Acetylcholine Receptor Clusters Research Articles

Congenital myasthenic syndrome due to mutations in MUSK suggests that the level of MuSK phosphorylation is crucial for governing synaptic structure.

MUSK encodes the muscle‐specific receptor tyrosine kinase (MuSK), a key component of the agrin‐LRP4‐MuSK‐DOK7 signaling pathway, which is essential for the formation and maintenance of highly specialized synapses between motor neurons and muscle fibers. We report a patient with severe early‐onset congenital myasthenic syndrome and two novel missense mutations in MUSK (p.C317R and p.A617V). Functional studies show that MUSK p.C317R, located at the frizzled‐like cysteine‐rich domain of MuSK, disrupts an integral part of MuSK architecture resulting in ablated MuSK phosphorylation and acetylcholine receptor (AChR) cluster formation. MUSK p.A617V, located at the kinase domain of MuSK, enhances MuSK phosphorylation resulting in anomalous AChR cluster formation. The identification and evidence for pathogenicity of MUSK mutations supported the initiation of treatment with β2‐adrenergic agonists with a dramatic improvement of muscle strength in the patient. This work suggests uncharacterized mechanisms in which control of the precise level of MuSK phosphorylation is crucial in governing synaptic structure.

Paediatric myasthenia gravis: Prognostic factors for drug free remission

Our aim was to identify clinical outcomes, serological features and possible prognostic indicators of paediatric myasthenia gravis (MG). We collected 74 MG patients with disease onset before the age of 16 years (73% pre-pubertal onset defined as ≤10 years), seen regularly at two UK specialist centres, over a period of 11 years. The cohort was multi-ethnic, with a high number of non-Caucasians (52%). Ocular presentation was seen in 38 (51%) and only 8 (21%) of these generalised. Fifty-two (70%) patients had antibodies to the acetylcholine receptor (AChR) measured by radioimmunoprecipitation, 10 (14%) had antibodies only to clustered AChRs detected by a cell based assay, 3 (4%) had muscle-specific kinase and one (1%) low-density lipoprotein receptor-related protein 4 antibody. Only 8 (11%) had no detectable antibodies. Seventeen patients attained drug free remission (Kaplan Meyer survival curve estimates 25% by 7 years). Several factors were associated with a higher likelihood of free remission: onset age ≤10 years, Asian and Caucasian races, lack of AChR antibodies on RIA, and normal repetitive nerve stimulation at diagnosis. However, in a multifactorial regression analysis, the antibody status was the only significant predictor for drug free remission, with 60% of patients with antibodies only to clustered AChR achieving this outcome. Complete drug free remission is not uncommon in paediatric MG and several factors appear to influence this outcome with antibody status being the most important. These factors can be easily evaluated at diagnosis, and may help to determine whose patients are likely to require more intensive treatments.

P.130MyoscreenTM, a drug discovery platform for disease modeling and the screening of compounds restoring muscle health

Effective treatments are missing for degenerative muscle disorders such as DMD because the current in vitro muscle pathological models lack physiology relevance and pharmacology predictivity. We recently reported MyoscreenTM human patient-derived skeletal myotubes that demonstrate sarcomeric organization, membrane-localized dystrophin and acetylcholine receptor (AChR) expression, response to chemical and electrical stimulation and pharmacologically relevant drug responses. The objective of the present study was to employ our extensive panel of patient derived myoblasts for disease modeling and assays pertinent to muscle biology such as myotube differentiation efficiency, AChR clustering, calcium homeostasis and contraction. To do so, a high-content screen for compounds that stimulate healthy myotube differentiation was performed. Hits were confirmed in several donors and potency determined in dose-response studies. Functional assays exploited calcium imaging techniques and the FDSS/µCELL from Hamamatsu for electrical stimulation. Out of 2560 pharmacologically active or FDA-approved drugs, 29 (1.1%) increased fusion index and/or myotube size by >40%. Molecules with previously established links to regeneration processes were identified (eg. thyroid hormone analogues), as well as CYT9733 that efficiently enhanced healthy and dystrophic myotube formation with an EC50 of 0.002-2µM. Other effects of CYT9733 include increased AChR clusters and intracellular calcium flux during excitation-contraction coupling upon ACh or electrical stimulation, intensifying myotube contraction. CYT9733 is an especially good candidate molecule for regulating changes in muscle regeneration phenotype and of further interest for preclinical testing. The results illustrate predictivity of this disease modeling and drug discovery platform, baptized MyoScreen, its capacity to select active compounds and provide early investigation of mechanisms of action involved.

