DNA strand breaks and TDP-43 mislocation are absent in the murine hSOD1G93A model of

22 Pages·2017·11.7 MB·English

RESEARCHARTICLE
DNA strand breaks and TDP-43 mislocation
G93A
are absent in the murine hSOD1 model of
amyotrophic lateral sclerosis in vivo and in
vitro
DianePenndorf☯,VedranaTadić☯,OttoW.Witte,JulianGrosskreutz‡,AlexandraKretz‡*
HansBergerDepartmentofNeurology,JenaUniversityHospital,Jena,Thuringia,Germany
a1111111111
☯Theseauthorscontributedequallytothiswork.
a1111111111
‡Theseauthorsalsocontributedequallytothiswork.
a1111111111
*[email protected]
a1111111111
a1111111111
Abstract
MutationsinthehumanCu/Znsuperoxidedismutasetype-1(hSOD1)genearecommonin
familialamyotrophiclateralsclerosis(fALS).Thepathophysiologyhasbeenlinkedto,e.g.,
OPENACCESS
organelledysfunction,RNAmetabolismandoxidativeDNAdamageconferredbySOD1
Citation:PenndorfD,TadićV,WitteOW,
malfunction.However,apartfrommetabolicallyevokedDNAoxidation,itisunclearwhether
GrosskreutzJ,KretzA(2017)DNAstrandbreaks
andTDP-43mislocationareabsentinthemurine severegenotoxicityincludingDNAsingle-strandbreaks(SSBs)anddouble-strandbreaks
hSOD1G93Amodelofamyotrophiclateralsclerosis (DSBs),originatesfromlossoffunctionofnuclearSOD1enzyme.Factorsthatendoge-
invivoandinvitro.PLoSONE12(8):e0183684.
nouslyinterferewithDNAintegrityandrepaircomplexesinhSOD1-mediatedfALSremain
https://doi.org/10.1371/journal.pone.0183684
similarlyunexplored.Inthisregard,uncontrolledactivationoftransposableelements(TEs)
Editor:EmanueleBuratti,InternationalCentrefor
mightcontributetoDNAdisintegrationandneurodegeneration.Theaimofthisstudywasto
GeneticEngineeringandBiotechnology,ITALY
elucidatetheroleofthefALS-causinghSOD1G93AmutationinthegenerationofsevereDNA
Received:June2,2017
damagebeyondwell-characterizedDNAbaseoxidation.Therefore,DNAdamagewas
Accepted:August9,2017 assessedinspinaltissueofhSOD1G93A-overexpressingmiceandincorrespondingmotor
Published:August23,2017 neuron-enrichedcellculturesinvitro.OverexpressionofthehSOD1G93Alocusdidnot
changethethresholdforsevereDNAdamageperse.WefoundthatlevelsofSSBsand
Copyright:©2017Penndorfetal.Thisisanopen
accessarticledistributedunderthetermsofthe DSBswereunalteredbetweenhSOD1G93Aandcontrolconditions,asdemonstratedinpost-
CreativeCommonsAttributionLicense,which mitoticmotorneuronsandinastrocytessusceptibletoreplication-dependentDNAbreak-
permitsunrestricteduse,distribution,and
age.Analogously,parametersindicativeofDNAdamageresponseprocesseswerenotacti-
reproductioninanymedium,providedtheoriginal
authorandsourcearecredited. vatedinvivoorinvitro.Evidenceforamutation-relatedelevationinTEactivationwasnot
detected,inaccordancewiththeabsenceofTARDNAbindingprotein43(TDP-43)protei-
DataAvailabilityStatement:Allrelevantdataare
withinthepaperanditsSupportingInformation nopathyintermsofcytoplasmicmislocationornuclearloss,asnuclearTDP-43issupposed
files. tosilenceTEsphysiologically.Conclusively,thesuperoxidedismutasefunctionofSOD1
Funding:ThisworkwassupportedbytheMinistry mightnotberequiredtopreserveDNAintegrityinmotorneurons,atleastwhenthefunction
forEconomics,SciencesandDigitalSocietyof ofTDP-43isunaltered.Ourdataestablishafoundationforfurtherinvestigationsaddressing
Thuringia(TMWWDG),intheframeworkofthe
functionalTDP-43interactionwithALS-relevantgeneticmutations.
ProExcellenceInitiativeRegenerAging
(RegenerAging-FSU-I-03/14toAK),andbythe
InterdisciplinaryCenterforClinicalResearch(IZKF)
Jena(ProjectFF01toAK).JGreceivedfunding
fromaBMBF(theBundesministeriumfu¨rBildung
PLOSONE|https://doi.org/10.1371/journal.pone.0183684 August23,2017 1/22
DNAdamageinthehSOD1G93AmousemodelofALS
undForschung)grantintheframeworkoftheE- Introduction
RAREprogram(PYRAMID),JPND(OnWebDUALS
Amyotrophiclateralsclerosis(ALS)representsthemostfrequentlethaldisorderofthemotor
andSOPHIA)oftheEuropeanUnionandthe
GermanAssociationforNeuromuscularDiseases systemandischaracterizedbyarelentlessandprogressivelossofupperandlowermotorneu-
(DGMe.V.).Thefundershadnoroleinstudy rons(MNs)ofthemotorcortex,brainstemandMNsofthespinalcord.Theprovenanceis
design,datacollectionandanalysis,decisionto complexandyetuntargetedbyefficienttherapeuticstrategies.Clinically,devastatingsymp-
publish,orpreparationofthemanuscript.
