Kazutoshi Nishiyama

Affinity-purification and characterization of caveolins from the brain: differential expression of caveolin-1, -2, and -3 in brain endothelial and astroglial cell types.

Caveolins 1, 2 and 3 are the principal protein components of caveolae organelles. It has been proposed that caveolae play a vital role in a number of essential cellular functions including signal transduction, lipid metabolism, cellular growth control and apoptotic cell death. Thus, a major focus of caveolae-related research has been the identification of novel caveolins, caveolae-associated proteins and caveolin-interacting proteins. However, virtually nothing is known about the expression of caveolins in brain tissue. Here, we report the purification and characterization of caveolins from brain tissue under non-denaturing conditions. As a final step in the purification, we employed immuno-affinity chromatography using rabbit polyclonal anti-caveolin IgG and specific elution at alkaline pH. The final purified brain caveolin fractions contained three bands with molecular masses of 52 kDa, 24 kDa and 22 kDa as visualized by silver staining. Sequencing by ion trap mass spectrometry directly identified the major 24-kDa component of this hetero-oligomeric complex as caveolin 1. Further immunocyto- and histochemical analyses demonstrated that caveolin 1 was primarily expressed in brain endothelial cells. Caveolins 2 and 3 were also detected in purified caveolin fractions and brain cells. The cellular distribution of caveolin 2 was similar to that of caveolin 1. In striking contrast, caveolin 3 was predominantly expressed in brain astroglial cells. This finding was surprising as our previous studies have suggested that the expression of caveolin 3 is confined to striated (cardiac and skeletal) and smooth muscle cells. Electron-microscopic analysis revealed that astrocytes possess numerous caveolar invaginations of the plasma membrane. Our results provide the first biochemical and histochemical evidence that caveolins 1, 2 and 3 are expressed in brain endothelial and astroglial cells.

NMDA-receptors 1 and 2A/b coassembly increased in human epileptic focal cortical dysplasia

Summary: Purpose: This study was designed to quantify the relation between expressions of NMDA receptor (NMDAR) subunits (1 and 2A/B) and the epileptogenicity in human focal cortical dysplasia.

Localization of Nav1.5 sodium channel protein in the mouse brain.

Nav1.5 or SCN5A is a member of the voltage-dependent family of sodium channels. The distribution of Nav1.5 protein was investigated in the mouse brain using immunohistochemistry. Immunostaining with a Nav1.5-specific antibody revealed that Nav1.5 protein was localized in certain distinct regions of brain including the cerebral cortex, thalamus, hypothalamus, basal ganglia, cerebellum and brain stem. Notably, we found that Nav1.5 protein co-localized with neurofilaments and clustered at a high density in the neuronal processes, mainly axons. These results suggest that Nav1.5 protein may play a role in the physiology of the central nervous system (generation and propagation of electrical signals by axons).

Claudin localization in cilia of the retinal pigment epithelium

Using immunocytochemistry and confocal microscopy we demonstrate that claudin‐immunoreactivity is a novel marker for retinal pigment epithelial cilia. Claudin‐immunoreactivity obtained by polyclonal anti‐claudin 1 antibody, which could crossreact with claudin 3, was colocalized with acetylated tubulin‐immunoreactivity in cultured human retinal pigment epithelial cells. Claudin‐immunoreactivity associated with the retinal pigment epithelium (RPE) cilia was more intense than was claudin‐immunoreactivity in the junctional complex. Approximately two‐thirds of the RPE cells in the rat contain cilia that are immunoreactive with acetylated tubulin on postnatal day 1, and a significant portion of these cilia label with the anti‐claudin 1 antibody. Cilia decrease in frequency over subsequent postnatal days, and are absent by postnatal day 30. As RPE cilia decrease in number during postnatal rat development, claudin‐immunoreactivity is lost earlier than acetylated tubulin, suggesting that the loss of claudin may initiate RPE cilium degeneration. Claudin‐immunoreactivity was not evident in cilia of photoreceptor cells, epithelia of nasal mucosa, small intestine, or colon, suggesting that claudin may be a unique molecule in RPE cilia. These data suggest that cilia of the RPE, unlike cilia on other cell types, contain claudin, and that this molecule may play an important and specific role in the function and/or maintenance of RPE cilia. Anat Rec 267:196–203, 2002.

Decreased calmodulin-NR1 co-assembly as a mechanism for focal epilepsy in cortical dysplasia.

The NMDA receptor is one of the ionotropic glutamate receptors essential for excitatory neurotransmission. The NMDAR1 subunit is inactivated by direct interaction with calmodulin. The protein levels of calmodulin, NMDAR1 and their complex were quantified in tissue resected from epileptogenic and non-epileptogenic cortical areas as determined by chronic subdural electrode recordings from three patients (aged 6, 14 and 18 years) with focal epilepsy associated with cortical dysplasia. In all patients, the co-assembly of calmodulin and NMDAR1 was decreased in epileptogenic dysplastic cortex compared with normal appearing non-epileptogenic cortex, while there was no significant difference in the total protein levels of calmodulin or NMDAR1 between the two EEG groups. These results suggest that decreased calmodulin-NMDAR1 co-assembly is a cellular mechanism that contributes to hyperexcitability in dysplastic cortical neurons and in focal seizure onsets.

