Hōʻike nā proteomic piha i nā biomarkers wai cerebrospinal e pili ana i ka lolo i ka maʻi asymptomatic a me ka maʻi Alzheimer.

ʻAʻole nele ka maʻi o Alzheimer (AD) i nā biomarkers protein e hōʻike ana i kāna mau pathophysiology kumu, keakeʻa nei i ka holomua o ka maʻi a me ka mālama ʻana. Maʻaneʻi, hoʻohana mākou i nā proteomic piha e ʻike ai i nā biomarkers cerebrospinal fluid (CSF) e hōʻike ana i kahi ākea ākea o AD pathophysiology. Ua ʻike ʻia ka nui o ka nui o ka spectrometry ma kahi o 3,500 a me 12,000 mau protein i AD CSF a me ka lolo. Ua hoʻoholo ka ʻikepili pūnaewele o ka proteome o ka lolo i nā modules biodiversity 44, ʻo 15 o ia mau mea i uhi ʻia me ka cerebrospinal fluid proteome. Hoʻopili ʻia nā māka CSF AD i loko o kēia mau module overlapping i ʻelima pūʻulu protein, e hōʻike ana i nā kaʻina pathophysiological like ʻole. ʻO nā synapses a me nā metabolites i loko o ka lolo AD e emi iho, akā hoʻonui ka CSF, aʻo ka glial-rich myelination a me nā pūʻulu immune i ka lolo a me ka CSF piʻi. Ua hōʻoia ʻia ke kūlike a me ka maʻi kikoʻī o nā hoʻololi panel ma mua o 500 mau hōʻailona CSF hou. Ua ʻike pū kēia mau pūʻulu i nā pūʻulu olaola i ka asymptomatic AD. ʻO ka holoʻokoʻa, ʻo kēia mau hopena he hana hoʻohiki i nā mea hana biomarker pūnaewele no nā noi lapaʻau ma AD.
ʻO ka maʻi o Alzheimer (AD) ke kumu maʻamau o ka neurodegenerative dementia ma ka honua holoʻokoʻa a ua ʻike ʻia e ka nui o nā ʻōnaehana ʻōnaehana biological, me ka synaptic transmission, glial-mediated immunity, a me ka mitochondrial metabolism (1-3). Eia nō naʻe, ke nānā mau nei kāna mau mea biomarkers protein i ka ʻike ʻana i ka protein amyloid a me tau, a no laila ʻaʻole hiki ke hōʻike i kēia pathophysiology like ʻole. ʻO kēia mau mea biomarkers protein "core" i hoʻopaʻa ʻia i ka wai cerebrospinal fluid (CSF) e komo pū me (i) amyloid beta peptide 1-42 (Aβ1-42), e hōʻike ana i ka hoʻokumu ʻana o nā papa amyloid cortical; (ii) huina tau, he hoailona o ke axon degeneration; (iii) phospho-tau (p-tau), he 'elele o ka pathological tau hyperphosphorylation (4-7). ʻOiai ua hoʻoikaika nui kēia mau mea biomarkers wai cerebrospinal i kā mākou ʻike ʻana i nā maʻi protein "marked" AD (4-7), ʻo lākou wale nō kahi ʻāpana liʻiliʻi o ka biology paʻakikī ma hope o ka maʻi.
ʻO ka nele o ka pathophysiological diversity o AD biomarkers ua alakaʻi i nā pilikia he nui, ʻo ia hoʻi (i) ka hiki ʻole ke ʻike a helu i ka heterogeneity biological o nā maʻi maʻi AD, (ii) lawa ʻole ke ana o ka maʻi nui a me ka holomua, ʻoi aku hoʻi i ka pae preclinical. iii) ka hoʻomohala ʻana i nā lāʻau lapaʻau i hiki ʻole ke hoʻopau piha i nā ʻano āpau o ka deterioration neurological. ʻO kā mākou hilinaʻi ʻana i ka pathology pae'āina e wehewehe iā AD mai nā maʻi pili e hoʻonui wale i kēia mau pilikia. ʻOi aku ka nui o nā hōʻike e hōʻike ana i ka hapa nui o ka poʻe ʻelemakule me ka dementia i ʻoi aku ma mua o hoʻokahi ʻano pathological o ka emi ʻana o ka cognitive (8). Ma kahi o 90% a ʻoi aʻe paha o ka poʻe me AD pathology i loaʻa pū kekahi maʻi vascular, TDP-43 inclusions, a i ʻole nā ​​​​maʻi degenerative ʻē aʻe (9). Ua hoʻopilikia kēia mau ʻāpana kiʻekiʻe o ka pathological overlap i kā mākou papa hana diagnostic i kēia manawa no ka dementia, a makemake ʻia kahi wehewehe pathophysiological o ka maʻi.
Ma muli o ka makemake nui o nā ʻano biomarkers AD, ke hoʻonui nei ke kahua i ke ʻano "omics" e pili ana i ka ʻōnaehana holoʻokoʻa e ʻike ai i nā biomarkers. Ua hoʻokumu ʻia ka Accelerated Pharmaceutical Partnership (AMP)-AD Alliance i ka makahiki 2014 a aia ma ke poʻo o ka papahana. ʻO kēia hana multidisciplinary e ka National Institutes of Health, academia, a me ka ʻoihana e manaʻo e hoʻohana i nā hoʻolālā e pili ana i ka ʻōnaehana e wehewehe maikaʻi i ka pathophysiology o AD a hoʻomohala i ka loiloi diagnostic biodiversity a me nā hoʻolālā lapaʻau (10). Ma keʻano he māhele o kēia papahana, ua lilo nā proteomics pūnaewele i mea hoʻohiki no ka holomua o nā biomarkers pūnaewele i AD. Hoʻonohonoho kēia ʻano ʻikepili i hoʻonohonoho ʻia i nā ʻikepili proteomic paʻakikī i loko o nā pūʻulu a i ʻole "modules" o nā protein co-expressed e pili pū ana me nā ʻano kelepona kikoʻī, organelles, a me nā hana biological (11-13). Ma kahi o 12 mau haʻawina proteomic pūnaewele waiwai nui i mālama ʻia ma ka lolo AD (13-23). Ma ke ʻano holoʻokoʻa, hōʻike kēia mau loiloi i ka mālama ʻana o ka proteome network brain AD i kahi hui modular mālama ʻia i nā cohorts kūʻokoʻa a me nā ʻāpana cortical. Eia kekahi, ʻo kekahi o kēia mau modules e hōʻike i nā loli reproducible i ka nui e pili ana i ka AD ma nā pūʻulu ʻikepili, e hōʻike ana i ka pathophysiology o nā maʻi he nui. ʻO ka hui pū ʻana, hōʻike kēia mau ʻike i kahi wahi heleuma hoʻohiki no ka loaʻa ʻana o ka proteome pūnaewele lolo ma ke ʻano he biomarker pūnaewele i AD.
I mea e hoʻololi ai i ka proteome o ka lolo AD i loko o nā biomarkers e pili ana i ka ʻōnaehana pono, ua hoʻohui mākou i ka pūnaewele i loaʻa i ka lolo me ka loiloi proteomic o AD CSF. ʻO kēia ala hoʻohui i alakaʻi ʻia i ka ʻike ʻana o ʻelima mau ʻōlelo hoʻohiki o nā biomarkers CSF e pili pū ana me kahi ākea o ka pathophysiology e pili ana i ka lolo, me nā synapses, nā kīʻaha koko, ka myelination, ka mumū, a me ka dysfunction o nā ala metabolic. Ua hōʻoia maikaʻi mākou i kēia mau panela biomarker ma o nā loiloi replication he nui, me ka ʻoi aku o 500 CSF samples mai nā maʻi neurodegenerative like ʻole. Hoʻopili kēia mau loiloi hōʻoia i ka nānā ʻana i nā pahuhopu pūʻulu i ka CSF o nā mea maʻi me ka asymptomatic AD (AsymAD) a i ʻole ka hōʻike ʻana i nā hōʻike o ka hōʻiliʻili amyloid maʻamau i kahi ʻano cognitive maʻamau. Hōʻike kēia mau kānana i ka heterogeneity biological nui i ka heluna AsymAD a ʻike i nā hōʻailona panel e hiki ke hoʻololi i nā poʻe i nā pae mua loa o ka maʻi. Ma ka holoʻokoʻa, hōʻike kēia mau hopena i kahi hana koʻikoʻi i ka hoʻomohala ʻana i nā mea hana biomarker protein e pili ana i nā ʻōnaehana lehulehu e hiki ke hoʻoponopono maikaʻi i nā pilikia lapaʻau i kū ʻia e AD.
ʻO ke kumu nui o kēia noiʻi ʻana ʻo ia ka ʻike ʻana i nā biomarkers wai cerebrospinal hou e hōʻike ana i nā ʻano pathophysiology e pili ana i ka lolo e alakaʻi ai i AD. Hōʻike ka Figure S1 i kā mākou noiʻi noiʻi, e komo pū ana me (i) kahi loiloi piha i alakaʻi ʻia e nā ʻike mua o AD CSF a me ka pale o ka lolo pūnaewele e ʻike ai i nā biomarkers maʻi CSF e pili ana i ka lolo, a (ii) ka hoʻopiʻi hou ʻana Aia kēia mau biomarkers i kekahi cerebrospinal kūʻokoʻa. nā pūʻulu wai. Ua hoʻomaka ka ʻimi noiʻi ʻimi me ka nānā ʻana i ka ʻōlelo ʻokoʻa o CSF ​​i 20 mau kānaka maʻamau a me 20 mau maʻi AD ma Emory Goizueta Alzheimer's Disease Research Center (ADRC). Ua wehewehe ʻia ka hōʻailona o AD ma ke ʻano he hōʻino koʻikoʻi koʻikoʻi ma ke alo o ka haʻahaʻa Aβ1-42 a me nā pae kiʻekiʻe o ka nui o ka tau a me ka p-tau i loko o ka wai cerebrospinal [Mean Montreal Cognitive Assessment (MoCA), 13.8 ± 7.0] [ELISA (ELISA). )]] (Pakaukau S1A). ʻO ka mana (mean MoCA, 26.7 ± 2.2) loaʻa nā pae maʻamau o nā biomarkers CSF.
