II II

III II II ^III II III III III

:

II III

lila

II II

St. John' s

I-IspBIProtectio nagainstArnylo id-BToxicity

By

([)Megan G. Conway (B.Sc.)

Athesis submitted tothe Schoo lof GraduateSt ud ies in partialfulfillmentof the requirementsforthedegreeof M.Sc.inMedicine(Neuroscie nces)

Division ofBiomedical Sciences Facultyof Medicine Memor ialUn iversityorNew fou nd land

January 20 12

Ne wfo und landand Labrador

Abstract

Alzhe imer'sdisease (AD) ishallmarkedbythe presenceofn euro fibrillarytanglesand amyloid-]! plaques.Amyloid-I Iplaques consistoftheamyloid-f) peptid e(AI))thathas beenshown to inducetoxieclkctsonneurons through theactivationofstress-re lated signallingand neuronalloss.The small heat shock protein. IIspBl(alsoreferredto as Hsp27and Hsp25 in mouse). is accumulatedin15%ofn eocort icalamyloid-]!plaquesin AD brains(Wilhclmus. 2( 06).Whetherthisrepresents apotentiallyprotective response to stress.or ispart ofthe disease processisunknown.Wehave previouslyreportedthat express io nof IlspBI not onlyprotects co rt ica lneuronsaga inst amyloidtox icity.butalso enhancestotalneurite growth in these neurons(King£'1al..2( 09 ).

Theamyloid-f) peptide isderivedfrom theproteolyticprocessingof theAmyloid PrecursorProtein(API')byf)-andy-sccrctascs.Mutationsin API' altersecretase cleavagesites. resultinginhigherproductionofthetoxic Af)(1-42) peptidethat undergoesaggre gationmore readily.Since Hspl3lhasbeenshown toprotectneurons againstamylo id toxicit y.itisconceivablethat HspB1mayinteractdirectlywith AI)or API'.Recent studieshavedemonstratedexogenousIlspBIbinding to synthetic Afl.We have replicated theseresultsin an attemptto determine the roleof HspBI in theinclusion of AI)aggregates.Wehypothesize thatHspl3l interactswithAftor its precursor API'.to eitheralter the distribution of AfVAl'1'withinthece ll.or itsreleasefrom the cell.

In order totest our hypothesis,weincubated His-tuggedHspB I withsynthetic Afl(1-42).

at physiologicaltemperature37°Covernight.lmmunopr ccipitation.using agaro sc protein A/Gbeadsincubatedwith themonoclonalanti-Hisprimary antibody.was used fill'ourprimary analysisofinteraction. Westernblotting ofthe nitrocellulosemembrane.

usingthe 6ElOp-amylo id (1-16) mo use monoclonalantibody.demonstrates thatAll is immunoprccipitatcdwithHis-HspBI, These resultspoint to a direct interactio nbetween IlspB1 andA[l(1-42). Wcinvestigatcd theinteractionofHspBIwithAPI' inIIEK293 cell line express ingwild-typeAPI' (AI'I'-wl) or AI'I'-swcd ish mutation(AI'I'-swclthat predom inate lyyieldsAII(1-42)through immunoprccipitation,His-tuggedHspBIwas incubated withthe conditio nedmediafro mthe AI'I'-wtandAI'I'-swccellsovernightat 37°C.lmmuno prcc ipitai io n wasperformed using magneticproteinA/Gbeads incubated with either 6E 10p-amyloid (1-16)mouse monoclonalantibody.or anti-HspBI human (SI'A-XOJ)rabbit polyclonal antibody.Subsequentwesternblottingofthe nitrocellulose memb raneusingthe 6EI0 and theXOJantibod icsdemonstratethat API' is immunoprccipitatcdwith His-HspBI.

These data suggestlispS I.perhaps via itschaperone activity.mayhealtering production of API'and/orAllwithin thecCIIpreventing secretion intothe extracellular env iro nments.

Acknow ledgeme nts

Thisworkwouldnot have beenpossiblewithouttheguida nce from mysupervisor.Dr.

KarenMcarow,Havingminimalexperience andknowledge oft he researchworld.I entered herlab asagraduate studentto lindan ambitio usatmosphere whereprecisehigh qualityresearchshinesthro ugh.Hcr constantstrivelorSl Kl:CSShasmadeherboth a rcmarkablc rcscardlcrand tcad lcrand Iam pro ud to havcstudicd undcr hcrsupcrvision.

Ithankyou Karenfill'providing me with this opportunity.

Tomysuperv isorycommittee.Dr. XihuaChen andDr. QiYuan. thankyou forthe support, encour agementandadvice,Possiblywithout knowing. youmademycommittee meetingslessdaunt ingthan the average graduate student.

Icannotsay enoughto FiroozchNafar,our lab technician,for herend lessinstr uctio nand countless hourshelpingme with thisresearch, l lcr insightandpositive attitudewere vcrywell recei vedon dayswhen the resultswerenot. Firoozch.Iam eternallygrate ful for yoursuppo rt.

Ialsowould like to thank mylabmatesfortheiradvice andsupport. Joe.Tor theplas mid work.witho ut Iwould notbe ableto complete thisproject andNeva forher technical advice.You both broughtlim andlaughter into thelab and it hasbeen a pleasure workingbesi deyou.

Outsideof the Mcarowlab Iha vetothankDr. Jackie Vande rl u itfill'organi zin gJournal Club withsuc h exciteme nt.Your eagernesstosharesc ie nce inspired me to broaden my vie w andsho wmycreativ ity.

Finallyto myfamil ywhohavesho w n undyin glo veandsupportthroughmyMasters programesp ec iall ymyparentsforthei rcont inuo us beliefin my ab ility.ToDa rcy.Ihope Ihaveove rco methe brick wall sproudly.To mybeautifuldaughter Syd neywhohas insp ired me everydayto strivetill'excellence,As yo ugro w older. sec thisasa testament to thepe rso n you ha ve helpe d me become. athankyo u. andawishfill'your future.

ForDad

Your guiding handonlUI"shoulderwillremain withmeforever.

TableofContents

Abstract...

Acknowledgements...

l.ist ofTablcs...

List of Figures...

Listof Abbrevia tio ns...

ChapterI:Introduction

1.1 FamilialAlzheimer's Disease...

1.2 CurrentHypotheses... . 1.2.1 Amyloid Cascade Hypothesis...

1.2.2 Hypcrphospho rylatcd TauHypothesis . 1.2.3 Inllamm at ion andoxidativestress inAD. 1.3 Amyloid PrecursorProtein...

1.3.1 Proico lyticprocessing of Amyloid PrecursorProtein...

1.3.2 Intracell ulartraffickingoftheAmyloid Precurso r Protein...

. ii

...iv

. xi

. xii

. 17

. 19

. 19

. 20

...23 ...25 ...27

...29

I.4Amylo id - r~Production andTox ic i ty 32 1.511catShockProtein beta- I (HspBI)inthe Brain 36

1.5.1 HspBI as achaperone protein... . 37

1.6 Alzheimer'sDisease andHspBI .41

1.7Proposed Therapies .42

I.XSummary... . n

1.9Hypothesis .45

1.10 Objectives... . .45

Chapter2:Materialsand Mcthods,.. . 52

2.1 HEK293Ce llCultureandConditioned Media... . 52

2.2Plasmid s andTrans!Cction... . 53

2.3Protein andConditio ned MediaCo llection... ....54

2.4 WesternB lolling... . 54

2.5Co-Inununoprccipitution.r.... . 56

2.6ELISA... . 5X

2.7Immunocytochem istry 60

2.XCo-localization Analysis... . (1 1

2.9StatisticalAnalysis 62

2.10Constructs....

Chapter 3:Results...

.. 63

. ...67

3.1Endog enous/exogeno us expressionofHspB I.APPwt and APPswein

HEK293naivecells ..clX

3.2Soluble API'(sAPP) is secreted intoconditionedmedia ofco-tra ns fcc tcdHEK

293cells ,(1)

3.3 Endogeno us/exogeno us expressionof HspBI in HEK 293 APPwt and APPswe

stable cells 70

3.4 AbsenceofAr~expressionin cellular lysateof AI'I'\\'tand Al' Pswcstable cell

3.5Increasedprotein expressionofA~( 1-42)aft er4X hrsisnotseen in the presence

ofl lspB I 72

3.6 SyntheticA~(1-42)peptideand recombinantHis-HspBIpeptideco-

irn munoprccipitatc... .. 73

3.7Recomb inantHis-HspBI co-innnunoprccipitutcswithso luble API'alpha 74

3.XDocsHspBI appcarto dccrcasc thcaccumulationofA PP products within

cells? ..

3.9Docs/-IspB I affect the productionandrelea seofa my lo idog cnic proteol ytic ...95

produ ctsofAf'Pswe?... . 95

3.9.1Expressio n ofmature andimmatureAPI' remainsunchangedin theprcscnccof

IIspBIinboth APPwtandAl' Pswccells... . 96

3.9.2CT FBle velsremainconstantwhenIIspB I isexpressedin llEK2()3 Al' Pwt/swc

sta ble cells.... ...97

3.9.3Isrelease ofs o lubleAPI' influenced bythe presenceof lls pB1 inAl'Pswc ce lls? ..

3.IOC cllular localizationof API'andIlsPB I...

Chaptcr 4: Discussion...

4.1TheSignifi ca nceof This Study...

4.2HEK293 CellsLackEndog eno usIIspBI....

