During programmed cell death, activation of caspase-3 leads to proteolysis of DNA repair proteins, cytoskeletal proteins, and the inhibitor of caspase-activated deoxyribonuclease, culminating in morphologic changes and DNA damage defining apoptosis. The participation of caspase-3 activation in the evolution of neuronal death after traumatic brain injury in rats was examined. Cleavage of pro-caspase-3 in cytosolic cellular fractions and an increase in caspase-3-like enzyme activity were seen in injured brain versus control. Cleavage of the caspase-3 substrates DNA-dependent protein kinase and inhibitor of caspase-activated deoxyribonuclease and co-localization of cytosolic caspase-3 in neurons with evidence of DNA fragmentation were also identified.

Programmed cell death occurs after ischemic, excitotoxic, and traumatic brain injury (TBI). Recently, a caspase-independent pathway involving intranuclear translocation of mitochondrial apoptosis-inducing factor (AIF) has been reported in vitro; but whether this occurs after acute brain injury was unknown. To address this question adult rats were sacrificed at various times after TBI. Western blot analysis on subcellular protein fractions demonstrated intranuclear localization of AIF in ipsilateral cortex and hippocampus at 2-72 h. Immunocytochemical analysis showed AIF labeling in neuronal nuclei with DNA fragmentation in the ipsilateral cortex and hippocampus. Immunoelectronmicroscopy verified intranuclear localization of AIF in hippocampal neurons after TBI, primarily in regions of euchromatin. Large-scale DNA fragmentation ( 50 kbp), a signature event in AIF-mediated cell death, was detected in ipsilateral cortex and hippocampi by 6 h. Neuron-enriched cultures exposed to peroxynitrite also demonstrated intranuclear AIF and large-scale DNA fragmentation concurrent with impaired mitochondrial respiration and cell death, events that are inhibited by treatment with a peroxynitrite decomposition catalyst. Intranuclear localization of AIF and large-scale DNA fragmentation occurs after TBI and in neurons under conditions of oxidative/nitrosative stress, providing the first evidence of this alternative mechanism by which programmed cell death may proceed in neurons after brain injury. Keywords: apoptosis, brain injury, controlled cortical impact, DNA damage, programmed cell death. Programmed cell death is an essential mechanism for the selective elimination of cells during development, homeostasis of tissues with cell turnover and removal of aging and abnormal cells (Steller 1995). In general, the process of programmed cell death is strictly regulated, with dysregulation occurring in, and contributing to, many acute and chronic pathological conditions (Thompson 1995). In the CNS, programmed cell death contributes to neuronal death after ischemia (Graham and Chen Abbreviations used: AIF, apoptosis inducing factor; CAD, of caspaseactivated deoxyribonuclease; kbp, kilo-basepair; PFGE, pulsed field gel electrophoresis; TBI, traumatic brain injury.

Poly-ADP-ribosylation is a post-translational modification performed by poly(ADP-ribose) polymerases (PARP), involved in many diverse cellular functions including DNA repair, transcription, and long-term potentiation. Paradoxically, PARP over-activation under pathologic conditions including traumatic brain injury (TBI) results in cell death. We previously demonstrated that intra-mitochondrial poly-ADP-ribosylation occurs following excitotoxic and oxidative injury in vitro. Here we sought to identify mitochondrial proteins modified by poly-ADP-ribosylation after TBI in vivo. Poly-ADP-ribosylation within mitochondria from injured brain after experimental TBI in rats was first verified using western blot and immunoelectron microscopy. Poly-ADP-ribosylated mitochondrial proteins identified using a targeted proteomic approach included voltage-dependent anion channel-1, mitofilin, mitochondrial stress proteins, and the electron transport chain components F 1 F 0 ATPase, cytochrome c oxidase, and cytochrome c reductase. To examine the functional consequences of mitochondrial poly-ADP-ribosylation, isolated rat brain mitochondria were exposed to conditions of nitrosative stress known to activate PARP. PARP activation-induced reductions in State 3 respiration were prevented by the PARP-1 inhibitor 5-iodo-6-amino-1,2-benzopyrone or exogenous poly(ADPribose) glycohydrolase. As the effects of PARP activation on mitochondrial respiration appear regulated by poly(ADPribose) glycohydrolase, a direct effect of poly-ADP-ribosylation on electron transport chain function is suggested. These findings may be of relevance to TBI and other diseases where mitochondrial dysfunction occurs.

