1 and Cathepsin&#X2010

This article isThis article by protected copyright. All rightsreserved. 10.1111/nan.12512doi: differences to lead between the of thisversion c and Version Record.Please copyediting, paginationbeen throughthe andproofreadingtypesetting, process,may which Accepted This article acceptedhas been for publication andundergone fullpeer review buthasnot Running title: Keywords: Corresponding author: Seniorauthors** equallycontributed authors * First equally contributed 1PJ, U.K. BrainBank,2. UCLInstitu Square Queen and Research building,Southmead Hospital,BS105NB, U.K. 1. ofBristol, University Group,Research Dementia Neurosciences, Clinical Level1, Learning Article Kiely A.P. degradation ofα Exploring theofkallikrein putative role Article :Original Article type : (Orcid ID 0000 MINERS SCOTT DR JAMES

, MinersS. multiple systemmultiple atrophy; alpha alpha - synuclein in multiple system atrophy in system synuclein multiple - synuclein proteolysissynuclein inmultiple systematrophy 1*

, Courtney R. , Holton [email protected] JL;

2 , Strand C.

- 0001 - te ofNeurology, London, Street, 1Wakefield WC1N 6, calpain - synuclein; kallikrei - 8594 2 - , Love S. , Love 1640) - 1 and cathepsin

1**

, HoltonJ L n - 6; calpain - D in theproteolyticD

2** -

1; cathepsin ite thisarticle as - D

This article isThis article by protected copyright. All rightsreserved. to we have Conclusions. regional distribution of However KLK6 Results. CTSDactivitywas in elevated MSAinthepontinebaseandcerebellar white matter. =20 MSA (n pontine nucleus,putamen,occipitaland cortex, base caudate cerebellar w and assessedinrelationto α M distribution ofα synucleinopathy, multiple atrophy system (MSA), this study ofsmall number proteasesthat overproduction and dementia with Aims. Abstract ethods. mRNA

Acceptedincreased Article

and There isevidencThere

studied ,

CAPN1 the or activity level oftheseproteolytic did enzymes was ) andage α Accumulation - to syn . - . We suggest

syn and levels K determine determine

allikrein in MSA Lewy

accumulation - protein matched e were α

that accumulation of - bodies syn . -

6 of whether

elevated . (KLK6)

syn load level that α

- post

cleave syn (DLB)

their their . and/or

, calpain - in MSA in mortem control mortem tissue protease protease activity

in in MSA results results from α upregulation - multiple multiple syn activity activity

- is 1 and are and α

not (CAPN1) - syn the

brain and of due impaired impaired removal uclein putamen and cerebellar whiteputamen andcerebellar matter KLK6, CAPN1 and CTSD were measuredKLK6, were CAPN1 and CTSD

is dysregulated

might thereby to

is altered regions (posteriorfrontalcortex, and cathepsin likely tolikely be

reduced

(α (n = (n = - syn) syn) no 20 in anotherα

in t correlate activity

). a in Parkinson’s disease

compensatory

modulate theregional PD and DLB. - of D α (CTSD) (CTSD)

- of syn hite

the proteases the proteases

with the -

rather than rather matter) are amongare a Our Our

response aim ,

(PD) in . its in

This article isThis article by protected copyright. All rightsreserved. UPS SNCA PD NNI NCI NAC MSA KLK6 GCIs DLB CTSD CSF CAPN1 α ALP AD Abbreviations - syn

Accepted Article

------

ubiquitin α Parkinson’sdisease nuclearinclusionneuronal cytoplasmicneuronal inclusion non systemmultiple atrophy kallikrein glial cytoplasmic inclusion withdementia D Cathepsin cerebrospinal fluid Calpain α autophagy Alzheimer’s disease - - synuclein synuclein gene synuclein - amyloid - 1 - -

proteasomal pathway 6 -

lysosomal pathway

- β component L ewy bodiesewy

This article isThis article by protected copyright. All rightsreserved. targeted it aggregation [ α α regulate zyme, probably ofreduces aggregation andtoxicity α responsiblelysosomalα protease for shown tobe autophagy pathogenesis of disorders. these expressionmRNA of thedisease feature numbers ofSmaller characterised by inclusionsneuritic (Lewy and dementia withLewybodies (DLB) hallmarkdefining neuropathological of ofAbnormal aggregates Introduction 15, - is Accepted the syn within Article

found within Lewy bod Lewy found within 16] protease A small number of A smallnumber non , and generate generate , and ,

as acompensatory viral - - syn leve lysosomal (ALP) pathway

in vitro

dysregulated inPDDLB and

vector M) non α

[ -

1 l, both syn [

[ 11 neuronal nuclear neuronal - - 8, 3] - amyloid mediated upregulation of KLK6protects againstα . - - cleavage fragments , suggesting In 11, 13] containing α bodies and α - in vitro

synuclein ( synuclein 12] - ie and synucleinopathies

s andGCIs response to elevated α - . KLK - β component( lysosomal calpain The ubiquitin The

and glial cytoplasmicglial inclusions (GCI

6 is expressed in neurons and oligodendrocytes is expressed inneuronsand 6 that impaired that impaired mayprotein clearance play arolein the

neurites), neurites), while multiple atrophy system are majorroutesare of - - α syn cleavage syn cleavage or cytoplasmic or cytoplasmic , theaggregates form syn both syn both in vivo - -

1 the syn) withinneurons, orglialsyn) neurites cells [ 18,

(reviewed in (CAPN1) p roteases

that α 20] . - NAC

synucleinopathies. In Parkinson’s disease KLK6 and CAPN1KLK6 ,

- α in vitro proteasomal

are lessneurotoxicare andinhibit and degrades extracellular - syn )

[ region 14] [

7 - including syn, in DLB andPD syn, inDLB - inclusionsNCI) (NNIor accumulates accumulates

9] [

have been shownhave been to and cleave and 4 . -

CTSD α 6] ,

- are ) s in vivo

. characteristic yn clearance yn clearance and havebeen

pathway (UPS)andthe pathway Cathepsin D (CTSD) isthe D(CTSD) Cathepsin

kallikrein - required for mediated mediated α proteases cleave monomeric cleave proteases [ without increase s 7, ) in oligodendrocytes. 8] - , 6 (KLK - [

syn pathologyin and isupregul 10] neuronal and neuronal - α α syn cleavage

. are alsoare a -

- syn

syn - (MSA) 6) (neurosin, α -

syn aggregation [ are the 13]

d [ 17

is SNCA . Brain - ated, ated, main 19] (PD)

; -

This article isThis article by protected copyright. All rightsreserved. for MSA cases control and7mixed MSA mixed cases protein/activity and 20 MSA selectionCase Materials and Methods load proteolytic support regions with KLK6, CAPN1 MSA and CTSDin of cleavage α [ models CAPN1 pathogenicity α α with α modelsofanimal DLB 23] - -

Accepted Articlesyn aggregation syn .

