Seok Joon Won

NADPH oxidase is the primary source of superoxide induced by NMDA receptor activation

Neuronal NMDA receptor (NMDAR) activation leads to the formation of superoxide, which normally acts in cell signaling. With extensive NMDAR activation, the resulting superoxide production leads to neuronal death. It is widely held that NMDA-induced superoxide production originates from the mitochondria, but definitive evidence for this is lacking. We evaluated the role of the cytoplasmic enzyme NADPH oxidase in NMDA-induced superoxide production. Neurons in culture and in mouse hippocampus responded to NMDA with a rapid increase in superoxide production, followed by neuronal death. These events were blocked by the NADPH oxidase inhibitor apocynin and in neurons lacking the p47phox subunit, which is required for NADPH oxidase assembly. Superoxide production was also blocked by inhibiting the hexose monophosphate shunt, which regenerates the NADPH substrate, and by inhibiting protein kinase C zeta, which activates the NADPH oxidase complex. These findings identify NADPH oxidase as the primary source of NMDA-induced superoxide production.

Chronic Treatment With Minocycline Preserves Adult New Neurons and Reduces Functional Impairment After Focal Cerebral Ischemia

Background and Purpose— Evidence suggests that activated microglia are detrimental to the survival of new hippocampal neurons, whereas blocking inflammation has been shown to restore hippocampal neurogenesis after cranial irradiation and seizure. The aim of this current study is to determine the effect of minocycline on neurogenesis and functional recovery after cerebral focal ischemia. Methods— Four days after temporary middle cerebral artery occlusion, minocycline was administered intraperitoneally for 4 weeks. BrdU was given on days 4 to 7 after middle cerebral artery occlusion to track cell proliferation. The number of remaining new neurons and activated microglia were quantified in the dentate gyrus. Infarct volume was measured to assess the treatment effect of minocycline. Motor and cognitive functions were evaluated 6 weeks after middle cerebral artery occlusion. Results— Minocycline delivered 4 days after middle cerebral artery occlusion for 4 weeks did not result in reduction in infarct size but significantly decreased the number of activated microglia in the dentate gyrus. Minocycline also significantly increased the number of newborn neurons that coexpressing BrdU and NeuN without significantly affecting progenitor cell proliferation in the dentate gyrus. Lastly, minocycline significantly improved motor coordination on the rotor rod, reduced the preferential use of the unaffected limb during exploration, reduced the frequency of footfalls in the affected limb when traversing on a horizontal ladder, and improved spatial learning and memory in the water maze test. Conclusions— Minocycline reduces functional impairment caused by cerebral focal ischemia. The improved function is associated with enhanced neurogenesis and reduced microglia activation in the dentate gyrus and possibly improved neural environment after chronic treatment with minocycline.

Ectopic expression of the catalytic subunit of telomerase protects against brain injury resulting from ischemia and NMDA-induced neurotoxicity.

The catalytic subunit of telomerase reverse transcriptase (TERT) protects dividing cells from replicative senescence in vitro. Here, we show that expression of TERT mRNA is induced in the ipsilateral cortical neurons after occlusion of the middle cerebral artery in adult mice. Transgenic mice that overexpress TERT showed significant resistance to ischemic brain injury. Among excitotoxicity, oxidative stress, and apoptosis comprising of routes of ischemic neuronal death, NMDA receptor-mediated excitotoxicity was reduced in forebrain cell cultures overexpressing TERT. NMDA-induced accumulation of cytosolic free Ca2+ ([Ca2+]c) was reduced in forebrain neurons from TERT transgenic mice, which was attributable to the rapid flow of [Ca2+]c into the mitochondria from the cytosol without change in Ca2+ influx and efflux through the plasma membrane. The present study provides evidence that TERT is inducible in postmitotic neurons after ischemic brain injury and prevents NMDA neurotoxicity through shift of the cytosolic free Ca2+ into the mitochondria, and thus plays a protective role in ameliorating ischemic neuronal cell death.

Decreased brain zinc availability reduces hippocampal neurogenesis in mice and rats

In the adult brain, neurogenesis occurs in the subgranular zone of the dentate gyrus (DG), where high levels of vesicular zinc are localized in the presynaptic terminals. To determine whether zinc has a role in modulating hippocampal neurogenesis under normal or pathologic conditions, we manipulated the level of vesicular zinc experimentally. To reduce hippocampal vesicular zinc, rats were either fed a zinc-deficient diet or treated with a zinc chelator, clioquinol (CQ). The number of progenitor cells and immature neurons was decreased significantly in the DG after 6 weeks of dietary zinc deprivation. Conversely, the number of progenitor cells and immature neurons was restored after a 2-week reversal to a normal zinc-containing diet. Similarly, a 1-week treatment with the zinc chelator, CQ, reduced the number of progenitor cells. The results of our previous study showed that hypoglycemia increased hippocampal neurogenesis. This study shows that zinc chelation reduced hypoglycemia-induced progenitor cell proliferation and neurogenesis. Finally, the role of vesicular zinc on neurogenesis was further assessed in zinc transporter 3 (ZnT3) gene deleted mice. Zinc transporter 3 knockout (KO) mice had significantly fewer proliferating progenitor cells and immature neurons after hypoglycemia. Our data provide converging evidence in support of the essential role zinc has in modulating hippocampal neurogenesis.

