Toshitaka Oohashi

Establishment by the rat lymph node method of epitope-defined monoclonal antibodies recognizing the six different α chains of human type IV collagen

A group of rat monoclonal antibodies recognizing the six different α chains of human type IV collagen have been established by our novel method. The method is designated the rat lymph node method in which enlarged medial iliac lymph nodes of a rat injected with an antigen emulsion via hind footpads are used as a source of B cells for cell fusion to produce hybridomas. The immunogens used were synthetic peptides having non-consensus amino acid sequences near the carboxyl termini of type IV collagen α chains. Hybridomas were screened both by ELISA with synthetic peptides and by indirect immunofluorescence with cryostat sections of human kidneys. Because the epitopes of all antibodies were determined by multipin-peptide scanning, they were confirmed to be isoform-specific. They are useful for identification of α chains of type IV collagen at the protein level in normal and abnormal conditions. The combined use of synthetic peptides as immunogens, the rat lymph node method as making monoclonal antibodies, and the multipin-peptide scanning as epitope mapping is found to be a strong tool for identification of peptides and proteins whose amino acid sequences are known or have been deduced.

Bral1, a brain-specific link protein, colocalizing with the versican V2 isoform at the nodes of Ranvier in developing and adult, mouse central nervous systems

Bral1, a brain-specific hyaluronan-binding protein, has been cloned recently. To gain insight into the role of Bral1, we generated a specific antibody against this protein. We have examined the detailed localization pattern of Bral1 protein and compared it with that of other members of the lectican proteoglycan family, such as brevican and versican, with which Bral1 is predicted to interact. The immunoreactivity of Bral1 antibody was predominantly observed in myelinated fiber tracts in the adult brain and could be detected at P20 in the white matter of the developing cerebellum, suggesting that expression starts when axonal myelination takes place. Furthermore, immunostaining demonstrated that Bral1 colocalized with the versican V2 isoform at the nodes of Ranvier. The present data suggest that Bral1 may play a pivotal role in the formation of the hyaluronan-associated matrix in the CNS that facilitates neuronal conduction by forming an ion diffusion barrier at the nodes.

Three Mechanisms Assemble Central Nervous System Nodes of Ranvier

Rapid action potential propagation in myelinated axons requires Na⁺ channel clustering at nodes of Ranvier. However, the mechanism of clustering at CNS nodes remains poorly understood. Here, we show that the assembly of nodes of Ranvier in the CNS involves three mechanisms: a glia-derived extracellular matrix (ECM) complex containing proteoglycans and adhesion molecules that cluster NF186, paranodal axoglial junctions that function as barriers to restrict the position of nodal proteins, and axonal cytoskeletal scaffolds (CSs) that stabilize nodal Na⁺ channels. We show that while mice with a single disrupted mechanism had mostly normal nodes, disruptions of the ECM and paranodal barrier, the ECM and CS, or the paranodal barrier and CS all lead to juvenile lethality, profound motor dysfunction, and significantly reduced Na⁺ channel clustering. Our results demonstrate that ECM, paranodal, and axonal cytoskeletal mechanisms ensure robust CNS nodal Na⁺ channel clustering.

Ten_m3 regulates eye-specific patterning in the mammalian visual pathway and is required for binocular vision.

Binocular vision requires an exquisite matching of projections from each eye to form a cohesive representation of the visual world. Eye-specific inputs are anatomically segregated, but in register in the visual thalamus, and overlap within the binocular region of primary visual cortex. Here, we show that the transmembrane protein Ten_m3 regulates the alignment of ipsilateral and contralateral projections. It is expressed in a gradient in the developing visual pathway, which is consistently highest in regions that represent dorsal visual field. Mice that lack Ten_m3 show profound abnormalities in mapping of ipsilateral, but not contralateral, projections, and exhibit pronounced deficits when performing visually mediated behavioural tasks. It is likely that the functional deficits arise from the interocular mismatch, because they are reversed by acute monocular inactivation. We conclude that Ten_m3 plays a key regulatory role in the development of aligned binocular maps, which are required for normal vision.