[ - 14] probably probably syn in thecytosol syn in In are are

[ and 20 were and control brains the 21] thi . D reduced inDLB tissue andPDinhuman clearance

, hypothesis that

s study egradation ofegradation varied and thatt - . We syn is responsible for syn isresponsible

were used for , physiological ineffective) (if ,

suggesting relationship CAPN1 suggesting a complex between and others

predilection for MSApathology we measurement measurement of . The upregulation of [ he extent of reduction he extent ofreduction 21] have

[ 23]

and MS fibrillar α fibrillar the accumulationof explored thehypothesisthatareduction in proteolytic .

, is CAPN1 capable

and assessedtheir demonstrated that theexpression andactivityof KLK6and NanoString nCountermRNA expression studies and7 A western immunoblottingwestern

the the used - [ syn 22] α - elevated α syn load (See Table (See syn load

. by CAPN by CAPN for biochemical assessment of enzyme CAPN1 is expressed neuronally CAPN1 isexpressedneuronally and

the the correlates response to three three of degradingboth α is association with α - - syn and CSF and CSF . syn ,

Unlike proteases however, however,

level inMSA.Wehavemeasured in MSA in MSA closely with

the increase in increase the

in DLB, in [ 1) . 21,

M . associated withaccelerated Subsets is associated with in regionswith high α ixed MSA cases were MSAcases ixed 23,

monomeric andfibrillar monomeric our 24] - α syn load - activity activity findings findings

syn accumulation of and and 6 α - control and and control in syn

co in brain animal animal and α do not do not . -

localises reduced

- - syn syn 6

This article isThis article by protected copyright. All rightsreserved. enzymes (NanoString Technologies We used analysisNanoString nCounter for paraffinwaxembedding, and8 France). Roche/Merck, UK mM NaCl,protease inhibitortabletand1%Triton andphosphatase occipital fromTissue wasdissected thefrontal a licenc approval UCL InstituteofNeurology.Thebraindonationprogramme andprotocolshaveethical All brainshadbeen H dataset. MSA cases) to allow findings compare usthe regionswithin in multiplebrain to a single For thecurrentstudy olivopontocerebell intothree of pathology subcategories: striatonigraldegeneration (SND), selected

Accepted Articleuman brain tissue The righthemisphere e issuedbythe TissueHuman Authority(No. 12198). , The lefthalf

lobe

NanoString nCounterHumanInflammat and α from from based on

and cerebellar white and matter homogenised the NRES the NRES Committee London - syn ), ina

ar atrophy of (OPCA), andacombination twoknownasmixed MSA. the . Total mRNA mRNA . Total Ozawa criteria Ozawa

donated to the Queen Squaredonated to the Queen BankforNeurologicalDisorders,Brain - , brain formalin, in10%was fixed buffered sliced,tissueblocksselected

we chose to use mixed MSA mixed (hereafterreferredwe chose touse cases simply as

Precellys , Seattle, WA, Seattle,

was sliced,

was 24

[ 25] μ homogeniser m sections prepared form sectionsprepared histology. isolated from lobe . )

MSA can be subdivided according to besubdivided MSA can thedistribution to quantify transcripts of α thethree and theslices , caudate nucleus, pontine putamen, – Central ion v2mRNAExpressionpanel Assay with ceramic frontal lobe andcerebellar whitematter frontal lobe ,

and tissueisst flash frozenandstored at

beads (Bertbeads lysis -

X (all reagents from X (all or buffer: 50mM Tris, buffer: 175 ed for research under ed forresearch under

in technologies, - syn

base, - - degrading degrading 80 ° C.

This article isThis article by protected copyright. All rightsreserved. instruction staining system (Menarini Diagnostics, Wokingham,UK) 1: Immunohistochemistry was performed Quantification ofα results cerebellar white matter significant difference RNA/reporter complex immobilisation.Two indifferences hybridization andprocessing efficiency and variables includingpurification contr fromcontrols excluded were further analysis. counts analysed GAPDH, GUSB, HPRT1,PGK1, TUBB biological normalized to mean the using geometric Software v2.0 nSolverAnalysis (NanoSt toatechnical subjected Rawcountswere randomly assignedtoplates. blinded tothediagnosis. Toavoid run probes withoptimal melting temperature screening for inter Probes were designdesigned accordingto manufacturer’s the principles of 6

Accepted1300 Article control and and control ols and tools positivecontrols and . ) were were

(

Abcam using nCounterexpression NanoString DatafromNanoString nSolver software. normalization s . inspected anddeterminedinspected anysamples relative tobeoutliers to negative

Sections were Sections , UK 6 MSA - -

syn load and intra ) for 1 h in cases . The , gene expressiongene

we used

pre and -

Benjamini (Table reporter andcaptureprobeinteractions, forand selection -

treated in the Menarini unit treated intheMenarini with anexcess ofsuper buffer used used according to the manufacturer’s instructions, tomanufacturer’s according the correcting the geometric for mean all house ) included in) included theCodeSet.

1). - a modified method automated a modifiedmethod for order bias, samples of cases or ofcases order bias,samples controls were - 150 with Yekutieli method falsepositiveYekutieli method wasusedto exclude between

[ 26]

- ng of total RNA from each samplewas totalof RNAfromeach ng tailed Student’s Student’s tailed anti The The .

The laboratory runningtheassa laboratory The - α MSA count data - syn ( and

Catalogue numberab15530Catalogue at as per

control frontal lobeand control frontal t The were were tests wereuse the

NanoString data NanoString manufacturer’s - normalised tonegative keeping genes normalisation

[ Intellipath FLX 26] d to test ford to test , including , including y was ring).

were were

analysed ( CLTC, and For

for

. This article isThis article by protected copyright. All rightsreserved. Accepted (R&D systems,UK)dilutedinPBS recombinant protein)and human (6.5 standards μgtotal blocked for3 h in1%BSA/P room (RT). temperature washedThe plateswere inPBS tween 0.01% high Aldrich,(Sigma Dorset,UK)dilut developed in KLK6 bysandwichlevel wasdeterminedinbraintissuehomogenates that ELISA Kallikrein in whichsignificancewas using quantified wasperformed Image J.Statisticalanalysis using a two immunoreactive bandsatthepredictedmolecularweightfor protein each of interest on ice. (GE membrane healthcare) Article Scientific) Samplessystem (Thermo FisherScientific). instructions. Protein was determination assay(Pierce)asperformed by BCA per immunoblotting Western thresholding macro. section area immunopositiveforα anddigital slidescanner and - mounted binding 96binding The blots The - 6 (KLK6) sandwich ELISA were run - For house, in DPX - well microplates (R&D systems, Abingdown, UK)andwasleftovernight (R&D at well microplates

SDS were analysed using the Odyssey system. using analysed theOdyssey were at 120

- as previously describedas with glass coverslips. with PAGE andwest PAGE images acquiredimages using Aperioa Leica Imagescope.The

set at

V. ProteinsweretransferredfromV. thegeltonitrocellulose BS at 26°C. Following a further washstep, Following afurther BS at26°C. tissue homogenates in 1X transfer buffer FisherScientific)(Thermo at37

p ed in PBS (1:200) wasusedtoed inPBS(1:200) Costar coatwellsinclear

syn syn 0.05. ern immunoblotting ern was

Immunolabelled Immunolabelled diluted determined by applyingdetermined by anImageJcolour [ 23] . Mouse monoclonalanti Mouse

