MuSK Research Articles

High sensitive detection and validation of neuronal specific autoantibodies in post COVID-19 patients with neurocognitive disorders

Abstract COVID-19 has affected tens of million patients worldwide and a significant portion of COVID-19 patients experience post-COVID syndrome characterized by a variety of persistent, debilitating symptoms including profound neurological symptoms. Studies have demonstrated the association between autoantibodies and the severity of COVID-19. but the role of autoantibodies in post-COVID-19 patients with neurological disorders has not yet well established. We screened the levels of anti-neuronal antibodies in 120 serum and CSF samples from 60 post-COVID-19 patients using an OmicsArray™ antigen panel containing 83 neuronal specific antigens (NSA). We identified 7 anti-NSA antibodies which were significantly elevated in serum of at least 1 post-COVID-19 patient with neurocognitive symptoms, including anti-CASPR2, anti-GAD65, anti-RCVRN, anti-mGluR2, anti-MuSK, anti-CSPG4 and anti-ELAVL2 (HuB). 3 anti-NSA antibodies (anti-MOG, anti-GAD65 and anti-mGluR2) were also detected positive in CSF samples of neurocognitive patients. Pre-incubation of the patient serum samples with the purified antigens inhibited the binding of the antibody with the specific NSA on array, validated the specificity of antigen array results. We further confirmed that anti-MuSK antibody in patient serum was targeting the extracellular domain of the MuSK protein using peptide array. Our results suggest anti-NSA antibodies may play a role in post-COVID neurological syndrom and warrant further study.

Structural insights into the assembly of the agrin/LRP4/MuSK signaling complex

MuSK is a receptor tyrosine kinase (RTK) that plays essential roles in the formation and maintenance of the neuromuscular junction. Distinct from most members of RTK family, MuSK activation requires not only its cognate ligand agrin but also its coreceptors LRP4. However, how agrin and LRP4 coactivate MuSK remains unclear. Here, we report the cryo-EM structure of the extracellular ternary complex of agrin/LRP4/MuSK in a stoichiometry of 1:1:1. This structure reveals that arc-shaped LRP4 simultaneously recruits both agrin and MuSK to its central cavity, thereby promoting a direct interaction between agrin and MuSK. Our cryo-EM analyses therefore uncover the assembly mechanism of agrin/LRP4/MuSK signaling complex and reveal how MuSK receptor is activated by concurrent binding of agrin and LRP4.

Genetic variation in the human olfactory receptor OR5AN1 associates with the perception of musks.

Humans have significant individual variations in odor perception, derived from their experience or sometimes from differences in the olfactory receptor (OR) gene repertoire. In several cases, the genetic variation of a single OR affects the perception of its cognate odor ligand. Musks are widely used for fragrance and are known to demonstrate specific anosmia. It, however, remains to be elucidated whether the OR polymorphism contributes to individual variations in musk odor perception. Previous studies reported that responses of the human musk receptor OR5AN1 to a variety of musks in vitro correlated well with perceptual sensitivity to those odors in humans and that the mouse ortholog, Olfr1440 (MOR215-1), plays a critical role in muscone perception. Here, we took advantage of genetic variation in OR5AN1 to examine how changes in receptor sensitivity are associated with human musk perception. We investigated the functional differences between OR5AN1 variants in an in vitro assay and measured both perceived intensity and detection threshold in human subjects with different OR5AN1 genotypes. Human subjects homozygous for the more sensitive L289F allele had a lower detection threshold for muscone and found macrocyclic musks to be more intense than subjects homozygous for the reference allele. These results demonstrate that the genetic variation in OR5AN1 contributes to perceptual differences for some musks. In addition, we found that the more functional variant of OR5A1, a receptor involved in β-ionone perception, is associated with the less functional variant of OR5AN1, suggesting that the perceived intensities of macrocyclic musks and β-ionone are inversely correlated.

From musk to body odor: Decoding olfaction through genetic variation.

