Ceramide induces neuronal apoptosis through the caspase-9/caspase-3 pathway.
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(2)-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(2)-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(2)-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.
Movsesyan
Vilen A
VA
Department of Neuroscience, Georgetown University Medical Center, 3970 Reservoir Road, N.W., Research Building, Rm. EP12, 20057, Washington, DC, USA.
Yakovlev
Alexander G
AG
Dabaghyan
Elvira A
EA
Stoica
Bogdan A
BA
Faden
Alan I
AI
D000818:Animals; D017209:Apoptosis; D053148:Caspase 3; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000166:cytology; D004305:Dose-Response Relationship, Drug; D006801:Humans; D007700:Kinetics; D009154:Mutation; D009474:Neurons / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000494:pharmacology*; D014407:Tumor Cells, Cultured
C064769:N-acetylsphingosine; C505407:CASP3 protein, human; C505530:CASP8 protein, human; C506433:CASP9 protein, human; C505410:Casp3 protein, rat; C505528:Casp8 protein, rat; C506435:Casp9 protein, rat; D053148:Caspase 3; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases; D013110:Sphingosine