Loss of Protein Kinase Csnk2b/CK2β at Neuromuscular Junctions Affects Morphology and Dynamics of Aggregated Nicotinic Acetylcholine Receptors, Neuromuscular Transmission, and Synaptic Gene Expression.

The protein kinase Csnk2/CK2 is important for cell proliferation, differentiation, and survival. Previously, we showed that CK2 binds distinctive proteins at neuromuscular junctions (NMJs) of mice and phosphorylates some of them. CK2 likely stabilizes clustered nicotinic acetylcholine receptors (AChRs). In the absence of the β-subunit of CK2 in skeletal muscle fibers, mice develop an age-dependent decrease of grip strength accompanied by NMJ fragmentation and impairments of neuromuscular transmission. However, the precise role of CK2β regarding the clustering of AChRs and downstream signaling at NMJs is unknown. Here, we compared conditional CK2β-deficient mice with controls and found in the mutants (1) a lower decrement of endplate potentials after repetitive stimulation and decrements of nerve-evoked compound muscle action potentials decayed more rapidly after synaptic transmission was partially blocked, (2) that their muscle weakness was partially rescued by administration of an acetylcholine esterase inhibitor, (3) fragmented NMJs and impaired AChR clustering was detected in muscles and cultured muscle cells, (4) enlarged myonuclei, (5) impaired synaptic gene expression, and (6) a high turnover rate of their AChR clusters in vivo. Altogether, our data demonstrate a role for CK2 at the NMJ by maintaining a high density of AChRs and ensuring physiological synaptic gene expression.

Loss of mitochondrial protein CHCHD10 in skeletal muscle causes neuromuscular junction impairment.

The neuromuscular junction (NMJ) is a synapse between motoneurons and skeletal muscles to control motor behavior. Acetylcholine receptors (AChRs) are restricted at the synaptic region for proper neurotransmission. Mutations in the mitochondrial CHCHD10 protein have been identified in multiple neuromuscular disorders; however, the physiological roles of CHCHD10 at NMJs remain elusive. Here, we report that CHCHD10 is highly expressed at the postsynapse of NMJs in skeletal muscles. Muscle conditional knockout CHCHD10 mice showed motor defects, abnormal neuromuscular transmission and NMJ structure. Mechanistically, we found that mitochondrial CHCHD10 is required for ATP production, which facilitates AChR expression and promotes agrin-induced AChR clustering. Importantly, ATP could effectively rescue the reduction of AChR clusters in the CHCHD10-ablated muscles. Our study elucidates a novel physiological role of CHCHD10 at the peripheral synapse. It suggests that mitochondria dysfunction contributes to neuromuscular pathogenesis.

Characterization of pathogenic monoclonal autoantibodies derived from muscle-specific kinase myasthenia gravis patients.

