APP基因

71

Co-localizationandDistributionofCerebralAPPandSP1anditsRelationshiptoAmyloidogenesis

BrianBrocka,RiyazBashaa,KatieDiPalmaa,AmyAndersona,G.JeanHarryb,DeborahC.Riced,BryanMaloneyc,DebomoyK.LahiricandNasserH.Zawiaa,∗

NeurotoxicologyandEpigenomicsLaboratory,DepartmentofBiomedicalandPharmaceuticalSciences,UniversityofRhodeIsland,Kingston,RI02881,USAb

NationalInstituteofEnvironmentalHealthSciences,ResearchTrianglePark,NC27709,USAc

LaboratoryofMolecularNeurogenetics,DepartmentofPsychiatry,InstituteofPsychiatricResearch,IndianaUniversitySchoolofMedicine,791UnionDrive,Indianapolis,IN46202,USAd

MaineDepartmentofHealthandHumanServices,11StateHouseStation,Augusta,ME04333,USA

a

Abstract.Alzheimer’sdiseaseischaracterizedbyamyloid-βpeptide(Aβ)-loadedplaquesinthebrain.Aβisacleavagefragmentofamyloid-βproteinprecursor(APP)andoverproductionofAPPmayleadtoamyloidogenesis.TheregulatoryregionoftheAPPgenecontainsconsensussitesrecognizedbythetranscriptionfactor,specificityprotein1(SP1),whichhasbeenshowntoberequiredfortheregulationofAPPandAβ.TounderstandtheroleofSP1inAPPbiogenesis,hereinwehavecharacterizedtherelativedistributionandlocalizationofSP1,APP,andAβinvariousbrainregionsofrodentandprimatemodelsusingimmunohistochemistry.WeobservedthatoveralldistributionandcellularlocalizationofSP1,APP,andAβaresimilarandneuronalinorigin.Theirdistributionisabundantinvariouslayersofneocortex,butrestrictedtothePurkinjecelllayerofthecerebellum,andthepyramidalcelllayerofhippocampus.ThesefindingssuggestthatoverproductionofAβinvivomaybeassociatedwithtranscriptionalpathwaysinvolvingSP1andtheAPPgene.

Keywords:Amyloid-β,amyloidogenesis,amyloid-βproteinprecursor,Brain,immunohistochemistry,monkey,SP1,transcription

INTRODUCTION

ThepresenceofAbundantdepositionofamyloid-β(Aβ)inthebrainisahallmarkpathologicalfeatureofAlzheimer’sdisease(AD),themostcommonneurode-generativedisorderfoundinelderlypopulations.Amy-loidogenesis,theprocessofAβformation,iscrucialinthedevelopmentofADpathology.Aβisgeneratedbytheproteolysisofatransmembraneproteincalledamyloid-βproteinprecursor(APP)[11,18,22,31,42].

author:NasserH.Zawia,Ph.D.,Departmentof

BiomedicalandPharmaceuticalSciences,UniversityofRhodeIs-land,Kingston,RI02881,USA.Tel.:+14018745909;Fax:+14018745787;E-mail:nzawia@uri.edu.

∗CorrespondingAnatomicalcharacterizationofADbrainshowsatro-phyofspecificregionsofthebrainduetothelossofneurons,neuronalprocessesandsynapsesinregionsas-sociatedwithhigherintellectualfunctioning,includingthecerebralcortexandhippocampus[6,53].

TounderstandtheregulationoftheAPPgene,the5’-untranslatedregion(5-UTR)aswellasthe5’-flankingregulatoryregion,includingthepromoter,oftheAPPgenehavebeencharacterized[21,27,30,39].The5’-flankingregulatoryregionoftheAPPgenelacksachar-acteristicTATAbox,isrichinGCboxelements,andcontainsconsensussitesthatarerecognizedbysever-altranscriptionfactors,includingspecificityprotein1(SP1)[21,27,39].SP1bindstothehuman[15],rhe-sus[43,44]andcynomolgus[33]monkey,andrat[4]

