Myasthenia Gravis: From the Viewpoint of Pathogenicity Focusing on Acetylcholine Receptor Clustering, Trans-Synaptic Homeostasis and Synaptic Stability.

Myasthenia gravis (MG) is a disease of the postsynaptic neuromuscular junction (NMJ) where nicotinic acetylcholine (ACh) receptors (AChRs) are targeted by autoantibodies. Search for other pathogenic antigens has detected the antibodies against muscle-specific tyrosine kinase (MuSK) and low-density lipoprotein-related protein 4 (Lrp4), both causing pre- and post-synaptic impairments. Agrin is also suspected as a fourth pathogen. In a complex NMJ organization centering on MuSK: (1) the Wnt non-canonical pathway through the Wnt-Lrp4-MuSK cysteine-rich domain (CRD)-Dishevelled (Dvl, scaffold protein) signaling acts to form AChR prepatterning with axonal guidance; (2) the neural agrin-Lrp4-MuSK (Ig1/2 domains) signaling acts to form rapsyn-anchored AChR clusters at the innervated stage of muscle; (3) adaptor protein Dok-7 acts on MuSK activation for AChR clustering from "inside" and also on cytoskeleton to stabilize AChR clusters by the downstream effector Sorbs1/2; (4) the trans-synaptic retrograde signaling contributes to the presynaptic organization via: (i) Wnt-MuSK CRD-Dvl-ß catenin-Slit 2 pathway; (ii) Lrp4; and (iii) laminins. The presynaptic Ca2+ homeostasis conditioning ACh release is modified by autoreceptors such as M1-type muscarinic AChR and A2A adenosine receptors. The post-synaptic structure is stabilized by: (i) laminin-network including the muscle-derived agrin; (ii) the extracellular matrix proteins (including collagen Q/perlecan and biglycan which link to MuSK Ig1 domain and CRD); and (iii) the dystrophin-associated glycoprotein complex. The study on MuSK ectodomains (Ig1/2 domains and CRD) recognized by antibodies suggested that the MuSK antibodies were pathologically heterogeneous due to their binding to multiple functional domains. Focussing one of the matrix proteins, biglycan which functions in the manner similar to collagen Q, our antibody assay showed the negative result in MG patients. However, the synaptic stability may be impaired by antibodies against MuSK ectodomains because of the linkage of biglycan with MuSK Ig1 domain and CRD. The pathogenic diversity of MG is discussed based on NMJ signaling molecules.

Synaptic Homeostasis and Its Immunological Disturbance in Neuromuscular Junction Disorders.

In the neuromuscular junction, postsynaptic nicotinic acetylcholine receptor (nAChR) clustering, trans-synaptic communication and synaptic stabilization are modulated by the molecular mechanisms underlying synaptic plasticity. The synaptic functions are based presynaptically on the active zone architecture, synaptic vesicle proteins, Ca2+ channels and synaptic vesicle recycling. Postsynaptically, they are based on rapsyn-anchored nAChR clusters, localized sensitivity to ACh, and synaptic stabilization via linkage to the extracellular matrix so as to be precisely opposed to the nerve terminal. Focusing on neural agrin, Wnts, muscle-specific tyrosine kinase (a mediator of agrin and Wnts signalings and regulator of trans-synaptic communication), low-density lipoprotein receptor-related protein 4 (the receptor of agrin and Wnts and participant in retrograde signaling), laminin-network (including muscle-derived agrin), extracellular matrix proteins (participating in the synaptic stabilization) and presynaptic receptors (including muscarinic and adenosine receptors), we review the functional structures of the synapse by making reference to immunological pathogenecities in postsynaptic disease, myasthenia gravis. The synapse-related proteins including cortactin, coronin-6, caveolin-3, doublecortin, R-spondin 2, amyloid precursor family proteins, glia cell-derived neurotrophic factor and neurexins are also discussed in terms of their possible contribution to efficient synaptic transmission at the neuromuscular junction.

Antibodies against Wnt receptor of muscle-specific tyrosine kinase in myasthenia gravis.

Muscle-specific tyrosine kinase (MuSK) antibodies are detected in a proportion of myasthenia gravis (MG) patients who are negative for acetylcholine receptor (AChR) antibodies and have prominent bulbar weakness and crises. In the MuSK ectodomains, the immunoglobulin-like 1 and 2 domains (Ig1/2) mediate the agrin-Lrp4-MuSK signaling and the cysteine-rich domain (CRD) mediates the Wnt-MuSK-Dishevelled signaling; both contribute to AChR clustering. Immunoblotting against recombinant proteins showed MuSK Ig1/2 antibodies in 33 anti-AChR-negative MG patients; 10 patients of them (30%) were additionally positive for MuSK CRD antibodies. The result suggests that MuSK antibodies have heterogeneity in their binding to functional domains of MuSK.

