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Paliperidone protects prefrontal cortical neurons from damages caused by MK-801 via Akt1/GSK3β signaling pathway

Schizophrenia Research, 1, 147, pages 14 - 23

Abstract

Recent studies have suggested that neurodegeneration is involved in the pathogenesis of schizophrenia, and some atypical antipsychotics appear to prevent or retard progressive morphological brain changes. However, the underlying molecular mechanisms are largely unknown. Whether changes in intracellular signaling pathways are related to their neuroprotective effects remains undefined. In the present study, we used mouse embryonic prefrontal cortical neurons to examine the neuroprotection of paliperidone against the neuronal damage induced by exposure to the NMDA receptor antagonist, MK-801. Paliperidone inhibited MK-801 induced neurotoxicity both in MTT metabolism assay (p < 0.01) and in lactate dehydrogenase (LDH) activity assay (p < 0.01). Time course studies reveled that paliperidone effectively attenuated the elevation of intracellular free calcium concentration ([Ca2 +]i) induced by exposure to MK-801 (p < 0.01). Moreover, paliperidone could significantly retard MK-801-mediated inhibition of neurite outgrowth (p < 0.01) and reverse MK-801-induced decreases of gene expression and phosphorylation of Akt1 and GSK3β (bothp < 0.01). Furthermore, these protective effects of paliperidone were blocked by pretreatment with a PI3K inhibitor LY294002. Taking together, our results demonstrated that paliperidone could protect prefrontal cortical neurons from MK-801-induced damages via Akt1/GSK3β signaling pathway.

Keywords: Paliperidone, Protection, Schizophrenia, Prefrontal cortical neurons.

1. Introduction

Schizophrenia is a heterogeneous psychotic disease that affects approximately 1% of the world's population ( Frangou and Murray, 1996 ). This mental disease is characterized by positive symptoms (hallucinations and delusional ideas), and negative and cognitive symptoms (low levels of emotional arousal, mental activity and social drive, decreased attention, memory and executive functions) ( Sadock et al., 2009 ). Schizophrenia patients with progressive ventricular dilation ( Johnstone and Frith, 1996 ), brain volume reductions, and cortical and subcortical gray matter loss ( Massana et al., 2005 ) are identified by Post hoc studies, suggesting that neurodegeneration may occur during the pathogenesis of schizophrenia.

A great number of evidences have shown that second generation antipsychotics (SGAs) might offer neuroprotective effects (Kurosawa et al, 2007 and Kim et al, 2008). In clinical practice, SGAs are reported to improve the long-term outcome of schizophrenia ( Wyatt and Henter, 1998 ), exhibit effective therapeutic efficacy for negative symptoms and cognitive deficits and have low incidence of extrapyramidal side effects (Kinon et al, 1996 and Shadach et al, 2000). However, the underling molecular mechanisms have not been well understood, especially the intracellular signaling pathways related to their neuroprotective effects which can reduce the progressive morphological brain change in schizophrenia.

Paliperidone, one of SGAs, has been reported to have neuroprotective effects against dopamine induced injury by reducing caspase-3 expression in SK-N-SH cell line ( Gassó et al., 2012 ). In addition, paliperidone protects SH-SY5Y cell from different stressors (MPP+, Aβ25-35, and hydrogen peroxide) which can mimic several models of psychosis ( Yang and Lung, 2011 ). Whether paliperidone has protective effects on cultured primary prefrontal cortical neurons and if so, what the underlying mechanisms are unclear.

Akt (also known as PKB), a member of the AGC kinase family, has become the focus of many signal transduction pathways, regulating complicated cellular processes including transcription, glucose metabolism, cell proliferation, apoptosis, angiogenesis, and cell motility ( Kandel and Hay, 1999 ). Akt has multiple roles in regulating neuronal cell size and survival (Franke, 2008 and Freyberg et al, 2009), preventing neuronal death, accelerating axonal regeneration ( Namikawa et al., 2000 ), and promoting axon elongation and branching ( Markus et al., 2002 ). At the same time, Akt also plays a crucial role in synaptic plasticity and memory formation ( Lin et al., 2001 ). Akt has three isoforms in mammalian cells: Akt1, Akt2 and Akt3, which play different roles in a variety of processes, such as development and metabolism ( Dummler and Hemmings, 2007 ). Akt1 is the most highly expressed isoform that is associated with schizophrenia so far ( Emamian et al., 2004 ). GSK3β as the major Akt1 downstream target was also found to be involved in the pathogenesis of schizophrenia (Kozlovsky et al, 2004 and Nadri et al, 2004). A great deal of researches of postmortem brains from patients with schizophrenia indicated that phosphorylation and protein levels of GSK3β in the frontal cortex, as well as GSK3β mRNA level in the dorsolateral prefrontal cortex were decreased (Emamian et al., 2004;Kozlovsky et al, 2004 and Koros and Dorner-Ciossek, 2007). Taken together, a large variety of studies showed the involvement of Akt1/GSK3β signaling pathway in schizophrenia, making them novel targets for treatment of schizophrenia. Thus, we propose that paliperidone may affect Akt1/GSK3β signaling pathway, and by which prevents and possibly reverses the neuron damage associated with schizophrenia.

