AChR Clustering Research Articles

Free essential amino acid feeding improves endurance during resistance training via DRP1-dependent mitochondrial remodelling.

Loss of muscle strength and endurance with aging or in various conditions negatively affects quality of life. Resistance exercise training (RET) is the most powerful means to improve muscle mass and strength, but it does not generally lead to improvements in endurance capacity. Free essential amino acids (EAAs) act as precursors and stimuli for synthesis of both mitochondrial and myofibrillar proteins that could potentially confer endurance and strength gains. Thus, we hypothesized that daily consumption of a dietary supplement of nine free EAAs with RET improves endurance in addition to the strength gains by RET. Male C57BL6J mice (9weeks old) were assigned to control (CON), EAA, RET (ladder climbing, 3 times a week), or combined treatment of EAA and RET (EAA+RET) groups. Physical functions focusing on strength or endurance were assessed before and after the interventions. Several analyses were performed to gain better insight into the mechanisms by which muscle function was improved. We determined cumulative rates of myofibrillar and mitochondrial protein synthesis using 2H2O labelling and mass spectrometry; assessed ex vivo contractile properties and in vitro mitochondrial function, evaluated neuromuscular junction (NMJ) stability, and assessed implicated molecular singling pathways. Furthermore, whole-body and muscle insulin sensitivity along with glucose metabolism, were evaluated using a hyperinsulinaemic-euglycaemic clamp. EAA+RET increased muscle mass (10%, P<0.05) and strength (6%, P<0.05) more than RET alone, due to an enhanced rate of integrated muscle protein synthesis (19%, P<0.05) with concomitant activation of Akt1/mTORC1 signalling. Muscle quality (muscle strength normalized to mass) was improved by RET (i.e., RET and EAA+RET) compared with sedentary groups (10%, P<0.05), which was associated with increased AchR cluster size and MuSK activation (P<0.05). EAA+RET also increased endurance capacity more than RET alone (26%, P<0.05) by increasing both mitochondrial protein synthesis (53%, P<0.05) and DRP1 activation (P<0.05). Maximal respiratory capacity increased (P<0.05) through activation of the mTORC1-DRP1 signalling axis. These favourable effects were accompanied by an improvement in basal glucose metabolism (i.e., blood glucose concentrations and endogenous glucose production vs. CON, P<0.05). Combined treatment with balanced free EAAs and RET may effectively promote endurance capacity as well as muscle strength through increased muscle protein synthesis, improved NMJ stability, and enhanced mitochondrial dynamics via mTORC1-DRP1 axis activation, ultimately leading to improved basal glucose metabolism.

Rapsyn overexpression improves recovery of the neuromuscular junction from tourniquet-induced ischemia-reperfusion injury in mouse hindlimb

The neuromuscular junction (NMJ) is an important factor to influence skeletal muscle contractile function. Tourniquet-induced ischemia-reperfusion (IR) results in the fragmentation of nicotinic acetylcholine receptors (nAChR) clusters in the NMJ and impairs neural controlled skeletal muscle contraction. Rapsyn, a cytoplasmic nAChR-associated protein concentrates and anchors nAChRs in the postsynaptic membrane and participates in the nAChR assembly at NMJ. Here, we observed the effect of overexpression of Rapsyn on morphological and functional recovery of the NMJ from tourniquet-induced IR injuries in mouse hindlimb. Unilateral hindlimb of C57/BL6 mice were subjected to 3 hours of ischemia by placing a rubber band and followed 6 weeks of reperfusion. Ad-m-RAPSN (5.2 x 1010 PFU/ml) was injected into gastrocnemius muscle (50 μl) by Hamilton microliter syringe after 2 weeks of tourniquet-induced IR. After 6 weeks of IR, the sciatic nerve-stimulated gastrocnemius muscle contraction and endplate potential (EPP) were recorded in in-situ muscle samples; the NMJs were labelled with alpha-bungarotoxin (BTX) and nerofilment-200 (NF-200) + synaptophysin (Syn) antibodies, and NMJ images were captured with Zeiss LSM 800. After 6 weeks of IR, NMJs in the gastrocnemius muscle showed significantly fragmented nAChR clusters (9.3 ± 0.7 vs. sham 1.7 ± 0.1 for numbers of fragments) with low rapsyn expression, and the EPP (15.29 ± 0.93 mV vs. sham 26.87 ± 0.89 mV) and nerve-stimulated muscle contraction (117.2 ± 8.5 g vs. sham 157.9 ± 4.9 g) were reduced. Ad-m-RAPSN injection increased rapsyn expression, mitigated fragmentation of nAChR clusters (3.2 ± 0.3), increased the EPP (20.35 ± 0.86) and nerve-stimulated muscle contraction (138.1 ± 3.5 g). The data suggest that tourniquet-related IR induces AChR cluster fragmentation and impairs NMJ function, and rapsyn overexpression improves the morphological and functional recovery of the NMJ from tourniquet-induced IR injuries.. This research is supported by the NIH/NIGMS 1R01GM145736 to Y-LL. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

