- Email: [email protected]
Inhibition of glycogen synthase kinase 3β is involved in the resistance to oxidative stress in neuronal HT22 cells
Brain Research 1005 (2004) 84 – 89 www.elsevier.com/locate/brainres
Research report
Inhibition of glycogen synthase kinase 3h is involved in the resistance to oxidative stress in neuronal HT22 cells Monika Scha¨fer a,1, Sharon Goodenough a,b, Bernd Moosmann a,2, Christian Behl a,b,* a
Independent Research Group Neurodegeneration, Max Planck Institute for Psychiatry, Munich, Germany b Department of Pathobiochemistry, Johannes Gutenberg University, Medical School, Mainz, Germany Accepted 2 January 2004
Abstract Oxidative stress is involved in several neurodegenerative diseases including Alzheimer’s disease, Parkinson’s disease and ischemic reperfusion injury (stroke). We have established clones of the murine hippocampal neuronal cell line HT22, which are resistant to the oxidative stress-causing agents glutamate and hydrogen peroxide, respectively. These cell clones show a mutual cross-resistance to other oxidative stressors, but not to essentially non-oxidative neurotoxins. We have discovered that the amount of phosphorylated, inactive glycogen synthase kinase (GSK) 3h is elevated in both resistant clones. Pharmacological inhibition of GSK-3h with lithium chloride in the sensitive parental neuronal cells results in an increased tolerance to glutamate and hydrogen peroxide, suggesting that GSK-3h is involved in the control of oxidative stress resistance in these cells. D 2004 Elsevier B.V. All rights reserved. Keywords: Oxidative stress; Oxidation-resistant cell; Glycogen synthase kinase 3h
1. Introduction Reactive oxygen species (ROS) like hydrogen peroxide and superoxide radicals are generated during mitochondrial respiration. In healthy cells, ROS are eliminated by several enzymes, e.g. superoxide dismutase, catalase and diverse peroxidases. Disruption of the mitochondrial respiratory chain by toxic insults or genetic defects and other causes can lead to a perturbation of the equilibrium between ROS generation and elimination. The resulting detrimental accumulation of ROS is called oxidative stress. Frequently, oxidative stress induces cell death through the oxidation of cellular proteins, lipids and DNA. The central nervous system is especially susceptible to oxidative stress because
* Corresponding author. Institute for Physiological Chemistry and Pathobiochemistry, Johannes Gutenberg University, Medical School, Duesbergweg 6, D-55099, Mainz, Germany. Tel.: +49-6131-3925890; fax: +49-6131-3925792. E-mail address: [email protected] (C. Behl). 1 Present address: Roche Diagnostics GmbH, 82377 Penzberg, Germany. 2 Present address: The Burnham Institute, La Jolla, CA, USA. 0006-8993/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2004.01.037
of its high oxygen turnover and possibly due to its high amount of unsaturated fatty acids. There is increasing evidence that oxidative stress plays an important role in several neurodegenerative diseases, e.g. Alzheimer’s disease, Parkinson’s disease and ischemic reperfusion injury (stroke) [3,4,6,13,15,20]. Oxidative stress may even play a causative role in these disorders. Therefore, signalling pathways leading to the resistance of neuronal cells to oxidative stress are of particular interest for neurodegenerative disease research. For this study, we have established clones of the murine hippocampal neuronal cell line HT22 which are resistant to the oxidative stress-causing agents glutamate and hydrogen peroxide. While HT22 cells lack functional glutamate receptors, in these cells, glutamate induces oxidative stress by its inhibitory action on a cysteineglutamate antiporter, which results in a depletion of intracellular cysteine and hence the antioxidant molecule glutathione (GSH) [17]. Indeed, HT22 cells are frequently employed for the analysis of disease-related oxidative neuronal cell death [5,11,19,22,26]. A direct comparison of oxidative stress-resistant and -sensitive neuronal cells may provide evidence about which signalling pathways may be involved in resistance to oxidative cell death and
M. Scha¨fer et al. / Brain Research 1005 (2004) 84–89
therefore unveil possible therapeutic targets. Several important proteins mediating glutamate-induced cell death in these cells have recently been identified, e.g. the translation initiation factor 2a [26]. Here, we have identified glycogen synthase kinase 3h (GSK-3h) as an important factor for the resistance of neuronal HT22 cells to oxidative stress.
2. Materials and methods 2.1. Cell culture Murine HT22 cells were cultured in DMEM containing 10% fetal calf serum, 1 mM sodium pyruvate and 1% of a penicillin/streptomycin solution (all from Life Technologies). For the generation of glutamate-resistant clones, cells were plated at low density on normal cell culture dishes and treated with gradually increasing concentrations of sodium glutamate over a time period of four months, starting with 2 mM (up to 40 mM). Single clones were isolated for clonal expansion. Hydrogen peroxideresistant cells were generated by cultivating the cells in medium containing 1.2 mM H2O2. After 3 weeks, resistant clones were isolated and expanded. To maintain the phenotype of the isolated cell clones, 40 mM glutamate (Sigma) was added to the culture medium of glutamateresistant cells and 450 AM H2O2 (Aldrich) was added to the culture of H2O2-resistant cells twice a week. Resistant cell clones were cultivated in fresh medium lacking glutamate or H2O2, respectively, for at least 6 days prior to the toxicity assays; medium was changed frequently to remove residual toxins. 2.2. Toxicity assay The influence of toxins on cell survival was measured via the ability of cells to reduce 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazoliumbromide (MTT) and were confirmed by microscopical evaluation and cell counting exactly as described [19]. Briefly, cells were plated into 96-well microtiter plates in a volume of 100 Al culture medium (1000 cells/well), incubated overnight, and were then treated with various concentrations of different toxins for 20 h. For pretreatment with lithium, 10 mM LiCl were applied to the cells 2 h before the toxins. After toxin treatment, 10 Al MTT solution (2.5 mg/ml) was added to each well, incubating for another 4 h at 37 jC. To dissolve the reduced MTT formazan, 100 Al solubilisation solution (0.1 g/ml SDS, 50% (vol/vol) dimethylformamide, pH 4.1 with acetic acid) was added to each well. After a few hours, absorbance was measured at 550 nm using a microtiter plate reader (Dynatech MR 7000). Each treatment group was measured at least fivefold. Negative control wells contained medium only. Absorbance of positive control wells (no toxin) was set as 100%.
