Effects of the C57BL/6 strain background on tauopathy progression in the rTg4510 mouse model

MISSING IMAGE

Material Information

Title:
Effects of the C57BL/6 strain background on tauopathy progression in the rTg4510 mouse model
Series Title:
Molecular Neurodegeneration
Physical Description:
Mixed Material
Creator:
Rachel M Bailey
John Howard
Joshua Knight
Naruhiko Sahara
Dennis W Dickson
Jada Lewis
Publisher:
Molecular Neurodegeneration
Publication Date:

Notes

Abstract:
Background: Cross-breeding of transgenic mice is commonly used to assess gene-gene interactions, particularly in the context of disease. Strain background changes can influence the phenotype of mouse models and can confound crossbreeding studies. We sought to determine if changing the strain background of a commonly used mouse model of tauopathy (rTg4510) would significantly impact the originally reported phenotype. On the original F1 FVB/N x 129S6 background, rTg4510 mice present with progressive cognitive decline, increased insoluble tau, robust tau pathology and age-dependent neurodegeneration. One of the most common strains in mouse modeling is C57BL/6. We and others have previously reported that this strain background alters the phenotypes of various models, including the JNPL3 model of tauopathy. To determine if the phenotype of rTg4510 mice was similarly affected by the introduction of the C57BL/6 background, we compared rTg4510 mice on the original F1 FVB/N x 129S6 background to rTg4510 mice on an F1 FVB/N x C57BL/6NTac (B6/NTac) background, herein termed rTg4510B6. Results: Despite a small, but significant increase in soluble human tau levels, young rTg4510B6 mice had equivalent levels of tau phosphorylation, aggregation and cognitive impairments as age-matched rTg4510 mice. At 6.5 months of age, rTg4510B6 mice displayed hyperphosphorylated insoluble tau and robust cortical tau neuropathology that was equivalent to age-matched rTg4510 mice; however, 10.5-month-old rTg4510B6 mice had greater amounts of phospho-tau in the cortex and hippocampus when compared to age-matched rTg4510 mice. Non-transgenic (NT) littermates of rTg4510B6 (NTB6) mice also had greater amounts of cortical and hippocampal phospho-tau at 10.5 months of age when compared to NT littermates of rTg4510 mice. Additionally, older rTg4510B6 mice had gross forebrain neurodegeneration that was equivalent to age-matched rTg4510 mice. Conclusions: Overall, our data shows that introduction of the C57BL/6 strain into the rTg4510 mouse background modestly alters the tau pathology that was originally reported in rTg4510 on the F1 FVB/129 background. In contrast, behavioral and neurodegenerative outcomes were not altered. These studies support the use of the rTg4510 mouse model on a partial C57BL/6 strain background without losing fidelity of the phenotype and suggest that the C57BL/6 background does not inherently protect against tauopathy.

Record Information

Source Institution:
University of Florida
Holding Location:
University of Florida
Rights Management:
All rights reserved by the source institution.
System ID:
AA00020068:00001

Full Text

PAGE 1

RESEARCHARTICLEOpenAccessEffectsoftheC57BL/6strainbackgroundon tauopathyprogressionintherTg4510mouse modelRachelMBailey1,2,JohnHoward1,2,JoshuaKnight1,2,NaruhikoSahara1,2,DennisWDickson2andJadaLewis1,2*AbstractBackground: Cross-breedingoftransgenicmiceiscommonlyusedtoassessgene-geneinteractions,particularlyin thecontextofdisease.Strainbackgroundchangescaninfluencethephenotypeofmousemodelsandcan confoundcrossbreedingstudies.Wesoughttodetermineifchangingthestrainbackgroundofacommonlyused mousemodeloftauopathy(rTg4510)wouldsignificantlyimpacttheoriginallyreportedphenotype.Ontheoriginal F1FVB/Nx129S6background,rTg4510micepresentwithprogressivecognitivedecline,increasedinsolubletau, robusttaupathologyandage-dependentneurodegeneration.Oneofthemostcommonstrainsinmousemodeling isC57BL/6.Weandothershavepreviouslyreportedthatthisstrainbackgroundaltersthephenotypesofvarious models,includingtheJNPL3modeloftauopathy.TodetermineifthephenotypeofrTg4510micewassimilarly affectedbytheintroductionoftheC57BL/6background,wecomparedrTg4510miceontheoriginalF1FVB/Nx 129S6backgroundtorTg4510miceonanF1FVB/NxC57BL/6NTac(B6/NTac)background,hereintermedrTg4510B6. Results: Despiteasmall,butsignificantincreaseinsolublehumantaulevels,youngrTg4510B6micehadequivalent levelsoftauphosphorylation,aggregationandcognitiveimpairmentsasage-matchedrTg4510mice.At6.5months ofage,rTg4510B6micedisplayedhyperphosphorylatedinsolubletauandrobustcorticaltauneuropathologythat wasequivalenttoage-matchedrTg4510mice;however,10.5-month-oldrTg4510B6micehadgreateramountsof phospho-tauinthecortexandhippocampuswhencomparedtoage-matchedrTg4510mice.Non-transgenic(NT) littermatesofrTg4510B6(NTB6)micealsohadgreateramountsofcorticalandhippocampalphospho-tauat 10.5monthsofagewhencomparedtoNTlittermatesofrTg4510mice.Additionally,olderrTg4510B6micehad grossforebrainneurodegenerationthatwasequivalenttoage-matchedrTg4510mice. Conclusions: Overall,ourdatashowsthatintroductionoftheC57BL/6strainintotherTg4510mousebackground modestlyaltersthetaupathologythatwasoriginallyreportedinrTg4510ontheF1FVB/129background.In contrast,behavioralandneurodegenerativeoutcomeswerenotaltered.Thesestudiessupporttheuseofthe rTg4510mousemodelonapartialC57BL/6strainbackgroundwithoutlosingfidelityofthephenotypeandsuggest thattheC57BL/6backgrounddoesnotinherentlyprotectagainsttauopathy. Keywords: Transgenicmousemodels,Strainbackground,C57BL/6,rTg4510,Tau,Tauopathy,Neurodegeneration, Behavior,Morriswatermaze *Correspondence: jada.lewis@ufl.edu1CenterforTranslationalResearchinNeurodegenerativeDiseaseand DepartmentofNeuroscience,UniversityofFlorida,Gainesville,FL32610,USA2DepartmentofNeuroscience,MayoClinic,Jacksonville,FL32224,USA 2014Baileyetal.;licenseeBioMedCentralLtd.ThisisanopenaccessarticledistributedunderthetermsoftheCreative CommonsAttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse,distribution,and reproductioninanymedium,providedtheoriginalworkisproperlycited.Bailey etal.MolecularNeurodegeneration 2014, 9 :8 http://www.molecularneurodegeneration.com/content/9/1/8

PAGE 2

Background Numeroustransgenicmiceexpr essingwild-typeormutant humantauhavebeencreatedto modeltheneuropathology oftauopathies – agroupofneurodegenerativediseasescharacterizedbytheaccumulation oftauproteinaggregates. Manyofthebehaviorsandpathologiesreportedforthese mousemodels,eventhosethatexpressthesametauprotein,aredifferent.Severalfactorsmaycontributetothese differences,includingthelev elsandregionsoftransgenic tauproteinexpression,thech aracteristicsofthepromoters usedtodrivetransgenictauexpression,andthebehavioraltestsusedtocharacterizethemodels.Thestrain backgroundoftransgenicmicecanalsoalterdisease progressionandpresentation. JNPL3[1]andrTg4510[2]micearetwowidelyused, independentmousemodelsofhumantauopathythat expressthesamehuman0N4Rtauproteinusingdifferent promotersystems.Previously,Bolmontetal.crossedthe JNPL3mousemodelfromamixedbackgroundontoan inbredC57BL/6Jbackgroundandfoundthattheregional distributionoftauopathywasalteredandthatthetiming ofphenotypewassignificantlydelayed[3].Studieshave alsosuggestedthattheC57BL/6backgroundisprotective againstneurotoxicityinotherdiseasemodels[4-7].Totest thehypothesisthatcrossingrTg4510micetoaC57BL/6 strainbackgroundisprotecti veagainsttauopathy,wehave comparedrTg4510miceontheoriginalF1FVB/Nx129S6 backgroundtorTg4510onanF1FVB/NxC57BL/6NTac background(rTg4510 B6 )(Figure1).Herewecomparethe phenotypesofbothyoungandoldrTg4510miceoneach strainbackground. Results IncreasedhumantaulevelsinyoungrTg4510 B6 mice Tauisnormallyahighlysolubleproteinandexpression levelsaregenerallymeasuredinsolubleproteinextracts. Inordertocomparehumantauexpressionlevelsin rTg4510 B6 andrTg4510mice,wepreparedsolublebrain extractsfrommiceat2.5monthsofage,priortorobust taupathologyandneuronalloss.rTg4510 B6 micehad asmall,butsignificantincrease(0.320.13;p<0.05) insolublehumantaulevelscomparedtorTg4510miceat 2.5monthsofage,suggestingthatrTg4510 B6 mice mayhavemoderatelyelevatedtautransgeneexpression (Figure2).UsingtheTau5antibodythatrecognizesboth thetransgenichumantauandendogenousmousetau, nodifferencesinsolubletotaltaulevelswerefoundbetweenrTg4510 B6 andrTg4510miceat2.5monthsofage. HumantauexpressionintherTg4510linesiscontrolled bythetetracyclinetransactivator(tTA).Althoughboththe tTA 129 miceusedforrTg4510miceandthetTA B6 mice usedforrTg4510 B6 miceoriginatedfromthesametTAline [8],thisminorincreaseintauexpressioncouldbedueto minorchangesintTAexpressiononthetwobackgrounds. Wewere,however,unabletoassesstTAexpression duetothelackofagoodtTAantibody,aproblemthat isconsistentwithdatafromothersworkingwithtTA mouselines[5,9,10]. Figure1 BreedingschemeforcomparisonofrTg4510miceondifferentstrainbackgrounds. BreedingschemeusedtoproducerTg4510 miceonanF1FVB/NxC57BL/6(FVB/B6)strainbackground(rTg4510 B6 )(Left)comparedtotheoriginalrT4510miceonanF1FVB/Nx129S6 (FVB/129)strainbackground(Right).MiceusedinthesestudiesincluderTg4510 B6 andrTg4510micethatcarryboththetauresponderandthe tTAactivatortransgenesandnon-transgenic(NT B6 andNT)mice.SingletransgenicmicegeneratedfromtheseF1crosseswerenotanalyzedin thesestudies. Bailey etal.MolecularNeurodegeneration 2014, 9 :8 Page2of20 http://www.molecularneurodegeneration.com/content/9/1/8