Bral1: Its Role in Diffusion Barrier Formation and Conduction Velocity in the CNS

At the nodes of Ranvier, excitable axon membranes are exposed directly to the extracellular fluid. Cations are accumulated and depleted in the local extracellular nodal region during action potential propagation, but the impact of the extranodal micromilieu on signal propagation still remains unclear. Brain-specific hyaluronan-binding link protein, Bral1, colocalizes and forms complexes with negatively charged extracellular matrix (ECM) proteins, such as versican V2 and brevican, at the nodes of Ranvier in the myelinated white matter. The link protein family, including Bral1, appears to be the linchpin of these hyaluronan-bound ECM complexes. Here we report that the hyaluronan-associated ECM no longer shows a nodal pattern and that CNS nerve conduction is markedly decreased in Bral1-deficient mice even though there were no differences between wild-type and mutant mice in the clustering or transition of ion channels at the nodes or in the tissue morphology around the nodes of Ranvier. However, changes in the extracellular space diffusion parameters, measured by the real-time iontophoretic method and diffusion-weighted magnetic resonance imaging (MRI), suggest a reduction in the diffusion hindrances in the white matter of mutant mice. These findings provide a better understanding of the mechanisms underlying the accumulation of cations due to diffusion barriers around the nodes during saltatory conduction, which further implies the importance of the Bral1-based extramilieu for neuronal conductivity.

Brevican distinctively assembles extracellular components at the large diameter nodes of Ranvier in the CNS

Brevican is known to be an abundant extracellular matrix component in the adult brain and a structural constituent of perineuronal nets. We herein show that brevican, tenascin‐R (TN‐R) and phosphacan are present at the nodes of Ranvier on myelinated axons with a particularly large diameter in the central nervous system. A brevican deficiency resulted in a reorganization of the nodal matrices, which was characterized by the shift of TN‐R, and concomitantly phosphacan, from an axonal diameter‐dependent association with nodes to an axonal diameter independent association. Supported by the co‐immunoprecipitation results, these observations indicate that the presence of TN‐R and phosphacan at nodes is normally brevican‐dependent, while in the absence of brevican these molecules can also be recruited by versican V2. The versican V2 and Bral1 distribution was not affected, thus indicating a brevican‐independent role of these two molecules for establishing hyaluronan‐binding matrices at the nodes. Our results revealed that brevican plays a crucial role in determining the specialization of the hyaluronan‐binding nodal matrix assemblies in large diameter nodes.

The murine Ten-m/Odz genes show distinct but overlapping expression patterns during development and in adult brain

The mouse TEN-M/ODZ proteins belong to a new family of type II transmembrane proteins with unknown function. The family consists of four members, which are expressed highly in brain and less in many other tissues. In the present study we have generated specific RNA probes and antibodies to characterize the expression of the 4 Ten-m/Odz genes in the developing and adult central nervous system (CNS) of mice. Ten-m/Odz3 and Ten-m/Odz4 mRNAs were first detectable at E7.5, Ten-m/Odz2 expression started at the 37 somite (E 10.5) stage, while Ten-m/Odz1 mRNA is not found before E15.5. In the adult mouse CNS mRNAs of the 4 Ten-m/Odzs were expressed in distinct patterns, which partially overlapped. Immunostaining and in situ hybridization localized proteins and mRNAs of Ten-m/Odzs in adjacent areas suggesting that TEN-M/ODZ proteins might be transported from the cell body along the axon or that they are shed from the cell surface and diffuse into distant regions.