85
2.3. Western blotting Cells were plated in 6-cm cell culture dishes overnight, were then scraped off in 0.5 ml PBS and lysed by sonification. Protein concentration was determined by Bradford assay. For Western analysis, 10 Ag of cellular protein per lane were separated by 10% SDS-PAGE and transferred to nitrocellulose membranes (Schleicher und Schuell) using Ponceau S staining to confirm equal loading. The blots were then blocked in 10 % non-fat milk in TBS-T for 2 h and subsequently incubated overnight in appropriate dilutions of primary antibody solution at 4 jC. Afterwards, horseradish peroxidase-conjugated secondary antibody was applied in TBS-T containing 1% low-fat milk for 2 h at ambient temperature. Immunoblots were visualized by Western blotting detection reagents from Amersham Pharmacia Biotech. Western blots were repeated three times at least and ODs of bands were calculated using Scion software. Anti-GSK antibody was purchased from Transduction Laboratories and anti-GSK-3h-Ser-P antibody from Cell Signaling; the latter is of proven specificity [9]. The gene expression arrays Mouse Pathway Finder-1, Mouse Apoptosis Q Series and Mouse Cancer Pathway Finder Q Series from GEArray were used exactly following the instructions of the supplier.
3. Results Glutamate-resistant (Glu-res) and H2O2-resistant (H2O2res) HT22 cells were established by long-term treatment with glutamate and H2O2, respectively. Survival assays confirmed the resistance of the selected clones (Glu-res and H2O2-res) against glutamate and H2O2 compared to the parental non-resistant cells (HT22 N). Interestingly, the two resistant cell clones remained their resistance phenotype even when the oxidative pressure was removed (not shown) and exhibited mutual cross-resistance: Glu-res neurons showed a higher tolerance for H2O2, and H2O2-res neurons showed a higher tolerance for glutamate than HT22 N cells (Fig. 1A,B). These findings indicate a more general resistance of the selected Glu-res and H2O2-res clones to oxidative insults. To prove the specificity of the observed resistance for oxidative insults, survival assays with several toxins that follow non-oxidative stress-inducing pathways were performed. The sphingolipid breakdown product sphingosine is an inhibitor of protein kinase C and a known inductor of apoptosis in neurons [10]. Staurosporine is a general inhibitor of protein kinases and leads to apoptosis in a variety of cells. Gramicidin A is a Na+/K+-channel, which causes cell death due to depolarisation [7]. The effect of these compounds on Glu-res and H2O2-res did not differ from their toxic effect on HT22 N cells; all toxins induced cell death to a similar degree in all clones tested
86
M. Scha¨fer et al. / Brain Research 1005 (2004) 84–89
Fig. 1. Relative resistance of three model cell clones HT22 N, Glu-res and H2O2-res with A: glutamate, B: hydrogen peroxide, C: sphingosine, D: staurosporine and E: gramicidin (A) Glu-res neurons show a higher resistance to all glutamate concentrations tested ( p < 0.05, two-way ANOVA) and to H2O2 concentrations between 300 and 600 AM ( p < 0.001, two-way ANOVA) than HT22 N neurons. H2O2-res neurons tolerate higher H2O2 concentrations ( p < 0.001 for all tested concentrations, two-way ANOVA) and higher glutamate concentrations ( p < 0.05 for all tested concentrations above 1 mM, two-way ANOVA) than HT22 N neurons. Sphingosine, staurosporine and gramicidin A all cause cell death to a similar degree in the three cell lines tested.
(Fig. 1C,D,E). These results indicate a specific resistance of Glu-res and H2O2-res neurons against oxidative stressinduced neurotoxicity. By the very nature of the experimental selection process for these clones, it may be possible that we have merely selected cells with an increased proliferation rate rather than cells specifically resistant to cell death. For this reason, the proliferation rates of the different cell clones were studied. As shown in Fig. 2, the growth rate of the Glu-res cells did not significantly differ from the
Fig. 2. Proliferation of HT22 N, Glu-res and H2O2-res neurons obtained by MTT assay. The curves for HT22 N and Glu-res cells are very similar; H2O2-res neurons grow slower than cells of the other two clones. The absorption values differ significantly from those of the other clones within the first 168 h ( p < 0.001, two-way ANOVA).
