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 to the protein kinase inhibitor staurosporine (30 nM, 24 hr). Treatment with the antioxidant (+/-)-alpha-tocopherol (100 microM) or the superoxide dismutase-mimetic manganese tetrakis (4-benzoyl acid) porphyrin (1 microM) significantly reduced staurosporine-induced cell death. Using hydroethidine-based digital videomicroscopy, we observed a significant increase in intracellular superoxide production that peaked 6-8 hr into the staurosporine exposure. This increase occurred in the absence of gross mitochondrial depolarization monitored with the voltage-sensitive probe tetramethylrhodamine ethyl ester. We then prepared extracts from staurosporine-treated hippocampal neurons and monitored cleavage of acetyl-Tyr-Val-Ala-Asp-aminomethyl-coumarin and acetyl-Asp-Glu-Val-Asp-AMC, fluorogenic substrates for caspase-1-like and caspase-3-like proteases, respectively. Staurosporine caused a significant increase in caspase-1-like activity that preceded intracellular superoxide production and reached a maximum after 30 min. Caspase-3-like activity paralleled intracellular superoxide production, with peak activity seen after 8 hr. Treatment with the corresponding caspase-3-like protease inhibitor acetyl-Asp-Glu-Val-Asp-aldehyde (10 microM) prevented the increase in caspase-3-like activity and staurosporine-induced nuclear fragmentation, but failed to prevent the rise in superoxide production and subsequent cell death. In contrast, treatment with caspase-1-like protease inhibitors reduced both superoxide production and cell death. Of note, antioxidants prevented superoxide production, caspase-3-like protease activity, and cell death even when added 4 hr after the onset of the staurosporine exposure. These results suggest a scenario of an early, caspase-1-like activity followed by a delayed intracellular superoxide production that mediates staurosporine-induced cell death of cultured rat hippocampal neurons.
Center for Interdisciplinary Clinical Research, Junior Research Group "Apoptosis and Cell Death," Westphalian Wilhelms-University, D-48149 Münster, Germany.
A J
AJ
Krohn
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E
Preis
J H
JH
Prehn
D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D000818:Animals; D000831:Animals, Newborn; D000975:Antioxidants / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects*; D020170:Caspase 1 / Q000378:metabolism*; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005456:Fluorescent Dyes; D006624:Hippocampus / Q000166:cytology; D008928:Mitochondria / Q000201:enzymology; D009474:Neurons / Q000166:cytology* / Q000187:drug effects / Q000201:enzymology; D009842:Oligopeptides / Q000494:pharmacology; D010617:Phenanthridines; D011480:Protease Inhibitors / Q000494:pharmacology; D051381:Rats; D011916:Rats, Inbred F344; D019311:Staurosporine / Q000494:pharmacology*; D013482:Superoxide Dismutase / Q000378:metabolism; D013481:Superoxides / Q000378:metabolism*; D014810:Vitamin E / Q000494:pharmacology
D000590:Amino Acid Chloromethyl Ketones; D000975:Antioxidants; D015853:Cysteine Proteinase Inhibitors; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; C098738:N-acetyl-tyrosyl-valyl-alanyl-aspartyl chloromethyl ketone; D009842:Oligopeptides; D010617:Phenanthridines; D011480:Protease Inhibitors; C099158:acetyl-aspartyl-glutamyl-valyl-aspartal; C106133:acetyl-tryptophyl-glutamyl-histidyl-aspartal; D013481:Superoxides; D014810:Vitamin E; C058428:hydroethidine; D013482:Superoxide Dismutase; C505410:Casp3 protein, rat; D053148:Caspase 3; D020169:Caspases; D020170:Caspase 1; D019311:Staurosporine