Brain-derived neurotrophic factor can act as a pronecrotic factor through transcriptional and translational activation of NADPH oxidase.

Several lines of evidence suggest that neurotrophins (NTs) potentiate or cause neuronal injury under various pathological conditions. Since NTs enhance survival and differentiation of cultured neurons in serum or defined media containing antioxidants, we set out experiments to delineate the patterns and underlying mechanisms of brain-derived neurotrophic factor (BDNF)–induced neuronal injury in mixed cortical cell cultures containing glia and neurons in serum-free media without antioxidants, where the three major routes of neuronal cell death, oxidative stress, excitotoxicity, and apoptosis, have been extensively studied. Rat cortical cell cultures, after prolonged exposure to NTs, underwent widespread neuronal necrosis. BDNF-induced neuronal necrosis was accompanied by reactive oxygen species (ROS) production and was dependent on the macromolecular synthesis. cDNA microarray analysis revealed that BDNF increased the expression of cytochrome b558, the plasma membrane-spanning subunit of NADPH oxidase. The expression and activation of NADPH oxidase were increased after exposure to BDNF. The selective inhibitors of NADPH oxidase prevented BDNF-induced ROS production and neuronal death without blocking antiapoptosis action of BDNF. The present study suggests that BDNF-induced expression and activation of NADPH oxidase cause oxidative neuronal necrosis and that the neurotrophic effects of NTs can be maximized under blockade of the pronecrotic action.

Hyperglycemia promotes tissue plasminogen activator-induced hemorrhage by Increasing superoxide production†‡

Risk of intracerebral hemorrhage is the primary factor limiting use of tissue plasminogen activator (tPA) for stroke. Clinical studies have established an association between admission hyperglycemia and the risk of hemorrhage with tPA use, independent of prior diabetes. Here we used an animal model of tPA‐induced reperfusion hemorrhage to determine if this clinical association reflects a true causal relationship.

Microglial activation induced by brain trauma is suppressed by post-injury treatment with a PARP inhibitor.

BackgroundTraumatic brain injury (TBI) induces activation of microglia. Activated microglia can in turn increase secondary injury and impair recovery. This innate immune response requires hours to days to become fully manifest, thus providing a clinically relevant window of opportunity for therapeutic intervention. Microglial activation is regulated in part by poly(ADP-ribose) polymerase-1 (PARP-1). Inhibition of PARP-1 activity suppresses NF-kB-dependent gene transcription and thereby blocks several aspects of microglial activation. Here we evaluated the efficacy of a PARP inhibitor, INO-1001, in suppressing microglial activation after cortical impact in the rat.MethodsRats were subjected to controlled cortical impact and subsequently treated with 10 mg/kg of INO-1001 (or vehicle alone) beginning 20 - 24 hours after the TBI. Brains were harvested at several time points for histological evaluation of inflammation and neuronal survival, using markers for microglial activation (morphology and CD11b expression), astrocyte activation (GFAP), and neuronal survival (NeuN). Rats were also evaluated at 8 weeks after TBI using measures of forelimb dexterity: the sticky tape test, cylinder test, and vermicelli test.ResultsPeak microglial and astrocyte activation was observed 5 to 7 days after this injury. INO-1001 significantly reduced microglial activation in the peri-lesion cortex and ipsilateral hippocampus. No rebound inflammation was observed in rats that were treated with INO-1001 or vehicle for 12 days followed by 4 days without drug. The reduced inflammation was associated with increased neuronal survival in the peri-lesion cortex and improved performance on tests of forelimb dexterity conducted 8 weeks after TBI.ConclusionsTreatment with a PARP inhibitor for 12 days after TBI, with the first dose given as long as 20 hours after injury, can reduce inflammation and improve histological and functional outcomes.