PAGE 3

Tauwasbiochemicallyequivalentbetweenyoung rTg4510 B6 andyoungrTg4510mice Normaltaufunctionandsolubilityisregulatedbyphosphorylation(reviewedin[11]);therefore,wecomparedthe phosphorylationofsolubletauinyoungrTg4510miceon bothstrainbackgrounds.Todetecttauphosphorylatedat S202andS396/404 – sitesthatarehyper-phosphorylatedin humantauopathies – weusedthephospho-tauantibodies CP13andPHF1,respectively[2,12-14].Analysisofsoluble brainextractwiththeCP13andPHF1antibodiesrevealed thatyoungrTg4510 B6 micehadsimilarphosphorylationof solubletauasrTg4510mice(Figure2). Acentralfeatureofhumantauopathiesisthelossoftau solubilityandtheaccumulationofhyper-phosphorylated tauinaggregatesthatcanbeisolatedfrombrainwith sarkosylextraction[15].Taudepositioninthesarkosylinsolublebrainfractioncanbedetectedasearlyas 2.5monthsofageinrTg4510mice.Withincreasing ageanddiseaseprogression ,insolubletaushiftsfrom a~55kDaspeciestoahyper-phosphorylated64kDa species[14]andwehavepreviouslydemonstratedthat 64kDatauisbiochemicallyequivalenttoNFTsintau transgenicmice[1].At2.5monthsofage,therewasno significantdifferenceintheaccumulationofinsoluble Figure2 2.5-month-oldrTg4510 B6 micehaveincreasedhumantauexpressioncomparedtorTg4510mice.(A-B) Westernblotanalysesof thesolublefractionsofwholebrainlysatesfromrTg4510 B6 andrTg4510miceat2.5monthsofage. (A) Representativewesternblotsofsoluble humantau(E1antibody),humanandmousetau(Tau5antibody),andphosphorylatedtau(CP13andPHF1antibodies)withaNTlittermate shownasanegativecontrol. (B) DensitometricquantificationofE1(humantau),Tau5(humanandmousetau),CP13(pS202tau)andPHF1 (pS396/404tau)normalizedtoGAPDH.rTg4510 B6 micehadincreasedsolublehumantau,indicatinggreatertautransgeneexpressionthan age-matchedrTg4510mice.Nodifferencesinsolublephospho-taulevelsweredetectedusingCP13andPHF1antibodies.Eachdotrepresentsan individualmousewiththemeanindicatedbytheblackline.n=9-10percohort.*P<0.05(Student ’ st-test). Bailey etal.MolecularNeurodegeneration 2014, 9 :8 Page3of20 http://www.molecularneurodegeneration.com/content/9/1/8

PAGE 4

humantauandthephosphorylationofinsolubletauin rTg4510 B6 micecomparedtorTg4510mice(Figure3). Importantly,weobservedtheinitialaggregationofa 64kDatauspeciesinrTg4510miceoneitherstrain background.Therewasconsiderablespreadinthedegree ofinsolubletaufoundwithineachgroup,consistentwith eachanimalbeinginslightlydifferentstagesoftheinitial, rapiddevelopmentoftauopathy.Wealsoexaminedthe insolublefractionwiththeTau5antibody,butwereunable toobtainsufficientTau5antibodyreactivitywiththeinsolublefractionfrom2.5month-oldrTg4510miceoneither strainbackground(datanotshown). ComparablecognitivedeficitsinyoungrTg4510 B6 and rTg4510mice TheinitialcharacterizationofrTg4510miceshowedthat humantauexpressingmicehadsimilarmotorperformancetotaunegativemiceontheF1FVB/129Sstrain backgroundat2.5monthsofage,beforewidespread neurodegenerationinthebrain[10].Studieshaveshown, however,thatnon-transgenic(NT)miceondifferent strainbackgroundscanhavevariablemotorperformance inavarietyofbehavioraltasks[16].Toassesseffectsof theC57BL/6strainbackgroundonsensorimotorfunction, 2.5-month-oldrTg4510 B6 ,rTg4510andNTmiceonboth strainbackgroundsperformedthecued(visibleplatform) versionofMWM.Nodifferencesinswimspeedwere observedacrosstrainingbetweenallgroups(Figure4A). Further,allgroupsimprovedperformanceovertraining, asindicatedbythedecreasedsearchpathtotheplatform [F(2,32)=35.91,p<0.001],butrTg4510miceonboth strainbackgroundshadlongersearchpathstoreach theplatformthanNTmice[F(1,16)=5.39,p<0.05] (Figure4B). Posthoc analysisrevealedthatbythethirdday ofvisibleplatformtraining,allgroupsswamcomparable distancestoreachtheplatform.Equivalentresultswere foundwithmeasurementsoftheescapelatencytoreach theplatform(datanotshown).Importantly,nodifferences betweenstrainsweredetected,signifyingthatmiceonan F1FVB/B6backgroundhadsimilarsensorimotorfunction asmiceontheF1FVB /129background. Spatiallearningandreferencememoryarehippocampal dependentfunctions[17].Thehippocampusisoneofthe firstregionsaffectedbytauopathyinrTg4510miceand Figure3 Equivalentaccumulationandphosphorylationofinsolubletauin2.5-month-oldrTg4510 B6 andrTg4510mice.(A-B) Western blotanalysesofthesarkosyl-insolublefractionsofwholebrainlysatesfromrTg4510 B6 andrTg4510miceat2.5monthsofage. (A) Representative westernblotsofinsolublehumantau(E1antibody)andphosphorylatedtau(CP13andPHF1antibodies)withaNTlittermateshownasanegative control. (B) DensitometricquantificationofE1(humantau),CP13(pS202tau)andPHF1(pS396/404tau).Phospho-tauisnormalizedtothe amountofhumantauaggregatedinthatfraction.InsolubletauaccumulationandphosphorylationweresimilarbetweenrTg4510 B6 andrTg4510 mice.Eachdotrepresentsanindividualmousewiththemeanindicatedbytheblackline.n=9-10percohort. Bailey etal.MolecularNeurodegeneration 2014, 9 :8 Page4of20 http://www.molecularneurodegeneration.com/content/9/1/8

PAGE 5

cognitivedeficitsinthehiddenplatformversionofMWM aredetectedasearlyas2.5monthsofageinrTg4510 ontheoriginalF1FVB/129strainbackground[2,14]. Analysisofour2.5-month-oldrTg4510andNTcohortsshowedimprovementinfindingthehiddenplatformforallgroupsacrosstrainingdays[escapelatency: F(4,64)=8.43,p<0.001),searchpath:F(4,64)=5.77, p<0.001](Figure5A-B).rTg4510miceshowedsignificantlylessimprovementinperformancethanNTmice asindicatedbylongerescapelatencies[F(1,16)=61.78, p<0.001)](Figure5A)andlongersearchpathstofindthe hiddenplatform[F(1,16)=48.83,p<0.001)](Figure5B). Additionally,nostrainbackgroundeffectwasdetected foreitherparameter.Irrespe ctiveofstrainbackground, rTg4510miceshowedincreasedthigmotaxic,orwallhugging,swimmingcomparedtoNTmice[F(1,16)= 24.70,p<0.001].ThethigomotaxicbehaviorofrTg4510 micedecreasedovertrainingdays[F(4,64)=19.07, p<0.001];however,itneverreachedthelowlevelsof thigomotaxisobservedinNTmice(Figure5C).Spatial learningperformancewasalsoassessedbythepercentageofdistanceswaminthetargetquadrantduringthe finalprobetrial – rTg4510miceonbothstrainbackgroundsweresignificantlyimpairedcomparedtoNT mice(p<0.0001)(Figure5D).Overall,wefoundthat crossingrTg4510micetoaC57BL/6strainbackground didnotaffectsensorimotorfunctionorattenuatethe cognitivedeclinethatisch aracteristicofrTg4510mice ontheoriginalstrainbackgroundat2.5monthsofage. CharacterizationoftauinolderrTg4510 B6 brainextracts Havingfoundthattheonsetoftauopathyisunchangedin rTg4510 B6 micecomparedtotheoriginalrTg4510mice, wethensoughttodetermineiftheC57BL/6background alterslatestagetauopathyinmiceat6.5and10.5months ofage.Unlikeyoungmice,oldrTg4510 B6 micehadsimilar levelsofsolublehumantauandincreasedlevelsoftotalsolubletau(humanandmouse)asrTg4510mice(p<0.01) (Figure6).Further,whiletherewasnotasignificantdifferenceinthephosphorylationofsolubletaudetectedwith CP13,analysiswithPHF1revealedthatoldrTg4510 B6 mice hadtwiceasmuchsolubletauphosphorylatedatS396/404 comparedtooldrTg4510mice(p<0.01)(Figure6).Bylate stagetauopathy,rTg4510micew erecharacterizedprimarily bya64kDaspeciesoftauinthesarkosyl-insolublebrain fraction,regardlessofstrainbackgroundandtheaccumulationofhumantauintheinsolublefractionwassimilar betweenrTg4510 B6 andrTg4510mice(Figure7).The accumulationofhumanandmousetauinthesarkosylinsolublefraction,asassessedwiththeTau5antibody, wasalsosimilarbetweenrTg4510 B6 andrTg4510mice (datanotshown).The64kDainsolubletaubandfrom rTg4510miceoneitherbackgroundwassimilarlyphosphorylatedatepitopesassociatedwithhumantauopathy whennormalizedtotheamountofhumantau(Figure7) orhumanandmousetau(datanotshown)inthesarkosylinsolublefraction.Resultsfromthebiochemicalanalysisof tauinolderrTg4510 B6 andrTg4510micearesummarized inTable1. rTg4510 B6 micehaveincreasedtaupathologycompared torTg4510mice Immunohistochemical(IHC)stainingwithCP13andPHF1 antibodieswasusedtocomparetauopathyinrTg4510 B6 andrTg4510mice.BothCP13andPHF1detectedstriking amountsoftauopathyinthecortexandhippocampusof Figure4 StrainbackgrounddoesnotalterswimspeedorsearchpathintheMWM.(A-B) PerformanceinthecuedMWMtaskwas equivalentamongstrTg4510andNTlittermatesoneitherstrainbackgroundat2.5monthsofage. (A) Swimspeedstothevisibleplatformwere equivalentacrossallgroups. (B) Allgroupsimprovedperformanceovertraining(p<0.001)withthesearchpathstoreachthevisibleplatform longerforrTg4510miceondays1and2,butcomparabletoNTmicebyday3.Nodifferencesbetweenstrainbackgroundsweredetected.Data expressedasmeanSEMandanalyzedviamultifactorial(genotypexstrain)RMANOVAwith posthoc Bonferonni ’ smultiplecomparisonstest. n=5percohort. Bailey etal.MolecularNeurodegeneration 2014, 9 :8 Page5of20 http://www.molecularneurodegeneration.com/content/9/1/8

