Isifo i-Alzheimer's (AD) asinazo iiprotheyini zebhayoloji ezibonisa i-pathophysiology yaso esisiseko, ethintela inkqubela yoxilongo kunye nonyango. Apha, sisebenzisa iiproteomics ezibanzi ukuchonga i-cerebrospinal fluid (CSF) biomarkers emele uluhlu olubanzi lwe-AD pathophysiology. I-Multiplex mass spectrometry ichongwe malunga ne-3,500 kwaye malunga ne-12,000 yeeprotheni kwi-AD CSF kunye nengqondo, ngokulandelelanayo. Uhlalutyo lothungelwano lweproteome yobuchopho lusombulule iimodyuli ezingama-44 zezinto ezahlukeneyo eziphilayo, ezili-15 zazo ezinxibelelene ne-cerebrospinal fluid proteome. Iimpawu ze-CSF ze-AD kwezi modyuli ezidibeneyo zigoqwe zibe ngamaqela amahlanu eprotheyini, emele iinkqubo ezahlukeneyo ze-pathophysiological. I-synapses kunye ne-metabolites kwi-AD yobuchopho iyancipha, kodwa i-CSF iyanda, ngelixa i-glial-rich myelination kunye namaqela omzimba kwingqondo kunye ne-CSF yanda. Ukuhambelana kunye nesifo esithile sotshintsho lwephaneli luqinisekisiwe ngaphezu kweesampuli ze-CSF ze-500 ezongezelelweyo. La maqela achonge amacandelwana ebhayoloji kwi-AD ye-asymptomatic. Ngokubanzi, ezi ziphumo linyathelo elithembisayo elibhekiselele kwi-web-based biomarker izixhobo zezicelo zeklinikhi kwi-AD.
Isifo se-Alzheimer's (AD) sesona sizathu sixhaphakileyo se-neurodeergenerative dementia kwihlabathi jikelele kwaye sibonakaliswe luluhlu olubanzi lwe-biological system dysfunctions, kubandakanywa ukuhanjiswa kwe-synaptic, i-glial-mediated immunity, kunye ne-mitochondrial metabolism (1-3). Nangona kunjalo, iiprotheyini zayo ezisekiweyo zebhayoloji zisagxile ekufumaneni iprotein ye-amyloid kunye ne-tau, kwaye ke ayinakukwazi ukubonisa le pathophysiology yahlukeneyo. Ezi "protein" zeprotheyini ze-biomarkers ezilinganiswa ngokuthembekileyo kwi-cerebrospinal fluid (CSF) ziquka (i) i-amyloid beta peptide 1-42 (Aβ1-42), ebonisa ukubunjwa kwe-cortical amyloid plaques; (ii) i-tau iyonke, uphawu lokuwohloka kwe-axon; (iii) phospho-tau (p-tau), ummeli we-pathological tau hyperphosphorylation (4-7). Nangona ezi biomarkers ze-cerebrospinal fluid ziye zaququzelela kakhulu ukufumanisa kwethu izifo zeprotheyini ze-AD "eziphawulweyo" (4-7), zimele kuphela inxalenye encinci ye-biology enzima emva kwesi sifo.
Ukungabikho kweentlobo ezahlukeneyo ze-pathophysiological ze-AD biomarkers kuye kwakhokelela kwimingeni emininzi, kubandakanywa (i) ukungakwazi ukuchonga kunye nokulinganisa i-biological heterogeneity yezigulane ze-AD, (ii) ukulinganisa okunganeleyo kobunzima besifo kunye nokuqhubela phambili, ngakumbi kwinqanaba le-preclinical, Kwaye (ii) iii) uphuhliso lwamachiza onyango aluphumelelanga ukusombulula ngokupheleleyo yonke imiba yokuwohloka kwemithambo-luvo. Ukuthembela kwethu kwi-pathology ephawulekayo ekuchazeni i-AD evela kwizifo ezinxulumene nayo kwandisa ezi ngxaki. Ubungqina obuninzi nangakumbi bubonisa ukuba uninzi lwabantu abadala abanesifo sengqondo esiyingozi baneempawu ezingaphezu kwesinye ze-pathological zokuncipha kwengqondo (8). Uninzi lwe-90% okanye ngaphezulu kwabantu abane-AD pathology nabo banesifo se-vascular, i-TDP-43 inclusions, okanye ezinye izifo eziphazamisayo (9). Le milinganiselo iphakamileyo yokudibana kwe-pathological iye yaphazamisa inkqubo yethu yangoku yokuxilonga kwi-dementia, kwaye inkcazo ebanzi ye-pathophysiological yesi sifo iyafuneka.
Ngokujonga imfuno engxamisekileyo yeendidi zebhayoloji ze-AD, intsimi iya isanda kwamkela indlela "ye-omics" esekwe kwinkqubo iyonke yokufumanisa i-biomarkers. I-Accelerated Pharmaceutical Partnership (AMP)-AD Alliance yasungulwa ngo-2014 kwaye ihamba phambili kule nkqubo. Lo mzamo wezinto ezininzi ngamaZiko ezeMpilo kaZwelonke, i-academia, kunye neshishini lijolise ekusebenziseni izicwangciso ezisekelwe kwinkqubo yokuchaza ngcono i-pathophysiology ye-AD kunye nokuphuhlisa uhlalutyo lokuxilonga kwezinto eziphilayo kunye nezicwangciso zonyango (10). Njengenxalenye yale projekthi, i-network proteomics ibe sisixhobo esithembisayo sokuqhubela phambili ii-biomarkers ezisekwe kwisistim kwi-AD. Le ndlela engenamkhethe yedatha iququzelela iiseti zedatha yeproteomics eyinkimbinkimbi ibe ngamaqela okanye "iimodyuli" zeeprotheni ezichazwe ngokubambisana ezidityaniswe neentlobo ezithile zeeseli, i-organelles, kunye nemisebenzi ye-biological (11-13). Phantse i-12 yolwazi olutyebileyo lwezifundo zeproteomics zenethiwekhi zenziwe kwi-AD brain (13-23). Ngokubanzi, olu hlalutyo lubonisa ukuba i-AD yenethiwekhi yobuchopho iproteome igcina umbutho ogcinwe kakhulu wemodyuli kumaqela azimeleyo kunye nemimandla emininzi ye-cortical. Ukongezelela, ezinye zezi modyuli zibonisa utshintsho olunokuphinda luveliselwe ubuninzi obunxulumene ne-AD kwiiseti zedatha, ezibonisa i-pathophysiology yezifo ezininzi. Ngokudibeneyo, ezi ziphumo zibonisa inqaku le-ankile elithembisayo lokufunyanwa kweproteome yenethiwekhi yobuchopho njengenkqubo esekwe kwi-biomarker kwi-AD.
Ukuze kuguqulwe i-AD ye-brain network proteome ibe yi-biomarker eluncedo ngokweklinikhi esekwe kwi-biomarker, sidibanise inethiwekhi evela kwingqondo kunye nohlalutyo lweproteomic ye-AD CSF. Le ndlela idibeneyo yakhokelela ekuchongweni kweeseti ezintlanu ezithembisayo ze-CSF ze-biomarkers ezinxulumene noluhlu olubanzi lwe-pathophysiology esekelwe ebuchosheni, kubandakanywa i-synapses, imithambo yegazi, i-myelination, ukuvuvukala, kunye nokungasebenzi kakuhle kweendlela ze-metabolic. Siqinisekise ngempumelelo ezi paneli ze-biomarker ngohlalutyo oluninzi lokuphindaphinda, kubandakanya neesampulu ze-CSF ezingaphezu kwama-500 ezivela kwizifo ezahlukeneyo ze-neurodeergenerative. Olu hlalutyo lokuqinisekiswa lubandakanya ukuhlola iinjongo zeqela kwi-CSF yezigulane ezine-AD engabonakaliyo (i-AsymAD) okanye zibonisa ubungqina bokuqokelela i-amyloid engaqhelekanga kwindawo eqhelekileyo yokuqonda. Olu hlalutyo lugxininisa i-heterogeneity ebalulekileyo yebhayoloji kubantu be-AsymAD kwaye ichonge iimpawu zephaneli ezinokuthi zikwazi ukuthoba abantu abakwinqanaba lokuqala lesi sifo. Ngokubanzi, ezi ziphumo zibonisa inyathelo eliphambili ekuphuhliseni izixhobo zeprotein biomarker ezisekelwe kwiinkqubo ezininzi ezinokusombulula ngempumelelo imingeni emininzi yekliniki ejongene ne-AD.
Eyona njongo yolu phononongo kukuchonga i-biomarkers entsha ye-cerebrospinal fluid ebonisa i-pathophysiology esekwe ebuchotsheni ekhokelela kwi-AD. Umzobo we-S1 uchaza indlela yethu yophando, ebandakanya (i) uhlalutyo olubanzi oluqhutywa ziziphumo zokuqala ze-AD CSF kunye ne-proteome yobuchopho bothungelwano ukuchonga i-biomarkers yesifo se-CSF enxulumene nengqondo, kunye (ii) nokuphindaphinda okulandelayo. amaqela olwelo. Uphando olujolise ekufumaneni luqale ngohlalutyo lwendlela yokubonakalisa umahluko we-CSF kubantu abangama-20 abaqhelekileyo ngokwengqondo kunye nezigulane ezingama-20 ze-AD kwiZiko loPhando ngeSifo se-Alzheimer's Emory Goizueta (ADRC). Ukuxilongwa kwe-AD kuchazwa njengento ebalulekileyo yokuphazamiseka kwengqondo phambi kwe-Aβ1-42 ephantsi kunye namanqanaba aphakamileyo e-tau epheleleyo kunye ne-p-tau kwi-cerebrospinal fluid [Mean Montreal Cognitive Assessment (MoCA), 13.8 ± 7.0] [ELISA (ELISA) )]] (Uluhlu S1A). Ulawulo (oluthethayo i-MoCA, 26.7 ± 2.2) lube namanqanaba aqhelekileyo e-CSF biomarkers.
I-CSF yomntu ibonakaliswe luluhlu oluguqukayo lobuninzi beprotheyini, apho i-albumin kunye nezinye iiproteni ezininzi kakhulu zinokuthintela ukubonwa kweeproteni ezinomdla (24). Ukwandisa ubunzulu bokufunyanwa kweprotheyini, sisuse iiprotheni zokuqala ezili-14 ezininzi kakhulu kwisampulu nganye ye-CSF phambi kohlalutyo lwe-mass spectrometry (MS) (24). Itotali ye-39,805 ye-peptides ichongiwe yi-MS, efakwe kwimephu ye-3691 yeeproteome kwiisampuli ezingama-40. Ubungakanani beprotheyini lwenziwa ngethegi yobuninzi be-tandem (TMT) ilebula (18, 25). Ukuze sisombulule idatha elahlekileyo, sibandakanye kuphela ezo ziprotheni eziye zabalwa ubuncinane kwi-50% yeesampuli kuhlalutyo olulandelayo, ngaloo ndlela ekugqibeleni ukulinganisa i-2875 proteomes. Ngenxa yomahluko obalulekileyo kumanqanaba obuninzi beeprotheyini ezipheleleyo, isampuli yokulawula ithathwa njengenkcazo ngaphandle (13) kwaye ayizange ibandakanywe kuhlalutyo olulandelayo. Amaxabiso obuninzi beesampulu ezingama-39 eziseleyo zahlengahlengiswa ngokobudala, isini, kunye nebhetshi covariance (13-15, 17, 18, 20, 26).
