[Show abstract][Hide abstract] ABSTRACT: Oxidative stress after ischemia/reperfusion has been shown to induce DNA damage and subsequent DNA repair activity. Apurinic/apyrimidinic endonuclease (APE) is a multifunctional protein in the DNA base excision repair pathway which repairs apurinic/apyrimidinic sites in DNA. We investigated the involvement of oxidative stress and expression of APE in neurons after oxygen-glucose deprivation and after global cerebral ischemia. Our results suggest that overexpression of human copper/zinc-superoxide dismutase reduced oxidative stress with a subsequent decrease in APE expression. Production of oxygen free radicals and inhibition of the base excision repair pathway may play pivotal roles in the cell death pathway after ischemia.
Journal of Neurochemistry 05/2005; 93(2):351-8. DOI:10.1111/j.1471-4159.2005.03039.x · 4.28 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cumulative evidence suggests that apoptosis plays a pivotal role in cell death in vitro after hypoxia. Apoptotic cell death pathways have also been implicated in ischemic cerebral injury in in vivo ischemia models. Experimental ischemia and reperfusion models, such as transient focal/global ischemia in rodents, have been thoroughly studied and the numerous reports suggest the involvement of cell survival/death signaling pathways in the pathogenesis of apoptotic cell death in ischemic lesions. In these models, reoxygenation during reperfusion provides a substrate for numerous enzymatic oxidation reactions. Oxygen radicals damage cellular lipids, proteins and nucleic acids, and initiate cell signaling pathways after cerebral ischemia. Genetic manipulation of intrinsic antioxidants and factors in the signaling pathways has provided substantial understanding of the mechanisms involved in cell death/survival signaling pathways and the role of oxygen radicals in ischemic cerebral injury. Future studies of these pathways may provide novel therapeutic strategies in clinical stroke.
[Show abstract][Hide abstract] ABSTRACT: The serine-threonine kinase Akt is activated by phosphorylation at serine-473. After phosphorylation, activated Akt inactivates BAD or caspase-9 or other apoptogenic components, thereby inhibiting cell death. In this study we examined the relationship between Akt phosphorylation and oxidative stress after transient focal cerebral ischemia (FCI) using copper-zinc superoxide dismutase (SOD1) transgenic (Tg) mice.
The mice were subjected to 60 minutes of middle cerebral artery occlusion by intraluminal suture blockade followed by 1, 4, and 24 hours of reperfusion. Phospho-Akt expression was examined by immunohistochemistry and Western blot analysis. Production of superoxide anion was assessed by the hydroethidine method in both wild-type mice and SOD1 Tg mice. DNA fragmentation was evaluated by terminal deoxynucleotidyl transferase-mediated uridine 5'-triphosphate-biotin nick end labeling (TUNEL).
Immunohistochemistry demonstrated that phospho-Akt was constitutively expressed and was decreased in the ischemic core as early as 1 hour after reperfusion, whereas it was temporally increased in the cortex at 4 hours. Phospho-Akt expression was enhanced in the SOD1 Tg mice. Western blot analysis showed that phospho-Akt was maximized 4 hours after reperfusion in the wild-type mice, whereas phospho-Akt was increased as early as 1 hour after ischemia in the SOD1 Tg mice. There was a significant decrease in TUNEL-positive cells in the SOD1 Tg mice compared with the wild-type mice.
The present study suggests that SOD1 may contribute to the early activation of the Akt cell survival signaling pathway and may attenuate subsequent DNA damage after transient FCI.
[Show abstract][Hide abstract] ABSTRACT: Oxidative stress plays a pivotal role in ischemic-reperfusion cell injury. Oxygen-derived free radicals trigger DNA strand damage, which is responsible for the activation of poly(ADP-ribose) polymerase (PARP). Recent studies have shown that peroxynitrite is the primary mediator of DNA damage and, hence, PARP activation after ischemia. PARP activation depletes NAD and ATP pools, ultimately resulting in necrotic cell death by loss of energy stores. Our study shows that PARP is upregulated as early as 15 min after 1 h of transient focal cerebral ischemia and remains for 8 h. We also examined the role of superoxide in PARP induction using copper/zinc-superoxide dismutase transgenic mice. Immunohistochemical and Western blotting data showed that there was no increased induction in PARP expression in these mice, suggesting that one of the mechanisms by which ischemic injury is attenuated in these mice might be by the inhibition of PARP induction. Furthermore, double staining of ischemic tissue with a PARP antibody and terminal deoxynucleotidyl transferase-mediated uridine 5'-triphosphate-biotin nick end labeling (TUNEL) indicated that most cells that are positive for TUNEL do not stain for the PARP antibody, confirming recent reports that PARP activation is involved in necrotic cell death rather than apoptosis during ischemic-reperfusion injury.