.. 97

. 9X

... ...117

. ...117

...1IX

4.3DecreasedRelease of AI)(1-42)fromAPP-swc CellswithIIspl3l 120

4,4IlspB I DirectlylntcractswithAf)(1-42)... .. 122

4.5IIspB1 Dircctlylntcractswith AmyloidPrecursor Protcin... ... 124

4.6Hspl3ldoesnotAffect ProcessingofAPP-s\\'~... . 124

4.7CellularCo- LocalizationofHspl31and API' Products... . 12()

4.1' Future Directions 127

Referen ces 129

ListofT ables

Tahle I Identifiedfunctions ofAPPproducts

Listof Figures

Fig u rc 1.1ProposedAmyloidCascadeHypothesis

Fig u rc 1.2Proposed Alzheimer's diseasehypotheses

Figure 1.3Arny loidog cnicversusNon-Amyloidog cnic processing of API'

Figu l'c1,4Traffickingof API'

Figure 1.5Possible effects ofHsps onncurodcgcnerativcdiseases

Figu rc2. 1Schematic of constructs

Figure2.2SchematicofAPI'antibodyrecog nitionsites

Fig u rc2.3Experimental Model

Figu rc3. 1TransfcctionEtliciencyofA PPw!,APPswe.EGFP-llspl3l-WtandEGFI'- EVC2 in naiveIIEK293cells

Fig u rc3.2Expressionofco-tra nsfcct cdAPI'",!,Al' PswcandEGFI'-HspB l- Wt fusion proteinin naiveIIEK 293cells

Figul'c3.3Expressio nofco-trans fcc tio nofnaiveIIEK 293 cellswithEGFI'- HspB l- Wt andAl' PwtorAl'Psw cconstruct sby immunocyt ochemistry

Figu rc3,4Levels of API' expressionincellular lysate andconditioned media ofnaive HEK 293 cellstransfcctcdwithAl' l'wtandAl' Pswcconstructover 72hrs

Figu re3.5Express ion oftruns fectcdpCIG-HspBI in Al' l'w tandAl' Pswc stable cell lines

Fi~lI rc3.6Express ion ofi lEK293stable Al'Pwtand Al'Pswc stable cell lines transfc ctcdwith pCIG-H aHspB1-WT- IRES- EG FI'byimmunocytoc hemistry

Figu r e 3.7Abse nceof AI\expressionin cellular lysate of AI'I'\\tandAl' Pswc stable cell lines

Fi~lI l'c3.8 Increasedproteinexpressionof AI\(J-42) after 4Xhrsisnot seen inthe prcscn ccofl lsplj l

Fi ~lIrc3.9Synth eti c

AI\

Fi~ lI rc3.1() Hsp B 1successfully co-immu noprccipitutcswithsoluble API'incellular condi t ionedmedia

Fi~IJrc3. 11 Immunocytochemi stry displayingdifferencesin cellular accumulatio n of API' in Al'Pswcstablecellstransfcc tcdwith pEG FI'-C2-HspBI-Wt

Figur e 3.12Processing ofAPI'in IIEK 293Al'PwtandAl' Pswestable cell lines translc ctcdwith pCIG-HspBIconstruct

Fi ~lI rc3.13SolubleAPI'levelsin pCIG-llspl3l positiveconditio nedmedia

FigurcLt -tlntraccllularMarkcrs

FigureJ.15Co-localizatio nanalysesof APPw!.Al' Pswcand Hspl3lwithinintracellular compartments.

AD ADAl\I lII ADDL

AIC D Akt APOE-I:~

APP Ask l ATP All BAC EI BBB BcI-2 Bip/GRP 7R Bi\I E BSA cAi\IP

Cdk-S CM

CNS CSF

List of Abbreviations

Alzheimer'sdisease alphasccretasc enzyme amyloidderiveddiffusibleligand API' intracellulardomain proteinkinaseB E4typeofa polipoprotein amyloid precursor protein apoptosis signal-rcgulating kinaseI adcnos inc tri-phosphatc amyloid beta beta sccrctasc enzyme bloodbrainbarrier B ccll lymphorna-Z glucose Regulator yProtein bctamcrcaptocthanol bovine serum albumin cyclic-adenos ine monophosphatc cyclin-dcpcndcnt protein kinase5 conditioned media central nervoussystem cerebral spina l fluid

CTFu CTFII DAPI DDT DLB Di\I Ei\1 DRC EC ECL ECFP ELISA ER ER K EV FAD Fas

FCS FTD P C4 ' X

CSK-J II IIEK IIRP

Cv tcrminalfragmentalpha Cvtcrminal fragme ntbeta nucleic acid sta in

dithithrcitol dcmcutinwithlcwybodies dulbcccosmodified eagle ' smedium dorsalrootganglion cntorhinalcortcx enzymatic chemiluminescence enha ncedgre en fluorescentprotein

enz ymelinked immunosorbantassay endoplasmic reticulum extrace llularsignalregulatedkinascs em ptyvector

familialalz hc im crs discasc

apoptosisantigenI/CD95/T NFRFS6 fet al cowserum

fronto -temporaldis ea sewith parkinsonism gcncticin disulfatc

glycog ensynthas e kinase3 beta humanembryonickidneycells horseradishperoxid ase

liS IIsf- 1 Hsp IC C II>E

IL-I/IL-6 II'

.INK kl>a

LTI>

LTP MA PKA PK2 I\IBI>

i\Ii\II}2/9 NF-kB NFT Ni\II>A NSAII>S

NTFu NTF'I PJX/i\IAPK PBS

heat shock

heat shock factorI heat shock protein immunocytochemistry insuli n degradingenzyme intcr lcukin- L'o

immunopr ccipi tation jun-N-terminalkinase kilodalton lon gterm depression long term potenti ation

mitogenact ivated proteinkinase-activate d protein microtubulebindingdomain

nuuri xmctallo protcinascs Zzv nuclear fact ork-bcra neurofibr illarytangles

N-mct hyl-/J-aspartate non-steroidalanti-infla nuna tory drugs Nvtcrminalfrngmcu t alpha Nvtcrminalfragmcntbeta p3X-mitogen activated proteinkinase phospha tebuffered saline

xvii

PF PKA PrP PS-I PS-2 SAU sAPPIl sAPPII-swc sAPPII-wt SUS SUS- PAGE

slls p SP SWE TBS TBST Tg TGN Tip61l TNF-Il WT

protolih ril protein kinaseA priouprotcin

prcscnilin I sccrctascenzyme prcscn ilin 2 sccrctasc cnzymc sporadicalzhcimcrs disease solubleamy lo id precursorproteinalpha solubleamyloidprecurso rproteinbctaswedish mutation solubleamylo idprecursor proteinbcta wildtype sodium dodccyl sulfate

sodium dodccyl sulphate-polyacrylamide gcl electroph oresi s

small heatshockprotein senileplaques swedish mutation trisbufferedsaline trisbuffcrcdsalincO.5'Yotwccn transgenicmice trans go lginetwork histoneucctyltransferasc tumor necrosisfactoralpha wild type

ChapterI

Introduction

1.1 FamilialAlzheimer's Disease

In25years. the now 500.000Canadiansaffected by ADwill do uble asthe first"baby boomer" turns 65 thisyear.Hcalthcarc costswillincrease tenfold fromS15 millionper annumtoS153million.While thewell-know nsporadic lim nismore common.4-5'X,or the reportedADcases arcunder age65presentingwith the familial fo rm of AD.

Diagnosisin these genetically predisposedindividual scanoccuras young as30yearsof age(www.alzheimer.ca). Early-onsetFamilialAD(FAD)sympto matically present s indistinguishablyfromsporadic orlate-onset Alzheimer'sdisease (SAD).Clinica lly.

both arc characterizedbyprogressiveneuronaldeterioration resultingin lossofme mory, spatialorientat ion. language defic its.mood as wellaspersonalitychanges(Mo rrisctal..

19X9).Physiologicallyhowever.thereexist differences in diseaseoriginbetween AD types.FAD isanautosomal dominant genetic formof AD.OCCUlTing throughmutations that usuallyresultin increased depositionofthe cytotoxicAmyloidbeta I-·Q (A]] 1-42) liagmcnt(A.Eckertctal..2003:A. Eckert.Marques,Kcil,Schusscl,&Muller.2003:

Citron ct al.,19l)2:Weidem ann ctal.,1( 97).Three well studied mutationsidentifiedin FAD arc:I) prcscnilin- Ion chromosome14(Alzheimer'sDiseaseCollaborative Group.

1(95) . 2)presenilin-Zon chromosomeI (Lcvy- Lahadctal.,1(95)and3) Amyloid PrecursorProtein (API')on chromosome 21 (R.E.Tanzictal..]9X7)acco unting tor 2%

ofallFADcases(Qucrfurth ctal.. 1(9 5).Trisomy-Z l cases arcshown to exhibitFAD pathology, associating an extra copy ofthe APrgcne with developmentor FAD (Qucrfurth, Wijsm an,StGeorge-Hyslop,&Selkoe,1( 95).

A majorfacto rinFAD comm onto all mutations is an altera tio n in All genera tio nor clearance(Goatcetal..1( 91).Oft he2\API'allelic mutation s ide nti fied,alloccur withinorimmediatel yborderin gon theA[l cod ing region(Goat c ctal.. \(91) suggesting that A[lplays acentralrole in ADpathogene sis(A.Eckert ct al..20(3 ).Mutationswithin theAll sequenceinfl uenceaggregationofA ]] bydisturbingthestructure ofthe accumulated peptide(Mori.C. 2(02) . Mutations borderingon theA[lcod ing region increase All product ionbyaff ectingAPI' processing,favou rin gtheamyloidogcnicII pathway(SeeFigure 1.3)(Cai, Go lde,&Younkin ,1993:Citro nct al.. 1996:Mo ri ctal..

2(02).Certa in populations appearto be exclusiveIll!'individualmutation s.For example, in myresearchIhavespecifically focusedon theso-called Swedishmutation . TheSwed ish do ublemutation consistsofsubstitutionsof theLysh711and Mcth71

residue s withAsn andLeu preced ingthe H~term inus or A[I.yield inga 5-Xfoldincreasein aggregateprone All(1-42 ) produ ctionthroughenhancedAPI' vulnerab ilitytoll-secretase cleavage (Citro net al..1992:Schcuncr,Eckman, Jensen,Song,Citron,Suzuki,Bird, Hard y,Hulton,Kukull,Larsonctal.. 1996: M.Mullanctul.,1992a).The propensityor A[l( 1-42)to aggrega te andformplaquesthat degenerateneuronsisthefocalpoint ofthe amyloid hypoth esis,oneofthe proposedprerequisite s ofAD.

1.2 Cur re nt Hypothe se s

Three hypothesesarcsugg es ted to accountIll!'the pathogenesisofAD witheach bein gexten sivel ystudied asthe primar yinsultthat initiatesthe disea se . Curre ntly. it appea rsAI3acc um u latio n ispresentinitially.1()IIO\\edbytau hypcrphospho rylation, in conj unc t ion within fla m mato ryres po nses thatcre ate acont inuumofins u lts.result ing ina disease state.

