Pharmacological inhibition of caspase-3-like protease activity has been shown to rescue neurons from apoptosis caused by withdrawal of trophic factors or excitotoxic injury ( Du et al., 1997 Eldadah et al., 1997). To date, more than 10 caspase family members have been identified, but their relative contribution to PCD still remains unclear ( Cohen, 1997 Salvesen and Dixit, 1997).ĭuring apoptosis, caspase-3 and related caspases cleave proteins of the cytoskeleton, the nuclear matrix, transcription factors, and DNA repair enzymes, and they activate apoptosis-specific deoxyribonucleases ( Cohen, 1997, Enari et al., 1998). Caspases are a family of cysteine proteases that specifically cleave proteins after Asp residues ( Alnemri et al., 1996). It participates in the activation of the protease caspase-3, a mammalian homolog of CED-3 ( Fernandes-Alnemri et al., 1994). Apaf-1 is the first identified mammalian homolog of CED-4 ( Zou et al., 1997). The proteins of the Bcl-2 family are the mammalian homologs of CED-9 and are believed to act upstream of CED-3 and CED-4 ( Hengartner and Horvitz, 1994). Studies in Caenorhabditis elegans have identified three genes that regulate PCD in the nematode: ced-3, ced-4, and ced-9 ( Horvitz et al., 1994). Growing evidence suggests that features of a conserved cell death program may also play a role in neuronal degeneration after stroke and trauma and in neurodegenerative disorders such as Alzheimer’s and Parkinson’s disease ( Bredesen, 1995 Thompson, 1995). 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.ĭuring the development of the nervous system, many neurons die because of a physiological process known as programmed cell death (PCD) ( Oppenheim, 1991). 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. In contrast, treatment with caspase-1-like protease inhibitors reduced both superoxide production and cell death. Treatment with the corresponding caspase-3-like protease inhibitor acetyl-Asp-Glu-Val-Asp-aldehyde (10 μ m) 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. Caspase-3-like activity paralleled intracellular superoxide production, with peak activity seen after 8 hr. Staurosporine caused a significant increase in caspase-1-like activity that preceded intracellular superoxide production and reached a maximum after 30 min. 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. This increase occurred in the absence of gross mitochondrial depolarization monitored with the voltage-sensitive probe tetramethylrhodamine ethyl ester. Using hydroethidine-based digital videomicroscopy, we observed a significant increase in intracellular superoxide production that peaked 6–8 hr into the staurosporine exposure. Treatment with the antioxidant (±)-α-tocopherol (100 μ m) or the superoxide dismutase-mimetic manganese tetrakis (4-benzoyl acid) porphyrin (1 μ m) significantly reduced staurosporine-induced cell death. ![]() We induced apoptosis in cultured rat hippocampal neurons by exposure to the protein kinase inhibitor staurosporine (30 n m, 24 hr).
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