Induction of CNS α-synuclein pathology by fibrillar and non-amyloidogenic recombinant α-synuclein

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Title:
Induction of CNS α-synuclein pathology by fibrillar and non-amyloidogenic recombinant α-synuclein
Physical Description:
Mixed Material
Language:
English
Creator:
Sacino, Amanda N.
Brooks, Mieu
McGarvey, Nicholas H.
McKinney, Alex B.
Thomas, Michael A.
Levites, Yona
Ran, Yong
Golde, Todd E.
Giasson, Benoit I.
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BioMed Central (Acta Neuropathologica Communications)
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Abstract:
Background: α-Synuclein (αS) is the major component of several types of brain inclusions including Lewy bodies, a hallmark of Parkinson’s disease. Aberrant aggregation of αS also is associated with cellular demise in multiple neurologic disorders collectively referred to as synucleinopathies. Recent studies demonstrate the induction of αS pathology by a single intracerebral injection of exogenous amyloidogenic αS in adult non-transgenic and transgenic mice expressing human αS. To further investigate the mechanism of pathology induction and evaluate an experimental paradigm with potential for higher throughput, we performed similar studies in neonatal mice injected with αS. Results: In non-transgenic mice, we observed limited induction of neuronal αS inclusions predominantly 8 months after brain injection of aggregated, amyloidogenic human αS. More robust inclusion pathology was induced in transgenic mice expressing wild-type human αS (line M20), and inclusion pathology was observed at earlier time points. Injection of a non-amyloidogenic (Δ71-82) deletion protein of αS was also able to induce similar pathology in a subset of M20 transgenic mice. M20 transgenic mice injected with amyloidogenic or non-amyloidogenic αS demonstrated a delayed and robust induction of brain neuroinflammation that occurs in mice with or without αS pathological inclusions implicating this mechanism in aggregate formation. Conclusions: The finding that a non-amyloidogenic Δ71-82 αS can induce pathology calls into question the simple interpretation that exogenous αS catalyzes aggregation and spread of intracellular αS pathology solely through a nucleation dependent conformational templating mechanism. These results indicate that several mechanisms may act synergistically or independently to promote the spread of αS pathology. Keywords: Amyloid, Neonatal, Parkinson’s disease, Pathology, α-Synuclein, Transgenic mice
General Note:
Sacino et al. Acta Neuropathologica Communications 2013, 1:38 http://www.actaneurocomms.org/content/1/1/38; Pages 1-14
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doi:10.1186/2051-5960-1-38 Cite this article as: Sacino et al.: Induction of CNS α-synuclein pathology by fibrillar and non-amyloidogenic recombinant α-synuclein. Acta Neuropathologica Communications 2013 1:38.

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RESEARCHOpenAccessInductionofCNS -synucleinpathologyby fibrillarandnon-amyloidogenicrecombinant -synucleinAmandaNSacino,MieuBrooks,NicholasHMcGarvey,AlexBMcKinney,MichaelAThomas,YonaLevites, YongRan,ToddEGolde*andBenoitIGiasson*AbstractBackground: -Synuclein( S)isthemajorcomponentofseveraltypesofbraininclusionsincludingLewybodies,a hallmarkofParkinson ’ sdisease.Aberrantaggregationof Salsoisassociatedwithcellulardemiseinmultiple neurologicdisorderscollectivelyreferredtoassynucleinopathies.Recentstudiesdemonstratetheinductionof S pathologybyasingleintracerebralinjectionofexogenousamyloidogenic Sinadultnon-transgenicand transgenicmiceexpressinghuman S.Tofurtherinvestigatethemechanismofpathologyinductionandevaluate anexperimentalparadigmwithpotentialforhigherthroughput,weperformedsimilarstudiesinneonatalmice injectedwith S. Results: Innon-transgenicmice,weobservedlimitedinductionofneuronal Sinclusionspredominantly8months afterbraininjectionofaggregated,amyloidogenichuman S.Morerobustinclusionpathologywasinducedin transgenicmiceexpressingwild-typehuman S(lineM20),andinclusionpathologywasobservedatearliertime points.Injectionofanon-amyloidogenic( 71-82)deletionproteinof Swasalsoabletoinducesimilarpathology inasubsetofM20transgenicmice.M20transgenicmiceinjectedwithamyloidogenicornon-amyloidogenic S demonstratedadelayedandrobustinductionofbrainneuroinflammationthatoccursinmicewithorwithout S pathologicalinclusionsimplicatingthismechanisminaggregateformation. Conclusions: Thefindingthatanon-amyloidogenic 71-82 Scaninducepathologycallsintoquestionthesimple interpretationthatexogenous Scatalyzesaggregationandspreadofintracellular Spathologysolelythrougha nucleationdependentconformationaltemplatingmechanism.Theseresultsindicatethatseveralmechanismsmay actsynergisticallyorindependentlytopromotethespreadof Spathology. Keywords: Amyloid,Neonatal,Parkinson ’ sdisease,Pathology, -Synuclein,TransgenicmiceBackgroundAcharacteristicofParkinson ’ sdisease,themostcommon neurodegenerativemovementdisorder,isthepresenceof intraneuronalLewybodies(LBs)inneurons.Theseinclusionsareformedfromtheamyloidogenicaggregationof thenormallysolublepresynapticprotein -synuclein( S). Sbraininclusionsalsoarepresentinaspectrumofneurodegenerativedisordersknownas -synucleinopathies [1-3].Adirectcausalrolefor Sinneurodegenerationis supportedbymissensemutationsorincreasedcopynumberofthe Sgene( SNCA )insomepatientswith Parkinson ’ sdiseaseandtherelateddisorderdementiawith Lewybodies[4-11].Despitealargenumberofexperimentalstudies,theprecisemechanism(s)of Stoxicityisstill notresolved,althoughmultiplelinesofevidencesupport thehypothesisthat Saggregationislinkedtocellular demise[1,12]. -Synucleinopathiesareprogressivediseasesandinrecentyearstherehavebeenincreasingeffortstoidentifythe mechanismsinvolvedinintracerebralspreadofpathology, asitisreasonedthattherapiesthatcouldsloworhaltpathologyspreadwouldlikelybediseasemodifying.Recently, *Correspondence: tgolde@ufl.edu ; bgiasson@ufl.edu CenterforTranslationalResearchinNeurodegenerativeDisease,Department ofNeuroscience,BMSBuildingJ-483,UniversityofFloridaCollegeof Medicine,1275CenterDrive,POBox100159,Gainesville,FL32610,USA 2013Sacinoetal.;licenseeBioMedCentralLtd.ThisisanOpenAccessarticledistributedunderthetermsoftheCreative CommonsAttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse,distribution,and reproductioninanymedium,providedtheoriginalworkisproperlycited.Sacino etal.ActaNeuropathologicaCommunications 2013, 1 :38 http://www.actaneurocomms.org/content/1/1/38

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severalexperimentalandpathologicalstudieshavesuggestedthatspreadingof Spathologymightoccurviaa seededconformational-templ atingproteinaggregation mechanism.Forexample,LBformationwasobservedin fetaldopaminergicneuronsofasubsetofPDpatientsthat receivedstriataltransplantsasanattemptedtherapeutic intervention[13-15].Aseedingmechanismwouldalso generallybeconsistentwiththeproposedBraakstagingof diseasethatappearstofollowneuroanatomicalpathways [16].Experimentallyitwasreportedthattheintracerebral injectionofextractsfrommoribundA53Thuman S transgenic(Tg)mice(lineM83)thatdevelopalateonset severemotorphenotypeassociatedwithwidespreadformationofneuronal SinclusionsintoyoungerhealthyM83 Tgmicecouldinducethesecellularandphenotypicpathologies[17-19].Furthermore,braininjectionofpre-formed recombinant SfibrilsintoM83Tgmicecanalsoinduce Spathologywithinbrainregionsthataredistantfromthe injectionsite[18],suggestingthatthese Sspeciescaninitiateandperhapsleadtotransmissionof Spathology.Inductionofbrain Spathologywasalsoreportedinnon-Tg (nTg)micefollowingintrastriatalinjectionofmurine fibrillar S[20].Morerecentlyitwasreportedthattheinjectionofeitherpreformedhumanormouse Sfibrilsin thesubstantianigraofnTgmicecouldalsoinduceneuronal Spathology,butthispathologycouldonlybeobserved3monthsormoreafterexposure[21].Collectively thesestudiesalongwithnumerous invitro andculture studiessupporttheconceptof Spathologyspreadwithin thebrainviaaconformationa ltemplatingmechanism. However,thismechanismofpathologyinductionremains tobeformallyproven invivo ,asotherpossiblemechanismscouldcontributeto Sinclusionpathologyinduction includingdisruptionofproteostasisandinnateimmune activation[22-25]. Tofurtherelucidatethemechanismsassociatedwiththe inductionofintraneuronal Sinclusionpathologyresultingfromexogenous Schallengeandtoevaluateapotentiallyhigherthroughputexperimentalparadigm,we injectedamyloidogenicandnon-amyloidogenicformsof SintothebrainofneonatalnTgandM20Tgmiceexpressingwild-typehuman S.Neonatalinjectionisasignificantlyeasierandfastersurgicalprocedurethan stereotacticinjectionintheadultbrain,mainlybecause cryo-anesthesiacanbeutilizedandtheskullisstillsoft andflexible.Thesestudiesrevealthatneonatalcerebral injectionofamyloidogenic Sresultsinlimitedneuronal SinclusionsinnTgmicethatareobservedpredominantly8monthsafterinjection.Similarstudiesin M20Tgmicealsorevealedalagtimeintheformation ofdetectable Spathology,butpathologywasmore widespreadthroughouttheneuroaxisandwasinduced bytheinjectionofbothamyloidogenicandnonamyloidogenicformsof S. Materialandmethods Antibodies pSer129isamousemonoclonalantibodyspecificto S phosphorylatedatSer129[26].Syn211andLB509are mousemonoclonalantibodiesspecificforhuman S [27,28].SNL-1isarabbitpolyclonalantibodyraised againstasyntheticpeptidecorrespondingtoaminoacids 104 – 119of Sandspecificallyreactswithbothmurine andhuman S[27].SNL-4isarabbitpolyclonalantibody raisedagainstasyntheticpeptidecorrespondingtoamino acids2 – 12of S[27].Syn506isaconformationalantiS mousemonoclonalantibodythatpreferentiallydetects S inpathologicalinclusions[29,30].Anti-p62(SQSTM1; Proteintech;Chicago,IL),anti-glialfibrillaryacidicprotein(GFAP;Promega;Madison,WI),andanti-ionized calcium-bindingadaptormolecule1(IBA-1;DAKO; Glostrio,Denmark)arerabbitpolyclonalantibodies.An anti-glyceraldehyde-3-phos phatedehydrogenase(GAPDH) mousemonoclonalantibodywasobtainedfromBiodesign (Memphis,TN). Figure1 Detectionofinjectedhuman Sintheneedletrack 2dayspostneonatalinjection. IHCstainingwithhuman Sspecific antibodyLB5092daysafterneonatalinjectionof25 g21 – 140human SfibrilsinnTgmice.Stainingshowsthepresenceofhuman Sinthe braininjectiontractadjacenttothelateralventricleinthecerebrum (blackarrows).Thetissuesectionwascounterstainedwithhematoxylin. Scalebar=250 m. Sacino etal.ActaNeuropathologicaCommunications 2013, 1 :38Page2of14 http://www.actaneurocomms.org/content/1/1/38

