Proteomics zuru oke na-ekpughe biomarkers cerebrospinal fluid biomarkers na asymptomatic na ọrịa ọrịa Alzheimer.

Ọrịa Alzheimer (AD) enweghị protein biomarkers nke na-egosipụta ọtụtụ pathophysiology dị n'okpuru ya, na-egbochi ọganihu nke nyocha na ọgwụgwọ. N'ebe a, anyị na-eji proteomics zuru oke iji chọpụta biomarkers cerebrospinal fluid (CSF) nke na-anọchite anya ọtụtụ AD pathophysiology. Multiplex mass spectrometry chọpụtara ihe dịka 3,500 na ihe dị ka protein 12,000 na AD CSF na ụbụrụ, n'otu n'otu. Nyocha netwọkụ nke ụbụrụ proteome kpebiri 44 modul biodiversity, 15 n'ime ha jikọtara ya na proteome mmiri cerebrospinal. A na-apịakọta akara CSF AD na modul ndị a jikọtara ọnụ n'ime otu protein ise, na-anọchite anya usoro pathophysiological dị iche iche. Synapses na metabolites dị na ụbụrụ AD na-ebelata, mana CSF na-abawanye, ebe glial-rich myelination na ndị na-alụso ọrịa ọgụ na ụbụrụ na CSF na-abawanye. A kwadoro ngbanwe na ọrịa kpọmkwem nke mgbanwe panel na ihe karịrị 500 ọzọ CSF samples. Otu ndị a chọpụtakwara otu obere ihe ndị dị ndụ na asymptomatic AD. N'ozuzu, nsonaazụ ndị a bụ nzọụkwụ na-ekwe nkwa na ngwaọrụ biomarker dabere na weebụ maka ngwa ụlọ ọgwụ na AD.
Ọrịa Alzheimer (AD) bụ ihe na-akpatakarị dementia neurodegenerative n'ụwa niile ma bụrụ nke a na-eji ọtụtụ usoro adịghị arụ ọrụ nke usoro ndu, gụnyere nnyefe synapti, mgbochi glial-mediated, na mitochondrial metabolism (1-3). Agbanyeghị, ndị na-ahụ maka protein biomarkers ka na-elekwasị anya n'ịchọpụta amyloid na protein tau, yabụ enweghị ike igosipụta pathophysiology dị iche iche. Ndị a "isi" protein biomarkers nke a na-atụkwa anya nke ọma na mmiri cerebrospinal (CSF) gụnyere (i) amyloid beta peptide 1-42 (Aβ1-42), nke na-egosipụta nguzobe nke cortical amyloid plaques; (ii) ngụkọta tau, ihe ịrịba ama nke axon degeneration; (iii) phospho-tau (p-tau), onye nnọchianya nke pathological tau hyperphosphorylation (4-7). Ọ bụ ezie na ndị a cerebrospinal fluid biomarkers enyerela anyị aka ịchọpụta ọrịa protein “akara akara” AD (4-7), naanị ha na-anọchi anya obere akụkụ nke usoro ihe omimi dị mgbagwoju anya n'azụ ọrịa ahụ.
Enweghị ụdị pathophysiological di iche iche nke AD biomarkers emewo ka ọtụtụ ihe ịma aka, gụnyere (i) enweghị ike ịchọpụta na ọnụ ọgụgụ dị iche iche nke ndu nke ndị ọrịa AD, (ii) ezughị oke nke oke ọrịa na ọganihu, karịsịa na ọkwa nke mbụ, na (ii) iii) mmepe nke ọgwụ ọgwụgwọ na-emezughị kpamkpam akụkụ niile nke mmebi akwara ozi. Ndabere anyị na pathology ala iji kọwaa AD site na ọrịa ndị metụtara ya na-eme ka nsogbu ndị a ka njọ. Ihe akaebe ndị ọzọ na-egosi na ọtụtụ ndị agadi nwere nkwarụ nwere ihe karịrị otu njirimara pathological nke mbelata ọgụgụ isi (8). Ihe dị ka 90% ma ọ bụ karịa nke ndị nwere ọrịa ọrịa AD nwekwara ọrịa vaskụla, ntinye TDP-43, ma ọ bụ ọrịa mmebi ndị ọzọ (9). Ọnụọgụ ndị a dị elu nke ndakọrịta ọrịa akpaghasịla usoro nyocha anyị ugbu a maka mgbaka, yana a chọkwara nkọwapụta pathophysiological karịa nke ọrịa ahụ.
N'iburu n'uche mkpa dị ngwa maka ụdị dị iche iche AD biomarkers, ubi ahụ na-arịwanye elu na-agbaso usoro "omics" dabere na usoro n'ozuzu iji chọpụta biomarkers. The Accelerated Pharmaceutical Partnership (AMP) -AD Alliance ewepụtara na 2014 ma bụrụ ụzọ n'ihu na mmemme ahụ. Mgbalị a multidisciplinary nke National Institutes of Health, academia, na ụlọ ọrụ na-achọ iji usoro dabeere na usoro iji kọwaa pathophysiology nke AD na ịmepụta nchọpụta nchọpụta diagnostics na usoro ọgwụgwọ (10). Dị ka akụkụ nke ọrụ a, netwọk proteomics aghọwo ngwá ọrụ na-ekwe nkwa maka ọganihu nke ndị na-emepụta ihe ndị dabeere na usoro na AD. Usoro data a na-adịghị ele mmadụ anya n'ihu na-ahazi data proteomics dị mgbagwoju anya n'ime otu ma ọ bụ "modul" nke protein egosipụtara nke jikọtara ya na ụdị cell, akụkụ ahụ, na ọrụ ndụ (11-13). Ihe fọrọ nke nta ka ọ bụrụ ọmụmụ ihe gbasara mgbasa ozi netwọkụ bara ụba 12 ka emegoro na ụbụrụ AD (13-23). N'ozuzu, nyocha ndị a na-egosi na proteome ụbụrụ ụbụrụ AD na-edobe nzukọ modular echekwara nke ukwuu na ndị otu nọọrọ onwe ha na mpaghara cortical. Na mgbakwunye, ụfọdụ n'ime modul ndị a na-egosi mgbanwe mgbanwe na ụbara metụtara AD n'ofe data, na-egosipụta pathophysiology nke ọtụtụ ọrịa. N'ịchịkọta, nchoputa ndị a na-egosipụta ihe arịlịka na-ekwe nkwa maka nchọpụta nke ụbụrụ netwọk proteome dị ka usoro biomarker dabeere na AD.
Iji gbanwee proteome ụbụrụ ụbụrụ AD ka ọ bụrụ ndị na-ahụ maka sistemu bara uru nke ụlọ ọgwụ, anyị jikọtara netwọkụ sitere na ụbụrụ yana nyocha proteomic nke AD CSF. Usoro a jikọtara ọnụ mere ka a chọpụta usoro ise na-ekwe nkwa nke CSF biomarkers nke jikọtara ya na ọtụtụ ụdị pathophysiology nke ụbụrụ, gụnyere synapses, arịa ọbara, myelination, mbufụt, na arụ ọrụ nke ụzọ metabolic. Anyị kwadoro ogwe biomarker ndị a nke ọma site n'ọtụtụ nyocha mmegharị, gụnyere ihe karịrị 500 CSF sitere na ọrịa neurodegenerative dị iche iche. Nyocha nkwado ndị a gụnyere nyochaa ebumnuche otu na CSF nke ndị ọrịa nwere asymptomatic AD (AsymAD) ma ọ bụ igosi ihe akaebe nke mkpokọta amyloid na-adịghị mma na gburugburu ọgụgụ isi. Ntụle ndị a na-akọwapụta ihe dị iche iche dị ndụ dị na AsymAD ma chọpụta akara ngosi panel nke nwere ike ịdepụta ndị mmadụ n'otu n'otu na mmalite nke ọrịa ahụ. N'ozuzu, nsonaazụ ndị a na-anọchite anya nzọụkwụ dị mkpa na mmepe nke protein biomarker ngwaọrụ dabere na ọtụtụ usoro nke nwere ike dozie ọtụtụ n'ime nsogbu ahụike nke AD chere ihu.
Ebumnuche bụ isi nke ọmụmụ a bụ ịchọpụta biomarkers cerebrospinal fluid ọhụrụ nke na-egosipụta ụdị pathophysiology dị iche iche nke ụbụrụ na-eduga na AD. Ọnụ ọgụgụ S1 na-akọwapụta usoro nyocha anyị, nke gụnyere (i) nyocha zuru oke nke nchọpụta mmalite nke AD CSF na ụbụrụ ụbụrụ netwọk iji chọpụta ọtụtụ ndị na-ahụ maka ọrịa CSF metụtara ụbụrụ, na (ii) mmeghari ọzọ na-esote ndị a biomarkers nọ n'ọtụtụ cerebrospinal nweere onwe ha. ìgwè mmiri. Nchọpụta gbadoro ụkwụ na nchọpụta malitere site na nyocha nke ngosipụta dị iche iche nke CSF n'ime mmadụ 20 nwere ọgụgụ isi na ndị ọrịa 20 AD na Emory Goizueta Alzheimer's Disease Research Center (ADRC). A kọwapụtara nchoputa nke AD dị ka nnukwu nkwarụ ọgụgụ isi na ọnụnọ Aβ1-42 dị ala na ọkwa dị elu nke ngụkọta tau na p-tau na mmiri cerebrospinal [Mean Montreal Cognitive Assessment (MoCA), 13.8 ± 7.0] [ELISA (ELISA) )]] (Obodo S1A). Njikwa (pụtara MoCA, 26.7 ± 2.2) nwere ọkwa nkịtị nke CSF biomarkers.
A na-eji CSF mmadụ nwere nnukwu protein dị ike, nke albumin na protein ndị ọzọ bara ụba nwere ike igbochi ịchọpụta protein nke mmasị (24). Iji mee ka omimi nke nchọpụta protein dịkwuo elu, anyị wepụrụ protein 14 nke mbụ buru ibu site na nlele CSF nke ọ bụla tupu nyocha spectrometry (MS) (24). Ngụkọta nke 39,805 peptides ka MS chọpụtara, bụ nke e debere na proteome 3691 na nlele 40. A na-eme ọnụọgụ protein site na ọtụtụ akara mkpado tandem mass (TMT) (18, 25). Iji dozie data na-efu efu, anyị gụnyere naanị protein ndị ahụ agbagoro na opekata mpe 50% nke ihe nlele na nyocha na-esote, yabụ n'ikpeazụ na-agụta proteome 2875. N'ihi nnukwu ọdịiche dị na mkpokọta protein n'ụba, a na-ahụta akara njikwa dị ka ihe na-apụ apụ (13) na etinyeghị ya na nyocha na-esote. A na-emezi ụkpụrụ n'ụba nke ihe atụ 39 fọdụrụnụ dabere na afọ, okike, na covariance batch (13-15, 17, 18, 20, 26).
