Neurotoxicimpactofprotein

fragmentationandaggregationin

tauopathymousemodels

Inauguraldissertationzur

ErlangungderWürdeeinesDoktorsderPhilosophie

vorgelegtder

Philosophisch-NaturwissenschaftlichenFakultät

derUniversitätBasel

von

FrederikSprenger

AusSalzkotten,Deutschland

Basel,2017

OriginaldokumentgespeichertaufdemDokumentenserverderUniversitätBasel

edoc.unibas.ch

II

GenehmigtvonderPhilosophisch-NaturwissenschaftlichenFakultät

aufAntragvon

Fakultätsverantwortlicher: Prof.Dr.MarkusRüegg

Dissertationsleiter: PDDr.Dr.DavidT.Winkler

Koreferent: Prof.Dr.BernhardBettler

Basel,den18.04.2017

____________________________________

UnterschriftdesFakultätsverantwortlichen

Prof.Dr.MartinSpiess

(Dekan)

Preface

III

Preface

The following dissertation was written by the author. The “Introduction” is based on an

extendedversionofareviewmanuscriptinpreparation(Sprenger,Winkler,2017expected).

The“Results”sectionconsistsoftwopublishedmanuscriptsandadditionalpreliminarydata.

Intheco-first-authorshippublication(Ozcelik,Sprengeretal.,2016)theauthorsignificantly

contributedtoexperiments,analysis,andwritingprocess.Inthesecondco-authorpublication

(Skachokova,Sprengeretal.,2015)theauthorcontributedtosomeanalysisandfinalwriting.

Theadditionaldatasectionistheresultofownwork.

Acknowledgements

V

Acknowledgements

Mymostheartfeltthanksgotomyboss,DavidWinkler,forhisvaluablementoringandhis

substantialsupport.

I would like to expressmy deep gratitude toMarkus Tolnay and Stephan Frank for their

supportofmywork.

Iamgratefultoourcollaborators,inparticularMichelGoedertandGrahamFraser,fortheir

expertise.

Iparticularlywishtothankcolleaguesandtechnicians,fortheirusefuldiscussions,advices,

andpracticalsupportthroughoutmywork.Myspecialthanksareextendedtothestaffatthe

ZLFanimalfacilityfortheirsupport.

Finally,Iwishtothankmyfamily,mywifeLimaandmysonSam.Thankyouforeverything

thatmakethispossible.

Abstract

VII

Abstract

The microtubule-associated protein tau and its pathological modification constitute the

central pathology of various human neurodegenerative diseases, including Alzheimer’s

disease (AD), collectively termed ‘tauopathies’. Abundant hyperphosphorylation and

aggregation of tau is a disease-defining hallmark, yet the underlying pathogenic and

pathophysiological processes have remained only partly understood. In addition, protein

fragmentation is a frequently observed phenomenon in the course of various

neurodegenerative diseases; however, the contribution of tau fragmentation to the

pathogenesisoftauopathiesisstillamatterofdebate.

Inournovel induciblemousemodel,co-expressionoftruncatedandfull-lengthhumantau

provokes axonal transport failure, mitochondrial mislocalization, disruption of the Golgi

apparatusanddysregulationofsynapticproteinsassociatedwithextensivenervecelllossand

asevereneurologicalphenotypeasearlyas3weeksofage.Ofnote,thiswasparalleledonly

by the formation of soluble oligomeric tau species, and no insoluble filamentous tau

aggregates;therewith,identifyingoligomerictauspeciesastoxickeyplayersintaupathology.

Despitecontinuousfull-lengthtauexpression,micerecoveredfromtheneurotoxicinsultonce

truncated tau expressionwas halted. The inductionof drastic but reversible neurotoxicity

highlights the neurotoxic potential of tau fragments as pathogenic mediators in

neurodegenerativedisorders.

Thepresentworkimplicatesthecomplexityofproteinfragmentationandoligomerizationand

theirneurotoxicimpactinthecontextoftauopathiesandaimsforabetterunderstandingof

thecellularmechanismsunderlyingtautoxicity.

VIII

Index

IX

Index

Preface III

Acknowledgements V

Abstract VII

Index IX

1 Introduction 13

1.1 Chapter1:Tauproteinandneurodegeneration............................................................13

1.1.1 Neurodegeneration.........................................................................................................131.1.2 Proteinfragmentationandneurodegeneration..............................................................141.1.3 Caspasesandcalpainsandneurodegeneration..............................................................151.1.4 Neurodegenerativedisease-associatedproteins............................................................171.1.5 Tauprotein......................................................................................................................18

1.1.5.1 Cellularlocalizationanddomainorganizationoftau..........................................................181.1.5.2 Tauisoforms........................................................................................................................191.1.5.3 Taufunction.........................................................................................................................201.1.5.4 Post-translationalmodificationsoftau................................................................................21

1.1.5.4.1 Tauphosphorylation.......................................................................................................221.1.5.4.2 Tautruncation.................................................................................................................23

1.1.6 Tauopathies.....................................................................................................................251.1.7 Prion-likeseedinginneurodegenerativeproteinopathies..............................................26

1.1.7.1 CSFAβ..................................................................................................................................26

1.2 Chapter2:Proteinfragmentationinneurodegenerativedisorders................................28

1.2.1 Neurodegenerativedisorders..........................................................................................281.2.2 ProteinfragmentationinAD:Aβ.....................................................................................30

1.2.2.1 Alzheimer’sdisease(AD)......................................................................................................301.2.2.2 Aβ.........................................................................................................................................31

1.2.3 ProteinfragmentationinfamilialCAA:ABriandADan....................................................331.2.3.1 Cerebralamyloidangiopathies:familialBritishandDanishdementia................................331.2.3.2 ABriandADan......................................................................................................................34

1.2.4 ProteinfragmentationinPD:α-synuclein.......................................................................351.2.4.1 Parkinson'sdisease(PD)......................................................................................................351.2.4.2 α-synuclein...........................................................................................................................36

1.2.5 ProteinfragmentationinTRD:Htt,ataxin,andatrophin................................................371.2.5.1 Huntington’sdisease(HD)...................................................................................................371.2.5.2 htt........................................................................................................................................371.2.5.3 Otherpolyglutaminediseases.............................................................................................38

1.2.6 ProteinfragmentationinPriondiseases:PrPCandPrPSc.................................................391.2.6.1 Priondiseases......................................................................................................................391.2.6.2 Prionprotein........................................................................................................................39

Index

X

1.2.7 ProteinfragmentationinFTLDandALS:TDP-43.............................................................411.2.7.1 TDP-43proteinopathies.......................................................................................................411.2.7.2 TDP-43..................................................................................................................................42

2 Aimsofthework 45

3 Results 47

3.1 PublicationNo.1:

Co-expressionoftruncatedandfull-lengthtauinducessevereneurotoxicity................47

3.2 Preliminarydata:

Protectiveeffectofearlytauburdenonlateneurotoxicdistresslevel–Mechanisms underlyingtauopathyandconsequencesforfuturetherapies.......................................67

3.2.1 DelayedmotorphenotypeinagedP301SxTAU62on-offmiceafterrecoveryofsevereneurotoxicity....................................................................................................................68

3.2.2 ReducedtaupathologyinagedP301SxTAU62on-offmiceafterrecoveryofsevereneurotoxicity....................................................................................................................70

3.2.3 ReducedtauproteinlevelsinagedP301SxTAU62on-offmiceafterrecoveryofsevereneurotoxicity....................................................................................................................71

3.2.4 Supplementalmaterial....................................................................................................73

3.3 PublicationNo.2:

Amyloid-betainthecerebrospinalfluidofAPPtransgenicmicedoesnotshowprion-like properties.....................................................................................................................75

4 Discussion 84

5 MaterialsandMethods 97

5.1 Animals.........................................................................................................................97

5.1.1 Housingoftransgenicmice.............................................................................................975.1.2 TAU62mice.....................................................................................................................975.1.3 P301Smice......................................................................................................................985.1.4 ALZ17mice......................................................................................................................985.1.5 ALZ31mice......................................................................................................................995.1.6 P301SxTAU62mice..........................................................................................................995.1.7 ALZ17xTAU62mice..........................................................................................................995.1.8 ALZ31xTAU62mice..........................................................................................................995.1.9 P301SxALZ31mice...........................................................................................................995.1.10 ALZ17xALZ31mice.....................................................................................................1005.1.11 APP23mice................................................................................................................100

5.2 DNAisolationandgenotyping.....................................................................................100

5.3 Histologyandimmunohistochemistry.........................................................................102

5.3.1 Tissuepreparationandprocessing:Brain,SpinalcordandSciaticnerve......................1025.3.2 HematoxylinandEosinStaining....................................................................................103

Index

XI

5.3.3 Gallyassilverstaining.....................................................................................................1045.3.4 HolmesSilverNitrate-LuxolFastBluestaining..............................................................1055.3.5 MassonTrichromestaining(Sciaticnerve)....................................................................1065.3.6 Musclespreparation......................................................................................................1065.3.7 Myosin-ATPase(Adenosintriphosphatase)staining(pH4.2).........................................1075.3.8 Semithinsections(Sciaticnerve)...................................................................................1085.3.9 Para-Phenylendiamine(Sciaticnerve)...........................................................................1085.3.10 Electronmicroscopy..................................................................................................108

5.4 Sarkosylextraction.....................................................................................................109

5.5 WesternBlot...............................................................................................................111

5.6 Antibodies..................................................................................................................112

5.7 Behavioralassessment................................................................................................113

5.7.1 Grid-test.........................................................................................................................1135.7.2 Rotarodtest...................................................................................................................1135.7.3 Objectrecognitiontest..................................................................................................113

5.8 Statistics.....................................................................................................................114

6 References 115

7 Abbreviations 135

Introduction

13

1 Introduction

1.1 Chapter1

Tauproteinandneurodegeneration

1.1.1 Neurodegeneration

In human beings, neurodegenerative diseases are commonly characterized by progressive

dysfunctionanddeceaseofneuronsassociatedwithpathologicaldepositsofalteredproteins

inthebrainaswellasinperipheralorgans.Inpatientswithneurologicaldisorders,theclinical

manifestationscausedbymalfunctionofindividualgeneexpressionproductscorrelatewith

theaffectedbrain regions, linkingaparticulardisease-type to itspredominantphenotype.

Unique pathological conformers or misfolded proteins with modified native physiological

properties are integral parts and the core concept of diverse human ’proteinopathies’;

nevertheless, the understanding of the cellular and molecular bases underlying the

pathogenesisofneurodegenerativediseasesgraduallywidenedovertheyears,yetbeingfar

fromfullydisclosed.

The pathological conformation and subsequent aggregation of proteins is not solely

responsible for neuronal degeneration, rather a complex network of molecular events

ultimatelyleadingtoprogressiveneuronaldysfunctionanddeath.Forinstance,theubiquitine

Introduction

14

proteasome system (UPS) and autophagy-lysosomal pathways, asmajormechanisms for

degradation of numerous proteins, have been found to be relevant for the genesis and

progressionofseveralneurodegenerativediseases(Kelleretal.,2000;Nixon,2007;reviewed

inOddo,2008;Pickfordetal.,2008).Indeed,variousaggregatedproteinsaswellasinduction

of proteasome inhibitors have been shown to interfere with the highly regulated cell

physiologyby impairingUPSfunction(Benceetal.,2001;Davidetal.,2002;Gregorietal.,

1995;Kecketal.,2003;Leeetal.,2010;Snyderetal.,2003;Tsengetal.,2007).Incontrastto

their ability of non-aggregated and soluble unfolded protein degradation, oligomeric or

aggregated species are rather inaccessible to the catalytic core of the UPS; an efficient

autophagy-lysosomalmachinery isneededtodegradeaggregation-proneproteins, thereby

preventingneuropathologicalprocesses(Angladeetal.,1997;Bolandetal.,2008;Qinetal.,

2003). In addition, stimulationof autophagyhasbeen shown to reduce the generationof

pathologicalproteininclusionsinnervecells(Ozceliketal.,2013;Schaefferetal.,2012;Wang

etal.,2009b).

Aside further neurodegenerative disease-causing processes such as glutamate-induced

exocytoxic insults (Marchetti et al., 2004) or neuroinflammatory processes (Harry et al.,

2000), another event detrimental to neuronal homeostasis is mitochondrial injury by

neurodegeneration-associatedproteins;mitochondrialdysfunction,i.e.intermsofimpaired

mitochondrial trafficking insideneurons (Ruietal.,2010;Ruietal.,2006)oralterations in

mitochondrialdynamics (Wangetal.,2009a), iscrucially linkedtooxidativeandnitrosativ

stress (Cho et al., 2009; Hirai et al., 2001; Lustbader et al., 2004), contributing to

neuropathologicalprocesses.

1.1.2 Proteinfragmentationandneurodegeneration

Protein fragmentation is a frequently observed phenomenon in the course of various

neurodegenerativeprocesses.Indeed,small,aggregationpronecleavedproteinsareintegral

partsofaplethoraofdisordersincludingAlzheimer’sdiseases(AD);familialBritishandDanish

dementia(FBD,FDD);Parkinson’sdiseases(PD);TDP-43relateddisorders;andinothertriplet

expansiondisorderssuchasHuntingtondisease(HD)andspinocerebellar-ataxias(SCAs).The

initiationoffragmentationremainsmostlyelusive;mutationsrepresentthecauseofaltered

Introduction

15

cleavage in some hereditary variants of neurodegenerative diseases such as presenilin

mutationsinADortheframeshiftmutationsinFBD;bycontrast,inmostsporadicformsthe

causeforincreasedoraberrantfragmentationissimplynotknown.

However,thecontributionofproteinfragmentationtothepathogenesisofproteinopathiesis

not always evident and the neurotoxic potential of cleavage products is still a matter of

debate.Cleavageoccursatmultiplesitesofsinglelargeproteinswithresearchersconsiderthe

question of a causal relationship between protein fragmentation and disease, orwhether

fragmentation just being an epiphenomenon. Protein aggregates in neurodegenerative

disordersmayeitherconsistof(I)fragmentsalonederivedfromlargerprecursorproteins:as

in case of amyloid-β (Aβ) in AD or amyloid-Bri (ABri) in FBD (Vidal et al., 1999); or they

comprise(II)full-lengthandfragmentedproteinsinparallel:asincaseofα-synuclein(α-Syn)

inPD(Duftyetal.,2007;Liuetal.,2005)orinTDP-43relateddisorders(Neumannetal.,2006;

Zhangetal.,2009b).

Neurodegeneration-associatedproteinscanbesubstratetoaplethoraofproteolyticenzymes

includingmembersoftheα-,β-,and!-secretasefamiliesaswellascysteineproteases.Aside

thrombin (Arai et al., 2005), cathepsins (Kenessey et al., 1997) and puromycin-sensitive

aminopeptidase(PSA)(Senguptaetal.,2006),membersofthecaspaseandcalpainfamilyare

themost prominent enzymes involved in tauprotein cleavage and therefore of particular

interestinthepresentwork.

1.1.3 Caspasesandcalpainsandneurodegeneration

Caspasesareintracellularcysteine-aspartatic-specificproteasesthatcleavetheirsubstrates

at specific sites. First expressed as latent zymogens, these pro-caspases get post-

translationallyactivatedthateithercanleadtotheinactivationofthesubstrateortoatoxic

gainoffunctionintheformofactiveproteinfragmentsintheproteolyticprocess.Asideother

non-apoptoticandpro-inflammatorymembers,apoptoticcaspasescanbesubdividedinto(I)

initiatorcaspases(caspases-2,8,9and10)that,inresponsetoastimulus,directthesignalto

(II) executioner caspases (caspases-3, 6, and 7) (Pop and Salvesen, 2009). These caspase

candidates are associated with programmed apoptotic cell death in various

neurodegenerative disorders including AD, HD, and PD. Also, calpains are intracellular

Introduction

16

calcium-activated (papain-like) neutral proteases and have been implicated in the

pathogenesisofneurodegenerativediseasessuchasADorPD;althoughtheircalcium-induced

apoptoticroleislesscharacterizedcomparedtothatofapoptoticcaspases.

Multipleneurodegeneration-relatedproteinsaresubstratetocaspase-andcalpain-mediated

cleavage. Indeed, caspases-3 (Metcalfe et al., 2012), caspase-6, and calpains are themain

enzymesinvolvedintauproteinfragmentation,withtheAsp421beingthemostprominent

caspasecleavagesite(forreviewseeAvila,2010;Fasuloetal.,2005;Guillozet-Bongaartset

al., 2005; Guo et al., 2004) (Figure 1.1). Furthermore, site-directed mutagenesis at two

caspase-7cleavagesitesofneurotoxicataxin-7proteininSpinocerebellarataxiatype7(SCA7)

polyglutamine(polyQ)disorderresults inanon-cleavableformofpolyQ-expandedataxin-7

displayingattenuatedneuronaldeath,aggregateformation,andtranscriptionalinterference

(Youngetal.,2007).Ofnote,caspase-3andcaspase-6havebeenimplicatedincytoskeletal

disintegrationthroughactinandtubulincleavageultimatelyleadingtoaxonaldegeneration

invitroandinvivo(Sokolowskietal.,2014).Aside,calpainactivationhasbeendemonstrated

to play a crucial role in Aβ-triggered pathological cascade in AD (Higuchi et al., 2012).

Moreover,N-terminalcalpaincleavedataxin-3fragmenthasbeenshowntoprovokealtered

behaviouralandmotorphenotypeassociatedwithpathologicalproteininclusionsandnerve

celldeathinvivo(Hubeneretal.,2011).

Figure1.1Proteolyticprocessingoftau.Caspaseandcalpainsarethemainproteasesinvolvedintauproteincleavage. Truncationof tau at distinct proteolytic cleavage sites can either lead to preservationof neuronalstructureand/orfunction,and/orexacerbationoftautoxicity.(Chesseretal.,2013)

Introduction

17

1.1.4 Neurodegenerativedisease-associatedproteins

Awidevarietyofcellularandmoleculareventscanbeascribedtotheindividualpathological

profile of a vast number of neurodegenerative diseases. However, pathologically altered

proteins including their characteristic structure and morphology remain to be the most

prominententity.Proteinsthatundergofragmentationinparalleltopathologicalaggregation

are not only found in neurodegenerative proteopathies, but also in systemic amyloidosis.

Proteinopathieswithassociatedproteinfragmentationinclude:

(I) Themicrotubule-associatedproteintauthatisencodedbyagene(MAPT)located

onchromosome17(Weingartenetal.,1975);

(II) The amyloid-beta peptide (Aβ) that is encoded by a gene (APP) located in

chromosome21(Alzheimer,1906,1907;Kangetal.,1987;Tanzietal.,1987);

(III) Theamyloid-Bri(ABri)andamyloid-Dan(ADan)peptidesthatarebothencoded

byagene(BRI)locatedonchromosome13(Vidaletal.,1999;Vidaletal.,2000);

(IV) Theneuronalproteinalpha-synuclein (α-Syn) that isencodedbyagene (SNCA)

locatedonchromosome4(Spillantinietal.,1997);

(V) Proteinsencodedbygeneslinkedtocytosine-adenine-guanine(CAG)trinucleotide

repeatsincludinghuntingtin(Htt),ataxins(1,2,3,6,7,and17),andatrophin-1

(Fanetal.,2014);

(VI) Prionprotein(PrP)thatisencodedbyagene(PRNP)locatedonchromosome20

(AguzziandO'Connor,2010);

(VII) Transactiveresponse(TAR)DNA-bindingprotein43(TDP-43)thatisencodedbya

gene(TARDBP)locatedonchromosome1(Ouetal.,1995);

(VIII) AndothersincludingproteinsthatbelongtotheFETfamilyincluding(F)usedin

sarcomaprotein(FUS),(E)wing’ssarcomaprotein(EWS),(T)ATA-bindingprotein-

associatedfactor15(TAF15)(Kwiatkowskietal.,2009;Lawetal.,2006);charged

multivescularbodyprotein2B(CHMP2B)(Ghazi-Noorietal.,2012);glycoprotein

reelin(D'Arcangeloetal.,1997);globularproteintransthyretin(TTR)(Conceicao

etal.,2016);actinbindingproteingelsolin(Chenetal.,2001;Solomonetal.,2012);

hormone islet amyloid peptide (IAPP) (Akter et al., 2016); and human serum

amyloidA(SAA)(Egashiraetal.,2011).

Introduction

18

Themostnotableneurodegeneration-associatedproteinswillbeaddressedinthefollowing

paragraphs;however,thetauproteinanditsroleintauopathiesandtheneuropathological

relevanceofproteinfragmentationwillbeofparticularinterest.

