Sgadòetal.MolecularAutism2013,4:51 http://www.molecularautism.com/content/4/1/51 RESEARCH Open Access Transcriptome profiling in engrailed-2 mutant mice reveals common molecular pathways associated with autism spectrum disorders Paola Sgadò1*†, Giovanni Provenzano1†, Erik Dassi2, Valentina Adami3, Giulia Zunino1, Sacha Genovesi1, Simona Casarosa4,5 and Yuri Bozzi1,5 Abstract Background: Transcriptome analysis has been used in autism spectrumdisorder(ASD) to unravel common pathogenic pathways based onthe assumption thatdistinct rare genetic variants or epigenetic modifications affect common biologicalpathways.To unravel recurrent ASD-related neuropathological mechanisms, wetook advantage ofthe En2−/−mouse model and performed transcriptome profiling on cerebellar and hippocampal adult tissues. Methods: Cerebellar and hippocampal tissue samples from three En2−/−and wild type(WT) littermate mice were assessed for differential gene expression using microarray hybridization followed by RankProd analysis. To identify functional categoriesoverrepresented inthedifferentially expressed genes, weused integrated gene-network analysis, geneontology enrichmentand mousephenotype ontology analysis. Furthermore, weperformed direct enrichment analysis of ASD-associated genes from theSFARIrepository inour differentially expressed genes. Results: Given thelimited numberof animals used inthe study, weused permissive criteria and identified 842 differentially expressed genes inEn2−/− cerebellum and 862 inthe En2−/−hippocampus. Our functional analysis revealed thatthe molecular signature ofEn2−/−cerebellum and hippocampus sharesconvergent pathological pathways with ASD, including abnormal synaptic transmission, altered developmental processes and increased immune response. Furthermore, when directly compared to therepository of theSFARI database, our differentially expressed genes in thehippocampus showed enrichment of ASD-associated genes significantly higher than previously reported.qPCR was performed for representative genes to confirm relative transcript levels compared to thosedetectedin microarrays. Conclusions: Despite the limited numberof animals used inthe study, our bioinformatic analysis indicates the En2−/− mouse is a valuable tool for investigating molecular alterations related to ASD. Keywords: En2,Neurodevelopmentaldisorders,Mousemodels,Immuneresponse,Synapticfunction,Scn1a,Grm5,Nrxn3 Background neuroimagingandneuropathologicalstudies[1-3].The Autism spectrum disorder (ASD) defines a complex most evident abnormality in ASD consists in an early groupofneurodevelopmentaldisabilitiescharacterizedbya (perinatal)brainovergrowthfollowedbyanarrestofgrowth wide range of impairments in social and communicative during the first year of age [4]. Neuropathological studies skills, stereotyped behaviors, and restricted mental flexi- onpost-mortemsamplesfromASDpatientsalsoshoweda bility [1]. The neurodevelopmental and neuroanatomical number of cellular and cytoarchitectural abnormalities at basesofASDhavebeenconfirmedbyanumberofclinical, the level of the cerebral cortex, cerebellum, amygdala and forebrain limbic structures. Anomalies in the cerebellum are the most reproducible neuropathological alterations in *Correspondence:sgado@science.unitn.it ASDpatients[3,5]. †Equalcontributors 1LaboratoryofMolecularNeuropathology,CentreforIntegrativeBiology A large series of evidence clearly indicates that neuro- (CIBIO),UniversityofTrento,ViadelleRegole101,38123Trento,Italy pathologicalfeaturesandbehavioraldeficitsofASDhave Fulllistofauthorinformationisavailableattheendofthearticle ©2013Sgadòetal.;licenseeBioMedCentralLtd.ThisisanopenaccessarticledistributedunderthetermsoftheCreative CommonsAttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse,distribution,and reproductioninanymedium,providedtheoriginalworkisproperlycited. Sgadòetal.MolecularAutism2013,4:51 Page2of12 http://www.molecularautism.com/content/4/1/51 a primarily genetic origin. However, the etiology of ASD expressed genes associated to ASD in this mouse model remainsessentiallyunknown[6,7].Transcriptomeanalysis ofthedisease. hasalsobeenusedtounravelcommonpathogenicpath- ways based on the assumption that distinct rare genetic Methods variants or epigenetic modifications affect common bio- Animals logical pathways dysregulated in ASD [6]. Several studies Experiments were conducted in conformity with the have analyzed genome-wide expression profiles of ASD EuropeanCommunitiesCouncilDirectiveof24November patients using lymphoblastoid cell lines and blood sam- 1986 (86/609/EEC) and were approved by the Italian ples,supportingupregulationofimmunegenesanddown- MinistryofHealthandEthicsCommitteeoftheUniversity regulation of neurodevelopmental genes as key players in ofTrento.Animalswerehousedina12hrlight/darkcycle the pathogenesis of ASD (see [8] for a review). Recently, with food and water available ad libitum. All surgery was geneco-expressionnetworkanalysisofautisticbrainareas performed under chloral hydrate anesthesia, and all efforts revealed defects in cortical patterning and an enrichment were made to minimize suffering. The generation of En2−/− ofdifferentiallyexpressedgenesassociatedwithASD[9]. micewaspreviouslydescribed[20].TheoriginalEn2mutants Thehomeobox-containingtranscriptionfactorengrailed-2 (mixed 129Sv x C57BL/6 and outbred genetic background) (En2) is crucially involved in the regionalization, patterning werecrossedatleastfivetimesintoaC57BL/6background. and neuronal differentiation of the midbrain and hindbrain