Dysregulation of Retinoid Transporters Expression in Body Fluids of Schizophrenia Patients Chunling Wan,†,‡,# Yifeng Yang,†,‡,# Huafang Li,§ Yujuan La,†,‡ Hui Zhu,†,‡ Lei Jiang,†,‡ Yongshuo Chen,†,‡ Guoyin Feng,§ and Lin He*,†,‡ Bio-X Life Science Research Center, Shanghai Jiao Tong University, 1954 Huashan Road, Shanghai 200030, China, Institutes for Nutritional Sciences, Shanghai Institute of Biological Sciences, Chinese Academy of Sciences, 320 Yueyang Road, Shanghai 200031, China, Shanghai Institute of Mental Health, 600 South Wan Ping Road, Shanghai, China Received April 18, 2006
This study aims to find the biomarkers or associated proteins in body fluids of schizophrenia patients so that we can further understand the etiology of schizophrenia. We applied proteomic technologies combining two-dimensional electrophoresis with Coomassie blue staining and mass spectrometry and identifyed a procedure for the clinical screening of disease-influenced body fluid proteins in two sets of samples, plasma from 19 schizophrenia patients and cerebrospinal fluid (CSF) from 35 drug-treated schizophrenic patients and 36 healthy controls. The expression of transthyretin (TTR) tetramer increased significantly in plasma of schizophrenic patients after a valid 2 months in-hospital antipsychotic treatment. Conversely, the expression of the TTR tetramer and apolipoprotein E (ApoE) was downregulated by up to 1.68 and 3.62 times, respectively, in the CSF of schizophrenia patients compared to that of normal controls, which has not been reported previously. Considering that the TTR tetramer and ApoE are both retinoid transporters, retinoid dysfunction might be involved in the pathology of schizophrenia. Keywords: Transthyretin • Retinoid Transporter • Schizophrenia • Plasma • Cerebrospinal Fluid
Introduction
Subjects
Schizophrenia is a complex and devastating brain disorder that affects 1% of the population and has serious financial consequences for the patients, their relatives, and the national economy.1,2 Its etiology is complex and mostly speculative, and its pathophysiology and psychopathology have a large degree of variability. As yet, there are no affirmative descriptive biological criteria for schizophrenia, and it is now defined solely by negative symptoms and disordered thinking. There is a prerequisite for the improved diagnosis, management, and treatment of this disorder. The composition of the cerebrospinal fluid (CSF) within and around the brain is an important physiological parameter that can be subject to disease processes with profound consequences for mental functions. In the present study, comparative proteome analysis was employed to screen for plasma protein alternations of the medically treated schizophrenia patients and CSF protein aberrations in schizophrenia patients to investigate the relationship of body fluids variables and negative symptomatology. * Address correspondence to Dr. Lin He, Shanghai Jiao Tong University, Bio-X Life Science Research Center, Hao Ran Building, 1954 Huashan Road, Shanghai 200030, China; or Institute for Nutritional Sciences, SIBS, Chinese Academy of Sciences, 294 Taiyuan Road, Shanghai 200031, China. E-mail:
[email protected]. Tel/fax: 86-21-62822491. † Shanghai Jiao Tong University. ‡ Chinese Academy of Sciences. # These two authors contributed equally. § Shanghai Institute of Mental Health. 10.1021/pr060176l CCC: $33.50
2006 American Chemical Society
The diagnosis of schizophrenia was assigned according to CCMD-II-R (a counterpart diagnostic criterion of DSM-III-R in China) and DSM-III.3 Plasma samples were from 19 schizophrenic patients, of whom 12 (63.2%) were men and 7 (36.8%) were women. The mean age was 38.8 years (range 15-57). These patients were given an 8-week course of chlorpromazine medication. Two plasma samples were obtained from the 19 schizophrenic patients enrolled in the study at baseline and after 8 weeks of treatment. A plasma protein expression profile was studied before and after antipsychiatric therapy in the patients. Patients showing a valid reduction in psychiatric symptoms based on the Positive and Negative Syndrome Scale for Schizophrenia (PANSS) score were labeled as responders, and those in whom psychiatric symptoms remained stable were labeled as nonresponders. Cerebrospinal fluid samples were collected by lumbar puncture from chlorpromazine-treated schizophrenic patients (n ) 35, age ) 63.2 ( 4.4) and age/sexmatched controls (n ) 36, age ) 60.3 ( 7.4). All of the recruited subjects were unrelated Han Chinese. Plasma and CSF samples were collected at the Shanghai Institute of Mental Health, following the guidelines of the local ethical committee. Written informed consent was obtained from all subjects after the procedure had been fully explained.