Myasthenia gravis (MG) is a chronic autoimmune disorder characterized by muscle weakness and caused by pathogenic autoantibodies that bind to membrane proteins at the neuromuscular junction. Most patients have autoantibodies against the acetylcholine receptor (AChR), but a subset of patients have autoantibodies against muscle-specific tyrosine kinase (MuSK) instead. MuSK is an essential component of the pathway responsible for synaptic differentiation, which is activated by nerve-released agrin. Through binding MuSK, serum-derived autoantibodies inhibit agrin-induced MuSK autophosphorylation, impair clustering of AChRs, and block neuromuscular transmission. We sought to establish individual MuSK autoantibody clones so that the autoimmune mechanisms could be better understood. We isolated MuSK autoantibody-expressing B cells from 6 MuSK MG patients using a fluorescently tagged MuSK antigen multimer, then generated a panel of human monoclonal autoantibodies (mAbs) from these cells. Here we focused on 3 highly specific mAbs that bound quantitatively to MuSK in solution, to MuSK-expressing HEK cells, and at mouse neuromuscular junctions, where they colocalized with AChRs. These 3 IgG isotype mAbs (2 IgG4 and 1 IgG3 subclass) recognized the Ig-like domain 2 of MuSK. The mAbs inhibited AChR clustering, but intriguingly, they enhanced rather than inhibited MuSK phosphorylation, which suggests an alternative mechanism for inhibiting AChR clustering.

Rapsyn facilitates recovery from desensitization in fetal and adult acetylcholine receptors expressed in a muscle cell line.

Key points The physiological significance of the developmental switch from fetal to adult acetylcholine receptors in muscle (AChRs) and the functional impact of AChR clustering by rapsyn are not well studied.Using patch clamp experiments, we show that recovery from desensitization is faster in the adult AChR isoform.Recovery from desensitization is determined by the AChR isoform‐specific cytoplasmic M3–M4 domain.The co‐expression of rapsyn in muscle cells induced AChR clustering and facilitated recovery from desensitization in both fetal and adult AChRs. In fetal AChRs, facilitation of recovery kinetics by rapsyn was independent of AChR clustering.These effects could be crucial adaptations to motor neuron firing rates, which, in rodents, have been shown to increase around the time of birth when AChRs cluster at the developing neuromuscular junctions. The neuromuscular junction (NMJ) is the site of a number of autoimmune and genetic disorders, many involving the muscle‐type nicotinic acetylcholine receptor (AChR), although there are aspects of normal NMJ development and function that need to be better understood. In particular, there are still questions regarding the implications of the developmental switch from fetal to adult AChRs, as well as how their functions might be modified by rapsyn that clusters the AChRs. Desensitization of human muscle AChRs was investigated using the patch clamp technique to measure whole‐cell currents in muscle‐type (TE671/CN21) and non‐muscle (HEK293) cell lines expressing either fetal or adult AChRs. Desensitization time constants were similar with both AChR isoforms but recovery time constants were shorter in cells expressing adult compared to fetal AChRs (P < 0.0001). Chimeric experiments showed that recovery from desensitization was determined by the M3–M4 cytoplasmic loops of the γ‐ and ε‐subunits. Expression of rapsyn in TE671/CN21 cells induced AChR aggregation and also, surprisingly, shortened recovery time constants in both fetal and adult AChRs. However, this was not dependent on clustering because rapsyn also facilitated recovery from desensitization in HEK293 cells expressing a δ‐R375H AChR mutant that did not form clusters in C2C12 myotubes. Thus, rapsyn interactions with AChRs lead not only to clustering, but also to a clustering independent faster recovery from desensitization. Both effects of rapsyn could be a necessary adjustment to the motor neuron firing rates that increase around the time of birth.

Development of a mouse nerve-transfer model for brachial plexus injury.

Nerve transfer involves the use of a portion of a healthy nerve to repair an injured nerve, and the process has been used to alleviate traumatic brachial plexus injuries in humans. Study of the neural mechanisms that occur during nerve transfer, however, requires the establishment of reliable experimental models. In this study, we developed an ulnar-musculocutaneous nerve-transfer model wherein the biceps muscle of a mouse was re-innervated using a donor ulnar nerve. Similar muscle action potentials were detected in both the end-to-end suture of the transected nerve (correctrepair) group and the ulnar-musculocutaneous nerve-transfer group. Also, re-innervated acetylcholine receptor (AChR) clusters and muscle spindles were observed in both procedures. There were fewer re-innervated AChR clusters in the nerve transfer group than in the correct repair group at 4 weeks, but the numbers were equal at 24 weeks following surgery. Thus, our ulnar-musculocutaneous nerve-transfer model allowed physiological and morphological evaluation for re-innervation process in mice and revealed the delay of this process during nerve transfer procedure. This model will provide great opportunities to study regeneration, re-innervation, and functional recovery induced via nerve transfer procedures.

