PS1dE9 Double Transgenic-Mice

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Keun Young Hwang and Insug Kang1 1 Kyung Hee University, Korea Hypoxia induces an inflammatory activation of microglia during cerebral ischemia. The transcription factor of hypoxia-inducible genes hypoxia-inducible factor-1 (HIF-1) is known to be involved in inflammation and immune response. JAK/STAT pathway is shown to activated microglial cells from cytokines(IFN-Ȗ). But its action mechanism is not understood in hypoxic microglia. Thus, we examined Role of JAK2/STAT signaling in HIF-1 activation and inflammatory responses of hypoxia-stimulated microglial cells. As expected that Hypoxia increases HIF-1 and inflammatory response gene in microglial. Also, Hypoxia induced JAK2/STAT phosphorylation and activation in microglial cells.JAK2/STAT3 pathway stimulates HIF-1 and hypoxic gene expression in hypoxia-stimulated microglial cells. whereas, treatment of AG490(JAK2 inhibitor) significantly inhibited hypoxia induced the expression of HIF-1Į accumulation. Next, the involvement of JAK2/STAT3 pathway in HIF-Į activation was determined by cotransfection of dominant-negative mutants of JAK2 (DN-JAK2) and JAK2,STAT3 siRNA.We found that downregulation of JAK2/STAT3 decreased hypoxic gene expression in microgilal cells. JAK2/STAT3 regulate hypoxia-induced expression of hypoxia responsive genes, iNOS, COX-2, and VEGF. Taken together, these results suggest that JAK2/STAT pathway plays an impotant role in hypoxia-induced HIF-1activation as well as expression of iNOS in BV2 microglia. And ROS appear to act upstream of JAK2 and JAK2 cross-talks with PI 3-kinase/Akt pathway in hypoxic BV2 microglia, we suggest that JAK2/STAT pathway regulates HIF1 activation and and hypoxic gene expression via ROS and PI 3-kinase/Akt dependent pathways in hypoxia-stimulated microglial cells

doi:10.1016/j.freeradbiomed.2012.10.201

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Chung Ju , Sunyoung Hwang1, Geum-Sil Cho1, In-Young Choi1, Yongseok Choi1, and Won-Ki Kim1 1 Korea University, Republic of Korea In cerebral ischemia, free radicals are greatly produced during initial energy failure, post-ischemic reperfusion and inflammatory responses. Thus, pharmacological manipulation of oxidative stress has been eagerly pursued for the therapy of cerebral ischemic injury. In the present study, we investigated antiischemic effects of trans- and cis-hinokiresinols, naturally occurring phenylpropanoids, using in vitro as well as in vivo experimental models. ORAC and DPPH assays showed that two isomers had similar free radical scavenging activities. However, only trans-hinokiresinol significantly decreased neuronal injury in cultured cortical neurons exposed to oxygen-glucose deprivation (OGD, 75 min) followed by re-oxygenation (R, 9 h). The differential neuroprotective effect could be due to stereo-specific augmentation of Cu/Zn-SOD activity by trans-hinokiresinol, compared to cis-hinokiresinol. In rats subjected to transient middle cerebral artery occlusion (1.5 h) followed by 24-h reperfusion, post-ischemic treatment of both hinokiresinols (2 and

7 h after onset of ischemia) significantly reduced cerebral infarct. Interestingly, however, preventative anti-ischemic cytoprotection was obtained only by pre-ischemic treatment with transhinokiresinol, but not with cis-isomer. Nitrotyrosine immunoreactivity in ischemic brain regions was reduced only by trans-hinokiresinol, but not cis-hinokiresinol. As treated after onset of ischemia, however, both hinokiresinols suppressed neutrophil infiltration into ischemic lesions to a similar extent. These differential anti-oxidant but comparable anti-inflammatory activities may explain stereo-specific anti-ischemic effects and differences in therapeutic time windows of hinokiresinols. More detailed delineation of anti-ischemic mechanism of hinokiresinols may provide a better strategy to develop efficacious regimen for cerebral ischemic stroke.