PAGE 6

latestagerTg4510miceoneitherbackgroundandtaupathologywasfoundinboththecellbodyandtheneuropil (Figures8and9).Thebrainstemwasminimallyaffected, reflectingthelowlevelsoftransgenictauexpression withinthisbrainstructure(Figures8and9).Results fromatwo-wayANOVA(strainbackgroundxage)indicatedthatrTg4510 B6 micehadincreasedcortical CP13-specificphospho-taucomparedtorTg4510mice [F(1,13)=8.60,p<0.05],buttherewasaninteraction ofstrainbackgroundandage[F(1,13)=8.40,p<0.05] (Figure8,Table2). Posthoc analysisrevealedthatcorticalCP13burdenwasincreasedinrTg4510 B6 miceat 10.5monthsofage(p<0.01),butnot6.5monthsof agewhencomparedtoage-matchedrTg4510mice.Independentofage,CP13burdenwasalsosignificantly higherinthehippocampusofrTg4510 B6 micecompared torTg4510mice[F(1,13)=15.00,p<0.01](Figure8, Table2).WealsoanalyzedthebrainstemofrTg4510 B6 andrTg4510miceandfoundthatCP13stainingwas increasedwithagebutunaffectedbythestrainbackground ofthemice(Figure8,Table2).Quantitationofregional PHF1stainingyieldedsimilarresults.CorticalPHF1 stainingwasincreasedinrTg4510 B6 micecomparedto rTg4510mice[F(1,13)=9.20,p<0.01],buttherewasan interactionofstrainbackgroundandage[F(1,13)=9.40, p<0.01]withPHF1burdenbeingsignificantlyhigherin rTg4510 B6 miceoverrTg4510miceonlyat10.5months ofage(p<0.01)(Figure9,Table2).Additionally,PHF1 wassignificantlyincreasedinthehippocampusof rTg4510 B6 micecomparedtorTg4510miceatboth6.5 and10.5monthsofage[F(1,13)=23.00,p<0.001] (Figure9).Interestingly,therewasalsoincreasedPHF1 immunoreactivitythatwasnotseenwithCP13inthe brainstemofrTg4510 B6 micewhencomparedtorTg4510 miceontheoriginalstrainbackground[F(1,13)=21.00, p<0.001](Figure9,Table2).Overall,IHCanalysis revealedage-dependentregionaldifferencesinphosphotaupathologyinmicecrossedtoaC57BL/6backgroundcomparedtomiceontheoriginalrTg4510strain background. Figure5 rTg4510 B6 andrTg4510micehaveequivalentcognitivedeficitsat2.5monthsofage. Spatiallearningandmemorywas equivalentlyimpairedinrTg4510 B6 andrTg4510miceat2.5monthsofage. (A) rTg4510micetooksignificantlymoretimetofindthehidden platformthanNTmice(p<0.001),withnodifferencebetweenthestrainbackgrounds. (B) Thesearchpathstoreachthehiddenplatformfor bothrTg4510 B6 andrTg4510micewereequivalentandsignificantlylongerthanthesearchpathsofNTmice(p<0.001). (C) rTg4510 B6 and rTg4510micealsohadsimilarthigmotaxicswimmingthatwassignificantlylongerthanthatofNTmice(p<0.001). (D) Spatialmemorywas equallyimpairedinrTg4510 B6 andrTg4510micecomparedtoage-matchedNTmiceasindicatedbythedecreasedpercentageofdistance traveledinthetargetquadrant(TQ)duringthefinalprobetrial. (A-C) DataexpressedasmeanSEMandanalyzedviamultifactorial(genotypex strain)RMANOVA. (D) Eachdotrepresentsanindividualmousewiththemeanindicatedbytheblackline.****p<0.0001[two-wayANOVA (genotypexstrain):maineffectofgenotypeindicated].n=5percohort. Bailey etal.MolecularNeurodegeneration 2014, 9 :8 Page6of20 http://www.molecularneurodegeneration.com/content/9/1/8

PAGE 7

Murinetauphosphorylationisinfluencedby strainbackground Ashumantautransgeneexpressioninthebrainstemof theoriginalrTg4510miceisverylow,itwassurprising todetectincreasedPHF1staininginthebrainstemof oldrTg4510 B6 mice.ThePHF1antibodyrecognizesboth phosphorylatedhumanandmousetau;therefore,itwas possiblethatthatadditionofC57BL/6intothestrain backgroundcouldincreasephosphorylationofendogenousmousetau.Totestthis,weanalyzedPHF1staining inNTmiceonboththeF1FVB/B6(NT B6 )andtheF1 FVB/129(NT)strainbackgrounds.SimilarlytorTg4510 mice,PHF1stainingwasincreasedinthecortexofNT B6 micecomparedtoNTmice[F(1,14)=18.00,p<0.001], buttherewasaninteractionofstrainbackgroundand age[F(1,14)=14.00,p<0.01]withPHF1burdenbeing significantlyhigherinNT B6 miceoverNTmiceonlyat 10.5monthsofage(p<0.001)(Figure10,Table3). Additionally,PHF1stainingwassignificantlyincreased inthehippocampusandbrainstemofNT B6 micecomparedtoNTmiceatbothtimepointsandindependent ofaninteractionwithage[hippocampus:F(1,14)= 33.00,p<0.0001;brainstem:F(1,14)=40.00,p<0.0001] (Figure10,Table3).AnalysiswiththeCP13antibody revealedthatoldNT B6 micehadsignificantlymoreCP13 staininginthecortex[F(1,14)=9.90,p<0.01],butnotin Figure6 OlderrTg4510 B6 micehaveincreasedsolubleTau5andPHF1levelscomparedtorTg4510mice.(A-B) Westernblotanalysesof thesolublefractionsofwholebrainlysatesfrom6.5-and10.5-month-oldrTg4510 B6 andrTg4510mice. (A) Representativewesternblotsof solublehumantau,humanandmousetau,andphosphorylatedtauandwithaNTlittermateshownasanegativecontrol. (B) Densitometric quantificationofE1(humantau),Tau5(humanandmousetau),CP13(pS202tau)andPHF1(pS396/404tau)normalizedtoGAPDH.rTg4510 B6 micehadequivalentlevelsofsolubleCP13andhumantau,andincreasedsolubletotaltau(humanandmoues)andPHF1taucomparedto rTg4510miceontheoriginalstrainbackground.Eachdotrepresentsanindividualmousewiththemeanindicatedbytheblackline.n=3-4per cohort.**p<0.01[two-wayANOVA(strainxage):maineffectofstrainindicated]. Bailey etal.MolecularNeurodegeneration 2014, 9 :8 Page7of20 http://www.molecularneurodegeneration.com/content/9/1/8

PAGE 8

thehippocampusorbrainstem,comparedtooldNTmice (datanotshown,Table3). EquivalentneurodegenerationinoldrTg4510 B6 and rTg4510mice ApathologicalhallmarkofrTg4510miceisage-dependent neurodegenerationofthefore brainregionsthatisdetected by5.5monthsofage[2,14].Consistentwiththis,6.5-and 10.5-month-oldrTg4510miceo nbothstrainbackgrounds hadsignificantlylowerhemi-brainweightsthanNTmice [F(1,24)=158.72,p<0.001](Figure11A).Therewasalso asmall,butsignificantdecreaseinthebrainweightofall miceonanF1FVB/B6backgroundcomparedtoallmice ontheoriginalF1FVB/129background[F(1,24)=4.60, Figure7 AccumulationandphosphorylationofinsolubletauisnotsignificantlychangedinolderrTg4510 B6 mice.(A-B) Westernblot analysesofthesarkosyl-insolublefractionsofwholebrainlysatesfromrTg4510 B6 andrTg4510miceat6.5and10.5monthsofage. (A) Representative westernblotsofinsolublehumanandphosphorylatedtauwithaNTlittermateshownasanegativecontrol. (B) Densitometricquantificationof E1(humantau),CP13(pS202tau)andPHF1(pS396/404tau).Phospho-t auisnormalizedtotheamountofhumantauaggregatedinthat fraction.Eachdotrepresentsanindividualmousewiththemeanindicatedbytheblackline.n=3-4percohort. Table1Two-wayANOVAofstrainbackgroundandageeffectsonsolubleandinsolubletauin6.5and10.5month-old rTg4510 B6 micecomparedtorTg4510mice StrainBackgroundAgeInteraction FractionstainF(DFn,DFd)PvalueF(DFn,DFd)PvalueF(DFn,DFd)Pvalue SolubleE1 1 F(1,10)=3.60P=0.09F(1,10)=3.20P=0.10F(1,10)=0.94P=0.36 SolubleTau5 1 F(1,10)=11.00P<0.01F(1,10)=3.20P=0.10F(1,10)=0.05P=0.82 SolubleCP13 1 F(1,10)=2.40P=0.15F(1,10)=0.06P=0.81F(1,10)=0.25P=0.63 SolublePHF1 1 F(1,10)=20.00P<0.01F(1,10)=4.80P=0.05F(1,10)=0.57P=0.47 InsolubleE1 F(1,10)=3.90P=0.08F(1,10)=0.002P=0.97F(1,10)=0.002P=0.97 InsolubleTau5 F(1,10)=2.70P=0.13F(1,10)=1.20P=0.29F(1,10)=0.50P=0.50 InsolubleCP13 2 F(1,10)=3.60P=0.09F(1,10)=0.29P=0.60F(1,10)=0.06P=0.81 InsolublePHF1 2 F(1,10)=0.04P=0.85F(1,10)=2.90P=0.12F(1,10)=0.75P=0.41 1 NormalizedtoGAPDH; 2 NormalizedtoinsolubleE1. Bailey etal.MolecularNeurodegeneration 2014, 9 :8 Page8of20 http://www.molecularneurodegeneration.com/content/9/1/8

PAGE 9

Figure8 (Seelegendonnextpage.) Bailey etal.MolecularNeurodegeneration 2014, 9 :8 Page9of20 http://www.molecularneurodegeneration.com/content/9/1/8

