Loss of Function of ATXN1 Increases Amyloid Я-Protein Levels by Potentiating Я-Secretase ...

13 Pages·2010·1.89 MB·English

THEJOURNALOFBIOLOGICALCHEMISTRY VOL.285,NO.12,pp.8515–8526,March19,2010
©2010byTheAmericanSocietyforBiochemistryandMolecularBiology,Inc. PrintedintheU.S.A.
(cid:1)
Loss of Function of ATXN1 Increases Amyloid -Protein Levels
(cid:1) (cid:1)
by Potentiating -Secretase Processing of -Amyloid
Precursor Protein*
Receivedforpublication,October24,2009,andinrevisedform,December30,2009 Published,JBCPapersinPress,January22,2010,DOI10.1074/jbc.M109.079079
CanZhang,AndrewBrowne,DanielChild,JasonR.DiVito,JesseA.Stevenson,andRudolphE.Tanzi1
FromtheGeneticsandAgingResearchUnit,MassGeneralInstituteforNeurodegenerativeDiseases,DepartmentofNeurology,
MassachusettsGeneralHospital,HarvardMedicalSchool,Charlestown,Massachusetts02129-2060
Alzheimerdisease(AD)isadevastatingneurodegenerativedis- tanglescomposedofhyperphosphorylatedTau.AlthoughA(cid:1)42
easewithcomplexandstronggeneticinheritance.Fourgeneshave andA(cid:1)40arethetwoprimaryA(cid:1)species,A(cid:1)42ismoreprevalent
been established to either cause familial early onset AD (APP, thanA(cid:1)40inamyloidplaques.Considerablegenetic,biochemical,
PSEN1,andPSEN2)ortoincreasesusceptibilityforlateonsetAD and molecular biological evidence suggests that the excessive
(APOE). To date (cid:1)80% of the late onset AD genetic variance accumulation of A(cid:1)is the primary pathological event leading
remains elusive. Recently our genome-wide association screen toAD(2,4,5).A(cid:1)isproducedbyasequentialproteolyticcleavage
identified four novel late onset AD candidate genes. Ataxin 1 ofatypeItransmembraneprotein,(cid:1)-amyloidprecursorprotein
(ATXN1)isoneofthesefourADcandidategenesandhasbeen (APP) (6). The initial cleavage of APP can occur through (cid:2)- or
indicatedtobethediseasegeneforspinocerebellarataxiatype1, (cid:1)-secretase (or BACE1). (cid:2)-Secretase cleavage produces sAPP(cid:2)
whichisalsoaneurodegenerativedisease.Mountingevidencesug- andthe(cid:2)-C-terminalfragment(C83);(cid:1)-secretasecleavagepro-
geststhattheexcessiveaccumulationofA(cid:1),theproteolyticprod- duces sAPP(cid:1)and the (cid:1)-C-terminal fragment (C99). Following
uctof(cid:1)-amyloidprecursorprotein(APP),istheprimaryADpath- trophic factor deprivation, sAPP(cid:1)can be further cleaved by an
ologicalevent.Inthisstudy,weaskwhetherATXN1mayleadtoAD unidentified protease, to produce N-APP, which contains the
pathogenesisbyaffectingA(cid:1)andAPPprocessingutilizingRNA N-terminal286aminoacidsofAPP(7).C83andC99canbefur-
interferenceinahumanneuronalcellmodelandmouseprimary thercleavedby(cid:3)-secretasetoproduceP3orA(cid:1).
cortical neurons. We show that knock-down of ATXN1 signifi- ADisageneticallycomplexdiseaseandonlyfourgeneshave
cantly increases the levels of both A(cid:1)40 and A(cid:1)42. This effect beenestablishedtoeithercauseearlyonsetautosomaldominant
could be rescued with concurrent overexpression of ATXN1. AD with complete penetrance (APP,PSEN1, andPSEN2) or to
Moreover,overexpressionofATXN1decreasedA(cid:1)levels.Regard- increasesusceptibilityforlateonsetADwithpartialpenetrance
ingtheunderlyingmolecularmechanism,weshowthattheeffect (APOE) (3). All four confirmed genes increase the absolute A(cid:1)
ofATXN1expressiononA(cid:1)levelsismodulatedvia(cid:1)-secretase levelsortheratiosofA(cid:1)42toA(cid:1)40,whichenhancestheoligomer-
cleavageofAPP.Takentogether,ATXN1functionsasageneticrisk izationofA(cid:1)intoneurotoxicassemblies(3,5).Todate(cid:1)80%of
modifierthatcontributestoADpathogenesisthroughaloss-of- thelateonsetADgeneticvarianceremainselusive(8).Ourlabo-
functionmechanismbyregulating(cid:1)-secretasecleavageofAPPand ratory recently performed a genome-wide association screen
A(cid:1)levels. (GWAS)andidentifiedfournovellateonsetADcandidategenes
thatachievedgenome-widestatisticalsignificance(beyondAPOE)
(9).AmongthefourADcandidategenes,Ataxin1(ATXN1)has
Alzheimer disease (AD)2 is a devastating neurodegenerative alsobeenshowntocausespinocerebellarataxiatype1(SCA1).As
disorderthatisclinicallycharacterizedbydeteriorationofmem- adifferentneurodegenerativediseasefromAD,SCA1ischarac-
oryandcognitivefunction,progressiveimpairmentofdailyliving terizedbyataxia,progressivemotordeterioration,andlossofPur-
activities,andseveralneuropsychiatricsymptoms(1).Onthecel- kinjecellsinthecerebellum(10,11).
lularandmolecularlevels,thepathophysiologyofADischaracter- IthasbeenshownthatATXN1leadstoSCA1throughaprimary
izedbytwodistinctivefeatures:amyloidplaquescomprisedpri- gain-of-function mechanism through the expanded polyglu-
marily of a small peptide named A(cid:1)(2–4), and neurofibrillary taminetractandfunctionaldomains(11).However,thecellular
andmolecularmechanismbywhichATXN1contributestoAD
*Thisworkwassupported,inwholeorinpart,bytheNIMH,theCureAlzhei- pathogenesisisstillunknown.Thus,inourcurrentstudy,weaim
mersFund,andtheMassachusettsGeneralHospitalFundforMedicalDis- to characterize the biological roles of ATXN1 and address the
covery(FMD). molecularmechanismbywhichATXN1affectsADpathogenesis.