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nTgmiceandM20 STgmiceAllprocedureswereperformedaccordingtotheNIH GuidefortheCareandUseofExperimentalAnimalsand wereapprovedbytheUniversityofFloridaInstitutional AnimalCareandUseCommittee.BL6C3HF1mice (CharlesRiverLaboratoriesInternationalInc,Wilmington, MA)havethesamestrainbackgroundas STgmice(line M20)andwereusedasnTgmice.TheM20Tgmiceexpresshumanwild-type Sunderthecontrolofthemouse PrPpromoterandthesemicedonotdevelopanyintrinsic phenotypeor Spathology[31,32].HemizygousM20Tg malemicewerematedwithfemaleBL6C3HF1miceand genotypedbyPCR,butalsoconfirmedbyimmunohistochemical(IHC)stainingofmousebrainsectionwithantihuman SantibodySyn211.Allanimalswerehoused3to 5toacageandmaintainedonadlibitumfoodandwater witha12hlight/darkcycle.Brain SinjectionintoneonatalmiceBilateralneonatal(P0)injectionsof Sproteinswere performedbyinsertingtheneedleabout0.5cmdeepinto thebrainjustlateraltothelateralventriclesinthecerebrum(seeFigure1)usingcryo-anesthesiaasdescribed previously[33].Inbrief,P0pupswerecryo-anesthetized oniceforupto5minutes.Eachpupreceivedbilateral injectionsof Sproteins.Injectionsweremadeusinga 10 LHamiltonsyringewitha30-gaugeneedle.Different syringeswereusedforeachtypeofproteintopreventany contamination.Post-injection,thepupswereplacedona heatingpadforrecoverybeforebeingreturnedtotheir homecage.Expressionandpurificationofrecombinant SproteinsThepRK172cDNAconstructionsexpressingfull-length human S,human Swithaminoacid71 – 82deletion ( 71-82),andN-terminaltruncated21 – 140 S(witha Metcodonaddedbeforeaminoacid21)werepreviously described[34,35]. SproteinswereexpressedinE.coli BL21(DE3)andpurifiedtohomogeneitybysizeexclusion (Superdex200gelfiltration)andionexchanged(MonoQ) chromatographiesaspreviouslydescribed[34,36].Fibrilpreparationofrecombinant Sformouse braininjection21 – 140 Sproteinwasassembledintofilamentsbyincubationat37Cat5mg/mlinsterilephosphatebuffered saline(PBS,Invitrogen)withcontinuousshakingat 1050rpm(ThermomixerR,Eppendorf,Westbury,NY). Samyloidfibrilassemblywasmonitoredaspreviously describedwithK114fluorometry[35,37]. Sfibrilswere dilutedinsterilePBSandtreatedbywaterbathsonication for2hours.Thesefibrilsweretestedforinductionof intracellularamyloidinclusionformationaspreviously described[38,39].ImmunohistochemicalanalysisMiceweresacrificedwithCO2euthanizationandperfused withPBS/heparin,followedbyperfusionwitheither70% Table1SummaryofneonatalnTgmiceinjectedwith SproteinsaMousestrainInoculumAgeatharvestNumberofmicePathologicalfindings C57BL6/C3Hfib S(2 lof1mg/ml)1month9Noinclusions C57BL6/C3Hfib S(2 lof1mg/ml)2months3Noinclusions C57BL6/C3Hfib S(2 lof1mg/ml)4months6Noinclusions C57BL6/C3Hfib S(2 lof1mg/ml)8months4Noinclusions C57BL6/C3Hfib S(5 lof5mg/ml)1month9Noinclusions C57BL6/C3Hfib S(5 lof5mg/ml)2months71of7miceshowrareinclusionsbC57BL6/C3Hfib S(5 lof5mg/ml)4months3Noinclusions C57BL6/C3Hfib S(5 lof5mg/ml)8months134of13miceshowrarecorticalinclusionscC57BL6/C3H 71-82 S(2 lof1mg/ml)1month4Noinclusions C57BL6/C3H 71-82 S(2 lof1mg/ml)2months3Noinclusions C57BL6/C3H 71-82 S(2 lof1mg/ml)4months2Noinclusions C57BL6/C3H 71-82 S(5 lof5mg/ml)1month9Noinclusions C57BL6/C3H 71-82 S(5 lof5mg/ml)2months8Noinclusions C57BL6/C3H 71-82 S(5 lof5mg/ml)8months6NoinclusionsanTgmicewereinjectedwith21 – 140human Sfibrils(fib)or 71-82human Satthedifferentdosagesindicatedandanalyzedfor Spathologyat1 – 8months post-injectionusingpSer129andSyn506antibodies.bSparseinclusionswereobservedinthemidbrainareaof1mouse.cSeeFigure 2 foraschematicneuroanatomicalmapshowingthedistributionof Spathology.Sacino etal.ActaNeuropathologicaCommunications 2013, 1 :38Page3of14 http://www.actaneurocomms.org/content/1/1/38

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ethanol/150mMNaClorPBSbufferedformalin.The brainandspinalcordwerethenremovedandfixedforat least24hoursintherespectivefixativesusedforperfusion.Aspreviouslydescribed,tissuesweredehydratedat roomtemperaturethroughaseriesofethanolsolutions, followedbyxyleneandthenwereinfiltratedwithparaffin at60C[40].Thetissueswerethenembeddedintoparaffin blocks,whichwerecutinto7 msections.Immunostaining ofthesectionswasperformedusingpreviouslydescribed methods[40]withtheavidin-biotincomplex(ABC)system (VectastainABCEliteKit,VectorLaboratories,Burlingame, CA),andwithimmunocomplexvisualizationviachromogen3,3 ’ -diaminobenzidine.Sectionswerecounterstained withhematoxylin.Allslides werescannedusinganAperio ScanScopeCS(40magnification;AperioTechnologies Inc.,Vista,CA),andimagesofrepresentativeareasof S pathologyweretakenusingtheImageScopeTMsoftware (40magnification;Aper ioTechnologiesInc.). Double-labelingimmunofluorescenceanalysisofmouse braintissue Paraffin-embeddedtissuesectionsweredeparaffinizedand hydratedthroughaseriesofgradedethanolsolutions followedby0.1MTris,pH7.6.Thesectionswereblocked with5%drymilk/0.1MTris,pH7.6,andwereincubated simultaneouslywithcombinationsofprimaryantibodies dilutedin5%drymilk/0.1MTris,pH7.6.Afterextensive washing,sectionswereincubatedwithsecondaryantibodiesconjugatedtoAlexa594orAlexa488(Invitrogen; Eugene,OR).Sectionswerepost-fixedwithformalin,incubatedwithSudanBlack,andstainedwith5 