Mikio Hirayama

Association of HTRA1 mutations and familial ischemic cerebral small-vessel disease

BACKGROUND The genetic cause of cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy (CARASIL), which is characterized by ischemic, nonhypertensive, cerebral small-vessel disease with associated alopecia and spondylosis, is unclear. METHODS In five families with CARASIL, we carried out linkage analysis, fine mapping of the region implicated in the disease, and sequence analysis of a candidate gene. We also conducted functional analysis of wild-type and mutant gene products and measured the signaling by members of the transforming growth factor beta (TGF-beta) family and gene and protein expression in the small arteries in the cerebrum of two patients with CARASIL. RESULTS We found linkage of the disease to the 2.4-Mb region on chromosome 10q, which contains the HtrA serine protease 1 (HTRA1) gene. HTRA1 is a serine protease that represses signaling by TGF-beta family members. Sequence analysis revealed two nonsense mutations and two missense mutations in HTRA1. The missense mutations and one of the nonsense mutations resulted in protein products that had comparatively low levels of protease activity and did not repress signaling by the TGF-beta family. The other nonsense mutation resulted in the loss of HTRA1 protein by nonsense-mediated decay of messenger RNA. Immunohistochemical analysis of the cerebral small arteries in affected persons showed increased expression of the extra domain-A region of fibronectin and versican in the thickened tunica intima and of TGF-beta1 in the tunica media. CONCLUSIONS CARASIL is associated with mutations in the HTRA1 gene. Our findings indicate a link between repressed inhibition of signaling by the TGF-beta family and ischemic cerebral small-vessel disease, alopecia, and spondylosis.

Coronavirus mouse hepatitis virus (MHV)-A59 causes a persistent, productive infection in primary glial cell cultures

Abstract MHV-A59 causes a chronic demyelinating disease in mice which is accompanied by persistence of viral genome in white matter. As part of the investigation into the mechanism of viral persistence, infection of glial cells, probable targets for chronic infection, was studied by the use of mixed glial, enriched oligodendrocyte and enriched astrocyte cultures. Following MHV-A59 infection in vitro, approximately 10% of oligodendrocytes and 30% of astrocytes expressed viral antigens in the absence of overt cytopathic effect. All cultures released infectious virus for the lifetime of the cultures, for at least 45 days in the case of mixed glial cultures. Cultures derived from previously infected mice were similar to those infected in vitro with respect to percentage of cells expressing viral antigen and levels of infectious virus produced. These results show (1) that glial cells are early sites of infection in vivo as well as sites of infection in in vitro cultures, and (2) that glial cells support a non-lytic but productive infection in vitro and thus may contribute to viral persistence in vivo.

Cultured human and rat oligodendrocytes and rat schwann cells do not have immune response gene associated antigen (Ia) on their surface

In order to determine if oligodendrocytes or Schwann cells had surface immune response gene associated antigen (Ia), we studied the binding of: (a) mouse monoclonal antibodies to rat Ia, to cultures of rat oligodendrocytes and Schwann cells; and, (b) mouse monoclonal antibodies to human Ia, to cultures of human oligodendrocytes employing radioimmunoassay and indirect immunofluorescence. Cells were identified using phenotypic markers; rabbit anti-galactocerebroside (GalC) for oligodendrocytes; rabbit anti-GalC and rabbit anti-Schwann cell for Schwann cells; rabbit anti-glial fibrillary acidic protein for astrocytes; rabbit anti-fibronectin for fibro-blasts and leptomeningeal cells, and the capacity to ingest latex particles for macrophage-microglia. Ia could not be detected on the surface of oligodendrocytes, Schwann cells, astrocytes, fibroblasts, or leptomeningeal cells. A small number of latex ingesting cells bound anti-Ia even after blocking of surface Fc receptors.

Serum-mediated oligodendrocyte cytotoxicity in multiple sclerosis patients and controls.

