Hirokazu Shiraishi

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.

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.

Increased T-cell immunity against aquaporin-4 and proteolipid protein in neuromyelitis optica

In neuromyelitis optica (NMO), B-cell autoimmunity to aquaporin-4 (AQP4) has been shown to be essential. However, the role of T cells remains ambiguous. Here, we first showed an increase in CD69+ activated T cells in PBMCs during NMO relapses. Next, T-cell responses to AQP4 and myelin peptides were studied in 12 NM0 patients, 10 multiple sclerosis (MS) patients and 10 healthy subjects (HS). Four hours after adding 1 of 28 overlapping AQP4 peptides, a mixture of AQP4 peptides (AQP4-M) or one of six distinct myelin peptides to 2-day cultured PBMC, CD69 expression on CD4+ T cells was examined. Data were analyzed by paired t-test, frequency of samples with 3-fold increase of CD69 on CD4+ cells (fSI3) and mean stimulation index (mSI). The T-cell response to AQP4-M was significantly increased in NMO (fSI3 = 10/12, mSI = 5.50), with AQP4 (11-30) and AQP4 (91-110) representing the two major epitopes (AQP4 (11-30), fSI3 = 11/12, mSI = 16.0 and AQP4 (91-110), fSI3 = 11/12, mSI = 13.0). Significant but less extensive responses to these two epitopes were also observed in MS and HS. Significant reactivities against AQP4 (21-40), AQP4 (61-80), AQP4 (101-120), AQP4 (171-190) and AQP4 (211-230) were exclusively found in NMO. In addition, responses to AQP4 (81-100) were higher and more frequently detected in NMO, without reaching statistical significance. Interestingly, among the six myelin peptides studied, proteolipid protein (95-116) induced a significant T-cell response in NMO (fSI3 = 7/12, mSI = 4.60). Our study suggests that cellular as well as humoral responses to AQP4 are necessary for NMO development and that the immune response to myelin protein may contribute to disease pathogenesis.

Antibodies to myelin oligodendrocyte glycoprotein in idiopathic optic neuritis

Objectives To investigate the differences of clinical features, cerebrospinal fluid (CSF), MRI findings and response to steroid therapies between patients with optic neuritis (ON) who have myelin oligodendrocyte glycoprotein (MOG) antibodies and those who have seronegative ON. Setting We recruited participants in the department of neurology and ophthalmology in our hospital in Japan. Methods We retrospectively evaluated the clinical features and response to steroid therapies of patients with ON. Sera from patients were tested for antibodies to MOG and aquaporin-4 (AQP4) with a cell-based assay. Participants Between April 2009 and March 2014, we enrolled serial 57 patients with ON (27 males, 30 females; age range 16–84 years) who ophthalmologists had diagnosed as having or suspected to have ON with acute visual impairment and declined critical flicker frequency, abnormal findings of brain MRI, optical coherence tomography and fluorescein fundus angiography at their onset or recurrence. We excluded those patients who fulfilled the diagnostic criteria of neuromyelitis optica (NMO)/NMO spectrum disorders (NMOSD), MS McDonalds criteria, and so on. Finally we defined 29 patients with idiopathic ON (14 males, 15 females, age range 16–84 years). Results 27.6% (8/29) were positive for MOG antibodies and 3.4% (1/29) were positive for AQP4. Among the eight patients with MOG antibodies, five had optic pain (p=0.001) and three had prodromal infection (p=0.179). Three of the eight MOG-positive patients showed significantly high CSF levels of myelin basic protein (p=0.021) and none were positive for oligoclonal band in CSF. On MRIs, seven MOG-positive patients showed high signal intensity on optic nerve, three had a cerebral lesion and one had a spinal cord lesion. Seven of the eight MOG-positive patients had a good response to steroid therapy. Conclusions Although not proving primary pathogenicity of anti-MOG antibodies, the present results indicate that the measurement of MOG antibodies is useful in diagnosing and treating ON.

