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1.  Potent and Selective Inhibitors of Human Reticulocyte 12/15-Lipoxygenase as Anti-Stroke Therapies 
Journal of medicinal chemistry  2014;57(10):4035-4048.
A key challenge facing drug discovery today is variability of the drug target between species, such as with 12/15-lipoxygenase (12/15-LOX), which contributes to ischemic brain injury, but its human and rodent isozymes have different inhibitor specificities. In the current work, we have utilized a quantitative high-throughput (qHTS) screen to identify compound 1 (ML351), a novel chemotype for 12/15-LOX inhibition, which has nanomolar potency (IC50 = 200 nM) against human 12/15-LOX and is protective against oxidative glutamate toxicity in mouse neuronal HT-22 cells. In addition, it exhibited greater than 250-fold selectivity versus related LOX isozymes, was a mixed inhibitor, and did not reduce the active-site ferric ion. Finally, 1 significantly reduced infarct size following permanent focal ischemia in a mouse model of ischemic stroke. As such, this represents the first report of a selective inhibitor of human 12/15-LOX with demonstrated in vivo activity in proof-of-concept mouse models of stroke.
PMCID: PMC4033661  PMID: 24684213
Human 15-lipoxygenase-1; high-throughput; inhibitor
2.  Prevention of JNK phosphorylation as a mechanism for rosiglitazone in neuroprotection after transient cerebral ischemia: activation of dual specificity phosphatase 
Rosiglitazone, a synthetic peroxisome proliferator-activated receptor-γ (PPARγ) agonist, prevents cell death after cerebral ischemia in animal models, but the underlying mechanism has not been clarified. In this study, we examined how rosiglitazone protects neurons against ischemia. Mice treated with rosiglitazone were subjected to 60 minutes of focal ischemia followed by reperfusion. Rosiglitazone reduced infarct volume after ischemia and reperfusion. We show that this neuroprotective effect was reversed with a PPARγ antagonist. Western blot analysis showed a significant increase in expression of phosphorylated stress-activated protein kinases (c-Jun N-terminal kinase (JNK) and p38) in ischemic brain tissue. Rosiglitazone blocked this increase. Furthermore, we observed that rosiglitazone increased expression of the dual-specificity phosphatase 8 (DUSP8) protein and messenger RNA in ischemic brain tissue. Dual-specificity phosphatase 8 is a mitogen-activated protein kinase phosphatase that can dephosphorylate JNK and p38. Another key finding of the present study was that knockdown of DUSP8 in primary cultured cortical neurons that were subjected to oxygen–glucose deprivation diminished rosiglitazone's effect on downregulation of JNK phosphorylation. Thus, rosiglitazone's neuroprotective effect after ischemia is mediated by blocking JNK phosphorylation induced by ischemia via DUSP8 upregulation.
PMCID: PMC3597369  PMID: 23032483
cerebral ischemia; c-Jun N-terminal kinase; dual-specificity phosphatase 8; p38 mitogen-activated protein kinase; rosiglitazone
3.  Induction of thioredoxin-interacting protein is mediated by oxidative stress, calcium, and glucose after brain injury in mice 
Neurobiology of Disease  2012;46(2):440-449.
Oxidative stress and glucose affect the expression of various genes that contribute to both reactive oxygen species generation and antioxidant systems. However, systemic alteration of oxidative stress-related gene expression in normal brains and in brains with a high-glucose status after ischemic reperfusion has not been explored. Using a polymerase chain reaction array system, we demonstrate that thioredoxin-interacting protein (Txnip) is induced by both oxidative stress and glucose. We found that Txnip mRNA is induced by ischemic-reperfusion injury and that Txnip is located in the cytoplasm of neurons. Moreover, in vitro oxygen-glucose deprivation (OGD) and subsequent reoxygenation without glucose and in vivo administration of 3-nitropropionic acid also promoted an increase in Txnip in a time-dependent manner, indicating that oxidative stress without glucose can induce Txnip expression in the brain. However, calcium channel blockers inhibit induction of Txnip after OGD and reoxygenation. Using the polymerase chain reaction array with ischemic and hyperglycemic-ischemic samples, we confirmed that enhanced expression of Txnip was observed in hyperglycemic-ischemic brains after middle cerebral artery occlusion. Finally, transfection of Txnip small interfering RNA into primary neurons reduced lactate dehydrogenase release after OGD and reoxygenation. This is the first report showing that Txnip expression is induced in neurons after oxidative or glucose stress under either ischemic or hyperglycemic-ischemic conditions, and that Txnip is proapoptotic under these conditions.
