Comparative gene expression study of the chronic exposure to clozapine and haloperidol in rat frontal cortex

Schizophrenia Research 134 (2012) 211–218

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Comparative gene expression study of the chronic exposure to clozapine and haloperidol in rat frontal cortex S. Hossein Fatemi a, b, c,⁎, Timothy D. Folsom a, Teri J. Reutiman a, Jessica Novak a, Rachelanne H. Engel a a b c

Department of Psychiatry, Division of Neuroscience Research, University of Minnesota Medical School, 420 Delaware St. SE, MMC 392, Minneapolis, MN 55455, United States Department of Pharmacology, University of Minnesota Medical School, 310 Church St. SE, Minneapolis, MN 55455, United States Department of Neuroscience, University of Minnesota Medical School, 310 Church St. SE, Minneapolis, MN 55455, United States

a r t i c l e

i n f o

Article history: Received 18 July 2011 Received in revised form 8 November 2011 Accepted 9 November 2011 Available online 10 December 2011 Keywords: Schizophrenia Frontal cortex Comt Clozapine Microarray

a b s t r a c t Antipsychotic drugs (APDs) are effective in treating some of the positive and negative symptoms of schizophrenia. APDs take time to achieve a therapeutic effect which suggests that changes in gene expression are involved in their efficacy. We hypothesized that there would be altered expression of specific genes associated with the etiology or treatment of schizophrenia in frontal cortex of rats that received chronic treatment with a typical APD (haloperidol) vs. an atypical APD (clozapine). Rats were administered clozapine, haloperidol, or sterile saline intraperitoneally daily for 21 days. Frontal cortices from clozapine-, haloperidol-, and saline-treated rats were dissected and subjected to microarray analysis. We observed a significant (1.5 fold, pb 0.05) downregulation of 278 genes and upregulation of 73 genes in the clozapine-treated brains vs. controls and downregulation of 451 genes and upregulation of 115 genes in the haloperidol-treated brains vs. control. A total of 146 genes (130 downregulated and 16 upregulated) were significantly altered by both clozapine and haloperidol. These genes were classified by functional groups. qRT-PCR (quantitative real-time polymerase chain reaction) analysis verified the direction and magnitude of change for a group of nine genes significantly altered by clozapine and 11 genes significantly altered by haloperidol. Three genes verified by qRT-PCR were altered by both drugs: Bcl2-like 1 (Bcl2l1), catechol-O-methyltransferase (Comt), and opioid-binding protein/cell adhesion molecule-like (Opcml). Our results show that clozapine and haloperidol cause changes in levels of many important genes that may be involved in etiology and treatment of schizophrenia. © 2011 Elsevier B.V. All rights reserved.

1. Introduction Antipsychotic drugs have proven efficacious in treating the positive symptoms (i.e., delusions, hallucinations, and disordered thought) and negative symptoms (i.e., social withdrawal, blunted affect, and anhedonia) of schizophrenia. Typical antipsychotic drugs such as haloperidol are dopamine D2 receptor antagonists while clozapine, the first atypical antipsychotic drug has a broader range of affinities including serotonin (5HT2A, 5HT2C, 5HT6, 5HT7), adrenergic (α1, α2), muscarinic (M1) and histaminergic receptors (Meltzer and Fatemi, 2000). Studies have shown the superiority of clozapine vs. typical agents in the treatment of total psychopathology, extrapyramidal symptoms (EPS), tardive dyskinesia (TD), and categorical response to treatment (Sharif et al., 2007). The time lag between the start of treatment and a therapeutic effect suggests that changes in gene expression contribute to their efficacy (Hyman and Nestler, 1996; Fatemi et al., 2008a,b).

⁎ Corresponding author at: 420 Delaware Street SE, MMC 392, Minneapolis, MN 55455, United States. Tel.: + 1 612 626 3633; fax: +1 612 624 8935. E-mail addresses: [email protected] (S.H. Fatemi), [email protected] (T.D. Folsom), [email protected] (T.J. Reutiman), [email protected] (R.H. Engel). 0920-9964/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.schres.2011.11.013

Previous microarray studies investigating the effects of chronic administration of clozapine and/or haloperidol in brains of rodents have been informative (Kontkanen et al., 2002; Thomas et al., 2003; Chong et al., 2004; Takahashi et al., 2004; MacDonald et al., 2005; Mehler-Wex et al., 2006; Sugata et al., 2007; Duncan et al., 2008). Consistent with the idea that gene expression changes are required to obtain a therapeutic response, members of the Fos family of transcription factors, which regulate the expression of other genes, have been shown to be upregulated by both clozapine (Nguyen et al., 1992; Roberson and Fibiger, 1992; Merchant and Dorsa, 1993) and haloperidol (Roberson and Fibiger, 1992; Merchant and Dorsa, 1993; Sebens et al., 1995). Thomas et al. (2003) demonstrated that 14-day treatment with haloperidol or clozapine increased expression of genes associated with lipid metabolism in frontal cortex of mice (Thomas et al., 2003). Genes coding for presynaptic proteins such as vesicle associated membrane protein 1 (Vamp1), syntaxin 1A, and synaptosomal associated protein 25 kDa (Snap25) were upregulated in the frontal cortex and striatum of mice following chronic exposure to haloperidol and clozapine revealing a potential effect on synaptic plasticity (MacDonald et al., 2005). Kontkanen et al. (2002) similarly found altered expression of presynaptic genes in rat cortex following chronic treatment with both drugs.