1Towhomcorrespondenceshouldbeaddressed:HarvardMedicalSchool,
Determining ATXN1-mediated pathological events in AD will
GeneticsandAgingResearchUnit,MassGeneralInstituteforNeurodegen-
erative Disease, Massachusetts General Hospital, 114 16th St., Charles- helpdevelopabetterunderstandingofADpathogenesisandiden-
town, MA 02129. Tel.: [phone]; Fax: [phone]; E-mail: tifynovelADtherapeutictargets.
[email protected].
2Theabbreviationsusedare:AD,Alzheimerdisease;APP,(cid:1)-amyloidpro-
teinprecursor;A(cid:1),amyloid(cid:1)-protein;ATXN1,ataxin1;SCA1,spinocer- EXPERIMENTALPROCEDURES
ebellarataxiatype1;GWAS,genome-wideassociationscreen;siRNA,
Cell Culture and Mouse Primary Cortical Neuron Culture—
small interfering RNA; ELISA, enzyme-linked immunosorbent assay;
CMV,cytomegalovirus. The H4 human neuroglioma cell line stably overexpressing
MARCH19,2010•VOLUME285•NUMBER12 JOURNALOFBIOLOGICALCHEMISTRY 8515
This is an Open Access article under the CC BY license.
ATXN1ModulatesAPPProcessingandA(cid:1)Levels
human APP751 (H4-APP751) has been reported previously represented as relative values by comparing to control
(12,13).ThestableH4-APP-C99celllinestablyoverexpressing treatment.
APP-C99 has been described previously (13). APP-C99, the Cell Lysis and Protein Amount Quantification—Cells were
product of (cid:1)-secretase, contains (cid:2)- and (cid:3)-secretase (but not lysedintheMammalianProteinExtractionReagent(Thermo-
(cid:1)-secretase)sites.ThestableH4-APP-C99celllineprovidesa scientific)with1(cid:2)Haltproteaseinhibitormixture(Thermo-
validsystemtoassesswhetheranyeffectsonAPPprocessing scientific). The lysates were collected and centrifuged at
aredependenton(cid:1)-secretase.Thesecelllineswereculturedin 13,000(cid:2)gfor20min.Pelletswerediscardedandsupernatants
Dulbecco’smodifiedEagle’smediumsupplementedwith10% weretransferredintoanewEppendorftube(16).Totalproteins
fetalbovineserum,2mML-glutamine,100units/mlofpenicil- werequantifiedbytheBCAproteinassaykit(Pierce).
lin,100(cid:4)g/mlofstreptomycin,and200(cid:4)g/mlofG418.Mouse
Western Blotting Analysis—Western blotting analysis was
primarycorticalneuronswerefromBrainbits(E18)andwere
carriedoutbythemethoddescribedpreviously(15,16).Briefly,
cultured in B27/Neurobasal medium supplemented with 1(cid:2)
after centrifugation and protein concentration measurement,
GlutaMAX(Invitrogen).
anequalamountofeachproteinsamplewasappliedforelec-
Plasmids, Chemicals, and Antibodies—The ATXN1-cDNA
trophoresisfollowedbymembranetransfer,antibodyincuba-
that overexpresses ATXN1 (Origene Inc. number SC314762)
tion, and signal development. The VersaDoc imaging system
wasinsertedintoapCMV-derivedvector(OrigeneInc.number
(Bio-Rad)wasusedtodeveloptheblotsandQuantityOnesoft-
PCMV6XL5).TheAPPC-terminalantibody(targetingthelast
ware(Bio-Rad)wasusedtoquantifytheproteinsofinterestby
19aminoacidsofAPP751,APP750,orAPP695;A8717;1:1000)
was purchased from Sigma. The sAPP(cid:1)antibody (targeting subtractingthebackground,followingtheprotocolsdescribed
ISEVKM,theCterminusofhumansAPP(cid:1)wildtype,2(cid:4)g/ml previously(15,16).
RNA Extraction and Quantitative Polymerase Chain Re-
or1:50)wasfromIBL.The6E10,anti-APPantibodywaspur-
chased from Covance and utilized for detection of sAPP(cid:2) action—RNAwasextractedusingtheRNeasyminikit(Qiagen
(1:1000).TheATXN1antibodies(76-3and76-8)werefromthe Inc.) and was described previously (15). RNA concentration
University of California, Davis/National Institutes of Health wasmeasuredusingtheNanoDropND-1000Spectrophotom-
NeuroMab Facility (1:1000). (cid:1)-Actin antibody (1:10,000) was eter(ThemofisherInc.).EqualquantitiesofRNAsampleswere
purchased from Sigma. The horseradish peroxidase-conju- subjected to cDNA synthesis using the SuperScript III first
gated secondary antibodies (anti-mouse and anti-rabbit) strand synthesis system (Invitrogen). We used a multiplex
(1:10,000)werepurchasedfromPierce. system to measure the relative amount of cDNA. Primers/
siRNAs—Small interfering RNA (siRNA) duplexes were probes that targeted our gene of interest were labeled with
obtained from Dharmacon, Inc. Four different individual on- FAM490 (Applied Biosystems, Inc.; ATXN1, Hs00165656_
target siRNAs were synthesized to target different regions of m1 or Mm00485928_m1; and APP, Hs01552283_m1 or
ATXN1: A, GGGAATAGGTTTACACAAA; B, GGTCTA- Mm00431827_m1).Thehousekeepinggene,(cid:1)-actin,wasused
ATGTAGGCAAGTA; C, CCAGCCAGCTCTTTGATTT; D, as the endogenous control and was labeled with a VIC/MGB
GAAGAACGGCTCTGTTAAA.Asmart-poolon-targetsiRNA probe (Applied Biosystems, Inc.; human, 4326315E; mouse,
was also obtained from Dharmacon, Inc. in which the four 4352341E). 1:10 diluted cDNAs were mixed with 2(cid:2) PCR
siRNAswerecombinedinequalmolarconcentrations(repre- UniversalMasterMix(AppliedBiosystems,Inc.)andamplified
sentedbyATXN1-consructEsiRNA).ThecontrolsiRNAwas
using an iCycler Real-time PCR System following the manu-
a scrambled siRNA from Dharmacon, Inc. The smart-pool
facturer’s directions (Bio-Rad). To determine differences in
accellsiRNAtargetingmouseATXN1andcontrolaccellsiRNA mRNA levels from the treatments, we utilized the (cid:3)(cid:3)C
wereobtainedfromDharmacon,Inc. t
method(17).