g/ml4 ’ ,6diamindino-2-phenylindole(DAPI).Thesectionswere coverslippedwithFluoromount-G(SouthernBiotech, Birmingham,AL)andvisualizedusinganOlympus BX51microscopemountedwithaDP71Olympus digitalcameratocaptureimages. Immunoblottinganalysis Mousebrainswerelysedin2%SDS/50mMTrispH7.5 bysonicationandheatedto100Cfor10minutes.Protein concentrationwasquantifiedusingthebicinchoninicacid (BCA)assayandbovineserumalbuminasastandard (PierceBiotechnology;Rockford,IL).15 goftotalprotein Figure2 Schematicsummaryshowingpredominantcorticaldistributionof SpathologyinnTgmice8monthsafterbrainneonatal injectionoffibrillar21 – 140 S. nTgmiceinjectedwith25 g21 – 140fibrillar(fib) S.Mapshowsrostral-caudaldistributionof Sinclusionsvia coronalsections.Equivalentdensityanddistributionof Spathologywasseenbilaterally.PathologywasdetectedwithantibodiespSer129and Syn506.Asshowninrepresentativeimages,small,roundedperinuclear Sinclusionandneuriticprofileswerefoundsparselydistributedinthe cortex.Thedistributionofinclusionswasverysimilarinallmicewithpathology.Scalebar=50 m. Figure3 Increasedpostnatalexpressionof Sinthebrainof nTgandM20Tgmice. TotalproteinmousebrainextractsfromP0, P2,P4,P8,P16,P30,andadult(3months)nTg(-)and(+)M20Tg micewasresolvedon13%SDSpolyacrylamidegelsandanalyzedby immunoblottingwithantiSantibodySNL-1,whichdetectsboth humanandmouse S,oranti-human SantibodySyn211. Immunoblottingwithananti-GAPDHantibodywasperformedasa loadingcontrol.Themobilityofmolecularmassmarkersisindicated ontheleft. Sacino etal.ActaNeuropathologicaCommunications 2013, 1 :38Page4of14 http://www.actaneurocomms.org/content/1/1/38

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wasresolvedbySDS-PAGEon13%polyacrylamidegels, followedbyelectrophoretictransferontonitrocellulose membranes.MembraneswereblockedinTrisbufferedsaline(TBS)with5%drymilk,andincubatedwithprimary antibodieswhichwerefollowedbyeithergoatanti-mouse conjugatedhorseradishperoxidase(HRP)(Amersham Biosciences;Piscataway,NJ)orgoatanti-rabbitHRP(Cell SignalingTechnology;Danvers,MA).Proteinbandswere detectedusingchemiluminescentreagent(NEN;Boston, MA)andaFluorChemEandMImager(Proteinsimple; SanJose,California). MALDI-TOFmassspectrometryoffull-length Sand 71-82 S Recombinanthumanfull-length Sand 71-82 S (~4mM)weredilutedto10 Mwith0.1%TFA(trifluoroaceticacid)solution.1 ldilutedsamplewasmixed with1 lsaturatedACCA( -cyano-4-hydroxycinnamic Table2SummaryofneonatalM20Tgmiceinjectedwith Sproteins a MousestrainInoculumsAgeatharvestNumberofmicePathologicalfindings M20(WT S)fib S(2 lof1mg/ml)1month7Noinclusions M20(WT S)fib S(2 lof1mg/ml)2months5Noinclusions M20(WT S)fib S(2 lof1mg/ml)4months3Noinclusions M20(WT S)fib S(2 lof1mg/ml)8months44of4miceshowsparsecorticalpathology M20(WT S)fib S(5 lof5mg/ml)1month43of4miceshowsparsecorticalpathology M20(WT S)fib S(5 lof5mg/ml)2months54of5miceshowsparsecorticalpathology M20(WT S)fib S(5 lof5mg/ml)4months32of3miceshowsparsecorticalpathology M20(WT S)fib S(5 lof5mg/ml)8months1212of12miceshowabundantpathology b M20(WT S) 71-82 S(2 lof1mg/ml)1month5Noinclusions M20(WT S) 71-82 S(2 lof1mg/ml)2months5Noinclusions M20(WT S) 71-82 S(2 lof1mg/ml)4months3Noinclusions M20(WT S) 71-82 S(2 lof1mg/ml)8months3Noinclusions M20(WT S) 71-82 S(5 lof5mg/ml)1month7Noinclusions M20(WT S) 71-82 S(5 lof5mg/ml)2months7Noinclusions M20(WT S) 71-82 S(5 lof5mg/ml)8months62outof6miceshowabundantpathology b a M20Tgmicewereinjectedwith21 – 140human Sfibrils(fib)or 71-82human Satthedifferentdosagesindicatedandanalyzedfor Spathologyat 1 – 8monthspost-injectionusingpSer129andSyn506antibodies. b SeeFigure 4 foraschematicneuroanatomicalmapshowingthedistributionof Spathology. Figure4 Schematicrepresentationofthedistributionof Spathologyat8monthsfollowingbrainneonatalinjectionof21 – 140 human Sfibrilsor 71-82humaninM20 STgmice. M20Tgmiceinjectedwith25 goffibrillar(fib)21 – 140human S( A )or 71-82 human S( B ).Mapsshowrostral-caudaldistributionof Sinclusionsviacoronalsections.Equivalentdensityanddistributionof Spathologywas seenbilaterally.PathologywasdetectedwithantibodiespSer129andSyn506.( A )P0injectionof21 – 140human Sfibrilsresultsintheformation of Sinclusionsthroughoutthecortex,hippocampus,midbrain,brainstem,andspinalcord.( B )P0injectionof 71-82human Salsoresultsin widespread Sinclusions.Thedistributionofinclusionswasverysimilarinallmicewithpathology. Sacino etal.ActaNeuropathologicaCommunications 2013, 1 :38Page5of14 http://www.actaneurocomms.org/content/1/1/38