tomsmanifestwithprogressivespasticandatrophicparalysis,impairmentsinspeechand
Competinginterests:Theauthorshavedeclared swallowingandfinallyleadtorespiratoryfailure3–5yearsaftertheonset[1].Amongthe
thatnocompetinginterestsexist. approximately20causativegenelociandabout30disease-modifyinggeneticfactorsidentified
todate[2],numerousmutationsinthehumanCu/Znsuperoxidedismutase1(hSOD1)gene
areaccountingforapproximately20%offamilialALS(fALS)and1–2%ofsporadicALS
(sALS)cases[1,2].SOD1mutationsinvolvingallfiveexonsarepredominantlysingle-base-
pairsubstitutions,whicharecommonlylinkedtoanaggressivecoursewithrapidclinicaldete-
rioration,includingthosevariantsimplicatingap.G93Amutation[2].Thoughdiseasepatho-
genesisgenerallyimplicatescellular,molecularandmetabolicalterations,theunderlying
etiologyisstilldebated.Likewise,theroleofsevereDNAdamageandendogenousDNArepair
strategiesinALSinitiationandprogressionareill-defined.Thistopicimpliesanotablerolefor
ventralhornspinalMNs,theneuronalpopulationthatismostafflictedinthediseasepathol-
ogy,whichmustcopewithnumerousDNAdamagingagents,suchasoxidizingmolecules,in
casesofbothfALSandsALS[3,4].Reactiveoxygenspecies(ROS)areamongthemostcrucial
noxiousmetabolitesimpactingMNsduetotheirimmenseenergeticrequirementsandmeta-
bolicturnover.IntracellularROSconcentrationsareincreasedinhSOD1-dependentfALSand
maycontributeconsiderablytogenomicinstability[5,6].ROSpreferentiallytargetDNAbases
orsugarresidues,resultinginDNAsingle-strandbreaks(SSBs).Theycanalsoinducethefor-
mationofDNAdouble-strandbreaks(DSBs),e.g.,ifthetranscriptionapparatusisaffectedat
locationswhereROS-mediatedlesionseventuateinanarrowsequence.
AccountingforthetoxicgainoffunctionassumedtooriginatefrommutanthSOD1
togetherwithalossoffunctionofnaïveSOD1enzymeinthenucleus[7],clinicalseverity
mightthusarisefromoxidativestress-inducedsevereDNAdamage.ThismayincludeDNA
SSBsandDSBs,inparticulariftheyfailtobecomerepaired,orremainmis-repaired.Inaccor-
dancewiththisnotion,thelevelsof8-hydroxy-deoxyguanosine,amarkerofoxidativeDNA
demise,areincreasedinspinalcordofsALSandfALSpatients[8].Likewise,DNAdamageis
risingduringROSexposureinculturedNSC34cellscarryingahSOD1G93Amutationcom-
paredwithwildtypetransfectants[7].However,whetherthismutationcausesapredisposition
toDNAstrandbreaksunderALS-likeconditionsinvivo,apartfromartificiallyhighROSlev-
els,hasremainedunexplored.
AsidefromROS-inducedDNAdamage,theroleofendogenouslygeneratedDNAdemise
inhSOD1-mediatedfALS,inparticularinnon-dividingMNslackingreplicationstressasa
considerablephysiologicalsourceofDNAbreakagehasbeensimilarlyuninvestigated.Uncon-
trolledactivationoftransposableelements(TEs)andtheirprogressivede-repressionarethus
discussedascriticallyinvolvedinDNAdamageandcentralnervoussystem(CNS)neurode-
generation[9–11].ThereareindicationsofacontributionofcertainTEstothepathologyof
ALS,atleastinsporadiccases[12].However,whetherTEactivationisimplicatedinhSOD1-
relatedMNdegenerationhasremainedanopenquestion.TEsrepresentrepetitiveDNAele-
mentsthataccountforapproximately30%-50%ofthemammaliangenome[13].Theyare
activelymobilewithintheDNAstrandandexhibitthehighestactivationinneuronaltissue,
forreasonsthatstillhavetobedefined[14,15].GenomicTEintegrationiscapableofdisturb-
inggenefunctionandcausingDNAstrandbreaks[16]ifnotproperlyprohibited,e.g.,byTE
DNAmethylation,histonemodificationsandpost-transcriptionalsilencing[9].Regardingthe
PLOSONE|https://doi.org/10.1371/journal.pone.0183684 August23,2017 2/22
DNAdamageinthehSOD1G93AmousemodelofALS
latter,thereisevidencethatnucleartransactiveresponseDNA-bindingprotein43(TDP-43)
targetsTE-derivedtranscriptsandthusplaysaphysiologicalroleinsilencingTEtransposition
[10].Pathologicalalterations,e.g.,nuclear-to-cytoplasmictranslocations,oftheRNA/DNA-
bindingproteinTDP-43areawell-acceptedhallmarkofbothfALSandsALS[17].However,
asoneexceptionalentity,hSOD1-relatedfALSisassumedtobedevoidofcytoplasmicTDP-43
aggregation[17].Intheabsenceofacytoplasmicproteinopathy,anuclearlossofTDP-43
mightcriticallyimpairitssuppressorfunctionontheactivationofTEmoieties.Todate,itis
unknownwhetherTDP-43stabilizationofDNAintegrityisalteredinMNscarryingahSOD1
mutationinspiteoftheputativeabsenceofaTDP-43mislocationorwhethertheextentofthe
hSOD1mutationimpactsongenomicintegrityingeneral.
Hence,theaimofthisstudywastoelucidatetheroleoftherapidlyprogressivefALS-caus-
ingp.G93AhSOD1mutationinthegenerationofsevereDNAdamage.Toallowanin-depth
evaluationbeyondwell-characterizedDNAbaseoxidation,e.g.,causedbyROSoverload,
DNASSBandDSBeventswereexaminedinthehSOD1G93Amurinemodelinvivoasafunc-
tionofthediseasestage,aswellasinMNsinvitro.ApartfromoverallDNAdamage,the
occurrenceofDNAbreakagewasspecifiedfornon-replicativeMNsanddividingastrocytesin
MN-enrichedcellcultureswithrespecttothehSOD1G93Amutation.Moreover,theDNAinteg-
rityandDNAdamageresponse(DDR)wereassessedinlightoftheDNA-stabilizingfunction
ofnuclearTDP-43,mediatedbyitsinteractionwithTEtranscripts,particularlyinspinalMNs.