Molecular Brain Research 06/2003; 113(1-2):28-36. DOI:10.1016/S0169-328X(03)00062-7 · 2.00 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Recent studies revealed that activation of extracellular signal-regulated kinase (ERK) may contribute to apoptosis, a cell death process involved in oxidative stress. We investigated phosphorylation of ERK1/2 and oxidative stress after transient focal cerebral ischemia (FCI) using transgenic (Tg) mice that overexpress copper/zinc-superoxide dismutase (SOD1). Mice were subjected to 60 min of middle cerebral artery (MCA) occlusion by intraluminal suture blockade followed by 1, 4 and 24 h of reperfusion. Immunohistochemistry showed that phospho-ERK1/2 was markedly increased in the cortex within the MCA territory at 1 h of reperfusion, followed by a decrease at 24 h. In SOD1 Tg mice, phospho-ERK1/2 was prominently reduced compared with non-ischemic controls, shown by immunohistochemistry. Western blot analysis confirmed a significant decrease in phospho-ERK1/2 1 h after FCI in the ischemic cortex (P
International Congress Series 06/2003; 1252:275-283. DOI:10.1016/S0531-5131(03)00072-4
[Show abstract][Hide abstract] ABSTRACT: Angiogenesis is an intricately regulated phenomenon. Its mechanisms in the ischemic brain have not been clearly elucidated. The authors investigated expression of angiogenesis-related genes using a complementary DNA (cDNA) array method as well as Western blotting and immunohistochemistry, and compared these studies with a temporal profile of angiogenesis in mouse brains after ischemia. The number of vessels significantly increased 3 days after injury, and proliferating endothelial cells increased as early as 1 day. This means that angiogenesis occurs immediately after the injury. Ninety-six genes implicated in angiogenesis were investigated with a cDNA array study. It was found that 42, 29, and 13 genes were increased at 1 hour, 1 day, and 21 days, respectively. Most of the well-known angiogenic factors increased as early as 1 hour. Vessel-stabilizing factors such as thrombospondins also increased. At 1 day, however, thrombospondins decreased to lower levels than in the control, indicating a shift from vascular protection to angiogenesis. At 21 days, many genes were decreased, but some involved in tissue repair were newly increased. Western blotting and immunohistochemistry showed findings compatible with the cDNA array study. Many molecules act in an orchestrated fashion in the brain after ischemia and should be taken into account for therapeutic angiogenesis for stroke.
[Show abstract][Hide abstract] ABSTRACT: Recent studies have revealed that activation of extracellular signal-regulated kinase (ERK) may contribute to apoptosis, a cell death process involved in oxidative stress. We examined phosphorylation of ERK1/2 and oxidative stress after transient focal cerebral ischemia (FCI) using transgenic (Tg) mice that overexpress copper/zinc superoxide dismutase (SOD1). The mice were subjected to 60 min of middle cerebral artery (MCA) occlusion by intraluminal suture blockade followed by 1, 4, and 24 hr of reperfusion. Immunohistochemistry and Western blot analysis showed that phospho-ERK1 was markedly increased in the cortex within the MCA territory at 1 hr of reperfusion (p < 0.01), followed by a decrease at 24 hr in wild-type mice. Double staining with phospho-ERK1/2 and neuron-specific nuclear protein showed that phospho-ERK1/2 was primarily expressed in neurons. In SOD1 Tg mice, phospho-ERK1/2 was prominently reduced compared with nonischemic controls, shown by immunohistochemistry. Western blot analysis confirmed a significant decrease in phospho-ERK1/2 1 hr after FCI in the ischemic cortex (p < 0.005). Apoptotic-related DNA fragmentation was reduced in the ischemic cortex of SOD1 Tg mice compared with wild-type mice using a cell death assay. These results suggest that phosphorylation of ERK1/2 may be involved in apoptosis or cell death after transient FCI and that SOD1 may attenuate apoptotic cell death mediated by the mitogen-activated protein kinase/ERK pathway.
