Postsynaptic Abnormalities at the Neuromuscular Junctions of Utrophin-deficient Mice

Utrophin is a dystrophin-related cytoskeletal protein expressed in many tissues. It is thought to link F-actin in the internal cytoskeleton to a transmembrane protein complex similar to the dystrophin protein complex (DPC). At the adult neuromuscular junction (NMJ), utrophin is precisely colocalized with acetylcholine receptors (AChRs) and recent studies have suggested a role for utrophin in AChR cluster formation or maintenance during NMJ differentiation. We have disrupted utrophin expression by gene targeting in the mouse. Such mice have no utrophin detectable by Western blotting or immunocytochemistry. Utrophindeficient mice are healthy and show no signs of weakness. However, their NMJs have reduced numbers of AChRs (α-bungarotoxin [α-BgTx] binding reduced to ~60% normal) and decreased postsynaptic folding, though only minimal electrophysiological changes. Utrophin is thus not essential for AChR clustering at the NMJ but may act as a component of the postsynaptic cytoskeleton, contributing to the development or maintenance of the postsynaptic folds. Defects of utrophin could underlie some forms of congenital myasthenic syndrome in which a reduction of postsynaptic folds is observed.     

Transplantation of Quail Collagen-tailed Acetylcholinesterase Molecules Onto the Frog Neuromuscular Synapse

The highly organized pattern of acetylcholinesterase (AChE) molecules attached to the basal lamina of the neuromuscular junction (NMJ) suggests the existence of specific binding sites for their precise localization. To test this hypothesis we immunoaffinity purified quail globular and collagen-tailed AChE forms and determined their ability to attach to frog NMJs which had been pretreated with high-salt detergent buffers. The NMJs were visualized by labeling acetylcholine receptors (AChRs) with TRITC-α-bungarotoxin and AChE by indirect immunofluorescence; there was excellent correspondence (>97%) between the distribution of frog AChRs and AChE. Binding of the exogenous quail AChE was determined using a speciesspecific monoclonal antibody. When frog neuromuscular junctions were incubated with the globular G₄/G₂ quail AChE forms, there was no detectable binding above background levels, whereas when similar preparations were incubated with the collagen-tailed A₁₂ AChE form >80% of the frog synaptic sites were also immunolabeled for quail AChE attached. Binding of the A₁₂ quail AChE was blocked by heparin, yet could not be removed with high salt buffer containing detergent once attached. Similar results were obtained using empty myofiber basal lamina sheaths produced by mechanical or freeze-thaw damage. These experiments show that specific binding sites exist for collagen-tailed AChE molecules on the synaptic basal lamina of the vertebrate NMJ and suggest that these binding sites comprise a “molecular parking lot” in which the AChE molecules can be released, retained, and turned over.     

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

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

AnkyrinG Is Required for Clustering of Voltage-gated Na Channels at Axon Initial Segments and for Normal Action Potential Firing

Voltage-gated sodium channels (NaCh) are colocalized with isoforms of the membrane-skeletal protein ankyrin_G at axon initial segments, nodes of Ranvier, and postsynaptic folds of the mammalian neuromuscular junction. The role of ankyrin_G in directing NaCh localization to axon initial segments was evaluated by region-specific knockout of ankyrin_G in the mouse cerebellum. Mutant mice exhibited a progressive ataxia beginning around postnatal day P16 and subsequent loss of Purkinje neurons. In mutant mouse cerebella, NaCh were absent from axon initial segments of granule cell neurons, and Purkinje cells showed deficiencies in their ability to initiate action potentials and support rapid, repetitive firing. Neurofascin, a member of the L1CAM family of ankyrin-binding cell adhesion molecules, also exhibited impaired localization to initial segments of Purkinje cell neurons. These results demonstrate that ankyrin_G is essential for clustering NaCh and neurofascin at axon initial segments and is required for physiological levels of sodium channel activity.     

