Neuroprotective effects of ebselen are associated with the regulation of Bcl-2 and Bax proteins in cultured mouse cortical neurons

Neuroscience Letters 399 (2006) 210–214

Neuroprotective effects of ebselen are associated with the regulation of Bcl-2 and Bax proteins in cultured mouse cortical neurons Jie-Hua Xu a , Hai-Tao Hu a , Yong Liu a,b , Yi-Hua Qian a,b , Zhao-Hui Liu a , Qing-Rong Tan c , Zhang-Jin Zhang a,b,c,d,∗ a

Department of Human Anatomy and Histoembryology, College of Medicine, Xi’an Jiaotong University, Xi’an, Shaanxi 710061, China b Neuroscience Research Program, College of Medicine, Xi’an Jiaotong University, Xi’an, Shaanxi 710061, China c Department of Psychiatry, Xijing Hospital, the Fourth Military Medical University, Xi’an, Shaanxi 710032, China d Department of Psychiatry, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Road, Bethesda, MD 20814, USA Received 6 November 2005; received in revised form 1 February 2006; accepted 2 February 2006

Abstract There is little information available on the mechanisms underlying the neuroprotective actions of the organoselenium compound ebselen. In this study, we sought to determine the relationship between alterations in the expression of Bcl-2 and Bax proteins and intracellular levels of calcium and the protective effects of ebselen with a concentration range of 0.01–20 ␮M against glutamate toxicity in cultured mouse cortical neurons. Pretreatment with ebselen at moderate doses (4–12 ␮M), but not at lower or higher doses, significantly improved glutamate-induced suppression of cell viability. Pretreatment with ebselen (8 ␮M) also prevented apoptotic alterations, completely reversed the suppression of Bcl-2 expression, and significantly inhibited Bax overexpression, but did not alter elevated intracellular concentrations of calcium induced by glutamate. Pre-, co-, and post-treatment with ebselen (8 ␮M) had similar potency in improving the decreased viability of glutamate-exposed cells. These results indicate that the neuroprotective effects of ebselen at low doses are associated with the regulation of Bcl-2 and Bax proteins but appear to be independent of glutamate-mediated elevation of intracellular calcium, suggesting that different mechanisms are involved in the actions of low and high dose regimens. Ebselen may be an effective agent used for early treatment of acute brain injuries. © 2006 Elsevier Ireland Ltd. All rights reserved. Keywords: Ebselen; Glutamate; Bcl-2; Bax; Calcium; Cultured cortical neurons

The organoselenium compound ebselen (2-phenyl-1,2-benzisoselenazol-3(2H)-one) is a mimic of glutathione peroxidase, which is known to have anti-oxidant and anti-inflammatory effects [3,19]. A large body of evidence has confirmed that ebselen also possesses neuroprotective effects [2,8,12,14,15,20,22]. The protective effects of high concentrations (100–400 ␮M) of ebselen against glutamate-induced neuronal injuries have been found to be related to the blockade of excess calcium ionic influx [2]. In addition, clinical studies have shown that early treatment with ebselen significantly improved the outcomes of acute brain ischemia and stroke [10,13,18], suggesting that ebselen, like other neuroprotective agents, have the therapeutic potential for acute brain injuries and chronic neurodegenerative disorders. However, little is known about the molecular mechanisms underlying the neuroprotective actions of this compound, particularly

∗

Corresponding author. Tel.: +1 301 295 1591; fax: +1 301 295 0923. E-mail address: [email protected] (Z.-J. Zhang).

0304-3940/$ – see front matter © 2006 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2006.02.024

in regard to whether Bcl-2 family members play a role for its actions. The Bcl-2 family is a group of proteins that regulate apoptotic and non-apoptotic forms of neuronal cell death in both normal cellular development and in acute and chronic pathological insults [1]. The Bcl-2 protein family can be divided into three major subgroups based on structure and function: anti-death members (e.g., Bcl-2 and Bcl-XL ), multi-domain pro-death members (e.g., Bax and Bak); and BH3 domain-only pro-death members. Increased expression of anti-death proteins promotes neuronal survival, whereas overexpression of pro-death proteins accelerates apoptotic process [1]. Changes in expression of Bcl-2 family mRNAs and proteins in the processes of neuroprotection and neurodegenerative diseases are well documented [1]. In the present study, we sought to characterize the effects of ebselen at lower concentrations (0.01–20 ␮M) to protect against glutamate-induced neurotoxicity, to block calcium ion influx, and to alter expression of Bcl-2 and Bax proteins in primarily cultured rat cortical neurons.