Structure of the neuromuscular junction: function and cooperative mechanisms in the synapse.

As an overview of the structure of the neuromuscular junction, three items are described focusing on cooperative mechanisms involving the synapse and leading to muscle contraction: (1) presynaptic acetylcholine release regulated by vesicle cycling (exocytosis and endocytosis); the fast-mode of endocytosis requires a large influx of external Ca(2+) and is promoted by the activation of G protein-coupled receptors and receptor tyrosine kinases; (2) postsynaptic acetylcholine receptor clustering mediated by the muscle-specific, Dok7-stimulated tyrosine kinase (MuSK) through two signaling mechanisms: one via agrin-Lrp4-MuSK (Ig1/2 domains) and the second via Wnt-MuSK (Frizzled-like cysteine-rich domain)-adaptor Dishevelled; Wnts/MuSK and Lrp4 direct a retrograde signal to presynaptic differentiation; (3) muscle contractile machinery regulated by Ca(2+) -release and Ca(2+) -influx channels, including the depolarization-activated ryanodine receptor-1 and the receptor- and/or store-operated transient receptor potential canonical. The first mechanism is dysfunctional in Lambert-Eaton myasthenic syndrome, the second in anti-acetylcholine receptor-negative myasthenia gravis (MG), and the third in thymoma-associated MG.

Randomised, double-blind, placebo-controlled study of tacrolimus in myasthenia gravis.

OBJECTIVES: To evaluate the ability of tacrolimus to reduce the corticosteroid dose in patients with myasthenia gravis (MG) and the drug's safety in a double-blind, placebo-controlled, parallel group study. METHODS: Patients being treated with oral prednisolone at doses equivalent to 10-20 mg/day, and with stable symptoms, were randomised to tacrolimus or placebo in a 28-week double-blind study. The dose of corticosteroid was tapered with the procedures specified in the protocol. The primary efficacy endpoint was the mean daily prednisolone dose given in the last 12 weeks of the study. RESULTS: Eighty patients received the study drug (40 patients in each group) and were included in the full analysis set. In the full analysis set, there was no significant difference in the primary efficacy endpoint between the two groups (p = 0.078). However, some secondary analyses suggested the steroid-sparing effect of tacrolimus. Tacrolimus was well tolerated, and no safety concerns were noted. CONCLUSIONS: This study suggests that tacrolimus has a potential advantage as a steroid-sparing agent in the treatment of MG patients. CLINICAL TRIAL REGISTRATION NUMBER: NCT00309088. Name of the trial registry: FK506 Phase 3 STUDY: A STUDY for Steroid Non-Resistant MG Patients.

[Editorial introduction: from the structure and functions of the neuromuscular junction to the diseases].

The neuromuscular junction has been recognized as a site for autoimmune and genetic disorders. Myasthenia gravis (MG) is mainly caused by postsynaptic nicotinic acetylcholine receptor (AChR) IgG1 antibodies that are directed against α-subunit 67-76 and 125-147 and activate complement. Thymic abnormalities are present in the autoimmune background. A proportion of MG patients without conformation-dependent AChR antibodies assayed by the cell-based method have muscle-specific tyrosine kinase (MuSK) antibodies which are largely IgG4 and partially IgG1. MuSK is activated by Dok-7 and Lrp4 (agrin receptor) and contributes to AChR clustering at the postsynaptic membrane via various kinase cascades in collaboration with Wnt-MuSK/Frizzled-Dishevelled signaling. Rapsyn interacts with MuSK-linked chaperones to stabilize postsynaptic architecture and also contributes to AChR phosphorylation. MG-associated thymomas express antigens that trigger antibody responses which play a part in disease generation and modification. Among these, ryanodine receptor-1 (RyR1; acts on sarcoplasmic Ca2+ release) antibodies cause muscle contractile weakness. Transient receptor potential canonical-3 (TRPC3) antibodies are also detected in thymoma-associated MG patients; they may participate in muscle contractile weakness because TRPC3 acts on RyR1, and may also impair the refill of sarcoplasmic Ca2+ stores since TRPCs contribute to the receptor-operated Ca2+ influx via the phospholipase C (PLC)-diacylglycerol (DAG) pathway in cooperation with the store-operated, STIM1/Orai1-mediated Ca2+ influx and TRPCs-Homerl-IP3R interaction. Lambert-Eaton myasthenic syndrome (LEMS) is caused by reduced ACh quantal release that occurs mainly because of presynaptic P/Q-type voltage-gated Ca2+ channel (VGCC) antibodies. Physicians should be vigilant for LEMS because it may predict an underlying malignancy, particularly small-cell lung carcinoma; SOX-1 antibodies are usually present in these patients and are absent in those who do not have caucer. Some patients with LEMS have antibodies against synaptotagmin-1, which associates with SNARE complex and functions as Ca2+ sensor for exocytosis. The stimulation of the M1-type presynaptic muscarinic AChR (mAChR)(G-proterin-coupled receptor) can compensate for the deficiency of Ca2+-mediated ACh quantal release via the PLC/DAG-mediated mechanism; This acts in a manner similar to the BDNF/NT4-TrkB interaction. The detection of M1 mAChR antibodies in LEMS suggests an impaired compensatory mechanism and corresponds, at least in part, to autonomic symptoms. Congenital myasthenic syndromes are classified into presynaptic, synaptic basal lamina and postsynaptic defects.

