title: Cloning and characterization of rat caspase-9: implications for a role in mediating caspase-3 activation and hippocampal cell death after transient cerebral ischemia.
abstract: 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.
authors.affiliation: Department of Neurology and Pittsburgh Institute of Neurological Disorders, University of Pittsburgh School of Medicine, Biomedical Science Tower, Pittsburgh, PA 15213, U.S.A.
authors.forename: Guodong
authors.initials: G
authors.lastname: Cao
authors.forename: Yumin
authors.initials: Y
authors.lastname: Luo
authors.forename: Tetsuya
authors.initials: T
authors.lastname: Nagayama
authors.forename: Wei
authors.initials: W
authors.lastname: Pei
authors.forename: R Anne
authors.initials: RA
authors.lastname: Stetler
authors.forename: Steven H
authors.initials: SH
authors.lastname: Graham
authors.forename: Jun
authors.initials: J
authors.lastname: Chen
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D017209:Apoptosis; D001921:Brain / Q000201:enzymology / Q000254:growth & development; D053148:Caspase 3; D053453:Caspase 9; D020169:Caspases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000502:physiology*; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015870:Gene Expression; D006624:Hippocampus / Q000201:enzymology / Q000473:pathology*; D002546:Ischemic Attack, Transient / Q000201:enzymology* / Q000473:pathology; D008297:Male; D008969:Molecular Sequence Data; D009474:Neurons / Q000201:enzymology / Q000473:pathology; D051381:Rats; D017207:Rats, Sprague-Dawley
chemical_list: D018076:DNA, Complementary; D004791:Enzyme Inhibitors; C505410:Casp3 protein, rat; C506435:Casp9 protein, rat; D053148:Caspase 3; D053453:Caspase 9; D020169:Caspases