The olfactory system combines input from multiple receptor types to represent odor information, but there are few explicit examples relating olfactory receptor (OR) activity patterns to odor perception. To uncover these relationships, we performed genome-wide scans on odor-perception phenotypes for ten odors in 1000 Han Chinese and validated results for six of these odors in an ethnically diverse population (n = 364). In both populations, consistent with previous studies, we replicated three previously reported associations (β-ionone/OR5A1, androstenone/OR7D4, cis-3-hexen-1-ol/OR2J3 LD-band), but not for odors containing aldehydes, suggesting that olfactory phenotype/genotype studies are robust across populations. Two novel associations between an OR and odor perception contribute to our understanding of olfactory coding. First, we found a SNP in OR51B2 that associated with trans-3-methyl-2-hexenoic acid, a key component of human underarm odor. Second, we found two linked SNPs associated with the musk Galaxolide in a novel musk receptor, OR4D6, which is also the first human OR shown to drive specific anosmia to a musk compound. We noticed that SNPs detected for odor intensity were enriched with amino acid substitutions, implying functional changes of odor receptors. Furthermore, we also found that the derived alleles of the SNPs tend to be associated with reduced odor intensity, supporting the hypothesis that the primate olfactory gene repertoire has degenerated over time. This study provides information about coding for human body odor, and gives us insight into broader mechanisms of olfactory coding, such as how differential OR activation can converge on a similar percept.

Evaluating an In-House Cell-Based Assay for Detecting Antibodies Against Muscle-Specific Tyrosine Kinase in Myasthenia Gravis.

Background and PurposeDetecting antibodies against muscle-specific tyrosine kinase (MuSK Abs) is essential for diagnosing myasthenia gravis (MG). We applied an in-house cell-based assay (CBA) to detect MuSK Abs.MethodsA stable cell line was generated using a lentiviral vector, which allowed the expression of MuSK tagged with green fluorescent protein in human embryonic kidney 293 (HEK293) cells. Serum and anti-human IgG antibody conjugated with red fluorescence were added. The presence of MuSK Abs was determined based on the fluorescence intensity and their colocalization in fluorescence microscopy. Totals of 218 serum samples collected from 177 patients with MG, 31 with other neuromuscular diseases, and 10 healthy controls were analyzed. The CBA results were compared with those of a radioimmunoprecipitation assay (RIPA) and an enzyme-linked immunosorbent assay (ELISA).ResultsThe MuSK-HEK293 cell line stably expressed MuSK protein. The CBA detected MuSK Abs in 34 (19.2%) of 177 samples obtained from patients with MG and in none of the participants having other neuromuscular diseases or in the healthy controls. The clinical characteristics of the patients with MuSK MG determined based on the CBA were strongly correlated with known clinical features of MuSK MG. There was an almost perfect agreement between the results of the CBA and those of the RIPA (Cohen's kappa=0.880, p<0.001) and ELISA (Cohen's kappa=0.982, p<0.001).ConclusionsThe results of the in-house CBA showed excellent agreement with both the RIPA and ELISA. Our in-house CBA can be considered a reliable method for detecting MuSK Abs.

Myopathy or Myasthenia Positive for Antibodies Against MuSK, Lrp4, and Titin.

Myopathy or Myasthenia Positive for Antibodies Against MuSK, Lrp4, and Titin.

A New Family of Rigid Dienone Musks Challenges the Perceptive Range of the Human Olfactory Receptor OR5AN1

A new family of dienone musks was discovered by alkylation of different aldehydes with but-3-en-1-yn-1-yllithium and subsequent domino reaction of a Saucy–Marbet transfer vinylation–Claisen rearrangement with an intramolecular Diels–Alder reaction, and concluding Lewis acid catalyzed double-bond isomerization. The newly synthesized dienone structures possess pleasant musk odors displaying fatty, slightly fruity and green facets. Although the dienone musks were predicted in silico to bind to the OR5AN1 receptor based on QM/MM calculations, they were found to be inactive in the in vitro assay. The latter results suggest that the OR5AN1 receptor is not the prime musk receptor but primarily responsible for the animalic character of certain macrocyclic ketones and nitro musks.