Heterozygousmatings(En2+/−xEn2+/−)wereusedtogener- [10-15].Humanstudiesindicatedassociationoftwointronic ate the En2+/+ (wild type, WT) and En2−/− littermates used single-nucleotide polymorphisms (SNPs) in the human inthisstudy.PCRgenotypingwasperformedaccordingto engrailed-2 (EN2) gene with ASD [16,17]. Furthermore, the protocol available at the Jackson Laboratory website the ASD associated A-C haplotype markedly affected (www.jax.org; mouse strain En2tm1Alj). WT and En2−/− EN2 promoter activity when tested with a luciferase re- age-matchedadult(3to5monthsold;weight=25to35g) porter assay in rat, mouse and human cell lines [18]. A littermatesmiceofbothsexeswereused. recentstudyoftheepigeneticevaluationofEN2inpost- mortem cerebellar samples from autistic patients indi- Microarrays cated a persistent upregulation of this homeobox gene RNAsfromdissectedhippocampiandcerebellafromthree inducedbyepigeneticabnormalitiesin histonemethyla- adult mice for each genotype were purified using standard tionpatternsthatmaycontributetoPurkinjecelllossin columnpurificationaccordingtothemanufacturer’sproto- someindividualswithautism[19]. col (RNAeasy Mini Kit, Qiagen, USA). RNA quality was Mice lacking the homeobox domain of En2 (En2hd/hd analyzed by microfluidic gel electrophoresis on RNA 6000 mice;[20],referredtoas En2−/−)havebeenproposed asa NanoChips using the Agilent 2100 Bioanalyzer. Only RNA model for ASD, due to their complex neuroanatomical with a high (>9) RNA integrity number was selected and and behavioral phenotype. En2−/− mice display cerebellar used for subsequent retro-transcription, labeling and array hypoplasia, including a reduced number of Purkinje cells, hybridization according to Agilent protocols. Mouse gene and a defect in the antero-posterior pattern of cerebellar expression arrays (Agilent 4X44K slides) were hybridized foliation[20-23].ThebehaviorofEn2−/−miceisalsorem- andscannedwiththeAgilentmicroarraystation. iniscent of some features of ASD individuals. Deficits in social behaviors were detected in En2−/− mice, including Bioinformatics decreased playand reduced socialinteractions;locomotor IntensityvalueswereprocessedwithAgi4x44PreProcess impairment,aswellasdefectivespatiallearningandmem- (http://bioconductor.org/packages/2.12/bioc/html/Agi4x44 ory,wasalsoreportedinthesemice[24-26].Furthermore, PreProcess.html) using default parameters to remove low- we reported dysfunctions in GABAergic interneurons in quality probes. Signals were then normalized by means of adultEn2−/−miceanddemonstratedengrailedproteinex- thequantilenormalizationmethod.Toevaluatedifferential pression in specific subpopulations of adult hippocampal expression, we used RankProd (http://www.bioconductor. andcorticalinterneurons[27]. org/packages/2.11/bioc/html/RankProd.html)[28].RankProd To unravel recurrent ASD-related neuropathological utilizes the Rank Product (RP) non-parametric method [29] mechanisms, we took advantage of the En2−/− mouse toidentifyup-ordownregulatedgenes.TheRPisequivalent modelandperformedgenome-wideexpressionprofiling tocalculatingthegeometric meanrankwitha statistical on cerebellar and hippocampal adult tissues. Our tran- method (average rank) that is slightly more sensitive to scriptome analysis of the cerebellum and hippocampus outlier data and puts a higher premium on consistency ofEn2−/−micesuggestsconvergentpathologicalpathways between the ranks in various lists. To assess for functional with ASD, including abnormal synaptic transmission categories overrepresented in the differentially expressed andincreasedimmuneresponse.Furthermore,weprovide genes, we used DAVID (http://david.abcc.ncifcrf.gov) and evidence for a significant enrichment of differentially IngenuityPathwayAnalysis(IngenuitySystems,Inc.,USA). Sgadòetal.MolecularAutism2013,4:51 Page3of12 http://www.molecularautism.com/content/4/1/51 To focus the functional analysis on brain expressed genes ventral-basal temporal region, both of which are involved weused,asbackgroundforourfunctionalanalyses,alistof indecodingsocialstimuliandthereforeareassociatedwith tissue specific ‘expressed genes’ for both the cerebellum the social deficits [32-34]. Most importantly, we previously and the hippocampus. Our ‘expressed genes’ listswere ob- showedanatomicaldefectsintheEn2−/−hippocampusthat tained by filtering the genes by the normalized expression might contribute to the behavioral deficits displayed by values and excluding the ones with the lowest expression thesemutants[27].Wethereforecombinedthecerebellar levels (<10th percentile), and include 13,652 genes for the gene expression profile to that of the hippocampus. cerebellumand13,141forthehippocampus.Thehypergeo- We found 862 differentially expressed genes in the hippo- metric test and the Student’s t-test were computed with R campus, among those 378 were upregulated and 484 were (http://www.r-project.org). downregulatedinEn2−/−micecomparedtotheirlittermate controls.Additionalfile2showstheentirelistofgenes QuantitativePCR differentiallyexpressedinthecerebellumandhippocampus TotalRNAswereextractedbyTrizol™reagent(Invitrogen ofEn2−/−micewiththedifferentialexpressionP valueand Life Technologies, USA) from dissected hippocampi and the percentage of false prediction (pfp) value calculated by cerebellafromfourWTandfourEn2−/−adultmice.RNAs RankProd.Giventheprofoundstructuralandcytoarchitec- were DNAse-treated and purified with RNeasy Mini turalphenotypeoftheEn2−/−cerebellumandthevariability Kit(Qiagen,USA).cDNAwassynthesizedfrompooled of the phenotype among individuals, we chose to be more RNAs(2 μg) using