IgG1-3 MuSK Antibodies Inhibit AChR Cluster Formation, Restored by SHP2 Inhibitor, Despite Normal MuSK, DOK7, or AChR Subunit Phosphorylation.

Up to 50% of patients with myasthenia gravis (MG) without acetylcholine receptor antibodies (AChR-Abs) have antibodies to muscle-specific kinase (MuSK). Most MuSK antibodies (MuSK-Abs) are IgG4 and inhibit agrin-induced MuSK phosphorylation, leading to impaired clustering of AChRs at the developing or mature neuromuscular junction. However, IgG1-3 MuSK-Abs also exist in MuSK-MG patients, and their potential mechanisms have not been explored fully. C2C12 myotubes were exposed to MuSK-MG plasma IgG1-3 or IgG4, with or without purified agrin. MuSK, Downstream of Kinase 7 (DOK7), and βAChR were immunoprecipitated and their phosphorylation levels identified by immunoblotting. Agrin and agrin-independent AChR clusters were measured by immunofluorescence and AChR numbers by binding of 125I-α-bungarotoxin. Transcriptomic analysis was performed on treated myotubes. IgG1-3 MuSK-Abs impaired AChR clustering without inhibiting agrin-induced MuSK phosphorylation. Moreover, the well-established pathway initiated by MuSK through DOK7, resulting in βAChR phosphorylation, was not impaired by MuSK-IgG1-3 and was agrin-independent. Nevertheless, the AChR clusters did not form, and both the number of AChR microclusters that precede full cluster formation and the myotube surface AChRs were reduced. Transcriptomic analysis did not throw light on the pathways involved. However, the SHP2 inhibitor, NSC-87877, increased the number of microclusters and led to fully formed AChR clusters. MuSK-IgG1-3 is pathogenic but seems to act through a noncanonical pathway. Further studies should throw light on the mechanisms involved at the neuromuscular junction.

Myasthenia Gravis: Types, Diagnosis and Treatment

The classic autoimmune disease, myasthenia gravis (MG), is brought on by certain autoantibodies at the neuromuscular junction. A classic illustration of an antibody-mediated autoimmune illness is MG. Autoantibodies against the acetylcholine receptors are present in the majority of MG patients (AChRs). Muscle-specific kinase (MuSK), low-density lipoprotein receptor-related protein 4 (Lrp4), and agrin are among the less frequently found autoantibodies. The downregulation, destruction, functional blockage, or disruption of AChR clustering in the postsynaptic membrane are all ways in which these autoantibodies interfere with cholinergic transmission between nerve terminals and muscle fibres. Fatigable muscle weakness, which may affect the ocular, bulbar, respiratory, and limb muscles, is the primary clinical symptom of MG. Depending on the autoantibody's kind and whether a thymoma is present, there are different clinical symptoms. By changing the immune homeostasis processes that stop the onset of autoimmune disorders like MG, a number of medications, including immune checkpoint inhibitors, penicillamine, tyrosine kinase inhibitors, and interferons, may cause de novo MG. Keywords: myasthenia, gravis, diagnosis, treatment

DOK7 Promotes NMJ Regeneration After Nerve Injury.