Hōʻike ʻia ka CSF kanaka e ka nui o ka nui o ka protein, kahi e hiki ai i ka albumin a me nā protein nui ʻē aʻe ke pale i ka ʻike ʻana i nā protein o ka hoihoi (24). No ka hoʻonui ʻana i ka hohonu o ka loaʻa ʻana o ka protein, ua wehe mākou i nā protein mua 14 nui loa mai kēlā me kēia laʻana CSF ma mua o ka nānā ʻana o mass spectrometry (MS) (24). Ua ʻike ʻia he 39,805 peptides e MS, i hoʻopaʻa ʻia i 3691 proteomes i 40 mau mea hoʻohālike. Hana ʻia ka helu ʻana o ka protein e ka nui o ka tandem mass tag (TMT) lepili (18, 25). I mea e hoʻoholo ai i ka ʻikepili nalo, ua hoʻokomo wale mākou i kēlā mau protein i helu ʻia ma ka liʻiliʻi o 50% o nā laʻana i ka nānā ʻana aʻe, no laila e helu ʻia nā proteomes 2875. Ma muli o ka ʻokoʻa koʻikoʻi o ka nui o ka nui o ka protein, ua manaʻo ʻia kahi ʻāpana hoʻomalu he outlier (13) a ʻaʻole i hoʻokomo ʻia i ka nānā ʻana ma hope. Ua hoʻoponopono ʻia nā waiwai nui o ke koena 39 e like me ka makahiki, ke kāne, a me ka covariance batch (13-15, 17, 18, 20, 26).
Ke hoʻohana nei i ka helu helu t-test analysis no ka loiloi ʻana i ka ʻōlelo ʻokoʻa ma ka hoʻonohonoho ʻikepili regression, ua ʻike kēia ʻike i nā protein nona ka nui o nā pae i hoʻololi nui ʻia (P <0.05) ma waena o ka mana a me nā hihia AD (Table S2A). E like me ka mea i hōʻike ʻia ma ka Figure 1A, ua hoʻemi nui ʻia ka nui o nā protein 225 i AD, a ua hoʻonui nui ʻia ka nui o nā protein 303. ʻO kēia mau protein i hōʻike ʻokoʻa ʻia he mau mea hōʻailona AD cerebrospinal fluid i ʻike mua ʻia, e like me ka microtubule-associated protein tau (MAPT; P = 3.52 × 10−8), neurofilament (NEFL; P = 6.56 × 10−3), ʻo Protein pili i ka ulu 43 (GAP43; P = 1.46 × 10−5), Fatty Acid Binding Protein 3 (FABP3; P = 2.00 × 10−5), Chitinase 3 like 1 (CHI3L1; P = 4.44 × 10−6), Neural Granulin (NRGN; P = 3.43 × 10-4) a me VGF nerve ulu kumu (VGF; P = 4.83 × 10-3) (4-6). Eia naʻe, ua ʻike pū mākou i nā pahuhopu koʻikoʻi ʻē aʻe, e like me ka GDP dissociation inhibitor 1 (GDI1; P = 1.54 × 10-10) a me SPARC pili i ka modular calcium binding 1 (SMOC1; P = 6.93 × 10-9). Hōʻike ʻo Gene Ontology (GO) o 225 i nā protein i hōʻemi nui ʻia i nā pilina pili me nā kaʻina wai o ke kino e like me ka metabolism steroid, ka coagulation koko, a me ka hana hormone (Figure 1B a me Table S2B). I ka hoʻohālikelike ʻana, pili loa ka protein i hoʻonui nui ʻia o 303 i ka hoʻolālā cell a me ka metabolism ikehu.
(A) Hōʻike ka ʻāpana lua pele i ka hoʻololi log2 paʻi (x-axis) pili i ka -log10 helu helu P waiwai (y-axis) i loaʻa ma ka hoʻāʻo-t, i hoʻohana ʻia no ka ʻike ʻana i ka ʻōlelo ʻokoʻa ma waena o ka mana (CT) a me Nā hihia AD o ka CSF proteome O nā protein a pau. Hōʻike ʻia nā protein me nā pae i hoʻemi nui ʻia (P <0.05) ma AD i ka uliuli, ʻoiai ʻo nā protein me ka piʻi nui ʻana o ka maʻi e hōʻike ʻia i ka ʻulaʻula. Hoʻopili ʻia ka protein i koho ʻia. (B) ʻO nā huaʻōlelo GO kiʻekiʻe e pili ana i ka protein e hoʻemi nui ʻia (uliuli) a hoʻonui ʻia (ʻulaʻula) i AD. Hōʻike i nā huaʻōlelo GO ʻekolu me nā helu-z kiʻekiʻe loa ma nā kahua o nā kaʻina olaola, nā hana molekala, a me nā ʻāpana kelepona. (C) Ua ana ʻo MS i ka pae MAPT ma ka laʻana CSF (hema) a me kona pilina me ka laʻana ELISA tau pae (akau). Hōʻike ʻia ka coefficient correlation Pearson me ka waiwai P pili. Ma muli o ka nele o ka ʻikepili ELISA no hoʻokahi hihia AD, aia kēia mau helu i nā waiwai no 38 o nā hihia i hoʻopaʻa ʻia he 39. (D) Ka nānā 'ana i ka hui 'ana (P <0.0001, Benjamini-Hochberg (BH) i ho'oponopono 'ia P <0.01) ma ka mana a me AD CSF i loa'a nā la'ana me ka ho'ohana 'ana i ka 65 i ho'ololi nui 'ia i loko o ka 'ikepili. Hoʻohālikelike, hana maʻamau.
ʻO ka pae proteomic o MAPT pili pili loa i ka pae ELISA tau i ana kūʻokoʻa (r = 0.78, P = 7.8 × 10-9; Kiʻi 1C), e kākoʻo ana i ka pono o kā mākou ana MS. Ma hope o ka ʻai ʻana o trypsin ma ke kiʻekiʻe o ka amyloid precursor protein (APP), ʻaʻole hiki ke hoʻopili maikaʻi ʻia nā peptides isoform-specific i ka C-terminus o Aβ1-40 a me Aβ1-42 (27, 28). No laila, ʻaʻohe pili o nā peptides APP a mākou i ʻike ai me nā pae ELISA Aβ1-42. I mea e loiloi ai i ka ʻōlelo ʻokoʻa o kēlā me kēia hihia, ua hoʻohana mākou i nā protein i hōʻike ʻokoʻa me ka P <0.0001 [fase discovery rate (FDR) i hoʻoponopono ʻia P <0.01] no ka hana ʻana i kahi kānana puʻupuʻu i mālama ʻia o nā laʻana (Table S2A). E like me ka mea i hōʻike ʻia ma ke Kiʻi 1D, hiki i kēia 65 mau protein koʻikoʻi ke hoʻohui pololei i nā laʻana e like me ke kūlana o ka maʻi, koe wale nō kahi hihia AD me nā ʻano like ʻole. ʻO kēia mau protein 65, ua hoʻonui ʻia ka 63 i AD, ʻoiai ʻelua wale nō (CD74 a me ISLR) i emi iho. I ka huina, ua ʻike ʻia kēia mau loiloi wai cerebrospinal i mau haneli o nā protein i AD hiki ke lilo i mau biomarkers maʻi.
A laila ua hana mākou i kahi loiloi pūnaewele kūʻokoʻa o ka proteome brain AD. ʻO ka cohort o ka lolo o kēia ʻike ua loaʻa ka dorsolateral prefrontal cortex (DLPFC) mai ka mana (n = 10), ka maʻi o Parkinson (PD; n = 10), hui pū ʻia ʻo AD / PD (n = 10) a me AD (n = 10). ) Laʻana. Emery Goizueta ADRC. Ua wehewehe mua ʻia ka heluna kanaka o kēia mau hihia 40 (25) a ua hōʻuluʻulu ʻia ma ka Papa S1B. Ua hoʻohana mākou i ka TMT-MS e nānā i kēia mau ʻiʻo lolo 40 a me ka cohort replication o nā hihia 27. I ka huina, ua hoʻokumu kēia mau ʻikepili lolo ʻelua i 227,121 peptides kūʻokoʻa, i kahakaha ʻia i 12,943 proteomes (25). ʻO kēlā mau protein wale nō i helu ʻia ma ka liʻiliʻi o 50% o nā hihia i hoʻokomo ʻia i nā hoʻokolokolo ma hope. Loaʻa i ka pūʻulu ʻikepili hope loa he 8817 mau protein i helu ʻia. E hoʻoponopono i ka nui o ka protein e pili ana i ka makahiki, ke kāne, a me ka post-mortem interval (PMI). Ua hōʻike ʻia ka hōʻike ʻokoʻa ʻana o ka ʻikepili i hoʻonohonoho ʻia ma hope o ka regression ua hoʻololi nui ʻia nā pae protein 2000 [P <0.05, analysis of variance (ANOVA)] i ʻelua a ʻoi aku ka nui o nā cohorts maʻi. A laila, hana mākou i ka nānā ʻana i ka cluster i mālama ʻia e pili ana i nā protein i hōʻike ʻia, a me ka P <0.0001 i ka hoʻohālikelike AD / control a / a i ʻole AD / PD (Figure S2, A a me B, Table S2C). ʻO kēia mau protein 165 i hoʻololi nui ʻia e hōʻike maopopo ana i nā hihia me AD pathology mai ka mana a me nā laʻana PD, e hōʻoia ana i nā loli ikaika AD-specific i ka proteome holoʻokoʻa.
