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PAGE 1 RESEARCHOpenAccessAlteredglialmarkerexpressioninautistic post-mortemprefrontalcortexandcerebellumCatherineEdmonson1,2 ,MarkNZiats1,3,4* andOwenMRennert1AbstractBackground: Thecellularmechanism(s)underlyingautismspectrumdisorders(ASDs)arenotcompletely understood,butASDsarethoughttoultimatelyresultfromdisruptedsynaptogenesis.However,studieshavealso shownthatglialcellnumbersandfunctionareabnormalinpost-mortembraintissuefromautisticpatients.Direct assessmentofglialcellsinpost-mortemhumanbraintissueistechnicallychallenging,limitingglialresearchin humanASDstudies.Therefore,weattemptedtodetermineifglialcell-typespecificmarkersmaybealteredin autisticbraintissueinamannerthatisconsistentwithknowncellularfindings,suchthattheycouldserveasa proxyforglialcellnumbersand/oractivationpatterns. Methods: Weassessedtherelativeexpressionoffiveglial-specificmarkersandtwoneuron-specificmarkersvia qRT-PCR.Westudiedtissuesamplesfromtheprefrontalcortex(PFC)andcerebellumofninepost-mortemautistic brainsamplesandnineneurologically-normalcontrols.Relativefold-changeingeneexpressionwasdetermined usingthe Ctmethodnormalizedtohousekeepinggene -actin,withatwo-tailedStudent s t -test P <0.05 betweengroupsconsideredassignificant. Results: Bothastrocyte-andmicroglial-specificmarkersweresignificantlymorehighlyexpressedinautisticPFCas comparedtomatchedcontrols,whileinthecerebellumonlyastrocytemarkerswereelevatedinautisticsamples.In contrast,neuron-specificmarkersshowedsignificantlylowerexpressioninboththePFCandcerebellumofautistic patientsascomparedtocontrols. Conclusions: Theseresultsareinlinewithpreviousfindingsshowingincreasedglialcellnumbersand up-regulationofglialcellgeneexpressioninautisticpost-mortembraintissue,particularlyinthePFC,aswellas decreasednumberofneuronsinboththePFCandcerebellumofautisticpatients.Theconcordanceofthese resultswithcell-levelstudiesinpost-mortemautisticbraintissuesuggeststhatexpressionofglialcell-type specificmarkersmayserveasausefulalternativetotraditionalcellularcharacterizationmethods,especially whenappropriately-preservedpost-mortemtissueislacking.Additionally,theseresultsdemonstrateabnormal glial-specificgeneexpressioninautisticbrains,supportingpreviousstudiesthathaveobservedalteredglialcell numbersoractivationpatternsinASDs.Futureworkshoulddirectlyassessthecorrelationbetweencell-type specificmarkerlevelsandcellnumberandactivationpatterns. Keywords: Astrocyte,Autisticdisorder,Geneexpression,Glia,Interneuron,Microglia,Neuron *Correspondence: ziatsm@mail.nih.govEqualcontributors1LaboratoryofClinicalandDevelopmentalGenomics,NationalInstituteof ChildHealthandHumanDevelopment,NationalInstitutesofHealth,49 ConventDrive,Building49,Room2C078,Bethesda,MD20814,USA3UniversityofCambridge,RobinsonCollege,GrangeRd,CambridgeshireCB3 9AN,UK Fulllistofauthorinformationisavailableattheendofthearticle 2014Edmonsonetal.;licenseeBioMedCentralLtd.ThisisanOpenAccessarticledistributedunderthetermsofthe CreativeCommonsAttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse, distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycited.TheCreativeCommonsPublic DomainDedicationwaiver(http://creativecommons.org/publicdomain/zero/1.0/)appliestothedatamadeavailableinthis article,unlessotherwisestated.Edmonson etal.MolecularAutism 2014, 5 :3 http://www.molecularautism.com/content/5/1/3 PAGE 2 BackgroundAutismspectrumdisorders(ASDs)areneurodevelopmentalsyndromesdefinedbyimpairmentsinlanguage, verbalandnon-verbalcommunication,andrestrictive/ repetitivepatternsofbehavior[1].ASDsymptomsmanifestwithinthefirsttwoyearsoflife,withtheirseverity andpresentationvaryingconsiderablybetweenindividuals,thusyieldingthe spectrum classification.ASDsare estimatedtoaffect1in88childrenintheUnitedStates, andtheprevalenceofASDsisatleastfourtimesmore