UNIVERSITÉ D’AIX-MARSEILLE FACULTE DE PHARMACIE ECOLE DOCTORALE SCIENCE DE LA VIE ET DE LA SANTÉ

INSTITUTE OF PHYSIOLOGICALLY ACTIVE COMPOUNDS LABORATORY OF GENETIC MODELING OF NEURODEGENERATIVE PROCESSES

Thèseprésentéepourobtenirlegradeuniversitairededocteur

Spécialité:Neurosciences

Andrei ROMAN

Tauproteinaggregationandα-synucleindysfunction:developmentofnewinvitroandinvivomodelstostudy

neurodegenerativediseases

Directeursdethese:VincentPeyrot&NataliaNinkina

Soutenuele06/07/2018devantlejury:

IsabelleArnal UniversitéGrenobleAlpes RapporteurGarnierCyrille UniversitédeMontpellier RapporteurVladimirBuchmanIPAC ExaminateurVincentPeyrot AMU Co-directeurdethèse

Numéronationaldethèse:2018AIXM0001/001ED62

Résumé

Les signatures histopathologiques de principalesmaladies neurodégénératives -maladied'AlzheimeretlamaladiedeParkinson-sontlesenchevêtrementsneurofibrillairesformés par la protéine tau et les corps de Lewy, formés par l'α-synucleine agrégée. Laformationetl'accumulationd'inclusionstauprovoquentdesperturbationsfonctionnellesducytosquelette, et conduit à une dégénérescence neuronale. Les mécanismes précis durepliement et de l'agrégation de ces protéines, pour la protéine tau comme pour l'α-synucleine,nesontpastotalementcomprisàcejour.

Ici,nousnoussommesintéressésàcettequestionenutilisantdesmodèlesinvitroetinvivo.Enétudiant l'agrégation tau invitro,nousavonsmisenévidenceunnouvelauto-assemblageréversibledetau,quidépenddelatempératureetdelaprésenced’ionszinc,etquiestaprioridifférentdel'agrégationdetauenprésenced'inducteursd'agrégationtelsquel'héparine.Ceprocessuspourraitnéanmoins être impliquédans lespremières étapesdel'agrégation pathologique de tau. Dans une deuxième partie nous avons développé desmodèlesmurinpourétudier lesdysfonctionnementde l’α-synucleine.Nousavonsmontréque l’α-synucleine est directement impliquée dans le développement embryonnaire derégionsspécifiquesdusystèmenerveux,etqu'elleadespropriétésmodulatricesseulementsurlesneuronesdopaminergiquesdelasubstantianigra,quisonttouchésdanslamaladiedeParkinson.

Lesrésultatsobtenusdansnosétudesdedeuxprotéinesquisubissentuneagrégationpathogèneetformentdesinclusionsintracellulairesontcontribuéàlacompréhensiondesprocessusmoléculairesetcellulairesassociésàladégénérescenceneuronale,cequifournirade nouvelles pistes pour développer de nouvelles stratégies de thérapies de maladiesneurodégénératives.Abstract

Thehistopathologicalhallmarksofthemostcommonneurodegenerativediseases–Alzheimer’s disease and Parkinson’s disease are neurofibrillary tangles formed by tauproteinandLewybodies inclusionsformedbyaggregatedα-synuclein.Theformationandaccumulation of these proteins into inclusions cause functional disruptions of thecytoskeleton and leads to neuronal degeneration. The precise mechanisms of tau andsynucleinmisfoldingandaggregationleadingtothosecellulare incluses,eventhoughverystudied, are not fully understood neither for tau protein nor for α-synuclein.

Here we have addressed this question using both in vitro and in vivo models.Investigatingtauaggregationinvitro,wehavefoundareversibleself-assemblyoftau,whichdepends on temperature and is induced by zinc ions, which is different from the tauaggregationinthepresenceofaggregation-inducerssuchasheparin.Thisprocesscouldbeimplicated in the first steps of tau pathological aggregation. In a second part,we havedevelopedamousemodelforstudyingtheα-synucleindysfunction.Wehaveshownthatα-synuclein isdirectly involved in theembryonicdevelopmentof thespecificregionsof thenervoussystem,andthatithasmodulatingeffectonlyonthepopulationsofdopaminergicneurons of substantia nigra, which are affected in Parkinson’s disease.

