Chirwere cheAlzheimer (AD) chinoshaya mapuroteni biomarkers anoratidza ayo akawanda ari pasi pathophysiology, achitadzisa kufambira mberi kwekuongororwa uye kurapwa. Pano, tinoshandisa proteomics yakazara kuona cerebrospinal fluid (CSF) biomarkers inomiririra huwandu hwakawanda hweAD pathophysiology. Multiplex mass spectrometry yakaonekwa ingangoita zviuru zvitatu nemazana mashanu uye ingangoita gumi nemaviri mapuroteni muAD CSF nehuropi, zvichiteerana. Kuongorora kwenetiweki kweproteome yeuropi kwakagadzirisa 44 biodiversity modules, 15 yacho yakapfuura ne cerebrospinal fluid proteome. Iwo CSF AD mamaki mune aya anopindirana mamodule anopetwa kuita mashanu mapuroteni mapoka, anomiririra akasiyana epathophysiological process. Iyo synapses uye metabolites muAD brain inodzikira, asi CSF inowedzera, nepo glial-rich myelination uye masoja ekudzivirira muuropi uye CSF achiwedzera. Kusagadzikana uye kutsanangurwa kwechirwere chekuchinja kwepaneli kwakasimbiswa mune anopfuura 500 ekuwedzera CSF samples. Aya mapoka akacherechedzawo mapoka ezvinyorwa zvipenyu mune asymptomatic AD. Pakazere, izvi mhedzisiro inhanho inovimbisa kune webhu-based biomarker maturusi ekiriniki application muAD.
Chirwere cheAlzheimer's (AD) ndicho chinonyanya kukonzera kukanganisa neurodegenerative pasi rose uye inoratidzwa nehuwandu hwehutachiona hwehutachiona hwehutachiona, kusanganisira synaptic transmission, glial-mediated immunity, uye mitochondrial metabolism (1-3). Nekudaro, mapuroteni ayo akamiswa ebiomarkers achiri kutarisa pakuona amyloid uye tau protein, uye saka haigone kuratidza iyi yakasiyana pathophysiology. Aya "core" protein biomarkers anonyanya kuyerwa mu cerebrospinal fluid (CSF) anosanganisira (i) amyloid beta peptide 1-42 (Aβ1-42), iyo inoratidza kuumbwa kwecortical amyloid plaques; (ii) tau yakazara, chiratidzo chekuderera kweaxon; (iii) phospho-tau (p-tau), mumiriri wepathological tau hyperphosphorylation (4-7). Kunyange zvazvo idzi cerebrospinal fluid biomarkers dzakafambisa zvikuru kuona kwedu "yakaratidza" AD mapuroteni zvirwere (4-7), inongomiririra chikamu chiduku chebiology yakaoma shure kwechirwere.
Kushaikwa kwepathophysiological siyana yeAD biomarkers kwakakonzera matambudziko mazhinji, kusanganisira (i) kusakwanisa kuziva uye kuverenga iyo biological heterogeneity yevarwere veAD, (ii) kusakwana kuyerwa kwekuoma kwechirwere uye kufambira mberi, kunyanya mudanho rekutanga, Uye ( iii) kugadzirwa kwemishonga yekurapa iyo yakatadza kugadzirisa zvachose zvikamu zvese zvekuora kwetsinga. Kuvimba kwedu nechirwere chepfungwa chekutsanangura AD kubva kuzvirwere zvinoenderana kunongowedzera matambudziko aya. Humbowo huzhinji hunoratidza kuti vanhu vakura vazhinji vane dementia vane zvinopfuura imwechete pathological maitiro ekuderera kwekuziva (8). Vanosvika 90% kana kupfuura vanhu vane AD pathology vanewo chirwere chevascular, TDP-43 inclusions, kana zvimwe zvirwere zvinopera (9). Izvi zvikamu zvakakwirira zvekupindirana kwepathological zvakakanganisa maitiro edu ezvino ekuongorora kwedementia, uye tsanangudzo yakazara yepathophysiological yechirwere inodiwa.
Tichifunga nezve kudiwa kwekukurumidzira kweakasiyana AD biomarkers, munda uri kuramba uchitora nzira ye "omics" yakavakirwa pahurongwa hwese hwekutsvaga biomarkers. Iyo Accelerated Pharmaceutical Partnership (AMP)-AD Alliance yakatangwa muna 2014 uye iri pamberi pechirongwa ichi. Uku kuedza kwakasiyana-siyana neNational Institutes of Health, academia, uye indasitiri ine chinangwa chekushandisa masisitimu-akavakirwa nzira kutsanangura zviri nani pathophysiology yeAD uye kugadzira biodiversity diagnostic analysis uye nzira dzekurapa (10). Sechikamu chechirongwa ichi, network proteomics yave chishandiso chinovimbisa chekusimudzira system-based biomarkers muAD. Iyi nzira isina kurerekera inofambiswa nedata inoronga yakaoma proteomics data seti mumapoka kana "modules" ye-co-inoratidzwa mapuroteni ayo anobatanidzwa neakananga masero marudzi, organelles, uye biological mabasa (11-13). Anenge gumi nemaviri eruzivo-akapfuma network proteomics zvidzidzo zvakaitwa paAD brain (13-23). Pakazere, ongororo idzi dzinoratidza kuti iyo AD brain network proteome inochengetedza yakachengetedzwa modular sangano mumasangano akazvimirira uye akawanda cortical matunhu. Pamusoro pezvo, mamwe emamodule aya anoratidza shanduko dzinodzokororwa muhuwandu hune hukama neAD pamaseti e data, zvichiratidza pathophysiology yezvirwere zvakawanda. Pamwe chete, izvi zvakawanikwa zvinoratidza chinovimbisa chinongedzo chekuwanikwa kwebrain network proteome sesystem-based biomarker muAD.
Kuti tishandure iyo AD brain network proteome kuita yekiriniki inobatsira system-based biomarkers, takabatanidza iyo brain-yakatorwa network neproteomic ongororo yeAD CSF. Iyi nzira yakabatanidzwa yakatungamirira mukuzivikanwa kwezvishanu zvinovimbisa seti yeCSF biomarkers iyo inobatanidzwa nehuwandu hwakawanda hwehuropi-based pathophysiology, kusanganisira synapses, tsinga dzeropa, myelination, kuzvimba, uye kusagadzikana kwemaitiro emagetsi. Isu takabudirira kusimbisa aya biomarker mapaneru kuburikidza neakawanda kudzokorora ongororo, kusanganisira anopfuura 500 CSF samples kubva kune dzakasiyana neurodegenerative zvirwere. Izvi zvinongororwa zvinongororwa zvinosanganisira kuongorora zvinangwa zveboka muCSF yevarwere vane asymptomatic AD (AsymAD) kana kuratidza humbowo hwekusajairika amyloid kuunganidza munzvimbo yakajairika yekuziva. Ongororo idzi dzinoratidza kukosha kwebiological heterogeneity muhuwandu hweAsymAD uye kuona mapaneru mamaki anogona kukwanisa kudzikisa vanhu mukutanga kwechirwere. Pakazere, mhedzisiro iyi inomiririra danho rakakosha mukugadzirwa kweprotein biomarker zvishandiso zvinoenderana neakawanda masisitimu anogona kubudirira kugadzirisa akawanda ekiriniki matambudziko akatarisana neAD.
Chinangwa chikuru chechidzidzo ichi ndechekuona mitsva yecerebrospinal fluid biomarkers inoratidza dzakasiyana siyana dzehuropi-based pathophysiology inotungamira kuAD. Mufananidzo S1 inotsanangura nzira yedu yekutsvagisa, iyo inosanganisira (i) ongororo yakazara inotungamirwa nekutanga zvakawanikwa zveAD CSF uye network brain proteome yekuona akawanda ane huropi ane chekuita nechirwere cheCSF biomarkers, uye (ii) inotevera kudzokorora Aya ma biomarker ari mune akati wandei akazvimirira cerebrospinal. fluid cohorts. Tsvagiridzo yakanangana nekuwanikwa yakatanga nekuongororwa kwekusiyanisa kutaura kweCSF muvanhu makumi maviri vane ruzivo rwakakwana uye makumi maviri evarwere veAD paEmory Goizueta Alzheimer's Disease Research Center (ADRC). Kuongororwa kweAD kunotsanangurwa sekukanganisa kwakakosha kwekunzwisisa muhupo hweAβ1-42 uye mazinga akakwirira etau uye p-tau mu cerebrospinal fluid [Mean Montreal Cognitive Assessment (MoCA), 13.8 ± 7.0] [ELISA (ELISA) )]] (Tafura S1A). Kudzora (kureva MoCA, 26.7 ± 2.2) yaive neyakajairwa mazinga eCSF biomarkers.
CSF yemunhu inoratidzirwa nehuwandu hwehuwandu hweprotein yakawanda, umo albumin uye mamwe mapuroteni akawandisa anogona kudzivirira kuonekwa kwemapuroteni ekufarira (24). Kuti tiwedzere kudzika kwekuwanikwa kweprotein, takabvisa mapuroteni gumi nemana ekutanga kubva kune yega yega yeCSF sampu isati yatanga misa spectrometry (MS) kuongororwa (24). Huwandu hwe39,805 peptides hwakaonekwa neMS, iyo yakamepurwa ku3691 maproteome mumasample makumi mana. Protein quantification inoitwa neakawanda tandem mass tag (TMT) yakanyorwa (18, 25). Kuti tigadzirise data yakashaikwa, isu takangobatanidza iwo mapuroteni akaverengerwa mune inokwana 50% yemasampuli muongororo yakatevera, nokudaro pakupedzisira quantifying 2875 proteomes. Nekuda kwemusiyano wakakura muhuwandu hwemapuroteni kuwanda mazinga, sampu yekudzora yakatorwa seyekunze (13) uye haina kuverengerwa muongororo yakatevera. Huwandu hunokosha hwemasamples makumi matatu asara akagadziridzwa zvichienderana nezera, murume kana mukadzi, uye batch covariance (13-15, 17, 18, 20, 26).