[Current status and future prospects of therapy for myasthenia gravis: considering thymectomy].

The benefits of thymectomy in non-thymomatous patients with myasthenia gravis (MG) remain controversial. The first detailed case of thymectomy in a patient with MG was reported in 1939, following which many cases were published. In 2000, Gronseth and Barohn reported the first meta-analysis of the effectiveness of thymectomy in MG patients without thymoma. They reviewed 28 papers systematically and reached these conclusions: (1) The benefit of thymectomy in non-thymomatous autoimmune MG has not been conclusively established, and (2) a well-designed controlled trial is essential. Following this report, Newsom-Davis et al. designed a thymectomy trial for non-thymomatous MG patients receiving prednisone (the MGTX study). Their study compared extended trans-sternal thymectomy (ETTX) combined with prednisone and prednisone alone groups with the aim to answer 3 questions: (1) Is the former more effective in improving myasthenic weakness? (2) Does the former require a lower total dose of prednisone, and thus decrease the likelihood of concurrent and long-term toxic effects? (3) Does the former enhance patients' quality of life by reducing adverse events and symptoms associated with the therapy? Currently, 67 centers, including our institute, are involved in this study. In total, 106 patients have been enrolled (the recruitment goal is 150). The patients are scheduled for a 5-year follow-up. The MGTX study will offer new information on the role of thymectomy in improving the quality of life of patients with MG.

[Recent advance in research for myasthenia gravis, in relation to various antibodies affecting synaptic structure and function].

Autoantibodies impair acetylcholine receptor (AChR) in myasthenia gravis (MG) and P/Q-type voltage-gated calcium channel (VGCC) in Lambert-Eaton myasthenic syndrome (LEMS). (1) Some of MG and LEMS patients are "seronegative" for respective antibodies or modified by antibodies that recognize other proteins than AChR and VGCC such as MuSK, AChR allosteric site, membrane Na+ channel and ryanodine receptor-1 (RyR1) in MG, and synaptotagmin-1 in LEMS. (2) Autoimmune responses affect the proteins participating in the mechanisms to compensate for synaptic disorders on the basis of presynaptic Ca2+ homeostasis provided by VGCC and non-VGCC (receptor-operated TRPCs): they act as enhancers of Ca(2+) -mediated ACh release via phospholipase C signaling pathways including M1-type presynaptic muscarinic AChR, neurotrophin receptor (TrkB), and fast-mode of synaptic vesicle recycling. (3) The pathophysiology contributive to contractile fatigue in MG includes RyR1 and also TRPC3. The TRPC3 also forms a complex with STIM1 and Orail to make up for Ca2+ after sarcoplasmic Ca2+ release. The prevalent detection of anti-TRPC3 antibodies in MG with thymoma could affect muscle contractile machineries in addition to anti-RyR1-induced affection. (4) When one faces "seronegative" MG, one should be cautious to conformation-specific antibodies and also congenital myasthenic syndromes.

Lambert-Eaton myasthenic syndrome: search for alternative autoimmune targets and possible compensatory mechanisms based on presynaptic calcium homeostasis.

