Abstract
The objective of this study is to elucidate the effect of a new glycogen synthase kinase-3β (GSK-3β) inhibitor in RA differentiated SH-SY5Y cells in oxygen and glucose deprivation (OGD) model. The pathway involved in GSK-3β signaling during OGD was measured to elucidate the mechanism of action. The differentiation of SH-SY5Y into mature neuronal cells was done with retinoic acid. During differentiation, upregulation of the growth-associated protein 43 (GAP43), neurogenin1 (NGN1), neuronal differentiation 2 (NeuroD2), and tripartite motif containing 11 (TRIM11) genes were observed. Twelve hours of optimal OGD exposure resulted in the alteration of GSK-3β functions of the neuron cells. Of the five molecules selected for this study, molecule G3 showed better effect in the initial phase of the study. Hence, G3 (0.5, 1, and 5 μM) was selected for further study in the OGD model. The standard GSK-3β inhibitor, AR-A014418 (1 μM), was used for comparison. Molecules were pretreated (30 min) and cotreated during OGD exposure. GSK-3β inhibitors showed antiapoptotic activity as evidenced by reduced caspase-3 enzyme activity and increased survivin transcription, as well as improved membrane integrity, evidenced by LDH assay. The inhibitor molecules also up-regulated survival AKT1/GSK-3β/β-catenin pathway and stabilized β-catenin. Inhibition of GSK-3β maintained neuronal survival by upregulating GAP43, Ngn1, and NeuroD2 gene transcription. Further GSK-3β inhibition reduced the TRIM11 gene transcription. In conclusion, both inhibitors have been found to control apoptosis and maintain neuronal functioning and this effect might have been mediated through AKT1/GSK-3β/β-catenin–TRIM11/survivin pathway.
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Embi N, Rylatt DB, Cohen P (1980) Glycogen synthase kinase-3 from rabbit skeletal muscle. Separation from cyclic-AMP-dependent protein kinase and phosphorylase kinase. Eur J Biochem 107:519–527
Woodgett JR (1990) Molecular cloning and expression of glycogen synthase kinase-3/factor A. EMBO J 9:2431–2438
Perez-Costas E, Gandy JC, Melendez-Ferro M, Roberts RC, Bijur GN (2010) Light and electron microscopy study of glycogen synthase kinase-3beta in the mouse brain. PLoS One 27:e8911
Ferrer I, Barrachina M, Puig B (2002) Glycogen synthase kinase-3 is associated with neuronal and glial hyperphosphorylated tau deposits in Alzheimer's disease, Pick's disease, progressive supranuclear palsy and corticobasal degeneration. Acta Neuropathol 104:583–591
Pandey GN, Dwivedi Y, Rizavi HS, Teppen T, Gaszner GL, Roberts RC, Conley RR (2009) GSK-3beta gene expression in human postmortem brain: regional distribution, effects of age and suicide. Neurochem Res 34:274–285
Sasaki C, Hayashi T, Zhang WR, Warita H, Manabe Y, Sakai K, Abe K (2001) Different expression of glycogen synthase kinase-3beta between young and old rat brains after transient middle cerebral artery occlusion. Neurol Res 23:588–592
Bhat RV, Shanley J, Correll MP, Fieles WE, Keith RA, Scott CW, Lee CM (2000) Regulation and localization of tyrosine216 phosphorylation of glycogen synthase kinase-3beta in cellular and animal models of neuronal degeneration. Proc Natl Acad Sci U S A 97:11074–11090
Chung H, Seo S, Moon M, Park S (2008) Phosphatidylinositol-3-kinase/Akt/glycogen synthase kinase-3 beta and ERK1/2 pathways mediate protective effects of acylated and unacylated ghrelin against oxygen-glucose deprivation-induced apoptosis in primary rat cortical neuronal cells. J Endocrinol 198:511–521
Mihara M, Erster S, Zaika A, Petrenko O, Chittenden T, Pancoska P, Moll UM (2003) p53 has a direct apoptogenic role at the mitochondria. Mol Cell 11:577–590