Methods After protease inhibitor cocktail and phenylmethylsulfonyl fluoride (PMSF) solution were added, CSF and plasma were Journal of Proteome Research 2006, 5, 3213-3216
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Table 1. Pretreatment and Post-treatment Values of Plasma Proteins in the Study Group (n ) responders (n ) 14)
19)a nonresponders (n ) 5)
protein name
pretreatment
post-treatment
P-valueb
TTR tetramer TTR monomer RBP Hp R2 ApoE Glutathione peroxidase precursor ApoA1 Retinoid (µmol/L)
0.156 ( 0.040 0.393 ( 0.045 0.139 ( 0.014 0.759 ( 0.181 0.128 ( 0.020 0.016 ( 0.002 3.187 ( 0.205 2.569 ( 0.178
0.222 ( 0.048 0.369 ( 0.035 0.164 ( 0.019 0.749 ( 0.159 0.134 ( 0.027 0.015 ( 0.003 3.238 ( 0.196 2.508 ( 0.217
0.002 0.628 0.049 0.927 0.674 0.552 0.824 0.802
pretreatment
post-treatment
P-value
0.218 ( 0.034 0.269 ( 0.020 0.117 ( 0.030 0.986 ( 0.163 0.107 ( 0.005 0.014 ( 0.001 1.84 ( 0.172
0.181 ( 0.034 0.295 ( 0.032 0.115 ( 0.0293 0.959 ( 0.151 0.090 ( 0.014 0.015 ( 0.001 1.892 ( 0.214
0.359 0.449 0.919 0.859 0.167 0.869 0.84
a Protein concentration was determined by the percentage of the volume of protein spot(s) in plasma total protein presented in the 2-DE gel. Values are shown as mean ( SE. b P-values < 0.01 are in boldface.
stored at -80 °C until two-dimensional gel electrophoresis (2-DE) was performed. To meet the requirements for the loading amount for a 2-DE analysis, the CSF samples from 36 controls and 35 patients were pooled to form a control CSF sample and a patient CSF sample. Then, these two pooled samples were divided into 2 and 4 wells, respectively (0.7 mg/ well). The proteomics operations were produced as described previously.4 Briefly, the plasma and pooled CSF samples for isoelectric focusing were applied onto immobilized pH 3-7 nonlinear gradient strips (24 cm). The second-dimensional separation was performed in 12.5% SDS polyacrylamide gels. After the second-dimensional separation, gels were stained with 0.1% Coomassie blue G-250. ImageMaster 2-D Platinum software was employed to the analysis of Coomassie blue-stained gels. The peptide-mass fingerprint method of matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) was used to identify the protein spots on 2-DE gels. Plasma vitamin A calculation was carried out with a PerkinElmer luminescence spectrometer. We used paired Student’s t-test to detect changes in plasma protein expression levels and retinoid level after medical treatment. We introduced a bivariate correlation model to estimate the correlation between vitamin A and TTR tetramer level. Statistical analysis was performed using Statistical Package of Social Science (SPSS) for Windows, version 10.0. All tests mentioned above were twotailed, and significance was accepted at P < 0.05. Differences in each of the CSF protein levels between schizophrenia patients and normal controls were evaluated by comparing the protein mean values of these two groups.
Results We compared 56 plasma protein spots in schizophrenia patients before and after medical treatment. Partial results are shown in Table 1. The expression levels of the TTR tetramer increased significantly in responders after medical treatment in comparison with the pretreatment levels (P ) 0.002). The up-regulated plasma expression of the TTR tetramer after antipsychotic treatment established by 2-DE gels was confirmed by Western blotting of native PAGE with rabbit polyclonal anti-TTR IgG (Figure 1). To reveal whether antipsychotic treatment is a contributing factor in the change of the TTR tetramer expression, we further screened the plasma protein expression patterns of five nonrespondent patients and found none of the investigated proteins showed significant changes. Although there were only five subjects included in this study, we can exclude an increasing trend of the TTR tetramer. This finding suggests that a modulation of the TTR gene expression is more likely to be associated with the disease process itself, 3214
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Figure 1. Western blot of TTR tetramer in the plasma. Western blot of TTR tetramer was implemented in the plasma of schizophrenia patients before and after antipsychiatric therapy (1 and 2, 3 and 4, 5 and 6 from the same individual, respectively). The expression of TTR tetramer increased significantly in responders after medical treatment (2, 4, and 6).