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In the current study, we used microarray technology to analyze the effects of chronic treatment with clozapine or haloperidol on brain gene expression in rats to identify potential genes of interest in the etiology of schizophrenia, which may have been missed in previous studies. We also used quantitative real-time polymerase chain reaction (qRT-PCR) to further investigate schizophrenia candidate genes and other genes of interest following treatment with these two APDs. 2. Materials and methods 2.1. Animals and drug treatment The University of Minnesota Institutional Animal Care and Use Committee approved all experimental procedures. Male Sprague–Dawley albino rats (Charles River) initially weighing approximately 250 g were housed in a temperature and humidity-controlled environment with a 12-hour light/dark cycle and had access to food and water ad libitum. Rats were randomly assigned to one of three groups: 1) clozapine (20 mg/kg/day, i.p., n = 20); 2) haloperidol (1.5 mg/kg/day, i.p., n = 20); or 3) sterile saline (n = 20), and administered drug or diluent in 1 mL volume for 21 days. The dosages for clozapine and haloperidol were chosen based on values obtained from literature that correspond to clinically relevant human-equivalent dosage (Gao et al., 1997). Animals were sacrificed 24 h after the last injection under deep anesthesia using ketamine (90 mg/kg) and Nembutal (40 mg/kg). Brains were removed and frontal cortex was dissected and flash frozen in liquid nitrogen and stored at −80 °C for future assays. 2.2. DNA microarray Rat frontal cortex (n= 4 per group) was homogenized and processed for microarray as described previously (Fatemi et al., 2006, 2009a,b). Data was analyzed as previously described (Fatemi et al., 2006, 2009a,b). 2.3. qRT-PCR qRT-PCR for selected genes was performed as previously described (Fatemi et al., 2005, 2009a,b, 2010a) using the TaqMan Gene Expression Assays (Applied Biosystems, Foster City, CA) and a glyceraldehyde 3phosphate dehydrogenase (Gapdh) endogenous control assay. 2.4. Statistical analysis For microarray and qRT-PCR data, all statistical analyses were performed using SPSS as described previously (Fatemi et al., 2006, 2009a,b, 2010a). Differences of the normalized mRNA expression levels of selected genes between clozapine-treated or haloperidol-treated and saline-treated rats were assayed using student's t-test. For microarray data, significant differences are defined as those with a fold change of at least 1.5 and a p valueb 0.05. For qRT-PCR, significant differences are defined as those with a p value b 0.05. 3. Results Following chronic treatment with clozapine, a total of 351 genes (278 downregulated, 73 upregulated) were significantly altered (p b 0.05, at least 1.5 fold change) in rat frontal cortex (Supplemental Table 1). A total of 566 genes (451 downregulated, 115 upregulated) displayed significantly altered (pb 0.05, at least 1.5 fold change) expression in rat frontal cortex following chronic treatment with haloperidol (Supplemental Table 2). Out of the total number of upregulated genes, 57 were unique to clozapine, 99 were unique to haloperidol, and 16 were common to both drugs (Fig. 1). Of the total number of

Fig. 1. Venn diagrams showing the number of genes that displayed significantly altered expression following treatment with clozapine and haloperidol.