commoninmalesthanfemales[2]. Theetiologyofautismiscomplexandtheneurobiologicalmechanism(s)thatresultintheclinicalphenotyperemaintobefullyunderstood.However,thereis strongevidencethattheautismphenotypeultimately resultsfromaberrantsynapticwiringinthedeveloping brain[3].Inparticular,studieshaveshownthatlongdistancecommunicationbetweendisparateneocortical areasmaybedisruptedinASDs,causingdelaysininformationprocessingwithinthebrainthatmanifestas thecommunication,language,andsocialdevelopment problemsseeninchildrenwithautism[4].Additionally, parallelresearchhasshownthatneuronalmicrocircuitrywithinbrainareasmayalsobedisruptedin ASDs,andthatthismayresultinlocalprocessingdeficitswithinbrainregionsrelatedtohigherfunctioning, suchastheprefrontalcortex(PFC)[5].Underlyingthese circuitdisruptionsisalargebodyofevidencethathas demonstrateddecreasednumbersofneurons(andtheir varioussubtypes)throughouttheautisticbrainbyearly childhoodinpost-mortemstudies[6]. Inadditiontothebodyofevidenceimplicating aberrantlocalandlong-distancesynapticdysfunctionin ASDs,manystudieshavedemonstratedmicroglialand astrocytedysfunctioninASDbrains.Forinstance,postmortempathologicalstudiesofautisticbrainusing immunocytochemistry(IHC)and/orstereologyhave identifiedmicroglialactivationpatterns[7-9],andhave demonstratedincreasedmicroglialcelldensityinmultiplebrainregions[8,10].Glialactivationreferstothe wellcharacterizedcascadeofeventsthatoccursupon reactionofgliatostimuli,resultingintheirmobilization, releaseofcytokines,andabilitytophagocytize,among otherprocesses[11];glialactivationisassociatedwith specificgeneexpressionpatternsthataredistinctfrom resting glialgeneexpression[12].Furthermore,positron emissiontomography(PET)usingamicroglial-specific radiotraceralsodemonstratedmicroglialactivationin multiplebrainregionsofautisticcases[13].Additionally, studiesinaRettsyndromemousemodel,asingle-gene deletiondisorderwithautismasacomponent,havealso demonstratedcellularmicroglialabnormalities[14],and aremarkablestudydemonstratedthatautistic-likephenotypescanbepartiallyreversedbyreplacingmutant Mecp2( / )microgliawiththeirrespectivewild-type cells[15]. Increasednumbersofastrocytes,withalteredcellsize andbranchingpatterns,havealsobeendemonstratedin post-mortemautisticbrains[16].Additionally,astrocytespecificcellmarkerproteinsareincreasedinmultiple autisticbrainregions[17,18].SimilartomicroglialstudiesinASDmousemodels,astrocyteshavebeenshown tobeabnormalinanumberofsingle-geneASDmodels, includingRett[19,20],FragileX[21],andTuberous Sclerosis[22].Inparalleltotheaforementionedmicroglial study,itwasalsoshownthatreplacingmutantastrocytes inMecp2( / )micecancorrectsomeaspectsofthe phenotype[23]. However,itisnotclearhowtheseseparatelinesof evidence onedemonstratingimmune/glialdysfunction inASDsandtheotherimplicatingsynapticabnormalities mayconvergeintoacommonmechanisminthe autisticbrainthatultimatelyresultsinthesharedclinical phenotype.Becauseseparatestudieshaveshownthat microgliaandastrocytesplaycriticalrolesinsculpting developingsynapsesduringnormalneurodevelopment [24,25],itisreasonabletohypothesizethatinherent defectsoraberrantnumbersofmicrogliaandastrocytes inthedevelopingautisticbrainmaybecausativeofthe synapticabnormalitiesbyaffectingtheproperwiringof developingneuronalconnections.However,because appropriately-preservedpost-mortemautisticbraintissueislacking[26],cellular-levelstudiesassessingglial numbersandactivationinhumanautisticbrainshave beenlimited.Moreover,quantificationofcellnumbers inpostmortemtissuebystereologyistechnicallychallenging,furtherlimitingtheabilityofresearchersto assessthefewappropriately-preservedtissuesamples thatareavailable.Finally,nostudieshaveconcurrently specificallyassessedformicroglia,astrocytes,andneurons inthesamesetofautisticbrainsamples.Asaconsequence,acomprehensiveunderstandingoftherelationshipbetweenglialandneuroncellsinautisticbrainsis