20 μ g/20

we used The integratedThe sections were processed by μ l inMESbuffer(ThermoFisher

the XCellsurelock blot the - 20 fiveand times manufacturer’s - tailed studenttailed - area KLK6 antibody KLK6 antibody - density percentage percentage

V for 80V for min - was t TM of test

This article isThis article by protected copyright. All rightsreserved. Accepted490 whichdark after wasread fluorescence which wasalsodilutedThe inassaybuffer. reaction wasleft toat37 proceed without inhibitor by Mittoo et al mercaptoethanol, 5CaCl2, 1 mM EGTA,1 mM mM EDTA)pre homogenate in40 humanCAPN1 purified Aldrich,diluted(Sigma Dorset,UK)were ( inassaybuffer of acalpain (Millipor CAPN1 fluorogenic substratefor CAPN1 CAPN1 ArticleCalpain Allsample measurementson eachplate. weremadeinduplicate. measurements on FLUOstar plate Optima reader. KLK6 concentration interpolationwas determined by against readat4 acid,The reaction with2andabsorbance wasstopped Nsulphuric washed, andchromogen systems, UK)dilutedinPBS/0.01% tween furtherwashes,streptavidin:HRPBSA/PBS, wasaddedfor2 After h at26°C. (1:200) (R&D polyclonal anti wereaddedovernightanotherwash,biotinylated goat at4°C.containing 1%BSA After nm and emission at518 sample Foreach nm and nm. the fluorescentsignal intheinhibited - activity - specific cleavagespecific sitein 1 (CAPN1) e , Hertfordshire, UK - 1 - specific inhibitor(10 specific -

was measured inbrain tissue homogenates [ KLK6 27] ,

and leftfor

serial dilutions of recombinant dilutionshumanKLK6(720serial of u fluorogenic activityfluorogenic assay . Each sample andstandardwasadded . Eachsample l assay buffer, containing buffer, l assay antibody (1 μg/ml) (R&D systems, (R&D (1μg/ml) Cambridge,antibody UK)dilutedin1% ic substrate (TMBS, R&Dsystems, UK)for ic substrate(TMBS, added 20the dark. minin ))

10 minat37

and the assays wereand theassays perf α , -

as previouslydescribed spectrin (1 spectrin μ M Tocris, Bristol,and tissuehomogenates UK). Brain in - 201 wasaddedfor h at26 a FLUOstar Optimaplatereader withexcitation at ° C, priortoof substrate addition the fluorogenic

μ 50 mMTris M, H - K(FAM) o

rmed - [ HCL, 50HCL, 5mM mN NaCL, 23] in by useof - EYY ~GMMK(DABCYL) duplicate duplicate - . The substrate . Thesubstrate warmed to 37

with or without with or without

- 11.25 ng/ml) included 11.25 ng/ml) included o an C, the plate wasthe plate C, to internally q internally wells, withor 50 ° included C as ° C for3hinthe

nM ina nM the 10 described described - β addition OH uenched uenched μ - l of

the

This article isThis article by protected copyright. All rightsreserved. plate.each dilutions from measurementsonserial of serially r measurements repeated ontwo were separateoccasions. activitywasinterpolated CTSD from thatintheuninhibitedwells.Each was sample and measured induplicate at was readusingaFLUOstar Optimaplatereader with excitation at Thewas buffer. left toproceedat37°Cfor3 reaction darkafter hinthe whichfluorescence 37 or standard wasaddedinduplicate withorwithoutand incubated buffer cathepsin (Pepstatin Exeter,UK) Sciences, methoxycoumarin peptide wasCTSD enzyme activity measured in braintissuehomogenates DactivityCathepsin (CTSD) fluorogenic assay these derived standard curve from occasions.two separate fromwells wassubtracted thatintheuninhibited wells. ° Accepted393 Article C

prior to addition of the fluorogenic substrate thefluorogenic prior to additionof

were included ineach plate

- made up of 0.1 420 nm 420

substrate - - D A at4μM)

from liver . For each sample thefluorescent. For sig sample each , -

Mca 4 - . Brain tissue homogenates tissuehomogenates . Brain yl)acetyl; Dnp=dinitrophenyl](BML

in the presence

M sodium acetate and0.1 sodiumM acetate ( - BML Gly The calpain - Lys - SE199, Enzo LifeSciences,Exeter,SE199, Enzo UK) measurements on serial dilutions onserial measurements of calpain - Pro

to - - Ile 1 activity control for or absence - Leu - Phe

of each sample each of - Phe variation acrossvariation plates.

M sodium chloride atpH M sodium chloride (1.25 of a . Allcomponents ecombinant CTSD that were includedwere ecombinant CTSDthat nal in the inhibited inhibited nal inthe wells wassubtracted - Arg

specific cathepsin specific

μl - - Measurements Measurements

P Leu in 1 - 145 -

Lys(Dnp) ml assay buffer) ml assaybuffer) was interpolated from - 001) by adding 320

pepstatin

was were

at 20 - - 340 nm D

diluted - were repeatedwere on - D inhibitor

- Arg diluted inassay

1 (20

μM 3.5 a fluorogenic a fluorogenic - - A NH2 [Mca= (7

. Each sample . Eachsample (Enzo Life (Enzo or and emission and emission

- for 10 at min in assay 1.25 purified

μ a g/ml)

n - ; This article isThis article by protected copyright. All rightsreserved. white matter(P<0.01) (Fig. increasedblot, was significantly (P < 0.05) inMSAtheputamen (Fig. but MSA andcontrols We KLK6, protein and CAPN1 CTSD 3] MSA betweenMSAandcontrol did notbrains differ lobe matter We KLK6, and CAPN1 CTSD Results 16. P correlation pairsbetween of variables. Statistical tests were performed using SPSS version by not normally distributed even after transformation,Kruskall logarithmicrequired transformation to normal distribution.variables obtain thatwere aFor postBonferroni Parametric statisticaltests used were forgroupscomparisons between Statistical analysis

Accepted, α Article Dunn’s testfor intergroupcomparisons. wa pairwise Pearson analysis

was assessed KLK6, CAPN1 and CTSD examined -

in MSA(4.53 - compared to controls syn ( values < 0.05 statisticallysignificant. considered were

from unchan SNCA MSA andcontrol

KLK6, CAPN1 and CTSD ) - ged inallother regions hoc test was used for multiple testwasused for hoc mRNA - .

fold increase) (P< increase) 0.01).fold KLK6 KLK6

mRNA expression was notexpression was protein

in any of the in anyof 3

). CTSD level ). CTSD brain

level s

. KLK6mRNA protein

was signific mRNA examined examined

region

was KLK6 mRNA level KLK6 mRNA altered altered

level level in . CTSD andCAPN1. CTSD - s studied unchanged inunchanged MSA group comparisons. In some cases group Insome comparisons. antly elevatedantly intheputamen

(Fig. (Fig. 2 level in asubse in MSA.

frontal lobe white andcerebellar was ).

(Fig. (Fig. 1

CAPN1 level, measured by level, measured western CAPN1

significantly elevatedinfrontal t of brains - Wallis testwasused, in thecerebellar white matter ) .