PAGE 10

p<0.05]thatwasindependentofgenotype.Wethen wantedtodetermineifthereweredifferencesinthesize ofregionsspecificallyvulnerabletotau-relatedneurodegenerationinthismodel.NeuronallossinforebrainregionsofrTg4510micehasbeenwellcharacterizedby workfromSpiresetal.thatusedstereologicalmethods todemonstratethattheoverallhippocampalformation hasthegreatestneuronallosswhencomparedtothe corticalandstriatalregions[18].Toassesshippocampal atrophyinrTg4510B6mice,wemeasuredtheareaofthe hippocampalformationinmatchedsagittalsectionsof 6.5-and10.5-month-oldrTg4510miceandNTlittermates onbothstrainbackgrounds.ThreefactorANOVA (agexstrainxgenotype)revealedthattheareaofthe hippocampiweresignifican tlyaffectedbyage,strain backgroundandgenotype[age:F(1,27)=7.57,p<0.01; strain:F(1,27)=5.07,p<0.05;genotype:F(1,27)=501.97, p<0.001],andthattherewasaninteractionbetweenall threefactors[F(1,27)=5.93,p<0.05]. Posthoc analysis revealedthatboth6.5-and10.5-month-oldrTg4510 micehadsignificantlysmallerhippocampalareascomparedtoNTmice(ps<0.0001),withnodifferencein sizebetweenrTg4510miceontheoriginalstrainbackgroundandrTg4510B6mice(Figure11B).Additionally, at6.5monthsofagetheareaofthehippocampiinNTB6micewassignificantlysmallerthanNTmice(p<0.01), butthisdifferencewasabrogatedby10.5monthsofage (Figure11B).WealsoexaminedtheCA1layer,asthis hippocampalsub-regionhasbeenreportedtohavean82% reductionofneuronsby8.5monthsofageinrTg4510mice ontheoriginalstrainbackground[18].Wecalculateda CA1indexvalueforeachmousewithasmallerindexvalue indicatingfewerCA1neurons(seeMethods).InagreementwithpreviousdatabySpiresetal.[18],wefound thatrTg4510micehadsignificantlysmallerCA1indices thanNTmice[F(1,27)=96.54,p<0.001],suggesting thatrTg4510micehavegreaterneuronalloss(Figure11C).Additionally,therewasnodifferencebetween strains,althoughtheCA1indexvaluewasfoundtosignificantlydecreasedwithage[F(1,27)=8.67,p<0.01], consistentwithprogressiveneuronallosspreviously reportedinthismodel.Overal l,histologicalanalysesof rTg4510B6brainsat6.5and10.5monthsofagerevealed substantialthinningoftheneuronalcelllayersoftheCA1 anddentategyrushippocampalsub-regions,grossatrophy ofthehippocampiandcorticesandventricleenlargement thatwasindistinguishablefromagematchedrTg4510 mice(Figure11D).DiscussionrTg4510micethatoverexpressmutanthumantauare widelyusedinbothacademiaandindustrytostudy modifiersofhumantauopathy[19-33],yetrTg4510mice onadifferentstrainbackgroundhavenotbeenfullycharacterized.Oneofthemostcommonlyusedmousestrains isC57BL/6andcrossingtothisbackgroundcanaffecta mousemodel ’ sphenotypebecauseofgeneticandbehavioraldifferencesthatexistamongstinbredmousestrains (reviewedin[34]).InthisstudyweanalyzedrTg4510mice onaC57BL/6NTacbackground(rTg4510B6)neartheonset oftauopathyat2.5monthsofageandduringlatestage tauopathyat6.5and10.5monthsofage.Wefoundthatat 2.5monthsofage,rTg4510B6micehadasmall,butsignificantincreaseinsolublehumantaulevelsandequivalent tauphosphorylationandagg regationascomparedtoagematchedrTg4510miceontheoriginalstrainbackground. Further,cognitionwasequa llyimpairedinyoungrTg4510B6andrTg4510miceascomparedtoNTlittermates.During latestagetauopathy,rTg4510B6micedisplayedhyperphosphorylatedinsolubletauand robustneurodegeneration thatwasequivalenttorTg4510mice.Additionally,solublephospho-taudetectedwiththePHF1antibodywas increasedinrTg4510B6micecomparedtorTg4510mice and10.5-month-oldrTg4510B6micehadgreateramounts ofphospho-tauinthecortexandhippocampuswhencomparedtoage-matchedrTg4510mice.Finally,NTlittermates ofrTg4510B6micealsohadgreateramountsofcorticaland hippocampalphospho-tauat 10.5monthsofagecompared toNTlittermatesofrTg4510mice.Interestingly,aged rTg4510andNTmicecontainingaC57BL/6background hadgreatervariationinmeasuredphospho-taulevels, therefore,agreaternumberofmiceareneededtoconfirmthatthefindingsofagedrTg4510B6micewerenot influencedbyanoutlier. ContrarytoourfindingswithrTg4510mice,previous workwithJNPL3miceshowedthatbackcrossingonto C57BL/6strainbackgroundsignificantlydelayedthe onsetofdiseaseandalteredthepathologicalpresentation (Seefigureonpreviouspage.) Figure8 OlderrTg4510B6micehaveregion-specificincreasesinCP13comparedtorTg4510mice. rTg4510B6micehadincreasedCP13 (pS202tau)staininginthecortexandhippocampus,butnotthebrainstemregionsofthebraincomparedtorTg4510miceat10.5monthsof age. (A) Representativeimagesofcortical,hippocampalandbrainstemregionsof6.5-and10.5-month-oldrTg4510B6andrTg4510micestained withtheCP13antibody. (B) QuantitativeanalysesofthestainedsectionsshowedthattheCP13burdeninthecortexofrTg4510B6micewas significantlygreaterthanthecorrespondingregionsofrTg4510miceat10.5months,butnot6.5monthsofage,andincreasedinthe hippocampusofrTg4510B6atbothagepoints.Eachdotrepresentsanindividualmousewiththemeanindicatedbytheblackline,n=3-5per cohort.***p<0.001[two-wayANOVA(strainxage):maineffectofstrainindicated].##p<0.01( posthoc Bonferonni ’ smultiplecomparisonstest). Scalebarrepresents200 m. Bailey etal.MolecularNeurodegeneration 2014, 9 :8 Page10of20 http://www.molecularneurodegeneration.com/content/9/1/8

PAGE 11

Figure9 (Seelegendonnextpage.) Bailey etal.MolecularNeurodegeneration 2014, 9 :8 Page11of20 http://www.molecularneurodegeneration.com/content/9/1/8

PAGE 12

oftheoriginalmodel[3].AlthoughJNPL3miceexpress thesameP301L0N4RtauasrTg4510mice,thereareseveralimportantdifferencesbetweenthemodelsthatmay explainthedisparateeffectsoftheC57BL/6strainon tauopathyineachmodel.Thetautransgenicintegration sitesinJNPL3andrTg4510micehavenotbeenpublished, butitislikelythattheincorporationofthehumantau cDNAintothemurinegenomeoccurredatdifferentlocationsforeachmodel.Basedontheintegrationsiteofthe transgenesandtheendogenousactivityofthatchromosomalregion,strainbackgroundcoulddifferentiallyaffect promoteractivityineachmodel[35].Moreover,thetransgenictauexpressioninJNPL3miceiscontrolledbythe mouseprionpromoter,resultinginhumanmutanttau thatpredominantlyaffectsneuronsinthespinalcordand hindbrain.Incontrast,tauexpressioninrTg4510miceis ultimatelycontrolledbytheCaMKII promoter,whichresultsinhightauexpressioninneuronsintheforebrainregion.Thepromoteraffectswhichpopulationsofneurons areexpressingthetransgeneanddifferentneuronalpopulationsmaybedifferentlyaffectedineachstrainbackground.Additionally,theC57BL/6sub-strainutilizedin eachstudycouldalsocontributetothedifference.Inthis study,wecrossedtau-expressingmicetoaC57BL/6NTac (B6/NTac)sub-strainwhileBolmontetal.crossedtauexpressingmicetoaC57BL/6J(B6/J)sub-strain.TheB6/ NTacsub-strainfromTaconicwasoriginallyderivedfrom theB6/JstrainfromJacksonlaboratory,butovertime geneticandphenotypicdifferenceshavedevelopedbetweenthetwosub-strains[36-38].Althoughwedetected significant,butrelativelymodestchangesinthetauopathy ofrTg4510miceonaB6/NTacbackground,itispossible thatmodifiersoftauopathyexis tontheB6/Jstrain.Finally, theC57BL/6strainzygosityhasbeenshowntoaffecttransgenicphenotype[4,5]andtheBolmontetal.studyused congenicC57BL/6micewhileourstudiesusedhybrid F1C57BL/6mice.Ifarecessivegenewereresponsible foralteringtauopathyinJNPL3,sucheffectswouldnot bedetectedinF1rTg4510B6mice. Inadditiontotheconcernsdetailedabove,differences intransgenicproteinlevelscouldconfoundcomparisonsofthesamemodelondiff erentstrainbackgrounds (i.e.rTg4510B6vs.rTg4510).Bycomparingtauprotein levelsbetweenrTg4510B6andrTg4510micepriorto substantialneuronalloss,wefoundthatrTg4510B6mice haveasmall,butsignificant,increaseinsolublehuman tau.Interestingly,thisdifferenceinsolublehumantau levelswasnotdetectedusinganantibodyspecificfor bothhumanandmousetau,whichmayreflectadifferenceinendogenoustaulevelsbetweenthetwostrain backgrounds.Itshouldbenotedthough,thatitiscurrentlyuncleariftheTau5antibodyhasequalaffinityfor humanandmousetau,confoundingsuchaninterpretation.IncreasedsolublehumantauinyoungrTg4510B6micecouldresultfromdecreasedtaudegradationorfrom increasedtautransgeneexpression.BecausethetauresponderlineutilizedintherTg4510B6andrTg4510 lineswereidentical,itisunlikelythatthedifferencein transgenictauexpressionar osefromthetauresponder line,perse.SincehumantauexpressionintherTg4510 modelisdependentonboththetauandthetTAtransgenes,elevatedlevelsoftTAexpressioncouldunderliethe (Seefigureonpreviouspage.) Figure9 IncreasedPHF1taustaininginmultiplebrainregionsofolderrTg4510B6mice. OlderrTg4510B6micehadincreasedPHF1 (pS396/404tau)stainingcomparedtorTg4510miceinthecortex,hippocampusandbrainstemregionsofthebrain. (A) Representativeimagesof cortical,hippocampalandbrainstemregionsfrom6.5-and10.5-month-oldrTg4510B6andrTg4510micestainedwiththePHF1antibody. (B) QuantitativeanalysesofthestainedsectionsshowedthatthePHF1burdenwassignificantlygreaterinthecortexofrTg4510B6miceat 10.5monthsandinthehippocampusandbrainstematboth6.5and10.5monthsofagecomparedtothecorrespondingregionsofrTg4510 mice.Eachdotrepresentsanindividualmousewiththemeanindicatedbytheblackline,n=3-5percohort.***p<0.001[two-wayANOVA (strainxage):maineffectofstrainindicated].##p<0.01( posthoc Bonferonni ’ smultiplecomparisonstest).Scalebarsrepresent200 m. CC=Corpuscallosum;7N=7thCranialnerve. Table2Two-wayANOVAofstrainbackgroundandageeffectsonphospho-tauburdenin6.5and10.5month-old rTg4510B6micecomparedtorTg4510miceStrainbackgroundAgeInteraction RegionstainF(DFn,DFd)PvalueF(DFn,DFd)PvalueF(DFn,DFd)Pvalue CortexCP13 F(1,13)=8.60P<0.05F(1,13)=0.08P=0.78F(1,13)=8.40P<0.05 CortexPHF1 F(1,13)=9.20P<0.001F(1,13)=0.05P=0.83F(1,13)=9.40P<0.001 HippCP13 F(1,13)=15.00P<0.01F(1,13)=0.05P=0.83F(1,13)=1.00P=0.33 HippPHF1 F(1,13)=23.00P<0.001F(1,13)=0.03P=0.88F(1,13)=1.40P=0.26 BSCP13 F(1,13)=1.20P=0.29F(1,13)=9.90P<0.01F(1,13)=2.80P=0.12 BSPHF1 F(1,13)=21.00P<0.001F(1,13)=1.10P=0.32F(1,13)=0.43P=0.52 Bailey etal.MolecularNeurodegeneration 2014, 9 :8 Page12of20 http://www.molecularneurodegeneration.com/content/9/1/8