Nuclear Factor Erythroid 2-Related Factor 2 Facilitates Neuronal Glutathione Synthesis by Upregulating Neuronal Excitatory Amino Acid Transporter 3 Expression

Astrocytes support neuronal antioxidant capacity by releasing glutathione, which is cleaved to cysteine in brain extracellular space. Free cysteine is then taken up by neurons through excitatory amino acid transporter 3 [EAAT3; also termed Slc1a1 (solute carrier family 1 member 1)] to support de novo glutathione synthesis. Activation of the nuclear factor erythroid 2-related factor 2 (Nrf2)-antioxidant responsive element (ARE) pathway by oxidative stress promotes astrocyte release of glutathione, but it remains unknown how this release is coupled to neuronal glutathione synthesis. Here we evaluated transcriptional regulation of the neuronal cysteine transporter EAAT3 by the Nrf2-ARE pathway. Nrf2 activators and Nrf2 overexpression both produced EAAT3 transcriptional activation in C6 cells. A conserved ARE-related sequence was found in the EAAT3 promoter of several mammalian species. This ARE-related sequence was bound by Nrf2 in mouse neurons in vivo as observed by chromatin immunoprecipitation. Chemical activation of the Nrf2-ARE pathway in mouse brain increased both neuronal EAAT3 levels and neuronal glutathione content, and these effects were abrogated in mice genetically deficient in either Nrf2 or EAAT3. Selective overexpression of Nrf2 in brain neurons by lentiviral gene transfer was sufficient to upregulate both neuronal EAAT3 protein and glutathione content. These findings identify a mechanism whereby Nrf2 activation can coordinate astrocyte glutathione release with neuronal glutathione synthesis through transcriptional upregulation of neuronal EAAT3 expression.

Sequential release of nitric oxide, zinc, and superoxide in hypoglycemic neuronal death

Oxidative stress and zinc release are both known to contribute to neuronal death after hypoglycemia; however, the cause—effect relationships between these events are not established. Here we found, using a rat model of profound hypoglycemia, that the neuronal zinc release and translocation that occur immediately after hypoglycemia are prevented by the nitric oxide synthase inhibitor 7-nitroindazole but not by overexpression of superoxide dismutase-1 (SOD-1). However, overexpression of SOD-1 prevented activation of poly(ADP-ribose) polymerase-1 (PARP-1) and neuronal death, suggesting that zinc release is upstream of superoxide production. Accordingly, zinc-induced superoxide production was blocked in neuronal cultures by the NADPH oxidase inhibitor apocynin and by genetic deficiency in the p47phox subunit of NADPH oxidase. A key role for the vesicular zinc pool in this process was suggested by reduced superoxide formation and neuronal death in mice deficient in zinc transporter 3. Together, these findings suggest a series of events in which nitric oxide production triggers vesicular zinc release, which in turn activates NADPH oxidase and PARP-1. This sequence may also occur in other central nervous system disorders in which zinc, nitric oxide, and oxidative stress have been linked.

Poly(ADP-ribose)polymerase-1 modulates microglial responses to amyloid β

BackgroundAmyloid β (Aβ) accumulates in Alzheimers disease (AD) brain. Microglial activation also occurs in AD, and this inflammatory response may contribute to disease progression. Microglial activation can be induced by Aβ, but the mechanisms by which this occurs have not been defined. The nuclear enzyme poly(ADP-ribose) polymerase-1 (PARP-1) regulates microglial activation in response to several stimuli through its interactions with the transcription factor, NF-κB. The purpose of this study was to evaluate whether PARP-1 activation is involved in Aβ-induced microglial activation, and whether PARP-1 inhibition can modify microglial responses to Aβ.MethodshAPPJ20 mice, which accumulate Aβ with ageing, were crossed with PARP-1-/- mice to assess the effects of PARP-1 depletion on microglial activation, hippocampal synaptic integrity, and cognitive function. Aβ peptide was also injected into brain of wt and PARP-1-/- mice to directly determine the effects of PARP-1 on Aβ-induced microglial activation. The effect of PARP-1 on Aβ-induced microglial cytokine production and neurotoxicity was evaluated in primary microglia cultures and in microglia-neuron co-cultures, utilizing PARP-1-/- cells and a PARP-1 inhibitor. NF-κB activation was evaluated in microglia infected with a lentivirus reporter gene.ResultsThe hAPPJ20 mice developed microglial activation, reduced hippocampal CA1 calbindin expression, and impaired novel object recognition by age 6 months. All of these features were attenuated in hAPPJ20/PARP-1-/- mice. Similarly, Aβ1-42 injected into mouse brain produced a robust microglial response in wild-type mice, and this was blocked in mice lacking PARP-1 expression or activity. Studies using microglial cultures showed that PARP-1 activity was required for Aβ-induced NF-κB activation, morphological transformation, NO release, TNFα release, and neurotoxicity. Conversely, PARP-1 inhibition increased release of the neurotrophic factors TGFβ and VEGF, and did not impair microglial phagocytosis of Aβ peptide.ConclusionsThese results identify PARP-1 as a requisite and previously unrecognized factor in Aβ-induced microglial activation, and suggest that the effects of PARP-1 are mediated, at least in part, by its interactions with NF-κB. The suppression of Aβ-induced microglial activation and neurotoxicity by PARP-1 inhibition suggests this approach could be useful in AD and other disorders in which microglial neurotoxicity may contribute.