Molecular Psychiatry (2003) 8, 983–993 & 2003 Nature Publishing Group All rights reserved 1359-4184/03 $25.00 www.nature.com/mp ORIGINAL RESEARCH ARTICLE Antipsychotic drug treatment alters expression of mRNAs encoding lipid metabolism-related EA Thomas1, RC George1, PE Danielson1, PA Nelson1, AJ Warren2,3,DLo2 and JG Sutcliffe1 1Department of Molecular Biology, The Scripps Research Institute, La Jolla, CA, USA; 2Digital Gene Technologies, La Jolla, CA, USA

Using an automated PCR-based genomics approach, TOtal Gene expression Analysis (TOGAs), we have examined gene expression profiles of mouse striatum and frontal cortex in response to clozapine and haloperidol drug treatment. Of 17 315 mRNAs observed, TOGAs identified several groups of related molecules that were regulated by drug treatment. The expression of some genes encoding proteins involved in neurotransmission, signal transduction, oxidative stress, cell adhesion, apoptosis and proteolysis were altered in the brains of both clozapine- and haloperidol-treated mice as recognized by TOGAs. Most notable was the differential expression of those genes whose products are associated with lipid metabolism. These include apolipoprotein D (apoD), the mouse homolog of oxysterol-binding -like protein 8 (OSBPL8), a diacylglycerol receptor (n-), and lysophosphatidic acid (LPA) acyltransferase. Real-time PCR analysis confirmed increases in the RNA expression of apoD (1.6–2.2-fold) and OSBPL8 (1.7–2.6-fold), and decreases in the RNA expression of n- chimerin (1.5–2.2-fold) and LPA acyltransferase (1.5-fold) in response to haloperidol and/or clozapine treatment. Additional molecules related to calcium homeostasis and signal transduction, as well as four sequences of previously unidentified mRNAs, were also confirmed by real-time PCR to be regulated by drug treatment. While antipsychotic drugs may affect several metabolic pathways, lipid metabolism/signaling pathways may be of particular importance in the mechanisms of antipsychotic drug action and in the pathophy- siology of psychiatric disorders. Molecular Psychiatry (2003) 8, 983–993. doi:10.1038/sj.mp.4001425 Keywords: clozapine; haloperidol; gene expression; striatum; mouse

Introduction ‘typical’ and ‘atypical’ antipsychotics. Typical anti- psychotic drugs, which include haloperidol, chlor- Neuropsychiatric disorders, including promazine and fluphenazine, are primarily dopamine and , are a heterogeneous group of D2 receptor antagonists, while the ‘atypical’ antipsy- devastating illnesses affecting 1–2% of people world- chotics, such as clozapine, risperidone and olanza- wide. Psychosis is a symptom in many psychiatric pine, have a range of affinities for several different illnesses, while specific symptoms of schizophrenia receptors in addition to those for include a diversity of both ‘positive’ symptoms, such dopamine.2–4 as delusions, hallucinations, disordered thinking and The major existing theories of psychiatric diseases bizarre behavior, and ‘negative’ symptoms, such as have been developed by exploring the mechanisms of social withdrawal, affective blunting, alogia (absence action of these antipsychotic drugs. For example, the of words) and anhedonia (decrease in the ability to ‘dopamine hypothesis’ of schizophrenia, formulated 1 experience pleasure). Most symptoms are treatable over 30 years ago, proposes that the hyperactivity of by the administration of antipsychotic drugs. The dopaminergic neurotransmission causes symptoms of efficacy of such medications has been well estab- schizophrenia.5,6 Major support for this hypothesis lished in that antipsychotic drugs reduce symptoma- comes from indirect pharmacological evidence, that tology and prevent relapse in a large percentage of is, the excellent correlation observed between the patients. The two main classes of drugs are the clinical efficacy of typical antipsychotic drugs and 7,8 their binding to the dopamine D2 receptor. Simi- Correspondence: J Gregor Sutcliffe, Department of Molecular larly, serotonergic systems have also been implicated Biology, The Scripps Research Institute, 10550 N. Torrey Pines in schizophrenia, largely because many of the newer Road, La Jolla, CA, USA. E-mail: [email protected] 3 atypical drugs have high affinity for the serotonin 2 Current address: Stratagene 11011 N. Torrey Pines Road, La Jolla, 9,10 CA, USA class of receptors. There is a substantial body of Received 26 March 2003; revised 20 June 2003; accepted 18 July evidence supporting neurotransmitter receptor ab- 2003 normalities in psychiatric disorders, and this concept Antipsychotic drugs and gene expression EA Thomas et al 984 has successfully guided the development of drugs to injection at 5 days, 12 days or 2 weeks. The dose of

