A specific immunological probe for the major myelin proteolipid. Confirmation of a deletion in DM-20.

Major myelin proteolipid (MMPL, also called PLP) and DM-20 are the two major intrinsic membrane proteins of CNS myelin. A specific immunological probe was obtained for MMPL by raising antibodies against the synthetic tridecapeptide 117-129 of MMPL. Antibodies against this peptide reacted with the MMPL but did not cross react with DM-20, while both proteolipids had been shown previously to be recognized by antibodies directed against the C-terminal hexapeptide of MMPL. This is in accordance with previous findings showing that DM-20 differs only from MMPL by a deletion of residues 100-140 (+/- few units). Furthermore, this site-specific immunological probe also recognizes MMPL in its native form in oligodendrocytes in primary glial cell cultures.

Molecular cloning and nucleotide sequence of a cDNA clone coding for rat brain myelin proteolipid.

A cDNA library from rat brain was constructed in pBR322 and screened with a 14-mer mixed oligonucleotide probe based on residues 231-235 of bovine proteolipid (PLP). A positive clone was isolated: it contained a 1334-base-pair cDNA insert and was subjected to DNA sequence analysis. The cDNA encoded information for the 276 amino acids of rat PLP. Comparison with bovine PLP sequence showed a complete amino acid sequence homology except for 4 amino acid residues.

Immunohistochemical study with an anti-myelin serum. A marker for all glial cells except 'dark' oligodendrocytes.

The usefulness of an anti-myelin antiserum as a possible marker for glial cells and related structures was investigated using rat brain. As expected, the myelin fibers were heavily stained but the neuronal cells and their processes were unreactive. The oligodendrocytes, identified on electron microscopy, revealed labelling of only the light and medium types, but not the dark cells. These results indicate that the suggested morphological classification of oligodendrocytes may be based on varying amounts of myelin antigen synthesis. Astrocytes from all areas, Golgi epithelial cells, Bergmann fibers and some subependymal cells also reacted with this anti-myelin antiserum but the staining was abolished completely by preabsorption with kidney powder. In contrast, the myelin fibers and the light and medium oligodendrocytes could still be labelled. We conclude that this anti-myelin antiserum should prove useful in studies of oligodendrocytes in the central nervous system.

Developmental expression of the C1G5F2 antigen in cultured rat oligodendroglial cells.

The C1G5F2 antigen is a newly described minor myelin antigen of the central nervous system. Its expression compared with that of some other main myelin protein components (Wolfgram W1 protein, myelin basic proteins (MBP) and proteolipids) was investigated in rat oligodendrocytes derived from 10-day-old primary glial cell cultures and subcultured for several days in a chemically defined medium. It was demonstrated immunocytochemically that this antigen is detected later than the major myelin markers. All cells immunoreactive with the monoclonal antibody C1G5F2 were always labeled either by W1-, MBP- or proteolipid-specific antisera. It was also shown at the electron microscopic level that this antigen is mainly expressed on the surface of the extremities of the fine oligodendroglial processes. All these observations suggest that the C1G5F2 antigen may be a useful marker for a specific step in the oligodendrocyte maturation stage.

Study of a 53 kDa cell surface antigen of oligodendrocytes in culture.

It was shown previously that pure oligodendrocytes release proteins when maintained in a chemically defined medium. Among these proteins, a 53 kDa glycoprotein was characterized as a component accessible from the external surface of these glial cells. Specific antibodies directed against this glycoprotein were obtained using two different procedures. They were tested on immunoblots of different cells; the protein was detected in C6 glioma cells and fibroblasts, but not in astrocytes. No immunoreactive band was observed on immunoblots of developing rat brain suggesting that this protein may be a minor constituent of the oligodendrocyte in vivo. These antibodies were also used on oligodendrocyte cultures to confirm our earlier finding that this glycoprotein is on the surface of the oligodendroglial plasma membrane. This protein appears to be a useful surface marker for oligodendrocytes in culture.

Oligodendrocyte cell surface recognized by a novel monoclonal antibody specific to sulfatide.

A rat monoclonal antibody (OL-1) was obtained by in-vitro immunization of rat spleenocytes with paraformaldehyde-fixed primary cultured glial cells derived from newborn rat brain and subsequent fusion with a rat myeloma cell line. The antibody secreted by the hybridoma immunostains live rat and mouse oligodendrocytes in primary and secondary cultures. The antibody binds specifically to oligodendrocytes and myelin structures in-situ. Radioimmunolabelling assays with a number of purified glycolipids offer thin layer chromatography separation show that OL-1 antibody binds strongly to sulfatide and to a lesser extent to galactosyl diglyceride.

Proteolipid DM-20 predominates over PLP in peripheral nervous system.