A laila ua hoʻohana mākou i kahi algorithm i kapa ʻia ʻo Weighted Gene Co-expression Network Analysis (WGCNA) e hana i ka nānā ʻana i ka pūnaewele ma ka proteome lolo i ʻike ʻia, nāna e hoʻonohonoho i ka ʻikepili i hoʻonohonoho ʻia i loko o nā modules protein me nā ʻano hōʻike like (11-13). Ua ʻike ʻia ka hōʻike ʻana he 44 modules (M) co-expressed proteins, hoʻokaʻawale ʻia a helu ʻia mai ka nui (M1, n = 1821 protein) a hiki i ka liʻiliʻi loa (M44, n = 34 proteins) (Figure 2A a me ka Papa S2D) ). E like me ka mea i ʻōlelo ʻia ma luna (13) E helu i ka hōʻike hōʻike ʻelele a i ʻole ka protein hiʻona o kēlā me kēia module, a hoʻopili pū me ke kūlana maʻi a me AD pathology, ʻo ia hoʻi, e hoʻokumu i ka hui ʻana o Alzheimer's Disease Registry (CERAD) a me Braak Score (Figure 2B). ʻO ka holoʻokoʻa, ua pili nui nā modula 17 i ka neuropathology AD (P <0.05). ʻO ka nui o kēia mau modula pili i ka maʻi i waiwai pū kekahi i nā hōʻailona kikoʻī ʻano cell (Figure 2B). E like me ka mea i ʻōlelo ʻia ma luna (13), hoʻoholo ʻia ka hoʻonui ʻia ʻana o ke ʻano cell ma ke kāʻili ʻana i ke kōpili overlap a me ka papa kuhikuhi o nā genes type-specific cell. Loaʻa kēia mau genes mai ka ʻikepili i paʻi ʻia i loko o nā neurons ʻiole kaʻawale, endothelial a me nā cell glial. Hoʻokolohua RNA sequencing (RNA-seq) (29).
(A) E ʻike i ka WGCNA o ka pale o ka lolo. (B) Biweight midcorrelation (BiCor) kālailai o ka modular signature protein (ka mea nui mua o ka modular protein expression) me AD neuropathological hiʻohiʻona (luna), me CERAD (Aβ plaque) a me Braak (tau tangles). Hōʻike ʻia ka ikaika o ka pilina maikaʻi (ʻulaʻula) a me ka maikaʻi ʻole (uliuli) e kahi palapala wela ʻelua kala, a hōʻike nā asterisk i ke koʻikoʻi helu (P <0.05). E hoʻohana i ka Hypergeometric Fisher's Exact Test (FET) (lalo) e nānā i ka hui ʻano cell o kēlā me kēia module protein. ʻO ka ikaika o ka pale ʻulaʻula e hōʻike ana i ke kiʻekiʻe o ka hoʻonui ʻana i ke ʻano o ke kelepona, a ʻo ka asterisk e hōʻike i ke koʻikoʻi helu (P <0.05). E hoʻohana i ke ala BH e hoʻoponopono i ka waiwai P i loaʻa mai ka FET. (C) Ka nānā 'ana i nā protein modular. Hōʻike ʻia nā kaʻina hana olaola pili loa no kēlā me kēia module a i ʻole pūʻulu module pili. oligo, oligodendrocyte.
ʻO kahi hoʻonohonoho o ʻelima mau astrocyte pili pili a me nā microglia-rich modules (M30, M29, M18, M24, a me M5) i hōʻike i kahi pilina maikaʻi maikaʻi me AD neuropathology (Figure 2B). Hoʻopili ka loiloi Ontology i kēia mau modula glial me ka ulu ʻana o ka cell, proliferation, a me ka palekana (Figure 2C a me ka Papa S2E). ʻElua mau modula glial hou, M8 a me M22, ua hoʻoikaika ikaika ʻia i ka maʻi. Pili loa ka M8 i ke ala o ka loaʻa ʻana o Toll-like, he cascade hōʻailona e pāʻani ana i ke kuleana koʻikoʻi i ka pane ʻana o ke kino (30). Ma ka manawa like, pili loa ka M22 i ka hoʻololi ʻana ma hope o ka unuhi ʻana. ʻO M2, ka mea waiwai i nā oligodendrocytes, e hōʻike ana i kahi pilina maikaʻi maikaʻi me ka AD pathology a me kahi pilina ontological me ka synthesis nucleoside a me ka replication DNA, e hōʻike ana i ka hoʻonui ʻana o ka cell proliferation i nā maʻi. Ma ke ʻano holoʻokoʻa, kākoʻo kēia mau ʻike i ke kiʻekiʻe o nā modula glial a mākou i ʻike mua ai i ka proteome network AD (13, 17). ʻIke ʻia i kēia manawa he nui nā modula glial pili AD i ka pūnaewele e hōʻike i nā pae haʻahaʻa haʻahaʻa i ka mana a me nā hihia PD, e hōʻike ana i kā lākou maʻi kikoʻī i hoʻokiʻekiʻe ʻia ma AD (Figure S2C).
ʻEhā mau modula wale nō i loko o kā mākou pūnaewele proteome (M1, M3, M10, a me M32) i hoʻopili maikaʻi ʻole ʻia me AD pathology (P <0.05) (Figure 2, B a me C). Loaʻa nā M1 a me M3 i nā māka neuronal. Ua pili loa ka M1 i nā hōʻailona synaptic, aʻo M3 e pili kokoke ana i ka hana mitochondrial. ʻAʻohe hōʻike o ka hoʻonui ʻana i ke ʻano cell no M10 a me M32. Hōʻike ka M32 i ka pilina ma waena o M3 a me ka metabolism cell, ʻoiai ʻo M10 e pili nui ana i ka ulu ʻana o ka cell a me ka hana microtubule. Hoʻohālikelike ʻia me AD, ua hoʻonui ʻia nā modula ʻehā i ka mana a me PD, e hāʻawi ana iā lākou i nā hoʻololi AD kikoʻī maʻi (Figure S2C). Ma ke ʻano holoʻokoʻa, kākoʻo kēia mau hopena i ka emi ʻana o ka nui o nā modula waiwai nui a mākou i ʻike mua ai ma AD (13, 17). I ka hōʻuluʻulu ʻana, ʻo ka loiloi pūnaewele o ka proteome o ka lolo i ʻike mākou i hana i nā modula AD-i hoʻololi ʻia e like me kā mākou ʻike mua.
Hōʻike ʻia ʻo AD e kahi pae asymptomatic mua (AsymAD), kahi e hōʻike ai nā kānaka i ka hōʻiliʻili amyloid me ka ʻole o ka emi ʻana o ka cognitive (5, 31). ʻO kēia kaʻina asymptomatic kahi puka makani koʻikoʻi no ka ʻike mua ʻana a me ka hana ʻana. Ua hōʻike mua mākou i ka mālama ʻana i ka modular ikaika o AsymAD a me AD proteome pūnaewele lolo ma waena o nā pūʻulu ʻikepili kūʻokoʻa (13, 17). I mea e hōʻoia ai ua kūlike ka ʻupena lolo a mākou i ʻike ai me kēia mau ʻike mua, ua loiloi mākou i ka mālama ʻana i nā modula 44 i ka ʻikepili i hoʻopili ʻia mai nā hui 27 DLPFC. Aia kēia mau hui i ka mana (n = 10), AsymAD (n = 8) a me AD (n = 9). Hoʻokomo ʻia nā hoʻohālike a me AD i ka nānā ʻana o kā mākou cohort brain discovery (Table S1B), aʻo nā hihia AsymAD he kū hoʻokahi wale nō i ka cohort replication. Ua loaʻa mai kēia mau hihia AsymAD mai ka waihona lolo Emory Goizueta ADRC. ʻOiai he mea maʻamau ka cognition i ka manawa o ka make, he kiʻekiʻe ke kiʻekiʻe o ka amyloid (mean CERAD, 2.8 ± 0.5) (Table S1B).
ʻO ka loiloi TMT-MS o kēia mau ʻiʻo lolo 27 i hopena i ka helu o 11,244 proteomes. Aia kēia helu hope i kēlā mau protein i helu ʻia ma ka liʻiliʻi o 50% o nā laʻana. Loaʻa i kēia pūʻulu ʻikepili hou he 8638 (98.0%) o nā protein 8817 i ʻike ʻia i kā mākou loiloi lolo, a ua aneane 3000 i hoʻololi nui i nā proteins ma waena o ka mana a me nā cohorts AD (P <0.05, ma hope o ka hoʻāʻo ʻana o Tukey i ka hoʻāʻo ʻana o ka ʻokoʻa) ( Papa S2F). Ma waena o kēia mau protein i hōʻike ʻia, ua hōʻike pū ʻo 910 i nā loli nui ma waena o AD a me nā hihia hoʻomalu proteome lolo (P <0.05, ma hope o ANOVA Tukey paired t-test). He mea pono e hoʻomaopopo i kēia mau hōʻailona 910 e kūlike loa i ke kuhikuhi o ka loli ma waena o nā proteomes (r = 0.94, P <1.0 × 10-200) (Figure S3A). Ma waena o nā protein i hoʻonui ʻia, ʻo nā protein me nā hoʻololi mau loa ma waena o nā pūʻulu ʻikepili he mau lālā o ka glial-rich M5 a me M18 modules (MDK, COL25A1, MAPT, NTN1, SMOC1, a me GFAP). Ma waena o nā protein i hōʻemi ʻia, ʻo ka poʻe me nā hoʻololi like ʻole he mau lālā wale nō ia o ka module M1 (NPTX2, VGF, a me RPH3A) pili me ka synapse. Ua hōʻoia hou mākou i nā hoʻololi e pili ana i ka AD o midkine (MDK), CD44, huna ʻia i pili i ka frizzled-related protein 1 (SFRP1) a me VGF ma ke komohana blotting (Figure S3B). Ua hōʻike ʻia ka nānā ʻana o ka mālama ʻana o ka module e pili ana i ka 80% o nā modules protein (34/44) i loko o ka proteome lolo i mālama nui ʻia i ka hoʻonohonoho ʻikepili replication (z-score> 1.96, hoʻoponopono ʻia ʻo FDR P <0.05) (Figure S3C). He ʻumikūmāhā o kēia mau modules i mālama kūikawā ʻia ma waena o nā proteome ʻelua (z-score> 10, FDR hoʻoponopono ʻia P <1.0 × 10−23). Ma ke ʻano holoʻokoʻa, ʻo ka ʻike a me ka hoʻopiʻi ʻana o ke kiʻekiʻe kiʻekiʻe o ka kūlike i ka ʻōlelo ʻokoʻa a me ka hoʻonohonoho modular ma waena o ka proteome o ka lolo e hōʻike ana i ka reproducibility o nā loli i nā protein cortex mua AD. Eia kekahi, ua hōʻoia pū ʻia ʻo AsymAD a me nā maʻi ʻoi aku ka nui o ka ʻōnaehana lolo.