treat these disorders. However, it has become clear haloperidol used was approximately the ED50 for that other, additional systems, components and/or inducing catalepsy in the C57BL/6J strain, while that factors are required to explain the complex nature of for clozapine was the same as that previously shown these diseases. Further examination of the mechan- to interrupt cognitive function in mice and one-tenth isms of antipsychotic drug action should reveal key that reported to induce catalepsy.28 Brains were information regarding the underlying pathophysiolo- rapidly removed and placed on ice. The striatum gical features of psychiatric diseases and may lead to and frontal cortex were dissected out and placed in novel targets for drug treatment. ice-cold phosphate-buffered saline. These striata and The administration of antipsychotic drugs has been cortical samples were pooled and the total and shown to elicit changes in gene expression. Several poly(A þ) RNA were prepared according to Thomas studies have shown increases in the expression of et al,29 and subjected to TOGAs and real-time PCR immediate-early genes (IEGs) after acute drug expo- analyses. RNA was also prepared from the striata and sure.11–15 Many IEGs are known to act as transcrip- cortex from a separate set of mice (n¼20/drug/time tional regulators, thereby linking receptor-mediated point) and used for Northern blot analysis. effects to changes in genomic activity. In addition, some studies have examined the expression of TOGAs data analysis and statistics candidate genes in animals after chronic exposure to Poly(A þ) RNA from control and drug-treated animals antipsychotic drugs. Alterations in the expression of were subjected to TOGAs methodology as described , including preprotachykinin, pre- previously30 and modified as described at www.dgt. proenkephalin and neurotensin, have been found in com. All data analyses were performed using TOGAs response to haloperidol treatment,16–19 while chronic proprietary software. To determine global variation in haloperidol and/or clozapine administration causes all data sets, correlations coefficients were deter- changes in the expression of nuclear receptors,20 mined by linear regression analysis of pairwise synaptic proteins21 and various neurotransmitter comparisons of normalized peak amplitudes. In the receptors, most reproducibly components of the cortical samples, the lowest correlation coefficient glutamatergic system.22–25 Two recent studies have was 0.971 among all samples, while the lowest used cDNA microarrays to investigate global changes correlation coefficient in the striatum samples was in gene expression in response to drug treatment: one 0.942, which represented the correlation between the examining the effects of chronic haloperidol treat- control and 2-week drug-treated time point, a com- ment on gene expression in rat striatum 26 and the parison in which the greatest number of genes would other looking at the effects of acute clozapine be expected to be regulated, hence giving rise to the treatment in the rat cortex.27 The results from both lower correlation coefficient. The software also studies have implicated synaptic function and cal- calculated a variance histogram to describe the cium regulation as being important to the mechan- amount of gene variation within a given sample set. isms of drug action. In this data set, 99.7% of all genes showed a variance In this study, we have surveyed gene expression less than 0.6. The remaining 0.3% of genes therefore changes in the mouse striatum and prefrontal cortex represent drug-regulated genes or random variation. both after clozapine and after haloperidol adminis- Using the criteria given below, we identified 51 genes tration, using the PCR-based, RNA display method, out of 17 315 screened, which represents 0.29% of the TOtal Gene expression Analysis (TOGAs). We have genes. identified several groups of molecules whose expres- All samples were run in duplicate and the average sion patterns are altered by drug treatment, most peak amplitude was used to determine fold regula- notably changes in the expression of genes related to tion. Genes were determined to have regulated phospholipids/fatty acid signaling/metabolism. expression levels in the drug-treated samples vs the control groups based on the following criteria: (1) a Z1.6-fold change in the expression level between the Materials and methods average peak amplitude of a given cDNA in the drug- Animal procedures and tissue preparation treated samples compared to controls; (2) each Male C57BL/6J mice (20–28 g) were housed in groups regulated cDNA must be consistently altered by of four on a standard 12/12 h light–dark cycle with ad Z1.6-fold in both chronic time points (12 days and libitum access to standard laboratory chow and tap 2 weeks) compared to both control time points and water. Groups of mice were treated with clozapine, (3) each cDNA must have a threshold abundance haloperidol or saline for different time points (n¼25 value of Z75 arbitrary fluorescence units. All peaks mice per drug per time point). For acute drug below this value were considered to be background treatments, mice received a single intraperitoneal signal. Furthermore, all computer-generated picks injection of saline or clozapine (7.5 mg/kg) and were were then manually screened to assess peak quality

killed by CO2 inhalation after 45 min or 7 h. For and integrity. cDNAs were eliminated when visual chronic treatments, mice received daily subcutaneous inspection suggested that they were spectral artifacts. injections of clozapine (7.5 mg/kg) or haloperidol A two-tailed permutation argument was used to (4 mg/kg) and were killed 24 h after their final determine the significance of a drug effect on the