Resultsobtainedinourstudiesoftwoproteinsthatundergopathogenicaggregationand form intracellular inclusions contributed to understanding ofmolecular and cellularprocessesassociatedwithneuronaldegeneration,whichisimportantforthedevelopmentofnewdisease-modifyingtherapiesofneurodegenerativedisorders.

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2. Results

2.1. In vitro model of tau aggregation

Zincisoneofthemostabundanttransitionmetalinthebodythatservestoregulatemany vital functions such as cell signaling, brain development and theresponsesofimmunecells(BarrandBurdette,2017;Frakeretal.,2000;Grabruckeret al., 2011). Despite vast importance, zinc, especially excess amounts of zinc, isneurotoxic, thus induceneuronal injury and cause celldeath (Wani et al.,2017).Growingevidencereportedthatzincmaycontributetoneurodegenerativediseasesby initiating proteins aggregation. In vivo study showed that dyshomeostasis ofintracellular zinc leads to lysosomal dysfunction and thus promotes α-synucleinaccumulation(TsunemiandKrainc,2014).Inaddition,zincwasshowntoinduceα-synucleinaggregationandspecificallyitsfibrillation(Breydoetal.,2012).Ourrecentdatashowed thatzincalsobinds toTDP-43andcause itsaggregation,pointing topotentialroleofzincinthedevelopmentofALS(seeannex).

Tau, the main component of neurofibrillary tangles in AD also appearedaffectedbyzinc.Tauhyperphosphorylation,thatbelievetobeaninitiatingeventinthegenesisofpairedhelicalfilaments,canbetriggeredbyzincdependentactivationoftheglycogensynthasekinase-3betaandinactivationofphosphataselikeproteinphosphatase2A(Boometal.,2009;Kwonetal.,2015).However,a growingbodyofevidence demonstrated that zinc directly binds to tau and thus induces itsaggregation(Huetal.,2017).Ithasbeenreportedthatzincdramaticallyacceleratethefibrillizationoftaufragment(244-372)andpathologicalmutantdK280ofhumantau,associatedwithFTDP-17,viabridgingCys-291andCys-322(Moetal.,2009).Invitro studies confirmed the ability of zinc to bind to another human taumutanthTauR406W,alsoassociatedwithsomecasesFTDP-17(Huangetal.,2014).Arecentstudyshowedthatzinc interactwithcysteineinR3repeatoftauandsignificantlyaccelerateaggregationofthreerepeattauconstruct(R1-R3-R4)(Jijietal.,2017).Alltheseresultsintroducedzincasanimportantfactorintauopathyandasatargetfortherapydevelopment.

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However,muchuncertainty stillexistsabout theprocessof zincpromotedaggregationoftauleadingtodevelopmentofneurodegeneration.Itisworthytonotethatallpresentstudiesabouttauaggregationperformedusingmutantformsoftauproteinand/orinpresenceofpolyanions,thatartificiallyaccelerateaggregationoftau.Herewehaveinvestigatedaggregationoffull-lengthhumanhTau40isoforminthe presence of zinc ions in a wide range of temperatures using turbidimetry,isothermaltitrationcalorimetry,dynamiclightscatteringandtransmissionelectronmicroscopy.WefoundthatcontrarytozincinducedaggregationofTDP-43andzincaccelerated aggregation of tau in the presence of heparin, zinc-initiatedoligomerizationof tau inabsenceofartificial aggregation inducers isa reversibleprocess,whichdependsontemperatureandconcentrationofzincions.