Positron emission tomography of reversible intellectual impairment induced by long-term anticholinergic therapy

Long-term oral anticholinergic (AC) therapy can occasionally produce intellectual impairment. We investigated a patient with Parkinsons disease accompanied by intellectual impairment induced by long-term AC therapy. The intellectual impairment of the patient disappeared after cessation of AC therapy. Positron emission tomography (PET), during and after long-term oral AC therapy, revealed that it causes bilateral diffuse decrease of glucose metabolism in the cortex, basal ganglia, thalamus, hippocampus and cerebellum. Cessation of the therapy resulted in diffuse increase of glucose metabolism in all of the above regions. Cranial CT and magnetic resonance imaging (MRI) showed no abnormalities. Our results suggest that long-term AC therapy causes reversible bilateral diffuse glucose hypometabolism.

Axial myoclonus mediated by the propriospinal tract: a case report.

We describe a patient with axial myoclonus. Myoclonus first occurred in the upper abdominal muscle and spread up to the neck muscle and down to the lower abdominal muscle. Physiological studies of the jerks revealed that the myoclonus arose in the thoracic spinal cord and spread slowly up and down the spinal cord at about 3 m/s. We conclude that the myoclonus is mediated by the propriospinal tract.

SPACRCAN in the developing retina and pineal gland of the rat: spatial and temporal pattern of gene expression and protein synthesis.

SPACRCAN is a hyaluronan‐binding proteoglycan that is present in the pineal gland and interphotoreceptor matrix of the retina. Here, we evaluate the pattern of SPACRCAN gene expression and protein appearance during retinal and pineal gland development in the rat. In situ hybridization histochemistry with SPACRCAN riboprobes indicates that hybridization signals are first evident in the retina over developing photoreceptor cells at embryonic day 16 (E16) and in the pineal gland at E21. Immunocytochemistry using a SPACRCAN antibody shows localization of SPACRCAN protein in the developing interphotoreceptor matrix by Postnatal day 5 (P5) and in the pineal gland by P6. These studies suggest that SPACRCAN mRNA expression may occur substantially earlier than the time when SPACRCAN protein is detectable in both the retina and the pineal gland. The period of retinal histogenesis when SPACRCAN is detected first is coincident with the time photoreceptors begin to extend from the outer retinal surface, suggesting that SPACRCAN may participate in the maturation and maintenance of the light‐sensitive photoreceptor outer segment. J. Comp. Neurol. 435:354–365, 2001.

Cryptogenic NORSE Its distinctive clinical features and response to immunotherapy

Objective: To report the distinctive clinical features of cryptogenic new-onset refractory status epilepticus (C-NORSE) and the C-NORSE score based on initial clinical assessments. Methods: A retrospective study was conducted for 136 patients with clinically suspected autoimmune encephalitis who underwent testing for autoantibodies to neuronal surface antigens between January 1, 2007, and August 31, 2016. Eleven patients with C-NORSE were identified. Their clinical features were compared with those of 32 patients with anti-NMDA receptor encephalitis (NMDARE). Results: The clinical outcome of 11 patients (median age, 27 years; 7 [64%] women) with C-NORSE was evaluated after a median follow-up of 11 months (range, 6–111 months). Status epilepticus was frequently preceded by fever (10/11 [91%]). Brain MRIs showed symmetric T2/fluid-attenuated inversion recovery hyperintensities (8/11 [73%]) and brain atrophy (9/11 [82%]). Only 2 of the 10 treated patients responded to the first-line immunotherapy, and 4 of the 5 patients treated with IV cyclophosphamide responded to the therapy. The long-term outcome was poor in 8 patients (73%). Compared with 32 patients with NMDARE (median age, 27 years; 24 [75%] women), those with C-NORSE had more frequent prodromal fever, status epilepticus, ventilatory support, and symmetric brain MRI abnormalities, had less frequent involuntary movements, absent psychobehavioral symptoms, CSF oligoclonal bands, or tumor association, and had a worse outcome. The C-NORSE score was higher in patients with C-NORSE than those with NMDARE. Conclusions: Patients with C-NORSE have a spectrum of clinical-immunological features different from those with NMDARE. The C-NORSE score may be useful for discrimination between them. Some patients could respond to immunotherapy.

Carcinomatous neuropathy associated with hepatic cell carcinoma: An autopsy case report

A 76-yr-old male patient with carcinomatous neuropathy associated with hepatic cell carcinoma, whose initial symptom was deep sensory disturbance followed by muscle weakness is described. The onset was subacute, followed by slow progression. Sural nerve biopsy, as well as electrophysiological examinations, revealed severe axonal degeneration without any evidence of demyelination. The autopsy findings were similar to findings described in the literature on carcinomatous neuropathy. Although carcinomatous neuropathy is usually associated with lung cancer, this report describes an association with hepatic cell carcinoma. The patient also had motor nerve involvement with positive serum anti-GM1 ganglioside antibody which decreased after immunosuppressant therapy in parallel with recovery of muscle weakness. The anti-GM1 ganglioside antibody may be involved in the pathogenesis of motor disturbance in the present case.