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acid)solution(acetonitrile:methanol=3:2).1 lsample mixturewasloadedtoACCApretreatedMSP96target (BrukerDaltonicsInc.;Billerica,MA).Thesampleswere analyzedwithaBrukerMicroflex(BrukerDaltonicsInc.; Billerica,MA)massspectrometerinlinearpositivemodel. SpectrawerecalibratedwithBrukerproteincalibrate standard. Results Tofurtherinvestigateinductionof Sinclusionformationfollowingbraininjectionofexogenous Sandto generateahigherthroughputexperimentalmodel,we injectednTgneonatalmousebrainswithexogenous preformedrecombinanthuman Samyloidfibrilscomprisedof21 – 140 S(Figure1).Weuseamino-truncated 21 – 140 S,asfibrilscomprisedofthisproteincanseed Ssimilarlytothefull-lengthproteininculturedcells [38,39,41,42]anditprovidestheabilitytodefinitivelyassessaggregationoftheendogenous Sbydetectionwith amino-terminalspecific Santibodies.Thepresenceof theexogenous S(25 ginjected)couldbereadily detectedintheneedletrack2dayspostinjectioninnTg mousebrainsusinganantibodytohuman S(Figure1). By4dayspost-injection,exogenous Swasnotdetectable,consistentwiththefindingsrecentlyreportedby Masuda-Suzukakeandcolleagues[21]whoalsoshowed exogenoushuman Sinjectedintothebrainwasdetectableonlywithinthefirst7dayspostinjection.Wedid notdetectlocalordistalinductionofintracellularpathologyat4,8,and16dayspost-injectionof25 gfibrillar S.AnalysisofnTgmousebrainsneonatallyinjected with2 gofexogenousfibrillar21 – 140 Sandagedup to8monthsdidnotrevealthepresenceofany Spathology(Table1).Injectionof25 gofexogenousfibrillar Figure5 Inductionof Spathologythroughouttheneuroaxis8monthsafterneonatalbraininjectionof25 gfibrillar21 – 140 Sin M20Tgmice. TissuesectionswerestainedwithpSer129.Dystrophicneuriteswerediffuselypresentthroughoutthebrainandspinalcord.The morerounded,Lewybody-likepathologywasseenpredominantlyintheolfactorybulb(OB),motorcortex(MC),amygdala(AMY),dentategyrus ofthehippocampus(DG),thalamus(TH),hypothalamus(HYP),substantianigra(SN),ventralpons(VP),andboththeventralanddorsalhornsof thespinalcord(VH-SCandDH-SC).Lewyneurite-likepathologyextendingintothecellularprocesseswasmorepredominantlyseeninthe piriformcortex(PC),entorhinalcortex(EC),striatum(ST),corpuscallosum(CC),andCA1ofthehippocampus(CA1).Tissuesectionswere counterstainedwithhematoxylin.Scalebar=50 m(OB),100 m(MC),50 m(PC),50 m(EC),100 m(ST),200 m(CC),50 m(AMY), 50 m(DG),50 m(CA1),50 m(TH),50 m(HYP),50 m(SN),50 m(VP),200 m(VH-SC),and200 m(DH-SC). Sacino etal.ActaNeuropathologicaCommunications 2013, 1 :38Page6of14 http://www.actaneurocomms.org/content/1/1/38

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21 – 140 Sresultedinrarepathologyinonlyonemouse at2monthspost-injection,butat8monthspostinjection,4outof13nTgmiceinjectedwiththisdoseof Sshowedsparse Sneuronalinclusionpathologyprimarilylocalizedtocorticalneurons(Table1andFigure2). InnTgcohortsinjectedwitheither2 gor25 gnonamyloidogenic 71-82 S[34,35,41],wedidnotobserve anypathologyinnTgmice(seeTable1).Inthesestudies 71-82 Swasusedasacontrolforconformational templatingmechanisms,asweandothershaveextensively studiedthisproteinandshowedthatitisdeficientinthe abilitytoformordirectlyaffect(induceorinhibit)theformationof Samyloidfibrils invitro orinculturemodels [34,35,41-43].Afterprolongedincubationsathighconcentrations, 71-82 -synucleincanformoligomersasobservedbynegativestainingelectronmicroscopy,butthese arenotamyloidogenicinnature.The 71-82 -synuclein usedforthecurrentstudies wasnotpre-incubatedandis inthesolubleformaspreviouslydescribed.Wehavealso recentlyshownthatthissamepreparationof 71-82 synucleincannotdirectlyseedtheformationof -synuclein inclusionsinprimaryneuronalcultures[42].Incontrast thesamepreparationoffibrillar -synucleincanseedinclusionformationveryeffici entlyinthosecultures. Toexaminewhetheroverexpressionof Scanincrease theefficiencyofinclusionpathologyformation invivo weperformedneonatalbraininjectionoffibrillar21 – 140 SinM20Tgmice,whichoverexpresswildtype human S.InadultM20Tgmicethereis~5-foldover expressionofhuman Sinthebrain,butthesemicedo notdevelop Spathologyduringtheirlifespanintheabsenceofadditionalmanipulations[31,32].Thesemice alsooverexpresstransgenichuman SduringdevelopmentthatcanbeobservedasearlyasP0(Figure3),and aspreviouslyreported,theexpressionof Sincreasesduringmousebraindevelopment[44].Thus,theymakean idealmodeltoexploreparadigmsforinductionof S pathology.Theneonatalbraininjectionof2 gfibrillar 21 – 140 Sdidnotinducetheformationofintraneuronal pathologyattimesupto4months,butby8months sparsecorticalpathologycouldbeobserved(Table2). Similarchallengeto 71-82 Sdidnotresultintheformationofpathology.Increasingthetreatmentto25 gfibrillar21 – 140 Sresultedinsparsebrain Spathologyas earlyas1monthand4months,butitwasextensivelydistributedthroughouttheneuroaxisby8months(Table2, Figures4and5)atahigherdensitythaninnTgmice showing Spathologyat8monthspost-injectionof25 g fibrillar21 – 140 S.Interestingly, Sinclusionswererarely observedinnigraldopaminergicneurons(Figure6).Similartothe SaggregatesinsymptomaticM83Tgmice, whichspontaneouslydevelopage-dependentpathology [31,32],theinclusionsinM20Tgmiceinducedbythe braininjectionoffibrillar21 – 140 Swerecomprisedof endogenouslyexpressed Sastheywerereactivewith amino-terminalspecificantibodiesSyn506andSNL-4 (Figures7and8).Theinclusionswerealsoreactivewith p62(sequestrosome;Figure9),arobustmarkerof Sinclusions[45].Unexpectedly,similarbraininjectionof 71-82 Salsoresultedinrobustandwidelydistributed S brainpathologyat8monthsinsomeoftheinjectedM20 Tgmice(Table2;Figures6,7,8,9and10).These Sinclusionswerealsocomprisedofendogenous S(i.e.reactive withantibodiesSyn506andSNL-4)hyperphosphorylated atSer129,andaccumulatedp62.Forcomparison,weshow thatsomeoftheM20Tgmice8monthspost-injection with25 gof 71-82 Saredevoidof Spathology(see Additionalfile1:FigureS1).AlthoughnoneofthenTgor Figure6 Themajorityof SinclusionsinthesubstantianigraofM20Tgmiceneonatallyinjectedwithexogenous SarenotinTH positiveneurons. M20Tgmice8monthsafterneonatalinjectionwith21 – 140human Sfibrils(fib)( A )or 71-82human S( B ).Double-labeled immunofluorescenceanalysisfortyrosinehydroxylase(TH;red)labelingthedopaminergicneuronsinthesubstantianigraarea,andpSer129 (green)labelingthehyperphosphorylated Sinclusionsshowminimalco-localization.Themajorityof Sinclusionswerenotfoundin TH+cells,exceptforafewneurons(whitearrows).CellnucleiwerecounterstainedwithDAPI.Scalebar=100 m. Sacino etal.ActaNeuropathologicaCommunications 2013, 1 :38Page7of14 http://www.actaneurocomms.org/content/1/1/38