The Lambert-Eaton myasthenic syndrome (LEMS) is a disease of neuromuscular transmission in which autoantibodies against the P/Q-type voltage-gated calcium channel (VGCC) at the presynaptic nerve terminal play a major role in decreasing quantal release of acetylcholine (ACh), resulting in skeletal muscle weakness and autonomic symptoms. It is associated with cancer, particularly small-cell lung carcinoma (SCLC), in 50-60% of LEMS patients; the nerve terminal and carcinoma cells apparently share a common antigen (VGCC), suggesting an immunological cross-reactivity that may lead to the neurological abnormality. Non-tumor LEMS has a strong association with HLA-DR3-B8. In approximately 15% of LEMS patients, no anti-P/Q-type VGCC antibodies are found, suggesting recognition of other targets(s). The VGCC-associated protein synaptotagmin could be one candidate, because it acts as an exocytotic calcium receptor, is implicated in fast ACh release; its N-terminus is exposed extracellularly during exocytosis and it is expressed in SCLC. Antibodies against synaptotagmin-1 were detected in both anti-VGCC-positive and -negative LEMS patients (20%), and it can be immunogenic, allowing induction of an animal model of LEMS. Another candidate target is the M1-type presynaptic muscarinic ACh receptor (M1 mAChR), also expressed extracellularly on motor nerve terminals; it modulates cholinergic transmission, linking to P/Q-type VGCC. In our series of 25 LEMS patients with and without SCLC, anti-M1 mAChR antibodies were prevalent in both anti-VGCC-positive and -negative LEMS patients. Autonomic symptoms seemed more frequent in the latter; serum from one of them passively transferred LEMS-type electrophysiological defects to mice. As a compensatory mechanism, researchers in Oxford suggested a shift in the dependence of ACh release from the P/Q-type to other types of VGCC. We have also focused on G protein-coupled mAChRs and neurotrophins, which may affect both P/Q-type VGCC and clathrin-independent "kiss-and-run" synaptic vesicle recycling (fast-mode of endocytosis) via protein kinase C activation. We hypothesize that these signaling cascades help to compensate for the immune-mediated defects in calcium entry in LEMS, compensation that may frequently be restricted by the coincident anti-M1 mAChR antibodies in this disease.

Autoantibodies against TRPC3 and ryanodine receptor in myasthenia gravis.

The transient receptor potential canonical type-3 (TRPC3, receptor- and store-operated Ca(2+) influx channel) participates in skeletal muscle contraction; its functional interactions with ryanodine receptor-1 (RyR1) are independent of sarcoplasmic Ca(2+) content and dihydropyridine receptor. In 25 generalized myasthenia gravis (MG), we detected antibodies against human TRPC3 peptide in 9 patients (8 with thymoma and one with hyperplastic thymus) and those against human RyR1 peptides in 16 patients (15 with thymoma and one with hyperplastic thymus). Both antibodies were found in patients with more severe myasthenia and could contribute to the contractile abnormalities in MG.

Is excitation-contraction coupling impaired in myasthenia gravis?

OBJECTIVE: To investigate whether excitation-contraction (E-C) coupling of muscle is impaired in patients with myasthenia gravis (MG). METHODS: In 51 patients with generalized MG and 35 normal subjects, compound muscle action potentials (CMAPs) of the abductor pollicis brevis, and movement-related potentials using an accelerometer placed at the thumb tip were simultaneously recorded after median nerve stimulation at the wrist. The E-C coupling time (ECCT) was estimated by a latency difference between CMAP and movement-related potential. Antibodies against acetylcholine receptor (AChR), ryanodine receptor (RyR), and muscle specific receptor tyrosine kinase (MuSK) were measured by immunoassays. RESULTS: The mean ECCT was significantly longer in patients with MG (mean+/-SEM; 2.79+/-0.1 ms; p=0.002) than in normal controls (2.52+/-0.1 ms). Among MG patients, the mean ECCT was longer for patients with thymoma than for those without it (P=0.04), and was shorter for patients treated with FK506 (an immunosuppressant and also an enhancer of RyR related Ca(2+) release) than for those not receiving this treatment (p=0.04). ECCT had no significant correlation with anti-AChR, anti-RyR, or anti-MuSK antibodies. CONCLUSIONS: In MG, E-C coupling appears to be impaired, particularly in patients with thymoma, and FK506 possibly facilitates E-C coupling. SIGNIFICANCE: The functional implication of impaired E-C coupling is not established, but it may contribute to muscle weakness in patients with MG.

Myasthenia gravis presenting with unusual neurogenic muscle atrophy.

We report a patient with myasthenia gravis who had neurogenic muscle atrophy in association with external ophthalmoplegia and weakness of the upper limbs. Neurogenic changes in the limb muscles were found on needle electromyography and histological studies. Symptoms improved and atrophy of the limbs diminished after intravenous immunoglobulin and oral corticosteroid therapy. We concluded that functional interruption of the neuromuscular junction caused the neurogenic muscle atrophy and that this was relieved by appropriate therapy.

Voglibose inhibits postprandial hypotension in neurologic disorders and elderly people.