1.1.5 Tauprotein

Tau belongs to the natively unfoldedmicrotubule-associated protein family (MAP) and is

abundantinthecentralandperipheralnervoussystem.Inthe1970s,amicrotubulebinding

activityoftauproteinhasfirstbeenshownbyWeingartenetal.,whoisolatedaheatstable

proteinmostabundantly found topromotemicrotubuleassemblyand stability in cell-free

conditions (Weingarten et al., 1975). Microtubules are protein polymers and a major

component of the cytoskeleton with an essential role in regulated motor-driven axonal

transport. Later, evidence frombrainsofpatientswithAD suggested that tau is actual an

integralpartofthepathologyinneurodegenerativedisorders(Goedertetal.,1988;Grundke-

Iqbaletal.,1986;Kondoetal.,1988;Kosiketal.,1986;Wischiketal.,1988a).Effortsfora

better understanding of the physiological role and identity of tau protein have been

intensifiedsince.

1.1.5.1 Cellularlocalizationanddomainorganizationoftau

Thetauprotein,synthesizedandproducedinallneurons, ispredominantlyfoundinaxons

(Binder et al., 1985). However, it also has been shown to be located in the dendritic

compartmentalbeit in lowerconcentrationaswellasunderpathologicalconditions in the

somatodendriticdomain(Ittneretal.,2010).Structurally,tauisanaturallyunfoldedprotein

thatcontainsfourmajorregions.Oncetauisboundtomultipletubulindimers,theN-terminal

acidicprojectionregionprotrudesoutwardfromthesurfaceofthemicrotubuleandinthis

way, being able to serve as a spacer between the individual components within the

microtubulenetwork(Chenetal.,1992;Frappieretal.,1994).Furthermore,itwasfoundthat

thisregionappearstointeractwithmembrane-bindingproteinssuchasannexinA2(AnxA2)

and thus retain the tauproteinat thedistal tipofneurites (Brandtetal., 1995;Gauthier-

Introduction

19

Kemperetal.,2011;Weissmannetal.,2009).Theproline-richregion(PRR)harboursmany

phosphorylationsitesandcontributestothemicrotubule-bindingaffinityoftau(Augustinack

et al., 2002; Biernat et al., 1992; Brandt and Lee, 1993;Goodeet al., 1997).Moreover, it

enablesinteractionwithotherproteinssuchastheSH3domaincontainingtyrosinekinaseFyn

(Leeetal.,1998).TheC-terminalregionoftaucontainsamicrotubulebindingdomain(MTB)

composedof18-aminoacid(aa)tandemrepeatsseparatedbysequencesof13-or14-aathat

encouragetautobindtothemicrotubulesandashorttailsequence,whichisinvolvedinthe

regulationofmicrotubulepolymerization(BrandtandLee,1993;Leeetal.,1988;Leeetal.,

1989).

1.1.5.2 Tauisoforms

Thetauproteinisencodedbyasinglegenethatcontainsatotalof16exons(Andreadisetal.,

1992).Six isoforms of tau are expressed in the adult humanbrain (Goedert et al., 1989).

ProducedbycomplexalternativemRNAsplicingoftheMAPTgenelocatedonchromosome

17q21.31,eachisoformdiffersinitsspecificrepresentationassuchinthepresenceorabsence

of both, amino-terminal inserts (0N, 1N, 2N) and carboxy-terminal microtubule-binding

repeatdomains(3R,4R).Thus,splicingoftheneuron-specifictautranscriptattwo29-or58-

aainsertsandone31-aarepeatdomainencodedbyexons2,3and10,respectively,resultsin

asetofproteinswiththerangefrom352-aainlengthfor3R0N,381for3R1Nand410for

3R2Nto383-aainlengthfor4R0N,412for4R1Nand441for4R2N(Figure1.2).

Introduction

20

Figure1.2SchematicrepresentationofthehumanTAUgene,TAUmRNA,andthesixtauproteinisoforms.Sixisoformsaregeneratedthroughalternativesplicingintheadulthumanbrain.(AdaptedfromBueeetal.,2000)

1.1.5.3 Taufunction

Tau,asanativelyunfoldedprotein,lacksawell-definedsecondaryortertiarystructurethat

allowstheproteintointeractwithavarietyofpartnersintheenvironmentofacell(Schweers

et al., 1994). The main physiological function of tau is stabilization of the microtubule

network bypromoting thepolymerizationof tubulinand thus themaintenanceofnormal

axonaltransport(Bohmetal.,1990;BrandtandLee,1993;Clevelandetal.,1977;Shahaniand

Brandt, 2002;Weingarten et al., 1975). As amain cytoskeleton component,microtubules

contributetomorphogenesis,divisionandintracellulartraffickinginthecell(Mitchisonand

Kirschner,1984).

Introduction

21

Atanygivenmoment,about80%oftauisindirectinteractionwithmicrotubules(Weissmann

etal.,2009).Taubindingtomicrotubulesisregulatedbyadelicateequilibriumofkinasesand

phosphatasesandboth,over-stabilizationbytauordetachedtaufromthemicrotubulecan

impaircellviability(Bramblettetal.,1993;Pandaetal.,2003;ThiesandMandelkow,2007;

Wangetal.,2007).

Infact,neuronalfunctioniscriticallydependentonanintactmicrotubulenetwork.Specific

cellular compartments suchas thepre-andpost-synaptic cell structureshavehighenergy

requirementsandaccumulatedwasteproductstomanage;therefore,cellularmotorsofthe

kinesinanddyneinsuperfamilyutilizeenergyderivedfromATPhydrolysistotransportcargo-

filledvesiclesoverlongdistancesonmicrotubuletracksintheaxon(DeVosetal.,2008).In

this way, mitochondria (Hollenbeck and Saxton, 2005), lysosomes (Harada et al., 1998),

peroxisomes(Walietal.,2016)andvariousotherorganellescanbelocalizedtodistinctareas

intheneuronalrealminordertoaccomplishtheirveryownfunction.Thetightbindingoftau

alters the intracellular traffic as well as ensures the dynamic instability of microtubules

(Mitchison and Kirschner, 1984; Trinczek et al., 1995). The latter is distinguished by their

capability to switch between slow growth and rapid shrinking duringmicrotubule growth

(Binder et al., 1985; Mitchison and Kirschner, 1984). Aside from its predominant

neurophysiological activity – binding to microtubule – numerous functions have been

attributedtotau.Amongthem,modulationofbiochemicalcascades,suchthattaucanactas

a protein scaffold (Brandt et al., 1995; Ittner et al., 2010; Reynolds et al., 2008) or direct

enzymeinhibitor(Perezetal.,2009).Inparallel,tauappearstointeractwithnucleicacidsas

wellasmitochondria(Jancsiketal.,1989;Kampersetal.,1996;Loomisetal.,1990;Sultanet

al.,2011);suggestingaroleasamultifunctionalcommunicationinstrumentwithinthecell.

1.1.5.4 Post-translationalmodificationsoftau

Inphysiologicalconditionsanduntypicallyformostcytosolicproteins,tauresistsacompact

foldedstructure;theentiretaumoleculeisconsideredtobeintrinsicallydisorderedandits

function is tightly regulated by a host of post-translational modification such as

phosphorylation(Grundke-Iqbaletal.,1986),acetylation(Cohenetal.,2011),glycosylation

(Gongetal.,2005),glycation(Ledesmaetal.,1994),sumoylation(DorvalandFraser,2006),

Introduction

22

ubiquitination(Morietal.,1987),nitration(Reynoldsetal.,2006),andtruncation(Gamblinet

al., 2003; Wischik et al., 1988b). The contribution of various cellular mechanisms to tau

pathogenesisarenotknownandithasyetremainedunclear,whichmodificationiscrucialfor

thedevelopmentoftauopathies.

Theindividualpost-translationalmodificationsoftaucanonlybeoutlinedinthepresentwork;

however, tauphosphorylation (I) and tau truncation (II)will be discussed in detail in the

followingparagraph.

1.1.5.4.1 Tauphosphorylation

Tauphosphorylationoccurs inbothpathologicalandphysiologicalconditions. Itwasfound

that isolated tau molecules from healthy human brains contained roughly two moles of

phosphate permole of tau,whereas tau proteins associatedwith paired helical filaments

(PHFs)ofpatientswithADcontainedsixtoeightmolesofphosphatepermoleoftau(Ksiezak-

Redingetal.,1992).

Giventheloosedisorderedcharacteroftau,manyknownpotentialphosphorylationsitesare

sensitive to numerous protein kinases and phosphatases; indeed, these phosphorylatable

domainsconsistof80serineandthreonineresiduesandfivetyrosineresiduesarekeyplayers

intheregulationofthemicrotubulebindingactivityoftau(Figure1.3).Themostprominent

candidatekinasesfortauphosphorylationincludeproline-directedkinasesglycogensynthase

kinase3(GSK3),cyclin-dependentproteinkinase(cdk5),thep38mitogen-activatedprotein

kinase(MAPK);orc-JunN-terminalkinases(JNK) families,aswellasotherstressactivated

kinases,suchascdc2;andnon-proline-directedkinasessuchasproteinkinaseA(PKA),protein

kinaseC(PKC),calmodulin(CaM)kinaseII,microtubule-affinityregulatingkinase(MARK),and

caseinkinaseII(CKII)(Correasetal.,1992).

Asidefromkinases,variousphosphatases(PP)havebeenidentifiedtodephosphorylatetau

protein(Sergeantetal.,2005).IthasbeenshownthatPP1,PP2AandPP2B,predominantly

dephosphorylate tau in vitro (Wangetal., 1995;Yamamotoetal., 1988);moreover,PP2A

expressionandactivitywasfoundtobereducedinthebrainsofADpatients,suggestingthat

dephosphorylation defects play a vital role in the pathological cascade in tau mediated

neurodegenerativedisorders(Gongetal.,1993).

Introduction

23

Indeed,abnormaltauphosphorylationisconsideredasanearlyeventinthepathologyoftau

(Bramblettetal.,1993).Asmentionedabove,theadulthumanbraintauharboursmorethan

80 potential phosphorylation sites and a disequilibrium in candidate protein kinase and

phosphatase activity results in tau hyperphosphorylation with subsequently increased

amountoftaudetachedfrommicrotubule.

Figure1.3Dysregulationofaxonaltransport.Destabilizationofmicrotubulesbydecreasedkinaseactivityresultsinhyperphoshorylatedandaggregatedtau.Subsequentdisintegrationofthemicrotubuletracksleadstokinesin-mediatedanterogradeanddynein-mediatedretrogradeaxonaltransportdysfunction.(AdaptedfromDeVosetal.,2008)

1.1.5.4.2 Tautruncation

Phosphorylationistypicallyregardedasoneofthemostrelevantmodificationresponsiblefor

changesoftauprotein.Alternatively,proteolyticprocessingoftaubyavarietyofendogenous

Introduction

24

proteaseshasbeenpostulatedtobe involved in thepathologicalcascade in taumediated

neurodegenerativedisorders(Gamblinetal.,2003;Rissmanetal.,2004).

Tauproteinexhibitaphysiologicalrandomcoilstructure;however,itisabletoassembleinto

orderedfilamentousaggregatesbytheformationofβ-sheetstructuralelements(vonBergen

et al., 2005). Given that the aggregation of tau correlateswith its propensity for β-sheet

structure,analteredshapeoftauproteinbyproteolyticfragmentationpotentiallyaffectits

aggregation capacity. The architecture of paired helical and straight filaments of AD is

predominantly defined by all six full-length isoforms of tau (Goedert et al., 1992);

nevertheless,studiesontauaggregationhadrevealedthattruncatedformsoftauare,infact,

presentinthecoreofPHFs(Gamblinetal.,2003;Menaetal.,1996;Rissmanetal.,2004).

Likewise,fragmentedtauhasbeenreportedtofacilitateandpromotefasterpolymerization

intofibrils,inthisway,highlightagreateraggregationpropensitycomparedtofull-lengthtau

(Abrahaetal.,2000).

InAlzheimer’sdisease, tau truncationwas found tobeanearlyeventandappearsbefore

neurofibrillary tangle (NFT) formation albeit after hyperphophorylation of tau (Guillozet-

Bongaartsetal.,2005;Mondragon-Rodriguezetal.,2008;Rohnetal.,2002;Saitoetal.,2010).

Various types of tau fragmentation have been reported in cells and brain tissue; cysteine

proteases suchascaspasesandcalpainshavebeen implicated inproteolysisof tauduring

apoptosis (Canuet al., 1998). In vitro and in vivoexperimentspredictedmultipleputative

cleavage sites of tau at both, its C-terminal aswell as N-terminal domain by a variety of

caspases(Delobeletal.,2008;Gamblinetal.,2003;Guillozet-Bongaartsetal.,2005;Horowitz

etal.,2004).However,notallproteolyticcleavagesitescanbeassignedtoadistinctprotease:

C-terminaltruncatedtauatglutamicacid391(E391)islinkedtoclinicaldementiayetcatalyzed

byanunknownproteolyticenzyme(Basurto-Islasetal.,2008;Novaketal.,1993).

However,presentlythemoststudied,caspasescleavetaupreferentiallyataspartaticacid421

(D421);both,caspase3andcaspase6havebeenfoundtobeinvolvedinthiscleavageatthe

C-terminal domain of tau (Guo et al., 2004; Rissman et al., 2004; Zhang et al., 2009a).

Moreover, accumulation of caspase-cleaved C-terminal fragments have been reported to

correlatewiththeprogressioninADandinvivomousemodelsoftauopathy(Basurto-Islaset

al.,2008;Centeetal.,2006;deCalignonetal.,2010;Delobeletal.,2008;Guillozet-Bongaarts

etal.,2005).

Introduction

25

1.1.6 Tauopathies

The term “Tauopathy” summarizes a heterogeneous group of disorders with

hyperphosphorylated,insoluble,filamentoustauproteininclusionsinneuronsandglialcells

(SpillantiniandGoedert,2013)(Figure1.4).Thesehumanneurodegenerativediseasesarein

most cases sporadic, and clinically characterized by dementia, often associated with

movement impairment. Most frequent tauopathies include AD (see paragraph 1.2.2.1),

Progressive supranuclearpalsy (PSP) (Steeleetal., 1964),Corticobasaldegeneration (CBD)

(Rebeizetal.,1968),Pick’sdisease(PiD)(Constantinidisetal.,1974),Argyrophilicgraindisease

(AgD) (Braak and Braak, 1989), and Frontotemporal dementia and parkinsonism linked to

chromosome17(FTDP-17T)(Wilhelmsenetal.,1994).

Figure1.4Differenttypesoftauimmunoreactivity intauopathies.Hyperphosphorylatedtau,AD(A);gallyaspositiveglobosetangle,AD(B);gallyaspositivetuftedastrocyte,PSP(C);hyperphosphorylatedtauinastrocyticplaques, CBD (D); hyperphosphorylated tau from pick bodies, PiD (E); hyperphosphorylated tau, AgD (F).(AdaptedfromNeumannetal.,2009)

Introduction

26

1.1.7 Prion-likeseedinginneurodegenerativeproteinopathies

Prion-like seedingor transmission isageneralmechanismobserved inneurodegeneration

proteinopathiesincludingCreutzfeldt-Jakobdisease(CjD),kuru,andscrapie.Inthisprocess

involvedaremisfoldedandaggregatedproteinsthatpotentiallyactasinfectiousagentsby

structurallycorruptingotherproteins,andthustriggertheirpathogenicaggregation(Jucker

andWalker,2013).Variousneurodegeneration-associatedproteinsincludingAβ,tau,α-Syn,

andTDP-43 are increasinglyemergingas considerable candidateswithpotential prion-like

properties(Figure1.5).IncaseofpotentialAβseeding,thisisreflectedinstudiesonmouse

models, where brain tissues from AD patients of APP transgenic mice can indeed seed

amyloidosisinvivo,indicatingaprion-likebehaviorofpathologicAβ(Kaneetal.,2000;Meyer-

Luehmannetal.,2006).Moreover, inoculationofbrainhomogenatesderivedfromhuman

tauopathypatientsandNFTsbearingP301Stautransgenicmiceintowild-typetauexpressing

ALZ17hostmicedemonstratedaprion-likespreadingpotentialof insolubletauaggregates

(Clavagueraetal.,2013;Clavagueraetal.,2009).

1.1.7.1 CSFAβ

PreviouslyithasbeenshownthatsmallandsolubleAβspeciesfoundinthebrainofAPP23

transgenicmiceexhibitprion-likebehaviour(Langeretal.,2011).GiventhatAβisalsopresent

in the cerebrospinal fluid (CSF), CSF biomarkers constitute a valuable tool for early AD

diagnosis(Cummings,2004;McKhannetal.,2011);currentCSFanalysis,though,suffersfrom

measurementsvariabilities(Scheltensetal.,2016).Studiesonpost-mortemCSFhavebeen

demonstratedthatAβ42levelsinverselycorrelatewithcorticalplaquedeposition(Tapiolaet

al.,2009).Further,inparalleltotheprogressionofsenileplaques,Aβ40andAβ42levelsinthe

CSFappeartodecreasewithage(Maiaetal.,2013).However,thepotencyofhumanCSFfor

amyloidaggregationinductioninmousemodelsofdementiaremainselusive.

Introduction

27

Figure 1.5 Spreading of neurodegeneration-associated proteins. Progressive prion-like propagation andspreadingofAβ(A),tau(B),α-Syn(C),andTDP-43(D)basedonbrainautopsystudiesofhumandiseasepatients.(AdaptedfromJuckerandWalker,2013)

Introduction

28

1.2 Chapter2:

Proteinfragmentationinneurodegenerativedisorders

FrederikSprenger,DavidT.Winkler

Reviewinpreparation

1.2.1 Neurodegenerativedisorders

The majority of neurodegenerative disorders is associated with pathological protein

aggregation.Theterm“proteinopathies”hasthereforebeenestablishedforthesediseases.

Themechanismsunderlyingproteinaggregation inproteinopathieshas inmanycasesonly

been partially understood. Different factors may eventually lead to pathological protein

aggregation.Firstofall,an increasedaggregationpropensityofagivenproteincan induce

pathological folding and aggregation. Proteins can be rendered aggregation prone by

patholocigal mutations, e.g. in the case of hereditary forms of proteinopathies, or by

truncation.Insporadicdiseases,factorsi.e.decreasedproteinclearance,e.g.byautophagy

disruption, are furthermore suspected to be underlying the aggregation process. These

processes involve protein cleavage and may thereby not only be beneficial by removing

misfoldingproteins,butalso indirectly contribute to render theaggregationpropensityby

cuttingproteinsintosmallpeptideswithseed-likecharacter.Inmostcases,theaggregating

proteins are rather small, often derived of a precursor protein. All this points towards a

unifying characteristic of protein cleavage as a key factor leading to aggregation prone

fragments. Such fragments often possess an inherently higher aggregation propensity

comparedtotheirprimaryfull-lengthproteinofwhichtheyarederived.

Proteincleavageisawidelyobservedprocessinneurodegenerativedisorders.Basedonthe

specificneurodegeneration-associatedproteins and theirmodifications leading todisease-

typicalhallmarksattheneuropathologicallevel,variousneurodegenerativediseasescanbe

linkedtooneanotherandassignedtothefollowingterms:

Introduction

29

(I) Tauopathy; including Alzheimer’s disease (AD), frontotemporal dementia and

parkinsonism linked to chromosome 17 (FTDP-17T), progressive supranuclear

palsy(PSP),cortocobasaldegeneration(CBD),andargyrophilicgraindisease(AGD);

Pickdisiease(PiD),andglobularglialtauopathy(GGT)(Fasuloetal.,2005;Ferreira

andBigio,2011;Guoetal.,2004;Parketal.,2007;Zhangetal.,2009a)(seealso

paragraph1.1.6.);

(II) Heriditary amyloidoses/Cerebral amyloid angiopathy (CAA); including AD and

familialCAArelatedtoAβvariantssuchasfamilialBritish(FBD)andDanish(FDD)

dementias;gelsolin;andtransthyterin(Chenetal.,2001;DeStrooperetal.,1999;

Ihseetal.,2013;Vidaletal.,1999;Vidaletal.,2000);

(III) α-Synucleinopathy;includingParkinsondisease(PD),dementiawithLewybodies

(DLB),andmultiplesystematrophy(MSA)(Duftyetal.,2007;Kessleretal.,2003;

Mishizen-Eberzetal.,2005);

(IV) Trinucleotiderepeatexpansiondisorder(TRD);includingHuntingtindisease(HD),

andspinocerebellar-ataxia-1,2,3,6,7,17(SCA3andSCA7)(Grahametal.,2006;

Hubeneretal.,2011;Mookerjeeetal.,2009;Wellingtonetal.,2002);

(V) Priondisease; includingCreutzfeldt-Jakobdisease(CjD)(Altmeppenetal.,2012;

Notarietal.,2008);

(VI) TDP-43proteinopathy;TDP-43relateddisorders including frontotemporal lobar

degeneration(FTDP-TDP)(Araietal.,2010;Igazetal.,2009;Nonakaetal.,2009);

(VII) FUS/FETproteinopathy; includingbasophilicinclusionbodydisease(BIBD)(Kent

etal.,2014;RademakersandRovelet-Lecrux,2009).

