Alterations in oligodendrocyte proteins, calcium homeostasis and new potential markers in schizophrenia anterior temporal lobe are revealed by shotgun proteome analysis

Daniel Martins-de-Souza 0 1 2 3 4 Wagner F. Gattaz 0 1 2 3 4 Andrea Schmitt 0 1 2 3 4 Christiane Rewerts 0 1 2 3 4 Sergio Marangoni 0 1 2 3 4 Jose C. Novello 0 1 2 3 4 Giuseppina Maccarrone 0 1 2 3 4 Christoph W. Turck 0 1 2 3 4 Emmanuel Dias-Neto 0 1 2 3 4 0 D. Martins-de-Souza S. Marangoni J. C. Novello Laboratorio de Proteomica, Departamento de Bioqumica, Instituto de Biologia , UNICAMP, Campinas, SP CEP 13083-970, Brazil 1 D. Martins-de-Souza C. Rewerts G. Maccarrone C. W. Turck (&) Max Planck Institute of Psychiatry , Kraepelinstrasse 2, 80804 Munich, Germany 2 D. Martins-de-Souza W. F. Gattaz E. Dias-Neto Laboratorio de Neurociencias, Faculdade de Medicina da USP, Instituto de Psiquiatria, Universidade de Sao Paulo , Rua Dr. Ovdio Pires de Campos, No 785, s/n Consolacao, Sao Paulo , SP CEP 05403-010, Brazil 3 Present Address: E. Dias-Neto (&) MD Anderson Cancer Center, University of Texas , 1515 Holcombe Blvd, Houston, TX 77030, USA 4 A. Schmitt Department of Psychiatry, University of Goettingen , Von Siebold Str. 5, 37075 Gottingen, Germany Global proteomic analysis of post-mortem anterior temporal lobe samples from schizophrenia patients and non-schizophrenia individuals was performed using stable isotope labeling and shotgun proteomics. Our analysis resulted in the identification of 479 proteins, 37 of which showed statistically significant differential expression. Pathways affected by differential protein expression include transport, signal transduction, energy pathways, cell growth and maintenance and protein metabolism. The collection of protein alterations identified here reinforces the importance of myelin/oligodendrocyte and calcium homeostasis in schizophrenia, and reveals a number of new potential markers that may contribute to the understanding of the pathogenesis of this complex disease. Introduction Dysfunctions in synaptogenesis and neural plasticity (Mirnics et al. 2000; Hakak et al. 2001; Vawter et al. 2001; Aston et al. 2004; Arion et al. 2007), energy metabolism (Vawter et al. 2001; Middleton et al. 2002; Prabakaran et al. 2004), cytoskeleton assembly (Hakak et al. 2001; Vawter et al. 2001; Tkachev et al. 2003) and oligodendrocyte metabolism (Tkachev et al. 2003; Aston et al. 2004; Katsel et al. 2005a, b; Arion et al. 2007) have been previously reported in studies of global gene expression in different brain regions of schizophrenia (SCZ) patients. However, as post-transcriptional mechanisms may prevent mRNA fluctuations to be directly translated into protein differential expression, proteomic studies are a nice complement to studies of differential gene expression. Some proteomic studies were performed in brain regions such as the anterior cingulate cortex (Clark et al. 2006) and the corpus callosum (Sivagnanasundaram et al. 2007), showing that the proteome alterations on these related pathways have been confirmed. Patterns of synchronization and desynchronization communicate between brain areas through specific neuronal activity (Singer 1999). In a complex disease such as SCZ it is possible that all brain areas play a role in the etiology since they are all connected. However, there are some areas which seem to be more involved based on their exerted functions: the pre-frontal cortex because of its executive functions (Miller and Cohen 2001), the basolateral amygdala because of its involvement in affective behavior (Davis and Whalen 2001), and the anterior cingulate cortex because of its participation in cognitive and affective processes (Carter et al. 1997). The temporal lobe concentrates important functions such as high-level auditory and visual processing, language, and transference from short- to long-term memory. All these functions can be compromised in SCZ in a process that apparently involves an imbalance of glutamate and gammaaminobutyric acid (GABA) leading to dopaminergic dysfunctions (Deakin and Simpson 1997). A reduction of the temporal lobe size in SCZ has been shown by several studies with magnetic resonance imaging (MRI) (Bogerts 1993; Suddath et al. 1989). The left temporal pole gray matter was smaller and left-greater than right asymmetry was reduced in SCZ patients (Kasai et al. 2003; Antonova et al. 2005), however, results are not consistent (Turetsky et al. 2003). Interestingly, the volume of the left anterior temporal cortex was negatively correlated with hallucinations (Crespo-Facorro et al. 2004). In the polar temporal cortex of SCZ patients, deficits were reported in glutamate presynaptic components (Deakin and Simpson 1997); glutamate and GABA uptake sites were reduced on the left side (Deakin et al. 1989; Simpson et al. 1989) with no losses of post-synaptic glutamate receptors (Nishikawa et al. 1983). Deakin and Simpson (1997) have shown that degenerated glutamate terminals in the anterior temporal lobe originate in the frontal cortex with important implications for SCZ. In the present work we performed a quantitative proteomic analysis of the left anterior temporal lobe (ATL) of SCZ and control samples using isotope-coded protein label (ICPL), a method for the accurate quantitative comparative analysis of protein regulation (Schmidt et al. 2005). ICPL is based on isotope labeling of free amino groups in intact proteins. After the modification, the heavy and light isotope labeled proteins are digested and analyzed by liquid chromatography (LC) followed by tandem mass spectrometry (MS/MS). Relative quantification of differential protein expression is based on the comparison of the peak intensities of the heavy- and light-labeled peptides from the mass spectra. The ICPL method is very reproducible and compatible with all known protein and peptide separation techniques, providing highly accurate quantification of regulated proteins. The identification of proteins differentially expressed in the ATL of SCZ patients was performed by MS/MS followed by subsequent database searches. After their validation in a large set of samples and patients, these proteins can provide valuable information not only for a better understanding of the biological basis of the disease, but can also serve as biomarkers for disease monitoring or as targets for pharmaceutical applications. Materials and methods All chemicals and solvents were from Bio-Rad (Hercules, CA, USA) and of the highest purity available. The ICPL kit was from Serva Electrophoresis (Heidelberg, Germany) and Prespotted AnchorChips were obtained from Bruker Daltonics (Bremen, Germany). Human anterior temporal lobe samples Frozen tissue blocks from the left anterior temporal lobe tissue, Brodmann Area (BA) 38, were collected postmortem from five SCZ patients and four controls free of psychiatric disorders. The left side was selected due to its importance in SCZ (DeLisi et al. 1989). All brain samples were obtained from the brain bank of the Central Institute of Mental Health (Mannheim, Germany), dissected by an experienced neuropathologist and deep-frozen in liqu (...truncated)