Young Astrocytic Mitochondria Attenuate the Elevated Level of CCL11 in the Aged Mice, Contributing to Cognitive Function Improvement.

Aging drives cognitive decline, and mitochondrial dysfunction is a hallmark of age-induced neurodegeneration. Recently, we demonstrated that astrocytes secrete functional mitochondria (Mt), which help adjacent cells to resist damage and promote repair after neurological injuries. However, the relationship between age-dependent changes in astrocytic Mt function and cognitive decline remains poorly understood. Here, we established that aged astrocytes secret less functional Mt compared to young astrocytes. We found the aging factor C-C motif chemokine 11 (CCL11) is elevated in the hippocampus of aged mice, and that its level is reduced upon systemic administration of young Mt, in vivo. Aged mice receiving young Mt, but not aged Mt improved cognitive function and hippocampal integrity. Using a CCL11-induced aging-like model in vitro, we found that astrocytic Mt protect hippocampal neurons and enhance a regenerative environment through upregulating synaptogenesis-related gene expression and anti-oxidants that were suppressed by CCL11. Moreover, the inhibition of CCL11-specific receptor C-C chemokine receptor 3 (CCR3) boosted the expression of synaptogenesis-related genes in the cultured hippocampal neurons and restored the neurite outgrowth. This study suggests that young astrocytic Mt can preserve cognitive function in the CCL11-mediated aging brain by promoting neuronal survival and neuroplasticity in the hippocampus.

Single-cell analysis identifies Ifi27l2a as a novel gene regulator of microglial inflammation in the context of aging and stroke.

Microglia are key mediators of inflammatory responses within the brain, as they regulate pro-inflammatory responses while also limiting neuroinflammation via reparative phagocytosis. Thus, identifying genes that modulate microglial function may reveal novel therapeutic interventions for promoting better outcomes in diseases featuring extensive inflammation, such as stroke. To facilitate identification of potential mediators of inflammation, we performed single-cell RNA sequencing of aged mouse brains following stroke and found that Ifi27l2a was significantly up-regulated, particularly in microglia. The increased Ifi27l2a expression was further validated in microglial culture, stroke models with microglial depletion, and human autopsy samples. Ifi27l2a is known to be induced by interferons for viral host defense, however the role of Ifi27l2a in neurodegeneration is unknown. In vitro studies in cultured microglia demonstrated that Ifi27l2a overexpression causes neuroinflammation via reactive oxygen species. Interestingly, hemizygous deletion of Ifi27l2a significantly reduced gliosis in the thalamus following stroke, while also reducing neuroinflammation, indicating Ifi27l2a gene dosage is a critical mediator of neuroinflammation in ischemic stroke. Collectively, this study demonstrates that a novel gene, Ifi27l2a, regulates microglial function and neuroinflammation in the aged brain and following stroke. These findings suggest that Ifi27l2a may be a novel target for conferring cerebral protection post-stroke.

Increased Expression of Interferon-Induced Transmembrane 3 (IFITM3) in Stroke and Other Inflammatory Conditions in the Brain.

Microglia, the resident innate immune cells of the brain, become more highly reactive with aging and diseased conditions. In collaboration with other cell types in brains, microglia can contribute both to worsened outcome following stroke or other neurodegenerative diseases and to the recovery process by changing their phenotype toward reparative microglia. Recently, IFITM3 (a member of the "interferon-inducible transmembrane" family) has been revealed as a molecular mediator between amyloid pathology and neuroinflammation. Expression of IFITM3 in glial cells, especially microglia following stroke, is not well described. Here, we present evidence that ischemic stroke causes an increase in IFITM3 expression along with increased microglial activation marker genes in aged brains. To further validate the induction of IFITM3 in post-stroke brains, primary microglia and microglial-like cells were exposed to a variety of inflammatory conditions, which significantly induced IFITM3 as well as other inflammatory markers. These findings suggest the critical role of IFITM3 in inducing inflammation. Our findings on the expression of IFITM3 in microglia and in aged brains following stroke could establish the basic foundations for the role of IFITM3 in a variety of neurodegenerative diseases, particularly those that are prevalent or enhanced in the aged brain.

