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GSK3β signaling pathway
Schizophrenia Research 157 (2014) 120–127
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Paliperidone protects SK-N-SH cells against glutamate toxicity via Akt1/GSK3β signaling pathway Lei Peng a, Xingzhen Zhang a, Xianping Cui b, Dexiao Zhu a, Jintao Wu a, Dong Sun c, Qingwei Yue a, Zeyan Li a, Haili Liu a, Guibao Li a, Jing zhang a, Hongyan Xu c, Fuchen Liu d, Chengkun Qin b, Mingfeng Li d, Jinhao Sun a,⁎ a
Department of Anatomy and Shandong Provincial Key Laboratory of Mental Disorders, School of Medicine, Shandong University, Jinan, Shandong 250012, PR China Department of General Surgery, Provincial Hospital Affiliated to Shandong University, Jinan, Shandong 250012, PR China Experimental Platform for Medical Function, School of Medicine, Shandong University, Jinan, Shandong 250012, PR China d Department of Neurobiology, Yale University School of Medicine, New Haven, CT 06510, USA b c
a r t i c l e
i n f o
Article history: Received 20 January 2014 Received in revised form 21 May 2014 Accepted 31 May 2014 Available online 22 June 2014 Keywords: Paliperidone Protection Schizophrenia SK-N-SH Glutamate
a b s t r a c t Schizophrenia is a heterogeneous psychotic illness and its etiology remains poorly understood. Recent studies have suggested that neurodegeneration is a component of schizophrenia pathology and some atypical antipsychotics appear to slow progressive morphological brain changes. In addition, the atypical antipsychotics were reported to have a superior therapeutic efficacy in treating schizophrenia and have a low incidence of extrapyramidal side effects (EPS) compared to typical antipsychotics. However, the mechanisms of atypical antipsychotics in treating schizophrenia and the basis for differences in their clinical effects were still totally unknown. In the present study, we investigated whether paliperidone shows protective effects on SK-N-SH cells from cell toxicity induced by exposure to glutamate. We examined the effects of the drugs on cell viability (measured by MTT metabolism assay and lactate dehydrogenase (LDH) activity assay), apoptosis rate, ROS levels and gene expression and phosphorylation of Akt1 and GSK3β. The results showed that paliperidone significantly increases the cell viability by MTT and LDH assays (p b 0.05), in contrast to the typical antipsychotic (haloperidol), which had little neuroprotective activity. Moreover, paliperidone retarded the glutamate-mediated promotion of ROS and the rate of apoptosis (p b 0.05). In addition, paliperidone also effectively reversed glutamate-induced decreases of gene expression and phosphorylation of Akt1 and GSK3β (both p b 0.05). Our results demonstrated that paliperidone could effectively protect SK-N-SH cells from glutamate-induced damages via Akt1/GSK3β signaling pathway. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Schizophrenia is a severe chronic debilitating disease with a lifetime prevalence of 1% of the world's population (Frangou and Murray, 1996). Although schizophrenia is considered to be a neurodevelopmental disorder, there is evidence that it is also progressive and possibly neurodegenerative. Post-hoc studies showed that schizophrenia patients may have progressive ventricular dilation (Johnstone and Frith, 1996). Moreover, brain volume reductions and cortical and subcortical gray matter loss were also found in patients (Massana et al., 2005). All of these observations strongly suggest that neurodegeneration occurred during the pathogenesis of schizophrenia. Antipsychotic drugs modulate dysfunction in chemical neurotransmission, which also plays an important role in relieving the symptoms of schizophrenia. Although typical antipsychotics, such as haloperidol, produce a marked reduction in the positive symptoms of schizophrenia, they do not overcome negative symptoms, such as confusion, apathy, ⁎ Corresponding author. E-mail address: [email protected] (J. Sun).
http://dx.doi.org/10.1016/j.schres.2014.05.037 0920-9964/© 2014 Elsevier B.V. All rights reserved.
and social withdrawal (Lindenmayer et al., 2007). Accumulated evidences suggested that some second generation antipsychotics (SGAs) might offer neuroprotective benefits (Kurosawa et al., 2007; Kim et al., 2008), due to therapeutic properties such as cognitive enhancement or prevention of disease progression and clinical deterioration (Lieberman et al., 2008). Studies in vitro have demonstrated that second-generation antipsychotics offer protection against SH-SY5Y cell apoptosis induced by serum withdrawal (Kim et al., 2008) and cytotoxin-induced apoptosis in the pheochromocytoma (PC12) cells (Qing et al., 2003). However, the underlying molecular mechanisms have not been fully understood. More research is needed to identify the effects of second-generation antipsychotics on cell survival and death, and to investigate the association between such effects and the drugs' therapeutic properties on schizophrenia. Paliperidone has been reported to have better efficacy and tolerability than other atypical antipsychotics in acute and long-term treatment of schizophrenia (Fowler et al., 2008). Also, paliperidone offers protective effects against dopamine induced SK-N-SH cell damages by decreasing caspase-3 levels (Gassó et al., 2012), and enhance the survival of SH-SY5Y and U937, two cell lines separately representing neural and
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immune cell types which are involved in the pathogenesis of schizophrenia (Schmidt et al., 2010). However, the protective effects of paliperidone on SK-N-SH cells and its underlying mechanisms are largely elusive. During the past few years several studies have shown direct evidences for the involvement of different signaling pathways in schizophrenia. Among those, the original study of Akt1/GSK3β signaling pathway exhibited evidence for a decrease in Akt1 protein levels and levels of phosphorylation of GSK3β in schizophrenia (Emamian et al., 2004). Akt1 is the most highly expressed isoform of Akt involved in complicated cellular processes, such as transcription, glucose metabolism, cell proliferation, apoptosis, angiogenesis and cell motility (Kandel and Hay, 1999). As the major Akt1 target, GSK3β was also found altered in individuals with schizophrenia (Emamian et al., 2004). Research also showed that phosphorylation and protein level of GSK3β in the frontal cortex, specifically the GSK3β mRNA level in the dorsolateral prefrontal cortex was changed in patients with schizophrenia by post-hoc studies (Kozlovsky et al., 2004; Koros and Dorner-Ciossek, 2007). Thus, we propose that paliperidone may protect SK-N-SH cells from damages induced by glutamate through Akt1/ GSK3β signaling pathway. In the current study, we investigated the protective effects of paliperidone against glutamate induced cell damages in SK-N-SH cell line. Cell viability was measured by MTT and LDH assays to demonstrate the effects of paliperidone on glutamate induced cell damages. Furthermore, real-time PCR and western blotting were applied to determine whether paliperidone offers protection via the Akt1/GSK3β signaling pathway. The results provide information on how paliperidone exerts protection effects and contribute to the understanding of better antipsychotic therapy effects of paliperidone. 2. Experimental procedures 2.1. Materials SK-N-SH cells were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS) were purchased from GIBCO BRL (Grand Island, NY, USA). Glutamate, haloperidol and MTT assay kit were purchased from Sigma-Aldrich (St. Louis, MO, USA). Paliperidone was synthesized by Jinan Weidu Chemical Company (Shandong, China). All other chemicals were purchased from commercial sources. 2.2. Establishment of neurotoxic cell model with glutamate SK-N-SH cells were grown in DMEM, which was supplied with 10% fetal bovine serum. Cultures were maintained at 37 °C, 5% CO2 until 80% confluence, and the medium was changed every three days. Confluent monolayers were passaged routinely by trypsinization. Before drug treatment, the cells were seeded at 2 × 104/cm2 in poly-L-lysine coated culture plates. After 24 h of in vitro culture, SK-N-SH cells were exposed to 1, 10 and 50 mM glutamate. 2.3. Protection of paliperidone on cultured cells SK-N-SH cells were cultured in 24-well plates at 37 °C in the presence of 5% CO2. The cultured cells were divided into the following groups: the first group was the normal control without the treatment of glutamate or paliperidone in culture medium. In the second group, 10 mM glutamate was used to establish the cell injury model. The third group, termed paliperidone protection group, was subdivided into three small groups that were co-treated with 50, 100 and 200 μM paliperidone at the same time as glutamate exposure. In the fourth group, i.e. haloperidol treated group, cells were co-cultured with 100 μM haloperidol and 10 μM glutamate. After 24 h incubation, cells were collected from all groups and subjected to the subsequent examinations.
