Agrin-induced Acetylcholine Receptor Research Articles

1α, 25(OH)2D3 regulates agrin-induced acetylcholine receptor clustering through upregulation of rapsyn expression in C2C12 myotubes

The active form of vitamin D (1α, 25-dihydroxyvitamin D3 [1α, 25(OH)2D3], referred to as 1,25D) has been suggested to play a pivotal role in skeletal muscle function and metabolism. However, the mechanisms through which 1,25D functions in this tissue remain to be elucidated. Recent studies have shown that vitamin D signaling regulates neuromuscular maintenance and improves locomotion in mice. In the present study, we examined the effects of 1,25D on neuromuscular synaptogenesis by measuring clustering of acetylcholine receptors (AChRs) in C2C12 myotubes. 1,25D treatment enhanced the agrin-induced AChR clustering in myotubes compared to treatment with agrin alone. Furthermore, siRNA-mediated knockdown of the vitamin D receptor (VDR) decreased the agrin-induced AChR clustering. 1,25D increased the expression of rapsyn, which is necessary for AChR clustering, while demonstrating no effect on other neuromuscular junction-related genes. In addition, rapsyn expression was dependent on 1,25D-VDR signaling. These results suggest that 1,25D-VDR signaling may regulate rapsin expression, resulting in the up-regulation of agrin-induced AChR clustering.

Sodium nitrate decreases agrin-induced acetylcholine receptor clustering.

BackgroundHumans are exposed to nitrate predominantly through diet with peak plasma concentrations within an hour after ingestion, but additional exposure is obtained from the environment, and minimally through de novo synthesis. Higher nitrate consumption has been associated with methemoglobinemia, spontaneous abortions, atherosclerosis, myocardial ischemia, septic and distressed lung, inflammatory bowel disease, amyotrophic lateral sclerosis, and neural tube defects. However, skeletal muscle development has not been examined.MethodsC2C12 skeletal muscle cell cultures were maintained, myoblasts were fused into myotubes, and then cultures were exposed to motor neuron derived agrin to enhance acetylcholine receptor (AChR) clustering. Untreated cultures were compared with cultures exposed to sodium nitrate at concentrations ranging from 10 ng/mL–100 μg/mL.ResultsThe results reported here demonstrate that 1 μg/mL sodium nitrate was sufficient to decrease the frequency of agrin-induced AChR clustering without affecting myotube formation. In addition, concentrations of sodium nitrate of 1 μg/mL or 100 μg/mL decreased gene expression of the myogenic transcription factor myogenin and AChR in correlation with the agrin-induced AChR clustering data.ConclusionsThese results reveal that sodium nitrate decreases the frequency of agrin-induced AChR clustering by a mechanism that includes myogenin and AChR gene expression. As a consequence sodium nitrate may pose a risk for skeletal muscle development and subsequent neuromuscular synapse formation in humans.

Chapter 24 - Neuromuscular junction disorders

Diseases of the neuromuscular junction comprise a wide range of disorders. Antibodies, genetic mutations, specific drugs or toxins interfere with the number or function of one of the essential proteins that control signaling between the presynaptic nerve ending and the postsynaptic muscle membrane. Acquired autoimmune disorders of the neuromuscular junction are the most common and are described here. In myasthenia gravis, antibodies to acetylcholine receptors or to proteins involved in receptor clustering, particularly muscle-specific kinase, cause direct loss of acetylcholine receptors or interfere with the agrin-induced acetylcholine receptor clustering necessary for efficient neurotransmission. In the Lambert-Eaton myasthenic syndrome (LEMS), loss of the presynaptic voltage-gated calcium channels results in reduced release of the acetylcholine transmitter. The conditions are generally recognizable clinically and the diagnosis confirmed by serologic testing and electromyography. Screening for thymomas in myasthenia or small cell cancer in LEMS is important. Fortunately, a wide range of symptomatic treatments, immunosuppressive drugs, or other immunomodulating therapies is available. Future research is directed to understanding the pathogenesis, discovering new antigens, and trying to develop disease-specific treatments.

