Acetylcholine Receptor Clusters Research Articles

Protein Tyrosine Phosphatase Receptor Type R (PTPRR) Reduces AChR Clustering by Dephosphorylating MuSK.

Neuromuscular junction (NMJ) formation and maintenance depend on the proper localization and concentration of various molecules at synaptic contact sites. Acetylcholine receptor (AChR) clustering on the postsynaptic membrane is a cardinal event in NMJ formation. Muscle-specific tyrosine kinase (MuSK), which functions depending on its phosphorylation, plays an essential role in AChR clustering. In the present study, we used plasmid-based biochemical screening and determined that protein tyrosine phosphatase receptor type R (PTPRR) is responsible for dephosphorylating MuSK on tyrosine residue 754. Furthermore, we showed that PTPRR significantly reduced MuSK-dependent AChR clustering in C2C12 myotubes. Collectively, these data illustrate a negative regulation function of PTPRR in AChR clustering.

ALS mutations in both human skeletal muscle and motoneurons differentially affects neuromuscular junction integrity and function

There is evidence for the involvement of human skeletal muscle (hSKM) in ALS neuromuscular junction (NMJ) dysfunction. However, the specific avenue by which the hSKM contributes to NMJ disruption is not well understood due to limited human-based studies performed to investigate the subject. Thus, hSKM and human motoneurons (hMN) generated from induced pluripotent stem cells of healthy individuals (WT) and ALS patients with two different SOD1 mutations were integrated into functional NMJ systems to investigate and compare the pathological contribution of the hSKM and hMN to ALS NMJ disruption. Morphological assessment of ALS NMJs demonstrated reduced acetylcholine receptor clustering in the post-synaptic membrane of co-cultures with ALS hSKM (hSKMSOD1-hMNWT and hSKMSOD1-hMNSOD1). Significantly reduced functional NMJ numbers, NMJ stability, contraction fidelity and increased fatigue index were observed in all ALS co-cultures compared to WT. However, these disease phenotypes were comparatively more severe in microphysiologic systems with hSKMSOD1-hMNWT or hSKMSOD1-hMNSOD1 than those with hSKMWT-hMNSOD1 co-cultures. Results from this study affirm that the inherent pathological defects in ALS hSKM, independent of motoneurons, significantly contributes to NMJ dysfunction. As such, therapeutically targeting the ALS hSKM may be just as, if not more critical than, the hMN in alleviating disease phenotypes and attenuating disease progression.

N-acetyl cysteine prevents arecoline-inhibited C2C12 myoblast differentiation through ERK1/2 phosphorylation.

Arecoline is known to induce reactive oxygen species (ROS). Our previous studies showed that arecoline inhibited myogenic differentiation and acetylcholine receptor cluster formation of C2C12 myoblasts. N-acetyl-cysteine (NAC) is a known ROS scavenger. We hypothesize that NAC scavenges the excess ROS caused by arecoline. In this article we examined the effect of NAC on the inhibited myoblast differentiation by arecoline and related mechanisms. We found that NAC less than 2 mM is non-cytotoxic to C2C12 by viability analysis. We further demonstrated that NAC attenuated the decreased number of myotubes and nuclei in each myotube compared to arecoline treatment by H & E staining. We also showed that NAC prevented the decreased expression level of the myogenic markers, myogenin and MYH caused by arecoline, using immunocytochemistry and western blotting. Finally, we found that NAC restored the decreased expression level of p-ERK1/2 by arecoline. In conclusion, our results indicate that NAC attenuates the damage of the arecoline-inhibited C2C12 myoblast differentiation by the activation/phosphorylation of ERK. This is the first report to demonstrate that NAC has beneficial effects on skeletal muscle myogenesis through ERK1/2 upon arecoline treatment. Since defects of skeletal muscle associates with several diseases, NAC can be a potent drug candidate in diseases related to defects in skeletal muscle myogenesis.