needed. Therefore,thepurposeofthisstudywastwo-fold. First,wesoughttodetermineifmicroglia,astrocyte,and neuron-specificmarkerswerealteredinpost-mortem autisticbraintissue,inordertofurtherinvestigatethe roleofgliainASDs.Then,wedeterminedifglialand neuronalcell-typespecificmarkerexpressionpatterns areconsistentwithknowncellular-levelfindings,becausegeneexpressionstudiesofpost-mortemhuman brainareofteneasiertoperformthancell-levelstudies, andthereforethisapproachmayserveasavaluable screening assaytoinferrelativecellproportions. Inthisstudy,wecomparedinternally-normalized mRNAexpressionlevelsofmicroglial,astrocyte,and neuronalcell-typespecificmarkergenesinpost-mortemEdmonson etal.MolecularAutism 2014, 5 :3 Page2of9 http://www.molecularautism.com/content/5/1/3 PAGE 3 braintissuefrompatientswithautismandhealthycontrols.Ourresultsprovidefurtherevidenceforaroleof gliainautismpathology,andsuggestthatassessmentof glialcell-typespecificmarkersmayserveasaproxyfor relativecellularnumbersoractivationpatterns.MethodsPost-mortembrainsamplesPost-mortembraintissuewasobtainedfromtheNational InstituteofChildHealthandHumanDevelopmentBrain andTissueBank,MD,USA.Thissourceobtainedconsent tousebraintissueforresearchfromeachpatientortheir guardianpriortohis/herdeath,andtheirprotocolwas approvedbytheirInstitutionalReviewBoard.Nopatientspecificidentifiableinformationwasobtained.Because multiplebrainregionshavebeenimplicatedinASDs,we performedouranalysisintwoseparateareasthathave beenconsistentlydemonstratedasabnormalinautism thePFCandthecerebellum.Weobtainedpost-mortem PFCbraintissuefromfiveindividualswithautismand fromfivehealthycontrols(Table1).Weobtainedpostmortemcerebellumbraintissuefromfourindividuals withautismandfourhealthycontrols.Themajorityof samplepairs(PFCandcerebellum)werederivedfromthe samedonorbrain.AllcaseswereCaucasianmales,and casecontrolswerematchedbyageascloselyaspossible.RNAisolationandqualitycontrolRNAisolationandqualitycontrolanalysiswere performedaspreviouslydescribed[27].Briefly,total RNAwasextractedusingTRIZOLReagent(Life Technologies,Carlsbad,CA,USA)accordingtothe manufacturer sprotocol.QuantificationofRNAwas performedusingaNanoDropND-1000,andRNA integritywasassessedusinganAgilentBioanalyzer 2100(Table2).ReversetranscriptasereactionTotalRNA(1 g)wasusedina20 LreversetranscriptasereactiontosynthesizecDNAwithSuperScript3 ReverseTranscriptase(LifeTechnologies)accordingto themanufacturer sprotocol.Briefly,1 goftotalRNA wasaddedtoanaqueoussolutioncontaining250ng/ L ofrandomhexamerand10mMdeoxyribonucleotide triphosphate.TheRNAwasdenaturedfor5minutesat 65Candthensnapcooledonicefor2minutes.After which0.1MDTT,5First-StrandBuffer(250mM Tris HCl,375mMKCl,15mMMgCl2),RNaseOUT RecombinantRibonucleaseInhibitor(40Units/ L),and SuperScript3ReverseTranscriptase(200Units/ L)were addedintoeachsamplemixture.Thereactionwas carriedoutunderthefollowingconditions:25Cfor5 minutes,50Cfor60minutes,and70Cfor15minutes. Table1ClinicalcharacteristicsandRNAqualityofautisticandcontrolbrainsamplesSample#UMB#DiagnosisBrain area Age(yr)Causeof death PMI(h)RNAquality A260/280A260/230RIN 15308*AutismPFC4.5Skullfracture212.0512.2664.9 21349AutismPFC5.6Drowning392.0442.2324.3 35144AutismPFC7.2Rhabdomyosarcoma32.0582.2715.4 45302*AutismPFC16.3DKA202.0312.2384 54999*AutismPFC20.8Cardiacarrhythmia142.0392.2326 64670*ControlPFC4.6CommotioCordis172.0482.2765.2 71185ControlPFC4.7Drowning172.0262.2434.7 84898ControlPFC7.7Drowning122.0562.1835.9 94848*ControlPFC16.7Drowning152.0442.1856.7 104727*ControlPFC20.5Multipleinjuries(MVA)52.0662.2096.5 115308*AutismCere4.5Skullfracture212.0871.7817.3 124899AutismCere14.3Drowning92.0772.3149.3 135302*AutismCere16.3DKA202.0831.6462.2 144999*AutismCere20.8Cardiacarrhythmia142.0812.1149.2 154670*ControlCere4.6CommotioCordis172.0882.1616.1 164722ControlCere14.5Multipleinjuries(ATV)162.0732.8286.5 174848*ControlCere16.7Drowning152.0872.3276.8 184727*ControlCere20.5MultipleInjuries(MVA)52.0672.3077.2PFC:Prefrontalcortex;Cere:Cerebellum;UMB:UniversityofMarylandBrainBanksamplenumber;PMI:Post-morteminterval;DKA:Diabeticketoacid osis;ATV: All-terrainvehicle;MVA:Motorvehicleaccident;RIN:RNAintegritynumber.