Disruption of Basal Lamina Components in Neuromotor Synapses of Children with Spastic Quadriplegic Cerebral Palsy

Cerebral palsy (CP) is a static encephalopathy occurring when a lesion to the developing brain results in disordered movement and posture. Patients present with sometimes overlapping spastic, athetoid/dyskinetic, and ataxic symptoms. Spastic CP, which is characterized by stiff muscles, weakness, and poor motor control, accounts for ∼80% of cases. The detailed mechanisms leading to disordered movement in spastic CP are not completely understood, but clinical experience and recent studies suggest involvement of peripheral motor synapses. For example, it is recognized that CP patients have altered sensitivities to drugs that target neuromuscular junctions (NMJs), and protein localization studies suggest that NMJ microanatomy is disrupted in CP. Since CP originates during maturation, we hypothesized that NMJ disruption in spastic CP is associated with retention of an immature neuromotor phenotype later in life. Scoliosis patients with spastic CP or idiopathic disease were enrolled in a prospective, partially-blinded study to evaluate NMJ organization and neuromotor maturation. The localization of synaptic acetylcholine esterase (AChE) relative to postsynaptic acetylcholine receptor (AChR), synaptic laminin β2, and presynaptic vesicle protein 2 (SV2) appeared mismatched in the CP samples; whereas, no significant disruption was found between AChR and SV2. These data suggest that pre- and postsynaptic NMJ components in CP children were appropriately distributed even though AChE and laminin β2 within the synaptic basal lamina appeared disrupted. Follow up electron microscopy indicated that NMJs from CP patients appeared generally mature and similar to controls with some differences present, including deeper postsynaptic folds and reduced presynaptic mitochondria. Analysis of maturational markers, including myosin, syntrophin, myogenin, and AChR subunit expression, and telomere lengths, all indicated similar levels of motor maturation in the two groups. Thus, NMJ disruption in CP was found to principally involve components of the synaptic basal lamina and subtle ultra-structural modifications but appeared unrelated to neuromotor maturational status.     

Neto-Mediated Intracellular Interactions Shape Postsynaptic Composition at the Drosophila Neuromuscular Junction

The molecular mechanisms controlling the subunit composition of glutamate receptors are crucial for the formation of neural circuits and for the long-term plasticity underlying learning and memory. Here we use the Drosophila neuromuscular junction (NMJ) to examine how specific receptor subtypes are recruited and stabilized at synaptic locations. In flies, clustering of ionotropic glutamate receptors (iGluRs) requires Neto (Neuropillin and Tolloid-like), a highly conserved auxiliary subunit that is essential for NMJ assembly and development. Drosophila neto encodes two isoforms, Neto-α and Neto-β, with common extracellular parts and distinct cytoplasmic domains. Mutations that specifically eliminate Neto-β or its intracellular domain were generated. When Neto-β is missing or is truncated, the larval NMJs show profound changes in the subtype composition of iGluRs due to reduced synaptic accumulation of the GluRIIA subunit. Furthermore, neto-β mutant NMJs fail to accumulate p21-activated kinase (PAK), a critical postsynaptic component implicated in the synaptic stabilization of GluRIIA. Muscle expression of either Neto-α or Neto-β rescued the synaptic transmission at neto null NMJs, indicating that Neto conserved domains mediate iGluRs clustering. However, only Neto-β restored PAK synaptic accumulation at neto null NMJs. Thus, Neto engages in intracellular interactions that regulate the iGluR subtype composition by preferentially recruiting and/or stabilizing selective receptor subtypes.     

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

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

Acetylcholine Receptor (AChR) Clustering Is Regulated Both by Glycogen Synthase Kinase 3β (GSK3β)-dependent Phosphorylation and the Level of CLIP-associated Protein 2 (CLASP2) Mediating the Capture of Microtubule Plus-ends

The postsynaptic apparatus of the neuromuscular junction (NMJ) traps and anchors acetylcholine receptors (AChRs) at high density at the synapse. We have previously shown that microtubule (MT) capture by CLASP2, a MT plus-end-tracking protein (+TIP), increases the size and receptor density of AChR clusters at the NMJ through the delivery of AChRs and that this is regulated by a pathway involving neuronal agrin and several postsynaptic kinases, including GSK3. Phosphorylation by GSK3 has been shown to cause CLASP2 dissociation from MT ends, and nine potential phosphorylation sites for GSK3 have been mapped on CLASP2. How CLASP2 phosphorylation regulates MT capture at the NMJ and how this controls the size of AChR clusters are not yet understood. To examine this, we used myotubes cultured on agrin patches that induce AChR clustering in a two-dimensional manner. We show that expression of a CLASP2 mutant, in which the nine GSK3 target serines are mutated to alanine (CLASP2–9XS/9XA) and are resistant to GSK3β-dependent phosphorylation, promotes MT capture at clusters and increases AChR cluster size, compared with myotubes that express similar levels of wild type CLASP2 or that are noninfected. Conversely, myotubes expressing a phosphomimetic form of CLASP2 (CLASP2–8XS/D) show enrichment of immobile mutant CLASP2 in clusters, but MT capture and AChR cluster size are reduced. Taken together, our data suggest that both GSK3β-dependent phosphorylation and the level of CLASP2 play a role in the maintenance of AChR cluster size through the regulated capture and release of MT plus-ends.     

