Hidenori Endo

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.

Activation of the Akt/GSK3β Signaling Pathway Mediates Survival of Vulnerable Hippocampal Neurons after Transient Global Cerebral Ischemia in Rats

Recent studies have revealed that the phosphatidylinositol 3-kinase (PI3-K) pathway is involved in apoptotic cell death after experimental cerebral ischemia. The serine—threonine kinase, Akt, functions in the PI3-K pathway and prevents apoptosis by phosphorylation at Ser473 after a variety of cell death stimuli. After phosphorylation, activated Akt inactivates other apoptogenic factors, including glycogen synthase kinase-3β (GSK3β), thereby inhibiting cell death. However, the role of Akt/GSK3β signaling in the delayed death of hippocampal neurons in the CA1 subregion after transient global cerebral ischemia (tGCI) has not been clarified. Transient global cerebral ischemia for 5 mins was induced by bilateral common carotid artery occlusion combined with hypotension. Western blot analysis showed a significant increase in phospho-Akt (Ser473) and phospho-GSK3β (Ser9) in the hippocampal CA1 subregion after tGCI. Immunohistochemistry showed that expression of phospho-Akt (Ser473) and phospho-GSK3β (Ser9) was markedly increased in the vulnerable CA1 subregion, but not in the ischemic-tolerant CA3 subregion. Double staining with phospho-GSK3β (Ser9) and terminal deoxynucleotidyl transferase-mediated uridine 5ʼ-triphosphate-biotin nick end labeling showed different cellular distributions in the CA1 subregion 3 days after tGCI. Phosphorylation of Akt and GSK3β was prevented by LY294002, a PI3-K inhibitor, which facilitated subsequent DNA fragmentation 3 days after tGCI. Moreover, transgenic rats that overexpress copper/zinc-superoxide dismutase, which is known to be neuroprotective against delayed hippocampal CA1 injury after tGCI, had enhanced and persistent phosphorylation of both Akt and GSK3β after tGCI. These findings suggest that activation of the Akt/GSK3β signaling pathway may mediate survival of vulnerable hippocampal CA1 neurons after tGCI.

Mitochondrial Translocation of p53 Mediates Release of Cytochrome c and Hippocampal CA1 Neuronal Death after Transient Global Cerebral Ischemia in Rats

Although p53 is a key modulator of cellular stress responses, the mechanism of p53-mediated apoptosis is ambiguous. p53 can mediate apoptosis in response to death stimuli by transcriptional activation of proapoptotic genes and transcriptional-independent mechanisms. Recent studies have shown that the p53 protein can directly induce permeabilization of the outer mitochondrial membrane by forming a inhibitory complex with a protective Bcl-2 family protein, resulting in cytochrome c release. However, how the mitochondrial p53 pathway mediates neuronal apoptosis after cerebral ischemia remains unclear. We examined the interaction between the mitochondrial p53 pathway and vulnerable hippocampal CA1 neurons in rats using a transient global cerebral ischemia (tGCI) model. Western blot analysis and immunofluorescent staining revealed mitochondrial p53 translocation after tGCI in the hippocampal CA1 neurons. Coimmunoprecipitation revealed that translocated p53 bound to Bcl-XL in the mitochondrial fraction. To examine the effect of a specific p53 inhibitor on the mitochondrial p53 pathway and apoptotic cell death after tGCI, we intravenously administered pifithrin-α (PFT). Mitochondrial p53 translocation and interaction between p53 and Bcl-XL were prevented by treatment with PFT. Moreover, cytochrome c release from mitochondria and subsequent apoptotic CA1 neuronal death were decreased with PFT treatment. These results suggest that the mitochondrial p53 pathway is one of the novel mechanisms mediating delayed death of vulnerable hippocampal CA1 neurons after tGCI.

Induction of MMP-9 Expression and Endothelial Injury by Oxidative Stress after Spinal Cord Injury

Matrix metalloproteinase-9 (MMP-9) activation plays an important role in blood-brain barrier (BBB) dysfunction after central nervous system injury. Oxidative stress is also implicated in the pathogenesis after cerebral ischemia and spinal cord injury (SCI), but the relationship between MMP-9 activation and oxidative stress after SCI has not yet been clarified. We examined MMP-9 expression after SCI using copper/zinc-superoxide dismutase (SOD1) transgenic (Tg) rats. Our results show that MMP-9 activity significantly increased after SCI in both SOD1 Tg rats and their wild-type (Wt) littermates, although the increase was less in the SOD1 Tg rats. This pattern of MMP-9 expression was further confirmed by immunostaining and Western blot analysis. In situ zymography showed that gelatinolytic activity increased after SCI in the Wt rats, while the increase was less in the Tg rats. Evans blue extravasation increased in both the Wt and Tg rats, but was less in the SOD1 Tg rats. Inhibitor studies showed that, with an intrathecal injection of SB-3CT (a selective MMP-2/MMP-9 inhibitor), the MMP activity, Evans blue extravasation, and apoptotic cell death decreased after SCI. We conclude that increased oxidative stress after SCI leads to MMP-9 upregulation, BBB disruption, and apoptosis, and that overexpression of SOD1 in Tg rats decreases oxidative stress and further attenuates MMP-9 mediated BBB disruption.

