A Comparative Proteomics Analysis of Rat Mitochondria from the Cerebral Cortex and Hippocampus in Response to Antipsychotic Medications Baohu Ji,‡,§,⊥,# Yujuan La,‡,§,⊥,# Linghan Gao,‡,§,⊥,# Hui Zhu,‡,§,⊥ Nan Tian,‡,§,⊥ Ming Zhang,‡,§,⊥ Yifeng Yang,‡,§,⊥ Xinzhi Zhao,‡,§,|,⊥ Ruqi Tang,‡,§,⊥ Gang Ma,‡,§,⊥ Jian Zhou,‡,§,⊥ Junwei Meng,‡,§,⊥ Jie Ma,‡,§,⊥ Zhao Zhang,‡,§,⊥ Huafang Li,† Guoyin Feng,† Yujiong Wang,‡,¶ Lin He,*,‡,§,|,⊥ and Chunling Wan*,‡,§,⊥ Bio-X Center, Shanghai Jiao Tong University, Shanghai, China, Institute for Nutritional Sciences, Shanghai Institutes of Biological Sciences, Chinese Academy of Sciences, Shanghai, China, Institutes of Biomedical Sciences, Fudan University, Shanghai, China, Shanghai Institute of Mental Health, Shanghai, China, School of Life Science, Ningxia University, Yinchuan, China, and Key Laboratory of Developmental Genetics and Neuropsychiatric Diseases (Shanghai Jiao Tong University), Ministry of Education, Shanghai, China Received October 19, 2008
An increasing number of experiments have found anomalies in mitochondria in the brains of psychotics, which suggests that mitochondrial dysfunction or abnormal cerebral energy metabolism might play an important role in the pathophysiology of schizophrenia (SCZ). We adopted a proteomic approach to identify the differential effects on the cerebral cortex and hippocampus mitochondrial protein expression of Sprague-Dawley (SD) rats by comparing exposure to typical and atypical antipsychotic medications. Differential mitochondrial protein expressions were assessed using two-dimensional (2D) gel electrophoresis for three groups with Chlorpromazine (CPZ), Clozapine (CLZ), quetiapine (QTP) and a control group. A total of 14 proteins, of which 6 belong to the respiratory electron transport chain (ETC) of oxidative phosphorylation (OXPHOS), showed significant changes in quantity including NADH dehydrogenase (ubiquinone) 1 alpha subcomplex 10 (Ndufa10), NADH dehydrogenase (ubiquinone) flavoprotein 2 (Ndufv2), NADH dehydrogenase (ubiquinone) Fe-S protein 3 (Ndufs3), F1-ATPase beta subunit (Atp5b), ATPase, H+ transporting, lysosomal, beta 56/58 kDa, isoform 2 (Atp6v1b2) and ATPase, H+ transporting, V1 subunit A, isoform 1 (Atp6v1a1). The differential proteins subjected to 2D were assessed for levels of mRNA using quantitative real time PCR (Q-RT-PCR), and we also made partial use of Western blotting for assessing differential expression. The results of our study may help to explain variations in SD rats as well as in human response to antipsychotic drugs. In addition, they should improve our understanding of both the curative effects and side effects of antipsychotics and encourage new directions in SCZ research. Keywords: schizophrenia • comparative proteomics • mitochondrial dysfunction • complex I • oxidative phosphorylation • antipsychotic medications
Introduction Schizophrenia (SCZ), the most severe of psychiatric disorders, affects 1% of the world population with broadly equal prevalence throughout diverse cultures and geographic areas. * To whom correspondence should be addressed. Lin He or Chunling Wan, Institutes of Biomedical Sciences Fudan University, 138 Yixueyuan Road, Shanghai 200032, PR China or Bio-X Center, Shanghai Jiao Tong University, 1954 Huashan Road, Shanghai 200030, China. Tel. and fax: 8621-62822491. E-mail: (C.W.)
[email protected] or (L.H.)