Transfection—Transfectionsofon-targetsiRNAswereper- Data Analysis—A(cid:1)40 and A(cid:1)42, as well as sAPP(cid:2) and
formedusingthe96-wellnucleofectionshuttlesystemfrom
sAPP(cid:1)levels were normalized to the BCA values from the
Lonza(previouslyAmaxa;SFsolution;DS137program)and
samecelllysates(14).(cid:1)-ActinwasusedintheWesternblot-
have been reported previously (12, 14). Cells were mixed
tinganalysisorquantitativePCRanalysistoaccountforany
with siRNA or plasmid DNA, and resuspended in transfec-
differences in loading. The levels of proteins, e.g. ATXN1,
tionsolutionaccordingtothemanufacturer’sprotocol.The
C83, and full-length APP, were normalized to the corre-
transfectedcellswereharvested48haftertransfection.The
spondingvaluesfromthesamelane.ThenormalizedsAPP(cid:2)
mouse primary cortical neurons were transfected with
and sAPP(cid:1)values were divided by normalized full-length
the accell ATXN1 siRNA or control siRNAs as recom-
APP levels from each sample. The values from the ATXN1
mendedbythemanufacturer’sprotocolsandharvested72h
aftertransfection. siRNA treatment were normalized to control siRNA treat-
A(cid:1)Measurement—A(cid:1)measurementwasperformedfollow- ment. The samples for each treatment were from at least
ing the manufacturer’s suggested protocols and as described threeforeachexperimentalgroupandweredemonstratedas
previously (15). In brief, A(cid:1)40 and A(cid:1)42 levels (pg/ml) were mean (cid:4) S.E. We used a two-tailed t test, as appropriate, to
quantified using a sandwich enzyme-linked immunosorbent compare the differences between two groups. The Bonfer-
(ELISA)assay(WakoandSignet).A(cid:1)40andA(cid:1)42levelswere roni correction analysis was used to correct for multiple
normalizedtotheproteinconcentrationsfromthecelllysates. comparisonswithinasingleexperiment.pvalue(cid:5)0.05was
NormalizedA(cid:1)40andA(cid:1)42valuesfromthetreatmentswere consideredstatisticallysignificant.
8516 JOURNALOFBIOLOGICALCHEMISTRY VOLUME285•NUMBER12•MARCH19,2010
ATXN1ModulatesAPPProcessingandA(cid:1)Levels
FIGURE1.Down-regulationofATXN1significantlyincreasesA(cid:1)40andA(cid:1)42levelsinH4-APP751cells.StableH4-APP751cellsweretransientlytrans-
fectedwithcontrolsiRNA(siCtrl)ordifferentATXN1siRNAconstructs(siATXN1)andharvested48hpost-transfection.CelllysatesweresubjectedtoWestern
blottinganalysistoassessATXN1protein,andconditionedmediumwasappliedtoELISAanalysistomeasureA(cid:1)40andA(cid:1)42levelsasdescribedunder
“ExperimentalProcedures.”AandB,quantitativeWesternblottinganalysisshowedthatallATXN1siRNAtreatmentssignificantlydecreasedATXN1protein
levelscomparedwithcontrolsiRNAtreatment.C,ATXN1siRNAtreatmentincreasedA(cid:1)40levelscomparedwithcontrolsiRNAtreatment.D,ATXN1siRNA
treatmentincreasedtheA(cid:1)42levelscomparedwithcontrolsiRNAtreatment.E,ATXN1siRNAtreatmentdidnotaltertheratiosofA(cid:1)42:A(cid:1)40comparedwith
controlsiRNAtreatment(p(cid:6)0.05).n(cid:7)4foreachexperimentgroup;mean(cid:4)S.E.;*,p(cid:5)0.05;**,p(cid:5)0.01versussiCtrl.
RESULTS protein levels by quantitative analysis compared with con-
Down-regulation of ATXN1 Increases A(cid:1)40 and A(cid:1)42 trol siRNA treatment (siATXN1-A, 79.5%; siATXN1-B,
Levels in H4-APP751 Cells—Our first aim was to study 85.0%; siATXN1-C, 74.5%; siATXN1-D, 78.3%; siATXN1-E,
whetherdown-regulationofendogenousATXN1couldalter 81.1%)(p(cid:5)0.01)(Fig.1,AandB).
A(cid:1)levels.Wefirstestablishedtheexperimentalconditions WenextmeasuredA(cid:1)40andA(cid:1)42levelsintheconditioned
under which ATXN1 siRNA treatment could significantly medium48hafterthetransfection.A(cid:1)40andA(cid:1)42levelswere
decrease ATXN1 protein levels in stable H4-APP751 cells. measuredusingELISAandnormalizedtothecelllysateprotein
H4-APP751 cells were transfected with different ATXN1 concentrationfromthesamesample.AllfivedifferentATXN1
siRNAs (siATXN1) or control siRNA (siCtrl) using the siRNAtreatmentsincreasedA(cid:1)40levelscomparedwithcon-
Amaxanucleofector(13,14).Cellswereharvested48hpost- trol siRNA treatment (43.3, 73.3, 99.0, 80.9, and 102.1% by
transfectionandcelllysateswerepreparedforWesternblot siATXN1constructsA,B,C,D,andE,respectively)(Fig.1C).In
analysis.TheATXN1antibody(76-3)hasbeenreportedpre- addition,allfivedifferentATXN1siRNAtreatmentsincreased
viouslyandwasusedtodetectATXN1protein(18).(cid:1)-Actin, A(cid:1)42 levels compared with control siRNA treatment (52.5,
ahousekeepinggene,wasusedasanegativecontrol.Allthe 72.9,152.6,174.0,and135.4%bysiATXN1constructsA,B,C,
ATXN1 siRNA treatments significantly decreased ATXN1 D, and E, respectively) (Fig. 1D). Collectively, these findings
MARCH19,2010•VOLUME285•NUMBER12 JOURNALOFBIOLOGICALCHEMISTRY 8517
ATXN1ModulatesAPPProcessingandA(cid:1)Levels
8518 JOURNALOFBIOLOGICALCHEMISTRY VOLUME285•NUMBER12•MARCH19,2010
ATXN1ModulatesAPPProcessingandA(cid:1)Levels
showed that knock-down of ATXN1 significantly increased treatment significantly increased A(cid:1)40 levels by 30.4% (p (cid:5)
bothA(cid:1)40andA(cid:1)42levels. 0.01)andtheA(cid:1)42levelsby88.0%(p(cid:5)0.05)(Fig.2E).Addi-
We also assessed whether ATXN1 siRNA treatment in- tionallyourdatashowedthatATXN1siRNAtreatmentdidnot
creasedtheratioofA(cid:1)42:A(cid:1)40.Ithasbeenshownthatmost significantlyaltertheratiosofA(cid:1)42:A(cid:1)40comparedwithcon-
familialADmutationsinAPP,PSEN1,andPSEN2increasethe trolsiRNAtreatment(p(cid:6)0.05)(Fig.2F).