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2.2. In vivo modelling of α-synuclein

dysfunction

AprogressivelossofDAneuronsthatoccursmostlyintheSNisthehallmarkofPD.It isbelievedthatα-synucleinisinvolvedinthisprocessbecauseitsdysfunctioncausedbymutations,abnormalpost-translationalmodificationsandaggregationisassociatedwithDAneuronspathology. InadditiontodysfunctionanddeathofDAneuronscausedbytoxicgainofα-synucleinfunction,itisfeasiblethatthelossofthisproteinfunctionalsocontributestothediseasepathogenesis.

Toprovethisidea,severalmouselineswithinactivationofthegeneencodingα-synucleinhavebeenproduced.However,studiesoftheselinesprovidedconvincingevidencethattheconstitutiveremovalofα-synucleinhasaverylimitedeffectonthenervous system of geneticallymodifiedmice. Since the synuclein family includesthreeproteins,whichhavehighdegreeofsimilaritytoeachotherandpotentially,partiallyoverlappingfunctions,thepossibilityofreplacingthefunctionofgeneticallyinactivated α-synuclein by two other functionally activemembers of the familycannot be excluded. Such functional redundancy is possible during embryonicdevelopmentatthestageofontogeneticselection,whentheplasticityofthenervoussystemisexceptionallyhigh.

Previousstudiesrevealedreducedcomplementofdopaminergicneurons intheSN ofearlypostnatalandadultα-synucleinknockoutmiceoriginallyproducedbyAbeliovichetal.(2000)comparedtotheirwildtypelittermates(~15%reduction),whereasdopaminergicneuronsoftheventraltegmentalarea(VTA)werenotaffectedin thesemice (Robertson et al., 2004). These resultswere later confirmed andextended to C57Bl6s linewith a large deletion encompassing Snca gene (Garcia-Reitboecketal.,2013).Ithasalsobeenshownthatthisreductionisalreadyobviousatembryonicday13(Garcia-Reitboecketal.,2013).

To study this phenomenon inmore details,we compared the number ofdopaminergicneuronsinSNandVTAofα-synucleinknockoutandwildtypemiceatearlierstagesofembryonicdevelopment,startingfromtheembryonicday11(E11),the earliest time point that allow confidently discriminate these two neuronal

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populations.SerialhistologicalsectionsthroughthemesencephalonregionofE11,E12andE13embryoswerepreparedand immunostainedwithantibodiesagainsttyrosinehydroxylase(TH),arate-limitingenzymeintheprocessofDAsynthesisandamarker commonly used for identification of dopaminergic neurons. Stereotaxiccounting of TH-positive neurons confirmed the previous observation about thedecreasednumberoftheseneuronsintheSNofknockoutmice.However,atE12nodifferenceinthenumberofTH-positiveneuronswasobservedintheSNandatE11the observed differencewas opposite,more neuronswere found in the SNof α-synucleinknockoutmice thanofwild typemice.At thesame time,nostatisticallysignificantdifferenceinthenumberofTH-positiveneuronswasobservedintheVTAofthesetwogroupsofanimalsatallthreestudieddevelopmentaltimepoints.

However, if α-synuclein deficiency in presynaptic terminal was indeeddevelopinginneuronsofPDpatientsashypothesised,significantlossofitsfunctionshalloccuronlyinadultorevenageingnervoussystem,whosecompensatoryabilityismuchlowercomparingtothedevelopingnervoussystem.Thus,inarelevantmodelof α-synuclein deficiency in PD, expression of α-synuclein should remain normalduring development to prevent activation any compensatorymechanisms at thatstageandbeswitchedoffinadultanimals.Thiscouldbeachieved inasophisticatedmouse model that combines conditional knockout of Snca gene and chemicallytriggerableexpressionofanactivatorofthegenedeletion.Productionofsuchsystemisdescribedin(Ninkinaetal.,2015)andtheenclosedpaper(Romanetal.,2017).

