Abstract
Sphingolipids are abundant constituents of neuronal membranes and have been implicated in intracellular signaling. We show that two analogs of glycosphingolipid biosynthetic intermediates, fumonisin B1 (which inhibits dihydroceramide synthesis) and DL-1-phenyl-2-palmitoylamino-3-morpholino-1-propanol (PPMP) (which inhibits glucosylceramide synthesis) decrease glycosphingolipid synthesis in rat sympathetic neurons. Although both fumonisin and PPMP inhibit glycosphingolipid synthesis, these inhibitors have differential effects on ceramide metabolism in axons. threo-PPMP, but not erythro-PPMP or fumonisin, induces an accumulation of [3H]palmitate-labeled ceramide and impairs axonal growth. Moreover, exogenously added, cell-permeable C6-ceramide, but not C6-dihydroceramide, mimicks the effect of PPMP. Our studies suggest that the lipid second messenger ceramide acts in distal axons, but not cell bodies, as a negative regulator of neurite growth.
Highlights
Glycosphingolipids (GSLs)1 are major components of eukaryotic cell membranes and are enriched in neuronal membranes
Inhibition of de Novo Sphingolipid Biosynthesis in Sympathetic Neurons—We examined the effects of fumonisin B1 (FB1) and PPMP on sphingolipid synthesis in rat sympathetic neurons. 11-Day-old neurons, cultured in 24-well dishes, were incubated with various concentrations of FB1, threo-PPMP, or erythro-PPMP for 4 days. [3H]Palmitate (10 Ci/ml) was added for the last 1 day of incubation, and incorporation of radiolabel into gangliosides was measured
A comparison of the effects of two inhibitors of the endogenous synthesis of GSLs (FB1 and PPMP) suggests that newly synthesized GSLs are not required for normal neurite elongation, rather the lipid second messenger ceramide negatively regulates neurite growth
Summary
Materials—[9,10-3H]Palmitate (specific activity 54 Ci/mmol) was purchased from Amersham Canada, Oakville, Ontario, Canada. Medium supplied to the center compartment containing the cell bodies was supplemented with 2.5% rat serum, 1 mg/ml ascorbic acid, 10 M cytosine arabinoside (to prevent growth of non-neuronal cells), and 10 ng/ml NGF. L15 medium supplied to neurons cultured in 24-well dishes was supplemented with 2.5% rat serum, 1 mg/ml ascorbic acid, and 100 ng/ml NGF. Gangliosides were separated, along with authentic standards, by high performance thin layer chromatography in the solvent system chloroform/methanol/15 mM CaCl2 (55:45:10, v/v). The water was aspirated and the wash repeated twice followed by addition of fresh culture medium This procedure effectively removes all visible traces of neurites from the side compartments [35]. Metabolism of Fluorescent Ceramide—13-Day-old neurons cultured in compartmented dishes were given 10 M N-{6-[(7-nitrobenz-2-oxa1,3-diazol-4-yl)amino]caproyl}-D-erythro-sphingosine (NBD-C6-ceramide) in the distal neurite-containing compartment. Other Methods—The phospholipid content of cells was measured by lipid phosphorous determination [38]
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