Journal of Neurochemistry, 2001, 78, 230±239 1,2-bis(2-Aminophenoxy)ethane-N , N ,N 0 , N 0 -tetraacetic acid induces caspase-mediated apoptosis and reactive oxygen species-mediated necrosis in cultured cortical neurons Kong Sook Han,* , ² Hyo Jung Kang,* , ² Eun Young Kim,² , ³ Won Joo Yoon,* , ² Seonghyang Sohn,§ Hyuk Jae Kwon§ and Byoung Joo Gwag* , ²³ *Department of Neuroscience, Ajou University School of Medicine, Suwon, Kyungkido, Korea ²Center for the Interventional Therapy of Stroke and Alzheimer's Disease, Ajou University School of Medicine, Suwon, Kyungkido, Korea ³Department of Pharmacology, Ajou University School of Medicine, Suwon, Kyungkido, Korea §Laboratory of Cell Biology, Institute for Medical Science, Ajou University School of Medicine, Suwon, Kyungkido, Korea Abstract Sustained alteration in [Ca 21 ] i triggers neuronal death. We examined morphological and signaling events of Ca 21 - de®ciency-induced neuronal death. Cortical cell cultures exposed to 20 mM 1,2-bis(2-aminophenoxy)ethane-N , N ,N 0 , N 0 -tetra- acetic acid (BAPTA-AM), an intracellular calcium chelator, underwent neuronal apoptosis within 12 h that was evident by shriveled cell bodies, aggregated and condensed nuclear chromatin, and disrupted nuclear membrane. Thereafter, surviving neurons revealed typical necrosis, accompanied by swelling of cell body and mitochondria, over 24 h. Both apoptosis and necrosis were prevented by inclusion of 1 mg/mL cycloheximide, a protein synthesis inhibitor. Treatment with BAPTA-AM induced translocation of Bax into mitochondria within 4 h and release of cytochrome c from mitochondria over 4±12 h. An active fragment of caspase-3, a downstream mediator of cytochrome c, was observed within 8h and cleaved PHF-1-positive tau. Administration of zVAD-fmk, a broad inhibitor of caspases, or DEVD-amc, a selective inhibitor of caspase-3, selectively prevented the apoptosis component of BAPTA-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 antioxidants blocked BAPTA-AM neurotoxicity. The present study suggests that neurons de®cient in [Ca 21 ] i undergo caspase-3-mediated apoptosis and reactive oxygen species (ROS)-mediated necrosis. Keywords: apoptosis, Ca 21 , caspase, cytochrome c, necro- sis, reactive oxygen species. J. Neurochem. (2001) 78, 230±239. The Ca 21 ion is an intracellular messenger essential for neuronal survival and function. The excitatory neurotrans- mitter, glutamate is released and accumulated in the synaptic cleft under pathological conditions such as hypoxic ischemia or trauma, which result in in¯ux and accumulation of Ca 21 through Ca 21 -permeable ionotropic glutamate receptors sensitive to NMDA and a-amino-3-hydroxy-5- methyl-4-isoxazolepropionic acid (AMPA)/kainate (Choi and Rothman 1990; McIntosh et al. 1996). Accumulated Ca 21 can trigger the production of various toxic substances such as NO through the activation of nitric oxide synthase, reactive oxygen species (ROS), and arachidonic acid (Lazarewicz et al. 1990; Dykens 1994; Dawson and Dawson 1996). Although internucleosomal DNA fragmentation and 230 q 2001 International Society for Neurochemistry, Journal of Neurochemistry, 78, 230±239 Received December 4, 2000; revised manuscript received March 20, 2001; accepted March 23, 2001. Address correspondence and reprint requests to Byoung Joo Gwag, Department of Pharmacology, Ajou University School of Medicine, San 5, Wonchon-dong, Paldal-gu, Suwon, Kyungki-do 442±749, Korea. E-mail: bjgwag@madang.ajou.ac.kr 1 K. S. Han and H. J. Kang equally contributed to this paper. Abbreviations used: AMPA, a-amino-3-hydroxy-5-methyl-4-isoxa- zolepropionic acid; Apaf-1, apoptotic protease-activating factor-1; Ara C, cytosine arabinofuranoside; BAPTA-AM, 1,2-bis(2-aminophe- noxy)ethane-N ,N ,N 0 , N 0 -tetraacetic acid, tetrakis(acetoxymethyl)ester; CDIA, Ca 21 de®ciency-induced apoptosis; CDIN, Ca 21 de®ciency- induced necrosis; DCDHF, 6-carboxy-2,7-dichlorodihydro¯uorescein diacetate; DTT, dithiothreitol; HCSS, HEPES-buffered control salt solution; LDH, lactate dehydrogenase; MEM, minimal essential media; PMSF, phenylmethylsulfonyl ¯uoride; ROS, reactive oxygen species; SDS, sodium dodecyl sulfate; TPEN, butoxycarbonylaspartate-¯uoro- methylketone-N ,N ,N 0 , N 0 -tetrakis-(2-pyridylmethyl)ethylenediamine.