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PatternsofNogomRNAandProteinExpressionintheDevelopingandAdultRatandAft


RegrowthofinjuredaxonsintheadultCNSofhighervertebratesisveryrestrictedMyelin-associatedneuritegrowthinhibitorsareatleastinpartresponsibleforthislackofregenerationSchwabandBartholdi1996Olson1997Amonocl

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Document on Subject : "PatternsofNogomRNAandProteinExpressionintheDevelopingandAdultRatandAft"— Transcript:

1 PatternsofNogomRNAandProteinExpressionin
PatternsofNogomRNAandProteinExpressionintheDevelopingandAdultRatandAfterCNSLesionsAndreaB.Huber,OliverWeinmann,ChristianBro¨samle,ThomasOertle,andMartinE.SchwabBrainResearchInstitute,UniversityofZurich,Zurich,8057Switzerland,andDepartmentofBiology,SwissFederalInstituteofTechnology,Zurich,8057SwitzerlandNogo-AisaneuritegrowthinhibitorinvolvedinregenerativefailureandrestrictionofstructuralplasticityintheadultCNS.Threemajorproteinproducts(Nogo-A,-B,and-C)arederivedfromthegene.HerewedescribetheembryonicandpostnatalexpressionofthethreeNogoisoformsintheratbyhybridizationandimmunohistochemistry.NorthernandWesternblotanalysisindicatedthatNogo-Aispredominantly RegrowthofinjuredaxonsintheadultCNSofhighervertebratesisveryrestricted.Myelin-associatedneuritegrowthinhibitorsareatleastinpartresponsibleforthislackofregeneration(SchwabandBartholdi,1996;Olson,1997).Amonoclonalantibody(mAb),IN-1,raisedagainstamajormyelin-associatedneuritegrowthinhibitor,calledNI-250,wasabletoneutralizethegrowth-inhibitingactivitiesofthesubstratetotheextentthat ReceivedSept.25,2000;revisedJan.31,2002;acceptedFeb.5,2002.ThisstudywassupportedbySwissNationalScienceFoundationGrant31-45549.95,theChristopherReeveParalysisFoundation(SpringŢeld,NJ),andthe expressedinmyelinofthematureCNSbutalsoinneurons,especiallyduringdevelopment.WeinvestigatedtheexpressionpatternofNogo-A/Baftercorticalandspinallesionsatseveraltimepointsandfoundnosignicantchangesofexpressioninoligodendrocytesafterinjury.Nogo-Bshowedwidespreadex-pressioninCNSaswellasinperipheraltissues,andNogo-Cwasfoundtobestronglyexpressedinskeletalmuscleandtolowerlevelsalsoinbrainandheart.Thisraisesthepossibilitythatbesidestheneuritegrowth-inhibitoryfunction,theNogofamilyofproteinsmighthaveadditional,sofarunknownphysiologicalMATERIALSANDMETHODSAnimalsandtissues.TissuesofembryonicandpostnatalLewisratswereusedinthisstudy.Thedayofvaginalplugdetectionwasconsideredembryonicday1(E1).Pregnantfemalesweredecapitated,andtheembryos(E14,E16,andE19)wererapidlyremoved,embeddedinTissueTek(OCTcompound;Zoeterwoude),andfrozeninisopentaneatCforsubsequentinsituhybridizationandimmunohistochemistry.Brain,spinalcord,andeyesofpostnatalanimalsfrompostnatalday0(P0),P1,P3,P5,P9,P14,andadultanimalswereremovedandprocessedasindicatedabove.Eachdevelopmentaltimepointwasanalyzedinatleastthreedifferentanimals.Inaddition,tissuesfromadultdecapitatedanimalswerecollectedforWesternandNorthernblotting.Forconfocalmicroscopy,adultLewisratswerekilledwithpentobarbital(500mg/kg)andperfusedtranscardiallywithRingerssolutionfollowedby4%para-formaldehydein0.15phosphatebufferwith5%sucrose.Thespinalcordswereremovedandpost-xedovernightin4%paraformaldehydeandphosphatebuffer.CNSlesions.AnimalexperimentalprocedureswereapprovedbytheVeterinaramtoftheCantonofZurich.Animalsweredeeplyanesthe-tizedbyintraperitonealinjectionoffentanylcitrate(0.0189mg/100gmbodyweight),uanisone(0.6mg/100gm,Hypnorm;JanssenBiochimica,Berse,Belgium),andmidazolam(0.6mg/100gm,Dormicum;Hoffmann-LaRoche,Basel,Switzerland).Forspinalcordlesions,theskinonthebackoftheanimalswasopened,andthevertebralcolumnwasexposed.AlaminectomywasperformedatlevelT8,andthedorsalhalfofthespinalcordwastransectedw

2 ithneiridectomyscissors.Thebackmuscleswe
ithneiridectomyscissors.Thebackmusclesweresutured;theskinwasclosedwithsurgicalstaples;andtheanimalswerelefttorecoveronaheatingpad.Forcerebralcortexlesions,thescalpoftheheadwasincised,andtheskullwasopenedwithadentaldrill.Alongitudinalcutof5mmwasperformedinonehemicortex2mmlateraltothemidline.Thescalpwassutured,andtheanimalswerelefttorecoveronaheatingpad.Aftersurvivaltimesof1and4dand1,2,and4weeks,theanimalswerekilledbydecapitation,andthetissuewasremoved,embeddedinTissueTek,andfrozenatCinisopentane.ASBrunawasgeneratedagainstapartialrecombinantNogo-Aprotein(aa7621163);ASBianca,specicfortheNterminusofNogo-Aand-B,wasraisedagainstbacteriallyproduced,immobilizedmetalafnitychromatography-puriedandgel-electroelutedfragmentsaa1172andaa131and59172ofratNogo-A;andAS472specicforNogo-AandAS818specicforNogo-CwereproducedagainstthesyntheticpeptidesSYDSIKLEPENPPPYEEA(bovinesequence),cor-respondingtoratsequenceaa623640withthreemismatches(Chenetal.,2000),andMDGQKKHWKDKVVD(ratsequence),respectively(ResearchGenetics,Huntsville,AL).Ascontrols,thecorrespondingpreimmuneseraandantiserapreincubatedwiththecorrespondingim-munogenicpeptideswereused.mAbIN-1wasraisedagainstapartiallypuriedCNSmyelinfraction(CaroniandSchwab,1988).BecauseitsbindingsiteontheNogoproteinisprobablyaconformationalepitope,ithasnotbeendeterminedsofar.Itsusefulnessforadetaileddifferentialexpressionanalysisisthereforelimited.ForimmunohistochemistrytheantibodiesweredilutedinPBS,pH7.4,containing1%normalgoatserumasfollows:mAbIN-1hybridomasupernatant,1:5;ASBruna,1:7500;ASBianca,1:2000;AS472,1:2000;AS818,1:2000;afnity-puriedAS818,1:50;anti-myelin-associatedglycoprotein(MAG),1:25(Roche);anti-myelinoligodendrocyteglycoprotein(MOG),1:25(Roche);anti-neurolament,1:100(Roche);andanti-myelinbasicpro-tein(MBP),1:250(Roche).ForWesternblotsthefollowingdilutionsinTrisbuffer,pH8.0,with0.1%TritonX-100wereused:ASBruna,1:5000;ASBianca,1:5000;AS472,1:2000;andafnity-puriedAS818,Northernblotanalysis.Poly(A)RNAwasextractedfromadultrattissuesusingtheFastTrackkit(Invitrogen,Groningen,TheNether-lands).RNAswereseparatedbyelectrophoresison1%formaldehydegelsandtransferredtoGenescreenmembranes(DuPont,Billerica,MA).Blotswerehybridizedwiththeantisenseriboprobeasdescribedearlier(Chenetal.,2000).Blothybridization,washing,andCDP-stardetectionweredoneasdescribedbythemanufacturer(Roche).SDSgelsandWesternblotting.Tissueswerehomogenizedandextractedonicein0.1Trisbuffer,pH8.0,with60mm()dimethylammonio]-1-propanesulfonicacid,10mEDTA,2.5mdoacetamide,1mphenylmethylsulfonyluoride,0.1g/mlaprotinin,1g/mlleupeptin,and1g/mlpepstatinA.Tissuedebriswaspelletedtogetaclearsupernatant.Twenty-vemicrogramsoftotalproteinweredissolvedinsamplebuffer,andSDS-PAGEandWesternblottingwereperformedasdescribed(Franketal.,1998).ThesecondaryantibodywasHRP-conjugatedanti-rabbitoranti-mouse(1:20,000;Pierce,Rockford,IL)andwasvisualizedusingachemiluminescencesystem(SuperSignal;Pierce).Tissuesfromveindividualanimalswereanalyzedseparately.InsituDigoxygenin-labeledsenseandantisenseRNAprobesweregeneratedasdescribed(Schaeren-WiemersandGerMoser,1993).Theprobewassynthesizedfroma2368bpratctemplate(nucleotides8153183);therecognizingallthreeisoformsof,con