The major central nervous system (CNS) myelin proteolipid (PLP) is also expressed in the peripheral nervous system (PNS). This paper gives evidence that DM-20, an isoform of PLP, also occurs in rat sciatic nerves, where, in contrast to what is seen in CNS myelin, it predominates over PLP. This conclusion was reached on the basis of results obtained by immunoblot analysis of a crude proteolipid extract from adult peripheral nerve with two site-specific anti-proteolipid (PLP and DM-20) antibodies. This finding was further corroborated by characterization of the products obtained by Polymerase Chain Reaction (PCR) amplification of cDNAs synthesized from total RNA of 14-day-old sciatic nerves. The significance of the occurrence of these proteolipids in PNS remains obscure.

A procedure for long-term culture of oligodendrocytes.

A procedure for long-term culture of oligodendrocytes is described, the starting material being 20-day-old primary mixed cultures of newborn rat brain. Cells were first incubated in a serum-free medium for 48 h before they were subcultured on poly-L-lysine coated plastic dishes. After this treatment, the oligodendrocytes developed well in Waymouth medium containing 10% (v/v) calf serum, while most of the astrocytes died. At 13 days in subculture more than 90% of the cells were identified as oligodendrocytes; the criteria for oligodendrocytes were based on their immunoreactivity to antisera against W1 Wolfgram protein, myelin basic proteins and the synthetic C-terminal hexapeptide of the major myelin proteolipid. At 13 and 19 days astrocytes were present, 7% and 20% respectively. The culture system described here may be useful to study the biochemical and immunological aspects of the oligodendrocytes.

Immunohistochemical studies of Wolfgram proteins in central nervous system of neurological mutant mice.

Immunohistochemical localization of Wolfgram proteins has been studied by the indirect immunoperoxidase technique with Wolfgram protein W1 antibodies in the nervous system of myelin deficient mutant mice: Jimpy, MSD and Quaking. In all these mutants, the myelinated fibers and the oligodendroglial cells (few in number) in the corpus callosum and the white matter of the cerebellum folium show a positive reaction to protein W1. These observations are in accordance with the immunological studies showing that the two major Wolfgram proteins, W1 and W2, of mutant mice have immunological similarities with that of the controls.

Demonstration of a specific localization of carbonic anhydrase C in the glial cells of rat CNS by an immunohistochemical method.

The localization of carbonic anhydrase C isoenzyme in the central nervous system (CNS) of the rat has been investigated using the indirect immunoperoxidase technique, at both optic and electron microscopic levels. Evidence is presented for a specific localization of the enzyme in the cytoplasm of the oligodendrocytes and astrocytes. Myelinated fibers show a weak staining. The positive reaction is restricted to the cytoplasmic areas of the myelin sheath and does not appear in the compact myelin. Neuronal cell bodies do not stain at all. A strong positive reaction to the antiserum was also observed in the choroid plexus.

An immunohistochemical study of two myelin-specific proteins in enriched oligodendroglial cell cultures combined with an autoradiographic investigation using [3H]thymidine.

The aim of the present work was to examine the possible relationship between proliferation and expression of 2 myelin specific proteins in cultured oligodendroglial cells. Mixed cultures of glial cells, from newborn rat brain, containing astroglia and oligodendroglia were grown in 2 different culture media, minimum Eagle's medium and Waymouth's medium both supplemented with 10% calf serum in presence or absence of adult rat brain soluble extract. The proliferative activity of the cells was followed over a 28-day period by autoradiography after radioactive thymidine incorporation. It was found that in cultures grown in Waymouth's medium the proportion of oligodendroglial cells was higher and that proliferation was more active than in minimum Eagle's medium. Addition of brain extract elicited a stimulation of the proliferation of the cells in the 2 basal media. Under all conditions W1 protein appeared earlier than MBP by immunofluorescent visualization. Some oligodendroglial cells synthesizing W1 protein were still able to proliferate. MBP appears to be a marker of a later stage of cell maturation since very few MBP-positive cells incorporated tritiated thymidine. More cells contained MBP in the presence of brain extract. These results suggest that oligodendroglial cell maturation proceeds by steps, the step of W1 protein expression is compatible with proliferation while that of MBP expression appears at the end of the proliferation phase.

UDPgalactose:ceramide galactosyltransferase in cultured oligodendrocytes: an enzymological and immunological study.

