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 b een a matter of much debate. Caspase-3 has been identified as a key proteas e in the execution of apoptosis, whereas calpains have mainly been implicat ed in excitotoxic neuronal injury. In a model of unilateral hypoxia-ischemi a in 7-day-old rats, caspase-3-like activity increased 16-fold 24 h postins ult, coinciding with cleavage of the caspase-3 proenzyme and endogenous cas pase-3 substrates, This activation was significantly decreased by pharmacol ogical calpain inhibition, using CX295, a calpain inhibitor that did not in hibit purified caspase-3 in vitro. Activation of caspase-3 by m-calpain, bu t not mu -calpain, was facilitated in a dose-dependent manner in vitro by i ncubating cytosolic fractions, containing caspase-3 preform, 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 i s the first report to our knowledge suggesting a direct link between the ea rly, excitotoxic, calcium-mediated activation of calpain after cerebral hyp oxia-ischemia and the subsequent activation of caspase-3, thus representing a tentative pathway of "pathological apoptosis.".