Clinical value of cell-based assays in the characterisation of seronegative myasthenia gravis

ObjectivePatients with myasthenia gravis without acetylcholine receptor (AChR) or muscle-specific kinase (MuSK) antibodies detected by radioimmunoprecipitation assays (RIAs) are classified as seronegative myasthenia gravis (SNMG). Live cell-based assays (l-CBAs) can...

Functional regeneration of the murine neuromuscular synapse relies on long-lasting morphological adaptations

BackgroundIn a broad variety of species, muscle contraction is controlled at the neuromuscular junction (NMJ), the peripheral synapse composed of a motor nerve terminal, a muscle specialization, and non-myelinating terminal Schwann cells. While peripheral nerve damage leads to successful NMJ reinnervation in animal models, muscle fiber reinnervation in human patients is largely inefficient. Interestingly, some hallmarks of NMJ denervation and early reinnervation in murine species, such as fragmentation and poly-innervation, are also phenotypes of aged NMJs or even of unaltered conditions in other species, including humans. We have reasoned that rather than features of NMJ decline, such cellular responses could represent synaptic adaptations to accomplish proper functional recovery. Here, we have experimentally tackled this idea through a detailed comparative study of the short- and long-term consequences of irreversible (chronic) and reversible (partial) NMJ denervation in the convenient cranial levator auris longus muscle.ResultsOur findings reveal that irreversible muscle denervation results in highly fragmented postsynaptic domains and marked ectopic acetylcholine receptor clustering along with significant terminal Schwann cells sprouting and progressive detachment from the NMJ. Remarkably, even though reversible nerve damage led to complete reinnervation after 11 days, we found that more than 30% of NMJs are poly-innervated and around 65% of postsynaptic domains are fragmented even 3 months after injury, whereas synaptic transmission is fully recovered two months after nerve injury. While postsynaptic stability was irreversibly decreased after chronic denervation, this parameter was only transiently affected by partial NMJ denervation. In addition, we found that a combination of morphometric analyses and postsynaptic stability determinations allows discriminating two distinct forms of NMJ fragmentation, stable-smooth and unstable-blurred, which correlate with their regeneration potential.ConclusionsTogether, our data unveil that reversible nerve damage imprints a long-lasting reminiscence in the NMJ that results in the rearrangement of its cellular components. Instead of being predictive of NMJ decline, these traits may represent an efficient adaptive response for proper functional recovery. As such, these features are relevant targets to be considered in strategies aimed to restore motor function in detrimental conditions for peripheral innervation.

Control of CRK-RAC1 activity by the miR-1/206/133 miRNA family is essential for neuromuscular junction function.

Formation and maintenance of neuromuscular junctions (NMJs) are essential for skeletal muscle function, allowing voluntary movements and maintenance of the muscle tone, thereby preventing atrophy. Generation of NMJs depends on the interaction of motor neurons with skeletal muscle fibers, which initiates a cascade of regulatory events that is essential for patterning of acetylcholine receptor (AChR) clusters at specific sites of the sarcolemma. Here, we show that muscle-specific miRNAs of the miR-1/206/133 family are crucial regulators of a signaling cascade comprising DOK7-CRK-RAC1, which is critical for stabilization and anchoring of postsynaptic AChRs during NMJ development and maintenance. We describe that posttranscriptional repression of CRK by miR-1/206/133 is essential for balanced activation of RAC1. Failure to adjust RAC1 activity severely compromises NMJ function, causing respiratory failure in neonates and neuromuscular symptoms in adult mice. We conclude that miR-1/206/133 serve a specific function for NMJs but are dispensable for skeletal muscle development.

