Oxidative stress and mitochondrial dysfunction as determinants of ischemic neuronal death and survival

Mitochondria are the powerhouse of the cell. Their primary physiological function is to generate adenosine triphosphate through oxidative phosphorylation via the electron transport chain. Reactive oxygen species generated from mitochondria have been implicated in acute brain injuries such as stroke and neurodegeneration. Recent studies have shown that mitochondrially‐formed oxidants are mediators of molecular signaling, which is implicated in the mitochondria‐dependent apoptotic pathway that involves pro‐ and antiapoptotic protein binding, the release of cytochrome c, and transcription‐independent p53 signaling, leading to neuronal death. Oxidative stress and the redox state of ischemic neurons are also implicated in the signaling pathway that involves phosphatidylinositol 3‐kinase/Akt and downstream signaling, which lead to neuronal survival. Genetically modified mice or rats that over‐express or are deficient in superoxide dismutase have provided strong evidence in support of the role of mitochondrial dysfunction and oxidative stress as determinants of neuronal death/survival after stroke and neurodegeneration.

Mitochondria are central integrators for and transducers of apoptotic signals in neurons. Mitochondria physiologically generate adenosine triphosphate through oxidative phosphorylation via the electron transport chain. Reactive oxygen species (ROS) generated from mitochondria mediate molecular signaling, such as p53. In mitochondria-dependent apoptosis, molecular signaling returns to mitochondria, then triggers the release of critical apoptotic activators and effectors of cell death, such as cytochrome c or apoptosisinducing factor, from the mitochondrial intermembrane space.
Recent findings demonstrate that p53 is involved in neuronal death that occurs with stroke and neurodegeneration (Crumrine et al. 1994;Li et al. 1994;Tomasevic et al. 1999;Saito et al. 2005;Endo et al. 2006a). Over-expression of copper/zinc-superoxide dismutase (SOD1) down-regulated PUMA (Niizuma et al. 2009), suggesting a functional relationship between oxidative stress and the p53 signaling pathway. Here, we discuss the role of mitochondrial dysfunction and oxidative stress as determinants of neuronal death after stroke and neurodegeneration, focusing on Bax, PUMA, PIDD, transcription-independent p53 translocation, and SOD1 over-expression.

Bax signaling pathway
Bax has an extensive amino acid homology with Bcl-2. Bax homodimerizes and forms heterodimers with Bcl-2 (Oltvai et al. 1993). Cell fractionation and confocal microscopy showed that Bax localized in the cytosol of most cells, although it has the C-terminal putative transmembrane domain, similar to that of Bcl-2 (Hsu et al. 1997). With apoptotic stimuli, Bax is post-transcriptionally activated, then it oligomerizes and translocates to mitochondria. Mitochondrial Bax triggers cytochrome c release from mitochondria (Gross et al. 1998;Fiskum et al. 1999).
In summary, apoptotic stimuli cause Bax to increase and translocate to mitochondria. Mitochondrial Bax interacts with other Bcl-2 family proteins, which triggers cytochrome c release (Fig. 1). precedes Bid truncation and translocation to mitochondria. Moreover, p53 translocates to the mitochondrial membrane and activates the mitochondria-dependent apoptotic pathway in a transcription-independent manner. BH3-only proteins and p53 interact with both pro-apoptotic Bax and anti-apoptotic Bcl-X L on the mitochondrial membrane. This interaction causes Bax oligomerization and activation, which trigger cytochrome c release, leading to neuronal death. tBid, truncated Bid.

