Muaj peev xwm tsim kua muag Biomarkers Rau Kev kuaj mob Parkinson's Disease—A Pilot Study Ⅰ

Paub meej: Parkinson's disease (PD) yog tus kab mob neurodegenerative thib ob tom qab Alzheimer's disease. Nyob rau hauv txoj kev tshawb no, lub kua muag proteome profile ntawm cov neeg mob idiopathic PD (iPD, n=24), cov neeg nqa khoom ntawm E46K-SNCA kev hloov pauv (n=3), thiab kev tswj kev noj qab haus huv (CT, n {{4}). }}) Cov ntsiab lus tau txheeb xyuas los txheeb xyuas cov neeg sib tw biomarkers rau kev kuaj mob ntawm PD. Kev soj ntsuam, kev cia siab, thiab kev tswj xyuas cov ntaub ntawv tau ua tiav, tshawb xyuas cov neeg koom nrog cov kua muag kua muag los ntawm nano-kua chromatography-mass spectrometry (nLC–MS / MS) thiab ntsuam xyuas lawv cov kev puas hlwb neurological. Cov ntaub ntawv proteomic tau txais muaj nyob ntawm ProteomeXchange nrog tus lej 10.6019/PXD028811.

Nyem rau cistanche Amway rau Parkinson tus kab mob

Cov kev soj ntsuam no tau coj mus rau kev txheeb xyuas ntawm 560 cov kua muag kua muag, qee qhov tau raug tswj hwm hauv cov neeg mob PD thiab tau cuam tshuam rau hauv kev tiv thaiv kab mob, o, apoptosis, collagen degradation, protein synthesis, tiv thaiv, lipid thauj, thiab hloov lysosomal muaj nuj nqi. Ntawm cov proteins no, rau qhov muaj feem xyuam rau cov txheej txheem neurodegenerative thiab pom tias muaj peev xwm zoo heev los faib cov neeg mob thiab kev tswj hwm. Cov kev tshawb pom no tau qhia tias qee cov proteins tau tswj hwm hauv cov kua muag ntawm cov neeg mob PD, feem ntau cov proteins koom nrog hauv kev ua haujlwm lysosomal. Yog li, hauv txoj kev tshawb no, cov kua muag kua muag tau pom tias cuam tshuam rau hauv neurodegeneration uas tej zaum yuav cuam tshuam rau tus kab mob phenotype hauv cov neeg mob PD.

Ntsiab lus: biomarkers; Tus kab mob Parkinson; kua muag zaj duab xis; lysosome

1. Taw qhia

Parkinson's disease (PD) yog tus kab mob neurodegenerative thib ob tshaj plaws tom qab Alzheimer's disease (AD), thiab nws yog tus cwj pwm los ntawm kev qeeb ntawm kev txav (bradykinesia), nce cov leeg nqaij (rigidity), co (tremor), thiab kev tswj tsis tau lub cev. Hauv pathologically lees paub PD, muaj qhov sib txawv thiab qhov tso tawm ntawm misfolded alpha-synuclein (-syn) hauv daim ntawv ntawm Lewy lub cev (LBs) thiab Lewy neurites hauv nruab nrab paj hlwb (CNS). Qhov kev hloov pauv no hauv kev sib sau ntawm cov qauv no yuav muaj feem cuam tshuam txog kev kho mob heterogeneity hauv tus kab mob no [1-3]. Tseeb, ntau qhov kev kho mob tsis zoo nyob rau hauv cov kab mob ntawm Lewy lub cev kab mob (LBDs), suav nrog cov neeg mob idiopathic (IPD) thiab caj ces PD thiab cov neeg mob uas muaj dementia nrog LBs. Ib qho ntawm cov teeb meem tseem ceeb hauv kev daws cov kab mob neurodegenerative yog nrhiav cov cim kev kho mob uas tso cai rau kev faib cov neeg mob thaum ntxov thiab pab saib xyuas cov kab mob.

