GSK3β signaling is involved in fipronil-induced apoptotic cell death of human neuroblastoma SH-SY5Y cells

Toxicology Letters 202 (2011) 133–141

Contents lists available at ScienceDirect

Toxicology Letters journal homepage: www.elsevier.com/locate/toxlet

Akt/GSK3␤ signaling is involved in fipronil-induced apoptotic cell death of human neuroblastoma SH-SY5Y cells Jeong Eun Lee a , Jin Sun Kang a , Yeo-Woon Ki a , Sang-Hun Lee b , Soo-Jin Lee c , Kyung Suk Lee d , Hyun Chul Koh a,∗ a

Department of Pharmacology, College of Medicine, Hanyang University, 133-791 Seoul, Republic of Korea Department of Biochemistry and Molecular Biology, College of Medicine, Hanyang University, 133-791 Seoul, Republic of Korea Department of Occupational & Environmental Medicine, College of Medicine, Hanyang University, 133-791 Seoul, Republic of Korea d Rural Development Administration, National Academy of Agricultural Science, Republic of Korea b c

a r t i c l e

i n f o

Article history: Received 23 November 2010 Received in revised form 27 January 2011 Accepted 31 January 2011 Available online 4 February 2011 Keywords: Fipronil Human dopaminergic neuroblastoma SH-SY5Y cells Apoptosis Reactive oxygen species Akt/GSK3␤

a b s t r a c t Fipronil (FPN) is a phenylpyrazole insecticide acted on insect gamma-aminobutyric acid (GABA) receptors. Although action of FPN is restricted on insect neuronal or muscular transmitter system, a few studies have assessed the effects of this neurotoxicant on neuronal cell death. To determine the mechanisms underlying FPN-induced neuronal cell death, we investigated whether reactive oxygen species (ROS) plays a role in FPN-induced apoptosis, using an in vitro model of human dopaminergic SH-SY5Y cells. FPN was cytotoxic to these cells and its cytotoxicity showed a concentration-dependent manner. Additionally, FPN treatment significantly decreased the tyrosine hydroxylase (TH) expression without change of glutamic acid decarboxylase 65 (GAD65) expression. FPN-induced dopaminergic cell death involved in increase of ROS generation since pretreatment with N-acetyl cysteine (NAC), an anti-oxidant, reduced cell death. After FPN treatment, dopamine (DA) levels decreased significantly in both cell and culture media, and oxidative effects of DA were blocked by NAC pretreatment. We showed that cell death in response to FPN was due to apoptosis since FPN increased cytochrome c release into the cytosol and activated caspase-3. It also led to nuclear accumulation of p53 and reduced the level of Bcl-2 protein in a concentration-dependent manner. Additionally, FPN altered the level of Akt/glycogen synthase kinase-3 (GSK3␤) phosphorylation. FPN reduced the Akt phosphorylation on Ser473, and in parallel with the inactivation of Akt, phosphorylation of GSK3␤ on Ser9 which inactivates GSK3␤, decreased after treatment with FPN. Furthermore, inhibition of the GSK3␤ signal protected the cell against FPN-induced cell death. These results suggest that regulation of GSK3␤ activity may control the apoptosis induced by FPN-induced oxidative stress associated with neuronal cell death. Crown Copyright © 2011 Published by Elsevier Ireland Ltd. All rights reserved.

1. Introduction Fipronil (FPN) is a phenylpyrazole insecticide widely used in agriculture, termite/fire ant control, and in pet ectoparasite treatment (Tingle et al., 2003). It acts on inotropic GABA (GABAA ) receptors of the insect nervous system and is less toxic, as they show higher affinity to insect than mammalian transmitterreceptors (Varró et al., 2009). The available findings show that FPN is a potent toxicant for mammals though less powerful than insects (Varró et al., 2009; Dominik et al., 1998), and causes disruption of thyroid function in rats (Julien et al., 2009), cytotoxicity in human hepatocytes (Das et al., 2006) and developmental neurotoxicity in zebrafish (Stehr et al., 2006). In addition, FPN can bind

∗ Corresponding author. Tel.: +82 2 2220 0653; fax: +82 2 2292 6686. E-mail address: [email protected] (H.C. Koh).

to mammalian GABAA and GABAC receptors and may concern its risk to human health (Ikeda et al., 2001; Ratra et al., 2002; Tingle et al., 2003). Interestingly, recent data show that FPN is a more potent disruptor of neuronal cell development than chlorpyrifos in PC12 cells used as a model of neuronal development, which lack the GABAA receptor (Lassiter et al., 2009). It suggests that FPN may cause neuronal cell toxicity through a different pathway than GABAA receptors. There is substantial evidence indicating increased oxidative stress during pesticide poisoning including increased lipid peroxidation, diminished energy metabolism and decreased cytochrome oxidase activity (DiCiero Miranda et al., 2000). Dopaminergic neurons may be preferentially targeted by pesticides such as paraquat (PQ) because of their vulnerability to reactive oxygen species-mediated oxidative injury (Bonneh-Barkay et al., 2005). Compared to other neuronal cells, dopaminergic cells are much more sensitive to oxida-

0378-4274/$ – see front matter. Crown Copyright © 2011 Published by Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.toxlet.2011.01.030

