Molecular Psychiatry (2004) 9, 684–697 & 2004 Nature Publishing Group All rights reserved 1359-4184/04 $30.00 www.nature.com/mp IMMEDIATE COMMUNICATION Mitochondrial dysfunction in schizophrenia: evidence for compromised brain metabolism and oxidative stress S Prabakaran*,1,2, JE Swatton*,1,2, MM Ryan*,1,2, SJ Huffaker*,1,2, JT-J Huang1,2, JL Griffin3, M Wayland4, T Freeman4, F Dudbridge4, KS Lilley5, NA Karp5, S Hester5, D Tkachev1,2, ML Mimmack1,2, RH Yolken6, MJ Webster7, EF Torrey7 and S Bahn1,2 1Department of Neurobiology, Babraham Institute, Cambridge, UK; 2Department of Psychiatry, University of Cambridge, Addenbrooke’s Hospital, Cambridge, UK; 3Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Sanger Building, Cambridge, UK; 4MRC Rosalind Franklin Centre for Genomics Research (formerly MRC Human Genome Mapping Project Resource Centre), Hinxton, Cambridge, UK; 5Department of Biochemistry, Cambridge Centre for Proteomics, University of Cambridge, Cambridge, UK; 6Stanley Division of Developmental Neurovirology, Johns Hopkins School of Medicine, Baltimore, MD, USA; 7Stanley Laboratory of Brain Research, Department of Psychiatry, Uniformed Services University for the Health Sciences, Bethesda, MD, USA The etiology and pathophysiology of schizophrenia remain unknown. A parallel transcrip- tomics, proteomics and metabolomics approach was employed on human brain tissue to explore the molecular disease signatures. Almost half the altered proteins identified by proteomics were associated with mitochondrial function and oxidative stress responses. This was mirrored by transcriptional and metabolite perturbations. Cluster analysis of transcrip- tional alterations showed that genes related to energy metabolism and oxidative stress differentiated almost 90% of schizophrenia patients from controls, while confounding drug effects could be ruled out. We propose that oxidative stress and the ensuing cellular adaptations are linked to the schizophrenia disease process and hope that this new disease concept may advance the approach to treatment, diagnosis and disease prevention of schizophrenia and related syndromes. Molecular Psychiatry (2004) 9, 684–697. doi:10.1038/sj.mp.4001511 Published online 20 April 2004 Keywords: schizophrenia; functional genomics; systems-based approach; gene ontology; mitochondria; oxidative stress The onset and etiology of schizophrenia has been problem with gene expression profiling studies in associated with a wide range of genetic and epigenetic neuropsychiatric disorders is that individual expres- factors with little agreement as to the true causality. sion changes are usually modest and that interindi- Despite the abundance of hypotheses, the list of vidual variability is high, which makes it difficult to reproducible findings is limited. Schizophrenia is distinguish true physiological differences from nor- clearly a genetically complex, if not a diverse, mal human variation. Thus, we employed an inte- disorder and it is possible that multiple etiologies grative approach on a large sample set to define could converge to the same disease phenotype and disease-specific abnormalities within schizophrenia pathophysiology. post-mortem brain tissue by analyses at the level of Microarray analysis is a powerful tool for identify- mRNA (transcriptome), protein (proteome) and low- ing gene expression alterations in disease tissue and molecular-weight intermediates (metabolome). The has been successfully employed to study a variety of investigation of gene and protein expression com- disorders, including complex neuropsychiatric dis- bined with measurements of metabolites provides orders. For example, two recent microarray studies on insights into disease-specific regulatory mechanisms schizophrenia prefrontal cortex found evidence for and metabolic networks, generating a more compre- metabolic alterations and myelin-related gene hensive and converging picture of the diseased brain. changes in schizophrenia.1,2 However, a prominent Using this approach, we identified several signifi- cantly altered metabolic pathways in schizophrenia brain tissue. In view of these findings, we propose Correspondence: S Bahn, Department of Psychiatry, University of that mitochondrial dysfunction and oxidative stress Cambridge, Cambridge CB2 2QQ, UK. are strongly involved in, and may underlie, the E-mail: [email protected] pathophysiology of schizophrenia. These abnormal- *These authors contributed equally to this work Received 10 December 2003; revised 24 February 2004; accepted ities may pertain to abnormal oxygen or glucose 04 March 2004 supply and/or growth factor signaling resulting in Mitochondrial dysfunction in schizophrenia S Prabakaran et al 685 global metabolic disturbances and precipitate the