ratio of A(cid:1)42:A(cid:1)40, which drives the aggregation of A(cid:1)into WealsovalidatedtheeffectofATXN1knock-downonA(cid:1)40
neurotoxic oligomeric assemblies (3, 5). The ratio of A(cid:1)42: andA(cid:1)42levelsinmouseprimarycorticalneurons.Themouse
A(cid:1)40 in cells treated with ATXN1 siRNAs had no significant corticalneuronsweretransfectedwithATXN1accellsiRNAor
differencecomparedwithcellstreatedwithcontrolsiRNA(p(cid:6) control siRNA and harvested 72 h post-transfection. Cell
0.05)(Fig.1E). lysateswereappliedtoquantitativeWesternblottinganalysis
ValidationofATXN1Knock-downEffectsonA(cid:1)40andA(cid:1)42 andATXN1antibody(76-8)wasutilizedtorevealtheATXN1
Levels—Next, we validated the down-regulation effect of protein. ATXN1 siRNA treatment significantly decreased
ATXN1onA(cid:1)levelsbyoverexpressionofATXN1.H4-APP751 ATXN1proteinlevelsby40.0%comparedwithcontrolsiRNA
cells were transfected with control siRNA (siCtrl) and/or treatment (p (cid:5) 0.05) (Fig. 2, G and H). Conditioned medium
ATXN1siRNA(siATXN1),aswellastheemptyvector(pCMV) wasappliedtoELISAanalysistomeasureA(cid:1)40andA(cid:1)42lev-
and/orATXN1-cDNAandappliedtoWesternblottinganalysis els. Normalized A(cid:1)40 and A(cid:1)42 levels from ATXN1 siRNA
andELISAasdescribedunder“ExperimentalProcedures.”The treatment were compared with control. The ATXN1 siRNA
combination of siATXN1/pCMV treatment significantly de- treatment significantly increased A(cid:1)40 levels by 49.8% (p (cid:5)
creasedATXN1proteinlevelscomparedwiththesiCtrl/pCMV 0.05) and the A(cid:1)42 levels by 19.5% compared with control
treatment,asexpected(Fig.2A).ThecombinationofsiATXN1/ siRNAtreatment(p(cid:5)0.05)(Fig.2I).Additionallythereexisted
ATXN1treatmentmarkedlyincreasedATXN1proteinlevels, nodifferencesontheratiosofA(cid:1)42:A(cid:1)40betweenthesamples
anddecreasedbothA(cid:1)40andA(cid:1)42levels,comparedwiththe fromATXN1siRNAtreatmentandthosefromcontrolsiRNA
siCtrl/pCMVtreatment(p(cid:5)0.05)(Fig.2,AandB).ThesiCtrl/ treatment (p (cid:6) 0.05) (Fig. 2J). Collectively, these data from
ATXN1treatmentdramaticallyincreasedATXN1proteinlev- naive H4 cells and mouse primary cortical neurons recapitu-
els,anddecreasedbothA(cid:1)40andA(cid:1)42levels,comparedwith latedthosefromH4-APP751cells,supportingthatthesilenc-
the siCtrl/pCMV treatment (Fig. 2, A and B). There was no ingofendogenousATXN1leadstoincreasedA(cid:1)40andA(cid:1)42
significantdifferenceineitherA(cid:1)40orA(cid:1)42levelsbetweenthe levels.
siATXN1/ATXN1andsiCtrl/ATXN1(p(cid:6)0.05).Thismightbe ATXN1 Loss of Function Elevates A(cid:1)Levels by Modulating
due to the effect of ATXN1, which is endogenously highly APP Processing—After we found that knock-down of ATXN1
expressed(11)andbecomessaturatedduringoverexpression. increased A(cid:1) levels, we studied whether knock-down of
Thus,theATXN1siRNAeffectonbothA(cid:1)40andA(cid:1)42levelsis ATXN1canaffectAPPproteinlevelsanditsproteolyticproc-
not an off-target effect, and can be rescued by concurrently essing. First, stable H4-APP751 cells were transfected with
introducingtheATXN1cDNA. different ATXN1 siRNAs and control siRNA. Conditioned
NextweaskedwhethertheeffectsofATXN1knock-downon mediumandcelllysateswerecollected48hpost-transfection
A(cid:1)40andA(cid:1)42levelsinH4-APP751cellscanalsobeobserved andsubjectedtoWesternblottinganalysis.AntibodyAPP8717
in naive H4 cells. Naive H4 cells were transiently transfected was used to detect full-length APP and its C-terminal frag-
withcontrolsiRNAortheATXN1siRNAandharvested48h ments.(cid:1)-Actinantibodywasusedastheloadingcontrol.The
post-transfection.CelllysateswereappliedtoWesternblotting ATXN1siRNAs(siATXN1-C,-D,and-E)didnotchangefull-
analysis. ATXN1 antibody (76-3) was utilized to reveal the length APP levels (p (cid:6) 0.05), whereas the ATXN1 siRNA
ATXN1 protein. Quantitative Western blotting analysis (siATXN1-Aand-B)modestlyincreasedfull-lengthAPPlevels
showedthatATXN1siRNAtreatmentsignificantlydecreased by(cid:1)30%(p(cid:5)0.05)(Fig.3,AandB).