Ukusebenzisa uhlalutyo lwe-t-test ye-statistical ukuvavanya ukubonakaliswa kokwahlula kwisethi yedatha yokubuyisela, olu hlalutyo luchonge iiprotheni ezinamazinga obuninzi atshintshiweyo kakhulu (P <0.05) phakathi kweemeko zolawulo kunye ne-AD (Itheyibhile S2A). Njengoko kuboniswe kwi-Figure 1A, ubuninzi beeprotheni ze-225 kwi-AD zancitshiswa kakhulu, kwaye ubuninzi beeprotheni ze-303 zanda kakhulu. Ezi proteni ezibonakaliswe ngokwahlukileyo ziquka amanqaku amaninzi asele echongiweyo kwi-cerebrospinal fluid AD, njenge-microtubule-associated protein tau (MAPT; P = 3.52 × 10-8), neurofilament (NEFL; P = 6.56 × 10-3), iProtein ehlobene nokukhula 43 (GAP43; P = 1.46 × 10-5), i-Fatty Acid Binding Protein 3 (FABP3; P = 2.00 × 10−5), Chitinase 3 efana ne-1 (CHI3L1; P = 4.44 × 10−6), Neural Granulin (NRGN; P = 3.43 × 10-4) kunye ne-VGF ye-nerve factor factor (VGF; P = 4.83 × 10-3) (4-6). Nangona kunjalo, siye safumanisa ezinye iithagethi ezibaluleke kakhulu, ezifana ne-GDP dissociation inhibitor 1 (GDI1; P = 1.54 × 10-10) kunye ne-SPARC enxulumene nemodyuli ye-calcium edibanisa i-1 (SMOC1; P = 6.93 × 10-9) . Uhlalutyo lwe-Gene Ontology (GO) lwe-225 oluncitshiswe kakhulu lweeprotheyini lubonakalise unxibelelwano olusondeleyo kunye neenkqubo zolwelo lomzimba ezifana ne-steroid metabolism, ukuhlangana kwegazi, kunye nokusebenza kwehomoni (Figure 1B kunye neTheyibhile S2B). Ngokwahlukileyo, iprotheni eyandiswe kakhulu ye-303 inxulumene ngokusondeleyo nokwakhiwa kweeseli kunye ne-metabolism yamandla.
(A) Iploti yentaba-mlilo ibonisa uguquko lwelog2 (x-axis) ngokunxulumene ne-log10 yexabiso le-P yeenkcukacha-manani (y-axis) efunyenwe luvavanyo lwe-t, olusetyenziselwa ukufumanisa intetho yokwahlula phakathi kolawulo (CT) kunye Iimeko ze-AD ze-CSF proteome yazo zonke iiproteni. Iiprotheyini ezinamazinga ancitshisiweyo kakhulu (P <0.05) kwi-AD ziboniswa nge-blue blue, ngelixa iiprotheyini ezinamazinga anyuka kakhulu kwizifo ziboniswa ngobomvu. Iprotheyini ekhethiweyo ibhaliwe. (B) Amagama aphezulu e-GO anxulumene neprotheyini ancitshiswa kakhulu (blue) kwaye anyuswe (bomvu) kwi-AD. Ibonisa amagama amathathu e-GO anawona manqaku aphezulu ngu-z kwiinkalo zenkqubo yebhayoloji, imisebenzi yeemolekyuli, kunye namalungu eselula. (C) I-MS ilinganisa inqanaba le-MAPT kwisampuli ye-CSF (ekhohlo) kunye nokulungelelaniswa kwayo kunye nesampula ye-ELISA inqanaba le-tau (ekunene). I-Pearson yolungelelwaniso i-coefficient enexabiso le-P elifanelekileyo iyaboniswa. Ngenxa yokunqongophala kwedatha ye-ELISA kwimeko enye ye-AD, la manani abandakanya amaxabiso angama-38 kumatyala angama-39 ahlalutyiweyo. (D) Uhlalutyo lweqela eligadiweyo (P <0.0001, uBenjamini-Hochberg (BH) ulungelelanise i-P <0.01) kulawulo kunye ne-AD CSF ifumene iisampulu zisebenzisa i-65 eyona nto ishintshile iiprotheni kwisethi yedatha. Yenza umgangatho, ulungelelanise.
Inqanaba leproteomic le-MAPT lihambelana ngokusondeleyo kwinqanaba le-ELISA tau elilinganiswe ngokuzimeleyo (r = 0.78, P = 7.8 × 10-9; Umzobo 1C), ukuxhasa ukuqinisekiswa komlinganiselo wethu we-MS. Emva kokugaya i-trypsin kwinqanaba le-amyloid precursor protein (APP), i-peptide ye-isoform-specific imephu kwi-C-terminus ye-Aβ1-40 kunye ne-Aβ1-42 ayikwazi ukuba i-ionized ngokufanelekileyo (27, 28). Ke ngoko, iipeptide ze-APP esizichongileyo azinanto yakwenza namanqanaba e-ELISA Aβ1-42. Ukuze sivavanye ukubonakaliswa kokwahlula kwimeko nganye, sasebenzisa iiprotheyini ezibonakaliswe ngokwahlukileyo kunye ne-P <0.0001 [izinga lokufumanisa ubuxoki (FDR) lilungiswe i-P <0.01] ukwenza uhlalutyo lweqela eligadiweyo leesampuli (Itheyibhile S2A). Njengoko kubonisiwe kwi-Figure 1D, ezi proteni ze-65 ezibaluleke kakhulu zinokudibanisa ngokuchanekileyo iisampulu ngokwemeko yesifo, ngaphandle kwemeko enye ye-AD eneempawu ezifana nolawulo. Kwezi proteni ze-65, i-63 yonyuka kwi-AD, ngelixa ezimbini kuphela (i-CD74 kunye ne-ISLR) zehla. Lilonke, olu hlalutyo lwe-cerebrospinal fluid ichonge amakhulu eeprotheyini kwi-AD enokuthi isebenze njengeempawu zesifo.
Emva koko senze uhlalutyo oluzimeleyo lwenethiwekhi ye-AD brain proteome. I-brain cohort yolu kufumanisa yayiquka i-dorsolateral prefrontal cortex (DLPFC) ukusuka kulawulo (n = 10), isifo sika-Parkinson (PD; n = 10), i-AD / PD exutywe (n = 10) kunye ne-AD (n = 10) iimeko. ) Isampulu. Emery Goizueta ADRC. Idemographics yala matyala angama-40 achazwe ngaphambili (25) kwaye ashwankathelwa kwiTheyibhile S1B. Sasebenzisa i-TMT-MS ukuhlalutya ezi zicubu zobuchopho ze-40 kunye neqela lokuphindaphinda iimeko ze-27. Lilonke, ezi datha zimbini zedatha yengqondo zivelise i-227,121 peptides ekhethekileyo, efakwe kwi-12,943 proteoms (25). Kuphela ezo proteins eziye zabalwa ubuncinane kwi-50% yamatyala afakiwe kuphando olulandelayo. Isethi yokugqibela yedatha yokufumanisa iqulethe i-8817 yeeprotheni ezilinganisiweyo. Lungisa amanqanaba obuninzi beprotheyini ngokusekelwe kwiminyaka yobudala, isini, kunye nesithuba sokufa emva kokufa (PMI). Uhlalutyo lwenkcazo eyahlukileyo yedatha ebekiweyo emva kokunciphisa ibonise ukuba> amanqanaba eprotheyini ye-2000 atshintshiwe kakhulu [P <0.05, uhlalutyo lokuhluka (ANOVA)] kwizibini ezimbini okanye ngaphezulu kwezifo. Emva koko, senze uhlalutyo lwe-cluster egadiweyo esekelwe kwiiprotheyini ezichazwe ngokwahlukileyo, kunye ne-P <0.0001 kwi-AD / ulawulo kunye / okanye i-AD / PD uthelekiso (Figure S2, A kunye ne-B, i-Table S2C). Ezi ziprotheni ze-165 eziguqulwe kakhulu zibonisa ngokucacileyo iimeko ezine-AD pathology ukusuka kulawulo kunye neesampuli zePD, eziqinisekisa utshintsho oluqinileyo lwe-AD kwiproteome yonke.
Emva koko sasebenzisa i-algorithm ebizwa ngokuba yi-Weighted Gene Co-expression Network Analysis (WGCNA) ukwenza uhlalutyo lwenethiwekhi kwiproteome yobuchopho efunyenweyo, eququzelela idatha ebekwe kwiimodyuli zeprotheni ezineepateni zokubonisa ezifanayo (11-13). Uhlalutyo oluchongiwe iimodyuli ze-44 (M) ezibonakaliswe ngokubambisana, zihlelwe kwaye zibalwe ukusuka kubukhulu (M1, n = 1821 iiprotheni) ukuya kumncinci (M44, n = 34 iiprotheyini) (Umfanekiso 2A kunye neTable S2D) ). Njengoko kukhankanyiwe ngasentla (13) Bala iprofayili yokubonisa ummeli okanye iprotheni yeempawu zemodyuli nganye, kwaye udibanise nesimo sesifo kunye ne-AD pathology, oko kukuthi, ukuseka umanyano lwe-Alzheimer's Registry Registry (CERAD) kunye ne-Braak Score (Figure 2B). Ngokubanzi, iimodyuli ze-17 zazinxulumene kakhulu ne-AD neuropathology (P <0.05). Uninzi lwezi modyuli ezinxulumene nesifo nazo zizityebi kwiimpawu ezikhethekileyo zeseli (Umfanekiso 2B). Njengoko kukhankanyiwe ngasentla (13), ukutyetyiswa kohlobo lweseli kumiselwa ngokuhlalutya ukugqithelana kwemodyuli kunye noluhlu lwereferensi yohlobo oluthile lweseli. Ezi zofuzo zithathwe kwidatha epapashiweyo kwii-neurons zemouse ezizimeleyo, iiseli ze-endothelial kunye ne-glial. Ulandelelwano lwe-RNA (RNA-seq) ulingelo (29).
(A) Fumanisa iWGCNA yeproteome yobuchopho. (B) Uhlalutyo lwe-Biweight midcorrelation (BiCor) lweprotheyini yesignesha yemodyuli (inxalenye yokuqala enkulu ye-modular protein expression) kunye neempawu ze-AD neuropathological (phezulu), kuquka i-CERAD (i-Aβ plaque) kunye ne-Braak (tau tangles) amanqaku. Ukuqina kokulungelelaniswa okuhle (obomvu) kunye nokubi (okuluhlaza okwesibhakabhaka) kuboniswa yimephu yokushisa enemibala emibini, kunye neenkwenkwezi zibonisa ukubaluleka kwamanani (P <0.05). Sebenzisa iHypergeometric Fisher's Exact Test (FET) (ezantsi) ukuvavanya unxulumano lohlobo lweeseli kwimodyuli nganye yeproteni. Ubunzima be-shading ebomvu bubonisa iqondo lokutyebisa uhlobo lweseli, kwaye i-asterisk ibonisa ukubaluleka kwamanani (P <0.05). Sebenzisa indlela ye-BH ukulungisa ixabiso le-P elivela kwi-FET. (C) Uhlalutyo lwe-GO lweeprotheni zemodyuli. Ezona nkqubo zebhayoloji ezisondeleleneyo ziboniswa kwimodyuli nganye okanye iqela lemodyuli enxulumeneyo. oligo, oligodendrocyte.
Isethi yeemodyuli ezintlanu ezinxulumene ngokusondeleyo ne-astrocyte kunye ne-microglia-rich (M30, M29, M18, M24, kunye ne-M5) ibonise ulungelelwaniso oluqinileyo olulungileyo kunye ne-AD neuropathology (Umfanekiso 2B). Uhlalutyo lwe-Ontology ludibanisa ezi modyuli zeglial kunye nokukhula kweeseli, ukwanda, kunye nokukhuseleka (Figure 2C kunye neThebhile S2E). Iimodyuli ezimbini ezongezelelweyo ze-glial, i-M8 kunye ne-M22, nazo zilawulwa ngamandla kwizifo. I-M8 inxulumene kakhulu ne-Toll-like receptor pathway, i-cascade yomqondiso edlala indima ephambili kwi-innate immune response (30). Ngelo xesha, i-M22 inxulumene ngokusondeleyo nokuguqulwa kwe-post-translation. I-M2, etyebileyo kwi-oligodendrocytes, ibonisa ukulungelelaniswa okuqinisekileyo okuqinileyo kunye ne-AD pathology kunye noqhagamshelwano lwe-ontological kunye ne-nucleoside synthesis kunye nokuphindaphinda kwe-DNA, ebonisa ukwanda kweeseli eziphuculweyo kwizifo. Ngokubanzi, ezi ziphumo zixhasa ukuphakama kweemodyuli ze-glial esizibonile ngaphambili kwi-proteome yenethiwekhi ye-AD (13, 17). Okwangoku kufunyenwe ukuba ezininzi iimodyuli ze-glial ezinxulumene ne-AD kwinethiwekhi zibonisa amanqanaba aphantsi okubonakalisa ekulawuleni kunye namatyala e-PD, ebonisa ukucaciswa kwesifo sabo esiphakamileyo kwi-AD (Figure S2C).