Gupta S, Campbell D, Dérijard B, Davis RJ (1995) Transcription factor ATF2 regulation by the JNK signal transduction pathway. Science 267:389–393
Hayashi T, Sakai K, Sasaki C, Zhang WR, Warita H, Abe K (2000) c-Jun N-terminal kinase (JNK) and JNK interacting protein response in rat brain after transient middle cerebral artery occlusion. Neurosci Lett 284:195–199
Barone FC, Parsons AA (2000) Therapeutic potential of anti-inflammatory drugs in focal stroke. Expert Opin Investig Drugs 9:2281–2306
Green SL, Kulp KS, Vulliet R (1997) Cyclin-dependent protein kinase 5 activity increases in rat brain following ischemia. Neurochem Int 31:617–623
Grimes CA, Jope RS (2001) The multifaceted roles of glycogen synthase kinase 3beta in cellular signalling. Prog Neurobiol 65:391–426
Logan CY, Nusse R (2004) The Wnt signalling pathway in development and disease. Annu Rev Cell Dev Biol 20:781–810
Shimizu H, Julius MA, Giarré M, Zheng Z, Brown AM, Kitajewski J (1997) Transformation by Wnt family proteins correlates with regulation of betacatenin. Cell Growth Differ 8:1349–1358
Aberle H, Bauer A, Stappert J, Kispert A, Kemler R (1997) beta-catenin is a target for the ubiquitin-proteasome pathway. EMBO J 16:3797–3804
Li L, Yuan H, Weaver CD, Mao J, Farr GH III, Sussman DJ, Jonkers J, Kimelman D et al (1999) Axin and Frat1 interact with dvl and GSK, bridging Dvl to GSK in Wnt-mediated regulation of LEF-1. EMBO J 18:4233–4240
Behrens J, von Kries JP, Kuhl M, Bruhn L, Wedlich D, Grosschedl R, Birchmeier W (1996) Functional interaction of beta-catenin with the transcription factor LEF-1. Nature 382:638–642
Zhang QG, Wang R, Khan M, Mahesh V, Brann DW (2008) Role of Dickkopf-1, an antagonist of the Wnt/beta-catenin signalling pathway, in estrogen-induced neuroprotection and attenuation of tau phosphorylation. J Neurosci 28:8430–8441
Chuang DM, Wang Z, Chiu CT (2011) GSK-3 as a target for lithium-induced neuroprotection against excitotoxicity in neuronal cultures and animal models of ischemic stroke. Front Mol Neurosci 9:4–15
Kelly S, Zhao H, Hua Sun G, Cheng D, Qiao Y, Luo J, Martin K, Steinberg GK et al (2004) Glycogen synthase kinase 3beta inhibitor Chir025 reduces neuronal death resulting from oxygen-glucose deprivation, glutamate excitotoxicity, and cerebral ischemia. Exp Neurol 188:378–386
Morales-Garcia JA, Luna-Medina R, Alonso-Gil S, Sanz-Sancristobal M, Palomo V, Gil C, Santos A, Martinez A et al (2012) Glycogen synthase kinase 3 inhibition promotes adult hippocampal neurogenesis in vitro and in vivo. ACS Chem Neurosci 3:963–971
Koh SH, Yoo AR, Chang DI, Hwang SJ, Kim SH (2008) Inhibition of GSK-3 reduces infarct volume and improves neurobehavioral functions. Biochem Biophys Res Commun 371:894–899
Valerio A, Bertolotti P, Delbarba A, Perego C, Dossena M, Ragni M, Spano P, Carruba MO et al (2011) Glycogen synthase kinase-3 inhibition reduces ischemic cerebral damage, restores impaired mitochondrial biogenesis and prevents ROS production. J Neurochem 116:1148–1159
Avrahami L, Farfara D, Shaham-Kol M, Vassar R, Frenkel D, Eldar-Finkelman H (2013) Inhibition of glycogen synthase kinase-3 ameliorates β-amyloid pathology and restores lysosomal acidification and mammalian target of rapamycin activity in the Alzheimer disease mouse model: in vivo and in vitro studies. J Biol Chem 288:1295–1306
Tapia-Rojas C, Schüller A, Lindsay CB, Ureta RC, Mejías-Reyes C, Hancke J, Melo F, Inestrosa NC (2015) Andrographolide activates the canonical Wnt signalling pathway by a mechanism that implicates the non-ATP competitive inhibition of GSK-3β: autoregulation of GSK-3β in vivo. Biochem J 466:415–430
