The presence of transthyretin in rat ependymal cells is due to endocytosis and not synthesis.

The presence and synthesis of transthyretin, a major carrier protein of thyroxine in rat cerebrospinal fluid, was investigated in choroid plexus epithelial cells and ependymal cells by immunocytochemistry, in situ hybridization, and analysis by Northern and Western blot using a specific oligonucleotide probe and a specific polyclonal antibody to transthyretin. Choroid plexus epithelial cells expressed transthyretin at high levels in developing rat cerebral hemispheres and in cultured cells. These cells secreted transthyretin into the cerebrospinal fluid. In the developing rat brain transthyretin was present in the cytoplasm of ependymal cells, in vesicles in contact with the apical membrane and in cilia. In ependymal cell cultures this protein was particularly abundant in the cilia of these cells. In contrast, ependymal cells did not synthesize transthyretin. It is postulated that transthyretin is transported to ependymal cells from the cerebrospinal fluid by endocytosis.

Expression of thyroid hormone receptors alpha and beta-1 messenger RNAs in human endothelial cells. The T3 hormone stimulates the synthesis of the messenger RNA of the intercellular adhesion molecule-1.

A study of the effect of the L-3,5,3'-triiodothyronine hormone on the expression of the mRNA of the adhesion molecule ICAM-1 led to the observation that the mRNA level is slightly up-regulated in human umbilical-cord endothelial cells. To analyze this induction at a molecular level, the search of T3 hormone receptors was undertaken. In this paper, we show that ECV 304 endothelial cells express the mRNAs encoding two thyroid hormone receptor isoforms alpha(alpha1 and alpha2) and one beta(beta1). This is, to our knowledge, a first important step towards the demonstration of the involvement of these receptors in the induction of the expression of ICAM-1 by the T3 hormone.

Expression of thyroid hormone receptor isoforms in rat oligodendrocyte cultures. Effect of 3,5,3'-triiodo-L-thyronine.

3,5,3'-Triiodo-L-thyronine (T3) acts at the genomic level by interacting with nuclear T3 receptors (T3Rs). We have used double immunostaining to follow the expression of T3Rs and oligodendrocytes (OL) lineage markers in rat secondary cultures consisting of 85-90% OL. Using antibodies against different synthetic peptides of T3Rs (alpha common: alpha 1 + alpha 2 and beta 1) we find that alpha-T3R is expressed in both O-2A progenitors and in mature OL, while beta 1-T3R is expressed only in mature OL. In cultured OL, beta 1-T3R mRNA is upregulated the most by T3. OL exhibit more numerous and longer processes when treated by T3.

Antibodies to cerebellar soluble lectin CSL in multiple sclerosis.

Cerebrospinal fluid samples from 239 patients with various neurological disorders were tested for the presence of autoantibodies to an endogenous mannose-binding protein, the cerebellar soluble lectin CSL, by means of an immunoblotting test with rat CSL as antigen. 47 of 51 patients with multiple sclerosis were positive for anti-CSL compared with 30 of 188 patients with other neurological disorders. 14 of the 30 false-positive patients were over 60 years old, an age group not typical of multiple sclerosis patients. The specificity of the test for multiple sclerosis was 85% and the sensitivity 93.5%. The possibility that CSL is an important immunological target in multiple sclerosis allows new insights into the possible causes and development of this disorder.

Carbohydrate and glycoprotein specificity of two endogenous cerebellar lectins.

Two endogenous cerebellar mannose binding lectins have been isolated in an active form by immunoaffinity chromatography employing their respective immobilized antibodies. One of them, termed cerebellar soluble lectin (CSL), was extracted in the absence of detergents, whereas the other, called Receptor 1 (R1), was soluble only in the presence of detergents. Tests of inhibition of agglutination of erythrocytes were performed with mono-, oligo and polysaccharides, as well as glycoconjugates of known structures. On the basis of agglutinating activities these 2 lectins are different from the previously reported lectins in brain, since they were not inhibited by galactosides and lactosides and were only marginally inhibited by glycosaminoglycans. CSL and R1 were better inhibited by mannose-rich glycopeptides as compared to the corresponding oligosaccharides. The different inhibition patterns obtained with glycans of known structures indicated that these lectins are very discriminative. Although CSL and R1 have similar specificities, they differed in their binding properties towards glycopeptides of ovalbumin. Both lectins showed considerable affinity for endogenous cerebellar glycopeptides, also rich in mannose. These glycopeptides belong to a few endogenous Con A-binding cerebellar glycoprotein subunits and are not present on other endogenous Con A-binding glycoproteins. In the forebrain, where CSL and R1 were also present, at least some of the glycoproteins interacting with the lectins were different from that observed in the cerebellum. Our data overall suggest that specific cell recognition in the nervous system could be invoked via the interactions between widely distributed lectins and cell-specific glycoproteins.

