1,2-bis(2-aminophenoxy)ethane-N,N,N ',N '-tetraacetic acid induces caspase-mediated apoptosis and reactive oxygen species-mediated necrosis in cultured cortical neurons

Sustained alteration in [Ca2+](i) triggers neuronal death. We examined morp hological and signaling events of Ca2+-deficiency-induced neuronal death. C ortical cell cultures exposed to 201 muM 1,2-bis(2-aminophenoxy)ethane-N,N, N ' ,N ' -tetraacetic acid (BAPTA-AM), an intracellular calcium chelator, u nderwent neuronal apoptosis within 12 h that was evident by shriveled cell bodies, aggregated and condensed nuclear chromatin, and disrupted nuclear m embrane. Thereafter, surviving neurons revealed typical necrosis, accompani ed by swelling of cell body and mitochondria, over 24 h. Both apoptosis and necrosis were prevented by inclusion of 1 mug/mL cycloheximide, a protein synthesis inhibitor. Treatment with BAPTA-AM induced translocation of Bax i nto mitochondria within 4 h and release of cytochrome c from mitochondria o ver 4-12 h. An active fragment of caspase-3, a downstream mediator of cytoc hrome c, was observed within 8 h and cleaved PHF-I-positive tau. Administra tion of zVAD-fmk, a broad inhibitor of caspases, or DEVD-amc, a selective i nhibitor of caspase-3, selectively prevented the apoptosis component of BAP TA-AM neurotoxicity. In contrast, BAPTA-AM-induced necrosis was propagated through sequential production of superoxide, mitochondrial and cytoplasmic reactive oxygen species, Combined treatment with caspase inhibitors and ant ioxidants blocked BAPTA-AM neurotoxicity. The present study suggests that n eurons deficient in [Ca2+](i) undergo caspase-3-mediated apoptosis and reac tive oxygen species (ROS)-mediated necrosis.