Shama Bhat

The isolation and long-term culture of oligodendrocytes from newborn mouse brain.

Using differential adhesion we successfully isolated relatively pure populations of mouse oligodendrocytes which can be maintained in vitro for more than two months. The highest percentage of galactocerebroside (GalC)-positive oligodendrocytes was 95% at 3 days after isolation. Thereafter, proliferation of astrocytes and fibroblasts occurred more quickly than did oligodendrocyte precursor division. GalC-positive oligodendrocytes rarely incorporate [3H]thymidine so that the use of a mitotic inhibitor (5 X 10(-6)M AraC) reduced the number of non-oligodendrocytes so as to maintain the purity of oligodendrocytes at more than 75% for 14 days in culture. This system will be of use for immunological and virological studies which require viable cultured mouse oligodendrocytes.

Developmental Expression of Neural Cell Adhesion Molecules of Oligodendrocytes In Vivo and in Culture

Abstract: Previously, we have shown that oligodendrocyte adhesion molecules are related to the 120,000–Mr neural cell adhesion molecule (NCAM‐120). In this report, we present further evidence that the oligodendrocyte adhesion molecule is NCAM‐120. Studies on the expression of NCAM‐120 and other molecular forms of NCAM in vivo in rat brain, in vitro in primary mixed cultures, and in cultures enriched for oligodendrocytes are described. Western blot analysis of rat brain using anti‐NCAM showed that NCAM‐120 first appears at postnatal day 7 and increases in quantity thereafter, coincident with the development of oligodendrocytes in vivo and comparable to the expression of myelin basic protein. Purified oligodendrocytes from 4‐week‐old rat brains expressed only NCAM‐120. Quantitation of various forms of NCAMs in rat brain showed marked age‐related differences in the expression of three molecular forms of NCAM. Immunofluorescence analysis showed that oligodendrocytes, at all ages tested, expressed NCAM, but in older oligodendrocytes, the intensity of staining was less. Western blot analysis of oligodendrocyte‐enriched cultures showed that from day 1 after isolation (12 days of age) through day 7 after isolation (18 days of age) only NCAM‐120 is seen. A possible role for NCAM in myelination and remyelination is discussed.

NCAM-180, the largest component of the neural cell adhesion molecule, is reduced in dysmyelinating quaking mutant mouse brain

To explore the role of the neural cell adhesion molecule (NCAM) in myelination and remyelination, we studied the developmental expression of various molecular forms of NCAM in dysmyelinating quaking mouse brain as compared with normal mouse brain. Normal mouse brain expressed the several molecular forms differentially during development. Quaking showed a marked reduction in the expression of NCAM-180, which represents the largest component of NCAM, in comparison with normal brain. This suggests that the defective myelin compaction in the quaking mutation may be the result of a deficiency of NCAM-180.

EXPRESSION OF NEURAL CELL ADHESION MOLECULE IN DYSMYELINATING MUTANTS

The possible role of neural cell adhesion molecule (NCAM) in myelination was studied in the dysmyelinating mouse mutants jimpy and shiverer, by characterizing the expression of the different molecular forms of brain NCAM as a function of age. In jimpy, the expression of NCAM-120 (120,000-Da NCAM) was low and in shiverer both NCAM-120 and NCAM-180 (180,000-Da NCAM) were reduced when compared to controls. In both jimpy and shiverer there was no significant change in the phospholipase C-sensitive NCAM-120. These data further support the possibility that NCAM may be involved in myelination.

Adult human brain expresses four different molecular forms of neural cell adhesion molecules.

Using antibodies to rat neural cell adhesion molecules (NCAM), we analyzed the NCAM of adult human brain. Various regions of the brain were analyzed quantitatively by Western blot. Grey matter showed four bands of NCAM with apparent molecular weights of 180,000, 170,000, 140,000 and 120,000. White matter showed one major band with an apparent M(r) of 120,000 and a minor band of 180,000. Cerebellar grey matter contained mainly 170,000, 140,000 and 120,000, white cerebellar white matter had only 180,000 and 120,000 M(1) NCAMS. Spinal cord showed mainly 120,000 M(r) NCAM. Deglycosylation using N-glycanase resulted in 170,000, 160,000, 130,000 and 110,000 M(r) proteins, suggesting that the four forms of human NCAM are derived from individual polypeptides. The presence of 170,000 M(1) NCAM is unique to human brain.