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2.4. Cell viability assay Cell viability was assessed by MTT assay, which reflects the intracellular redox state through mitochondrial metabolic activity of viable cells. The neuronal viability was measured using the MTT assay first. Briefly, after treated with different drugs, the cultured cells were subjected to MTT solution (0.1 mg/ml) at 37 °C for 4 h. The culture medium was then removed and dimethyl sulfoxide was added into medium to dissolve the formazan product while shaking for 10 min at room temperature. The fluorescent absorbance was then quantified spectrophotometrically at 492 nm using a microplate reader (Multiskan MK3, Thermo Labsystem, USA). The quantity of living cells was indicated indirectly by the optical density (OD) of absorbance of formazan. To confirm the assessment of cell viability and the neurotoxicity of glutamate, LDH activity assay was also performed. LDH assay measures the extracellular release of cytosolic lactate dehydrogenase due to cytotoxicity. The amount of LDH applied to the cultured media was determined according to the manufacturer's protocol (Jiancheng Bioengineering Institute, Nanjing, China) and OD was measured at 450 nm via a microplate reader (Multiskan MK3, Thermo Labsystem, USA). The mean value of each condition was normalized to the normal group. 2.5. Apoptosis analysis In order to determine whether paliperidone protects SK-N-SH cells against glutamate induced cell apoptosis, Hoechst 33342-PI double staining was performed. The cultured cells were treated with 10 μg/ml Hoecst33342 and PI for 15 min at 37 °C in the dark. Then labeled cells were observed through a LSM 780 Laser Scanning Confocal Microscope (Carl Zeiss SAS, Jena, Germany) with 200× magnifications. The apoptosis rate was calculated according to a previous report (Xiao Q. et al., 2010). Moreover, flow cytometry with Annexin V-FITC and propidium iodide (PI) double staining was carried out to give a quantitative analysis of apoptosis. In short, SK-N-SH cells cultured in different groups were collected with 0.125% trypsin, then washed with PBS for three times. 5 × 105 cells were stained with Annexin V-FITC and PI for 15 min in the dark afterwards. Subsequently, the fluorescence of each group was analyzed by flow cytometry (Becton Dickinson, Franklin Lakes, NJ, USA). 2.6. Determination of reactive oxygen species (ROS) levels Intracellular ROS was detected using the non-fluorescent probe 2′, 7′-dichlorofluorescein diacetate (DCFH-DA). DCFH-DA is oxidized into the fluorescent compound 2′, 7′-dichlorofluorescein (DCFH) under the presence of ROS in SK-N-SH cells. Briefly, 10 μM (the volume was 0.5 ml) DCFH-DA was added to the SK-N-SH cell suspension prepared freshly before the assay. The mixture was incubated at 37 °C in the dark for 20 min and mixed every 5 min. After the incubation period, the cells were washed three times with serum-free DMEM. Fluorescence intensity was measured with 485 nm excitation and 530 nm emissions using flow cytometry (Cantoll, BD, USA). The increasing production of ROS was expressed as a percentage to the control. 2.7. mRNA expressions of Akt1 and GSK3β To explore the mechanisms of paliperidone in protecting SK-N-SH cells from glutamate induced cell damage, the mRNA expression of Akt1 and GSK3β in different experimental groups was assayed. Total RNA was extracted from SK-N-SH cells in each group using TRIzol (Sangon Biotech Co., Ltd. Shanghai, China) and reverse transcribed into cDNA according to the manufacturer's instruction. Then, the cDNA was subjected to real-time PCR on Mastercycler ep realplex (Eppendorf, Hamburg, Germany). PCR was performed at the conditions of 95 °C for 2 min, followed by 35 cycles of 95 °C for 15 s, 60 °C for 15 s,
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and 68 °C for 20 s. The following primers were used in this: Akt1 primer pairs 5′-GGTATTTTGATGAGGAGTTCACG-3′ and 5′-GAGTAGGAGAACTG GGGGAAGT-3′ and GSK3β primer pairs 5′-GGCTACCATCCTTATTCCTC CT-3′ and 5′-GTCTGTCCACGGTCTCCAGTAT-3′, as well as control GAPDH primer pairs 5′-GAAGGTGAAGGTCGGAGTC-3′ and 5′-GAAGAT GGTGATGGGATTTC-3′, respectively. Akt1 and GSK3β mRNA abundance was calculated using comparative threshold cycle (2−ΔΔCT) method and normalized to internal control, GAPDH. 2.8. Protein levels of phospho-Akt1 (Ser 473) and phospho-GSK3β (Ser 9) SK-N-SH cells were treated with 10 mM glutamate in the presence or absence of 100 μM paliperidone for 2 h. Then, the cells were washed with ice-cold PBS and harvested in RIPA lysis buffer (Beyotime Institute of Biotechnology, Shanghai, China) containing protease inhibitors, which prevent protein degradation and dephosphorylation. After the total protein concentrations were measured with BCA method (Boster Biological Technology, Wuhan, China), 20 μg proteins in each group were subjected to 10% sodium dodecyl sulfate (SDS) polyacrylamide gel/tris-glycine electrophoresis, then transferred to nitrocellulose (NC) membranes and blocked with 5% nonfat milk in PBS containing 0.1% Tween 20 (PBS-T) for 2 h. Subsequently, the membranes were incubated with primary antibodies against Akt (1:1000, Beyotime Institute of Biotechnology), GSK3β (1:1000, Beyotime Institute of Biotechnology), phosphorylated Akt1 (p-Akt1) (1:100, Cell signaling Technology) and phosphorylated GSK3β (p-GSK3β) (1:100, Cell signaling Technology) at 4 °C overnight. Then the membranes were incubated with HRP labeled secondary IgG (1:2000) for 1 h at room temperature. Finally, the membranes were assayed with a chemiluminescence system (ECL system) and protein level was quantified by Image J software (National Institute of Health, USA). The levels of phosphorylated Akt and GSK3β were normalized to total Akt and GSK3, respectively. 2.9. Statistical analysis Statistical analysis was performed using the SPSS 17.0 software. Data were expressed as means ± S.E.M relative to that of vehicle for each condition. Analysis of variance (ANOVA) or student's t-test was applied,
followed by the Limited Slip Differential (LSD) post-hoc tests. A p value b 0.05 was regarded as statistically significant.