Infantile onset myasthenia gravis with MuSK antibodies

Approximately 80% of patients with myasthenia gravis (MG) show a high antibody (Ab) titer against acetylcholine receptor (AChR). Patients with MG who do not have high titer of anti-AChR are called seronegative MG (SNMG) and their clinical characteristics are shown to be rather different from anti-AChR positive MG in the literature.1 It was recently reported that 70% of sera from generalized SNMG patients contained Ab to a muscle-specific receptor tyrosine kinase (MuSK).2 The frequency of anti-MuSK Ab in generalized SNMG is reportedly from 38% to 47% of cases.3–5 Anti-MuSK MG mainly affects young to middle-aged adults. We describe a case of late infantile onset MG with anti-MuSK Ab. A 29-year-old man first experienced bilateral ptosis after febrile seizures when he was 2 years old. His parents soon noticed abnormal phonation. At age 3 years, the diagnosis of MG was made based on a positive edrophonium test. Treatment was initiated with oral prednisolone. His ocular symptoms responded well to …

Vocal cord paralysis in myasthenia gravis with anti-MuSK antibodies

About 20% of patients with generalized myasthenia gravis (MG) have undetectable serum antibodies to the nicotinic acetylcholine receptor (AChR). These patients are referred to as having seronegative MG. Recently, it has been demonstrated that about 50% of patients with generalized seronegative MG have antibodies to the surface membrane enzyme muscle-specific tyrosine kinase (MuSK) in Caucasian populations and a high frequency of respiratory crises.1 Here, we report a case of MG with anti-MuSK antibodies presenting with vocal cord paralysis, facial muscle weakness, and bulbar palsy. A 56-year-old man showed mild upper limb muscle weakness and fatigue on swallowing and chewing that worsened at the end of days in April 2000. He was admitted to our hospital in February 2001. Neurologic examination revealed mild muscle weakness and atrophy of his proximal muscles in the neck, shoulder, upper arm, and thigh. Nasal voice and dysphagia and facial muscle weakness were also observed, There was no ptosis or diplopia. Although repetitive …

Thymus histology and concomitant autoimmune diseases in Japanese patients with muscle-specific receptor tyrosine kinase-antibody-positive myasthenia gravis.

The differences in the characteristics of thymus histology, coexisting autoimmune diseases and related autoantibodies between anti‐muscle‐specific receptor tyrosine kinase (MuSK)‐antibody (Ab)‐positive myasthenia gravis (MG) patients, and anti‐acetylcholine receptor (AChR)‐Ab‐positive MG patients are not clearly defined.

Abnormal distribution of AQP5 in labial salivary glands is associated with poor saliva secretion in patients with Sjögren's syndrome including neuromyelitis optica complicated patients

ABSTRACT Objective: To investigate whether aquaporins (AQPs) are involved in salivary gland dysfunction in patients with neuromyelitis optica (NMO) complicated with Sjögren’s syndrome (SS). Methods: Eight primary SS (pSS) patients, four NMO spectrum disorder (NMOsd) patients complicated with SS (NMOsd-SS), and three control subjects were enrolled. Immunohistochemistry of labial salivary glands (LSGs) was performed to determine the expressions of AQP4, AQP5, and tumor necrosis factor-alpha (TNF-α). In vitro expression of AQP5 was examined by Western blotting in cultured primary salivary gland epithelial cells (SGECs). Results: No expression of AQP4 was shown in all LSGs. AQP5 was clearly expressed in the all acini, but the predominant localization of AQP5 in the apical side was diminished in the patients with pSS or NMOsd-SS compared with the controls and tended to be even lower in NMOsd-SS than pSS. The abnormal localization of AQP5 was associated with poor saliva secretion. No difference was found in TNF-α expression in the LSGs between patients with pSS and NMOsd-SS. AQP5 expression of SGECs in vitro was not changed by TNF-α or interleukin-10. Conclusions: Our results suggest that AQP5 but not AQP4 contributes to salivary secretion in patients with SS including those with NMO complicated with SS.