Kushandisa nhamba yekuongorora t-test kuongorora kuongorora kusiyanisa kutaura pamusoro pekugadzirisa data set, kuongorora uku kwakaratidza mapuroteni ane huwandu hwakawanda hwakashandurwa zvakanyanya (P <0.05) pakati pekutonga uye AD kesi (Table S2A). Sezvakaratidzwa muMufananidzo 1A, kuwanda kwehuwandu hwemapuroteni e225 muAD yakaderedzwa zvakanyanya, uye kuwanda kwemapuroteni e303 kwakawedzera zvakanyanya. Aya mapuroteni anotsanangurwa zvakasiyana anosanganisira akati wandei akambozivikanwa cerebrospinal fluid AD mamaki, akadai semicrotubule-associated protein tau (MAPT; P = 3.52 × 10-8), neurofilament (NEFL; P = 6.56 × 10-3), mapuroteni ane hukama nekukura 43. (GAP43; P = 1.46 × 10-5), Fatty Acid Binding Protein 3 (FABP3; P = 2.00 × 10-5), Chitinase 3 se1 (CHI3L1; P = 4.44 × 10−6), Neural Granulin (NRGN; P = 3.43 × 10-4) uye VGF nerve growth factor (VGF; P = 4.83 × 10-3) (4-6). Zvisinei, takaonawo zvimwe zvinangwa zvakakosha zvikuru, zvakadai seGDP dissociation inhibitor 1 (GDI1; P = 1.54 × 10-10) uye SPARC-inoenderana nemodular calcium binding 1 (SMOC1; P = 6.93 × 10-9). Gene Ontology (GO) ongororo ye225 yakaderedzwa zvakanyanya mapuroteni yakaratidza hukama hwepedyo nemaitiro emvura yemuviri senge steroid metabolism, ropa coagulation, uye kuita kwehomoni (Mufananidzo 1B uye Tafura S2B). Kusiyana neizvi, protein yakawedzera zvakanyanya ye303 ine hukama nesero chimiro uye simba metabolism.
(A) The volcano plot inoratidza log2 fold change (x-axis) maererano ne -log10 statistical P value (y-axis) inowanikwa net-test, iyo inoshandiswa kuona mutsauko wekutaura pakati pekutonga (CT) uye AD makesi eiyo CSF proteome Yese mapuroteni. Mapuroteni ane mazinga akaderedzwa zvakanyanya (P <0.05) muAD anoratidzwa mubhuruu, nepo mapuroteni ane mazinga akanyanya kuwedzera muchirwere anoratidzwa mutsvuku. Protein yakasarudzwa yakanyorwa. (B) Matemu eGO epamusoro ane chekuita neprotein akanyanya kuderedzwa (blue) uye akawedzera (tsvuku) muAD. Inoratidza iwo matatu eGO mazwi ane epamusoro z-zvibodzwa muminda yebiological process, mamorekuru mabasa, uye maseru zvinhu. (C) MS akayera MAPT mwero muCSF sampuli (kuruboshwe) uye kuwirirana kwayo nemuenzaniso ELISA tau level (kurudyi). Iyo Pearson corelation coefficient ine yakakosha P kukosha inoratidzwa. Nekuda kwekushaikwa kweELISA data yeimwe AD kesi, manhamba aya anosanganisira kukosha kwemakumi matatu nemasere emakumi matatu nemapfumbamwe akaongororwa makesi. (D) Inotarisirwa kuongororwa kwechikwata (P <0.0001, Benjamini-Hochberg (BH) yakagadzirisa P <0.01) pakutonga uye AD CSF yakawana sampuli dzinoshandisa 65 zvakanyanya kuchinjwa mapuroteni mu data set. Standardize, normalize.
Iyo proteomic level yeMAPT yakabatana zvakanyanya kune yakazvimiririra kuyerwa ELISA tau level (r = 0.78, P = 7.8 × 10-9; Mufananidzo 1C), ichitsigira kutendeseka kweMS yedu kuyerwa. Mushure mokunge trypsin digestion pamwero weamyloid precursor protein (APP), isoform-specific peptides yakarongwa kuC-terminus yeAβ1-40 uye Aβ1-42 haigoni ionized zvakanaka (27, 28). Naizvozvo, iyo APP peptides yatakaona haina chekuita neELISA Aβ1-42 mazinga. Kuti tiongorore mutsauko wekutaura kwechiitiko chimwe nechimwe, takashandisa mapuroteni akaratidzwa zvakasiyana-siyana neP <0.0001 [false discovery rate (FDR) yakagadziriswa P <0.01] kuti iite tsvakurudzo yeboka rinotarisirwa remasampuli (Table S2A). Sezvinoratidzwa muMufananidzo 1D, aya makumi matanhatu neshanu mapuroteni akakosha anogona kunyatso kuunganidza samples zvinoenderana nemamiriro echirwere, kunze kwekesi imwe yeAD ine hunhu hwakafanana. Pamapuroteni aya makumi matanhatu neshanu, makumi matanhatu nenhatu akawedzera muAD, nepo maviri chete (CD74 neISLR) akadzikira. Pakazara, idzi cerebrospinal fluid ongororo dzakaratidza mazana emapuroteni muAD anogona kushanda sechirwere biomarkers.
Ipapo takaita yakazvimirira network ongororo yeAD brain proteome. Uropi hwehuropi hwekuwanikwa uku hwaisanganisira dorsolateral prefrontal cortex (DLPFC) kubva kutonga (n = 10), chirwere cheParkinson (PD; n = 10), yakasanganiswa AD / PD (n = 10) uye AD (n = 10) kesi. ) Muenzaniso. Emery Goizueta ADRC. Huwandu hwevanhu vemakumi mana emakesi aya akambotsanangurwa (25) uye akapfupikiswa muTafura S1B. Isu takashandisa TMT-MS kuongorora aya makumi mana ehuropi tishu uye replication cohort yemakumi maviri nenomwe. Pakazara, aya maviri ehuropi data seti akagadzira 227,121 akasiyana peptides, ayo akamepurwa kune 12,943 maproteomes (25). Izvo chete mapuroteni ayo akaverengerwa mune inokwana 50% yenyaya akaverengerwa mukuferefeta kwakatevera. Yekupedzisira kuwanikwa data set ine 8817 quantified proteins. Gadzirisa mazinga eprotein akawandisa zvichienderana nezera, murume kana mukadzi, uye post-mortem interval (PMI). Kuongororwa kwekutaura kwakasiyana kweiyo data yakatarwa mushure mekudzora kwakaratidza kuti > 2000 mapuroteni mazinga akashandurwa zvakanyanya [P <0.05, kuongororwa kwekusiyana (ANOVA)] mune maviri kana anopfuura echirwere cohorts. Zvadaro, takaita ongororo yakatariswa yemasumbu zvichienderana nemapuroteni akaratidzwa zvakasiyana, uye P <0.0001 muAD / control uye / kana AD / PD kuenzanisa (Mufananidzo S2, A uye B, Tafura S2C). Aya mapuroteni 165 akashandurwa zvakanyanya anoratidza zvakajeka nyaya dzine AD pathology kubva pakutonga uye PD samples, zvichisimbisa kusimba kwakasimba kweAD-chaiyo shanduko muproteome yese.
Isu takazoshandisa algorithm inonzi Weighted Gene Co-expression Network Analysis (WGCNA) kuita network ongororo pane yakawanikwa brain proteome, iyo inoronga iyo data yakaiswa mumaprotein module ane akafanana ekutaura mapatani (11-13). Kuongorora kwakaratidza 44 modules (M) mapuroteni akabatanidzwa, akarongwa uye akaverengwa kubva kune yakakura (M1, n = 1821 mapuroteni) kusvika kune zviduku (M44, n = 34 mapuroteni) (Mufananidzo 2A uye Tafura S2D)). Sezvambotaurwa pamusoro (13) Verenga mumiriri wekutaura chimiro kana hunhu hweprotein yemodule yega yega, uye iwirirane nayo nemamiriro echirwere uye AD pathology, ndiko kuti, gadza mubatanidzwa weAlzheimer's Disease Registry (CERAD) uye Braak Score (Figure 2B). Pakazara, 17 modules dzaive dzakabatana zvakanyanya neAD neuropathology (P <0.05). Mazhinji emamodule ane hukama nechirwere aya zvakare akapfuma mumasero emhando-chaiwo mamaki (Mufananidzo 2B). Sezvambotaurwa pamusoro (13), kupfumisa rudzi rwesero kunotemerwa nekuongorora iyo module inopindirana uye rondedzero yereferenzi yemhando-yakatarwa genes. Aya majini anotorwa kubva kune yakadhindwa data mune yakasarudzika mbeva neurons, endothelial uye glial masero. RNA sequencing (RNA-seq) kuyedza (29).
(A) Tsvaga iyo WGCNA yeuropi proteome. (B) Biweight midcorrelation (BiCor) kuongororwa kweprotein modular siginecha (yekutanga chikamu chikuru chemodular protein expression) ine AD neuropathological maitiro (kumusoro), kusanganisira CERAD (Aβ plaque) uye Braak (tau tangles) zvibodzwa. Kusimba kwezvakanaka (zvitsvuku) uye zvakashata (bhuruu) kuwirirana kunoratidzwa nemavara maviri ekupisa mepu, uye asterisks inoratidza kukosha kwenhamba (P <0.05). Shandisa Hypergeometric Fisher's Exact Test (FET) (pazasi) kuongorora iyo sero rudzi kushamwaridzana kweprotein yega yega. Kusimba kwemumvuri mutsvuku kunoratidza dhigirii yemhando yemasero ekupfumisa, uye asterisk inoratidza kukosha kwenhamba (P <0.05). Shandisa nzira yeBH kugadzirisa kukosha kweP kunobva kuFET. (C) GO kuongorora kwe modular mapuroteni. Iwo akanyanya hukama maitiro ebhayoloji anoratidzwa kune yega module kana yakabatana module boka. oligo, oligodendrocyte.