1.2.1 AmyloidCas c adeHypo thesis

TheAmy lo idCasca de Hypothes is(Figu re1.1)wasintrodu cedin 199 2 soo n allcr the1\ 13peptide wasfoundto be the primar y compo ne ntofse ni le plaqu es(SP s)believed tobe one ofthepath o geniclesion sfoundinAD brain s(Mast er sctal.,19 X5).This propo sedthatAll accumulati onwasthecruc ia l measureofAD patholog y(J.A.lIardy8:

Higgin s.1( 92).Thcdiscoverythat AI3 was aproductof API'metabo lism(Sclkoc.

2006a) and thatmutation swithinAPI'alte redAI3deposition(Haa ssct al.,1992:Seube rt ctal.,1992:Haass.C.1( 9 2 ).furt herstreng the ne d thecasefixAf3beingat thecor eofAD.

Adva nces inthcseq ue nc eofe ve nts in dementiapa th ol og yha ve offeredsuppor t to TheAmy lo idCascadeHypo thesis.Firstly. analte rna telimn ofde me nt ia,Fro nto te mpo ra l dementia withPa rk inson is m (FTDI').ischaracterizedbytauneurofibr illarytangles similar tothosefoundinAD.yet AI3plaque s arcabse nt (Goedert,Cro wther.8:

Spi llanti ni. 199 X).Th issugg cs ts thatsevere taumisfoklin gisnot su ffic ien t totriggerthe fornmtionofSl'sfoundinAD.Seco nd ly.tran sge nicmice ovcrcxprcssi ng bo thmutant

API'and mutanttau exhibit escalatedneurofibrillarytangles inco mpariso n tomice overcxprcss ing mutanttaualone(1.Lewisctal..2( 01)suggesting API'mutationsasan enhancer of further neuro-toxic insults.Thirdly.recentevidence has shown Alldimers generate tau hyperposphor ylationcausingneuritedegeneration(Jin,Shepardson.Yang.

Chen. Walsh.&Sc lkoc,201Ia).Lastly. abuildupof l\[l is observed in the brain before symptomsarise (D.M.Walsh.Klyub in.Fadeeva.Rowan.&Sclkoc,2(02)suggesting that Allaccumulation precedesclinical signsofAD.

However.the Amylo id Hypo thesishasfallenunder criticismastheinitiatoror ADsinceclinical trialsfound noimprovement in cognitive functio n inpatient s immunizedagainstAll. despiteevidence of plaqueclearance in the post-mortembrains (Golde.Petrucelli.&Lewis.2010: Holme set al.. 200S). While amyloid loadappearsto poorly correlatewithseverity ofcognitivedeclinein AD.SPs are presentinsymptomatic AD.Furthermore. thediscrepancy inamyloid load and degreeof cognitivedeclineis controversialbecause adefin ite toxic All specieshas yet to be determinedillvivo (discussed furtherunder"Amyloid-]!ProductionandToxicity").

1.2.2 HypcrphosphorylatcdTauHypothesis

Theother majorhypothesis surroundingAD onsetand progressionisthe disrupti on of microtubuleassemblyandfunctio nduetohypcrphosphorylatcd,inso luble.

filamentous Tau protein.Alterationof tau phosphor ylationis the principle causeof FTs thought to playakeyrole inADonset(Hernandez,GomezdeBarreda.Fuster-Mata nzo.

Lucas.8:Avila.2(10).Tau isexpressed in the centraland peripheral nervous systemand.

to alesser extentin kidney.lungandtestis(Gu.Oyama.8:lhara,1( 96 ).It ismost abunda nt in neuron al axons (Lee8:Trojanowski.200 Ia). but can also be found in sornatodcndritic compart ments(Tashiro.Hasegawa.lhara,8:lwatsuho, 1(97)and oligodcndrocytcs(c.Klein et al.,20(2 ).

TauconsistsofanN-terminal projection region.aproline-rich domain.a C- terminalregionandamicrotubul ebindingdoma in (MBD) through which tauprotein bindsto and thus stabilizes microtubule assembly(Mandclkow ctal.,1(96) (Kar.Fan.

Smith.Goedert.8:Amos.2003: Santarc llact al.,2(04).Thetandemrepeatsequences within the MBD are thought to directlybind microtubulcs throughtheir positivenet charge. which interactswith negativelychargedresidues in tubulin(Jho, Zhulina,Kim.8:

Pincus.2010:Kar cr ul.,20(3).Phosphorylat ion oftuu directlyinfluencesits abilityto regulate microtubule assemblybyneutralizingthepositive chargeandaltering conformationoftheMI3D.detachingtaufromthemicrotubulcs (Fischer ctal.,2(09).

The detachedtau can accumulateinneuronal cellbodies and ncuritcs. formin ginsoluble fi laments andNf Ts(Lee8:Trojanowski.2001 b:von Bergen.Barghorn ,Biernat.

Mandclkow.z;Mandclkow,2005:von Bergen.Li,8:Mandclkow,2( 05).In addition.

the iVIBD ortau containscritica lsequencesthat can assumethel~-sheetstructures requiredfortau aggregation and formation or pathologicalinclusions (von Bergenctal..

200I:vonBergenctal.,20(5).

SimilartoAll. taumutationshave been ide ntifie d whichheightenproduct ion of one isoformofta uoveranother. Mutationslead ingto changesin the ratioofthree-repeat tolour-r epeatisoformsmayresultin anexcessoftauin relationto microtubulebinding sites.allowingtil!'excesstauavailabilityfor filament assembly(Hultonetal..199 X:

Spillantinietal..199X:Spillantini.Crowther.Karnpho rst.I-1eutink.&vanSwictcn.

199 X).Missensemutationsthat affect alliso forms of tauresultinreducedmicrotubule assembly(Hasegawa.Smith.&Goedert,199X).thus causingmicrotubul edestab ilization and detrimentaleffectson axonaltran sp ort.

Inaddition.sincetauactivityisregulatedbythe phosphor ylation state.

hypcrphospho rylatio noftau as seeninAD depressestheabilityof tautobindto microt ubulcs (Lindwall&Cole.19X4:Hampel etal..2(10).Enquiryinto themechan ism behindtauhypcrphosphorylat ionhasleadto thediscoveryofmanytau kinasesincluding cyclin-dcpcndcnt proteinkinase 5(cdk-S).cyclicadenos ine monopho sphate (cAMI')- dependentproteinkinase(I'KA)and themosttherapeuticallypromising glycogen synthase kinasc-J]!(G SK -3r~ )(Mendesetal..2009:Schaffer etal..200X),Dysregulation of thcsckinascs and phosphatascsisthoughttoresultintauhyperphosphorylat ion.Alli s thoug ht topromoteGSK- 3 r~activationasitisup-regulatedinAD brains anditsactivity isshown to increasewithaging (Schafferetal..200X).Trialsimplement ingtheGSK- 31~

inhibitor.lithium .failed toshowalterationsin tau.phosphotau. All(1-42)levelsor cognitiveperform ancein mild AD patients (Hampel etal..2010:Mendes etal..20m ).

however.thismaybe owingtolithium' snon-specific ity as aninhibitor. Interestingly.

Hspl3lhasbeensho wnto bind hypcrphosphorylatcdtau and promote its

dephosphorylationand degradation.therebypromoting cellular surviva l (Shimura,Miura - Sh im ura . 8.:.Kosik. 2( 04 ).

The usc of trans genicmice hasdemonstr atedthat reductionor bothso luble All and tauisneeded to amelioratecognitivedeficits seenin tripletransgen icsthat haveSl's aswell as NT Fs (Oddo, Caccamo . Smit h.Green.8.:.La Feria.2( 06 ) concludingthatAD pathogenesiscannot beresolved witho ut consideri ngbothproteins.

1.2.3 Intlamm ati on and Oxida ti veStressinAD

Stud iesfromthe IlJlJO'sfollo wed infla mmat ionasa causative facto rin AD. yet toda yit remains unc learwhet her inllamm a tio nrep rese nts acauseor a conseq ue nceor AD.Clin icalevidence in supportoraroletil!'inflammationincludeselevatedcytokincs, infla m ma to ry agent sfoundin acuteinflamm ator yrespon se s.and activatedmicrogli ain po st-mortemADbraincomparedto age-matchedcontrols(Filli tct'II..1991:Frank- Canno n. Alto.McAlpinc. 8.:.Tanse y.200 lJ).Retro sp ect ivestud ies or individualsunder lon g-termtreatment with SAIDs(Ion-steroidal ant i-in tla m ma to ry drugs) have demonstrateddelayed onsetandred uce dse ve r ity or ADsym ptoms. altho ughrecent trial s havefa iled to confi rm this(Szekel y 8.:.Zandi.2(10).

Activa ted mic ro gl ia ce llssurroundAll plaqu esin bot hhuman AI)and tran sgeni c mouse ADbrai ns.ho we ve r the ir exact contribu t iontoplaque formationisunkn ow n (Johnston.Bou tin.8.:.Allan.2(11).Overall.it appea rsthat moderateactivat io nmaybe be ne fi cia l through anincreaseinAllclearance(Boissonneaultet'II..2009: K.Chen ct'II..

2(06).whereas stro ng activationmig ht slow do wntheabi lity toclearAll. increase prod uctionofpro-inflammatory cyto kincs and accelerateneuronal damage(Arnaud.

Robaki s.&Figueiredo -Pereira, 2(06).Microglialcellsimmunoreactivefo rintc rlc uki n-I were increasedin tissue sect io ns ofAD patient s(Griffin etal.. 1( 89) and inAI' I'/I'S 1 transgeni c mice(Hickm an . Alliso n.&EI Khou ry. 2(08). Interleu kin -6immunoreact ivit y in Sl'sand neurons in AD brains in itiallywasthou ght to be increased(Baue r etal..1992:

Heyser. Maslia h,Samim i.Cam pbe ll.&Gold. 1(9 7). yetrecent paper s sho waprotective roleofi ntcrleuk in-o in plaq ueclearance(Chakrabartyetal.. 2(10).Interestin gl y.

intcrl eukin-6increase sAPI'synthesis inneuron alcells(Altsticl&Sperber.1(91) and inte rleukin- Ist imu latesAPI'synthes isin endotheli al cells(Goldg ahcr et al..1( 89) . Increase inan alte rna tecytokinc . TlF«. resu ltedinincreased tauhypc rph osph orylat ion . intra-neuro na lAr~deposi ts andalso neur on aldeat hin tripletran sgen ic AD min : (Janc ls ins et al.,2(08 ).One theorybehind theroleofintlammat ion inAD isthata local insultin the brain perh ap s amy loidaggreg atio n willstimulate produ ction ofcytokinc s andwhi le initiatin gAl~clearance.prol on ged activation(co upled with prolongedcytokinc releas e)inc reases product ion ofAP I'.Thiscombi nedwit haltere dAPI'proc essi ng.

whether fromgenetic mutation s orotherwise.brings abo uta cyclicpatt ern of immunorea ctiv ity(Figure 1.2 ).