3. Results 3.1. Paliperidone promotes cell growth and increased cell viability against glutamate In order to establish the neurotoxic cell model with glutamate, SK-NSH cells were subjected to a series concentration of glutamate concentrations (1, 10, 50 mM). At 1 mM, glutamate (Fig. 1B) did not display a significant effect on cell morphology compared to cultured cells in normal medium (Fig. 1A) which grew well with long neuritis and bright glow. When the cells were exposed to 10 mM glutamate, neuritis retracted and cell debris appeared (Fig. 1C). As the concentration of glutamate rises to 50 mM, almost all the neuritis were destroyed and more cell debris were present in the culture (Fig. 1D). In order to give a quantitative analysis of damage effects induced by glutamate, MTT and LDH assays were performed (Fig. 1E and F). MTT measurement showed that cell viability gradually decreased as the concentration of glutamate increased from 1 mM to 50 mM. Consistent with morphological observations, cells treated with 10 mM and 50 mM glutamate were obviously damaged compared to control groups (p b 0.05). The results of LDH assay confirmed the observations by the MTT assay. Based on our experiment, 10 mM glutamate was selected to induce cell damages of SK-N-SH cells. In order to investigate the protective effects of paliperidone, SK-N-SH cells were treated with a series concentration of paliperidone (50 μM, 100 μM, and 200 μM) in the presence of 10 mM glutamate (Fig. 2). The results showed that 100 μM and 200 μM paliperidone treatment effectively increased cell viability and few cell debris appeared. However, 50 μM of paliperidone's protection effects was not obvious, a considerable number of cell debris still appeared in the cell culture. MTT and LDH release assays were also performed to investigate the protective effects of paliperidone. As shown in Fig. 2, paliperidone treatment reversed the glutamate-induced the decrease of cell viability in MTT assay and LDH release (Fig. 2E, F), suggesting that 100 μM paliperidone effectively protected SK-N-SH cells from the damages induced by glutamate (p b 0.05 for MTT, p b 0.01 for LDH).
Fig. 1. Survival of SK-N-SH cells exposed to glutamate. SK-N-SH cells were cultured for 12 h with 0 mM (A), 1 mM (B), 10 mM (C), and 50 mM (D) glutamate, respectively. (E) Effects of glutamate on the viability of SK-N-SH measured by MTT assay. (F) Effects of glutamate on the release of LDH by LDH release assay. OD values were mean ± S.E.M. from five independent experiments. *p b 0.01 vs control group. Scale bars: A–D, 100 μm.
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Fig. 2. Protection of paliperidone against glutamate induced neuronal damage. (A–D) The cells were treated with 0 (A), 50 μM (B), 100 μM (C), 200 μM (D) paliperidone as 10 mM glutamate exposure, respectively. As the concentration of paliperidone increased, the protective effects of paliperidone against cell damage induced by glutamate gradually enhanced. Cell viability was further assessed by MTT (E) and LDH (F) assays. The comparison of protective effects against glutamate between paliperidone and haloperidol was performed by MTT (G) and LDH (H) measures. OD values represent mean ± S.E.M. from five independent experiments. *p b 0.05 vs 0 μM paliperidone group, **p b 0.01 vs 0 μM paliperidone group, and #p b 0.05 vs 100 μM paliperidone group. Scale bars: A–D, 100 μm.
In order to study whether paliperidone has a superior protective effect against glutamate compared to typical antipsychotics (haloperidol), the protective effects of 100 μM haloperidol were examined. The concentration of haloperidol was selected according to a previous study (Lindenmayer et al., 2007). The MTT and LDH results showed that 100 μM paliperidone has a better protective effect than 100 μM haloperidol (Fig. 2G, H, both p b 0.05). 3.2. Paliperidone prevent glutamate induced cell apoptosis Hoechst 33342 and PI double staining were carried out to investigate the effects of paliperidone on glutamate induced apoptosis. Surviving cells were stained with bright blue integrated nuclei (Fig. 3) while the
apoptotic cells had bright red fragmented nuclei (Fig. 3). In glutamate injury group, the number of cells with bright red nuclei was increased compared to normal control group with only few apoptotic cells observed. By contrast, the number of apoptotic nucleoli decreased in paliperidone protection group. To further investigate the effects of paliperidone on apoptosis, flow cytometry was deployed with Annexin V-FITC and PI double staining. Fig. 3 showed that 10 mM glutamate increased the average rate of apoptosis from 3.2% to 8.4%, indicating that 10 mM glutamate effectively induces cell apoptosis. However, when co-cultured with 100 μM paliperidone, apoptotic cells were largely decreased in number, demonstrating that paliperidone can protect SK-N-SH cells from apoptosis induced by glutamate.
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Fig. 3. Paliperidone prevents glutamate induced cell apoptosis. SK-N-SH cells were exposed to glutamate with or without paliperidone for 24 h and double stained with Hoechst 33342 (blue) and PI (red) (C, F, and I) or subjected to flow cytometry assay (J, K, and L) to determine cell apoptosis. (A, B, C) Normal control group: cells in DMEM medium; (D, E, F) glutamate treated group: cells in 10 mM glutamate; (G, H, I) paliperidone protection group: cells in 10 mM glutamate + 100 μM paliperidone; J, K and L were the results of flow cytometry of the control, glutamate injury group and paliperidone protection group, respectively. Statistical analysis suggested that paliperidone significantly reduced cell apoptosis induced by glutamate treatment (p b 0.01, n = 5). Pictures of A–I were captured with LSM 780 laser confocal Zeiss microscope at 200× magnification. Scale bars: A–I, 100 μm.
3.3. Paliperidone decreases ROS levels ROS levels can reflect the degree of oxidative damage induced by glutamate. Excessive ROS may cause several kinds of irreversible oxidative damage to cellular components (such as proteins, lipids and nucleic acids), and activate signaling pathways ultimately leading to cell death. Fig. 4 showed the level of ROS treated with different drugs. In glutamate injury group, the ROS level was significantly increased compared to control group (from 9.22% to 16.48%, p b 0.05). By contrast, the addition of paliperidone effectively attenuated the fluorescence intensity (p b 0.05). Co-treatment with paliperidone and glutamate only slightly increased the level of ROS (Fig. 4C), suggesting
that paliperidone can effectively protect SK-N-SH cells from glutamateinduced damage by inhibiting the generation of ROS. 3.4. Paliperidone up-regulating Akt1-GSK3β expressions To study the molecular mechanism of paliperidone on protecting cells from glutamate-induced damages, we measured the gene expression level of Akt1 and GSK3β, both of which were involved in the pathogenesis of schizophrenia. Real-time PCR and Western blot were both performed to detect Akt1 and GSK3β expression. When SK-N-SH cells were exposed to glutamate, Akt1 and GSK3β mRNA expression was decreased and this inhibition effect can be reversed by paliperidone
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Fig. 4. Paliperidone decreases ROS levels. SK-N-SH cells were treated with glutamate and paliperidone for 24 h for ROS detection. (A–C) The fluorescence intensity of the cells stained with DCFH-DA and recorded by flow cytometer. There is significantly statistical difference between cells in control group and cells treated with glutamate (p b 0.01, n = 5), and between glutamate group and paliperidone protection group (p b 0.05, n = 5).
(p b 0.05, Fig. 5D). The level of phosphorylated Akt1 and GSK3β, which was commonly used as indicator of functional Akt1 and GSK3β in schizophrenia, was detected by Western blot (Fig. 5A–C). Results showed that glutamate treatment significantly decreased the levels of phosphorylated Akt1 and GSK3β (p b 0.05); and these effects diminished with paliperidone treatment (p b 0.05).
3.5. Akt1 inhibitor LY294002 partly attenuates the protection effect of paliperidone on SK-N-SH cells We then explored whether paliperidone achieves protective effects via Akt1/GSK3β signaling pathway. LY294002, an Akt1 inhibitor, was used to co-culture with paliperidone and glutamate. MTT and LDH assays were conducted 24 h after drug treatment. As shown in Fig. 6, the viability of SK-N-SH cells was dramatically decreased when LY294002 was applied. The result of LDH release assay was consistent with that of MTT, confirming that the protection effect of paliperidone was attenuated by Akt1 inhibitor-LY294002. All the results suggested that paliperidone may modulate Akt1/GSK3β pathway to protect SK-N-SH cells from glutamate-induced damages.