doi:10.1016/j.freeradbiomed.2012.10.202

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Ige Joseph Kade , Vinicio Granados-Soto2, and Hector Isaac RochaGonzalez3 1 2 Federal University of Technology, Nigeria, CINVESTAV, 3 Mexico, Escu. Super. de Med. Inst. Politéc. Nac.. Mexico The purpose of this study was to obtain sesamin from oil seeds of Sesamum indicum and assess its profile as a drug with antinociceptive and antioxidant properties. The purity of the 11 13 sesamin was confirmed using HPLC, NMR and NMR and gas chromatography-mass spectrometry techniques. The antioxidant profile was determined in vitro by evaluating its free radical scavenging ability, reducing property and ability to prevent betacarotene bleaching whereas that antinociception was assessed in the formalin test. Our data showed that sesamin (0.03 – 30 mg/kg, p.o.) exhibit maximum antinociceptive effect at a dose of 3 mg/kg in rats. Pre-treatment with methiothepin (a non-selective 5HT receptor antagonist, 0.1 mg/kg, i.p.) abolished sesamininduced antinociception. In contrast, pre-treatment with 4-(2’methoxy-phenyl)-1-[2’-(n-2’’-pyridinyl)-p-iodobenzamino-]ethylpiperazine (a selective 5-HT1A receptor antagonist, 0.1 mg/kg, i.p.) did not abolish the antinociceptive effect of sesamin. Furthermore, sesamin did not scavenge free radicals, reduce iron (III) nor prevent beta-carotene bleaching in vitro. Taken together, these results suggest that sesamin-induced antinociception may be mediated in part by the activation of serotonergic receptors which definitely exclude the participation of 5-HT1A receptors. In addition, data suggest that the antinociceptive effect of sesamin is not due to its antioxidant properties. Acknowledgements: Partially supported by a TWAS-Conacyt fellowship.

doi:10.1016/j.freeradbiomed.2012.10.203

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Hyoung-Chun Kim , Zhengyi Li1, Thuy-Ty Lan Nguyen1, The-Vinh Tran1, Hoang-Lan Thi Le1, Jong-Seok Chae2, Jong-Ryul Ha2, and EunJoo Shin1 1 2 Kangwon National University, Korea, Yuyu Inc., Suwon, Korea

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EGb 761, a standardized Ginkgo biloba extract which contains flavonoids and terpene trilactones as two major constituents, possess free radical scavenging antioxidant activities and antiinflammatory properties by antagonizing platelet-activating factor receptor (PAFR). Moreover, EGb 761 has been shown to protect against ȕ-amyloid (Aȕ)-induced neurotoxicity. In the present study, we observed that YY 1224, a terpene strengthened Gingko biloba extract and EGb 761, a standard extract of Ginkgo biloba were prevented Aȕ (1-42)-induced learning impairments and increases in platelet activating factor receptor (PAF-R) as well as decreases in the expression of PAF-acetylhydrolase (AH) in mice. Aȕ (1-42)-induced reduction in muscarinic M1 acetylcholine receptor (M1 mAChR) gene expression was also attenuated by YY 1224 or EGb 761. In addition, YY-1224 or EGb 761 blocked Aȕ (1-42)-induced decreases in p-ERK 1/2. Dicyclomine, an M1 mAChR antagonist, reversed YY 1224- or EGb 761-mediated protective effects in response to Aȕ (1-42). Moreover, YY 1224 or EGb 761 significantly attenuated increases in oxidative burdens (as measured by lipid peroxidation, protein oxidation and reactive oxygen species) and in pro-inflammatory factors [as evaluated by the protein expression of the tumor necrosis factor (TNF)-Į, interleukin (IL)-1ȕ, IL-6, interferon (INF)-Ȗ and inducible nitric oxide synthase (iNOS)] induced by Aȕ (1-42). Consistently, repeated treatment with YY-1224 or EGb 761 significantly ameliorated the learning impairment and Aȕ plaque formation in the APPswe/PS1dE9 double-transgenic mice. The protective action of YY-1224 was consistently more effective than that of EGb 761 in response to Aȕ insult. Therefore, above signaling networks are important for neuroprotective potential mediated by YY-1224 [Supported by a grant (2012K001115) from brain research center of the 21 st Century Frontier Research Program, Ministry of Science and Technology, Korea. Z. Li, T-T. L. Nguyen, T.-V. Tran, and H.-L. T. Le are involved in BK 21 program, National Research Foundation of Korea].