Aggregatesofaberrantmodifiedproteinsmayeitherconsistofmainlyfragmentedprecursor

protein remnants, or contain co-aggregated full-length proteins and cleaved fragments in

parallel. Cleavage derived species of themost notable entities and their potential role in

neurodegeneration will be addressed separately in the following paragraphs; a detailed

discussion, though, of other individual protein modifications is beyond the scope of the

presentwork.

Introduction

30

1.2.2 ProteinfragmentationinAD:Aβ

1.2.2.1 Alzheimer’sdisease(AD)

In2015,theworldwideprevalenceofdementiawasestimatedtobe46.8million(Princeet

al.,2015).Thisnumberisforecasttodoubleevery20years,reachingover130millionaffected

people in 2050.Over90%of allAD casesoccur sporadically; foremost amongothers, age

constitutesthemainriskfactorfordementia(Blennowetal.,2006);inaddition,thefrequency

oftheapolipoproteinE4(ApoE4)allele,amajorgeneticriskfactor,onchromosome19inlate-

onsetAD(LOAD)patientshasbeenshowntobesignificantly increased(Strittmatteretal.,

1993).Incontrast,about5to10%arefamilialcases(FAD)andresultsinaggressiveearly-onset

progression of AD (EOAD) (Tanzi, 1999).Mutations in genes coding for amyloid precursor

protein (APP; located on chromosome21; presenilin 1 (PS1; located on chromosome14),

presenilin2(PS2;locatedonchromosome1)havebeenreportedinFAD(Goateetal.,1991;

Sherringtonetal.,1995).

Alzheimer’s disease is a most probably heterogeneous neurodegenerative disorder. It is

clinically characterizedby progressive cognitive decline, typically delineatedby short-term

andlong-termmemoryimpairment,aswellaslossofsocialabilitiesincludingsymptomslike

confusion,irritability,aggressionandlanguagebreakdown(Tabertetal.,2005;Waldemaret

al.,2007);inparallel,patientsareaffectedbymusculardeteriorationandmotordisabilities

(Scarmeasetal.,2004).Furthermore,AD isneuropathologicallydefinedbyextracellularβ-

amyloid (Aβ; see paragraph 1.2.2.2) plaques, vascular Aβ deposits (cerebral amyloid

angiopathy;CAA;seeparagraph1.2.3.)andintracellularaggregatesoftauprotein(NFTs;see

paragraph1.1.6)(Alzheimer,1906,1907)(Figure1.6).

Massive neuronal and dendritic loss is the primary cause of cortical atrophy during the

progressionofAD.Atrophicchangesresultsincorticalthinningandenlargementofthelateral

cerebral ventricles. Reduced neuronal numbers in a variety of brain regions such as the

temporal, parietal and enthorhinal cortex (Gomez-Isla et al., 1997), theCA1 regionof the

hippocampus (West et al., 1994) and amygdala (Vereecken et al., 1994) have been

documented;however,thecauseofneurondeathisdisputable.

Infact,thepathogenicmechanismsarepoorlyunderstood,withsomestudiessuggestthat

Introduction

31

intraneuronaland/oroligomericAβ-peptidesactaskeymediatorsofneurotoxicityandcell

death(BayerandWirths,2010;LarsonandLesne,2012).However,pathologicaltauprotein

increasinglygainedattentioninthepathogenesisoftauopathiesincludingAD.Otherstudies

provide evidence of an intimate coherence between neuron loss and the appearance of

neurofibrillarytangles(Crasetal.,1995;Gomez-Islaetal.,1997);indeed,incontrasttothe

degreeofAβpathologyinformofsenileplaques,theextentoftaupathologyhasbeenshown

tocorrelatebestwiththeclinicalstateofAD(Arriagadaetal.,1992;Giannakopoulosetal.,

2007;Landauetal.,2016;Ossenkoppeleetal.,2016).

Figure 1.6 The pathological hallmarks of AD. Senile Aβ plaque (A), cerebrovascular amyloid (B), andneurofibrillarytangles(C).(AandBadaptedfromCastellanietal.,2010)

1.2.2.2 Aβ

Amyloid-beta(Aβ)isgeneratedfromtheamyloidprecursorprotein(APP)(Kangetal.,1987;

Tanzi et al., 1987), which is a single transmembrane glycoprotein consisting of an

exocytoplasmic domain and a short cytoplasmic tail. This large precursor protein is

sequentially processed releasing distinct secreted derivatives into vesicle lumens and the

extracellularspace.AssummarizedinFigure1.7,APPisproteolyticprocessedvia(I)thenon-

amyloidogenicpathway;or(II)theamyloidogenicpathway(Selkoe,2001b);ofnote,thelatter

iscrucialforAβliberationandthusofneuropathologicalrelevance.

Introduction

32

(I) Thenon-amyloidogenicpathway

This processing prevents an accumulation of Aβ peptides by demolishing the complete

amyloid sequence. APP is sequentially cleaved by α-and !-secretases to release a smaller

fragment(p3),thathasnoneuropathologicalrecognizedrole.Theα-sitecleavageofAPPby

adamalysin protease (ADAM) cuts 12-aa at the N-terminal single transmembrane domain

(Roberts et al., 1994); consequently, a C-terminally truncated formof soluble ectodomain

fragment (α-sAPP) is released from the membrane. The remaining 83-residue C-terminal

fragment(C83;CTFα)inthemembraneisfurtherprocessedby!-secretaseswhichthenleads

tothereleaseoftheshortextracellularp3fragment(Selkoe,2001b)andtheAPPintracellular

domain(AICD)(Zhangetal.,2011).

(II) Theamyloidogenicpathway

Aβ protein is liberated by sequential proteolytic processing of APP involving β-and !-

secretases. Initially,proteolytic cleavageoccurs16-aa residues to theN-terminalof theα-

cleavagesitebytheβ-siteAPPcleavingenzyme1(BACE1)(Hussainetal.,1999;Sinhaetal.,

1999;Vassaretal.,1999),generatingasolubleaminoterminalAPPderivate(β-sAPP)anda

membrane-associated 99-residue C-terminal (C99, CTFβ). Further, !-secretase activity at

different intramembranousCTFβsitesgeneratesAβspeciesofvarying lengthbetween35-

and43-aa.ForemostamongthemarepeptidesofAβ40orAβ42aainlengths,thatare,together

with theAICD,most abundantly produced (Citron et al., 1995;Haass et al., 1994; Selkoe,

2001a;Tang,2009).The!-secretaseisahighlyhydrophobiccatalyticenzymeandcomposed

ofdistinctcomponents:presenilinproteins(PS1orPS2),nicastrin,anteriorpharynxdefective

1 (APH1)andpresenilinenhancer2 (PEN-2).Asideofbeing involved inAPPprocessing,!-

secretase cleaves further substrates, such as Notch, cadherins, CD44 and neuregulin (De

StrooperandAnnaert,2010).

Introduction

33

Figure1.7SchematicrepresentationofAPPprocessing. Innon-amyloidogenicprocessing,APPissequentiallycleavedbyα-secretaseand!-secretases to releasethep3 fragment. Inamyloidogenicprocessing,sequentialcleavagebyβ-and!-secretasesreleasestheAβpeptide.(ThathiahandDeStrooper,2011)

1.2.3 ProteinfragmentationinfamilialCAA:ABriandADan

1.2.3.1 Cerebralamyloidangiopathies:familialBritishandDanishdementia

Cerebralamyloidangiopathy(CAA)summarizesentitieswithdepositionofamyloidwithinthe

wallsofbloodvesseloftheCNS(Ghisoetal.,2001;Mandybur,1986);itoccursassporadic

and/orfamilialdiseaseformswithvariousproteinsinvolvedincludingAPPinAD,amyloid-Bri

precursor protein (ABriPP) in familial British dementia (FBD), and amyloid-Dan precursor

protein(ADanPP)infamilialDanishdementia(FDD)(Rensinketal.,2003;Reveszetal.,1999;

Reveszetal.,2002).

Introduction

34

FBDandFDDare late-onsetdiseasesand clinically characterizedbyprogressivedementia,

spastictetraparesis,andcerebellarataxia(Meadetal.,2000;Stromgrenetal.,1970);with

neuropathologicalhallmarkssimilartothoseinADincludingsevereCAA,neuroinflammation,

and neurofibrillary tangle formation (Coomaraswamy et al., 2010; Revesz et al., 2002;

RostagnoandGhiso,2008).

1.2.3.2 ABriandADan

MutationintheintegralmembraneproteinBRI2leadstoaccumulationofhighlysolubleABri

andADanpeptides.BRI2isatype-IIsingle-spanningtrans-membraneprecursorproteinof266-

aainlength.Amissensemutationordecamerduplicationmutationproducesaframe-shiftin

theBRIgenesequencearegeneratingtwolarger,277-residueprecursorproteinsABriPPand

ADanPP,respectively(Vidaletal.,1999;Vidaletal.,2000).

TheimmatureBRI2precursorproteiniscleavedinamulti-stepproteolyticprocessresultingin

distinct intermediate- and end-products: (I) several proteases such as furin and other

subtilisin/kexin-likeproproteinconvertases(PPCs) leadstothesecretionofa23-residueC-

terminalpeptide(Bri2-23)(Kimetal.,1999).(II)theremainingmembraneboundmatureBRI2

proteinisfurtherprocessedbyα-secretasesADAM10releasingtheBRICHOSdomaintothe

extracellularspace(Martinetal.,2008).(III)finally,anN-terminalfragment(NTF)undergoes

proteolyticcleavagebysignalpeptidepeptidase-like2(SPPL2)resultinginasmallextracellular

BRI2-C-terminalpeptideandinparalleltoanintracellulardomain(ICD)(Martinetal.,2008).

Notably, instead of Bri2-23, proteolytic processing of ABriPP or ADanPP by PPCs leads to

liberationofhighlysoluble34-residueC-terminalpeptidesABriandADan,respectively(Figure

1.8).

Unliketheshorterwild-typepeptideBri,cleavedABrishowedanincreasedpropensityinthe

formationoftoxicoligomersthroughinter-moleculedisulfidebonds invitro(Cantlonetal.,

2015; El-Agnaf et al., 2001).Overexpressionof different amyloid peptides in the retinaof

DrosophilademonstratedthatADanappearstobemoretoxiccomparedtoe.g.Aβ42peptides

(Marcoraetal.,2014).Thetwoamyloidogenicpeptides,ABriandADan,whichdonotoccurin

nature intheabsenceof thediseasecausingchanges intherespectiveprecursorproteins,

demonstrate that the de novo creation of short peptides is sufficient to induce amyloid

Introduction

35

formation and associated neurodegeneration. ABri and ADan therefore serve as principal

models for anupstream roleofprotein cleavage inneurodegeneration. In addition to the

inductionofamyloiddeposits,thesenovelpeptidesalsoinducedownstreamtaupathologyin

FBDandFDD(DelCampoetal.,2015).Inmurinemodels,co-expressionofsolubleADanand

P301Stauexacerbatestautoxicityassociatedsynapticdysfunction,andresultsinincreased

solublehyperphosphorylatedandinsolubleaggregatedtauspeciesaswellastautruncation

atAsp421(Coomaraswamyetal.,2010;Garringeretal.,2013).

Figure1.8SchematicrepresentationofBRI2processing.(Cantlonetal.,2015)

1.2.4 ProteinfragmentationinPD:α-synuclein

1.2.4.1 Parkinson’sdisease(PD)

Parkinson’s disease (PD) is characterized by tremors and locomotion abnormalities and

constitutes the most frequent movement disorder among α-synucleinopathies including

dementiawith Lewy body (DLB) andMultiple SystemAtrophy (MSA). PD is pathologically

defined by the presence of aberrantα-synuclein (α-Syn) in intracellular deposits of Lewy

bodies (LB) and Lewy neurites (LN) and by neuronal degeneration in the substantia nigra

(Forno,1996;reviewedinTofarisandSpillantini,2005).

Introduction

36

1.2.4.2 α-synuclein

Proteolyticprocessingofα-synucleinisthoughttobeconsiderablyrelevantfortheformation

offibrillogenicproteininclusionsandneurotoxicity(Duftyetal.,2007)(Figure1.9); indeed,

truncatedα-synucleinspecieshavebeenfound inbrainsPDandDLBpatients (Babaetal.,

1998;Liuetal.,2005).Infact,C-terminallytruncatedα-Synhasbeenfoundtoincreasethe

aggregationpropensityoffull-lengthα-Syn,resultinginenhancedneurotoxicityinvivoandin

vitro(Giassonetal.,2002;Kandaetal.,2000;Lietal.,2005).Italsohasbeendemonstrated

that C-terminal truncated α-Syn aggregates more rapidly compared to its full-length

counterpart;moreover,truncatedα-Synspeciesexhibitseedingpotentialoffull-lengthα-Syn

aggregation in vitro (Murray et al., 2003). Of note, manganese (Mn), a cofactor for

homeostaticandtrophicenzymesintheCNS,hasbeenshowntoinducecleavageofα-Syn

proteininvitroandprovoketoxicα-Synoligomers,ultimatelyleadingtoneuronalinjury(Xu

etal.,2015).Aside,matrixmetalloproteinases(MMPs)generatingaggregation-enhancingα-

SynfragmentsinvitroarebelievedtobealsorelevantforPDpathogenesisinvivo(Levinetal.,

2009).Further,N-terminaltruncationofα-Synproteinpreventsβ-sheetandfibrilformation

(Kessler et al., 2003). Also, in transgenic mice overexpressing a calpain-specific inhibitor,

reducedproteolyticcleavageofα-Synproteinleadtoadecreaseofα-Syn-positiveaggregates

andastrogliosis;indicatingacrucialroleoftruncatedα-Synspecies,butalsoofcalpainsinthe

pathogenesisofPD(Diepenbroeketal.,2014).

Figure1.9Schematic representationofα-Synproteinprocessing. (Adapted fromEmanueleandChieregatti,2015)

Introduction

37

1.2.5 ProteinfragmentationinTRD:Htt,ataxin,andatrophin

1.2.5.1 Huntington’sdisease(HD)

Huntington’s disease (HD) is a late-onset, autosomal dominant, and progressive

neurodegenerativedisordercausedbyaCAGtrinucleotiderepeatsexpansioninthecoding

region of the huntingtin (htt) gene,which encodes an polyglutamine (polyQ) stretch that

altersthefunctionofthehttprotein;indeed,themutanthttpossiblyaffectscellularprocesses

includingmitochondrialdysfunctionandvesicletransportfailure(Browne,2008).Inaddition,

polyQ induced conformational changemakes thehtt proteinmoreaggregation-proneand

causeinclusionsinthecytoplasm,dendritesandaxonterminalsofneurons(Fanetal.,2014).

Interestingly,ithasbeenshownthatwild-typehuntingtincandiminishtheneurotoxicityof

mutanthuntingtin(Leavittetal.,2001;Leavittetal.,2006;Zhangetal.,2003)andalsoactas

anautophagyscaffold(Ruietal.,2015).

The clinical picture of HD includes psychiatric features and dementia; progressive chorea

developmentandothermovementabnormalities.Inparallel,severedegenerationoccursin

thestriatalmedium-sizedspinyneurons,andsubsequentlyinthedeeplayersofthecortex

(NovakandTabrizi,2011;VonsattelandDiFiglia,1998).

1.2.5.2 htt

Thecorrelationbetweenhuntingtinlengthandneurotoxicityispoorlyunderstood;however,

httfragmentshavebeenfoundinthebrainsofHDpatients,aswellasintransgenicmouse

modelsofHD(Kimetal.,2001;Wellingtonetal.,2002).Indeed,proteolyticcleavageofmutant

httbycaspases,calpainsandotherproteasessuchasMMPs(Milleretal.,2010)releasean

aggregation-pronepolyQ tract containingN-terminal fragment, andultimately leading to

intracellularinclusionsandneuronaldysfunction(Wellingtonetal.,2000)(Figure1.10);aswell

as to impairedmitochondrial trafficking,precedingthe formationofaggregates (Orretal.,

2008). Further,mousemodels transgenic for truncated formsofhtthave showna rapidly

progressiveandlethalphenotype(Mangiarinietal.,1996;Schillingetal.,1999);incontrast,

miceexpressingfull-lengthmutantconstructsexhibitonlyamildpathologicalphenotype(Van

Raamsdonketal.,2005).

Introduction

38

1.2.5.3 Otherpolyglutaminediseases

Other trinuleotide repeat expansion disorders, including various forms of Spinocerebellar

ataxia (SCA types1,2,3,6,7, and17)andDentatorubral-pallidoluysianatrophy (DRPLA)

sharecommonpropertiesofHD,albeitbeing less frequent;mutations inpolyQexpansion

altersthenormalfunctionofataxinproteins(MargolisandRoss,2001;Palhanetal.,2005)

and the transcription co-regulator atrophin-1 (Sato et al., 2009), respectively, leading to

intranuclearaggregationincerebellarPurkinjecellsandcorticalneurons(Rolfsetal.,2003).

Among the other types, an important neuropathological role of ataxin cleavage has been

showninaDrosophilamodelofSCA3andforSCA7 invitroand invivo.Specifically,polyQ-

containing fragments derived from caspase-dependent cleavage of ataxin-3 have been

demonstratedtoinduceneurotoxicity,thuscontributetoSCA3diseaseprogression(Junget

al.,2009)Inaddition,ithasbeenshownthattoxicpolyQ-containingfragmentsgeneratedby

caspase-7 mediated N-terminal cleavage of ataxin-7 are subject to regulated clearance

mechanisms; however, distinct post-translational modifications such as

acetylation/deacetylationappear tomodulate fragmentstability/clearance,and thusbeing

crucialformediatingpolyQ-fragmentinducedtoxicity.(Mookerjeeetal.,2009).

Figure1.10Schematicrepresentationofhttproteinprocessing.(AdaptedfromRossandTabrizi,2011)

Introduction

39

1.2.6 ProteinfragmentationinPriondiseases:PrPCandPrPSc

1.2.6.1 Priondiseases

Priondiseasesareagroupofneurodegenerativedisordersthatmayoccursporadically,but

also manifest familial and, infectious forms. In human beings, prion diseases such as

Creutzfeldt-Jakobdisease(CjD)andkuruareclinicallycharacterizedbyprogressivedementia

andmotordysfunction(Prusiner,1991).

Prions are infectious, pathogenic proteins that, in contrast to viruses, are encoded by a

chromosomal gene (PRNP) and are devoid of nucleic acid; they are crucially linked to the

conversion of the physiological cellular prion protein (PrPC), a membrane-associated

extracellularglycoproteinanchoredbyglycosylphosphatidylinositol(GPI),intotheabnormal,

self-propagating‘scrapie’prionisoform(PrPSc)(Prusineretal.,1998).Thepost-translational

modification froma structural α-helical sheet to insoluble β-sheet structures results in an

increased aggregation propensity of PrPSc; ultimately, polymerizing into amyloidogenic

depositsandencouragingprionpropagation(DeArmondetal.,1985).

1.2.6.2 Prionprotein

Biologically active fragments derived from proteolytic processing are thought to have

implicationsinthecourseofpriondiseases.PrPCissubjecttodiverseproteolyticeventsunder

physiologicalconditions:(I)α-cleavagewithintheneurotoxicdomainproducessolubleN1-

andamembraneboundC1-fragment;(II)β-cleavagegivesrisetoaN2-andC2-fragment;and

(III) shedding close to the plasma membrane releases almost full-length PrPC into the

extracellularspace(reviewedinAltmeppenetal.,2012)(Figure1.11).