134

J.E. Lee et al. / Toxicology Letters 202 (2011) 133–141

tive injury (Dinis-Oliveira et al., 2006; Lotharius and O’Malley, 2000) Neurons have several protective mechanisms against various apoptotic stimuli that activate cellular signaling pathways. The phosphatidylinositol 3-kinase (PI3K)/Akt pathway is a critical anti-apoptotic pathway (Datta et al., 1997). Akt is an upstream component of anti-apoptotic processes related to the activation of PI3K. Phosphorylated Akt promotes cell survival by inhibiting proapoptotic proteins and stimulating anti-apoptotic proteins such as caspase-9, Bad, cAMP-response element-binding protein (CREB) and NF-кB (Yoshimoto et al., 2001). Akt also phosphorylates and inhibits glycogen synthase kinase-3 (GSK3␤). GSK3␤ is a major target of PI3K/Akt and inhibition of GSK3␤ contributes to the survival promoting function of PI3K/Akt (Pap and Cooper, 1998; Hetman et al., 2000). GSK3␤ is a serine/threonine kinase that was first recognized as an enzyme capable of phosphorylating and inactivating glycogen synthase (Embi et al., 1980; Woodgett and Cohen, 1984). It is a multifunctional enzyme, affecting numerous biological functions including gene expression, cellular architecture, and apoptosis. There are two isoforms: GSK3␣ and GSK3␤, encoded by different genes (Frame and Cohen, 2001). GSK3␤ is activated by phosphorylation of tyrosine 216 and inactivated by phosphorylation of serine 9 residues. Recently, it has been shown that GSK3␤ plays a critical role in oxidative stress-induced neuronal apoptosis and the pathogenesis of neurodegenerative diseases such as Alzheimer’s disease and Parkinson’s disease (PD) (Muyllaert et al., 2008; Avila and Hernandez, 2007; Martinez et al., 2002). Moreover, 6-hydroxydopamine (6-OHDA) and rotenone induced neuronal cell death through the activation of GSK3␤ (Chen et al., 2004; King and Jope, 2005). Inhibition of GSK3␤ by lithium chloride, a GSK3␤ inhibitor, protects dopaminergic neurons from 1-methyl4-phenyl-1,2,5,6-tetrahydropyridine (MPTP)-induced dopamine (DA) depletion (Wang et al., 2007; King et al., 2001; Youdim and Arraf, 2004), suggesting that GSK3␤ may play an important role in dopaminergic cell death. In this study, we evaluated the mechanisms involved in in vitro FPN cytotoxicity. To measure FPN-induced apoptosis, we examined the activation of caspase-3, release of cytochrome c, and expression of Bcl-2 and p53 levels in human dopaminergic neuroblastoma SH-SY5Y (SH-SY5Y) cells. We focused on the Akt/GSK3␤ signaling because this has been implicated in cell survival and death.

2. Materials and methods 2.1. Cell culture SH-SY5Y cells were obtained from the American Type Culture Collection (ATCC, VA) and cultured in Dulbecco’s Modified Eagles Medium (DMEM) supplemented with 2 mM l-glutamine and 10% heat-inactivated fetal bovine serum. Cells were maintained at 37 ◦ C in a humidified 5% CO2 atmosphere. Cells used for western blot analysis were grown in 6-well cluster dishes, whereas those used for cell viability assays were grown in 96-well plates. Cells were plated at a density of 5 × 104 cells (96-well plates) and allowed to attach overnight. 40 mM FPN stock was used to make the dilutions for cell treatment. Immediately before treatment FPN addition, dilutions of FPN were made in DMSO and added to fresh cell medium to achieve the required concentration.

2.2. Reagents and antibodies FPN (Sigma–Aldrich, MO) was dissolved in DMSO. Akt, phospho-Akt (ser 473), GSK3␤, phospho-GSK3␤ (ser9), caspase-3, p53, Bcl2 and Lamin A/C were purchased from Cell Signaling Technologies, ␤-actin from Abcam and cytochrome c from Biovision. All other chemicals were obtained from Sigma–Aldrich. As FPN and Kenpaullone (Cayman, MI) were dissolved in dimethyl sulfoxide, controls were made with the highest concentration used 0.005% final concentration of DMSO. Dimethyl sulfoxide addition did not affect the viability values of control plates.

2.3. Cell viability Cell viability was measured by MTS assay (CellTiter96® AQueous One Solution Cell Proliferation Assay, Promega, WI). Briefly, MTS was added to SH-SY5Y cells in 96well plates and the plates were incubated the plate at 37 ◦ C for 4 h in a humidified 5% CO2 atmosphere. Metabolically active cells convert the yellow MTS tetrazolium compound to a purple formazan product. The latter is soluble in tissue culture medium and the quantity of formazan product as measured by the absorbance at 490 nm is directly proportional to the number of living cells in culture. Results were expressed as a percentage of the controls.

2.4. Measurement of LDH release LDH assays were used to measure the leakage of soluble cytoplasmic LDH into the extracellular medium due to cell death, using a LDH cytotoxicity detection kit (Takara, MK401, Japan). LDH converts pyruvate to lactic acid in the presence of reduced ␤-nicotinamide adenine dinucleotide (NADH), and pyruvate not converted to lactic acid produces a highly colored phenylhydrazone when treated with 2,4-dinitrophenylhydrazine. After incubation in the presence of either FPN or vehicle, culture medium was collected and centrifuged at 4000 × g for 10 min at 4 ◦ C. The resulting supernatant was used to measure LDH activity, following the manufacturer’s instructions. The reaction was run in the dark for 30 min prior to measurement, and the absorbance was measured with a multiplate reader at 492 nm. Results are expressed as percentages of the control.

2.5. Measurement of intracellular reactive oxygen species (ROS) Production of ROS was measured using an oxidation sensitive fluorescent probe, 2 ,7 -dichlorofluorescin diacetate (DCF-DA), based on the ROS-dependent oxidation of DCF-DA to DCF. Cells plated in coated 6-well plates were grown in DMEM medium and treated with 100 ␮M FPN or DMSO as control for 6 h, with or without pretreatment with the antioxidant, N-acetyl cysteine (NAC). The medium was removed and cells were washed with PBS. Then, 200 ␮l DCF-DA (10 ␮M) was added for 30 min at 37 ◦ C in the dark and the cells were washed with PBS. In general, ROS production shows concentration-dependent manner and ROS itself has short half-life (Saulsbury et al., 2009; Hu and Zhu, 2007). Therefore, for the study of FPN-induced cytotoxicity was detected at 6 h after treatment. Intracellular ROS production was measured from the fluorescence intensity. Fluorescent images were taken with an Olympus microscope.

2.6. Western blot analysis To determine levels of protein expression, we prepared extracts from the SHSY5Y cells. Adherent cells were scraped off the culture dishes and lysed by incubation with the radio-immunoprecipitation assay (RIPA) lysis buffer (50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 1% sodium deoxycholate, 1% NP-40, 0.1% SDS) containing 1 mM phenylmethylsulfonyl fluoride (PMSF), protease inhibitor cocktail, and phosphatase inhibitor cocktail (Roche, IN) on ice. Collected cells were broken by sonication on ice and centrifuged at 10,000 × g for 20 min at 4 ◦ C. Protein concentrations were determined with the Bradford reagent and 30 ␮g samples of extracted protein were resolved on SDS–polyacrylamide gels and then transferred to nitrocellulose membranes. The membranes were incubated in the presence of different primary antibodies at 4 ◦ C overnight and then the membranes were incubated with secondary antibody coupled to horseradish peroxidase. Immunoreactivity was visualized using enhanced chemiluminescence (Amersham, Buckinghamshire, England, UK). Protein bands were quantified with a densitometer (Molecular Devices, VERSAmax, CA).

