Conformational specificity of the C4F6 SOD1 antibody; low frequency of reactivity in sporadic ALS cases

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Title:
Conformational specificity of the C4F6 SOD1 antibody; low frequency of reactivity in sporadic ALS cases
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Mixed Material
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English
Creator:
Ayers, Jacob I
Xu, Guilian
Pletnikova, Olga
Troncoso, Juan C.
Hart, John P
Borchelt, David R.
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Bio Med Central (Acta Neuropathologica Communications)
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Abstract:
Greater than 160 missense mutations in copper-zinc superoxide dismutase-1 (SOD1) can cause amyotrophic lateral sclerosis (ALS). These mutations produce conformational changes that reveal novel antibody binding epitopes. A monoclonal antibody, clone C4F6 - raised against the ALS variant G93A of SOD1, has been identified as specifically recognizing a conformation shared by many ALS mutants of SOD1. Attempts to determine whether non-mutant SOD1 adopts a C4F6-reactive conformation in spinal tissues of sporadic ALS (sALS) patients has produced inconsistent results. To define the epitope recognized by C4F6, we tested its binding to a panel of recombinant ALS-SOD1 proteins expressed in cultured cells, producing data to suggest that the C4F6 epitope minimally contains amino acids 90–93, which are normally folded into a tight hairpin loop. Multiple van der Waals interactions between the 90–93 loop and a loop formed by amino acids 37–42, particularly a leucine at position 38, form a stable structure termed the β-plug. Based on published modeling predictions, we suggest that the binding of C4F6 to multiple ALS mutants of SOD1 occurs when the local structure within the β-plug, including the loop at 90–93, is destabilized. In using the antibody to stain tissues from transgenic mice or humans, the specificity of the antibody for ALS mutant SOD1 was influenced by antigen retrieval protocols. Using conditions that showed the best discrimination between normal and misfolded mutant SOD1 in cell and mouse models, we could find no obvious difference in C4F6 reactivity to spinal motor neurons between sALS and controls tissues. Keywords: Superoxide dismutase 1, Amyotrophic lateral sclerosis, C4F6 epitope, Conformational antibodies
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Ayers et al. Acta Neuropathologica Communications 2014, 2:55 http://www.actaneurocomms.org/content/2/1/55; Pages 1-13
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doi:10.1186/2051-5960-2-55 Cite this article as: Ayers et al.: Conformational specificity of the C4F6 SOD1 antibody; low frequency of reactivity in sporadic ALS cases. Acta Neuropathologica Communications 2014 2:55.

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University of Florida
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University of Florida
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© 2014 Ayers et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
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Electronic Supplementary Material 3! ! !ESM3 Antigen retrieval impacts SEDI immunoreactivity to hSOD1 in transgenic mouse spinal cord tissues. Tissue sections from mice overexpressing WT, G93A, and G37R, or non transgenic mice for controls, were stained with SEDI following either no antigen retrieval or formic acid and 6M guanidine hydrochloride (FA & GdnHCl). All tissues from mice expressing mutant hSOD1 (G93A and G37R) were harvested from paralyzed mice. The WT hSOD1 tissue w as from mice ~18 months of age. Scale bar 100 m!



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RESEARCHOpenAccessConformationalspecificityoftheC4F6SOD1 antibody;lowfrequencyofreactivityinsporadic ALScasesJacobIAyers1,GuilianXu1,OlgaPletnikova2,JuanCTroncoso2,PJohnHart3,4andDavidRBorchelt1*AbstractGreaterthan160missensemutationsincopper-zincsuperoxidedismutase-1(SOD1)cancauseamyotrophiclateral sclerosis(ALS) Thesemutationsproduceconformationalchangesthatrevealnovelantibodybindingepitopes.A monoclonalantibody,cloneC4F6-raisedagainsttheALSvariantG93AofSOD1,hasbeenidentifiedasspecifically recognizingaconformationsharedbymanyALSmutantsofSOD1.Attemptstodeterminewhethernon-mutant SOD1adoptsaC4F6-reactiveconformationinspinaltissuesofsporadicALS(sALS)patientshasproducedinconsistent results.TodefinetheepitoperecognizedbyC4F6,wetesteditsbindingtoapanelofrecombinantALS-SOD1proteins expressedinculturedcells,producingdatatosuggestthattheC4F6epitopeminimallycontainsaminoacids90 – 93, whicharenormallyfoldedintoatighthairpinloop.MultiplevanderWaalsinteractionsbetweenthe90 – 93loopanda loopformedbyaminoacids37 – 42,particularlyaleucineatposition38,formastablestructuretermedthe -plug. Basedonpublishedmodelingpredictions,wesuggestthatthebindingofC4F6tomultipleALSmutantsofSOD1 occurswhenthelocalstructurewithinthe -plug,includingtheloopat90 – 93,isdestabilized.Inusingtheantibodyto staintissuesfromtransgenicmiceorhumans,thespecificityoftheantibodyforALSmutantSOD1wasinfluencedby antigenretrievalprotocols.Usingconditionsthatshowedthebestdiscriminationbetweennormalandmisfolded mutantSOD1incellandmousemodels,wecouldfindnoobviousdifferenceinC4F6reactivitytospinalmotor neuronsbetweensALSandcontrolstissues. Keywords: Superoxidedismutase1,Amyotrophiclateralsclerosis,C4F6epitope,ConformationalantibodiesIntroductionAmyotrophiclateralsclerosis(ALS)isafatalneurodegenerativediseasecharacterizedbythelossofupperand lowermotorneurons.Approximately90%ofthecases aresporadic(sALS)inoriginwhereas10%arefamilial (fALS)andcausedbymutationsidentifiedinmorethan 10differentgenes{http://alsod.iop.kcl.ac.uk/}.Oneof thefirstdominantly-inheritedfALS-associatedgenesto beidentifiedwasthe SOD1 gene;mutationsin SOD1 accountfor10-20%ofallfALScases.Therearenow morethan160missensemutationswithinthisgenethat havebeendescribedinALSpatients{http://alsod.iop.kcl. ac.uk/}.Onthebasisofstudiesinvariousanimal models,itisthoughtthatthemutationsin SOD1 causea gainoftoxicpropertiestoproducetheprogressiveparalyticsymptomsobservedinfALSpatients.Importantly, thesymptomsandCNSpathologyobservedinpatients harboringSOD1mutationsareverysimilartothoseobservedinnon-inheritedformsofdisease,suggestingthat therecouldberelatedmechanismsofpathogenesis. ThetoxicpropertiesofmutatedSOD1arethoughtto arisefrommutation-inducedconformationalchanges leadingtoSOD1misfoldingandaggregation.Wild-type SOD1(WT)canacquiresomeofthesamepropertiesas mutantSOD1whenoxidizedandstrippedofmetal cofactors;thesepreparationshavealsobeenshowntobe toxicwhenadministeredtocells[1-5].Indeed,transgenic micethatarehomozygousforWTSOD1transgenesand expressingveryhighlevelsofproteinformaggregatepathologysimilartowhatisseeninmutantSOD1micewith paralyticsymptoms[6-8].Additionally,co-expressionof *Correspondence: borchelt@mbi.ufl.edu1DepartmentofNeuroscience,CenterforTranslationalResearchin NeurodegenerativeDisease,McKnightBrainInstitute,UniversityofFlorida, Box100159,Gainesville,FL32610,USA Fulllistofauthorinformationisavailableattheendofthearticle 2014Ayersetal.;licenseeBioMedCentralLtd.ThisisanOpenAccessarticledistributedunderthetermsoftheCreative CommonsAttributionLicense(http://creativecommons.org/licenses/by/4.0),whichpermitsunrestricteduse,distribution,and reproductioninanymedium,providedtheoriginalworkisproperlycredited.TheCreativeCommonsPublicDomain Dedicationwaiver(http://creativecommons.org/publicdomain/zero/1.0/)appliestothedatamadeavailableinthisarticle, unlessotherwisestated.Ayers etal.ActaNeuropathologicaCommunications 2014, 2 :55 http://www.actaneurocomms.org/content/2/1/55

