Isifo i-Alzheimer's (AD) asinazo izimpawu ze-protein biomarkers ezibonisa i-pathophysiology yaso eyisisekelo eningi, evimbela ukuqhubeka kokuxilongwa nokwelashwa. Lapha, sisebenzisa ama-proteomics abanzi ukuhlonza ama-biomarker e-cerebrospinal fluid (CSF) amelela uhla olubanzi lwe-AD pathophysiology. I-Multiplex mass spectrometry ikhombe cishe amaprotheni angu-3,500 futhi acishe abe yi-12,000 ku-AD CSF nasengqondweni, ngokulandelana. Ukuhlaziywa kwenethiwekhi yeproteome yobuchopho kuxazulule amamojula angama-44 ezinhlobonhlobo zezinto eziphilayo, angu-15 awo agqine ne-cerebrospinal fluid proteome. Omaka be-CSF AD kulawa mamojula agqagqene agoqwe aba amaqembu amahlanu amaprotheni, amele izinqubo ezahlukene ze-pathophysiological. Ama-synapse kanye nama-metabolite ebuchosheni be-AD ayancipha, kodwa i-CSF iyanda, kuyilapho i-glial-rich myelination kanye namaqembu amasosha omzimba ebuchosheni kanye ne-CSF akhula. Ukungaguquguquki kanye nokucaciswa kwezifo kwezinguquko zephaneli kwaqinisekiswa kumasampula e-CSF engeziwe angaphezu kuka-500. Lawa maqembu aphinde akhombe ama-subgroups e-biological ku-AD engabonakali. Sekukonke, le miphumela iyisinyathelo esithembisayo esibheke kumathuluzi e-biomarker asekelwe kuwebhu wezinhlelo zokusebenza zomtholampilo ku-AD.
Isifo i-Alzheimer's (AD) siyimbangela evame kakhulu yokuwohloka komqondo okubangelwa yi-neurodeergenerative emhlabeni wonke futhi ibonakala ngokungasebenzi kahle kwesistimu yezinto eziphilayo, okuhlanganisa ukudluliswa kwe-synaptic, i-glial-mediated immune, kanye ne-mitochondrial metabolism (1-3). Kodwa-ke, ama-protein biomarker ayo asunguliwe asagxile ekutholeni amaprotheni e-amyloid ne-tau, ngakho-ke awakwazi ukukhombisa le pathophysiology ehlukahlukene. Lezi zimpawu ze-biomarker zamaprotheni "eziyinhloko" ezikalwa ngokuthembekile kakhulu ku-cerebrospinal fluid (CSF) zihlanganisa (i) i-amyloid beta peptide 1-42 (Aβ1-42), ebonisa ukwakheka kwama-cortical amyloid plaques; (ii) i-tau ephelele, uphawu lokuwohloka kwe-axon; (iii) i-phospho-tau (p-tau), ummeleli we-pathological tau hyperphosphorylation (4-7). Nakuba lezi zimpawu ze-biomarker zoketshezi lwe-cerebrospinal zisize kakhulu ekutholeni kwethu izifo zamaprotheni e-AD "emakiwe" (4-7), zimele kuphela ingxenye encane yezinto eziphilayo eziyinkimbinkimbi ngemuva kwalesi sifo.
Ukuntuleka kokuhlukahluka kwe-pathophysiological yezimpawu ze-AD biomarker kuye kwaholela ezinseleleni eziningi, okuhlanganisa (i) ukungakwazi ukuhlonza kanye nokulinganisa i-biological heterogeneity yeziguli ze-AD, (ii) ukulinganisa okunganele kobunzima besifo nokuqhubekela phambili, ikakhulukazi esigabeni sokuqala, Futhi ( iii) ukuthuthukiswa kwemithi yokwelapha ehlulekile ukuxazulula ngokuphelele zonke izici zokuwohloka kwemizwa. Ukuthembela kwethu ku-pathology eyingqopha-mlando ukuchaza i-AD evela ezifweni ezihlobene kwandisa lezi zinkinga. Ubufakazi obuningi bukhombisa ukuthi iningi labantu asebekhulile abanokuwohloka komqondo banesici esingaphezu kwesisodwa se-pathological sokuncipha kwengqondo (8). Cishe u-90% noma ngaphezulu wabantu abane-AD pathology nabo banesifo semithambo, i-TDP-43 inclusions, noma ezinye izifo eziwohlokayo (9). Lezi zilinganiso eziphezulu zokunqwabelana kwe-pathological ziphazamise uhlaka lwethu lwamanje lokuxilonga lokuwohloka komqondo, futhi kudingeka incazelo ebanzi ye-pathophysiological yalesi sifo.
Ngenxa yesidingo esiphuthumayo sezinhlobonhlobo zama-biomarker e-AD, inkambu iya ngokuya yamukela indlela “ye-omics” esuselwe ohlelweni oluphelele lokuthola ama-biomarker. I-Accelerated Pharmaceutical Partnership (AMP)-AD Alliance yethulwa ngo-2014 futhi ihamba phambili ohlelweni. Lo mzamo wemikhakha eminingi owenziwe yiNational Institutes of Health, academia, kanye nezimboni uhlose ukusebenzisa amasu asekelwe ohlelweni ukuchaza kangcono i-pathophysiology ye-AD kanye nokuthuthukisa ukuhlaziywa kokuxilonga kwezinhlobonhlobo zezinto eziphilayo kanye namasu okwelapha (10). Njengengxenye yale phrojekthi, i-network proteomics isiphenduke ithuluzi elithembisayo lokuthuthukisa ama-biomarker asekelwe ohlelweni ku-AD. Le ndlela eqhutshwa idatha engachemile ihlela amasethi wedatha eyinkimbinkimbi ye-proteomics ibe ngamaqembu noma "amamojula" amaprotheni achazwe ngokuhlanganyela ahlotshaniswa nezinhlobo ezithile zamaseli, ama-organelles, nemisebenzi yebhayoloji (11-13). Cishe izifundo eziyi-12 zenethiwekhi ezicebile ze-proteomics zenziwe ebuchosheni be-AD (13-23). Sekukonke, lokhu kuhlaziya kubonisa ukuthi i-proteome yenethiwekhi yobuchopho ye-AD igcina inhlangano emodulayo elondolozwe kakhulu kumaqoqo azimele kanye nezifunda eziningi zekhohlo. Ngaphezu kwalokho, amanye alawa mamojula abonisa izinguquko eziphindaphindekayo ngobuningi obuhlobene ne-AD kuwo wonke amasethi wedatha, abonisa i-pathophysiology yezifo eziningi. Sekukonke, lokhu okutholakele kubonisa iphoyinti eliqinisekisayo lokutholwa kwe-proteome yenethiwekhi yobuchopho njenge-biomarker esekelwe ohlelweni ku-AD.
Ukuze siguqule i-proteome yenethiwekhi yobuchopho ye-AD ibe ama-biomarker asekelwe kusistimu awusizo emtholampilo, sihlanganise inethiwekhi esuselwe ebuchosheni nokuhlaziywa kwe-proteomic kwe-AD CSF. Le ndlela edidiyelwe yaholela ekuhlonzweni kwamasethi amahlanu athembisayo we-CSF biomarkers ahlotshaniswa nohlu olubanzi lwe-pathophysiology esekelwe ebuchosheni, okuhlanganisa ama-synapses, imithambo yegazi, i-myelination, ukuvuvukala, kanye nokungasebenzi kahle kwezindlela ze-metabolic. Siqinisekise ngempumelelo lawa maphaneli e-biomarker ngokusebenzisa ukuhlaziya okuningiliziwe okuphindaphindayo, okuhlanganisa amasampula e-CSF angaphezu kwama-500 avela ezifweni ezihlukahlukene ze-neurodeergenerative. Lokhu kuhlaziywa kokuqinisekisa kufaka phakathi ukuhlola okuhlosiwe kweqembu ku-CSF yeziguli ezine-AD engabonakali (AsymAD) noma ukukhombisa ubufakazi bokuqoqwa kwe-amyloid okungavamile endaweni evamile yokuqonda. Lokhu kuhlaziya kugqamisa ukuhlukahluka kwebhayoloji okubalulekile kubantu be-AsymAD futhi kuhlonze omaka bephaneli abangakwazi ukuhlukanisa abantu ngabanye ezigabeni zokuqala zesifo. Sekukonke, le miphumela imele isinyathelo esibalulekile ekuthuthukisweni kwamathuluzi e-protein biomarker asekelwe ezinhlelweni eziningi ezingaxazulula ngempumelelo izinselele eziningi zomtholampilo ezibhekene ne-AD.
Inhloso eyinhloko yalolu cwaningo ukuhlonza ama-biomarker amasha e-cerebrospinal fluid abonisa i-pathophysiology esekelwe ebuchosheni eholela ku-AD. Umfanekiso S1 uveza indlela yethu yocwaningo, ehlanganisa (i) ukuhlaziya okuphelele okuqhutshwa ukutholwa kokuqala kwe-AD CSF kanye nenethiwekhi yobuchopho proteome ukuhlonza izimpawu eziningi zezifo ze-CSF ezihlobene nobuchopho, kanye (ii) nokuphindaphinda okulandelayo Lezi zimpawu ze-biomarker ziku-cerebrospinal eziningana ezizimele. ama-fluid cohorts. Ucwaningo olugxile ekutholakaleni luqale ngokuhlaziywa kokuvezwa komehluko kwe-CSF kubantu abangama-20 abajwayelekile ngokwengqondo kanye neziguli ezingama-20 ze-AD e-Emory Goizueta Alzheimer's Research Center (ADRC). Ukuxilongwa kwe-AD kuchazwa njengokukhubazeka okuphawulekayo kwengqondo lapho kukhona i-Aβ1-42 ephansi kanye namazinga aphakeme e-tau ephelele ne-p-tau ku-cerebrospinal fluid [Mean Montreal Cognitive Assessment (MoCA), 13.8 ± 7.0] [ELISA (ELISA) )]] (Ithebula S1A). Ukulawula (okusho i-MoCA, i-26.7 ± 2.2) kube namazinga ajwayelekile ama-biomarker e-CSF.
I-CSF yomuntu ibonakala ngohlu oluguquguqukayo lokuchichima kwamaprotheni, lapho i-albumin namanye amaprotheni amaningi kakhulu engavimbela ukutholwa kwamaprotheni anentshisekelo (24). Ukwandisa ukujula kokutholwa kwamaprotheni, sisuse amaprotheni okuqala ayi-14 anala kakhulu kusampula ngayinye ye-CSF ngaphambi kokuhlaziywa kwe-mass spectrometry (MS) (24). Isamba sama-peptide angama-39,805 akhonjwe yi-MS, aklanywa kuma-proteome angama-3691 kumasampula angama-40. Ukulinganisa amaprotheni kwenziwa nge-multiple tandem mass tag (TMT) ilebula (18, 25). Ukuze kuxazululwe idatha engekho, sifake kuphela lawo maprotheni abalwe okungenani ku-50% wamasampuli ekuhlaziyweni okwalandela, ngaleyo ndlela sagcina silinganisela ama-proteome angu-2875. Ngenxa yomehluko obalulekile kumaleveli obuningi bamaprotheni esewonke, isampula lokulawula liye labhekwa ngokwezibalo njengelingaphandle (13) futhi alizange lifakwe ekuhlaziyweni okwalandela. Amanani amaningi wamasampula angama-39 asele alungiswa ngokuya ngeminyaka, ubulili, kanye ne-batch covariance (13-15, 17, 18, 20, 26).