3 tainstranscriptAsequencebetweennucleotid
tainstranscriptAsequencebetweennucleotides2535and4678(Fig.1).Cryostatsections(15werecollectedonSuperfrost-Plusslides(Menzel-Glaser,Braunschweig,Germany),andinsituhybridizationwasperformedasdescribedearlier(Schaeren-WiemersandGern-Moser,1993).Inbrief,sectionswerexedin4%paraformaldehydeandPBS,acetylatedin0.1trieth-anolamineand0.25%acidanhydride,andpermeabilizedforeither20min(postnataltissues)or5min(embryonictissues)in1%TritonX-100andPBS.Hybridizationwasperformedovernightin5SSCbuffercontaining50%formamideand2%blockingreagent(Roche)at68Twostringentwasheswereperformedin0.2SSCatthesametemper-aturefor1hr,andsignalsweredetectedwithalkalinephosphatase-coupledanti-digoxigeninantibodies(Roche)usingnitrobluetetrazolium(NBT)and5-bromo-4-chloro-3-indolylphosphate(BCIP)ascolorreac-tionsubstrates.Immunohistochemistryandhistoblot.Myelinproteins,e.g.,MBP,aresensitivetoxationandtissuepermeabilization.Differentxationmeth-odswereusedtooptimizetissuestructureandpreservationofthevariousantigenstomatchtheimmunohistochemicalsignaltotherelevantdistributionofNogoasdetectedbytheinsituhybridizationandhistoblotprocedure.FifteenmicrometerserialcryostatsectionsweremountedonSuperfrost-Plusslidesandeitherxedwithethanolandaceticacidasdescribedearlier(Rubinetal.,1994),exceptthatthequenchingstepwasomitted,orincubatedfor30secinCmethanolormethanolandDMSO(10%).ForEGTA-ethanolandaceticacidtreatment,sectionswereincubatedfor5minat4Cin0.1PIPES,5mEGTA,and2mMgCl,pH6.8,beforeethanolandaceticacidxation.Theprimaryantibodieswereincubatedovernightat4C,andsubsequenttreatmentwasasdescribed(Rubinetal.,1994).Signaldetectionwasdonewithbiotinylatedgoatanti-rabbitantibodies(VectorLaboratories,Burlin-game,CA)andtheABCkit(VectorLaboratories)using3,3diaminobenzidineasachromogen.Peptidecompetitionoftheantibodysignalwasperformedbypreincubationoftheantibodyfor2hrinthepresenceoftheimmunogenicpeptide(0.25l).ThesectionswereevaluatedusingaZeiss(Oberkochen,Germany)Axiophotmicroscope.Forconfocallaserscanningmicroscopy,20mserialcryostatsectionsweremountedonSuperfrost-Plusslidesandpretreatedfor20mineitherinethanolandaceticacidfordoublestainingforMBPandNogo-AorinKryox(Merck,Darmstadt,Germany)fordoublestainingforMAGorMOGandNogo-Aandprocessedasindicatedabove.ThesectionswereanalyzedusingaZeissLSM410microscope.Toconrmthesemiquan-titativeimmunohistochemistrysignals,weusedamodiedhistoblotmethodfordirecttransferofproteinsfromafreshfrozensectiontoanimmobilizedmatrix.Briey,anitrocellulosemembranewaswettedinSDS-containingtransferbuffer(Benkeetal.,1995).Twelvemicrometercryostatsectionswerequicklythawed,pressedontothemembranefor30sec,andinspectedforcompletetransfer.Theproteinsboundtonitro-cellulosemembraneswereimmunostainedwithrespectiveantiserausingtheprocedureofconventionalWesternblotting.Immunoreactivitywasvisualizedusingthealkalinephosphatasereactionsubstratesystem(NBTandBCIP).Electronmicroscopicimmunohistochemistry.Animalsweretranscardi-allyperfusedbyRingerssolution,followedby4%formaldehyde,0.25%J.Neurosci.,May1,2002,Huberetal.ExpressionPatternofNogo glutaraldehyde,and70mgofCaClin0.1phosphatebuffer.Opticnervesweredissectedandpost-xedovernightinthesamesolution.Thetissuewaswashedin0.1cacody