parental cell line, while H2O2-res cells grew even slower than HT22 N and Glu-res cells. This suggests that the increased resistance of the selected cell clones is not due to an increased growth rate. The expression of various intracellular signalling molecules in sensitive versus resistant HT22 cells were studied employing different commercially available signal transduction DNA arrays, but no significant differences could be observed with respect to the signalling molecules that were spotted on the small-scale arrays used including p65/ NF-nB, AKT2, bcl-2 (data not shown). Consequently, the expression of additional signal factors that are known to be involved in cell survival was investigated. Along this line, Western blot analysis for total GSK revealed a reduced level of this enzyme in both Glu-res and H2O2-res neurons compared to the sensitive HT22 N cells (Fig. 3A). Using an antibody that specifically recognizes GSK-3h phosphorylated at serine-9 (representing inactive GSK-3h) as previously shown [9], we found that the level of inactive GSK-3h is significantly elevated in resistant HT22 cells compared to sensitive clones (Fig. 3B) indicating an overall reduction in GSK-3h activity in the resistant clones. In order to test whether the observed GSK-3h inactivation is involved in the development of oxidative stress resistance, we blocked GSK-3h activity in sensitive HT22 N neurons pharmacologically, by adding LiCl, a welldescribed inhibitor of GSK-3h [16,23]; multiple indepen-
M. Scha¨fer et al. / Brain Research 1005 (2004) 84–89
87
4. Discussion
Fig. 3. Western blot results for GSK protein. (A) Total GSK levels are reduced in both resistant clones when compared to the parental cell line. (* corresponds to p < 0.001, one-way ANOVA). (B) The level of serine phophorylated GSK-3h protein is higher both resistant clones compared to sensitive cells (* corresponds to p < 0.001, one-way ANOVA). Foldexpression levels of resistant cells compared to sensitive cells are shown.
dent cell clones have been used. Inhibition of GSK-3h did indeed lead to an increased tolerance of HT22 neurons to hydrogen peroxide and glutamate (Fig. 4A,B), indicating that GSK-3h inhibition mediates oxidative stress resistance in these cells.
Oxidative stress is believed to occur during the pathogenesis of many neurodegenerative diseases. Mechanisms leading to oxidative stress resistance may therefore provide a promising target for the development of compounds for the prevention and treatment of neurodegenerative conditions. The molecular comparison of oxidative stress-resistant and -sensitive cell clones is a powerful tool to study these mechanisms. In the present work, neuronal cell clones were established which are exclusively resistant to oxidative insults, but still susceptible to toxins essentially not related to oxidations such as protein kinase inhibitors. The validity of the cell model used is stressed further by the fact that the growth rate in the resistant clones is not accelerated compared to sensitive parental neurons, indicating that their resistance is mediated by a change in signalling pathways and expression patterns, which are unrelated to their clonal behaviour as cell lines. Of special interest is the finding that neuronal HT22 cells selected for their resistance to H2O2 are also resistant to glutamate and vice versa. In these cells, therefore, the rate-limiting biochemical step towards cell death must either be the same for both toxins, or must be counteracted by the same ratelimiting defense mechanism. In addition, the observed cross-resistance to other oxidative stressors is consistent with other reports employing oxidative stress-resistant cells, proposing that such toxins against which certain selected cells are resistant are inducing a common cell death pathway (oxytosis pathway). Resistant clones would then be characterized by a block or downregulation of components of this pathway [11]. The elucidation of these factors may reveal novel therapeutic targets for the prevention of oxidative stress-associated neurodegenerative diseases. Examination of the established resistant clones revealed similar changes in the total levels of GSK in both types
Fig. 4. Toxicity assay for HT22 N neurons with and without LiCl pretreatment. Pretreatment of cells with LiCl in order to inhibit GSK-3h activity enhances the tolerance of HT22 N neurons to hydrogen peroxide (A) and glutamate (B) (* corresponds to p < 0.05, one-way ANOVA).
88
M. Scha¨fer et al. / Brain Research 1005 (2004) 84–89
of resistant cell clones compared to sensitive HT22 neurons. Further, the levels of serine-phosphorylated (inactive) GSK-3h were elevated compared to sensitive cells, indicating that inhibition of GSK-3h may play a role in mediating oxidative stress resistance in HT22 cells. These findings are highly interesting in the light of the wellestablished link between cell survival and GSK-3h: While GSK-3h activation is involved in apoptotic processes [14,21], its inhibition is part of antiapoptotic signalling pathways like Wnt-signalling, the PI-3 kinase and the MAP-kinase pathway [1,8,9,21,27]. In fact, it has been shown that inhibition of GSK-3h activity by over expression of the GSK binding protein or kinase-dead dominant negative mutant of GSK-3h results in a decreased susceptibility of cortical neurons to trophic-withdrawal induced cell death [14]. Changes in these pathways are currently discussed to be connected with oxidative stress-associated neurodegenerative diseases. The Wnt-pathway, for example, has been suspected to be the so far missing link between the two pathological hallmarks of AD, amyloid plaques and neurofibrillary tangles [12]. In addition, the activation of GSK-3h itself was already shown to be a consequence of incubating cells with the main component of the senile plaques in AD: amyloid h (Ah) [25]. Inhibition of GSK3h activity or blocking its expression, on the other hand, protected cortical and hippocampal primary cell cultures from Ah-induced cell death [2,24]. We and others were able to show that Ah can induce oxidative stress [3,4,18]. Nevertheless, data suggesting a direct link between oxidative stress and changes in GSK-3h activity were so far missing. In summary, we present oxidative stress-resistant neuronal cell lines for the investigation of intracellular signalling under oxidative stress conditions. This study provides evidence for GSK-3h to be directly involved in pathways leading to oxidative cell death since its inhibition could be shown to lead to an increased resistance of neurons to the oxidative stressors glutamate and H2O2. These results indicate that GSK-3h could be a central factor for oxidative stress-related disorders and that GSK-3h may have the potential of being an interesting therapeutical target.
Acknowledgements This work was supported by a grant of the Deutsche Forschungsgemeinschaft to CB (DFG Be 1475/2-2, Alzheimer Priority Program).
References [1] H. Aberle, A. Bauer, J. Stappert, A. Kispert, R. Kemler, Beta-catenin is a target for the ubiquitin – proteasome pathway, EMBO J. 16 (1997) 3797 – 3804.
