Ci a ion: Ace a, A.;
Gómez-Es eban, J.C.;
Mu ue a-Goyena, A.; Galdos, M.;
Azka go a, M.; Elo za, F.;
Ruza a, N.; Iba ondo, O.; Pe ei o, X.;
Vecino, E. Po en ial Tea Bioma ke s
o he Diagnosis o Pa kinson’s
Disease—A Pilo S udy. P o eomes
2022,10, 4. h ps://doi.o g/10.3390/
p o eomes10010004
Academic Edi o s: Jens R. Coo ssen
and Ma hew P. Padula
Recei ed: 7 Oc obe 2021
Accep ed: 10 Janua y 2022
Published: 13 Janua y 2022
Publishe ’s No e: MDPI s ays neu al
wi h ega d o ju isdic ional claims in
published maps and ins i u ional a il-
ia ions.
Copy igh : © 2022 by he au ho s.
Licensee MDPI, Basel, Swi ze land.
This a icle is an open access a icle
dis ibu ed unde he e ms and
condi ions o he C ea i e Commons
A ibu ion (CC BY) license (h ps://
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4.0/).
p o eomes
A icle
Po en ial Tea Bioma ke s o he Diagnosis o Pa kinson’s
Disease—A Pilo S udy
A an xa Ace a 1,2 , Juan Ca los Gómez-Es eban 3,4,5, Ane Mu ue a-Goyena 4,5 , Ma a Galdos 6,
Mikel Azka go a 7, Felix Elo za 7, Noelia Ruza a 1,6, Oli e Iba ondo 8, Xand a Pe ei o 1,6 and Elena Vecino 1,6,*
1
Expe imen al Oph halmo-Biology G oup, Depa men o Cell Biology and His ology, Uni e si y o he Basque
Coun y UPV/EHU, 48940 Leioa, Spain; [email p o ec ed] (A.A.); [email p o ec ed] (N.R.);
xand a.pe ei [email p o ec ed] (X.P.)
2Depa men o Neu oscience, Biodonos ia Heal h Resea ch Ins i u e, 20014 Donos ia-San Sebas ian, Spain
3Depa men o Neu ology, C uces Uni e si y Hospi al, 48903 Ba akaldo, Spain;
[email p o ec ed]
4Neu odegene a i e Diseases G oup, BioC uces Heal h Resea ch Ins i u e, 48903 Ba akaldo, Spain;
[email p o ec ed]
5Depa men o Neu osciences, Facul y o Medicine and Nu se y, Uni e si y o he Basque Coun y
UPV/EHU, 48940 Leioa, Spain
6Begike -Oph halmology Resea ch G oup, BioC uces Heal h Resea ch Ins i u e, 48903 Ba akaldo, Spain;
[email p o ec ed]
7Depa men o P o eomics, Cen e o Coope a i e Resea ch in Biosciences, 48160 De io, Spain;
[email p o ec ed] (M.A.); [email p o ec ed] (F.E.)
8RS-S a is ics, 20500 A asa e, Spain; [email p o ec ed]
*Co espondence: [email p o ec ed]; Tel.: +34-94-601-2820
Abs ac :
Pa kinson’s disease (PD) is he second mos common neu odegene a i e disease a e
Alzheime ’s disease. In his s udy, he ea p o eome p o ile o pa ien s wi h idiopa hic PD (iPD,
n= 24), ca ie s o he E46K-SNCA mu a ion (n= 3) and heal hy con ol (CT, n= 27) subjec s was
analyzed o iden i y candida e bioma ke s o he diagnosis o PD. An obse a ional, p ospec i e
and case-con ol pilo s udy was ca ied ou , analyzing he pa icipan s ea samples by nano-liquid
ch oma og aphy–mass spec ome y (nLC–MS/MS) and assessing hei neu ological impai men .
The p o eomic da a ob ained a e a ailable a P o eomeXchange wi h iden i ie 10.6019/PXD028811.
These analyses led o he iden i ica ion o 560 ea p o eins, some o which we e de egula ed in PD
pa ien s and ha ha e been implica ed in immune esponses, in lamma ion, apop osis, collagen
deg ada ion, p o ein syn hesis, de ense, lipid anspo and al e ed lysosomal unc ion. O hese
p o eins, six we e ela ed o neu odegene a i e p ocesses and showed a good capaci y o classi y
pa ien s and con ols. These indings e ealed ha ce ain p o eins we e up egula ed in he ea s o
PD pa ien s, mainly p o eins in ol ed in lysosomal unc ion. Thus, in his s udy, ea p o eins we e
iden i ied ha a e implica ed in neu odegene a ion and ha may be ela ed o an agg essi e disease
pheno ype in PD pa ien s.
Keywo ds: bioma ke s; Pa kinson’s disease; ea ilm; lysosome
1. In oduc ion
Pa kinson’s disease (PD) is he second mos common neu odegene a i e disease a e
Alzheime ’s disease (AD), and i is cha ac e ized by slowness o mo emen (b adyki-
nesia), inc eased muscle one ( igidi y), shaking ( emo ) and impai ed pos u al con ol.
In pa hologically con i med PD, he e is a a iable amoun and deposi ion o mis olded
alpha-synuclein (
α
-syn) in he o m o Lewy bodies (LBs) and Lewy neu i es in he cen al
ne ous sys em (CNS). This a iabili y in he accumula ion o hese s uc u es is likely o
e lec he clinical he e ogenei y in his disease [
1
–
3
]. Indeed, se e al clinical diso de s lie
in he spec um o Lewy body diseases (LBDs), including pa ien s wi h idiopa hic (iPD)
P o eomes 2022,10, 4. h ps://doi.o g/10.3390/p o eomes10010004 h ps://www.mdpi.com/jou nal/p o eomes
P o eomes 2022,10, 4 2 o 15
and gene ic PD and pa ien s su e ing demen ia wi h LBs. One o he main challenges in
dealing wi h neu odegene a i e diseases is o ind clinical ma ke s ha allow o he ea ly
classi ica ion o pa ien s and help o moni o disease p og ession.