The authors examined blood pressure, glucose, insulin, and neurotensin before and after intake of 75 g glucose with or without voglibose in 28 neurologic patients and 20 healthy controls. Voglibose significantly prevented hypotension and neurotensin increment after glucose intake and had no influence on glucose or insulin increment. These results suggest that voglibose benefits postprandial hypotension.

Elevation of IL-12 p40 and its antibody in myasthenia gravis with thymoma.

We examined the serum levels of cytokines, interferon (IFN)-alpha, IFN-gamma, interleukin (IL)-4, IL-12 p40, and IL-12 p70; those that affect the T helper 1 and 2 balance in patients with myasthenia gravis (MG). Among the cytokines tested, only IL-12 p40, together with the serum titer of anti-IL-12 p40 antibody, was significantly elevated in MG with thymoma. Their elevation was independent of the histopathology of thymoma. Thymectomy decreased the levels of IL-12 p40 accompanied by the anti-acetylcholine receptor antibody, but not anti-IL-12 p40 antibodies. These data strongly suggest the association of IL-12 p40 and its autoantibody with the immunopathology of MG with thymoma.

Lambert-Eaton myasthenic syndrome as an autoimmune calcium channelopathy.

Lambert-Eaton myasthenic syndrome, often associated with small-cell lung carcinoma, is a disease of neuromuscular transmission in which antibodies directed against voltage-gated calcium channel (VGCC)(P/Q-type) in the motor nerve terminal play a crucial role in causing a deficient quantal release of acetylcholine. The motor nerve terminal and carcinoma cell may share a common antigen. The study using synthetic peptides and recombinant protein specified the extracellular S5-S6 linker regions in 3 of 4 domains as immunodominant sites in the molecular structure of P/Q-type VGCC alpha1 subunit. Also, the study by use of peptides and recombinant protein corresponding to synaptotagmin I suggested that in this functionally VGCC-associated presynaptic protein, the segment which exposes extracellularly during exocytosis can be immunogenic for the syndrome.

Comparative study of pre-operative thymic imaging and pathology in patients with myasthenia gravis.

The objective of our study is to compare the diagnostic significance of mediastinal CT and (201)Tl-single-photon emission computed tomography (SPECT) with thymic histology in patients with myasthenia gravis (MG). The subjects were 30 patients with MG who were scheduled to receive thymectomy. They did not receive immunosuppressive therapy. The mediastinal CT and (201)Tl-SPECT were performed before the thymectomy. As a consequence, 9 patients had thymoma, 11 had lymphoid follicular hyperplasia (LFH), and 10 had normal thymus on histologic examination after thymectomy. Retrospectively, CT diagnosed the histology of all 9 patients with thymoma, and 5 of 11 with LFH. (201)Tl-SPECT could detect abnormal accumulations in only 6 of 9 cases of thymoma, and 6 of 11 cases of LFH. (201)Tl-SPECT could not distinguish thymoma from LFH. We conclude that a CT study is recommended as an essential study for the detection of thymic abnormalities in pre-operative patients with MG.

Clinical study of FK506 in patients with myasthenia gravis.

To investigate the usefulness of low-dose FK506 for the treatment of myasthenia gravis (MG), we treated 19 patients with generalized MG in a 16-week open clinical trial of FK506 (3-5 mg/day). At the end of the trial, total MG scores (range: 0-27 points) improved by 3 points or more in 7 of 19 patients (37%), and activities of daily living (ADL) scores (range: 0-6 points) also improved by 1 point or more in 8 of 19 patients (42%). Nine of 19 patients (47%) showed improvement in either MG or ADL scores. Significant reduction of anti-acetylcholine receptor antibody titers and interleukin 2 production were observed at the end of this study. Minor but commonly observed side effects were an increase in neutrophil count and a decrease in lymphocyte count. No serious adverse events such as renal toxicity or diabetes mellitus were observed during the 16-week treatment period. FK506 could safely serve as an adjunct to steroid therapy for MG at low dosage.

Sensorimotor polyneuropathy with 5-aminosalicylic acid: a case report.

A 69-year-old man with ulcerative colitis (UC) developed sensorimotor polyneuropathy. First, he received salazosulphapyridine (SASP) as treatment for the UC. The symptoms of UC disappeared immediately, but he developed skin eruptions and dysesthesia in his lower limbs. When SASP was changed to 5-aminosalicylic acid (5-ASA), his skin eruptions were resolved, however, he developed weakness and atrophy in his right arm as well as progressive worsening of the dysesthesia in his legs and gait disturbance. Deep tendon reflexes (DTR) were absent in all extremities. After 5-ASA was discontinued, the polyneuropathy symptoms recovered gradually. This clinical course suggests that the sensorimotor polyneuropathy may have been caused by 5-ASA.