[2] G. Alvarez, J.R. Munoz-Montano, J. Satrustegui, J. Avila, E. Bogonez, J. Diaz-Nido, Lithium protects cultured neurons against beta-amyloidinduced neurodegeneration, FEBS Lett. 453 (1999) 260 – 264. [3] C. Behl, Amyloid beta-protein toxicity and oxidative stress in Alzheimer’s disease, Cell Tissue Res. 290 (1997) 471 – 480. [4] C. Behl, J.B. Davis, R. Lesley, D. Schubert, Hydrogen peroxide mediates amyloid beta protein toxicity, Cell 77 (1994) 817 – 827. [5] C. Behl, T. Skutella, F. Lezoualc’h, A. Post, M. Widmann, C. Newton, F. Holsboer, Neuroprotection against oxidative stress by estrogens: structure – activity relationship, Mol. Pharmacol. 51 (1997) 535 – 541. [6] D.A. Butterfield, J. Kanski, Brain protein oxidation in age-related neurodegenerative disorders that are associated with aggregated proteins, Mech. Ageing Dev. 1222 (2001) 945 – 962. [7] Z. Chen, C. Alcayaga, B.A. Suarez-Isla, B. O’Rourke, G. Tomaselli, E.A. Marban, ‘‘Minimal’’ sodium channel construct consisting of ligated S5-P-S6 segments forms a toxin-activatable ionophore, J. Biol. Chem. 277 (2002) 24653 – 24658. [8] P. Cohen, S. Frame, The renaissance of GSK3, Nat. Rev., Mol. Cell Biol. 2 (2001) 769 – 776. [9] R.J. Crowder, R.S. Freeman, Glycogen synthase kinase-3 beta activity is critical for neuronal death caused by inhibiting phosphatidylinositol 3-kinase or Akt but not for death caused by nerve growth factor withdrawal, J. Biol. Chem. 275 (2000) 34266 – 34271. [10] O. Cuvillier, Sphingosine in apoptosis signaling, Biochim. Biophys. Acta 1585 (2002) 153 – 162. [11] R. Dargusch, D. Schubert, Specificity of resistance to oxidative stress, J. Neurochem. 81 (2002) 1394 – 1400. [12] G.V. DeFerrari, N.C. Inestrosa, Wnt signaling function in Alzheimer’s disease, Brain Res. Rev. 33 (2000) 1 – 12. [13] B. Halliwell, Role of free radicals in the neurodegenerative diseases: therapeutic implications for antioxidant treatment, Drugs Aging 18 (2001) 685 – 716. [14] M. Hetman, J.E. Cavanaugh, D. Kimelman, Z. Xia, Role of glycogen synthase kinase-3h in neuronal apoptosis induced by trophic withdrawal, J. Neurosci. 20 (2000) 2567 – 2574. [15] P. Jenner, C.W. Olanow, Oxidative stress and the pathogenesis of Parkinson’s disease, Neurology 47 (1996) S161 – S170. [16] P.S. Klein, D.A. Melton, A molecular mechanism for the effect of lithium on development, Proc. Natl. Acad. Sci. U. S. A. 93 (1996) 8455 – 8459. [17] P. Maher, J.B. Davis, The role of monoamine metabolism in oxidative glutamate toxicity, J. Neurosci. 16 (1996) 6394 – 6401. [18] R.J. Mark, E.M. Blanc, M.P. Mattson, Amyloid beta-peptide and oxidative cellular injury in Alzheimer’s disease, Mol. Neurobiol. 12 (1996) 211 – 224. [19] B. Moosmann, C. Behl, The antioxidant neuroprotective effects of estrogens and phenolic compounds are independent from their estrogenic properties, Proc. Natl. Acad. Sci. 96 (1999) 8867 – 8872. [20] B. Moosmann, C. Behl, Antioxidants as treatment for neurodegenerative disorders, Expert Opin. Investig. Drugs 11 (2002) 1407 – 1435. [21] M. Pap, G.M. Cooper, Role of glycogen synthase kinase-3 in the phosphatidylinositol 3-kinase/Akt cell survival pathway, J. Biol. Chem. 273 (1998) 19929 – 19932. [22] Y. Sagara, S. Hendler, S. Khoh-Reiter, G. Gillenwater, D. Carlo, D. Schubert, J. Chang, Propofol hemisuccinate protects neuronal cells from oxidative injury, J. Neurochem. 73 (1999) 2524 – 2530. [23] V. Stambolic, L. Ruel, J.R. Woodgett, Lithium inhibits glycogen synthase kinase-3 activity and mimics wingless signalling in intact cells, Curr. Biol. 6 (1996) 1664 – 1668. [24] A. Takashima, K. Noguchi, K. Sato, T. Hoshino, K. Imahori, Tau protein kinase I is essential for amyloid beta-protein-induced neurotoxicity, Proc. Natl. Acad. Sci. U. S. A. 90 (1993) 7789 – 7793. [25] A. Takashima, K. Noguchi, G. Michel, M. Mercken, M. Hoshi, K. Ishiguro, K. Imahori, Exposure of rat hippocampal neurons to amyloid beta peptide (25-35) induces the inactivation of phosphatidyl inositol-3 kinase and the activation of tau protein kinase
M. Scha¨fer et al. / Brain Research 1005 (2004) 84–89 I/glycogen synthase kinase-3 beta, Neurosci. Lett. 203 (1996) 33 – 36. [26] S. Tan, N. Somia, P. Maher, D. Schubert, Regulation of antioxidant metabolism by translation initiation factor 2alpha, J. Cell Biol. 152 (2001) 997 – 1006.
89
[27] C. Yost, M. Torres, J.R. Miller, E. Huang, D. Kimelman, R.T. Moon, The axis-inducing activity, stability, and subcellular distribution of beta-catenin is regulated in Xenopus embryos by glycogen synthase kinase 3, Genes Dev. 10 (1996) 1443 – 1454.
