biomarkers of alzheimer-associated endosomal dysfunction
TRANSCRIPT
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BiomarkersofAlzheimer-AssociatedEndosomalDysfunction
JessicaNeufeld
Submittedinpartialfulfillmentofthe
requirementsforthedegreeofDoctorofPhilosophy
intheGraduateSchoolofArtsandSciences
COLUMBIAUNIVERSITY
2018
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©2018
JessicaNeufeld
Allrightsreserved
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ABSTRACT
BiomarkersofAlzheimer-AssociatedEndosomalDysfunction
JessicaNeufeld
EndosomaldysfunctionhasbeenmechanisticallylinkedtoAlzheimer’sDisease(AD).Todate,no
invivobiomarkersforthiscellulardeficitexist.Yetsuchbiomarkersarerequiredfordetermining
its prevalence in AD and tracking its time course—both in disease progression and potential
clinicaltrials.Withthisgoalinmind,wemadeuseofanassortmentofmousemodelsbearing
AD-relatedendosomaltraffickingdefectsthroughselectivedeletionofretomercoreproteins.
WecollectedCSFandbrainexosomesfromtheseretromer-deficientmodelsandperformeda
battery of molecular inquiries which included lipidomic and proteomic screens, as well as
hypothesis-drivenbiochemistry.Theresultsofthiscomprehensiveinvestigationincludethefirst
characterization of themurine CSF lipidome and the deepest characterization to date of the
murineCSFproteome.
Herein,we report that VPS26a haploinsufficiency in the brain imparts no detectable protein
changesintheCSFasmeasuredbylabeledLC-MS/MSatthreemonthsofage.Thisdeficitdoes,
however,causeareliablereductionofCSFsphingomyelind18:1/18:1,whichisexacerbatedby
age,extendingtoothersphingomyelinsandotherlipidclassesincludingdihydrosphingomyelins
andmonohexosylceramides.
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CompleteknockoutofitsparalogVPS26bpromotesanenrichmentofBACE1-cleavedAPPCTFs
(b-CTFs)inbrain-derivedexosomesandmayalterexosomalbiogenicpathways.Similartrends
wereseeninaneuronal-specificknockout(viaCamk2a-Crerecombinase)ofretromer’slinchpin,
VPS35.
Most importantly, anunbiasedproteomic screenofCSF collected frommicewitha selective
knockoutofVPS35 in forebrainneurons(engineeredusingtheCamk2a system)uncovereda
totalof71hits(52parametricand19nonparametric)fromthe1505proteinsdetected.Pathway
analysis and follow-up studies identified two distinct molecular categories with previously
establishedrelevancetoAD:BACE1substratesandMAPT(morecommonlyreferredtoastau).
Wereportthat,bothinvivoandinvitro,neuronal-selectiveknockoutofVPS35causesincreased
secretionoftheN-terminalfragments(NTFs)ofBACE1substratesAPLP1andCHL1aswellastotal
tau, and importantly, that these events occur independent of cell death. Further, we find
evidenceofconvergenceofthesepathwaysinbothmouseandhumanCSF.However,asthese
BACE1 substrates likely accumulate in plaques, we propose CSF total tau as a biomarker of
endosomaldysfunctionwithutilityovertheentirecourseofADprogression.
We have identified and validated a series of in vivo biomarkers that are reflective of AD-
associatedendosomaldysfunction.Whileclearlysensitivetothiscellularpathology,futurework
isrequiredtodeterminetheirspecificity.Additionally,follow-upstudiesarerequiredtoshow
that interventions which rescue endosomal dysfunction affect this molecular profile. The
identifiedbiomarkersholdgreatpromiseforearlydetectionofendosomaldysfunctioninADand
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fortrackingitscourse,duringthediseaseprogressionandforclinicaltrials. Furthermore,the
unexpectedbutvalidatedfinding,showingthatincreasedCSFtauisreflectiveofAD-associated
endosomaldysfunction,suggeststhatendosomaldysfunctionisauniversaldeficitsharedamong
ADpatientsinitsearlieststagesofdisease.
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TableofContents
ListofFigures………….………………………………………………………………………………………………………….…….iiiListofTables.……………………………………………………………………………………………………………………..…….iv
1 Chapter1:Introduction 11.1 Alzheimer’sDisease 1
1.1.1 Epidemiology..........................................................................................................................11.1.2 Riskfactors.............................................................................................................................21.1.3 Pathologicalhallmarks...........................................................................................................41.1.4 Diagnosticchallenges.............................................................................................................51.1.5 Theamyloidhypothesis.........................................................................................................61.1.6 Theendosomeasanemergingtherapeutictarget.............................................................11
1.2 Theretromerassembly 141.2.1 Retromerasamasterconductorofendosomaltrafficking................................................141.2.2 RetromerinAD....................................................................................................................181.2.3 RetromerdeficiencyasamodelofAD-relatedendosomaldysfunction............................19
1.3 Thesearchforbiomarkersofendosomaldysfunction 20
2 Chapter2:CSFLipidomics 252.1 Introduction 25
2.1.1 Lipidsandtheendosome.....................................................................................................252.1.2 LipiddysregulationinAD.....................................................................................................252.1.3 Massspectrometryoflipids................................................................................................262.1.4 CSFlipidomicsstrategy........................................................................................................262.1.5 Choiceoftheretromer-deficientmodel.............................................................................27
2.2 Results 272.2.1 CollectionofhighqualityCSF..............................................................................................272.2.2 ThemurineCSFlipidomecloselyresemblesthatofhumans.............................................292.2.3 LipidomicscreenrevealssphingomyelinchangesintheCSFofVPS26a-haploinsufficient
mice.....................................................................................................................................312.2.4 ReductionsinVPS26a+/-CSFsphingomyelinsaremorepronouncedwithageandextend
tootherlipidgroups...........................................................................................................362.2.5 SphingomyelindysregulationintheVPS26a+/-brainisnon-neuronal...............................38
2.3 Discussion 40
3 Chapter3:BrainExosomes 423.1 Introduction 42
3.1.1 Exosomebiology..................................................................................................................423.1.2 Exosomesasreportersofendosomaldysfunction.............................................................423.1.3 Brainexosomebiomarkerstrategy.....................................................................................43
3.2 Results 443.2.1 Isolationofbrain-derivedexosomesfromwildtypemice:validationoftechnique..........443.2.2 Retromercoreproteinsarepresentinbrainexosomefractions.......................................463.2.3 Brainexosomesderivedfromretromer-deficientmicehavereducedretromerprotein
levels...................................................................................................................................473.2.4 Brainexosomesderivedfromretromerknockoutmiceareenrichedinb-CTFs...............49
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3.2.5 Brainexosomesfromretromerknockoutmicehavedecreasedlevelsofexosomalmarkersalixandflotillin-1..................................................................................................52
3.3 Discussion 54
4 Chapter4:CSFProteomics 564.1 Introduction 56
4.1.1 Endosomaldysfunctionandthesecretedproteome..........................................................564.1.2 CSFProteomicsbiomarkerstrategy....................................................................................574.2.1 CSFfromVPS26a+/-micerevealsnoapparentproteinalterationsasmeasuredviaLC/MS-
MS.......................................................................................................................................584.2.2 MassspectrometryrevealsenrichmentofBACE1substrateN-terminalfragmentsand
totaltauinCSFofVPS35Camk2aknockoutmice.............................................................634.2.3 BACE1SubstrateNTFsandTotalTauareconfirmedashitsinanewcohortofVPS35
Camk2aknockoutmice......................................................................................................714.2.4 TranslationtohumanspositionsCSFtotaltauasabiomarkerofendosomaldysfunction
inAD....................................................................................................................................754.3 Discussion 79
4.3.1 VPS26a+/-CSFProteomics....................................................................................................804.3.2 VPS35Camk2aCSFProteomics...........................................................................................80
5. Chapter5:Conclusionsandfuturedirections 865.1 Overview 865.2 CSFlipidomics 865.3 Brainexosomestudies 875.4 CSFproteomics 89
6. MaterialsandMethods 93
7. References 102
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ListofFigures
Chapter1Figure1.1ThegeneticsofAD. ....................................................................................................... 3Figure1.2ThepathologicalhallmarksofAD. ............................................................................... 5Figure1.3Theprocessingpathwaysofamyloidprecursorprotein(APP). .................................. 7Figure1.4Theamyloidcascadehypothesis. ................................................................................. 8Figure1.5EndosomaltraffickinginAD. ...................................................................................... 12Figure1.6Theretromerassembly. .............................................................................................. 15Figure1.7ThesearchforbiomarkersofAD-relatedendosomaldysfunction. .......................... 21Figure1.8Mousemodelsofretromer-dependentendosomaldysfunction. ............................ 24Figure2.1CollectionofhighqualityCSF. .................................................................................... 28Figure2.2ComparisonofthemouseandhumanCSFlipidome. ............................................... 30Figure2.3VPS26a+/-CSFlipidomicsstudyoverview. .................................................................. 32Figure2.4LipidomicscreenrevealsalteredsphingomyelinprofileinVPS26a+/-CSF. .............. 33Figure2.5Follow-upstudyinnewcohortconfirmsdecreaseofSMd18:1/18:1inVPS26a+/-
CSF. ........................................................................................................................................ 35Figure2.6AgeexacerbatessphingomyelinprofileinVPS26a+/-CSFandextendseffectsto
otherlipidclasses. ................................................................................................................ 37Figure2.7SphingomyelinalterationsinVPS26a-haploinsufficientmicederivefromnon-
neuronalorigins. .................................................................................................................. 39Figure3.1Isolationofhighqualityexosomesfromadultmurinebrain. .................................. 45Figure3.2Retromercargorecognitioncoreproteinsarepresentinbrainexosomefractions.47Figure3.3Murinebrainexosomesreflectretromerdeficiencyinthebrain. ........................... 48Figure3.4Retromerdeficiencyinvivopromotesb-CTFaccumulationinexosomes. ............... 51Figure3.5BrainexosomesfromVPS26bknockoutmicecontainreducedlevelsofcanonical
exosomemarkers ................................................................................................................. 52Figure3.6DescriptiveelectronmicroscopyrevealsenrichmentofsmallervesiclesinVPS26b-/-
brainexosomes. ................................................................................................................... 53Figure4.1OverviewoflabeledLC-MS/MSapproachforproteomicanalysisofVPS26a+/-CSF.
............................................................................................................................................... 59Figure4.2Qualityassessmentrevealsnormallydistributedproteinquantitationforall
samples. ................................................................................................................................ 60Figure4.3GlobalCSFprofileinVPS26a+/-micelargelyresemblesthatoflittermatecontrols.
............................................................................................................................................... 61Figure4.4PAFAH1B3isunchangedinVPS26a+/-CSF. ................................................................. 63Figure4.5OverviewoflabeledLC-MS/MSapproachforproteomicanalysisofVPS35Camk2α
knockoutCSF. ....................................................................................................................... 64Figure4.6ComparisonwithpriormurineCSFproteomicstudies. ............................................ 65Figure4.7Qualityassessmentrevealshighlevelsofcorrelationbetweenreplicates. ............. 66Figure4.8BACE1substrateNTFsareelevatedinCSFfromVPS35Camk2αknockoutmice. ... 69Figure4.9NonparametricanalysisrevealspresenceofMapt(tau)inonlyretromerknockout
CSF. ........................................................................................................................................ 70
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Figure4.10IngenuityPathwayAnalysis(IPA)implicatesAD-relatedproteinsasupstreamregulatorsinVPS35knockoutCSF. ...................................................................................... 71
Figure4.11ElevationofBACE1substrateNTFsinVPS35knockoutsisconfirmedinvivoandinvitro. ...................................................................................................................................... 72
Figure4.12TotaltauissignificantlyelevatedinVPS35Camk2αknockoutCSF. ....................... 74Figure4.13TotaltauisenrichedinmediafromVPS35-/-neuroncultures. ............................. 75Figure4.14NTFsofBACE1substratesarecorrelatedinmouseandhumanCSF. ..................... 76Figure4.15APLP1NTFsarehighlycorrelatedwithAβ42andtotaltauinhumanCSF. ............ 77Figure4.16CSFLevelsofAPLP1NTFsarelowerinADpatients. ............................................... 79
ListofTables
Table4.1ParametricHitsofVPS35Camk2aCSFProteomics. ................................................... 66
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Acknowledgments
Thescientificpursuitisasubstantialundertaking,asthepathfrombeginningtoendisrarely
straightorentirelyprescribed.Accordingly,itrequirespassion,dedication,andoftenasizeable
supportnetworktofulfillone’sinitialobjective.Iamblessedtolayclaimtosuchanextensive
andunwaveringcast,withoutwhich,thisjourneywouldnothavebeenpossible.
Ifirstwishtothankmyfellowlabmateswhohavesharedtheirknowledge,expertise,andbench
spacewithmeoverthepastfewyears.Inparticular,IwishtoacknowledgeSabrinaSimoes,
whoassistedwiththeelectronmicroscopyforthisprojectandwhohastaughtmemoreabout
scienceandlifethanIevercouldhaveasked.ImustalsothankAndreaUrban,whose
exceptionaladministrativesupportandholidaygoodieskeptallturbulenceatbay.Finally,I
wouldliketoacknowledgeMilankumarKothiyaforhisoutstandingtechnicalassistanceinthe
finalphasesofthisproject.
ToourcontributingcollaboratorsEmilyChen,EstelaAreaGomez,RobinChan,YimengXu,Larry
Honig,DavidKulick,LaurelProvencher,andPurvishPatel,Iextendmyutmostgratitude.Iwould
alsoliketoacknowledgeBeverlySheltonandXiaolanShen,whoprovidedexperttrainingin
murineCSFcollectiontechniques,thecornerstoneofthisproject.
IamindebtedtothePathologyDepartmentforgrantingmethisexceptionaltraining
opportunity,andtoZaiaSivoforherunendingadvocacy.Ialsowishtoacknowledgemythesis
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committeemembers,CarolTroy,ClarissaWaites,GilDiPaolo,fortheirexceptionaladvisement
throughoutthecourseofthisproject,andtoAndrewTeichforhiswillingnesstoserveas
additionalcounselforthedefense.
Iwishtothankmyfamilywhohasalwaysencouragedmetoshootforthestars,andsupported
mydedicationingettingthere.Andinparticular,mybrotherJason,whoseweeklyphonecalls
kepteverythinginperspective.
Myworkhasbeenfurtherblessedbythesupportofcountlessfriends.Iamparticularly
indebtedtoNoopurKhobrekarandAidanQuinnforthegiftsoftheirtimeandscientific
wisdom,andtoLyudaKovalchukeandYelenaAkhtiorskaya,towhomtheword‘friendship’
knowsnobounds.IalsowishtothankFabriceMercier,wholearnedmoreaboutretromerin
thepastthreeyearsthanheeverintended,andwhoseloveandgheegotmethroughallof
life’sgreatestchallenges.
Finally,Iwouldliketothankmythesisadvisor,ScottSmall,forhisinvaluablementorshipover
thecourseofthisproject.Scotthasalwaysprovidedboththetoolsandthecounselnecessary
tosucceed.Andimportantly,hisconfidenceinmyabilitytodosohasneverwavered.Scotthas
shownmewhatitmeanstobebotharigorousscientistandadiligentscholar,andhisunending
supportalongbothtrajectorieshasaffordedmetremendousgrowth.
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1 Chapter1:Introduction1.1 Alzheimer’sDisease
1.1.1 Epidemiology
Alzheimer’sdisease (AD) isadevastatingneurodegenerativedisease,affectingmore than5.5
millionAmericans.itiscurrentlytheleadingcauseofdementia(Hebert,Weuveetal.2013)and
thesixthleadingcauseofdeath(KochanekKD2016).ADdiagnosishingesuponaconvergenceof
pathologicalandcognitivesymptoms;yet,heterogeniccomplexitiesposechallengetodiagnostic
accuracy and therapeutic intervention. Nevertheless, AD is broadly categorized into two
subgroupsbasedonageofonset:early (EOAD)and lateonset (LOAD).The formercomprises
thosebelowtheageof65,whileLOADincludespatients65andolder.
MostADcasesappearsporadicallywithoutanyclearinheritancepattern.However,dominantly
inheritedpatternshavebeenreported,andareestimatedtorepresentbetween1%and5%of
all cases (Bird 1993). This dominantly inherited AD often presents at an earlier age and is
thereforeoftentermedearlyonsetfamilialAD(eFADorEOFAD).
Nearlytwo-thirdsofthoselivingwithADarewomen(Hebert,Weuveetal.2013).Thereasonfor
this sex-based discrepancy remains unclear, although social (e.g. inequitable educational
attainment for thecurrentADpopulation)andbiologicaldistinctions (e.g. higherassociation
withtheAPOE-4genotype)havebeenproposedascontributingfactors (Carter,Resnicketal.
2012,Altmann,Tianetal.2014).
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1.1.2 Riskfactors
ThemajorityofADsufferers(96%)are65orolder(Hebert,Weuveetal.2013).Infact,oneinten
adults over 65hasAD and theprevalence increaseswith age (up to 32%of adults over 85),
renderingagethebiggestriskfactorforthedisease(Hebert,Weuveetal.2013).
Asecondriskfactorisgeneticpredisposition,involvingtwotypesofassociations:deterministic
genes and risk genes. Deterministic genes are dominantly inherited and disease-causing.
MutationsinAPP,PSEN1,andPSEN1comprisethelargemajorityofthesecases(Bird1993);more
recently,rarebutcausalmutationsinSORL1havealsobeenreported(Pottier,Hannequinetal.
2012,Vardarajan,Zhangetal.2015,Verheijen,VandenBosscheetal.2016).
Riskgenes,ontheotherhand,increaseone’slikelihoodtodevelopthedisease,asdetermined
bygenomewideassociationstudies(GWAS).Suchgenes includeAPOE,whichiscurrentlythe
largestgeneticriskfactorforsporadicAD.HeterozygosityfortheAPOE4alleleincreasesone’s
risk for developing AD roughly 3-fold, while homozygosity increases the risk 12- to 15-fold
(Michaelson2014,WuandZhao2016).Interestingly,therareAPOE2alleleappearstolowerthis
risk(WuandZhao2016).AgrowingnumberofothergeneslinkedtoADhavebeenidentified
throughgeneticsstudiesinrecentyears(Figure1.1)(Guerreiro,Brasetal.2013,KarchandGoate
2015),thoughnoneconferashighariskfordiseasedevelopmentasAPOE4.
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Figure1.1ThegeneticsofAD.
AgrowingnumberofgeneshavebeenlinkedtoAD,includingbothdeterministicandriskgenes
(adaptedfrom:(Guerreiro,Brasetal.2013,KarchandGoate2015)).
Numerousenvironmentalriskfactorsincludingdiet,exercise,education,andlifeactivitieshave
beenproposedinthelasttwodecadesastheresultofepidemiologicalinvestigations(Luchsinger,
Tangetal.2001,Scarmeas,Zarahnetal.2003,Scarmeas,Sternetal.2006,Luchsinger2008,
Scarmeas,Luchsingeretal.2009,Duncan,Nikelskietal.2017).Thoughthemechanisticimpact
of such factors remains unclear, there is growing consensus that they might impact one’s
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‘cognitivereserve’whichisthoughttoprotectthebrainagainstAD-relatedpathology(Scarmeas,
Zarahnetal.2003,Scarmeas,Zarahnetal.2004,Stern2006).
1.1.3 PathologicalhallmarksADwasfirstdescribedover100yearsagobyGermanphysicianAloisAlzheimer.Whileexamining
the post-mortem brain of a patient with severe memory defects, he noticed abnormal
accumulationswhichhe termedplaquesand tangles (Figure1.2).Thesehallmarkshavesince
comprisedthegeneralhistopathologicalcriteriaforADdiagnosis.In1911,theamyloidnatureof
theseextracellularplaqueswasdescribedbytheGermanneuropathologistMaxBielschowsky,
butitwasn’tuntilthe1980sthatscientistsidentifiedbetaamyloid(Ab),thepeptideatthecore
ofamyloidplaques(Kirschner,Inouyeetal.1987).In1986,biochemicalstudiesuncoveredtau
(Mapt)asthepathologicalbuildingblockofneurofibrillarytangles(Grundke-Iqbal, Iqbaletal.
