Expression of mutant TDP-43 induces neuronal dysfunction in transgenic mice

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Title:
Expression of mutant TDP-43 induces neuronal dysfunction in transgenic mice
Series Title:
Molecular Neurodegeneration
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English
Creator:
Xu, Ya-Fei
Zhang, Yong-Jie
Lin, Wen-Lang
Cao, Xiangkun
Stetler, Caroline
Dickson, Dennis W.
Lewis, Jada
Petrucelli, Leonard
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BioMed Central
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Abstract:
Background: Abnormal distribution, modification and aggregation of transactivation response DNA-binding protein 43 (TDP-43) are the hallmarks of multiple neurodegenerative diseases, especially frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-U) and amyotrophic lateral sclerosis (ALS). Researchers have identified 44 mutations in the TARDBP gene that encode TDP-43 as causative for cases of sporadic and familial ALS http://www.molgen.ua.ac.be/FTDMutations/. Certain mutant forms of TDP-43, such as M337V, are associated with increased low molecular weight (LMW) fragments compared to wild-type (WT) TDP-43 and cause neuronal apoptosis and developmental delay in chick embryos. Such findings support a direct link between altered TDP-43 function and neurodegeneration. Results: To explore the pathogenic properties of the M337V mutation, we generated and characterized two mouse lines expressing human TDP-43 (hTDP-43M337V) carrying this mutation. hTDP-43M337V was expressed primarily in the nuclei of neurons in the brain and spinal cord, and intranuclear and cytoplasmic phosphorylated TDP-43 aggregates were frequently detected. The levels of TDP-43 LMW products of ~25 kDa and ~35 kDa species were also increased in the transgenic mice. Moreover, overexpression of hTDP-43M337V dramatically down regulated the levels of mouse TDP- 43 (mTDP-43) protein and RNA, indicating TDP-43 levels are tightly controlled in mammalian systems. TDP-43M337V mice displayed reactive gliosis, widespread ubiquitination, chromatolysis, gait abnormalities, and early lethality. Abnormal cytoplasmic mitochondrial aggregates and abnormal phosphorylated tau were also detected in the mice. Conclusion: Our novel TDP-43M337V mouse model indicates that overexpression of hTDP-43M337V alone is toxic in vivo. Because overexpression of hTDP-43 in wild-type TDP-43 and TDP-43M337V mouse models produces similar phenotypes, the mechanisms causing pathogenesis in the mutant model remain unknown. However, our results suggest that overexpression of the hTDP-43M337V can cause neuronal dysfunction due to its effect on a number of cell organelles and proteins, such as mitochondria and TDP-43, that are critical for neuronal activity. The mutant model will serve as a valuable tool in the development of future studies designed to uncover pathways associated with TDP-43 neurotoxicity and the precise roles TDP-43 RNA targets play in neurodegeneration. Keywords: aggregation, ALS, mitochondria, mouse model, tau
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Publication of this article was funded in part by the University of Florida Open-Access publishing Fund. In addition, requestors receiving funding through the UFOAP project are expected to submit a post-review, final draft of the article to UF's institutional repository, IR@UF, (www.uflib.ufl.edu/ufir) at the time of funding. The Institutional Repository at the University of Florida (IR@UF) is the digital archive for the intellectual output of the University of Florida community, with research, news, outreach and educational materials

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RESEARCHARTICLE OpenAccessExpressionofmutantTDP-43inducesneuronal dysfunctionintransgenicmiceYa-FeiXu1,Yong-JieZhang1,Wen-LangLin1,XiangkunCao1,CarolineStetler1,DennisWDickson1,JadaLewis1,2,3andLeonardPetrucelli1*AbstractBackground: Abnormaldistribution,modificationandaggregationoftransactivationresponseDNA-binding protein43(TDP-43)arethehallmarksofmultipleneurodegenerativediseases,especiallyfrontotemporallobar degenerationwithubiquitin-positiveinclusions(FTLD-U)andamyotrophiclateralsclerosis(ALS).Researchershave identified44mutationsinthe TARDBP genethatencodeTDP-43ascausativeforcasesofsporadicandfamilialALS http://www.molgen.ua.ac.be/FTDMutations/.CertainmutantformsofTDP-43,suchasM337V,areassociatedwith increasedlowmolecularweight(LMW)fragmentscomparedtowild-type(WT)TDP-43andcauseneuronal apoptosisanddevelopmentaldelayinchickembryos.SuchfindingssupportadirectlinkbetweenalteredTDP-43 functionandneurodegeneration. Results: ToexplorethepathogenicpropertiesoftheM337Vmutation,wegeneratedandcharacterizedtwomouse linesexpressinghumanTDP-43(hTDP-43M337V)carryingthismutation.hTDP-43M337Vwasexpressedprimarilyinthe nucleiofneuronsinthebrainandspinalcord,andintranuclearandcytoplasmicphosphorylatedTDP-43aggregates werefrequentlydetected.ThelevelsofTDP-43LMWproductsof~25kDaand~35kDaspecieswerealsoincreasedin thetransgenicmice.Moreover,overexpressionofhTDP-43M337VdramaticallydownregulatedthelevelsofmouseTDP43(mTDP-43)proteinandRNA,indicatingTDP-43levelsaretightlycontrolledinmammaliansystems.TDP-43M337Vmice displayedreactivegliosis,widespreadubiquitination,chromatolysis,gaitabnormalities,andearlylethality.Abnormal cytoplasmicmitochondrialaggregatesandabnormalphosphorylatedtauwerealsodetectedinthemice. Conclusion: OurnovelTDP-43M337VmousemodelindicatesthatoverexpressionofhTDP-43M337Valoneistoxic in vivo .BecauseoverexpressionofhTDP-43inwild-typeTDP-43andTDP-43M337Vmousemodelsproducessimilar phenotypes,themechanismscausingpathogenesisinthemutantmodelremainunknown.However,ourresults suggestthatoverexpressionofthehTDP-43M337Vcancauseneuronaldysfunctionduetoitseffectonanumberof cellorganellesandproteins,suchasmitochondriaandTDP-43,thatarecriticalforneuronalactivity.Themutant modelwillserveasavaluabletoolinthedevelopmentoffuturestudiesdesignedtouncoverpathwaysassociated withTDP-43neurotoxicityandthepreciserolesTDP-43RNAtargetsplayinneurodegeneration. Keywords: aggregation,ALS,mitochondria,mousemodel,tauBackgroundTDP-43isthemajorcomponentofubiquitinatedinclusionsinmostcasesofALSandFTLD-U[1,2],andthe linkbetweenTDP-43mutationsandneurodegeneration wasfirstestablishedin2008[3,4].Autosomaldominant mutationsin TARDBP ,thegeneencodingTDP-43,are associatedwithsporadicandfamilialALS[3-7].TDP-43 isaubiquitouslyexpressed414-aminoacidnuclearproteinandahighlyconservedheterogeneousnuclearribonucleoprotein(hnRNP).TDP-43hashigh-binding affinityforthe(TG)nmotifandisinvolvedingenetranscription,pre-mRNAsplicing,mRNAstability,and mRNAtransport[8,9].Underdiseaseconditions,TDP43istruncated,phosphorylated,ubiquitinatedand aggregatedbothinthenucleusandcytoplasm.Under suchconditions,cytoplasmicTDP-43aggregation *Correspondence:petrucelli.leonard@mayo.edu1DepartmentofNeuroscience,MayoClinic,(4500SanPabloRoad), Jacksonville,(32224),USA FulllistofauthorinformationisavailableattheendofthearticleXu etal MolecularNeurodegeneration 2011, 6 :73 http://www.molecularneurodegeneration.com/content/6/1/73 2011Xuetal;licenseeBioMedCentralLtd.ThisisanOpenAccessarticledistributedunderthetermsoftheCreativeCommons AttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse,distribution,andreproductionin anymedium,providedtheoriginalworkisproperlycited.