GiventhattheneurotoxicdomainofPrPCisessentialfortheabnormalconversiontothePrPSc

isoform,α-cleavageandtheresultinginactivationofthisstructuralpartisassumedtobea

protectivemechanismastoprionpropagation(Lewisetal.,2009;Turnbaughetal.,2012).In

addition,α-cleavedC1-fragmenthasbeen linked toapoptotic caspase-3activation in vitro

(Sunyachetal.,2007).Ofnote,thecleavagederivedN1-fragmenthasbeendemonstratedto

beneuroprotectiveinvitroandinvivo(Guillot-Sestieretal.,2009).

Introduction

40

The role of PrPC shedding by a desintegrin and metalloproteinase 10 (ADAM10) in

neurodegenerativediseasesremainunclear.However,thereisevidencethatsheddingofnot

onlyPrPC,butalsoofalreadymisfoldedPrPScleadstoaccumulationofanchorlessPrPSc;thus,

encouragingneurotoxicspreading inthebrain(Chesebroetal.,2005;Rogersetal.,1993).

Notably,sheddedPrPScinthecerebrospinalfluid(CSF)isalsothoughttoenhancepathological

transmission(Tagliavinietal.,1992).

N-terminallytruncatedPrPspecieshasbeenfoundtoco-localizedwithfull-lengthformsin

infectiousPrPScaggregatesinmousebrains(Panetal.,2005).Indeed,varioustruncatedforms

offull-lengthPrPSchasbeenshowntobeinvolvedinpriondiseases:themostcommonPrPSc

fragment PrP27-30 (Parchi et al., 1996); PrP7-8 truncated forms in Gerstmann-Sträussler-

Scheinker(GSS)diseases(Parchietal.,1998);PrP16-17truncatedspeciesinscrapie-infected

animals(Caugheyetal.,1998);orPrP-CTF12/13insporadicCjD(Zouetal.,2003).Aside,itis

thoughtthatnon-fibrillarPrPoligomershaveimplicationsintheprocessofpriondiseases.

However,N-terminaltruncatedPrPleadtonon-specificaggregates,butfailedtoformtoxic

oligomericspecies invitroand invivo; indicatingthatthestructuralN-terminusplaysakey

roleintheinfectiousandneurotoxicprocess(Trevittetal.,2014).Moreover,transgenicmice

expressing a truncated form of mutant PrP lacking a short N-terminal segment of 9-aa

displayedaneurotoxicphenotype(Westergardetal.,2011).

Introduction

41

Figure1.11SchematicrepresentationofPrPprocessing.(AdaptedfromAltmeppenetal.,2012)

1.2.7 ProteinfragmentationinFTLDandALS:TDP-43

1.2.7.1 TDP-43proteinopathies

TDP-43proteinopathies including sporadic and familial frontotemporal lobardegeneration

(FTLD)(Araietal.,2006)andamyotrophiclateralsclerosis(ALS)(Neumannetal.,2006)area

groupof neurodegenerativedisorder causedbypathological neuritic inclusions containing

transactiveresponse(TAR)DNA-bindingprotein43.AsideAD,FTLDisthemostcommonform

ofprogressivedementiainhumanbeingsundertheageof65years(Nearyetal.,1998).ALS,

however, is the most common cause of motor neuron degeneration accompanied by

progressivemusclewasting.Moreover,FTLDpatientsmayalsodevelopALS;andviceversa

(Murphyetal.,2007).Aside,mutationintheprogranulingene(PGRN)arethoughttocause

familialformofFTLD(Ghidonietal.,2008).

Introduction

42

1.2.7.2 TDP-43

Physiologically,TDP-43isthoughttoplayaroleintheregulationofvariouscellularprocesses

including alternate splicing, transcription, apoptosis, microRNA biogenesis, and mRNA

transportandstability(reviewedinBurattiandBaralle,2008;Ouetal.,1995).HumanTDP-43

is a 414-aa nuclear protein, ubiquitously expressed and highly conserved; structurally, it

harbors two RNA-recognition motifs (RRM1; RRM2) and a protein-protein interaction

mediating C-terminal glycine-rich region (Wang et al., 2004). Furthermore, the TDP-43

moleculeexhibitthreepotentialcaspase-3cleavagesites(Zhangetal.,2007).

Both,hyperphosphorylatedandubiquitinatedfull-lengthandN-terminallytruncatedTDP-43

are themajor component ofneuritic inclusions in brain tissue of FTLD and ALS patients.

Caspase 3/7-mediated proteolytic cleavage at Asp89 and Asp220 of wild-type TDP-43

generatesa35-kDa(CTF35)anda25-kDaC-terminal fragment (CTF25) respectively (Figure

1.12); moreover, different cleavage sites at Arg208, Asp219, and Asp247 of CTF25 were

further identified(Igazetal.,2009;Neumannetal.,2006;Nonakaetal.,2009).Moreover,

eachindividualTDP-43CTFhasbeenshowntopossessdistinctmolecularpropertiesincluding

intracellulardistributionandphosphorylationstatus,contributingtothepathogenicdiversity

ofTDP-43proteinopathies(Furukawaetal.,2011).

Sofar,accumulationoftruncatedspeciesmightmediatetoxicity,orissimplyaconsequence

inthecourseofneurodegeneration;inanycase,thepathologicalroleofTDP-43fragments

remains elusive. However, several studies implicate a neurotoxic relevance of TDP-43

fragmentation. Indeed, expressionofcaspase-cleavedCTF25 fragment inhumancell lines

lead tocelldeathaccompaniedby the formationof toxic insolubleaggregates;aside, this

fragmentdidnotinteractwithfull-lengthnuclearTDP-43oraffectitsfunction,thus,indicating

atoxicgain-of-function(Zhangetal.,2009b).Furthermore,thisfragmentshowedanincreased

hyperphosphorylation propensity at sites not required for neurotoxic inclusion formation

comparedtofull-lengthTDP-43.Incontrast,anotherstudydemonstratedthatTDP-43CTFs

provokeboth,first,theformationofaberrantphosphorylatedandubiquitinatedaggregates,

and second, the incorporation of newly synthesized endogenous full-lengths species into

thesecytoplasmicinclusions(Nonakaetal.,2009).Interestingly,giventhatregulationofexon

splicingisadefinedfunctionofTDP-43,aberrantsplicinghasbeendemonstratedincultured

Introduction

43

cellsexpressingTDP-43CTFscleavedatArg208; thus, strengthenedthepathogenic roleof

TDP-43fragmentsinFTLDandALS(Igazetal.,2009).

However,thereisalsoevidenceoffragmentation-mediatedclearanceoffull-lengthTDP-43:

the activation of endoplasmatic reticulum (ER) membrane-bound caspase-4 cleavage

diminishesERstresscausedbyabundantaccumulationofTDP-43;subsequentactivationof

the downstream caspase-3/7 pathway ultimately results in reduced full-length TDP-43

cytotoxicityandnecroticcelldeath(Lietal.,2015).

Figure1.12SchematicrepresentationofTDP-43domainprocessing.(AdaptedfromChiang2016)

Introduction

44

Wehaveoutlinedaboveseveralexamplesofproteinopathies,whereproteincleaveageresults

in aggregation prone fragments, pointing towards a general pathogenic role of protein

fragmentation in neurodegenerative diseases. This can’t be said without mentioning the

ongoingdebateonthetoxicityofaggregatesinproteinopathies.

Thereexisttwoopposingviewsonamyloidlesionsinmostneurodegenerativediseases:high

molecular,fibrillaryaggregatescaneitherbeconsideredasinertintracellulargarbageor,as

specifically toxic entities. In most proteinopathies, the current evidence points towards

considerableneurotoxicityofearly,non-fibrillaryoligomericaggregates(Bergeretal.,2007;

Gersonetal.,2014;Pattersonetal.,2011;Usenovicetal.,2015).Thisconceptofoligomer

toxicity in neurodegenerative disorders is compatible with a primary pathogenic role of

proteinfragmentsthatinducetheaggregationofproteinsintooligomericspecies.Wehave

recentlyreportedsuchatoxicityinducingeffectofataufragmentintautransgenicmouse

models(Ozceliketal.,2016),providingexperimentalsupportofthishypothesis.

Aimsofwork

45

2 Aimsofthework

Attributabletotheincreaseinlifeexpectancy,theprevalenceoftauopathiesincludingADis

continually growingwith underlying pathologicalmechanisms partly unknown. Given that

currenttherapeutictreatmentsarelimitedtosymptomaticapproaches,globalsocietyisfaced

withenormoussocioeconomicchallenges.

Yet,neurodegenerativediseaseresearchhighlightedvariousaspectsofthepathogenesisof

tauopathiessuchasproteinfragmentationandspreading.

Protein fragmentation is increasinglyemergingasan integralevent in thepathogenesisof

neurodegenerativediseases.Numerousinvitroandinvivostudieshaveaddressedwhether

cleavageofdisease-associatedproteinsissufficienttoexacerbateanongoingdiseaseprocess.

Indeed, the relevance of protein fragmentation has been highlighted in the course of a

plethora of proteinopathies with an emphasis on the neurotoxic potential of protein

fragments (Altmeppen et al., 2012; Furukawa et al., 2011; Masters and Selkoe, 2012;

Mookerjeeetal.,2009;Vidaletal.,1999;Wellingtonetal.,2000).Asfortauprotein,truncated

formshavebeenshowntofacilitatetauaggregation;andthus,appeartopossessanincreased

neurotoxicpotentialcomparedtofull-lengthtauspecies(Abrahaetal.,2000).Intauopathies,

thedynamicsofvariousneurotoxictauspeciesisstillamatterofdebate.Todate, it isnot

knownwhichtauspeciesisthemosttoxicandhowthetoxicityismediated(Goedert,2015).

However, hyperphosphorylated and aggregated tau species are considered as the

fundamentalneurodegenerativecomponentsintauopathies(SpillantiniandGoedert,2013).

Aimsofwork

46

Directlydeterminingthepathologicalfunctionofdistincttauspeciesandcellularmechanisms

underlyingtautoxicity,invivo,remainschallenging.Numeroustautransgenicmousemodels

havebeencreatedtoexplainchangesintheregulationoftaumetabolism(Duyckaertsetal.,

2008). Accordingly, we generated multiple transgenic mouse lines in order to study the

relevanceoftaufragmentationfortheneurodegenerativeprocessintauopathies.Thesemice

eitherexpressatruncatedtausequence(3RΔtau151–421)aloneorincombinationwithwild-

type(0N3Ror2N4R)ormutant(0N4R)full-lengthtauisoforms.Ofnote,animalmodelsthat

expresswild-type full-length formsof taugenerallystaydevoidof insoluble tau inclusions,

whereasmodelsthatoverexpressmutatedfull-lengthtauexhibitNFTs(Allenetal.,2002;Gotz

etal.,1995).

Aside tauprotein, theaggregationofAβspeciesgeneratedbyproteolyticcleavageofAPP

constitutesakeypathologicalhallmarkofAD.Lately,aprion-likeroleofmousebrainderived

Aβ species has been demonstrated (Langer et al., 2011); thus indicating that Aβ species

presentintheCSFmayexhibitsimilarseed-likepropertiesinvivo.

Within this framework, we sought, to longitudinally analyze the effects of a human tau

fragmentinamurinemodelandspecificallyitspotentialinteractionwithhumanfull-length

tau,andinparallel,toanalyzepotentialseed-potentCSFderivedAβspeciesinvivo.

Results

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3 Results

3.1 PublicationNo.1

Co-expression of truncated and full-length tau inducessevereneurotoxicity

Ozcelik S*,Sprenger F*, Skachokova Z, FraserG, Abramowski D, Clavaguera F,ProbstA,FrankS,MüllerM,StaufenbielM,GoedertM,TolnayM,WinklerDT.

*Bothauthorscontributedequallytothework

MolPsychiatry.2016

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SupplementalInformation

SupplementaryInformationaccompaniesthepaperontheMolecularPsychiatrywebsite

(http://www.nature.com/mp)

SupplementaryVideos:

VideoS1

A3-week-oldP301SxTAU62ondoubletransgenicmouse isshown.Notetheparalysisof the

hindlimbs,whilethemouseisstillcapableofmovingbyusingtheforelimbs.

VideoS2

Videoofa5-month-oldhomozygousP301Smouse.HomozygousP301Smicedevelopovert

motor impairmentsstartingatagesof3-4months.Thismouse iswalkingslowly,however,

thereisnoseverehindlimbpalsyoccurringinP301Smiceuptoagesof5months.

VideoS3

ThesameP301SxTAU62on-offmouseseeninVideoS1isshown,butnow38daysafterDtau

expressionhasbeenstoppedbywithdrawalofdoxycycline.Thehindlimbpalsyissignificantly

improvedandthemouseisagainwalkingalmostnormally.

VideoS4

A3-week-oldALZ17xTAU62ondoubletransgenicmouse isshown.Notetheparalysisof the

hindlimbs,whilethemouseisstillcapableofmovingbyusingtheforelimbs.

VideoS5

ArecoveredALZ17xTAU62on-offmouse isshownonemonthafterDtauexpressionhasbeen

stoppedbywithdrawalofdoxycycline.

VideoS6

ParalyzedALZ31xTAU62onmouseagedthreeweeks.

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VideoS7

12-month-oldP301SxALZ31mouse.Notethenormalgridclimbingcapabilityofthisdouble

transgenicmouse.

VideoS8

4-month-oldALZ17SxALZ31mouse.Notethenormalgridclimbingcapabilityofthisdouble

transgenicmouse.

Results

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SupplementaryTableandFigures:

TableS1

(a-c) Overview on the transgenic and co-transgenicmouse lines used for the present studies. Tau isoformsexpressedareshown.Darkgreyboxesindicatethetaurepeatdomains(3Ror4Risoforms),andlightgreyboxesN-terminalinserts(e.g.ALZ17�2N4R).The tau cDNA constructs are either driven by a standard Thy1.2minigene (Thy1.2) or by amodified Thy1.2minigene that contains a tetracycline controlled transcriptional silencer element (Thy1.2-tTS) in case of theTAU62mouse. (a) shows the tau isoformsexpressed in single-transgenic lines. (b) shows the tau formsof3mouselinesco-expressingfull-lengthtauwithDtau.(c)depicts2mouselinesco-expressingdifferentfull-lengthtauisoforms.

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FigureS1

TAU62micedevelopaslowlyprogressivemotorphenotype.Motorfitnesswasassessedonaverticalmeshgrid.TimespentonthegridprogressivelydeclinedinTAU62mice,whileB6controlswereunimpairedat12monthsandshowedamilddeclineat18monthsofage.

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FigureS2

(a)ExtensivehyperphosphorylationoftauwasseeninthebrainstemofoldhomozygousP301Smicebystainingwithantibodiestargetinglatephospho-epitopes.MultipletautanglesandgranularaggregatesweredetectableinthesemicebyGallyassilverstain.Thescalebarinacorrespondsto50µm.(b)Westernblottingundernon-reducingconditionsrevealedhigh-moleculartauspeciesinparalyzedP301SxTAU62onmice(lanes6&7);similartauspecieswereseeninagedtanglebearinghomozygousP301Smice(lane5).Whentauexpressionwashalted,nomorehigh-moleculartauformsweredetectableinP301SxTAU62on-offmice(lane3;Westernblotperformedwithanti-tauantibodyHT7).(c)StainingwiththeRD3antibodytargetingAsp421showsthepresenceofDtauinthe highmolecular weight tau species. (d) Sarkosyl-extraction detects only soluble tau species in paralyzedP301SxTAU62on mice (“sol”: sarkosyl-soluble tau; “insol”: sarkosyl-insoluble fraction). (e-g)Dtau was widelyexpressed in the spinal cord of P301SxTAU62on mice (e) and phosphorylated at the AT8 epitope (f). Uponcessation of Dtau expression,Dtau- and AT8-positive tau was no longer detectable (g). The scale bar in ecorrespondsto100µmine-g.

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FigureS3

(a-m)P301SxTAU62miceexhibitsignsofGolgidisruption,proteinmissortingandmitochondrialclustering.ThesesignsarereversibleuponcessationofDtauexpression.ImmunohistochemistryusingantibodiesagainstMG160(a-f), synaptophysin (g-i), VAMP2 (k-m), and cytochromeCoxidase (COX) (n-p), in thehippocampusof non-transgenicmice(B6),3-week-oldparalyzedmice(P301SxTAU62on)andrecoveredmice6weeksaftercessationofDtauexpression(P301SxTAU62on-off).Thescalebarinacorrespondsto19µmind-f,63µmina-candg-i,and400µm in k-m. The scale bar in n corresponds to 30µm for n-p. Arrows in (e) indicate fragmented Golgistructures.

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FigureS4

(a-c)YoungTAU62miceexhibitnormalsciaticnerves(Masson’strichromstain(a);para-Phenylenediamine(b);immunohistochemistry using 2f11 antibody (c). The scale bar in c corresponds to 30 µm in a-c. (d-g) M.gastrocnemiusstainedforATPase(pH4.2).Darktype1fibres(1)andlighttype2fibres(2).Thescalebaringcorresponds to 50µm (ford-g). P301S: heterozygousmice transgenic for humanmutant P301S tau, aged 3weeks;TAU62:heterozygousmiceexpressing3Rtau151-421,aged3weeks;P301SxTAU62

on:paralyzedmice,aged3weeks;P301SxTAU62on-off:recoveredmice,6weeksaftercessationoftheexpressionofDtau.

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FigureS5

(a-i)Co-expressionof3Rwild-typetauandDtau(ALZ31xTAU62mice)causesparalysisandneuropathy,whicharenotreverseduponcessationofDtauexpression.(a)Paralyzed(aged3weeks)andnon-recovered(3weeksaftercessationofDtauexpression)ALZ31xTAU62mice (seealsovideoS6). (b)Absenceof recoveryofmotorfunctionasassessedbyagrid-testofALZ31xTAU62micefollowingtheremovalofdoxycyclinebetween14and16days(blueline;trianglesindicatethetimesofeuthanasia,n=6).MotorfunctionofheterozygousALZ31mice(green line,n=7). (c)WesternblotwithHT7ofbrainstemtissue fromnon-transgenicmice (B6),TAU62mice,ALZ31miceandALZ31xTAU62mice.Actinstainingwasusedastheloadingcontrol.(d-i)HistologicalanalysisofparalyzedALZ31xTAU62miceaged3weeks,usingAT8(d),AT100(e),NF200(f),Holmes-Luxol(HL)(g),Masson’strichrome(h),andHematoxylin-eosin(HE)staining(i).Thearrowin(g)pointstoaspheroid.Thescalebarindcorrespondsto200µmindande;100µminf;33µming;50µminh,i.

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FigureS6

(a,b)Robustexpressionofthetwofull-lengthtauisoformsinP301SxALZ31(a)andALZ17xALZ31(b)co-transgenicmice.Forcomparison,expressionofmiceco-transgenicforDtauwithfull-lengthtau,aswellastherespectivesingletransgenicmiceisshown.WesternblotsrununderreducingconditionsusingHT7antibody.

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SupplementalExperimentalProcedures Antibodiesusedforimmunohistochemistry(IHC)andWesternblotting(WB)(speciesismouse,unlessindicatedotherwise): Antibody Target Dilution SourceHT7 humantau

aa159-163WB1:4000IHC1:800

Pierce,Rockford,IL#MN1000

BR134 humantau WB1:1000 (Goedertetal.,1989)

Tau-C3 TaucleavedatresidueAsp421

WB1:1000IHC1:1000

SantaCruzBiotechnology,Inc,Dallas,TX#sc-32240

AT8 TaupSer202/Thr205

WB1:1000IHC1:800

Pierce,Rockford,IL#MN1020

AT100 TaupThr212/Ser214

WB1:1000IHC1:500

Pierce,Rockford,IL#MN1060

PHF-1 TaupSer396/404

WB1:2000IHC1:1000

PeterDavies,AlbertEinsteinCollegeofMedecine,Bronx,NY

MC1 Tauaa5-15,312-322

IHC1:100 PeterDavies,AlbertEinsteinCollegeofMedecine,Bronx,NY

2F11 neurofilament(NF)NF-L,NF-H(70kD)

IHC1:800 Dako,Glostrup,DK#M0762

NF200 neurofilament(200kD) IHC1:100 (Probstetal.,2000)GFAP glialfibrillaryacidic

proteinIHC1:500 ThermoFisherScientificInc.,Kalamazoo,

MI#MS-1407-R7

Synaptophysin synaptophysin IHC1:1000 MilliporeCorporation,Billerica,MA#MAB5258

MG160(rabbit) Golgiapparatus IHC1:1000 NicholasGonatas,PathologyandLaboratoryMedicine,UniversityofPennsylvania,PA

VAMP2/Synaptobrevin2(rabbit)

transportvesicles IHC1:1000 Synapticsystem,Goettingen,Germany#104202

GAPDH(6C5) GAPDH WB1:1000 SantaCruzBiotechnology,SantaCruz,CA,#32233

ß-actin actin WB1:5000 Sigma-Aldrich,SaintLouis,MO#A5316Coxsubunit1a mitochondrial

stainingIHC1:200 Abcamplc,Cambridge,UK

#ab14705

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3.2 Preliminarydata

Protective effect of early tau burden on late neurotoxicdistress level – Mechanisms underlying tauopathy andconsequencesforfuturetherapies

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Protectiveeffectofearlytauburdenonlateneurotoxicdistresslevel–Mechanisms

underlyingdelayedtauopathyandconsequencesforfuturetherapies

Preliminaryresults

Wehaverecentlyprovidedevidencethatassemblyoffilamentoustauaggregatesisnotsolely

responsibleforthecharacteristictautoxicityinneurodegenerativedisorders.Wecouldshow

thatboth,co-expressionoffull-lengthP301Smutant,aswellasfull-lengthwildtypetauwith

Δtau151-421resultsinahighinvivoneurotoxicityassociatedwiththeformationofsolublehigh

molecular weight tau oligomers, extensive nerve cell dysfunction and severemotor palsy

albeitinabsenceofinsolubletauaggregatesortangles(Ozceliketal.,2016).