1986,Kosik,Joachimetal.1986).Debatelingersregardingwhichpost-translationallymodified
formscontributemosttotheseaberrantintracellularaccumulations(Grundke-Iqbal,Iqbaletal.
1986, Askanas, Engel et al. 1994, Novak 1994); however, it is largely accepted that
hyperphosyphorylationoftauisapathologiceventwhichpromotestangleformation(Alonso,
Zaidietal.2001,GongandIqbal2008,Wang,Xiaetal.2013,Hu,Zhangetal.2016).
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Figure1.2ThepathologicalhallmarksofAD.
Silver staining of an AD brain reveals extracellular amyloid plaques (a) and intracellular
neurofibrillarytangles(b)(Serrano-Pozo,Froschetal.2011).
1.1.4 Diagnosticchallenges
AmajorhurdleinthetreatmentandresearchofADoverthepastseveraldecadeshasbeenthe
absenceofreliableclinicalbiomarkers.Commonpresentingsymptomsincludememorydeficits,
personalitychanges,anddisorientation(APA2017).Yet,notallpatientspresentwiththesame
constellation of symptoms and many of these symptoms are shared among other types of
transient or progressive neuropathies, rendering behavioral criteria alone insufficient. In the
early1990s,nearly90yearsafteritsrecognitionasadisease,acerebrospinalfluid(CSF)-based
biomarker profile (centering on Ab and tau levels) for AD provided amajor step forward in
antemortemdiagnosticsanddiseasemanagement.Tonote,itwasinitiallyhypothesizedthatCSF
fromADpatientswouldcontainhigherlevelsofAb,reflectingitsincreasedaccumulationinthe
brain;surprisingly,however,studiesrevealedthatADCSFcontainedstrikingly lessAb (Fagan,
Mintun et al. 2006),whichwas retrospectively attributed to its preferential sequestration in
plaques.
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Recent technological advancements have paved the way for the development of diagnostic
biomarkersusingadditionalmodalities.Imagingbiomarkers,forexample,relyuponradiolabeled
compoundswhichcanbetargetedtowardsspecificbiologicalcomponentssuchasextracellular
amyloidandneurofibrillarytau.Acompoundnamed11C-PIBhasdemonstratedearlypromisein
thelabelingofamyloidplaques(Archer,Edisonetal.2006).Additionalplaque-targetingtracers
bearingthemorestableF18radioisotopehavealsobeendeveloped.Theseincludeflorbetapir,
florbetaben, flutemetamol, and the more recent fluselenamyl. Radioligands targeting
neurofibrillarytangles includeAV-1451(T807/flortaucipir),MK-6240,PI-2620,THK5317,PBB3,
andJNJ-067.Thegrowingarsenalofneuroimagingtoolstargetingthesetwohistopathological
hallmarksrendersimminenttheirinvivovisualization,whichcouldimprovediagnosticacuityand
histopathologicalmonitoring.
1.1.5 Theamyloidhypothesis
ThepredominanttheoryofADpathogenesis,termedtheamyloidhypothesis(oramyloidcascade
hypothesis),wasproposedin1991(HardyandAllsop1991)followingthediscoveryofdisease-
causingmutationsinAPPinearly-onsetAD(Chartier-Harlin,Crawfordetal.1991).Thistheory
positsthatincreasedaccumulationofextracellularAbpeptideaggregatesistheinitialpathogenic
eventwhichgivesrisetoalldisease-relatedneuropathology(HardyandAllsop1991).Despite
loomingspeculation,thishypothesishasremainedthedominanttheoryfornearlytwodecades,
motivatingextensivestudyoftheunderlyingbiochemistryandprovidingthefieldalong-awaited
moleculardrugtarget.
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APP,theholoproteinofAb,isatype-Imembraneproteinhighlyexpressedinthebrain,whereit
isthoughttocontributetothestructuralandfunctionalintegrityofneuronalsynapses(Priller,
Baueretal.2006,Tyan,Shihetal.2012).Itcanbecleavedbyvariousenzymesalongtwogeneral
pathways, yielding fragmentswithdistinct functional properties. These twopathways are (1)
amyloidogenicand(2)nonamyloidogenic,namedfortheircapacitytogeneratetheAbpeptide
(Figure1.3).
Figure1.3Theprocessingpathwaysofamyloidprecursorprotein(APP).
TwopathwaysdefinetheprocessingfateofAPP.Thenon-amyloidogenicpathwayisinitiatedby
enzymaticcleavageofADAM9,ADAM10,orADAM17toyieldsAPPaandthe83-aminoacidC-
terminal fragment (C83ora-CTF).Amyloidogenic cleavagebeginswith cleavagebyBACE1 to
yield sAPPb and the 99-amino acid C-terminal fragment (C99 or b-CTF). This C99 fragment
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undergoessubsequentcleavagebyag-secretasecomplex(composedofpresinilin1or2,nicastrin,
PEN2,andAPH-1)toyieldAbandtheAPPintracellulardomain(AICD)(LaFerla,Greenetal.2007).
TheamyloidogenicpathwaygivesrisetoAbviasequentialcleavageofAPPbyabeta-secretase
(BACE1)andag-secretase complex, yieldingapeptideofeither40aminoacids (Ab40)or42
aminoacids(Ab42)dependingonthesiteofgammacleavage.StudieshaveshownthatAb42is
more prone to aggregation (i.e. more pathogenic) than Ab40 and accordingly serves as the
centralmolecularculpritoftheamyloidhypothesis(Bitan,Kirkitadzeetal.2003).
Figure1.4Theamyloidcascadehypothesis.
This leading theory of ADpathogenesis asserts that the accumulation of Ab42 is the central,
initiatingeventwhichgivesrisetoallsubsequentpathology.Thoughvariousaggregateformsof
Ab42 may have differential pathologic effects, their accumulation is presumed causal of
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downstreameventsincludingpairedhelicalformation(PHF)oftau,neuronalloss,andcognitive
decline(Karran,Merckenetal.2011).
Thisleadingtheory,informedbyagrowingbodyofresearch,assertsthattheaggregationofAb42
promotes a cascade of cytotoxic effects (Figure 1.4) which include tau dysregulation,
neurofibrillarytangleformation,andultimatelyneuronaldeath.Severaldetails,however,remain
unclear.Forexample,debatehasensuedregardingwhichaggregateformofAb42(monomeric,
oligomeric, or deposited plaque) contributemost to disease pathology (Walsh, Klyubin et al.
2002,Glabe 2006), and in general, themechanism throughwhich these events promote tau
dysregulation(Zheng,Bastianettoetal.2002,ManczakandReddy2013,Bloom2014).
The amyloid hypothesis is strengthened by several key findings. First, autosomal dominant,
disease-causingmutationsinAPPandgamma-secretasepresenilinsPSEN1andPSEN2havebeen
identified (Levy-Lahad,Wasco et al. 1995, Sherrington, Rogaev et al. 1995). The pathogenic
impactofthesemutationsisattributedtoincreasedAb42generationthrougheitheranincreased
productionofAb (bothAb40andAb42)altogetheroran increase in theratioofAb42/Ab40.
Second,Down’sSyndromepatientsareatsubstantiallyhigherriskforAD,withmorethanhalf
ultimately developing the disease. This has been attributed to the extra copy of APP (on
chromosome21), and therefore substantially elevated levels of itsmetabolic productswhich
include Ab (Teller, Russo et al. 1996). Third, genome wide association studies (GWAS) have
identifiedassociationsinseveralgeneswhichhavepurportedrolesinAbclearance(suchasAPOE
andclusterin)toAD(Zlokovic,Marteletal.1996,Tanzi,Moiretal.2004).Infact,APOEstatusis
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currentlythestrongestgeneticriskfactorforsporadicAD,withtheAPOE4alleleaccountingfor
roughly half of all AD cases (Ashford 2004). Though the precise pathological contribution of
APOE4isdebated,studieshavedemonstrateditsdecreasedclearancecapacityforextracellular
AbascomparedwithAPOE2andAPOE3(Strittmatter,Weisgraberetal.1993,Bales,Verinaetal.
1999,Holtzman,Balesetal.2000,Shibata,Yamadaetal.2000,Holtzman2001,Ashford2004).
Yet, despite its rise as thepredominant theory ofADpathogenesis in the past twodecades,
several challenges to the amyloid hypothesis remain. First, there is an anatomicalmismatch
betweenextracellularplaquesandneurotoxicityinADbrains.Specifically,themedialtemporal
lobe, which undergoes the primary and most extreme neurotoxicity (Khan, Liu et al. 2014,
Altmann,Ngetal.2015)bears the leastplaqueburden in thecortex (BraakandBraak1991,
Altmann,Ngetal.2015).Second,theoveralldepositionofAbintheADbraindoesnotcorrelate
with cognitive decline; in fact, extensive amyloid plaques have been documented in non-
demented, cognitively ‘normal’ individuals (Rentz, Locascio et al. 2010). Third, other
histopathological events such as endocytic abnormalities appear to precede local amyloid
depositioninADbrains(Cataldo,Peterhoffetal.2000),raisingquestionastothepositioningof
amyloidaccumulationalongthepathologicaltimelineofAD.Andfourth,todate,agrowinglist
oftherapiestargetingextracellularamyloidhavelargelyfailedtoshowefficacyintreatingthe
disease. One must consider that the failure of Ab-targeting therapies could derive from
neurodegenerationoccurringpriortointervention.However,takentogether,theseobservations
pose challenge to the predominant theory of AD pathogenesis and motivate the quest for
additionaldrugtargets.
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1.1.6 TheendosomeasanemergingtherapeutictargetThoughnot considereda canonicalbiomarkerofAD,endosomaldysfunctionhas increasingly
beenrecognizedasasalientfeatureofitscytopathology.Cataldo,etal.describedanabnormal
enlargement (2.5-fold) of neuronal endosomes as an early event in AD pathology (Cataldo,
Barnettetal.1997)andlaterreportedthatthiscellulareventprecedeslocalizedAbdeposition
inbothsporadicADandDownSyndromepatients(Cataldo,Peterhoffetal.2000).Othergroups
havealsodescribedendosomalenlargementasafeatureofIPSC-derivedneuronsfrompatients
with both sporadic (Israel, Yuan et al. 2012) and autosomal-dominant forms of AD (Raja,
Mungenastetal.2016).
Furthermore, genetic studies have linked a growing number of genes involved in endosomal
traffickingtoAD.TheseincludeSORL1,BIN1,PICALM,ABCA7,CD2AP,SNX1,SNX3,RAB7A,and
KIAA1033(Rogaeva,Mengetal.2007,Vardarajan,Bruesegemetal.2012,Guerreiro,Brasetal.
2013,Lambert,Ibrahim-Verbaasetal.2013,KarchandGoate2015,Steinberg,Stefanssonetal.
2015).As such, endosomal traffickinghas risenasoneof themajor categoriesofAD-related
genes (Small, Simoes-Spassov et al. 2017) (Figure 1.5). And, asmentioned previously, causal
mutationsintheendosomaltraffickingreceptorSORL1havebeenidentified(Pottier,Hannequin
etal.2012,Vardarajan,Zhangetal.2015,Verheijen,VandenBosscheetal.2016),underscoring
apathogenicroleforendosomaldysfunctioninAD.
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Figure1.5EndosomaltraffickinginAD.
(a)Geneticstudieshaveidentifiedvariantsinagrowingnumberofgeneswhichconferincreased
risk of AD. These genes broadly coalesce into four functional categories including endosomal
trafficking. As studies have shown defects in the other three gene categories can lead to
endosomaltraffickingdefects,thiscategoryrepresentsacommonfinalpathwayfordownstream
neurotoxicevents.(b)EndosomaltraffickinggeneslinkedtoADperformdistinctfunctionswhich
canaffectsortingdynamicsattheendosomalhub.Endosomaltrafficjams,nowthoughttoplay
a role in AD pathogenesis, result from defects to the degradation pathway (1), the recycling
pathway(2),and/ortheretrogradepathway(3)(Small,Simoes-Spassovetal.2017).
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Finally, expression profiling studies of post-mortem brain tissue have identified a significant
reductionofendosomaltraffickingproteins(VPS35,VPS26a,andBeclin-1)andlipids(PI3P)inthe
areasof thebrainmostaffected inAD (Small,Kentetal.2005,Pickford,Masliahetal.2008,
Morel,Chamounetal.2013).Thoughexpressionprofilingofpost-mortembrainsisoftenlimited
tolatestageAD,Beclin-1reductionwasdescribedinapost-mortemstudyofpatientsintheinitial
symptomaticstageofmildcognitiveimpairment(MCI),positioningitsdeclineasanearlyevent
inADpathology(Pickford,Masliahetal.2008).
ThegeneticandbiochemicalstudieslinkingendosomaltraffickingdeficitstoADhavemotivated
mechanisticexplorationoftheresultingmolecularpathology.Asaresultofthisresearch,there
is growing consensus that endosomal dysfunction can promote Ab accumulation through a
varietyofmechanisms(Wen,Tangetal.2011,Morel,Chamounetal.2013,Nixon2017,Small,
Simoes-Spassovetal.2017);theseincludetraffickingdefectswhichincreasetheretentiontime
ofAPPand/or itsamyloidogeniccleavageenzymesintheendosome,therebypromotingtheir
interaction(Wen,Tangetal.2011,Bhalla,Vetanovetzetal.2012).Interestingly,thereversehas
also been demonstrated: that amyloidogenic APP processing can promote endosomal
dysfunctionduetocytotoxiceffectsofamyloidogenicAPPproducts(Jiang,Mullaneyetal.2010,
Xu, Weissmiller et al. 2016, Small, Simoes-Spassov et al. 2017). Accordingly, endosomal
dysfunctionhasbeenproposedasacommoncellularpathwaycapableofmediatingsubsequent
AD-relatedpathology(Small,Simoes-Spassovetal.2017)(Figure1.5)andanoveldrugtargetfor
discovery(Berman,Ringeetal.2015,Small,Simoes-Spassovetal.2017).
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1.2 Theretromerassembly
1.2.1 Retromerasamasterconductorofendosomaltrafficking
Retromerhasbeentermedamasterconductorofendosomaltrafficking,asitcoordinatesthe
transportofproteincargooutoftheendosomealongtwopathways:theretrogradepathway
towardthetrans-golginetwork(TGN)ortherecyclingpathwaytowardtheplasmamembrane
(Figure1.6a).Retromerwasfirstdescribedinyeastininthelate1990s(Seaman,Marcussonet
al. 1997, Seaman, McCaffery et al. 1998); its mammalian orthologs were identified shortly
thereafter (Edgar and Polak 2000, Haft, de la Luz Sierra et al. 2000) and have been studied
extensivelyintheyearssince.
Earlystudiesinyeastrevealedacriticalroleforretromerinendolysosomalhealth.Specifically,
Seaman et al. demonstrated that retromer traffics receptor VPS10p along the retrograde
pathway,ensuringcontinuousdeliveryofitsligandcarboxypeptidaseY(CPY)fromtheTGNto
theprevacuolarendosome (Seaman,Marcussonet al. 1997, Seaman,McCafferyet al. 1998).
They further showed that mutations in retromer caused an increased vacuolar retention of
VPS10pandanabnormalsecretionofvacuolarproteaseCPYintotheextracellularspace.Follow-
upstudiesconfirmedasimilarroleforretromerinthemammaliansystem:lysosomaldeliveryof
proteasecathepsinDviaretrogradetraffickingofitsreceptor,thecation-independentmannose-
6-phosphatereceptor(CIM6PR)(Arighi,Hartnelletal.2004).Numerousstudiesintheyearssince
have unveiled a multitude of other retromer cargo including mammalian VPS10 domain-
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containingreceptors(e.g.SORL1)andglutamatereceptors(e.g.GluR1)(SmallandGandy2006,
Zhao,Nothwehretal.2007,Lane,Rainesetal.2010).
Theterm‘retromer’refersnotsimplytooneprotein,butinsteadamulti-modularassemblyof
proteins which must organize biochemically to traffic endosomal cargo to the appropriate
destination(SmallandPetsko2015).Thesemodulesofretromerassemblyhavebeengrouped
into five categories (Figure 1.6b) which coalesce around their functional contribution: cargo
recognition,membranerecruiting,tubulation,actin-remodeling,andthecargotobetrafficked
toitsdestination(SmallandPetsko2015).
Figure1.6Theretromerassembly.
(a)Retromertrafficscargointwoofthethreeroutesoutoftheendosome:therecyclingpathway
and the retrograde pathway. (b) The five modules of retromer assembly include the cargo
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recognitioncore (which includesVPS35,VPS29,andeitherVPS26aorVPS26b), themembrane
recruitingmodule(whichincludesbothproteinsandlipidssuchasPI3P),thetubulationmodule,
actin-remodelingcomponents,andthecargoandreceptorstobetraffickedoutoftheendosome
(adaptedfrom(SmallandPetsko2015)).
Thoughindividualcomponentsofthisassemblymayvaryinacontext-dependentmanner,the
cargorecognitioncore,whichconsistsof3vacuolarproteinsortingproteins(VPS35,VPS29,and
either VPS26a or VPS26b), is an essential and unifying component of the retromer assembly
(Figure1.6b).Infact,expressionofthesecoreproteinsisoftentightlyregulatedwithinthecell,
andadeficiency inanyoneof theseproteins results in concomitant reductionof its trimeric
bindingpartners(unpublisheddata).RecentstudiessuggestthatcoreproteinparalogsVPS26a
andVPS26bformdistincttrimerswithVPS35andVPS29andthattheseassembliesmayperform
differentfunctionsinthebrain(Kerr,Bennettsetal.2005,Kim,Leeetal.2010,Bugarcic,Vetter
etal.2015).
This core trimer is recruited to theendosomalmembrane via componentsof themembrane
recruitingmodule,whichcanincludeproteinssuchassortingnexin-3(SNX3)(Harterink,Portet
al.2011)andRAB7A(Rojas,vanVlijmenetal.2008,Seaman,Harbouretal.2009,Harrison,Hung
et al. 2014). The endosomal-enriched lipid phosphatidylinositol-3-phosphate (PI3P) also
contributestomembranerecruitmentoftheretromerassemblythroughbindingofSNXsviatheir
phoxhomologydomain(Cozier,Carltonetal.2002)andisthereforeincludedinthisfunctional
category.
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Essential also for this trafficking process is the generation of a nascent tubule toward the
directionofcargotransport.Thisfunction isperformedbythemembranetubulationmodule,
comprisedofsortingnexinsSNX1,SNX2,SNX5,andSNX6(Rojas,Kametakaetal.2007,Wassmer,
Attaretal.2007).Heterodimersofthesesortingnexinsdirectlybindthecargorecognitioncore
while stabilizing membrane curvature via carboxy- terminal BIN–amphiphysin–RVS (BAR)
domains(AttarandCullen2010,MimandUnger2012).
Anactin-remodelingmodulebindstotheretromerassemblyviaVPS35whereitgivessupportto
thebuddingtubuleviathegenerationofactinpatches(Harbour,Breusegemetal.2010).The
proteins involved inthisprocess includemembersof theWASHcomplex:WASprotein family
homologue1(WASH1),FAM21,strumpellin,coiled-coildomain-containingprotein53(CCDC53)
andKIAA1033(GomezandBilladeau2009,DeriveryandGautreau2010,Harbour,Breusegemet
al.2010).
The fifth module of retromer assembly consists of the cargo and/or cargo receptors to be
transportedoutoftheendosome.Whilevariouscargoofretromercontinuetobeunearthed,
severalbearingneurologicalrelevancehavebeenidentifiedtodate.TheseincludeVPS10-domain
containingreceptors(SORL1,SORCS1,SORCS2,SORCS3,andsortilin),severalofwhichhavebeen
recognizedfortheirroleinthetransportofAPPoutoftheendosome(Small,Kentetal.2005,
Rogaeva,Mengetal.2007,Bhalla,Vetanovetzetal.2012,Fjorback,Seamanetal.2012,Lane,St
George-Hyslop et al. 2012). Asmentioned above, CIM6PR,whichdelivers cathepsinD to the
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endolysosomalsystem,hasalsobeenverifiedasaretromercargo(Arighi,Hartnelletal.2004,
Seaman2004). Finally, severalneuronal signaling (GluR1 (Temkin,Morishitaetal.2017))and
microgliaphagocytic (CD36andTREM2 (Lucin,O'Brienetal.2013)) receptorshavealsobeen
showntorelyonretromerfortransportalongtherecyclingpathway(Lucin,O'Brienetal.2013,
Choy,Parketal.2014).