4 latebuffer,osmicatedfor1hrin1%incacodyla
latebuffer,osmicatedfor1hrin1%incacodylatebuffer,dehydratedthroughanascendingseriesofalcoholfollowedby10mininpropyleneoxidetwice,andthenembeddedinEponaraldite.Aftercuring,ultrathinsectionsof7090nmwerecut,takenuponnickelgrids,andprocessedforNogo-A,MAG,andMOGimmunoreactivitybyovernightincubationat4CwithAS472(afnity-puried,diluted1:20inblockingbuffercontaining10%goatserumand2.5%ovalbumin),anti-MAG(1:2),andanti-MOG(1:2),followedbygold-coupledsecondaryantibodies(1:50;BritishBiocell,Cardiff,UK)andstainingwithuranylacetate.Onsomesections,etchingbysodiumethoxide(3NaOHinethanoldiluted1:300andincubatedfor20sec)wasusedtoexposetheantigenandtoincreasespeciclabeling(Trappetal.,1989).Theabsolutedensityofthegoldlabelwasincreasedafteretching,buttherelativedensitybetweendifferentcompartmentswasverysimilartothatofnonetchedsections.Controlsectionsstainedwitheitherpreimmuneserumorsecondaryantibodyonlydidnotshowsig-cantamountsofgoldlabeling.ThesectionswereanalyzedinaZeissEM902microscope.Twoindependentobservers,blindedtotheexper-imentalconditions,countedthedistributionofgoldgrainsinthefollow-ingcompartments:axonalcytoplasm,innerloopofmyelinsheathandaxonmembrane(thetwocompartmentscannotbedistinguished),com-pactmyelin,andouterloopofmyelinsheath.Goldgrainsina2030nminterfacezonethatcouldnotbeunequivocallyassignedtoonecompart-mentwerecountedseparatelyasanoverlapcompartmentandlaterdistributedaccordingtothecalculatedratiosbetweenthetwointerfacingcompartments.Thevolumedensitiesofthefourcompartmentsweredeterminedstereologicallybyapointgridandusedtocalculatearelativedensityofimmunogoldlabelingforeachcompartment(Weibeletal.,1966).Approximately750eldswerecountedperanimalforfourdif-ferentanimals,andameanrelativedensityofNogo-Aimmunoreactivitywascalculated.RESULTSThedistributionpatternsofNogomRNAsandproteinsduringdevelopmentandintheadultwereanalyzedusinginsituhybrid-izationandimmunohistochemistryonratbrainandspinalcordsectionsbetweenE14andadult.SeveralprobesandantibodiesagainstdifferentepitopesoftheNogosequenceallowedustodistinguishbetweenthetwoisoformsNogo-Aand-C(Fig.1).BecauseNogo-Bhasnouniquesequence(Chenetal.,2000),itwasonlypossibletodetermineitsexpressionbyexclusion:asignalobservedwithASBruna,ASBianca,andtheprobebutnotwithAS472,theprobe,orAS818,specicforNogo-C,indicatedexpressionofthemiddle-sizedtranscript,Nogo-B.WethereforeusethetermNogo-A/Bwhennodistinc-tionwaspossible.Matchedpreparationsusingsenseprobes,preimmunesera,orantiserapreincubatedwiththerelevantim-munogenicpeptidewereusedascontrols;theygavenosignisignalsinanyofthetissuesstudied.Forimmunohistochemistry,weuseddifferentxationprotocolsofthefreshfrozensections:ethanolandaceticacidvisualizedNogoexpressioninmyelin,butexposuretothisxativereducedthesignalinnonmyelinatedtissues(e.g.,spinalmotorneuronsandinterneuronsinFig.),presumablybyextractionoftheantigen.FixationwithmethanolshowedexpressionofNogoincellbodiesofoligoden-drocytesandneurons(seeFig.4),butthemyelinstainingwasweakened.ExposuretoEGTAbeforeethanolandaceticacidxationallowedvisualizationofNogoexpressedbothinmyelin Figure1.Nogoisoforms,probes,andantisera.Thethreescriptswiththeregion(),Nogo-A-specicregion(Nogo-A/B-specicNterminus(),

5 andNogo-C-specicNterminus11aa)areshown.T
andNogo-C-specicNterminus11aa)areshown.Twolonghydrophobicstretches(35and36aa)intheregioncommontoallthreeisoforms,servingaspotentialtrans-membranedomains,aremarked(gray).RiboprobesusedforinsituhybridizationandNorthernblotareshown:AS472andAS818wereraisedagainstpeptidesrecognizingNogo-Aand-C,respectively.ASBiancawasraisedagainsttheNogo-A/B-speciterminus,andASBrunawasraisedagainstabacterialrecombinantproteinincludingthecommonpartpresentinallthreeNogoisoformsandtheC-terminalportionoftheNogo-A-specicregion.NotethatNogo-Bhasnouniquesequence. Table1.RegionaldistributionofNogo-A,-B,-Cproteinexpressionintheadultrat LocationNogo-ANogo-A/BSpinalcordWhitematterOligodendrocytecellbodiesAstrocytesMotorneuronsCerebralcortexLayerILayerII/IIILayerIVLayerV/VIDentategyrusCerebellumGranulecellsPurkinjecellsMolecularlayerDeepcerebellarnucleiRetinaRetinalganglioncellsInnerandouternuclearlayerPeripheralgangliaDorsalrootgangliaSemiquantitativeanalysisoftheNogoexpressioninthenervoussystemasfoundinimmunohistochemistryontissuesections.,Undetectable;(),veryweak;,strong;,verystrong.AS472.ASBrunaorASBianca.AS818.Freshfrozensectionspost-treatedwithethanolandaceticacidorEGTA-ethanolandaceticacid.Freshfrozensectionspost-treatedwithmethanolormethanol-DMSO.Freshfrozensectionspost-treatedwith0.1%paraformaldehydeforimmunohistochemicalvisualizationofthethreeNogoisoformsindifferenttissues.Granulecelllayerofthecerebellumshowsweakunspecicstaining(negativeinsituhybridizationandhistoblot).Huberetal.ExpressionPatternofNogoJ.Neurosci.,May1,2002, andincellbodies;however,thetissuepreservationwassomewhatimpairedcomparedwiththeothermethods.Table1summarizestheexpressionofthethreeNogoisoformsintheadultratandtheuseofthedifferentxatives.NorthernandWesternblotanalysisNorthernBlotanalysisofadultrattissueswiththeriboproberevealedthatthelongestisoform,,wasmainlytranscribedinmyelinatedtissuesoftheCNS:opticnerve,spinalcord,andbrainshowedaveryprominentbandat4.6kb(Fig.2SmalleramountsofmRNAcouldalsobedetectedindorsalrootganglion(DRG),sciaticnerve,heart,andtestis.HighmRNA(2.6kb)levelswerefoundinopticnerve,spinalcord,andbrainandalsoinsciaticnerve,lung,andkidney(Fig.).LowerlevelsofmRNAwerefoundinDRG,testis,spleen,heart,andliver,whereasnomRNAwasdetectableinskeletalmuscle.mRNA(1.7kb)washighlyexpressedinskeletalmuscle,opticnerve,spinalcord,andbrain.Lowerlevelswerepresentinheart,liver,spleen,andkidney.OnWesternblotsofadulttissueextracts,Nogo-Awaspresentinbrainandspinalcord,andlowlevelswerealsofoundintestisandheart(Fig.2).NoneoftheotherperipheraltissuestestedexpresseddetectablelevelsofNogo-A.Nogo-B,revealedbyin-cubationoftheWesternblotwithASBianca,waspresentinbrain,spinalcord,testis,heart,lung,spleen,andkidney.Lowlevelswerealsofoundinliver.SkeletalmuscledidnotexpressdetectableamountsofNogo-Bprotein.HighlevelsofNogo-CweredetectedwithAS818inskeletalmuscle,whereasaweakersignalwasfoundinbrain,spinalcord,andheart.InsituhybridizationandimmunohistochemistryDuringdevelopment,expressionofNogo-Cprotein,thesmallestofthethreeNogos,wasprominentinperipheraltissuessuchasskeletalmuscle,skin,andintestinalepithelium,whereasexpres-sioninthenervoussystemwasundetectablewiththecurrentlyavailableant

