1. Regulation of synaptic BACE1 trafficking and Aβ generation
through late endocytic transport
David Wilkes, Xuan Ye, Qian Cai.
2. Wilkes2
Abstract
Cumulative evidence suggeststhat -amyloid(A) peptidesplayakeyrole insynapticdamage
and affectmemoryprocessesearlyinthe course of Alzheimer'sdisease (AD). Amyloid-β(Aβ) peptide is
producedviaβ-secretase,mainlybyβ-site amyloidprecursorprotein(APP)-cleavingenzyme1(BACE1).
BACE1 aberrantly accumulatesatthe distal axonsand synapsesinADneurons and reliesonlate
endocytictransporttobe degraded withinlysosomesinthe soma. APPisprimarilyprocessedinthe late
endocyticpathway,butthe mechanismthrough whichBACE1istraffickedfromthe synapse tothe
lysosome isstill largely unknown. We hypothesizedthat snapin deficiencyresultsindefective
retrograde transportof late endosomesandcausesaccumulationof BACE1at the nerve terminal,thus
enhancingAPPamyloidogenicprocessing. Totestthishypothesis,synaptosomeswerepreparedfrom
conditional snapin knockoutmice andproteinlevelswere assayedbyWesternblotting. We showed
that deletionof snapin causesanabnormal accumulationof synapticBACE1and APP,increasing APP
processingandAβgeneration atthe nerve terminal. Altogether,ourstudy indicated thatSnapin-
mediatedretrograde transportisimportantforregulatingsynapticBACE1traffickinganditscleavage of
APP.
Background
Alzheimer’sdisease(AD) isaprogressive neurodegenerativedisease characterizedbymemory
lossand impairedcognitive function. The disease beginsprimarilyinthe highlyplasticneuronsof the
hippocampus,andthenspreadstothe cerebral cortex andotherbrainregions. The pathologyof this
disease ischaracterized bysynapticdysfunction,axonal degeneration, neuronal loss,andthe formation
of neurofibrillarytangles andsenileplaques thatare composedmostlyof amyloid-beta(Aβ) peptide.
However,ithasbeenfoundthatthe degree of cognitive impairmentinADismost correlatedtothe level
of the soluble formof oligomericAβ inneurons,andnottothe amountor size of the extracellular
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plaquesthemselves(HardyandSelkoe,2002). Aβis generatedthroughthe sequential cleavingof
amyloidprecursorprotein(APP) byaβ-secretase andthenbya γ-secretase.β-site amyloidprecursor
proteincleavingenzyme 1(BACE1) isthe prominentβ-secretaseinvolvedinamyloidogenesis inneurons
(Huse etal., 2000; Vassar etal., 2009).
The initial cleavage stepinvolvingBACE1andAPPisthe rate-limitingstep inAβproduction,
whichoccurs predominantlyin late endosomes [alsocalled“multi vesicularbodies”(MVBs)].The lowpH
and extensive surface areawithinlate endosomesisoptimal forBACE1activity(Vassaretal.,2009; Wu
et al., 2011). BACE1 is a transmembrane aspartyl protease (Huseetal.2000), whichis endocytosed
fromthe plasmamembrane intothe endocyticpathway,where itiseventuallytraffickedbymeansof
retrograde transportto lysosomesfordegradation(Sannerudetal.,2011). In AD brains,increasedlevels
of BACE1,especiallywhencolocalizedwithAPPinthe endocyticpathway(Dasetal.,2013), have been
directlycorrelatedtoarise in Aβproduction(Vassaretal.,2009; Ye andCai, 2014), as well asto a
greateraccumulationinacidiccompartments—probablyendosomes(Kandalepas etal., 2013).