N'iji nyocha t-ule ọnụ ọgụgụ nyocha iji nyochaa okwu dị iche iche na nhazi data regression, nyocha a chọpụtara protein ndị nwere nnukwu mgbanwe gbanwere (P <0.05) n'etiti njikwa na AD ikpe (Table S2A). Dịka e gosiri na eserese 1A, ụbara ngụkọta nke protein 225 dị na AD belatara nke ukwuu, na ụbara protein 303 mụbara nke ukwuu. Protein ndị a egosipụtara dị iche iche gụnyere ọtụtụ akara AD cerebrospinal achọpụtara, dị ka protein tau jikọtara microtubule (MAPT; P = 3.52 × 10-8), neurofilament (NEFL; P = 6.56 × 10-3), Protein metụtara uto 43. (GAP43; P = 1.46 × 10−5), Fatty Acid Binding Protein 3 (FABP3; P = 2.00 × 10-5), Chitinase 3 dị ka 1 (CHI3L1; P = 4.44 × 10-6), Neural Granulin (NRGN; P = 3.43 × 10-4) na VGF ụbụrụ na-eto eto (VGF; P = 4.83 × 10-3) (4-6). Otú ọ dị, anyị chọpụtakwara ihe mgbaru ọsọ ndị ọzọ dị oke mkpa, dị ka GDP dissociation inhibitor 1 (GDI1; P = 1.54 × 10-10) na SPARC modular calcium binding 1 (SMOC1; P = 6.93 × 10-9). Gene Ontology (GO) nyocha nke 225 budata protein belatara gosipụtara njikọ chiri anya na usoro mmiri ahụ dị ka metabolism steroid, coagulation ọbara, na ọrụ homonụ (Ọnyocha 1B na tebụl S2B). N'ụzọ dị iche, protein mụbara nke ukwuu nke 303 nwere njikọ chiri anya na nhazi cell na ike metabolism.
(A) Mkpebi ugwu mgbawa na-egosi mgbanwe mgbanwe log2 (x-axis) metụtara -log10 statistical P value (y-axis) nwetara site na t-ule, nke a na-eji achọpụta ọdịiche dị n'etiti njikwa (CT) na AD ikpe nke CSF proteome nke protein niile. A na-egosi protein ndị nwere ọkwa dị ala nke ukwuu (P <0.05) na AD na-acha anụnụ anụnụ, ebe a na-egosipụta na-acha uhie uhie. A na-akpọ protein ahọpụtara. (B) Okwu GO kacha elu metụtara protein na-ebelata nke ukwuu (acha anụnụ anụnụ) na ụbara (ọbara ọbara) na AD. Na-egosi usoro GO atọ nwere akara z- kacha elu n'ọhịa nke usoro ndu, ọrụ molekụla, na akụkụ cellular. (C) MS tụrụ ọkwa MAPT na nlele CSF (aka ekpe) yana njikọ ya na ọkwa ELISA tau (n'aka nri). E gosipụtara ọnụọgụ njikọ Pearson yana uru P dabara adaba. N'ihi enweghị data ELISA maka otu ikpe AD, ọnụ ọgụgụ ndị a gụnyere ụkpụrụ maka 38 n'ime ikpe 39 nyochara. (D) Nyocha ụyọkọ na-elekọta (P <0.0001, Benjamini-Hochberg (BH) gbanwere P <0.01) na njikwa na AD CSF chọtara ihe nlele site na iji 65 kachasị gbanwere protein na data data. Hazie, normalize.
Ọkwa proteomic nke MAPT nwere njikọ chiri anya na ọkwa ELISA tau tụrụ onwe ya (r = 0.78, P = 7.8 × 10-9; Ọgụgụ 1C), na-akwado nkwado nke nha MS anyị. Mgbe mgbaze trypsin na ọkwa nke amyloid precursor protein (APP), peptides isoform-kpọmkwem nke e debere na C-terminus nke Aβ1-40 na Aβ1-42 enweghị ike ionized nke ọma (27, 28). Ya mere, peptides APP anyị chọpụtara enweghị ihe jikọrọ ya na ọkwa ELISA Aβ1-42. Iji nyochaa okwu dị iche iche nke ikpe ọ bụla, anyị na-eji protein dị iche iche egosipụtara na P <0.0001 [ọnụego nchọpụta ụgha (FDR) dezie P <0.01] iji mee nyocha ụyọkọ na-elekọta nke ihe nlele (Table S2A). Dị ka egosiri na eserese 1D, protein 65 ndị ​​a dị oke mkpa nwere ike ịchịkọta nlele n'ụzọ ziri ezi dịka ọnọdụ ọrịa siri dị, belụsọ otu ikpe AD nwere njirimara njikwa. N'ime protein 65 ndị ​​a, 63 mụbara na AD, ebe naanị abụọ (CD74 na ISLR) belatara. Na mkpokọta, nyocha nke mmiri cerebrospinal a achọpụtala ọtụtụ narị protein na AD nke nwere ike bụrụ ndị na-ahụ maka ọrịa.
Mgbe ahụ, anyị mere nyocha netwọkụ nke nọọrọ onwe ya nke proteome ụbụrụ AD. Otu ụbụrụ nke nchọpụta a gụnyere dorsolateral prefrontal cortex (DLPFC) site na njikwa (n = 10), ọrịa Parkinson (PD; n = 10), agwakọta AD/PD (n = 10) na AD (n = 10). ) Ihe atụ. Emery Goizueta ADRC. A kọwapụtala ihe ngosi igwe mmadụ nke ikpe 40 ndị a na mbụ (25) wee chịkọta ya na tebụl S1B. Anyị jiri TMT-MS nyochaa anụ ahụ ụbụrụ 40 ndị a yana ngbanwe nke ikpe 27. Na mkpokọta, usoro data ụbụrụ abụọ a rụpụtara 227,121 peptides pụrụ iche, nke edobere na proteome 12,943 (25). Naanị protein ndị ahụ agbagoro na opekata mpe 50% nke ikpe ka etinyere na nyocha ndị na-esote. Ntọala nchoputa ikpeazụ nwere protein dị 8817. Gbanwee ọkwa ụbara protein dabere na afọ, okike, na etiti oge ọnwụ (PMI). Ntụle okwu dị iche iche nke data setịpụrụ mgbe nlọghachi azụ gosiri na> 2000 protein protein gbanwere nke ukwuu [P <0.05, analysis of variance (ANOVA)] na ọrịa abụọ ma ọ bụ karịa. Mgbe ahụ, anyị mere nyocha ụyọkọ nke a na-elekọta na-adabere na protein ndị egosipụtara dị iche iche, yana P <0.0001 na AD / njikwa na / ma ọ bụ AD / PD ntụnyere (Ọnọdụ S2, A na B, Tebụl S2C). Protein 165 ndị ​​a gbanwere nke ukwuu gosipụtara n'ụzọ doro anya okwu nwere ọrịa pathology AD site na njikwa yana ihe nlele PD, na-akwado mgbanwe AD-kpọmkwem siri ike na proteome niile.
Anyị wee jiri algọridim akpọrọ Weighted Gene Co-expression Network Analysis (WGCNA) iji mee nyocha netwọkụ na proteome ụbụrụ achọpụtara, nke na-ahazi data etinyere n'ime modul protein nwere usoro okwu yiri ya (11-13). Nyocha ahụ chọpụtara 44 modul (M) protein ndị jikọtara ọnụ, na-ahazi na ọnụ ọgụgụ site na ndị kasị ibu (M1, n = 1821 protein) na nke kacha nta (M44, n = 34 proteins) (Figure 2A na Table S2D) ). Dị ka e kwuru n'elu (13) Gbakọọ profaịlụ okwu nnọchiteanya ma ọ bụ protein njirimara nke modul ọ bụla, ma jikọta ya na ọnọdụ ọrịa na AD pathology, ya bụ, guzobe njikọ nke ndekọ ọrịa Alzheimer (CERAD) na Braak Score (Figure 2B). N'ozuzu, modul 17 nwere njikọ chiri anya na AD neuropathology (P <0.05). Ọtụtụ n'ime modul ndị a metụtara ọrịa nwekwara ọgaranya na ụdị mkpụrụ ndụ akọwapụtara kpọmkwem (Njirimara 2B). Dịka ekwuru n'elu (13), a na-ekpebi nkwalite ụdị cell site n'ịtụle mgbakwụnye modul na ndepụta ntụaka nke mkpụrụ ndụ ụdị sel akọwapụtara. A na-enweta mkpụrụ ndụ ihe nketa ndị a site na data ebipụtara na neurons mouse dịpụrụ adịpụ, endothelial na glial cell. Nnwale usoro RNA (RNA-seq) (29).
(A) Chọpụta WGCNA nke ụbụrụ proteome. (B) Nyocha midcorrelation dị arọ (BiCor) nke protein mbinye aka modular (ihe mbụ bụ isi nke ngosipụta protein modular) nwere njirimara AD neuropathological (n'elu), gụnyere CERAD (Aβ plaque) na akara Braak (tau tangles). A na-egosipụta ike nke njikọ dị mma (acha ọbara ọbara) na nke na-adịghị mma (acha anụnụ anụnụ) site na map okpomọkụ nke agba abụọ, na asterisks na-egosi mkpa ndekọ ọnụ ọgụgụ (P <0.05). Jiri Hypergeometric Fisher's Exact Test (FET) (ala) iji chọpụta njikọ ụdị cell nke modul protein ọ bụla. Ike nke ndo na-acha uhie uhie na-egosi ogo ụdị mmụba cell, na akara mmuke na-egosi mkpa ndekọ ọnụ ọgụgụ (P <0.05). Jiri usoro BH dozie uru P ewepụtara na FET. (C) GO nyocha nke protein modular. E gosipụtara usoro ndu ndị nwere njikọ chiri anya maka modul ọ bụla ma ọ bụ otu modul metụtara ya. oligo, oligodendrocyte.
