Jean-Louis Nussbaum

Arrest of proteolipid transport through the Golgi apparatus in Jimpy brain

SummaryImmunocytochemical investigations were performed on Jimpy and control mouse brains using three specific anti-myelin proteolipids antisera: immunoaffinity purified multivalent anti-(PLP + DM-20) proteolipid antibodies, anti-C-terminal hexapeptide 271–276 and anti-tridecapeptide 117–129 antisera. The results show that oligodendrocytes and myelin sheaths in normal mouse brain are labelled to the same extent by the three specific antisera; in contrast, in Jimpy brain these cellular structures are only stained by the multivalent antibodies and the site-specific, anti-tridecapeptide antiserum. The absence of labelling with C-terminal hexapeptide antiserum in mutant brain is interpreted as the result of either a large deletion or a point mutation producing a frameshift in the C-terminal part of the sequences of the proteolipids PLP and DM-20. Furthermore, we show that this mutation prevents the normal transport of proteolipid molecules through the Golgi apparatus. The existence of a minor, extra-Golgi apparatus metabolic route for proteolipids to myelin structures is also discussed.

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 dit 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 diners 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.

UDPgalactose:Ceramide Galactosyltransferase of Rat Brain: A New Method of Purification and Production of Specific Antibodies

Abstract A new method for purification of UDPgalactose: ceramide galactosyltransferase (EC 2.4.1.45) is described. The principal steps involved solvent extraction at—70°, Triton X‐100 extraction, and DEAE‐Sephadex and Blue Sepharose chromatography. The active configuration of the enzyme was stabilized by phospholipids and a rapid loss of enzymatic activity was observed after removal of these lipids. The inactive enzyme could be fully reactivated in the presence of brain phospholipids dispersed in a Triton X‐100‐containing buffer. The purified enzyme preparation showed two major components by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate with apparent molecular weights of 50–70,000. The 53,000‐dalton protein was isolated by preparative gel electrophoresis in the presence of sodium dodecyl sulfate and used to produce antibodies against UDPgalactosexeramide galactosyltransferase.

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.

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 Eagles medium and Waymouths 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 Waymouths medium the proportion of oligodendroglial cells was higher and that proliferation was more active than in minimum Eagles 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.

Immunocytochemical localization of UDP-galactose: ceramide galactosyltransferase in myelin and oligodendroglial cells of rat brain

SummarySpecific antibodies were prepared against rat-brain UDP-galactose:ceramide galactosyltransferase (CGalT) and used to study the localization of this enzyme at light and electron microscopic levels. Using an immunocytochemical technique the presence of CGalT was revealed in the cytoplasm and processes of oligodendrocytes and in myelin sheaths of developing and adult rat brain. No immunostaining was detected in neurons or astrocytes. At the ultrastructural level the immunostaining of oligodendrocytes was most intense at the periphery of cytoplasm and probably included plasma membrane. Among the intracellular organelles of oligodendrocytes, specific labelling was occasionally seen in the stacks of Golgi apparatus membranes. In myelin sheaths anti-CGalT staining seems to be restricted to the outermost and innermost lamellae. The finding of CGalT in distant portions of oligodendrocyte processes and in loosely wrapped myelin membranes might indicate that myelin galactocerebrosides are synthesized in the proximity of the site of their incorporation into the newly formed myelin.

Endogenous retroviruses and multiple sclerosis. II. HERV-7q

The search for new endogenous retroviral sequences, on the basis of sequence homologies with the pol gene of the recently reported multiple sclerosis associated retrovirus (MSRV), allowed us to identify a full length endogenous retrovirus sequence located on the long arm of human chromosome 7. This retrovirus, HERV-7q, includes in its env region, within a single 1,620 bp open reading frame, a 664 bp domain almost identical to a 3 non-coding region of the rab7 gene. Transcripts encompassing both the env and the 3 LTR regions of HERV-7q have already been identified as expressed sequence tags, suggesting that this env-like gene might code for a 538 amino acid long deduced protein.

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.

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

The developmental expression of UDPgalactosexeramide galactosyltransferase (CGalT), an enyme 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 subultures prepared from 20‐day‐old primary cultures the activity of CGalT per oligodendrocyte increased from 1.3 × 10−6 nmol/hr on day 4 to 3.7 × −6nmol/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.

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.