The Journal of Neuroscience : The Official Journal of the Society for Neuroscience 09/2002; 22(18):7923-30. · 6.34 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The serine-threonine kinase, Akt, is involved in the survival signaling pathways in many cell systems. The present study examined phosphorylation of Akt at serine-473 and DNA fragmentation after traumatic brain injury (TBI) in mice. Immunohistochemistry showed phospho-Akt was decreased in the injured cortex 1 h after TBI, whereas it was temporally increased at 4 h in the perifocal damaged cortex. In the CA1 region of the hippocampus, phospho-Akt was increased after TBI. Western blot analysis showed that Akt was significantly decreased as early as 1 h after trauma; however, the phosphorylation was accelerated at 4 h. Double staining with phospho-Akt and phospho-BAD or phospho-GSK-3beta revealed the colocalization of phospho-Akt and downstream elements. Double staining with phospho-Akt and terminal deoxynucleotidyl transferase-mediated uridine 5'-triphosphate-biotin nick end-labeling showed different cellular distributions after TBI. The present study implicates Akt phosphorylation in the signaling pathways that are involved in cell survival after TBI.
Neurobiology of Disease 05/2002; 9(3):294-304. DOI:10.1006/nbdi.2002.0482 · 5.08 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Superoxide anion radicals (O2*-) are implicated in ischemia/reperfusion injury, although a direct relationship has not been elucidated. Recently, a specific method of hydroethidine (HEt) oxidation by O2*- was developed to detect O2*- production in a variety of experimental brain injury models. To clarify the role of O2*- in the mechanism of ischemia/reperfusion, we investigated O2*- production after ischemia/reperfusion and ischemia/reperfusion injury in mutant mice deficient in mitochondrial manganese superoxide dismutase (MnSOD) and in wild-type littermates.
Ischemia/reperfusion was performed for 60 minutes using intraluminal suture blockade of the middle cerebral artery in the mutant or wild-type mice. We evaluated fluorescent kinetics of HEt or ethidium, the oxidized form of HEt, in brains after an intravenous injection of HEt, followed by measurement of cellular O2*- production using specific HEt oxidation by O2*- before and after ischemia/reperfusion. Furthermore, we compared O2*- production and subsequent infarct volume in the mice using triphenyltetrazolium chloride after ischemia/reperfusion.
HEt oxidation to ethidium is primarily a result of mitochondrially produced O2*- under physiological conditions. Cerebral ischemia/reperfusion produced O2*- prominently in neurons shortly after reperfusion, followed by a delayed increase in endothelial cells. A deficiency in MnSOD in mutant mice increased mitochondrial O2*- production and exacerbated cerebral infarction, worsening neurological deficits after ischemia/reperfusion.
These results suggest that mitochondrial O2*- production may be a critical step underlying the mechanism of ischemia/reperfusion injury and that MnSOD may protect against ongoing oxidative cell death after ischemia/reperfusion.
[Show abstract][Hide abstract] ABSTRACT: The hippocampal CA1 neurons are selectively vulnerable to global ischemia, and neuronal death occurs in a delayed manner. The threshold of global ischemia duration that induces neuronal death has been studied, but the relationship between ischemia duration and glial death in the hippocampal CA1 area has not been fully studied. We examined neuronal/glial viability and morphological changes in the CA1 subregion after different durations of global ischemia. Global ischemia was induced in Sprague-Dawley rats by 10, 5, and 3 min of bilateral common carotid artery occlusion and hypotension. At 1-56 days after ischemia, the morphological reactions of neurons, astrocytes, oligodendrocytes, and microglia were immunohistochemically evaluated. Most of the hippocampal CA1 pyramidal neurons underwent delayed death at 3 days after 10/5 min of ischemia, but not after 3 min of ischemia. The number of astrocytes gradually declined after 10/5 min of ischemia, and viable astrocytes showed characteristic staged morphological reactions. Oligodendrocytes also showed morphological changes in their processes after 10/5 min of ischemia. Microglia transformed into a reactive form at 5 days only after 10/5 min of ischemia. These data suggest that some morphological changes in glial cells were not dependent on neuronal cell death, but their own reactions to the different severity of ischemia.