J.-H. Xu et al. / Neuroscience Letters 399 (2006) 210–214

Cortical neuronal cell suspension was prepared from 15day-old fetal mice as previously described [16] and seeded on 96-well plates or 22-mm dishes precoated with poly-d-lysine (100 ␮g/ml) at a density of 1 × 106 cells/ml in Dulbecco’s Modified Eagles’s Medium (DMEM) containing 10% fetal bovine serum, 50 ␮g/ml gentamicin, and 25 mM KCl. To prevent the proliferation of non-neuronal cells, cytosine arabinofuranoside (10 ␮M) was added at 12 h after seeding. The medium was replaced periodically during the culture period. Cultures were maintained at 37 ◦ C in humidified air containing 5% CO2 . Experiments were conducted when the cultured cells reached the desired density (in general 6–7 days after seeding). Cell survival was determined by an MTT assay [7]. To determine dose–response curves of ebselen in the absence and the presence of glutamate, ebselen (Calbiochem, San Diego, CA, USA) at a concentration between 0.01 and 20 ␮M was directly added to the medium and incubated for 12 h. After that a portion of the ebselen-treated cultures were prepared for cell survival analysis and others were exposed to glutamate. To add glutamate, the cells were washed twice with Locke’s solution (154 mM NaCl, 5.6 mM KCl, 3.6 mM NaHCO3 , 2.3 mM CaCl2 , 5.6 mM glucose, and 5 mM HEPES at pH 7.4) and incubated with glutamate (1 mM) for 20 min at 20–22 ◦ C in Locke’s solution. The glutamate was then washed out and the cells continued to be cultured in the medium for 12 h, followed by harvest for the survival analysis. Meanwhile, double TUNEL and DAPI fluorescence staining was used to detect neuronal apoptosis as described previously [6]. In addition, a separate experiment was conducted to compare the effects of ebselen pre-, post-, and co-treatment in glutamateexposed cells. For these treatment regimens, the optimal concentration of ebselen, which was determined by the dose–response curve, was co-treated with glutamate (1 mM) at the same time or added at 12 h before or immediately after washout of glutamate. After treatment, cell survival was analyzed using MTT assay. Western blot analysis was conducted to determine the expression of Bcl-2 and Bax proteins in response to pretreatment with ebselen in the absence and the presence of glutamate. Cultured cells were pretreated with ebselen at an optimal concentration as described above for 12 h, followed by exposure to glutamate (1 mM) for 20 min. Cell proteins were then extracted as described previously [5]. Extracted proteins equivalent to 50–100 ␮g were resolved on 12% SDS–PAGE gels and transferred onto PVDF membranes. Non-specific binding was blocked by incubating with 5% low fat milk in TBST overnight at 4 ◦ C. Immunodetection was performed with polyclonal antibodies against Bcl-2 and Bax (1:1000) (PharMingen, Canada). The membranes were washed twice with TBST and incubated with horseradish-peroxidase conjugated anti-rabbit IgG (1:5000) for 30 min at room temperature. After washing, protein bands were quantified with the image digitizing software Un-Scan-It version 5.1 (Silk Scientific, Orem, UT, USA). ␤-Actin level was measured to ensure equal protein loading. To determine whether ebselen could prevent an increase in intracellular calcium levels stimulated by glutamate, cultured cells were pretreated with either vehicle or ebselen at an optimal concentration for 12 h and then received vehicle or 1 mM glu-

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Fig. 1. Dose–response curves of ebselen at a concentration between 0.01 and 20 ␮M in the absence (open circles) and in the presence (filled circles) of glutamate (1 mM) in cultured mouse cortical neurons. Following treatment with ebselen for 12 h, some cultured cells were exposed to glutamate for 20 min. Cell survival was measured using MTT assay. Data were expressed as mean ± S.E.M. (n = 5). * p < 0.05 vs. controls (100%); # p < 0.05 vs. glutamate-treated group (open square).

