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RESEARCHOpenAccessEvaluatingtherelationshipbetweenamyloid-βandα-synucleinphosphorylatedatSer129indementiawithLewybodiesandParkinson'sdiseaseMartaSwirski1,JScottMiners1,RohandeSilva2,TammarynLashley2,HelenLing2,JaniceHolton2,TamasRevesz2andSethLove1*AbstractIntroduction:LewybodyandAlzheimer-typepathologiesoftenco-exist.
Severalstudiessuggestasynergisticrelationshipbetweenamyloid-β(Aβ)andα-synuclein(α-syn)accumulation.
WehaveexploredtherelationshipbetweenAβaccumulationandthephosphorylationofα-synatserine-129(pSer129α-syn),inpost-mortemhumanbraintissueandinSH-SY5Yneuroblastomacellstransfectedtooverexpresshumanα-syn.
Methods:WemeasuredlevelsofAβ40,Aβ42,α-synandpSer129α-synbysandwichenzyme-linkedimmunosorbentassay,insolubleandinsolublefractionsofmidfrontal,cingulateandparahippocampalcortexandthalamus,fromcasesofParkinson'sdisease(PD)with(PDD;n=12)andwithoutdementia(PDND;n=23),dementiawithLewybodies(DLB;n=10)andage-matchedcontrols(n=17).
Wealsoexaminedtherelationshipofthesemeasurementstocognitivedecline,asmeasuredbytime-to-dementiaandthemini-mentalstateexamination(MMSE)scoreinthePDpatients,andtoBraaktanglestage.
Results:Inmostbrainregions,theconcentrationofinsolublepSer129α-syncorrelatedpositively,andsolublepSer129α-synnegatively,withthelevelsofsolubleandinsolubleAβ.
InsolublepSer129α-synalsocorrelatedpositivelywithBraakstage.
Inmostregions,thelevelsofinsolubleandsolubleAβandtheproportionofinsolubleα-synthatwasphosphorylatedatSer129weresignificantlyhigherinthePDandDLBgroupsthanthecontrols,andhigherinthePDDandDLBgroupsthanthePDNDbrains.
InPD,theMMSEscorecorrelatednegativelywiththelevelofinsolublepSer129α-syn.
ExposureofSH-SY5YcellstoaggregatedAβ42significantlyincreasedtheproportionofα-synthatwasphosphorylatedatSer129(aggregatedAβ40exposurehadasmaller,non-significanteffect).
Conclusions:Together,thesedatashowthattheconcentrationofpSer129α-syninbraintissuehomogenatesisdirectlyrelatedtothelevelofAβandBraaktanglestage,andpredictscognitivestatusinLewybodydiseases.
IntroductionAlzheimer'sdisease(AD),Parkinson'sdisease(PD)anddementiawithLewybodies(DLB)arethemostcommonage-relatedneurodegenerativediseasesandtogetherac-countfor80%to90%ofpatientswithdementia[1,2].
ThepathologicalhallmarksofADareextracellularaccu-mulationsofamyloid-β(Aβ)asplaquesandintracellularaggregatesofhyperphosphorylatedtauthatformneuro-fibrillarytanglesandneuropilthreads.
ThepathologicalhallmarksofPDandDLBareLewybodiesandLewyneurites,composedofα-synuclein(α-syn)[3-5].
Althoughthesedefiningabnormalitiesarecharacteristicanddis-tinct,manydementiacaseshavemixedpathology:alargeproportionofADpatients(>50%)hasadditionalLewybodypathologyinadditiontoplaquesandtangles[6-16].
InParkinson'sdiseasewithdementia(PDD)andDLBap-proximately40%ofcaseshavesignificantnumbersofAβplaquesandneurofibrillarytangles[17].
Patientswithmixedpathologytendtopursueamoreaggressivedis-easecourse[18],withmorepronouncedcognitivedys-functionthaninpatientswithpureAD[19-24].
InPDandDLB,thenumberofcorticalα-synaggregatesissignificantlyhigherinpatientswhohaveAβplaquesinthecortex[25,26]andα-synaccumulateswithinsome*Correspondence:seth.
love@bris.
ac.
uk1DementiaResearchGroup,InstituteofClinicalNeurosciences,SchoolofClinicalSciences,UniversityofBristol,Bristol,UKFulllistofauthorinformationisavailableattheendofthearticle2014Swirskietal.
;licenseeBioMedCentralLtd.
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org/licenses/by/4.
0),whichpermitsunrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycredited.
TheCreativeCommonsPublicDomainDedicationwaiver(http://creativecommons.
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0/)appliestothedatamadeavailableinthisarticle,unlessotherwisestated.
Swirskietal.
Alzheimer'sResearch&Therapy2014,6:77http://alzres.
com/content/6/9/77plaque-associateddystrophicneurites[27].
TransgenicmiceexpressingbothAβandα-synhadmoreLewybodypathologyandmoreseveredeficitsinlearningandmemorythandidmiceexpressingα-synalone[28].
ThesestudiessuggestasynergisticrelationshipbetweenAβandα-syn.
However,thereasonsforthefrequentpathologicaloverlapbetweenADandLewybodydiseasesarepoorlyunderstood.
Arecentmeta-analysisofgenome-wideasso-ciationstudiesofADandPDdidnotdetectanygenelocithatincreasedtheriskofbothdiseasesandconcludedthatthepathologicaloverlapislikelytoresultfromprocessesdownstreamofthesusceptibilitygenesfortheindividualdiseases[29].
α-syncaninducethehyperphosphorylationoftauthroughtheactivationofproteinkinaseA[30]andglycogensynthasekinase3β[31,32]and,thereby,promotetheformationofneurofibrillarytangles.
However,itisnoteworthythatthemostfrequentformofpathologicaloverlapbetweenLewybodydiseasesandADisthepres-enceofincreasednumbersofAβplaquesinPDDandDLB[25,33],withlimitedformationoftanglesandthein-teractionsbetweenα-synandAβwere,therefore,thepri-maryfocusofthisstudy.
Thepredominantmodificationofα-syninLewybodydis-easesisphosphorylationatSer129[34,35].
Approximately90%ofα-synwithinLewybodiesandneuritesisphosphory-latedatSer129,comparedto4%inthenormalbrain[35].
Thepreciseroleofα-synphosphorylationatSer129remainsunclear:most[36-39],butnotall,studies[40-42]suggestthatphosphorylationmediatestheaggregationandneuro-toxicityofα-syn.
IrrespectiveofwhetherthesechangesprecedethedevelopmentofLewybodiesoroccuratalaterstage,itiswellestablishedthatpSer129α-synlevelscorrelatewithdiseaseseverity[43-45].
Obietal.
[44]found,thatinDLBcaseswithADpathology,pSer129α-synlevelscorrelatedstronglywithparenchymalAβload(asassessedbyimmunohistochemistry).
Theaimofourstudywastoexplorethisrelationshipfurther,inmultipleregionsofbrainfromParkinson'sdiseasewithoutdementia(PDND),PDDandDLBpatientsandage-matchedcontrols,bymeasuringtheconcentrationsofthetwomajorformsofsolubleandinsolubleAβ(Aβ40,Aβ42)bysandwichELISA,aspreviously[46-49],andofsolubleandinsolubleα-syn(bothtotalandpSer129α-syn)alsobyELISA.
InPDpatientswealsoanalyzedtherelationshipbetweenAβ,totalα-syn,pSer129α-synandante-mortemcognitivefunction,asindicatedbymini-mentalstateexamination(MMSE)scores.
Lastly,inSH-SY5Ycellsthatstablyexpressedhighlevelsofendogenousα-syn,weassessedthedirectinfluenceofdifferentformsofAβonthephosphorylationofα-synatSer129invitro.
MethodsCaseselectionWestudied35casesofPD(23PDNDand12PDD)fromtheQueenSquareBrainBank(QSBB)forNeuro-logicalDisorders,UCLInstituteofNeurology,London,and10casesofDLBand17age-matchedcontrolsfromtheSouthWestDementiaBrainBank(SWDBB),Univer-sityofBristol(Table1).
ProtocolsforbrainbankingattheQSBBwereapprovedbytheLondonMulti-CentreResearchEthicsCommittee(RECreference08/H0718/54+5)andwrittenconsentfortheuseofbraintissueandforaccesstothemedicalrecordforresearchwasobtainedfromallcases.
TheSouthWestDementiaBrainBankhadethicalapprovalfromtheNorthSomersetandSouthBristolResearchEthicsCommittee(RECreference08/H0106/28).
Alldiseasecaseswerediagnosedusingwidelyacceptedneuropathologicalcriteria[50,51].
CaseswereexcludedfromthestudyiftheyhadaneuropathologicaldiagnosisofAD(thatis,ifhistologyshowedADneuropathologicalchangethatwasconsideredasufficientexplanationfordementiaaccordingtotheNationalInstituteonAging-Alzheimer'sAssociationguidelinesfortheneuropathologicalassess-mentofAD[51]oranyotherneurodegenerativediseaseapartfromPDorDLB.
Theywerealsoexcludedifneuro-histologyrevealedseverecerebralamyloidangiopathyorothersignificantcerebrovasculardisease.
ToassessthepossibleinfluenceofAβ-inducedphosphor-ylationofα-synoncognitivedeclineinPDpatients,ourTable1Control,Parkinson'sdiseasenon-dementia(PDND),Parkinson'sdiseasedementia(PDD)anddementiawithLewybodies(DLB)cases:demographicandclinicaldataControl(n=17)PDND(n=23)PDD(n=12)DLB(n=10)Meanageatonset(years)±SDN/A61.
1±9.
158.
2±7.
769.
7±7.
3Meanageatdeath(years)±SD79.
2±8.
777.
7±6.
277.
85±6.
177.
0±9.
0Meandiseaseduration(years)±SDN/A16.
6±6.
719.
7±6.
57.
3±2.
0Gender(%)3(18)female13(57)female6(50)female4(40)femaleMeanpost-mortemdelay(hours)±SD37.
0±16.
663.
6±27.
038.
2±21.
728.
0±10.
9Meantimetodementia(years)±SDN/AN/A14.
6±6.
9N/AMedianBraaktanglestage(range)II(0toIII)II(ItoIV)II(ItoIV)II(0toIII)N/A,notavailable;n,number;SD,standarddeviation.
Swirskietal.
Alzheimer'sResearch&Therapy2014,6:77Page2of17http://alzres.
com/content/6/9/77analysesincludedthetimetodementiainpatientswithPDD,andthescoreontheMMSEwithinthelastyearoflife,whereavailable.
Inallcasesconsenthadbeengivenfortheuseofbraintissueandforaccesstothepatients'clinicalrecordsforresearch.
TissuepreparationBraintissue(200mg)samplesofmidfrontal,cingulateandparahippocampalcortexandthalamusweresequen-tiallyextractedin1%NP-40buffer(140mMNaCl,3mMKCl,25mMTRIS,5mMethylenediaminetetraace-ticacid(EDTA),2mM1,10phenanthroline)aspreviouslydescribedforAβmeasurementsinhumanpost-mortemtissue[46,48,49,52].