GiventhatourP301SxTAU62on-offmicewereexposedtodrasticneurotoxicstressduringearly

postnatallife,weaskedwhetherthiseventhasanypossibleimpactontaupathologylaterin

life,evenaftertheexpressionoftruncatedtauishalted.Initially,wehypothesizedthatthe

observedprevalentoligomerictauspeciesmightactasaformoftoxicseedformoredrastic

tau pathology in later stages, ultimately manifesting in earlier tau tangle formation.

Interestingly, compared to their heterozygous P301S tau transgenic littermates that have

neverbeenparalyzed,recoveredP301SxTAU62on-offmicethat,incontrast,experiencedearly

neurotoxicity tau burden however exhibit a better motor phenotype associated with

considerablylesstauproteinlevelsandanoverallattenuatedtaupathology.

3.2.1 DelayedmotorphenotypeinagedP301SxTAU62on-offmiceafterrecovery

ofsevereneurotoxicity

TomonitorthebehaviouralphenotypeinP301SxTAU62on-offmiceaftercessationofΔtau151-421

butcontinuousfull-lengthP301Stauexpression,weperformedabatteryofmotorfunction

testsatagesof12to16months.Giventhatthepathologicalhindlimbpostureisnormalized

inrecoveredP301SxTAU62on-offmice(Ozceliketal,2016,Figure3.1a),wechosetorepeatthe

tailsuspensiontest:14-month-oldheterozygousP301Stransgeniclittermatesshowedalready

first signsof hind limb clasping that aggravated at 16monthsof age,while thehind limb

spreadingofP301SxTAU62on-offmicestayedalmostnormalupto16monthsofage(Figure3.1

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bandc).Indeed,comparedtotheirheterozygousP301Slittermates,P301SxTAU62on-offmice

maintainasignificantlybettermotorstrength,coordinationandbalanceat14monthsofage

thatisstillpronounced,butnon-significantattheageof16months(Figure3.1dande).Of

note,bothtransgeniclinesshowedcomparableperformancesonthegridandrotarodatthe

age of 12months, aswell as no differences in sex-specific bodyweightwere found at 16

monthsofage(Figure3.1d,eandf).

months months 16 months

0

10

20

30

40

50

male female

Bodyweight

(g)

0

30

60

90

120

150

180

161412

***

***

***

Tim

e on

Grid

(sec

)

0

10

20

30

40

***

*** ***

Tim

e on

Rot

arod

(sec

)

161412

d e f

P301S heterozygous P301SxTAU62on-off P301SxTAU62on-offP301S heterozygous

aged 14 months

P301SxTAU62onparalyzed,

P301SxTAU62on-offrecovered,

aged 16 monthsaged 6 weeksaged 3 weeks

a b c

P301S heterozygous

P301SxTAU62on-off

B6

Figure3.1.FormerlyparalyzedP301SxTAU62on-offmicedevelopasignificantdelayedmotorphenotype. Incontrast to their heterozygous P301S transgenic littermates, P301SxTAU62on-off mice exhibit a better gridreflex,motorstrengthandmotorcoordinationatagesof12,14,and16months.(a-c)Pathologicalhindlimbspreadingwasassessedbyatailsuspensiontest.(a)ParalyzedP301SxTAU62onmiceatageof3weeksexhibitpathological hind limb posture. 3 weeks after cessation of Δtau151-421 expression, P301SxTAU62

on-off micerecoverfromtheirpalsy,albeitcontinuativeexpressionofP301Smutanttau(fordetailsseeOzceliketal2016).(bandc)LessvisibleuponP301SxTAU62on-offmice,pathologicalhindlimbspreadingmanifestedpredominantlyinheterozygousP301Stransgenic littermatesat14to16monthsofage. (d-f)Basedonthegrid(d,P301Sheterozygous:12months,n=15;14months,n=21;16months,n=21;P301SxTAU62on-off:12months,n=5;14months,n=11;16months,n=13;non-transgenicB6:12months,n=5;14months,n=13;16months,n=12)androtarod(e,P301Sheterozygous:12months,n=15;14months,n=25;16months,n=21;P301SxTAU62on-off:12months,n=5;14months,n=14;16months,n=13;non-transgenicB6:12months,n=5;14months,n=13;16months,n=12)performancetest,P301SxTAU62on-offmiceexhibitsignificantlybettermotorfitnessatagesof14 to 16 months, compared to heterozygous P301S transgenic littermates. (f) Sex-sorted body weightmonitoringat16monthsofage(P301Sheterozygous:male,n=6;female,n=17;P301SxTAU62on-off:male,n=5;female,n=6).Datarepresentsthemeanands.d.ofindicatedanimalspergroup.***P<0.001.

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3.2.2 ReducedtaupathologyinagedP301SxTAU62on-offmiceafterrecoveryof

severeneurotoxicity

InordertoelucidatewhethertheconsiderablydelayedmotorphenotypeinP301SxTAU62on-

offmicewasassociatedwithavaryingdegreeofseverityoftaupathology,wescreenedbrain

sections of both, 16-months-old P301SxTAU62on-off mice and their heterozygous P301S

littermates,forthepresenceofcertaintauspecies.Indeed,wecouldshowthatneurofibrillary

tanglesparalleledthepreviouslyobservedimpairedmotorphenotypeinheterozygousP301S

littermates;specifically,GallyasBraakSilverstainingshowedmassivetautangleformationin

the brainstem of heterozygous P301S mutant littermates, whereas formerly paralyzed

P301SxTAU62on-offmiceremainedalmostdevoidoffibrillarytauinclusions(Figure3.2a,band

e).

Furthermore, in linewith these histopathological findings, anti-tau antibody AT8 revealed

attenuated tau hyperphosphorylation in P301SxTAU62on-off mice when compared to their

Figure3.2.FormerlyparalyzedP301SxTAU62on-offmicerevealsignificantlesstaupathology.ComparedtotheirheterozygousP301Slittermates,P301SxTAU62on-offmicethatunderwentsevere,earlytaustressremainalmostdevoid of tau tangle pathology in the brainstem and exhibit attenuated tau hyperphosphorylation. (a - d)Histological analysis of 16-month-old P301SxTAU62on-off mice (b and d) and heterozygous P301S transgeniclittermates(aandc)usingGallyas–Braaksilver(aandb)andanti-tauantibodyAT8(candd).Thescalebarin(d)correspondsto500μmin(aandb)and200μmin(candd).QuantificationofGallyaspositivetautanglesinthebrainstemofP301Sheterozygous(n=10)andP301SxTAU62on-off(n=10)mice.***P<0.001.

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heterozygousP301Slittermates(Figure3.2candd).Though, tauproteinismostexpressedin

thebrainstemareaunderthecontrolofneuron-specificThy1.2-promoterelement, wealso

foundsignificantlesstaupathologyinthecervicalspinalcord(SupplementalFigureS3.1c-d)

andtheforebrainarea(SupplementalFigureS3.1d-f)ofP301SxTAU62on-off;asopposedtothis,

nosignificantdifferenceinfilamentoustauformationcouldbedetectedinthehippocampal

area(SupplementalFigureS3.1g-i).

Further,onlyheterozygousP301Slittermatesshowedhyperphosphorylationoflateepitopes

inthebrainstem,visualizedbyanti-tauAT100antibody,consistentwiththepresenceoftau

filaments (SupplementalFigureS3.2aandc);ofnote,Δtau151-421wasnotexpressed inthe

brainstemofeitherthetransgenicmouselines(SupplementalFigureS3.2bandd).

3.2.3 ReducedtauproteinlevelsinagedP301SxTAU62on-offmiceafterrecovery

ofsevereneurotoxicity

Giventheobtaineddatasofaravailableweconsideredthatchangesintauproteinexpression

levels correspond to the present histopathological phenotypes in formerly paralyzed

P301SxTAU62on-offmiceandtheirheterozygousP301Smutantlittermates.ThroughWestern

Blot analysis,we confirmed, asexpected, a significant reduction inboth the total tauand

sarkosyl-soluble tauexpression level inP301SxTAU62on-offmice (Figure3.3a-d);notably, in

heterozygous P301S littermates, the marked histopathological tau tangle pathology is

reflected in a significant increase of the sarkosyl-insoluble tau fraction expression levels

(Figure3.3aandb,stars).

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0.0

0.5

1.0

1.5

2.0

2.5

3.0 *

Total tau

HT7

/GAP

DH

0.0

0.2

0.4

0.6

0.8***

Insoluble tau fraction

HT7

/GAP

DH

aP301S heterozygous P301SxTAU62on-off

aged 16 months

P301Shomo

B6

HT7

GAPDH

HT7

GAPDH

c

0.0

0.5

1.0

1.5

2.0 *

Soluble tau

HT7

/GAP

DH

P301S heterozygousP301SxTAU62on-off

totaltau

solubletau

*

*

b

d

Figure3.3.FormerlyparalyzedP301SxTAU62on-offmicerevealsignificantlesstotal, insolubleandsolubletauproteinlevels.(a-d)Westernblottingwithhuman-specificanti-tauantibodyHT7ofbrainstemtissuefromP301Shomozygous(lane1),non-transgenicB6(lane2),P301Sheterozygous(lanes3-7)andP301SxTAU62on-off(lanes8-12)mice (a and c); GAPDH staining was used for protein normalization (b andd). At the age of 16months,significantly increased total tau protein levels were detected in heterozygous P301S transgenic littermates;associatedwithelevatedinsolubletaufractionlevels(aandb,stars).(candd)Sarkosyl-extractiondetectsonlysolubletauspeciesthatwerefoundtobesignificantlyattenuatedinP301SxTAU62on-offmice.Datarepresentsthemeanands.d.of5animalspergroup.*P<0.05and***P<0.001.

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3.2.4 Supplementalmaterial

P301S

heter

ozyg

ous

P301S

xTAU62

on-o

ff0.00000

0.00005

0.00010

0.00015

0.00020

0.00025 ***

Cervical Spinal Cord

Gal

lyas

pos

itive

par

ticle

/pix

el

P301S

heter

ozyg

ous

P301S

xTAU62

on-o

ff0.00000

0.00002

0.00004

0.00006

0.00008 **

Forebrain

Gal

lyas

pos

itive

par

ticle

/pix

el

P301S

heter

ozyg

ous

P301S

xTAU62

on-of

f0.000000

0.000001

0.000002

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0.000004 ns

Hippocampus

Gal

lyas

pos

itive

par

ticle

/pix

el

P301S heterozygous P301SxTAU62on-off

aged 16 months

a b

g

c

fd e

h iGallyas

FigureS3.1.FormerlyparalyzedP301SxTAU62on-offmicerevealsignificantlesstaupathologyinthecervicalspinalcordandforebrainarea.(a-i)Histologicalanalysisof16-month-oldP301SxTAU62on-offmice(b,e,andh)andheterozygousP301Stransgeniclittermates(a,dandc)usingGallyas–Braaksilverstainofthecervicalspinalcord(a-c),forebrain(d-f)andhippocampalarea(g-i).Dottedsquaresindicateareaofmagnificationinthelower-right corner (a,b,d ande). The scalebar in (h) corresponds to500μm in (a,b,d,e, gand h). (c, f and i)QuantificationofGallyaspositivetautanglesinthecervicalspinalcord(c),forebrain(f)andhippocampus(i)ofP301Sheterozygous(n=10)andP301SxTAU62on-off(n=10)mice.ns=non-significant,**P<0.01and***P<0.001.

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FigureS3.2.(aandb) Immunohistochemistrywithanti-tauantibodiestargeting latephospho-epitopesandDtau.AftercessationofDtauexpression,extensivehyperphosphorylationofAT100-positivetauwasonlyseeninthebrainstemofheterozygousP301Slittermates(a),butnotinP301SxTAU62on-offmice(b).Inthebrainstemofbothtransgeniclines,noC3-positiveDtaucouldbedetectedattheageof16months(aandb).Thescalebarin(d)correspondsto100μmin(aandd).

Results

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3.3 PublicationNo.2

Amyloid-beta in the cerebrospinal fluid of APP transgenicmicedoesnotshowprion-likeproperties

Skachokova Z, Sprenger F, Breu K, Abramowski D, Clavaguera F, Hench J,StaufenbielM,TolnayM,WinklerDT.

CurrAlzheimerRes.2015

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SupplementaryInformation1.1. ELISA

Abeta40andsAPPlevelsweremeasuredusingHuman6E10andAPPalpha/sAPPbetaKitsrespectively,bothfromMesoScaleDiscovery,accordingtothemanufacturer’sinstructions.1.2. Mice

Genotype Seed Seeddonorage,mo

Seedingtime,mo

App23 tgCSF 18 14App23 tgCSF 24 14App23 tgCSF 24 14App23 tgCSF 24 14App23 tgCSF 18 21App23 tgCSF 18 21App23 tgCSF 24 21App23 tgCSF 24 21App23 tgCSF 24 21App23 wtCSF 3 21App23 wtCSF 24 21App23 wtCSF 24 21App23 FB 24 20App23 FB 24 20C57BL6 tgCSF 24 21C57BL6 tgCSF 18 21C57BL6 tgCSF 18 21

App23conc. tgCSF

24 11

App23conc. wtCSF

24 11

App23conc. tgCSF

24 20

Table S1. Table of all mice used for quantitative analysis in the study. Abbreviations used: tg=transgenic,wt=wildtype,FB=forebrainhomogenate,conc.=concentrated.

Results

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Supplementaryresults1.1. ELISA

Sample Aβ40,pg/μlCSF sAPPα,pg/μlCSF sAPPβ,pg/μlCSFAPP23CSF 4.2 92 121ConcentratedAPP23CSF 92.6 2594 3659

TableS2.ELISAtableofresults.

1.2. QuantificationofAβpathologycomparinginjectedvsnon-injectedhippocampus

Genotype Treatmentgroup

Seedingtime,mo N

Lowerratio GMR Upper

ratio Pvalue

APP23 tgCSF 14 4 0.77 0.94 1.15 0.56

APP23 tgCSF 21 5 0.79 0.91 1.05 0.19

APP23 wtCSF 21 3 0.65 0.81 1.01 0.06

APP23 FB 20 2 0.41 0.54 0.70 0.00***

C57BL6 tgCSF 21 3 0.00 0.00 0.00 NA

APP23conc. tgCSF

11 1 0.59 0.88 1.30 0.52

APP23conc. wtCSF

11 1 0.72 1.10 1.67 0.66

APP23conc. tgCSF

20 1 0.61 0.92 1.40 0.70

TableS3.Geometricmeanratios(GMR)ofamyloid-βratioscomparingnon-injectedvsinjectedhippocampus.N indicates the number of mice used; tg=transgenic, wt=wild type, FB=forebrain homogenate,conc.=concentrated;***indicatesp<0.001.

Discussion

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4 Discussion

Alzheimer’s disease represents the most prevalent form of a major subclass of various

neurodegenerativediseases:thetauopathies.InSwitzerlandalone,over100’000peopleare

estimatedtosufferfromdementiaincludingAD;andthisnumberisexpectedtotripleby2050

(Blankmanet.al, 2012). Todate, the lackof therapeutic treatments limitspharmacological

therapies to symptomatic interventions and given that millions of people have been

diagnosed worldwide, AD constitutes a crucial source of social and economic problems

(Scheltensetal.,2016).

ThediseasespectruminpatientswithneurodegenerativedisordersincludingADextendsfrom

genetic alterations to protein modifications and altered inner life of a cell in affected

anatomical regions through to clinical manifestations. Years of research has led to the

discovery of a plethora of proteins being crucial in the course of neurodegenerative

syndromes;buteversinceresearchersarepuzzledbypiecesofthebigpicture.Thecleavage

of larger, neurodegeneration-associated proteins into smaller, potentially neurotoxic

fragmentshasbeensubjectofnumerousstudies; indeed,academicresearchsuggeststhat

fragmentationandothersubstantialeventsleadingtoneuronaldysfunctionatthesametime

haveledmanypeopletosufferfromvarietyofneurodegenerativediseasessuchasADand

other tauopathies, PD, HD, or CjD. However, the relevance of protein fragmentation in

neurodegenerationisstillunderdebate.

Discussion

85

Inhuman tauopathies, thedistinctmolecular and cellularmechanisms contributing to the

pathogenesisandprogressionoftauopathiesremainstillopaque.Precisely, the initiatorof

neurotoxiceventsandhowneurotoxicity ismediated isnotknown indetail.Aside theAβ

peptide and its pathological role in AD, the closer correlation between tau and disease

progressionarouseattentionandpromotedtau-focusedresearch.Thus,thedevelopmentof

transgenic mouse models allowed to investigate core aspects of the pathogenesis of

tauopathies by modeling particular disease steps in vivo; ultimately aiming for a better

understanding of the neuropathological mechanisms behind the complex nature of

neurodegenerativeprocesses.

Thepursuitofneurotoxictauspecies

The neuropathological identity of AD falls into two broad categories: extracellular senile

plaquescomposedof theAPPcleavageproductAβand filamentous tau inclusionsderived

fromaccumulationofmodifiedtauspecies.

Tauplaysacentralroleinmultipleneurodegenerativedisorders;however,itscontributionto

theonsetandprogressionofADisstillamatterofdebate.ButastaugenemutationinFTDP-

17T(Huttonetal.,1998;Poorkajetal.,1998;Spillantinietal.,1998)pavedthewayforinvivo

expression ofmutant human tau protein, transgenicmousemodels were generated that

exhibit distinct aspects of tau pathology such as age-related filamentous tau deposits,

neuronaldegenerationandaxonaltransportdysfunction(Gotzetal.,2004).

The formation of tau aggregates is depending on the conformational switch to β-sheet

structure;indeed,pointmutationsbutalsovariouspost-translationalmodificationsincluding

phosphorylation,acetylation,glycosylation,ubiquitination,andtruncationstructurallyalter

tauandthusfavouritsaccumulation.Whileaberrantaggregationandhyperphosphorylation

oftauareproposedtobethepivotalneurotoxicelementsinhumantauopathies,themost

toxicspeciesandhowtoxicityismediatedisofongoingdebate(BraakandDelTredici,2011;

ColemanandYao,2003;Goedert,2015;Terryetal.,1991).

Notably,proteolyticcleavageoftauhasbeenshowntobeanearlyeventinthepathological

cascadewithconsiderableimpactontautoxicity;indeed,fragmentationoftauoccursbefore

Discussion

86

toxictangleformationandfacilitateandpromotetauaggregationcomparedtofull-lengthtau

species (Abraha et al., 2000; Berger et al., 2007; Cowan et al., 2010). Various proteases

includingcaspase3,caspase6andcalpainshavebeenidentifiedtocleavefull-lengthtauinto

fragments which accumulation correlates with the progression of AD; notably, D421

constitutesthemostprominentcaspasecleavagesite inall formsof tauopathies (Basurto-

Islasetal.,2008;Fasuloetal.,2005;Guillozet-Bongaartsetal.,2005;Guoetal.,2004).