ATXN1proteinlevelsby85.7%(p(cid:5)0.01versuscontrol)(Fig.2, We next examined whether ATXN1 down-regulation af-
C and D). Conditioned medium from the treatments was fectedAPPprocessingbyassessingtheratiosofitsproteolytic
applied to ELISA analysis to measure A(cid:1)40 and A(cid:1)42 levels, cleavage fragment levels to full-length APP levels. ATXN1
and then normalized to the corresponding cell lysate protein siRNAtreatmentsdidnotsignificantlyaltertheabsoluteC83
concentrations. Normalized A(cid:1)40 and A(cid:1)42 levels from levelscomparedwithcontrol(p(cid:6)0.05)(Fig.3,AandB).But
ATXN1siRNAtreatmentswerecomparedwiththevaluesfrom ATXN1siRNAtreatmentssignificantlydecreasedtheratioof
control siRNA treatment. It was shown that ATXN1 siRNA C83:APP-FL compared with control siRNA treatment (18.0,
FIGURE2.ValidationofATXN1loss-of-functioneffectonA(cid:1)levels:itcanberescuedbyATXN1overexpressioninH4-APP751cells;andadditionally
ATXN1siRNAelevatedA(cid:1)levelsinnaiveH4cellsandmouseprimarycorticalneurons.A,H4-APP751cellsweretransfectedwithcontrolsiRNA(siCtrl)
and/orATXN1siRNA(siATXN1),aswellastheemptyvector(pCMV)and/orATXN1-cDNAandappliedtoWesternblottinganalysisasdescribedunder“Experi-
mentalProcedures.”B,samplesinAwereappliedtoELISAasdescribedunder“ExperimentalProcedures.”ATXN1cDNAnotonlydecreasedA(cid:1)40andA(cid:1)42
levels(comparingsiCtrl/pCMVandsiCtrl/ATXN1),butalsorescuedtheATXN1siRNAeffectonA(cid:1)40andA(cid:1)42levels(comparingsiATXN1/pCMVandsiATXN1/
ATXN1).C–F,naiveH4cellsweretransfectedwithcontrolsiRNA(siCtrl)orATXN1siRNA(siATXN1)andappliedtoWesternblottinganalysisorELISAasdescribed
under“ExperimentalProcedures.”ATXN1siRNAtreatmentsignificantlydecreasedATXN1proteinlevels(CandD),aswellasincreasedbothA(cid:1)40andA(cid:1)42
levels(E),butdidnotsignificantlychangetheratiosofA(cid:1)42:A(cid:1)40(F).G–J,mouseprimarycorticalneuronsweretransfectedwithcontrolsiRNAorATXN1siRNA
andappliedtoWesternblottinganalysisorELISAasdescribedunder“ExperimentalProcedures.”ATXN1siRNAtreatmentsignificantlydecreasedATXN1protein
levels(GandH),andincreasedbothA(cid:1)40andA(cid:1)42levels(I),butdidnotsignificantlychangetheratiosofA(cid:1)42:A(cid:1)40(J)comparedwithcontrolsiRNA
treatment.n(cid:7)3foreachexperimentalgroup;mean(cid:4)S.E.;*,p(cid:5)0.05;**,p(cid:5)0.01versuscontrol.
MARCH19,2010•VOLUME285•NUMBER12 JOURNALOFBIOLOGICALCHEMISTRY 8519
ATXN1ModulatesAPPProcessingandA(cid:1)Levels
FIGURE3.Down-regulationofATXN1altersAPPprocessingactivity.A,H4-APP751cellsweretransfectedwithATXN1siRNAorcontrolsiRNA(siCtrl)
andharvested48hpost-transfection.CelllysateswereappliedtoWesternblottinganalysisasdescribedunder“ExperimentalProcedures.”B,graphic
representationofdatafromAasdescribedunder“ExperimentalProcedures.”TheATXN1siRNAs(siATXN1-C,-D,and-E)didnotchangefull-lengthAPP
levels(p(cid:6)0.05),whereastheATXN1siRNA(siATXN1-Aand-B)modestlyincreasedfull-lengthAPPlevels(p(cid:5)0.05).TheATXN1siRNAtreatmentdidnot
alterC83levelsalone,butdecreasedtheratioofC83:full-lengthAPPcomparedwithcontrolsiRNAtreatment(n(cid:7)4).C,naiveH4cellstreatedwithATXN1
siRNAorcontrolsiRNAwereappliedtoWesternblottinganalysisasdescribedunder“ExperimentalProcedures.”D,graphicrepresentationofdatafrom
Casdescribedunder“ExperimentalProcedures.”ATXN1siRNAtreatmentdidnotsignificantlychangefull-lengthAPPlevels(n(cid:7)3).E,mouseprimary
corticalneuronsweretreatedwithATXN1siRNAorcontrolsiRNAandappliedtoWesternblottinganalysisasdescribedunder“ExperimentalProce-
dures.”F,graphicrepresentationofdatafromE.ATXN1siRNAtreatmentdidnotsignificantlychangefull-lengthAPPlevels.n(cid:7)3foreachexperimental
group;mean(cid:4)S.E.;*,p(cid:5)0.05;**,p(cid:5)0.01versuscontrol.
32.6,28.9,23.5,and26.6%byATXN1constructsA,B,C,Dand ThenweassessedtheeffectsofATXN1down-regulationin
E,respectively)(Fig.3,AandB).Thus,down-regulationofwild naiveH4cellsandmouseprimarycorticalneurons.NaiveH4
typeATXN1modulatesAPPprocessing. cells were transfected with human ATXN1 siRNA or control
8520 JOURNALOFBIOLOGICALCHEMISTRY VOLUME285•NUMBER12•MARCH19,2010
ATXN1ModulatesAPPProcessingandA(cid:1)Levels
FIGURE4.Knock-downofATXN1potentiates(cid:1)-secretaseprocessingofAPP.A,H4-APP751cellsweretransfectedwithdifferentATXN1siRNAsand
controlsiRNA(siCtrl)andharvested48hpost-transfection.ConditionedmediumwasappliedtoWesternblottinganalysisasdescribedunder“Exper-
imentalProcedures.”B,graphicrepresentationofdatafromA.ThesAPP(cid:2)levelsweredividedbyfull-lengthAPPlevelsfromthesamesamplesto
representtheratioofsAPP(cid:2):APP-FL.ATXN1siRNAtreatmentdidnotchangethesAPP(cid:2)levels(p(cid:6)0.05versuscontrol).ATXN1siRNAtreatmentdidnot
significantlyaltertheratioofsAPP(cid:2)tofull-lengthAPP(p(cid:6)0.05versuscontrol).C,H4-APP751cellsweretransfectedwithdifferentATXN1siRNAsand
controlsiRNAandharvested48hpost-transfection.ThesAPP(cid:1)-specificantibodywasusedtodetectsAPP(cid:1)intheconditionedmedium.D,graphic
representationofdatafromC.ThesAPP(cid:1)levelswerecomparedwithfull-lengthAPPlevelsfromthesamesamples.ATXN1siRNAtreatmentelevated
boththesAPP(cid:1)levelsandtheratioofsAPP(cid:1)tofull-lengthAPP.n(cid:7)4foreachexperimentalgroup;mean(cid:4)S.E.;*,p(cid:5)0.05;**,p(cid:5)0.01versus
correspondingcontrols.