*Indicatesbothprefrontalcortexandcerebellumsampleswereassess edfromthe samedonorbrain.Edmonson etal.MolecularAutism 2014, 5 :3 Page3of9 http://www.molecularautism.com/content/5/1/3 PAGE 4 ThecDNAproducedfromthereactionwasdilutedto 0.25withnucleasefreewater.RealtimequantitativePCRSYBRGreenExpressionAssaySystem(AppliedBiosystems,FosterCity,CA,USA)wasusedtomeasurerelative, normalized,mRNAexpressionlevels.Weassessedfour separatemicroglial-specificcellsurfacegenes:Triggering receptorexpressedonmyeloidcells2( TREM2 ), DAP12 CX3Cchemokinereceptor1( CX3CR1 ),andallograft inflammatoryfactor1( AIF1 )[28].Twocelltypespecificintermediatefilaments,glialfibrillaryacidicprotein( GFAP ),whichisastrocyte-specific[29],andthe pan-neuronalcellmarker NEFL [30],wereusedto assessforastrocytesandneurons,respectively.Additionally,weassessedforGABAergicinterneurons specificallywithparvalbumin( PVL )[31].Theintermediatefilamenthousekeepinggenebeta-actin( ACTB )was usedasacontrol.Forwardandreverseprimersequences weregeneratedusingPrimer3softwareandsynthesizedby EurofinsMWGOperon(Huntsville,AL,USA)(Table2). Quantitativereversetranscriptasepolymerasereaction (qRT-PCR)wasperformedusinganABIPrism7900SequenceDetectionSystem(LifeTechnologies)witha 96-wellformat.EachqRT-PCRreactioncontained6.5 L water,12.5 LSYBRGreenmastermix(AppliedBiosystems),1 Lforwardprimer(10 M),1 Lreverseprimer (10 M),and4 LofcDNA(0.25).Datawascollected usingtheSDS2.3Program(AppliedBiosystems)underthe followingrunparameters:48Cfor30minutes,95Cfor 10minutes,40cyclesof95Cfor15seconds,60Cfor1 minute,andafinaldissociationstage.DataanalysisThetargetgenesandtheendogenouscontrolswere measuredwithtechnicaltriplicatesineachqRT-PCR reaction,andallgeneswereassessedinthreeseparate, independentqRT-PCRruns.Thecyclethresholdnumber(Ct)wascalculatedusingRQManager1.2Software (AppliedBiosystems).Relativeexpressionofeachtarget genewasnormalizedto ACTB usingthe Ctmethod. All P valuesreportedarebasedonatwo-tailedStudent s t -test.Onlyresultswitha P valuelessthan0.05were consideredsignificant.ResultsTheaveragepost-morteminterval(PMI)wasnotsignificantlydifferentbetweenautisticandcontroltissue samples(Table1;ASD=17.9h,ctrl=13.2h, P =0.16). Thisremainedtrueaftersub-stratifyingbybrainregion (ASDPFC=19.4h,ctrlPFC=13.2h, P =0.28;andASD cerebellum=16.0h,ctrlcerebellum=13.25h, P =0.48). RNAisolatedfrompost-mortembraintissuewasgenerallyofhighquality,andtheRNAIntegrityNumber (RIN)wasnotsignificantlydifferentbetweenautismand controls(ASD=5.84,ctrl=6.18, P =0.67).TheRINwas alsonotsignificantlydifferentaftersub-stratifyingby brainregion(ASDPFC=4.92,ctrlPFC=5.80, P =0.13; andASDcerebellum=7.00,Ctrlcerebellum=6.65, P =0.85). Table2PrimersusedforqRT-PCRPrimernamePrimersequence(5 to3 )ODMW%GCcontentTm(C) ActinB-FAGAAAATCTGGCACCACACC4.160645060.4 ActinB-RAGAGGCGTACAGGGATAGCA4.36240.15562.4 Trem2-FCCGGCTGCTCATCTTACTCT3.359955562.4 Trem2-RAGTCATAGGGGCAAGACACC4.261605562.4 Dap12-FGAGACCGAGTCGCCTTATCA3.861025562.4 Dap12-RGTCATGATTCGGGCTCATTT3.76114.14558.4 Cx3cr1-FGCAGATCCAGAGGTTCCCTT3.760935562.4 Cx3cr1-RTAACAGGCCTCAGCCAAATC3.960555060.4 Gfap-FCTGCGGCTCGATCAACTCA3.55748.857.962.3 Gfap-RTCCAGCGACTCAATCTTCCTC3.66277.152.462.7 Nefl-FAGCTGGAGGACAAGCAGAAC4.46209.15562.4 Nefl-RTGCCATTTCACTCTTTGTGG3.560654558.4 Parvalbumin-FCTGGAGACAAAGATGGGGAC4.36240.15562.4 Parvalbumin-RCAGAGAGGTGGAAGACCAGG4.46265.16064.5 Aif1-FAGCAGTGATGAGGATCTGCC4.06182.15562.4 Aif1-RAGCATTCGTTTCAGGGACAT3.96132.14558.4F:Forward;R:Reverse;OD:Opticaldensity;MW:Molecularweight;Tm:Meltingtemperature.Edmonson