Neuroblastoma Cell - Oligodendrocyte Interaction Is Mediated by Neural Cell Adhesion Molecules

The role of neural cell adhesion molecule (NCAM) in the interaction between neurons and oligodendrocytes was studied using N2A neuroblastoma cells, which express only the 180,000 Mr NCAM and rat oligodendrocytes which express only 120,000 Mr NCAM. Oligodendrocytes bound to neuroblastoma cells, and the binding was inhibited by anti-NCAM, suggesting that NCAM is involved in oligodendrocyte-neuron interaction. The possible role of NCAM in myelination and remyelination is discussed.

Rat oligodendrocytes have cell adhesion molecules

The aggregation of isolated oligodendrocytes appears to be mediated by cell adhesion molecules. The cell adhesion molecules are inactivated by trypsin. Regeneration of the adhesion molecules is prevented by inhibitors of either protein synthesis or glycosylation. An immunological assay is described to quantitate the molecules involved in oligodendrocyte adhesion based on the following criteria: (a) treatment of oligodendrocytes with rabbit immunoglobulins against oligodendrocyte-conditioned medium prevented their aggregation; and (b) incubation of these immunoglobulins with oligodendrocyte-conditioned medium neutralized their ability to inhibit aggregation.

Expression and localization of Lewisx glycolipids and GD1a ganglioside in human glioma cells

We analysed the glycolipid composition of glioma cells (N-370 FG cells), which are derived from a culture of transformed human fetal glial cells. The neutral and acidic glycolipid fractions were isolated by column chromatography on DEAE-Sephadex and analysed by high-performance thin-layer chromatography (HPTLC). The neutral glycolipid fraction contained 1.6 µg of lipid-bound glucose/galactose per mg protein and consisted of GlcCer (11.4% of total neutral glycolipids), GalCer (21.5%), LacCer (21.4%), Gb4 (21.1%), and three unknown neutral glycolipids (23%). These unknown glycolipids were characterized as Lewisx (fucosylneolactonorpentaosyl ceramide; Lex), difucosylneolactonorhexaosyl ceramide (dimeric Lex), and neolactonorhexaosyl ceramide (nLc6) by an HPTLC-overlay method for glycolipids using specific mouse anti-glycolipid antibodies against glycolipid and/or liquid-secondary ion (LSI) mass spectrometry. The ganglioside fraction contained 0.6 µg of lipid-bound sialic acid per mg protein with GD1a as the predominant ganglioside species (83% of the total gangliosides) and GM3, GM2, and GM1 as minor components. Trace amounts of sialyl-Lex and the complex type of sialyl-Lex derivatives were also present. Immunocytochemical studies revealed that GD1a and GalCer were primarily localized on the surface of cell bodies. Interestingly, Lex glycolipids and sialyl-Lex were localized not only on the cell bodies but also on short cell processes. Especially, sialyl-Lex glycolipid was located on the tip of fine cellular processes. The unique localization of the Lex glycolipids suggests that they may be involved in cellular differentiation and initiation of cellular growth in this cell line.

Galactose to ceramide linkage is essential for the binding of a polyclonal antibody to galactosyl ceramide

Characterization of a polyclonal antibody to galactosyl ceramide (Gal-Cer) which inhibits the internalization and infection of HIV-1 in neural cell lines was carried out. Polyclonal antibody to Gal-Cer was produced by injecting rabbits with Gal-Cer liposomes. The specificity of anti-Gal-Cer binding was studied by high performance thin layer chromatography (HPTLC)-based immunoassay. Using natural and semisynthetic lipids, the specificity of anti-Gal-Cer interaction was studied. The antibody bound to Gal-Cer and its derivatives. The antibody did not bind to glucosyl ceramide or lactosyl ceramide. Glucosyl ceramide differs from Gal-Cer by a hydroxyl group at the fourth carbon and in lactosyl ceramide galactose is linked to ceramide by an intervening glucose molecule. This indicates that D-galactose linked to ceramide is essential for binding. Removal of fatty acid from Gal-Cer, as seen with N-palmitoyl- and N-oleoyl Gal-Cer, had no effect on the binding. It appears that the third carbon of Gal-Cer is not involved in the binding. This is supported by the binding of anti-Gal-Cer to sulfatide or GM4 in which sulfate or sialic acid are added at the third carbon of Gal-Cer, respectively.

C6 glioma cells express modified neural-cell adhesion molecule-like glycoproteins

We have studied the expression of neural-cell adhesion molecule (N-CAM)-like glycoproteins in a C6 glioma cell line. We found that: (a) C6 cells express N-CAM-like proteins on the cell surface, (b) the N-CAM-like proteins in C6 cells have apparent molecular weights of 130,000 and 150,000 kDa which are not seen in rodent brain and (c) deglycosylation of C6 N-CAMs suggest that the modifications are both in the carbohydrate and protein parts of the N-CAM. The expression of modified N-CAM glycoproteins is of interest in relation to the regulation of N-CAM expression.