ʻO kahi hōʻike kikoʻī hou aʻe o ka hōʻike ʻokoʻa i loko o ka ʻikepili replication o ka lolo e hōʻike ana i ke kiʻekiʻe koʻikoʻi o nā hoʻololi protein AsymAD, me ka huina o 151 i hoʻololi nui ʻia ma waena o AsymAD a me ka mana (P <0.05) (Figure S3D). E like me ka ukana amyloid, ua piʻi nui ka APP i ka lolo o AsymAD a me AD. Hoʻololi nui wale ʻo MAPT i AD, i kūlike me ka piʻi ʻana o nā kuʻekuʻe a me kona pilina ʻike ʻia me ka emi ʻana o ka cognitive (5, 7). Hōʻike nui ʻia nā modules glial-rich (M5 a me M18) i ka hoʻonui ʻia ʻana o nā protein i AsymAD, ʻoiai ʻo ka module M1 pili i ka neuron ka mea i hōʻike ʻia i nā protein i emi iho i AsymAD. Hōʻike ka nui o kēia mau hōʻailona AsymAD i nā loli nui i nā maʻi hōʻailona. Ma waena o kēia mau hōʻailona ʻo SMOC1, he protein glial nona ka M18, e pili ana i ka lolo lolo a me ka ulu ʻana o nā maka a me nā lālā (32). ʻO ka MDK kahi mea ulu heparin-binding e pili ana i ka ulu ʻana o ka cell a me ka angiogenesis (33), kekahi lālā o M18. Ke hoʻohālikelike ʻia me ka pūʻulu mana, ua hoʻonui nui ʻia ʻo AsymAD, a ma hope o ka piʻi nui ʻana o AD. I ka hoʻohālikelike ʻana, ua hoʻemi nui ʻia ka synaptic protein neuropentraxin 2 (NPTX2) i ka lolo AsymAD. Ua pili mua ʻo NPTX2 me ka neurodegeneration a he kuleana koʻikoʻi i ka mediating excitatory synapses (34). Ma ke ʻano holoʻokoʻa, ʻike ʻia kēia mau hopena i nā ʻano hoʻololi protein preclinical like ʻole i AD e ʻike ʻia e holomua me ka paʻakikī o ka maʻi.
Hāʻawi ʻia ua loaʻa iā mākou kahi hohonu hohonu o ka uhi ʻana o ka protein i ka ʻike ʻana i ka proteome o ka lolo, ke hoʻāʻo nei mākou e hoʻomaopopo piha i kona uhi ʻana me ka transcriptome AD-level. No laila, ua hoʻohālikelike mākou i ka proteome o ka lolo a mākou i ʻike ai me ka module a mākou i hana mua ai mai ke ana microarray o 18,204 genes ma AD (n = 308) a me ka mana (n = 157) mau kiko DLPFC (13). ke kau ana. Ma ka huina holoʻokoʻa, ua ʻike mākou i nā modula RNA ʻokoʻa 20, ʻo ka nui o ia mau mea i hōʻike i ka hoʻonui ʻana i nā ʻano cell kūikawā, me nā neurons, oligodendrocytes, astrocytes, a me microglia (Figure 3A). Hōʻike ʻia nā hoʻololi lehulehu o kēia mau modula ma AD ma ke Kiʻi 3B. Kūlike me kā mākou protein-RNA overlap analysis ma mua o ka hoʻohana ʻana i ka MS proteome hohonu ʻole i kapa ʻia (e pili ana i 3000 proteins) (13), ʻo ka hapa nui o nā modules 44 i loko o ka pūnaewele proteome lolo a mākou i ʻike ai aia i loko o ka pūnaewele transcriptome ʻAʻohe mea nui i loko. ʻO kā mākou ʻike a me ka hana hou ʻana i nā modules protein 34 i mālama nui ʻia i loko o ka proteome o ka lolo, ʻo 14 wale nō (~ 40%) i hala i ka hoʻāʻo pololei a Fisher (FET) i hōʻoia i ka nui o ka helu ʻana me ka transcriptome (Figure 3A). Hoʻohālikelike me ka hoʻoponopono ʻana i ka pōʻino DNA (P-M25 a me P-M19), ka unuhi ʻana i ka protein (P-M7 a me ka P-M20), RNA binding/splicing (P-M16 a me P-M21) a me ka huli ʻana i ka protein (P-M13 a me P- M23) ʻaʻole pili me nā modula i ka transcriptome. No laila, ʻoiai ua hoʻohana ʻia kahi ʻikepili proteome hohonu i ka loiloi overlap o kēia manawa (13), ʻo ka hapa nui o ka proteome network AD ʻaʻole i palapala ʻia i ka pūnaewele transcriptome.
(A) Hōʻike ka Hypergeometric FET i ka hoʻonui ʻia ʻana o nā hōʻailona kikoʻī ʻano cell i loko o ka module RNA o ka transcriptome AD (luna) a me ke ʻano o ka overlap ma waena o nā modules RNA (x-axis) a me ka protein (y-axis) o ka lolo AD (lalo) . ʻO ka ikaika o ka pale ʻulaʻula e hōʻike i ke kiʻekiʻe o ka hoʻonui ʻana i nā ʻano cell i ka papa luna a me ka ikaika o ka overlap o nā modules i ka papa lalo. Hōʻike nā asterisk i ke koʻikoʻi helu helu (P <0.05). (B) Ka degere o ka pilina ma waena o nā ʻano genes o kēlā me kēia transcriptome module a me ke kūlana AD. ʻO nā modula ma ka hema ka pili maikaʻi loa me AD (uliuli), a ʻo nā mea ma ka ʻaoʻao ʻākau ka pili maikaʻi loa me AD (ʻulaʻula). Hōʻike ka waiwai P i hoʻololi ʻia i ka log-transformed BH-corrected i ke degere o ka nui o ka helu helu o kēlā me kēia pilina. (C) ʻO nā modula ʻāwili nui me ka hoʻonui ʻana i ke ʻano o ke kelepona. (D) Ka hoʻopili ʻana i ka hoʻololi ʻana i ka log2 paʻi o ka protein i kapa ʻia (x-axis) a me RNA (y-axis) i loko o ka module overlapping. Hōʻike ʻia ka coefficient correlation Pearson me ka waiwai P pili. Micro, microglia; kino lani, astrocytes. CT, mana.
ʻO ka hapa nui o ka protein a me nā modula RNA e kaʻana like i nā profile hoʻohuihui ʻano cell like a me nā kuhikuhi hoʻololi AD maʻamau (Figure 3, B a me C). I nā huaʻōlelo ʻē aʻe, ua paʻi ʻia ka modula M1 pili i ka synapse o ka proteome o ka lolo (PM1) i ʻekolu mau neuronal-rich homologous RNA modules (R-M1, R-M9 a me R-M16), aia i AD hōʻike ʻia ʻelua. he pae hoemi. Pēlā nō, ʻoi aku ka nui o nā modula protein M5 a me M18 me nā modula RNA waiwai i nā astrocytes a me nā māka microglial (R-M3, R-M7, a me R-M10) a pili nui i nā maʻi Hoʻonui. Ke kākoʻo hou nei kēia mau hiʻohiʻona modular ma waena o nā pūʻulu ʻikepili ʻelua i ka hoʻonui ʻana i ke ʻano cell a me nā loli pili i ka maʻi a mākou i ʻike ai i loko o ka proteome lolo. Eia nō naʻe, ʻike mākou i nā ʻokoʻa koʻikoʻi ma waena o ka pae RNA a me nā pae protein o nā māka hoʻokahi i kēia mau modula. ʻO ka hoʻoponopono ʻana i ka ʻike ʻokoʻa o nā proteomics a me nā transcriptomics o nā molekole i loko o kēia mau modula overlapping (Figure 3D) e hōʻike ana i kēia kūlike ʻole. No ka laʻana, ua hōʻike ʻo APP a me kekahi mau protein glial module (NTN1, MDK, COL25A1, ICAM1, a me SFRP1) i ka piʻi nui ʻana o ka proteome AD, akā ʻaʻohe loli i ka transcriptome AD. ʻO kēia mau hoʻololi kikoʻī protein e pili kokoke ana i nā pā amyloid (23, 35), e hōʻike ana i ka proteome ke kumu o nā loli pathological, a ʻaʻole i ʻike ʻia kēia mau loli i ka transcriptome.
