RESEARCHARTI CLE Open AccessOlanzapine induced DNA methylation changessupport the dopamine hypothesis of psychosisMelkaye G Melka1, Christina A Castellani1, Benjamin I Laufer1, N Rajakumar2, Richard OReilly2and Shiva M Singh1*AbstractBackground: The dopamine (DA) hypothesis of schizophrenia proposes the mental illness is caused by excessivetransmission of dopamine in selected brain regions. Multiple lines of evidence, including blockage of dopaminereceptors by antipsychotic drugs that are used to treat schizophrenia, support the hypothesis. However, thedopamine D2 receptor (DRD2) blockade cannot explain some important aspects of the therapeutic effect ofantipsychotic drugs. In this study, we hypothesized that antipsychotic drugs could affect the transcription of genesin the DA pathway by altering their epigenetic profile.Methods: To test this hypothesis, we examined the effect of olanzapine, a commonly used atypical antipsychoticdrug, on the DNA methylation status of genes from DA neurotransmission in the brain and liver of rats. GenomicDNA isolated from hippocampus, cerebellum, and liver of olanzapine treated (n = 2) and control (n = 2) rats wereanalyzed using rat specific methylation arrays.Results: Our results show that olanzapine causes methylation changes in genes encoding for DA receptors(dopamine D1 receptor, dopamine D2 receptor and dopamine D5 receptor), a DA transporter (solute carrier family 18member 2), a DA synthesis (differential display clone 8), and a DA metabolism (catechol-O-methyltransferase). Weassessed a total of 40 genes in the DA pathway and found 19 to be differentially methylated between olanzapinetreated and control rats. Most (17/19) genes showed an increase in methylation, in their promoter regions with insilico analysis strongly indicating a functional potential to suppress transcription in the brain.Conclusion: Our results suggest that chronic olanzapine may reduce DA activity by altering gene methylation. Itmay also explain the delayed therapeutic effect of antipsychotics, which occurs despite rapid dopamine blockade.Furthermore, given the common nature of epigenetic variation, this lends insight into the differential therapeuticresponse of psychotic patients who display adequate blockage of dopamine receptors.Keywords: Dopamine, Psychosis, Olanzapine, DNA methylation, EpigeneticsBackgroundThe cause of schizophrenia remains unclear, thoughmuch evidence suggests that it may be produced by anexcess transmission of dopamine (DA) in selectedbrain regions [1]. The DA hypothesis is supported by anumber of observations. First, there is evidence thatpsychosis is associated with a hyper-dopaminergicstate [2,3]. Second, all antipsychotic drugs block dopa-mine receptors, particularly D2 receptors [4]. Third,patients with schizophrenia have elevated D2 receptorsproducing a behavioral hypersensitivity to dopamine[4,5]. Furthermore, despitethedifficultiesof replicat-ing genome wide association studies and candidategene studies inschizophrenia, a number of genes inthedopaminepathway, includingdopamineD1recep-tor(DRD1), dopamineD2receptor(DRD2), dopamineD5receptor(DRD5), differential displayclone8(DDC),catechol-O-methyltransferase(COMT)andsolutecar-rier family 18member 2(SLC18A2), whichis alsoavesicularmonoaminetransporter, havebeenidentifiedasassociatedwithschizophrenia[6]. Thesegeneshavediverse functions, which include dopamine synthesis* Correspondence: ssingh@uwo.ca1Department of Biology, Molecular Genetics Unit, Western Science Centre,The University of Western Ontario, London, Ontario N6A 5B7, CanadaFull list of author information is available at the end of the articleJMP 2013 Melka et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the CreativeCommons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, andreproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedicationwaiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwisestated.Melka