CD59 Expression in Skeletal Muscles and Its Role in Myasthenia Gravis.

BACKGROUND AND OBJECTIVES: Complement regulatory proteins at the neuromuscular junction (NMJ) could offer protection against complement-mediated damage in myasthenia gravis (MG). However, there is limited information on their expression at the human NMJ. Thus, this study aimed at investigating the expression of the cluster of differentiation 59 (CD59) at the NMJ of human muscle specimens and demonstrating the overexpression of CD59 mRNA and protein in the muscles of patients with MG. METHODS: In this observational study, muscle specimens from 16 patients with MG (9 and 7 patients with and without thymoma, respectively) and 6 nonmyopathy control patients were examined. Immunohistochemical stains, Western blot analysis, and quantitative real-time reverse transcription PCR were used to evaluate the CD59 expression. RESULTS: A strong localized expression of CD59 was observed at the NMJ in both patients with and without MG. Moreover, the CD59/glyceraldehyde-3-phosphate dehydrogenase protein ratio in patients with MG was significantly higher than that in the nonmyopathy controls (MG; n = 16, median 0.16, interquartile range (IQR) 0.08-0.26 and nonmyopathy controls; n = 6, median 0.03, IQR 0.02-0.11, p = 0.01). The proportion of CD59 mRNA expression relative to AChR mRNA expression (ΔCtCD59/AChR) was associated with the quantitative MG score, MG activities of daily living score, and MG of Foundation of America Clinical Classification (r = 0.663, p = 0.01; r = 0.638, p = 0.014; and r = 0.715, p = 0.003, respectively). DISCUSSION: CD59, which acts as a complement regulator, may protect the NMJ from complement attack. Our findings could provide a basis for further research that investigates the underlying pathogenesis in MG and the immunomodulating interactions of the muscle cells.

Metformin inhibits the pathogenic functions of AChR-specific B and Th17 cells by targeting miR-146a.

Myasthenia gravis (MG) is characterized by fatigable skeletal muscle weakness with a fluctuating and unpredictable disease course and is caused by circulating autoantibodies and pathological T helper cells. Regulation of B-cell function and the T-cell network may be a potential therapeutic strategy for MG. MicroRNAs (miRNAs) have emerged as potential biomarkers in immune disorders due to their critical roles in various immune cells and multiple inflammatory diseases. Aberrant miR-146a signal activation has been reported in autoimmune diseases, but a detailed exploration of the relationship between miR-146a and MG is still necessary. Using an experimental autoimmune myasthenia gravis (EAMG) rat model, we observed that miR-146a was highly expressed in the spleen but expressed at low levels in the thymus and lymph nodes in EAMG rats. Additionally, miR-146a expression in T and B cells was also quite different. EAMG-specific Th17 and Treg cells had lower miR-146a levels, while EAMG-specific B cells had higher miR-146a levels, indicating that targeted intervention against miR-146a might have diametrically opposite effects. Metformin, a drug that was recently demonstrated to alleviate EAMG, may rescue the functions of both Th17 cells and B cells by reversing the expression of miR-146a. We also investigated the downstream target genes of miR-146a in both T and B cells using bioinformatics screening and qPCR. Taken together, our study identifies a complex role of miR-146a in the EAMG rat model, suggesting that more caution should be paid in targeting miR-146a for the treatment of MG.

Decreased expression of miR-29 family associated with autoimmune myasthenia gravis.