Despiteitswell-establishedtoxicgainoffunctiononSOD1enzymeactivity,ourresults
indicatethatthep.G93AmutationatthehSOD1locusdoesnotchangethethresholdforsevere
DNASSBsorDSBsperse,atleastwhenassessedinamurinecontext.Thiswasdemonstrated
inpost-mitoticMNsandinastrocytessusceptibletoreplication-dependentDNAbreakage.
Analogously,parametersindicativeofDDRprocesseswerenotactivatedinvivoorinvitro.
TheabsenceofTDP-43proteinopathyintermsofcytoplasmicmislocationornuclearlossin
thehSOD1G93Amodel,irrespectiveofthediseasestage,mightunderscoretheroleofTDP-43
inDNAstabilization.ConsistentwiththelackofTDP-43decompartmentalizationandnuclear
loss,wedidnotobtainevidenceofelevatedTEactivationduringtheseverediseasestage.
WeconcludethatthesuperoxidedismutasefunctionofSOD1mightnotberequiredtopre-
serveDNAintegrityinMNs,atleastwhenthefunctionofTDP-43isunaltered.Ourdata
mightestablishthefoundationforfurtherinvestigationsaddressingtheinteractionofthe
DNAstabilizingfunctionofTDP-43withALS-relevantgeneticmutations.
Materialsandmethods
Mice
TransgenicmiceoverexpressingmutanthSOD1G93A(B6.Cg-Tg(SOD1(cid:3)G93A)1Gur/J;Jackson
Laboratory,BarHarbor,ME,USA;stocknumber004435)wereusedtomimicALS-likecondi-
tions[18]priortoandaftertheonsetofdisease-relatedsymptoms.Thediseasestagewas
assessedbyageinparallelwithaclinicalscoreasdetailedinS1Tableincludingthedefinition
ofhumaneendpoints.Clinicalstatewasexaminedatleastweekly.Asassessedfromathree-
yearscharacterizationofapproximately200malehSOD1G93Amiceinourfacility,theonsetof
diseasemanifestationbyhindlimbparesisoccurredatanaverageageof18.8±0.1weeks.In
thisstudy,exclusivelymaleanimalswereincludedanddesignatedas‘diseased’assoonasa
clinicalscoreof1–2wasreached,usuallyoccurringatanageof19–23weeks.Presymptomatic
micewerephenotypicallynormalandwereincludedat8–9weeksofage.Sincetheywerebred
onanidenticalbackground,11-13-week-oldmaleC57BL/6Jmiceservedascontrols.Forin
vitroexperiments,B6.Cg-Tg(SOD1(cid:3)G93A)1Gur/Jtransgenicandnon-transgenicembryosat
E13wereusedascelldonors.GenotypingwasperformedbygenomicPCRimmediatelybefore
PLOSONE|https://doi.org/10.1371/journal.pone.0183684 August23,2017 3/22
DNAdamageinthehSOD1G93AmousemodelofALS
cellplating.ConditionsofcellreplicationandimpairedDNArepairweregainedinbraintis-
suefromadultNOD.CB17-Prkdcscid/NCrHsdmicedisplayingdefectiveDDRresponsesand
repairofDSBs,withorwithoutfronto-corticalxenograftingofhighlyproliferativeLN229
humanglioblastomacells(kindlyprovidedbyJ.Walter,DepartmentofNeurosurgeries,Jena
UniversityHospital).Micewerehousedandmatedunderstandardized,pathogen-freecondi-
tionswithunlimitedaccesstostandarddietandwaterandexposedtoa14/10-hourslight/dark
cycle.AllanimalinterventionswereconductedincompliancewiththeDirectivesofthePro-
tectionofAnimalsActandwereapprovedbytheanimalwelfareauthoritiesofThuringia
(accreditationnumber:02-046/14).
Cometassay
AdultmicedeeplyanesthetizedwithvaporizedIsoflurane(IsolfuraneCP1,CPPharma,Burg-
dorf,NI,Germany)inaspecializedchamberweretranscardiallyperfusedwithice-coldPBS
anddecapitated.Topreparesinglecellsuspensions,thecervicalandthoracicspinalcordswere
micro-dissectedandincubatedincalcium-freeHibernate1A(BrainBitsLLC,Springfield,IL,
USA)supplementedwith0.5mMGlutaMAX™(Gibco™/ThermoFisherScientific,Waltham,
ME,USA),0.132MD-(+)-trehalosedihydrate(Sigma-Aldrich,Munich,BY,Germany),310
U/mlDNaseI(Sigma-Aldrich)and1mg/mlcollagenasetypeIA(Sigma-Aldrich)for30min
at37˚Cundergentleagitation.Theworkingsolutionwasthenreplacedwithasolutioncon-
taining2mg/mlpapain(Worthington,Lakewood,NJ,USA)insteadofcollagenase.Celldis-
seminatesweremaintainedat37˚Cfor30minunderrepeatedtriturationusingafire-polished
Pasteurpipettetoobtainasinglecellsuspension.Enzymaticdigestionwithpapainwasfinally
blockedbytheadditionofice-cold0.1%ovomucoid(Sigma-Aldrich)inHibernate1Awork-
ingsolution,inwhichtheenzymeswerereplacedwith0.1%BSA(Serva,Heidelberg,BW,Ger-
many).ThissuspensionwassequentiallypassedthroughFalcon™cellstrainerswithamesh
sizeof100μmand40μm(ThermoFisherScientific,Waltham,ME,USA)beforebeingcentri-
fugedat500xgfor5min.Asapositivecontrol,corticalcellsfromtheNOD.CB17-Prkdcscid/
