GSK-3β as a target for protection against transient cerebral ischemia

Wei Wang, Mingchang Li, Yuefei Wang, Zhongyu Wang, Wei Zhang, Fangxia Guan, Qianxue Chen, Jian Wang, Wei Wang, Mingchang Li, Yuefei Wang, Zhongyu Wang, Wei Zhang, Fangxia Guan, Qianxue Chen, Jian Wang

Abstract

Stroke remains the leading cause of death and disability worldwide. This fact highlights the need to search for potential drug targets that can reduce stroke-related brain damage. We showed recently that a glycogen synthase kinase-3β (GSK-3β) inhibitor attenuates tissue plasminogen activator-induced hemorrhagic transformation after permanent focal cerebral ischemia. Here, we examined whether GSK-3β inhibition mitigates early ischemia-reperfusion stroke injury and investigated its potential mechanism of action. We used the rat middle cerebral artery occlusion (MCAO) model to mimic transient cerebral ischemia. At 3.5 h after MCAO, cerebral blood flow was restored, and rats were administered DMSO (vehicle, 1% in saline) or GSK-3β inhibitor TWS119 (30 mg/kg) by intraperitoneal injection. Animals were sacrificed 24 h after MCAO. TWS119 treatment reduced neurologic deficits, brain edema, infarct volume, and blood-brain barrier permeability compared with those in the vehicle group. TWS119 treatment also increased the protein expression of β-catenin and zonula occludens-1 but decreased β-catenin phosphorylation while suppressing the expression of GSK-3β. These results indicate that GSK-3β inhibition protects the blood-brain barrier and attenuates early ischemia-reperfusion stroke injury. This protection may be related to early activation of the Wnt/β-catenin signaling pathway.

Keywords: TWS119; Wnt/β-catenin signaling; blood-brain barrier; ischemic stroke.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interest exists.

Figures

Figure 1
Figure 1
A. Neurologic deficit score at 24 h after transient middle cerebral artery occlusion (MCAO). Rats were treated with vehicle or TWS119 (n=24 rats/group). The data are expressed as means ± SD. *P<0.05 vs. vehicle group. B. Brain water content at 24 h after transient middle cerebral artery occlusion (MCAO). The data are expressed as means ± SD. *P<0.05, **P<0.01 vs. sham group.
Figure 2
Figure 2
Infarct volume at 24 h after transient middle cerebral artery occlusion (MCAO). Rats were treated with vehicle or TWS119 (n=6 rats/group). A. Representative TTC-stained, 2-mm coronal brain sections. B. Quantification of the infarct volume in groups that underwent MCAO and treatment with vehicle or TWS119. Infarct volume was calculated as a percentage as follows: infarct area of the ipsilateral hemisphere/total area of the ipsilateral hemisphere × 100%. No infarction was detected in the sham group. The results are expressed as means ± SD. *P<0.05 vs. vehicle group.
Figure 3
Figure 3
Blood-brain barrier permeability as measured by Evans blue leakage at 24 h after transient middle cerebral artery occlusion (MCAO). Rats were treated with vehicle or TWS119 (n=6 rats/group). The Evans blue index (ratio of absorbance intensity in the ischemic hemisphere to that in the nonischemic hemisphere) was used to evaluate blood-brain barrier permeability. The results are expressed as means ± SD. *P<0.05, **P<0.01 vs. sham group.
Figure 4
Figure 4
Western blot analysis of proteins in the Wnt/β-catenin signaling pathway and tight junctions of the blood-brain barrier 24 h after transient MCAO (n=6 rats/group). Representative Western blots and densitometric analysis of (A) total β-catenin, (B) phosphorylated β-catenin, (C) The ratio of p-β-catenin to the total β-catenin, (D) GSK-3β and (E) zonula accludens-1 (ZO-1). GAPDH was used as a loading control. The expression of β-catenin and ZO-1 increased in the TWS119-treated group compared to that in the vehicle-treated group. TWS119 treatment reduced the expression of phosphorylated β-catenin and GSK-3β. Data are representative of at least three independent experiments. The results are expressed as means ± SD.