BACKGROUND: Myasthenia gravis (MG) is a rare autoimmune disease mainly mediated by autoantibodies against the acetylcholine receptor (AChR) at the neuromuscular junction. The thymus is the effector organ, and its removal alleviates the symptoms of the disease. In the early-onset form of MG, the thymus displays functional and morphological abnormalities such as B cell infiltration leading to follicular hyperplasia, and the production of AChR antibodies. Type-I interferon (IFN-I), especially IFN-ß, is the orchestrator of thymic changes observed in MG. As Dicer and miR-29 subtypes play a role in modulating the IFN-I signalization in mouse thymus, we investigated their expression in MG thymus. METHODS: The expression of DICER and miR-29 subtypes were thoroughly investigated by RT-PCR in human control and MG thymuses, and in thymic epithelial cells (TECs). Using miR-29a/b-1-deficient mice, with lower miR-29a/b-1 expression, we investigated their susceptibility to experimental autoimmune MG (EAMG) as compared to wild-type mice. RESULTS: DICER mRNA and all miR-29 subtypes were down-regulated in the thymus of MG patients and DICER expression was correlated with the lower expression of miR-29a-3p. A decreased expression of miR-29 subtypes was similarly observed in MG TECs; a decrease also induced in TECs upon IFN-ß treatment. We demonstrated that miR-29a/b-1-deficient mice were more susceptible to EAMG without higher levels of anti-AChR IgG subtypes. In the thymus, if no B cell infiltration was observed, an increased expression of Ifn-ß associated with Baff expression and the differentiation of Th17 cells associated with increased expression of Il-6, Il-17a and Il-21 and decreased Tgf-ß1 mRNA were demonstrated in miR-29a/b-1-deficient EAMG mice. CONCLUSIONS: It is not clear if the decreased expression of miR-29 subtypes in human MG is a consequence or a causative factor of thymic inflammation. However, our results from the EAMG mouse model indicated that a reduction in miR-29a/b1 may contribute to the pathophysiological process involved in MG by favoring the increased expression of IFN-ß and the emergence of pro-inflammatory Th17 cells.

Efgartigimod improves muscle weakness in a mouse model for muscle-specific kinase myasthenia gravis.

Myasthenia gravis is hallmarked by fatigable muscle weakness resulting from neuromuscular synapse dysfunction caused by IgG autoantibodies. The variant with muscle-specific kinase (MuSK) autoantibodies is characterized by prominent cranial and bulbar weakness and a high frequency of respiratory crises. The majority of MuSK MG patients requires long-term immunosuppressive treatment, but the result of these treatments is considered less satisfactory than in MG with acetylcholine receptor antibodies. Emergency treatments are more frequently needed, and many patients develop permanent facial weakness and nasal speech. Therefore, new treatment options would be welcome. The neonatal Fc receptor protects IgG from lysosomal breakdown, thus prolonging IgG serum half-life. Neonatal Fc receptor antagonism lowers serum IgG levels and thus may act therapeutically in autoantibody-mediated disorders. In MuSK MG, IgG4 anti-MuSK titres closely correlate with disease severity. We therefore tested efgartigimod (ARGX-113), a new neonatal Fc receptor blocker, in a mouse model for MuSK myasthenia gravis. This model involves 11 daily injections of purified IgG4 from MuSK myasthenia gravis patients, resulting in overt myasthenic muscle weakness and, consequently, body weight loss. Daily treatment with 0.5 mg efgartigimod, starting at the fifth passive transfer day, reduced the human IgG4 titres about 8-fold, despite continued daily injection. In muscle strength and fatigability tests, efgartigimod-treated myasthenic mice outperformed control myasthenic mice. Electromyography in calf muscles at endpoint demonstrated less myasthenic decrement of compound muscle action potentials in efgartigimod-treated mice. These substantial in vivo improvements of efgartigimod-treated MuSK MG mice following a limited drug exposure period were paralleled by a tendency of recovery at neuromuscular synaptic level (in various muscles), as demonstrated by ex vivo functional studies. These synaptic improvements may well become more explicit upon longer drug exposure. In conclusion, our study shows that efgartigimod has clear therapeutic potential in MuSK myasthenia gravis and forms an exciting candidate drug for many autoantibody-mediated neurological and other disorders.

MicroRNA-653 Inhibits Thymocyte Proliferation and Induces Thymocyte Apoptosis in Mice with Autoimmune Myasthenia Gravis by Downregulating TRIM9.