NCrHsdstraindevoidofLN229xenograftswereused.Thecellpelletwasresuspendedinice-
coldPBSanddilutedtoafinalconcentrationof100,000cells/ml.Thecometassaywasper-
formedusingtheOxiSelect™CometAssayKit(3-wellslides;CellBiolabs,SanDiego,CA,
USA),andallstepswereperformedaccordingtothemanufacturer’smanual.Briefly,the
dilutedsinglecellsuspensionwasmixedata1:10ratiowithOxiSelect™Cometagarose,plated
onpre-coatedslidesandincubatedfor15minat4˚C.Allstepspriortosinglecellgelelectro-
phoresiswereperformedinthedarktoavoidadditionalUVlight-inducedDNAdamage.For
celllysisandDNAunwinding,theslidesweretransferredto4˚Cconditions,incubatedinlysis
bufferfor50minandthenimmersedinalkalinesolutionfor30min.TodetectbothDNA
SSBsandDSBs,singlecellgelelectrophoresiswasperformedunderalkalineconditions(15V,
300mA,30min).Theslideswererinsedinwater,dehydratedin70%ethanolfor5minand
thenstainedwith1xVistaGreenDNA-dyesolutionfor15min.Randomimagesofatleast60
cometsoutofapproximately2,250cellsdistributedamong3separatedwellsperanimalwere
obtainedusinganAxioplan2Imagingmicroscope(20xairobjective;Zeiss,Oberkochen,BW,
Germany)coupledtoanAxioCamHRccamera(Zeiss).Forsemi-automatedcometanalysis,
theCASPsoftwarewasemployed[19].Forcometscoring,thefeatures‘PercentofTailDNA’
(%TailDNA),whichrepresentstherelativetailintensity,andthe‘TailMoment’wereselected
ascomparativeparameters.The‘TailMoment’providesinformationonthelengthofthe
comettail,whichismultipliedwiththe%TailDNA.Althoughthe‘TailMoment’isfrequently
used,theapplicationof%TailDNAbearstheadvantagethatitislinearlycorrelatedwiththe
breakfrequencyandallowsforavisualcometscoringgradationasdescribedbyCollins(2004)
PLOSONE|https://doi.org/10.1371/journal.pone.0183684 August23,2017 4/22
DNAdamageinthehSOD1G93AmousemodelofALS
[20].Basedonthisvisualcometscoringalgorithm,cometswereclassifiedbytheir%TailDNA
accordingtothefollowingcategories:category0,0–20%TailDNA;category1,20–40%
TailDNA;category2,40–60%TailDNA;category3,60–80%TailDNA;andcategory4,80–
100%TailDNA.Intotal,cometsfrom3animalspergroupwereinvestigated.
Motorneuron-enrichedcellculture
Pregnantmiceweresacrificedbycervicaldislocationwithsubsequentdecapitation.MNs
derivedfromspinalcordsofE13mouseembryoswereculturedonanastrocyticfeederlayeras
previouslydescribed[21,22].Briefly,spinalcordsoftransgenicandnon-transgenicembryos
weredissected,cutintopiecesandpooledinHBSS(Gibco™/ThermoFisherScientific)contain-
ing1%penicillin-streptomycin(Gibco™/ThermoFisherScientific)and1MHEPES(Roth,
Karlsruhe,BW,Germany),accordingtothegenotype.Thetissuewasdigestedin0.1%trypsin
inHBSSfor15minat37˚Candtrituratedwithafire-polishedPasteurpipettesubsequentto
DNase(AppliChem,Darmstadt,HE,Germany)treatmenttoobtainasinglecellsolution.The
MNsandglialcellswereseparatedbycentrifugationina6.2%OptiPrepdensitygradientsolu-
tion(Axis-Shield/AlereTechnologiesAS,Oslo,Norway)dilutedinL-15medium(Sigma-
Aldrich).Separatedastrocytes(50,000cells/coverslip)wereseededonpoly-D-lysinehydrobro-
mide(Sigma-Aldrich)-coated12-mmcoverslips(MarienfeldGmbH&Co.KG,Lauda-Ko¨nig-
shofen,BW,Germany)tocreateafeedermonolayer.Duringthefirstweek,theastrocytic
feederlayermediumwascomposedofL-glutamine-freeDMEM/Ham’sF-12medium
(Gibco™/ThermoFisherScientific)ata1:1ratiosupplementedwith1%penicillin-streptomy-
cinand10%fetalcalfserum(PANBiotech,Aidenbach,BY,Germany).Thefetalcalfserum
wasthenreplacedwithequalamountsofhorseserum.Afterconfluencewasreached,astro-
cyticcelldivisionwasblockedbyadditionof5μM1-β-D-Arabinofuranosylcytosine(Calbio-
chem1/MerckChemicals,Darmstadt,HE,Germany),whichwasremovedbywashingafter24
h.MNs(15,000–30,000cells/coverslip)wereseededonthepreparedfeedermonolayerofthe
matchinggenotype.MNsweregrownfor14DIVinL-glutamine-freeNeurobasalmedium
(Gibco™/ThermoFisherScientific)supplementedwith2%50xB271Supplement(Gibco™/
ThermoFisherScientific),0.2%100xN-2Supplement(Gibco™/ThermoFisherScientific),1
mML-glutamine(Gibco™/ThermoFisherScientific),2%horseserum(Gibco™/ThermoFisher
Scientific),1%penicillin-streptomycinand2ng/mlhumanrecombinantBDNF(PeproTech,
RockyHill,NJ,USA).Forallculturingsteps,the5%-CO -humidifiedincubatorwassetat
2
37˚C.
