Takeshi Tabira

An experimental analysis of interlamellar tight junctions in amphibian and mammalian C.N.S. myelin

SummaryThe distribution of interlamellar tight junctions was examined in myelin sheaths ofXenopus tadpole optic nerve and rabbit epiretinal tissue fixed with aldehydes, postfixed with osmium ferrocyanide and embedded in a water-soluble medium, Durcupan. Intramyelinic zonulae occludentes were clearly formed by fusion of adjacent intraperipd lines which corresponded to the external leaflets of oligodendrocytes. These occurred in register with other tight junctions present within successive lamellae and appeared as a series of radial lines extending either partially or totally across the thickness of the myelin sheath. This distribution of zonulae occludentes corresponded with that of tight junctional particle strands observed in freeze-fracture replicas.Analysis of intramyelinic vacuolation induced by hexachlorophene (HCP) intoxication indicated that lamellar splitting was frequently limited by the tight junctions. The intramyelinic zonulae occludentes also restricted the diffusion of colloidal lanthanum which had penetrated the myelin intraperiod gap followingin vivo perineural injection. The results of this study provide evidence favouring a correspondence between interlamellar tight junctions and the ‘radial component’ of myelin described earlier by other investigators. Furthermore, observations of swollen myelin sheaths, resulting from HCP intoxication, suggest that these junctions may play a major role in maintaining myelin sheath integrity and limiting the extent of breakdown during certain pathological conditions.

HLA-DR2–restricted responses to proteolipid protein 95-116 peptide cause autoimmune encephalitis in transgenic mice

In multiple sclerosis (MS) patients who carry the Class II major histocompatibility (MHC) type HLA-DR2, T cells specific for amino acids 95-116 in the proteolipid protein (PLP) are activated and clonally expanded. However, it remains unclear whether these autoreactive T cells play a pathogenic role or, rather, protect against the central nervous system (CNS) damage. We have addressed this issue, using mice transgenic for the human MHC class II region carrying the HLA-DR2 (DRB1* 1502) haplotype. After stimulating cultured lymph node cells repeatedly with PLP95-116, we generated 2 HLA-DR2-restricted, PLP95-116-specific T-cell lines (TCLs) from the transgenic mice immunized with this portion of PLP. The TCLs were CD4+ and produced T-helper 1 (Th1) cytokines in response to the peptide. These TCLs were adoptively transferred into RAG-2/2 mice expressing HLA-DR2 (DRG1* 1502) molecules. Mice receiving 1 of the TCLs developed a neurological disorder manifested ataxic movement without apparent paresis on day 3, 4, or 5 after cell transfer. Histological examination revealed inflammatory foci primarily restricted to the cerebrum and cerebellum, in association with scattered demyelinating lesions in the deep cerebral cortex. These results support a pathogenic role for PLP95-116-specific T cells in HLA-DR2+ MS patients, and shed light on the possible correlation between autoimmune target epitope and disease phenotype in human CNS autoimmune diseases.

Heterogeneous induction of 72-kDa heat shock protein (HSP72) in cultured mouse oligodendrocytes and astrocytes

The expression of 72-kDa heat shock protein (HSP72) in cultured mouse oligodendrocytes and astrocytes exposed to heat shock was investigated by double immunolabelling with anti-HSP72 monoclonal antibody (C92F3B-1) and antibodies against galactocerebroside (GalC) or glial fibrillary acidic protein (GFAP). After 3 h recovery from heat shock, an intermediate level of HSP72 immunolabelling was localized in the nucleolus and cytoplasm of astrocytes (less than 25%) and to a lesser extent in oligodendrocytes (less than 2%). After 8-48 h, HSP72 was expressed intensely in the cytoplasm and nuclear matrix of oligodendrocytes (20-40%), while weak/intermediate immunostaining was detectable in astrocytes (5-15%). The levels of HSP72 expressed in oligodendrocytes and astrocytes decreased around 72-120 h, but a few oligodendrocytes (4%) remained intensely immunolabelled. These results indicate that heat shock induces HSP72 in both oligodendrocytes and astrocytes. However, this response is heterogeneous.