Research report
Inhibition of glycogen synthase kinase 3h is involved in the resistance to oxidative stress in neuronal HT22 cells Monika Scha¨fer a,1, Sharon Goodenough a,b, Bernd Moosmann a,2, Christian Behl a,b,* a
Independent Research Group Neurodegeneration, Max Planck Institute for Psychiatry, Munich, Germany b Department of Pathobiochemistry, Johannes Gutenberg University, Medical School, Mainz, Germany Accepted 2 January 2004
Abstract Oxidative stress is involved in several neurodegenerative diseases including Alzheimer’s disease, Parkinson’s disease and ischemic reperfusion injury (stroke). We have established clones of the murine hippocampal neuronal cell line HT22, which are resistant to the oxidative stress-causing agents glutamate and hydrogen peroxide, respectively. These cell clones show a mutual cross-resistance to other oxidative stressors, but not to essentially non-oxidative neurotoxins. We have discovered that the amount of phosphorylated, inactive glycogen synthase kinase (GSK) 3h is elevated in both resistant clones. Pharmacological inhibition of GSK-3h with lithium chloride in the sensitive parental neuronal cells results in an increased tolerance to glutamate and hydrogen peroxide, suggesting that GSK-3h is involved in the control of oxidative stress resistance in these cells. D 2004 Elsevier B.V. All rights reserved. Keywords: Oxidative stress; Oxidation-resistant cell; Glycogen synthase kinase 3h
1. Introduction Reactive oxygen species (ROS) like hydrogen peroxide and superoxide radicals are generated during mitochondrial respiration. In healthy cells, ROS are eliminated by several enzymes, e.g. superoxide dismutase, catalase and diverse peroxidases. Disruption of the mitochondrial respiratory chain by toxic insults or genetic defects and other causes can lead to a perturbation of the equilibrium between ROS generation and elimination. The resulting detrimental accumulation of ROS is called oxidative stress. Frequently, oxidative stress induces cell death through the oxidation of cellular proteins, lipids and DNA. The central nervous system is especially susceptible to oxidative stress because
* Corresponding author. Institute for Physiological Chemistry and Pathobiochemistry, Johannes Gutenberg University, Medical School, Duesbergweg 6, D-55099, Mainz, Germany. Tel.: +49-6131-3925890; fax: +49-6131-3925792. E-mail address: [email protected] (C. Behl). 1 Present address: Roche Diagnostics GmbH, 82377 Penzberg, Germany. 2 Present address: The Burnham Institute, La Jolla, CA, USA. 0006-8993/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2004.01.037
of its high oxygen turnover and possibly due to its high amount of unsaturated fatty acids. There is increasing evidence that oxidative stress plays an important role in several neurodegenerative diseases, e.g. Alzheimer’s disease, Parkinson’s disease and ischemic reperfusion injury (stroke) [3,4,6,13,15,20]. Oxidative stress may even play a causative role in these disorders. Therefore, signalling pathways leading to the resistance of neuronal cells to oxidative stress are of particular interest for neurodegenerative disease research. For this study, we have established clones of the murine hippocampal neuronal cell line HT22 which are resistant to the oxidative stress-causing agents glutamate and hydrogen peroxide. While HT22 cells lack functional glutamate receptors, in these cells, glutamate induces oxidative stress by its inhibitory action on a cysteineglutamate antiporter, which results in a depletion of intracellular cysteine and hence the antioxidant molecule glutathione (GSH) [17]. Indeed, HT22 cells are frequently employed for the analysis of disease-related oxidative neuronal cell death [5,11,19,22,26]. A direct comparison of oxidative stress-resistant and -sensitive neuronal cells may provide evidence about which signalling pathways may be involved in resistance to oxidative cell death and
M. Scha¨fer et al. / Brain Research 1005 (2004) 84–89
therefore unveil possible therapeutic targets. Several important proteins mediating glutamate-induced cell death in these cells have recently been identified, e.g. the translation initiation factor 2a [26]. Here, we have identified glycogen synthase kinase 3h (GSK-3h) as an important factor for the resistance of neuronal HT22 cells to oxidative stress.
2. Materials and methods 2.1. Cell culture Murine HT22 cells were cultured in DMEM containing 10% fetal calf serum, 1 mM sodium pyruvate and 1% of a penicillin/streptomycin solution (all from Life Technologies). For the generation of glutamate-resistant clones, cells were plated at low density on normal cell culture dishes and treated with gradually increasing concentrations of sodium glutamate over a time period of four months, starting with 2 mM (up to 40 mM). Single clones were isolated for clonal expansion. Hydrogen peroxideresistant cells were generated by cultivating the cells in medium containing 1.2 mM H2O2. After 3 weeks, resistant clones were isolated and expanded. To maintain the phenotype of the isolated cell clones, 40 mM glutamate (Sigma) was added to the culture medium of glutamateresistant cells and 450 AM H2O2 (Aldrich) was added to the culture of H2O2-resistant cells twice a week. Resistant cell clones were cultivated in fresh medium lacking glutamate or H2O2, respectively, for at least 6 days prior to the toxicity assays; medium was changed frequently to remove residual toxins. 2.2. Toxicity assay The influence of toxins on cell survival was measured via the ability of cells to reduce 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazoliumbromide (MTT) and were confirmed by microscopical evaluation and cell counting exactly as described [19]. Briefly, cells were plated into 96-well microtiter plates in a volume of 100 Al culture medium (1000 cells/well), incubated overnight, and were then treated with various concentrations of different toxins for 20 h. For pretreatment with lithium, 10 mM LiCl were applied to the cells 2 h before the toxins. After toxin treatment, 10 Al MTT solution (2.5 mg/ml) was added to each well, incubating for another 4 h at 37 jC. To dissolve the reduced MTT formazan, 100 Al solubilisation solution (0.1 g/ml SDS, 50% (vol/vol) dimethylformamide, pH 4.1 with acetic acid) was added to each well. After a few hours, absorbance was measured at 550 nm using a microtiter plate reader (Dynatech MR 7000). Each treatment group was measured at least fivefold. Negative control wells contained medium only. Absorbance of positive control wells (no toxin) was set as 100%.
