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

Abstract

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 -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."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 (1). Recent studies suggest that cell death after HI is different from developmentally regulated cell death in most cases and cannot appropriately be described as apoptotic (2-5). Nevertheless, HI cell death shares important morphological and biochemical features with apoptotic cell death, such as activation of caspases and nucleosomal DNA fragmentation (6 -17). Caspases, a family of cysteine proteases with an unusual substrate specificity, requiring an aspartate residue in the P1 position, have been identified as key executors of apoptosis (18). Calpains, another family of cysteine proteases, are calcium-activated and are proposed to participate in the turnover of cytoskeletal proteins and regulation of kinases, transcription factors, and receptors (19,20). Calpains have mainly been implicated in excitotoxic neuronal injury and necrosis (21-23). Pharmacological inhibitors of calpains and caspases exert cerebroprotective effects (9, 14 -16, 24 -26). A growing body of literature has emerged, demonstrating functional connections between calpains and caspases (27). Common substrate proteins have been identified, such as fodrin (28 -31), calpastatin (32, 33), actin (34), PARP (35), and tau (36). There are reports demonstrating calpain-mediated cleavage of caspase-3 (35, 37) and caspase-7 (38, 39) as well as caspase-8 and -9 (39). Furthermore, the proapoptotic protein Bax was cleaved by calpain during drug-induced apoptosis of HL-60 cells (40), and calpain may be responsible for cleaving the loop r...

Multiple death pathways in retina-derived 661W cells following growth factor deprivation: crosstalk between caspases and calpains

“…Accordingly, caspase-3 underwent cleavage into a 17-kDa active form that could be identified by immunoblot from 24 h of starving. An additional 29-kDa band, previously described as a calpainmediated breakdown product, 21,34 was detectable at 96 h, suggesting crosstalk between these two families of proteases ( Figure 2b). We also determined the activation status of caspase-12, an endoplasmic reticulum-associated caspase, previously related to serum deprivation-induced apoptosis in other systems.…”

“…For instance, caspases facilitate calpain activity through the cleavage of its endogenous inhibitor calpastatin. 18,19 At the same time, calpains directly proteolyse executioner caspases, mediating their activation, 20,21 as well as proapoptotic Bcl-2 family members that eventually cause the release of apoptogenic factors from the mitochondria involved in the triggering of the caspase cascade. [22][23][24] While the time course of developmental apoptosis in the mouse retina has been extensively described in several studies, 10,[25][26][27] the biochemical death pathways involved are not fully understood.…”

Irradiation-induced progenitor cell death in the developing brain is resistant to erythropoietin treatment and caspase inhibition

“…22 Downstream of these events, activation of both Epo has been demonstrated to be antiapoptotic in other models of neuronal injury both in vitro 38 and in vivo. 39,40 Using a well-characterized model of neonatal hypoxia-ischemia (HI), 41,42 we observed that Epo reduced the infarct volumes by approximately 50% (unpublished observations; manuscript under revision). In this study, we were unable to detect any reduction in the loss of SVZ, GCL or myelination after IR.…”

NMNAT3 is protective against the effects of neonatal cerebral hypoxia‐ischemia

“…Furthermore, we also observed that NMNAT3 upregulation was associated with a significant decrease in the degradation of calpastatin (Fig. 5), a calcium‐sensitive protease known to inhibit the activity of both calpains and caspase‐3 23, 24, 25, 26, 27. It is thus tempting to suggest that the biological mechanism by which NMNAT3 protects neurons against the effects of neonatal H‐I involves an increase in the capacity of mitochondria to buffer the rapid increases in intracellular calcium as a result of ischemia‐induced glutamate‐dependent excitotoxicity, thereby preventing calpastatin degradation and the subsequent activation of calpain and caspase‐3.…”

“…Calpastatin (CASTN) is a calcium‐activated cysteine protease responsible for the proteolytic inhibition of calpains 23, 24. Increases in intracellular calcium after neonatal cerebral ischemia lead to the activation of calpains and subsequent degradation of CASTN resulting in unopposed activation of calpain‐mediated neuronal cell death 25, 26, 27. Caspase‐3 also promotes the degradation of CASTN in early apoptotic neurodegeneration.…”