et al. Journal of Molecular Psychiatry 2013, 1:19http://www.jmolecularpsychiatry.com/content/1/1/19andrelease, receptoroccupancy, sensitivityof thedopa-mine receptors, and hyper-response of the receptor-signalling cascade [1]. Because all antipsychotic drugsblocktheD2receptor, it iswidelybelievedthat theD2blockade is central to the therapeutic efficacy of anti-psychoticdrugs. However, several empirical observationsseemtobeatoddswiththisassumption. First, whileD2blockadeoccurs withinseveral hours, it takes days andsometimes weeks for patients torespondtotreatmentwith antipsychotics [7]. Second, many patients showlimited or no therapeutic response despite markedblockadeoftheD2receptor[8]. Finally, somepatientsfail to respondto standarddoses of a specific anti-psychotic drug, which occurs despite adequate D2blockade, while interestingly the patient will respondto anequivalent(interms ofD2blockade)of aseconddrug [9,10]. These clinical observations are comple-mented by imaging studies, which show that anti-psychotic D2 blockade can only contribute a smallpercentageof thevariationinantipsychoticeffective-ness [11]. Epigenetic mechanisms, particularly DNAmethylation, which is responsive to environmental fac-tors [12,13], have the potential toalter the levels ofbrain molecules and may contribute to psychoticsymptomsandcognitivedeficitsinschizophrenia[14].Indeed, recentreportshaveidentifiedmethylationdif-ferencesinsomegenesimplicatedinschizophrenia, asreviewed by Dempster et al. [15]. We propose that anti-psychoticdrugsaffectthetranscriptionofgenesintheDApathway by altering their epigenetic profile. Wefurther hypothesizethat this alterationcontributes tothetherapeuticeffect of antipsychoticdrugs. Inordertotestthishypothesis, wehavedesignedthisstudytoassess the effect of a therapeutic dose of an anti-psychotic drug (olanzapine) onDNAmethylationintwoneural (hippocampus, cerebellum) andone non-neural (liver) control tissue in vivo.MethodsThe study useda rat model andgenome-wide DNAmethylationfollowing MethylatedDNAImmunopre-cipitation(MeDIP). Adult maleSpragueDawleyratsof12weeksofage(250-300g)werepurchasedfromCharles River, PQ, Canada. Upon arrival, rats wereseparated into individual cages and housed in con-trolledhumidityandtemperatureona12-hourlight/darkcycle (lights onat 7:00a.m.). The InstitutionalAnimal Care Committee of the University of WesternOntariohadapprovedallanimal-relatedproceduresusedinthisstudyfollowingtheCanadianandNational Insti-tuteof HealthGuides onanimal experimentation. Ani-mals were weighed and divided into two treatment groupswithcomparablemeans of weight. Their stress-inducedlocomotor activity (following a 5 min tail pinch) wasrecorded for 30 min using an automated open-field ac-tivity chamber (San Diego Instruments, San Diego,CA, USA). Acomputerthat detectsthedisruptionofphotocell beamsrecordedthenumberof beambreaksperfiveminutesforhalfanhouraseachanimal moves,andtheaveragebeambreaks/5minbins was reported.Theratsthenreceivedinjectionsofolanzapine(Zyprexa,Lilly, IN, USA; 2.5 mg/kg, i.m.; n = 8) or vehicle (phosphatebuffered saline (PBS); n = 8) between 1:30 pm and 3:00 pmdailyfor19days. Onday20, eighteenhoursafterthelast olanzapine/vehicle injection, rats were subjectedtostress-induced locomotor activity to assess the therapeuticefficacyofchronicolanzapine. Twenty-fourhoursafterthey were decapitated without anaesthesia, micro-punchesof brainareasorliversampleswerecollectedandflashfrozen.Genomic DNAisolated fromhippocampus, cerebel-lumandliverfromolanzapinetreated(n = 2) andcon-trol (n = 2) rats were analyzed using rat methylationarrays. ThegenomicDNAwasisolatedfromolanzapinetreated and saline control samples of hippocampus,cerebellum, and liver to analyze DNA methylationusingrat methylationarrays. GenomicDNAwasiso-lated from the interphase layer of TRIzol using sodiumcitrate, followedbyethanolprecipitationandpurifica-tionusing the QIAamp DNAMicro Kit (QIAGEN,Valencia, CA). DNA was then quantified using a Nano-Drop ND-1000 spectrophotometer (Thermo FisherScientificInc., Wilmington, DE) andall samples hadOD260/OD280 nmratios of 1.82.0andOD260/OD230nmratiosof 2.02.4. ThemethylatedDNAimmuno-precipitation(MeDIP), samplelabeling, hybridization,and processing were performed at Arraystar Inc.