sequence databases using Mascot search engine clinical syndrome of schizophrenia. (www.matrixscience.com). Processing of proteomics data Following gel-to-gel Materials and methods matching of spots, statistical analysis (Student’s t-test) of normalized protein abundance changes between Proteomics samples was performed using BVA software module as described.4 Tissue collection and sample preparation Fresh- frozen prefrontal cortex tissue (Brodmann area 9) Metabolomics from white and gray matter of 10 schizophrenia and 10 well-matched control individuals was obtained Acquisition of metabolomics data For NMR-based from the Neuropathology Consortium of the Stanley metabolomics analysis, high-resolution magic angle brain collection (Stanley Medical Research Institute, spinning (HRMAS) 1H NMR spectroscopy was USA).3 Samples were homogenized in amido performed on white and gray matter brain tissue as sulfobetaine 14 (ASB14) buffer (8 M urea, 2% described previously.5 HRMAS 1H NMR spectra were ASB14, 5 mM Mg acetate, 20 mM Tris base, pH 8) acquired using a Bruker 700 MHz (Bruker Avance, containing complete protease inhibitor cocktail Bruker GmBH, Rheinstetten, Germany) at þ 41C using (Roche) and phosphatase inhibitors (1 mM Na a conventional solvent suppressed pulse/acquire pyrophosphate, 1 mM Na orthovanadate, 10 mM sequence (RD Àp/2Àt1Àp/2ÀtmÀp/2 TR ¼ 3s, b-glycerophosphate, 50 mM NaF and 1% Triton). SW ¼ 10 kHz, 32k data points, solvent suppression Proteins were extracted by precipitation using applied during the preparation time (t1)of4ms 100 mM ammonium acetate in methanol and and mixing time (tm) of 150 ms, number of resuspended in ASB14 buffer. The concentration of acquisitions ¼ 128, 7 kHz spinning rate). Spectra protein was determined using a detergent-compatible were also acquired with a Carr Purcell Meiboom and protein assay kit (BioRad). Gill (CPMG) pulse sequence to attenuate selectively lipid resonances and bound metabolites relative to aqueous metabolites. CPMG spectra were obtained 2D fluorescence difference gel electrophoresis (2D- using a 40 ms total spin echo time (spin echo DIGE) 2D-DIGE was performed as described delay ¼ 500 ms, total number of spin echoes ¼ 40, previously.4 In short, individual protein samples with other parameters identical to those described (100 mg) were minimally labeled with Cy3 or Cy5 (200 pmol, Amersham Biosciences). A protein pool for the above NOESY pulse sequence). consisting of all protein samples included in the Processing of metabolomics data Following Fourier study was generated for use as an internal standard, transformation, spectra were integrated across t and was minimally labeled with Cy2. All CyDyes 0.04 ppm spectral regions between 0.4 and 9.4 ppm were supplied as charge-balanced NHS esters. using a routine with Matlab (Mathworks, CA, USA). Proteins labeled with Cy2 (pool), Cy3 and Cy5 were The output vector representing each spectrum was mixed and separated by isoelectric focusing using normalized across the integral regions, excluding the 24 cm nonlinear IPG DryStrips, pH 3–10 (Amersham water resonance, and this formed the input to the Biosciences) according to the manufacturer’s pattern recognition routine. instructions. Proteins were further separated according to molecular weight using SDS- Genomics polyacrylamide gels (12%, Ettan DALT Twelve apparatus, Amersham Biosciences). Following Tissue collection and RNA extraction The procedure electrophoresis, gels were scanned at appropriate has been described previously.6 In brief, fresh-frozen wavelengths for Cy2, Cy3 and Cy5 fluorescence prefrontal cortex tissue from 54 schizophrenia and 50 using Typhoont 9400 (Amersham Biosciences). Gel well-matched control individuals were obtained from images were cropped using ImageQuantt V5.2 the Neuropathology Consortium of the Stanley brain (Amersham Biosciences), and protein expression collection (Stanley Medical Research Institute, USA). was quantified using DeCyder Batch Processor and Key demographic characteristics are summarized Biological Variation Analysis (BVA) software V5.02, in Table 3. Total RNA was extracted from post- (Amersham Biosciences). The Cy2 internal standard mortem prefrontal cortices (Brodmann region 9) of included in all experiments for normalization schizophrenia and control brains using Tri-reagent purposes allowed both intra- and intergel analyses. (Sigma, UK) or Trizol (Gibco-Brl). RNA quality was Protein spots showing significant changes (Pr0.05) assessed using a high-resolution electrophoresis in schizophrenia with respect to controls were system (Agilent Technologies, Palo Alto, CA, USA). picked from a colloidal Coomassie-stained gel, digested with trypsin (MassPrep Station, Micromass) Microarray analysis Isolated total RNA