InthegenomeofacoremouselinethefirstcodingexonoftheSncagenehasbeen flanked by LoxP-sites (“floxed”), which allowed Cre-recombinase-drivendeletion of the flanked region leading to complete inactivation of α-synucleinexpression.AnimalsofthislinecanbebredwithanimalsofanotherlinethatcarriesatransgeniccassetteinwhichexpressionofCre-recombinaseisdrivenbyatissue-orcell-specific promoter, for example neuron-specific enolase (NSE) promoter torestricttheCre-expressiontoneuronsorTHpromotertorestricttheCre-expressionto catecholamine neurons only. Moreover, transgenic lines expressing Cre-recombinasemodifiedbyconjugationwithestrogenreceptor(ER)couldbeused.Themodified Cre is sequestered in the cell cytoplasm and therefore is inactive untilbindingestrogenanalogtamoxifen,whichtriggersitstranslocationtothecellnucleus

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whereitcanexertitsrecombinaseactivity.Inourstudyweusedatransgenicmouseline expressingmodified Cre-ERT2 recombinase under control ofNSE promoter,which allow deletion of “floxed” sequences in neurons only after tamoxifenadministration to experimental animals. For this type of conditional inactivationexperimentsitisvitaltohaveanabilitytocontroltheefficiencyofthetheLoxP/Crerecombination and identify cells inwhich this recombination took place. This iscommonly achievedby introducing into genome of animalswith “floxed” gene ofinterest of a reporter locus, for example a “floxed-stop”-lacZ cassette intomouseendogenousRosa26 locus.For further functionalexperiments itwas important togeneratemouse linesthatwillcarrymodificationsofSncagene(either“floxed”orcompletely inactivated)andareporterRosa26 locus.Productionofsuch lineswascomplicatedbylocalizationofSncaandRosa26atthesamemousechromosome6,approximately 54Mb apart. To overcome this problem, we carried out a largebreedingandselectionprogrammetoobtainfounderanimalswithSncaandRosa26appearon thesamechromosomeincisastheresultofnaturalchromosomecrossoverinmeiosis.TwolineswithdesiredchromosomalarrangementofSncaandRosa26locihavebeenproduced.OneoftheselinescarriedtheSncagenewith“floxed”firstcodingexon and in another one this exon has been already deleted by LoxP/Crerecombinationinthepreviousbreedingprogramandtherefore,thislinerepresentedanewvariantofconstituentα-synucleinknockout.Importantly,deletionofthefirstcodingexon,eitherconstitutiveorconditional,leftonlyminimal“footprint”,i.e.onlyone22-nucleotideLoxPsiteintheSncageneintronandastheresultexpressionofnoothergene isaffected inmicecarrying thesegenomicmodifications,whichmakesthemsignificantlybettermodelsofα-synucleindeficiencythanotherlinespreviouslyproducedinotherlaboratories.

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3. Discussion & perspectives

3.1. A new Tau self-assembly model

SofarmostoftheinvitrostudiesabouttauaggregationintoPHFshavebeenperformed using establishedmodelswhich involve aggregation cofactors such asheparinorcongored.While itmightbeagoodmodel tostudy the impacton thisprocessofpotentialinhibitors,ortheeffectofsomepost-translationalmodificationon tau,webelieve that itmightnotbe thebestmodel to study the process itself.Indeed, heparin and congo red by accelerating the aggregation processmay alsomodifyitandthusmaskorenhancetheimpactofknownmodulatorsofaggregationsuchaszincions.Thisiswhywedecidedtoworkinabufferdevoidofanyaggregationco-factors,inreducingconditions.Intheseconditionswehavebeenabletoget3newinsightsaboutzincimpactontauself-assembly.

