Inhibition of caspase-mediated apoptosis by peroxynitrite in traumatic brain injury.
In traumatic brain injury (TBI), neurons surviving the primary insult may succumb through poorly understood secondary mechanisms. In vitro, cortical neurons exposed to stretch injury exhibited enhanced vulnerability to NMDA, apoptotic-like DNA fragmentation, peroxynitrite (PN) formation, and cytoplasmic cytochrome c accumulation. Surprisingly, caspase-3 activity was undetectable by both immunoblotting and fluorogenic activity assays. Therefore, we hypothesized that PN directly inhibits caspases in these neurons. Consistent with this, stretch injury in cultured neurons elicited tyrosine nitration of procaspase-3, but not caspase-9 or Apaf-1, suggesting a direct interaction of PN with caspase-3. In an ex vivo system, PN inhibited the activity of caspase-3, and this inhibition was reversible with the addition of the sulfhydryl reducing agent dithiothreitol, indicating that PN inhibits caspases by cysteinyl oxidation. Moreover, in cultures, the PN donor 3-morpholinosydnonimine (SIN-1) blocked staurosporine-induced caspase-3 activation and its downstream effects including PARP-1 [poly-(ADP-ribose) polymerase-1] cleavage and phosphotidylserine inversion, suggesting that peroxynitrite can inhibit caspase-3-mediated apoptosis. To examine these mechanisms in vivo, rats were exposed to a lateral fluid percussion injury (FPI). FPI caused increased neuronal protein nitration that colocalized with TUNEL staining, indicating that PN was associated with neurodegeneration. Caspase-3 activity was inhibited in brain lysates harvested after FPI and was restored by adding dithiothreitol. Our data show that caspase-mediated apoptosis is inhibited in neurons subjected to stretch in vitro and to TBI in vivo, mostly because of cysteinyl oxidation of caspase-3 by PN. However, this is insufficient to prevent cell death, indicating that the TBI therapy may, at a minimum, require a combination of both anti-apoptotic and anti-oxidant strategies.
Lau
Anthony
A
Division of Applied and Interventional Research, Toronto Western Research Institute, Toronto, Ontario, Canada M5T 2S8.
Arundine
Mark
M
Sun
Hong-Shuo
HS
Jones
Michael
M
Tymianski
Michael
M
D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D015153:Blotting, Western / Q000379:methods; D001930:Brain Injuries / Q000139:chemically induced / Q000473:pathology*; D020169:Caspases / Q000502:physiology*; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000166:cytology; D045304:Cytochromes c / Q000378:metabolism; D004195:Disease Models, Animal; D004622:Embryo, Mammalian; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007150:Immunohistochemistry / Q000379:methods; D020287:In Situ Nick-End Labeling / Q000379:methods; D008297:Male; D051379:Mice; D008981:Molsidomine / Q000031:analogs & derivatives / Q000494:pharmacology; D016202:N-Methylaspartate / Q000633:toxicity; D009433:Neural Inhibition / Q000187:drug effects*; D009474:Neurons / Q000187:drug effects / Q000473:pathology*; D020030:Nitric Oxide Donors / Q000494:pharmacology; D030421:Peroxynitrous Acid; D010812:Physical Stimulation / Q000009:adverse effects; D051381:Rats; D017207:Rats, Sprague-Dawley; D019311:Staurosporine / Q000494:pharmacology
D004791:Enzyme Inhibitors; D020030:Nitric Oxide Donors; D030421:Peroxynitrous Acid; C002385:linsidomine; D016202:N-Methylaspartate; D045304:Cytochromes c; D008981:Molsidomine; D020169:Caspases; D019311:Staurosporine