6 isera(Fig.3).However,Nogo-Cwaspresentinp
isera(Fig.3).However,Nogo-CwaspresentinpostnatalPurkinjecells(seeFig.7),butonlyverylowproteinlevelsweredetectedinotherpartsoftheCNS(e.g.,cortex;seeFig.5SpinalcordandperipheralgangliaNogo-A/Bwasexpressedattheearliesttimepointanalyzed,E14,inneuronsofthespinalcordwiththehighestlevelinventral Figure2.NorthernandWesternblotanalysis.,NorthernhybridizationwiththeC-terminalprobeonmRNAofadultrattissuesrevealed(4.6kb)tobestronglyexpressedinbrain,spinalcord(),andopticnerve().LowlevelsofmRNAwerefoundinDRG,sciaticnerve),testis,andheart.NotethatforDRGandsciaticnerve,lessmRNAwasloadedthanfortheothertissues.mRNA(2.6kb)washighintheCNSbutwasalsodetectedinDRG,sciaticnerve,lung,andkidneyandatlowerlevelintestis,liver,andspleen.Strongexpressionof(1.7kb)wasobservedinspinalcord,brain,opticnerve,andskeletalmuscle.Insciaticnerve,heart,liver,spleen,andkidney,expressionwaslower.Forloadingcontrol,theblotwasreprobedwithaglyceraldehyde-3-phosphatedehydrogenaseriboprobe.,Intheadultrat,Nogo-A(190kDa)wasstronglyexpressedinbrainandspinalcord,asrevealedbyAS472(andASBrunaandASBianca;datanotshown).Apartfromthenervoussystem,Nogo-Awasonlyexpressedindetectableamountsintestisandheart.Thebandpresentinliver()wasalsodetectedbypreimmuneseraandsecondaryantibodyonlyandrepresentsthereforeanunspecicsignal.ASBiancashowedexpressionofNogo-B(55kDa)inbrain,spinalcord,testis,heart,lung,liver,spleen,andkidney.AS818detectedastrongNogo-Cbandat25kDainskeletalmuscle.ThesmallestNogoisoformwasalsopresentinbrainandheart,40and5%,respectively,oftheamountpresentinskeletalmuscle,asrevealedbydensitometricanalysisonWesternblotswiththesameamountoftotalproteJ.Neurosci.,May1,2002,Huberetal.ExpressionPatternofNogo motorneurons.PeripheralgangliasuchasDRGandsympatheticgangliawerealsoexpressingNogo-A/B(Fig.4).InP3andP5animals,largeneuronsinintermediatelaminaearoundthecen-tralcanalwerestronglyexpressingNogo,inadditiontotheventralmotorneurons.AtageslaterthanP5,aslightdownregu-lationofAS472immunoreactivityparticularlyinmorecaudalmotorneuronswasobserved,whereasthesignalwiththecorre-spondingRNAproberemainedstrong(datanotshown).ThiseffectwaslesspronouncedwithASBrunaandASBianca,possiblyreectingadifferentialexpressionofNogo-Aand-B.Interneuronsonalllevelsofthespinalcordshowedstrongex-pressionofmRNAintheadult,comparablewiththeinten-sityofmotorneurons,whereasNogoproteinexpressionwaslow(Fig.4).Fixationwithethanolandaceticacidresultedina Figure3.NogoexpressionintheE16ratembryo.Insituhybridizationwithproberevealedstrongex-pressioninthemantlelayerofpostmi-toticneuronsinthedevelopingfore-braincortex().Inthetrigeminalganglion(C,D),highlevelsofNogo-Aweredetected(,AS472).Nogo-Aproteinwasalsofoundinthetrigeminalnervebers,extraocularmuscles(arrows),andopticnerve(arrowhead).NotethatNogo-C(AS818;)wasonlypresentinextraocularmuscles(arrows)butnotinthetrigeminalganglionoropticnerve.StrongexpressionofNogo-CwasfoundwithAS818inintestinalepithelium(arrowsG,H,Nogo-AmRNA()andprotein()expressioninthespinalcord()andDRG(asterisks).Again,proteinexpressionrevealedbyAS472wasalsofoundinnervebers(H,ar-),whereasmRNAexpressionprobe)wasrestrictedtoneuro-nalcellbodiesintheDRGandspinalcord.ThelevelofmRNAexpressioninDRGwashigherth

7 aninthespinalcord;however,proteinlevelsw
aninthespinalcord;however,proteinlevelswerecompara-ble.Scalebar:B–E,G,H,550F,B,,140Huberetal.ExpressionPatternofNogoJ.Neurosci.,May1,2002, strongNogosignalinmyelin,whereastheneuronalNogowasextractedtoalargedegreebythismethod;therefore,theneuro-nalsignalisveryweak(Fig.4).However,expressionofNogo-AinmotorneuronswasobservedinsectionsxedwithmethanolbyAS472(Fig.4)verysimilartostainingpatternsobtainedbymAbIN-1(Fig.4).Postnatally,thestrongestNogo-Aproteinexpressionwasfoundinmyelinandoligodendro-cytecellbodies,asrevealedwithAS472(Fig.4)andmAbIN-1(Fig.4DRGswerestainedwithboththeAS472/probeandASBruna/probefromtheearliesttimepointobserved,E14,untiltheadultstage(Figs.3).AlthoughNogo-Apro-teinwasdetectedinperipheralnerve,nomRNAwasfound,pointingtoanaxonallocationandlackofNogo-AexpressioninSchwanncellsandperineurium(Fig.4arrows).Nogo-A/BwasalsodetectedathighlevelsinperipheralgangliaotherthanDRG,e.g.,thetrigeminalganglion(Fig.3NeocortexandhippocampusAtnodevelopmentalstageanalyzedwasanydifferentialmRNAexpression(probe)ordifferenceinstainingwithASBrunaandASBiancaorAS472detected.ItisthereforenotpossibletomakeastatementaboutspecicexpressionofNogo-B,andwearereferringtoNogo-A/B.Nogo-CwasnotdetectablewithAS818duringdevelopment,andonlyverylowexpressionwasfoundinthecortexintheadult(Fig.5).InE16animals,postmitoticneuronsinthemantlelayerwerestronglyexpressingNogo-A/B,whereasthematrixlayer,comprisingthedividingcells,wasstainedlessintensely(Fig.3).Threedayslater,neuronsinthecorticalplate,mantlelayer,andmatrixlayerwereexpressingNogo-A/B.LargesubplateneuronswereNogo-A/B-positiveatE19P5(Fig.5).Nogo-A/Bwasfoundinneuronsofthecorticalplateandadditionallyinthesubplateneurons.AtP3,whentheneocortexalreadyconsistedofseverallayers,neuronsinlayerIVwereprimarilynegative,whereas Figure4.Nogo-Aexpressioninthespinalcordanddorsalrootganglia.Insituhybridizationwiththeprobeshowedthepresenceofthistranscriptinneuronalsubtypesofthespinalcord,DRG,andsympatheticganglia()atE14()andE16().Intheadult,mRNAwasfoundinoligodendrocytecellbodiesinthewhitematter(arrowheads).ThecorrespondingimmunohistochemistrywithAS472(E,I)revealedNogo-Aexpressionmainlyinthemyelinatedareasofthespinalcordandveryintenselyinoligodendrocytecellbodies(E,Iarrowheads).Lessstronglymyelinatedareassuchastheregionofthecorticospinaltract(arrow)werealsostainedless.Fixationofthetissuewithethanolandaceticacid(A,B,E,G,IrevealedtheNogo-Alocalizedtomyelinandoligodendrocytecellbodies.NeuronalNogo-Awasvisualizedbyxationwithmethanol(F,H,J).SpinalneuronsinthegraymatterwereexpressingmRNA()andNogo-Aprotein(,highmagnicationofmotorneurons).StrongNogo-AexpressionwasfoundinadultDRGneuronsandtheirneurites(arrows),whereastheneuriteswerenotstainedwiththeprobe(arrows).mAbIN-1stronglystainedwhitematterandoligodendrocytecellbodies()aswellasmotorneurons(methanol).Scalebar:A,C,E,G,460D,F,H,140J.Neurosci.,May1,2002,Huberetal.ExpressionPatternofNogo neuronsinlayerVandinthecorticalplatewereexpressingNogo-A/B.AllneuronsmigratedtotheirlayersatP5,whereNogowasfoundtobeexpressedinsomecellsoflayerVI,butmoreneuronswerepositiveinlayersIIIVandV.Inadultanimals,insituhybridizationwithbothprobesrevealedmRNAsignalsinneuronsofallcorticallaye