Tacrolimus inhibits Th1 and Th17 responses in MuSK-antibody positive myasthenia gravis patients

Muscle specific tyrosine kinase antibody positive myasthenia gravis (MuSK- MG) is characterized by autoantibodies against the MuSK protein of the neuromuscular junction resulting in weakness of bulbar and proximal muscles. We previously demonstrated that patients with MuSK-MG have increased pro-inflammatory Th1 and Th17 responses. Tacrolimus, an immunosuppressant used in AChR-MG and transplantation patients, inhibits T cell responses through interference with IL-2 transcription. The therapeutic efficacy and immunological effect of tacrolimus in MuSK-MG is unclear. In the current study we examined the proliferation, phenotype and cytokine production of CD4+ and CD8+ T cells in peripheral blood mononuclear cells of MuSK-MG following a 3-day in vitro culture with or without tacrolimus. We determined that tacrolimus profoundly suppressed CD4 and CD8 T cell proliferation and significantly suppressed Th1 and Th17 responses, as demonstrated by a reduced frequency of IFN-γ, IL-2, and IL-17 producing CD4 T cells and reduced frequencies of IFN-γ and IL-2 producing CD8 T cells. Tacrolimus also inhibits pathogenic Th17 cells coproducing IL-17 and IFN-γ. In addition, tacrolimus suppressed follicular T helper cell (Tfh) and regulatory T helper cell (Treg) subsets. These findings provide preliminary support for tacrolimus as a potential alternative immunosuppressive therapy for MuSK-MG.

Clinical differences between early-and late-onset myasthenia

Objective To explore the clinical differences between patients with early-onset myasthenia gravis(EOMG)and those with late-onset myasthenia gravis(LOMG). Methods This was a retrospective study enrolling 157 MG patients.Based on the age of onset, patients were divided into the EOMG group(n=85)and the LOMG group(n=72). The groups were compared on clinical characteristics, including clinical manifestations, MG classification, electrophysiological findings on repetitive nerve stimulation(RNS), single fiber electromyography(SFEMG), levels of antibody against acetylcholine receptors(Ach-R Ab), antibody to muscle-specific kinase(MuSK Ab), titin antibody(Titin Ab), ryanodine receptor antibody(RyR Ab), thyroid function, thymectomy, thymus pathology and responses to treatment. Results The mean ages of onset were markedly different〔(40.9±9.7)years vs.(62.0±12.2)years, P 0.05). Moreover, patients in the EOMG group were more likely to have abnormal thyroid function and higher percentages of receiving steroids, tacrolimus, plasma exchange therapy, and thymectomy(P<0.05). Conclusions The clinical profiles of LOMG are different from those of EOMG in clinical manifestations, thyroid function, thymectomy frequency, striational antibody levels and disease-modifying drug options. Key words: Myasthenia gravis; Recepors, cholinergic

Homeostasis and Disorder of Musculoskeletal System.Molecular signaling and its pathogenic alterations in neuromuscular junction formation.

The neuromuscular junction(NMJ)is the synapse between a motor neuron and the skeletal muscle that is essential for muscle contraction. Impairments at the NMJ lead to neuromuscular-transmission pathologies characterized by fatigable muscle weakness. Muscle-specific receptor tyrosine kinase MuSK plays key roles in NMJ formation. Over the past decade, studies examining the NMJ formation signals have identified molecules involved in the signaling pathways and have promoted a better understanding of characteristic molecular mechanisms for MuSK activation. Unlike many other receptor tyrosine kinases, MuSK is regulated by the cytoplasmic activator Dok-7 in addition to the extracellular activator agrin. It is well established that all of these molecules are indispensable in the formation and maintenance of the NMJ. In this chapter, we review molecular signaling, particularly MuSK signaling, in the formation of the NMJ and the altered molecular signaling associated with neuromuscular disorders.

