Reduction of low-density lipoprotein receptorrelated protein (LRP1) in hippocampal neurons does not proportionately redu...

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Title:
Reduction of low-density lipoprotein receptorrelated protein (LRP1) in hippocampal neurons does not proportionately reduce, or otherwise alter, amyloid deposition in APPswe/PS1dE9 transgenic mice
Series Title:
Alzheimer’s Research & Therapy
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English
Creator:
Xu, Guilian
Green, Cameron C.
Fromholt, Susan E.
Borchelt, David R.
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BioMed Central
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Introduction: The low-density lipoprotein receptor-related protein (LRP1) and its family members have been implicated in the pathogenesis of Alzheimer’s disease. Multiple susceptibility factors converge to metabolic pathways that involve LRP1, including modulation of the processing of amyloid precursor protein (APP) and the clearance of Ab peptide. Methods: We used the Cre-lox system to lower LRP1 levels in hippocampal neurons of mice that develop Alzheimer-type amyloid by crosses between mice that express Cre recombinase under the transcriptional control of the GFAP promoter, mice that harbor loxp sites in the LRP1 gene, and the APPswe/PS1dE9 transgenic model. We compared amyloid plaque numbers in APPswe/PS1dE9 mice lacking LRP1 expression in hippocampus (n = 13) to mice with normal levels of LRP1 (n = 12). Student t-test was used to test whether there were significant differences in plaque numbers and amyloid levels between the groups. A regression model was used to fit two regression lines for these groups, and to compare the rates of Ab accumulation. Results: Immunohistochemical analyses demonstrated efficient elimination of LRP1 expression in the CA fields and dentate gyrus of the hippocampus. Within hippocampus, we observed no effect on the severity of amyloid deposition, the rate of Ab40/42 accumulation, or the architecture of amyloid plaques when LRP1 levels were reduced. Conclusions: Expression of LRP1 by neurons in proximity to senile amyloid plaques does not appear to play a major role in modulating the formation of these proximal deposits or in the appearance of the associated neuritic pathology.

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University of Florida
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University of Florida
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doi - 10.1186/alzrt110
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AA00012622:00001


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RESEARCH OpenAccessReductionoflow-densitylipoproteinreceptorrelatedprotein(LRP1)inhippocampalneurons doesnotproportionatelyreduce,orotherwise alter,amyloiddepositioninAPPswe/PS1dE9 transgenicmiceGuilianXu1*,CameronCGreen1,2,SusanEFromholt1andDavidRBorchelt1*AbstractIntroduction: Thelow-densitylipoproteinreceptor-relatedprotein(LRP1)anditsfamilymembershavebeen implicatedinthepathogenesisofAlzheimer sdisease.Multiplesusceptibilityfactorsconvergetometabolic pathwaysthatinvolveLRP1,includingmodulationoftheprocessingofamyloidprecursorprotein(APP)andthe clearanceofA b peptide. Methods: WeusedtheCre-loxsystemtolowerLRP1levelsinhippocampalneuronsofmicethatdevelop Alzheimer-typeamyloidbycrossesbetweenmicethatexpressCrerecombinaseunderthetranscriptionalcontrolof theGFAPpromoter,micethatharborloxpsitesintheLRP1gene,andtheAPPswe/PS1dE9transgenicmodel.We comparedamyloidplaquenumbersinAPPswe/PS1dE9micelackingLRP1expressioninhippocampus(n=13)to micewithnormallevelsofLRP1(n=12).Studentt-testwasusedtotestwhetherthereweresignificant differencesinplaquenumbersandamyloidlevelsbetweenthegroups.Aregressionmodelwasusedtofittwo regressionlinesforthesegroups,andtocomparetheratesofA b accumulation. Results: ImmunohistochemicalanalysesdemonstratedefficienteliminationofLRP1expressionintheCAfieldsand dentategyrusofthehippocampus.Withinhippocampus,weobservednoeffectontheseverityofamyloid deposition,therateofA b 40/42accumulation,orthearchitectureofamyloidplaqueswhenLRP1levelswere reduced. Conclusions: ExpressionofLRP1byneuronsinproximitytosenileamyloidplaquesdoesnotappeartoplaya majorroleinmodulatingtheformationoftheseproximaldepositsorintheappearanceoftheassociatedneuritic pathology.IntroductionAmyloiddepositionisakeypathologicaleventinAlzheimer sdisease.Alargebodyofevidencesuggeststhat lowdensitylipoprotein(LDL)receptorrelatedprotein (LRP1),maybeakeymediatorofamyloiddeposition. AsamemberoftheLDLreceptorfamily,LRP1isa large,multifunctional,endocyticreceptorthatishighly expressedinneurons(reviewedbyAndersen[1]),activatedastrocytes[2-4],andmi croglia[5].Directbinding ofLRP1totheamyloidprecursorprotein(APP)has beenshowntoaffectendoproteolyticprocessingofAPP toincreasetheproductionofA b 42peptides[6],which arethemajorconstituentofamyloiddeposits[7].LRP1 canpromoteA b productionbyalteringtheprocessing ofAPPthroughinteractionsviatheKunitzprotease inhibitor(KPI)domain(isoformsAPP751orAPP770) [6].Althoughthenon-KPI-APPisoformcanweakly bindtoLRP1throughcytoplasmicadaptorproteins, *Correspondence:xugl@ufl.edu;drb1@ufl.edu1DepartmentofNeuroscience,SantaFeHealthCareAlzheimer sDisease Center,CenterforTranslationalResearchinNeurodegenerativeDisease, McKnightBrainInstitute,UniversityofFlorida,Gainesville,FL32610,USA FulllistofauthorinformationisavailableattheendofthearticleXu etal Alzheimer sResearch&Therapy 2012, 4 :12 http://alzres.com/content/4/2/12 2012Xuetal.;licenseeBioMedCentralLtd.ThisisanopenaccessarticledistributedunderthetermsoftheCreativeCommons AttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse,distribution,andreproductionin anymedium,providedtheoriginalworkisproperlycited.

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suchasFE65[8],APP695processingmaynotbeinfluencedassignificantlybyLRP1.Additionally,LRP1has alsobeenreportedtomediateexportofA b acrossthe blood-brainbarrier(reviewedby[9]);LRP1inendothelialcellsmaybinddirectlytoA b 1-40andexportit acrosstheblood-brainbarrier[10].Consistentwiththis notion,reducingLRP1levelsbyantisensetreatment reducesblood-brainbarrierclearanceofA b 42[11]. PolymorphismsintheLRP1genehavebeenassociated withincreasedriskforAlzheimer sdisease[12]and LRP1levelsaresignificantlyreducedinADpatients [13].Morerecently,Sehgalandcolleaguesreportedthat anextractof Withaniasomnifera inducesarapidclearanceofamyloidpathologywithaco-incidentinduction ofLRP1expressioninliver[14].OtherproteinsimplicatedinamyloidogenesishavebeenidentifiedasinteractingwithLRP1[15],suchasapolipoprotienE(ApoE) and a 2-macroglobulin.Bothoftheseproteinsare ligandsofLRP1andhavebeenreportedtomediatethe clearanceofA b [13,16-19].Moreover,ApoE,whichisa majorligandforLRP1,modulatesthedistributionand characterofamyloiddepositio n[20].Collectively,these datasuggestthatLRP1couldbeacrucialfactorinAlzheimer sdiseaseandthereceptorcharacteristicsof LRP1makethisproteinapotential drugable target whereinfluencingthebindingofLRP1toanyofits ligandscouldbeausefultherapeutic. Todate,therehavebeenfewstudiesthatexaminethe roleofLRP1 invivo usinganimalmodelsbygenetic manipulationofLRP1expression.ConstitutiveinactivationofLRP1islethal[21].We[22]andothers[23]have usedmicewithtargeteddeletionofLRP1receptorassociatedprotein(RAP),whichregulatesLRP1maturation, asameanstoreduceLRP1levelsinmousemodelsof Alzheimer sdisease.OnegrouphasreportedthathomozygousdeletionofRAP,whichlowersLRP1levelsby 75%[24],resultsinincreasedA b depositioninPDAPP mice[23].Similarly,weobservedthatheterozygous deletionofRAPalsoincreasedamyloiddeposition,in theAPPswe/PS1dE9model;however,inourcaseLRP1 levelswerenotsignificantlylowered[22].Expressionof anLRP1mini-receptoratapproximately3.7-foldover endogenousLRP1levelsinthePDAPPtransgenicmodel wasreportedtohavenoeffectonamyloidburden[25]. Collectively,thesestudiesillustratetheinconsistent in vivo findingsregardingLRP1andamyloidogenesisin transgenicmicethatover-expressmutantAPP(withor withoutco-expressionofmutantpresenilin). TofurtherprobetheroleofLRP1inamyloidogenesis, weusedCre/Loxtechnologytoconditionallyreduce LRP1expressioninthebrainsofmicethatco-express mutantAPP(APPswe)andmutantpresenilin1 (PS1dE9).Forthesestudiesweusedmicethatexpress Creunderthecontroloftheglialfibrillaryacidicprotein (GFAP)[26].Inpreviousstudieswiththeseanimals, othershavedemonstratedCre mediatedrecombination inbothneuronsandglialthroughoutthebrain[27].In ourhands,GFAP-mediatedexpressionofCreproduced incompleterecombination,suchthatthelevelsofLRP1 intotalbrainwerereducedapproximately50%.However,wedeterminedthatLRP1immunostaininginCA andgranuleneuronsofthehippocampuswasvirtually eliminated.Withinthehippocampus,weobservedno obviouschangeinthelevelofneuriticpathologyor amyloidburdenbylossofLRP1.MaterialsandmethodsAnimalsMicewithtargetedLoxPsitesintroducedintointronic andpromoterregionsoftheLRP1genewereobtained fromJoachimHerz[28,29],calledLRP1lox/lox.TransgenicmiceexpressingGFAP-Crewereobtainedfrom JacksonLaboratories(BarHarbor,ME,USA;Strain: FVB-Tg(GFAP-cre)25Mes /J;StockNumber:004600) [26].Theseanimalswereback-crossedtoC57BL/6J miceforatleastfourgenerationstoreducetheFVB straincontributionbeforebreedingtoLRP1lox/lox mice.LRP1lox/loxmice wereinparallelbredto APPswe/PS1dE9(line85)mice[30](bothwithC57BL/ 6Jbackground)toproducemicehomozygousforthe LRP1loxplocus.Togeneratetripletransgenicmice,we matedmicetransgenicforGFAP-Creandhomozygous forLRP1loxptomicetransgenicforAPPswe/PS1dE9 andhomozygousforLPRloxp.FVB/NJmaleandfemale micewerepurchasedfromJacksonLaboratories(stock number:001800)toproducefostermothers.Eventually, animalswiththegenotypesofAPPswe/PS1dE9(+)/LRP1 lox/loxwith/withouttheGF AP-Cregenewereusedfor thestudy.AllofthemicewerehousedinstandardSPF cagesfor9to18monthsbeforeharvesting.Allproceduresinvolvinganimalhandlingandprocessingwere approvedbytheUniversityofFloridaInstitutionalAnimalCareandUseCommitteeandincompliancewith theNationalInstitutesofHealthguidelines.WesternblotanalysisLevelsofLRP1wereassessedbyWesternblot,using standardmethodswehavepreviouslydescribed[31]. AliquotsofthePBSbrainhomogenates(100 l)from 14-to15-month-oldmiceweremixedwith10 lof 10%SDS,centrifugedatthet opspeed(approximately 10,000xg)inabenchtopmicrofugefor10minutes, andthen30 lofthesupernatantwasmixedwith10 l 4xLaemmlibufferandboiledfor5minutes.Anamount ofsampleequalto50 gtotalprotein(byBCAassay) wereloadedperlaneforSDS-PAGEandtransferredto nitrocellulosemembranes. Blotswereincubatedwith rabbitpolyclonalantibody377 a LRP1(1:1,000)andXu etal Alzheimer sResearch&Therapy 2012, 4 :12 http://alzres.com/content/4/2/12 Page2of14

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thenreprobedwiththem/hSOD1antibody(1:3,000)to gaugeproteinloading[32].TheECLsignalwascaptured andquantifiedusingtheL AS-3000ima gingsystem (FUJIFILMLifeScience,Tokyo,Japan).TissuepreparationforhistologyMiceweredeeplyanesthetizedbyinjectionanesthetic (3%bodyweightof1.2%Tribromoethanol(Avertin Sigma,St.Louis,MO,USA)beforetranscardialperfusionwithcoldphosphatebu fferedsaline(PBS,pH7.4). Thebrainswerethenremovedandcutsagittallydown themidline;onehemi-brainwasfrozenondryicefor biochemistryassay,theotherhemi-brainwasimmersion fixedin4%paraformaldehydeinPBS.After48hoursin fixationat4C,brainsweretransferredtoPBS,andthen 30%sucroseinPBSforseveraldaysbeforecryostatsectioningat30 mthickness.Fromthefirstcompletesectionclosesttomid-line,10sectionspergroupwere storedineachwellof24-well-platesandtheentire hemi-brainwassectioned.Sectionswerekeptinantifreezesolution(100mMsodiumacetate,250mMpolyvinylpyrrolidone,40%ethyleneglycol,pH6.5)at-20C untilstaining.ImmunostainingImmunostainingof30 mfreefloatingsectionswasperformedaccordingtoastandardprotocol.Sectionswere washedinPBStoremovetheanti-freezesolution,and thenblockedinPBScontaining5%normalgoatserum with0.1%TritonX-100before incubatingwithantibodiesagainstLRP1(rabbitpolyclonal377 a LRP1 (1:1,000)),glialfibrillaryacidicprotein(GFAP)(mouse monoclonal,1:1,000,Millip ore,Billerica,MA,USA), neuronalantigenNeuN(mousemonoclonal,1:1000, Millipore)or6E10(mousemonoclonal,1:1,000,Covance,Princeton,NJ,USA).Secondaryantibodiesofgoat anti-rabbitandgoatanti-mouse,conjugatedwithAlexa 594orAlexa488(Invitrogen,Carlsbad,CA,USA)were usedtovisualizeprimaryantibodybinding.Afterwashingtoremoveunboundsecondaryantibody,thesections werethenmountedonmicroscopeslidesanddriedin air.Aquickdipin0.1%SudanBlackBsolution(in70% ethanol)for5to10minuteswasusedtoblockthe auto-fluorescenceoflipofuscinthentheslideswere washedin70%ethanol,thenwater,driedandcovered byProLongGoldantifadereagentwithDapi(Invitrogen).AnOlympusDSU-IX81SpinningDiscConfocal microscope(Tokyo,Japan)wasusedtocapturethe images.ThioflavinSstainingandsilverstainingofamyloid plaquesOnesectionfromeachwell(seeaboveTissuepreparationforhistology)wasrandomlyselectedforThioflavin SstainingaccordingtotheGunternstandardprotocol [33]withsmallmodificatio n[34].Fluorescentphotos weretakenusing5xobjectivesbyaCanondigitalcamera(Tokyo,Japan).Silverimpregnationstainingwas performedonthesame30 mfloatingsectionsbymodifiedHirano smethod[35,36].Briefly,sectionswere washedandmountedonslidestodryasthoseforThioflavinSstaining,andthenthestepsdescribedforsilver impregnationmethodsofparaffinslideswerefollowed [36].AmyloidplaquequantificationFoursectionsperanimal(from300to1,500 mlateral tomidline)stainedwithThioflavinSwereusedtoquantifytheamyloidplaquelevels.Theimagesofthehippocampuswereoutlinedandthenumbersofamyloid plaquesweremanuallycountedbyanobserverblinded togenotype.Theimagesofeachhippocampuswereoutlinedandquantifiedthreetimesonthreedifferentdays toreducebiasandthenanalyzed.Thenumbersofamyloidplaquesineachsectionwereaveragedandthen numbersofplaquesfromthefoursectionsfromdifferentlevelsofthebrainwereaveragedtoproducethe finalnumberofamyloidplaques/sectioninthehippocampusofeachanimal.A b ELISAThefrozenhemi-brainfromeachtransgenicanimalwas weighed,thenhomogenizedbysonicationinfive volumesbyweightinPBSwith1proteaseinhibitor cocktail(SigmaCat.P8340).A100 laliquotofthisPBS brainhomogenatewasusedfo rELISAanalysis.Protein aggregatesinthePBShomo genateweredenaturedby theadditionof150 lof8.0Mguanidine-HCltoafinal concentrationof4.8M,andmixedatroomtemperature forthreetofourhoursbeforestorageovernightat-20 C.Thedenaturedsampleswerethenfurtherdiluted 200-foldinreactionbuffer(PBScontaining5%BSA, 0.03%Tween-20,and1proteaseinhibitorcocktail (Sigma)).Sampleswerefinallypreparedforapplication totheELISAmicroplatebymixingwithanequal volumeofStandardDilutionBufferprovidedbythe manufacturersupplementedwith1proteaseinhibitor cocktail.Aliquotsofthesesampleswereassayedfor bothAb 40andAb 42usingcommerciallyavailablesandwich-ELISAsaccordingtothemanufacturer smanual (Invitrogen).StatisticalanalysisoftheELISAdataWecomparedthedifferencesamongthemeansofAb 40 and42levelsamongthesameagegroupsandthedistributionofthelevelsacrossallages.Thecomparisonin thesameagegroupbetweentwodifferentgenotypes (GFAP-Crepositiveornegative)wascarriedoutusingXu etal Alzheimer sResearch&Therapy 2012, 4 :12 http://alzres.com/content/4/2/12 Page3of14