Fac o s such as oxida i e s ess, p o eolysis, al e ed a y acid o phospholipid con-
cen a ions ha e been associa ed wi h s uc u al changes o
α
-syn, and pos - ansla ional
modi ica ions can also al e he size, s uc u e o p o ein load o
α
-syn [
4
]. De egula ing
mu a ions and mul iplica ions in he
α
-syn gene (SNCA) cause au osomal-dominan amil-
ial o ms o PD [
5
]. Pe haps he mos pa hogenic mu a ion known o induce PD is he E46K
mu a ion in
α
-syn (E46K-SNCA), which unde lies a g oup o agg essi e, gene ic LBDs
wi h clinical and pa hological ea u es eminiscen o demen ia wi h LBs [
6
,
7
]. Al hough
mu a ions in
α
-syn a e a e, hey p o ide a unique oppo uni y o iden i y pa ien s wi h
PD and o be e unde s and he p og ession o hese pa hologies.
I is known ha PD pa ien s de elop hallucina ions, changes in eye and eyelid mo e-
men , and al e ed ea composi ion and in educed amoun s [
8
,
9
]. In addi ion o eye-su ace
damage, hese pa ien s can expe ience changes in accommoda ion, educed isual acui y,
sco oma o ma ion (a eas whe e hei ield o ision is pa ially diminished o comple ely
degene a ed) and a hinning o he e inal laye s, pa icula ly due o a educ ion in he num-
be o ne e ibe s [
10
,
11
]. De iciencies in neu o ansmission and monoamine me abolism
also in luence he pa hological changes o he isual sys em o PD pa ien s [
12
]. These
changes a e qui e ypical, since monoamines a e in ol ed in he ansmission o isual
in o ma ion in he e ina [
13
]. Mo eo e , PD pa ien s may expe ience al e a ions o he
inne a ion o he an e io pa o he eye ha can ul ima ely al e he composi ion o ea s.
Many neu o ansmi e s and ophic subs ances a e sec e ed in ea s, o igina ing
om he aqueous humo o om ne es unning h ough he co nea [
14
]. Howe e , he
cellula and molecula mechanisms ha unde lie hese associa ions ha e ye o be ully
elucida ed. As PD a ec s se e al non-mo o sys ems and pe iphe al ne es, lac imal
sec e ion could be al e ed in hese pa ien s, and he ea p o ein composi ion migh exhibi
a cha ac e is ic p o ile in hese pa ien s ha could se e as a diagnos ic bioma ke . In
his sense, analyzing he ea p o eins o pa ien s wi h E46K-SNCA and iPD could help o
iden i y use ul candida e bioma ke s o he diagnosis and p og ession o PD. The e o e,
he aim o he p esen s udy was o analyze he ea p o eome p o ile in iPD pa ien s and
ca ie s o he E46K-SNCA mu a ion in o de o ind a bioma ke ha could help in he
u u e diagnosis o PD, using non-in asi e samples.
2. Ma e ials and Me hods
2.1. S udy Coho
We designed an obse a ional, p ospec i e, case-con ol pilo s udy in which
24 pa ien s
wi h iPD, 3 ca ie s o he E46K-SNCA mu a ion and 27 heal hy subjec s (CTs) we e en olled.
This esea ch was conduc ed by medically quali ied pe sonnel a e ecei ing app o al om
he OSI Ezke alde-Enka e i-C uces E hics Commi ee CEIC E18/47. The s udy was ca ied
ou in s ic acco dance wi h he ene s o he Helsinki Decla a ion on Biomedical Resea ch
In ol ing Human Subjec s. Be o e sample collec ion, signed in o med consen was ob ained
om all he subjec s a e ha ing explained he na u e and possible consequences o
he s udy. The pa ien s we e ec ui ed a he Neu ology and Oph halmology Se ice
Uni s o he C uces Uni e si y Hospi al (Ba akaldo, Bizkaia, Spain) du ing ou pa ien
consul a ions, based on he ag eed inclusion and exclusion c i e ia. Pa ien s wi h iPD
ul illed he Pa kinson’s UK B ain Bank c i e ia o he diagnosis o PD and did no ca y
any known mu a ions. The inclusion c i e ia o CTs we e o be o e 40 yea s o age, o
ei he gende and wi hou a clinical his o y o any neu ological diso de . Indi iduals
wi h (o wi h a his o y o ) any sys emic o ocula diso de /condi ion we e included in he
s udy. The exclusion c i e ia consis ed o eye su ge y in he p e ious h ee mon hs, ch onic
eye medica ion (e.g., o glaucoma), a his o y o alle gies, using any opical medica ion
(o he han a i icial ea s) o o al medica ion con aining co icos e oids, a opy and pa ien s
wi h Sjög en’s synd ome. Con ac -lens use s we e also excluded o a oid any possible
P o eomes 2022,10, 4 3 o 15
in e e ence wi h he in e p e a ion o he esul s. P elimina y ocula es s and sample
collec ion we e pe o med on he same day, while pa ien s wi h PD we e also seen on a
sepa a e day by membe s o he esea ch eam who a e expe s in mo emen diso de s o
assess he Pa kinson’s Scales.
2.2. Oph halmological Examina ion
The clinical oph halmological examina ion included he Schi me ’s es I (SCH), which
de e mines whe he he eye p oduces enough ea s o keep i mois . The es allows he
wa e in ea s o a el along he leng h o a pape es s ip, and no mal alues a e
≥
10 mm
we ing o he pape a e 5 min. Tea - ilm ins abili y was assessed by using ea b eakup
ime (TBUT) wi h luo escein. TBUT is he ime aken o he i s d y spo o appea on
he co nea a e a comple e blinking. A alue less han 10 s usually e lec s some ea - ilm
ins abili y. Any sys emic disease o any o he condi ion/medica ion use ha could in e e e
wi h he in e p e a ion o he esul s was conside ed a c i e ion o exclusion.
