Regulation of Synaptic Structure by Ubiquitin C-Terminal Hydrolase L1

12 Pages·2009·3.76 MB·English

TheJournalofNeuroscience,June17,2009•29(24):7857–7868•7857
NeurobiologyofDisease
Regulation of Synaptic Structure by Ubiquitin C-Terminal
Hydrolase L1
AnnaE.Cartier,1,2,3StevanN.Djakovic,1AfshinSalehi,1ScottM.Wilson,4EliezerMasliah,2,3andGentryN.Patrick1
1SectionofNeurobiology,DepartmentofBiologicalSciences,UniversityofCalifornia,SanDiego,LaJolla,California92093-0347,Departmentsof
2Neurosciencesand3Pathology,UniversityofCalifornia,SanDiego,LaJolla,California92093-0624,and4DepartmentofNeurobiology,CivitanResearch
Center,UniversityofAlabama,Birmingham,Alabama35294
UbiquitinC-terminalhydrolaseL1(UCH-L1)isadeubiquitinatingenzymethatisselectivelyandabundantlyexpressedinthebrain,andits
activityisrequiredfornormalsynapticfunction.Here,weshowthatUCH-L1functionsinmaintainingnormalsynapticstructureinhippocam-
palneurons.WefoundthatUCH-L1activityisrapidlyupregulatedbyNMDAreceptoractivation,whichleadstoanincreaseinthelevelsoffree
monomericubiquitin.Conversely,pharmacologicalinhibitionofUCH-L1significantlyreducesmonomericubiquitinlevelsandcausesdramatic
alterationsinsynapticproteindistributionandspinemorphology.InhibitionofUCH-L1activityincreasesspinesizewhiledecreasingspine
density.Furthermore,thereisaconcomitantincreaseinthesizeofpresynapticandpostsynapticproteinclusters.Interestingly,however,ectopic
expressionofubiquitinrestoresnormalsynapticstructureinUCH-L1-inhibitedneurons.ThesefindingspointtoasignificantroleofUCH-L1in
synapticremodeling,mostlikelybymodulatingfreemonomericubiquitinlevelsinanactivity-dependentmanner.
Introduction disease(Leroyetal.,1998),leadstoa50%reductionincatalyticof
Theubiquitinproteasomesystem(UPS)isamajorcellularpath- UCH-L1activityinvitro,implyingthatlossofUCH-L1activity
wayforproteindegradationineukaryoticcells.TheUPSisin- mayreducetheavailabilityoffreeubiquitinandcontributetoan
volvedinthedevelopment,maintenance,andremodelingofsyn- impairedclearanceofproteinsbytheUPS.Furthermore,trans-
apticconnectionsinthemammalianCNS(Patrick,2006;Yiand genicmice,whichexpresstheI93Mmutant,exhibitasignificant
Ehlers,2007).UbiquitinC-terminalhydrolaseL1(UCH-L1)be- reductionindopaminergicneuronsinthesubstantianigraand
longstoafamilyofdeubiquitinatingenzymes(DUBs)compris- thedopaminecontentinthestriatum(Setsuieetal.,2007).
ingUCH-L1–5.Itisahighlyconservedproteinthatisselectively UCH-L1isalsocommonlyfoundintheneurofibrillarytan-
andabundantlyexpressedinneurons,representing1–2%oftotal gles observed in Alzheimer’s Disease (AD) brains in which the
solubleproteininthebrain(Wilkinsonetal.,1989).UCH-L1is levels of soluble UCH-L1 are decreased (Choi et al., 2004). A
known to generate free monomeric ubiquitin from ubiquitin recent study revealed that pharmacological inhibition of
precursors(Finleyetal.,1989;Larsenetal.,1998).Recentinvitro UCH-L1activityleadstoanimpairmentinsynaptictransmission
studieshaveshownthatUCH-L1possessesubiquitinligaseactivity andmaintenanceoflong-termpotentiation(LTP)(Gongetal.,
(Liuetal.,2002).Inadditiontoitsenzymaticactivities,UCH-L1 2006), a form of synaptic plasticity that is involved in learning
associates with ubiquitin to inhibit its degradation and therefore and memory in the hippocampus. Moreover, transduction of
maintainmonomericubiquitinlevels(Osakaetal.,2003). UCH-L1 protein into the hippocampus restored both synaptic
NumerouslinesofevidencehavelinkedUCH-L1toneurode- andbehavioraldefectsobservedintheAPP/PS1mousemodelof
generative disorders. The gracile axonal dystrophy (gad) mice AD(Gongetal.,2006).
haveanaturallyoccurring,spontaneousmutationintheUch-l1 Here,weinvestigatedtheroleofUCH-L1atsynapses.Wefind
genethatcausesalossofdetectableUCH-L1expression(Saigoh thatUCH-L1activityisregulatedbysynapticactivity.Synaptic
etal.,1999).Gadmiceexhibitseveresensoryataxiaatearlystages activationofUCH-L1iscorrelatedwithanincreaseinthelevels
of pathogenesis caused by axonal degeneration in the gracile of free monomeric ubiquitin. Pharmacological suppression of
tract, followed by motor paresis at later stages (Kikuchi et al., UCH-L1activityreducesmonomericubiquitinlevelsandleads
1990).TheI93MmutationintheUCH-L1gene,whichwasre- todramaticalterationstosynapticstructure.Strikingly,overex-
portedinaGermanfamilywithautosomaldominantParkinson’s pressionofubiquitinrescuestheeffectsofUCH-L1inhibition.
ThesedatasuggestthatUCH-L1isoneofthemajorDUBsinthe
brainthatcontrolsubiquitinhomeostasis.Moreover,ourfind-
ReceivedApril15,2009;revisedMay14,2009;acceptedMay15,2009. ingsindicatealteredUCH-L1activityleadstodeleteriouseffects
ThisworkwassupportedbyaNationalInstitutesofHealth(NIH)postdoctoraltraininggrant(A.E.C.);NIHGrants onsynapsestructureandfunction.
AG18440,AG10435,andAG22074(E.M.);TheRayThomasEdwardsFoundation(G.N.P.);andUniversityofCalifornia,
SanDiegostartupfunds(G.N.P.).WethankMariaMoribito,DarwinBerg,AnirvanGhosh,andthePatrickLaboratory MaterialsandMethods
foradviceandcriticalreviewofthismanuscript.WealsothankAlanOkadafortechnicalassistance.
CorrespondenceshouldbeaddressedtoGentryN.Patrickattheaboveaddress.E-mail:[email protected]. Reagents. UCH-L1 [LDN-57444 (LDN)] and UCH-L3 (4,5,6,7-
DOI:10.1523/JNEUROSCI.1817-09.2009 tetrachloroindan-1,3-dione) inhibitors were purchased from Calbio-
Copyright©2009SocietyforNeuroscience 0270-6474/09/297857-12$15.00/0 chem.NMDAandD((cid:1))-2-amino-5-phosphonopentanoicacid(APV)
7858•J.Neurosci.,June17,2009•29(24):7857–7868 Cartieretal.•RegulationofSynapticStructurebyUCH-L1
(NMDAreceptorantagonist)werepurchasedfromTocrisBioscience. cloned in the pEGFP-N1 vector. The single point mutations in the
The hemagglutinin (HA)-tagged ubiquitin probe (HAUb-VME; vinyl UCH-L1DNAwereintroducedbyPCR-basedsite-directedmutagenesis
methylesterfunctionalizedprobe)wassynthesizedasdescribedprevi- oftemplateplasmidcDNAusingprimersdesignedtointroducespecific
ously(Borodovskyetal.,2002)andwasprovidedbyDr.H.Ovaa(The mutations (C90S, 5(cid:4)-CCATTGGGAATTCCTCTGGCATCGGAC-3(cid:4),
NetherlandsCancerInstitute,Amsterdam,TheNetherlands). and D30A, 5(cid:4)-TTCGTGGCCCTGGGGCTG-3(cid:4)). All constructs were
UCH-L1-deficientmice.TheUCH-L1-deficientandwild-typelitter- verified by sequencing and by expression of proteins of the expected
matemouse(Uch-L1nm3419)brains(8weeksofage)wereobtainedfrom molecularweightinHEK293Tcells.