85
2.3. Western blotting Cells were plated in 6-cm cell culture dishes overnight, were then scraped off in 0.5 ml PBS and lysed by sonification. Protein concentration was determined by Bradford assay. For Western analysis, 10 Ag of cellular protein per lane were separated by 10% SDS-PAGE and transferred to nitrocellulose membranes (Schleicher und Schuell) using Ponceau S staining to confirm equal loading. The blots were then blocked in 10 % non-fat milk in TBS-T for 2 h and subsequently incubated overnight in appropriate dilutions of primary antibody solution at 4 jC. Afterwards, horseradish peroxidase-conjugated secondary antibody was applied in TBS-T containing 1% low-fat milk for 2 h at ambient temperature. Immunoblots were visualized by Western blotting detection reagents from Amersham Pharmacia Biotech. Western blots were repeated three times at least and ODs of bands were calculated using Scion software. Anti-GSK antibody was purchased from Transduction Laboratories and anti-GSK-3h-Ser-P antibody from Cell Signaling; the latter is of proven specificity [9]. The gene expression arrays Mouse Pathway Finder-1, Mouse Apoptosis Q Series and Mouse Cancer Pathway Finder Q Series from GEArray were used exactly following the instructions of the supplier.
3. Results Glutamate-resistant (Glu-res) and H2O2-resistant (H2O2res) HT22 cells were established by long-term treatment with glutamate and H2O2, respectively. Survival assays confirmed the resistance of the selected clones (Glu-res and H2O2-res) against glutamate and H2O2 compared to the parental non-resistant cells (HT22 N). Interestingly, the two resistant cell clones remained their resistance phenotype even when the oxidative pressure was removed (not shown) and exhibited mutual cross-resistance: Glu-res neurons showed a higher tolerance for H2O2, and H2O2-res neurons showed a higher tolerance for glutamate than HT22 N cells (Fig. 1A,B). These findings indicate a more general resistance of the selected Glu-res and H2O2-res clones to oxidative insults. To prove the specificity of the observed resistance for oxidative insults, survival assays with several toxins that follow non-oxidative stress-inducing pathways were performed. The sphingolipid breakdown product sphingosine is an inhibitor of protein kinase C and a known inductor of apoptosis in neurons [10]. Staurosporine is a general inhibitor of protein kinases and leads to apoptosis in a variety of cells. Gramicidin A is a Na+/K+-channel, which causes cell death due to depolarisation [7]. The effect of these compounds on Glu-res and H2O2-res did not differ from their toxic effect on HT22 N cells; all toxins induced cell death to a similar degree in all clones tested
86
M. Scha¨fer et al. / Brain Research 1005 (2004) 84–89
Fig. 1. Relative resistance of three model cell clones HT22 N, Glu-res and H2O2-res with A: glutamate, B: hydrogen peroxide, C: sphingosine, D: staurosporine and E: gramicidin (A) Glu-res neurons show a higher resistance to all glutamate concentrations tested ( p < 0.05, two-way ANOVA) and to H2O2 concentrations between 300 and 600 AM ( p < 0.001, two-way ANOVA) than HT22 N neurons. H2O2-res neurons tolerate higher H2O2 concentrations ( p < 0.001 for all tested concentrations, two-way ANOVA) and higher glutamate concentrations ( p < 0.05 for all tested concentrations above 1 mM, two-way ANOVA) than HT22 N neurons. Sphingosine, staurosporine and gramicidin A all cause cell death to a similar degree in the three cell lines tested.
(Fig. 1C,D,E). These results indicate a specific resistance of Glu-res and H2O2-res neurons against oxidative stressinduced neurotoxicity. By the very nature of the experimental selection process for these clones, it may be possible that we have merely selected cells with an increased proliferation rate rather than cells specifically resistant to cell death. For this reason, the proliferation rates of the different cell clones were studied. As shown in Fig. 2, the growth rate of the Glu-res cells did not significantly differ from the
Fig. 2. Proliferation of HT22 N, Glu-res and H2O2-res neurons obtained by MTT assay. The curves for HT22 N and Glu-res cells are very similar; H2O2-res neurons grow slower than cells of the other two clones. The absorption values differ significantly from those of the other clones within the first 168 h ( p < 0.001, two-way ANOVA).