Juvenile Myasthenia Gravis- A rare case report

Background : It is an acquired autoimmune mediated postsynaptic disorder that is rare in childhood with prevalence of 5 in 1,00,000. First case was reported in the year 1644 in USA of an Indian chief Opechankanough. The disease is due to circulating antibodies which bind to acetylcholine receptors at neuromuscular junction and prevent a normal action of acetylcholine in opening the calcium channel in muscle fibers. There are two types of the disease: ocular (corresponding to 10% of cases) [1] or generalized (90% of cases), although both may occur under different temporal evolution in the same patient. Myasthenia gravis geoepidemiology shows that it is a rare disorder with similar incidence and prevalence in the world, except for infantile MG, which is more common in Asia. Methods : With progressive ptosis towards the end of the day, Juvenile Myasthenia gravis was suspected. Anti cholinesterase (AChE) test was done under ECG monitoring using intravenous injection of neostigmine (Prostigmine test) which showed a dramatic response i.e. immediate elevation of both upper eyelids with resolution of ptosis[Fig. 1 and 2]. Bed side icepack test was also performed which was positive. Computerized tomography (CT scan) of thorax was done to rule out thymic hyperplasia or thymoma. We diagnosed this child as a case of juvenile myasthenia gravis and treated with intravenous neostigmine(0.04mg/ kg/dose) 4 hourly followed by atropine(0.05mg/kg/dose) to counteract ill muscarinic side effects of neostigmine. Intravenous methylprednisolone was also given as pulse therapy (30mg/ kg/day for 3 consecutive days) with multipara monitoring (for hypertension and hyperglycemia). Plasma anti-AChR antibodies though advised was not affordable. Results : The cardinal symptom in MG is muscle weakness that is typically fluctuating and fatigable. The most common initial presentation involves a specific muscle weakness, and not a generalized weakness. Symptoms are exacerbated with exercise (it may be caused by sustained muscle action) and heat, and decreased with relaxation and cooling. The disease involves striated or voluntary muscles, with a typical anatomical distribution. The weakness is usually ocular, bulbar, proximal muscle groups (most commonly the upper limbs) and neck, but may also in some patients involve the respiratory muscles. The disease can progress from mild to moderate to severe in weeks and months with episodes of exacerbations and remissions. The Myasthenia Gravis Foundation of America# proposed a clinical classification of the disease into five main classes and several subclasses, according to the location of muscle weakness and its intensity. Ptosis or extra ocular muscle weakness is the initial presentation in 75% of patients [1] and occurs during the course of the disease in 90% of cases (remaining exclusively ocular only 16% of cases [2-4]). When anti AChR antibodies are negative, MuSK receptor antibodies should be advised. Table 1 : Classification of MG according to Ossermann Scale Type 1 Ocular myasthenia characterized by ptosis and diplopia Type 2a Slow onset, frequently ocular, with gradual evolution to skeletal musculature Type 2b Slow onset with dysarthria, dysphagia, and change in mastication Type 3 Fast onset with severe bulbar and skeletal muscle fatigue with respiratory muscle compromise Type 4 Severe MG that manfests in two years Conclusion : Repetitive ulnar nerve stimulation (RNS) studies show significant decremental response in abductor digiti minimi suggestive of post synaptic neuromuscular junction (NMJ) receptor disorder[5]. Other differentials which should be considered include neuroparalytic snake bite, Miller-Fischer type of GBS and botulism. Our patient was classified as type III i.e. fast onset with severe bulbar and skeletal muscle fatigue with respiratory muscle compromise, so he had to be ventilated for 3 days. He was weaned from IPPV to T-piece over 12 hours and then to CPAP for 6 hours maintaining normal saturations and vitals. Thereafter he was referred to higher center for plasmapharesis but since then he is lost to follow up.