We examined serum-mediated cytotoxicity on cultured rat oligodendrocytes, using serum from patients with acute or chronic progressive multiple sclerosis and normal controls. We found heat-labile serum factors in serum from MS and also in controls. The cytotoxic effects of MS and normal sera were not restored by adding a source of complement. Despite apparent lack of disease specificity, such factors might damage oligodendrocytes if they gained access to these cells through a damaged blood-brain barrier in MS or other disorders.

The ultrastructure of isolated rat oligodendroglial cell cultures

SummaryCultured rat oligodendrocytes were studied by transmission electron microscopy. Cells were identified by an immunocytochemical stain for galactocerebroside, a specific cell surface marker for oligodendroglial cells.Oligodendroglial cell perikarya contained numerous ribosomal rosettes, microtubules, prominent networks of cisternae of the Golgi apparatus and residual bodies. Glycogen and intermediate filaments were absent. Oligodendrocytes gave rise to numerous processes. Pentalaminar and heptalaminar profiles, consistent with tight and gap junctions, were seen between plasma membranes of processes and between perikarya and processes.The cell surface of processes showed numerous gross ruffles which stained for galactocerebroside. Similar membranous profiles appeared in the vicinity of oligodendroglial processes and suggested to us that the plasma membrane or certain of its components may be released into the medium.We concluded that cultured rat oligodendrocytes maintain many similarities with oligodendrocytesin situ and, therefore, are valid models for morphologic, physiologic and biochemical studies.

Characteristic features and progression of abnormalities on MRI for CARASIL.

Objectives: The objective of this study was to clarify the characteristic brain MRI findings for genetically diagnosed CARASIL (cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy). Methods: Seven patients with CARASIL carrying HTRA1 mutations (representing 6 Japanese families) were included in this study. Eighteen brain MRIs were reviewed and evaluated with a new rating scale based on scoring for abnormal hyperintense lesions and atrophy. Results: At the last follow-up MRI, all patients had hyperintense lesions on T2-weighted images of the frontal white matter, anterior temporal lobe, external capsules, and thalami. Patients with longer time from the onset of cognitive impairment had higher MRI severity score. The atrophy advanced, followed by white matter lesion progression. During the early stage, hyperintense lesions were observed in the frontal white matter, external capsule, and pons. During the late stage, the arc-shaped hyperintense lesion from the pons to the middle cerebellar peduncles, which we designated the “arc sign,” became evident. The arc sign was a characteristic finding for CARASIL in the advanced stage. Conclusions: These characteristic MRI findings for CARASIL are useful for selecting patients for genetic testing. The rating scale correlates well with disease duration and might be useful for assessing disease progression.

MK-801 is cytotoxic to microglia in vitro and its cytotoxicity is attenuated by glutamate, other excitotoxic agents and atropine: Possible presence of glutamate receptor and muscarinic receptor on microglia

We examined the cytotoxicity of MK-801 on cultured microglia and demonstrated its cytotoxicity. Cytotoxicity of MK-801 was reduced by the addition of L-glutamate, kainate and NMDA. The action of MK-801 was due to the direct action of microglia. It suggested the existence of glutamate receptor in microglia. Cytotoxicity of MK-801 was reduced by the addition of atropine sulfate which suggested the presence of muscarinic receptor in microglia.

Generation and biological properties of a monoclonal antibody to Galactocerebroside

Galactocerebroside ( GalC ) is a major glycolipid of myelin and myelin-forming cells. We have generated a mouse IgM monoclonal antibody to GalC (M-anti- GalC ) which bound only to oligodendrocytes in rat and bovine central nervous system cultures as assessed by immunofluorescence. Double staining with rabbit anti-glial fibrillary acidic protein and anti-fibronectin antisera revealed no binding of M-anti- GalC to astrocytes or fibroblasts. Schwann cells, but not fibroblasts, were stained in short-term cultures of rat Schwann cells. M-anti- GalC exhibited in vitro cytotoxicity to rat and bovine oligodendrocytes in the presence of complement. This monoclonal antibody with its monospecificity, consistent titer, and capacity to induce cell lysis should be useful for in vitro and in vivo investigations concerning myelination and demyelination.