Ma hope o ka hoʻokaʻawale kūʻokoʻa ʻana i ka lolo a me nā proteomes CSF i ʻike mākou, ua hana mākou i kahi loiloi piha o nā pūʻulu ʻikepili ʻelua e ʻike ai i nā biomarkers AD CSF e pili ana i ka pathophysiology o ka pūnaewele lolo. Pono mākou e wehewehe mua i ka overlap o nā proteome ʻelua. ʻOiai ua ʻae nui ʻia ka hōʻike ʻana o CSF ​​i nā loli neurochemical i ka lolo AD (4), ʻaʻole maopopo ka degere o ka overlap ma waena o ka lolo AD a me ka proteome CSF. Ma ka hoʻohālikelike ʻana i ka helu o nā huahana gene i ʻike ʻia i loko o kā mākou mau proteomes ʻelua, ua ʻike mākou ma kahi o 70% (n = 1936) o nā protein i ʻike ʻia i loko o ka wai cerebrospinal ua helu pū ʻia ma ka lolo (Figure 4A). Hoʻopili ʻia ka hapa nui o kēia mau protein overlapping (n = 1721) i kekahi o 44 co-expression modules mai ka hoʻonohonoho ʻike ʻike lolo (Figure 4B). E like me ka mea i manaʻo ʻia, ua hōʻike ʻia nā modula lolo nui ʻeono (M1 a M6) i ka nui o ka nui o ka CSF overlap. Eia nō naʻe, aia nā ʻāpana lolo liʻiliʻi (no ka laʻana, M15 a me M29) hiki ke loaʻa kahi kiʻekiʻe kiʻekiʻe o ka overlap, ʻoi aku ka nui ma mua o ka module lolo ʻelua o kona nui. Hoʻoikaika kēia iā mākou e hoʻohana i kahi ʻano kikoʻī a me ka helu ʻana i ka helu ʻana i ka overlap ma waena o ka lolo a me ka wai cerebrospinal.
(A a me B) ʻO nā protein i ʻike ʻia i loko o ka lolo ʻike a me nā pūʻulu ʻikepili CSF overlap. Hoʻopili ʻia ka hapa nui o kēia mau protein overlapping me kekahi o ka 44 co-expression modules o ka lolo co-expression network. (C) E ʻike i ka overlap ma waena o ka proteome wai cerebrospinal a me ka proteome pūnaewele lolo. Hōʻike kēlā me kēia lālani o ka palapala ʻāina wela i ka nānā ʻana o ka hypergeometric FET. Hōʻike ka lālani luna i ka uhi ʻana (ke poʻo hina/ʻeleʻele) ma waena o ka module lolo a me ka proteome CSF holoʻokoʻa. Hōʻike ka laina ʻelua i ka hoʻopili ʻana ma waena o nā modula lolo a me ka protein CSF (i uhi ʻia i ka ʻulaʻula) i hoʻoponopono nui ʻia i AD (P <0.05). Hōʻike ka lālani ʻekolu i ka overlap ma waena o nā modula lolo a me ka protein CSF (blue shading) i hoʻoponopono nui ʻia ma AD (P <0.05). E hoʻohana i ke ala BH e hoʻoponopono i ka waiwai P i loaʻa mai ka FET. (D) ʻO ka papa kōpana kōpili e pili ana i ka hui ʻano kelepona a me nā huaʻōlelo GO pili. Loaʻa i kēia mau panela he 271 mau protein pili i ka lolo, he ʻano ʻokoʻa koʻikoʻi i ka CSF proteome.
Ke hoʻohana nei i nā FET hoʻokahi huelo, ua loiloi mākou i ke koʻikoʻi o ka protein overlap ma waena o ka proteome CSF a me nā modula lolo pākahi. Ua hōʻike ʻia ka hōʻike ʻana he 14 mau modula lolo i ka CSF data set he nui nā overlaps (FDR adjusted P <0.05), a me kahi module hou (M18) kahi kokoke i ke koʻikoʻi (FDR adjusted P = 0.06) (Figure 4C , lālani luna). Makemake pū mākou i nā modula i hoʻopili ikaika ʻia me nā protein CSF i hōʻike ʻia. No laila, ua hoʻohana mākou i ʻelua mau loiloi FET hou e hoʻoholo ai i kahi o (i) CSF protein i hoʻonui nui ʻia i AD a (ii) CSF protein i hoʻemi nui ʻia i AD (P <0.05, paired t test AD/control) Brain modules me ka uhi pono. mawaena o lakou. E like me ka mea i hōʻike ʻia ma nā lālani waena a me lalo o ke Kiʻi 4C, hōʻike kēia mau loiloi hou i ka 8 o nā modula lolo 44 i uhi nui ʻia me ka protein i hoʻohui ʻia i AD CSF (M12, M1, M2, M18, M5, M44, M33, a me M38) . ), ʻoiai ʻelua mau modula wale nō (M6 a me M15) i hōʻike i kahi ʻano nui me ka protein i hōʻemi ʻia i AD CSF. E like me ka mea i manaʻo ʻia, aia nā modula 10 āpau i nā modules 15 me ka overlap kiʻekiʻe loa me ka proteome CSF. No laila, ke manaʻo nei mākou ʻo kēia mau modula he 15 mau kumu kiʻekiʻe o nā biomarkers CSF i loaʻa i ka lolo.
Hoʻopili mākou i kēia mau modula 15 overlapping i ʻelima mau panela protein nui e pili ana i ko lākou kokoke i ka kiʻi kumu lāʻau WGCNA a me kā lākou hui pū ʻana me nā ʻano cell a me ka gene ontology (Figure 4D). Aia ka papa mua i nā modula waiwai i nā māka neuron a me nā protein pili i ka synapse (M1 a me M12). Aia i loko o ka papa synaptic he 94 mau protein, a ua loli nui nā pae i loko o ka CSF proteome, i lilo ia i kumu nui loa o nā māka CSF pili i ka lolo ma waena o nā panela ʻelima. Ua hōʻike ka lua o ka hui (M6 a me M15) i ka pilina pili me nā hōʻailona cell endothelial a me ke kino vascular, e like me ka "ho'ōla ʻeha" (M6) a me "ka hoʻoponopono ʻana i ka pane ʻana o ka humoral immune" (M15). Ua pili loa ka M15 i ka metabolism lipoprotein, pili loa i ka endothelium (36). Aia i loko o ka panel vascular he 34 CSF markers pili i ka lolo. ʻO ke kolu o ka hui e komo i nā modula (M2 a me M4) e pili nui ana i nā hōʻailona oligodendrocyte a me ka hoʻonuiʻana o nā pūnaewele. No ka laʻana, ʻo nā huaʻōlelo ontology o ka pae kiʻekiʻe o M2 ka "hoʻoponopono maikaʻi o ka hana hou ʻana o DNA" a me ka "kaʻina biosynthesis purine". Eia nō naʻe, ʻo nā mea o M4 ka "glial cell differentiation" a me ka "chromosome segregation". Aia i loko o ka panel myelination he 49 CSF markers pili i ka lolo.
Loaʻa i ka hui ʻehā ka nui o nā modules (M30, M29, M18, M24, a me M5), a kokoke i nā modula āpau i waiwai nui i nā microglia a me nā māka astrocyte. E like me ka myelination panel, ʻo ka ʻehā o ka panel i loaʻa nā modula (M30, M29, a me M18) e pili kokoke ana i ka hoʻonui ʻana o ke kelepona. ʻO nā modula ʻē aʻe i loko o kēia pūʻulu pili loa i nā huaʻōlelo immunological, e like me ka "kaʻina hana imune" (M5) a me "ka hoʻoponopono pane ʻana" (M24). Aia i loko o ka hui glial immune he 42 CSF markers pili i ka lolo. ʻO ka mea hope loa, ʻo ka papa hope loa he 52 mau māka pili i ka lolo ma nā modula ʻehā (M44, M3, M33, a me M38), aia nā mea āpau ma ke kino e pili ana i ka mālama ʻana i ka ikehu a me ka metabolism. ʻO ka mea nui loa o kēia mau modula (M3) pili pili i ka mitochondria a waiwai nui i nā kaha kikoʻī neuron. ʻO M38 kekahi o nā lālā liʻiliʻi liʻiliʻi o kēia metabolome a hōʻike pū i ka kikoʻī neuron haʻahaʻa.
ʻO ka holoʻokoʻa, hōʻike kēia mau paneli ʻelima i kahi ākea o nā ʻano cell a me nā hana i loko o ka AD cortex, a hui pū ʻia nā 271 mau māka CSF pili i ka lolo (Table S2G). I mea e loiloi ai i ka pono o kēia mau hualoaʻa MS, ua hoʻohana mākou i ka proximity extension assay (PEA), kahi ʻenehana orthogonal antibody-based me nā mana multiplexing, kiʻekiʻe kiʻekiʻe a me ka kikoʻī, a nānā hou i nā ʻano wai cerebrospinal i loaʻa iā mākou kahi ʻāpana o kēia mau biomarkers 271. (n = 36). Hōʻike kēia mau pahuhopu 36 i ka hoʻololi ʻana i ka helu AD o PEA, kahi pili pili i kā mākou mau ʻike MS-based (r = 0.87, P = 5.6 × 10-12), ʻo ia ka mea i hōʻoia ikaika i nā hopena o kā mākou loiloi MS piha (Figure S4 ).