Journal of Neurotrauma 02/2002; 19(1):85-98. DOI:10.1089/089771502753460268 · 3.71 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Mitochondria are known to be involved in the early stage of apoptosis by releasing cytochrome c, caspase-9, and the second mitochondria-derived activator of caspases (Smac). We have reported that overexpression of copper/zinc superoxide dismutase (SOD1) reduced superoxide production and ameliorated neuronal injury in the hippocampal CA1 subregion after global ischemia. However, the role of oxygen free radicals produced after ischemia/reperfusion in the mitochondrial signaling pathway has not been clarified. Five minutes of global ischemia was induced in male SOD1-transgenic (Tg) and wild-type (Wt) littermate rats. Cytosolic expression of cytochrome c and Smac and activation of caspases were evaluated by immunohistochemistry, Western blot, and caspase activity assay. Apoptotic cell death was characterized by DNA nick end and single-stranded DNA labeling. In the Wt animals, early superoxide production, mitochondrial release of cytochrome c, Smac, and cleaved caspase-9 were observed after ischemia. Active caspase-3 was subsequently increased, and 85% of the hippocampal CA1 neurons showed apoptotic DNA damage 3 d after ischemia. Tg animals showed less superoxide production and cytochrome c and Smac release. Subsequent active caspase-3 expression was not evident, and only 45% of the neurons showed apoptotic DNA damage. A caspase-3 inhibitor (N-benzyloxycarbonyl-val-ala-asp-fluoromethyl ketone) reduced cell death only in Wt animals. These results suggest that overexpression of SOD1 reduced oxidative stress, thereby attenuating the mitochondrial release of cytochrome c and Smac, resulting in less caspase activation and apoptotic cell death. Oxygen free radicals may play a pivotal role in the mitochondrial signaling pathway of apoptotic cell death in hippocampal CA1 neurons after global ischemia.
The Journal of Neuroscience : The Official Journal of the Society for Neuroscience 02/2002; 22(1):209-17. · 6.34 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The serine-threonine kinase, Akt, prevents apoptosis by phosphorylation at serine-473 in several cell systems. After phosphorylation, activated Akt inactivates other apoptogenic factors, such as Bad or caspase-9, thereby inhibiting cell death. The present study examined phosphorylation of Akt at serine-473 and DNA fragmentation after transient focal cerebral ischemia in mice subjected to 60 minutes of focal cerebral ischemia by intraluminal blockade of the middle cerebral artery. Phospho-Akt was analyzed by immunohistochemistry and Western blot analysis. The DNA fragmentation was evaluated by terminal deoxynucleotidyl transferase-mediated uridine 5-triphosphate-biotin nick end-labeling (TUNEL). Immunohistochemistry showed the expression of phospho-Akt was markedly increased in the middle cerebral artery territory cortex at 4 hours of reperfusion compared with the control, whereas it was decreased by 24 hours. Western blot analysis showed a significant increase of phospho-Akt 4 hours after focal cerebral ischemia in the cortex, whereas phospho-Akt was decreased in the ischemic core. Double staining with phospho-Akt and TUNEL showed different cellular distributions of phospho-Akt and TUNEL-positive staining. Phosphorylation of Akt was prevented after focal cerebral ischemia by LY294002, a phosphatidylinositol 3-kinase inhibitor, which facilitated subsequent DNA fragmentation. These results suggest that phosphorylation of Akt may be involved in determining cell survival or cell death after transient focal cerebral ischemia.
[Show abstract][Hide abstract] ABSTRACT: Oxidative stress after ischemia/reperfusion has been shown to induce DNA damage and subsequent DNA repair activity. Ku 70/86, multifunctional DNA repair proteins, bind to broken DNA ends and trigger a DNA repair pathway. We investigated the involvement of these proteins in the development of neuronal tolerance to global cerebral ischemia after ischemic preconditioning.
Adult male Sprague-Dawley rats were subjected to either 5 minutes of lethal global ischemia with or without 3 minutes of sublethal ischemic preconditioning or 3 minutes of ischemia only. Neuronal injury was histologically assessed, and DNA damage was visualized by in situ labeling of DNA fragmentation and DNA gel electrophoresis. Ku expression was also examined by immunohistochemistry and Western blot analysis.
Hippocampal CA1 neurons underwent DNA-fragmented cell death 3 days after 5 minutes of ischemia. However, these neurons showed a strong tolerance to 5 minutes of ischemia 1 to 3 days after ischemic preconditioning. Immunohistochemistry showed virtually no constitutive expression of Ku proteins in CA1 neurons; however, ischemic preconditioning induced neuronal Ku 70 expression 1 to 3 days later. Western blot confirmed an increase in Ku 70 in this region at the same time.
The temporal and spatial expression of Ku 70 corresponded to tolerance of the hippocampal CA1 neurons to subsequent ischemia, suggesting the involvement of Ku proteins in the development of neuronal tolerance after ischemic preconditioning.
