Ceramide induces apoptosis to immature cerebellar granule cells in culture

A recent study revealed that ceramide acts as a second messenger in the sph ingomyelin pathway and thus plays an important regulatory role in programme d cell death (apoptosis) to cell the lines induced by tumor-necrosis factor (TNF)-alpha and interleukin (IL)-1 beta, although its effect remains contr oversial regarding primary neuronal culture. We investigated the effect of a cell-permeable ceramide analog (Cz-ceramide) on cultures of cerebellar gr anule cells, which is thought to have active sphingomyelin pathway during d evelopment. The presence of C-2-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 mu M After DIV2, CGCs became less sens itive to C-2-ceramide and the ED50 was 200 mu M at DIV 7. DNA staining with Hoechst 33258 showed the morphology of apoptotic nuclei in the degeneratin g neurons. Internucleosomal DNA degradation could also be observed by gel e lectrophoresis. Protein and RNA synthesis inhibitors prevented the death of neurons. C-2-dihydroceramide, which lacks the 4-5 trans double bond and fa iled to induce neuronal death. These results thus demonstrated that C-2-cer amide induces apoptosis to the CGCs at the early stage in vitro, however th e CGCs were found to be less sensitive to C-2-ceramide at the later stage i n vitro.