Location of a transiently expressed glycoprotein in developing cerebellum delineating its possible ontogenetic roles.

The development pattern of a 31,000 mol. wt phosphatidyl inositol-anchored membrane glycoprotein was followed during development in mouse and rat cerebellum using monoclonal antibody 194-653. The epitope was developmentally regulated and particularly abundant in post mitotic precursors of granule cells, newly formed parallel fibres and unmyelinated axons of the white matter between the 5th and the 15th postnatal days. It decreased considerably thereafter. In the adult, a significant although relatively low staining was observed only in white matter. Observation at the ultrastructural level showed that most of the 31,000 mol. wt glycoprotein was very concentrated on neuronal plasma membranes. A little immunoreactivity was also found intracellularly at the perinuclear membrane of neuroblasts of the external germinal layer. The antigen was present in the coated pits and intracellularly in coated vesicles. Immunochemical studies indicated that 31,000 mol. wt antigen was very likely to be a previously identified transient concanavalin A-binding glycoprotein insoluble in neutral detergents (Reeber et al., 1981; Brain Res. 229, 53-65). It appeared to be one of the glycoprotein ligands for two endogenous mannosyl-lectins isolated from rat cerebellum (Zanetta et al., 1985, Devl. Brain Res. 17, 233-243, Zanetta et al., 1987, J. Neurochem. 49, 1250-1257). The affinity of the 31,000 mol. wt glycoprotein for the two endogenous lectins, together with its developmental pattern and localization indicate that it could be an important molecule for contact guidance during migration of neurons and for myelination and could take part in other ontogenetic steps.

Con A-binding glycoproteins in the developing cerebellum of control and hypothyroid rats.

Concanavalin A (Con A)-binding glycoproteins were studied during the postnatal development of the cerebellum of control and hypothyroid rats. Only 4 glycoprotein bands have a transient behavior in control animals. They progressively increase until the 13th day and markedly decline between the 15th and the 18th postnatal day. In the cerebellum of hypothyroid rats, the level of these compounds is greatly reduced and the previous decrease observed in controls is not found again. This defect of Con A-binding glycoproteins mainly localized on the plasma membrane of parallel fibers might be related to the reduced synaptogenesis observed in the molecular layer of hypothyroid rats between parallel fibers and Purkinje cell dendritic spines.

Evidence for the presence of lectins with mannose specificity in the rat cerebellum.

Two different methods were set up to detect the possible presence of lectin-like molecules with a specificity for mannose-rich glycans in the rat cerebellum. The first, affinity histochemistry, involved the isolation of a particular class of glycoproteins from the cerebella of 11-day-old rats followed by the formation of covalent complexes with horseradish peroxidase and then incubation with cerebellar slices. The second used in vitro interactions between [3H]leucine-labeled proteins, kept in solution, with insolubilized [14C]glucosamine-labeled glycoproteins. The results of both methods are compatible with the presence of lectin-like activities inhibited by high mannose concentrations, but not other sugars. However, the binding sites preferred by these molecules seem to be more than a single mannose residue.

Arguments in favour of endocytosis of glycoprotein components of the membranes of parallel fibers by Purkinje cells during the development of the rat cerebellum.

Chloroquine (a drug known to induce a dysfunction of lysosomes) was used to study the behavior of Concanavalin A binding glycoproteins located on the axolemma of parallel fibers in young rat cerebella, and abundant on these membranes at a period preceding synaptogenesis with the dendrites of Purkinje cells. Chloroquine induces in Purkinje cells a large accumulation of grains consisting of membrane whorls in lysosomes. These grains stain for Concanavalin A, and do not stain either for a mitochondrial marker (aspartate aminotransferase mitochondrial isoenzyme) or for a marker of the Purkinje cell internal membrane (PSG). It is suggested that the material accumulating in the Purkinje cells under the effect of chloroquine comes from the parallel fibers. Together with the observation that alpha-D-mannosidase (involved in the degradation of these glycoproteins) is exclusively located inside Purkinje cells, these results provide a firm indication that this material enters the Purkinje cells through pinocytosis. The absence of ATPase activity (ATPase is a glycoprotein plasma membrane marker highly concentrated on parallel fibers) within these grains suggested that not all the components of these membranes are pinocytosed, but that the process is specific for certain molecules. These results are compatible with the ultrastructural observations of others, and support the arguments in favour of the pinocytosis phenomenon being one of the first steps of synapse formation. The observed specificity of pinocytosis for certain molecules suggests that a receptor-mediated recognition of some glycans of glycoproteins is the preliminary event in the establishment of synapses.