ThetissuewashomogenizedinaPre-cellys24homogenizer(StrettonScientific,Derbyshire,UK)with2.
3mmceramicbeads(Biospec,Stratech,Suffolk,UK).
Thehomogenateswerespunat13,000*gfor15minutesat4°Candthesupernatantwasremovedandstoredat80°C.
Insolublematerialwassolubilizedbyvigorousagitationin6MGuHCl,re-homogenizedandleftforfourhoursatroomtemperature(RT)beforestorageat80°C.
Totalα-synsandwichELISATotalα-synlevelwasdeterminedbysandwichELISA.
Mousemonoclonalanti-α-synantibody(0.
5μg/ml;BDBiosciences,Oxford,UK)wascoatedontoaNUNCMaxisorp96-wellplateovernightatRT.
TheplatewaswashedinPBS/0.
01%tween-20andblockedfor1.
5hoursin1%BSA/PBS.
Tissuesamples(insolubleandsolubleex-tractsdiluted1:200inPBS)wereaddedfortwohoursatRTwithconstantshaking.
Theplatewasrinsed,tappeddryandbiotinylatedpolyclonalanti-α-synuclein(1μg/ml;R&DSystems,Oxford,UK)dilutedinPBSwasaddedfortwohoursatRT.
Theplatewasrinsedandtappeddry,streptavidin-horseradishperoxidase(HRP)(1:200,R&DSystems)wasaddedfor20minutesand,afterfurtherwashing,chromogenicsubstrate(TMBS,R&DSystems)wasaddedfor20minutesinthedark.
Thereactionwasstoppedwith2Nsulfuricacidandabsorbanceat450nMreadinaFLUOstarOptimaplatereader(BMGLabtech,Aylesbury,UK).
Totalα-synlevelswereinterpolatedfrommeasurementsmadeonserialdilutionsofrecombinanthumanα-synrangingfrom62.
5to0.
98ng/ml(rPeptide,Stratech,Suffolk,UK).
Measurementsforeachsamplewererepeatedinduplicate.
pSer129α-synsandwichELISAMousemonoclonalanti-α-synantibody(0.
5μg/ml;BDBiosciences)wascoatedontoaNUNCMaxisorp96-wellplateovernightatRT.
TheplatewaswashedinPBS/0.
01%tween-20andblockedfortwohoursin1%BSA/PBS.
Tissuesamples(insolubleextractsdiluted1:99inPBS,solubleextractsdiluted1:3)wereaddedforfivehoursatRTwithconstantshaking.
Theplatewasrinsedandtappeddryandanti-pSer129α-syn(0.
8μg/ml;Abcam,Cambridge,UK)dilutedinPBSwasaddedandlefttoincu-bateat4°Covernight.
Followingwashingoftheplate,biotinylatedhorseanti-rabbitantibody(1.
5μg/ml;Vectorlabs,Peterborough,UK)dilutedinPBSwith0.
01%tween-20wasaddedforonehouratRT.
Theplatewasrinsedandtappeddry,streptavidin-HRPwasaddedforonehourfollowedbychromogenicsubstratefor20minutesinthedark.
Thereactionwasstoppedwith2Nsulfuricacidandabsorbanceat450nMreadinaFLUOstarOptimaplatereader(BMGLabtech).
TheconcentrationofpSer129α-synwasdeterminedasdescribedpreviously[53],byinterpolationfrommeasurementsofserialdilutions(200to3.
125ng/ml)ofrecombinantα-synthathadbeenphos-phorylatedatSer129byincubatingwithcaseinkinaseII(seebelow).
SpecificityofthepSer129α-synantibodyWeconductedapreliminarystudytoconfirmthespeci-ficityofthepSer129α-synantibody.
Full-lengthrecom-binanthumanα-syn(1mg/ml;rPeptide,Statech)wasincubatedwithcaseinkinaseI(CKI)(1,000units,NewEnglandBiolabs,Hitchin,UK)orcaseinkinaseII(CKII)(500units,NewEnglandBiolabs,oneunitbeingdefinedastheamountofCKIIrequiredtocatalyzethetransferof1ρmolofphosphateto100μMCKIIpeptidese-quenceRRRADSDDDDDinoneminuteat30°C)foronehourat30°Cinthepresenceof200μMATP(NewEnglandBiolabs)(protocoladaptedfromLeeetal.
[54];Walkeretal.
[45]).
Asacontrol,anothersamplewastreatedinthesamemannerintheabsenceofeitherCKIorCKII.
Samplesweredilutedin1%Tris-bufferedsaline(TBS)(1:400)andappliedtoapre-wetted(in1%TBS)nitro-cellulosemembraneandincubatedatroomtemperatureforonehour.
Themembranewaswashedin0.
3%Tris-bufferedsalinewithTween20(TBST)thenincubatedwith10%non-fatmilkin0.
3%TBSTforonehouratRTwithagitationtopreventnon-specificbinding.
AfterwashingthemembraneinTBST,primaryantibodies(totalα-syn,0.
5μg/ml,BDBiosciences;pSer129α-syn,0.
8μg/ml,Abcam;pSer87α-syn,200μg/ml,SantaCruz,Dallas,TX,USA)dilutedin5%non-fatmilkinTBSTwereappliedovernight.
Thefol-lowingdaythemembranewasagainwashedinTBSTandincubatedwithperoxidase-conjugatedsecondaryantibodydilutedin5%non-fatmilkinTBSTforonehouratRTwithagitation.
ThemembranewaswashedandthendevelopedonphotographicfilmusingImmobi-lonchemiluminescencereagents(Millipore,Danvers,MA,USA)accordingtothemanufacturer'sguidelines.
ThepSer129α-synantibodylabelledα-synfollowingin-cubationwithCKII,andtoalesserextentCKI,butdidnotlabelrecombinantα-synthathadnotbeenphosphorylatedSwirskietal.
Alzheimer'sResearch&Therapy2014,6:77Page3of17http://alzres.
com/content/6/9/77withCKIorCKII.
Incontrast,anon-phosphorylation-specificα-synantibody(BDBiosciences)detectedallformsofα-syn,andapSer87-specificα-synantibodydetectedasignalonlyafterincubationofα-synwithCKI(asexpectedfrompreviousstudiesbyOkochietal.
[55]andPaleologouetal.
[40]).
ThesefindingsconfirmedthespecificityofthepSer129α-synantibody(seeAdditionalfile1:FigureS1).
Aβ40sandwichELISAThelevelofAβ40wasmeasuredinpost-mortembraintissuesamplesbysandwichELISAasdescribed[49,56].
High-bindingCostar96-wellplates(R&DSystems)werecoatedwithanti-humanAβ(2μg/ml;clone6E10,raisedagainstaminoacids4–7,Covance,Maidenhead,UK)di-lutedinPBSandincubatedovernightatRT.
AfterfivewasheswithPBScontaining0.
05%tween-20,theplateswereblockedwith300μLprotein-freePBSblockingbuffer(ThermoFisherScientific,Loughborough,UK)fortwohoursatRT.
Afterafurtherfivewashes,brainhomogenatesamples(insolubleextractsdiluted1:49,solubleextractsdiluted1:3)andserialdilutionsofrecombinanthumanAβ1-40(SigmaAldrich,Dorset,UK)inPBScontaining1%1,10phenanthroline(SigmaAldrich)(topreventdegradationofAβ[57])wereincubatedfortwohoursatRTwithrocking.
Afterafurtherwashstep,theplateswereincubatedwithanti-humanAβ1-40(1μg/ml;Covance)fortwohoursatRT.
Theantibodywaspre-paredusingtheLightning-Linkbiotinylationkit(InnovaBiosciences,Cambridge,UK)accordingtothemanu-facturer'sguidelines.
Afterfurtherwashes,theplatewasrinsedandtappeddry,streptavidin-HRPaddedfor20minutes,andchromogenicsubstratefor20minutesinthedark.
Thereactionwasstoppedwith2Nsulfuricacidandabsorbanceat450nMreadinaFLUOstarOptimaplatereader(BMGLabtech).
TheAβ1–40levelinthebraintissuesampleswasinterpolatedfromastandardcurvegeneratedbyserialdilutionofrecombinanthumanAβ1–40(SigmaAldrich)intherange16,000to1.
024nM.
Eachsamplewasassayedinduplicate.
Aβ42sandwichELISAThelevelofAβ42wasmeasuredinpost-mortembraintissuesamplesbysandwichELISAasoutlinedabovewithafewmodifications.
Anti-humanAβ1-42(0.
5μg/ml;12F4,Covance)wasusedasthecaptureantibody.
Tissuesamples(insolubleextractsdiluted1:9,solubleextractsdiluted1:3)wereincubatedatRTforfourhours.
Biotinyl-atedanti-humanAβ(0.
1μg/ml;ThermoFisherScientific)dilutedinPBSwasusedfordetectionandincubatedover-nightat4°C.
Followingwashing,rinsinganddrying,streptavidin-HRPwasaddedtotheplateforonehourandchromogenicsubstratefor20minutesinthedark.
Aβ1-42concentrationinbraintissuewasinterpolatedfromastandardcurvegeneratedbyserialdilution(16,000to1.
024nM)ofrecombinanthumanAβ1–42(SigmaAldrich).
Eachsamplewasassayedinduplicate.
TheAβ1-42ELISAdidnotdetectAβ1-40,andtheAβ1-40ELISAdidnotdetectAβ1-42.
SandwichELISAvalidationIntra-assayandinter-assaycoefficientsofvariationwerecalculatedfortheELISAsaswellasspikeandrecoverytests(seeAdditionalfile2:TableS3),inwhichserialdi-lutionsofAβ40,Aβ42,α-synorpSer129α-synwereaddedtobrainhomogenatesratherthanassaydiluent.
Therecovered:addedratioforeachaddedprotein(aratiosometimestermedtheresponserate)andthecorrelationbetweenthecalculatedconcentrationandmeasuredcon-centrationofaddedproteinwereassessedintheinsolubleandsolublefractionsofthehomogenates.
Therecovered:addedratiosofsolubleandinsolublepSer129α-syn(theformerwellbelow1,thelatterwellabove1)suggeststhatonadditiontobrainhomogenates,whichalreadycon-tainedrelativelyhighbaselineamountsofα-synaswellassomepSer129α-syn,mostoftheaddedsolublepSer129α-synrapidlyaggregatedandenteredtheinsolublefrac-tioninthehomogenate.
Datafromthetotalα-synandAβ42assaysindicatedagoodrecoveryrate,withrecoveryofAβ40being50%.
Inalloftheassaystherewasaverycloselinearcorrelationbetweentheconcentrationofaddedproteinandtheconcentrationofproteindeterminedbytheassay(asshownbythePearsonrandPvalues),enablingvalidcomparisonstobemadebetweenbrainsandalsobetweencohorts.