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coincideswiththedepletionofnuclearTDP-43.The mannerthroughwhichTDP-43causesneurodegenerationhasnotbeenidentified;however,recentlyTDP-43 mousemodelsgeneratedbyourgroupandothers demonstratethatoverexpressionofTDP-43[either wild-type,A315Tmutant,G348Cor NLS(defective nuclearlocalizationsignalTDP-43)]istoxicandcan causeneurodegenerationinthecentralnervoussystem [10-16].Thatbeingsaid,thevariousTDP-43transgenic modelsexhibitothersimilaritiesaswellasdifferences. Forinstance,mosttransgenicmodelsshowedincreased ubiquitinlevels,TDP-43fragmentation,phosphorylation, gliosis,motorfunctionalimpairments,andshortened lifespan.Ontheotherhand,neuronalloss,caspaseactivation,redistributionofTDP-43fromnucleitocytoplasm,cytoplasmicTDP-43inclusions,downregulation ofendogenousmTDP-43andabnormalmitochondrial aggregationwereeitheronlyseeninsomeofthose TDP-43transgenicmice,ornotexamined.Tofurther confirmthetoxiceffectofTD P-43overexpressionand tospecificallystudymutantTDP-43,wegenerated transgenicmiceexpressinghTDP-43M337Vundercontrol ofthemouseprion(PrP)promoter[17]. IntheTDP-43M337Vmice,hTDP-43M337Vismainly expressedinthebrainandspinalcord,afindingthatis consistentwithtransgenicmiceoverexpressingwildtypeTDP-43thatwepreviouslyreported[11].TDP43M337Vmiceexhibitedcertainfeaturessimilartothose seeninALS,suchasTDP-43cleavage,phosphorylation, aggregation,increasedubiquitination,gliosis,gaitdisturbances,andearlylethality;however,themicealsoexhibitedotherfeaturesnotyetreportedinhumans,which maybeduetotheeffectofhTDP-43M337Voverexpression.Suchfeaturesinclude:downregulationofmTDP43,abnormalmitochondrialaggregationandabnormal tauphosphorylation.ResultsGenerationoftransgenicmiceoverexpressinghTDP-43M337VproteinToexplorethepathogenicpropertiesofmutant (M337V)TDP-43,wegeneratedtransgenicmiceusing themouseprionpromotertoconstitutivelydrive expressionoffull-lengthhTDP-43carryingtheM337V mutation.Three(Lines1,4,and6)ofeightindependent founderlinesshowedgermlinetransmission.The expressionlevelofhumanTDP-43waslowinline1 (~20%ofendogenouslevels),whilelines4andline6 hadsimilarhTDP-43expressionlevelstothatofwildtypeTDP-43mice(TDP-43WTline3chereaftertermed TDP-43WT),whichwepreviouslygeneratedwiththe samepromoter[11](Figure1A,B).Biochemicalanalyses ofhTDP-43M337Vshowedthatproteinexpressionwas highestinthebrainandspinalcord,withlowlevelsin othertissues(Figure1C).Theimmunohistochemistry (IHC)ofhemizygousmiceshowedthathTDP-43M337Vexpressionwasprimarilyinnucleianddistributed throughoutthegraymatterofthespinalcordandbrain (Figure1D,E).Hemizygou smicefromalllineswere phenotypically,histologica lly,andimmunohistochemicallyindistinguishablefromNTmiceupto12months ofage,currentlytheoldestageavailable.Giventhesimilarlevelsofexpressionbetweenlines4and6,thefull characterizationofline4isshownandallsubsequent descriptionofTDP-43M337Vmicerefertohomozygous micefromline4unlessotherwisenoted.Theexpression levelofhTDP-43M337Vinthebrainsofhomozygous micewasabout1.90.06foldoverthatofhemizygous mice(Figure2A,B).ThetotallevelsofTDP-43protein (humanandmouseTDP-43)inthebrainsofhemizygousandhomozygousmicewere1.60.03and2.7 0.08foldthatofendogenousmouseTDP-43inthenontransgenicmice(NT),respectively(Figure2A,C).These resultsobtainedfromhTDP-43M337Vcomparedwith totalTDP-43indicatedthatendogenousmTDP-43proteinlevelswerelikelydownregulatedinTDP-43M337Vmice,inadose-dependentfashion.Weconfirmedthat mTDP-43wassimilarlydownregulatedatthemRNA level(~20%and30%reductionofmTDP-43,respectively,comparedtoNTmice)usingrealtime-PCRanalysesofhemizygousandhomozygousbrains(Figure 2D).ThemRNAlevelsofhTDP-43inthebrainof homozygousmicewere1.860.14foldofthatinthe hemizygousmice(Figure2E),consistentwiththe hTDP-43proteinlevels.Moreover,the~25kDaand ~35kDaTDP-43LMWspeciesalsoincreasedsignificantlyinthehemizygousandhomozygousmice,and theirlevelscorrelatedwiththelevelsoffull-lengthTDP43(Figure2F,G). Atapproximatelypost-natalday21,homozygous TDP-43M337Vmicebegantohavebodytremorsanddifficultyrecruitingtheirhindlimbs(notshown).They failedtoshowproperescapeextensionbysplayingtheir hindlimbsuponelevation,asshownbytheirNTcounterparts(Figure3A,B).Theydisplayedanirregular, dragginggaitpattern,dueatleastinparttolimbweakness(Figure3C,D).By~1monthold,homozygous TDP-43M337Vmicehadsignificantlylowerbrainand bodyweightcomparedwiththeirNTandhemizygous littermates(Figure3E,F).Dueinparttomuscleweakness,homozygousTDP-43M337Vmicewereunableto feedfromafoodhopper,losttheabilitytorightthemselves,becamemoribundandrequiredeuthanasia. Approximately70%ofthehomozygousTDP-43M337Vmicebecamemoribundby1monthofage,whichwas statisticallysignificantcomparedwithNTandhemizygouslittermates(Figure3G) .Wehadprevioussuccess (unpublished)extendingthelifespanofmousemodelsXu etal MolecularNeurodegeneration 2011, 6 :73 http://www.molecularneurodegeneration.com/content/6/1/73 Page2of14