Transplantation of astrocytic mitochondria modulates neuronal antioxidant defense and neuroplasticity and promotes functional recovery after intracerebral hemorrhage.

Astrocytes release functional mitochondria (Mt) that play regulatory and pro-survival functions upon entering adjacent cells. We recently demonstrated that these released Mt could enter microglia to promote their reparative/pro-phagocytic phenotype that assists in hematoma cleanup and neurological recovery after intracerebral hemorrhage (ICH). However, a relevance of astrocytic Mt transfer into neurons in protecting brain after ICH is unclear. Here, we found that ICH causes a robust increase in superoxide generation and elevated oxidative damage that coincides with loss of the mitochondrial enzyme manganese superoxide dismutase (Mn-SOD). The damaging effect of ICH was reversed by intravenous transplantation of astrocytic Mt that upon entering the brain (and neurons), restored Mn-SOD levels and reduced neurological deficits in male mice subjected to ICH. Using an in vitro ICH-like injury model in cultured neurons, we established that astrocytic Mt upon entering neurons prevented reactive oxygen species-induced oxidative stress and neuronal death by restoring neuronal Mn-SOD levels, while at the same time promoted neurite extension and upregulation of synaptogenesis-related gene expression. Furthermore, we found that Mt genome-encoded small peptide humanin (HN) that is normally abundant in Mt, could simulate Mt-transfer effect on neuronal Mn-SOD expression, oxidative stress, and neuroplasticity under ICH-like injury. This study demonstrates that adoptive astrocytic Mt transfer enhances neuronal Mn-SOD-mediated anti-oxidative defense and neuroplasticity in the brain, which potentiate functional recovery following ICH.SIGNIFICANCE STATEMENTMitochondrial dysfunction and antioxidant defense play essential role in brain damage after intracerebral hemorrhage (ICH). Astrocytes release functional mitochondria (Mt) that enter adjacent cells to help brain homeostatic function. Here, we show that systemic transplantation of astrocytic Mt restores ICH-impaired neuronal anti-oxidative defense, enhances neurite outgrowth, and improves stroke recovery after ICH. Our study suggests that systemic transplantation of astrocytic Mt could be considered as a novel and potentially promising strategy for ICH treatment.

Determining the effect of aging, recovery time, and post-stroke memantine treatment on delayed thalamic gliosis after cortical infarct.

Secondary injury following cortical stroke includes delayed gliosis and eventual neuronal loss in the thalamus. However, the effects of aging and the potential to ameliorate this gliosis with NMDA receptor (NMDAR) antagonism are not established. We used the permanent distal middle cerebral artery stroke model (pdMCAO) to examine secondary thalamic injury in young and aged mice. At 3 days post-stroke (PSD3), slight microgliosis (IBA-1) and astrogliosis (GFAP) was evident in thalamus, but no infarct. Gliosis increased dramatically through PSD14, at which point degenerating neurons were detected. Flow cytometry demonstrated a significant increase in CD11b+/CD45int microglia (MG) in the ipsilateral thalamus at PSD14. CCR2-RFP reporter mouse further demonstrated that influx of peripheral monocytes contributed to the MG/Mϕ population. Aged mice demonstrated reduced microgliosis and astrogliosis compared with young mice. Interestingly, astrogliosis demonstrated glial scar-like characteristics at two years post-stroke, but not by 6 weeks. Lastly, treatment with memantine (NMDAR antagonist) at 4 and 24 h after stroke significantly reduced gliosis at PSD14. These findings expand our understanding of gliosis in the thalamus following cortical stroke and demonstrate age-dependency of this secondary injury. Additionally, these findings indicate that delayed treatment with memantine (an FDA approved drug) provides significant reduction in thalamic gliosis.

Aging lowers PEX5 levels in cortical neurons in male and female mouse brains.