Iimodyuli ezine kuphela kwi-proteome yethu yothungelwano (M1, M3, M10, kunye ne-M32) zihambelana kakhulu kakubi ne-AD pathology (P <0.05) (Umfanekiso 2, B kunye no-C). Zombini i-M1 kunye ne-M3 zizityebi kwiimpawu ze-neuronal. I-M1 inxulumene kakhulu neempawu ze-synaptic, ngelixa i-M3 inxulumene ngokusondeleyo nomsebenzi we-mitochondrial. Akukho bungqina bokutyetyiswa kohlobo lweeseli ze-M10 kunye ne-M32. I-M32 ibonisa uxhulumaniso phakathi kwe-M3 kunye ne-cell metabolism, ngelixa i-M10 ihambelana kakhulu nokukhula kweseli kunye nomsebenzi we-microtubule. Xa kuthelekiswa ne-AD, zonke iimodyuli ezine zonyuswa kulawulo kunye nePD, zibanika utshintsho oluthile lwe-AD lwesifo (Umfanekiso we-S2C). Ngokubanzi, ezi ziphumo zixhasa ukuhla kobuninzi beemodyuli ezizityebi ze-neuron esizibonile ngaphambili kwi-AD (13, 17). Isishwankathelo, uhlalutyo lwenethiwekhi yeproteome yobuchopho esiyifumene yavelisa iimodyuli eziguqulwe ngokuthe ngqo ze-AD ezihambelana neziphumo zethu zangaphambili.
I-AD ibonakaliswe ngesigaba sokuqala se-asymptomatic (AsymAD), apho abantu babonisa ukuqokelela kwe-amyloid ngaphandle kokuncipha kwengqondo yeklinikhi (5, 31). Eli nqanaba le-asymptomatic limele ifestile ebalulekileyo yokufunyanwa kwangaphambili kunye nokungenelela. Ngaphambili siye sabonisa ukugcinwa kwemodyuli eyomeleleyo ye-AsymAD kunye ne-AD ye-proteome yenethiwekhi yobuchopho kwiiseti zedatha ezizimeleyo (13, 17). Ukuze siqinisekise ukuba inethiwekhi yobuchopho esiyifumene ngoku iyahambelana nezi ziphumo zangaphambili, sihlalutye ukugcinwa kweemodyuli ze-44 kwisethi yedatha ephindaphindiweyo evela kwimibutho ye-27 DLPFC. Le mibutho ibandakanya ulawulo (n = 10), i-AsymAD (n = 8) kunye ne-AD (n = 9) iimeko. Ulawulo kunye neesampuli ze-AD zibandakanyiwe kuhlalutyo lweqela lethu lobuchopho (Itheyibhile S1B), ngelixa iimeko ze-AsymAD zazikhethekileyo kuphela kwiqela lokuphindaphinda. Ezi meko ze-AsymAD nazo zavela kwi-Emory Goizueta ADRC bank bank. Nangona ukuqonda kwakuqhelekileyo ngexesha lokufa, amanqanaba e-amyloid ayephezulu ngokungaqhelekanga (ithetha i-CERAD, i-2.8 ± 0.5) (i-Table S1B).
Uhlalutyo lwe-TMT-MS kwezi zicubu zengqondo ze-27 zibangele ubungakanani be-proteoms ye-11,244. Olu balo lokugqibela lubandakanya kuphela ezo proteni ezibalwe ubuncinci kwi-50% yeesampulu. Le seti yedatha ephindwe kabini iqulethe i-8638 (98.0%) yeeprotheni ze-8817 ezifunyenwe kuhlalutyo lwethu lobuchopho, kwaye phantse i-3000 yatshintsha kakhulu iiprotheyini phakathi kolawulo kunye ne-AD cohorts (P <0.05, emva kovavanyo lwe-Tukey oludibeneyo t ukuhlalutya ukuhluka) ( Uluhlu lwe-S2F). Phakathi kwezi proteni ezibonakaliswe ngokwahlukileyo, i-910 iphinde yabonisa utshintsho olubalulekileyo phakathi kwe-AD kunye neemeko zokulawula iproteome yengqondo (P <0.05, emva kwe-ANOVA Tukey edibeneyo yovavanyo lwe-t-test). Kuyafaneleka ukuba uqaphele ukuba aba bamakishi be-910 bahambelana kakhulu kwicala lokutshintsha phakathi kweproteomes (r = 0.94, P <1.0 × 10-200) (Figure S3A). Phakathi kweeprotheyini ezandisiweyo, iiprotheyini ezinotshintsho oluhambelana kakhulu phakathi kweeseti zedatha ikakhulu ngamalungu e-glial-rich M5 kunye ne-M18 modules (MDK, COL25A1, MAPT, NTN1, SMOC1, kunye ne-GFAP). Phakathi kweeprotheni ezincitshisiweyo, abo banotshintsho oluhambelanayo phantse babe ngamalungu kuphela emodyuli ye-M1 (NPTX2, VGF, kunye ne-RPH3A) ehambelana ne-synapse. Siye saqinisekisa ngakumbi utshintsho olunxulumene ne-AD ye-midkine (MDK), i-CD44, i-protein enxulumene ne-frizzled 1 (SFRP1) kunye ne-VGF nge-western blotting (Figure S3B). Uhlalutyo lokugcinwa kwemodyuli lubonise ukuba malunga ne-80% yeemodyuli zeprotheni (34/44) kwiproteome yengqondo zigcinwe kakhulu kwiseti yedatha yokuphindaphinda (z-score> 1.96, i-FDR ilungiswe i-P <0.05) (Umfanekiso we-S3C). Ishumi elinesine kwezi modyuli zigcinwe ngokukodwa phakathi kweeproteomes ezimbini (z-score> 10, i-FDR ilungisiwe P <1.0 × 10-23). Ngokubanzi, ukufunyanwa kunye nokuphindaphinda iqondo eliphezulu lokuhambelana kwintetho yokwahlula kunye nokwakheka kweemodyuli phakathi kweproteome yobuchopho kuqaqambisa ukuveliswa kotshintsho kwiiproteni ze-AD ze-cortex yangaphambili. Ukongeza, iphinde yaqinisekisa ukuba i-AsymAD kunye nezifo eziphambili kakhulu zinesakhiwo senethiwekhi yengqondo efanayo.
Uhlalutyo oluthe kratya lwenkcazo eyahlukileyo kwiseti yedatha yokuphindaphinda ingqondo igxininisa iqondo elibalulekileyo lotshintsho lweprotheyini ye-AsymAD, kubandakanywa inani elipheleleyo le-151 elitshintshileyo kakhulu kwiiprotheni phakathi kwe-AsymAD kunye nolawulo (P <0.05) (Umfanekiso we-S3D). Ngokuhambelana nomthwalo we-amyloid, i-APP kwingqondo ye-AsymAD kunye ne-AD yanda kakhulu. I-MAPT itshintsha kuphela ngokuphawulekayo kwi-AD, ehambelana namanqanaba okwanda kwe-tangles kunye nokulungelelaniswa kwayo okwaziwayo kunye nokuncipha kwengqondo (5, 7). Iimodyuli ezityebileyo ze-glial (i-M5 kunye ne-M18) zibonakaliswe kakhulu kwiiprotheni ezandisiweyo kwi-AsymAD, ngelixa imodyuli ye-M1 enxulumene ne-neuron iyona nto imele iiprotheni ezinciphileyo kwi-AsymAD. Uninzi lwala maphawu e-AsymAD abonisa utshintsho olukhulu kwizifo ezineempawu. Phakathi kwezi ziphawuli yi-SMOC1, iprotheni ye-glial ye-M18, ehambelana nezicubu zengqondo kunye nokuphuhliswa kwamehlo kunye nemilenze (32). I-MDK yinto yokukhula kwe-heparin-binding ehambelana nokukhula kweeseli kunye ne-angiogenesis (33), elinye ilungu le-M18. Xa kuthelekiswa neqela lolawulo, i-AsymAD yanda kakhulu, ilandelwa kukunyuka okukhulu kwe-AD. Ngokwahlukileyo, i-synaptic protein neuropentraxin 2 (NPTX2) yancitshiswa kakhulu kwingqondo ye-AsymAD. I-NPTX2 yayikade inxulunyaniswa ne-neurodegeneration kwaye inendima eyaziwayo ekulawulweni kwe-synapses evuselelayo (34). Ngokubanzi, ezi ziphumo zibonakalisa iindidi ezahlukeneyo zeeprotheyini eziguqukayo kwi-AD ezibonakala ziqhubela phambili ngobunzima besi sifo.
Ngenxa yokuba sizuze ubunzulu obubalulekileyo bokugubungela iprotheyini ekubhaqweni kweproteome yobuchopho, sizama ukuqonda ngakumbi ukugqithelana kwayo kunye ne-network-level AD transcriptome. Ke ngoko, sithelekisa iproteome yobuchopho esiyifumene kunye nemodyuli esiye sayenza ngaphambili kwi-microarray yokulinganisa i-18,204 genes kwi-AD (n = 308) kunye nolawulo (n = 157) izicubu ze-DLPFC (13). ukugqithelana. Iyonke, sichonge iimodyuli ze-RNA ezahlukeneyo ze-20, ezininzi zazo zibonise ukutyetyiswa kweentlobo ezithile zeeseli, ezibandakanya i-neurons, i-oligodendrocytes, i-astrocytes, kunye ne-microglia (Umfanekiso 3A). Utshintsho oluninzi lwezi modyuli kwi-AD luboniswe kuMfanekiso 3B. Ngokuhambelana neprotheyini yethu yangaphambili-i-RNA yohlalutyo olugqithisiweyo usebenzisa i-MS proteome enzulu engabhalwanga (malunga neeprotheyini ze-3000) (13), uninzi lweemodyuli ze-44 kwinethiwekhi yeproteome yobuchopho esiyifumene kwinethiwekhi ye-transcriptome Akukho kudlula okubalulekileyo. ukufumanisa kwethu kunye nokuphindaphinda kweemodyuli ze-34 zeprotheyini ezigcinwe kakhulu kwiproteome yobuchopho, kuphela i-14 (~40%) ephumelele uvavanyo oluchanekileyo lukaFisher (FET) lubonakalise ukudityaniswa okubalulekileyo kwezibalo kunye ne-transcriptome (Figure 3A). Iyahambelana nokulungiswa komonakalo we-DNA (P-M25 kunye ne-P-M19), ukuguqulelwa kweprotheyini (P-M7 kunye ne-P-M20), i-RNA idibanisa / i-splicing (P-M16 kunye ne-P-M21) kunye nokujoliswa kweprotheni (P-M13 kunye ne-P- M23) ayidibani neemodyuli kwi-transcriptome. Ngoko ke, nangona isethi yedatha yeproteome enzulu isetyenziselwa uhlalutyo lwangoku lwangoku (13), ininzi ye-proteome yenethiwekhi ye-AD ayinamaphu kwi-network transcriptome.
