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The Journal of Neuroscience, October 15, 1998, 18(20):8186-8197
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
Aaron J.
Krohn1,
Elke
Preis2, and
Jochen H. M.
Prehn1, 2
1 Center for Interdisciplinary Clinical Research,
Junior Research Group "Apoptosis and Cell Death," Westphalian
Wilhelms-University, D-48149 Münster, Germany, and
2 Department of Pharmacology and Toxicology,
Philipps-University, D-35032 Marburg, Germany
We induced apoptosis in cultured rat hippocampal neurons by
exposure to the protein kinase inhibitor staurosporine (30 nM, 24 hr). Treatment with the antioxidant
(±)- -tocopherol (100 µM) or the superoxide
dismutase-mimetic manganese tetrakis (4-benzoyl acid) porphyrin (1 µM) 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 µM) 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.
Key words:
oxygen free radicals; superoxide; programmed cell death; apoptosis; caspase-1; caspase-3; mitochondria; hydroethidine; TMRE; vitamin E; superoxide dismutase; neuroprotection
Copyright © 1998 Society for Neuroscience 0270-6474/98/18208186-12$05.00/0
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