Dr.ScottWilson(UniversityofAlabama,Birmingham,AL).Thisisa Drugtreatmentsandvirusinfections.Forproteinexpressionanalysisby
spontaneousmousemutationthataroseatTheJacksonLaboratoryand Westernblottingorimmunofluorescencestainingexperiments,cultured
subsequentlymappedbytheScottWilsongroup(Waltersetal.,2008). neuronsweretreatedwith10(cid:1)MUCH-L1(LDN)orUCH-L3inhibitor
Antibodies.Thefollowingantibodieswereusedinthisstudy:mouse for24h.InexperimentsinwhichneuronsweresubjectedtoLDNtreat-
anti-Myc, rabbit anti-CDK5, rabbit anti-green fluorescent protein mentandinfections,neuronswerefirsttreatedwithLDNandthenin-
(GFP),andrabbitanti-guanylatekinase-associatedprotein(GKAP)an- fectedbyaddingvirionsdirectlytotheculturemediumandallowing
tibodies (purchased from Santa Cruz Biotechnology); mouse anti- proteinexpressionfor12–14h.ThetotaltimeofexposuretoLDNwas
postsynapticdensity-95(PSD-95)andrabbitanti-GluR1(obtainedfrom keptconstant(24h).Activitystimulationexperimentswereperformed
Calbiochem);rabbitanti-Shankantibody(agenerousgiftfromDr.Eu- bytreatingcultureswithNMDAandglycineat50and10(cid:1)M,respec-
njoonKim,KoreaAdvancedInstituteofScienceandTechnology,Dae- tively,for10minat37°C.Whereindicated,neuronswerepretreatedwith
jeon,Korea);rabbitanti-GluR1,mouseanti-NR1,andrabbitanti-NR2A UCH-L1inhibitor(10(cid:1)M)for24horAPV(50(cid:1)M)for10minbefore
antibodies;ratanti-Homerandrabbitanti-SynapsinI(MilliporeBio- additionofNMDA/glycinetotheculturemedia.
scienceResearchReagents);mouseanti-Bassoon(NventaBiopharma- Immunostaining.Attheendofeachexperiment,hippocampalneu-
ceuticals); chicken anti-Map2 (Abcam); rabbit anti-UCH-L1 (BI-
rons plated on coverslips or 35 mm glass-bottom dishes were rinsed
OMOL);rabbitanti-ubiquitin(Dako);andrabbitanti-Vamp2(Synaptic
brieflyinPBSandfixedwith4%paraformaldehyde(PFA)and4%su-
Systems).
Primaryneuronalcultures.Hippocampalneuroncultureswerepre- croseinPBS-MC(PBSwith1mMMgCl2and0.1CaCl2)for10minat
roomtemperature.NeuronswerethenrinsedthreetimeswithPBS-MC
pared from postnatal day 1 (P1) or P2 rat hippocampi as previously
andsubsequentlyblockedandpermeabilizedwithblockingbuffercon-
described(Patricketal.,2003).Briefly,forimmunostainingexperiments,
taining2%BSAand0.2%TritonX-100inPBS-MCfor20min.After
rathippocampiweredissected,dissociatedbypapaintreatmentandme-
rinsing neurons three times with PBS-MC, primary antibodies were
chanicaltrituration,andplatedatmediumdensity(45,000cells/cm2)on
addedinblockingbufferandcultureswereincubatedovernightat4°C.
poly-D-lysine-coated coverslips (12 mm in diameter) or glass-bottom Thefollowingantibodiesanddilutionswereusedforimmunofluores-
dishes (MatTek; 35 mm). For biochemical experiments, mixed hip-
cencestainings:mouseanti-PSD-95(1:500),rabbitanti-SynapsinI(1:
pocampalandcorticalneuronswereplatedathighdensityonsix-well
2000),mouseanti-Bassoon(1:2000),rabbitanti-Shank(1:2000),rabbit
plates((cid:2)500,000cellsperwell)coatedwithpoly-D-lysine.Cultureswere anti-GluR1 (1:20), chicken anti-Map2 (1:5000), and mouse anti-Myc
maintainedinB27supplementedNeurobasalmedia(Invitrogen)until
(1:1000).AfterthreewasheswithPBS-MC,neuronswereincubatedin
14–21dinvitro(DIV).
goatanti-rabbit,-mouse,or-chickensecondaryantibodiesconjugatedto
FractionationsandDUBlabelingassay.Fractionsfromratbrainswere
Alexa 488, Alexa 568, or Alexa 678 (1:500 each; Invitrogen) at room
preparedaspreviouslydescribed(Carlinetal.,1980;Choetal.,1992).
TheDUBactivityassaywasdonebyincubating20(cid:1)goflysatesfrom temperaturefor1h.NeuronswerewashedthreetimeswithPBS-MCand
mountedonslideswithAquaPoly/Mount(Polysciences).Forlivelabel-
neuronalculturesorratbrainfractionswiththeHAUb-VMEsubstratein
ingofsurfaceGluR1,theanti-GluR1antibodyagainsttheNterminus
labelingbuffer(50mMTris,pH7.4,5mMMgCl2,250mMsucrose,1mM extracellular epitope of the receptor was added to neurons in culture
DTT,and1mMATP)for1hat37°C.Proteinswerethenresolvedby
mediumat1:20dilutionfor10minbeforewashingoutexcessantibody
SDS-PAGE4–20%gradientgels,andblotsweresubsequentlyprobed
andfixingwith4%PFA/4%sucrose.
with anti-HA monoclonal antibody. Labeled proteins were identified
Electronmicroscopy.Maturehippocampalneurons((cid:5)21DIV)were
basedontheirmigrationonSDS-PAGEgels,andbycomparisonwith
platedin35mmglass-bottomdishesandtreatedwithDMSO(control)
previouslypublisheddatainwhichthespecificbandswereanalyzedby
massspectroscopy(Borodovskyetal.,2002). orLDN(10(cid:1)M).After24h,cellswerefixedin2%paraformaldehydeand
1%glutaraldehyde,andthenfixedinosmiumtetraoxideandembedded
RecombinantDNAandSindbisconstructs.TheSindbisenhancedgreen
inEponAraldite.Oncetheresinhardened,blockswiththecellswere
fluorescent protein (EGFP) viral construct was made by cloning the
detachedfromeachdishandmountedforsectioningwithanultramic-
EGFP(Clontech)openreadingframedirectlyintopSinRep5(Invitro-
gen).GFPu(inpEGFP-C1plasmidbackbone;Clontech),afusionofthe rotome(Leica).Gridswerestainedwith1%uranylacetateandanalyzed
CL1degron(degradationsignal)ontheCterminusofGFP,waskindly withaZeissOM10electronmicroscopeaspreviouslydescribed(Rock-
providedbyDr.RonKopito(StanfordUniversity,PaloAlto,CA).GFPu ensteinetal.,2001).Manualanalysisofpresynapticterminaldiameter,
isubiquitinatedandspecificallydegradedbytheUPS(Gilonetal.,1998; vesiclenumber,andsynapticcontactzonewasperformed.Atotalof10
Benceetal.,2001,2005).TheAgeI–BsrGIfragmentfromphotoactivat- micrographswasobtainedandfromeachgrid(ninegridspercondition)
able(pa)GFP(akindgiftprovidedbyJenniferLipponcott-Schwartz, foratotalof90electronmicrographsanalyzedpercondition.Analysis
National Institutes of Health, Bethesda, MD) was subcloned into the wasperformedusingImageQuant.Magnificationwas30,000(cid:6).Statisti-
GFPuplasmid.paGFPuwasthensubclonedintopSinRep5(Invitrogen). calsignificancewasdeterminedbyunpairedtwo-tailedStudent’sttest.