(Rockville, Maryland, USA). Data analysisinvolvedthecomparisonof differentiallyenrichedregionsbetweendrugexposed(E) andcontrol (C) rats, thelog2-ratiovalueswereaveragedandthenusedtocalculatetheMvalue [M = Average (log2 MeDIPE/InputE) - Average(log2MeDIPC/InputC)] for eachprobe. NimbleScansliding-windowpeak-findingalgorithmwasrunonthisdatatofindthedifferentialenrichmentpeaks(DEP). UsinganRscript program, a hierarchical clustering analysis wascompleted. The probe data matrix was obtained byusing PeakScores fromdifferentially methylated re-gions selectedby DEPanalysis. This analysis usedaPeakScore 2 to define the DEPs, which is equivalenttotheaveragep-value0.01, forall probeswithinthepeak. Thisanalysisdealswithatotal of 40candidategenesof thedopaminergicpathway. GeneList VennDiagramwasusedtoassessthedistributionof genesaffectedacross tissuetypes [16]. Transcriptionfactorbindingsitesof DRD5thatshowedincreasedmethyla-tion in hippocampus, cerebellum and liver were identi-fied using CTCFBSDB 2.0 [17].Melka et al. Journal of Molecular Psychiatry 2013, 1:19 Page 2 of 7http://www.jmolecularpsychiatry.com/content/1/1/19ResultsStress-inducedlocomotor activity was significantly de-creased (p = 0.001) inolanzapine treated rats 21 dayspost-stress (32.6 4.4beambreaks/5minbins) as com-pared to that of matched-control (85.6 7.3 beam breaks/5 min bins) (Table 1). Further, Table 2 shows 19 of the 40genes that were differentially methylated(p < 0.01) as aresult of olanzapinetreatment inthethreetissues (hip-pocampus, cerebellumandliver) studied. Themajorfea-turesof theseresultsarefourfold. First, themethylationchanges are specific to the promoter regions of the genes.Second, most changes represent an increase in methylationas a result of olanzapine treatment. Third, the methylationchanges are tissue (hippocampus, cerebellum, andliver)specific. Fourthandfinally, most changesarespecifictothehippocampus (Figure1). For example, 15of the19genesthatshowedsignificantincreaseinmethylationarespecific to hippocampus of olanzapine treated as comparedtothematchedcontrol rats(Table2). Also, 3out of 40genes showedsignificant increase (DRD5, SLC18A2andDDC8)andone(ATP2A2)showedsignificantdecreaseinmethylationinthecerebellumof olanzapinetreatedrats(Table 2). Furthermore, 5 of the 40 genes were diffe-rentially methylatedinolanzapine treatedliver as com-paredtothematched-controls(Table2). Theseincludedthe DA receptor genes (DRD1, DRD2 and DRD5),PPP3CAandCAMKK2. Also, onegene, COMT, showeddecreased methylation in olanzapine treated liver samples.Overall, anumberofgenesrelatedtoDAfunctioningaredifferentiallymethylatedas aconsequenceof olanzapinetreatmentinvivo. Theyaretissue(hippocampus)specificwithonly2ofthe19genesaffectedinhippocampusandliver(PPP3CAandCAMKK2); withone(DRD5)affectedin all three tissues in the same direction (Figure 1).Furthermore, DARPP32, ADCY3 (AC3), DA receptor genesand a DA transporter gene (SLC18A2/VMAT2) weresignifiedintheDAreceptorsignallingpathwayamongstgenes that showed significant increases in methylation fol-lowing olanzapine treatment (Figure 2). Also, we assessedgene-specific sequences that were methylated in the olan-zapine treated samples. The region of DRD5 that was dif-ferentiallymethylated following olanzapine treatmenthasthe necessary features of a functioning promoter, as illus-trated by the identified CTCF transcription factor bindingsites (Figure 3).DiscussionThe significant increase in weight gain of olanzapinetreatedanimals inthepresent studyandinpreviouslyreported studies [18] has suggested that the paradigmadapted was capable