OBJECTIVES: Myasthenia gravis (MG) is an organ-specific autoimmune neuromuscular disorder that occurs as a result of the impairment in neuromuscular junction and autoantibody attack on the postsynaptic receptors. Increasing evidence suggests that microRNAs (miRs) might be involved in the development of MG. Therefore, the present study aimed to investigate the regulatory function of miR-653 on MG and its relationship with tripartite motif 9 (TRIM9). METHODS: The thymic tissues obtained from MG patients with thymic hyperplasia were prepared for establishing an MG mouse model in BALB/c mice. Afterwards, the miR-653 and TRIM9 expressions were determined in thymic tissues. A dual-luciferase reporter assay was carried out to validate whether miR-653 directly targets TRIM9. Finally, the thymocytes were exposed to mimics or inhibitors of miR-653, or siRNA against TRIM9 with the use of MTT assays and flow cytometry for the verification of the gain or loss function of miR-653 and TRIM9 on viability, cell cycle progression, and apoptosis of thymocytes. RESULTS: There was a decrease in thymocyte miR-653 and an increase in TRIM9 in thymic tissues of MG mice. miR-653 was found to negatively regulate TRIM9. Overexpression of miR-653 or depletion of TRIM9 resulted in the inhibition of cell viability, suppression of cell cycle progression, and induction of apoptosis rate in thymocytes. CONCLUSION: The findings from the present study provided evidence that miR-653 impairs proliferation and promotes apoptosis of thymocytes of MG mice by suppressing TRIM9, indicating that miR-653 could be used as potential therapeutic target in the treatment of autoimmune MG.

Toll-like receptor 9 antagonist suppresses humoral immunity in experimental autoimmune myasthenia gravis.

Recent studies have demonstrated the important role of toll-like receptor 9 (TLR9) signalling in autoimmune diseases, but its role in myasthenia gravis (MG) has not been fully established. We show herein that blocking TLR9 signalling via the suppressive oligodeoxynucleotide (ODN) H154 alleviated the symptoms of experimental autoimmune myasthenia gravis (EAMG). With the downregulation of dendritic cells (DCs), TLR9 interruption reduced follicular helper T cells (Tfh) and germinal centre (GC) B cells, leading to decreased antibody production. In addition, TLR9+ B cells as well as total B cells in the spleen were inhibited by H154. These findings highlight the critical role of TLR9 in EAMG and suggest that the inhibition of the TLR9 pathway might be a potential pharmacological strategy for the treatment of myasthenia gravis.

NFAT1 Regulates Systemic Autoimmunity through the Modulation of a Dendritic Cell Property.

The transcription factor NFAT1 plays a pivotal role in the homeostasis of T lymphocytes. However, its functional importance in non-CD4+ T cells, especially in systemic immune disorders, is largely unknown. In this study, we report that NFAT1 regulates dendritic cell (DC) tolerance and suppresses systemic autoimmunity using the experimental autoimmune myasthenia gravis (EAMG) as a model. Myasthenia gravis and EAMG are T cell-dependent, Ab-mediated autoimmune disorders in which the acetylcholine receptor is the major autoantigen. NFAT1-knockout mice showed higher susceptibility to EAMG development with enhanced Th1/Th17 cell responses. NFAT1 deficiency led to a phenotypic alteration of DCs that show hyperactivation of NF-κB-mediated signaling pathways and enhanced binding of NF-κB (p50) to the promoters of IL-6 and IL-12. As a result, NFAT1-knockout DCs produced much higher levels of proinflammatory cytokines such as IL-1ß, IL-6, IL-12, and TNF-α, which preferentially induce Th1/Th17 cell differentiation. Our data suggest that NFAT1 may limit the hyperactivation of the NF-κB-mediated proinflammatory response in DCs and suppress autoimmunity by serving as a key regulator of DC tolerance.

Silencing of Dok-7 in Adult Rat Muscle Increases Susceptibility to Passive Transfer Myasthenia Gravis.

Myasthenia gravis (MG) is an autoimmune disease mediated by autoantibodies that target proteins at the neuromuscular junction, primarily the acetylcholine receptor (AChR) and the muscle-specific kinase. Because downstream of kinase 7 (Dok-7) is essential for the full activation of muscle-specific kinase and consequently for dense clustering of AChRs, we hypothesized that reduced levels of Dok-7 increase the susceptibility to passive transfer MG. To test this hypothesis, Dok-7 expression was reduced by transfecting shRNA-coding plasmids into the tibialis anterior muscle of adult rats by in vivo electroporation. Subclinical MG was subsequently induced with a low dose of anti-AChR monoclonal antibody 35. Neuromuscular transmission was significantly impaired in Dok-7-siRNA-electroporated legs compared with the contralateral control legs, which correlated with a reduction of AChR protein levels at the neuromuscular junction (approximately 25%) in Dok-7-siRNA-electroporated muscles, compared with contralateral control muscles. These results suggest that a reduced expression of Dok-7 may play a role in the susceptibility to passive transfer MG, by rendering AChR clusters less resistant to the autoantibody attack.