1.2.2 RetromerinAD
Retromerwas first linked to AD by our lab in 2005 through amicroarray experimentwhich
revealed that the entorhinal cortex of AD brains are deficient in retromer core components
VPS35andVPS26(Small,Kentetal.2005).ThoughthereasonfordecliningretromerlevelsinAD
wasunclearatthetime,recentworksuggeststhatbothgeneticsandenvironmentcanplayarole
(Rogaeva,Mengetal.2007,Morabito,Bermanetal.2014).
Follow-upstudiesalsorevealedthatsilencingVPS35invitroincreasesAbproduction,suggesting
that this defect might be upstream of AD pathology (Small, Kent et al. 2005). A cellular
mechanismwas lateruncovereduponthe identificationofAPPreceptorSORL1asaretromer
cargo,highlightingtheamyloidogeniceffectsofextendedendosomalretentionofAPP(i.e.that
traffickingmatters)(SmallandGandy2006,Muhammad,Floresetal.2008,SmallandDuff2008,
Lane,Rainesetal.2010,Lane,StGeorge-Hyslopetal.2012).Follow-upstudieshaveconfirmed
that retromer deficiency (via knockdown of VPS35 or VPS26a) can lead to increased
amyloidogenic processing through trafficking defects in APP (Muhammad, Flores et al. 2008,
Bhalla,Vetanovetzetal.2012,Choy,Chengetal.2012).Interestingly,retromerhasalsobeen
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showntotrafficBACE1outoftheendosome(Wen,Tangetal.2011)(whereitisthoughttoimpart
themajority of its amyloidogenic effects on APP (Small and Gandy 2006)). This suggests an
additional mechanism through which retromer dysfunction enhances Ab accumulation,
increasingretentionoftheinitiatingamyloidogenicenzymeaswellasitssubstrateAPPinthe
neuronalendosome.
GeneticsstudieshavefurthersupportedapathologicalroleofretromerinAD.Todate,variants
havebeenidentifiedingeneticcomponentsofall5retromermoduleswhichconferanincreased
riskforAD(Rogaeva,Mengetal.2007,Vardarajan,Bruesegemetal.2012,Lambert, Ibrahim-
Verbaasetal.2013).Asmentionedpreviously,causalmutationsinretromerreceptorSORL1has
alsobeendiscovered(Pottier,Hannequinetal.2012,Vardarajan,Zhangetal.2015),positioning
retromerdysfunctionupstreamoftheresultingpathologyinatleastasubsetofcases.
1.2.3 RetromerdeficiencyasamodelofAD-relatedendosomaldysfunction
RetromerdeficiencyinvitrorecapitulatesseveralaspectsofAD-relatedendosomaldysfunction.
First, knockdown of retromer core proteins promotes Ab accumulation through increased
retentionofAPPandBACE1intheendosome(Wen,Tangetal.2011,Bhalla,Vetanovetzetal.
2012). Second, it results in an enlargement of endosomes (Offe, Dodson et al. 2006, Bhalla,
Vetanovetzetal.2012),purportedlythroughanaccumulationofcargoatthelimitingmembrane.
Third, retromer deficiency disrupts delivery of protease cathepsin D to the endolysosomal
system,resultinginitsincreasedextracellularsecretion(andincreasedcathepsinDsecretioninto
theCSFhasbeenreported inADbrains) (Schwagerl,Mohanetal.1995).Fourth, itcausesan
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increaseinfreecholesterol(alsoreportedinAD(Cutler,Kellyetal.2004,Xiong,Callaghanetal.
2008)),anditsredistributiontolateendocyticcompartments(Marquer,Tianetal.2016).Finally,
recentworkinhumaniPSC-derivedneuronsdemonstratesthatretromerdeficiencypromotes
hyperphosphorylationoftau,andthatthiseffectisindependentofitseffectsonAb(Young,Fong
etal.2018).
Mousemodelsofretromerdeficiency,generatedbyselectivedeletionofthecargorecognition
core,alsoexhibitenhancedaccumulationofAbinthebrain(Muhammad,Floresetal.2008,Wen,
Tangetal.2011).Furthermore,theydemonstratedefectsinlong-termpotentiation(whichmay
be reversed using gamma-secretase inhibitors (Muhammad, Flores et al. 2008)) and
hippocampal-dependentmemorytasks(Muhammad,Floresetal.2008,Wen,Tangetal.2011).
1.3 Thesearchforbiomarkersofendosomaldysfunction
1.3.1 Rationale
Endosomal dysfunction is clearly linked toADpathology, and represents a novel therapeutic
targetfordiscovery(Berman,Ringeetal.2015,Small,Simoes-Spassovetal.2017).Criticaltothis
pursuit,however, istheidentificationofabiomarkerofendosomaldysfunction,servingthree
primarypurposes:1)addressingtheepidemiologyofendosomaldysfunctioninAD;2)tracking
thediseasecourse;and3)properlystratifyingandmonitoringpatientsinupcomingclinicaltrials.
Theprojectdescribedhereinwasdesignedtoaddressthisunmetneedthroughidentificationof
aclinicallyrelevantbiomarkerofAD-relatedendosomaldysfunction.
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1.3.2 Hypothesis
ThegeneralhypothesismotivatingthisinvestigationisthatAD-relatedendosomaldysfunctionin
themurinebrainwillimpartmolecularchangesintheextracellularmilieuwhichcanbedetected
viaclinicallyusefulbiofluids.Prior invitrostudieshavedemonstratedthatendosomaldefects
canpromotebothproteinandlipidalterationsinthecellularsecretome(Rojas,vanVlijmenetal.
2008,Yuyama,Yamamotoetal.2008,Sullivan,Jayetal.2011).Toourknowledge,thisstudyis
thefirsttofullytranslatesuchlogicintoadiscovery-driveninvestigationofAD-relatedendosomal
dysfunctioninthemurinebrain.
1.3.3 Strategy
Ourstudydesignbroadlyinvolvesthegenerationofmousemodelsofendosomaldysfunction,
thecollectionandanalysisofbiologicalreservoirsmolecularbiomarkersusingunbiasedscreens,
andultimatelytheirtranslationtohumansubjects(Figure1.7).Thisstrategyisoutlinedinmore
detailinthesectionsthatfollow.
Figure1.7ThesearchforbiomarkersofAD-relatedendosomaldysfunction.
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Thisproject involves the collectionofCSFandbrainexosomes from retromer-deficientmouse
modelsforinvestigationofproteomicandlipidomicchangeswhichcanbetranslatedtohuman
subjects. Utility of such a biomarker includes uncovering the epidemiology of endosomal
dysfunctioninAD,aswellasproperpatientstratificationandmonitoringforupcomingclinical
trialsofendosome-targetingagents.
1.3.3.1 Techniques
WehaveoptedtoinvestigatemolecularchangesthatariseinboththeCSFandinpurifiedbrain
exosomes fromourmousemodelsof endosomaldysfunction.CSF is an intuitive reservoir as
retromerdysfunctionhasbeenshowntoimpactthecellularsecretome(e.g.enhancedsecretion
ofcathepsinDandAb,proteinswhicharereadilydetected inCSF) (Muhammad,Floresetal.
2008,Rojas,vanVlijmenetal.2008,Bhalla,Vetanovetzetal.2012).Itisalsonowatechnically
feasiblehaven fordiscoveryasprotocols formurineCSFcollectionhavebeendevelopedand
successfullyreportedinagrowingnumberofstudies(Barten,Cadelinaetal.2011,Schelle,Hasler
et al. 2017). Biomarkers identified in this fluid could therefore directly translate to human
subjectsasCSFcollectioninhumansisawell-establishedclinicalprocedure.
Brain-derivedexosomesrepresentthesecondreservoirfordiscoveryinthisproject.Asexosomes
derivefromthelimitingendosomalmembrane(whichisalteredinretromerdysfunction)(Bhalla,
Vetanovetz et al. 2012), it follows that these changes might be reflected in the secreted
exosomes.Infact,invitrostudieshaveconfirmedelevationofC-terminalfragmentsofretromer
cargoAPPinexosomesfollowingknockdownofcoreproteinVPS35(Sullivan,Jayetal.2011).The
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recent development of two critical techniques position brain-derived exosomes as a feasible
havenforbiomarkerdiscovery.Thefirst isaprotocol for isolatingexosomesfromthemurine
brain(Perez-Gonzalez,Gauthieretal.2012).Thesecondisaprotocolforisolatingbrain-derived
exosomesfromhumanserum(Goetzl,Boxeretal.2015,Goetzl,Mustapicetal.2016).Together
thesemakepossibletheindirecttranslationfromfindingsinmurinebrainexosomestohuman
brain-derivedserumexosomes,whichcanbeobtainedclinicallythroughvenipuncture.
Wehave chosen to investigate both protein and lipid changes as both can be altered in the
cellularsecretomeasaresultofendosomaldysfunction(Rojas,vanVlijmenetal.2008,Yuyama,
Yamamotoetal.2008,Strauss,Goebeletal.2010,Sullivan,Jayetal.2011,Bhalla,Vetanovetzet
al.2012).Thesemolecularapproacheswillbedescribedfurtherintheensuingchapters.
1.3.3.2 MouseModels
We have amassed an assortment of retromer-deficientmousemodels, distinct in either the
deletedcargorecognitioncoreproteinorintheregionofgeneticdefect(Figure1.8).TheCamk2a
knockoutmodels(VPS35andVPS26a),restrictretromerdeficiencytoforebrainneurons.Andthe
useofVPS26a inparticular restricts themoleculardefect to theVPS26a-containingassembly
(whileVPS35depletionaffectsbothVPS26a-andVPS26b-containingassemblies).Knockoutof
VPS26b—thebrain-enrichedVPS26paralog(Kerr,Bennettsetal.2005)—providesanothermodel
ofregionalretromerdeficiency(inthiscase,ofonlytheVPS26b-containingassembly).Finally,
useoftheVPS26a-haploinsufficient(knockoutisembryoniclethal)modelallowsexplorationof
retromerdeficiencyinallbraincells,asstudieshavesuggestedthatdeficiencyoftheVPS26a-
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containingretromerassemblyinADmaynotbeentirelylimitedtoneurons(Lucin,O'Brienetal.
2013).
Figure1.8Mousemodelsofretromer-dependentendosomaldysfunction.
(a)Retromer-deficientmodelshavebeengeneratedbydeletionofcargorecognitioncoreproteins
VPS35,VPS26a,orVPS26b.(b)Thesemodelsdiffernotonlyinthechoiceofcoreprotein,butalso
thelevelandselectivityofdeletion.
Finally,ourdesignincludesthetranslationofthemostcompellingCSForexosomalfindingsto
humansubjects,eitherdirectly inthecaseofCSForviaextractionofbrain-derivedexosomes
fromhumanserum(Goetzl,Boxeretal.2015).
We acknowledge that the analysis of two classes of molecules (lipids and proteins) in two
biologicalreservoirs(CSFandexosomes)forthefourretromer-deficientmodelslistedaboveis
anambitiousundertaking,requiringsubstantialtime,materials,andanimals.Assuch,wehave
chosenaself-refiningapproach,limitingourefforttothosestudiesdeemedthemostfruitfuland
eliminatingthecontinueduseof techniquesoranimalmodelswhichcarry limitedpromiseof
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biomarkerdiscovery.Thus,notalltechniqueswereperformedforallrodentmodels,andnotall
findingswerefullytranslatedtohumansubjects.
2 Chapter2:CSFLipidomics
2.1 Introduction
2.1.1 Lipidsandtheendosome
Lipids comprise a diverse class of molecules which are critical for many aspects of cellular
function including membrane formation, energy metabolism, signaling, and intracellular
trafficking.Furthermore,lipidsformtheenclosingstructurefornotonlycellularentitiesbutan
entirehostofmicrovesicles(e.g.ectosomes,exosomes)theyreleaseintotheextracellularspace.
Infact,studieshavedemonstratedthatgeneticdefectswhichimpedeendolysosomaldynamics
canaffectnotonlyinternallipidmetabolism(Chen,Gordonetal.2005),butalsothepopulation
of secreted lipids (Strauss, Goebel et al. 2010), thus providing an extracellular signal of
intracellularendosomaldysregulation.Itfollows,therefore,thatretromerdysfunctionmaylead
toalterationsinintracellularlipidlocalization,andultimatelyextracellularsecretion.
2.1.2 LipiddysregulationinAD
LipiddysregulationhasbeenobservedinvariouspathologiesincludingAD(Foley2010,DiPaolo
and Kim 2011,Morel, Chamoun et al. 2013). These observations include altered cholesterol
metabolismanddeficienciesofendosomaltraffickingsubstituentssuchasPI3P(Foley2010,Di
PaoloandKim2011,Morel,Chamounetal.2013),anessentialcomponentforretromerassembly
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(Rojas,vanVlijmenetal.2008).Assuchdysregulationcanresultfromendosomaldysfunction
(Marquer,Tianetal.2016), it is reasonable topostulate that thesetwoobservationsmaybe
linkedintheADbrain.
2.1.3 Massspectrometryoflipids
Theadventofmassspectrometryinrecentdecadeshaspavedthewayforlipidomicinvestigation
ofcells,tissues,andbiofluids,andhasfurtheredourunderstandingoftheirbiologicalrelevance.
LC-MS allows both qualitative and quantitative analyses and has shown great promise in
biomarker discovery of various biological fluids (Capodivento, Visigalli et al. 2017, Zarrouk,
Debbabietal.2017).
2.1.4 CSFlipidomicsstrategy
CSF collection inmice is not trivial. Roughly 5uL of fluid can be extracted from eachmouse
throughthecisternamagnaviaapost-mortemtechnique,thoughtheamountcanvarybasedon
amultitudeoffactorssuchasage,gender,anddiet.Specialcareisrequiredtominimizeblood
contamination as an intricate network of blood vessels overlay this tiny pocket of fluid, and
substantialpracticeiswarrantedtoattaintherequisiteprecisionandconsistencyformolecular
profiling.Accordingly,thefirststepofthisprojectinvolvedmasteryofhighqualitymurineCSF
collection,whichisreportedintheresultsthatfollow.
Uponcommencementofthisproject,onlyahandfulofmurineCSFstudieshadbeenpublished,
andnoneincludedlipidomics.Therefore,consideringtherelativelylowconcentrationoflipidsin
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humanCSF(roughly10-13ug/mL)(Seyer,Boudahetal.2016),weoptedforapoolingapproach,
intendedtomaximizetheamountoffluidfordiscoverywhilestilloperatingwithinthelimitsof
ourmousehusbandrycapacity.CSFcollectionfromthisretromermodelyieldedroughly5-10uL
permouse(versusseveralmillilitersfromhumans).Here,wechosetopoolsamplestoafinal
volume of 30uL per biological replicate in order to boost the signal/noise ratio for the less
abundant lipid species and ultimately expand the array of detectable lipids available for
biomarkerdiscovery.
2.1.5 Choiceoftheretromer-deficientmodel
We began our studies with the VPS26a-haploinsufficient mice for several reasons. First,
haploinsufficiency is likelymorereflectiveof theextentof retromerdeficiency inADpatients
thanacompleteknockoutofretromercoreproteins.Second,asretromerdeficiencyinADmay
notbelimitedtoneurons(Lucin,O'Brienetal.2013),wechosetobeginwithamodelinwhich
thegeneticdeficitissharedamongallcelltypesinthebrain.
2.2 Results
2.2.1 CollectionofhighqualityCSF
Murine CSF collection is a highly-skilled procedure which requires much practice to yield
consistentresults.InordertoperformlipidomicinvestigationsofmurineCSF,wefirstneededto
ensure that we could collect high quality samples from our rodent models. As blood
contaminationcouldhavetremendousimpactontheresultsofouranalyses(duetothedistinct
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molecular profiles of these twobiofluids),we implemented a sensitive hemoglobin ELISA for
qualityassessmentofindividualsamples.
CSFwascollectedfrom40agoutimiceusingapost-mortemprocedure(Figure2.1)andassessed
forquality.PreviouslypublishedbloodcontaminationthresholdsformurineCSFstudiesinclude
0.003%(Barten,Cadelinaetal.2011)androughly0.01%(Dislich,Wohlrabetal.2015)(reported
threshold for visible contamination). Using these two criteria we are able to isolate
contamination-free CSF in these mice in 87% of samples (at a threshold of 0.003% blood
contamination)or95%(atathresholdof0.01%bloodcontamination).Samplespassingthemost
stringentofthresholdswasusedfordownstreamanalyses.
Figure2.1CollectionofhighqualityCSF.
(a)MurineCSFwascollectedthroughthecisternamagnainapost-mortemprocedure.(b)Forty
sampleswereobtainedfromagoutimiceandassessedforqualityusingtwobloodcontamination
thresholdswhichhavebeenpreviously reported:0.003%and0.01%(Levy-Lahad,Wascoetal.
1995,Scarmeas,Sternetal.2006,Barten,Cadelinaetal.2011,Dislich,Wohlrabetal.2015).
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2.2.2 ThemurineCSFlipidomecloselyresemblesthatofhumans
Utilityofthisstrategyforpurposesofbiomarkerdiscoveryhingedgreatlyuponbothourability
todetectlipidsinasmallvolumeofCSFandonsubstantialsimilarityinthecompositionofthe
mouseandhumanCSFlipidome.Asmurinelipidomicstudieshadnotyetbeenreportedatthe
timeofthisstudy,wefeltitimperativetofirstcharacterizethemurineCSFlipidomeandcompare
itscompositiontothatofhumanCSF.
Tothisend,weperformedapilotofLC-MSonlipidsextractedfrombiologicalreplicatesof30uL
ofpooledwildtypeagoutiCSF.First, it isworthnoting thatwewereable todetect signal for
nearlyalllipidclassesthatarepresentinhumanCSFandthattheiroverallcontributiontothe
CSF lipidome is remarkably similar to that of humans (Figure 2.2a; results from independent
runs).Thelimitedvolume(30uLformiceversus300uLforhumans),however, largelyrestricts
discovery to the most highly abundant lipid species (cholesteryl esters, free cholesterol,
phosphatidylcholine, sphingomyelins, diacylglycerol, triacylglycerol, and
lysophosphatidylcholine). And often, within each lipid class, not all individual lipid species
detectableinhumanCSFaredetectableinsuchasmallvolumeofwildtypemouseCSF(Figure
2.2b).
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Figure2.2ComparisonofthemouseandhumanCSFlipidome.
(a)HighqualityCSFfromagoutiwildtypemiceunderwentlipidextractionandwasrunviaLC-MS
in parallel (using six pooled biological replicates of 30uL each); internal standards provided
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absolutequantitation.Separately,human(non-dementedcontrol)CSFsampleswererunviaLC-
MSinparallelusing300uLpersample.Lipidclassesarereportedasapercentageoftotaldetected
lipid species (mol %); FC=free cholesterol; CE=cholesteryl ester; AC= acyl carnitine ;
MG=monoacylglycerol; DG=diacylglycerol; TG=triacylglycerol; Cer=ceramide; dhCer=
dihidroceramide;SM=sphingomyelin;dhSM=dihidrosphingomyelin;GalCer=galactosylceramide;
Sulf=sulfatide;PA= phosphatidicacid;PC=phosphatidylcholine;PCEe=phosphatidylcholineether;
PS=phosphatidylserine; PI=phosphatidylinositol; PG=phosphatidylglycerol;
BMP=bismonoacylglycerophosphate; AcylPG=acyl-phosphatidylglycerol;
LPC=lysophosphatidylcholine; LPCe=lysophosphatidylcholine ether; NAPE=N-acyl
phosphatidylethanolamine;NSer=N-acylserine.(b)Lipidclassesarefurtherbrokendowninto
theindividualmoleculeswhichcomprisethatclass.Oftheeighteencholesterylestersidentified
inhumanCSF, theeightmosthighlyabundantwerealsodetected in30uLofmurineCSF (red
boxes),suggestingahighlevelofsimilarityinthemurineandhumanCSFlipidome.