As in mRNA mRNA in all

previous studies previous , 3 ,

regions (Fig. ) and cerebellar ) andcerebellar were unchangedwere in and ANOVAwith s used toassess the western western

( P < 0.05) , followed followed

the data blot, 4 )

. (

[ in 2,

This article isThis article by protected copyright. All rightsreserved. Accepted elevated lower inregions α withhigher significantly with or CTSD Lastly, we region Brain cerebellar white matter (P< matter 0.001) enzyme activity was ArticleMSA in regions We region Brain frontal highest in ofindicator regionalvariation of in severity We determined the distributionRegional ofa measured cortex , the putamen (P (P < 0.05)the putamen cerebellar andwhite matter (P<0.0001)

level oractivity in the

(Fig. 7B the assessed

variation in variation in variation , andcaudatenucleus, (Fig.and lowestintheoccipital cortex 5).

pontine base and putamen, intermediatepontine baseandputamen, inthe cerebellar whitematter, KLK6 complete -

C) KLK6

(Fig. (Fig.

, whether protein level protein α but but significantly (Pelevated intheputamen and cerebellar white < 0.0001) -

syn load( level, orwith 6B .

in MSA

none of KLK6, CAPN1 andCTSD KLK6, CAPN1 andCTSD Across thesixregionsexamined,α MSA andcontrolcohorts - syn load inMS syn load ) .

CTSD activity regional differences in - syn load (Fig. (Fig. percentage sectionpercentage areaimmunopositive forα

these trends trends these by ELISA

CAPN1 6 C

). (Fig. (Fig. 7A) A

, was significantly elevatedinthepontinebaseand

and

GCI or reached CTSD CAPN1

pathology in MSA.Thepathology of level in α , level or activity level in relation to relation α in and .

KLK6 level w KLK6 level α level or - CAPN1 and CTSDactivity CAPN1 and syn load were related were syn load to

and statistical significance

CTSD - syn loaddidnotcorrelate activity. activity. - as significantly elevated in syn load in MSA enzyme activities, KLK6 was generally KLK6 was generally

(Fig. (Fig. in MSA .

- tended to be KLK6, CAPN1 syn

) - . syn was CAPN1

) in

as an

all

This article isThis article by protected copyright. All rightsreserved. synucleinopathies of PD, D [ proteases ofthat should course, benoted, andα high GCI burden wehavetested.α ofproteases Theupregulation these that CTSD. α white matter CAPN1CTSD and α Discussion was activity p <0.05) 8C). No (Fig. 0.44, (Fig.8B), andCAPN1activity p<0.01) correlated In Upregulation ofKLK6,CTSD an 28] - - syn load syn homeostasis, Accepted Article cerebellar white matterfromthe

a accumulation of nd endothelin The findings Abnormal accumulation of W LB and MSA. LB and e have examined the e haveexamined expression of , and itremains possibleothers that

positively (r=0.64, withCTSDactivity 0.0001) 8A) p< (Fig. between between

observed –

regions of were were

[ contrast withourprevious observationsin contrast 1 - in MSA regions converting enzyme (ECE) converting enzyme - There is There 3] α significant correlation betweensignificant KLK6 activitylevel, CTSD andCAPN1 in anyofthe - - increased in syn loadistosyn likely beacompensatory responsetoelevated

syn inMSAis syn suggesting

high GCI burden ,

in relationα to the were were no d CAPN1 inMSA

we of have onlytestedasmallnumber potential evidence that evidence α not α that other regions other

MSA - combined cohortcombined controls andMSA of syn in neurons andglialcellsisacharacteristicsyn in hallmark unlikely tounlikely resultfrom correlated

failure failure with

and α correlated three proteases, previously proteases, three to shown regulate in the , [ 29]

of proteolysis or removal byothermeans - such as syn load - -

with that weexamined syn syn load , may bedownregulatedinMSA may putamen, pontine base andcerebellputamen, pontine base mRNA level mRNA the level or the level activity of

positively (r withCTSDactivity =0.37, - matrix metalloproteinase syn degrading enzymes inregio enzymes syn degrading

in different regionsin different brain of

in MSA in

reduced

PD and DLB PD and DLB . is However, differences differences However,

. increased in

activity of the

and

CAPN1 (r= activity [

cases 23] KLK6, CAPN1 KLK6, or CAPN1 , and α , KLK6 level - 3 (MMP - α .

indicate syn α . KLK6, - syn. ns of ar in - 3)

This article isThis article by protected copyright. All rightsreserved. [ KLK6whereas interacts α with anddegrades in vitro [ oligodendrocytes CTSD MSA results accumulation in MSA. to hypothesise cortexcerebral that human brain ismost tissue andDLBthatthereduction inPD marked of regions effects of mouse models DLB monomeric forrequired and CTSDall oligodendrocytes in regulated PD and DLB that cleave and degrade intracellular andextracellular poolsof α PD and DLB arepathways routesof major contributemay to Accepted13, 11] Article , are partlyco , are 36] , CAPN1

in In thepresent study,we [ .

35 A reduction in proteolytic ofactivity A reduction KLK6

vivo -

39] , atleastinpart, α [

α 4 have major cleavage siteshave majorcleavage

- and KLK6 in MSA becauseand KLK6inMSA

- - syn [

syn . CTSD the primary is enzyme responsiblelysosomal for α 6, 8] that areduction inproteolyticcleavage . 30]

- and arelikely t [

[ We localised withα the pathogenesisofMSA 17 a 15, ggregation contain

and MSA and - ,

19] [

16] and others and 21] [ . These proteases . These 23] .

and MSA Brain

the highestlevel from defective proteolytic clearance. . KLK6, CAPN1 and CTSD KLK6, andCTSD CAPN1 α , [

31 - and have syn clearance syn clearance - o impacton α specific inductionspecific protectiveagainstα of is KLK6 , - -

34] syn inSH showed cleavage cleavage [ 22] explore .

within thenon small A these these , and there isevidence thatCTSD there has , and

are found within Lewy body inclusions and GCIsare bodyinclusionsand foundwithinLewy previously . Thee - - SY5 s syn both intracellularly syn bothintracellularly d was

enzymes areenzymes withinneuronsand expressed and havebeen shown be to -

of α number the hypothesis the hypothesis that α syn loadinPD,DLBand Y cells, andregulate cells, Y α

shown to

ndosomal - has been proposedto playaroleinthe has been syn are are - that KLK6andCAPN1 reducedin are Aβ component (NAC) region (NAC) whichis Aβ component

might [ of proteases havebeeni 23]

expressed neuronally, andwithin . inhibit - It therefo seemed lysosomal andautophagic

contribute tocontribute α - syn

We have chosento aggregation o - [ syn accumulation in 11] in vitro -

syn levels MSA -

syn degradation syn degradation and extracellularly and extracellularly

dysregulated in .