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thatshowedearlylethalityt hroughtheuseofintensive caresuchasdelayedweaningandsupplementingdiets withgelanddough.Wehavenowemployedsimilar measuresinanefforttoprolongthelivesofhomozygousTDP-43M337Vtransgenicmice.Thephenotypeof thehomozygousTDP-43M337Vmicefromline6(not shown)wassimilartothatdescribedforline4.PathologicalalterationofTDP-43Immunohistochemistrywasperformedonbothline4 andline6ofTDP-43M337VandNTmice.Theresults showedthatTDP-43wasmostlyinnucleiinthebrain andspinalcordofTDP-43M337VandNTmiceandthat theTDP-43M337Vmicespecificallyexpressedhighlevels ofhTDP-43thatwerenotseeninthecontrols(Figure 4A-C).CytoplasmicTDP-43wasdetectedwitheithera humanTDP-43specificantibodyortotalTDP-43 antibodythatrecognizedbothhumanandmouseTDP43.CytoplasmicTDP-43wasdetectedinspinalcord neuronsandlessfrequentlyinthebrainstemandcortex ofTDP-43M337Vmice(Figure4A,B,Dand4Earrowhead,Table1).Abnormallyphosphorylated(pS403/ pS404)TDP-43(pTDP-43)wasfrequentlylocated withinnuclearbodiesordif fuselywithinthecytoplasm ofmotorneuronsintheanteriorhornsofthespinal cordofTDP-43M337Vmice(Figure4G,Harrowheads, arrows)butnotintheNTmice(Figure4I).Farless cytoplasmicpTDP-43wasdetectedintheposterior hornofTDP-43M337Vmiceandinthebrainstem(<5 neurons/section;Table1).pTDP-43-immunoreactive nuclearbodieswerenotdetectedinthebrainsofTDP43M337Vmice(Table1).Multiplesmall,distinct,cytoplasmicinclusionsthatwereimmunoreactiveforpTDP43werefrequentlyobservedwithinneuronsinlayerV Figure1 TDP-43expressioninTDP-43M337Vmice .(A)Westernblotsofbrainlysatesfromnon-transgenic(NT)mice,founderline1,4,6of TDP-43M337Vhemizygousmiceandline3cofTDP-43WThemizygousmiceshowthatTDP-43M337V(lines4,6)andTDP-43WTmiceareexpression matchedforbothtotal(mTDP-43andhTDP-43)andhTDP-43levels,whichwassubsequentlyconfirmed(B)bydensitometricmeasurements. ComparedwithNTmice(10.04),thelevelsoftotalTDP-43inlines1,4,6and3care1.20.05;1.60.07;1.30.17;1.90.22fold respectively.Comparedtoline1(10.06),thelevelsofhTDP-43ofline4,6and3care8.31.2;7.71.0and8.31.3fold,respectively.Data shownin(B)aremeansSEMofsixdifferentmiceofeachgenotype.(C)WesternblotoftissuelysatesofTDP-43M337Vmicefrombrain(Br), spinalcord(SC),thighmuscle(TM),heart(He),stomach(St),kidney(Ki),live(Li)andspleen(Sp)probedwithanantibodyagainsthTDP-43 revealshighexpressioninthebrainandspinalcord(line4).(D-E)ImmunohistochemistryshowshTDP-43distributedthroughoutthegraymatter ofthespinalcord(A)andbrain(B)inthehemizygousTDP-43M337Vmice. Xu etal MolecularNeurodegeneration 2011, 6 :73 http://www.molecularneurodegeneration.com/content/6/1/73 Page3of14

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ofthecortexofTDP-43M337Vmice(Figure4J,K, arrows)thatwerenotobserv edinNTcontrols(Figure 4L).Theextentanddistributionofthesefeaturesinthe TDP-43M337Vmicewassimilartothatobservedinour previouslyreportedTDP-43WTmice[11].Cytoplasmiceosinophilicaggregates,ubiquitination,and gliosisinTDP-43M337VmiceAstrikinghistologicalfeatureinTDP-43M337Vmicewas thepresenceofcytoplasmiceosinophilicaggregates(Figure5A)locatedwithintheneuronsoftheanteriorhorn ofthespinalcordandlessfrequentlyintheposterior hornandbrainstem.Theseaggregateswereabsentfrom NTmice(Figure5B).Neuronallossduetoapoptosis wasnotdetectedinTDP-43M337Vmice,asassessedby TUNELstainingandstainingforactivatedcaspase3 (datanotshown);however,increasedubiquitinationand reactivegliosiswereobservedinTDP-43M337Vmiceand notinNTmice(Figure5C-J).Ubiquitinationwasboth widespread,andgenerallyincreased,inboththenucleus andcytoplasmofneuronsinspinalcordandbrain(Figure5C,E).Ubiquitinationwasmorewidespreadthan cytoplasmicTDP-43orphospho-TDP-43aggregates (Table1),andco-immunoprecipitationstudiesindicated thathTDP-43wasnotubiquitinated(Additionalfile1). ReactivegliosiswasalsoseenintheTDP-43M337Vmice, asGFAP-positiveastrocytes andIBA-1-positivemicrogliawereelevatedinthespinalcordandbrainstemof TDP-43M337VmicecomparedwithNTmice(Figure5GJ;Table1).EachofthesefeaturesobservedintheTDP43M337VmicewassimilartothatobservedinourpreviouslyreportedTDP-43WTmodel[11].Homozygous TDP-43M337Vmicefromline4and6(notshown) showedsimilarpathologicalchanges.AbnormalmitochondrialaggregatesinTDP-43M337VmiceTheeosinophilicaggregates inspinalmotorneuronsof TDP-43M337Vmicewereimmunoreactiveforthemitochondrialmarker,COX-IV(Figure6A),whichindicates theaggregateswerecomposedofabnormalclustersof Figure2 DownregulationofmouseTDP-43andproductionoflowermolecularweight(LMW)TDP-43speciesinTDP-43M337Vmice Westernblots(A)ofbrainlysatesfromNT,hemizygous(Hemi)andhomozygous(Homo)TDP-43M337VmiceprobedforhTDP-43ortotalTDP-43 levelswerequantifiedby(B)densitometrytoshowthatmutanthTDP-43expressionisapproximately90%higherinhomozygousmice comparedtohemizygousmice(Hemi=10.04;Homo=1.90.06),**p<0.001.Densitometricanalysis(C)showsthattotalTDP-43levelsin eachgenotypegroup(NT=10.05;Hemi=1.60.03;Homo=2.70.08).**p<0.001.DatashownaremeansSEMofsixdifferentmice ofeachgenotype.(D)QuantitativerealtimePCR(qPCR)usingmTDP-43-specificprimersdemonstratethemRNAlevelsofmTDP-43inNT, hemizygousandhomozygousmicebrain(NT=10.07;Hemi=0.80.05;Homo=0.70.01).*p<0.05,**p<0.01.(E)qPCRusinghTDP-43 -specificprimersshowsthemRNAlevelsofhTDP-43inhemizygousandhomozygousmicebrain(Hemi=10.09;Homo=1.860.14).**p< 0.01.Datashownin(D)and(E)aremeansSEMoffourdifferentmiceofeachgenotype.(F)DarkerexposureoftheWesternblotshownin(A) revealedincreasedLMWspeciesofTDP-43inhemi-andhomozygousTDP-43M337Vmice.(G)Densitometricanalysisof~25kDaand~35kDa TDP-43speciesindicatesthatbothspeciescorrelatewithtotalTDP-43expression(For~25kDaspecies,NT=10.14;Hemi=1.40.21;Homo =3.40.26.For~35kDaspecies,NT=10.32;Hemi=1.60.28;Homo=2.40.13).**p<0.01.DatashownaremeansSEMofsix differentmiceofeachgenotype. Xu etal MolecularNeurodegeneration 2011, 6 :73 http://www.molecularneurodegeneration.com/content/6/1/73 Page4of14