Orientationwasverifiedbyrestrictiondigestandconstructswerecon- LiveimagingofpaGFPudegradation.Culturedhippocampalneurons
firmedbyDNAsequencing.TheHis6-Myc-ubiquitinwasprovidedby ((cid:5)21DIV)on35mmglass-bottomdisheswereincubatedfor24hbefore
Dr.RonKopitoandwasclonedintopSinRep5.Theyellowfluorescent imaginginmediacontainingeitherDMSO(control)orLDN(10(cid:1)M).
protein(YFP)-actinpSinRep5plasmidswaskindlyprovidedbyDr.E. paGFPuvirionswereaddeddirectlytoculturemediaafter12hofLDN
Schuman(CaliforniaInstituteofTechnology,Pasadena,CA).Forpro- treatmentandproteinexpressionwasallowedtocontinuefor12–14h.
ductionofrecombinantSindbisvirions,RNAwastranscribedusingthe CulturemediawasthenreplacedwithwarmHBS[HEPES-bufferedsa-
SP6mMessagemMachineKit(Ambion)andelectroporatedintoBHK linesolutioncontainingthefollowing(inmM):119NaCl,5KCl,2CaCl2,
cellsusingaBTXECM600at220V,129(cid:3),and1050(cid:1)F.Virionswere 2MgCl ,30glucose,10HEPES].Cellsweremaintainedat(cid:2)35°Cusing
2
collected after 24–32 h and stored at (cid:1)80°C until use. For UCH-L1 aceramicheatlamp(ZooMed),andbathtemperaturewascontinually
expressionconstructs,theUCH-L1openreadingframewasobtained monitoredbyadigitalprobethermometer.Infectedneurons(identified
fromIncytefull-lengthhumancDNAclone(OpenBiosystems)encoding bymCherryexpression)werethenphotoactivatedfor10–15swith100
wild-typeUCH-L1andwasamplifiedbyPCRwitha5(cid:4)-oligocontaining W Hg2(cid:7) lamp and a D405/40(cid:6) with 440 DCLP dichroic filter set
anXhoIsiteanda3(cid:4)-oligocontaininganAgeIsite,andsubsequently (Chroma).Forliveimaging,pyramidal-likeneuronswereselectedina
Cartieretal.•RegulationofSynapticStructurebyUCH-L1 J.Neurosci.,June17,2009•29(24):7857–7868•7859
microplate fluorometer (HTS 7000 Plus;
PerkinElmer) every 5 min for 2 h at room
temperature.
Westernblotanalysis.Culturedneuronswere
lysed in radioimmunoprecipitation assay
(RIPA)lysisbuffer(50mMTris,pH7.4,150mM
NaCl, 1% Triton X-100, 0.1% SDS, 1 mM
EDTA)containingproteaseinhibitors(Roche).
Rat or mouse brains were homogenized in
RIPAbufferat900rpminTeflon-glasshomog-
enizers.Neuronalcelllysatesorbrainhomoge-
nateswerecentrifugedat14,000rpm,andsu-
pernatants were removed and protein
concentrationwasdeterminedbyBSAprotein
assaykit(Pierce)usingbovineserumalbumin
asastandard.Proteinsampleswereresolvedby
SDS-PAGEandelectrophoreticallytransferred
tonitrocellulosemembranes.Membraneswere
thenblockedfor1hinTBSTblockingbuffer
(TBS,0.1%Tween20,and5%milk)atroom
temperatureandthenincubatedwithprimary
antibodiesinblockingbufferovernightat4°C.
Theantibodiesusedwereatthefollowingdilu-
tions:mouseanti-PSD-95(1:5000),rabbitanti-
Synapsin I (1:10,000), rabbit anti-GluR1
(1:5000), rabbit anti-Shank (1:10,000), rabbit
anti-GFP (1:10,000), rabbit anti-UCH-L1
(1:5000),rabbitanti-GKAP(1:2000),ratanti-
Homer (1:2000), mouse anti-NR1 (1:2000),
rabbit anti-NR2A (1:2000), rabbit anti-
ubiquitin (1:2000), rabbit anti-Vamp2
(1:5000), and rabbit anti-CDK5 (1:10,000).
Blots were then washed three times in TBST
washingbuffer(TBS,0.1%Tween20)andin-
cubatedwithgoatanti-rabbit,-mouse,or-rat
IgG conjugated to horseradish peroxidase (1:
5000).Proteinbandswerevisualizedbychemi-
luminescenceplusreagent(PerkinElmer)and
weredigitizedandquantifiedusingNIHImageJ
software.Forstatisticalanalysis,unpairedStu-
dent’s t test was performed between any two
conditions.
Imageacquisitionandquantification.Confo-
Figure1. UCH-L1expressionandactivitylevelsinratbrainandmatureculturedhippocampalneurons.A,Representative cal images were acquired using a Leica
imageofahippocampalneuronimmunolabeledwithanti-PSD-95andanti-UCH-L1antibodies.B,GFP-expressinghippocampal DMI6000invertedmicroscopeoutfittedwitha
neuronsimmunostainedwithanti-PSD-95andanti-UCH-L1antibodies.ThestraighteneddendritesinAandBcorrespondtothe Yokogawaspinningdiskconfocalhead,anOrca
regionsindicatedbyarrowsinthewhole-cellimages.ThearrowheadsindicateselectedregionsinwhichUCH-L1colocalizeswith ER high-resolution black-and-white cooled
PSD-95andGFP-filledspines.Representativemaximumz-projectedconfocalimages(cellandstraighteneddendrites)aredepicted. CCD camera (6.45 (cid:1)m/pixel at 1(cid:6))
Whole-cellscalebar,20(cid:1)m;dendritescalebar,5(cid:1)m.C,ActiveDUBprofilingwithHAUb-VMEinsubcellularfractionsofratbrain.The (Hamamatsu),PlanApochromat40(cid:6)/1.25nu-
immunoblotwasprobedwithanti-HAantibodytodetectactiveDUBs.D,Comparisonoflabeled(active)andunlabeled(inactive)UCH-L1 mericalaperture(NA)and63(cid:6)/1.4NAobjec-
(HAUb-UCH-L1andUCH-L1,respectively)inratbrainfractionsinanimmunoblotthatwasprobedwithanti-UCH-L1antibody.E,The tive,andaMellesGriotargon/krypton100mW
postsynapticmarker,PSD-95,wasusedascontrolforfractionation.Numberscorrespondtothefollowingfractions:(1)whole-brain air-cooled laser for 488/568/647 nm excita-
homogenate(H);(2)crudesynaptosomalfraction(P2);(3)cytosolpluslightmembranes(S2);(4)lightmembranes(Golgi,ER)(P3);(5) tions.Exposuretimeswereheldconstantdur-
cytosolicfraction(S3);(6)PSD-1T(oneTritonX-100extraction);(7)PSD-2T(twoTritonX-100extraction). ing acquisition of all images for each experi-
ment. Pyramidal-like cells were chosen in a
randommanner.Confocalz-stackimages(with0.5(cid:1)msections)were randommanner.Confocalz-stacksweretaken
acquiredwitha63(cid:6)objectiveevery2min. at0.4–0.5(cid:1)mdepthintervalsinallexperiments.Forimageanalysis,
Invitroproteasomeactivityassay.Proteasomeactivitywasmeasuredas maximum z-projections were used. Images were thresholded equally
previouslydescribedwithslightmodifications(KisselevandGoldberg, 1.5–2timesabovebackground.Dendritesfromindividualneuronswere
2005).Briefly,culturedneuronswereincubatedfor24hinmediacon- thenstraightenedandusedforanalysis.Fluorescenceintensityassociated
taining either DMSO (control) or LDN (10 (cid:1)M). Neurons were then withpresynapticandpostsynapticproteinpunctawasmeasuredtode-
lysed in proteasome assay buffer (50 mM Tris-HCl, pH 7.5, 250 mM terminethesizeandnumberofpuncta(normalizedtodendriticlength)
sucrose,5mMMgCl2,0.5mMEDTA,2mMATP,1mMDTT,and0.025% incontrolandLDN-treatedneurons.GFP-expressinghippocampalneu-
digitonin)for15minonice.Lysateswereclearedbycentrifugationat ronswereusedforspinemorphologyanalysisusingtheEdgefitterNIH
14,000rpmfor15min.Onehundredmicromolarfluorogenicprotea- ImageJplug-in(GhoshLaboratory,UniversityofCalifornia,SanDiego,
some peptide substrate N-succinyl-Leu-Leu-Val-Tyr-7-amido-4- LaJolla,CA).Todeterminethelengthofaspine,thedistancefromthetip
methylcoumarin(Suc-LLVY-AMC)(BIOMOLInternational)wasthen oftheprotrusiontothedendriticshaftwasmeasuredmanually.Tomea-
addedtoequalamountsofclearedlysatesina96-wellmicrotiterplate. surethewidthofaspine,themaximallengthofthespineheadperpen-
Fluorescence(380nmexcitation;460nmemission)wasmonitoredona diculartothelongaxisofthespineneckwasmeasured.Thenumberof
7860•J.Neurosci.,June17,2009•29(24):7857–7868 Cartieretal.•RegulationofSynapticStructurebyUCH-L1
spinesvisiblealongthedendritewascounted
manuallyper1(cid:1)mdendriticlength.Measure-
ments were then automatically logged from
NIH ImageJ into Microsoft Excel. Statistical
significancewasdeterminedbyunpairedtwo-
tailedStudent’sttest.Allimagingandanalysis
of ubiquitin rescue experiments (see Fig. 8)
wereperformedinablindedmanner.Forquan-
tificationoftheproteasomereporterdegrada-
tion, images were thresholded above back-
ground equally between conditions. Total
integratedfluorescenceintensitywasmeasured
from dendrites at each time interval and ex-
pressedasthepercentagechangefromtime0.
