Synergistic activation of caspase-3 by m-calpain after neonatal hypoxia-ischemia: a mechanism of "pathological apoptosis"?
The relative contributions of apoptosis and necrosis in brain injury have been a matter of much debate. Caspase-3 has been identified as a key protease in the execution of apoptosis, whereas calpains have mainly been implicated in excitotoxic neuronal injury. In a model of unilateral hypoxia-ischemia in 7-day-old rats, caspase-3-like activity increased 16-fold 24 h postinsult, coinciding with cleavage of the caspase-3 proenzyme and endogenous caspase-3 substrates. This activation was significantly decreased by pharmacological calpain inhibition, using CX295, a calpain inhibitor that did not inhibit purified caspase-3 in vitro. Activation of caspase-3 by m-calpain, but not mu-calpain, was facilitated in a dose-dependent manner in vitro by incubating cytosolic fractions, containing caspase-3 proform, with calpains. This facilitation required the presence of some active caspase-3 and could be abolished by including the specific calpain inhibitor calpastatin. This indicates that initial cleavage of caspase-3 by m-calpain, producing a 29-kDa fragment, facilitates the subsequent cleavage into active forms. This is the first report to our knowledge suggesting a direct link between the early, excitotoxic, calcium-mediated activation of calpain after cerebral hypoxia-ischemia and the subsequent activation of caspase-3, thus representing a tentative pathway of "pathological apoptosis."
Blomgren
K
K
Perinatal Center, Institute of Physiology and Pharmacology, Göteborg University, Sweden. klas.blomgren@fysiologi.gu.se
Zhu
C
C
Wang
X
X
Karlsson
J O
JO
Leverin
A L
AL
Bahr
B A
BA
Mallard
C
C
Hagberg
H
H
D000818:Animals; D000831:Animals, Newborn; D017209:Apoptosis; D001921:Brain / Q000378:metabolism / Q000473:pathology; D002154:Calpain / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004151:Dipeptides / Q000494:pharmacology; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000378:metabolism; D005260:Female; D006801:Humans; D000860:Hypoxia; D015151:Immunoblotting; D007150:Immunohistochemistry; D020128:Inhibitory Concentration 50; D007511:Ischemia; D008297:Male; D008840:Microfilament Proteins / Q000378:metabolism; D011480:Protease Inhibitors / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D011994:Recombinant Proteins / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013379:Substrate Specificity; D013997:Time Factors; D013049:Spectrin
D002154:Calpain; D002352:Carrier Proteins; D053148:Caspase 3; D020169:Caspases; D015853:Cysteine Proteinase Inhibitors; D004151:Dipeptides; D004792:Enzyme Precursors; D008840:Microfilament Proteins; D011480:Protease Inhibitors; D012333:RNA, Messenger; D011994:Recombinant Proteins; D013049:Spectrin; C090138:AK 295; C505407:CASP3 protein, human; C505410:Casp3 protein, rat; C098484:mu-calpain proenzyme