theSuperScript™ VILO™ (Invitrogen permissive and include genes with smaller differential Life Technologies, USA) according to the manufacturer’s expressionfold changesapplyingamoderate cutofftothe instructions. Individual PCR reactions were conducted in uncorrected P value [see Additional file 2]. Differentially a volume of 20 μl using the KAPA FAST SYBR qPCR kit expressed genes, which are common in the hippocampus (KAPABiosystems, USA) according to manufacturer’s andthecerebellum,aresummarizedinFigure1andlisted instructions. Mouse mitochondrial ribosomal protein in Additional file 3. Remarkably, En2 was not among the L41 (Mrpl41) was used as a standard for quantification differentially expressed genes. The microarray probe for as previously shown [30]. Primers (MWG, Germany) En2(A_51_P397876)wasdesignedonthe3′untranslated were designed on different exons to avoid amplification of region of the gene that is not deleted in the En2−/− locus genomic DNA. A list of primer sequences is reported in [20].Ourdataconfirmthepreviousreportsindicatingthe Additionalfile1.EachPCRcycleconsistedofdenaturation presenceofaresidualtranscriptfromtheEn2−/−locus[20]. for 10 s at 94°C, annealing for 20 s at 60°C and extension Furthermore, real-time quantitative PCR (qPCR) ana- for30sat72°C.ThefluorescenceintensityofSYBRgreenI lysisofEn2expressionusinghomeoboxspecificprimers was read and acquired at 72°C after completion of the ex- revealed no expression of the full-length gene in En2−/− tension step of each cycle. PCR conditions for individual mice[seeAdditionalfile4]. primersetswereoptimizedbyvaryingtemplatecDNAand primerconcentrationinordertoobtainasinglePCRprod- Functionalanalysis uct and amplification efficiency >90%. Relative expression To explore the biological processes most relevant to the valueswerecalculatedusingthePfafflmethod[31]. phenotype of the En2−/− mice, the differentially expressed Results Differentialgeneexpressionincerebellumand hippocampusofEn2−/−mice The cerebellum of En2−/− mice shows Purkinje cell loss and structural abnormalities resembling the neuropathological features observed in ASD patients [20,21,23]. To identifygenesandpathwaysalteredinEn2−/−mice,weini- tially performed transcriptome profiling of the whole cerebellar tissue. En2−/− and WT cerebella from adult mice wereassessedfordifferentialgeneexpressionbymicroarray and bioinformatical analysis (see Methods). We found 842 differentially expressed genes in the cerebellum of En2−/− Figure1Venndiagramofdifferentiallyexpressedgenesin mice compared to their control littermates. Among these, En2−/−adultcerebellumandhippocampus.Atotalof842and 407 and 435 were up- and downregulated, respectively. 862differentiallyexpressedgeneswereidentifiedintheadult Alterations in limbic structures have also been shown in cerebellum(CB,blue)andhippocampus(Hippo,red)ofEn2−/− mice,respectively.Amongthese,238showdifferentialexpression thetemporallobesofautisticpatients.Themainabnormal- inbothtissues. ities were shown in the superior temporal sulcus and the Sgadòetal.MolecularAutism2013,4:51 Page4of12 http://www.molecularautism.com/content/4/1/51 geneswereanalyzedthroughintegratedgene-networkana- cellularcomponentssynapse,synapticvesicleandneuronal lysis using the curated Ingenuity Pathway Analysis (IPA) projection. Within the biological processes, the most database and the Database for Annotation, Visualization representedtermswere‘neuronalactivities’and‘calcium and Integrated Discovery(DAVID).Additional file5shows mediated signal’. Interestingly, the keyword ‘phospho- themostsignificantlyenricheddiseaseandcellularfunction protein’ was significantly enriched, suggesting a generic categories obtained with IPA and gene ontology analysis. alteration in the protein phosphorylation state in the Functional categories included increased seizure and En2−/−hippocampus. decreased neurotransmission release in the hippocampus, To assess the functional consequences of En2 ablation, and decreased cancer-related diseases and development we analyzed enrichment of the differentially expressed of lymphocytes as cellular function in the cerebellum. genesbasedonthemousephenotypefromtheMammalian To identify statistically significant over-representation of Phenotype Ontology (MPO) project [35], using ToppGene key neurobiological processes, functional annotation ana- [36]. In the En2−/− cerebellum the only significantly lysiswasperformedwithDAVIDusingatissuespecificlist enriched mouse phenotype was ‘loss of dopaminergic of ‘expressedgenes’asbackground(seeMethods).Tover- neurons’, a term associated with the cerebellar phenotype ify the tissue-expression pattern of the samples, we first bytheroleofEn2inmidbrain/hindbraindevelopmentand classified the differentially expressed genes based on their dopaminergic neuron survival [37,38]. For the hippocam- tissue expression (P <0.05, calculated using Benjamini pus we found significant enrichment for terms related to multiple testing correction). We only found significant seizureandalteredsynaptictransmission(Figure4).These tissue expression terms for En2−/− hippocampus show- data are in accordance with our previously described in- ingsignificantenrichmentinexpressionofgenesrelated crease in seizure susceptibility and decrease in GABAergic to‘braincortex’,‘brain’,‘hypothalamus’,‘eye’and‘hippocam- neuronsubpopulationsinEn2−/−mice[27,30]. pus’[seeAdditionalfile6].Wethenanalyzedthefunctional Remarkably, when analyzed specifically for ASD- annotationusinggenesetsfromthegeneontology(GO) associatedgenes,thedifferentiallyexpressedgenesshowed