Seti yezvishanu zvine hukama neastrocyte uye microglia-rich modules (M30, M29, M18, M24, uye M5) yakaratidza kuwirirana kwakasimba kwakanaka neAD neuropathology (Mufananidzo 2B). Ontology ongororo inobatanidza aya glial modules nekukura kwesero, kuwanda, uye kusadzivirirwa (Mufananidzo 2C uye Tafura S2E). Maviri ekuwedzera glial modules, M8 uye M22, zvakare akadzorwa zvakanyanya muchirwere. M8 ine hukama zvakanyanya neToll-like receptor nzira, inosaina cascade inoita basa rakakosha mukupindura kwemuviri immune system (30). Panguva imwecheteyo, M22 yakanyatsobatana nekushandurwa kwemashure ekushandura. M2, iyo yakapfuma mune oligodendrocytes, inoratidza kuwirirana kwakasimba kwakanaka neAD pathology uye ontological connection ne nucleoside synthesis uye DNA replication, zvichiratidza kuwedzera kwesero muzvirwere. Pakazara, izvi zvakawanikwa zvinotsigira kukwidziridzwa kweglial modules yatakamboona muAD network proteome (13, 17). Izvozvi zvinoonekwa kuti akawanda AD-ane hukama neglial modules munetiweki anoratidza yakaderera matauriro mazinga mukutonga uye PD kesi, zvichiratidza hunhu hwavo hwechirwere hwakasimudzwa muAD (Mufananidzo S2C).
Mamodule mana chete mumambure edu eproteome (M1, M3, M10, uye M32) akabatana zvakanyanya neAD pathology (P <0.05) (Mufananidzo 2, B uye C). Ose M1 uye M3 akapfuma mune neuronal mamaki. M1 ine hukama zvakanyanya nesynaptic masiginecha, nepo M3 iri padhuze nekuita mitochondrial. Iko hakuna humbowo hwesero rudzi hupfumi hweM10 neM32. M32 inoratidza kuwirirana pakati peM3 nesero metabolism, nepo M10 yakabatana zvakanyanya nekukura kwesero uye microtubule basa. Kuenzaniswa neAD, ese mamodule mana akawedzerwa mukutonga uye PD, achivapa chirwere-chaiyo AD shanduko (Mufananidzo S2C). Pakazere, mhedzisiro iyi inotsigira kudzikira kwakawanda kwemauron-rich modules yatakamboona muAD (13, 17). Muchidimbu, kuongororwa kwenetiweki kweproteome yehuropi yatakawana yakagadzira AD-chaiyo yakashandurwa mamodules inoenderana nezvatakawana kare.
AD inoratidzirwa neyekutanga asymptomatic nhanho (AsymAD), umo vanhu vanoratidza amyloid kuunganidza pasina kiriniki yekuziva kuderera (5, 31). Iyi asymptomatic nhanho inomiririra hwindo rakakosha rekuona nekukurumidza uye kupindira. Isu takamboratidza kuchengetedza kwakasimba kweAsymAD uye AD brain network proteome pane yakazvimirira data seti (13, 17). Kuitira kuti tive nechokwadi chekuti network yehuropi yatakawana ikozvino inopindirana nezvakawanikwa izvi zvakapfuura, takaongorora kuchengetedzwa kwemamodule makumi mana nemana mune yakadzokororwa data yakaiswa kubva ku27 DLPFC masangano. Aya masangano anosanganisira kutonga (n = 10), AsymAD (n = 8) Uye AD (n = 9) nyaya. Kudzora uye masampula eAD akaverengerwa mukuongororwa kwedu kwekuwanikwa kwehuropi cohort (Table S1B), nepo AsymAD makesi aive akasiyana chete mune yekudzokorora cohort. Aya maAsymAD makesi akauyawo kubva kuEmory Goizueta ADRC brain bank. Kunyangwe cognition yaive yakajairika panguva yerufu, mazinga eamyloid aive akakwira zvisingaite (kureva CERAD, 2.8 ± 0.5) (Table S1B).
TMT-MS kuongororwa kweaya 27 uropi matishu akakonzera kuwanda kwe11,244 proteinomes. Kuverenga kwekupedzisira uku kunosanganisira chete iwo mapuroteni akaverengerwa mune inokwana 50% yemasampuli. Iyi yakadzokororwa seti yedata ine 8638 (98.0%) yemapuroteni e8817 akawanikwa mukuongororwa kwedu kwehuropi, uye angangoita 3000 akachinja zvakanyanya mapuroteni pakati pekutonga neAD cohorts (P <0.05, mushure meTukey's paired t bvunzo yekuongorora mutsauko) ( Tafura S2F). Pakati peaya mapuroteni akaratidzwa zvakasiyana, 910 yakaratidzawo shanduko yakakosha pakati peAD uye brain proteome control kesi (P <0.05, mushure meANOVA Tukey paired t-test). Zvinokosha kucherechedza kuti aya 910 mamakisi akanyatsoenderana munzira yekuchinja pakati peproteomes (r = 0.94, P <1.0 × 10-200) (Mufananidzo S3A). Pakati pemapuroteni akawedzera, mapuroteni ane kuchinja kwakanyanya pakati pemaseti e data anonyanya kuve nhengo dze glial-rich M5 uye M18 modules (MDK, COL25A1, MAPT, NTN1, SMOC1, uye GFAP). Pakati pemapuroteni akaderedzwa, avo vane shanduko dzakanyanya kuenderana vangangove nhengo dzeM1 module (NPTX2, VGF, uye RPH3A) yakabatana ne synapse. Isu takaona zvakare shanduko ine chekuita neAD ye midkine (MDK), CD44, yakavanzwa-ine chekuita neprotein 1 (SFRP1) uye VGF nekumadokero blotting (Mufananidzo S3B). Kuongororwa kwekuchengetedza Module kwakaratidza kuti inenge 80% yeprotein modules (34/44) muproteome yeuropi yakanyatsochengetedzwa muzvinyorwa zvekudzokorora data (z-score> 1.96, FDR yakagadziriswa P <0.05) (Mufananidzo S3C). Gumi nemana emamodule aya akanyatsochengetedzwa pakati pemaproteomes maviri (z-score> 10, FDR yakagadziriswa P <1.0 × 10-23). Pakazere, kuwanikwa uye kudzokororwa kweiyo yepamusoro dhigirii yekuenderana mukusiyanisa kutaura uye modular kuumbwa pakati peuropi proteome inosimbisa kuberekana kweshanduko muAD frontal cortex mapuroteni. Pamusoro pezvo, yakasimbisawo kuti AsymAD uye zvimwe zvirwere zvepamberi zvine chimiro chakafanana chehuropi network.
Kuongororwa kwakadzama kwekutaura kwekusiyanisa muuropi yekudzokorora data seti inosimbisa dhigirii yakakosha yeAsymAD mapuroteni shanduko, kusanganisira huwandu hwe151 hwakachinja zvakanyanya mapuroteni pakati peAsymAD uye kutonga (P <0.05) (Mufananidzo S3D). Zvinoenderana neamyloid mutoro, APP muuropi yeAsymAD uye AD yakawedzera zvakanyanya. MAPT inongoshandura zvakanyanya muAD, iyo inopindirana nekuwedzera kwemazinga etangles uye kuwirirana kwayo kunozivikanwa nekuderera kwekuziva (5, 7). Iyo glial-rich modules (M5 uye M18) inoratidzwa zvakanyanya mumapuroteni akawedzera muAsymAD, nepo neuron-inoenderana neM1 module ndiyo inomiririra yakanyanya kuderera mapuroteni muAsymAD. Mazhinji eaya mamaki eAsymAD anoratidza shanduko huru muzvirwere zvezviratidzo. Pakati peaya mamakisi pane SMOC1, glial protein yeM18, inosanganiswa nemamota ehuropi uye kukura kwemaziso nemakumbo (32). MDK is heparin-binding growth factor inoenderana nekukura kwesero uye angiogenesis (33), imwe nhengo yeM18. Kuenzaniswa neboka rekutonga, AsymAD yakawedzera zvakanyanya, ichiteverwa nekuwedzera kukuru kweAD. Kusiyana neizvi, synaptic protein neuropentraxin 2 (NPTX2) yakaderedzwa zvakanyanya muuropi hweAsymAD. NPTX2 yaimbove yakabatana neurodegeneration uye ine basa rinozivikanwa mukuyananisa excitatory synapses (34). Pakazere, mhedzisiro iyi inoratidza akasiyana akasiyana preclinical mapuroteni shanduko muAD anoita seanofambira mberi nekuoma kwechirwere.
Tichifunga kuti takawana hudzamu hwakakosha hwekuvhara mapuroteni mukuwanikwa kweproteome yeuropi, tiri kuyedza kunzwisisa zvizere kupindirana kwayo netiweki-level AD transcriptome. Naizvozvo, takafananidza huropi hweproteome hwatakawana nemodule yatakambogadzira kubva kuchiyero che microarray ye18,204 genes muAD (n = 308) uye kutonga (n = 157) DLPFC tishu (13). overlapping. Pakazara, takaona makumi maviri akasiyana eRNA modules, mazhinji ayo airatidza kupfumiswa kwemamwe maseru marudzi, anosanganisira neurons, oligodendrocytes, astrocytes, uye microglia (Mufananidzo 3A). Kuchinja kwakawanda kwemamodule aya muAD kunoratidzwa muMufananidzo 3B. Zvinoenderana neprotein yedu yapfuura-RNA overlap analysis tichishandisa yakadzika isina kunyorwa MS proteome (inenge 3000 mapuroteni) (13), mazhinji e44 modules mubrain proteome network yatakawana ari mu transcriptome network Hapana chakakosha kupindirana. kuwanikwa kwedu uye kudzokorora kwe34 mapuroteni modules akachengetwa zvakanyanya muuropi proteome, 14 chete (~ 40%) yakapasa bvunzo yaFisher chaiyo (FET) yakaratidza kuve nenhamba yakakosha kupindirana neiyo transcriptome (Mufananidzo 3A). Inopindirana neDNA kukuvara kugadzirisa (P-M25 uye P-M19), shanduro yeprotein (P-M7 uye P-M20), RNA inosunga / splicing (P-M16 uye P-M21) uye mapuroteni akananga (P-M13 uye P- M23) haina kupindirana nemamodule mune iyo transcriptome. Nokudaro, kunyange zvazvo yakadzika proteome data seti inoshandiswa mukuongorora kwezvino kupindirana (13), yakawanda yeAD network proteome haina kunyorwa kune transcriptome network.