4. Discussion In the present study, we investigated the protective effects of paliperidone on SK-N-SH cell during glutamate exposure and attempted to clarify the intracellular mechanisms by which paliperidone exerts superior antipsychotic therapeutic effects in clinic. We mainly assessed the effects of paliperidone against the cytotoxicity of glutamate by assaying the cell viability, LDH release, ROS formation, and the rate of apoptosis. We found that paliperidone can increase cell viability, decrease the levels of LDH and ROS, inhibit cell apoptosis and thus, effectively prevent SK-N-SH cells from glutamate induced apoptosis and oxidative stress. Paliperidone exhibited a better protective effect against glutamate than haloperidol. Results also showed that paliperidone reversed glutamate-induced decline of Akt1 and GSK3β expression level, indicating that paliperidone may achieve its protection effects against glutamate in SK-N-SH cells via Akt1/GSK3β signaling pathway. Glutamate is the main excitatory neurotransmitter in the brain, and is involved in different activities and pathways in the central nervous system (CNS) (Kishida and Naka, 1967; Danbolt, 2001). Excitotoxicity, which is considered as an overactivation of glutamate receptors triggering neuronal cell death, has been associated with several acute and
Fig. 5. Paliperidone increases Akt (Ser 473) and GSK3β (Ser 9) expression against glutamate treatment. The levels of Akt1 and GSK3β were determined by Real-time PCR (D) and western blotting (A, B, C). Paliperidone could obviously attenuate glutamate-induced down-regulation of Akt1 and GSK3β. Scattergrams of five independent experimental data points were presented as mean value. *p b 0.01 compared with the control group, #p b 0.01, compared with glutamate injury group.
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Fig. 6. LY294002 partly attenuates paliperidone's protective effects against glutamate. SK-N-SH cells were treated with glutamate and paliperidone for 24 h in the presence of Akt1 inhibitor LY294002 and subjected to MTT metabolism assay (A) and LDH release assay (B). Paliperidone group: cells in 10 mM glutamate + 100 μM paliperidone; LY294002 group: cells in 10 mM glutamate + 100 μM paliperidone + 10 μM LY294002. The OD values were expressed as mean value from 5 independent experiments. *p b 0.05, compared with paliperidone treated group. The results showed that LY294002 can obviously inhibit the viability of SK-N-SH cells treated by paliperidone, indicating that the protective effects of paliperidone were mediated, at least partially, through Akt1/GSK3β signaling pathway.
chronic neurodegenerative disorders like multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), Parkinson's disease (PD) and Alzheimer's disease (AD) (Coyle and Puttfarcken, 1993; Danbolt, 2001). Meanwhile, previous studies showed that excessive or sustained stimulation by glutamate can lead to mitochondrial dysfunction, accumulation of ROS, and oxidative stress, which makes neuron become predisposed or hypersensitive to glutamate-mediated damage and leads to excitotoxic neuronal death (Coyle and Puttfarcken, 1993; Ghosh and Greenberg, 1995; Wang and Qin, 2010). Vuk Koprivica investigated the protective effects of aripiprazole in a neuronal glutamate toxicity assay, which may model aspects of neurodegeneration occurring in schizophrenia (Koprivica V et al., 2011). In our present study, 10 mM glutamate was used to establish the toxic model of SK-N-SH cells. MTT and LDH assays showed that glutamate can injure SK-N-SH cells, leading to decreased cell viability as well as increased LDH release. The levels of ROS also dramatically rose during glutamate exposure. Moreover, Annexin V-FITC and PI double staining showed that 10 mM glutamate increased the rate of apoptosis from 3.2% to 8.4%, indicating that glutamate can induce cell apoptosis. All the results suggested that glutamate can effectively injure SK-N-SH cells and model neurodamages in schizophrenia to certain degree. Recent studies reported that some SGAs have neuroprotective and neurogenetic effects against several kinds of injuries (Dickerson and Sharp, 2006; Ono et al., 2010; Koprivica V et al., 2011; Park et al., 2011). Some of them demonstrated the ability to decrease the rate of brain volume reduction in schizophrenics, which might be associated with their clinical efficacy for schizophrenia (Scherk and Falkai, 2006; Yulug et al., 2006). In previous studies, paliperidone exhibited some neuroprotective functions, such as protecting human neuroblastoma SK-K-SH cells from injuries induced by dopamine, alleviating oxidative stress induced by Aβ23-35 and MPP+, and providing neuroprotection against hydrogen peroxide (Yang and Lung, 2011; Gassó et al., 2012). In our present study, we also examined the protective effects of paliperidone on SK-N-SH cells injured by glutamate. The results showed that paliperidone exhibited strong protective effects by MTT and LDH assays. It was noticed that paliperidone could prevent SK-N-SH cells from glutamate induced oxidative stress by reducing the ROS production. Apoptosis related assays suggested that paliperidone also exert some anti-apoptosis effects and the underlying molecular mechanisms should be investigated in the future. Accumulating evidence suggested that the pathogenesis of schizophrenia may include multiple mechanisms which regulate neuron survival, neurite outgrowth and expression of genes that were associated with schizophrenia. Akt1/GSK3β signaling pathway has been implicated in both the pathogenesis and in the therapeutic mechanisms associated with psychosis (Emamian et al., 2004; Franke, 2008). In order to investigate the molecular mechanisms through which paliperidone achieves its protective effects, the expression of Akt1 and GSK3β was
assayed by RT-PCR and western blot. We found that paliperidone can effectively prevent glutamate induced reduction of Akt1 and GSK3β expression and phosphorylation in SK-N-SH cells. In our previous study, we reported that paliperidone protects prefrontal cortical neurons from damages caused by MK-801 via up-regulating Akt1/GSK3β signaling pathway. Both of the findings suggested that paliperidone can exert cell protective effects through modulating Akt1/GSK3β signaling pathway. In summary, we investigated the protective effects of paliperidone on SK-N-SH cell damage induced by glutamate exposure. We found that paliperidone exhibited its protective effects via Akt1/GSK3β signaling pathway, suggesting that paliperidone might be effective in regulating altered brain structure of schizophrenia. Moreover, our results also showed that paliperidone may exert anti-apoptosis effects to protect SK-N-SH cells from glutamate induced cell apoptosis. The current study may lead to new insights into pathogenesis and therapeutic treatment for schizophrenia. Role of funding source This study was supported by grants of the National Natural Science Foundation of China (No. 81071081, No. 81371471), Natural Science Foundation of Shandong Province (ZR2010HM051 and ZR2012HM026), and a grant of Shandong Provincial Science and Technology Development Plan (2011GSF11810). Contributors This study was designed by J. Sun. The experiments were done by L. Peng, X. Zhang, D. Zhu, J. Wu, H. Liu and Q. Yue. X. Cui, D. Sun. G. Li, Y. Li and J. Zhang were responsible for the statistical analysis. H. Xu, C. Qin and J. Sun provided writing support for this publication. J. Sun and M. Li reviewed and edited the manuscript for scientific accuracy. All authors were involved in the discussion and interpretation of the data. All authors agree that this paper is an accurate representation of the study results. Conflict of interest None. Acknowledgments We sincerely thank Nenad Šestan (Yale University) for his support and the use of the laboratory. We also thank Ms. Xingzhen Zhang for her continuous support.