Myostatin Inhibitors Affect MyoD and Myogenin Expression

Myostatin (growth differentiation factor 8, GDF8) inhibits myogenesis and is a potential therapeutic target for muscular dystrophy and other myopathies. Animals with mutations in myostatin genes have increased muscle mass resulting from hypertrophy and hyperplasia. In this project three common myostatin inhibitors, GDF8 antibody, ActRIIB-Fc, and Alk4/5 inhibitor, were examined for their effects on agrin-induced acetylcholine receptor (AChR) clustering and expression of myogenic regulatory factors MyoD and myogenin. C2C12 cell cultures were maintained, myoblasts were differentiated into myotubes, and then cultures were exposed to motor neuron derived agrin to enhance AChR clustering. Untreated cultures were compared with cultures exposed to myostatin inhibitors at various concentrations. GDF8 antibody and Alk4/5 inhibitor decreased the frequency of agrin-induced AChR clustering with a dose response, while ActRIIB-Fc had no effect. Moreover, GDF8 antibody and ActRIIB-Fc increased MyoD expression and decreased myogenin expression, while Alk4/5 inhibitor decreased MyoD expression and increased myogenin expression. The differential effect of myostatin inhibitors on these events may be dependent on the different mechanisms of inhibition. Additional research could reveal if a myostatin inhibitor is an appropriate treatment for myopathies like muscular dystrophy. Jacqueline Sohn was supported by the Midwestern University Summer Fellowship Program. Wade A. Grow was supported by Midwestern University intramural funds.

Plastic Additives Decrease Agrin-Induced Acetylcholine Receptor Clusters and Myotube Formation in C2C12 Skeletal Muscle Cell Culture

Common additives in plastics such as bisphenol A (BPA) or phthalates like di-(2-ethylhexyl) phthalate (DEHP) are environmental estrogens that have been shown to be endocrine disruptors in some experimental animal models. This project used the C2C12 cell culture model to examine how exposure to BPA or DEHP affects two aspects of skeletal muscle development, the fusion of myoblasts into myotubes and agrin-induced clustering of acetylcholine receptors (AChRs). During myotube formation AChRs cluster spontaneously. Treatment with motor neuron derived agrin increases the frequency of AChR clusters through an agrin signaling pathway that also clusters other postsynaptic components of the neuromuscular synapse. For this project C2C12 cells were exposed to BPA or DEHP while myoblasts fused into myotubes. After exposure to 10 μM BPA or 100 μM DEHP the frequency of agrin-induced AChR clusters decreased. In addition, myotube formation decreased as a higher percentage of nuclei remained in myoblasts. Furthermore, BPA or DEHP reduced the amount of the myogenic regulatory factor myogenin. This suggests that BPA and DEHP decrease AChR clustering by reducing myogenin. Moreover, plastic additives like BPA and DEHP may pose a risk for skeletal muscle development in humans.

APP interacts with LRP4 and agrin to coordinate the development of the neuromuscular junction in mice

ApoE, ApoE receptors and APP cooperate in the pathogenesis of Alzheimer's disease. Intriguingly, the ApoE receptor LRP4 and APP are also required for normal formation and function of the neuromuscular junction (NMJ). In this study, we show that APP interacts with LRP4, an obligate co-receptor for muscle-specific tyrosine kinase (MuSK). Agrin, a ligand for LRP4, also binds to APP and co-operatively enhances the interaction of APP with LRP4. In cultured myotubes, APP synergistically increases agrin-induced acetylcholine receptor (AChR) clustering. Deletion of the transmembrane domain of LRP4 (LRP4 ECD) results in growth retardation of the NMJ, and these defects are markedly enhanced in APP(-/-);LRP4(ECD/ECD) mice. Double mutant NMJs are significantly reduced in size and number, resulting in perinatal lethality. Our findings reveal novel roles for APP in regulating neuromuscular synapse formation through hetero-oligomeric interaction with LRP4 and agrin and thereby provide new insights into the molecular mechanisms that govern NMJ formation and maintenance. DOI:http://dx.doi.org/10.7554/eLife.00220.001.

