The developmental expression of the myelin basic protein (MBP) gene was studied in rat cultured oligodendrocytes using immunofluorescence and in situ hybridization. In newborn rat brain cultures, which contain only glial cells, large amounts of MBP-specific mRNA (as assayed by grain counts in autoradiograms) abruptly accumulated within immature oligodendrocytes 5 to 6 days postnatal. MBP always emerged 6 to 8 days after birth; thus, a week after, galactocerebroside (GC), an early oligodendrocyte marker, had appeared. The percentage of MBP mRNA and MBP-positive cells peaked at about 15 days postnatal and decreased thereafter. The time of emergence of MBP in these cultured oligodendrocytes appears to be determined at a very early stage in their development and independent of continuous neuronal influences. There is a striking correspondence between the times of appearance of MBP in cultured oligodendrocytes and those in the intact animal. Thus, primary cultures made from 5-day prenatal, newborn, and 2-day postnatal animals all express MBP at about the same developmental stage, namely, after 14, 8, and 6 days in culture, respectively. Furthermore, cultured oligodendrocytes obtained from the spinal cord express MBP before those obtained from midbrain or hemispheres, as they would in the intact animal. Thus, the developmental expression of the MBP gene occurs in a similar time frame in vitro and in vivo. In oligodendrocyte-enriched cultures, where 60% to 80% of the cells express MBP, in situ hybridization with the cDNA clone revealed MBP-specific mRNA in the cell body and sometimes in the processes of the differentiated oligodendrocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

In multiple sclerosis, a demyelinating disease of young adults, there is a paucity of myelin repair in the central nervous system (CNS) which is necessary for the restoration of fast saltatory conduction in axons. Consequently, this relapsing disease often causes marked disability. In similar diseases of small rodents, however, remyelination can be quite extensive, as in the demyelinating disease caused by the A59 strain of mouse hepatitis virus (MHV-A59), a coronavirus of mice. To investigate when and where oligodendrocytes are first triggered to repair CNS myelin in such disease, we have used a complementary DNA probe specific for one major myelin protein gene, myelin basic protein (MBP), which hybridizes with the four forms of MBP messenger RNA in rodents. Using Northern blot and in situ hybridization techniques, we previously found that MBP mRNA is first detected at about 5 days after birth, peaks at 18 days and progressively decreases to 25% of the peak levels in the adult. We now report that in spinal cord sections of adult animals with active demyelination and inflammatory cells, in situ hybridization reveals a dramatic increase in probe binding to MBP-specific mRNA at 2-3 weeks after virus inoculation and before remyelination can be detected by morphological methods. This increase of MBP-specific mRNA is found at the edge of the demyelinating area and extends into surrounding areas of normal-appearing white matter. Thus, in situ hybridization with myelin-specific probes appears to be a useful method for detecting the timing, intensity and location of myelin protein gene reactivation preceding remyelination. This method could be used to elucidate whether such a reactivation occurs in multiple sclerosis brain tissue. Our results suggest that in mice, glial cells react to a demyelinating process with widespread MBP mRNA synthesis which may be triggered by a diffusible factor released in the demyelinated areas.

Abstract. We have immortalized rat central nervous system (CNS) cells of primary cultures of rat optic nerve with murine leukemia virus w2,SV-40-6, which is defective in assembly and contains the SV-40 large T antigen and neomycin resistance genes, to produce a cell line that we named A7. After drug selection, >90% of the growing cells expressed nuclear SV-40 large T cells and a fraction of these contained the astrocyte-specific marker, glial fibrillary acidic protein.The majority of these cells also expressed surface marker A4 (specific for neural tube derivatives), Ran 2, p185 (the 185-kD phosphoprotein product of the neu oncogene), and fibronectin, but did not express the astrocyte enzymes glutamine synthetase and monoamine oxidase B. Surface markers characteristic of glial progenitors (A2B5) and oligodendrocytes (galactocerebroside) were not detected. After two rounds of cell cloning, subclone A7.6-3 expressed Ran 2, fibronectin, and the neural cell adhesion molecule (N-CAM) but not glial fibrillary acidic protein and A4. The A7 cell line and subclones also displayed certain functions of type 1 astrocytes: the conditioned medium of these cells had a potent mitogenic activity for glial progenitor cells which could be neutralized by anti-platelet-derived growth factor antibodies and monolayers of these cells supported the growth of embryonic hypothalamic neurons. We conclude that a retrovirus containing SV-40 large T antigen can immortalize rat CNS cells and that such immortalized glial cells retain at least two important functions of type 1 astrocytes: the ability to secrete platelet-derived growth factor and to support the growth of embryonic CNS neurons. Moreover, such stable immortalized clonal cell lines can be used to study gene regulation in glial cells.

The developmental program controlling the expression of myein basic protein (MBP) gene was studied in the rat using the technique of in situ hybridization. A "S-labeled cDNA done of mouse MBP encoding an amino acid sequence present in all four ofthe major forms ofrodent MBP was used. The probe hybridized to the tracts ofwhite matter with different intensities, depending on the age of the animal and the region of the brain examined. In the medulla oblongata, maximal hybridization was found in 5and 7-day-old rats and was confined to the tectospinal tracts, fibers ofthe seventh cranial nerve, and the spinocerei This study was made possible by grants from the American Multiple Sclerosis Society and the Swedish Medical Research Council (grant No.

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