Role of the p38 mitogen-activated protein kinase/cytosolic phospholipase A2 signaling pathway in blood-brain barrier disruption after focal cerebral ischemia and reperfusion

Cytosolic phospholipase A2 (cPLA2) is a key enzyme that mediates arachidonic acid metabolism, which causes cerebral ischemia-induced oxidative injury, blood—brain barrier (BBB) dysfunction, and edema. Recent reports have shown that p38 mitogen—activated protein kinase (MAPK) is related to phosphorylation and activation of cPLA2 and release of arachidonic acid. However, involvement of the p38 MAPK pathway in cPLA2 activation and of reactive oxygen species in expression of p38 MAPK/cPLA2 after ischemia—reperfusion injury in the brain remains unclear. To address these issues, we used a model of transient focal cerebral ischemia (tFCI) in rats. Western blot analysis showed a significant increase in expression of phospho-p38 MAPK and phospho-cPLA2 in rat brain cortex after tFCI. Activity assays showed that both p38 MAPK and cPLA2 activation markedly increased 1 day after reperfusion. Intraventricular administration of SB203580 significantly suppressed activation and phosphorylation of cPLA2 and attenuated BBB extravasation and subsequent edema. Moreover, overexpression of copper/zinc-superoxide dismutase remarkably diminished activation and phosphorylation of both p38 MAPK and cPLA2 after reperfusion. These findings suggest that the p38 MAPK/cPLA2 pathway may promote BBB disruption with secondary vasogenic edema and that superoxide anions can stimulate this pathway after ischemia—reperfusion injury.

Reduction in Oxidative Stress by Superoxide Dismutase Overexpression Attenuates Acute Brain Injury after Subarachnoid Hemorrhage via Activation of Akt/Glycogen Synthase Kinase-3β Survival Signaling

Recent studies have revealed that oxidative stress has detrimental effects in several models of neurodegenerative diseases, including subarachnoid hemorrhage (SAH). However, how oxidative stress affects acute brain injury after SAH remains unknown. We have previously reported that overexpression of copper/zinc-superoxide dismutase (SOD1) reduces oxidative stress and subsequent neuronal injury after cerebral ischemia. In this study, we investigated the relationship between oxidative stress and acute brain injury after SAH using SOD1 transgenic (Tg) rats. SAH was produced by endovascular perforation in wild-type (Wt) and SOD1 Tg rats. Apoptotic cell death at 24 h, detected by a cell death assay, was significantly decreased in the cerebral cortex of the SOD1 Tg rats compared with the Wt rats. The mortality rate at 24 h was also significantly decreased in the SOD1 Tg rats. A hydroethidine study demonstrated that superoxide anion production after SAH was reduced in the cerebral cortex of the SOD1 Tg rats. Moreover, phosphorylation of Akt and glycogen synthase kinase-3β (GSK3β), which are survival signals in apoptotic cell death, was more enhanced in the cerebral cortex of the SOD1 Tg rats after SAH using Western blot analysis and immunohistochemistry. We conclude that reduction in oxidative stress by SOD1 overexpression may attenuate acute brain injury after SAH via activation of Akt/GSK3β survival signaling.

Bad as a converging signaling molecule between survival PI3-K/Akt and death JNK in neurons after transient focal cerebral ischemia in rats

Bad, a proapoptotic Bcl-2 family protein, plays a critical role in determining cell death/survival. The phosphatidylinositol 3-kinase (PI3-K)/Akt pathway and the c-Jun N-terminal kinase (JNK) pathway are thought to be involved in regulation of Bad. Therefore, the present study was performed to clarify the role of Bad as a common target of the PI3-K/Akt and JNK pathways after transient focal cerebral ischemia (tFCI) in rats. We found that Akt activity increased at 3 h and then decreased, whereas JNK activity increased 7 to 24 h in the peripheral area after tFCI. Administration of LY294002, a PI3-K-specific inhibitor, exacerbated DNA fragmentation, whereas administration of SP600125, a JNK-specific inhibitor, attenuated it. Inhibited by LY294002, phospho-Bad (Ser136) expression increased in the peripheral area 3 h after tFCI, with suppression of Akt activity. Furthermore, phospho-Bad (Ser136) and phospho-Akt (Ser473) were colocalized. Decreases in phospho-Bad (Ser136) and Bad/14-3-3 dimerization and increases in Bcl-XL/Bad or Bcl-2/Bad dimerization observed 7 to 24 h after tFCI, were prevented by SP600125 administration, with inhibition of JNK activity. The present study indicates that signal predominance varies from PI3-K/Akt-mediated survival signaling to JNK-mediated death signaling with the development of neuronal damage in the peripheral area after tFCI. This study also suggests that PI3-K/Akt has a role in Bad inactivation, whereas the JNK pathway is involved in Bad activation. We conclude that Bad may be an integrated checkpoint of PI3-K/Akt-mediated survival signaling and JNK-mediated death signaling and that it contributes to cell fate in the peripheral area after cerebral ischemia.