(A) I-Hypergeometric FET ibonisa ukutyetyiswa kweziphawuli zodidi oluthile lweseli kwimodyuli ye-RNA ye-AD transcriptome (phezulu) kunye neqondo lokuxinana phakathi kwe-RNA (x-axis) kunye neemodyuli zeprotheyini (y-axis) zobuchopho beAD. (ezantsi). Ubuninzi be-shading ebomvu bubonisa iqondo lokutyebisa iintlobo zeseli kwipaneli ephezulu kunye nokuqina kokugqithiswa kweemodyuli kwipaneli engezantsi. Iinkwenkwezi zibonisa ukubaluleka kwamanani (P <0.05). (B) Iqondo lonxulumano phakathi kweempawu zofuzo zemodyuli nganye ye-transcriptome kunye nobume be-AD. Iimodyuli ezisekhohlo zezona zihambelana kakubi kunye ne-AD (eluhlaza okwesibhakabhaka), kwaye ezo zisekunene zezona zihambelana ne-AD (ebomvu). I-log-transformed BH-corrected value P ibonisa iqondo lokubaluleka kwamanani olungelelwaniso ngalunye. (C) Iimodyuli ezidityanisiweyo ezibalulekileyo ezinohlobo lweeseli ekwabelwana ngazo. (D) Uhlalutyo oluhambelanayo lwenguqu ye-log2 yeprotheyini ebhaliweyo (x-axis) kunye ne-RNA (y-axis) kwimodyuli edibeneyo. I-Pearson yolungelelwaniso i-coefficient enexabiso le-P elifanelekileyo iyaboniswa. Micro, microglia; imizimba yasezulwini, ii-astrocytes. CT, ulawulo.
Uninzi lweemodyuli zeprotheyini kunye neemodyuli ze-RNA zabelana ngeeprofayili zohlobo lweeseli ezifanayo kunye nezikhokelo zokutshintsha kwe-AD (Umfanekiso 3, B kunye noC). Ngamanye amazwi, imodyuli ye-M1 enxulumene ne-synapse yeproteome yobuchopho (PM1) izotywe kwiimodyuli ezintathu ze-RNA ezine-neuronal-rich homologous (R-M1, R-M9 kunye ne-R-M16), ezikwi-AD Zombini zibonisiwe. inqanaba elincitshisiweyo. Ngokufanayo, i-glial-rich M5 kunye ne-M18 iimodyuli zeprotheyini zidibanisa kunye neemodyuli ze-RNA ezityebileyo kwi-astrocytes kunye ne-microglial markers (R-M3, R-M7, kunye ne-R-M10) kwaye zibandakanyeka kakhulu kwizifo Ukunyuka. Ezi mpawu zemodyuli ekwabelwana ngazo phakathi kweeseti zedatha ezimbini zixhasa ngakumbi uhlobo lweseli lokutyebisa kunye notshintsho olunxulumene nesifo esilubonileyo kwiproteome yobuchopho. Nangona kunjalo, siye sabona iiyantlukwano ezininzi ezibalulekileyo phakathi kwe-RNA kunye namanqanaba eprotheyini yabamakishi ngabanye kwezi modyuli ekwabelwana ngazo. Uhlalutyo oluhambelanayo lwentetho ehlukeneyo yeproteomics kunye ne-transcriptomics yeemolekyuli ngaphakathi kwezi modyuli ezidibeneyo (Umfanekiso we-3D) ugxininisa oku kungahambelani. Ngokomzekelo, i-APP kunye nezinye iiprotheni zemodyuli ye-glial (NTN1, MDK, COL25A1, ICAM1, kunye ne-SFRP1) zibonise ukwanda okukhulu kwe-AD proteome, kodwa akukho phantse utshintsho kwi-AD transcriptome. Olu tshintsho oluthile lweprotheyini lunokuthi luhambelane ngokusondeleyo kwiiplagi ze-amyloid (23, 35), ezigqamisa iproteome njengomthombo wotshintsho lwe-pathological, kwaye olu tshintsho lungenakubonakaliswa kwi-transcriptome.
Emva kokuhlalutya ngokuzimeleyo ingqondo kunye neeproteomes ze-CSF esizifumeneyo, senze uhlalutyo olubanzi lweeseti zedatha ezimbini ukuchonga iimpawu zebhayoloji ze-AD CSF ezinxulumene ne-pathophysiology yothungelwano lobuchopho. Kufuneka siqale sichaze ukudityaniswa kweeproteome ezimbini. Nangona yamkelwa ngokubanzi ukuba i-CSF ibonisa utshintsho lwe-neurochemical kwingqondo ye-AD (4), iqondo elichanekileyo lokudlulana phakathi kwengqondo ye-AD kunye ne-CSF proteome ayicacanga. Ngokuthelekisa inani leemveliso zofuzo ezabelwana ngazo ezifunyenwe kwiiproteome zethu ezimbini, sifumene ukuba phantse i-70% (n = 1936) yeeprotheyini ezichongiweyo kwi-cerebrospinal fluid nazo zilinganiswe kwingqondo (Figure 4A). Uninzi lwezi proteni ezidlulanayo (n = 1721) zifakwe kwimephu enye yeemodyuli ze-44 zokubonisa ngokubambisana ukusuka kwiseti yedatha yengqondo (Figure 4B). Njengoko bekulindelekile, iimodyuli ezintandathu ezinkulu zobuchopho (M1 ukuya kwi-M6) zibonise isixa esikhulu se-CSF esidlulayo. Nangona kunjalo, kukho iimodyuli ezincinci zobuchopho (umzekelo, i-M15 kunye ne-M29) ezifikelela kwinqanaba eliphezulu elingalindelekanga lokugqithana, elikhulu kunemodyuli yengqondo kabini ubukhulu bayo. Oku kusikhuthaza ukuba samkele indlela eneenkcukacha ngakumbi, eqhutywa ngokwezibalo ukubala ukuhlangana phakathi kwengqondo kunye nolwelo lwe-cerebrospinal.
(A kunye ne-B) Iiprotheyini ezichongiweyo kwingqondo yokufumanisa kunye neeseti zedatha ye-CSF ziyadlulana. Uninzi lwezi proteni ezidlulayo zihambelana nenye yeemodyuli ze-44 zokubonisa ngokubambisana kwengqondo. (C) Fumanisa ukudibana phakathi kwe-cerebrospinal fluid proteome kunye neproteome yenethiwekhi yobuchopho. Umqolo ngamnye wemephu yobushushu umele ucazululo olwahlukileyo lwe-FET ye-hypergeometric. Umqolo ophezulu ubonisa ukugqithela (grey / black shading) phakathi kwemodyuli yobuchopho kunye neproteome yonke ye-CSF. Umgca wesibini ubonisa ukuba ukugqithwa phakathi kweemodyuli zobuchopho kunye neprotheni ye-CSF (i-shaded ebomvu) ilawulwa kakhulu kwi-AD (P <0.05). Umqolo wesithathu ubonisa ukuba ukugqithwa phakathi kweemodyuli zobuchopho kunye neprotheni ye-CSF (i-blue shading) iphantsi kakhulu-ilawulwa kwi-AD (P <0.05). Sebenzisa indlela ye-BH ukulungisa ixabiso le-P elivela kwi-FET. (D) Iphaneli yemodyuli esongwayo esekwe kunxulumano lohlobo lweseli kunye nemigaqo yeGO enxulumeneyo. Ezi ziphaneli ziqulethe iiprotheni ze-271 ezinxulumene nengqondo, ezinentsingiselo ecacileyo eyahlukileyo kwiproteome ye-CSF.
Sisebenzisa ii-FET ezinomsila omnye, siye savavanya ukubaluleka kokudibana kweprotheyini phakathi kwe-CSF proteome kunye neemodyuli zobuchopho bomntu ngamnye. Uhlalutyo lubonise ukuba i-modyuli ye-14 yobuchopho kwiseti yedatha ye-CSF ine-statistically overlaps (i-FDR ilungisiwe P <0.05), kunye nemodyuli eyongezelelweyo (M18) i-overlap yayo isondele nokubaluleka (i-FDR ihlengahlengiswe P = 0.06) (Umfanekiso 4C , umqolo ophezulu). Sikwanomdla kwiimodyuli ezinxibelelana ngamandla neeproteni ze-CSF ezichazwe ngokwahlukileyo. Ke ngoko, siye sasebenzisa ucazululo olongezelelweyo lweFET ezimbini ukufumanisa ukuba yeyiphi (i) iprotein ye-CSF eyonyuswe kakhulu kwi-AD kunye (ii) neprotein ye-CSF yehliswe kakhulu kwi-AD (P <0.05, idityanisiwe t uvavanyo lwe-AD/control) iimodyuli zobuchopho ezinokudibana okunentsingiselo. phakathi kwabo. Njengoko kuboniswe kwimiqolo ephakathi kunye nezantsi yoMzobo we-4C, olu hlalutyo olongezelelweyo lubonisa ukuba i-8 yeemodyuli ze-44 zengqondo zidibanisa kakhulu kunye neprotheni eyongeziweyo kwi-AD CSF (M12, M1, M2, M18, M5, M44, M33, kunye ne-M38) . ), ngelixa iimodyuli ezimbini kuphela (i-M6 kunye ne-M15) zibonise ukugqithiswa okunentsingiselo kunye neprotheni encitshisiweyo kwi-AD CSF. Njengoko kulindelekile, zonke iimodyuli ezili-10 zikwimodyuli ezili-15 ezinowona mgangatho uphezulu we-CSF proteome. Ke ngoko, sicinga ukuba ezi modyuli ze-15 ziyimithombo yemveliso ephezulu ye-AD ye-biomarkers ye-CSF ephuma ebuchotsheni.
Sisonge ezi modyuli zidibanayo zili-15 zaba ziipaneli zeprotheyini ezinkulu ezintlanu ngokusekwe kukusondela kwazo kumzobo womthi we-WGCNA kunye nokunxulumana kwazo neentlobo zeeseli kunye ne-ontology yemfuza (Figure 4D). Iphaneli yokuqala iqulethe iimodyuli ezizityebi kwiimpawu ze-neuron kunye neeprotheni ezinxulumene ne-synapse (M1 kunye ne-M12). Iphaneli ye-synaptic iqulethe i-protein ye-94 iyonke, kwaye amanqanaba kwi-proteome ye-CSF atshintshile kakhulu, okwenza kube ngumthombo omkhulu weempawu ze-CSF ezinxulumene nengqondo phakathi kweepaneli ezintlanu. Iqela lesibini (i-M6 kunye ne-M15) libonise uxhulumaniso olusondeleyo kunye nabamakishi beeseli ze-endothelial kunye nomzimba we-vascular, njengokuthi "ukuphulukiswa kwesilonda" (M6) kunye "nokulawulwa kwe-humoral immune response" (M15). I-M15 nayo inxulumene kakhulu ne-lipoprotein metabolism, ehambelana ngokusondeleyo ne-endothelium (36). Iphaneli ye-vascular ineempawu ze-34 CSF ezinxulumene nengqondo. Iqela lesithathu libandakanya iimodyuli (i-M2 kunye ne-M4) ehambelana kakhulu nabamakishi be-oligodendrocyte kunye nokwanda kweeseli. Umzekelo, amagama akumgangatho ophezulu we-ontology we-M2 abandakanya "ulawulo olulungileyo lokuphindaphinda kwe-DNA" kunye "nenkqubo ye-purine biosynthesis". Okwangoku, ezo ze-M4 ziquka "ukuhlukana kweeseli ze-glial" kunye "nokwahlukana kwe-chromosome". Iphaneli ye-myelination iqulethe iimpawu ze-CSF ze-49 ezinxulumene nengqondo.
Iqela lesine liqulethe iimodyuli ezininzi (M30, M29, M18, M24, kunye ne-M5), kwaye phantse zonke iimodyuli zizityebi kakhulu kwi-microglia kunye ne-astrocyte markers. Ngokufana nephaneli ye-myelination, iphaneli yesine nayo iqulethe iimodyuli (M30, M29, kunye ne-M18) ehambelana ngokusondeleyo nokwanda kweeseli. Ezinye iimodyuli ezikweli qela zihambelana kakhulu nemigaqo ye-immunological, efana "nenkqubo yempembelelo ye-immune" (M5) kunye "nokulawulwa kwempendulo ye-immune" (M24). Iqela elikhuselayo le-glial liqulethe iimpawu ze-CSF ze-42 ezinxulumene nengqondo. Ekugqibeleni, iphaneli yokugqibela ibandakanya iimpawu ze-52 ezinxulumene nengqondo kwiimodyuli ezine (M44, M3, M33, kunye ne-M38), zonke ezikumzimba ohambelana nokugcinwa kwamandla kunye ne-metabolism. Ezona modyuli zinkulu (M3) zinxulumene ngokusondeleyo ne-mitochondria kwaye zizityebi kwiimpawu ezithile ze-neuron. I-M38 lelinye lamalungu emodyuli amancinci kule metabolome kwaye ikwabonisa ubume be-neuron ephakathi.