of causing the metabolic disturbancesobserved in patients taking chronic olanzapine [19].Furthermore, thesignificantlyreducedlocomotoractivityof olanzapine treated rats indicated the therapeuticefficacyof the drug administered, which was comparableto the dosage appliedinprevious studies [20]. Thisargument was further supported by the observed changesin methylation of DA pathway genes.Of special interest to this report are the genes involvedindopamine synthesis, transport, receptor, metabolism,interaction, andfunction[6]. Therationaleforthisfocusstemmed from the fact that although antipsychotics inter-act with some dopamine receptors (D2), the actual mech-anism of clinical effect behind antipsychotic efficacy in thetreatment of psychosis isnot fullyunderstood. What ismissing from the dopamine hypothesis of psychosis is theunderstanding of the underlying molecular mechanism(s)that may begin to reveal the full spectrum of the antipsy-chotics effects. Specifically, the results of the present studysuggestaninvolvementofDNAmethylationingenesofthe dopamine pathway as an essential epigenetic mechan-ismintreatingpsychosis [21]. Our results showedthatolanzapinecausesanincreaseinDNAmethylationinasignificant (~40%) number of genes withanimportantrole in dopamine neurotransmission. These include genesinvolvedinthedopaminesynthesis, transport, receptor,andmetabolism(Figure2). Further, themajorityof DApathway genes affected by olanzapine treatment werefound to be hippocampus-specific, which is viewed as oneoftheprimarysitesforschizophreniasymptoms[21-23].Also, anumberof genesidentifiedinthecurrent studyhave been previously implicated in schizophrenia [24-27].This increase in methylation in the DA pathway candidategenes is expected to interfere with transcription and sup-press the functional gene product [28]. We also assessed ifthe methylated regions of individual genes have the poten-tialtointerferewithtranscription. Theresultsarguethatgene-specific differentially methylated regions (DMRs)have necessary features of active promoters. All the affectedDApathwaygenesweredifferentiallymethylatedintheirpromoter regions and therefore could result in altered geneTable 1 Locomotor activity (average number of beam breaks/5 minutes stderr) of olanzapine treated rats and theirmatched-controls during day 0 and 21 as measured baseline (before stress) and post-stress (n = 8 per each group)Day 0 baseline Day 0 post-stress Day 21 baseline Day 21 post-stressControl 22.0 2.64 101.9 4.00 17.1 2.47 85.6 7.28Olanzapine 22.0 2.65 94.7 6.54 16.9 2.67 32.6 4.42p-values 1 0.452 0.973 0.001T-test was used to compute the significance of differences between olanzapine treated and control groups.Melka et al. Journal of Molecular Psychiatry 2013, 1:19 Page 3 of 7http://www.jmolecularpsychiatry.com/content/1/1/19expression[29]. Forexample, thepromoterregionoftheDRD5 gene is differentially methylatedin all three tissues.Thisgenehasbeenimplicatedincognitivefunctionsthatinclude working memory [30]. Furthermore, DRD1andDRD5 have been reported to have distinct regulatory roleson synaptic plasticity, spontaneous motor activity, memoryandtheinformationbeingprocessedbythehippocampus[31,32]. HereweshowthatDRD5, whichencodestheD5subtype DA receptor, and has been previously described asasusceptibilitygeneforschizophrenia[33,34], mayresultindiminishedD5subtypebyincreaseinmethylationfol-lowingolanzapinetreatment. Thisisexpectedinallthreetissuesstudied. ItisalsoknownthatDRD5interactswithDRD2 in the process of augmenting or suppressing cellularfunctions [35]. Further, the DRD5 gene region differentiallymethylated in response to olanzapine is compatible withmethylation specific interference of transcription, as wehave identifiedinsilicopredictedCTCFtranscriptionTable 2 Candidate genes of the dopamine pathway, showing significantly increased methylation (p < 0.01) followingolanzapine treatment in hippocampus, cerebellum