Differentiation of Motor Neuron-Like Cells from Tonsil-Derived Mesenchymal Stem Cells and Their Possible Application to Neuromuscular Junction Formation.

Human tonsil-derived mesenchymal stem cells (T-MSCs) are newly identified MSCs and present typical features of MSCs, including having the differentiation capacity into the three germ layers and excellent proliferation capacity. They are easily sourced and are useful for stem cell therapy in various disease states. We previously reported that T-MSCs could be differentiated into skeletal myocytes and Schwann-like cells; therefore, they are a promising candidate for cell therapies for neuromuscular disease. Motor neurons (MNs), which regulate spontaneous behavior, are affected by a wide range of MN diseases (MNDs) for which there are no effective remedies. We investigated the differentiation potential of MN-like cells derived from T-MSCs (T-MSC-MNCs) for application to therapy of MNDs. After the process of MN differentiation, the expression of MN-related markers, including Islet 1, HB9/HLXB9 (HB9), and choline acetyltransferase (ChAT), was increased when compared with undifferentiated T-MSCs. The secretion of acetylcholine to the conditioned medium was significantly increased after MN differentiation. We cocultured T-MSC-MNCs and human skeletal muscle cells, and confirmed the presence of the acetylcholine receptor clusters, which demonstrated the formation of neuromuscular junctions. The potential functional improvements afforded by these T-MSC-MNCs could be useful in the treatment of MNDs caused by genetic mutation, viral infection, or environmental problems.

Efficient gene transfer into primary muscle cells to analyze nerve-independent postsynaptic organization in vitro

Acetylcholine receptor (AChR) clustering on the surface of muscle cells is a hallmark of postsynaptic differentiation at the vertebrate neuromuscular junction (NMJ). Even though the assembly of complex postsynaptic apparatuses is known to rely on both, pre- and postsynaptic signals, the identity of muscle-derived proteins modulating postsynaptic assembly and maintenance is still to be fully elucidated. Efficient gene transfer into muscle cells represents a powerful tool to analyze the contribution of muscle proteins on postsynaptic assembly and maintenance. Here, we describe a protocol that combines efficient electroporation of primary muscle satellite cells with the formation of aneural complex postsynaptic structures on the surface of myotubes. In vitro formed postsynaptic structures share various similarities with in vivo postsynaptic NMJ domains. While primary myotubes express increasing amounts of the ε AChR subunit, associated with NMJ maturation, surface AChR aggregates lack this AChR subunit. Our results also validate the functional expression of a luciferase reporter gene, as well as the response of complex postsynaptic structures to pharmacological treatment. Together, these methods in primary muscle cells are a valuable tool to perform a detailed and accurate analysis of the potential role of muscle-derived proteins on the maintenance of complex postsynaptic structures and to identify nerve-derived signals regulating functional NMJ maturation.

Cullin-3 dependent deregulation of ACTN1 represents a new pathogenic mechanism in nemaline myopathy.