tamate treatment. Intracellular levels of calcium were measured every 5 min for 50 min after the addition of glutamate using a microspectrofluorimetry method with the sensitive calcium ion indicator fura-3/AM as described previously [9]. Data derived from cell survival and Western blot analyses were transformed to percent of controls and expressed as mean ± S.E.M. All data were obtained from four or more independent experiments. One- or two-way variance analysis (ANOVA) was used to detect statistical significance, followed by Bonferrori t-test for multiple comparisons. Statistical significance was defined as p ≤ 0.05. The dose–response curves of ebselen in the absence and the presence of glutamate are shown in Fig. 1. Treatment with ebselen alone at any concentration did not improve cell survival. Conversely, cell viability was significantly suppressed by the two highest concentrations of 15 and 20 ␮M compared to controls (F(7,32) = 70.540, p < 0.001). The exposure to glutamate resulted in a nearly 64% decrease of cell survival (shown as open square in Fig. 1) compared to controls. However, the decrease in survival rate elicited by glutamate exposure was significantly prevented by pretreatment with ebselen at moderate concentrations (4–12 ␮M, F(6,28) = 31.617, p < 0.001), but either lower (1 ␮M) or higher (16 and 20 ␮M) doses of ebselen was ineffective in preventing the glutamate-induced suppression of cell viability. Among the effective concentrations, 8 ␮M of ebselen displayed the most potent effect and was then chosen as an optimal dose for subsequent experiments. Pretreatment with 8 ␮M ebselen significantly inhibited apoptotic alterations in neuronal cells induced by glutamate (Fig. 2A–C). The number of apoptotic cells pretreated with ebselen was significantly lower than cells treated with glutamate alone, but did not differ from vehicle controls (F(2,12) = 29.238, p = 0.001, Fig. 2D). Either co- or post-treatment with ebselen (8 ␮M) did not significantly differ from pretreatment regimen in the viability of

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Fig. 2. Anti-apoptotic effects of ebselen (Ebs, 8 ␮M) in glutamate (Glu)-exposed neuronal cells examined using double TUNEL and DAPI fluorescence staining method. Cells were treated with vehicle (A) or 1 mM glutamate (B) for 20 min. (C) Cells pretreated with ebselen (8 ␮M) for 12 h and then exposed to glutamate for 20 min. Cells stained with pink fluorescence are apoptotic. The number of apoptotic cells of the three groups is plotted in (D). Data are expressed as mean ± S.E.M. (n = 5). * p < 0.05 vs. controls; # p < 0.05 vs. glutamate-treated group.

cells exposed to glutamate, despite the post-treated cells displayed a statistically significant decrease in viability compared to controls (F(4,20) = 11.703, p < 0.001, Fig. 3). Fig. 4 illustrates the changes in expression of Bcl-2 and Bax proteins observed in cultured cells exposed to ebselen (8 ␮M) or glutamate (1 mM) alone or the combination. The expression of the two proteins was unchanged in cells treated with ebselen alone compared to controls, whereas exposure to glutamate produced a significant decrease in Bcl-2 expression (F(3,12) = 21.126, p < 0.001) and a significant increase in Bax expression (F(3,12) = 68.942, p < 0.001). However, the

glutamate-induced alterations in the expression of both proteins were completely reversed by the pretreatment with ebselen. In untreated cells, the baseline concentration of intracellular Ca2+ was 60–75 nM (Fig. 5), but this rapidly increased to a peak of 360–390 nM and sustained to the endpoint of experiment when glutamate (1 mM) was added. In cultured cells, pretreated with ebselen with 8 ␮M, the changes in intracellular calcium concentration induced by glutamate was similar to cells untreated with ebselen. The present study demonstrated that pretreatment with ebselen at moderate doses (4–12 ␮M) significantly improved survival

Fig. 3. A comparison of neuroprotective effects of pre-, co-, and post-treatment with ebselen (8 ␮M) in glutamate-exposed cells. Ebselen was given at 12 h before exposed to, immediately after withdrawal of, or co-treated with glutamate (1 mM) at the same time. Data are expressed as mean ± S.E.M. (n = 5). * p < 0.05 vs. controls; # p < 0.05 vs. glutamate-treated group.

Fig. 4. Effects of 8 ␮M ebselen pretreatment with and without glutamate (1 mM) exposure on expression of Bcl-2 and Bax proteins. Western blotting bands are shown in the upper panel. Quantitative data are plotted in low panel and expressed as mean ± S.E.M. (n = 5). * p < 0.05 vs. controls; # p < 0.05 vs. glutamate-treated group.

J.-H. Xu et al. / Neuroscience Letters 399 (2006) 210–214

Fig. 5. The time course of intracellular levels of Ca2+ in cells exposed to glutamate (1 mM) following pretreatment with vehicle or 8 ␮M ebselen. Data are expressed as mean ± S.E.M. (n = 4).