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Excelwiththetwo-tailStudent t -testwithbothequal andunequalvariance.TheA b levelswithineachage groupweregraphedinExcelandlinearregressionlines wereadded.ExcelAnalysisToolPakadd-inwasusedfor regressionanalysis.IBMSPSSStatistics19[(trialversion),Somers,NY,USA]wasusedtotestthesignificancebetweentheregressioncurvesbygenotypes.A P valueof<0.05wasconsideredstatisticallysignificant.ResultsAstrainofGFAP-CremicethatexpressesCreearlyin neurogenesiswasusedtocrosstothehomozygousmice withLRP1loxpsiteswiththeexpectationofcomplete recombinationofflox edgenesinneuronsaswellasglia [27].TheGFAP-Cremicethatwereusedforthisstudy fromtheJacksonLaboratoryweremaintainedintheFVB strain.Tomakethemcompatiblewithourstrainsofmice, weback-crossedthemtoC57BL/6JmiceforfourgenerationsbeforecrossingtoLRP1lox/loxmice,andthenwe continuedtocrossthemtoC57BL/6JcongenicAPPswe/ PS1dE9mice(line85).Micethatweretransgenicfor APPswe/PS1dE9andLRP1loxp/loxpwerematedtomice thatweretransgenicforGFAP-CreandLRP1loxp/loxp, ormicethatweretransgenicforallthreealleles(APPswe/ PS1dE9,GFAP-CreandLRP1loxp/loxp)werematedto miceonlytransgenicforLRP1loxp/loxp.Atotalof54 breedingcagesweresetupoverthecourseofthestudyto generatethemiceweultimatelyexamined.Morethan20 ofthesecagesfailedtogenerateanyoffspring,5cagesgeneratedafewoffspring,butthemothersfailedtocarefor thelittersandnoneoftheoffspringlivedtoweaningage (fourweeksold).Oftheanimalsofthedesiredgenotype (APPswe/PS1dE9/LRP1loxp/loxp/GFAP-Cre)thatwere produced,almosthalfdiedsuddenlyinthefirstnine monthsoflife(seeAdditionalfile1TableS1). Becauseofthereducedviabilityofthemiceofthe desiredgenotypeandthepoormotheringoftheC57BL/ 6Jfemales,webeganafosterbreedingprogram.FVB/NJ females,(whichareverygoodmothers),wereusedto fosterallnewbornoffspringinanefforttoincreasethe frequencyofanimalsthatsu rvivetoweaning.Thisfoster-motheringprogramwassuccessfulinproducinga greaternumberofanimalsofthedesiredgenotypethat survivedtoweaning.Surprisingly,thesurvivalrateof theoffspringthatwerefoste r-motheredwasalsomuch improved.Atotalof167offspringwereproducedinthe foster-motheringprogramofwhichonly7diedbefore ninemonthsofageandonly1ofthese7wastriple transgenicgenotype(seeAdditionalfile1TableS1). Overall,thedistributionofthemicewithallfourofthe genotypeswasmuchclosertoMendeliandistribution (25%ofeachgenotypeexpected).Atpresent,wehave noexplanationforthisobservation,butitappearsthat thefoster-motheringthatimprovedsurvivaltoweaning agetransferredtoimprovedresistancetotheunderlying problemthatcausedthesuddenearlydeathafterweaning.Ultimatelymostofthemicethatwerestudiedin thisreportweregeneratedbyfoster-mothering.GFAP-CreexpressioninLRP1lox/loxmicelowersLRP1 levelsinhippocampusUsingthebreedingscheme describedabove,weproducedmicetransgenicforGFAP-Cre,APPswe/PS1dE9, andhomozygousfortheflox edLRP1alleles.Allmice describedinthisworkwerehomozygousforthe LRP1loxallele.Noobviousdevelopmentalabnormalities wereobservedinthebrainsofmicethatweretransgenic forGFAP-Crewhetherornottheywerealsotransgenic fortheadditionalAPPswe/PS1dE9transgenes(not shown).Immunoblotsofhemi-brainhomogenates demonstratedthatLRP1levelswerereducedbyapproximately50%inmicethatweretransgenicforAPPswe/ PS1dE9andGFAP-Creascomparedtomicethatwere transgeniconlyforAPPswe/PS1dE9(Figure1). Inimmunohistochemicalstainsofhippocampusof micethatweredoubletransgenicforAPPswe/PS1dE9 andGFAP-CreintheLRP1lox/loxbackground,we observedverylittlestainingforLRP1inhippocampal neuronsintheCAfieldsanddentategyrus(DG)(Figure 2;Additionalfile2FigureS1).Incontrast,inAPPswe/ PS1dE9/LRP1lox/loxmiceweobservedstrongimmunoreactivityforLRP1intheseregions(Figure2).This patternofreactivitymatcheswhatwouldbeexpected basedonthelevelsofmRNAinthesestructures(Additionalfile3FigureS2).Inthecortexofmiceofthe samegenotype,weobservedthatmostneurons,identifiedbyNeuNstaining,remainedpositiveforLRP1 immunoreactivity,albeitatwhatappearedtobeslightly lowerlevels,indicatingthattherecombinationofthe flox edLRP1allelewaslesscompleteincorticalneurons (Figure3).Thus,althoughthelevelofrecombinationin thecortexwaslessthanreportedforotherflox edalleles [27],thelevelofrecombinationinthehippocampuswas muchmorerobustandthuswefocusedouranalysison amyloiddepositionwithinthehippocampus. Basedonpreviousstudies,weexpectedthatLRP1would beexpressedatsomelevelinastrocytes,particularly activatedastrocytes[2-4].NoLRP1immunoreactivity couldbedetectedinrestingastrocytesofanygenotype (Additionalfile4FigureS3).Inthehippocampusof nine-month-oldGFAP-Cre/APPswe/PS1dE9/LRP1lox/ loxmice,weobservedthatamyloiddepositsstillformed atarelativelyhighfrequency(seebelow)andaround thesedepositsweobservedactivatedastrocytes.However,wefailedtodetectLRP1inactivatedastrocytes surroundingtheseplaques(Figure4).Withinthese depositsweobservedLRP1immunoreactivitythatwas reminiscentofneuriticprofiles(Figure4).TheseLRP1Xu etal Alzheimer sResearch&Therapy 2012, 4 :12 http://alzres.com/content/4/2/12 Page4of14

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immunoreactiveneuriticprofilesweremostlyabsent withinthemolecularlayerofthehippocampusof APPswe/PS1dE9/LRP1lox/loxmicethatwerepositive forGFAP-Cre. Collectively,thesedataindicatethatthelevelsofLRP1are inherentlylowinastrocytes,includingactivatedastrocytes, andthatexpressionofCreviatheGFAPpromoterproducesefficientrecombinationoftheLRP1geneinneurons Figure1 ThelevelofLRP1issignificantlylowerinthemiceexpressingGFAP-CrewithLRP1lox/lox .NineAPPswe/PS1dE9GFAP-Cre LRP1lox/loxmicewithdifferentgenotypeswereusedtodeterminethelevelsofLRP1.TheSDS-solublefractionsofwholebrainlysates (homogenizedinPBS)containinganequalamountoftotalprotein(50 g)wasloadedineachlane.Antibody a 377toLRP1(1:1,000,agiftfrom Dr.Herz,UniversityofTexasSouthwesternMedicalCenter)wasusedtodetectLRP1level.The85kDabandistheendoproteolyticallycleaved matureLRP1.MouseSOD1(14kDbands)wasusedasendogenousloadingcontrol.TheratioofbandintensityofLRP1toSOD1wasquantified, standardizedandplottedbelow.Thestandarddeviationsofthemeasurementsareshownbyerrorbars. Xu etal Alzheimer sResearch&Therapy 2012, 4 :12 http://alzres.com/content/4/2/12 Page5of14

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ofthehippocampus.Wepresumethatanyexpressionof LRP1inastrocyteswasalsoreducedinLRP1lox/loxmice thatweretransgenicforGFAP-Crebuttheabsenceof detectablestainingintheabsenceofGFAP-Cremakesit impossibletoconfirmthisassumption.HippocampaldepositionofA b isnotalteredbyreduction inLRP1levelsInthehippocampusofnine-month-oldAPPswe/ PS1dE9/LRP1lox/loxmice,weobservednoobviousdifferenceinthenumberofAb immunoreactivedeposits Figure2 LRP1levelsarereducedinhippocampalneuronsofLR P1lox/loxmicethataretransgenicforGFAP-Cre .Eight-month-old APPswe/PS1dE9GFAP-CreLRP1lox/loxlittermateswereusedforimmunostaining.AllmicewerehomozygousforLRP1loxpallelesand positiveforAPPswe/PS1dE9transgenes.ThepresenceorabsenceofGFAP-Creisnotedabovetheimages.LRP1 a 377antibody(1:1,000,red)was usedtostainLRP1;NeuN(1:1,000,green)wasusedtostainneurons;Dapiinthemountingreagentwasusedtodisplaynuclei.Confocal microscopeimagesshowedmostoftheneuronsinCAfields (A) andthedentategyrus (C and E) ofAPPswe/PS1dE9LRP1lox/loxGFAP-Cre micedonotexhibitLRP1immunoreactivity.ControlmicelackingtheGFAP-CretransgeneshowedintensestainingofLRP1inhippocampal neurons (B,D ,and F) .Threetofourpairsofmicepergenotypewereusedforimmunostainingandfourtofivesectionsperanimalwere analyzed. Xu etal Alzheimer sResearch&Therapy 2012, 4 :12 http://alzres.com/content/4/2/12 Page6of14

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inmicethatwerepositiveforGFAP-Creascomparedto micelackingGFAP-Cre(Figure5).Withthelackofan obviousdifferenceinamyloidplaqueburdenin APPswe/PS1dE9micelackingLRP1expressioninthe hippocampus,wesoughtto carefullyquantifyplaque numbersinthehippocampusofmultiplesectionsat multiplelevels.TissuesectionswerestainedwithThioflavinSasameansofdetectingamyloiddepositsand thenthehippocampuswasoutlinedandthenumberof depositscounted(seeAdditionalfile5FigureS4).The numberofdepositsinthehippocampusofAPPswe/ PS1dE9/LRP1loxp/loxpmice(n=13)weresimilarto APPswe/PS1dE9/LRP1loxp/loxp/GFAP-Cremice(n= 12),meaningthatsignificantlyreducingLRP1expression inthehippocampushadnoobviouseffectonthenumberordistributionofamyloiddepositsinthisstructure. Femalemiceofallgenotypesshowedslightlyhigher levelsofamyloiddepositsascomparedtomales(Figure 5).WeassessedwhetherlossofLRP1alteredanyofthe morphologicalfeaturesoftheplaquesuchasdensityof neuriticstructures,findingnoobviousdifferencebysilverstaining(Additionalfile6FigureS5).TotalA b levelsdonotchangewhenLRP1levelsare reducedSandwichELISAswereusedtomeasurethetotal amountofA b 40andA b 42inthebrainsofthesemice asanothermeanstoassessamyloidburden.Tocompletelysolubilizedifferentformsofthepeptides,aggregated orotherwise,homogenateswereincubatedforseveral hoursinguanidine-HClbeforebeingdilutedinbuffer forELISA.Consistentwithresultsfromtheamyloidplaquecounting,ELISAmeasurementsconfirmedthatthe levelsofA b 40and42inAPPswe/PS1dE9micewith Figure3 LRP1immunostainingofcorticalneuronsinGFAP-CreLRP1lox/loxmice .Imagesfromthesamesetsofmicedescribedinthe legendtoFigure2werecapturedtoshowcorticallevelsofLRP1.LRP1 a 377antibody(1:1,000,red)wasusedtostainLRP1;NeuN(1:1,000, green)wasusedtostainneurons;Dapiinthemountingreagentwasusedtodisplaynuclei.Therewasnoobviousdifferencebetweenmice transgenicforGFAP-CreandthoselackingGFAP-Cre. Xu etal Alzheimer sResearch&Therapy 2012, 4 :12 http://alzres.com/content/4/2/12 Page7of14

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Figure4 LRP1isbelowthelevelofdetectioninastrocytes .ThesamesetsofmicedescribedinthelegendtoFigure2were immunostained.LRP1 a 377antibody(1:1,000,red)wasusedforLRP1stainingandantibodytoGFAP(1:1,000,green)wasusedtomark astrocytes.ReactiveastrocytescouldbeseenaroundamyloidplaquesinAPPswe/PS1dE9LRP1lox/lox(rightcolumn)butthesecellsdidnot showdetectablelevelsofLRP1.ImmunoreactivitytoLRP1wasevidentwithintheplaqueasneuriticprofiles.LRP1immunostaininginthese neuriticstructureswasdiminishedinmosthippocampalamyloidplaquesofAPPswe/PS1dE9LRP1lox/loxGFAP-Cremice(leftcolumn). Xu etal Alzheimer sResearch&Therapy 2012, 4 :12 http://alzres.com/content/4/2/12 Page8of14

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reducedlevelsofLRP1werenodifferentfromthatof APPswe/PS1dE9micewithnormallevelsofLRP1 (Table1andFigure6).Further,aregressionanalysisto plotA b levelsatdifferentagesshowedthattherateof A b accumulationdidnotchangebythereductionof LRP1levels(Additionalfile7FigureS6).DiscussionInthepresentstudy,wehaveusedgeneticmanipulation toreducetheexpressionlevelsofLRP1inthehippocampusofmicethatco-expressmutantAPPand mutantPS1.Wedemonstratethatloweringthelevelsof LRP1byapproximately50%throughoutthebrainand Figure5 HippocampalamyloidplaquenumbersdonotchangewhenLRP1isreduced. (A and B) Representativeexamplesof immunohistochemicalstainswith6E10antibody(1:1,000,Covance)forAb inAPPswe/PS1dE9/LRP1lox/loxmicethatwereeitherpositiveor negativeforGFAP-Cre. (C) Toquantifyamyloiddeposits,westainedsectionswithThioflavin-S,whichproducesasharperdistinctionbetween tissueandamyloidplaques(seeAdditionalfile5FigureS4foranexample)andcapturedimages.Thehippocampuswasoutlinedandthenthe numberofamyloidplaquesinsidethehippocampuswascountedmanually.AdotplotofamyloidplaquenumbersinmicethatwereCre+is comparedtothenumbersinmicethatwereCre-.Animalsthatwereusedasbreedersarenoted.Nosignificantdifferencesbetweengenotypes eitherCre+(n=12)vs.Cre-(n=13)groups( P =0.75)orbetweenbreeders(n=10)vs.non-breeders(n=15)( P =0.69)wasnoted. Xu etal Alzheimer sResearch&Therapy 2012, 4 :12 http://alzres.com/content/4/2/12 Page9of14

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possiblybyamuchgreaterdegreeinhippocampushad nosignificantimpactontherate,severityorcharacter ofamyloiddeposition.Wefoundnoevidenceofaproportionalchangeinhippo campalamyloidburdenby reducingLRP1expressioninCAandDGneuronswith noobviousmorphologicalchangeintheappearanceof theplaquesthatformed. ToreduceLRP1levelsinthebrainsofAPPswe/ PS1dE9mice,weemployedmicethatexpressCre recombinaseunderthetranscriptionalregulationofthe GFAPpromoterandmiceinwhichtheLRP1allelehas beenmodifiedbyintroductionofloxpsites.The GFAP-Cremicehavepreviouslybeenusedinseveral studiesandtheseinvestigationshavepreviouslydemonstratedthatCreisexpressedearlyinneurogenesissuch thattargetedgenesmayrecombineinbothneuronsand glia[26,27].InourcrossoftheGFAP-Cremicewith LRP1lox/loxmice,weobservedefficienttargetingof LRP1expressioninthehippocampalneurons.However, corticalneuronscontinuedtoshowdemonstrableLRP1 immunoreactivity.Wenote,thatinthehippocampus,a subsetofneuronsinthemolecularlayercontinuedto expressLRP1inGFAP-Cre/loxp/loxpmice;however, thelevelsinCAandDGneuronsweredramatically reduced.ThelevelsofLRP1inastrocytesappearedto beinherentlylowandwecouldnotconfirmwhetheror notexpressionofLRP1wasreducedfurtherinLRP1lox/ loxmiceharboringtheGFAP-Cre. Weobservednoobviousdevelopmentalabnormality orreductionsinthenumbersofCAandDGneuronsin micetransgenicforGFAP-CreandLRP1loxp/loxp,or miceofthisgenotypethatwerealsotransgenicfor APPswe/PS1dE9.However,asnotedinResults,we encounteredsignificantdifficultyinbreedingtheseanimalsandnotedahighdegreeoflethalityearlyinthe process.Forreasonsthatremainunknown,theseissues wereovercomeusingastrategyoffostermotheringwith FVB/NJfemales.TheAPPswe/PS1dE9miceandthe LRP1loxpmicewerebothcongenicintheC57BL/6J backgroundandwebredtheGFAP-CreintoC57BL/6J miceforfourgenerationsbeforebeginningthecrosses toproduceexperimentalanimals.Inourhands,miceof theC57BL/6Jstrainarepoorermothersthancommonly usedhybridstrainsortheinbredFVB/NJstrain.Aswe expected,fostermotheringsolvedissueswehadwith poormotheringbyC57BL/6Jfemales.Unexpectedly,offspringthatwerefoster-motheredbyFVB/NJfemales survivedmuchbetterthanmiceofthesamegenotype motheredbyC57BL/6Jfemales.Wepresentlyhaveno explanationforthisoutcome.WenotethatinapreviousefforttocrosscongenicC57BL/6JAPPswe/ PS1dE9micetocongenicC57BL/6JRAPknockoutmice weobservedahighleveloflethalityinmicethatwere transgenicforAPPswe/PS1dE9andhomozygousknockoutforRAP[22].RAPisadirectERchaperonefor LRP1andlossofRAPlowersthelevelofmatureLRP1 thatreachesthecellsurface[37].Notably,others observednoissuesincrossesofthePDAPPmouse modeltoRAPknockoutmice[23],soweconcluded thatthelethalityweobserve dourearlierworkwasdue tosomeinteractionbetweenmutantPS1andRAP.Our experienceinthematingoftheLRP1loxpmiceto APPswe/PS1dE9miceleadsustobelievethattheremay besomemorecomplexinteractionbetweenPS1dE9and RAP/LRP1thatreducesviability.Thiscurrentstudy definesthesourceofthisdefecttothenervoussystem becauseLRP1isonlyreducedinthebrainbythe expressionofGFAP-Cre.Althoughlikelytobeunrelated toAD,furtherexaminationofthebasisofthislethality mayrevealfunctionsofAPPsweorPS1dE9thatimpact CNSfunction. BecausethereductioninneuronalLRP1wasgreatest inthehippocampus,wefocusedourpathologicanalyses onthehippocampus,findingnosignificantchangein theseverityofamyloiddepositionwhenLRP1levels werereducedintheAPPswe/PS1dE9mice.Thedata showninFigure5Cdocumentthevariabilityinthe numberofamyloiddepositsinhippocampusbetween Table1ThelevelsofAb inthebrainsofmiceofeachgenotypeandageAge(month)CreN A b40(pg/mgbrain) A b42(pg/mgbrain) ratio40/42 AverageSTDEV.P P -valueAverageSTDEV.P P -value 8 -31,2571,0220.402711930.45 4.6 +3481315 142 75 3.4 9 -172,0241,1920.903991920.71 5.1 +122,074746 421116 4.9 15 -44,2359460.651,0151040.74 4.2 +44,582823 972188 4.7 16to18 -44,4451,1350.749711810.79 4.6 +54,8301,777 1,020289 4.7TherewasnodifferenceinthelevelsofA b 40and42inthebrainsofmouseexpressingAPPswe/PS1dE9geneintheLRP1lox/loxhomozygousbackgroundin theabsenceorpresenceoftheGFAP-Cretransgeneinanygivenagegroup.Xu etal Alzheimer sResearch&Therapy 2012, 4 :12 http://alzres.com/content/4/2/12 Page10of14

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micethatarepositiveandnegativeforCrerecombinase. Inbothgroups,thenumberofanimalsthatwerebelow andabovethemean(horizontalline)wassimilar.Notably,ifthenumberofdepositshadbeenreducedbyjust 30%(withthesamelevelofvariance),thenwewould havebeenabletohavereachedstatisticalsignificance( P =0.05)withjust10animalsineachgroup(ourn swere 12vs.13).Basedonquantificationofimmunoblotsfrom Figure6 A b ELISAassayofwholebrainhomogenatesshownodifferencesinA b levelsbygenotype .A b levelsin8-,9-,15-and16to 18-month-oldwithnormalLRP1levels(APPswe/PS1dE9(+)/LRP1lox/lox)werecomparedtothosewithlowerLRP1levels(GFAP-Cre+).Therewas nodifferenceinAb levelsbygenotypebyage(pvaluesrange0.40to0.90basedonStudent s t -test).ELISAdatausedtogeneratethegraphare showninTable1,whichincludesinformationonthenumberofanimalsusedateachage. Xu etal Alzheimer sResearch&Therapy 2012, 4 :12 http://alzres.com/content/4/2/12 Page11of14

