Developmental increase in the amount of rapsyn per acetylcholine receptor promotes postsynaptic receptor packing and stability

Neuromuscular synaptic transmission depends upon tight packing of acetylcholine receptors (AChRs) into postsynaptic AChR aggregates, but not all postsynaptic AChRs are aggregated. Here we describe a new confocal Fluorescence Resonance Energy Transfer (FRET) assay for semi-quantitative comparison of the degree to which AChRs are aggregated at synapses. During the first month of postnatal life the mouse tibialis anterior muscle showed increases both in the number of postsynaptic AChRs and the efficiency with which AChR was aggregated (by FRET). There was a concurrent two-fold increase in immunofluorescent labeling for the AChR-associated cytoplasmic protein, rapsyn. When 1-month old muscle was denervated, postsynaptic rapsyn immunostaining was reduced, as was the efficiency of AChR aggregation. In vivo electroporation of rapsyn-EGFP into muscle fibers increased postsynaptic rapsyn levels. Those synapses with higher ratios of rapsyn-EGFP to AChR displayed a slower metabolic turnover of AChR. Conversely, the reduction of postsynaptic rapsyn after denervation was accompanied by an acceleration of AChR turnover. Thus, a developmental increase in the amount of rapsyn targeted to the postsynaptic membrane may drive enhanced postsynaptic AChRs aggregation and AChR stability within the postsynaptic membrane.     

Combination of agrin and laminin increase acetylcholine receptor clustering and enhance functional neuromuscular junction formation In vitro

Clustering of acetylcholine receptors (AChR) at the postsynaptic membrane is a crucial step in the development of neuromuscular junctions (NMJ). During development and after denervation, aneural AChR clusters form on the sarcolemma. Recent studies suggest that these receptors are critical for guiding and initiating synaptogenesis. The aim of this study is to investigate the effect of agrin and laminin‐1; agents with known AChR clustering activity; on NMJ formation and muscle maturation. Primary myoblasts were differentiated in vitro on collagen, laminin or collagen and laminin‐coated surfaces in the presence or absence of agrin and laminin. The pretreated cells were then subject to innervation by PC12 cells. The number of neuromuscular junctions was assessed by immunocytochemical co‐localization of AChR clusters and the presynaptic marker synaptophysin. Functional neuromuscular junctions were quantitated by analysis of the level of spontaneous as well as neuromuscular blocker responsive contractile activity and muscle maturation was assessed by the degree of myotube striation. Agrin alone did not prime muscle for innervation while a combination of agrin and laminin pretreatment increased the number of neuromuscular junctions formed and enhanced acetylcholine based neurotransmission and myotube striation. This study has direct clinical relevance for treatment of denervation injuries and creating functional neuromuscular constructs for muscle tissue repair. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 76: 551–565, 2016     

A quantum of neurotransmitter causes minis in multiple postsynaptic cells at the Caenorhabditis elegans neuromuscular junction

The polyadic synapse, where a single presynaptic active zone associates with two or more postsynaptic cells, exists in both mammals and invertebrates. An important but unresolved question is whether synaptic transmission occurs between the presynaptic site and its various postsynaptic partners. Using the dual whole-cell voltage clamp technique, we analyzed miniature postsynaptic currents (mPSCs or minis) at the C. elegans neuromuscular junction (NMJ), which is a polyadic synapse. We found that neighboring muscle cells at the same position along the body axis had high frequencies of concurrent mPSCs, which could not be explained by pure chance. Although body-wall muscle cells are electrically coupled, the high frequency of concurrent mPSCs was not due to electrical coupling because there was no correlation between the frequency of concurrent mPSCs and the degree of electrical coupling; the rise time of concurrent mPSCs was identical to that of nonconcurrent mPSCs but distinct from that of junctional currents (I(j)); and a mutant defective in electrical coupling showed normal frequency of concurrent mPSCs. Our analyses suggest that a single quantum of neurotransmitter may cause mPSCs in multiple postsynaptic cells at polyadic synapses, and that high-fidelity synaptic transmission occurs between the presynaptic site and its various postsynaptic partners. Thus, polyadic synapses could be a distinct mechanism for synaptic divergence and for synchronizing activities of postsynaptic cells.     