Nemaline myopathy is a congenital neuromuscular disorder characterized by muscle weakness, fiber atrophy and presence of nemaline bodies within myofibers. However, the understanding of underlying pathomechanisms is lacking. Recently, mutations in KBTBD13, KLHL40 and KLHL41, three substrate adaptors for the E3-ubiquitin ligase Cullin-3, have been associated with early-onset nemaline myopathies. We hypothesized that deregulation of Cullin-3 and its muscle protein substrates may be responsible for the disease development. Using Cullin-3 knockout mice, we identified accumulation of non-muscle alpha-Actinins (ACTN1 and ACTN4) in muscles of these mice, which we also observed in KBTBD13 patients. Our data reveal that proper regulation of Cullin-3 activity and ACTN1 levels is essential for normal muscle and neuromuscular junction development. While ACTN1 is naturally downregulated during myogenesis, its overexpression in C2C12 myoblasts triggered defects in fusion, myogenesis and acetylcholine receptor clustering; features that we characterized in Cullin-3 deficient mice. Taken together, our data highlight the importance for Cullin-3 mediated degradation of ACTN1 for muscle development, and indicate a new pathomechanism for the etiology of myopathies seen in Cullin-3 knockout mice and nemaline myopathy patients.

The clinical spectrum of the congenital myasthenic syndrome resulting from COL13A1 mutations.

Next generation sequencing techniques were recently used to show mutations in COL13A1 cause synaptic basal lamina-associated congenital myasthenic syndrome type 19. Animal studies showed COL13A1, a synaptic extracellular-matrix protein, is involved in the formation and maintenance of the neuromuscular synapse that appears independent of the Agrin-LRP4-MuSK-DOK7 acetylcholine receptor clustering pathway. Here, we report the phenotypic spectrum of 16 patients from 11 kinships harbouring homozygous or heteroallelic mutations in COL13A1. Clinical presentation was mostly at birth with hypotonia and breathing and feeding difficulties often requiring ventilation and artificial feeding. Respiratory crisis related to recurrent apnoeas, sometimes triggered by chest infections, were common early in life but resolved over time. The predominant pattern of muscle weakness included bilateral ptosis (non-fatigable in adulthood), myopathic facies and marked axial weakness, especially of neck flexion, while limb muscles were less involved. Other features included facial dysmorphism, skeletal abnormalities and mild learning difficulties. All patients tested had results consistent with abnormal neuromuscular transmission. Muscle biopsies were within normal limits or showed non-specific changes. Muscle MRI and serum creatine kinase levels were normal. In keeping with COL13A1 mutations affecting both synaptic structure and presynaptic function, treatment with 3,4-diaminopyridine and salbutamol resulted in motor and respiratory function improvement. In non-treated cases, disease severity and muscle strength improved gradually over time and several adults recovered normal muscle strength in the limbs. In summary, patients with COL13A1 mutations present mostly with severe early-onset myasthenic syndrome with feeding and breathing difficulties. Axial weakness is greater than limb weakness. Disease course improves gradually over time, which could be consistent with the less prominent role of COL13A1 once the neuromuscular junction is mature. This report emphasizes the role of collagens at the human muscle endplate and should facilitate the recognition of this disorder, which can benefit from pharmacological treatment.

Establishment of efficient differentiation conditions from tonsil-derived mesenchymal stem cells to motor neuron cells

Background & Aim Tonsil-derived MSCs (T-MSCs) present typical features of MSCs, including the ability to differentiate into tissues of the three primary germ layers, and have excellent proliferation capacity and ease of source supply are useful for stem cell therapy in various disease states. Previously, we reported that T-MSCs can be differentiated into skeletal myocytes and Schwann-like cell, which are therefore a promising candidate in cell therapies for neuromuscular disease. Motor neurons are differentiated cells that control voluntary actions and are affected primarily by a wide spectrum of neurological disorders, generally indicated as motor neuron diseases (MNDs). MNDs may present with a range of symptoms deriving from muscular weakness/atrophy and leading to death, but no effective treatment exists for these illnesses. Therefore, we investigated the potential of motor neuron-like cells (MNCs) differentiated from T-MSCs for overcome of MNDs. Methods, Results & Conclusion After the MN differentiating in our own methods, we confirmed that the expression of MN-related markers, including ISL1, HB9, ChAT in T-MSC-MNCs has increased compared to T-MSCs. Also, to test their functional efficiency, the change of acetylcholine secretion in the culture medium under the differentiation into T-MSC-MNCs was measured. Furthermore, by confirming the acetylcholine receptor clusters when co-culturing T-MSC-MNCs and human skeletal muscle cells, which was demonstrated the T-MSC-MNCs could form neuromuscular junctions. The functional improvements afforded by these T-MSC-MNCs are potentially useful in the treatment of human MNDs caused by genetic, viral, and environmental problems.