Ast rog lios is isfoundinAD brain s aswell(Akiyam aet al., 2(00). Previou s studies haverevealedAl~presentinastrocy te processe s(Kurt. Davies.&Kidd,1(99 )and lyses ofAr~loaded astrocyt es resultedintheform ationofSPs( ageIe.D'Andr ea. Lee.

Venkataraman.&Wang.2(03). Studies ha ve show n eviden ce otccllu larAll

internalizationfrom degenera ting synapscs anddendritesopeningthe possibilityof externa l uptake ofAr~byastrocytcsviaendocytosis(Nagelectal.,200.1).Also.

extracellularsoluble oligo mericformsofA r~maystimulateAr~productionwithin astrocytcs(1.L.Perezct al.,2(10)( DaRocha-So utoctal.,2(11).l-xtruccllularAI~is tho ught to limnporesor ionchannels in the cellular membr anetherebyallowi nginfluxof excessCa²'subsequentlyactivat ingdetrimentalsignalling cascades such ascaspasc and calpainactivat ion (Kawahara&Kuroda. 2000:.1.L.Perezct al.,2(10).Treatment with AI~canactivate both.INKand p.1liMAI'Ksignallingpathwaysthus activatingapo ptoiic signalling (A. Eckertctal.,2(0 1).

Taken together,itappearsaccumulat ionofi\f~intooligomcrs.althoughyet unknownif throughinternalor externalproduction.can triggerastrocyteactivation.

possibl ythroughincreasedcalciuminflux.in turnsynthes izingandsecreting toxic substances contributing to AD neurotoxicity(Dalcocha-Sou to ct ul., 2(11).

1..1Amyloid Precu rsorProtein

Amy lo idPrecursorProtein (APP)is atype-Itransmembraneprotein,witha large extrace llularglycosy lutcdN-tcrminus anda shortercyto plasmicC-tcnn inus. ubiquitously expressedin many celltypesand highlyconservedacross spec ies (Figure1..1) (Kangct al.,19li7).TheAI~porti onof API' islocated at the cellsurfacewith part or thcpeptide embeddedinthe membr ane(Mattson.2( 04).Alternativesplicing createsthreeiso forms ofAPI'in human: 770.751and695 (R.E.Tanzictal.,19lili). withthc695isof o rm preferentially expressed inneurons(Sisodia .Koo.Hoff man,Perry.&Price,199.1).API'

hasbeensho w n tobe concentra tedinthe synapsesof neuro ns. andincreasesinthe ratio ofthe neuronalAp p⁶'15isoformto others.is associated withAD (Matsui et al..2( 07).

API'undergoespost-tra nslational modificationsincluding glyco sy latio n, tyrosine sulfat ion and phosph o rylat io n.Mutatio nsof AP I'(incl udingtheSwedishmutat ion)are notthoughttobeloss-o f- fun ct ionmutat io ns meaningsuchmutation sdo not interf ere withApp'sbiol ogicalfunct ion (M.Mullanct al.. 1l}l}2a).l lcn cc,AI\isthoughttobe a by-productof API' met ab olism and appe ar s toha venodirect associat ion with Ap p's bio log ica l role(Cit ronctal.. !l}l}2;Citron.Tc plow.8.:Sclkoc.!l}l}5;Citro nctal.. 1l}!)6:

Goatcctal..1l}l}I;SclkocctaI.,1l}!)6).

API' is tho ug ht toha venumerou s bio log ica lfu nc t io ns.As a cellsur face recept or (Ho8.:Sudhof2004;Kan g cr al..Il}S7).API'mayha ve theabilitytoinduce cell sig na lling (I'.R.Turner.O'Co nnor.Tate.8.:Abraham.20(3). functio ningas a rece pto r- likemodulatoryprotein inne uro na l processes(Ashle y.Pac ka rd.Ata man.8.: Budnik.

2( 05).infl uencingWntsigna lling.API'issuggestedto mediate I\-catenin downrcgulati onin primaryne uro ns byspec ifi c phosphory lationorl)..cateni nresidues(Y.

Che n8.: Bodies.2(07).In addi tio n.Ap p's large extrace llu lardomainis capableor bindingtose ve ra l extracell u lar ma trix proteins.allud ingto a roleince ll-ce llandcell- matrixad hes io n (Mattso n.1l}l(7).In neuron s. API' ispresent ingrowt hcones ofncuritcs andbo thpresynapti c andpostsynapt icsites (Fe rrei ra. Cac e res.8.: Ko sik.1l}l}J;Saho.

lk in,Bux ba um.8.:Grcc ngard,20(3).API'is sho wntoinc reas esyna ptic formation and repair after inj ury(Pri llcrctal.. 2(06).ne ur ite outgro wth(R.G.Perez.Squazzo .8.: Koo , 1l}l(6)and playsa role incell mig ra t io n (Re inha rd. Hebe rt.8.:De Stroopcr,20(5).

Knock-downofAPI'throughilluteroclcctro porationin rodentdevelopingcortex.

revealedadependency onAPI'fill'co rrectmigration of neuronal precurso rce llsinto the cortica lplate(Young- Pearsectal.. 2(07).Whereasknock-downor API' inhibited corticalplate entry.over-expressionofAPI'causedacceleratedmigrationofcellspastthe cortical plateboundary,confirmingtherequire mentofnormalAPI' levelsfill'COITect neuronal migration (Ye ung-Pearsectal..2( 07 ).

1.3.1 Proteolyticprocessingof AmyloidPrecursorProtein

API'undergoestwotypesofproteolyticprocessingby sccrctasc enzymes differinginwhetherAllpeptide isproduced (Figure 1.3). Non-arny loidogcnicprocessing heginswiththe release oftheextracellular N-terminusorAPI' hyu-sccrcrascor ADAMIObetween residuesArlit>and Arl^l7withintheAfl regionthereby precluding All production(Sisodia,1(92).The released N-terminusofAl'P fromthecellsurface producedby u-sccrcta scisdenotedas solubleAPI'alphaorsAl' l'u .Thisleavesanli3 aminoacid cytoplasmic membrane-boundfragment(OU/CTFu).whichconstitutesthe middlep3 fragment (cxtraccllularlyreleased )andAPI'intrace llulardomain(AlfD) (intracellular).TheCTFiscleavedbyv-sccrctasc,generating the non-aggregatingp3 fragment whichsubstitutes1'01'thetoxicArl(Frigcrio ctal..20I0;S.A.Small&Gandy.

20( 6).AllAPI' fragmentsgenerated byAPI' processinghave been identifiedin one or morebiologicalprocesses. yet detailson the specific functionsofthe fragmentsare

unkno wn since investi gati onhasfocusedprimaril y on theA~peptide in itsrelation to AD(Summarized in TableI).

Activationofm usca rin icacety lcho line recepto rsincrease sproduct ionor sA l' l' u.

sugges ting that non-amyloido genieprocessin gis favouredbyneuronalact ivity (Buckne r.

And rews-Hanna.&Schactcr, 200X).Activit ydependence on API'pro cessingisno ta novel finding(Cirritoctal.,2005;Kamcnctzct al.,2003: Nitsch.Farber.Growden,&

Wurtrnan,1993;Sclkoc,2006b).however.whycerta inneuronsarcrobus tlyaffected by AI~accumulat ion while othersappea r unsuscc pt iblc,is afairl yne wfocus, Supportis gro w ing(o ralink bet we eni\r~dep osition,vulnera blehcteromodalassoci ationareasand co rt ica l dysfunction in AD (Bucknerct al.,2005: Bucknerct al.,2009:Klun k ct al., 2( 04 ).Cort ica l region simplicatedin AD havebeen identifiedas connectional"hubs"or areasthat act ascommunicationstations for informationprocessin gconnectingotherwise seg reg ated brainsyste msacro ss taskstates(S porns.Tononi .8:Edel ma n.2000:Spo rns8:

Kott er. 2004;Spo rns.Honey.8:Kotter.2(07).Moreover,in restin ghumans.increa sed activi tyin the defaultnetwork (Maw yerct al.,2(01).an area which correspond s to identifiedhubs(medial prefrontalcortex.medial temporal lobe.posterior cing ulatc cort ex ),positivelycorrelateswithSI' lo ad (Bucknerct al.,2( 0 5).

Conversely.Ar~produ cti onfrom API' processingoccursthro ughthe amyloidogen ie path wayrequirin g the BAC E enzymeorI~-sccrct ase(Vassar ctal..1999).

BAC Ecleaves API'at the N-te rmi na lend or theAr~seque nce thusenab lingAr~

production and secretionofsoluble API'beta(sAppr~).Clcuvugc by y-sccrcta sc cleaves

the membranecytop las mic terminal fragme nt(C99/CTFII) atpositionAfl⁴¹¹⁴²libcrating theC-tc rm inus or Aft allo wingentryintothe extr ace llu la rmilieu . There mai n ingAICD cantran sloca tc tothenucle uswhere itregul ates gcncexpressionincl udi ngind uc tio nor apo pto ticgenesthro ug hbind ing withFc6 5 andTip60(13'10 ct '11..2007:Cao8:Sud ho f 200I:D.M.Wal sh et'11.. 2(02).Amylo idogcnic pro cessingcan bemod ul atedby alte ri ngAPP' s susceptib ility to BAC E cleavage through cellu lar locali zation.