[Show abstract][Hide abstract] ABSTRACT: Ku70 and Ku86, multifunctional DNA repair proteins, bind to broken DNA ends, including double-strand breaks, and trigger a DNA repair pathway. To investigate the involvement of these proteins in DNA fragmentation after ischemia/reperfusion, Ku protein expression was examined before and after transient focal cerebral ischemia (FCI) in mice.
Adult male CD-1 mice were subjected to 60 minutes of FCI by intraluminal suture blockade of the middle cerebral artery. Ku protein expression was studied by immunohistochemistry and Western blot analysis. DNA fragmentation was evaluated by gel electrophoresis and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL). The spatial relationship between Ku expression and DNA fragmentation was examined by double labeling with Ku and TUNEL after reperfusion.
Immunohistochemistry showed constitutive expression of Ku proteins in control brains. The number of Ku-expressing cells was decreased in the entire middle cerebral artery territory as early as 4 hours after reperfusion and remained reduced until 24 hours. Western blot analyses confirmed the significant reduction of these proteins (59.4% and 57.7% reduction in optical density at 4 hours of reperfusion from the normal level of Ku70 and Ku86 bands, respectively; P<0.001). DNA gel electrophoresis demonstrated DNA laddering 24 hours after reperfusion, but not at 4 hours. Double staining with Ku and TUNEL showed a concomitant loss of Ku immunoreactivity and TUNEL-positive staining.
These results suggest that the early reduction of Ku proteins and the loss of defense against DNA damage may underlie the mechanism of DNA fragmentation after FCI.
[Show abstract][Hide abstract] ABSTRACT: Release of cytochrome c from mitochondria to cytosol is a critical step in the mitochondrial-dependent signaling pathways of apoptosis. The authors have reported that manganese superoxide dismutase (Mn-SOD) attenuated cytochrome c release and apoptotic cell death after focal cerebral ischemia (FCI). To investigate downstream to the cytochrome c-dependent pathway, the authors examined caspase-9 activation after transient FCI by immunohistochemistry and Western blotting in both wild-type and Sod2 -/+ mice. Mice were subjected to 60 minutes of middle cerebral artery occlusion followed by 1, 2, 4, or 24 hours of reperfusion. Two hours after reperfusion, cytochrome c and caspase-9 were observed in the cytosol and significantly increased in Sod2 -/+ mutants compared with wild-type mice as shown by Western blotting. Immunofluorescent double labeling for cytochrome c and caspase-9 showed cytosolic cytochrome c 1 hour after transient FCI. Cleaved caspase-9 first appeared in the cytosol at 2 hours and colocalized with cytochrome c. Terminal deoxynucleotidyl transferase-mediated uridine 5;-triphosphate-biotin nick and labeling (TUNEL) showed significant increase of positive cells in Sod2 -/+ mice compared with the wild-type in the cortex, but not in the caudate putamen. The current study revealed Mn-SOD might affect cytochrome c translocation and downstream caspase activation in the mitochondrial-dependent cell death pathway after transient FCI.
[Show abstract][Hide abstract] ABSTRACT: Reactive oxygen species (ROS) are implicated in reperfusion injury after focal cerebral ischemia (FCI). Reactive oxygen species regulate activity of transcription factors like NF-kappaB. The authors investigated the role of ROS in NF-kappaB activity after FCI using transgenic mice that overexpressed human copper/zinc-superoxide dismutase (SOD1) and that had reduced infarction volume after FCI. Superoxide dismutase transgenic and wild-type mice were subjected to 1 hour of middle cerebral artery occlusion (MCAO) and subsequent reperfusion. Immunohistochemistry showed SOD1 overexpression attenuated ischemia-induced NF-kappaB p65 immunoreactivity. Colocalization of NF-kappaB and the neuronal marker, microtubule-associated proteins (MAPs), showed that NF-kappaB was up-regulated in neurons after FCI. Electrophoretic mobility shift assays showed that SODI overexpression reduced ischemia-induced NF-kappaB DNA binding activity. Supershift assays showed that DNA-protein complexes contained p65 and p50 subunits. Immunoreactivity of c-myc, an NF-kappaB downstream gene, was increased in the ischemic cortex and colocalized with NF-kappaB. Western blotting showed that SOD1 overexpression reduced NF-kappaB and c-Myc protein levels in the ischemic brain. Colocalization of c-Myc and TUNEL staining was observed 24 hours after FCI. The current findings provide the first evidence that SOD1 overexpression attenuates activation of NF-kappaB after transient FCI in mice and that preventing this early activation may block expression of downstream deleterious genes like c-myc, thereby reducing ischemic damage.