public databases and our ‘expressed genes’ list as back- significant over-representation ofknown ASDsusceptibil- ground. Figure 2 shows all GO terms enriched in the ity genes when compared directly to the repository of the differentially expressed genes of En2−/− cerebellum with SimonsFoundationandAutismResearchInitiative(SFARI) thegenecountsandtherelativePvalue(P<0.05,calculated database (gene.sfari.org). Since the SFARI database com- using Benjamini multiple testing correction). Among prisesgenesrelatedtoneurodevelopmentaldisorders,there- the most represented functional categories were several fore expressed in the brain, we filtered the SFARI database terms related to the major histocompatibility complex genelistwithourlistof ‘expressedgenes’inthecerebellum (MHC)-mediated immunity and immune response. The andinthehippocampus(seeMethodsfordetails).Weused GOtermsrelatedtotheEn2−/−hippocampusareshown these SFARI gene lists to calculate enrichment with our inFigure3withthegenecountsandtherelativePvalue differentially expressed genes. Table 1 shows the list of (P <0.05, calculated using Benjamini multiple testing the ASD-associated differentially expressed genesforthe correction). Of the enriched terms, many related to the cerebellum and the hippocampus. To compute significant P-value 0.00 0.01 0.02 0.03 0.04 0.05 major histocompatibility complex antigen MHC class I-like antigen recognition MHC protein complex MHC class I, alpha chain, alpha1 and alpha2 MHC I-mediated immunity plasma membrane part antigen processing and presentation P value cell surface Gene counts external side of plasma membrane IPNATNETRHPERRO_MOLECULAR FUNCTION immunoglobulin C-type GOTERM_CELLULAR COMPONENT immunoglobulin C1-set GPAONTTEHREMR__BBIOIOLLOOGGICICAALL P PRROOCCEESSSS graft-versus-host disease SMART KEGG_PATHWAY allograft rejection SP_KIR_KEYWORDS immune response 0 20 40 60 80 100 gene counts Figure2OverrepresentedgeneontologycategoriesfordifferentiallyexpressedgenesintheEn2−/−adultcerebellum.Differentially expressedgenes(n=842)wereanalyzedforenrichmentingeneontologycategoriesusingDAVIDwithaBenjaminicorrectedPvaluecutoffof 0.05.Categoriesarearrangedfrommostsignificantanddownwards(grayline),foreachcategorythenumberofgenesisindicatedbythelength ofthehorizontalbars(genecounts).Tohighlightdistinctontologicalcategories,barsarecolor-codedasindicatedintheinsettothefigure.Data detailsarealsoreportedinAdditionalfile5. Sgadòetal.MolecularAutism2013,4:51 Page5of12 http://www.molecularautism.com/content/4/1/51 P-value 0.00 0.01 0.02 0.03 0.04 0.05 synapse voltage-gated channel plasma membrane plasma membrane part clathrin-coated vesicle cell junction cell projection synaptic vesicle cytoskeletal protein actin-binding P value neuron projection Gene counts voltage-gated ion channel GOTERM_CELLULAR COMPONENT SP_KIR_KEYWORDS neuronal activities PANTHER_MOLECULAR FUNCTION voltage-gated sodium channel PANTHER_BIOLOGICAL PROCESS cortical cytoskeleton KEGG_PATHWAY potassium channel potassium transport coated vesicle extrinsic to membrane dendrite vascular smooth muscle contraction melanogenesis phosphoprotein tight junction alternative splicing cytoskeleton cell cortex part calmodulin-binding calcium mediated signaling 0 50 100 150 200 250 300 350 gene counts Figure3OverrepresentedgeneontologycategoriesfordifferentiallyexpressedgenesintheEn2−/−adulthippocampus.Differentially expressedgenes(n=862)wereanalyzedforenrichmentingeneontologycategoriesusingpublicdatabaseswithaBenjaminicorrectedPvalue cutoffof0.05.FurtherdetailsregardingthefigureandinsetaredescribedinthelegendtoFigure2.DatadetailsarealsoreportedinAdditionalfile5. enrichment between our differentially expressed genes and separated in tissue-specific groups, and the comparison theSFARIgenes,weemployedthehypergeometrictest.The betweenthestudies.ForVoineaguetal.[9]were-analyzed statistical analysis indicated significant enrichment only for thecerebellumdatausingtheGEO2Rtoolwithdefaultpa- the hippocampus (P <0.05), whereas no significant enrich- rametersandusedtheseresultstoevaluatethecorrespond- mentwasobservedforthecerebellum. encewith our study. The results for cerebellum show a Tocompareour findingswiththethreemajor genome- significant enrichment with ASD-associated genes only for wide expression studies on ASD brain tissue [9,39,40],we the Voineagu et al. study (4.75% enrichment, P=0.0343). matched the publicly available differentially expressed Ourstudy,however,wastheonlyonetodisplaysignificant genes with the same ASD-related gene lists that we enrichment of ASD-related genes (4.24% enrichment, used for our analysis. To increase accuracy, we computed, p=0.0265)inthelimbicregions. for each study, the hypergeometric test and obtained an To validate microarray findings with qPCR analysis, we enrichment P value that we used for direct comparison. selected eight and eighteen representative genes from the Table2summarizestheresultsoftheenrichmentanalyses, cerebellum and the hippocampus differentially expressed P value Gene counts MOUSE PHENOTYPE P-value 0.00 0.01 0.02 0.03 0.04 0.05 abnormal CNS synaptic transmission abnormal synaptic transmission seizures abnormal excitatory postsynaptic currents 0 10 20 30 40 50 Gene counts Figure4MousephenotypecategoriesassociatedwithdifferentiallyexpressedgenesintheEn2−/−hippocampus.Differentiallyexpressed genesinEn2−/−hippocampuswereanalyzedforenrichmentinmousephenotypesusingToppGenewithacorrectedPvaluecutoffof0.05. Categoriesarearrangedfrommostsignificantanddownwards(grayline);numberofgenesinthecategoryisindicatedbythelengthofthe