1.3. 2Intracellu la rTraffickingoftheAmyloidPrecursorProt ein

Nas ce nt APP,tran scribedin thenucleus,istraffickedthroughtheendopl asmi c reticu lum(ER)tothe go lgiappa ra tus.wherepo st-translati on almodification s.N- glycos lylationandO-glycoslyat ionoccurs.andconti nues throughthetra nsgo lgi net work (T G ) to yield mature APP protein(Fig ure1.4).API' tra vels thro ug h the secretory pathwa yto theplas ma membranewhe re ADA1\'110(u-sccrciasc)initia tes no n- arnyloidogcnicAPI' pro cessing.A pool of APP doc s notunde rgoim mediate processing.

andas aconseq ue nce isinternalizedbythecell(Ha asset'11.. 1992:Haass.Ko o,Mellon . Hung.8:Sc lkoc,1( 92).API' then eith er recy cl es back to the cellsurfa ce or istargetedto the lat e endoso mes/lysos o mes (Marquez-Sterling.Lo.Sisod ia,8:Koo.1997:Pasternak, Calla ha n.8: Mahuran.2004:Yam aza ki,Koo,8:Sclko c,1(96).He re AI'Pencounter s tra nsmcmbrancousBACEin thecndosomcs of theearlycndoc yti cpathway,thefirstste p in AI) prod uc tio n(Haass et'11..1992:Lor enzenct'11..2( 10 ).Ide nt ifiedsort ing motif sin API'( -P-X- Y) and BAC E(D-X-X-L-L) cytoplas m ic tailstar getbothprot einsfor

transport to cndosomcsvia clathr incoated vesicles(BAC Efrom TGN-en dosomes :AI'I' fro m cellsurf acc-cndoso rn cs)(W,1.Chen,Goldstein,&Bro wn,199 0:He , l.i, Chang .&

Tang,20(5). Fina lizing the produ cti on orA II.v-sc crcta sc(amult imc ri cpro tein com p lex consistingofI'S-I andPS-2and the siteforFADprese n ilin mutatio ns ).hasbeen fou nd enr ic hed in the latecndosomcs/lysosornc s(Pasternaketal.,20 0-1).Evid ence for the cndosomal/lyso sornallocalizationof theamy lo idog c n icsc c rctus cs is providedbyreduced All productionwhenblockingthe interna lizationof cellsurf ace AI' I'(Cirritoetal.,20 0 5 :

Koo&Squazzo ,I(94). and the ac idi ficationofthecndo som al-I yso som alpath w a y

(Kuether, Haa ss,&Ste ine r.2006:Schrader-Fischer&Pag an ctt i.199 6 : Vingt de u xct al., 2(07). Reportersyste m studiescouplingy-sc crctas cto fluorescenceha ve indicated the pre sen ce olv-scc rc ta sc in the cndocyticpathway.suggestingthe endosomes as the major sites 1l1l'AIlproduction(Kact hcrctal.,2(06).

Theoriesexistthat mutati on s inAI' I'resultin a greaterpo ol ofcndocytoscdAI' I'.

resultingin increasedavaila b ilityorA P I' forBACEclea vag e with in the cndocytic pathway.thusexplaini ngthe increasedA[lproduc tio nobserved(Hartmannct ul.,19(n).

The reis also evidencelorBA C E andso lub le Allco-localizationto the TGI.giving the op por tun ity1111'amy lo id og cnic cleavageof AI'I'10occur beforeit encounters u-secrctasc at the pla sm amembrane(Gree nfie ldetal.,I(99).TheSwedishmutationincreasesthe rateor [l-clcavagcby 5-10fold(Sin ha&Lic bc rb urg, I(99) andispropo sedto alter the traffickingof AI' I'(Al' Pswc).Thismutation partlydisrup tssort ingofsecretedAI' I'(De Stroo pc r et al.,I(95) andap pears to undergoI{-cleavage duringtrans itto the cellsurface (Haass.Hung ,Schlo s smachcr,Tcplow.&Sclkoc, ]993:Haa s s.Hung,Sclkoc,S: Tcplow.

1994: Thinakaran. Tcplow,Siman.Greenberg.&Sisodia. 1(96).Amyloidogcnically cleavedAI'I'/Al~is then traffickedthrough the cndocytic pathway to the Iysosomcswhere they-sccrclasc complex finalizesAl~production.Since thesite lor processing is thought to differ inrclat ion to the type ofAl'P(wtversus mutated). the exact details ofthe transportationpro cessback tothe cell surface forreleaseof the solubleAI'I'forms and All arc unclearatthistime.

Anovelpath wayinAI'I'rc-uptakc by cells directl yfrom cel lsurfaceinto the lyso so rncs,thereb ybypass ing thecndoso rna lcompart men ts,hasbeen sho wn by Lorenze n and colleagues.Fluorcscc ntly labelled Al' Pwtis show nenriched inthelysosomcs,and thedirect pathwayfrom cellsurfacetolyso som e appearsto be blocke dby theSwedish mutation(Lorenzen etal.. 2(10).A possible parallel to this trafficking pathway maybe theprion protein (Prf') which no rmally traffics throughthecndocytic compartme nts.

except when pathologicallymisfoldcd,appcarsto traffic directlyto theIysoso mcs (Magalhacs ctal.. 2(02)-Giventhatthe Swedishmutationis not believedto alterthe foldingof'the API'prot ei n (rathertheAll production).Al' Pswcmaybenormally traffickedthro ughthecndocyt iccompart me ntsevadingdirectentryintoIysom som cs fromthe cellsurf ace,Ifthc pathwa y chose nhas aninfluence on thetypeorAPI' pro cessin g(i.c.amylc idog cnicversusnon-umyloido gcnic).endo cyto sisthroughthe cndocyt iccompart ment mayleadtomor eefficient AI'I'cleavagebythe sccrc tusc enzymesand thushigh erAll production.whereas rapidtran sporttotheIysosom csmay

lead to degrada tionbylysosomal protcases (Lorenzenct al..:W10 ). Thishighlightsthe complexityyettoberesolvedinAPI'traffic kingand processingofbothnormal and mutated API'.

1.4 Arnyloid-fProductionand Toxicity

Antyloidisdefine d as aheterogenous class oftissueproteinprecipitates which share a common [i-shcct seco ndarystructure (Gandy. 2(02).Amylo idcan be produced throughoutvariousareas ofthe body (systemicamyloid)orbeconfinedtoa parti cular area such as renal amyloidorcerebralamyloidas seenin AD(Gandy&Pctunccska.

2(00).Increases in eithertotal Allor the relativeconcentrationof both All (1-40) (more concentratedincerebrovascular plaques) and All (1-42)(more concentratedin neuritic plaques) havebeen implicated inFADandSAD (Luc ct al..llJlJlJ).Multipledisease associationsexist withamyloid depositionincludinginclusionbod ymyosit is(muscle disease) andcerebralamyloidangiopathy(Rcvcsz, Holton.&Lashley.2(02). Altho ugh Allis acknowledged as a toxicspecies of AD.All docs not exist specificallyto cause AD (Lahiri&Maloney. 2(10).Allhas beenshown to11<1\'1:a multitudeolnonnalbiological processessuchas activationoI'kinase enzymes(Bogoycv itch,Boehm.Oakley. Keller man.&Barr.2004;Tabaton,Zhu,Perry. Smith.&Gilibcrto,2(10).regulationof cholestero ltranspo rt(Igbavboa,Sun.Weisman.He.&Wood.200lJ;Yao&

Papadopoulos.2(02). functioningas a transcri pt ion factor (Baileyct al.. 2011:B.

Maloney&Lahiri, 2(11).andanti-microbialactivity which may be associated with A[rs

pro-in fla mm at o ry activ ity(Sosciactal..2010). lienee. AIl(I-40 )and(1-42)arcprodu ced under normalcircu ms tancesinagingbrains witho utde me ntia.butarc bel ie vedto bein lower ratioand altered con fo rm at io n tha nobservedinADbrains.

Abno rma lacc um ulat ionofamyloidmaylead to amy lo idosisresu lti ng in to xic ity.

Afltoxi eityind ucesacti nstressfibre forma tio nand isprop osedtoinhibitacti n dynam ics.

trigger ingactin pol ym erization and diso rgan ization of act infil amentsin neuronal dendr ites (Mendo za- Naranj o , Gonza lcz- B illault.&Macc ioni, 2007:Song.Pcridc s, Wang.&Liu, 20(2 ).Axona l transpo rtincu lturedneuronsis inhibitedby A[lthro ughits effects upon thepolymerizat ionandaggreg ationofintracellu laract in (Hiruma.Kat akur a.

Taka has hi.Ichika w a.&Kawa kam i.20OJ).Intracellular aggrcgationof act inhasbeen desc ribedpre viou slyin ncurudcgcnc rat ivcdise a ses as rod-li kestruc tures fo rm edin hippocampa l neu ron s inres pon setostres s suc hasAfltreatment(M.T.Maloney.

Minarnidc,Kinle y.Boyle .&Bamburg. 2(05).Dis rup tio n of theac tin cyt o skele to n can lead togro wthconecollapseand degene ra t io n of neuronaldendr ites(Mobe rg&

Bamburg.2000).Initiall y.inves t iga tioninto the toxicnature of AIll<lCusedonthe insolubl efib rillarform found inthesenile plaq uespresent in AD bra ins. Theseplaqu es arctho ught tobege ne rated throu ghhydrophob ic interac tio ns between Allfragments whichassoc iateintorod-likestruct ures,so-called fibri ls.Duetoitsmor e hydrophob ic natu re . A[l(1-42 )isthemo reaggre gate proneform oft hepeptide(011ctal.,20II).

WhileSI"sarc thediagnosticlesio nsorADneuron aldamage.theins oluble fi bril la r AI\

associated hasbeenpropo sed as arese rvoirtill'smalloligomc rs(cg.dimcr s)withinthe plaq uecore. curo na l to xicit yhasrece nt lybeenassociatedwith the conve rsionof non-

toxicAI)monomerstotoxic oligorncrs(dimcrs.trimcrs,ctc.),inadditionto thefibrillar form foundinSPs(Selkoc.2007:D.M.Walsh&Selkoc.2(04).(.I.Hardy&Selkoc.

2( 02).These smalloligomcrs can diffuse to neighbo uringneuronscausing synaptic injury(Shankarct al.,2(08).Dendritic spine loss of-60'%is observed inhippocampal pyramida l neurons after15 daysofexposuretosub-nanomolarconcentrationsof A[) smalloligo rncrs,reflecting aloss of excitator y synapses.Spine densityreturnedto almost norm allevelswith cessation ofAI) treatment(Shankar ctul.,2( 07).