Immunohistochemicalassessmentofα-syn,pSer129α-syn,Aβ42andAβ40Formalin-fixedparaffin-embeddedsectionsofmid-frontal,cingulate,parahippocampalcortexandthalamusinallDLBcaseswereimmunolabelledforAβ1-42(0.
5μg/ml;Covance),Aβ1-40(1μg/ml;Covance),pSer129α-syn(0.
8μg/ml;Abcam)andα-syn(80mg/l;VectorLabs,Peterborough,UK)byuseofastandardstreptavidin-biotin-HRPimmunohistochemistryprotocol[58].
Theextentofimmunolabellingofeachantigenwasmea-suredbyfieldfractionanalysiswiththehelpofImageProPlussoftware(MediaCybernetics,Marlow,UK)drivingaLeicaDMmicroscopewithamotorizedstage.
Thesoftwaremadeanunbiasedselectionoftwelve*20-objectivefieldsandthepercentageareaimmunopo-sitivefortherelevantantigenwasdeterminedforeachsection,asoutlinedpreviously[59,60].
CellcultureSH-SY5YneuroblastomacellsweretransfectedwithapCDNA3.
1vector(LifeTechnologies,UK)containingwild-typehumanSNCAcDNAunderthecontrolofacytomegalovirus(CMV)promoter.
TransfectionwascarriedSwirskietal.
Alzheimer'sResearch&Therapy2014,6:77Page4of17http://alzres.
com/content/6/9/77outwithTransFast(Promega,Southampton,UK),followedbyselectionofclones(andtheirsubsequentmaintenance)inculturemediumcontaining0.
3mg/mlG418(Geneticin,LifeTechnologies,Paisley,UK).
TheculturemediumforSH-SY5Ycells,eitheruntransfectedorstablyexpressinghu-manwild-typeα-syn,consistedof42%vol/volHam'sF12nutrientmixture(F12)(Sigma)and42%vol/volEagle'sminimumessentialmedium(Sigma),supplementedwith15%vol/volfetalcalfserum(Sigma),2mML-glutamine(Sigma),1%vol/volnon-essentialaminoacidssolution(Sigma),20units/mLpenicillin,20mg/mLstreptomycin(Sigma)and250ng/mLamphotericinB(LifeTechnologies)at37°Cin5%CO2(21%O2).
AdditionofAβtocellculturesBeforetreatmentwithAβ,theculturemediumwasre-placedwithserum-freemedium(nofetalbovineserumandnoG418)for24hous.
Aβsolutionswerealsoprepared24hoursinadvance.
Stocksolutionsof1mMAβ42andAβ40(CambridgeBiosciences,Cambridge,UK)in35%acetonitrileweredilutedinserum-freemediumat1μMand10μM.
TheAβwaseitherleftovernighttoaggregateat26°Cfor24hours(aspreviouslydescribed[61])orim-mediatelyplacedovernightina80°Cfreezer.
Aβ(eitheraggregatedorfresh)wasaddedtoflasksthefollowingday(10μMacetonitrilewasaddedtocontrolflasks)andincu-batedfor24hours.
PreparationofcelllysatesforsandwichELISACellswereincubatedwithDulbecco'sPBSwithoutcal-ciumchlorideandmagnesiumchloride(Sigma-Aldrich)at37°Cforfiveminutesandthenremovedfromtheflask,transferredintoaFalcontube,andspunforthreeminutesat13,000rpm.
ThecellswerewashedinPBSandlysedin100μlnon-denaturingproprietarycelllysisbuffer(Sigma-Aldrich,Dorset,UK)accordingtotheFigure1CorrelationbetweenAβandinsolublepSer129α-syn.
Eachpointrepresentsaseparatecase.
Thebest-fitlinearregression(solidlines)and95%confidenceintervals(interruptedlines)aresuperimposed.
OnlysignificantP-values(andassociatedcorrelationcoefficients)areshowninthefigure.
SignificantpositivecorrelationsbetweeninsolublepSer129α-synandsolubleAβ42/Aβ40werefoundintheparahippocampal(PH)cortexandthalamus(TH).
SignificantpositivecorrelationswerealsofoundbetweeninsolublepSer129α-synandinsolubleAβ42inthecingulate(CG)andPHcortex.
Inaddition,significantcorrelationswerefoundbetweeninsolublepSer129α-synandinsolubleAβ40inthemidfrontalandCGcortex.
Aβ,amyloid-β;pSer129α-syn,alpha-synucleinphosphorylatedatserine129.
Swirskietal.
Alzheimer'sResearch&Therapy2014,6:77Page5of17http://alzres.
com/content/6/9/77manufacturer'sguidelines,andspunat13,000rpmfor15minutesat4°C.
Cellsupernatants(solublefraction)wereremovedandstoredat80°Cuntilused.
Atotalof6MGuHCl(100μl)wasaddedtotheremainingin-solublepelletandleftatRTfor1.
5hours(insolublefraction)beforethetubewasstoredat80°C.
StatisticalanalysisWheneverpossible,parametricstatisticaltestswereusedforcomparisonsbetweengroups(insomecasesthisrequiredlogarithmictransformationofthedatatoobtainanormaldistribution):analysisofvariance(ANOVA)withDunnett'stestforpairwiseintergroupcomparisons,orrepeatedmea-suresANOVAfortheanalysisofinvitromeasurementsoncellsexposedtodifferentconcentrationsofAβduringthesameexperiment.
Forvariablesthatwerenotnormallydistributedevenaftertransformation,theKruskall-Wallistestwasused,withDunn'stestforpairwiseintergroupcom-parisons.
PearsonorSpearmananalysiswasusedasappro-priatetoassessthecorrelationbetweenpairsofvariables.
StatisticaltestswereperformedusingGraphPadPrismv5.
P-values<0.
05wereconsideredstatisticallysignificant.
ResultspSer129α-syncorrelateswithinsolubleandsolubleAβInmostregionsthelevelofinsolublepSer129α-synin-creasedasthelevelsofAβ40andAβ42increased(Figure1,Additionalfile3:TableS1),withsignificantpositivecorre-lationsbetweeninsolublepSer129α-synandsolubleAβ40andAβ42intheparahippocampalcortex(solubleAβ40:r=0.
376,P=0.
003;solubleAβ42:r=0.
287,P=0.
024)andthalamus(solubleAβ40:r=0.
398,P=0.
002;solubleFigure2CorrelationbetweenAβandsolublepSer129α-syn.
Eachpointrepresentsaseparatecase.
Thebest-fitlinearregression(solidlines)and95%confidenceintervals(interruptedlines)aresuperimposed.
OnlysignificantP-values(andassociatedcorrelationcoefficients)areshowninthefigure.
SignificantnegativecorrelationsbetweensolubleAβ42andsolublepSer129α-synwerefoundinthecingulate(CG)cortexandthalamus(TH).
Incontrast,asignificantpositivecorrelationwasfoundbetweensolubleAβ42andsolublepSer129α-syninthemidfrontal(MF)cortex.
SignificantnegativecorrelationswerefoundbetweensolubleAβ40andsolublepSer129α-synintheCG,parahippocampal(PH)cortexandTH.
SignificantnegativecorrelationswerealsofoundbetweensolublepSer129α-synandinsolubleAβ42intheMFcortexandTH.
AsignificantpositivecorrelationwasobservedbetweensolublepSer129α-synandinsolubleAβ40inthePHcortex.
Aβ,amyloid-β;pSer129α-syn,alpha-synucleinphosphorylatedatserine129.
Swirskietal.
Alzheimer'sResearch&Therapy2014,6:77Page6of17http://alzres.
com/content/6/9/77Aβ42:r=0.
404,P=0.
002).
Significantpositivecorrela-tionswerealsofoundbetweeninsolublepSer129α-synandinsolubleAβ42inthecingulate(r=0.
293,P=0.
022)andparahippocampalcortex(r=0.
314,P=0.
013)aswellasbetweeninsolublepSer129α-synandinsolubleAβ40inthemidfrontal(r=0.
719,P<0.
0001)andcingulatecortex(r=0.
304,P=0.
017).
SignificantnegativecorrelationsbetweensolublepSer129α-synandsolubleAβ42werefoundinthecingulatecortexandthalamus(Figure2;Additionalfile3:TableS1)(cingulatecortex:r=0.
287,P=0.
035;thalamus:r=0.
373,P=0.
0036).
Incontrast,asignificantpositivecorrelationwasobservedbetweensol-ubleAβ42andsolublepSer129α-syninthemidfrontalcortex(r=0.
443,P=0.
0015).
Significantnegativecorrela-tionsbetweensolubleAβ40andsolublepSer129α-synwerefoundinthreeoffourregions(cingulatecortex:r=0.
313,P=0.
021;parahippocampalcortex:r=0.
286,P=0.
028;andthalamus:r=0.
376,P=0.
0033).
Sig-nificantnegativecorrelationswerealsofoundbetweensolublepSer129α-synandinsolubleAβ42inthemid-frontalcortexandthalamus(midfrontalcortex:r=0.
475,P=0.
0005;thalamus:r=0.
399,P=0.
0018).
AsignificantpositivecorrelationbetweensolublepSer129α-synandinsolubleAβ40wasalsofoundintheparahippocampus(r=0.
316,P=0.
015).
Fieldfractionanalysisoftheextentofimmunohisto-chemicallabelingoftheseantigensintheDLBcasesre-vealedasignificantpositivecorrelationbetweenpSer129α-synandAβ42inthemid-frontalregiononly(r=0.
849,P=0.
0019)(seeAdditionalfile4:TableS2).
Therewasonlyweak,non-significantcorrelationbetweentheleveloftheseantigensinthebrainhomogenatesandthepercent-agearealabeledinparaffinsectionsfromthecorrespon-dingregionsinthecontralateralcerebralhemisphere.
InsolublepSer129α-synlevelcorrelateswithBraaktanglestageTheinsolublepSer129α-synlevelcorrelatedpositivelywiththeBraaktanglestageonlyinthemid-frontalcor-tex(Figure3)(r=0.
526,P=0.
0002).
SolublepSer129α-synlevelscorrelatednegativelywiththeBraakstageinthecingulateregiononly(r=0.
335,P=0.
028).
InsolubleAβishigherindiseasegroupsthancontrolsandinDLBthanPDinmostregionsThelevelofinsolubleAβ42inthecingulateandpara-hippocampalcortexwassignificantlyhigherinalldis-easecohortsthancontrols(Figure4).
TheDLBcohorthadasignificantlyhigherlevelofinsolubleAβ42inthemidfrontalcortexthandidanyoftheothergroups.
Incontrast,thelevelofinsolubleAβ42inthethalamuswashigherinthePDcohortsthaninDLBorcontrols.
SimilarresultswereobservedforinsolubleAβ40,withsomeregionaldifferences.
Inthemidfrontalandcingu-latecortexitwaspresentatasignificantlyhigherlevelinalldiseasegroupsthancontrols.