Molecular Psychiatry Antipsychotic drugs and gene expression EA Thomas et al 985 regulation of cDNA levels. We partitioned the chronic In situ hybridization analysis drug time points and both control time points into In situ hybridization was performed on free-floating every possible combination of two (representing the sections from brains of control mice and those treated requirement for a given cDNA to be regulated in both with saline, haloperidol (4 mg/kg) or clozapine chronic time points compared to both controls) and (7.5 mg/kg) (n¼4 for each treatment) as described then determined how many of these partitions previously.31 Coronal sections were hybridized at resulted in ‘hits’, where a ‘hit’ is Z1.6-fold change 551C for 16 h with a 35S-labeled, single-stranded in the peak amplitude between partitioned sets. From antisense cRNA probe against 12 different cDNA these permutations, we could determine the signifi- clones (107 cpm/ml). 35S-labeled sense-strand probes cance of our observed number of hits due to drug were used as controls. Excess probe was removed by treatment (the chronic time points relative to the washing with 2  saline-sodium citrate buffer (SSC; controls) compared to the expected number of hits 1  SSC¼0.015 M NaCl/0.0015 M sodium citrate) under the null hypothesis of no drug effect (ie every containing 14 mM b-mercaptoethanol (RT; 30 min), combination of time points producing an equal followed by incubation with 4 mg/ml ribonuclease in number of hits). The following formula describes the 0.5 M NaCl/0.05 M EDTA/0.05 M Tris-HCl (pH 7.5) for approximate standard normal random variable under 1 h at 371C. High-stringency washes were carried out these conditions: at 551C for 2 h in 1  SSC/50% formamide/0.8 mM b- pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi mercaptoethanol, and then at 681C for 1 h in ½Obs À k=3Š= nÃpÃð1 À pÞ; 0.2  SSC/1.4 mM b-mercaptoethanol/0.5% sarkosyl. where Obs is the observed number of hits we Slices were mounted onto Superfrost slides (Fisher identified according to the above criteria; k is the Scientific) and dehydrated with ethanol and chloro- total number of hits from every permutation of time form before autoradiography. Slides were exposed for points; n is the total number of genes visualized. In 1–4 days to a Kodak X-AR film and then dipped in an the above formula, p¼k/3n. We calculated P-values Ilford K-5 emulsion. After 4 weeks, slides were for different TOGAs fold changes (1.4–1.8-fold) and developed with Kodak D19 developer, fixed and different threshold values (25–100 arbitrary fluores- counterstained with Richardson’s blue. cence units), resulting in a range of P-values from 0.0003 to 0.0013, all indicating a significant effect of Results drug at these criteria. Groups of mice (n¼25) were treated with clozapine, Real-time PCR validation haloperidol or saline for 45 min, 7 h, 5 days, 12 days Real-time PCR experiments were performed using and 2 weeks. Poly(A) þ-enriched RNA from the the ABI PRISMs 7700 Sequence Detection System striatum and frontal cortex were analyzed using (Applied Biosystems), according to the manufac- TOGAs, an automated, systematic, PCR-based RNA turer’s guidelines with the SYBR Green Reagent display technique. Complete TOGAs analysis in- as the fluorescent reporter molecule. PCR products volves 256 different PCR reactions with a given were amplified from first-strand cDNA prepared restriction enzyme (MspI, in this case) performed on from 1 mg polyA þ-selected RNA from the striatum each RNA sample and divides PCR fragments from of pooled control and drug-treated mice. This RNA the 30 ends of mRNAs into 256 nonoverlapping pools. was the same as that used in the initial TOGAs The fluorescent PCR products, each of which derives analysis. Primers specific to each cDNA were used in from a different mRNA in the starting population, are combination with primers to amplify housekeeping detected by capillary electrophoresis. Each peak genes. The housekeeping gene, hypoxanthine:gua- whose amplitude corresponds to greater that one part nine phosphoribosyl transferase (HPRT), was used to in 106 of the input RNA is entered into an electronic normalize the expression levels in these studies. The database, which can be queried by proprietary soft- absence of nonspecific amplification and the pre- ware (for details, see Sutcliffe et al,30 www.dgt.com). sence of a single PCR product are confirmed for each A representative PCR profile highlighting expression primer pair by agarose-gel electrophoresis. For the regulation by clozapine and haloperidol is shown in clozapine-regulated candidate genes, 68% of the Figure 1. Approximately 17 315 TOGAs cDNA pro- cDNAs (11 out of 16) were validated by real-time ducts were detected and databased in the present PCR analysis. study. The database was queried electronically for TOGAs Northern blot analysis product amplitudes that differed in duplicate drug The total (15 mg/lane) and/or poly(A þ) RNA (2 mg/ experiments by 1.6-fold or more in both the 12-day lane) were separated by electrophoresis and trans- and 2-week drug- vs saline-treated samples (see ferred onto Biotrans membrane using standard tech- ‘Materials and methods’ for further details). A total niques. of 51 candidate cDNAs (‘digital sequence tags’, DSTs) Hybridization bands were quantified by densito- were identified to be regulated by clozapine and/or metry. Data were normalized to cyclophilin hybridi- haloperidol (drug effect; P¼0.0006 at 1.6-fold regula- zation and are expressed as fold stimulation over tion). A total of 46 were found to be regulated by basal. clozapine (23 upregulated and 23 downregulated) in