References

    1. Takeuchi S, Nagatani K, Otani N, Nawashiro H, Sugawara T, Wada K. et al. Hydrogen improves neurological function through attenuation of blood-brain barrier disruption in spontaneously hypertensive stroke-prone rats. BMC Neurosci. 2015;16:22.
    1. Reeson P, Tennant KA, Gerrow K, Wang J, Weiser Novak S, Thompson K. et al. Delayed inhibition of VEGF signaling after stroke attenuates blood-brain barrier breakdown and improves functional recovery in a comorbidity-dependent manner. The Journal of neuroscience: the official journal of the Society for Neuroscience. 2015;35:5128–43.
    1. Fang W, Sha L, Kodithuwakku ND, Wei J, Zhang R, Han D. et al. Attenuated Blood-Brain Barrier Dysfunction by XQ-1H Following Ischemic Stroke in Hyperlipidemic Rats. Molecular neurobiology. 2015;52:162–75.
    1. Weiner GM, Ducruet AF. CEACAM1: a novel adhesion molecule that regulates the secretion of matrix metalloproteinase-9 in neutrophils and protects the blood-brain barrier after ischemic stroke. Neurosurgery. 2014;75:N21–2.
    1. Wu L, Jiang Y, Zhu J, Wen Z, Xu X, Xu X. et al. Orosomucoid1: Involved in vascular endothelial growth factor-induced blood-brain barrier leakage after ischemic stroke in mouse. Brain research bulletin. 2014;109:88–98.
    1. Rabiei Z, Rafieian-Kopaei M. Neuroprotective effect of pretreatment with Lavandula officinalis ethanolic extract on blood-brain barrier permeability in a rat stroke model. Asian Pacific journal of tropical medicine. 2014;7:S421–6.
    1. Liebner S, Corada M, Bangsow T, Babbage J, Taddei A, Czupalla CJ. et al. Wnt/beta-catenin signaling controls development of the blood-brain barrier. The Journal of cell biology. 2008;183:409–17.
    1. Liu L, Wan W, Xia S, Kalionis B, Li Y. Dysfunctional Wnt/beta-catenin signaling contributes to blood-brain barrier breakdown in Alzheimer's disease. Neurochemistry international. 2014;75:19–25.
    1. Shruster A, Ben-Zur T, Melamed E, Offen D. Wnt signaling enhances neurogenesis and improves neurological function after focal ischemic injury. PloS one. 2012;7:e40843.
    1. Ding S, Wu TY, Brinker A, Peters EC, Hur W, Gray NS. et al. Synthetic small molecules that control stem cell fate. Proceedings of the National Academy of Sciences of the United States of America. 2003;100:7632–7.
    1. Chen Y, Zheng L, Liu J, Zhou Z, Lv X, Zhang J. et al. [Activation of Wnt/beta-catenin pathway in NK cells by glycogen synthase kinase-3beta inhibitor TWS119 promotes the expression of CD62L] Xi bao yu fen zi mian yi xue za zhi = Chinese journal of cellular and molecular immunology. 2015;31:44–8.
    1. Wang W, Li M, Chen Q, Wang J. Hemorrhagic Transformation after Tissue Plasminogen Activator Reperfusion Therapy for Ischemic Stroke: Mechanisms, Models, and Biomarkers. Molecular neurobiology. 2015;52:1572–9.
    1. Wang W, Li M, Wang Y, Li Q, Deng G, Wan J. et al. GSK-3beta inhibitor TWS119 attenuates rtPA-induced hemorrhagic transformation and activates the Wnt/beta-catenin signaling pathway after acute ischemic stroke in rats. Molecular neurobiology. 2016;53:7028–36.