Kusetshenziswa ukuhlaziya kokuhlolwa kwe-t kwezibalo ukuze kuhlolwe inkulumo ehlukile kusethi yedatha yokuhlehla, lokhu kuhlaziya kuhlonze amaprotheni amaleveli awo okuchichima ashintshwe kakhulu (P <0.05) phakathi kwezimo zokulawula kanye ne-AD (Ithebula S2A). Njengoba kuboniswe ku-Figure 1A, ukuchichima kwengqikithi yamaprotheni angu-225 ku-AD kwancishiswa kakhulu, futhi ukuchichima kwamaprotheni angu-303 kwanda kakhulu. Lawa maprotheni avezwa ngokwehlukana ahlanganisa izimpawu ze-AD ze-cerebrospinal fluid ezimbalwa ezihlonzwe ngaphambilini, njenge-microtubule-associated protein tau (MAPT; P = 3.52 × 10−8), neurofilament (NEFL; P = 6.56 × 10−3), Amaprotheni ahlobene nokukhula 43 (GAP43; P = 1.46 × 10−5), I-Fatty Acid Binding Protein 3 (FABP3; P = 2.00 × 10−5), Chitinase 3 like 1 (CHI3L1; P = 4.44 × 10−6), Neural Granulin (NRGN; P = 3.43 × 10-4) kanye ne-VGF nerve growth factor (VGF; P = 4.83 × 10-3) (4-6). Nokho, siphinde sahlonza ezinye izinjongo ezibaluleke kakhulu, ezifana ne-GDP dissociation inhibitor 1 (GDI1; P = 1.54 × 10-10) kanye ne-SPARC ehlobene ne-modular calcium binding 1 (SMOC1; P = 6.93 × 10-9) . Ukuhlaziywa kwe-Gene Ontology (GO) kwamaphrotheni angama-225 anciphe kakhulu kwembula ukuxhumana okusondelene nezinqubo zoketshezi lomzimba njenge-steroid metabolism, ukuhlangana kwegazi, nomsebenzi wamahomoni (Umfanekiso 1B kanye neThebula S2B). Ngokuphambene, iphrotheni ekhuphuke kakhulu ye-303 ihlobene eduze nesakhiwo seseli kanye ne-energy metabolism.
(A) Isakhiwo sentaba-mlilo sibonisa ukuguquguquka kokugoqa kwelogi2 (x-eksisi) ngokuhlobene nenani elingu-P lezibalo le--log10 (y-eksisi) elitholwe ukuhlolwa kuka-t, okusetshenziselwa ukuthola inkulumo ehlukile phakathi kokulawula (CT) kanye Izimo ze-AD ze-CSF proteome Yawo wonke amaprotheni. Amaprotheni anamazinga anciphe kakhulu (P <0.05) ku-AD aboniswa ngombala oluhlaza okwesibhakabhaka, kuyilapho amaprotheni anamazinga akhuphuka kakhulu ezifo aboniswa ngokubomvu. Iphrotheni ekhethiwe ilebuli. (B) Amagama aphezulu e-GO ahlobene namaprotheni ancishiswa kakhulu (aluhlaza okwesibhakabhaka) futhi anda (abomvu) ku-AD. Ibonisa amagama amathathu e-GO anezikolo eziphakeme kakhulu zika-z emikhakheni yezinqubo zebhayoloji, imisebenzi yamangqamuzana, nezingxenye zamaselula. (C) I-MS ilinganise ileveli ye-MAPT kusampula ye-CSF (kwesokunxele) kanye nokuhlotshaniswa kwayo nesampula ELISA ileveli ye-tau (kwesokudla). I-Pearson coefficient ehambisanayo enevelu engu-P iyaboniswa. Ngenxa yokuntuleka kwedatha ye-ELISA yecala elilodwa le-AD, lezi zibalo zifaka amanani wamacala angama-38 kwangu-39 ahlaziyiwe. (D) Ukuhlaziywa kweqoqo eligadiwe (P <0.0001, Benjamini-Hochberg (BH) kulungiswe i-P <0.01) ekulawuleni futhi i-AD CSF ithole amasampula asebenzisa amaprotheni angu-65 ashintshe kakhulu kusethi yedatha. Linganisa, lungisa.
Izinga le-proteomic le-MAPT lihlobene eduze nezinga le-ELISA tau elilinganiswa ngokuzimela (r = 0.78, P = 7.8 × 10-9; Umfanekiso 1C), elisekela ukufaneleka kokulinganisa kwethu kwe-MS. Ngemuva kokugaya kwe-trypsin ezingeni le-amyloid precursor protein (APP), ama-peptide e-isoform-specific afakwe kumephu ku-C-terminus ye-Aβ1-40 kanye ne-Aβ1-42 ayikwazi ukwenziwa i-ionized kahle (27, 28). Ngakho-ke, ama-peptide e-APP esiwahlonze awahlanganise lutho namazinga e-ELISA Aβ1-42. Ukuze sihlole inkulumo ehlukile yesimo ngasinye, sisebenzise amaprotheni avezwa ngokuhlukile nge-P <0.0001 [izinga lokutholwa okungamanga (FDR) elilungiswe P <0.01] ukuze senze ukuhlaziya kweqoqo eligadiwe lamasampuli (Ithebula S2A). Njengoba kuboniswe kuMfanekiso 1D, lawa maprotheni angu-65 abaluleke kakhulu angahlanganisa kahle amasampula kuye ngesimo sesifo, ngaphandle kwekesi elilodwa le-AD elinezici ezifana nokulawula. Kulawa maprotheni angama-65, angama-63 anyuke ku-AD, kuyilapho amabili kuphela (i-CD74 ne-ISLR) ehlile. Sekukonke, lokhu kuhlaziya kwe-cerebrospinal fluid kuhlonze amakhulukhulu amaprotheni ku-AD angase asebenze njengezimpawu zesifo.
Bese senza ukuhlaziya okuzimele kwenethiwekhi ye-AD brain proteome. Iqembu lobuchopho lalokhu kutholwa lalihlanganisa i-dorsolateral prefrontal cortex (DLPFC) kusukela ekulawuleni (n = 10), isifo sika-Parkinson (PD; n = 10), amacala e-AD/PD (n = 10) kanye ne-AD (n = 10) exubile. ) Isampula. Emery Goizueta ADRC. Izibalo zabantu zalezi zimo ezingama-40 zichazwe ngaphambilini (25) futhi zifingqiwe kuThebula S1B. Sisebenzise i-TMT-MS ukuhlaziya lezi zicubu zobuchopho ezingama-40 kanye neqembu lokuphindaphinda lamacala angama-27. Sekukonke, lawa masethi edatha yobuchopho amabili akhiqize ama-peptide ahlukile angama-227,121, aklanywe kuma-proteoms ayi-12,943 (25). Yilawo maprotheni kuphela ayebalwe okungenani ku-50% wamacala afakiwe ophenyweni olwalandela. Isethi yokugcina yedatha yokutholwa iqukethe amaprotheni angu-8817 anenani. Lungisa amazinga okuchichima kwamaprotheni ngokusekelwe eminyakeni yobudala, ubulili, nesikhawu sangemuva kokufa (PMI). Ukuhlaziywa kwesimo esihlukile sedatha esethwe ngemva kokuhlehla kubonise ukuthi > amazinga we-protein angu-2000 ashintshwe kakhulu [P <0.05, ukuhlaziywa kokuhluka (ANOVA)] kumaqoqo amabili wezifo noma ngaphezulu. Bese, senze ukuhlaziya kweqoqo eligadiwe ngokusekelwe kumaprotheni avezwa ngokuhlukile, kanye ne-P <0.0001 ku-AD/control kanye/noma iziqhathaniso ze-AD/PD (Umfanekiso S2, A no-B, Ithebula S2C). Lawa maprotheni angu-165 ashintshe kakhulu abonisa ngokucacile amacala ane-AD pathology kusukela ekulawuleni namasampuli e-PD, eqinisekisa izinguquko eziqinile eziqondene ne-AD kuyo yonke i-proteome.
Sibe sesisebenzisa i-algorithm ebizwa ngokuthi i-Weighted Gene Co-expression Network Analysis (WGCNA) ukuze senze ukuhlaziya inethiwekhi ku-proteome yobuchopho etholiwe, ehlela idatha esethwe ibe amamojula amaprotheni anamaphethini wokukhuluma afanayo (11-13). Ukuhlaziywa kuhlonze amamojula we-44 (M) amaprotheni e-co-expressed, ahlungwe futhi abalwe kusukela kwamakhulu (M1, n = 1821 amaprotheni) kuya kokuncane kakhulu (M44, n = 34 amaprotheni) (Umfanekiso 2A kanye neThebula S2D) ). Njengoba kushiwo ngenhla (13) Bala iphrofayili yenkulumo emele noma isici sephrotheni yemojuli ngayinye, futhi uyihlobanise nesimo sesifo kanye ne-AD pathology, okungukuthi, sungula umfelandawonye we-Alzheimer's Disease Registry (CERAD) kanye ne-Braak Score (Figure 2B). Sekukonke, amamojula we-17 ayehlobene kakhulu ne-AD neuropathology (P <0.05). Eziningi zalawa mamojula ahlobene nesifo nawo anothe ngomaka ohlobo lweseli (Umfanekiso 2B). Njengoba kushiwo ngenhla (13), ukucebisa kohlobo lweseli kunqunywa ngokuhlaziya ukugqagqana kwemojuli kanye nohlu lwereferensi lwezakhi zofuzo zohlobo oluthile lweseli. Lezi zakhi zofuzo zithathwe kudatha eshicilelwe kuma-neurons egundane ahlukene, amaseli e-endothelial kanye ne-glial. Ukulandelana kwe-RNA (i-RNA-seq) ukuhlolwa (29).
(A) Zitholele i-WGCNA yeproteome yobuchopho. (B) Ukuhlaziywa kwe-Biweight midcorrelation (BiCor) kwephrotheni yesiginesha ye-modular (ingxenye yokuqala enkulu ye-modular protein expression) enezici ze-AD neuropathological (phezulu), okuhlanganisa i-CERAD (i-Aβ plaque) kanye nezikolo ze-Braak (tau tangles). Ukuqina kokuhlobana okuhle (okubomvu) nokunegethivu (okuluhlaza okwesibhakabhaka) kuboniswa imephu yokushisa enemibala emibili, futhi izinkanyezi zibonisa ukubaluleka kwezibalo (P <0.05). Sebenzisa I-Hypergeometric Fisher's Exact Test (FET) (phansi) ukuze uhlole ukuhlotshaniswa kohlobo lweseli yemojula ngayinye yephrotheni. Ukuqina kokufiphaza okubomvu kubonisa izinga lokunothisa kohlobo lweseli, futhi inkanyezi ikhombisa ukubaluleka kwezibalo (P <0.05). Sebenzisa indlela ye-BH ukulungisa inani elingu-P elitholakala ku-FET. (C) Ukuhlaziywa kwe-GO kwamaprotheni ajwayelekile. Izinqubo zebhayoloji ezihlobene kakhulu zikhonjiswa kumojuli ngayinye noma iqembu lemojuli ehlobene. i-oligo, i-oligodendrocyte.