False-positive acetylcholine receptor antibody results in patients without myasthenia gravis

Acetylcholine receptor antibodies are very specific for myasthenia. During a large prospective cohort study of myasthenia, we encountered five patients, positive for acetylcholine receptor (AChR) antibodies by radioimmunoprecipitation assay (RIA), whose clinical course revealed diagnoses other than myasthenia. Two patients had transiently raised AChR antibodies associated with Guillain-Barré syndrome. Antibodies to clustered AChRs, in a live cell-based assay, were negative in all five patients, suggesting that results from the RIAs were false-positives. It is possible that the AChR antibodies detected by RIA in these cases were non-pathogenic, and directed to intracellular epitopes of the AChR.

Dexamethasone Improves Neuromuscular Junction Recovery From Ischemia‐Reperfusion Injury Induced by Tourniquet Application in Mouse Hindlimb

Tourniquet‐induced ischemia‐reperfusion (IR) injuries impede long‐term recovery of skeletal muscle contraction. The neuromuscular junction (NMJ) is an important factor to influence skeletal muscle contractile function. Here, we observed the effect of dexamethasone (Dex) 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 different periods of reperfusion (1 week, 2 weeks, 4 weeks, and 6 weeks). Dex treatment (1 mg/kg/day, i.p.) began on the day of IR induction and lasted for 7 days. After 3 hours of ischemia and different periods of reperfusion, NMJs in the gastrocnemius muscle showed significantly the morphological and functional damage. In the gastrocnemius muscle, TNFα and IL‐1β (two inflammatory cytokines) were increased after 1, 2 and 6 weeks of tourniquet‐induced IR. Treatment with Dex inhibited the elevation of TNFα and IL‐1β, mitigated fragmentation of nicotinic acetylcholine receptor (nAChR) clusters, promoted re‐innervation of nAChR clusters, and increased nerve‐stimulated end plate potentials (EPPs).The data suggested that tourniquet‐related IR induced serious skeletal muscle inflammation for a long period. Dex treatment for 1 week inhibits IR‐caused inflammation in the early period of IR, and improves the morphological and functional recovery of the NMJ from tourniquet‐induced IR injuries.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Neuromuscular Junction Structure and Redox Status of Skeletal Muscle Fiber: Effect on Schwann Cells