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mitochondria.NTmice(Figure6B)didnotshowaltered redistributionofCOX-IVimmunostainingintothejuxtanuclearpatternobservedintheTDP-43M337Vmice. Electronmicroscopic(EM)resultsconfirmedthatthe majorityofneuronalcytoplasmicinclusionsinthespinal motorneuronsofTDP-43M337Vmicecontainedaggregatesofmitochondria(Figure6C)thatwereoccasionally surroundedbyacoreofmicrotubules(20nm)inrandomorientations(Figure6D).Theaggregatedmitochondriashowedvariabledegreesoflossofinnercristae Figure3 ReducedbrainandbodyweightandmotordysfunctioninTDP-43M337Vmice .(A-B)Upontailelevation,NTmice(A)showednormal escaperesponsebysplayingtheirhindlimbswhilehomozygousTDP-43M337Vmice(B)heldtheirhindlimbsclosetotheirbodyandfailedtoshow properescapeextension.(C-D)GaitofNTandTDP-43M337Vmicewasevaluatedbyinkedfootplacementonpaper.1monthold(C)NTmiceshow normalfootplacementandgait;whereas,(D)homozygousTDP-43M337Vmiceshowedanirregular,inwardly-placedfootfallswithadragging pattern.Forepawsandhindpawswerecoatedinredandblueink,respectively,toevaluateplacementofpawsduringtravel.(E)At1month,brain weightand(F)bodyweightofhomozygousTDP-43M337Vmicewassignificantlylowerthanthatofage-matchedNTandhemizygousmice.Brain weight:NT=4289mg;Hemi=42911mg;Homo=33829mg.Bodyweight:NT=12.31g;Hemi=122g;Homo=62g.Data shownarethemeansSEMof6-8micepergroup.***p<0.0001.(G)HemizygousTDP-43M337Vmiceweremated,andthesurvivaloftheresulting pupsofeachgenotypewasdetermined.Theresultsareplottedasapercentageofpupsaliveperpostnataldayoflife.Survivalrateforallcohorts werecalculatedusingKaplan-Meiermethods(p<0.001foroveralllog-ranktest).HomozygousTDP-43M337Vmicehadhighermortality(about70% death)around1monthofage,whichwasstatisticallysignificantcomparedwithNTandhemizygouslittermates(p<0.001). Xu etal MolecularNeurodegeneration 2011, 6 :73 http://www.molecularneurodegeneration.com/content/6/1/73 Page5of14

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Figure4 DistributionofTDP-43andphosphorylatedTDP-43(pTDP-43)aggregatesinTDP-43M337Vmice .Immunostaininginspinalcord sectionsofa1-montholdTDP-43M337Vfrom(A)line4,(B)line6and(C)NTmiceshowshTDP-43innuclei,withoccasionalcytoplasmicstaining (arrowhead);hTDP-43wasnotobservedinNTmice.(D-F)ImmunostainingofspinalcordsectionsfortotalTDP-43showsthatTDP-43 immunoreactivityinbothnucleiandcytoplasminTDP-43M337Vmice(arrowhead)(D-E),butonlyinnucleiinNTmice(F).Immunostainingof spinalcord(G-I)orcortical(J-L)neuronsforphosphorylated(p403/404)TDP-43.(G-H)Nuclearbodies(arrowheads)withinspinalmotorneurons ofTDP-43M337VmiceimmunostainedforpTDP-43.Occasionally,diffusecytoplasmicpTDP-43wasfoundwithinthemotorneuronsintheanterior hornofTDP-43M337Vmice(arrows).(I)NTmicelackedsimilarpTDP-43staininginthespinalcord.(J-K)CytoplasmicaggregatesofpTDP-43 (arrows)wereoftenseenincorticalneuronsofTDP-43M337Vmicethatwereabsentfrom(L)NTmice.Scalebars:50 M. Table1RegionaldistributionofpathologiesinTDP-43M337VmiceCortex,layer V HippocampusStriatumBrainstemCerebellumAnteriorhornofspinal cord Posteriorhornofspinal cord CytoplasmicTDP-43+--+-+++ NuclearpTDP-43---+/--+++ CytoplasmicpTDP-43++--+-+++ Ubiquitination++++++-++++ IBA-1-positive microglia +--++-+++ GFAP-positive astrocytes +--++-+++ Mitochondrial aggregates +--+-+++ p-tau++++++-+/-+/Xu etal MolecularNeurodegeneration 2011, 6 :73 http://www.molecularneurodegeneration.com/content/6/1/73 Page6of14

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andvacuolization(Figure6E).ThedegeneratingmitochondriainTDP-43M337Vmice(Figure6E)weresmaller thanthoseinNTmice(Figure6F).InNTmice,mitochondriawereusuallyadmi xedwithothercytoplasmic organellessuchasendoplasmicreticulumandribosome (Figure6F);however,thisinterveningorganelleswere absentintheneuronalaggregatesofTDP-43M337Vmice (Figure6E).ThemitochondrialaggregatesintheTDP43M337Vmicewerefrequentinthespinalcordand brainstem,lessfrequentinthecortex,andrareinother brainregions(Table1).HomozygousTDP-43M337Vmicefromline6alsodevelopedjuxtanuclearmitochondrialaggregates(notshown).M337VTDP-43doesnotaltermitochondrialfusionand fissionproteinsWehavepreviouslydemonstratedthatmitochondrial clusteringinneuronsofTDP-43WTmiceisaccompaniedbychangesinproteinlevelsand/orphosphorylationofproteinsthatregulatemitochondrialfissionand fusion[11].Giventhesimilaritybetweenmitochondrial aggregatesinTDP-43M337Vmiceandourpreviously describedTDPWTmice,wesoughttodetermineifmitochondrialfissionandfusionproteinswerealteredinthe TDP-43M337Vmice.Surprisingly,thealterationsto pDLP1(Ser616),Fis1,andMFN1thatwereobservedin theTDP-43WTmice,regardlessofline,werenotpresent intheTDP-43M337Vmice,despitethecomparablemitochondrialaggregationinea chtransgenicmodel(Additionalfile2).ChromatolysisinTDP-43M337VmiceInthespinalmotorneuronofTDP-43M337Vmice,Nissl bodiesbecamedispersed(Figure7A)comparedtoNT mice(Figure7B).Therewasdisintegrationofchromophil substanceprimarilywithinthecellbodiesofthespinal cordsofTDP-43M337Vmice(Figure7C)thatwasnot observedinNTmice(Figure7D).Ultrastructuralanalysis showedreducedcytoplasmicdensityandlesscytoplasmic organellesinTDP-43M337Vmice(Figure7E)comparedto NTmice(Figure7F).ThesefindingareindicativeofchromatolysisandsuggestthatneuronsinTDP-43M337Vmice areundergoingperikaryalresponsetoaxonaldegeneration.HyperphosphorylatedtauaccumulationinTDP-43M337VandTDP-43WTmiceAbnormal,phosphorylatedtauwasfoundinthebrains ofbothTDP-43WTandTDP-43M337Vmice.ImmunohistochemicalanalysesofthecorticesofTDP-43M337V(Figure8A)andTDP-43WTmice(Figure8B)showed significantlyelevatedphosphorylatedtau(CP13)immunoreactivitythroughouttheneuropilofthebraincomparedwithNTmice(Figure8C).Additionally, cytoplasmictauaccumulationswerefoundintheTDP43WTmiceandmuchlessfrequentlyintheTDP43M337Vmice(Figure8A,B).Westernblotsofbrain lysatesshowedthatthelevelsofphosphorylatedtau (CP13)weresignificantlyincreasedinboththeTDP43WTandTDP-43M337Vmice(Figure8D),whilethe levelofdephosphorylatedtau(Tau-1)wasdramatically decreased.Therewasnosignificantchangeinthelevel Figure5 NeuropathologyinTDP-43M337Vmice .Hematoxylinandeosinstainingrevealed(A,arrows)eosinophilicaggregatesinspinalmotor neuronsfromTDP-43M337Vmicethatarenotobservedin(B)NTmice.Abnormalubiquitinimmunoreactivitywaspresentinthecytoplasmand nucleusofneuronsinthe(C)spinalcordofTDP-43M337Vmice,butnotin(D)NTmice.Similarubiquitinationwasobservedinthe(E)cortexof TDP-43M337Vmice,butnotin(F)NTmice.Enhanced(G-H)GFAPand(I-J)IBA-1immunoreactivityindicativeofreactiveastrogliosisandactivated microglia,respectively,wereobservedinTDP-43M337Vmice(G,I),butnotNTmice(H,J).Scalebars:100 M. Xu etal MolecularNeurodegeneration 2011, 6 :73 http://www.molecularneurodegeneration.com/content/6/1/73 Page7of14