ISSN1387-2877/08/$17.002008–IOSPressandtheauthors.Allrightsreserved

72B.Brocketal./Co-localizationandDistributionofCerebralAPPandSP1anditsRelationshiptoAmyloidogenesis

Fig.1.SP1consensussitesonhuman,rhesusandcynomolgusmonkey,andratAPPpromotersequences.Availablesequencesforhuman(GenBank#D87675),rhesus(GenBank#AF067971),cynomolgus(GenBank#M58727)andrat(RGSCv3.4)APPpromotersequencesfrom-1000basesupstreamofthe+1transcriptionstartsitetothetranslationstart“ATG”werescannedwiththe“TESS”utility[41]forthepresenceofSP1consensussequencesat95%minimumhomology.Sitesareindicatedontherespectivesequences.(A)Humansequence,experimentally-validatedsitesareindicatedatpositions−57/−52[36],+54/+59[8],and+63/+83[51].(B)Rhesusmonkeysequence,(C)Availablecynomolgusmonkeysequence,(D)Ratsequence.Experimentally-validatedSP1siteat−215/−206[16]isindicated.

APPpromoters(Fig.1)andacceleratestheproductionofAPPmRNA,whichcanbefurthersplicedtogener-ateseveralcell-specificspecies[16,35].SP1belongstoafamilyofzincfingerprotein(ZFP)transcriptionfactorsthatincludesothermemberssuchasSP2,SP3andSP4[13,14,17,20,38].Severalstudieshavedemon-stratedthepresenceandimportanceofSP1intheex-pressionofAPPaswellasβ-APPcleavingenzyme1(BACE1),anenzymethatparticipatesinthefirststepofAPPproteolysisandiscrucialfortheformationofAβ[1,5,10,23,26,40].Notably,theprofilesofSP1DNA-bindingactivityoverthelifespanofrodentsarecommensuratewiththepatternsofAPPmRNAexpres-sion,anddepletionofSP1levelsinvitrogreatlydimin-ishesAPPpromoteractivity[1].Inaddition,elevatedlevelsofSP3andSP4haverecentlybeendiscoveredinADbrainsamples[2].

AseriesofstudieshavemappedSP1,APPandAβinrodent,primate,andhumanbrains;however,theseexperimentslimitedanalysistooneortwoofthesepro-teinsatatimeandtospecificregionsofthebrain[3,19,28,45,49,50].PreviousstudieshavealsolocalizedAPPandAβonthetransmembrane,withinorganellesandinthecytoplasmiccompartmentofcorticalneurons[3,16,32,49,50].InadditiontoanAPP,AβandSP1con-nection,theco-localizationofAβandapolipoproteinE(apoE)hasrecentlybeendemonstratedintheperivascu-

lardrainagechannelsofAPP-transgenicmicesuggest-inganassociationofapoEintheperivascularclearanceofAβ[48].

Inthecurrentstudy,weexaminedtheregionaldis-tributionandcellularlocationofSP1,APP,andAβinbothratandnon-humanprimatebrains.WefurtherinvestigatedthehypothesisthatcellsrichinSP1con-tainhighlevelsofAPPanditsamyloidogeniccleavageproduct,Aβ,therebysuggestingtheroleoftranscrip-tionalregulationinamyloidogenesis.MATERIALSANDMETHODSAnimals&tissuepreparation,rats

Timed-pregnantLong-Evanshoodedratswereob-tainedfromCharlesRiverLaboratories(Wilmington,MA).Dayofbirthwasconsideredpostnatalday0(PND0).Torandomizeprenatalandgeneticfactors,pupsfromalllitterswerepooledandnewlitterswerereconstitutedbytherandomselectionof9–10malepupsperlitteronPND1.Littersweremaintainedataconstantlittersizeoverthecourseoflactationwiththeadditionoffemalefillerpupsifoneoftheoriginalmalepupsdied.Theanimalswerehousedatambienttemper-ature(21±2◦C)andrelativehumidity(50±10%)with

B.Brocketal./Co-localizationandDistributionofCerebralAPPandSP1anditsRelationshiptoAmyloidogenesis73

a12-hourlight/darkcycle(0700–1900hours).Foodandwaterwerefreelyavailablethroughoutthestudy.PupswereweanedonPND21andplacedingrouphous-ing(3animals/cage)forthreemonthsandthenhousedindividually.AllanimalprocedureswereconductedaccordingtoaprotocolapprovedbytheInstitutionalAnimalCareandUseCommitteeoftheUniversityofRhodeIsland.

