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

J Cereb Blood Flow Metab. 2002 May;22(5):534-46. doi: 10.1097/00004647-200205000-00005.


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 pathway 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 gene 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 were extremely low in the adult brain, whereas they 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 overexpressed and proteolytically activated in the hippocampal CA1 neurons at 8 to 72 hours of reperfusion. The temporal profile of caspase-9 activation coincided with that of cytochrome c release and caspase-3 activation, but preceded CA1 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 relatively specific caspase-9 inhibitor N-benzyloxycarbonyl-Leu-Glu-His-Asp-fluoro-methylketone before ischemia attenuated caspase-3-like activity and significantly enhanced neuronal survival in the CA1 sector. In contrast, inhibition of caspase-8 activity had no significant effect on caspase-3 activation or neuronal survival. These results suggest that the caspase-9-dependent intrinsic pathway may be the primary mechanism responsible for the activation of caspase-3 in ischemic hippocampal neurons.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Apoptosis*
  • Brain / enzymology
  • Brain / growth & development
  • Caspase 3
  • Caspase 9
  • Caspases / chemistry
  • Caspases / genetics
  • Caspases / metabolism*
  • Caspases / physiology*
  • Cloning, Molecular*
  • DNA, Complementary / genetics
  • Enzyme Activation
  • Enzyme Inhibitors / pharmacology
  • Gene Expression
  • Hippocampus / enzymology
  • Hippocampus / pathology*
  • Ischemic Attack, Transient / enzymology*
  • Ischemic Attack, Transient / pathology
  • Male
  • Molecular Sequence Data
  • Neurons / enzymology
  • Neurons / pathology
  • Rats
  • Rats, Sprague-Dawley


  • DNA, Complementary
  • Enzyme Inhibitors
  • Casp3 protein, rat
  • Casp9 protein, rat
  • Caspase 3
  • Caspase 9
  • Caspases