Transcriptome profiling in engrailed-2 mutant mice reveals common molecular pathways ...

12 Pages·2013·0.63 MB·Italian

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:[email protected] 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.
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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).
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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 neuropatho-
logical features observed in ASD patients [20,21,23]. To
identifygenesandpathwaysalteredinEn2−/−mice,weini-
tially performed transcriptome profiling of the whole cere-
bellar 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
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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.
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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).
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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
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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.
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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
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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
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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),