PMCID: PMC3323710  PMID: 22366181
Middle cerebral artery occlusion; Oxygen-glucose deprivation; Polymerase chain reaction array; Reactive oxygen species; Thioredoxin; Thioredoxin-interacting protein
4.  Release of mitochondrial apoptogenic factors and cell death are mediated by CK2 and NADPH oxidase 
Activation of the NADPH oxidase subunit, NOX2, and increased oxidative stress are associated with neuronal death after cerebral ischemia and reperfusion. Inhibition of NOX2 by casein kinase 2 (CK2) leads to neuronal survival, but the mechanism is unknown. In this study, we show that in copper/zinc-superoxide dismutase transgenic (SOD1 Tg) mice, degradation of CK2α and CK2α′ and dephosphorylation of CK2β against oxidative stress were markedly reduced compared with wild-type (WT) mice that underwent middle cerebral artery occlusion. Inhibition of CK2 pharmacologically or by ischemic reperfusion facilitated accumulation of poly(ADP-ribose) polymers, the translocation of apoptosis-inducing factor (AIF), and cytochrome c release from mitochondria after ischemic injury. The eventual enhancement of CK2 inhibition under ischemic injury strongly increased 8-hydroxy-2′-deoxyguanosine and phosphorylation of H2A.X. Furthermore, CK2 inhibition by tetrabromocinnamic acid (TBCA) in SOD1 Tg and gp91 knockout (KO) mice after ischemia reperfusion induced less release of AIF and cytochrome c than in TBCA-treated WT mice. Inhibition of CK2 in gp91 KO mice subjected to ischemia reperfusion did not increase brain infarction compared with TBCA-treated WT mice. These results strongly suggest that NOX2 activation releases reactive oxygen species after CK2 inhibition, triggering release of apoptogenic factors from mitochondria and inducing DNA damage after ischemic brain injury.
PMCID: PMC3318149  PMID: 22146192
casein kinase 2; middle cerebral artery occlusion; NADPH oxidase; NOX2; reactive oxygen species
5.  Neuroprotection by IL-6 Is Mediated by STAT3 and Antioxidative Signaling in Ischemic Stroke 
Background and Purpose
Interleukin-6 (IL-6) has been shown to have a neuroprotective effect in brain ischemic injury. However, its molecular mechanisms are still poorly understood. In this study, we investigated the neuroprotective role of the IL-6 receptor (IL-6R) by IL-6 in the reactive oxygen species defense system after transient focal cerebral ischemia (tFCI).
IL-6 was injected in mice before and after middle cerebral artery occlusion. Co-immunoprecipitation assays were performed for analysis of IL-6R association after tFCI. Primary mouse cerebral cortical neurons were transfected with small interfering RNA probes targeted to IL-6Rα or gp130 and were used for chromatin-immunoprecipitation assay, luciferase promoter assay, and cell viability assay. Reduction in infarct volumes by IL-6 was measured after tFCI.
IL-6R was disrupted via a disassembly between IL-6Rα and gp130 associated by protein oxidation after reperfusion following tFCI. This suppressed phosphorylation of signal transducer and activator of transcription 3 (STAT3), and finally induced neuronal cell death via a decrease in manganese-superoxide dismutase (Mn-SOD). However, IL-6 injections prevented disruption of IL-6R against reperfusion after tFCI, consequently restoring activity of STAT3 through recovery of the binding of STAT3 to gp130. Moreover, IL-6 injections restored the transcriptional activity of the Mn-SOD promoter through recovery of the recruitment of STAT3 to the Mn-SOD promoter, and reduced infarct volume after tFCI.
This study demonstrates that IL-6 has a neuroprotective effect against cerebral ischemic injury through IL-6R-mediated STAT3 activation and Mn-SOD expression.