In our current study, we used MK-801 to injure cultured mouse embryonic prefrontal cortical neurons that could mimic neurodegeneration of schizophrenia in a certain degree in vitro ( Dickerson and Sharp, 2006 ). MTT metabolism, LDH activity and time course of [Ca2 +]iassays were performed to investigate the effects of paliperidone on MK-801 induced these damages. Also, real-time PCR and western blotting were applied to determine whether paliperidone offer neuroprotection via Akt1/GSK3β signaling pathway.

2. Experimental procedures

2.1. Animals and materials

Pregnant Kunming mice were purchased from Laboratory Animal Center of Shandong University in Jinan, China. The animals were maintained on a 12-h light–dark cycle with food and water available ad libitum. All protocols involving animal handling were in accordance with the China Animal (Scientific) Procedures Act.

Dulbecco's modified Eagle's medium F12 (DMEM-F12) and fetal bovine serum (FBS) were purchased from GIBCO BRL (Grand Island, NY, USA). Paliperidone was synthesized by Jinan Weidu Chemical Company, Shandong, China. MK-801 (dizocilpine) and MTT assay kit were purchased from Sigma-Aldrich (St. Louis, MO, USA). All other chemicals were purchased from commercial sources.

2.2. Culture of cortical neurons and drug treatment

Primary prefrontal cortical neurons were prepared according to the method reported previously ( Kurosawa et al., 2007 ). In brief, mouse embryos at embryonic day 15 were removed and transferred into ice-cold magnesium- and calcium-free Hanks Solution. Cortices were dissected from embryonic brains, dissociated mechanically and digested with trypsin. Dissociated neurons were plated at a density of 2.0 × 105/cm2(for neuroprotection studies) or 2.0 × 104/cm2(for neurite outgrowth studies) into 6-well plates pre-coated with poly-l-lysine (5 mg/ml). The cultured neurons were grown in humidified 5% CO2/95% air atmosphere at 37 °C for 5 days. After 24 h of incubation, 10 μM cytarabine was added to prevent the proliferation of non-neuronal cells.

According to a previous report ( Ono et al., 2010 ), 100 μM MK-801 was selected to injury prefrontal cortical neurons to mimic the neuronal damage occurred in schizophrenia in vitro. To investigate the protective effect, paliperidone of different concentrations were added to the medium at the same time as MK-801 exposure. The Akt1 inhibitor LY294002 was used to verify whether the protective effects of paliperidone were mediated through Akt1/GSK3β signaling pathway.

2.3. Neuron viability

Prefrontal cortical neurons were plated on 96-well plates at a density of 2 × 105cells/cm2and treated with paliperidone at concentrations of 25 μM, 50 μM, 100 μM or 200 μM either alone or in combination with a series of concentrations of MK-801 and 10 μM LY294002 for 48 h. Each condition was tested at least three times. The neuron viability was measured using the MTT assay. In brief, prefrontal cortical neurons were incubated with MTT (0.5 mg/ml) at 37 °C for 4 h. Then, the culture medium was removed and neurons in each well were incubated with 100 μL for 10 min on a shaker at room temperature and quantified spectrophotometrically at 492 nm using a microplate reader (Multiskan MK3, Thermo Labsystem, USA).

2.4. LDH release assay

LDH release assay was further performed to solidify the measurement of cell viability and the neurotoxicity of MK-801. In brief, LDH released in the cultured media of prefrontal cortical neurons treated as mentioned above was measured with a LDH assay kit (Jiancheng Bioengineering Institute, Nanjing, China) by measuring the optical density (OD) value of each well at 450 nm using a microplate reader (Multiskan MK3, Thermo Labsystem, USA) according to the protocol provided by the manufacturer. The mean value of each condition was normalized as percentage of the normal group.