and prevented apoptosis provoked by the neurotoxin glutamate in cultured mouse cortical neurons. These results suggest that even at relatively low doses, ebselen possesses neuroprotective effects, confirming the results of previous studies obtained in HT22 cells [15], cultured rat cerebellar granule neurons [12], and chick embryonic retinal cells [2]. In this study, we demonstrated that changes in the expression of Bcl-2 and Bax induced by glutamate were completely reversed by pretreatment with ebselen at the maximally effective dose of 8 ␮M. Similar results have also been observed in PC12 cells exposed to the nitric oxide donor sodium nitroprusside (SNP), in which an acute treatment with 2.5 ␮M ebselen inhibited the down-regulation of Bcl-2 expression [14]. Bcl-2 and Bax are central proteins with contrary roles in the regulation of apoptosis [1]. Previous studies have shown that glutamate neurotoxicity is reduced by enhancement of Bcl-2 expression, but Bax overexpression worsens glutamate-associated neural injuries [11,21]. Obviously, the protective effects of ebselen against glutamate toxicity observed here are, at least in part, attributed to its ability to reverse down-regulation of the antiapoptotic protein Bcl-2 and suppress up-regulation of the proapoptotic protein Bax. These findings suggest a role of Bcl-2 family members in the neurprotective actions of selenium compounds. On the other hand, the present study reveals that, despite the most apparent effects of ebselen in protecting cells from glutamate toxicity were observed at the maximally effective dose of 8 ␮M, pretreatment with this dose failed to suppress the elevation of intracellular calcium levels provoked by glutamate. This is consistent with a recent study demonstrating that high doses of ebselen (100–400 ␮M) completely block glutamate-driven calcium influx, but low doses (4–40 ␮M) do not [2], suggesting that the effects of low doses of ebselen may be unrelated to the blockade of calcium influx. There seem to be different mechanisms responsible for the actions of different dosing regimens. It has been demonstrated that glutamate excitotoxicity is mainly associated with two mechanisms—overactivation of glutamate receptors and oxidative stress—which cause an exces-

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sive influx of calcium and sodium ions and an overproduction of oxygen free radicals, resulting in cell swelling and subsequent neuronal death [4,17]. In addition to modulating glutamateactivated cation channels, ebselen is an anti-oxidant agent that has the capacity to suppress reactive oxygen species production, enhance glutathione synthesis and oxygenase activity [15,22]. Thus, it might be postulated that the anti-oxidant effects of ebselen dominate at low doses of ebselen, whereas the effects of high doses may be mainly derived from the blockade of calcium influx or both. Interestingly, the effects of ebselen between 0.01 and 20 ␮M, either in the absence or the presence of glutamate, did not appear in a dose-dependent fashion (see Fig. 1). Ebselen treatment also did not enhance the viability or alter the expression of Bcl-2 proteins in cells unexposed to glutamate. Conversely, higher doses (>12 ␮M) resulted in a significant decrease both in cell viability and protection against glutamate toxicity, suggesting that the agent lacks neurotrophic effects and, at higher concentrations, is toxic to cells. These results confirm the different effects of lower and high doses on calcium concentrations mentioned earlier. The differences in the effects between low and high dosing regimens of ebselen observed in the present study warrant further study, particularly using molecular approaches. We noticed that ebselen added after glutamate withdrawal was almost as efficient as ebselen pretreatment in enhancing cell survival. This may have important therapeutic implications, i.e., ebselen may be effective in the treatment of various brain injuries at early stage. Indeed, clinical studies have shown the effectiveness of ebselen in the early treatment of acute brain ischemia and stroke [10,13,18]. In summary, pretreatment with ebselen at moderate doses (4–12 ␮M), but not at lower or higher doses, significantly protects cells from glutamate toxicity. The protection of the moderate doses is associated with the regulation of Bcl-2 and Bax proteins, but unrelated to the blockade of glutamate-medicated calcium influx. Ebselen lacks neurotrophic effects. Pre- and post-treatment with ebselen have similar protective effects. Ebselen may be an effective agent for early treatment of brain injuries. Acknowledgements Zhang-Jin Zhang gratefully acknowledges the financial support of the Stanley Medical Research Institute, Bethesda, MD, USA. We thank Dr. Christopher J. Hough (USUHS, Bethesda, MD, USA) for reading the manuscript. References [1] R.S. Akhtar, J.M. Ness, K.A. Roth, Bcl-2 family regulation of neuronal development and neurodegeneration, Biochim. Biophys. Acta 1644 (2004) 189–203. [2] F.B. Centuriao, C.L. Corte, M.W. Paixao, A.L. Braga, G. Zeni, T. Emanuelli, J.B. Rocha, Effect of ebselen and organochalcogenides on excitotoxicity induced by glutamate in isolated chick retina, Brain Res. 1039 (2005) 146–152. [3] I.A. Cotgreave, S.K. Duddy, D. Kass, D. Thompson, P. Moldeus, Studies on the anti-inflammatory activity of ebselen: ebselen interferes with

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