Motor function recovery from injury requires the regeneration of not only muscle fibers, but also the neuromuscular junction-the synapse between motor nerve terminals and muscle fibers. However, unlike muscle regeneration which has been extensively studied, little is known about the molecular mechanisms of NMJ regeneration. Recognizing the critical role of agrin-LRP4-MuSK signaling in NMJ formation and maintenance, we investigated whether increasing MuSK activity promotes NMJ regeneration. To this end, we evaluated the effect of DOK7, a protein that stimulates MuSK, on NMJ regeneration. Reinnervation, AChR cluster density, and endplate area were improved, and fragmentation was reduced in the AAV9-DOK7-GFP-injected muscles compared with muscles injected with AAV9-GFP. These results demonstrated expedited NMJ regeneration associated with increased DOK7 expression and support the hypothesis that increasing agrin signaling benefits motor function recovery after injury. Our findings propose a potentially new therapeutic strategy for functional recovery after muscle and nerve injury, i.e., promoting NMJ regeneration by increasing agrin signaling.

MiR-142a-3p: A novel ACh receptor transcriptional regulator in association with peripheral nerve injury

Long-term denervation leads to the disintegration of nicotinic acetylcholine receptor (nAChR) located at the endplate structure, which translates to deficits in functional activation despite nerve repair. Because of a lack of effective measures to protect AChR expression, we explored the effect of alterations in muscular miR-142a-3p on nAChR. In this study, we constructed a model of miR-142a-3p knockdown by transfecting a miR-142a-3p inhibitor short hairpin RNA (shRNA) into C2C12 myotubes, and we injected this miR-142a-3p inhibitor shRNA into the tibialis anterior (TA) muscle in uninjured mice and in denervated mice by transecting the sciatic nerve. Our results showed that miR-142a-3p knockdown led to an increased number and area of AChR clusters in myotubes invitro and larger neuromuscular endplates in adult mice. Furthermore, miR-142a-3p knockdown delayed the disintegration of motor endplates after denervation. Last, upon miR-142a-3p knockdown in uninjured and denervated mice, we observed an increase in the mRNA levels of five AChR subunits as well as mRNAs of genes implicated in AChR transcription and AChR clustering. Together, these results suggest that miR-142a-3p may be a potential target for therapeutic intervention to prevent motor endplate degradation following peripheral nerve injury.

Severe congenital myasthenic syndromes caused by agrin mutations affecting secretion by motoneurons

Congenital myasthenic syndromes (CMS) are predominantly characterized by muscle weakness and fatigability and can be caused by a variety of mutations in genes required for neuromuscular junction formation and maintenance. Among them, AGRN encodes agrin, an essential synaptic protein secreted by motoneurons. We have identified severe CMS patients with uncharacterized p.R1671Q, p.R1698P and p.L1664P mutations in the LG2 domain of agrin. Overexpression in primary motoneurons cultures in vitro and in chick spinal motoneurons in vivo revealed that the mutations modified agrin trafficking, leading to its accumulation in the soma and/or in the axon. Expression of mutant agrins in cultured cells demonstrated accumulation of agrin in the endoplasmic reticulum associated with induction of unfolded protein response (UPR) and impaired secretion in the culture medium. Interestingly, evaluation of the specific activity of individual agrins on AChR cluster formation indicated that when secreted, mutant agrins retained a normal capacity to trigger the formation of AChR clusters. To confirm agrin accumulation and secretion defect, iPS cells were derived from a patient and differentiated into motoneurons. Patient iPS-derived motoneurons accumulated mutant agrin in the soma and increased XBP1 mRNA splicing, suggesting UPR activation. Moreover, co-cultures of patient iPS-derived motoneurons with myotubes confirmed the deficit in agrin secretion and revealed a reduction in motoneuron survival. Altogether, we report the first mutations in AGRN gene that specifically affect agrin secretion by motoneurons. Interestingly, the three patients carrying these mutations were initially suspected of spinal muscular atrophy (SMA). Therefore, in the presence of patients with a clinical presentation of SMA but without mutation in the SMN1 gene, it can be worth to look for mutations in AGRN.