Action of nicotine and analogs on acetylcholine receptors having mutations of transmitter-binding site residue αG153

A primary target for nicotine is the acetylcholine receptor channel (AChR). Some of the ability of nicotine to activate differentially AChR subtypes has been traced to a transmitter-binding site amino acid that is glycine in lower affinity and lysine in higher affinity AChRs. We studied the effects of mutations of this residue (αG153) in neuromuscular AChRs activated by nicotine and eight other agonists including nornicotine and anabasine. All of the mutations increased the unliganded gating equilibrium constant. The affinity of the resting receptor (K_d) and the net binding energy from the agonist for gating (ΔG_B) were estimated by cross-concentration fitting of single-channel currents. In all but one of the agonist/mutant combinations there was a moderate decrease in K_d and essentially no change in ΔG_B. The exceptional case was nicotine plus lysine, which showed a large, >8,000-fold decrease in K_d but no change in ΔG_B. The extraordinary specificity of this combination leads us to speculate that AChRs with a lysine at position αG153 may be exposed to a nicotine-like compound in vivo.     

Spectrin Tetramer Formation Is Not Required for Viable Development in Drosophila

The dominant paradigm for spectrin function is that (αβ)₂-spectrin tetramers or higher order oligomers form membrane-associated two-dimensional networks in association with F-actin to reinforce the plasma membrane. Tetramerization is an essential event in such structures. We characterize the tetramerization interaction between α-spectrin and β-spectrins in Drosophila. Wild-type α-spectrin binds to both β- and β_H-chains with high affinity, resembling other non-erythroid spectrins. However, α-spec^R22S, a tetramerization site mutant homologous to the pathological α-spec^R28S allele in humans, eliminates detectable binding to β-spectrin and reduces binding to β_H-spectrin ∼1000-fold. Even though spectrins are essential proteins, α-spectrin^R22S rescues α-spectrin mutants to adulthood with only minor phenotypes indicating that tetramerization, and thus conventional network formation, is not the essential function of non-erythroid spectrin. Our data provide the first rigorous test for the general requirement for tetramer-based non-erythroid spectrin networks throughout an organism and find that they have very limited roles, in direct contrast to the current paradigm.     

Meltrin β/ADAM19 Interacting with EphA4 in Developing Neural Cells Participates in Formation of the Neuromuscular Junction

Background

Development of the neuromuscular junction (NMJ) is initiated by the formation of postsynaptic specializations in the central zones of muscles, followed by the arrival of motor nerve terminals opposite the postsynaptic regions. The post- and presynaptic components are then stabilized and modified to form mature synapses. Roles of ADAM (A Disintegrin And Metalloprotease) family proteins in the formation of the NMJ have not been reported previously.

Principal Findings

We report here that Meltrin β, ADAM19, participates in the formation of the NMJ. The zone of acetylcholine receptor α mRNA distribution was broader and excess sprouting of motor nerve terminals was more prominent in meltrin β–deficient than in wild-type embryonic diaphragms. A microarray analysis revealed that the preferential distribution of ephrin-A5 mRNA in the synaptic region of muscles was aberrant in the meltrin β–deficient muscles. Excess sprouting of motor nerve terminals was also found in ephrin-A5 knockout mice, which lead us to investigate a possible link between Meltrin β and ephrin-A5-Eph signaling in the development of the NMJ. Meltrin β and EphA4 interacted with each other in developing motor neurons, and both of these proteins localized in the NMJ. Coexpression of Meltrin β and EphA4 strongly blocked vesicular internalization of ephrin-A5–EphA4 complexes without requiring the protease activity of Meltrin β, suggesting a regulatory role of Meltrin β in ephrin-A5-Eph signaling.

Conclusion

Meltrin β plays a regulatory role in formation of the NMJ. The endocytosis of ephrin-Eph complexes is required for efficient contact-dependent repulsion between ephrin and Eph. We propose that Meltrin β stabilizes the interaction between ephrin-A5 and EphA4 by regulating endocytosis of the ephrinA5-EphA complex negatively, which would contribute to the fine-tuning of the NMJ during development.     