Cov yam ntxwv xws li oxidative kev nyuaj siab, proteolysis, thiab hloov pauv fatty acid lossis phospholipid concentrations tau cuam tshuam nrog cov qauv kev hloov pauv rau -syn, thiab kev hloov pauv tom qab hloov pauv tuaj yeem hloov qhov loj, qauv, lossis protein ntau ntawm -syn [4]. Deregulating kev hloov pauv thiab ntau ntxiv hauv -syn gene (SNCA) ua rau autosomal-dominant tsev neeg cov ntaub ntawv ntawm PD [5]. Tej zaum qhov kev hloov pauv pathogenic tshaj plaws uas paub tias ua rau PD yog E46K kev hloov pauv hauv -syn (E46K-SNCA), uas cuam tshuam rau ib pawg ntawm kev txhoj puab heev, noob caj noob ces LBDs nrog kev kho mob thiab cov yam ntxwv ntawm kev nco txog kev dementia nrog LBs [6,7]. Txawm hais tias kev hloov pauv hauv -syn tsis tshua muaj, lawv muab lub sijhawm tshwj xeeb los txheeb xyuas cov neeg mob nrog PD thiab kom nkag siab zoo dua qhov kev loj hlob ntawm cov kab mob no.

Nws paub tias PD cov neeg mob tsim kev hnov ​​​​lus, kev hloov pauv ntawm qhov muag thiab daim tawv muag, thiab hloov cov kua muag muaj pes tsawg leeg hauv cov nqi txo [8,9]. Ntxiv rau qhov kev puas tsuaj ntawm lub qhov muag, cov neeg mob no tuaj yeem muaj kev hloov pauv hauv kev pabcuam, txo qhov pom kev pom, tsim scotoma (qhov chaw uas lawv qhov kev pom kev tsis pom kev yog ib nrab lossis tag nrho degenerated), thiab thinning ntawm retinal txheej, tshwj xeeb tshaj yog vim kev txo qis. nyob rau hauv tus naj npawb ntawm paj fibers [10,11]. Kev tsis txaus ntawm neurotransmission thiab monoamine metabolism kuj cuam tshuam rau cov kev hloov pauv pathological rau qhov pom ntawm cov neeg mob PD [12].

Cov kev hloov pauv no zoo heev vim tias monoamines koom nrog hauv kev xa cov ntaub ntawv pom hauv retina [13]. Tsis tas li ntawd, cov neeg mob PD tuaj yeem muaj kev hloov pauv mus rau innervation ntawm lub qhov muag sab hauv uas tuaj yeem hloov pauv cov kua muag thaum kawg. Ntau cov neurotransmitters thiab trophic tshuaj yog secreted nyob rau hauv lub kua muag, originating los ntawm aqueous lom zem los yog paj hlwb khiav los ntawm lub cornea [14].

Txawm li cas los xij, cellular thiab molecular mechanisms uas underlie cov koom haum no tseem tsis tau paub meej. Raws li PD cuam tshuam rau ntau lub cev tsis yog lub cev muaj zog thiab cov hlab ntsha peripheral, lacrimal secretion tuaj yeem hloov pauv hauv cov neeg mob no, thiab cov kua muag protein muaj pes tsawg leeg tuaj yeem pom cov yam ntxwv ntawm cov neeg mob no uas tuaj yeem ua tus kuaj biomarker. Hauv qhov kev nkag siab no, kev tshuaj xyuas cov kua muag kua muag ntawm cov neeg mob E46K-SNCA thiab iPD tuaj yeem pab txheeb xyuas cov neeg sib tw biomarkers muaj txiaj ntsig rau kev kuaj mob thiab kev loj hlob ntawm PD.

Yog li, txoj kev tshawb fawb tam sim no tsom los tshuaj xyuas cov kua muag proteome profile hauv cov neeg mob iPD thiab cov neeg nqa khoom ntawm E46K-SNCA kev hloov pauv kom pom cov biomarker uas tuaj yeem pab rau kev kuaj mob yav tom ntej ntawm PD, siv cov qauv tsis cuam tshuam.