    1. Zan L, Zhang X, Xi Y, Wu H, Song Y, Teng G. et al. Src regulates angiogenic factors and vascular permeability after focal cerebral ischemia-reperfusion. Neuroscience. 2014;262:118–28.
    1. Li Y, Xu XL, Zhao D, Pan LN, Huang CW, Guo LJ. et al. TLR3 ligand Poly IC Attenuates Reactive Astrogliosis and Improves Recovery of Rats after Focal Cerebral Ischemia. CNS neuroscience & therapeutics. 2015;21:905–13.
    1. Wang M, Hong X, Chang CF, Li Q, Ma B, Zhang H, Simultaneous detection and separation of hyperacute intracerebral hemorrhage and cerebral ischemia using amide proton transfer MRI. Magnetic resonance in medicine; 2015.
    1. Gattinoni L, Zhong XS, Palmer DC, Ji Y, Hinrichs CS, Yu Z. et al. Wnt signaling arrests effector T cell differentiation and generates CD8+ memory stem cells. Nat Med. 2009;15:808–13.
    1. Wang W, Li M, Wang Y, Li Q, Deng G, Wan J, GSK-3beta inhibitor TWS119 attenuates rtPA-induced hemorrhagic transformation and activates the Wnt/beta-catenin signaling pathway after acute ischemic stroke in rats. Molecular neurobiology; 2015.
    1. Wang J, Yu L, Jiang C, Chen M, Ou C, Wang J. Bone marrow mononuclear cells exert long-term neuroprotection in a rat model of ischemic stroke by promoting arteriogenesis and angiogenesis. Brain, behavior, and immunity. 2013;34:56–66.
    1. Zan L, Wu H, Jiang J, Zhao S, Song Y, Teng G. et al. Temporal profile of Src, SSeCKS, and angiogenic factors after focal cerebral ischemia: correlations with angiogenesis and cerebral edema. Neurochemistry international. 2011;58:872–9.
    1. Zhao X, Wu T, Chang CF, Wu H, Han X, Li Q. et al. Toxic role of prostaglandin E2 receptor EP1 after intracerebral hemorrhage in mice. Brain, behavior, and immunity. 2015;46:293–310.
    1. Wang J, Liu X, Lu H, Jiang C, Cui X, Yu L. et al. CXCR4(+)CD45(-) BMMNC subpopulation is superior to unfractionated BMMNCs for protection after ischemic stroke in mice. Brain, behavior, and immunity. 2015;45:98–108.
    1. Zhang W, Davis CM, Edin ML, Lee CR, Zeldin DC, Alkayed NJ. Role of endothelial soluble epoxide hydrolase in cerebrovascular function and ischemic injury. PloS one. 2013;8:e61244.
    1. Wang J, Yu L, Jiang C, Fu X, Liu X, Wang M. et al. Cerebral ischemia increases bone marrow CD4+CD25+FoxP3+ regulatory T cells in mice via signals from sympathetic nervous system. Brain, behavior, and immunity. 2015;43:172–83.
    1. Wu H, Wu T, Hua W, Dong X, Gao Y, Zhao X. et al. PGE2 receptor agonist misoprostol protects brain against intracerebral hemorrhage in mice. Neurobiology of aging. 2015;36:1439–50.
    1. Borlongan CV. The blood brain barrier in stroke. Curr Pharm Des. 2012;18:3613–4.
    1. Wang B, Tian S, Wang J, Han F, Zhao L, Wang R. et al. Intraperitoneal administration of thioredoxin decreases brain damage from ischemic stroke. Brain research. 2015;1615:89–97.
    1. Li TT, Fan ML, Hou SX, Li XY, Barry DM, Jin H. et al. A novel snake venom-derived GPIb antagonist, anfibatide, protects mice from acute experimental ischaemic stroke and reperfusion injury. British journal of pharmacology. 2015;172:3904–16.
    1. Kim EH, Tolhurst AT, Szeto HH, Cho SH. Targeting CD36-mediated inflammation reduces acute brain injury in transient, but not permanent, ischemic stroke. CNS neuroscience & therapeutics. 2015;21:385–91.