2.7. Cell fractionation Cells were lysed in buffer A (0.25 M sucrose, 10 mM Tris–HCl (pH 7.5), 10 mM KCl, 1.5 mM MgCl2 , 1 mM EDTA, 1 mM dithiothreitol, 0.1 mM PMSF) by homogenizer. Homogenates were centrifuged at 750 × g for 10 min at 4 ◦ C and supernatants were collected and centrifuged at 10,000 × g for 20 min at 4 ◦ C. The supernatants were used as cytosolic fraction, and the pellet as mitochondrial fraction. The pellets were resuspended in buffer B (0.25 M sucrose, 10 mM Tris–HCl (pH 7.5), 10 mM KCl, 1.5 mM MgCl2 , 1 mM EDTA, 1 mM dithiothreitol, 0.1 mM PMSF, 1% NP40). To prepare nuclear extracts, the cells were washed twice with cold PBS and detached from plates with detaching buffer (150 mM NaCl, 1 mM EDTA (pH 8.0), 40 mM Tris–HCl (pH 7.6)) for 5 min at room temperature. The cells were then transferred to microcentrifuge tubes and centrifuged at 300 × g for 4 min at 4 ◦ C. The supernatants were discarded, and the pellets resuspended in 400 ␮l cold buffer A (10 mM Hepes (pH 7.9), 10 mM KCl, 0.1 mM EDTA, 1 mM DTT, 0.5 mM PMSF) and incubated on ice for 15 min. 10 ␮l of 10% Nonidet P-40 was added, and the mixtures were vortex briefly. Nuclei were pelleted by centrifugation at 2800 × g for 4 min at 4 ◦ C and then resuspended in 50 ␮l of ice-cold buffer B (20 mM Hepes (pH 7.9), 0.4 M NaCl, 1 mM EDTA, 1 mM DTT, 1 mM PMSF). The mixtures were shaken vigorously

J.E. Lee et al. / Toxicology Letters 202 (2011) 133–141

135

for 15 min at 4 ◦ C, centrifuged at 15,000 × g for 5 min, and the supernatants were collected. 2.8. Quantification of DA and its metabolites The levels of DA and its metabolites 3,4-dihydroxyphenylacetic acid (DOPAC), and homovanillic acid (HVA) were determined by a modified method (Mori et al., 2005). Cells in 10-cm dishes were washed 3 times with phosphate-buffered saline, and collected. Cells were homogenized in 0.2 N perchloric acid (PCA) containing 0.1 M EDTA with internal standard, centrifuged at 13,000 rpm for 10 min, and filtered though minispin filters for an additional 5 min at 13,000 rpm. Samples (culture media and cell lysate) preformed the alumina extraction and eluted in 0.1 N PCA. Samples (20 ␮l) were injected with a Rheodyne injector for separation on a reverse phase ␮-Bondapak C18 column (150 mm × 3.0 mm, Eicom, Japan) maintained at 30 ◦ C with a column heater (Waters, Cotland, NY). The mobile phase consisted of 0.05 M citric acid, 0.05 M disodium phosphate (pH 3.1), 3.2 mM 1-octanesulfonic acid (sodium salts), 0.3 mM EDTA and 12% methanol pumped at a flow rate of 0.5 ml/min with a Waters solvent delivery system. Electroactive compounds were analyzed at +750 mV using an analytical cell with an amperometric detector (Eicom, Model ECD300, Japan). Elution peaks were processed using the DS Chromatographic Software (Donam, Korea). DA and its metabolites peaks were corrected by comparison to the respective internal standard, and concentrations were calculated from external standard injected immediately before and after each experiment. The pellet was dissolved in 1 ml 0.5 N NaOH. Protein content was determined by DC protein assay kit (Bio-Rad). For further analysis, amounts of intracellular DA and its metabolites were corrected for sample protein concentration and expressed as ng/mg protein. The DA and its metabolites of medium sample were corrected for media volume and expressed as ng/ml. 2.9. Data analysis For all experiments using cell lines, data were results from at least three independent experiments, each with triplicate determinations (n ≥ 3). For western analysis, blots from at least three independent experiments were used for densitometry (n ≥ 3). Statistical comparisons were performed by one-way ANOVA followed by Student’s t-test. Error bars represent SEM. *p < 0.05; **p < 0.01; ns, not statistically significant (p > 0.05).

3. Results 3.1. Effect of FPN exposure on SH-SY5Y cell viability To examine the effects of FPN on dopaminergic cell death, SH-SY5Y cells were treated for 24 h to various concentrations of FPN or to vehicle (DMSO; final concentration 0.005%). Microscopic

Fig. 1. The FPN-induced morphological changed in SH-SY5Y cells. SH-SY5Y cells were treated for 24 h to various concentrations of FPN (0–200 ␮M) and morphological changes were analyzed. The cellular morphological changes were observed using inverted microscope (20×).

observations showed that the FPN treatment caused a significant decrease in the number of SH-SY5Y cells on concentration dependent manner (Fig. 1). Moreover, cells exposed to FPN were more rounded in appearance and had lost their projections. As shown in Fig. 2A, cell viability was significantly diminished in the presence of 50–200 ␮M of FPN, and 200 ␮M FPN was found to completely impair the cell viability (87.6%). Release of LDH into the medium was detected in FPN-treated cultures but not in control. Cell death increased with time in the presence 100 ␮M FPN (Fig. 2B). In the concentration-dependent curves shown in Fig. 2A, the IC50 value for FPN was about 100 ␮M in SH-SY5Y cells. Therefore, for the study of FPN-induced cytotoxicity, FPN was used at 100 ␮M.

Fig. 2. Effects of FPN exposure on SH-SY5Y cell viability. SH-SY5Y cells exposed to FPN after 24 h treated with serum-free media. FPN was added into the culture media in triplicate wells. SH-SY5Y cells were treated for 24 h to various concentrations of FPN (0–200 ␮M). The FPN-induced toxicity showed a concentration- and time-dependent effect in these cells. Cell viability as measured by MTS (A) after 24 h (n = 5). (B) LDH assay performed at individual time after 100 ␮M FPN was added into the media. (C) Western blot analysis of tyrosine hydroxylase (TH) and glutamic acid decarboxylase (GAD) 65 with whole cell. Error bars are standard error of the mean (SE). *p < 0.05, **p < 0.01 with the respect to the control (DMSO).