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

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

A critical step for postsynaptic F‐actin organization: Regulation of Baz/Par‐3 localization by aPKC and PTEN

Actin remodeling has emerged as a critical process during synapse development and plasticity. Thus, understanding the regulatory mechanisms controlling actin organization at synapses is exceedingly important. Here, we used the highly plastic Drosophila neuromuscular junction (NMJ) to understand mechanisms of actin remodeling at postsynaptic sites. Previous studies have suggested that the actin‐binding proteins Spectrin and Coracle play a critical role in NMJ development and the anchoring of glutamate receptors most likely through actin regulation. Here, we show that an additional determinant of actin organization at the postsynaptic region is the PDZ protein Baz/Par‐3. Decreasing Baz levels in postsynaptic muscles has dramatic consequences for the size of F‐actin and spectrin domains at the postsynaptic region. In turn, proper localization of Baz at this site depends on both phosphorylation and dephosphorylation events. Baz phosphorylation by its binding partner, atypical protein kinase C (aPKC), is required for normal Baz targeting to the postsynaptic region. However, the retention of Baz at this site depends on its dephosphorylation mediated by the lipid and protein phosphatase PTEN. Misregulation of the phosphorylation state of Baz by genetic alterations in PTEN or aPKC activity has detrimental consequences for postsynaptic F‐actin and spectrin localization, synaptic growth, and receptor localization. Our results provide a novel mechanism of postsynaptic actin regulation through Baz, governed by the antagonistic actions of aPKC and PTEN. Given the conservation of these proteins from worms to mammals, these results are likely to provide new insight into actin organization pathways. © 2009 Wiley Periodicals, Inc. Develop Neurobiol 2009     

Synapse elimination from the mouse neuromuscular junction in vitro: A non-hebbian activity-dependent process

The effect of action potentials on elimination of mouse neuromuscular junctions (NMJ) was studied in a three compartment cell culture preparation. Axons from superior cervical ganglion or ventral spinal cord neurons in two lateral compartments formed multiple neuromuscular junctions with muscle cells in a central compartment. The loss of synapses over a 2–7-day period was determined by serial electrophysiological recording and a functional assay. Electrical stimulation of axons from one side compartment during this period, using 30-Hz bursts of 2-s duration, repeated at 10-s intervals, caused a significant increase in synapse elimination compared to unstimulated cultures (p< 0.001). The extent of homosynaptic and heterosynaptic elimination was comparable, i. e., of the 226 functional synapses of each type studied, 111 (49%) of the synapses that had been stimulated were eliminated, and 87 (39%) of unstimulated synapses on the same muscle cells were eliminated. Also, simultaneous bilateral stimulation caused significantly greater elimination of synapses than unilateral stimulation (p< 0.005). These observations are contrary to the Hebbian hypothesis of synaptic plasticity. A spatial effect of stimulus-induced synapse elimination was also evident following simultaneous bilateral stimulation. Prior to stimulation, most muscle cells were innervated by axons from both side compartments, but after bilateral stimulation, muscle cells were predominantly unilaterally innervated by axons from the closer compartment. These experiments suggest that synapse elimination at the NMJ is an activity-dependent process, but it does not follow Hebbian or anti-Hebbian rules of synaptic plasticity. Rather, elimination is a consequence of postsynaptic activation and a function of location of the muscle cell relative to the neuron. An interaction between spatial and activity-dependent effects on synapse elimination could help produce optimal refinement of synaptic connections during postnatal development. © 1993 John Wiley & Sons, Inc.     