Molecular Psychiatry Antipsychotic drugs and gene expression EA Thomas et al 986 Table 1 Summary of TOGA-identified genes grouped according to drug-specific regulation

Clozapine regulated Haloperidol regulated

ApoD Striatin LPA acyltransferase Hypoxia-induced gene 1 L1-like cell adhesion molecule Neurod4 BPTF Zinc-binding protein TNIK-8 Nucleolin TRAP240 GEF

C-myc-binding protein MM-1 Neu12 Neu18 Neu24 Neu37 Neu43

Coregulated by both drugs

Glutamate receptor g2 subunit Ribosomal protein P2 Cystatin C Ribosomal protein S10 Ca þ 2 channel a1A subunit Ribonuclear protein D2 DJ-1 (ras related) Actin-related protein PDE1B1 Neuroendocrine peptide MAP/Erk kinase Precursor OSBPL-8 Histone H2A n-Chimerin NARF Steroid dehydrogenase Ke6 Clz62 GSHPx Clz40 ALL-1 Neu4 MEK-binding partner Neu13 Protein phosphatase 5 Neu17 Ubiquitin/60S fusion protein Neu44 Ubiquitin-conjugated enzyme Neu104

s E2R Figure 1 A portion of one of 256 TOGA expression Ubiquinol Neu112 profiles for clozapine and haloperidol. Each peak represents L1 repetitive sequence Neu136 a cDNA PCR product that is separated by size (in nucleotides) along the x-axis and abundance (in fluores- cence units) along the y-axis and each panel reflects a different stage of drug treatment: 45 min, 7 h, 12 days and 2 weeks. In all, 256 different display panels allows for the conjugating enzyme E2R was increased by clozapine visualization of nearly all RNAs in a given sample. Here, the in the striatum, but decreased in the cortex (Table 2). TOGAs primer was ACAG. Only the region surrounding Many of the DSTs had candidate identities pre- this peak in the electropherogram is shown. Tick marks dicted electronically by their TOGAs addresses. along the top of the panels represent the migration positions These identities were verified by extended specific of mRNAs in the mouse portion of the GenBank database, primers as described previously.30 For those DSTs that whose TOGAs addresses have been electronically pre- dicted. did not correspond to a species with a predicted identity, cDNA clones were isolated and their nucleo- tide sequences were determined. The sequences of these DSTs were blasted in the GenBank and mouse the striatum and/or cortex and 38 by haloperidol genome databases to determine the identity of the (28 upregulated and 10 downregulated) in the molecules. The identities of these candidate cDNA striatum only. Out of the 51 DSTs in total, 13 were clones fell into several distinct classes, while 14 DSTs clozapine specific in their regulation, five were were additionally identified, which corresponded to haloperidol specific and 33 were regulated by both presently unknown sequences (Table 2). This infor- drugs (Table 1). The differential regulation of expres- mation reveals which systems or pathways may be sion was also apparent in the striatum vs cortex for affected by antipsychotic drug treatment and, possi- some of the DSTs. The expression patterns of the bly, which systems may be dysfunctional in psychia- glutamate receptor g2 subunit, Neu17 and Neu136, tric disorders. were altered by clozapine only in the cortex and not To validate the TOGAs methodology, real-time PCR the striatum, while the expression of ubiquitin- analysis was performed on selected cDNAs using the

Molecular Psychiatry Table 2 Classes of genes identified by TOGAs and fold regulation by clozapine and haloperidol in the striatum and cortex

Fold regulation by TOGAs Fold regulation by TOGAs

Cloz Cloz Hal Acc.# Cloz Cloz Hal Acc.# (striat) (cortex) (striat) (striat) (cortex) (striat)

Neurotransmitter receptors Ribosomal Glutamate receptor g2 subunit 1.23 0.45 2.02 D10011 Ribosomal protein S10 1.70 1.02 2.03 BC003853 Ca þ 2 channel a1A subunit 0.21 0.95 0.60 AB025352 Ribosomal protein P2 2.08 1.08 2.40 AK012402