downregulated genes, 148 were unique to clozapine, 321 were unique to haloperidol, and 130 were common to both drugs, and (Fig. 1) in total, there were 146 total genes that were significantly altered by both clozapine and haloperidol (Supplemental Table 3). The most highly downregulated and upregulated genes (based on fold change) are listed in Tables 1 (clozapine) and 2 (haloperidol). Genes upregulated by clozapine included cyclin D1 (Ccnd1), adrenergic receptor alpha 2b (Adra2b), gap junction protein alpha 1 (Gja1; also known as connexin 43), and synuclein gamma (Sncg) (Table 1). Genes downregulated by clozapine included heat shock 70 kDa protein 1a (Hspa1a), early B-cell factor 1, and potassium voltage-gated channel, shal related family, member 2 (Kcndw) (Table 1). Genes upregulated by haloperidol included quaking homolog (Qk) and beta catenin (Catnb1) while genes downregulated by haloperidol included hairless protein (Hr) and Lamin B1 (Lmnb1) (Table 2). Six of the most significantly altered genes were by altered by both clozapine and haloperidol: Hspa1a, Fructose-6-phosphate2-kinase/fructose-2,6-bisphosphatase (Pfkb3), ATP-binding cassette, sub-family C (CFTR/MRP), member 8 (Abcc8), Lmnb1 and vertebrate Lin7 homolog 3 (Lin7c) and STEAP family member 3 (Steap3) (Tables 1 and 2). Tabulation of the significantly affected genes, based on functional relevance showed involvement of genes from multiple biological pathways. These most common functional groups for genes altered by clozapine were: 1) signal transduction and cell communication (26.2%; i.e., phosphodiesterase 4a and gamma-aminobutyric acid (GABA) A receptor beta 1); 2) regulation of nucleobase, nucleoside, nucleotide, and nucleic acid metabolism (13.1%; i.e., forkhead box E1 and cAMP response binding element protein like-2); 3) transport (11.7%; i.e., glutamate receptor ionotropic 4 and gap junction protein, alpha 1); 4) metabolism and energy pathways (10.6%; i.e. Comt and glutamic acid decarboxylase 2); 5) cell growth and maintenance (7.7%; i.e., dystrobrevin beta and fibrinogen like-2); 6) protein metabolism (6.8%; i.e., calpain 8 and gamma synuclein); and 7) immune system (2.6%; i.e., interleukin 6 signal transducer and complement component 3) (Supplemental Table 1). The most common functional groups for genes altered by haloperidol were: 1) signal transduction and cell communication (26.7%; i.e., neural cell adhesion molecule 1 and benzodiazapine receptor associated protein 1); 2) regulation of nucleobase, nucleoside, nucleotide, and nucleic acid metabolism (14%; i.e., nuclear factor 1B and FBJ murine osteosarcoma viral oncogene homolog); 3) transport (12.7%; i.e., synaptic vesicle protein 2b and glutamate receptor ionotropic 2); 4) cell growth and maintenance

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Table 1 Top downregulated and upregulated genes in rat frontal cortex following chronic clozapine treatment. Name

Gene symbol (Genedata)

UniGene

Fold change

Function

Fructose-6-phosphate2-kinase/fructose-2,6-bisphosphatase 3 Early B-cell factor 1

Pfkfb3 Ebf

Rn.10791 Rn.11257

0.10 0.14

KH domain containing, RNA binding, signal transduction associated 2

Khdrbs2

Rn.154423

0.14

Dicer1, Dcr-1 homolog (Drosophila) Heat shock 70kD protein 1A Lamin B1 Potassium voltage gated channel, Shal-related family, member 2 Small glutamine rich protein with tetratricopeptide repeats 2 STEAP family member 3 ATP-binding cassette, sub-family C (CFTR/MRP), member 8 Inter-alpha-inhibitor H4 heavy chain Synuclein, gamma Seminal vesicle secretory protein 1 A disintegrin-like and metalloprotease (reprolysin type) with thrombospondin type 1 motif, 7 (predicted) Gap junction protein, alpha 1, 43 kD (connexin 43) Transcribed locus Adrenergic receptor, alpha 2b Lin-7-C Cyclin D1 Chemokine (C-C motif) ligand 4

Dicer1 Hspa1a Lmnb1 Kcnd2 Sgt2 Steap3 Abcc8 Itih4 Sncg Svs1 Adamts7

Rn.205881 Rn.1950 Rn.11362 Rn.87841 Rn.23400 Rn.16304 Rn.11187 Rn.11308 Rn.2883 Rn.56148 Rn.23850

0.16 0.16 0.16 0.19 0.19 0.19 0.20 2.07 2.10 2.16 2.22

Metabolism; energy pathways Regulation of nucleobase, nucleoside, nucleotide and nucleic acid metabolism Regulation of nucleobase, nucleoside, nucleotide and nucleic acid metabolism Gene silencing Protein metabolism Cell growth and/or maintenance Transport Miscellaneous Cell communication; signal transduction Transport Protein metabolism Protein metabolism Miscellaneous Protein metabolism

Gja1

Rn.10346 Rn.35594 Rn.10296 Rn.44269 Rn.22279 Rn.37880

2.22 2.27 2.31 2.38 2.52 3.82

Transport Miscellaneous Cell communication; signal transduction Cell communication; signal transduction Cell Communication; Signal transduction Immune response

Adra2b Lin7c Ccnd1 Ccl4

(9.5%; i.e., vitronectin and myelin-associated oligodendrocytic basic protein); 5) metabolism and energy pathways (9.5%; i.e., peroxiredoxin 6 and hexokinase 2); 6) protein metabolism (6.5%; i.e., plasminogen activator, tissue and parkin); 7) immune response (2.1%; i.e., CD59 antigen and natural cytotoxicity triggering receptor 3); and 8) apoptosis (1.1%; i.e., bcl-x short form and BH3 interacting (with BCL2 family) domain, apoptosis agonist) (Supplemental Table 2). We performed qRT-PCR analysis of 43 genes of interest (16 altered by clozapine, 27 altered by haloperidol), many of which were candidate genes for schizophrenia. We found that 10 genes were significantly altered following chronic treatment with clozapine (9 downregulated, 1 upregulated; Table 3), nine of which matched the direction and

magnitude of change from the microarray analysis. Significantly altered genes included Comt, arrestin, beta 2 (Arrb2), synaptotagmin 13 (Syt13), and phosphodiesterase 1c (Pde1c) (Table 2). qRT-PCR identified 16 genes that showed significantly reduced mRNA following chronic treatment with haloperidol, 11 of which that matched the direction and magnitude of microarray analysis (Table 4). Significantly altered genes included Comt, glutamate receptor, ionotropic AMPA 2 (Gria2), glutamate receptor, ionotropic, AMPA 3 (Gria3), and synapsin 3 (Syn3) (Table 4). Three genes which demonstrated concordance between microarray and qRT-PCR analyses were common to both lists: B-Cell CLL/Lymphoma 2 like 1 (Bcl2l1), Comt, and Opioid-binding protein/ cell adhesion molecule-like (Opcm1) (Tables 3 and 4).