Otu setịpụ nke astrocyte ise nwere njikọ chiri anya na modul microglia-rich (M30, M29, M18, M24, na M5) gosipụtara mmekọrịta dị mma na AD neuropathology (Njirimara 2B). Nyocha Ontology na-ejikọta modul glial ndị a na uto cell, mmụba, na mgbochi (Figure 2C na Tebụl S2E). Modul glial abụọ ọzọ, M8 na M22, na-achịkwa nke ọma na ọrịa. M8 nwere njikọ chiri anya na ụzọ nnabata nke Toll-like, cascade nke na-arụ ọrụ dị mkpa na nzaghachi mgbochi ebumpụta ụwa (30). N'otu oge ahụ, M22 nwere njikọ chiri anya na mgbanwe ntụgharị asụsụ. M2, nke bara ụba na oligodendrocytes, na-egosi njikọ dị mma siri ike na pathology AD na njikọ ontological na njikọ nucleoside na DNA replication, na-egosi mmụba cell na ọrịa. N'ozuzu, nchoputa ndị a na-akwado nkwalite nke modul glial nke anyị hụburu na AD network proteome (13, 17). Achọpụtara ugbu a na ọtụtụ modul glial metụtara AD na netwọkụ na-egosi ọkwa ngosipụta dị ala na njikwa yana ikpe PD, na-egosipụta nkọwapụta ọrịa ha na-ebuli elu na AD (Figure S2C).
Naanị modul anọ dị na proteome netwọk anyị (M1, M3, M10, na M32) na-ejikọta ya na AD pathology (P <0.05) (Figure 2, B na C). Ma M1 na M3 bara ụba na akara neuronal. M1 nwere njikọ chiri anya na mgbama synapti, ebe M3 nwere njikọ chiri anya na ọrụ mitochondrial. Enweghị ihe akaebe maka nkwalite ụdị cell maka M10 na M32. M32 na-egosipụta njikọ dị n'etiti M3 na cell metabolism, ebe M10 nwere njikọ chiri anya na uto cell na ọrụ microtubule. E jiri ya tụnyere AD, modul anọ niile na-abawanye na njikwa na PD, na-enye ha mgbanwe AD kpọmkwem ọrịa (Ọgụgụ S2C). N'ozuzu, nsonaazụ ndị a na-akwado mbelata mbelata nke modul bara ụba neuron nke anyị hụburu na AD (13, 17). Na nchịkọta, nyocha netwọkụ nke ụbụrụ ụbụrụ nke anyị chọpụtara mepụtara AD-kpọmkwem modul gbanwere nke kwekọrọ na nchọpụta anyị gara aga.
A na-eji AD mara ọkwa asymptomatic n'oge (AsymAD), nke ndị mmadụ n'otu n'otu na-egosipụta mkpokọta amyloid na-enweghị mbelata ọgụgụ isi ụlọ ọgwụ (5, 31). Ọkwa asymptomatic a na-anọchite anya windo dị oke mkpa maka nchọpụta mmalite na ntinye aka. Anyị egosipụtala mbụ nchekwa modular siri ike nke AsymAD na proteome ụbụrụ ụbụrụ AD n'ofe data nọọrọ onwe ya (13, 17). Iji hụ na netwọkụ ụbụrụ anyị chọpụtara ugbu a kwekọrọ na nchoputa ndị a gara aga, anyị nyochara nchekwa nke modul 44 na data emegharịgharịrị sitere na otu 27 DLPFC. Òtù ndị a gụnyere njikwa (n = 10), AsymAD (n = 8) Na AD (n = 9). Agụnyere ihe nlele njikwa na AD na nyocha nke ndị otu ụbụrụ nchọpụta anyị (Table S1B), ebe okwu AsymAD pụrụ iche naanị na otu ntugharị. Okwu AsymAD ndị a bịakwara site na ụlọ akụ ụbụrụ Emory Goizueta ADRC. Ọ bụ ezie na cognition bụ ihe nkịtị n'oge ọnwụ, ọkwa amyloid dị elu nke ukwuu (pụtara CERAD, 2.8 ± 0.5) (Table S1B).
Nyocha TMT-MS nke anụ ahụ ụbụrụ 27 ndị a rụpụtara na ọnụọgụ nke 11,244 proteome. Ọnụọgụ ikpeazụ a gụnyere naanị protein ndị ahụ agbagoro na opekata mpe 50% nke nlele. Ntọala data a emegharịgharịrị nwere 8638 (98.0%) nke protein 8817 achọpụtara na nyocha ụbụrụ anyị chọpụtara, ma ihe fọrọ nke nta ka ọ bụrụ 3000 gbanwere protein n'etiti njikwa na ndị otu AD (P <0.05, mgbe Tukey's paired t test for analysis of variance) Okpokoro S2F). N'ime protein ndị a egosipụtara dị iche iche, 910 gosikwara mgbanwe dị ukwuu n'etiti AD na ikpe njikwa proteome ụbụrụ (P <0.05, mgbe ANOVA Tukey jikọtara t-ule). Ọ dị mma ịmara na akara ngosi 910 ndị a na-agbanwe agbanwe na ntụgharị mgbanwe n'etiti proteome (r = 0.94, P <1.0 × 10-200) (Figure S3A). N'ime protein ndị na-abawanye ụba, protein ndị nwere mgbanwe na-agbanwe agbanwe n'etiti usoro data bụ ndị otu glial-rich M5 na M18 modul (MDK, COL25A1, MAPT, NTN1, SMOC1, na GFAP). N'ime protein ndị e belatara, ndị nwere mgbanwe na-agbanwe agbanwe bụ ihe fọrọ nke nta ka ọ bụrụ nanị ndị òtù M1 modul (NPTX2, VGF, na RPH3A) jikọtara ya na synapse. Anyị gara n'ihu nyochaa mgbanwe AD metụtara midkine (MDK), CD44, protein frizzled metụtara 1 (SFRP1) na VGF site na blotting ọdịda anyanwụ (Ọgụgụ S3B). Nyocha nchekwa modul gosiri na ihe dị ka 80% nke modul protein (34/44) na proteome ụbụrụ ka echekwara nke ọma na nhazi data mmụgharị (z-score> 1.96, FDR gbaziri P <0.05) (Figure S3C). Edobere iri na anọ n'ime modul ndị a nke ọma n'etiti proteome abụọ (z-score> 10, FDR emeziri P <1.0 × 10-23). N'ozuzu, nchoputa na mmụgharị nke ogo dị elu nke nkwekọ n'ụdị okwu dị iche iche na nhazi modular n'etiti ụbụrụ proteome na-egosipụta ngbanwe nke mgbanwe na protein cortex AD n'ihu. Na mgbakwunye, o gosikwara na AsymAD na ọrịa ndị ka dị elu nwere usoro netwọkụ ụbụrụ yiri nke ahụ.
Ntụle zuru ezu nke okwu dị iche iche na data ngbanwe nke ụbụrụ na-egosipụta ọkwa dị ịrịba ama nke mgbanwe protein AsymAD, gụnyere ngụkọta nke 151 gbanwere protein n'etiti AsymAD na njikwa (P <0.05) (Figure S3D). N'ikwekọ na ibu amyloid, APP na ụbụrụ AsymAD na AD mụbara nke ukwuu. MAPT na-agbanwe naanị na AD, nke dabara n'ọkwa dị elu nke tangles yana njikọ ya na mbelata ọgụgụ isi (5, 7). Modul-ọgaranya glial (M5 na M18) na-egosipụta nke ọma na protein na-abawanye na AsymAD, ebe M1 modul metụtara neuron bụ onye nnọchite anya protein ndị belatara na AsymAD. Ọtụtụ n'ime akara AsymAD ndị a na-egosi mgbanwe dị ukwuu na ọrịa mgbaàmà. N'ime ihe nrịbama ndị a bụ SMOC1, protein glial nke M18, nke jikọtara ya na ụbụrụ ụbụrụ na mmepe nke anya na aka (32). MDK bụ ihe na-akpata heparin na-ejikọta ya na uto cell na angiogenesis (33), onye ọzọ so na M18. E jiri ya tụnyere otu njikwa, AsymAD mụbara nke ukwuu, na-esote mmụba dị ukwuu na AD. N'ụzọ dị iche, protein synapti neuropentraxin 2 (NPTX2) belatara nke ukwuu na ụbụrụ AsymAD. Ejikọtara NPTX2 na mbụ na neurodegeneration ma nwee ọrụ a ma ama na mgbasa ozi synapses excitatory (34). N'ozuzu, nsonaazụ ndị a na-ekpughe mgbanwe dị iche iche nke protein preclinical dị iche iche na AD nke yiri ka ọ na-aga n'ihu na oke ọrịa ahụ.
Nyere na anyị enwetala nnukwu mkpuchi mkpuchi protein na nchọpụta nke ụbụrụ proteome, anyị na-agbalị ịghọta nke ọma nkwekọ ya na ọkwa AD transcriptome. Ya mere, anyị jiri proteome ụbụrụ anyị chọpụtara na modul anyị mepụtara na mbụ site na nha microarray nke mkpụrụ ndụ ihe nketa 18,204 na AD (n = 308) na njikwa (n = 157) anụ ahụ DLPFC (13). ndakota. Na mkpokọta, anyị chọpụtara 20 ụdị RNA dị iche iche, ọtụtụ n'ime ha gosipụtara mmụba nke ụdị sel akọwapụtara, gụnyere neurons, oligodendrocytes, astrocytes, na microglia (Nyocha 3A). Ọtụtụ mgbanwe nke modul ndị a na AD ka egosiri na eserese 3B. N'ikwekọ na nyocha protein-RNA anyị gara aga site na iji MS proteome nke enweghị akara (ihe dị ka protein 3000) (13), ọtụtụ n'ime modul 44 dị na netwọkụ proteome ụbụrụ anyị hụrụ na netwọk transcriptome Ọ nweghị nnukwu ndakọrịta na. nchọpụta anyị na mmụgharị nke modul protein protein 34 nke a na-edobere nke ọma na proteome ụbụrụ, naanị 14 (~ 40%) gafere ule Fisher (FET) gosipụtara na ọ nwere nchịkọta ọnụ ọgụgụ dị ịrịba ama na transcriptome (Figure 3A) . Dakọtara na nrụzi mmebi DNA (P-M25 na P-M19), ntụgharị protein (P-M7 na P-M20), njikọ RNA / splicing (P-M16 na P-M21) na ntinye protein (P-M13 na P- M23) anaghị ejikọta na modul na transcriptome. Ya mere, ọ bụ ezie na a na-eji usoro data proteome miri emi mee ihe na nyocha nke overlap ugbu a (13), ọtụtụ n'ime proteome netwọk AD adịghị edepụta na netwọk transcriptome.