Ngokubanzi, ezi panels zintlanu zibonisa uluhlu olubanzi lweentlobo zeeseli kunye nemisebenzi kwi-cortex ye-AD, kwaye ngokudibeneyo iqulethe i-271 enxulumene nengqondo ye-CSF markers (Itheyibhile S2G). Ukuze sivavanye ubunyani bezi ziphumo ze-MS, sisebenzise i-proximity extension assay (PEA), itekhnoloji esekwe kwi-orthogonal antibody enekhono lokuphindaphinda, ubuntununtunu obuphezulu kunye neenkcukacha ezithile, kwaye siphinde sahlalutya iisampulu ze-cerebrospinal fluid safumana i-subset yezi zimpawu ze-biomarker ezingama-271. (n = 36). Ezi thagethi ze-36 zibonisa utshintsho kwi-AD ye-multiple ye-PEA, ehambelana ngokusondeleyo neziphumo zethu ezisekelwe kwi-MS (r = 0.87, P = 5.6 × 10-12), Eye yaqinisekisa ngokuqinisekileyo iziphumo zohlalutyo lwethu olubanzi lwe-MS (Figure S4 ).
Imixholo yezinto eziphilayo ezigxininiswe ngamaqela ethu amahlanu, ukusuka kwi-synaptic signaling ukuya kwi-metabolism yamandla, zonke zihambelana ne-pathogenesis ye-AD (1-3). Ngoko ke, zonke iimodyuli ze-15 eziqulethe ezi panels zihambelana ne-AD pathology kwiproteome yengqondo esiyifumene (Umfanekiso 2B). Eyona nto iphawulekayo kukulungelelaniswa okuphezulu kwe-pathological phakathi kweemodyuli zethu ze-glial kunye ne-negative pathological correlation phakathi kweemodyuli zethu ezinkulu ze-neuronal (M1 kunye ne-M3). Uhlalutyo lwentetho ehlukeneyo yeproteome yethu yobuchopho ephindaphindwayo (Umfanekiso we-S3D) uphinda ubonise i-M5 kunye ne-M18-derived glial proteins. Kwi-AsymAD kunye ne-AD ye-symptomatic, i-protein ye-glial eyandisiweyo kakhulu kunye ne-M1 enxulumene ne-synapses Iprotheni iyancitshiswa kakhulu. Olu qwalaselo lubonisa ukuba iimpawu ze-271 ze-cerebrospinal fluid esizichongileyo kumaqela amahlanu zihambelana neenkqubo zesifo kwi-AD cortex, kubandakanywa nezo zenzeka kwizigaba zokuqala ze-asymptomatic.
Ukuze uhlalutye ngcono indlela yokutshintsha kweeprotheyini zephaneli kwingqondo kunye nolwelo lomgogodla, sizobe oku kulandelayo kwimodyuli nganye ye-15 ehambayo: (i) ifumene inqanaba lobuninzi bemodyuli kwiseti yedatha yengqondo kunye (ii) nemodyuli. iprotheni Umahluko ubonakaliswa kwi-cerebrospinal fluid (Figure S5). Njengoko bekutshiwo ngaphambili, i-WGCNA isetyenziselwa ukumisela ubuninzi bemodyuli okanye ixabiso leprotheyini ebuchotsheni (13). Imephu ye-volcano isetyenziselwa ukuchaza ukubonakaliswa okwahlukileyo kweeprotheni zemodyuli kwi-cerebrospinal fluid (AD / control). La manani abonisa ukuba ezintathu kwiiphaneli ezintlanu zibonisa iindlela ezahlukeneyo zokuthetha kwingqondo kunye nolwelo lomgogodla. Iimodyuli ezimbini zepaneli ye-synapse (i-M1 kunye ne-M12) ibonisa ukuhla kwinqanaba lobuninzi kwingqondo ye-AD, kodwa idibanisa kakhulu kunye neprotheni eyandisiweyo kwi-AD CSF (Figure S5A). Iimodyuli ezinxulumene ne-neuron eziqulethe i-metabolome (i-M3 kunye ne-M38) ibonise ubuchopho obufanayo kunye neepatheni zokubonisa i-cerebrospinal fluid ezingahambelaniyo (Figure S5E). Iphaneli ye-vascular nayo ibonise iindlela ezahlukeneyo zokubonisa, nangona iimodyuli zayo (i-M6 kunye ne-M15) zanda ngokulinganayo kwingqondo ye-AD kwaye zehla kwi-CSF enesifo (Figure S5B). Iipaneli ezimbini eziseleyo ziqulethe iinethiwekhi ezinkulu ze-glial ezineeprotheni ezihlala zilawulwa ngokuqhubekayo kumacandelo omabini (Umfanekiso S5, C kunye no-D).
Nceda uqaphele ukuba ezi ntsingiselo azixhaphakanga kubo bonke abamakishi kwezi panel. Ngokomzekelo, iphaneli ye-synaptic ibandakanya iiprotheni ezininzi ezincitshiswe kakhulu kwi-AD brain kunye ne-CSF (Figure S5A). Phakathi kwezi ziphawuli ze-cerebrospinal fluid eziphantsi zilawulwa yi-NPTX2 kunye ne-VGF ye-M1, kunye ne-chromogranin B ye-M12. Nangona kunjalo, ngaphandle kwezi ngaphandle, uninzi lweempawu zethu ze-synaptic ziphakanyiswe kwi-AD spinal fluid. Ngokubanzi, olu hlalutyo lukwazile ukwahlula iindlela ezibalulekileyo zezibalo kwingqondo kunye namanqanaba e-cerebrospinal fluid kwiphaneli yethu nganye ezintlanu. Ezi ndlela ziqaqambisa ubudlelwane obuntsonkothileyo kwaye buhlala obahlukileyo phakathi kwengqondo kunye ne-CSF yeprotheyini yokubonakalisa kwi-AD.
Emva koko, sasebenzisa uhlalutyo oluphezulu lwe-MS lokuphindaphinda (i-CSF replication 1) ukunciphisa isethi yethu ye-271 yee-biomarkers kwiithagethi ezithembisayo kunye nokuveliswa kwakhona (Umfanekiso 5A). Ikopi ye-CSF ye-1 iqulethe iisampulu ze-96 zizonke ezivela ku-Emory Goizueta ADRC, kubandakanywa nolawulo, i-AsymAD, kunye neqela le-AD (Itheyibhile S1A). Ezi meko ze-AD zibonakaliswe ngokuncipha kwengqondo (i-MoCA ithetha, i-20.0 ± 3.8), kunye notshintsho kwii-biomarkers ze-AD eziqinisekisiweyo kwi-cerebrospinal fluid (I-Table S1A). Ngokuchaseneyo nohlalutyo lwe-CSF esilufumeneyo, olu phindo lwenziwa kusetyenziswa indlela ye-MS esebenzayo kunye nephezulu "ye-single-shot" (ngaphandle kolwahlulo olungaphandle kwe-line), kubandakanywa neprotocol yokulungiselela isampula eyenziwe lula ephelisa imfuno yokukhutshwa komzimba kwiisampuli zomntu ngamnye. . Endaweni yoko, "ijelo lokuphucula" elinye eliphelelwe ngumzimba lisetyenziselwa ukukhulisa umqondiso weeprotheyini ezincinci (37). Nangona inciphisa i-proteome epheleleyo, le ndlela yokudubula enye iyanciphisa kakhulu ixesha lomatshini kwaye inyuse inani leesampuli ezibhalwe nge-TMT ezinokuhlalutywa ngokusebenzayo (17, 38). Lilonke, uhlalutyo luchonge i-6,487 peptides, ebonisa i-proteom ye-1,183 kwiimeko ezingama-96. Njengoko kuhlalutyo lwe-CSF esilufumeneyo, kuphela ezo ziprotheni ezibalwe ubuncinane kwi-50% yeesampuli zifakwe kwizibalo ezilandelayo, kwaye idatha yabuyiselwa kwimiphumo yobudala kunye nesini. Oku kukhokelele kumlinganiselo wokugqibela weeproteome ezingama-792, ezingama-95% zazo nazo zachongwa kwiseti yedatha ye-CSF efunyenweyo.
(A) Iithagethi zeprotheyini ze-CSF ezinxulumene nobuchopho eziqinisekisiweyo kwiqela lokuqala le-CSF eliphindwe kabini kwaye lifakwe kwiqela lokugqibela (n = 60). (B ukuya ku-E) Amanqanaba ephaneli yebhayomarker (composite z-amanqaku) alinganiswa kumaqela amane ophindaphindo lweCSF. Iimvavanyo ezidityanisiweyo ze-t okanye i-ANOVA ene-post-correction ye-Tukey yayisetyenziselwa ukuvavanya ukubaluleka kwezibalo zokutshintsha kobuninzi kuhlalutyo ngalunye lokuphindaphinda. CT, ulawulo.
Kuba sinomdla ngakumbi ekuqinisekiseni iithagethi zethu ze-CSF ze-271 ezinxulumene nengqondo ngohlalutyo olubanzi, siya kunciphisa uviwo olungakumbi lwale proteome iphindaphindwayo kwaba bamakishi. Phakathi kwezi proteni ze-271, i-100 yafunyanwa kwi-CSF yokuphindaphinda 1. Umzobo we-S6A ubonisa ukubonakaliswa okungafaniyo kwezi mpawu ze-100 ezidibeneyo phakathi kokulawula kunye neesampuli zokuphindaphinda kwe-AD. I-Synaptic kunye ne-metabolite histones yandisa kakhulu kwi-AD, ngelixa iiprotheni ze-vascular zinciphisa kakhulu kwisifo. Uninzi lwabamakishi be-100 abadlulayo (n = 70) bagcina ulwalathiso olufanayo lwenguqu kwiiseti zedatha ezimbini (Figure S6B). Ezi ziphawuli ze-CSF ze-70 eziqinisekisiweyo ezinxulumene nengqondo (Itheyibhile S2H) zibonisa ubukhulu becala iindlela zokubonisa iipaneli zangaphambili, oko kukuthi, ukulawulwa kweeprotheyini zemithambo kunye nokulawulwa okuphezulu kwazo zonke ezinye iipaneli. I-10 kuphela yale 70 yeeprotheyini eziqinisekisiweyo zibonise utshintsho kwi-AD yobuninzi ephikisana nale mizila yeepaneli. Ukuze kuveliswe iphaneli ebonisa kakuhle indlela yonke yobuchopho kunye ne-cerebrospinal fluid, asizibandakanyi ezi proteni ze-10 kwiphaneli yomdla esiye saqinisekisa ekugqibeleni (Umfanekiso 5A). Ke ngoko, iphaneli yethu ekugqibeleni ibandakanya iyonke yeeprotheyini ze-60 eziqinisekisiweyo kumaqela amabini azimeleyo e-CSF e-AD kusetyenziswa ukulungiswa kweesampulu ezahlukeneyo kunye nohlalutyo lweqonga le-MS. Iiplani zokubonisa i-z-score zala maphaneli okugqibela kwi-CSF ikopi yokulawula i-1 kunye neemeko ze-AD ziqinisekisile i-panel trend trend ebonwe kwiqela le-CSF esiyifumene (Umfanekiso 5B).