Molecular regulation of postsynaptic differentiation at the neuromuscular junction

The neuromuscular junction (NMJ) is a synapse that develops between a motor neuron and a muscle fiber. A defining feature of NMJ development in vertebrates is the re-distribution of muscle acetylcholine (ACh) receptors (AChRs) following innervation, which generates high-density AChR clusters at the postsynaptic membrane and disperses aneural AChR clusters formed in muscle before innervation. This process in vivo requires MuSK, a muscle-specific receptor tyrosine kinase that triggers AChR re-distribution when activated; rapsyn, a muscle protein that binds and clusters AChRs; agrin, a nerve-secreted heparan-sulfate proteoglycan that activates MuSK; and ACh, a neurotransmitter that stimulates muscle and also disperses aneural AChR clusters. Moreover, in cultured muscle cells, several additional muscle- and nerve-derived molecules induce, mediate or participate in AChR clustering and dispersal. In this review we discuss how regulation of AChR re-distribution by multiple factors ensures aggregation of AChRs exclusively at NMJs.     

Laminins promote postsynaptic maturation by an autocrine mechanism at the neuromuscular junction

A prominent feature of synaptic maturation at the neuromuscular junction (NMJ) is the topological transformation of the acetylcholine receptor (AChR)-rich postsynaptic membrane from an ovoid plaque into a complex array of branches. We show here that laminins play an autocrine role in promoting this transformation. Laminins containing the α4, α5, and β2 subunits are synthesized by muscle fibers and concentrated in the small portion of the basal lamina that passes through the synaptic cleft at the NMJ. Topological maturation of AChR clusters was delayed in targeted mutant mice lacking laminin α5 and arrested in mutants lacking both α4 and α5. Analysis of chimeric laminins in vivo and of mutant myotubes cultured aneurally demonstrated that the laminins act directly on muscle cells to promote postsynaptic maturation. Immunohistochemical studies in vivo and in vitro along with analysis of targeted mutants provide evidence that laminin-dependent aggregation of dystroglycan in the postsynaptic membrane is a key step in synaptic maturation. Another synaptically concentrated laminin receptor, Bcam, is dispensable. Together with previous studies implicating laminins as organizers of presynaptic differentiation, these results show that laminins coordinate post- with presynaptic maturation.     

The dynamics of recycled acetylcholine receptors at the neuromuscular junction in vivo

At the peripheral neuromuscular junction (NMJ), a significant number of nicotinic acetylcholine receptors (AChRs) recycle back into the postsynaptic membrane after internalization to intermingle with not-yet-internalized ;pre-existing' AChRs. However, the way in which these receptor pools are maintained and regulated at the NMJ in living animals remains unknown. Here, we demonstrate that recycled receptors in functional synapses are removed approximately four times faster than pre-existing receptors, and that most removed recycled receptors are replaced by new recycled ones. In denervated NMJs, the recycling of AChRs is significantly depressed and their removal rate increased, whereas direct muscle stimulation prevents their loss. Furthermore, we show that protein tyrosine phosphatase inhibitors cause the selective accumulation of recycled AChRs in the peri-synaptic membrane without affecting the pre-existing AChR pool. The inhibition of serine/threonine phosphatases, however, has no effect on AChR recycling. These data show that recycled receptors are remarkably dynamic, and suggest a potential role for tyrosine dephosphorylation in the insertion and maintenance of recycled AChRs at the postsynaptic membrane. These findings may provide insights into long-term recycling processes at less accessible synapses in the central nervous system in vivo.     

A Highlights from MBoC Selection: Neuromuscular synapse integrity requires linkage of acetylcholine receptors to postsynaptic intermediate filament networks via rapsyn–plectin 1f complexes

Mutations in the cytolinker protein plectin lead to grossly distorted morphology of neuromuscular junctions (NMJs) in patients suffering from epidermolysis bullosa simplex (EBS)-muscular dystrophy (MS) with myasthenic syndrome (MyS). Here we investigated whether plectin contributes to the structural integrity of NMJs by linking them to the postsynaptic intermediate filament (IF) network. Live imaging of acetylcholine receptors (AChRs) in cultured myotubes differentiated ex vivo from immortalized plectin-deficient myoblasts revealed them to be highly mobile and unable to coalesce into stable clusters, in contrast to wild-type cells. We found plectin isoform 1f (P1f) to bridge AChRs and IFs via direct interaction with the AChR-scaffolding protein rapsyn in an isoform-specific manner; forced expression of P1f in plectin-deficient cells rescued both compromised AChR clustering and IF network anchoring. In conditional plectin knockout mice with gene disruption in muscle precursor/satellite cells (Pax7-Cre/cKO), uncoupling of AChRs from IFs was shown to lead to loss of postsynaptic membrane infoldings and disorganization of the NMJ microenvironment, including its invasion by microtubules. In their phenotypic behavior, mutant mice closely mimicked EBS-MD-MyS patients, including impaired body balance, severe muscle weakness, and reduced life span. Our study demonstrates that linkage to desmin IF networks via plectin is crucial for formation and maintenance of AChR clusters, postsynaptic NMJ organization, and body locomotion.     