ThelevelofinsolubleFigure3CorrelationbetweenpSer129α-synandBraakstageincombinedPD(n=35)andDLB(n=10)patients.
Eachpointrepresentsaseparatecase.
Thebest-fitlinearregression(solidlines)and95%confidenceintervals(interruptedlines)aresuperimposed.
OnlysignificantP-values(andassociatedcorrelationcoefficients)areshowninthefigure.
InsolublepSer129α-synlevelcorrelatedpositivelywithBraakstageinthemidfrontalcortex.
SolublepSer129α-synlevelcorrelatednegativelywithBraakstageinthecingulatecortex.
DLB,dementiawthLewybodes;PD,Parkinson'sdisease;pSer129α-syn,alpha-synucleinphosphorylatedatserine129.
Swirskietal.
Alzheimer'sResearch&Therapy2014,6:77Page7of17http://alzres.
com/content/6/9/77Aβ40intheparahippocampuswassignificantlyhigherinDLBthancontrols.
SolubleAβishigherindiseasegroupsthancontrolsandindementiagroupsthanPDNDinseveralregionsThelevelofsolubleAβ42wassignificantlyhigherinthePDcohortsthancontrolsinallregions(Figure5).
ThelevelintheparahippocampalcortexwassignificantlyhigherinDLBthanPDNDorcontrols,andinthemid-frontalandcingulatecortexitwashigherinPDDthanDLB.
AlldiseasegroupshadasignificantlyhigherlevelofsolubleAβ40thancontrolsinmostregions.
Totalinsolubleα-synvariesmodestlybetweendiseasegroupsThelevelofinsolubleα-syninthemidfrontalandparahip-pocampalcortex(Figure4)wassignificantlyhigherinPDDthaninPDNDorcontrolswhereasintheDLBcohortthelevelwassignificantlyhigherinthecingulateregiononly.
Nosignificantdifferenceswerefoundbetweengroupsinthethalamus.
NosignificantdifferencesbetweencontrolsandPDNDwerefoundinanyregion.
Totalsolubleα-synishigherinPDthancontrolsbutlowerinDLBthanPDThelevelofsolubleα-syn(Figure5)wassignificantlyhigherinPDNDthancontrolsinallregions,andinPDDthancontrolsinthemidfrontalandparahippocam-palcortexandthalamus.
Unexpectedly,theDLBcohorthadsignificantlylowersolubletotalα-synthanthePDgroupsinmostregions.
InsolublepSer129α-synishigherandsolublepSer129α-synislowerindiseasegroupsthancontrolsInmostregions,theproportionofinsolubleα-synthatwasphosphorylatedatSer129wassignificantlyhigherinthePDDandDLBgroupsthanthecontrols(Figure6)andinseveralregionstheproportionwassignificantlyhigherinbothPDDandDLBthanPDND.
Conversely,inthesolublefraction,theproportionofα-synthatwasphosphorylatedatSer129wassignificantlyhigherincontrolsthandiseasegroupsinmostregions(Figure7).
Irrespectiveofcohort,asignificantlyhigherproportionofα-synwasphosphorylatedatSer129intheinsolublethanthesolublefractions(datanotshown;P<0.
001).
Figure4InsolubleAβandα-synlevelsincontrols,PDND,PDDandDLB.
Box-and-whiskerplotsindicatethefullrange,interquartilerangeandmedianvalueineachgroup.
InsolubleAβ42levelwassignificantlyhigherinDLBthancontrolsinallregionsexceptthethalamus.
ItwasalsosignificantlyhigherinDLBthanPDNDorPDDinthemidfrontalregionbutlowerinthethalamus.
Inallregionsexceptthemidfrontal,theinsolubleAβ42levelwassignificantlyhigherinbothPDNDandPDDthancontrols.
TheinsolubleAβ40levelwassignificantlygreaterinmidfrontalandcingulatecortexandthalamusinPDND,PDDandDLBthancontrols,andintheparahippocampusthelevelwassignificantlyhigherinDLBthaninPDNDorPDDbutnotcontrols.
Thetotalinsolubleα-synlevelwassignificantlyhigherinPDDthancontrolsorPDNDinmidfrontalcortexandhigherthaninDLBorcontrolsintheparahippocampus.
Totalinsolubleα-syninthecingulatecortexwassignificantlyhigherinDLBthaninPDNDorcontrols.
Nosignificantdifferencesbetweengroupswereobservedinthethalamus.
Aβ,amyloid-β;DLB,dementiawithLewybodies;PDD,Parkinson'sdiseasewithdementia;PDND,Parkinson'sdiseasewithoutdementia;α-syn,α-synuclein.
Swirskietal.
Alzheimer'sResearch&Therapy2014,6:77Page8of17http://alzres.
com/content/6/9/77AbsoluteproteinlevelsofpSer129α-synaredisplayedinAdditionalfile5:FigureS2.
MMSEscorecorrelatesnegativelywithinsolublepSer129α-syn,totalinsolubleα-synandinsolubleAβ42Inallregionsapartfromthethalamus,theMMSEscorecorrelatednegativelywiththelevelofinsolublepSer129α-syn(Figure8)(midfrontal:r=0.
555,P=0.
017;cin-gulate:r=0.
816,P<0.
0001;parahippocampalcortex:r=0.
752,P=0.
0003).
Furthermore,inthemidfrontalregiontherewasasignificantnegativecorrelationbetweentheMMSEscoreandbothinsolubleAβ42(r=0.
591,P=0.
0098)andtotalinsolubleα-syn(r=0.
498,P=0.
036).
NosignificantcorrelationswereobservedbetweenMMSEscoreandsolubleAβ42,Aβ40,totalα-synorpSer129α-syn.
Timetodementiacorrelatednegativelywithsol-ubleAβ40inthisregion(r=0.
58,P=0.
048)butdidnotshowanyothersignificantcorrelations(datanotshown).
AβtreatmentinducedphosphorylationatSer129inα-syn-overexpressingSHSY-5YcellsExposureofcellstoaggregatedAβ1-42(10μM)sig-nificantlyincreasedthepercentageofα-synintheinsolublefractionthatwasphosphorylatedatSer129.
Ser129phosphorylationwasalsohigherafterexposuretosoluble10μMAβ1-42andaggregated(butnotfresh)10μMAβ1-40buttheseincreasesdidnotreachstatis-ticalsignificance(Figure9).
Therewasatrendtowardsapositivecorrelation(Spearmanr=0.
49,P=0.
06)betweentheconcentrationofaggregatedAβandthepercentageofα-synphosphorylatedatSer129.
Inthesolublefraction,thepercentageofα-synthatwasphosphorylatedatSer129wasmuchlowerandtendedtodeclineafterexposuretoaggregatedAβ1-42andAβ1-40butnotafterexposuretofreshAβ(Figure10).
DiscussionAlthoughoverlapbetweenADandDLBpathologyoccursmuchmoreoftenthanwouldbeexpectedbychance,themolecularbasisispoorlyunderstood.
Furthermore,themolecularchangesunderlyingthedevelopmentofdemen-tiainpatientswithPDarenotfullyunderstood.
Wefoundthatmostpartsofthecerebralcortexexaminedshowed:(1)significantcorrelationsbetweenphosphorylationofα-synatSer129andtheamountofsolubleandinsolubleAβ;(2)significantcorrelationsbetweenphosphorylationFigure5SolubleAβandα-synlevelsincontrols,PDND,PDDandDLB.
Box-and-whiskerplotsindicatethefullrange,interquartilerangeandmedianvalueineachgroup.
SolubleAβ42levelwassignificantlyhigherinPDNDandPDDthancontrolsinallregions,andinthemidfrontalandcingulatecortex,itwassignificantlyhigherinPDDthanDLB.
InDLBthelevelofsolubleAβ42wassignificantlyhigherthanincontrolsandPDNDintheparahippocampusandhigherthancontrolsinthethalamus.
ThesolubleAβ40levelwassignificantlyelevatedinPDNDandPDDcomparedwithcontrolsinallregions.
ThelevelwasalsosignificantlyhigherinDLBthanincontrolsinallregionsexceptthecingulatecortex.
Solubletotalα-synlevelsweresignificantlygreaterinPDNDthancontrolsinallregions,andsignificantlyhigherinPDDthancontrolsinthemidfrontalcortex,parahippocampusandthalamus.
*P<0.
05,**P<0.
01,***P<0.
001.
Aβ,amyloid-β;DLB,dementiawithLewybodies;PDD,Parkinson'sdiseasewithdementia;PDND,Parkinson'sdiseasewithoutdementia;α-syn,α-synuclein.
Swirskietal.
Alzheimer'sResearch&Therapy2014,6:77Page9of17http://alzres.
com/content/6/9/77ofα-synatSer129andBraakstage;(3)higherlevelsofsol-ubleandinsolubleAβinPDandDLBthancontrols,andPDDandDLBthanPDND;and(4)ahigherproportionofα-synphosphorylatedatSer129inPDandDLBthancon-trols,andPDDandDLBthanPDND.
Ourstudyalsoshowedthattheproportionofα-synphosphorylatedatSer129correlatedwithante-mortemMMSE.
Lastly,ourinvitrostudiesshowedthatexposureofSH-SY5Ycellsoverexpressingwild-typeα-syntoAβ42significantlyincreasedtheproportionofα-synthatwasphosphory-latedatSer129.
Thesebiochemicalstudiesextendpre-viousfindingsofasynergisticrelationshipbetweenAβandα-synandsuggestthatAβ,particularlyAβ42,pro-motesthephosphorylationofα-synatSer129.
Ourbiochemicalstudiessupportpreviousimmunohis-tochemicalfindingsofapositivecorrelationbetweenin-solubleα-synandAβinLewybodydisease[25,26,62-64].
Inaddition,ourfindingofacorrelationwithBraakstage,althoughmorerestrictedintermsofregionsofthecortex,isinkeepingwithotherstudiesshowingassociationsbetweenα-synandtanglepathology[7,44]andsuggestthattherearemultipleinteractionsbetweenAlzheimer-typeandLewybody-typepathology.
Deramecourtetal.
[7]reportedthatallpatientswithsporadicDLBhadabundantdepositsofAβ42.
Inaddition,infamilieswithautosomal-dominantADcausedbyamyloidpre-cursorprotein(APP)orpresenilingenemutations,ahighproportionofpatientsshowLBpathologyataut-opsy[65,66].
Furthermore,patientswithmixedLBandAβplaquepathologyhaveamoreaggressivediseasecourseandmorepronouncedcognitivedysfunctionthandopatientswithpureAD[19,21-23].
TransgenicmiceexpressingbothhumanAβandα-synalsohavemoreseveredeficitsinlearningandmemory,andmoreFigure6Percentageofinsolubleα-synphosphorylatedatSer129.
Box-and-whiskerplotsindicatethefullrange,interquartilerangeandmedianvalueineachgroup.