PMCID: PMC3395465  PMID: 21940958
IL-6; STAT3; Mn-SOD; cerebral ischemia; neuroprotection
6.  Minocycline-Preconditioned Neural Stem Cells Enhance Neuroprotection after Ischemic Stroke in Rats 
The Journal of Neuroscience  2012;32(10):3462-3473.
Transplantation of neural stem cells (NSCs) offers a novel therapeutic strategy for stroke; however, massive grafted-cell death following transplantation, possibly due to a hostile host-brain environment, lessens the effectiveness of this approach. Here, we have investigated whether reprogramming NSCs with minocycline, a broadly-used antibiotic also known to possess cytoprotective properties, enhances survival of grafted cells and promotes neuroprotection in ischemic stroke. NSCs harvested from the subventricular zone of fetal rats were preconditioned with minocycline in vitro and transplanted into rat brains 6 h after transient middle cerebral artery occlusion. Histological and behavioral tests were examined from days 0–28 after stroke. For in vitro experiments, NSCs were subjected to oxygen-glucose deprivation and reoxygenation. Cell viability and antioxidant gene expression were analyzed. Minocycline preconditioning protected the grafted NSCs from ischemic reperfusion injury via up-regulation of Nrf2 and Nrf2-regulated antioxidant genes. Additionally, preconditioning with minocycline induced the NSCs to release paracrine factors, including brain-derived neurotrophic factor, nerve growth factor, glial cell-derived neurotrophic factor, and vascular endothelial growth factor. Moreover, transplantation of the minocycline-preconditioned NSCs significantly attenuated infarct size and improved neurological performance, compared with non-preconditioned NSCs. Minocycline-induced neuroprotection was abolished by transfecting the NSCs with Nrf2-small interfering RNA before transplantation. Thus, preconditioning with minocycline, which reprograms NSCs to tolerate oxidative stress after ischemic reperfusion injury and to express higher levels of paracrine factors through Nrf2 up-regulation, is a simple and safe approach to enhance the effectiveness of transplantation therapy in ischemic stroke.
PMCID: PMC3315362  PMID: 22399769
7.  Oxidative Stress in Ischemic Brain Damage: Mechanisms of Cell Death and Potential Molecular Targets for Neuroprotection 
Antioxidants & Redox Signaling  2011;14(8):1505-1517.
Significant amounts of oxygen free radicals (oxidants) are generated during cerebral ischemia/reperfusion, and oxidative stress plays an important role in brain damage after stroke. In addition to oxidizing macromolecules, leading to cell injury, oxidants are also involved in cell death/survival signal pathways and cause mitochondrial dysfunction. Experimental data from laboratory animals that either overexpress (transgenic) or are deficient in (knock-out) antioxidant proteins, mainly superoxide dismutase, have provided strong evidence of the role of oxidative stress in ischemic brain damage. In addition to mitochondria, recent reports demonstrate that NADPH oxidase (NOX), an important pro-oxidant enzyme, is also involved in the generation of oxidants in the brain after stroke. Inhibition of NOX is neuroprotective against cerebral ischemia. We propose that superoxide dismutase and NOX activity in the brain is a major determinant for ischemic damage/repair and that these major anti- and pro-oxidant enzymes are potential endogenous molecular targets for stroke therapy. Antioxid. Redox Signal. 14, 1505–1517.