2.2.3 LipidomicscreenrevealssphingomyelinchangesintheCSFofVPS26a-haploinsufficient
mice
We therefore applied this pooling approach to our investigation of CSF lipid changes in the
VPS26a+/-mice(Figure2.3).HighqualityCSFfrom3-month-oldVPS26a+/-malesandlittermate
controlswere pooled into six biological replicates. Following lipid extraction and addition of
internalstandards,LC-MSwasperformedforalltwelvereplicatesinparallel.
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Figure2.3VPS26a+/-CSFlipidomicsstudyoverview.
CSFwascollectedfromVPS26a+/-miceandcontrollittermates.Contamination-freesampleswere
pooledintobiologicalreplicatesandrunviaLC-MSfollowinglipidextractionandtheadditionof
internalstandardsforabsolutequantitation.
AglobalcomparisonofthegenerallipidclassesrevealednosignificantchangesintheCSFfrom
3-month-oldVPS26a+/-miceascomparedwiththeirlittermatecontrols(Figure2.4a).However,
acloserlookattheindividuallipidspeciessuggestedalteredsphingomyelinsecretionintothe
CSFbytheVPS26a-haploinsufficientbrain(Figure2.4b).Inparticular,foursphingomyelinspecies
(d18:1/18:1,d18:1/22:1,d18:1/24:1,andd18:1/26:0)weredecreasedinCSFfromtheVPS26a
knockdownmice.Interestingly,SMd18:1/18:1wasabletodistinguishthetwogenotypeswitha
92%diagnosticaccuracy(p<0.01)inaregressionmodel.
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Figure2.4LipidomicscreenrevealsalteredsphingomyelinprofileinVPS26a+/-CSF.
(a)VPS26ahaploinsufficiencyimpartsnosignificantchangestotheclassesofCSFlipids.Values
arenormalizedtototallipidcontentandthentothewildtypeandrepresentthemean+/-SEM
(n=6); FC=free cholesterol; CE=cholesteryl ester; AC= acyl carnitine ; MG=monoacylglycerol;
DG=diacylglycerol; TG=triacylglycerol; Cer=ceramide; dhCer= dihidroceramide;
SM=sphingomyelin; dhSM=dihidrosphingomyelin; GalCer=galactosylceramide; Sulf=sulfatide;
PA= phosphatidic acid; PC=phosphatidylcholine; PCEe=phosphatidylcholine ether;
PS=phosphatidylserine; PI=phosphatidylinositol; PG=phosphatidylglycerol;
BMP=bismonoacylglycerophosphate; AcylPG=acyl-phosphatidylglycerol;
LPC=lysophosphatidylcholine; LPCe=lysophosphatidylcholine ether; NAPE=N-acyl
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phosphatidylethanolamine; NSer=N-acyl serine. (b)Abreakdownof individual sphingomyelin
speciesrevealsdecreasesinSMd18:1/18:1,d18:1/22:1,d18:1/24:1,andd18:1/26:0.Valuesare
normalizedtototallipidcontentandthentothewildtypeandrepresentthemean+/-SEM(n=6;
*P-values<0.5)
WesoughttovalidatethesefindingsfromourinitialscreeninanewcohortofmiceusingCSF
fromindividualsamples.Here,weselectedamoretargetedapproach,assessingonlypositive-
modelipidswhichincludesthesphingomyelinclass.Thisallowedustoconcentratesignalinthe
lipid category of interest instead of splitting into three modes. We were hopeful that this
approach would compensate for the smaller volume available for this follow-up validation.
Despite our efforts, however, we were unable to detect two of the sphingomyelin species
(d18:1/26:0 and d18:1/26:1) from the first cohort of pooled CSF. Among the remaining ten,
d18:1/18:1 was once again markedly reduced in the retromer-deficient CSF, confirming its
decreaseintheVPS26a-deficientbrain,whilemostremainingsphingomyelinspeciesshoweda
trendtowarddecrease(Figure2.5).
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Figure2.5Follow-upstudyinnewcohortconfirmsdecreaseofSMd18:1/18:1inVPS26a+/-CSF.
Afollow-upanalysisofCSFfromindividual3-month-oldVPS26a+/-miceandlittermatecontrols
was conducted. (a)No changes in the positivemode lipid classeswere observed. Values are
normalizedtototallipidcontentandthentothewildtypeandrepresentthemean+/-SEM(n=6);
Cer=ceramide; SM=sphingomyelin; dhSM=dihidrosphingomyelin; GalCer=galactosylceramide;
PC=phosphatidylcholine; PCEe=phosphatidylcholine ether; LPC=lysophosphatidylcholine;
LPCe=lysophosphatidylcholineether.(b)Abreakdownofindividualsphingomyelinspeciesreveals
decreasesinSMd18:1/18:1,d18:1/22:1,d18:1/24:1,andd18:1/26:0.Valuesarenormalizedto
totallipidcontentandthentothewildtypeandrepresentthemean+/-SEM(n=6;*P-values<0.5)
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(b) Among the ten sphingomyelins detected in these samples, SM d18:1/18:1 was again
significantlyreducedinVPS26a+/-CSF.Valuesarenormalizedtothewildtypeandrepresentthe
mean+/-SEM(n=8;*P-value<0.05).
2.2.4 ReductionsinVPS26a+/-CSFsphingomyelinsaremorepronouncedwithageandextend
tootherlipidgroups
CuriousastowhateffectagemighthaveontheCSFsphingomyelinprofile inthesemice,we
repeatedthisexperimentusing individualsamples in6-month-oldmice.Again,sphingomyelin
d18:1/18:1 was reduced in the CSF, as well as d18:1/20:1, and d18:1/20:0, d18:1/22:0, and
d18:1/24:0(Figure2.6b).Generally,allsphingomyelinspeciesshowedadownwardtrendinthe
VPS26a+/-mice,andinfacttheentireclassofsphingomyelinswasreduced(Figure2.6a),atrend
whichdidnotreachsignificanceinyoungercohorts.
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Figure2.6AgeexacerbatessphingomyelinprofileinVPS26a+/-CSFandextendseffectstootherlipidclasses.
Inatargetedinvestigationofpositive-modeCSFlipids,threeentirelipidclassesaresignificantly
reduced: sphingomyelins (SM), dihydrosphingomyelins (dhSM), and monohexosylceramides
(MhCer). Values are normalized to thewildtype and represent themean +/- SEM (n=3-5; *P-
value<0.05,**P-value<0.01);dhCer=dihidroceramide;Cer=ceramide;LacCer= lactosylceramide;
GalCer=galactosylceramide; PC=phosphatidylcholine; PCEe=phosphatidylcholine ether;
LPC=lysophosphatidylcholine;LPCe=lysophosphatidylcholineether.(b)Abreakdownofindividual
sphingomyelinspeciesrevealsreductionof five lipids intheVPS26a+/-CSF:SMd18:1/18:1,SM
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d18:1/20:0, SM d18:1/20:1, SM d18:1/22:0, SM d18:1/24:0. Values are normalized to the
wildtypeandrepresentthemean+/-SEM(n=3-5;*P-value<0.05,**P-value<0.01).
Unexpectedly,alterationsinotherlipidclassesappearedinthe6-month-oldVPS26a+/-CSF;these
included reductions inbothdihydrosphingomyelins andmonohexosylceramides (Figure2.6a).
Taken together, these studies indicate progressive sphingolipid dysregulation in the VPS26a-
haploinsufficient brain, which can be detected via CSF reporters such as sphingomyelin
d18:1/18:1atthreemonths(andlikelyotherspeciesatlatertimepoints).
2.2.5 SphingomyelindysregulationintheVPS26a+/-brainisnon-neuronal
VPS26haploinsufficiencyresultsinaknockdownofthiscoreretromerproteininallcellsofthe
brain.AsVPS26aisexpressedinallbraincelltypes(Figure2.7a)(Zhang,Chenetal.2014),itis
difficult to speculate which cell type might be responsible for imparting these sphingoliid
changes.Inordertoexplorethisinvivo,wemadeuseofourVPS26aneuron-specificknockout
(via Camk2a-Cre recombinase). Since VPS26a deficiency in thismousemodel is restricted to
neurons,acomparisonofCSFlipidomicsfromthetwoVPS26amodelscanindicatewhetherthe
sphingomyelindysregulationisdrivenbyneuronalretromerdeficiencyorinsteadbyothercell
types. (We acknowledge this isn’t a perfect comparison since the VPS26a+/- model is
haploinsufficientforVPS26awhiletheVPS26aCamk2aknockoutmodelisfullydepletedofthe
geneinneurons.However,ifthesphingomyelinchangeswerecomingfromneurons,wewould
expectanevenmorepronouncedphenotypeintheCamk2aknockouts.)
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Figure 2.7 Sphingomyelin alterations in VPS26a-haploinsufficient mice derive from non-neuronalorigins.
(a) VPS26a is expressed in many cell types in the brain (Zhang, Chen et al. 2014). A global
assessment of lipidomic changes reveals no changes in sphingomyelins (SM),
dihydrosphingomyelins(dhSMs),ormonohexosylceramides(MhCer)intheVPS26aCamk2amice,
asnotedat thisage in theVPS26a-haploinsufficientmice. (b)Additionally,no changes inany
sphingomyelinsareobservedintheVPS26aCamk2aCSF.Valuesarenormalizedtothewildtype
andrepresentthemean+/-SEM(n=4-6;*P<0.05).
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Tothisend,wecollectedCSFfrom6-month-oldVPS26aCamk2aknockoutmiceandlittermate
controlsandrepeatedthelipidomicsexperiment.Acomparisonoflipidclassesrevealsadistinct
globalprofileintheCSFoftheseretromer-deficientmiceascomparedwiththeVPS26a+/-model.
Changesinsphingomyelins,dihydrosphingomyelins,andmonohexosylceramidesobservedinCSF
from6-month-oldVPS26a+/-micearenotsharedinthe6-month-oldVPS26aCamk2aknockouts
(Figure2.7b).Furthermore,abreakdownofthesphingomyelinclassrevealsnochangesinany
individual lipid species (Figure 2.7c).We therefore conclude that the sphingomyelin changes
observedinVPS26a+/-CSFaretheresultofretromerdeficiencyinanon-neuronalcelltype.
Anadditionalconclusionfromthiscomparisonisthatretromerdeficiencyindifferentcelltypes
ofthebraincanleadtodistinctCSFlipidprofiles,andthatcompleteknockdownofVPS26ain
neuronsimpartsnodetectablechangestotheCSFlipidome.
2.3 Discussion
Thestudyoflipidsinthecontextofpathologyhasgainedincreasingmomentumoverthepast
fewdecades,propelledbyadvancementsinmassspectrometrytechnology.Toourknowledge,
thisstudyrepresentsthefirstattemptinutilizingthistechnologytocharacterizethemouseCSF
lipidome,and togauge its feasibility in reportingonendosomaldysfunction in thebrain.The
overwhelmingsimilarityinthelipidprofileofmouseandhumanCSFsuggeststhistechniqueasa
fruitfulmeansofbiomarkerdiscovery.
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DespitethesmallvolumeofmouseCSFavailableformassspectrometry,172lipidspecieswere
detectedandquantifiedinourinitialscreenusingpooledreplicatesof30uLeach.This isover
50%ofthenumberdetectedin300uLofhumanCSF(318species)usingthesametechnology.It
is worth mentioning that pooling CSF from mice of the same genotype (30uL volume) was
favorable over analyzing individual samples (7uL volume) in maximizing the number of
quantifiable lipids (by 20% in the case of sphingomyelins). In the end,we deemmurine CSF
lipidomicsafeasiblemeansofbiomarkerdetectioninatleasthalfofthehumanCSFlipidome.
In this study,we conclude that VPS26a haploinsufficiency alters the sphingomyelin profile in
murine CSF and that the profile intensifies with age, extending into other lipid classes
(dihydrosphingomyelinsandmonohexosylceramides).Themostconsistentofthesechangesis
sphingomyelinisSMd18:1/18:1,whichwasdepictedinallcohortsfromeachagegroup.
It is interestingtonote,however,thatallofthesechangesseemtoderiveentirelyfromnon-
neuronalcells.Infact,completedeletionofVPS26ainneurons(viaCamk2a-Cre)resultedinno
significantalterationsintheCSF.Wewonderwhetherthismightindicateamoreprominentrole
fortheVPS26a-containingassemblyinaglialcelltype,atleastinitseffectsontheextracellular
lipidome.
Determining the source of these sphingomyelin changes in the brain would require the
generation of alternative cell type-specific knockdown models, or alternatively, in vitro
knockdownof individualcell types for lipidomicassessmentof theirsecretome.Furthermore,
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studiesofCSFsphingomyelinsinADpatientshaveprovidedconflictingresults(thoughamajority
suggestelevationofthislipidclassinAD)(Kosicek,Zetterbergetal.2012,Fonteh,Ormsethetal.
2015),renderingthedevelopmentofasphingomyelinbiomarkerapotentiallycomplicatedtask.
Asthisprojectprogressedsimultaneouslyalongmultiplefronts,weoptedtoinsteadfocusour
effortsonourparallelproteomicstudieswhichyieldedmorecompellingresults.
3 Chapter3:BrainExosomes
3.1 Introduction
3.1.1 Exosomebiology
Exosomesaresmall(30-150nm),secreted,membrane-boundproductsoftheendosomalsystem.
They can travel via bodily fluids within and between organs, carrying with them preserved
material fromtheiroriginatingcells (Costa-Silva,Aielloetal.2015,Hoshino,Costa-Silvaetal.
2015,Zhang,Zhangetal.2015),andarethoughttobefairlyresistanttocirculatingproteasesin
biologicalfluids.Assuch,theyhavebeenincreasinglystudiedinthepastdecadefortheirability
toreportondiseasestates(Goetzl,Boxeretal.2015,Abner,Jichaetal.2016,Goetzl,Mustapic
etal.2016).Morerecently,theyhaveevenbeenimplicatedinvariouspathologies,asprimers
formetastasisofprimarytumorsandvehiclesforthespreadofproteinaggregatesinAD(Perez-
Gonzalez,Gauthieretal.2012,Asai,Ikezuetal.2015,Xiao,Zhangetal.2017).
3.1.2 Exosomesasreportersofendosomaldysfunction
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Fourkeyobservationssupportthehypothesisthatretromer-mediatedendosomaldysfunctionin
themurinebrainmightalterthecontentofsecretedexosomes.First,ithasbeendemonstrated
byseveralindependentstudiesthatendolysosomaldysfunctioncanaltertheproteinand/orlipid
profileofexosomes(Yuyama,Yamamotoetal.2008,Strauss,Goebeletal.2010,Alvarez-Erviti,
Seowetal.2011).Second,retromerdysfunctionspecificallyhasbeenshowntoaltertheprotein
contentofexosomesinvitro(Sullivan,Jayetal.2011).Third,proteinswhichcomprisethecargo
recognitioncoreofretromerhavebeendetectedinexosomesofbodilyfluids(Reinhardt,Sacco
et al. 2013). And fourth, a recent study of neuronally-derived exosomes from AD patients
revealedchangesinagroupofendolysosomalproteins(Goetzl,Boxeretal.2015).
3.1.3 Brainexosomebiomarkerstrategy
Importantly,tworecentadvancementsinexosomeisolationmakefeasiblethestudyofmurine
brainexosomesforbiomarkerdiscovery.First,atechniqueforisolatingbrain-derivedexosomes
inhumanserumwasdescribed(Goetzl,Boxeretal.2015,Goetzl,Boxeretal.2015,Abner,Jicha
etal.2016),providingthefirsteveracquisitionofbrain-specificexosomesinvivo.Andsecond,
the development of a protocol for isolating adult mouse brain exosomes (Perez-Gonzalez,
Gauthieretal.2012,Asai,Ikezuetal.2015)allowstheinterrogationofrodentmodelsofdisease,
reducingthetranslationalhurdlesandboostingtheexosomeyieldoverconventionalcellculture
studies.Thus,at least in theory,biomarkers identified inmurinebrainexosomescannowbe
developedforevaluationinhumanserum-circulatingexosomesofbrainorigin.
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Asaresult,weendeavoredtocharacterizethebrainexosomesfromseveralofourretromer-
deficientmodels.InformedbyourlipidomicstudyintheVPS26a-haploinsufficientmice(where
wenotedamildCSFlipidomicphenotypeofnon-neuronalorigin),weoptedtoperformourwork
incompleteretromerknockoutmodels:theVPS35fl/flCamk2a-CreKOandtheVPS26bknockout.
3.2 Results
3.2.1 Isolationofbrain-derivedexosomesfromwildtypemice:validationoftechnique
Exosomeisolationfromrodentbrainsisnotatrivialtask(Perez-Gonzalez,Gauthieretal.2012).
Itincludesmultipleroundsofultracentrifugations,andan18-houriodixanol(optiprep)gradient
separation(Figure3.1a).Weinitiallysoughttovalidatethisprotocolusingpreviouslyestablished
biochemicalandphysicalpropertiesofexosomes(Figure3.1b-d).Inapilotofwildtypemice,we
determined that gradient fractions 4-7 are enriched in exosomalmarkers alix and flotillin-1,
contain 30-150nm vesicles of exosomal morphology, and float between 1.07 and 1.11g/mL,
similartothosereportedpreviouslyforiodixanolgradientisolations(Collino,Pomattoetal.2017,
Klingeborn,Dismukeetal.2017).Assuch,weutilizedtheseexosome-enrichedfractionsforall
downstreamanalysesdescribedherein.
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Figure3.1Isolationofhighqualityexosomesfromadultmurinebrain.
(a)Exosomeisolationfrombraintissuerequiresmultiplesteps.(b)Exosome-enrichedfractions
are positive for markers alix and flotillin-1 with limited contamination from other cellular
compartments(golgiasmeasuredbyGM130andERasmeasuredbycalnexin).(c)Thesefractions
floatatadensityof1.07-1.11g/mL,similar to thatpreviously reported for iodixanolgradients
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(Collino,Pomattoetal.2017,Klingeborn,Dismukeetal.2017).(d)Electronmicroscopyreveals
vesicles30-150nmindiameterdisplayingtheconventionalexosomemorphology.
3.2.2 Retromercoreproteinsarepresentinbrainexosomefractions
Several groups have reported the presence of retromer cargo recognition core proteins in
exosomes of various tissues or fluids (Gonzales, Pisitkun et al. 2009, Demory Beckler,
Higginbotham et al. 2013, Skogberg, Gudmundsdottir et al. 2013). Therefore, we wondered
whetherwecoulddetecttheseretromerproteinsinbrain-derivedexosomesofadultmice.Using
the technique described above,we isolated exosomes from the brains ofwildtypemice and
probedforretromercoreproteinsVPS35andVPS26a(Figure3.2a).Infact,bothoftheseproteins
were present preferentially in the exosome-enriched fractions. In a separate study, we also
detectedretromercoreproteinVPS26aonintraluminalvesicles(ILVs)ofmulti-vesicularbodies
(MVBs)inneuronalcultures(Figure3.2b);asMVBscanultimatelygiverisetoexosomesviafusion
withtheplasmamembrane,wepresentthisobservationinsupportofthenotionthatretromer
cargoproteinscanassociatewithexosomesinthebrain.
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Figure3.2Retromercargorecognitioncoreproteinsarepresentinbrainexosomefractions.
(a)CargorecognitioncoreproteinsVPS35andVPS26aaredetectedinexosomefractionsisolated
fromwildtypemousebrain.(b)RetromercoreproteinVPS26aisfoundonintraluminalvesicles
(ILVs)ofamultivesicularbody(MVB)inwildtypemouseneurons.