8 rswithlowerlevelsinlayerIVneurons(Fig.5,
rswithlowerlevelsinlayerIVneurons(Fig.5,commonprobe,Gprobe),whereasthelevelofproteinexpressionseemedrelativelylowcomparedwitholigodendrocytecellbodiesinthecorpuscallosum(Fig.5,ASBruna,,AS472).Inthehippocampus,strongexpressionofNogo-AwasobservedintheregionsCA1CA4bybothinsituhybridizationandimmu-nohistochemistryatbirth(Fig.6).ThegranulecellsofthedentategyrusexpressedlessNogo-AmRNAandprotein.Intheadult,expressionofNogo-Aand-BwasseeninthepyramidalcellsofCA1CA4,whereaslesssignalcouldbedetectedinthedentategyrus(Fig.6).Inadditiontothestratumpyramidale,Nogo-Awasalsodetectedinhippocampalberlayersbyimmu-nohistochemistry(Fig.6)andhistoblotting(Fig.6).Overall,theproteinexpressioninneuronswaslow,however,comparedwiththewhitematterofthembriafornixandthecorpuscallo-sum.Interestingly,parvalbumin-andcalbindin-positivehip-pocampalinterneuronswerealsofoundtoexpressNogo-A(Fig.,respectively).Inthecerebellum,differentialexpressionofNogo-A,-B,and-Cwasobserved.PurkinjecellswereexpressingNogo-A/Bfrombirthon,asrevealedbyASBruna,ASBianca,andAS472andcorrespondingRNAprobes(Fig.7).Nogo-C,however,wasonlyfoundatlowlevelsinPurkinjecellsatP3(Fig.7),butexpressionincreasedduringpostnataldevelopment(Fig.7).Inadditiontohighlevelsinthesoma,theNogoproteinswerealsofoundinPurkinjecelldendrites(Fig.7).Theexternalgranularlayer(EGL)showedmRNAexpressionofatP3,whichstartedtodecreasefromP5onandwasnearlyundetectableatP14(Fig.7).Interestingly,thestrongestimmunoreactivityatP9wasfoundinthepremigratoryzoneoftheEGL,whereastheoutermostsublayer,theproliferativezone,wasstainedless(Altman,1972a)(Fig.7).OncethegranulecellshadmigratedpastthePurkinjecellstotheirnalpositioninthegranulecelllayer,noneoftheNogoisoformswasexpressedinthesecellsanymore.Consistentwiththeappearanceofdifferentiatedandmyelinat-ingoligodendrocytes(ReynoldsandWilkins,1988),Nogo-A-positivesmall,non-neuronalcellswererstdetectedatP5intheregionofthedeepcerebellarnuclei.Withtimetheyextendedtowardtheendofthefolia(P9)andcouldalsobedetectedinthegranulecelllayerfromP14on(Fig.7).Nogo-AproteinwashighestinwhitematterfromP14toadult.NeuronsinthedeepcerebellarnucleiexpressedNogo-A/Bproteinatbirthandthroughoutdevelopment,withveryhighlevelspresentintheadult(Fig.7).Nogo-A/BmRNAandproteinwerefoundinallneuronalcelltypesoftheretinafromembryonicageson.Atbirth,Nogo-A/Bwasexpressedinretinalganglioncellsaswellasinthecytoblastlayergivingrisetotheinnerandouternuclearlayer(Fig.8ImmunoreactivityforAS472andASBrunaandhybridizationsignalsfortheprobeswereequallystrongatalltimepointsobserved(datanotshown),thereforeprohibitinganystatementaboutspecicexpressionofNogo-B.Nogo-A,Nogo-A/BmRNAandproteinexpression,orboth,inretinalganglioncellswasstrongintheadult(Fig.8).Nogo-AandNogo-A/Bproteinsseemtobetargetedtoneurites,becausetheinnerandouterplexiformlayerswerestainedwithASBruna,whereasnoinsituhybridizationsignalwasdetectedintheseSubcellularlocalizationofNogo-AinwhitematterConfocalmicroscopicanalysisofsectionsofadultratspinalcordandopticnerverevealedthatNogo-Awasmainlyfoundin Figure5.Nogo-A,-B,and-Cexpressionintheneocortex.Insituhybridizationswiththe,ASBruna;,AS,AS818;,Nisslstaining.InE19(),astrongsignalwasobservedinthematrixlayer(),mantlela