PMCID: PMC3061196  PMID: 20812869
8.  Oxidative stress increases phosphorylation of IκB kinase-α by enhancing NF-κB-inducing kinase after transient focal cerebral ischemia 
The IκB kinase (IKK) complex is a central component in the classic activation of the nuclear factor-κB (NF-κB) pathway. It has been reported to function in physiologic responses, including cell death and inflammation. We have shown that IKK is regulated by oxidative status after transient focal cerebral ischemia (tFCI) in mice. However, the mechanism by which oxidative stress influences IKKs after tFCI is largely unknown. Nuclear accumulation and phosphorylation of IKKα (pIKKα) were observed 1 h after 30 mins of tFCI in mice. In copper/zinc-superoxide dismutase knockout mice, levels of NF-κB-inducing kinase (NIK) (an upstream kinase of IKKα), pIKKα, and phosphorylation of histone H3 (pH3) on Ser10 were increased after tFCI and were higher than in wild-type mice. Immunohistochemistry showed nuclear accumulation and pIKKα in mouse brain endothelial cells after tFCI. Nuclear factor-κB-inducing kinase was increased, and it enhanced pH3 by inducing pIKKα after oxygen–glucose deprivation (OGD) in mouse brain endothelial cells. Both NIK and pH3 interactions with IKKα were confirmed by coimmunoprecipitation. Treatment with IKKα small interfering RNA significantly reduced cell death after OGD. These results suggest that augmentation of NIK, IKKα, and pH3 in response to oxidative stress is involved in cell death after cerebral ischemia (or stroke).
PMCID: PMC2888847  PMID: 20125184
focal cerebral ischemia; histone H3 phosphorylation; IκB kinase-α; NF-κB-inducing kinase; oxidative stress; oxygen–glucose deprivation
9.  Reperfusion and Neurovascular Dysfunction in Stroke: From Basic Mechanisms to Potential Strategies for Neuroprotection 
Molecular neurobiology  2010;41(2-3):172-179.
Effective stroke therapies require recanalization of occluded cerebral blood vessels. However, reperfusion can cause neurovascular injury, leading to cerebral edema, brain hemorrhage, and neuronal death by apoptosis/necrosis. These complications, which result from excess production of reactive oxygen species in mitochondria, significantly limit the benefits of stroke therapies. We have developed a focal stroke model using mice deficient in mitochondrial manganese-superoxide dismutase (SOD2−/+) to investigate neurovascular endothelial damage that occurs during reperfusion. Following focal stroke and reperfusion, SOD2−/+ mice had delayed blood-brain barrier breakdown, associated with activation of matrix metalloproteinase and high brain hemorrhage rates, whereas a decrease in apoptosis and hemorrhage was observed in SOD2 overexpressors. Thus, induction and activation of SOD2 is a novel strategy for neurovascular protection after ischemia/reperfusion. Our recent study identified the signal transducer and activator of transcription 3 (STAT3) as a transcription factor of the mouse SOD2 gene. During reperfusion, activation of STAT3 and its recruitment into the SOD2 gene were blocked, resulting in increased oxidative stress and neuronal apoptosis. In contrast, pharmacological activation of STAT3 induced SOD2 expression, which limits ischemic neuronal death. Our studies point to antioxidant-based neurovascular protective strategies as potential treatments to expand the therapeutic window of currently approved therapies.
PMCID: PMC2877155  PMID: 20157789
Cerebral ischemia; Oxidative stress; Reactive oxygen species; Mitochondria; Mn-SOD; STAT3; NADPH oxidase; CK2; Neuroprotective signaling
10.  Mitochondrial and apoptotic neuronal death signaling pathways in cerebral ischemia 
Biochimica et biophysica acta  2009;1802(1):92-99.
Mitochondria play important roles as the powerhouse of the cell. After cerebral ischemia, mitochondria overproduce reactive oxygen species (ROS), which have been thoroughly studied with the use of superoxide dismutase transgenic or knockout animals. ROS directly damage lipids, proteins, and nucleic acids in the cell. Moreover, ROS activate various molecular signaling pathways. Apoptosis-related signals return to mitochondria, then mitochondria induce cell death through the release of pro-apoptotic proteins such as cytochrome c or apoptosis-inducing factor. Although the mechanisms of cell death after cerebral ischemia remain unclear, mitochondria obviously play a role by activating signaling pathways through ROS production and by regulating mitochondria-dependent apoptosis pathways.