Schwann Cell Derived Signals Enhance AChR Clustering in C2C12 Myotubes

The neuromuscular junction (NMJ) is a tripartite structure made up of the pre‐synaptic motor neuron terminal, the post‐synaptic muscle end plate, and the perisynaptic Schwann cells (SC). Degeneration of the NMJ is strongly associated with age‐associated muscle mass and strength declines as well as other neurodegenerative diseases; however, the cellular interactions and signals that initiate the degeneration are poorly understood. In particular, understanding of the role of perisynaptic SCs in NMJ maintenance is scant, whereas the signaling between motor neurons and muscle fibers has been extensively studied. For example, agrin is a motor neuron derived secreted factor that is the primary signal for the induction of acetylcholine receptor (AChR) clustering in myofibers, and treatment of myotubes in culture with agrin has been shown to similarly promote AChR clustering in the absence of neurons. The presence of SCs at motor nerve terminals implicates signaling from these cells in both NMJ formation and maintenance. Thus, the goal was to determine if SC derived signals impact AChR clustering. We hypothesized that SC derived factors would potentiate agrin‐induced aneural AChR clustering in C2C12 myotubes. Differentiated C2C12 myotubes were treated with agrin for either 10 minutes or 4 hours to minimally or maximally induce AChR clustering, respectively. Additional myotubes were treated with agrin for 10 minutes followed by treatment with conditioned media (CM) derived from SW10 Schwann cell line cultures for the remaining 4 hours. Finally, additional myotube cultures were treated for 4 hours with CM alone or normal complete media as controls. The number of clusters and mean cluster area were increased in myotube cultures treated with the combination of a 10‐minute agrin pulse followed by and CM from SW10 cells compared to cultures treated for either 10 minutes or 4 hours with agrin. Neither cluster number nor area were elevated compared with controls in cultures treated with CM alone. These data suggest that SC‐derived signals can potentiate agrin‐induced post‐synaptic AChR clustering in vitro. We are currently investigating the critical SC‐derived signals that potentiate AChR clustering to determine factors that may provide therapeutic benefit in neurodegenerative phenotypes including aging.

Neuromuscular Synapse Dynamics in Aged and Injured Skeletal Muscle

It is evident that both aged and diseased skeletal muscle exhibit interesting neuromuscular dynamics, characterized by changes in morphology and functionality of the synapse. Although they display a high regenerative capacity, skeletal muscles are still susceptible to nervous system perturbations and impacts of aging. Aged neuromuscular junctions (NMJs) demonstrate a diseased phenotype following some form of muscle atrophy, indicating the presence of paracrine signaling between myofibers and neuronal cells. In this study, we investigated differences in neuromuscular dynamics in injured and aged skeletal muscle using whole‐mount immunostaining and imaging. It has previously been shown that disruption of one component of the muscle microenvironment, such as vasculature, disrupts the structure of NMJs, however it is unclear if aged skeletal muscle experiences similar changes. We hypothesized that both aged and injured NMJs would display similar morphological dynamics, characterized by denervation and fragmentation of postsynaptic acetylcholine receptors (AChRs). We demonstrated that aged and injured NMJs exhibit fragmentation of AChRs, Wallerian degeneration of the presynaptic motor neurons, decreased overlap of pre‐ and post‐synaptic sides, and decreased area of AChR clusters. Due to the prevalence of neuromuscular disorders in the aging population, it is pertinent to understand nerve‐muscle interactions in the healthy aging state in order to hypothesize how these dynamics may be altered in the age‐induced injury states. Ultimately, this will allow us to better comprehend the molecular and cellular mechanisms that are responsible for neuromuscular degeneration in order to develop strategies to improve mobility and quality of life.

Impaired signaling for neuromuscular synaptic maintenance is a feature of Motor Neuron Disease