Isethi yamamojula amahlanu ahlobene eduze ne-astrocyte kanye ne-microglia-rich (M30, M29, M18, M24, kanye ne-M5) ibonise ukuhlobana okuqinile okuhle ne-AD neuropathology (Figure 2B). Ukuhlaziywa kwe-Ontology kuxhumanisa lawa mamojula we-glial nokukhula kwamaseli, ukwanda, kanye nokuzivikela komzimba (Umfanekiso 2C kanye neThebula S2E). Amamojula amabili engeziwe e-glial, i-M8 ne-M22, nawo alawulwa ngokuqinile ezifweni. I-M8 ihlobene kakhulu ne-Toll-like receptor pathway, i-cascade ekhombisayo edlala indima ebalulekile ekuphenduleni kokuzivikela komzimba okungaphakathi (30). Ngesikhathi esifanayo, i-M22 ihlobene eduze nokuguqulwa kwangemuva kokuhumusha. I-M2, ecebile ngama-oligodendrocyte, ikhombisa ukuhlobana okuqinile okuhle ne-AD pathology kanye nokuxhumana kwe-ontological ne-nucleoside synthesis kanye nokuphindaphinda kwe-DNA, okubonisa ukwanda kwamaseli okuthuthukisiwe ezifweni. Sekukonke, lokhu okutholakele kusekela ukuphakama kwamamojula we-glial esike sawabona kunethiwekhi ye-AD proteome (13, 17). Njengamanje kutholakala ukuthi amamojula amaningi ahlobene ne-AD ahlobene ne-glial kunethiwekhi abonisa amazinga aphansi enkulumo ekulawuleni kanye namacala e-PD, agqamisa ukucaciswa kwesifo kwawo okuphakeme ku-AD (Figure S2C).
Amamojula amane kuphela kunethiwekhi yethu ye-proteome (M1, M3, M10, ne-M32) ahlobene kakhulu ne-AD pathology (P <0.05) (Umfanekiso 2, B no-C). Kokubili i-M1 ne-M3 inothile ngomaka be-neuronal. I-M1 ihlobene kakhulu nezimpawu ze-synaptic, kuyilapho i-M3 ihlobene eduze nomsebenzi we-mitochondrial. Abukho ubufakazi bokuthi uhlobo lweseli lunothisiwe lwe-M10 ne-M32. I-M32 ibonisa ukuxhumana phakathi kwe-M3 kanye ne-cell metabolism, kuyilapho i-M10 ihlobene kakhulu nokukhula kwamangqamuzana nokusebenza kwe-microtubule. Uma kuqhathaniswa ne-AD, wonke amamojula amane anyuswa ekulawuleni kanye ne-PD, okuwanika izinguquko ze-AD eziqondene nesifo (Figure S2C). Sekukonke, le miphumela isekela insada enciphile yamamojula anothe nge-neuron esiwabonile ngaphambilini ngo-AD (13, 17). Kafushane, ukuhlaziya kwenethiwekhi ye-proteome yobuchopho esiyitholile kukhiqize amamojula aguqulwe ngokukhethekile e-AD ahambisana nokutholwe kwethu kwangaphambilini.
I-AD ibonakala ngesigaba sokuqala se-asymptomatic (AsymAD), lapho abantu bebonisa ukuqoqwa kwe-amyloid ngaphandle kokuncipha komqondo womtholampilo (5, 31). Lesi sigaba se-asymptomatic simelela iwindi elibalulekile lokutholwa kusenesikhathi nokungenelela. Phambilini sikhombise ukulondolozwa okuqinile kwe-modular ye-AsymAD ne-AD yenethiwekhi yobuchopho proteome kuwo wonke amasethi edatha azimele (13, 17). Ukuze siqinisekise ukuthi inethiwekhi yobuchopho esiyitholile njengamanje iyahambisana nalokhu okutholwe ngaphambilini, sihlaziye ukulondolozwa kwamamojula angama-44 kusethi yedatha ephindaphindwe evela ezinhlanganweni ezingama-27 ze-DLPFC. Lezi zinhlangano zihlanganisa amacala okulawula (n = 10), i-AsymAD (n = 8) kanye ne-AD (n = 9). Amasampula okulawula kanye ne-AD afakwe ekuhlaziyweni kweqoqo lethu lobuchopho bokutholwa (Ithebula S1B), kuyilapho izimo ze-AsymAD zazihlukile kuphela eqenjini eliphindaphindayo. Lawa macala e-AsymAD nawo avela ebhange lobuchopho lase-Emory Goizueta ADRC. Nakuba ukuqonda kwakujwayelekile ngesikhathi sokufa, amazinga e-amyloid ayephezulu ngokungavamile (kusho i-CERAD, i-2.8 ± 0.5) (Ithebula S1B).
Ukuhlaziywa kwe-TMT-MS kwalezi zicubu zobuchopho ze-27 kuholele ekulinganisweni kwama-proteome angu-11,244. Lesi sibalo sokugcina sihlanganisa kuphela lawo maprotheni abalwe okungenani ku-50% wamasampuli. Le sethi yedatha ephindaphindwayo iqukethe ama-8638 (98.0%) amaprotheni angu-8817 atholwe ekuhlaziyeni kwethu ubuchopho obutholakele, futhi cishe i-3000 ishintshe kakhulu amaprotheni phakathi kokulawula kanye namaqoqo e-AD (P <0.05, ngemva kokuhlolwa kuka-Tukey okubhanqiwe ukuze kuhlaziywe ukuhluka) ( Ithebula S2F). Phakathi kwalawa maprotheni avezwe ngokwehlukana, i-910 iphinde yabonisa izinguquko eziphawulekayo zezinga phakathi kwe-AD kanye nezimo zokulawula i-proteome yobuchopho (P <0.05, ngemva kokuhlolwa kwe-T kwe-ANOVA Tukey). Kuyaphawuleka ukuthi lezi zimpawu ze-910 zihambisana kakhulu endleleni yoshintsho phakathi kwama-proteoms (r = 0.94, P <1.0 × 10-200) (Figure S3A). Phakathi kwamaprotheni anda, amaprotheni anezinguquko ezingaguquki kakhulu phakathi kwamasethi edatha ngokuyinhloko angamalungu amamojula we-glial-rich M5 kanye ne-M18 (MDK, COL25A1, MAPT, NTN1, SMOC1, ne-GFAP). Phakathi kwamaprotheni ancishisiwe, labo abanezinguquko ezingaguquki kakhulu cishe babengamalungu kuphela we-module ye-M1 (NPTX2, VGF, ne-RPH3A) ehambisana ne-synapse. Siphinde saqinisekisa izinguquko ezihlobene ne-AD ze-midkine (MDK), i-CD44, i-protein 1 ehlobene nefrizzled (SFRP1) kanye ne-VGF nge-western blotting (Figure S3B). Ukuhlaziywa kokugcinwa kwemojula kubonise ukuthi cishe u-80% wamamojula amaprotheni (34/44) ku-proteome yobuchopho alondolozwe kakhulu kusethi yedatha yokuphindaphinda (z-score> 1.96, i-FDR ilungiswe P <0.05) (Figure S3C). Ayishumi nane kulawa mamojula agcinwe ngokukhethekile phakathi kwama-proteome amabili (z-score> 10, i-FDR elungisiwe P <1.0 × 10−23). Sekukonke, ukutholakala nokuphindaphinda kwezinga eliphezulu lokungaguquguquki ekukhulumeni okuhlukile kanye nokwakheka kwemodular phakathi kweproteome yobuchopho kugqamisa ukuphindaphindeka kwezinguquko kumaprotheni e-AD frontal cortex. Ngaphezu kwalokho, kuphinde kwaqinisekisa ukuthi i-AsymAD kanye nezifo ezithuthuke kakhulu zinesakhiwo senethiwekhi yobuchopho efanayo kakhulu.
Ukuhlaziywa okuningiliziwe kwenkulumo ehlukile kusethi yedatha yokuphindaphinda kobuchopho kugqamisa izinga elibalulekile lezinguquko ze-AsymAD amaprotheni, okuhlanganisa isamba samaprotheni angu-151 ashintshe kakhulu phakathi kwe-AsymAD nokulawula (P <0.05) (Figure S3D). Ngokuhambisana nomthwalo we-amyloid, i-APP ebuchosheni be-AsymAD ne-AD inyuke kakhulu. I-MAPT ishintsha kakhulu kuphela ku-AD, ehambisana namazinga anyukile ama-tangles kanye nokuhlobana kwayo okwaziwayo nokwehla kwengqondo (5, 7). Amamojula acebile nge-glial (M5 ne-M18) abonakala kakhulu kumaprotheni anda ku-AsymAD, kuyilapho imojula ye-M1 ehlobene neuron imelela kakhulu amaprotheni anciphile ku-AsymAD. Eziningi zalezi zimaki ze-AsymAD zibonisa izinguquko ezinkulu ezifweni ezinezimpawu. Phakathi kwalezi zomaka kukhona i-SMOC1, iphrotheni ye-glial ye-M18, ehlotshaniswa nezimila zobuchopho nokuthuthukiswa kwamehlo nezitho (32). I-MDK iyisici sokukhula esibopha i-heparin esihlobene nokukhula kwamaseli kanye ne-angiogenesis (33), elinye ilungu le-M18. Uma kuqhathaniswa neqembu lokulawula, i-AsymAD yanda kakhulu, ilandelwa ukwanda okukhulu kwe-AD. Ngokuphambene, i-synaptic protein neuropentraxin 2 (NPTX2) yehliswe kakhulu ebuchosheni be-AsymAD. I-NPTX2 phambilini ibihlotshaniswa ne-neurodegeneration futhi inendima eyaziwayo ekulamuleni ama-synapses ajabulisayo (34). Sekukonke, le miphumela yembula izinguquko ezahlukahlukene zamaprotheni e-preclinical ku-AD abonakala ethuthuka ngobulukhuni besifo.
Uma sibheka ukuthi sizuze ukujula okubalulekile kokumbozwa kwamaprotheni ekutholweni kwe-proteome yobuchopho, sizama ukuqonda ngokugcwele ukugqagqana kwayo ne-network-level AD transcriptome. Ngakho-ke, siqhathanise i-proteome yobuchopho esiyitholile nemojula esiyikhiqize ngaphambilini kusukela esilinganisweni se-microarray sezakhi zofuzo eziyi-18,204 ku-AD (n = 308) nokulawula (n = 157) izicubu ze-DLPFC (13). ukweqa. Sekukonke, sihlonze amamojula e-RNA ahlukene angama-20, amaningi awo abonisa ukunothiswa kwezinhlobo ezithile zamaseli, okuhlanganisa ama-neurons, ama-oligodendrocyte, ama-astrocyte, nama-microglia (Umfanekiso 3A). Izinguquko eziningi zalawa mamojula ku-AD ziboniswa kuMfanekiso 3B. Ngokuhambisana nokuhlaziywa kokugqagqana kwe-protein-RNA yethu yangaphambilini kusetshenziswa i-MS proteome engabhaliwe ejulile (cishe amaprotheni angu-3000) (13), iningi lamamojula angu-44 kunethiwekhi ye-proteome yobuchopho esiwatholile akunethiwekhi ye-transcriptome Akukho ukugqagqana okubalulekile. ukutholakala kwethu nokuphindaphinda kwamamojula angu-34 amaprotheni agcinwe kakhulu ku-proteome yobuchopho, angu-14 (~40%) kuphela aphumelele ukuhlolwa okunembile kukaFisher (FET) okubonakale kunokugqagqana okuphawulekayo kwezibalo ne-transcriptome (Umfanekiso 3A) . Ihambisana nokulungisa umonakalo we-DNA (i-P-M25 ne-P-M19), ukuhumusha kwamaprotheni (P-M7 kanye ne-P-M20), i-RNA ebophayo/i-splicing (P-M16 ne-P-M21) nokukhomba amaprotheni (P-M13 kanye ne-P- M23) ayidluleli namamojula ku-transcriptome. Ngakho-ke, nakuba isethi yedatha ye-proteome ejulile isetshenziswa ekuhlaziyweni kwamanje kokugqagqana (13), iningi le-AD yenethiwekhi ye-proteome ayenziwe imephu kunethiwekhi ye-transcriptome.