rather than a consequence of the body’s response to an antipsychotic therapy. We obtained the plasma retinoid data of 14 responder patients, and their retinoid level did not show any obvious changes after valid medical treatment (P ) 0.802) (Table 1). Results of a bivariate correlation analysis revealed no correlation between the TTR tetramer and retinoid level (r ) -0.05, P ) 0.807). Another important finding in our study concerned the significant decrease in TTR and ApoE in the CSF of schizophrenia patients, which has not previously been reported. To establish whether TTR tetramer abnormalities in plasma could be confirmed, we further investigated CSF protein expression in 35 patients and 36 controls. We determined the expression levels of 80 protein spots including TTR, ApoE, retinol-binding protein (RBP), Ig kappa, Ig gamma, haptoglobin (Hp), apolipoprotein J (ApoJ), apolipoprotein A1 (ApoA1), and so on, and found ApoE and TTR tetramer were the two most obvious differentially expressed proteins between schizophrenia patient sample pool and control pool (Figure 2). The expression levels of the TTR tetramer and ApoE in the CSF samples were reduced 1.68 and 3.62 times, respectively, in the disease group compared to the controls (Table 2). This result confirmed our previous observation of changed TTR tetramer expression in schizophrenia patient plasma. Meanwhile, an even greater depressed expression of ApoE was found in the CSF of schizophrenia patients.
Discussion In humans, TTR serves as an important retinoid transporter, which carries 20% of retinoid.5 Because of the changes in TTR tetramer level in both plasma and CSF, and the observed significant lower ApoE (another retinoid transporter) level in the CSF of schizophrenia patients, we were especially interested in whether there was a reaction responsible for the reduction of retinoid level in schizophrenia patients. On account of the same protein concentration loaded on each gel (0.7 mg) and each protein spot evaluated as a percentage of total CSF
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anisms underlying vitamin A dysfunction. Gorman’s research group found that the TTR concentration in CSF was significantly lower in the depressed patients than in the comparison subjects.8 Retinoid dysregulation may be an important factor in the etiology of schizophrenia, and Goodman gave three independent lines of evidence suggesting retinoid as a causative factor in schizophrenia.9 Further evidences that TTR and ApoE have profound effects on brain function comes from functional studies. TTR-null mice display increased exploratory activity and reduced signs of depressive behavior.10 Apolipoprotein E-knockout mice showed severe learning deficits in spatial learning and memory abilities.11
Figure 2. Typical 2-DE picture of human CSF. The gel was separated on a 26 × 20 cm plate and Coomassie blue-stained. The horizontal axis represents the IEF dimension, which stretches from pH 3-7 (nonlinear). The vertical axis represents 12.5% SDSPAGE gel. Table 2. Expression Analysis of CSF Proteins in Schizophrenia Patients and Controlsa protein name
TTR tetramer TTR monomer TRFE (Serotransferrin precursor) TETN (Tetranectin precursor) RBP Ig kapa Ig gama Hp R2 ApoJ ApoE ApoA1 A1AT (R1-antitrypsin precursor) Albumin A1AH (R1-acid glycoprotein 2 precursor)
schizophrenia controls ratiob
0.76 2.04 10.22 0.077 0.23 9.23 15.41 0.13 3.43 0.28 0.32 5.89 23.34 3.01
1.29 1.52 11.29 0.055 0.23 10.01 22.38 0.16 2.4 1.00 0.38 4.74 16.67 3.96
-1.68 +1.34 -1.11 +1.41 -1.02 -1.08 -1.45 -1.17 +1.43 -3.62 -1.17 +1.24 +1.4 -1.31
a Pooled CSF of well-matched schizophrenia patients (n ) 35) and controls (n ) 36) were analyzed by two-dimensional electrophoresis. Protein concentration was determined by the percentage of the volume of protein spot(s) in CSF total protein presented in the 2-DE gel. b Fold differences represent the ratio of disease/control (+) and control/disease (-) values.