and liver, in a rat model in vivoGene name Accession Chr. Strand TSS TTS EH-CH EC-CC EL-CLDrd5 NM_012768 chr14 - 77769487 77768059 1 1 1Camkk2 NM_031338 chr12 + 34772493 34938479 1 1Calm3 NM_012518 chr1 - 77252717 77245608 1Kcnj2 NM_017296 chr10 + 100574984 100576268 1Kcnj4 NM_053870 chr7 - 117603827 117601722 1Cacna1c NM_012517 chr4 - 155517389 154895690 1Ppp3ca NM_017041 chr2 + 234130175 234409232 1 1Adcy3 NM_130779 chr6 + 27118324 27202275 1Plcd1 NM_017035 chr8 - 124052193 124023089 1Ppp2r2d NM_144746 chr1 + 198640770 198674758 1Ppp1r1b NM_138521 chr10 + 87121888 87131587 1Nos1 NM_052799 chr12 - 39869484 39811720 1Itpr1 NM_001007235 chr4 + 143705359 144030051 1Ppp1r14b NM_172045 chr1 + 209648656 209650767 1Crem NM_001110860 chr17 + 62770632 62837670 1Slc18a2 NM_013031 chr1 + 265789916 265824551 1Ddc8 NM_001017481 chr10 - 108349394 108336343 1Drd2 NM_012547 chr8 + 52641159 52707749 1Drd1a NM_012546 chr17 + 16655925 16658161 1Chr chromosome, TSS transcription start site, TTS transcription termination site, EH-CH, EC-CC, EL-CL the number of differentially enriched peaks thatshowed increased in methylation in Olanzapine treated experimental (E) rats as compared to matched-controls (C) in hippocampus (H), cerebellum (C)and liver (L), respectively.Adcy3Cacna1cCalm3CremItpr1Kcnj2Kcnj4Nos1Plcd1Ppp1r14bPpp1r1bPpp2r2dGenes showing increased methylation in hippocampus onlyDdc8Slc18a2Genes showing increased methylation in cerebellum onlyDrd1aDrd2Genes showing increased methylation inliver onlyDrd5Genes showing increased methylation in hippocampus, cerebellum and liverGene showing increased methylation in hippocampus and liverCamkk2Ppp3caFigure 1 Genes showing increased methylation in hippocampus, cerebellum and liver, following Olanzapine treatment. DRD5 was theonly candidate gene that was found to be affected by Olanzapine treatment in both brain regions and the liver.Melka et al. Journal of Molecular Psychiatry 2013, 1:19 Page 4 of 7http://www.jmolecularpsychiatry.com/content/1/1/19factorbindingsites(Figure3) that warrant furthercon-firmation. Similarly, the expression of DRD2 could be reg-ulated through methylation or demethylation of cytosinesat theputative promoter region of DRD2 [36-38]. Thus,the results included in this report offer a unifying mechan-ism of DNA methylation, which may represent the molecu-lar basis for response to olanzapine, in a manner where thedrug affects transcription of candidate genes fromthedopamine pathway in addition to its effect on D2 blockade.This suggests that alterations to DNA methylation, in par-ticular, and epigenetic changes, in general, may be used todevelopnovel strategies for thetreatment of psychosis.We acknowledge the added value of confirming themethylation changes in the promoter regions of DA genesusinganadditional techniquepossiblyinvolvingalargernumber of rats in our future study. Further, we intend toexplore the expression of mRNA and proteins of relevantgenes that are affected by DNAmethylationso as toinvestigate the efficacy of olanzapine in treating psychosisvia altering methylation status. Various confoundingfactors suchas gene-diet/drug interactions couldaffectmethylationchanges[12,39,40]. Therefore, inthepresentPresynaptic terminal Postsynaptic terminalReceptor bindingDRD1LDRD2LDRD3DRD4DRD5HCLNorepinephrineTyrosine DopamineSynthesisTHDDCCTransporterSLC6A3SLC6A2SLC18A1SLC18A2CDBHMetabolismMAOAMAOBCOMTLGenes involved in DA pathway with unknown functions GRIN3A, PKC (PRKCG), AMP(TMPRSS5), GMP (GMPS), PKG, ATF1, CBP, CAMK4, CALY, PIP2, CAMKK2HL, CALM3H, KCNJ2H, KCNJ4H, CACNA1CH, PPP3CAH, ADCY3H, PLCD1H, PPP2R2DH, PPP1R1BH(DARPP32)H, NOS1H, ITPR1H, PPP1R14BH, CREMH, ATP2A2CFigure 2 Candidate genes in the dopaminergic pathway (adapted and modified from Yeh et al. Brain and Cognition 2012; 80: 282289). Superscripts (H, C and L) represent the genes that showed increased methylation in hippocampus, cerebellum, or