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WTSOD1withmutantSOD1almostinvariablyacceleratestheonsetofparalysiswithevidencethatWTSOD1 hasbeeninducedtoaggregatewithmutantSOD1[9-13]. ThesestudiespointtoWTSOD1asapotentialpathogeniclinkbetweenfALSandsALSandmoreimportantly, implicateSOD1asatargetfortherapeuticinterventionin themajorityofALScases.Throughthecourseofthese studies,conformation-specificantibodiestoSOD1have emergedascriticalreagentstodistinguishmisfolded,presumablytoxicSOD1,fromproteinthatachievesamore nativeconformation.Examplesoftheseantibodiesinclude aseriesofmonoclonalantibodiesgeneratedbyimmunizingmicewitharecombinantapoformofG93AhSOD1, yieldingantibodiesdesignatedC4F6,A5C3,andD3H5 [14].Todate,however,theepitoperecognizedbythese antibodieshasnotbeencompletelycharacterized. Theanti-hSOD1antibody,C4F6,whichhasbeenwidely usedinstudiestoidentifymisfoldedSOD1,wasreportedto showstrongimmunoreactivitytodenaturedG93A,significantreactivity(butmuchlower)tootherhSOD1mutants, andverylowreactivitytodenaturedWThSOD1[14].WT hSOD1canbeinducedtobindC4F6byoxidationinvitro, andsuchreactivitywaslinkedtosporadicALSbydemonstratingC4F6immunoreactivitytospinalmotorneuronsin sALScases[5].However,whenBrothertonetal.usedC4F6 tostainspinalcordtissuefromsALScasesandanA4V fALScase,theyobservedthatC4F6reactedwithinclusions intheA4VcasebutnotinthesALScases[15].MorerecentlySaxenaetal.[16]linkedtheaccumulationofA5C3 reactivemutantSOD1tomotorneurontoxicityinthe G93AmousemodelofALS.Withtheemergenceofthese antibodiesasimportantresear chtools,andthepossibledevelopmentoftheseantibodiesastherapies[17],itisincreasinglyimportanttobetterunderstandthenatureofthe conformationalepitoperecognizedbytheseantibodies. Theonlyinformationcurrentlyavailableonthenature oftheepitopeisthatitislocatedinexon4ofthehSOD1 protein[18],whichcomprisesaminoacids80 – 119of SOD1.Weuseacombinationofimmunohistochemical andbiochemicaltechniquestodemonstratethatamino acids90 – 93ofthehSOD1protein,whichcomprisealoop domainbetween -strands5and6,arecriticalcomponentsoftheC4F6epitope.Thekeyresiduesidentifiedin theepitopeincludeanasparticresidueatposition90that beginsasequenceofAsp-Lys-Aspandposition93(AlafavoredbutGlytolerated).AcomparisonofX-raycrystal structuresofWTandG93ASOD1didnotrevealanobviousconformationalchangeinthe90 – 93loopelement thatcouldproduceitsapparentconformationalspecificity. Instead,ourdatafitbestwithamodelinwhichanymutationormodificationthatincreasesflexibilityinthe90 – 93 loopenablesC4F6reactivity.Furthermore,wesystematicallyassesstheimpactofstandardantigenretrievalproceduresontheimmunoreactivityofC4F6,andrelated antibodies,towardsmutantandWTSOD1infixedtissue specimensfromtransgenicmice,findingthatstandard antigenretrievaltechniquesgreatlyinfluencereactivity. Usingconditionsoptimizedinthemousetissues,we examinedthefrequencyofreactivitywiththisantibodyin post-mortemspinalcordtissuefrom25sALScases.In ourhands,noneofthesALScasesweexaminedshowed reactivitythatwasdistinctfromwhatcouldbeobserved incontrols.MaterialsandmethodsTransgenicmiceAllthestrainsoftransgenicmiceusedinthisstudyhave beenpreviouslydescribed:WThSOD1over-expressing miceusedhereweretheB6SJL-Tg(SOD1)2Gur/Jhybrid line[19],G93AhSOD1inB6SJL-TgN(SOD1-G93A)1Gur mice[19],G37RhSOD1inGn.G37RLine29mice[20], andL126ZhSOD1intheL126ZLine45mice[21].These micewereallmaintainedinahybridbackgroundof C57BL/6JandC3H/HeJ.Allstudiesinvolvingmicewere approvedbytheInstitutionalAnimalCareandUseCommitteeattheUniversityofFlorida.Foridentificationof genotype,DNAwasextractedfrommousetailbiopsies andanalyzedbyPCRaspreviouslydescribed[12].SubjectsTissuesfromsALScaseswerecollectedwithpatient consentandhandledunderprotocolsapprovedbythe JohnsHopkinsInstitutionalReviewBoard.Allsamples werecodedandde-identified.Spinalcordtissuesfroma totalof25sALScasesand5non-diseasecontrolswere madeavailableforanalysis(Additionalfile1:TableS1 providesdetailsonthepatientsandtissuesanalyzed). Spinalcordtissueswerepreservedbyimmersionfixation in10%formalinforatleast14daysandthenprocessed forparaffinembeddingandsectioningat10 m.Tissues fromALScaseswereanalyzedforthepresenceofrelevant pathologicfeaturesincludingmotorneuronloss,Bunina bodyinclusions,CystatinCpositiveneuronalinclusions, skein-likeinclusions,andsphericalinclusions.Forthese pathologiesasemi-quantitativeassessmentofabundance wasused.Wealsodeterminedwhichcasesshowedcorporaamylaceainclusions(presence+orabsence-).TransgenicmousetissuecollectionSpinalcordswerecollectedfrommiceforimmunohistochemistry(IHC)analysis.Animalswereanesthetized withisofluraneandperfusedtranscardiallywith20mlof phosphate-bufferedsalinefollowedby20mlof4%paraformaldehyde.Spinalcordswereimmediatelyremoved andplacedin4%paraformaldehydefor24 – 48hoursat 4Cpriortoparaffinprocessing.Ayers etal.ActaNeuropathologicaCommunications 2014, 2 :55Page2of13 http://www.actaneurocomms.org/content/2/1/55