9 yer(),andcorticalplate(Alsoatbirth(),str
yer(),andcorticalplate(Alsoatbirth(),strongexpressionincorticalplateandsubplatewasfound,whichwaspersistinginP3()andP5().NeuronsinlayerVwerestronglyexpressinginP3,whereaslayerVIwasonlyweaklystained.Intheadult(),neuronsinlayerIVofthecortexwerestainedweakerforNogo-A/BthanneuronsinlayersIIandIIIandVandVI.VerylowlevelsofNogo-CproteinweredetectedwithAS818().Imagewidth:,210,420Huberetal.ExpressionPatternofNogoJ.Neurosci.,May1,2002, oligodendrocytecellbodiesandprocessesaswellasintheoutermost(outerloop)andinnermost,adaxonal(innerloop)myelinmembrane(Fig.9).ColocalizationwithMBPwasmi-nor,showingthatNogo-Aisnotdetectablyexpressedinthecompactmyelin(Fig.9).Nogo-AdidcolocalizewithMAG,knowntobelocalizedspecicallyintheinnermostloopofthemyelinmembrane(Trappetal.,1989)(Fig.9),andMOG,localizedintheoutermostloopofthemyelinsheath(Fig.9Doubleimmunohistochemistrywithanantibodyagainsttheastrocytemarkerglialbrillaryacidicprotein(GFAP)didnotresultinanycolocalization,demonstratingthatNogo-Aisnotpresentinastrocytes(Fig.9Postembeddingimmunoelectronmicroscopyofadultratopticnervesconrmedtheseobservations(Fig.10).Thehighestim-munoreactivityforNogo-Awasfoundinanareathatcomprisedtheinnermostloopofthemyelinsheathandtheaxonalmem-brane.Lowerbutspeciclabelingwaspresentovertheoutermyelinloop,andonlyverylowlabelingwaspresentovercompact Figure6.Nogo-A/Bexpressioninthehip-pocampus.Atbirth,expressionofbothNogo-AmRNA()andprotein()wasseeninpyrami-dalcellsofhippocampalregionsCA1whereasweakerstainingwasfoundinthegran-uleneuronsofthedentategyrus(A,,AS472).Nogo-Awasalsofoundinthembria().Intheadulthippocampus,Nogo-A/BwasexpressedinpyramidalcellsofCA1CA4(C,,ASBruna;,AS472).Thedistinctlyweakersignalsoftheseneuronsinindicatethatthepre-dominantformmightbeNogo-B.NotethehighNogo-Aproteinlevelsinthemyelinatedtractsofthecorpuscallosum()andmbriafornix().AhistoblotprobedwithAS472re-vealedexpressionofNogo-Aalsoinhippocam-berlayers(,HighNogo-Aexpression;lowNogo-Aexpression.Nogo-A(G,I,AS472)wasexpressedinparvalbumin-positive()andcalbindin-positive)interneuronsinthehilus.Scalebar:A,B,275CĐF,K,550G,H,10I,J,20J.Neurosci.,May1,2002,Huberetal.ExpressionPatternofNogo Figure7.Nogoexpressioninthecerebellum.AtP3(),Nogo-A/BwasexpressedinPurkinjecells(,ASBianca;,AS472),whereasverylittleNogo-Cwasfoundintheseneurons(,AS818).Nogo-CrevealedbyimmunohistochemistrywithAS818wasfoundinPurkinjecellsinP14andadult(J,O).Nogo-A-positiveneuronsinthepremigratorylayeroftheEGLweremorestronglystainedthantheoutermost,proliferativesublayer(C,inset).Nogo-A/BexpressionremainedhighinoligodendrocytesinthewhitematterandinoligodendrocytesinthegranulecelllayerinP14andadult.NeuronsinthedeepcerebellarnucleiwerestronglyexpressingNogo-A/Batalltimepoints.SectionsstainedwithASBianca(B,G,L)andAS472(D,I,N)werexedwithEGTA-ethanolandaceticacid,revealingtheprominentexpressionofNogo-A/Binwhitematter,Purkinjecells,andtheirdendrites.Scalebar:275,35Huberetal.ExpressionPatternofNogoJ.Neurosci.,May1,2002, myelinandtheaxonalcytoplasm.TherelativedensityforNogo-Aintheinnerloopwas8.80.12(SEM)timeshigherthanincompactmyelin.Intheouterloop,therelativeNogo-Aimmu-noreactivitywas4.00.12timeshigherthanincompactmyelin(Fig.10).Confocalmicro

10 scopicanalysisrevealedcolocalizationofNo
scopicanalysisrevealedcolocalizationofNogo-Awithneurolamentinaxons(Fig.9).Immunoelec-tronmicroscopydemonstratedonlya1.20.21timeshigherrelativeNogo-Adensityintheaxoplasmthanincompactmyelin(Fig.10).Becauseaxonalandinnerloopmembranesareclosetogether,theresolutionoftheimmunogoldtechniquedoesnotallowcleardistinctionbetweenthem.However,whenopticnervesectionsveryclosetotheeyeballwhereaxonsarenotmyelinatedwerestainedwithAS472andanalyzedbyelectronmicroscopy,nosignicantlabelingwasfound.ThissuggeststhatthebulkofNogo-Aisindeedexpressedintheinnermostloopofthemyelinsheathandnotoronlyatlowlevelsintheaxonalmembrane(datanotshown).NogoexpressionafterCNSlesionsNogo-Ahasinitiallybeenidentiedasamyelin-associatedneu-ritegrowthinhibitorinvolvedinregenerativefailureafterinjury.Therefore,itsexpressionafterCNSlesionsisparticularlyinter-esting.WestudiedexpressionofNogo-A/Bafterbothcorticalandspinallesionsafter1,4,7,14,and28d.Atalltimepointsstudied,noobviouschangesinexpressionoftranscriptswerefoundinoligodendrocytes.NonaffectedtissueinthevicinityofthelesioncontinuedtoexpressNogo-A/Batnormallevelsinbothwhiteandgraymatter(Fig.11).Withtime,thelesionarealledwithinltratingcells,andaglialscardeveloped.Neitherltratingcellsnorscar-formingastrocytesandbroblastsex-pressedNogo-A/BmRNAorproteinatdetectablelevels(Fig.11).WethereforeconcludethatinhibitionofberregenerationbyNogo-AisexertedbytheintacttissueadjacenttoCNSlesionsitesratherthanbyincreasedexpressionatthesiteofinjury.At4dafterlesion,slightupregulationofNogo-A/BmRNAandproteinexpressionwasobservedincorticalneuronsinthevicinityoftheinjurybutnotinspinalcordandatanyothertimepoint(Fig.11WehaveinvestigatedtheexpressionpatternofthethreeNogoisoforms,Nogo-A,-B,and-C,inthedevelopingandadultratnervoussystem.Nogo-Awaspresentinoligodendrocytesandtheinnermostandoutermostmyelinmembrane,consistentwithitsfunctionasamyelin-associatedneuritegrowthinhibitor.Inad- Figure8.Nogoexpressionintheretina.Atbirth(),strongexpressionofNogo-A/BmRNA(andprotein(,ASBruna)inthegan-glioncelllayer()aswellasthecyto-blastlayer()wasobserved.Expressionwashighestattheinterfacebetweentheganglioncelllayerandcytoblastlayer.TheganglioncellswerealsoNogo-A/B-positiveintheadult(),andbothin-nerandouternuclearlayers(inl,onlwereexpressingmRNA(probe)andprotein(,ASBruna).Nogo-A/Bproteinseemedtobetargetedtoneurites,becausetheinnerandouterplexiformlayers(ipl,opl)werepositiveforASBruna(A,D,Nisslstaining.Scalebar,70J.Neurosci.,May1,2002,Huberetal.ExpressionPatternofNogo dition,Nogo-A,Nogo-B,orbothaswellasNogo-Cwerefoundinthedevelopingandadultnervoussystem,inparticularinseveraltypesofneurons.Thiswideexpressionpattern,includingearlydevelopmentalstages,suggestsadditionalfunctionsfortheNogoproteins.Inarecentpaper(Josephsonetal.,2001),similarresultswerereportedforNogo-A/BmRNAexpressioninspinalcordandinperipheralneurons.However,proteinexpressionoftheNogoisoformsandsubcellularlocalizationwerenotaddressedinthatstudy.Nogo-AinoligodendrocytesandmyelinNogo-AwasfoundinoligodendrocytecellbodiesandwhitematteroftheadultCNS.Confocalanalysisrevealednocolocal-izationwithMBPincompactmyelinbutshowedthepresenceofNogo-Aintheinnerloopofthemyelinsheath,intheoutermyelinloop,a