siRNA.Mouseprimarycorticalneuronsweretransfectedwith malizedthemtothecelllysateproteinlevels.Wethencom-
mouseaccellATXN1siRNAorcontrolsiRNA.Cellswerehar- paredthenormalizedsAPP(cid:1)levelstofull-lengthAPPlevels
vested and applied to Western blotting analysis. Antibody (normalized to (cid:1)-actin). Each of the ATXN1 siRNA treat-
APP8717wasusedtodetectfull-lengthAPP.(cid:1)-Actinantibody ments increased sAPP(cid:1) levels and the ratio of sAPP(cid:1):
wasusedtodetect(cid:1)-actin,whichwasusedaloadingcontrol. APP-FL compared with control siRNA treatment (Fig. 4, C
TheATXN1siRNAtreatmentdidnotsignificantlychangefull- and D). Specifically, sAPP(cid:1) levels were increased 118.3,
lengthAPPlevelsinnaiveH4cells(p(cid:6)0.05)(Fig.4,CandD)or 167.0,98.1,81.1,and157.0%bytreatmentofsiATXN1con-
inmouseprimarycorticalneurons(p(cid:6)0.05)(Fig.3,EandF). structsA,B,C,D,andE,respectively.TheratiosofsAPP(cid:1):
Takentogether,down-regulationofwildtypeATXN1increases APP-FLwereincreasedby75.3,108.2,67.8,62.0,and95.6%
A(cid:1)levels via modulating APP processing, rather than APP bytreatmentofsiATXN1constructsA,B,C,DandE,respec-
levels. tively(Fig.4,CandD).
ATXN1LossofFunctionPotentiatesBACE1CleavageofAPP— Validation of ATXN1 Knock-down Affecting A(cid:1)Levels and
To further study APP proteolytic processing, we assessed APP Processing through (cid:1)-Secretase—We have shown that
secretedAPPproducts,includingsAPP(cid:2)andsAPP(cid:1),fromthe knock-downofATXN1significantlypotentiated(cid:1)-secretase
conditionedmedium.6E10antibodywasusedtodetectsAPP(cid:2) cleavageofAPP,whichincreasesbothA(cid:1)40andA(cid:1)42levels.
protein. sAPP(cid:2)levels were quantified and normalized to the Next, we utilized stable H4-APP-C99 cells to validate this
cell lysate protein levels from the same samples. The ratio of finding. The stable H4-APP-C99 cell line has saturated
sAPP(cid:2)tofull-lengthAPPwascalculatedbydividingnormal- (cid:1)-secretase activity and provides a valid system to assess
ized sAPP(cid:2)values by full-length APP values (normalized to whether any effects on APP processing are dependent on
(cid:1)-actin)fromthesamesamples.ATXN1siRNAtreatmentdid (cid:1)-secretase (12). First, H4-APP-C99 cells were transiently
notchangetheabsolutesAPP(cid:2)levelscomparedwithcontrol transfected with ATXN1 siRNA and control siRNA. Cells
siRNA treatment (p (cid:6) 0.05) (Fig. 4, A and B). Each of the wereharvested48hpost-transfectionandappliedtoWest-
ATXN1siRNAtreatmentsdecreasedtheratioofsAPP(cid:2)tofull- ern blotting analysis and ELISA. ATXN1 siRNA treatment
lengthAPPlevels,butdidnotreachastatisticallysignificance markedly decreased ATXN1 protein levels (Fig. 5A). ELISA
level(p(cid:6)0.05)(Fig.4B). revealedthattherewasnodifferenceinA(cid:1)40orA(cid:1)42levels
ThesAPP(cid:1)antibodywasusedtodetectthesAPP(cid:1)protein inthecellstreatedwithATXN1siRNAcomparedwiththose
fromthecellmedium.WequantifiedsAPP(cid:1)levelsandnor- treatedwithcontrol(p(cid:6)0.05)(Fig.5B).Next,H4-APP-C99
MARCH19,2010•VOLUME285•NUMBER12 JOURNALOFBIOLOGICALCHEMISTRY 8521
ATXN1ModulatesAPPProcessingandA(cid:1)Levels
FIGURE5.ModulationofATXN1levelsdoesnotalterAPPprocessingorA(cid:1)levelsinH4-APP-C99cells.AandB,H4-APP-C99cellsweretransfectedwith
ATXN1siRNAorcontrolsiRNA(siCrtl)andharvested48hpost-transfection.CelllysatesandconditionedmediumwereappliedtoWesternblottinganalysisand
ELISA,respectively,asdescribedunder“ExperimentalProcedures.”ATXN1siRNAtreatmentmarkedlydecreasedtheATXN1proteinlevel.B,therewereno
differencesinA(cid:1)40orA(cid:1)42levelsbetweenthecellstreatedwithATXN1siRNAandthosetreatedwithcontrolsiRNA.CandD,H4-APP-C99cellswere
transfectedwithATXN1-cDNAorpCMVemptyvectorandharvested48hpost-transfection.CelllysatesandconditionedmediumwereappliedtoWestern
blottinganalysisandELISA.C,ATXN1-cDNAtreatmentmarkedlyincreasedATXN1proteinlevels.D,ATXN1-cDNAdidnotchangeeitherA(cid:1)40orA(cid:1)42levels(p(cid:6)
0.05;versuscontrol).EandF,celllysatesfromA(H4-APP-C99cellstreatedwithATXN1orcontrolsiRNAs)wereappliedtoWesternblottinganalysis,andprobed
withAPP8717and(cid:1)-actinantibodies.F,graphicrepresentationofdatafromE.ThereexistednosignificantdifferencesinthelevelsofAPP-C83orAPP-C99.n(cid:7)
3foreachexperimentalgroups;mean(cid:4)S.E.;*,p(cid:5)0.05;**,p(cid:5)0.01versuscorrespondingcontrols.