PAGE 13

Figure10 (Seelegendonnextpage.) Bailey etal.MolecularNeurodegeneration 2014, 9 :8 Page13of20 http://www.molecularneurodegeneration.com/content/9/1/8

PAGE 14

increasedhumantaulevels.TheCaMKII -tTAmiceused inrTg4510andrTg4510B6miceoriginatedfromthesame lineandlaboratory[8]andtheyweresubsequentlybackcrossedtodifferentmousestrainsbythetwodifferentlaboratoriesfromwhichweacquiredthemice.Itispossible thatchangeoftransgenecopynumbermayhaveoccurred betweenthetwotTAsub-lines,andthiscouldinfluence ourfindings.Thiscanoccurifthereisinherentinstability ofthetransgenelocus[39].WeattemptedtousecommerciallyavailableantibodiesraisedagainsttTAtodetermine iftTAlevelswerechangedbetweenrTg4510B6and rTg4510mice,butlikeothers,wewereunsuccessfulin detectingtTAproteinusingtheseantibodies[5,9,10]. We,therefore,wereunabletodetermineifalteredtTA expressionunderliesthemodestlyincreasedtaulevels inyoungrTg4510B6mice. Despiteincreasedhumantauexpressionin2.5-montholdrTg4510B6mice,wedidnotdetectdifferencesintau phosphorylationoraggregationatthisage.Furthermore, rTg4510B6andrTg4510miceweresimilarlycognitively impaired.ThehiddenplatformversionofMWMrevealed thatcognitionwasequivalentlyimpairedin2.5-month-old rTg4510miceacrossstrainbackgrounds.Interestingly, performanceinthecuedversionoftheMWMtask showedadifferenceintheswim pathtothevisibleplatform betweenrTg4510andNTmiceacrossalldays.Closer analysisofindividualtrainingdaysrevealedthatalthough rTg4510miceonbothbackgroundstooksignificantly longerpathstoreachtheplatformondays1and2,by day3theyperformedcomparablytoNTlittermates. ThissuggeststhatrTg4510micetookalongertimeto learnthecue,butoncelearned,wereabletoperform comparablytoNTmicesothatthedeficitsobservedin rTg4510miceonbothbackgroundsstrainscanbe accreditedtocognitivedeficitsratherthananinabilityto completethetask.Overall,althoughbehavioraldifferences havebeenreportedbetweenC57BL/6and129S6inbred strains[40,41],wefoundnodifferencesinsensorimotor functionorspatialdependentlearninginmiceofthesame genotypeandonanF1FVB/B6versusanF1FVB/129 strainbackground. Interestingly,aged(6.5-and10.5-month-old)rTg4510B6micedidnothavesignificantlydifferenthumantaulevels, asevaluatedbysolublehumantau.Totalsolubletaulevels, though,weresignificantlyincreasedinagedrTg4510B6mice ascomparedtorTg4510micewh enanantibodyspecificto bothhumanandmousetauwasused.Assessmentoftau proteinproductioninoldmice,however,iscomplicatedby significantneurodegenerati onasthoseneuronsexpressing humantauarelikelytheneuronsthathavediedorwilldie. Giventhis,themodestbutsignificantlyelevatedlevelsof humantauexpressioninth e2.5-month-oldrTg4510B6micecomparedtorTg4510arelikelytobemoreaccurate. Inoldmice,thebiochemicalanalysisoftaurevealed increasedphospho-tauinthesolublebrainfractionof rTg4510B6micecomparedtorTg4510miceusingthe PHF1antibody.Additionally,IHCanalysisrevealedthat PHF1stainingwasincreasedinboththeforebrainand hindbrainofrTg4510B6mice,whileCP13burdenwasincreasedinforebrainregions(cortexandhippocampus), butnotinthebrainstemofoldrTg4510B6micecompared tooldrTg4510mice.Furthermore,NTB6micealsohad (Seefigureonpreviouspage.) Figure10 IncreasedPHF1staininginolderNTB6micecomparedtoNTmice. NTmiceonanF1FVB/B6background(NTB6)hadincreased PHF1(pS396/404tau)staininginthecortex,hippocampusandbrainstemregionsofthebraincomparedtoNTmiceonanF1FVB/129 background(NT). (A) Representativeimagesfromtheprimarysensorycortex,hippocampalformationandbrainstemofNTB6andNTmicestained withPHF1. (B) QuantificationofPHF1burdeninthecortex,hippocampus,andbrainstemof6.5-and10.5-month-oldNTB6andNTmice.Cortical PHF1stainingwassignificantlyincreasedinNTB6micecomparedtoNTmiceonlyat10.5monthsofage,whileat6.5and10.5monthsofage PHF1burdenwasincreasedinthehippocampalformationandbrainstem.Eachtrianglerepresentsanindividualmousewiththemeanindicated bytheblackline,n=3-7percohort.***p<0.0001[two-wayANOVA(strainxage):maineffectofstrainindicated].###p<0.001 ( posthoc Bonferonni ’ smultiplecomparisonstest).Scalebarsrepresent200 m.CC=Corpuscallosum;7N=7thCranialnerve. Table3Two-wayANOVAofstrainbackgroundandageeffectsonphospho-tauburdenin6.5and10.5month-oldNTB6micecomparedtoNTmiceStrainbackgroundAgeInteraction RegionstainF(DFn,DFd)PvalueF(DFn,DFd)PvalueF(DFn,DFd)Pvalue CortexCP13 F(1,14)=9.90P<0.01F(1,14)=1.50P=0.24F(1,14)=3.30P=0.09 CortexPHF1 F(1,14)=18.00P<0.001F(1,14)=9.30P<0.01F(1,14)=14.00P<0.01 HippCP13 F(1,14)=1.30P=0.28F(1,14)=0.04P=0.84F(1,14)=0.01P=0.92 HippPHF1 F(1,14)=33.00P<0.0001F(1,14)=0.32P=0.58F(1,14)=4.50P=0.05 BSCP13 F(1,14)=0.03P=0.86F(1,14)=0.28P=0.61F(1,14)=3.20P=0.10 BSPHF1 F(1,14)=40.00P<0.0001F(1,14)=9.80P<0.01F(1,14)=1.20P=0.29 Bailey etal.MolecularNeurodegeneration 2014, 9 :8 Page14of20 http://www.molecularneurodegeneration.com/content/9/1/8

PAGE 15

Figure11 (Seelegendonnextpage.) Bailey etal.MolecularNeurodegeneration 2014, 9 :8 Page15of20 http://www.molecularneurodegeneration.com/content/9/1/8