[email protected]. ‡ Shanghai Jiao Tong University. § Shanghai Institutes of Biological Sciences. ⊥ Key Laboratory of Developmental Genetics and Neuropsychiatric Diseases. # These authors contributed equally to this work. | Fudan University. † Shanghai Institute of Mental Health. ¶ Ningxia University. 10.1021/pr800876z CCC: $40.75
2009 American Chemical Society
The symptoms of SCZ are generally divided into two categories, namely, ‘positive’ symptoms, such as hallucinations, delusions, disorganization of thought, bizarre and incongruous behavior, and ‘negative’ symptoms, such as loss of motivation, restricted range of emotional experience and expression, alogia and reduced hedonic capacity.1 Although a great deal of work has been done in this area, the molecular mechanism triggering SCZ has, so far, remained elusive. The etiology of SCZ appears to be multifaceted, with genetic, nutritional, environmental, and developmental factors all implicated.2 Antipsychotic drugs remain the current standard of care for mental disorders including schizophrenia.3 Chlorpromazine (CPZ) was the first of the first-generation of antipsychotics (the so-called “typical antipsychotics”). It operates on central dopaminergic pathways, and although effective, it has serious side effects.4 A number Journal of Proteome Research 2009, 8, 3633–3641 3633 Published on Web 05/14/2009
research articles of new antipsychotic drugs (the so-called “atypical antipsychotics”) have been introduced since 1990. Clozapine (CLZ) is the first atypical antipsychotic drug, so designated because it has more antipsychotic effects without the adverse mobility effects of the first-generation drugs.4 Subsequent antipsychotic drugs have followed, such as quetiapine (QTP), risperidone and olanzapine, and so forth. The atypical antipsychotic drugs usually act on both dopamine receptors and serotonin receptors.5,6 There is increasing evidence that mitochondrial energy metabolism might be disturbed by antipsychotic drugs. The research results of Burkhardt et al. showed that the typical and atypical antipsychotics have a direct inhibitory effect on the respiratory electron transport chain (ETC), especially on complex I enzyme activity, in freeze-thawed preparations of rat brain mitochondria.7,8 Maurer and Mo¨ller9 analyzed the activity of the mitochondrial respiratory chain enzyme complexes, which produce ATP via oxidative phosphorylation (OXPHOS), and found that antipsychotics may inhibit the mitochondrial complex I in human brain tissue.10 The mechanisms underlying the antipsychotic-associated neurotoxicity, and the toxic effects on mitochondria, have not yet been fully identified. Proteomics is a powerful tool for identifying protein expression alterations in disease tissue and has been successfully employed to study a variety of disorders, including SCZ.11-13 In this study, we used comparative proteomics to analyze expression changes of all mitochondrial proteins from the cerebral cortex and hippocampus of Sprague- Dawley (SD) rats in response to antipsychotic medication. The rats were divided into four groups, three of which were treated with CPZ, CLZ and QTP, respectively, and one control group. The goal of this study was to evaluate the effects of antipsychotic drugs on mitochondria function, and to provide further understanding of the curative effects and the side effects of antipsychotics.
Materials and Methods Animals and Treatment. Male SD rats (6 weeks old, n ) 50) weighing 200 ( 10 g on arrival were obtained from the Shanghai Laboratory Animal Co., Ltd. (SLAC, Shanghai, China). The rats were randomly divided into one of four groups: a CPZtreatment group (11 rats), a CLZ-treatment group (12 rats), a QTP-treatment group (14 rats) and a control group (13 rats). Three or four rats were housed in one cage and kept on a 12/ 12 h light/dark cycle with food and water freely available. The animal room was air conditioned and the ambient temperature was maintained at 23.5 ( 3.5 °C and relative humidity at 55 ( 15%. After 2 days of acclimatization, all rats were given their respective antipsychotics which were dissolved in 0.9% (w/v) saline and delivered by oral gavage using an injector with a metal ball tip, either CPZ (90 mg/(kg of body weight/day)),14 CLZ (45 mg/(kg/day))15 or QTP (50 mg/(kg/day)).16 Control animals were treated in the same way with 0.9% (w/v) saline. At the beginning of each week, all four groups were weighed in order to adjust the dose according to the weight gained the previous week. After a 34-day exposure to the antipsychotics, the rats were killed by cervical dislocation. This procedure was conducted in compliance with the Guide for the Care and Use of Laboratory Animals as approved by the local animal ethics committee. Isolation of Mitochondria. Fresh mitochondria samples were collected from the cerebral cortex and hippocampus of the three treated groups and the control group. The cerebral cortex were rapidly removed, placed on ice, and divided into 3634