Skeletal muscle sympathetic denervation disrupts the neuromuscular junction postterminal organization: A single-cell quantitative approach

The sympathetic nervous system (SNS) regulates skeletal muscle motor innervation and stabilizes the NMJ in health, disease and aging. Previous studies using both chemical (6-hydroxydopamine, 6-OHDA) and microsurgically-induced sympathetic denervation examined the NMJ organization and transmission in the mouse; however, a detailed quantification of the postterminal on larger hindlimb muscles involved in gait mechanics and posture is lacking. The purpose of this study was to determine whether targets of the sympathetic neuron (SN) exhibiting different intrinsic composition such as the fast-twitch extensor digitorum longus (EDL) and the slow-twitch soleus muscles differ in their response to SN deprivation, and to develop a strategy to accurately quantify the impact of sympathectomy on the NMJ postterminal including those fibers located deeper in the muscle. This approach included muscle fixed ex vivo or through transcardial perfusion in mice treated with 6-OHDA or control ascorbic acid. We measured NMJ postterminal mean terminal total area, number of postterminal fragments, mean fragment area, and mean distance between fragments in free-floating alpha-bungarotoxin-stained in 1038 isolated muscle fibers. We found that muscle fiber sympathetic innervation plays a crucial role in the structural organization of the motorneuron-myofiber synapse postterminal and its deprivation leads to AChR cluster dispersion or shrinking as described in various neuromuscular diseases and aging.

Counteractive and cooperative actions of muscle β-catenin and CaV1.1 during early neuromuscular synapse formation

SummaryActivity-dependent calcium signals in developing muscle play a crucial role in neuromuscular junction (NMJ) formation. However, its downstream effectors and interactions with other regulators of pre- and postsynaptic differentiation are poorly understood. Here, we demonstrate that the skeletal muscle calcium channel CaV1.1 and β-catenin interact in various ways to control NMJ development. They differentially regulate nerve branching and presynaptic innervation patterns during the initial phase of NMJ formation. Conversely, they cooperate in regulating postsynaptic AChR clustering, synapse formation, and the proper organization of muscle fibers in mouse diaphragm. CaV1.1 does not directly regulate β-catenin expression but differentially controls the activity of its transcriptional co-regulators TCF/Lef and YAP. These findings suggest a crosstalk between CaV1.1 and β-catenin in the activity-dependent transcriptional regulation of genes involved in specific pre- and postsynaptic aspects of NMJ formation.

Involvement of neuronal and muscular Trk-fused gene (TFG) defects in the development of neurodegenerative diseases

Trk-fused gene (TFG) mutations have been identified in patients with several neurodegenerative diseases. In this study, we attempted to clarify the effects of TFG deletions in motor neurons and in muscle fibers, using tissue-specific TFG knockout (vMNTFG KO and MUSTFG KO) mice. vMNTFG KO, generated by crossing TFG floxed with VAChT-Cre, showed deterioration of motor function and muscle atrophy especially in slow-twitch soleus muscle, in line with the predominant Cre expression in slow-twitch fatigue-resistant (S) and fast-twitch fatigue-resistant (FR) motor neurons. Consistently, denervation of the neuromuscular junction (NMJ) was apparent in the soleus, but not in the extensor digitorum longus, muscle. Muscle TFG expressions were significantly downregulated in vMNTFG KO, presumably due to decreased muscle IGF-1 concentrations. However, interestingly, MUSTFG KO mice showed no apparent impairment of muscle movements, though a denervation marker, AChRγ, was elevated and Agrin-induced AChR clustering in C2C12 myotubes was inhibited. Our results clarify that loss of motor neuron TFG is sufficient for the occurrence of NMJ degeneration and muscle atrophy, though lack of muscle TFG may exert an additional effect. Reduced muscle TFG, also observed in aged mice, might be involved in age-related NMJ degeneration, and this issue merits further study.