Peroxisomes exist in nearly every cell, oxidizing fats, synthesizing lipids and maintaining redox balance. As the brain ages, multiple pathways are negatively affected, but it is currently unknown if peroxisomal proteins are affected by aging in the brain. While recent studies have investigated a PEX5 homolog in aging C. elegans models and found that it is reduced in aging, it is unclear if PEX5, a mammalian peroxisomal protein that plays a role in peroxisomal homeostasis and degradation, is affected in the aging brain. To answer this question, we first determined the amount of PEX5, in brain homogenates from young (3 months) and aged (26 through 32+ months of age) wild-type mice of both sexes. PEX5 protein was decreased in aged male brains, but this reduction was not significant in female brains. RNAScope and real-time qPCR analyses showed that Pex5 mRNA was also reduced in aged male brain cortices, but not in females. Immunohistochemistry assays of cortical neurons in young and aged male brains showed that the amount of neuronal PEX5 was reduced in aged male brains. Cortical neurons in aged female mice also had reduced PEX5 levels in comparison to younger female mice. In conclusion, total PEX5 levels and Pex5 gene expression both decrease with age in male brains, and neuronal PEX5 levels lower in an age-dependent manner in the cortices of animals of both sexes.

Inhibition of Calcium/Calmodulin-Dependent Protein Kinase Kinase ß Is Detrimental in Hypoxia⁻Ischemia Neonatal Brain Injury.

Neonatal hypoxia-ischemia (HI) is a major cause of death and disability in neonates. HI leads to a dramatic rise in intracellular calcium levels, which was originally thought to be detrimental to the brain. However, it has been increasingly recognized that this calcium signaling may also play an important protective role after injury by triggering endogenous neuroprotective pathways. Calcium/calmodulin-dependent protein kinase kinase ß (CaMKK ß) is a major kinase activated by elevated levels of intracellular calcium. Here we evaluated the functional role of CaMKK ß in neonatal mice after HI in both acute and chronic survival experiments. Postnatal day ten wild-type (WT) and CaMKK ß knockout (KO) mouse male pups were subjected to unilateral carotid artery ligation, followed by 40 min of hypoxia (10% O2 in N2). STO-609, a CaMKK inhibitor, was administered intraperitoneally to WT mice at 5 minutes after HI. TTC (2,3,5-triphenyltetrazolium chloride monohydrate) staining was used to assess infarct volume 24 h after HI. CaMKK ß KO mice had larger infarct volume than WT mice and STO-609 increased the infarct volume in WT mice after HI. In chronic survival experiments, WT mice treated with STO-609 showed increased tissue loss in the ipsilateral hemisphere three weeks after HI. Furthermore, when compared with vehicle-treated mice, they showed poorer functional recovery during the three week survival period, as measured by the wire hang test and corner test. Loss of blood-brain barrier proteins, a reduction in survival protein (Bcl-2), and an increase in pro-apoptotic protein Bax were also seen after HI with CaMKK ß inhibition. In conclusion, inhibition of CaMKK ß exacerbated neonatal hypoxia-ischemia injury in mice. Our data suggests that enhancing CaMKK signaling could be a potential target for the treatment of hypoxic-ischemic brain injury.

Critical role of sphingosine-1-phosphate receptor-2 in the disruption of cerebrovascular integrity in experimental stroke.

The use and effectiveness of current stroke reperfusion therapies are limited by the complications of reperfusion injury, which include increased cerebrovascular permeability and haemorrhagic transformation. Sphingosine-1-phosphate (S1P) is emerging as a potent modulator of vascular integrity via its receptors (S1PR). By using genetic approaches and a S1PR2 antagonist (JTE013), here we show that S1PR2 plays a critical role in the induction of cerebrovascular permeability, development of intracerebral haemorrhage and neurovascular injury in experimental stroke. In addition, inhibition of S1PR2 results in decreased matrix metalloproteinase (MMP)-9 activity in vivo and lower gelatinase activity in cerebral microvessels. S1PR2 immunopositivity is detected only in the ischemic microvessels of wild-type mice and in the cerebrovascular endothelium of human brain autopsy samples. In vitro, S1PR2 potently regulates the responses of the brain endothelium to ischaemic and inflammatory injury. Therapeutic targeting of this novel pathway could have important translational relevance to stroke patients.

TrkB agonist antibody pretreatment enhances neuronal survival and long-term sensory motor function following hypoxic ischemic injury in neonatal rats.