3.2.3 Brainexosomesderivedfromretromer-deficientmicehavereducedretromerprotein
levels
Wenextaskedwhetherthelevelsofretromerproteinsinbrain-derivedexosomeswouldreflect
thoseinthebraintissue,andassuch,provideameansofreportingonretromerdeficiencyinthe
brain.Toinvestigate,weisolatedbrainexosomesfromtworetromerknockoutmodels(VPS26b
knockoutandVPS35Camk2aknockout)andcomparedthelevelsofretromercargoproteinswith
thoseinexosomesofthecontrols(Figure3.3).
Micelackingneuronal-enrichedcoreproteinVPS26bexhibitadeficiencyofcoreproteinsVPS35
andVPS29inthebrain,whilelevelsofitsparalogVPS26aarelargelyunchanged,attributedto
theirnonreduntantinclusionindistinctretromerassemblies(Figure3.3a).Brainexosomesfrom
thesemicerevealanoticeablysimilarpatternamongretromerproteinlevels(Figure3.3a).
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Figure3.3Murinebrainexosomesreflectretromerdeficiencyinthebrain.
(a)MicebearinghomozygousdeletionofcoreproteinVPS26bhavereducedlevelsofcoreproteins
VPS35,VPS26a,andVPS29butnochangesinVPS26ainbothbraintissueandinbrain-derived
exosomes. (b)Micewith neuron-specific deletion of VPS35 (via Camk2a-Cre) showdecreased
levels of other core proteins in both the brain and in brain-derived exosomes. Values are
normalized to the wildtype and represent the mean +/- SEM (n=3-9; *P-value<0.05, **P-
value<0.01,***P-value<0.001,****P-value<0.0001;withbrainlysatecontributionfromSabrina
Simoes).
KnockoutoftheVPS35viaCamk2a-Crerecombinasecausesalossofallretromercoreproteins
inthebrain(Figure3.3b).Ofthecoreproteinsmeasuredinexosomesfromthesemice(VPS35,
VPS26b,andVPS29),wefoundasimilarpatternofdeficiency(Figure3.3b).Collectively,these
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datasuggestthatbrain-derivedexosomefractions,whichcontainretromercoreproteins,may
offerutilityasareporterofretromerlevelsinthebrain.
3.2.4 Brainexosomesderivedfromretromerknockoutmiceareenrichedinb-CTFs
Whileabiomarkerofretromerdeficiencymayproveusefulforasubsetofpatients,itispossible
thatAD-relatedretromerdysfunctioncanresultfromgeneticmutationswhichhavenoeffecton
overall retromer levels. Therefore, we sought to investigate other functional readouts of
retromerdeficiencywhichmightbereleasedinassociationwithexosomes.
APP, as described above, is a transmembrane protein which can be cleaved by several
membrane-associated proteases in various cellular compartments. The resulting C-terminal
fragments(CTFs)havebeenfoundinexosomesfromculturedcells(Sullivan,Jayetal.2011),as
well as brain-derived exosomes (Perez-Gonzalez, Gauthier et al. 2012). Retromer deficiency
increasesretentionofAPPintheendosome(Bhalla,Vetanovetzetal.2012),where it ismost
likelytobecleavedbyBACE1(SmallandGandy2006),andendosomalmembrane(bearingthe
BACE1-cleaved CTF) serves as starting material for exosomal biogenesis. Therefore, we
hypothesizedthatexosomesfromretromer-deficientbrainswouldbeenrichedintheseBACE1-
cleavedCTFs(b-CTFs).Infact,theelevationofCTFsinexosomeshaspreviouslybeendescribed
asaresultofretromerdeficiencyinHEK-293cells(Sullivan,Jayetal.2011).
To this end,we isolatedexosomes from the twomodels describedaboveand theirwildtype
littermatesandprobedthesampleswithanantibodydirectedtowardanepitopewithintheb-
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CTF region of APP (M3.2). Interestingly, brain exosomes fromboth the neuron-enriched and
neuron-selectiveknockouts(VPS26bknockoutandVPS35Camk2aknockout)revealedastriking
elevationinthelevelsofb-CTFs(Figure3.4a).Wewereunabletodetectfull-lengthAPPinthe
brainexosomesofeitherthewildtypeorknockoutmice,whichcorroboratespriorreportsthat
full-lengthAPPisscarcelyfoundinexosomesascomparedwithitscleavedC-terminalfragments
(Perez-Gonzalez,Gauthieretal.2012).AcomparisonwithhippocampallysatefromtheVPS26b
knockouts(Figure3.4b)indicatesthatthiseffectisconcentratedintheendosomalcompartments
whichgiverisetotheseexosomes.Takentogether,thesefindingsdemonstratethatincreased
amyloidogenic processing of APP in the endosome of neuronal retromer knockouts can be
detectedinbrain-derivedexosomesviaenrichmentofb-CTFs.
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Figure3.4Retromerdeficiencyinvivopromotesb-CTFaccumulationinexosomes.
(a)FullknockoutofVPS26bandneuronal-selectiveknockoutofVPS35(viaCamk2aCre)increase
theaccumulationofb-CTFsdetectedinpurifiedbrainexosomes.Valuesarefirstnormalizedto
ponceauandthenthewildtypeaverageandrepresentthemean+/-SEM(n=8-9;*P-value<0.05,
**P-value<0.01).(b)HippocampaltissuelysatesfromVPS26bknockoutmiceshowonlyamodest
trendtowardincreaseinb-CTFs(n=4;tissuelysatecontributedbySabrinaSimoes).
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3.2.5 Brain exosomes from retromer knockout mice have decreased levels of exosomal
markersalixandflotillin-1
Inthecourseofourbrainexosomestudies,wehappeneduponanentirelyunexpectedfinding:
that brain exosomes from retromer knockoutmice contain lower levels of two conventional
exosomemarkersalixandflotillin-1(Figure3.5).Thoughthistrenddidnotreachsignificancein
theVPS35Camk2aknockoutmice(perhapsduetohighvariabilitybetweensamples),itwasclear
intheVPS26bknockoutmodel,witharoughly50%reductionasmeasuredbyimmunoblot.
Figure 3.5 Brain exosomes fromVPS26b knockoutmice contain reduced levels of canonicalexosomemarkers
(a)BrainexosomesisolatedfromVPS26bknockoutmicehavemarkedlyreducedlevelsofexosome
markersalixandflotillin-1asmeasuredbywesternblot.(b)Atrendtowarddecreaseofalixand
flotillin-1,thoughnotsignificant,isalsoobservedintheVPS35Camk2amodel.Valuesarefirst
normalizedtoponceauandthenthewildtypeaverageandrepresentthemean+/-SEM(n=8;*P-
value<0.05,**P-value<0.01).
As alix and flotillin are often used to confirm exosomal identity, wewonderedwhether the
decrease intheseproteins intheexosomal fractionssuggestchanges inexosomalbiogenesis,
andpossibly theirmorphology.Accordingly,weexamined thebrainexosomes fromretromer
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knockout (VPS26b-/-) and control (VPS26b+/+)mice using electronmicroscopy. Though overall
exosomalmorphologyseemedtobefairlyconsistentbetweenthetwogroups,wenotedwhat
seemedtobeashifttowardsmaller(<50nMindiameter)exosomesintheretromerknockout
fractionsascomparedwiththecontrol(Figure3.6).Thisdescriptiveobservationwasnottested
statisticallyusingbiologicalreplicates.However,iftrue,thisfindingwouldfurthersuggestthat
retromer-dependentendosomaldysfunctioncanalter thebiogenicpathwaysofexosomes, in
additiontoregulatingtheircontent.
Figure3.6DescriptiveelectronmicroscopyrevealsenrichmentofsmallervesiclesinVPS26b-/-brainexosomes.
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A descriptive analysis (n=1) of exosome-containing fractions from VPS26-/- vs control brains
revealsaperceivedenrichmentofsmaller(<50nmvesicles,examplesindicatedwithblackarrows)
intheknockoutcondition.Statisticswerenotperformed.
3.3 Discussion
Ourstudiesconfirmthepresenceofcargorecognitioncoreproteinsintheexosome-containing
fractionsfrommurinebrainandrevealthattheirlevelsreflectthatofthecellsfromwhichthey
originate.Thus,atleastinspecificcasesofretromerdeficiency,theymaybesuitedtoreporton
overall retromer levels in the brain. We opted not to immediately pursue this finding for
biomarker development, however, given that AD-related endosomal dysfunctionmay not be
limitedtoareductionofretromerlevels.
Priorstudieshavedemonstratedthatendolysosomaldysfunctioncanalterthecompositionof
secretedexosomes(Yuyama,Yamamotoetal.2008,Strauss,Goebeletal.2010,Sullivan,Jayet
al.2011).Ourworkisamongthefirsttoshowconfirmationofthisphenomenoninvivoandthat
brain-derivedexosomesmightreportonbrain-specificendosomaldysfunction.Givenretromer’s
roleindivertingAPPfromamyloidogenicprocessingbyBACE1intheendosome(Small,Kentet
al.2005,SmallandGandy2006,Wen,Tangetal.2011,Bhalla,Vetanovetzetal.2012),itisn’t
surprisingthatretromerdeficiencypromotesaccumulationofb-CTFs,whichcanbeloadedonto
ILVsandsecretedasexosomesintotheextracellularspace(Perez-Gonzalez,Gauthieretal.2012).
Itisreasonabletopostulate,however,thatanycellulardefectresultinginincreasedAPP(orits
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cleavageenzymeBACE1)intheneuronalendosomemightpromotetheincreasedaccumulation
oftheseamyloidogenicfragmentsinbrain-derivedexosomes.
Unfortunately,wewereunabletotranslatethisfindingtohumanpatients,aswefailedtodetect
b-CTFsinserumexosomesofbrainoriginviaimmunoblot.Thisresultcouldarisefromatleast
three different possibilities. First, this lack of detection could simply be due to inadequate
sensitivityoftheavailabletechnologies;inthiscase,thedevelopmentofamoresensitiveassay
(suchasasimoa(Childs-Disney,Wuetal.2007,Wu,Milutinovicetal.2015))forthedetectionof
b-CTFscouldbeuseful.Thesecondpossibility isthatexosome-associatedb-CTFsarenotfully
retainedinexosomesthatcrosstheblood-brainbarrier.Ascleavageenzymessuchasa-secretase
(ADAM10), BACE1, and g-secretase (nicastrin) have been detected in brain-derived exosome
fractions (Perez-Gonzalez,Gauthier et al. 2012), onemust consider that CTFs from thebrain
mightbeprogressivelyprocessedthroughouttheirtransportintothebloodstream.Andthird,it
remainsentirelypossiblethatexosomeswhichhouseb-CTFssimplydonotcrosstheblood-brain
barrier. While a growing number of studies have shown delivery of exosomes from the
bloodstream into the brain (Yang, Martin et al. 2015, Khalyfa, Gozal et al. 2017), few have
describedthereversetransport(Goetzl,Boxeretal.2015,Goetzl,Mustapicetal.2016),andthe
mechanisms for exosomal traverse of the blood-brain remain largely unclear. (Whether
exosomesaredeliveredacrossthisbarrierinacontent-specificmanneriscurrentlyunknownbut
shouldbeconsideredasonepotentialexplanation for the lackofb-CTFs incirculatingserum
exosomes.) If either of the latter two possibilities are true, then continued work for the
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of transport into the bloodstream. Accordingly, collection and interrogation (e.g. via mass
spectrometry)ofneuronally-derivedexosomesalreadycirculating in thebloodstreamofmice
withneuron-specificretromerdeficiencycouldprovideanoptimalhavenfordiscovery.
Finally,wereportanunexpected finding: that retromerdeficiency invivomayalterexosome
biogenic pathways. This conclusion is supported by a decrease in exosomemarkers alix and
flotillin-1 in the exosome fractions from retromer-deficient mice and a potential (as yet
descriptive)shiftinexosomesize.RecentstudiesbyotherlabshavedemonstratedthatAlixmay
be functionally regulated by Arf6 (Ghossoub, Lembo et al. 2014), which can also regulate
retromer (Marquer,Tianetal.2016).Thoughthedirectionalityof theserelationshipsdoesn’t
clarify the observed transcriptional effects of retromer deficiency on alix in our studies, it
supports the notion that these processes of retromer-dependent endosomal trafficking and
exosomebiogenesisaretightlylinkedinthemammalianbrain.Interestingly,workbyoneofour
collaboratorsrevealedthatneuronally-derivedexosomesfromADpatientsaresmallerandare
deficientincanonicalexosomemarkers(inthiscaseTSG101andCD81)thanthosefromcontrols
(Tran2017).
4 Chapter4:CSFProteomics
4.1 Introduction
4.1.1 Endosomaldysfunctionandthesecretedproteome
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Itiswelldocumentedthatdefectsinendolysosomalfunctioncanalterthesecretedproteome
(Rojas, van Vlijmen et al. 2008, Tholen, Biniossek et al. 2011, Tholen, Biniossek et al. 2014).
Moreover, recent work has demonstrated that retromer-dependent endosomal dysfunction
resultsinanincreasedsecretionofAb(Bhalla,Vetanovetzetal.2012)andcathepsinD(Rojas,
vanVlijmenetal.2008).BothofthesemoleculesmigrateintotheCSF,andinfactaccumulatein
ADpatientsalongwithotherkeycomponentsoftheendolysosomalsystem(Schwagerl,Mohan
etal.1995,Armstrong,Mattssonetal.2014). Takentogether, theseobservations leadus to
hypothesize that retromer deficiency in the rodent brain will induce measurable proteomic
alterationsintheCSF.
4.1.2 CSFProteomicsbiomarkerstrategy
Asmentionedpreviously,CSFcollectioninrodentsisahighlytechnicalprocedurewhichyields
minimalfluid(roughly5uL).Nonetheless,severalgroupshavesuccessfullyperformedproteomics
onmurineCSF,detectingover700proteinsinaslittleas2uLoffluidperreplicate(Smith,Angel
etal.2014,Dislich,Wohlrabetal.2015).Wethereforeoptedtousesuchanunbiasedapproach
to maximize our biomarker discovery efforts in favor of hypothesis-driven immunoblotting
(which isempiricallybiased, reliesonavailabilityof reactiveantibodies,andcanrequireeven
moreCSFperreplicatetodetectsignal).
CSFisrelativelylowinproteincontent(0.2-0.8mg/mLinhumans(Rozek,Ricardo-Dukelowetal.
2007,ShoresandKnapp2007))ascomparedwithotherbiofluidssuchasserum(60-80mg/mL).
Furthermore,CSFproteinsspanawidedynamicrange,upto12ordersofmagnitudeinhumans
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(Huhmer, Biringer et al. 2006, Schutzer, Liu et al. 2010),with albumin and immunoglobulins
occupyingalargemajorityofthetotal(YuanandDesiderio2005).Therefore,asinthelipidomics
study,we selecteda strategy that involved thepoolingof individual samples in thehopesof
increasingthenumberofdetectableproteinsavailableforbiomarkerdiscovery.
Ourstudydesigninvolvedthecollectionofhighquality(bloodcontamination-free)CSFsamples
tobepooledintobiologicalreplicatesof30uLeach.ThesewererunviaLCMS/MSusingeithera
labeled or unlabeled approach, and any resulting hits from the screen were followed up in
biochemicalanalysesofnewcohorts.
WeelectedtobeginwiththeVPS26a-haploinsufficientmodelaswefeltthelevelsofretromer
knockdownwouldbemorereflectiveofthatseeninADpatientsandasthismodeldoesnotlimit
retromerdysfunctiontoneurons.We laterperformedasimilarstudyontheVPS35knockout
model,whichexhibitsfullneuronaldeletion(viaCamk2a-Cre)ofthislinchpintoretromer’score.
Resultsfromeachstudyareincludedinthissection.
4.2 Results
4.2.1 CSF from VPS26a+/- mice reveals no apparent protein alterations as measured via
LC/MS-MS
HighqualityCSFwascollectedfrom3-month-oldVPS26a+/-miceandwildtypelittermatesand
pooledinto4biologicalreplicatespergenotype.Thesesampleswereeachlabeledwithdistinct
isobaricTMTtagsandrunviaLC/MS-MSalongwithacompositereferencesample,whichwas
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used for normalization (Figure 4.1). This strategy interprets signal from each isobaric tag as
relativesamplecontribution(i.e.relativequantitation)fortheproteinsidentified.
Figure4.1OverviewoflabeledLC-MS/MSapproachforproteomicanalysisofVPS26a+/-CSF.
CSFwascollectedfrom3-month-oldVPS26+/-malesandlittermatecontrolsandassessedforblood
contaminationviahemoglobinELISA.Contamination-freesamplesweresubsequentlypooledinto
biologicalreplicatesof30uLeach(n=4pergenotype).Proteinsweredigestedandlabeledwitha
distinct isobaricTMTtag,combined,andfractionatedbypHtoreducesamplecomplexity.LC-
MS/MSwassubsequentlyperformedonthefractionatedsamplesfollowedbystatisticalanalyses.
Using this labeled approach, 1215 quantifiable proteins were positively identified in the
combinedCSF.Histogramsofthelogratios(sample/reference)forallidentifiedproteinsreveal
fairlynormaldistributionsamongthebiologicalreplicates(Figure4.2).
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Figure4.2Qualityassessmentrevealsnormallydistributedproteinquantitationforallsamples.
Foreachsample,relativequantitationforeachprotein(measuredassample/referencesignal)
wasperformedandreportedlogarithmically(logratio).Ahistogramofthese1215logratiosis
normallydistributedforeachofthefourVPS26a+/-(KD1-4)andVPS26a+/+(WT1-4)samples.
Surprisingly, however, of all 1215 proteins detected, none were significantly altered in the
VPS26a+/-CSFaftercontrollingformultipletesting.Infact,acorrelationplotofthelogratiosfor
the retromer-deficient (KD)CSFversuscontrol (WT) revealsanR-squaredof0.95 (Figure4.3)
indicatingaveryhighlevelofcorrelationofCSFproteinsbetweenthetwogenotypes.
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Figure4.3GlobalCSFprofileinVPS26a+/-micelargelyresemblesthatoflittermatecontrols.
AcomparisonofmeanlogratiosbetweentheVPS26a+/-(KD)andlittermatecontrols(WT)reveals
ahighlevelofcorrelationbetweenthetwosamples.
Wewonderedwhetherperhapsthestatistical‘penalty’formultiplecomparisonwasmaskingreal
butmodestproteinchangeswithinourdataset.Therefore,welooseneddiscoveryrestrictions
andperformedaregressionanalysistorevealthetopstatisticalcandidates(thoughnonemet
the multiple testing-corrected significance threshold). Among the handful of statistical
candidateswere severalwhich have been previously linked to the endosomal system. These
includedfolatereceptor1(FOLR1),GDNFfamilyreceptoralpha1(GFRA1),profilin(PFN1),and
platelet-activatingfactoracetylhydrolase1b3(PAFAH1B3).GFRA1isknowntobetraffickedby
SORL1,a retromer cargo,outof theendosome (Glerup, Lumeetal. 2013). FOLR1undergoes
endosomal recyclingback to thecell surface, though theprecisemechanismremainsunclear
(Wibowo,Singhetal.2013).PFN1isinvolvedinactinremodeling,oneofthefivefunctionsofthe
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retromerassembly,andinfluencesCD36recyclingwhichhasbeenshowntodependonretromer
(Romeo,Moultonetal.2007,Harbour,Breusegemetal.2010,Lucin,O'Brienetal.2013).And
PAFAH1B3playsacriticalroleinendosomaltubuleformation,anotherfunctionoftheretromer
assembly (Bechler, Doody et al. 2011). All of these proteins were elevated in the VPS26a
knockdownCSFexceptforPAFAH1B3.Asthese‘hits’wereonlyidentifiedthroughalooseningof
thestatisticalparameters,itwasimperativethatweassesstheirCSFlevelsinanewcohortof
VPS26a+/-mice.Wethusturnedtoimmunoblotinpursuitofantibodieswithreactivityinmurine
CSF.
