Activation of the caspase 8 pathway mediates seizure-induced cell death in cultured hippocampal neurons.
In response to harmful stresses, cells induce programmed cell death (PCD) or apoptosis. Seizures can induce neural damage and activate biochemical pathways associated with PCD. Since seizures trigger intracellular calcium overload, it has been presumed that the intrinsic cell death pathway mediated by mitochondrial dysfunction would modulate cell death following seizures. However, previous work suggests that the extrinsic cell death pathway may initiate the damage program. Here we investigate intrinsic versus extrinsic cell death pathway activation using caspase cleavage as a marker for activation of these pathways in a rat in vitro model of seizures. Hippocampal cells, chronically treated with kynurenic acid, had kynurenic acid withdrawn to induce seizure-like activity for 40 min. Subjecting rat hippocampal cultures to seizures increased cell death and apoptosis-like DNA fragmentation using TUNEL staining. Seizure-induced cell death was blocked by both MK801 (10 microM) and CNQX (40 microM), which suggests multiple glutamate receptors regulate seizure-induced cell death. Cleavage of the initiator caspases, caspase 8 and 12 were increased 4h following seizure, and cleavage of the quintessential executioner caspase, caspase 3 was increased 4h following seizure. In contrast, caspase 9 cleavage only increased 24h following seizure. Using an affinity labeling approach to trap activated caspases in situ, we show that caspase 8 is the apical caspase activated following seizures. Finally, we show that the caspase 8 inhibitor Ac-IETD-CHO was more effective at blocking seizure-induced cell death than the caspase 9 inhibitor Ac-LEHD-CHO. Taken together, our data suggests the extrinsic cell death pathway-associated caspase 8 is activated following seizures in vitro.
Meller
R
R
Robert S. Dow Neurobiology Laboratories, Legacy Research, Portland, OR 97232, USA. rmeller@downeurobiology.org
Clayton
C
C
Torrey
D J
DJ
Schindler
C K
CK
Lan
J Q
JQ
Cameron
J A
JA
Chu
X P
XP
Xiong
Z G
ZG
Simon
R P
RP
Henshall
D C
DC
D018750:6-Cyano-7-nitroquinoxaline-2,3-dione / Q000494:pharmacology; D000818:Animals; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases / Q000378:metabolism*; D016923:Cell Death / Q000187:drug effects / Q000502:physiology*; D002478:Cells, Cultured; D004249:DNA Damage / Q000502:physiology*; D016291:Dizocilpine Maleate / Q000494:pharmacology; D018691:Excitatory Amino Acid Antagonists; D006624:Hippocampus / Q000473:pathology*; D007736:Kynurenic Acid; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D009474:Neurons / Q000473:pathology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D012640:Seizures / Q000139:chemically induced / Q000473:pathology*
D018691:Excitatory Amino Acid Antagonists; D016291:Dizocilpine Maleate; D018750:6-Cyano-7-nitroquinoxaline-2,3-dione; D007770:L-Lactate Dehydrogenase; C505528:Casp8 protein, rat; C506435:Casp9 protein, rat; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases; D007736:Kynurenic Acid