11 ndinoligodendrocytecellbodiesandprocesse
ndinoligodendrocytecellbodiesandprocesses.Immunoelectronmicroscopyconrmedtheseobservations:Nogo-Aimmunoreactivitywashighintheinnerandouterloopsofthemyelinsheathandlowinthecompactmyelin. Figure9.LocalizationofNogo-AinCNSwhitematterbyconfocalmicroscopicanalysis.,NocolocalizationofNogo-A(shownbyAS472ingreen)wasfoundwithMBP(),whichisamajorconstituentofcompactmyelin.Nogo-Awasex-pressedinoligodendrocytecellbodies,theirpro-cesses,andtheinnerloop(B,Carrowheads)andouterloop(B,Carrows)ofthemyelinsheath.ThexationprotocolrequiredtodemonstrateMBPexpressionloweredtheNogo-Asignalintheinnerloopofthemyelinmembrane,whereasthedifferentprotocolusedforMAGandMOGimmunohistochemistryshowedstrongexpressionofNogo-Aintheinnerandouterloopsofthemyelinsheath.,Nogo-A(AS472,green)isex-pressedintheouterloop(arrows)ofthemyelinsheathandwasfoundtocolocalizetherewithMOG(,AstrongNogo-Asignal(AS472,green)wasalsofoundintheinnerloop(arrow-),whereMAG()isexpressed.C,DSomeNogo-A(AS472,green)wasalsopresentinaxons,aswasdemonstratedbycolocalization()ofAS472withanantibodyagainstneuroment(,Nogo-A(AS472,green)wasnotpresentinastrocyteprocessesstainedbyananti-bodyagainstGFAP().Scalebar:A,C,85B,D,45Huberetal.ExpressionPatternofNogoJ.Neurosci.,May1,2002, Inthedevelopingcerebellum,appearanceofNogo-Ainoligo-dendrocytescoincidedwithmyelination:mRNAintheoligodendrocytesofthedevelopingcerebellumwasrstdetectedatP5inthedeepcerebellarregions,andNogo-AproteinwasstronglyexpressedatP9inoligodendrocytesofthewhitematter.AtP9,Nogo-A-positivecellbodieswerefoundtowardtheendsofthefoliainthewhitematter,andsomelabeledcellswerefoundinthegranulecelllayer.ComparingtheappearanceofNogo-AwiththetimecourseofexpressionofthemajormyelinproteinMBP,itbecameapparentthatNogo-AisjustprecedingMBPexpressionandmyelination(ReynoldsandWilkins,1988).Nogo-Ahasbeenclonedasamyelin-associatedinhibitorofregenerativeaxongrowth.WehavestudiedNogo-Aexpressionlevelsatseveraltimepointsafterbrainandspinalcordlesionsandfoundnoevidencefordecreasedorincreasedexpressioninoligodendrocytes,unlikeMBPandproteolipidprotein,whichareupregulatedafterinjury(BartholdiandSchwab,1998;Freietal.,2000).Also,wedidnotnddetectableexpressionofNogo-Ainthescartissueinandaroundthelesionsite,aswasdescribedforotherrepulsivemolecules,e.g.,Sema3Ainbroblast-likecells(Pasterkampetal.,1999)orproteoglycansinastrocytes(Levine,1994;McKeonetal.,1995).Ratherthanbeingalesion-induced Figure10.LocalizationofNogo-Aintheopticnervebyimmunoelectronmicroscopy.Nogo-Awasdetectedonopticnerveultrathinsectionsbyimmunogoldelectronmicroscopy.A,B,AS472goldgrains(arrows)werefoundattheinnerloopandlessfrequentlyattheouterloopofthemyelinsheath.ExpressionofMAG(arrowhead)wasdetectedintheinnermostloop,andMOG(arrowheadimmunoreactivitywasdetectedintheouter,DoublelabelingforNogo-A(smallgrains;arrows)andMAG(largegrains;arrowheads)conrmedthisdistribution.,QuanticationandstatisticalanalysisofthedistributionofNogo-Aproteininadultratopticnerve.Scalebar:,0.25J.Neurosci.,May1,2002,Huberetal.ExpressionPatternofNogo factor,Nogo-Athereforelikelyrestrictsbergrowthintheintacttissueinthevicinityofthelesionandfartheraway,whereitisnormallyexpressedinoligodendrocytes.Thisisconsistentwithrecen