Oftheseproteins,onlyPAFAH1B3hadacommerciallyavailableantibodywhichshowedreliable
reactivityinmurineCSF.InitialoptimizationstudiesinmouseCSFindicatedthatlargevolumesof
CSF(20uLperreplicate)wererequiredtodetectsignal.Wethereforeperformedawesternblot
usingpooledCSFfromVPS26a+/-miceandlittermatecontrolstoassessPAFAH1B3levelsinthe
CSF.Due to the largevolumerequiredper lane,only twobiological replicateswereusedper
condition.Whilewerealizethissmallsamplesizeisinsufficientformostbiochemicalassays,our
goal was the identification of a distinctive biomarker of endosomal dysfunction. Thus, we
reasoned that this follow-up validation (of pooled samples which should minimize variance
among sampleswithin each genotype) should properly informon the purporteddecrease of
PAFAH1B3intheCSFofthesemice.Resultsoftheimmunoblotrevealed,however,thatthelevels
of this protein are unchanged in the CSF of VPS26a+/-mice (Figure 4.4), rendering the initial
statisticalassessmentofthisprotein(asunchanged)correct.
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Figure4.4PAFAH1B3isunchangedinVPS26a+/-CSF.
ImmunoblotofpooledCSFrevealsnochangeinPAFAH1B3levels inretromerknockdownmice
(n=2 due to limited availability of CSF). Values are first normalized to ponceau and then the
wildtypeaverageandrepresentthemean+/-SEM.
We ultimately conclude that VPS26a haploinsufficiency results in a mild CSF proteomic
phenotype,withnodetectablechangesvisLC-MS/MSattheageofthreemonths.Perhaps,asis
thecasefortheCSFlipidome,agemightexacerbateproteomicchanges,allowingdetectionata
latertimepoint(e.g.sixmonths),orperhapsacompleteknockoutisbettersuitedforsuchhigh-
throughputbiomarkerdiscovery.
4.2.2 MassspectrometryrevealsenrichmentofBACE1substrateN-terminalfragmentsand
totaltauinCSFofVPS35Camk2aknockoutmice
4.2.2.1MassspectrometryofVPS35Camk2amiceprovidesdeepestcharacterizationofmurine
CSFproteometodate
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Informedby theabove-mentionedwork inourVPS26a+/-model,weopted to repeat theCSF
proteomicscreenusinganeuron-specificretromerknockoutmodelatsixmonthsofagehoping
thatthesetwoadjustmentswouldhelpexacerbateaproteomicphenotypewemightbeableto
detect.
Inthisinstance,weemployedasimilarpoolingstrategy,butowingtothelowlevelsofproteinin
CSF collected from thismodel (0.10-0.16µg/mLas comparedwith0.68-0.8µg/mL in theprior
model),weoptedforanunlabeledapproach(Figure4.5),whichrequiressequentialrunsofeach
biologicalreplicate.
Figure4.5OverviewoflabeledLC-MS/MSapproachforproteomicanalysisofVPS35Camk2αknockoutCSF.
CSFwascollectedfrom6-month-oldVPS35knockoutmalesandlittermatecontrolsandassessed
forbloodcontaminationviahemoglobinELISA.Contamination-freesamplesweresubsequently
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pooledintobiologicalreplicatesof30uLeach(n=4-5pergenotype).Proteinsweredigestedand
labeledwithadistinct isobaricTMT tag, combined,and fractionatedbypH to reduce sample
complexity. LC-MS/MSwas subsequently performed on the fractionated samples followed by
statisticalanalyses.
Usingthisstrategy,1505proteinswerepositivelyidentifiedintheCSFby1uniquepeptide(1048
proteinsby2uniquepeptides).Todate,priorproteomicsstudiesofmurineCSF(usingasimilar
unlabeledapproachonsmallervolumes)havedetectedasmanyas817proteinsbyoneunique
peptideand715withatleasttwouniquepeptidesequences(Smith,Angeletal.2014,Dislich,
Wohlrabetal.2015)(Figure4.6),renderingthisyetthedeepestcharacterizationtodateofthe
murineCSFproteome.
Figure4.6ComparisonwithpriormurineCSFproteomicstudies.
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Usingapooled,unlabeledapproachintheVPS35Camk2aknockoutmodel,weidentifiednearly
doubletheproteinsreportedpreviouslywithoneuniquepeptide,androughlyonethirdmorethan
previouslyreportedwithtwouniquepeptides(Smith,Angeletal.2014,Dislich,Wohlrabetal.
2015).
Technicalreplicateswerehighlycorrelated(Figure4.7a)aswerethemeanscoresforbiological
replicates of the same genotype (Figure 4.7b), indicating robustness of technique. Quality
assessmentsperformedbyourProteomicCoreidentifiedoneoutlierfromeachgenotypewhich
wasremovedpriortodownstreamanalyses.
Figure4.7Qualityassessmentrevealshighlevelsofcorrelationbetweenreplicates.
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(a) A comparison of label-free quantification (LFQ) intensities for each protein reveal a high
correlationbetweentechnicalreplicates.(b)AcomparisonofLFQintensitiesforeachproteinin
biological replicates (represented as the mean of technical duplicates) demonstrates high
correlationbetweenbiologicalreplicates.
InourquestforaCSFbiomarkerofretromerdysfunction,wedecidedtoemploytwoanalytical
strategies: 1) a standard parametric analysis of proteins detected in samples from both
genotypes,and2)anonparametricanalysisinwhichweaskedwhichproteinsarepresentinone
condition but not the other (as such a categorical distinction might provide even greater
biomarkerpotential).
4.2.2.2Parametric analysis reveals changes in BACE1 substrate N-terminal fragments and
apolipoproteins
Ofthe1505proteinsidentifiedinthisstudy,52weresignificantlyalteredintheVPS35knockout
CSFasdeterminedbystandardANOVA(Table4.1).Withinthislist,aclassofelevatedproteins
wereofparticularinterest:BACE1substrates(CHL1,APLP1,andAPLP2,andAPP)(Figure4.8a).
PeptidesfromtheseBACE1substratesmappedontotheN-terminalfragments(NTFs),suggesting
mechanisticconvergenceuponBACE1cleavage(Figure4.8b),whichliberatestheNTFintothe
endosomallumen(andultimatelytheextracellularspace).
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Table4.1ParametricHitsofVPS35Camk2aCSFProteomics.
Accession Symbol p-value q-value Direction P40142 Tkt 6.26E-08 0.0001 ↓ Q06335 Aplp2 2.83E-07 0.0002 ↑ P17182 Eno1 9.96E-07 0.0003 ↓ P17563 Selenbp1 1.12E-06 0.0003 ↓ P70296 Pebp1 1.14E-06 0.0003 ↓ Q06890 Clu 1.11E-06 0.0003 ↑ G3UYU6 Ptprd 2.47E-06 0.0005 ↑ O70362 Gpld1 3.30E-06 0.0005 ↑ P01029 C4b 2.79E-06 0.0005 ↑ P06909 Cfh 4.44E-06 0.0005 ↑ P08905 Lyz2 4.10E-06 0.0005 ↑ P09411 Pgk1 3.31E-06 0.0005 ↓ P70232 Chl1 2.69E-06 0.0005 ↑ P45376 Akr1b1 5.86E-06 0.0006 ↓ P63054 Pcp4 7.80E-06 0.0008 ↑ Q8VCM7 Fgg 7.99E-06 0.0008 ↓ A0A0R4J0X7 Gapdhs 1.05E-05 0.0009 ↓ H7BX99 F2 1.16E-05 0.0009 ↑ P08226 Apoe 1.31E-05 0.0009 ↑ P32848 Pvalb 1.00E-05 0.0009 ↓ Q9D0F9 Pgm1 1.32E-05 0.0009 ↓ P55065 Pltp 1.48E-05 0.001 ↑ A0A087WR50 Fn1 1.84E-05 0.0011 ↑ P08249 Mdh2 1.78E-05 0.0011 ↓ P97290 Serping1 1.63E-05 0.0011 ↑ Q61361 Bcan 1.74E-05 0.0011 ↑ P01887 B2m 2.25E-05 0.0012 ↑ P13020 Gsn 2.10E-05 0.0012 ↑ Q9CPU0 Glo1 2.35E-05 0.0012 ↓ P15626 Gstm2 3.20E-05 0.0016 ↓ P17183 Eno2 3.34E-05 0.0016 ↓ Q62000 Ogn 3.19E-05 0.0016 ↑ Q640N1 Aebp1 4.04E-05 0.0018 ↑ A0A075B664 Iglv2 4.42E-05 0.0019 ↓ Q03157 Aplp1 4.36E-05 0.0019 ↑ Q8R480 Nup85 5.36E-05 0.0022 ↓ P50247 Ahcy 6.07E-05 0.0025 ↓ E9QPX1 Col18a1 6.45E-05 0.0026 ↑ P21550 Eno3 7.23E-05 0.0028 ↓ A0A0R4J107 Apeh 7.85E-05 0.003 ↓ P51910 Apod 8.21E-05 0.003 ↑ P00920 Ca2 8.89E-05 0.0032 ↓ P02802 Mt1 9.13E-05 0.0032 ↓ P29699 Ahsg 9.68E-05 0.0033 ↑ Q9DCD0 Pgd 9.75E-05 0.0033 ↓ P12023 App 1.09E-04 0.0036 ↑ Q04447 Ckb 1.16E-04 0.0037 ↓ Q5NC80 Nme1 1.24E-04 0.0039 ↓ P02089 Hbb-b2 1.31E-04 0.004 ↓ P12960 Cntn1 1.32E-04 0.004 ↑ P14152 Mdh1 1.35E-04 0.004 ↓ Q62165 Dag1 1.39E-04 0.004 ↑
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Figure4.8BACE1substrateNTFsareelevatedinCSFfromVPS35Camk2αknockoutmice.
(a)Mass spectrometryofVPS35Camk2a knockoutCSF reveals significant increases inBACE1
substratesAPLP1,APLP2,CHL1,andAPP.(b)PeptidesmaptotheN-terminalfragment(NTF)for
eachBACE1substratedetected.ValuesarereportedasLFQintensityandrepresentthemeanof
technicalduplicates.Barsrepresentmean+/-SD(n=3-4;*P-value<0.05**P<0.01,***P<0.001).
4.2.2.3NonparametricAnalysisUncoversChangesinCSFTotalTau
We then performed a nonparametric analysis, looking for proteins which present in one
conditionbutnottheother,assuchproteins(whichwouldbeoverlookedusingstandardANOVA
analyses) could carry high biomarker potential. We first employed this strategy looking for
proteins present in at least four technical replicates (of eight total) only the VPS35 Camk2a
knockoutCSFbutcompletelyabsentincontrols.Thisqueryresultedinalistof11proteins(Figure
4.9)spanningvariousfunctionalcategories.Interestingly,amongthesecategoricalhitswasMapt
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(tau),aproteinwhichhasclearpathologictiestoAD(Grundke-Iqbal,Iqbaletal.1986,Bejanin,
Schonhautetal.2017)andisreliablyelevatedintheCSFofADpatients(Andreasen,Minthonet
al.2001).Wethenusedasimilarstrategytoidentifyproteinspresentinatleastfourtechnical
replicates of the control CSF but absent from all knockout samples. This yielded a list of 8
additionalnonparametrichits(Figure4.9).
Figure4.9NonparametricanalysisrevealspresenceofMapt(tau)inonlyretromerknockoutCSF.
InanonparametricanalysisoftheVPS35Camk2aCSFmassspectrometrydata,eightproteins
were detected in at least four technical replicates of the wildtype CSF and absent from all
knockoutreplicatesand11proteins inat least fourtechnical replicatesofcontrolsbutabsent
fromallwildtypereplicates.Notably,mapt(tau)ispresentinonlytheretromerknockoutCSF.
4.2.2.4Ingenuity Pathway Analysis (IPA) indicates APP, MAPT, and PSEN1 among the top
upstreamregulatorsofCSFchangesinVPS35neuronalknockoutmice
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To further clarify the wealth of information gained from this CSF study (which includes 52
parametric hits and 19 nonparametric hits), we turned to ingenuity pathway analysis (IPA)
(Kramer, Green et al. 2014) to determine which genes might be ‘regulating’ the global CSF
changes in our VPS35 knockoutmodel. This software relies upon established transcriptional
interactions to predict upstreammolecular convergence of global changes in large datasets
(Kramer,Greenetal.2014).Ofthetopsixpotentialregulators,threeofthem(APP,MAPT,and
PSEN1)arepathologicallylinkedtoAD,andthetoptwo(APPandMAPT)areaberrantlyprocessed
proteinswhichcomprisethecurrentCSFdiagnosticprofile(Figure4.10).
Figure 4.10 Ingenuity Pathway Analysis (IPA) implicates AD-related proteins as upstreamregulatorsinVPS35knockoutCSF.
IPArevealsAPP,Mapt,andPSEN1amongthetopsixupstream‘regulators’ofthepotential843
identifiedinthisset.
4.2.3 BACE1SubstrateNTFsandTotalTauareconfirmedashits inanewcohortofVPS35
Camk2aknockoutmice
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4.2.3.1BACE1substrateNTFsareconfirmedviaimmunoblotinvivoandinvitro
IntriguedbytheBACE1substratehits,wesoughttoconfirmtheirelevationinvivoinanewcohort
ofmiceusingimmunoblottechnique.Ofthislist(CHL1,APLP1,APLP2,andAPP),weidentified
availableantibodiesforbothCHL1andAPLP1(directedattheN-terminalregion)thatthatwere
reactiveinmouseCSF.BothoftheseproteinswereagainelevatedintheCSFofthenewcohort,
confirming the initialmass spectrometry results (Figure4.11a).Molecularweight comparison
withcorticallysates(viaimmunoblotmigration)suggeststhattheseareindeedtheNTFs,notthe
full-lengthprotein(datanotshown).Invitroworksupportedthesefindings,aslevelsofCHL1and
APLP1NTFswere increased in themedia fromVPS35fl/fl neurons infectedwith Cre.GFP virus
versusthatofthecontrol(Figure4.11b).
Figure4.11ElevationofBACE1substrateNTFsinVPS35knockoutsisconfirmedinvivoandinvitro.
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(a)CSF fromanewcohortofVPS35Camk2a knockoutmiceand littermatecontrols revealsa
significantincreaseintheNTFsofBACE1substratesAPLP1andCHL1,confirmingthesehitsfrom
the initial proteomic screen. Bars represent mean +/- SD (n=6-8; *P<0.05). (b) Media from
culturedprimaryVPS35fl/flneuronsinfectedwithCre.GFP(VPS35+/+)orD-Cre.GFP(VPS35-/-)is
enrichedinNTFsfromBACE1substratesAPLP1andCHL1.Barsrepresentmean+/-SD(n=6-7from
2independentexperiments;*P<0.05).
4.2.3.2Single molecule array (simoa) confirms elevation of total tau in VPS35 Camk2a
knockoutCSF
DetectionoftauinmouseCSFhasbeenachallengingtask,aslevelsinCSFareroughly50,000-
foldlowerthanthatofbraintissue(Barten,Cadelinaetal.2011).Asaresult,toourknowledge,
noonehasmanagedtodetectthisproteininmurineCSFusingconventionalimmunoblotorELISA
inwildtypemice.Infact,effortsonourparttodetecttauinmurineusingbothimmunoblotand
MSDELISAwereunsuccessful.Therefore,weturnedtosinglemoleculearray(simoa),arecently
developedtechnologywhichboastsdetectiondowntothelevelofasinglemolecule.Usingthis
approach,wewereabletoreliablydetecttotaltauinaslittleas5uLofmurineCSF.Furthermore,
a comparison of VPS35 Camk2a-Cre knockoutmice and their littermate controls revealed a
strikingincreaseintheknockoutCSF(Figure4.12),confirmingthisnonparametrichitfromour
initialscreen.
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Figure4.12TotaltauissignificantlyelevatedinVPS35Camk2αknockoutCSF.
In a new cohort ofVPS35Camk2a knockoutmice, CSF total tau ismarkedly increased in the
knockoutascomparedwithcontrol.Dotsrepresentrawvalues.Barsrepresentmean+/-SD(n=5-
9;*P<0.05,**P<0.01,***P<0.001).
WefurthervalidatedthisfindinginvitrothroughknockoutofVPS35(vialentiviraldeliveryofCre-
recombinase in VPS35fl/fl primary neurons). Here, a comparison with controls revealed an
elevation in secreted total tau from the VPS35 knockouts while levels of secreted lactate
dehydrogenase(LDH)wasunchanged(Figure4.13),indicatingthattheincreaseinextracellular
tauresultsfromanactivemechanismindependentofcelldeath.
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Figure4.13TotaltauisenrichedinmediafromVPS35-/-neuroncultures.
VPS35fl/flprimaryneuronsweretreatedwithCre.GFP(VPS35-/-)orD-Cre.GFP(VPS35+/+).(a)Total
tauandLDHweremeasuredinthemediausingMSDtechnologyandpromegaCyto-toxassay,
respectively. Values were normalized to total protein and then to the control. Bar graphs
representmean +/- SEM (n=7, *P<0.05; **P<0.01; ***P<0.001). (b) Immunoblot of neuronal
culturelysatedemonstrateslentiviralknockdownofVPS35.Valuesarenormalizedtotheloading
controlandtothewildtypemean.Bargraphsrepresentmean+/-SEM(n=5-6,*P<0.05;**P<0.01;
***P<0.001;****P<0.0001).
4.2.4 TranslationtohumanspositionsCSFtotaltauasabiomarkerofendosomaldysfunction
inAD
4.2.4.1BACE1SubstrateNTFsarehighlycorrelatedinmouseandhumanCSF
During the courseofourmurineCSF validation studies,we stumbleduponan intriguingand
unanticipatedfinding:thatBACE1substrateNTFsAPLP1andCHL1arehighlycorrelatedinmouse
CSF(Figure4.14a).WethereforeturnedtohumanCSF(non-dementedcontrols)toseewhether
thisrelationshipheld(Figure4.14b).Usingasimilarimmunoblotapproach,wediscoveredthat
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APLP1andCHL1NTFsarealsohighlycorrelatedintheCSFofhumansubjects.AstheseNTFsare
liberatedbythesamecleavageenzyme(BACE1),wereasonthissuggeststhatthelevelsofAPLP1
andCHL1NTFsintheCSFdependprimarilyontheactivityand/orlocalizationofBACE1.
Figure4.14NTFsofBACE1substratesarecorrelatedinmouseandhumanCSF.
CSF from mice (n=27) (a) and healthy control (n=39) CSF (b) reveals a striking correlation
(p<0.0001 for mouse and human) between the NTFs of APLP1 and CHL1 as measured by
immunoblot.Valuesarenormalizedtoalbuminandthegroupaveragebeforecombiningcohorts
fromdifferentblots.
4.2.4.2APLP1iscorrelatedtoAb42andtotaltauinhumanCSF
Next,wewonderedwhetherthelevelsoftheseBACE1substrateNTFswerecorrelatedwithother
AD-relatedproteins(Ab42,totaltau,andptau-181)intheCSF.Weexploredtheserelationships
inhumanCSFassimoaassayswithmousereactivityhadnotyetbeendevelopedforAb42and
ptau-181.Remarkably,analysisof38non-dementedcontrolsrevealedthatCSFlevelsofAPLP1
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NTFsaresignificantlycorrelatedwithAb42,totaltauandptau-181(Figure4.15;Ab42:p<0.01;
totaltau:p<0.0001;ptau-181:p<0.05;n=38).
ThecorrelationwithAb42isperhapsnotentirelysurprisingasbothAb42andAPLP1NTFsare
bothdownstreamproductsofBACE1cleavage.Yet,thecorrelationsbetweenAPLP1NTFsand
bothtotaltauandptau-181areunexplainedbycurrentknowledge,althoughrecentinvitrowork
has shownthat retromerdeficiencypromotes tauphosphorylation (Young,Fongetal.2018).
TheseassociationsintheCSFsuggestthatthepathwayscontrollingsecretionofBACE1-cleaved
substratesandtaumaybelinkedintheplaque-freemammalianbrain.
Figure4.15APLP1NTFsarehighlycorrelatedwithAβ42andtotaltauinhumanCSF.
Astudyof38non-dementedhealthycontrolsrevealedsignificantcorrelationsbetweenCSFN-
APLP1andAb42(p<0.01),totaltau(p<0.0001),andptau-181(p<0.05).
