The brain hippocampus region has been known to possess markedly higher susceptibility to ischemic hypoxia and proceed to the specific neuronal apoptosis. Recently, relevance of p53 tumor suppressor gene to the induction of neuronal cell death, especially in central nervous system (CNS), has been focused. To elucidate the mechanism of the specific p53 dependent/independent apoptosis in hippocampus neurons, we established the mouse model induced neuronal apoptosis in hippocampus regions by bilateral common carotid artery occlusion (BCCAO), and analyzed the specific hippocampus proteins related to the ischemic apoptosis, using p53 gene knockout mice (p53-/-) comparing with those of p53 wild mice (p53+/+), by two proteomic strategies; 1) Differential display (comparative 2DE) by 2D-DIGE (fluorescence difference 2-D gel electrophoresis technology), 2) Differential display (comparative LC-MASS) by cICAT (cleavable isotope-coded affinity tags) method. In 2D-DIGE, about 4450 protein spots were detected in total, and 213 spots were found as ischemic stress sensitive or p53 specific proteins, such as p53 specific but ischemic apoptosis independnt;93, p53 independent but apoptosis dependent;53, p53 and apoptosis dependent but these factors are not interacted;14, and p53 and apoptosis dependent and these factors are interacted;39 (p53+/+specific apoptosis related;24, p53-/-specific apoptosis related;15). On the other hand, in cICAT method, 722 proteins were identified in 1074 unique peptides in 16000 peptides analyzed from hippocampus, and 467 (216 increased and 234 decreased) proteins were identified as the p53 specific apoptosis related proteins. These unique proteins identified by both methods included proteins related to apoptosis and cell cycle, redox related proteins, structure proteins, primarily chaperone proteins, kinases, small GTPases, cellular metabolism enzymes, and unknown proteins. These data suggest that the neuronal ischemic apoptosis in mouse hippocampus results from complex interactions between p53 dependent pathways involving redox enzyme reduction, induction of stress-induced proteins and apoptosis/cell cycle related proteins, and p53 independent pathway involving increased metabolism, disruption and reproduction of membrane and structural proteins, and ,ultimately, cell integrity breakdown. The prospective ability of those strategies for functional study of the neuronal apoptosis in central nervous system will be discussed.