Immunofluorescence
AdultmiceweredeeplyanesthetizedwithvaporizedIsofluraneinaspecializedchamber,trans-
cardiallyperfusedwithice-coldPBSandwithfreshlyprepared0.1Msodiumcacodylatetrihy-
drate(Sigma-Aldrich)buffercontaining4%PFA(pH7.3)andsubsequentlydecapitated.The
cervicalspinalcordsweremicro-dissectedandkeptovernightintheperfusionsolutionat4˚C
forpost-fixation.Theywerethencryoprotectedin15%sucrose(Roth)for48hat4˚Candfro-
zenin2-methylbutane.Tissuesliceswerecut(25μmforcervicalspinalcord;16μmforbrain)
withacryotome(LeicaBiosystems,Wetzlar,HE,Germany),driedat37˚Candsubjectedtoan
antigenretrievalstepbyincubatingthetissueslicesin10mMsodiumcitratedihydrate
(Sigma-Aldrich)buffer(pH9.0)at80˚Cfor30minorincubatingtheMN-enrichedculturesin
1%SDS(Serva)inPBSfor10minsubsequenttocellfixationin4%PFA/PBSfor20min.Non-
specificepitopebindingwasblockedbyincubationin10%NDS(MerckChemicals)orNGS
(Gibco™/ThermoFisherScientific)in3%BSA/PBScontainingpermeabilizing0.3%TritonX-
100foratleast2h.
PLOSONE|https://doi.org/10.1371/journal.pone.0183684 August23,2017 5/22
DNAdamageinthehSOD1G93AmousemodelofALS
Primaryantibodiescomprisedmouseanti-SMI32(1:500intissue,1:1,000inMNculture;
monoclonal;Covance,Princeton,NJ,USA,RRID:AB_2564642)asaMNmarkerandrabbit
anti-βIII-tubulin(1:200intissue,1:250inMNculture;polyclonal;Sigma-Aldrich,RRID:
AB_262133)asageneralneuronalmarker.Apolyclonalrabbitanti-TDP-43wasusedinvivo
andinvitro(1:1,000intissue,1:100inMNculture;polyclonal;AcrisAntibodies,Herford,
NW,Germany,RRID:AB_615042).Rabbitanti-53BP1(1:1,000intissue,1:8,000inMNcul-
ture;polyclonal;Abcam,Cambridge,ENG,UK,RRID:AB_722497)andrabbitanti-γH2AX
(1:500inMNculture;polyclonal;Abcam,RRID:AB_303388)weretakenasindicatorsof
DSBs.Mouseanti-PCNA(1:500intissue,1:100inMNculture;monoclonal;Covance,RRID:
AB_663239)servedasamarkerforcellreplicationandDNArepair.Primaryantibodieswere
dilutedin2%NDSorNGS/PBSin3%BSAcontaining0.3%TritonX-100.Tissuespecimens
andMN-enrichedcellcultureswereincubatedwithprimaryantibodysolutionfor2nightsat
4˚C.AfterwashinginPBS,thesampleswerekeptinthedarkandincubatedintheappropriate
secondaryantibodysolutionfor2h.Afterawashingstep,thecellnucleiwerecounterstained
with40,6-Diamidino-2-phenylindoledihydrochloride(DAPI;Sigma-Aldrich)for5min,
washedagainwithPBSandmountedwithFluoromount-G(SouthernBiotech,Birmingham,
AL,USA).
Foranalyses,thesampleswereimagedasz-stacksusingconfocallaserscanningmicroscopy
(LSM710;Zeiss)with40xand63ximmersionoilobjectivesforthetissuespecimensandthe
MNcultures,respectively.Forthecervicalspinalcordspecimens,thefocuswassetontheven-
tralhorn,whichwasidentifiedaccordingtoitstypicalmorphology.Forimageprocessing,
includingadjustmentsofgammasettings,ZEN2012software(Zeiss)wasused.Z-stackimages
oftheventralhornwereoverlaidas“maximumintensityprojection”.Forquantitativeanalysis
ofnuclearγH2AXfociinculturedastrocytesofthefeederlayer,astrocyteswereselected
accordingtotheirsizeandmorphology,andtheirnucleiwerediscriminatedastheregionof
interest(ROI)bytheDAPIsignal.TheextentoftheγH2AXsignalwithintheROIwascalcu-
latedas‘integrateddensitypernucleus’usingImageJsoftware[23]andispresentedinarbi-
traryunits(AU)±SEM.
Quantitativereal-timePCR(qPCR)
AdultcontrolandhSOD1G93AtransgenicmicewereanesthesizedwithvaporizedIsofluranein
aspecializedchamberandsacrificedbydecapitation.Thespinalcordwasmicro-dissectedand
theextractedtissuesweresnap-frozeninliquidnitrogen.RNAwasisolatedaccordingtostan-
dardprotocolsusingQIAzollysisreagent(Qiagen,Hilden,NW,Germany).TheRNAsolution
wasthentreatedwiththeTURBODNA-free™Kit(Invitrogen™/ThermoFisherScientific)to
removeputativegenomicDNAcontamination.TheRNAconcentrationandpuritywere
determinedbyspectrophotometry(NanoDrop2000c;Peqlab/VWR,Darmstadt,HE,Ger-
many).Meanratiosof2.02±0.002and2.04±0.004forthe260nm/280nmand260nm/230
nmabsorbancevalues,respectively,revealedtheabsenceofproteinandphenolcontamination.
TheabsenceofDNAcontaminationandtheRNAintegrity,assessedbytheRNAIntegrity
Score,weredeterminedusingtheQIAxcelRNAQCKitv2.0(Qiagen)viatheQIAxcel
Advancedsystem(Qiagen).Afterdilutionto100ng/μl,RNAwastranscribedintocDNAuti-
lizingtheRevertAidFirstStrandcDNASynthesisKit(ThermoFisherScientific).