2.3. Neu ological Examina ion
A neu ologis expe in he ield o mo emen diso de s eco ded he age o he PD
pa ien s a ea collec ion, hei disease du a ion and hei sco e on he Uni ied Pa kinson’s
Disease Ra ing Scale (UPDRS) and Hoehn Yah scale. These es s se e as s a i ica ion
c i e ia o ack he p og ession o he disease. The con iden iali y o pa ien da a was
ensu ed h ough he use o he Basque Biobank ((www.biobanco asco.o g (accessed on
2 No embe 2019).
2.4. Tea -Sample Collec ion
All ea samples we e collec ed by using calib a ed 10
µ
L glass mic ocapilla y ubes
(BLAUBRAND in aMa k, We heim, Ge many). Tea samples we e ob ained om he
in e io empo al ea meniscus, minimizing i i a ion o he ocula su ace o lid ma gin,
and wi hou ins alla ion o anes hesia. The ea samples we e collec ed om bo h eyes
o each pa icipan and immedia ely placed in p ecooled Eppendo ubes. A e ea
collec ion, he samples we e s o ed a −80 ◦C un il hei analyses.
2.5. P o eomics Analyses
The p o eomic analyses we e ca ied ou a he CIC bioGUNE P o eomics Se ice
(De io, Bizkaia, Spain), using he Fil e Aided Sample P epa a ion (FASP) p o ocol [
15
] o
sample p ocessing and diges ion. T ypsin (Sigma Ald ich, S . Louis, MO, USA) was added
a a ypsin:p o ein a io o 1:50, and he mix u e was incuba ed o e nigh a 37
◦
C, d ied
in a RVC2 25 Speed ac concen a o (Ch is , Vienna, Aus ia). The pep ides ob ained we e
desal ed and esuspended in 0.1% Fo mic Acid (FA) (Sigma Ald ich, S . Louis, MO, USA)
using C18 s age ips (Millipo e, S . Louis, MO, USA).
Samples we e analyzed in a no el hyb id apped ion mobili y spec ome y quad upole
ime o ligh mass spec ome e ( imsTOF P o wi h PASEF, B uke Dal onics, B emen, Ge -
many), coupled online o a nanoElu e liquid ch oma og aph (B uke , Co en y, UK). This
mass spec ome e akes ad an age o a no el scan mode e med pa allel accumula ion
se ial agmen a ion (PASEF), which mul iplies he sequencing speed wi hou any loss
in sensi i i y, and i has been p o en o p o ide ou s anding analy ical speed and sensi-
i i y o p o eomics analyses [
16
]. Samples (200 ng) we e di ec ly loaded on o a 15 cm
B uke nanoelu e FIFTEEN C18 analy ical column (B uke , Co en y, UK) and esol ed a
400 nL/min. The column was hea ed o 50 ◦C in an o en.
P o ein iden i ica ion and quan i ica ion we e ca ied ou by using he PEAKS so wa e
(Bioin o ma ics solu ions Inc., Wa e loo, CA, USA) Sea ches we e ca ied ou agains a
da abase o canonical human Unip o /Swissp o en ies (no iso o ms conside e ), wi h
p ecu so and agmen ole ances o 20 ppm and 0.05 Da. An a ea-based label- ee p o ein
quan i ica ion was pe o med by using he PEAKS Q module a ailable in he PEAKS
so wa e. Only p o eins iden i ied wi h a leas wo pep ides a a False Disco e y Ra e
P o eomes 2022,10, 4 4 o 15
(FDR) < 1% and p esen in a leas 70% o he samples om one o he expe imen al
g oups unde analysis we e conside ed o u he analysis. The da a we e loaded on o he
Pe seus pla o m [
17
] and u he p ocessed (log2 ans o ma ion, impu a ion) be o e he
applica ion o a S uden ’s - es o di e en ial p o ein-exp ession analysis. The p o eomics
da a a e summa ized in Supplemen a y Ma e ials File S1, and he mass spec ome y
p o eomics da a we e deposi ed o he P o eomeXchange Conso ium ia he PRIDE [
18
]
pa ne eposi o y, wi h he da ase iden i ie s PXD028811 and 10.6019/PXD028811.
2.6. S a is ical Analyses
S a is ical analyses we e ca ied ou by using he IBM SPSS S a is ics package o
Windows, 23.0 (IBM-SPSS, A monk, New Yo k, NY, USA). The no mali y o con inuous
da a was assessed wi h a Shapi o–Wilk es . The p- alues calcula ed de e mine he p oba-
bili y ha he associa ion be ween p o eins in he da ase and a gi en canonical pa hway,
unc ional ne wo k o ups eam egula o is explained by chance alone, based on a Fishe ’s
exac es , wi h a p- alue < 0.05 being conside ed signi ican .
Uni a ia e logis ic eg essions we e used o iden i y he s a is ically signi ican cha ac-
e is ics associa ed wi h PD-pa ien disc imina ion. A e a iable selec ion, mul i a ia e
logis ic eg essions we e used o cons uc a PD disc imina ion model and o iden i y he
clinical and p o eomic a iables ele an o PD iden i ica ion. Logis ic models allow he
ela ionship be ween a disc e e dependen a iable and a se o con inuous o disc e e
independen a iables o be es ima ed. The models allow he classi ica ion o pa ien s as
wi h o wi hou PD, calcula ing he p obabili y o belonging o each g oup. The p- alue o
each co a ia e desc ibes he s a is ical signi icance o he associa ion be ween he esponse
and each e m included in he model.