protein, the observed lower TTR tetramer and ApoE protein concentrations in CSF of schizophrenia patients were not related to the abnormalities of the total protein content in patients’ CSF, which has been addressed in several studies.6 We speculated that the decreased CSF TTR tetramer and ApoE observed in patients with schizophrenia reflects a decreased transport of vitamin A to the brain. Unfortunately, we could not obtain retinoid data in CSF to test this hypothesis, because no more CSF samples of these patients were collected. It is somehow strange that the average content of TTR (monomer and tetramer) observed in our 2-DE gels is only 2.81% of CSF protein, which is greatly lower than the 25% reported.7 TTR is synthesized de novo by epithelial cells of the choroid plexus in humans. Although our data do not suggest any reason for the decrease in TTR and ApoE in the CSF of schizophrenia patients, a defect in protein synthesis in the CSF is a possibility. Comparative analysis of deregulated proteins in both body fluids revealed a pattern of related functions, which fits well into the existing knowledge of the toxic processes and mech-
The CSF samples have been pooled to obtain a sufficient amount of proteins for the 2-DE analysis; thus, we cannot make any statistical conclusions about any proteins being differentially expressed. Pooling of heterogeneous samples is not the first choice when analyzing disease-related proteins, because it will compensate the individual differences of the interesting proteins. More sensitive methods to evaluate the suggestive result of ApoE and TTR tetramer down-regulation in the CSF of schizophrenia patients are required. The other limitation of the present study is that the protein quantification is based on 2-DE gels. Most of the proteins have multiple isoforms that differ in electrophoretic mobility. Protein spots volumes are therefore insufficient to present certain protein’s estimation, although we estimated as many isoforms of the same protein as we could to form this protein expression value. Furthermore, because of the small sample number for 2-DE, these results will need to be reproduced in a larger cohort in the future. Two important findings of our study were the downregulation of two retinol transporter proteins, ApoE and TTR tetramer, in the CSF of schizophrenia patients, and TTR tetramer corresponding alterations in schizophrenia patients’ plasma after valid medical treatment. These results imply an insufficient transport of retinol to the brain. We believe that measurement of TTR and ApoE in the CSF and plasma has the promise to provide a surrogate marker that will aid in diagnosis of schizophrenia in high-risk populations and in identification of its optimal treatments. However, we will have to await results of future studies that more fully define the mechanism of these retinoid transporters involved in schizophrenia.
Acknowledgment. We thank all the patients who took part in the study. We also thank Dr. Ann B. Goodman for critical reading of the manuscript and helpful discussions. This work was supported by grants from the national 973 and 863 Programs of China, Shanghai Postdoctoral Science Foundation, the National Natural Science Foundation of China, the Ministry of Education, PRC. References (1) Haro, J. M.; Salvador-Carulla, L.; Cabases, J.; Madoz, V.; VazquezBarquero, J. L. Br. J. Psychiatry 1998, 173, 334-40. (2) Rossler, W.; Salize, J.; Knapp, M. Fortschr. Neurol. Psychiatr. 1998, 66 (11), 496-504. (3) Spitzer, R. L.; Williams, J. B.; Gibbon, M.; First, M. B. Arch. Gen. Psychiatry 1992, 49 (8), 624-9. (4) Yang, Y.; Wan, C.; Li, H.; Zhu, H.; La, Y.; Jiang, L.; Feng, G.; He, L. Anal. Chem. 2006, 78 (11). 3571-6. (5) Blake, C. C.; Geisow, M. J.; Oatley, S. J.; Rerat, B.; Rerat, C. J. Mol. Biol. 1978, 121 (3), 339-56. (6) Torrey, E. F.; Albrecht, P.; Behr, D. E. Am. J. Psychiatry 1985, 142 (5), 657-8.
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letters (7) Herbert, J.; Wilcox, J. N.; Pham, K. T.; Fremeau, R. T., Jr.; Zeviani, M.; Dwork, A.; Soprano, D. R.; Makover, A.; Goodman, D. S.; Zimmerman, E. A.; et al. Neurology 1986, 36 (7), 900-11. (8) Sullivan, G. M.; Hatterer, J. A.; Herbert, J.; Chen, X.; Roose, S. P.; Attia, E.; Mann, J. J.; Marangell, L. B.; Goetz, R. R.; Gorman, J. M. Am. J. Psychiatry 1999, 156 (5), 710-5. (9) Goodman, A. B. Proc. Natl. Acad. Sci. U.S.A. 1998, 95 (13), 7240-4.
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Wan et al. (10) Sousa, J. C.; Grandela, C.; Fernandez-Ruiz, J.; de Miguel, R.; de Sousa, L.; Magalhaes, A. I.; Saraiva, M. J.; Sousa, N.; Palha, J. A. J. Neurochem 2004, 88 (5), 1052-8. (11) Oitzl, M. S.; Mulder, M.; Lucassen, P. J.; Havekes, L. M.; Grootendorst, J.; de Kloet, E. R. Brain Res. 1997, 752 (1-2), 189-96.
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