(A) Hypergeometric FET inoratidza kuvandudzwa kwesero mhando-chaiwo mamaki muRNA module yeAD transcriptome (yepamusoro) uye dhigirii rekupindirana pakati peRNA (x-axis) uye protein (y-axis) modules yehuropi hweAD. (pasi) . Kusimba kwemumvuri mutsvuku kunoratidza dhigirii yekupfumisa emhando dzemasero mumusoro wepamusoro uye kusimba kwekupindirana kwemamodule muchikamu chepasi. Nyeredzi dzinoratidza kukosha kwenhamba (P <0.05). (B) Chiyero chekubatana pakati pehunhu hwemajini ega ega transcriptome module uye AD mamiriro. Iwo ma modules ari kuruboshwe ndiwo anonyanya kukanganiswa neAD (blue), uye ari kurudyi ndiwo anonyanya kuwirirana neAD (tsvuku). Iyo log-yakashandurwa BH-yakagadziriswa P kukosha inoratidza dhigirii yehuwandu hwehuwandu hwekubatana kwega kwega. (C) Akakosha anopindirana mamodule ane akagovaniswa sero rudzi kusimudzira. (D) Correlation analysis ye log2 fold change yeprotein yakanyorwa (x-axis) uye RNA (y-axis) mumodule inopindirana. Iyo Pearson corelation coefficient ine yakakosha P kukosha inoratidzwa. Micro, microglia; mitumbi yokudenga, ma<em>astrocyte. CT, kutonga.
Mazhinji anopindirana mapuroteni uye maRNA mamodule anogovera akafanana sero rudzi rwekupfumisa mafaera uye anowirirana AD shanduko nzira (Mufananidzo 3, B uye C). Mune mamwe mazwi, iyo synapse-inoenderana neM1 module yeuropi proteome (PM1) inomepu kune matatu neuronal-rich homologous RNA modules (R-M1, R-M9 uye R-M16), ari muAD Ose akaratidzwa. mwero wakaderedzwa. Saizvozvowo, glial-rich M5 uye M18 protein modules inopindirana neRNA modules yakapfuma mune astrocytes uye microglial markers (R-M3, R-M7, uye R-M10) uye inobatanidzwa zvakanyanya muzvirwere Kuwedzera. Aya akagovaniswa ma modular maficha pakati pemaseti maviri e data anoenderera mberi nekutsigira sero rudzi kupfumisa uye shanduko dzine hukama nechirwere dzatakaona muuropi proteome. Nekudaro, isu takaona misiyano yakawanda yakakosha pakati peRNA uye mapuroteni mazinga emunhu anomaka mune aya akagovaniswa mamodule. Correlation ongororo yekusiyanisa kutaura kweproteomics uye transcriptomics yemamorekuru mukati meaya anopindirana mamodule (Mufananidzo 3D) inosimbisa iyi kusagadzikana. Semuenzaniso, APP uye akati wandei mamwe mapuroteni eglial module (NTN1, MDK, COL25A1, ICAM1, uye SFRP1) yakaratidza kuwedzera kukuru kweAD proteome, asi pakanga pasina shanduko muAD transcriptome. Idzi shanduko dzeprotein-chaiyo dzinogona kunge dzine hukama hwepedyo nemaamyloid plaques (23, 35), inoratidzira iyo proteome senzvimbo yekuchinja kwepathological, uye shanduko idzi dzinogona kusaratidzwa mune zvinyorwa.
Mushure mekuzvimiririra kuongorora huropi uye CSF maproteomes atakawana, takaita ongororo yakazara yemaseti maviri e data kuti tizive AD CSF biomarkers ine chekuita neiyo pathophysiology yebrain network. Tinofanira kutanga tatsanangura kupindirana kwemaproteome maviri. Kunyangwe zvichigamuchirwa zvakanyanya kuti CSF inoratidza shanduko yeurourochemical muAD brain (4), iyo chaiyo dhigirii yekupindirana pakati peAD brain neCSF proteome haina kujeka. Nokuenzanisa nhamba yezvigadzirwa zvemajini zvakagoverwa zvakaonekwa mumaproteome edu maviri, takaona kuti inenge 70% (n = 1936) yemapuroteni akaonekwa mu cerebrospinal fluid akaverengwawo muuropi (Mufananidzo 4A). Mazhinji eaya anopindirana mapuroteni (n = 1721) akaiswa mepu kune imwe ye44 co-kutaura modules kubva pakuwanikwa kwehuropi data set (Mufananidzo 4B). Sezvaitarisirwa, iwo matanhatu makuru ehuropi modules (M1 kusvika M6) yakaratidza huwandu hukuru hweCSF inopindirana. Nekudaro, kune madiki europi mamodule (semuenzaniso, M15 uye M29) anowana zvisingatarisirwe yakakwirira dhigirii yekupindirana, yakakura kupfuura yeuropi module kaviri saizi yayo. Izvi zvinotikurudzira kuti titore nzira yakadzama, inofambiswa nenhamba yekuverenga kupindirana pakati pehuropi uye cerebrospinal fluid.
(A uye B) Mapuroteni akaonekwa muhuropi hwekuwanikwa uye CSF data seti inopindirana. Mazhinji emapuroteni aya anopindirana akabatanidzwa neimwe ye44 co-expression modules yeuropi co-expression network. (C) Tsvaga kupindirana pakati pecerebrospinal fluid proteome neuropi network proteome. Mutsara wega wega wemepu yekupisa inomiririra yakaparadzana yakatarisana kuongororwa kweiyo hypergeometric FET. Mutsara wepamusoro unoratidza kupindirana (grey / dema shading) pakati peuropi module uye yese CSF proteome. Mutsara wechipiri unoratidza kuti kupindirana pakati peuropi modules uye CSF mapuroteni (shaded mutsvuku) akanyanya kukwira-inodzorwa muAD (P <0.05). Mutsara wechitatu unoratidza kuti kupindirana pakati peuropi modules uye CSF protein (blue shading) inonyanya kuderera-yakagadziriswa muAD (P <0.05). Shandisa nzira yeBH kugadzirisa kukosha kweP kunobva kuFET. (D) Kupeta module pani yakavakirwa pasero rudzi kushamwaridzana uye ane hukama GO mazwi. Aya mapaneru ane huwandu hwe271 mapuroteni ane hukama nehuropi, ane zvinoreva mutsauko wekutaura muCSF proteome.
Tichishandisa single-tailed FETs, takaongorora kukosha kweprotein inopindirana pakati peCSF proteome nemamodule ehuropi ega ega. Kuongorora kwakaratidza kuti huwandu hwe14 brain modules muCSF data set ine statistically akakosha overlaps (FDR yakagadziridzwa P <0.05), uye imwezve module (M18) iyo inopindirana iri pedyo nekukosha (FDR yakagadziridzwa P = 0.06) (Mufananidzo 4C , mutsara wepamusoro). Isu tiri zvakare kufarira mamodule anopindirana zvakanyanya nemapuroteni eCSF anoratidzwa zvakasiyana. Naizvozvo, takaisa ongororo mbiri dzeFET kuti tione kuti ndeipi ye (i) CSF protein yakawedzera zvakanyanya muAD uye (ii) CSF protein yakadzikira zvakanyanya muAD (P <0.05, paired t test AD/control) Brain modules ine hukama hunoenderana. pakati pavo. Sezvinoratidzwa pakati nepakati mitsara yeFigure 4C, iyi ongororo yakawedzerwa inoratidza kuti 8 ye44 brain modules inopindirana zvakanyanya neprotein yakawedzerwa muAD CSF (M12, M1, M2, M18, M5, M44, M33, uye M38) . ), nepo ma modules maviri chete (M6 neM15) airatidza kupindirana kunoreva neprotein yakaderedzwa muAD CSF. Sezvinotarisirwa, ese gumi mamodules ari mumamodule gumi nemashanu ane akanyanya kupindirana neCSF proteome. Naizvozvo, isu tinofungidzira kuti aya gumi nemashanu mamodule ndiwo akakwira-goho masosi eAD brain-yakatorwa CSF biomarkers.
Isu takapeta aya gumi neshanu anopindirana mamodule kuita mashanu makuru mapuroteni mapaneru zvichienderana nekuva pedyo kwavo mudhiyagiramu yemuti weWGCNA uye kushamwaridzana kwavo nemhando dzemasero uye gene ontology (Mufananidzo 4D). Iyo yekutanga pani ine mamodule akapfuma mune neuron mamaki uye synapse-ane hukama mapuroteni (M1 uye M12). Iyo synaptic panel ine huwandu hwe94 mapuroteni, uye mazinga ari muCSF proteome achinja zvakanyanya, zvichiita kuti ive iyo huru sosi yehuropi-inoenderana CSF mamaki pakati pemapaneru mashanu. Boka rechipiri (M6 neM15) rakaratidza kushamwaridzana kwepedyo ne endothelial cell markers uye vascular body, yakadai se "kuporeswa kwemaronda" (M6) uye "mutemo wehumoral immune response" (M15). M15 inobatanidzawo zvakanyanya ne lipoprotein metabolism, iyo yakanyatsobatana ne endothelium (36). Iyo vascular panel ine 34 CSF mamaki ane hukama nehuropi. Boka rechitatu rinosanganisira ma modules (M2 neM4) ayo anonyanya kuenderana nemafuta e oligodendrocyte uye kuwedzera kwesero. Semuenzaniso, iwo epamusoro-level ontology mazwi eM2 anosanganisira "positive regulation yeDNA replication" uye "purine biosynthesis process". Zvichakadaro, izvo zveM4 zvinosanganisira "glial cell differentiation" uye "chromosome segregation". Iyo myelination pani ine 49 CSF mamaki ane hukama nehuropi.
Boka rechina rine akawanda mamodule (M30, M29, M18, M24, uye M5), uye anenge ese mamodule akapfuma zvakanyanya mu microglia uye astrocyte mamaki. Zvakafanana nepaneti yemyelination, pani yechina inewo mamodules (M30, M29, uye M18) ayo ane hukama zvakanyanya nekuwanda kwesero. Mamwe ma modules ari muboka iri ane hukama zvakanyanya nemashoko ekudzivirira zvirwere, zvakadai se "immune effect process" (M5) uye "immune response regulation" (M24). Glial immune immune group ine 42 CSF mamaki ane hukama nehuropi. Pakupedzisira, chikwata chekupedzisira chinosanganisira 52 zviratidzo zvine chokuita neuropi pamamodules mana (M44, M3, M33, uye M38), zvose zviri pamuviri zvine chokuita nekuchengetedza simba uye metabolism. Iyo yakakura kwazvo yeaya mamodule (M3) ane hukama zvakanyanya nemitochondria uye akapfuma mune neuron-chaiwo mamaki. M38 ndeimwe yenhengo diki dzemodule mune iyi metabolome uye zvakare inoratidza zvine mwero neuron chaiyo.