3. Resul s
3.1. Pa ien s and Clinical Pa ame e s
Be ween Ma ch 2019 and Oc obe 2019, 24 pa ien s wi h iPD, h ee symp oma ic ca i-
e s o he E46K-SNCA mu a ion and 27 CT subjec s we e included in his pilo s udy, whose
gene al cha ac e is ics a e p esen ed in Table 1. The PD pa ien s’ mean disease du a ion
was 9.36 yea s, and hey had mild bila e al mo o disabili y (UPDRS sco e) and Hoehn and
Yah sco es ha sugges ed mild- o-mode a e mo o impai men . A cha ac e is ic o iPD
pa ien s was ha 40% o hem had mild- o-mode a e blepha i is.
Table 1. Demog aphics, PD cha ac e is ics and eye condi ions.
Va iables, Uni s iPD E46K-SNCA Con ol
n24 3 27
Age, yea s 60.4 (9.21) 45 (14.73) 49.69 (12.17)
Gende (F/M) (%) 38/62 0/100 54/46
Disease du a ion, yea s 9.36 (6.66) 3.33 (3.51) N/A
Hoehn and Yah sco e 2 (0.82) 2 (1) N/A
UPDRS sco e (%) 32.39 (9.03) 16.56 (11.12) N/A
Pa I UPDRS sco e 17.19 (12.6) 6.25 (0.0) N/A
Pa II UPDRS sco e 27.52 (10.07) 11.54 (8.38) N/A
Pa III UPDRS sco e 45.13 (15.09) 25.00 (16.7) N/A
Pa IV UPDRS sco e 16.30 (9.46) 10.14 (9.05) N/A
Blepha i is (%) 40.74 0 0
The esul s a e displayed as he means (s anda d de ia ion), excep o he sex and Hoehn and Yah sco es ha a e
shown as he pe cen age and median: UPDRS, Uni ied Pa kinson’s Disease Ra ing Scale; n, numbe o subjec s; F,
emale; iPD, idiopa hic Pa kinson’s disease; M, male.
3.2. nLC MS/MS Da a
A o al o 560 ea p o eins we e iden i ied in he samples analyzed he e, in acco -
dance wi h p e ious p o eomic s udies on ea s, and se e al g oup-speci ic ea p o eome
al e a ions we e e iden in he PD pa ien s ela i e o he CTs. P o eins wi h di e en
P o eomes 2022,10, 4 5 o 15
abundances we e de ec ed in bo h g oups, al hough mos o he de egula ed p o eins in he
ea p o eome o PD pa ien s we e o e exp essed. An analysis o he STRING PPI ne wo k
was pe o med o e alua e he in e ac ions be ween p o eins ha di e ed signi ican ly in
he PD pa ien s and CTs (Figu e 1). P o eins ha we e o e exp essed in PD pa ien s we e
implica ed in signi ican ly mo e in e ac ions han expec ed, indica ing ha he inc eases
in hose p o eins ha e some biological connec ion (Figu e 1A). Howe e , weake ela ion-
ships we e obse ed be ween he p o eins ha we e down egula ed, ep esen ing a clea ly
di e en ia ed clus e (Figu e 1B). Mos o he di e en ially exp essed p o eins could be
localized o ei he he ex acellula and in acellula compa men s.
P o eomes 2022, 10, x FOR PEER REVIEW 5 o 15
Blepha i is (%) 40.74 0 0
The esul s a e displayed as he means (s anda d de ia ion), excep o he sex and Hoehn and Yah
sco es ha a e shown as he pe cen age and median: UPDRS, Uni ied Pa kinson’s Disease Ra ing
Scale; n, numbe o subjec s; F, emale; iPD, idiopa hic Pa kinson’s disease; M, male.
3.2. nLC MS/MS Da a
A o al o 560 ea p o eins we e iden i ied in he samples analyzed he e, in acco d-
ance wi h p e ious p o eomic s udies on ea s, and se e al g oup-speci ic ea p o eome
al e a ions we e e iden in he PD pa ien s ela i e o he CTs. P o eins wi h di e en
abundances we e de ec ed in bo h g oups, al hough mos o he de egula ed p o eins in
he ea p o eome o PD pa ien s we e o e exp essed. An analysis o he STRING PPI ne -
wo k was pe o med o e alua e he in e ac ions be ween p o eins ha di e ed signi i-
can ly in he PD pa ien s and CTs (Figu e 1). P o eins ha we e o e exp essed in PD pa-
ien s we e implica ed in signi ican ly mo e in e ac ions han expec ed, indica ing ha he
inc eases in hose p o eins ha e some biological connec ion (Figu e 1A). Howe e , weake
ela ionships we e obse ed be ween he p o eins ha we e down egula ed, ep esen ing
a clea ly di e en ia ed clus e (Figu e 1B). Mos o he di e en ially exp essed p o eins
could be localized o ei he he ex acellula and in acellula compa men s.
(A)
P o eomes 2022, 10, x FOR PEER REVIEW 6 o 15
(B)
Figu e 1. A STRING PPI ne wo k analysis showing he di ec in e ac ions o he bioma ke s iden i-
ied. The associa ions be ween he p o eins shown a e in ended o be speci ic and signi ican , such
ha he p o eins ha in e ac pa icipa e in a sha ed ac i i y. Splicing iso o ms o pos - ansla ional
modi ica ions collapse, such ha each node ep esen s all p o eins p oduced by a single p o ein-
coding gene locus. (A) P o ein–p o ein in e ac ion (PPI) ne wo k o he p o eins up egula ed in PD
pa ien s ela i e o he CTs. (B) PPI ne wo k o he p o eins down egula ed in he ea luid o PD
pa ien s.