(A) I-Hypergeometric FET ibonisa ukunothiswa kwezimaka zohlobo oluthile lweseli kumojuli ye-RNA ye-AD transcriptome (phezulu) kanye nezinga lokugqagqana phakathi kwamamojula e-RNA (x-axis) kanye neprotheyini (y-axis) yobuchopho be-AD. (ngezansi). Ukuqina kokufiphaza okubomvu kubonisa izinga lokunothisa kwezinhlobo zamaseli kuphaneli ephezulu kanye nokuqina kokugqagqana kwamamojula kuphaneli engezansi. Izinkanyezi zibonisa ukubaluleka kwezibalo (P <0.05). (B) Izinga lokuhlobana phakathi kwezakhi zofuzo zemojuli ngayinye ye-transcriptome nesimo se-AD. Amamojula angakwesokunxele yiwona ahlotshaniswa kabi kakhulu ne-AD (aluhlaza okwesibhakabhaka), futhi lawo angakwesokudla yiwona ahlotshaniswa kahle kakhulu ne-AD (obomvu). Inani elingu-P eliguqulelwe ngelogi elingu-BH libonisa izinga lokubaluleka kwezibalo lokuhlobana ngakunye. (C) Amamojula agqagqene abalulekile anohlobo lweseli okwabelwana ngalo. (D) Ukuhlaziywa kokuhlobana koshintsho lwelogi2 olugoqiwe lwephrotheni enelebula (x-axis) kanye ne-RNA (y-eksisi) kumojuli egqagqene. I-Pearson coefficient ehambisanayo enevelu engu-P iyaboniswa. I-microglia; izindikimba zasezulwini, ama-astrocyte. CT, ukulawula.
Amaprotheni amaningi agqagqene namamojula e-RNA abelana ngamaphrofayili wohlobo lweseli afanayo kanye nezikhombisi-ndlela zokushintsha ze-AD (Umfanekiso 3, B no-C). Ngamanye amazwi, imojula ye-M1 ehlobene ne-synapse ye-brain proteome (PM1) yenziwe imephu yaba amamojula amathathu e-RNA e-neuronal-rich homologous (R-M1, R-M9 kanye ne-R-M16), aku-AD Womabili abonisiwe. izinga elincishisiwe. Ngokufanayo, amamojula we-glial-rich M5 kanye ne-M18 amaprotheni ahambisana namamojula e-RNA anothe nge-astrocyte nama-microglial marker (R-M3, R-M7, kanye ne-R-M10) futhi ahileleke kakhulu ezifweni Ukwanda. Lezi zici ze-modular okwabelwana ngazo phakathi kwamasethi amabili wedatha ziqhubeka zisekela ukunothiswa kohlobo lweseli kanye nezinguquko ezihlobene nesifo esizibonile ku-proteome yobuchopho. Kodwa-ke, sibone umehluko omkhulu obalulekile phakathi kwe-RNA namazinga amaprotheni omaka ngabanye kulawa mamojuli abiwe. Ukuhlaziywa kokuhlobana kokuvezwa okuhlukile kwe-proteomics kanye ne-transcriptomics yama-molecule ngaphakathi kwala mamojula agqagqene (Umfanekiso we-3D) kugqamisa lokhu kungahambisani. Isibonelo, i-APP namanye amaphrotheni amamojula e-glial ambalwa (NTN1, MDK, COL25A1, ICAM1, ne-SFRP1) abonise ukwanda okukhulu kwe-AD proteome, kodwa cishe alukho ushintsho ku-AD transcriptome. Lezi zinguquko eziqondene namaprotheni zingase zihlobane eduze nama-amyloid plaques (23, 35), zigqamisa i-proteome njengomthombo wezinguquko ze-pathological, futhi lezi zinguquko zingase zingaboniswa ku-transcriptome.
Ngemva kokuhlaziya ngokuzimela ubuchopho nama-CSF proteomes esiwatholile, senze ukuhlaziya okuphelele kwamasethi amabili wedatha ukuze sihlonze ama-biomarker e-AD CSF ahlobene ne-pathophysiology yenethiwekhi yobuchopho. Kufanele siqale sichaze ukugqagqana kwama-proteome amabili. Nakuba kwamukelwa kabanzi ukuthi i-CSF ikhombisa izinguquko ze-neurochemical ebuchosheni be-AD (4), izinga eliqondile lokudlulana phakathi kobuchopho be-AD ne-CSF proteome alicacile. Ngokuqhathanisa inani lemikhiqizo yofuzo okwabelwana ngayo etholwe kuma-proteome ethu amabili, sithole ukuthi cishe ama-70% (n = 1936) amaprotheni akhonjwe ku-cerebrospinal fluid nawo alinganiswa ebuchosheni (Umfanekiso 4A). Iningi lala maprotheni agqagqene (n = 1721) afakwe kumephu eyodwa yamamojula angama-44 e-co-expression asuka kusethi yedatha yobuchopho bokutholwa (Umfanekiso 4B). Njengoba bekulindelekile, amamojula ayisithupha amakhulu obuchopho (i-M1 kuya ku-M6) abonise inani elikhulu kakhulu lokugqagqana kwe-CSF. Kodwa-ke, kukhona amamojula amancane obuchopho (isibonelo, i-M15 ne-M29) afinyelela izinga eliphakeme ngokungalindelekile lokugqagqana, elikhulu kunemojula yobuchopho kabili ubukhulu bayo. Lokhu kusishukumisela ukuba sisebenzise indlela enemininingwane eyengeziwe, eqhutshwa izibalo yokubala ukugqagqana phakathi kobuchopho noketshezi lwe-cerebrospinal.
(A no-B) Amaprotheni atholwe ebuchosheni bokutholwa kanye namasethi edatha ye-CSF ayagqagqana. Iningi lala maprotheni agqagqene ahlotshaniswa neyodwa yamamojula angama-44 e-co-expression yenethiwekhi yokuxhumana kobuchopho. (C) Thola ukugqagqana phakathi kwe-cerebrospinal fluid proteome ne-brain network proteome. Umugqa ngamunye wemephu yokushisa umele ukuhlaziywa okugqagqene okuhlukile kwe-hypergeometric FET. Umugqa ophezulu ubonisa ukugqagqana (ukufiphala okumpunga/okumnyama) phakathi kwemojula yobuchopho nayo yonke i-CSF proteome. Umugqa wesibili ubonisa ukuthi ukugqagqana phakathi kwamamojula obuchopho nephrotheni ye-CSF (efakwe umbala obomvu) kulawulwa ngokuphawulekayo ku-AD (P <0.05). Umugqa wesithathu ubonisa ukuthi ukugqagqana phakathi kwamamojula obuchopho kanye nephrotheni ye-CSF (i-blue shading) kulawulwa phansi kakhulu ku-AD (P <0.05). Sebenzisa indlela ye-BH ukulungisa inani elingu-P elitholakala ku-FET. (D) Iphaneli yemojuli egoqayo esekelwe ekuhlotshaneni kohlobo lweseli kanye nemigomo ye-GO ehlobene. Lawa maphaneli aqukethe isamba samaphrotheni ahlobene nobuchopho angama-271, anenkulumo ehlukile enengqondo ku-CSF proteome.
Sisebenzisa ama-FET anomsila owodwa, sihlole ukubaluleka kokunqwabelana kwamaprotheni phakathi kwe-CSF proteome kanye namamojula obuchopho ngamanye. Ukuhlaziywa kwembula ukuthi ingqikithi yamamojula obuchopho be-14 kusethi yedatha ye-CSF inokugqagqana okuphawulekayo (i-FDR ilungiswe P <0.05), kanye nemojuli eyengeziwe (M18) ukugqagqana kwayo kuseduze nokubaluleka (i-FDR ilungiswe P = 0.06) (Umfanekiso 4C , umugqa ophezulu). Futhi sinentshisekelo kumamojula agqagqana kakhulu namaprotheni e-CSF avezwe ngokuhlukile. Ngakho-ke, sisebenzise ukuhlaziya okubili kwe-FET ukuze sithole ukuthi yimaphi (i) amaprotheni e-CSF anyuswe kakhulu ku-AD futhi (ii) amaprotheni e-CSF ehle kakhulu ku-AD (P <0.05, amamojula abhanqiwe t wokuhlola AD/control) amamojula obuchopho anokugqagqana okunenjongo. phakathi kwabo. Njengoba kuboniswe emigqeni emaphakathi nephansi yoMfanekiso 4C, lokhu kuhlaziya okwengeziwe kubonisa ukuthi i-8 yamamojula obuchopho angu-44 adlulana kakhulu namaprotheni angezwe ku-AD CSF (M12, M1, M2, M18, M5, M44, M33, kanye ne-M38) . ), kuyilapho amamojula amabili kuphela (i-M6 ne-M15) abonisa ukunqwabelana okunenjongo neprotheyini encishisiwe ku-AD CSF. Njengoba kulindelekile, wonke amamojula ayi-10 akumamojula ayi-15 anokugqagqana okuphezulu ne-CSF proteome. Ngakho-ke, sicabanga ukuthi lawa mamojula ayi-15 ayimithombo enesivuno esikhulu se-AD etholakala ebuchosheni be-CSF biomarkers.
Sigoqe lawa mamojuli agqagqene angu-15 aba amaphaneli amahlanu amakhulu amaprotheni ngokusekelwe ekubeni seduze kwawo kumdwebo wesihlahla se-WGCNA kanye nokuhlotshaniswa kwawo nezinhlobo zamaseli kanye ne-ontology yofuzo (Umfanekiso 4D). Iphaneli yokuqala iqukethe amamojula acebile kumaka we-neuron namaprotheni ahlobene ne-synapse (M1 ne-M12). Iphaneli ye-synaptic iqukethe ingqikithi yamaprotheni angu-94, futhi amazinga e-CSF proteome ashintshe kakhulu, okwenza kube umthombo omkhulu wezimpawu ze-CSF ezihlobene nobuchopho phakathi kwamaphaneli amahlanu. Iqembu lesibili (i-M6 ne-M15) libonise ukuxhumana okuseduze nama-endothelial cell markers kanye nomzimba we-vascular, njengokuthi "ukuphulukiswa kwesilonda" (M6) kanye "nokulawulwa kwe-humoral immune response" (M15). I-M15 nayo ihlobene kakhulu ne-lipoprotein metabolism, ehlobene eduze ne-endothelium (36). Iphaneli yemithambo iqukethe izimpawu ze-CSF ezingama-34 ezihlobene nobuchopho. Iqembu lesithathu lihlanganisa amamojula (i-M2 ne-M4) ahlobene kakhulu nezimpawu ze-oligodendrocyte kanye nokwanda kwamaseli. Isibonelo, imigomo yezinga eliphezulu ye-ontology ye-M2 ihlanganisa "ukulawulwa okuhle kokuphindaphinda kwe-DNA" kanye "nenqubo ye-purine biosynthesis". Phakathi naleso sikhathi, lezo ze-M4 zihlanganisa "ukuhlukaniswa kwe-glial cell" kanye "nokuhlukaniswa kwe-chromosome". Iphaneli ye-myelination iqukethe izimpawu ze-CSF ezingama-49 ezihlobene nobuchopho.
Iqembu lesine liqukethe amamojula amaningi (M30, M29, M18, M24, kanye ne-M5), futhi cishe wonke amamojula acebile kakhulu kuma-microglia kanye nama-astrocyte marker. Ngokufana nephaneli ye-myelination, iphaneli yesine nayo iqukethe amamojula (M30, M29, kanye ne-M18) ahlobene eduze nokwanda kwamaseli. Amanye amamojula kuleli qembu ahlobene kakhulu namagama e-immunological, njengokuthi "inqubo yomphumela wokuzivikela komzimba" (M5) kanye "nokulawulwa kokusabela kwamasosha omzimba" (M24). Iqembu le-glial immune system liqukethe izimpawu ze-CSF ezingama-42 ezihlobene nobuchopho. Ekugcineni, iphaneli yokugcina ihlanganisa omaka be-52 abahlobene nobuchopho kumamojula amane (M44, M3, M33, kanye ne-M38), wonke asemzimbeni ahlobene nokugcinwa kwamandla kanye ne-metabolism. Inkulu kulawa mamojula (M3) ihlobene eduze ne-mitochondria futhi inothe ngomaka be-neuron ethize. I-M38 ingelinye lamalungu emojula amancane kule metabolome futhi ikhombisa ukucaciswa kwe-neuron okumaphakathi.