OnPND600(20months),fiveanimals(onefromeachlitter)wereusedforthestudy.Theseani-malsweredeeplyanesthetizedwithanintraperitonealinjectionofsodiumpentobarbital(40mg/kgbodyweight)andtranscardiallyperfusedwith100ccofper-fusionwash(0.8%sodiumchloride,0.4%dextrose,0.8%sucrose,0.023%calciumchlorideand0.034%anhydrous-sodiumcacodylate)followedby100ccper-fusionfix(4%sucrose,4%paraformaldehydeand1.07%anhydroussodiumcacodylate,pH7.2).Thebrainswereremovedandpost-fixedinthesamefix-ative(perfusionfix)overnight.Thecollectedbrainsweresubjectedtosectioning.Coronalsections(40µm)werecollectedusingMultiBrainTechnology(Neuro-ScienceAssociates,Knoxville,TN).Sectionsspan-ningthecerebellumandhippocampusweremaintainedinapreservativefixativeat−20◦C.Thesesectionsweresubjectedtoimmunohistochemicalanalysisasde-scribedinsubsequentsections.Animals&tissuepreparation,monkeys

Twenty-threeyear-oldcynomolgus(Macacafascic-ularis)femalemonkeys,housedattheNIHPoolesville,MD,primatefacilityoverthelastdecadeoflife,wereeuthanizedviaoverdoseofpentobarbital,withaveteri-naryconfirmationofdeath.Thebrainswereexcised,cutalongthemid-sagittalplane,andeachhemispherewascutinto10mmsectionsandrapidlyimmersion-fixedin10%neutralbufferedformalin.Thebrainsectionscontainingthefrontalassociationcortexwererinsedandtakenthroughagradedseriesofethanolconcentrations,processed,andembeddedinparaffin.Eight-micronserialsectionsthroughtheregionwerecollectedonchargedslides.Immunohistochemistry

TodeterminethecellulardistributionofAPP,SP1,andAβ,immunohistochemistrywasconductedus-ingfree-floatingbraintissueof20month-oldrodentsandtheparaffinembeddedbraintissueof23year-oldcynomolgusmonkeys.Free-floatingsectionsof

braintissuesobtainedfromrodentsweretakenfromthepreservativefixativeandbrieflywashedindistilledwatertorinseawaythefixativepriortoPBSrins-es.Bothfree-floatingrodentandparaffinembeddedmonkeybraintissuesweresubjectedtobriefwash-esin1XPhosphateBufferSaline(PBS)and3%Hy-drogenperoxide(H2O2).Afterrinsing,thesectionswereincubatedinPBSwith2%BovineSerumAlbu-min(BSA)and1%TritonX-100blockingsolutionfor30min.,andincubatedinthepresenceofeitherrab-bitIgGspecifictotheNterminus(Fig.2A)ofAPP(1:200;Sigma-Aldrich,St.Louis,MO),monoclonalantibody6E10forAPP/Aβ(Fig.2A)(1:50;Sigma-Aldrich),orrabbitantiserumtoaninternalepitopewithinSP1(Fig.2B)(1:100;SantaCruzBiotechnolo-gy,SantaCruz,CA),overnightat4◦C.SectionswerewashedwithPBSandincubatedalongwiththeappro-priatespecies-specificbiotinylatedsecondaryantibodyagainstmouse/rabbit(1:200;VectorLabs,Burlingame,CA)for30min.ThesectionswereincubatedwithStreptavidin(VectorLabs)for30min,rinsedbrieflywithPBS,andimmunoreactivitydetectedwiththesubstrate,3-3󰀂Diaminobenzidinetetrahydrochloride(VectorLabs).Thefree-floatingrodentsectionsweremountedonmicroscopeslidesusingprocedurespro-videdbyNeuroscienceAssociates.CoverslipsweremountedwithPermanentMountingMedium(VectorLabs)ontherodentandmonkeymicroslides.Immunofluorescentstaining