parental cell line, while H2O2-res cells grew even slower than HT22 N and Glu-res cells. This suggests that the increased resistance of the selected cell clones is not due to an increased growth rate. The expression of various intracellular signalling molecules in sensitive versus resistant HT22 cells were studied employing different commercially available signal transduction DNA arrays, but no significant differences could be observed with respect to the signalling molecules that were spotted on the small-scale arrays used including p65/ NF-nB, AKT2, bcl-2 (data not shown). Consequently, the expression of additional signal factors that are known to be involved in cell survival was investigated. Along this line, Western blot analysis for total GSK revealed a reduced level of this enzyme in both Glu-res and H2O2-res neurons compared to the sensitive HT22 N cells (Fig. 3A). Using an antibody that specifically recognizes GSK-3h phosphorylated at serine-9 (representing inactive GSK-3h) as previously shown [9], we found that the level of inactive GSK-3h is significantly elevated in resistant HT22 cells compared to sensitive clones (Fig. 3B) indicating an overall reduction in GSK-3h activity in the resistant clones. In order to test whether the observed GSK-3h inactivation is involved in the development of oxidative stress resistance, we blocked GSK-3h activity in sensitive HT22 N neurons pharmacologically, by adding LiCl, a welldescribed inhibitor of GSK-3h [16,23]; multiple indepen-
M. Scha¨fer et al. / Brain Research 1005 (2004) 84–89
87
4. Discussion
Fig. 3. Western blot results for GSK protein. (A) Total GSK levels are reduced in both resistant clones when compared to the parental cell line. (* corresponds to p < 0.001, one-way ANOVA). (B) The level of serine phophorylated GSK-3h protein is higher both resistant clones compared to sensitive cells (* corresponds to p < 0.001, one-way ANOVA). Foldexpression levels of resistant cells compared to sensitive cells are shown.
dent cell clones have been used. Inhibition of GSK-3h did indeed lead to an increased tolerance of HT22 neurons to hydrogen peroxide and glutamate (Fig. 4A,B), indicating that GSK-3h inhibition mediates oxidative stress resistance in these cells.
Oxidative stress is believed to occur during the pathogenesis of many neurodegenerative diseases. Mechanisms leading to oxidative stress resistance may therefore provide a promising target for the development of compounds for the prevention and treatment of neurodegenerative conditions. The molecular comparison of oxidative stress-resistant and -sensitive cell clones is a powerful tool to study these mechanisms. In the present work, neuronal cell clones were established which are exclusively resistant to oxidative insults, but still susceptible to toxins essentially not related to oxidations such as protein kinase inhibitors. The validity of the cell model used is stressed further by the fact that the growth rate in the resistant clones is not accelerated compared to sensitive parental neurons, indicating that their resistance is mediated by a change in signalling pathways and expression patterns, which are unrelated to their clonal behaviour as cell lines. Of special interest is the finding that neuronal HT22 cells selected for their resistance to H2O2 are also resistant to glutamate and vice versa. In these cells, therefore, the rate-limiting biochemical step towards cell death must either be the same for both toxins, or must be counteracted by the same ratelimiting defense mechanism. In addition, the observed cross-resistance to other oxidative stressors is consistent with other reports employing oxidative stress-resistant cells, proposing that such toxins against which certain selected cells are resistant are inducing a common cell death pathway (oxytosis pathway). Resistant clones would then be characterized by a block or downregulation of components of this pathway [11]. The elucidation of these factors may reveal novel therapeutic targets for the prevention of oxidative stress-associated neurodegenerative diseases. Examination of the established resistant clones revealed similar changes in the total levels of GSK in both types
Fig. 4. Toxicity assay for HT22 N neurons with and without LiCl pretreatment. Pretreatment of cells with LiCl in order to inhibit GSK-3h activity enhances the tolerance of HT22 N neurons to hydrogen peroxide (A) and glutamate (B) (* corresponds to p < 0.05, one-way ANOVA).
88
M. Scha¨fer et al. / Brain Research 1005 (2004) 84–89
of resistant cell clones compared to sensitive HT22 neurons. Further, the levels of serine-phosphorylated (inactive) GSK-3h were elevated compared to sensitive cells, indicating that inhibition of GSK-3h may play a role in mediating oxidative stress resistance in HT22 cells. These findings are highly interesting in the light of the wellestablished link between cell survival and GSK-3h: While GSK-3h activation is involved in apoptotic processes [14,21], its inhibition is part of antiapoptotic signalling pathways like Wnt-signalling, the PI-3 kinase and the MAP-kinase pathway [1,8,9,21,27]. In fact, it has been shown that inhibition of GSK-3h activity by over expression of the GSK binding protein or kinase-dead dominant negative mutant of GSK-3h results in a decreased susceptibility of cortical neurons to trophic-withdrawal induced cell death [14]. Changes in these pathways are currently discussed to be connected with oxidative stress-associated neurodegenerative diseases. The Wnt-pathway, for example, has been suspected to be the so far missing link between the two pathological hallmarks of AD, amyloid plaques and neurofibrillary tangles [12]. In addition, the activation of GSK-3h itself was already shown to be a consequence of incubating cells with the main component of the senile plaques in AD: amyloid h (Ah) [25]. Inhibition of GSK3h activity or blocking its expression, on the other hand, protected cortical and hippocampal primary cell cultures from Ah-induced cell death [2,24]. We and others were able to show that Ah can induce oxidative stress [3,4,18]. Nevertheless, data suggesting a direct link between oxidative stress and changes in GSK-3h activity were so far missing. In summary, we present oxidative stress-resistant neuronal cell lines for the investigation of intracellular signalling under oxidative stress conditions. This study provides evidence for GSK-3h to be directly involved in pathways leading to oxidative cell death since its inhibition could be shown to lead to an increased resistance of neurons to the oxidative stressors glutamate and H2O2. These results indicate that GSK-3h could be a central factor for oxidative stress-related disorders and that GSK-3h may have the potential of being an interesting therapeutical target.
Acknowledgements This work was supported by a grant of the Deutsche Forschungsgemeinschaft to CB (DFG Be 1475/2-2, Alzheimer Priority Program).
References [1] H. Aberle, A. Bauer, J. Stappert, A. Kispert, R. Kemler, Beta-catenin is a target for the ubiquitin – proteasome pathway, EMBO J. 16 (1997) 3797 – 3804.