2.5. [Ca2 +]i measurement

The prefrontal cortical neurons were seeded on 24-well plates at the density of 2 × 105 cells/cm2. After 5 days of culture, the neurons were treated with 100 μM MK-801 for 48 h. After removal of DMEM/F12 medium, the neurons were washed with sterile cold PBS and incubated with 10 μM fluo-3/Am solution for 1 h at 37 °C. Then the mixed solution was aspirated and cells were rinsed with PBS to eliminate non-specific staining. Zeiss 780 laser scanning confocal microscope (Carl Zeiss SAS, Jena, Germany) was used for image acquisition with the detection wavelength at 488 nm. Intracellular calcium fluorescence intensity was analyzed using ZEN software package. Five views of each condition were randomly selected and their mean fluorescence intensity was calculated for further data analysis.

2.6. Time course of [Ca2 +]i measurement

To further determine the time course effect of paliperidone on [Ca2 +]i, prefrontal cortical neurons were cultured for 7 days and then incubated with 10 μM fluo-3/AM for 1 h at 37 °C. After rinsed twice with Hank's Balanced Salt Solution (HBSS), Cells were separated into three groups and continuously observed with a Cell Observer SD (Carl Zeiss SAS, Jena, Germany) with excitation wavelength of 488 nm as previously reported ( Tao-Cheng et al., 2001 ). In the normal control group, neurons were incubated in HBSS for 720 s. In MK-801 group, neurons were incubated in HBSS for 360 s and then treated with 100 μM MK-801 for another 360 s. In MK-801 plus paliperidone group, neurons were incubated in HBSS for 360 s and then treated with 100 μM MK-801 plus 100 μM paliperidone for another 360 s. During the entire observation, cells were maintained at 37 °C, and time lapse images were collected in 12-s intervals. Solution changes were accomplished by adding fresh solutions containing different drugs. The fluorescence intensity over each individual neuronal cell body at each time point was acquired using ZEN software. Ten neurons at 0, 360 and 720 s were selected for further statistical analysis. All data used for statistical analysis were standardized previously. Comparison of [Ca2 +]ifluorescence variances between 720 and 360 s as well as between 360 and 0 s was determined by paired-sample Mann–WhitneyUtest in each group with SPSS 17.0 software. A value ofp < 0.05 was regarded as statistical significance.

2.7. Hoechst 33342 and PI double staining

Hoechst 33342-PI staining was performed to investigate whether paliperidone can protect prefrontal cortical neurons against MK-801 induced neurotoxicity by ameliorating cell apoptosis. Cells were assigned into four groups. In normal control group, neurons were cultured in DMEM/F12 medium. In MK-801 group, neurons were treated with 100 μM MK-801. In paliperidone group, neurons were treated with 100 μM MK-801 plus 100 μM Paliperidone. In LY294002 group, neurons were treated with 100 μM MK-801 plus 100 μM Paliperidone and 10 μM LY294002. The experiments were carried out according to the procedure of a previous report ( Xiao et al., 2010 ). In brief, the cultured neurons were treated with 10 μg/ml Hoecst33342 and PI for 15 min at 37 °C in the dark. After washed with PBS for three times, the stained neurons were viewed under a LSM 780 laser scanning confocal microscope (Carl Zeiss SAS, Jena, Germany) with 200 × magnifications. Apoptotic nuclei were considered to be those with bright red fragmented nuclei containing one or multiple lobes of condensed chromatin, and survival nuclei were considered to be those with bright blue fluorescence. The apoptosis rate was considered as the ratio of the apoptotic and survival nuclei in five randomly selected fields.

2.8. Measurement of neurite outgrowth

In order to assess the effect of paliperidone on neurite outgrowth, the prefrontal cortical neurons were plated on 6-well plates at the density of 2 × 104cells/cm2. According to the preliminary study ( Klimaviciusa et al., 2008 ), neurite outgrowth was measured 12 h after the cultured neurons were treated with 100 μM paliperidone in the presence or absence of 100 μM MK-801. Images of the cultured neurons were obtained with an inverted phase contrast microscope. Five randomly selected fields were observed for each condition. The total length and number of neuritis as well as the total number of neurons in each image were measured using IPP image analysis software. The mean neurite length was calculated as the ratio of total neurite length to the number of neurons, and the neurite density was calculated as the ratio of total neurite number to the number of neurons. The results were analyzed statistically.

2.9. Real-time PCR for detection of the mRNA levels of Akt1 and GSK3β

After treated with different drugs for 48 h, the cultured prefrontal cortical neurons were collected, RNA was extracted from neurons in each well of 6-well plates using TRIzol reagent (Sangon Biotech Co., Ltd. Shanghai, China) and reverse transcribed into cDNA using cDNA synthesis kit according to the manufacturer's instruction.

