UnderstandingthePathologyofAlzheimer’s

DiseaseintheSubstantiaNigraoftheBrainstem

throughUnbiasedStereologyHimaRajana

LeaT.GrinbergLab

UCSFMemoryandAgingCenter

November2014

StructuredAbstract:

Background:Alzheimer’sDisease(AD)affectsmillionsofpeopleworldwide,and

withouragingpopulation,theprevalenceofADisonlyincreasing.Recentresearchshows

thatADpathologybeginsinthebrainstemevenbeforeclinicalsymptoms,suchasmemory

lossandimpairedcognition,appear.Thesubstantianigra(SN)isadopamineproducing

nucleusinthebrainstemcloselytiedtoothernucleithathaveshownchangesinearlyAD,

suchasthelocuscoeruleus.ThepresentstudyanalyzeschangesintheSNbywayoftau-

proteinaccumulation,ahallmarkofAD,inbrainsofearlyADpatientsusingunbiased

stereology.Methods:Humanbrainstemsfromsevensubjectsaged46-71withearlyAD

wereobtainedfromtheBrainBankoftheBrazilianAgingBrainStudyGroup,fixedin

celloidin,andprocessedusingimmunohistochemistryforunbiasedstereologicalanalysis

toquantitativelycharacterizethetau-proteinburden.Results:Althoughtherewasasmall

samplesizeofonlysevenbrains,thereappearstobeapositiverelationshipbetweentau

burdenandageofpatients,whichistrendingtowardsignificance.Therewasnocorrelation

betweenBraakstageandtauburden.ConclusionsandFurtherResearch:Thefindings

areinlinewithrecentlypublishedworkdescribinganincreaseintauburdenwithage,

independentofAD.However,thesmallsamplesizegivesuslittlepowerwhenmaking

conclusions,andforthisreason,animmediateexpansionofsamplesizeisnecessaryto

createanormativebaseforfurtherstudiesofearlyADintheSN.

I. Introduction

Asof2014,over5millionindividualsaged65andolderinAmericahavebeen

diagnosedwithAlzheimer’sdisease,alongwithanadditional200,000individualsunder65

diagnosedwithearly-onsetAlzheimer’sdisease(Alz.org).Currently,oneinninepeople,or

11%ofthepopulationaged65orolderhasorwillhaveAlzheimer’sdisease(Alz.org).By

2030,seniorcitizensareexpectedtomakeup19%oftheAmericanpopulation,compared

to12.4%intheyear2000,withthenumberofseniorsdoublingto72.1millionindividuals

(AdministrationonAging).Becauseoftheincreasingnumberofindividualsaged65and

olderintheUnitedStates,thenumberofnewcasesforAlzheimer’sandotherdementiasis

predictedtodoublebytheyear2050(Alz.org).

Aseveryindividualages,thebrainchanges,andtheybegintodevelopplaquesand

neurofibrillarytangles.Theprevalenceofplaquesandneurofibrillarytanglesin

Alzheimer’sdisease,however,ispurportedlymuchhigher(Hardy2002),andincreases

withtheprogressionofthedisease.Plaquesoccurwhenpiecesofthebeta-amyloidprotein,

whichisapartofthemembranesurroundingneurons,startstocomeoffthemembrane

andclumptogetheroutsidethecells.Sincethereisnogeneticchangeintheamyloidbeta

precursorprotein,weknowthatplaquesarecausedbytranscriptionalorposttranslational

changes(Selkoeet.all1988).Neurofibrillarytanglesarecausedbyhyperphosphorylated

tauproteinsinsidecells.ThetaugenecodesforRNA,whichinturn,codesforthetau

protein.Theproteinhasseveralepitopes,whichareactivatedandinactivatedby

phosphorylation.Thiscanbecomparedtothecockpitofaplane;eachofthemanybuttons

controlsaspecificfunction,andcanbeturnedonandoff.Theabnormallyfunctioningtau

protein,whichusuallysupportsthemicrotubules(MTs)inneurons,nolongerhasthesame

affinityfortheMTs(Geschwind2003).Subsequently,theMTsinneuronscollapse,

combinationofplaquesandtanglesimpedesnormalfunctionsincells,andeventually

precipitatescelldeath.

