Altered glial marker expression in autistic post-mortem prefrontal cortex and cerebellum

MISSING IMAGE

Material Information

Title:
Altered glial marker expression in autistic post-mortem prefrontal cortex and cerebellum
Series Title:
Molecular Autism
Physical Description:
Mixed Material
Creator:
Catherine Edmonson
Mark N Ziats
Owen M Rennert
Publisher:
Molecular Autism
Publication Date:

Notes

Abstract:
Background: The cellular mechanism(s) underlying autism spectrum disorders (ASDs) are not completely understood, but ASDs are thought to ultimately result from disrupted synaptogenesis. However, studies have also shown that glial cell numbers and function are abnormal in post-mortem brain tissue from autistic patients. Direct assessment of glial cells in post-mortem human brain tissue is technically challenging, limiting glial research in human ASD studies. Therefore, we attempted to determine if glial cell-type specific markers may be altered in autistic brain tissue in a manner that is consistent with known cellular findings, such that they could serve as a proxy for glial cell numbers and/or activation patterns. Methods: We assessed the relative expression of five glial-specific markers and two neuron-specific markers via qRT-PCR. We studied tissue samples from the prefrontal cortex (PFC) and cerebellum of nine post-mortem autistic brain samples and nine neurologically-normal controls. Relative fold-change in gene expression was determined using the ΔΔCt method normalized to housekeeping gene β-actin, with a two-tailed Student’s t-test P <0.05 between groups considered as significant. Results: Both astrocyte- and microglial-specific markers were significantly more highly expressed in autistic PFC as compared to matched controls, while in the cerebellum only astrocyte markers were elevated in autistic samples. In contrast, neuron-specific markers showed significantly lower expression in both the PFC and cerebellum of autistic patients as compared to controls. Conclusions: These results are in line with previous findings showing increased glial cell numbers and up-regulation of glial cell gene expression in autistic post-mortem brain tissue, particularly in the PFC, as well as decreased number of neurons in both the PFC and cerebellum of autistic patients. The concordance of these results with cell-level studies in post-mortem autistic brain tissue suggests that expression of glial cell-type specific markers may serve as a useful alternative to traditional cellular characterization methods, especially when appropriately-preserved post-mortem tissue is lacking. Additionally, these results demonstrate abnormal glial-specific gene expression in autistic brains, supporting previous studies that have observed altered glial cell numbers or activation patterns in ASDs. Future work should directly assess the correlation between cell-type specific marker levels and cell number and activation patterns.

Record Information

Source Institution:
University of Florida
Holding Location:
University of Florida
Rights Management:
All rights reserved by the source institution.
System ID:
AA00020066:00001