horizontalbars(genecounts). Sgadòetal.MolecularAutism2013,4:51 Page6of12 http://www.molecularautism.com/content/4/1/51 Table1Enrichmentofautismspectrumdisorder(ASD)-relatedgenesinEn2−/−cerebellumandhippocampus differentiallyexpressedgenes (a)Cerebellum GeneSymbol GeneName Pvalue Foldchange Ada adenosinedeaminase 3.50E-03 1.705 Ahi1 Abelsonhelperintegrationsite1 4.70E-03 1.690 Cacna1g calciumchannel,voltage-dependent,Ttype,alpha1Gsubunit 8.20E-03 0.670 Cdh10 cadherin10 4.40E-03 1.662 Eml1 echinodermmicrotubuleassociatedproteinlike1 2.80E-03 0.581 Erbb4 v-erb-aerythroblasticleukemiaviraloncogenehomolog4(avian) 9.90E-03 0.653 Glo1 glyoxalase1 6.00E-03 0.625 Gnas GNAS(guaninenucleotidebindingprotein,alphastimulating)complexlocus 5.60E-03 1.565 Grm5 glutamatereceptor,metabotropic5 3.70E-03 1.656 Itgb7 integrinbeta7 1.00E-04 0.453 Kdm5c lysine(K)-specificdemethylase5C 8.50E-03 0.660 Kit kitoncogene 3.00E-03 0.591 Nrp2 neuropilin2 0.00 2.503 Nrxn3 neurexinIII 1.80E-03 1.843 Park2 Parkinsondisease(autosomalrecessive,juvenile)2,parkin 5.20E-03 1.723 Pinx1 PIN2/TERF1interacting,telomeraseinhibitor1 1.00E-03 1.871 Plcb1 phospholipaseC,beta1 3.60E-03 1.677 Rb1cc1 RB1-induciblecoiled-coil1 6.90E-03 1.635 Rpp25 ribonucleaseP25subunit(human) 8.00E-03 0.619 Stk39 serine/threoninekinase39,STE20/SPS1homolog(yeast) 9.40E-03 1.548 Th tyrosinehydroxylase 7.90E-03 1.564 (b)Hippocampus GeneSymbol GeneName Pvalue Foldchange Aff4 AF4/FMR2family,member4 1.30E-03 4.490 Atp2b2 ATPase,Ca++transporting,plasmamembrane2 2.80E-03 0.544 Baiap2 brain-specificangiogenesisinhibitor1-associatedprotein2 8.00E-04 0.478 Camta1 calmodulinbindingtranscriptionactivator1 4.40E-03 0.561 Dab1 disabledhomolog1(Drosophila) 4.10E-03 0.552 Dctn5 dynactin5 4.10E-03 1.733 Dlg4 discs,largehomolog4(Drosophila) 0.00 0.346 Egr2 earlygrowthresponse2 7.00E-04 2.099 Eif4ebp2 eukaryotictranslationinitiationfactor4Ebindingprotein2 3.90E-03 0.551 Ep400 E1Abindingproteinp400 2.00E-04 2.361 Foxp1 forkheadboxP1 3.00E-04 0.425 Gabra4 gamma-aminobutyricacid(GABA)Areceptor,subunitalpha4 4.00E-04 2.151 Gnas GNAS(guaninenucleotidebindingprotein,alphastimulating)complexlocus 3.90E-03 0.555 Gsk3b glycogensynthasekinase3beta 3.50E-03 0.541 Gtf2i generaltranscriptionfactorIII 3.80E-03 0.552 Kit kitoncogene 2.00E-04 0.417 Klc2 kinesinlightchain2 4.80E-03 0.565 Lrrc1 leucinerichrepeatcontaining1 4.20E-03 1.742 Nrcam neuron-glia-CAM-relatedcelladhesionmolecule 3.10E-03 1.777 Ntng1 netrinG1 1.90E-03 1.842 Sgadòetal.MolecularAutism2013,4:51 Page7of12 http://www.molecularautism.com/content/4/1/51 Table1Enrichmentofautismspectrumdisorder(ASD)-relatedgenesinEn2−/−cerebellumandhippocampus differentiallyexpressedgenes(Continued) Ntrk3 neurotrophictyrosinekinase,receptor,type3 1.00E-04 0.426 Park2 Parkinsondisease(autosomalrecessive,juvenile)2,parkin 1.00E-04 2.402 Plcb1 phospholipaseC,beta1 8.00E-04 0.487 Prkcb proteinkinaseC,beta 8.00E-04 0.490 Rpp25 ribonucleaseP25subunit(human) 1.10E-03 0.487 Sbf1 SETbindingfactor1 4.40E-03 0.562 Scn1a sodiumchannel,voltage-gated,typeI,alpha 1.00E-04 0.410 Scn8a sodiumchannel,voltage-gated,typeVIII,alpha 1.00E-04 0.414 Syn1 synapsinI 5.00E-04 0.469 Syne1 synapticnuclearenvelope1 4.80E-03 0.562 Thra thyroidhormonereceptoralpha 1.00E-04 0.419 Ube2h ubiquitin-conjugatingenzymeE2H 4.00E-03 0.559 Ubl7 ubiquitin-like7(bonemarrowstromalcell-derived) 3.70E-03 0.557 ASD-relatedgenesenrichedintheEn2−/−(a)cerebellumand(b)hippocampuswiththeirdifferentialexpressionPvalueandfoldchange. genelists,respectively.Theselectedgenesreporteddif- homolog 4 (Erbb4) P=0.003; calcium channel, voltage- ferentialexpressionvaluesinmicroarrayexperimentsran- dependent,T type, alpha 1G subunit (Cacna1g) P=0.003] gingbetween0.41folddecreaseto2.5foldincrease.With (Figure 5a). Fifteen genes of the eighteen selected were qPCR, five of the eight selected genes showed statistically instead significantly changed in the En2−/− hippocam- significantdifferentialexpressionintheEn2−/−cerebellum pus [netrin G1 (Ntng1) P=0.008; Parkinson disease [glutamate receptor, metabotropic 5 (Grm5) P=0.031; (autosomalrecessive,juvenile)2,parkin,(Park2)P=0.004; neurexinIII(Nrxn3)P=0.032;neuropilin2(Nrp2)P= gamma-aminobutyric acid (GABA) A receptor, subunit 0.009; v-erb-a erythroblastic leukemia viral oncogene alpha4(Gabra4)P=0.007;earlygrowthresponse2(Egr2) Table2CorrelationofSimonsFoundationandAutismResearchInitiative(SFARI)databasegeneswithpublished transcriptomestudiesinAutismSpectrumDisorder(ASD)brainandourstudy Cerebellum #SFARI %enrichment Pvalue Genenames genes Thisstudy(3En2−/−,3WT) 21 2.79% 4.98E-01 Ada,Ahi1,Cacna1g,Cdh10,Eml1,Erbb4,Glo1,Gnas,Grm5,Itgb7, Kdm5c,Kit,Nrp2,Nrxn3,Park2,Pinx1,Plcb1,Rb1cc1,Rpp25,Stk39,Th Voineaguetal.[9] 16 4.75% 3.43E-02(*) AHI1,ANK3,CACNA1G,CBS,EN2,EPHB6,FAT1,FOXP1,GAP43,GRIN2A, (11autism,10controls) HSD11B1,NLGN3,NTNG1,RAB11FIP5,SLC30A5,UBE3A Purcelletal.[39] 1 3.33% 5.85E-01 CNR1 (9autism,9controls) Limbicregions #SFARI %enrichment Pvalue Genenames genes Thisstudy(3En2−/−,3WT) 33 4.24% 2.65E-02(*) Aff4,Atp2b2,Baiap2,Camta1,Dab1,Dctn5,Dlg4,Egr2,Eif4ebp2, Ep400,Foxp1,Gabra4,Gnas,Gsk3b,Gtf2i,Kit,Klc2,Lrrc1,Nrcam, Ntng1,Ntrk3,Park2,Plcb1,Prkcb,Rpp25,Sbf1,Scn1a,Scn8a,Syn1, Syne1,Thra,Ube2h,Ubl7 Voineaguetal.[9] 36 3.70% 1.04E-01 AHI1,APBA2,ATP2B2,ATRNL1,AUTS2,BCL2,BTAF1,CADM1,CD99L2, (13autism,13controls) DNM1L,DPP10,EIF4EBP2,FAT1,GRIN2A,ICA1,MAOA,MSN,NTRK3, PCDH9,PPFIA1,PRKCB,PTCHD1,RAB11FIP5,RGS7,RPP25,SLC16A3, SLC25A12,SLC9A9,STXBP1,SYT17,TOMM20,TSC2,TUBGCP5,UBE2H, UBR5,UPF3B Garbettetal.