Thehyperphosphorylated,andsubsequentlymisfolded,tauproteinspreadsthroughout

thebrainfollowingacharacteristicpattern.TheBraakstagingsystem,initiallypublishedin

1991,isaqualitativemethodofcategorizingthedegreeofADpathologyinpostmortem

brainsfromstagesItoVI(BraakandBraak1991).Forthisstudyofchangesinthebrainin

earlyAD,brainswithBraakstages0toIIwereused.In1991,itwasestablishedthatthere

islittletonotau-positiveneurofibrillarytangleburdenintheneocortex,theupperpartof

thecerebralcortexinearlystages.Becausethecerebralcortexhousesareasdealingwith

cognitivefunction,suchaslanguage,memory,motorcommands,andspatiallanguage,the

disease-definingsymptomsofADarenotmanifesteduntilthelaterstages.TheBraak

stagingsystemisstillusedtoday,butwasreviewedandmodifiedin2011toinclude

changesinthelocuscoeruleus,asubcorticalnucleus,intheearlystages(Grinberget.al

2011).Thisexplainshowdiseasepathologydevelopsevenbeforeclinicalsymptoms

appear,andfurtherresearchcouldleadtoaneffectivetreatmentthattargetsADbeforeit

spreadsintotheneocortex.

TheneuronallossinADoriginatesinanareaofthebrainstemknownasthe

isodendriticcore.Theisodendriticcoreismadeupoffourmainnuclei:thedorsalraphe,

locuscoeruleus,parabrachialnucleus,,andthesubstantianigra(SN).Thesubstantianigra

isaffectedinseveralneurodegenerativediseases,includingintheearlystagesof

Parkinson’sdisease(Braak2003),andproducestheneurotransmitterdopamine,whichis

knowntoplayaroleinhappinessandthebrain’srewardsystem.Mostdopamine-producer

neuronsoftheSNalsoharborneuromelanin,apigmentthatcausesthecharacteristicdark

coloroftheSN.

Asthediseaseprogresses,thedegreeofdementiaiscloselyrelatedtoneuronallossin

ADpatients.Whilewedoknowthatneurofibrillarytanglesandneuronallossarehallmarks

ofAD,wedonotknowif,orhow,theyarerelated.Thereisagapofstudiesdoneofwell-

characterizedindividualstounderstandthetwolesionsinthecontextofoneanother.The

overallgoalofthelabthatthisstudywasconductedat,theGrinbergLab(Memoryand

AgingCenter,UCSF),istoquantitativelystudythisrelationshipbetweenproteinbuildup

andneuronallossthroughoutthefourmajornucleioftheisodendriticcoreusing

innovativemethods.Myproject,specifically,focusesonanucleusoftheisodendriticcore

knownasthesubstantianigra,andthisisfurtherdiscussedbelow.

DespitealltheeffortsandmoneytoputintounderstandingandcuringADoverthelast

threedecades,includingallkindsofsophisticatedexperimentalmodelsdesignedtomimic

thedisease,excellenttreatmentresultsondrugstriedintheseanimalmodelsandhuge

effortsinhumanclinicaltrialsusingthesamedrugs,weareyettofindsomethingtocureor

evendelaytheprogressionofAD.Atthispoint,wemustgobacktothefundamentalsofAD

pathology,andlookathowthediseaseprogressesinhumans.Becausetherehasbeenlittle

successintranslatingpositiveresultsfromanimalmodelstohumanmodelsbeyondearly

stageclinicaltrials,itisimportantthatwegainathoroughunderstandingofthewayAD

functionsinthehumanbrainbeforeidentifyingtherapeutictargetsanddeveloping

treatments.

Currently,littledataisavailableasabaselineofnormativechangesintheSNthrough

aging,sothisstudyseekstocreatealibraryofdataforthepathologyofADintheSN.

Additionally,thechangesintheSNduringaging,suchasthenaturaloccurrenceoftangles

andplaques,aswellasneuronalloss,arestillcontroversial.SincetheSNisbilateral,itis

alsonotknownifthebuild-upoftanglesandplaquesdiffersontheleftandrightsides

(Alho2014)

InlookingattheSNthroughthelensofAD,weemployadoublestainingtechniqueto

identifyboththetau-negativeneurons,stainedwithgallocyanine,andthetau-positive

neurons,stainedwithCP13toanalyzechangesintheSNwithageandwithprogressionof

AD(Theofilas2014).WelackdataonwhatcharacterizesthepathologyofearlyADinthe

brainstem.Theresultscontributetoabaseofknowledgeforimaging,clinical,and

anatomicalstudiesoftheSNintheearlystagesofAD.MyhypothesisisthatbecausetheSN

isapartoftheisodendriticcoreandconnectedtoothernucleioftheisodentriticcore

vulnerabletoAD,therewillalsobechangesintheSNinearlyAD.However,becauseof

whatisknownofBraakstages,wherethereislittleproteinburdenintheearlystagesof

AD,therewillbecorrelationbetweenageandtauburden.