Full Text

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.gov†Equalcontributors1LaboratoryofClinicalandDevelopmentalGenomics,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? Science 2003, 5646: 826 – 830. 4.JustMA,CherkasskyVL,KellerTA,KanaRK,MinshewNJ: Functionaland anatomicalcorticalunderconnectivityinautism:evidencefromanFMRI studyofanexecutivefunctiontaskandcorpuscallosummorphometry. CerebCortex 2007, 17 (4):951 – 961. 5.RubensteinJL,MerzenichMM: Modelofautism:increasedratioof excitation/inhibitioninkeyneuralsystems. GenesBrainBehav 2003, 2 (5):255 – 267. 6.CourchesneE,PierceK,SchumannCM,RedcayE,BuckwalterJA,Kennedy DP,MorganJ: Mappingearlybraindevelopmentinautism. Neuron 2007, 56 (2):399 – 413. 7.VargasDL,NascimbeneC,KrishnanC,ZimmermanAW,PardoCA: Neuroglialactivationandneuroinflammationinthebrainofpatients withautism. AnnNeurol 2005, 57 (1):67 – 81. 8.MorganJT,ChanaG,PardoCA,AchimC,SemendeferiK,BuckwalterJ, CourchesneE,EverallIP: Microglialactivationandincreasedmicroglial densityobservedinthedorsolateralprefrontalcortexinautism. BiolPsychiatry 2010, 68 (4):368 – 376. 9.MorganJT,ChanaG,AbramsonI,SemendeferiK,CourchesneE,EverallIP: Abnormalmicroglial-neuronalspatialorganizationinthedorsolateral prefrontalcortexinautism. BrainRes 2012, 1456: 72 – 81. 10.TetreaultNA,HakeemAY,JiangS,WilliamsBA,AllmanE,WoldBJ,Allman JM: Microgliainthecerebralcortexinautism. JAutismDevDisord 2012, 42 (12):2569 – 2584. 11.WatkinsLR,MilliganED,MaierSF: Glialactivation:adrivingforcefor pathologicalpain. TrendsNeurosci 2001, 24 (8):450 – 455. 12.HickmanSE,KingeryND,OhsumiTK,BorowskyML,WangLC,MeansTK,El KhouryJ: ThemicroglialsensomerevealedbydirectRNAsequencing. NatNeurosci2013, 16 (12):1896 – 1905. 13.SuzukiK,SugiharaG,OuchiY,NakamuraK,FutatsubashiM,TakebayashiK, YoshiharaY,OmataK,MatsumotoK,TsuchiyaKJ,IwataY,TsujiiM,Sugiyama T,MoriN: Microglialactivationinyoungadultswithautismspectrum disorder. JAMAPsychiatry 2013, 70 (1):49 – 58. 14.MaezawaI,JinLW: Rettsyndromemicrogliadamagedendritesand synapsesbytheelevatedreleaseofglutamate. JNeurosci 2010, 30 (15):5346 – 5356. 15.DereckiNC,CronkJC,LuZ,XuE,AbbottSB,GuyenetPG,KipnisJ: Wild typemicrogliaarrestpathologyinamousemodelofRettsyndrome. Nature 2012, 484 (7392):105 – 109. 16.CaoF,YinA,WenG,SheikhAM,TauqeerZ,MalikM,NagoriA,SchirripaM, SchirripaF,MerzG,BrownWT,LiX: Alterationofastrocytesand Wnt/ -cateninsignalinginthefrontalcortexofautisticsubjects. JNeuroinflammation 2012, 9 (1):223. 17.LaurenceJA,FatemiSH: Glialfibrillaryacidicproteiniselevatedinthe superior,frontal,parietalandcerebellarcorticesofpatientswithautism. Cerebellum 2005, 4: 206 – 210. 18.FatemiSH,FolsomTD,ReutimanTJ,LeeS: Expressionofastrocytic markersaquaporin4andconnexin43isalteredinbrainsofsubjects withautism. Synapse 2008, 62 (7):501 – 507. 19.MaezawaI,SwanbergS,HarveyD,LaSalleJM,JinLW: Rettsyndrome astrocytesareabnormalandspreadMeCP2deficiencythroughgap junctions. JNeurosci 2009, 29 (16):5051 – 5061. 20.YasuiDH,XuH,DunawayKW,LasalleJM,JinLW,MaezawaI: MeCP2 modulatesgeneexpressionpathwaysinastrocytes. MolAutism 2013, 4 (1):3. 21.YuskaitisCJ,BeurelE,JopeRS: Evidenceofreactiveastrocytes butnotperipheralimmunesystemactivationinamouse modelofFragileXsyndrome. BiochimBiophysActa 2010, 1802 (11):1006 – 1012. 22.UhlmannEJ,ApicelliAJ,BaldwinRL,BurkeSP,BajenaruML,OndaH, KwiatkowskiD,GutmannDH: Heterozygosityforthetuberoussclerosis complex(TSC)geneproductsresultsinincreasedastrocytenumbers anddecreasedp27-Kip1expressioninTSC2+/ cells. Oncogene 2002, 21 (25):4050 – 4059.23.LioyDT,GargSK,MonaghanCE,RaberJ,FoustKD,KasparBK,HirrlingerPG, KirchhoffF,BissonnetteJM,BallasN,MandelG: Aroleforgliainthe progressionofRett ’ ssyndrome. Nature 2011, 475 (7357):497 – 500. 24.ErogluC,BarresBA: Regulationofsynapticconnectivitybyglia. Nature 2010, 468: 223 – 231. 25.PaolicelliRC,BolascoG,PaganiF,MaggiL,ScianniM,PanzanelliP,Giustetto M,FerreiraTA,GuiducciE,DumasL,RagozzinoD,GrossCT: Synaptic pruningbymicrogliaisnecessaryfornormalbraindevelopment. Science 2011, 6048: 1456 – 1458. 26.AbbottA: Tissue-bankshortage:brainchild. Nature 2011, 478 (7370):442 – 443. 27.ZiatsMN,RennertOM: AberrantexpressionoflongnoncodingRNAsin autisticbrain. JMolNeurosci 2013, 49 (3):589 – 593.Edmonson etal.MolecularAutism 2014, 5 :3 Page8of9 http://www.molecularautism.com/content/5/1/3