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ImmunohistochemistryandperiodicacidSchiffstainingTocharacterizethepathologicfeaturesofhumansALS tissues,10 msectionswereimmunostainedwithubiquitin (Rabbitanti-ubiquitin(1:500)fromDAKO,CA),whichrevealedskein-likeinclusions,cys tatin-C(Rabbitanti-cystatin C(1:100)fromMillipore/Upst ateBiotech,Catalog#ABC20., Billerica,MA),whichrevealed Bunina-likeinclusions,orby H&Estaining,whichrevealed Lewy-body-likeinclusions andBuninabodyinclusions.Al lstainingofhumantissues usedasteam/citratebufferant igenretrievalprotocol.Immunohistochemistryonmousetissueswasperformedon 5 – 7 msections.Sectionsweredeparaffinizedandeither leftinwater,incubatedin95%formicacidfor20minutes followedbyovernightincubationin6Mguanidinehydrochlorideatroomtemperature,orsteamedin10mMcitrate bufferwith0.05%Tween20,pH6.0for30minutes. Sectionswereblockedofendogenousperoxidasesby immersionin0.3%H2O2inPBSfollowingmultiplePBS washesforthesectionsthatunderwentantigenretrieval.Followingblockingofnon-specificstainingwith 10%normalgoatseruminPBScontaining0.5%Tween20(PBST),sectionswereincubatedovernightat4C witheithertheC4F6antibody(Medimabs,Montreal, Quebec,Canada)ata1:500dilutionortheSEDIantibody(kindgiftfromJaniceRobertson)ata1:250dilutioninPBSTwith3%normalgoatserum.Thesections werethenincubatedwithabiotinylatedsecondaryantimouseantibody(VectorLaboratories,Burlingame,CA) diluted1:500inPBSTwith3%normalgoatserum followedbyincubationwiththeABC-horseradishperoxidasestainingkit(VectorL aboratories,Burlingame, CA).Sectionsweredevelopedusing0.05%w/v3,3 ’ -diaminobenzidine(Sigma-Aldrich,St.Louis,MO)inPBS containing0.0015%H2O2andcounterstainedwith hematoxylin.ImagesweretakenusinganOlympus BX60microscope. TissuesectionswerestainedwithperiodicacidSchiffby firstoxidizingthemin0.5%periodicacid(FisherScientific, Pittsburgh,PA)solutioninwaterfor5minutes.Following arinseindistilledwatersectionswereplacedinSchiff ’ s reagent(Sigma-Aldrich,St.Louis,MO)for15minutes. Sectionswerewashedinwarmtapwaterfor5minutesand thencounterstainedinhematoxylinfor1minuteand washedagaininwaterfor5minutes.Slideswerethen dehydratedandcoverslipped.SOD1cDNAexpressionplasmids,celllines,and transfectionsWTandmutanthSOD1untaggedproteinswereexpressed fromplasmidsbasedonthemammalianpEF-BOSexpressionvector,andhavebeenpreviouslydescribed [11,22-26].YFPtaggedSOD1cDNAvariantswerecreated fromawormexpressionvector(pPD30.38)thatcontains WThSOD1fusedtoeYFP(yellowfluorescentprotein) kindlyprovidedbyDr.RickMorimoto(Northwestern University).MutantfluorescentlytaggedSOD1variants wereconstructedfollowingsimilarproceduresandhave beenpreviouslydescribed[12,27].AllcDNAgenesand pEF-BOSvectorsencodingthesecDNAswereverifiedby sequencingpriortotheiruseinexperimentation.CHO cells(ATCC,Manassas,VA)wereusedforallcellculture studiesinwhichimmunocytochemistry(ICC)wastobe performed,andHEK293FTcells(Invitrogen,Carlsbad, CA)wereusedifbiochemicalanalysiswastobeperformed.AllcelllinesweremaintainedfollowingATCC recommendations. TransfectionofcellsforICCwasperformedonglass coverslipsthatwerepreviouslycoatedwith0.5mg/ml poly-L-lysinein1phosphatebufferedsalinesolution (PBS).Atotalof0.8 gofvectorDNAwastransfectedper well,usingLipofectamine2000(Invitrogen,Carlsbad, CA).Forbiochemicalanalyses,atotalof4 gofvector DNAwasusedtotransfectcellsin60mmpoly-L-lysine coateddishes(BDBiosciences,Bedford,MA).Eachtransfectionexperimentwasrepeatedaminimumof3times.ImmunocytochemistryAfterrinsingthecells3timesin1PBS,transfectedcells werefixedwith4%paraformaldehydein1PBSfor15minutes.Cellswerethenpermeabilizedusingice-cold100% methanolfor5minutesfollowedbyincubationin20% normalgoatserumin1PBS.Immunostainingwasthen performedwithhSOD1(1:1000)andC4F6(1:1000)antibodiesin1PBSwith10%normalgoatserumandincubationovernightat4C.Cellswerethenincubatedfor 1hourwithsecondaryantibodies(Invitrogen,Carlsbad, CA)Alexafluorgoatanti-rabbit568forhSODandAlexafluorgoatanti-mouse568forC4F6diluted1:2000in1 PBSwith10%normalgoatserum.Cellsweretreatedwith 4 ’ ,6-diamidino-2-phenylindole,dihydrochloride,stock 14.3mM(DAPI)(Invitrogen,Carlsbad,CA)diluted 1:2000in1PBSfor10minutestovisualizenuclei.FluorescencewasvisualizedonanepifluorescenceOlympus BS60microscope.RecombinantSOD1expressionandpurificationRecombinanthSOD1proteinswereexpressedandpurifiedaspreviouslydescribed[28].Dot-blotwithrecombinantSOD1proteinRecombinanthSOD1proteins(1.5 g),asindicatedinfigurelegends,werespottedontoanitrocellulosemembranes. Theproteinswereallowedtodryonthemembranefor 30minutesat25CandthenthemembranewassubmergedinPBSfor5minutes.Themembraneswerethen incubatedin4%paraformaldehydefor30minutesfollowed by5washesinPBSfor5minuteseach.Themembranes werethentreatedwithvaryingconditions:1)submergedinAyers etal.ActaNeuropathologicaCommunications 2014, 2 :55Page3of13 http://www.actaneurocomms.org/content/2/1/55

