Sphingolipid biosynthesis is necessary for dendrite growth and survival of cerebellar Purkinje cells in culture.

The requirement of complex sphingolipid biosynthesis for growth of neurons was examined in developing rat cerebellar Purkinje neurons using a dissociated culture system. Purkinje cells developed well-differentiated dendrites and axons after 2 weeks in a serum-free nutrient condition. Addition of 2 microM fumonisin B1, a fungal inhibitor of mammalian ceramide synthase, inhibited incorporation of [3H]galactose/glucosamine and [14C]-serine into complex sphingolipids of cultured cerebellar neurons. Under this condition, the expression of Purkinje cell-enriched sphingolipids, including GD1 alpha, 9-O-acetylated LD1 and GD3, and sphingomyelin, was significantly decreased. After 2 weeks' exposure to fumonisin B1, dose-dependent measurable decreases in the survival and visually discernible differences in the morphology were seen in fumonisin-treated Purkinje cells. The Purkinje cell dendrites exhibited two types of anomalies; one population of cells developed elongated but less-branched dendrites after a slight time lag, but their branches began to degenerate. In some cells, formation of elongated dendrite trees was severely impaired. However, treatment with fumonisin B1 also led to the formation of spinelike protrusions on the dendrites of Purkinje cells as in control cultures. In contrast to the alterations observed in Purkinje cells, morphology of other cell types including granule neurons appeared to be almost normal after treatment with fumonisin B1. These observations indicated strongly that membrane sphingolipids participate in growth and maintenance of dendrites and in the survival of cerebellar Purkinje cells. Indeed, these effects of fumonisin B1 were reversed, but not completely, by the addition of 6-[[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-amino dcaproyl]sphingosine (C6-NBD-ceramide), a synthetic derivative of ceramide. Thus, we conclude that deprivation of membrane sphingolipids in a culture environment is responsible for aberrant growth of Purkinje cells.