlike junctional AChR accumulations, which disappeared within a day of denervation, both junctional and extrajunctional proteoglycan deposits were stable in size and morphology. Junctional proteoglycan deposits appeared to correspond to discrete ultrastructural plaques of basal lamina, which were initially separated by broad expanses of lamina-free muscle surface. The extent of this basal lamina, and a corresponding thickening of the postsynaptic membrane, also increased during the accumulation of AChR and proteoglycan along the path of nerve contact. Presynaptic differentiation of synaptic vesicle clusters became detectable at the developing neuromuscular junction only after the formation of postsynaptic plaques containing both AChR and proteoglycan. It is concluded that motor nerves induce a gradual formation and growth of AChR aggregates and stable basal lamina proteoglycan deposits on the muscle surface during development of the neuromuscular junction.     

Localization of dystrophin relative to acetylcholine receptor domains in electric tissue and adult and cultured skeletal muscle

Two high-affinity mAbs were prepared against Torpedo dystrophin, an electric organ protein that is closely similar to human dystrophin, the gene product of the Duchenne muscular dystrophy locus. The antibodies were used to localize dystrophin relative to acetylcholine receptors (AChR) in electric organ and in skeletal muscle, and to show identity between Torpedo dystrophin and the previously described 270/300-kD Torpedo postsynaptic protein. Dystrophin was found in both AChR-rich and AChR-poor regions of the innervated face of the electroplaque. Immunogold experiments showed that AChR and dystrophin were closely intermingled in the AChR domains. In contrast, dystrophin appeared to be absent from many or all AChR-rich domains of the rat neuromuscular junction and of AChR clusters in cultured muscle (Xenopus laevis). It was present, however, in the immediately surrounding membrane (deep regions of the junctional folds, membrane domains interdigitating with and surrounding AChR domains within clusters). These results suggest that dystrophin may have a role in organization of AChR in electric tissue. Dystrophin is not, however, an obligatory component of AChR domains in muscle and, at the neuromuscular junction, its roles may be more related to organization of the junctional folds.     

Wnt/beta-catenin signaling suppresses Rapsyn expression and inhibits acetylcholine receptor clustering at the neuromuscular junction

The dynamic interaction between positive and negative signals is necessary for remodeling of postsynaptic structures at the neuromuscular junction. Here we report that Wnt3a negatively regulates acetylcholine receptor (AChR) clustering by repressing the expression of Rapsyn, an AChR-associated protein essential for AChR clustering. In cultured myotubes, treatment with Wnt3a or overexpression of beta-catenin, the condition mimicking the activation of the Wnt canonical pathway, inhibited Agrin-induced formation of AChR clusters. Moreover, Wnt3a treatment promoted dispersion of AChR clusters, and this effect was prevented by DKK1, an antagonist of the Wnt canonical pathway. Next, we investigated possible mechanisms underlying Wnt3a regulation of AChR clustering in cultured muscle cells. Interestingly, we found that Wnt3a treatment caused a decrease in the protein level of Rapsyn. In addition, Rapsyn promoter activity in cultured muscle cells was inhibited by the treatment with Wnt3a or beta-catenin overexpression. Forced expression of Rapsyn driven by a promoter that is not responsive to Wnt3a prevented the dispersing effect of Wnt3a on AChR clusters, suggesting that Wnt3a indeed acts to disperse AChR clusters by down-regulating the expression of Rapsyn. The role of Wnt/beta-catenin signaling in dispersing AChR clusters was also investigated in vivo by electroporation of Wnt3a or beta-catenin into mouse limb muscles, where ectopic Wnt3a or beta-catenin caused disassembly of postsynaptic apparatus. Together, these results suggest that Wnt/beta-catenin signaling plays a negative role for postsynaptic differentiation at the neuromuscular junction, probably by regulating the expression of synaptic proteins, such as Rapsyn.     

Novel mouse model reveals distinct activity-dependent and -independent contributions to synapse development

The balanced action of both pre- and postsynaptic organizers regulates the formation of neuromuscular junctions (NMJ). The precise mechanisms that control the regional specialization of acetylcholine receptor (AChR) aggregation, guide ingrowing axons and contribute to correct synaptic patterning are unknown. Synaptic activity is of central importance and to understand synaptogenesis, it is necessary to distinguish between activity-dependent and activity-independent processes. By engineering a mutated fetal AChR subunit, we used homologous recombination to develop a mouse line that expresses AChR with massively reduced open probability during embryonic development. Through histological and immunochemical methods as well as electrophysiological techniques, we observed that endplate anatomy and distribution are severely aberrant and innervation patterns are completely disrupted. Nonetheless, in the absence of activity AChRs form postsynaptic specializations attracting motor axons and permitting generation of multiple nerve/muscle contacts on individual fibers. This process is not restricted to a specialized central zone of the diaphragm and proceeds throughout embryonic development. Phenotypes can be attributed to separate activity-dependent and -independent pathways. The correct patterning of synaptic connections, prevention of multiple contacts and control of nerve growth require AChR-mediated activity. In contrast, myotube survival and acetylcholine-mediated dispersal of AChRs are maintained even in the absence of AChR-mediated activity. Because mouse models in which acetylcholine is entirely absent do not display similar effects, we conclude that acetylcholine binding to the AChR initiates activity-dependent and activity-independent pathways whereby the AChR modulates formation of the NMJ.     