II. Methods:

Priortomyanalysisofthetissueusingunbiasedstereology,thebrainstemwascutinto60

micrometersectionsandprocessedusingimmunohistochemistryandspecificbrainareas

weresampledaccordingtoBBBABSGprotocol(Grinbergetal.2007).Ashortsummaryof

thismethodfollows.

a. Participants:

Ageoftimeofdeath Case BraakStage Gender

44 7020.12 0 Female

46 9379.13 2 Female

47 7678.13 0 Female

56 9526.12 1 Male

70 6664.12 1 Female

71 6366.13 0 Male

71 6931.12 2 Female

Brainstemtissuefrom7individualsobtainedfromtheBBBABSGwasused.Grinbergand

colleagueshavedescribedtheBBBABSGprotocolindetail.

b.EmbeddingandSectioning

Thebrainstemwasembeddedincelloidintoreducetissuedistortion,byfirst

dehydratingitwithprogressivelystrongerethanolsolutionsandthengoingthrougha

dessicationprocesstostronglysecurethetissue.Whenenoughliquidhasbeenremoved

fromthecelloidintoreachanIndiarubberconsistency,theblocksweresectionedusinga

slidingmicrotome.Brainstemsweresectionedalternatelyintoone300micrometerand

five60micrometersections.Thethicksectionsaretheodd-numberedsections,usedto

calculateanunbiasedestimatefortheneuronalpopulationofthesixnucleiinthe

isodendriticcore.Theeven60micrometersections,usedtoevaluatethetauprotein

burdenineachofthenuclei.AcomparisonoftheburdenintheearlyoflatestagesofAD,as

wellaswithcontrols,isdoneinthe60micrometersections.Duringthecuttingprocess,

eachsectionwasphotographedusinganEOS5DMarkII.

b. StainingandImmunohistochemistry

`Thethin60micrometersectionswerefirststainedusingamonoclonalCP13

antibody,whichstainedforbothcytoplasmicboundandextracellularphosphorylatedtau.

Beyondthis,theywerecounterstainedovernightin2.0pHgallocyaninetostainthe

nucleoliandcreatefurthercontrastbetweentaupositiveneuronsandsurroundingcells.

Then,thesectionsweremountedtoslides,coverslipped,andlabeledbynumber.

c. StereologyintheSubstantiaNigra

Intermsofphysicalorientation,theSNisabilateralnucleuslocatedinthemidbrain,

adjacenttothecerebralpeduncles.Itisdividedintothreeparts:theparcompacta,parts

diffusa,andparsreticulata.Theparscompactahasthehighestneurondensityandismade

upofthelargest,mostpigmentedneurons.Theparsdiffusaisnotasdense,andhassmaller,

lesspigmentedneurons,althoughtherearesomeclustersofneuronsresemblingthose

foundintheparscompacta.Finally,theparsreticulatacontainsthickdendritesofthe

neuronsintheparscompactaregion,aswellasafewspread-outneuronslackingthe

neuromelaninpigment.

StereologicalanalyseswereperformedusingtheStereoInvestigatorprogram(MBF

StereoInvestigatorv.10,MBFBioscience,Williston,VT,USA).Thisset-upwascomprisedof

amotorizedstagesystem,whichallowedmovementofthestagetoviewadifferentsection

oftheslideifnecessary,abrightfieldmicroscope(AxioA2,ZeissMicroscopy,Thornwood,

NY,USA),andacolorcameratofacilitateuseofthemicroscopeonanexternalmonitor.

Figure1:Thestereologyset-uprequiresabrightfieldmicroscopewithamotorizedstageanda

motorizedstagecontrollertoadjustviewingframe.

Basedonpreviousstereologicalanalyses,itwasdeterminedthatcountingevery

otherevensection,asopposedtoeveryevensection,producedaccurateresults,sothis

processhasbeenimplementedinordertoincreaseefficiency.Priortobeginning

stereologicalanalysis,itisimperativetofindtheoptimalparameterstoestimateneuronal

numbers.Indoingso,thesubstantianigrawasfirstdelineatedusingtwoseparatecontours

forthetwoseparatesidesusingthe5x/0.16objective,asoutlinedintheoptical

fractionatorworkflowofStereoInvestigator.Theboundariesoftheregionofinterestwere

decidedbasedonvisualcuessuchasincreasinglyspareneuronsandtheAtlasofthe

CytoarchitectureoftheHumanBrainstem(OlszewskiandBaxter1982)CP13positivecells

weremarkedinthe40x/1.30oilobjective.BothobjectivesarefromZeissMicroscopy.The

cellswerecountedusingtheStereoInvestigatorOpticalFractionatorworkflow,as

describedinpreviousworks(SchmitzandHof2007).

d. Resample-OversampleProbe

Followingtheinitialcountingofcellsinthepilotstudy,theStereoInvestigator

Resample-Oversampleprobeisruntohelpdeterminetheoptimalparameters.Thegoalof

thepilotstudyistodeterminetheminimumcountingandsamplingrequiredtoensure

accuracyofsampling.Inotherwords,thegoalistocountenoughcellswithminimalwork.