Ocular myasthenia gravis induced by human acetylcholine receptor ϵ subunit immunization in HLA DR3 transgenic mice.

Extraocular muscles (EOM) are preferentially involved in myasthenia gravis (MG) and acetylcholine receptor (AChR) antibody positive MG patients may occasionally present with isolated ocular symptoms. Although experimental autoimmune myasthenia gravis (EAMG) induced by whole AChR immunization closely mimics clinical and immunopathological aspects of MG, EOM are usually not affected. We have previously developed an EAMG model, which imitates EOM symptoms of MG by immunization of human leukocyte antigen (HLA) transgenic mice with α or γ-subunits of human AChR (H-AChR). To investigate the significance of the ϵ-subunit in ocular MG, we immunized HLA-DR3 and HLA-DQ8 transgenic mice with recombinant H-AChR ϵ-subunit expressed in Escherichia coli. HLA-DR3 transgenic mice showed significantly higher clinical ocular and generalized MG severity scores and lower grip strength values than HLA-DQ8 mice. H-AChR ϵ-subunit-immunized HLA-DR3 transgenic mice had higher serum anti-AChR antibody (IgG, IgG1, IgG2b, IgG2c and IgM) levels, neuromuscular junction IgG and complement deposit percentages than ϵ-subunit-immunized HLA-DQ8 transgenic mice. Control mice immunized with E. coli extract or complete Freund adjuvant (CFA) did not show clinical and immunopathological features of ocular and generalized EAMG. Lymph node cells of ϵ-subunit-immunized HLA-DR3 mice showed significantly higher proliferative responses than those of ϵ-subunit-immunized HLA-DQ8 mice, crude E. coli extract-immunized and CFA-immunized transgenic mice. Our results indicate that the human AChR ϵ-subunit is capable of inducing myasthenic muscle weakness. Diversity of the autoimmune responses displayed by mice expressing different HLA class II molecules suggests that the interplay between HLA class II alleles and AChR subunits might have a profound impact on the clinical course of MG.

Silencing miR-146a influences B cells and ameliorates experimental autoimmune myasthenia gravis.

MicroRNAs have been shown to be important regulators of immune homeostasis as patients with aberrant microRNA expression appeared to be more susceptible to autoimmune diseases. We recently found that miR-146a was up-regulated in activated B cells in response to rat acetylcholine receptor (AChR) α-subunit 97-116 peptide, and this up-regulation was significantly attenuated by AntagomiR-146a. Our data also demonstrated that silencing miR-146a with its inhibitor AntagomiR-146a effectively ameliorated clinical myasthenic symptoms in mice with ongoing experimental autoimmune myasthenia gravis. Furthermore, multiple defects were observed after miR-146a was knocked down in B cells, including decreased anti-R97-116 antibody production and class switching, reduced numbers of plasma cells, memory B cells and B-1 cells, and weakened activation of B cells. Previously, miR-146a has been identified as a nuclear factor-κB-dependent gene and predicted to base pair with the tumour necrosis factor receptor-associated factor 6 (TRAF6) and interleukin-1 receptor-associated kinase 1 (IRAK1) genes to regulate the immune response. However, our study proved that miR-146a inhibition had no effect on the expression of TRAF6 and IRAK1 in B cells. This result suggests that the function of miR-146a in B cells does not involve these two target molecules. We conclude that silencing miR-146a exerts its therapeutic effects by influencing the B-cell functions that contribute to the autoimmune pathogenesis of myasthenia gravis.

DBC1 is a suppressor of B cell activation by negatively regulating alternative NF-κB transcriptional activity.

CD40 and BAFFR signaling play important roles in B cell proliferation and Ig production. In this study, we found that B cells from mice with deletion of Dbc1 gene (Dbc1(-/-)) show elevated proliferation, and IgG1 and IgA production upon in vitro CD40 and BAFF, but not BCR and LPS stimulation, indicating that DBC1 inhibits CD40/BAFF-mediated B cell activation in a cell-intrinsic manner. Microarray analysis and chromatin immunoprecipitation experiments reveal that DBC1 inhibits B cell function by selectively suppressing the transcriptional activity of alternative NF-κB members RelB and p52 upon CD40 stimulation. As a result, when immunized with nitrophenylated-keyhole limpet hemocyanin, Dbc1(-/-) mice produce significantly increased levels of germinal center B cells, plasma cells, and Ag-specific Ig. Finally, loss of DBC1 in mice leads to higher susceptibility to experimental autoimmune myasthenia gravis. Our study identifies DBC1 as a novel regulator of B cell activation by suppressing the alternative NF-κB pathway.