Thepercentageofinsolubleα-synphosphorylatedatSer129wassignificantlyhigherinPDDthancontrolsinallregionsandinDLBinregionsexceptthemidfrontalcortex.
Inaddition,thepercentagewassignificantlyhigherinPDDthanPDNDinthemidfrontalandparahippocampalcortex,andinDLBthanPDNDinthecingulatecortex.
Inthethalamusandparahippocampalcortex,thepercentageofinsolubleα-synphosphorylatedatSer129wassignificantlyhigherinPDNDthancontrols.
DLB,dementiawithLewybodies;PDD,Parkinson'sdiseasewithdementia;PDND,Parkinson'sdiseasewithoutdementia;α-syn,α-synuclein.
Swirskietal.
Alzheimer'sResearch&Therapy2014,6:77Page10of17http://alzres.
com/content/6/9/77intraneuronalα-syninclusionsthandomicetransgenicforα-synalone[28].
OtherevidencecomesfromtheobservationbyKurataetal.
[67],ofenhancedaccumu-lationofbothAβandphosphorylatedα-syninmicedoublytransgenicformutantAPPandpresenilin-1com-paredtothatinmicetransgenicforAPPalone.
Obietal.
[44]demonstratedanassociationbetweenAβandpSer129α-syndetectedimmunohistochemicallyinthehumantemporalneocortexhumantissue,andwefoundasimilarcorrelationinthemid-frontalcortex.
However,itwasnoteworthythatthecorrelationbetweenAβandpSer129α-synwaslessconsistentindifferentbrainregionswhenwequantifiedtheseantigensimmu-nohistochemicallythanbyELISA,andonlyaweak,non-significantcorrelationwasdemonstratedbetweentheimmunohistochemicalandbiochemicalmeasurements.
SeveralpreviousstudieshavehighlighteddisparitiesbetweenELISAandimmunohistochemistry[68-70].
Someofthesedisparitiesarethoughttoreflecttheef-fectsofformalinfixationandtissueprocessingonthepreservationofantigenicepitopes,andothersmayrelatetoadegreeofcross-linkingofsolubleandinsolublepro-teins,preventingtheirseparateanalysisinthefixed,paraffin-embeddedtissue.
Inaddition,sandwichELISAsprovideanobjectivemeasureoftheactualconcentrationoftheanalyteinamuchlarger,morerepresentativevol-umeoftissuethanisincludedinaparaffinsection,andreliesonacombinationoftwodifferentantibodiesforspecificity.
Ourbiochemicalmethodsalsoallowedustomeasuresolubleprotein.
Thesignificantnegativecorre-lationsbetweensolublepSer129α-synandAβareinkeepingwithanenhancedshiftofpSer129α-synintotheinsolublefractionasaconsequenceofAβ.
ThepresentfindingshighlighttheimportanceofcombiningFigure7Percentageofsolubleα-synphosphorylatedatSer129.
Thepercentageofsolubleα-synphosphorylatedatSer129wassignificantlyhigherincontrolsthaninthePDgroupsinallregionsexceptmidfrontal.
Thepercentageofsolubleα-synphosphorylatedatSer129wasalsosignificantlyhigherincontrolsthanDLBinthecingulate.
Conversely,thepercentagewassignificantlyhigherinDLBthanPDNDorPDDintheparahippocampusandthalamusandsignificantlyhigherinPDNDthanDLBinthemidfrontalcortex.
*P<0.
05,**P<0.
01,***P<0.
001.
DLB,dementiawithLewybodies;PDD,Parkinson'sdiseasewithdementia;PDND,Parkinson'sdiseasewithoutdementia;α-syn,α-synuclein.
Swirskietal.
Alzheimer'sResearch&Therapy2014,6:77Page11of17http://alzres.
com/content/6/9/77biochemicalassessmentwithimmunohistochemicalme-thodswhenstudyingthequantitativerelationshipbet-weendifferentproteins.
Directmolecularinteractionbetweenα-synandAβwasdemonstratedinvitro,bymultidimensionalnuclearmag-neticresonance(NMR)spectroscopy[71].
Aβ42interactedmorestronglythanAβ40withα-syn,leadingtomajorstructuralchangestoα-syn,anditsoligomerizationandprecipitationwithinfourhours.
ThesefindingsmayberelevanttotheobservationbyBateetal.
[72]whoob-servedthatAβ42(butnotAβ40)enhancedα-syn-induceddamagetosynapses.
Aβ42morestronglypromotedtheformationofhighermolecularweightα-synpolymersinvitro[28].
Inkeepingwiththis,wefoundthatAβ42levelgenerallycorrelatedmorestronglywithpSer129α-syninhumanbraintissueextractsthanwithAβ40.
WealsoshowedthatAβ42hadamorepronouncedeffectthanAβ40onthephosphorylationofα-syninSH-SY5Ycells.
Invitrostudieshaveshownthatα-syncanbephos-phorylatedatSer129byCKI,CKII[55],severalGprotein-coupledreceptorkinases(GRKs1,2,5,6)[73],leucine-richrepeatkinase2(LRRK2)[74]andPolo-likekinases[75,76].
ThelevelsofCKIandCKIIexpressionareelevatedinbothADandDLB[76,77],raisingthepossibilitythattheseenzymesmaybeinvolvedinAβ-inducedphosphoryl-ationofα-synatSer129,similartotheAβ-inducedphosphorylationoftau[78-80].
Morethan90%ofα-syninLewybodiesandneuritesisphosphorylatedatSer129[34,35].
TheimportanceofFigure8CorrelationbetweenMMSEscoreandinsolubleAβorα-syn.
Thebest-fitlinearregression(solidlines)and95%confidenceintervals(interruptedlines)aresuperimposed.
OnlysignificantP-values(andassociatedcorrelationcoefficients)areshowninthefigure.
Inallregionsexceptthethalamus,thelevelofinsolublepSer129α-synshowedsignificantnegativecorrelationwiththeMMSEscore.
ThescorealsoshowedasignificantnegativecorrelationwithmidfrontalinsolubleAβ42andtotalinsolubleα-syn.
Aβ,amyloid-β;MMSE,mini-mentalstateexamination;pSer129α-syn,alpha-synucleinphosphorylatedatserine129;α-syn,α-synuclein.
Swirskietal.
Alzheimer'sResearch&Therapy2014,6:77Page12of17http://alzres.
com/content/6/9/77pSer129α-synwasrecognizedintheUnifiedLewy-typeSynucleinopathyStagingSchemeofBeachetal.
[43],basedontheabundanceanddistributionofpSer129α-syn.
WehaveshownthatbiochemicalmeasurementofpSer129α-synbysandwichELISAisanexcellentmarkerofLewybodydiseasesubtype.
PreviousstudieshavedemonstratedtheutilityofpSer129α-synmeasure-mentasamarkerofdiseasestage[43,44]andshownthatthelevelisgenerallyhigherinDLBandPDDthaninPDND[45].
TheaccumulationofpSer129anticipatesthedevelopmentofLewybodypathology[45,81].
Theparti-tioningandenrichmentofpSer129α-syninmembraneandinsolublebrainfractionsprobablyreflectschangesintheconformationandsolubilityofα-synthatpromoteitsassociationwithmembranestructures[82-84].
OurdatashowthatexposureofSH-SY5Ycellsoverexpressingwild-typeα-syntoAβresultsinashifttowardsinsolublepSer129α-syn,withatrendtowardslossofsolublepSer129α-syn.
Infuturestudiesitwouldbeofinteresttoinvestigatethedistributionofα-synandpSer129α-synfol-lowingAβexposureinthiscellmodelandtodeterminetheenzymesresponsible.
WehavefoundasignificantnegativecorrelationbetweenthelevelofinsolublepSer129α-synandtheMMSEscore.
ThissupportspreviousworksuggestingthatSer129phos-phorylationincreasestheneurotoxicityofα-synandisdet-rimentaltocognitivefunction.
Satoetal.
[85]showedthatpSer129α-synacceleratedA53Tα-syn-inducedneurode-generation;thiseffectwasabolishedbyinactivationofG-protein-coupledreceptorkinase6(GRK6)–respon-sibleforphosphorylationofα-synatSer129.
Incon-trast,enhancementofphosphataseactivityinα-syntransgenicmicecausedareductionofphosphorylatedα-syn,increaseddendriticarborizationofneuronsinthecerebralcortexandreducedastroglialandmicro-glialactivation[39].
Thesemorphologicaleffectswereassociatedwithimprovedmotorperformance.
Phos-phorylationofα-synatSer129wasalsoshowntoreduceFigure9Percentageofinsolubleα-synphosphorylatedatSer129afterexposureofSH-SY5YcellstoaggregatedorsolubleAβ1-42andAβ1-40.
Thepercentageofinsolubleα-synthatwasphosphorylatedatSer129wassignificantlyincreasedafter24hoursexposureofthecellsto10μMaggregatedAβ1-42(P=0.
009).
Ser129phosphorylationwasalsohigherafterexposuretosoluble10μMAβ1-42andaggregated10μMAβ1-40buttheincreasesdidnotreachsignificance.
Aβ,amyloid-β.
Swirskietal.
Alzheimer'sResearch&Therapy2014,6:77Page13of17http://alzres.
com/content/6/9/77α-syn-mediatedinhibitionoftyrosinehydroxylase,anen-zymeinvolvedincatecholaminesynthesis;therefore,phosphorylationofα-synmayinfluencedopaminelevels[86].
AlthoughAβ42,Aβ40andtotalα-synlevelsinsev-eralbrainregionsallcorrelatednegativelywithMMSEscore,thosecorrelationswerenotasstrongasthatbe-tweenMMSEscoreandpSer129α-syn.
OurfindingsunderscorethecloseassociationbetweenpSer129α-synaccumulationandcognitiveimpairment,anddopointtoapathogeneticrelationshipbetweenSer129phosphorylationofα-synanddiseaseprogression.
ConclusionsOurfindingsinthisstudy,thefirsttoexaminetherela-tionshipbetweenα-syn,pSer129,Aβ1-40andAβ1-42levelsinhumanpost-mortembraintissuebysandwichELISA,supporttheexistenceofapathogenicrelationshipbetweentheaccumulationofAβ,particularlyAβ42,andthephosphorylationofα-synatSer129,increasingthese-verityofLewybodydiseaseandthelikelihoodofdemen-tia.
Furtherinvestigationsarerequiredtodeterminetheprecisebiochemicalpathwaysresponsibleforthisinter-action,therelativecontributionsofdifferentprocesses(in-cludingAβ-associatedSer129phosphorylationofα-synandα-syn-associatedphosphorylationoftau)onthede-velopmentofcombinedADandLewybodypathologyandtheprogressionofneurodegeneration,andalsothepos-sibleinfluenceofAβonotherpotentialsitesofα-synphosphorylation.
AdditionalfilesAdditionalfile1:FigureS1.
Dotblotsdemonstratingthespecificityofthephosphorylation-specificα-synantibodies.