Darshit BS, Balaji B, Rani P, Ramanathan M (2014) Identification and in vitro evaluation of new leads as selective and competitive glycogen synthase kinase-3β inhibitors through ligand and structure based drug design. J Mol Graph Model 53:31–47
Cheung YT, Lau WK, Yu MS, Lai CS, Yeung SC, So KF, Chang RC (2009) Effects of all-trans-retinoic acid on human SH-SY5Y neuroblastoma as in vitro model in neurotoxicity research. Neurotoxicology 30:127–135
Liu Y, Encinas M, Comella JX, Aldea M, Gallego C (2004) Basic helix-loop-helix proteins bind to TrkB and p21(Cip1) promoters linking differentiation and cell cycle arrest in neuroblastoma cells. Mol Cell Biol 24:2662–2672
Strittmatter SM, Vartanian T, Fishman MC (1992) GAP-43 as a plasticity protein in neuronal form and repair. J Neurobiol 23:507–520
Kim S, Ghil SH, Kim SS, Myeong HH, Lee YD, Suh-Kim H (2002) Overexpression of neurogenin1 induces neurite outgrowth in F11 neuroblastoma cells. Exp Mol Med 34:469–475
Agholme L, Lindström T, Kågedal K, Marcusson J, Hallbeck M (2010) An in vitro model for neuroscience: differentiation of SH-SY5Y cells into cells with morphological and biochemical characteristics of mature neurons. J Alzheimers Dis 20:1069–1082
Constantinescu R, Constantinescu AT, Reichmann H, Janetzky B (2007) Neuronal differentiation and long-term culture of the human neuroblastoma line SH-SY5Y. J Neural Transm Suppl 72:17–28
López-Carballo G, Moreno L, Masiá S, Pérez P, Barettino D (2002) Activation of the phosphatidylinositol 3-kinase/Akt signalling pathway by retinoic acid is required for neural differentiation of SH-SY5Y human neuroblastoma cells. J Biol Chem 277:25297–25304
Seo S, Lim J-W, Yellajoshyula D, Chang L-W, Kroll KL (2007) Neurogenin and NeuroD direct transcriptional targets and their regulatory enhancers. EMBO J 26:5093–5108
Zhao S, Fu J, Liu X, Wang T, Zhang J, Zhao Y (2012) Activation of Akt/GSK-3beta/beta-catenin signalling pathway is involved in survival of neurons after traumatic brain injury in rats. Neurol Res 34:400–407
Slee EA, Harte MT, Kluck RM, Wolf BB, Casiano CA, Newmeyer DD, Wang HG, Reed JC et al (1999) Ordering the cytochrome c-initiated caspase cascade: hierarchical activation of caspases-2, −3, −6, −7, −8, and −10 in a caspase-9-dependent manner. J Cell Biol 144:281–292
Newcomb-Fernandez JK, Zhao X, Pike BR, Wang KK, Kampfl A, Beer R, DeFord SM, Hayes RL (2001) Concurrent assessment of calpain and caspase-3 activation after oxygen-glucose deprivation in primary septo-hippocampal cultures. J Cereb Blood Flow Metab 21:1281–1294
Namura S, Zhu J, Fink K, Endres M, Srinivasan A, Tomaselli KJ, Yuan J, Moskowitz MA (1998) Activation and cleavage of caspase-3 in apoptosis induced by experimental cerebral ischemia. J Neurosci 18:3659–3668
Deveraux QL, Reed JC (1999) IAP family proteins--suppressors of apoptosis. Genes Dev 13:239–252
O'Brien WT, Huang J, Buccafusca R, Garskof J, Valvezan AJ, Berry GT, Klein PS (2011) Glycogen synthase kinase-3 is essential for β-arrestin-2 complex formation and lithium-sensitive behaviors in mice. J Clin Invest 121:3756–3762
Acknowledgments
This project was funded by the Medicine Division, Department of Biotechnology (DBT), New Delhi, India (reference no. BT/PR14062/MED/30/357/2010). The author would like to thank Dr. Sivaram Hariharan for his contribution in correcting the grammar of this manuscript.
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Darshit, B.S., Ramanathan, M. Activation of AKT1/GSK-3β/β-Catenin–TRIM11/Survivin Pathway by Novel GSK-3β Inhibitor Promotes Neuron Cell Survival: Study in Differentiated SH-SY5Y Cells in OGD Model. Mol Neurobiol 53, 6716–6729 (2016). https://doi.org/10.1007/s12035-015-9598-z
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DOI: https://doi.org/10.1007/s12035-015-9598-z