Akt/GSK3β Survival Signaling Is Involved in Acute Brain Injury After Subarachnoid Hemorrhage in Rats

Background and Purpose— Apoptotic cell death is associated with acute brain injury after subarachnoid hemorrhage (SAH). The Akt/glycogen synthase kinase-3&bgr; (GSK3&bgr;) pathway plays an important role in the cell death/survival pathway after a variety of cell death stimuli. However, its role in acute brain injury after SAH remains unknown. Methods— We used an endovascular perforation model of SAH in rats. Phospho-Akt and phospho-GSK3&bgr; expression was examined by Western blot analysis and immunohistochemistry. Terminal deoxynucleotidyl transferase–mediated uridine 5′-triphosphate-biotin nick end-labeling (TUNEL) and a cell death assay were used for detection of apoptosis. We administered LY294002 to examine the role of the Akt/GSK3&bgr; pathway in the phosphoinositide 3-kinase pathway after SAH. Results— Phosphorylation of Akt and GSK3&bgr; was accelerated after SAH. In the cerebral cortex, where acute brain injury was the most severe, phosphorylation of these proteins was observed in the early phase after SAH. Cortical neurons with continuous Akt phosphorylation did not colocalize with TUNEL-positive cells at 24 hours. LY294002 reduced Akt and GSK3&bgr; phosphorylation and increased brain injury after SAH. Conclusions— The present study suggests that the Akt/GSK3&bgr; pathway might be involved in neuronal survival in acute brain injury after SAH.

Potential Role of PUMA in Delayed Death of Hippocampal CA1 Neurons After Transient Global Cerebral Ischemia

Background and Purpose— p53-upregulated modulator of apoptosis (PUMA), a BH3-only member of the Bcl-2 protein family, is required for p53-dependent and -independent forms of apoptosis. PUMA localizes to mitochondria and interacts with antiapoptotic Bcl-2 and Bcl-XL or proapoptotic Bax in response to death stimuli. Although studies have shown that PUMA is associated with pathomechanisms of cerebral ischemia, clearly defined roles for PUMA in ischemic neuronal death remain unclear. The purpose of this study was to determine potential roles for PUMA in cerebral ischemia. Methods— Five minutes of transient global cerebral ischemia (tGCI) were induced by bilateral common carotid artery occlusion combined with hypotension. Results— PUMA was upregulated in vulnerable hippocampal CA1 neurons after tGCI as shown by immunohistochemistry. In Western blot and coimmunoprecipitation analyses, PUMA localized to mitochondria and was bound to Bcl-XL and Bax in the hippocampal CA1 subregion after tGCI. PUMA upregulation was inhibited by pifithrin-α, a specific inhibitor of p53, suggesting that PUMA is partly controlled by the p53 transcriptional pathway after tGCI. Furthermore, reduction in oxidative stress by overexpression of copper/zinc superoxide dismutase, which is known to be protective of vulnerable ischemic hippocampal neurons, inhibited PUMA upregulation and subsequent hippocampal CA1 neuronal death after tGCI. Conclusions— These results imply a potential role for PUMA in delayed CA1 neuronal death after tGCI and that it could be a molecular target for therapy.

Minocycline Prevents Focal Neurological Deterioration Due to Cerebral Hyperperfusion After Extracranial-Intracranial Bypass for Moyamoya Disease

BACKGROUND Cerebral hyperperfusion (CHP) is a potential complication of superficial temporal artery-middle cerebral artery (STA-MCA) anastomosis for moyamoya disease (MMD), and optimal postoperative management has not yet been established. Minocycline, a neuroprotective antibiotic agent, plays a role in blocking matrix metalloproteinase 9 (MMP-9), which contributes to edema formation and hemorrhagic conversion after cerebral ischemia-reperfusion. Patients with MMD have been shown to have increased serum MMP-9 levels. OBJECTIVE To examine the effect of minocycline on the prevention of postoperative CHP after STA-MCA anastomosis for MMD. METHODS N-isopropyl-p-[I]iodoamphetamine single-photon emission computed tomography was performed 1 and 7 days after STA-MCA anastomosis on 109 hemispheres in 86 consecutive patients with MMD (ages, 9-69 years; mean, 37.2 years). Postoperative systolic blood pressure was strictly maintained at lower than 130 mm Hg in all 109 surgeries. The most 60 recent hemispheres were managed by the intraoperative and postoperative intravenous administration of minocycline hydrochloride (200 mg/d). The incidence of focal neurological deterioration (FND) due to CHP was then compared with that in 36 patients undergoing 49 surgeries managed without minocycline. RESULTS FND due to CHP was observed in 4 operated hemispheres in patients treated without minocycline (4/49, 8.16%), and in none in the minocycline-treated group (0/60) (P = .0241). Multivariate analysis revealed that minocycline administration (P < .001), surgery on the left hemisphere (P = .031), and a smaller recipient artery diameter (P < .001) significantly correlated with FND due to CHP. CONCLUSION The administration of minocycline with strict blood pressure control may represent secure and effective postoperative management to prevent symptomatic CHP after STA-MCA anastomosis for MMD.