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6Mguanidinehydrochloridefor30minutesor2)submergedin10mMcitratebufferwith0.05%Tween20, pH6.0andsteamedfor30minutes.Aftereachtreatment membraneswereagainwashedinPBS5timesfor5minuteseach.Odysseyblockingbuffer(LI-COR,Lincoln, NE)wasthenusedtoblockthemembranesfor1hour.ImmunostainingwasperformedbyincubatingthemembranesinasolutionofC4F6(1:1000)andhSOD(1:2500) antibodiesdilutedtogetherinOdysseyblockingbufferwith 0.1%Tween20at4Covernight.Membraneswererinsed inPBST5timesfor5minuteseachandthenincubatedin asolutioncontainingboththeIRDye680RDgoatantimouseandIRDye800CWgoatanti-rabbitantibodies (LI-COR,Lincoln,NE)dilutedinOdysseyblockingbuffer containing0.1%Tween20and0.01%sodiumdodecylsulfatefor45minutesatroomtemperature.Membranes werethenrinsed4timesinPBS-Tfor5minuteseach,1 timeinPBSfor5minutes,andthenimagedusingthe OdysseyInfraredImagingSystems(LI-COR,Lincoln, NE).DensitometricanalysiswasperformedusingOdyssey softwareversion3.0.ImmunoblottingFollowingtransienttransfectionfor48hourswith SOD1constructsasdescribedabove,cellswereharvestedinPBSwith1:100v/vproteaseinhibitorcocktail(Sigma,St.Louis,MO).Thecellswerethenlysed bysonicatingthesamplestwotimesfor15seconds eachbeforelowspeedcentrifugation(~800g)for 10minutes.Proteinconcentrationsofthesupernatant werethendeterminedbybicinchoninicacidassayas describedbythemanufacturer(PierceBiotechnology, Rockford,IL).Variousproteinconcentrations,asindicatedinfigurelegends,wereboiledfor5minutesin Laemmlisamplebufferwith -mercaptoethanoland electrophoresedin18%Tris-Glycinegels(Invitrogen, Carlsbad,CA).Followingtransfer,membraneswere blockedinOdysseyblockingbuffer(Odyssey)andsubsequentprocessingandimagingusingtheOdysseyInfraredImagingSystems(LI-COR)wasperformedas describedfortherecombinantdot-blots.MolecularmodellingTheconformations(rotamers)adoptedbythesidechains ofresiduesE40,D90,K91,D92,andG93forWTSOD1 werevisualizedbysuperimposing7structuresof32subunitsthatareavailableintheproteindatabank(PDB) (Additionalfile2:TableS2)usingPyMOL(ThePyMOL MolecularGraphicsSystem,Version1.7.0,Schrdinger, LLC).ThestructuralconsequencesofmutationofG93to Awasexaminedinsilicobysuperimposing3structuresof 16subunitsforG93ASOD1thatareavailableinthePDB bank(Additionalfile2:TableS2)usingPyMOL.StatisticalanalysisAllstatisticalanalyseswereanalyzedinGraphPad PRISM5.01Software(laJolla,CA)asexplainedinfigure legends.ResultsCharacterizationoftheC4F6epitopeFrompreviouscharacterizationoftheC4F6antibody,it hadbeenestablishedthatitdoesnotrecognizemouse SOD1[5].InthesequencesadjacenttotheG93Aresidue, thereisasequencedifferenceatposition90,whichinhumanisDandinmouseisG.AmutationoftheDat90to alaninecausesALSandhasbeenpreviouslyshownto causetheproteintoaggregate[22].Wetherefore,tested theimmunoreactivityofC4F6totheD90AhSOD1mutationfollowingtransienttransfectionandimmunocytochemistry.RobustexpressionofD90A,alongwithG93A andWTSOD1wasindicatedbyimmunostainingwiththe controlhSODantibodythatcandetectallvariantsof hSOD1(Figure1a-c).However,C4F6immunoreactivity wasabsentincellstransfectedwithbothD90Aandthe controlWThSOD1(Figure1e,f).AsexpectedG93A showedstrongstainingwithC4F6(Figure1d).Thisfindingindicatesthattheasparticacidataminoacid90isa criticalcomponentoftheC4F6epitope. Tofurtherdemonstratetheimportanceofthisamino acid,weanalyzedimmunoblotsofcelllysatescontaining theWT,G93A,andD90AproteinsseparatedbydenaturingSDS-PAGEusingthehSODantibodyforassessing proteinloadingandtheC4F6antibodytodeterminerelativeaffinity(Figure1).Threedifferentamountsofcelllysatewereloadedonthegel.AllthreehSOD1proteins revealedastrongsignalwhenprobedwiththehSODantibody,withdecreasingintensityofreactivitywhenlessproteinwasloaded(Figure1g,asteriskmarkspositionof primarygeneproduct).TheD90Avariantexhibitsslightly fasterelectrophoreticmobilityinSDS-PAGEbecausethe lossofthechargedresidueincreasesSDS-bindingtothe protein[29].Usingfluorescently-labeledsecondaryantibodiesandtheOdysseyImagingSystem(seeMaterials andmethods),thesameblotwasthenprobedwiththe C4F6antibody.AlthoughtheG93Aproteindisplayedhigh immunoreactivityforC4F6,theWTandD90Aproteins werelessreactive(Figure1g).Whennormalizedtothe signalintensityofG93A,theaffinityofC4F6forboththe WTandD90AhSOD1proteinswas5or25-foldlower, respectively(Figure1g,h).Togetherwiththeimmunocytochemicalstudies,theseoutcomesprovidestrongevidence thattheAspatposition90isanessentialresidueinthe epitopeforC4F6.ConsideringalsothatreactivityfordenaturedG93ASOD1ismuchhigherthanthatfordenaturedWTSOD1,wealsoconcludethattheepitope extendstoposition93.Thus,theminimalepitopefor C4F6ispredictedtobeD-K-D-G/A.Importantly,C4F6Ayers etal.ActaNeuropathologicaCommunications 2014, 2 :55Page4of13 http://www.actaneurocomms.org/content/2/1/55

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possessesreactivitytotheWTsequenceofDKDGand thuscouldreactwithSOD1encodingmutationsother thanG93Aiftheonlyconsequenceofthemutationwas toexposetheDKDGsequencetoallowantibodybinding. MolecularmodelingoftheC4F6epitope TobetterunderstandthestructureofhSOD1around thisstretchofaminoacidsandtodeterminethestructuralconsequencesofmutationsthataffecttheC4F6 epitope,wealignedthecrystalstructuresof32subunits from7distinctcrystalstructuresthatareinthePDB database(Additionalfile2:TableS2)andmergedthem intoonemoleculetorevealthestructuralheterogeneity ofourregionofinterest(Figure2a).Astrikingobservationwasthehighdegreeofalignmentthatcouldbeobtainedinthesestructuresdespitethefactthatdifferent crystallizationconditionswereusedtoobtainthesedata. Aminoacids90 – 93arelocatedinthebeta-turn Figure1 TheD90AALSmutantbindsC4F6weakly. Transiently-transfectedcellsexpressinghSOD1proteinsG93A (a,d) ,WT (b,e) ,orD90A (c,f) wereimmunostainedwitheitherhSODantibodyorC4F6antibody.NucleiwerestainedwithDAPI(blue). (g) Forimmunoblotanalysis, HEK293FTcellsweretransientlytransfectedwithG93A,WT,D90A,orleftuntransfected(UT)for48hoursand50,10,and2 goftotalprotein fromhomogenateswereanalyzedbySDS-PAGE. (h) Immunoblotssimilartothoseshownin (g) werequantified.Ateachproteinconcentration, themostintenseband(asterisk)wasquantifiedandtheintensitywasnormalizedtotheintensityforthebandproducedincellsexpressingthe G93Avariant.ThepositionsofendogenousSOD1fromCHOcellsismarkedasisthepositionofanunknowncross-reactiveband.Incells transfectedwithconstructsforG93ASOD1,asecondfastermigratingbandwasdetectedbybothhSOD1andC4F6antibody(doubleasterisk). ThisbandmaybeacleavageproductofG93ASOD1orrepresentproteinmodifiedinsomemanner.Datafrom3replicateexperimentswere quantifiedandgraphed(meanratioS.E.(errorbars)). Scalebar 50 m.*P 0.05,***P 0.001(unpaired t -test). Ayers etal.ActaNeuropathologicaCommunications 2014, 2 :55Page5of13 http://www.actaneurocomms.org/content/2/1/55