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Schizophrenia Research journal homepage: www.elsevier.com/locate/schres
Paliperidone protects SK-N-SH cells against glutamate toxicity via Akt1/GSK3β signaling pathway Lei Peng a, Xingzhen Zhang a, Xianping Cui b, Dexiao Zhu a, Jintao Wu a, Dong Sun c, Qingwei Yue a, Zeyan Li a, Haili Liu a, Guibao Li a, Jing zhang a, Hongyan Xu c, Fuchen Liu d, Chengkun Qin b, Mingfeng Li d, Jinhao Sun a,⁎ a
Department of Anatomy and Shandong Provincial Key Laboratory of Mental Disorders, School of Medicine, Shandong University, Jinan, Shandong 250012, PR China Department of General Surgery, Provincial Hospital Affiliated to Shandong University, Jinan, Shandong 250012, PR China Experimental Platform for Medical Function, School of Medicine, Shandong University, Jinan, Shandong 250012, PR China d Department of Neurobiology, Yale University School of Medicine, New Haven, CT 06510, USA b c
a r t i c l e
i n f o
Article history: Received 20 January 2014 Received in revised form 21 May 2014 Accepted 31 May 2014 Available online 22 June 2014 Keywords: Paliperidone Protection Schizophrenia SK-N-SH Glutamate
a b s t r a c t Schizophrenia is a heterogeneous psychotic illness and its etiology remains poorly understood. Recent studies have suggested that neurodegeneration is a component of schizophrenia pathology and some atypical antipsychotics appear to slow progressive morphological brain changes. In addition, the atypical antipsychotics were reported to have a superior therapeutic efficacy in treating schizophrenia and have a low incidence of extrapyramidal side effects (EPS) compared to typical antipsychotics. However, the mechanisms of atypical antipsychotics in treating schizophrenia and the basis for differences in their clinical effects were still totally unknown. In the present study, we investigated whether paliperidone shows protective effects on SK-N-SH cells from cell toxicity induced by exposure to glutamate. We examined the effects of the drugs on cell viability (measured by MTT metabolism assay and lactate dehydrogenase (LDH) activity assay), apoptosis rate, ROS levels and gene expression and phosphorylation of Akt1 and GSK3β. The results showed that paliperidone significantly increases the cell viability by MTT and LDH assays (p b 0.05), in contrast to the typical antipsychotic (haloperidol), which had little neuroprotective activity. Moreover, paliperidone retarded the glutamate-mediated promotion of ROS and the rate of apoptosis (p b 0.05). In addition, paliperidone also effectively reversed glutamate-induced decreases of gene expression and phosphorylation of Akt1 and GSK3β (both p b 0.05). Our results demonstrated that paliperidone could effectively protect SK-N-SH cells from glutamate-induced damages via Akt1/GSK3β signaling pathway. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Schizophrenia is a severe chronic debilitating disease with a lifetime prevalence of 1% of the world's population (Frangou and Murray, 1996). Although schizophrenia is considered to be a neurodevelopmental disorder, there is evidence that it is also progressive and possibly neurodegenerative. Post-hoc studies showed that schizophrenia patients may have progressive ventricular dilation (Johnstone and Frith, 1996). Moreover, brain volume reductions and cortical and subcortical gray matter loss were also found in patients (Massana et al., 2005). All of these observations strongly suggest that neurodegeneration occurred during the pathogenesis of schizophrenia. Antipsychotic drugs modulate dysfunction in chemical neurotransmission, which also plays an important role in relieving the symptoms of schizophrenia. Although typical antipsychotics, such as haloperidol, produce a marked reduction in the positive symptoms of schizophrenia, they do not overcome negative symptoms, such as confusion, apathy, ⁎ Corresponding author. E-mail address: [email protected] (J. Sun).
http://dx.doi.org/10.1016/j.schres.2014.05.037 0920-9964/© 2014 Elsevier B.V. All rights reserved.
and social withdrawal (Lindenmayer et al., 2007). Accumulated evidences suggested that some second generation antipsychotics (SGAs) might offer neuroprotective benefits (Kurosawa et al., 2007; Kim et al., 2008), due to therapeutic properties such as cognitive enhancement or prevention of disease progression and clinical deterioration (Lieberman et al., 2008). Studies in vitro have demonstrated that second-generation antipsychotics offer protection against SH-SY5Y cell apoptosis induced by serum withdrawal (Kim et al., 2008) and cytotoxin-induced apoptosis in the pheochromocytoma (PC12) cells (Qing et al., 2003). However, the underlying molecular mechanisms have not been fully understood. More research is needed to identify the effects of second-generation antipsychotics on cell survival and death, and to investigate the association between such effects and the drugs' therapeutic properties on schizophrenia. Paliperidone has been reported to have better efficacy and tolerability than other atypical antipsychotics in acute and long-term treatment of schizophrenia (Fowler et al., 2008). Also, paliperidone offers protective effects against dopamine induced SK-N-SH cell damages by decreasing caspase-3 levels (Gassó et al., 2012), and enhance the survival of SH-SY5Y and U937, two cell lines separately representing neural and
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immune cell types which are involved in the pathogenesis of schizophrenia (Schmidt et al., 2010). However, the protective effects of paliperidone on SK-N-SH cells and its underlying mechanisms are largely elusive. During the past few years several studies have shown direct evidences for the involvement of different signaling pathways in schizophrenia. Among those, the original study of Akt1/GSK3β signaling pathway exhibited evidence for a decrease in Akt1 protein levels and levels of phosphorylation of GSK3β in schizophrenia (Emamian et al., 2004). Akt1 is the most highly expressed isoform of Akt involved in complicated cellular processes, such as transcription, glucose metabolism, cell proliferation, apoptosis, angiogenesis and cell motility (Kandel and Hay, 1999). As the major Akt1 target, GSK3β was also found altered in individuals with schizophrenia (Emamian et al., 2004). Research also showed that phosphorylation and protein level of GSK3β in the frontal cortex, specifically the GSK3β mRNA level in the dorsolateral prefrontal cortex was changed in patients with schizophrenia by post-hoc studies (Kozlovsky et al., 2004; Koros and Dorner-Ciossek, 2007). Thus, we propose that paliperidone may protect SK-N-SH cells from damages induced by glutamate through Akt1/ GSK3β signaling pathway. In the current study, we investigated the protective effects of paliperidone against glutamate induced cell damages in SK-N-SH cell line. Cell viability was measured by MTT and LDH assays to demonstrate the effects of paliperidone on glutamate induced cell damages. Furthermore, real-time PCR and western blotting were applied to determine whether paliperidone offers protection via the Akt1/GSK3β signaling pathway. The results provide information on how paliperidone exerts protection effects and contribute to the understanding of better antipsychotic therapy effects of paliperidone. 2. Experimental procedures 2.1. Materials SK-N-SH cells were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS) were purchased from GIBCO BRL (Grand Island, NY, USA). Glutamate, haloperidol and MTT assay kit were purchased from Sigma-Aldrich (St. Louis, MO, USA). Paliperidone was synthesized by Jinan Weidu Chemical Company (Shandong, China). All other chemicals were purchased from commercial sources. 2.2. Establishment of neurotoxic cell model with glutamate SK-N-SH cells were grown in DMEM, which was supplied with 10% fetal bovine serum. Cultures were maintained at 37 °C, 5% CO2 until 80% confluence, and the medium was changed every three days. Confluent monolayers were passaged routinely by trypsinization. Before drug treatment, the cells were seeded at 2 × 104/cm2 in poly-L-lysine coated culture plates. After 24 h of in vitro culture, SK-N-SH cells were exposed to 1, 10 and 50 mM glutamate. 2.3. Protection of paliperidone on cultured cells SK-N-SH cells were cultured in 24-well plates at 37 °C in the presence of 5% CO2. The cultured cells were divided into the following groups: the first group was the normal control without the treatment of glutamate or paliperidone in culture medium. In the second group, 10 mM glutamate was used to establish the cell injury model. The third group, termed paliperidone protection group, was subdivided into three small groups that were co-treated with 50, 100 and 200 μM paliperidone at the same time as glutamate exposure. In the fourth group, i.e. haloperidol treated group, cells were co-cultured with 100 μM haloperidol and 10 μM glutamate. After 24 h incubation, cells were collected from all groups and subjected to the subsequent examinations.