Sekukonke, lawa maphaneli amahlanu abonisa uhla olubanzi lwezinhlobo zamaseli nemisebenzi ku-AD cortex, futhi ngokuhlangene aqukethe omaka be-CSF abahlobene nobuchopho abangu-271 (Ithebula S2G). Ukuze sihlole ukufaneleka kwale miphumela ye-MS, sisebenzise i-proximity extension assay (PEA), ubuchwepheshe obusekelwe ku-orthogonal antibody obunamandla okuphindaphinda, ukuzwela okuphezulu nokucaciswa, futhi sahlaziya kabusha amasampula oketshezi lwe-cerebrospinal sithole Isethi engaphansi yalezi zimpawu ezingama-271. (n = 36). Lezi zinhloso ze-36 zibonisa ushintsho ku-AD multiple of PEA, ehlobene eduze nemiphumela yethu esekelwe ku-MS (r = 0.87, P = 5.6 × 10-12) , Okuqinisekise ngokuqinile imiphumela yokuhlaziya kwethu okuphelele kwe-MS (Figure S4 ).
Izindikimba zebhayoloji ezigcizelelwa ngamaqembu ethu amahlanu, kusukela ekuboniseni i-synaptic kuya ku-metabolism yamandla, zonke zihlobene ne-pathogenesis ye-AD (1-3). Ngakho-ke, wonke amamojula angu-15 aqukethe la maphaneli ahlobene ne-AD pathology ku-proteome yobuchopho esiyitholile (Umfanekiso 2B). Okuphawuleka kakhulu ukuhlobana okuphezulu okuhle kwe-pathological phakathi kwamamojula ethu e-glial kanye nokuhlangana okunamandla okungekuhle kwe-pathological phakathi kwamamojula wethu amakhulu we-neuronal (M1 ne-M3). Ukuhlaziywa kwenkulumo ehlukile kwe-proteome yethu yobuchopho ephindaphindwayo (Umfanekiso we-S3D) futhi kugqamisa amaprotheni e-glial asuselwa ku-M5 kanye ne-M18. Ku-AsymAD ne-symptomatic AD, amaprotheni e-glial anda kakhulu kanye nama-synapses ahlobene ne-M1 Iphrotheni iyancipha kakhulu. Lokhu kuhlola kubonisa ukuthi izimpawu ze-cerebrospinal fluid ezingama-271 esizihlonze emaqenjini amahlanu zihlobene nezinqubo zezifo ku-AD cortex, kuhlanganise nalezo ezenzeka ezigabeni zokuqala ze-asymptomatic.
Ukuze sihlaziye kangcono indlela yoshintsho lwamaphaneli amaprotheni ebuchosheni nasoketshezini lomgogodla, sidwebe okulandelayo kumamojula agqagqene angu-15: (i) sithole ileveli yobuningi bemojula kusethi yedatha yobuchopho kanye (ii) nemojula. amaprotheni Umehluko uvezwa ku-cerebrospinal fluid (Figure S5). Njengoba kushiwo ngaphambili, i-WGCNA isetshenziselwa ukunquma ubuningi bemojuli noma inani lamaprotheni ebuchosheni (13). Imephu yentaba-mlilo isetshenziselwa ukuchaza ukuvezwa okuhlukile kwamaphrotheni e-modular ku-cerebrospinal fluid (AD/control). Lezi zibalo zibonisa ukuthi amaphaneli amathathu kwamahlanu akhombisa ukuthambekela kokukhuluma okuhlukile ebuchosheni nasoketshezini lomgogodla. Amamojula amabili ephaneli ye-synapse (i-M1 ne-M12) abonisa ukwehla kwezinga lokuchichima ebuchosheni be-AD, kodwa anqwabelana kakhulu nephrotheni ekhuphukile ku-AD CSF (Figure S5A). Amamojula ahlobene ne-neuron aqukethe i-metabolome (i-M3 ne-M38) abonise ubuchopho obufanayo kanye namaphethini e-cerebrospinal fluid expression angahambisani (Figure S5E). Iphaneli ye-vascular iphinde ibonise izitayela ezahlukene zokukhuluma, nakuba amamojula ayo (i-M6 ne-M15) anyuswe ngokulinganisela ebuchosheni be-AD futhi ehla ku-CSF enesifo (Figure S5B). Amaphaneli amabili asele aqukethe amanethiwekhi amakhulu e-glial amaprotheni awo ahlala elawulwa phezulu kuwo womabili amagumbi (Umfanekiso S5, C kanye no-D).
Sicela uqaphele ukuthi lawa mathrendi awavamile kubo bonke omaka kulawa maphaneli. Isibonelo, iphaneli ye-synaptic ihlanganisa amaprotheni amaningana ancishiswe kakhulu ebuchosheni be-AD kanye ne-CSF (Figure S5A). Phakathi kwalezi zimpawu zoketshezi lwe-cerebrospinal ezilawulwa phansi kukhona i-NPTX2 ne-VGF ye-M1, kanye ne-chromogranin B ye-M12. Kodwa-ke, ngaphandle kwalokhu okuhlukile, iningi lomaka bethu be-synaptic liphakeme ku-AD uketshezi lomgogodla. Sekukonke, lokhu kuhlaziya kukwazile ukuhlukanisa amathrendi abalulekile ngokwezibalo kubuchopho namazinga oketshezi lwe-cerebrospinal kumaphaneli ethu amahlanu. Lawa mathrendi agqamisa ubudlelwano obuyinkimbinkimbi futhi obuvame ukuhluka phakathi kobuchopho nokubonakaliswa kwamaprotheni e-CSF ku-AD.
Bese, sasebenzisa ukuhlaziywa kokuphindaphinda kwe-MS okudlulele okuphezulu (i-CSF replication 1) ukuze sinciphise isethi yethu yama-biomarker angu-271 kuya kokuhlosiwe okuthembisayo nokuphindaphindeka (Umfanekiso 5A). Ikhophi 1 ye-CSF iqukethe isamba samasampuli angama-96 avela ku-Emory Goizueta ADRC, okuhlanganisa nokulawula, i-AsymAD, neqoqo le-AD (Ithebula S1A). Lezi zimo ze-AD zibonakala ngokuncipha komqondo okumaphakathi (kusho i-MoCA, 20.0 ± 3.8), kanye nezinguquko kuma-biomarker e-AD aqinisekiswa ku-cerebrospinal fluid (Ithebula S1A). Ngokuphambene nokuhlaziywa kwe-CSF esikutholile, lokhu kuphindaphinda kwenziwa kusetshenziswa indlela ye-MS esebenza kahle kakhulu futhi ephezulu kakhulu (ngaphandle kokuhlukaniswa okungaxhunyiwe ku-inthanethi), okuhlanganisa isampula yokulungiselela iphrothokholi eyenziwe lula eqeda isidingo sokuncipha komzimba kwamasampula ngamanye. . Esikhundleni salokho, "isiteshi sokuthuthukisa" esisodwa esiphelelwe amandla omzimba sisetshenziselwa ukukhulisa isignali yamaprotheni amaningi amancane (37). Nakuba kunciphisa ukumbozwa kwe-proteome okuphelele, le ndlela yokudubula okukodwa inciphisa kakhulu isikhathi somshini futhi inyusa inani lamasampuli anelebula ye-TMT angahlaziywa asebenzayo (17, 38). Sekukonke, ukuhlaziya kuhlonze ama-peptide angama-6,487, afaka imephu kuma-proteom ayi-1,183 ezimeni ezingama-96. Njengokuhlaziya kwe-CSF esikutholile, yilawo maprotheni kuphela abalwe okungenani ku-50% wamasampuli afakiwe ekubalweni okwalandela, futhi idatha yahlehliswa ngenxa yemiphumela yobudala nobulili. Lokhu kuholele ekulinganisweni kokugcina kwama-proteome angama-792, ama-95% awo nawo ahlonzwa kusethi yedatha ye-CSF etholakele.
(A) Okuqondiwe kwamaprotheni e-CSF ahlobene nobuchopho aqinisekiswa kuqoqo lokuqala le-CSF eliphindaphindwe futhi afakwe kuphaneli yokugcina (n = 60). (B kuya ku-E) Amaleveli wephaneli ye-biomarker (inhlanganisela engu-z-izikolo) akalwa ngamaqoqo amane okuphindaphinda e-CSF. Ukuhlolwa kuka-t okubhangqiwe noma i-ANOVA enokulungiswa kwangemuva kuka-Tukey kusetshenziswe ukuhlola ukubaluleka kwezibalo zezinguquko ngobuningi ekuhlaziyweni kwempinda ngakunye. CT, ukulawula.
Njengoba sinentshisekelo ikakhulukazi yokuqinisekisa izinjongo zethu ze-CSF eziphathelene nobuchopho ezingama-271 ngokuhlaziya okuphelele, sizokhawulela ukuhlolwa okwengeziwe kwale proteome ephindaphindwayo kulabamaka. Phakathi kwalawa maprotheni angu-271, i-100 itholwe ekuphindaphindweni kwe-CSF 1. Umfanekiso we-S6A ubonisa ukuvezwa okuhlukile kwalezi zimpawu ezigqagqene eziyi-100 phakathi kokulawula kanye namasampula okuphindaphinda kwe-AD. I-Synaptic ne-metabolite histones anda kakhulu ku-AD, kuyilapho amaprotheni e-vascular ehla kakhulu ezifweni. Iningi lezimpawu ezigqagqene eziyi-100 (n = 70) zigcine inkomba efanayo yoshintsho kumasethi amabili wedatha (Umfanekiso S6B). Lezi zimaki ze-CSF ezihlobene nobuchopho ezingama-70 (Ithebula le-S2H) zikhombisa kakhulu amathrendi enkulumo yephaneli ebonwe ngaphambilini, okungukuthi, ukulawulwa phansi kwamaprotheni emithambo kanye nokulawulwa phezulu kwawo wonke amanye amaphaneli. Angu-10 kuphela kulawa maprotheni angu-70 aqinisekisiwe abonise izinguquko ngobuningi be-AD obuphikisana nalawa mathrendi ephaneli. Ukuze sikhiqize iphaneli ebonisa kahle yonke inkambo yobuchopho noketshezi lwe-cerebrospinal, asiwahlanganisi lawa maprotheni ayi-10 kuphaneli yentshisekelo esigcine siyiqinisekisile (Umfanekiso 5A). Ngakho-ke, iphaneli yethu ekugcineni ihlanganisa isamba samaprotheni angama-60 aqinisekiswe kumaqoqo amabili azimele e-CSF AD kusetshenziswa ukulungiswa kwesampula okuhlukile kanye nokuhlaziywa kwenkundla ye-MS. Iziqephu zenkulumo engu-z-score zalawa maphaneli okugcina kusilawuli sekhophi engu-1 ye-CSF kanye nezimo ze-AD ziqinisekise ithrendi yephaneli ebonwe kuqoqo le-CSF esilitholile (Umfanekiso 5B).