Free-floatingsections(40microns)ofrathippocam-pus,cortexandcerebellumwereremovedfromstoragesolution,washedwithdistilledwater,1XPBSand3%H2O2andincubatedin2%BSA/1%TritonX-100/PBSblockingsolutionfor30min.Sectionswereincu-batedovernightwiththeprimaryantibodyforeitherAPP(1:200),SP1(1:100),orAβ(1:50),andwashedwithPBS.Thesectionswereincubatedwiththeap-propriatespecies-specificbiotinylatedsecondaryanti-bodyagainstmouse/rabbit(1:200;VectorLabs)for30min.,andrinsedwithbuffer.Sectionswereincubat-edwithTexasRedStreptavidin(1:100;VectorLabs)for30mintofacilitatetheattachmentofthefluorescenttagtotargetproteins.AfterabriefrinseinPBS,sec-tionsweremountedonmicroscopeslidesaccordingtostandardproceduresofNeuroscienceAssociates.TheslidesweresecuredwithcoverslipsusingtheFluores-cenceMountingMedium(VectorLabs)topreservethequalityoffluorescenceimmunoreactivity.

74B.Brocketal./Co-localizationandDistributionofCerebralAPPandSP1anditsRelationshiptoAmyloidogenesis

Fig.2.Epitopesofantisera/antibodiesusedinthisstudy.Schematicdiagramsof(A)APP(indicatinglocationofAβpeptide)and(B)SP1proteinswithbindingsitesofantisera/antibodiesshown.

DoublefluorescencestainingwasconductedforSP1andAPPinthemonkeybrainsections.Thesesec-tionswerefirstimmunostainedforSP1andthenlabeledwithTexasRedusingtheimmunofluorescentproce-dureasdescribedabove.AfterabriefrinseinPBS,thepreviousstepswererepeatedusingtheprimaryAPPantibodylabeledwithFluorescein(green)streptavidin(1:200;VectorLabs)todistinguishthepresenceofAPPfromSP1.AfterabriefrinseinPBS,sectionsweredriedandcover-slippedwithFluorescenceMountingMediumtopreservethequalityoffluorescenceim-munoreactivity.ThisdoublelabelingofSP1(TexasRed)andAPP(Fluorescein)wasperformedinthesamesectiontoshowco-localizationintheneurons.

Inallcases,negativecontrolswererunonslidesfromeachanimalbyomittingtherespectiveprimaryantibodyincubationstep.Nosignalwasevidentafterincubationoftissuesinsecondaryantibodyalone.Microscopy

AllsectionswereexaminedusingaNikonEclipseE600microscopewithanattachedDiagnosticInstru-mentsdigitalcamera,usingSPOTDiagnosticInstru-mentssystemandsoftware(Nikon,Melville,NY).Flu-orescentimageswereviewedusingFITC(green)andRhodamine(red)filtersintheNikonEpi-fluorescencefiltersettovisualizetheimmunoreactivityoftargetpro-teins.Furtherstudiesareneededtoquantifyproteinexpression,cellloss,anddamage.

gusmonkey,andratAPPpromotersequences(Fig.1).Thedatapresentedinthefollowingsubsequentstudieswerebasedontheobservationsmadewithatleast3sections/slidespereachstainingprocessinthepresenceofdifferentprimaryantibodiesraisedagainstepitopesofAPP,Aβ,andSP1proteinsasshowninFig.2.RodentstudiesofSP1,APP,andAβimmunoreactivityindifferentregionsofthebrain