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2.4. Cell viability assay Cell viability was assessed by MTT assay, which reflects the intracellular redox state through mitochondrial metabolic activity of viable cells. The neuronal viability was measured using the MTT assay first. Briefly, after treated with different drugs, the cultured cells were subjected to MTT solution (0.1 mg/ml) at 37 °C for 4 h. The culture medium was then removed and dimethyl sulfoxide was added into medium to dissolve the formazan product while shaking for 10 min at room temperature. The fluorescent absorbance was then quantified spectrophotometrically at 492 nm using a microplate reader (Multiskan MK3, Thermo Labsystem, USA). The quantity of living cells was indicated indirectly by the optical density (OD) of absorbance of formazan. To confirm the assessment of cell viability and the neurotoxicity of glutamate, LDH activity assay was also performed. LDH assay measures the extracellular release of cytosolic lactate dehydrogenase due to cytotoxicity. The amount of LDH applied to the cultured media was determined according to the manufacturer's protocol (Jiancheng Bioengineering Institute, Nanjing, China) and OD was measured at 450 nm via a microplate reader (Multiskan MK3, Thermo Labsystem, USA). The mean value of each condition was normalized to the normal group. 2.5. Apoptosis analysis In order to determine whether paliperidone protects SK-N-SH cells against glutamate induced cell apoptosis, Hoechst 33342-PI double staining was performed. The cultured cells were treated with 10 μg/ml Hoecst33342 and PI for 15 min at 37 °C in the dark. Then labeled cells were observed through a LSM 780 Laser Scanning Confocal Microscope (Carl Zeiss SAS, Jena, Germany) with 200× magnifications. The apoptosis rate was calculated according to a previous report (Xiao Q. et al., 2010). Moreover, flow cytometry with Annexin V-FITC and propidium iodide (PI) double staining was carried out to give a quantitative analysis of apoptosis. In short, SK-N-SH cells cultured in different groups were collected with 0.125% trypsin, then washed with PBS for three times. 5 × 105 cells were stained with Annexin V-FITC and PI for 15 min in the dark afterwards. Subsequently, the fluorescence of each group was analyzed by flow cytometry (Becton Dickinson, Franklin Lakes, NJ, USA). 2.6. Determination of reactive oxygen species (ROS) levels Intracellular ROS was detected using the non-fluorescent probe 2′, 7′-dichlorofluorescein diacetate (DCFH-DA). DCFH-DA is oxidized into the fluorescent compound 2′, 7′-dichlorofluorescein (DCFH) under the presence of ROS in SK-N-SH cells. Briefly, 10 μM (the volume was 0.5 ml) DCFH-DA was added to the SK-N-SH cell suspension prepared freshly before the assay. The mixture was incubated at 37 °C in the dark for 20 min and mixed every 5 min. After the incubation period, the cells were washed three times with serum-free DMEM. Fluorescence intensity was measured with 485 nm excitation and 530 nm emissions using flow cytometry (Cantoll, BD, USA). The increasing production of ROS was expressed as a percentage to the control. 2.7. mRNA expressions of Akt1 and GSK3β To explore the mechanisms of paliperidone in protecting SK-N-SH cells from glutamate induced cell damage, the mRNA expression of Akt1 and GSK3β in different experimental groups was assayed. Total RNA was extracted from SK-N-SH cells in each group using TRIzol (Sangon Biotech Co., Ltd. Shanghai, China) and reverse transcribed into cDNA according to the manufacturer's instruction. Then, the cDNA was subjected to real-time PCR on Mastercycler ep realplex (Eppendorf, Hamburg, Germany). PCR was performed at the conditions of 95 °C for 2 min, followed by 35 cycles of 95 °C for 15 s, 60 °C for 15 s,
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and 68 °C for 20 s. The following primers were used in this: Akt1 primer pairs 5′-GGTATTTTGATGAGGAGTTCACG-3′ and 5′-GAGTAGGAGAACTG GGGGAAGT-3′ and GSK3β primer pairs 5′-GGCTACCATCCTTATTCCTC CT-3′ and 5′-GTCTGTCCACGGTCTCCAGTAT-3′, as well as control GAPDH primer pairs 5′-GAAGGTGAAGGTCGGAGTC-3′ and 5′-GAAGAT GGTGATGGGATTTC-3′, respectively. Akt1 and GSK3β mRNA abundance was calculated using comparative threshold cycle (2−ΔΔCT) method and normalized to internal control, GAPDH. 2.8. Protein levels of phospho-Akt1 (Ser 473) and phospho-GSK3β (Ser 9) SK-N-SH cells were treated with 10 mM glutamate in the presence or absence of 100 μM paliperidone for 2 h. Then, the cells were washed with ice-cold PBS and harvested in RIPA lysis buffer (Beyotime Institute of Biotechnology, Shanghai, China) containing protease inhibitors, which prevent protein degradation and dephosphorylation. After the total protein concentrations were measured with BCA method (Boster Biological Technology, Wuhan, China), 20 μg proteins in each group were subjected to 10% sodium dodecyl sulfate (SDS) polyacrylamide gel/tris-glycine electrophoresis, then transferred to nitrocellulose (NC) membranes and blocked with 5% nonfat milk in PBS containing 0.1% Tween 20 (PBS-T) for 2 h. Subsequently, the membranes were incubated with primary antibodies against Akt (1:1000, Beyotime Institute of Biotechnology), GSK3β (1:1000, Beyotime Institute of Biotechnology), phosphorylated Akt1 (p-Akt1) (1:100, Cell signaling Technology) and phosphorylated GSK3β (p-GSK3β) (1:100, Cell signaling Technology) at 4 °C overnight. Then the membranes were incubated with HRP labeled secondary IgG (1:2000) for 1 h at room temperature. Finally, the membranes were assayed with a chemiluminescence system (ECL system) and protein level was quantified by Image J software (National Institute of Health, USA). The levels of phosphorylated Akt and GSK3β were normalized to total Akt and GSK3, respectively. 2.9. Statistical analysis Statistical analysis was performed using the SPSS 17.0 software. Data were expressed as means ± S.E.M relative to that of vehicle for each condition. Analysis of variance (ANOVA) or student's t-test was applied,
followed by the Limited Slip Differential (LSD) post-hoc tests. A p value b 0.05 was regarded as statistically significant.