136

J.E. Lee et al. / Toxicology Letters 202 (2011) 133–141

Fig. 3. Generation of ROS by FPN causes cell death in SH-SY5Y cells. SH-SY5Y cells were labeled with DCF-DA (10 ␮M) and examined for ROS generation. The fluorescence images were taken by an Olympus microscope. Treatment with FPN (100 ␮M) produced an increase in the fluorescence due to generation of ROS, and the accumulation of ROS was reversed by the 5 mM NAC. (A) Each group was treated DMSO, FPN (100 ␮M) only and pre-treatment of 5 mM (NAC) as an anti-oxidant before the treatment of FPN, respectively. (B) Measurement of SH-SY5Y cell viability by MTS assay after 24 h (n = 4). (C and D) Changes in dopamine (DA) and its metabolites by FPN in SH-SY5Y cells. SH-SY5Y cells were incubated with 100 ␮M FPN for 12 h with or without 5 mM NAC pretreatment and then amounts of DA, DOPAC and HVA were measured by HPLC. Intracellular amounts (C) and media concentrations (D) of DA and its metabolites were measured. Error bars are standard error of the mean (SE). *p < 0.05, **p < 0.01, with the respect to the control condition. # p < 0.05, ## p < 0.01, with respect to the FPN treatment.

3.2. Effect of FPN exposure on dopaminergic and GABAergic neuron of SH-SY5Y cells To determine the involvement of dopaminergic neuron in FPNinduced neurotoxicity, we examined the effect of FPN on expression of tyrosine hyroxylase (TH), a marker for dopaminergic neuron, and glutamic acid decarboxylase 65 (GAD65), a marker for GABAergic neuron in these cells. As shown in Fig. 2C, FPN treatment significantly decreased the TH expression as compared to controls, but did not change the GAD65 expression. These results suggested that FPN affects dopaminergic neuronal component of SH-SY5Y cells without GABAergic neuronal toxicity. 3.3. FPN-induced oxidative stress in SH-SY5Y cells We measured levels of ROS in cells with DCF-DA. Treatment with FPN (100 ␮M) produced an increase of the fluorescence due to generation of intracellular ROS (Fig. 3A), and the accumulation of ROS was reversed by the simultaneous presence of 5 mM of the antioxidant NAC (Fig. 3A). In addition, we investigated the effect on cell viability of FPN after pre-treatment with NAC (5 mM). NAC partially reversed the killing effect of FPN (Fig. 3B). These results suggest that SH-SY5Y cells undergo cell death as a result of ROS produced in response to FPN treatment. 3.4. Effects of FPN on DA and its metabolites in SH-SY5Y cells To clarify the involvement of oxidative stress in FPN-induced neurotoxicity, we examined the effect of FPN on DA metabolism. Levels of DA and its metabolites were analyzed on 12 h after the treatment of FPN. As shown in Fig. 3C and D, FPN treatment significantly reduced DA levels in both cell and media to about 40–45% of

control values (p < 0.01). The treatment of FPN increased DOPAC and HVA levels in media, but intracellular HVA level did not affect (n = 5, Fig. 3C and D). NAC pretreatment partially blocked the oxidative effects of DA contents in both cell and media (Fig. 3C and D).

3.5. FPN-induced apoptosis in SH-SY5Y cells Increased ROS production has been shown to trigger release of mitochondrial cytochrome c into the cytosol, which is considered a key step in apoptosis. The released cytochrome c forms a complex with apoptotic protease activating factor-1 (Apaf-1) and caspase-9, and this initiates the apoptotic signal. Caspase-3 is a downstream effecter of caspase-9 and plays a critical role in the execution of apoptosis. As shown in Fig. 4A, FPN significantly increased the cytochrome c release. To see whether caspase-3 was involved in FPN-induced apoptosis in the SH-SY5Y cells, we measured its activation using an anti-caspase-3 antibody that recognizes both the intact and cleaved caspase-3. We found that FPN treatment activated caspase-3 in a concentration-dependent manner (Fig. 4B). Bcl-2 family members are major regulators of mitochondrial integrity and mitochondria-initiated cytochrome c release and caspase activation. The Bcl-2 family includes anti-apoptotic members such as Bcl-2, and proapoptotic members such as Bax. Bcl-2 prevents cytochrome c release by heterodimerizing with Bax. Our results showed that treatment of cell with FPN decreased the protein level of Bcl-2 in a concentration-dependant manner (Fig. 4B). The p53 expression known to be involved in apoptotic signaling in neuronal cell death was measured after treatment with FPN to determine the signaling pathway that was activated by this treatment. As shown in Fig. 4C, nuclear accumulation of p53 increased following FPN treatment and the level of cytosolic p53 enhanced.

J.E. Lee et al. / Toxicology Letters 202 (2011) 133–141

137

Fig. 4. Effect of FPN on intracellular apoptotic signaling. Western blot analysis of cytochrome c with mitochondrial/cytosolic proteins (A) capase-3, cleaved caspase-3 and Bcl-2 with whole cell (B) and p53 with nuclear/cytosolic proteins (C). Each sample (n = 3–5) was incubated 24 h with DMSO or FPN (25–100 ␮M). Samples containing 30 ␮g protein were loaded onto 15% SDS–PAGE gels, and the blots were probed with the corresponding antibodies.

3.6. FPN treatment alters the phosphorylation of Akt and GSK3ˇ We examined the level of GSK3␤ (phospho-Ser9, inactive GSK3␤) and Akt (phospho-Akt; Ser473, active Akt) by immunoblot assay. The cells treated with DMSO or FPN (25, 50 and 100 ␮M) for 24 h in serum-free media. Akt phosphorylation on Ser473, indicative of Akt activation, was reduced after 24 h of treatment with FPN (Fig. 5). As shown in Fig. 5B, when normalized to ␤-actin and compared to the DMSO, there were 36%, 56% and 82% reduction in phospho-Ser473 Akt in FPN-treated cells to each concentration, respectively. Total amounts of Akt were unaltered by treatment with FPN. FPN treatment increased the level of phospho-Akt at 3 h but it declined below baseline by 24 h (Fig. 6). GSK3␤ is a phosphorylated downstream substrate of Akt, which in its activated (dephosphorylated Ser9) state promotes cell death in response to oxidative stress. Phosphorylation levels of GSK3␤ on Ser9 decreased after 24 h of FPN treatment (Fig. 5A and B). The highest phospho-GSK3␤ level also was detectable at 3 h and decreased under base line until at least 24 h after treatment (Fig. 6). These results represented an activity effect of FPN on GSK3␤ activity correlated with inactivation of Akt. These observations are consistent with previous findings that GSK3␤ is inactivated by phosphorylation on Ser9 by Akt, and that GSK3␤ activation promotes neuronal cell death in response to 6-OHDA. 3.7. The effect of GSK3ˇ inhibition on FPN-treated SH-SY5Y cells We also examined whether inhibition of the GSK3␤ signaling pathway attenuated apoptosis, by pre-treatment with specific