Groupedanalysisofdendriticfluorescencede-
cayovertimefromeachtreatmentgroupwas
plottedaslinegraphs(mean(cid:8)SEM).Thedeg-
radationrateofreporterfluorescencedecaywas
obtainedbytakingthedifferenceoftotalfluo-
rescenceloss[arbitraryunits(AU)]overtime
(F (cid:1)F /time )fromindividualexperiments.
i n n
Themeanrate(cid:8)SEMpertreatedgroupwas
thennormalizedtothecontrolrates.Forstatis-
ticalanalysis,groupeddegradationrateswere
analyzed by unpaired two-tailed t tests. One-
halfofthepaGFPureporterdegradationexper-
iments were performed in a blinded manner.
Therewasnosignificantdifferencebetweenex-
periments performed blind and unblind, and
thereforethedatawerecombined.
Results
CharacterizationofUCH-L1
distributionandactivityinneurons
To determine the subcellular localization
of UCH-L1, cultured hippocampal neu-
ronswereimmunostainedforendogenous
UCH-L1 and the postsynaptic marker
PSD-95(Fig.1A,B).Insomeexperiments,
GFP was used as a cell-filling marker to Figure2. Activity-dependentupregulationoffreemonomericubiquitinlevelsinneuronsisdependentonUCH-L1activity.
visualizespines(Fig.1B).UCH-L1expres- A–C,Culturedneuronsweretreatedwith50(cid:1)MNMDA/10(cid:1)Mglycineor50(cid:1)MNMDA/10(cid:1)Mglycineplus50(cid:1)MAPVfor10min.
sionwasdetectedinbothsomaandden- LysatesfromcontrolandtreatedneuronswerelabeledwithHAUb-VMEsubstrateandsubjectedtoWesternblotanalysis.A,A
drites of hippocampal neurons. UCH-L1 representativeimmunoblotprobedwithanti-HAantibodydemonstratesthelevelsoflabeled(active)UCH-L1incontroland
isdistributedinamicropunctatemanner treatedneuronsinthepresenceorabsenceoftheDUBlabelingreagent(toppanel).Theblotwassubsequentlystrippedand
reprobedwithanti-UCH-L1antibodytodemonstratethelevelsoflabeledandunlabeledUCH-L1(HAUb-UCH-L1andUCH-L1,
andpartiallycolocalizedwithPSD-95(Fig.
respectively)(bottompanel).B,Arepresentativeimmunoblotprobedwithanti-HAantibodydemonstratesactivitylevelsof
1A,B).Moreover,UCH-L1wasfoundto
UCH-L1inresponsetodrugtreatments(toppanel).ThebottompanelofBshowsequalamountsofUCH-L1betweentreatments.
belocalizedtodendriticspinesofneurons
C,DensitometryanalysisofsixindependentexperimentsfromDublabelingassaysisshown.D–F,Culturedneuronsweretreated
(Fig.1B).Tofurthercharacterizethesub- with50(cid:1)MNMDA/10(cid:1)Mglycinefor10min(D)or10(cid:1)MUCH-L1inhibitor(LDN)for24hwithorwithoutanadditional10min
cellulardistributionofUCH-L1,weused treatmentwith50(cid:1)MNMDA/10(cid:1)Mglycine(E,F),respectively.Lysateswereresolvedon15%SDS-PAGEandimmunoblotswere
differentialanddensitygradientcentrifu- probedwithanti-ubiquitinantibody.Representativeblotsfromeachexperimentareshown.Relativebandintensitiesofcorre-
gationtoenrichforvarioussynapticcom- spondingmonomericubiquitinwerequantifiedandaredepictedinthebottompaneloffigureF.Meanvalues(cid:8)SEMoffourto
partments from rat brain. In addition, fiveindependentexperimentsareshown.Forstatisticalanalysis,one-wayANOVAwithposthocBonferroni’smultiple-comparison
testwasused.*p(cid:9)0.05;**p(cid:9)0.01;***p(cid:9)0.001.
we used a novel hemagglutinin-tagged
ubiquitin-vinyl methyl ester-derived ac-
fractionscomparedwiththeotherDUBsdetectedandispresent
tivesite-directedprobe(HAUb-VME)thatcovalentlymodifies
inanactiveform.AcomparisonbetweenactiveUCH-L1modi-
DUBsincludingUCHs(Borodovskyetal.,2002)toprofileactive
fiedbythesubstrate(upperband)andtheunmodified,inactive
DUBspresentinthebrain.Thisassayprovidesahighlysensitive
approachfordetectingUCH-L1activityasourprobeisspecifi- UCH-L1(lowerband)isdemonstratedinFigure1Dandindi-
callytargetedtoUCH-L1whenitisinanactiveform.Usingthis catesthat(cid:2)50%ofUCH-L1ispresentinactiveforminthetotal
probe,wemonitoredtheactivityofUCH-L1inlysatesgenerated homogenate (Fig. 1D, lane 1). Interestingly, we found that
fromratbrainfractions.TheprofileofactiveDUBspresentin UCH-L1associatedwiththePSDisprimarilyinanactiveform
thesevariousratbrainfractionsisshowninFigure1C.Alower (Fig.1D,lanes6,7).ToverifythattheHAUb-VMElabelingof
exposureoftheblotpresentedinFigure1Cisgiveninsupple- the DUB at (cid:2)35 kDa is primarily modified UCH-L1 and not
mentalFigure1A(availableatwww.jneurosci.orgassupplemen- UCH-L3,weperformedtheDUBlabelingassayonlysatesfrom
tal material). As observed, UCH-L1 is highly expressed in all UCH-L1-deficient mice (nm3419) (Walters et al., 2008). Here,
Cartieretal.•RegulationofSynapticStructurebyUCH-L1 J.Neurosci.,June17,2009•29(24):7857–7868•7861
wefoundvirtuallynolabelingofanyotherDUBthatrunsator monoubiquitinlevelsinLDN-treatedneurons(Fig.2E)(con-
nearthesamemolecularweightofUCH-L1(supplementalFig. trol, 1.0 (cid:8) 0.11; LDN-treated, 0.59 (cid:8) 0.01). Moreover, the
S1B,toppanel,availableatwww.jneurosci.orgassupplemental levelsoffreemonomericubiquitininneuronalculturespre-
material). Together, our data demonstrate that UCH-L1 is ex- treated with LDN before NMDA receptor stimulation were
pressed ubiquitously in neurons with a subpopulation distrib- reduced to the levels observed in control untreated neurons
utedtospinesandpostsynapticdensities. (Fig. 2F) (control, 1.0 (cid:8) 0.02; LDN-treated, 0.64 (cid:8) 0.05;
NMDA/Gly-treated, 2.0 (cid:8) 0.13; NMDA/Gly plus LDN-
treated,1.04(cid:8)0.08;p(cid:9)0.001,one-wayANOVA).Thissug-
NMDAreceptoractivationupregulatesUCH-L1activity
geststhatNMDAreceptoractivationinneuronsincreasesfree
Arecentstudyhasdemonstratedthatpharmacologicalinhibi-
monomeric ubiquitin in an UCH-L1-dependent manner. In
tion of UCH-L1 activity by 40% is sufficient to significantly
addition, we tested whether LDN affected the ability of
attenuate LTP in rat hippocampal slices (Gong et al., 2006).
UCH-L1 to bind ubiquitin. We performed in vitro ubiquitin
ThissuggeststhatUCH-L1functionmayberequiredforsyn-
binding assays with either bacterially expressed glutathione
apticplasticityandmayitselfberegulatedbysynapticactivity.