Myastenia gravis s protilátkami proti MuSK

Myastenia gravis je autoimunitni onemocněni s tvorbou protilatek proti postsynapticke casti nervosvalove plotenky. Při průkazu protilatek proti acetylcholinovým receptorům se jedna o seropozitivni myastenia gravis a pokud tyto protilatky nejsou přitomny, pak je to seronegativni myastenia gravis. U 35 % (0–49 %) seronegativnich forem byla prokazana protilatka proti svalove specificke tyrozin-kinaze (MuSK). Tyto anti-MuSK myasthenia gravis se vyznacuji převahou postiženi žen, casnějsich vznikem, charakteristickým klinickým nalezem, horsi a nekonstantni reakci na lecbu inhibitory cholinesterazy. Z neurofyziologických vysetřeni je důležite vysetřit repetitivni stimulaci i proximalni svaly (m. trapezius, m. deltoides) a mimicke svaly (m. nasalis, m. orbicularis oculi). Při vysetřeni SF EMG (single-fiber EMG) je podstatně vyssi pozitivita při vysetřeni m. frontalis ci m. orbicularis oculi. Neni indikovana thymektomie. Ucinna je imunoterapie – podani imunoglobulinů, plazmafereza, kortikoidy v kombinaci s dalsimi imunosupresivy. Myastenia gravis s anti-MuSK protilatkami se vyznacuje nestabilnim průběhem, větsi frekvenci myastenických krizi a větsi terapeutickou narocnosti.

Molecular mechanisms underlying the formation and maintenance of neuromuscular junctions and a possible therapeutic approach.

The mammalian neuromuscular junction(NMJ), a cholinergic synapse between a motor neuron and a skeletal muscle fiber, is essential for neural control of muscle contraction. Impaired formation and/or maintenance of NMJs results in disorders of neuromuscular transmission such as myasthenia gravis(MG)and congenital myasthenic syndromes(CMSs). The formation and maintenance of NMJs are orchestrated by the muscle-specific receptor tyrosine kinase MuSK. Activation of MuSK involves its essential cytoplasmic activator Dok-7, the MuSK co-receptor Lrp4, and the motor neuron-derived MuSK activator agrin. Indeed, CMS-associated mutations are identified in the genes encoding these 4 proteins, and autoantibodies against MuSK, Lrp4, or agrin are found in MG patients, demonstrating the pathophysiological significance of the MuSK activation machinery. However, Lrp4 and agrin also play crucial roles separate from MuSK activation in NMJ formation and maintenance. Based on the finding that forced expression of Dok-7 in muscle activates MuSK and enlarges NMJs, we recently developed DOK7 gene therapy as a therapy aimed at enlarging the NMJ, which has potential for treating various neuromuscular disorders with defective NMJ structure.

Multiscale Simulations Suggest a Mechanism for the Association of the Dok7 PH Domain with PIP-Containing Membranes.

Dok7 is a peripheral membrane protein that is associated with the MuSK receptor tyrosine kinase. Formation of the Dok7/MuSK/membrane complex is required for the activation of MuSK. This is a key step in the complex exchange of signals between neuron and muscle, which lead to neuromuscular junction formation, dysfunction of which is associated with congenital myasthenic syndromes. The Dok7 structure consists of a Pleckstrin Homology (PH) domain and a Phosphotyrosine Binding (PTB) domain. The mechanism of the Dok7 association with the membrane remains largely unknown. Using multi-scale molecular dynamics simulations we have explored the formation of the Dok7 PH/membrane complex. Our simulations indicate that the PH domain of Dok7 associates with membranes containing phosphatidylinositol phosphates (PIPs) via interactions of the β1/β2, β3/β4, and β5/β6 loops, which together form a positively charged surface on the PH domain and interact with the negatively charged headgroups of PIP molecules. The initial encounter of the Dok7 PH domain is followed by formation of additional interactions with the lipid bilayer, and especially with PIP molecules, which stabilizes the Dok7 PH/membrane complex. We have quantified the binding of the PH domain to the model bilayers by calculating a density landscape for protein/membrane interactions. Detailed analysis of the PH/PIP interactions reveal both a canonical and an atypical site to be occupied by the anionic lipid. PH domain binding leads to local clustering of PIP molecules in the bilayer. Association of the Dok7 PH domain with PIP lipids is therefore seen as a key step in localization of Dok7 to the membrane and formation of a complex with MuSK.

Ligand Specificity and Evolution of Mammalian Musk Odor Receptors: Effect of Single Receptor Deletion on Odor Detection.