- and aredown KLK6, CAPN1

syn protective protective re reasonable dentified f full in vivo - syn in syn - length study

and [ 9] -

This article isThis article by protected copyright. All rightsreserved. pathology accumulation becomesbut eventually overwhelmed oreven contribute that is couldproteases be KLK6 increasedand CAPN1 be inMSA should of individual the highestα is incontrastto findings previous PDand in DLB theproteases.strong between correlation levels to abnormal proteinaccumulationinAlzheimer’s disease response to [ be CTSD patholog increasedwere prot MMP pathogenesis of 41]

Accepted neuronal Article induced ininduced animalmodelsofPD . ection against ection disease These - in thepontinebaseandcerebellar white matter are allare elevated 3 not specific for not specific D T ,

he present findings do not support our hypothesis findings he present donotsupport are activeextracellularlyare espite y :

regions in MSA in

KLK6 and CAPN1 data indicate CTSD in elevated α elevated - syn burden

the in MSA in regions that in MSAregions that α

- (data not shown) (data is unknown. The synucleinopathies, including marked up marked a the pons protective protective

in MSA α - - syn syn , that

we failed we failed anto detect association and are usuallyand are

in PD .

were Whether this response affords any protection thisresponseany against Whether affords α , particularlyinthe cerebellar white we matter where noticeda in MSA in MSA an - regulation ofKLK6,CAPN1 andCTSDin mechanism , probablysimilartotheupregulationof CTSDinresponse CTSD isinducedasan

and havebeen elevated findings [

or 10] was independent ofα was independent have a across regions

and co upregulated Our

in support o support ,

or ;

high the putamen and the putamen ce our datasuggest that - localises with α localises finding MSA perhaps a perhaps [

21, implicated inimplicated thepathogenesisrather than

α - 23] syn loa [ . ther 21 . A recent studyrevealed thatinductionA recent

of CTSD shown been haspreviously to early early compensa in PD andDLB . -

24]

elevated

[ response studies (reviewed in studies 42, . - d and syn load syn Instead, Instead, . with

Other 43] - syn in nigral neurons syn innigral

. α pronounced pronounced rebellar whitematter,and

CTSD, CTSD, KLK6 and CAPN1 in MSA -

α to protein accumulation to protein syn load within syn load induction of

KLK6 [ - 41] syn proteases syn [ MSA in 40] tory lysosomal s to disease . KLK6 and CAPN1 ,

. It why is unclear

CAPN1 and regions with MSA [ 44, all three three all 45] , suchas - in syn CTSD ) PD ,

This article isThis article by protected copyright. All rightsreserved. inthemarked cerebellum, avulnerable region in MSA post of themRNA,proteinlevel and cohort other regions. The was activity significantly increased inthecerebellum andprotein CTSD mRNA protein level, measured by ELISA, across regions,particularlyinthecerebellum. For instanc tofragments to remains promote bedetermined. aggregation α suggest protective of fibrillarα thatfragments the seed propagationoffull α intermediate synuc indifferentbrainregionsproteases MSA indicating - -

Acceptedsyn Articlesyn - translational modification compared to leinopathies aggregation and and size for some of the analyses size forsome analyses of the There were There differences Assessing thepotential s that this s that enzyme has

that accelerate in - syn an experimental mousean experimental model partially cleaved α α - [ syn 14,

is PD divergence divergence

- further further

46, degrading pathology

and DLB and toxicity 47] level . complicated CAPN1 is , assessed western by blot,

a protective functiona protective may and facilitates

role of enzymesthese inthe between

and . of the protein of the ofactivity the proteases mayhaveproteases different roles inthepathogenesisof - For instance, instance, For syn fragments syn fragments was partly bean partly artefact ofsampling, . that there is that there variation intheregulation of these

but significantly increased significantly increased also

could mRNA and level proteinandenzyme activities

by their potentialroleintheproductionof increased in increased - s length α length

clearance. of PDof . Thev enzyme CAPN1 also reflect also reflect that may drivethat may

[ : ariability expression inmRNA was most 49, - i.e. proteolytic cleavagei.e. proteolytic reduces

syn in MSA. generate s, e.g. ,

particularly

PD 51] The potentialforCTSD cleavage

were were following incomplete degradation incomplete following e, regional inregulation differences

. [

KLK6 mRNAwasreduced but

48 pathogenesis of α due to Most Most in the Differences Differences

- s the unchanged whereas CTSDunchanged whereas 50]

C - evidence forKLK6evidence terminally truncated

aggregation and mRNA processingormRNA in patients cerebellum inMSA.

reflect CAPN1 CAPN1 were alsofoundwere in ing thesmaller - with the inhibitors are inhibitors of full - α length

- syn

This article isThis article by protected copyright. All rightsreserved. mRNAexpr synuclein 2 Alpha 1 References proteasesof thesethree inrelationto theα MSA. studies arerequiredtodeterminethe preciserol CAPN1CTSD inMSA and accumulate in MSAduetoreduced activity in regionswith a highα are CTSDproteases We haveshownthatKLK6,CAPN1 and Conclusion. ne studiesusingofpathological numbers stratified process.larger cases Future for mRNAequally, whi of thedisease. However,itremains possible stage that,evenattheend of thedisease for thisarisingfromtheneuropathological inanefforttoavoid possiblestudy bias subtype OPCA

Accepted Articleuropathological willberequired to subtype explore thisfurther. ch - - Determining whether thereareDetermining whether cell predominant pa predominant synuclein mRNA expression in inMSA.oligodendrocytes Glia 62: 2014; 964 lossneuronal olivopontocerebellarandaffects structures striatonigral regions Kingsbury AE, Daniel SE, Sangha H, Eisen S, Lees S, Lees DanielSE,Kingsbury AE, SanghaH,Eisen AJ, FosterOJ.in Alteration alpha Asi YT, Simpso

expression ession Movin Parkinson's2004; disease. Disord 19: 162 n JE, Heath PR, Wharton SB, Lees AJ, Revesz T,n JE,Heath PR,WhartonSB,LeesAJ,Revesz HouldenH, HoltonJL. ttern of ttern loss1).neuronal Wehadselected MSA (Figure - syn burden. Thesyn burden. may be may may vary regionsmay between vulnerabilityto withdifferential the a response compensatory to elevated α

findings - specific in changes thedistributionandexpression of theseenzymes. of Theupregulation - syn would informative.also be syn

e oftheseenzymesinthe pathogenesisof indicate thatindicate α

upregulated upregulated - syn is unlikely to syn isunlikely

in MSA - syn load. Further Further syn load.

- - 70 mixed mixed cases , particularly

70 KLK6, KLK6,

in - This article isThis article by protected copyright. All rightsreserved. Biochemistryspecies. 2008; 9678 47: the 9 Dprotectsagainstcathepsin alpha Zhou Y,L, Peng LD, Speake Parks R,CrabtreeD,Liang Q,Crimmins S,SchneiderL,Uchiyama Y,Iwatsubo T, 8 5 level affectsalpha Woulfe J,Feany MB,Myllykangas L,MG, Schlossmacher Tyynela J. Cathepsin D expression 7 reciprocal relationship.Mol Neurobiol 2013;537 47: 6 curseParkinson'sdisease: orblessing. ActaNeuropathol2012;124: 153 5 14508 autophagy PJ, McLean Unni rolesinvivoubiquitin VK. Distinct forthe 4 Mol BrainRes2001; 92:58 of Expression alpha 3 Accepted Article

degradation of alpha - 20 Sevlever D,JiangSevlever Cathepsin P, SH. Dis Yen lysosomal enzyme involvedthe main in L, Qiao S,CaldwellHamamichi KA,CaldwellGA,YacoubianTA, ZL, Wilson S, Xie V, Cullen Lindfors M, NgJ,PaetauA, Swinton E,KolodziejP, P, H,Saftig Boston Xilouri M,BrekkOR,Stefanis L. alpha Ebrahimi Ebrahimi Wirdefeldt K, Bogdanovic N,WesterbergL,Payami H,Schalling M, Murdoch G.