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Ji et al. three parts. Part I was placed in a freezing tube containing 0.8 mL of TRIzol reagent (Invitrogen, CA) and kept in liquid nitrogen to isolate total RNA; Part II was stored in a freezing tube and placed in liquid nitrogen to isolate all the proteins; Part III contained most of the cerebral cortex and hippocampus and these were examined immediately after collection. The hippocampus was divided into two parts, Part I and Part III. First, the tissues were washed three times using a mitochondrial isolation homogenization buffer (MIB) consisting of 320 mM sucrose, 1 mM EGTA, 1 mM EDTA, 0.23 mM PMSF, 10 mM Hepes-KOH (PH 7.4) and a correspondingly superfluous protease inhibitor cocktail set I (Merck-Calbiochem, Darmstadt, Germany). The tissue was then pooled (two random rats from the same group) and minced with scissors and gently homogenized with ultrasonic vibrations on ice. After diluting the homogenate to 5 mL with the MIB, large cellular debris and nuclei were eliminated by centrifuging for 3 min at 1500g, 4 °C, and the supernatant was transferred into a new centrifuge tube. The deposition was resuspended in 3 mL of MIB, after centrifuging for 3 min at 1500g, 4 °C and the supernatant was combined with that of the first step. The combined supernatant was centrifuged for 3 min at 1500g for 5 min, 4 °C. The supernatant was transferred into a new centrifuge tube and centrifuged for 10 min at 15 000g, 4 °C. The resultant deposit was resuspended in 2 mL of MIB and layered onto 1.5 mL of 7.5% (w/v) Ficoll-sucrose medium (7.5% Ficoll-70 mM sucrose, 210 mM mannitol, 1 mM EDTA-K2, 0.23 mM PMSF and 5 mM Hepes-Tris, pH 7.4) on top of 1 mL of 10% (w/v) Ficoll-sucrose medium and centrifuged in a Beckman Optima MAX-E Ultracentrifuge (Beckman Coulter, CA) at 100 000g for 90 min. Mitochondrial pellets were enriched below the 10% Ficoll-sucrose medium. The pellets were suspended in 4.5 mL of MIB and then centrifuged for 5 min at 15 000g, 4 °C. The pellets were gently mixed and resuspended in 4.5 mL of fatty acid-free bovine serum albumin (BSA) containing MIB and centrifuged at 9800 rpm (revolutions per minute) for 10 min. The nonsynaptic purified mitochondrial pellets were then washed once in MIB alone, pelleted at 15 000g for 5 min (the mitochondrial pellets were evaluated by electron microscope) and resuspended in an appropriate volume of MIB and stored at -80 °C for subsequent analyses.17 Protein Extraction and 2D Electrophoresis. Total mitochondrial protein was prepared from each specimen. The specimen was centrifuged at 4 °C, 15 000g for 5 min. After removal of the supernatant, the deposit was suspended in 1 mL of sample buffer containing of 7 M Urea, 2 M Thiourea, 4% CHAPS, 65 mM 1,4-dithioerythritol, 40 mM Tris, 10 µL of 100 mM EDTA, 10 µL of 0.1 mM PMSF, and 10 µL of protease inhibitor cocktail set I (Merck-Calbiochem). The mitochondria pellets were gently homogenized with ultrasonic vibrations on ice until the sample buffer was limpid. After 1 h of incubation at room temperature, the sample was centrifuged at 12 °C, 14 000g for 1 h to remove insoluble material. The supernatant protein was added to 4 mL of sample buffer and treated for salt removal three times using the Amicon Ultra-4 (Millipore, Billerica, MA) at 25 °C, 4000g for 20 min. The protein content in the supernatant was determined by the Coomassie blue method. A total of 450 µL of sample buffer containing 1.6 mg of total protein was added to the 2.4 µL of IPG buffer (pH 3-10, GE Healthcare, Piscataway, NJ) and centrifuged at 4 °C, 14 000 rpm for 30 min. Supernatant was used for the first dimension. Hydration was carried out using PH 3-10 nonlinear Immobiline DryStrips (GE Healthcare) on an Ettan IPGphor apparatus
research articles
Proteomics Analysis of Rat Mitochondria (GE Healthcare) for 14 h at 100 V/h, followed by 1 h at 500 V/h, 1 h at 1000 V/h, and 1 h at 4 000 V/h, and then focused for 12 h at 8 000 V/h until 96 000 V/h. After the isoelectric focusing, the Immobiline DryStrips were incubated for 15 min in a solution containing 50 mM Tris-HCl (pH 8.8), 6 M urea, 30% glycerol, 2% SDS and 60 mM DTT at room temperature. The strips were then alkylated for 15 min in a similar solution with DTT replaced by 100 mM iodoacetamide.18 The seconddimensional separation was performed in 12.5% SDS polyacrylamide gels. Gel was fixed in a 400 mL fix buffer containing 200 mL of methanol, 20 mL of Orthophosphoric acid and 180 mL of deionized H2O. Protein