doi:10.1016/j.freeradbiomed.2012.10.204

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Rui-Ming Liu , Hasina Akhter1, Carol Ballinger1, Thomas van Groen1, Inga Kadish1, Michelle Fanucchi1, and Edward M. Postlethwait1 1 University of Alabama at Birmingham Alzheimer’s Disease (AD), an aging-related neurodegenerative disease, is a major cause of dementia in the elderly. Although early-onset (familial) AD is attributed to gene mutations, the cause for late-onset (sporadic) AD, which accounts for 95% of AD cases, is unknown. In this study, we show that exposure of 6week old wild type (WT) C57BL/6 mice and APP/PS1 mice, a well-established animal model of AD, to a cyclic O3 exposure protocol, which mimics human exposure, impaired learning/memory function of APP/PS1 mice although it had no significant effect on WT mice. Furthermore, we show that O3 exposure induced neuronal cell apoptosis in male, but suppressed the expression of growth associated protein 43 and therefore potentially neuron growth in female, APP/PS1 mice although it had no significant effect on brain amyloid beta peptide accumulation in neither gender. Consistent with our previous observations in AD patients, male APP/PS1 mice had lower levels of antioxidants in their brain and, consequently, showed higher sensitivity to O3-induced oxidative stress than female mice. In vitro studies further show that 4-hydroxynonenal, a lipid peroxidation product that was increased in the plasma and brain of O3 exposed male APP/PS1 mice, induced neuroblastoma cell

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apoptosis. Together, our data suggest that O3 inhalation induces oxidative stress and alters gene expression in the brain and is a risk factor for the development of AD, especially in genetic predisposed populations. This study also reveals for the first time that different mechanisms may underlie cognitive function decline in men and women associated with O3 exposure.

doi:10.1016/j.freeradbiomed.2012.10.205

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Jan Lewerenz1 and Pamela Maher 1 2 University of Ulm,Germany, Salk Institute The cystine/glutamate antiporter system xc imports cystine into cells while exporting glutamate in a 1:1 ratio. Intracellularly, cystine is reduced to cysteine, which is the rate-limiting substrate for the antioxidant glutathione. As a consequence, system xc has been implicated in processes like cell proliferation, neuroprotection and brain tumor growth. In vitro, inhibition of system xc by glutamate induces oxidative glutamate toxicity, a form of cell death due to glutathione depletion. The specific subunit of system xc , xCT, is regulated by the transcription factors ATF4 and Nrf2. ATF4 translation is facilitated by phosphorylation of the translation initiation factor eIF2Į. Phosphoinositide 3kinases (PI3Ks), which act downstream of receptor tyrosine kinases, are also known to play an important role in neuroprotections and tumor cell proliferation. We show that in hippocampal HT22 cells inhibition of PI3Ks decreases eIF2Į phosphorylation and ATF4 expression. This effect is counteracted by inhibition of glycogen synthase kinase-3ȕ (GSK3ȕ), a kinase activated following PI3K inhibition. Furthermore, transient transfection of HT22 cells with a luciferase construct containing the ATF4 5’UTR results in an identical regulation of luciferase activity upon pharmacological manipulation of the PI3K pathway. Moreover, inhibition of PI3K down-regulates while inhibition of GSK3ȕ up-regulates system xc activity in HT22 cells and mouse embryonic fibroblasts (MEFs). In HT22 cells, this regulation is paralleled by increased and decreased sensitivity to oxidative glutamate toxicity, respectively, indicating that it has functional consequences. Importantly, we observed that PI3K inhibition down-regulated eIF2Į phosphorylation and ATF4 levels in some glioblastoma cell cultures derived from human tumor biopsies. In summary, we show that the PI3K/GSK3ȕ pathway regulates system xc activity via modulation of eIF2Į phosphorylation and subsequently ATF4 but not Nrf2 levels. Thus, this pathway mediates the resistance of neuronal cell lines against oxidative stress and may also be active in some glioblastomas, providing a novel therapeutic target.

doi:10.1016/j.freeradbiomed.2012.10.206

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Ken-ichiro Matsumoto , Megumi Ueno1, Ikuo Nakanishi1, Ken-ichi Yamada2, and Ichio Aoki1 1 2 National Institute of Radiological Sciences Japan, Kyushu University, Japan

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