Particularly of interest,BACE1seemsto concentrate aberrantlyindystrophicneuronsaroundamyloid
plaquesinAPPtransgenicmouse brains,whichsuggestsanincrease insynapticAPPprocessing.BACE1
levelsincrease withage andin the brainsof patients withsporadicAD(Fukumotoetal.,2004). It is
importantto note thata rise inBACE1 levelsisdue to itsenhancedstability and/orreducedturnover,
and notby an increase inproductionof BACE1 (Lefortetal.,2012). Consequently, BACE1levels
potentially couldbe primarily controlledby modulationof late endosomal retrogradetransport.
Neuronal endocytosismoderates cellsignalingbycontrollinghow manyreceptorscanactively
interactat the membrane surface (Yuzaki,2010). Endocytosisbeginswithbuddingof the membrane,
whichcontainssurface proteins(includingBACE1),toformvesicles,whichfuse withearlyendosomes.
Early endosomes mature intolate endosomesbyaregulated inwardbuddingprocess toforma “multi
vesicularbody”(late endosome),increasing membranesurface areaandallowinglysosome-delivered
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hydrolase access(Nixon,2005). The late endosome thenfuseswitha lysosomeinordertodegrade its
proteincargo. The processby whichlate endosomesanditsBACE1 contentsmigrate toand thenfuse
withlysosomesisstillpoorlyunderstood(Ye andCai,2014). Upon fusion,acidhydrolases fromthe
lysosome become activeanddigestlate endosomal cargo(Nixon,2005). Mature lysosomesmainly
reside inthe neuronal soma,farfromthe axonterminal (Cai etal.,2010). Therefore,late endosomes
containingBACE1dependonretrograde transportalongthe axonal microtubulesinordertodeliver
theircargoesto the lysosomesfordegradation inthe soma(Cai etal.,2010).
Retrograde transportisdrivenbydynein, the majormicrotubule-basedminus-enddirected
motor proteininneurons. Deficitsindynein-relatedproteinssuchasdyneinintermediate chain(DIC)
and Snapinhave beenshowntodisruptlate-endocytictransportandleadto excessproductionof Aβ
(Cai et al.,2010; Ye and Cai,2014). Snapinis an importantadaptorproteinfor dyneinmotors,asithas
beenfoundtocoordinate late endocytictransportandmaturationof lysosomes(Cai etal.,2010).
Snapinis alsothoughtto interactwithdyneinintermediate chain(DIC),whichallowsrecruitmentof
dyneinmotorstolate endocyticorganellesfor retrograde transport. Experimentsinvolvingsnapin
knockout(KO) mice orDIC substitutionresultedindecreaseddynein-assistedtransportof BACE1
containingvesicles,andenhancedAPPprocessing(Ye andCai,2014). Distal accumulationof late
endosomes containingAβ42hasalsobeenfound inAD mouse neurons,possiblydue to impaired
retrograde transport of APPand BACE1, whichmay accountfor the excessproductionof Aβ observed in
AD neurons (Ye andCai,2014). In dendrites,APPandBACE1converge mostlyduringthe endocytic
pathway,triggeringamyloidogenesis (Dasetal.,2013).
One of the earliestsymptomsinADbrains isthe lossof synapticfunctionality (Selkoe,2002).
The lossof synapseshasbeendiscovered tobe the bestpredictorof cognitive impairmentin ADbrains
(Tampellini etal.,2011). BACE1 has beenfoundtolocalize invesiclesat the presynapticterminalsnear
active zones(Kandalepasetal.,2013). Excessive Aβproductionandrelease atsynapticsitesare tightly
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correlatedwithincreasedamyloiddeposition andsynapticdysfunction(StokinandGoldstein,2006;
Takahashi etal.,2002). Consideringthatabnormal accumulationof BACE1and APPat presynaptic
terminalsoccursbefore the onsetof plaque (Zhangetal.,2009), interventioninthe BACE1trafficking
pathwaycouldbe a reliablemeansfor haltingamyloidpathologyearlyinAD. Thisraisesthe central
questionof whetherenhancementof Snapin-mediateddynein motor-assistedBACE1transport can
decrease itssynapticaccumulationandAβgenerationatnerve terminals,reducingordelayingsynaptic
pathologyinADmouse brains.
