Masakatsu Motomura

The Muscle Protein Dok-7 Is Essential for Neuromuscular Synaptogenesis

The formation of the neuromuscular synapse requires muscle-specific receptor kinase (MuSK) to orchestrate postsynaptic differentiation, including the clustering of receptors for the neurotransmitter acetylcholine. Upon innervation, neural agrin activates MuSK to establish the postsynaptic apparatus, although agrin-independent formation of neuromuscular synapses can also occur experimentally in the absence of neurotransmission. Dok-7, a MuSK-interacting cytoplasmic protein, is essential for MuSK activation in cultured myotubes; in particular, the Dok-7 phosphotyrosine-binding domain and its target in MuSK are indispensable. Mice lacking Dok-7 formed neither acetylcholine receptor clusters nor neuromuscular synapses. Thus, Dok-7 is essential for neuromuscular synaptogenesis through its interaction with MuSK.

Autoantibodies to low‐density lipoprotein receptor–related protein 4 in myasthenia gravis

Myasthenia gravis (MG) is an autoimmune disease of the neuromuscular junction, where acetylcholine receptor (AChR), muscle‐specific kinase (MuSK), and low‐density lipoprotein (LDL) receptor‐related protein 4 (Lrp4) are essential. About 80% and 0% to 10% of patients with generalized MG have autoantibodies to AChR and MuSK, respectively, but pathogenic factors are elusive in others. Here we show that a proportion of AChR antibody‐negative patients have autoantibodies to Lrp4. These antibodies inhibit binding of Lrp4 to its ligand and predominantly belong to the immunoglobulin G1 (IgG1) subclass, a complement activator. These findings together indicate the involvement of Lrp4 antibodies in the pathogenesis of AChR antibody‐negative MG. Ann Neurol 2011

Dok-7 Mutations Underlie a Neuromuscular Junction Synaptopathy

Congenital myasthenic syndromes (CMSs) are a group of inherited disorders of neuromuscular transmission characterized by fatigable muscle weakness. One major subgroup of patients shows a characteristic “limb girdle” pattern of muscle weakness, in which the muscles have small, simplified neuromuscular junctions but normal acetylcholine receptor and acetylcholinesterase function. We showed that recessive inheritance of mutations in Dok-7, which result in a defective structure of the neuromuscular junction, is a cause of CMS with proximal muscle weakness.

An improved diagnostic assay for Lambert-Eaton myasthenic syndrome.

A new immunoprecipitation assay has been established for detecting antibodies to voltage-gated calcium channels (VGCCs) in Lambert-Eaton myasthenic syndrome (LEMS), using 125I-omega-conotoxin MVIIC, which binds to P-type VGCCs, to label extracts of human cerebellum. Fifty six of 66 serum samples (85%) from patients with clinically and electrophysiologically definite LEMS were positive for the presence of VGCC antibodies, defined as a titre > 3 SD above the mean for the healthy controls (n = 10). All disease controls (n = 40) were negative. This sensitive immunoassay should prove valuable in the diagnosis of LEMS.

Acetylcholine receptors loss and postsynaptic damage in MuSK antibody-positive myasthenia gravis

Muscle‐specific tyrosine kinase (MuSK) antibodies are found in some patients with “seronegative” myasthenia gravis (MG), but how they cause myasthenic symptoms is not clear. We visualized acetylcholine receptors (AChRs) and complement component 3 (C3) in muscle biopsies from 10 Japanese MG patients with MuSK antibodies, compared with 42 with AChR antibodies. The AChR density was not significantly decreased in MuSK antibody (Ab)–positive end‐plates compared with AChR antibody–positive end‐plates, and C3 was detected in only two of eight MuSK Ab–positive patients. MuSK antibodies do not appear to cause substantial AChR loss, complement deposition, or morphological damage. Effects on MuSK function need to be explored. Ann Neurol 2005;57:289–293

Reduction of P/Q-type calcium channels in the postmortem cerebellum of paraneoplastic cerebellar degeneration with Lambert-Eaton myasthenic syndrome.

The aim of this study was to clarify whether autoimmunity against P/Q‐type voltage‐gated calcium channels (VGCCs) in the cerebellum was associated with the pathogenesis of paraneoplastic cerebellar degeneration (PCD) with Lambert‐Eaton myasthenic syndrome (LEMS). We used human autopsy cerebellar tissues from three PCD‐LEMS patients and six other disease patients including one with LEMS as the controls. We compared cerebellar P/Q‐type VGCC in these patients and controls for the amount and ratio of autoantibody‐channel complex using an 125I‐ω‐conotoxin MVIIC‐binding assay with Scatchard analysis, and their distribution using autoradiography. The quantity of cerebellar P/Q‐type VGCC measured by Scatchard analysis were reduced in PCD‐LEMS patients (63.0 ± 7.0fmol/mg, n = 3), compared with the controls (297.8 ± 38.9fmol/mg, n = 6). The ratio of autoantibody‐VGCC complexes to total P/Q‐type VGCCs measured by immunoprecipitation assay were increased in PCD‐LEMS patients. We analysed cerebellar specimens by autoradiography using 125I‐ω‐conotoxin MVIIC, which specifically binds to P/Q‐type VGCCs. In PCD‐LEMS cerebellum, the toxin binding sites of P/Q‐type VGCCs were markedly reduced compared with controls, especially in the molecular layer, which is the richest area of P/Q‐type VGCCs in the normal cerebellum. This suggests that P/Q‐type VGCCs of the cerebellar molecular layer is the immunological target in developing PCD‐LEMS.