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theM20Tgmicewereextensivelyanalyzedforbehavioral changes,thepresenceof Spathologicalinclusionswasnot associatedwithanyovertbehavioralabnormalities. Toassessiftherewasanassociationbetweenneuroinflammation(astrogliosisormicrogliosis)andinductionof Saggregation,tissuesectionsfromallinjected nTgandM20Tgmicewerestainedwithantibodiesto GFAPandIBA-1.Asexpected,controluntreatedM20Tg miceat8monthsofageshowedbasallevelsofastrocytes andmicroglia(Figure11Aand11H)[17,32].Mostinjected nTgandM20Tgmiceat1,2or4monthpost-injection didnotdisplayincreasedastrogliosisormicrogliosisas Figure7 IHCshowingsimilar Spathologyinducedbyneonatalbraininjectionoffibrillarhuman21 – 140 Sand 71-82human Sin M20TgmicecomparedtoasymptomaticM83Tgmouse. Brainstemtissuesectionsfroma15month-oldsymptomaticM83Tgmouse( A ) and8month-oldM20Tgmiceneonatallyinjectedinthebrainwith25 g21 – 140fibrillar(fib) S( B )or25 g 71-82( C )showsimilarstainingof SinclusionsasdetectedwithpSer129byIHC. SinclusionsarealsodetectedwithSyn506andp62antibodies.Syn506isamousemonoclonal antibodythatconformationallydetects Sinclusions;andp62isarabbitpolyclonalantibody,whichnon-specificallyrecognizesintracellular proteinaggregates.Scalebar=100 m. Figure8 Detectionof SinclusionsinM20Tgmice8monthsafterPObraininjectionofexogenous Swithbothamino-terminal S antibodySNL-4andpSer129. Double-labeledimmunofluorescenceofmidbrainwithSNL-4(red)andpSer129(green)showsthatpSer129+ hyperphosphorylated SinclusionsareSNL-4+inasymptomatic15month-oldM83Tgmouse( A )and8month-oldM20Tgmiceneonatally injectedinthebrainwith25 g21 – 140fibrillar(fib) S( B )or25 g 71-82( C ).CellnucleiwerecounterstainedwithDAPI.Scalebar=100 m. Sacino etal.ActaNeuropathologicaCommunications 2013, 1 :38Page8of14 http://www.actaneurocomms.org/content/1/1/38

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shownforM20Tgmice2monthspost-injectionwith 25 gfibrillar S(Figure11Band11I).Attheseagessome oftheM20Tgmicewithorwithoutbrain Spathology alsorevealedamodestincreaseinastrogliosis.In8month oldnTgmiceinjectedwithfibrillar Sandwithmodest S pathology,onlyminimalinductionofastrocytesand microgliawasobserved(Figure11C,11D,11Jand11K). Conversely,in8montholdM20Tgmicewithsignificant Spathologyinducedbyneonatalinjectionof25 g fibrillar S,robustastrogliosisandmodestmicrogliosis wereobserved(Figure11E,11L).Furthermore,in8month oldM20Tgmiceinjectedwith25 g 71-82 S,there wasalsorobustastrogliosis(Figure11F,11G,11M,and 11N)regardlessofwhether Spathologyhaddeveloped. Thesefindingsindicatethattreatmentwithfibrillaror non-amyloidogenic Scaninduceadelayedactivationof neuroinflammationthatissignificantlyaccentuatedin M20TgmicerelativetonTgmice. Figure9 Co-localizationofp62with Sinclusionsin8month-oldM20Tgmicefollowingneonatalbraininjectionofexogenous S. Double-labeledimmunofluorescenceanalysisinthemidbrainregionforp62(red)andpSer129(green)showingthatmostpSer129+ hyperphosphorylated Sinclusionsarep62+inasymptomatic15month-oldM83Tgmouse( A )and8month-oldM20Tgmiceneonatally injectedinthebrainwith25 g21 – 140fibrillar(fib) S( B )or25 g 71-82( C ).CellnucleiwerecounterstainedwithDAPI.Scalebar=100 m. Figure10 Inductionof Spathologythroughouttheneuroaxis8monthsafterneonatalbraininjectionof25 g 71-82 SinM20Tg mice. TissuesectionswerestainedwithpSer129.Roundperikaryalinclusionsanddystrophicneuriteswerediffuselyspreadthroughoutthebrain andspinalcord.Themorerounded,Lewybody-likepathologywasseenpredominantlyinthemedialpreopticarea(MPA),striatum(ST),thalamus (TH),hypothalamus(HYP),substantianigra(SN),andventralpons(VP).Lewyneurite-likepathologyextendingintothecellularprocesseswas morepredominantlyseenintheentorhinalcortex(EC),amygdala(AMY),andCA3regionofthehippocampus(CA3).Tissuesectionswere counterstainedwithhematoxylin.Scalebars=100 m(MPA),50 m(EC),100 m(ST),100 m(AMY),200 m(CA3),50 m(TH),100 m(HYP), 50 m(SN),and100 m(VP). Sacino etal.ActaNeuropathologicaCommunications 2013, 1 :38Page9of14 http://www.actaneurocomms.org/content/1/1/38

