STAUROSPORINE-INDUCED APOPTOSIS OF CULTURED RAT HIPPOCAMPAL-NEURONS INVOLVES CASPASE-1-LIKE PROTEASES AS UPSTREAM INITIATORS AND INCREASED PRODUCTION OF SUPEROXIDE AS A MAIN DOWNSTREAM EFFECTOR

We induced apoptosis in cultured rat hippocampal neurons by exposure t o the protein kinase inhibitor staurosporine (30 nM, 24 hr), Treatment with the antioxidant (+/-)-alpha-tocopherol (100 mu M) or the superox ide dismutase-mimetic manganese tetrakis (4-benzoyl acid) porphyrin (1 mu M) significantly reduced staurosporine-induced cell death. Using h ydroethidine-based digital videomicroscopy, we observed a significant increase in intracellular superoxide production that peaked 6-8 hr int o the staurosporine exposure. This increase occurred in the absence of gross mitochondrial depolarization monitored with the voltage-sensiti ve probe tetramethylrhodamine ethyl ester. We then prepared extracts f rom staurosporine-treated hippocampal neurons and monitored cleavage o f acetyl-Tyr-Val-Ala-Asp-aminomethyl-coumarin and acetyl-Asp-Glu-Val-A sp-AMC, fluorogenic substrates for caspase-1-like and caspase-3-like p roteases, respectively. Staurosporine caused a significant increase in caspase-1-like activity that preceded intracellular superoxide produc tion and reached a maximum after 30 min. Caspase-3-like activity paral leled intracellular superoxide production, with peak activity seen aft er 8 hr. Treatment with the corresponding caspase-3-like protease inhi bitor acetyl-Asp-Glu-Val-Asp-aldehyde (10 mu M) prevented the increase in caspase-3-like activity and staurosporine-induced nuclear fragment ation, but failed to prevent the rise in superoxide production and sub sequent cell death, In contrast, treatment with caspase-1-like proteas e inhibitors reduced both superoxide production and cell death. Of not e, antioxidants prevented superoxide production, caspase-3-like protea se activity, and cell death even when added 4 hr after the onset of th e staurosporine exposure. These results suggest a scenario of an early , caspase-1-like activity followed by a delayed intracellular superoxi de production that mediates staurosporine-induced cell death of cultur ed rat hippocampal neurons.