Withinthisframework,wehaveestablishedaninduciblemouseline(TAU62)overexpressing

a short, human 3R tau151–421 fragment (Δtau) that develop a mildly progressive motor

phenotypewithataxiaatabout18monthsofage.Interestingly,TAU62micedidnotdevelop

filamentous tau inclusions but exhibited neuron specific pretangle pathology in form of

abnormallyphosphorylatedtau;thisisinlinewithfindingsinothertaufragmentexpressing

mousemodels (McMillanetal.,2011),butcontradicts thepredominantneuropathological

phenotypedefinedbycorticalinsolubletaufilamentsinratmodelsoverexpressingtruncated

tau(Filipciketal.,2012).However,theobservedmildneurotoxicitywascomparabletothat

seen inmice expressing full-length wild-type forms of tau (Probst et al., 2000, 2N4R tau

transgenicALZ17mice;0N3RtautransgenicALZ31).

Inthecourseofthepresentwork,Ihavefocusedonthedrastic,butreversibleneurotoxicity

inourownnovelmousemodel co-expressing truncatedand full-length tau (Ozceliket al.,

2016). In linewith this,we set out to complete the neuropathological characterization of

several transgenic mouse models that recapitulate different aspects of AD to study the

neurodegenerativerelevanceoftaufragmentationandoligomerizationinvivo.

Truncatedtauinducessevereneurotoxicityinpresenceoffull-lengthformsoftau

InAD,PHFsarecomposedofnotonlyfull-lengthtauspeciesbutalsotruncatedformsoftau

(Gamblinetal.,2003;Goedertetal.,1992;Menaetal.,1996;Rissmanetal.,2004;Wischiket

al.,1988b).Wehaveapproachedthisneuropathologicalconditionwithco-expressionofΔtau

anddifferentfull-lengthformsoftauinvivo.

Discussion

87

First,we generateddouble transgenic P301SxTAU62onmice co-expressingΔtau andP301S

mutant 0N4R tau. Aside, full-length tau expressing human mutant P301S heterozygous

transgenicmicedevelopinsolubletaufilamentsandparalysisaround12monthsofage(Allen

etal., 2002). Strikingly,ourP301SxTAU62on exhibitedextensivenerve cell dysfunctionand

severemotorpalsyalreadybytheageof3weeks;consistently,inspinalcordneurons,axonal

transportdisruptionwasassociatedwithaccumulationofneurofilamentandaxonalspheroids

formation;furthermore,peripheralnervesshowedsignsofWalleriandegenerationwithfiber

lossandmyelindebrisfollowedbydrasticneurogenicmuscleatrophy.Itisinterestingtonote

thatdespitemassivefunctionalandstructuraldefects,onlyoligomerictauandnoformation

ofinsolubletauaggregatesortanglescouldhavebeendetected;infact,thepresenceoftoxic

tauoligomersaloneisastronghintthattheneurotoxiccascadeleadingtoneurodegeneration

mightnotdependonfilamentoustauinclusions.

Remarkably,thepronounceddrasticneurotoxicityinP301SxTAU62onmicewasphenotypically

andpathohistologicallyreversible:despitecontinuousexpressionoffull-lengthP301Stau,we

could confirm the reversibility of Δtau induced neuronal dysfunction in severely affected

P301SxTAU62on mice. When only Δtau expression was halted, former paralyzed

P301SxTAU62on-off mice rapidly regained full motor control and re-established nerve fiber

function and restored muscle fiber atrophy was observed. Moreover, the functional and

structuralrecoverywasparalleledbythedisappearanceofoligomerictauspecies.

Second,giventheartificialnatureofP301Smutanttau,weaimedforan invivoanalysisof

Δtau inpresenceof full-lengthwild-typetau. Indeed,wecould reproducethepronounced

reversible neurotoxicity in double transgenic ALZ17xTAU62onmice co-expressing Δtau and

full-lengthwild-type2N4Rtau.

Third,weanalyzedTAU62xALZ31onmiceexpressing3RΔtauandtheshortestformofwild-

type human 0N3R tau; however, these mice exhibited the most pronounced neurotoxic

phenotypethatwasnotreversiblewhenΔtauexpressionwasceased.Apossibleexplanation

isthatfunctionalandstructuralrecoverywouldrequirethepresenceofmixed3R/4Rratioof

tauoligomericspecies.

Of note, co-expression of only full-length tau species was not sufficient to induce severe

neurotoxicity: both, P301SxALZ31 and ALZ17xALZ31 double transgenic mice only exhibit

pretangletaupathologyanddidnotdisplayparalysis.

Discussion

88

Overall,thesefindingshighlighttwoessentialaspectsoftauopathies:taufragmentationand

oligomerization. In accordance with previous findings, we could highlight a neurotoxic

potentialoftaufragmentsaspathogenicmediatorsintauopathies.Indeed,recentworkhas

shownthattruncationoftaunotonlyfacilitatestauassemblyintofilaments(Abrahaetal.,

2000;Mocanuetal.,2008;Sydowetal.,2011),butalsoexacerbatesthetoxicityoffull-length

tau(Fasuloetal.,2005).Inparallel,weidentifiedoligomericformsoftauasakeyneurotoxic

species contributing to the neurodegenerative process in tauopathies; in fact, tau toxicity

precedestheformationof insolubleaggregates inourmousemodelandthus fibrillarytau

formation appears to be not required for tau toxicity, similar to findings in other

proteinopathies (Eisenberg and Jucker, 2012). Indeed, this confirms the recognized

importanceofsolubleoligomerictauspeciesforthepathogenesisoftauopathies(Lasagna-

Reevesetal.,2010;Spires-Jonesetal.,2011;Spires-Jonesetal.,2009).

Targetingtruncatedtauspecies

Theneurotoxicpotential of protein fragments in the courseof variousneurodegenerative

disorders remains to be established in detail. However, neurodegeneration-associated

proteins are substrate to various proteolytic enzymes that cleave designated proteins at

individuallydifferentsites(Figure4.1).Theneurotoxiceffectofproteinfragmentshasbeen

showni.e.fortheintramebranouslocatedAPP(Selkoe,2001a),thetransmembranouslocated

BRI2 (RostagnoandGhiso, 2008;Vidal et al., 1999;Vidal et al., 2000), intracellular and/or

extracellularcleavedα-Syn(Duftyetal.,2007;Kimetal.,2012;Mishizen-Eberzetal.,2005),

intracellularcleavedhtt(Grahametal.,2006;Waldron-Robyetal.,2012;Wellingtonetal.,

2002) and ataxins (Guyenet et al., 2015; Hubener et al., 2013; Mookerjee et al., 2009),

membraneanchoredPrP(Altmeppenetal.,2012;Trevittetal.,2014),andnuclearTDP-43

(Furukawa et al., 2011; Nonaka et al., 2009). Across the different neurodegenerative

disorders,majorenzymesinvolvedintheproteolyticprocessesarecaspasesandcalpains;in

particular, tau protein is cleaved by caspases-3/6 (Fasulo et al., 2005; Guo et al., 2004;

Metcalfeetal.,2012)andcalpains(FerreiraandBigio,2011;Higuchietal.,2012;Parketal.,

2007).

Discussion

89

Inlightofthis,inhibitionofcaspasesandcalpainscleavageemergedasapotentialtargetto

prevent disease pathology andmight be a promising therapeutic strategy considering the

observed neurotoxicity in ourmousemodel. Indeed, calpain-specific inhibitors have been

showntoattenuateAD-likepathologyin3xTgADmice(Medeirosetal.,2012);furtherreduce

ataxin cleavage (Haacke et al., 2007) and attenuate α-Syn induced synaptic impairments

(Diepenbroeketal.,2014).Inaddition,caspaseinhibitionhasalsobeenshowntomitigateα-

Synpathology(Bassiletal.,2016).

Nevertheless,thereisalsoevidencethatcleavageofneurodegeneration-associatedprotein

speciesmayconstitutearegularcellularprocess(Lietal.,2005).Forinstance,N-terminally

truncatedα-SynspeciesarepresentincaseswithorwithoutLewypathologyandcorrelating

withthetotalamountofα-Syn(Muntaneetal.,2012).

Figure4.1Overviewof the cellular locationofneurodegeneration-associatedproteins and their individualcleavagesites.

Discussion

90

Targetingtoxicoligomerictauspecies

The spectrumof neurotoxic potentials of tau species is still under debate.Oligomeric tau

formationoccursearlyintheprogressionofADpathology(Pattersonetal.,2011).InAD,late

insolubletauaggregatesarebelievedtocontributetonervecelldeathandcloselycorrelate

withcognitivedecline(Arriagadaetal.,1992;Gomez-Islaetal.,1997).Incontrast,thetoxicity

of oligomeric tau has been shown to correlate betterwith neuronal dysfunction than the

extentoffilamentoustau(Bergeretal.,2007).

Ourdataprovideevidencethatsolubleoligomerictauspeciesplayakeyroleintheneurotoxic

cascadeof taupathology leading toneuronal loss (Blairetal.,2013;Brundenetal.,2008;

Gerson and Kayed, 2013; Gerson et al., 2014; Usenovic et al., 2015). Indeed, progressive

neuronal degeneration in absence of tau filament formation was reported not only in

tauopathymousemodels (Oddo et al., 2003; Spires et al., 2006; Sydow andMandelkow,

2010), but also in tau transgenic drosophilamodels (Cowanet al., 2010;Wittmann et al.,

2001).Aside,oligomericproteinspecieshavebeenimplicatedinthepathogenesisofvarious

neurodegenerativediseases: inbrainsofADpatients, oligomericAβpeptides appeared to

participateintheneurotoxiccascadeevenbeforetheonsetofsymptoms(Lesneetal.,2013);

moreover, toxic extracellular α-Syn oligomers have been shown to favour prion-like

propagationinPDinvitro(Danzeretal.,2011);further,doperminergiclossinthesubstantia

nigracausedbyα-Synoligomerformationratherthanamyloidfibrilsinvivohighlightedthe

toxicityofsolubleoligomers(Winneretal.,2011);notably,oligomericAβandα-Synspecies

werepostulatedtobecompetenttoprovokeneurotoxictauoligomerization(Lasagna-Reeves

etal.,2010);inaddition,toxicoligomersformsofPrPhavebeensuggestedtomediatethe

infectiousprocessinpriondiseases(Silveiraetal.,2005).

Oligomeric tau species are considerably interesting as a potential immune target. The

observedsevereneurotoxicityassociatedwithabundanttauoligomerformationsuggestsour

mouse model of special relevance for this type of pharmacological treatment. Indeed, a

growingbodyofevidencesupportsthestrategyofdiminishingtaupathologywiththerapeutic

antibodies.Studiesonactiveimmunizationusingphospho-taupeptideshasbeenshownto

Discussion

91

mitigate tau aggregation in P301L transgenicmice (Asuni et al., 2007).Moreover, passive

immunization approaches with anti-tau directed monoclonal antibodies were shown to

reduce taupathology,whenadministratedprior to thediseaseonset (Boutajangoutetal.,

2011;Chaietal.,2011).However,therapidonsetoftautoxicityinourmousemodelfavours

it greatly for immunotherapeutic approaches and investigating clearance mechanisms of

pathologicaltauproteininvivo.

Targetingtheaxonaltransportsystemandoligomerictauinducedneurotoxicity

Herewereportthat fragmentedtauexacerbatesthetoxicityof full-lengthtau invivo.Our

mousemodelexhibitpronounceddrastic,butremarkably,reversibletoxiceffectofoligomeric

tau species associated with extensive neuronal dysfunction and a severe neurological

phenotype. In P301SxTAU62on mice, apparent mitochondrial dislocation and clumping,

disruption of the Golgi network and missorting of synaptic proteins strongly suggests a

widespreaddisruptionofthecellulartransportsystem(Kopeikinaetal.,2011;Liazoghlietal.,

2005). Interestingly, comparable perinuclear mitochondrial clumping associated with the

presence of only soluble tau specieswas reflected in studies on rTg4510 transgenicmice

(Kopeikinaetal.,2011).FragmentationoftheGolgiapparatus(GA)disturbsthecentralsorting

machineryforallnewlysynthesizedproteinsdestinedforfastaxonaltransportandisbelieved

to be an early event in various neurodegenerative disorders (Gonatas et al., 2006;

Hammerschlagetal.,1982;Liazoghlietal.,2005).InAD,giventhattheGAwasfoundtobe

fragmenteduponcdk5dysregulation,thisprocessappearstodirectlyprecedecelldeath(Sun

etal.,2008).Ofnote,overexpressionofα-SynhasbeenshowntocauseGolgifragmentation

andblockER-Golgitraffickinginvitroandinvivo(Cooperetal.,2006;Winslowetal.,2010).

However,somaticaccumulationofsynaptophysininthehippocampusofP301SxTAU62onmice

and degradation of VAMP2, a binding partner of synaptophysin, indicate congested Golgi

pathways and synaptic transmission disturbances (Nakashiba et al., 2008; Pennuto et al.,

2003).

Inaccordancewithourfindings,damagetotheaxonaltransportsystemhavebeendescribed

inmultipleneurodegenerativedisordersincludingAD(Kopeikinaetal.,2011),ALS(DeVoset

Discussion

92

al.,2007),PD(Sahaetal.,2004),andHD(Changetal.,2006).Further,inAD,Aβwasreported

tointerferewithmitochondrialtransportinculturedhippocampalneurons(Ruietal.,2006).

Intauopathies,over-expressionoftheshortesttauisoformhasbeenshowntoimpairaxonal

transportinvivo;inaddition,thiswasparalleledwithinsolubletauinclusions(Ishiharaetal.,

1999).Incontrast,solubletauspeciesweresufficienttoinducedisruptionofthemicrotubule

cytoskeletonindrosophila(Cowanetal.,2010).

Inphysiologicalconditions,tauistightlyassociatedwithmicrotubulesandcriticallyinvolved

in the dynamically assembly and stability of this cytoskeleton component; and thus,

instrumental for viability of the intracellular transport system (Kosik, 1993). However,

modificationsoftauarelikelyresponsibleforthedysregulationofthemicrotubulenetwork.

Therearetwopossibleexplanationsforthedisruptionofpolarizedmicrotubuletracks:one

source of interference might constitute abnormal high levels of tau and its concomitant

excessive binding to microtubules results in the detachment of kinesin; thus, ultimately

preventsanterogradetransportofindividualcargoes(BaasandQiang,2005;Dixitetal.,2008;

Dubeyetal.,2008). Inthiscase,hyperphosphorylationandsubsequentdissociationoftau,

typicallyresponsibleforthedestabilizationofmicrotubulesinthefirstplace,mightbeeven

of benefit for themicrotubule network operability.Of note, co-expression of various full-

lengthsformsoftauinourP301SxALZ31andALZ17xALZ31revealedcomparablelevelsoftau

hyperphosphorylation, without a pronounced motor phenotype; thus, tau

hyperphosphorylationappearnot to correlatedirectlywithnerve cell death inourmouse

modelsandstrengthenthepossibilitythatabnormalphosphorylationmightnotbeaprimary

pathoglogicalevent.

Microtubulenetworkdisruptionisamajorpathogeniceventintheevolutionoftauopathies.

Our findings inP301SxTAU62onmice suggest that, initiatedbyΔtauoverexpression, highly

toxicoligomerictauspeciesinterferewithmicrotubuleassemblyandstabilization,ultimately

contributetoaxonaltransportdisruption;however,theunderlyingmechanismsremaintobe

establishedindetail.Apossibleapproachtodissecttheprocessofaxonaltransportdisruption

wouldbetoassessvariouspotentmicrotubulestabilizingcompoundssuchasepothiloneD

Discussion

93

andpaclitaxelthathavebeenshowntopartlyrescuetauinducedaxonaltransportdysfunction

(Ballatoreetal.,2012;Bartenetal.,2012;Brundenetal.,2012;DasandMiller,2012).

Anotherapproachwouldbetotargettauoligomericformationdirectlythroughaggregation

inhibitors including methylene blue (MB) (Violet et al., 2015; Wischik et al., 1996) and

curcumin(Maetal.,2013).However,whileMBhasbeenshowntoblocktauproteinassembly

throughtherepeatdomainandbeingcompetenttoreverseearlytauaggregation,however,

itfailedtoremoverobusttanglepathologyintherTG4510mousemodeloftauopathy(Spires-

Jonesetal.,2014).Inaddition,areductionofAβlevelsandimprovedcognitivedeficitsupon

MBtreatmentwasreported(Medinaetal.,2011).

Protectiveeffectsofearlytauburdenonlateneurotoxicdistresslevel

ThisworkoriginatesfromourmostrecentpublishedobservationthatoverexpressionofΔtau

inpresenceoffull-lengthtaucausesdrastic,butreversibleneurotoxicityinvivo(Ozceliketal.,

2016).Basedonour results,we setout to investigate the long-termconsequencesof the

extensiveneurotoxicstressduringearlypostnatallifeinformerlyparalyzedP301SxTAU62on-

offmice.Remarkably,wefoundthatthesemiceexhibitalesspronouncedmotorphenotype

at14 to16monthsofagecomparedto theirheterozygousP301S littermates thatdidnot

experiencecompellingneurotoxicityearlyinlife.Thisisparalleledbysignificantreducedtau

pathology in aged P301SxTAU62on-off mice: the histopathological phenotype of formerly

paralyzedmiceshowedmitigatedtauhyperphosphorylationandalmostnosignsofinsoluble

tau inclusions. In linewith these findings, significant less total, insoluble, and soluble tau

proteinlevelscouldbedetected.

OurobservationsadvocatealatepreventiveeffectofearlyΔtauinducedneurotoxicityand

raisesthepossibilityofdiversesources,aloneor incombination,beingresponsibleforthe

underlying delayed tau pathology in P301SxTAU62on-off mice. As aforementioned, various

active immunizationattemptsusingrecombinanttaupeptideshavebeenshowntoreduce

tau pathology in various tau transgenicmousemodels (Asuni et al., 2007; Bi et al., 2011;

Boutajangoutetal.,2010).Apossiblescenarioisthattheexcessivetoxictauoligomerization

inyoungP301SxTAU62on-offmicewouldtriggeranautoimmunereactionintheformofarising

anti-tauantibodies.Alternatively,otherclearancemachineriesmaybeinvolvedinremoving

Discussion

94

oligomericorhyperphosphorylatedtaufromthecell.Forinstance,cytosolicproteasessuch

as puromycin sensitive aminopeptidase (PSA) were observed to attenuate tau levels and

motordeficitsinvariousmodelsoftauopathyincludingDrosophila(Karstenetal.,2006)and

mice(Kudoetal.,2011).Consideringthis,PSAmightregulatetaudegradationintwoways:

directlyviaproteolyticdegradationoftauasreportedinotherinvitrostudies(Senguptaetal.,

2006),orindirectlybyinducingautophagy(Menziesetal.,2010);specifically,theinhibitionof

PSAhasledtoincreasedlevelsoftoxicpolyQ-containinghttandataxin-3fragmentsaswellas

mutantα-Synassociatedwithadecreaseinautophagosomeformation.Indeed,theprotective

effects of early tau stress in P301SxTAU62on-offmice could point towards the induction of

autophagicpathways ingeneral.The removalofaggregation-proneproteins throughbasal

autophagyhasbeenshowntobeofimportanceforcellsurvival;indeed,theconsensusinthe

field is that impairmentof theautophagyprocessandsubsequent lossofacytoprotective

functionofautophagyleadstoneurodegeneration(Haraetal.,2006;Komatsuetal.,2006).