Arecoline inhibits and destabilizes agrin-induced acetylcholine receptor cluster formation in C2C12 myotubes

Areca nut (Areca catechu) is chewed as a medical and psychoactive food by roughly 10% of the world population. Areca nut chewing may lead to low birth weight, premature delivery and impaired muscle development. Our previous study showed that arecoline, a major alkaloid in the areca nut, inhibited the myogenic differentiation of C2C12 myoblastic cells. The clustering of acetylcholine receptors (AChRs) in the postsynaptic membrane at the neuromuscular junction (NMJ) by agrin, a signaling protein released by motor neurons, is critical for the development of functional muscles. Here, we further investigate whether arecoline affects the AChR clustering using cultured C2C12 myotubes. Rhodamine-conjugated α-bungarotoxin was used to detect the presence of AChR clusters. Our results showed that arecoline inhibited the formation of agrin-induced AChR clusters and destabilized agrin-induced or spontaneous AChR cluster formation. In addition, arecoline inhibited the expression of myogenin in C2C12 myotubes. These results shed light on the important role of arecoline on the detrimental effect of areca nut to muscle development.

Antibody to MyoD or Myogenin Decreases Acetylcholine Receptor Clustering in C2C12 Myotube Culture

Skeletal muscle development is influenced by myogenic regulatory factors, including the expression of MyoD and myogenin. Our objective was to use the C2C12 cell culture model to test the hypothesis that both MyoD and myogenin were required for agrin-induced acetylcholine receptor (AChR) clustering and the fusion of myoblasts into myotubes. We induced fusion of myoblasts into myotubes by switching from growth medium (GM) to differentiation medium (DM). During myotube formation AChRs cluster spontaneously, but treatment with motor neuron derived agrin increases clustering of AChRs and other postsynaptic components of the neuromuscular synapse. We examined the normal expression pattern of MyoD and myogenin in C2C12 cell culture using immunofluorescence. MyoD was highly expressed while myoblasts were in GM, but expression declined within 72 hours after cell cultures were switched to DM. Myogenin expression was low in GM, but increased when cell cultures were switched to DM. Next we used antibodies to decrease MyoD and/or myogenin function. Fluorescence microscopy images were captured and then analyzed to assess agrin-induced AChR clustering with or without antibody treatment. Finally we calculated the proportion of nuclei in myotubes and myoblasts by creating digital overlays of phase contrast and DAPI stained microscopy images. This allowed the comparison of myotube formation with or without antibody treatment. We report that antibody to either MyoD or myogenin decreases the frequency of agrin-induced AChR clustering without affecting myotube formation. We conclude that agrin-induced AChR clustering requires both MyoD and myogenin.

Agrin regulates CLASP2-mediated capture of microtubules at the neuromuscular junction synaptic membrane

Agrin is the major factor mediating the neuronal regulation of postsynaptic structures at the vertebrate neuromuscular junction, but the details of how it orchestrates this unique three-dimensional structure remain unknown. Here, we show that agrin induces the formation of the dense network of microtubules in the subsynaptic cytoplasm and that this, in turn, regulates acetylcholine receptor insertion into the postsynaptic membrane. Agrin acted in part by locally activating phosphatidylinositol 3-kinase and inactivating GSK3β, which led to the local capturing of dynamic microtubules at agrin-induced acetylcholine receptor (AChR) clusters, mediated to a large extent by the microtubule plus-end tracking proteins CLASP2 and CLIP-170. Indeed, in the absence of CLASP2, microtubule plus ends at the subsynaptic muscle membrane, the density of synaptic AChRs, the size of AChR clusters, and the numbers of subsynaptic muscle nuclei with their selective gene expression programs were all reduced. Thus, the cascade linking agrin to CLASP2-mediated microtubule capturing at the synaptic membrane is essential for the maintenance of a normal neuromuscular phenotype.