RNA expression analysis of passive transfer myasthenia supports extraocular muscle as a unique immunological environment.

PURPOSE: Myasthenia gravis demonstrates a distinct predilection for involvement of the extraocular muscles (EOM), and we have hypothesized that this may be due to a unique immunological environment. To assess this hypothesis, we took an unbiased approach to analyze RNA expression profiles in EOM, diaphragm, and extensor digitorum longus (EDL) in rats with experimentally acquired myasthenia gravis (EAMG). METHODS: Experimentally acquired myasthenia gravis was induced in rats by intraperitoneal injection of antibody directed against the acetylcholine receptor (AChR), whereas control rats received antibody known to bind the AChR but not induce disease. After 48 hours, animals were killed and muscles analyzed by RNA expression profiling. Profiling results were validated using qPCR and immunohistochemical analysis. RESULTS: Sixty-two genes common among all muscle groups were increased in expression. These fell into four major categories: 12.8% stress response, 10.5% immune response, 10.5% metabolism, and 9.0% transcription factors. EOM expressed 212 genes at higher levels, not shared by the other two muscles, and a preponderance of EOM gene changes fell into the immune response category. EOM had the most uniquely reduced genes (126) compared with diaphragm (26) and EDL (50). Only 18 downregulated genes were shared by the three muscles. Histological evaluation and disease load index (sum of fold changes for all genes) demonstrated that EOM had the greatest degree of pathology. CONCLUSIONS: Our studies demonstrated that consistent with human myasthenia gravis, EOM demonstrates a distinct RNA expression signature from EDL and diaphragm, which is based on differences in the degree of muscle injury and inflammatory response.

Drosophila Nesprin-1 controls glutamate receptor density at neuromuscular junctions.

Nesprin-1 is a core component of a protein complex connecting nuclei to cytoskeleton termed LINC (linker of nucleoskeleton and cytoskeleton). Nesprin-1 is anchored to the nuclear envelope by its C-terminal KASH domain, the disruption of which has been associated with neuronal and neuromuscular pathologies, including autosomal recessive cerebellar ataxia and Emery-Dreifuss muscular dystrophy. Here, we describe a new and unexpected role of Drosophila Nesprin-1, Msp-300, in neuromuscular junction. We show that larvae carrying a deletion of Msp-300 KASH domain (Msp-300 (∆KASH) ) present a locomotion defect suggestive of a myasthenia, and demonstrate the importance of muscle Msp-300 for this phenotype, using tissue-specific RNAi knock-down. We show that Msp-300 (∆KASH) mutants display abnormal neurotransmission at the larval neuromuscular junction, as well as an imbalance in postsynaptic glutamate receptor composition with a decreased percentage of GluRIIA-containing receptors. We could rescue Msp-300 (∆KASH) locomotion phenotypes by GluRIIA overexpression, suggesting that the locomotion impairment associated with the KASH domain deletion is due to a reduction in junctional GluRIIA. In summary, we found that Msp-300 controls GluRIIA density at the neuromuscular junction. Our results suggest that Drosophila is a valuable model for further deciphering how Nesprin-1 and LINC disruption may lead to neuronal and neuromuscular pathologies.

Delivery of an miR155 inhibitor by anti-CD20 single-chain antibody into B cells reduces the acetylcholine receptor-specific autoantibodies and ameliorates experimental autoimmune myasthenia gravis.

MicroRNA-155 (miR155) is required for antibody production after vaccination with attenuated Salmonella. miR155-deficient B cells generated reduced germinal centre responses and failed to produce high-affinity immunoglobulin (Ig)G1 antibodies. In this study, we observed up-regulation of miR155 in the peripheral blood mononuclear cells (PBMCs) of patients with myasthenia gravis (MG), and miR155 was also up-regulated in torpedo acetylcholine receptor (T-AChR)-stimulated B cells. We used an inhibitor of miR155 conjugated to anti-CD20 single-chain antibody to treat both the cultured B cells and the experimental autoimmune MG (EAMG) mice. Our results demonstrated that silencing of miR155 by its inhibitor impaired the B cell-activating factor (BAFF)-R-related signalling pathway and reduced the translocation of nuclear factor (NF)-κB into the nucleus. Additionally, AChR-specific autoantibodies were reduced, which may be related to the altered amounts of marginal zone B cells and memory B cells in the spleens of EAMG mice. Our study suggests that miR155 may be a promising target for the clinical therapy of MG.