Table 2 Top downregulated and upregulated genes in rat frontal cortex following chronic haloperidol treatment. Name

Gene symbol (Genedata)

UniGene

Fold change

Function

Fructose-6-phosphate2-kinase/ fructose-2,6-bisphosphatase 3 Lamin B1 Hairless protein

Pfkfb3

Rn.10791

0.09

Metabolism; energy pathways

Lmnb1 Hr

Rn.11362 Rn.41543

0.15 0.16

Heat shock 70kD protein 1B Ribonuclease III, nuclear

Hspa1a Rnasen

Rn.1950 Rn.41113

0.17 0.17

STEAP family member 3 ATP-binding cassette, sub-family C (CFTR/MRP), member 8 Abelson helper integration site 1 Beta-transducin repeat containing Lymphocyte cytosolic protein 1 Beta-catenin Polo like kinase 2 Quaking NTE-related protein Lin-7-C Cold inducible RNA binding protein Mitochondrial very-long-chain acyl-CoAthioesterase Bone morphogenetic protein receptor, type 1A Basic helix-loop-helix domain containing, classB2

Steap3 Abcc8

Rn.16304 Rn.11187

0.17 0.17

Cell growth and/or maintenance Regulation of nucleobase, nucleoside, nucleotide and nucleic acid metabolism Protein metabolism Regulation of nucleobase, nucleoside, nucleotide and nucleic acid metabolism Cell communication; signal transduction Transport

Ahi1 Btrc Lcp1 Catnb1 Plk2 Qk Netl Lin7c Cirbp Acot2 Bmpr1a Bhlhb2

Rn.23637 Rn.21800 Rn.14256 Rn.112601 Rn.12100 Rn.85462 Rn.35594 Rn.44269 Rn.28931 Rn.37524 Rn.44965 Rn.81055

0.18 0.18 0.18 2.14 2.16 2.18 2.23 2.25 2.55 2.55 2.58 2.67

DEAD (Asp-Glu-Ala-Asp) box polypeptide 3, X-linked

Ddx3x

Rn.95841

2.72

Cell communication; signal transduction Protein metabolism Cell communication; signal transduction Signal transduction Cell communication; signal transduction RNA metabolism Miscellaneous Cell communication; signal transduction Cell communication; signal transduction Miscellaneous Cell communication; signal transduction Regulation of nucleobase, nucleoside, nucleotide and nucleic acid metabolism Regulation of nucleobase, nucleoside, nucleotide and nucleic acid metabolism

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Table 3 Microarray and qRT-PCR results for selected affected genes in rat frontal cortex following chronic treatment with clozapine. Gene

Symbol

Microarray fold changea

Gene relative to normalizer (qRT-PCR)

QRT-PCR p value

Arrestin, beta 2 B-Cell CLL/Lymphoma 2 like 1 Catecholamine-Omethyltransferase Colony stimulating factor 2 receptor, beta 1, low-affinity (granulocyte–macrophage) Glycosylphosphatidylinositol specific phospholipase D1 Opioid-binding protein/cell adhesion molecule-like (Opcml) Phosphodiesterase 1C Polycystic kidney disease 1 Synaptotagmin binding, cytoplasmic RNA interacting protein

Arrb2 Bcl2l1 Comt

0.32 0.32 0.24

0.287 0.550 0.569

0.013 0.002 0.00048

Csf2rb1

0.46

0.436

0.008

Gpld1

0.55

0.505

0.020

Opcml

0.50

0.517

0.001

a

Pde1c Pkd1 Syncrip

0.42 0.62 0.48

0.539 0.572 0.607

0.041 0.004 0.004

p b 0.05.