S-transferase (GST) or GST-UCH-L1 and ubiquitin. We
Indeed,depolarization-dependentCa2(cid:7)influxintosynapto-
found that pretreatment of GST-UCH-L1 with LDN greatly
somes has been shown to produce a rapid decrease in ubiq-
diminished its ability to bind ubiquitin (supplemental Fig.
uitinconjugates(Chenetal.,2003).Totestthepossibilitythat
S2B,toppanel,availableatwww.jneurosci.orgassupplemen-
UCH-L1 might be regulated by synaptic activity, we stimu-
tal material). Together, this indicates LDN affects both the
lated neuronal cultures (DIV 21) with NMDA receptor ago-
catalyticandubiquitinbindingactivitiesofUCH-L1.
nist.WefoundthatstimulationofsynapticactivitybyNMDA/
glycinesignificantlyupregulatedUCH-L1activityincultured
InhibitionofUCH-L1activityaffectssynaptic
neurons(Fig.2A,toppanel).Acomparisonoflabeled(active)
proteinclusters
and unlabeled (inactive) UCH-L1 in response to NMDA re-
Recent electrophysiological studies on gad mice and hip-
ceptorstimulationisshowninFigure2A,bottompanel.On
pocampal slices treated with LDN have demonstrated that
average, the activity of UCH-L1 increased by (cid:2)1.5-fold in
UCH-L1 is required for LTP and maintenance of memory
responsetoNMDAreceptorstimulation(Fig.2B,C)(control,
(Gongetal.,2006;Sakuraietal.,2008).Thebrainsofgadmice
1.0 (cid:8) 0.07; NMDA/Gly, 1.63 (cid:8) 0.16; p (cid:10) 0.003, one-way
shownogrossstructuralabnormalities;however,itispossible
ANOVA). Moreover, the NMDA-induced upregulation of
that discrete alterations in neuronal morphology or synaptic
UCH-L1 activity was efficiently blocked by pretreatment of
structure occur because of the lack of UCH-L1 activity. To
neurons with the NMDA receptor antagonist APV demon-
examinethispossibility,wecomparedtheimmunocytochem-
strating the specificity of our treatment (Fig. 2B,C). This
ical distribution of synaptic proteins in control and LDN-
novel and potentially important finding demonstrates that
treatedneurons,severalofwhicharetargetsforubiquitination
synapticactivitycanmodulatetheactivityofUCH-L1.
and degradation by the UPS (Colledge et al., 2003; Ehlers,
2003; Guo and Wang, 2007; Lee et al., 2008). Moreover, the
UCH-L1activityisrequiredforNMDA-inducedupregulation postsynapticproteinsweexaminedaremajorcomponentsof
offreemonomericubiquitin thePSD,whichisahighlydynamicstructureanditsmolecular
MultiplefunctionshavebeenascribedtoUCH-L1invitroand composition and biochemical stability is very responsive to
invivo.ItisknownthatUCH-L1canactasaubiquitinhydro- changes in synaptic activity. Furthermore, these activity-
laseandgeneratefreeubiquitinspeciesfromprecursorubiq- dependentmolecularchangesareregulatedinpartbytheUPS
uitinpolypeptides(Wilkinsonetal.,1989).UCH-L1canalso (Ehlers,2003).Wefoundthatexposureofmaturehippocam-
bindtoubiquitinandactasaubiquitinstabilizertopreventits palneuronstoLDNleadstodramaticalterationsinsynaptic
degradationbylysosomes(Osakaetal.,2003).Therefore,we structure (Fig. 3). We observed a significant increase in the
wereinterestedtodeterminewhetherNMDAreceptorstimu- size of several synaptic protein puncta in LDN-treated neu-
lationhadanyeffectonthelevelsoffreemonomericubiquitin ronscomparedwiththoseincontrolneurons.Onaverage,the
(alsoreferredtoasmonoubiquitin)andwhetherUCH-L1ac- size of postsynaptic proteins PSD-95, Shank, and surface
tivityplayedaroleinmodulatingubiquitinlevelsinresponse GluR1punctaincreasedby77,70,and39%,respectively(Fig.
toNDMAreceptoractivation.Wefoundthatstimulationwith 3A–D) (PSD-95 puncta, control, 1.0 (cid:8) 0.03; LDN-treated,
NMDA/glycine(10min)increasedfreemonomericubiquitin 1.77(cid:8)0.04;Shankpuncta,control,1.0(cid:8)0.04;LDN-treated,
levels approximately twofold in cultured neurons (Fig. 2D) 1.69(cid:8)0.04;surfaceGluR1puncta,control,1.0(cid:8)0.07;LDN-
(control, 1.0 (cid:8) 0.11; NMDA, 1.8 (cid:8) 0.14). We next asked treated,1.39(cid:8)0.08).Wealsodetectedanincreaseinthesize
whether inhibiting UCH-L1 activity blocks the upregulation ofpresynapticproteinpunctaasmeasuredbyimmunolabel-
ofmonoubiquitininNMDAreceptor-stimulatedneurons.To ingforpresynapticnerveterminalswithSynapsinIandBas-
assessthis,weusedapreviouslydescribedUCH-L1inhibitor, soon. We found that, on average, there was a 34 and 25%
LDN,whichspecificallyinhibitsUCH-L1whilehavingnoef- increaseinthesizeofSynapsinIandBassoonpuncta,respec-
fectonotherUCHfamilymembers(Liuetal.,2003;Gonget tively (Fig. 3A,B,D) (Synapsin I puncta, control, 1.0(cid:8) 0.05;
al.,2006).TodemonstratetheefficacyofUCH-L1inhibition LDN-treated, 1.34 (cid:8) 0.07; Bassoon puncta, control, 1.0 (cid:8)
by LDN, neuronal lysates were preincubated with increasing 0.03; LDN-treated, 1.25 (cid:8) 0.02). Interestingly, we found no
amountsofLDNbeforeDUBlabelingassays.Wefoundthat observable difference in the dendritic protein marker MAP2
10 (cid:1)M LDN significantly inhibited UCH-L1 activity in vitro stainingbetweencontrolandLDN-treatedneurons(Fig.3C).
(supplemental Fig. S2A, top panel, available at www. This indicates that UCH-L1 inhibition preferentially affects
jneurosci.orgassupplementalmaterial).Neuronstreatedwith synapses while having no effect on the overall integrity of
LDNat10(cid:1)Mhadsignificantlydecreasedmonoubiquitinlev- dendrites.Wealsoexaminedwhethertherewasaconcomitant
els (Fig. 2E). On average, we observed a 40% reduction in alterationinthedensityofsynapticproteinpuncta.Wefound
7862•J.Neurosci.,June17,2009•29(24):7857–7868 Cartieretal.•RegulationofSynapticStructurebyUCH-L1
that the density of PSD-95 puncta was
decreased by 30% (Fig. 3E) (control,
1.0 (cid:8) 0.04; LDN-treated, 0.8 (cid:8) 0.3).
However, we did not observe any
changesinthenumberofShank,surface
GluR1, Synapsin I, and Bassoon puncta
(Fig.3E).Todemonstratethespecificity
ofourUCH-L1inhibitor,neuronswere
treatedwithaUCH-L3inhibitor(LDN-
L3). UCH-L3 is a closely related DUB
andhasalsobeenshowntobeinvolved
ingeneratingfreemonomericubiquitin.
However,wedidnotfindanyalterations
in synaptic structures in LDN-L3-
treated neurons (supplemental Fig.
S3A–C, available at www.jneurosci.org
as supplemental material). Together,
thesedatashowthatUCH-L1activityis
specifically involved in maintenance of
synapsestructure.
UCH-L1regulatesspinemorphology
anddensity
Abundant evidence demonstrates that
spines undergo activity-dependent
changesinshapeandnumber,andthere-
fore, spines could serve as a cellular sub-
strateforchemicalandstructuralsynaptic
plasticity (Segal, 2002, 2005). Therefore,
weaskedwhethertheobservedalterations
inthesynapticproteinclusterscouldpos-
sibly be accompanied by any changes in
spine size and density. To detect alter-
ations in spine morphology, we analyzed
spines from GFP-expressing neurons
treated with vehicle or LDN. We found
Figure3. InhibitionofUCH-L1activityalterssynapticstructure.A–C,CulturedneuronsweretreatedwithLDNfor24h.Atthe
strikingalterationsinthesizeofspinesin
endofLDNtreatments,neuronswerefixed,permeabilized,andimmunolabeledwithanti-PSD-95andanti-SynapsinI(A),
LDN-treatedneurons.Spinesexhibitedan
anti-Shankandanti-Bassoon(B),orlive-labeledwithanti-GluR1forsurfaceGluR1staining,andthenfixedandstainedwith
enlargementof(cid:2)80%inspineheadwidth
anti-PSD-95andanti-MAP2antibodies(C).Representativemaximumz-projectedconfocalimagesandstraighteneddendrites
and 37% increase in spine length (Fig. fromcontrolandLDN-treatedneuronsaredepicted.Scalebar,5(cid:1)m.D,E,Presynapticandpostsynapticproteinpunctasizeand
4C,F) (spine head width, control, 1.0 (cid:8) numberwereanalyzedincontrolandLDN-treatedneurons.ThemeanpunctasizeandnumberinLDN-treatedneuronswere
0.09; LDN-treated, 1.8 (cid:8) 0.22) (Fig. normalizedtothoseofcontrolneuronsfromthreetofourindependentexperiments.Thenumberofpunctawascalculatedper10
4D,G)(spinelength,control,1.0(cid:8)0.03; (cid:1)mdendritelength.Measurementsforindividualstainingsweremadeonthefollowingnumberofdendritescorrespondingto
LDN-treated, 1.37 (cid:8) 0.08). We also ob- controlandLDN-treatedneurons,respectively:PSD-95,84and99;Shank,70and102;SynapsinI,46and55;Bassoon,66and93;
servedthatblockingUCH-L1activitydra- andGluR1,51and56.Meanvalues(cid:8)SEMareshown.Forstatisticalanalysis,unpairedStudent’sttestwasperformedbetween
anytwoconditions.*p(cid:9)0.001.
