Inhibition of Caspase-Mediated Apoptosis by Peroxynitrite in Traumatic Brain Injury

Abstract

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.

PICK1-mediated GluR2 endocytosis contributes to cellular injury after neuronal trauma

“…Â area (1.49 in 2 ), we calculated that at 2.9 p.s.i., J (on cells) E9.6 N s. We have shown earlier that this sublethal trauma does not confer delayed cell death on its own -as assessed by propidium iodide uptake 20 h after injury -or alter membrane permeability, as assessed by carboxyfluorescein uptake and retention of fluorescein di-acetate. 9 This model is highly consistent with other paradigms of in vitro TBI in the literature, 30,32 which have frequently been used to delineate the intracellular mechanisms that contribute to progressive neuronal injury after mechanical trauma.…”

“…We used the cells for experiments 11-14 days after isolation consistent with earlier in vitro stretch assays. 24,29,30 In vitro model of mild TBI. Before stretch, the culture medium was replaced with 2 ml HEPES buffered saline (concentrations in mM: 121 NaCl, 5 KCl, 20 glucose, 10 HEPES acid, 7 HEPES-Na salt, 3 NaHCO 3 , 1 Na-pyruvate, 1.8 CaCl 2 and 0.01 glycine, adjusted to pH 7.4 with NaOH).…”

Temporal relationship of peroxynitrite-induced oxidative damage, calpain-mediated cytoskeletal degradation and neurodegeneration after traumatic brain injury

“…Consistent with this hypothesis, it has been shown that ROS can inhibit calpain activity via oxidation of the sulfhydryl groups of cysteine residues at the active site of the enzyme (Benuck et al, 1992) (Guttmann et al, 1997) (Guttmann and Johnson, 1998). Recent work by another group has shown that another cysteine protease caspase-3 in the traumatized brain is similarly inhibited by PN specifically, and that this is prevented by application of the sulfhydryl reducing agent dithiothreitol (Lau et al, 2006). The latter is also relevant since the SBDP150 fragment which also showed a biphasic time course is partially generated by caspase 3 as well as calpain (Wang, 2000b).…”

Characterization of hereditary inclusion body myopathy myoblasts: possible primary impairment of apoptotic events

“…Alternatively, it might be that upregulation of caspases activation in HIBM cells does not result in a concordant upregulation of PS externalization, although this apoptotic phenomenon is known to be caspase dependent. 26 Interestingly, there is accumulating data pointing to caspaseindependent PS externalization occurring during apoptosis, for example in STS-treated primary T lymphocytes, 27 possibly indicating that in some conditions those two processes may be independently activated. Therefore, it may well be that caspases activation, but not PS externalization, is involved in HIBM pathology.…”