ACTIVATION AND CLEAVAGE OF CASPASE-3 IN APOPTOSIS INDUCED BY EXPERIMENTAL CEREBRAL-ISCHEMIA

We examined the expression, activation, and cellular localization of c aspase-3 (CPP32) using immunohistochemistry, immunoblots, and cleavage of the fluorogenic substrate carbonyl-Asp-Glu-Val-Asp-7-amino-4-trifl uoromethyl methyl coumarin (zDEVD-afc) in adult mouse brain after temp orary (2 hr) middle cerebral artery occlusion produced by filament ins ertion into the carotid artery. Immunoreactive caspase-3p32 but not it s cleavage product caspase-3p20 was constitutively expressed in neuron s throughout brain and was most prominent in neuronal perikarya within piriform cortex. Caspase-like enzyme activity was elevated in brain h omogenate 0-3 hr after reperfusion and reached a peak within 30 to 60 min. Caspase-3p20 immunoreactivity became prominent in neuronal perika rya within the middle cerebral artery territory at the time of reperfu sion and on immunoblots 1-12 hr later. DNA laddering (agarose gels) an d terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL)stained cells were detected 6-24 hr after reperfusion, At 12-24 hr, immunoreactive p20 was visualized in TUNEL-positive cell s, a finding also observed in apoptotic mouse cerebellar granule cells on postnatal day 5. Together, these observations suggest the existenc e of a time-dependent evolution of ischemic injury characterized by th e close correspondence between caspase-like enzyme activation and an a ssociated increase in immunoreactive product (caspase-3p20) beginning at or before reperfusion and followed several hours later by morpholog ical and biochemical features of apoptosis.