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structureoftheloopbetweenthe5 th and6 th betasheets ofhSOD1andareincontactwiththeloopbetweenthe 3 rd and4 th beta-sheets(Figure2a).IntheWTstructure, thesidechainofthelysineatposition91ishighlysolvent exposedandcanbefoundinmultiplebackboneindependentrotamers(81possible;Figure2a).Todetermine howtheG93Amutationmayalterstructureinthe90 – 93 loop,weperformedthesametypeofstructurealignments fortheavailablestructuresoftheG93Amutant(16subunitsfrom3distinctcrystalstructures;Figure2b).The overallarchitectureoftheG93Avariantisverysimilarto thatofWTSOD1,particularlyintheloopregionof90 – 93(Figure2b).SuperimpositionofthestructuresofWT andG93Ashowsthattheoverallconformationofthe90 – 93loop,andtheadjacentloopbetween -strands3and4, intheG93Avariantwasnotobviouslydifferentfromthat ofWT(Figure2candd). Condition-specificimmunoreactivityofC4F6tohSOD1 variants Todeterminetheutilityofthisantibodyintissuepreparations,wetestedtheimmunoreactivityoftheantibody onspinalcordtissuefromseverallinesoftransgenic miceexpressinghSOD1variantsfollowingdifferent antigenretrievaltechniques.WecomparedtheC4F6 immunoreactivityin4differe ntlinesofhSOD1expressing mice:theGurneyWTlinewhichdisplaynoclinicalsymptomsbutexhibitsomeofthevacuolarchangesseenin G93Amutantmice[30],theG93Alineofmicethatsuccumbtodiseasein5 – 7monthsofage(inourcolony)[19], theG37RLine29micewhichdevelopdiseasein7 – 9 monthsofage,andtheL126ZLine45micethatexpressa truncatedhSOD1andsuccumbtodiseaseat8 – 10months ofage.OurforgoingimmunoblottingdatasuggestthatdenaturationofSOD1proteincouldrevealtheepitoperequiredforC4F6immunoreactivity.Totestthishypothesis, weemployedatreatmentwithformicacidtobreakthe proteincrosslinksformedfromfixingthetissuewithparaformaldehydefollowedbyanovernightincubationin6M GdnHCltodenaturetheprotein.Immunoreactivitytothe SEDIantibodythatrecognizesanepitopeburiedinthe SOD1dimerinterfacewasusedonthesametissueasa positivecontrol[31].Thisantigenretrievaltechniquewas observedtogreatlyincreasethereactivityoftheSEDIantibody,andindicatesthatthistreatmenthasthepotentialto disruptSOD1structureandexposeburiedepitopes (Additionalfile3:FigureS1).Inadditiontothistechnique wealsotestedtheimmunoreactivityofC4F6following steamingthetissuesectionsincitratebufferfor30minutes, whichisacommonlyusedantigenretrievaltechnique.TissuefromhSOD1non-transgenicmicedisplayedlittleorno immunoreactivityforC4F6regardlessoftreatment (Figure3a-c).Inuntreatedtissuefrommiceexpressingthe G93AvariantofhSOD1,towhichtheantibodywasproduced,strongC4F6immunoreactivitywasobservedinthe neuropil,includingstainingaroundthevacuolarstructures thatarecommonlypresentinthislineofmiceatendstage (Figure3d).Thepatternandintensityofreactivitywas changedlittlebytreatmentwithformicacidandGdnHCl (Figure3e).Fortissuessteamedincitratebuffer,themajor differenceobservedwasincreasedintra-neuronalstaining inadditiontotheneuropilreactivity.Inuntreatedtissue frommiceoverexpressingWThSOD1at~18monthsof age,rarepunctateC4F6immunoreactivitywasobservedin theneuropilandwhitematter(Figure3g).Followingeither denaturationprotocol,C4F6immunoreactivitygreatlyincreasedintheneuropilinadditiontostainingaroundthe rimofvacuolardeposits,identicaltothosefoundinG93A tissuefromsickmice(Figure3h,i).Treatmentwithcitrate bufferseemedtoslightlyenhancetheoverallstainingwhen comparedtoGdnHCltreatmentwhilealsoenhancing neuronalcellbodystaining. TheevidencethattheC4F6antibodyrecognizesa conformationalepitopederivesfromitshighreactivity tofALSmutantSOD1encodingotherdisease-associated mutations;apropertythatweobserveinfixed,but otherwiseuntreated,transfectedculturedcells[27].In ourhands,C4F6stainingofspinalcordsfromparalyzed G37Rmicewasrelativelyweakwithonlydiffusestaining ab cd Figure2 StructureofWTandG93AhSOD1inthecriticalC4F6 bindingsite.(a) Thecriticalaminoacidsthoughttobeinvolvedin C4F6bindingarelocatedinaloopstructureformedbyaminoacids 90 – 93. (b) AlignmentofstructuresofG93AhSOD1from3different crystalswith16subunitsinthePDBdatabase. (c) Mergedalignment ofWTandG93AhSOD1. (d) Viewofalignedcompletestructuresfor WTandG93AhSOD1. Ayers etal.ActaNeuropathologicaCommunications 2014, 2 :55Page6of13 http://www.actaneurocomms.org/content/2/1/55

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intheneuropilandoccasionalstainingofcellsresemblingastrocytes(Figure3j).Inthesemice,theoverall levelofC4F6stainingwasgreatlyincreasedfollowingeitherantigenretrievaltechnique,withincreasedstaining inneuropil,robustneuronalcellbodystaining,and vacuolarstainingobservedinthegraymatter(Figure3k andl).ImmunoreactivityofC4F6tospinalcordsofparalyzedmiceexpressingthetruncatedhSOD1mutant L126Zwasevident,butlimited,inuntreatedtissues,but appearedtoincreaseinintensityfollowingeitherantigen retrievaltechnique(Figure3m-o).IntheseL126Zmice, theantibodyappearedtostainintra-cellularfibrillar structuresaswellasfibrillarstructuresintheneuropil. Thismodelshowedverylittleofthediffuseneuropil stainingthatwasobservedintissuewiththeother SOD1variants;afindingconsistentwithpreviousobservationsthatthismutationisshort-livedandprimarily accumulatesonlyinaggregates[21].Takentogether, thesedatarevealthatantigenretrievaltreatmentsthat couldpotentiallydenatureSOD1produceC4F6immunoreactivityforeverySOD1varianttested,including over-expressedWThSOD1. Figure3 (a-o)Asnotedinthemarginsofthefigure,tissuesectionsfrommiceoverexpressingWT,G93A,G37R,andL126ZhSOD1,or nontransgenicmiceforcontrols,werestainedwithC4F6followingeithernoantigenretrieval,formicacidand6Mguanidine hydorchloride(FA&GdnHCL),orsteamingincitratebuffer. AlltissuesfrommiceexpressingmutanthSOD1(G93A,G37R,andL126Z)were harvestedfromparalyzedmice.TheWThSOD1tissuewasfrommice~18monthsofage.Arrowsinpanels d and f highlightstainingaroundthe vacuolarstructurescommonlypresentinG93Amice. Scalebar 100 m. Ayers etal.ActaNeuropathologicaCommunications 2014, 2 :55Page7of13 http://www.actaneurocomms.org/content/2/1/55