[Show abstract][Hide abstract] ABSTRACT: Neuronal death in the hippocampal CA1 subregion has been shown to occur in a delayed manner after transient global ischemia. The 2-vessel occlusion model is one of the most frequently used global ischemia paradigms in rodents. Although researchers often fail to induce bilateral delayed CA1 neuronal death, the importance of hypotension severity has not been fully discussed. We induced 10 min of global ischemia with 2-vessel occlusion and various severities of hypotension in rats, and the subsequent neuronal damage and neurogenesis in the hippocampal CA1 pyramidal cell layer were immunohistochemically studied. Neuronal apoptosis after global ischemia was also characterized by terminal deoxynucleotidyl transferase-mediated uridine 5'-triphosphate-biotin nick end labeling (TUNEL). The mean arterial blood pressure of 31-35 mmHg was the most appropriate range of hypotension in this model because of low mortality and consistent bilateral CA1 injury. Most of the neurons in the CA1 pyramidal cell layer lost neuron specific nuclear protein and became TUNEL-positive 3 days after ischemia. There was no evidence of apoptosis or neurogenesis at 7-28 days. There were ischemia-tolerant neurons in the CA1 pyramidal cell layer that survived delayed neurodegeneration, however, further studies are necessary to characterize the property of these neurons.
Brain Research 10/2000; 877(2):281-7. DOI:10.1016/S0006-8993(00)02684-6 · 2.84 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The X-ray repair cross-complementing group 1 (XRCC1) protein plays a central role in the DNA base excision repair pathway by interacting with DNA ligase III and DNA polymerase beta. The present study examined the protein expression of XRCC1 and DNA fragmentation before and after cold injury-induced brain trauma (CIBT) in mice, in which apoptosis is assumed to participate. Immunohistochemistry showed the nuclear expression of XRCC1 in the entire region of the control brains. Fifteen minutes after CIBT, nuclear immunoreactivity was predominantly decreased in the inner boundary of the lesion, followed by a significant reduction of XRCC1 in the entire lesion 4 h after CIBT. A characteristic 70-kDa band was detected in the non-traumatic area, and was markedly decreased after CIBT as shown by Western blot analysis. DNA fragmentation was also observed after CIBT, and double staining with XRCC1 immunohistochemistry and terminal deoxynucleotidyl transferase-mediated uridine 5'-triphosphate-biotin nick end labeling showed a spatial relationship between XRCC1 loss and DNA fragmentation 24 h after CIBT. These data indicate that early decrease of XRCC1 and failure of the DNA repair mechanism may contribute to DNA-damaged neuronal cell death after CIBT.
Brain Research 07/2000; 869(1-2):105-11. DOI:10.1016/S0006-8993(00)02375-1 · 2.84 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Release of mitochondrial cytochrome c into the cytosol is a critical step in apoptosis. We have reported that early release of cytochrome c in vivo occurs after permanent focal cerebral ischemia (FCI) and is mediated by the mitochondrial antioxidant manganese superoxide dismutase (SOD). However, the role of reactive oxygen species produced after ischemia-reperfusion in the mitochondrial apoptosis process is still unknown, although overexpression of copper/zinc-SOD (SOD1), a cytosolic isoenzyme, protects against ischemia-reperfusion. We now hypothesize that the overexpression of SOD1 also prevents apoptosis after FCI. To address this issue, we examined the subcellular distribution of the cytochrome c protein in both wild-type mice and in SOD1 transgenic (Tg) mice after transient FCI. Cytosolic cytochrome c was detected as early as 2 hr after reperfusion, and correspondingly, mitochondrial cytochrome c was significantly reduced after FCI. Cytosolic cytochrome c was significantly lower in the SOD1 Tg mice compared with wild types 2 (p < 0.0001) and 4 (p < 0.05) hr after FCI. Apaf-1, which interacts with cytochrome c and activates caspases, was constitutively expressed in both groups of animals, with no alteration after FCI. Double staining with cytochrome c immunohistochemistry and terminal deoxynucleotidyl transferase-mediated uridine 5'-triphosphate-biotin nick end labeling showed a spatial relationship between cytosolic cytochrome c expression and DNA fragmentation. A significant amount of DNA laddering was detected 24 hr after ischemia and was reduced in SOD1 Tg mice. These data suggest that SOD1 blocks cytosolic release of cytochrome c and could thereby reduce apoptosis after transient FCI.
The Journal of Neuroscience : The Official Journal of the Society for Neuroscience 04/2000; 20(8):2817-24. · 6.34 Impact Factor