Abstract
1-Methylthiodihydroceramide (10 microM) decreased de novo ceramide biosynthesis by about 90% in primary cultured cerebellar neurons. Accordingly, 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 synthesis was restored upon addition of dihydroceramide or ceramide, in micromolar concentrations, to the culture medium, suggesting that none of the glycosyltransferases involved in glycosphingolipid biosynthesis is inhibited by this analog. Assays of the enzymes catalyzing sphinganine 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 therefore due to its ability to deplete cells of newly formed free sphinganine. As a consequence of depletion of sphinganine levels, 1-methylthiodihydroceramide disrupted axonal growth in cultured hippocampal neurons in a manner similar to that reported for direct inhibitors of sphingolipid 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 microM) antagonized the inhibition of growth. Interestingly, the D-erythro and the L-threo isomere were equally effective, but the corresponding free base as well as other structurally related compounds did not affect either sphingolipid biosynthesis or neuronal growth.
Highlights
1-Methylthiodihydroceramide (10 M) decreased de novo ceramide biosynthesis by about 90% in primary cultured cerebellar neurons
We demonstrate that treatment of cerebellar neurons with 1-methylthiodihydroceramide (1-MSDH-Cer) strongly interferes with de novo Cer synthesis, and SL formation, by stimulating the catabolism of sphinganine, a vital precursor of medium; FB1, fumonisin B1, GlcCer, glucosylceramide; GSLs, glycosphingolipids; HPLC, high performance liquid chromatography; MEM, minimum essential medium; PDMP, DL-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol, SM, sphingomyelin; SPP, sphingosine 1-phosphate; the term sphinganine is used for dihydrosphingosine
We have described an SL analog that stimulated the activity of sphinganine kinase in vivo, resulting in increased rates of sphinganine degradation and, as a consequence, significantly reduced rates of SL synthesis
Summary
Materials—Six-day-old NMRI (Navy Marine Research Institute) mice were obtained from Dr Brigitte Schmitz from the Institut fur Anatomie und Physiologie der Haustiere of the University of Bonn, Germany. Sphingolipid Labeling, Extraction, and Analysis—After 4 to 5 days in culture, cerebellar neurons were rinsed two times with serum-free MEM and incubated in the presence of the DH-Cer analogs added as complexes with bovine serum albumin to the culture medium (MEM) containing 0.3% heat-inactivated horse serum. The reaction contained 0.1 M Tris buffer (pH 7.4), 0.5 mM dithiothreitol, 100 M stearoyl-CoA, 50 M labeled sphinganine (0.5 Ci), which was previously sonicated for 2 min on ice in the buffer solution, and 120 g of cell protein mixture, in a total volume of 80 l. After incubation for 15 min at 37 °C, the lipids were extracted, separated by TLC (chloroform/methanol/water, 80:10:1, by volume), and radiolabeled Cer was determined by scanning or cutting out the regions
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