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hemi-brains,weconservativelyestimatethattheminimumreductioninLRP1levelswouldbeintherangeof 50%.Bypowercalculation,weshouldhavebeenpoweredtoobservea30%decreaseinamyloidplaquenumbersandthusweconcludethatreducingLRP1levels doesnotproduceaproportionaldecreaseinamyloid plaquenumbers.Consistentwithourfindings,Zerbinatti andcolleaguesreportedthatincreasingthelevelsof LRP1,viaexpressionofarecombinantmini-receptor, didnotproduceaproportionalincreaseinamyloidburden[25].However,thisstudydidnotesmallincreases oflevelsofbothsolubleandinsolubleA b inthecortex withamoreselectiveincreaseinsolubleA b inthehippocampusofoldermicewithhighamyloidburden.Our measuresoftotalAb didnotfindevidencethatreducing LRP1levelsproducedachangeintheaccumulated levelsofA b .Zerbinattiandcolleaguesalsonotedthe appearanceofA b 42dimersafterextractioninbuffers containing0.1Mcarbonate.Wehavenotbeenableto reliablydetectdimericA b inourAPPswe/PS1dE9mice atanyageandthuswecannotcommentastowhether reducingLRP1reducesthelevelsofthesedimers. Interpretingtheoutcomeofthisworkisnotas straightforwardasonewouldlikebecausetheLRP1 levelswerenotreducedtozero.Oneissueisthatwedo notknowhowmuchLRP1mustbeexpressedtosustain amyloidogenesisorwhetherweshouldexpectchanges inamyloidformationinproportiontochangesinLRP1 levels.Additionally,thesourceoftheAb peptidesthat producedepositsmaybeneuronsthatprojectaxons intothehippocampusaswellaslocalneurons.Within thehippocampus,weobservedaneareliminationof LRP1immunoreactivityofneuronsoftheCAfields. However,thehippocampusreceivesaxonalprojections fromtheentorhinalcortexmakingitpossiblethatan activityofLRP1incorticalneuronalprojectionsmasked anyeffectoflocalreductionsinLRP1levelsinhippocampus.Ultimately,themostconservativeinterpretation isthatsignificantsuppressionofLRP1activityinlocal hippocampalneuronsdoesnotproduceaproportional decreaseinamyloidplaquenumbersthisstructure. ApartfromthepotentialeffectsofLRP1reductionson amyloidplaquelevels,wehadanticipatedthatwemight seesomechangeinneuriticpathology.NearlyallamyloidplaquesinAPPswe/PS1dE9micecontainapolipoproteinE(ApoE)[31]andApoEisknowntobea ligandforLRP1thatmodulatesneuriteoutgrowth[38]. Thus,wehadthoughtitpossiblethatreducingLRP1 locallyinthehippocampuscouldhavereducedneuritic pathologyaroundamyloidplaques.Notably,inAPPswe/ PS1dE9/LRP1lox/loxmicethatwerepositiveforGFAPCreweobservedlargeneuriticplaquesinwhichthe neuritesweredepletedofLRP1immunoreactivity.ConclusionsThisstudyusedgenetictoolstoreduceLRP1levelsin thebrainsofmicethatco-expressAPPswe/PS1dE9and developamyloidpathology,producingamodelinwhich LRP1levelsinmosthippocampalneuronswerelowered tobelowthelevelofdetectionbyimmunohistochemistry.InthehippocampusofmicewithreducedLRP1 levels,weobservednoobviouschangeintherateof deposition,severity,orch aracterofamyloiddeposits. Obviously,thisstudydoesnotprovideanyinsightinto whatLRP1expressedinnon-neuralcelltypes,suchas endothelialcells,otherorgansmaydoinregardtomodulatingamyloiddeposition.Itisalsoverypossiblethat homologuesofLRP1mayhavecompensatedforany effectofreducingthelevelsofLRP1.However,atminimum,ourstudyindicatesthatreducingthelevelsof LRP1doesnotproduceproportionalreductionsinamyloidplaquenumbersinthehippocampusofthe APPswe/PS1dE9model.Thus,thisgenetictestdoesnot producesupportiveevidenceforthenotionthatmodulatingLRP1functioninneuronsorglialcouldbebeneficialinmodulatingamyloidosis.Whetherthisoutcomeis uniquetotheAPPswe/PS1dE9modelisofcoursean issueinassessingthepotentialimportanceofthispathwayinhumandisease.AdditionalmaterialAdditionalfile1:Genotypedistributionofthetransgenicmiceused inthisstudy .Intotal,325transgenicmicewithLRP1lox/lox backgroundweregenerated.Themicelistedinthetablearethosethat reachedweaningageandsurvivedtoatleast1.5monthsofage.The actualandexpectedpercentagesofmiceofeachgenotypearelisted. ThepredicatedfrequencyrateisbasedonMendeliandistribution.Asis typicalforAPPswe/PS1dE9micethatarefounddead,therewereno obviousbirthdefectstoaccountfortheearlylethality(TableS1). Additionalfile2:LowpowerimageofLRP1immunostainingin hippocampusofAPPswe/PS1dE9/LRP1lox/loxmicethatare positiveornegativeforGFAP-Cre .Free-floatingfrozensectionswere immunostainedasdescribedinMethodsofthemaintext.Theimages shownarerepresentativeofwhatwasobservedinatleastthreeanimals ofeachgenotype(FigureS1). Additionalfile3:LowpowerimagecapturedfromtheAllenBrain Atlas .Theimageshownherewascapturedfromthewebsiteforthe AllenBrainAtlasfortheadultmousebrain.Thegenenamesearchedfor wasLRP1.Theviewshowndisplaysfalsecolorforexpressionlevelsofa sagittalview(FigureS2). Additionalfile4:Restingastrocytesarenotimmunoreactiveto LRP1antibodies .Theimagesshownarehighpowerimagesof hippocampusfromAPPswe/PS1dE9/LRP1lox/loxmicethatwereeither positiveornegativeforGFAP-Cre.Weobservednoobviousreactivityin cellsshowingamorphologyidentifiedbyGFAPstainingandtherewas noindicationofsignificantco-localizationofGFAPandLRP1 immunoreactivity.Theimagesshownarerepresentativeofwhatwas observedinatleastthreeanimalsofeachgenotype(FigureS3). Additionalfile5:Thioflavin-Sstainingofhippocampalsections .The numbersofamyloidplaquesinthehippocampusasidentifiedby Thioflavin-Sstainingsectionweremanuallybyoutliningtheborderof thehippocampusontheimageandthencountingthenumberofXu etal Alzheimer sResearch&Therapy 2012, 4 :12 http://alzres.com/content/4/2/12 Page12of14

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plaquesinhippocampus.Theseimagesshowrepresentativeexamplesof animalsofbothgenotypes(FigureS4). Additionalfile6:Themorphologyofhippocampalamyloiddeposits isunchangedbythelackofLRP1 .Silverstainsof30 msectionsfrom APPswe/PS1dE9LRP1lox/loxmicethatwereeitherpositiveor negativeforGFAP-Cre.Whitearrowsmarkneuritesandthewhitearrow headsmarktothecoreoftheplaques(FigureS5). Additionalfile7:RegressionanalysisofA b levelsinmiceofeach genotypebyage .NodifferenceintherateofA b accumulationwas evident.Theregressioncurveoftheslopesbetweentwogroupsof animalsisnotsignificantforA b 40( P =0.409)orA b 42( P =0.864)(Figure S6). Abbreviations ApoE:apolipoprotienE;APP:amyloidprecursorprotein;APPswe/PS1dE9:a tandemintegrationoftransgenesencodingamyloidprecursorproteinwith theSwedishmutationandpresenilin1genewithexon9deletion;BCA: bicinchoninicacidassay:abiochemicalassayforproteinconcentration;CA: cornuammonisinhippocampus;Cre-lox:Crerecombinase-LoxPsitesystem, agenetictooltocontrolsitespecificrecombinationeventsingenomic DNA;DG:dentategyrusinhippocampus;ELISA:enzyme-linked immunosorbentassay;GFAP-Cre:transgenicmicewithCrerecombinase drivenbyglialfibrillaryacidicprotein(GFAP)promoter;LRP1:low-density lipoproteinreceptor-relatedprotein. Acknowledgements WegratefullythankJoachimHerz(UniversityofTexasSouthwesternMedical Center,Dallas,Texas,USA)forprovidingLRP1antibodyandmicewith targetedLoxPsites(LRP1lox/loxmice),andWeiHou(Departmentof Biostatistics,UniversityofFlorida)forhelpwithstatistics.Thisstudywas supportedbyAmericanHealthAssistanceFoundation(AHAF)[PilotAwards (GX)]andSantaFeHealthCareAlzheimer sDiseaseResearchCenter. Authordetails1DepartmentofNeuroscience,SantaFeHealthCareAlzheimer sDisease Center,CenterforTranslationalResearchinNeurodegenerativeDisease, McKnightBrainInstitute,UniversityofFlorida,Gainesville,FL32610,USA.2DepartmentofEducationalPsychologyandLearningSystems,Collegeof Education,FloridaStateUniversity,Tallahassee,FL32306,USA. Authors contributions Allauthorscontributedtothismanuscript.GXdesignedtheexperiments, participatedtogeneratethecohortsofmice,carriedoutmostofthe experiments,performedthestatisticalanalysis,anddraftedthemanuscript. CGcontributedtothehistology,immunostainingworkandcollecteddataof amyloidplaques.SFgenotypedthemiceandrecordedthebreedingdata. DRBconceivedofthewholestudy,participatedindetailedexperimental designandpreparedthemanuscript.Allauthorsreadandapprovedthe finalmanuscript. Competinginterests Theauthorsdeclarethattheyhavenocompetinginterests. Received:25February2012Revised:19April2012 Accepted:26April2012Published:26April2012 References1.AndersenOM,WillnowTE: LipoproteinreceptorsinAlzheimer sdisease. TrendsNeurosci 2006, 29 :687-694. 2.RebeckGW,ReiterJS,StricklandDK,HymanBT: ApolipoproteinEin sporadicAlzheimer sdisease:allelicvariationandreceptorinteractions. Neuron 1993, 11 :575-580. 3.BuG,MaksymovitchEA,NerbonneJM,SchwartzAL: Expressionand functionofthelowdensitylipoproteinreceptor-relatedprotein(LRP)in mammaliancentralneurons. 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JClinInvest 2001, 108 :779-784. 20.HoltzmanDM,FaganAM,MackeyB,TenkovaT,SartoriusL,PaulSM, BalesK,AsheKH,IrizarryMC,HymanBT: ApolipoproteinEfacilitates neuriticandcerebrovascularplaqueformationinanAlzheimer sdisease model. AnnNeurol 2000, 47 :739-747. 21.HerzJ,ClouthierDE,HammerRE: LDLreceptor-relatedproteininternalizes anddegradesuPA-PAI-1complexesandisessentialforembryo implantation. Cell 1992, 71 :411-421. 22.XuG,KarchC,LiN,LinN,FromholtD,GonzalesV,BorcheltDR: Receptorassociatedprotein(RAP)playsacentralroleinmodulatingAbeta depositioninAPP/PS1transgenicmice. PLoSOne 2008, 3 :e3159. 23.VanUdenE,MalloryM,VeinbergsI,AlfordM,RockensteinE,MasliahE: Increasedextracellularamyloiddepositionandneurodegenerationin humanamyloidprecursorproteintransgenicmicedeficientinreceptorassociatedprotein. JNeurosci 2002, 22 :9298-9304.Xu etal Alzheimer sResearch&Therapy 2012, 4 :12 http://alzres.com/content/4/2/12 Page13of14

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24.WillnowTE,ArmstrongSA,HammerRE,HerzJ: Functionalexpressionof lowdensitylipoproteinreceptor-relatedproteiniscontrolledby receptor-associatedprotein invivo ProcNatlAcadSciUSA 1995, 92 :4537-4541. 25.ZerbinattiCV,WozniakDF,CirritoJ,CamJA,OsakaH,BalesKR,ZhuoM, PaulSM,HoltzmanDM,BuG: Increasedsolubleamyloid-betapeptide andmemorydeficitsinamyloidmodelmiceoverexpressingthelowdensitylipoproteinreceptor-relatedprotein. ProcNatlAcadSciUSA 2004, 101 :1075-1080. 26.ZhuoL,TheisM,Alvarez-MayaI,BrennerM,WilleckeK,MessingA: hGFAPcretransgenicmiceformanipulationofglialandneuronalfunction in vivo Genesis 2001, 31 :85-94. 27.MooreSA,SaitoF,ChenJ,MicheleDE,HenryMD,MessingA,CohnRD, Ross-BartaSE,WestraS,WilliamsonRA,HoshiT,CampbellKP: Deletionof braindystroglycanrecapitulatesaspectsofcongenitalmuscular dystrophy. Nature 2002, 418 :422-425. 28.RohlmannA,GotthardtM,WillnowTE,HammerRE,HerzJ: Sustained somaticgeneinactivationbyviraltransferofCrerecombinase. Nat Biotechnol 1996, 14 :1562-1565. 29.RohlmannA,GotthardtM,HammerRE,HerzJ: Inducibleinactivationof hepaticLRPgenebycre-mediatedrecombinationconfirmsroleofLRP inclearanceofchylomicronremnants. JClinInvest 1998, 101 :689-695. 30.JankowskyJL,SluntHH,RatovitskiT,JenkinsNA,CopelandNG,BorcheltDR: Co-expressionofmultipletransgenesinmouseCNS:acomparisonof strategies. BiomolEng 2001, 17 :157-165. 31.LesuisseC,XuG,AndersonJ,WongM,JankowskyJ,HoltzG,GonzalezV, WongPC,PriceDL,TangF,WagnerS,BorcheltDR: Hyper-expressionof humanapolipoproteinE4inastrogliaandneuronsdoesnotenhance amyloiddepositionintransgenicmice. HumMolGenet 2001, 10 :2525-2537. 32.BorcheltDR,LeeMK,SluntHS,GuarnieriM,XuZS,WongPC,BrownRH, PriceDL,SisodiaSS,ClevelandDW: Superoxidedismutase1with mutationslinkedtofamilialamyotrophiclateralsclerosispossesses significantactivity. ProcNatlAcadSciUSA 1994, 91 :8292-8296. 33.GunternR,BourasC,HofPR,ValletPG: AnimprovedthioflavineSmethod forstainingneurofibrillarytanglesandsenileplaquesinAlzheimer s disease. Experientia 1992, 48 :8-10. 34.TebbenkampAT,GreenC,XuG,Denovan-WrightEM,RisingAC, FromholtSE,BrownHH,SwingD,MandelRJ,TessarolloL,BorcheltDR: Transgenicmiceexpressingcaspase-6-derivedN-terminalfragmentsof mutanthuntingtindevelopneurologicabnormalitieswithpredominant cytoplasmicinclusionpathologycomposedlargelyofasmaller proteolyticderivative. HumMolGenet 2011, 20 :2770-2782. 35.HiranoA,ZimmermanHM: Silverimpregnationofnervecellsandfibers incelloidinsections.Asimpleimpregnationtechnique. ArchNeurol 1962, 6 :114-122. 36.YamamotoT,HiranoA: AcomparativestudyofmodifiedBielschowsky, BodianandthioflavinSstainsonAlzheimer sneurofibrillarytangles. NeuropatholApplNeurobiol 1986, 12 :3-9. 37.BuG,GeuzeHJ,StrousGJ,SchwartzAL: 39kDareceptor-associated proteinisanERresidentproteinandmolecularchaperoneforLDL receptor-relatedprotein. EmboJ 1995, 14 :2269-80. 38.HoltzmanDM,PitasRE,KilbridgeJ,NathanB,MahleyRW,BuG, SchwartzAL: Lowdensitylipoproteinreceptor-relatedproteinmediates apolipoproteinE-dependentneuriteoutgrowthinacentralnervous system-derivedneuronalcellline. ProcNatlAcadSciUSA 1995, 92 :9480-9484.doi:10.1186/alzrt110 Citethisarticleas: Xu etal .: Reductionoflow-densitylipoprotein receptor-relatedprotein(LRP1)inhippocampalneuronsdoesnot proportionatelyreduce,orotherwisealter,amyloiddepositionin APPswe/PS1dE9transgenicmice. Alzheimer sResearch&Therapy 2012 4 :12. 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 Xu etal Alzheimer sResearch&Therapy 2012, 4 :12 http://alzres.com/content/4/2/12 Page14of14