Programmed cell death involves a complex and interrelated cascade of cysteine proteases termed caspases that are synthesized as inactive zymogens, which are proteolytically processed to active enzymes. Caspase-8 is an initiator caspase that becomes activated when Fas death receptor-Fas ligand (FasL) coupling on the cell surface leads to coalescence of a "death complex" perpetuating the programmed cell death cascade. In this study, brain tissue samples removed from adult patients during the surgical management of severe intracranial hypertension after traumatic brain injury (TBI; n=17) were compared with postmortem control brain tissue samples (n=6). Caspase-8 mRNA was measured by semiquantitative reverse transcription and polymerase chain reaction, and caspase-8 protein was examined by Western blot and immunocytochemistry. Fas and FasL were also examined using Western blot. Caspase-8 mRNA and protein were increased in TBI patients vs. controls, and caspase-8 protein was predominately expressed in neurons. Proteolysis of caspase-8 to 20-kDa fragments was seen only in TBI patients. Fas was also increased after TBI vs. control and was associated with relative levels of caspase-8, supporting formation of a death complex. These data identify additional steps in the programmed cell death cascade involving Fas death receptors and caspase-8 after TBI in humans.

DNA damage is a common sequela of traumatic brain injury (TBI). Available techniques for the in situ identification of DNA damage include DNA polymerase I-mediated biotin-dATP nick-translation (PANT), the Klenow fragment of DNA polymerase I-mediated biotin-dATP nick-end labeling (Klenow), and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL). While TUNEL has been widely utilized to detect primarily double-strand DNA breaks, the use of PANT to detect primarily single-strand DNA breaks and Klenow to detect both single- and double-strand DNA breaks has not been reported after TBI. Accordingly, coronal brain sections from naive rats and rats at 0, 0.5, 1, 2, 6, 24, and 72 h (n = 3-5/group) after controlled cortical impact with imposed secondary insult were processed using the PANT, Klenow, and TUNEL methods. Cells with DNA breaks were detected by PANT in the ipsilateral hemisphere as early as 0.5 h after injury and were maximal at 6 h (cortex = 66.3+/-15.8, dentate gyrus 58.6+/-12.8, CA1 = 15.8+/-5.9, CA3 = 12.8+/-4.2 cells/x 400 field, mean +/- SEM, all p < 0.05 versus naive). Cells with DNA breaks were detected by Klenow as early as 30 min and were maximal at 24 h (cortex = 56.3+/-14.3, dentate gyrus 78.0+/-16.7, CA1 = 25.8+/-4.7, CA3 = 29.3+/-15.1 cells/x 400 field, all p < 0.05 versus naive). Cells with DNA breaks were not detected by TUNEL until 2 h and were maximal at 24 h (cortex = 47.7+/-21.4, dentate gyrus 63.0+/-11.9, CA1 = 5.6+/-5.4, CA3 = 6.9+/-3.7 cells/x 400 field, cortex and dentate gyrus p < 0.05 versus naive). Dual-label immunofluorescence revealed that PANT-positive cells were predominately neurons. These data demonstrate that TBI results in extensive DNA damage, which includes both single- and double-strand breaks in injured cortex and hippocampus. The presence of multiple types of DNA breaks implicate several pathways in the evolution of DNA damage after TBI.

