C-2-ceramide, a cell-permeable analog of ceramide, caused cell death in cultured rat cortical neuronal cells. C-2-ceramide-induced neuronal loss was accompanied by upregulation of caspase-3 activity, measured by cleavage of its fluorogenic substrate Ac-DEVD-AMC. Similar results were obtained when cortical neuronal cultures were treated with sphingomyelinase, an enzyme responsible for ceramide formation in the cell. Morphological evaluation of C-ceramide-treated cortical neurons showed nuclear condensation and fragmentation as visualized by Hoechst 33258 staining. Co-administration of the selective caspase-3 inhibitor z-DEVD-fmk or caspase-9 inhibitor z-LEHD-fmk significantly reduced C-2-ceramide-induced cell death, while co-application of the caspase-8, inhibitor z-IETD-fmk. was without effect. Immunoblot analysis of protein extracts from C-2-ceramide-treated cortical neuronal cultures revealed upregulation of active caspase-9 and caspase-3 protein levels, whereas presence of active caspase-8 immunoreactivity was undetectable in this system. Administration of C-ceramide to SH-SY5Y human neuroblastoma cells also caused apoptotic cell death. Moreover. ceramide-induced cell death was significantly decreased in caspase-9 dominant-negative SH-SY5Y cells. while both caspase-8 dominant-negative cultures and mock-transfected cells showed equally high levels of cell death following C-ceramide treatment. Taken together, these data suggest that neuronal death induced by ceramide may be linked to the caspase-9/caspase-3 regulated intrinsic pathway of cellular apoptosis. (C) 2002 Elsevier Science (USA), All rights reserved.