Perinatal hypoxic ischemia (H-I) causes brain damage and long-term neurological impairments, leading to motor dysfunctions and cerebral palsy. Many studies have demonstrated that the TrkB-ERK1/2 signaling pathway plays a key role in mediating the protective effect of brain-derived neurotrophic factor (BDNF) following perinatal H-I brain injury in experimental animals. In the present study, we explored the neuroprotective effects of the TrkB-specific agonist monoclonal antibody 29D7 on H-I brain injury in neonatal rats. First, we found that intracerebroventricular (icv) administration of 29D7 in normal P7 rats markedly increased the levels of phosphorylated ERK1/2 and phosphorylated AKT in neurons up to 24 h. Second, P7 rats received icv administration of 29D7 and subjected to H-I injury induced by unilateral carotid artery ligation and exposure to hypoxia (8% oxygen). We found that 29D7, to a similar extent to BDNF, significantly inhibited activation of caspase-3, a biochemical hallmark of apoptosis, following H-I injury. Third, we found that this 29D7-mediated neuroprotective action persisted at least up to 5 weeks post-H-I injury as assessed by brain tissue loss, implicating long-term neurotrophic effects rather than an acute delay of cell death. Moreover, the long-term neuroprotective effect of 29D7 was tightly correlated with sensorimotor functional recovery as assessed by a tape-removal test, while 29D7 did not significantly improve rotarod performance. Taken together, these findings demonstrate that pretreatment with the TrkB-selective agonist 29D7 significantly increases neuronal survival and behavioral recovery following neonatal hypoxic-ischemic brain injury.

Critical role of sphingosine-1-phosphate receptor 2 (S1PR2) in acute vascular inflammation.

The endothelium, as the interface between blood and all tissues, plays a critical role in inflammation. Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid, highly abundant in plasma, that potently regulates endothelial responses through interaction with its receptors (S1PRs). Here, we studied the role of S1PR2 in the regulation of the proadhesion and proinflammatory phenotype of the endothelium. By using genetic approaches and a S1PR2-specific antagonist (JTE013), we found that S1PR2 plays a key role in the permeability and inflammatory responses of the vascular endothelium during endotoxemia. Experiments with bone marrow chimeras (S1pr2(+/+) → S1pr2(+/+), S1pr2(+/+) → S1pr2(-/-), and S1pr2(-/-) → S1pr2(+/+)) indicate the critical role of S1PR2 in the stromal compartment, in the regulation of vascular permeability and vascular inflammation. In vitro, JTE013 potently inhibited tumor necrosis factor α-induced endothelial inflammation. Finally, we provide detailed mechanisms on the downstream signaling of S1PR2 in vascular inflammation that include the activation of the stress-activated protein kinase pathway that, together with the Rho-kinase nuclear factor kappa B pathway (NF-kB), are required for S1PR2-mediated endothelial inflammatory responses. Taken together, our data indicate that S1PR2 is a key regulator of the proinflammatory phenotype of the endothelium and identify S1PR2 as a novel therapeutic target for vascular disorders.

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.

Minocycline-preconditioned neural stem cells enhance neuroprotection after ischemic stroke in rats.

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 upregulation 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 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.

Induction of thioredoxin-interacting protein is mediated by oxidative stress, calcium, and glucose after brain injury in mice.

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.

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 CK2ß 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.

Neuroprotection by interleukin-6 is mediated by signal transducer and activator of transcription 3 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). METHODS: IL-6 was injected in mice before and after middle cerebral artery occlusion. Coimmunoprecipitation assays were performed for analysis of an 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. RESULTS: IL-6R was disrupted through a disassembly between IL-6Rα and gp130 associated by protein oxidation after reperfusion after tFCI. This suppressed phosphorylation of signal transducer and activator of transcription 3 (STAT3) and finally induced neuronal cell death through a decrease in manganese-superoxide dismutase. 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 manganese-superoxide dismutase promoter through recovery of the recruitment of STAT3 to the manganese-superoxide dismutase promoter and reduced infarct volume after tFCI. CONCLUSIONS: This study demonstrates that IL-6 has a neuroprotective effect against cerebral ischemic injury through IL-6R-mediated STAT3 activation and manganese-superoxide dismutase expression.

NADPH oxidase is involved in post-ischemic brain inflammation.