Theresample-oversampleprocessbeginswithanexhaustiveanalysisofeachandevery

siteinthetissueinpredeterminedblocks.Afterthis,theprogramusesaformulato

determinetheparameterssuchthatthecounterisgettingthemostaccurateresultswith

theleastamountofworkpossible.

Intheoversampleparameters,thegridsizewasautomaticallygenerated,andthe

opticalfractionatortopguardzonewassetas5micrometersanddissectorheightat25

micrometers,basedontheminimumsectionthickness.Sectionthicknesswasmeasured

manuallyusingamotortogothroughthez-axiseachtimeaneuronwascounted,andthe

measureusedincalculationswasanaverageofallthicknessmeasurements.Newresample

parameterswereestablisheduponrunningtheprobeandevaluatingthedataplottedinthe

resampleoversamplegraph.

Figure2:Thisgraph,generatedusingtheResampleOversampleprobein

StereoInvestigator,wasgeneratedinMicrosoftExcel.Itshowshowclosethe

neuronalestimatesareincountingeveryintervalofdissectorsites,fromevery

sitetoevery20thsite.Accordingtothisgraph,theoptimalintervalisevery3rd

dissectorsite,becausetheestimatesusingone,two,andthreecountersare

closesttoeachother.Therefore,countingeverythirddissectorsitewillallowus

tomaintainaccuracywithminimalwork.

Thepilotstudyisrecountedaccordingtotheparametersgivenbytheresample

oversamplegraph,andthedataiscomparedtodeterminehowclosetheresampleand

oversampleestimatesofneuronalpopulationsare.Theoretically,theyshouldbethesame,

becausetheparametersarecalculatedsuchthatthesamelevelofaccuracyismaintained,

evenwithfewerdissectorsites.Iftheestimatesaresignificantlydifferent,theoversample-

resampleprocessisrepeateduntiltheestimatesarereliableandcanbeusedtocount

additionalcasesusingthesameparameters.

ACP13-negativeneuronisstainedlightbrownbecauseoftheneuromelanintheSN,

whilethenucleusandnucleolusarestainedbluebecauseofthegallocyaninestain.Only

neuronswherethenucleusandnucleolusareclearlyvisiblearecounted,becausethe

sectioningprocesscancutoffneuronsalongthez-axis,creatingfragmentsthatcanskew

neuronalestimatesifcountedasneurons.Additionally,usingthenucleolusandnucleusas

markersinstereologyisinlinewiththestainingtechniquesdescribedinthestaining

section.IncomparisontotheCP13-negativeneurons,CP13-postiveneuronsarecoveredin

adarkbrownblanketofCP13stain.Thestainingisapproximatelyuniformacrossthecell

Figure2:Thesubstantianigraistracedat5x.Thisimageisthecompleteright

sideofthenucleus.Thecharacteristicwavystructure,wherethecontourgoes

inandout,followingthelinesoftheSN,isvisiblehere.

body,andthenucleolusmaynotbevisible.Inmostcases,granulesarevisible.TheCP13

stainalsopenetratesintothecellprocessesastheneuronsinteractwithoneanother,

makingthedendritesoftheneuronclearlyvisibleastheyreachbeyondthecellbody.

Figure4:CP13-stainedneuron,whereCP13blanketsthecellevenlyand

granulesarevisible.ThepresenceofCP13stainincellprocessesdelineatesthe

dendritesoftheneuron.

Inordertoensurethateachneuroniscountedamaximumofonetimewhenthe

programplacesthedissectorrandomly,anyneuronsincontactwiththered-edgedpartof

thesquare(seebelowfigureforreference)werenotcounted,eveniftheymeetthecriteria

accordingtothepreviousparagraph.Anyneuronstouchingthegreen-edgedpartofthe

square,whethercompletelyorpartiallyinsidethesquare,arecounted.Additionally,

severaldissectorsintersectedwiththeborderofthecontour,asshownintheimagebelow.

Figure5:Thesubstantianigracontourisbrokenintomanycountingframes,andthe

dissectorsite,wheretheusercountsneurons,isconsistentlyinthebottomleft

corner.Thesmallerthecountingframe,themoredissectorsitestocount.