In thisstudy,we soughtto address whetherdisruption of retrograde transport accumulates
BACE1 at the synapse, thereby enhancingamyloidogenicprocessing andamplifyingsynapticAD
pathology.Snapin KOmice will serveasa unique genetictool that allowsustoaddresswhethersnapin
deficiency heightens synapticretentionof BACE1,exacerbatingsynapticAgeneration.
Materials and Methods
Genotyping
PCR genotypingassaywasperformedtoidentify homozygous snapin flox mice (snapinflox/flox
;
Thy-1 Cre Tg) afterthe tissue-specificCre wasconfirmed. DNA was extracted frommouse tail snips
usingPCRlysate reagent(Viagen). The snapin loxPsiteforwardprimer5′- GTGCAGCAGCTCGACTCTC -3′
and loxPsite reverse primer5’- AGCCAACCTCAACTTCAAGG– 3’ were usedtoflox the snapin gene
codingregion. The DNA segmentswere amplifiedbyusingthe followingPCRreactionsystem:9.46µl
H2O, 1.25µl 10X PCR Buffer,0.25µl 10mM dNTPs,0.38µl 50mM MgCl2, 0.3µl 10µM forwardprimer,
0.3µl 10µM reverse primer,0.06µl (5U/µl) Taq Polymerase,and0.5µl tail DNA. Thirty three cyclesof
PCR were used withthe followingcycles:94° C for3 minutes(onlyfirsttime),94°C for 30 seconds,55°
C for 30 seconds,72° C for 45 seconds,repeat33 times,72° C for5 minutes(onlylasttime),thenkeepat
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16° C. By runningPCRproductson 2% agarose gel,homozygoussnapin flox mice show abandat 275bp,
while wild-type mice canbe identifiedby abandat 188bp.
Synaptosome Preparation
Synaptosome preparationsfromWTand snapin conditional KOmouse brains (snapinflox/flox
;Thy-
1 Cre Tg) were collectedusingPercoll gradientcentrifugationasdescribedinthe protocol (Leenderset
al.,2004). Cortex tissue fromWTand snapin mutantmice were homogenizedinice coldIsolationBuffer
(IB) [10mM Tris-HCl,1mMEGTA, 1mM EDTA, 0.25 M sucrose and protease inhibitors(Roche),pH7.4].
Homogenateswere centrifugedat1,330 g for 3 minutes,the supernatantwasgathered,andthe pellet
was resuspendedwithIBandcentrifugedagainat1,330 g for 3 minutes. The firstandsecond
supernatantwere combinedandthencentrifugedat21,000 g for 10 minutesandthenresuspendedin
12 ml of 15% Percoll. 2 ml of the 15% Percoll suspensionwasoverlaidonPercoll gradientthathas 3.5
ml of 15% Percoll gradientlayeredover23% Percoll gradient.The gradientwasthenseparatedby
centrifugationfor5minutesat30,700 g. The synaptosomal fractionwascollectedfromthe 15%/23%
Percoll layers,andcombinedwith0.5ml of 10 mg/ml bovine serumalbumin(BSA)in3ml IB. The
mixture wasthencentrifugedat16,700g for10 minutesandresuspendedinIB.Proteinquantification
was performedbyBCA assay(Pierce Chemical Co.). 15µg of proteinfromsynaptosome andpostnuclear
supernatant(PNS) homogenateswere resolvedby4-12% SDS-PAGEfor sequentialWesternblotsonthe
same membranesafterstrippingbetweeneachapplicationof antibody.
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Figure 1
Percoll gradient centrifugation (Sims, 1990)
Westernblotting
Westernblotwasperformedbyfirst blockingfor30 minuteswith10% non-fatmilk/TBST
solution,followedbyincubationwithprimaryandsecondaryantibodiesfor1 houreach. SuperSignal
WestPico Chemiluminescencesubstrate (ThermoScientific) wasusedtoperformenhanced
chemiluminescence (ECL) usingscientificimagingfilm(LabScientific,Inc.). NIHImageJsoftware was
usedforquantitative analysis. All immunoblotswere fromexperimentsdone atleast 4 times. Statistical
analyseswere completedwith unpairedStudent'sttestand are showninfigures asmean± SEM. H. Cai
providedpolyclonal anti-BACE1antibody. Polyclonal anti-SnapinantibodywassuppliedbyZ.H.Sheng.