PAGE 16

increasedPHF1andCP13stainingcomparedtoNT micethatwasspatiotemporallyparalleltotheincreased PHF1andCP13staininginrTg4510B6miceversusrTg4510 mice.Importantly,analysisof64kDatauinthebiochemicallyabnormal,sarko syl-insolublefractionand grossassessmentsofneurodegenerationthroughhemibrainweights,hippocampalareas,andCA1neuronal thickness,indicatednodifferencesbetweenrTg4510mice ondifferentstrainbackgrounds.Takentogether,these resultssuggestthatthedifferencesweobservedinsolublephosphorylatedtaulevelsandphospho-tauburden ofrTg4510B6micecouldbeattributedtoincreased phosphorylationofendogenousmousetauratherthan transgenichumantau.Regardless,theenhancedmurine tauphosphorylationdoesnotappeartoaltertoultimate neurodegenerativephenotypeobservedinrTg4510B6mice.Interestingly,ithasbeenreportedthat Dab1 deficient miceonaC57BL/6strainbackgroundhaveincreased murinetauphosphorylationcomparedto Dab1 deficient miceonaBALB/cByJstrainbackground[42].Further,the increasedmurinephospho-tauhasbeencorrelatedwith higherexpressionofanovelmodifieroftauphosphorylation, Stk25 ,inC57BL/6mice[43].In Dab1 deficientmice onaC57BL/6background,increasedphosphorylationof mousetauwasnotassociatedwithovertneurodegeneration.Thesefindingshighlightacriticalconsiderationwhen studyingphospho-taualterat ionsinmicethatcontainboth mouseandhumantau.ConclusionsHerewereportthatintroductionoftheC57BL/6strain intotherTg4510mousebackgroundminimallyaltersthe presentationoftaupathologythatwasoriginallyreported inrTg4510ontheF1FVB/129background.At2.5months ofage,rTg4510B6micehadminorincreasesinhumantau expressioncomparedtorTg4 510mice,butcognitivedeclineandphosphorylationoftauwasequivalentinrTg4510 miceonbothstrainbackgrounds.Latestagetauopathyin rTg4510B6micewasrobustwithequivalentinsolubletau aggregationandgrossneurodegenerationtorTg4510mice. Additionally,weobservedminor,butsignificant,increases intauphosphorylationinbotholderrTg4510B6miceas comparedtorTg4510mice,andinolderNTB6miceas comparedtoNTmice.Overall,thesestudiesprovide evidencethatrTg4510micecanbecrossedtoaB6/NTac backgroundwithoutlossordelayoftheoriginalphenotype.MethodsMiceThegenerationoftherTg4510mousemodelthatusesa systemofresponderandactivatortransgenestoexpress humanmutant(P301L)tauhasbeenpreviouslydescribed [2].Briefly,micecarryingarespondergenewithhuman tauP301LcDNAdownstreamofatetracyclineresponse element(TRE)weremaintainedonanFVB/Nbackground whilemicecarryinganactivatorgenewiththetetracycline transactivator(tTA)downstreamofacalcium/calmodulin kinaseII (CaMKII )promoterweremaintainedona 129S6background(tTA129S).TRE-taurespondermiceare crossedwithtTA129SactivatormicetoproducerTg4510 miceonanF1FVB/129Sbackgroundwithhumanmutant tauexpressionfocusedwithinforebrain.tTA129Smicewere obtainedbycrossingoffspringcarryingtheCaMKII -tTA transgene[8]with129S6/SvEvmicepurchasedfrom Taconic[2](Figure1).TheoriginaltTA129Smicewere acquiredfromGeorgeCarlson.Concurrently,tTA miceweremaintainedonaB6background(tTAB6)by crossingtheCaMKII -tTAmice[8]toC57BL/6NTac mice(Taconic)formorethantengenerations[44].The originaltTAB6micewereacquiredfromLi-HueiTsai. TotesttheeffectsoftheB6strainbackgroundonthe progressionoftauopathyintherTg4510mousemodel, wecrossedTRE-taurespondermicetotTAB6activatormice toproducerTg4510B6miceonanF1FVB/B6geneticbackground(Figure1).TheinitialcharacterizationofrTg4510 indicatedthatsingletransgenic(tauonlyandtTAonly)and non-transgenic(NT)littermateshavesimilarperformances inMorriswatermaze,sotomaximizethenumberof rTg4510micethatcouldberunwithinthesametest, onlyNTmicewereusedascontrols.Cohortsincluded 2.5-,6.5-and10.5-month-oldrTg4510,rTg4510B6and NTlittermatesofbothsexes(seeTable4fordetails). Grubb ’ sanalysisofthewesternblotofsarkosyl-insoluble tau(seeprotocolbelow)ide ntifiedone2.5-month-old (Seefigureonpreviouspage.) Figure11 OlderrTg4510B6micehaveequivalentgrossneurodegeneration. Grossbrainandhippocampalatrophywasequivalentin rTg4510B6andrTg4510miceat6.5and10.5monthsofage. (A) AllmiceonanF1FVB/B6backgroundhadsmallerhemi-brainsthanallmiceon anF1FVB/129Sbackground(p<0.05).Independentofstrainbackground,hemi-brainweightsforrTg4510miceweresignificantlydecreased comparedtoNTmice. (B) Hippocampalatrophy,asassessedbydecreasedhippocampalarea,wassignificantlyincreasedinage-matchedrTg4510 micecomparedtoNTlittermates,withnodifferencebetweenrTg4510miceondifferentstrainbackgrounds. (C) rTg4510B6andrTg4510mice hadsimilarCA1indexvaluesthatweresignificantlylessthanthatofNTmice,indicatingincreasedneuronallossintheCA1hippocampal sub-region. (D) Overall,rTg4510micehadgrossatrophyoftheforebrainandventricleenlargementcomparedtoNTmicethatwasindistinguishable betweenage-matchedrTg4510miceondifferentstrainbackgrounds.In rTg4510mice,thehippocampalformationwasvisiblyreducedinsize withsubstantialthinningoftheneuronallayersintheCA1anddentategyrussub-regions. (A-C) Eachdotrepresentsanindividualmousewith themeanindicatedbytheblackline.Datawasanalyzedviamultifactorial( agexgenotypexstrain)ANOVA.***p<0. 001(maineffectofgenotypeindica ted). n=2-7percohort.Scalebarsrepresent200 m. Bailey etal.MolecularNeurodegeneration 2014, 9 :8 Page16of20 http://www.molecularneurodegeneration.com/content/9/1/8

PAGE 17

malerTg4510mouseasanoutlierduetoextremely hightauaggregation.Allwesternblotdatafromthis mousewasexcludedfromthefinalresultsasthismay havebeenatissuepreparationerror. Micewerehousedandtreatedinaccordancewiththe NIHGuidefortheCareandUseofLaboratoryAnimals. Allanimalprocedureswereapprovedandconductedin accordancewiththeMayoClinicInstitutionalAnimal CareandUsecommitteeandtheUniversityofFlorida AnimalCareandUsecommittee.Miceweremaintained inapathogen-freefacilityona12hourlight/darkcycle withwaterandfoodprovided adlibitum .MorriswatermazerTg4510,rTg4510B6andNTlittermatesofbothsexes underwentMorriswatermaze(MWM)testingat 2.5monthsofage.Micewerehandledfor6daysprior totheinitiationofbehavioraltraining.MWMtesting wasperformedina60cmhighand1.5mdiameter circularpoolfilledwithwa termaintainedbetween24 and26Candthatwasmadeopaqueusingnon-toxic paint.Theswimpathofeachmousewasrecordedbya videocamerasuspended2.5moverthecenterofthepool andconnectedtothevideotrackingsystem(HVSImage AdvancedTrackerVP2000,HVSImage,Buckingham,UK). Curtainssurroundingaportionofthepoolwereusedto blocktheviewofthecomputer.Boththecurtainsand wallsoftheroomcontaineddark,geometricshapesto beusedasvisualcues. MWMtestingoccurredover6consecutivedayswith taskacquisitiontrainingondays1through5andwith probetrialstoevaluatespatialmemoryonday3,priorto theonsetofthetrainingsession,andonday6.Fortraining sessions,a15cmdiameterplatformwassubmerged1cm underthesurfaceofthewater inthemiddleofthetarget quadrant.Thehiddenescapeplatformwaslocatedinthe sametargetquadrantforeachsessionforeachmouse.The mousewastransportedtothepoolinaholdingcontainer andreleasedfacingthewallofthepoolfromeachof thecardinaldirectionsinasemi-randomfashion.Once released,themousehad60secondstolocatethehidden platform.Ifamousefailedtofindtheplatform,thenit wasgentlyguidedtotheplatformandeachmousewas allowedtoremainontheplatformfor10seconds.Each mousereceived4trainingtrialsperday.Duringtheprobe trialsondays3and6,thehiddenplatformwasremoved fromthepoolandeachanimalsearchedforthehidden platformfor60seconds. PriortothestartoftheMWM,the2.5-month-oldcohort underwentavisiblecuedtestt oevaluatesensorimotorfunction.AcurtainsurroundedtheMWMtanksothatspatial cuescouldnotbereferencedandaplatform0.5cmabove thewaterthatcontainedavisualcue(atallblackrod)was placedinthetank.Boththequa drantlocationofthevisible cuedplatformandthemousereleasepointswerechanged semirandomlyforeachtrialtopreventhabituationtoaparticularquadrant.Miceweregiven90secondstoreachthe platformbeforebeinggentlyg uidedtotheplatformwhere themiceremainedfor10seconds.Eachmousewastested4 timesperdayfor3days.TissueharvestandpreparationMicewereeuthanizedbycervicaldislocationandthe brainswererapidlyremovedandcutdownthemidline. Thelefthemispherewasdropfixedin10%formalinfor immunohistochemicalanalysisandtherighthemisphere wasfrozenondryiceandthenstoredat 80C.Fortau biochemicalanalysis,sarkosylfractionationwasperformed onthefrozenbrainsaspreviouslydescribed[2].Specifically, eachwholehemispherewashomogenizedin10volumes ofhomogenatebuffer[50mMTris – HCl,274mMNaCl, 5mMKCl,1%proteaseinhibitormixture(Sigma),1% phosphataseinhibitorcocktailsIandII(Sigma),and1mM phenylmethylsulfonylfluoride(PMSF),(pH8.0)]and200 l ofhomogenatewasthencentrifugedat150,000xgfor 15minutesat4C.Thesupernatant,whichcontains thesolubletauspecies,wascollectedandtheprotein concentrationdeterminedusingastandardBCAprotein assay(Pierce).Pelletswerefurtherhomogenizedin3 volumes(600 l)ofBufferB[10mMTris – HCl(pH7.4), 0.8MNaCl,10%Sucrose,1mMEGTAand1mMPMSF] andcentrifugedfor15minutesat150,000xgat4C.The supernatantswereincubatedwith1%sarkosyl(Sigma)for onehourat37Candthencentrifugedat150,000xgfor 30minutesat4C.Thesarkosyl-insolublepelletwasresuspendedin20 lTEbuffer[10mMTris – HCl(pH8.0), 1mMEDTA]toobtainthebiochemicalequivalentof neurofibrillarytangles.AntibodiesTauantibodiesusedincludedthepolyclonalantibodyE1 (specificforaminoacids19-33ofhumantau)[45]and themonoclonalantibodyTau5(providedbyDr.LesterI. Binder,NorthwesternUnive rsityMedicalSchool)for westernblotanalysisandthemonoclonaltauantibodies Table4Age,numberandsexofmiceusedineach experimentalprocedureAgeAnalysisrTg4510B6NTB6rTg4510NT 2.5Months MWM5F3F,2M2F,3M2F,3M WB6F,4M1F,1M5F,4M1F,1M 6.5Months WB2F,1M-4FIHC2F,1M3M5F5F,2M 10.5Months WB1F,2M2M2F,2M1F,1M IHC1F,3M4M2F,3M3F,2MKey:MWM=Morriswatermaze;WB=Westernblot;IHC=Immunohistochemistry; NT=Non-transgenic;F=Female;M=Male.Bailey etal.MolecularNeurodegeneration 2014, 9 :8 Page17of20 http://www.molecularneurodegeneration.com/content/9/1/8