Poly(ADP-ribose) polymerase-1 (PARP-1) is an enzyme best known for its role in DNA repair and as a mediator of NAD+ depletion and energy failure-induced cell death. We tested the effect of the potent and selective ideno-isoquinolone PARP-1 inhibitor INO-1001 after controlled cortical impact (CCI) in mice. Anesthetized adult male mice were subjected to moderate CCI (velocity 6 m/sec, depth 1.2 mm) or sham-injury. Immediately after CCI or sham-injury mice received either INO-1001 (1.6 mg/kg) or vehicle via intracerebral injection (5 microl over 5 min) in a randomized fashion. At 2 h, contused brain tissue was dissected and NAD+ levels were measured. Separate mice underwent neuropathological outcome tests that included spatial memory acquisition (Morris water maze days 14-20), and assessment of contusion volume and hippocampal cell death at day 21. Local treatment with INO-1001 preserved brain NAD+ levels 2 h after CCI (vehicle = 67 +/- 7.6, INO-1001 = 95.8 +/- 4.4 % uninjured hemisphere; n = 6/group, p = 0.03). In the Morris water maze, treatment with INO-1001 reduced the latency to find the hidden platform and increased the time spent in the target quadrant versus vehicle after CCI (n = 11/group, p < or = 0.05). Histological damage did not differ between vehicle and INO-1001-treated mice after CCI. Treatment with INO-1001 prevented NAD+ depletion and improved outcome, although modestly, identifying PARP-mediated energy failure as a contributor to the pathological sequelae of TBI. Further study testing the effects of PARP inhibitors is warranted, specifically in models of brain injury where energy failure is seen.

The pathobiology of traumatic brain injury (TBI) includes activation of multiple caspases followed by cell death with a spectrum of apoptotic phenotypes. There are initiator (e.g. caspase-2, -8, and -9) and effector (e.g. caspase-3 and -7) caspases. Recently, caspase-2 and -8 have been shown to regulate cell death via provoking cytochrome c release from the mitochondria upstream of caspase-9. Here, we show that an intracerebral injection of the pan-caspase inhibitor boc-Aspartyl(OMe)-fluoromethylketone (BAF; 1 lmol) 1 min after TBI in rats reduces caspase-3-like activity, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) and tissue damage, and cytochrome c release in ipsilateral cortex at 24 h versus vehicle. To investigate whether either caspase-2 and/or caspase-8 activation may contribute to cytochrome release, the effect of BAF treatment on caspase-2 and caspase-8 proteolysis was also examined. boc-aspartyl(OMe)-fluoromethylketone treatment inhibited proteolysis of caspase-2 but not caspase-8 24 h after TBI in rats versus vehicle. However, BAF with or without nerve growth factor (12.5 ng/h¾14 days intracerebrally via osmotic pump) did not result in differences in motor function, Morris water maze performance, hippocampal neuron survival, nor contusion volume at 14 days. These data suggest that BAF treatment reduces acute cell death after TBI by inhibiting mitochondrial release of cytochrome c, possibly via a mechanism involving initiator caspases; however, BAF appears to delay cell death, rather than result in permanent protection.

Activation of protein kinase B (PKB, also known as Akt) by phosphorylation at serine-473 and threonine-308 promotes cell survival in multiple in vitro and in vivo models where neuronal death is seen, including traumatic brain injury (TBI); however, whether PKB is activated in humans after TBI was heretofore unknown. Activated PKB inhibits apoptogenic factors and is involved in the regulation of several transcription factors. Accordingly, we examined phosphorylation of the PKB signaling pathway in humans as well as rats after TBI using phosphospecific antibodies. Increased phosphorylation of PKB and PKB substrates was detected in injured brain from both humans and rats. In humans, increased phosphorylation of the PKB signaling pathway-related proteins Bad and forkhead transcription factor (FKHR) was detected in patients with TBI versus controls. In rats, increased phosphorylation of FKHR, inhibitor of jBa, and cyclic adenosine monophosphate responsive element binding protein (CREB) was detected after TBI versus controls. The deoxyribonucleic acid-binding activity of CREB was also enhanced after TBI in rats. Increased phosphorylation of PKB and PKB substrates was identified in neurons and other cell types by immunohistochemistry in both humans and rats. These data show increased phosphorylation of PKB, PKB substrates, and related proteins after both experimental and clinical TBI, suggesting either activation of the PKB signaling pathway or reduced phosphatase activity in both species.