Pakazere, aya mapaneru mashanu anoratidza huwandu hwakawanda hwemhando dzemasero uye mabasa muAD cortex, uye pamwe chete ane 271 ane hukama nehuropi CSF mamaki (Table S2G). Kuti tiongorore huchokwadi hweiyi mhedzisiro yeMS, takashandisa proximity extension assay (PEA), orthogonal antibody-based tekinoroji ine multiplexing kugona, kunzwisiswa kwepamusoro uye hunhu, uye tikaongororazve masamples ecerebrospinal fluid takawana A subset yeaya mazana maviri nemakumi manomwe ane biomarkers. (n = 36). Izvi zvinangwa zve36 zvinoratidza shanduko yeAD yakawanda yePEA, iyo inowirirana zvakanyanya kune yedu MS-based zvakawanikwa (r = 0.87, P = 5.6 × 10-12) , Iyo yakasimbisa zvakasimba migumisiro yekuongorora kwedu kwakazara MS (Mufananidzo S4 )
Iwo madingindira ebhayoloji anosimbiswa nemapoka edu mashanu, kubva kune synaptic kusaina kusvika kune simba metabolism, ese ane hukama neiyo pathogenesis yeAD (1-3). Naizvozvo, ese gumi nemashanu mamodule ane aya mapaneru ane hukama neAD pathology muuropi proteome yatakawana (Mufananidzo 2B). Iyo inonyanya kukosha ndeyepamusoro yakanaka pathological correlation pakati pe glial modules uye yakasimba isina kunaka pathological correlation pakati pemakuru edu neuronal modules (M1 neM3). Iko kusiyanisa kutaura kwekuongorora kweyedu yakadzokororwa brain proteome (Mufananidzo S3D) zvakare inosimbisa M5 uye M18-yakatorwa glial mapuroteni. MuAsymAD uye chiratidzo cheAD, iyo yakanyanya kuwedzera glial mapuroteni uye M1-yakabatana synapses Iyo puroteni inoderedzwa zvakanyanya. Aya maratidziro anoratidza kuti 271 cerebrospinal fluid mamaki atakaona mumapoka mashanu ane hukama nemaitiro echirwere muAD cortex, kusanganisira ayo anoitika mumatanho ekutanga asymptomatic.
Kuti tiongorore zvirinani shanduko yemapuroteni epaneti muuropi uye musana wemvura, takadhirowa zvinotevera kune imwe neimwe yemamodule gumi nemashanu anopokana: (i) akawana iyo module kuwanda nhanho muuropi data set uye (ii) module. mapuroteni Musiyano unoratidzwa mu cerebrospinal fluid (Mufananidzo S5). Sezvambotaurwa, WGCNA inoshandiswa kuona iyo module kuwanda kana hunhu hweprotein kukosha muuropi (13). Mepu yegomo rinoshandiswa kutsanangura mutsauko wekutaura kwe modular mapuroteni mu cerebrospinal fluid (AD/control). Aya manhamba anoratidza kuti matatu pamashanu mapaneru anoratidza akasiyana ekutaura maitiro muuropi uye musana wemvura. Iwo maviri modules epaneti ye synapse (M1 uye M12) inoratidza kuderera kwehuwandu hwehuwandu muhuropi hweAD, asi zvakanyanya kupindirana neprotein yakawedzera muAD CSF (Mufananidzo S5A). Iyo neuron-related modules ine metabolome (M3 uye M38) yakaratidza huropi hwakafanana uye cerebrospinal fluid kutaura maitiro asingaenderane (Mufananidzo S5E). Iro vascular panel yakaratidzawo maitiro akasiyana-siyana ekutaura, kunyange zvazvo ma modules (M6 neM15) akawedzera zvishoma muuropi yeAD uye akaderera muCSF inorwara (Mufananidzo S5B). Iwo maviri mapaneru asara ane mahombe glial network ane mapuroteni anogara achikwiridzirwa mumakamuri ese ari maviri (Mufananidzo S5, C uye D).
Ndokumbira utarise kuti maitiro aya haawanikwe kune ese mamark mumapanera aya. Semuenzaniso, iyo synaptic panel inosanganisira akati wandei mapuroteni akaderedzwa zvakanyanya muhuropi hweAD uye CSF (Mufananidzo S5A). Pakati peaya anodzika-regulated cerebrospinal fluid markers pane NPTX2 neVGF yeM1, uye chromogranin B yeM12. Nekudaro, zvisinei nekusarudzika uku, mazhinji emamaki edu esynaptic akakwidziridzwa muAD spinal fluid. Pakazere, ongororo idzi dzakakwanisa kusiyanisa maitiro akakosha muhuropi uye cerebrospinal fluid mazinga mune yega yega mapaneru edu mashanu. Aya maitiro anosimbisa hukama hwakaoma uye kazhinji hwakasiyana pakati pehuropi neCSF mapuroteni kutaura muAD.
Zvadaro, takashandisa yakakwira-kuburikidza MS kudzokorora ongororo (CSF replication 1) kupfupisa yedu 271 seti yema biomarkers kune zvakanyanya kuvimbisa uye zvinogoneka tariswa (Mufananidzo 5A). CSF kopi 1 ine huwandu hwemakumi mapfumbamwe nematanhatu esampuli kubva kuEmory Goizueta ADRC, kusanganisira kutonga, AsymAD, uye AD cohort (Table S1A). Aya makesi eAD anoratidzwa nekuderera kwekuziva kuderera (kureva MoCA, 20.0 ± 3.8), uye shanduko muAD biomarkers yakasimbiswa mucerebrospinal fluid (Table S1A). Kusiyana nekuongorora kweCSF kwatakawana, kudzokororwa uku kunoitwa pachishandiswa nzira yakanyatsonaka uye yepamusoro "single-shot" MS nzira (isina kupatsanurwa kwe-off-line), kusanganisira yakareruka sampuli yegadziriro yeprotocol inobvisa kudiwa kwekudzivimbika kwemasamples ega ega. . Pane kudaro, imwe chete inodzivirirwa ne "inosimudzira chiteshi" inoshandiswa kukwidziridza chiratidzo chemapuroteni mashoma (37). Kunyange zvazvo ichideredza kuvharwa kweproteome yose, iyi nzira yepfuti imwe chete inoderedza nguva yemuchina uye inowedzera nhamba yemasampuli akanyorwa neTMT anogona kuongororwa kushanda (17, 38). Pakazara, ongororo yakaratidza 6,487 peptides, iyo yakaratidza 1,183 mapuroteni mumakumi mapfumbamwe nematanhatu. Sezvakaita nekuongorora kweCSF kwatakawana, iwo mapuroteni chete akaverengerwa mune inokwana 50% yemasampuli akaverengerwa mukuverenga kwaitevera, uye iyo data yakadzoserwa kumashure kwemhedzisiro yezera uye murume kana murume. Izvi zvakakonzera kuverengerwa kwekupedzisira kwe792 maproteomes, 95% ayo akaonekwa zvakare muCSF data set yakawanikwa.
(A) Brain-inoenderana neCSF mapuroteni zvinangwa zvakasimbiswa mune yekutanga yakadzokororwa CSF cohort uye inosanganisirwa mune yekupedzisira mapaneru (n = 60). (B kusvika E) Panel biomarker mazinga (composite z-scores) akayerwa mune ina CSF replication cohorts. Paired t-tests kana ANOVA ine Tukey's post-correction yakashandiswa kuongorora kukosha kwenhamba yekuchinja kwehuwandu mukuwongorora kwega kwega. CT, kutonga.
Sezvo isu tichinyanya kufarira kuona zvatinovavarira 271 zvine chekuita nehuropi CSF kuburikidza nekuongorora kwakadzama, isu tichadzikamisa kuongororwa kweiyi proteome yakadzokororwa kune aya mamaki. Pakati peaya mapuroteni e271, 100 yakaonekwa muCSF replication 1. Mufananidzo S6A inoratidza mutsauko wekutaura kweiyo 100 inopindirana mamakisi pakati pekutonga uye AD kudzokorora samples. Synaptic uye metabolite histones inowedzera zvakanyanya muAD, nepo mapuroteni evascular anoderera zvakanyanya muchirwere. Mazhinji e100 anopindirana mamakisi (n = 70) akachengetedza nzira imwechete yekuchinja mumaseti maviri edata (Mufananidzo S6B). Aya makumi manomwe akasimbiswa ehuropi ane chekuita neCSF mamaki (Table S2H) anonyanya kuratidza yaimbocherechedzwa mapaneru ekutaura mafambiro, kureva, kudzika-mutemo wevascular mapuroteni uye kumusoro-mutemo wemamwe ese mapaneru. 10 chete yeaya makumi manomwe akasimbiswa mapuroteni airatidza shanduko muhuwandu hweAD hwakapesana nemaitiro aya epaneru. Kuti tigadzire pani inonyatso ratidza maitiro ese ehuropi uye cerebrospinal fluid, isu takabvisa aya mapuroteni gumi kubva papaneru yekufarira iyo yatakazosimbisa (Mufananidzo 5A). Naizvozvo, mapaneru edu anozopedzisira asanganisira huwandu hwemapuroteni makumi matanhatu akasimbiswa mune maviri akazvimirira CSF AD cohorts achishandisa akasiyana ekugadzirira sampuli uye MS papuratifomu kuongororwa. Iyo z-score kutaura zvirongwa zveaya mapaneru ekupedzisira muCSF kopi 1 control uye AD makesi akasimbisa iyo mapaneru maitiro akaonekwa muCSF cohort yatakawana (Mufananidzo 5B).