liver, respectively. Geneswithout a superscript were not affected by olanzapine treatment. Subscript (C) represents the gene that showed reduced methylationin the cerebellum.ABFigure 3 The genomic location and promoter region of the dopamine D5 receptor gene (DRD5). (A) Chromosomal position of the DRD5(from http://www.ensembl.org/index.html), which showed significantly increased methylation (p < 0.01) following olanzapine treatment. (B) Sequence ofthe promoter region of DRD5, with CTCF transcription factor binding sites highlighted in blue (identified using CTCFBSDB version 2.0 by Bao et al.Nucleic Acids Research 2008; 36, D83-D87). The frequency of A/C/G/T in the promoter sequence was 0.174/0.308/0.370/0.148, respectively. The CpGcount in the promoter region was 50 out of 250 bp.Melka et al. Journal of Molecular Psychiatry 2013, 1:19 Page 5 of 7http://www.jmolecularpsychiatry.com/content/1/1/19study, propercautionwastakentoexcludeconfoundingfactors that couldpossiblyleadtomethylationchanges.For example, all experimental animals were kept in a uni-formenvironmentandwerenotexposedtootherdrugsor environments including diets, which may contribute todifferences inmethylationstatus of the treatment andcontrolgroups. Allratswereofthesamebreed, sex, ageand comparable body weight. They showed similar signifi-cant effects fromolanzapine onweight gainandloco-motor activity.ConclusionsThe results of the present study suggest that chronicolanzapinemayreduceDAactivityinthelong-termbyalteringgene methylation. Furthermore, olanzapine in-ducedgenemethylationmayexplainthedelayedthera-peuticeffectof thedrug, whichoccursdespitetherapiddopamine blockade and differential therapeutic re-sponses of psychotic patients showing adequate D2blockade.Competing interestsThe authors declare that they have no competing interests.Authors contributionsMGM: participated in designing this study, analyzed and interpreted the data,wrote the first draft and revised the manuscript. CAC and BIL revised themanuscript for critical intellectual contents. RNR and RO: participated from thevery conception and design of study to revising the manuscript for criticalintellectual contents. SMS: conceived and designed the study, interpreted thedata, critically reviewed the manuscript for important intellectual contents. Allauthors have read and approved the manuscript as submitted.AcknowledgementsThis research was supported by grants from the Canadian Institute of HealthResearch and Dr. Shiva M. Singh held a Senior Research Fellowship from theOntario Mental Health Foundation during the course of this study.Author details1Department of Biology, Molecular Genetics Unit, Western Science Centre,The University of Western Ontario, London, Ontario N6A 5B7, Canada.2Department of Psychiatry, The University of Western Ontario, London,Ontario N6A 5B7, Canada.Received: 6 September 2013 Accepted: 16 October 2013Published: 4 November 2013References1. Madras BK: History of the discovery of the antipsychotic dopamine D2receptor: a basis for the dopamine hypothesis of schizophrenia.J Hist Neurosci 2013, 22:6278.2. 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Singh S, Li SS: Epigenetic effects of environmental chemicals bisphenol aand phthalates. Int J Mol Sci 2012, 13:1014310153.doi:10.1186/2049-9256-1-19Cite this article as: Melka et al.: Olanzapine induced DNA methylationchanges support the dopamine hypothesis of psychosis. Journal ofMolecular Psychiatry 2013 1:19.Submit your next manuscript to BioMed Centraland take full advantage of: Convenient online submission Thorough peer review No space constraints or color gure charges Immediate publication on acceptance Inclusion in PubMed, CAS, Scopus and Google Scholar Research which is freely available for redistributionSubmit your manuscript at www.biomedcentral.com/submitMelka et al. Journal of Molecular Psychiatry 2013, 1:19 Page 7 of 7http://www.jmolecularpsychiatry.com/content/1/1/19