4. Discussion Our studies have demonstrated, at the molecular level, the chronic effects of clozapine and haloperidol on multiple gene families in frontal cortex of the rat. We found significant overlap in genes altered by both drugs. This overlap is consistent with previous findings (Thomas et al., 2003; MacDonald et al., 2005). We verified the change in direction and magnitude of 9 genes, via qRT-PCR for clozapine, and 11 genes for haloperidol. Five of these genes altered by clozapine, Arrb2, Bcl2-like 1 (Bcl2l1), Comt, colony stimulating factor 2 receptor beta 1, and Opioid-binding protein/cell adhesion molecule-like (Opcml), have been previously associated with schizophrenia (He et al., 2006; Le-Niculescu et al., 2007; Chen et al., 2008; Liou et al., 2008; Moskovina et al., 2009). Six of the genes altered by haloperidol and verified by qRT-PCR have previously been associated with schizophrenia: Bcl2l1, Comt, Opcm1, Glutamate receptor, ionotropic, 2 (Gria2), glutamate receptor 3 (Gria3), and midkine (Mdk) (Shimizu et al., 2003; Le-Niculescu et al., 2007; Magri et al., 2008). Of particular interest, as they were verified for both drugs, are Comt, Opcml, and Bcl2l1. Comt is involved in the dopamanergic system (Karoum et al., 1994; Männistö and Kaakkola, 1999; Tunbridge et al., 2004). Multiple studies have identified COMT as being involved in cognition and as a Table 4 Microarray and qRT-PCR results for selected affected genes in rat frontal cortex following chronic treatment with haloperidol. Gene

Symbol Microarray Gene relative QRT-PCR fold changea to normalizer p value (qRT-PCR)

B-Cell CLL/Lymphoma 2 like 1 Bone morphogenetic protein 4 Catecholamine-Omethyltransferase Fibroblast activation protein Glutamate receptor, ionotropic, 2 Glutamate receptor subunit 3 Midkine Multidrug resistance protein 1a Opioid-binding protein/cell adhesion molecule-like Synapsin III Vesicle-associated membrane protein 1

Bcl2l1 Bmp4 Comt

0.36 0.28 0.27

0.502 0.684 0.504

0.001 0.043 0.0002

Fap Gria2

0.38 0.51

0.597 0.477

0.004 0.001

Gria3 Mdk Abcb1a Opcml

0.39 0.29 0.26 0.60

0.516 0.328 0.605 0.505

0.0002 0.001 0.03 0.0001

Syn3 Vamp1

0.54 0.62

0.502 0.521

0.00004 0.007

a

p b 0.05.

Fig. 2. Effect of clozapine, haloperidol, or saline (control) on COMT expression in rat frontal cortex. The 24 kDa of COMT/-actin is significantly reduced (p b 0.014) following chronic treatment with clozapine while neither isoform displayed altered protein expression following treatment with haloperidol. Reprinted from Molecular Psychiatry 12(4):322–323, Fatemi, S.H., Folsom, T.D., Catechol-o-methyltransferase gene regulation in rat frontal cortex, Fig. 1 (Copyright 2007) with permission from Nature Publishing Group.

schizophrenia susceptibility gene (i.e., Männistö and Kaakkola, 1999; Le-Niculescu et al., 2007; Goghari and Sponheim, 2008). Moreover, COMT variants have been associated with changes in gray matter volume in hippocampus and dorsolateral PFC in healthy individuals (Honea et al., 2009), two areas that show reduced volume in subjects with schizophrenia (Weiss et al., 2005; Wolf et al., 2008). Our laboratory has previously shown that protein levels for the 24 kDa cytosolic isoform of COMT were significantly downregulated in rat frontal cortex following chronic treatment with clozapine (Fig. 2; pb 0.014) while haloperidol had no significant effect (Fig. 2) (Fatemi and Folsom, 2007). Bcl2l1 (otherwise known Bclx) has two splice variants: anti-apoptotic Bclxl and pro-apoptotic Bclxs (Boise et al., 1993). Rats administered phencyclidine (PCP) display impaired spatial memory and decreased the ratio of Bclxl to Bclxs in posterior cingulate cortex (He et al., 2006). However, treatment with the atypical antipsychotic quetiapine reversed the ratio of Bclxl to Bclxs, providing a neuroprotective effect, and improved spatial memory (He et al., 2006). It may be that clozapine and haloperidol have similar effects, although we did not examine mRNA levels of the splice variants in the current study. Opcml expression is enriched in cerebral cortex and hippocampus (Miyata et al., 2003) and localized to postsynaptic spines (Yamada et al., 2007). Inhibition of Opcml function via an anti-Opcml antibody or antisense oligodeoxynucleotide results in a significant decrease in the number of synapses and dendrites while overexpression of Opcml mRNA has the opposite effect, suggesting a role for Opcml in synaptic plasticity (Yamada et al., 2007). Recently, in a gene-wide analysis of genome-wide association (GWA) data set for schizophrenia, OPCLM was identified as a gene of interest (O'Donovan et al., 2008; Moskovina et al., 2009). Further study is required to determine what the role of OPCLM might have in schizophrenia. Although not verified by qRT-PCR, we observed an increase of mRNA for Gap junction protein alpha 1, 43 kDa (Gja1) (also known as connexin 43) following treatment with both clozapine (Table 1), and haloperidol (Supplemental Table 2). Our laboratory has previously shown significantly reduced expression of connexin 43 in neocortex of adult mice that were exposed to viral infection in utero (Fatemi et al., 2008a). Connexin 43 has recently been shown to be reduced in dentate gyrus in rats exposed to chronic unpredictable mild stress (Li et al., 2010). The increase in connexin 43 mRNA expression induced by clozapine treatment may be related to clozapine's known effects to reduce depression and suicidality. Three myelination genes – myelin basic protein (Mbp), myelin oligodendrocyte protein (Mog), and myelin oligodendrocytic basic protein (Mobp) – were significantly upregulated by chronic treatment with haloperidol (Supplemental Table 2). Mbp mRNA was also significantly increased following chronic treatment with clozapine (Supplemental Table 1). Our laboratory has previously found significantly reduced mRNA for all three genes as measured by microarray and verified by qRT-PCR in cerebella of adult mice from dams exposed to influenza virus on day 16 of pregnancy (Fatemi et al., 2009c). MBP, MOG, and MOBP have been implicated as candidate genes for schizophrenia (Lewis et al., 2003; Tkachev et al., 2003; Chambers and Perrone-