Figure5:Thegallocyanine-stainedneuronsaresmaller,andadarkerareaof

thenucleusandnucleolus,wherethestainisdeeper,isvisible.

A

At40x,itisquitedifficulttotellwherethedissectorisinrelationtothecontourasawhole

andrelativetothepositionoftheslide,sohavinganoverallmapofthecontoursindicating

wherethedissectoristellsushowthedissectorfallsonthecontour,andconsequently

whichneuronscanbecountedandwhichonesfalloutsidethepreviouslydelineated

contourisnecessary.TheMacroviewfeatureonStereologyinvestigatorhelpsovercome

thisissue,asshownintheimagebelow.

e. StatisticalAnalysis

Theresampleoversampleprobeusedaformulaembeddedintheprogramtocalculate

theappropriateintervalfortheresample.Thecoefficientsoferrorforthetau-positive

neuronalestimateswerecalculatedusingthepredictionmethodsfromthe

StereoInvestigator,theGundersenandSchmitz-Hof’scoefficientoferrormeasurements.

Figure6:TheMacroviewfeature,locatedintheupperlefthandcorner,shows

theentirecontour,withasmallerboxindicatingwherethedissectorsite,

displayedontherightsideat40x,isinrelationtothewholecontour.Becauseof

this,weknowtocountonlytheneuronsontheleftsideoftheorangecontour

line,sincetheyaretheonlyonesinsidethecontour.

Thesemeasurementsareusefulfordeterminingtheprecisionofneuronalestimates,and

havebeendescribedpreviously(Gundersenetal.1999;Schmitz,1998).Plannedstatistical

analysisforfurtherstudiesfollowsinthefurtherresearchsection.

III. Results

The60micometergallocyanine-stainedsectionsfulfilledthebasicrequirementsof

unbiaseddesign-basedstereology,whichhasproventobetheoptimalmethodforcell

countingtimeandagainforitsabilitiestodetectthemostminutecellgroupdifferences

(SchmitzandHof2005).Everycellinsidetheregionofinteresthadequalopportunitytobe

selectedforcountingfortwomainreasons.First,theserialsectioningofthebrainstem

allowedustoevaluatethefullthicknessoftheSNalongthez-axis,sowedidnotmissany

neurons.Second,theboundariesoftheSNwereeasilydetectable,andwewereableto

drawcontoursaroundallofthem.Althoughthesectionsweresupposedlyall60

micrometersthick,thicknesswasmeasuredtoaccountforanyshrinkageordiscrepancies

incutting.Thesectionthicknessofeachcasewasanaverageofthemeasuredsection

thicknessesoreachdissectorsites.Thishelpedaccountforanyfolding,waviness,or

Table1:Thistableshowsallofthedta

warpingofthetissue.Themeansectionthicknessacrossallcaseswas48.7micrometers

withastandarddeviationof0.28micrometers.

Throughtheresampleoversampleprobe,IdeterminedthatIcouldcounteverythird

dissectorsitewithoutlosingaccuracy.Duetotheexperimentalnatureofstereology,Ihad

toconducttheoversample-resamplefourtimesbeforereceivingsatisfactoryresults.Iwas

thenabletousetheseparameterstocountsixothercases.Originally,IcountednotCP13-

negativecells,inadditiontothedataofCP13positiveneuronspresentedinthispaper.

Unfortunately,therewereproblemswiththecounterstainintheimmunohistochemistry

process,sotheseresultshavebeendeemedunreliable.Boththeresample-oversampleand

theCP13-negativecellissueswillbeexplainedfurtherinthediscussionsection.

Ofthesevencasescounted,fourappearedtohavenotauburden,andtheremaining

threehadsimilarburdens.Afterblindcounting,theBraakstagesandageofpatientwere

matchedwiththedata,andwetestedforcorrelation.Basedonthisdata,thereisno

correlationbetweentauburdenandBraakstage,asallofthecasesthathadtauburdens

wereBraakstage0or1.Whilethesamplesizeistoosmalltoclaimacorrelationbetween

tauburdenandage,thereisapositivetrendbetweenageandtauburden.Outofthree

patientsolderthan65attimeofdeath,eachhadadifferentBraakstageof0,1,and2,but

twoofthethreeshowedsimilarpresenceofneurofibrillarytangles.Outofthe4patients

underage65attimeofdeath,only1patientshowedanytauburden,andthelevelwas

similartothetwopatientsover65.Thus,thepositivecorrelationbetweenageandtau

burdenistrendingtowardssignificance,andtheimmediatecourseofactionwouldbeto

expandthesamplesizeofthisstudy.Thiswouldallowustodetermineifthecorrelationis

simplytheresultofasmallsamplesizeorcanbesupportedwithfurtherdata.