[40] 4 3.05% 5.52E-01 AHI1,MSN,SDC2,SLC9A9 (6autism,6controls) PercentageofenrichmentcalculatedonSFARIASD-associatedgenescomparedtothenumberofdifferentiallyexpressedgenes.Differentiallyexpressedgenesin Voineaguetal.[9]calculatedusingGEO2Ranalysis.Genespresentinatleasttwoofthestudiesareshowninbold.EnrichmentPvaluescalculatedwiththe hypergeometrictest,(*)P<0.05. Sgadòetal.MolecularAutism2013,4:51 Page8of12 http://www.molecularautism.com/content/4/1/51 a b Cerebellum Hippocampus 2.6 2.6 2.4 Microarray 2.4 Microarray qPCR qPCR 2.2 2.2 2 2 1.8 1.8 1.6 1.6 1.4 1.4 1.2 1.2 1 1 0.8 0.8 0.6 GRM5 NRXN3 NRP2 ERBB4 CACNA1G 000...246 NTNG1 PARK2 GABRA4 EGR2 SCN1 A NTRK3 PLCB1 SYN1 PRKCB GNAS CTTN FXR2 GABBR1 NLGN2 GSK3b Figure5QuantitativePCRvalidationofdifferentiallyexpressedgenes.AselectednumberofdifferentiallyexpressedgenesintheEn2−/− cerebellum(a)andhippocampus(b)werevalidatedbyqPCR.RelativemRNAexpressionlevel(foldexpression)asobtainedbyqPCRperformed onwholecerebellumorhippocampusextractsofadultwildtype(WT)andEn2−/−mice.CorrelationoffoldexpressionfromqPCR(lightcolorbars)and microarray(darkcolorbars)resultswascalculatedusingPearson’sCorrelation.ExceptforEgr2,qPCRresultsforalltheevaluatedgenesshowed significantcorrelationwithmicroarrayresults(R=0.84;P<0.05).Valuesareexpressedaseachgene/L41comparativequantitationratiosnormalizedon theexpressionofWT(mean±s.e.mofthreereplicatesfrompoolsoffouranimalspergenotype;P<0.01,Student’st-test,WTversusEn2−/−. P=0.007; sodium channel, voltage-gated, type I, alpha with ASD, including abnormal synaptic transmission (Scn1a) P=0.001; neurotrophic tyrosine kinase, receptor, and increased immune response. Furthermore, when type3(Ntrk3) P=0.0003;phospholipaseC, beta 1(Plcb1) directlycomparedtotherepositoryoftheSFARIdatabase P=0.0001; cortactin (Cttn) P=0.0005; synapsin I (Syn1) (gene.sfari.org), our differentially expressed genes in the P=0.007; fragile X mental retardation, autosomal homolog hippocampusshowanenrichmentofASD-associatedgenes 2(Fxr2)P=0.0004;proteinkinaseC,beta(Prkcb)P=0.001; significantlyhigherthanpreviouslyreported[41]. gamma-aminobutyric acid (GABA) B receptor, 1, (Gabbr1) Transcriptome analysis has been employed to unravel P=0.03; neuroligin 2 (Nlgn2) P=0.001; glycogen synthase common pathways based on the assumption that the kinase3beta(Gsk3b)P=0.003;GNAS(guaninenucleotide core phenotypes of ASD may be caused by convergent binding protein, alpha stimulating) complex locus (Gnas) molecular mechanisms [6]. Several studies have analyzed P=0.01] (Figure 5b). Except for Egr2, in all tested genes genome-wide expression profiles of lymphoblastoid cell the expression differences reported by qPCR correlated lines and blood samples from ASD patients, pointing to withthemicroarraydata(Pearsonr=0.84,P<0.00005). an upregulation of immune genes as key mechanisms in the pathogenesis of ASD [8]. Despite the limited source Discussion of brain tissue samples from ASD cases and the technical Todate,morethan500autism-associatedgeneshavebeen restrictions,studiesofASDbraintranscriptomeareemer- identified (SFARI Gene; gene.sfari.org; updated mar/2013); gingasstrategicforuncoveringfunctionallyrelevantalter- yet the etiology of ASD remains essentially unknown [6,7]. ations in gene expression. A previous microarray study The significance of animal models in ASD research has found alterations of glutamatergic neurotransmission in been widely recognized as important for unraveling the ASD cerebellum [39], and expression profiles from ASD molecular, cellular, anatomical, electrophysiological and patient temporal cortices showed upregulation of genes behavioralconsequencesofgenedysfunctioninASD.Here, involved in innate immune response and downregulation we present a transcriptome analysis in a mouse model of several neurodevelopmental genes [40]. Moreover, the of ASD of two brain areas, the cerebellum and the transcriptome profiles from three different brain regions hippocampus, areas that are profoundly affected in ASD (frontal cortex, temporal cortex and cerebellum) of nine- patients. Despite the small number of samples used for teenautismcasesandseventeencontrolswereinvestigated themicroarrayanalysisandthesamplegenderheterogen- recentlyusingclassicaldifferentialexpressionanalysisand eity,thelowgenetic varianceamongindividualsalloweda anetwork-basedapproach[9].Theseanalysesshowedup- reasonablestatisticalpowerforourbioinformaticanalysis. regulation of genes involved in immune response and Our study revealed that the molecular signature of these downregulationofgenesinvolvedinsynapticfunctionand two brain regions shares convergent pathological pathways vesiculartransport[9].Ourresultsareinaccordancewith Sgadòetal.MolecularAutism2013,4:51 Page9of12 http://www.molecularautism.com/content/4/1/51 these findings. Using gene ontology enrichment,integrated strong support for a causative link between the loss of gene-network analysis and mouse phenotypes analysis, we NRXN3 and the development of ASD [47]. Our results of report significantly enriched functions and pathways that anincreasedexpressionofNrxn3inthecerebellumsuggest werepreviouslyassociatedtoASD[42].Indetail,wefound alterations in Purkinje cell synaptic formation, where increased immune response and major histocompatibility NRXNs have been shown to participate to the forma- complex-related immunity in the En2−/− cerebellum; tion of glutamatergic synapses through interaction with decreased and abnormal neurotransmission and increased Cerebellin 1 precursor protein (also downregulated in seizures in the En2−/− hippocampus [see Additional file 5 theEn2−/−cerebellum)andGluR∂2[48,49]. fordetails].Moreover,bydirectcomparisonwiththeSFARI