inhibitors of this signaling pathway, namely LiCl and Kenpaullone. LiCl and Kenpaullone significantly reduced FPN-induced toxicity (Fig. 7), and cell viability increased at the 100 ␮M treatment with inhibitors about 19% and 65%, respectively. No significant toxicity was found when cells were incubated with 25 mM LiCl or 20 ␮M Kenpaullone alone (Fig. 7A). To further characterize the intracellular pathways involved in the cell death induced by FPN, we examined activation of caspase3, phosphorylation of GSK3␤ and Akt by western blot assays after incubation with FPN, with and without pre-treatment with LiCl and Kenpaullone (Fig. 7B). In accordance with the MTS results, FPN treatment led to significant activation of caspase-3 after 24 h and pre-incubation with 25 mM LiCl prevented the caspase-3 activation. Similar results were found for 20 mM Kenpaullone. As expected, phosphorylation of GSK3␤ on Ser9 as well as phosphorylation of Akt on Ser473 increased after pre-treatment with the GSK3␤ inhibitors. 4. Discussion The objectives of this study were to determine if FPN was toxic to SH-SY5Y cells and, if so, to examine the mechanism of FPN-induced cell death. Our results indicate that FPN induced cell death in these cells and its cytotoxic effects involved an increase in ROS generation. Furthermore, FPN activated the caspase-3 apoptotic pathway system via cytochrome c release from mitochondria. FPN-induced apoptotic cell death in SH-SY5Y cells is due to the alteration of several proteins including Bcl2, p53, Akt and GSK3␤ as a result of oxidative stress and that GSK3␤ plays a key role in this apoptotic cell death.

138

J.E. Lee et al. / Toxicology Letters 202 (2011) 133–141

Fig. 5. Effect of the down-regulation of Akt/GSK3␤ activity on FPN-induced SH-SY5Y cell death. Western blot analysis of Akt and GSK3␤ phosphorylation in SH-SY5Y cells after exposure FPN for 24 h and control. (A) Samples loaded into 12% SDS–PAGE gels 30 ␮g protein per lane. Each western blot is representative of six different experiments. (B and C) Bands of protein were quantified using scanning densitometry. p-Akt and Akt (B), p-GSK3␤ and GSK3␤ (C) ratios are shown in each panel, expressed as the percentage of the control. Error bars are standard error of the mean (SE). *p < 0.05, **p < 0.01 compared with control.

In the present study, we observed that FPN was cytotoxic to these cells and its cytotoxicity showed a concentration- and timedependent from the cell viability tests using MTS and LDH assay. SH-SY5Y cells consist of various neuronal cell components. To clar-

ify the neuronal toxicity of dopaminergic neuron, we examined the TH and GAD65 expression at 12 h after FPN treatment. As a result, we found that FPN treatment induced decrease of TH expression however, GAD65 expression did not alter. These results suggest that

Fig. 6. The time-course effect of the Akt/GSK3␤ activity on FPN-induced SH-SY5Y cell death. SH-SY5Y cells were incubated with DMSO (control, 0 h) and FPN (100 ␮M) for the indicated times (1, 3, 6, 12 and 24 h). Total proteins (30 ␮g) were analyzed for p-Akt, p-GSK3␤, total Akt, total GSK3␤ and ␤-actin by western blotting. Each blot from three independent experiments performed on SH-SY5Y cells.

J.E. Lee et al. / Toxicology Letters 202 (2011) 133–141

139

Fig. 7. The protective effect of GSK3␤-inhibitors on the FPN-induced toxicity in SH-SY5Y cells. (A) SH-SY5Y cells were pre-treated with LiCl or Kenpaullone (25 mM and 20 ␮M, respectively), then incubated with FPN (100 ␮M) for 24 h at 37 ◦ C and cell viability was assessed using MTS assay. The data are expressed as the percentage of the control (DMSO), and are the mean of 4 independent experiments with triplicate samples. *p < 0.05, **p < 0.01 compared with control. (B) After treatment with LiCl or Kenpaullone (25 mM and 20 ␮M, respectively), changes of p-GSK3␤, p-Akt and cleaved caspase-3 were monitored by western blotting. Samples loaded into 12% SDS–PAGE gels 30 ␮g protein per lane. Total proteins were analyzed for p-Akt, p-GSK3␤, cleaved caspase-3 and ␤-actin by western blotting. Each western blot is representative of three different experiments.

FPN-induced cytotoxicity affects only dopaminergic neuronal component among the other neuronal components of SH-SY5Y cells. We found that incubation of SH-SY5Y cells with FPN increased ROS production and that the FPN-induced cell death and ROS generation were attenuated by pre-treatment of antioxidant, NAC. These results indicate that oxidative stress is involved in the FPNinduced neurotoxicity in SH-SY5Y cells and is consistent with the ROS-mediated toxicity of pesticides in the same cell line. Accumulating evidence suggests that oxidative stress is one of the most important pathways leading to neuronal cell death (Jenner, 2003). Several studies have reported that ROS production by 6-OHDA and MPTP plays a primary role in the induction of neuronal damage and cause apoptosis via cytochrome c release and activation of caspase by inhibiting mitochondrial complex I (Lotharius et al., 1999; Przedborski and Vila, 2003; Perier et al., 2005). In addition, the herbicide PQ and the pesticides dieldrin and rotenone also induce the formation of ROS and induce nigrostriatal dopaminergic degeneration through a mitochondrial dysfunction (Kang et al., 2009;

Kitazawa et al., 2003; Klintworth et al., 2007; Richardson et al., 2005). These studies suggest that pesticides and environmental toxins may be involved in the neuronal cell death and implicate a systemic defect in mitochondrial complex I. In addition, FPN treatment decreased the DA levels, and increased its metabolites, and NAC treatment partially blocked the acceleration of oxidative metabolism by FPN. These studies suggest that FPN may be involved in the neuronal cell death by oxidative stress. In our previous in vivo study, exposure to PQ, a strong redox agent, increased susceptibility of dopaminergic neurons to oxidative stress and the neurotoxic mechanism involved enhancement of the oxidative pathway of DA metabolism (Kang et al., 2009). Also, in the other study demonstrated that rotenone can induced ROS formation and involved in DA redistribution to the cytosol, which in turn may plays a role in rotenone-induced apoptosis of dopaminergic cells (Watabe and Nakaki, 2007). We also showed that FPN caused caspase-3 activation and cytochrome c release from mitochondria, thus inducing apoptosis