ProteinclearancesystemsandautophagydysfunctioninADhavebeensubjecttonumerous

studies(Nixon,2013;Nixonetal.,2005;Pirasetal.,2016);specifically,thereductionofBeclin1

expressionhasbeenshowntoincreaseAβpathologyanddisruptintheautophagyfluxinvivo

(Pickfordetal.,2008).InPD,α-Synoverexpressioncausesmislocalizationoftheautophagy

protein Atg9, which controls the formation of phagophore in the process of autophagy,

throughRab1ainhibition;however,Rab1aoverexpressionhasbeenshowntoreverseα-Syn

inducedautophagyimpairment(Winslowetal.,2010).Inaddition,stimulationofinsulinand

insulin-likegrowth factor1 signaling cascades results in increasedautophagic clearanceof

toxic huntingtin aggregates (Yamamoto et al., 2006). Furthermore, mTORC1-independent

activationofautophagybytrehaloseand inhibitionof themTORC1pathwaybyrapamycin

havenbeenshowntopromotetheclearanceofmutanthuntingtinandα-Syninvitro(Sarkar

etal.,2007).Inlinewiththesefindings,ourgroupcouldpreviouslyshowthattreatmentwith

theseautophagy-inducingagentsresultsindelayedprogressionoftaupathologyandsignsof

a restored autophagic flux associated with reduced accumulation of autophagy related

proteinsp62andLC3inP301Smutantmice(Ozceliketal.,2013;Schaefferetal.,2012).Of

note, microtubule-associated protein 1 light chain 3 protein (LC3) is required in the

autophagosomeformationprocess(MizushimaandKomatsu,2011);andp62isanubiquitin-

LC3-binding protein that links various substrates including protein aggregates to the

Discussion

95

autophagosome-lysosome degradation machinery (Kim et al., 2008). However, we lack a

completeunderstandingoftheautophagymachineryinourmousemodelsatthistimepoint;

thus,itwouldbeinterestingtoinvestigateapotentialautophagic,rectifyingresponsetoΔtau

inducedsevereneurotoxicityassociatedwiththeformationoftoxictauoligomers.Forthis,

experimentswithautophagyinducingagentsincludingrapamycinwouldyieldnewinsightinto

the degradation process of toxic tau species and the potential late preventive,

neuroprotectiveeffectofearlytaustress inagedP301SxTAU62on-offmice. Inparticular,the

assessmentofautophagicmarkersincludingLC3,p62,andBeclin1wouldallowtoanalyzeand

defineaclearerpictureoftheautophagyfluxphenomenon.

MouseCSFderivedAβspeciesdonotinduceprion-likeamyloidogenicpropagation

inAPP23transgenichostmice

ThisworkextendsrecentpublishedfindingsreportedonbrainandCSFderivedAβseeding

(Fritschi et al., 2014). Here we set out to inoculate forebrain homogenate or CSF in the

hippocampusofAPP23transgenicmice,andanalysetheseedingeffectofAβintheCSFand

brain.Consistentwiththispreviousreport,wefoundthatAβspeciesinbrainhomogenates

harbours seed-like potential, whereas CSF derived Aβ species lack prion-like propagation

activity(Skachokovaetal.,2015).Indeed,APP23transgenicmiceseededwithbrainderived

Aβfor20monthsexhibitedandincreasedplaque-loadinthehippocampus;incontrast,inthe

sameexperimentalset-up,CSFderivedAβdidnotresultinachangeinplaque-load.Apossible

explanationmightconstitutetheabsenceorstructuraldifferenceofseed-potentAβspecies

in the CSF. Whereas small and soluble Aβ species derived from APP23 mice have been

demonstratedtobehighlyseed-potent(Langeretal.,2011),theseAβspeciesappearnotto

bepresentintheCSF;possiblyduetodownregulatedtransporttotheCSFcompartment,or

upregulated degradation within the CSF compartment. Further, N-terminal truncated Aβ

specieshavebeenfoundlargelyinthebrain,butlessintheCSFofADpatients(Langeretal.,

2011).However,ifitsstructuraldiversityaffectstheseedingpotentialofAβremainselusive.

Inaddition,analysisoftheselectiveAβtransportandpotentialco-factorsinvolvedmightbe

neededtoexplainthelackofseed-potentAβspeciesinADCSF.

Discussion

96

Conclusionandperspectives

Ourdatashowthattruncatedtau,whenco-expressedwithfull-lengthformsoftau,resultsin

drastic,butreversibleearlytaustress.Wefurtherhave identifiedsolubletauoligomersas

toxickeyspecies,ratherthaninsolubletauaggregates,intheneuropathologicalcascadeof

tauopathies. This information is in line with the neurotoxic potential of protein cleavage

products and oligomeric species observed in diverse neurodegenerative disorders and

strengthenstheideathattruncatedtauindeedcontributestothepathogenesisoftauopathies

(HangerandWray,2010;RossandPoirier,2004).

In fact, ourmousemodel offers a remarkable setting to asses therapeutic strategies and

elucidatefurtherthepathomechanismsleadingtotauopathy.Forinstance,modulationoftau

fragmentationandassociated formationofoligomeric tauspeciesmaypotentiallyprevent

neurodegenerativeprocesses;ultimately,beingofcrucialrelevanceforthedevelopmentof

noveltherapeuticapproaches.

WefurthershowthatCSFderivedAβspeciesisnotcompetentenoughtoinduceprion-like

propagation. Instead, an interesting issue emerged from our work is that abundant toxic

oligomerictauspeciespresentinourmousemodelmightactuallyexhibitprion-likepotential.

Thishypothesisraisesthepossibilitytobetterunderstandprion-likeprocessesintauopathies

andneedstobecorroboratedbyfurtherexperiments.

In conclusion, ourwork underlines the role of fragmented tau and soluble oligomeric tau

aggregationintheneurodegenerativeprocessoftauopathies.

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5 MaterialsandMethods

5.1 Animals

Alltransgenicmicewereheterozygousforthetransgenesofinterest,unlessspecifically

mentionedotherwise.ThenumberofmiceusedwasminimizedaccordingtotheSwiss

regulation on Animal Experimentation. All animal experiments were conducted in

accordancewiththeSwissguidelinesforanimalcareandapprovedbythe local legal

authorities.

5.1.1 Housingoftransgenicmice

Femalemiceweregroup-housedwithamaximumoffiveindividualspercage.Malemice

werehousedindividuallyastoavoidaggressive interactions.Freeaccesstofoodand

waterwasprovidedandanimalswerekeptona12hour/12hourinvertedlightcycle.

5.1.2 TAU62mice

Inducible and neuron-specific 3R tau151–421 (Δtau) expressing TAU62 mice were

generated by co-injection of two Thy 1.2 minigene-based constructs into C57BL/6J

oocytes.TheThy1.2tTSconstructwasobtainedbyreplacingexon2ofthemurineThy

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98

1.2promoterbyatetracyclinecontrolledtranscriptionalsilencerelement(tTS).TheThy

1.2-TRE-Δtau construct contained a tetracycline responsive element (TRE) �800bp

upstreamofthehumanwild-typeΔtaucDNAencodingaminoacids151to421ofa3-

repeatdomainspanninghumanwild-typetaufragment(0N3Rtau151-421).Atotalofsix

positive transgenic founder TAU62 mice (C57BL/6J-TgN(tTS-Thy1-Δtau151-421) were

identifiedandtheinducibleexpressionofhumanΔtauwasassessedbywesternblotand

immunohistochemistry. The Lines 62-2 and 62-48 expressed comparable and robust

Δtau(‘on’)andstoppedexpressionfollowingtheremovalofdoxycycline(‘on-off’).Most

experimentswereperformedusing theTAU62/48 line,abbreviatedTAU62.TAU62/2

micewereusedtoruleoutaninsertionsiteeffect.

5.1.3 P301Smice

The production of P301S mutant 0N4R tau transgenic mice (C57BL/6J-TgN (Thy1

hTauP301S))hasbeenpreviouslydescribed(Allenetal.,2002).Miceweregeneratedby

subcloningP301SmutatedcDNAencodingtheshortesthumanfour-repeattauisoform

(383aminoacids isoformsofhuman tau) into amurineThy1.2 genomicexpression

vectorusingXhoIrestrictionsite.Transgenicmiceweregeneratedbymicroinjectioninto

pronucleiof(C57BL/6JxCBA/ca)F1generation.PCRanalysiswasperformedtoidentify

the founders, which then were interbred with C57BL/6J mice. Homozygous and

heterozygousP301Smicewereselectedaccordingtospecificexperiment.

5.1.4 ALZ17mice

Theproductionoffull-lengthwild-type2N4RtautransgenicALZ17mice(C57BL/6J-TgN

(Thy1hTau)17)hasbeenpreviouslydescribed(Probstetal.,2000).Miceweregenerated

bysubcloningcDNAencodingthelongesthumantauisoformintotheXhoIrestriction

siteof themurineThy1.2minigene.Aftermicroinjection intopronucleiofB6D2F1x

B6D2F1embryos,thefounderswereanalyzedbyPCRandtheninterbredwithC57BL/6J

mice.

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99

5.1.5 ALZ31mice

ForthegenerationofALZ31wild-typehuman0N3Rtautransgenicmice(C57BL/6J-TgN

(Thy1hTau)31), 0N3R human tau complementary DNA was cloned into the neuron-

specificThy1.2promoterelementandinjectedintoC57BL/6Joocytes.

5.1.6 P301SxTAU62mice

P301SxTAU62doubletransgenicmiceco-expressatau151-421fragment(Δtau)withfull-

lengthmutantP301S(0N4R)tauas longas500mgkg−1doxycyclinewasprovidedad

libitum.

5.1.7 ALZ17xTAU62mice

ALZ17xTAU62doubletransgenicmiceco-expressatau151-421fragment(Δtau)withfull-

lengthwildtype(2N4R)tauaslongas500mgkg−1doxycyclinewasprovidedadlibitum.

5.1.8 ALZ31xTAU62mice

ALZ31xTAU62doubletransgenicmiceco-expressatau151-421fragment(Δtau)withfull-

lengthwildtype(0N3R)tauaslongas500mgkg−1doxycyclinewasprovidedadlibitum.

5.1.9 P301SxALZ31mice

P301SxALZ31doubletransgenicmicehavebeenobtainedbycrossingfull-lengthmutant

P301S(0N4R)tauwithfull-lengthwildtype(0N3R)tauexpressingmice.

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100

5.1.10 ALZ17xALZ31mice

ALZ17xALZ31 double transgenic mice have been obtained by crossing full-length

wildtype(2N4R)tauwithfull-lengthwildtype(0N3R)tauexpressingmice.

5.1.11 APP23mice

APP23miceoverexpressthehumanfull-lengthAPPgene(751-aaisoform)harbouring

theSwedishdoublemutationatpositions670/671(KM->NL),undercontroloftheThy

1.2promoter.TheAPP751cDNAwasinsertedintotheblunt-endedXhoIrestrictionsite

of themurine Thy 1.2minigene that wasmicroinjected into C57BL/6J oocytes. The

founderswereanalyzedbyPCRandtheninterbredwithC57BL/6Jmice.

5.2 DNAisolationandgenotyping

GenomicDNAisolatedfromtoebiopsieswasusedtoidentifyandanalyzethetransgene

inheritance of the respective mouse line. Biopsies were incubated in lysis buffer

containing0.1mg/mlProteinaseK (Macherey-Nagel,Germany) incubatedovernight

(O/Nat55°Cand600rpminEppendorfThermomixercomfortshaker.Thesolutionwas

centrifuged(13000rpm,5min)and750μlofthesupernatantwasmixedwith750μl

isopropanolbyinvertingthetubes.Thesolutionwascentrifuged(13000rpm,10min),

thesupernatantwasdiscardedandthepelletwaswashedwith200μlof75%ethanol

(EtOH).After thesolutionwascentrifugedagain (13000rpm,10min), theremaining

DNApelletwasdriedonEppendorfThermomixercomfortshaker(55°C,5-10min)and

dissolved in 250μl dH2O (50°C, 1h).DNA sampleswere kept at 4°C until analysis of

transgeneinheritancebyPCR.

Reagent Concentration SupplierTris 100mM(pH8.0) Biomol#08003EDTA 5mM FlukaBioChemika#03690NaCl 200mM Merck#1.06404.10000SDS 0.20% BioRad#1610301

Table5.1:Lysisbuffercomposition

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101

PCR conditionswere determined independently for each transgenes of interest and

eachindividualPCRprogramwasrunonaPTC100machine(MJResearch,Canada).PCR

productswererunon1.5%agarosegelsat150Vfor90min.

Reagent VolumeForwardprimer(10pmol/μl) 2μlReverseprimer(10pmol/μl) 2μlTopTaqMastermix(Qiagen,Germany) 12.5μlSterileH2O 6.5μlDNA 2μl

Table5.2:PCRmixture

Temperature Time Cycles95°C 2min 195°C 1min

3060°C 1min72°C 2min72°C 10min 14°C ∞

Table5.3:TAUF151PCRprogram

Temperature Time Cycles95°C 4min 195°C 1min

3060°C 1min72°C 3min72°C 10min 14°C ∞

Table5.4:P301SPCRprogram

Temperature Time Cycles95°C 10min 195°C 20s

3054°C 15s72°C 1min72°C 10min 14°C ∞

Table5.5:P301SxALZ31PCRprogram

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102

Temperature Time Cycles94°C 2min 194°C 45s 160°C 1min

3572°C 45s94°C 45s72°C 5min 14°C ∞

Table5.6:APP23PCRprogram

Primersusedforgenotypingweredesignedindividuallyfortransgeneofinterest.PCRprogram Forwardprimer ReverseprimerTAUF151 GTGGATCTCAAGCCCTCAAG GGCGACTTGGGTGGAGTAP301S GGTTTTTGCTGGAATCCTGG GGAGTTCGAAGTGATGGAAGALZ31xP301S CCTCTCCCGTCCTCGCCTCTG

TCGAAGACAGACCACGGGGCGGAGATC

APP23 CCGATGGGTAGTGAAGCAATGGTT

GAATTCCGACATGACTCAGG

Table5.7:Designedprimersequences(5’-3’)

5.3 Histologyandimmunohistochemistry

Micewereanesthetizedwithamixtureof100mgkg−1ketamine(Ketalar®,Pfizer)and

10mg kg−1 xylazine (Rompun® 2%, Bayer) intraperitoneally and after deep sleeping,

micewereinjectedby100mgkg−1sodiumpentobarbital(Pentothal®0.5g,Ospedalia

AG)andtranscardiallyperfusedwith0.01Mcoldphosphate-bufferedsaline(PBS).

5.3.1 Tissue preparation and processing: Brain, Spinal cord and Sciatic

nerve

After perfusion, the spinal cord, sciatic nerve and the brain were quickly removed,

immersionfixedin4%paraformaldehydeO/N,andembeddedinparaffin.Brainswere

cuteithersagittally(4–20μmtransverseserialsections)orincoronalplaneanteriorto

the hippocampus (4–20 μm transverse serial sections starting at -2 Bregma to -3

Bregma,asdefinedbytheMouseBrainAtlasbyG.PaxinosandK.Franklin)byasliding

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103

microtome(LeicaSM2000R,Leica,Germany).Paraffinsectionsweretransferredona

floatingbath(60°C),mountedonhistologicalslides(LeicaIPS,Leica,Germany)anddried

O/Nat37°Cbeforefurtherusage.ParaffinsectionsweredeparaffinisedinXylol(20min;

fromBiosystems,Switzerland),rehydratedin100%EtOH(3min),96%EtOH(2x3min)

and70%EtOH(2x3min),followedbywashinginPBS.Inordertomaskantigenicsites,

antigenretrievalwasperformedinCitricacidbufferpH6.0(ProTaps®)for30minat90

°C.Afterblockingin2.5%normalhorseserum(VectorLaboratories)for30minatroom

temperature(RT),sectionswereincubatedwithprimaryantibodydilutedinPBSO/Nat

4°C.Thenextday,thesectionswerewashed inTris/PBSsolution(3x5min)andthen

incubated with ImmPRESTM Reagent peroxidase for anti-mouse antibody (Vector

Laboratories, USA) for 1h at RT. After the incubation, sections were washed with

Tris/PBS (3x5 min) and developed by using chromogen ImmPACTTM NovaREDTM

Peroxidasesubstratekit(VectorLaboratories,USA).Thedevelopingtimewascontrolled

underamicroscopeandsectionswashedwithH2Otostopthereaction.Additionally,

slideswerethencounterstainedwithhematoxyline(J.T.Baker).Finally,sectionswere

rehydratedin70%EtOH(1min),96%and100%EtOH(each2x1min)andkeptinxylol,

beforeusingPertex®mountingmedium(Biosystems,Switzerland).Picturesofsection

were takenusing anOlympusDP73 (Olympus,USA)microscope. The content of the

originalimageswasnotneglectedbychangesofprocessedimages.

5.3.2 HematoxylinandEosinStaining

Afterdeparaffinisingandrehydration,sectionswererinsedincoldtapwaterandstained

inhematoxylinfor5-8min.Afterwashingincoldtapwateranddecolorizingshortlyin

alcohol-HCl,theslideswerewashedagainincoldtapwaterandkeptinwarmwateruntil

bluecolourappears.Then,sectionswereimmersedin1%erythrosineBsolution(RAL

diagnosis)for2-3min,washedagainshortlyincoldtapwater,followedbydehydration

inEtOH(70%,96%and100%)andmountingaspreviouslydescribed.

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104

5.3.3 Gallyassilverstaining

Sectionsweresilver-impregnatedbyapreviouslydescribedmethod(Braaketal.,1988;

Gallyas,1971).Afterdeparaffinizationandrehydrationthesectionswereincubatedin

3%PeriodicAcid(Sigma-Aldrich,USA)for30minatRT.SlidesthenwerewashedindH2O

(2 min) and incubated in 1% Alkaline Silver solution (1M sodium hydroxide; 0.6M

potassiumiodide;1%silvernitratesolution;allfromMerck,Germany),for10minatRT.

After incubation in ABC solution and simultaneousmonitoring of the reaction time,

sectionswere treatedwith0.5%AceticAcid (Merck,Germany) toblock the reaction

during30mininRT.TreatedslideswerewashedindH2Oandincubatedin5%Sodium

thiosulfate(Merck,Germany)for5minatRT.Then,slideswerewashedincoldtapwater

andnucleistained(hematoxylineosinstaining),followedbydehydrationinEtOH(70%,

96%and100%)andmountingaspreviouslydescribed.

Solution Composition Concentration Supplier

A SodiumCarbonateAnhydride 0.5M Merck,Germany

BAmmoniumnitrate 24mM Merck,GermanySilverNitrate 0.01M Merck,GermanyTungstosilicicAcidHydrate 3.5mM Sigma-Aldrich,USA

C

Ammoniumnitrate 24mM Merck,GermanySilverNitrate 0.01M Merck,GermanyTungstosilicicAcidHydrate 3.5mM Sigma-Aldrich,USAFormalinSolution 0,26% Merck,Germany

Table5.8:ABCsolution

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105

5.3.4 HolmesSilverNitrate-LuxolFastBluestaining

Reagents Concentration Supplier

Impregnationsolution

Boricacid 1,24% Merck,GermanyDinatriumtetraborat(Borax)

1,90% Merck,Germany

Silvernitrate 1% Merck,GermanyPyridinsolution 10% J.T.Baker

Reductionsolution

Sodiumsulfite 0.8M Merck,GermanyHydrochinon 90mM Merck,GermanyGoldchloride 0,25% Sigma-Aldrich,USAAcidoxalic 2% Merck,GermanySodiumthiosulfate 5% Merck,Germany

LuxolFastBluesolution

Luxolfastblue 24mM Medite

Ethanol 0.01M PharmacyUSBAceticacid 3.5mM Merck,Germany

Table5.9:HolmesSilverNitrate-LuxolFastBluestainingsolutions

Sectionsweredeparaffinisedandrehydratedaspreviouslydescribedandplacedin20%

Silvernitratesolution(Merck,Germany),for90mininthedarkatRT.Slidesthenwere

washed indH2O (3x)and incubated in impregnation solutionat37°CO/N.Nextday,

sections were placed onto filter paper to remove superfluous fluid and transferred

directlyinreductionsolutionfor10min.Thesectionswerethenwashedintapwater(5

min)andplacedindH2O.After,thesectionsweremovedin0.25%Goldchloridesolution

(5min)andthenrinsed indH2O(10min).Washedslideswere incubated in2%Acid

oxalic(10min)andthereactionwerestoppedbyrinsingthesectionsindH2O.Slides

werethenplacedin5%sodiumthiosulfatesolutionfor5minandrinsedintapwater

beforeplacingtheslidesbrieflyin70%(1x)and96%EtOH(2x).Next,thesectionswere

incubatedfor2hoursat60°CinLuxolfastbluesolutionandbrieflywashedin96%EtOH

(2x) to remove excess stain, then placed in running cold tap water for 5 min and

transferred into dH2O. Slides then were placed in 0.1% Lithium carbonate for few

secondsanddistainedin70%EtOHbeforetobewashedindH2O.Finally,theslideswere

immersed in Cresyl violet solution (10min; at RT) andwashed in 96% EtOH (2x) to

removeexcessstainandthenin100%EtOH(2x)beforetoplaceinxylolandapplyPertex

mountingmedium.