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dochead Research
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title p Reduction of low-density lipoprotein receptor-related protein (LRP1) in hippocampal neurons does not proportionately reduce, or otherwise alter, amyloid deposition in APPswe/PS1dE9 transgenic mice
aug
au id A1 ca yes snm Xufnm Guilianinsr iid I1 email xugl@ufl.edu
A2 Greenmi CCameronI2 cgreen@stetson.edu
A3 FromholtESusansfromhol@ufl.edu
A4 BorcheltRDaviddrb1@ufl.edu
insg
ins Department of Neuroscience, SantaFe HealthCare Alzheimer's Disease Center, Center for Translational Research in Neurodegenerative Disease, McKnight Brain Institute, University of Florida, Gainesville, FL 32610, USA
Department of Educational Psychology and Learning Systems, College of Education, Florida State University, Tallahassee, FL 32306, USA
source Alzheimer's Research & Therapy
issn 1758-9193
pubdate 2012
volume 4
issue 2
fpage 12
url http://alzres.com/content/4/2/12
xrefbib pubidlist pubid idtype doi 10.1186/alzrt110pmpid 22537779
history rec date day 25month 2year 2012revrec 1942012acc 2642012pub 2642012
cpyrt 2012collab Xu et al.; licensee BioMed Central Ltd.note This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
abs
sec st Abstract
Introduction
The low-density lipoprotein receptor-related protein (LRP1) and its family members have been implicated in the pathogenesis of Alzheimer's disease. Multiple susceptibility factors converge to metabolic pathways that involve LRP1, including modulation of the processing of amyloid precursor protein (APP) and the clearance of Aβ peptide.
Methods
We used the Cre-lox system to lower LRP1 levels in hippocampal neurons of mice that develop Alzheimer-type amyloid by crosses between mice that express Cre recombinase under the transcriptional control of the GFAP promoter, mice that harbor loxp sites in the LRP1 gene, and the APPswe/PS1dE9 transgenic model. We compared amyloid plaque numbers in APPswe/PS1dE9 mice lacking LRP1 expression in hippocampus (n = 13) to mice with normal levels of LRP1 (n = 12). Student t-test was used to test whether there were significant differences in plaque numbers and amyloid levels between the groups. A regression model was used to fit two regression lines for these groups, and to compare the rates of Aβ accumulation.
Results
Immunohistochemical analyses demonstrated efficient elimination of LRP1 expression in the CA fields and dentate gyrus of the hippocampus. Within hippocampus, we observed no effect on the severity of amyloid deposition, the rate of Aβ40/42 accumulation, or the architecture of amyloid plaques when LRP1 levels were reduced.
Conclusions
Expression of LRP1 by neurons in proximity to senile amyloid plaques does not appear to play a major role in modulating the formation of these proximal deposits or in the appearance of the associated neuritic pathology.
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Introduction
Amyloid deposition is a key pathological event in Alzheimer's disease. A large body of evidence suggests that low density lipoprotein (LDL) receptor related protein (LRP1), may be a key mediator of amyloid deposition. As a member of the LDL receptor family, LRP1 is a large, multifunctional, endocytic receptor that is highly expressed in neurons (reviewed by Andersen abbrgrp abbr bid B1 1), activated astrocytes B2 2B3 3B4 4, and microglia B5 5. Direct binding of LRP1 to the amyloid precursor protein (APP) has been shown to affect endoproteolytic processing of APP to increase the production of Aβ42 peptides B6 6, which are the major constituent of amyloid deposits B7 7. LRP1 can promote Aβ production by altering the processing of APP through interactions via the Kunitz protease inhibitor (KPI) domain (isoforms APP751 or APP770) 6. Although the non-KPI-APP isoform can weakly bind to LRP1 through cytoplasmic adaptor proteins, such as FE65 B8 8, APP695 processing may not be influenced as significantly by LRP1. Additionally, LRP1 has also been reported to mediate export of Aβ across the blood-brain barrier (reviewed by B9 9); LRP1 in endothelial cells may bind directly to Aβ1-40 and export it across the blood-brain barrier B10 10. Consistent with this notion, reducing LRP1 levels by antisense treatment reduces blood-brain barrier clearance of Aβ42 B11 11. Polymorphisms in the LRP1 gene have been associated with increased risk for Alzheimer's disease B12 12 and LRP1 levels are significantly reduced in AD patients B13 13. More recently, Sehgal and colleagues reported that an extract of it Withania somnifera induces a rapid clearance of amyloid pathology with a co-incident induction of LRP1 expression in liver B14 14. Other proteins implicated in amyloidogenesis have been identified as interacting with LRP1 B15 15, such as apolipoprotien E (ApoE) and α2-macroglobulin. Both of these proteins are ligands of LRP1 and have been reported to mediate the clearance of Aβ 13B16 16B17 17B18 18B19 19. Moreover, ApoE, which is a major ligand for LRP1, modulates the distribution and character of amyloid deposition B20 20. Collectively, these data suggest that LRP1 could be a crucial factor in Alzheimer's disease and the receptor characteristics of LRP1 make this protein a potential "drugable" target where influencing the binding of LRP1 to any of its ligands could be a useful therapeutic.
To date, there have been few studies that examine the role of LRP1 in vivo using animal models by genetic manipulation of LRP1 expression. Constitutive inactivation of LRP1 is lethal B21 21. We B22 22 and others B23 23 have used mice with targeted deletion of LRP1 receptor associated protein (RAP), which regulates LRP1 maturation, as a means to reduce LRP1 levels in mouse models of Alzheimer's disease. One group has reported that homozygous deletion of RAP, which lowers LRP1 levels by 75% B24 24, results in increased Aβ deposition in PDAPP mice 23. Similarly, we observed that heterozygous deletion of RAP also increased amyloid deposition, in the APPswe/PS1dE9 model; however, in our case LRP1 levels were not significantly lowered 22. Expression of an LRP1 mini-receptor at approximately 3.7-fold over endogenous LRP1 levels in the PDAPP transgenic model was reported to have no effect on amyloid burden B25 25. Collectively, these studies illustrate the inconsistent in vivo findings regarding LRP1 and amyloidogenesis in transgenic mice that over-express mutant APP (with or without co-expression of mutant presenilin).
To further probe the role of LRP1 in amyloidogenesis, we used Cre/Lox technology to conditionally reduce LRP1 expression in the brains of mice that co-express mutant APP (APPswe) and mutant presenilin 1 (PS1dE9). For these studies we used mice that express Cre under the control of the glial fibrillary acidic protein (GFAP) B26 26. In previous studies with these animals, others have demonstrated Cre mediated recombination in both neurons and glial throughout the brain B27 27. In our hands, GFAP-mediated expression of Cre produced incomplete recombination, such that the levels of LRP1 in total brain were reduced approximately 50%. However, we determined that LRP1 immunostaining in CA and granule neurons of the hippocampus was virtually eliminated. Within the hippocampus, we observed no obvious change in the level of neuritic pathology or amyloid burden by loss of LRP1.
Materials and methods
Animals
Mice with targeted Lox P sites introduced into intronic and promoter regions of the LRP1 gene were obtained from Joachim Herz B28 28B29 29, called LRP1 lox/lox. Transgenic mice expressing GFAP-Cre were obtained from Jackson Laboratories (Bar Harbor, ME, USA; Strain: FVB-Tg(GFAP-cre)25Mes/J; Stock Number: 004600) 26. These animals were back-crossed to C57BL/6J mice for at least four generations to reduce the FVB strain contribution before breeding to LRP1 lox/lox mice. LRP1 lox/lox mice were in parallel bred to APPswe/PS1dE9 (line 85) mice B30 30 (both with C57BL/6J background) to produce mice homozygous for the LRP1 loxp locus. To generate triple transgenic mice, we mated mice transgenic for GFAP-Cre and homozygous for LRP1 loxp to mice transgenic for APPswe/PS1dE9 and homozygous for LPR loxp. FVB/NJ male and female mice were purchased from Jackson Laboratories (stock number: 001800) to produce foster mothers. Eventually, animals with the genotypes of APPswe/PS1dE9(+)/LRP1 lox/lox with/without the GFAP-Cre gene were used for the study. All of the mice were housed in standard SPF cages for 9 to 18 months before harvesting. All procedures involving animal handling and processing were approved by the University of Florida Institutional Animal Care and Use Committee and in compliance with the National Institutes of Health guidelines.
Western blot analysis
Levels of LRP1 were assessed by Western blot, using standard methods we have previously described B31 31. Aliquots of the PBS brain homogenates (100 μl) from 14- to 15-month-old mice were mixed with 10 μl of 10% SDS, centrifuged at the top speed (approximately 10,000 xg) in a bench top microfuge for 10 minutes, and then 30 μl of the supernatant was mixed with 10 μl 4x Laemmli buffer and boiled for 5 minutes. An amount of sample equal to 50 μg total protein (by BCA assay) were loaded per lane for SDS-PAGE and transferred to nitrocellulose membranes. Blots were incubated with rabbit polyclonal antibody 377 αLRP1 (1:1,000) and then reprobed with the m/h SOD1 antibody (1:3,000) to gauge protein loading B32 32. The ECL signal was captured and quantified using the LAS-3000 imaging system (FUJIFILM Life Science, Tokyo, Japan).
Tissue preparation for histology
Mice were deeply anesthetized by injection anesthetic (3% body weight of 1.2% Tribromoethanol (Avertin Sigma, St. Louis, MO, USA) before transcardial perfusion with cold phosphate buffered saline (PBS, pH7.4). The brains were then removed and cut sagittally down the midline; one hemi-brain was frozen on dry ice for biochemistry assay, the other hemi-brain was immersion fixed in 4% paraformaldehyde in PBS. After 48 hours in fixation at 4°C, brains were transferred to PBS, and then 30% sucrose in PBS for several days before cryostat sectioning at 30 μm thickness. From the first complete section closest to mid-line, 10 sections per group were stored in each well of 24-well-plates and the entire hemi-brain was sectioned. Sections were kept in anti-freeze solution (100 mM sodium acetate, 250 mM polyvinyl pyrrolidone, 40% ethylene glycol, pH6.5) at -20°C until staining.
Immunostaining
Immunostaining of 30 μm free floating sections was performed according to a standard protocol. Sections were washed in PBS to remove the anti-freeze solution, and then blocked in PBS containing 5% normal goat serum with 0.1% Triton X-100 before incubating with antibodies against LRP1 (rabbit polyclonal 377 αLRP1 (1:1,000)), glial fibrillary acidic protein (GFAP) (mouse monoclonal, 1:1,000, Millipore, Billerica, MA, USA), neuronal antigen NeuN (mouse monoclonal, 1:1000, Millipore) or 6E10 (mouse monoclonal, 1:1,000, Covance, Princeton, NJ, USA). Secondary antibodies of goat anti-rabbit and goat anti-mouse, conjugated with Alexa 594 or Alexa 488 (Invitrogen, Carlsbad, CA, USA) were used to visualize primary antibody binding. After washing to remove unbound secondary antibody, the sections were then mounted on microscope slides and dried in air. A quick dip in 0.1% Sudan Black B solution (in 70% ethanol) for 5 to 10 minutes was used to block the auto-fluorescence of lipofuscin then the slides were washed in 70% ethanol, then water, dried and covered by ProLong Gold antifade reagent with Dapi (Invitrogen). An Olympus DSU-IX81 Spinning Disc Confocal microscope (Tokyo, Japan) was used to capture the images.
Thioflavin S staining and silver staining of amyloid plaques
One section from each well (see above Tissue preparation for histology) was randomly selected for Thioflavin S staining according to the Guntern standard protocol B33 33 with small modification B34 34. Fluorescent photos were taken using 5x objectives by a Canon digital camera (Tokyo, Japan). Silver impregnation staining was performed on the same 30 μm floating sections by modified Hirano's method B35 35B36 36. Briefly, sections were washed and mounted on slides to dry as those for Thioflavin S staining, and then the steps described for silver impregnation methods of paraffin slides were followed 36.
Amyloid plaque quantification
Four sections per animal (from 300 to 1,500 μm lateral to midline) stained with Thioflavin S were used to quantify the amyloid plaque levels. The images of the hippocampus were outlined and the numbers of amyloid plaques were manually counted by an observer blinded to genotype. The images of each hippocampus were outlined and quantified three times on three different days to reduce bias and then analyzed. The numbers of amyloid plaques in each section were averaged and then numbers of plaques from the four sections from different levels of the brain were averaged to produce the final number of amyloid plaques/section in the hippocampus of each animal.
Aβ ELISA
The frozen hemi-brain from each transgenic animal was weighed, then homogenized by sonication in five volumes by weight in PBS with 1 × protease inhibitor cocktail (Sigma Cat.P8340). A 100 μl aliquot of this PBS brain homogenate was used for ELISA analysis. Protein aggregates in the PBS homogenate were denatured by the addition of 150 μl of 8.0 M guanidine-HCl to a final concentration of 4.8 M, and mixed at room temperature for three to four hours before storage overnight at -20°C. The denatured samples were then further diluted 200-fold in reaction buffer (PBS containing 5% BSA, 0.03% Tween-20, and 1 × protease inhibitor cocktail (Sigma)). Samples were finally prepared for application to the ELISA microplate by mixing with an equal volume of Standard Dilution Buffer provided by the manufacturer supplemented with 1 × protease inhibitor cocktail. Aliquots of these samples were assayed for both Aβ40 and Aβ42 using commercially available sandwich-ELISAs according to the manufacturer's manual (Invitrogen).
Statistical analysis of the ELISA data
We compared the differences among the means of Aβ40 and 42 levels among the same age groups and the distribution of the levels across all ages. The comparison in the same age group between two different genotypes (GFAP-Cre positive or negative) was carried out using Excel with the two-tail Student t-test with both equal and unequal variance. The Aβ levels within each age group were graphed in Excel and linear regression lines were added. Excel Analysis ToolPak add-in was used for regression analysis. IBM SPSS Statistics 19 [(trial version), Somers, NY, USA] was used to test the significance between the regression curves by genotypes. A P-value of < 0.05 was considered statistically significant.
Results
A strain of GFAP-Cre mice that expresses Cre early in neurogenesis was used to cross to the homozygous mice with LRP1 loxp sites with the expectation of complete recombination of flox'ed genes in neurons as well as glia 27. The GFAP-Cre mice that were used for this study from the Jackson Laboratory were maintained in the FVB strain. To make them compatible with our strains of mice, we back-crossed them to C57BL/6J mice for four generations before crossing to LRP1 lox/lox mice, and then we continued to cross them to C57BL/6J congenic APPswe/PS1dE9 mice (line 85). Mice that were transgenic for APPswe/PS1dE9 and LRP1 loxp/loxp were mated to mice that were transgenic for GFAP-Cre and LRP1 loxp/loxp, or mice that were transgenic for all three alleles (APPswe/PS1dE9, GFAP-Cre and LRP1 loxp/loxp) were mated to mice only transgenic for LRP1 loxp/loxp. A total of 54 breeding cages were set up over the course of the study to generate the mice we ultimately examined. More than 20 of these cages failed to generate any offspring, 5 cages generated a few offspring, but the mothers failed to care for the litters and none of the offspring lived to weaning age (four weeks old). Of the animals of the desired genotype (APPswe/PS1dE9/LRP1loxp/loxp/GFAP-Cre) that were produced, almost half died suddenly in the first nine months of life (see Additional file supplr sid S1 1 Table S1).
suppl
Additional file 1
text b Genotype distribution of the transgenic mice used in this study. In total, 325 transgenic mice with LRP1 lox/lox background were generated. The mice listed in the table are those that reached weaning age and survived to at least 1.5 months of age. The actual and expected percentages of mice of each genotype are listed. The predicated frequency rate is based on Mendelian distribution. As is typical for APPswe/PS1dE9 mice that are found dead, there were no obvious birth defects to account for the early lethality (Table S1).
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Because of the reduced viability of the mice of the desired genotype and the poor mothering of the C57BL/6J females, we began a foster breeding program. FVB/NJ females, (which are very good mothers), were used to foster all newborn offspring in an effort to increase the frequency of animals that survive to weaning. This foster-mothering program was successful in producing a greater number of animals of the desired genotype that survived to weaning. Surprisingly, the survival rate of the offspring that were foster-mothered was also much improved. A total of 167 offspring were produced in the foster-mothering program of which only 7 died before nine months of age and only 1 of these 7 was triple transgenic genotype (see Additional file 1 Table S1). Overall, the distribution of the mice with all four of the genotypes was much closer to Mendelian distribution (25% of each genotype expected). At present, we have no explanation for this observation, but it appears that the foster-mothering that improved survival to weaning age transferred to improved resistance to the underlying problem that caused the sudden early death after weaning. Ultimately most of the mice that were studied in this report were generated by foster-mothering.
GFAP-Cre expression in LRP1 lox/lox mice lowers LRP1 levels in hippocampus
Using the breeding scheme described above, we produced mice transgenic for GFAP-Cre, APPswe/PS1dE9, and homozygous for the flox'ed LRP1 alleles. All mice described in this work were homozygous for the LRP1lox allele. No obvious developmental abnormalities were observed in the brains of mice that were transgenic for GFAP-Cre whether or not they were also transgenic for the additional APPswe/PS1dE9 transgenes (not shown). Immunoblots of hemi-brain homogenates demonstrated that LRP1 levels were reduced by approximately 50% in mice that were transgenic for APPswe/PS1dE9 and GFAP-Cre as compared to mice that were transgenic only for APPswe/PS1dE9 (Figure figr fid F1 1).
fig Figure 1caption The level of LRP1 is significantly lower in the mice expressing GFAP-Cre with LRP1 lox/lox
The level of LRP1 is significantly lower in the mice expressing GFAP-Cre with LRP1 lox/lox. Nine APPswe/PS1dE9 × GFAP-Cre × LRP1 lox/lox mice with different genotypes were used to determine the levels of LRP1. The SDS-soluble fractions of whole brain lysates (homogenized in PBS) containing an equal amount of total protein (50 μg) was loaded in each lane. Antibody α377 to LRP1 (1:1,000, a gift from Dr. Herz, University of Texas Southwestern Medical Center) was used to detect LRP1 level. The 85 kDa band is the endoproteolytically cleaved mature LRP1. Mouse SOD1 (14 kD bands) was used as endogenous loading control. The ratio of band intensity of LRP1 to SOD1 was quantified, standardized and plotted below. The standard deviations of the measurements are shown by error bars.
graphic alzrt110-1 hint_layout double
In immunohistochemical stains of hippocampus of mice that were double transgenic for APPswe/PS1dE9 and GFAP-Cre in the LRP1lox/lox background, we observed very little staining for LRP1 in hippocampal neurons in the CA fields and dentate gyrus (DG) (Figure F2 2; Additional file S2 2 Figure S1). In contrast, in APPswe/PS1dE9/LRP1lox/lox mice we observed strong immunoreactivity for LRP1 in these regions (Figure 2). This pattern of reactivity matches what would be expected based on the levels of mRNA in these structures (Additional file S3 3 Figure S2). In the cortex of mice of the same genotype, we observed that most neurons, identified by NeuN staining, remained positive for LRP1 immunoreactivity, albeit at what appeared to be slightly lower levels, indicating that the recombination of the flox'ed LRP1 allele was less complete in cortical neurons (Figure F3 3). Thus, although the level of recombination in the cortex was less than reported for other flox'ed alleles 27, the level of recombination in the hippocampus was much more robust and thus we focused our analysis on amyloid deposition within the hippocampus.
Figure 2LRP1 levels are reduced in hippocampal neurons of LRP1lox/lox mice that are transgenic for GFAP-Cre
LRP1 levels are reduced in hippocampal neurons of LRP1lox/lox mice that are transgenic for GFAP-Cre. Eight-month-old APPswe/PS1dE9 × GFAP-Cre × LRP1 lox/lox littermates were used for immunostaining. All mice were homozygous for LRP1loxp alleles and positive for APPswe/PS1dE9 transgenes. The presence or absence of GFAP-Cre is noted above the images. LRP1 α377 antibody (1:1,000, red) was used to stain LRP1; NeuN (1:1,000, green) was used to stain neurons; Dapi in the mounting reagent was used to display nuclei. Confocal microscope images showed most of the neurons in CA fields (A) and the dentate gyrus (C and E) of APPswe/PS1dE9 × LRP1lox/lox × GFAP-Cre mice do not exhibit LRP1 immunoreactivity. Control mice lacking the GFAP-Cre transgene showed intense staining of LRP1 in hippocampal neurons (B, D, and F). Three to four pairs of mice per genotype were used for immunostaining and four to five sections per animal were analyzed.
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Additional file 2
Low power image of LRP1 immunostaining in hippocampus of APPswe/PS1dE9/LRP1 lox/lox mice that are positive or negative for GFAP-Cre. Free-floating frozen sections were immunostained as described in Methods of the main text. The images shown are representative of what was observed in at least three animals of each genotype (Figure S1).
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Additional file 3
Low power image captured from the Allen Brain Atlas. The image shown here was captured from the website for the Allen Brain Atlas for the adult mouse brain. The gene name searched for was LRP1. The view shown displays false color for expression levels of a sagittal view (Figure S2).
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Figure 3LRP1 immunostaining of cortical neurons in GFAP-Cre × LRP1lox/lox mice
LRP1 immunostaining of cortical neurons in GFAP-Cre × LRP1lox/lox mice. Images from the same sets of mice described in the legend to Figure 2 were captured to show cortical levels of LRP1. LRP1 α377 antibody (1:1,000, red) was used to stain LRP1; NeuN (1:1,000, green) was used to stain neurons; Dapi in the mounting reagent was used to display nuclei. There was no obvious difference between mice transgenic for GFAP-Cre and those lacking GFAP-Cre.
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Based on previous studies, we expected that LRP1 would be expressed at some level in astrocytes, particularly activated astrocytes 234. No LRP1 immunoreactivity could be detected in resting astrocytes of any genotype (Additional file S4 4 Figure S3). In the hippocampus of nine-month-old GFAP-Cre/APPswe/PS1dE9/LRP1lox/lox mice, we observed that amyloid deposits still formed at a relatively high frequency (see below) and around these deposits we observed activated astrocytes. However, we failed to detect LRP1 in activated astrocytes surrounding these plaques (Figure F4 4). Within these deposits we observed LRP1 immunoreactivity that was reminiscent of neuritic profiles (Figure 4). These LRP1 immunoreactive neuritic profiles were mostly absent within the molecular layer of the hippocampus of APPswe/PS1dE9/LRP1lox/lox mice that were positive for GFAP-Cre.
Additional file 4
Resting astrocytes are not immunoreactive to LRP1 antibodies. The images shown are high power images of hippocampus from APPswe/PS1dE9/LRP1 lox/lox mice that were either positive or negative for GFAP-Cre. We observed no obvious reactivity in cells showing a morphology identified by GFAP staining and there was no indication of significant co-localization of GFAP and LRP1 immunoreactivity. The images shown are representative of what was observed in at least three animals of each genotype (Figure S3).
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Figure 4LRP1 is below the level of detection in astrocytes
LRP1 is below the level of detection in astrocytes. The same sets of mice described in the legend to Figure 2 were immunostained. LRP1 α377 antibody (1:1,000, red) was used for LRP1 staining and antibody to GFAP (1:1,000, green) was used to mark astrocytes. Reactive astrocytes could be seen around amyloid plaques in APPswe/PS1dE9 × LRP1lox/lox (right column) but these cells did not show detectable levels of LRP1. Immunoreactivity to LRP1 was evident within the plaque as neuritic profiles. LRP1 immunostaining in these neuritic structures was diminished in most hippocampal amyloid plaques of APPswe/PS1dE9 × LRP1lox/lox × GFAP-Cre mice (left column).