PMCID: PMC2790539  PMID: 19751828
Mitochondria; Cerebral ischemia; SOD1; Reactive oxygen species; Neuronal death; PIDD
11.  Oxidative stress increases phosphorylation of IκB kinase α by enhancing NF-κB–inducing kinase after transient focal cerebral ischemia 
The IκB kinase (IKK) complex is a central component in the classic activation of the NF-κB pathway. IKK has been reported to function in physiological responses including cell death and inflammation. We have shown that IKK is regulated by oxidative status after transient focal cerebral ischemia (tFCI) in mice. However, how oxidative stress influences IKKs after tFCI is largely unknown. Nuclear accumulation and phosphorylation of IKKα (pIKKα) were observed 1 h after 30 mins of tFCI in mice. In copper/zinc-superoxide dismutase knock-out mice, the levels of NF-κB–inducing kinase (NIK) (an upstream kinase of IKKα), pIKKα, and phosphorylation of histone H3 (pH3) on Ser10 were increased after tFCI and were higher than in wild-type mice. Immunohistochemistry showed nuclear accumulation and phosphorylation of IKKα in mouse brain endothelial cells after tFCI. NIK was increased and it enhanced pH3 by inducing pIKKα after oxygen-glucose deprivation in mouse brain endothelial cells. NIK and pH3 interactions with IKKα were confirmed by coimmunoprecipitation. Treatment with IKKα small interfering RNA significantly reduced cell death after oxygen-glucose deprivation. These results suggest that augmentation of NIK, IKKα, and pH3 in response to oxidative stress is involved in cell death after cerebral ischemia (or stroke).
PMCID: PMC2888847  PMID: 20125184
focal cerebral ischemia; histone H3 phosphorylation; IκB kinase α; NF-κB–inducing kinase; oxidative stress; oxygen-glucose deprivation
12.  CK2 Is a Novel Negative Regulator of NADPH Oxidase and a Neuroprotectant in Mice after Cerebral Ischemia 
NADPH oxidase is a major complex that produces reactive oxygen species (ROSs) during the ischemic period and aggravates brain damage and cell death after ischemic injury. Although many approaches have been tested for preventing production of ROSs by NADPH oxidase in ischemic brain injury, the regulatory mechanisms of NADPH oxidase activity after cerebral ischemia are still unclear. In this study, we identified casein kinase 2 (CK2) as a critical modulator of NADPH oxidase and elucidated the role of CK2 as a neuroprotectant after oxidative insults to the brain. We found that the protein levels of the catalytic subunits CK2α and CK2α', as well as the total activity of CK2, are significantly reduced after transient focal cerebral ischemia (tFCI). We also found this deactivation of CK2 caused by ischemia/reperfusion increases expression of Nox2 and translocation of p67phox and Rac1 to the membrane after tFCI. Interestingly, we found that the inactive status of Rac1 was captured by the catalytic subunit CK2α under normal conditions. However, binding between CK2α and Rac1 was immediately diminished after tFCI, and Rac1 activity was markedly increased after CK2 inhibition. Moreover, we found that deactivation of CK2 in the mouse brain enhances production of ROSs and neuronal cell death via increased NADPH oxidase activity. The increased brain infarct volume caused by CK2 inhibition was restored by apocynin, a NADPH oxidase inhibitor. This study suggests that CK2 can be a direct molecular target for modulation of NADPH oxidase activity after ischemic brain injury.
PMCID: PMC2786083  PMID: 19940173
CK2; transient focal ischemia; NADPH oxidase; Rac1; tetrabromocinnamic acid; reactive oxygen species
13.  Regulation of Mn-SOD Activity and Neuroprotection by STAT3 in Mice after Cerebral Ischemia 
Cerebral ischemia and reperfusion increase superoxide anions (O2•−) in brain mitochondria. Manganese superoxide dismutase (Mn-SOD; SOD2), a primary mitochondrial antioxidant enzyme, scavenges superoxide radicals and its overexpression provides neuroprotection. However, the regulatory mechanism of Mn-SOD expression during cerebral ischemia and reperfusion is still unclear. In this study, we identified the signal transducer and activator of transcription 3 (STAT3) as a transcription factor of the mouse Mn-SOD gene, and elucidated the mechanism of O2 •− overproduction after transient focal cerebral ischemia (tFCI). We found that Mn-SOD expression is significantly reduced by reperfusion in the cerebral ischemic brain. We also found that activated STAT3 is usually recruited into the mouse Mn-SOD promoter and upregulates transcription of the mouse Mn-SOD gene in the normal brain. However, at early post-reperfusion periods after tFCI, STAT3 was rapidly downregulated and its recruitment into the Mn-SOD promoter was completely blocked. In addition, transcriptional activity of the mouse Mn-SOD gene was significantly reduced by STAT3 inhibition in primary cortical neurons. Moreover, we found that STAT3 deactivated by reperfusion induces accumulation of O2 •− in mitochondria. The loss of STAT3 activity induced neuronal cell death by reducing Mn-SOD expression. Using SOD2-/+ heterozygous knock-out mice, we found that Mn-SOD is a direct target of STAT3 in reperfusion-induced neuronal cell death. Our study demonstrates that STAT3 is a novel transcription factor of the mouse Mn-SOD gene and plays a crucial role as a neuroprotectant in regulating levels of reactive oxygen species in the mouse brain.