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106

5.3.5 MassonTrichromestaining(Sciaticnerve)

Reagents Concentration Supplier

Weigert'sIronHematoxylinsolutionI

SolutionI Hematoxyline 33mM Merck,Germany

Ethanol 96% PharmacyUSB

Ferricchloridesolution

SolutionII Iron(III)chloride

32mM Merck,Germany

Acid-HCl 25% Merck,Germany

AcidfuchsinPonceau

SolutionI Fuchsinacid 17mM Merck,Germany

Aceticacid 1% Merck,Germany

SolutionII Ponceau 23mM Chroma,Germany

Aceticacid 1% Merck,Germany

Anilinebluesolution

Anilineblue 34mM Chroma,Germany

Aceticacid 2,5% Merck,Germany

Table5.10:MassonTrichromestainingsolutions

ThesectionsweredeparaffinisedandincubatedinWeigert’sIronHematoxylinsolution

(1min)andwashed inrunningcoldwaterfollowedbywarmwater(5-10min).Next,

slideswereplacedinAcidfuchsinPonceausolution(5min),followedbywashingintap

water.Thesectionswerethenincubatedin1%AcidPhosphomolibdic(Merck,Germany)

for5minandAnilinebluesolutionwasaddeddirectlyontheslidesfor3min(without

discardingthe1%AcidPhosphomolibdic)undershaking.Then,thesectionswererinsed

three-fivetimes indH2Oandtransferred in1%aceticacid (5sec).Finally, therinsed

slidesweredehydratedinabsoluteEtOH(2x),theninxylol,andcoverslipped.

5.3.6 Musclespreparation

After perfusion, gastrocnemius (GC), soleus (Sol), tibialis anterior (TA) and extensor

digitorum longus (EDL) were removed and snap frozen in liquid nitrogen cooled

isopentane.MuscleswereembeddedonacorkdisconO.C.TTMcompound(Tissue-Tek®

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107

SakuraTM,USA)andstoredat-80°C.Coronalcryosections(10-12μm)werecutwitha

cryostat(HydraxC,HistocomAG,Switzerland)maintainedat-20°C.

5.3.7 Myosin-ATPase(Adenosintriphosphatase)staining(pH4.2)

Reagents Concentration SupplierVeronalacetatebuffer(stocksolution)

Sodiumacetate

0.1M Merck,Germany

Sodiumbarbiturate(Veronal)

0.1M Sigma-Aldrich,USA

ATPpH4.2solution

VeronalacetatebufferpH4.2

0.1M Sigma-Aldrich,USA

HCl 0.1M VeronalCabuffer(stocksolution)

Sodiumbarbiturate(Veronal)

0.1M Sigma-Aldrich,USA

CaCl2

0.03M

Merck,Germany

VeronalCaATPpH9.4solution

Adenosin-5-triphosphatedisodium(ATP)

4.5mM

Fluka#02060

Cobaltchloride

2%

Merck,Germany

HCl 0.1M

NaOH 1M Merck,Germany

Ammoniumsulfidesolution

21%

Sigma-Aldrich,USA

Table5.11:Myosin-ATPasestainingsolutions

FrozensectionsofO.C.TembeddedsampleswereequilibratedO/Nat-20°C,andthen

placedintothecryostatforminimum20minutes.Thesamplesweremountedonthe

cryostatwithO.C.Tand10μmsectionswerecutandcollectedonwarmslidesatRT.

Beforetoproceedthestaining,thesectionsmustbedryat least1hatRT.Theslides

weretransferredinATPpH4.2solution(10min;RT)andplacedshortlyforwashingin

Veronal-CapH9.4buffer.Next,thesectionswereincubatedinVeronal-Ca-ATPpH9.4

solution(45minatRT).Theslideswerethenwashedintapwater(2x)andmovedin

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108

dH2Obeforetransferringin2%CobaltChloridefor5min.Afterwashingintapwater

(3x),thesectionswereplacedinAmmoniumsulfidesolution(14sec)andrinsedbefore

to be rehydrated in ascending EtOH (1x in 70%, 2x in 96% and 2x in 100%) and

transferredinxylolandapplyPertexmountingmedium.

5.3.8 Semithinsections(Sciaticnerve)

Sciaticnervesweredissectedandfixedforatleast2hin2.5%ofGlutaraldehydeatRT,

followed bywashing samples in 10mM PBSO/N. The tissueswere reduced in 1%

Osmium tetroxide (Oxkem Limited 10x1g) for 2h at RT. After a dehydration step in

histologicalgradeEtOH(70%,80%,90%and2x100%)for20mineachand2xinAcetone

for30min,thetissuewasincubated,first,inAcetone-Durcupan(1:1)solutionfor60min

and,second,inAcetone-Durcupane(1:3)O/Nat4°C.Then,thetissuesweremountedin

Durcupanresin,containing150mlofDurcupanA(Fluka),150mlofDurcupanB(Fluka),

3.1mlofDurcupanC(Fluka),4mlofDurcupanD(Fluka),andcookedat60°Cfor2-3

days,beforeprocessedforlightmicroscopy.Semithinsectionswerecut(1.5μm)using

aglasstripthatwasequippedwithaReichert-Jungapparatus.

5.3.9 Para-Phenylendiamine(Sciaticnerve)

Ultrathincryosectionsontheslidesweredriedonawarmplatebeforeincubatedin1%

p-Phenylenediamine(Sigma-Aldrich,USA)solutionfor2-3h.Theslidesthenwererinsed

6-10timesindH2OanddriedatRT.

5.3.10 Electronmicroscopy

Micewereanesthetizedaspreviouslydesrcibed.AftertranscardiallyperfusionwithPBS

for2-4min,animalswereperfusedfurtherwithafixativesolutioncomposedofwith2%

paraformaldehyde,2%glutaraldehydeand10mMPBS(pH7.4)for1h.Brainsandspinal

cordswereremovedandpostfixedfor1h,followedbyrinsingofthetissuesin10mM

PBS.

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Thetissueswerereducedin1%Osmiumtetroxideand1.5%PotassiumFerrocyanide

for40min;after,thetissuesweretransferredin1%Osmiumtetroxidfor40min.The

tissues were dehydrated as previously described and embedded in Epon. During

dehydration, the sectionswere treatedwith 1% uranyl acetate in 70% EtOH for 1h.

Ultrathin sections from selected areaswere cutwith amicrotome (Ultracut E; Leica

MicrosystemsGmbH,Wetzlar,Germany),collectedonsingle-shotgrids,stainedin6%

uranylacetatefor1h.SectionswereexaminedandphotographedwithaMorgagniFEI

80kVelectronmicroscope(FEICompany,Eindhoven,TheNetherlands).

5.4 Sarkosylextraction

Reagent Concentration SupplierTris 25mM(pH7.4) BiomolNaCl 150mM Merck,GermanyEDTA 1mM FlukaBioChemikaEGTA 1mM Sigma-Aldrich,USASodiumpyrophosphate 5mM Sigma-Aldrich,USAPhosSTOP®, Phosphatase inhibitor cocktailtablet

1tablet Roche,Switzerland

Sodiumfluoride 30mM Merck,GermanyComplete Mini, Protease inhibitor cocktailtablets

1tablet Roche,Switzerland

Phenylmethylsulfonylfluoride(PMSF) 1mM Sigma-Aldrich,USALeupeptine 10μg/ml Sigma-Aldrich,USAAprotinine 10μg/ml Sigma-Aldrich,USAPepstatine 10μg/ml Sigma-Aldrich,USA

Table5.12:Extractionbuffer

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110

Reagent Concentration Supplier

Tris 10mM(pH7.4) Biomol

NaCl 800mM Merck,Germany

Sucrose 10% FlukaBioChemika

EGTA 1mM Sigma-Aldrich,USA

Phenylmethylsulfonylfluoride(PMSF) 1mM Sigma-Aldrich,USA

Leupeptine 10μg/ml Sigma-Aldrich,USA

Aprotinine 10μg/ml Sigma-Aldrich,USA

Pepstatine 10μg/ml Sigma-Aldrich,USA

Table5.13:A68buffer

Phosphataseandproteaseinhibitorcocktailtabletswerefreshlyaddedtoeachbuffer.

Sarkosyl extraction was performed as described previously (Delobel et al., 2008).

FollowingPBSperfusion,onehalfofthemousebrainwasdissectedintoforebrainand

brainstemandfrozeninliquidnitrogenorondryice.Braintissuewashomogenizedin

coldExtractionbuffer1:3(w/v)byusingUltraturraxT8(IKA labortechnik)andbriefly

sonicated(BandelinSONOPULS,90

%power,10%cycle,10secpulses).Thesampleswerecentrifugedat4000g(5000rpm)

for15minand10%ofthesupernatant(=totaltau)wascollectedandaliquoted.Then,

samples were further centrifuged at 80 000g (28 000 rpm) for 15 min by using

ultracentrifuge (Beckman Coulter, OptimaTM L-70K Ultracentrifuge) by using SW55Ti

rotor(BeckmanCoulter).Thesupernatant(=solubletau)wascollectedandaliquoted.

TheremainingpelletswerehomogenizedinA68buffer1:3(w/v)andcentrifugedat4

000g(5000rpm)for20minand1%ofsarkosyl(N-laurylsarcosine,Sigma-Aldrich,USA)

added for 1h30 at 37 °C in thermoshaker (Eppendorf Thermomixer comfort shaker)

undershaking(maxrpm).Thesampleswerefurthercentrifugedat80000g(28000rpm)

for30minandthepelletswasresuspendedin150μlg−1of50mMTris-HCl,pH7.4and

aliquoted(=sarkosylinsolubletau).

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111

5.5 WesternBlot

Reagent Concentration SupplierTris 20mM(pH7.5) BiomolNaCl 137mM Merck,GermanyComplete Mini, Protease inhibitor cocktailtablets

1tablet Roche,Switzerland

Table5.14:TBS-Completebuffer

Reagent Reducedsample(μl)

Non-reducedsample(μl)

Sample x xNuPAGE®LDSSampleBuffer(4X) 5 xNuPAGE®ReducingAgent(10X) 2 —DeionizedWater x xTotalVolume 20 20

Table5.15:Reducedandnon-reducedsamplepreparation

FollowingPBSperfusion,onehalfofthemousebrainwasdissectedintoforebrainand

brainstemandfrozeninliquidnitrogenorondryice.Thebraintissuewashomogenized

in1:10volumeofTBS-CompletebufferbyusingUltraturraxT8(IKAlabortechnik)and

briefly sonicated (Bandelin SONOPULS, 90% power, 10% cycle, 10 sec pulses). The

sampleswerespundownat4000g(5000rpm)for30minandthesupernatantwas

collectedandaliquoted.Westernblotswereperformedeitherunderreducingornon-

reducingconditions(seeTable5.15).Followingappropriatepreparation,sampleswere

heatedfor5minat95°Candshortlyspindownandloadedontoa7%NuPAGE®Tris-

acetate gel. Gels were run first at 100V for 30min, then subsequently at 120V for

additional60min.AftertheremovalofgelsfromthecassetteandactivationofPVDF

membrane (AmershamBiosciences) for first, 30 sec inmethanol and then, 5min in

transferbuffer,samplesweretransferredonthePVDFmembraneat30Vfor2hbyusing

theXCellIITMBlotModule.Next,Unspecificbindingepitopeswereblockedwith5%non-

fat milk in PBS-T (0.01M PBS pH 7.4; 0.05 % Tween-20) for 1h at RT, followed by

incubationwithprimaryantibodyO/Nat4°Conashaker.AfterwashingwithPBS-T(3x5

min)atRT,themembranewasincubatedwithhorseradishperoxidase(HRP)-conjugated

anti-mouseor-rabbitsecondaryantibodyfor1hatRT.Then,themembranewaswashed

again inPBS-T (3x5min) atRT anddetectedbyelectrochemiluminescence (ECL) (GE

Healthcare, USA). Western blot was performed with the NuPAGE® System from

Invitrogen.

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112

5.6 Antibodies

Antibody Target Dilution SourceHT7 humantau

aa159-163WB1:4000IHC1:800

Pierce,Rockford,IL#MN1000

BR134 humantau WB1:1000 (Goedertetal.,1989)

Tau-C3 TaucleavedatresidueAsp421

WB1:1000IHC1:1000

SantaCruzBiotechnology,Inc,Dallas,TX#sc-32240

AT8 TaupSer202/Thr205

WB1:1000IHC1:800

Pierce,Rockford,IL#MN1020

AT100 TaupThr212/Ser214

WB1:1000IHC1:500

Pierce,Rockford,IL#MN1060

PHF-1 TaupSer396/404

WB1:2000IHC1:1000

PeterDavies,AlbertEinsteinCollegeofMedecine,Bronx,NY

MC1 Tauaa5-15,312-322

IHC1:100 PeterDavies,AlbertEinsteinCollegeofMedecine,Bronx,NY

2F11 neurofilament(NF)NF-L,NF-H(70kD)

IHC1:800 Dako,Glostrup,DK#M0762

NF200 neurofilament(200kD) IHC1:100 (Probstetal.,2000)

GFAP glialfibrillaryacidicprotein

IHC1:500 ThermoFisherScientificInc.,Kalamazoo,MI#MS-1407-R7

Synaptophysin synaptophysin IHC1:1000 MilliporeCorporation,Billerica,MA#MAB5258

MG160(rabbit) Golgiapparatus IHC1:1000 NicholasGonatas,PathologyandLaboratoryMedicine,UniversityofPennsylvania,PA

VAMP2/Synaptobrevin2(rabbit)

transportvesicles IHC1:1000 Synapticsystem,Goettingen,Germany#104202

GAPDH(6C5) GAPDH WB1:1000 SantaCruzBiotechnology,SantaCruz,CA,#32233

ß-actin actin WB1:5000 Sigma-Aldrich,SaintLouis,MO#A5316

Coxsubunit1a mitochondrialstaining

IHC1:200 Abcamplc,Cambridge,UK#ab14705

RD3,clone8E6/C11 Humantau,recognize3R,residue209-224

WB1:4000;IHC1:3000

MilliporeCorporation,Billerica,MA#05-803

RD4,clone1E1/A6 Humantauandmouse,recognize4R,aa275-291

WB1:4000;IHC1:100

MilliporeCorporation,Billerica,MA#05-804

T49 Specificforrodenttau

WB1:10000

VirginiaLee,CNDR,UniversityofPennsylvaniaSchoolofMedicine,Philadelphia,PA

Table 5.16: Antibodies used for immunohistochemistry (IHC) and Western blotting (WB). Species ismouse,unlessindicatedotherwise)

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113

5.7 Behavioralassessment

5.7.1 Grid-test

Grid reflex andmotor strengthofmicewas assessedby the grid test.Animalswere

placedonaverticalmeshgridandthe latencyto falloff fromthegridwasrecorded

duringamaximumtimeof180sec.Eachmousewastested3timeswithatleasta5min

restintervalinbetweentrails.Anaveragescoreperdaywasmade.

5.7.2 Rotarodtest

ThemotorcoordinationandbalanceofmicewereassessedusingthePanlabHarvard

Rotarod.TheRotarodstartsataspeedof4rpmandacceleratesconsistentlywith1rpm

every3sec.Thetestingphaseconsistedof4consecutivedays.Witheachanimal,an

acclimation period consisting of 3 sessions with a rest interval of at least 5 min in

betweentrailswasperformed.Subsequently,miceweretestedfor3consecutivedays

with3trialseachdayandarestintervalof5minminimumandthemeanlatencytofall

wasdocumented.

5.7.3 Objectrecognitiontest

Shorttermmemorywasassessedbytheobjectrecognitiontest.Animalswereplacedin

asquaredopenfieldbox(48×48×40cm)underdimlightconditions.Micewereallowed

tofreelyexploretheboxduringahabituationphaseover3consecutivedaysfor15min,

untilnosignsofstresswerepresent.Duringatrainingphaseover2days,twoidentical

objectswereintroducedatdiagonalcornersofthefieldfortrainingsessionsof10min

duration.Thetrainingwashaltedwhenthemicehadcloselyexploredtheobjectsfor20

sec,foramaximumtrainingdurationof10min(Legeretal.,2013)).Inthetestphase,

theanimals’short-termmemorywastestedbyreplacingoneofthefamiliarobjectswith

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114

anovelone,andthetimespentexploringeachobjectduringaperiodof6minwasvideo

recorded.VideoscoringVLCvideoplayer,VideoLAN,France)wasdonebyaresearcher

blindtothegenotype,andasexplorationcriterianosesniffing/touchingoftheobjectat

2cmorlessdistance(Legeretal.,2013))wereused.Forbothtrainingandtestphases,

10cmhighobjectscomposedofthesamematerialwereused,andthepositionofthe

novelandfamiliarobjectswererandomizedacrossgroups.

5.8 Statistics

All statistical analysiswasperformedusingone-wayanalysisof variance followedby

Bonferroni’s multiple comparison test and Student’s t-tests with GraphPad Prism

SoftwareVersion5.0a(GraphPadSoftware,LaJolla,CA,USA).P-valuesareestablished

and outlined as follows: *P<0.05, **P<0.01 and ***P<0.001. Themean and s.d. are

indicated.

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Abbreviations

135

7 Abbreviations

α-SynaaAβABriABriPPADAMADAM10ADanADanPPADAgDAICDALSAnxA2APH1ApoE4APPAspATPBRI2C-terminalCAACAGCaMCBDcdk;cdcCHMP2BCjDCKCNSCSFCTF

α-synucleinaminoacidsamyloidbetaamyloid-Briamyloid-Briprecursoradamalysinproteaseadesintegrinandmetalloproteinase10amyloid-Danamyloid-DanprecursorAlzheimer’sdiseaseargyrophilicgraindiseaseAPPintracellulardomainamyotrophiclateralsclerosisannexinA2anteriorpharynxdefective1apolipoproteinE4amyloidprecursorproteinasparticacidadenosinetriphosphateBRICHOSdomaincontaining2Bcarboxyterminalcerebralamyloidangiopathycytosine-adenine-guaninetrinucleotiderepeatscalmodulincorticobasaldegenerationcyclin-dependentkinasechargedmultivescularbodyprotein2BCreutzfeldt-JakobdiseasecaseinkinasecentralnervoussystemcerebrospinalfluidC-terminalfragment

Abbreviations

136

DLBDNADRPLAE391ECLEOADERgGAGGTGPIGSK3GSSFADFBDFDDFTDFTLDFTDP-17TGSK3hHDhttIAPPIHCJNKkDaLBLNLOADMAPKMAPTMARKmRNAMSAMTBµmmmmMMMPMnN-terminalNTFnmnmolNFTPBSPCR

dementiawithLewybodiesdeoxyribonucleicacidDentatorubral-pallidoluysianatrophyC-terminaltruncatedtauatGlutamicAcid391enhancedchemiluminescenceearly-onsetADendoplasmaticreticulumgramgolgiapparatusglobularglialtauopathyglycosylphosphatidylinositolglycogensynthasekinase3Gerstmann-Sträussler-ScheinkerdiseasesfamilialADfamilialBritishdementiafamilialDanishdementiafrontotemporaldementiafrontotemporallobardegenerationFTDandparkinsonismlinkedtochromosome17glycogensynthasekinase3hourHuntingtondiseasehuntingtinhormoneisletamyloidpeptideimmunohistochemistryc-JunN-terminalkinasekilodaltonsLewybodiesLewyneuriteslate-onsetADmicrotubuleassociatedproteinkinasemicrotubuleassociatedproteintaumicrotubule-affinityregulatingkinasemessengerribunucleicacidmultiplesystematrophymicrotubulebindingdomainmicrometer(s)millimeter(s)millimolarmatrixmetalloproteinasemanganeseaminoterminalaminoterminalfragmentnanometer(s)nanomolarneurofibrillarytanglephosphatebufferedsalinepolymerasechainreaction

Abbreviations

137

PCsPDPEN-2PGRNPHFPiDPKPolyQPPPRNPPrPCPrPScPRRPS1/2PSAPSPRRMsAPPSAASCASH3SPPL2TARDBPTDP-43TRDTTRUPStTsVAMP2

proproteinconvertasesParkinson’sdiseasepresenilinenhancer2progranulinpairedhelicalfilamentsPick’sdiseaseproteinkinasePolyglutaminediseasephosphatasePRioNProteincellularprionprotein scrapieprionproteinpronline-richregionpresenilin1and2puromycin-sensitiveaminopeptidaseprogressivesupranuclearpalsyRNA-recognitionmotifssolubleectodomainofAPPserumamyloidAspinocerebellar-ataxiaSCRHomology-3signalpeptidepeptidase-like2transactiveresponseDNAbindingproteintransactiveresponseDNAbindingprotein43kDATrinucleotiderepeatexpansiondisorderGlobularproteintransthyretinubiquitineproteasomesystemtetracyclinecontrolledtranscriptionalsilencerelementvesicleassociatedmembraneprotein