Blotsofrecombinantα-synincubatedwithdistilledwater(α-syn)caseinkinaseI(α-syn+CKI)orcaseinkinaseII(α-syn+CKII)andprobedwithpan-α-syn(column1),pSer129α-syn(column2)orpSer87α-syn(column3)antibody.
ThepSer129α-synantibodylabelledα-synfollowingincubationwithCKII,andtoalesserextentCKI,butdidnotlabelrecombinantα-synthathadnotbeenphosphorylatedwithCKIorCKII.
LabelingwiththepSer87-specificα-synantibodyoccursonlyafterincubationofα-synwithCKI.
Thesefindingsareaspredictedfromtheknownpatternsofphosphorylationofα-synwithCKIandCKII.
Figure10Percentageofsolubleα-synphosphorylatedatSer129afterexposureofSH-SY5YcellstoaggregatedorsolubleAβ1-42andAβ1-40.
Thepercentageofsolubleα-synphosphorylatedatSer129wasmuchlowerthanthefigureforinsolubleα-syn.
ThepercentagetendedtofallafterexposuretoaggregatedAβ1-42andAβ1-40,particularlyat10μM,butnotsignificantlyso,anddidnotchangenoticeablyafterexposuretosolubleAβ.
ThebarsshowthemeanandSEofmeasurementsfromfiveseparateexperiments.
Aβ,amyloid-β.
Swirskietal.
Alzheimer'sResearch&Therapy2014,6:77Page14of17http://alzres.
com/content/6/9/77Additionalfile2:TableS3.
Intra-assaycoefficientofvariation(CV)frominsoluble(shaded)andsolubleAβ42,Aβ40,totalα-synandpSer129α-synELISAs.
Additionalfile3:TableS1.
CorrelationbetweenlevelsofAβandα-syninthemidfrontal,cingulateandparahippocampalcortexandthethalamus.
Additionalfile4:TableS2.
CorrelationbetweenlevelsofAβandα-syninthemidfrontal,cingulateandparahippocampalcortexandthethalamusfromimmunohistochemistryfieldfractionanalyses.
Additionalfile5:FigureS2.
pSer129α-synlevelsincontrols*,PDND,PDDandDLB.
Box-and-whiskerplotsindicatethefullrange,interquartilerangeandmedianvalueineachgroup.
InsolublepSer129α-synlevelsweresignificantlyhigherinPDgroupscomparedwithcontrolsinthemidfrontalregion.
PDDpatientsalsoshowedsignificantlyhigherlevelsofinsolublepSer129α-syncomparedwithPDNDandDLBinthesameregion.
AlldiseasegroupsshowedsignificantlyhigherlevelsofinsolublepSer129α-synlevelscomparedwithcontrolsinthecingulateregion.
NosignificantdifferenceininsolublepSer129α-synlevelswereobservedintheparahippocampalcortexandthalamus.
SolublepSer129α-synlevelsweresignificantlyhigherinPDgroupscomparedwithcontrolsandDLBinthemidfrontalregion.
Incontrast,solublepSer129levelsweresignificantlyhigherincontrolsandDLBcomparedwithPDgroupsintheparahippocampalregion.
*Controlnnumbersvariedinthefollowingassaysduetolimitedtissueavailability(solublefraction:totalα-syn:midfrontaln=13,cingulaten=10,parahippocampaln=16,thalamusn=16;pSer129α-syn:midfrontaln=5,cingulaten=12,parahippocampaln=14;Aβ42:midfrontaln=13,cingulaten=16;Aβ40:midfrontaln=9,cingulaten=14,parahippocampaln=16).
AbbreviationsAD:Alzheimer'sdisease;ANOVA:analysisofvariance;APP:amyloidprecursorprotein;Aβ:amyloid-beta;BSA:bovineserumalbumin;CKI:caseinkinaseI;CKII:caseinkinaseII;CMV:cytomegalovirus;DLB:dementiawithLewybodies;EDTA:ethylenediaminetetraaceticacid;ELISA:enzyme-linkedimmunosorbentassay;GuHCl:guanidiniumchloride;HRP:horseradishperoxidase;MMSE:mini-mentalstateexamination;PBS:phosphate-bufferedsaline;PD:Parkinson'sdisease;PDD:Parkinson'sdiseasewithdementia;PDND:Parkinson'sdiseasewithoutdementia;pSer129α-syn:alpha-synucleinphosphorylatedatserine129;QSBB:QueenSquareBrainBank;RT:roomtemperature;Ser129:serine129;SWDBB:SouthWestDementiaBrainBank;TBS:Tris-bufferedsaline;TBST:Tris-bufferedsalinewithTween20;TRIS:tris(hydroxymethyl)aminomethane;α-syn:alpha-synuclein.
CompetinginterestsTheauthorsdeclarethattheyhavenocompetinginterests.
Authors'contributionsSL,JSM,JHandTRdesignedthestudy.
RdSdevelopedandcharacterizedtheSH-SY5Ycellsexpressinghumanwild-typeα-syn,MSandJSMperformedalloftheotherlaboratorystudiesontheSH-SY5Ycellsandonbraintissue;TL,JHandTRperformedtheneuropathologicalcharacterizationofmostthePDNDandPDDcases;HLreviewedtheclinicalrecordsandretrievedtheMMSEscores;MS,JSMandSLanalyzedthedataanddraftedthemanuscript.
Alloftheauthorsreadandapprovedthefinalmanuscript.
AcknowledgementsThisresearchwassupportedbygrantsfromAlzheimer'sResearchUKandBRACE(BristolResearchintoAgeingandCareoftheElderly).
TheSouthWestDementiaBrainBankwasalsosupportedbyABBUK(Alzheimer'sBrainBankUK,supportingBrainsforDementiaResearch).
TheQueenSquareBrainBankissupportedbytheRetaLilaWestonInstituteofNeurologicalStudies,UCLInstituteofNeurologyandthePSPAssociation.
PartofthisworkwasundertakenatUCLH/UCLwhoreceivedaproportionoffundingfromtheDepartmentofHealth'sNIHRBiomedicalResearchCentresfundingschemeand,inpart,funded/supportedbytheNationalInstituteforHealthResearch(NIHR)BiomedicalResearchUnitinDementiabasedatUniversityCollegeLondonHospitals(UCLH),UniversityCollegeLondon(UCL).
RdeSwasfundedbytheRetaLilaWestonTrustforMedicalResearch.
Authordetails1DementiaResearchGroup,InstituteofClinicalNeurosciences,SchoolofClinicalSciences,UniversityofBristol,Bristol,UK.
2DepartmentofMolecularNeuroscience,RetaLilaWestonInstituteofNeurologicalStudiesandQueenSquareBrainBankforNeurologicalDisorders,InstituteofNeurology,UniversityCollegeLondon,London,UK.
Received:26April2014Accepted:14October2014References1.
MayeuxR,SternY:EpidemiologyofAlzheimerdisease.
ColdSpringHarbPerspectMed2012,2:a006239.
2.
NowrangiMA,RaoV,LyketsosCG:Epidemiology,assessment,andtreatmentofdementia.
PsychiatrClinNorthAm2011,34:275.
3.
FornoLS:NeuropathologyofParkinson'sdisease.
JNeuropatholExpNeurol1996,55:259–272.
4.
PollanenMS,DicksonDW,BergeronC:PathologyandbiologyoftheLewybody.
JNeuropatholExpNeurol1993,52:183–191.
5.
SpillantiniMG,SchmidtML,LeeVM,TrojanowskiJQ,JakesR,GoedertM:α-SynucleininLewybodies.
Nature1997,388:839–840.
6.
BergeronC,PollanenM:LewybodiesinAlzheimerdisease–oneortwodiseasesAlzheimerDisAssocDisord1989,3:197.
7.
DeramecourtV,BomboisS,MaurageCA,GhestemA,DrobecqH,VanmechelenE,LebertF,PasquierF,DelacourteA:BiochemicalstagingofsynucleinopathyandamyloiddepositionindementiawithLewybodies.
JNeuropatholExpNeurol2006,65:278–288.
8.
DicksonD,CrystalH,MattiaceL,KressY,SchwagerlA,Ksiezak-RedingH,DaviesP,YenSH:DiffuseLewybodydisease:lightandelectronmicroscopicimmunocytochemistryofsenileplaques.
ActaNeuropathol1989,78:572–584.
9.
GibbW,MountjoyC,MannD,LeesA:ApathologicalstudyoftheassociationbetweenLewybodydiseaseandAlzheimer'sdisease.
JNeurolNeurosurgPsychiatry1989,52:701–708.
10.
HamiltonRL:LewybodiesinAlzheimer'sdisease:aneuropathologicalreviewof145casesusingα-synucleinimmunohistochemistry.
BrainPathol2000,10:378–384.
11.
IrwinDJ,LeeVM,TrojanowskiJQ:Parkinson'sdiseasedementia:convergenceofα-synuclein,tauandamyloid-βpathologies.
NatRevNeurosci2013,14:626–636.
12.
IrwinDJ,WhiteMT,ToledoJB,XieSX,RobinsonJL,VanDeerlinV,LeeVM,LeverenzJB,MontineTJ,DudaJE,HurtigHI,TrojanowskiJQ:NeuropathologicsubstratesofParkinsondiseasedementia.
AnnNeurol2012,72:587–598.
13.
MikolaenkoI,PletnikovaO,KawasCH,O'BrienR,ResnickSM,CrainB,TroncosoJC:Alpha-synucleinlesionsinnormalaging,Parkinsondisease,andAlzheimerdisease:evidencefromtheBaltimoreLongitudinalStudyofAging(BLSA).
JNeuropatholExpNeurol2005,64:156–162.
14.
ParkkinenL,PirttilT,AlafuzoffI:Applicabilityofcurrentstaging/categorizationofα-synucleinpathologyandtheirclinicalrelevance.
ActaNeuropathol2008,115:399–407.
15.
SaitoY,KawashimaA,RuberuNN,FujiwaraH,KoyamaS,SawabeM,AraiT,NaguraH,YamanouchiH,HasegawaM,IwatsuboT,MurayamaS:Accumulationofphosphorylatedα-synucleininaginghumanbrain.
JNeuropatholExpNeurol2003,62:644–654.
16.
UchikadoH,LinWL,DeLuciaMW,DicksonDW:AlzheimerdiseasewithamygdalaLewybodies:adistinctformofα-synucleinopathy.
JNeuropatholExpNeurol2006,65:685–697.
17.
GalpernWR,LangAE:Interfacebetweentauopathiesandsynucleinopathies:ataleoftwoproteins.
AnnNeurol2006,59:449–458.
18.
HallidayG,HelyM,ReidW,MorrisJ:TheprogressionofpathologyinlongitudinallyfollowedpatientswithParkinson'sdisease.
ActaNeuropathol2008,115:409–415.
19.