Real-timeqPCRwasperformedwithBrilliantIISYBRGreenQPCRMasterMix(Agilent
Technologies,SantaClara,CA,USA).AfinalcDNAconcentrationof25ngper20μlofsample
volumeincluding500nMspecificprimerswasused.Theprimersequencesweredesignedto
exclusivelyrecognizespecifictargetsequencesusingthePrimer-BLASTOnlinetool[24].The
PLOSONE|https://doi.org/10.1371/journal.pone.0183684 August23,2017 6/22
DNAdamageinthehSOD1G93AmousemodelofALS
primerspecificityandpredictedtargetsizewereconfirmedbycapillaryelectrophoresis,and
thesequenceswereasfollows:
L1 (GenBank:D84391.1;ampliconsize:131bp):
orl
5’-AGTCTGTACCACCTGGGAAC-3’;5’-GGCAAGCTCTCTTACAGGGAA-3’
L1 (GenBank:AF016099.1;ampliconsize:123bp):
spa
5’-ATTGAAGCTCACGGCACATTTC-3’;5’-GATGACCACTTATTCTGCATGTCTT-3’
MusrsL1r(GenBank:J02793.1;ampliconsize:133bp):
5’-CCGCAAGCTGGAAGTTCATTAG-3’;5’-AATGGTGGCAGTAGGAGCACA-3’
Piwil1(NM_021311.3;ampliconsize:93bp):
5’-CACGACGATCAGGGAGTGACC-3’;5’-TTCCAGTCAGCTCAGGTGTTC-3’
ArgonauteRISCcatalyticsubunit2(Ago2)(NM_153178.4;ampliconsize:100bp):
5’-ACGCTCTGTGTCAATACCCG-3’;5’-TCCTTCAGCGCTGTCATGTT-3’
Ki67(NM_001081117.2;ampliconsize:80bp):
5’-TGGTCACCATCAAGCGGAG-3’;5’-AATACTCCTTCCAAACAGGCAG-3’
Glyceraldehyde-3-phosphate dehydrogenase(Gapdh)([25];ampliconsize:164bp):
5‘-GGAAGTGTCAGGGGAGGAGA-3‘;5‘-GGCTACTTGGCGGTGTACAT-3‘.
ForqPCR,thefollowingparameterswereapplied:heatingto95˚C(10min),DNAdenatur-
ationat95˚C(15s),annealingat60˚C(30s)andproductelongationat72˚C(30s),withthe
lastthreestepsbeingrepeatedfor40cycles.Attheterminationofeachrun,ameltcurveanaly-
siswasperformedtoevaluatetheproductspecificity.DatawereobtainedusingRotor-GeneQ
SeriesSoftware2.3.1(Qiagen).Therelativetranscriptfoldchangeswereassessedaccordingto
Pfaffletal.[26]usingthedelta-deltathresholdcycle(ΔΔC).Thevalueswerenormalized
t
againstthehousekeepinggeneGapdh,whichwasco-amplifiedinthesamerun.Biologicalrep-
licateswereassessedastechnicalduplicatesthatincludeda‘no-templatecontrol’ineachrun.
Relativetranscriptfoldchangeswereassessedfromthemeanvalueoftwoduplicates.
Subcellularproteinfractionationandwesternblotanalysis
Adultmiceweresacrificed,theventralcervicalandthoracicspinalcordsweremicro-dissected,
andproteinswereisolatedwithrespecttotheirsubcellularlocalizationusingtheSubcellular
ProteinFractionationKitforTissues(ThermoFisherScientific),accordingtothemanufactur-
er’srecommendations.Theproteinconcentrationsofthedifferentsubcellularfractionswere
analyzedbyBradfordAssayusingQuickStart™BradfordDyeReagent(Bio-Rad,Hercules,CA,
USA).Forthewesternblotanalysis,10μgofadistinctsubcellularproteinfractionwassub-
jectedtostandardSDS-PAGEandblottedontoaHybond-PPVDFmembrane(GEHealth-
care,LittleChalfont,ENG,UK).Toblocknon-specificepitopes,themembranewasincubated
in5%low-fatpowderedmilk(Roth)inTBScontaining0.1%Tween120for1hpriortothe
applicationofprimaryantibodysolution.Thefollowingprimaryantibodieswereused:rabbit
anti-TDP-43(1:2,500;polyclonal;AcrisAntibodies,RRID:AB_615042),rabbitanti-NF-κB
p65(1:1,000;polyclonal;SantaCruzBiotechnology,Dallas,TX,US,RRID:AB_632037)and
mouseanti-NF-κBp65-activesubunit(1:1,000;monoclonal;MerckChemicals,RRID:
AB_2178887).Primaryantibodieswereincubatedin2%low-fatpowderedmilk(Roth)inTBS
PLOSONE|https://doi.org/10.1371/journal.pone.0183684 August23,2017 7/22
DNAdamageinthehSOD1G93AmousemodelofALS
containing0.1%Tween120overnightat4˚C.AfterwashingwithTBScontaining0.1%
Tween120,themembraneswereincubatedwiththeappropriateHRP-conjugatedsecondary
antibodies(1:5,000;SantaCruzBiotechnology).Afterawashingstep,bandswererevealedby
chemiluminescenceusingImmobilonWesternHRPSubstratesolutions(MerckChemicals)
anddocumentedusingtheLAS3000(Fujifilm,Du¨sseldorf,NW,Germany)withthecorre-
spondingsoftware.Thesolubleandchromatin-boundnuclearfractionswerequantifiedusing
ImageJsoftware[23].TDP-43proteinlevelswerenormalizedtop65levels,andallvalueswere
relativizedtomeancontrolvalues.
Statisticalanalyses
Allvaluesareexpressedasthemean±SEM,withtheexceptionofthetranscriptleveldata,
whicharepresentedasthegeometricmean±SEM.Formultiplecomparisonsofnormallydis-
tributeddatasets,one-wayANOVAandtwo-wayANOVAfollowedbytheposthocHolm-
Sˇ´ıda´ktestwereperformedaccordingtotheparametersaddressed.Fornon-parametricsingle
andmultiplecomparisons,theMann-WhitneyUandKruskal-Wallistestswereapplied,
respectively.Apvalueof0.05wasdefinedasthethresholdforsignificance.Statisticswerecon-
ductedwithSigmaPlot13.0softwaresystems(Systat).