PAGE 18

CP13(specificforpS202tau)andPHF1(specificfor pS396/404tau)(providedbyDr.PeterDavies,The EinsteinInstituteforMedicalResearch,Manhasset,NY) forbothwesternblotandimmunohistochemicalanalysis. Anti-GAPDH(Biodesign)wasusedasaloadingcontrol forwesternblotanalysis.WesternblottingFortauproteinanalysis,1-5 gofthesolublefractionand 3 l(6.5-and10.5-month-oldmice)or4.5 l(2.5-montholdmice)ofthesarkosyl-insolublefractionwereloadedin eachwell.BrainlysatesweredilutedinNovexTris-glycine SDS-samplebuffer(Invitrogen)with -mercaptoethanol andheatdenaturedat95Cfor5minutesbeforebeing loadedonto26-well10%Tris-g lycinegels(Invitrogen)and separatedbySDS-PAGE.ProteinwastransferredinCAPS transferbuffer(Sigma)toPVDFmembranes.Membranes werethenblockedinTrisbufferedsalineplus0.1% TritonX-100(TBS-T)with5%non-fatmilkandincubated overnightwithantibodydilutedinTBS-T/5%milk.MembraneswerewashedinTBS-T,incubatedwithperoxidaseconjugatedgoatanti-mouseHRPorgoatanti-rabbitHRP secondaryantibodies(Jack sonImmunoResearch)for1 houratroomtemperatureandwashedagaininTBS-T. MembranesweredevelopedusingWesternLightning Plus(PerkinElmer)andim agedusingaFluorChemE System(ProteinSimple).Therelativelevelsofimmunoreactivityweredeterminedbydensitometryusing thesoftwareAlphaViewSA(ProteinSimple).Relative solubletauandphospho-taulevelswerequantifiedby normalizingproteinlevelstoGAPDHlevels.Therelative levelsofphospho-tauinthesarkosyl-insolublefractionwere determinedbynormalizingCP13andPHF1levelswithE1 levelsandTau5levels(datanotshown)foreachmouseto correctfortheamountoftau aggregatedinthesarkosylinsolublefractionofthatanimal.Tauandphospho-tau levelsarepresentedrelativeto6.5-month-oldrTg4510mice fortheoldercohorts(6.5and10.5monthsofage)and relativeto2.5-month-ol drTg4510micefortheyoung cohort(2.5monthsofage).ImmunohistochemicalanalysisFixedmousebrainswereparaffinembeddedandcutinto 5 msagittalsections.Hematoxylinandeosin(H&E)stainingwasperformedonatleasttwobrainsectionsfromeach mousetoalignallbrainstoapproximately1.3mmlateral tothemidlineusingamousebrainatlas[46].Stainedslides weredigitallyscannedusingaScanscopeXTscanner. Hippocampalareameasure mentswereperformedona sagittalsectionstainedwithH&Eobtainedforeachmouse. Thehippocampalformationwasthenoutlinedusing ImageScopeversion10software(Aperio,Vista,CA)and theresultantareavalueforeachanimalwasusedtogenerategroupmeans.Thesamesectionusedforhippocampal areameasurementsperanimalwasalsousedtocalculate theCA1index.Threelineswe repseudo-randomlydrawn perpendiculartothemainaxisoftheCA1celllayerand thenumberofintactneuronalcellbodiestouchingeach linewasblindlycounted.Eachofthecountswassummed toobtaina “ CA1index ” valueperanimal. Standardimmunohistochemicalprocedureswereused toimmunostaintissueswithPHF1andCP13antibodies andcounterstainwithhematoxylinusingtheDako UniversalAutostainerwithDAKOEnvision+HRP system(Dako).Stainedslidesweredigitallyscanned usingaScanscopeXTscannerandwereanalyzedusing ImageScopesoftware.Positivepixelcountalgorithmswere createdtomeasurethepercentpositivityofthesecondary antibody,specificallychromogenDAB,inaselectedregion.Distinctalgorithmswereusedforburdenanalysisof eachprimaryantibodyineitherrTg4510orNTlittermates.Thebrainregionsanalyzedincludedthecortexand thehippocampalformation,whichexpressedhighlevels ofhumantau,andthebrainstem,whichhasverylow humantauexpressioninrTg4510mice.StatisticalanalysisResultsforMWMtrainingaredisplayedasthegroup meanSEMandwereanalyzedusingmultifactorialrepeatedmeasures(RM)ANOVA,withstrainbackground andgenotypeasbetweensubjectfactorsandtrainingdays aswithinsubjectfactors.Whenappropriate, posthoc comparisonswereperformed.TheprobetrialforMWM wasanalyzedusingtwo-wayANOVA(genotypexstrain) with posthoc Bonferroni ’ smultiplecomparisonstest. Westernblotresultsfrom 2.5-month-oldmicewere analyzedwithanunpaired,twotailedStudent ’ st-test. Assessmentsofgrossneurop athologyin6.5-and10.5month-oldrTg4510andrTg4510B6miceandNTB6littermateswereanalyzedusingone-wayANOVAwith posthoc analysiswithBonferroni ’ smultiplecomparisontests. WesternblotandIHCdatafrom6.5-and10.5-month-old rTg4510andrTg4510B6micewereanalyzedusingtwo-way ANOVAwithstrainbackgroundandageasindependent variablesand posthoc analysiswithBonferroni ’ smultiple comparisontest.Grubb ’ sanalysiswasusedtoidentifyoutliersinthewesternblotanalysi sofsarkosyl-insolubletau. AnalyseswereperformedusingGraphPadPrismversion 6.00software(GraphPadSof tware)andSPSSversion20.0 (IBM).Inallcases,p<0.05was consideredtobestatisticallysignificant.Abbreviations TRE: Tetracyclineresponseelement;CaMKII :Calcium/calmodulinkinaseII ; B6:C57BL/6mousestrain;NT:Non-transgenicmiceonanFVB/Nx129S6 strainbackground;NTB6:Non-transgenicmiceonanFVB/NxC57BL/6N strainbackground;tTA:Tetracyclinetransactivator;tTA129S:MicewithatTA transgeneona129S6strainbackground;tTAB6:MicewithatTAtransgene onaC57BL/6Nstrainbackground;rTg4510:Micewithtauresponderand tTAtransgenesonanFVB/Nx129S6strainbackground;rTg4510B6:MicewithBailey etal.MolecularNeurodegeneration 2014, 9 :8 Page18of20 http://www.molecularneurodegeneration.com/content/9/1/8