PAGE 9

28.RansohoffR,PerryV: Microglialphysiology:uniquestimuli,stimuli, specializedresponses. AnnuRevImmunol 2009, 27: 119 – 145. 29.BabaH,NakahiraK,MoritaN,TanakaF,AkitaH,IkenakaK: GFAPgene expressionduringdevelopmentofastrocyte. DevNeurosci 1997, 19 (1):49 – 57. 30.Lpinoux-ChambaudC,EyerJ: Reviewonintermediatefilamentsofthe nervoussystemandtheirpathologicalalterations. HistochemCellBiol 2013, 140 (1):13 – 22. 31.CondeF,LundJS,JacobowitzDM,BaimbridgeKG,LewisDA: Localcircuit neuronsimmunoreactiveforcalretinin,calbindinD-28korparvalbumin inmonkeyprefrontalcortex:distributionandmorphology. JCompNeurol 1994, 341 (1):95 – 116. 32.ImaiY,IbataI,ItoD,OhsawaK,KohsakaS: AnovelgeneIba1inthemajor histocompatibilitycomplexclassIIIregionencodinganEFhandprotein expressedinamonocyticlineage. BiochemBiophysResCommun 1996, 224: 855 – 862. 33.CasanovaMF: Theneuropathologyofautism. BrainPathol 2007, 17 (4):422 – 433. 34.SmithAM,GibbonsHM,OldfieldRL,BerginPM,MeeEW,CurtisMA,Faull RL,DragunowM: M-CSFincreasesproliferationandphagocytosiswhile modulatingreceptorandtranscriptionfactorexpressioninadult microglia. JNeuroinflammation 2013, 10: 85. 35.ZiatsMN,RennertOM: Thecerebelluminautism:pathogenicoran anatomicalbeacon? Cerebellum 2013, 12 (5):776 – 777. 36.BaileyA,LuthertP,DeanA,HardingB,JanotaI,MontgomeryM, RutterM,LantosP: Aclinicopathologicalstudyofautism. Brain 1998, 121 (5):889 – 905. 37.PurcellAE,JeonOH,ZimmermanAW,BlueME,PevsnerJ: Postmortem brainabnormalitiesoftheglutamateneurotransmittersysteminautism. Neurology 2001, 57 (9):1618 – 1628. 38.AnithaA,NakamuraK,ThanseemI,YamadaK,IwayamaY,ToyotaT, MatsuzakiH,MiyachiT,YamadaS,TsujiiM,TsuchiyaKJ,MatsumotoK, IwataY,SuzukiK,IchikawaH,SugiyamaT,YoshikawaT,MoriN: Brain region-specificalteredexpressionandassociationofmitochondriarelatedgenesinautism. MolAutism 2012, 3 (1):12. 39.KernJK,GeierDA,SykesLK,GeierMR: Evidenceofneurodegenerationin autismspectrumdisorder.TranslNeurodegener 2013, 2 (1):17. 40.CourchesneE,MoutonPR,CalhounME,SemendeferiK,Ahrens-BarbeauC, HalletMJ,BarnesCC,PierceK: Neuronnumberandsizeinprefrontal cortexofchildrenwithautism. JAMA 2011, 306 (18):2001 – 2010. 41.CourchesneE,CampbellK,SolsoS: Braingrowthacrossthelifespanin autism:age-specificchangesinanatomicalpathology. BrainRes 2011, 1380: 138 – 145. 42.FatemiSH,BlattGJ: AlterationsinGABAergicbiomarkersintheautism brain:researchfindingsandclinicalimplications. AnatRec(Hoboken) 2011, 294 (10):1646 – 1652. 43.WhitneyER,KemperTL,RoseneDL,BaumanML,BlattGJ: Densityof cerebellarbasketandstellatecellsinautism:evidenceforalate developmentallossofPurkinjecells. JNeurosciRes 2009, 87 (10):2245 – 2254. 44.OblakAL,RoseneDL,KemperTL,BaumanML,BlattGJ: Alteredposterior cingulatecorticalcyctoarchitecture,butnormaldensityofneuronsand interneuronsintheposteriorcingulatecortexandfusiformgyrusin autism. AutismRes 2011, 4 (3):200 – 211. 