Othersourcesof antibodiesare asfollows:polyclonal anti-APPc-terminal andpolyclonal anti-APP-CTF
antibodies(Millipore/Chemicon),monoclonal anti-rab7andpolyclonal anti-SNAP25antibodies(Sigma),
and monoclonal anti-p115antibody(BDbiosciences).
Synaptosome
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Results
Figure 2
Gel electrophoresis,performedafterconfirmationof the tissue specificCre, of constitutional
knockoutPCRproducts obtainedfrom mouse tails. Cre-Lox recombinationwasusedonmice embryos
to create a constitutionalsite-specificdeletionatthe snapin codingregion. Homozygous snapin KO
mutantmice (snapinflox/flox
;Thy-1Cre Tg) show a single bandat275bp (Lanes:3 and 6),while WT mice
(snapin+/+
; Thy-1 Cre Tg) displayaband at 188bp (Lanes:8, 9 and 10). Heterozygous mutantmice
(snapinflox/+
;Thy-1 Cre Tg) showa bandat both 188bp and275bp (Lanes:1, 2, 4, 5, 7 and11). Lane L isa
100bp DNA ladderand lane Ctrl isa positive control forsnapin KO.
Previously,site-specificCre wastestedforandconfirmedinthe brainsof the mice usedin the
followinginvestigation. Inordertoconfirmhomozygous snapin-mutantmice,agel electrophoresisassay
was performed forthe purpose of visuallydistinguishingmouse mutations (Figure 2). AfterDNA
separation,asingle bandat275bp for lanes3 and 6 (Figure 2) reveals thatthe snapin gene encoding
regionwascompletelyfloxed inbothchromosomes of the mice meaningtheyare homozygous
(snapinflox/flox
;Thy-1Cre Tg). Lanes 1, 2, 4, 5, 7 and 11 each showed twobandsat both188bp and 275bp,
indicatingthatthe mice are heterozygous (snapinflox/+
;Thy-1Cre Tg),and thustheyare not viable for
experimental use. Inlanes8,9, and10, there isa single bandat188bp whichspecifies thatthese mice
containan unaffected codingregion (snapin+/+
;Thy-1 Cre Tg) andare therefore wild-type mice thatcan
be utilizedas experimental controls.
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Figure 3
Representative blotsandquantitative analysisshowingincreasedsynapticaccumulationof APP,BACE1,
and Rab7 in the brainof snapin KOmice. Followingsynaptosome fractionation,equal amounts(15µg) of
synaptosome (SS) andpost-nuclearsupernatant(PNS)fromWTand snapin KOmice were sequentially
immunoblotted. The purityof the synaptosome wasconfirmedbythe absence of p115,a Golgi marker
protein. Snapinisshowntobe absent inthe synaptosome of snapin KOmice,confirmingthe precision
of thismouse model. Relativeproteinlevelsinsynaptosomesof snapin KOmice were comparedwith
those of WT littermates. Datawere analyzedfrom4 pairsof mice for each genotype andexpressedas
mean± SEMwithStudent’sttest: ∗∗p < 0.01; ∗p < 0.05.
To examine whethersynapticBACE1accumulationoccursin snapin-deficientmice,sequential
immunoblotswereperformedonsynaptosomesfromthe brainsof snapin KOmice andWT littermates.