AI)dimcrsextractedfrom humanCS F havebeensho wn todisrupt synaptic plasticit y(Klyubinctal.,2( 08). Longterm potentiati on(LTP)isinhibited by so luble AI\

oligorncrs throughexcessive activationofl MDA-receptors similar toinhibitionof glutamaterc-upt akc (W.L.Klein.Kra ft!.&Finch.200I:Lictal.,20II:D.M.Walsh ct al.,20(2). Theseresults suggest that AI\oligomcrsshinI:'v1DAR-dependa ntsignalling cascadestowardpathwaysinvolved inthe inductionof LTD(long term depressio n)in contrastto LTP(Sclkoc,2(08).Chronicactivationofthese pathwaysby solubleAI) oligorncrsmay underlie the synapse lossinhippocampus thatoccurs earlyinthedisease processin brainsof AD patients(Masliahct al.,2001:Sclkoc,2(08).

Recentl y. subnanomolar concentrations(0.5nlvl) ofsoluble AI\ dimcrsisolated fr ornAl) cort exhavebeen shown todirectlyinduce tauhyperph osphor ylationin hippocampalneuronsdisruptingthemicrotubulecytoske leto n and resultin gin neuritic degene ration(Jin,Shepardson. Yang. Chen. Walsh.&Sclkoc,20 1Ib).Thisfindingis significantas it describes effects by endoge nous levels ofAI\on neuronal cells found

within theAD brain,in comparisontoexogenouslyincreasedlevels and presents evidenceor directinfluence on tau functionhyArt Moreover.the isolateddimcrswere principallycomposedor A I~(1-42 )(Jin, Shepardso n. Yang. Chen. Walsh.&Sc lkoc, 20II h).Finally.transgenicmice that express only oligornc rshut not plaques(AI'I'II>".II)) develop ADsympto mssimilarto mice engineered to convertoligomcrsinto plaques (AI' I' II>'>31)X I'SII\E9 )(Gandyct al.,2( 10).Eventhough a growing amountofevide nce is emergingin regards tosolubleAf~oligorncrs.thisdocsnot discounttherole orplaques inneurondegener ation.There is evidence thatperi-plaqueAf~assembliesarc associated with neuriticdystro phy (Knowlesct ul.,1( 99 ).and localdendritic spine loss((Kofliect ul.,2( 09 ).thereforeplacingsoluble oligomcrsand insolublefibri lsasco-contributo rs to Al~neuronal toxicity(Sclkoe.2(11).

A0can be degradedbydifferentsystems. whichincludeenzymaticdegradation or receptor-mediated clearance(R.E.Tanzi.Moir.&Wagner.2(04 ).Severalpcptidascs havebeendescribedto possessthe abilitytodegradeAll including insulindegradin g enzyme (IDE) (Qiu ct al.,1998:Vckrcllisctal.,2(00 )and ncprylisin(Sastrc&

Gentleman.20 10:A.J.Turner.Isaac.&Coates.20( 1).

Inadd ition.Aflcanhe clearedthroughphagocyto sisbyactivatedmicroglia and viatransportthrough thebloodbrainbarr ier (BBB) whichcan he enhancedbybindingto chaperones suchasApol.and«-macroglobulin(Suzuki&Nakaya, 20(8) .

1.5 HeatShock Proteinbeta-I(HspBI) in the Brain

Heat shock proteins arc up-regulatedin responseto cellular stress including elevated temperatures.exposureto heavymetals.ethanoland anoxia(Lindquist.19X6).

Inductionof Hspsisregulate dbyheat-shockfactors andaheatshock clement present at the promoter regionof the heatshockgene (Morimoto . Kline. Bimston,8:Cotto.1( 97).

Ileatshockproteinscanbedivided into two famili esbasedonsize and function:classic lispssuchaslisp100.Ilsp90.Ilsp70.Ilsp60. and the small heatshockfamily:uB- crystallin. HspBI.Hsp20.IIspB2and IIspB 3(Kapp cet'II..2003;Wilhelmusct'II..

2(06).Hsps withamolecularweightof60kDaor more possessan ATP-binding site and arcactivelyinvolvedinthe processof reiIIIdingmisfoldcdproteins(Gusc vet'II..2( 02 ).

Smalllisps.withamolecularweightof40 kDa orless.lack thisATI'-bind ingdomain and assistthe largerlispsintheir refoldingfunction(Mackac,2( 00).

HspBI (alsoreferred to asHsp27/25) ishighl yexpressedin muscle(Wilhclmuset 'II..2(06) andglialcel ls.spec ific a lly reactive astrocytcs (Renkawck.Voortcr,Bosman.

vanWorkum ,8:delong.1994: Shinoh ara.lnaguma,Goto,lnagaki.8:Kato,1(93). In the nervoussystem.Hsp BIexpressionis seen inspinalcord motorneuro nsand peripheralsensory neurons.whereit playsa keyrolein promotingneuro nalsurvivaland axonalgro wth.particularlyinadult neuron s (Costiganet'II..199X;Dodge ct'II..2006;S.

E.Lewiset'II..1999;William s.Rahimtula,8:Mcarow,2005:Williams.Rahinuula,8:

Mearow .2(06) .While HspBI isexpressedinso me areasof thebrain suchascranial nerve nuclei andhypothalam us (Armstro ng.Krueger-Nang. Currie.8:Hawkes. 2001:

Plumier,Hopkins,Robertson, &Currie,1997),it is not expressedin corticalneurons(M.

King, afar,Clarke, & Mcarow, 2(09).HspBI has beenco- localizedto post-synapt ic stru cturesin rat cerebellum and peri- synapticglial proce ssesfollowinghyperthermiaand this incre asewasnot seenin unstressedanimals (Bechtold & Brown. 20(0).lispsmay be secretedbyastrocytcsand maybe takenup byneighbourin gneuronsthuspossibly providing acompensato rymechanismtill'lack ofcorticalexpression(Ouyang .Xu, Sun.

&Gi fl~mL2006:Taylor.Robinson.Gifondo rwa,TytclL8.:Milliga n.2(07).Thc commo nfunct ion s ofHsp BIarcchaperoneactivity,thcrmoto lcru ncc,inhibitio nof apoptosis,regulat ionofcell development andcell differentiation,and interact ion with cytos kc lctalclementsin cellgrowth (Arrigo,2005:Arrigo . 2007: Charette8.:Landry, 2000a:Parccllic rct al..2003 : Vargas-Roi g,Fanelli,Lopez.Gago.Tello,Aznar,8.:

Ciocca.1997b).

1.5.1 IIspBI asa chaperoneprotein

Heatshoc kproteins(Hsp)arcthe cell' sdefencemech an ismin timcsofstrcssand arc thustermed "chaperones",Chapero nescan be definedas proteinsthat:I)havea role in the intracellularmanagementolmisfoldcdproteins,2) induce conlormationa lchanges ofproteins,3)actasatransport er of proteins(Hendrick& Hartl.1¹)93:Wilhclmus,dc WaaL&Vcrbcck.2(07).Hsps can bindunfold edprotei nsand keep themin their nativc state (Walter& Buchner,2(02).or recognizemisfoldcdproteinsand transport thcmto theprotcosomcfordegradation(Wilhclmusct al..2006:Wilhclmusct ul..2007).

Molecularchaperones workbyrecognizing and binding tohydrophobic aminoac ids expose dto thesurface ofthe substrate protein therebypreventingunproductive aggregatio n (Hartl, 19(6).

IlspB1conta insahighlyconservedamino acidsequence.the u-crystallindomain attheC-tcrminus. whichform sl~- shcct simportantfortheforma t io nof stablcdimcrs (VanMont for t,Slingsby.&Vierling,2(01).TheI-tcrminusisessentialforthe deve lo pment ofhighmolecular oligorncrs(Haslbcck ,2002:Theriault ct al..2( 04).which exclusively have chaperone functionillvitro,Theseoligorncrsconsist ofstable dimcrs of neighboringmonomers (Guscv ctal..2( 02 ).Oligomerizationof HspB I dependson exposureto stressand its phosphorylat ion state.Stressinduces an increase ofex pressio n (afterhours)and phosphor ylation(aftersevera lminutes )ofHspB I.HspBI in unstre ssed cellsexistsaslarge oligomcrs, while upon phosphor ylationHspBI dissociates into smallerspec ies,includingdimcrsand monomer s(Lambert,Charette, Bernier,Guimo nd.

&Landry.1999:Rousect ul.,1994:Williamsct al.,2(05).Stimulationofthe p]XMAl'

kinasecascade lead stothe activation ofMAPKAPK2 which isreportedto directly phosphorylateHspBI (Kyriakis&Avruch. 1996:Rogallactal.. I99(». HspBIis phosphory latcdon] serine residuesinthe human HspBI(S15.S7X.SX2)and2in the rodent HspBI(515and SS6in mouseor S90in hamsterHspBI).Oligomcrization appears to be connected to chaperoneactivity: aggregatesof largeoligorncrshave high chaperoneactivity. whereasdimcrs have nochaperoneactivity(Guscvct al..2( 02 ).

lisp sha vehccninvol vedin rescue ofaggrega te pron cprotein sinman )'ins ta nccs.

InDem en tia withI.C\\)'Bodics (D Ll 3).u-synuc lci n prote ininclusi on s ado pta[i-shcct struc ture leadin gto aggreg ationsuc hasoligomeri cassemblyillco rt ica l and limbicarcas (Sp illa nt in i&Go ede rt.2( 00).until it reaches a sta h lc fibrill arf(JrI11(Conw a yct al..2(0 0 ) sim ila r to AflinAD.Previou sly.IlspBIand uB-cr yst allinha vebeen sho w n topro tect again stu-synuc lci n-i nd uccd to xici tyand aggr egation(Mel.c anct al.. 2002:Rckasctal..

2004:Waud hyct al.. 2( 10 ).Thishashccn furtherindicat ed as evide nce ofrccomhinanl slls ps,inc lud ing HspBI.weresho w n to bind (thoughthrougha weak and transicnt intc rac t io n) to u-synuc lc in thus inhibitingmatureu-sy nuc lc in fihril formation and aggre gation(Bru ins ma ctal.,2( 11). Moreo ve r,mutant atax in- I tran sfcctcdHcL acells displaylar genuc lea r aggreg a tes thatco-Ioca lizcwithendo gen ou sHsp70.uhiquitin and 20 Sprot co som c.suggest ing that thc ce ll's endo genou spro te indcgrad ati onsystc m is insu ffi cientto suppress theseaggre gate s.Ove r-ex pres sio nof Hs p70 resultedina sign ifica ntred uct io n of nuclearaggre gate s(Cummin gs ct al.,199X).sho w ing thatan exogeno usove r-e xpress io nofHspwasbetterah lc toprotect tha nan endogenou s one.