Results
ThehSOD1G93Amutationdoesnotincreasespinalcordsusceptibility
towardsDNAstrandbreaks
ToassesstheimpactofthehSOD1G93Amutation,implicatingatoxicgainofSOD1activity,on
DNAintegritybeyondbasepairoxidation,theamountofdeleteriousDNASSBsandDSBs
wasinvestigatedinthespinalcordofcontrolandseverelydiseasedhSOD1G93Amice.Cell
nucleifromventralpartsofthecervicalandthoracicspinalcordweresubjectedtothe‘comet
assay’,inwhichvoltage-basedremovalofnuclearDNAintoaso-called‘comettail’isconsid-
eredtobedirectlyproportionaltotheamountofDNAstrandbreaks.Theextentofnuclear
DNAdistortionwasdeterminedas‘TailMoment’andasthe‘percentofTailDNA’(%
TailDNA)andclassifiedaccordingtotheseverityofDNAdamage.Themean‘TailMoment’
didnotdifferbetweencontrol(9.44±0.98)anddiseased(6.58±0.51)hSOD1G93Atransgenic
mice(Fig1A,leftbars;p=0.429;control:n=282comets,hSOD1G93A:n=425comets,quanti-
fiedfrom3animalspergroup).Similarly,themean%TailDNAremainedlowandaccounted
for13.39±0.9%and11.59±0.57%inthecontrolanddiseasedhSOD1G93Atransgenicgroups,
respectively,therebyexhibitingnoobviousdifferences(Fig1A,rightbars;p=0.778;control:
n=282comets,hSOD1G93A:n=425comets,quantifiedfrom3animalspergroup).Toassess
gradualeffectsonDNAintegrityandexcludeapossiblemaskingeffectarising,e.g.,fromthe
mutualequilibrationofverystrongandweakDNAdamage,the%TailDNAresultswerefur-
therclassifiedintocategoriesrangingfrom0to4accordingtotheseverityofDNAdisintegra-
tion,asrecentlysuggestedbyCollins[20](Fig1B).Bothundercontrolanddiseaseconditions,
cometsdedicatedtocategory0(0–20%TailDNA)representedthelargestfraction,accounting
for79.38±2.31%and81.31±1.01%oftheisolatedcells,respectively(p=0.618).Accordingly,
themeanvaluesof%TailDNAwerealsoallocatedtothiscategory.Ofallcometsanalyzed,
13.38±2.53%inthecontrolspecimensand15.88±1.09%inthehSOD1G93Atransgenicspeci-
menswerefurtherattributedtocategory1ofmildDNAdamageandwerethusnotsignifi-
cantlydifferentfromeachother(p=0.520).Similarly,incategories2and3,whichcollectively
comprisedamarginalportionwithinthecomet-positivecellpopulation(category2:
4.64±0.2%inthecontrolgroupversus2.09±0.28%inthetransgenicgroup,p=0.512;
PLOSONE|https://doi.org/10.1371/journal.pone.0183684 August23,2017 8/22
DNAdamageinthehSOD1G93AmousemodelofALS
Fig1.AssessmentofDNASSBsandDSBsinspinalcordtissueusingthecometassay.(A)Theextent
ofoverallDNAdamage,asexpressedbyTailMoment(leftbars)and%TailDNA(rightbars)incervicaland
thoracicspinalcordtissues,wassimilarbetweencontrol(ctrl)anddiseasedhSOD1G93Atransgenicmice.(B)
Specificationofcometmorphologybyattributiontosubclasses0–4,accordingtoincreasing%TailDNA
values(seeMethodsfordetails),revealedsimilarproportionsofcometsdedicatedtoeachcategorybetween
controlanddiseasedhSOD1G93Atransgenicmice.AdistinctpatternofDNAdamage,however,wasallocated
tocorticalcellsfrommurineNOD.CB17-Prkdcscid/NCrHsdspecimensdisplayingdefectiveDNArepair
(positivecontrol;rightbar),thusconfirmingassaysensitivity.n.s.,notsignificant.
https://doi.org/10.1371/journal.pone.0183684.g001
category3:2.6±0.2%inthecontrolgroupversus0.72±0.18%inthetransgenicgroup,
p=0.627),nodifferencesweredetectedbetweencontrolandALS-likeconditions.Ineither
group,cometsfulfillingcategory4criteria,indicatingthatalmosttheentireDNAcontenthad
shiftedtothecomettail,werenotpresent.Thesuitabilityofthe‘cometassay’tocategorize
DNAdamageinCNStissuewasconfirmedbyco-analysesofcorticalneuralcellsderivedfrom
murineNOD.CB17-Prkdcscid/NCrHsdspecimensexhibitingsevereDNArepairdeficits.The
overallpatternofcometsdedicatedtotheDNAdamagecategoriesinthisreferencetissue
clearlydifferedfromthecontrolandhSOD1G93Atransgenicspecimens(Fig1B,rightbar),as
reflectedbyareducedproportionofcellsthatweredevoidofDNAdamageconcomitantwith
anincreasedportionofcometsassignedtoDNAdamagecategories1–4.Thisfindingdemon-
stratesthatthesensitivityofthe‘cometassay’wassufficienttosubtlydetectanddiscriminate
thedemiseofDNA.