Structural basis of agrin–LRP4–MuSK signaling

Synapses are the fundamental units of neural circuits that enable complex behaviors. The neuromuscular junction (NMJ), a synapse formed between a motoneuron and a muscle fiber, has contributed greatly to understanding of the general principles of synaptogenesis as well as of neuromuscular disorders. NMJ formation requires neural agrin, a motoneuron-derived protein, which interacts with LRP4 (low-density lipoprotein receptor-related protein 4) to activate the receptor tyrosine kinase MuSK (muscle-specific kinase). However, little is known of how signals are transduced from agrin to MuSK. Here, we present the first crystal structure of an agrin-LRP4 complex, consisting of two agrin-LRP4 heterodimers. Formation of the initial binary complex requires the z8 loop that is specifically present in neuronal, but not muscle, agrin and that promotes the synergistic formation of the tetramer through two additional interfaces. We show that the tetrameric complex is essential for neuronal agrin-induced acetylcholine receptor (AChR) clustering. Collectively, these results provide new insight into the agrin-LRP4-MuSK signaling cascade and NMJ formation and represent a novel mechanism for activation of receptor tyrosine kinases.

Divalent and monovalent autoantibodies cause dysfunction of MuSK by distinct mechanisms in a rabbit model of myasthenia gravis

Muscle-specific kinase (MuSK), a receptor tyrosine kinase, is required for the formation and maintenance of neuromuscular junctions (NMJs). Although autoantibodies against MuSK have been demonstrated to cause myasthenia gravis (MG), the underlying pathogenic mechanism remains unclear because a major subclass of these antibodies is functionally monovalent. We investigated the pathogenic role of MuSK antibodies in the onset of MG in vivo and in vitro. Ultrastructural visualization of NMJs in paretic rabbits with MuSK antibodies indicated that postsynaptic membranes were preserved, despite a significant loss of complexity in the convoluted synaptic folds. In addition, an in vitro assay indicated that both divalent and monovalent antibodies from paretic rabbits could interfere with agrin-induced acetylcholine receptor (AChR) clustering in cultured myotubes. Furthermore, in the absence of agrin, divalent antibodies induced MuSK phosphorylation and accelerated downregulation of Dok-7, an essential intracellular MuSK binding protein, while monovalent antibodies inhibited agrin-induced phosphorylation of MuSK, thus demonstrating distinct molecular mechanisms underlying the MuSK dysfunction induced by these two types of antibodies. Taken together, these findings suggest that complement activation is not necessary for the MG onset and that both divalent and monovalent antibodies may cause MG in vivo by inducing MuSK dysfunction.

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

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

Ethanol decreases agrin-induced acetylcholine receptor clustering in C2C12 myotube culture

We investigated the effect of ethanol on skeletal muscle development using C2C12 cell culture. The ethanol concentrations of 10 mM, 25 mM, and 100 mM, were tested because plasma samples of alcohol-dependent individuals fall within this range. We assessed two specific events in skeletal muscle development, the fusion of myoblasts to form myotubes and the acetylcholine receptor (AChR) clustering associated with neuromuscular synapse formation. We report that ethanol does not effect myotube formation or the viability of myoblasts or myotubes in C2C12 cell culture. However, ethanol does effect AChR clustering on C2C12 myotubes. As motor neurons approach skeletal muscle during development, agrin is released by motor neurons and induces AChR clustering on muscle fibers. In our experiments, agrin was applied to cell cultures during the period when myoblasts fuse to form myotubes. In cell cultures exposed to ethanol during myotube formation, agrin-induced AChR clustering was decreased compared to untreated cultures. In cell cultures exposed to ethanol during myoblast proliferation, with ethanol removed during myotube formation, agrin-induced AChR clustering was unaffected. We conclude that exposure to a physiologically relevant concentration of ethanol during the specific period of myotube formation decreases agrin-induced AChR clustering.