Signal transduction Ribonuclear protein D2 2.05 1.26 1.70 AK007389

GEF (guanine nucleotide exchange factor) 0.62 0.56 0.86 AW530801 Other DJ-1 1.76 1.27 1.90 BC002187 Neuroendocrine 1.53 0.84 1.98 BC012263 PDE1B1 1.51 1.31 1.56 L01695 Peptide precursor Striatin 1.38 1.07 1.60 BC004025 Nucleolin 1.27 1.15 2.07 AF318184 MAP/Erk kinase 1.77 1.19 2.14 S68267 Histone H2A 2.95 1.36 2.91 BE138257 Lipid related Actin-related protein 1.86 0.96 2.02 AK017993 Apo D 2.4 ND 1.33 L39123 Cystatin C 2.14 1.26 1.95 AK014368 OSBPL8 2.0 ND 2.3 XM_137248 NARF 0.62 0.82 0.59 AB043550 LPA acyltransferase 0.62 0.72 0.81 AB005623 TRAP240 0.62 0.55 0.86 AW530801 n-Chimerin 0.57 0.81 0.61 AF332070 C-myc-binding protein MM-1 1.50 3.89 1.16 AF108357 Steroid dehydrogenase Ke6 1.88 1.09 2.36 M32010 L1 repetitive sequence 0.35 0.59 0.51 BF136402

Oxidative Novel sequences Thomas expression EA gene and drugs Antipsychotic GSHPx 2.68 1.18 2.42 X03920 Clz40 0.33 ND 0.34 BC037646 Hypoxia-induced gene 1 1.12 1.29 1.64 AK018360 Clz62 1.52 ND 2.66 BC034895 tal et

Cell adhesion NEU4 0.53 0.59 0.55 NOVEL L1-like molecule 0.61 0.75 0.69 X94310 NEU12 0.63 0.58 0.81 BF138011

Transcription regulation NEU13 0.60 0.84 0.47 AQ934098 BPTF 0.49 0.78 0.69 AB032251 NEU17 1.07 0.47 1.75 AA590162 Neurod4 1.38 0.80 2.17 AF108133 NEU18 0.61 0.72 0.77 NOVEL Zinc-binding protein 1.45 1.02 1.87 X64053 NEU24 0.54 0.47 0.76 BG062546 All-1 0.30 0.40 0.59 L17069 NEU37 0.55 0.69 0.83 BF787389

Apoptosis NEU43 0.51 0.35 1.28 BE979587 Traf2-Nck interacting kinase 8 0.54 0.54 0.73 AW045551 NEU44 0.62 0.84 0.62 AK004618 MEK-binding partner 1 1.92 1.33 1.90 AF082526 NEU104 2.10 0.85 3.38 BC010095 Protein phosphatase 5 0.52 1.14 0.64 AF018262 NEU112 1.91 1.20 2.11 AA277826

Proteolysis NEU136 1.28 1.81 1.60 AA030405 Ubiquitin/60S fusion protein 2.21 1.82 2.82 AF118402 oeua Psychiatry Molecular Ubiquitin-conjugated enzyme E2R 2.05 0.45 6.95 BC011112 Ubiquinol complex 2.06 0.70 3.02 AK003443

Accession numbers for the DST sequences are listed. For Neu4 and Neu18, no matches in the databases were found. ND, not determined. 987 Antipsychotic drugs and gene expression EA Thomas et al

988 s Clz40 n-Chimaerin same striatal RNA samples as in the initial TOGA 1.5

analysis. To validate the drug-induced expression 1.0 regulation, Northern blot analysis was performed on 1.0 striatum and cortical RNA from a different set of 0.5

equivalently treated mice (n¼20). Both analyses Abundance 0.5 (fold-change) revealed expression patterns consistent with the s 0.0 0.0 initial TOGA regulation patterns for clozapine of   TOGA PCR Northern TOGA PCR Northern the following cDNAs: apolipoprotein D (apoD), Ca+2 channel α1A subunit Clz62 oxysterol-binding protein-like protein-8 (OSBPL-8), 1.5 3 n-chimerin, P/Q-type calcium channel a1A subunit, lysophosphatidic acid (LPA) acyltransferase, Traf2- 1.0 2 and Nck-interacting kinase-8 (TNIK-8), L1-like cell

Abundance 0.5 1

adhesion molecule, neural activity-related ring finger (fold-change) protein (NARF), calmodulin-dependent phosphodies- 0.0  0 terase (PDE1B1) and four unidentified sequences, TOGA PCR Northern TOGA PCR Northern Clz40, Clz62, Neu13 and Neu44 (Figure 2). For all but TNIK-8 two DSTs, NARF and Neu44, real-time PCR analysis LPA-acyltransferase confirmed the expression regulation for haloperidol 1.0 1.0 (Figure 2). mRNA expression was not detected on

