Mannose dependent tightening of the rat ependymal cell barrier. In vivo and in vitro study using neoglycoproteins.

The possible role of carbohydrate binding proteins (lectins) and glycoconjugates in the formation of junctions ensuring tightening between ependymal cells was studied using synthetic glycoconjugates, the neoglycoproteins. These compounds are prepared by substituting bovine serum albumin with sugar residues and additional labelling (or not) with fluorescein or biotin. Injections of these components into the cerebral ventricles of adult rats resulted in a binding pattern which could be related to their carbohydrate composition. Mannose-containing neoglycoproteins were bound to ependymal cell cilia and penetrated rapidly the brain tissue. Such phenomenon was not seen with glucose- or galactose-containing neoglycoprotein molecules. In contrast, mannose-, galactose- and glucose-containing neoglycoproteins bound strongly to some endothelial cells around blood vessels. Fluorescent unglycosylated serum albumin did not bind to any brain structures. In contrast, co-injection of mannose-containing non-fluorescent neoglycoproteins with the other fluorescent compounds (including fluorescent sugar-free BSA) resulted in the penetration of the fluorescent compounds into the brain tissue. This internalization into brain was attributed to disaggregation of junctions between ependymal cells. Cultured ependymal cells behaved likewise. In short term experiments (5 min-1 h), only the mannose-containing neoglycoproteins bound strongly to the ependymal cells, particularly to the cilia. In long term experiments (1-9 days), mannose-containing neoglycoproteins specifically induced the disappearance of junctions between the cultured cells. These results emphasize the importance of mannose-dependent recognition system in the maintenance of junctions between ependymal cells, where a mannose-binding lectin has been previously detected.

The alpha and beta thyroid receptors are expressed by cultured ependymal cells. Correlation with the effect of L-3,5,3'-triiodothyronine on glutamine synthetase mRNAs.

It is generally accepted that L-3,5,3'-triiodothyronine (L-T3) acts at the genomic level through an interaction with specific nuclear L-T3 receptors (NT3R). Using antibodies raised against different peptides of NT3R, we report here the immunocytochemical localization of the alpha, alpha 2, beta 1 NT3R subtypes in ependymal cell primary cultures. The alpha and beta thyroid hormone receptors are both expressed. While the alpha and alpha 2 subtypes are found in almost all cells, the beta 1 receptors are present in few cells only. The possibility that alpha and beta receptors are colocalized is discussed. We also demonstrate that ependymal cells respond to L-T3 with a marked increase of the expression of the glutamine synthetase messenger RNAs.

A study of in vitro and in vivo morphological changes of ependymal cells induced by galactocerebrosides.

Ependymal cells in culture and in vivo were treated with mixture of galactocerebrosides. Galactocerebroside is the major glycolipid of myelin and in demyelinating diseases is found in cerebrospinal fluid. Morphological changes induced by this treatment were examined by microscopy at both optical and ultrastructural levels. In vitro, cilia, microvilli, and junctions between the cells disappeared, processes containing intermediate filaments developed, and the cells lost characteristics typical of ependymal cells and became more astrocyte-like. As shown by vital staining with a fluorescent compound and by nuclear incorporation of bromodeoxyuridine, cells did not proliferate during the period of galactocerebroside treatment and the morphological transformation was restricted to the ependymal cells. In contrast, asialoganglioside-GM1 and sulfatides had no effect on ependymal cell morphology. Some of the in vitro observations could be reproduced in vivo. Junctions between ependymal cells disappeared and intercellular spaces appeared between these cells and the cerebral parenchyma at the basolateral side of the ependymal layer. At the apical side, morphological modifications of junctions and cilia were less evident. As these experimental conditions resemble those existing during demyelination the morphological changes described may account for perturbations of the physiological functions of the ependymal cell.