Pakati pemapuroteni makumi matanhatu aya, pane mamorekuru anozivikanwa kuve akabatana neAD, akadai seosteopontin (SPP1), inova pro-inflammatory cytokine iyo yakabatanidzwa neAD muzvidzidzo zvakawanda (39-41), uye GAP43, A synaptic protein. izvo zvakanyatsobatanidzwa neurodegeneration (42). Mapuroteni akanyanyo nyatso nyatso ongororwa zviratidzo zvine chekuita nezvimwe zvirwere zveurourodegenerative, zvakaita seamyotrophic lateral sclerosis (ALS) ine chekuita ne superoxide dismutase 1 (SOD1) uye chirwere cheParkinson desaccharase (PARK7). Isu takaona zvakare kuti mamwe akawanda mamaki, akadai se SMOC1 uye uropi-rakapfuma membrane yekunamatira inosaina protein 1 (BASP1), ine mashoma ekare ma link kune neurodegeneration. Zvinokosha kucherechedza kuti nekuda kwekuderera kwavo kwese kwakawanda mu CSF proteome, zvakatiomera kuti tishandise iyi yepamusoro-yepamusoro-pfuti yekuona nzira yekuona yakavimbika MAPT uye mamwe mapuroteni ane hukama neAD (semuenzaniso, NEFL neNRGN. ) ( 43, 44).
Isu takazotarisa aya makumi matanhatu ekutanga mapaneru mamaki mune mamwe matatu ekuwedzera kudzokorora ongororo. MuCSF Copy 2, takashandisa imwechete TMT-MS kuongorora yakazvimirira cohort ye297 control uye AD samples kubva Emory Goizueta ADRC (17). CSF replication 3 yaisanganisira reanalysis yeanowanikwa TMT-MS data kubva ku120 control uye AD varwere kubva kuLausanne, Switzerland (45). Takaona zvinopfuura zvikamu zviviri muzvitatu zvemakasi makumi matanhatu ekutanga mudhata rega rega. Kunyange zvazvo chidzidzo cheSwiss chakashandisa mapuratifomu eMS akasiyana uye TMT quantification nzira (45, 46), isu takanyatso dhinda yedu mapaneru maitiro mune maviri akadzokororwa ongororo (Mufananidzo 5, C uye D, uye Matafura S2, I, uye J). Kuti tiongorore hunhu hwechirwere cheboka redu, takashandisa TMT-MS kuongorora yechina replication data set (CSF replication 4), iyo yaisanganisira kwete kutonga chete (n = 18) uye AD (n = 17) kesi, asiwo PD ( n = 14)), ALS (n = 18) uye frontotemporal dementia (FTD) samples (n = 11) (Table S1A). Isu takabudirira kuverenga zvingangoita zviviri muzvitatu zvemapuroteni epaneru mune ino cohort (38 kunze kwe60). Mhedzisiro iyi inoburitsa AD-chaiyo shanduko mune ese mashanu biomarker mapaneru (Mufananidzo 5E uye Tafura S2K). Kuwedzera kweboka remetabolite kwakaratidza kusimba kwakasimba kweAD, kuteverwa nemyelination uye glial boka. Kusvika padiki, FTD inoratidzawo kuwedzera pakati peaya mapaneru, ayo anogona kuratidza akafanana angangoita network shanduko (17). Kusiyana neizvi, ALS nePD yakaratidza zvakada kufanana myelination, glial, uye metabolome profiles seboka rinodzora. Pakazere, kunyangwe paine misiyano mukugadzirira kwemuenzaniso, MS chikuva, uye TMT quantification nzira, idzi ongororo dzakadzokororwa dzinoratidza kuti edu ekutanga mapaneru mamaki ane anowirirana zvakanyanya AD-chaiwo shanduko mune anopfuura mazana mashanu akasarudzika eCSF samples.
AD neurodegeneration yave ichizivikanwa zvakanyanya makore akati wandei kusati kwatanga zviratidzo zvekuziva, saka pane kudiwa kwekukurumidzira kwema biomarkers eAsymAD (5, 31). Zvisinei, humwe humwe uchapupu hunoratidza kuti biology yeAsymAD iri kure nehomogeneous, uye kupindirana kwakaoma kwengozi uye kusimba kunotungamirira kune misiyano mikuru yemunhu mukufambira mberi kwechirwere (47). Kunyange zvazvo yaishandiswa kuzivisa nyaya dzeAsymAD, mazinga e-core CSF biomarkers (Aβ1-42, tau yakazara uye p-tau) haana kuratidza kukwanisa kunyatsofanotaura kuti ndiani achafambira mberi kune dementia (4, 7), zvichiratidza zvimwe Zvingave. zvinodikanwa kusanganisa azere biomarker maturusi akavakirwa pane akati wandei maficha ehuropi physiology kunyatso kurongedza njodzi yehuwandu uhu. Nokudaro, isu takazoongorora yedu AD-yakagadziriswa biomarker panel muAsymAD yevanhu veCSF kopi 1. Aya 31 AsymAD makesi akaratidza abnormal core biomarker levels (Aβ1-42 / total tau ELISA ratio, <5.5) uye cognition yakakwana (kureva MoCA, 27.1). ± 2.2) (Tafura S1A). Pamusoro pezvo, vanhu vese vane AsymAD vane kiriniki yedementia mamakisi e0, zvichiratidza kuti hapana humbowo hwekudzikira mukuziva kwemazuva ese kana kushanda kwekuita.
Takatanga kuongorora mazinga emapanera akasimbiswa mune ese 96 CSF inodzokorora 1, kusanganisira iyo AsymAD cohort. Takaona kuti mapaneru akati wandei muboka reAsymAD aive neakakosha AD-yakafanana nekuwanda shanduko, iyo vascular panel yakaratidza kuderera kweAsymAD, nepo mamwe mapaneru airatidza kukwira kumusoro (Mufananidzo 6A). Naizvozvo, mapaneru ese airatidza kuwirirana kwakakosha neELISA Aβ1-42 uye yakazara tau mazinga (Mufananidzo 6B). Kusiyana neizvi, kuwirirana pakati peboka neMoCA mamakisi kwakaderera. Chimwe chezvinhu zvinonyanya kushamisa zvakawanikwa kubva mukuwongorora uku ndiko kuwanda kwakakura kwemapaneru akawanda muAsymAD cohort. Sezvinoratidzwa muMufananidzo 6A, nhanho yepaneru yeboka reAsymAD kazhinji inoyambuka padanho reboka rekutonga neboka reAD, richiratidza kusiyanisa kwakanyanya. Kuti tiwedzere kuongorora iyi heterogeneity yeAsymAD, takashandisa Multidimensional Scaling (MDS) ongororo kune 96 CSF replication 1 kesi. Ongororo yeMDS inobvumira kuona kufanana pakati pemakesi akavakirwa pane zvimwe zvinosiyana mune data set. Kune iyi cluster ongororo, isu tinongoshandisa iwo akasimbiswa mapaneru mamaki ane shanduko yakakosha (P <0.05, AD/control) mukutsvaga kweCSF uye kudzokorora 1 proteome (n = 29) (Table S2L) level. Kuongorora uku kwakaburitsa pachena kusanganisa kwepakati pakati pekutonga kwedu uye makesi eAD (Mufananidzo 6C). Mukupesana, mamwe maAsymAD makesi akanyatso kuunganidzwa muboka rekutonga, nepo mamwe ari mumakesi eAD. Kuenderera mberi nekuongorora iyi AsymAD heterogeneity, takashandisa mepu yedu yeMDS kutsanangura mapoka maviri eaya maAsymAD kesi. Boka rekutanga raisanganisira maAsymAD makesi akaunganidzwa padyo nekutonga (n = 19), nepo boka rechipiri raizivikanwa nemakesi eAsymAD ane marker profile padyo neAD (n = 12).
(A) Chiyero chekutaura (z-score) cheboka reCSF biomarker mune ese makumi mapfumbamwe nematanhatu samples muCSF replication 1 cohort, kusanganisira AsymAD. Ongororo yekusiyana neyeTukey's post-correction yakashandiswa kuongorora kukosha kwenhamba yekuchinja kwepanel kuwanda. (B) Correlation analysis of panel protein abundance level (z-score) ine MoCA score uye total tau level muELISA Aβ1-42 uye CSF copy 1 samples. Iyo Pearson corelation coefficient ine yakakosha P kukosha inoratidzwa. (C) Iyo MDS ye96 CSF kopi 1 kesi yaive yakavakirwa pakuwanda kwemazinga e29 yakasimbiswa mapaneru mamaki, ayo akashandurwa zvakanyanya mune zvese kuwanikwa uye CSF kopi 1 data seti [P <0.05 AD/control (CT)]. Kuongorora uku kwakashandiswa kupatsanura boka reAsymAD mukutonga (n = 19) uye AD (n = 12) madiki. (D) The volcano plot inoratidza mutsauko wekutaura kwese CSF replication 1 mapuroteni ane log2 fold shanduko (x-axis) inoenderana ne -log10 nhamba yeP kukosha pakati peaviri maAsymAD madiki. Iyo mapaneru biomarkers ane mavara. (E) CSF kudzokorodza 1 kuwanda nhanho yeboka rekusarudza biomarkers inoratidzirwa zvakasiyana pakati peAsymAD subgroups. Tukey's post-adjusted analysis of variance yakashandiswa kuongorora kukosha kwenhamba.
Takaongorora musiyano weprotein kutaura pakati pekutonga uku uye AD-seAsymAD kesi (Mufananidzo 6D uye Tafura S2L). Mepu yegomo inoputika inoratidza kuti 14 mapanera mamaki achinja zvakanyanya pakati pemapoka maviri aya. Zvizhinji zveizvi mamakisi inhengo dze synapse uye metabolome. Zvisinei, SOD1 uye myristoylated alanine-rich protein kinase C substrate (MARCKS), iyo inhengo dzemyelin uye glial immune groups, maererano, zvakare inhengo yeboka iri (Mufananidzo 6, D uye E) . Iro vascular panel yakapawo zviratidzo zviviri zvakaderedzwa zvakanyanya muAD-seAsymAD boka, kusanganisira AE inosunga mapuroteni 1 (AEBP1) uye inobatanidza nhengo yemhuri C9. Pakanga pasina musiyano wakakosha pakati pekutonga uye AD-seAsymAD subgroups muELISA AB1-42 (P = 0.38) uye p-tau (P = 0.28), asi pakanga paine mutsauko wakakosha muyero yakazara tau (P = 0.0031 ) (Fig. S7). Kune akati wandei mapenera mamaki anoratidza kuti shanduko pakati peaviri maAsymAD madiki akakosha kupfuura ese emazinga etau (semuenzaniso, YWHAZ, SOD1, uye MDH1) (Mufananidzo 6E). Pakazere, mhedzisiro iyi inoratidza kuti yedu yakasimbiswa pani inogona kunge iine biomarkers inogona subtype uye inogona kuita njodzi stratification yevarwere vane asymptomatic chirwere.