Pre‐synaptic nerve terminal and post‐synaptic acetylcholine receptor clusters of neuromuscular junctions (NMJs) perfectly overlap in muscles of healthy adult animals. These junctions are supported by Schwann cells (SCs). NMJs undergo drastic alterations under certain circumstances in which redox status of the muscle is altered, including ageing, disease and peripheral nerve injury. SCs have been implicated in NMJ stability and re‐modelling. In non‐mammalian models hydrogen peroxide (H2O2) has been reported to control axonal guidance through activation of SCs.This study has examined changes in redox status of the muscle fibers in relation to changes in SC distribution and disruption of NMJ structure.Hindlimb skeletal muscles from adult and old WT mice, muscles from Sod1 KO (a model of accelerated muscle loss) and denervated muscles were used in this study. Changes in the redox state of the muscles were examined by use of the Amplex red assay to study mitochondrial peroxide production by permeabilised fiber bundles and intravital‐imaging of the ratio‐metric fluorescent probe cyto‐Hyper2 to specifically assess cytosolic H2O2 in muscle fibers. Fixed, whole EDL muscles were stained with s100‐beta antibody to visualise SCs alongside alpha‐bungarotoxin staining to visualise the NMJs within the intact muscle.Muscles from old mice had an increased rate of mitochondrial peroxide production compared with those from adult mice and this increased significantly when the muscle was denervated. In adult mice peroxide generation in muscle was increased significantly at 7 day time point post‐denervation and although it remained elevated, levels began to fall at 14 and 21 days post‐denervation.Immuno‐staining revealed s100‐beta expression along the axons and overlapped with the post‐synaptic bungarotoxin staining in muscle from adult and old WT mice, however s100‐beta expression appeared reduced in samples from old mice in comparison to adult. In denervated muscle, SC staining in the form of neuronal ‘tubes’ were evident for several days following surgery but staining was reduced at 14 and 21 days. When considered against the profile of peroxide generation in this model of denervation, these data suggest that at around 14 days post‐denervation, the potential initial protective adaptations have given way to degenerative processes.The pattern of SC staining in muscle of Sod1 KO mice was unlike the pattern observed in either the old mice or the denervated model despite evidence of an altered redox status and disrupted NMJs.These data suggest that the redox environment of the muscle influences SC location and potentially function which could contribute to the NMJ structural alterations seen in these models.Support or Funding InformationSupported by NIA AG051442This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

MuSK myasthenia gravis monoclonal antibodies: Valency dictates pathogenicity.

ObjectiveTo isolate and characterize muscle-specific kinase (MuSK) monoclonal antibodies from patients with MuSK myasthenia gravis (MG) on a genetic and functional level.MethodsWe generated recombinant MuSK antibodies from patient-derived clonal MuSK-specific B cells and produced monovalent Fab fragments from them. Both the antibodies and Fab fragments were tested for their effects on neural agrin-induced MuSK phosphorylation and acetylcholine receptor (AChR) clustering in myotube cultures.ResultsThe isolated MuSK monoclonal antibody sequences included IgG1, IgG3, and IgG4 that had undergone high levels of affinity maturation, consistent with antigenic selection. We confirmed their specificity for the MuSK Ig-like 1 domain and binding to neuromuscular junctions. Monovalent MuSK Fab, mimicking functionally monovalent MuSK MG patient Fab-arm exchanged serum IgG4, abolished agrin-induced MuSK phosphorylation and AChR clustering. Surprisingly, bivalent monospecific MuSK antibodies instead activated MuSK phosphorylation and partially induced AChR clustering, independent of agrin.ConclusionsPatient-derived MuSK antibodies can act either as MuSK agonist or MuSK antagonist, depending on the number of MuSK binding sites. Functional monovalency, induced by Fab-arm exchange in patient serum, makes MuSK IgG4 antibodies pathogenic.

Correct expression and localization of collagen XIII are crucial for the normal formation and function of the neuromuscular system.

Transmembrane collagen XIII has been linked to maturation of the musculoskeletal system. Its absence in mice (Col13a1-/- ) results in impaired neuromuscular junction (NMJ) differentiation and function, while transgenic overexpression (Col13a1oe ) leads to abnormally high bone mass. Similarly, loss-of-function mutations in COL13A1 in humans produce muscle weakness, decreased motor synapse function and mild dysmorphic skeletal features. Here, analysis of the exogenous overexpression of collagen XIII in various muscles revealed highly increased transcript and protein levels, especially in the diaphragm. Unexpectedly, the main location of exogenous collagen XIII in the muscle was extrasynaptic, in fibroblast-like cells, while some motor synapses were devoid of collagen XIII, possibly due to a dominant negative effect. Concomitantly, phenotypical changes in the NMJs of the Col13a1oe mice partly resembled those previously observed in Col13a1-/- mice. Namely, the overall increase in collagen XIII expression in the muscle produced both pre- and postsynaptic abnormalities at the NMJ, especially in the diaphragm. We discovered delayed and compromised acetylcholine receptor (AChR) clustering, axonal neurofilament aggregation, patchy acetylcholine vesicle (AChV) accumulation, disrupted adhesion of the nerve and muscle, Schwann cell invagination and altered evoked synaptic function. Furthermore, the patterns of the nerve trunks and AChR clusters in the diaphragm were broader in the adult muscles, and already prenatally in the Col13a1oe mice, suggesting collagen XIII involvement in the development of the neuromuscular system. Overall, these results confirm the role of collagen XIII at the neuromuscular synapses and highlight the importance of its correct expression and localization for motor synapse formation and function.