α-Dystrobrevin isoforms differ in their colocalization with and stabilization of agrin-induced acetylcholine receptor clusters

In skeletal muscle, alpha-dystrobrevin (alphaDB) is expressed throughout the sarcolemma with high concentrations at the neuromuscular junction. Mice lacking alphaDB display a mild muscular dystrophy and perturbations at the neuromuscular junction that include disruptions to acetylcholine receptor (AChR) cluster stability and patterning. In adult skeletal muscle, three alternatively spliced isoforms (alphaDB1, alphaDB2, alphaDB3) are expressed, while two other splice variants (alphaDB1(-) and alphaDB2(-)) are expressed only during early development. alphaDB is clearly important in AChR stabilization; however, the degree to which individual alphaDB isoforms and their specific functional domains contribute to AChR cluster stability is not fully understood. To investigate this, we established a primary muscle cell culture system from alphaDB knockout mice and stably expressed individual alphaDB isoforms using retroviral infection. A comparison between wild-type and alphaDB knockout muscle cells showed that in the absence of alphaDB, fewer AChR clusters formed in response to agrin treatment, and these AChR clusters were very unstable. Retroviral expression studies revealed that the largest isoforms (alphaDB1, alphaDB1(-), alphaDB2, alphaDB2(-)) colocalized with agrin-induced AChR clusters and rescued the AChR cluster formation defects back to wild-type levels, while only the first three isoforms fully rescued AChR cluster stability back to wild-type levels. alphaDB2(-) conferred an intermediate level of stability to the AChR clusters. In contrast, alphaDB3 showed no specific colocalization with AChR clusters and little effect on AChR cluster formation or stabilization. Twice as much syntrophin was found associated with alphaDB2 compared with alphaDB2(-) in myotubes suggesting that increased recruitment of syntrophin by alphaDB may enhance the stability of AChR clusters. Taken together, these data demonstrate that different alphaDB isoforms have different functional capabilities in the formation and maintenance of AChR clusters in muscle cells, and that these differences are likely due to the presence of different functional domains in each isoform.

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.

An Extracellular Pathway for Dystroglycan Function in Acetylcholine Receptor Aggregation and Laminin Deposition in Skeletal Myotubes

The dystroglycan (DG) complex is involved in agrin-induced acetylcholine receptor clustering downstream of muscle-specific kinase where it regulates the stability of acetylcholine receptor aggregates as well as assembly of the synaptic basement membrane. We have previously proposed that this entails coordinate extracellular and intracellular interactions of its two subunits, alpha- and beta-DG. To assess the contribution of the extracellular and intracellular portions of DG, we have used adenoviruses to express full-length and deletion mutants of beta-DG in myotubes derived from wild-type embryonic stem cells or from cells null for DG. We show that alpha-DG is properly glycosylated and targeted to the myotube surface in the absence of beta-DG. Extracellular interactions of DG modulate the size and the microcluster density of agrin-induced acetylcholine receptor aggregates and are responsible for targeting laminin to these clusters. Thus, the association of alpha- and beta-DG in skeletal muscle may coordinate independent roles in signaling. We discuss how DG may regulate synapses through extracellular signaling functions of its alpha subunit.

Lipid rafts are involved in C95 (4,8) agrin fragment-induced acetylcholine receptor clustering

During development of the neuromuscular junction, high densities of acetylcholine receptors accumulate beneath the overlying nerve terminal. A defining feature of mature synapses is the sharp demarcation of acetylcholine receptor density, which is ∼1000-fold higher in the postsynaptic as compared with the contiguous extrasynaptic muscle membrane. These high densities of receptors accumulate by at least four mechanisms, re-distribution of existing surface receptors, local synthesis of new receptors, decreased turnover of synaptic receptors, and limitation of diffusion of sub-neural, aggregated receptors. The limitation of receptor diffusion within the membrane is likely in part due to the anchoring of acetylcholine receptor complexes to components of the cytoskeleton. Here we have tested the idea that lipid rafts—mobile, cholesterol enriched microdomains within the lipid bilayer—are another mechanism by which acetylcholine receptors are clustered in the postsynaptic apparatus. Using mouse C2C12 cells, a muscle cell line, we show that a carboxy terminal 95 amino acid fragment [C95 (4,8)] of the extracellular matrix molecule agrin that is essential for nerve-induced postsynaptic differentiation, promotes the redistribution of acetylcholine receptors into lipid rafts. Disruption of lipid rafts before agrin treatment largely inhibits de novo agrin-induced acetylcholine receptor clustering. Moreover, mature acetylcholine receptor clusters are destabilized if lipid rafts are disrupted. These results show that lipid rafts are important in both the initial clustering and later stabilization of agrin-induced acetylcholine receptor clusters and also suggest that lipid rafts may contribute to the postsynaptic localization of acetylcholine receptors in vivo.

