Inhibition of caspase-3-mediated cell death via peroxynitrite formation following traumatic brain injury

Psychological and neurological deficits experienced by TBI victims are caused by neurodegeneration which can be classified under two headings: primary or secondary. Although primary injury is considered unsalvageable, secondary injury includes neurons slowly degenerating from a complex combination of pathological events following the initial impact and is potentially treatable by pharmacology. Currently, however, no pharmacological interventions for attenuating secondary injury are available. Studies have demonstrated many cell death pathways activated following TBI, including oxidative and proteolytic mechanisms. Oxidants can decrease proteolysis in vitro, thus it was postulated that oxidants might inhibit proteolytic death following TBI.;We examined a cortical culture model of secondary injury for possible interactions between death pathways mediated by caspase-3 and peroxynitrite representing archetypical proteolytic and oxidative mechanisms respectively. Peroxynitrite formation was readily evident after injury but, while cytoplasmic cytochrome c release indicated initiation of Type II apoptosis, no caspase-3 cleavage or activity was observed. We further demonstrated that peroxynitrite interacted directly with caspase-3.;To isolate the effect of peroxynitrite on caspases, we next developed recombinant cell-free caspase systems. Treatment with pure peroxynitrite or peroxynitrite donors decreased caspase-mediated proteolysis. Furthermore, this inhibition could be alleviated using a sulphydryl reducing agent, suggesting cysteinyl oxidation as a mechanism of inhibition.;To study the effects of the simultaneous activation of cell death pathways, we induced artificial caspase-3 activation and peroxynitrite formation in cortical cultures. Application of staurosporine alone induced caspase-3 activation and cell death, but subsequent application of the peroxynitrite donor SIN-1 prevented caspase-3-mediated effects. Ultimately, no neuroprotection was observed.;To test relevance in vivo, we assayed a whole animal model of TBI for similar hallmarks. We determined that peroxynitrite was associated with neuronal death and an oxidative inhibition of caspase-3 activity, but not caspase-9 activity. These findings support the notion that oxidative inhibition of caspase-3 occurs following TBI.;Overall, we observed an inhibition of caspase-3 by peroxynitrite in multiple models of TBI of increasing complexity. This interaction may not only explain the failure of caspase inhibitors and anti-oxidant treatments in the treatment of TBI, but might also suggest novel neuroprotective pharmacological approaches.