(A) Hypergeometric FET na-egosiputa mmụba nke ụdị cell-kpọmkwem nrịbama na RNA modul nke AD transcriptome (n'elu) na ogo nke overlap n'etiti RNA (x-axis) na protein (y-axis) modul nke AD ụbụrụ. (n'okpuru) . Ike nke ndò na-acha uhie uhie na-egosi ogo nke ịba ụba nke ụdị cell dị n'elu panel na ike nke nkpuchi nke modul na ala panel. Asterisks na-egosi mkpa ọnụọgụgụ (P <0.05). (B) Ogo nke mmekọrịta dị n'etiti mkpụrụ ndụ ihe nketa njirimara nke modul transcriptome ọ bụla na ọkwa AD. Modul ndị dị n'aka ekpe bụ ndị kacha emetụta AD (acha anụnụ anụnụ), na ndị nọ n'aka nri na-ejikọta ya na AD (acha uhie uhie). Uru P nke BH-gbanwere log-gbanwere na-egosi ogo mkpa ndekọ aha nke njikọ ọ bụla. (C) Modul mkpirisi dị ịrịba ama nwere nkwalite ụdị cell nkekọrịta. (D) Ntụle njikọ nke log2 fold mgbanwe nke protein akpọrọ (x-axis) na RNA (y-axis) na modul na-ekpuchi. E gosipụtara ọnụọgụ njikọ Pearson yana uru P dabara adaba. Micro, microglia; anụ ahụ nke eluigwe, astrocytes. CT, njikwa.
Imirikiti protein na modul RNA na-ekerịta ụdị profaịlụ nkwalite ụdị cell na ntụzịaka mgbanwe AD na-agbanwe agbanwe (Foto 3, B na C). N'ikwu ya n'ụzọ ọzọ, a na-edepụta modul M1 metụtara synapse nke ụbụrụ proteome (PM1) na modul RNA homologous atọ nke neuronal (R-M1, R-M9 na RM16), nke dị na AD abụọ gosipụtara. a belatara larịị. N'otu aka ahụ, modul protein glial-rich M5 na M18 jikọtara ya na modul RNA bara ụba na astrocytes na akara microglial (R-M3, RM-M7, na RM10) ma na-etinye aka na ọrịa na-abawanye. Ihe njirimara modular ndị a na-ekekọrịta n'etiti usoro data abụọ a na-akwado nkwalite ụdị cell na mgbanwe ndị metụtara ọrịa anyị hụrụ na proteome ụbụrụ. Agbanyeghị, anyị hụrụ ọtụtụ ọdịiche dị ịrịba ama n'etiti ọkwa RNA na protein nke ihe nrịbama n'otu n'otu na modul ndị a ekekọrịtara. Ntụle njikọ nke ngosipụta dị iche iche nke proteomics na transcriptomics nke ụmụ irighiri ihe dị n'ime modul ndị a na-ekpuchi anya (Njirimara 3D) na-eme ka enweghị nkwekọrịta a pụta ìhè. Dịka ọmụmaatụ, APP na ọtụtụ protein glial modul ndị ọzọ (NTN1, MDK, COL25A1, ICAM1, na SFRP1) gosipụtara mmụba dị ukwuu na proteome AD, mana ọ fọrọ nke nta ka ọ bụrụ mgbanwe ọ bụla na AD transcriptome. Mgbanwe ndị a akọwapụtara nke ọma nwere ike jikọta ya na plaques amyloid (23, 35), na-eme ka proteome pụta ìhè dị ka isi iyi nke mgbanwe pathological, mgbanwe ndị a nwere ike ọ gaghị egosipụta na transcriptome.
Mgbe anyị nyochachara onwe ya na ụbụrụ ụbụrụ na proteome CSF anyị chọpụtara, anyị mere nyocha zuru oke nke usoro data abụọ ahụ iji chọpụta ndị na-ahụ maka biomarkers AD CSF metụtara pathophysiology nke netwọkụ ụbụrụ. Anyị ga-ebu ụzọ kọwaa nchikota nke proteome abụọ ahụ. Ọ bụ ezie na a na-anabata nke ọma na CSF na-egosipụta mgbanwe neurochemical na ụbụrụ AD (4), kpọmkwem ogo nke ndakọrịta n'etiti ụbụrụ AD na proteome CSF edoghị anya. Site n'ịtụle ọnụ ọgụgụ nke ngwaahịa mkpụrụ ndụ ihe nketa achọpụtara na proteome abụọ anyị, anyị chọpụtara na ihe fọrọ nke nta ka ọ bụrụ 70% (n = 1936) nke protein ndị a chọpụtara na mmiri cerebrospinal bụkwa nke a na-atụle na ụbụrụ (Nyocha 4A). Ọtụtụ n'ime protein ndị a jikọtara ọnụ (n = 1721) bụ nke edobere n'otu n'ime modul nkwupụta okwu 44 sitere na ntọala data ụbụrụ nchọta (Njirimara 4B). Dị ka a tụrụ anya ya, modul isii kacha ibu ụbụrụ (M1 ruo M6) gosipụtara ọnụ ọgụgụ CSF kacha ukwuu. Agbanyeghị, enwere modul ụbụrụ ndị pere mpe (dịka ọmụmaatụ, M15 na M29) na-enweta ogo dị elu na-atụghị anya ya, buru ibu karịa modul ụbụrụ ugboro abụọ nha ya. Nke a na-akpali anyị ịnakwere usoro zuru ezu karị, nke na-agbakọ ọnụ ọgụgụ iji gbakọọ ndakọta n'etiti ụbụrụ na mmiri mmiri cerebrospinal.
(A na B) Ndị na-edozi ahụ achọpụtara na ụbụrụ nchọpụta na data CSF na-ejikọta. Ọtụtụ n'ime protein ndị a jikọtara ọnụ na-ejikọta ya na otu n'ime modul nkwupụta okwu 44 nke netwọk mmekọrịta ụbụrụ. (C) Chọpụta ihe nkedo dị n'etiti proteome mmiri mmiri na ụbụrụ ụbụrụ. Ahịrị nke ọ bụla nke maapụ okpomọkụ na-anọchi anya nyocha nkpuchi dị iche nke hypergeometric FET. Ahịrị elu na-egosi ndakọ (ncha ntụ ntụ/oji) n'etiti modul ụbụrụ yana proteome CSF dum. Ahịrị nke abụọ na-egosi na a na-ahazi njikọ dị n'etiti modulu ụbụrụ na protein CSF (nke na-acha uhie uhie) na AD (P <0.05). Ahịrị nke atọ na-egosi na ndakọrịta n'etiti modulu ụbụrụ na protein CSF (shading blue) na-achịkwa nke ọma na AD (P <0.05). Jiri usoro BH dozie uru P ewepụtara na FET. (D) Ogwe mpịaji modul dabere na njikọ ụdị cell yana okwu GO metụtara ya. Ogwe ndị a nwere ngụkọta nke protein ndị metụtara ụbụrụ 271, nke nwere okwu dị iche iche bara uru na proteome CSF.
N'iji FET otu ọdụdụ, anyị tụlere mkpa ọ dị nchikota protein n'etiti proteome CSF na modul ụbụrụ onye ọ bụla. Nyocha ahụ gosipụtara na ngụkọta nke ụbụrụ ụbụrụ 14 dị na data CSF nwere nchịkọta dị ịrịba ama (FDR gbanwere P <0.05), yana modul ọzọ (M18) nke nkwekọ ya dị nso (FDR gbanwee P = 0.06) (Nyocha 4C). , ahịrị elu). Anyị nwekwara mmasị na modul na-ejikọta ya na protein CSF dị iche iche. Ya mere, anyị tinyere nyocha abụọ FET ọzọ iji chọpụta nke (i) protein CSF mụbara nke ukwuu na AD na (ii) protein CSF belatara nke ukwuu na AD (P <0.05, paired t test AD/control) Brain modules with bara uru overlap. n'etiti ha. Dị ka egosiri na ahịrị etiti na ala nke Figure 4C, nyocha ndị ọzọ na-egosi na 8 nke ụbụrụ ụbụrụ 44 na-ejikọta ya na protein agbakwunyere na AD CSF (M12, M1, M2, M18, M5, M44, M33, na M38). . ), ebe naanị modul abụọ (M6 na M15) gosipụtara ndakọrịta bara uru na protein belatara na AD CSF. Dị ka a tụrụ anya ya, modul 10 niile dị na modul iri na ise nke nwere nkwekọ kachasị elu na proteome CSF. Ya mere, anyị na-eche na modul 15 ndị ​​a bụ isi mmalite dị elu nke ụbụrụ AD sitere na CSF biomarkers.
Anyị na-apịaji modul iri na ise ndị a na-atụgharị n'ime nnukwu protein ise nke dabere na nso ha dị na eserese osisi WGCNA na mkpakọrịta ha na ụdị cell na mkpụrụ ndụ ontology (Nyocha 4D). Ogwe nke mbụ nwere modul bara ụba na akara neuron na protein metụtara synapse (M1 na M12). Ogwe synapti nwere ngụkọta nke protein 94, na ọkwa dị na proteome CSF agbanweela nke ukwuu, na-eme ka ọ bụrụ isi iyi nke akara CSF metụtara ụbụrụ n'etiti akụkụ ise ahụ. Òtù nke abụọ (M6 na M15) gosipụtara njikọ chiri anya na ihe nrịbama mkpụrụ ndụ endothelial na ahụ vaskụla, dị ka "ọgwụgwọ ọnya" (M6) na "iwu nke nzaghachi mmeghachi omume humoral" (M15). M15 nwekwara njikọ chiri anya na metabolism lipoprotein, nke nwere njikọ chiri anya na endothelium (36). Ogwe vaskụla nwere akara 34 CSF metụtara ụbụrụ. Otu nke atọ na-agụnye modul (M2 na M4) bụ ndị metụtara nke ọma na oligodendrocyte markers na cell proliferation. Dịka ọmụmaatụ, usoro ontology dị elu nke M2 gụnyere "usoro nhazi DNA dị mma" na "usoro biosynthesis purine". Ka ọ dị ugbu a, ndị nke M4 gụnyere "iche nke glial cell" na "nkewa chromosome". Ogwe myelination nwere akara 49 CSF metụtara ụbụrụ.
Otu nke anọ nwere ọtụtụ modul (M30, M29, M18, M24 na M5), na ihe fọrọ nke nta ka ọ bụrụ modul niile bara ụba nke ukwuu na microglia na akara astrocyte. Yiri ogwe myelination, panel nke anọ nwekwara modul (M30, M29, na M18) ndị nwere njikọ chiri anya na mmụba cell. Modul ndị ọzọ dị n'otu a nwere njikọ chiri anya na okwu immunological, dị ka "usoro mmetụta mgbochi" (M5) na "usoro nzaghachi mgbochi" (M24). Otu mgbochi glial nwere akara 42 CSF metụtara ụbụrụ. N'ikpeazụ, ogwe ikpeazụ gụnyere akara 52 metụtara ụbụrụ na modul anọ (M44, M3, M33, na M38), ihe niile dị na ahụ metụtara nchekwa ike na metabolism. Nke kachasị n'ime modul ndị a (M3) nwere njikọ chiri anya na mitochondria ma bụrụ ọgaranya na akara akara neuron. M38 bụ otu n'ime ndị otu modul pere mpe na metabolome a ma na-egosipụtakwa nkọwapụta neuron na-agafeghị oke.