Sequentialimmunoblotsforboththe post-nuclearsupernatant(PNS)—ahomogenate of neural cells
withthe nuclearcompartmentremoved—andthe purifiedsynaptosome(SS) were performedon
separate membranes againstthe followingantibodies:APP, CTFs[carboxyl-terminal fragments:C83,
C89, C99 (byproductsof APPprocessing)],BACE1,late-endosomal markerRab7,presynapticmembrane
associatedindicatorSNAP25,andGolgi markerp115. The purityof the synaptosomeswasdetermined
by the absence of p115. Relative proteinlevelsinthe synaptosomeof WTand snapin KOmice were
quantitativelycompared(Figure 3). The APP level detectedinthe synaptosomeof snapin-deficientmice
was 4.08 ± 0.45 (p< 0.01) timesmore thanthe amountidentifiedin WTlittermates(Figure 3). Likewise,
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BACE1 levelsinthe synaptosomewere markedlyincreased inthe snapin KOmice, 5.1 ± .27 (p < 0.01)
timeshigherrelativetothe WT mice (Figure 3). Rab7 exhibitedasignificant rise inthe synaptosome of
snapin-mutantmice,withaproteinlevel1.7± 0.12 (p < 0.05) timeshigherthaninthe WT control
(Figure 3).CTFs,a by-productof amyloidogenesis, appearedtooccurin the highestdensityatthe
synaptosomesof snapin KOmice. Additionally, there wasahigherconcentration of BACE1in snapin-
deficientmice atthe synaptosome thaninthe PNS.
Discussion
In previousstudies,Snapinwasshowntomediate late-endosomal traffickingthrough
interactionswithdynein(Cai etal.,2010), and alsoto modulate BACE1retrograde transport and
turnover(Ye and Cai,2014). In thisstudy,we hypothesized thatdisruptionof late-endocytictransport
viasnapin deletioncausesaccumulationof BACE1at synapticterminals,leadingtoenhancedAPP
processing. Thisstudyexpandedfurtheronpreviouswork,demonstratingthatthe amountof BACE1 in
snapin-mutantmouse brainswas elevatedinthe synaptosome comparedwithinthe PNS,lending
credence tothisstudy’shypothesis.
The snapin KO mice usedinthisstudy,identifiedbygenotyping,were confirmedtofunctionas
intendedbythe immunoblotinfigure 3,showingasubstantial decrease in Snapinexpression level.
Small amountsof Snapincan still be seeninthe immunoblotforthe PNSknockoutbecause the
homogenate included glial cells,whichwerenotaffectedbyCre-Lox recombination. Furthervalidation
for thisstudy’s hypothesis isprovidedbythe elevated amountof late-endosomal markers(rab7) inthe
snapin-deficientsynaptosome,indicatinganaccumulationof late endosomesinthe KOcondition. This
evidence suggeststhatwithoutSnapin, late endosomeswouldbe unabletoassociate withdyneinand
be deliveredto lysosomesfordegradation. Itisnow clearthat late-endocytictraffickingisvital tothe
regulationof amyloidogenesisnearthe synapse, inagreementwithpreviousstudiesthatconfirmedthis
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pathwayto be the primarylocationof APPprocessingandto be crucial inBACE1 turnover(Ye and Cai,
2014). The mostdirectpiece of evidence tosupportthisstudy’shypothesisis the profoundincreasein
BACE1, as well asin APP,inthe synaptosomesof snapin KOmice whencompared toWT mice.
Previously,anincrease of Aβin snapin-deficientmice wasdemonstratedbyusinganELISA assay (Ye and
Cai,2014). Consistently,ELISA assayresultshave shownthatAβlevelsin snapin KOmice are
significantlyelevatedinsynapticpreparationscomparedtoPNSfromthe total cortex (datanot shown).
These findingsimplicate snapin deficiencyinimpaired late endocytictransportandBACE1 to
accumulation inlate endosomes,enhancingBACE1cleavage of APPandAβproductionat nerve
terminals.
The significant increase insynapticamyloid-relatedproteins due toanabsence of Snapin
functionalityisasolidconfirmationthatSnapinhasa majorrole inregulating Aβproduction through
late endocytictransport. These results,inconjunctionwiththe previouslydescribedstudies,supporta
potential mechanismforsynapticADpathologyinwhichAβcausesSnapin-dyneinmotoruncoupling.