PMCID: PMC2712132  PMID: 19474327
STAT3; Mn-SOD; transcription; superoxide; MCAO; apoptosis
14.  The role of Akt signaling in oxidative stress mediates NF-κB activation in mild transient focal cerebral ischemia 
Reactive oxygen species, derived from hypoxia and reoxygenation during transient focal cerebral ischemia (tFCI), are associated with the signaling pathway that leads to neuronal survival or death, depending on the severity and duration of the ischemic insult. The Akt survival signaling pathway is regulated by oxidative stress and is implicated in activation of nuclear factor-κB (NF-κB). We used mild cerebral ischemia in mice to induce increased levels of Akt phosphorylation in the cortex and striatum. To clarify the role of Akt activation via NF-κB after tFCI, we injected the specific Akt inhibitor IV, which inhibits Akt phosphorylation/activation. Inhibition of Akt phosphorylation induced decreases in sequential NF-κB signaling after 30 mins of tFCI at 1 h. Furthermore, the downstream survival signals of the Akt pathway were also decreased. Akt inhibitor IV increased ischemic infarct volume and apoptotic-related DNA fragmentation. Superoxide production in the ischemic brains of mice pretreated with the Akt inhibitor was higher than in vehicle-treated mice. In addition, those pretreated mice showed a reduction of approximately 33% in copper/zinc-superoxide dismutase expression. We propose that Akt signaling exerts its neuroprotective role via NF-κB activation in oxidative cerebral ischemia in mice.
PMCID: PMC2605287  PMID: 18628779
Akt pathway; focal cerebral ischemia; NF-κB signaling; superoxide
15.  STAT3 inhibits the degradation of HIF-1α by pVHL-mediated ubiquitination 
Experimental & Molecular Medicine  2008;40(5):479-485.
Hypoxia-inducible factor 1α (HIF-1α) is rapidly degraded by the ubiquitin-proteasome pathway under normoxic conditions. Ubiquitination of HIF-1α is mediated by interaction with von Hippel-Lindau tumor suppressor protein (pVHL). In our previous report, we found that hypoxia-induced active signal transducer and activator of transcription3 (STAT3) accelerated the accumulation of HIF-1α protein and prolonged its half-life in solid tumor cells. However, its specific mechanisms are not fully understood. Thus, we examined the role of STAT3 in the mechanism of pVHL-mediated HIF-1α stability. We found that STAT3 interacts with C-terminal domain of HIF-1α and stabilizes HIF-1α by inhibition of pVHL binding to HIF-1α. The binding between HIF-1α and pVHL, negative regulator of HIF-1α stability, was interfered dose-dependently by overexpressed constitutive active STAT3. Moreover, we found that the enhanced HIF-1α protein levels by active STAT3 are due to decrease of poly-ubiquitination of HIF-1α protein via inhibition of interaction between pVHL and HIF-1α. Taken together, our results suggest that STAT3 decreases the pVHL-mediated ubiquitination of HIF-1α through competition with pVHL for binding to HIF-1α, and then stabilizes HIF-1α protein levels.
PMCID: PMC2679355  PMID: 18985005
anoxia; hypoxia-inducible factor1, α subunit; neoplasms; STAT3 transcription factor; ubiquitination; von Hippel-Lindau tumor suppressor protein

Results 1-15 (15)