IV. Discussion

Asourworldwidepopulationages,theprevalenceofneurodegenerativediseases,

especiallydementiaandAlzheimer’s,isincreasing.Atthesametime,thescientific

community’seffortstoworktowardsacure,oreventreatmentofAlzheimer’sdisease,have

beenstalledbythedifficulttransitionfromanimalmodelstohumanmodels.Something

abouthumanbrainsisdifferentenoughthatwhateversemblanceofprogressisachievedin

animalmodelsdoesnottranslatetohumanmodels.Forthisreason,itisnecessarytostudy

thewayADprogressesinhumansspecifically,becausethereisagapofknowledgethatcan

onlybefilledbyusingpostmortembraintissuehelpcharacterizeearlystagesofAD.

Currently,theseisalackofunbiaseddataonthesubstantianigra,akeynuceusofthe

isodendriticcore,knownchieflyforthedarkpigmentasaresultofdopamineproduction.

Althoughweknowofsomechangesintheothernucleioftheisodendriticcore,including

thelocuscoeruleus,littleisknownabouttheSNinthecontextofAD.Becauseoneofthe

hallmarksofADistauproteinbuildup,weanalyzedproteinbuildupintheSNusing

unbiasedstereologyinapost-mortemsampleof7adultsaged46-71attimeofdeath.

Quantitativeneuropathologicalmethodsaretimeconsuming,tediousandusuallybiased

duetotheenourmousamontofneuronsinthebrain.Design-basedstereologyis

transformingthewayquantitativeneuropathologyisperformed,allowingustomake

predictionsfortheneuronnumberanddensityofatissuewithoutcountingeachandevery

neuronthroughanestablishmentofparametersusingaresample-oversampleprocess.In

addition,therandomnessofstereologyallowsustomakeunbiasedmeasurementsandbe

abletotrustinthevalidityofresults.

Althoughthereseemedtobenodiscernablerelationshipbetweentauburdenand

Braakstage,theredoesappeartobeapositiverelationshipbetweenageandtauburden,

trendingtowardsignificance.Thisisinlinewithaveryrecentlypublishedpaperontau

buildupasrelatedtoageinthebrainstem.Craryandcolleaguessuggesttheuseofanew

term:primaryage-relatedtauopathy(PART)todescribetaubuildupinthebrainthat

seemstobetiedonlytoage(Craryet.al.2014).Pathologically,thiscanbedistinguished

fromAD,frontotemporaldementia,orotherneurodegenerativediseasesbythelackofthe

beta-amyloidplaques.Althoughthelackofbeta-amyloidplaquespointsoadiagnosesis

earlyAD,therearesomeclinicaldifferences,sincePARTusuallyhasalessercognitive

impact.LookingatPARTthroughthelensofAD,Craryandcolleaguesfoundthatmany

patientswithmild-to-moderateneurofibrillaryburdensimilartoearlystageADlackedthe

betaamyloidplaquescharacteristicofAD.Thus,theneurofibrillarytanglesmaybe

involvedinanonADagingrelatedprocess.TheincidenceofPARTwasmuchhigherin

olderpatients,whichsupportsthepossiblepositiverelationshipbetweentauburdenand

age.

EventhoughmyfindingsaresupportedbyCrary’swork,itisdifficulttoconfirm

anythingworkingwithsuchasmallsamplesize,especiallyinhumans.Humansaresuch

diverse,varied,anduniquebeingsthatvariationbecomesnottheexceptionbutthenorm.

Forthisreason,manycasesarenecessarytodrawageneralconclusionthatcanbeapplied

totheentirepopulation,andthisstudydoesnothavethehighsamplesize.Becauseofthe

gapofknowledgeconsideringthesubstantianigraandAD,weareunabletocalculatethe

exactnumberofcasesneededtohavepowerintermsofstatistics.Thebiggestreasonfor

thesmallsamplesizeinthisstudyisthedifficultyofprocuringhumanbrainswiththeearly

ADdiagnosis.Thiskindofanalysiscanonlybedonepostmortem,andprocessingthe

brainsforstereologyinhumanmodelstakesaverylongtimecomparedtoratbrains,

whicharemuchsmaller.Itisstillimportanttostudythisinhumansbecausethedisease

manifestsitselfinhumans.Therefore,themaingoalofthisstudyisnotnecessarilytomake

sweepingconclusionaboutthenatureofADintheSN,butrathertobuildaknowledgebase

tocharacterizetheSNinearlyAD.AsIwritethispaper,Iamintheprocessofincreasing

samplesize,andbytheendofthiswinter,Ishouldhavearound20cases,whichgivesmuch

morepowerintermsofbeingrepresentativeofearlyADbrains,thanseven.