RNA interference targeting Bcl-6 ameliorates experimental autoimmune myasthenia gravis in mice.

Follicular helper T (Tfh) cells are dedicated to providing help to B cells and are strongly associated with antibody-mediated autoimmune disease. B cell lymphoma 6 (Bcl-6) is a key transcription factor of Tfh cells, and IL-21 is known to be a critical cytokine produced by Tfh cells. We silenced Bcl-6 gene expression using RNA interference (RNAi) delivered by a lentiviral vector, to evaluate the therapeutic role of Bcl-6 short hairpin RNAs (shRNAs) in experimental autoimmune myasthenia gravis (EAMG). Our data demonstrate that CD4(+)CXCR5(+)PD-1(+) Tfh cells, Bcl-6 and IL-21 were significantly increased in EAMG mice, compared with controls. In addition, we found that frequencies of Tfh cells were positively correlated with the levels of serum anti-AChR Ab. In-vivo transduction of lenti-siRNA-Bcl6 ameliorates the severity of ongoing EAMG with decreased Tfh cells, Bcl-6 and IL-21 expression, and leads to decreased anti-AChR antibody levels. Furthermore, we found that siRNA knockdown of Bcl-6 expression increases the expression of Th1(IFN-γ, T-bet) and Th2 markers (IL-4 and GATA3), but failed to alter the expression of Th17-related markers (RORγt, IL-17) and Treg markers (FoxP3). Our study suggests that Tfh cells contribute to the antibody production and could be one of the most important T cell subsets responsible for development and progression of EAMG or MG. Bcl-6 provides a promising therapeutic target for immunotherapy not only for MG, but also for other antibody-mediated autoimmune diseases.

Identification of novel MicroRNA signatures linked to experimental autoimmune myasthenia gravis pathogenesis: down-regulated miR-145 promotes pathogenetic Th17 cell response.

Emerging evidence demonstrates that miRNAs, a new family of key mRNA regulatory molecules, have crucial roles in controlling and modulating immunity. Their contribution to myasthenia gravis (MG), a T cell-dependent, antibody-mediated nervous system autoimmune disease, has not been thoroughly investigated. In the present study, using a highly sensitive microarray-based approach, we identified 11 miRNAs with differential expression between Peripheral Blood Mononuclear Cells (PBMC) from experimental autoimmune MG (EAMG) rats and control rats. miR-145 is one of the most significantly down-regulated miRNAs in PBMC from EAMG rats. Down-regulation of miR-145 expression was confirmed in PBMC and CD4+CD25- T cells (T effector cells) from both EAMG rats and MG patients by real-time PCR. Bioinformatics target prediction identified two crucial immune-related molecules-CD28 and NFATc1, as putative targets of miR-145. Furthermore, miR-145 inhibited CD28 and NFATc1 expression by directly targeting their 3'-UTRs, which was abolished by mutation of the miR-145 and CD28/NFATc1 binding sites. In vitro up-regulation of miR-145 in CD4+ T cells can significantly reduce CD28 protein levels accompanied by decreased proliferative response. In a dendritic cell (DC)-T cell coculture system, overexpression of miR-145 in AChR-specific CD4+ T cells suppresses NFATc1 expression and T Helper 17 cells level. Finally, we observed that administration of lentiviral-miR-145 decreased the severity of ongoing, established EAMG with decreased IL-17 production, and also decreased serum anti-AChR IgG levels. Our studies provide an important new insight into the pathogenesis of EAMG and MG, which may open a new perspective for the development of effective gene therapy for EAMG/MG.

Proteomic analysis of rat tibialis anterior muscles at different stages of experimental autoimmune myasthenia gravis.