Time Lapse in Vivo Visualization of Developmental Stabilization of Synaptic Receptors at Neuromuscular Junctions

The lifetime of nicotinic acetylcholine receptors (AChRs) in neuromuscular junctions (NMJs) is increased from <1 day to >1 week during early postnatal development. However, the exact timing of AChR stabilization is not known, and its correlation to the concurrent embryonic to adult AChR channel conversion, NMJ remodeling, and neuromuscular diseases is unclear. Using a novel time lapse in vivo imaging technology we show that replacement of the entire receptor population of an individual NMJ occurs end plate-specifically within hours. This makes it possible to follow directly in live animals changing stabilities of end plate receptors. In three different, genetically modified mouse models we demonstrate that the metabolic half-life values of synaptic AChRs increase from a few hours to several days after postnatal day 6. Developmental stabilization is independent of receptor subtype and apparently regulated by an intrinsic muscle-specific maturation program. Myosin Va, an F-actin-dependent motor protein, is also accumulated synaptically during postnatal development and thus could mediate the stabilization of end plate AChR.     

Anterograde Activin Signaling Regulates Postsynaptic Membrane Potential and GluRIIA/B Abundance at the Drosophila Neuromuscular Junction

Members of the TGF-β superfamily play numerous roles in nervous system development and function. In Drosophila, retrograde BMP signaling at the neuromuscular junction (NMJ) is required presynaptically for proper synapse growth and neurotransmitter release. In this study, we analyzed whether the Activin branch of the TGF-β superfamily also contributes to NMJ development and function. We find that elimination of the Activin/TGF-β type I receptor babo, or its downstream signal transducer smox, does not affect presynaptic NMJ growth or evoked excitatory junctional potentials (EJPs), but instead results in a number of postsynaptic defects including depolarized membrane potential, small size and frequency of miniature excitatory junction potentials (mEJPs), and decreased synaptic densities of the glutamate receptors GluRIIA and B. The majority of the defective smox synaptic phenotypes were rescued by muscle-specific expression of a smox transgene. Furthermore, a mutation in actβ, an Activin-like ligand that is strongly expressed in motor neurons, phenocopies babo and smox loss-of-function alleles. Our results demonstrate that anterograde Activin/TGF-β signaling at the Drosophila NMJ is crucial for achieving normal abundance and localization of several important postsynaptic signaling molecules and for regulating postsynaptic membrane physiology. Together with the well-established presynaptic role of the retrograde BMP signaling, our findings indicate that the two branches of the TGF-β superfamily are differentially deployed on each side of the Drosophila NMJ synapse to regulate distinct aspects of its development and function.     

Acute Activation,Desensitization and Smoldering Activation of Human Acetylcholine Receptors

The behavioral effects of nicotine and other nicotinic agonists are mediated by AChRs in the brain. The relative contribution of acute activation versus chronic desensitization of AChRs is unknown. Sustained “smoldering activation” occurs over a range of agonist concentrations at which activated and desensitized AChRs are present in equilibrium. We used a fluorescent dye sensitive to changes in membrane potential to examine the effects of acute activation and chronic desensitization by nicotinic AChR agonists on cell lines expressing human α4β2, α3β4 and α7 AChRs. We examined the effects of acute and prolonged application of nicotine and the partial agonists varenicline, cytisine and sazetidine-A on these AChRs. The range of concentrations over which nicotine causes smoldering activation of α4β2 AChRs was centered at 0.13 µM, a level found in smokers. However, nicotine produced smoldering activation of α3β4 and α7 AChRs at concentrations well above levels found in smokers. The α4β2 expressing cell line contains a mixture of two stoichiometries, namely (α4β2)₂β2 and (α4β2)₂α4. The (α4β2)₂β2 stoichiometry is more sensitive to activation by nicotine. Sazetidine-A activates and desensitizes only this stoichiometry. Varenicline, cytisine and sazetidine-A were partial agonists on this mixture of α4β2 AChRs, but full agonists on α3β4 and α7 AChRs. It has been reported that cytisine and varenicline are most efficacious on the (α4β2)₂α4 stoichiometry. In this study, we distinguish the dual effects of activation and desensitization of AChRs by these nicotinic agonists and define the range of concentrations over which smoldering activation can be sustained.     

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.