PUMA signaling pathway
PUMA was originally identified as a direct target of p53 with two putative p53 binding sites (Nakano and Vousden 2001;Yu et al. 2001). PUMA has been reported to be a strong inducer of apoptosis. In an in vitro study, PUMA expression caused rapid apoptosis (Yu et al. 2001) and PUMA inhibition by antisense oligonucleotide reduced apoptosis (Nakano and Vousden 2001). PUMA induced apoptosis through a mitochondria-dependent pathway (Nakano and Vousden 2001;Yu et al. 2001).
Recent reports have demonstrated that PUMA induces apoptosis by interacting with anti-or pro-apoptotic proteins (Nakano and Vousden 2001;Yu et al. 2001;Chen et al. 2005;Kim et al. 2006;Steckley et al. 2007). PUMA can interact with multiple Bcl-2 family members through the BH3 domain (Chen et al. 2005). It localized to mitochondria and interacted with both pro-apoptotic Bax (Kim et al. 2006;Steckley et al. 2007) and anti-apoptotic Bcl-2 or Bcl-X L (Nakano and Vousden 2001;Yu et al. 2001) through a BH3 domain, followed by cytochrome c release and caspase activation.
PUMA is also known to have important roles in neuronal apoptosis. Its over-expression induced apoptosis in primary neurons (Cregan et al. 2004), and PUMA nullizygous neurons are resistant to araC-induced apoptosis (Wyttenbach and Tolkovsky 2006). PUMA mediated oxidative stress-induced neuronal apoptosis through cytochrome c release and caspase activation in a primary culture of mouse neurons (Steckley et al. 2007). It also mediated camptothecin-induced neuronal death in a primary mouse neuron culture (Uo et al. 2007). PUMA regulated neuronal death after tGCI (Reimertz et al. 2003;Niizuma et al. 2009) (Fig. 2).
In summary, PUMA is induced by p53, and interacts with pro-apoptotic and anti-apoptotic Bcl-2 family proteins, resulting in cytochrome c release (Fig. 1).

PIDD signaling pathway
p53-Induced protein with a death domain was also identified as a target of p53 (Lin et al. 2000). Since PIDD overexpression in p53-deficient human cell lines induces cellcycle arrest and apoptosis, PIDD is considered to act downstream of p53. Full length PIDD is constitutively cleaved into an N-terminal fragment and a C-terminal fragment (PIDD-C) by autoproteolysis. PIDD-C is further cleaved into PIDD-CC by autoproteolysis (Tinel and Tschopp 2004;Tinel et al. 2007).
Evidence for the role of PIDD-CC in the activation of caspase-2 has been accumulating (Tinel and Tschopp 2004;Berube et al. 2005;Ren et al. 2005;Seth et al. 2005). PIDD-CC, receptor-interacting protein-associated ICH-1/CED-3 homologous protein with a death domain (RAIDD), and procaspase-2 form a large protein complex, which is referred to as the PIDDosome, similar to the caspase-9-activating apoptosome complex (Tinel and Tschopp 2004). PIDD interacts with RAIDD through the death domain, and RAIDD interacts with caspase-2 through the caspase recruitment domain, resulting in the crystal structure of the PIDDosome (Park et al. 2007). Procaspase-2 was schematically dimerized and activated by the PIDDosome (Park et al. 2007). Similar to the caspase-9-activating apoptosome complex, the PIDDosome regulates stress-induced apoptosis (Tinel and Tschopp 2004).
In contrast to PIDD-CC, PIDD-C is thought to have an anti-apoptotic role. In a recent study, PIDD-C formed a protein complex with a nuclear factor-jB essential modulator and receptor-interacting protein 1. This activated the transcription factor nuclear factor-jB pathway in response to genotoxic stress (Janssens et al. 2005).
In neuronal death, PIDD-CC increased after tGCI, followed by caspase-2 activation and Bid cleavage (Niizuma et al. 2008). Truncated Bid interacts with Bax, exposing the N-terminus of Bax and inducing its oligomerization followed by release of pro-apoptotic proteins from mitochondria (Desagher et al. 1999).
In summary, PIDD is transcriptionally induced by p53, then cleaved by autoproteolysis. PIDD-CC forms the PID-Dosome, which activates caspase-2. Caspase-2 cleaved Bid, followed by the interaction of Bax and truncated Bid, resulting in cytochrome c release (Fig. 1).