cellsweretransientlytransfectedwithATXN1-cDNAorthe AlterationinsAPP(cid:1)LevelsCorrelateswithChangesinA(cid:1)40
emptyvector.ATXN1-cDNAtreatmentmarkedlyincreased Levels,butNotA(cid:1)42Levels—Collectively,ourdatashowedthat
ATXN1proteinlevels,butdidnotchangethelevelsofA(cid:1)40 ATXN1significantlyalteredthelevelsofA(cid:1)andsAPP(cid:1).Here
orA(cid:1)42comparedwithcontrol(p(cid:6)0.05)(Fig.5,CandD). weaskedwhethertherewasacorrelationbetweenthelevelsof
Additionally,Westernblottinganalysiswithanti-APPanti- A(cid:1)40orA(cid:1)42,andsAPP(cid:1),byconsolidatingthepreviousdata
bodyAPP8717revealednodetectabledifferenceinthepro- inaregressionstudy.LevelsofsAPP(cid:1)wereplottedwithlevels
tein levels of C83 and C99 in cells treated with ATXN1 ofA(cid:1)40orA(cid:1)42fromthesamesamples,andrepresentedasa
siRNAcomparedwiththosetreatedwithcontrolsiRNA(p(cid:6) -foldchangebycomparingthesiATXN1-treatedsamplestothe
0.05)(Fig.5,EandF).Thus,knock-downofATXN1hadno control. The x axis was represented by the -fold changes in
effectonAPPprocessingorA(cid:1)levelsinH4-APP-C99cells, sAPP(cid:1)levels,andtheyaxiswasrepresentedbythe-foldchange
whichsuggestedthatATXN1levelsmodulateAPPprocess- inA(cid:1)40orA(cid:1)42levels.Thisanalysisrevealedasignificantcor-
ing and amyloid (cid:1)-protein levels via a (cid:1)-secretase-depen- relationbetweensAPP(cid:1)andA(cid:1)40(p(cid:5)0.05;Fig.6A),butnot
dent/(cid:3)-secretase-independentmechanism. betweensAPP(cid:1)andA(cid:1)42(p(cid:6)0.05;Fig.6B).Theresultsug-
8522 JOURNALOFBIOLOGICALCHEMISTRY VOLUME285•NUMBER12•MARCH19,2010
ATXN1ModulatesAPPProcessingandA(cid:1)Levels
FIGURE6.AlterationinsAPP(cid:1)levelssignificantlycorrelateswithchangesinA(cid:1)40levels,butnotA(cid:1)42levels.LevelsofsAPP(cid:1)wereplottedtogether
withlevelsofA(cid:1)40andA(cid:1)42fromthesamesamplesinpreviousexperiments.Thedatawererepresentedasapercentagechangebycomparingthe
siATXN1-treatedsamplestocontrol.ThexaxiswasrepresentedbythepercentagechangeinsAPP(cid:1)levels,andtheyaxisrepresentedbythepercentagechange
inA(cid:1)40orA(cid:1)42levels.Thelinerepresentsthelinearregressionforthisdata.TherewasasignificantcorrelationbetweenthechangesinsAPP(cid:1)levelsandthe
changesinA(cid:1)40levels(p(cid:5)0.05),butnotA(cid:1)42levels(p(cid:6)0.05).
gestedthatthechangesinsAPP(cid:1)andA(cid:1)40maysharecommon DISCUSSION
mechanismsmediatedbymodulationofATXN1.
Mounting evidence has shown that extended polyglu-
ATXN1 Knock-down Does Not Affect APP mRNA Levels or
taminemutantATXN1isassociatedwiththediseaseSCA1,
Protein Turnover Rate—It has been reported that both wild
however, little is known about its endogenous functions.
typeandextendedpolyglutaminemutantATXN1proteinscan
Matillaandcolleagues(21)haveperformedastudyfocused
function as transcriptional regulators (19, 20), therefore, we on assessing whether loss of function of ATXN1 affects the
asked whether ATXN1 knock-down increased A(cid:1)levels by pathophysiology of SCA1. Interestingly, mice homozygous
affectingAPPtranscription.NaiveH4cellsormouseprimary and heterozygous for ATXN1 (or Sca1 in the paper) null
cortical neurons were transfected with control siRNA or mutationwereviableandfertile(consistentwithourinvitro
ATXN1siRNAasdescribedunder“ExperimentalProcedures.” study that ATXN1 knock-down does not reveal cytotoxici-
CelllysatesweresubjectedtoRNAextractionandquantitative ty),3 and they did not display any signs of ataxia or loss of
PCR analysis utilizing the corresponding human or mouse
cerebellar Purkinje cells. Intriguingly, they found that
ATXN1probetodetectATXN1mRNAlevels.(cid:1)-Actinwasuti- ATXN1wasimportantinlearningtasksmediatedbythehip-
lizedastheinternalcontrol.ATXN1mRNAlevelsfromATXN1 pocampus and the cerebellum. First, these Sca1 null mice
siRNAtreatmentwerecomparedwiththelevelsfromcontrol displayed neurobehavioral abnormalities and were severely
siRNAtreatment.OurdatashowedthatATXN1siRNAtreat- impairedinthespatialversionoftheMorriswatermazetest,
mentsignificantlydecreasedATXN1mRNAlevelsby82.0%in suggesting the presence of motor learning deficits. Second,
naiveH4cells(p(cid:5)0.01)(Fig.7A),butdidnotalterAPPmRNA paired-pulsefacilitationwassignificantlydecreasedinSca1
levelscomparedwithcontrol(p(cid:6)0.05)(Fig.7B).Additionally, null mice of both homozygous and heterozygous genetic
inmouseprimarycorticalneurons,ATXN1siRNAtreatment backgrounds, although they revealed normal long-term
significantly decreased ATXN1 mRNA levels by 51.6% (p (cid:5) potentiation and post-tetanic potentiation analyses in the
0.01) (Fig. 7C), but did not significantly change APP mRNA CA1areaofthehippocampuscomparedwithcontrolmice.