ʻO nā kumuhana olaola i hoʻokūpaʻa ʻia e kā mākou mau hui ʻelima, mai ka hōʻailona synaptic a hiki i ka metabolism ikehu, pili i ka pathogenesis o AD (1-3). No laila, ʻo nā modula 15 āpau i loaʻa i kēia mau panela e pili ana i ka pathology AD i loko o ka proteome lolo a mākou i ʻike ai (Figure 2B). ʻO ka mea kaulana loa ʻo ka correlation pathological maikaʻi kiʻekiʻe ma waena o kā mākou mau modula glial a me ka correlation pathological maikaʻi ʻole ma waena o kā mākou mau modula neuronal nui loa (M1 a me M3). ʻO ka hōʻike hōʻike ʻokoʻa o kā mākou proteome lolo hou (Figure S3D) e hōʻike ana i nā protein glial i loaʻa iā M5 a me M18. I ka AsymAD a me ka hōʻailona AD, ʻoi aku ka nui o nā protein glial i hoʻonui ʻia a me nā synapses pili i M1 Ua hoʻemi nui ʻia ka protein. Hōʻike kēia mau ʻike e pili ana ka 271 cerebrospinal fluid markers a mākou i ʻike ai i loko o nā hui ʻelima i nā kaʻina hana maʻi i loko o ka cortex AD, me nā mea i hana ʻia i nā pae mua asymptomatic.
I mea e noʻonoʻo maikaʻi ai i ka hoʻololi ʻana o nā protein panel i loko o ka lolo a me ka wai spinal, ua huki mākou i kēia no kēlā me kēia o nā modules overlapping 15: (i) ʻike i ka pae nui o ka module i ka hoʻonohonoho ʻikepili lolo a (ii) ka module pūmua Hōʻike ʻia ka ʻokoʻa i ka wai cerebrospinal (Figure S5). E like me ka mea i ʻōlelo ʻia ma mua, hoʻohana ʻia ka WGCNA e hoʻoholo i ka nui o ka module a i ʻole ka waiwai protein i loko o ka lolo (13). Ho'ohana 'ia ka palapala 'āina pele no ka wehewehe 'ana i ka 'ike 'oko'a o nā protein modular i loko o ka wai cerebrospinal (AD/control). Hōʻike kēia mau kiʻi i ʻekolu o nā paneli ʻelima e hōʻike i nā ʻano hōʻike like ʻole i ka lolo a me ka wai spinal. Hōʻike nā modula ʻelua o ka synapse panel (M1 a me M12) i ka emi ʻana o ka nui o ka nui o ka lolo AD, akā ʻoi aku ka nui o ka nui o ka protein i ka AD CSF (Figure S5A). ʻO nā modula pili neuron e loaʻa ana i ka metabolome (M3 a me M38) i hōʻike like ʻole i ka lolo a me ka cerebrospinal fluid expression pattern inconsistent (Figure S5E). Ua hōʻike pū ka panel vascular i nā ʻano hōʻike like ʻole, ʻoiai ua hoʻonui ʻia kona mau modula (M6 a me M15) i loko o ka lolo AD a ua emi iho i ka CSF maʻi (Figure S5B). Aia nā ʻaoʻao ʻelua i nā ʻupena glial nui nona nā protein i hoʻoponopono mau ʻia ma nā keʻena ʻelua (Figure S5, C a me D).
E ʻoluʻolu, ʻaʻole maʻamau kēia mau ʻano i nā māka āpau i kēia mau panela. No ka laʻana, ʻo ka synaptic panel ka nui o nā protein i hoʻemi nui ʻia i loko o ka lolo AD a me CSF (Figure S5A). Ma waena o kēia mau hōʻailona wai cerebrospinal i hoʻoponopono ʻia ʻo NPTX2 a me VGF o M1, a me chromogranin B o M12. Eia nō naʻe, ʻoiai kēia mau ʻokoʻa, ua hoʻokiʻekiʻe ʻia ka hapa nui o kā mākou synaptic markers i ka wai spinal AD. Ma ke ʻano holoʻokoʻa, ua hiki i kēia mau kānana ke hoʻokaʻawale i nā hiʻohiʻona koʻikoʻi i ka lolo a me nā pae wai cerebrospinal i kēlā me kēia o kā mākou ʻelima panela. Hōʻike kēia mau hiʻohiʻona i ka pilina paʻakikī a ʻokoʻa ma waena o ka lolo a me ka hōʻike protein CSF ma AD.
A laila, ua hoʻohana mākou i ka loiloi hoʻopiʻi MS kiʻekiʻe (CSF replication 1) e hōʻemi i kā mākou 271 set of biomarkers i nā mea hoʻohiki maikaʻi loa a hiki ke hana hou ʻia (Figure 5A). Loaʻa i ka CSF kope 1 he 96 mau laʻana mai Emory Goizueta ADRC, me ka mana, AsymAD, a me AD cohort (Table S1A). Hōʻike ʻia kēia mau hihia AD e ka emi ʻana o ka cognitive haʻahaʻa (mean MoCA, 20.0 ± 3.8), a me nā loli i nā biomarkers AD i hōʻoia ʻia i ka wai cerebrospinal (Table S1A). Kūlike ʻole i ka loiloi CSF a mākou i ʻike ai, hana ʻia kēia hoʻopiʻi me ka hoʻohana ʻana i kahi ala MS "hoʻokahi-pana" kiʻekiʻe a ʻoi aku ka maikaʻi (me ka ʻole o ka hoʻokaʻawale ʻana o ka laina), me kahi protocol hoʻomākaukau laʻana maʻalahi e hoʻopau i ka pono no ka immunodepletion o kēlā me kēia laʻana. . Akā, hoʻohana ʻia kahi "channel hoʻonui" i hoʻopau ʻia i ka immune e hoʻonui i ka hōʻailona o nā protein liʻiliʻi (37). ʻOiai e hōʻemi ana ia i ka uhi proteome holoʻokoʻa, ʻo kēia ʻano pana hoʻokahi e hōʻemi nui i ka manawa mīkini a hoʻonui i ka helu o nā laʻana i hōʻailona ʻia e TMT i hiki ke nānā pono ʻia (17, 38). I ka huina, ua ʻike ʻia ka loiloi he 6,487 peptides, i hoʻopaʻa ʻia i 1,183 proteomes i 96 mau hihia. E like me ka CSF analysis a mākou i ʻike ai, ʻo kēlā mau protein wale nō i helu ʻia ma ka liʻiliʻi o 50% o nā laʻana i hoʻokomo ʻia i loko o nā helu ma hope, a ua hoʻihoʻi ʻia ka ʻikepili no nā hopena o ka makahiki a me ke kāne. Ua alakaʻi kēia i ka helu hope loa o 792 proteomes, 95% o ia mau mea i ʻike pū ʻia ma ka ʻikepili CSF i loaʻa.
(A) Ua hōʻoia ʻia nā pahuhopu protein CSF pili i ka lolo i ka cohort CSF replicated mua a hoʻokomo ʻia i ka papa hope (n = 60). (B a hiki i E) Nā pae biomarker panel (huihui z-mau helu) i ana ʻia i loko o nā ʻehā CSF replication cohorts. Ua hoʻohana ʻia nā t-tests a i ʻole ANOVA me ka hoʻoponopono hope a Tukey no ka loiloi i ke koʻikoʻi helu helu o nā loli i ka nui o kēlā me kēia loiloi replicate. CT, mana.
No ka mea makemake nui mākou i ka hōʻoia ʻana i kā mākou mau pahuhopu CSF e pili ana i ka lolo ma o ka nānā ʻana piha, e kaupalena mākou i ka nānā hou ʻana i kēia proteome replicated i kēia mau māka. Ma waena o kēia mau proteins 271, ua ʻike ʻia ʻo 100 i ka CSF replication 1. Hōʻike ʻo S6A i ka hōʻike ʻokoʻa o kēia 100 overlapping markers ma waena o ka mana a me nā hōʻailona hoʻopiʻi AD. Hoʻonui ka synaptic a me ka metabolite histones i ka AD, aʻo nā protein vascular e hoʻemi nui i ka maʻi. ʻO ka hapa nui o ka 100 overlapping markers (n = 70) i ​​mālama i ka ʻaoʻao like o ka hoʻololi ʻana i nā pūʻulu ʻikepili ʻelua (Figure S6B). ʻO kēia 70 mau hōʻailona CSF pili i ka lolo (Table S2H) e hōʻike nui ana i nā hiʻohiʻona hōʻike panel i ʻike mua ʻia, ʻo ia hoʻi, ka hoʻohaʻahaʻa ʻana i nā protein vascular a me ka hoʻoponopono ʻana o nā panela āpau. ʻO 10 wale nō o kēia 70 mau protein i hōʻoia ʻia i hōʻike i nā loli i ka nui o AD i kūʻē i kēia mau ʻano papa. No ka hana ʻana i kahi panel e hōʻike maikaʻi loa i ke ʻano holoʻokoʻa o ka lolo a me ka wai cerebrospinal, ua haʻalele mākou i kēia mau protein 10 mai ka papa hoihoi a mākou i hōʻoia hope ai (Figure 5A). No laila, loaʻa i kā mākou papa he 60 mau protein i hōʻoia ʻia i loko o ʻelua mau pūʻulu CSF AD kūʻokoʻa me ka hoʻohana ʻana i nā hoʻomākaukau laʻana like ʻole a me ka nānā ʻana i ka platform MS. Ua hōʻoia ka manaʻo o ka z-score o kēia mau panela hope loa i ka mana CSF kope 1 a me nā hihia AD i ke ʻano o ka panel i ʻike ʻia ma ka cohort CSF i loaʻa iā mākou (Figure 5B).
Ma waena o kēia mau proteins 60, aia nā molekole i ʻike ʻia e pili pū me AD, e like me osteopontin (SPP1), he cytokine pro-inflammatory i pili pū me AD i nā haʻawina he nui (39-41), a me GAP43, A synaptic protein. pili pono i ka neurodegeneration (42). ʻO nā protein i hōʻoia piha ʻia he mau hōʻailona e pili ana i nā maʻi neurodegenerative ʻē aʻe, e like me amyotrophic lateral sclerosis (ALS) pili superoxide dismutase 1 (SOD1) a me ka maʻi o Parkinson e pili ana i ka desaccharase (PARK7). Ua hōʻoia pū mākou he nui nā mea hōʻailona ʻē aʻe, e like me SMOC1 a me ka brain-rich membrane attachment signaling protein 1 (BASP1), i kaupalena i nā loulou mua i ka neurodegeneration. He mea pono ke hoʻomaopopo ʻia ma muli o ko lākou haʻahaʻa haʻahaʻa holoʻokoʻa i ka CSF proteome, he paʻakikī iā mākou ke hoʻohana i kēia ala kiʻekiʻe-throughput hoʻokahi kiʻi kiʻi kiʻekiʻe e ʻike pono ai i ka MAPT a me kekahi mau protein pili pili AD (e like me NEFL a me NRGN. ) ( 43, 44).