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Collectively, these data indicate that the levels of LRP1 are inherently low in astrocytes, including activated astrocytes, and that expression of Cre via the GFAP promoter produces efficient recombination of the LRP1 gene in neurons of the hippocampus. We presume that any expression of LRP1 in astrocytes was also reduced in LRP1lox/lox mice that were transgenic for GFAP-Cre but the absence of detectable staining in the absence of GFAP-Cre makes it impossible to confirm this assumption.
Hippocampal deposition of Aβ is not altered by reduction in LRP1 levels
In the hippocampus of nine-month-old APPswe/PS1dE9/LRP1lox/lox mice, we observed no obvious difference in the number of Aβ immunoreactive deposits in mice that were positive for GFAP-Cre as compared to mice lacking GFAP-Cre (Figure F5 5). With the lack of an obvious difference in amyloid plaque burden in APPswe/PS1dE9 mice lacking LRP1 expression in the hippocampus, we sought to carefully quantify plaque numbers in the hippocampus of multiple sections at multiple levels. Tissue sections were stained with Thioflavin S as a means of detecting amyloid deposits and then the hippocampus was outlined and the number of deposits counted (see Additional file S5 5 Figure S4). The number of deposits in the hippocampus of APPswe/PS1dE9/LRP1loxp/loxp mice (n = 13) were similar to APPswe/PS1dE9/LRP1 loxp/loxp/GFAP-Cre mice (n = 12), meaning that significantly reducing LRP1 expression in the hippocampus had no obvious effect on the number or distribution of amyloid deposits in this structure. Female mice of all genotypes showed slightly higher levels of amyloid deposits as compared to males (Figure 5). We assessed whether loss of LRP1 altered any of the morphological features of the plaque such as density of neuritic structures, finding no obvious difference by silver staining (Additional file S6 6 Figure S5).
Figure 5Hippocampal amyloid plaque numbers do not change when LRP1 is reduced
Hippocampal amyloid plaque numbers do not change when LRP1 is reduced. (A and B) Representative examples of immunohistochemical stains with 6E10 antibody (1:1,000, Covance) for Aβ in APPswe/PS1dE9/LRP1lox/lox mice that were either positive or negative for GFAP-Cre. (C) To quantify amyloid deposits, we stained sections with Thioflavin-S, which produces a sharper distinction between tissue and amyloid plaques (see Additional file 5 Figure S4 for an example) and captured images. The hippocampus was outlined and then the number of amyloid plaques inside the hippocampus was counted manually. A dot plot of amyloid plaque numbers in mice that were Cre+ is compared to the numbers in mice that were Cre-. Animals that were used as breeders are noted. No significant differences between genotypes either Cre+ (n = 12) vs. Cre- (n = 13) groups (P = 0.75) or between breeders (n = 10) vs. non-breeders (n = 15) (P = 0.69) was noted.
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Additional file 5
Thioflavin-S staining of hippocampal sections. The numbers of amyloid plaques in the hippocampus as identified by Thioflavin-S staining section were manually by outlining the border of the hippocampus on the image and then counting the number of plaques in hippocampus. These images show representative examples of animals of both genotypes (Figure S4).
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Additional file 6
The morphology of hippocampal amyloid deposits is unchanged by the lack of LRP1. Silver stains of 30 μm sections from APPswe/PS1dE9 × LRP1 lox/lox mice that were either positive or negative for GFAP-Cre. White arrows mark neurites and the white arrow heads mark to the core of the plaques (Figure S5).
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Total Aβ levels do not change when LRP1 levels are reduced
Sandwich ELISAs were used to measure the total amount of Aβ40 and Aβ42 in the brains of these mice as another means to assess amyloid burden. To completely solubilize different forms of the peptides, aggregated or otherwise, homogenates were incubated for several hours in guanidine-HCl before being diluted in buffer for ELISA. Consistent with results from the amyloid plaque counting, ELISA measurements confirmed that the levels of Aβ40 and 42 in APPswe/PS1dE9 mice with reduced levels of LRP1 were no different from that of APPswe/PS1dE9 mice with normal levels of LRP1 (Table tblr tid T1 1 and Figure F6 6). Further, a regression analysis to plot Aβ levels at different ages showed that the rate of Aβ accumulation did not change by the reduction of LRP1 levels (Additional file S7 7 Figure S6).
tbl Table 1The levels of Aβ in the brains of mice of each genotype and agetblbdy cols 10
r
c center
Age (month)
Cre
N
cspan 3
Aβ40 (pg/mg brain)
Aβ42 (pg/mg brain)
ratio 40/42
Average
STDEV.P
P-value
Average
STDEV.P
P-value
hr
8
-
3
1,257
1,022
0.40
271
193
0.45
4.6
+
3
481
315
142
75
3.4
9
-
17
2,024
1,192
0.90
399
192
0.71
5.1
+
12
2,074
746
421
116
4.9
15
-
4
4,235
946
0.65
1,015
104
0.74
4.2
+
4
4,582
823
972
188
4.7
16 to 18
-
4
4,445
1,135
0.74
971
181
0.79
4.6
+
5
4,830
1,777
1,020
289
4.7
tblfn
There was no difference in the levels of Aβ40 and 42 in the brains of mouse expressing APPswe/PS1dE9 gene in the LRP1 lox/lox homozygous background in the absence or presence of the GFAP-Cre transgene in any given age group.
Figure 6Aβ ELISA assay of whole brain homogenates show no differences in Aβ levels by genotype
Aβ ELISA assay of whole brain homogenates show no differences in Aβ levels by genotype. Aβ levels in 8-, 9-, 15- and 16 to 18-month-old with normal LRP1 levels (APPswe/PS1dE9(+)/LRP1lox/lox) were compared to those with lower LRP1 levels (GFAP-Cre +). There was no difference in Aβ levels by genotype by age (p values range 0.40 to 0.90 based on Student's t-test). ELISA data used to generate the graph are shown in Table 1, which includes information on the number of animals used at each age.
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Additional file 7
Regression analysis of Aβ levels in mice of each genotype by age. No difference in the rate of Aβ accumulation was evident. The regression curve of the slopes between two groups of animals is not significant for Aβ40 (P = 0.409) or Aβ42 (P = 0.864) (Figure S6).
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Discussion
In the present study, we have used genetic manipulation to reduce the expression levels of LRP1 in the hippocampus of mice that co-express mutant APP and mutant PS1. We demonstrate that lowering the levels of LRP1 by approximately 50% throughout the brain and possibly by a much greater degree in hippocampus had no significant impact on the rate, severity or character of amyloid deposition. We found no evidence of a proportional change in hippocampal amyloid burden by reducing LRP1 expression in CA and DG neurons with no obvious morphological change in the appearance of the plaques that formed.
To reduce LRP1 levels in the brains of APPswe/PS1dE9 mice, we employed mice that express Cre recombinase under the transcriptional regulation of the GFAP promoter and mice in which the LRP1 allele has been modified by introduction of lox p sites. The GFAP-Cre mice have previously been used in several studies and these investigations have previously demonstrated that Cre is expressed early in neurogenesis such that targeted genes may recombine in both neurons and glia 2627. In our cross of the GFAP-Cre mice with LRP1lox/lox mice, we observed efficient targeting of LRP1 expression in the hippocampal neurons. However, cortical neurons continued to show demonstrable LRP1 immunoreactivity. We note, that in the hippocampus, a subset of neurons in the molecular layer continued to express LRP1 in GFAP-Cre/loxp/loxp mice; however, the levels in CA and DG neurons were dramatically reduced. The levels of LRP1 in astrocytes appeared to be inherently low and we could not confirm whether or not expression of LRP1 was reduced further in LRP1lox/lox mice harboring the GFAP-Cre.
We observed no obvious developmental abnormality or reductions in the numbers of CA and DG neurons in mice transgenic for GFAP-Cre and LRP1 loxp/loxp, or mice of this genotype that were also transgenic for APPswe/PS1dE9. However, as noted in Results, we encountered significant difficulty in breeding these animals and noted a high degree of lethality early in the process. For reasons that remain unknown, these issues were overcome using a strategy of foster mothering with FVB/NJ females. The APPswe/PS1dE9 mice and the LRP1 loxp mice were both congenic in the C57BL/6J background and we bred the GFAP-Cre into C57BL/6J mice for four generations before beginning the crosses to produce experimental animals. In our hands, mice of the C57BL/6J strain are poorer mothers than commonly used hybrid strains or the inbred FVB/NJ strain. As we expected, foster mothering solved issues we had with poor mothering by C57BL/6J females. Unexpectedly, offspring that were foster-mothered by FVB/NJ females survived much better than mice of the same genotype mothered by C57BL/6J females. We presently have no explanation for this outcome. We note that in a previous effort to cross congenic C57BL/6J APPswe/PS1dE9 mice to congenic C57BL/6J RAP knockout mice we observed a high level of lethality in mice that were transgenic for APPswe/PS1dE9 and homozygous knockout for RAP 22. RAP is a direct ER chaperone for LRP1 and loss of RAP lowers the level of mature LRP1 that reaches the cell surface B37 37. Notably, others observed no issues in crosses of the PDAPP mouse model to RAP knockout mice 23, so we concluded that the lethality we observed our earlier work was due to some interaction between mutant PS1 and RAP. Our experience in the mating of the LRP1 loxp mice to APPswe/PS1dE9 mice leads us to believe that there may be some more complex interaction between PS1dE9 and RAP/LRP1 that reduces viability. This current study defines the source of this defect to the nervous system because LRP1 is only reduced in the brain by the expression of GFAP-Cre. Although likely to be unrelated to AD, further examination of the basis of this lethality may reveal functions of APPswe or PS1dE9 that impact CNS function.
Because the reduction in neuronal LRP1 was greatest in the hippocampus, we focused our pathologic analyses on the hippocampus, finding no significant change in the severity of amyloid deposition when LRP1 levels were reduced in the APPswe/PS1dE9 mice. The data shown in Figure 5C document the variability in the number of amyloid deposits in hippocampus between mice that are positive and negative for Cre recombinase. In both groups, the number of animals that were below and above the mean (horizontal line) was similar. Notably, if the number of deposits had been reduced by just 30% (with the same level of variance), then we would have been able to have reached statistical significance (P = 0.05) with just 10 animals in each group (our n's were 12 vs. 13). Based on quantification of immunoblots from hemi-brains, we conservatively estimate that the minimum reduction in LRP1 levels would be in the range of 50%. By power calculation, we should have been powered to observe a 30% decrease in amyloid plaque numbers and thus we conclude that reducing LRP1 levels does not produce a proportional decrease in amyloid plaque numbers. Consistent with our findings, Zerbinatti and colleagues reported that increasing the levels of LRP1, via expression of a recombinant mini-receptor, did not produce a proportional increase in amyloid burden 25. However, this study did note small increases of levels of both soluble and insoluble Aβ in the cortex with a more selective increase in soluble Aβ in the hippocampus of older mice with high amyloid burden. Our measures of total Aβ did not find evidence that reducing LRP1 levels produced a change in the accumulated levels of Aβ. Zerbinatti and colleagues also noted the appearance of Aβ42 dimers after extraction in buffers containing 0.1 M carbonate. We have not been able to reliably detect dimeric Aβ in our APPswe/PS1dE9 mice at any age and thus we cannot comment as to whether reducing LRP1 reduces the levels of these dimers.
Interpreting the outcome of this work is not as straightforward as one would like because the LRP1 levels were not reduced to zero. One issue is that we do not know how much LRP1 must be expressed to sustain amyloidogenesis or whether we should expect changes in amyloid formation in proportion to changes in LRP1 levels. Additionally, the source of the Aβ peptides that produce deposits may be neurons that project axons into the hippocampus as well as local neurons. Within the hippocampus, we observed a near elimination of LRP1 immunoreactivity of neurons of the CA fields. However, the hippocampus receives axonal projections from the entorhinal cortex making it possible that an activity of LRP1 in cortical neuronal projections masked any effect of local reductions in LRP1 levels in hippocampus. Ultimately, the most conservative interpretation is that significant suppression of LRP1 activity in local hippocampal neurons does not produce a proportional decrease in amyloid plaque numbers this structure.
Apart from the potential effects of LRP1 reductions on amyloid plaque levels, we had anticipated that we might see some change in neuritic pathology. Nearly all amyloid plaques in APPswe/PS1dE9 mice contain apolipoprotein E (ApoE) 31 and ApoE is known to be a ligand for LRP1 that modulates neurite outgrowth B38 38. Thus, we had thought it possible that reducing LRP1 locally in the hippocampus could have reduced neuritic pathology around amyloid plaques. Notably, in APPswe/PS1dE9/LRP1lox/lox mice that were positive for GFAP-Cre we observed large neuritic plaques in which the neurites were depleted of LRP1 immunoreactivity.
Conclusions
This study used genetic tools to reduce LRP1 levels in the brains of mice that co-express APPswe/PS1dE9 and develop amyloid pathology, producing a model in which LRP1 levels in most hippocampal neurons were lowered to below the level of detection by immunohistochemistry. In the hippocampus of mice with reduced LRP1 levels, we observed no obvious change in the rate of deposition, severity, or character of amyloid deposits. Obviously, this study does not provide any insight into what LRP1 expressed in non-neural cell types, such as endothelial cells, other organs may do in regard to modulating amyloid deposition. It is also very possible that homologues of LRP1 may have compensated for any effect of reducing the levels of LRP1. However, at minimum, our study indicates that reducing the levels of LRP1 does not produce proportional reductions in amyloid plaque numbers in the hippocampus of the APPswe/PS1dE9 model. Thus, this genetic test does not produce supportive evidence for the notion that modulating LRP1 function in neurons or glial could be beneficial in modulating amyloidosis. Whether this outcome is unique to the APPswe/PS1dE9 model is of course an issue in assessing the potential importance of this pathway in human disease.
Abbreviations
ApoE: apolipoprotien E; APP: amyloid precursor protein; APPswe/PS1dE9: a tandem integration of transgenes encoding amyloid precursor protein with the Swedish mutation and presenilin 1 gene with exon 9 deletion; BCA: bicinchoninic acid assay: a biochemical assay for protein concentration; CA: cornu ammonis in hippocampus; Cre-lox: Cre recombinase-Lox P site system, a genetic tool to control site specific recombination events in genomic DNA; DG: dentate gyrus in hippocampus; ELISA: enzyme-linked immunosorbent assay; GFAP-Cre: transgenic mice with Cre recombinase driven by glial fibrillary acidic protein (GFAP) promoter; LRP1: low-density lipoprotein receptor-related protein.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
All authors contributed to this manuscript. GX designed the experiments, participated to generate the cohorts of mice, carried out most of the experiments, performed the statistical analysis, and drafted the manuscript. CG contributed to the histology, immunostaining work and collected data of amyloid plaques. SF genotyped the mice and recorded the breeding data. DRB conceived of the whole study, participated in detailed experimental design and prepared the manuscript. All authors read and approved the final manuscript.
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Acknowledgements
We gratefully thank Joachim Herz (University of Texas Southwestern Medical Center, Dallas, Texas, USA) for providing LRP1 antibody and mice with targeted Lox P sites (LRP1 lox/lox mice), and Wei Hou (Department of Biostatistics, University of Florida) for help with statistics. This study was supported by American Health Assistance Foundation (AHAF) [Pilot Awards (GX)] and SantaFe HealthCare Alzheimer's Disease Research Center.
refgrp Lipoprotein receptors in Alzheimer's diseaseAndersenOMWillnowTETrends Neurosci200629687lpage 69410.1016/j.tins.2006.09.002link fulltext 17000013Apolipoprotein E in sporadic Alzheimer's disease: allelic variation and receptor interactionsRebeckGWReiterJSStricklandDKHymanBTNeuron19931157558010.1016/0896-6273(93)90070-88398148Expression and function of the low density lipoprotein receptor-related protein (LRP) in mammalian central neuronsBuGMaksymovitchEANerbonneJMSchwartzALJ Biol Chem199426918521185287518435Multiple, diverse senile plaque-associated proteins are ligands of an apolipoprotein E receptor, the alpha 2-macroglobulin receptor/low-density-lipoprotein receptor-related proteinRebeckGWHarrSDStricklandDKHymanBTAnn Neurol19953721121710.1002/ana.4103702127531418Expression of alpha(2)-macroglobulin receptor/low density lipoprotein receptor-related protein (LRP) in rat microglial cellsMarzoloMPvon BernhardiRBuGInestrosaNCJ Neurosci Res20006040141110.1002/(SICI)1097-4547(20000501)60:3<401::AID-JNR15>3.0.CO;2-L10797543Association of membrane-bound amyloid precursor protein APP with the apolipoprotein E receptor LRPRebeckGWMoirRDMuiSStricklandDKTanziREHymanBTBrain Res Mol Brain Res20018723824511245926Visualization of A beta 42(43) and A beta 40 in senile plaques with end-specific A beta monoclonals: evidence that an initially deposited species is A beta 42(43)IwatsuboTOdakaASuzukiNMizusawaHNukinaNIharaYNeuron199413455310.1016/0896-6273(94)90458-88043280FE65 constitutes the functional link between the low-density lipoprotein receptor-related protein and the amyloid precursor proteinPietrzikCUYoonISJaegerSBusseTWeggenSKooEHJ Neurosci2004244259426510.1523/JNEUROSCI.5451-03.200415115822Clearing amyloid through the blood-brain barrierZlokovicBVJ Neurochem20048980781110.1111/j.1471-4159.2004.02385.x15140180RAGE (yin) versus LRP (yang) balance regulates alzheimer amyloid beta-peptide clearance through transport across the blood-brain barrierDeaneRWuZZlokovicBVStroke2004352628263110.1161/01.STR.0000143452.85382.d115459432Testing the neurovascular hypothesis of Alzheimer's disease: LRP-1 antisense reduces blood-brain barrier clearance, increases brain levels of amyloid-beta protein, and impairs cognitionJaegerLBDohguSHwangMCFarrSAMurphyMPFleegal-DemottaMALynchJLRobinsonSMNiehoffMLJohnsonSNKumarVBBanksWAJ Alzheimers Dis200917553570pmcid 280283319433890Genetic association of the low-density lipoprotein receptor-related protein gene (LRP), an apolipoprotein E receptor, with late-onset Alzheimer's diseaseKangDESaitohTChenXXiaYMasliahEHansenLAThomasRGThalLJKatzmanRNeurology199749566110.1212/WNL.49.1.569222170Modulation of amyloid beta-protein clearance and Alzheimer's disease susceptibility by the LDL receptor-related protein pathwayKangDEPietrzikCUBaumLChevallierNMerriamDEKounnasMZWagnerSLTroncosoJCKawasCHKatzmanRKooEHJ Clin Invest20001061159116610.1172/JCI1101330142211067868Withania somnifera reverses Alzheimer's disease pathology by enhancing low-density lipoprotein receptor-related protein in liverSehgalNGuptaAValliRKJoshiSDMillsJTHamelEKhannaPJainSCThakurSSRavindranathVProc Natl Acad Sci USA20121093510351510.1073/pnas.111220910922308347LDL receptor-related protein (LRP) in Alzheimer's disease: towards a unified theory of pathogenesisVan UdenEKangDEKooEHMasliahEMicrosc Res Tech20005026827210.1002/1097-0029(20000815)50:4<268::AID-JEMT3>3.0.CO;2-110936878The low-density lipoprotein receptor-related protein, a multifunctional apolipoprotein E receptor, modulates hippocampal neurite developmentNaritaMBuGHoltzmanDMSchwartzALJ Neurochem1997685875959003044Alpha2-macroglobulin enhances the clearance of endogenous soluble beta-amyloid peptide via low-density lipoprotein receptor-related protein in cortical neuronsQiuZStricklandDKHymanBTRebeckGWJ Neurochem1999731393139810501182Clearance of Alzheimer's amyloid-ss(1-40) peptide from brain by LDL receptor-related protein-1 at the blood-brain barrierShibataMYamadaSKumarSRCaleroMBadingJFrangioneBHoltzmanDMMillerCAStricklandDKGhisoJZlokovicBVJ Clin Invest20001061489149910.1172/JCI1049838725411120756LRP: a multifunctional scavenger and signaling receptorHerzJStricklandDKJ Clin Invest200110877978420093911560943Apolipoprotein E facilitates neuritic and cerebrovascular plaque formation in an Alzheimer's disease modelHoltzmanDMFaganAMMackeyBTenkovaTSartoriusLPaulSMBalesKAsheKHIrizarryMCHymanBTAnn Neurol20004773974710.1002/1531-8249(200006)47:6<739::AID-ANA6>3.0.CO;2-810852539LDL receptor-related protein internalizes and degrades uPA-PAI-1 complexes and is essential for embryo implantationHerzJClouthierDEHammerRECell19927141142110.1016/0092-8674(92)90511-A1423604Receptor-associated protein (RAP) plays a central role in modulating Abeta deposition in APP/PS1 transgenic miceXuGKarchCLiNLinNFromholtDGonzalesVBorcheltDRPLoS One20083e315910.1371/journal.pone.0003159252228618776935Increased extracellular amyloid deposition and neurodegeneration in human amyloid precursor protein transgenic mice deficient in receptor-associated proteinVan UdenEMalloryMVeinbergsIAlfordMRockensteinEMasliahEJ Neurosci2002229298930412417655Functional expression of low density lipoprotein receptor-related protein is controlled by receptor-associated protein in vivoWillnowTEArmstrongSAHammerREHerzJProc Natl Acad Sci USA1995924537454110.1073/pnas.92.10.4537419797538675Increased soluble amyloid-beta peptide and memory deficits in amyloid model mice overexpressing the low-density lipoprotein receptor-related proteinZerbinattiCVWozniakDFCirritoJCamJAOsakaHBalesKRZhuoMPaulSMHoltzmanDMBuGProc Natl Acad Sci USA20041011075108010.1073/pnas.030580310132715314732699hGFAP-cre transgenic mice for manipulation of glial and neuronal function in vivoZhuoLTheisMAlvarez-MayaIBrennerMWilleckeKMessingAGenesis200131859410.1002/gene.1000811668683Deletion of brain dystroglycan recapitulates aspects of congenital muscular dystrophyMooreSASaitoFChenJMicheleDEHenryMDMessingACohnRDRoss-BartaSEWestraSWilliamsonRAHoshiTCampbellKPNature200241842242510.1038/nature0083812140559Sustained somatic gene inactivation by viral transfer of Cre recombinaseRohlmannAGotthardtMWillnowTEHammerREHerzJNat Biotechnol1996141562156510.1038/nbt1196-15629634821Inducible inactivation of hepatic LRP gene by cre-mediated recombination confirms role of LRP in clearance of chylomicron remnantsRohlmannAGotthardtMHammerREHerzJJ Clin Invest199810168969510.1172/JCI12405086149449704Co-expression of multiple transgenes in mouse CNS: a comparison of strategiesJankowskyJLSluntHHRatovitskiTJenkinsNACopelandNGBorcheltDRBiomol Eng20011715716510.1016/S1389-0344(01)00067-311337275Hyper-expression of human apolipoprotein E4 in astroglia and neurons does not enhance amyloid deposition in transgenic miceLesuisseCXuGAndersonJWongMJankowskyJHoltzGGonzalezVWongPCPriceDLTangFWagnerSBorcheltDRHum Mol Genet2001102525253710.1093/hmg/10.22.252511709540Superoxide dismutase 1 with mutations linked to familial amyotrophic lateral sclerosis possesses significant activityBorcheltDRLeeMKSluntHSGuarnieriMXuZSWongPCBrownRHPriceDLSisodiaSSClevelandDWProc Natl Acad Sci USA1994918292829610.1073/pnas.91.17.8292445928058797An improved thioflavine S method for staining neurofibrillary tangles and senile plaques in Alzheimer's diseaseGunternRBourasCHofPRValletPGExperientia19924881010.1007/BF019235941371102Transgenic mice expressing caspase-6-derived N-terminal fragments of mutant huntingtin develop neurologic abnormalities with predominant cytoplasmic inclusion pathology composed largely of a smaller proteolytic derivativeTebbenkampATGreenCXuGDenovan-WrightEMRisingACFromholtSEBrownHHSwingDMandelRJTessarolloLBorcheltDRHum Mol Genet2011202770278210.1093/hmg/ddr17621515588Silver impregnation of nerve cells and fibers in celloidin sections. A simple impregnation techniqueHiranoAZimmermanHMArch Neurol1962611412210.1001/archneur.1962.0045020002800313907612A comparative study of modified Bielschowsky, Bodian and thioflavin S stains on Alzheimer's neurofibrillary tanglesYamamotoTHiranoANeuropathol Appl Neurobiol1986123910.1111/j.1365-2990.1986.tb00677.x242258039 kDa receptor-associated protein is an ER resident protein and molecular chaperone for LDL receptor-related proteinBuGGeuzeHJStrousGJSchwartzALEmbo J1995142269803983347774585Low density lipoprotein receptor-related protein mediates apolipoprotein E-dependent neurite outgrowth in a central nervous system-derived neuronal cell lineHoltzmanDMPitasREKilbridgeJNathanBMahleyRWBuGSchwartzALProc Natl Acad Sci USA1995929480948410.1073/pnas.92.21.9480408257568158