Hence,usingthe‘cometassay,’whichissensitivetobothDNASSBandDSBwhenper-
formedunderalkalineconditions[27],weobtainednoevidenceforincreasedDNAdamage
underconditionsofhSOD1G93A-relatedALS-likepathology.SincethehSOD1G93Amutationis
presentinallsomaticcellsandislikelytoinfluenceeithercelltypeofthenervoussystem,
cometsampleswerenotselectedforneurons.Indeed,althoughMNsarelikelytodisplaythe
strongestvulnerability,ALS-relatedpathomechanismsarenotrestrictedtothiscellentity[28].
DNAdamageresponseisnotactivatedinmotorneuronscarryingthe
hSOD1G93Amutation
SinceitisnotpossibletodiscriminatebetweenSSBsandDSBsinDNAbythecometproce-
dure,atleastinanalkalinemilieu[27],wehypothesizedthatanequalizingshiftinthepropor-
tionofeitherentityofDNAdamagemightmaskDNAinstability.Toexcludethispossibility
andtoexploretheactivationofDDRresponsesunderhSOD1G93A-relatedALSconditions,the
PLOSONE|https://doi.org/10.1371/journal.pone.0183684 August23,2017 9/22
DNAdamageinthehSOD1G93AmousemodelofALS
appearanceofnuclear53BP1-positivefociwasanalyzedbyimmunofluorescenceasafunction
ofdiseaseprogression.The53BP1multi-domainmarkeroperatesasanearlycomponentof
theDDRcascadebycontributingtotheprocessingofandsignalingfromsitesofDNAdamage
[29].Additionally,itservesasarobustmarkerspecificallyforDNADSBs[29].Therefore,
53BP1activationwasassessedinthecervicalspinalcordtissueofcontrol(n=5),presymptom-
atic(n=2)andseverelydiseased(n=4)hSOD1G93Amice,aswellasinanα-MN-enrichedcell
culturesystem(Fig2;n=90cells/preparation,3preparations).Ingeneral,the53BP1foci
remainedassingleeventsbothinvivo(Fig2A–2C’)andinvitro(Fig2Dand2E’),andthey
werenotincreasedinhSOD1G93AMNs.SimilarresultswereobtainedforγH2AX(Fig2F–2I;
n=90cells/preparation,3preparations),anotherwell-establishedmarkerforDNADSBsand
DDRactivationthatco-localizesandinteractswithseveralDDRmediators[30].Again,
γH2AX-positivefociremaineddiscreteinculturedMNsderivedfrombothcontrolandtrans-
genicmice(Fig2Fand2G’).However,inreplication-competentfeederlayerastrocytes,there
wasagreaterabundanceofγH2AX-positivefoci(Fig2Hand2I).Quantificationofnuclear
γH2AXfocibyassessmentoftheintegrateddensityofγH2AX-positivesignalfrequenciesper
nucleus(Fig2J)revealed19.55AU±2.00AUand16.35AU±0.59AUinthecontrol(n=600
cells/preparation,3preparations)andtransgenic(n=600cells/preparation,3preparations)
astrocytecultures,respectively,andwerethussimilaramongthegroups(p=0.216).
Takentogether,wecouldnotidentifyevidenceforalterationsintheratioofSSBs:DSBs(Fig
2)inthehSOD1G93AanimalmodelofALS,eitherunderinvivoorinvitroconditions.More-
over,eventsindicatingtherelocationofDDRproteinssuchas53BP1tonuclearfociandthe
generationofγH2AXpositivenuclearfoci,e.g.,toalignbrokenDNAstrands,werenot
increased.
DNArepairisnotincreasedinthespinalcordofhSOD1G93Amutants
TofurtherexcludethattheproportionbetweenSSBsandDSBsisinfluencedbymolecular
strategiesthatcompensateforDNAdamage,whichareevenbeyondDDRmediatorprocesses,
weanalyzedproliferatingcellnuclearantigen(PCNA)asapotentmarkerforongoingDNA
damagerepair[31,32].PCNAactsasaslidingclamp,whichprovidesaplatformforthe
recruitmentoffactorsnecessaryforDNAreplicationandrepairandisinvolvedinthemedia-
tionofDNAdamagetolerancepathways[32,33].Ingeneral,inimmuno-stainedcells,PCNA
activationisindicatedbytheappearanceofnuclearfoci.Thus,inquiescentcells,asneurons
thataredevoidofSphaseactivity,positivePCNAfociareindicativeofactiveDNArepairpro-
cesses[32].Regardingthisfeature,weanalyzedtheformationofnuclearPCNA-positivefoci
intheventralcervicalhornofcontrol,presymptomaticandseverelydiseasedhSOD1G93Amice.
ThesedatawereassessedtodeterminewhetherDNArepairinthistransgenicstrainwas
alteredgenericallyorwhetheritchangedeitherbeforeoraftertheonsetofsymptoms.Inboth
controltissue(n=6)andcervicalspinalcordscarryingthehSOD1G93Amutation(presymp-
tomatic:n=3;diseased:n=5),non-replicativeneuronsdisplayedanuclearPCNAsignal
devoidofanyfoci,indicatingthattherewerenoactiveDNArepairprocesses(Fig3A–3C’).
Correspondingobservationswereobtainedinvitro(Fig3Dand3E’;n=90cells/preparation,3
preparations).SimilartranscriptlevelsoftheproliferationmarkerKi67undercontrolanddis-
easeconditions(S2Fig)furtherprovedthelackofSphaseinduction.Toconfirmthecapability
ofthePCNAantibodytogeneratefoci-likesignals,eitherinSphaseorasasignofDNArepair,
humanLN229cellline-derivedbrainglioblastomacellsxenograftedintoNOD.
CB17-Prkdcscid/NCrHsdmicewerestainedwithanidenticalantibodydirectedagainst
PCNA(Fig3Fand3F’).Thus,highlyreplicativetumorcellsshowedstrongnuclearPCNA
foci.ConsistentwiththedefectintheDDRapparatusinthismousemodel,cellsintumor-free
PLOSONE|https://doi.org/10.1371/journal.pone.0183684 August23,2017 10/22