matically reduced the number of spines
(Fig. 4E). LDN-treated neurons had a
(cid:2)50% reduction in the number of spines compared with the changes observed in spines were accompanied by alterations
controluntreatedneurons(spines/micrometer:control,0.72(cid:8) intheactincytoskeleton.Tovisualizeactin-filledspines,neu-
0.05; LDN-treated, 0.35 (cid:8) 0.05). These data demonstrate that ronswereinfectedwithYFP-actinSindbisvirus(supplemen-
alterations in synaptic structure induced by inhibition of tal Fig. S4, available at www.jneurosci.org as supplemental
UCH-L1activityarealsoaccompaniedbychangesinspinemor- material).Inlinewithourpreviousobservation,wefoundthat
phology.Indeed,weobserveda30%decreaseinthedensityof YFP-actin-filled spines were enlarged in neurons that were
PSD-95 puncta in LDN-treated neurons. This potentially indi- treated with LDN. In addition, neurons were stained with
catesthatspinelossprecedesthedisassemblyofthepostsynaptic phalloidin to label F-actin in dendritic spines (supplemental
densityinUCH-L1-inhibitedneurons. Fig. S4B,C, available at www.jneurosci.org as supplemental
Filamentous actin (F-actin) accumulates at high concen- material).WefoundthatF-actinpunctatoalsobeenlargedin
tration in dendritic spines, and actin filaments provide the LDN-treatedneuronsandthattheycolocalizedwithsynaptic
structuralbasisforcytoskeletalorganizationinspines.Actin- markers PSD-95 (supplemental Fig. S4B, available at www.
based changes in the morphology of spines are regulated by jneurosci.org as supplemental material) or Shank (supple-
synaptictransmissionandareknowntocontributetosynaptic mental Fig. S4C, available at www.jneurosci.org as supple-
plasticity (Fischer et al., 1998, 2000; Fukazawa et al., 2003; mentalmaterial).Together,ourdataindicatethatalterations
Okamoto et al., 2004). We examined whether structural to the actin cytoskeleton occur concomitantly with altered
Cartieretal.•RegulationofSynapticStructurebyUCH-L1 J.Neurosci.,June17,2009•29(24):7857–7868•7863
sitieswithanaveragelengthof0.64(cid:8)0.04
(cid:1)m (Fig. 5I–K). The synaptic terminals
were irregular and contained abundant
clearsynapticvesiclesandcoatedpits(Fig.
5F).Thedendriteswerealsoirregularand
displayedenlargedmitochondria,andfo-
cal vacuolization (Fig. 5G,H). Together,
ourEMstudiescorroborateourimmuno-
fluorescencestudiesandfurthersubstanti-
atearoleforUCH-L1functioninmain-
tainingnormalsynapticstructure.
AlteredUCH-L1activityaffectssynaptic
proteincomposition
Since inhibition of UCH-L1 activity re-
sulted in a reduction in the levels of free
monomericubiquitinandstructuralalter-
ationsatsynapses,weaskedwhetherthese
changeswereaccompaniedbyalterations
inthestabilityofsynapticproteins(Fig.6).
Oftheproteinsexamined,weonlyfound
alteredPSD-95expressionlevelsinLDN-
treated neurons. We observed a 60% in-
creaseinthelevelsofPSD-95inneurons
treated with LDN (Fig. 6A,C) (control,
1.0(cid:8)0.12;LDN-treated,1.6(cid:8)0.16).We
alsoexaminedproteinexpressionlevelsin
brain homogenates obtained from wild-
type and UCH-L1-deficient mice (Fig.
6B). Interestingly, we found that protein
expression levels of PSD-95 and Shank
were increased by 70 and 47% in mouse
brain homogenates deficient in UCH-L1
activity (Fig. 6D) (PSD-95, wild type,
1.0 (cid:8) 0.15; UCH-L1 null, 1.7 (cid:8) 0.19;
Shank,wildtype,1.0(cid:8)0.09;UCH-L1null,
1.47(cid:8)0.10).Thesedatashowthatlossof
Figure4. InhibitionofUCH-L1activityaltersspinesizeanddensity.A,B,CulturedneuronsweretreatedwithLDNfor24h.
UCH-L1activityaffectsthestabilityofcer-
EGFPSindbisvirionswereaddeddirectlytoculturemediaafter10–12hofLDNtreatment,andproteinexpressionwasallowedto
continuefor12–14h.AttheendofLDNtreatments,neuronswerefixed,permeabilized,andimmunolabeledwithanti-PSD-95 tainscaffoldproteinsbutnotallsynaptic
andanti-SynapsinIantibodies.ThestraighteneddendritesinBcorrespondtotheregionsindicatedbyarrowsinthewhole-cell proteins.
imagesinA.C–G,Quantificationofthenumber,widths,andlengthsofGFP-filledspinesincontrolandLDN-treatedneuronsis
shown.C,D,Measurementsforspineheadwidthsandspinelengthswerenormalizedtothoseofcontrolvalues.F,G,Cumulative EffectsofUCH-L1inhibitiononglobal
frequencyplotsshowingdistributionofspineheadwidth(inmicrometers)andspinelength(inmicrometers)incontroland UPSactivity
LDN-treatedneuronsareshown.E,Quantificationofspinedensityisrepresentedasthenumberofspinesper1(cid:1)mdendrite Since our data clearly demonstrate that
length.Thequantifieddatawereobtainedfromthreeindependentexperiments,inwhich(cid:5)20dendritesand(cid:5)900spineswere
blockingUCH-L1activityreducesthelev-
analyzedpercondition.Meanvalues(cid:8)SEMareshown.*p(cid:9)0.05,**p(cid:9)0.01,unpairedStudent’sttest.
elsoffreemonomericubiquitin,wesetout
todeterminewhetherinhibitingUCH-L1
presynapticandpostsynapticproteinpunctaandspinesizein activityplaysaroleinmodulatingglobalUPSfunction.Weas-
UCH-L1-inhibitedneurons. sessedproteasomeactivityinvitrobymonitoringthecleavageof
TofurtheranalyzetheeffectsofUCH-L1inhibitiononsyn- a synthetic fluorogenic substrate (Suc-LLVY-AMC) in total ly-
aptic structure, we performed electron microscopy on LDN- sates from control and LDN-treated neurons and detected no
treatedneurons(Fig.5).Inuntreatedhippocampalcultures,as differencesbetweenconditions(Fig.7A).Sinceoneofthemajor
expected,abundantandwellorganizedneuriticprocesseswere functions of UCH-L1 is generating free ubiquitin, we assessed
identified (Fig. 5A–D). Most synaptic contacts were synapto- whetheralteringubiquitinlevelsbyoverexpressingUCH-L1has
dendritricratherthanaxo-somatic.Onaverage,presynapticter- anyeffectonUPSactivity.Wegeneratedwild-typeandmutant
minalscontained81.4(cid:8)6.2vesiclespersynapseandmeasured GFP-taggedUCH-L1mammalianexpressionconstructs.Allof
0.89(cid:8)0.07(cid:1)mindiameter,displayingsymmetricalpostsynaptic ourconstructswereequallyexpressedinHEK293Tcells(Fig.7B,
densitiesof(cid:2)0.59(cid:8)0.06(cid:1)minlength(Fig.5I–K).Incontrast, top panel). Furthermore, HAUb-VME labeling assay on these
hippocampalculturestreatedwithLDNdisplayedabnormalpre- lysatesshowedwild-typeUCH-L1-GFPtobeactive,whereasno
synapticandpostsynapticterminals(Fig.5E–H).Thepresynap- activitywasdetectedforeitherthecatalyticallyinactiveC90Sor
ticterminalscontainedonaverage107.2(cid:8)6.2vesiclespersyn- ubiquitin binding-deficient D30A mutants (Fig. 7B, bottom
apseandwereenlarged,averagingindiameterfrom1.64(cid:8)0.14 panel).Ourdataareconsistentwithpreviousreportsdescribing
(cid:1)mindiameter,anddisplayedthickenlargedpostsynapticden- theseUCH-L1mutants(Sakuraietal.,2006).Wenextexamined
7864•J.Neurosci.,June17,2009•29(24):7857–7868 Cartieretal.•RegulationofSynapticStructurebyUCH-L1
Figure5. UltrastructuralsynapticalterationsinprimaryneuronstreatedwithLDN.A–H,Representativeelectronmicrographsofculturedneuronstreatedwithvehicleor10(cid:1)MLDNfor24h.