3.1.1. Existence of two types of zinc binding sites

on tau

Mutations in MAPT gene, abnormal PTM and interaction with someaggregation-prone proteins, polyanions and metal ions can induce tau proteinaggregation.Recently,invitrostudiesrevealedthatmetalions,especiallyzinc,couldimpacttaufibrillization(Huetal.,2017;Moetal.,2009).In2009Moetal.(Moetal.JBC2009)showedthatZn2+acceleratesthefibrillizationofataupeptidecoveringthethe244to372aminoacids.InthisstudyconductedinthepresenceofHeparintheyconcludetotheexistenceof1typeofbindingsite.Theyalsoshow thatCys291andCys322playacentralroleinthisbinding.Whiletheyconcludetotheexistenceofonehighaffinitybindingsite(Kd=3.8µM),theyobserveabiphasiceffectofzinc.WhileatlowmicromolarconcentrationsZn2+dramaticallyacceleratesfibrilformation,higherconcentrationsofZn2+inhibitit.Amorerecentstudyconductedthistimeaonefulllengthtauwithapointmutation(ΔK280)stillinpresenceofheparinalsoconcludedthatZn2+ dramaticallyacceleratedtauaggregation(Huetal.BBA2017).Inthisstudythey also determined a single binding site with a high affinity (Kd=0.7 µM).

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Nevertheless,toobtainthefittingtheyhadtoremovemanyexperimentalpointsfromtheirisothermaltitrationcalorimetrydata.IntheirdiscussiontheyevenadmitthatthepossibilitythatZn2+hastwoindependentsetsofbindingsitesfortau:astrongbindingsiteatlowermolarratio(theycall“specific”)andaweakbindingsiteathighmolarratio(theycall“nonspecific”).

This is why we conducted a similar ITC experiment but with moreexperimentalpoints(forthiswe fusedtwoconsecutive ITCtitrations).Ourresultsclearlydemonstratethatzincbindtotau2typesofsites,onesitewithahighaffinity(Kd=0.5 µM) and three siteswith a low affinity (Kd= 16 µM),which could havedifferentfunctions.FirstsitewouldcorrespondtoCys-291andCys-322.OtherthreesitesmightimplicatesHis(H-268,H-299,H-329,H-330orH-362)whichhavebeenshown tobe involved in taubinding (Moetal.,2009).This interaction,as clearlydemonstratedbyourDLSdataleadtosignificantstructuralchangesintau.Indeed,bindingofthefirstzincionleadstocompactizationoftheinitiallydisorderedprotein,whichreflectsinthedropofthehydrodynamicdiameterofTauspeciesfrom12.2to10.8nm.Moreover,itisaccompaniedbyadecreaseofpolydispersityindex,indicatingthatthepopulationoftauconformations insolutionbecomesmorehomogeneous.TheconformationalchangesoftauuponfirstzincbindinghasbeenalsoshownbyYunpengHuangetal.usingCDexperiments thatallowedtheauthors tosuggestapotentialphysical linkbetweenzincandtau(Huangetal.,2014).The furtherzincbinding to low affinity sites results in evenmore compact structure of tauwithhydrodynamicdiameterabout8nm,allowingusinturntohypothesizetheexistenceof physiological role of low affinity binding sites. This role could be significantlydifferent from the one of high affinity site, but to reveal these roles, furtherinvestigationsarenecessarily.

This study raise new questions about mechanism of zinc-induced tauaggregationinparticularabouttherole,playedbyhighandlowaffinitysitesinthisprocess,thelocalizationofthelowaffinitysites,andwhichaminoacidsparticipateinzincchelation.Thestructurationoftauweobserveduponzincbindingtobothhighandlowaffinitysitesopensthechallengeofresolutionof3DstructureofzincbindingsitesusingNMR,whichcouldbepossibleatlowtemperatures.

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3.1.2. Zinc induces fast reversible non-PHF

aggregates

Contrary to thewelldescribed fibrillarystructureofPHFsdescribed in theliterature, the aggregateswe have observed do not seem to have a homogenousstructure.Theyare thioflavinnegative,whichmean theydonothavebeta sheetsstructure, and consistently with our DLS result they appear as huge granularaggregates.Yetitisnotthefirsttimethatsuchgranularaggregatesareobservedwithtau constructs. Indeed, it has been shown that tau244-372 form similar granularaggregates in presence of high concentrations of zinc (Mo et al., 2009). Similarstructureshaveevenbeenobservedfromcellpreparations(Huetal.,2017).