2. Cov ntaub ntawv thiab cov txheej txheem

2.1. Kawm Cohort

Peb tau tsim ib qho kev soj ntsuam, yav tom ntej, kev tswj xyuas cov ntaub ntawv uas 24 tus neeg mob nrog iPD, 3 tus neeg nqa khoom ntawm E46K-SNCA kev hloov pauv, thiab 27 yam kev noj qab haus huv (CTs) tau cuv npe. Qhov kev tshawb fawb no tau ua los ntawm cov neeg ua haujlwm kho mob uas tsim nyog tom qab tau txais kev pom zoo los ntawm OSI Ezkerralde-Enkaterri-Cruces Ethics Committee CEIC E18/47. Txoj kev tshawb no tau ua raws li nruj raws li cov cai ntawm Helsinki Tshaj Tawm ntawm Kev Tshawb Fawb Txog Kev Tshawb Fawb Txog Tib Neeg. Ua ntej sau cov qauv, kos npe pom zoo tau txais los ntawm txhua yam kev kawm tom qab tau piav qhia qhov xwm txheej thiab qhov tshwm sim ntawm qhov kev kawm. Cov neeg mob tau raug xaiv los ntawm Neurology thiab Ophthalmology Service Units ntawm Cruces University Tsev Kho Mob (Barakaldo, Bizkaia, Spain) thaum muaj kev sib tham sab nraud, raws li kev pom zoo suav nrog thiab cais tawm. Cov neeg mob nrog IPD tau ua tiav Parkinson's UK Brain Bank cov txheej txheem rau kev kuaj mob PD thiab tsis muaj kev hloov pauv uas paub.

Cov txheej txheem suav nrog CTs yuav tsum muaj hnub nyoog tshaj 40 xyoo, ntawm poj niam txiv neej, thiab tsis muaj keeb kwm kho mob ntawm ib qho kev puas hlwb. Cov tib neeg uas muaj (los yog muaj keeb kwm ntawm) ib qho kab mob los yog qhov muag tsis pom kev / mob tau suav nrog hauv txoj kev tshawb no. Cov txheej txheem cais tawm muaj xws li kev phais qhov muag hauv peb lub hlis dhau los, tshuaj kho qhov muag ntev (xws li, rau glaucoma), keeb kwm ntawm kev ua xua, siv cov tshuaj pleev ib ce (tshwj tsis yog kua muag dag) lossis tshuaj noj qhov ncauj uas muaj corticosteroids, atopy, thiab cov neeg mob Sjögren's. syndrome.

Cov neeg siv lub ntsej muag tiv thaiv kuj raug cais tawm kom tsis txhob muaj kev cuam tshuam nrog kev txhais cov txiaj ntsig. Kev kuaj pom qhov muag ua ntej thiab cov qauv sau tau ua nyob rau tib hnub, thaum cov neeg mob nrog PD kuj tau pom nyob rau ib hnub cais los ntawm cov tswv cuab ntawm pab pawg tshawb fawb uas yog cov kws tshaj lij hauv kev txav mus los los ntsuas Parkinson's Scales.

2.2. Ophthalmological Examination

Kev kuaj mob ophthalmological suav nrog Schirmer's test I (SCH), uas txiav txim siab seb qhov muag puas tsim kua muag txaus kom nws ntub. Qhov kev ntsuam xyuas tso cai rau cov dej hauv cov kua muag mus raws qhov ntev ntawm daim ntawv xeem strip, thiab cov nqi ib txwm muaj ntau dua lossis sib npaug li 10 mm wetting ntawm daim ntawv tom qab 5 min. Tear-film instability raug soj ntsuam los ntawm kev siv lub sij hawm tsim kua muag (TBUT) nrog fluorescein. TBUT yog lub sij hawm coj mus rau thawj qhov chaw qhuav tshwm rau ntawm lub pob kws tom qab ua tiav blinking. Tus nqi tsawg dua 10 s feem ntau qhia txog qee qhov kua muag-zaj duab xis tsis ruaj khov. Txhua yam kab mob hauv lub cev lossis lwm yam kev mob / kev siv tshuaj uas tuaj yeem cuam tshuam nrog kev txhais cov txiaj ntsig tau suav tias yog qhov ntsuas rau kev cais tawm.