S.H. Fatemi et al. / Schizophrenia Research 134 (2012) 211–218 Table 5 Genes that displayed altered expression by both clozapine and haloperidol. Downregulated 3,5-cyclic AMP phosphodiesterase 5-hydroxytryptamine (serotonin) receptor 2 A Adaptor-related protein complex AP-3, mu 1subunit Adducin 1, alpha AKAP95 Aldehyde dehydrogenase family 1, subfamily A2 Alpha actinin 4 Alpha thalassemia/mental retardation syndrome X-linked homolog (human) Androgen-responsive gene encoding an ARD-like protein Arginase 2 Arrestin, beta 2 ATP-binding cassette, sub-family A (ABC1), member 1 B-box and SPRY domain containing Bcl2-like 1 Benzodiazapine receptor associated protein 1 Beta-galactoside-binding lectin Beta-transducin repeat containing BMP/retinoic acid-inducible neural-specific protein 2 Bone morphogenetic protein 4 Brain and reproductive organ-expressed protein Calcium channel beta 4 subunit cAMP responsive element binding protein 3-like 1 cAMP-regulated guanine nucleotide exchangefactor I (cAMP-GEFI) Casein kinase 1 delta Catecholamine-O-methyltransferase CCAAT/enhancer binding protein gamma CD36 antigen CGI-146 protein Claudin 1 Cln3p Collagen, type V, alpha 2 (Col5a2) Colony stimulating factor 2 receptor, beta 1, low-affinity (granulocyte-macrophage) Core binding factor beta Cyclin E Cytoplasmic dynein heavy chain 2 Dre1 protein Dystrophin-related protein 2 splice variant ELAV (embryonic lethal, abnormal vision, Drosophila)-like 2 (Hu antigen B) Fibroblast activation protein FKBP-associated protein Flavin-containing monooxygenase 1 FMS-like tyrosine kinase 1 Fructose-6-phosphate2-kinase/ fructose-2,6-bisphosphatase G protein-binding protein CRFG G protein-coupled receptor 108 Gamma-aminobutyric acid receptor beta 1 Gap junction membrane channel protein beta 2 General transcription factor IIB GIRK2 Glucosidase, alpha; acid (Pompe disease, glycogen storage disease type II) Glutamate decarboxylase 2 (islet) Glutathione-S-transferase, mu type 2 (Yb2) Glycosylphosphatidylinositol specific phospholipase D1

Upregulated Benzodiazapine receptor associated protein 1 Bone morphogenetic protein receptor, type 1A Camello-like 3 CD209b antigen Complement component 3 DEAD (Asp-Glu-Ala-Asp) box polypeptide 3, X-linked FXYD domain-containing ion transport regulator 6 Gap junction protein, alpha 1, 43 kD (connexin 43) iGb3 synthase Lin-7-C Myelin basic protein NTE-related protein Parkin Phosphodiesterase10a Polo like kinase 2 Synaptotagmin 13

(continued on next page)

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Table 5 (continued) Downregulated

Upregulated

Guanine nucleotide binding protein, alpha stimulating, olfactory type Hairless protein Hairy/enhancer-of-split related with YRPW motif 1 Heat shock 10 kD protein 1 (chaperonin 10) High molecular-weight neurofilament Histone H4 variant H4-v.1 Insulin-like growth factor 1 receptor Interleukin 6 signal transducer KH-type splicing regulatory protein lamin B receptor Lamin B1 Lectin, galactose binding, soluble 8 Lectin, galactoside-binding, soluble 2 Low density lipoprotein receptor-related protein 3 MAD homolog 2 MAP kinase kinase-related protein Max interacting protein 1 Meningioma expressed antigen 5 (hyaluronidase) Mid-1-related chloride channel 1 Midkine Myxovirus (influenza) resistance, homolog ofmurine Mx (also interferon-inducible protein IFI78) N-acylaminoacyl-peptide hydrolase Natural cytotoxicity triggering receptor 3 Neural cell adhesion molecule 1 Nuclear fragile X mental retardation protein interacting protein 1 Nuclear receptor co-repressor 1 Opioid-binding protein/cell adhesion molecule-like Origin recognition complex, subunit 4 Peptidyl arginine deiminase, type II Peroxiredoxin 6 Peroxisomal acyl-CoA thioesterase 1 Peroxisomal biogenesis factor 11A P-glycoprotein Phosphodiesterase 1C Plasminogen activator, tissue Polycystic kidney disease 1 Potassium channel, subfamily K, member 3 Potassium voltage gated channel, Shal-related family, member 2 Potassium voltage-gated channel, subfamily H(eag-related), member 2 Protein phosphatase 1, regulatory (inhibitor) subunit 12A Rab geranylgeranyl transferase, a subunit RAB3A interacting protein (rabin3)-like 1 RAS guanyl releasing protein 1 RAS, dexamethasone-induced 1 Reticulon 4 receptor Ribosomal protein s25 ROD1 regulator of differentiation 1 (S. pombe) RT1 class Ib gene S100 calcium binding protein A11 (calgizzarin) Scrapie responsive protein 1 Sex hormone binding globulin or androgen-bindingprotein (other gene product from ABP gene) Shab-related delayed-rectifier K+channel(Kv9.3) Small glutamine-rich protein with tetratricopeptide repeats 2 Sodium-dependent high-affinity dicarboxylatetransporter 3 Solute carrier family 2 (facilitated glucosetransporter), member 13 Solute carrier family 2 (facilitated glucosetransporter), member 4 (continued on next page)