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oftotaltau(Tau-5)(Figure8D).Toexplorethe mechanismfortauphosphorylation,weexaminedproteinkinasesandphosphatasesandfoundsignificant increasesinphospho-(Ser)PKCsubstrateinbothTDP43WTandTDP-43M337Vmice,indicatingPKCactivation mayberesponsibleforsuchabnormaltauphosphorylation(Figure8D).Thelevelsofothertaukinases,suchas GSK-3 b andCDK5,andthetauphosphatasePP2Awere notsignificantlychangedintheTDP-43M337VandTDP43WTmice(datanotshown).Abnormallyphosphorylatedtauwasrarelyfoundinthespinalcord(datanot shown).TheregionaldistributionofhyperphosphorylatedtauissummarizedinTable1.DiscussionInthecurrentstudy,wegeneratedandcharacterizeda noveltransgenicmousemodelthatoverexpresses hTDP-43carryingtheM337Vmutationunderthe mouseprionpromoter.HomozygousTDP-43M337V mice(referredtoasTDP-43M337Vmice)developphenotypicandpathologicfeaturesincludinggaitdisturbances, gliosis,increasedubiquitination,TDP-43truncation, phosphorylation,andbot hnuclearandcytoplasmic phosphorylatedinclusions. Importantly,thenovelfindingsdescribedinthecurrentmanuscriptandthecounterpartTDP-43WTmice manuscript[11],demonst rateexpressionofhuman TDP-43atsimilarlevelsresultsinremarkablysimilar phenotypesandpathologies,suggestingthatthephenotypesofbothmodelsareduetoTDP-43overexpression, andnotspecificallyduetothismutation.Previously publishedfindingsregardingTDP-43overexpressionin mousemodelsstronglysuggestedthatoverexpressionof TDP-43itselfistoxic;however,noneofthesestudies Figure6 AbnormalmitochondrialaggregationsinTDP-43M337Vmice .COX-IVimmunoreactivityillustratesdenselystainedaggregatesina spinalmotorneuronof(A)TDP-43M337Vmice,butnotin(B)NTmice.(C)ElectronmicrographofaspinalmotorneuronfromaTDP-43M337Vmouseshowsaclusterofmitochondriasurroundingafilamentouscore(boxedarea).(D)Enlargementofthefilamentouscoreshowing20nm filamentsandvariousvesicles.Smalldensegranulesarestainprecipitates.(E)Electronmicrographofamitochondrialaggregateinanotherspinal motorneuronoftheTDP-43M337Vmouse.Notethecloseproximityofmitochondriawithvaryingdegreesofdegeneratinginnercristae (arrowheads),comparedtonormalmitochondriainmotorneuronsof(F)NTmice.(E)ThedegeneratingmitochondriaofTDP-43M337Vmicealso appearsmallerinsizecomparedwiththoseof(F)NTmice.Asteriskin(E)indicatesavacuolatedmitochondrion.(F)InNTmice,mitochondria (arrows)wereusuallyseparatedbyothercytoplasmicorganelles,e.g.roughendoplasmicreticulum(arrowheads)andribosomes.Scalebars:50 MinAandB. Xu etal MolecularNeurodegeneration 2011, 6 :73 http://www.molecularneurodegeneration.com/content/6/1/73 Page8of14

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Figure7 ChromatolysisinTDP-43M337Vmice .Nisslstainingofmotorneuronsof(A)TDP-43M337Vmiceismuchweakerthanthatin(B)NT mice.(C)ElectronmicrographofachromatolyticneuronofTDP-43M337Vmicecomparedtothe(D)normalneuronoftheNTmouseshows rarefactionofcytoplasmicorganelles(E),comparedwiththenormalcytoplasmicdensity(D)andpackedorganelles(F)ofNTmice.Scalebars:20 MinAandB;5 MinCandD;and1 MinEandF. Xu etal MolecularNeurodegeneration 2011, 6 :73 http://www.molecularneurodegeneration.com/content/6/1/73 Page9of14

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haveincludedmutantTDP43linesthatwerecomparableinexpressionlevelandpattern,promotersystem, andstraintoWTTDP-43counterparts[10-16].Inconstitutivetransgenicratst udies,Zhouandcolleagues reportedthatmutantM337VTDP-43appearedtobe moretoxicthanwild-typeTDP-43[18]eventhoughthe animalsexpressedthehTDP-43proteinatsimilarlevels. Itisunclearwhythereisadiscrepancybetweenour mutantandwild-typeTDP-43transgenicmodelsand thoseintherat.Onepossibilitycouldbethedifference ofpromotersutilizedinthes tudies.Additionally,Zhou andcolleaguesdidnotlookatendogenousratTDP-43 levelsinresponsetothehTDP-43overexpression[18]; therefore,itispossiblethatdifferencesinendogenous TDP-43playedaroleintheratphenotype.Moreover, incontrasttoourhTDP-43cDNAconstruct,theconstitutivehTDP-43constructutilizedbyZhouandcolleaguesisahTDP-43minigene[18],whichappearsto contain3 UTRandbindingregionsthathavebeen shownrecentlytoberequir edforautoregulationof TDP-43[19,20].Giventhis,itispossiblethatthehTDP43intheconstitutiveratmo delisalsoautoregulated andthatthepresenceofthemutationinthehTDP-43 minigenepreventsautoregulationofthemutant,butnot thewild-type,hTDP-43.Finally,intheTDP-43ratmodels,thereappearstobeextensivehTDP-43 Figure8 TaupathologyinTDP-43M337Vmice .Immunohistochemistryofthecorticesof(A)TDP-43M337Vand(B)TDP-43WTmiceshowed elevatedlevelsofphosphorylatedtau(CP13)inbothTDP-43M337VandTDP-43WTmicecomparedto(C)NTmice.(D)Westernblottingofbrain lysatesfromNT,TDP-43M337VandTDP-43WTmiceprobedwithCP13antibodyshowedincreasesinmurinetauphosphorylationatserine residues202inbothlinesofTDP-43transgenicmice.Stainingwithtau-1antibodyshowedreduceddephosphorylatedtaulevelsinbothlinesof TDP-43transgenicmicewhencomparedtoNTmice.Stainingwithantibodytau-5showedthatoveralltaulevelswereequivalentacrossnontransgenicandtransgenicmice.Therewasadramaticincreaseofphospho-(Ser)PKCsubstrateinbothTDP-43transgeniclinesindicatingPKC activationthatwasnotpresentinNTmice.Scalebars:50 MinA,BandC. Xu etal MolecularNeurodegeneration 2011, 6 :73 http://www.molecularneurodegeneration.com/content/6/1/73 Page10of14