Phakathi kwezi proteni ze-60, kukho iimolekyuli ezaziwa ukuba zidibene ne-AD, njenge-osteopontin (SPP1), eyi-cytokine e-pro-inflammatory edibene ne-AD kwizifundo ezininzi (39-41), kunye ne-GAP43, i-synaptic protein. oko kuhambelana ngokucacileyo ne-neurodegeneration (42). Ezona proteni ziqinisekiswe ngokupheleleyo ziziphawuli ezinxulumene nezinye izifo ze-neurodeergenerative, ezifana ne-amyotrophic lateral sclerosis (ALS) enxulumene ne-superoxide dismutase 1 (SOD1) kunye nesifo sika-Parkinson enxulumene ne-desaccharase (PARK7). Siye saqinisekisa ukuba ezinye iimpawu ezininzi, ezinje nge-SMOC1 kunye ne-membrane etyebileyo ebuchotsheni i-protein ye-1 (BASP1), inomda wamakhonkco angaphambili kwi-neurodegeneration. Kuyaphawuleka ukuba ngenxa yobuninzi babo obuphantsi kwiproteome ye-CSF, kunzima ngathi ukusebenzisa le ndlela ephezulu yokukhangela i-single-shot yokufumanisa ngokuthembekileyo i-MAPT kunye nezinye iiprotheni ezinxulumene ne-AD (umzekelo, i-NEFL kunye ne-NRGN ) ( 43, 44).
Emva koko siye sajonga abamakishi bephaneli ephambili ye-60 kuhlalutyo olongezelelweyo oluphindwe kathathu. Kwi-CSF Copy 2, sasebenzisa i-TMT-MS eyodwa ukuhlalutya iqela elizimeleyo lolawulo lwe-297 kunye neesampuli ze-AD ezivela ku-Emory Goizueta ADRC (17). I-CSF yokuphindaphinda i-3 iquka ukuhlaziywa kwakhona kwedatha ye-TMT-MS ekhoyo evela kulawulo lwe-120 kunye nezigulane ze-AD ezivela eLausanne, eSwitzerland (45). Sichonge ngaphezu kwesibini esithathwini sabamakishi abaphambili abangama-60 kwidatha nganye. Nangona uphando lwaseSwitzerland lusebenzisa iiplatifti ezahlukeneyo ze-MS kunye neendlela zokulinganisa i-TMT (i-45, i-46), saphinda savelisa ngokugqithiseleyo iindlela zethu zephaneli kuhlalutyo oluphindaphindiweyo (Umfanekiso 5, C kunye no-D, kunye neeThebhile S2, I, kunye no-J) . Ukuvavanya isifo esithile seqela lethu, sasebenzisa i-TMT-MS ukuhlalutya iseti yesine yokuphindaphinda idatha (i-CSF replication 4), engabandakanyi kuphela ukulawula (n = 18) kunye ne-AD (n = 17) iimeko, kodwa kunye ne-PD ( n = 14)), i-ALS (n = 18) kunye neesampuli ze-frontotemporal dementia (FTD) (n = 11) (Itheyibhile S1A). Silinganise ngempumelelo phantse isibini kwisithathu seeproteni zephaneli kweli qela (38 ngaphandle kwama-60). Ezi ziphumo zigxininisa utshintsho oluthile lwe-AD kuzo zonke iiphaneli ze-biomarker ezintlanu (Umfanekiso 5E kunye neThebhile S2K). Ukunyuka kweqela le-metabolite kubonise i-AD enamandla kakhulu, ilandelwa yi-myelination kunye neqela le-glial. Kwinqanaba elingaphantsi, i-FTD iphinda ibonise ukwanda phakathi kwezi panels, ezinokuthi zibonise utshintsho olufanayo lwenethiwekhi (17). Ngokwahlukileyo, i-ALS kunye ne-PD zibonise phantse i-myelination efanayo, i-glial, kunye neeprofayili ze-metabolome njengeqela lokulawula. Ngokubanzi, ngaphandle kokungafani kulungiselelo lwesampulu, iqonga le-MS, kunye neendlela zokulinganisa ubungakanani be-TMT, olu hlalutyo oluphindaphindiweyo lubonisa ukuba iimpawu zethu zephaneli eziphambili zineenguqu ezichanekileyo ze-AD kwiisampuli ze-CSF ezingaphezulu kwe-500.
I-AD neurodegeneration iye yamkelwa ngokubanzi kwiminyaka eliqela ngaphambi kokuqala kweempawu zokuqonda, ngoko kukho imfuneko engxamisekileyo yee-biomarkers ze-AsymAD (5, 31). Nangona kunjalo, ubungqina obuninzi nangakumbi bubonisa ukuba i-biology ye-AsymAD ikude kwi-homogeneous, kwaye intsebenziswano eyinkimbinkimbi yomngcipheko kunye nokomelela kukhokelela ekuhlukeni okukhulu komntu ngamnye ekuqhubekeni kwesifo esilandelayo (47). Nangona isetyenziselwa ukuchonga iimeko ze-AsymAD, amanqanaba ee-biomarkers ze-CSF ezingundoqo (Aβ1-42, i-tau iyonke kunye ne-p-tau) azibonakalisi ukuba zikwazi ukuqikelela ngokuthembekileyo ukuba ngubani oza kuqhubela phambili kwi-dementia (4, 7), ebonisa ngakumbi Kuyimfuneko ukubandakanya izixhobo ezipheleleyo ze-biomarker ezisekwe kwimiba emininzi ye-brain physiology ukucwangcisa ngokuchanekileyo umngcipheko waba bantu. Ngoko ke, emva koko sihlalutye iphaneli yethu ye-biomarker eqinisekisiweyo ye-AD kwi-AsymAD yabemi be-CSF ikopi 1. Ezi ziganeko ze-31 ze-AsymAD zibonise amanqanaba angaqhelekanga e-biomarker ye-biomarker (Aβ1-42 / i-total tau ELISA ratio, <5.5) kunye nengqiqo epheleleyo (ithetha i-MoCA, i-27.1) ± 2.2) (Itheyibhile S1A). Ukongeza, bonke abantu abane-AsymAD banamanqaku ekliniki ye-dementia eyi-0, ebonisa ukuba akukho bungqina bokuhla kokuqonda okanye ukusebenza kwemihla ngemihla.
Siqale sahlalutya amanqanaba eepaneli eziqinisekisiweyo kuzo zonke ii-96 CSF eziphindaphinda i-1, kuquka i-AsymAD cohort. Sifumene ukuba iipaneli ezininzi kwiqela le-AsymAD zineenguqu ezibalulekileyo ze-AD-ezifana nobuninzi, iphaneli ye-vascular ibonise ukuhla kwe-AsymAD, ngelixa zonke ezinye iipaneli zibonisa ukunyuka okuphezulu (Umfanekiso 6A). Ke ngoko, zonke iiphaneli zibonise ulungelelwaniso olubaluleke kakhulu kunye ne-ELISA Aβ1-42 kunye namanqanaba e-tau ewonke (Umfanekiso 6B). Ngokwahlukileyo, unxulumano phakathi kweqela kunye nenqaku le-MoCA lihle kakhulu. Esinye seziphumo ezimangalisayo kolu hlahlelo luluhlu olukhulu lwephaneli yobuninzi kwiqela le-AsymAD. Njengoko kubonisiwe kuMfanekiso 6A, inqanaba lephaneli yeqela le-AsymAD lidla ngokuwela inqanaba lephaneli yeqela lolawulo kunye neqela le-AD, libonisa ukuhluka okuphezulu. Ukuphonononga ngakumbi le ntlukwano ye-AsymAD, sisebenzise uhlalutyo lwe-Multidimensional Scaling (MDS) kwi-96 CSF yokuphindaphinda iimeko ze-1. Uhlalutyo lwe-MDS luvumela ukujonga ukufana phakathi kwamatyala asekelwe kwiinguqu ezithile kwisethi yedatha. Kolu hlalutyo lweqela, sisebenzisa kuphela ezo zimakishi zephaneli eziqinisekisiweyo ezinotshintsho olubalulekileyo (P <0.05, AD/control) kwinqanaba lokufunyanwa kwe-CSF kunye ne-replication 1 proteome (n = 29) (Itheyibhile S2L). Olu hlalutyo luvelise ukudibanisa okucacileyo kwendawo phakathi kolawulo lwethu kunye neemeko ze-AD (Umfanekiso 6C). Ngokwahlukileyo, ezinye iimeko ze-AsymAD zihlanganiswe ngokucacileyo kwiqela lolawulo, ngelixa ezinye zifumaneka kwiimeko ze-AD. Ukuphonononga ngakumbi le AsymAD heterogeneity, sisebenzise imephu yethu ye-MDS ukuchaza amaqela amabini ezi meko ze-AsymAD. Iqela lokuqala libandakanya iimeko ze-AsymAD ezihlanganiswe ngokusondeleyo kulawulo (n = 19), ngelixa iqela lesibini libonakaliswe ngamatyala e-AsymAD kunye neprofayili yokumakisha kufuphi ne-AD (n = 12).
(A) Inqanaba lenkcazo (i-z-score) yeqela le-biomarker ye-CSF kuzo zonke iisampuli ze-96 kwi-CSF replication 1 cohort, kuquka i-AsymAD. Uhlalutyo lokungafani kunye nokulungiswa kwe-Tukey emva kokulungiswa kusetyenziswe ukuvavanya ukubaluleka kwezibalo zokutshintsha kobuninzi bephaneli. (B) Uhlalutyo lonxulumano lwenqanaba leprotheyini yobuninzi (z-score) kunye ne-MoCA yamanqaku kunye nenqanaba le-tau elipheleleyo kwi-ELISA Aβ1-42 kunye ne-CSF ikopi ye-1 isampuli. I-Pearson yolungelelwaniso i-coefficient enexabiso le-P elifanelekileyo iyaboniswa. (C) I-MDS ye-96 ye-CSF ikopi ye-1 yamatyala yayisekelwe kumanqanaba obuninzi be-29 abamakishi bephaneli abaqinisekisiweyo, abatshintshwe kakhulu kuzo zombini ukufumanisa kunye ne-CSF ikopi yedatha ye-1 [P <0.05 AD/control (CT)]. Olu hlalutyo lwalusetyenziselwa ukwahlula iqela le-AsymAD ekulawuleni (n = 19) kunye ne-AD (n = 12) ama-subgroups. (D) Isakhiwo se-volcano sibonisa ukubonakaliswa okungafaniyo kwazo zonke iiprotheni ze-CSF zokuphindaphinda i-1 iiprotheni ezine-log2 fold fold (x-axis) ngokumalunga ne-log10 yexabiso le-P yezibalo phakathi kwamaqela amabini e-AsymAD. Iphaneli zebhayomarkers zinemibala. (E) I-CSF yokuphindaphinda i-1 inqanaba lobuninzi beqela lokukhetha i-biomarkers ibonakaliswe ngokwahlukileyo phakathi kwama-subgroups e-AsymAD. Uhlalutyo lukaTukey lwasemva kokuhlengahlengiswa kokungafani lusetyenziselwe ukuvavanya ukubaluleka kwamanani.
Sihlolisise ukubonakaliswa kweprotheyini eyahlukileyo phakathi kolu lawulo kunye neemeko ze-AD ezifana ne-AsymAD (Umfanekiso we-6D kunye neThebhile S2L). Imephu ye-volcano enesiphumo ibonisa ukuba i-14 panel markers zitshintshe kakhulu phakathi kwamaqela amabini. Uninzi lwaba bamakishi ngamalungu e-synapse kunye ne-metabolome. Nangona kunjalo, i-SOD1 kunye ne-myristoylated alanine-rich protein kinase C substrate (MARCKS), engamalungu e-myelin kunye namaqela e-immune e-glial, ngokulandelanayo, nawo akweli qela (Umfanekiso 6, D kunye no-E). Iphaneli ye-vascular nayo inikele ngamanqaku amabini ancitshiswe kakhulu kwiqela le-AD elifana ne-AsymAD, kubandakanywa ne-AE ebopha iprotheyini ye-1 (AEBP1) kunye nokuncedisa ilungu lentsapho ye-C9. Kwakungekho nantlukwano ebalulekileyo phakathi kolawulo kunye ne-AD-njenge-AsymAD subgroups kwi-ELISA AB1-42 (P = 0.38) kunye ne-p-tau (P = 0.28), kodwa ngokwenene kwakukho umehluko omkhulu kwinqanaba elipheleleyo le-tau (P = 0.0031 ) (Umfanekiso S7). Kukho iziphawuli zephaneli ezininzi ezibonisa ukuba utshintsho phakathi kwamaqela angaphantsi e-AsymAD abaluleke kakhulu kunamanqanaba e-tau ewonke (umzekelo, YWHAZ, SOD1, kunye ne-MDH1) (Umfanekiso 6E). Ngokubanzi, ezi ziphumo zibonisa ukuba iphaneli yethu eqinisekisiweyo inokuqulatha ii-biomarkers ezinokuthi zifake i-subtype kunye nomngcipheko onokuthi ubekho kwizigulana ezinesifo esingabonakaliyo.
