Opposing roles for caspase and calpain death proteases in L-glutamate-induced oxidative neurotoxicity.
Oxidative glutamate toxicity in HT22 murine hippocampal cells is a model for neuronal death by oxidative stress. We have investigated the role of proteases in HT22 cell oxidative glutamate toxicity. L-glutamate-induced toxicity was characterized by cell and nuclear shrinkage and chromatin condensation, yet occurred in the absence of either DNA fragmentation or mitochondrial cytochrome c release. Pretreatment with the selective caspase inhibitors either benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (pan-caspase), N-acetyl-Leu-Glu-His-Asp-aldehyde (caspase 9) or N-acetyl-Ile-Glu-Thr-Asp-aldehyde (caspase 8), significantly increased L-glutamate-induced cell death with a corresponding increase in observed nuclear shrinkage and chromatin condensation. This enhancement of glutamate toxicity correlated with an increase in L-glutamate-dependent production of reactive oxygen species (ROS) as a result of caspase inhibition. Pretreating the cells with N-acetyl-L-cysteine prevented ROS production, cell shrinkage and cell death from L-glutamate as well as that associated with the presence of the pan-caspase inhibitor. In contrast, the caspase-3/-7 inhibitor N-acetyl-Asp-Glu-Val-Asp aldehyde was without significant effect. However, pretreating the cells with the calpain inhibitor N-acetyl-Leu-Leu-Nle-CHO, but not the cathepsin B inhibitor CA-074, prevented cell death. The cytotoxic role of calpains was confirmed further by: 1) cytotoxic dependency on intracellular Ca(2+) increase, 2) increased cleavage of the calpain substrate Suc-Leu-Leu-Val-Tyr-AMC and 3) immunoblot detection of the calpain-selective 145 kDa alpha-fodrin cleavage fragment. We conclude that oxidative L-glutamate toxicity in HT22 cells is mediated via calpain activation, whereas inhibition of caspases-8 and -9 may exacerbate L-glutamate-induced oxidative neuronal damage through increased oxidative stress.
Elphick
Lucy M
LM
Division of Biochemical Sciences, Faculty of Health and Medical Sciences, University of Surrey, Guildford, GU2 7XH, UK.
Hawat
Mohammad
M
Toms
Nick J
NJ
Meinander
Annika
A
Mikhailov
Andrey
A
Eriksson
John E
JE
Kass
George E N
GE
D000818:Animals; D002154:Calpain / Q000037:antagonists & inhibitors / Q000502:physiology*; D061945:Caspase Inhibitors; D020169:Caspases / Q000502:physiology*; D016923:Cell Death / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D018698:Glutamic Acid / Q000633:toxicity*; D051379:Mice; D009474:Neurons / Q000187:drug effects / Q000201:enzymology* / Q000473:pathology; D018384:Oxidative Stress / Q000187:drug effects / Q000502:physiology*; D011480:Protease Inhibitors / Q000494:pharmacology
D061945:Caspase Inhibitors; D011480:Protease Inhibitors; D018698:Glutamic Acid; D002154:Calpain; D020169:Caspases