etal.MolecularAutism 2014, 5 :3 Page4of9 http://www.molecularautism.com/content/5/1/3 PAGE 5 InthePFC,quantificationofmicroglialmarkersdemonstratedsignificantlyincreasedexpressioninautistic samplesof TREM2 DAP12 ,and CX3CR1 ,butnot AIF1 (Figure1).Theexpressionof TREM2 washighestofall microglialmarkers,approximately1.75-foldhigherin autismbraintissuethancontrols( P =0.0016).Thelevels of CX3CR1 and DAP12 were1.50-fold( P =0.0092)and 1.34-fold( P =0.0086)higherinautisticsamplesrelative tocontrols,respectively.Similarly,theexpressionof astrocytemarker GFAP wassignificantlyhigherinautisticbrains(1.70-fold, P =0.0049).Conversely,however, boththepan-neuronalmarker NEFL ,andtheGABAergicinterneuron-specificmarker PVA ,weresignificantly lowerinautisticsamplescomparedtocontrols(0.68fold, P =0.0034;and0.52-fold, P =0.0020,respectively). Inpost-mortemcerebellum,theexpressionofastrocytemarker GFAP wasalsosignificantlyhigherinautism samplesthaninhealthycontrols(2.63-fold, P =0.0022; Figure2).Incontrast,theexpressionofmicroglial markers TREM2 DAP12 CX3CR1 ,and AIF1 werelower inautismtissuethanincontroltissue,withfoldchangesof 0.780( P =0.0056),0.797( P =0.0083),0.659( P =0.0029), and0.808( P =0.0052),respectively.Expressionofneuronal markers PVA and NEFL werealsolowerinautismsamples thanincontrolsamples(0.862-fold, P =0.033;and0.798fold, P =0.013,respectively),aswasfoundinthePFC.DiscussionWhiletherehavebeenmultiplestudiesassessingRNA expressionlevelsinautistictissue,herewereportonthe expressionofmicroglial,astrocyte,andneuron-specific cellmarkersconcurrentlyintworegionsofautistic brains.Ourresultsshowthatglial-specificmarkers demonstratealteredexpressioninautisticbrains.Moreover,theexpressionpatternofthesecell-typespecific markersparallelspreviousfindingsofglialcellnumber andactivationpatternsinautisticbrainassessedvia cell-leveltechniques(discussedbelow).Therefore,this approachmaybeausefulalternativeforassessing activationand/orcellnumbersinpost-mortembrain tissuestudiesofASDpatients. Microgliacell-markerresearchisstillarelativelynew area,andthusthemarkersusedtoquantifymicroglial cellnumberandactivationarestilldebated.Toaddress thisissue,weusedfourdifferentmarkersthatare putativelymicroglial-specific.Ourresultsdemonstrate thatinthePFC,thereisincreasedexpressionofall microglialmarkersassessed,although AIF1 didnot reachstatisticalsignificance.However,previousreports haveshownthat AIF1 expressioninthebrainislow [32],potentiallycontributingtothisresult.Thefinding ofincreasedmicroglialcellmarkersinautisticPFCisin agreementwithanumberofstudiesthathavefoundincreasednumbersandactivationofmicrogliainautistic brains.Forinstance,Morganetal showedincreased microglialdensityindorsolateralPFCgreymatterof ASDbrainsviaIHCandstereology[8],andtheyalso demonstratedthatmicrogliaaremorecloselyassociated withneuronsinautisticdorsolateralPFCthanincontrols[9].Similarly,Tetreaultetal alsodemonstrated increasedmicroglialdensityinthefrontoinsularand visualcortexofautisticbrainsascomparedtocontrols [10].Additionally,anumberofstudieshavespecifically identifiedmicroglialactivationinautisticfrontalcortex, throughbothPETradiotracerimaging[13]andIHC/ cytokineprofilingapproaches[7].Ourresultsprovide furthersupporttothegrowingbodyofevidencedemonstratingincreasedmicroglialnumbersandactivationin autisticPFC,andourcellmarkergeneexpressionresults arelargelyinconcordancewiththesecelllevelstudies. Incontrast,ourcerebellarresultsshowsignificantly lowerexpressionofallfourmicroglialcellspecific Figure1 Expressionofcell-typespecificmarkersinprefrontalcortexsamplesofautisticcasesrelativetocontrols. Errorbarsrepresent 95%confidenceintervals.* P <0.05,** P <0.005,n.s.