ATP potentiates the formation of AChR aggregate in the co-culture of NG108-15 cells with C2C12 myotubes

The role of adenosine 5′-triphosphate (ATP) and P2Y1 nucleotide receptor in potentiating agrin-induced acetylcholine receptor (AChR) aggregation is being demonstrated in a co-culture system of NG108-15 cell, a mouse neuroblastoma X rat glioma hybrid cell line that resembles spinal motor neuron, with C2C12 myotube. In the co-cultures, antagonized P2Y1 receptors showed a reduction in NG108-15 cell-induced AChR aggregation. Parallel to this observation, cultured NG108-15 cell secreted ATP into the conditioned medium in a time-dependent manner. Enhancement of ATP release from the cultured NG108-15 cells by overexpression of active mutants of small GTPases increased the aggregation of AChRs in co-culturing with C2C12 myotubes. In addition, ecto-nucleotidase was revealed in the co-culture, which rapidly degraded the applied ATP. These results support the notion that ATP has a role in directing the formation of post-synaptic apparatus in vertebrate neuromuscular junctions.

Neuregulin potentiates agrin-induced acetylcholine receptor clustering in myotubes

Agrin and neuregulin (HRG-beta1) play complementary roles in synapse formation. While HRG-beta1 induces transcriptional up-regulation of postsynaptic proteins, here we present evidence that it can potentiate agrin-induced acetylcholine receptor (AChR) clustering in C2 myotubes. Agrin induced maximal AChR clustering in 4 h. HRG-beta1 treatment for 4 h produced no increase over basal AChR cluster numbers. When myotubes were treated for 4 h with 100 pM agrin, HRG-beta1 augmented AChR cluster numbers by 2-fold compared to myotubes treated with 100 pM agrin alone. Thus, HRG-beta1 can potentiate agrin-induced AChR clustering.

ATP Potentiates Agrin-induced AChR Aggregation in Cultured Myotubes: ACTIVATION OF RHOA IN P2Y1 NUCLEOTIDE RECEPTOR SIGNALING AT VERTEBRATE NEUROMUSCULAR JUNCTIONS

At vertebrate neuromuscular junctions, ATP is known to stabilize acetylcholine in the synaptic vesicles and to be co-released with it. We have shown previously that a nucleotide receptor, P2Y(1) receptor, is localized at the nmjs, and we propose that this mediates a trophic role for synaptic ATP there. In cultured myotubes, the activation of P2Y(1) receptors modulated agrin-induced acetylcholine receptor (AChR) aggregation in a potentiation manner. This potentiation effect in agrin-induced AChR aggregation was reduced by antagonizing the P2Y(1) receptors. The guanosine triphosphatase RhoA was shown to be responsible for this P2Y(1)-potentiated effect. The localization of RhoA in rat and chicken skeletal muscles was restricted at the neuromuscular junctions. Application of P2Y(1) agonists in cultured myotubes induced RhoA activation, which showed an additive effect with agrin-induced RhoA activation. Over-expression of dominant-negative mutant of RhoA in cultured myotubes diminished the agrin-induced AChR aggregation, as well as the potentiation effect of P2Y(1)-specific agonist. Application of UTP in the cultures also triggered similar responses as did 2-methylthioadenosine 5'-diphosphate, suggesting the involvement of other subtypes of P2Y receptors. These results demonstrate that RhoA could serve as a downstream mediator of signaling mediated by P2Y(1) receptor and agrin, which therefore synergizes the effects of the two neuron-derived trophic factors in modulating the formation and/or maintenance of post-synaptic apparatus at the neuromuscular junctions.