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Table 5 (continued) Downregulated

Upregulated

Solute carrier family 3, member 1 Solute carrier family 5 (sodium iodidesymporter), member 5 Sortilin 1 Src associated in mitosis, 68 kDa Sulfite oxidase SYAP1 protein Synapsin III Synaptogyrin 1 Tafazzin (cardiomyopathy, dilated 3A (X-linked); endocardial fibroelastosis 2; Barth syndrome) T-cell, immune regulator 1, ATPase, H+transporting, lysosomal V0 protein a isoform 3 Testis-specific farnesyl pyrophosphatesynthetase THAP domain containing 4 Transcription termination factor, mitochondrial Transcriptional repressor CREM Tropomyosin 1 (alpha) Tumor suppressor pHyde Ubiquitin conjugating enzyme V-maf musculoaponeurotic fibrosarcoma (avian) oncogene homolog (c-maf) V-myc myelocytomatosis viral related oncogene, neuroblastoma derived (avian) Zinc finger protein 91

Bizzozero, 2004). Importantly, MOG mRNA has previously been shown to be reduced in prefrontal cortex of subjects with schizophrenia (Tkachev et al., 2003) and MBP mRNA has been shown to be reduced in hippocampus of subjects with schizophrenia (Chambers and Perrone-Bizzozero, 2004). The genes that displayed altered expression by both clozapine and haloperidol (including the previously discussed Bcl2l1, Comt, Gja1, and Mbp) show that both typical and atypical APDs may work through similar pathways. Cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) mediated signaling may be targets of APD action, as evidenced by increased expression of mRNA for phosphodiesterase 10a (Pde10a) and reduced mRNA expression of 3,5-cyclic AMP phosphodiesterase (Pde4a) and phosphodiesterase 1C (Pde1C) (Table 5; Supplemental Table 3). Phosphodiesterases are a superfamily of enzymes that hydrolyze cAMP and cGMP and play important roles in signal transduction in brain (Menniti et al., 2006). We have previously demonstrated increased expression of PDE4A in frontal cortex of subjects with autism (Braun et al., 2007) and decreased expression of PDE4B in cerebella of subjects with schizophrenia and bipolar disorder (Fatemi et al., 2008b). Currently, inhibitors of PDE10A are being proposed to treat psychosis in schizophrenia (Chappie et al., 2009). Serotonin receptor 5A (Htr2a), was reduced by both drugs. Polymorphisms of HTR2A have been linked to schizophrenia (Abdolmaleky et al., 2004) and with metabolic abnormalities associated with clozapine and olanzapine treatments (Gunes et al., 2009). GABAergic and glutamatergic signaling may also be affected by both drugs as shown by reductions in GABAA receptor beta 1 (GABRβ1) and glutamic decarboxylase 2 (Gad2, also known as GAD65). GAD2 converts glutamate to GABA and GAD2 has been associated with schizophrenia (LeNiculescu et al., 2007). Our laboratory has preliminary data showing significant reductions in GABRβ1 in cerebella of subjects with schizophrenia and bipolar disorder (Fatemi, unpublished observations). Ncam1 has multiple roles in brain development including dendritic growth and synaptic plasticity (Brennaman and Maness, 2009). A number of single nucleotide polymorphisms (SNPs) for NCAM have been associated with schizophrenia (Sullivan et al., 2007) and increased levels of a soluble fragment containing the extracellular domain