The long-sought musk odor receptor family in mammals was discovered and found to be well conserved and narrowly tuned to musk odors. In mice, deletion of the most sensitive musk receptor resulted in drastic reduction in sensitivity to muscone, demonstrating a strong link between receptor and odor perception. In humans, we found one musk receptor that recognized both macrocyclic and nitro musks that had distinct chemical structures. The structure-activity relationships were in a good agreement with human sensory perception and therefore may be used to develop novel musk aroma in fragrance fields. Finally, identification of a natural ligand(s) for musk receptors in mammals other than musk deer would reveal an evolutionarily pivotal role in each species in the future.

Implausibility of the vibrational theory of olfaction

The vibrational theory of olfaction assumes that electron transfer occurs across odorants at the active sites of odorant receptors (ORs), serving as a sensitive measure of odorant vibrational frequencies, ultimately leading to olfactory perception. A previous study reported that human subjects differentiated hydrogen/deuterium isotopomers (isomers with isotopic atoms) of the musk compound cyclopentadecanone as evidence supporting the theory. Here, we find no evidence for such differentiation at the molecular level. In fact, we find that the human musk-recognizing receptor, OR5AN1, identified using a heterologous OR expression system and robustly responding to cyclopentadecanone and muscone, fails to distinguish isotopomers of these compounds in vitro. Furthermore, the mouse (methylthio)methanethiol-recognizing receptor, MOR244-3, as well as other selected human and mouse ORs, responded similarly to normal, deuterated, and (13)C isotopomers of their respective ligands, paralleling our results with the musk receptor OR5AN1. These findings suggest that the proposed vibration theory does not apply to the human musk receptor OR5AN1, mouse thiol receptor MOR244-3, or other ORs examined. Also, contrary to the vibration theory predictions, muscone-d30 lacks the 1,380- to 1,550-cm(-1) IR bands claimed to be essential for musk odor. Furthermore, our theoretical analysis shows that the proposed electron transfer mechanism of the vibrational frequencies of odorants could be easily suppressed by quantum effects of nonodorant molecular vibrational modes. These and other concerns about electron transfer at ORs, together with our extensive experimental data, argue against the plausibility of the vibration theory.

Diagnostic and clinical classification of autoimmune myasthenia gravis

Myasthenia gravis is characterized by muscle weakness and abnormal fatigability. It is an autoimmune disease caused by the presence of antibodies against components of the muscle membrane localized at the neuromuscular junction. In most cases, the autoantibodies are against the acetylcholine receptor (AChR). Recently, other targets have been described such as the MuSK protein (muscle-specific kinase) or the LRP4 (lipoprotein related protein 4). Myasthenia gravis can be classified according to the profile of the autoantibodies, the location of the affected muscles (ocular versus generalized), the age of onset of symptoms and thymic abnormalities.The disease generally begins with ocular symptoms (ptosis and/or diplopia) and extends to other muscles in 80% of cases. Other features that characterize MG include the following: variability, effort induced worsening, successive periods of exacerbation during the course of the disease, severity dependent on respiratory and swallowing impairment (if rapid worsening occurs, a myasthenic crisis is suspected), and an association with thymoma in 20% of patients and with other autoimmune diseases such as hyperthyroidism and Hashimoto's disease. The diagnosis is based on the clinical features, the benefit of the cholinesterase inhibitors, the detection of specific autoantibodies (anti-AChR, anti-MuSK or anti-LRP4), and significant decrement evidenced by electrophysiological tests.In this review, we briefly describe the history and epidemiology of the disease and the diagnostic and clinical classification. The neonatal form of myasthenia is explained, and finally we discuss the main difficulties of diagnosis.

Urothelial differentiation of human amniotic fluid stem cells by urothelium specific conditioned medium

Human amniotic fluid stem cells (HAFSCs) have a high proliferative capacity and a good differentiation potential, and may thus be suitable for regenerative medicine. To date, urothelial differentiation mechanisms of HAFSCs are poorly understood. We have investigated the urothelial differentiation potential of HAFSCs so that they can be therapeutically applied to cure defective diseases of bladder. To induce the stem cell differentiation, HAFSCs were cultured in a bladder cancer-derived conditioned medium. After 2 weeks of culture, HAFSCs began to express the urothelial lineage-specific markers (UPII, CK8 and FGF10). Meanwhile, pluripotency markers (Oct-4, Sox-2 and Nanog) were downregulated at both RNA and protein levels in the differentiated HAFSCs. Immunocytochemistry data revealed that differentiated HAFSCs expressed urothelial markers of UPII and CK8. We have screened the receptor tyrosine kinase arrays with the differentiated HAFSCs. The screening showed that MuSK, Tie-1 and EphA4 receptor tyrosine kinases were upregulated, whereas EphA7 and FGF R1 kinases were downregulated in HAFSCs. The data suggest that HAFSCs can be an important urothelium cell source, which can be used for urinary tract engineering.