- lysosomal inthe of pathway degradation alpha Lu Y, Standaert DG, Walls KC, Shacka JJ,Roth DG,Walls KC, Shacka Lu Y,KA,ZhangJ.Lysosomalenzyme Standaert - - Fakhari D,Wahlster L,McLean PJ. Proteindegradation pathwaysin Fakhari D,Cantuti - synuclein - synuclein inthe synuclein human brain:to relation Lewybodydisease.Brain Res - synuclein synuclein and generation of carboxy its - 6

processing, invivo. aggregation,andtoxicity MolBrain2009; 2: 5

- - synuclein aggregationsynuclein Mol andtoxicity. Brain2008;1: 17 Castelvetri I, Fan Z, Rockenstein E, Masliah E, Hyman BT, I,FanZ,Rockenstein E,MasliahHyman Castelvetri - 87

- Synuclein and a Synuclein proteindegradationsystems: - 51

- - proteasome system andtheproteasome system synuclein. JNeurosci 2 synuclein. - terminally truncated - 72

011; 31:

This article isThis article by protected copyright. All rightsreserved. Diamandis EP. ofhumankallikrein Thespectrum 6 (zyme/proteaseM/neurosin)expression 17 2001;276: 2380 residues acid in themiddleofalpha 16 J2001;toxicity. Neurochem 78:384 ofAlzheimer'scomponent (NAC) disease amyloid responsiblef 15 forms of alpha Distinct VM, DR. Lee TrojanowskiJQ,Lynch cleavage pat 14 341 Extracellular neurosindegradesalpha 13 Lett 2008; 436:52 of Cleavage normal andpathological formsof alpha 12 synucleinopathies. HumMolGenet2003; 12:2625 degradationbyserineproteasesynuclein neurosin: implication offor pathogenesis 11 52 nucleusof with caudate primates experimental 10

Accepted Article - 6

Petraki CD, Petraki CD, VN,Karavana SkoufogiannisPT, GM, SP,Howarth DJ,Yousef Little IV,Giasson BI,Murray Trojanowski VM. JQ,Lee Ahydrophobic of stretch 12 amino B,IrvineBodles AM,GuthrieDJ,Greer GB.Identification of region of non the Mishizen H,WatanabeTatebe Y,KasaiT, Mizuno M,Ta T, Nakagawa F,NakagawaKasai T, N,WatanabeY,Kametani Tokuda T,Yamaguchi M,Mizuno T. M, Iwata A,Maruyama Akagi T, T, I, Hashikawa S,NukinaN.Kanazawa Tsuji Alpha Yelamanchili SV, ChaudhuriAD,Flynn CT,FoxHS.Upregulation of cathepsinDinthe - synuclein by calpain I synuclein bycalpain in vitro.2003; J Neurochem 86: 836 - - 6 Eberz AJ,GuttmannEberz RP,GiassonH, GA,3rd,HodaraR,Ischiropoulos BI, Day -

- - synuclein is synuclein J forassembly. Biolessential filament Chem 95 - synuclein in cells.synuclein cultured Neurosci Res2010; 67:

parkinsonism. Mol Neurodegener 2011;parkinsonism. Mol Neurodegener 6: - 35 - synuclein byneurosin invitro.Neurosci

terns pathologic of normaland

or its aggregationand naka M,TokudaT. - 47

- Abeta Abeta - This article isThis article by protected copyright. All rightsreserved. AJ,HoltonLees ofpathological JL,T. involvementRevesz Thespectrum ofthestriatonigr 25 PLoSneurosin e53250 inpatientswithsynucleinopathy. One 2013; 8: levelsofbothHM. Low alpha CSF 24 enzymes kallikrein withdementia bodies Lewy isassociated withdeclineinth 23 model of systemmultiple atrophy.Mol 2015;Neurodegener 10:48 modifiedtargeted, neurosin (kallikr 22 and reduces thepathologyinanalpha deliveryE. Lentivirusmediated of neurosin 21 synucleinopathies. HumMolGenet2003; 12:2625 synuclein 20 preferentially expressed Biophysin brain.Biochim Acta1997; 1350: 11 H, 19 is expressed inmatureoligodendrocytes. BrainResMol 1999; BrainRes 217 71: 18 1 in humantissuesas assessed byimmunohistochemistry. Cytochem 2001; JHistochem 49: 431

Accepted ArticleYamaguchi N. Molecularcloning of anoveltrypsin - 41 Ozawa T, DG, Paviour D,QuinnNP, KA,SanghaH,KilfordL,Healy Josephs Wood NW, Wennstrom M,SurovaY, F,Hansson HallS,Nilsson L, O, C,Minthon Bostrom Nielsen JS,RenfrewR,SwirskiMiners M,Love S.Accumulation of alpha B,Valera E, Spencer Rockenstein E, Trejo B, Spencer S,ShenMichael J,KosbergK,Rockenstein E,Patrick Adame C, A, Masliah M, Iwata A,Maruyama Akagi T, T, I, Hashikawa S,NukinaN.Kanazawa Tsuji Alpha Yamashiro K,Tsuruoka N,KodamaS,TsujimotoM, YamamuraY,TanakaT, Nakazato H,Yamanaka X,Matsumoto He M/neurosin K,ShiosakaS,YoshidaS.Protease mRNA

degradation protease byserine neurosin: implication offor pathogenesis - 6 and calpain - 1. Neuropathol Acta Commun 2014; 2:164 - synuclein and synuclein the alpha ein - - synuclein Mol modelofTher 2013; LBD. 21: 31 6) reducesalpha

promotes clearance of clearance wild promotes - Morales M,AdameA,Masliah E.Abrain -

35 - like serineprotease (neurosin)

- accumulationsynuclein inamouse - synuclein cl e alpha - synuclein

eaving enzyme - - - synuclein in type alpha type 4

degrading degrading - 24 - synuclein

- 41 al

- - This article isThis article by protected copyright. All rightsreserved. of atrophy.alteration lysosomes multiplesystem in Neuroreport 2012; 23:270 33 2007;22: 766 postmortemthe rostralpons tissue fromof 32 2010;and progressivesupranuclear palsy.BrainRes 1326: 174 insu subunit expression the 31 byboth proteasome.JBiol degraded 2003; andthe autophagy Chem 278: 25009 30 with indementia reduced 2017; Lewybodies.JNeurochem 141:275 29 Themetalloproteinases. Journalofbiological chemistry 2005; 280: 25216 Proteolytic cleavage of extracellular secreted{alpha} 28 the detection of activity. Anal calpain 2003;Biochem 319: 234 27 expression withcolor AL, Webster PJ,Hood DavidsonEH, L, DimitrovK.Directmultiplexed measurement of gene George RD, Grogan JJ,MaysuriaM,Mi T, James 26 correlations. Brain2004;127: 2657 and olivopontocerebellar systems inmultiplesystem atrophy: clinicopathological