To be e unde s and he po en ial ole o he di e en ially exp essed p o eins in a
biological con ex , unc ional anno a ion analyses we e pe o med wi h di e en gene on-
ology (GO) e ms. As a esul , de egula ed p o eins we e seen o be in ol ed in di e en
biological p ocesses (Figu e 2): he immune esponse (lipocalin 2—LCN 2, se pin B3—
SPB3, ollicula dend i ic cell sec e ed pep ide—FDSCP, lac adhe in—MFGM and neu o-
phil gela inase-associa ed lipocalin—NGAL); apop osis (ol ac omedin-4—OLFM4,
caspase-14—CASPE and gamma-glu amylcyclo ans e ase—GGCT); in lamma ion (Cal-
p o ec in-S100A8, S100A9); collagen deg ada ion (myeloblas in—PRTN3 and me allop o-
einase 9—MMP9); p o ein syn hesis (he e ogeneous nuclea ibonucleop o ein A3—
ROA3); ke a inocy e di e en ia ion (calmodulin-like p o ein 5—CALML5); lipid
anspo (apolipop o ein D—APOD, apolipop o ein A2—APOA2 and low-densi y-lipo-
p o ein- ela ed p o ein 2—LRP2); and de ense (myelope oxidase—MPO, de ensin alpha
3—DEFA 3, glu a hione pe oxidase 3—GPX3, chi inase-3-like p o ein 2—CH3L2 and ly-
sozyme C—LYSC).
Figu e 2. Rela i e biological unc ions o he de egula ed p o eins iden i ied in PD ea s exp essed
as a %.
I he PD pa ien s we e conside ed all oge he , and sepa a ely as iPD pa ien s and
pa ien s wi h he E46K-SNCA mu a ion, up egula ed ea p o eins we e e iden in all
P o ein
syn hesis
Immune
esponse
Apop osis
De ence
Ke a inocy e
di e en ia ion
Neu odegene a ion
O he s
In lamma ion
Collagen
deg ada ion
Lipid anspo
Figu e 1.
A STRING PPI ne wo k analysis showing he di ec in e ac ions o he bioma ke s iden i ied.
The associa ions be ween he p o eins shown a e in ended o be speci ic and signi ican , such ha
he p o eins ha in e ac pa icipa e in a sha ed ac i i y. Splicing iso o ms o pos - ansla ional
modi ica ions collapse, such ha each node ep esen s all p o eins p oduced by a single p o ein-
coding gene locus. (
A
) P o ein–p o ein in e ac ion (PPI) ne wo k o he p o eins up egula ed in
PD pa ien s ela i e o he CTs. (
B
) PPI ne wo k o he p o eins down egula ed in he ea luid o
PD pa ien s.
P o eomes 2022,10, 4 6 o 15
To be e unde s and he po en ial ole o he di e en ially exp essed p o eins in a bio-
logical con ex , unc ional anno a ion analyses we e pe o med wi h di e en gene on ology
(GO) e ms. As a esul , de egula ed p o eins we e seen o be in ol ed in di e en biological
p ocesses (Figu e 2): he immune esponse (lipocalin 2—LCN 2, se pin B3—SPB3, ollicula
dend i ic cell sec e ed pep ide—FDSCP, lac adhe in—MFGM and neu ophil gela inase-
associa ed lipocalin—NGAL); apop osis (ol ac omedin-4—OLFM4,
caspase-14—CASPE
and
gamma-glu amylcyclo ans e ase—GGCT); in lamma ion (Calp o ec in-S100A8, S100A9);
collagen deg ada ion (myeloblas in—PRTN3 and me allop o einase 9—MMP9); p o ein
syn hesis (he e ogeneous nuclea ibonucleop o ein A3—ROA3); ke a inocy e di e en i-
a ion (calmodulin-like p o ein 5—CALML5); lipid anspo (apolipop o ein D—APOD,
apolipop o ein A2—APOA2 and low-densi y-lipop o ein- ela ed p o ein 2—LRP2); and
de ense (myelope oxidase—MPO, de ensin alpha 3—DEFA 3, glu a hione pe oxidase
3—GPX3, chi inase-3-like p o ein 2—CH3L2 and lysozyme C—LYSC).
P o eomes 2022, 10, x FOR PEER REVIEW 6 o 15
(B)
Figu e 1. A STRING PPI ne wo k analysis showing he di ec in e ac ions o he bioma ke s iden i-
ied. The associa ions be ween he p o eins shown a e in ended o be speci ic and signi ican , such
ha he p o eins ha in e ac pa icipa e in a sha ed ac i i y. Splicing iso o ms o pos - ansla ional
modi ica ions collapse, such ha each node ep esen s all p o eins p oduced by a single p o ein-
coding gene locus. (A) P o ein–p o ein in e ac ion (PPI) ne wo k o he p o eins up egula ed in PD
pa ien s ela i e o he CTs. (B) PPI ne wo k o he p o eins down egula ed in he ea luid o PD
pa ien s.
To be e unde s and he po en ial ole o he di e en ially exp essed p o eins in a
biological con ex , unc ional anno a ion analyses we e pe o med wi h di e en gene on-
ology (GO) e ms. As a esul , de egula ed p o eins we e seen o be in ol ed in di e en
biological p ocesses (Figu e 2): he immune esponse (lipocalin 2—LCN 2, se pin B3—
SPB3, ollicula dend i ic cell sec e ed pep ide—FDSCP, lac adhe in—MFGM and neu o-
phil gela inase-associa ed lipocalin—NGAL); apop osis (ol ac omedin-4—OLFM4,
caspase-14—CASPE and gamma-glu amylcyclo ans e ase—GGCT); in lamma ion (Cal-
p o ec in-S100A8, S100A9); collagen deg ada ion (myeloblas in—PRTN3 and me allop o-
einase 9—MMP9); p o ein syn hesis (he e ogeneous nuclea ibonucleop o ein A3—
ROA3); ke a inocy e di e en ia ion (calmodulin-like p o ein 5—CALML5); lipid
anspo (apolipop o ein D—APOD, apolipop o ein A2—APOA2 and low-densi y-lipo-
p o ein- ela ed p o ein 2—LRP2); and de ense (myelope oxidase—MPO, de ensin alpha
3—DEFA 3, glu a hione pe oxidase 3—GPX3, chi inase-3-like p o ein 2—CH3L2 and ly-
sozyme C—LYSC).