One of the main differences between these granular aggregates compared to

PHF is that they are fully reversible. Removing of Zn2+ using EDTA induces aimmediatedisassemblyoftheseaggregates.Inaddition,thefactthattheprocessisalsofullyreversiblewhendecreasingthetemperatureseemstoindicatethattheself-assemblyisaintrinsicpropertyoftau.InthatsenseZn2+impactontaustructureandself-assemblycouldhaveaphysiologicalrole,inasimilarfashionthatMg2+impactstubulin self-assembly. Interestingly, hydrophobic/hydrophilic balance withinmolecule changes during tau aggregation, that could even lead to transition ofaggregatesinseparatephaseandformationofdroplets(Wegmannetal.,2018).

Figure22(seenextpage)summarizethephysiologicalhypothesisofthisnewpathway.Indeed,duetoitsreversibility,theformationoftheseoligomerscouldbeinducedincellsbyzincsignalsunderphysiologicalconditionsandplayaroleintauregulation.Aswehaveshowntheexistenceoftwotypeofsites,bothimpactingtaustructure, would permit a fine regulation of tau functions, would that be itsinteractionwithtubulinorsomeofitslessstudiedtargetssuchasDNAforexample(Asadollahietal.,2017).

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Figure22:Zinc-inducedreversibleself-assemblyvs.PHFformation.TherightpartoftheschemecorrespondstothepathologicalpathwayleadingtoPHFsformationandNFTformation.Thispathwayisgloballyirreversibleandfavoredbyposttranslationalmodifications suras (hyper)phosphorylationofTau,andZn2+. The leftpartof theschemecorrespondstoaphysiologicalpathwaywhichisfullyreversible.Inthepresenceof lowZn2+ concentrations,Tauundergoes compaction; in thepresenceofhighZn2+concentrationsitleadstotheformationofgranularTauoligomers.

Another hypothesis would be that this new pathway could be a way ofsequesteringtauinordertopreventitfromgoingintothePHFformationirreversiblepathway.Orontheoppositeitcouldbeawayofbufferingzincinto thecellsothatitdoesnotreachconcentration thatwould thatwouldbe toxic forcell functions.Ofcourse,wecannotexcludethatthosegranularaggregatesarepartofthereversibleinitialstepsleadingtoPHFformation,butthefactthatsimilarmechanismhavebeenproposed for other prone-to-aggregate proteins such asAmyloid beta is anotherargumentinfavorofourhypothesis.

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Figure 23. Effect of zinc on amyloid-beta structure. [A] Amyloid-beta peptides form

ordered fibrillar aggregates. In presence of zinc amyloid-beta peptides form amorphous

aggregates. [B] Amino-acid sequence of amyloid-beta 40 and amyloid-beta 42, with the

main coordination sites of zinc; [C] Long fibrillar structures formed by amyloid-beta 42

(top) and disturbed amyloid aggregates of amyloid-beta 42 formed in presence of zinc

(bottom). Adopted from (Mithu et al., 2011; Rezaei-Ghaleh et al., 2011; Zhang et al.,

2018).Zincisoneofthemostabundantmetalioninhumanbodywhereitisinvolved

invariousphysiologicalfunctions.However,abnormalzincaccumulationisfoundinNDD.IncreasedlevelofzincobservesinamyloidplaquesandNFTsinAD,pointtoaroleofzincinformationofpathogenicinclusions.Recentevidencedemonstratethatzinc promotes amyloid-beta aggregation in non-fibrillary way. Equimolarconcentrationofzinceffect toaggregationkineticsofamyloid-beta,however,zincleadstochangesinstructureandmorphologyofamyloid-betaaggregates(Figure23).

Thus,aninterestingperspectiveofthisworkwouldbetotestourhypothesisthatthistaureversibleaggregationisindeedaconcurrentpathwaytoPHFformation.One of the first things to do would be to test whether tau with specific posttranslational modifications (for example PHF specific phosphorylation) is stillcapable ofundergoingsuchazincinducedreversibleself-assembly.Anotherpossibleexperimentwouldbe toseewhathappenswhenheparin isaddedtothegranularaggregates.