140

J.E. Lee et al. / Toxicology Letters 202 (2011) 133–141

in the SH-SY5Y cells. Moreover, FPN altered the levels of nuclear p53 and Bcl2, also involved in apoptotic signaling. It is shown in this report that FPN accumulated nuclear p53 in these cells. p53 is known to lead to apoptosis in addition to its well known function as a transcription factor that promotes apoptosis (Vogelstein et al., 2000). Activated p53 can cause a rapid decrease in the steady-state levels of Akt and facilitate the cells more prone to apoptosis (Shen et al., 2007). Bcl2 expression correlated with the alteration of PI3K/Akt signaling, being significantly decreased after 24 h of FPN treatment. Bcl2 as an anti-apoptotic protein prevents cytochrome c release and acts as an antioxidant to block ROS-mediated apoptosis (Hockenbery et al., 1993; Kane et al., 1993; Steinman, 1995). To evaluate the role of Akt/GSK3␤ in FPN-induced neuronal cell death, we measured phospho-GSK3␤ and Akt levels. We found that FPN decreased phospho-GSK3␤ (Ser9) in parallel with inactivation of Akt (Ser473) in concentration dependent manner. Thus, inactivation of Akt in FPN treatment cells was correlated with the activation of GSK3␤. Total GSK3␤ amount and total Akt did not appear to be affected by FPN. Increased levels of phospho-Akt were seen after 3 h and 6 h with FPN but had fallen below baseline by 24 h. This is, as expected, a response to overcome external insults. Increased expression of phosphorylated Akt can occur in a variety of nervous system insults, such as during free radical exposure, whereas loss of Akt activity leads to cellular death. Concomitant with Akt activation, dephosphorylation of GSK3␤ (Ser9) was also decreased with similar pattern of Akt inactivation by FPN treatment. Namely, the treatment of FPN leads to activation of GSK3␤ and reduced Akt activity. It therefore appeared that reduction of GSK3␤ activity was an important mechanism in FPN-induced dopaminergic cell death. A recent study demonstrated that 6-OHDA (Chen et al., 2004) and MPTP (Petit-Paitel et al., 2009) can also promote GSK3␤ dephosphorylation (Ser9). To confirm the role of GSK3␤ in FPN-related cell death, we inhibited it using two selective GSK3␤ inhibitors, LiCl and Kenpaullone. Lithium is commonly used to treat bipolar disorder and is a well known GSK3␤ inhibitor (Li et al., 2007). Lithium has direct inhibitory effect of ser9-phosphorylation of GSK3␤ (Jope, 2003), and Kenpaullone is known to be a selective ATP-competitive inhibitor of GSK3␤ (Kunick et al., 2004). These agents inhibit GSK3␤, a target of PI3K/Akt signaling system. Therefore, these agents fortify the inhibitory effects of the PI3K/Akt signaling pathway on GSK3␤ (De Sarno et al., 2002). Cell viability increased as a result of LiCl and Kenpaullone treatment of SH-SY5Y cells exposed to FPN. Furthermore, cleaved caspase-3 also decreased in GSK3␤, indicating that inactivation of GSK3␤ prevents apoptotic cell death in response to FPN. A role of GSK3␤ in apoptotic processes leading to neuronal cell death has been demonstrated in several studies (Avila and Hernandez, 2007; Martinez et al., 2002; Grimes and Jope, 2001). The best known mechanism for inactivation of GSK3␤ involves phosphorylation at Ser9 by the kinase Akt (Lizcano and Alessi, 2002). Recent work demonstrates that GSK3␤ is a key determinant of apoptosis leading to neuronal cell death following ␤-amyloid, MPTP, rotenone and 6-OHDA treatments (Chen et al., 2004; King and Jope, 2005; Wang et al., 2007; Koh et al., 2008). Also, accumulating data shows that GSK3␤ contributes to the cell death of dopaminergic cells including SH-SY5Y and PC12 cells in response to various toxic insults or oxidative stress (Chen et al., 2004; King and Jope, 2005; Lee et al., 2006) and GSK3␤ activity may be relevant for Lewy body formation (Avraham et al., 2005). In addition, GSK3␤ is an important mediator of DA, and the Akt/GSK3␤ pathway might be implicated in DA-related disorders (Beaulieu et al., 2004). All these findings suggest that GSK3␤ may be involved in the neuronal cell death. In conclusion, our results help to better characterize FPNinduced neuroblastoma cell death. FPN was found to induce