    1. Luo Y, Zhou Y, Xiao W, Liang Z, Dai J, Weng X. et al. Interleukin-33 ameliorates ischemic brain injury in experimental stroke through promoting Th2 response and suppressing Th17 response. Brain research. 2015;1597:86–94.
    1. Liang J, Wang P, Wei J, Bao C, Han D. Nicotinamide Mononucleotide Adenylyltransferase 1 Protects Neural Cells Against Ischemic Injury in Primary Cultured Neuronal Cells and Mouse Brain with Ischemic Stroke Through AMP-Activated Protein Kinase Activation. Neurochemical research. 2015;40:1102–10.
    1. Hacke W, Kaste M, Bluhmki E, Brozman M, Davalos A, Guidetti D. et al. Thrombolysis with alteplase 3 to 4.5 hours after acute ischemic stroke. The New England journal of medicine. 2008;359:1317–29.
    1. Ajmone-Cat MA, D'Urso MC, di Blasio G, Brignone MS, De Simone R, Minghetti L. Glycogen synthase kinase 3 is part of the molecular machinery regulating the adaptive response to LPS stimulation in microglial cells. Brain, behavior, and immunity. 2016;55:225–35.
    1. Lin CF, Tsai CC, Huang WC, Wang YC, Tseng PC, Tsai TT. et al. Glycogen Synthase Kinase-3beta and Caspase-2 Mediate Ceramide- and Etoposide-Induced Apoptosis by Regulating the Lysosomal-Mitochondrial Axis. PloS one. 2016;11:e0145460.
    1. Artus C, Glacial F, Ganeshamoorthy K, Ziegler N, Godet M, Guilbert T. et al. The Wnt/planar cell polarity signaling pathway contributes to the integrity of tight junctions in brain endothelial cells. J Cereb Blood Flow Metab. 2014;34:433–40.
    1. Reis M, Liebner S. Wnt signaling in the vasculature. Experimental cell research. 2013;319:1317–23.
    1. Polakis P. Formation of the blood-brain barrier: Wnt signaling seals the deal. The Journal of cell biology. 2008;183:371–3.
    1. Zhou J, Chen Y, Cao C, Chen X, Gao W, Zhang L. Inactivation of glycogen synthase kinase-3beta up-regulates beta-catenin and promotes chondrogenesis. Cell and tissue banking. 2015;16:135–42.
    1. Ji XK, Xie YK, Zhong JQ, Xu QG, Zeng QQ, Wang Y. et al. GSK-3beta suppresses the proliferation of rat hepatic oval cells through modulating Wnt/beta-catenin signaling pathway. Acta pharmacologica Sinica. 2015;36:334–42.
    1. Taurin S, Sandbo N, Qin Y, Browning D, Dulin NO. Phosphorylation of beta-catenin by cyclic AMP-dependent protein kinase. The Journal of biological chemistry. 2006;281:9971–6.
    1. Li Y, Zhu J, Liu Y, Chen X, Lei S, Li L. et al. Glycogen Synthase Kinase 3beta Influences Injury Following Cerebral Ischemia/Reperfusion in Rats. International journal of biological sciences. 2016;12:518–31.
    1. Ha TS, Park HY, Seong SB, Ahn HY. Puromycin aminonucleoside increases podocyte permeability by modulating ZO-1 in an oxidative stress-dependent manner. Experimental cell research. 2016;340:139–49.
    1. Tornavaca O, Chia M, Dufton N, Almagro LO, Conway DE, Randi AM. et al. ZO-1 controls endothelial adherens junctions, cell-cell tension, angiogenesis, and barrier formation. The Journal of cell biology. 2015;208:821–38.

Source: PubMed

Sponsoren und Mitarbeiter

Krankheiten

Drogeninterventionen

3
Abonnieren