N'ozuzu, ogwe ise ndị a na-egosipụta ụdị dịgasị iche iche nke ụdị cell na ọrụ dị na AD cortex, na mkpokọta nwere akara CSF metụtara ụbụrụ 271 (Table S2G). Iji nyochaa izi ezi nke nsonaazụ MS ndị a, anyị na-eji nyocha ndọtị nso nso (PEA), teknụzụ na-emegide mmadụ nke nwere ike ịgbakọ ọnụ, ikike dị elu na nkọwapụta, wee nyochaa nlele mmiri nke cerebrospinal anyị chọtara akụkụ nke ndị a 271 biomarkers. (n = 36). Ihe mgbaru ọsọ 36 ndị a na-egosipụta mgbanwe na AD multiple nke PEA, nke nwere njikọ chiri anya na nchọpụta MS anyị (r = 0.87, P = 5.6 × 10-12) , Nke kwadoro nke ọma nsonaazụ nke nyocha MS zuru ezu (Nyocha S4). ).
Isiokwu ndị dị ndụ nke otu ise anyị kwusiri ike, site na nrịbama synapti na ike metabolism, niile metụtara pathogenesis nke AD (1-3). Ya mere, modul 15 niile nwere akụkụ ndị a metụtara ọrịa AD na ụbụrụ proteome nke anyị chọpụtara (Njirimara 2B). Ihe kachasị ama bụ njikọ dị mma nke ọrịa pathological dị n'etiti modul glial anyị yana njikọ siri ike na-adịghị mma n'etiti modul neuronal anyị (M1 na M3). Ntụle okwu dị iche iche nke proteome ụbụrụ anyị emegharịgharịrị (Ọgụgụ S3D) na-egosipụtakwa protein glial sitere na M5 na M18. Na AsymAD na symptomatic AD, protein glial kachasị na-abawanye na synapses metụtara M1 Protein na-ebelata nke ukwuu. Nleba anya ndị a na-egosi na akara 271 cerebrospinal fluid anyị chọpụtara n'ime otu ise ahụ metụtara usoro ọrịa na cortex AD, gụnyere ndị na-eme na mmalite nke asymptomatic.
Iji nyochaa nke ọma mgbanwe ntụziaka nke protein panel na ụbụrụ na mmiri ọkpụkpụ, anyị na-esetịpụ ihe ndị a maka nke ọ bụla n'ime 15 modul overlapping: (i) hụrụ modul ụbara larịị na ụbụrụ data set na (ii) modul. protein A na-egosipụta ihe dị iche na mmiri cerebrospinal (Njirimara S5). Dị ka e kwuru na mbụ, a na-eji WGCNA chọpụta ụbara modul ma ọ bụ uru protein njirimara na ụbụrụ (13). A na-eji maapụ mgbawa ugwu kọwaa nkọwa dị iche iche nke protein modular na mmiri cerebrospinal (AD/control). Ọnụ ọgụgụ ndị a na-egosi na atọ n'ime akụkụ ise ahụ na-egosi usoro okwu dị iche iche na ụbụrụ na mmiri ọkpụkpụ azụ. Modul abụọ nke synapse panel (M1 na M12) na-egosi mbelata n'ọkwa ụba dị na ụbụrụ AD, mana ọ na-adaba nke ọma na protein na-abawanye na AD CSF (Figure S5A). Modul metụtara neuron nwere metabolome (M3 na M38) gosipụtara ụdị okwu ụbụrụ na ụbụrụ cerebrospinal na-ekwekọghị ekwekọ (Ọnyocha S5E). Ogwe vaskụla ahụ gosikwara usoro okwu dị iche iche, ọ bụ ezie na modul ya (M6 na M15) na-abawanye nke ọma na ụbụrụ AD wee belata na CSF na-arịa ọrịa (Figure S5B). Ogwe abụọ fọdụrụnụ nwere nnukwu netwọkụ glial nke a na-ahazi protein ya na ngalaba abụọ ahụ (Ọnyocha S5, C na D).
Biko mara na usoro ndị a abụghị ihe nrịbama niile dị na paịlị ndị a. Dịka ọmụmaatụ, panel synapti gụnyere ọtụtụ protein ndị na-ebelata nke ukwuu na ụbụrụ AD na CSF (Ọnọdụ S5A). N'ime ihe nrịbama mmiri cerebrospinal a na-achịkwa ala bụ NPTX2 na VGF nke M1, na chromogranin B nke M12. Otú ọ dị, n'agbanyeghị ihe ndị a, ọtụtụ n'ime akara synapti anyị na-ebuli elu na mmiri spinal AD. N'ozuzu, nyocha ndị a nwere ike ịmata ọdịiche dị ịrịba ama na ọnọdụ ụbụrụ na ụbụrụ cerebrospinal na nke ọ bụla n'ime akụkụ ise anyị. Usoro ndị a na-akọwapụta mgbagwoju anya na mmekọrịta dị iche iche n'etiti ụbụrụ na nkwupụta protein CSF na AD.
Mgbe ahụ, anyị na-eji nyocha mmegharị MS dị elu (CSF replication 1) iji mee ka 271 setịpụ ihe ndị na-emepụta ihe na-eme ka ọ bụrụ ihe mgbaru ọsọ kachasị mma na nke a pụrụ imepụtaghachi (Nyocha 5A). Mpempe akwụkwọ CSF 1 nwere ngụkọta nke nlele 96 sitere na Emory Goizueta ADRC, gụnyere njikwa, AsymAD, na AD otu (Table S1A). A na-eji ọnọdụ AD ndị a site na mbelata ọgụgụ isi dị nro (pụtara MoCA, 20.0 ± 3.8), yana mgbanwe na AD biomarkers kwadoro na mmiri cerebrospinal (Table S1A). N'adịghị ka nyocha CSF anyị chọtara, a na-eme mmegharị a site na iji usoro MS "otu-shot" nke ọma na nke dị elu (na-enweghị akụkụ dị n'ahịrị), gụnyere usoro nkwadebe nlele dị mfe nke na-ewepụ mkpa nke immunodepletion nke ihe nlele nke onye ọ bụla. . Kama nke ahụ, a na-eji otu “ọwa nkwalite” mebie ihe na-alụso ọrịa ọgụ iji kwalite mgbama nke protein ndị na-adịchaghị ukwuu (37). Ọ bụ ezie na ọ na-ebelata mkpokọta proteome mkpuchi, usoro otu a na-agbapụ na-ebelata oge igwe ma na-abawanye ọnụ ọgụgụ nke akara TMT nke enwere ike nyochaa ike (17, 38). Na mkpokọta, nyocha ahụ chọpụtara 6,487 peptides, nke debere na 1,183 proteome n'ime ikpe 96. Dị ka ọ dị na nyocha CSF anyị chọtara, ọ bụ naanị protein ndị ahụ agbagoro na opekata mpe 50% nke ihe nlele ahụ ka etinyere na ngụkọ ndị sochirinụ, eweghaghachi data ahụ maka mmetụta nke afọ na okike. Nke a butere na njedebe ikpeazụ nke 792 proteome, 95% nke a chọpụtakwara na data CSF achọtara.
(A) Ebumnobi protein CSF metụtara ụbụrụ enwetara n'ime ndị otu CSF nke mbụ emegharịrị ma tinye ya na panel ikpeazụ (n = 60). (B ruo E) Ọkwa biomarker panel (composite z-scores) tụrụ na mkpokọta CSF anọ. Ejikọtara t-ule ma ọ bụ ANOVA nwere nzizi Tukey iji nyochaa mkpa mgbakọ na mwepụ nke mgbanwe n'ụba na nyocha nke ọ bụla. CT, njikwa.
Ebe ọ bụ na anyị nwere mmasị karịsịa n'ịkwado ebumnuche CSF anyị metụtara ụbụrụ 271 site na nyocha zuru oke, anyị ga-amachi nyocha ọzọ nke proteome a na-atụgharị na akara ndị a. N'ime protein 271 ndị a, ahụrụ 100 na CSF replication 1. Foto S6A na-egosi ọdịiche dị iche iche nke ihe nrịbama 100 ndị a na-ekpuchi n'etiti njikwa na ihe nlele AD. Synapti na metabolite histones na-abawanye na AD, ebe protein vaskụla na-ebelata nke ukwuu na ọrịa. Ọtụtụ n'ime akara 100 overlapping (n = 70) jigidere otu ntụzịaka nke mgbanwe na nhazi data abụọ (Ọgụgụ S6B). Ihe nrịbama CSF metụtara ụbụrụ 70 a kwadoro (Table S2H) na-egosipụtakarị usoro okwu okwu panel nke a na-ahụbu na mbụ, ya bụ, usoro nchịkwa nke protein vaskụla na nhazi elu nke akụkụ ndị ọzọ niile. Naanị 10 n'ime protein 70 a kwadoro gosipụtara mgbanwe na ụba AD nke megidere usoro panel ndị a. Iji mepụta otu panel nke na-egosipụta nke ọma n'ozuzu usoro nke ụbụrụ na ụbụrụ cerebrospinal, anyị wepụrụ protein 10 ndị a na panel nke mmasị nke anyị mechara nyochaa (Nyocha 5A). Ya mere, panel anyị mechara gụnye ngụkọta nke protein 60 akwadoro na ndị otu CSF AD abụọ nọọrọ onwe ha site na iji nkwadebe nlele dị iche iche na nyocha ikpo okwu MS. Atụmatụ okwu z-score nke akụkụ ikpeazụ ndị a na njikwa CSF 1 na AD gosipụtara omume panel a hụrụ na ndị otu CSF anyị chọtara (Nyocha 5B).