cytotoxicity in SH-SY5Y cells via apoptotic pathways and Akt/GSK3␤ may play a key role in FPN-mediated neuronal cell death. Further investigation needs to be revised to precisely determine the role of GSK3␤ in FPN-induced neuronal cell death and potential toxic effect of several popular used agents including FPN against in vivo and in vitro for screening of injurious to human. Conflict of interest statement The authors state that they have no financial interest in the products mentioned within this article. Acknowledgements This work was supported by a grant from “Research Program for Agriculture Science & Technology Development (Agenda No. 4-1227, 2009)” Rural Development Administration, Republic of Korea, and by a grant from the Korea Science and Engineering Foundation (2010-0029506) through the MRC for Regulation of Stem Cell Behaviors at Hanyang University, College of Medicine, Republic of Korea. References Avila, J., Hernandez, F., 2007. GSK-3 inhibitions for Alzheimer’s disease. Expert. Rev. Neurother. 7, 1527–1533. Avraham, E., Szargel, R., Eyal, A., Rott, R., Engelender, S., 2005. Glycogen synthase kinase 3beta modulates synphilin-1 ubiquitylation and cellular inclusion formation by SIAH: implications for proteasomal function and Lewy body formation. J. Biol. Chem. 280, 42877–42886. Beaulieu, J.M., Sotnikova, T.D., Yao, W.D., Kockeritz, L., Woodgett, J.R., Gainetdinov, R.R., Caron, M.G., 2004. Lithium antagonizes dopamine-dependent behaviors mediated by an AKT/glycogen synthase kinase 3 signaling cascade. Proc. Natl. Acad. Sci. USA 101, 5099–5104. Bonneh-Barkay, D., Langston, W.J., Di Monte, D.A., 2005. Toxicity of redox cycling pesticides in primary mesencephalic cultures. Antioxid. Redox. Signal. 7, 649–653. Chen, G., Bower, K.A., Ma, C., Fang, S., Thiele, C.J., 2004. Glycogen synthase kinase 3beta(GSK3beta) mediates 6-hydroxydopamine-induced neuronal death. FASEB J. 18, 1162–1164. Das, P.C., Cao, Y., Cherrington, N., Hodgson, E., Rose, R.L., 2006. Fipronil induces CYP isoforms and cytotoxicity in human hepatocytes. Chem. Biol. Interact. 164, 200–214. Datta, S.R., Dudek, H., Tao, S., Masters, S., Fu, H., Gotoh, Y., Greenberg, M.E., 1997. Akt phosphorylation of BAD couples survival signals to the cell intrinsic death machinery. Cell 91, 231–241. De Sarno, P., Li, X., Jope, R.S., 2002. Regulation of Akt and glycogen synthase kinase-3 beta phosphorylation by sodium valproate and lithium. Neuropharmacology 43, 1158–1164. DiCiero Miranda, M., de Bruin, V.M., Vale, M.R., Viana, G.S., 2000. Lipid peroxidation and nitrite plus nitrate levels in brain tissue from patients with Alzheimer’s disease. Gerontology 46, 179–184. Dinis-Oliveira, R.J., Remião, F., Carmo, H., Duarte, J.A., Navarro, A.S., Bastos, M.L., Chavalho, F., 2006. Paraquat exposure as an etiological factor of Parkinson’s disease. Neurotoxicology 27, 1110–1122. Dominik, H., Loretta, M.C., Casida, J.E., 1998. Mechanisms for selective toxicity of fipronil insecticide and its sulfone metabolite and desulfinyl photoproduct. Chem. Res. Toxicol. 11, 1529–1535. Embi, N., Rylatt, D.B., Cohen, P., 1980. Glycogen synthase kinase-3 from rabbit skeletal muscle. Separation from cyclin-AMP-dependent protein kinase and phosphorylase kinase. Eur. J. Biochem. 107, 519–527. Frame, S., Cohen, P., 2001. GSK3 takes centre stage more than 20 years after its discovery. Biochem. J. 359, 11–16. Grimes, C.A., Jope, R.S., 2001. The multifaceted roles of glycogen synthase kinase 3beta in cellular signaling. Prog. Neurobiol. 65, 391–426. Hetman, M., Cavanaugh, J.E., Kimelman, D., Xia, Z., 2000. Role of glycogen synthase kinase-3␤ in neuronal apoptosis induced by trophic withdrawal. J. Neurosci. 20, 2567–2574. Hockenbery, D.M., Oltvai, Z.N., Yin, X.M., Milliman, C.L., Korsmeyer, S.J., 1993. Bcl-2 functions in an antioxidant pathway to prevent apoptosis. Cell 75, 241–251. Hu, J.M., Zhu, X.Z., 2007. Rotenone-induced neurotoxicity of THP-1 cells requires production of reactive oxygen species and activation of phosphatidylinositol-3kinase. Brain Res. 1153, 12–19. Ikeda, T., Zhao, X., Nagata, K., Kono, Y., Yeh, J.Z., Narahashi, T., 2001. Fipronil modulation of gamma-aminobutyric acid(A) receptors in rat dorsal root ganglion neurons. J. Pharmacol. Exp. Ther. 296, 914–921. Jenner, P., 2003. Oxidative stress in Parkinson’s disease. Ann. Neurol. 3, 26–36. Jope, R.S., 2003. Lithium and GSK-3: one inhibitor, two inhibitory actions, multiple outcomes. Trends Pharmacol. Sci. 24, 441–443.

J.E. Lee et al. / Toxicology Letters 202 (2011) 133–141 Julien, L., Véronique, G., Nicole, P.H., Marion, C., 2009. Fipronil-induced disruption of thyroid function in rats is mediated by increased total and free thyroxine clearances concomitantly to increased activity of hepatic enzymes. Toxicology 255, 38–44. Kane, D.J., Sarafian, T.A., Anton, R., Hahn, H., Gralla, E.B., Valentine, J.S., Ord, T., Bredesen, D.E., 1993. Bcl-2 inhibition of neural death: decreased generation of reactive oxygen species. Science 262, 1274–1277. Kang, M.J., Gil, S.J., Koh, H.C., 2009. Paraquat induces alternation of the dopamine catabolic pathways and glutathione levels in the substantia nigra of mice. Toxicol. Lett. 188, 148–152. King, T.D., Bijur, G.N., Jope, R.S., 2001. Caspase-3 activation induced by mitochondrial complex I is facilitated by glycogen synthase kinase-2beta and attenuated by lithium. Brain Res. 919, 106–114. King, T.D., Jope, R.S., 2005. Inhibition of glycogen synthase kinase-3 protects cells from intrinsic but not extrinsic oxidative stress. Neuroreport 16, 597–601. Kitazawa, M., Anantharam, V., Kanthasamy, A.G., 2003. Dieldrin induces apoptosis by promoting caspase-3-dependent proteolytic cleavage of protein kinase Cdelta in dopaminergic cells: relevance to oxidative stress and dopaminergic degeneration. Neuroscience 119, 945–964. Klintworth, H., Newhouse, K., Li, T., Choi, W.S., Faigle, R., Xia, Z., 2007. Activation of c-Jun N-terminal protein kinase is a common mechanism underlying paraquat- and rotenone-induced dopaminergic cell apoptosis. Toxicol. Sci. 97, 149–162. Koh, S.H., Noh, M.Y., Kim, S.H., 2008. Amyloid-beta-induced neurotoxicity is reduced by inhibition of glycogen synthase kinase-3. Brain Res. 1188, 254–262. Kunick, C., Lauenroth, K., Leost, M., Meijer, L., Lemcke, T., 2004. 1-Azakenpaullone is a selective inhibitor of glycogen synthase kinase-3 beta. Bioorg. Med. Chem. Lett. 14, 413–416. Lassiter, T., Mackillop, E.A., Ryde, I.T., Seidler, F.J., Slotkin, T.A., 2009. Is fipronil safer than chlorpyrifos? Comparative developmental neurotoxicity modeled in PC12 cells. Brain Res. Bull. 78, 313–322. Lee, C.S., Park, W.J., Ko, H.H., Han, E.S., 2006. Differential involvement of mitochondrial permeability transition in cytotoxicity of 1-methyl-4-phenylpyridinium and 6-hydroxydopamine. Mol. Cell. Biochem. 289, 193–200. Li, X., Friedman, A.B., Zhu, W., Wang, L., Boswell, S., May, R.S., Davis, L.L., Jope, R.S., 2007. Lithium regulates glycogen synthase kinase-3beta in human peripheral blood mononuclear cells: implication in the treatment of bipolar disorder. Biol. Psychiatry 61, 216–222. Lizcano, J.M., Alessi, D.R., 2002. The insulin signalling pathway. Curr. Biol. 12, 236–238. Lotharius, J., Dugan, L.L., O’Malley, K.L., 1999. Distinct mechanisms underlie neurotoxin-mediated cell death in cultured dopaminergic neurons. J. Neurosci. 19, 1284–1293. Lotharius, J., O’Malley, K.L., 2000. The parkinsonism-inducing drug 1-methyl4-phenylpyridinium triggers intracellular dopamine oxidation. A novel mechanism of toxicity. J. Biol. Chem. 275, 38581–38588. Martinez, A., Alonso, M., Castro, A., Pérez, C., Moreno, F.J., 2002. First non-ATP competitive glycogen synthase kinase 3 beta (GSK-3beta) inhibitors: thiadiazolidinones (TDZD) as potential drugs for the treatment of Alzheimer’s disease. J. Med. Chem. 45, 1292–1299. Mori, A., Ohashi, S., Nakai, M., Moriizumi, T., Mitsumoto, Y., 2005. Neural mechanisms underlying motor dysfunction as detected by the tail suspension test in MPTPtreated C57BL/6 mice. Neurosci. Res. 51, 265–274.