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Genotypes APP/PS1 + + Total GFAP Cre + + LRP Lox/lox Lox/lox Lox/lox Lox/lox Expected frequency of offspring 25% 25% 25% 25% 100% Non fostered Number of mice identified by genotype 56 31 21 50 158 Observed frequency 35% 20% 13% 32% 100% Estimated number of mice that died before 1.5 months of age (age of genotyping ) ~20 ~30 ~50 Number of mice found dead (< 9mos old) 2 3 9 1 15 (Premature death rate in group after weaning) (4%) (10%) (43%) (2%) (9%) Age range of mice found dead (months) (2.3 6.3) (1.9 8.8) (2.4 4.6) 3.3 Fostered Number of Mice 45 41 32 49 167 Observed frequency 27% 25% 19% 29% 100% Estimated number of mice that died before 1.5 months of age (age of genotyp ing ) ~5 ~15 ~20 Number of mice found dead (< 9mos old) 4 2 1 0 7 (Premature death rate in group after weaned) (9%) (5%) (3%) (0%) (4%) Age range of mice found dead (months) (2.0 9.0) (2.8 7.7) 3.7


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Abstract
Introduction
The low-density lipoprotein receptor-related protein (LRP1) and its family members have been implicated in the pathogenesis of Alzheimer's disease. Multiple susceptibility factors converge to metabolic pathways that involve LRP1, including modulation of the processing of amyloid precursor protein (APP) and the clearance of Aβ peptide.
Methods
We used the Cre-lox system to lower LRP1 levels in hippocampal neurons of mice that develop Alzheimer-type amyloid by crosses between mice that express Cre recombinase under the transcriptional control of the GFAP promoter, mice that harbor loxp sites in the LRP1 gene, and the APPswe/PS1dE9 transgenic model. We compared amyloid plaque numbers in APPswe/PS1dE9 mice lacking LRP1 expression in hippocampus (n = 13) to mice with normal levels of LRP1 (n = 12). Student t-test was used to test whether there were significant differences in plaque numbers and amyloid levels between the groups. A regression model was used to fit two regression lines for these groups, and to compare the rates of Aβ accumulation.
Results
Immunohistochemical analyses demonstrated efficient elimination of LRP1 expression in the CA fields and dentate gyrus of the hippocampus. Within hippocampus, we observed no effect on the severity of amyloid deposition, the rate of Aβ40/42 accumulation, or the architecture of amyloid plaques when LRP1 levels were reduced.
Conclusions
Expression of LRP1 by neurons in proximity to senile amyloid plaques does not appear to play a major role in modulating the formation of these proximal deposits or in the appearance of the associated neuritic pathology.
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Alzheimer's Research & Therapy. 2012 Apr 26;4(2):12
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RESEARCH OpenAccessReductionoflow-densitylipoproteinreceptorrelatedprotein(LRP1)inhippocampalneurons doesnotproportionatelyreduce,orotherwise alter,amyloiddepositioninAPPswe/PS1dE9 transgenicmiceGuilianXu1*,CameronCGreen1,2,SusanEFromholt1andDavidRBorchelt1*AbstractIntroduction: Thelow-densitylipoproteinreceptor-relatedprotein(LRP1)anditsfamilymembershavebeen implicatedinthepathogenesisofAlzheimer sdisease.Multiplesusceptibilityfactorsconvergetometabolic pathwaysthatinvolveLRP1,includingmodulationoftheprocessingofamyloidprecursorprotein(APP)andthe clearanceofA b peptide. Methods: WeusedtheCre-loxsystemtolowerLRP1levelsinhippocampalneuronsofmicethatdevelop Alzheimer-typeamyloidbycrossesbetweenmicethatexpressCrerecombinaseunderthetranscriptionalcontrolof theGFAPpromoter,micethatharborloxpsitesintheLRP1gene,andtheAPPswe/PS1dE9transgenicmodel.We comparedamyloidplaquenumbersinAPPswe/PS1dE9micelackingLRP1expressioninhippocampus(n=13)to micewithnormallevelsofLRP1(n=12).Studentt-testwasusedtotestwhetherthereweresignificant differencesinplaquenumbersandamyloidlevelsbetweenthegroups.Aregressionmodelwasusedtofittwo regressionlinesforthesegroups,andtocomparetheratesofA b accumulation. Results: ImmunohistochemicalanalysesdemonstratedefficienteliminationofLRP1expressionintheCAfieldsand dentategyrusofthehippocampus.Withinhippocampus,weobservednoeffectontheseverityofamyloid deposition,therateofA b 40/42accumulation,orthearchitectureofamyloidplaqueswhenLRP1levelswere reduced. Conclusions: ExpressionofLRP1byneuronsinproximitytosenileamyloidplaquesdoesnotappeartoplaya majorroleinmodulatingtheformationoftheseproximaldepositsorintheappearanceoftheassociatedneuritic pathology.IntroductionAmyloiddepositionisakeypathologicaleventinAlzheimer sdisease.Alargebodyofevidencesuggeststhat lowdensitylipoprotein(LDL)receptorrelatedprotein (LRP1),maybeakeymediatorofamyloiddeposition. AsamemberoftheLDLreceptorfamily,LRP1isa large,multifunctional,endocyticreceptorthatishighly expressedinneurons(reviewedbyAndersen[1]),activatedastrocytes[2-4],andmi croglia[5].Directbinding ofLRP1totheamyloidprecursorprotein(APP)has beenshowntoaffectendoproteolyticprocessingofAPP toincreasetheproductionofA b 42peptides[6],which arethemajorconstituentofamyloiddeposits[7].LRP1 canpromoteA b productionbyalteringtheprocessing ofAPPthroughinteractionsviatheKunitzprotease inhibitor(KPI)domain(isoformsAPP751orAPP770) [6].Althoughthenon-KPI-APPisoformcanweakly bindtoLRP1throughcytoplasmicadaptorproteins, *Correspondence:xugl@ufl.edu;drb1@ufl.edu1DepartmentofNeuroscience,SantaFeHealthCareAlzheimer sDisease Center,CenterforTranslationalResearchinNeurodegenerativeDisease, McKnightBrainInstitute,UniversityofFlorida,Gainesville,FL32610,USA FulllistofauthorinformationisavailableattheendofthearticleXu etal Alzheimer sResearch&Therapy 2012, 4 :12 http://alzres.com/content/4/2/12 2012Xuetal.;licenseeBioMedCentralLtd.ThisisanopenaccessarticledistributedunderthetermsoftheCreativeCommons AttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse,distribution,andreproductionin anymedium,providedtheoriginalworkisproperlycited.

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suchasFE65[8],APP695processingmaynotbeinfluencedassignificantlybyLRP1.Additionally,LRP1has alsobeenreportedtomediateexportofA b acrossthe blood-brainbarrier(reviewedby[9]);LRP1inendothelialcellsmaybinddirectlytoA b 1-40andexportit acrosstheblood-brainbarrier[10].Consistentwiththis notion,reducingLRP1levelsbyantisensetreatment reducesblood-brainbarrierclearanceofA b 42[11]. PolymorphismsintheLRP1genehavebeenassociated withincreasedriskforAlzheimer sdisease[12]and LRP1levelsaresignificantlyreducedinADpatients [13].Morerecently,Sehgalandcolleaguesreportedthat anextractof Withaniasomnifera inducesarapidclearanceofamyloidpathologywithaco-incidentinduction ofLRP1expressioninliver[14].OtherproteinsimplicatedinamyloidogenesishavebeenidentifiedasinteractingwithLRP1[15],suchasapolipoprotienE(ApoE) and a 2-macroglobulin.Bothoftheseproteinsare ligandsofLRP1andhavebeenreportedtomediatethe clearanceofA b [13,16-19].Moreover,ApoE,whichisa majorligandforLRP1,modulatesthedistributionand characterofamyloiddepositio n[20].Collectively,these datasuggestthatLRP1couldbeacrucialfactorinAlzheimer sdiseaseandthereceptorcharacteristicsof LRP1makethisproteinapotential drugable target whereinfluencingthebindingofLRP1toanyofits ligandscouldbeausefultherapeutic. Todate,therehavebeenfewstudiesthatexaminethe roleofLRP1 invivo usinganimalmodelsbygenetic manipulationofLRP1expression.ConstitutiveinactivationofLRP1islethal[21].We[22]andothers[23]have usedmicewithtargeteddeletionofLRP1receptorassociatedprotein(RAP),whichregulatesLRP1maturation, asameanstoreduceLRP1levelsinmousemodelsof Alzheimer sdisease.OnegrouphasreportedthathomozygousdeletionofRAP,whichlowersLRP1levelsby 75%[24],resultsinincreasedA b depositioninPDAPP mice[23].Similarly,weobservedthatheterozygous deletionofRAPalsoincreasedamyloiddeposition,in theAPPswe/PS1dE9model;however,inourcaseLRP1 levelswerenotsignificantlylowered[22].Expressionof anLRP1mini-receptoratapproximately3.7-foldover endogenousLRP1levelsinthePDAPPtransgenicmodel wasreportedtohavenoeffectonamyloidburden[25]. Collectively,thesestudiesillustratetheinconsistent in vivo findingsregardingLRP1andamyloidogenesisin transgenicmicethatover-expressmutantAPP(withor withoutco-expressionofmutantpresenilin). TofurtherprobetheroleofLRP1inamyloidogenesis, weusedCre/Loxtechnologytoconditionallyreduce LRP1expressioninthebrainsofmicethatco-express mutantAPP(APPswe)andmutantpresenilin1 (PS1dE9).Forthesestudiesweusedmicethatexpress Creunderthecontroloftheglialfibrillaryacidicprotein (GFAP)[26].Inpreviousstudieswiththeseanimals, othershavedemonstratedCre mediatedrecombination inbothneuronsandglialthroughoutthebrain[27].In ourhands,GFAP-mediatedexpressionofCreproduced incompleterecombination,suchthatthelevelsofLRP1 intotalbrainwerereducedapproximately50%.However,wedeterminedthatLRP1immunostaininginCA andgranuleneuronsofthehippocampuswasvirtually eliminated.Withinthehippocampus,weobservedno obviouschangeinthelevelofneuriticpathologyor amyloidburdenbylossofLRP1.MaterialsandmethodsAnimalsMicewithtargetedLoxPsitesintroducedintointronic andpromoterregionsoftheLRP1genewereobtained fromJoachimHerz[28,29],calledLRP1lox/lox.TransgenicmiceexpressingGFAP-Crewereobtainedfrom JacksonLaboratories(BarHarbor,ME,USA;Strain: FVB-Tg(GFAP-cre)25Mes /J;StockNumber:004600) [26].Theseanimalswereback-crossedtoC57BL/6J miceforatleastfourgenerationstoreducetheFVB straincontributionbeforebreedingtoLRP1lox/lox mice.LRP1lox/loxmice wereinparallelbredto APPswe/PS1dE9(line85)mice[30](bothwithC57BL/ 6Jbackground)toproducemicehomozygousforthe LRP1loxplocus.Togeneratetripletransgenicmice,we matedmicetransgenicforGFAP-Creandhomozygous forLRP1loxptomicetransgenicforAPPswe/PS1dE9 andhomozygousforLPRloxp.FVB/NJmaleandfemale micewerepurchasedfromJacksonLaboratories(stock number:001800)toproducefostermothers.Eventually, animalswiththegenotypesofAPPswe/PS1dE9(+)/LRP1 lox/loxwith/withouttheGF AP-Cregenewereusedfor thestudy.AllofthemicewerehousedinstandardSPF cagesfor9to18monthsbeforeharvesting.Allproceduresinvolvinganimalhandlingandprocessingwere approvedbytheUniversityofFloridaInstitutionalAnimalCareandUseCommitteeandincompliancewith theNationalInstitutesofHealthguidelines.WesternblotanalysisLevelsofLRP1wereassessedbyWesternblot,using standardmethodswehavepreviouslydescribed[31]. AliquotsofthePBSbrainhomogenates(100 l)from 14-to15-month-oldmiceweremixedwith10 lof 10%SDS,centrifugedatthet opspeed(approximately 10,000xg)inabenchtopmicrofugefor10minutes, andthen30 lofthesupernatantwasmixedwith10 l 4xLaemmlibufferandboiledfor5minutes.Anamount ofsampleequalto50 gtotalprotein(byBCAassay) wereloadedperlaneforSDS-PAGEandtransferredto nitrocellulosemembranes. Blotswereincubatedwith rabbitpolyclonalantibody377 a LRP1(1:1,000)andXu etal Alzheimer sResearch&Therapy 2012, 4 :12 http://alzres.com/content/4/2/12 Page2of14

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thenreprobedwiththem/hSOD1antibody(1:3,000)to gaugeproteinloading[32].TheECLsignalwascaptured andquantifiedusingtheL AS-3000ima gingsystem (FUJIFILMLifeScience,Tokyo,Japan).TissuepreparationforhistologyMiceweredeeplyanesthetizedbyinjectionanesthetic (3%bodyweightof1.2%Tribromoethanol(Avertin Sigma,St.Louis,MO,USA)beforetranscardialperfusionwithcoldphosphatebu fferedsaline(PBS,pH7.4). Thebrainswerethenremovedandcutsagittallydown themidline;onehemi-brainwasfrozenondryicefor biochemistryassay,theotherhemi-brainwasimmersion fixedin4%paraformaldehydeinPBS.After48hoursin fixationat4C,brainsweretransferredtoPBS,andthen 30%sucroseinPBSforseveraldaysbeforecryostatsectioningat30 mthickness.Fromthefirstcompletesectionclosesttomid-line,10sectionspergroupwere storedineachwellof24-well-platesandtheentire hemi-brainwassectioned.Sectionswerekeptinantifreezesolution(100mMsodiumacetate,250mMpolyvinylpyrrolidone,40%ethyleneglycol,pH6.5)at-20C untilstaining.ImmunostainingImmunostainingof30 mfreefloatingsectionswasperformedaccordingtoastandardprotocol.Sectionswere washedinPBStoremovetheanti-freezesolution,and thenblockedinPBScontaining5%normalgoatserum with0.1%TritonX-100before incubatingwithantibodiesagainstLRP1(rabbitpolyclonal377 a LRP1 (1:1,000)),glialfibrillaryacidicprotein(GFAP)(mouse monoclonal,1:1,000,Millip ore,Billerica,MA,USA), neuronalantigenNeuN(mousemonoclonal,1:1000, Millipore)or6E10(mousemonoclonal,1:1,000,Covance,Princeton,NJ,USA).Secondaryantibodiesofgoat anti-rabbitandgoatanti-mouse,conjugatedwithAlexa 594orAlexa488(Invitrogen,Carlsbad,CA,USA)were usedtovisualizeprimaryantibodybinding.Afterwashingtoremoveunboundsecondaryantibody,thesections werethenmountedonmicroscopeslidesanddriedin air.Aquickdipin0.1%SudanBlackBsolution(in70% ethanol)for5to10minuteswasusedtoblockthe auto-fluorescenceoflipofuscinthentheslideswere washedin70%ethanol,thenwater,driedandcovered byProLongGoldantifadereagentwithDapi(Invitrogen).AnOlympusDSU-IX81SpinningDiscConfocal microscope(Tokyo,Japan)wasusedtocapturethe images.ThioflavinSstainingandsilverstainingofamyloid plaquesOnesectionfromeachwell(seeaboveTissuepreparationforhistology)wasrandomlyselectedforThioflavin SstainingaccordingtotheGunternstandardprotocol [33]withsmallmodificatio n[34].Fluorescentphotos weretakenusing5xobjectivesbyaCanondigitalcamera(Tokyo,Japan).Silverimpregnationstainingwas performedonthesame30 mfloatingsectionsbymodifiedHirano smethod[35,36].Briefly,sectionswere washedandmountedonslidestodryasthoseforThioflavinSstaining,andthenthestepsdescribedforsilver impregnationmethodsofparaffinslideswerefollowed [36].AmyloidplaquequantificationFoursectionsperanimal(from300to1,500 mlateral tomidline)stainedwithThioflavinSwereusedtoquantifytheamyloidplaquelevels.Theimagesofthehippocampuswereoutlinedandthenumbersofamyloid plaquesweremanuallycountedbyanobserverblinded togenotype.Theimagesofeachhippocampuswereoutlinedandquantifiedthreetimesonthreedifferentdays toreducebiasandthenanalyzed.Thenumbersofamyloidplaquesineachsectionwereaveragedandthen numbersofplaquesfromthefoursectionsfromdifferentlevelsofthebrainwereaveragedtoproducethe finalnumberofamyloidplaques/sectioninthehippocampusofeachanimal.A b ELISAThefrozenhemi-brainfromeachtransgenicanimalwas weighed,thenhomogenizedbysonicationinfive volumesbyweightinPBSwith1proteaseinhibitor cocktail(SigmaCat.P8340).A100 laliquotofthisPBS brainhomogenatewasusedfo rELISAanalysis.Protein aggregatesinthePBShomo genateweredenaturedby theadditionof150 lof8.0Mguanidine-HCltoafinal concentrationof4.8M,andmixedatroomtemperature forthreetofourhoursbeforestorageovernightat-20 C.Thedenaturedsampleswerethenfurtherdiluted 200-foldinreactionbuffer(PBScontaining5%BSA, 0.03%Tween-20,and1proteaseinhibitorcocktail (Sigma)).Sampleswerefinallypreparedforapplication totheELISAmicroplatebymixingwithanequal volumeofStandardDilutionBufferprovidedbythe manufacturersupplementedwith1proteaseinhibitor cocktail.Aliquotsofthesesampleswereassayedfor bothAb 40andAb 42usingcommerciallyavailablesandwich-ELISAsaccordingtothemanufacturer smanual (Invitrogen).StatisticalanalysisoftheELISAdataWecomparedthedifferencesamongthemeansofAb 40 and42levelsamongthesameagegroupsandthedistributionofthelevelsacrossallages.Thecomparisonin thesameagegroupbetweentwodifferentgenotypes (GFAP-Crepositiveornegative)wascarriedoutusingXu etal Alzheimer sResearch&Therapy 2012, 4 :12 http://alzres.com/content/4/2/12 Page3of14