A laila nānā mākou i kēia 60 mau hōʻailona papa kuhikuhi i loko o ʻekolu mau loiloi hou. Ma ka CSF Copy 2, ua hoʻohana mākou i hoʻokahi TMT-MS e nānā i kahi hui kūʻokoʻa o ka mana 297 a me nā hōʻailona AD mai Emory Goizueta ADRC (17). Hoʻopili hou ʻia ka CSF 3 i kahi reanalysis o nā ʻikepili TMT-MS i loaʻa mai ka mana 120 a me nā maʻi AD mai Lausanne, Switzerland (45). Ua ʻike mākou i ʻoi aku ma mua o ʻelua hapakolu o nā hōʻailona koʻikoʻi 60 i kēlā me kēia waihona. ʻOiai ua hoʻohana ka haʻawina Swiss i nā ʻano ʻano MS like ʻole a me nā ʻano helu helu TMT (45, 46), ua hoʻopuka ikaika mākou i kā mākou papa hana i loko o ʻelua mau loiloi hou (Figure 5, C a me D, a me nā Papa S2, I, a me J). No ka loiloi i ke kiko o ka maʻi o kā mākou hui, ua hoʻohana mākou i ka TMT-MS e kālailai i ka ʻehā o ka replication data set (CSF replication 4), ʻaʻole i hoʻokomo wale i ka mana (n = 18) a me AD (n = 17) mau hihia, akā pū kekahi PD ( n = 14)), ALS (n = 18) a me frontotemporal dementia (FTD) hōʻailona (n = 11) (S1A Papa). Ua hoʻonui maikaʻi mākou i kahi kokoke i ʻelua hapakolu o nā protein panel i kēia cohort (38 mai 60). Hōʻike kēia mau hopena i nā hoʻololi kikoʻī AD i nā paneli biomarker ʻelima (Figure 5E a me ka Papa S2K). ʻO ka hoʻonui ʻana i ka pūʻulu metabolite i hōʻike i ka kikoʻī AD ikaika loa, ukali ʻia e ka myelination a me ka hui glial. I ka liʻiliʻi liʻiliʻi, hōʻike pū ʻo FTD i ka piʻi ʻana ma waena o kēia mau panela, e hōʻike ana i nā loli pūnaewele like like (17). I ka hoʻokaʻawale ʻana, hōʻike ʻo ALS a me PD i ka like like o ka myelination, glial, a me ka metabolome profiles e like me ka pūʻulu mana. Ma ke ʻano holoʻokoʻa, ʻoiai nā ʻokoʻa o ka hoʻomākaukau laʻana, MS platform, a me nā ʻano helu helu TMT, ua hōʻike ʻia kēia mau loiloi hou ʻana i ko mākou mau hōʻailona papa kuhikuhi i nā loli kikoʻī AD i ʻoi aku ma mua o 500 mau laʻana CSF kūʻokoʻa.
Ua ʻike nui ʻia ka neurodegeneration AD i kekahi mau makahiki ma mua o ka hoʻomaka ʻana o nā hōʻailona cognitive, no laila aia kahi koi wikiwiki no nā biomarkers o AsymAD (5, 31). Eia naʻe, ʻoi aku ka nui o nā hōʻike e hōʻike ana he mamao loa ka biology o AsymAD mai ka homogeneous, a ʻo ka pilina paʻakikī o ka pilikia a me ke kūpaʻa e alakaʻi i nā ʻokoʻa nui o kēlā me kēia kanaka i ka holomua o ka maʻi (47). ʻOiai ua hoʻohana ʻia e ʻike i nā hihia AsymAD, ʻaʻole i hōʻoia ʻia nā pae o nā biomarkers CSF koʻikoʻi (Aβ1-42, total tau a me p-tau) hiki ke wānana pono i ka mea e holomua i ka dementia (4, 7), e hōʻike ana i nā mea hou aʻe. pono e hoʻokomo i nā mea hana biomarker holistic e pili ana i nā ʻano he nui o ka physiology lolo e hoʻopaʻa pololei i ka pilikia o kēia heluna. No laila, ua nānā mākou i kā mākou panel biomarker AD-validated i ka heluna AsymAD o CSF ​​kope 1. ʻO kēia mau hihia 31 AsymAD i hōʻike i nā pae biomarker maʻamau (Aβ1-42 / total tau ELISA ratio, <5.5) a me ka cognition piha (mean MoCA, 27.1 ± 2.2) (Pakaukau S1A). Eia kekahi, ʻo ka poʻe āpau me AsymAD he helu dementia maʻi o 0, e hōʻike ana ʻaʻohe hōʻike o ka emi ʻana o ka hana cognitive a i ʻole ka hana hana.
Ua nānā mua mākou i nā pae o nā panela i hoʻopaʻa ʻia i nā 96 CSF replicates 1, me ka cohort AsymAD. Ua ʻike mākou he nui nā paneli i ka hui AsymAD i loaʻa nā loli nui e like me AD, ua hōʻike ka panel vascular i kahi hiʻohiʻona i lalo i AsymAD, aʻo nā panela āpau i hōʻike i kahi ʻano piʻi (Figure 6A). No laila, ua hōʻike nā panela a pau i kahi pilina koʻikoʻi loa me ELISA Aβ1-42 a me ka nui o nā pae tau (Figure 6B). Ma ka ʻokoʻa, ʻaʻole maikaʻi ka pilina ma waena o ka hui a me ka helu MoCA. ʻO kekahi o nā mea i ʻike nui ʻia mai kēia mau loiloi ʻo ka nui o ka nui o nā panela i ka cohort AsymAD. E like me ka mea i hōʻike ʻia ma ka Figure 6A, ʻo ka pae panel o ka hui AsymAD maʻamau e hele i ka pae panel o ka pūʻulu mana a me ka pūʻulu AD, e hōʻike ana i ka loli kiʻekiʻe. No ka ʻimi hou ʻana i kēia heterogeneity o AsymAD, ua noi mākou i ka loiloi Multidimensional Scaling (MDS) i 96 CSF replication 1 hihia. Hiki i ka loiloi MDS ke ʻike i ka like ma waena o nā hihia e pili ana i kekahi mau ʻokoʻa i ka hoʻonohonoho ʻikepili. No kēia kānana puʻupuʻu, hoʻohana wale mākou i kēlā mau hōʻailona panel i hōʻoia ʻia he loli koʻikoʻi (P <0.05, AD/control) i ka ʻike CSF a me ka replication 1 proteome (n = 29) (Table S2L) pae. Ua hoʻopuka kēia hōʻuluʻulu manaʻo i ka clustering spatial ma waena o kā mākou mana a me nā hihia AD (Figure 6C). I ka hoʻokaʻawale ʻana, ʻike maopopo ʻia kekahi mau hihia AsymAD i ka pūʻulu hoʻomalu, aʻo nā mea ʻē aʻe aia i nā hihia AD. No ka ʻimi hou ʻana i kēia heterogeneity AsymAD, ua hoʻohana mākou i kā mākou palapala MDS e wehewehe i nā pūʻulu ʻelua o kēia mau hihia AsymAD. ʻO ka hui mua i loaʻa nā hihia AsymAD i hoʻopili kokoke i ka mana (n = 19), aʻo ka lua o ka hui i hōʻike ʻia e nā hihia AsymAD me kahi ʻaoʻao marker kokoke i AD (n = 12).
(A) ʻO ka pae hōʻike (z-score) o ka pūʻulu biomarker CSF ma nā 96 a pau ma ka CSF replication 1 cohort, me AsymAD. Ua hoʻohana ʻia ka ʻikepili o ka ʻokoʻa me ka hoʻoponopono hope a Tukey e loiloi i ke koʻikoʻi helu o nā loli nui o ka panel. (B) Ka hoʻoponopono hoʻoponopono ʻana o ka pae kiʻekiʻe o ka protein panel (z-score) me ka helu MoCA a me ka nui o ka pae tau ma ELISA Aβ1-42 a me CSF kope 1 nā laʻana. Hōʻike ʻia ka coefficient correlation Pearson me ka waiwai P pili. (C) ʻO ka MDS o 96 CSF kope 1 hihia i hoʻokumu ʻia ma luna o nā pae nui o 29 validated panel markers, i hoʻololi nui ʻia i ka ʻike a me ka CSF kope 1 mau pūʻulu ʻikepili [P <0.05 AD/control (CT)]. Ua hoʻohana ʻia kēia loiloi e hoʻokaʻawale i ka pūʻulu AsymAD i ka mana (n = 19) a me AD (n = 12). (D) Hōʻike ka ʻāpana lua pele i ka hōʻike ʻokoʻa o nā protein CSF replication 1 āpau me ka log2 fold change (x-axis) pili i ka -log10 helu helu P ma waena o nā pūʻulu AsymAD ʻelua. Ua kala ʻia nā biomarkers panel. (E) Hōʻike ʻokoʻa ʻia ka pae hoʻopiʻi CSF 1 o nā pūʻulu koho biomarkers ma waena o nā pūʻulu AsymAD. Ua hoʻohana ʻia ka loiloi hope-hoʻoponopono ʻana o Tukey no ka loiloi i ke koʻikoʻi helu.