Scn1aencodes the voltage-gatedsodium channelalpha repositoryofASD-relatedgenes,weshowthatthegeneex- subunit.DenovonullmutationsinSCN1Aresultinsevere pressionchangesobservedintheEn2−/−hippocampuswere myoclonicepilepsyofinfancy[50].SCN1Amutationshave significantly enriched in ASD-related genes. Furthermore, been associated to a number of neurological disorders, the proportion of ASD-associated genes enrichment in including generalized epilepsy with febrile seizures plus, En2−/−hippocampuswassignificantlyhigherthanprevious Dravetsyndrome,borderlinemyoclonicepilepsyininfancy, studies (Table 2) when compared withVoineagu et al. [9], intractablechildhoodepilepsywithgeneralizedtonic-clonic likelythemostcomprehensivetranscriptomestudyofASD seizures, familial hemiplegic migraine, and a number of post-mortem brain to date. In the case of the cerebellum, cryptogenic focal and generalized epilepsies. Recently, incontrasttoVoineaguetal.[9]wedidnotfindsignificant de novo mutations in SCN1A have been associated with enrichment of ASD-associated genes in En2−/− mice. Such ASD[51],andareportofarecognizedmutationinSCN1A difference could be the result of the complex structural suggestsawidephenotypic variationofthegenemutations and cytoarchitectural abnormalities in En2−/− cerebellum causing a variety of neurologic disorders, including ASD [20,21] and the consequent phenotypical variability, or [52]. In mice, heterozygous loss-of-function mutation in couldsimplyreflectdifferencesbetweenmouseandhuman Scn1a(Scn1a+/−),reproducesseveralofthesymptomsasso- phenotypes, as the incidence of cerebellar hypoplasia was ciated to the human mutation, such as thermally induced notreportedinthediagnosticcriteriausedinthestudy[9]. and spontaneous seizures, premature death, ataxia and Remarkably, EN2 was among the differentially expressed sleep disorder[53,54].Scn1a+/− mice show both cognitive genesfoundinVoineaguetal.[9],confirmingourevidence deficits and autistic traits that are caused by impaired abouttheroleofEn2intheneuropathologyofASD,andin GABAergicneurotransmissionandcanberescuedbydrug anteriorbrainstructures[27]. treatment. Scn1a down-regulation in the En2−/− hippo- Among the differentially expressed genes, Grm5, Nrxn3 campus could contribute to the abnormal excitability and andScn1aareofparticularinterestforASD.Grm5encodes alteredGABAergicneurotransmissionshowninthesemice mGluR5, a G-protein coupled receptor for the neuro- by our previous studies [27,30]. Pharmacological rescue of transmitter glutamate [43]. In a recent study, mGluR5 thehippocampalphenotypeintheEn2−/−withGABAergic hasbeenshowntoparticipateinthepathogenesisoffragile drugsiscurrentlyunderinvestigation. X syndrome (FXS) while genetic downregulation of Grm5 Anomalies in the cerebellum are the most reproducible was able to compensate for some of the symptoms in a neuropathologicalalterationsinASDpatients.Severalcere- mouse model of FXS [44]. Furthermore, Grm5 was shown bellar abnormalities have been observed in mouse models to be downregulated in hippocampal neurons lacking ofbothEn2gain-andloss-of-function.Ectopicoverexpres- Shank3, another ASD-associated gene [45]. These data sion of En2 in Purkinje cells during late embryonic and supportacentralroleforGrm5inneurobiologicalpathways postnatal cerebellar development results in reduced cere- relatedtoASDpathogenesis.Ourresultsshowanincreased bellarvolumeandlossofPurkinjecellsandothercerebellar expressionofGrm5inthe cerebellumofEn2−/−mice,sug- neurons[55,56].Interestingly,En2knock-outcausesdefect- gestingaroleofGrm5inthecerebellarphenotypeofthese ivecerebellarpatterning,reducedPurkinjecellnumberand mice. The contribution of Grm5 and its interaction with abnormal dendritic foliation [10,57], indicating that alter- Fmr1intheEn2−/−hippocampusremainstobeestablished ations in En2 expression levels during development cause and could open new perspective of pharmacological and similar phenotypes. Furthermore, deficits in social behav- geneticrescueoftheASD-relatedphenotypeofthesemice. iors as well as defective spatial learning and memory were Nrxn3 encodes neuronal adhesion proteins of the also reported in En2−/− mice [24-26]. A recent epigenetic Neurexin (NRXN) family. NRXNs are presynaptic cell analysisofEN2promotermethylationinthe cerebellumof adhesion proteins that form trans-synaptic complexes ASD individuals indicated hypermethylation of the pro- with their postsynaptic counterpart neuroligins (NLGNs) moter region and persistent upregulation of the gene. The and have important roles in synapse development and authorsreportthatpromoterhypermethylationisnormally function[46].Recently,areportofhemizygousanddenovo associated with a decrease in gene expression and suggest deletions involving NRXN3 in ASD families provided the possibility of a developmental mechanism intended to Sgadòetal.MolecularAutism2013,4:51 Page10of12 http://www.molecularautism.com/content/4/1/51 support downregulation of EN2 during perinatal develop- foldchangeexpressioncomparedtoWTlittermates,differential ment [19]. Taken together, this evidence suggests that an expressionPvalueandpercentageoffalseprediction(pfp)calculated overall imbalance in EN2 expression may be relevant for withRankProd.Pvaluecut-offP<0.01forthecerebellumandP<0.005 forthehippocampus. ASDpathogenesis,asitcouldproducealterationsincritical Additionalfile3:GenesdifferentiallyexpressedinbothEn2−/− brain functions. Comparable evidence of a similar dosage cerebellumandhippocampus.Genescommonlyregulatedinthe effect has been reported