A central event in the pathogenesis of motor neuron disease (MND) is the loss of neuromuscular junctions (NMJs), yet the mechanisms that lead to this event in MND remain to be fully elucidated. Maintenance of the NMJ relies upon neural agrin (n-agrin) which, when released from the nerve terminal, activates the postsynaptic Muscle Specific Kinase (MuSK) signaling complex to stabilize clusters of acetylcholine receptors. Here, we report that muscle from MND patients has an increased proportion of slow fibers and muscle fibers with smaller diameter. Muscle cells cultured from MND biopsies failed to form large clusters of acetylcholine receptors in response to either non-MND human motor axons or n-agrin. Furthermore, levels of expression of MuSK, and MuSK-complex components: LRP4, Caveolin-3, and Dok7 differed between muscle cells cultured from MND patients compared to those from non-MND controls. To our knowledge, this is the first time a fault in the n-agrin-LRP4-MuSK signaling pathway has been identified in muscle from MND patients. Our results highlight the n-agrin-LRP4-MuSK signaling pathway as a potential therapeutic target to prolong muscle function in MND.

Association of a newly identified lncRNA LNC_000280 with the formation of acetylcholine receptor clusters in vitro

Peripheral nerve injury (PNI) can disintegrate acetylcholine receptor (AChR) clusters in the postsynaptic membrane. In our previous research, lncRNAs that were differentially expressed in the whole transcriptome sequencing of denervated muscle atrophy after PNI were screened. By utilizing Gene Ontology (GO) analysis and protein-protein interaction (PPI) networks, a novel lncRNA LNC_000280 was predicted to be associated with neuromuscular junction (NMJ). The myotubes were used to assess the connection between LNC_000280 and AChR cluster formation in vitro by overexpression and knockdown of LNC_000280 in the C2C12 cell line. Our findings demonstrated that the overexpression of LNC_000280 repressed the gene expression and protein level of AChR subunits in myotubes and further reduced the area of AChR aggregates on the cell membrane. In contrast, the knockdown of LNC_000280 brought about opposite results. In addition, the transcriptional level of Sorbs2 changed inversely with the quantity change of LNC_000280. In conclusion, LNC_000280 may associate with the formation of AChR clusters.

The CMS19 disease model specifies a pivotal role for collagen XIII in bone homeostasis

Mutations in the COL13A1 gene result in congenital myasthenic syndrome type 19 (CMS19), a disease of neuromuscular synapses and including various skeletal manifestations, particularly facial dysmorphisms. The phenotypic consequences in Col13a1 null mice (Col13a1−/−) recapitulate the muscle findings of the CMS19 patients. Collagen XIII (ColXIII) is exists as two forms, a transmembrane protein and a soluble molecule. While the Col13a1−/− mice have poorly formed neuromuscular junctions, the prevention of shedding of the ColXIII ectodomain in the Col13a1tm/tm mice results in acetylcholine receptor clusters of increased size and complexity. In view of the bone abnormalities in CMS19, we here studied the tubular and calvarial bone morphology of the Col13a1−/− mice. We discovered several craniofacial malformations, albeit less pronounced ones than in the human disease, and a reduction of cortical bone mass in aged mice. In the Col13a1tm/tm mice, where ColXIII is synthesized but the ectodomain shedding is prevented due to a mutation in a protease recognition sequence, the cortical bone mass decreased as well with age and the cephalometric analyses revealed significant craniofacial abnormalities but no clear phenotypical pattern. To conclude, our data indicates an intrinsic role for ColXIII, particularly the soluble form, in the upkeep of bone with aging and suggests the possibility of previously undiscovered bone pathologies in patients with CMS19.

The clinical need for clustered AChR cell-based assay testing of seronegative MG

Trial eligibility in myasthenia gravis (MG) remains largely dependent on a positive autoantibody serostatus. This significantly hinders seronegative MG (SNMG) patients from receiving potentially beneficial new treatments. In a subset of SNMG patients, acetylcholine receptor (AChR) autoantibodies are detectable by a clustered AChR cell-based assay (CBA). Of 99 SNMG patients from two academic U.S. centers, 18 (18.2%) tested positive by this assay. Autoantibody positivity was further validated in 17/18 patients. In a complementary experiment, circulating AChR-specific B cells were identified in a CBA-positive SNMG patient. These findings corroborate the clinical need for clustered AChR CBA testing when evaluating SNMG patients.