of NCAM have been observed in CSF, prefrontal cortex, and hippocampus of individuals with schizophrenia (Vawter et al., 1998, 2001). The observed reduction of Ncam1 mRNA suggests that APD treatment may reverse this pathological finding. Nuclear fragile X mental retardation protein interacting protein 1 (Nufip1) is involved in shuttling of mRNAs between the nucleus and ribosomes in the cytoplasm, in association with fragile X mental retardation protein (Fmrp), potentially regulating protein synthesis near synapses (Bardoni et al., 2003). Our laboratory has demonstrated significant reductions in FMRP in cerebella of subjects with schizophrenia (Fatemi et al., 2010b). Interleukin 6 signal transducer (Il6st) is a transducer chain shared by multiple cytokines including interleukin 6 (IL6), IL11, ciliary neurotrophic factor (CNTF), and leukemia inhibitory factor (LIF) (Kishimoto, 1994). IL6 expression has been shown to be elevated in first episode psychosis and acutely relapsed inpatients with schizophrenia (Miller et al., 2011). The observed reduction in Il6st mRNA by both clozapine and haloperidol may ameliorate cytokine dysregulation. Insulin-like growth factor 1 receptor (Igf1r) mediates the multiple physiological effects of the IGF1 signaling system. While a study showed that SNPs of IGF1R had no association with schizophrenia (Gunnell et al., 2007), our results of Igf1r mRNA downregulation suggests that APDs may correct imbalances in this system in subjects with schizophrenia. S100 calcium binding protein A11 (S100A11) is a member of the superfamily of S100 EF-hand calcium binding proteins with possible roles in inflammation, regulation of enzyme activity, and cell growth and regulation (He et al., 2009). Our laboratory has previously demonstrated increased mRNA for S100A8 and S100A9 in response to chronic administration of olanzapine (Fatemi et al., 2006) suggesting that these proteins are targets of multiple APDs. Fructose6-phosphate2-kinase 3 (Pfkfb3) regulates the steady state concentration of fructose-2,6-bisphosphate, which in turn is an activator of phosphofructokinase, an important regulatory enzyme of glycolysis (Sakakibara et al., 1997). A number of genes that are involved with glycolysis including Pfkfb2 show associations with schizophrenia (Stone et al., 2004). Gene expression has been measured in a number of animal models of schizophrenia (reviewed by Van Schijndel and Martens, 2010). These models include neurodevelopmental models, such the model developed in our own laboratory of prenatal viral infection (Fatemi et al., 2005, 2008a, 2009c); pharmacological models such as use of phencyclidine (PCP); or genetic models such as knockout mice. Due to the heterogeneity of such approaches, microarray results are variable although genes that code for sodium and potassium voltage-gated channels have appeared in multiple gene lists (Van Schijndel and Martens, 2010). We similarly observed altered expression of a number of potassium channel genes: five were downregulated by clozapine, two were upregulated by haloperidol, and seven were downregulated by haloperidol (Supplemental Tables 1 and 2). Of particular interest are Kcnk3, Kcnd2, and Kcnh2 which were downregulated by both haloperidol and clozapine. KCNH2 has previously been associated with schizophrenia (Huffaker et al., 2009). Studies that combine an intervention designed to produce phenotypes of schizophrenia such as neonatal hypoxia (Sommer et al., 2010) combined with pharmacological treatment have provided important information on the effects of APDs on gene expression changes. Sommer et al. (2010) identified a number of genes upregulated in frontal cortex, two of which (syntaxin 1 and complexin 1) were reversed by treatment with clozapine. As both syntaxin 1 and complexin 1 are presynaptic proteins, the action of clozapine may help restore deficits in synaptic transmission (Sommer et al., 2010). Our results have demonstrated that chronic administration of clozapine and haloperidol impact a variety of gene families in rat frontal cortex and genes that have been associated with schizophrenia, potentially explaining the efficacy of clozapine. Most importantly, we identified 145 genes that were altered by both APDs including Bcl2l1, Comt, Gja1, Htr2a, Mbp, and Pde4a. These genes may hold the key

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to understanding how the APDs operate. Future studies to further elucidate gene expression changes as a result of APD treatment could include: 1) measuring the acute or sub-chronic effects of APD treatment (1 day, or 7 days) to obtain a better idea of the time course of APD effect on gene expression; 2) testing chronic APD treatment on gene expression using animal models of schizophrenia; 3) including behavioral testing to verify whether the observed gene expression changes translate to correction of cognitive or behavioral deficits observed in animal models of schizophrenia. Supplementary materials related to this article can be found online at doi:10.1016/j.schres.2011.11.013. Role of funding source Funding for this study was provided by the Stanley Medical Research Institute (SMRI), (Grant # 02R-232) (SHF). SMRI had no further role in study design; in the collection, analysis, and interpretation of data; in the writing of the report; and in the decision to submit the paper for publication.

Contributors Author SHF designed the study and supervised all aspects of the experiments and writing of the manuscript. Author TDF performed dissections, analyzed microarray and qRT-PCR data, performed literature searches, and contributed to the writing of the manuscript. Author TJR performed the animal experiments and dissections, and analyzed microarray and qRT-PCR data. Authors JN and RHE performed literature searches.

Conflict of interest All authors declare that they have no conflicts of interest.

Acknowledgments Grant support from the Stanley Medical Research Institute (Grant # 02R-232) to SHF is gratefully acknowledged. Microarray analyses were performed by the BioMedical Genomics Center at the University of Minnesota. qRT-PCR analysis was provided by D. Patel and R. Rooney at Genome Explorations, Inc.

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