Pane kudiwa kwekukurumidzira kwesystem-based biomarker maturusi ekuyera zvirinani uye kunanga akasiyana pathophysiology kuseri kweAD. Zvishandiso izvi zvinotarisirwa kuti zvisangoshandura AD diagnostic framework, asi zvakare kukurudzira kugamuchirwa kwemaitiro anobudirira, anonyatsotarisana nemurwere (1, 2). Kuti izvi zviitike, takashandisa nzira isina kurerekera yakazara yeproteomics kuhuropi hweAD neCSF yekuona webhu-based CSF biomarkers inoratidza huwandu hwakawanda hwehuropi-based pathophysiology. Ongororo yedu yakaburitsa mashanu CSF biomarker mapaneru, ayo (i) anoratidza synapses, tsinga dzeropa, myelin, immune uye metabolic dysfunction; (ii) ratidza kubereka kwakasimba pamapuratifomu akasiyana eMS; ( iii) Ratidza kuchinja kunofambira mberi kwechirwere mukati mekutanga nekunonoka kweAD. Pakazere, izvi zvakawanikwa zvinomiririra nhanho inovimbisa yekuvandudza kwakasiyana, yakavimbika, yewebhu-yakatarisana biomarker maturusi ekutsvagisa kweAD uye makiriniki mashandisirwo.
Mhedzisiro yedu inoratidza sangano rakachengetedzwa zvakanyanya reAD brain network proteome uye inotsigira kushandiswa kwayo sechitsigiro chekusimudzira-based biomarker development. Ongororo yedu inoratidza kuti maviri akazvimirira TMT-MS dhataseti ane AD uye AsymAD brain ane yakasimba modularity. Izvi zvakawanikwa zvinowedzera basa redu rekare, zvichiratidza kuchengetedzwa kwemamodule ane simba anopfuura 2,000 brain tissues kubva kune akawanda akazvimiririra cohorts mu frontal, parietal, uye temporal cortex (17). Iyi network yekubvumirana inoratidza shanduko dzakasiyana-siyana dzine chekuita nechirwere dzakaonekwa mukutsvagisa kwazvino, kusanganisira kuwedzera kweglial-rich inflammatory modules uye kudzikira kwemamodule neuron-rich. Kufanana nekutsvagisa kwazvino, iyi yakakura-yakakura network ine zvakare yakakosha modular shanduko muAsymAD, ichiratidza akasiyana akasiyana preclinical pathophysiology (17).
Zvakadaro, mukati meiyi inochengetedza zvakanyanya sisitimu-yakavakirwa hurongwa, pane yakanyatso-grained biological heterogeneity, kunyanya pakati pevanhu vari mukutanga kweAD. Yedu biomarker pani inokwanisa kuratidza maviri madiki muAsymAD, ayo anoratidza kukosha kwakasiyana kutaura kweakawanda CSF mamaki. Boka redu rakakwanisa kuratidza mutsauko webhayoloji pakati pezvikamu zviviri izvi, izvo zvaive zvisiri pachena padanho reiyo AD biomarkers. Kuenzaniswa neboka rekutonga, iyo Aβ1-42 / yakazara tau ratios yeava maAsymAD vanhu vaive vakaderera zvisingaite. Zvisinei, mazinga etau chete akanga akasiyana zvakanyanya pakati pezvikwata zviviri zveAsymAD, nepo Aβ1-42 uye p-tau mazinga akasara akaenzana. Sezvo yakakwira CSF tau ichiita kunge iri nani inofanotaura zviratidzo zvekuziva kupfuura Aβ1-42 mazinga (7), isu tinofungidzira kuti maviri eAsymAD cohorts anogona kunge aine njodzi dzakasiyana dzekuenderera kwechirwere. Tichifunga nezve mashoma emuenzaniso saizi yeAsymAD yedu uye kushomeka kweiyo longitudinal data, kumwe kutsvagisa kunodiwa kuti utore izvi mhedzisiro. Nekudaro, mhedzisiro iyi inoratidza kuti system-yakavakirwa CSF pani inogona kusimudzira kugona kwedu kunyatso stratify vanhu panguva yeasymptomatic nhanho yechirwere.
Pakazara, zvatinowana zvinotsigira basa rezvakawanda zvebhayoloji mabasa mupathogenesis yeAD. Nekudaro, dysregulated simba metabolism rakava iro dingindira rine mukurumbira reese mashanu edu akasimbiswa ekunyora mapaneru. Mapuroteni eMetabolic, akadai se hypoxanthine-guanine phosphoribosyltransferase 1 (HPRT1) uye lactate dehydrogenase A (LDHA), ndiwo anonyanya kusimbiswa synaptic biomarkers, zvichiratidza kuti kuwedzera kweAD CSF kune zvepabonde zvakanyanya. Mitsipa yedu yeropa uye glial mapaneru zvakare ane akati wandei mamaki anobatanidzwa mumetabolism yeoxidative zvinhu. Izvi zvakawanikwa zvinopindirana nebasa rakakosha rinoitwa nemetabolism muhuropi hwese, kwete kungosangana nepamusoro pesimba rinodiwa neuroni, asiwo kusangana nehukuru hwesimba hunodiwa hweastrocytes nemamwe masero eglial (17, 48). Mhedzisiro yedu inotsigira humbowo huri kukura hwekuti shanduko mune redox kugona uye kuvhiringwa kwesimba nzira inogona kunge iri musimboti wekubatanidza pakati akati wandei maitiro akakosha anosanganisirwa pathogenesis yeAD, kusanganisira mitochondrial kusagadzikana, glial-mediated kuzvimba, uye Vascular kukuvara (49). Pamusoro pezvo, iyo metabolic cerebrospinal fluid biomarkers ine huwandu hukuru hwemapuroteni akapfuma akasiyana pakati pekutonga kwedu uye AD-seAsymAD subgroups, zvichiratidza kuti kukanganiswa kweaya simba uye redox nzira kunogona kuve kwakakosha mune preclinical nhanho yechirwere.
Huropi hwakasiyana uye cerebrospinal fluid panel mafambiro atakaona zvakare ane zvinonakidza biological zvinoreva. Synapses uye metabolomes akapfuma mueuroni anoratidza kuderera kwemazinga muhuropi hweAD uye kuwedzera kuwanda mucerebrospinal fluid. Tichifunga kuti neuroni dzakapfuma mumitochondria inogadzira simba pamasynapses kuti ipe simba kune akawanda masaini masaini (50), kufanana kwemazita emaprofiles emapoka maviri euron anotarisirwa. Kurasikirwa kwemaneuroni uye kuwedzererwa kwemasero akakuvadzwa kunogona kutsanangura huropi uhwu uye CSF mapaneru mafambiro mune gare gare chirwere, asi havagone kutsanangura yekutanga mapaneru shanduko yatinoona (13). Imwe tsananguro inogoneka yezviwanikwa izvi mukutanga asymptomatic chirwere ndeye abnormal synaptic pruning. Humbowo hutsva mumakonzo mamodheru hunoratidza kuti microglia-mediated synaptic phagocytosis inogona kunge yakaitwa zvisina kujairika muAD uye inotungamira mukurasikirwa kwekutanga kwe synapse muuropi (51). Izvi zvakaraswa synaptic zvinhu zvinogona kuungana muCSF, ndosaka tichiona kuwedzera kweCSF mune neuron panel. Immune-mediated synaptic pruning inogonawo kutsanangura zvishoma kuwedzera kweglial proteins yatinoona muuropi uye cerebrospinal fluid mukati mekuita kwechirwere. Pamusoro pekuchekerera kwesynaptic, kukanganisa kwakazara munzira ye exocytic kunogonawo kutungamira kune akasiyana uropi uye CSF mataurirwo euronal mamaki. Tsvakurudzo dzinoverengeka dzakaratidza kuti zvinyorwa zveexosomes mupathogenesis yeAD brain zvakachinja (52). Iyo extracellular nzira inobatanidzwawo mukupararira kweAβ (53, 54). Izvo zvakakosha kucherechedza kuti kudzvanywa kwe exosomal secretion kunogona kuderedza AD-senge pathology muAD transgenic mbeva modhi (55).
Panguva imwecheteyo, mapuroteni ari muvascular panel akaratidza kuwedzera kwepakati muuropi AD, asi zvakanyanya kuderera muCSF. Kusashanda zvakanaka kweropa-brain barriers (BBB) kunogona kutsanangura zvakawanikwa izvi. Zvizhinji zvakazvimiririra postmortem zvidzidzo zvevanhu zvakaratidza kuparara kweBBB muAD (56, 57). Zvidzidzo izvi zvakasimbisa zviitiko zvakasiyana-siyana zvisina kujairika zvakatenderedza iyi yakavharwa zvakasimba yemasero endothelial, anosanganisira uropi capillary leakage uye perivascular kuunganidzwa kwemapuroteni anotakurwa neropa (57). Izvi zvinogona kupa tsananguro yakapfava yeakakwira mapuroteni evascular muuropi, asi haigone kutsanangura zvizere kupera kwemapuroteni aya mu cerebrospinal fluid. Imwe mukana ndeyekuti iyo yepakati tsinga sisitimu iri kushingaira kupatsanura aya mamorekuru kugadzirisa dambudziko rekuwedzera kuzvimba uye oxidative kusagadzikana. Kuderedzwa kwemamwe mapuroteni akanyanya eCSF mupaneru ino, kunyanya ayo anobatanidzwa mumutemo we lipoprotein, ane chekuita nekudzivirirwa kwemazinga anokuvadza ekuzvimba uye neuroprotective process ye reactive oxygen marudzi. Izvi ndezvechokwadi kune Paroxonase 1 (PON1), iyo lipoprotein inosunga enzyme inokonzera kuderedza oxidative kusagadzikana mazinga mukutenderera (58, 59). Alpha-1-microglobulin/bikunin precursor (AMBP) imwe yakanyatsodzika-yakagadziriswa chiratidzo cheboka revascular. Ndiyo inotangira lipid transporter bikunin, iyo inobatanidzwawo mukudzvinyirirwa kwekuzvimba uye neurological Protection (60, 61).