Myasthenia gravis (MG) is an autoimmune disease in which autoantibodies, most commonly directed against the acetylcholine receptor (AChR), impair neuromuscular transmission and cause muscle weakness. In this study, we utilized two-dimensional difference in-gel electrophoresis (2D-DIGE) to analyze the muscle's proteomic profile at different stages of experimental autoimmune myasthenia gravis (EAMG). We identified twenty-two differentially expressed proteins, mainly related to metabolic and stress-response pathways. Interestingly, these identified proteins have also been associated with other contraction-impairing muscle pathologies (e.g. inclusion body myositis), suggesting a similar response of the muscle to such conditions.

Specific binding of collagen Q to the neuromuscular junction is exploited to cure congenital myasthenia and to explore bases of myasthenia gravis.

Acetylcholinesterase (AChE) at the neuromuscular junction (NMJ) is anchored to the synaptic basal lamina via a triple helical collagen Q (ColQ) in the form of asymmetric AChE (AChE/ColQ). The C-terminal domain of ColQ binds to MuSK, the muscle-specific receptor tyrosine kinase, that mediates a signal for acetylcholine receptor (AChR) clustering at the NMJ. ColQ also binds to heparan sulfate proteoglycans including perlecan. Congenital defects of ColQ cause endplate AChE deficiency. A single intravenous administration of adeno-associated virus serotype 8 (AAV8)-COLQ to Colq-/- mice rescued motor functions, synaptic transmission, and the ultrastructure of NMJ. We also injected AAV1-COLQ-IRES-EGFP to the left tibialis anterior and observed colocalization of AChE/ColQ at all the examined NMJs of the non-injected limbs. Additionally, injection of purified recombinant AChE/ColQ protein complex into gluteus maximus accumulated AChE in non-injected forelimbs. These observations suggest that the tissue-targeting signal of ColQ can be exploited to specifically deliver the transgene product to the target tissue. MuSK antibody-positive myasthenia gravis (MG) accounts for 5-15% of autoimmune MG. As AChR deficiency is typically mild and as cholinesterase inhibitors are generally ineffective or worsen myasthenic symptoms, we asked if the patient's MuSK-IgG interferes with binding of ColQ to MuSK. In vitro overlay of AChE/ColQ to muscle sections of Colq-/- mice revealed that MuSK-IgG blocks binding of ColQ to the NMJ. In vitro plate-binding of MuSK to ColQ disclosed that MuSK-IgG exerts a dose-dependent block of MuSK-ColQ interaction. In addition, passive transfer of MuSK-IgG to mice reduced the size and density of ColQ to ∼10% of controls and had a lesser effect on the sizes and densities of AChR and MuSK. Elucidation of molecular mechanisms of specific binding of ColQ to the NMJ enabled us to ameliorate devastating myasthenic symptoms of Colq-/- mice and to reveal bases of anti-MuSK MG.

Ocular and generalized myasthenia gravis induced by human acetylcholine receptor γ subunit immunization.

INTRODUCTION: HLA-DQ8 transgenic mice develop ocular myasthenia gravis (oMG), which then progresses to generalized MG (gMG) when immunized with the human acetylcholine receptor (H-AChR) α subunit. Because the fetal AChR γ subunit is expressed in adult extraocular muscles, we anticipated that γ subunit immunization would generate an immune response to mouse AChR and induce MG in mice. RESULTS: H-AChR γ subunit immunization in HLA-DQ8 mice induced an autoimmune response to mouse AChR and led to the destruction of AChR in the neuromuscular junction (NMJ) by anti-AChR antibody and complement activation, and it triggered upregulation of AChR gene transcription. CONCLUSION: Our findings indicate that oMG may be induced by immunity to the AChR γ subunit.

Genetic deficiency of estrogen receptor alpha fails to influence experimental autoimmune myasthenia gravis pathogenesis.

Autoimmune myasthenia gravis (MG) is characterized by T cell and antibody responses to muscle nicotinic acetylcholine receptor (AChR). It is well known that MG as other autoimmune diseases is more prevalent in women than men and estrogen administration enhances experimental autoimmune MG (EAMG) severity. To determine whether estrogen influences EAMG pathogenesis through estrogen receptor alpha (ERα) activation, ERα knockout (KO) and wild-type (WT) C57BL/6 mice were immunized with AChR. ERα KO mice were equally susceptible to EAMG as WT mice and exhibited comparable antibody and immunopathological responses to AChR, suggesting a lack of involvement of ERα in EAMG pathogenesis.