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Excelwiththetwo-tailStudent t -testwithbothequal andunequalvariance.TheA b levelswithineachage groupweregraphedinExcelandlinearregressionlines wereadded.ExcelAnalysisToolPakadd-inwasusedfor regressionanalysis.IBMSPSSStatistics19[(trialversion),Somers,NY,USA]wasusedtotestthesignificancebetweentheregressioncurvesbygenotypes.A P valueof<0.05wasconsideredstatisticallysignificant.ResultsAstrainofGFAP-CremicethatexpressesCreearlyin neurogenesiswasusedtocrosstothehomozygousmice withLRP1loxpsiteswiththeexpectationofcomplete recombinationofflox edgenesinneuronsaswellasglia [27].TheGFAP-Cremicethatwereusedforthisstudy fromtheJacksonLaboratoryweremaintainedintheFVB strain.Tomakethemcompatiblewithourstrainsofmice, weback-crossedthemtoC57BL/6JmiceforfourgenerationsbeforecrossingtoLRP1lox/loxmice,andthenwe continuedtocrossthemtoC57BL/6JcongenicAPPswe/ PS1dE9mice(line85).Micethatweretransgenicfor APPswe/PS1dE9andLRP1loxp/loxpwerematedtomice thatweretransgenicforGFAP-CreandLRP1loxp/loxp, ormicethatweretransgenicforallthreealleles(APPswe/ PS1dE9,GFAP-CreandLRP1loxp/loxp)werematedto miceonlytransgenicforLRP1loxp/loxp.Atotalof54 breedingcagesweresetupoverthecourseofthestudyto generatethemiceweultimatelyexamined.Morethan20 ofthesecagesfailedtogenerateanyoffspring,5cagesgeneratedafewoffspring,butthemothersfailedtocarefor thelittersandnoneoftheoffspringlivedtoweaningage (fourweeksold).Oftheanimalsofthedesiredgenotype (APPswe/PS1dE9/LRP1loxp/loxp/GFAP-Cre)thatwere produced,almosthalfdiedsuddenlyinthefirstnine monthsoflife(seeAdditionalfile1TableS1). Becauseofthereducedviabilityofthemiceofthe desiredgenotypeandthepoormotheringoftheC57BL/ 6Jfemales,webeganafosterbreedingprogram.FVB/NJ females,(whichareverygoodmothers),wereusedto fosterallnewbornoffspringinanefforttoincreasethe frequencyofanimalsthatsu rvivetoweaning.Thisfoster-motheringprogramwassuccessfulinproducinga greaternumberofanimalsofthedesiredgenotypethat survivedtoweaning.Surprisingly,thesurvivalrateof theoffspringthatwerefoste r-motheredwasalsomuch improved.Atotalof167offspringwereproducedinthe foster-motheringprogramofwhichonly7diedbefore ninemonthsofageandonly1ofthese7wastriple transgenicgenotype(seeAdditionalfile1TableS1). Overall,thedistributionofthemicewithallfourofthe genotypeswasmuchclosertoMendeliandistribution (25%ofeachgenotypeexpected).Atpresent,wehave noexplanationforthisobservation,butitappearsthat thefoster-motheringthatimprovedsurvivaltoweaning agetransferredtoimprovedresistancetotheunderlying problemthatcausedthesuddenearlydeathafterweaning.Ultimatelymostofthemicethatwerestudiedin thisreportweregeneratedbyfoster-mothering.GFAP-CreexpressioninLRP1lox/loxmicelowersLRP1 levelsinhippocampusUsingthebreedingscheme describedabove,weproducedmicetransgenicforGFAP-Cre,APPswe/PS1dE9, andhomozygousfortheflox edLRP1alleles.Allmice describedinthisworkwerehomozygousforthe LRP1loxallele.Noobviousdevelopmentalabnormalities wereobservedinthebrainsofmicethatweretransgenic forGFAP-Crewhetherornottheywerealsotransgenic fortheadditionalAPPswe/PS1dE9transgenes(not shown).Immunoblotsofhemi-brainhomogenates demonstratedthatLRP1levelswerereducedbyapproximately50%inmicethatweretransgenicforAPPswe/ PS1dE9andGFAP-Creascomparedtomicethatwere transgeniconlyforAPPswe/PS1dE9(Figure1). Inimmunohistochemicalstainsofhippocampusof micethatweredoubletransgenicforAPPswe/PS1dE9 andGFAP-CreintheLRP1lox/loxbackground,we observedverylittlestainingforLRP1inhippocampal neuronsintheCAfieldsanddentategyrus(DG)(Figure 2;Additionalfile2FigureS1).Incontrast,inAPPswe/ PS1dE9/LRP1lox/loxmiceweobservedstrongimmunoreactivityforLRP1intheseregions(Figure2).This patternofreactivitymatcheswhatwouldbeexpected basedonthelevelsofmRNAinthesestructures(Additionalfile3FigureS2).Inthecortexofmiceofthe samegenotype,weobservedthatmostneurons,identifiedbyNeuNstaining,remainedpositiveforLRP1 immunoreactivity,albeitatwhatappearedtobeslightly lowerlevels,indicatingthattherecombinationofthe flox edLRP1allelewaslesscompleteincorticalneurons (Figure3).Thus,althoughthelevelofrecombinationin thecortexwaslessthanreportedforotherflox edalleles [27],thelevelofrecombinationinthehippocampuswas muchmorerobustandthuswefocusedouranalysison amyloiddepositionwithinthehippocampus. Basedonpreviousstudies,weexpectedthatLRP1would beexpressedatsomelevelinastrocytes,particularly activatedastrocytes[2-4].NoLRP1immunoreactivity couldbedetectedinrestingastrocytesofanygenotype (Additionalfile4FigureS3).Inthehippocampusof nine-month-oldGFAP-Cre/APPswe/PS1dE9/LRP1lox/ loxmice,weobservedthatamyloiddepositsstillformed atarelativelyhighfrequency(seebelow)andaround thesedepositsweobservedactivatedastrocytes.However,wefailedtodetectLRP1inactivatedastrocytes surroundingtheseplaques(Figure4).Withinthese depositsweobservedLRP1immunoreactivitythatwas reminiscentofneuriticprofiles(Figure4).TheseLRP1Xu etal Alzheimer sResearch&Therapy 2012, 4 :12 http://alzres.com/content/4/2/12 Page4of14

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immunoreactiveneuriticprofilesweremostlyabsent withinthemolecularlayerofthehippocampusof APPswe/PS1dE9/LRP1lox/loxmicethatwerepositive forGFAP-Cre. Collectively,thesedataindicatethatthelevelsofLRP1are inherentlylowinastrocytes,includingactivatedastrocytes, andthatexpressionofCreviatheGFAPpromoterproducesefficientrecombinationoftheLRP1geneinneurons Figure1 ThelevelofLRP1issignificantlylowerinthemiceexpressingGFAP-CrewithLRP1lox/lox .NineAPPswe/PS1dE9GFAP-Cre LRP1lox/loxmicewithdifferentgenotypeswereusedtodeterminethelevelsofLRP1.TheSDS-solublefractionsofwholebrainlysates (homogenizedinPBS)containinganequalamountoftotalprotein(50 g)wasloadedineachlane.Antibody a 377toLRP1(1:1,000,agiftfrom Dr.Herz,UniversityofTexasSouthwesternMedicalCenter)wasusedtodetectLRP1level.The85kDabandistheendoproteolyticallycleaved matureLRP1.MouseSOD1(14kDbands)wasusedasendogenousloadingcontrol.TheratioofbandintensityofLRP1toSOD1wasquantified, standardizedandplottedbelow.Thestandarddeviationsofthemeasurementsareshownbyerrorbars. Xu etal Alzheimer sResearch&Therapy 2012, 4 :12 http://alzres.com/content/4/2/12 Page5of14

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ofthehippocampus.Wepresumethatanyexpressionof LRP1inastrocyteswasalsoreducedinLRP1lox/loxmice thatweretransgenicforGFAP-Crebuttheabsenceof detectablestainingintheabsenceofGFAP-Cremakesit impossibletoconfirmthisassumption.HippocampaldepositionofA b isnotalteredbyreduction inLRP1levelsInthehippocampusofnine-month-oldAPPswe/ PS1dE9/LRP1lox/loxmice,weobservednoobviousdifferenceinthenumberofAb immunoreactivedeposits Figure2 LRP1levelsarereducedinhippocampalneuronsofLR P1lox/loxmicethataretransgenicforGFAP-Cre .Eight-month-old APPswe/PS1dE9GFAP-CreLRP1lox/loxlittermateswereusedforimmunostaining.AllmicewerehomozygousforLRP1loxpallelesand positiveforAPPswe/PS1dE9transgenes.ThepresenceorabsenceofGFAP-Creisnotedabovetheimages.LRP1 a 377antibody(1:1,000,red)was usedtostainLRP1;NeuN(1:1,000,green)wasusedtostainneurons;Dapiinthemountingreagentwasusedtodisplaynuclei.Confocal microscopeimagesshowedmostoftheneuronsinCAfields (A) andthedentategyrus (C and E) ofAPPswe/PS1dE9LRP1lox/loxGFAP-Cre micedonotexhibitLRP1immunoreactivity.ControlmicelackingtheGFAP-CretransgeneshowedintensestainingofLRP1inhippocampal neurons (B,D ,and F) .Threetofourpairsofmicepergenotypewereusedforimmunostainingandfourtofivesectionsperanimalwere analyzed. Xu etal Alzheimer sResearch&Therapy 2012, 4 :12 http://alzres.com/content/4/2/12 Page6of14

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inmicethatwerepositiveforGFAP-Creascomparedto micelackingGFAP-Cre(Figure5).Withthelackofan obviousdifferenceinamyloidplaqueburdenin APPswe/PS1dE9micelackingLRP1expressioninthe hippocampus,wesoughtto carefullyquantifyplaque numbersinthehippocampusofmultiplesectionsat multiplelevels.TissuesectionswerestainedwithThioflavinSasameansofdetectingamyloiddepositsand thenthehippocampuswasoutlinedandthenumberof depositscounted(seeAdditionalfile5FigureS4).The numberofdepositsinthehippocampusofAPPswe/ PS1dE9/LRP1loxp/loxpmice(n=13)weresimilarto APPswe/PS1dE9/LRP1loxp/loxp/GFAP-Cremice(n= 12),meaningthatsignificantlyreducingLRP1expression inthehippocampushadnoobviouseffectonthenumberordistributionofamyloiddepositsinthisstructure. Femalemiceofallgenotypesshowedslightlyhigher levelsofamyloiddepositsascomparedtomales(Figure 5).WeassessedwhetherlossofLRP1alteredanyofthe morphologicalfeaturesoftheplaquesuchasdensityof neuriticstructures,findingnoobviousdifferencebysilverstaining(Additionalfile6FigureS5).TotalA b levelsdonotchangewhenLRP1levelsare reducedSandwichELISAswereusedtomeasurethetotal amountofA b 40andA b 42inthebrainsofthesemice asanothermeanstoassessamyloidburden.Tocompletelysolubilizedifferentformsofthepeptides,aggregated orotherwise,homogenateswereincubatedforseveral hoursinguanidine-HClbeforebeingdilutedinbuffer forELISA.Consistentwithresultsfromtheamyloidplaquecounting,ELISAmeasurementsconfirmedthatthe levelsofA b 40and42inAPPswe/PS1dE9micewith Figure3 LRP1immunostainingofcorticalneuronsinGFAP-CreLRP1lox/loxmice .Imagesfromthesamesetsofmicedescribedinthe legendtoFigure2werecapturedtoshowcorticallevelsofLRP1.LRP1 a 377antibody(1:1,000,red)wasusedtostainLRP1;NeuN(1:1,000, green)wasusedtostainneurons;Dapiinthemountingreagentwasusedtodisplaynuclei.Therewasnoobviousdifferencebetweenmice transgenicforGFAP-CreandthoselackingGFAP-Cre. Xu etal Alzheimer sResearch&Therapy 2012, 4 :12 http://alzres.com/content/4/2/12 Page7of14

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Figure4 LRP1isbelowthelevelofdetectioninastrocytes .ThesamesetsofmicedescribedinthelegendtoFigure2were immunostained.LRP1 a 377antibody(1:1,000,red)wasusedforLRP1stainingandantibodytoGFAP(1:1,000,green)wasusedtomark astrocytes.ReactiveastrocytescouldbeseenaroundamyloidplaquesinAPPswe/PS1dE9LRP1lox/lox(rightcolumn)butthesecellsdidnot showdetectablelevelsofLRP1.ImmunoreactivitytoLRP1wasevidentwithintheplaqueasneuriticprofiles.LRP1immunostaininginthese neuriticstructureswasdiminishedinmosthippocampalamyloidplaquesofAPPswe/PS1dE9LRP1lox/loxGFAP-Cremice(leftcolumn). Xu etal Alzheimer sResearch&Therapy 2012, 4 :12 http://alzres.com/content/4/2/12 Page8of14

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reducedlevelsofLRP1werenodifferentfromthatof APPswe/PS1dE9micewithnormallevelsofLRP1 (Table1andFigure6).Further,aregressionanalysisto plotA b levelsatdifferentagesshowedthattherateof A b accumulationdidnotchangebythereductionof LRP1levels(Additionalfile7FigureS6).DiscussionInthepresentstudy,wehaveusedgeneticmanipulation toreducetheexpressionlevelsofLRP1inthehippocampusofmicethatco-expressmutantAPPand mutantPS1.Wedemonstratethatloweringthelevelsof LRP1byapproximately50%throughoutthebrainand Figure5 HippocampalamyloidplaquenumbersdonotchangewhenLRP1isreduced. (A and B) Representativeexamplesof immunohistochemicalstainswith6E10antibody(1:1,000,Covance)forAb inAPPswe/PS1dE9/LRP1lox/loxmicethatwereeitherpositiveor negativeforGFAP-Cre. (C) Toquantifyamyloiddeposits,westainedsectionswithThioflavin-S,whichproducesasharperdistinctionbetween tissueandamyloidplaques(seeAdditionalfile5FigureS4foranexample)andcapturedimages.Thehippocampuswasoutlinedandthenthe numberofamyloidplaquesinsidethehippocampuswascountedmanually.AdotplotofamyloidplaquenumbersinmicethatwereCre+is comparedtothenumbersinmicethatwereCre-.Animalsthatwereusedasbreedersarenoted.Nosignificantdifferencesbetweengenotypes eitherCre+(n=12)vs.Cre-(n=13)groups( P =0.75)orbetweenbreeders(n=10)vs.non-breeders(n=15)( P =0.69)wasnoted. Xu etal Alzheimer sResearch&Therapy 2012, 4 :12 http://alzres.com/content/4/2/12 Page9of14

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possiblybyamuchgreaterdegreeinhippocampushad nosignificantimpactontherate,severityorcharacter ofamyloiddeposition.Wefoundnoevidenceofaproportionalchangeinhippo campalamyloidburdenby reducingLRP1expressioninCAandDGneuronswith noobviousmorphologicalchangeintheappearanceof theplaquesthatformed. ToreduceLRP1levelsinthebrainsofAPPswe/ PS1dE9mice,weemployedmicethatexpressCre recombinaseunderthetranscriptionalregulationofthe GFAPpromoterandmiceinwhichtheLRP1allelehas beenmodifiedbyintroductionofloxpsites.The GFAP-Cremicehavepreviouslybeenusedinseveral studiesandtheseinvestigationshavepreviouslydemonstratedthatCreisexpressedearlyinneurogenesissuch thattargetedgenesmayrecombineinbothneuronsand glia[26,27].InourcrossoftheGFAP-Cremicewith LRP1lox/loxmice,weobservedefficienttargetingof LRP1expressioninthehippocampalneurons.However, corticalneuronscontinuedtoshowdemonstrableLRP1 immunoreactivity.Wenote,thatinthehippocampus,a subsetofneuronsinthemolecularlayercontinuedto expressLRP1inGFAP-Cre/loxp/loxpmice;however, thelevelsinCAandDGneuronsweredramatically reduced.ThelevelsofLRP1inastrocytesappearedto beinherentlylowandwecouldnotconfirmwhetheror notexpressionofLRP1wasreducedfurtherinLRP1lox/ loxmiceharboringtheGFAP-Cre. Weobservednoobviousdevelopmentalabnormality orreductionsinthenumbersofCAandDGneuronsin micetransgenicforGFAP-CreandLRP1loxp/loxp,or miceofthisgenotypethatwerealsotransgenicfor APPswe/PS1dE9.However,asnotedinResults,we encounteredsignificantdifficultyinbreedingtheseanimalsandnotedahighdegreeoflethalityearlyinthe process.Forreasonsthatremainunknown,theseissues wereovercomeusingastrategyoffostermotheringwith FVB/NJfemales.TheAPPswe/PS1dE9miceandthe LRP1loxpmicewerebothcongenicintheC57BL/6J backgroundandwebredtheGFAP-CreintoC57BL/6J miceforfourgenerationsbeforebeginningthecrosses toproduceexperimentalanimals.Inourhands,miceof theC57BL/6Jstrainarepoorermothersthancommonly usedhybridstrainsortheinbredFVB/NJstrain.Aswe expected,fostermotheringsolvedissueswehadwith poormotheringbyC57BL/6Jfemales.Unexpectedly,offspringthatwerefoster-motheredbyFVB/NJfemales survivedmuchbetterthanmiceofthesamegenotype motheredbyC57BL/6Jfemales.Wepresentlyhaveno explanationforthisoutcome.WenotethatinapreviousefforttocrosscongenicC57BL/6JAPPswe/ PS1dE9micetocongenicC57BL/6JRAPknockoutmice weobservedahighleveloflethalityinmicethatwere transgenicforAPPswe/PS1dE9andhomozygousknockoutforRAP[22].RAPisadirectERchaperonefor LRP1andlossofRAPlowersthelevelofmatureLRP1 thatreachesthecellsurface[37].Notably,others observednoissuesincrossesofthePDAPPmouse modeltoRAPknockoutmice[23],soweconcluded thatthelethalityweobserve dourearlierworkwasdue tosomeinteractionbetweenmutantPS1andRAP.Our experienceinthematingoftheLRP1loxpmiceto APPswe/PS1dE9miceleadsustobelievethattheremay besomemorecomplexinteractionbetweenPS1dE9and RAP/LRP1thatreducesviability.Thiscurrentstudy definesthesourceofthisdefecttothenervoussystem becauseLRP1isonlyreducedinthebrainbythe expressionofGFAP-Cre.Althoughlikelytobeunrelated toAD,furtherexaminationofthebasisofthislethality mayrevealfunctionsofAPPsweorPS1dE9thatimpact CNSfunction. BecausethereductioninneuronalLRP1wasgreatest inthehippocampus,wefocusedourpathologicanalyses onthehippocampus,findingnosignificantchangein theseverityofamyloiddepositionwhenLRP1levels werereducedintheAPPswe/PS1dE9mice.Thedata showninFigure5Cdocumentthevariabilityinthe numberofamyloiddepositsinhippocampusbetween Table1ThelevelsofAb inthebrainsofmiceofeachgenotypeandageAge(month)CreN A b40(pg/mgbrain) A b42(pg/mgbrain) ratio40/42 AverageSTDEV.P P -valueAverageSTDEV.P P -value 8 -31,2571,0220.402711930.45 4.6 +3481315 142 75 3.4 9 -172,0241,1920.903991920.71 5.1 +122,074746 421116 4.9 15 -44,2359460.651,0151040.74 4.2 +44,582823 972188 4.7 16to18 -44,4451,1350.749711810.79 4.6 +54,8301,777 1,020289 4.7TherewasnodifferenceinthelevelsofA b 40and42inthebrainsofmouseexpressingAPPswe/PS1dE9geneintheLRP1lox/loxhomozygousbackgroundin theabsenceorpresenceoftheGFAP-Cretransgeneinanygivenagegroup.Xu etal Alzheimer sResearch&Therapy 2012, 4 :12 http://alzres.com/content/4/2/12 Page10of14