Multiple sclerosis cerebrospinal fluid produces myelin lesions in tadpole optic nerves.

To investigate the myelinotoxicity of cerebropsinal fluid in multiple sclerosis, we used an in vivo model of the myelinated central-nervous-system tract of tadpoles for quantitative double-blind tests of 46 cerebrospinal-fluid samples. Groups of xenopus tadpoles were injected with cerebrospinal fluid near the optic nerve. Forty-eight hours later, whole mounts of optic nerves were prepared, and a differential interference microscope was used to count myelin lesions. Cerebrospinal-fluid samples from 60 per cent of the patients with an acute attack of definite multiple sclerosis had myelinotoxic activity. This activity correlated best with the severity and duration of the disease, rather than with gamma-globulin or total protein concentrations. Activity was negative in 85 per cent of cerebrospinal-fluid samples from a control group with other neurologic diseases. This assay is a useful method for investigating myelinotoxic factors of cerebrospinal fluid in patients with multiple sclerosis, but was not helpful diagnostically.

Developmental and aging changes in the expression patterns of beta-amyloid in the brains of normal and down syndrome cases

Immunohistochemical staining with polyclonal antibodies to synthetic amyloid (residues 1-28 of A4) was performed on normal and Down syndrome brains from fetuses to adults. Positive staining appeared in the cytoplasmic processes of astrocytes in the subpial layer and white matter of developing brains, and reappeared in astrocytic fibers of the subpial layer as well as in cerebrovascular and plaque core amyloid in elderly brains. The reappearance of positively stained astrocytes and amyloid occurred earlier in adult Down syndrome patients. The results indicate that the A4 protein is a developmental protein, and its reappearance in Alzheimer and adult Down syndrome brains may be related to the regeneration process.

Hexachlorophene-induced myelin lesions in the amphibian central nervous system. A freeze-fracture study.

Abstract Myelin membranes in the optic nerves and spinal cord of Xenopus laevis tadpoles were studied with the freeze-fracture technique during the pathogenesis of edematous lesions induced by hexachlorophene (HCP) intoxication. Freeze-fracture replicas of myelin exposed to HCP for as long as 12 days revealed that even chronic intoxication did not significantly alter particle strands associated with zonulae occludentes between tongue processes and their adjacent layers of myelin. Furthermore, experiments using electron-dense tracers demonstrated that the permeability characteristics of these junctions were unchanged. Following subcutaneous perineural injection of lanthanum or horseradish peroxidase (HRP), the tracers easily penetrated the optic nerve and were uniformly distributed throughout its parenchyma. While only limited amounts of lanthanum could penetrate myelin and enter the vacuoles, no HRP was found within the large myelin vacuoles after either brief or long-term exposure to HCP. In contrast to the tight junctions, some modifications were observed in the distribution of other intramembranous particles associated with myelin lamellae adjacent to swollen regions of the sheath. These alterations primarily involved formation of multifocal, particle-free elevations which corresponded to areas in which the fused inner glial leaflets, forming the major dense line, had separated. Such particle-deficient areas were absent in myelin of control as well as normal sheaths of HCP-intoxicated animals. Thus, these findings demonstrated that the structural and physiological characteristics of a tight junctional component of myelin were not affected by HCP. Sparing of these junctions may account for the relatively low incidence of demyelination and reversible nature of HCP lesions. The development of particle-deficient blisters on glial leaflets suggests, however, that HCP may be capable of modifying other aspects of the chemical organization of myelin, thereby producing local alterations in the compact arrangement of myelin lamellae.