A–D,Control,untreatedneuronsexhibitnormalcharacteristicsforpresynapticterminals(pst),postsynapticdensities(psd),mitochondria(m),anddendriticprocesses(dp).E–H,LDN-treated
neuronsdisplayabnormalsynapticstructuresdemonstratedbyenlargedpresynapticterminals,irregularpostsynapticdensities,abundantcoatedpits(cp)andclearsynapticvesicles(sv),and
vacuolizationofdendrites.Magnification,30,000(cid:6).QuantificationsofPSDdiameter(I),numberofvesicles(J),andsynapticzonecontact(K)incontrolandLDN-treatedneuronsareshown.
Averagedresultsfromatotalof10micrographsforeachgrid(9gridspercondition)wereusedfordataanalysis.Meanvalues(cid:8)SEMareshown.*p(cid:9)0.01,unpairedStudent’sttest.
thelevelsofpolyubiquitinconjugatesbyWesternblotanalysisas UCH-L1(supplementalFig.S5A,B,availableatwww.jneurosci.
itisknownthatinhibitionoftheproteasomeactivitycandirectly org as supplemental material). Together, these experiments
affectthelevelsofubiquitin-conjugatedproteins.Interestingly, suggest that altered UCH-L1 activity does not directly affect
althoughwefoundthatoverexpressionofwild-typeormutant proteasomefunction.Furthermore,anincreasedmonomeric
UCH-L1-GFP had no effect on the levels of polyubiquitinated ubiquitinlevelsgeneratedbyoverexpressionofUCH-L1does
proteins(Fig.7C,toppanel),therewasanapproximatelyfour- nothaveaneffectonthelevelofpolyubiquitinconjugatesand
foldtofivefoldincreaseinthelevelsoffreemonomericubiquitin proteasomefunction.
in cells overexpressing wild-type and the C90S mutant of However,tobetterdeterminewhetherglobalUPSfunctionis
UCH-L1 (Fig. 7C, middle panel; D) (GFP, 1.0 (cid:8) 0.16; wild- affected,wemonitoredthedegradationofGFPuinhippocampal
typeUCH-L1,4.3(cid:8)0.23;C90S-UCH-L1,4.7(cid:8)0.67;D30A- neuronsusingtime-lapseconfocalmicroscopy.Weusedapho-
UCH-L1, 0.97 (cid:8) 0.07). These data are quite interesting be- toactivatablevariantofGFPu(paGFPu),whichisaUPS-specific
cause although both the C90S and D30A mutants lack reporter protein substrate and requires polyubiquitination for
hydrolytic activity, the C90S mutant still maintains its ubiq- degradationbytheproteasome(Benceetal.,2005).Weobserved
uitin binding ability, which is thought to be important for a 50% decrease in the degradation rate of paGFPu in LDN-
stabilizing ubiquitin levels (Osaka et al., 2003; Sakurai et al., treatedneurons(Fig.7E,F).Together,thissuggeststhatthecata-
2006).Wealsomeasuredproteasomeactivityinvitroinlysates lytic activity of the proteasome is not affected by decreased
from wild-type and mutant UCH-L1 overexpressing HEK UCH-L1activity.However,UCH-L1inhibitioncanaffectglobal
293Tcells.Wedidnotdetectanychangesinproteasomeac- ubiquitin-dependent UPS function, most likely because of de-
tivitylevelsbetweenwild-typeandC90SorD30Amutantsof creasedmonomericubiquitinlevels.
Cartieretal.•RegulationofSynapticStructurebyUCH-L1 J.Neurosci.,June17,2009•29(24):7857–7868•7865
there was no significant difference in the density of PSD-95
punctabetweencontrol(GFPandDMSO-treatedneurons)and
myc-ubiquitin-expressingneurons(Fig.8A–D,F)(GFP-DMSO,
1.0(cid:8)0.04;ubiquitin-DMSO,0.97(cid:8)0.05;GFPplusLDN,0.89(cid:8)
0.03;ubiquitinplusLDN,1.1(cid:8)0.03).Moreover,expressionof
ubiquitinitselfhadnoeffectonsynapticstructure.Together,our
datademonstratethatreductionsinthelevelsoffreemonomeric
ubiquitinbecauseofthelackofUCH-L1activityleadtomajor
structuralsynapticalterationsinhippocampalneurons.
Discussion
Activity-dependent remodeling of synaptic connections in the
brain is thought to be crucial for modulations in synaptic
strength.Recently,theUPShasbeenshowntobeanimportant
factor for this remodeling and for synaptic plasticity. Interest-
ingly, very little is understood about the UPS components in-
volvedandtheirregulationatsynapses.Inthepresentstudy,we
setouttounderstandthefunctionofUCH-L1,thehighestex-
pressed DUB in the brain, at synapses to uncover regulatory
mechanismswhichexistinneuronstocontrolitsfunction.Our
data demonstrate that UCH-L1 is distributed throughout so-
maticanddendriticcompartmentsofhippocampalneurons.Us-
ing a specific substrate that can monitor UCH-L1 activity, we
showthatUCH-L1ispartiallyactiveintotallysatesfromcultured
neuronsandinthepostsynapticdensityfractionspreparedfrom
rat brain. Strikingly, however, we found that NMDA receptor
activationrapidlyincreasesUCH-L1activityandconcomitantly
increasesfreemonomericubiquitinlevels.Furthermore,block-
ingUCH-L1activitybyLDNsignificantlyreducedthelevelsof
monomeric ubiquitin. Together, these findings suggest that
UCH-L1 may be responsible for modulating the levels of free
monomeric ubiquitin pools available for various cellular pro-
cesses. Furthermore, NMDA receptor-dependent activation of
UCH-L1 may potentially have a significant effect on synaptic
transmission by controlling ubiquitin levels in neurons. This
novelfindingisthefirstreportdemonstratingthatthefunctionof
UCH-L1ismodulatedbyneuronalactivity.
ManytargetsoftheUPSexistatsynapsesincludingscaffold
Figure6. ComparisonofsynapticproteinexpressionlevelsincontrolandLDN-treatedneu-
and structural proteins whose function is critical for several
ronsandinwild-typeandUCH-L1-deficientmousebrains.A,B,LysatesfromcontrolandLDN-
forms of plasticity (Patrick, 2006; Yi and Ehlers, 2007). Since
treatedculturedneurons(A)orhomogenatesfromwild-typeandUCH-L1-deficientmouse
decreased UCH-L1 activity is associated with several neurode-
brains(B)weresubjectedtoWesternblotanalysis.C,D,Relativebandintensitieswerequan-
generativedisorders,andpharmacologicalinhibitionofUCH-L1
tifiedandnormalizedtothoseofcontrolneuronsorwild-typemousebrains.Thequantified
datawereobtainedfromtwoandfourindependentexperimentsforbrainhomogenatesand activitysubstantiallyreducesLTP,weassessedanystructuralal-
LDN-treatedneurons,respectively.Meanvalues(cid:8)SEMareshown.*p(cid:9)0.01,unpairedStu- terationstosynapsesinUCH-L1-inhibitedneurons.Wefound
dent’sttest. that spines became greatly enlarged in LDN-treated neurons
whiletheirdensitysignificantlydecreased.Theseenlargedspines
were associated with presynaptic and postsynaptic proteins, in
Ectopicexpressionofubiquitinrestoressynapticstructurein particular, important scaffold proteins such as PSD-95 and
LDN-treatedneurons Shank.Wefoundapositivecorrelationbetweenthesizeofspines
ThemajorfunctionofUCH-L1isthoughttomaintainthelevels and the size of synaptic protein clusters associated with those
offreemonomericubiquitinusedforvariouscellularprocesses. spines. Furthermore, we observed alterations to the actin cy-
Todeterminewhetheralterationinsynapticstructureinducedby toskeletonastherewasasignificantaccumulationofF-actinin
inhibitionofUCH-L1weremainlyattributabletoareductionin spines. The increased spine size together with the striking de-
thelevelsoffreemonomericubiquitin,weperformedubiquitin crease in spine number potentially reveals heterogeneity in the
rescueexperimentsinLDN-treatedneurons(Fig.8).Wefound sensitivitytoalteredmonomericubiquitinlevelsbetweenspines.