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micethatarepositiveandnegativeforCrerecombinase. Inbothgroups,thenumberofanimalsthatwerebelow andabovethemean(horizontalline)wassimilar.Notably,ifthenumberofdepositshadbeenreducedbyjust 30%(withthesamelevelofvariance),thenwewould havebeenabletohavereachedstatisticalsignificance( P =0.05)withjust10animalsineachgroup(ourn swere 12vs.13).Basedonquantificationofimmunoblotsfrom Figure6 A b ELISAassayofwholebrainhomogenatesshownodifferencesinA b levelsbygenotype .A b levelsin8-,9-,15-and16to 18-month-oldwithnormalLRP1levels(APPswe/PS1dE9(+)/LRP1lox/lox)werecomparedtothosewithlowerLRP1levels(GFAP-Cre+).Therewas nodifferenceinAb levelsbygenotypebyage(pvaluesrange0.40to0.90basedonStudent s t -test).ELISAdatausedtogeneratethegraphare showninTable1,whichincludesinformationonthenumberofanimalsusedateachage. Xu etal Alzheimer sResearch&Therapy 2012, 4 :12 http://alzres.com/content/4/2/12 Page11of14

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hemi-brains,weconservativelyestimatethattheminimumreductioninLRP1levelswouldbeintherangeof 50%.Bypowercalculation,weshouldhavebeenpoweredtoobservea30%decreaseinamyloidplaquenumbersandthusweconcludethatreducingLRP1levels doesnotproduceaproportionaldecreaseinamyloid plaquenumbers.Consistentwithourfindings,Zerbinatti andcolleaguesreportedthatincreasingthelevelsof LRP1,viaexpressionofarecombinantmini-receptor, didnotproduceaproportionalincreaseinamyloidburden[25].However,thisstudydidnotesmallincreases oflevelsofbothsolubleandinsolubleA b inthecortex withamoreselectiveincreaseinsolubleA b inthehippocampusofoldermicewithhighamyloidburden.Our measuresoftotalAb didnotfindevidencethatreducing LRP1levelsproducedachangeintheaccumulated levelsofA b .Zerbinattiandcolleaguesalsonotedthe appearanceofA b 42dimersafterextractioninbuffers containing0.1Mcarbonate.Wehavenotbeenableto reliablydetectdimericA b inourAPPswe/PS1dE9mice atanyageandthuswecannotcommentastowhether reducingLRP1reducesthelevelsofthesedimers. Interpretingtheoutcomeofthisworkisnotas straightforwardasonewouldlikebecausetheLRP1 levelswerenotreducedtozero.Oneissueisthatwedo notknowhowmuchLRP1mustbeexpressedtosustain amyloidogenesisorwhetherweshouldexpectchanges inamyloidformationinproportiontochangesinLRP1 levels.Additionally,thesourceoftheAb peptidesthat producedepositsmaybeneuronsthatprojectaxons intothehippocampusaswellaslocalneurons.Within thehippocampus,weobservedaneareliminationof LRP1immunoreactivityofneuronsoftheCAfields. However,thehippocampusreceivesaxonalprojections fromtheentorhinalcortexmakingitpossiblethatan activityofLRP1incorticalneuronalprojectionsmasked anyeffectoflocalreductionsinLRP1levelsinhippocampus.Ultimately,themostconservativeinterpretation isthatsignificantsuppressionofLRP1activityinlocal hippocampalneuronsdoesnotproduceaproportional decreaseinamyloidplaquenumbersthisstructure. ApartfromthepotentialeffectsofLRP1reductionson amyloidplaquelevels,wehadanticipatedthatwemight seesomechangeinneuriticpathology.NearlyallamyloidplaquesinAPPswe/PS1dE9micecontainapolipoproteinE(ApoE)[31]andApoEisknowntobea ligandforLRP1thatmodulatesneuriteoutgrowth[38]. Thus,wehadthoughtitpossiblethatreducingLRP1 locallyinthehippocampuscouldhavereducedneuritic pathologyaroundamyloidplaques.Notably,inAPPswe/ PS1dE9/LRP1lox/loxmicethatwerepositiveforGFAPCreweobservedlargeneuriticplaquesinwhichthe neuritesweredepletedofLRP1immunoreactivity.ConclusionsThisstudyusedgenetictoolstoreduceLRP1levelsin thebrainsofmicethatco-expressAPPswe/PS1dE9and developamyloidpathology,producingamodelinwhich LRP1levelsinmosthippocampalneuronswerelowered tobelowthelevelofdetectionbyimmunohistochemistry.InthehippocampusofmicewithreducedLRP1 levels,weobservednoobviouschangeintherateof deposition,severity,orch aracterofamyloiddeposits. Obviously,thisstudydoesnotprovideanyinsightinto whatLRP1expressedinnon-neuralcelltypes,suchas endothelialcells,otherorgansmaydoinregardtomodulatingamyloiddeposition.Itisalsoverypossiblethat homologuesofLRP1mayhavecompensatedforany effectofreducingthelevelsofLRP1.However,atminimum,ourstudyindicatesthatreducingthelevelsof LRP1doesnotproduceproportionalreductionsinamyloidplaquenumbersinthehippocampusofthe APPswe/PS1dE9model.Thus,thisgenetictestdoesnot producesupportiveevidenceforthenotionthatmodulatingLRP1functioninneuronsorglialcouldbebeneficialinmodulatingamyloidosis.Whetherthisoutcomeis uniquetotheAPPswe/PS1dE9modelisofcoursean issueinassessingthepotentialimportanceofthispathwayinhumandisease.AdditionalmaterialAdditionalfile1:Genotypedistributionofthetransgenicmiceused inthisstudy .Intotal,325transgenicmicewithLRP1lox/lox backgroundweregenerated.Themicelistedinthetablearethosethat reachedweaningageandsurvivedtoatleast1.5monthsofage.The actualandexpectedpercentagesofmiceofeachgenotypearelisted. ThepredicatedfrequencyrateisbasedonMendeliandistribution.Asis typicalforAPPswe/PS1dE9micethatarefounddead,therewereno obviousbirthdefectstoaccountfortheearlylethality(TableS1). Additionalfile2:LowpowerimageofLRP1immunostainingin hippocampusofAPPswe/PS1dE9/LRP1lox/loxmicethatare positiveornegativeforGFAP-Cre .Free-floatingfrozensectionswere immunostainedasdescribedinMethodsofthemaintext.Theimages shownarerepresentativeofwhatwasobservedinatleastthreeanimals ofeachgenotype(FigureS1). Additionalfile3:LowpowerimagecapturedfromtheAllenBrain Atlas .Theimageshownherewascapturedfromthewebsiteforthe AllenBrainAtlasfortheadultmousebrain.Thegenenamesearchedfor wasLRP1.Theviewshowndisplaysfalsecolorforexpressionlevelsofa sagittalview(FigureS2). Additionalfile4:Restingastrocytesarenotimmunoreactiveto LRP1antibodies .Theimagesshownarehighpowerimagesof hippocampusfromAPPswe/PS1dE9/LRP1lox/loxmicethatwereeither positiveornegativeforGFAP-Cre.Weobservednoobviousreactivityin cellsshowingamorphologyidentifiedbyGFAPstainingandtherewas noindicationofsignificantco-localizationofGFAPandLRP1 immunoreactivity.Theimagesshownarerepresentativeofwhatwas observedinatleastthreeanimalsofeachgenotype(FigureS3). Additionalfile5:Thioflavin-Sstainingofhippocampalsections .The numbersofamyloidplaquesinthehippocampusasidentifiedby Thioflavin-Sstainingsectionweremanuallybyoutliningtheborderof thehippocampusontheimageandthencountingthenumberofXu etal Alzheimer sResearch&Therapy 2012, 4 :12 http://alzres.com/content/4/2/12 Page12of14

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plaquesinhippocampus.Theseimagesshowrepresentativeexamplesof animalsofbothgenotypes(FigureS4). Additionalfile6:Themorphologyofhippocampalamyloiddeposits isunchangedbythelackofLRP1 .Silverstainsof30 msectionsfrom APPswe/PS1dE9LRP1lox/loxmicethatwereeitherpositiveor negativeforGFAP-Cre.Whitearrowsmarkneuritesandthewhitearrow headsmarktothecoreoftheplaques(FigureS5). Additionalfile7:RegressionanalysisofA b levelsinmiceofeach genotypebyage .NodifferenceintherateofA b accumulationwas evident.Theregressioncurveoftheslopesbetweentwogroupsof animalsisnotsignificantforA b 40( P =0.409)orA b 42( P =0.864)(Figure S6). Abbreviations ApoE:apolipoprotienE;APP:amyloidprecursorprotein;APPswe/PS1dE9:a tandemintegrationoftransgenesencodingamyloidprecursorproteinwith theSwedishmutationandpresenilin1genewithexon9deletion;BCA: bicinchoninicacidassay:abiochemicalassayforproteinconcentration;CA: cornuammonisinhippocampus;Cre-lox:Crerecombinase-LoxPsitesystem, agenetictooltocontrolsitespecificrecombinationeventsingenomic DNA;DG:dentategyrusinhippocampus;ELISA:enzyme-linked immunosorbentassay;GFAP-Cre:transgenicmicewithCrerecombinase drivenbyglialfibrillaryacidicprotein(GFAP)promoter;LRP1:low-density lipoproteinreceptor-relatedprotein. Acknowledgements WegratefullythankJoachimHerz(UniversityofTexasSouthwesternMedical Center,Dallas,Texas,USA)forprovidingLRP1antibodyandmicewith targetedLoxPsites(LRP1lox/loxmice),andWeiHou(Departmentof Biostatistics,UniversityofFlorida)forhelpwithstatistics.Thisstudywas supportedbyAmericanHealthAssistanceFoundation(AHAF)[PilotAwards (GX)]andSantaFeHealthCareAlzheimer sDiseaseResearchCenter. Authordetails1DepartmentofNeuroscience,SantaFeHealthCareAlzheimer sDisease Center,CenterforTranslationalResearchinNeurodegenerativeDisease, McKnightBrainInstitute,UniversityofFlorida,Gainesville,FL32610,USA.2DepartmentofEducationalPsychologyandLearningSystems,Collegeof Education,FloridaStateUniversity,Tallahassee,FL32306,USA. Authors contributions Allauthorscontributedtothismanuscript.GXdesignedtheexperiments, participatedtogeneratethecohortsofmice,carriedoutmostofthe experiments,performedthestatisticalanalysis,anddraftedthemanuscript. CGcontributedtothehistology,immunostainingworkandcollecteddataof amyloidplaques.SFgenotypedthemiceandrecordedthebreedingdata. DRBconceivedofthewholestudy,participatedindetailedexperimental designandpreparedthemanuscript.Allauthorsreadandapprovedthe finalmanuscript. Competinginterests Theauthorsdeclarethattheyhavenocompetinginterests. Received:25February2012Revised:19April2012 Accepted:26April2012Published:26April2012 References1.AndersenOM,WillnowTE: LipoproteinreceptorsinAlzheimer sdisease. TrendsNeurosci 2006, 29 :687-694. 2.RebeckGW,ReiterJS,StricklandDK,HymanBT: ApolipoproteinEin sporadicAlzheimer sdisease:allelicvariationandreceptorinteractions. Neuron 1993, 11 :575-580. 3.BuG,MaksymovitchEA,NerbonneJM,SchwartzAL: Expressionand functionofthelowdensitylipoproteinreceptor-relatedprotein(LRP)in mammaliancentralneurons. 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JClinInvest 2001, 108 :779-784. 20.HoltzmanDM,FaganAM,MackeyB,TenkovaT,SartoriusL,PaulSM, BalesK,AsheKH,IrizarryMC,HymanBT: ApolipoproteinEfacilitates neuriticandcerebrovascularplaqueformationinanAlzheimer sdisease model. AnnNeurol 2000, 47 :739-747. 21.HerzJ,ClouthierDE,HammerRE: LDLreceptor-relatedproteininternalizes anddegradesuPA-PAI-1complexesandisessentialforembryo implantation. Cell 1992, 71 :411-421. 22.XuG,KarchC,LiN,LinN,FromholtD,GonzalesV,BorcheltDR: Receptorassociatedprotein(RAP)playsacentralroleinmodulatingAbeta depositioninAPP/PS1transgenicmice. PLoSOne 2008, 3 :e3159. 23.VanUdenE,MalloryM,VeinbergsI,AlfordM,RockensteinE,MasliahE: Increasedextracellularamyloiddepositionandneurodegenerationin humanamyloidprecursorproteintransgenicmicedeficientinreceptorassociatedprotein. JNeurosci 2002, 22 :9298-9304.Xu etal Alzheimer sResearch&Therapy 2012, 4 :12 http://alzres.com/content/4/2/12 Page13of14

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24.WillnowTE,ArmstrongSA,HammerRE,HerzJ: Functionalexpressionof lowdensitylipoproteinreceptor-relatedproteiniscontrolledby receptor-associatedprotein invivo ProcNatlAcadSciUSA 1995, 92 :4537-4541. 25.ZerbinattiCV,WozniakDF,CirritoJ,CamJA,OsakaH,BalesKR,ZhuoM, PaulSM,HoltzmanDM,BuG: Increasedsolubleamyloid-betapeptide andmemorydeficitsinamyloidmodelmiceoverexpressingthelowdensitylipoproteinreceptor-relatedprotein. ProcNatlAcadSciUSA 2004, 101 :1075-1080. 26.ZhuoL,TheisM,Alvarez-MayaI,BrennerM,WilleckeK,MessingA: hGFAPcretransgenicmiceformanipulationofglialandneuronalfunction in vivo Genesis 2001, 31 :85-94. 27.MooreSA,SaitoF,ChenJ,MicheleDE,HenryMD,MessingA,CohnRD, Ross-BartaSE,WestraS,WilliamsonRA,HoshiT,CampbellKP: Deletionof braindystroglycanrecapitulatesaspectsofcongenitalmuscular dystrophy. Nature 2002, 418 :422-425. 28.RohlmannA,GotthardtM,WillnowTE,HammerRE,HerzJ: Sustained somaticgeneinactivationbyviraltransferofCrerecombinase. Nat Biotechnol 1996, 14 :1562-1565. 29.RohlmannA,GotthardtM,HammerRE,HerzJ: Inducibleinactivationof hepaticLRPgenebycre-mediatedrecombinationconfirmsroleofLRP inclearanceofchylomicronremnants. JClinInvest 1998, 101 :689-695. 30.JankowskyJL,SluntHH,RatovitskiT,JenkinsNA,CopelandNG,BorcheltDR: Co-expressionofmultipletransgenesinmouseCNS:acomparisonof strategies. BiomolEng 2001, 17 :157-165. 31.LesuisseC,XuG,AndersonJ,WongM,JankowskyJ,HoltzG,GonzalezV, WongPC,PriceDL,TangF,WagnerS,BorcheltDR: Hyper-expressionof humanapolipoproteinE4inastrogliaandneuronsdoesnotenhance amyloiddepositionintransgenicmice. HumMolGenet 2001, 10 :2525-2537. 32.BorcheltDR,LeeMK,SluntHS,GuarnieriM,XuZS,WongPC,BrownRH, PriceDL,SisodiaSS,ClevelandDW: Superoxidedismutase1with mutationslinkedtofamilialamyotrophiclateralsclerosispossesses significantactivity. ProcNatlAcadSciUSA 1994, 91 :8292-8296. 33.GunternR,BourasC,HofPR,ValletPG: AnimprovedthioflavineSmethod forstainingneurofibrillarytanglesandsenileplaquesinAlzheimer s disease. Experientia 1992, 48 :8-10. 34.TebbenkampAT,GreenC,XuG,Denovan-WrightEM,RisingAC, FromholtSE,BrownHH,SwingD,MandelRJ,TessarolloL,BorcheltDR: Transgenicmiceexpressingcaspase-6-derivedN-terminalfragmentsof mutanthuntingtindevelopneurologicabnormalitieswithpredominant cytoplasmicinclusionpathologycomposedlargelyofasmaller proteolyticderivative. HumMolGenet 2011, 20 :2770-2782. 35.HiranoA,ZimmermanHM: Silverimpregnationofnervecellsandfibers incelloidinsections.Asimpleimpregnationtechnique. ArchNeurol 1962, 6 :114-122. 36.YamamotoT,HiranoA: AcomparativestudyofmodifiedBielschowsky, BodianandthioflavinSstainsonAlzheimer sneurofibrillarytangles. NeuropatholApplNeurobiol 1986, 12 :3-9. 37.BuG,GeuzeHJ,StrousGJ,SchwartzAL: 39kDareceptor-associated proteinisanERresidentproteinandmolecularchaperoneforLDL receptor-relatedprotein. EmboJ 1995, 14 :2269-80. 38.HoltzmanDM,PitasRE,KilbridgeJ,NathanB,MahleyRW,BuG, SchwartzAL: Lowdensitylipoproteinreceptor-relatedproteinmediates apolipoproteinE-dependentneuriteoutgrowthinacentralnervous system-derivedneuronalcellline. ProcNatlAcadSciUSA 1995, 92 :9480-9484.doi:10.1186/alzrt110 Citethisarticleas: Xu etal .: Reductionoflow-densitylipoprotein receptor-relatedprotein(LRP1)inhippocampalneuronsdoesnot proportionatelyreduce,orotherwisealter,amyloiddepositionin APPswe/PS1dE9transgenicmice. Alzheimer sResearch&Therapy 2012 4 :12. 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 Xu etal Alzheimer sResearch&Therapy 2012, 4 :12 http://alzres.com/content/4/2/12 Page14of14

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Genotypes APP/PS1 + + Total GFAP Cre + + LRP Lox/lox Lox/lox Lox/lox Lox/lox Expected frequency of offspring 25% 25% 25% 25% 100% Non fostered Number of mice identified by genotype 56 31 21 50 158 Observed frequency 35% 20% 13% 32% 100% Estimated number of mice that died before 1.5 months of age (age of genotyping ) ~20 ~30 ~50 Number of mice found dead (< 9mos old) 2 3 9 1 15 (Premature death rate in group after weaning) (4%) (10%) (43%) (2%) (9%) Age range of mice found dead (months) (2.3 6.3) (1.9 8.8) (2.4 4.6) 3.3 Fostered Number of Mice 45 41 32 49 167 Observed frequency 27% 25% 19% 29% 100% Estimated number of mice that died before 1.5 months of age (age of genotyp ing ) ~5 ~15 ~20 Number of mice found dead (< 9mos old) 4 2 1 0 7 (Premature death rate in group after weaned) (9%) (5%) (3%) (0%) (4%) Age range of mice found dead (months) (2.0 9.0) (2.8 7.7) 3.7