Isolation and immunohistochemical localization of a "Purkinje cell specific glycoprotein subunit from rat cerebellum.

The indirect immunohistochemical method has been used to determine the cellular localization of a Con A-binding glycoprotein subunit insoluble in Triton X-100, isolated from a rat cerebellar membrane pellet by Con A-Sepharose affinity chromatography and preparative PAGE electrophoresis in the presence of sodium dodecyl sulfate. This glycoprotein subunit (N-terminal amino acid Ala; MW 24,000 daltons) contains 24% carbohydrate (w/w) mainly mannose but also N-acetylglucosamine, galactose and sialic acid. At the optical level, the antiserum raised against this glycoprotein stains the whole Purkinje cell (dendrite, cell body and even the axon) but not the other cerebellar neurons. This staining is not eliminated when the antiserum is absorbed by rat forebrain or brain stem homogenates but is, in contrast, completely abolished by absorption with rat cerebellum homogenates. Thus, this antigen appears to be in the central nervous system exclusive to the cerebellum, and in the cerebellum specific for Purkinje cells. This antigen is present throughout the postnatal development of the rat cerebellum and the staining is already very strong at the fifth postnatal day.

Glycoproteins from adult rat brain synaptic vesicles. Fractionation on four immobilized lectins.

Glycoproteins obtained from large amounts of highly purified synaptic vesicles isolated from adult rat brain were fractionated by sequential affinity chromatography in the presence of SDS on four different immobilized lectins: concanavalin A, Ulex europeus lectin, Ricinus sanguinis lectin and wheat germ agglutinin. 83% of the total protein-bound sugar of synaptic vesicles can be adsorbed on the lectins and separated from the bulk of carbohydrate free proteins. Nine fractions containing only glycoproteins and differing by their terminal sugars were separated by analysed for their carbohydrate composition and electrophoretic profiles. A considerable heterogeneity of the glycoprotein population was observed which cannot be explained solely by the microheterogeneity of the glycans of the synaptic vesicle glycoproteins.

Transient concanavalin A-binding glycoproteins of the parallel fibres of the developing rat cerebellum: evidence for the destruction of their glycans.

The lifetime of the glycoprotein glycans of the rat cerebellum was followed in the 2nd and 3rd weeks of postnatal age, after injection of labelled glucosamine. It appears that a particular class of glycans binding to Concanavalin A synthesized at an early age has a short lifetime. These results indicate that the glycans of the concanavalin A-binding glycoproteins abundant on the newly formed parallel fibres are rapidly degraded between the 14th and the 18th postnatal day.

Synaptosomal plasma membrane glycoproteins. II. Isolation of fucosyl-glycoproteins by affinity chromatography on the Ulex europeus lectin specific for L-fucose.

After solubilization in sodium dodecyl sulphate, almost 90% of synaptosomal plasma membrane glycoproteins were separated from the bulk of synaptosomal plasma membrane proteins by sequential affinity chromatography on two immobilized lectins: concanavalin A and the Ulex europeus lectin specific for L-fucose. Four fractions were obtained and their sugar composition and electrophoretic patterns were determined. Fucosyl-glycoproteins contain more than 26% of protein and 85% of the protein-bound sugar of synaptosomal plasma membrane; hence they constitute a major class of glycoproteins in these membranes. The presence of some glucose in glycoproteins fractions obtained after affinity chromatography on the two lectins suggests that this sugar could be a structural component of some brain glycoproteins. Polyacrylamide gel electrophoresis revealed at least 28 major bands in fucosylglycoprotein fractions, and 11 in other fractions. Several of these major bands appear to contain more than one glycoprotein each. This heterogeneity appears to be mostly the result of the heterogeneity of the neuronal population in the central nervous system. Microheterogeneity of glycoprotein sugar chains and possible contamination of synaptosomal plasma membranes play, in our opinion, only a minor role.