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Toevaluatethepurity,biophysicalproperties,andintegrityoftherecombinant 71-82 S,weperformedMS analysis(Figure12).WeconfirmedbyK114fluorometry that 71-82 Sproteinwasnotamyloidogenicaspreviouslydescribed[35],andthattheadditionofexogenous 71-82 Scouldnotinduce Sinclusionformationin culturedcells[42]. Discussionandconclusion Ourstudiesdemonstratethatthebraininjectionofexogenous Scaninduceintraneuronal Spathologyafter prolongedincubationtimes.Withindaystheinjected S israpidlyclearedandtheinclusionpathologythatarises fromendogenouslyexpressed Stakesmonthstoform. ThesefindingsareconsistentwiththoseofMasudaSuzukakeandcolleagueswhoshowedthatexogenously injectedhuman Sfibrils(10 g)intothebrainsofnTg micecanbedetectedforlessthan1week,butinduction of Spathologyisobserved3monthslater[21].Allprior studiesofintraneuronalinductionof Sbycerebralchallengetoexogenousfibrilshavebeeninterpretedas beingindicativeofa “ prion-like ” spreadof Spathology [18,20,21].Indeedthedelayedinductionof Spathology byexogenous SobservedhereandbyMasuda-Suzukake etal.[21]maybeinterpretedasstable Sseedsthatare presentbelowdetectablelevels.Overtimetheseseedsinducepathology,whichthenmayspreadviaacycleofinclusionpathologygivingrisetoadditionalnucleationevents thatcanbespreadfromcelltocell.Ourfindingsthatan injectionofanon-amyloidogenicformof S( 71-82)can inducesimilardelayedpathologyindicatethatitmaybe prematuretoconcludethatthepathologyinducedissolely attributabletoconformationaldependenttemplating events.InboththeMasuda-Suzukakeetal.[21]andLuk etal.[20]studiesusingnTgmice,soluble Swasinjected ascontrolsandnoinductionofpathologywasreported.As wefindthatatahigherdoseof Sthereismorerobustinductionof Spathologybothintermsofextentofpathologyandtimetoonsetofpathologyinduction,andthat injectionof SinM20Tgmicealsoenhancestheresultant inclusionpathologyphenotyp e,itispossiblethatthelower doses(5 – 10 g)ofinjectedsoluble Sinthosestudies [20,21]mayaccountforthelackofpathologyinduction reported.Notably,asoluble Scontrolinjectionwasnot reportedinthestudydemonstratingpathologyinduction inadultM83Tgmice[18].Hereusingneonatal STg mice,weobservedthatinjectionofsoluble 71-82 Sis capableofinducing Spathologysimilartoamyloidogenic S.Inductionofrobustintraneuronal Spathologybyexogenous 71-82 Schallengedoesnotappeartobeattributabletotheneonatalinjectionparadigmaswehave observedsimilarfindingsinadultmice(Sacinoetal.,in preparation).ThefindingthatM20Tgmicearemore pronetoinclusionformationresultingfromtreatmentwith eitherexogenousfibrillar Sorsoluble 71-82 Sislikely duetoadosageeffectof Sexpression,whichcouldbe akintopatientswithduplicationortriplicationofthe SNCA gene.Althoughthecohortsofmiceusedhereare notlarge,theyarecomparabletothoseusedbyothersto Figure11 Delayedinductionofastrogliosisandmicrogliosisin miceneonatallyinjectedwithsoluble 71-82 Sorfibrillar S at8monthspost-injection. TissuesectionswerestainedwithGFAP antibody( A G ),whichdetectsastrocytes,andIBA-1antibody( H-N ), whichdetectsmicroglia.Representativeimagesweretakenofthe entorhinalcortex,whereahighdensityof Spathologytendstoform duetoneonatalinjection(seeFigures2,4,5and10).An8-month-old controluntreatedM20Tgmouse( A H )andaM20Tgmouseinjected with25 gof21 – 140fibrillar(fib) Sat2monthspost-injection( B I ) showbasallevelsofastrocytesandmicroglia.Therewasnosignificant increasefrombasallevelsinthebrainsofanTgmousewithpathology ( C J )relativetoasimilarnTgmousewithoutpathology( D K )at 8monthspost-injectionof25 gof21 – 140fibrillar S.Robust astrocyteandmicrogliaactivationasobserved8monthsafterinjection inM20Tgmicewithbrain Spathologytreatedwith25 gof21 – 140 fibrillar S( E L ).Inaddition,robustastrogliosiswasalsoobservedin M20Tgmice8monthsafterinjectionof25 gof 71-82 Swith ( F M ),orwithout( G N )brain Spathology.Tissuesectionswere counterstainedwithhematoxylin.Scalebar=50 m. Sacino etal.ActaNeuropathologicaCommunications 2013, 1 :38Page10of14 http://www.actaneurocomms.org/content/1/1/38

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studytheinductionofbrainpathologyusinginjectedexogenous S[18,20,21].Largercohortsofmiceinjected withvariousdosagesof Sarecurrentlybeingagedtolongertimepointstofurtherunderstandthemechanismsinvolvedinexogenous Sinductionofbrainpathology. Circumstantialevidencefrompost-mortemstudiesof thedistributionof SpathologyinthebrainsofPD patientsaswellastheinductionof Spathologyin transplantedneuronsinthebrainsofsomePDpatients hasbeenusedtosupportthehypothesisthat Spathology mayspreadfromcell-to-cellbya “ prion-like ” mechanism [13,14,46-48].However,manyalternativeexplanationsincludingchronicneuroinflammation,oxidativestresstriggeredbyexcitotoxicity,andlossofhomeostasisfrom cellularstress,mayleadtothefailureofmolecularchaperonesandothermachinerytoeffectivelycontrolthelevel ofmisfolded S[22-25]. Ourdatathatsolublenon-amyloidogenic Scaninducewidespread Spathologyraisesquestionsregarding the “ prion-like ” spreadofpathologythathasbeen reported,butitisprematuretoconcludethatourstudiesdefinitivelyrefutethatmechanism.Itisplausiblethat inthebrainanon-amyloidogenic Scouldbeconverted intoamyloidogenicseedsthroughadditionalmodificationsorinteractionswithlipidsorproteinchaperones. Thus,studiesthattrackthefateofinjectedexogenous Swillbenecessarytoevaluatethesepossibilities. Alternatively,thesestudiesdostronglysuggestthat othermechanism(s)ofinductionofpathologybyexogenous Sshouldbeconsidered.Forexample,there areextensivereportsonhowextracellular Scanleadto activationoftheinnateimmuneresponseviatoll-likereceptorpathwaysakintolipopolysaccharideactivation [49-62],andsingleintracerebellarorintraperitoneal