PAGE 19

tauresponderandtTAtransgenesonanFVB/NxC57BL/6Nstrain background;MWM:Morriswatermaze;IHC:Immunohistochemical. Competinginterests JLholdsIPandhasreceivedroyaltiesinexcessof$10,000NIHthresholdfor significantconflictofinterestinthepastyearfromtherTg4510mouse model. Authors ’ contributions RMB,carriedoutanimalbreedingandharvests,tissueprocessing, immunohistochemicalstudies,dataanalysis,manuscriptwriteupandaided inconceptualizationofstudy.JH,performedanimalbehavioraltests,western blotting,dataanalysisandcontributedtomanuscriptwriteup.JK,carried outanimalbreedingandharvesting.NS,contributedtowesternblotting. DWD,contributedtothegenerationofstainedmousebrainsectionsfor immunohistochemicalanalysisandmanuscriptediting.JL,conceivedstudy andinterpreteddata,contributedresearchanimalsandperformed manuscriptediting.Allauthorsreadandapprovedthefinalmanuscript. Acknowledgements WewouldliketoacknowledgesupportbyNINDS(NIH1F31NS078896-01-A1) toRMBand(P50NS072187)toDWD.Additionalsupportwasprovidedfrom MayoClinictoDWDandJLandtheUniversityofFloridaDepartmentof NeuroscienceandCenterforTranslationalResearchinNeurodegenerative DiseasetoNSandJL. Received:30July2013Accepted:10January2014 Published:15January2014 References1.LewisJ,McGowanE,RockwoodJ,MelroseH,NacharajuP,VanSlegtenhorstM, Gwinn-HardyK,PaulMurphyM,BakerM,YuX, etal : Neurofibrillarytangles, amyotrophyandprogressivemotordisturbanceinmiceexpressingmutant (P301L)tauprotein. NatGenet 2000, 25: 402 – 405. 2.SantacruzK,LewisJ,SpiresT,PaulsonJ,KotilinekL,IngelssonM,Guimaraes A,DeTureM,RamsdenM,McGowanE, etal : Tausuppressionina neurodegenerativemousemodelimprovesmemoryfunction. Science(NewYork,NY) 2005, 309: 476 – 481. 3.BolmontT,ClavagueraF,Meyer-LuehmannM,HerzigMC,RaddeR, StaufenbielM,LewisJ,HuttonM,TolnayM,JuckerM: Inductionoftau pathologybyintracerebralinfusionofamyloid-beta-containingbrain extractandbyamyloid-betadepositioninAPPxTautransgenicmice. AmJPathol 2007, 171: 2012 – 2020. 4.McKinneyBC,SchneiderJS,SchaferGL,LowingJL,MohanS,ZhaoMX, HengMY,AlbinRL,SeasholtzAF,AkilH,MurphyGG: Decreasedlocomotor activityinmiceexpressingtTAundercontroloftheCaMKIIalpha promoter. Genes,Brain,Behavior 2008, 7: 203 – 213. 5.HanHJ,AllenCC,BuchoveckyCM,YetmanMJ,BornHA,MarinMA,Rodgers SP,SongBJ,LuHC,JusticeMJ, etal : Strainbackgroundinfluences neurotoxicityandbehavioralabnormalitiesinmiceexpressingthe tetracyclinetransactivator. JNeurosci 2012, 32: 10574 – 10586. 6.QiaoG,LiZ,MintoAW,ShiaJ,YangL,BaoL,TschoppJ,GaoJX,WangJ, QuiggRJ,ZhangJ: Alteredthymicselectionbyoverexpressingcellular FLICEinhibitoryproteininTcellscauseslupus-likesyndromeina BALB/cbutnotC57BL/6strain. CellDeathDiffer 2010, 17: 522 – 533. 7.ClarkAG,FanQ,BradyGF,MackinKM,CoffmanED,WestonML,FosterMH: Regulationofbasementmembrane-reactiveBcellsinBXSB,(NZBxNZW) F1,NZB,andMRL/lprlupusmice. Autoimmunity 2013, 46: 188 – 204. 8.MayfordM,BachME,HuangYY,WangL,HawkinsRD,KandelER: Controlof memoryformationthroughregulatedexpressionofaCaMKIItransgene. Science(NewYork,NY) 1996, 274: 1678 – 1683. 9.KrestelHE,MayfordM,SeeburgPH,SprengelR: AGFP-equippedbidirectional expressionmodulewellsuitedformonitoringtetracycline-regulatedgene expressioninmouse. NucleicAcidsRes 2001, 29: E39. 10.ZhouH,HuangC,YangM,LandelCP,XiaPY,LiuYJ,XiaXG: Developing tTAtransgenicratsforinducibleandreversiblegeneexpression. IntJBiol Sci 2009, 5: 171 –181. 11.IqbalK,LiuF,GongCX,AlonsoAdelC,Grundke-IqbalI: Mechanismsof tau-inducedneurodegeneration. ActaNeuropathol 2009, 118: 53 – 69. 12.OtvosLJr,FeinerL,LangE,SzendreiGI,GoedertM,LeeVM: Monoclonal antibodyPHF-1recognizestauproteinphosphorylatedatserineresidues 396and404. JNeurosciRes 1994, 39: 669 – 673. 13.WeaverCL,EspinozaM,KressY,DaviesP: Conformationalchangeasone oftheearliestalterationsoftauinAlzheimer ’ sdisease. NeurobiolAging 2000, 21: 719 – 727. 14.RamsdenM,KotilinekL,ForsterC,PaulsonJ,McGowanE,SantaCruzK, GuimaraesA,YueM,LewisJ,CarlsonG, etal : Age-dependentneurofibrillary tangleformation,neuronloss,andmemoryimpairmentinamousemodel ofhumantauopathy(P301L). JNeurosci 2005, 25: 10637 – 10647. 15.GreenbergSG,DaviesP: ApreparationofAlzheimerpairedhelical filamentsthatdisplaysdistincttauproteinsbypolyacrylamidegel electrophoresis. ProcNatlAcadSciUSA 1990, 87: 5827 – 5831. 16.O ’ LearyTP,GunnRK,BrownRE: Whatarewemeasuringwhenwetest straindifferencesinanxietyinmice? BehavGenet 2013, 43: 34 – 50. 17.AggletonJP,SandersonDJ,PearceJM: Structurallearningandthe hippocampus. Hippocampus 2007, 17: 723 – 734. 18.SpiresTL,OrneJD,SantaCruzK,PitstickR,CarlsonGA,AsheKH,HymanBT: Region-specificdissociationofneuronallossandneurofibrillarypathology inamousemodeloftauopathy. AmJPathol 2006, 168: 1598 – 1607. 19.BergerZ,RoderH,HannaA,CarlsonA,RangachariV,YueM,WszolekZ, AsheK,KnightJ,DicksonD, etal : Accumulationofpathologicaltau speciesandmemorylossinaconditionalmodeloftauopathy. JNeurosci 2007, 27: 3650 – 3662. 20.RamalhoRM,VianaRJ,CastroRE,SteerCJ,LowWC,RodriguesCM: ApoptosisintransgenicmiceexpressingtheP301Lmutatedformof humantau. MolMed 2008, 14: 309 –317. 21.Spires-JonesTL,deCalignonA,MatsuiT,ZehrC,PitstickR,WuHY,OsetekJD, JonesPB,BacskaiBJ,FeanyMB, etal : Invivoimagingrevealsdissociation betweencaspaseactivationandacuteneuronaldeathintangle-bearing neurons. JNeurosci 2008, 28: 862 – 867. 22.DickeyC,KraftC,JinwalU,KorenJ,JohnsonA,AndersonL,LebsonL,Lee D,DicksonD,deSilvaR, etal : Aginganalysisrevealsslowedtauturnover andenhancedstressresponseinamousemodeloftauopathy. AmJPathol 2009, 174: 228 – 238. 23.LeeDC,RizerJ,SelenicaML,ReidP,KraftC,JohnsonA,BlairL,GordonMN, DickeyCA,MorganD: LPS-inducedinflammationexacerbatesphospho-tau pathologyinrTg4510mice. JNeuroinflammation 2010, 7: 56. 24.RocherAB,CriminsJL,AmatrudoJM,KinsonMS,Todd-BrownMA,LewisJ, LuebkeJI: Structuralandfunctionalchangesintaumutantmiceneurons arenotlinkedtothepresenceofNFTs. ExpNeurol 2010, 223: 385 – 393. 25.FoxLM,WilliamCM,AdamowiczDH,PitstickR,CarlsonGA,Spires-JonesTL, HymanBT: Solubletauspecies,notneurofibrillaryaggregates,disrupt neuralsystemintegrationinatautransgenicmodel. JNeuropatholExp Neurol 2011, 70: 588 – 595. 26.BartenDM,FanaraP,AndorferC,HoqueN,WongPY,HustedKH,Cadelina GW,DecarrLB,YangL,LiuV, etal : Hyperdynamicmicrotubules,cognitive deficits,andpathologyareimprovedintautransgenicmicewithlow dosesofthemicrotubule-stabilizingagentBMS-241027. JNeurosci 2012, 32: 7137 – 7145. 27.LjungbergMC,AliYO,ZhuJ,WuCS,OkaK,ZhaiRG,LuHC: CREB-activity andnmnat2transcriptionaredown-regulatedpriortoneurodegeneration, whileNMNAT2over-expressionisneuroprotective,inamousemodelof humantauopathy. HumMolGenet 2012, 21: 251 – 267. 28.Spires-JonesTL,FoxLM,RozkalneA,PitstickR,CarlsonGA,KazantsevAG: Inhibitionofsirtuin2withsulfobenzoicacidderivativeAK1isnon-toxic andpotentiallyneuroprotectiveinamousemodeloffrontotemporal dementia. FrontPharmacol 2012, 3: 42. 29.AbisambraJF,JinwalUK,BlairLJ,O ’ LearyJCIII,LiQ,BradyS,WangL,Guidi CE,ZhangB,NordhuesBA, etal : Tauaccumulationactivatestheunfolded proteinresponsebyimpairingendoplasmicreticulum-associated degradation. JNeurosci 2013, 33: 9498 – 9507. 30.NashKR,LeeDC,HuntJBJr,MorgantiJM,SelenicaML,MoranP,ReidP, BrownlowM,Guang-YuYangC,SavaliaM, etal : Fractalkineoverexpressionsuppressestaupathologyinamousemodeloftauopathy. Neurobiol Aging 2013, 34: 1540 – 1548. 31.SelenicaML,BrownlowM,JimenezJP,LeeDC,PenaG,DickeyCA,Gordon MN,MorganD: Amyloidoligomersexacerbatetaupathologyinamouse modeloftauopathy. NeurodegenerDis 2013, 11: 165 – 181. 32.ClippingerAK,D ’ AltonS,LinWL,GendronTF,HowardJ,BorcheltDR, CannonA,CarlomagnoY,ChakrabartyP,CookC, etal : RobustcytoplasmicBailey etal.MolecularNeurodegeneration 2014, 9 :8 Page19of20 http://www.molecularneurodegeneration.com/content/9/1/8

PAGE 20

accumulationofphosphorylatedTDP43intransgenicmodelsoftauopathy. ActaNeuropathol 2013, 126: 39 – 50. 33.PerezPD,HallG,KimuraT,RenY,BaileyRM,LewisJ,FeboM,SaharaN: In vivofunctionalbrainmappinginaconditionalmousemodelofhuman tauopathy(tau(P301L))revealsreducedneuralactivityinmemory formationstructures. MolNeurodegeneration 2013, 8: 9. 34.BotheGW,BolivarVJ,VedderMJ,GeistfeldJG: Geneticandbehavioral differencesamongfiveinbredmousestrainscommonlyusedinthe productionoftransgenicandknockoutmice. Genes,Brain,Behavior 2004, 3: 149 – 157. 35.KrezowskiJ,KnudsonD,EbelingC,PitstickR,GiriRK,SchenkD,WestawayD, YounkinL,YounkinSG,AsheKH,CarlsonGA: Identificationofloci determiningsusceptibilitytothelethaleffectsofamyloidprecursor proteintransgeneoverexpression. HumMolGenet 2004, 13: 1989 – 1997. 36.BryantCD,ZhangNN,SokoloffG,FanselowMS,EnnesHS,PalmerAA, McRobertsJA: BehavioraldifferencesamongC57BL/6substrains: implicationsfortransgenicandknockoutstudies. JNeurogenet 2008, 22: 315 – 331. 37.MekadaK,AbeK,MurakamiA,NakamuraS,NakataH,MoriwakiK,ObataY, YoshikiA: GeneticdifferencesamongC57BL/6substrains. ExpAnim 2009, 58: 141 – 149. 38.ZuritaE,ChagoyenM,CanteroM,AlonsoR,Gonzalez-NeiraA,Lopez-Jimenez A,Lopez-MorenoJA,LandelCP,BenitezJ,PazosF,MontoliuL: Genetic polymorphismsamongC57BL/6mouseinbredstrains. TransgenicRes 2011, 20: 481 – 489. 39.PravtchevaDD,WiseTL: Transgeneinstabilityinmiceinjectedwithan invitromethylatedIgf2gene. MutationRes 2003, 529: 35 – 50. 40.HolmesA,WrennCC,HarrisAP,ThayerKE,CrawleyJN: Behavioralprofiles ofinbredstrainsonnovelolfactory,spatialandemotionaltestsfor referencememoryinmice. Genes,Brain,Behavior 2002, 1: 55 – 69. 41.ClapcoteSJ,RoderJC: Surveyofembryonicstemcelllinesourcestrains inthewatermazerevealssuperiorreversallearningof129S6/SvEvTac mice. BehavBrainRes 2004, 152: 35 – 48. 42.BrichJ,ShieFS,HowellBW,LiR,TusK,WakelandEK,JinLW,MumbyM, ChurchillG,HerzJ,CooperJA: Geneticmodulationoftauphosphorylation inthemouse. JNeurosci 2003, 23: 187– 192. 43.MatsukiT,ZakaM,GuerreiroR,vanderBrugMP,CooperJA,CooksonMR, HardyJA,HowellBW: IdentificationofStk25asageneticmodifierofTau phosphorylationinDab1-mutantmice. PloSone 2012, 7: e31152. 44.CruzJC,TsengHC,GoldmanJA,ShihH,TsaiLH: AberrantCdk5activation byp25triggerspathologicaleventsleadingtoneurodegenerationand neurofibrillarytangles. Neuron 2003, 40: 471 – 483. 45.CroweA,Ksiezak-RedingH,LiuWK,DicksonDW,YenSH: TheNterminal regionofhumantauispresentinAlzheimer ’ sdiseaseproteinA68andis incorporatedintopairedhelicalfilaments. AmJPathol 1991, 139: 1463 – 1470. 46.PaxinosG,FranklinKBJ: Themousebraininstereotaxiccoordinates. 2nd edition.SanDiego:AcademicPress;2001.doi:10.1186/1750-1326-9-8 Citethisarticleas: Bailey etal. : EffectsoftheC57BL/6strainbackground ontauopathyprogressionintherTg4510mousemodel. Molecular Neurodegeneration 2014 9 :8. Submit your next manuscript to BioMed Central and take full advantage of: € Convenient online submission € Thorough peer review € No space constraints or color “gure charges € Immediate publication on acceptance € Inclusion in PubMed, CAS, Scopus and Google Scholar € Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Bailey etal.MolecularNeurodegeneration 2014, 9 :8 Page20of20 http://www.molecularneurodegeneration.com/content/9/1/8