Northern blots for Neu44, the L1-like cell adhesion 0.5 0.5 Abundance molecule and OSBPL-8. (fold-change) We next investigated the CNS expression patterns 0.0 0.0  of drug-regulated genes using in situ hybridization TOGA PCR Northern TOGA PCR Northern analysis. In untreated mouse brains, some mRNAs exhibited relatively ubiquitous expression patterns, PDE1B1 Neu13 while others exhibited considerable anatomical se- lectivity (Table 3). Relatively abundant and ubiqui- 1.0 1.0 tous expression patterns were detected for NARF, LPA

acyltransferase, n-chimerin and TNIK-8, while Neu44 0.5 0.5 Abundance

was ubiquitously expressed in low abundance (Table (fold-change) 3). Striatal-predominant expression was detected for 0.0 0.0 the mouse homolog of OSBPL-8 and PDE1B1, but TOGA PCR TOGA PCR Northern mRNA expression was also detected in the cortex and NARF Neu44 hippocampus (Table 3, Figure 3). Clz40 displayed 2.0 1.5

enriched expression in the nucleus accumbens and 1.5 dentate gyrus over other brain regions, but also 1.0 showed low-abundance ubiquitous hybridization sig- 1.0

Abundance 0.5 nals (Table 3). The novel species, Neu13, was (fold-change) 0.5 detected in sparsely distributed individual cells, 0.0  0.0 predominantly in the cortex, ventricular lining, TOGA PCR TOGA PCR L1-like molecule OSBPL-8 thalamus and hippocampus (Table 3, Figure 3). 3 The expression patterns of OSBPL-8 and Neu13 1.0 were further examined by in situ hybridization using 2 brains of saline-, clozapine- and haloperidol-treated 0.5 mice (n¼4 per drug pair). OSBPL-8 mRNA was found Abudance 1 (fold-change) to be increased in the striatum and cortex by both clozapine and haloperidol treatment (Figure 3). This 0.0 0 TOGA PCR TOGA PCR effect was evident primarily in the striatum, olfactory Figure 2 Bar graphs demonstrating the quantitative corre- tubercle and cortex. In contrast, Neu13 exhibited an lations for the indicated genes as determined by TOGAs, overall decreased expression in the brains of mice real-time PCR analysis and Northern blot analysis. Open treated with either clozapine or haloperidol, being bars represent saline treatment; closed bars, clozapine barely detectable in the brains of clozapine-treated treatment (7.5 mg/kg) and stippled bars, haloperidol treat- mice (Figure 3). The increase in the expression of ment (4 mg/kg). Details of these three methods, TOGAs, apoD in drug-treated animals as detected by in situ real-time PCR and Northern blot analysis, are described in hybridization has been demonstrated and discussed ‘Materials and methods’. Data are shown for the following þ 2 a in our previous studies.32 DSTs: Clz40, n-chimerin, Ca channel subunit 1A sub- unit, Clz62, TNIK-8, LPA-acyltransferase, PDE1B1, Neu13, NARF, Neu44, L1-like molecule and OSBPL-8. For some Discussion genes, Northern blot and/or real-time PCR data are not available. Data are expressed as percent of control after s In this study, we utilized TOGA to investigate normalization using HPRT for the real-time PCR studies and changes in gene expression in mouse striatum and cyclophilin for the Northern blot studies.

Molecular Psychiatry Antipsychotic drugs and gene expression EA Thomas et al 989 Table 3 Anatomical distributions of mRNAs of clozapine- and haloperidol-regulated clones based on in situ hybridization analysis

Clone #

Clz40 Clz43 Clz62 Neu1 Neu7 Neu13 Neu19 Neu20 Neu23 Neu26 Neu28 Neu44

Brain region Cortex Layer II/III þ þ þ þ þþÀ þþþþþþþþþþ Layer IV þ þ þ þþ þþ þ þþ À þþ À þ þ Layer V þ þþ þ þþ þþ À þþþ À þþ þþ þ þ Layer VI þ þ þ þþ þþ þ þþþ þ þþ À þ þ Septum þþþþþÀÀþþþÀþÀþ Striatum þ þþþ À þ þþ þ þþ þþþ þþ À þ þ Nucleus acc. þþ þþ À þ þþ À þþ þþþ þþ À þ þ Olfactory tubercle þþ þþ þþ þ þþ À þþþ þþ þþ À À þ

Hippocampus Dentate gyrus þþþ þ þ þþþ þþþ þ þþ þ þþ þþ þ þþ CA1 þ þ þ þþþ þþþ þþ þþ þ þþ þþ þ þþ CA2/3 þ þ þ þþþ þþþ þþ þþ þ þþ þþ þ þþ Hypothalamus þ À À þ þ À À þþ þþ À þ Thalamus ÀÀÀÀþþþþÀÀþþþÀÀ Amygdala þþ À þ þ þþ À þþ À þþ þþ À þ

In situ hybridization studies were performed using 35S-labeled riboprobes on free-floating brain sections from control mice (see Materials and methods for details). Slices were exposed to autoradiographic film for 1–5 days and then dipped in silver emulsion and exposed for 4 weeks. The abundance of hybridization signals was based on the density of the signal from the autoradiographic data or by high-magnification examination of emulsion-dipped slides: þ, low abundance; þþ, medium abundance; þþþ, high abundance; À, not detected.