2.3. Neurological Examination

Ib tus kws tshaj lij ntawm cov kws kho mob hlwb hauv kev ua haujlwm ntawm kev txav mus los tau sau tseg lub hnub nyoog ntawm cov neeg mob PD ntawm kev sau kua muag, lawv cov kab mob ntev, thiab lawv cov qhab nia ntawm Unified Parkinson's Disease Rating Scale (UPDRS) thiab Hoehn Yahr scale. Cov kev ntsuam xyuas no ua raws li cov txheej txheem stratification los taug qab qhov kev loj hlob ntawm tus kab mob. Kev tsis pub lwm tus paub ntawm tus neeg mob cov ntaub ntawv tau lees paub los ntawm kev siv Basque Biobank.

2.4. Tear-Sample Collection

Tag nrho cov qauv tsim kua muag tau sau los ntawm kev siv calibrated 10 µL iav microcapillary hlab (BLAUBRAND intraMark, Wertheim, Lub teb chaws Yelemees). Cov qauv tsim kua muag tau txais los ntawm qhov tsis zoo ntawm lub cev lub cev tsim kua muag meniscus, txo qhov khaus ntawm qhov muag lossis lub hau npog, thiab tsis muaj kev siv tshuaj loog. Cov qauv tsim kua muag tau sau los ntawm ob lub qhov muag ntawm txhua tus neeg koom nrog thiab tam sim ntawd muab tso rau hauv cov raj Eppendorf precooled. Tom qab sau cov kua muag, cov qauv tau muab khaws cia ntawm −80 ◦C kom txog rau thaum lawv ntsuas.

2.5. Proteomics Analyzes

Cov kev soj ntsuam proteomic tau ua tiav ntawm CIC bioGUNE Proteomics Service (Derio, Bizkaia, Spain), siv Filter Aided Sample Preparation (FASP) raws tu qauv [15] rau kev ua qauv thiab kev zom zaub mov. Trypsin (Sigma Aldrich, St. Louis, MO, USA) tau ntxiv ntawm trypsin: protein ratio ntawm 1:50, thiab cov sib tov tau incubated hmo ntuj ntawm 37 ◦C, qhuav nyob rau hauv ib tug RVC{{4} } Speedvac concentrator (Christ, Vienna, Austria). Cov peptides tau txais raug tshem tawm thiab rov ua dua hauv 0.1 feem pua ​​​​Formic Acid (FA) (Sigma Aldrich, St. Louis, MO, USA) siv C18 cov lus qhia theem (Millipore, St. Louis, MO, USA).

Cov qauv raug soj ntsuam hauv ib qho tshiab hybrid trapped ion mobility spectrometry quadrupole lub sij hawm ntawm davhlau huab hwm coj spectrometer (timesTOF Pro nrog PASEF, Bruker Daltonics, Bremen, Lub teb chaws Yelemees), ua ke hauv online rau nanoElute kua chromatograph (Bruker, Coventry, UK). Qhov loj spectrometer no yuav siv sij hawm kom zoo dua ntawm ib qho tshiab scan hom hu ua parallel accumulation serial fragmentation (PASEF), uas muab cov kev sib txuas ceev yam tsis muaj kev poob rau hauv rhiab heev, thiab nws tau raug pov thawj los muab kev soj ntsuam ceev thiab rhiab heev rau kev soj ntsuam proteomics [16].