The levels of Akt1 and GSK3β mRNA were determined by SYBR green-based real-time PCR on Mastercycler ep realplex (Eppendorf, Hamburg, Germany) using Akt1 primer pairs 5′-AAACCTGGCGGCCACGCTAC-3′ and 5′-GACCGTGGTCCACTGCAGGC-3′ and GSK3β primer pairs 5′-CCGCCCGGCTAACACCACTG-3′ and 5′-TGGCGTTTGCAGGCGGTGAA-3′, as well as control GAPDH primer pairs 5′-ACCACAGTCCATGCCATCAC-3′ and 5′-TCCACCACCCTGTTGCTGTA-3′, respectively. PCR was performed at the conditions of 95 °C for 2 min, followed by 35 cycles of 95 °C for 15 s, 55 °C for15s, and 68 °C for 20s. The relative levels of Akt1 and GSK3β mRNA were calculated using comparative threshold cycle (2− ΔΔCT) method and normalized to the control GAPDH.

2.10. Western blotting assay for detection of the protein levels of phospho-Akt1 (Ser 473) and phospho-GSK3β (Ser 9)

Prefrontal cortical neurons were plated on 6-well plates at a density of 2 × 105cells/cm2. After 7 days of incubation, the cells were treated with 100 μM paliperidone in the presence or absence of 100 μM MK-801 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 as well as phosphatase inhibitors sodium orthovanadate and sodium fluoride to inhibit protein degradation and dephosphorylation. The supernatants were collected by centrifugation at 12000 rpm for 20 min. After the protein concentrations were measured with BCA method (Boster Biological Technology, Wuhan, China), equal amounts of proteins (20 μg) from the cell extracts in each treatment condition were subjected to 10% sodium dodecyl sulfate (SDS) polyacrylamide gel/Tris-glycine electrophoresis and transferred electrophoretically onto nitrocellulose (NC) membranes. After blocking with 5% nonfat milk in PBS containing 0.1% Tween 20 (PBS-T) for 2 h, the membranes were incubated with 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), respectively, overnight at 4 °C. After washed with PBS-T, the membranes were then incubated with HRP labeled secondary IgG (1: 2000) for 1 h at room temperature. The immunoreactive bands were detected with a chemiluminescence system (ECL system) and 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.

2.11. Statistical analysis

Data were expressed as means ± SD relative to that of vehicle for each condition. All data were statistically analyzed with SPSS software 17.0 using one-way ANOVA followed by the Limited Slip Differential (LSD) post hoc tests. A value ofp < 0.05 was regarded as statistical significance.

3. Results

3.1. MK-801 treatment decreases neuronal cell viability

Exposure of cultured prefrontal cortical neurons to MK-801 of different concentrations (25 μM, 50 μM, 100 μM, and 200 μM) induced varying degrees of neuron damages: 25 μM and 50 μM MK-801 did not display a significant effect on neuron morphology, while 100 μM and 200 μM MK-801 decreased the number of neurites and partially collapsed network ( Fig. 1 A–E).

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Fig. 1 MK-801-induced damages to prefrontal cortical neurons. (A–E) The photomicrographs of prefrontal cortical neurons exposed for 48 h to MK-801 at concentrations of 0 (A), 25 μM (B), 50 μM (C), 100 μM (D) and 200 μM (E), respectively. As the concentration of MK-801 increased, the number of neurites and intensity of neuronal network gradually decreased. When incubated with 200 μM MK-801, the number of neurites was largely decreased and the neuronal network was collapsed. (F) Effects of MK-801 on the viability of prefrontal cortical neurons measured by MTT assay. (G) Effects of MK-801 on the release of LDH by LDH release assay. OD values were means ± SD from five independent experiments. *p < 0.01 vs control group. Scale bars: A–E, 100 μm.

To evaluate the degree of neuronal damages induced by exposure to MK-801, MTT assay was performed. Although the OD values of cells treated with 25 μM and 50 μM MK-801 were comparable to that of control (p > 0.05), the OD values of the cells treated with 100 μM and 200 μM MK-801 declined significantly (p < 0.01), indicating that neuron viability was damaged ( Fig. 1 F). These results were consistent with morphological observations.

To further confirm the effects of MK-801 on neuron viability, activity of LDH released to the media by the neurons after MK-801 treatment was measured. As shown in Fig. 1 G, the results were consistent with those of MTT assay.