Nicotinamide adenine dinucleotide phosphate oxidase (NOX) is widely expressed in brain tissue including neurons, glia, and endothelia in neurovascular units. It is a major source of oxidants in the post-ischemic brain and significantly contributes to ischemic brain damage. Inflammation occurs after brain ischemia and is known to be associated with post-ischemic oxidative stress. Post-ischemic inflammation also causes progressive brain injury. In this study we investigated the role of NOX2 in post-ischemic cerebral inflammation using a transient middle cerebral artery occlusion model in mice. We demonstrate that mice with NOX2 subunit gp91(phox) knockout (gp91 KO) showed 35-44% less brain infarction at 1 and 3 days of reperfusion compared with wild-type (WT) mice. Minocycline further reduced brain damage in the gp91 KO mice at 3 days of reperfusion. The gp91 KO mice exhibited less severe post-ischemic inflammation in the brain, as evidenced by reduced microglial activation and decreased upregulation of inflammation mediators, including interleukin-1ß (IL-1ß), tumor necrosis factor-α, inducible nitric oxide synthases, CC-chemokine ligand 2, and CC-chemokine ligand 3. Finally, we demonstrated that an intraventricular injection of IL-1ß enhanced ischemia- and reperfusion-mediated brain damage in the WT mice (double the infarction volume), whereas, it failed to aggravate brain infarction in the gp91 KO mice. Taken together, these results demonstrate the involvement of NOX2 in post-ischemic neuroinflammation and that NOX2 inhibition provides neuroprotection against inflammatory cytokine-mediated brain damage.

Oxidative stress in ischemic brain damage: mechanisms of cell death and potential molecular targets for neuroprotection.

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.

NADPH oxidase mediates striatal neuronal injury after transient global cerebral ischemia.

Medium spiny neurons (MSNs) constitute most of the striatal neurons and are known to be vulnerable to ischemia; however, the mechanisms of the vulnerability remain unclear. Activated forms of nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase (NOX), which require interaction between cytosolic and membrane-bound subunits, are among the major sources of superoxide in the central nervous system. Although increasing evidence suggests that NOX has important roles in neurodegenerative diseases, its roles in MSN injury after transient global cerebral ischemia (tGCI) have not been elucidated. To clarify this issue, C57BL/6 mice were subjected to tGCI by bilateral common carotid artery occlusion for 22 minutes. Western blot analysis revealed upregulation of NOX subunits and recruitment of cytosolic subunits to the cell membrane at early (3 to 6 hours) and late (72 hours) phases after tGCI. Taken together with immunofluorescent studies, this activation arose in MSNs and endothelial cells at the early phase, and in reactive microglia at the late phase. Pharmacological and genetic inhibition of NOX attenuated oxidative injury, microglial activation, and MSN death after tGCI. These findings suggest that NOX has pivotal roles in MSN injury after tGCI and could be a therapeutic target for brain ischemia.

Oxidative stress increases phosphorylation of IkappaB kinase-alpha by enhancing NF-kappaB-inducing kinase after transient focal cerebral ischemia.

The IkappaB kinase (IKK) complex is a central component in the classic activation of the nuclear factor-kappaB (NF-kappaB) 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 IKKalpha (pIKKalpha) were observed 1 h after 30 mins of tFCI in mice. In copper/zinc-superoxide dismutase knockout mice, levels of NF-kappaB-inducing kinase (NIK) (an upstream kinase of IKKalpha), pIKKalpha, 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 pIKKalpha in mouse brain endothelial cells after tFCI. Nuclear factor-kappaB-inducing kinase was increased, and it enhanced pH3 by inducing pIKKalpha after oxygen-glucose deprivation (OGD) in mouse brain endothelial cells. Both NIK and pH3 interactions with IKKalpha were confirmed by coimmunoprecipitation. Treatment with IKKalpha small interfering RNA significantly reduced cell death after OGD. These results suggest that augmentation of NIK, IKKalpha, and pH3 in response to oxidative stress is involved in cell death after cerebral ischemia (or stroke).

Reperfusion and neurovascular dysfunction in stroke: from basic mechanisms to potential strategies for neuroprotection.

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.