Cov qauv (200 ng) tau ncaj qha thauj mus rau 15 cm Bruker nano elute FIFTEEN C18 analytical kem (Bruker, Coventry, UK) thiab daws ntawm 400 nL / min. Kem tau rhaub rau 50 ◦C hauv qhov cub. Kev txheeb xyuas cov protein thiab kev ntsuas tau ua tiav los ntawm kev siv PEAKS software (Bioinformatics Solutions Inc., Waterloo, CA, USA) Kev tshawb nrhiav tau tawm tsam cov ntaub ntawv ntawm canonical human Uniprot / Swissprot nkag (tsis muaj isoforms considerer), nrog precursor thiab fragment tolerances ntawm 20 ppm thiab 0.05 Da.

Ib cheeb tsam-raws li daim ntawv lo tsis muaj protein ntau tau ua los ntawm kev siv PEAKS Q module muaj nyob rau hauv PEAKS software. Tsuas yog cov proteins uas muaj tsawg kawg yog ob lub peptides ntawm False Discovery Rate (FDR) < 1 feem pua ​​​​thiab tam sim no nyob rau hauv tsawg kawg yog 70 feem pua ​​​​ntawm cov qauv los ntawm ib qho ntawm cov kev sim hauv kev soj ntsuam tau txiav txim siab rau kev tshuaj xyuas ntxiv. Cov ntaub ntawv raug thauj mus rau Perseus platform [17] thiab ua tiav ntxiv (log2 transformation, imputation) ua ntej daim ntawv thov ntawm Tus Tub Kawm Ntawv t-test rau qhov sib txawv protein-expression tsom xam. Cov ntaub ntawv proteomics tau sau tseg hauv Cov Khoom Siv Ntxiv S1, thiab cov ntaub ntawv loj spectrometry proteomics tau muab tso rau hauv ProteomeXchange Consortium los ntawm PRIDE [18] tus khub repository, nrog rau dataset identifiers PXD028811 thiab 10.6018/PXD0.

2.6. Kev Tshawb Fawb Txog Kev Tshawb Fawb

Kev txheeb xyuas txheeb cais tau ua los ntawm kev siv IBM SPSS Statistics pob rau Windows, 23.0v (IBM-SPSS, Armonk, New York, NY, USA). Qhov zoo ib yam ntawm cov ntaub ntawv txuas ntxiv tau soj ntsuam nrog kev sim Shapiro-Wilk. Cov p-values ​​xam txiav txim seb qhov tshwm sim ntawm kev sib raug zoo ntawm cov proteins nyob rau hauv cov ntaub ntawv thiab ib tug muab canonical txoj kev, functional network, los yog upstream regulator yog piav los ntawm lub sij hawm ib leeg, raws li ib tug Fisher qhov tseeb xeem, nrog p-tus nqi < {{5 }}.05 suav tias yog qhov tseem ceeb.

Univariate logistic regressions tau siv los txheeb xyuas cov yam ntxwv tseem ceeb ntawm PD-tus neeg mob kev ntxub ntxaug. Tom qab xaiv qhov sib txawv, ntau qhov sib txawv logistic regressions tau siv los tsim tus qauv PD kev ntxub ntxaug thiab txheeb xyuas cov tshuaj kho mob thiab cov tshuaj tiv thaiv kab mob cuam tshuam rau kev txheeb xyuas PD. Cov qauv logistic tso cai rau kev sib raug zoo ntawm qhov sib txawv ntawm qhov sib txawv thiab cov txheej txheem txuas ntxiv lossis tsis sib haum xeeb los ntawm kev kwv yees. Cov qauv tso cai rau kev faib tawm ntawm cov neeg mob uas muaj lossis tsis muaj PD, suav txog qhov muaj feem cuam tshuam rau txhua pawg. Tus nqi p-tus nqi ntawm txhua qhov sib txawv piav qhia txog qhov tseem ceeb ntawm kev sib koom ua ke ntawm cov lus teb thiab txhua nqe lus suav nrog hauv tus qauv.

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