Phakathi kwalawa maprotheni angama-60, kunama-molecule aziwa ukuthi ahlotshaniswa ne-AD, njenge-osteopontin (SPP1), okuyi-cytokine evuselela ukuvuvukala ehlotshaniswa ne-AD ezifundweni eziningi (39-41), kanye ne-GAP43, i-synaptic protein. lokho kuxhumene ngokusobala ne-neurodegeneration (42). Amaprotheni aqinisekiswe ngokugcwele amamaki ahlobene nezinye izifo ze-neurodegenerative, njenge-amyotrophic lateral sclerosis (ALS) ehlobene ne-superoxide dismutase 1 (SOD1) kanye ne-desaccharase ehlobene nesifo sika-Parkinson (PARK7). Siphinde saqinisekisa ukuthi abanye omaka abaningi, njenge-SMOC1 kanye ne-membrane enothe ebuchosheni ebonisa iphrotheni 1 (BASP1), banezixhumanisi zangaphambilini ezilinganiselwe zokuguqulwa kwe-neurodegeneration. Kubalulekile ukuqaphela ukuthi ngenxa yobuningi babo obuphansi ku-CSF proteome, kunzima ngathi ukusebenzisa le ndlela yokuthola isibhamu esisodwa esiphezulu ukuze sithole ngokuthembekile i-MAPT namanye amaprotheni athile ahlobene ne-AD (isibonelo, i-NEFL ne-NRGN ) ( 43, 44).
Sibe sesihlola laba bamaka bephaneli ababalulekile abangu-60 ekuhlaziyeni okuphindwe kathathu okwengeziwe. Ku-CSF Copy 2, sisebenzise i-TMT-MS eyodwa ukuze sihlaziye iqoqo elizimele lokulawula okungu-297 namasampuli e-AD avela ku-Emory Goizueta ADRC (17). I-CSF replication 3 ihlanganisa ukuhlaziywa kabusha kwedatha ye-TMT-MS etholakalayo evela ku-120 control kanye neziguli ze-AD ezivela e-Lausanne, Switzerland (45). Sithole ngaphezu kwezingxenye ezimbili kwezintathu zomaka ababalulekile abangama-60 kudathasethi ngayinye. Nakuba ucwaningo lwaseSwitzerland lusebenzise izinkundla ezihlukene ze-MS nezindlela zokulinganisa ze-TMT (45, 46), sikhiqize kabusha amathrendi ethu ephaneli ekuhlaziyeni okuphindaphindiwe okubili (Umfanekiso 5, C no-D, kanye namaThebula S2, I, no-J) . Ukuze sihlole ukucaciswa kwesifo seqembu lethu, sisebenzise i-TMT-MS ukuhlaziya isethi yedatha yokuphindaphinda yesine (i-CSF replication 4), engafaki amacala okulawula kuphela (n = 18) kanye ne-AD (n = 17), kodwa ne-PD ( n = 14)), i-ALS (n = 18) kanye namasampula okuwohloka komqondo we-frontotemporal (FTD) (n = 11) (Ithebula S1A). Silinganise ngempumelelo cishe izingxenye ezimbili kwezintathu zamaphrotheni ephaneli kuleli qoqo (38 kwangu-60). Le miphumela igqamisa izinguquko eziqondene ne-AD kuwo wonke amaphaneli ama-biomarker amahlanu (Umfanekiso 5E kanye neThebula S2K). Ukwanda kweqembu le-metabolite kubonise ukucaciswa okunamandla kwe-AD, okulandelwa yi-myelination neqembu le-glial. Ngokwezinga elincane, i-FTD iphinde ibonise ukwanda phakathi kwalawa maphaneli, angase abonise izinguquko ezifanayo zenethiwekhi ezingase zibe khona (17). Ngokuphambene, i-ALS ne-PD zibonise cishe amaphrofayili afanayo e-myelination, i-glial, ne-metabolome njengeqembu lokulawula. Sekukonke, naphezu komehluko ekulungiseleleni isampula, inkundla ye-MS, nezindlela zokulinganisa ze-TMT, lokhu kuhlaziya okuphindaphindiwe kubonisa ukuthi omaka bethu bephaneli ababalulekile banezinguquko eziqondile ze-AD kumasampuli angaphezu kuka-500 e-CSF ahlukile.
I-AD neurodegeneration iye yaqashelwa kabanzi eminyakeni eminingana ngaphambi kokuqala kwezimpawu zokuqonda, ngakho-ke kunesidingo esiphuthumayo sama-biomarker e-AsymAD (5, 31). Kodwa-ke, ubufakazi obuningi bubonisa ukuthi i-biology ye-AsymAD ikude kakhulu ne-homogeneous, futhi ukusebenzisana okuyinkimbinkimbi kwengozi nokuqina kuholela ekuhlukeni okukhulu komuntu ngamunye ekuqhubekeni kwesifo okulandelayo (47). Nakuba esetshenziselwa ukukhomba amacala e-AsymAD, amazinga ama-biomarker angumgogodla we-CSF (Aβ1-42, i-tau ephelele ne-p-tau) awazange afakazele ukuthi akwazi ukubikezela ngokuthembekile ukuthi ubani ozothuthukela ekuwohlokeni komqondo (4, 7), okubonisa okwengeziwe kudingekile ukufaka amathuluzi ama-biomarker aphelele asekelwe ezicini eziningi ze-physiology yobuchopho ukuze kuhlelwe ngokunembile ubungozi balesi sibalo. Ngakho-ke, kamuva sahlaziya iphaneli yethu ye-biomarker eqinisekisiwe ye-AD kubantu base-AsymAD bekhophi ye-CSF engu-1. Lezi zimo ze-31 ze-AsymAD zibonise amazinga angajwayelekile e-biomarker angumongo (Aβ1–42/total tau ELISA ratio, <5.5) kanye nokuqonda okuphelele (mean MoCA, 27.1) ± 2.2) (Ithebula S1A). Ngaphezu kwalokho, bonke abantu abane-AsymAD banesilinganiso sokuwohloka komqondo emtholampilo esingu-0, okubonisa ukuthi abukho ubufakazi bokwehla kokusebenza kwengqondo noma ukusebenza kwansuku zonke.
Siqale sahlaziya amazinga amaphaneli aqinisekisiwe kuwo wonke ama-96 CSF aphindaphinda 1, okuhlanganisa neqembu le-AsymAD. Sithole ukuthi amaphaneli amaningana eqenjini le-AsymAD anezinguquko ezibalulekile zobuningi be-AD, iphaneli ye-vascular ibonise ukwehla kwe-AsymAD, kuyilapho wonke amanye amaphaneli abonisa ukuthambekela okuphezulu (Umfanekiso 6A). Ngakho-ke, wonke amaphaneli abonise ukuhlobana okubaluleke kakhulu ne-ELISA Aβ1-42 kanye namazinga e-tau ephelele (Umfanekiso 6B). Ngokuphambene, ukuhlobana phakathi kweqembu kanye nesikolo se-MoCA kubi kakhulu. Okunye okutholwe okuphawuleka kakhulu okuvela kulokhu kuhlaziya ububanzi obukhulu bobuningi bephaneli kuqoqo le-AsymAD. Njengoba kuboniswe kuMfanekiso 6A, izinga lephaneli leqembu le-AsymAD livamise ukweqa izinga lephaneli yeqembu lokulawula neqembu le-AD, libonisa ukuhlukahluka okuphezulu. Ukuze siqhubeke sihlola lokhu kuhlukahluka kwe-AsymAD, sisebenzise ukuhlaziya kwe-Multidimensional Scaling (MDS) kumakesi angu-96 we-CSF wokuphindaphinda 1. Ukuhlaziywa kwe-MDS kuvumela ukubona ngeso lengqondo ukufana phakathi kwamacala okususelwe kokuguquguqukayo okuthile kusethi yedatha. Kulokhu kuhlaziywa kweqoqo, sisebenzisa kuphela labomaka bephaneli abaqinisekisiwe abanoshintsho olubalulekile ngokwezibalo (P <0.05, AD/control) kuzinga lokutholwa kwe-CSF nokuphindaphinda 1 proteome (n = 29) (Ithebula S2L). Lokhu kuhlaziya kukhiqize ukuhlangana okucacile kwendawo phakathi kokulawula kwethu kanye namacala e-AD (Umfanekiso 6C). Ngokuphambene, amanye amacala e-AsymAD ahlanganiswe ngokucacile eqenjini lokulawula, kanti amanye atholakala kuma-AD. Ukuqhubeka nokuhlola lokhu kwe-AsymAD heterogeneity, sisebenzise imephu yethu ye-MDS ukuchaza amaqembu amabili alawa macala e-AsymAD. Iqembu lokuqala lalihlanganisa amacala e-AsymAD ahlanganiswe eduze kokulawula (n = 19), kuyilapho iqembu lesibili libonakala ngamacala e-AsymAD anephrofayela yomaka eduze ne-AD (n = 12).
(A) Izinga lenkulumo (z-score) yeqembu le-CSF biomarker kuwo wonke amasampuli angu-96 kuqoqo le-CSF lokuphindaphinda 1, okuhlanganisa i-AsymAD. Ukuhlaziywa kokuhluka nokulungiswa kwangemuva kuka-Tukey kwasetshenziswa ukuze kuhlolwe ukubaluleka kwezibalo zezinguquko zobuningi bephaneli. (B) Ukuhlaziywa kokuhlobana kweleveli ye-protein abundance level (z-score) nesikolo se-MoCA kanye nesamba sezinga le-tau kumasampuli we-ELISA Aβ1-42 kanye ne-CSF yekhophi engu-1. I-Pearson coefficient ehambisanayo enevelu engu-P iyaboniswa. (C) I-MDS yamakesi angu-96 wekhophi engu-1 ye-CSF yayisekelwe emazingeni amaningi omaka bephaneli abaqinisekisiwe abangu-29, ashintshe kakhulu kukho kokubili ukutholwa kanye ne-CSF ikhophi yamasethi wedatha engu-1 [P <0.05 AD/control (CT)]. Lokhu kuhlaziya kusetshenziswe ukuhlukanisa iqembu le-AsymAD ekulawuleni (n = 19) kanye nama-AD (n = 12) ama-subgroups. (D) Isakhiwo sentaba-mlilo sibonisa ukubonakaliswa okuhlukile kwawo wonke amaprotheni e-CSF aphindaphindayo angu-1 anoshintsho olugoqiwe lwe-log2 (x-eksisi) ngokuhlobene nenani elingu-P lezibalo le--log10 phakathi kwamaqembu amancane e-AsymAD. Amaphaneli biomarker anemibala. (E) Izinga le-CSF lokuphindaphinda 1 lokuchichima kwama-biomarker weqembu lokukhetha livezwa ngokwehlukana phakathi kwamaqembu amancane e-AsymAD. Ukuhlaziywa kwangemuva kokulungiswa kukaTukey kokuhluka kwasetshenziswa ukuze kuhlolwe ukubaluleka kwezibalo.
Sihlole umehluko wokuvezwa kwamaprotheni phakathi kwalokhu kulawula kanye nezimo ze-AsymAD ezifana ne-AD (Umfanekiso 6D kanye neThebula S2L). Imephu yentaba-mlilo ewumphumela ibonisa ukuthi omaka bephaneli abayi-14 bashintshe kakhulu phakathi kwamaqembu amabili. Iningi lalabamaka lingamalungu e-synapse kanye ne-metabolome. Kodwa-ke, i-SOD1 kanye ne-myristoylated alanine-rich protein kinase C substrate (MARCKS), okungamalungu e-myelin kanye namaqembu omzimba we-glial, ngokulandelanayo, nawo angaphansi kwaleli qembu (Umfanekiso 6, D no-E) . Iphaneli yemithambo yegazi iphinde yanikela ngezimpawu ezimbili ezincishiswe kakhulu eqenjini le-AD le-AsymAD, okuhlanganisa i-AE ebopha iphrotheni 1 (AEBP1) kanye nelunga lomndeni elihambisana ne-C9. Kwakungekho mehluko obalulekile phakathi kokulawula kanye ne-AD-like AsymAD subgroups ku-ELISA AB1-42 (P = 0.38) kanye ne-p-tau (P = 0.28), kodwa ngempela kube khona umehluko obalulekile enanini le-tau eliphelele (P = 0.0031 ) (Fig. S7). Kukhona omaka bephaneli abambalwa ababonisa ukuthi izinguquko phakathi kwamaqembu amancane e-AsymAD zibaluleke kakhulu kunamazinga engqikithi ye-tau (isibonelo, YWHAZ, SOD1, ne-MDH1) (Umfanekiso 6E). Sekukonke, le miphumela ikhombisa ukuthi iphaneli yethu eqinisekisiwe ingaqukatha ama-biomarker angafaka uhlobo oluncane kanye nokuhlukaniswa kwengozi okungaba khona kweziguli ezine-asymptomatic disease.