Figure 3 In situ hybridization analysis demonstrating increased OSBPL-8 mRNA expression and decreased Neu13 mRNA expression in mouse brain after clozapine and haloperidol administration. Free-floating coronal sections from mouse brains of saline and drug-treated mice were probed with an antisense 35S-labeled OSBPL-8 and Neu13 riboprobes. NC, neocortex; CPu, caudate putamen; NAcc, nucleus accumbens; Hipp, hippocampus; Thal, thalamus; Hyp, hypothalamus.

cortex induced by clozapine and haloperidol admin- time points (12 days and 2 weeks) compared to saline- istration. We profiled expression patterns at five treated controls. The expression of genes encoding different time points of drug treatment (45 min, 7 h, proteins involved in diverse functions, including in 5 days, 12 days and 2 weeks), and, in this study, have neurotransmission, calcium homeostasis, signal focused on those genes regulated at the long-term transduction, oxidative stress, cell adhesion,

Molecular Psychiatry Antipsychotic drugs and gene expression EA Thomas et al 990 apoptosis and proteolysis, were found to be altered in to inhibit arachidonic acid-induced aggregation of the brains of both clozapine- and haloperidol-treated rabbit platelets, suggesting an inhibition of the mice. Notable was the differential expression of genes generation of prostaglandin endoperoxides or throm- whose products are associated with phospholipids/ boxanes47 and elevate arachidonic and docosahexae- fatty acid signaling and metabolism. These include noic acid levels in red cell membranes of the mouse homolog of OSBPL-8, n-chimerin, LPA schizophrenic patients.48 Clozapine administration acyltransferase, steroid dehydrogenase Ke6 and apoD is also associated with other serum lipid abnormal- (which we have already described in a previous ities in psychiatric patients.49 study).32 The final lipid-related molecule identified in our Oxysterols are 27-carbon products of cholesterol study, steroid dehydrogenase Ke6, has significant oxidation that display several biological activities, homology to mammalian 11 beta-hydroxysteroid including cytotoxic effects, perturbation of membrane dehydrogenases (11 beta-HSDs). These enzymes cat- structure and function of membrane-associated pro- alyze the interconversion of active glucocorticoids teins, as well as disturbance of cellular cholesterol (cortisol and corticosterone) into their inactive 11- homeostasis.33,34 Oxysterol-binding proteins are a keto products (cortisone and 11-deoxycorticoster- family of eucaryotic intracellular lipid receptors that one).50,51 This is consistent with the fact that bind oxysterols and regulate cholesterol and lipid glucoregulatory abnormalities, including weight gain, homeostasis.34,35 Our mouse sequence was found to have been associated with the use of antipsychotic be the homolog of the human OSBPL-8, whose medications.49,52,53 Overall, these findings suggest function is not precisely known,35 but likely functions that the antipsychotic drugs may exert their effects, in a manner similar to other family members. beneficial or otherwise, by targeting pathways asso- Interestingly, the oxysterol, 25-hydroxycholesterol, ciated with lipid metabolism and homeostasis. This which is a potent regulator of cellular cholesterol notion is consistent with a growing body of literature homeostasis in mammalian cells, has been shown to that has implicated abnormalities in various aspects elevate apoD levels in cultured astrocytes.36 This of lipid metabolism, signal transduction, cholesterol provides a potential link towards a common function and glucocorticoid homeostasis in psychiatric dis- for these two drug-regulated genes. orders.54–57,58 N-chimerin (a1-chimerin) was originally identified In the current study, we also identified genes that as a 34-kDa protein highly expressed in the brain and are related to calcium signaling and homeostasis to be then later defined as a member of a new family of regulated by antipsychotic drug treatment. These ‘nonprotein kinase C’ phorbal ester/diacylglycerol include the P/Q-type calcium channel a1A subunit, (DAG) receptors.37,38 DAG is a lipid second messenger the calcium/calmodulin-dependent PDE1B1 and two generated directly by the action of phospholipase C candidate genes, glutamate receptor g2 subunit and isozymes or indirectly via the activation of phospho- striatin, a calmodulin-binding protein 4. These find- lipase D and subsequent release of phosphatidic acid ings are consistent with a recent study investigating (PA), a phospholipid signaling molecule. These DAG global gene expression changes in response to drug receptors act as GTPase accelerating proteins, which treatment using cDNA microarray analysis, in which enhance cellular G-protein signal transduction.38 We several genes involved in the regulation of intracel- also confirmed the expression regulation of LPA lular calcium stores were recognized.27 In this study, acyltransferase, which catalyzes the conversion of candidate genes were first identified in a microarray LPA to PA.39 LPA, another phospholipids signaling screen of genes regulated by acute clozapine treat- mediator, acts via a specific G-protein-coupled recep- ment, and then additional in situ hybridization tor (lysophospholipid receptors), several of which analyses were performed on selected genes to exam- have been recently identified.40,41 Hence, n-chimerin ine the expression levels in response to chronic and LPA acyltransferase appear to also function in a clozapine and haloperidol treatment.27 Increased common pathway of lipid signal transduction. Inter- expression patterns of visinin-like proteins, which estingly, membrane lysophosphatidylcholine levels belong to a family of intracellular calcium sensor has been shown to be elevated in platelets of proteins, , a calcium-binding protein schizophrenic patients.42 enriched in large dense core vesicles, and calcineurin Antipsychotic drugs have previously been shown A, a calcium and calmodulin-dependent serine– to alter phospholipid-related second messenger path- threonine protein phosphatase, were observed in ways, such as those involved in phosphatidylinositol response to both clozapine and haloperidol.27 Inter- (PI) hydrolysis, by blocking neurotransmitter receptor estingly, microarray analysis of gene expression in active sites. Chronic haloperidol, chlorpromazine and schizophrenic brain tissue has identified an increased clozapine administration to rats has been shown to expression of calcineurin A in the prefrontal cortex of cause decreases in norepinephrine- and dopamine- neuroleptic-medicated patients.59 and serotonin-induced PI hydrolysis in rat brain.43–46 Since it is not possible to perform drug-induced Many studies, however, have observed effects of gene expression analysis on human subjects, we antipsychotic drugs on pathways involving fatty employed a mouse model in this study. However, acids, which may not be related to blockade of the mouse brain is comparatively different receptors. Clozapine administration has been shown from primate brain, especially the frontal cortex.