N'ime protein 60 ndị a, e nwere ụmụ irighiri ihe ndị a maara na ha na AD, dị ka osteopontin (SPP1), nke bụ cytokine pro-inflammatory nke e jikọtara ya na AD n'ọtụtụ ọmụmụ (39-41), na GAP43, protein synaptic. nke ahụ jikọtara nke ọma na neurodegeneration (42). Ndị na-edozi ahụ kacha kwenye n'ụzọ zuru ezu bụ akara ndị metụtara ọrịa ndị ọzọ na-adịghị ahụkebe, dị ka amyotrophic lateral sclerosis (ALS) metụtara superoxide dismutase 1 (SOD1) na ọrịa Parkinson metụtara desaccharase (PARK7). Anyị enyochalakwa na ọtụtụ nrịbama ndị ọzọ, dị ka SMOC1 na protein protein 1 (BASP1) bara ụba nke ụbụrụ, nwere oke njikọ gara aga na neurodegeneration. Ọ dị mma ịmara na n'ihi ụbara ha dị ala na proteome CSF, ọ na-esiri anyị ike iji usoro nchọpụta otu ihe dị elu iji chọpụta MAPT na ụfọdụ protein ndị ọzọ metụtara AD (dịka ọmụmaatụ, NEFL na NRGN). ) ( 43, 44 ).
Anyị wee lelee akara nrịbama 60 ndị a na nyocha ntugharị ọzọ atọ ọzọ. Na CSF Copy 2, anyị jiri otu TMT-MS nyochaa otu ndị nọọrọ onwe ha nke njikwa 297 na ihe atụ AD sitere na Emory Goizueta ADRC (17). Ntugharị CSF 3 gụnyere nyochagharị nke data TMT-MS dị site na njikwa 120 yana ndị ọrịa AD sitere na Lausanne, Switzerland (45). Anyị achọpụtala ihe karịrị ụzọ abụọ n'ụzọ atọ nke akara 60 dị mkpa na nhazi data ọ bụla. Ọ bụ ezie na ọmụmụ ihe Switzerland na-eji usoro MS dị iche iche na ụzọ ọnụọgụ TMT (45, 46), anyị na-emepụtaghachi usoro panel anyị na nyocha abụọ ugboro ugboro (Njirimara 5, C na D, na Tebụl S2, I, na J). Iji nyochaa nkọwapụta ọrịa nke otu anyị, anyị jiri TMT-MS nyochaa usoro nhazi nke anọ nke anọ (CSF replication 4), nke gụnyere ọ bụghị naanị njikwa (n = 18) na AD (n = 17), kamakwa PD. n = 14)), ALS (n = 18) na frontotemporal dementia (FTD) samples (n = 11) (Table S1A). Anyị gụpụtara ihe fọrọ nke nta ka ọ bụrụ ụzọ abụọ n'ụzọ atọ nke protein panel dị na otu a (38 n'ime 60). Nsonaazụ ndị a na-akọwapụta mgbanwe AD-kpọmkwem na akụkụ ise biomarker ise niile (Ọnọdụ 5E na Tebụl S2K). Mmụba nke otu metabolite gosipụtara nkọwapụta AD siri ike, nke myelination na glial sochiri ya. Ruo n'ókè dị nta, FTD na-egosikwa mmụba n'etiti akụkụ ndị a, nke nwere ike igosipụta mgbanwe netwọk nwere ike ime (17). N'ụzọ dị iche, ALS na PD gosipụtara ihe fọrọ nke nta ka ọ bụrụ otu profaịlụ myelination, glial, na metabolome dị ka otu njikwa. N'ozuzu, n'agbanyeghị ọdịiche dị na nkwadebe nlele, ikpo okwu MS, na usoro ọnụọgụ TMT, nyocha ndị a ugboro ugboro na-egosi na ihe nrịbama panel anyị nwere mgbanwe dị iche iche AD na-agbanwe agbanwe na ihe karịrị 500 CSF pụrụ iche.
AD neurodegeneration ka amatala nke ọma ọtụtụ afọ tupu mmalite nke mgbaàmà uche, yabụ enwere mkpa ngwa ngwa maka ndị na-ahụ maka biomarkers nke AsymAD (5, 31). Otú ọ dị, ihe àmà ndị ọzọ na-egosi na ihe ndị dị ndụ nke AsymAD dị anya site na otu, na mmekọrịta dị mgbagwoju anya nke ihe ize ndụ na nkwụsịtụ na-eduga n'ọtụtụ dị iche iche nke onye ọ bụla na ọganihu ọrịa na-esote (47). Ọ bụ ezie na ejiri ya iji chọpụta okwu AsymAD, ọkwa nke isi CSF biomarkers (Aβ1-42, total tau na p-tau) egosighi na ha nwere ike ịkọwa onye ga-enwe ọganihu na dementia (4, 7), na-egosi karịa Ọ nwere ike ịbụ. ọ dị mkpa ịgụnye ngwaọrụ biomarker holistic dabere n'ọtụtụ akụkụ nke physiology ụbụrụ iji kọwaa nke ọma ihe ize ndụ nke ndị a. Ya mere, anyị mechara nyochaa AD-validated biomarker panel na ọnụ ọgụgụ ndị AsymAD nke CSF oyiri 1. Ihe ndị a 31 AsymAD gosipụtara ọkwa dị iche iche nke biomarker (Aβ1-42 / ngụkọta tau ELISA ratio, <5.5) na cognition zuru oke (pụtara MoCA, 27.1). ± 2.2) (Obodo S1A). Na mgbakwunye, ndị niile nwere AsymAD nwere akara dementia ụlọ ọgwụ nke 0, na-egosi na ọ nweghị ihe akaebe na-egosi mbelata n'ime ọgụgụ isi kwa ụbọchị ma ọ bụ arụmọrụ arụ ọrụ.
Anyị buru ụzọ nyochaa ọkwa nke paịlị akwadoro na 96 CSF megharịrị 1, gụnyere ndị otu AsymAD. Anyị chọpụtara na ọtụtụ ogwe dị na otu AsymAD nwere mgbanwe dị ukwuu AD-dị ka mgbanwe dị ukwuu, panel vaskụla gosipụtara ọnọdụ dị ala na AsymAD, ebe akụkụ ndị ọzọ niile gosipụtara ọganihu elu (Nyocha 6A). Ya mere, akụkụ niile gosipụtara mmekọrịta dị oke mkpa na ELISA Aβ1-42 na mkpokọta tau (Figure 6B). N'ụzọ dị iche, njikọ dị n'etiti otu na akara MoCA adịghị mma. Otu n'ime nchoputa kacha pụta ìhè site na nyocha ndị a bụ nnukwu ọnụ ọgụgụ panel dị na otu AsymAD. Dịka e gosiri na eserese 6A, ọkwa panel nke otu AsymAD na-agafekarị ọkwa panel nke otu njikwa na otu AD, na-egosi mgbanwe dị elu. Iji nyochaa ụdị dị iche iche nke AsymAD, anyị tinyere nyocha Multidimensional Scaling (MDS) na 96 CSF replication 1. Nyocha MDS na-enye ohere iji anya nke uche hụ myirịta dị n'etiti ikpe dabere na ụfọdụ mgbanwe dị na nhazi data. Maka nchịkọta ụyọkọ a, anyị na-eji naanị akara nrịbama panel ndị ahụ nwere mgbanwe dị ịrịba ama (P <0.05, AD/control) na nchọpụta CSF na mmụgharị 1 proteome (n = 29) (Table S2L). Nyocha a weputara mkpokọta oghere doro anya n'etiti njikwa anyị na ikpe AD (Njirimara 6C). N'ụzọ dị iche, ụfọdụ okwu AsymAD na-agbakọta nke ọma na otu njikwa, ebe ndị ọzọ dị na ikpe AD. Iji nyochaa ụdị dị iche iche nke AsymAD, anyị jiri maapụ MDS kọwaa otu abụọ nke ikpe AsymAD ndị a. Otu nke mbụ gụnyere okwu AsymAD gbakọtara nso na njikwa (n = 19), ebe otu nke abụọ bụ nke AsymAD nwere profaịlụ akara nso AD (n = 12).
(A) ọkwa ọkwa (z-score) nke CSF biomarker group na 96 sample na CSF replication 1 cohort, gụnyere AsymAD. A na-eji nyocha nke iche na nzizi Tukey wee nyochaa mkpa mgbakọ na mwepụ nke mgbanwe njupụta panel. (B) Nyocha njikọ nke ọkwa protein nke ukwuu (z-score) yana akara MoCA na mkpokọta tau na ELISA Aβ1-42 na CSF oyiri 1 sample. E gosipụtara ọnụọgụ njikọ Pearson yana uru P dabara adaba. (C) MDS nke 96 CSF oyiri 1 ikpe dabere na ọkwa dị ukwuu nke akara ngosi panel 29 kwadoro, bụ nke gbanwere nke ukwuu na nchọpụta ahụ na CSF oyiri 1 data set [P <0.05 AD/control (CT)]. A na-eji nyocha a kewaa otu AsymAD n'ime njikwa (n = 19) na AD (n = 12). (D) Mkpesa ugwu mgbawa na-egosi ụdị dị iche iche nke protein CSF 1 niile nwere mgbanwe mgbanwe log2 (x-axis) metụtara uru -log10 statistical P n'etiti otu abụọ AsymAD. The panel biomarkers bụ agba. (E) CSF mmeghari 1 n'ọkwa dị ukwuu nke ndị otu biomarkers ka egosipụtara iche n'etiti otu obere AsymAD. A na-eji nyocha mgbanwe nke Tukey emezigharịrị nke mgbanwe iji chọpụta mkpa mgbakọ na mwepụ.
Anyị enyochala okwu protein dị iche n'etiti njikwa ndị a na AD-dị ka okwu AsymAD (Njirimara 6D na tebụl S2L). Map mgbawa ugwu na-apụta na-egosi na akara ngosi panel 14 agbanweela nke ukwuu n'etiti otu abụọ ahụ. Ọtụtụ n'ime akara ndị a bụ akụkụ nke synapse na metabolome. Otú ọ dị, SOD1 na myristoylated alanine-rich protein kinase C substrate (MARCKS), bụ ndị so na myelin na glial dịghịzi otu, n'otu n'otu, sokwa na otu a (Nyocha 6, D na E). Ogwe vaskụla ahụ nyekwara akara abụọ nke belatara nke ukwuu na AD-dị ka AsymAD otu, gụnyere AE binding protein 1 (AEBP1) yana onye otu ezinụlọ C9. Enweghị nnukwu ọdịiche dị n'etiti njikwa na AD-dị ka AsymAD subgroups na ELISA AB1-42 (P = 0.38) na p-tau (P = 0.28), mana enwere nnukwu ọdịiche dị na mkpokọta tau (P = 0.0031). ) (Fig. S7). Enwere ọtụtụ nrịbama panel nke na-egosi na mgbanwe dị n'etiti nkeji abụọ nke AsymAD dị ịrịba ama karịa ngụkọta tau (dịka ọmụmaatụ, YWHAZ, SOD1, na MDH1) (Figure 6E). N'ozuzu, nsonaazụ ndị a na-egosi na panel anyị akwadoro nwere ike ịnwe biomarkers nke nwere ike ịdepụta ihe ize ndụ nke ndị ọrịa nwere ọrịa asymptomatic.