thattheexpressionofubiquitinfor12hcompletelyrescuedthe ThesealterationsinsynapticstructuremaycontributetotheLTP
effects of UCH-L1 inhibition on PSD-95 size and distribution. defects observed in UCH-L1-inhibited neurons (Gong et al.,
AlthoughPSD-95punctawasincreasedinLDN-treatedneurons, 2006).Wealsoanalyzedthedensityofsynapticproteinpuncta
theexpressionofmyc-ubiquitincompletelyblockedtheeffectof andfoundadecreaseonlyinPSD-95.Thiscouldreflectchanges
LDN (Fig. 8A–E) (GFP-DMSO, 1.0 (cid:8) 0.05; ubiquitin-DMSO, inthestabilityortraffickingofPSD-95.Indeed,theretentionof
0.93 (cid:8) 0.04; GFP plus LDN, 1.6 (cid:8) 0.04; ubiquitin plus LDN, PSD-95inindividualspineshasbeenshowntobehighlydynamic
1.0(cid:8)0.03).Aspreviouslyobserved,therewasaslightdecreasein (Grayetal.,2006).Wealsoanalyzedproteinexpressionlevelsfor
thedensityofPSD-95punctainLDN-treatedneurons;however, severalsynapticproteinstodeterminewhethertheobservedal-
7866•J.Neurosci.,June17,2009•29(24):7857–7868 Cartieretal.•RegulationofSynapticStructurebyUCH-L1
terationsinthesizeordensityofsynaptic
punctacorrelatedwiththeirstability.We
only detected changes in the protein ex-
pressionlevelsofPSD-95andShank.Al-
though the levels of PSD-95 increased in
both LDN-treated neurons and brain ly-
sates from UCH-L1-deficient mice, the
levels of Shank protein expression were
only elevated in the brains of UCH-L1-
deficientmice.However,itisquiteplausi-
blethatthestabilityofsynapticproteinsis
not equally sensitive to altered UCH-L1
activityand/orlevelsofmonomericubiq-
uitin. Alternatively, UCH-L1 may have
specifictargetsinneuronsasotherinter-
actingpartnersforUCH-L1havebeenre-
ported (Caballero et al., 2002; Liu et al.,
2002;Kabutaetal.,2008).However,itis
alsofeasibletohypothesizethatdecreased
UCH-L1activityresultsindecreasedmo-
nomeric ubiquitin levels, which elicits a
pathogenicprogramthatinitiateswiththe
lossofunstablespineswithaconcomitant
redistributionandaccumulationofsynap-
ticproteinssuchasPSD-95andShankinto
more stable spines. Strikingly, we found
that overexpression of ubiquitin restored
normalsynapticstructureinLDN-treated
neurons.Thisindicatesmonomericubiq-
uitin levels to be regulatory for normal
synaptic structure. Together, these find-
ingspointtoasignificantroleofUCH-L1
insynapticremodelingbymodulatingfree
Figure7. EffectsofalteredUCH-L1activityonUPSfunction.A,CulturedneuronsweretreatedwithvehicleorLDNfor24h.
ubiquitinlevelsmostlikelyinanactivity- ProteasomeactivitywasmeasuredintotalcelllysatesfromcontrolandLDN-treatedneuronsinvitrobyfluorimetricassayusinga
dependentmanner. Suc-LLVY-AMCfluorogenicsubstrate.RelativefluorescenceunitsovertimeincontrolandLDN-treatedneuronsareshowninaline
Does altered UCH-L1 activity affect graph.Meanvalues(cid:8)SEMoffiveindependentexperimentsareshown.B–D,HEK293Tcellsweretransfectedwithwild-type,
UPS function? To address this question, C90SandD30AUCH-L1-GFPconstructsfor48h.B,ExpressionlevelsofGFP,wild-type,andmutantUCH-L1-GFPwereassessedby
weexaminedproteasomeactivitylevelsin Westernblotanalysis.Immunoblotswereprobedwithanti-GFPantibody(toppanel).Activitylevelsofwild-typeandmutant
vitroincontrolandLDN-treatedneurons. UCH-L1-GFPwereassessedbyDUBlabelingassay(HAUb-VME)oflysatesfromHEK293T-transfectedcells.Arepresentative
Westernblotoflabeledlysatesisdemonstrated(bottompanel).C,Expressionlevelsoffreemonomericubiquitinandpolyubiq-
Wedidnotdetectanychangesinprotea-
uitinconjugatesintransfectedHEK293Tcellsareshown.Westernblotprobedwithanti-UCH-L1andanti-CDK5(loadingcontrol)
someactivitylevelsmeasuredintotalcell
antibodiesdemonstratingequalexpressionlevelsofwild-typeandmutantUCH-L1-GFPandequalproteinloading,respectively.
lysates from control and UCH-L1-
D,RelativeintensitiesoffreemonomericubiquitinlevelsinGFP,wild-type,andmutantUCH-L1-GFPtransfectedcellsobtainedby
inhibitedneurons.Kyratzietal.(2008)re- densitometryanalysisofubiquitinblotsfromthreeindependentexperiments.Meanvalues(cid:8)SEMareshown.*p(cid:9)0.01,
ported similar findings. They found that unpairedStudent’sttestbetweencontrol(GFP)andanytestgroup.E–G,CulturedneuronsweretreatedwithLDNfor24h.paGFPuvirions
the loss of UCH-L1 in neurons derived wereaddeddirectlytoculturemediaafter10–12hofLDNtreatment,andproteinexpressionwasallowedtocontinuefor12–14h.E,
fromgadmicedidnotaffectproteasome Representativestraighteneddendritesoftime-lapseexperimentsfromcontrolorLDN-treatedculturedneuronsexpressingpaGFPuare
activity when assayed in vitro. Further- shown.Scalebar,10(cid:1)m.F,Groupedanalysis(plottedaslinegraphs;mean(cid:8)SEM)ofdendriticpaGFPufluorescenceintensitynormalized
more,theyalsofoundthatoverexpression totime0forcontrol(paGFPualone)orLDN-treatedneurons.LiveimagingofpaGFPudegradationwasmadeon6and11cells,two
ofwild-typeUCH-L1,whichsignificantly dendritespercell,fromcontrolandLDN-treatedneurons,respectively.RFU,Relativefluorescenceunit.Thecolorlook-upscaleforpaGFPu
degradationisblack(lowest)towhite(highest).*p(cid:9)0.01,unpairedStudent’sttest.
upregulated free monomeric ubiquitin
levels,didnotalterproteasomeactivityin
vitro (Kyratzi et al., 2008). This is consistent with our findings uitination(Benceetal.,2005).Indeed,thestabilityofPSD-95
(supplemental Fig. 5A,B, available at www.jneurosci.org as and Shank, two PSD scaffolds shown to be regulated by
supplemental material). This would suggest that the excess ubiquitin-mediatedproteasomaldegradation,issignificantly
availabilityofmonomericubiquitindoesnotincreasethepro- increasedinUCH-L1-deficientmice.
teolytic activity of the proteasome. Interestingly, however, a Together,ourfindingshaveuncoveredanovellinkbetween
significant decrease in the degradation rate of the paGFPu neuronal activity, UCH-L1 function, the maintenance of free
reporter was found in neurons that were treated with LDN. monomericubiquitinlevels,andtheregulationofsynapticstruc-
This could potentially reflect the sensitivity of our live- ture.Importantly,thisstudysuggeststhatalteredsynapsestruc-
imagingreporterassaystoalteredubiquitinlevelsinUCH-L1- turecausedbythemisregulationofUCH-L1activityandmono-
inhibitedneurons.Althoughourinvitromeasurementofpro- meric ubiquitin homeostasis may serve as an underlying
teasome activity is independent of substrate ubiquitination, mechanismfordefectsinsynapticplasticityseeninneurodegen-
paGFPudegradationbytheproteasomeisdependentonubiq- erativedisease.