Contraction Control of Aligned Myofiber Sheet Tissue by Parallel Oriented Induced Pluripotent Stem Cell-Derived Neurons.

Fabrication and application of engineered complex tissues composed of different types of cells is a crucial milestone in the next phase of tissue engineering. The delicate organization structure of each tissue component and its physiological connections enable all the functions in the human body. In this study, cell sheet-based engineering allowed us to fabricate a complex myofiber sheet tissue using motor neurons derived from human-induced pluripotent stem cells. In contrast with previous studies of other groups, a myofiber sheet with a biomimetic aligned structure was produced from human myoblasts using a striped-patterned thermoresponsive dish, which enabled manipulation of the sheet tissue by simply lowering the culture temperature. The myofiber sheet was transferred onto a gel that promotes functional maturation of human myofibers, resulting in production of contractile human muscle tissue. Just by seeding motor neurons onto the sheet tissue, all the neurons physically contacted to the aligned myofibers, and autonomously elongated in parallel to the myofiber orientation. In addition, the neurite outgrowth was enlarged by coculturing on the myofiber sheet. The presence of the neurons enhanced clustering of myofiber acetylcholine receptors (AChRs), typically found at the neuromuscular junctions (NMJs). Consequently, contraction behaviors of the myofiber sheet were regulated by neuronal signal transduction through NMJs. Muscle contraction was induced when the motor neurons were stimulated by glutamic acid, and effectively blocked by administration of d-tubocurarine as an antagonistic inhibitor for the AChR. The fibrin-based gel was useful as a culture environment for tissue maturation and as a favorable substrate for unobstructed contractions. Our neuron-muscle sheet tissue will be scalable by simply enlarging the micropatterned substrate and manipulable three dimensionally; fabrication of a thick tissue and a bundle-like structured tissue will be possible just by layering multiple sheets or rolling up the sheet. Simplified control over self-orientation of neurite elongation will be advantageous for fabrication of such a large and complex tissue. Therefore, our methodology, established in this study, will be instrumental in future applications of regenerative medicine for locomotion apparatus. Impact Statement A complex tissue containing skeletal myofibers and induced pluripotent stem cell-derived motor neurons was fabricated from human cells based on the cell sheet engineering technology. A micropatterned thermoresponsive culture dish and a fibrin-based gel substrate enabled production of aligned, and functionally matured myofiber sheet tissue. The motor neurons were autonomously oriented simply by seeding on the aligned myofiber sheet tissue. Induction and inhibition of the muscle contraction were effectively controlled by neuronal signal transduction. Considering the potential scalability and manipulability of the neuron-muscle sheet tissue, our methodology will contribute to future applications of regenerative medicine for locomotion apparatus.

Transport and Secretion of the Wnt3 Ligand by Motor Neuron-like Cells and Developing Motor Neurons.

The vertebrate neuromuscular junction (NMJ) is formed by a presynaptic motor nerve terminal and a postsynaptic muscle specialization. Cumulative evidence reveals that Wnt ligands secreted by the nerve terminal control crucial steps of NMJ synaptogenesis. For instance, the Wnt3 ligand is expressed by motor neurons at the time of NMJ formation and induces postsynaptic differentiation in recently formed muscle fibers. However, the behavior of presynaptic-derived Wnt ligands at the vertebrate NMJ has not been deeply analyzed. Here, we conducted overexpression experiments to study the expression, distribution, secretion, and function of Wnt3 by transfection of the motor neuron-like NSC-34 cell line and by in ovo electroporation of chick motor neurons. Our findings reveal that Wnt3 is transported along motor axons in vivo following a vesicular-like pattern and reaches the NMJ area. In vitro, we found that endogenous Wnt3 expression increases as the differentiation of NSC-34 cells proceeds. Although NSC-34 cells overexpressing Wnt3 do not modify their morphological differentiation towards a neuronal phenotype, they effectively induce acetylcholine receptor clustering on co-cultured myotubes. These findings support the notion that presynaptic Wnt3 is transported and secreted by motor neurons to induce postsynaptic differentiation in nascent NMJs.