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.

Characterization of LRP4/Agrin Antibodies From a Patient With Myasthenia Gravis.

To determine whether human anti-LRP4/agrin antibodies are pathogenic in mice and to investigate underpinning pathogenic mechanisms. Immunoglobulin (Ig) was purified from a patient with myasthenia gravis (MG) with anti-LRP4/agrin antibodies and transferred to mice. Mice were characterized for body weight, muscle strength, twitch and tetanic force, neuromuscular junction (NMJ) functions including compound muscle action potential (CMAP) and endplate potentials, and NMJ structure. Effects of the antibodies on agrin-elicited muscle-specific tyrosine kinase (MuSK) activation and AChR clustering were studied and the epitopes of these antibodies were identified. Patient Ig-injected mice had MG symptoms, including weight loss and muscle weakness. Decreased CMAPs, reduced twitch and tetanus force, compromised neuromuscular transmission, and NMJ fragmentation and distortion were detected in patient Ig-injected mice. Patient Ig inhibited agrin-elicited MuSK activation and AChR clustering. The patient Ig recognized the β3 domain of LRP4 and the C-terminus of agrin and reduced agrin-enhanced LRP4-MuSK interaction. Anti-LRP4/agrin antibodies in the patient with MG is pathogenic. It impairs the NMJ by interrupting agrin-dependent LRP4-MuSK interaction.

Congenital myasthenic syndrome-associated agrin variants affect clustering of acetylcholine receptors in a domain-specific manner.

Congenital myasthenic syndromes (CMS) are caused by mutations in molecules expressed at the neuromuscular junction. We report clinical, structural, ultrastructural, and electrophysiologic features of 4 CMS patients with 6 heteroallelic variants in AGRN, encoding agrin. One was a 7.9-kb deletion involving the N-terminal laminin-binding domain. Another, c.4744G>A - at the last nucleotide of exon 26 - caused skipping of exon 26. Four missense mutations (p.S1180L, p.R1509W, p.G1675S, and p.Y1877D) expressed in conditioned media decreased AChR clusters in C2C12 myotubes. The agrin-enhanced phosphorylation of MuSK was markedly attenuated by p.Y1877D in the LG3 domain and moderately attenuated by p.R1509W in the LG1 domain but not by the other 2 mutations. The p.S1180L mutation in the SEA domain facilitated degradation of secreted agrin. The p.G1675S mutation in the LG2 domain attenuated anchoring of agrin to the sarcolemma by compromising its binding to heparin. Anchoring of agrin with p.R1509W in the LG1 domain was similarly attenuated. Mutations of agrin affect AChR clustering by enhancing agrin degradation or by suppressing MuSK phosphorylation and/or by compromising anchoring of agrin to the sarcolemma of the neuromuscular junction.

Identification of the downstream molecules of agrin/Dok-7 signaling in muscle.

The development of the neuromuscular junction depends on signaling processes that involve protein phosphorylation. Motor neuron releases agrin to activate muscle protein Dok-7, a key tyrosine kinase essential for the formation of a mature and functional neuromuscular junction. However, the signaling cascade downstream of Dok-7 remains poorly understood. In this study, we combined the clustered regularly interspaced short palindromic repeats/Cas9 technique and quantitative phosphoproteomics analysis to study the tyrosine phosphorylation events triggered by agrin/Dok-7. We found tyrosine phosphorylation level of 36 proteins increased specifically by agrin stimulation. In Dok-7 mutant myotubes, however, 13 of the 36 proteins failed to be enhanced by agrin stimulation, suggesting that these 13 proteins are Dok-7-dependent tyrosine-phosphorylated proteins, could work as downstream molecules of agrin/Dok-7 signaling. We validated one of the proteins, Anxa3, by in vitro and in vivo assays. Knocking down of Anxa3 in the cultured myotubes inhibited agrin-induced AChR clustering, whereas reduction of Anxa3 in mouse muscles induced abnormal postsynaptic development. Collectively, our phosphoproteomics analysis provides novel insights into the complicated signaling network downstream of agrin/Dok-7.