Molecular cloning of Bral2, a novel brain-specific link protein, and immunohistochemical colocalization with brevican in perineuronal nets

The hyaluronan binding chondroitin sulphate proteoglycans, called lecticans, are the abundant extracellular matrix molecules in the developing and/or adult brain. The link proteins (LPs) are also known to be coordinately present in brain. We report here the molecular cloning and expression analysis of a novel member of LPs: Bral2, predominantly expressed in brain. The Bral2 mRNA expression is first detected at P20 and continued through adulthood, suggesting its functional importance and association with adult-type lecticans. The substantial immunoreactivity of Bral2 is found in several nuclei throughout the midbrain and hindbrain in a perineuronal net pattern. In situ hybridization revealed that Bral2 is synthesized by these neurons themselves, especially by the GABAergic neurons in the cerebellar cortex. Interestingly, the colocalization and synergic importance of Bral2 and brevican in the perineuronal nets is indicated by the comparative immunohistochemical analysis using wild-type and brevican-deficient mouse brain. Our results suggest that Bral2 is involved in the formation of extracellular matrix contributing to perineuronal nets and facilitate the understanding of a functional role of these extracellular matrices.

DIFFERENTIAL EXPRESSION OF ALPHA 1 (IV), ALPHA 2(IV), ALPHA 5(IV) AND ALPHA 6(IV) COLLAGEN CHAINS IN THE BASEMENT MEMBRANE OF BASAL CELL CARCINOMA

Type IV collagen, the major component of basement membrane, consists primarily of ·1(IV) and ·2(IV) chains. Recently, other types of collagen IV chains, i.e. ·3(IV), ·4(IV), ·5(IV) and ·6(IV) chains, have been identified by protein chemistry and molecular cloning. We have examined the diversity of the assembly of ·(IV) chains of the basement membrane surrounding tumour nests of basal cell carcinomas, in tissues from 11 patients, by immunohistochemical analysis using specific monoclonal antibodies to six ·(IV) chain. The immunostaining profile of each chain differed with respect to the histological subtypes of basal cell carcinoma. In the morphea-like subtype, which was more invasive, ·1(IV) and ·2(IV) chains were discontinuously stained, and ·5(IV) and ·6(IV) chains were entirely absent. However, in the superficial subtype, which was non-aggressive, ·1(IV), ·2(IV), ·5(IV) and ·6(IV) chains were well stained compared with the other subtypes of basal cell carcinoma. In addition, in the solid subtype, which showed slow growth and ulceration, ·1(IV) and ·2(IV) chains were continuously stained, and ·5(IV) and ·6(IV) chains were discontinuous or absent. The assembly of ·5(IV) and ·6(IV) chains into the basement membrane was inhibited in the solid and morphea subtypes of BCC. This differential expression of type IV collagen chains seems to be associated with the invasive potential of basal cell carcinoma

Mechanical Stretch Enhances COL2A1 Expression on Chromatin by Inducing SOX9 Nuclear Translocalization in Inner Meniscus Cells

The meniscus plays an important role in controlling the biomechanics of the knee. However, the mechanical stress‐related response in meniscus cells remains unclear. We investigated mechanical stretch‐regulated gene expression in human meniscus cells. Human inner and outer meniscus cells were prepared from the inner and outer halves of the lateral meniscus. The gene expressions of Sry‐type HMG box (SOX) 9 and α1(II) collagen (COL2A1) were assessed by real‐time PCR analyses after cyclic tensile strain (CTS) treatment (0.5 Hz, 5% stretch). The localization and phosphorylation of SOX9 were evaluated by immunohistochemical and Western blot (WB) analyses. Chromatin immunoprecipitation (IP) analysis was performed to assess the stretch‐related protein–DNA complex formation between SOX9 and the COL2A1 enhancer on chromatin. Type II collagen deposition and SOX9 production were detected only in inner menisci. CTS treatments increased expression of the COL2A1 and SOX9 genes in inner meniscus cells, but not in outer meniscus cells. In addition, CTS treatments stimulated nuclear translocalization and phosphorylation of SOX9 in inner meniscus cells. Chromatin IP analyses revealed that CTS increased the association between SOX9 and its DNA‐binding site, included in the COL2A1 enhancer, on chromatin. Our results indicate that inner and outer meniscus cells have different properties in mechanical stretch‐induced COL2A1 expression. In inner meniscus cells, mechanical stretch may have an essential role in the epigenetic regulation of COL2A1 expression.