An immortalized jimpy oligodendrocyte cell line: defects in cell cycle and cAMP pathway.

Normal and jimpy oligodendrocytes in secondary cultures were transfected with plasmids containing the SV40 T-antigen gene expressed under the control of the mouse metallothionein-I promoter. Two immortalized stable cell lines, a normal (158N) and jimpy (158JP) cell line, expressed transcripts and proteins of oligodendrocyte markers, including proteolipid protein (PLP), myelin basic protein (MBP), and carbonic anhydrase II (CAII). Galactocerebroside and sulfatide were also detected with immunocytochemistry. Immunoelectron microscopy using gold particles showed that the truncated endogenous jimpy PLP was distributed throughout the cytoplasm and in association with the plasma membrane of cell bodies and processes. The length of the cell cycle in the jimpy oligodendrocytes in the absence of zinc was 31 h, about a 4-h longer cell cycle than the normal line. In the presence of 100 microM zinc, the cell cycle became 3 h shorter for both cell lines, with the jimpy cell cycle duration remaining 4 h longer than the normal line. Interestingly, the jimpy cell line showed a significant deficiency in stimulation via the cAMP pathway. While the level of oligodendrocyte markers (PLP, MBP, and CAII) were significantly increased by dibutyryl cAMP (dbcAMP) treatment in the normal cell line, no changes were observed in the jimpy cell lines. This observation, together with previous results showing jimpy oligodendrocyte's failure to respond to basic fibroblast growth factor (bFGF), suggests a role for PLP in a signal transduction pathway. Jimpy and normal oligodendrocytes transfected with the SV40T antigen gene, driven by the wild-type promoter of mouse metallothionein-I, continue to express properties of oligodendrocytes and therefore provide a powerful model to explore the function of myelin proteins and to dissect the complexity of the jimpy phenotype.

Involvement of de novo ceramide biosynthesis in lymphotoxin-induced oligodendrocyte death.

Both experimental and clinical studies suggest that lymphotoxin (LT) plays an important role in multiple sclerosis (MS) by inducing oligodendrocyte (OL) depletion. However, the mechanism of LT cytotoxicity is unknown. Because of the role of ceramide as a cell death mediator for a large variety of cytotoxic molecules, we have investigated the possible role of this second messenger in LT-induced cytotoxicity on SV40 immortalized new-born mice OL. Human recombinant LT exposure (50 ng/ml) resulted in intracellular ceramide accumulation which peaked at 48 h (approximately 170% increase) and paralleled LT-induced cytotoxicity. Moreover, fumonisin B1, a potent and specific ceramide synthase inhibitor, not only inhibited ceramide accumulation but also protected OL from LT cytotoxicity. These results suggest that LT-induced ceramide synthase stimulation and subsequent increased intracellular ceramide concentration are implicated in oligodendrocyte death.

Isolation and characterization of defective jimpy oligodendrocytes in culture.

This study characterizes jimpy oligodendrocyte-enriched secondary cultures isolated from 10-12 days in vitro primary glial cell cultures derived from 1-2-day-old jimpy mouse brains. Proliferation of defective oligodendrocytes was carefully investigated with regard to the expression of myelin basic protein and proteolipid protein and their respective mRNAs. Less than 5% of contaminating astrocytes (GFAP+ cells) were usually present. The identity of jimpy oligodendrocytes was confirmed using an antibody directed against a peptide from the wild type proteolipid protein C-terminal sequence for immunocytochemistry and an oligonucleotide complementary to mRNA derived from exon 5 of the proteolipid protein gene for in situ hybridization. Both the antibody and the probe recognize only normal oligondedrocytes while jimpy oligodendrocytes always remain unstained. Proteolipid protein in normal and jimpy oligodendrocytes was detected with antibody recognizing normal and mutated forms. Between 80 and 95% of the cells in normal and jimpy cultures at 2 and 4 days in vitro in secondary cultures express myelin basic protein and proteolipid protein and their respective mRNAs. The percentage of oligodendrocytes (PLP+ or MBP+) in S phase of the cell cycle was 7-10% for both normal and jimpy oligodendrocytes. This contrasts with the in vivo situation where the proliferation rate of oligodendrocytes in jimpy brains is higher than in normal brains. In addition, jimpy oligodendrocytes remain unresponsive to basic fibroblast growth factor treatment while a similar treatment stimulates the proliferation of normal oligodendrocytes.