title: Cloning of a novel Apaf-1-interacting protein: a potent suppressor of apoptosis and ischemic neuronal cell death.
abstract: Cytochrome c-initiated activation of apoptotic protease activating factor-1 (Apaf-1) is a key step in the mitochondrial-signaling pathway for the activation of death-executing caspases in apoptosis. This signaling pathway has been implicated in the pathophysiology of various neurological disorders, including ischemic brain injury. In this study, we have cloned a novel rat gene product, designated as Apaf-1-interacting protein (AIP), which functions as a dominant-negative inhibitor of the Apaf-1-caspase-9 pathway. AIP is constitutively expressed in the brain, but at substantially lower levels than Apaf-1 and caspase-9. AIP can directly bind to Apaf-1 in vitro through its N-terminal caspase-recruiting domain, and this protein interaction was increased in cells undergoing apoptosis. Cytosolic extracts from cells overexpressing AIP were highly resistant to cytochrome c- dATP-induced activation of caspase-9 and caspase-3. Gene transfection of AIP into cell lines, including the neuronal-differentiated PC12 cells, potently suppressed apoptosis induced by various pro-apoptotic stimuli. To further investigate the functional role of AIP in primary neurons and in the brain, an adeno-associated virus (AAV) vector carrying the AIP cDNA was constructed. AAV-mediated overexpression of AIP in primary cortical- hippocampal neurons markedly reduced cell death and caspase-3 activation triggered by protein kinase C inhibition, DNA damage, or oxygen- glucose deprivation. Moreover, intracerebral infusion of the AAV vector resulted in robust AIP expression in the hippocampus and significantly promoted CA1 neuronal survival after transient global cerebral ischemia. These results suggest that molecular targeting of the Apaf-1-caspase-9 signaling pathway may be a feasible neuroprotective strategy to enhance the endogenous threshold for caspase activation and prevent neuronal loss in stroke and related disorders.
authors.affiliation: Department of Neurology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261, USA.
authors.forename: Guodong
authors.initials: G
authors.lastname: Cao
authors.forename: Michael
authors.initials: M
authors.lastname: Xiao
authors.forename: Fengyan
authors.initials: F
authors.lastname: Sun
authors.forename: Xiao
authors.initials: X
authors.lastname: Xiao
authors.forename: Wei
authors.initials: W
authors.lastname: Pei
authors.forename: Juan
authors.initials: J
authors.lastname: Li
authors.forename: Steven H
authors.initials: SH
authors.lastname: Graham
authors.forename: Roger P
authors.initials: RP
authors.lastname: Simon
authors.forename: Jun
authors.initials: J
authors.lastname: Chen
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D053477:Apoptotic Protease-Activating Factor 1; D001921:Brain / Q000378:metabolism; D002545:Brain Ischemia / Q000235:genetics / Q000378:metabolism* / Q000628:therapy; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism* / Q000494:pharmacology; D053453:Caspase 9; D020169:Caspases / Q000235:genetics* / Q000378:metabolism* / Q000494:pharmacology; D002470:Cell Survival / Q000187:drug effects / Q000502:physiology; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D004195:Disease Models, Animal; D005786:Gene Expression Regulation; D005822:Genetic Vectors / Q000235:genetics / Q000494:pharmacology; D008969:Molecular Sequence Data; D009474:Neurons / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D018696:Neuroprotective Agents / Q000494:pharmacology; D011485:Protein Binding / Q000502:physiology; D011506:Proteins / Q000378:metabolism*; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D051381:Rats; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D015854:Up-Regulation
chemical_list: C506594:Apaf1 protein, rat; D053477:Apoptotic Protease-Activating Factor 1; D002352:Carrier Proteins; D018076:DNA, Complementary; D018696:Neuroprotective Agents; D011506:Proteins; D012333:RNA, Messenger; C506435:Casp9 protein, rat; D053453:Caspase 9; D020169:Caspases