Enwere mkpa ngwa ngwa maka ngwaọrụ biomarker dabere na sistemụ iji tụọ ma lekwasịrị anya pathophysiology dị iche iche n'azụ AD. Ngwa ndị a na-atụ anya na ọ bụghị naanị na ọ ga-agbanwe usoro nchọpụta AD anyị, kamakwa ọ na-akwalite nnabata nke usoro ọgwụgwọ dị irè, nke akọwapụtara onye ọrịa (1, 2). Iji mezuo nke a, anyị tinyere usoro proteomics zuru oke na-enweghị isi na ụbụrụ AD na CSF iji chọpụta ndị na-ahụ maka biomarkers CSF nke na-egosipụta ọtụtụ ụdị pathophysiology nke ụbụrụ. Nyocha anyị mepụtara panel CSF biomarker ise, nke (i) na-egosipụta synapses, arịa ọbara, myelin, mgbochi na metabolic dysfunction; (ii) na-egosipụta ike reproducibility na dị iche iche MS nyiwe; (iii) Gosi mgbanwe ọrịa na-aga n'ihu n'oge mmalite na ngwụcha nke AD. N'ozuzu, nchoputa ndị a na-anọchite anya nzọụkwụ na-ekwe nkwa maka mmepe nke ngwaọrụ biomarker dị iche iche, nke a pụrụ ịdabere na ya, nke dabeere na weebụ maka nyocha AD na ngwa ụlọ ọgwụ.
Nsonaazụ anyị gosipụtara nzukọ echekwara nke ukwuu nke AD ụbụrụ netwọk proteome ma kwado iji ya dị ka arịlịka maka mmepe biomarker dabere na sistemụ. Nyocha anyị na-egosi na TMT-MS dataset abụọ nọọrọ onwe ha nwere AD na ụbụrụ AsymAD nwere modularity siri ike. Nchọpụta ndị a na-agbatị ọrụ anyị gara aga, na-egosipụta ichekwa modul dị ike nke ihe karịrị ụbụrụ ụbụrụ 2,000 sitere na ọtụtụ ndị otu nọọrọ onwe ha na frontal, parietal, and temporal cortex (17). Netwọk nkwekọrịta a na-egosipụta mgbanwe dị iche iche metụtara ọrịa a na-ahụ na nyocha ugbu a, gụnyere mmụba nke glial-rich modul inflammatory na mbelata nke modul bara ụba neuron. Dị ka nyocha ugbu a, netwọkụ a buru ibu na-egosipụtakwa mgbanwe modular dị ịrịba ama na AsymAD, na-egosi ụdị pathophysiology dị iche iche dị iche iche (17).
Otú ọ dị, n'ime usoro a na-agbanwe agbanwe nke ukwuu, a na-enwe ụdị ọdịdị dị iche iche nke ndụ, karịsịa n'etiti ndị mmadụ n'otu n'otu na mmalite nke AD. Ogwe biomarker anyị nwere ike igosipụta otu subgroups abụọ na AsymAD, nke gosipụtara ngosipụta dị iche iche nke ọtụtụ akara CSF. Ndị otu anyị nwere ike igosi ọdịiche dị n'ime ndụ dị n'etiti otu abụọ a, nke na-apụtaghị ìhè na ọkwa nke isi AD biomarkers. E jiri ya tụnyere otu njikwa, ọnụọgụ Aβ1-42/ mkpokọta tau nke ndị AsymAD ndị a dị obere nke ọma. Agbanyeghị, ọ bụ naanị mkpokọta tau dị iche iche dị iche n'etiti otu abụọ AsymAD, ebe ọkwa Aβ1-42 na p-tau ka dị ka nke a. Ebe ọ bụ na CSF dị elu yiri ka ọ bụ amụma dị mma nke mgbaàmà uche karịa ọkwa Aβ1-42 (7), anyị na-eche na ndị otu AsymAD abụọ nwere ike ịnwe ihe ize ndụ dị iche iche nke ọganihu ọrịa. N'inye ntakịrị ihe nlele nke AsymAD anyị yana enweghị data ogologo oge, achọrọ nyocha ọzọ iji jiri obi ike nweta nkwubi okwu ndị a. Agbanyeghị, nsonaazụ ndị a na-egosi na panel CSF nke dabere na sistemụ nwere ike ịkwalite ikike anyị ịhazi ndị mmadụ n'otu n'otu nke ọma n'oge ọrịa asymptomatic nke ọrịa ahụ.
N'ozuzu, nchọpụta anyị na-akwado ọrụ nke ọtụtụ ọrụ ndu na pathogenesis nke AD. Agbanyeghị, metabolism ike na-adịghị agwụ agwụ ghọrọ isiokwu pụtara ìhè nke akụkụ ise anyị niile enwetara nkwado. Ndị na-edozi ahụ na-edozi ahụ, dị ka hypoxanthine-guanine phosphoribosyltransferase 1 (HPRT1) na lactate dehydrogenase A (LDHA), bụ ihe ndị na-emepụta ihe na-emepụta ihe na-emepụta ihe na-eme ka ọ dịkwuo ike, na-egosi na mmụba nke AD CSF na-enwe mmekọahụ nke ukwuu. Ọbara ọbara anyị na akụkụ glial nwekwara ọtụtụ akara na-etinye aka na metabolism nke ihe oxidative. Nchọpụta ndị a kwekọrọ na isi ọrụ usoro metabolic na-arụ na ụbụrụ dum, ọ bụghị naanị iji gboo mkpa ike dị elu nke neurons, kamakwa iji gboo mkpa ike dị elu nke astrocytes na sel glial ndị ọzọ (17, 48). Nsonaazụ anyị na-akwado ihe akaebe na-eto eto na mgbanwe na ikike redox na nkwụsị nke ụzọ ike nwere ike ịbụ njikọ dị n'etiti ọtụtụ usoro isi na-etinye aka na pathogenesis nke AD, gụnyere ọrịa mitochondrial, mbufụt mgbasa ozi glial, na mmebi Vascular (49). Tụkwasị na nke ahụ, ndị na-emepụta ihe ndị na-emepụta mmiri nke cerebrospinal metabolic nwere ọnụ ọgụgụ dị ukwuu nke protein dị iche iche dị iche iche n'etiti njikwa anyị na AD-dị ka AsymAD subgroups, na-atụ aro na nkwụsị nke ike ndị a na ụzọ redox nwere ike dị oke egwu na ọkwa mbụ nke ọrịa ahụ.
Ụbụrụ dị iche iche na usoro akụkụ mmiri cerebrospinal anyị hụrụ nwekwara mmetụta dị ndụ na-atọ ụtọ. Synapses na metabolomes bara ụba na neurons na-egosi mbelata ọkwa dị na ụbụrụ AD yana ịba ụba nke mmiri cerebrospinal. Nyere na neurons bara ụba na mitochondria na-emepụta ume na synapses iji nye ike maka ọtụtụ akara ngosi pụrụ iche (50), a na-atụ anya myirịta nke profaịlụ okwu nke otu neuron abụọ a. Ọnwụ nke neurons na extrusion nke mkpụrụ ndụ mebiri emebi nwere ike ịkọwa ụbụrụ ndị a na usoro CSF ​​na ọrịa na-esote, ma ha enweghị ike ịkọwa mgbanwe mgbanwe nke mbụ anyị na-ahụ (13). Otu nkọwa enwere ike maka nchoputa ndị a n'oge ọrịa asymptomatic mbụ bụ ịkwachaa synapti na-adịghị mma. Ihe akaebe ohuru na ụdị òké na-egosi na phagocytosis na-emekọrịta ihe na microglia nwere ike ịgbalite nke ọma na AD wee bute mfu synapse n'isi na ụbụrụ (51). Ihe synaptik a tụfuru nwere ike ịgbakọta na CSF, nke mere anyị ji hụ mmụba nke CSF na panel neuron. Ịkwachaa synapti nke na-eme ka ihe mgbochi ọrịa nwekwara ike ịkọwa akụkụ nke mmụba nke protein glial anyị na-ahụ na ụbụrụ na mmiri ụbụrụ cerebrospinal n'oge usoro ọrịa a. Na mgbakwunye na ịkwachaa synapti, ihe na-adịghị mma n'ozuzu na ụzọ exocytic nwekwara ike iduga ụbụrụ dị iche iche na okwu CSF nke akara akara neuronal. Ọtụtụ ọmụmụ egosila na ọdịnaya nke exosomes na pathogenesis nke ụbụrụ AD agbanweela (52). Ụzọ extracellular na-etinyekwa aka na mmụba nke Aβ (53, 54). Ọ dị mma ịmara na nkwụsị nke nzuzo nzuzo nwere ike belata AD-dị ka pathology na AD transgenic òké ụdị (55).
N'otu oge ahụ, protein dị na vascular panel gosipụtara mmụba na-agafeghị oke na ụbụrụ AD, mana ọ na-ebelata nke ukwuu na CSF. Ihe mgbochi ụbụrụ ọbara (BBB) ​​nwere ike ịkọwa nchoputa ndị a akụkụ. Ọtụtụ nchọpụta mmadụ nwere onwe ya egosila ndakpọ BBB na AD (56, 57). Ọmụmụ ihe ndị a gosipụtara ihe omume dị iche iche na-adịghị mma nke gbara oyi akwa nke mkpụrụ ndụ endothelial a mechiri emechi, gụnyere mgbapu ụbụrụ ụbụrụ na mkpokọta perivascular nke protein na-ebute ọbara (57). Nke a nwere ike inye nkọwa dị mfe maka protein vaskụla dị elu na ụbụrụ, ma ọ pụghị ịkọwa n'ụzọ zuru ezu nbibi nke otu protein ndị a na mmiri cerebrospinal. Otu ihe nwere ike ime bụ na usoro nhụjuanya nke etiti na-arụsi ọrụ ike ikewapụ ụmụ irighiri ihe ndị a iji dozie nsogbu nke ịba ụba nke mbufụt na nrụgide oxidative. Mbelata nke ụfọdụ n'ime protein CSF kachasị njọ na panel a, karịsịa ndị na-etinye aka na nhazi nke lipoprotein, metụtara mgbochi nke nsị na-emerụ ahụ na usoro neuroprotective nke ụdị oxygen na-emeghachi omume. Nke a bụ eziokwu maka Paroxonase 1 (PON1), enzyme na-ejikọta lipoprotein na-ahụ maka ibelata nrụgide oxidative na mgbasa (58, 59). Alfa-1-microglobulin/bikunin precursor (AMBP) bụ akara ọzọ na-agbadata nke ọma nke otu vaskụla. Ọ bụ ihe mmalite nke onye na-ebugharị lipid bikunin, nke na-etinyekwa aka na nkwụsị ọkụ na Nchedo akwara (60, 61).
