Synergistic Activation of Caspase-3 by m-Calpain after Neonatal Hypoxia-Ischemia

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 μ-calpain, was facilitated in a dose-dependent manner in vitroby 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.” 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 μ-calpain, was facilitated in a dose-dependent manner in vitroby 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.” hypoxia-ischemia aminomethylcoumarin DNA fragmentation factor 45 α-fodrin breakdown product inhibitor of caspase-activated DNase poly(ADP-ribose) polymerase dithiothreitol 3-(3-cholamidopropyl)dimethylammonio-1-propanesulfonic acid benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone t-butoxycarbonyl-Asp-(OMe)-fluoromethyl ketone phosphate-buffered saline polymerase chain reaction glyceraldehyde-3-phosphate dehydrogenase Leu-Tyr-AMC The relative contributions of necrosis and apoptosis to the injury that develops after cerebral hypoxia-ischemia (HI)1 has been a matter of much debate (1Lee J.M. Zipfel G.J. Choi D.W. Nature. 1999; 399: 7-14Crossref PubMed Scopus (1004) Google Scholar). 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The of by the increased and was 24 h h A and The in of but a of after HI A and of caspase-3 and of and active is of using the active caspase-3 and and 24 h of demonstrating the of caspase-3 and calpain activity 24 h of the calpain activity has but the of active caspase-3 has of did not the cleavage the caspase-3 A and was a 29-kDa to the after HI in This did not an of was was with calpastatin purified CX295, HI in was the the of the that was and This was not by calpain purified by but could be by and calpain The caspase-3-like activity increased after of from different with and could be abolished calpastatin was in the was in the the activity was not significantly increased of active caspase-3 to cytosolic fractions, the active caspase-3 could be as a as an of active caspase-3 the of the 29-kDa from the The of the 29-kDa was by including of active caspase-3 to the the activity increased caspase-3 was in the of This could be abolished by including in the Caspase-3 could not m-calpain, incubating with of caspase-3 did not the calpain activity between and A cytosolic containing caspase-3 proform, was with and different of caspase-3 and as active caspase-3 was the of a in the demonstrating the the relative in caspase-3-like activity in the is in the a caspase-3 that the active caspase-3 be as a as the of active caspase-3 an increased of the 29-kDa but was active as that the some endogenous m-calpain, in to caspase-3 a demonstrating that the of caspase-3 cleavage in the was not to is that in a producing an the The cleavage of the from to was could be by and calpastatin not inhibit m-calpain, but not μ-calpain, could cleavage of the of producing a 29-kDa could be This is the report to our knowledge demonstrating a functional in substrate between the calpains. Recent studies have cleavage of caspase-3 by producing an cleavage product (35McGinnis N. Wang K.K. Biochem. Biophys. Res. 1999; PubMed Scopus Google Scholar, H.R. H. Salvesen G.S. 1999; 94: PubMed Google Scholar), but another report to such cleavage A. G. J. 1999; 18: PubMed Scopus Google Scholar). H.R. H. Salvesen G.S. 1999; 94: PubMed Google identified the cleavage in the of In was not m-calpain, of could functional that calpains are to and that calpains as of B.T. K. Li P. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). of studies calpastatin to the the first to that calpains H.R. H. 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Res. 1989; PubMed Scopus Google and caspase-3 is in the that proteases be important in the In is the first report to our knowledge demonstrating a functional in the substrate of the calpains and as as facilitated activation of caspase-3 by Caspase-3 has been identified as a key protease in the execution of apoptosis, whereas calpains have mainly been implicated in excitotoxic neuronal injury. suggest a direct link between the early, excitotoxic, calcium-mediated activation of calpains after cerebral HI and the subsequent activation of caspase-3, thus representing a tentative pathway of “pathological apoptosis.” This be important in the brain of the of calpains and caspase-3 are to the active caspase-3 and to Wang of the