Stereologyisanexperimentalprocessinandofitself,becausetheResample

Oversampleprobeallowstheusertotailortheprogramtothetypeoftissuebeingcounted.

Priortoanystereologicalanalysisofmultiplecases,itisnecessarytorunacomplexpilot

studytoensurethatresultsarenotonlyreliable,butcanalsobereplicatedinotherlabs.As

mentionedintheresultssections,Ihadtoconducttheoversampleprobefourtimestoget

satisfactoryresults.ThealgorithminStereoInvestigatorrecommendedthatIcountevery

thirddissectorsitethreetimesinallfouroccurrences.Theoretically,theoverallneuronal

estimatesfortheoversampleandtheresampleshouldbethesame,butthatwasn’tthecase

intheinitialoversample.Throughouttheprocess,issueswithinconsistentneuron

estimates,inconsistentthicknessmeasurements,andinaccuratelydrawncontours

promptedmetoconducttheanalysisagain.Finally,onthefourthround,Ihadreliable

parameters,whichIthenusedtocountsixadditionalcases.

IoriginallycountedbothCP13positiveandCP13negativeneurons.Uponreviewofthe

resultsandcomparisonstootherstereologicalestimatesofsubstantianigraneuron

population,mynumberswereonly10-20%ofothers’results.Weevaluatedmycriteriafor

countingneuronsandthecountingparametersandeventuallyconcludedthatthe

inaccurateresultswerearesultofafaultycounterstain.AftertheCP13stainwasappliedto

thesections,theywerecounterstainedinagallocyaninewashtoilluminatetheCP13

negativeneurons.The60micrometersectionsmayhavebeenslightlytoothinforthe

harshchemicals,ormayhavereactednegativelytothetestedconcentrationofdetergentin

thesolution.Inordertoaccountforthisinfurtherstudies,neuronalestimateswillnowbe

measuringfromonlythethick300micrometersectionsofeachcase.Asofnow,the

stainingandstereologyprotocolhasbeenoptimizedforthe300micrometersections,with

consistentlyreliableresults.Aswehavelearnedfromthisstudy,theCP13staindone

throughimmunohistochemistryworkswellonthethin60micrometersections.Asperthe

cuttingprotocolfollowedbytheGrinberglab,300micrometersectionsand60micrometer

sectionsarecutalternately,sothe300micrometersectionsareanaccuraterepresentation

ofthe60micrometersections.Inordertounderstandtherelationshipanddynamic

betweenneuronallossandtauproteinaccumulation,wewillnowutilizeacombinationof

datafromthe300micrometersections,forneuronalestimates,and60micrometer

sections,forproteinburdenestimates.

V. ConclusionsandFurtherResearch

Thisstudywasaninvestigationofthehumansubstantianigraintheearlystagesof

AD.Thereisapositivetrendbetweenageandtauproteinaccumulation,andasofnow,no

relationshipbetweenBraakstageandtauburden.However,thesmallsamplesizegivesus

verylittlepowerinmakingapplicableconclusions.Theimmediatenextstepistoincrease

thesamplesizeinordertorunappropriatestatisticalanalysis.Theanalysiswilltestthe

individualcorrelationsbycomparinganatomicalchanges,suchasinclusionburden,with

ageandBraakstageusinglinearregressionanalysis.Theregressionmodelswillinclude

indicatorsforADgroupaswellasageatdeath.

Additionally,inordertoinvestigatetheroleofthesubstantianigrainAD,and

perhapslookfurtherintothestillundiscoveredrelationshipbetweenSNproteinburden

andBraakstage,wecanuseneuronalestimatesfromthe300micrometersections.

Comparingtheoverallneuronalestimatestothetau-positiveneuronestimateswould

enableustocalculateafractionoftheneuronsintheSNaretaupositiveatdifferentstages

ofearlyAD.ThisfractioncouldserveasametricusedtocharacterizeeachstageofADand

understandthepathwaythatADtakesthroughtheSN.Beyondthis,comparingthedata

fromtheSNtoconclusionsdrawninothernucleioftheisodendriticcorecouldshedlight

onADthroughoutthebrainstem,leadingtopotentialtherapeutictargetsfordrug

development.

Bibliography

"AdministrationonAging(AoA)."AgingStatistics.AdministrationforCommunityLiving,31

Dec.2000.Web.09Nov.2014.