4.2.4.3APLP1NTFslikelybindtoamyloidplaques
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ThepathologicprogressionofADgenerallyinvolvesthedevelopmentofextensiveextracellular
plaques,whichhavebeenknowntosopupsecretedproteins,ultimatelyaffectingtheirdetection
intheCSF(Fagan,Mintunetal.2006).Todate,thepropertieswhichrenderproteinslikelyto
sticktoplaquesarenotfullyunderstood,thoughsomehaveattemptedtoaddclaritytothisissue
(Gozal,Chengetal.2006).Theultimateaimofourprojectisthetranslationoffindingstohuman
subjects in order to identify all individualswith endosomal dysfunction (both pre- and post-
plaque development). As such, it is imperative that levels of our biomarker of endosomal
dysfunctionnotbeconfoundedbythepresenceofplaques.
AqueryoftheliteratureturneduponereportthatinfactAPP-relatedproteinssuchasAPLP1
andAPLP2canaccumulateinneuriticplaquesincontrolandADbrains(McNamara,Ruffetal.
1998).WeturnedagaintohumanCSFtoseewhetherAPLP1NTFsmightbestickingtoplaques
inhuman subjects.WeobtainedCSF from5patientswithMCIwhowerebiomarker-positive
(Ab42>210 and Tau/Ab42<0.39) for AD (i.e. ‘plaque-positive’). Using this limited set, we
performedwasastandardt-testofCSFAPLP1inplaque-positiveADpatientsvsage-andgender-
matchedcontrols.TheunderlyingassumptionforthisquerywasthatifAPLP1isnotstickingto
plaques, the levelswouldbeeitherunchangedorelevated inplaque-positivepatients versus
controls.Whatwefoundinsteadwasamarkeddecreaseintheplaque-positivecohort(Figure
4.16), suggesting that like Ab42, APLP1 NTFsmight be binding plaques in the AD brain.We
followedupwithacorrelationanalysisofAPLP1andAb42inCSFfromthe5ADpatientswiththe
assumptionthatifAPLP1isnotaccumulatinginplaquesasisAb42,thiscorrelationshouldbreak.
Despitethe lowstatisticalpowerofsuchasmalldataset, thecorrelationbetweenAPLP1and
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Ab42 remained quite strong (p=.003) in AD brains (data not shown). Taken together, these
observationssupportpriorfindingsandsuggestthatCSFlevelsofotherBACE1cleavageproducts
suchasAPLP1NTFsmaybeconfoundedbyplaques intheADbrain,thuscompromisingtheir
potentialforbiomarkerutility.
Figure4.16CSFLevelsofAPLP1NTFsarelowerinADpatients.
N-APLP1islowerintheCSFfromplaque-positiveADpatientsthaninage-andgender-matched
controls(n=5,22;*P<0.05).
4.3 Discussion
CSFcollectioninmiceisahighlytechnicalprocedure,oftenyieldingnomorethan5uLoffluidper
mouse. Nonetheless, other groups have successfully characterized this fluid via LC-MS/MS
(Smith,Angeletal.2014,Dislich,Wohlrabetal.2015),identifyingover700proteinsinaslittleas
2uL. As such, we sought out to uncover CSF protein biomarkers in two different models of
retromerdeficiencyusingasimilarstrategy.
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4.3.1 VPS26a+/-CSFProteomics
ThatwefoundnoproteinchangesintheVPS26a-haploinsufficientCSFwassurprising,particularly
given the number of positively identified proteins (1215 in pooled replicates) available for
discovery. There could be several explanations for this. First, it is entirely possible that a
heterozygousmodelisinsufficienttogenerateaclearprotein-basedprofileintheCSF(oratleast
onestrongenoughtoovercomethemultiplecomparison‘penalties’insuchalargedataset).As
welearnedthroughourlipidomicsstudiesofthismodel,perhapsusingalatertimepointinthese
mice (e.g. six months instead of three—the age used for our VPS35 Camk2a model) would
increasethelikelihoodofdiscoverythroughanexacerbationofcellularpathology.
A second explanation for is that the VPS26a-containing assembly is less critical to trafficking
eventswhichalter the cellular secretome.Yet,weknow from in vitro studies thatdefects in
VPS26aincreasethesecretionofproteinssuchascathepsinD(Rojas,vanVlijmenetal.2008);
thus,weargueagainstsuchanexplanation.
4.3.2 VPS35Camk2aCSFProteomics
OurunbiasedCSFscreenofneuronal-selectiveVps35knockoutmiceyieldedacomplementof
proteins that most reliably accumulate in the CSF in a model of AD-associated endosomal
dysfunction.Aftervalidatingthemostrelevantproteins,weturnedtohumanCSFtoinvestigate
whethertheproposedrelationshipbetweentheseproteinsoccursinthehumanbrain.Themain
advantage of starting with a mouse model, besides greater experimental control, is that
endosomaldysfunctioncanberestrictedtocorticalneurons,obviatinganyconfusionofcelltype
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specificity.Althoughthereisasignificantoverlapinendosomalmachinerybetweenmiceand
humans, cross-species differencesmight exist. Thus,while our positive findings are deemed
reliable,wecannotexcludetheimportanceofproteinsthatdidnotemergefromourprimary
mousescreen.
OurVPS35Camk2aknockoutCSFstudydemonstratesthedeepestprobeintothemurineCSF
proteome to date (by roughly 100% using a threshold of 1 unique peptide and 50%with a
threshold of 2 unique peptides)with an unlabeled approach. This featmay be attributed to
technicaldifferences(e.g.equipmentandhandling),butitislikelyatleastinpartaresultofthe
poolingstrategywedesigned,allowingalargervolumepersampleavailableforproteomicstudy
thanthoseusedinpriorstudies.
4.3.2.1BACE1SubstrateNTFsinMiceandHumans
ThefactthatAPPfragmentswereamongthehitsfromthisscreenprovidesadditionalunbiased
evidencelinkingretromertoAD.Morebroadly,anumberofpreviousstudies,includingaCSF
proteomic screen inBACE1KOmice,haveestablished thatAPP,APLP2,APLP1,andCHL1are
substrates cleavedbyBACE1 in theneuronal endosome, generating and secretingn-terminal
fragments of each. Our screen identified fragments of all of these proteins, fromwhichwe
concludethatretromer-dependentendosomaldysfunctionpromotestheiracceleratedcleavage
and secretion. All four of these proteins, including BACE1 itself, are type I transmembrane
proteins that traffic through theendosome. Retromer typically functions in trafficking these
typesoftransmembraneproteins,andindeed,thusfar,bothAPPandBACE1havebeenfoundto
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betraffickedbyretromer,withretromerdeficiencyincreasingtheirresidenttimeinendosomal
membranes.Thus,theobservedaccumulationofthefourfragmentsmayreflectacombination
ofmistraffickedsubstrateandtheircleavingenzyme.WenotethatwedidnotdetectAβinthe
CSFofWTorKOmice,eventhoughretromerdeficiencydoescauseanincreaseinAβinmouse
brains,andinthemediaofculturedneurons.Weconsideritlikely,therefore,thatendogenous
murine Aβ is below detection of the methods used. Our human studies support this
interpretation, suggesting that in plaque-free individuals a marker of retromer-dependent
endosomaldysfunctionispositivelyassociatedwithCSFAβ.
Unfortunately, our studies corroborate other reports that BACE1 substrate NTFs may bind
plaques inthebrain (Bayer,Cappaietal.1999), thuscompromisingtheirbiomarkerpotential
overthecourseofAD.Itispossible,however,thattheycouldprovideutilityinearly(pre-plaque)
ADpatientsorinplaque-freeneurodegenerativedisorderssuchasParkinson’sdisease(PD).
4.3.2.2TotalTauasaBiomarkerofAD-RelatedEndosomalDysfunction
Although tau is known to be secreted from the endolysosomal pathway, we consider the
observationthatretromer-dependentendosomaldysfunctioncausesanincreaseinCSFtauthe
more importantandnovelresult fromourstudies. Priorpatientstudiesprovidedsomeclues
aboutthisrelationship.CSFstudiesinADpatientscarryingdeleteriousgeneticvariantsencoding
theretromerreceptorSorl1haveshownthatthesevariantsareassociatedwithrelativeincreases
inCSFtau.However,nostudies,toourknowledge,haveprofiledtheCSFinSorl1carrierswho
are plaque free. Since amyloid plaques are avid binders of APP fragments andmany other
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extracellular proteins, itwould be interesting to probe the CSF of carriers of Sorl1 causative
mutations, inpre-plaquestagesofdisease. Neimann-PicktypeC1(NPC1) isadisorderthat is
interesting in this regard, since it is adisorderwhoseabnormal cholesterol trafficking causes
primaryendosomaldysfunction,withoutcausingplaqueformation.Interestingly,thesepatients
havebeenfoundtohaveCSFelevationsinbothtauandAb. WhileNPC1patientsdodevelop
neurofibrillarytanglesitisunlikelythattheirintraneuronaltaupathologyaccountsfortheCSF
taufindings,sinceFTDpatientswithaggressivetaupathologydonottypicallyhaveincreasesin
CSFtau.
UnlikefragmentsofAPPandperhapsitshomologues,taudoesnotbindamyloidplaques,which
commonlyexist inevennormalaging individuals. Thus,amongtheproteinswe investigated,
elevation in CSF tau has the best potential of being a useful biomarker of AD-associated
endosomal dysfunction. Our studies show that CSF tau can act as a sensitive marker of
endosomaldysfunction,butsayslittleaboutitsspecificity.AsabiomarkerofAD,chronicCSF
elevationintotaltauandptaudoesnotonlyhavehighsensitivity,butwhenobservedchronically
itappearstobespecific.Otherneurodegenerativediseases,withjustasmuchneuronaldeath
orNFTburdenasAD,donottypicallymanifestelevationsinCSFtau(Zetterberg2017),andso
AD’suniquepathophysiologymustdrivethiselevationandnotcelldeathortaupathology.While
acute events, such as strokes or seizures, can cause an increase in CSF tau, this elevation is
transientandnotchronic.
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Whataspect,then,ofAD’spathophysiologyisthedriverofchronicelevationsinCSFtau?Since
tauissecretedfromtheendolysosomalsystemweanticipatethatanydefectthatincreasestau
accumulation in this compartment will play a role. Our studies permit us to conclude that
retromer-dependentendosomaltraffickingregulatesCSFtau.Itislikelythatotherdefectsinthe
endolysosomalsystemwillhaveasimilareffect.Additionally,defectsintauclearance,whilestill
poorlyunderstood,mustalsobeconsidered. Withtheavailabilityofnewdetectiontoolsand
reagentsthatmeasuremouseCSFtau,itisnowpossibletoinvestigatemultiplemousemodelsin
order to map the full range of molecular defects that might regulate CSF tau. With the
developmentoffuturereagentstomeasurenotonlytotaltau,butlevelsofitsfragmentsand
phosphorylation state, the uniquemolecular profile of CSF tau linked to differentmolecular
defectswillalsobeinformative.
AseriesofrecentstudiesillustratehowthecurrentabilitytodetecttotaltauinmouseCSFcan
elegantlybeusedtobeginclarifyingthesequestions.Thesestudieshaveshownthatmicethat
expressmutationsinAPPandPS1developelevationsinCSFtauovertime(Maia,Kaeseretal.
2013,Schelle,Hasleretal.2017).Sincethesemicedonotdevelopovertneurodegenerationor
tanglepathology, theyprovidedirect evidence that cell deathor taupathologyper se is not
requiredforCSFtauelevations.WhilethesestudieshaveshownthatblockingAPPcleavagewith
aBACE1inhibitorreducestauelevation(Schelle,Hasleretal.2017),implicatingAPPfragments,
howthesefragmentsleadtoincreasedtausecretionremainsunknown.Interestingly,however,
independent studies have shown that mice bearing these mutations develop retromer-
dependentdefectsovertime(ChuandPratico2017),andmorebroadlythattheaccumulationof
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intraneuronal APP fragments cause endosomal dysfunction (Jiang,Mullaney et al. 2010, Xu,
Weissmilleretal.2016).
As recently reviewed,endosomaldysfunction representsaunifiedcellbiologicaldefect inAD
(Small, Simoes-Spassovetal.2017),whichcanbecausedprimarilybymoleculardefects that
directly target the endosome, or secondarily via defects that elevate intraneuronal APP
fragments. Moreover, evidence suggests that whether directly or indirectly, endosomal
dysfunction can act a pathogenic hub, mediating common downstream abnormalities. Our
resultswithretromerknockoutmiceshow,forthefirsttime,thatmoleculesthatdirectlytarget
the endosome lead to established abnormalities in CSF tau. The prior studieswithAPP/PS1
mutant mice established that increasing APP fragments phenocopies the same abnormality.
Whether in the latter case this occurs through the effect on APP fragments on endosomal
dysfunction,remainstobetested.Forthereasonslistedabove,thisseemshighlyplausible.If
so, then elevation in CSF tauwould be considered a sensitive and specific biomarker of AD-
associatedendosomaldysfunction.
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5. Chapter5:Conclusionsandfuturedirections
5.1 Overview
AgrowingnumberofADtherapeuticshavefailedinclinicaltrials,promptingthequestfornew
drugtargets.Asaresult,endosomaltraffickinghasemergedasanewlyvalidatedtargetfordrug
discovery.Currently lacking,however,arebiomarkersofendosomaldysfunction,essential for
addressingtheepidemiologyofthiscellulardeficitinADandfortrackingitscourseoverdisease
progressionandinclinicaltrials.
We sought to fulfill this urgent need using retromer deficiency as a model of AD-related
endosomaldysfunction.Accordingly,weperformedabatteryofbiochemicalanalysesonCSFand
brainexosomesofmicewithselectivedeletionofretromer’scoreproteins.Ourstudiesinclude
the first characterizationof themouseCSF lipidomeand thedeepest characterizationof the
mouseCSFproteometodate,andproposeaCSFproteinbiomarkerwithclinicalutility.
5.2 CSFlipidomics
VPS26ahaploinsufficiencyresultsinamildCSFsphingolipidprofileofnon-neuronalorigin,while
completeknockoutofVPS26ainforebrainneuronsyieldsnolipidomicprofileatall.Whetherthis
indicatesthattheVPS26a-containingassemblyismorecriticaltonon-neuronalcellsorthatnon-
neuronalcellscontributemoretothemurineCSFlipidomecannotbeconcludedfromthisstudy.
Yet,itwarrantsconsiderationthattranslationofthisprofiletohumanCSFislimitedentirelyto
retromerdysfunctioninnon-neuronalcells(ofayetundeterminedtype).Furtherclarificationof
suchabiomarkerrequiresdissectionoftheextracellularlipidomiccontributionbyvariousglial
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types.Thiscouldpotentiallybeexploredinvitrothroughlentiviralknockdownofretromerincell
type-specificcultures(e.g.astrocytes,microglia).Alternatively,invivostudiesofVPS26a-floxed
miceusingCre-recombinaseexpressionunderothercelltype-specificpromoters(e.g.CX3CR1for
microglia)arewarranted.AssphingomyelinstudiesinhumanCSFtodatehaveyieldedconflicting
results and our CSF proteomic experiments unearthedmore compelling leads, this lipidomic
profileofnon-neuronalretromerdysfunctionwasnotpursuedfurther.
Possiblemechanisms for these changesmight includemistraffickingofbiosyntheticenzymes.
Neutralsphingomyelinase,whichconvertssphingomyelinintoceramide,alsoregulatesexosomal
budding (Trajkovic,Hsuet al. 2008).Asourbrain-derivedexosomal studies suggest retromer
mightimpactexosomalbiogenesis,enhancedendosomalretentionofthissphingomyelinaseis
anintriguinghypothesis.
5.3 Brainexosomestudies
Exploration of two retromer knockoutmodels (full body knockout of VPS26b and forebrain-
selective neuronal knockout of VPS35) revealed that the amyloidogenic effects of retromer
knockoutinneurons(viaincreasedretentionofbothBACE1andAPPintheendosome)canbe
detectedinbrainexosomes.Thisenrichmentofb-CTFsinexosomes,whichtakeoriginfromthe
endosomalmembrane,isperhapsunsurprisinggiventheabove-mentionedeffectsofretromer
dysfunctiononamyloidogenicprocessingpathways.
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Follow-upstudiesinhumanserumexosomesofbrainorigin,however,failedtodetectb-CTFsin
thesevesicles.Whether(a)thesefragmentsdonotendupinserum-circulatingexosomesor(b)
they simply cannot be detected using current technology is unknown. Studies to clarify this
questionarewarrantedinthepursuitofabrainexosomebiomarker.Theformerwouldimpose
adifferentstrategy,perhapsusingserumexosomesinneuronal-selectiveretromerknockoutsas
a starting material for biomarker discovery to overcome the challenges associated with
circulation entry. The latterwould prompt development ofmore sensitive technologies (e.g.
simoa)fordetectionofbrain-derivedproteinsinserumexosomes.
Anunexpectedfindingduringthecourseofthesestudieswasthepotentialimpactofretromer
deficiencyonexosomebiogenesis.Thiswassuggestedbyreductionofexosomalmarkersalixand
flotillin-1andapossible(asyetdescriptive)shifttowardsmallervesicles. Interestingly,recent
workbyanothergroupindicatesthatbrain-derivedexosomesfromADpatientsaresmallerand
bearfewercanonicalexosomalmarkersthanthoseofmatchedcontrols(Tran,Mustapicetal.).
While theseparallels are intriguing, further clarificationof retromer’s role in theseexosomal
biogenic pathways is required. Future studies would likely include electron microscopy and
nanotracker analysis to precisely assess changes in morphology of both exosomes and the
originatingMVBs.Inaddition,proteomicstudiescoulduncoverchangesinexosomalcontentand
perhapsprovidemechanisticclues.Thoughourbiomarkerstudiesbecamefocusedonthemore
compellingCSFproteomicfindings,theimpactofretromerdeficiencyonexosomalbiogenesisis
acontinuedinterestofthelab,largelymotivatedbytheresultsofthisproject.
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5.4 CSFproteomics
ItisinterestingtonotethathaploinsufficiencyofVPS26ayieldsnodetectableproteinchangesin
theCSFatthreemonths.Perhaps,as inlipidomics,a latertimepointwouldintensifychanges
whichmayhavebeenmasked in this screen.Orperhapsa completeknockout is required to
detectproteomicchangesintheCSFofthesemice.Consideringthesepossibillities,weoptedto
makebothoftheseadjustmentsthroughanalysisofacompleteknockoutofVPS35inforebrain
neuronsatsixmonthsofage,summarizedbelow.
KnockoutofVPS35inforebrainneuronsresultsinasignificantchangeinnumerousCSFproteins,
includingtwocategoriesparticularlyrelevanttoADpathology:BACE1substrate(APLP1,APLP2,
CHL1, and APP) NTFs and Mapt (tau). Follow-up studies in vivo and in vitro confirmed the
elevation of BACE1 substrate NTFs and total tau in retromer knockout CSF. Interestingly,
IngenuityPathwayAnalysispositionedthesetwocategories(APPandtau)asthetopupstream
‘regulators’ofallCSFchanges,suggestingtheirrelevanceinthedownstreameffectsofretromer-
dependent endosomal dysfunction. Follow-up studies in CSF from both mice and humans
revealed striking correlations between levels of BACE1-cleaved fragments, implicating BACE1
modulation(e.g.byretromerdysfunction)asaprimarydriveroftheirextracellularrelease,which
canbedirectlymeasuredinCSF.
Perhapsmostremarkable,however,wasthecorrelationbetweentheNTFofBACE1substrate
APLP1 and total tau in plaque-free human CSF, suggesting biological convergence (at the
intracellularendosome)oftheseseeminglydistinctcategoriesofhits.Yet,follow-upstudiesina
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smallcohortofplaque-positiveADpatientssupportearlierreportsthattheseAPP-relatedBACE1
substrateNTFsmaybindextracellularamyloid.Andalthoughthesefragmentsmightstillcarry
biomarker potential in early (pre-plaque) patients or in other plaque-free neurodegenerative
diseases such as PD, thier utility is therefore compromised in the progressive AD brain.