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immunoreactivityinboththenucleusandthecytoplasm,regardlessofmutation;whereas,TDP-43mainly localizedinthenucleiinbothofourTDP-43M337Vand TDP-43WTmice.Thetoxicitythatweobserveinour TDP-43transgenicmiceisthereforelikelytobedue,at leastinpart,totheimpactofhighTDP-43levelson nuclearfunctions.OverexpressionofTDP-43inthe nucleiofTDP-43micemayinterferewithinteraction withitsDNAandRNAtargets,disruptnormalsubstrate metabolismandleadtoneuronaldysfunction.Recent effortstoidentifyneuronalRNAtargetsofTDP-43 implicatedinneurodegenerationledtotheidentification ofFUS/TLS,progranulin,tauandataxin1and-2and TDP-43itself[20-22].Additionalstudiesareneededto explorethepreciserolesofTDP-43DNA/RNAtargets atworkinthesemousemodels.BothTDP-43LMW speciesandcytoplasmicTDP-43arepresentinour TDP-43mice,andtheymayalsocontributetothe pathologiesandabnormalbehavior. OurresultsshowedthatoverexpressionofhTDP43M337VcandownregulateendogenousmouseTDP-43 levels,whichisconsistentwiththepropertyofTDP-43 autoregulation.TDP-43c anautoregulateitsmRNA level,inpartbydirectlybindingtothe3 UTRofitsown transcript,therebytriggeringexosome-mediateddegradationornonsense-mediatedRNAdegradation[19,20]. TheC-terminalregion(a.a321-366)ofTDP-43is requiredforautoregulation[19].Recentreportsallow forthepossibilitythatmutationofTDP-43withinthe C-terminusmayaffecttheefficiencyofautoregulation; however,thepresenceoftheM337Vmutantwithin hTDP-43didnotimpairitsabilitytoregulateendogenousmTDP-43.ThelossofnuclearmTDP-43in responsetohTDP-43M337Voverexpressionhasbeen proposedtoplayaroleinthepathogenesisobservedin anotherTDP-43transgenicmousemodel[14].ConsideringthesubstantialhomologybetweenthehTDP43M337VandmTDP-43proteins,itwouldseemlikely thathTDP-43M337Vmightfunctionallycompensatefor thelossofmTDP-43,butwecannotexcludethepossibilitythatlossofnuclearmTDP-43maycontributeto thephenotypeofhTDP-43M337Vtransgenicmice. Abnormalmitochondrialaccumulationhasnowbeen observedinbothourTDP-43M337VandTDP-43WTmice [11]andinanotherwild-typeTDP-43overexpressing mousemodeldrivenbyThy1.2 promoter[13],suggestingthatTDP-43canregulatemitochondrialdynamics. Inadditiontoclusteringofmitochondria,thesizeand theultrastructuralintegrityofneuronalmitochondria wasreducedinourTDP-43micecomparedwithmitochondriaofNTmice.IntheTDP-43WTmice,wehad previouslyidentifiedchangesinlevelsandthephosphorylationstateforproteinsthatcriticallyregulatemitochondrialfissionandfusio nandsuggestedthatthese changesmaycontributetotheabnormalaggregation andmorphologyofmitochondria.Surprisingly,wedid notfindsimilarchangesinmitochondrialfissionand fusionproteinsintheTDP-43M337Vmice,suggesting thatmitochondrialaggregationobservedinbothTDP43modelsmayresultfromanotherpathogenicprocesses,suchasaxonaldegeneration. Onoccasion,mitochondrialclusterswithintheneurons oftheTDP-43M337Vmicewereassociatedwithmicrotubules.TDP-43mayalterthemicrotubule-basedmitochondrialtransportationsystemandleadtobothmicrotubule andmitochondrialaggregationanddysfunction.While thereareanumberofwaysthroughwhichthiscould occur,itwasinterestingtofindthatthemicrotubuleassociatedproteintauwasabnormallyhyperphosphorylatedin thebrainofbothourTDP-43WTandTDP-43M337Vmice. Taufunctionstostabilizemic rotubulesandtofacilitate axonaltransport.Abnormal phosphorylationoftaucan reduceitsabilitytobindmicrotubules[23].Anumberof kinasescanphosphorylatetau,andoneofthesekinases, PKC,wasactivatedinbothTDP-43M337VandTDP-43WTmice.Tauhyperphosphorylationmightbepartiallydueto PKCactivationinthemice;however,theparadigm throughwhichTDP-43overexpressionresultedintau hyperphosphorylationand/ orPKCactivationisstill unclear.OnepossibilityisthatexacerbatedTDP-43autoregulationdisruptsthehomeostasisofotherkeyproteins andeventuallyleadstodysfun ction.Alternatively,PKC activationandtauhyperphosphorylationcouldbesecondaryorunrelatedtomitochondrialclustering.Thereisa largebodyofevidencesuggestingthataxonaltransportis disruptedinALS[24,25],whichincludesevidencethat SOD1mutationsimpairaxonaltransport[26,27].Interestingly,depletionofkinesinheavychain(kif5B)resultsin mitochondrialperinuclearclusterssimilartothose describedinourTDP-43models[28]. Onelimitationofourmodelisthatalthoughmanyof thefeaturesareconsistentwiththoseobservedinALS, thereareseveralfeaturesofthismodelthatareincongruouswiththehumandisease.Phenotypically,our miceshowretardedgrowthfromearlylife;whereas, humanswithALStypicallyshowprogressivedisease afterinitiallynormaldevelopmentintoadulthood.While ourTDP-43M337Vmiceshowearlylethality,theageat deathforthemiceissignificantlyyoungerincomparisontothelifespanofhumanswithALSduetomutationsinTDP-43.WedidnotseelossofnuclearTDP-43 immunoreactivitysimilartothatobservedinaffected neuronsinALS,evenincellswithcytoplasmicTDP-43 inclusions.ItispossiblethatmTDP-43underwentredistributionasrecentlyreportedinanotherTDP-43mouse model[14];however,weareunabletoconfirmthisdue tounavailabilityofmTDP-43-specificantibody.Wealso observedconsiderablechromatolysisintheTDP-Xu etal MolecularNeurodegeneration 2011, 6 :73 http://www.molecularneurodegeneration.com/content/6/1/73 Page11of14