T cell repertoire in DQ5-positive MuSK-positive myasthenia gravis patients

Myasthenia gravis (MG) is a prototypical antibody-mediated disease characterized by muscle weakness and fatigability. Serum antibodies to the acetylcholine receptor and muscle-specific tyrosine kinase receptor (MuSK) are found in about 85% and 8% of patients respectively. We have previously shown that more than 70% of MG patients with MuSK antibodies share the HLA DQ5 allele. The aim of the present study was to analyze the T cell receptor (TCR) repertoire specific for recombinant human MuSK protein. We used the CDR3 TRBV-TRBJ spectratyping (immunoscope) to analyze the T cell response to MuSK from 13 DQ5+ MuSK-MG patients and from 7 controls (six DQ5+ MuSK negative subjects and one DQ5− DQ3+ MuSK positive patient). DQ5+ MuSK-MG patients but not controls used a restricted set of TCR VJ rearrangements in response to MuSK stimulation. One semiprivate (TRBV29-TRBJ2.5) rearrangement was found in 5/13 patients, while 4 other semiprivate (one in TRBV28-TRBJ2.1 and in TRBV3-TRBJ1.2, and two in TRBV28-TRBJ1.2) rearrangements were differently shared by 4/13 patients each and were absent in controls. When we sequenced the TRBV29-TRBJ2.5 rearrangement, we obtained 26 different sequences of the expected 130 bp length from 117 samples of the 5 positive patients: two common motifs GXGQET/TEHQET were shared in 4 patients as semiprivate motifs. Thus, the MuSK-specific T-cell response appears to be restricted in DQ5+ MuSK-MG patients, with a semiprivate repertoire including a common motif of TRBV29. This oligoclonal restriction of T cells will allow the identification of immunodominant epitopes in the antigen, providing therefore new tools for diagnosis and targeted therapy.

Potential mechanisms in MuSK-myasthenia gravis

Figure 3: A,C: Coomassie gel with non reducing conditions of puri#ed IgG (1), papain digested IgG (2) and puri#ed FAB fragments (3). B,D: RIPA of Plasma, puri#ed IgG and puri#ed FAB fragments as well as healthy control serum. Figure 7: A= IP of MuSK by commercial antibodies against MuSK and patient antibodies, and co-precipitated Lrp4 from cells transfected with MuSK and Lrp4, and transfection control (Lrp4, but no MuSK). B=Healthy control serum. C=IP with patient F or commercial antibody, western blot with antibody against human IgG, FAB speci#c, or against MuSK. D,E= quanti#cation of band size from 5 patients, normalized as % of commercial antibody against MuSK (N=3). 1-way ANOVA + Dunnett’s multiple comparison test. D= MuSK (all ns). E=Lrp4. F= Ratio Lrp4/MuSK. 1-way ANOVA+ Dunnett’s multiple comparison test. Conclusions • FAB-fragments can block agrin induced AChR clustering but do not appear to induce agrinindependent AChR clusters • Therefore, agrin independent AChR clustering by MuSK antibodies appears to be dependent on MuSK dimerization • Binding of MuSK antibodies decreases the interaction between MuSK and Lrp4 Summary of Results • MuSK ab effect on AChR clustering correlate broadly • with MuSK ab titre • FAB fragments as well as IgG reduce agrin induced AChR clustering • FAB fragments, in contrast to IgG, do not induce agrinindependent AChR clustering • Patient antibodies co-precipitate less Lrp4 than commercial antibodies against MuSK • All results are still preliminary, more data required