Figu e 2. Rela i e biological unc ions o he de egula ed p o eins iden i ied in PD ea s exp essed
as a %.
I he PD pa ien s we e conside ed all oge he , and sepa a ely as iPD pa ien s and
pa ien s wi h he E46K-SNCA mu a ion, up egula ed ea p o eins we e e iden in all
P o ein
syn hesis
Immune
esponse
Apop osis
De ence
Ke a inocy e
di e en ia ion
Neu odegene a ion
O he s
In lamma ion
Collagen
deg ada ion
Lipid anspo
Figu e 2.
Rela i e biological unc ions o he de egula ed p o eins iden i ied in PD ea s exp essed
as a %.
I he PD pa ien s we e conside ed all oge he , and sepa a ely as iPD pa ien s and
pa ien s wi h he E46K-SNCA mu a ion, up egula ed ea p o eins we e e iden in all h ee
o hese g oups, wi h hei exp ession a ying in each (Figu e 3). I should be no ed ha
only h ee pa ien s ca ying he E46K-SNCA mu a ion we e s udied, and he dispe sion o
he da a om hen was g ea e . The same p o eins we e mo e s ongly exp essed in he
ea s o pa ien s wi h he E46K-SNCA mu a ion han in he ea s o pa ien s wi h iPD. All
o hese compa isons we e made on he alues ela i e o hose o he CT ea samples.
P o eomes 2022, 10, x FOR PEER REVIEW 7 o 15
h ee o hese g oups, wi h hei exp ession a ying in each (Figu e 3). I should be no ed
ha only h ee pa ien s ca ying he E46K-SNCA mu a ion we e s udied, and he dispe -
sion o he da a om hen was g ea e . The same p o eins we e mo e s ongly exp essed
in he ea s o pa ien s wi h he E46K-SNCA mu a ion han in he ea s o pa ien s wi h
iPD. All o hese compa isons we e made on he alues ela i e o hose o he CT ea
samples.
Figu e 3. Rep esen a ion o he old change in exp ession o he signi ican p o eins when he pa-
ien s included in he s udy we e conside ed as iPD pa ien s (o ange), E46K-SCNA ca ie s (g ay)
o all PD pa ien s oge he (blue). The same de egula ed p o eins appea in all h ee g oups, bu he
changes in exp ession we e s onge in he g oup o E46K-SCNA ca ie s.
Table 2 shows he p o eins ha we e up egula ed ( old > 1.5) and down egula ed
( old < 0.5) in he iPD g oup wi h blepha i is when compa ed wi h iPD pa ien s wi hou
blepha i is. Pa ien s wi h he E46K-SNCA mu a ion did no de elop blepha i is.
Table 2. De egula ed p o eins compa ing iPD wi h blepha i is s. iPD wi hou blepha i is.
En y Name P o ein Name p-Value Fold
OLFM4 Ol ac omedin-4 7.08 × 10−5 4.25
SPB3 Se pin B3 5.29 × 10−4 3.99
SPRR3 Small p oline- ich p o ein 3 1.77 × 10−2 3.31
MMP9 Ma ix me allop o einase-9 7.33 × 10−3 3.08
CASPE Caspase-14 4.88 × 10−2 2.67
PRTN3 Myeloblas in 1.76 × 10−2 2.63
GGCT Gamma-glu amylcyclo ans e ase 2.56 × 10−2 2.40
CATD Ca hepsin D 1.24 × 10−3 2.31
CALL5 Calmodulin-like p o ein 5 3.48 × 10−2 1.91
TIG1 Re inoic acid ecep o esponde p o ein 1 3.87 × 10−2 1.90
MDHM Mala e dehyd ogenase mi ochond ial 8.59 × 10−3 1.66
NGAL Neu ophil gela inase-associa ed lipocalin 3.51 × 10−2 1.54
NQO1 NAD(P)H dehyd ogenase (quinone) 1 1.92 × 10−2 0.49
PLST Plas in-3 1.15 × 10−2 0.40
MYH14 Myosin-14 4.80 × 10−2 0.37
AMPL Cy osol aminopep idase 3.78 × 10−2 0.36
AK1A1 Alcohol dehyd ogenase (NADP(+)) 3.53 × 10−2 0.35
ADH1G Alcohol dehyd ogenase 1C 2.64 × 10−3 0.35
PRDX5 Pe oxi edoxin-5 mi ochond ial 4.71 × 10−2 0.35
RINI Ribonuclease inhibi o 2.21 × 10−2 0.34
VATA V- ype p o on ATPase ca aly ic subuni A 1.49 × 10−4 0.25
0
10
20
30
40
50
60
0 1020304050
Fold change
Numbe o p o eins
PD iPD E46K-SNCA
Figu e 3.
Rep esen a ion o he old change in exp ession o he signi ican p o eins when he pa ien s
included in he s udy we e conside ed as iPD pa ien s (o ange), E46K-SCNA ca ie s (g ay) o all PD
pa ien s oge he (blue). The same de egula ed p o eins appea in all h ee g oups, bu he changes in
exp ession we e s onge in he g oup o E46K-SCNA ca ie s.
P o eomes 2022,10, 4 7 o 15
Table 2shows he p o eins ha we e up egula ed ( old > 1.5) and down egula ed
( old < 0.5) in he iPD g oup wi h blepha i is when compa ed wi h iPD pa ien s wi hou
blepha i is. Pa ien s wi h he E46K-SNCA mu a ion did no de elop blepha i is.
Table 2. De egula ed p o eins compa ing iPD wi h blepha i is s. iPD wi hou blepha i is.