Kukho imfuno engxamisekileyo yezixhobo ze-biomarker ezisekelwe kwinkqubo yokulinganisa ngcono kunye nokujolisa kwii-pathophysiology ezahlukeneyo emva kwe-AD. Ezi zixhobo zilindeleke ukuba zingatshintshi kuphela isakhelo sokuxilonga se-AD, kodwa sikhuthaze nokwamkelwa kweendlela zonyango ezisebenzayo, ezichanekileyo zesigulane (1, 2). Ukuza kuthi ga ngoku, sisebenzise indlela engakhethi cala yeproteomics kwingqondo ye-AD kunye ne-CSF ukuchonga ii-biomarkers ze-CSF ezisekwe kwiwebhu ezibonisa uluhlu olubanzi lwe-pathophysiology esekwe ebuchotsheni. Uhlalutyo lwethu luvelise iiphaneli ze-biomarker ze-CSF ezintlanu, ezithi (i) zibonise i-synapses, imithambo yegazi, i-myelin, i-immune kunye ne-metabolic dysfunction; (ii) bonisa ukuveliswa okunamandla kwiiplatifti ezahlukeneyo ze-MS; (iii) Bonisa utshintsho oluqhubekayo lwesifo kumanqanaba okuqala nasekupheleni kweAD. Ngokubanzi, ezi ziphumo zimele inyathelo elithembisayo ekuphuhliseni izinto ezahlukeneyo, ezithembekileyo, ezijoliswe kwiwebhu ze-biomarker izixhobo zophando lwe-AD kunye nezicelo zeklinikhi.
Iziphumo zethu zibonisa umbutho ogcinwe kakhulu we-AD yenethiwekhi yobuchopho proteome kunye nokuxhasa ukusetyenziswa kwayo njengeankile yophuhliso lwenkqubo-based biomarker. Uhlalutyo lwethu lubonisa ukuba iiseti ezimbini ezizimeleyo ze-TMT-MS ezine-AD kunye nobuchopho be-AsymAD zinemodyuli eyomeleleyo. Ezi ziphumo zandisa umsebenzi wethu wangaphambili, zibonisa ukugcinwa kweemodyuli ezinamandla ezingaphezu kwe-2,000 yezicubu zengqondo ezivela kumaqela amaninzi azimeleyo kwi-frontal, parietal, kunye ne-temporal cortex (17). Olu nxibelelwano lwemvumelwano lubonisa utshintsho olunxulumene nesifo olubonwa kuphando lwangoku, kubandakanywa nokunyuka kweemodyuli ezityebileyo ze-glial kunye nokuncipha kweemodyuli ze-neuron-rich. Njengophando lwangoku, le nethiwekhi inkulu ineenguqu ezibalulekileyo zemodyuli kwi-AsymAD, ebonisa iindidi ezahlukeneyo ze-pathophysiology ye-preclinical (17).
Nangona kunjalo, ngaphakathi kwesi sikhokelo sisekelwe kwinkqubo yolondolozo, kukho ukungafani kwebhayoloji ecolekileyo ngakumbi, ngakumbi phakathi kwabantu abakwizigaba zokuqala ze-AD. Iphaneli yethu ye-biomarker iyakwazi ukubonisa amaqela amancinci amabini kwi-AsymAD, ebonisa ukubonakaliswa okuphawulekayo kwamanqaku amaninzi e-CSF. Iqela lethu liye lakwazi ukugqamisa umahluko webhayoloji phakathi kwala macandelwana amabini, awayengabonakali kwinqanaba le-biomarkers ze-AD eziphambili. Xa kuthelekiswa neqela lolawulo, i-Aβ1-42/totali ye-tau ratios yaba bantu be-AsymAD yayiphantsi ngokungaqhelekanga. Nangona kunjalo, kuphela amanqanaba e-tau ewonke ahluke kakhulu phakathi kwamaqela amabini e-AsymAD, ngelixa i-Aβ1-42 kunye ne-p-tau amanqanaba ahlala ethelekiswa. Ekubeni i-CSF ephezulu ibonakala ngathi yi-predictor engcono yeempawu zengqondo kunamanqanaba e-Aβ1-42 (7), siyakrokra ukuba amaqela amabini e-AsymAD anokuba nemingcipheko eyahlukeneyo yokuqhubeka kwesifo. Ngenxa yobungakanani besampulu elinganiselweyo ye-AsymAD yethu kunye nokunqongophala kwedatha ye-longitudinal, uphando olongezelelweyo luyafuneka ukwenza ezi zigqibo ngokuzithemba. Nangona kunjalo, ezi ziphumo zibonisa ukuba iphaneli ye-CSF esekwe kwinkqubo inokuphucula amandla ethu okuphatha abantu ngokufanelekileyo ngexesha lenqanaba lesi sifo.
Ngokubanzi, iziphumo zethu zixhasa indima yemisebenzi emininzi yebhayoloji kwi-pathogenesis ye-AD. Nangona kunjalo, i-dysregulated energy metabolism yaba ngumxholo obalaseleyo wazo zonke iiphaneli zethu zokulebula eziqinisekisiweyo ezintlanu. Iiprotheni ze-Metabolic, ezifana ne-hypoxanthine-guanine phosphoribosyltransferase 1 (HPRT1) kunye ne-lactate dehydrogenase A (LDHA), zezona zingqinisiso ze-synaptic biomarkers eziqinisekisiweyo, ezibonisa ukuba ukwanda kwe-AD CSF kukwabelana ngesondo kakhulu. Imithambo yethu yegazi kunye neepaneli zeglial nazo zineempawu ezininzi ezibandakanyekayo kwimetabolism yezinto ezine-oxidative. Ezi ziphumo zihambelana nendima ephambili edlalwa ziinkqubo zemetabolism kwingqondo iphela, kungekuphela nje ukuhlangabezana nemfuno ephezulu yamandla e-neuron, kodwa kunye nokuhlangabezana nemfuno ephezulu yamandla e-astrocyte kunye nezinye iiseli zeglial (17, 48). Iziphumo zethu zixhasa ubungqina obukhulayo bokuthi utshintsho kwi-redox enokwenzeka kunye nokuphazamiseka kweendlela zamandla kunokuba lunxibelelwano oluphambili phakathi kweenkqubo ezininzi eziphambili ezibandakanyekayo kwi-pathogenesis ye-AD, kubandakanywa ukuphazamiseka kwe-mitochondrial, ukuvuvukala kwe-glial, kunye nomonakalo we-Vascular (49). Ukongeza, i-metabolic cerebrospinal fluid biomarkers iqulethe inani elikhulu leeprotheyini ezityebileyo ngokwahlukileyo phakathi kolawulo lwethu kunye ne-AD-like AsymAD subgroups, ebonisa ukuba ukuphazamiseka kwezi ndlela zamandla kunye ne-redox kunokuba kubaluleke kakhulu kwinqanaba lokuqala lesi sifo.
Ubuchopho kunye neendlela zephaneli ye-cerebrospinal fluid esizibonileyo nazo zinefuthe elinomdla kwibhayoloji. Ii-synapses kunye ne-metabolomes ezityebileyo kwii-neurons zibonisa amanqanaba anciphileyo kwingqondo ye-AD kunye nokwanda kobuninzi kwi-cerebrospinal fluid. Ngenxa yokuba ii-neuron zizityebi kwi-mitochondria evelisa amandla kwii-synapses ukunika amandla kwiimpawu zazo ezininzi ezikhethekileyo (50), ukufana kweeprofayili zokubonisa zala maqela mabini e-neuron kulindeleke. Ukulahleka kwee-neurons kunye nokukhutshwa kweeseli ezonakalisiweyo kunokuchaza obu buchopho kunye ne-CSF iindlela zepaneli kwisifo samva, kodwa abanakuchaza utshintsho lwangaphambili lwephaneli esilubonayo (13). Enye ingcaciso enokubakho yezi zinto zifunyanisiweyo kwisifo sokuqala esingenazimpawu zesifo kukuthenwa okungaqhelekanga kwe-synaptic. Ubungqina obutsha kwiimodeli zempuku bucebisa ukuba i-microglia-mediated synaptic phagocytosis inokuthi isebenze ngokungaqhelekanga kwi-AD kwaye ikhokelele ekulahlekeni kwe-synapse kwangethuba kwingqondo (51). Esi sixhobo esilahliweyo se-synaptic sinokuqokelela kwi-CSF, yingakho sibona ukwanda kwe-CSF kwiphaneli ye-neuron. Ukuthenwa kwe-synaptic ye-immune-Mediated synaptic kunokuchaza ngokuyinxenye ukonyuka kweeproteni ze-glial esizibona kwingqondo kunye nolwelo lwe-cerebrospinal kuyo yonke inkqubo yesifo. Ukongeza kwi-synaptic pruning, ukungaqhelekanga ngokubanzi kwindlela ye-exocytic kunokukhokelela kwingqondo eyahlukeneyo kunye nokubonakaliswa kwe-CSF yeempawu ze-neuronal. Izifundo ezininzi zibonise ukuba umxholo we-exosomes kwi-pathogenesis yengqondo ye-AD utshintshile (52). Indlela ye-extracellular nayo ibandakanyeka ekwandeni kwe-Aβ (53, 54). Kuyafaneleka ukuba uqaphele ukuba ukunyanzeliswa kwe-exosomal secretion kunokunciphisa i-AD-like pathology kwi-AD transgenic mouse model (55).
Ngelo xesha, iprotheni kwiphaneli ye-vascular ibonise ukunyuka okuphakathi kwingqondo ye-AD, kodwa iyancipha kakhulu kwi-CSF. Ukungasebenzi kakuhle kwe-blood-brain barrier (BBB) kunokucacisa ngokuyinxenye ezi ziphumo. Izifundo ezininzi ezizimeleyo ze-postmortem zabantu zibonise ukuphuka kwe-BBB kwi-AD (56, 57). Olu phononongo luqinisekise imisebenzi eyahlukeneyo engaqhelekanga ejikeleze lo maleko utywinwe ngokungqongqo weeseli ze-endothelial, kubandakanya ukuvuza kwe-capillary yobuchopho kunye nokuqokelelana kwe-perivascular yeeproteni ezihanjiswa ngegazi (57). Oku kunokubonelela ngengcaciso elula yeeprotheni eziphakamileyo ze-vascular kwingqondo, kodwa ayikwazi ukuchaza ngokupheleleyo ukuchithwa kwezi proteni ezifanayo kwi-cerebrospinal fluid. Enye into enokwenzeka kukuba inkqubo ye-nervous central ihlukanisa ngokusebenzayo ezi molekyuli ukusombulula ingxaki yokwanda kokuvuvukala kunye noxinzelelo lwe-oxidative. Ukunciphisa ezinye zeeprotheyini ze-CSF ezinzima kakhulu kule phaneli, ngokukodwa ezo zibandakanyeka kulawulo lwe-lipoprotein, zihambelana nokuvinjelwa kwamanqanaba ayingozi okuvuvukala kunye nenkqubo ye-neuroprotective yeentlobo ze-oksijini ezisebenzayo. Oku kuyinyaniso kwi-Paroxonase 1 (PON1), i-lipoprotein ebopha i-enzyme ejongene nokunciphisa amanqanaba oxinzelelo lwe-oxidative kwi-circulation (58, 59). I-Alpha-1-microglobulin/i-bikunin precursor (AMBP) yenye i-marker ephantsi kakhulu elawulwayo yeqela le-vascular. I-precursor ye-lipid transporter bikunin, ekwabandakanyeka ekunyanzeleni ukuvuvukala kunye noKhuseleko lwe-neurological (60, 61).