ThestainingpatternsofSP1,APP,andAβwerevisualizedinvariousbrainregionsoftheratincludingcerebralcortex,hippocampus,andcerebellum.Thegeneralpatternofimmunoreactivity,asvisualizedbydensityofstaining,inthecerebralcortexwassimilarforSP1,APP,andAβwithadistributionthroughoutthecorticallayers(Fig.3)andsparsestaininginthemolecularlayer(Fig.3A,B,C).Thatistosay,thecellsthatshowintenseSP1stainingalsoshowasimilargreaterintensityofAPPstaining,whereasareaswhereSP1stainingisabsentarealsodeficientinAPPstaining.Highermagnification(60Xobjective)revealedthatSP1waslocalizedinthesomaofpyramidalandglobularneuronsofmultiplecorticallayers(Fig.3D).APPandAβstainingsweredetectedatthecellmembraneandwithintheorganellesandcytoplasmiccompartmentofcorticalneurons(Fig.3E&F)aspreviouslyreported[3,16,32,49,50].

Inthehippocampus,immunoreactivityforSP1,APP,andAβwasrestrictedtothepyramidalcelllayerofCAregions1–3(Fig.4A–C);howeverdistinctstainingwasnotfoundinthecorpuscallosum(Figure2D–F).DistinctlocalizationofSP1,APP,andAβexpressionwasfoundintheglobularPurkinjeCells(PC)ofthecerebellarlobule;somescatteredstainingwasalsonot-edinthemolecularlayer(Fig.5A–C).Fluorescencela-

RESULTS

ThepremiseofthepresentworkisthatSP1consen-sussitesarepresentonhuman,rhesusandcynomol-

B.Brocketal./Co-localizationandDistributionofCerebralAPPandSP1anditsRelationshiptoAmyloidogenesis75

Fig.3.SP1,APPandAβimmunoreactivityinratcortex.(A–C)SimilarityofstainingpatternofSP1,APPandAβ;(D–F).CellularcharacteristicsofSP1,APPandAβathighermagnification.

Fig.4.SP1,APPandAβimmunoreactivityinhippocampusandcorpuscallosumofrat.(A–C)SimilarityofstainingintheCAregionoftheHippocampus;(D–F)AbsenceofstainingforSP1,APPandAβinCorpusCallosum.

belinginthecerebellumfurtherconfirmedthedefinedneuronalexpressionofthesecomponentsinthePurk-injecelllayers(Fig.5D–F).Itisnoteworthythattheregionsofthecerebrum,hippocampus,andcerebelluminwhichthecellsover-expressedSP1alsocontainedhigherlevelsofAPPandanoverabundanceofAβ.

76B.Brocketal./Co-localizationandDistributionofCerebralAPPandSP1anditsRelationshiptoAmyloidogenesis

Fig.5.SP1,APPandAβimmunoreactivityinratcerebellum.(A–C)SimilarityofSP1,APP,andAßstaininginthePurkinjecelllayerofthecerebellum;(D–F)FluorescentmicrographsofthePurkinjecelllayer.ML=molecularlayer.PL=Purkinjecelllayer.GL=granularcelllayer.

PrimatestudiesofSP1,APP,andAβ

immunoreactivityindifferentregionsofthebrainToinvestigatewhethertheaforementionedresultswitharodentspeciesarespecies-specific,immunos-tainingpatternforSP1,APP,andAβwasfurtheranalyzedbyfluorescencetagginginparaffin-fixedslidespreparedfromthefrontalassociationcortexofcynomolgusmonkeys.Thefluorescencetaggingcon-firmedthattheexpressionoftheseproteinswaslocal-izedprimarilyinneuronalcells(Fig.4).Texasred(SP1)andAPPfluorescein(APP)wereusedtodistin-guishthepresenceofeachprotein,withthemergedim-age(yellow)indicatingoverlappingpresenceforSP1andAPPinthesamecells(Fig.6A–C).6E10stain-ing(APP/Aβ)highlightedareasofplaqueformationinthecorticalsections(Fig.6EandF),indicatingthatasignificantportionofthe6E10-generatedsignalwasextracellularanddistinctfromN-terminalAPPsignal.