The penetration of fluorescein-conjugated and electron-dense tracer proteins into Xenopus tadpole optic nerves following perineural injection

The permeability of Xenopus tadpole optic nerves to macromolecules was studied in order to evaluate the usefulness of this system for studying mechanisms of serum-induced CNS demyelination in vivo. Single injections of either horseradish peroxidase (HRP), ferritin or fluorescein-conjugated human IgG were injected around the right optic nerve and tadpoles were then sacrificed between 15 min and 48 h. Each of the tracers had penetrated the nerve parenchyma by 30 min. Entry of HRP and ferritin occurred mainly via extracellular clefts between adjacent astrocytic endfeet in the glia limitans region. A similar mode of passage was suggested for IgG. Once within the nerve, the tracers became rapidly associated with myelinated axons. HRP was also seen in the periaxonal space but did not directly penetrate the myelin sheath. By 24 h, extracellular localization of tracer was virtually absent with nearly all of the tracer now being concentrated in vesicles within astrocytic processes and perikarya. The distribution of the tracers was not confined to the optic nerve on the injected side; some was seen in adjacent cranial peripheral nerves and surrounding extraocular musculature. Also, tracers eventually penetrated the pial sheath of the contralateral optic nerve. The results of this study indicate that tadpole optic nerves are permeable to a wide range of macromolecules. Furthermore, the distribution of these tracers to nearby cranial peripheral nerves may provide an important opportunity for testing the differential effect of various substances on central and peripheral myelin sheaths.

Constitutive expression of 65-kDa heat shock protein (HSP65)-like immunoreactivity in cultured mouse oligodendrocytes

The expression of mycobacterial 65-kDa heat shock protein (HSP65)-like immunoreactivity in cultured mouse oligodendrocytes and astrocytes was investigated using three monoclonal antibodies (ML30, IA1, 3A) specific for the mycobacterial HSP65. In western blot analysis, these antibodies recognized the proteins with molecular weights approximately of 50-, 60-, and 70-kDa expressed in both heat-stressed and unstressed glial cells. When the cells were exposed to heat stress, the expression of both 50- and 70-kDa proteins was attenuated, whereas that of the 60-kDa protein was not affected. On immunocytochemical studies, an appreciable level of HSP65 immunolabelling was identified in most (> 90%) oligodendrocytes under both heat-stressed and unstressed conditions but only marginally detectable in most (> 95%) astrocytes. These results indicate that mouse oligodendrocytes in vitro express the mycobacterial HSP65-like immunoreactivity constitutively.

Binding of an SJL T cell clone specific for myelin basic protein to SJL brain microvessel endothelial cells is inhibited by anti-VLA-4 or its ligand, anti-vascular cell adhesion molecule 1 antibody

Adhesion molecules probably are required for the migration of T lymphocytes to inflamed tissues, but the roles of these molecules have yet to be understood in the pathogenesis of inflammatory diseases such as multiple sclerosis. The adhesion of an SJL murine T cell clone specific for myelin basic protein (MBP) to endothelial cells (ECs) from SJL newborn brain microvessels was examined. Sixty percent of the 2 x 10(4) T cell clones stimulated once every 2 weeks with MBP were bound to ECs, whereas less than 5% of the same number of lymphocytes from peripheral lymph nodes were bound. However, binding was not central nervous system (CNS)-specific. Monoclonal antibody to VLA-4 or VCAM-1 partially inhibited the binding of the T cell clone to ECs. Binding of the T cell clone to ECs increased when the latter were incubated with IL-1 or TNF, but was not inhibited by anti-VLA-4 or VCAM-1. We suggest that the VLA-4/VCAM-1 pathway functions in the binding of the T cell clone specific for MBP to brain ECs but that adhesion molecules other than VLA-4/VCAM-1 are involved because anti-VLA-4 and anti-VCAM-1 did not produce complete inhibition.

Baló's concentric sclerosis in a 4-year-old Japanese infant

We report the youngest known case with Balós concentric sclerosis (Balós disease), a variant of multiple sclerosis. This 4-year-old Japanese boy was diagnosed by clinical manifestations and by characteristic findings on magnetic resonance imaging (MRI). Dexamethasone was given intravenously, 3 mg twice daily for 10 days. The clinical manifestations were resolved within 2 weeks, and the MRI findings were markedly improved after 3 weeks. Following the initiation of steroid therapy, he showed remarkable clinical and physical improvement. It cannot be excluded that the clinical improvement resulted from the steroid therapy.