Accordingly, CSF total tau emerges as the top validated hit as a biomarker of AD-related
endosomaldysfunctionwithutilitythroughoutthecourseofdiseaseprogression.Remarkably,
studies from other groups have shown that APPmice, which undergo progressive retromer
deficiency,alsoexhibitage-relatedelevationsinCSFtotaltau(Maia,Kaeseretal.2013).
Nonetheless, follow-up studies are required to confirm the specificity of CSF total tau as a
biomarkerofendosomaldysfunctionandthatagentswhichrescuethiscellulardeficitreduceCSF
total tau. To address this question, investigationsofothermodelsofAD-relevant endosomal
dysfunction are required. Various models might include neuronal-selective knockouts of
Phosphatidylinositol3-kinaseVPS34(Miranda,Lasieckaetal.2018)andmicebearingtheAPOE4
allele (Nuriel, Peng et al. 2017). Additionally, treatmentwith retromer-stabilizing chaperones
(Berman,Ringeetal.2015)orviraldeliveryofretromercoreproteinswillaidthismission.
Post-translational modifications of tau are numerous and include various cleavage and
phosphorylationevents.WorkinbothmiceandhumanssuggeststhatthemajorityofCSFtauis
at least partially truncated at the C-terminal (and perhaps the N-terminal, as well) (Barten,
Cadelinaetal.2011,Meredith,Sankaranarayananetal.2013).Thetoolsweusedinthisstudy
rely upon detection of a central epitope, as do standard assessments of CSF total tau in AD
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patients. Future work to clarify the exact sequencemay impart additional specificity to this
markerwhilealso informingon theunderlyingbiology.Mass spectrometryofneuronculture
mediawillgreatlyassistthisinquiry(asmurineCSFtauisnearlyundetectable,eveninretromer
knockoutmice).
WhileatleastaportionofCSFtauisphosphorylated,thestrikingcorrelationsbetweenAPLP1
NTFsandtotaltauwenotedinhumanCSFwerelesssignificantinptau.Therefore,wepostulate
that thepathways involved in the secretionofAPLP1NTFsandphosphorylated taumightbe
more distinct andwe predict that the elevation in extracellular tau we see in our retromer
knockoutisnotdrivenentirelybyanincreaseinptau181.Follow-upworkinneuronalcultureis
requiredtoconfirmwhetherthisisindeedtrue.
Although the general objective of this project was limited to biomarker identification, the
identificationofsuchanintriguingandAD-relevantbiomarkermotivatesanincreasinginterest
in theunderlyingbiology.Theendolysosomal systemhasbeenclearly linked to tausecretion
(Saman,Kimetal.2012,Rodriguez,Mohamedetal.2017,Caballero,Wangetal.2018).Yet,it
remainsunclearwhichofthevariouscompartmentscontributemosttothisprocess.Recentwork
hasdemonstrated that tau canaccumulate in lateendosomes (Caballero,Wanget al. 2018),
compartmentswhichcanfusewiththeplasmamembrane,liberatingtheirlumenalcontentsinto
theextracellularspace.Othergroupshavedemonstratedthattaucanaccumulateinexosomes
(Wang,Balajietal.2017);yet,thisvesicle-boundpopulationcomprisesaverysmallpercentage
(2%)ofextracellulartau(Wang,Balajietal.2017).Thus,weproposethatthemorethan5-fold
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increaseinextracellulartauinretromerknockoutCSFislikelynon-exosomal.Nonetheless,we
hypothesizethatitistheselateendosomes/MVBswhichgiverisetothetauaccumulationwesee
inbothCSFandculturemedia.Furtherinvestigationstopinpointthismechanismarewarranted
andincludebothimmunocytochemistryandelectronmicroscopy.
Thiscomprehensivebiomarkerdiscoveryprojectmadeuseofanassortmentofmousemodelsof
AD-relevantendosomaldysfunctiontouncovervariousbiomarkercandidatesinbrainexosomes
andCSF.Translationstohumans,however, isnotalwaysstraight-forward,despitesubstantial
genomic homology. The hurdle is further challenged here by the presence of extracellular
plaques which are known to alter the CSF proteome of AD patients. Our translational
explorationsofvariouscandidatessuggestthatCSFtotaltau—unaffectedbythepresenceofAD
plaques—is themost reliable biomarker of retromer-dependent endosomal dysfunctionwith
clinicalutility.Furthermore,iffollow-upstudiesconfirmsensitivityandspecificitythismolecular
profile,thenperhapsthemostremarkableconclusionofthisstudyisthatendosomaldysfunction
isauniversalfeatureofADpathology,presentingattheearliestofstages.
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6. MaterialsandMethods
AntibodiesandReagents
Thefollowingantibodieswereusedforimmunoblot:VPS35(ab10099,mouse,1:1000,Abcam),
VPS26a (ab23892, rabbit, 1:1000, Abcam), VPS26b (NBP1-92575, rabbit, 1:500, Novus
Biologicals),VPS29(SAB2501105,goat,1:500,SigmaAldrich),Actin(NB600-535,mouse,1:10000,
NovusBiologicals),Albumin(NB600-41532,goat,1:5000,NovusBiologicals),CHL1(AF2147-SP,
goat,1:500,NovusBiologicals),CHL1(25250-1-AP,rabbit,1:1000,ProteinTech),APLP1(AF3179-
SP, rabbit, 1:500, R&D Systems), Tau (ab80579, mouse, 1:500, Abcam), beta-III tubulin
(ab107216,chicken,1:5000,Abcam),anti-mouseHRP(170-6516,Bio-Rad),anti-goatHRP(172-
1034, rabbit, 1:3000,Bio-Rad), anti-rabbitHRP (1706515, goat, Bio-Rad), anti-chicken800CW
(925-32218, donkey, 1:20000, Licor), anti-mouse 680LT (925-68022, donkey, 1:20000, Licor),
anti-rabbit(925-32211,goat,1:20000,Licor),ProteinG-HRP(18-161,1:10000,SigmaAldrich),
ProteinA-HRP(101023,1:10000,ThermoFisher).
ThefollowingELISAkitswereusedformouseandhumanCSFexperiments:HemoglobinMouse
ELISA Kit (ab157715, Abcam), human APLP1 (LS-F8957-1, LS Bio). Roche lumi-light substrate
(12015200001,SigmaAldrich)wasusedforchemiluminescence.
Mousemodels
AllmicewerehousedinabarrierfacilityandusedinaccordancewithNationalInstituteofHealth
andColumbiaUniversityInstitutionalAnimalCareandUseCommitteeregulations.
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VPS35fl/fl;Cam2a-Creknockoutmice
MiceexpressingloxP-flankedVPS35(VPS35fl/fl)onaC57BL/6backgroundwerecrossedwithmice
expressing Cre-recombinase under the Camk2a promoter to knock-out VPS35 in forebrain
neurons.VPS35fl/flmicelackingCre-recombinaseservedasthecontrolcondition.Miceages6-12
monthswereusedforallexperiments.
VPS26afl/fl;Cam2a-Creknockoutmice
MiceexpressingloxP-flankedVPS26a(VPS26afl/fl)onaC57BL/6backgroundwerecrossedwith
miceexpressingCre-recombinaseundertheCamk2apromotertoknock-outVPS26ainforebrain
neurons.VPS26afl/flmicelackingCre-recombinaseservedasthecontrolcondition.Six-month-old
micewereusedforalllipidomicsexperiments.
VPS26b-/-Mice
VPS26b heterozygote were generated by a targeting vector containing a neomycin-resistant
expressioncassetteflankedbygenomicregionsofVPS26b,negativeselection,electroporation
intoembryonicstemcells,andinjectionintomouseblastocyst(asdescribedpreviously)(Kim,Lee
etal.2010).ChimericmaleswerematedwithC57BL/6femalemicetogenerateaheterozygote
colony(Kim,Leeetal.2010).Heterozygoteswerethenmatedtogeneratethecompleteknockout
andthewildtypecontrols.Twelve-month-oldmicewereusedforallexosomeexperiments.
VPS26a+/-Mice
CongenicVPS26aheterozygoteswerecrossedona120/SvEvagoutibackground
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for10generationsandmaintainedthroughsiblingmating(Muhammad,Floresetal.2008).Mice
ages3-6monthswereusedforalllipidomicsandproteomicsexperiments.
MurineCSFcollection
MurineCSFwascollected inapost-mortemprocedure followingCO2overdose inaccordance
with IACUCguidelines.Briefly,euthanizedmicewereplaced inapronepositionand the skin
coveringthebackoftheneckwassurgicallyexcised.Acottonswabcontaining70%ethanolwas
usedtoremoveanyhairorskin fromtheexposedtissue.Then,a27-gaugeneedle (SV*27EL,
TerumoMedicalProducts)attachedtoa1-mlsyringe(329650,BDBiosciences)wasinsertedinto
thecisternamagnaallowingflowofCSFintothebutterflyneedle.After10-15seconds,theneedle
wasremovedandtheCSFaspiratedintomicrocentrifugetubes(1605-0000,USAScientific)which
wereimmediatelyplacedondryiceandsubsequentlystoredat-80C.Roughly5-10uLoffluidwas
collectedpermouse.CSFwhichwasvisiblycontaminatedwithbloodwasdiscarded.Allremaining
samplesunderwentmorestringentassessmentforbloodcontaminationviahemoglobinELISA
using0.5uL ina1:200dilution. Samplesbelow0.01%bloodcontaminationwere retained for
biochemicalanalyses.Foreachassay,bothindividualandpooledreplicatesbetweenconditions
were matched for hemoglobin content to reduce the likelihood of a blood contamination
confound.
Lipidomicanalyses
Lipidswereextracted fromequalvolumesofmurineCSF (30ul forpooledsamplesor7uL for
individual samples). Lipid extractswere prepared via chloroform-methanol extraction, spiked
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withappropriateinternalstandards,andanalyzedusinga6490TripleQuadrupoleLC/MSsystem
(Agilent Technologies, Santa Clara, CA) as described previously(Chan, Oliveira et al. 2012).
Glycerophospholipids and sphingolipids were separated with normal-phase HPLC using an
Agilent Zorbax Rx-Sil column (inner diameter 2.1 x 100mm) under the following conditions:
mobilephaseA(chloroform:methanol:1Mammoniumhydroxide,89.9:10:0.1,v/v/v)andmobile
phaseB(chloroform:methanol:water:ammoniumhydroxide,55:39.9:5:0.1,v/v/v);95%Afor2
min,lineargradientto30%Aover18minandheldfor3min,andlineargradientto95%Aover
2minandheldfor6min.Quantificationoflipidspecieswasaccomplishedusingmultiplereaction
monitoring(MRM)transitionsthatweredevelopedinearlierstudies(Chan,Oliveiraetal.2012)
in conjunction with referencing of appropriate internal standards: ceramide d18:1/17:0 and
sphingomyelind18:1/12:0(AvantiPolarLipids,Alabaster,AL).Valuesarerepresentedasmole
fractionwithrespecttototallipid(%molarity).Forthis,lipidmass(inmoles)ofanyspecificlipid
isnormalizedbythetotalmass(inmoles)ofallthelipidsmeasured(Chan,Oliveiraetal.2012).In
addition,allofourresultswerefurthernormalizedbyproteincontent.
Proteomicanalyses:LabeledLC-MS/MSofVPS26a+/-CSF
HighqualityCSF(<0.01%bloodcontamination)collectedfromVPS26+/-andwildtypelittermates
was pooled into four biological replicates per genotype of 30uL each. Each replicate was a
composite of CSF from roughly 4-6 mice. Protein concentrations from each sample were
determined by a fluorescent-based protein assay (Q33211, Life Technology). Concentrations
rangedfrom0.68-0.8µg/mLpersample.Equalamounts(20μg)fromeachsampleweredigested
by trypsinand labeledwithTMT isobaricmass tags.A reference sample (comprisedofequal
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amounts fromeachsample)wasgeneratedforquantification.AfterTMT labeling,allsamples
were mixed and peptides were fractionated by C18 reverse phase with a pH gradient (Life
Technologies).PeptidemixtureswereanalyzedbyOrbitrapFusionTribridmass spectrometer
(Thermo Fisher) and searched against the mouse Uniprot protein database using Proteome
Discoverer2.1 (ThermoFisher).Overall intensityofeachTMTchannelwasnormalized to the
referencesample.Minitab17wasusedtoperformstatisticalanalysesandgeneratestatistical
plots. Qlucore Omic Exporer software was used to perform statistical analysis on
phosphopeptides.
Proteomicanalyses:UnlabeledLC-MS/MSofVPS35Camk2aKnockoutCSF
High quality CSF (<0.01% blood contamination) collected from VPS35 knockout and control
littermateswaspooledintobiologicalreplicatesof30uLeach,whichwereroughlymatchedfor
hemoglobin content. Each replicate was a composite of CSF from 4-7 mice. The protein
concentrationofeachsamplewasdeterminedviaQubitProteinQuantificationAssay(Q33211,
LifeTechnology).Concentrationsforeachsamplerangedfrom0.10-0.16mg/ml.Eachsamplewas
loadedontoanS-trapcolumn,digestedwithtrypsin,elutedwithtrifluoroaceticacid,anddried.
Each sample was divided into two technical replicates (each containing 1.5ug of digested
protein).MS/MSwasperformedoneachsampleusingaThermoOrbitrapFusionTribridMass
Sepctrometer(ThermoFisher).ProteomeDiscoverersoftware(version1.4)wasusedtosearch
theacquitedMS/MSdataagainstamouseproteindatabasedownloadedfromUniProt.Positive
identificationwassetat1%proteinFDRforthoseproteinshavingatleasttwoquantifiablevalues
detected.Anyduplicatedprotein identifications from thedatabasewere removed.A totalof
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1505 mouse proteins were detected and included in the final data set. For each protein,
quantification was determined by averaging the peak area of the threemost abundant and
distinctpeptides.Totalareaswereusedfornormalization.QlucoreOmicsExplorerandMinitab17
Softwarewereused toperformcorrelationand statistical analyses. Spearmancorrelationsof
technical replicates were used to determine group outliers. One outlier per genotype was
identifiedbytheProteomicsCoreandremovedpriortodownstreamanalyses.
GenerationofGFP.CreandGFP.D-CreVirusforVPS35KnockoutinCellCulture
HEK293Tcellsweregrownto70%confluence inmediacontainingDMEM,5%glutaMAx,10%
FBS,andpen/streppriortolipofectamine(LTX)transfectionwithplasmidscontainingD8.2,VSVG,
andeitherGFP.CreorGFP.D-Cre.After72hours,mediawascollectedandfiltered(SE1M003M00,
Millipore)beforeincubationwithlenti-xconcentrator(631231,Clontech)andcentrifugationto
collecttheviralparticles.
VPS35KnockoutinPrimaryNeuronCultures
HippocampiorcerebralcorticesfromVPS35fl/flP0pupsweredissectedincoldHBSSanddigested
in 0.5% Trypsin-EDTA (25200056, Invitrogen) for 20minutes at 37C. The digested tissuewas
rinsed in HBSS and homogenized gently with a flame-polished glass pipette in NBA media
containing Glutamax (35050-061, Life Technologies), B-27 (17504044, Thermo Fisher), and
pen/strep(15140-122,ThermoFisher).Neuronswereplatedinsix-wellplates(83.3921,Sarstedt)
containing poly-D-lysine-coated 12mm glass coverslips (NC9702891, Thermo Fisher) for
immunofluorescence or in poly-L-ornithine-coated 12-well plates (83.3921, Sarstedt) for
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immunoblotandmediacollectionexperiments.Mediawasreplacedat24hoursand1/3media
replacements were conducted every 5 days thereafter. On DIV5, lentivirus containing either
GFP.CreorGFP.D-Creviruswasaddedtoeachwell.OnDIV15,mediawasremovedandneurons
fixed in 4% paraformaldehyde (15710-S, Electron Microscopy Science) for downstream
immunofluorescenceorlysedincoldRIPAbuffer(89901,ThermoFisher)forimmunoblot.
Immunoblotting
SampleswerepreparedinLDSsamplebuffer(NP0007,ThermoFisher)andreducingagent(161-
0792,Bio-Rad),boiled for5minutesat90Cand runviaelectrophoresis in4-12%bis-tris gels
(NP0336orNP0335, Thermo Fisher) inMESbuffer (NP0002-02, Thermo Fisher). For CSF and
mediaexperiments,equalvolumes(5-15uL)wereloadedineachlane;forlysate,equalmasses
wereloadedineachlane.wereloadedperlane.Proteinswerethentransferredtonitrocellulose
membranes(IB3010-31,Invitrogen)viaiBlot(P3;7:40)andblockedfor1hourinbovineserum
albumin (A30075-100gm, Research Product International) or odyssey blocking buffer (927-
40000, Licor) diluted 1:1 in PBS-T. Membranes were then probed with primary antibodies
overnight at 4C, washed in PBS-T, probedwith secondary antibodies (HRP-conjugated or IR-
conjugated) foronehouratroomtemperature.Membraneswereagainwashed inPBS-Tand
imagedinRochesubstrate(12015196001,SigmaAldrich)forECLoralonewiththeLicorodyssey
system.
TotalTauQuantificationinCultureMedia
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Total tauwasquantified fromculturemediausing theMSDmouse total tau kit (K151DSD-1,
MesoScaleDiscovery)accordingtothemanufacturer’sguidelines.Wellswerefirstblockedfor
onehourandwashed.Then,25uLofculturemediawasaddedinduplicatewellsalongwiththe
total taustandardsprepared inNBAmedia(withglutamax,B-27,andpen-strep,asdescribed
above),andincubatedforonehour.Wellswerewashedandincubatedwithdetectionantibody
for an additional hour before washing and analyzing in read buffer T solution on the MSD
instrument.CelldeathwasassessedviaLDHlevelsintheculturedmediausingCytoTox-ONE™
HomogeneousMembraneIntegrityAssay(G7890,Promega).ValuesfortotaltauandLDHwere
normalizedtototalprotein,whichwasmeasuredusingBCA(23227,Pierce).
TotalTauQuantificationinMurineCSF
CSF total tauwasquantifiedby SIMOAusing themouse total tauassay (102209,Quanterix).
Individual CSF samples (5uL)werediluted1:80 in samplebuffer and then split into technical
duplicates.Standardsandsampleswererunaccordingtothemanufacturer’s instructionsand
readontheHD-1analyzer(Quanterix).
HumanCSFStudies
CSFfrompatientswithAlzheimer’sdiseaseorMildCognitiveImpairment,andNormalControls,
wasrequestedfromtheColumbiaUniversityAlzheimer’sDiseaseResearchCenter(ADRC)CSF
bank. This CSF bank includes CSF obtained by lumbar puncture and banked for research
accordingtoprotocolsapprovedbytheColumbiaUniversityIRB.CSFwasstoredfrozenin400uL
aliquots,inpolypropylenetubes,at-80C.FrozenCSFaliquotswerelaterthawed,andanalyzed
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forAb42,totaltau,andphospho-tau,bytheADRCClinicalCore,usingamultiplexfluorimetric
microspherexMAPbead-basedsandwichimmunoassayusingmonoclonalantibodiescovalently
coupledtospectrallyspecificfluorescentbeads.Thisassay(Inno-BIAAlzBio3,FujirebioUSInc,
Alpharetta,GA)wasperformedusingaLuminex100machine.IdenticalfrozenCSFaliquotswere
thawedandusedfortheexperimentsdescribedhere.N-APLP1andN-CHL1weremeasuredvia
immunoblotasdescribedabove.N-APLP1werealsomeasuredviaELISA(LS-F8957-1,LSBio)in
duplicateaccordingtothemanufacturer’sinstructionsfollowinga1:20dilutionasinformedby
early optimizations in control CSF. Total protein was calculated via BCA (23227, Pierce).
Thresholds implementedtoconfirmplaquepositivity inADpatients includesAb42<210pg/mL
andtotaltau/Ab42>0.39.
Statisticalmethods
Student’s t-testwas used for all biochemical experiments and immunohistochemical analysis
usingtwo-taileddistributionwithequalvariance(P<0.05).Limmasoftwarewasusedforanalysis
ofCSFproteomics,andFDR-correctedq-valuesare reported.Significance is indicatedas*P<
0.05,**P<0.01and***P<0.001;****P<0.0001).
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