Kunesidingo esiphuthumayo samathuluzi e-biomarker asekelwe ohlelweni ukuze kulinganiswe kangcono futhi kuqondiswe ku-pathophysiology ehlukahlukene ngemuva kwe-AD. Lawa mathuluzi alindeleke ukuthi angashintshi nje kuphela uhlaka lwethu lokuxilonga lwe-AD, kodwa futhi akhuthaze ukwamukelwa kwamasu okwelapha asebenzayo, aqondene nesiguli (1, 2). Kuze kube manje, sisebenzise indlela engachemi ebanzi ye-proteomics ebuchosheni be-AD kanye ne-CSF ukuze sihlonze izimpawu ze-CSF ezisekelwe kuwebhu ezibonisa ububanzi obubanzi be-pathophysiology esekelwe ebuchosheni. Ukuhlaziywa kwethu kukhiqize amaphaneli amahlanu e-biomarker e-CSF, okuthi (i) abonise ama-synapses, imithambo yegazi, i-myelin, ukungasebenzi kahle kwe-immune kanye ne-metabolic; (ii) khombisa ukukhiqiza kabusha okuqinile kumapulatifomu ahlukene we-MS; (iii) Bonisa izinguquko eziqhubekayo eziqondene nesifo kuzo zonke izigaba zokuqala nezakamuva ze-AD. Sekukonke, lokhu okutholakele kumelela isinyathelo esithembisayo ekuthuthukisweni kwamathuluzi ama-biomarker agxile kuwebhu ahlukahlukene, athembekile, ocwaningo lwe-AD kanye nezinhlelo zokusebenza zomtholampilo.
Imiphumela yethu ibonisa inhlangano elondolozwe kakhulu ye-AD brain network proteome futhi isekela ukusetshenziswa kwayo njengehange lokuthuthukiswa kwe-biomarker esekelwe ohlelweni. Ukuhlaziya kwethu kukhombisa ukuthi amasethi wedatha amabili azimele we-TMT-MS aqukethe ubuchopho be-AD kanye ne-AsymAD anokushintshashintsha okuqinile. Lokhu okutholakele kunweba umsebenzi wethu wangaphambilini, okubonisa ukulondolozwa kwamamojula anamandla angaphezu kwezicubu zobuchopho ze-2,000 ezivela kumaqoqo amaningi azimele ku-frontal, parietal, kanye ne-temporal cortex (17). Le nethiwekhi yokuvumelana ibonisa izinguquko ezihlukahlukene ezihlobene nesifo ezibonwa ocwaningweni lwamanje, okuhlanganisa ukwanda kwamamojula okuvuvukala anothe nge-glial kanye nokuncipha kwamamojula anothe nge-neuron. Njengocwaningo lwamanje, le nethiwekhi enkulu futhi ifaka izinguquko ezibalulekile ze-AsymAD, ekhombisa i-pathophysiology eyahlukene ye-preclinical (17).
Kodwa-ke, ngaphakathi kwalolu hlaka olusekelwe kusistimu olulondoloza kakhulu, kukhona ukuhlukahluka kwebhayoloji okusanhlamvu okuhle kakhulu, ikakhulukazi phakathi kwabantu ngabanye abasezigabeni zokuqala ze-AD. Iphaneli yethu ye-biomarker iyakwazi ukukhombisa amaqenjana amabili ku-AsymAD, abonisa ukuvezwa okubalulekile okuhlukile komaka abaningi be-CSF. Iqembu lethu likwazile ukugqamisa umehluko webhayoloji phakathi kwala maqembu amancane amabili, obekungabonakali kahle ezingeni lama-biomarker e-AD ayisisekelo. Uma kuqhathaniswa neqembu lokulawula, izilinganiso ze-Aβ1-42/total tau zalaba bantu be-AsymAD beziphansi ngokungavamile. Kodwa-ke, amazinga e-tau kuphela ayehluke kakhulu phakathi kwama-subgroups amabili e-AsymAD, kuyilapho amazinga e-Aβ1-42 kanye ne-p-tau ahlala eqhathaniswa. Njengoba i-CSF ephezulu ibonakala iyisibikezelo esingcono sezimpawu zokuqonda kunamazinga we-Aβ1-42 (7), sisola ukuthi amaqoqo amabili e-AsymAD angase abe nezingozi ezihlukene zokuqhubeka kwesifo. Uma kubhekwa usayizi wesampula olinganiselwe we-AsymAD yethu kanye nokuntuleka kwedatha ye-longitudinal, ucwaningo olwengeziwe luyadingeka ukuze ufinyelele lezi ziphetho ngokuzethemba. Kodwa-ke, le miphumela ikhombisa ukuthi iphaneli ye-CSF esekelwe ohlelweni ingathuthukisa ikhono lethu lokuhlukanisa abantu ngendlela ephumelelayo ngesikhathi sesigaba se-asymptomatic sesifo.
Sekukonke, okutholakele kwethu kusekela indima yemisebenzi eminingi yezinto eziphilayo ku-pathogenesis ye-AD. Kodwa-ke, i-dysregulated energy metabolism yaba yindikimba evelele yawo wonke amaphaneli ethu okulebula aqinisekisiwe amahlanu. Amaprotheni e-Metabolic, afana ne-hypoxanthine-guanine phosphoribosyltransferase 1 (HPRT1) kanye ne-lactate dehydrogenase A (LDHA), ayizimpawu ze-synaptic biomarker eziqinisekiswe ngokuqinile, okubonisa ukuthi ukwanda kwe-AD CSF ubulili obuphindaphindekayo. Imithambo yethu yegazi namaphaneli e-glial nawo aqukethe izimpawu ezimbalwa ezibandakanyeka kumetabolism yezinto ezithinta i-oxidative. Lokhu okutholakele kuhambisana neqhaza elibalulekile elidlalwa izinqubo ze-metabolic kubo bonke ubuchopho, hhayi nje ukuhlangabezana nesidingo esikhulu samandla sama-neurons, kodwa futhi nokuhlangabezana nesidingo esikhulu samandla sama-astrocyte namanye amaseli e-glial (17, 48). Imiphumela yethu isekela ubufakazi obukhulayo bokuthi izinguquko ezingaba khona ze-redox kanye nokuphazamiseka kwemigudu yamandla kungase kube isixhumanisi esiyinhloko phakathi kwezinqubo ezimbalwa ezibalulekile ezihilelekile ku-pathogenesis ye-AD, okuhlanganisa ukuphazamiseka kwe-mitochondrial, ukuvuvukala kwe-glial-mediated, nokulimala kwe-Vascular (49). Ngaphezu kwalokho, i-metabolic cerebrospinal fluid biomarkers iqukethe inani elikhulu lamaprotheni acebile ngokwehlukana phakathi kokulawula kwethu kanye nama-subgroups afana ne-AD-AsymAD, okuphakamisa ukuthi ukuphazamiseka kwalezi zindlela zamandla kanye ne-redox kungase kubaluleke kakhulu esigabeni sokuqala sesifo.
Amathrendi ahlukene ephaneli yobuchopho ne-cerebrospinal fluid esiwabonile nawo anemithelela ethokozisayo yebhayoloji. Ama-Synapses kanye nama-metabolomes acebile kuma-neurons akhombisa amazinga anciphile ebuchosheni be-AD futhi anda ngobuningi ku-cerebrospinal fluid. Njengoba kunikezwe ukuthi ama-neurons acebe nge-mitochondria ekhiqiza amandla kuma-synapses ukuze anikeze amandla kumasiginali awo akhethekile (50), ukufana kwamaphrofayili wenkulumo yala maqembu amabili ama-neuron kulindeleke. Ukulahleka kwama-neurons kanye nokukhishwa kwamaseli alimele kungachaza lobu buchopho kanye nezitayela zephaneli ye-CSF ezifweni zakamuva, kodwa azikwazi ukuchaza izinguquko zephaneli zakuqala esizibonayo (13). Enye incazelo engaba khona yalokhu okutholakele ekuqaleni kwesifo esingenazimpawu ukuthenwa okungavamile kwe-synaptic. Ubufakazi obusha kumamodeli wamagundane buphakamisa ukuthi i-microglia-mediated synaptic phagocytosis ingase isebenze ngokungavamile ku-AD futhi iholele ekulahlekeni kwe-synapse yangaphambi kwesikhathi ebuchosheni (51). Le nto elahliwe ye-synaptic ingase iqongelele ku-CSF, yingakho sibona ukwanda kwe-CSF kuphaneli ye-neuron. Ukuthenwa kwe-synaptic kokuzivikela komzimba kungase kuchaze kancane ukwanda kwamaprotheni e-glial esiwabona ebuchosheni nasoketshezini lwe-cerebrospinal kuyo yonke inqubo yesifo. Ngaphezu kokuthenwa kwe-synaptic, ukungahambi kahle okuphelele kwendlela ye-exocytic kungase futhi kuholele ebuchosheni obuhlukile kanye nezinkulumo ze-CSF zomaka be-neuronal. Ucwaningo oluningi lubonise ukuthi okuqukethwe kwe-exosomes ku-pathogenesis ye-AD yobuchopho kushintshile (52). Umzila ongaphandle kwamaselula nawo uyabandakanyeka ekwandeni kwe-Aβ (53, 54). Kuyaphawuleka ukuthi ukucindezelwa kokukhishwa kwe-exosomal kunganciphisa i-AD-like pathology kumamodeli egundane e-AD transgenic (55).
Ngesikhathi esifanayo, iphrotheni ephaneli le-vascular ibonise ukwanda okulinganiselwe ebuchosheni be-AD, kodwa yehla kakhulu ku-CSF. Ukungasebenzi kahle kwe-blood-brain barrier (BBB) kungachaza ngokwengxenye lokhu okutholakele. Izifundo eziningi ezizimele ze-postmortem zabantu zibonise ukuwohloka kwe-BBB ku-AD (56, 57). Lezi zifundo ziqinisekise imisebenzi ehlukahlukene engavamile ezungeze lolu ngqimba oluvalwe ngokuqinile lwamaseli e-endothelial, okuhlanganisa ukuvuza kwe-capillary yobuchopho kanye nokunqwabelana kwemithambo yegazi yamaprotheni aphethwe yigazi (57). Lokhu kunganikeza incazelo elula yamaprotheni emithambo yegazi ephakeme ebuchosheni, kodwa ayikwazi ukuchaza ngokugcwele ukuncipha kwala maprotheni afanayo ku-cerebrospinal fluid. Okunye okungenzeka ukuthi isimiso sezinzwa esimaphakathi sihlukanisa ngenkuthalo la ma-molecule ukuze kuxazululwe inkinga yokwanda kokuvuvukala nokucindezeleka okwenziwe nge-oxidative. Ukwehliswa kwamanye amaprotheni anzima kakhulu e-CSF kuleli phaneli, ikakhulukazi lawo ahilelekile ekulawuleni i-lipoprotein, kuhlobene nokuvinjelwa kwamazinga ayingozi okuvuvukala kanye nenqubo ye-neuroprotective yezinhlobo ze-oxygen esebenzayo. Lokhu kuyiqiniso nge-Paroxonase 1 (PON1), i-enzyme ebopha i-lipoprotein enesibopho sokunciphisa amazinga okucindezeleka okwenziwe nge-oxidative ekujikelezeni (58, 59). I-Alpha-1-microglobulin/bikunin precursor (AMBP) ingenye umaka olawulwa phansi ngokuphawulekayo weqembu lemithambo. Yisandulela se-lipid transporter bikunin, ehilelekile ekucindezelweni kokuvuvukala kanye nokuvikelwa kwemizwa (60, 61).