Seronegative Lambert-Eaton myasthenic syndrome Study of 110 Japanese patients

The authors characterized the clinical and immunologic features of 110 patients with Lambert-Eaton myasthenic syndrome (LEMS). Anti-P/Q-type voltage-gated calcium channels (VGCC) antibodies were detected in 85% of the patients (seropositive) but not in the rest (seronegative). Except for the indication that small cell lung carcinoma is less common in seronegative patients, no significant differences were found in the clinical characteristics of patients who had or did not have anti-P/Q-type VGCC antibodies. The results of passive transfer experiments suggest that seronegative LEMS is also an autoantibody-mediated disorder.

Autoimmune Targets of Heart and Skeletal Muscles in Myasthenia Gravis

OBJECTIVE To investigate the clinical, histological, and immunological features of patients with myasthenia gravis (MG) who also developed myocarditis and/or myositis. DESIGN Observational and retrospective case series. SETTING Keio University, Hanamaki General Hospital, Kanazawa University, Nagasaki University, and Juntendo University. PATIENTS A cohort of 8 patients with MG with clinically defined inflammatory myopathies. INTERVENTIONS Clinical and histological features were described. Serological analyses included MG-related antistriational autoantibodies (those to titin, ryanodine receptor, muscular voltage-gated potassium channel Kv1.4) and myositis-specific autoantibodies. RESULTS Of 924 patients with MG, 8 (0.9%) had inflammatory myopathies. The mean (SD) onset age of MG was 55.3 (10.3) years. All patients showed severe symptoms with bulbar involvement; 5 patients had myasthenic crisis and 4 had invasive thymoma. Myocarditis was found in 3 patients and myositis in 6. Myocarditis, developing 13 to 211 months after the MG onset, was characterized by heart failure and arrhythmias. Myositis, developing before or at the same time as MG, affected limb and paraspinal muscles. Histological findings of skeletal muscles showed CD8(+) lymphocyte infiltration. Seven patients had 1 of these antistriational autoantibodies but not myositis-specific autoantibodies. Immunomodulatory therapy was required for all patients and was effective for both MG and inflammatory myopathies, although 1 patient died. CONCLUSIONS Heart and skeletal muscles are autoimmune targets in some patients with MG. This autoimmunity has a broad clinical spectrum with antistriational autoantibodies.

Characteristics of myasthenia gravis according to onset-age: Japanese nationwide survey

OBJECTIVE To clarify the prevalence and clinical characteristics of myasthenia gravis (MG) in Japan. METHODS We performed a nationwide epidemiological survey of MG in Japan. The clinical features were compared among five groups of patients, divided according to onset age. A generalized additive model (GAM) was used to assess the linearity of these relationships. RESULTS A total of 8542 patients were reported, and detailed data were analyzed for 3141 patients. The estimated number of MG patients in Japan was 15,100, giving a prevalence of 11.8 per 100,000. Elderly-onset MG (≥ 65 years) accounted for 7.3% in 1987 (adjusted for population in 2005), but this had increased to 16.8% in 2006. Infantile-onset MG (0-4 years) accounted for 10.1% in 1987, and was still as high as 7.0% in 2006. The rate of ocular MG was highest (80.6%) in infantile-onset and lowest (26.4%) in early-onset disease, but the rate rose again in the late-onset group. GAM analysis of the ocular form showed a U-shaped curve, with a dip in the 20s. Anti-acetylcholine receptor antibodies were positive in only 50% of infantile-onset, but nearly 90% of elderly-onset patients. GAM analyses assessing the concurrence of thymoma and hyperplasia both showed reversed U-shapes, with peaks in the 50s and 20s-40s, respectively. CONCLUSIONS Persistent high incidence of infantile-onset disease and clinical heterogeneity according to onset age are characteristic features of MG in Japan.

Effects of reduced joint mobility on sarcomere length, collagen fibril arrangement in the endomysium, and hyaluronan in rat soleus muscle.

Immobilization is often associated with a decrease in muscle elasticity. This condition is called muscle contracture, but the mechanism is not yet clear. We examined changes in ankle joint mobility, sarcomere length, collagen fibril arrangement in the endomysium, and hyaluronic acid (HYA) in muscular tissue 1, 2, 4, 8, and 12 weeks after immobilization of rat soleus muscles in shortened position. Ankle joint mobility decreased with the duration of immobilization. Sarcomere length had shortened 1week after immobilization, but did not show further change 2, 4, 8, and 12 weeks after immobilization. Collagen fibril arrangement in the endomysium 1 and 2 weeks after immobilization was longitudinal to the axis of the muscle fibers, whereas 4, 8, and 12 weeks after immobilization it was circumferential. HYA in muscular tissue had increased 1 week after immobilization but remained at the same level at weeks 2, 4, 8, and 12. Histochemically, HYA in the endomysium of immobilized muscular tissue was stained more strongly and widely than that in the control tissue. Increased HYA in muscular tissue may induce muscle stiffness, but the significance of how HYA is related to the mechanism of muscle contracture was not clear. The findings suggest that muscle contracture started 1 week after immobilization and increased with the length of immobilization. Consequently, muscle contracture is affected by the shortening muscle fibers during the early stage of immobilization, after which the collagen adapts by the fibril arrangement in the endomysium becoming more circumferential. This change in collagen fibril arrangement may cause advanced muscle contracture in the late stage of immobilization.