Evaluation of a conducting elastomeric composite material for intramuscular electrode application

Electrical stimulation of the muscle has been proven efficacious in preventing atrophy and/or reanimating paralyzed muscles. Intramuscular electrodes made from metals have significantly higher Young's Moduli than the muscle tissues, which has the potential to cause chronic inflammation and decrease device performance. Here, we present an intramuscular electrode made from an elastomeric conducting polymer composite consisting of PEDOT-PEG copolymer, silicone and carbon nanotubes (CNT) with fluorosilicone insulation. The electrode wire has a Young's modulus of 804 (±99) kPa, which better mimics the muscle tissue modulus than conventional stainless steel (SS) electrodes. Additionally, the non-metallic composition enables metal-artifact free CT and MR imaging. These soft wire (SW) electrodes present comparable electrical impedance to SS electrodes of similar geometric surface area, activate muscle at a lower threshold, and maintain stable electrical properties in vivo up to 4 weeks. Histologically, the SW electrodes elicited significantly less fibrotic encapsulation and less IBA-1 positive macrophage accumulation than the SS electrodes at one and three months. Further phenotyping the macrophages with the iNOS (pro-inflammatory) and ARG-1 (pro-healing) markers revealed significantly less presence of pro-inflammatory macrophage around SW implants at one month. By three months, there was a significant increase in pro-healing macrophages (ARG-1) around the SW implants but not around the SS implants. Furthermore, a larger number of AchR clusters closer to SW implants were found at both time points compared to SS implants. These results suggest that a softer implant encourages a more intimate and healthier electrode-tissue interface. Statement of significanceIntramuscular electrodes made from metals have significantly higher Young's Moduli than the muscle tissues, which has the potential to cause chronic inflammation and decrease device performance. Here, we present an intramuscular electrode made from an elastomeric conducting polymer composite consisting of PEDOT-PEG copolymer, silicone and carbon nanotubes with fluorosilicone insulation. This elastomeric composite results in an electrode wire with a Young's modulus mimicking that of the muscle tissue, which elicits significantly less foreign body response compared to stainless steel wires. The lack of metal in this composite also enables metal-artifact free MRI and CT imaging.

SHP2 inhibitor protects AChRs from effects of myasthenia gravis MuSK antibody

ObjectiveTo determine whether an SRC homology 2 domain-containing phosphotyrosine phosphatase 2 (SHP2) inhibitor would increase muscle-specific kinase (MuSK) phosphorylation and override the inhibitory effect of MuSK-antibodies (Abs).MethodsThe effect of the SHP2 inhibitor NSC-87877 on MuSK phosphorylation and AChR clustering was tested in C2C12 myotubes with 31 MuSK-myasthenia gravis (MG) sera and purified MuSK-MG IgG4 preparations.ResultsIn the absence of MuSK-MG Abs, NSC-87877 increased MuSK phosphorylation and the number of AChR clusters in C2C12 myotubes in vitro and in DOK7-overexpressing C2C12 myotubes that form spontaneous AChR clusters. In the presence of MuSK-MG sera, the AChR clusters were reduced, as expected, but NSC-87877 was able to protect or restore the clusters. Two purified MuSK-MG IgG4 preparations inhibited both MuSK phosphorylation and AChR cluster formation, and in both, clusters were restored with NSC-87877.ConclusionsStimulating the agrin-LRP4-MuSK-DOK7 AChR clustering pathway with NSC-87877, or other drugs, could represent a novel therapeutic approach for MuSK-MG and could potentially improve other NMJ disorders with reduced AChR numbers or disrupted NMJs.