Pasinei nekufungidzira kwakasiyana-siyana kunofadza, kusakwanisa kuona zvakananga maitiro ezvirwere zvebiochemical ndiyo inonyanyozivikanwa muganhu wekutsvaga-inotungamirirwa neproteomics kuongorora. Naizvozvo, kumwe kutsvagisa kwakakosha kutsanangura nenzira ine chivimbo nzira dziri kuseri kweizvi biomarker mapaneru. Kuti tifambe takananga kukuvandudzwa kweMS-based clinical analysis, kutungamira kweramangwana kunodawo kushandiswa kwenzira dzakanangwa dzehuwandu hwekuongororwa kwakakura kwebiomarker, sekusarudza kana kufanana kwekutarisa maitiro (62). Isu munguva pfupi yapfuura takashandisa parallel reaction monitoring (63) kusimbisa akawanda eCSF mapuroteni shanduko anotsanangurwa pano. Zvinangwa zvakati wandei zvinotariswa zvinoverengerwa nekurongeka kwakakosha, kusanganisira YWHAZ, ALDOA, uye SMOC1, iyo mepu kune yedu synapse, metabolism, uye kuzvimba mapaneru, zvichiteerana (63). Independent Data Acquisition (DIA) uye mamwe maMS-akavakirwa mazano anogona zvakare kubatsira kune chinangwa chekuongorora. Bud et al. (64) Munguva pfupi yapfuura zvakaratidzwa kuti pane kupindirana kwakakosha pakati peAD biomarkers akaonekwa mune yedu CSF yekuwana data set uye yakazvimirira DIA-MS data set, iyo ine angangoita mazana maviri eCSF samples kubva kune matatu akasiyana eEuropean cohorts. Izvi zvidzidzo zvichangoburwa zvinotsigira kugona kwemapaneru edu kuti ashandure kuita yakavimbika MS-based yekuona. Chinyakare masoja ekudzivirira chirwere uye aptamer-yakavakirwa kucherechedzwa kwakakosha zvakare kune imwe budiriro yeakakosha AD biomarkers. Nekuda kwekudzikira kwakawanda kweCSF, zvinonyanya kunetsa kuona izvi biomarkers uchishandisa yakakwirira-kuburikidza MS nzira. NEFL neNRGN mienzaniso miviri yakadai yeyakaderera-yakawanda CSF biomarkers, iyo yakamisikidzwa kune mapaneru muongororo yedu yakazara, asi haigone kuwonekwa nekuvimbika tichishandisa yedu imwe MS zano. Matanho ekunangisa anoenderana neakawanda masoja ekudzivirira chirwere, akadai sePEA, anogona kusimudzira shanduko yekiriniki yezvimaka izvi.
Pakazere, chidzidzo ichi chinopa yakasarudzika nzira yeproteomics yekuzivikanwa nekusimbisa CSF AD biomarkers zvichibva pane akasiyana masisitimu. Kukwenenzvera aya mapaneru emakaka pane mamwe ekuwedzera AD cohorts uye MS mapuratifomu anogona kuratidza kuvimbisa kufambisira mberi AD ngozi stratification uye kurapwa. Zvidzidzo zvinoongorora hurefu hwemapaneru aya nekufamba kwenguva zvakakoshawo kuona kuti ndeupi musanganiswa wemamarker unonyatso gadzirisa njodzi yechirwere chekutanga uye shanduko mukuoma kwechirwere.
Kunze kwemasamples matatu akakopwa neCSF, ese masampula eCSF akashandiswa muchidzidzo ichi akaunganidzwa pasi pechitsigiro cheEmory ADRC kana masangano ekutsvakurudza ane hukama. Huwandu hweseti ina dzeEmory CSF samples dzakashandiswa mune izvi zvidzidzo zveproteomics. Iyo CSF cohort yakawanikwa iine sampuli kubva makumi maviri ane hutano kudzora uye makumi maviri AD varwere. CSF kopi 1 inosanganisira samples kubva makumi matatu nemaviri ekutonga ane hutano, makumi matatu nevaviri AsymAD vanhu, uye 33 AD vanhu. CSF kopi 2 ine 147 zvidzori uye zana nemakumi mashanu AD samples. Iyo yakawanda-zvirwere CSF replication 4 cohort yaisanganisira gumi nemasere zvinodzora, 17 AD, 19 ALS, 13 PD, uye gumi neshanu FTD samples. Zvinoenderana nechibvumirano chakabvumidzwa neEmory University Institutional Ongororo Board, vese vatori vechikamu vedzidzo yeEmory vakawana mvumo ine ruzivo. Zvinoenderana ne2014 National Institute of Aging Best Practice Guidelines for Alzheimer's Centers (https://alz.washington.edu/BiospecimenTaskForce.html), cerebrospinal fluid yakaunganidzwa uye yakachengetwa nekubaya lumbar. Kudzora uye varwere veAsymAD uye AD vakagamuchira yakamisikidzwa cognitive ongororo paEmory Cognitive Neurology Clinic kana Goizueta ADRC. Mienzaniso yavo yecerebrospinal fluid yakaedzwa neINNO-BIA AlzBio3 Luminex yeELISA Aβ1-42, yakazara tau uye p-tau kuongorora (65). ELISA tsika dzinoshandiswa kutsigira kurongwa kwekuongorora kwezvidzidzo zvichibva pane yakamiswa AD biomarker yekucheka-kubvisa maitiro (66, 67). Basic demographic uye diagnostic data kune mamwe maCSF ekuongororwa (FTD, ALS, uye PD) anowanikwawo kubva kuEmory ADRC kana akabatana masangano ekutsvagisa. Iyo pfupiso kesi metadata yeaya Emory CSF makesi inogona kuwanikwa muTebhura S1A. Hunhu hweSwitzerland CSF replication 3 cohort dzakamboburitswa (45).
CSF yakawana muenzaniso. Kuti tiwedzere kudzika kwekuwanikwa kwedu kweiyo CSF data set, kushandiswa kwekudzivirira kwehuwandu hwemapuroteni akawandisa kwakaitwa isati yasvika trypsinization. Muchidimbu, 130 μl yeCSF kubva kumakumi mana ega ega eCSF samples uye yakaenzana vhoriyamu (130 μl) yePamusoro Sarudza Top14 Yakawanda Protein Depletion Resin (Thermo Fisher Scientific, A36372) yakaiswa mu spin column (Thermo Fisher Scientific, A89868) Tembiricha Incubate). Mushure mekutenderera kwemaminetsi gumi nemashanu, isa centrifuge sampu pa1000g kwemaminetsi maviri. A 3K ultracentrifugal sefa mudziyo (Millipore, UFC500396) yakashandiswa kutarisisa effluent sample ne centrifuging pa14,000g kwemaminetsi makumi matatu. Diraisa ese mavhoriyamu emuenzaniso kusvika ku75 μl nephosphate buffered saline. Kuwanda kweprotein kwakaongororwa nebicinchoninic acid (BCA) nzira zvichienderana neprotocol yemugadziri (Thermo Fisher Scientific). Iyo immunodepleted CSF (60 μl) kubva kune ese makumi mana samples yakagayiwa ne lysyl endopeptidase (LysC) uye trypsin. Muchidimbu, sampu yakaderedzwa uye alkylated ne 1.2 μl 0.5 M tris-2 (-carboxyethyl) -phosphine uye 3 μl 0.8 M chloroacetamide pa 90 ° C kwemaminitsi gumi, uye zvino sonicated mumvura yekugeza kwemaminitsi gumi nemashanu. Sampuli yacho yakaderedzwa ne 193 μl 8 M urea buffer [8 M urea uye 100 mM NaHPO4 (pH 8.5)] kusvika kuhuwandu hwekupedzisira hwe6 M urea. LysC (4.5 μg; Wako) inoshandiswa kugaya kwehusiku patembiricha yekamuri. Sample yacho yakabva yadirwa kuita 1 M urea ine 50 mM ammonium bicarbonate (ABC) (68). Wedzera huwandu hwakaenzana (4.5 μg) hwetrypsin (Promega), wobva waisa sampuro kwemaawa gumi nemaviri. Acidify the digested peptide solution kusvika pachikamu chekupedzisira che1% formic acid (FA) uye 0.1% trifluoroacetic acid (TFA) (66), uyezve desalt ne50 mg Sep-Pak C18 column (Mvura) sezvatsanangurwa pamusoro (25) . Iyo peptide yakabva yaburitswa mu1 ml ye50% acetonitrile (ACN). Kumisa mapuroteni quantification pamabhechi (25), 100 μl aliquots kubva kumasampuli makumi mana eCSF akasanganiswa kuti abudise sampuro yakasanganiswa, iyo yakabva yakamurwa kuita mashanu epasi rose mukati (GIS) (48) samples. Ese masampuli ega ega uye akasanganiswa zviyero akaomeswa ne-high-speed vacuum (Labconco).
CSF inokopa muenzaniso. Dayon nevamwe vaaishanda navo vakambotsanangura kudzikira kwekudzivirira uye kugaya kweCSF kopi 3 samples (45, 46). Iwo akasara kudzokorora samples aive asiri ega ega immunodepleted. Gaya aya masampuli asina kubviswa mu trypsin sezvakatsanangurwa kare (17). Kune imwe neimwe inodzokororwa kuongororwa, 120 μl aliquots ye eluted peptide kubva kumuenzaniso wega wega akaiswa pamwe chete uye akakamurwa kuita akaenzana vhoriyamu aliquots kuti ishandiswe seTMT-yakanyorwa pasi rose mukati mwero (48). Ese masampuli ega ega uye akasanganiswa zviyero akaomeswa ne-high-speed vacuum (Labconco). Kuitira kukwidziridza chiratidzo cheyakaderera-yakawanda CSF protein, nekubatanidza 125 μl kubva kune yega yega sampu, "yakakwidziridzwa" sampu yakagadzirirwa yega yega yekudzokorora ongororo [kureva, sampuli yebhayoloji inotevedzera sampu yekutsvagisa, asi mari iripo ndeye. yakakura zvikuru (37, 69)] yakabatanidzwa musanganiswa weCSF sampuli (17). Sample yakasanganiswa yakabva yabviswa immunoremoved ichishandisa 12 ml yeHigh Select Top14 Yakawanda Protein Removal Resin (Thermo Fisher Scientific, A36372), yakagayiwa sezvatsanangurwa pamusoro, uye inosanganisirwa mune yakatevera yakawanda TMT kunyora.
Nguva yekutumira: Aug-27-2021