levelscomparedwithcontrol(p(cid:6)0.05)(Fig.7D).Thus,these Although the underlying molecular mechanism behind
data suggested that the down-regulation of ATXN1 increases the learning and memory loss seen with these SCA1 null
A(cid:1)40andA(cid:1)42levelsbyamechanismotherthanmodulating miceisstillunidentified,theroleofATXN1inlearningand
APPtranscription. memoryisfurthersupportedbyourmostrecentADGWAS
Becauseweobservedthatknock-downofATXN1hadatrend genetics study (9). We showed that ATXN1 may be a risk
to increase APP protein levels in H4-APP751 cells, we asked factoroflateonsetAD(9).Interestingly,ourcurrentinvitro
whether it may be due to altering APP turnover rate. functional study has found that ATXN1 loss of function
H4-APP751cellsweretransfectedwithATXN1siRNAorcon- potentiates APP processing and increases A(cid:1)levels. Addi-
trolsiRNAfor42h,andthentreatedwith40(cid:4)g/mlofcyclo- tionally,emergingevidencehassuggestedthatalterationin
heximide for a different time period (0, 3, or 6 h). Cells were A(cid:1)levels may cause learning and neurobehavioral abnor-
harvestedandcelllysatesweresubjectedtoWesternblotting
malities(3,22).Therefore,wehypothesizethatthelearning
analysis as described under “Experimental Procedures.” Our and memory loss in SCA1 null mice may be caused by ele-
datashowedthatthereexistednosignificantdifferencesinthe vated A(cid:1)levels. A further examination on these SCA1 null
proteinlevelsoffull-lengthAPPat3and6hofcycloheximide
micewillberequiredtotestthishypothesis.
treatment (p (cid:6) 0.05 versus controls at corresponding time
point) (Fig. 7, E and F). Collectively, our data showed that
ATXN1knock-downdoesnotaffectAPPmRNAlevelsorAPP 3C.Zhang,A.Browne,D.Child,J.R.DiVito,J.A.Stevenson,andR.E.Tanzi,
proteinturnoverrate. unpublisheddata.
MARCH19,2010•VOLUME285•NUMBER12 JOURNALOFBIOLOGICALCHEMISTRY 8523
ATXN1ModulatesAPPProcessingandA(cid:1)Levels
FIGURE7.Knock-downofATXN1doesnotalterAPPmRNAlevelsorproteinturnoverrate.A,naiveH4cellsweretransfectedwithATXN1siRNAor
controlsiRNA.Cellswereharvested48hpost-transfectionandappliedtoquantitativePCRanalysis.ATXN1siRNAtreatmentsignificantlydecreased
ATXN1mRNAlevels.B,APPmRNAlevelsinthesamesamplesfromA.ATXN1siRNAtreatmentdidnotsignificantlyalterAPPmRNAlevels.C,mouse
primarycorticalneuronsweretransfectedwithATXN1siRNAorcontrolsiRNAasdescribedunder“ExperimentalProcedures.”Cellswereharvested72h
post-transfectionandappliedtoquantitativePCRanalysis.ATXN1siRNAtreatmentsignificantlydecreasedATXN1mRNAlevels.D,APPmRNAlevelsin
thesamesamplesfromC.ATXN1siRNAtreatmentdidnotsignificantlyalterAPPmRNAlevels(p(cid:6)0.05).E,H4-APP751cellsweretransfectedwithATXN1
siRNAorcontrolsiRNAfor42h,andthentreatedwith40(cid:4)g/mlofcycloheximideforadifferenttimeperiod(0,3,or6h).Cellswereharvestedandcell
lysatesweresubjectedtoWesternblottinganalysisasdescribedunder“ExperimentalProcedures.”F,graphicrepresentationofdatafromE.There
existednosignificantdifferencesintheproteinlevelsoffull-lengthAPPat3and6hofcycloheximidetreatment. n(cid:6)3foreachexperimentalgroup;
mean(cid:4)S.E.;*,p(cid:5)0.05;**,p(cid:5)0.01versuscorrespondingcontrols.
Although further study on SCA1 null mice is required to matelyleadingtoAD.Thismechanismisdifferentfromits
elucidatetheunderlyingmechanismbywhichATXN1leads primaryroleinSCA1,whichisprimarilyagainoffunction
to learning and memory loss and possibly other AD patho- causedbyitsextendedpolyglutaminetract(11).Additionally
physiology,ourrecentGWASstudyshowedthatATXN1is modulating ATXN1 levels by overexpression may be an
an important AD candidate gene (9). The mechanism by effective AD therapeutic approach because ATXN1 cDNA
which the polymorphism in ATXN1 confers AD risk is still overexpression leads to decreases in both A(cid:1)40 and A(cid:1)42
unknown. The polymorphism identified in our GWAS is levels. Due to the complex network that exists between
located in an intron region. ATXN1 has been shown to ATXN1anditsinteractingpartners(11,18,26),theATXN1-
undergo alternative splicing (10), and defects of alternative mediatedpathogenesisinADandSCA1iscomplexandmay
splicing can alter protein functions, and are emerging as be affected by multiple other risk components, e.g. genetic
majorcontributorstoADandotherneurodegenerativedis- andenvironmentalfactors.
eases(23,24).ItispossiblethatthepolymorphisminATXN1 Emerging evidence indicates that neurodegenerative dis-
mayaffectthealternativesplicingofATXN1,whichleadtoa easesmaysharecommoncellularandmolecularpathophysio-
loss-of-functioneffectintheATXN1proteinbyanunknown logicalfeatures,e.g.intracellularaggregatesandneurotoxicity.
mechanism. The loss of function of ATXN1 can potentiate Importantly, these features are commonly caused by protein
(cid:1)-secretaseprocessingofAPPandincreasesA(cid:1)levels,ulti- misfoldingandubiquitin-proteasomedegradationsystemdys-
8524 JOURNALOFBIOLOGICALCHEMISTRY VOLUME285•NUMBER12•MARCH19,2010