[2] G. Alvarez, J.R. Munoz-Montano, J. Satrustegui, J. Avila, E. Bogonez, J. Diaz-Nido, Lithium protects cultured neurons against beta-amyloidinduced neurodegeneration, FEBS Lett. 453 (1999) 260 – 264. [3] C. Behl, Amyloid beta-protein toxicity and oxidative stress in Alzheimer’s disease, Cell Tissue Res. 290 (1997) 471 – 480. [4] C. Behl, J.B. Davis, R. Lesley, D. Schubert, Hydrogen peroxide mediates amyloid beta protein toxicity, Cell 77 (1994) 817 – 827. [5] C. Behl, T. Skutella, F. Lezoualc’h, A. Post, M. Widmann, C. Newton, F. Holsboer, Neuroprotection against oxidative stress by estrogens: structure – activity relationship, Mol. Pharmacol. 51 (1997) 535 – 541. [6] D.A. Butterfield, J. Kanski, Brain protein oxidation in age-related neurodegenerative disorders that are associated with aggregated proteins, Mech. Ageing Dev. 1222 (2001) 945 – 962. [7] Z. Chen, C. Alcayaga, B.A. Suarez-Isla, B. O’Rourke, G. Tomaselli, E.A. Marban, ‘‘Minimal’’ sodium channel construct consisting of ligated S5-P-S6 segments forms a toxin-activatable ionophore, J. Biol. Chem. 277 (2002) 24653 – 24658. [8] P. Cohen, S. Frame, The renaissance of GSK3, Nat. Rev., Mol. Cell Biol. 2 (2001) 769 – 776. [9] R.J. Crowder, R.S. Freeman, Glycogen synthase kinase-3 beta activity is critical for neuronal death caused by inhibiting phosphatidylinositol 3-kinase or Akt but not for death caused by nerve growth factor withdrawal, J. Biol. Chem. 275 (2000) 34266 – 34271. [10] O. Cuvillier, Sphingosine in apoptosis signaling, Biochim. Biophys. Acta 1585 (2002) 153 – 162. [11] R. Dargusch, D. Schubert, Specificity of resistance to oxidative stress, J. Neurochem. 81 (2002) 1394 – 1400. [12] G.V. DeFerrari, N.C. Inestrosa, Wnt signaling function in Alzheimer’s disease, Brain Res. Rev. 33 (2000) 1 – 12. [13] B. Halliwell, Role of free radicals in the neurodegenerative diseases: therapeutic implications for antioxidant treatment, Drugs Aging 18 (2001) 685 – 716. [14] M. Hetman, J.E. Cavanaugh, D. Kimelman, Z. Xia, Role of glycogen synthase kinase-3h in neuronal apoptosis induced by trophic withdrawal, J. Neurosci. 20 (2000) 2567 – 2574. [15] P. Jenner, C.W. Olanow, Oxidative stress and the pathogenesis of Parkinson’s disease, Neurology 47 (1996) S161 – S170. [16] P.S. Klein, D.A. Melton, A molecular mechanism for the effect of lithium on development, Proc. Natl. Acad. Sci. U. S. A. 93 (1996) 8455 – 8459. [17] P. Maher, J.B. Davis, The role of monoamine metabolism in oxidative glutamate toxicity, J. Neurosci. 16 (1996) 6394 – 6401. [18] R.J. Mark, E.M. Blanc, M.P. Mattson, Amyloid beta-peptide and oxidative cellular injury in Alzheimer’s disease, Mol. Neurobiol. 12 (1996) 211 – 224. [19] B. Moosmann, C. Behl, The antioxidant neuroprotective effects of estrogens and phenolic compounds are independent from their estrogenic properties, Proc. Natl. Acad. Sci. 96 (1999) 8867 – 8872. [20] B. Moosmann, C. Behl, Antioxidants as treatment for neurodegenerative disorders, Expert Opin. Investig. Drugs 11 (2002) 1407 – 1435. [21] M. Pap, G.M. Cooper, Role of glycogen synthase kinase-3 in the phosphatidylinositol 3-kinase/Akt cell survival pathway, J. Biol. Chem. 273 (1998) 19929 – 19932. [22] Y. Sagara, S. Hendler, S. Khoh-Reiter, G. Gillenwater, D. Carlo, D. Schubert, J. Chang, Propofol hemisuccinate protects neuronal cells from oxidative injury, J. Neurochem. 73 (1999) 2524 – 2530. [23] V. Stambolic, L. Ruel, J.R. Woodgett, Lithium inhibits glycogen synthase kinase-3 activity and mimics wingless signalling in intact cells, Curr. Biol. 6 (1996) 1664 – 1668. [24] A. Takashima, K. Noguchi, K. Sato, T. Hoshino, K. Imahori, Tau protein kinase I is essential for amyloid beta-protein-induced neurotoxicity, Proc. Natl. Acad. Sci. U. S. A. 90 (1993) 7789 – 7793. [25] A. Takashima, K. Noguchi, G. Michel, M. Mercken, M. Hoshi, K. Ishiguro, K. Imahori, Exposure of rat hippocampal neurons to amyloid beta peptide (25-35) induces the inactivation of phosphatidyl inositol-3 kinase and the activation of tau protein kinase
M. Scha¨fer et al. / Brain Research 1005 (2004) 84–89 I/glycogen synthase kinase-3 beta, Neurosci. Lett. 203 (1996) 33 – 36. [26] S. Tan, N. Somia, P. Maher, D. Schubert, Regulation of antioxidant metabolism by translation initiation factor 2alpha, J. Cell Biol. 152 (2001) 997 – 1006.
89
[27] C. Yost, M. Torres, J.R. Miller, E. Huang, D. Kimelman, R.T. Moon, The axis-inducing activity, stability, and subcellular distribution of beta-catenin is regulated in Xenopus embryos by glycogen synthase kinase 3, Genes Dev. 10 (1996) 1443 – 1454.