Accepted Article Makioka K,Yamazaki T, M,Nakazato Y,OkamotoTakatama Activationand K. Langerveld AJ, MihalkoD,DeLong J,IdeCF.Gene expression changesin C, Walburn S,Bukhatwa ZengBY,RoseS,Jenner P. inproteasomal Acomparisonofchanges Webb JL,Ra JS,LoveMiners S.Endothelin CH, Kim SM,Lee Sung JY, UmJW,LeeHJ, Park J, Oh YJ,ST,PaikLee ChungKC. SR, M. Mittoo L, Synthesis Bradley S,Sundstrom Geiss GK,RE, Bumgarner BirdittB,DahlT,DowidarN,DunawayDL,FellHP, S, Ferree - 77

vikumar B,Atkins JN,Rubinsztein J,Skepper DC.Alpha - coded probepairs.NatBiotechnol 2008; 26: 317 bstantia nigrainParkinson's multiple atrophy system disease, - 71 - converting alpha enzymes degrade

multiple systematrophymultiple patients. Mov Disord tton JD, P, Oliveri OsbornJL, PengT, Ratcliffe

and evaluationoffluorescent probesfor - synucle

in via matrix - - 83

- 86 - 25

- synuclein and are synuclein andare - -

24 Synuclein is Synuclein

- 6 -

This article isThis article by protected copyright. All rightsreserved. chemistry 2011; 286: 14168 3 40 2 the andReduces Synuclein Pathology inan[alpha] E. LentivirusMediatedDelivery of NeurosinPromotes ofWild Clearance 39 2: 5 alphaaffects Woulfe J,Feany M,Myllykangas L,M,Tyynela J. D Schlossmacher Cathepsin expression level 38 andin synuclein Bactivity cathepsin SH N, ZhangJ.Over 37 JournalFASEB 2014;3146 28: Sotiropoulou G,Vekrellis K.of naturally Resistance secreted α 36 againstprotects alpha D,Walls L, LuY, K,ShackaJ,RothZhangJ.Lysosomalenzyme cathepsin Standaert D Parks R,CrabtreeD,Liang Q,Crimmins S,SchneiderL,Uchiyama Y,Iwatsubo Y,Peng T, Zhou 35 formation inoligodendrocytes. JMol Neurosci2012; 47:256 Involvement system ofmacroautophagyinmultiple atrophy andproteinaggregate 34 - 1: 31 mediated mediated alpha

Accepted Article

- 41 Choi DH, Kim YJ, Kim YG, Joh TH, Beal MF,Kim Choi DH,KimYJ,YG,JohTH, metalloproteinaseYS. Beal Roleof matrix Sp V, Cullen Lindfors M D, Crabtree Dodson M, Ximerakis M,Pampalakis G, Roumeliotis TI,SykiotiV L, Qiao S,CaldwellHamamichi K,CaldwellG, Yacoubian T, S,XieZ Wilson L,GoldbaumBergmann Schwarz O,M,

encer encer B, S,ShenMichael J,KosbergK,Rockenstein E,Patrick Adame C, A, Masliah - synuclein synuclein processing, aggregation,andtoxicity invivo.Brain Molecular 2009; - expression of an inactive mutantexpression cathepsin ofaninactive Dincreases alpha endogenous - synuclein cleavage cell death. indopaminergic of TheJournal biological - synuclein aggregationsynuclein Mole andtoxicity. , NgJ,PaetauA, Swinton E,KolodziejP, P, H,Saftig Boston - 77 - 58

Ouyang X,Boyer

- SY5Y cells.2014;J Neurochem 128:950 Probst - Guittaut Ballestas M,LiangQ, ME, Fineberg - Synuclein ModelMolTher 2013; ofLBD. - Cousin S,Richter Cousin

- S, GarbisSD,StefanisL, - 66 - synuclein toproteolysis. The synuclein cular Braincular 2008;1: 17

- Landsberg C. - type [alpha] type - - 61 L, Speake L,L, Speake

- This article isThis article by protected copyright. All rightsreserved. DisordRelat 2008; 14: 309 inthegirus cinguliin of thecalpain/cdk5/p25pathway Parkinson's Parkinsonism disease. 48 7818 ofdegradation of fibrillizedandnitrated species alpha DR.Trojanowski JQ,Lynch Cleavage of alpha 47 processing to disease MB, Feany Masliah E,Rohn TT. Calpain 46 movement disorder.NatRevNeurol2017; 13: 232 45 neurodegenerati 44 up for early exprGene 43 activation. Res2005; Rejuvenation 227 8: Cellular responses accumulationinvolve autophagyandlysosomal enzyme to protein 42 systemdisease andmultiple atrophy. NeurobiolDis 2018; 114:140 Lysosomal responseinrelationtoalpha 41

Accepted Article - 29 Alvira D,FerrerI,Gutierrez Mishizen LR, Dufty BM,Warner HouST, Jiang SX,Gomez F, Krismer insightsWenning GK. atrophy: into anddebilitatingMultiple system arare D,WenningBruck L.Glia GK, N,Fellner Stefanova andalpha AM,Cataldo JL, SA, Barnett Berman LiJ,Quarless S,BursztajnLippaC,NixonRA. D,Brown L,Butler QB,ChinDJ,Batey KarimS,Mu Puska MI, K,Regelsberg G,Lutz Molnar

ession content and cellular ofD disease cathepsin inAlzheimer's evidence brain: - regulation regulation of theendosomal - on: Acomplex interaction. Neurobiol Dis 2016; 85: 262 Eberz AJ,NorrisEberz EH,GiassonBI,HodaraR,IschiropoulosH - linked aggregation.170: AmJPathol2007; 1725 - 13

- Cuesta J, Cuesta Garcia - cleavage of alpha - synuclein pathologydiffersbetweenParkinson's - - lysosomal Neuron 1995; system. 14: 671 37 -

synuclein by calpain: by synuclein potential rolein er G,Ricken G,Pirker W, KovacsLaszlo GG.L, - - 43 Castro B,Pal

- - synuclein. Biochemistry2005; 44: Isla T,Le

- tneja MS, tneja DA,Karanian BahrBA. synuclein: proteolytic connecting synuclein: enhouts KM,OxfordJT, las M, Camins A.M, Camins las Activation - - 52 synuclein in synuclein

- 38 - 74

, LeeVM,

- 80

This article isThis article by protected copyright. All rightsreserved. Biomedical Centre.Research supported theNationalInstitute University by forHealthResearch College London Hospitals Research InstituteNeurological StudiesandtheMedical for Council UK.Thisresearch was Square AtrophySystem Trust.BankisBrain Reta Queen by the supported LilaWest Foundation; Alzheimer’s bytheMultiple UK Research Solutions. AKissupported and CBD bytheKingBaudouin managed Multiple System AtrophyCoalition;FundSophia, MSAcoalitionHoLo) andthe This work was Acknowledgements 2014;23: 3975 aggregation andsynaptic im of thecalpain Melki R,Pieri S,L, Helling K, Marcus Krueger RiessR, MasliahE, O,S.Overexpression Nuber 51 979 disorders involving mesencephalon: 1996; aroleinnervecell Neuroscience the death? 73: the mesencephalon withParkinson's diseaseof patients butnot other neurodegenerative in 50 Parkinson'sdisease. JNeurosci2003; 23:4081 prevents calpains Inhibitionof neuronalandbehavioraldeficits in anMPTPmousemodelof RS, SM, Slack Melloni E,P 49

Accepted Article - 87

Diepenbroek M,Casadei H,N, Esmer S Mouatt SJ,Crocker Smith PD, Jackson - specific inhibitorspecific calpastatin reduceshumanalpha funded - - 89 Prigent A,KarlssonPrigent Incr JO, EC. AgidY,Hirsch

by the Multiple System Atrophy rzedborski rzedborski S, Robertson GS,AnismanH,Merali Z,ParkDS. pairment in [A30P]alphaSyn transgenicpairment in[A30P]alphaSyn mice.HumMol Genet .