3.2. Paliperidone protects prefrontal cortical neurons from MK-801-induced damages

According to a previous report ( Ono et al., 2010 ) and our own experimental results, 100 μM MK-801 was chosen to induce schizophrenia-like neuronal damage to the cultured neurons. To investigate whether the paliperidone has protective effect, paliperidone of different concentrations (25 μM, 50 μM, 100 μM, and 200 μM) was added to the medium in the presence of MK-801. As shown in Fig. 2 , 100 μM and 200 μM paliperidone treatment could largely increase the number of neurites and enhance the neuronal network formation compared with those treated with MK-801 exposure alone ( Fig. 2 B, E, F), while 25 μM and 50 μM paliperidone treatment had no such protective effect ( Fig. 2 C, D).

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Fig. 2 Protection of paliperidone against MK-801 induced neuronal damage. (A) Neurons of control group with long neuritis and complicated neuronal network. (B–E) The neurons treated with 0 (B), 25 μM (C), 50 μM (D), 100 μM (E) and 200 μM (F) paliperidone, respectively. Paliperidone exerted protective effects against MK-801 damage in a dose-dependent manner. The neurite and intensity of neuronal network were enhanced as the concentration of paliperidone increased. Cell viability was further assessed by MTT (G) and LDH (H) assays. The effects of paliperidone alone on cell viability were also measured by MTT (I) and LDH (J) assays. OD values represent means ± SD from five independent experiments. *p < 0.01 vs control group;#p < 0.01 vs MK-801 group. Scale bars: A–F, 100 μm.

The MTT and LDH release assays were also performed to investigate the protective effects of paliperidone. As shown in Fig. 2 , paliperidone treatment dose dependently reversed the MK-801-induced changes of OD values of neurons in MTT assay and LDH release ( Fig. 2 G, H), showing the most significant effects with 100 μM paliperidone treatment (p < 0.01 for MTT,p < 0.001 for LDH). Therefore, 100 μM paliperidone was selected for the following experiments in our study.

3.3. Effects of MK-801 and paliperidone on [Ca2 +]i

Changes in [Ca2 +]iconcentration were monitored by using fluo-3/AM, which was usually used as the indicator of [Ca2 +]i. The fluorescence of Ca2 +was much brighter in MK-801 treated cells than that in control cells ( Fig. 3 A, B). In the presence of paliperidone, the fluorescence intensity was effectively decreased compared with that of cells treated with MK-801 alone ( Fig. 3 C). The statistical results showed that neurons treated with MK-801 alone showed obviously increased levels of [Ca2 +]i(p < 0.01), which could be inhibited by 100 μM paliperidone (p < 0.01) ( Fig. 3 D).

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Fig. 3 The changes of [Ca2 +]iconcentration in prefrontal cortical neurons treated with paliperidone and MK-801. (A–C) The fluorescence images of the neurons stained with Fluo-3/AM ester and recorded by LSCM (× 200). (A) In normal control group, the neurons emit weak green fluorescence. (B) Neurons cultured with 100 μM MK-801 had high intensity of green fluorescence. (C) Neurons co-treated with MK-801 and paliperidone. The increased [Ca2 +]iby MK-801 on was attenuated by paliperidone. (D) Quantification of fluorescence intensity of prefrontal cortical neurons treated with MK-801 and paliperidone. Scattergrams of five independent experimental data points were presented as mean value. There is significantly statistical difference between neurons in control group and neurons treated with MK-801 (*p < 0.01, n = 5), and between neurons treated with MK-801 and treated with paliperidone (#p < 0.01, n = 5). Scale bars: A–C: 100 μm.

To further determine the effects of paliperidone on [Ca2 +]i, time course studies were performed. Fig. 4 showed that the fluorescence intensity (0–720 s) of the normal control group was maintained at a horizontal level. While when MK-801 were added into the medium at 360 s, the fluorescence intensity of the neurons was increased dramatically, that was 1.6-fold of the initial values at 720 s (p < 0.01) ( Fig. 4 D). By contrast, addition of paliperidone at 360 s effectively attenuated the fluorescence intensity (p < 0.01). Co-treatment with paliperidone and MK-801 only slightly increased the fluorescence intensity ( Fig. 4 F), suggesting that paliperidone can effectively protect prefrontal cortical neurons from MK-801-induced damage by decreasing [Ca2 +]i.

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Fig. 4 Time course effects of paliperidone and MK-801 on [Ca2 +]iindicated by Fluo-3/AM. (A, B) The normalized fluorescence intensity of control neurons in HBSS (0–720 s). (C, D) The normalized fluorescence intensity of neurons treated with 100 μM MK-801 at 360 s. (E, F) The normalized fluorescence intensity of neurons in co-treated with paliperidone and MK-801 at 360 s. Panels A, C, and E show the changes of [Ca2 +]iin representative single neuronal cell, respectively, and panels B, D and F show the average alternation of [Ca2 +]iin ten randomly selected neurons.

