Ceramide induces apoptosis to immature cerebellar granule cells in culture

A recent study revealed that ceramide acts as a second messenger in the sphingomyelin pathway and thus plays an important regulatory role in programmed cell death (apoptosis) to cell the lines induced by tumor-necrosis factor (TNF)-α and interleukin (IL)-1β, although its effect remains controversial regarding primary neuronal culture. We investigated the effect of a cell-permeable ceramide analog (C₂-ceramide) on cultures of cerebellar granule cells, which is thought to have active sphingomyelin pathway during development. The presence of C₂-ceramide decreased the number of cerebellar granule cells (CGCs) in a concentration-dependent manner when added at DIV 1 (1 day in vitro). The ED₅₀ was 60 μM. After DIV2, CGCs became less sensitive to C₂-ceramide and the ED₅₀ was 200 μM at DIV 7. DNA staining with Hoechst 33258 showed the morphology of apoptotic nuclei in the degenerating neurons. Internucleosomal DNA degradation could also be observed by gel etectrophoresis. Protein and RNA synthesis inhibitors prevented the death of neurons. C₂-dihydroceramide, which lacks the 4-5 trans double bond and failed to induce neuronal death. These results thus demonstrated that C₂-ceramide induces apoptosis to the CGCs at the early stage in vitro, however the CGCs were found to be less sensitive to C₂-ceramide at the later stage in vitro.