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ToconfirmtheeffectsthetwoantigenretrievaltechniqueshadontheaffinityofC4F6forthehSOD1protein,wespottedpurified,recombinantWT,G37R,and G93AhSOD1proteinsontonitrocellulose,andfixed themwithparaformaldehyde.Themembraneswerethen treatedwithGdnHClorcitratewithsteam,beforetestingimmunoreactivitytoC4F6.Asacontrol,wealso testedtheimmunoreactivityoftheseproteinswiththe hSODantibody.Inpreviouswork,wehavedetermined thatthisantibodycannotimmunoprecipitatenatively foldedWTSOD1,butishighlyreactivetoWTSOD1in fixedtissues[32]andtoWTSOD1over-expressedin culturedcells[27].Followingtreatmentofthenitrocellulosemembranewith6MGdnHCl,wefoundtheimmunoreactivityforbothhSODandC4F6antibodieswas notsignificantlyalteredforanyofthehSOD1proteins tested(Figure4a-d).ThisfindingindicatesthatGdnHCl aloneisnotsufficienttodenaturetheprotein.Unfortunately,thecombinationofformicacidwithGdnHCl couldnotbeusedbecauseitdissolvesnitrocellulose,and thuswecannoteasilyassaywhetherformicacidtreatmentwithGdnHClefficientlydenaturesSOD1.When themembranewassteamedincitratebuffer,theimmunoreactivityofallthreeproteinswassignificantlyincreasedforbothhSODandC4F6antbodies.This findingindicatesthatthecombinationofheatandcitratebufferefficientlyexposestheepitopeforhSOD1 antibody(apeptideantibody)andsimilarlyexposesan epitoperecognizedbyC4F6. C4F6immunoreactivitytomisfoldedWTSOD1in postmortemtissuefromsALScases Aspreviouslydiscussed,tworecentstudieshaveinvestigatedwhetherWTSOD1inspinalmotorneuronsof sALScasesbecomesreactivetotheC4F6antibody,producingdatathatwasnotentirelycomparablebecause onestudyomittedantigenretrievalwhereastheother usedcitrate/heattreatments[5,15].Havingnowachieved abetterunderstandingofthenatureoftheepitopeand theeffectsofantigenretrievalonantibodybinding,we performedC4F6immunohistochemistryonpostmortem spinalcordtissuefromasetof25sALSpatientsand5 controls.Wedirectlycomparedthetwoimmunohistochemicalprotocols:eithernoantigenretrievalorsteamingofthesectionsincitratebufferthatwereusedby Boscoetal.andBrothertonetal.[5,15].Inourhandsand inourpatientsamples,nosignificantdifferencewasseen inthepatternofC4F6stainingwhetherthetissueswere untreatedpriortostaining(Additionalfile4:FigureS3)or steamedincitrate(Figure5).Asnotedabove,citrate Figure4 Condition-specificimmunoreactivityofthehSODandC4F6antibodiestopurifiedrecombinanthSOD1.(a) 1.5 gofthe indicatedrecombinanthSOD1proteinwasspottedontoanitrocellulosemembrane.Allmembranesweretreatedwith4%paraformaldehyde followedbyeithernotreatment,6Mguanidinehydrochloride(Para/GdnHCl),orsteamedincitratebuffer(Para/Cit.Buff.)asdescribedinMethods. MembraneswerethenblottedwiththehSOD (a) andC4F6 (c) antibodies. (b and d) TheintensitiesofthespotswerequantifiedusinganOdyssy ImagingSystemwiththevaluesnormalizedtotheWTsignal,forthehSODblots,ortotheG93Asignal,fortheC4F6blots;thedatafrom3 independentexperimentsaregraphed(meanratioS.E.(errorbars)).*P 0.05,***P 0.001(unpaired t -test). Ayers etal.ActaNeuropathologicaCommunications 2014, 2 :55Page8of13 http://www.actaneurocomms.org/content/2/1/55

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treatmentofmutantSOD1miceclearlyaugmentedreactivitytopathologicaccumulationsofmutantSOD1in miceandhenceweshowimagesofthecitratetreatedhumantissuesforcomparison.AscontrolsforC4F6staining,weusedmurinetissuefromnon-transgenicor hSOD1over-expressingmice(Figure5a-c).InbothsALS andnon-ALShumantissueexamined,sphericalC4F6immunoreactivestructureswereobserved(Figure5d-f). Thesespheroids,whichoccasionallyshowedanimmunopositiveringaroundaclearcenter,werefoundextracellularlythroughoutthegrayandwhitematterofthespinal cordsectionstovaryingdegrees(Figure5e-h).Occasionally,weobservedextracellularpunctatestaininginthe graymatter(Figure5f,g).Eighteenofthe25sALScases andallfiveofthecontrolcasesweexaminedpossessed theseC4F6-reactivespheroids(Additionalfile1:TableS1). Althoughthemajorityofcasesrevealedmassiveneurodegenerationandmotorneuronloss,thosetissuesections thatretainedmotorneuronsweredevoidofintracellular C4F6immunoreactivity.Theregularshapeofthespheroid structureswasreminiscentofacommonfeatureofadult humantissuestermedcorporaamylacea[33].Todeterminethefrequencyofthispathologyinourtissues,we performedaperiodicacidSchiffstain,whichisusedfor thedetectionofthesedeposits[33].Thisstainrevealed thatthesestructureswerefrequentinoursetofcaseswith thelocationandshapeofthestructurebeingidenticalto theC4F6immunopositivespheroiddeposits.Importantly, ourcontrolcasesalsohadcorporaamylaceadepositsand thefrequencyofthesedepositsalignedwiththefrequency ofC4F6reactivespheroidsinourcontrolcases.Therefore, weconcludedthatthespheroidinclusion-likereactivity withC4F6waseithersometypeofcrossreactivitywith thecorporaamylaceadepositsorsomenon-diseaserelatedassociationofSOD1withthesestructures.Inany case,thestructureswerenotspecifictosALScases. Discussion Inthisstudywesoughttomorepreciselydefinetheepitopeoftheanti-hSOD1C4F6antibodyandelucidatethe basisforitsabilitytospecificallyrecognizemanydifferent ALSmutantsofSOD1.Previousstudieshaddetermined thattheepitopeofC4F6islocatedinexon4ofthe hSOD1protein,whichencompassesaminoacids80to 119[5].Here,weusedapproachesinimmunocytochemistryandimmunoblottingtodemonstratethatresidues 90 – 93oftheprotein(DKDG)comprise,atleastinpart,a criticalelementoftheepitoperecognizedbyC4F6.The Figure5 LackofC4F6immunoreactivityintissuefromsALScases. Forapointofreference,tissuesfromnon-transgenicmice (a) ,WT (b) andL126Z (c) hSOD1overexpressingmicewereco-stained.ThesetissuesweresteamedincitratebuffertoenhanceC4F6immunoreactivityas describedinFigure5. (d-f) C4F6stainingofhumantissuefromnon-diseasecontrolsrevealedoccasionalspheroid(arrowinpanele)andpunctate immunoreactivedeposits(3casesshown). (g,h) SimilarstructureswereobservedinhumantissuefromsALScases(2casesshown).Forboththe controlandsALScasestheimagesshownarefromtissuessteamedincitrate.Lessstainingwasobservedinuntreatedtissues(Additionalfile4: FigureS3). (i) Thespheroiddepositswereidenticalinappearanceandfrequencytostructuresidentifiedascorporaamylacea(highlightedwith arrows)bythePeriodicAcid-Schiffstain. Scalebar 100 m. Ayers etal.ActaNeuropathologicaCommunications 2014, 2 :55Page9of13 http://www.actaneurocomms.org/content/2/1/55