Therefore,introducingexcess SnapinintoanADbrain couldbe a possible methodforovercomingthe
disruptionof motorcouplinginducedbyAβ.
Conclusion and Future Plans
The resultsfromthisexperimentdemonstrate thatdyneinmotor-driventransportof late
endosomesservesanimportantrole inregulatingBACE1accretionand,consequently, synapticAPP
processing. Excessive amyloidogenesis,especiallyinaxonterminals,isthoughttocause axonal
degenerationandsynapticdysfunction(StokinandGoldstein, 2006). Additionally, synapticlosshas
beenfoundtobe one of the earliestandbestpredictorsof cognitive decline inAD(Tampellini etal.,
2011). Consideringthis, synapticBACE1traffickingisapotentially invaluabletherapeutictargetfor
treatingADpathology early andeffectively.
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Furtherexperimentswill need tobe conductedtoconfirmthe findingsinthisstudy, andtoshed
more lighton the regulatorymechanisms of synapticBACE1traffickingthroughdynein-associated
transport. AD neuronsshow reducedretrograde transportof BACE1,whichis causedby Snapin-dynein
motor uncoupling(Ye andCai,2014). Dr. QianCai’slab will nextstudythe effectsof defective late
endocyticBACE1transport onsynapticretentionof BACE1and whetheritwill increaseBACE1
processingof APPat the nerve terminal inADmouse models andADpatientbrains. Thislabwill alsobe
investigatingthe effectsof enhancing Snapin-mediatedsynapticBACE1traffickinginADtransgenic
mouse models by overexpressionof Snapin. Thisapproachwill potentially be usedtoreduce synaptic
Aβ generation andsynapse loss. NeitherADmouse brainsnorhumanAD synaptosomeshave been
isolatedandstudiedforsynapticBACE1accumulationordefectsinSnapin-dyneincoupling. Looking
furtherahead,humanADbrainswill mostlikelybe studiedinorderto determineif there ispotential for
Snapin-targetedorlate-endocytic-relatedtherapiesinrepressingorameliorating the symptomsof AD
patients. The goal for thisresearchisthat humanAD patientswill respondpositivelytoSnapin or
relatedtreatments,asisthe case so far in mouse models. However, currentmouse models only
representthe hereditary formof AD,as opposed tothe sporadicformof AD that isseenprevalently in
humans. SporadicAD patientsmayhave variedanddistinct underlyingcausesfortheirdisease notyet
considered,andsothese patientsmaydifferinthe typesof treatmentthatare effectiveforthem.
Despite possible complicationsintargetingsporadicformsof AD, understandingabnormalitiesin
retrograde transport showspromise forthe developmentof earlierdiagnosesandmore beneficial
interventionsinthe future.
Acknowledgements
I wouldlike tothankDr. QianCai forgivingme the opportunitytobe a memberof her lab,and
for hertakingthe time out of her verybusyschedule tobe patientwithme,andhercommitment to
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helpingme expandmythinking andsucceed. Iwouldalsolike tothankmylabmentor,Dr. Xuan Ye, as
well asall the lab members, forprovidinginsight intolaboratorytechniquesandexperimental design. I
am alsoverygrateful forthe 2015 Neuroscience SummerUndergraduate ResearchProgram,forthe
program’sco-directors,Dr.JanetAlder,Dr.Michael Matise,andDr. Mladen-RokoRasin,andforthe
NeuroSURPprogramadministrativecoordinator,JoanMordes,all of whomhave shownphenomenal
dedicationtothe studentsinthe program. I appreciate the accommodations, resources,and
commitmenttoscientificresearch providedby The RobertWoodJohnsonMedical School andby
Rutgers,The State University of NewJersey. Finally,Iwouldlike tothankall those whohave helped
supportme and have contributed positively tomylife:family, friends,professorsandmentors.
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