spots were visualized using 0.1% Coomassie Blue G-250 staining overnight. Each gel was twice discolored for 2 h using 400 mL of 20% Ammonium sulfate. Gel Image Analysis. Stained gels were scanned on a UMAX PowerLook III scanner (resolution 300 DPI, GE Healthcare), and the resulting TIFF images were analyzed using ImageMaster 2D Platinum software 5.0 (GE Healthcare). The automatic detection of the spot from the gel images included spot detection, modification, warping, background subtraction and matching. Spot volumes were normalized against the total volume of all the spots in the gel.18 Protein Identification using Mass Spectrometry. Protein spots of interest were excised manually from the Coomassie blue-stained gel and diced into small pieces (∼ 4 mm3) with a sheared tip and placed into a 96 hole board. The gel fragments were destained and dehydrated by washing twice for 30 min with 25 mM NH4HCO3 in 50% acetonitrile until shrunken and white. The destained gel particles were then dried for 19 min at 37 °C. Then, every dry gel particle was incubated in 10 µL of 25 mM NH4HCO3 with trypsin (GE Healthcare) for digestion for 3 h. Total peptides were extracted twice with 50% acetonitrile (ACN), 5% Trifluoroacetic acid (TFA) (Sigma-Aldrich, St Louis, MO), and then, the peptides supernatants were dried at 37 °C for 2 h. The dried peptides were added to a 3 µL buffer A (0.3% TFA, 50% ACN) and then thoroughly crushed with pipettes. The mix was then added to 3 µL of saturated R-Cyano4-hydroxycinnamic acid (Fluka, Milwaukee, WI) and again crushed. A total of 0.3 µL of the mixture was then subject to mass spectrometric analysis (MALDI-ToF/MS Pro, GE Healthcare). The peptide fingerprinting was compared using the GE Healthcare database and a mascot database. RNA Isolation and Q-RT-PCR. The cerebral cortex and hippocampus samples with added 0.8 mL of TRIzol reagent were removed from the liquid nitrogen and thawed on ice, then homogenized on a Mini-Bead Beater (Biospec, Bartlesville, OK). Total RNA of the sample was extracted using a TRIzol reagent recommended protocol, and quantities from 260/280 were measured to adjust the initial total RNA. The cDNA was synthesized by random hexamers using the Superscript III FirstStrand Synthesis System for RT-PCR (Invitrogen). We measured the gene expression level of objective genes which code those proteins with the most significant changes using a Q-RT-PCR system on SyberGreen I (Molecular Probe, Inc.) and ABI PRISM 7900 (Applied Biosystems, Los Angeles, CA). Q-RT-PCR was performed using gene-specific primers designed by Primer Express 2.0. A comparative Ct method was employed for quantification according to the manufacturer’s protocol. Glyceraldehyde 3-phosphate dehydrogenase (Gapdh) was used for normalization.19 The relative gene expression was evaluated using the comparative cycle threshold method.20 Protein Isolation and Western Blotting. Total proteins were extracted from the cerebral cortex and the accessory of RNA
was extracted from the hippocampus using TRIzol reagent. We applied Western blotting to some of the differential expression proteins of ETC and Gfap, Cyb5b assessed by 2-D. Western blotting was performed with the following primary antibodies: mouse monoclonal anti-Gfap (2E1), sc-33673; Atp6v1b1/2 (F6), sc-55544; Atp5b (3D5), sc-58618; Ndufs3 (17D95), sc-58393; goat polyclonal anti-Atp6v1a1 (C-16), sc-31462; Cyb5b (K-18), sc-48470 (Santa Cruz Biotechnology, CA) and using Polyacrylamide gradient (7.5-15%) SDS gels. A total of 50 µg of soluble protein was loaded per lane. Gels were blotted onto PVDF membranes and blocked with 5% nonfat dry milk in Trisbuffered saline containing 0.05% Tween 20 (TBST) overnight at 4 °C. After primary antibody (1:1000) incubation for 3 h at room temperature, membranes were washed three times using TBST for 5 min, and then incubated with corresponding horseradish peroxidase (HRP)-conjugated secondary antibody (1:8000) for 2 h at room temperature. Membranes were then washed using TBST for 5 min, three times. Blots were developed with 1 mL of Western blotting luminol reagent for 3 min (Santa Cruz-2048, Santa Cruz, CA) and visualized by autoradiography. HRP-conjugated monoclonal mouse anti-glyceraldehyde 3-phosphate dehydrogenase (Gapdh) was introduced as a reference gene. Western blots were digitized using Image Quant 5.2 (Molecular Dynamics). Statistical Analysis. All the data were tested for normal distribution using the Kolmogorov-Smirnov Test before calculation of differences. Normally distributed data were analyzed using one-way ANOVA followed by a Post Hoc Dunnett Test for comparison with the control group.21 Data that was not distributed normally were analyzed using nonparametric Wilcoxon Mann-Whitney Test.21 P-values