Naphezu kwemibono ehlukahlukene ethakazelisayo, ukungakwazi ukubona ngokuqondile izindlela zezifo ze-biochemical kuwumkhawulo owaziwayo wokuhlaziywa kwe-proteomics eqhutshwa ukutholwa. Ngakho-ke, ucwaningo olwengeziwe luyadingeka ukuze kuchazwe ngokuzethemba izindlela ezingemuva kwala maphaneli e-biomarker. Ukuze siqhubekele ekuthuthukisweni kokuhlaziywa komtholampilo okusekelwe ku-MS, isiqondiso sesikhathi esizayo sidinga futhi ukusetshenziswa kwezindlela zobuningi ezihlosiwe zokuqinisekiswa kwe-biomarker enkulu, njengokuqapha kokusabela okukhethiwe noma okufanayo (62). Sisanda kusebenzisa ukuqapha kokusabela okufanayo (63) ukuze siqinisekise izinguquko eziningi zamaprotheni e-CSF ezichazwe lapha. Okuhlosiwe okuningana okubalulekile kwephaneli kubalwa ngokunemba okubalulekile, okuhlanganisa i-YWHAZ, i-ALDOA, ne-SMOC1, ekhomba i-synapse yethu, i-metabolism, namaphaneli okuvuvukala, ngokulandelana (63). I-Independent Data Acquisition (DIA) namanye amasu asekelwe ku-MS angase abe usizo ekuqinisekiseni okuqondiwe. Bud et al. (64) Muva nje kuboniswe ukuthi kunokugqagqana okubalulekile phakathi kwama-AD biomarker ahlonzwe kusethi yethu yedatha yokutholwa kwe-CSF kanye nesethi yedatha ezimele ye-DIA-MS, ehlanganisa cishe amasampula e-CSF angu-200 avela kumaqoqo amathathu ahlukene aseYurophu . Lezi zifundo zakamuva zisekela amandla amaphaneli ethu ukuze aguqulele ekutholeni okuthembekile okusekelwe ku-MS. I-antibody evamile kanye nokutholwa okusekelwe ku-aptamer nakho kubalulekile ekuthuthukisweni okuqhubekayo kwama-AD biomarker. Ngenxa yobuningi obuphansi be-CSF, kunzima kakhulu ukuthola lezi zimpawu zemvelo zisebenzisa izindlela ze-MS ze-high-throughput. I-NEFL kanye ne-NRGN yizibonelo ezimbili ezinjalo zama-biomarker e-CSF anensada ephansi, ezifakwe kumephu kuphaneli ekuhlaziyeni kwethu okuphelele, kodwa azikwazi ukutholwa ngokuthembekile kusetshenziswa isu lethu elilodwa le-MS. Amasu okukhomba asekelwe kumasosha omzimba amaningi, njenge-PEA, angase akhuthaze ukuguqulwa komtholampilo kwalezi zomaka.
Sekukonke, lolu cwaningo luhlinzeka ngendlela ehlukile ye-proteomics yokuhlonza nokuqinisekiswa kwama-biomarker e-CSF AD asekelwe kumasistimu ahlukene. Ukuthuthukisa lawa maphaneli omaka kuwo wonke amaqoqo e-AD engeziwe nezinkundla ze-MS kungase kuthembise ukuqhubekisela phambili ukuhlukaniswa kwengozi ye-AD nokwelashwa. Ucwaningo oluhlola izinga lobude lalawa maphaneli ngokuhamba kwesikhathi nalo lubalulekile ukuze kutholwe ukuthi iyiphi inhlanganisela yezimpawu ezibonisa kangcono ingozi yesifo sangaphambi kwesikhathi kanye nezinguquko kubunzima besifo.
Ngaphandle kwamasampuli angu-3 akopishwe yi-CSF, wonke amasampula e-CSF asetshenziswe kulolu cwaningo aqoqwe ngaphansi kwe-Emory ADRC noma izikhungo zocwaningo ezihlobene eduze. Isamba samasethi amane amasampula e-Emory CSF asetshenziswe kulezi zifundo ze-proteomics. Iqembu le-CSF litholakale liqukethe amasampula avela kuzilawuli ezinempilo ezingama-20 kanye neziguli ezingama-20 ze-AD. Ikhophi 1 ye-CSF ihlanganisa amasampula asuka kuzilawuli ezinempilo ezingu-32, abantu abangabodwana be-AsymAD abangu-31, kanye nabantu abangu-33 AD. Ikhophi 2 ye-CSF iqukethe izilawuli ezingu-147 namasampuli angu-150 AD. Iqembu le-CSF lokuphindaphinda 4 lezifo eziningi lihlanganisa izilawuli eziyi-18, 17 AD, 19 ALS, 13 PD, kanye namasampuli ayi-11 FTD. Ngokwesivumelwano esivunywe Ibhodi Lokubuyekeza Isikhungo Se-Emory University, bonke ababambiqhaza bocwaningo lwe-Emory bathole imvume enolwazi. Ngokwe-National Institute of Aging Best Practice Guidelines yango-2014 yezikhungo ze-Alzheimer's (https://alz.washington.edu/BiospecimenTaskForce.html), uketshezi lwe-cerebrospinal lwaqoqwa futhi lwagcinwa ngokubhoboza i-lumbar. Ukulawula kanye neziguli ze-AsymAD kanye ne-AD zithole ukuhlolwa kwengqondo okujwayelekile e-Emory Cognitive Neurology Clinic noma e-Goizueta ADRC. Amasampula abo e-cerebrospinal fluid ahlolwe yi-INNO-BIA AlzBio3 Luminex ye-ELISA Aβ1-42, ingqikithi yokuhlaziywa kwe-tau kanye ne-p-tau (65). Amanani e-ELISA asetshenziselwa ukusekela ukuhlukaniswa kwezifundo ngokususelwa kunqubo emisiwe ye-AD biomarker cut-off (66, 67). Idatha eyisisekelo ye-demographic kanye nokuxilonga yokunye ukuxilonga kwe-CSF (FTD, ALS, and PD) nayo itholakala ku-Emory ADRC noma izikhungo zocwaningo ezixhumene nayo. Imethadatha yesifinyezo yalezi zimo ze-Emory CSF ingatholakala kuThebula S1A. Izici ze-Swiss CSF replication 3 cohort zishicilelwe ngaphambilini (45).
I-CSF ithole isampula. Ukuze kwandiswe ukujula kokutholakala kwethu kwesethi yedatha ye-CSF, ukusetshenziswa kwamasosha omzimba kwamaprotheni anala kakhulu kwenziwa ngaphambi kwe-trypsinization. Ngamafuphi, i-130 μl ye-CSF evela kumasampuli e-CSF angu-40 kanye nevolumu elinganayo (130 μl) ye-High Select Top14 Abundance Protein Depletion Resin (Thermo Fisher Scientific, A36372) ibekwe kukholomu yokuphotha (Thermo Fisher Scientific, A89868) ekamelweni) izinga lokushisa Fukamela). Ngemuva kokuphotha imizuzu eyi-15, beka isampula ku-1000g imizuzu emi-2. Idivayisi yesihlungi ye-3K ultracentrifugal (Millipore, UFC500396) yasetshenziswa ukugxilisa isampula yokulahlwa nge-centrifuging ku-14,000g imizuzu engu-30. Nciphisa wonke amasampula amavolumu abe ngu-75 μl nge-phosphate buffered saline. Ukugcwala kwamaprotheni kuhlolwe ngendlela ye-bicinchoninic acid (BCA) ngokuya ngephrothokholi yomkhiqizi (i-Thermo Fisher Scientific). I-CSF (60 μl) engenawo amandla okuzivikela kuwo wonke amasampula angama-40 yagaywa nge-lysyl endopeptidase (LysC) kanye ne-trypsin. Ngamafuphi, isampula yancishiswa futhi i-alkylated ne-1.2 μl 0.5 M tris-2 (-carboxyethyl) -phosphine kanye ne-3 μl 0.8 M i-chloroacetamide ku-90 ° C imizuzu engu-10, bese i-sonicated emanzini okugeza imizuzu engu-15. Isampula lihlanjululwe nge-193 μl 8 M urea buffer [8 M urea kanye ne-100 mM NaHPO4 (pH 8.5)] kuya ekuhlanganiseni kokugcina kwe-6 M urea. I-LysC (4.5 μg; Wako) isetshenziselwa ukugaya ebusuku ekamelweni lokushisa. Isampula libe selihlanjululwa laba ku-1 M urea ne-50 mM ammonium bicarbonate (ABC) (68). Engeza inani elilinganayo (4.5 μg) le-trypsin (i-Promega), bese ufukamela isampula amahora angu-12. Faka i-acidi isisombululo se-peptide egayiwe ekuhlanganiseni kokugcina kwe-1% formic acid (FA) kanye no-0.1% trifluoroacetic acid (TFA) (66), bese ukhipha usawoti ngekholomu ye-Sep-Pak C18 engu-50 mg (Amanzi) njengoba kuchazwe ngenhla (25) . I-peptide yabe isikhishwa ku-1 ml we-50% acetonitrile (ACN). Ukuze kulinganiswe ubuningi bamaprotheni kuwo wonke amaqoqo (25), ama-aliquots angu-100 μl kuwo wonke amasampula e-CSF angu-40 ahlanganiswa ukuze kukhiqizwe isampula exubile, eyabe isihlukaniswa yaba amasampuli angaphakathi emhlabeni wonke amahlanu (GIS) (48). Wonke amasampula angawodwana namazinga ahlanganisiwe omiswa nge-vacuum enesivinini esikhulu (Labconco).
I-CSF ikopisha isampula. U-Dayon nozakwabo baye bachaza ngaphambilini ukuwohloka kwamasosha omzimba nokugaya kwe-CSF amasampula ama-3 (45, 46). Amasampula aphindaphindayo asele awazange abe ne-immunodepleted. Gaya lawa masampuli angakhishiwe ku-trypsin njengoba kuchazwe ngaphambilini (17). Ekuhlaziyeni ngakunye okuphindaphindiwe, ama-aliquots angu-120 μl we-peptide eluted kusukela kusampula ngayinye ahlanganiswe ndawonye futhi ahlukaniswa abe ama-aliquots evolumu alinganayo azosetshenziswa njengezinga langaphakathi lomhlaba elilebulwe yi-TMT (48). Wonke amasampula angawodwana namazinga ahlanganisiwe omiswa nge-vacuum enesivinini esikhulu (Labconco). Ukuze kuthuthukiswe isignali yeprotheni ye-CSF enensada ephansi, ngokuhlanganisa i-125 μl kusampula ngayinye, isampula “ethuthukisiwe” yalungiselelwa ukuhlaziywa okuphindaphindwayo ngakunye [okungukuthi, isampula yebhayoloji elingisa isampula yocwaningo, kodwa inani elitholakalayo okukhulu kakhulu (37, 69)] kuhlanganiswe kusampula ye-CSF exubile (17). Isampula elixutshiwe libe selikhishwa amasosha omzimba kusetshenziswa u-12 ml we-High Select Top14 Abundance Protein Removal Resin (Thermo Fisher Scientific, A36372), yagaywa njengoba kuchazwe ngenhla, futhi yafakwa ekulebulani okwalandela okuningi kwe-TMT.
Isikhathi sokuthumela: Aug-27-2021