Cloning and characterization of rat caspase-9: Implications for a role in mediating caspase-3 activation and hippocampal cell death after transient cerebral ischemia

Delayed hippocampal neurodegeneration after transient global ischemia is mediated. at least in part. through the activation of terminal caspases, particularly caspase-3. and the subsequent proteolytic degradation of critical Cellular proteins. Caspase-3 may be activated by the membrane receptor-initiated caspase-8-dependent extrinsic path ay and the mitochondria-initiated caspase-9-dependent intrinsic pathway: however the precise role of these deduced apoptosis-signaling pathways in activating caspase-3 in ischemic neurons remains elusive. The authors cloned the caspase-9-ene from the rat brain and investigated its potential role in mediating ischemic neuronal death in a rat model of transient global ischemia. Caspase-9 gene expression and protease activity ere extremely low in the adult brain. whereas them were developmentally upregulated in newborn rats, especially at postnatal 12 weeks. a finding consistent with the theory of an essential role for caspase-9 in neuronal apoptosis during brain development. After 15-minute transient global ischemia. caspase-9 was over-expressed and protcolytically activated in the hippocampal CAI neurons at 8 to 72 hours of reperfusion. The temporal profile of capase-9 activation coincided with that of cytochrome c release and caspase-3 activation, but preceded CAI neuronal death. Immunoprecipitation experiments, revealed that there was enhanced formation of Apaf-1/caspase-9 complex in the hippocampus 8 and 24 hours after ischemia. Furthermore. intracerebral ventricular infusion of the related specific caspase-9 inhibitor N-benzyloxycarbonyl-Leu-Glu-His-Asp- fluoro-methylketone before ischemic attenuated caspase-3-like activity and significantly enhanced neuronal survival in the CAI sector. In contrast, inhibition of caspase-8 activity had no significant effect on caspase-3 activation or neuronal survival. These results that the caspase-9-dependent intrinsic pathway may be the primary mechanism responsible for the activation of caspase-3 in ischemic hippocampal neurons.