Tks5 Regulates Synaptic Podosome Formation and Stabilization of the Postsynaptic Machinery at the Neuromuscular Junction.

Currently, the etiology of many neuromuscular disorders remains unknown. Many of them are characterized by aberrations in the maturation of the neuromuscular junction (NMJ) postsynaptic machinery. Unfortunately, the molecular factors involved in this process are still largely unknown, which poses a great challenge for identifying potential therapeutic targets. Here, we identified Tks5 as a novel interactor of αdystrobrevin-1, which is a crucial component of the NMJ postsynaptic machinery. Tks5 has been previously shown in cancer cells to be an important regulator of actin-rich structures known as invadosomes. However, a role of this scaffold protein at a synapse has never been studied. We show that Tks5 is crucial for remodeling of the NMJ postsynaptic machinery by regulating the organization of structures similar to the invadosomes, known as synaptic podosomes. Additionally, it is involved in the maintenance of the integrity of acetylcholine receptor (AChR) clusters and regulation of their turnover. Lastly, our data indicate that these Tks5 functions may be mediated by its involvement in recruitment of actin filaments to the postsynaptic machinery. Collectively, we show for the first time that the Tks5 protein is involved in regulation of the postsynaptic machinery.

Agrin/Lrp4 signal constrains MuSK-dependent neuromuscular synapse development in appendicular muscle.

The receptor tyrosine kinase MuSK, its co-receptor Lrp4 and the Agrin ligand constitute a signaling pathway that is crucial in axial muscle for neuromuscular synapse development, yet whether this pathway functions similarly in appendicular muscle is unclear. Here, using the larval zebrafish pectoral fin, equivalent to tetrapod forelimbs, we show that, similar to axial muscle, developing appendicular muscles form aneural acetylcholine receptor (AChR) clusters prior to innervation. As motor axons arrive, neural AChR clusters form, eventually leading to functional synapses in a MuSK-dependent manner. We find that loss of Agrin or Lrp4 function, which abolishes synaptic AChR clusters in axial muscle, results in enlarged presynaptic nerve regions and progressively expanding appendicular AChR clusters, mimicking the consequences of motoneuron ablation. Moreover, musk depletion in lrp4 mutants partially restores synaptic AChR patterning. Combined, our results provide compelling evidence that, in addition to the canonical pathway in which Agrin/Lrp4 stimulates MuSK activity, Agrin/Lrp4 signaling in appendicular muscle constrains MuSK-dependent neuromuscular synapse organization. Thus, we reveal a previously unappreciated role for Agrin/Lrp4 signaling, thereby highlighting distinct differences between axial and appendicular synapse development.

Drebrin Regulates Acetylcholine Receptor Clustering and Organization of Microtubules at the Postsynaptic Machinery.

Proper muscle function depends on the neuromuscular junctions (NMJs), which mature postnatally to complex “pretzel-like” structures, allowing for effective synaptic transmission. Postsynaptic acetylcholine receptors (AChRs) at NMJs are anchored in the actin cytoskeleton and clustered by the scaffold protein rapsyn, recruiting various actin-organizing proteins. Mechanisms driving the maturation of the postsynaptic machinery and regulating rapsyn interactions with the cytoskeleton are still poorly understood. Drebrin is an actin and microtubule cross-linker essential for the functioning of the synapses in the brain, but its role at NMJs remains elusive. We used immunohistochemistry, RNA interference, drebrin inhibitor 3,5-bis-trifluoromethyl pyrazole (BTP2) and co-immunopreciptation to explore the role of this protein at the postsynaptic machinery. We identify drebrin as a postsynaptic protein colocalizing with the AChRs both in vitro and in vivo. We also show that drebrin is enriched at synaptic podosomes. Downregulation of drebrin or blocking its interaction with actin in cultured myotubes impairs the organization of AChR clusters and the cluster-associated microtubule network. Finally, we demonstrate that drebrin interacts with rapsyn and a drebrin interactor, plus-end-tracking protein EB3. Our results reveal an interplay between drebrin and cluster-stabilizing machinery involving rapsyn, actin cytoskeleton, and microtubules.