DISCUSSION

AseriesofstudieshaveprovidedevidencethatSP1isinvolvedintheregulationoftheAPPgene,withSP1proposedasanessentialtranscriptionfactorintheex-pressionofAPP[1,8,29,34,37,51].Ourpreviousstudysuggestedthat,withaconcurrentactivationofAPP(Fig.7)andBACE1expression,SP1couldalsobein-

volvedinamyloidogenesis.Notably,siRNAknock-downofSP1abolishedabout70%oftheactivityofthehumanAPPpromoterintransfectedPC12cellsandSP1activityacrosslifespanmirrorsthatofAPPgeneexpression[1].Furthermore,theabilityofSP1toreg-ulatetheexpressionofBACE1,thecrucialβ-secretaseassociatedwiththeonsetofAD,hasalsobeendemon-strated[5,46].Recentstudiesalsosuggestedthatintra-cellularaccumulationofAβinneuronalcellsmaypro-moteADpathology[6,7,9,19,47,52].However,theseexperimentslimitedanalysistooneortwoofthesepro-teinsatatimeandtospecificregionsofthebrainofonespecies.Tobridgethisgapofknowledgeandex-tendthestudyfromrodenttoprimatemodels,thecur-rentinvestigationwasdesignedtoexaminethelocal-izationofSP1,Aβ,andAPPindistinctbrainregionsofbothrodentandprimate.OurimmunohistologicalcharacterizationsoughtnottobetakenassoleevidencefortherelationshipbetweentheseproteinsbutaretoprovidecorroborationoftherelationshipbetweenSP1andAPPestablishedinpreviousstudies.Thestainingpatterndisplayedintherodentbrainwassuggestiveofaprimaryneuronallocalization,withanotablelackofSP1andAPPsignalsinglia.Thisneuronallocaliza-tionwaspresentinallregionsexamined.Inaddition,mergedimagesforbothAPPandSP1demonstratedlocalizationwithinasubpopulationofneurons.

ThisstudyshowedthatSP1,APP,andAβimmunore-activityintheratcerebralcortex,hippocampus,and

B.Brocketal./Co-localizationandDistributionofCerebralAPPandSP1anditsRelationshiptoAmyloidogenesis77

Fig.6.SP1,APPandAβimmunoreactivityinmonkeycortex.Intra-neuronalstainingofSP1,APPandAβinthefrontalassociationcortexofcynomolgusmonkey(Macacafascicularis).Thefluorescenceimmunoreactivityof(A)SP1,(B)APP,and(D)Aβ;(C)showstheco-localizationofSP1andAPP;(E&F)revealsthestainingpatternsofAβinsenileplaques.

cerebellumaresimilarandappeartobelocalizedex-clusivelytoneurons(Figs3–6).Inaddition,withinthehippocampus,thehighlydensegranulecellsdonotex-hibitanystainingbeyondbackground.Similarly,inthecerebellum,bothSP1andAPPproteinsareexpressedonlyinthePurkinjecelllayer,withnostaininginthecerebellargranulecelllayer.Ourstudiesalsosuggest-edthattherelationshipbetweenSP1andAPPisnotspecies-specificandisalsopresentinnon-humanpri-mates.Theco-localizationofSP1andAPPinprimatesishighlyrelevantbecause,unlikemuridrodents,agingprimatesareknowntoexpressAβplaques.SP1hasadevelopmentalpatternofexpressionanditsactiva-tionisdependentoncertainstimuli[55].Furthermore,wereSP1ageneraltranscriptionfactor,onewouldex-pectwidespreadinitiationinallitstargetgenes,how-ever,wehavepreviouslyfoundthatSP1targetgenesareselectivelyactivatedundercertainconditions[1,55].Thus,thesedatastronglysuggestthatamyloido-genesisandplaqueformationmaybedriveninpartbyatranscriptionalpathwaywhichinvolvesinteractionsbetweenSP1andtheAPPgene.