3.4. Paliperidone antagonizes the inhibitory effect of MK-801 on neurite outgrowth

To assess the effect of paliperidone on neurite outgrowth, prefrontal cortical neurons were treated with different drugs immediately after plating and the total length and number of neurites per neuron were measured 12 h after plating ( Fig. 5 ). The results showed that MK-801 obviously decreased the average neurite length and density as compared with control group (p < 0.01). Paliperidone could effectively block the inhibitory effect of MK-801 as demonstrated by dramatically promoted average neurite length (p < 0.001) ( Fig. 5 D) and enhanced neurite density (p < 0.01) ( Fig. 5 E).

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Fig. 5 Effects of MK-801 and paliperidone on neurite outgrowth. (A–C) The photomicrographs of prefrontal cortical neurons exposed to different drugs for 12 h after plating. Compared to control group (A), MK-801 treatment could effectively decrease the total length and number of neurite (B), and this negative effect to neurons was dramatically blocked by paliperidone (C). (D–E) Quantitative analysis of neurite outgrowth of prefrontal cortical neurons treated with different drugs. The mean values of neutrite outgrowth were presented.#p < 0.01 vs MK-801 group.

3.5. Paliperidone enhances the expression of Akt1 and GSK3β in prefrontal cortical neurons treated with MK-801

Real-time PCR and Western blotting were performed to detect Akt1 and GSK3β expression in prefrontal cortical neurons treated with MK-801 in combination with or without paliperidone. Akt1 and GSK3β mRNA expression was dramatically decreased by MK-801 treatment, which was reversed by co-treatment with paliperidone (p < 0.01) ( Fig. 6 A, B, and D). The level of phosphorylated Akt1 and GSK3β were commonly used as the functional phenotype of Akt1 and GSK3β in schizophrenia. Thus, the effects of MK-801 and paliperidone on the level of phosphorylated Akt1 and GSK3β were examined using Western blotting. Similar to the mRNA expressions of Akt1 and GSK3β, MK-801 treatment significantly decreased the protein levels of phosphorylated Akt1 and GSK3β, and these effects were reversed by paliperidone treatment (p < 0.01) ( Fig. 6 C, E), again indicating that paliperidone could protect prefrontal cortical neurons from MK-801-induced damages.

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Fig. 6 Paliperidone increases Akt1 and GSK3β expression against MK-801 treatment. Real-time PCR (A, B) and western blotting (E, F) were used to determine the levels of Akt1 and GSK3β. Paliperidone could effectively attenuate MK-801-induced down-regulation of Akt1 and GSK3β. In photographs A and B, pound represents GAPDH group, pentagram represents control group, triangle represents paliperidone protection group, and the asterisk represents MK-801 damage group. (C, D, G and H) Quantitative analysis of Akt1 and GSK3β mRNA and protein levels of different groups. Scattergrams of data points were expressed as mean value from 3 independent experiments. *p < 0.01 compared with control group. There is significantly statistical difference between paliperidone group and MK-801 treated group (#p < 0.01).

3.6. Akt1 inhibitor LY294002 partly attenuates the neuroprotection effect of paliperidone on prefrontal cortical neurons

To explore whether the protective effects of paliperidone was mediated by Akt1/GSK3β signaling pathway, prefrontal cortical neurons were pretreated with the Akt1 inhibitor LY294002 for 1 h before treated with MK-801 and paliperidone. 48 h later, cells were double stained with Hoechst 33342 and PI, or subjected to MTT metabolism assay. The Hoechst 33342 and PI staining showed that surviving cells had bright blue integrated nuclei ( Fig. 7 A, D, G, J) while the apoptotic cells had bright red fragmented nuclei ( Fig. 7 B, E, H, K). Only few apoptotic cells with bright red nuclei were found in the control and paliperidone protection groups ( Fig. 7 A–C, G–I). By contrast, the numbers of cells with bright red nuclei were increased in MK-801 group and LY294002 group, indicating there were more apoptotic cells compare with control and paliperidone treatment groups. MTT metabolism assay also manifested that the protection effect of paliperidone against MK-801 induced damage of prefrontal cortical neurons was attenuated by LY294002. All the results suggested that paliperidone could protect prefrontal cortical neurons from MK-801-induced damages partly via Akt1/GSK3β pathway. In addition, the effects of LY294002 alone and paliperidone alone on apoptosis rate of prefrontal cortical neurons were also measured in our experiments. The results showed that both treatments had no significant effect on apoptosis rate compared to normal control group.