HansenL,SalmonD,GalaskoD,MasliahE,KatzmanR,DeteresaR,ThalL,PayMM,HofstetterR,KlauberM,RiceV,ButtersN,AlfordM:TheLewybodyvariantofAlzheimer'sdisease-aclinicalandpathologicalentity.
Neurology1990,40:1–8.
20.
KosakaK,YoshimuraM,IkedaK,BudkaH:DiffusetypeofLewybodydisease-progressivedementiawithabundantcorticalLewybodiesandsenilechangesofvaryingdegree-anewdisease.
ClinNeuropathol1984,3:185–192.
Swirskietal.
Alzheimer'sResearch&Therapy2014,6:77Page15of17http://alzres.
com/content/6/9/7721.
KraybillML,LarsonEB,TsuangDW,TeriL,McCormickWC,BowenJD,KukullWA,LeverenzJB,CherrierMM:Cognitivedifferencesindementiapatientswithautopsy-verifiedAD,Lewybodypathology,orboth.
Neurology2005,64:2069–2073.
22.
LanglaisPJ,ThalL,HansenL,GalaskoD,AlfordM,MasliahE:NeurotransmittersinbasalgangliaandcortexofAlzheimer'sdiseasewithandwithoutLewybodies.
Neurology1993,43:1927–1934.
23.
OlichneyJM,GalaskoD,SalmonDP,HofstetterCR,KatzmanR,ThalLJ:CognitivedeclineisfasterinLewybodyvariantthaninAlzheimer'sdisease.
Neurology1998,51:351–357.
24.
TrojanowskiJQ:EmergingAlzheimer'sdiseasetherapies:focusingonthefuture.
NeurobiolAging2002,23:985–990.
25.
LashleyT,HoltonJL,GrayE,KirkhamK,O'SullivanSS,HilbigA,WoodNW,LeesAJ,ReveszT:Corticalα-synucleinloadisassociatedwithamyloid-βplaqueburdeninasubsetofParkinson'sdiseasepatients.
ActaNeuropathol2008,115:417–425.
26.
PletnikovaO,WestN,LeeMK,RudowGL,SkolaskyRL,DawsonTM,MarshL,TroncosoJC:Aβdepositionisassociatedwithenhancedcorticalα-synucleinlesionsinLewybodydiseases.
NeurobiolAging2005,26:1183–1192.
27.
WirthsO,WeickertS,MajtenyiK,HavasL,KahlePJ,OkochiM,HaassC,MulthaupG,BeyreutherK,BayerTA:LewybodyvariantofAlzheimer'sdisease:α-synucleinindystrophicneuritesofAβplaques.
Neuroreport2000,11:3737–3741.
28.
MasliahE,RockensteinE,VeinbergsI,SagaraY,MalloryM,HashimotoM,MuckeL:β-Amyloidpeptidesenhanceα-synucleinaccumulationandneuronaldeficitsinatransgenicmousemodellinkingAlzheimer'sdiseaseandParkinson'sdisease.
ProcNatlAcadSciUSA2001,98:12245–12250.
29.
MoskvinaV,HaroldD,RussoG,VedernikovA,SharmaM,SaadM,HolmansP,BrasJM,BettellaF,KellerMF,NicolaouN,Simón-SánchezJ,GibbsJR,SchulteC,DurrA,GuerreiroR,HernandezD,BriceA,StefánssonH,MajamaaK,GasserT,HeutinkP,WoodN,MartinezM,SingletonAB,NallsMA,HardyJ,OwenMJ,O'DonovanMC,WilliamsJ,etal:Analysisofgenome-wideassociationstudiesofAlzheimerdiseaseandofParkinsondiseasetodetermineifthese2diseasesshareacommongeneticrisk.
JAMANeurol2013,70:1268–1276.
30.
JensenPH,HagerH,NielsenMS,HjrupP,GliemannJ,JakesR:α-SynucleinbindstotauandstimulatestheproteinkinaseA-catalyzedtauphosphorylationofserineresidues262and356.
JBiolChem1999,274:25481–25489.
31.
DukaT,DukaV,JoyceJN,SidhuA:α-SynucleincontributestoGSK-3β-catalyzedtauphosphorylationinParkinson'sdiseasemodels.
FASEBJ2009,23:2820–2830.
32.
KawakamiF,SuzukiM,ShimadaN,KagiyaG,OhtaE,TamuraK,MaruyamaH,IchikawaT:Stimulatoryeffectofα-synucleinonthetau-phosphorylationbyGSK-3β.
FEBSJ2011,278:4895–4904.
33.
EdisonP,RoweCC,RinneJO,NgS,AhmedI,KemppainenN,VillemagneVL,O'KeefeG,NgrenK,ChaudhuryK,MastersCL,BrooksDJ:AmyloidloadinParkinson'sdiseasedementiaandLewybodydementiameasuredwith[11C]PIBpositronemissiontomography.
JNeurolNeurosurgPsychiatry2008,79:1331–1338.
34.
AndersonJP,WalkerDE,GoldsteinJM,deLaatR,BanducciK,CaccavelloRJ,BarbourR,HuangJ,KlingK,LeeM,DiepL,KeimPS,ShenX,ChatawayT,SchlossmacherMG,SeubertP,SchenkD,SinhaS,GaiWP,ChilcoteTJ:PhosphorylationofSer-129isthedominantpathologicalmodificationofα-synucleininfamilialandsporadicLewybodydisease.
JBiolChem2006,281:29739–29752.
35.
FujiwaraH,HasegawaM,DohmaeN,KawashimaA,MasliahE,GoldbergMS,ShenJ,TakioK,IwatsuboT:α-Synucleinisphosphorylatedinsynucleinopathylesions.
NatCellBiol2002,4:160–164.
36.
ChauKY,ChingHL,SchapiraAH,CooperJM:Relationshipbetweenαsynucleinphosphorylation,proteasomalinhibitionandcelldeath:relevancetoParkinson'sdiseasepathogenesis.
JNeurochem2009,110:1005–1013.
37.
ChenL,FeanyMB:α-SynucleinphosphorylationcontrolsneurotoxicityandinclusionformationinaDrosophilamodelofParkinsondisease.
NatNeurosci2005,8:657–663.
38.
ChenL,PeriquetM,WangX,NegroA,McLeanPJ,HymanBT,FeanyMB:Tyrosineandserinephosphorylationofα-synucleinhaveopposingeffectsonneurotoxicityandsolubleoligomerformation.
JClinInvest2009,119:3257–3265.
39.
LeeKW,ChenW,JunnE,ImJY,GrossoH,SonsallaPK,FengX,RayN,FernandezJR,ChaoY:Enhancedphosphataseactivityattenuatesα-synucleinopathyinamousemodel.
JNeurosci2011,31:6963–6971.
40.
PaleologouKE,OueslatiA,ShakkedG,RospigliosiCC,KimHY,LambertoGR,FernandezCO,SchmidA,CheginiF,GaiWP,ChiappeD,MoniatteM,SchneiderBL,AebischerP,EliezerD,ZweckstetterM,MasliahE,LashuelHA:PhosphorylationatS87isenhancedinsynucleinopathies,inhibitsα-synucleinoligomerization,andinfluencessynuclein-membraneinteractions.
JNeurosci2010,30:3184–3198.
41.
SchreursS,GerardM,DeruaR,WaelkensE,TaymansJM,BaekelandtV,EngelborghsY:InvitrophosphorylationdoesnotinfluencetheaggregationkineticsofWTα-synucleinincontrasttoitsphosphorylationmutants.
IntJMolSci2014,15:1040–1067.
42.
WaxmanEA,GiassonBI:Specificityandregulationofcaseinkinase-mediatedphosphorylationofalpha-synuclein.
JNeuropatholExpNeurol2008,67:402–416.
43.
BeachTG,AdlerCH,LueLF,SueLI,BachalakuriJ,Henry-WatsonJ,SasseJ,BoyerS,ShirohiS,BrooksR,EschbacherJ,WhiteCL3rd,AkiyamaH,CavinessJ,ShillHA,ConnorDJ,SabbaghMN,WalkerDG,ArizonaParkinson'sDiseaseConsortium:UnifiedstagingsystemforLewybodydisorders:correlationwithnigrostriataldegeneration,cognitiveimpairmentandmotordysfunction.
ActaNeuropathol2009,117:613–634.
44.
ObiK,AkiyamaH,KondoH,ShimomuraY,HasegawaM,IwatsuboT,MizunoY,MochizukiH:Relationshipofphosphorylatedα-synucleinandtauaccumulationtoAβdepositioninthecerebralcortexofdementiawithLewybodies.
ExpNeurol2008,210:409–420.
45.
WalkerDG,LueLF,AdlerCH,ShillHA,CavinessJN,SabbaghMN,AkiyamaH,SerranoGE,SueLI,BeachTG,ArizonaParkinsonDiseaseConsortium:Changesinpropertiesofserine129phosphorylatedα-synucleinwithprogressionofLewy-typehistopathologyinhumanbrains.
ExpNeurol2013,240:190–204.
46.
MinersJS,vanHelmondZ,RaikerM,LoveS,KehoePG:ACEvariantsandassociationwithbrainAβlevelsinAlzheimer'sdisease.
AmJTranslRes2010,3:73–80.
47.
TaylerH,FraserT,MinersJS,KehoePG,LoveS:OxidativebalanceinAlzheimer'sdisease:relationshiptoAPOE,Braaktanglestage,andtheconcentrationsofsolubleandinsolubleamyloid-β.
JAlzheimersDis2010,22:1363–1373.
48.
vanHelmondZ,MinersJS,KehoePG,LoveS:HighersolubleamyloidβconcentrationinfrontalcortexofyoungadultsthaninnormalelderlyorAlzheimer'sdisease.
BrainPathol2010,20:787–793.
49.
MinersJS,JonesR,LoveS:DifferentialchangesinAβ42andAβ40withage.
JAlzheimersDis2014,40:727–735.
50.
McKeithIG,DicksonD,LoweJ,EmreM,O'BrienJ,FeldmanH,CummingsJ,DudaJ,LippaC,PerryE,AarslandD,AraiH,BallardCG,BoeveB,BurnDJ,CostaD,DelSerT,DuboisB,GalaskoD,GauthierS,GoetzCG,Gomez-TortosaE,HallidayG,HansenLA,HardyJ,IwatsuboT,KalariaRN,KauferD,KennyRA,KorczynA,etal:DiagnosisandmanagementofdementiawithLewybodies:thirdreportoftheDLBconsortium.
Neurology2005,65:1863–1872.
51.
MontineTJ,PhelpsCH,BeachTG,BigioEH,CairnsNJ,DicksonDW,DuyckaertsC,FroschMP,MasliahE,MirraSS,NelsonPT,SchneiderJA,ThalDR,TrojanowskiJQ,VintersHV,HymanBT,NationalInstituteonAging,Alzheimer'sAssociation:NationalInstituteonAging–Alzheimer'sAssociationguidelinesfortheneuropathologicassessmentofAlzheimer'sdisease:apracticalapproach.