Motor function recovery: deciphering a regenerative niche at the neuromuscular synapse

The coordinated movement of many organisms relies on efficient nerve–muscle communication at the neuromuscular junction (NMJ), a peripheral synapse composed of a presynaptic motor axon terminal, a postsynaptic muscle specialization, and non-myelinating terminal Schwann cells. NMJ dysfunctions are caused by traumatic spinal cord or peripheral nerve injuries as well as by severe motor pathologies. Compared to the central nervous system, the peripheral nervous system displays remarkable regenerating abilities; however, this capacity is limited by the denervation time frame and depends on the establishment of permissive regenerative niches. At the injury site, detailed information is available regarding the cells, molecules, and mechanisms involved in nerve regeneration and repair. However, a regenerative niche at the final functional step of peripheral motor innervation, i.e. at the mature neuromuscular synapse, has not been deciphered. In this review, we integrate classic and recent evidence describing the cells and molecules that could orchestrate a dynamic ecosystem to accomplish successful NMJ regeneration. We propose that such a regenerative niche must ensure at least two fundamental steps for successful NMJ regeneration: the proper arrival of incoming regenerating axons to denervated postsynaptic muscle domains, and the resilience of those postsynaptic domains, in morphological and functional terms. We here describe and combine the main cellular and molecular responses involved in each of these steps as potential targets to help successful NMJ 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.     

Regulation of ACh receptor clustering by the tyrosine phosphatase Shp2

At the vertebrate neuromuscular junction (NMJ), postsynaptic aggregation of muscle acetylcholine receptors (AChRs) depends on the activation of MuSK, a muscle-specific tyrosine kinase that is stimulated by neural agrin and regulated by muscle-intrinsic tyrosine kinases and phosphatases. We recently reported that Shp2, a tyrosine phosphatase containing src homology two domains, suppressed MuSK-dependent AChR clustering in cultured myotubes, but how this effect of Shp2 is controlled has remained unclear. In this study, biochemical assays showed that agrin-treatment of C2 mouse myotubes enhanced the tyrosine phosphorylation of signal regulatory protein alpha1 (SIRPalpha1), a known activator of Shp2, and promoted SIRPalpha1's interaction with Shp2. Moreover, in situ experiments revealed that treatment of myotubes with the Shp2-selective inhibitor NSC-87877 increased spontaneous and agrin-induced AChR clustering, and that AChR clustering was also enhanced in myotubes ectopically expressing inactive (dominant-negative) Shp2; in contrast, AChR clustering was reduced in myotubes expressing constitutively active Shp2. Significantly, expression of truncated (nonShp2-binding) and full-length (Shp2-binding) forms of SIRPalpha1 in myotubes also increased and decreased AChR clustering, respectively, and coexpression of truncated SIRPalpha1 with active Shp2 and full-length SIRPalpha1 with inactive Shp2 reversed the actions of the exogenous Shp2 proteins on AChR clustering. These results suggest that SIRPalpha1 is a novel downstream target of MuSK that activates Shp2, which, in turn, suppresses AChR clustering. We propose that an inhibitory loop involving both tyrosine kinases and phosphatases sets the level of agrin/MuSK signaling and constrains it spatially to help generate high-density AChR clusters selectively at NMJs.     

Guanylate cyclase and cyclic GMP-dependent protein kinase regulate agrin signaling at the developing neuromuscular junction

During formation of the neuromuscular junction (NMJ), agrin secreted by motor axons signals the embryonic muscle cells to organize a postsynaptic apparatus including a dense aggregate of acetylcholine receptors (AChRs). Agrin signaling at the embryonic NMJ requires the activity of nitric oxide synthase (NOS). Common downstream effectors of NOS are guanylate cyclase (GC), which synthesizes cyclic GMP, and cyclic GMP-dependent protein kinase (PKG). Here we show that GC and PKG are important for agrin signaling at the embryonic NMJ of the frog, Xenopus laevis. Inhibitors of both GC and PKG reduced endogenous AChR aggregation in embryonic muscles by 50-85%, and blocked agrin-induced AChR aggregation in cultured embryonic muscle cells. A cyclic GMP analog, 8-bromo-cyclic GMP, increased endogenous AChR aggregation in embryonic muscles to 3- to 4-fold control levels. Overexpression of either GC or PKG in embryos increased AChR aggregate area by 60-170%, whereas expression of a dominant negative form of GC inhibited endogenous aggregation by 50%. These results indicate that agrin signaling in embryonic muscle cells requires the activity of GC and PKG as well as NOS.     