Ngaphandle kweengqikelelo ezahlukeneyo ezinomdla, ukungakwazi ukubona ngokuthe ngqo iindlela zesifo se-biochemical ngumda owaziwayo wohlalutyo oluqhutywa yiproteomics. Ke ngoko, uphando olongezelelweyo luyimfuneko ukuchaza ngokuzithemba iindlela ezisemva kwezi panel biomarker. Ukuze uqhubele phambili ekuphuhliseni uhlalutyo lweklinikhi olusekwe kwi-MS, isalathiso sexesha elizayo sikwafuna ukusetyenziswa kweendlela ezichongiweyo zobungakanani bokuqinisekiswa kwe-biomarker enkulu, njengokujonga okukhethiweyo okanye okuhambelanayo (62). Kutshanje sisebenzise esweni ukusabela okufanayo (63) ukuqinisekisa uninzi lweenguqu zeprotheyini ze-CSF ezichazwe apha. Iithagethi ezininzi zephaneli eziphambili zibalwe ngokuchaneka okubalulekileyo, kubandakanywa i-YWHAZ, i-ALDOA, kunye ne-SMOC1, ebonisa imephu ye-synapse yethu, i-metabolism, kunye neepaneli zokudumba, ngokulandelanayo (63). I-Independent Data Acquisition (DIA) kunye nezinye izicwangciso ze-MS-based nazo zinokuba luncedo ekuqinisekiseni okujoliswe kuyo. Bud et al. (64) Kutshanje kubonakaliswe ukuba kukho ukudityaniswa okubalulekileyo phakathi kwee-biomarkers ze-AD ezichongiweyo kwiseti yethu yedatha yokufunyanwa kwe-CSF kunye neseti yedatha ye-DIA-MS ezimeleyo, equka phantse iisampulu ze-CSF ezingama-200 ezivela kumaqela amathathu ahlukeneyo aseYurophu. Olu phononongo lwakutsha nje luxhasa amandla eepaneli zethu ukuguqula abe yi-MS-based ethembekileyo yokuchongwa. I-antibody yesiNtu kunye nobhaqo olusekwe kwi-aptamer lukwabalulekile kuphuhliso olongezelelweyo lweempawu ze-AD eziphambili. Ngenxa yobuninzi obuphantsi be-CSF, kunzima kakhulu ukufumanisa ezi biomarkers usebenzisa iindlela ze-MS eziphezulu. I-NEFL kunye ne-NRGN yimizekelo emibini enjalo yee-biomarkers ze-CSF ezinobuninzi obuphantsi, ezifakwe kwimaphu kwiphaneli kuhlalutyo lwethu olubanzi, kodwa ayikwazi ukufunyanwa ngokuthembekileyo kusetyenziswa isicwangciso sethu se-MS esisodwa. Izicwangciso zokujolisa ezisekelwe kwii-antibodies ezininzi, ezifana ne-PEA, zinokukhuthaza ukuguqulwa kweklinikhi kwaba bamakishi.
Ngokubanzi, olu phononongo lubonelela ngendlela ekhethekileyo yeproteomics yokuchongwa kunye nokuqinisekiswa kwee-biomarkers ze-CSF ze-AD ezisekelwe kwiinkqubo ezahlukeneyo. Ukuphucula ezi phaneli zokumakisha kuzo zonke iindawo ezongezelelweyo ze-AD kunye namaqonga e-MS kunokungqina ukuba kuyathembisa ukuqhubela phambili umngcipheko we-AD kunye nonyango. Amaphononongo avavanya inqanaba le-longitudinal la maphaneli ekuhambeni kwexesha nawo abaluleke kakhulu ukufumanisa ukuba yeyiphi indibaniselwano yabamakishi eyona ndlela ibalaseleyo yomngcipheko wesifo sakwangoko kunye notshintsho kubukhali besifo.
Ngaphandle kweesampuli ze-3 ezikopishwe yi-CSF, zonke iisampuli ze-CSF ezisetyenziswe kolu phononongo ziqokelelwe phantsi kwe-Emory ADRC okanye amaziko ophando ahlobene ngokusondeleyo. Zizonke iiseti ezine zeesampulu ze-Emory CSF zisetyenzisiwe kwezi zifundo zeproteomics. Iqela le-CSF lifunyenwe liqulethe iisampuli ezivela kwi-20 yokulawula okunempilo kunye nezigulane ze-20 ze-AD. Ikopi ye-CSF ye-1 ibandakanya iisampuli ezivela kwi-32 yolawulo olunempilo, abantu be-31 be-AsymAD, kunye ne-33 AD ngabanye. Ikopi ye-CSF 2 iqulethe ulawulo lwe-147 kunye neesampuli ze-AD ezili-150. I-multi-desease CSF replication 4 cohort iquka ulawulo lwe-18, i-17 AD, i-19 ALS, i-13 PD, kunye ne-11 iisampulu zeFTD. Ngokwesivumelwano esivunyiweyo yiBhodi yokuHlola yeziko leYunivesithi yase-Emory, bonke abathathi-nxaxheba bokufunda i-Emory bafumana imvume enolwazi. Ngokwe-2014 National Institute of Aging Best Practice Guidelines for Alzheimer's Centres (https://alz.washington.edu/BiospecimenTaskForce.html), i-cerebrospinal fluid yaqokelelwa kwaye yagcinwa ngokuhlatywa kwe-lumbar. Ulawulo kunye ne-AsymAD kunye nezigulana ze-AD zifumene uvavanyo lwengqondo olusemgangathweni kwiKliniki ye-Emory Cognitive Neurology okanye i-Goizueta ADRC. Iisampuli zabo ze-cerebrospinal fluid zavavanywa yi-INNO-BIA AlzBio3 Luminex ye-ELISA Aβ1-42, i-tau iyonke kunye nohlalutyo lwe-p-tau (65). Amaxabiso e-ELISA asetyenziselwa ukuxhasa ulwahlulo loxilongo lwezifundo ngokusekwe kwiikhrayitheriya ezimiselweyo ze-AD biomarker cut-off (66, 67). Idatha esisiseko ye-demographic kunye ne-diagnostic kwezinye izifo ze-CSF (FTD, ALS, kunye ne-PD) nazo zifunyenwe kwi-Emory ADRC okanye amaziko ophando adibeneyo. Imeko yesishwankathelo imethadatha yezi meko ze-Emory CSF inokufumaneka kwiThebhile S1A. Iimpawu ze-Swiss CSF replication 3 cohort ziye zapapashwa ngaphambili (45).
I-CSF ifumene isampuli. Ukuze kwandiswe ubunzulu bokufumanisa kwethu idatha ye-CSF, ukusetyenziswa kwe-immune yeeprotheyini eziphezulu zenziwa ngaphambi kokuba i-trypsinization. Ngamafutshane, i-130 μl ye-CSF esuka kwiisampulu ze-CSF ezingama-40 kunye nomthamo olinganayo (130 μl) we-High Select Top14 Ubuninzi beProtein yokuncipha kweResin (i-Thermo Fisher Scientific, A36372) zibekwe kwikholamu ye-spin (Thermo Fisher Scientific, A89868) kwigumbi iqondo lobushushu Incubate). Emva kokujikeleza imizuzu eyi-15, i-centrifuge isampuli kwi-1000g imizuzu emi-2. Isixhobo sokucoca i-3K ye-ultracentrifugal (i-Millipore, i-UFC500396) isetyenziselwe ukugxila kwisampulu yamanzi amdaka nge-centrifuging kwi-14,000g imizuzu engama-30. Nciphisa zonke iisampulu zesampulu zibe yi-75 μl kunye ne-phosphate buffered saline. Uxinzelelo lweprotheyini luye lwavavanywa yi-bicinchoninic acid (BCA) indlela ngokweprotocol yomenzi (i-Thermo Fisher Scientific). I-CSF ye-immunodepleted (60 μl) kuzo zonke iisampulu ezingama-40 zotyiswa nge-lysyl endopeptidase (LysC) kunye ne-trypsin. Ngamafutshane, isampuli yancitshiswa kwaye i-alkylated kunye ne-1.2 μl 0.5 M tris-2 (-carboxyethyl) -phosphine kunye ne-3 μl 0.8 M i-chloroacetamide kwi-90 ° C imizuzu eyi-10, kwaye emva koko i-sonicated kwindawo yokuhlambela amanzi imizuzu eyi-15. Isampulu yahlanjululwa nge-193 μl 8 M urea buffer [8 M urea kunye ne-100 mM NaHPO4 (pH 8.5)] ukuya kugxininiso lokugqibela lwe-6 M urea. I-LysC (4.5 μg; Wako) isetyenziselwa ukugaya ebusuku kwiqondo lokushisa. Isampulu iye yahlanjululwa yaba yi-1 M urea kunye ne-50 mM ammonium bicarbonate (ABC) (68). Yongeza isixa esilinganayo (4.5 μg) se-trypsin (i-Promega), kwaye emva koko ufukamele isampuli iiyure ezili-12. I-Acidify isisombululo se-peptide egayiweyo kwi-concentration yokugqibela ye-1% ye-asidi ye-formic (FA) kunye ne-0.1% ye-trifluoroacetic acid (TFA) (66), kwaye emva koko i-desalt kunye ne-50 mg yekholomu ye-Sep-Pak C18 (Amanzi) njengoko kuchazwe ngasentla (25) . Emva koko i-peptide yahluthwa kwi-1 ml ye-50% ye-acetonitrile (ACN). Ukulinganisa ubungakanani beprotheyini kuzo zonke iibhetshi (25), i-100 μl ye-aliquots kuzo zonke iisampulu ze-CSF ezingama-40 zadityaniswa ukuvelisa isampulu exutyiweyo, eyathi ke yahlulwa yaba ziisampuli ezintlanu zomgangatho wangaphakathi (GIS) (48). Zonke iisampulu zomntu ngamnye kunye nemigangatho edibeneyo yomiswa nge-vacuum yesantya esiphezulu (i-Labconco).
I-CSF ikhuphela isampuli. I-Dayon kunye noogxa baye bachaza ngaphambili ukuchithwa kwe-immune kunye nokugaya kwe-CSF ikopi ye-3 isampuli (45, 46). Iisampulu eziphindaphindayo eziseleyo azizange ziphelelwe ngumzimba. Yetyisa ezi sampuli zingasuswanga kwi-trypsin njengoko bekuchaziwe ngaphambili (17). Kucazululo ngalunye oluphindaphindiweyo, i-120 μl ye-aliquots ye-peptide eluted ukusuka kwisampulu nganye yadityaniswa kunye kwaye yahlulahlulwe kwi-aliquots yevolumu elinganayo ukuze isetyenziswe njengomgangatho wangaphakathi wehlabathi obhalwe nge-TMT (48). Zonke iisampulu zomntu ngamnye kunye nemigangatho edibeneyo yomiswa nge-vacuum yesantya esiphezulu (i-Labconco). Ukuze kunyuswe umqondiso weprotein ye-CSF yobuninzi obuphantsi, ngokudibanisa i-125 μl kwisampulu nganye, isampulu "eyongeziweyo" yalungiselelwa kuhlalutyo lokuphindaphinda ngalunye [okt, isampuli yebhayoloji elinganisa isampulu yophando, kodwa isixa esikhoyo. inkulu kakhulu (37, 69)] idityaniswe kwisampulu exubeneyo yeCSF (17). Isampulu exubeneyo emva koko i-immunoremoved kusetyenziswa i-12 ml ye-High Khetha i-Top14 Ubuninzi beProtein yokususa i-Resin (i-Thermo Fisher Scientific, i-A36372), igaywe njengoko kuchaziwe ngasentla, kwaye ifakwe kwi-labeling ye-TMT eninzi elandelayo.
Ixesha lokuposa: Aug-27-2021