Temporal relationships between de novo protein synthesis, calpain and caspase 3‐like protease activation, and DNA fragmentation during apoptosis in septo‐hippocampal cultures

Abstract

Caspase 3-like proteases are key executioners in mammalian apoptosis, and the calpain family of cysteine proteases has also been implicated as an effector of the apoptotic cascade. However, the influence of upstream events on calpain/caspase activation and the role of calpain/caspase activation on subsequent downstream events are poorly understood. This investigation examined the temporal profile of apoptosis-related events after staurosporine-induced apoptosis in mixed glial-neuronal septo-hippocampal cell cultures. Following 3 hr exposure to staurosporine (0.5 μM), calpain and caspase 3-like proteases processed α-spectrin to their signature proteolytic fragments prior to endonuclease-mediated DNA fragmentation (not evident until 6 hr), indicating that endonuclease activation is downstream from calpain/caspase activation. Cycloheximide, a general protein synthesis inhibitor, completely prevented processing of α-spectrin by calpains and caspase 3-like proteases, DNA fragmentation and cell death, indicating that de novo protein synthesis is an upstream event necessary for activation of calpains and caspase 3-like proteases. Calpain inhibitor II and the pan-caspase inhibitor Z-D-DCB each inhibited their respective protease-specific processing of α-spectrin and attenuated endonuclease DNA fragmentation and cell death. Thus, activation of calpains and caspase 3-like proteases is an early event in staurosporine-induced apoptosis, and synthesis of, as yet, unknown protein(s) is necessary for their activation. J. Neurosci. Res. 52:505–520, 1998. © 1998 Wiley-Liss, Inc.