in the case of mutations of other En2−/−cerebellumandhippocampuswithdifferentialexpressionPvalue genes critically involved in gene expression regulation and andfoldchange. maintenance of synaptic and neuronal homeostasis, such Additionalfile4:En2full-lengthexpression.QuantitativePCRanalysis ofEn2full-lengthexpressionintheWTandEn2−/−cerebellumand as MECP2 and ARX [58,59]. It remains to be established hippocampus.ValuesareexpressedasEn2/L41comparativequantitation whether En2 overexpressing mice display abnormal be- ratiosnormalizedontheexpressionofWTinthecerebellum haviors relevant to autism. Microarray data have been (mean±s.e.mofthreereplicatesfrompoolsofthreeanimalsper genotype;P<0.01,Student’st-test,WTversusEn2−/−). producedforEn2overexpressingPurkinje cellsonadiffer- Additionalfile5:Integratedgene-networkanalysisandgene ent platform; however, the results overlap only marginally ontologyenrichment.Tablesa-fshowthetenmostsignificantdisease withthehereinreportedstudy[60]. andfunctionalannotationsobtainedwithIngenuityPathwaysAnalysis (IPA)forEn2−/−cerebellum(a-c)andEn2−/−hippocampus(d-f)differentially expressedgenes.Functionalcategoriesandannotationsareshownwiththeir Conclusions Pvalue,thepredictedactivationstate,theactivationz-score,theassociated Using transcriptome analysis, we identified over 800 genes genesandthenumberofenrichedgenes.Activationz-score>2or<−2 differentiallyexpressedinthecerebellumandhippocampus indicatessignificantlyincreasedordecreasedannotations.Tablesg-jshow of En2−/− mice. Despite the small number of samples used geneontologyanalysisdetailsforEn2−/−cerebellum(g,h)andEn2−/− hippocampus(i,j)differentiallyexpressedgenes.Geneontologyannotations and the relatively small statistical power, our study is the areshownwithgenecountsandcorrectedPvalues.Significantdiseaseand first to analyze molecular changes occurring in two brain functionsforup-(c,f,h)anddownregulated(b,e,j)genesareshown separately. structures with neuroanatomical alterations relevant to Additionalfile6:Tissueexpressionofdifferentiallyexpressed ASD in a mouse model of this disease. Our bioinformatic genesinEn2−/−hippocampus.Tableshowinggeneontologytissue analysis of the molecular signature of En2−/− cerebellum expressionannotationsfordifferentiallyexpressedgenesintheEn2−/− and hippocampus shows a significant convergence of hippocampus.Nosignificanttissueexpressionannotationswerefound fordifferentiallyexpressedgenesinEn2−/−cerebellum. neurobiological pathways previously linked to ASD path- ology in brain samples from ASD patients. Overall, the Abbreviations present study points to a strong impact of transcriptome ASD:Autismspectrumdisorders;pfp:Percentageoffalsepositives; analysis on mouse models for identifying neurobiological DAVID:DatabaseforAnnotation,VisualizationandIntegratedDiscovery; pathwayscommonlyalteredwhenASDgenesaredisrupted FXS:FragileXsyndrome;GABA:Gammaaminobutyricacid;GO:Gene ontology;IPA:Ingenuitypathwayanalysis;MHC:Majorhistocompatibility in a human patient and in a mouse model alike. Further- complex;MPO:Mammalianphenotypeontology;NLGN:Neuroligin; more, together with the frequent association of cerebellar NRXN:Neurexin;RP:Rankproduct;SFARI:SimonsFoundationandAutism neuroanatomicalalterationstotheneuropathologyofASD, ResearchInitiative;SNP:Single-nucleotidepolymorphism;WT:Wildtype. our molecular analysis suggests a contribution also for the Competinginterests hippocampus, where molecular changes relevant to ASD Theauthorsdeclarethattheyhavenocompetinginterests. mayoccuralsoinhumanpatients.Thisnotionissupported Authors’contribution by the consistent enrichment of ASD-related genes in the En2−/− hippocampus compared to the cerebellum PmSi,cGroPararnaydaYnBdcqoPnCceRiveexdpetrhimeestnutds.yP.PS,SG,GPPa,nVdA,EGDZpaenrfdorSmGepdebrfioorinmfoedrmatic and to other similar studies performed on ASD patient analysis.PS,GPandYBanalyzeddata.PSandYBwrotethepaper.PS,SCand YBprovidedfunding.Allauthorsreadandapprovedthefinalmanuscript. tissue samples [41]. Finally, our results confirm the En2−/− mouse model of ASD as a valuable tool for in- Acknowledgements vestigating neuroanatomical, behavioral,as well as mo- P.S.issupportedbyProvinciaAutonomadiTrentoandtheEuropean lecularalterationsrelatedtoASD. Community’sFP7/2007-2013undergrantagreementMarieCurieFP7- PCOFUND-GA-2008-226070‘progettoTrentino’,projectEnCort.Thiswork wasfundedbytheItalianMinistryofUniversityandResearch(PRIN2008 Availability of supporting data grant#200894SYW2_002andPRIN2010–2011grant#2010N8PBAA_002toY. B.)andtheUniversityofTrento(CIBIOstart-upgranttoS.C.andY.B.).Wethank The data sets supporting the results of this article are AndreaMessina,FedericoVaggiandTommasoSchiavinottoforhelpful availableintheGEOrepository,GSE51612. discussions,andPatriziaPaoliforadministrativesupport. Authordetails Additional files 1LaboratoryofMolecularNeuropathology,CentreforIntegrativeBiology (CIBIO),UniversityofTrento,ViadelleRegole101,38123Trento,Italy. Additionalfile1:RT-qPCRprimersusedinthestudy. 2LaboratoryofTranslationalGenomics,CentreforIntegrativeBiology(CIBIO), UniversityofTrento,ViadelleRegole101,38123Trento,Italy.3High Additionalfile2:Differentiallyexpressedgeneinthecerebellum andinthehippocampusofEn2−/−mice.Tablesshowingdifferentially ThroughputScreeningCoreFacility,CentreforIntegrativeBiology(CIBIO), expressedgenesintheEn2−/−(a)cerebellumand(b)hippocampuswith UniversityofTrento,ViadelleRegole101,38123Trento,Italy.4Laboratoryof DevelopmentalNeurobiology,CentreforIntegrativeBiology(CIBIO),

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