3. Results 3.1. Paliperidone promotes cell growth and increased cell viability against glutamate In order to establish the neurotoxic cell model with glutamate, SK-NSH cells were subjected to a series concentration of glutamate concentrations (1, 10, 50 mM). At 1 mM, glutamate (Fig. 1B) did not display a significant effect on cell morphology compared to cultured cells in normal medium (Fig. 1A) which grew well with long neuritis and bright glow. When the cells were exposed to 10 mM glutamate, neuritis retracted and cell debris appeared (Fig. 1C). As the concentration of glutamate rises to 50 mM, almost all the neuritis were destroyed and more cell debris were present in the culture (Fig. 1D). In order to give a quantitative analysis of damage effects induced by glutamate, MTT and LDH assays were performed (Fig. 1E and F). MTT measurement showed that cell viability gradually decreased as the concentration of glutamate increased from 1 mM to 50 mM. Consistent with morphological observations, cells treated with 10 mM and 50 mM glutamate were obviously damaged compared to control groups (p b 0.05). The results of LDH assay confirmed the observations by the MTT assay. Based on our experiment, 10 mM glutamate was selected to induce cell damages of SK-N-SH cells. In order to investigate the protective effects of paliperidone, SK-N-SH cells were treated with a series concentration of paliperidone (50 μM, 100 μM, and 200 μM) in the presence of 10 mM glutamate (Fig. 2). The results showed that 100 μM and 200 μM paliperidone treatment effectively increased cell viability and few cell debris appeared. However, 50 μM of paliperidone's protection effects was not obvious, a considerable number of cell debris still appeared in the cell culture. MTT and LDH release assays were also performed to investigate the protective effects of paliperidone. As shown in Fig. 2, paliperidone treatment reversed the glutamate-induced the decrease of cell viability in MTT assay and LDH release (Fig. 2E, F), suggesting that 100 μM paliperidone effectively protected SK-N-SH cells from the damages induced by glutamate (p b 0.05 for MTT, p b 0.01 for LDH).
Fig. 1. Survival of SK-N-SH cells exposed to glutamate. SK-N-SH cells were cultured for 12 h with 0 mM (A), 1 mM (B), 10 mM (C), and 50 mM (D) glutamate, respectively. (E) Effects of glutamate on the viability of SK-N-SH measured by MTT assay. (F) Effects of glutamate on the release of LDH by LDH release assay. OD values were mean ± S.E.M. from five independent experiments. *p b 0.01 vs control group. Scale bars: A–D, 100 μm.
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Fig. 2. Protection of paliperidone against glutamate induced neuronal damage. (A–D) The cells were treated with 0 (A), 50 μM (B), 100 μM (C), 200 μM (D) paliperidone as 10 mM glutamate exposure, respectively. As the concentration of paliperidone increased, the protective effects of paliperidone against cell damage induced by glutamate gradually enhanced. Cell viability was further assessed by MTT (E) and LDH (F) assays. The comparison of protective effects against glutamate between paliperidone and haloperidol was performed by MTT (G) and LDH (H) measures. OD values represent mean ± S.E.M. from five independent experiments. *p b 0.05 vs 0 μM paliperidone group, **p b 0.01 vs 0 μM paliperidone group, and #p b 0.05 vs 100 μM paliperidone group. Scale bars: A–D, 100 μm.
In order to study whether paliperidone has a superior protective effect against glutamate compared to typical antipsychotics (haloperidol), the protective effects of 100 μM haloperidol were examined. The concentration of haloperidol was selected according to a previous study (Lindenmayer et al., 2007). The MTT and LDH results showed that 100 μM paliperidone has a better protective effect than 100 μM haloperidol (Fig. 2G, H, both p b 0.05). 3.2. Paliperidone prevent glutamate induced cell apoptosis Hoechst 33342 and PI double staining were carried out to investigate the effects of paliperidone on glutamate induced apoptosis. Surviving cells were stained with bright blue integrated nuclei (Fig. 3) while the
apoptotic cells had bright red fragmented nuclei (Fig. 3). In glutamate injury group, the number of cells with bright red nuclei was increased compared to normal control group with only few apoptotic cells observed. By contrast, the number of apoptotic nucleoli decreased in paliperidone protection group. To further investigate the effects of paliperidone on apoptosis, flow cytometry was deployed with Annexin V-FITC and PI double staining. Fig. 3 showed that 10 mM glutamate increased the average rate of apoptosis from 3.2% to 8.4%, indicating that 10 mM glutamate effectively induces cell apoptosis. However, when co-cultured with 100 μM paliperidone, apoptotic cells were largely decreased in number, demonstrating that paliperidone can protect SK-N-SH cells from apoptosis induced by glutamate.
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Fig. 3. Paliperidone prevents glutamate induced cell apoptosis. SK-N-SH cells were exposed to glutamate with or without paliperidone for 24 h and double stained with Hoechst 33342 (blue) and PI (red) (C, F, and I) or subjected to flow cytometry assay (J, K, and L) to determine cell apoptosis. (A, B, C) Normal control group: cells in DMEM medium; (D, E, F) glutamate treated group: cells in 10 mM glutamate; (G, H, I) paliperidone protection group: cells in 10 mM glutamate + 100 μM paliperidone; J, K and L were the results of flow cytometry of the control, glutamate injury group and paliperidone protection group, respectively. Statistical analysis suggested that paliperidone significantly reduced cell apoptosis induced by glutamate treatment (p b 0.01, n = 5). Pictures of A–I were captured with LSM 780 laser confocal Zeiss microscope at 200× magnification. Scale bars: A–I, 100 μm.
3.3. Paliperidone decreases ROS levels ROS levels can reflect the degree of oxidative damage induced by glutamate. Excessive ROS may cause several kinds of irreversible oxidative damage to cellular components (such as proteins, lipids and nucleic acids), and activate signaling pathways ultimately leading to cell death. Fig. 4 showed the level of ROS treated with different drugs. In glutamate injury group, the ROS level was significantly increased compared to control group (from 9.22% to 16.48%, p b 0.05). By contrast, the addition of paliperidone effectively attenuated the fluorescence intensity (p b 0.05). Co-treatment with paliperidone and glutamate only slightly increased the level of ROS (Fig. 4C), suggesting
that paliperidone can effectively protect SK-N-SH cells from glutamateinduced damage by inhibiting the generation of ROS. 3.4. Paliperidone up-regulating Akt1-GSK3β expressions To study the molecular mechanism of paliperidone on protecting cells from glutamate-induced damages, we measured the gene expression level of Akt1 and GSK3β, both of which were involved in the pathogenesis of schizophrenia. Real-time PCR and Western blot were both performed to detect Akt1 and GSK3β expression. When SK-N-SH cells were exposed to glutamate, Akt1 and GSK3β mRNA expression was decreased and this inhibition effect can be reversed by paliperidone
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Fig. 4. Paliperidone decreases ROS levels. SK-N-SH cells were treated with glutamate and paliperidone for 24 h for ROS detection. (A–C) The fluorescence intensity of the cells stained with DCFH-DA and recorded by flow cytometer. There is significantly statistical difference between cells in control group and cells treated with glutamate (p b 0.01, n = 5), and between glutamate group and paliperidone protection group (p b 0.05, n = 5).
(p b 0.05, Fig. 5D). The level of phosphorylated Akt1 and GSK3β, which was commonly used as indicator of functional Akt1 and GSK3β in schizophrenia, was detected by Western blot (Fig. 5A–C). Results showed that glutamate treatment significantly decreased the levels of phosphorylated Akt1 and GSK3β (p b 0.05); and these effects diminished with paliperidone treatment (p b 0.05).
3.5. Akt1 inhibitor LY294002 partly attenuates the protection effect of paliperidone on SK-N-SH cells We then explored whether paliperidone achieves protective effects via Akt1/GSK3β signaling pathway. LY294002, an Akt1 inhibitor, was used to co-culture with paliperidone and glutamate. MTT and LDH assays were conducted 24 h after drug treatment. As shown in Fig. 6, the viability of SK-N-SH cells was dramatically decreased when LY294002 was applied. The result of LDH release assay was consistent with that of MTT, confirming that the protection effect of paliperidone was attenuated by Akt1 inhibitor-LY294002. All the results suggested that paliperidone may modulate Akt1/GSK3β pathway to protect SK-N-SH cells from glutamate-induced damages.