141

Muyllaert, D., Kremer, A., Jaworski, T., Borghgraef, P., Devijver, H., 2008. Glycogen synthase kinase-3beta, or a link between amyloid and tau pathology? Genes Brain Behav. 7 (Suppl. 1), 57–66. Pap, M., Cooper, G.M., 1998. Role of glycogen synthase kinase-3 in the phosphatidylinositol 3-kinase/Akt cell survival pathway. J. Biol. Chem. 273, 19929–19932. Perier, C., Tieu, K., Guégan, C., Caspersen, C., Jackson-Lewis, V., Carelli, V., Martinuzzi, A., Hirano, M., Przedborski, S., Vila, M., 2005. Complex I deficiency primes Baxdependent neuronal apoptosis through mitochondrial oxidative damage. Proc. Natl. Acad. Sci. U. S. A. 102, 19126–19131. Petit-Paitel, A., Brau, F., Cazareth, J., Chabry, J., 2009. Involvement of cytosolic and mitochondrial GSK-3beta in mitochondrial dysfunction and neuronal cell death of MPTP/MPP-treated neurons. PLoS One 4, e5491. Przedborski, S., Vila, M., 2003. The 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine mouse model: a tool to explore the pathogenesis of Parkinson’s disease. Ann. N. Y. Acad. Sci. 991, 189–198. Ratra, G.S., Erkkila, B.E., Weiss, D.S., Casida, J.E., 2002. Unique insecticide specificity of human homomeric rho 1 GABA(C) receptor. Toxicol. Lett. 129, 47–53. Richardson, J.R., Quan, Y., Sherer, T.B., Greenamyre, J.T., Miller, G.W., 2005. Paraquat neurotoxicity is distinct from that of MPTP and rotenone. Toxicol. Sci. 88, 193–201. Saulsbury, M.D., Heyliger, S.O., Wang, K., Johnson, D.J., 2009. Chlorpyrifos induces oxidative stress in oligodendrocyte progenitor cells. Toxicology 259, 1–9. Shen, J.H., Zhan, Y., Wu, N.H., Shen, Y.F., 2007. Resistance to geldanamycin-induced apoptosis in differentiated neuroblastoma SH-SY5Y cells. Neurosci. Lett. 414, 110–114. Stehr, C.M., Linbo, T.L., Incardona, J.P., Scholz, N.L., 2006. The developmental neurotoxicity of fipronil: notochord degeneration and locomotor defects in zebrafish embryos and larvae. Toxicol. Sci. 92, 270–278. Steinman, H.M., 1995. The Bcl-2 oncoprotein functions as a pro-oxidant. J. Biol. Chem. 270, 3487–3490. Tingle, C.C., Rother, C.F., Lauer, D.S., King, W.J., 2003. Fipronil: environmental fate, ecotoxicology, and human health concerns. Rev. Environ. Contam. Toxicol. 176, 1–66. ˝ J., Világi, I., 2009. In vitro effects of fipronil on neuronal excitability Varró, P., Gyori, in mammalian and molluscan nervous systems. Ann. Agric. Environ. Med. 16, 71–77. Vogelstein, B., Lane, D., Levine, A.J., 2000. Surfing the p53 network. Nature 408, 307–310. Wang, W., Yang, Y., Ying, C., Li, W., Ruan, H., Zhu, X., You, Y., Han, Y., Chen, R., Wang, Y., Li, M., 2007. Inhibition of glycogen synthase kinase-3beta protects dopaminergic neurons from MPTP toxicity. Neuropharmacology 52, 678–684. Watabe, M., Nakaki, T., 2007. Mitochondrial complex 1 inhibitor rotenone-elicited dopamine redistribution from vesicles to cytosol in human dopaminergic SHSY5Y cells. J. Pharmacol. Exp. Ther. 323, 499–507. Woodgett, J.R., Cohen, P., 1984. Multisite phosphorylation of glycogen synthase Molecular basis for the substrate specificity of glycogen synthase kinase-3 and casein kinase-II(glycogen synthase kinase-5). Biochem. Biophys. Acta 788, 339–347. Yoshimoto, T., Uchino, H., He, Q.P., Li, P.A., Siesjo, B.K., 2001. Cyclosporin A, but not FK506, prevents the downregulation of phosphorylated Akt after transient focal ischemia in the rat. Brain Res. 899, 148–158. Youdim, M.B., Arraf, Z., 2004. Prevention of MPTP (N-methyl-4-phenyl-1,2,3,6tetra-hydropyridine) dopaminergic neurotoxicity in mice by chronic lithium: involvements of Bcl-2 and Bax. Neuropharmacology 46, 1130–1140.