Molecular Psychiatry Antipsychotic drugs and gene expression EA Thomas et al 991 Interspecies differences in the cortex exist in terms of allowing only single expression dichotomies to be the complexity of neuronal connectivity, the organi- investigated. Unlike microarray techniques, these zation and elaboration of dopaminergic innervation, methods have not been widely used to study receptor subtype expression, colocali- neuropsychiatric disorders; however, one study has zation and development processes.60–62 The dynamics used SAGE to identify interesting candidate genes of dopamine release, uptake kinetics, regulation by associated with bipolar disorder.67 firing frequency and presynaptic autoreceptor func- High-throughput analyses have revealed complex tion in the human striatum, which consists of a gene expression patterns induced by antipsychotic distinct caudate nucleus and putamen, also differs drug treatment. With these emerging studies, we are considerably from rodent striatum.63,64 Hence, the beginning to gain an understanding about how drugs pharmacological response in cortical or striatal sites may be working to correct underlying abnormalities to antipsychotic drugs may be different in primate in psychiatric and other diseases that use antipsycho- brain than in mouse brain resulting in potentially tic drugs as part of their treatment regimen. Some of different transcriptome profiles. these molecules may represent new drug targets for While microarray analysis has been a popular improved therapeutics for these disorders and sup- approach for the study of differentially expressed port the involvement of lipid pathways in the genes, its application has successfully identified pathophysiology of schizophrenia and other psychia- relevant candidate genes in many disease and drug tric disorders. paradigms, TOGAs offers several advantages over presently used microarray methods. The first is sensitivity. Whereas microarrays have sensitivity that Acknowledgements ranges from one part in 105 to one part in 104, depending upon the nucleotide sequence and com- This work was supported by National Institute of position of each particular element in the array, Health Grants #NS44169 (EAT) and #GM32355 (JGS) TOGAs has a sensitivity of one part in 106.We and Digital Gene Technologies. We thank James identified several transcripts of low abundance in the Koziol for statistical advice and comments. current study, such as the glutamate receptor g2 subunit and the Ca þ 2 channel a1A subunit, and many of which could not be detected by Northern blot References analysis and might not have been detected by microarrays. Secondly, TOGAs is not limited by the 1 Lewis DA, Lieberman JA. Catching up on schizophrenia: natural history and neurobiology. Neuron 2000; 28: 325–334. current state of knowledge, which can restrict the 2 Kerwin R, Taylor D. 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