N'agbanyeghị echiche dị iche iche na-adọrọ mmasị, enweghị ike ịchọpụta kpọmkwem usoro ọrịa nke biochemical bụ mmachi ama ama nke nyocha proteomics na-akpata nchoputa. Ya mere, nyocha ọzọ dị mkpa iji jiri obi ike kọwaa usoro dị n'azụ ogwe biomarker ndị a. Iji gaa n'ihu na mmepe nke nyocha ụlọ ọgwụ dabeere na MS, ntụziaka n'ọdịnihu na-achọkwa iji usoro ọnụọgụ nke ezubere iche maka nnukwu nkwenye biomarker, dị ka nhọrọ ma ọ bụ nleba anya mmeghachi omume yiri ya (62). N'oge na-adịbeghị anya, anyị jiri nleba anya mmeghachi omume yiri ya (63) iji kwado ọtụtụ mgbanwe protein CSF akọwara ebe a. A na-agụta ọtụtụ ebumnuche ndị kacha mkpa n'ụzọ ziri ezi, gụnyere YWHAZ, ALDOA, na SMOC1, bụ ndị na-esepụta eserese nke synapse anyị, metabolism, na akụkụ mbufụt, n'otu n'otu (63). Nnweta Data Independent (DIA) na atumatu MS ndị ọzọ nwere ike ịba uru maka nkwenye ebumnuche. Bud et al. (64) E gosipụtara ya n'oge na-adịbeghị anya na enwere nnukwu ndakọrịta n'etiti AD biomarkers nke achọpụtara na nhazi data CSF anyị yana setịpụrụ data DIA-MS nọọrọ onwe ya, nke nwere ihe fọrọ nke nta ka ọ bụrụ 200 CSF sample sitere na otu ndị otu European atọ dị iche iche. Ọmụmụ ihe ndị a na-adịbeghị anya na-akwado ikike nke akụkụ anyị ịgbanwe ka ọ bụrụ nchọpụta dabeere na MS nke a pụrụ ịdabere na ya. Omenala mgbochi mgbochi na nchọpụta dabere na aptamer dịkwa mkpa maka mmepe n'ihu nke ndị isi AD biomarkers. N'ihi ụbara CSF dị ala, ọ na-esiri ike ịchọpụta ndị biomarkers site na iji ụzọ MS dị elu. NEFL na NRGN bụ ihe atụ abụọ dị otú ahụ nke CSF biomarkers dị ala, nke edobere na panel na nyocha anyị zuru oke, mana enweghị ike ịchọta ya site na iji otu atụmatụ MS anyị. Atụmatụ ezubere iche dabere na ọtụtụ ọgwụ mgbochi ọrịa, dị ka PEA, nwere ike ịkwalite mgbanwe ụlọ ọgwụ nke akara ndị a.
N'ozuzu, ọmụmụ a na-enye ụzọ proteomics pụrụ iche maka njirimara na nkwenye nke CSF AD biomarkers dabere na usoro dị iche iche. Ịkwalite ogwe nrịbama ndị a n'ofe ndị otu AD ndị ọzọ na nyiwe MS nwere ike igosi na ha na-ekwe nkwa ịkwalite nhụsianya na ọgwụgwọ AD. Nnyocha ndị na-enyocha ogo ogologo ogologo nke akụkụ ndị a ka oge na-aga dịkwa oke mkpa iji chọpụta nchikota akara kacha mma na-akọwapụta ihe ize ndụ nke ọrịa mmalite na mgbanwe na oke ọrịa.
Ewezuga ihe nlele 3 nke CSF depụtaghachiri, anakọtara ụdị CSF niile ejiri mee ihe n'ọmụmụ ihe a n'okpuru nkwado Emory ADRC ma ọ bụ ụlọ ọrụ nyocha nwere njikọ chiri anya. A na-eji ngụkọta nke ụdị anọ nke Emory CSF mee ihe na ọmụmụ ihe gbasara ahụike. Achọpụtara ndị otu CSF nwere ihe nlele sitere na njikwa ahụike 20 yana ndị ọrịa 20 AD. Mpempe akwụkwọ CSF 1 gụnyere ihe nlele sitere na njikwa ahụike 32, ndị mmadụ 31 AsymAD, na ndị 33 AD. CSF oyiri 2 nwere njikwa 147 na ihe atụ 150 AD. Otu ìgwè 4 nke CSF nwere ọtụtụ ọrịa gụnyere njikwa 18, 17 AD, 19 ALS, 13 PD, na ụdị 11 FTD. Dika nkwekorita nke ndi Emory University Institutional Review Board kwadoro, ndi nile n'eme ihe omumu Emory nwetara nkwenye amara. Dị ka 2014 National Institute of Aging Best Practice Guidelines for Alzheimer's Centers (https://alz.washington.edu/BiospecimenTaskForce.html) si kwuo, a na-anakọta ma chekwaa mmiri mmiri cerebrospinal site na nkwụsị lumbar. Njikwa na ndị ọrịa AsymAD na AD nwetara ntule ọgụgụ isi na Emory Cognitive Neurology Clinic ma ọ bụ Goizueta ADRC. A nwalere ihe nlele mmiri nke cerebrospinal ha site na INNO-BIA AlzBio3 Luminex maka ELISA Aβ1-42, mkpokọta tau na p-tau analysis (65). A na-eji ụkpụrụ ELISA na-akwado nhazi nchọpụta nchọpụta nke isiokwu dabere na AD biomarker njirisi njedebe (66, 67). A na-enwetakwa data gbasara igwe mmadụ yana data nyocha maka nyocha CSF ndị ọzọ (FTD, ALS, na PD) site na Emory ADRC ma ọ bụ ụlọ ọrụ nyocha metụtara. Enwere ike ịhụ metadata nchịkọta akụkọ maka ikpe Emory CSF na tebụl S1A. Ebipụtala njiri mara nke Switzerland CSF replication 3 cohort (45).
CSF chọtara nlele ahụ. Ka iwelie omimi nke nchọpụta anyị nke nhazi data CSF, a na-eme oriri na-edozi ahụ nke protein dị ukwuu tupu trypsinization. Na nkenke, 130 μl nke CSF sitere na 40 CSF samples na nha nhata (130 μl) nke High Select Top14 Abundance Protein Depletion Resin (Thermo Fisher Scientific, A36372) ka etinyere na kọlụm ntụgharị (Thermo Fisher Scientific, A89868) n'ime ụlọ. okpomọkụ Incubate). Mgbe ị na-atụgharị maka nkeji 15, centrifuge sample na 1000g maka nkeji 2. A na-eji ngwaọrụ nzacha ultracentrifugal 3K (Millipore, UFC500396) tinye uche n'omume na-asọ asọ site na centrifuging na 14,000g maka nkeji iri atọ. Gwakọta mpịakọta niile na 75 μl na saline buffered phosphate. Ejiri usoro bicinchoninic acid (BCA) nyochaa ntinye protein dị ka usoro onye nrụpụta (Thermo Fisher Scientific). CSF (60 μl) na-egbochi ọrịa ahụ sitere na nlele 40 niile ejiri lysyl endopeptidase (LysC) na trypsin gbarie. Na nkenke, sample e belatara na alkylated na 1.2 μl 0.5 M tris-2 (-carboxyethyl) -phosphine na 3 μl 0.8 M chloroacetamide na 90 Celsius C maka 10 nkeji, na mgbe ahụ sonicated na a mmiri saa maka 15 nkeji. Ejiri ihe nkpuchi urea 193 μl 8M [8 M urea na 100 mM NaHPO4 (pH 8.5)] gbazere ihe nlele ahụ na ntinye ikpeazụ nke 6 M urea. A na-eji LysC (4.5 μg; Wako) maka mgbari abalị n'ime ụlọ okpomọkụ. A na-agbanye ihe nlele ahụ na 1 M urea na 50 mM ammonium bicarbonate (ABC) (68). Tinye nha nha (4.5 μg) nke trypsin (Promega), wee tinye ihe nlele ahụ maka awa iri na abụọ. Tinye ihe ngwọta peptide gbazere na ntinye ikpeazụ nke 1% formic acid (FA) na 0.1% trifluoroacetic acid (TFA) (66), wee desat na kọlụm 50 mg Sep-Pak C18 (Mmiri) dị ka akọwara n'elu (25) . E wepụrụ peptide ahụ na 1 ml nke 50% acetonitrile (ACN). Iji mee ka ọnụọgụ protein dị n'ofe batches (25), 100 μl aliquots sitere na ihe atụ 40 CSF niile jikọtara ọnụ iji mepụta ihe atụ agwakọtara, nke kewara n'ime ọkọlọtọ ise zuru ụwa ọnụ (GIS) (48). A na-akpọnwụ n'otu n'otu na ụkpụrụ agwakọtara site na oghere dị elu (Labconco).
CSF na-eṅomi ihe nlele ahụ. Dayon na ndị ọrụ ibe akọwala mbụ mbelata mgbochi na mgbaze nke CSF oyiri 3 sample (45, 46). Emebighị ihe mgbochi ọrịa ndị ọzọ n'otu n'otu. Gwakọta ihe nlele ndị a na-ewepụghị na trypsin dị ka akọwara na mbụ (17). Maka nyocha ọ bụla ugboro ugboro, 120 μl aliquots nke peptide eluted sitere na nlele ọ bụla ka agbakọtara ọnụ wee kee ya n'otu nha nha nha nha nke a ga-eji dị ka ọkọlọtọ ime ụwa TMT akpọrọ (48). A na-akpọnwụ n'otu n'otu na ụkpụrụ agwakọtara site na oghere dị elu (Labconco). Iji kwalite mgbaàmà nke protein CSF dị ala, site na ijikọta 125 μl site na nlele ọ bụla, a kwadebere ihe nlele "emelitere" maka nyocha nke ọ bụla [ya bụ, ihe nlele nke ndụ nke na-eṅomi nyocha nyocha, mana ego dịnụ bụ. buru ibu (37, 69)] jikọtara n'ime ihe nlele CSF agwakọtara (17). E wepụrụ ihe atụ agwakọta agwakọta site na iji 12 ml nke High Select Top14 Abundance Protein Removal Resin (Thermo Fisher Scientific, A36372), gbarie dị ka akọwara n'elu, ma tinye ya na ntinye aha ọtụtụ TMT na-esote.