Postsynaptic CaV1.1-driven calcium signaling coordinates presynaptic differentiation at the developing neuromuscular junction

Proper formation of neuromuscular synapses requires the reciprocal communication between motor neurons and muscle cells. Several anterograde and retrograde signals involved in neuromuscular junction formation are known. However the postsynaptic mechanisms regulating presynaptic differentiation are still incompletely understood. Here we report that the skeletal muscle calcium channel (CaV1.1) is required for motor nerve differentiation and that the mechanism by which CaV1.1 controls presynaptic differentiation utilizes activity-dependent calcium signaling in muscle. In mice lacking CaV1.1 or CaV1.1-driven calcium signaling motor nerves are ectopically located and aberrantly defasciculated. Axons fail to recognize their postsynaptic target structures and synaptic vesicles and active zones fail to correctly accumulate at the nerve terminals opposite AChR clusters. These presynaptic defects are independent of aberrant AChR patterning and more sensitive to deficient calcium signals. Thus, our results identify CaV1.1-driven calcium signaling in muscle as a major regulator coordinating multiple aspects of presynaptic differentiation at the neuromuscular synapse.

Podosome-directed MT1-MMP trafficking and surface insertion regulate AChR clustering & remodeling

Podosome-directed MT1-MMP trafficking and surface insertion regulate AChR clustering & remodeling

α−Calcitonin gene-related peptide inhibits autophagy and calpain systems and maintains the stability of neuromuscular junction in denervated muscles

ObjectiveAlthough it is well established that a-calcitonin gene-related peptide (CGRP) stabilizes muscle-type cholinergic receptors nicotinic subunits (AChR), the underlying mechanism by which this neuropeptide regulates muscle protein metabolism and neuromuscular junction (NMJ) morphology is unclear. MethodsTo elucidate the mechanisms how CGRP controls NMJ stability in denervated mice skeletal muscles, we carried out physiological, pharmacological, and molecular analyses of atrophic muscles induced by sciatic nerve transection. ResultsHere, we report that CGRP treatment in vivo abrogated the deleterious effects on NMJ upon denervation (DEN), an effect that was associated with suppression of skeletal muscle proteolysis, but not stimulation of protein synthesis. CGRP also blocked the DEN-induced increase in endocytic AChR vesicles and the elevation of autophagosomes per NMJ area. The treatment of denervated animals with rapamycin blocked the stimulatory effects of CGRP on mTORC1 and its inhibitory actions on autophagic flux and NMJ degeneration. Furthermore, CGRP inhibited the DEN-induced hyperactivation of Ca2+-dependent proteolysis, a degradative system that has been shown to destabilize NMJ. Consistently, calpain was found to be activated by cholinergic stimulation in myotubes leading to the dispersal of AChR clusters, an effect that was abolished by CGRP. ConclusionTaken together, these data suggest that the inhibitory effect of CGRP on autophagy and calpain may represent an important mechanism for the preservation of synapse morphology when degradative machinery is exacerbated upon denervation conditions.

I.2New genes and better treatment for congenital myasthenic syndromes

The congenital myasthenic syndromes (CMS) are rare inherited disorders of neuromuscular transmission characterised by fatiguable muscle weakness. Their overall prevalence is uncertain but is thought to be in the order of 1 in 100 000 of the population in the UK. They are genetically determined (usually autosomal recessive - so a history of consanguinity is common), non-autoimmune disorders. Remarkable differences in severity occur even within families harboring the same mutation. Although impairment of neuromuscular transmission may often give rise to similar clinical presentation many distinct molecular and cellular mechanisms may be involved. It has become evident over the last ten years that impaired synaptic structure and stability are disease features of equal importance as defects in proteins directly involved in signal transmission. Understanding the molecular mechanisms that underlie impaired synaptic transmission helps guide appropriate and often life-transforming therapy. What has also become evident is that for forms of CMS in which synaptic structure is disrupted, treatment with β2-adrengic receptor agonists is highly effective. Cases where components of the AGRN-LRP4-MUSK-DOK7 AChR clustering pathway are mutated usually respond well to this form of medication. Moreover, in CMS patients and in animal models of CMS long term treatment with pyridostigmine may be detrimental to synaptic structure but can be partially alleviated by treatment with β2-adrengic receptor agonists. The benefit of β2-adrenergic agonists alone or combined with pyridostigmine or 3,4-Diaminopyridine is increasingly becoming an effective part of the repertoire for treating many different subtypes of CMS.