ActaNeuropathol2012,123:1–11.
52.
MinersJS,MorrisS,LoveS,KehoePG:Accumulationofinsolubleamyloid-βinDown'ssyndromeisassociatedwithincreasedBACE-1andneprilysinactivities.
JAlzheimersDis2011,23:101–108.
53.
MinersJS,MouldingH,deSilvaR,LoveS:ReducedvascularendothelialgrowthfactorandcapillarydensityintheoccipitalcortexindementiawithLewybodies.
BrainPathol2014,24:334–343.
54.
LeeG,TanakaM,ParkK,LeeSS,KimYM,JunnE,LeeSH,MouradianMM:CaseinkinaseII-mediatedphosphorylationregulatesα-synuclein/synphilin-1interactionandinclusionbodyformation.
JBiolChem2004,279:6834–6839.
55.
OkochiM,WalterJ,KoyamaA,NakajoS,BabaM,IwatsuboT,MeijerL,KahlePJ,HaassC:ConstitutivephosphorylationoftheParkinson'sdiseaseassociatedα-synuclein.
JBiolChem2000,275:390–397.
56.
BaruaNU,MinersJS,BienemannAS,WyattMJ,WelserK,TaborAB,HailesHC,LoveS,GillSS:Convection-enhanceddeliveryofneprilysin:anovelamyloid-β-degradingtherapeuticstrategy.
JAlzheimersDis2012,32:43–56.
57.
QiuWQ,YeZ,KholodenkoD,SeubertP,SelkoeDJ:Degradationofamyloidβ-proteinbyametalloproteasesecretedbymicrogliaandotherneuralandnon-neuralcells.
JBiolChem1997,272:6641–6646.
Swirskietal.
Alzheimer'sResearch&Therapy2014,6:77Page16of17http://alzres.
com/content/6/9/7758.
AshbyEL,KehoePG,LoveS:Kallikrein-relatedpeptidase6inAlzheimer'sdiseaseandvasculardementia.
BrainRes2010,1363:1–10.
59.
ChalmersK,WilcockGK,LoveS:APOEε4influencesthepathologicalphenotypeofAlzheimer'sdiseasebyfavouringcerebrovascularoverparenchymalaccumulationofAβprotein.
NeuropatholApplNeurobiol2003,29:231–238.
60.
ChalmersKA,WilcockGK,VintersHV,PerryEK,PerryR,BallardCG,LoveS:CholinesteraseinhibitorsmayincreasephosphorylatedtauinAlzheimer'sdisease.
JNeurol2009,256:717–720.
61.
vanHelmondZ,HeesomK,LoveS:CharacterisationoftwoantibodiestooligomericAβandtheiruseinELISAsonhumanbraintissuehomogenates.
JNeurosciMethods2009,176:206–212.
62.
Colom-CadenaM,GelpiE,CharifS,BelbinO,BlesaR,MartíMJ,ClarimónJ,LleóA:Confluenceofα-synuclein,tau,andβ-amyloidpathologiesindementiawithLewybodies.
JNeuropatholExpNeurol2013,72:1203–1212.
63.
ComptaY,ParkkinenL,O'SullivanSS,VandrovcovaJ,HoltonJL,CollinsC,LashleyT,KallisC,WilliamsDR,deSilvaR,LeesAJ,ReveszT:Lewy-andAlzheimer-typepathologiesinParkinson'sdiseasedementia:whichismoreimportantBrain2011,134:1493–1505.
64.
DicksonDW,FujishiroH,OrrC,DelleDonneA,JosephsKA,FrigerioR,BurnettM,ParisiJE,KlosKJ,AhlskogJE:Neuropathologyofnon-motorfeaturesofParkinsondisease.
ParkinsonismRelatDisord2009,15:S1–S5.
65.
LeverenzJB,FishelMA,PeskindER,MontineTJ,NochlinD,SteinbartE,RaskindMA,SchellenbergGD,BirdTD,TsuangD:LewybodypathologyinfamilialAlzheimerdisease:evidencefordisease-andmutation-specificpathologicphenotype.
ArchNeurol2006,63:370.
66.
RosenbergCK,Pericak-VanceMA,SaundersAM,GilbertJR,GaskellPC,HuletteCM:LewybodyandAlzheimerpathologyinafamilywiththeamyloid-βprecursorproteinAPP717genemutation.
ActaNeuropathol2000,100:145–152.
67.
KurataT,KawarabayashiT,MurakamiT,MiyazakiK,MorimotoN,HtaY,TakehisaY,NagaiM,IkedaM,MatsubaraE,WestawayD,HyslopPS,HarigayaY,KamiyaT,ShojiM,AbeK:Enhancedaccumulationofphosphorylatedalpha-synucleinindoubletransgenicmiceexpressingmutantbeta-amyloidprecursorproteinandpresenilin-1.
JNeurosciRes2007,85:2246–2252.
68.
PappotH,SkovBG,PykeC,Grondahl-HansenJ:Levelsofplasminogenactivatorinhibitortype1andurokinaseplasminogenactivatorreceptorinnon-smallcelllungcancerasmeasuredbyquantitativeELISAandsemiquantitativeimmunohistochemistry.
LungCancer1997,17:197–209.
69.
deWitteH,PappotH,BrunnerN,Grondahl-HansenJ,Hoyer-HansenG,BehrendtN,Guldhammer-SkovB,SweepF,BenraadT,DanoK:ELISAforcomplexesbetweenurokinase-typeplasminogenactivatoranditsreceptorinlungcancertissueextracts.
IntJCancer1997,72:416–423.
70.
FerrierCM,deWitteHH,StraatmanH,vanTienovenDH,vanGeloofWL,RietveldFJ,SweepCG,RuiterDJ,vanMuijenGN:Comparisonofimmunohistochemistrywithimmunoassay(ELISA)forthedetectionofcomponentsoftheplasminogenactivationsysteminhumantumourtissue.
BrJCancer1999,79:1534–1541.
71.
MandalPK,PettegrewJW,MasliahE,HamiltonRL,MandalR:InteractionbetweenAβpeptideandαsynuclein:molecularmechanismsinoverlappingpathologyofAlzheimer'sandParkinson'sindementiawithLewybodydisease.
NeurochemRes2006,31:1153–1162.
ApublishederratumappearsinNeurochemRes2007,32:2002.
72.
BateC,GentlemanS,WilliamsA:α-Synucleininducedsynapsedamageisenhancedbyamyloid-beta(1–42).
MolNeurodegener2010,5:55.
73.
ProninAN,MorrisAJ,SurguchovA,BenovicJL:SynucleinsareanovelclassofsubstratesforGprotein-coupledreceptorkinases.
JBiolChem2000,275:26515–26522.
74.
QingH,WongW,McGeerEG,McGeerPL:Lrrk2phosphorylatesαsynucleinatserine129:Parkinsondiseaseimplications.
BiochemBiophysResCommun2009,387:149–152.
75.
InglisKJ,ChereauD,BrighamEF,ChiouSS,SchobelS,FrigonNL,YuM,CaccavelloRJ,NelsonS,MotterR,WrightS,ChianD,SantiagoP,SorianoF,RamosC,PowellK,GoldsteinJM,BabcockM,YednockT,BardF,BasiGS,ShamH,ChilcoteTJ,McConlogueL,Griswold-PrennerI,AndersonJP:Polo-likekinase2(PLK2)phosphorylatesα-synucleinatserine129incentralnervoussystem.
JBiolChem2009,284:2598–2602.
76.
MbefoMK,PaleologouKE,BoucharabaA,OueslatiA,SchellH,FournierM,OlschewskiD,YinGW,ZweckstetterM,MasliahE,KahlePJ,HirlingH,LashuelHA:PhosphorylationofsynucleinsbymembersofthePolo-likekinasefamily.
JBiolChem2010,285:2807–2822.
77.
YasojimaK,KuretJ,DeMaggioAJ,McGeerE,McGeerPL:Caseinkinase1deltamRNAisupregulatedinAlzheimerdiseasebrain.
BrainRes2000,865:116–120.
78.
BaigS,vanHelmondZ,LoveS:TauhyperphosphorylationaffectsSmad2/3translocation.
Neuroscience2009,163:561–570.
79.
GtzJ,ChenF,VanDorpeJ,NitschR:FormationofneurofibrillarytanglesinP301LtautransgenicmiceinducedbyAβ42fibrils.
Science2001,293:1491–1495.
80.
LewisJ,McGowanE,RockwoodJ,MelroseH,NacharajuP,VanSlegtenhorstM,Gwinn-HardyK,MurphyMP,BakerM,YuX,DuffK,HardyJ,CorralA,LinWL,YenSH,DicksonDW,DaviesP,HuttonM:Neurofibrillarytangles,amyotrophyandprogressivemotordisturbanceinmiceexpressingmutant(P301L)tauprotein.
NatGenet2000,25:402–405.
ApublishederratumappearsinNatGenet2000,26:127.
81.
LueLF,WalkerDG,AdlerCH,ShillH,TranH,AkiyamaH,SueLI,CavinessJ,SabbaghMN,BeachTG:Biochemicalincreaseinphosphorylatedα‐synucleinprecedeshistopathologyofLewy‐typesynucleinopathies.
BrainPathol2012,22:745–756.
82.
AuluckPK,CaraveoG,LindquistS:α-Synuclein:membraneinteractionsandtoxicityinParkinson'sdisease.
AnnuRevCellDevBiol2010,26:211–233.
83.
BartelsT,AhlstromLS,LeftinA,KampF,HaassC,BrownMF,BeyerK:TheN-terminusoftheintrinsicallydisorderedproteinα-synucleintriggersmembranebindingandhelixfolding.
BiophysJ2010,99:2116–2124.
84.
UverskyVN,LiJ,FinkAL:Metal-triggeredstructuraltransformations,aggregation,andfibrillationofhumanα-synuclein-apossiblemolecularlinkbetweenParkinson'sdiseaseandheavymetalexposure.
JBiolChem2001,276:44284–44296.
85.
SatoH,ArawakaS,HaraS,FukushimaS,KogaK,KoyamaS,KatoT:Authenticallyphosphorylatedα-synucleinatSer129acceleratesneurodegenerationinaratmodeloffamilialParkinson'sdisease.
JNeurosci2011,31:16884–16894.
86.
LouH,MontoyaSE,AlerteTN,WangJ,WuJ,PengX,HongCS,FriedrichEE,MaderSA,PedersenCJ:Serine129phosphorylationreducestheabilityofα-synucleintoregulatetyrosinehydroxylaseandproteinphosphatase2Ainvitroandinvivo.
JBiolChem2010,285:17648–17661.
doi:10.
1186/s13195-014-0077-yCitethisarticleas:Swirskietal.
:Evaluatingtherelationshipbetweenamyloid-βandα-synucleinphosphorylatedatSer129indementiawithLewybodiesandParkinson'sdisease.
Alzheimer'sResearch&Therapy20146:77.
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