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Fig. 7 LY294002 partly attenuates paliperidone's protective effects against MK-801. Prefrontal cortical neurons were exposure to MK-801 with or without paliperidone for 48 h in the presence of Akt1 inhibitor LY294002 and double stained with Hoechst33342 (blue) and PI (red) (C, F, I, and L) to determine cell apoptosis (N) or subjected to MTT metabolism assay (M). (A, B, C) Normal control group: neurons in DMEM/F12 medium; (D, E, F) MK-801 group: neurons in 100 μM MK-801; (G, H, I) Paliperidone group: neurons in 100 μM MK-801 + 100 μM paliperidone; (J, K, L) LY294002 group: neurons in 100 μM MK-801 + 100 μM Paliperidone + 10 μM LY294002. Scattergrams of data points were expressed as mean value from 5 independent experiments. *p < 0.01, compared with MK-801 treated group;#p < 0.01, compared with Paliperidone treated group. Scale bars: A–L, 100 μm. The results indicated that the protective effects of paliperidone were mediated, at least partially, through Akt1/GSK3β signaling pathway.

4. Discussion

In the present study, an in vitro model of schizophrenia was established with MK-801 to investigate the protective effects of paliperidone against the neuronal damage of MK-801 on mouse prefrontal cortical neurons and clarify the intracellular mechanisms by which the second antipsychotic drugs attenuate the brain structure changes occurred in schizophrenia. The results showed that paliperidone treatment could alleviate MK-801 induced neuronal damage by increasing cell viability, promoting neurite outgrowth, and inhibiting cell apoptosis and [Ca2 +]irelease. More importantly, paliperidone could reverse MK-801-induced decreases of expression and phosphorylation of Akt1 and GSK3β, indicating that paliperidone exerts its neuroprotection effects against MK-801 in prefrontal cortical neurons partly via Akt1/GSK3β signaling pathway.

MK-801, also known as dizocilpine, is a noncompetitive glutamate receptors (N-methyl-d-aspartic acid receptor, NMDA) antagonist ( Tiedtke et al., 1990 ) and has been widely used to establish animal models of schizophrenia (Tiedtke et al, 1990, Hoffman, 1992, Corbett, 1995, Deutsch et al, 2002, and Manahan-Vaughan et al, 2008). MK-801 could block NMDA receptors and mimic the positive (hallucinations and delusions), negative and cognitive symptoms (decreased attention, memory and executive functions) occurred in animals with schizophrenia. Behavior researches showed that MK-801 could damage mice's learning and working memory as well as spatial orientation capacity. Moreover, vacuolization of prefrontal cortical neurons, atrophy of neuronal cell bodies and dendrites fracture have been found in the brain of mice treated with MK-801 (Olney et al, 1991 and de Olmos et al, 2009). Many studies have reported that MK-801 could damage a series of cultured cells in vitro (Hwang et al, 1999, Klimaviciusa et al, 2008, and Leung et al, 2008). According to previous study ( Ono et al., 2010 ), neuronal damage of schizophrenia was partly simulated with MK-801 in vitro. Both the MTT metabolism and LDH activity assays showed that MK-801 could induce neuronal damage in a dose-dependent manner. Cell viability decreased gradually (showing OD values in MTT assay declined from 0.214 to 0.129) with MK-801 concentration increasing from 25 μM to 200 μM, showing significant difference at 100 μM and 200 μM MK-801 compared with that of control. LDH measurement showed consistent results with MTT assay. It was worth noting that LDH release was not further changed as MK-801 increased from 100 μM to 200 μM, which should be further elucidated in the future.

Recently developed SGAs have been reported to have neuroprotective and neurogenetic effects against several kinds of injuries (Dickerson and Sharp, 2006, Ono et al, 2010, Koprivica et al, 2011, and Park et al, 2011). Some of them demonstrated the ability to decrease the rate of brain volume reductions in schizophrenics, which might be associated with their clinical efficacy for schizophrenia (Scherk and Falkai, 2006 and 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 and Gassó et al, 2012). In our present study, the effects of paliperidone alone on prefrontal cortical neurons were performed by MTT and LDH assays. The outcome of MTT indicated that only 100 μM and 200 μM paliperidone exhibited a protection effect and the positive effect of 200 μM paliperidone was slightly declined compared with that of 100 μM paliperidone.