Overall,theseinvivoimmunohistochemicalfindingsvalidatetheexistenceofarelationshipbetweenAPPexpressionandSP1andsuggestthatAβproductionoccursincellswhereSP1isstronglypresent.ThepredominantlocalizationofthesethreeproteinsinthesameneuronsandinareasprimarilyaffectedbyADpathologysuggeststhatSP1mayrendersomeneuronsmoresusceptibletoamyloidogenicdamage.Further-more,SP1isalsothoughttohavearoleintheregula-tionofthetaugene(MAPT)andthuscouldalsocon-tributetotheformationofneurofibrillarytangles.Tan-gleshavebeenshowntoco-existwithAβ[12].Thus,environmentalmodelsofADshouldconsiderSP1asapotentialtargetthatcouldinfluencetheprocessofboth

78B.Brocketal./Co-localizationandDistributionofCerebralAPPandSP1anditsRelationshiptoAmyloidogenesis

Fig.7.ModelofSP1actionupontheAPPgene,requiringco-localization.(A)Non-pathogenicmodelofSP1effectsonAPPexpression.SP1transcriptionfactor(blackoval)interactswiththeAPPgenepromotertostimulatetranscriptionatlevelsleavingpotentialSP1consensussites(emptyoval)unstimulated.APPmRNAistranslatedtoAPPprotein,whichisprocessedbysecretasestoproduce(amongotherproducts)Aβpeptide.(B)Inthepathogenicpathway,SP1promoteractivityisincreasedeitheracutelyorinalatent(LEARn)fashionbyearly-lifeexposuretoenvironmentalinsult[1,25].IncreasedSP1expressionleadstogreaterSP1TFavailability,whichstimulateshigherlevelsofAPPtranscription.IncreasedAPPlevelsleadtomoreavailableAPP,whichisprocessedbysecretasestoAβ.

amyloidogenesisandtaudeposition.Takentogethertheseresults,alongwiththerecentlyproposedroleofSP1inalteringlatentearly-lifeassociatedregulation(LEARn)thataffectstheexpressionofgenesassoci-atedwithalater-manifestcondition,couldexplaintheprogressionofamyloidogenesisseeninAD[25].

Basedontheseresults,weproposethefollowingmodelofSP1actionupontheAPPgene,requiringco-localization,asshowninFig.7.Underanon-pathogenicmodelofSP1effectsonAPPexpression,SP1transcriptionfactorinteractswiththeAPPgenepromotertostimulatetranscriptionatnormallevels,leavingsomepotentialSP1consensussitesunstimulat-ed.APPmRNAistranslatedtoAPPprotein,whichisprocessedbysecretasestoproduce(amongotherprod-ucts)Aβpeptide.Underthepathogenicpathway,SP1promoteractivitywouldbeincreasedeitherimmedi-atelyorinalatent(LEARn)[25]fashionbyearly-lifeexposuretoenvironmentalinsult.Suchinsultcouldincludelead(Pb),asshownbyourgroupinmice[1]andmorerecentlyinmonkeys[54].Thiswouldbethroughapotentiallypathogenicsomaticepitype[24].IncreasedSP1expressionleadstogreaterSP1TFavail-ability,whichstimulateshigherlevelsofAPPtranscrip-tion.IncreasedAPPlevelsleadtomoreavailableAPP,whichisprocessedbysecretasestoAβ.

ACKNOWLEDGEMENTS

ThisresearchwassupportedbytheIntramuralRe-searchProgramoftheNIH,NationalInstituteofEn-vironmentalHealthSciences(NIEHS)andbygrants(ES013022andAG027246)fromtheNationalInsti-tutesofHealth(NIH)awardedtoNHZ.Theresearchcorefacilitywasfunded(P20RR016457)bytheNa-tionalCenterforResearchResources(NCRR),acom-ponentofNIH.WorkatDKL’slaboratorywasfund-edbytheAlzheimer’sassociation(ZenithAward)andNIH(AGR0118379andAGR0118884).TheauthorsthankDr.GordonFlakeandDr.Dixie-AnnSawinfortheirhelpfulcommentsonanearlierversionofthismanuscript.References

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