JLH is supported by the Multiple issupportedbythe JLH System Atrophy - Lewis V, Lamba WR, Hayley SP,V, WR,Hayley Lamba Lewis GrimmE,Callaghan aido TC, J,KahlePJ,NixonRA,RaoMV, Takano - 91

(MSA) (MSA)

eased eased M trust - Synuclein processing, Synuclein

(Grant number1402(Grant - calpain expressionin calpain

Trust; the on This article isThis article by protected copyright. All rightsreserved. tocompared control. CTSD (E expression ofKLK6 (D), indetectedis detected difference inMSAfrontallobe.No inthemRNA significant tocompared control No inCTSD (B) (A). significantdifference or (C) mRNA SNCA expression tocompared control Figure Legends: authorship, The author(s)declare Conflict of interest is storedunderresearch for alicense Tissue (No.issued bytheHuman Authority 12198). received ethical approvalforCommitteeLondon bytheNRES research Disorders, UCL Instituteof TheNeurology. donation and brain programme tissuedonated SquareWe usedbrain to fromcases Brainthe Queen Bank for Neurological Ethical approval and JH analy responsible foracquisitionofdata; SL Author contributions Figure 1.Figure

Accepted Articleand s ed and interpreted thedata;

JH revis

Graphical representation ofexpressionGraphical inmRNA fold change were responswere

ed andreviewedfinal the article forintellectualcontentapproval. andfinal and/or publication of this and/or publication

(n = 6) (n

Bars indicate indicate andBars the mean

no potential with conflictsofinterest ible fortheconception and designofexperiments; JSM . Significantly ) or SNCA (F) inthe) orSNCA(F) cerebellar whitematterof MSA cases RC andCSperformed theRC immunohistochemistry; all authorscontributedto

article.

greater KLK6 is mRNA detected lobe inMSAfrontal

standard deviation.

respect to theresearch,respect

draft ing

the manuscript; SL the –

Central andCentral tissue MSA protocols have

(n= 6)(n=

and AK

JSM

were were

This article isThis article by protected copyright. All rightsreserved. **P < 0.01 ***P < 0.001****P < 0.0001. occipital(CBM), andfrontallobe Barsindicate lobe(OCC) (FCX)). themean andSEM. (PUT)putamen pontinebase (PNS),cerebellar nucleus(caudate), caudate white load represents thepercentagesection areaimmunopositive forα Figure the and indicate mean caudate nucleus,base, cerebellarputamen, white pontineand matter lobe.occipital immunoblots performed intriplicatewere to andnormalised 7 4.Figure Graphical representation of levelsof cathepsin and pontine nucleus, base, cerebellar white matter andoccipital lobe. inandperformed triplicate normalisedto MSA (M) 3.Figure Graphical representation of levelsof calpain and pontine nucleus, base, cerebellar white matter andoccipital lobe. inandperformed triplicate normalisedto MSA (M) 2.Figure Graphical representation of levelsof kallikrein )

Acceptedand MSA(M) Article standard deviation standard deviation 5 .

Bar chartshowing Bar regional (n = (n = 7 7 ) )

(n = 7)(n = following westernb following western blot analysis. Densitometry analysis offollowing westernblotanalysis.Densitometryanalysis immunoblotswere

standard devia

following we stern blot analysis. Densitometry analysisstern blotanalysis.Densitometry of lot analysis. Densitometry analysis oflot analysis.Densitometry analysis immunoblotswere α tion - syn load in multiple system atrophy multiplesystem syn loadin

β β - - actin for frontal lobe, forfrontal actin puta lobe forfrontal actin - 1 (CAPN1) incontrol(C) - - 6 (KLK6) incontrol(C)

D (CTSD) β - actin forfrontalactin lobe, , putamen, caudate, putamen,

(n = 6)(n = Bars indicate the indicate Bars mean the indicate Bars mean - syn determined syn in men, caudate

in control(C)

(n = 20)(n =

(n = (n = (n = (n =

matter . 7 7 α ) )

Bars Bars - and and syn

(n = (n = This article isThis article by protected copyright. All rightsreserved. Accepted respectively line andouter (rfu).activity represent The circles individual. mean solid the level fromeach The thick CAPN1 enzyme activity CAPN1 (rfu).(C) plotted enzyme activity against CTSDenzyme (relativeactivity unitsKLK6 fluorescence (rfu)).(B) protein lev KLK6 of MSA(n=20)20). (A) proteinlevel(ng/ml)(n = andcontrols plottedagainstCTSD 8.Figure Relationship KLK6,between inacombinedcohort CAPN1CTSDincerebellum and regression and 95% confidence andprotease(verticalbars).Thethicksolidbars) thebest anddottedlinesindicate themeanThe solid andthinbarsindicate circles valuesandSEMfor cerebellar whiteoccipital matter (CBM), a lobe(OCC) Articleagainst 20)(n = Figure *P < 0.05 ***P < 0.001 ****P < 0.0001. (CBM), matter occipital andfrontallobe lobeindicate (FCX)). Bars mean (OCC) the and SEM. (PUT)(n=20) inputamen cau substrateactivityfluorogenic peptide assaysweremeasured inMSA(n=20)andcontrols levels, measured bysandwich andCAPN ELISA (n=20) and controls Figure 7 6

α . . Regional association between ( between . Regionalassociation

and controls - Bar chartsshowing Bar syn load in putamen (PUT)pontine nucleus(caudate), (PNS), loadcaudate base in putamen syn .

lines indicatethebest

across (n = 20) (n regions with differentpredilectionregions for pathology date nucleusdate (caudate), pontine (PNS),cerebellar base white differences . Thelevel or activityofeachproteasewasplotted protein

intervals. - fit linear

A in

) KLK6, ( (A) (A)

1 and CTSD 1 and regression intervals and95% confidence KLK6 B ) CAPN1, ( (B) (B) nd frontallobe inMSAcases. (FCX)) CAPN1 and

enzyme enzyme C el (ng/ml) plottedagainst el (ng/ml) )

CTSD and activities using (C) α - CTSD inMSA syn load(horizontal syn α - syn load . KLK6 protein KLK6 protein -

fit linear fit linear (n = 20)(n =

in MSA

inner

This article isThis article by protected copyright. All rightsreserved. Accepted Article