Bip/GRP7X.part ofthc IIsp70familyfoundin thcER. hind sdirectlyto Al' Pswc impairingmaturat ionand decreasing sAPf'. Afl(lAO)andAll(1-42)recoveryin condi tio ncd medium ofhuman embryo nic kidney293 cells(IIEK 2(3)('I'.Yan g. Turne r.

&Gaut,199 X),The mechanismhchind thcred uc tio n inAPI' mctaholitcsin conditioncd

med iu m mayhc retentionof API' in thcER thusweakeningthc chance ofll/yscc rc tasc cleav ageand/orsuhs tratc depletion.

HspSI may act to inhibitapo ptosisthrou ghinteractingwith theouter mitochondrial memb raneand interfering withtheactivationofthecytochro mec/Apaf- l/dATPcomp lex inhibit ingtheactivatio nofprocaspasc-O(Brucyctal.,2000:

Concannon.Orrcnius,&Samali.200I:Samali ctal.,200I:Sarto.Binz,&Moc arclli, 2( 00).ThephosphorylatedformofHspBI hasbeenseen to inhibit Daxxapoptotic proteinand preventtheassociationofDaxx withFasandAsk I (Charette&Landry.

2000b:Charette,Lavoie,Lambert,&Landry.2(00 ).Apopt osismayalsobesto pped by activation of Akt orERK and pro-sur vivaltranscriptionfacto r,nuclear factor-«li(NF- KB )(Kennedy.Kandel, Cross.&Hay.1999:Lcvrcssc,Butterfield.Zcntrich,&Heasley, 20( 0). all ofwhichHspBI hasbeenshown to enhancethc activationof:thusacting ina pro-survival natur e(Parccllicr,Gurbuxani ,Schmitt.So lary,&Garrido.2003).

A0toxic ityinducesactinstress fibreform ati onlead ingtoinhibition of axonal transpo rt ,growthconecollapseanddegenerationofncuro nal ncuro nal dend rites.Actin sta bilizat ion by sHspshasbeen shown throughtheir abilitytoactas anacti ncapping protein thuspromot ingsurvival (Guayct al., 1997:Guscv,Bogarchc va.&Marston.

2(02 ).

The pro-survi valandchapero nefunctionsof HspBI maybe potentialmechani smsin which HspBI caneffect API' processingor distribut ionwithinthc cell and influencethc productionofA ji thcrcby impedingAlloxidativedamage,

1.6 Alzheimer'sDiseaseandIlspB I

IlspBIhas been recognizedin15%ofSl'sin neoco rtical areasof AD patientsbra ins (Wilhclrnus ct al.,2006:Wilhelmu s ct al.,2(07). Whether thislocalizatio nis anattempt byIlspBIto ame liora te thedetrimen ta leffectsorA[~on cellsorpart orthe disease processisunknow n.how everpreviou s work hum our lab has observed increased neurona lsurvivalincellsexposed toAr~(1-42) when transfcctcd with Hsp BI (M.Kingct al.,200lJ) pointin gto apossible protective mechanism.Insupport ofthis.Hsp131can decrease the leveloI'phosphorylatedtauand rescue tau-mediated celldeath (Shimuraet al.,2( 04 )andhasbeen implicated in neurite growthandcytoskclctulstabilityinDRG neurons(Williamsct al., 2006:William s&Mcarow,2(11).

Direct interac tionbetweenuB-crystallinand IlspB I withAr~hasbeen demonstratedin vitro(Kudva, Hidd inga,Butler.Mucskc,&Eberhardt. IlJlJ7:Liang.

2(00 ).Wilhclmusct al.,2006investigatedthe rolesorsl lspsonAllcytotoxicity.They roundIlspBI.Hsp20 and uB-erysta llin.butnot IlspB2Jn.bind toAr~

(

decrease its aggregationbyreducingthe propensityorAr~to limn [}.-sheetconformatio n.

Furthe rmore.IlspB1.IIsp20 anduB-erysta llin inhibited cerebrovascularAr~toxicity.

probablybyreducingtheaccumulationorAr~at thecellsurface (Wilhelmusctul.,2( 06).

Amore recentstudy foundIlspl3l .API'and I3cl-2limna complexwithin mitochondria ofserumstarvedN2acellswhichisassociatedwithapo ptosis.Thiseff ectwasincreased in cellscontainingthe Al' l's we mutationand API'over-expressio n increasedHsp BI

presencein mitochondr ia.Itisimpo rtanttonot e that APPovcr-cxprcssion aloncdidnot induceapoptos is instead a seco nd insult inthcfo rm ofserumsta rvation wasneeded(I'.

T.Yang.Hsu,&Kuo. 2(09). "

PerhapsHspBI is recruited to thc mitochond riaunder stress ful conditionsto provide

chapero ne acti vity andactivatesapoptos is throu ghmitochond rialdysfunctionor converse ly, thesequest eringof HspBlinsidethc mitochondri aimpair it from bindingto thccytoso lic cytochromec.thusinhibitingHspBlfromprovidingitsanti-apoptotic effects.

Whilc HspBlhasbeen implicated in neuronal protection and reductionofp rotein aggrega tio n, thc questionremainswhether HspBIlocalized inSPs of AD brainsis a fa iled aucmpt to counterac t AI)aggrega tio norpart ofthc diseaseproccss.

1.7Pro posed Therapies

ResearchersinAD haveidentifi ed variousareasaspossibleintervent ionsagainst amy loidaggregationgive n eviden ceforAI)toxicity(Cit ron.2(04 ):

I.)[)-sccrct ascinhibitors (c.g.McmapsinZ).These block thc firstamyloido gcni c cleavageof 1\1'1'. inhibitingthe formationof 1\/\N-tcnninus(Chan gct al..

2(04)howeverthesehavebeen provenineffective in clinical trials"

2.)Y-sccrctascinhibito rs (e.g.scmagaccstat).These block theseco nd cleavage orAPI'in the cellmembranethatwould stop the formation or AllC-te rminus.

Thesehavenotbeensuccessfulto dateasv-sccrctusc binds ligands otherthan APPsuchas otch,hence inhibition hasother negativeeffects(Basi ct al., 2010:lmbimbo&Pcrctto, 2009: Shih&Wang.2(07).

3.)SelectiveAIJ(1-42) loweringagents(e.g.tarcn tlurbi l).Thesemod ulatey- sccrctasctoreduce All(1-42)production in favour of shorterAIJversions, Tarcnflurbilunfort unatclywas foundtohaveno beneficiallyeffectsonAD patientsuponcompletionofthePhaseIIIclinicaltrial(Greenctal.,200l)).

4.)Immunotherapie s.These stimulate the hostimmune system torecognizeand attack Allor provide antibodiesthatprevent plaquedeposition orenhance clearanceof plaques.Unfortunately. AfJ immunotherapiescancausemicro- haemorrhagesdue to activation ofmatrixmctallop rotcinascs,MMI'2 and MMPl)(Wilcock ctal.,2(11).

5.)Anti-aggregationagents(c.g.apomorphine).Theseprevent AfJ fragments from aggregating orclearaggregatesonce theyarc formed(Lashucl ct ul., 2002:Parker et al., 20(2).IlspBI may 1:111underthis categoryas an endogenousanti-aggregation agent throughits chaperoneactivity.

I.XSummary

FAD isthegeneticformof ADacco untingfi ll'4-5%of ADcases.Mutations. such as the Swedish mutation in the API'genehave been shO\\"I1 to increaseAlltoxicity

throughfavouringthearnylo idogcnic pathway.hasteningproduction of aggregateprone AI\(1-42)(Citronct al..1992;£3.Maloney&Lahiri.20II ).Allcan alsoassembleinto oligo mcrswhich appear to betheIocus ofAli toxic ityrecently.Thcsc oligomcrsincrease apoptosis. disruptcell signallingandinhibitLTI' andsynaptic plasticity(D.M.Walsh&

Sclkoc,2(04 ).Thishasbroughtthe focusof Allresearchawaytrom clearingSlsto disrupting amyloid oligomerization.

A possible mechanism fill'disruption ofmisfoldcdor aggregated proteinsisthrough theworkofmolecular chaperones.Exog eno usHspBI protectsneuronsfrom celldeath causedby All treatment (M.King etal.. 2(09).HspB I thoughnot found endogenouslyin co rtica lneuron s.issecreted byneighbourin g glia l cellsand maybe takenup by adjacent neuronstoelicit itsprotectiveeffects,Throughitschaperonefunction.HspB I may sequestermutatedor misfoldcdAPI'taggingit for degradation befo re process ingoccurs or sequester API'withinthecell inhibiting its secretion into the extrace llular environment.Since bothAll and IlspB Iarc involvedin thecell deathpathway.IlspBI couldinhibitAllactivation ofapoptosis.The possibilityalsoexistsforIlspBI10exert changeson cytoskclet ulelementsthusinterferingwith tau misfolding and inhibiting production of FTs.

HspBI hasmanytherapeutic possibilitiesin ncurodcgcncrativcdiseasessuch as AD.

Myfocuson interactionsbetween IlspB I andAI'I'/Aflexplore thepotential effectsofthe small heat shock protein on API'processingand distributi onwithinthece ll. As

mentionedpreviously. locatio nof API' withinthe cell plays a rolein the type of processing thatwilloccur and thusthe productionor Aft To further myiuvcstigarion. I compare the wild type API'againstthe Swedishmutated API'in regards to distribution and release of proteolytic products (includingi\f~I--IOandAI~ I--I2)to determine possible modificationsinthe presence ofHsp B I.

1.9Hypothesis

HspSI interact swithAI~oritsprecursor API'and throughthisinteractionisable to alter API' processingand hence the distribution/release oftoxicAI~peptide.

l.tO Objcct ivc

Investigatethecorrelation betweenHspBI andAI'I'/All

HspB I andAI~(1-42)

ObjectiveI:Is thereaninteraction between AllandIlsp B1'!

Objective2:Canwemanipulate the relationship betweenHspB1andAll to decreasetheamountofreleased All'!

HspBI andAPI'

Objective3:Isthere aninteract io n betweenHspBIandAPI''!