LDH release was not significantly different among control group and paliperidone groups at different concentrations. When prefrontal cortical neurons were co-treated with 100 μM MK-801 and paliperidone with different concentrations, MK-801 induced neuronal damage were attenuated. MTT detection revealed that 50 μM paliperidone exerted an effective protective effect, and 100 μM paliperidone exhibited more significant protective influence. Similar to a previous study ( Schmidt et al., 2010 ), the effect of paliperidone on LDH release was dose-depended: when paliperidone concentration was 200 μM or higher, its protection effects were declined, possibly due to toxicity of excessive paliperidone to prefrontal cortical neurons.

To further investigate the role of Ca2 +on protective effects of paliperidone on prefrontal cortical neurons injured by MK-801, changes of intracellular free calcium concentration ([Ca2 +]i) with time were studied. The results showed that paliperidone could effectively retard [Ca2 +]iincrease over time induced by exposure to MK-801. Further study is needed to clarify the intracellular mechanism for this change.

Akt1/GSK3β signaling pathway played an important role in embryonic brain development ( Yang et al., 2005 ). Recent studies showed that both Akt1 and GSK3β also involved in cellular survival and pathogenesis of schizophrenia (Emamian et al., 2004; Franke, 2008). To explore the molecular mechanisms by which paliperidone exhibits its neuroprotective effects, expression and phosphorylation levels of Akt1 and GSK3β were explored by using real-time PCR and western blotting. The results showed that paliperidone could reverse MK-801-induced reduction of Akt1 and GSK3β expression and phosphorylation in prefrontal cortical neurons, in consistence with previous reports showing MK-801 reduced Akt1 expression ( Ono et al., 2010 ), and changed Akt signaling pathway in prefrontal cortex ( Xi et al., 2011 ). It has been reported that Akt1/GSK3β signaling pathway plays an important role in neuron synaptic plasticity, axon regeneration and branching (Namikawa et al., 2000; Markus et al., 2002;Lai et al, 2006 and Peineau et al, 2008). In accordance with the molecular analysis results, paliperidone could effectively reverse MK-801 exposure reduced neurite outgrowth including total neurite length and neurite density.

To substantiate our assumption that paliperidone exerts its neuroprotection via Akt1/GSK3β signaling pathway, the effects of LY294002 on the protection of paliperidone against MK-801 in prefrontal cortical neurons were evaluated. As a specific inhibitor of phosphatidylinositol 3-kinase (PI3K) ( Vlahos et al., 1994 ), LY294002 was also used to inhibit Akt1 activity ( Walker et al., 2000 ). The results showed that LY2940002 effectively attenuated the neuroprotection of paliperidone against MK-801 induced damages, which supported our original inference.

In summary, we initially investigated the neuroprotection of paliperidone on mouse prefrontal cortical neurons injured by exposure to MK-801. Our results showed that paliperidone exhibited its protective effects via Akt1/GSK3β signaling pathway, suggesting that paliperidone might have a strong neuroplasticity and could be effective in regulating altered brain structure of schizophrenia.

Role of funding source

This work was financially supported by grants of National Natural Science Foundation of China (No.81071081), Natural Science Foundation of Shandong Province (ZR2010HM051 and ZR2012HM026), Shandong Provincial Science and Technology Development Plan (2011GSF11810), Specialized Research Fund for the Doctoral Program of Higher Education (20120131110039) and Independent Innovation Foundation of Shandong University (2012TS113).

Contributors

All authors contributed to and have approved the final manuscript.

Conflict of interest

All authors declare that they have no conflicts of interest.

Acknowledgments

We thank Dr. Lihua Bao for moral and technical supports of this study. This work was supported by grants of National Natural Science Foundation of China (No. 81071081), Natural Science Foundation of Shandong Province (ZR2010HM051 and ZR2012HM026), and Shandong Provincial Science and Technology Development Plan (2011GSF11810), Specialized Research Fund for the Doctoral Program of Higher Education (20120131110039) and Independent Innovation Foundation of Shandong University (2012TS113).

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Footnotes

a Key Laboratory for Experimental Teratology of the Ministry of Education and Department of Anatomy, Shandong University School of Medicine, Jinan, Shandong 250012, China

b Department of Histology and Embryology, Shandong University School of Medicine, Jinan, Shandong 250012, China

c Department of Pharmacy, Shandong University School of Pharmacy, Jinan, Shandong 250012, China

d Department of Psychiatry, Shandong University School of Medicine, Jinan, Shandong 250012, China

lowast Corresponding author. Tel./fax: + 86 531 88382093.