Sphingolipid depletion increases formation of the scrapie prion protein inneuroblastoma cells infected with prions

Sphingolipid-rich rafts play an essential role in the posttranslational (Bo rchelt, D. R., Scott, M., Taraboulos, A, Stahl, N., and Prusiner, S. B. (19 90) J. Cell Biol. 110, 743-752)) formation of the scrapie prion protein PrP Sc from its normal conformer PrPc (Taraboulos, A., Scott, M., Semenov, A., Avrahami, D., Laszlo, L., Prusiner, S. B., and Avraham, D. (1995) J. Cell B iol. 129, 121-132). We investigated the importance of sphingolipids in the metabolism of the PrP isoforms in scrapie-infected ScN2a cells. The ceramid e synthase inhibitor fumonisin B-1 (BB,) reduced both sphingomyelin (SM) an d ganglioside GM1 in cells by up to 50%, whereas PrPSc increased by 3-4-fol d. Whereas FB1 profoundly altered the cell lipid composition, the raft resi dents PrPc, PrPSc, caveolin 1, and GM1 remained insoluble in Triton X-100. Metabolic radiolabeling demonstrated that PrPc production was either unchan ged or slightly reduced in FB1-treated cells, whereas PrPSc formation was a ugmented by 3-4-fold. To identify the sphingolipid species the decrease of which correlates with increased PrPSc, we used two other reagents. When cel ls were incubated with sphingomyelinase for 3 days, SM levels decreased, GM 1 was unaltered, and PrPSc increased by 3-4-fold. In contrast, the glycosph ingolipid inhibitor PDMP reduced PrPSc while increasing SM. Thus, PrPSc see ms to correlate inversely with SM levels. The effects of SM depletion contr asted with those previously obtained with the cholesterol inhibitor lovasta tin, which reduced PrPSc and removed it from detergent-insoluble complexes.