Activation of Muscle-Specific Kinase (MuSK) Reduces Neuromuscular Defects in the Delta7 Mouse Model of Spinal Muscular Atrophy (SMA).

Spinal muscular atrophy (SMA) is a motor neuron disease caused by insufficient levels of the survival motor neuron (SMN) protein. One of the most prominent pathological characteristics of SMA involves defects of the neuromuscular junction (NMJ), such as denervation and reduced clustering of acetylcholine receptors (AChRs). Recent studies suggest that upregulation of agrin, a crucial NMJ organizer promoting AChR clustering, can improve NMJ innervation and reduce muscle atrophy in the delta7 mouse model of SMA. To test whether the muscle-specific kinase (MuSK), part of the agrin receptor complex, also plays a beneficial role in SMA, we treated the delta7 SMA mice with an agonist antibody to MuSK. MuSK agonist antibody #13, which binds to the NMJ, significantly improved innervation and synaptic efficacy in denervation-vulnerable muscles. MuSK agonist antibody #13 also significantly increased the muscle cross-sectional area and myofiber numbers in these denervation-vulnerable muscles but not in denervation-resistant muscles. Although MuSK agonist antibody #13 did not affect the body weight, our study suggests that preservation of NMJ innervation by the activation of MuSK may serve as a complementary therapy to SMN-enhancing drugs to maximize the therapeutic effectiveness for all types of SMA patients.

Ankyrin-dependent Na+ channel clustering prevents neuromuscular synapse fatigue

Skeletal muscle contraction depends on activation of clustered acetylcholine receptors (AchRs) and muscle-specific Na+ channels (Nav1.4). Some Nav1.4 channels are highly enriched at the neuromuscular junction (NMJ), and their clustering is thought to be essential for effective muscle excitation. However, this has not been experimentally tested, and how NMJ Na+ channels are clustered is unknown. Here, using muscle-specific ankyrinR, ankyrinB, and ankyrinG single, double, and triple-conditional knockout mice, we show that Nav1.4 channels fail to cluster only after deletion of all three ankyrins. Remarkably, ankyrin-deficient muscles have normal NMJ morphology, AchR clustering, sarcolemmal levels of Nav1.4, and muscle force, and they show no indication of degeneration. However, mice lacking clustered NMJ Na+ channels have significantly reduced levels of motor activity and their NMJs rapidly fatigue after repeated nerve-dependent stimulation. Thus, the triple redundancy of ankyrins facilitates NMJ Na+ channel clustering to prevent neuromuscular synapse fatigue.

The HSPG syndecan is a core organizer of cholinergic synapses.

The extracellular matrix has emerged as an active component of chemical synapses regulating synaptic formation, maintenance, and homeostasis. The heparan sulfate proteoglycan (HSPG) syndecans are known to regulate cellular and axonal migration in the brain. They are also enriched at synapses, but their synaptic functions remain more elusive. Here, we show that SDN-1, the sole orthologue of syndecan in C. elegans, is absolutely required for the synaptic clustering of homomeric α7-like acetylcholine receptors (AChRs) and regulates the synaptic content of heteromeric AChRs. SDN-1 is concentrated at neuromuscular junctions (NMJs) by the neurally secreted synaptic organizer Ce-Punctin/MADD-4, which also activates the transmembrane netrin receptor DCC. Those cooperatively recruit the FARP and CASK orthologues that localize α7-like-AChRs at cholinergic NMJs through physical interactions. Therefore, SDN-1 stands at the core of the cholinergic synapse organization by bridging the extracellular synaptic determinants to the intracellular synaptic scaffold that controls the postsynaptic receptor content.