4. Discussion In the present study, we investigated the protective effects of paliperidone on SK-N-SH cell during glutamate exposure and attempted to clarify the intracellular mechanisms by which paliperidone exerts superior antipsychotic therapeutic effects in clinic. We mainly assessed the effects of paliperidone against the cytotoxicity of glutamate by assaying the cell viability, LDH release, ROS formation, and the rate of apoptosis. We found that paliperidone can increase cell viability, decrease the levels of LDH and ROS, inhibit cell apoptosis and thus, effectively prevent SK-N-SH cells from glutamate induced apoptosis and oxidative stress. Paliperidone exhibited a better protective effect against glutamate than haloperidol. Results also showed that paliperidone reversed glutamate-induced decline of Akt1 and GSK3β expression level, indicating that paliperidone may achieve its protection effects against glutamate in SK-N-SH cells via Akt1/GSK3β signaling pathway. Glutamate is the main excitatory neurotransmitter in the brain, and is involved in different activities and pathways in the central nervous system (CNS) (Kishida and Naka, 1967; Danbolt, 2001). Excitotoxicity, which is considered as an overactivation of glutamate receptors triggering neuronal cell death, has been associated with several acute and
Fig. 5. Paliperidone increases Akt (Ser 473) and GSK3β (Ser 9) expression against glutamate treatment. The levels of Akt1 and GSK3β were determined by Real-time PCR (D) and western blotting (A, B, C). Paliperidone could obviously attenuate glutamate-induced down-regulation of Akt1 and GSK3β. Scattergrams of five independent experimental data points were presented as mean value. *p b 0.01 compared with the control group, #p b 0.01, compared with glutamate injury group.
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Fig. 6. LY294002 partly attenuates paliperidone's protective effects against glutamate. SK-N-SH cells were treated with glutamate and paliperidone for 24 h in the presence of Akt1 inhibitor LY294002 and subjected to MTT metabolism assay (A) and LDH release assay (B). Paliperidone group: cells in 10 mM glutamate + 100 μM paliperidone; LY294002 group: cells in 10 mM glutamate + 100 μM paliperidone + 10 μM LY294002. The OD values were expressed as mean value from 5 independent experiments. *p b 0.05, compared with paliperidone treated group. The results showed that LY294002 can obviously inhibit the viability of SK-N-SH cells treated by paliperidone, indicating that the protective effects of paliperidone were mediated, at least partially, through Akt1/GSK3β signaling pathway.
chronic neurodegenerative disorders like multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), Parkinson's disease (PD) and Alzheimer's disease (AD) (Coyle and Puttfarcken, 1993; Danbolt, 2001). Meanwhile, previous studies showed that excessive or sustained stimulation by glutamate can lead to mitochondrial dysfunction, accumulation of ROS, and oxidative stress, which makes neuron become predisposed or hypersensitive to glutamate-mediated damage and leads to excitotoxic neuronal death (Coyle and Puttfarcken, 1993; Ghosh and Greenberg, 1995; Wang and Qin, 2010). Vuk Koprivica investigated the protective effects of aripiprazole in a neuronal glutamate toxicity assay, which may model aspects of neurodegeneration occurring in schizophrenia (Koprivica V et al., 2011). In our present study, 10 mM glutamate was used to establish the toxic model of SK-N-SH cells. MTT and LDH assays showed that glutamate can injure SK-N-SH cells, leading to decreased cell viability as well as increased LDH release. The levels of ROS also dramatically rose during glutamate exposure. Moreover, Annexin V-FITC and PI double staining showed that 10 mM glutamate increased the rate of apoptosis from 3.2% to 8.4%, indicating that glutamate can induce cell apoptosis. All the results suggested that glutamate can effectively injure SK-N-SH cells and model neurodamages in schizophrenia to certain degree. Recent studies reported that some SGAs have neuroprotective and neurogenetic effects against several kinds of injuries (Dickerson and Sharp, 2006; Ono et al., 2010; Koprivica V et al., 2011; Park et al., 2011). Some of them demonstrated the ability to decrease the rate of brain volume reduction in schizophrenics, which might be associated with their clinical efficacy for schizophrenia (Scherk and Falkai, 2006; Yulug et al., 2006). In previous studies, paliperidone exhibited some neuroprotective functions, such as protecting human neuroblastoma SK-K-SH cells from injuries induced by dopamine, alleviating oxidative stress induced by Aβ23-35 and MPP+, and providing neuroprotection against hydrogen peroxide (Yang and Lung, 2011; Gassó et al., 2012). In our present study, we also examined the protective effects of paliperidone on SK-N-SH cells injured by glutamate. The results showed that paliperidone exhibited strong protective effects by MTT and LDH assays. It was noticed that paliperidone could prevent SK-N-SH cells from glutamate induced oxidative stress by reducing the ROS production. Apoptosis related assays suggested that paliperidone also exert some anti-apoptosis effects and the underlying molecular mechanisms should be investigated in the future. Accumulating evidence suggested that the pathogenesis of schizophrenia may include multiple mechanisms which regulate neuron survival, neurite outgrowth and expression of genes that were associated with schizophrenia. Akt1/GSK3β signaling pathway has been implicated in both the pathogenesis and in the therapeutic mechanisms associated with psychosis (Emamian et al., 2004; Franke, 2008). In order to investigate the molecular mechanisms through which paliperidone achieves its protective effects, the expression of Akt1 and GSK3β was
assayed by RT-PCR and western blot. We found that paliperidone can effectively prevent glutamate induced reduction of Akt1 and GSK3β expression and phosphorylation in SK-N-SH cells. In our previous study, we reported that paliperidone protects prefrontal cortical neurons from damages caused by MK-801 via up-regulating Akt1/GSK3β signaling pathway. Both of the findings suggested that paliperidone can exert cell protective effects through modulating Akt1/GSK3β signaling pathway. In summary, we investigated the protective effects of paliperidone on SK-N-SH cell damage induced by glutamate exposure. We found that paliperidone exhibited its protective effects via Akt1/GSK3β signaling pathway, suggesting that paliperidone might be effective in regulating altered brain structure of schizophrenia. Moreover, our results also showed that paliperidone may exert anti-apoptosis effects to protect SK-N-SH cells from glutamate induced cell apoptosis. The current study may lead to new insights into pathogenesis and therapeutic treatment for schizophrenia. Role of funding source This study was supported by grants of the National Natural Science Foundation of China (No. 81071081, No. 81371471), Natural Science Foundation of Shandong Province (ZR2010HM051 and ZR2012HM026), and a grant of Shandong Provincial Science and Technology Development Plan (2011GSF11810). Contributors This study was designed by J. Sun. The experiments were done by L. Peng, X. Zhang, D. Zhu, J. Wu, H. Liu and Q. Yue. X. Cui, D. Sun. G. Li, Y. Li and J. Zhang were responsible for the statistical analysis. H. Xu, C. Qin and J. Sun provided writing support for this publication. J. Sun and M. Li reviewed and edited the manuscript for scientific accuracy. All authors were involved in the discussion and interpretation of the data. All authors agree that this paper is an accurate representation of the study results. Conflict of interest None. Acknowledgments We sincerely thank Nenad Šestan (Yale University) for his support and the use of the laboratory. We also thank Ms. Xingzhen Zhang for her continuous support.
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