Antipsychotic Treatment Alters Protein Expression Associated with Presynaptic Function and Nervous System Development in Rat Frontal Cortex Dan Ma,†,# Man K. Chan,†,# Helen E. Lockstone,† Sandra R. Pietsch,† Declan N. C. Jones,‡ Jackie Cilia,‡ Mark D. Hill,‡ Melanie J. Robbins,‡ Isabel M. Benzel,‡ Yagnesh Umrania,† Paul C. Guest,† Yishai Levin,† Peter R. Maycox,‡ and Sabine Bahn*,† Institute of Biotechnology, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QT, U.K., and Psychiatry CEDD, New Frontiers Science Park, GlaxoSmithKline, Third Avenue, Harlow, CM19 5AW, U.K. Received November 12, 2008
Haloperidol and olanzapine are widely used antipsychotic drugs in the treatment of schizophrenia and other psychotic disorders. Despite extensive research efforts within the biopharmaceutical industry and academia, the exact molecular mechanisms of their action remain largely unknown. Since the response of patients to existing medications can be variable and often includes severe side effects, it is critical to increase our knowledge on their mechanism of action to guide clinical usage and new drug development. In this study, we have employed the label-free liquid chromatography tandem mass spectrometry (LC-MSE) to identify differentially expressed proteins in rat frontal cortex following subchronic treatment with haloperidol or olanzapine. Subcellular fractionation was performed to increased proteomic coverage and provided insight into the subcellular location involved in the mechanism of drug action. LC-MSE profiling identified 531 and 741 annotated proteins in fractions I (cytoplasmic-) and II (membrane enriched-) in two drug treatments. Fifty-nine of these proteins were altered significantly by haloperidol treatment, 74 by olanzapine and 21 were common to both treatments. Pathway analysis revealed that both drugs altered similar classes of proteins associated with cellular assembly/organization, nervous system development/function (particularly presynaptic function) and neurological disorders, which indicate a common mechanism of action. The top affected canonical signaling pathways differed between the two treatments. The haloperidol data set showed a stronger association with Huntington’s disease signaling, while olanzapine treatment showed stronger effects on glycolysis/gluconeogenesis. This could either relate to a difference in clinical efficacy or side effect profile of the two compounds. The results were consistent with the findings reported previously by targeted studies, demonstrating the validity of this approach. However, we have also identified many novel proteins which have not been found previously to be associated with these drugs. Further study of these proteins could provide new insights into the etiology of the disease or the mechanism of antipsychotic medications. Keywords: haloperidol • olanzapine • synaptic • development • proteomics • rat
Introduction Antipsychotic drugs can help control psychotic symptoms and induce remission in patients with schizophrenia and other related illnesses. Haloperidol is a typical antipsychotic, widely used in the treatment of positive symptoms of schizophrenia and for reduction of agitation in patients.1 Olanzapine is an atypical antipsychotic effective in the reduction of not only * To whom correspondence should be addressed. Dr. Sabine Bahn, M.D. Ph.D. MRCPsych, Cambridge Centre for Neuropsychiatric Research, Institute of Biotechnology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QT. Tel., +44 1223 334151; fax, +44 1223 334162; e-mail, sb209@ cam.ac.uk. † University of Cambridge. # These authors contributed equally to this work. ‡ GlaxoSmithKline.
3284 Journal of Proteome Research 2009, 8, 3284–3297 Published on Web 04/28/2009
positive symptoms, but also negative and cognitive symptoms.2 However, both compounds can induce severe and often debilitating side effects, such as weight gain, type II diabetes and tardive dyskinesia.3-7 The development of new antipsychotic drugs for treatment of schizophrenia and other psychotic disorders is an important ongoing area of research. This is critical since the response of patients to existing medications can be variable including side effects. Therefore, the most appropriate drug for individual patients requires careful consideration. However, the exact molecular mechanisms of their action remain largely unknown. Previous psychopharmacological studies have shown that antipsychotic drugs block a range of neurotransmitter receptors.8 Haloperidol targets mainly dopamine D2 receptors in basal ganglia and upper brainstem, while olanzapine acts on 10.1021/pr800983p CCC: $40.75
2009 American Chemical Society
Proteomic Study of Antipsychotic Drugs in Rat Brain
research articles
Figure 1. Fontal cortex region and LC-MSE workflow. (a) Frontal cortex region (red highlight) of rat brain used for proteomic studies. (b) Theoretical peak showing the concept of integration across the mass spectrometric and chromatographic volumes of each detected peptide ion. The quantitative information is obtained from the low energy scans of intact peptides. (c) Summary of LC-MSE, data processing and (d) R script workflows.
both dopamine D2 and 5-HT2A receptors8-10 in the cerebral cortex.10,11 In addition, haloperidol and olanzapine have other neuronal or cellular functions including stimulation of cell proliferation in the prefrontal cortex12-15 and improvement of brain energy/glucose metabolism.9,16 Many genes and proteins modulated by antipsychotic drugs are involved in synaptic function and energy metabolism,17,18 and this could relate to the clinical efficacy and/or side effect profile as well as the delayed onset of therapeutic action of these drugs. Therefore, the aim of this study was to gain further insight into these mechanisms by identifying the brain proteomic alterations associated with subchronic treatment of rats with common antipsychotics, using liquid chromatography tandem mass spectrometry (LC-MSE) profiling as an approach to qualitative and quantitative proteomic analyses. The methodology also employed a differential detergent extraction technique to increase proteomic coverage and facilitate the study of membrane and membrane-associated proteins.19 This resulted in the identification of common and distinct proteins and pathways altered by haloperidol and olanzapine, many of which have neuronal functions.
Materials and Methods Animals and Drug Treatment. Adult male Wistar rats (Charles River, Margate, U.K.) weighing 280-300 g were used. All experiments were conducted in full compliance with the Home Office Guidance (U.K. Animals Scientific Procedures Act 1986) and the ethical policies of GlaxoSmithKline. Rats were housed in a temperature-controlled room (21 ( 1 °C) on a 12: 12 h light/dark cycle (lights on at 0600 h). Food and water were available ad libitum. Antipsychotic drugs were administrated by oral gavage at 2 mL/kg once per day for 21 days with either vehicle (0.5 mg/
mL tartaric acid in 1% methylcellulose water, n ) 8), 1 mg/kg haloperidol (n ) 8) or 2 mg/kg olanzapine (n ) 8). The doses administered have been shown previously to have biological activity (GSK in-house studies20 and other studies21,22). Two hours after treatment on day 21, rats were killed by decapitation and the brains were dissected on ice to collect frontal cortices (defined as the anterior portion of the cortex up to 2.15 mm rostral from Bregma,23 and as in other reference24) (Figure 1a) which were frozen in liquid nitrogen and stored at -80 °C until use. Differential Detergent Fractionation. Approximately 30 mg of frontal cortex tissue was subjected to proteome subcellular fractionation using the in-house optimized Qproteome Cell Compartment Kit (Qiagen, West Sussex, U.K.) to obtain cytosolic- and membrane-enriched fractions. In brief, tissues were minced using a metal blade to 0.5) were summed to obtain a ‘protein intensity’. Student’s t test and false discovery rate (FDR, Benjamin-Hochberg) control procedures were applied to identify significant alterations in protein expression levels between different experimental groups. Significance was set to p < 0.05 and q (corrected p-values)