En y Name P o ein Name p-Value Fold
OLFM4 Ol ac omedin-4 7.08 ×10−54.25
SPB3 Se pin B3 5.29 ×10−43.99
SPRR3 Small p oline- ich p o ein 3 1.77 ×10−23.31
MMP9 Ma ix me allop o einase-9 7.33 ×10−33.08
CASPE Caspase-14 4.88 ×10−22.67
PRTN3 Myeloblas in 1.76 ×10−22.63
GGCT Gamma-glu amylcyclo ans e ase 2.56 ×10−22.40
CATD Ca hepsin D 1.24 ×10−32.31
CALL5 Calmodulin-like p o ein 5 3.48 ×10−21.91
TIG1
Re inoic acid ecep o esponde p o ein 1
3.87 ×10−21.90
MDHM Mala e dehyd ogenase mi ochond ial 8.59 ×10−31.66
NGAL
Neu ophil gela inase-associa ed lipocalin
3.51 ×10−21.54
NQO1 NAD(P)H dehyd ogenase (quinone) 1 1.92 ×10−20.49
PLST Plas in-3 1.15 ×10−20.40
MYH14 Myosin-14 4.80 ×10−20.37
AMPL Cy osol aminopep idase 3.78 ×10−20.36
AK1A1 Alcohol dehyd ogenase (NADP(+)) 3.53 ×10−20.35
ADH1G Alcohol dehyd ogenase 1C 2.64 ×10−30.35
PRDX5 Pe oxi edoxin-5 mi ochond ial 4.71 ×10−20.35
RINI Ribonuclease inhibi o 2.21 ×10−20.34
VATA V- ype p o on ATPase ca aly ic subuni A 1.49 ×10−40.25
No e: p< 0.05 s a is ical signi icances; old change > 1.5 o up egula ed p o eins; old change < 0.5 o down egu-
la ed p o eins.
Among he ea p o eins de egula ed in PD pa ien s, a g oup o six p o eins s ood
ou , i e o which ha we e o e exp essed in he PD g oup ela i e o he CTs (p elamin
A/C—LMNA, ca hepsin D—CATD, acid ce amidase—ASAH1, ansi ional endoplasmic
e iculum ATPase—TERA and cy oplasmic dynein 1—DYHC1), whe eas one o hem was
down egula ed ( ipep idyl-pep idase 1—TPP1: Table 3). These p o eins we e associa ed
wi h neu odegene a i e p ocesses, mainly al e a ions in lysosomal au ophagy, apop osis,
e og ade axonal anspo and demyelina ing p ocesses. I was in e es ing o no e he
ela ionship o hese p o eins wi h al e a ions in lysosomal unc ion. O he six p o eins,
h ee p esen ed a good capabili y o co ec ly classi y PD pa ien s and CTs, as e lec ed
by he AUC alues o he ROC cu es, and hey we e ela ed o al e a ions in lysosomal
unc ion: CATD, ASAH1 and DYHC1.
Table 3. De egula ed ea p o eins ela ed o neu odegene a i e unc ions.
En y
Name P o ein Name Pep ides Unique Pep ides p-Value Fold Change AUC %
LMNA P elamin-A/C 25 24 4.00 ×10−22.25 67.1
CATD Ca hepsin D 19 19 1.48 ×10−31.82 75.4
ASAH1 Acid ce amidase 6 6 2.98 ×10−31.80 72.3
TERA T ansi ional endoplasmic
e iculum ATPase 18 18 1.99 ×10−21.60 65
DYHC1
Cy oplasmic dynein 1 hea y chain 1
9 9 7.50 ×10−31.32 70.2
TPP1 T ipep idyl-pep idase 1 11 11 4.13 ×10−20.64 69
AUC = a ea unde he ROC cu e.
P o eomes 2022,10, 4 8 o 15
The uni a ia e logis ic eg ession analysis iden i ied ou a iables wi h s a is ical
signi icance a
α
= 0.05: he age o he pa ien s and he old change in CATD, ASAH1 and
DYHC1 p o eins (Table 4).
Table 4. Uni a ia e logis ic eg ession models.
Unique Va iable Used in Model Es ima e p-Value
Sex 1.163 0.072
Age 0.088 0.00 *
LMNA 0.351 0.055
CATD 1.426 0.010 *
ASAH1 0.990 0.020 *
TERA 0.801 0.040 *
DYHC1 1.197 0.085
TPP1 −0.527 0.147
Time wi h disease 9.483 0.995
UPDRS sco e 488.104 0.995
Pa I UPDRS sco e 311.136 0.994
Pa II UPDRS sco e 964.522 0.995
Pa III UPDRS sco e 313.201 0.995
Pa IV UPDRS sco e 398.234 0.995
HY Sco e 20.013 0.995
No e: * p< 0.05 s a is ical signi icance. Pa UPDRS, he Uni ied Pa kinson’s disease a ing scale’s domains.
In addi ion, he LMNA and DYHC1 p o eins we e seen o ha e s a is ical signi icances
a
α
= 0.1. A mul i a ia e logis ic eg ession model was es ablished by conside ing age and
p o eins wi h any signi icance le el. While he gene al model only showed signi icance in
he case o he CATD p o ein (Table 5), he model was likely o ha e been a ec ed by he
small numbe o samples a ailable, and inc easing he numbe o samples s udied migh
well iden i y o he signi ican a iables.
Table 5. Mul i a ia e logis ic eg ession model.
Mul i a ia e Model Va iables Es ima e p-Value
(In e cep ) −50.576 0.053
Age 0.166 0.040 *
LMNA 0.495 0.181
CATD 1.670 0.040 *
ASAH1 0.141 0.912
TERA 0.470 0.655
DYHC1 0.798 0.602
No e: * p< 0.05 s a is ical signi icances.