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3.2. Modelling of α-synuclein pathology

Incontrasttotauthemodellingofα-synucleinpathologiesismorechallenging(Benskeyetal.,2016).Animalmodelsofoverexpressionoftendonotrecapitulateallspecific features ofPDpathology such as selective lossofdopaminergicneurons,substantia nigra degeneration and decreased dopamine level which are keypathologicaleventsinPD(Giassonetal.,2002;Gomez-Islaetal.,2003;vanderPuttenetal.,2000).Knockoutmodelsthatreproducesynapticdeprivationofα-synucleinasa consequence of changed compartmentalization caused by its cytoplasmicaggregation, have demonstrated mild abnormalities in presynaptic terminalsfunction(Giassonetal.,2002;Gomez-Islaetal.,2003;vanderPuttenetal.,2000).Knockdownofα-synucleinexpressionbyviraldeliveryofα-synuclein-specificshRNAintoSNofadultanimalsledtothedegenerationinthisregion(Collieretal.,2016;Gorbatyuketal.,2010;KanaanandManfredsson,2012)thatindicatesanimportantrole of this protein inmaintaining the functional state of SN in normal and itsinvolvement in thepathology.Geneticassociationbetweendecreased α-synucleinexpressionandriskofPDhasbeenalsodemonstrated(Markopoulouetal.,2014).Therefore, it is feasible that the loss of function of α-synuclein caused by itscytoplasmic aggregation and withdrawing from the synaptic terminals play thepathogenicroleinthedevelopmentofPD.Thelessprominentpathologyincaseofconstituentgeneknockoutcomparingtogeneknockdowninadultanimalscouldbeexplained by functional compensation for α-synuclein loss during embryonicdevelopmentwhentheplasticityofnervoussystemisveryhigh.

3.2.1. α-synuclein participate in the development

of DA neurons in substantia nigra

To reveal the involvement of α-synuclein in embryonic development ofdopaminergicneuronswehavecarriedoutcomparativemorphometricanalysesofthedopaminergicneuronsintheSNandVTAofα-synucleinknockoutandwildtypemice. SN and VTA are two neighboring anatomical structures that containpopulations of dopaminergic neurons. Importantly, in PD brains the loss of

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dopaminergic neurons predominantly accrue in SN whereas VTA dopaminergicneurons aremuch less affected. We have studied the number of dopaminergicneuronsatdays11.5,12.5and13.5ofmouseembryogenesis,aftertheonsetofα-synuclein expression at E10.5. At this stage the postmitotic precursor cells aremigratedfromtheventricularzoneattheborderofthemidbrainandforebrainintotheanatomicalareaofSNandVTAwheretheyundergofurtherdifferentiationintomatureneuronsthatexpressaspecificsetofmarkerproteinsincludingTH.Duringthisperiodofembryonicdevelopment,aprogressive increase inexpressionof α-synucleinisobservedinbothstudiedstructures(AbeliovichandHammond,2007).

OurresultshaveshownthatthedynamicsoftheappearanceofTH-positiveneuronsintheembryonicmesencephalonisdifferentforα-synucleinknockoutandwildtypemice.Inthepresenceofα-synuclein,asharpincreaseinthenumberofTH-positiveneuronsatE12.5followedbyamoderatedeclineatE13.5wasobservedintheSN,whereasinknockoutmicethenumberofTH-positiveneuronsappearedtobehigheratE11.5butdidnotchangeasmuchas inwild typeSNover thenext twoembryonicdays.As the result, atE13.5 thenumber ofTH-positiveneurons in α-synucleinknockoutmicebecomeslowerthaninwildtypemice,whichisconsistentwiththepreviouslypublishedobservations(Garcia-Reitboecketal.,2013).ApossibleexplanationoftheseobservationisthatprecursorsofSNneuronslackingα-synucleinmigratefastertothemesencephalonbuttheirconsequentdifferentiationintomatureneuronsispartiallycompromised.

