L-METHYLTHIODIHYDROCERAMIDE, A NOVEL ANALOG OF DIHYDROCERAMIDE, STIMULATES SPHINGANINE DEGRADATION RESULTING IN DECREASED DE-NOVO SPHINGOLIPID BIOSYNTHESIS

1-Methylthiodihydroceramide (10 mu M) decreased de novo ceramide biosy nthesis by about 90% in primary cultured cerebellar neurons, According ly, de novo formation of sphingomyelin and of glycosphingolipids, all of which contain ceramide in their backbone, was reduced in a time-and concentration-dependent manner by up to 80%. Complex sphingolipid syn thesis was restored upon addition of dihydroceramide or ceramide, in m icromolar concentrations, to the culture medium, suggesting that none of the glycosyltransferases in volved in glycosphingolipid biosynthesi s is inhibited by this analog, Assays of the enzymes catalyzing sphing anine biosynthesis, as well as its N-acylation to form dihydroceramide , revealed that they were also not affected, In contrast, there was a 2.5-fold increase in the activity of sphinganine kinase, Reduction of de novo sphingolipid biosynthesis by 1-methylthiodihydroceramide is th erefore due to its ability to deplete cells of newly formed free sphin ganine. As a consequence of depletion of sphinganine levels, 1-methylt hiodihydroceramide disrupted axonal growth in cultured hippocampal neu rons in a manner similar to that reported for direct inhibitors of sph ingolipid synthesis; thus, there was essentially no axon growth after incubation with 1-methylthiodihydroceramide between days 2 and 3, and co-incubation with short acyl chain analogs of ceramide (5 mu m) antag onized the inhibition of growth, Interestingly, the D-erythro and the L-threo isomere were equally effective, but the corresponding free bas e as well as other structurally related compounds did not affect eithe r sphingolipid biosynthesis or neuronal growth.