=notsignificant. Edmonson etal.MolecularAutism 2014, 5 :3 Page5of9 http://www.molecularautism.com/content/5/1/3 PAGE 6 markersinautisticbrains.Whileotherstudieshave identifiedmicroglialactivationinthecerebellumof autistictissue[33],nostudyhasattemptedtospecifically quantifymicroglialcellsinthecerebellumusingthe markersassessedhere,andthereforehistopathologic studiesinthecerebellumareneededtoconfirmthese findings.Onereportdescribedincreasedmicroglialcell activationinthecerebellum,assessedviaHLA-DRstaininginthewhitematterandgranularcelllayerofthe cerebellum[7],andanothershowedincreasedmicroglial activationthroughoutthebrain(althoughmostprominentlyinthecerebellum)usingan invivo PETmetabolic radiotracer[13].However,HLA-DRexpressionin humanmicroglialcellshasbeenshowntobehighly variablebetweenindividuals,anditsexpressionactually decreasesuponcytokinestimulation[34].Moreover,as wepreviouslydiscussed[35],thecerebellumisanatomicallyandphysiologicallyunique;thusmetabolicand pathologicalfindingsinthecerebellummustbeinterpretedwithcaution.Furthermore,ourtissuesamples fromcerebellumcontainedallthreelayersofthecerebellarcortex,asopposedtothemolecularlayeronly. Untildirecthistologicassessmentofthesemicroglial markersareperformedinpost-mortemautisticcerebellum,ourresultsmustbeinterpretedcautiously.However,ourresultsdosuggestthattherearesignificant differencesinmicroglialcellmarkersbetweenASDPFC andcerebellum,perhapsreflectingdifferencesinmicroglialactivationand/orcellnumbersbetweentheseareas inASDbrain. InboththePFCandthecerebellum,therewassignificantlyincreasedexpressionoftheastrocyte-specific marker GFAP inautisticbrains.Thistrendwasmost prominentinthecerebellum,where GFAP expression wasovertwo-foldhigherinASDbrainsthaninhealthy controls.Ourfindingsparallelthoseofpreviousstudies, whichhaveshownincreasedexpressionofGFAPprotein inthecerebellumandcortexofpatientsofautism throughIHCstaining,westernblotting,andmRNAexpression[7,17,36,37].Whilestudieshavenotbeendone toquantifyastrocytenumbersintheautisticcerebellum, ourresultsandthoseofpreviousstudiesprovideevidenceforastroglialreactioninautism. Interestingly,wealsofoundsignificantlydecreased expressionofthepan-neuronalmarker NEFL inboth thePFCandthecerebellumofautisticbrains.This resultisalsosupportedbypreviousstudies,whichhave showndecreased NEFL mRNAexpressionintheanteriorcingulategyrus,motorcortex,andthalamusofautisticbrains[38].However,cell-levelstudiesinautistic brainhaveproducedconflictingresultsaboutneuron numbers.Whilealargebodyofevidencehassuggested thereisalossofneuronsinmanyareasofautistic brains,asrecentlyreviewedin[39],otherstudieshave shownthatyoungautisticbrainsmayhave70%more neuronsinthePFC[40].Importantly,though,istheage ofthepatientattimeofdeath,aslongitudinalstudies havesuggestedthatearlybrainovergrowthinASDs quicklyreversestoaphenotypeofneuronalloss[41]. Consequently,theolderageofpatientsinthisstudymay biasourfindingstowardstheneuronallossspectrumof thedisease. Similarly,wefoundsignificantdecreasesintheGABAergicinterneuron-specificmarker PVA inboththePFCand cerebellumofautisticsamples Despitemanystudies demonstratingdecreasedGABAergiccomponentsacross differentareasoftheautisticbrain,asreviewedin[42],the onepathologicalanalysisofparvalbumin-positiveinterneuronsinASDdidnotidentifydifferencesintheautistic cerebellum[43].However,thisstudyonlyassessedthe molecularlayerofthecerebellarcortex,whereasourtissue samplescontainedallthreelayers.Additionally,while Figure2 Expressionofcell-typespecificmarkersincerebellumsamplesofautisticcasesrelativetocontrols. Errorbarsrepresent 95%confidenceintervals.