The developmental expression of UDPgalactose:ceramide galactosyltransferase (CGalT), an enzyme marker of one myelinogenic activity in nervous tissue, was studied in cultured oligodendrocytes. The activity of CGalT in cultures followed a characteristic pattern of developmental changes. In the primary cultures these changes could be represented by a biphasic curve with a maximum of enzymatic activity at about the 25th day in culture. After purifying the oligodendrocytes from the primary cultures and replating them in culture dishes, similar developmental changes of CGalT were observed. In the subcultures prepared from 20-day-old primary cultures the activity of CGalT per oligodendrocyte increased from 1.3 x 10(-6) nmol/hr on day 4 to 3.7 x -6 nmol/hr on day 21. Immunocytochemical studies with the antiserum against rat brain CGalT showed the presence of CGalT+ oligodendrocytes after 7 days in the primary culture (earliest time studied), later on the number of CGalT+ oligodendrocytes increased until 28 days (latest time examined). In the subcultures of purified oligodendrocytes the bulk of oligodendrocytes was stained by the anti-CGalT antibodies after 15 days. These results suggest that the initial expression of CGalT in oligodendroglial cultures involves an increase of the number of CGalT+ oligodendrocytes and of the amount of enzyme protein per cell.

A morphological and biochemical study of the myelin-like membrane structures formed in cultures of pure oligodendrocytes.

This study reports the production of myelin-like membranes in oligodendrocyte subcultures derived from 20-day-old primary glial cell cultures of newborn rat brain. These multi-layered structures show a variable number of membrane turns; up to 10 concentric lamellae are found in 3- to 4-week-old subcultures. When they are compacted, alternate dense and intraperiodic lines with a periodicity of 11.2 nm are noticeable. The most typical myelin proteins were detected straight on the multi-lammellar structures by a gold immunocytochemical method. Subcellular fractions containing these myelin-like structures were isolated by ultracentrifugation on a discontinuous sucrose gradient. They were analysed by sodium dodecylsulfate-polyacrylamide gel electrophoresis and immunoblotting; UDP-galactose: ceramide galactosyltransferase and 2',3'-cyclic nucleotide 3'-phosphohydrolase activities were also measured. The results indicate that the multi-layered membrane profiles have many characteristics of the myelin found in vivo; nevertheless some differences were still apparent. Our data support the concept of the cultured oligodendrocytes expressing the intrinsic myelinogenic properties and possessing a basic developmental program of myelination, apparently in the absence of stimuli coming from other brain cells.

Comparative immunohistochemical study of C1G5F2 antigen and myelin/oligodendrocyte glycoprotein (MOG) expression in brain of several animal species.

The localisation of the myelin/oligodendrocyte specific antigen C1G5F2 and the myelin/oligodendrocyte glycoprotein (MOG) was studied in parallel in the brain of various species throughout the phylogenetic line of vertebrates. This immunofluorescence study was performed on unfixed brain sections by using the newly described monoclonal antibody C1G5F2 and polyclonal anti-MOG antibody. The antigen C1G5F2 is detected from the reptilian class onwards whereas MOG is only found in mammals. Both antibodies clearly stained only the myelin sheaths in the brain of adult animals. The phylogenetic distribution of the C1G5F2 antigen compared to the other well known myelin proteins may indicate that it has a specific function during myelination. Moreover, evidence is given that the C1G5F2 antigen is a new minor myelin protein distinct from the glycoprotein MOG.

Detection of Wolfgram W1 protein, myelin basic proteins and proteolipids in cultured oligodendrocytes by the electro-immunoblotting method.

Oligodendrocytes, mechanically obtained from primary cultures of newborn rat brain, were investigated by a sensitive electro-immunoblotting method for the presence of the characteristic myelin proteins: Wolfgram W1 protein, basic proteins and proteolipids. These three major myelin protein types were detectable from the 20th to the 40th day in culture. The present biochemical findings are in accordance with previous immunohistochemical data and provide additional evidence that oligodendrocytes in culture are capable of synthesizing every myelin and oligodendrocyte marker found in vivo, in the absence of neurones.

Site-specific antibodies to rat myelin proteolipids directed against the C-terminal hexapeptide.

A site-specific antiserum against the rat myelin proteolipids was produced in rabbits by injection of a synthetic polypeptide composed of the C-terminal amino acids of the proteolipid sequence. The immunogenic hexapeptide H-Gly-Arg-Gly-Thr-Lys-Phe-OH was coupled to chicken egg-albumin with dimethylsuberimidate. Antibodies specific for this peptide reacted with the 2 myelin proteolipid protein bands after SDS polyacrylamide gel electrophoresis and electrophoretic transfer onto nitrocellulose. Immunocytochemical investigations with this anti-peptide antiserum showed that the Golgi complexes of the oligodendrocytes were highly labeled as noted previously with multivalent antibodies. Labeling of vesicles and discontinuous staining of the plasmalemma were also observed in the most actively myelinating oligodendrocytes. In contrast to previous results, the major dense line was free of staining; this may indicate that at this site the C-terminal hexapeptide is inaccessible to these antibodies and perhaps buried in the lipid bilayer, in disagreement with the proposed organization of the myelin proteolipid in the myelin membrane.