Objective 4:Canwemanipul ate the relationshipbetweenIlspBIand API' to decrease theamountof AIIproduction'!

Amyloid Cascade Hypothesis Altered APP processing

•

Increased A13(1-42)production and accumulation

•

Oligomerizationof A13(1-42)and depositionas diffuse plaques

•

Neurotoxiceffects of oligomeric and protofibr illar A13(1-42)on synaptic projections

•

Inflammatory response with activation of microglia and astrocytes

Increasing synaptic a ! neuronalinj ury

•

Progressive oxidativeinjury signalling (kinase and phosphatase activity)

Tau hyperPhOSPhOry!tion and tangle formation

•

Extensive neuronal dysfunct ion, transmitter release deficitsand cell death

•

DEMENTIA

Fi:,:u rc1.1Pro po sed Amy loidCascadeHypo thes is (From(J.HardyS:Sclkoc.2(02).

Reprinted with permissionfrom AAAS.

risk factors ,

altered proteol vsis of A PP

+ . .. . .

extracellular secretion . ofA betapeptide

+ .

.. diffuse amyloid plaq ues ".

... .. / (ill l rHcell il~~lreffects':')

.' ... {fibri llogCllcsis)·"· .". .(kinase/phosphatase '.

:: ." J'

". " . amy I

O ct

.Ineuritic

':.. plaque s. '. -"' plaques ~.a.n.l~~~:?. ~ .~

,

(oxic1at~vestress)".

(inc.cytokinerelease)

, ^{. .}

Inflammatory Respon se

Fi~u rc1.2Sc he ma t icofthe ma inhypo th esesimplicat ed inADpath olog y andthe ir interactions or depen de nceon eac h component for ADdevelop ment.

A Cytoso l

[I' n Y40Y42 E

V y ¥

n

.EVKMn.ana:I!l!iIiHCI3 i i ' ">il i 4 i l " d.' l iri l h i ii 6 i i eTVIVITlVtilKKKQ

Transmembrane domain

B Non-Amyloidogenic C Amyloidog enic

p3 I

~APP'"

* !

!

APP fl·CTF

Fi~lIrc1.3Sc he ma ticotAl' Psho w ing the(A)All peptid ewithin the transm emb rane region alo ng withthe scc rc tascenzyme cleav age sites.Notice the alte redy-scc rc tusc cleav agesite usedtoproduceAll (1-4 2)which isup-re gul ated in the A!'I'-swemutation.

API' pro ce ssingoccursthrough (B) thenon-arnyloidogcnic(u-sccrctasc)pathwa yeluding theformation or Allor(e)theamyloido gcni c(fl-seeretase)pathwayfavouringthe forma tionoft heAll peptide.From (Thinakar an&Ko o. 200X).Reprinted with pe rmi s s io n fromASBM B.

-~

0 /

'---\

LYWWj

Fig ur e1.4Schematic ofcellular traffickingof A!'!'frommatu rationwithin thego lgi to transportto the plasma membranevia thesecreto rypathwayand continuingthrough recyclinginto thecndocytic/Iysosornalpathwayfordegradation.From (Thinakaran&

Koo. 2(08).Reprintedwith permissionfromASI3MI3.

Neurodegenerative Stress

DyingNeurons

I

APP

Clearance SenilePlaques Inflammation

Heat-ShockProteins

(HSP90.HSP71etc)

~

/ TLR 4

A ~

Chaperone / ! Aggregation

P38M T ^{K N~}

1

(IL-6. TNF-a) Oeposrts

/ +

r---.,

Fi!-:u rc 1.5Sche mat ic of po ssibleHspeffect son ncurodcgcncrutivcdisease.

TahleI.identifiedfunct ionsofAI'I' products

sAP P

AIC D

CT F

Product Fu nc tion Binding Partn ers Refe r en ces

Neurite outgrowth Undefined (Mattson,1997)

Synaptogenesis (S.A.Small &

Synaptic plast icity Gandy,2006;D.H.

Neurona lexcitability SmalletaI.,1999)

Regul ator of (Caille,Allinqua nt,

neuronal ste mcell Dupont,Bou illot,

division Langer,Muller,&

Proch iant z,2004b;

P.R.Turner et al., 2003)

Surface receptor ApoE (LaFcrla. 20m:

Ccllad hesion ECM Malison.1997:D.

Neurite outgrowth Undefined H.Small ct al..

Rcgulato r ofC2+ 1999:1'.R.Turner

homeostasis ctaI..200-')

Axonal transport Kinesin,JIP (BaoetaI.,2007;G.

cargoreceptor D.King & Scott,

Kinase mediated Shc/Grb2,H P 2004;Lazaro v etal.,

signalling 2005;Tarr,

Gene transcription Fe65,HP,Numb Roncarati,Pelicci, Pelicci,&

D'Adamio,2002;

Taru etal.,2002) (M urcsanS;

Murcsan. 2(04 )

Chapter2

Materials and Methods

2.1CellCulture

Nafve HumanEmbryonicKidney 293 (HEK293)andstable celllines ofwild-typc Amyloid PrecursorProtein (APPwt )andmutated swedishAPI'(Al' Pswc )(akind gill from Dr.Paul Fraser-University of Toronto) were grown in high glucose Dulbcccos ModifiedEag le's Medium(DMEM.Invitrogen. Carlsbad.CAlsupplemented withI'/';, pen-strep/L-glutamine (P/S/G).10% Fetal BovineSerum (Fe S) and 400 pg Gcncticin Disulfate (G418.Gibco, Invitrogen. CA). to 80%confluencyin IOml petridishes. G4 18 suppleme ntation wasused only fo r stable cell lines andstopped uponinitiationof experimen tal procedures. Cellswereincubatedat37°Cwith 5%CO2supplementat ion.

2.2I'lasmid s andTransfcction

As sho wn in Fig.2.3Experimenta l model.I performed experiments aslollo ws:i) HEK293naive cellsco-transfcctcd withAPl'wtorAl'Pswc construct. pmaxG FI' (positivecontrolvector withenhancedgreen fluorescent protein) (VSC-IOOI.Lonza.

Basel

Switzerla nd)and pEGFI'-C2-HaHspl3l-W Tfusionproteinwhich hasbeenpreviously shown tobefunctional inI'CI2cells(Mearow ctal.,20(2 ) or the control emptyvector (EVC2)and ii)Al'PwtandAl' PswcHEK 293 stablecelllinestransfcctcdwithpCIG- HaHspl3l-WT - IRES-EGFPconstruct.Theseexperi mentalmodels were initiallyused

interchangea bly.howe ver. uponexperimen tationit wasnote dthe largeEGFI' tag fusedto theIIspB I-Wt protein may causeinter ferenceinthe normal metabolismofthe IIspBI protein thus affecting the results obtained.Inadditio n. interferencein resultsmayoccur fromthe stressof over-expressing twoproteins (lIs pB I- WtandAI'I') bytransfcction within the cell. Therefore,to address thesediscrepancies.theAl'Pwt/swcIIEK 293 stable celllinestransfcctcdwith the smaller pCIG-l lallspB1-WT-IRES- EGFP construc t was usedforthemajo rity ofthe experimentationunlessotherwise noted.

Confluent HEK293cells. Al' l'wtandAPPswestablecell linesweretransiently transfcctcdin low serum mediausingLipo fcctami nc20001 ~1(Invitrogen.Carlsbad.CA.

USA) as perrnanulacturcrsprotocol.Briefly. 20 piLipofcctaminc2000reagentwas added to an aliquotor 500 pi Opti-Mcm Reduced Serum media (GibcoInvitrogen.

Carlsbad.CA.USA ) whileX.O~lgDNA wasaddedto a second aliquotcontaining500~"

Opti-Mcm Reduced Serum med ia andincubatedfor5 min.Since theAl'P wtandAl' Pswc constructsarc not tagged proteins.itis imposs ible tovisualize thetransfccti onefficiency oftheseproteinsincomparisonto the taggedIlspBI-Wt that wastran sfc cted.To overco me this issue.IaddedpmaxG FP to theAPI'co-transfcctio nreactionin a ratioor 1:4since the pmaxGFP istranstcctcd at a muchlower concentration than API'.

There fo re.if2 pi of pmaxGl-Pyield.50'%transfccti onefficiency thenIcanassumeXpi olAl'Pwillgive at least 50%,transfcctionefficiency.TheLipof cct umi ncsolutionwas addedto the DNA slowlyand incubatedfor20minuponwhichthesolutionwasadded to the IIEK293cells.Mediawas changed4-6 hI'Sposttransfcction !i'OIl1thereduced serum media to mediacontaining DMEM withI'YoI'/S/G.O.5'X,ITS.0.5XB-27(Gibco.

Invitrogen.Carlsbad.CA.US/\)supplementand theA[{degradat ion inhibitor 0.1'Yc, phosphoramid ondisodium salt (17.7mM.Sigma-Aldr ich. Oakville. Ont.,Canada ).

2.3Protein andCond itioned Media Collection

Celllysate andcellularconditio ned mediumwerecollected24and4Xhrspost mediumchange. Cellularconditioned medium was collectedon ice with additionor protease inhibitorcocktail tablet(RocheDiagno stics.Laval. Quebec,Canada) immediately uponco llect io n. Mediawascentrifugedat 14.000gIorSminat 4°Cto discardanycell debris.HEK293cellswere collected byadding I ml icecoldTBS with 200mM sodiumvanadateandscrapingcellsoffthe plate witharubberpoliceman.Cells were pclletcd for :;min at4000 g at4°Cand re-suspende d inicecold proteinlysisbuff er (I'X,NP40. 10%glycerol.O-l{-thioglucopyranoside. protease inhibitortablet.200mM sodium vanadate. sodium fluorideand magnesium chlor idein TBS) andstoredat-20°C until experimentation.

2.4 WesternBlotAnalysis

Western blot analysiswasperformedwithsamplesoftotal cellularlysat eand cellularcondit ioned medium.Sampleswerethawed on icc andcentrifugedat 14.000g ti ll'10 minat4°Candsupernatants were usedtodetermine proteinco ncentratio ns usinga BSAproteinassaykit(Pierce Chemica ls.Rockford,IL.USA). Lacmmlisample buffer (10%SDS. glycerol.1M TrispH6.X.dl leO.0.0I%Bromoph enolblue) containingfresh l{-mercaptocthanol(13MI:)wasaddedto50 pg or eithercellularlysateorcellular