* P <0.05,** P <0.005. Edmonson etal.MolecularAutism 2014, 5 :3 Page6of9 http://www.molecularautism.com/content/5/1/3 PAGE 7 parvalbumininterneuronshavebeenshowntobe unchangedintheautisticposteriorcingulatecortexand fusiformgyrus[44],andincreasedintheautistichippocampus[45],theyhavenotbeendirectlyassessedinthe autisticPFC. Inadditiontopotentiallyservingasmarkersofglial cellnumbersand/oractivation,manyofthegenes assessedhavespecificputativebiologicalrelevanceto ASDsthemselves.Forinstance,inbrain, Dap12 (also knownas TYROBP )encodesamicroglial-specifictransmembranesignalingpolypeptide[46].Theencoded proteinactsasanactivatingsignaltransductionelement withknownrolesinbrainmyelinationandinflammation [47].Itsreceptor, TREM2 ,encodesamembraneprotein thatfunctionsinmodulatingthebrain simmuneresponseviaproductionofconstitutivecytokines,andis criticalforactivatingmicroglialphagocytosis[48].Rare mutationswithinthesetwogeneshavebeenassociated withNasu-Hakoladisease[49].Nasu-Hakoladiseaseis characterizedneurologicallybynewonsetpsychiatric andcognitivesymptomsinthefourthdecadeoflife, evolvingtomemorylossandcognitivedeclineresemblingAlzheimer sdisease[50].Interestingly,singlenucleotidepolymorphismsinthisreceptorpathwaywere alsorecentlylinkedtoasignificantlyincreasedriskof Alzheimer sdisease,andarethoughttorelatetothe inabilityofmicrogliatoproperlyremoveneurodegenerativedebrissuchasbeta-amyloid[51].Itisintriguingto speculatethatdefectsinthissamepathwayinneurodevelopmentaldisorderssuchasASDsmayalsoresultin defectsinmicroglialphagocytosis,butinthedevelopmentalcontextitistheinabilitytopruneoverabundant synapses,asopposedtoneurodegenerativedebris,that resultsinthebehavioralphenotype. Similarly, CX3CR1 ,alsoknownasthefractalkine receptor,encodesaproteinreceptoronthesurfaceof microgliathatbindstothechemokineCX3CL1(also calledfractalkine);fractalkinefunctionstoinducemicroglialmigrationandadhesionduringphagocytosis[52]. Thispathwaywasrecentlydemonstratedtoplaya criticalroleinthedevelopingbrainofmiceallowingfor migrationofmicrogliatotheirsynaptictargets,where phagocytosisandsynapticrefinementoccur. CX3CR1 knockoutmicehadmoresynapsesoncorticalneurons thanwild-typemice,anddisplayedsubtleneurological deficits[25]. AIF1 ,alsoknownas IBA1 ,encodesaproteinthatis consistentlyup-regulatedinexpressionduringmicroglial activation,andthereforehasbeenusedtodiscriminate betweenrestingandactivatedmicroglia[53].The AIF1 geneislocatedwithinthehighlyvariablelocuscontainingpartsofthemajorhistocompatibilitycomplex,which itselfhasbeenlinkedtoASDsanddysfunctionalmicrogliaphagocytosis[54]. Glialfibrillaryacidicprotein(GFAP)isanintermediate filamentproteinthatisexpressedbyastrocytes,andas discussedabove,hasbeenpreviouslyshowntobeupregulatedinautisticbrainsaswasalsoshownhere. Interestingly,decreasedexpressionofGFAPhasbeen reportedinotherneurodevelopmentaldisorders,suchas Schizophreniaandbipolardisorder[55].GFAP,like allintermediatedfilamentproteins,isimportantin maintainingthecellularcytoskeletoninastrocytes. Thecytoskeletonplaysanumberofkeyfunctional rolesinadditiontomaintainingcellshape,including inter-cellularcommunication,mitosis,andcellularmigrationduringphagocytosis.Mutationsin GFAP are responsibleforAlexander sdisease,araredisorder characterizedbyseveredevelopmentaldelay,increased headsize,andseizures[56]. Overall,ourfindingsdemonstratethattheautistic brainbymid-childhoodhasmolecularchangesconsistentwithincreasedastrocyteexpressionanddecreased neuronalandinterneuronexpressioninboththePFC andcerebellum,withPFC-specificincreasedmicroglial markerexpression.Furthermore,thespecificglial moleculesfoundtobeabnormalinautisticbrainsare intimatelyinvolvedinglialmobilizationandphagocytosispathways,theyhavepreviouslybeenshowntobe criticalfornormalneurodevelopment,andareknown tobecausativeofotherrareneurodevelopmental phenotypes.Thesefindingssupportthenotionthata complexinterplaybetweenglialdysfunctionand neurogenesismayunderlietheclinicalmanifestations ofASDs. Thisstudyhasanumberoflimitationsofnote.Foremostistherelativelymodestsamplesize.Unfortunately, post-mortemhumanbrainresearchingeneralishamperedbythelackofaccessibilitytotissuesamples,and