45.LawrenceYA,KemperTL,BaumanML,BlattGJ: Parvalbumin-, calbindin-,andcalretinin-immunoreactivehippocampalinterneuron densityinautism. ActaNeurolScand 2010, 121 (2):99 – 108. 46.Paradowska-GoryckaA,JurkowskaM: Structure,expressionpatternand biologicalactivityofmolecularcomplexTREM-2/DAP12. HumImmunol 2013, 74 (6):730 – 737. 47.KaifuT,NakaharaJ,InuiM,MishimaK,MomiyamaT,KajiM,SugaharaA, KoitoH,Ujike-AsaiA,NakamuraA,KanazawaK,Tan-TakeuchiK,IwasakiK, YokoyamaWM,KudoA,FujiwaraM,AsouH,TakaiT: Osteopetrosisand thalamichypomyelinosiswithsynapticdegenerationinDAP12-deficient mice. JClinInvest 2003, 111 (3):323 – 332. 48.TakahashiK,RochfordCD,NeumannH: Clearanceofapoptoticneurons withoutinflammationbymicroglialtriggeringreceptorexpressedon myeloidcells-2. JExpMed 2005, 201 (4):647 – 657. 49.KanekoM,SanoK,NakayamaJ,AmanoN: Nasu-Hakoladisease:thefirst casereportedbyNasuandreview. Neuropathology 2010, 30 (5):463 – 470. 50.BianchinMM,MartinKC,deSouzaAC,deOliveiraMA,RiederCR: Nasu-Hakoladiseaseandprimarymicroglialdysfunction.NatRevNeurol 2010, 6 (9):2pfollowing523. 51.JonssonT,StefanssonH,SteinbergS,JonsdottirI,JonssonPV,SnaedalJ, BjornssonS,HuttenlocherJ,LeveyAI,LahJJ,RujescuD,HampelH,Giegling I,AndreassenOA,EngedalK,UlsteinI,DjurovicS,Ibrahim-VerbaasC,HofmanA,IkramMA,vanDuijnCM,ThorsteinsdottirU,KongA,StefanssonK: VariantofTREM2associatedwiththeriskofAlzheimer ’ sdisease. NEnglJ Med 2013, 368 (2):107 – 116. 52.WolfY,YonaS,KimKW,JungS: Microglia,seenfromtheCX3CR1angle. FrontCellNeurosci 2013, 18: 7 – 26. 53.SchwabJM,FreiE,KlusmanI,SchnellL,SchwabME,SchluesenerHJ: AIF-1 expressiondefinesaproliferatingandalertmicroglial/macrophage phenotypefollowingspinalcordinjuryinrats. JNeuroimmunol 2001, 119 (2):214 – 222. 54.NeedlemanLA,McAllisterAK: Themajorhistocompatibilitycomplexand autismspectrumdisorder. DevNeurobiol 2012, 72 (10):1288 – 1301. 55.Johnston-WilsonNL,SimsCD,HofmannJP,AndersonL,ShoreAD,Torrey EF,YolkenRH: Disease-specificalterationsinfrontalcortexbrainproteins inschizophrenia,bipolardisorder,andmajordepressivedisorder.The StanleyNeuropathologyConsortium. MolPsychiatry 2000, 5 (2):142 – 149. 56.LiR,JohnsonAB,SalomonsG,GoldmanJE,NaiduS,QuinlanR,CreeB, RuyleSZ,BanwellB,D ’ HoogheM,SiebertJR,RolfCM,CoxH,ReddyA, Gutirrez-SolanaLG,CollinsA,WellerRO,MessingA,vanderKnaapMS, BrennerM: Glialfibrillaryacidicproteinmutationsininfantile,juvenile, andadultformsofAlexanderdisease. AnnNeurol 2005, 57 (3):310 – 326.doi:10.1186/2040-2392-5-3 Citethisarticleas: Edmonson etal. : Alteredglialmarkerexpressionin autisticpost-mortemprefrontalcortexandcerebellum. MolecularAutism 2014 5 :3. 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