PKC and PKA Regulate AChR Dynamics at the Neuromuscular Junction of Living Mice

The steady state of the acetylcholine receptor (AChR) density at the neuromuscular junction (NMJ) is critical for efficient and reliable synaptic transmission. However, little is known about signaling molecules involved in regulating the equilibrium between the removal and insertion of AChRs that establishes a stable postsynaptic receptor density over time. In this work, we tested the effect of activities of two serine/threonine kinases, PKC and PKA, on the removal rate of AChRs from and the re-insertion rate of internalized recycled AChRs into synaptic sites of innervated and denervated NMJs of living mice. Using an in vivo time-lapse imaging approach and various pharmacological agents, we showed that PKC and PKA activities have antagonistic effects on the removal and recycling of AChRs. Inhibition of PKC activity or activation of PKA largely prevents the removal of pre-existing AChRs and promotes the recycling of internalized AChRs into the postsynaptic membrane. In contrast, stimulation of PKC or inactivation of PKA significantly accelerates the removal of postsynaptic AChRs and depresses AChR recycling. These results indicate that a balance between PKA and PKC activities may be critical for the maintenance of the postsynaptic receptor density.     

Agrin induces phosphorylation of the nicotinic acetylcholine receptor.

Agrin causes acetylcholine receptors (AChRs) on chick myotubes in culture to aggregate, forming specializations that resemble the postsynaptic apparatus at the vertebrate skeletal neuromuscular junction. Here we report that treating chick myotubes with agrin caused an increase in phosphorylation of the AChR beta, gamma, and delta subunits. H-7, a potent inhibitor of several protein serine kinases, blocked agrin-induced phosphorylation of the gamma and delta subunits, but did not prevent either agrin-induced AChR aggregation or phosphorylation of the beta subunit. Experiments with anti-phosphotyrosine antibodies demonstrated that agrin caused an increase in tyrosine phosphorylation of the beta subunit that began within 30 min of adding agrin to the myotube cultures, reached a plateau by 3 hr, and was blocked by treatments known to block agrin-induced AChR aggregation. Anti-phosphotyrosine antibodies labeled agrin-induced specializations as they do the postsynaptic apparatus. These results suggest that agrin-induced tyrosine phosphorylation of the beta subunit may play a role in regulating AChR distribution.     

Formation of complex AChR aggregates in vitro requires α‐dystrobrevin

Efficient function at the neuromuscular junction requires high‐density aggregates of acetylcholine receptors (AChRs) to be precisely aligned with the motor nerve terminal. A collaborative effort between the motor neuron and muscle intrinsic factors drives the formation and maintenance of these AChR aggregates. α‐Dystrobrevin (αDB), a cytoplasmic protein found at the postsynaptic membrane, has been implicated in the regulation of AChR aggregate density and patterning. To investigate the contribution of αDB to the muscle intrinsic program regulating AChR aggregate development, we analyzed the formation of complex, pretzel‐like AChR aggregates on primary muscle cell cultures derived from αDB knockout (αDB‐KO) mice in the absence of nerve or agrin. In myotubes lacking αDB, complex AChR aggregates failed to form, whereas aggregates formed readily in wildtype myotubes. Five major isoforms of αDB are expressed in skeletal muscle: αDB1, αDB1(?), αDB2, αDB2(?), and αDB3. Expression of αDB1 or αDB1(?) in αDB‐KO myotubes restored formation of complex AChR aggregates similar to those in wildtype myotubes. In contrast, individual expression of αDB2, αDB2(?), αDB3, or an αDB1 phosphorylation mutant resulted in the formation of few, if any, complex AChR aggregates. Collectively, these data suggest that αDB is a significant component of the muscle intrinsic program that mediates the formation of complex AChR aggregates and that αDB's tyrosine phosphorylation sites are of particular functional importance to this program. Although the muscle intrinsic program appears to influence synaptogenesis, the formation of complex mature AChR aggregates in αDB‐KO mice (with the motor neuron present) suggests the motor neuron, not the muscle intrinsic program, is the major stimulus driving the maturation of AChRs from plaque to pretzel in vivo. © 2009 Wiley Periodicals, Inc. Develop Neurobiol 2009     

Shp2 is dispensable in the formation and maintenance of the neuromuscular junction

SHP2, a protein tyrosine phosphatase with two SH2 domains, has been implicated in regulating acetylcholine receptor (AChR) gene expression and cluster formation in cultured muscle cells. To understand the role of SHP2 in neuromuscular junction (NMJ) formation in vivo, we generated mus cle-specific deficient mice by using a loxP/Cre strategy since Shp2 null mutation causes embryonic lethality. Shp2(floxed/floxed) mice were crossed with mice expressing the Cre gene under the control of the human skeletal alpha-actin (HSA) promoter. Expression of SHP2 was reduced or diminished specifically in skeletal muscles of the conditional knockout (CKO) mice. The mutant mice were viable and fertile, without apparent muscle defects. The mRNA of the AChR alpha subunit and AChR clusters in CKO mice were localized in a narrow central region surrounding the phrenic nerve primary branches, without apparent change in intensity. AChR clusters colocalized with markers of synaptic vesicles and Schwann cells, suggesting proper differentiation of presynaptic terminals and Schwann cells. In comparison with age-matched littermates, no apparent difference was observed in the size and length of AChR clusters in CKO mice. Both the frequency and amplitude of mEPPs in CKO mice were similar to those in controls, suggesting normal neurotransmission when SHP2 was deficient. These results suggest that Shp2 is not required for NMJ formation and/or maintenance.