injectionsoflipopolysaccharidehavebeenshownto resultinthelong-lastinginductionof Sneuronalinclusionformation[63,64].Importantly, Slackingresidues 71 – 82hasbeenshowntoinduceinflammationsimilarto full-length S[54,59].Inourcurrentstudy,wehave observedadelayed,long-termactivationofneuroinflammationinducedbybraintreatmenttobothsoluble andfibrillar SthatisaccentuatedinM20TgmicecomparedtonTgmice.Thesefindingsareconsistentwithcell culturestudiesthatshowedthatbothsolubleandaggregated Sarepotentactivatorsofinflammation[49-62].It ispossiblethattheexogenoustreatmentof Smaytrigger aslowpositivefeedbackloopofinflammationandsecretionfollowedbyaggregationthatmayrequireacertain thresholdofinflammationthatbuildsovertime.Therefore, someoftheM20Tgmicewithneuroinflammation,but without Sinclusions8monthsaftertreatmentwith 7182 Smaynotyethavereachedthenecessarythreshold. Exogenous 71-82 Smaynotbeaspotentaninducerof thisprocessasisfibrillar S,becauseitmayhaveashorter half-lifethanaggregated S,whichcouldexplainwhyit wasnotaspotentasfibrillar S;however,thispossibility willbeinvestigatedinfuturestudies.However,thehypothesisthatinflammationmayplayanimportantroleinthe spreadof Spathologyinducedbyexogenous Sisonly oneofseveralpossiblemechanismsthatmayactsynergisticallyorindependentlytopromotethespreadof Spathology[22-25]. Furthermore,thereisabundantevidencethatprionoid self-proteinaggregatesrepresentwhatarereferredtoimmunologicallyasDangerAssociatedMolecularPatterns (DAMPs)andarecapableofinducingrobustimmuneresponses[65].Anumberofstudiesshowthatwhen prionoidsassociatedwithCNSproteinopathiesareapplied Figure12 Massspectrometricanalysisofthe 71-82 Susedforneonatalbraininjection. Toverifythat 71-82 Swasthecorrectprotein anditsintegrity,weperformedmassspectrometryandcomparedthemolecularmasstofulllength S.Uppanel,recombinantfull-lengthhuman Swildtype;bottompanel,recombinanthuman 71-82 S. Sacino etal.ActaNeuropathologicaCommunications 2013, 1 :38Page11of14 http://www.actaneurocomms.org/content/1/1/38

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exogenouslytoglialcellsthey canactivateinnateimmunity throughpatternrecognitionreceptors(PRR)andinducea proinflammatoryresponse[65-68].Thisinnateimmuneresponseinturncouldtriggerin clusionpathology.Notably, thesemechanismsarenotmutuallyexclusiveandmaybe mutuallyself-reinforcing[25].Ourimpressionfromthese studiesisthatamyloidogenic Sisamoreefficientinducer ofpathologythanthenon-amyloidogenic S.Thisdifferentialefficiencycouldbeattributedtoamyriadofdifferent propertiesrangingfromstabilityoftheaggregated S,differentialabilitytotemplatepathology,immunogenicity,or somecombinationofthesefactors. Ourrecentstudiesinculturedcellprovidestrongevidencethatamyloidogenicconformationaltemplatingof Scanreadilyoccurundercertainconditions[42],but thesituation invivo islikelymorecomplexandaggregateformationcaninvolveseveralmechanisms.Tothis point,treatmentwith 71-82 Sdidnotinduce Saggregationinculturedcells,whilefibrillar Swasableto readilydoso.Alimitationofstudiesinculturecellsis thedurationoftime(afewweeks)thatthecellscanbe maintainedexperimentally,whichmaynotbeefficient tostudymechanismsthatareslowerandmoreprogressive.Collectivelythesedataindicatethatexposuretoexogenous Scaninduceintracellularaggregateformation byatleast2mechanismsthatarenotmutuallyexclusive andcouldlikelybesynergistic. Furtherstudieswillbeneededtodeterminetherelative contributionof “ prion-like ” proteinself-templatingversus othermechanismsintheinductionandpropagationof S pathology.Notably,theneonatalinjectionparadigmthat wehavedevelopedcanacceleratethesemechanisticstudiesbyreducingthetimeneededtoestablishcohorts ofmicenecessarytoconductthosestudies.Asnonamyloidogenic Scaninduce Spathologysimilartofibrillaramyloidogenic S,itispossiblethatanyformof braininjurythatpromotesreleaseofnormalcellular S couldtriggerintraneuronal Spathology.Extracellular S releasecouldalsooccurduringneurodegenerationwhen neuronsdieandthiscouldbeexacerbatediftheproteinis notclearedrapidly.Moredefinitiveelucidationofthe mechanism(s)thatunderlieinductionandspreadof S pathologyarelikelytoprovidekeyinsightsintoongoing effortsdesignedtotarget S invivo andtherebyultimately leadtonoveldiseasemodifyingtherapiesforPDandother -synucleinopathies.AdditionalfileAdditionalfile1:FigureS1. Lackofinductionof Spathology throughouttheneuroaxis8monthsafterneonatalbraininjectionof 25 g 71-82 SinM20Tgmice. Tissuesectionswerestainedwith pSer129.BrainregionsthattypicallyshowedLewybody/neurite-like pathologyafterinjectionof25 g 71-82 SinM20Tgmice,wereblankin anunaffectedmouse:themedialpreopticarea(MPA),striatum(ST), thalamus(TH),hypothalamus(HYP),substantianigra(SN),ventralpons(VP), entorhinalcortex(EC),amygdala(AMY),andCA3regionofthe hippocampus(CA3).Tissuesectionswerecounterstainedwithhematoxylin. Scalebars=100 m(MPA),50 m(EC),100 m(ST),100 m(AMY),200 m (CA3),50 m(TH), 100 m(HYP),50 m(SN),and100 m(VP). Competinginterest Theauthorsdeclarethattheyhavenoconflictofinterest. Authors ’ contributions ANSdesignedthestudy,performedtheinjections,analyzedthedata,and draftedthemanuscript.MBcollectedthesamplesandanalyzedthedata. 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