Evidence for activation of caspase-3–like protease in excitotoxin- and hypoxia/hypoglycemia-injured neurons

Abstract: Caspase activation has been shown to be a critical step in several models of neuronal apoptosis such as staurosporine treatment of human neuroblastoma SH-SY5Y cells and potassium deprivation of rat cerebellar granule neurons. One common event is the appearance of caspase-mediated 1 20-kDa nonerythroid ea-spectrin breakdown product (SBDP1 20). Second, inhibitors of the caspase family are effective blockers of such neuronal death. In this study, we report the appearance of caspase-mediated SBDP1 20 in excitotoxin-challenged fetal rat cerebrocortical neurons [N-methyl-o-aspartate (NMDA), a-amino-3-hydroxy-5-methyl-4-isoxazolepro-pionic acid, and kainate] and rat cerebellar granule neu-rons (NMDA and kainate). A general caspase inhibitor, carbobenzoxy-Asp-CH2OC (0)-2,6-dichlorobenzene (Z-D-DCB), blocked the formation of SBDP1 20 under these conditions and attenuated the observed NMDA-induced lactate dehydrogenase (LDH) release in both cell types. Furthermore, hydrolytic activity toward a caspase-3-pre-ferred synthetic peptide substrate, acetyl-DEVD-7-amido-4-methylcoumarin, was significantly elevated in NM DA-treated granule neurons. Lastly, oxygen—glucose deprivation (OGD)-challenged cerebrocortical cultures also showed the appearance of SBDP12O. Again, Z-D-DCB blocked the SBDP12O formation as well as attenu-ated the LDH release from the OGD-challenged neurons. Taken together, the presence of caspase-specific SBDP1 20 and the neuroprotective effects of Z-D-DCB strongly suggest that caspase activation contributes at least in part to excitotoxin- and OGD-induced neurona