Alho,AnaTerezaDiLorenzo."Three-dimensionalandStereologicalCharacterizationofthe

HumanSubstantiaNigraduringAging."(2014):n.pag.Web.2Nov.2014.

Association,Alzheimer's."2014Alzheimer’sDiseaseFactsandFigures."Alzheimer’s&

Dementia10.2(2014):n.pag.Alz.org.Alzheimer'sAssociation.Web.21Sept.

2014.

Braak,H.,andE.Braak."NeuropathologicalStageingofAlzheimer-relatedChanges."Acta

Neuropathologica82.4(1991):239-59.Web.21Oct.2014.

Braak,Heiko,K.DelTredi,U.Rub,andRADeVos."StagingofBrainPathologyRelatedto

SporadicParkinson'sDisease."TheNeurobiologyofAging24.2(2003):197-211.

Web.7Oct.2014.

Buttner-Ennever,JeanA.,A.K.E.Horn,andJerzyOlszewski.OlszewskiandBaxter's

CytoarchitectureoftheHumanBrainstem.N.p.:Karger,n.d.Print.

Crary,JohnF.,JohnQ.Trojanowski,JulieA.Scheider,andJoseF.Abisambra."PrimaryAge-

relatedTauopathy(PART):ACommonPathologyAssociatedwithHumanAging."

ActaNeuropathologica(2014):n.pag.Web.1Nov.2014.

Geschwind,DanielH."TauPhosphorylation,Tangles,andNeurodegeneration:TheChicken

ortheEgg?"Neuron40(2003):457-60.Web.15Sept.2014.

Grinberg,LeaTenenholz,RenataEloahLucenaFerretti,JoséMarceloFarfel,RenataLeite,

CarlosAugustoPasqualucci,SérgioRosemberg,RicardoNitrini,PauloHilário

NascimentoSaldiva,andWilsonJacobFilho."BrainBankoftheBrazilianAging

BrainStudyGroup—aMilestoneReachedandMorethan1,600CollectedBrains."

CellandTissueBanking8.2(2007):151-62.Web.18Sept.2014.

Grinberg,LeaTenenholz,UdoRueb,andHelmutHeinsen."Brainstem:NeglectedLocusin

NeurodegenerativeDiseases."FrontiersinNeurology2(2011):n.pag.Web.3Oct.

2014.

Gundersen,H.J.G.,E.B.V.Jensen,K.Kieu,andJ.Nielsen."TheEfficiencyofSystematic

SamplinginStereology-Reconsidered."JournalofMicroscopy193.3(1999):199-

211.Web.1Nov.2014.

Hardy,J."TheAmyloidHypothesisofAlzheimer'sDisease:ProgressandProblemsonthe

RoadtoTherapeutics."Science297.5580(2002):353-56.Web.10Sept.2014.

Kazee,AnnMarie,ChristopherCox,andEricK.Richfield."SubstantiaNigraLesionsin

AlzheimerDiseaseandNormalAging."AlzheimerDisease&AssociatedDisorders

9.2(1995):61-67.Web.19Oct.2014.

"MedicalNeurosciences."MedicalNeurosciences.UniversityofWisconsinMadison,n.d.

Web.13Oct.2014.

Schmitz,C.,andP.r.Hof."Design-basedStereologyinNeuroscience."Neuroscience130.4

(2005):813-31.Web.29Oct.2014.

Schmitz,Christoph."VariationofFractionatorEstimatesandItsPrediction."Anatomyand

Embryology198.5(1998):371-97.Web.1Nov.2014.

Selkoe,DennisJ."F3-AmyloidPrecursorProteinofAlzheimerDiseaseOccursas110-to

135-kilodaltonMembrane-associatedProteinsinNeuralandNonneuralTissues."

MedicalSciences85(1988):7341-345.Web.14Oct.2014.

Theofilas,Panos,LiviaPolichiso,XuehuaWang,LuziaC.Lima,AnaT.l.Alho,RenataE.p.

Leite,ClaudiaK.Suemoto,CarlosA.Pasqualucci,WilsonJacob-Filho,Helmut

Heinsen,andLeaT.Grinberg."ANovelApproachforIntegrativeStudieson

NeurodegenerativeDiseasesinHumanBrains."JournalofNeuroscienceMethods

226(2014):171-83.Web.27Sept.2014.

Yoshiyama,Yasumasa,BinZhang,andJenniferBruce."ReductionofDetyrosinated

MicrotubulesandGolgiFragmentationAreLinkedtoTau-inducedDegeneration

inAstrocytes."JournalofNeuroscience23.33(2003):10662-0671.Web.16Aug.

2014.