pediatricsamplesinparticulararescarce[26].Therefore, replicationwithalargenumberofsampleswillbe important.However,wechoseqRT-PCRtechniquesin thispilotstudybecauseoftheincreasedsensitivity comparedtowhole-genomemicroarrayorsequencing approaches,andthereforesomeaspectsofthesmall samplesizelimitationareaddressed.Secondly,dueto theinter-individualheterogeneityofthebrain,andin thebrain-bankingmethodologiesusedindistinguishing areasofpost-mortembraintissue,itcannotbeassumed thatallsampleswillderivefromtheexactsame anatomicsitewithinthePFCorcerebellum;thislimitationislargelyunavoidable.Lastly,theapproachofusing cell-typespecificmarkerexpressionasaproxyforcell numberand/oractivationstillneedsverification,by assessingthemconcurrentlywithtraditionalhistopathologic/stereologyanalysis.However,theconcordanceofourresultswithpreviouslypublishedstudies,andthe scarcityofappropriateASDbraintissueandtechnicalEdmonson etal.MolecularAutism 2014, 5 :3 Page7of9 http://www.molecularautism.com/content/5/1/3 PAGE 8 expertise,suggestthismaybeavaluableandsimple alternative screening approach.ConclusionsInsummary,assessmentofglialnumbersandactivation inautismpost-mortembrainresearchishamperedbythe scarcityofappropriately-preservedtissue,andthetechnicalchallengeoftraditionalstereotacticmethods.We showthatglialandneuroncell-typespecificmarkershave mRNAexpressionpatternsthatparallelknowncellular aberrationsinASDs.Ourresultsprovidefurtherevidence thatglialcellsmayplayaroleinthepathogenesisof ASDs,andsuggestthatassessingforglialcell-typespecific markerexpressionmayrepresentaviableapproachto relativelyquantifyglialcellpatternsinASDpost-mortem research.Abbreviations AIF1 : Allograftinflammatoryfactor1;ASD:Autismspectrumdisorder; CX3CR1 :CX3Cchemokinereceptor1;IHC:Immunocytochemistry; PET:Positronemissiontomography;PFC:Prefrontalcortex;PMI:Post-mortem interval;qRT-PCR:Quantitativereal-timepolymerasechainreaction;RIN:RNA integritynumber; TREM2 :Triggeringreceptorexpressedonmyeloidcells2. Competinginterests Theauthorsdeclarethattheyhavenocompetinginterests. Authors contributions CE,MNZ,andOMRconceivedofanddesignedtheanalysis.CEperformed theexperiments.CE,MNZ,andOMRanalyzedresultsandwrotethe manuscript.Allauthorsreadandapprovedthefinalmanuscript. Acknowledgements TheIntramuralResearchProgramattheNationalInstituteofChildHealth andHumanDevelopmentsupportedthiswork.MNZwasalsosupportedby BaylorCollegeofMedicineMSTPandtheNIH-UniversityofCambridgeBiomedicalScholarsProgram.CEwasadditionallysupportedbytheUniversity ofFloridaCollegeofMedicine.Thefundershadnoroleinstudydesign,data collectionandanalysis,decisiontopublish,orpreparationofthemanuscript. Authordetails1LaboratoryofClinicalandDevelopmentalGenomics,NationalInstituteof ChildHealthandHumanDevelopment,NationalInstitutesofHealth,49 ConventDrive,Building49,Room2C078,Bethesda,MD20814,USA.2UniversityofFloridaCollegeofMedicine,1600SWArcherRd,Gainesville,FL 32603,USA.3UniversityofCambridge,RobinsonCollege,GrangeRd, CambridgeshireCB39AN,UK.4BaylorCollegeofMedicineMSTP,OneBaylor Plaza,Houston,TX77030,USA. Received:14September2013Accepted:23December2013 Published:10January2014 References1.AmericanPsychiatricAssociation: DiagnosticandStatisticalManualofMental Disorders. 5thedition.Arlington:AmericanPsychiatricPublishing;2013. 2.CentersforDiseaseControlandPrevention: Prevalenceofautism spectrumdisorders autismanddevelopmentaldisabilitiesmonitoring network,14sites,UnitedStates,2008. MMWR 2012, 3: 1 24. 3.ZoghbiHY: Postnatalneurodevelopmentaldisorders:meetingatthe synapse? 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Submit your next manuscript to BioMed Central and 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 redistribution Submit your manuscript at www.biomedcentral.com/submit Edmonson etal.MolecularAutism 2014, 5 :3 Page9of9 http://www.molecularautism.com/content/5/1/3 |