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43M337Vmice;however,chromatolysisisinfrequentin endstagehumanALS,andoftenonlydetectedincases withrapiddiseaseprogression[29].TDP-43M337Vmice alsoexhibitotherpathologiesnotfrequentlyseenin humans,suchasabnormalmitochondrialaggregation. Interestingly,increasedphospho-tauhasbeenreported inALSpatients,especiallythosewithcognitiveimpairment[30,31],whichsuggeststhatfurtheranalysisofa potentialrelationshipbetweenTDP-43andtauin mousemodelsandinhumancasesmightbewarranted. Themotor,biochemical,andpathologicalfeatures observedinTDP-43M337Vmice(line4)werealsonoted inasecondindependentlineofTDP-43M337Vmice(line 6),suggestingthatthesefeatureswerenotsimplydueto transgeneinsertionaleffects. WeobservedsomeregionaldifferencesinourTDP-43 transgenicmodels,despitetherelativelyuniformexpressionoftheTDP-43proteinsthroughoutthegraymatter ofthebrainandspinalcord.Forexample,nuclearpTDP43inclusions,abnormalmit ochondrialaggregationand chromatolysisweremainlyobservedinspinalcord,while cytoplasmpTDP-43inclusionsandtauphosphorylation wereprimarilyseeninthebrain.Thisindicatesthatthe mechanismsregulatingTDP43andbeingregulatedby TDP-43arelikelytobedifferentinthebrainandspinal cordandwarrantfurtherinvestigation.ConclusionInsummary,thisnovelTDP-43M337VmousemodelindicatesthatoverexpressionofhTDP-43M337Valoneistoxic invivo .TDP-43M337Vmicerecapitulatecertainpathologicfeaturesseeninneurodegenerativediseases,including TDP-43fragmentation,phos phorylation,aggregation, increasedubiquitinationandgliosis.Whilethesefeatures couldalsoindicategeneralneuronaldysfunction,the micealsoexhibitdownregulationofmouseTDP-43, abnormalmitochondriaaggr egationandabnormaltau phosphorylation.Becaus eoverexpressionofmutant hTDP-43producesphenotype ssimilartothewild-type TDP-43model,themechanismscausingpathogenesisin themutantmodelremainunknown.However,these resultsshouldnotbesurprising,giventhemutantTDP43andwild-typeTDP-43bioc hemicallyaresimilarin humandisease.Assuch,theTDP-43M337Vmicemay serveasavaluabletoolforf uturestudiesofyet-to-beexamineddiseasepathwaysandthepreciserolesTDP-43 RNAtargetsplayinneurodegeneration.MethodsGenerationofTDP-43M337VTransgenicMiceThehumanwild-typeTDP-43cDNAwasgeneratedas previouslydescribed[11]andwasinsertedintothe Xho I siteofthepcDNA3.1.TogeneratetheM337Vmutation,sitedirectedmutagenesiswasperformedusing Quikchangekit(Strategene ).Theprimersusedforthe mutationwere:5 -CAGTTGGGGTATGGTGGGCATGTTAGC-3 and5 -GCTAACATGCCCACCATACCCCAACTG-3 .Afterconfirmingthemutationby sequencing,theM337VTDP-43cDNAwasinserted intothe Xho IsiteofMoPrPvector[17].Following sequencing,theconstructwaslinearizedwith Not I,gel purifiedanddigestedwith b -agarose.DNAwasfiltered, concentratedanddilutedto3ng/ linmicroinjection buffer.Thetransgenewasmicroinjectedintofertilized C57BL/6(B6)mouseeggsandre-implantedintopseudopregnantfemales.EightfounderswerematedwithB6 micetodeterminegermlinetransmissionandestablish expressionlevels.TDP-43founderlines(line4and6) thathavesimilartransgenelevelstotheTDP-43WTmicewepreviouslydescribed[11],wereusedforallsubsequentexperiments.Homozygousmicewereproduced throughacrossbreedingoftransgenicmicefromthe sameline.Atnotimewerelines4and6intercrossed. Procedureswereperformedinaccordancewiththe MayoInstitutionalAnimalCareandUseCommittee.GenotypingTransgenicmicewereidentifiedbyPCRusinghTDP43-specificprimers:5 -TGGAGAAGTTCTTATGGTGCAGGTC-3 and5 -GGTATTAGCCTATGGGGGACAC-3 againstcontrolactin-specificprimers(5 CGGAACCGCTCATTGCC-3 and5 -ACCCACACTGTGCCCATCTA-3 ).Homozygousmicewere identifiedbyQuantitativereal-timePCR(seeQuantitativereal-timePCRsection).Quantitativereal-timePCRLevelsofhumanandmouseTDP-43transcriptswere determinedviaTaqManGeneExpressionAssays (AppliedBiosystems,Carlsbad,CA).TotalRNAwasisolatedeitherfromtailsamplesforidentificationofhomozygousmiceorfrombrainorspinalcordtissueto determinethelevelsofhumanandmouseTDP-43transcripts.TRIzol(Invitrogen,Carlsbad,CA)andPure Link RNAMiniKit(Invitrogen,Carlsbad,CA)were usedforRNAextraction.3 gRNAwereusedto synthesizecDNAusingtheHighCapacitycDNA ReverseTranscriptionKit(AppliedBiosystems,Carlsbad,CA).TheqPCRassayusedthefollowing:hTDP-43 Hs00606522_m1,mTDP-43Mm00523866_mand18S rRNAHs99999901_s1.ThePCRwasrunontheABI 7900anddataanalyzedusingSoftwareRQManager1.2 (AppliedBiosystems,Carlsbad,CA).TissuepreparationSagittalhalfbrainandspinalcolumnwereimmersion fixedin10%formalinforimmunohistochemistry,and theotherhalfbrainwasfrozenondryiceforXu etal MolecularNeurodegeneration 2011, 6 :73 http://www.molecularneurodegeneration.com/content/6/1/73 Page12of14