Plasma vitronectin is reduced in patients with myasthenia gravis: Diagnostic and pathophysiological potential.

Myasthenia gravis (MG) is an autoimmune disease leading to varying degrees of skeletal muscle weakness. It is caused by specific antibodies directed against definite components in the postsynaptic membrane at the neuromuscular junction (NMJ), such as the acetylcholine receptor (AChR) and the muscle-specific kinase (MUSK) receptor. In clinical practice, MG patients may be classified into three main subgroups based on the occurrence of serum autoantibodies directed against AChR or MUSK receptor or antibody-negative. As the MG subgroups differ in terms of clinical characteristics, disease pathogenesis, prognosis, and response to therapies, they could benefit from targeted treatment as well as the detection of other possible disease biomarkers. We performed proteomics on plasma fractions enriched in low-abundance proteins to identify potential biomarkers according to different autoimmune responses. By this approach, we evidenced a significant reduction of vitronectin in MG patients compared to healthy controls, irrespective of the autoantibodies NMJ target. The obtained results were validated by mono- and two-dimensional Western blotting analysis. Vitronectin is a multifunctional glycoprotein involved in the regulation of several pathophysiological processes, including complement-dependent immune response, coagulation, fibrinolysis, pericellular proteolysis, cell attachment, and spreading. The pathophysiological significance of the reduction of plasma vitronectin in MG patients has yet to be fully elucidated. It could be related either to a possible deposition of vitronectin at NMJ to counteract the complement-mediated muscle damage at this level or to a parallel variation of this glycoprotein in the muscle extracellular matrix with secondary induced alteration in clustering of AChRs at NMJ, as it occurs with variation in concentrations of agrin, another extracellular matrix component. The clinical value of measuring plasma vitronectin has yet to be defined. According to present findings, significantly lower plasma values of this glycoprotein might be indicative of an impaired complement-dependent immune response.

Differential effects of spinal motor neuron-derived and skeletal muscle-derived Rspo2 on acetylcholine receptor clustering at the neuromuscular junction

We recently reported that R-spondin 2 (Rspo2), a secreted activator of Wnt/β-catenin signaling, promotes acetylcholine receptor (AChR) clustering and neuromuscular junction (NMJ) formation via its receptor, Lgr5. Rspo2 is expressed highly in spinal motor neurons (SMNs) and marginally in the skeletal muscle, but the origin of Rspo2 at the NMJ remains elusive. We rescued Rspo2-deficient (Rspo2−/−) mice by specifically expressing Rspo2 in the skeletal muscle and SMNs. SMN-specific Rspo2 mitigated or over-corrected abnormal features of the NMJs and AChR clusters observed in Rspo2−/− mice including (i) abnormal broadening of enlarged AChR clusters, (ii) three of six abnormal ultrastructural features, and (iii) abnormal expression of nine genes in SMNs and the diaphragm. In contrast, muscle-specific Rspo2 normalized all six abnormal ultrastructural features, but it had no effect on AChR clustering and NMJ formation at the light microscopy level or on abnormal gene expression in SMNs and the diaphragm. These results suggest that SMN-derived Rspo2 plays a major role in AChR clustering and NMJ formation in the postsynaptic region, and muscle-derived Rspo2 also plays a substantial role in juxtaposition of the active zones and synaptic folds.