4. Discussion
The p esen s udy was ca ied ou o analyze p o eins by nano-liquid ch oma og aphy–
mass spec ome y (nLC–MS/MS) and selec hose ela ed o neu odegene a i e diseases
ha had a highe disc imina o y powe be ween he PD and TC g oups s udied. Se e al
sea ches o candida e bioma ke s in he ea s o PD pa ien s ha e been pe o med in ecen
yea s; howe e , he conclusion was ha u he alida ion s udies we e needed o ind
he bes candida e diagnos ic o p ognos ic bioma ke o his pa hology. The diagnosis
o PD is cu en ly based on he p esence o mo o and non-mo o symp oms, including
sleep dis u bances o ol ac o y de ici s. Mo o symp oms appea mainly when he loss o
dopamine gic neu ons eaches a ound 50–60%. The e o e, achie ing an ea ly diagnosis will
open he way o modi y he cou se o he disease and hope ully delay he se e e disabili y
caused by his pa hology. This issue has become a key aspec in PD esea ch, al hough
he lack o alid bioma ke s ep esen s a majo obs acle ha p e en s he iden i ica ion o
pa ien s in p eclinical o p od omal s ages, as well as he moni o ing o he e olu ion o he
P o eomes 2022,10, 4 9 o 15
disease and he e ec s o ea men s. Fo his eason, he p esen s udy se ou o sea ch o
a eliable bioma ke o PD in a nonin asi e luid, such as ea s. The eye has many neu al
and ascula elemen s ha a e also ound in he b ain, making i ideal o he disco e y o
new bioma ke s ha could be used o diagnose PD o o he neu odegene a i e diseases, as
well as in he disco e y o new he apeu ic a ge s.
He e, ea s om PD and CT pa ien s we e ob ained h ough glass capilla ies wi hou
p io anes hesia and wi hou ouching he ocula su ace. P e ious pilo s udies on pa ien
ea s ha e been pe o med wi h a g oup o pa ien ea s [
19
], and ou analysis is he
i s indi idual ea analysis ha will allow o he iden i ica ion o speci ic indi idual
ma ke s. By using capilla ies o ob ain he ea samples, we a oid ouching he conjunc i al
epi helium, hus disc imina ing p o eins ound in he ea om conjunc i al p o eins, as
occu s wi h he use o Schi me s ips.
In ou s udy, we ound signi ican ly de egula ed p o eins in PD pa ien s. These p o-
eins a e mainly in ol ed in in lamma o y and neu odegene a i e p ocesses, apop osis and
immune esponses. In lamma ion is ubiqui ous in neu odegene a i e diseases, including
PD, and ecen s udies ha e iden i ied ha
α
-syn-de i ed T-cell epi opes a e p e e en ially
ecognized in PD pa ien s, as well as T cells in PD a ge egions, possibly sugges ing an
au oimmune componen o PD [
20
]. Neu ons we e hough o be p o ec ed om au oim-
mune a ack, bu dopamine gic neu ons a e ulne able because hey ha e p o eins on hei
cell su ace ha help he immune sys em ecognize o eign subs ances [
21
]. T cells can
po en ially mis ake disease damaged neu ons o o eign subs ances, and, as a esul , Sulze
e al. p oposed a model in which
α
-syn-speci ic T cells cause neu onal dea h in neu odegen-
e a i e diseases associa ed wi h speci ic mis olding o his p o ein. In ou s udy, g oups o
de egula ed p o eins ela ed o immune esponse, such as LCN2, SPB3 and FDSCP, among
o he s, we e obse ed in ea samples. Howe e , ce ain immunoglobulins in ol ed in
immune esponse, such as IgA2, did no p esen s a is ically signi ican di e ences be ween
he PD g oup and he CT g oup. Fu he s udies a e needed o elucida e whe he his
ela ionship be ween immune esponse and p o eins ound in ea s is di ec ly ela ed o PD.
Neu oin lamma ion can be de ined as a non-speci ic in lamma o y e en in he b ain,
and he en i e CNS has a neu oin lamma o y componen , which is e iden in diseases such
as Mul iple Scle osis (MS), b ain aneu ysms and ce eb o ascula acciden s, epilepsy, AD
and PD, and in which he ac i i y o ex acellula ma ix (ECM) deg ading enzymes o
me allop o eases (MMPs) has been implica ed. MMPs pa icipa e in many physiological
and pa hological p ocesses in he b ain and blood. The blood–b ain ba ie (BBB) is o med
by he endo helial capilla ies ha sepa a e he blood supply o he b ain, and i s unc ion
esides in h ee s uc u es ha a e c i ical o i s in eg i y: he endo helial cells o he b ains
capilla ies, he igh junc ions (TJs) be ween hese cells, and he basemen memb ane. The
endo helium is a ba ie o small hyd ophilic compounds as he TJs seal he gaps be ween
he adjacen endo helial cells, impeding he uncon olled passage o solu es be ween hese
cells and con e ing he b ain endo helium in o a ela i ely impe meable
ba ie [22,23].
Howe e , he basemen memb ane connec s hese endo helial cells o pe icy es and as-
ocy es o o m he neu o ascula uni ha acili a es communica ion be ween he cells.
This memb ane is undamen al o he BBB o unc ion adequa ely and, as such, o b ain
homeos asis and i s gene al heal h. I has been p oposed ha MMPs speci ically dis up
TJs, and hey diges he basemen memb ane o he endo helium, he eby con ibu ing o
ce ain b ain diseases [
24
]. While i is echnically di icul o demons a e MMP ac i i y
in i o
, s onge MMP ac i i y was associa ed wi h g ea e BBB pe meabili y ollowing a
ce eb o ascula acciden and du ing epe usion
in i o
[
25
]. An inc ease in MMP-2 and
MMP-9 mRNA and ac i i y has been desc ibed a e epe usion in hype ensi e a s wi h
medial ce eb al a e y occlusion (MCAO) [
26
]. Mo eo e , BBB leakage was also de ec ed in
he pi i o m co ex o hese a s, in conjunc ion wi h he dis up ion o TJs, indica ing ha
hese MMPs al e BBB in eg i y by deg ading TJ p o eins [
26
]. As such, and despi e he
echnical challenges, he e is some e idence ha MMPs diges TJ and ECM p o eins
in i o
.
The exp ession and ac i i y o MMP-1, -2 and -9 ha e been de e mined in pos mo em