Transcription-independent p53 translocation
Most of the effects of p53 are ascribed to its function as a transcription factor. However, reports have suggested that p53 can also induce apoptosis independently of its transcriptional activity (Caelles et al. 1994;Bennett et al. 1998;Mihara et al. 2003). In response to certain death stimuli, a fraction of stabilized p53 rapidly translocates to mitochondria in some cell types (Marchenko et al. 2000;Mihara et al. 2003;Erster et al. 2004). In p53 null cancer cells, exogenous p53 targeted to mitochondria induced apoptosis and suppressed colony formation in a transcription-independent manner (Mihara et al. 2003). Furthermore, endogenous mitochondrial p53 forms inhibitory complexes with antiapoptotic Bcl-X L and Bcl-2 proteins, which cause cytochrome c release and caspase activation (Mihara et al. 2003). Mitochondrial translocation of p53 launches a rapid proapoptotic response in a transcription-independent manner that jump-starts and amplifies the slower transcriptiondependent response (Erster et al. 2004). This translocation may be regulated by the Akt-Mdm2 pathway through monoubiquitylation of p53 (Marchenko et al. 2007).
In neuronal cell death, p53 translocated to mitochondria and interacted with anti-apoptotic Bcl-X L , followed by cytochrome c release after tGCI (Endo et al. 2006a). Inhibition of p53 translocation caused by a specific dosage of the p53 inhibitor, pifithrin-a, resulted in neuroprotection of the hippocampal CA1 subregion against cerebral ischemia and reperfusion (Endo et al. 2006a).
In summary, p53 can induce apoptosis in a transcriptionindependent manner by interacting with Bcl-X L after ischemia (Fig. 1).

SOD1 over-expression
Evidence is accumulating in support of the idea that activation of p53 signaling pathways, which precedes release of pro-apoptotic proteins from mitochondria, can cause apoptosis in ischemic neurons. However, the upstream events that lead to p53 signaling and neuronal death are unclear. ROS formation during reperfusion after cerebral ischemia in the mitochondria appears to be one such event. This is supported by the finding that p53 target genes up-regulated in response to elevated oxidative stress in liver samples (Han et al. 2008). ROS cause DNA damage, which activates DNA-dependent kinase and ataxia telangiecta protein, resulting in phosphorylation of p53 at specific serine residues (Nakagawa et al. 1999;Shangary et al. 2000).
Specific scavengers of ROS, such as SOD1, may play a major role in modulating death signaling. SOD1 is an antioxidant isoenzyme mainly localized in the cytosol that dismutates superoxide anions to hydrogen peroxide (Fridovich 1975). SOD1 is constitutively present in all cells (Huang et al. 1999). In animals that over-express SOD1, cytochrome c release and neuronal death were highly inhibited after FCI (Kinouchi et al. 1991;Chan 1996;Fujimura et al. 2000), tGCI (Murakami et al. 1997;Chan et al. 1998;Endo et al. 2006b), and subarachnoid hemorrhage (Endo et al. 2007). The phospho-Akt survival pathway was significantly up-regulated in SOD1-over-expressing animals compared with wild-type animals (Noshita et al. 2003;Endo et al. 2006bEndo et al. , 2007. In contrast to the survival pathway, p53 upregulation was inhibited by SOD1 over-expression after FCI (Saito et al. 2005). Moreover, PUMA up-regulation was inhibited in SOD1-over-expressing animals after tGCI (Niizuma et al. 2009), suggesting that oxidative stress may modulate pro-survival Akt signaling and pro-death p53 signaling that determine death or survival of ischemic neurons.
These results cumulatively suggest that oxidative stress, known to be generated during reperfusion following an ischemic event, is associated with cell survival signaling such as Akt, cell death pathways such as p53, and the determination of subsequent neuronal survival or death.

Conclusion
Oxidative stress may regulate p53-dependent transcription, p53 translocation, and pro-survival Akt signaling through phosphorylation, at least in part. Decreasing oxidative stress by SOD1 over-expression results in neuroprotection. Mitochondrial dysfunction and oxidative stress may determine neuronal death/survival after stroke and neurodegeneration. assistance with figure preparation. Supported by National Institutes of Health grants P50 NS014543, R01 NS025372, R01 NS036147, and R01 NS038653.

Conflicts of interest
All authors declare no conflicts of interests.