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importanceoftheAspresidueatposition90wasconfirmedbydemonstratinglowimmunoreactivityofC4F6 fortheD90AvariantofhSOD1(eitherinfixedcellsoron immunoblots).WhenC4F6ispresentedwithdenatured proteinonimmunoblots,amuchhigheraffinityofthe antibodyforSOD1containingtheD-K-D-Asequenceat 90 – 93(aswouldbethecasefortheG93Avariant)over D-A-D-G(forWThSOD1)wasrevealed.Thus,ourdata stronglyimplicateaminoacids90and93ofthehSOD1 proteinasessentialelementsoftheepitoperecognizedby C4F6withresiduesDataminoacids90andG/Aat93 comprisingtheminimalresiduesresponsibleforantibody binding. Structurally,aminoacids90 – 93arelocatedinthe beta-turnstructureformedbytheloopbetweenthe5thand6thbetastrandsofhSOD1.Analignmentof32distinctWThSOD1subunitsfromthePDBdatabaseof7 independentcrystalstructures,revealedtheconsistent featuresoftheprotein ’ sconformation.Thetertiary structureofWThSOD1bringsthe90 – 93loopinclose proximitytotheglutamicacidatposition40,whichsits attheapexoftheloopbetweenbetastrands3and4. Thisregionoftheproteinhasbeenreferredtoasthe plugbecauseL38capsoneendofthe -barrelstructure [34,35].Attheapexofthisplug,thesidechainofLys91 showstwomajorpopulations;inoneorientationofthe sidechaintowardsAsp92andinalesspopulatedorientationtowardGlu40.ThelatercasewouldfavorhydrogenbondingbetweentheaminogroupofLys91andthe carbonyloftheGluresidueatposition40.Additionally, duetotheircloseproximity,numerousvanderWaals interactionsbetweenthesetwoloopstructureswould stabilizethenativeconformationregardlessofwhether thesidechainofLys91ispositionedoverGlu40or Asp92.Collectively,thesenon-covalentinteractionsbetweenthetwoloopsstabilizeatightbendinthebackboneoftheproteintoformtheloopcontainingamino acids90 – 93.Weproposethatinthestructureofthe WTnativeprotein,therigidityofthisstructuremost likelypreventstheC4F6antibodyfrombindingtoan epitopethatconsistsminimallyofresiduesD-K-D-G/A atacids90 – 93. ThestructuresavailableforSOD1-G93A(3distinct structureswith16subunits)showthatresidues90 – 93 aregenerallyfoundinasimilarconformationasobservedinWTSOD1.Thus,incomparingtheWTand G93Avariantsintheregionthatcontainsacriticalcomponentoftheepitopethereisnotanobviousconformationalsignaturethatweseeasprovidingspecificityfor C4F6binding.TheC4F6antibodywasraisedagainstrecombinantapoG93Aprotein,thestructureofwhich wasincludedinthestructuresweanalyzedandwhich completelyalignswiththatofmetallatedG93Aprotein. Thus,theconformationalelementcausedbytheG93A mutationthatenablesC4F6bindingdoesnotappearto beduetoastablechangeinlocalstructurearoundthe 90 – 93loop. InstudiesofWTandmutantSOD1expressedinculturedcells,ourstudiesreportedhere,andpreviously [27],indicatethattheC4F6antibodycanbeusedto detectmisfoldedmutantSOD1andthatmostifnotall fALSmutantsshareacommonmisfoldedconformation aroundtheloopbetween90 – 93.Bysomemannermutationsdistantfromthe90 – 93loop,suchasA4V[27], produceastructuralchangethatcausesaminoacids90 – 93toadoptaconformationthatenablesthebindingof C4F6despitethelackofthefavoredAatposition93. NMRstudiesoftheG93Amutanthaveprovidedevidenceforlocalchangesinproteinstructure,withthe mutationcausingaminoacidsD90andV94tohavea muchhigherexposuretosolventthanexistsintheWT protein[36,37].Molecular-dynamicsimulationsofthe A4V,G37R,andH46RvariantsofSOD1havesuggested destabilizationofthe -plugregion,whichincludesthe 90 – 93loop[38,39].Thus,multiplemutationscaninduce destabilizationofthe90 – 93loopandmayexplainwhy C4F6showsgreaterreactivitytomutantSOD1overWT proteinwhenusedforimmunocytochemistryontransientlytransfectedcells[27].Onepossibleexplanationfor theabilityofC4F6torecognizemanydifferentmutants ofSOD1withgreateravidityisthatmanymutationsincreasemobilityincriticalelementsofthestructureand thisincreasedflexibilityispropagatedthroughthebackbonetoreducetherigidityofthe -plugandallowthe antibodygreateraccesstotheweakerepitopeofD-K-D-G. InregardtotheconformationalspecificityofC4F6, ourdataindicatethattheconditioninwhichwehave thegreatestconfidencethatnon-nativeconformationof themutantSOD1isdrivingreactivityiswhenusedfor immunocytochemistryincellmodels.Inourstudiesof cellsover-expressinghumanSOD1encodingALSmutationsotherthanG93A[27],theantibodyconsistently recognizesasubsetofcellsthatover-expressmutant SOD1.Inthesestudies,cellsarefixedbrieflyinparaformaldehydeandnoparticularantigenretrievalisrequired. Overall,ourexperiencewiththecellmodelssuggeststhat C4F6candiscriminatebetweenWTandmutantSOD1by immunocytochemistryoffixedcells. Todeterminetheutilityoftheantibodyinfixedtissues andtheeffectofcommonantigenretrievalprocedureson binding,weexaminedC4F6reactivitytospinalcordsfrom miceoverexpressingWTandmutanthumanSOD1. TreatmentsthatweassumedwoulddenatureSOD1,treatmentinformicacidand6MGdnHClorheattreatment incitratebuffer,resultedinasignificantincreaseinC4F6 staininginspinalcordsfromallSOD1variantstested. Similarly,weobservedincreasedC4F6reactivitytopurifiedSOD1spottedontonitrocellulosemembraneafterAyers etal.ActaNeuropathologicaCommunications 2014, 2 :55Page10of13 http://www.actaneurocomms.org/content/2/1/55

