Activation and cleavage of caspase-3 in apoptosis induced by experimental cerebral ischemia.
We examined the expression, activation, and cellular localization of caspase-3 (CPP32) using immunohistochemistry, immunoblots, and cleavage of the fluorogenic substrate N-benzyloxycarbonyl-Asp-Glu-Val-Asp-7-amino-4-trifluoromethyl coumarin (zDEVD-afc) in adult mouse brain after temporary (2 hr) middle cerebral artery occlusion produced by filament insertion into the carotid artery. Immunoreactive caspase-3p32 but not its cleavage product caspase-3p20 was constitutively expressed in neurons throughout brain and was most prominent in neuronal perikarya within piriform cortex. Caspase-like enzyme activity was elevated in brain homogenate 0-3 hr after reperfusion and reached a peak within 30 to 60 min. Caspase-3p20 immunoreactivity became prominent in neuronal perikarya within the middle cerebral artery territory at the time of reperfusion and on immunoblots 1-12 hr later. DNA laddering (agarose gels) and 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 cells, a finding also observed in apoptotic mouse cerebellar granule cells on postnatal day 5. Together, these observations suggest the existence of a time-dependent evolution of ischemic injury characterized by the close correspondence between caspase-like enzyme activation and an associated increase in immunoreactive product (caspase-3p20) beginning at or before reperfusion and followed several hours later by morphological and biochemical features of apoptosis.
Namura
S
S
Stroke and Neurovascular Regulation, Neurosurgical Service, Departments of Surgery and Neurology, Massachusetts General Hospital, Harvard Medical School, Charlestown, Massachusetts 02129, USA.
Zhu
J
J
Fink
K
K
Endres
M
M
Srinivasan
A
A
Tomaselli
K J
KJ
Yuan
J
J
Moskowitz
M A
MA
D000367:Age Factors; D000818:Animals; D000918:Antibody Specificity; D017209:Apoptosis / Q000502:physiology*; D001710:Biotin; D001921:Brain / Q000098:blood supply / Q000201:enzymology*; D053148:Caspase 3; D020169:Caspases; D003374:Coumarins / Q000494:pharmacology; D003546:Cysteine Endopeptidases / Q000032:analysis / Q000276:immunology / Q000378:metabolism*; D053938:DNA Fragmentation; D003856:Deoxyuracil Nucleotides; D004789:Enzyme Activation / Q000187:drug effects; D004792:Enzyme Precursors / Q000378:metabolism; D002546:Ischemic Attack, Transient / Q000378:metabolism*; D008297:Male; D051379:Mice; D008815:Mice, Inbred Strains; D009842:Oligopeptides / Q000494:pharmacology; D013194:Staining and Labeling
D003374:Coumarins; D003856:Deoxyuracil Nucleotides; D004792:Enzyme Precursors; D009842:Oligopeptides; C106566:benzyloxycarbonyl-aspartyl-glutamyl-valyl-aspartyl-7-amino-4-trifluoromethylcoumarin; D001710:Biotin; C505409:Casp3 protein, mouse; D053148:Caspase 3; D020169:Caspases; D003546:Cysteine Endopeptidases