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biochemistry.After24hours,thespinalcordwas removedfromthevertebralcolumnandfixedovernight.WesternBlottingTissueswerehomogenizedat10ml/g(volume/weight)in lysisbuffer(50mMTris-HCl,pH7.4,300mMNaCl,1% TritonX-100,5mMEDTA,2%SDS,PMSF,andproteaseandphosphataseinhibitor).Followingcentrifugation,supernatantwasassessedbyBCAassay(Pierce, Rockford,IL).Followingwesternblotting,membranes wereincubatedwithmousemonoclonalTDP-43antibody,whichwasgeneratedusingaminoacids1to261of hTDP-43astheimmunogenandwasfoundtorecognize aminoacids205-222ofhTDP-43byepitopemapping [32];rabbitpolyclonalTDP-43antibodyagainstamino acids288-441;mouseCP13;mouseTau5;mousetau-1; mousemonoclonalglyceraldehyde-3-phosphatedehydrogenase(GAPDH)antibody;Phospho-(Ser)PKCSubstrate Antibody;mouseDLP1antibody;rabbitphospho-DLP1 (Ser616)antibody;rabbitFis1antibody;ormousemitofusin1antibody.SeeAdditionalfile3foracompletelistof primaryantibodiesused.Followingincubationwithan appropriatesecondaryantibody,immunoreactivitywas visualizedbyECLandexposuretofilm.Immunohistochemistry(IHC)andhistochemistryTissueswereembeddedinparaffin,sectioned(5 mthick) andmountedonglassslides.Sectionsweredeparaffinized inxyleneandrehydratedinagradedseriesofalcohol,followedbydH20.Antigenretrievalwasperformedina dH2Osteambathfor30min.TissueswereimmunostainedwithmonoclonalTDP43antibodyorantibodies towardpS403/S404-phosphorylatedTDP-43,ubiquitin, glialfibrillaryacidicprotein(GFAP),ionizedcalcium-bindingadaptormolecule1(IBA-1),cytochromeoxidasesubunitIV(COX-IV),orphospho-tau(CP13;pS202tau)using theDAKOAutostainer(DakoAutoMachineCorporation) andtheDAKOEnVision+HRPsystem.DAKOLiquid DABSubstrate-Chromogensystemwasthechromogen. SeeAdditionalfile3foracompletelistofprimaryantibodiesused.Afterimmunostaining,sectionswerebriefly counterstainedwithhematoxylintostaincellnucleiand coverslipped.Paraffin-embeddedsectionswerealso stainedwithhematoxylinandeosin.ElectronMicroscopySpinalcordsfrom4%-paraformaldehyde-perfusedmice wereimmersedin2.5%glutaraldehyde-0.1Mcacodylate buffer,post-fixedin1%OsO4,dehydratedinalcohol andpropyleneoxide,andfinallyinfiltratedand embeddedinEpon812.Ultrathinsectionsmountedon coppergridswerestainedwithuranylacetateandlead citrate.ImageswereobtainedwithaGatanCCDcamera usingaPhilips208Selectronmicroscope.GaitAnalysisMethodsFrontpawswerepaintedred,andhindfeetwerepainted bluewithnontoxicpaint.Themicewereplacedina plastictunnelwithastripofwhitepapercoveringthe floor.Attheendofthetunnelwasanenclosedblack boxtoencouragemicetocrossthepaperandintothe box.Thetestwasrepeateduntilmiceleftatleastfive pairsofadjacentfootprints.StatisticsOne-wayANOVAwithTukey sposthocanalysiswereused tocomparemeasuresamong3groups.Student st-testanalysiswasusedtocomparemeasuresbetween2groups. Kaplan-Meiermethodswereusedforsurvivalanalysis.For datapresentation,normaliz edvalueswereaveragedand presentedasmeanstandarderrorofmeans(SEM). Valuesof p <0.05wereconsideredstatisticallysignificant.AdditionalmaterialAdditionalfile1:FigureA1:IncreasedubiquitinlevelsinTDP43M337VmicebuthTDP-43itselfisnotubiquitinated .HumanTDP-43 wasimmunoprecipitatedfrombrainhomogenatesderivedfrom nontransgenicandhomozygousTDP-43m337vmice.Briefly,brain homogenatescontaining500 gproteinwereincubatedwith1.5 g mousemonoclonalTDP-43antibodyovernightat4Cwithgentle shaking.ProteinGagarosewasaddedfor4hat4Cthenpelletedby centrifugation.Proteinthuscapturedwaselutedusingsampleloading bufferandresolvedbySDS/PAGEforWesternblotanalysis.Shownare immunoblotsoftheinputsandtheimmunoprecipitatedproteins, probedusinganantibodytoubiquitinortototalTDP-43.Notethata markedincreaseinubiquitinlevelsisobservedinthehomogenates derivedfromhomozygousmicepriortoimmunoprecipitation. Nonetheless,theimmunoprecipitatedhumanTDP-43isnot immunopositiveforubiquitin.Arrow=IgGHeavyChain. Additionalfile2:FigureA2:Nomitochondrialfissionandfusion proteinchangesinTDP-43M337Vmice .ImmunoblotanalysisofSer616phosphorylatedDLP1,DLP1,Fis1,andmitofusin1(MFN1)expression levelinbrainlysatesofnontransgenic(NT),hemizygous(Hemi),and homozygous(Homo)TDP-43M337Vmiceofbothline4andline6.There arenosignificantproteinchangesamongdifferentmicegroups. Additionalfile3:AdditionalTable:PrimaryAntibodyList .Fulllistof theprimaryantibodiesusedinthisstudy. Listofabbreviations ALS:amyotrophiclateralsclerosis;FTLD-U:frontotemporallobar degenerationwithubiquitin-positiveinclusions;TDP-43:TARDNAbinding protein-43;hTDP-43:humanTDP-43;mTDP-43:mouseTDP-43.pTDP-43: phospho-TDP-43. Acknowledgements ThisworkwassupportedbytheAFARAffiliateResearchGrantProgram(YZ), MayoClinicFoundation(DWD,JL,LP),NationalInstitutesofHealth/National InstituteonAging[5R01AG026251-04andP01-AG17216-08(LP)],National InstitutesofHealth/NationalInstituteofNeurologicalDisordersandStroke [R01NS063964-01(LP)and1R21NS071097-01(JL)],AmyotrophicLateral SclerosisAssociation(LP,JL)andDepartmentofDefense[USAMRMC PR080354(LP,JL)andAL093108(LP)].WewouldliketothankJimeiTong, CindyYu,MonicaCastanedes-Casey,LindaRousseauandVirginiaPhillipsfor technicalsupport.Xu etal MolecularNeurodegeneration 2011, 6 :73 http://www.molecularneurodegeneration.com/content/6/1/73 Page13of14

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Authordetails1DepartmentofNeuroscience,MayoClinic,(4500SanPabloRoad), Jacksonville,(32224),USA.2CenterforTranslationalResearchin NeurodegenerativeDisease(CTRND),CollegeofMedicine,Universityof Florida,(1275CenterDrive),Gainesville,(32610),USA.3Departmentof Neuroscience,CollegeofMedicine,UniversityofFlorida,(1275CenterDrive), Gainesville,(32610),USA. Authors contributions YXandYZperformedexperiments,dataanalysisandco-wrotethe manuscript.XCperformedexperiments,WLperformedElectronMicroscopy study.CSeditedthemanuscript.JL,DWDandLPconceivedofthestudy, participatedinitsdesignandcoordinationandeditedthemanuscript.All authorsreadandapprovedthefinalmanuscript. Competinginterests YX,YZ,JL,andLPareinventorsofthisandrelatedmousemodels;however, noroyaltieshavebeengeneratedfromthisinvention. Received:29April2011Accepted:26October2011 Published:26October2011 References1.AraiT,HasegawaM,AkiyamaH,IkedaK,NonakaT,MoriH,MannD, TsuchiyaK,YoshidaM,HashizumeY,OdaT: TDP-43isacomponentof ubiquitin-positivetau-negativeinclusionsinfrontotemporallobar degenerationandamyotrophiclateralsclerosis. BiochemBiophysRes Commun 2006, 351 :602-611. 2.NeumannM,SampathuDM,KwongLK,TruaxAC,MicsenyiMC,ChouTT, BruceJ,SchuckT,GrossmanM,ClarkCM, etal : UbiquitinatedTDP-43in frontotemporallobardegenerationandamyotrophiclateralsclerosis. Science 2006, 314 :130-133. 3.SreedharanJ,BlairIP,TripathiVB,HuX,VanceC,RogeljB,AckerleyS, DurnallJC,WilliamsKL,BurattiE, etal : TDP-43mutationsinfamilialand sporadicamyotrophiclateralsclerosis. Science 2008, 319 :1668-1672. 4.KabashiE,ValdmanisPN,DionP,SpiegelmanD,McConkeyBJ,Vande VeldeC,BouchardJP,LacomblezL,PochigaevaK,SalachasF, etal : TARDBP mutationsinindividualswithsporadicandfamilialamyotrophiclateral sclerosis. 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Additional Table. Primary Antibody List AntibodyCatologue #VendorWB DilutionIHC Dilution Description mouse monoclonal TDP-432E2-D3Novus 1:10001:3000 recognizing human TDP-43 (hTDP-43)Biologicals at amino acids 202-222 rabbit polyclonal TDP-4312892-1-APProteinTech 1:10001:3000 made to amino acids 260414 (Total TDP-43)Group recognize both human and mouse TDP-43 rabbit phospho-TPD-43 TIP-PTDP05Cosmo Bio1:2000 (pS403/404) ubiquitin05-944Chemicon/ Millipore1:60000 ubiquitinZ0458Dako1:1000 cytochrome c oxidaseab16056Abcam1:3000 subunit IV (COX-IV) GFAPPU020-UPBiogenex1:2500 Iba1019-19741Wako1:2000 GAPDHA8634OHBiodesign International1:10000 mouse CP13Provided by Peter Davis1:10001:1000 recognizing pS202 tau Albert Einstein College of Medicine mouse Tau 5 Provided by Lester Binder1:1000 recognizing total tau Northwestern University mouse Tau1MAB3420CHEMICON1:1000 anti-unphosphorylatedS202/205 tau Phospho-(Ser) PKC 2261Cell Signaling1:500 Substrate Antibody mouseDLP1 antibody 611112BD 1:1000 rabbit phospho-DLP1(Ser616) 3455Cell Signaling1:1000 rabbit Fis1 antibodyIMG-5113AIMGENEX1:500 mouse mitofusin 1 (MFN1)H00055669-A01Novus Biologicals1:500