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heat/citratetreatment.Atleastforthecaseinwhichheat/ citratetreatmentwereused,wehaveconfidencethatsome oftheincreasedreactivityoftheantibodytotissueisdue todirecteffectsontheproteinratherthanfacilitatingbetteraccessoftheantibodytoitsepitope.Becauseofthe muchgreateraffinityoftheC4F6antibodyfortheG93A protein,wecannotbesurewhethertheimmunoreactivity detectedinthesemiceisindicativeofconformational change.Notably,incontrasttonon-transgenicmiceand miceexpressinghighlevelsofWThSOD1,untreated spinalcordsofparalyzedmiceexpressingtheG37Rand L126ZmutantsshowedatleastsomeC4F6immunoreactivitythroughoutthegreymatter;eitherintheformof diffuseneuropilreactivityintheG37Rmiceorasdiscrete punctaintheL126Zmice.Inallthreemutantmouse models,antigen-retrievaltreatmentssignificantlyincreasedtheintensityofreactivityand,intheL126Zmice, elaboratedfibrillarinclusions.However,thesetreatments alsoincreasedreactivityintheWThSOD1miceandthus weattributeC4F6reactivityinthiscasetorelaxationof theWTSOD1structuretoloosenthetightbendinthe backbonearoundaminoacids90 – 93.Thus,althoughantigenretrievalmethodsdoappeartoenhanceC4F6binding toaccumulationsofSOD1inpathologicstructures,indicatingtheutilityoftheantibody,usersshouldnotethat antigenretrievalmethodsthatcoulddenatureSOD1may produceC4F6reactivitythatisnotnecessarilyindicative oftheconformationalstatethatexistedinlivingtissue. TheC4F6monoclonalantibodyisoneofseveralthat havebeenusedthatweregeneratedbyimmunizingmice withhumanG93ASOD1[14,40].Todeterminewhether ourfindingsforC4F6mayextendtoothermonoclonal antibodiesofthistype,weexaminedthebindingofthe A5C3monoclonal[40]tocellstransfectedwithvectors toexpressWTandtheG85R,G93A,andD90Avariants (Additionalfile5:FigureS2).Somewhatsurprisingly,unlikeC4F6,theA5C3antibodyshowednosignificantcross reactivitytoG85RhSOD1(Additionalfile5:FigureS2). ThisfindingsuggeststhattheA5C3antibodymayhavea greaterdependencyonthepresenceofanalanineatposition93thantheC4F6antibody. AsdescribedintheIntroduction,therehavebeentwo priorstudiesofwhetherspinalmotorneuronsfromcases ofsALSshowspecificimmunoreactivitytoC4F6antibody [5,15].Oneofthesestudiesdidnotuseantigenretrieval (Boscoetal.)whiletheotherusedaheat/citrateprotocol similartowhatwedescribehere(Brothertonetal.).Inthe Boscoetal.study,theydemonstratedthatasubsetof motorneuronsinasubsetofcases(4of9)showedaberrantC4F6immunoreactivityofspinalmotorneuroncell bodies[5].IntheBrothertonetal.study,whichcompared sALScasestoanA4VfALScaseandcontrols,C4F6recognizedskeinlikeinclusions(similartowhatweobserved intheL126Zmousemodelhere)inthespinalmotor neuronsofA4VfALScases.InsALScases,aswellascontrols,noC4F6reactiveinclusionswereobservedand insteadamorediffusecellbodystainingwasobserved.In ourhandsandinourpatientsamples,whethertissues werestainedwithoutantigenretrievalorafterheat/citrate treatment,thepatternofimmunoreactivityinspinalcords ofsALSandcontrolpatientsweresimilar.Inboth,we observedthatthemostimmunoreactivestructureswere extracellularspheroidandpunctatedeposits.Nostaining ofmotorneuroncellbodieswasobservedthatresembled theimmunoreactivecellbodiesidentifiedascontaining misfoldedhSOD1intheBoscostudy[5].Wenoteda directcorrelationbetweenthefrequencyofC4F6reactive spheroidstructuresandthefrequencyofcorporaamylaceadeposits,whicharecommonglycoproteinacousinclusionsthatarefrequentlyfoundintheCNStissuesof elderlypeople[33,41].Thesestructuresimmunostainfor multipleproteinsincludingubiquitin,hemeoxygenase, MnSOD1,anda-synuclein[42].WhethertheSOD1 reactivitypresentinthesestructuresisindicativeofabiologicallyrelevantconcentrationoftheproteinorissome non-specificinteractionwithothercomponentsofthe structureisunknown.ThesameC4F6reactivestructures wereobservedinnon-ALSpatientsandthusthesestructuresdonotrepresentadisease-specificpathology.Thus, whetherusingantigenretrieval,whichcouldrevealpathologicaccumulationsofSOD1,ornoantigenretrieval, whichcouldrevealSOD1possessingaconformational signatureproducedbyALSmutantSOD1,wefailedto detectrobustC4F6reactivitythatisspecifictosALS spinalmotorneurons.OurdatasuggestthatWThuman SOD1inspinalmotorneuronsinpost-mortemtissuesof sALScasesisinfrequentlypresentinaconformationthat exposestheC4F6epitope.AdditionalfilesAdditionalfile1:TableS1. HumanTissueCharacteristics. Additionalfile2:TableS2. DetailsforSOD1structuralanalysis. Additionalfile3:FigureS1. Antigen-retrievalimpactsSEDI immunoreactivitytohSOD1intransgenicmousespinalcordtissues. TissuesectionsfrommiceoverexpressingWT,G93A,andG37R,ornontransgenicmiceforcontrols,werestainedwithSEDIfollowingeitherno antigenretrievalorformicacidand6Mguanidinehydrochloride(FA& GdnHCl).AlltissuesfrommiceexpressingmutanthSOD1(G93Aand G37R)wereharvestedfromparalyzedmice.TheWThSOD1tissuewas frommice~18monthsofage. Scalebar 100 m. Additionalfile4:FigureS3. C4F6immunoreactivityintissuefromsALS caseswithoutantigenretrieval.Tissuesfromnontransgenicmice (a) ,WT (b) ,andG93A (c) hSOD1overexpressingmicewerestainedwithC4F6as controls. (d) C4F6stainingofhumantissuefromnon-diseasecontrols revealedverylittleimmunoreactivity. (e,f) InhumantissuefromsALS cases(2casesshown),occasionalspheroiddepositsappearedtobeC4F6 immunopositive.ForboththecontrolandsALScasestheimagesshown arefromtissuesnottreatedforantigenretrieval.Thespheroiddeposits wereidenticalinappearanceandfrequencytostructuresidentifiedasAyers etal.ActaNeuropathologicaCommunications 2014, 2 :55Page11of13 http://www.actaneurocomms.org/content/2/1/55

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corporaamylacea(highlightedwitharrows)bythePeriodicAcid-Schiff stain. Scalebar 200 m. Additionalfile5:FigureS2. C4F6andA5C3differintheirspecificity forSOD1.C4F6revealednoimmunoreactivitytoWTSOD1 (c) but stainedcellstransfectedwithG85RSOD1 (d) .Followingtransient transfectionwithWT (e,i) ,G93A (f,j) ,G85R (g,k) ,orD90A (h,l) cells immunostainedwiththeA5C3antibodyonlyshowedreactivitytoG93A (j) .NucleiwerestainedwithDAPI(blue). Scalebar 50 m. Competinginterests Theauthorsdeclaretheyhavenocompetinginterests. Acknowledgments WewouldliketothankDr.JaniceRobertsonfortheuseoftheanti-SOD1 monoclonalSEDIantibody.WearegratefulforhelpfuladvicefromDrs.Julian WhiteleggeandJoanS.Valentine.Thisworkwassupportedbyagrantfrom theNationalInstitutesofNeurologicalDiseaseandStroke(P01NS049134 – ProgramProject).Thenon-diseasedcontrolhumantissuewasaccessioned viatheJohnsHopkinsUniversityAlzheimer ’ sDiseaseResearchCenterthatis supportedbyagrantbytheNationalInstitutesofHealth(P50AG05146). Authordetails1DepartmentofNeuroscience,CenterforTranslationalResearchin NeurodegenerativeDisease,McKnightBrainInstitute,UniversityofFlorida, Box100159,Gainesville,FL32610,USA.2DepartmentofPathology,TheJohns HopkinsUniversitySchoolofMedicine,Baltimore,MD,USA.3Departmentof BiochemistryandX-rayCrystallographyCoreLaboratory,UniversityofTexas HealthScienceCenter,SanAntonio,USA.4DepartmentofVeteransAffairs, GeriatricResearch,Education,andClinicalCenter,SouthTexasHealthCare System,SanAntonio,TX,USA. 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! ESM2 C4F6 and A5C3 differ in their specificity for SOD1 C4F6 revealed no immunoreactivity to WT SOD1 ( c ) but stained cells transfected with G85R SOD1 ( d ) Following transient transfection with WT ( e, i ), G93A ( f, j ), G85R ( g, k ), or D90A ( h, l ) cells immunostained with the A5C3 antibody only showed reactivity to G93A ( j ) Nuclei were stained with DAPI (blue). Scale bar 50 m.



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Supplemen tary Figure 3 Figure S3 C4F6 immunoreactivity in tissue from sALS cases without antigen retrieval. Tissues from non transgenic mice ( a ), WT ( b ), and G93A ( c ) hSOD1 overexpressing mice were stained with C4F6 as controls. ( d ) C4F6 staining of human tissue from non disease controls revealed very little immunoreactivity. ( e, f ) In human tissue from sALS cases (2 cases shown), occasional spheroid deposits appeared to be C4F6 immunopositive. For both the control and sALS cases the image s shown are from tissues not treated for antigen retrieval. The spheroid deposits were identical in appearance and frequency to structures identified as corpora amylacea (highlighted with arrows) by the Periodic Acid Schiff stain. Scale bar 200 m