SignificantlossofdopaminergicneuronsintheSNhavebeen observedinPDpatientswhereasthepathologyofdopaminergicneuronsinVTAisnotsoprofound(Alberico et al., 2015).We did not find statistically significant differences in thenumberofVTAneuronsbetweenα-synucleinknockoutandwildtypemiceatanyofthethreestudiedembryonicdays.TheseresultssuggestthatdopaminergicneuronsofSNareintrinsicallymoresensitivetochangesofα-synucleinexpressionthanVTAneurons,which is consistentwith differential sensitivity of these two groups ofneuronstoPDpathology(Dudaetal.,2016;Maingayetal.,2006;Mosharovetal.,2009).

Obtaineddatarevealedaprominentmodulatingeffectofα-synucleinontheearlydevelopmentandmaturationofdopaminergicneuronsintheSNandnosuch

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effectondopaminergicneuronsinVTA,suggestingthatthelossoramelioratingofα-synucleinfunctionintheadultnervoussystemmighthavenegativeeffectspecificallyon SN neurons andmight contribute to theirdifferential dysfunction in PD. Thisfurtheremphasizesanurgentneedfortransgenicmodelsrecapitulatingalate-onsetdepletion of α-synuclein, which better recapitulates pathological changes in thenervoussystemofPDpatients.

3.2.2. Generation of new mouse lines for

conditional inactivation of Snca

Recently,acoremouseline forconditional inactivationofSnca,α-synucleinencoding gene, has been produced (Ninkina et al., 2015). In studies that involveconditional inactivationofanygenebyLoxP/Crerecombination it is important tomonitor the efficiency of recombination and identify cells in which thisrecombinationhasactuallyhappened.TomakethismonitoringpossibleforstudieswithconditionalinactivationofSncagene,weproducedtwonewmouselinesthatcarry in their genomes bothmodified Snca gene, either “floxed” or already fullygenetically inactivated, and a “floxed” reporter cassette in the Rosa26 locus. TheavailabilityofthesetwonewlinesmakesitpossibletoproducemicecarryingvariouscombinationsofgeneticallymodifiedSncageneandRosa26locustogetherwithCre-expressingtransgeniccassettes.Thisshallalloweasygenerationofexperimentalandcontrol animal cohorts for conditional inactivation of Snca gene by LoxP/CrerecombinationthatcanbemonitoredusingareporterconstructintheRosa26locusforfastidentificationofcellswithinactivationofSncaandtherefore,depletedofα-synuclein.

Thisconditionalα-synucleinknockoutmodelswillbeaveryusefultool forstudying various aspects of α-synuclein function and dysfunction, such asseeding/transmissionofitsaggregation.Itwillbeinstrumentalfor revealingindetailhowthelossofnormalα-synucleinfunctionthatoccursintheadultbraincontributestothedevelopmentofthenigrostriatalpathologyinPD.Todatewearedoingbiginvivostudyusingournewconditionalknockoutmodels.Wealreadyproducedlargecohorts ofanimals,inpartofthemweinduceddepletionofα-synucleininadultage(6 months). General phenotype and motor behavior of experimental mice are

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controlled at regular intervals. Brain samples from representative groups ofexperimentaland controlanimalswillbe takenat4,8,12and18monthsafter6month injection point. Expression of α-synucleinwill bemeasured by qRT-PCR,Westernblottingand immunohistochemistry (inparallelwithbeta-Galstainingofbrain sections to monitor efficiency of Cre/LoxP recombination). Expression ofvariousneuronalandsynapticmarkersinthebrainofanimalswithspecialattentiontomarkersofnigrostriatalsystemfunctionwillbeperformedbyqRT-PCR,Westernblottingand immunohistochemistry.Levelsofdopamineand itsmetabolites inthestriatumwillbemeasuredbyHPLC.Possibly,lossofα-synucleinfunctionswillleadto lossofdopaminergicneurons.Wewill investigate thiseventbymorphometricanalysisofneuronsonaseriesofsectionsacrossthewholeanatomicalstructureofSNandVTA.Thus,wewillknowwhattheimpactofα-synucleindepletionintheadultbrainis.