12 texperimentsthatpointedtowardtonicinhibi
texperimentsthatpointedtowardtonicinhibitionofaninternalgrowthprogramintheneuronbyNogo-A:afterappli-cationofNogo-A-neutralizingantibodiestotheintactadultratcerebellum,profusesproutingofuninjuredPurkinjecellaxonsandupregulationofgrowth-associatedgeneswereobserved(Zagrebelskyetal.,1998;Buffoetal.,2000).DissociatedDRGneuronstransplantedintomyelinatedbertractshavebeenshowntobeabletogrowlongaxonalprocesses(Daviesetal.,1997).Itisunclearhowthesecells,whichareclearlyresponsivetoNogo-Ainculture(Chenetal.,2000),canovercomethisinhibi-invivo.Becauselesioneddorsalrootsnormallydonotregen-erateintothespinalcord,alteredsensitivityofthesetrans-planted,ectopicneuronstogrowthinhibitorsappearslikely,possiblythroughalteredreceptororsecondmessengerlevels(Caietal.,1999;Fournieretal.,2001).NogoexpressioninneuronsNogo-AmRNAandproteinwasexpressedinneuronalcellbod-iesandneuritesasearlyasE14.NofunctionoftheneuronalNogo-Aisknown;however,itseemsunlikelythattheneuritegrowthinhibitionismediatedbyNogo-Aexpressedinneurons.ExtensivefunctionalstudiesinvitrorevealedconsistenteffectsoftheneutralizingantibodyIN-1onthesubstrateonly;myelinpreparationsorliving,culturedoligodendrocytesubstrateswerepreincubatedwithIN-1andwashed,andneuronsweresubse-quentlyadded(Caronietal.,1988).CultureofDRGneuronsinthepresenceofIN-1onalamininsubstratehadnoeffectonneuritegrowth(A.B.HuberandM.E.Schwab,unpublishedobservations).Inoligodendrocytes,onlyasmallportionofNogo-Aistransportedtothecellsurface,whereasthemajorityisassociatedwithendoplasmicreticulum(ER)andGolgi(vanderHaaretal.,2001).ItremainstobedeterminedwhetherneuronsareexpressinganyNogoisoformatthecellsurface.Dependingonsubcellularlocalization,severalpossibilitiesofaneuronalfunctionofNogo-Aareconceivable:(1)Nogo-Acouldactasacellsurfacesignal,repulsive,attractive,orother,forotherneu-rons,neurites,ornon-neuronalcells.Differentreceptorsordown-streampathwaysmaymediatethespecicityofaction,ashasbeenshown,e.g.,fornetrin-1(Hongetal.,1999).Strikingly,wefoundNogo-AtobehighlyexpressedinthepremigratoryzoneoftheEGLinthedevelopingcerebellum.CerebellargranulecellsaregeneratedintheproliferativezoneoftheEGLstartingaroundP0,accumulateinthepremigratorylayer,andthenmigratein-wardpastthePurkinjecellstoformthegranulecelllayer(Alt-man,1972a,b).ThegranulecellsoftheinternalgranulecelllayerwerenotexpressingNogo-Aanymore,indicatingpossiblein-volvementofNogo-Aingranulecellmigration.Thatmoleculesinvolvedinaxonguidancecanalsoinuenceneuronalmigrationhasbeenshownrecently,e.g.,fornetrin-1(Blellochetal.,1999;Yeeetal.,1999;Alcantaraetal.,2000),semaphorins(HuandRutishauser,1996;Eickholtetal.,1999),andSlit(Wuetal.,1999).Nogo-Amaythusbeanotherexampleofamultifunctionalsignalmoleculesuchasmembersoftheneurotrophinfamily,whichhavebeenshowntobeinvolvedinsuchdiversefunctionsasneuriteoutgrowth,cellsurvivalordeathdecisions,andsynapticplasticity(Casaccia-Bonneletal.,1999;KlintsovaandGreenough,1999;Davies,2000).(2)Althoughsignal-transducingmotifsonNogo-Ahavenotyetbeenfound,afunctionofNogo-AorNogo-Basareceptorforanasyetunknownligandiswellconceivable.Bidirectionalsignalingisknownforanotherfamilyof,inpart,repulsivemolecules,theephrinsandEphreceptors(Klein,1999).(3)N

13 ogo-A,-B,and-Ccouldalsohaveanintra-cellu
ogo-A,-B,and-Ccouldalsohaveanintra-cellularfunctioninneurons,possiblyinadditiontotheneuritegrowth-inhibitoryactivityonthecellsurfaceofoligodendrocytes. Figure11.ExpressionofNogomRNAandproteinisunchangedaftertraumaticCNSinjury.InsituhybridizationwithNogoprobftercortical,Correspondingimmunohisto-chemistry(ASBruna)onanadjacentsec-tion.AslightupregulationofNogo-A/Bincorticalneuronsinthevicinityoftheinjurywasobservedatthistimepointbutnotatanyothertimepointorinspinalcord.NotetheNogo-freelesionareaandlesionborders.Insituhybridizationwiththe,Immunohis-tochemistrywithASBruna4weeksafterspinallesion.Nodetectableupregulationordownregulationcanbeobserved.Thelesionareasareandmarkedbyasterisks.Scalebar,550Huberetal.ExpressionPatternofNogoJ.Neurosci.,May1,2002, Intracellularly,Nogo-Aisexpressedinareticularpatternetal.,2000;A.B.Huber,M.E.vanderHaar,M.E.Schwab,unpublishedobservations),asareNogo-Band-Candothermembersofthereticulonfamilyofproteins(vandeVeldeetal.,1994).Thefunctionofthereticulons,towhichNogo-A,-B,and-Carerelatedinthecommon,C-terminaldomain,isun-known.RolesinproteintransferthroughtheER,proteinpack-aging,trafcking,orboth,andregulationofintracellularcalciumlevelshavebeensuggested(vandeVeldeetal.,1994).ThetwoshorterNogoformshaveawidespreadexpressionpattern,includ-ingperipheraltissues.Todate,thephysiologicalfunctionsofNogo-Band-Careunknown.InadditiontothepotentinhibitoryactivityfoundintheNogo-A-specicpartofthemolecule(Oertleetal.,2000;Prinjhaetal.,2000),membranesofNogo-A-andNogo-C-transfectedhumanembryonickidneycellsanda66-residueregionintheC-terminaldomainofNogoexpressedasglutathione-transferasefusionproteinsinbacteriawereabletoinduceDRGgrowthconecollapseinvitroetal.,2000).Recently,aneuron-specicreceptorforthis66-residueportionofNogowasidentied(Fournieretal.,2001).Recombi-nantNogo-Cdidnotinhibitaxonoutgrowthinvitro(Oertleetal.,2000),butitremainstobedemonstratedwhetherNogo-Band-Cgettransportedtothecellsurfaceandexposetheir66-residueloopbetweenthetwotransmembranedomains,ashasbeenshownforNogo-A(GrandPreetal.,2000).Inconclusion,ourresultsdemonstratethatNogo-AislocalizedinCNSmyelininawaythatisconsistentwithitsdescribedfunctionasaneuritegrowthinhibitor.Atthesametime,theexpressionofNogo-Aduringdevelopment,inparticularinneu-rons,andthewidespreadexpressionofitsisoformsNogo-Band-Csuggestadditional,yetunknownfunctionsofthisprotein.AlcantaraS,RuizM,DeCastroF,SorianoE,SoteloC(2000)Netrin-1actsasanattractiveofrepulsivecuefordistinctmigrationneuronsduringthedevelopmentofthecerebellarsystem.DevelopmentAltmanJ(1972a)Postnataldevelopmentofthecerebellarcortexintherat.I.Theexternalgerminallayerandthetransitionalmolecularlayer.JCompNeurol145:353AltmanJ(1972b)Postnataldevelopmentofthecerebellarcortexintherat.III.Maturationofthecomponentsofthegranularlayer.JCompNeurol145:465BartholdiD,SchwabME(1998)Oligodendroglialreactionfollowingspinalcordinjuryinrat:transientupregulationofMBPmRNA.GliaBenkeD,WenzelA,ScheuerL,FritschyJM,MohlerH(1995)Immu-nobiologicalcharacterizationoftheNMDA-receptorsubunitNR1inthedevelopingandadultratbrain.JReceptSignalTransductResBlellochR,NewmanC,KimbleJ(1999)Controlofcellmigrationdur-ingCaenorhabditiselegansdevelopment

14 .CurrOpinCellBiolBregmanBS,Kunkel-Bagden
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