Evidence for a presynaptic action of chlordimeform at the insect neuromuscular junction

The mechanism of action of chlordimeform on the mealworm nerve-muscle preparation was studied with microelectrodes. Chlordimeform affected neither the mean amplitude nor the frequency of spontaneous miniature excitatory postsynaptic potentials. Extracellular focal recordings show that in the presence of 0.8 mM chlordimeform the presynaptic spike is almost unchanged, but the quantal content for evoked transmitter release is reduced. It is suggested that chlordimeform decreases the influx of calcium at the presynaptic terminal during the active phase of the nerve terminal action potential, thereby inhibiting evoked transmitter release.     

Reduction in glutamate uptake is associated with extrasynaptic NMDA and metabotropic glutamate receptor activation at the hippocampal CA1 synapse of aged rats

This study aims to determine whether the regulation of extracellular glutamate is altered during aging and its possible consequences on synaptic transmission and plasticity. A decrease in the expression of the glial glutamate transporters GLAST and GLT‐1 and reduced glutamate uptake occur in the aged (24–27 months) Sprague–Dawley rat hippocampus. Glutamatergic excitatory postsynaptic potentials recorded extracellularly in ex vivo hippocampal slices from adult (3–5 months) and aged rats are depressed by DL‐TBOA, an inhibitor of glutamate transporter activity, in an N‐Methyl‐d‐ Aspartate (NMDA)‐receptor‐dependent manner. In aged but not in young rats, part of the depressing effect of DL‐TBOA also involves metabotropic glutamate receptor (mGluRs) activation as it is significantly reduced by the specific mGluR antagonist d‐methyl‐4‐carboxy‐phenylglycine (MCPG). The paired‐pulse facilitation ratio, a functional index of glutamate release, is reduced by MCPG in aged slices to a level comparable to that in young rats both under control conditions and after being enhanced by DL‐TBOA. These results suggest that the age‐associated glutamate uptake deficiency favors presynaptic mGluR activation that lowers glutamate release. In parallel, 2 Hz‐induced long‐term depression is significantly decreased in aged animals and is fully restored by MCPG. All these data indicate a facilitated activation of extrasynaptic NMDAR and mGluRs in aged rats, possibly because of an altered distribution of glutamate in the extrasynaptic space. This in turn affects synaptic transmission and plasticity within the aged hippocampal CA1 network.     

γ-secretase promotes Drosophila postsynaptic development through the cleavage of a Wnt receptor

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Agrin-induced acetylcholine receptor clustering is mediated by the small guanosine triphosphatases Rac and Cdc42

During neuromuscular junction formation, agrin secreted from motor neurons causes muscle cell surface acetylcholine receptors (AChRs) to cluster at synaptic sites by mechanisms that are insufficiently understood. The Rho family of small guanosine triphosphatases (GTPases), including Rac and Cdc42, can mediate focal reorganization of the cell periphery in response to extracellular signals. Here, we investigated the role of Rac and Cdc42 in coupling agrin signaling to AChR clustering. We found that agrin causes marked muscle-specific activation of Rac and Cdc42 in differentiated myotubes, as detected by biochemical measurements. Moreover, this activation is crucial for AChR clustering, since the expression of dominant interfering mutants of either Rac or Cdc42 in myotubes blocks agrin-induced AChR clustering. In contrast, constitutively active Rac and Cdc42 mutants cause AChR to aggregate in the absence of agrin. By indicating that agrin-dependent activation of Rac and Cdc42 constitutes a critical step in the signaling pathway leading to AChR clustering, these findings suggest a novel role for these Rho-GTPases: the coupling of neuronal signaling to a key step in neuromuscular synaptogenesis.