Ua nānā mākou i ka ʻōlelo protein ʻokoʻa ma waena o kēia mau mana a me nā hihia AsymAD e like me AD (Figure 6D a me Table S2L). Hōʻike ʻia ka palapala ʻāina lua pele he 14 mau hōʻailona papa i loli nui ma waena o nā hui ʻelua. ʻO ka hapa nui o kēia mau māka he mau lālā o ka synapse a me ka metabolome. Eia nō naʻe, ʻo SOD1 a me ka myristoylated alanine-rich protein kinase C substrate (MARCKS), ʻo ia nā lālā o ka myelin a me nā pūʻulu immune glial, i kēlā me kēia hui (Figure 6, D a me E). Ua hāʻawi pū ka panel vascular i ʻelua mau māka i hoʻemi nui ʻia i ka hui AsymAD like me AD, me ka AE binding protein 1 (AEBP1) a hoʻokō i ka lālā o ka ʻohana C9. ʻAʻohe ʻokoʻa koʻikoʻi ma waena o ka mana a me AD-like AsymAD subgroups ma ELISA AB1-42 (P = 0.38) a me p-tau (P = 0.28), akā aia maoli ka ʻokoʻa koʻikoʻi i ka nui o ka pae tau (P = 0.0031). ) (Fig. S7). Aia kekahi mau hōʻailona panel e hōʻike ana he ʻoi aku ka nui o nā loli ma waena o nā pūʻulu AsymAD ʻelua ma mua o ka nui o nā pae tau (no ka laʻana, YWHAZ, SOD1, a me MDH1) (Figure 6E). Ma ke ʻano holoʻokoʻa, hōʻike kēia mau hopena e loaʻa i kā mākou paneli hōʻoia ʻia nā biomarkers hiki ke subtype a me ka stratification pilikia o nā maʻi me ka maʻi asymptomatic.
Loaʻa kahi koi wikiwiki no nā mea hana biomarker e pili ana i ka ʻōnaehana e ana maikaʻi a hoʻopaʻa i nā ʻano pathophysiology ma hope o AD. Manaʻo ʻia kēia mau mea hana ʻaʻole e hoʻololi wale i kā mākou AD diagnostic framework, akā hoʻolaha hoʻi i ka hoʻohana ʻana i nā hoʻolālā lapaʻau kūpono, hoʻomanawanui-specific (1, 2). I kēia hopena, ua noi mākou i kahi ala proteomic piha ʻole i ka lolo AD a me CSF e ʻike i nā biomarkers CSF e pili ana i ka pūnaewele e hōʻike ana i kahi ākea o ka pathophysiology e pili ana i ka lolo. ʻO kā mākou hōʻuluʻulu ʻana i hana i ʻelima mau paneli biomarker CSF, (i) hōʻike i nā synapses, nā kīʻaha koko, myelin, immune and metabolic dysfunction; (ii) hōʻike i ka reproducibility ikaika ma nā kahua MS like ʻole; (iii) Hōʻike i nā hoʻololi ʻokoʻa o ka maʻi i ka wā mua a me ka hope o AD. ʻO ka holoʻokoʻa, hōʻike kēia mau ʻike i kahi hana hoʻohiki i ka hoʻomohala ʻana i nā mea hana biomarker like ʻole, hilinaʻi, pūnaewele no ka noiʻi AD a me nā noi lapaʻau.
Hōʻike kā mākou mau hopena i ka hoʻonohonoho mālama nui ʻia o ka proteome network brain AD a kākoʻo i kona hoʻohana ʻana ma ke ʻano he heleuma no ka hoʻomohala ʻana i ka biomarker. Hōʻike kā mākou hōʻike ʻana i ʻelua mau ʻikepili TMT-MS kūʻokoʻa i loaʻa i ka lolo AD a me AsymAD i ka modularity ikaika. Hoʻonui kēia mau ʻike i kā mākou hana mua, e hōʻike ana i ka mālama ʻana i nā modula ikaika o nā ʻiʻo o ka lolo ma mua o 2,000 mai nā cohorts kūʻokoʻa he nui i ka frontal, parietal, a me ka cortex kino (17). Ke hōʻike nei kēia ʻenehana ʻokoʻa i nā loli e pili ana i nā maʻi i ʻike ʻia i ka noiʻi o kēia manawa, me ka hoʻonui ʻana o nā modules inflammatory glial-rich a me ka emi ʻana o nā modula waiwai waiwai neuron. E like me ka noiʻi o kēia manawa, ʻike pū ʻia kēia pūnaewele nui i nā loli modular koʻikoʻi i AsymAD, e hōʻike ana i nā ʻano like ʻole preclinical pathophysiology (17).
Eia nō naʻe, i loko o kēia ʻōnaehana conservative hoʻokumu ʻia, ʻoi aku ka nui o ka heterogeneity biological maikaʻi, ʻoi aku ma waena o nā poʻe i ka wā mua o AD. Hiki i kā mākou papa biomarker ke hōʻike i ʻelua mau pūʻulu ma AsymAD, e hōʻike ana i ka hōʻike ʻokoʻa koʻikoʻi o nā māka CSF lehulehu. Ua hiki i kā mākou hui ke hōʻike i nā ʻokoʻa olaola ma waena o kēia mau pūʻulu ʻelua, ʻaʻole i ʻike ʻia ma ke kiʻekiʻe o nā biomarkers koʻikoʻi AD. Hoʻohālikelike ʻia me ka pūʻulu mana, ʻo ka Aβ1-42/total tau ratios o kēia mau poʻe AsymAD he haʻahaʻa haʻahaʻa. Eia nō naʻe, ʻo ka nui o nā pae tau wale nō i ʻokoʻa loa ma waena o nā pūʻulu AsymAD ʻelua, aʻo nā pae Aβ1-42 a me ka p-tau i hoʻohālikelike ʻia. No ka mea, ʻoi aku ka maikaʻi o ka CSF tau kiʻekiʻe i nā hōʻailona cognitive ma mua o nā pae Aβ1-42 (7), ke manaʻo nei mākou he ʻokoʻa paha nā pilikia o ka piʻi ʻana o ka maʻi i nā cohorts AsymAD ʻelua. Hāʻawi ʻia i ka liʻiliʻi liʻiliʻi o kā mākou AsymAD a me ka nele o ka ʻikepili longitudinal, pono e noiʻi hou e huki i kēia mau hopena. Eia naʻe, hōʻike kēia mau hopena e hiki i kahi papa CSF e pili ana i ka ʻōnaehana ke hoʻonui i ko mākou hiki ke hoʻopaʻa pono i nā kānaka i ka wā asymptomatic o ka maʻi.
Ma ka holoʻokoʻa, kākoʻo kā mākou ʻike i ke kuleana o nā hana olaola he nui i ka pathogenesis o AD. Eia nō naʻe, ua lilo ka dysregulated energy metabolism i poʻomanaʻo koʻikoʻi o kā mākou papa inoa inoa ʻelima. ʻO nā protein metabolic, e like me ka hypoxanthine-guanine phosphoribosyltransferase 1 (HPRT1) a me ka lactate dehydrogenase A (LDHA), ʻo ia nā mea biomarkers synaptic i hōʻoia ʻia, e hōʻike ana i ka piʻi ʻana o AD CSF he wahine hiki ke hana hou. Loaʻa i kā mākou mau kīʻaha koko a me nā paneli glial kekahi mau māka i komo i ka metabolism o nā mea oxidative. Kūlike kēia mau ʻike me ke kuleana nui o nā kaʻina hana metabolic i loko o ka lolo holoʻokoʻa, ʻaʻole wale no ka hoʻokō ʻana i ka koi ikaika nui o nā neurons, akā no ka hoʻokō ʻana i ka koi ikaika kiʻekiʻe o nā astrocytes a me nā cell glial ʻē aʻe (17, 48). Kākoʻo kā mākou mau hopena i nā hōʻike ulu e hiki ke hoʻololi i ka hiki redox a me ka hoʻopau ʻana i nā ala o ka ikehu ke kumu kumu ma waena o nā kaʻina hana nui i pili i ka pathogenesis o AD, me nā maʻi mitochondrial, glial-mediated inflammation, a me Vascular damage (49). Eia kekahi, ʻo ka metabolic cerebrospinal fluid biomarkers ka nui o nā proteins waiwai like ʻole ma waena o kā mākou mana a me AD-like AsymAD subgroups, e hōʻike ana he mea koʻikoʻi ka hoʻopau ʻana o kēia mau ala ikaika a me redox i ka pae preclinical o ka maʻi.
ʻO nā ʻano loina ʻokoʻa o ka lolo a me ka cerebrospinal fluid panel a mākou i ʻike ai he mau hopena olaola hoihoi. ʻO nā synapses a me nā metabolomes waiwai i nā neurons e hōʻike ana i ka emi ʻana o nā pae i ka lolo AD a hoʻonui i ka nui o ka wai cerebrospinal. Ma muli o ka waiwai o nā neurons i ka mitochondria e hana ana i ka ikehu ma nā synapses e hāʻawi i ka ikehu no kā lākou mau hōʻailona kūikawā (50), ua manaʻo ʻia ke ʻano like o nā ʻōlelo hōʻike o kēia mau pūʻulu neuron. ʻO ka nalowale o nā neurons a me ka hoʻokuʻu ʻana o nā cell i hōʻino ʻia hiki ke wehewehe i kēia mau lolo a me CSF panel au i ka maʻi ma hope, akā ʻaʻole hiki iā lākou ke wehewehe i nā hoʻololi o ka paneli mua a mākou e ʻike ai (13). ʻO kahi wehewehe kūpono no kēia mau ʻike i ka maʻi asymptomatic mua ʻo ia ka ʻoki ʻana i ka synaptic. Hōʻike nā hōʻike hou i nā hiʻohiʻona ʻiole e hiki ke hoʻoneʻe ʻia ka microglia-mediated synaptic phagocytosis i AD a alakaʻi i ka nalowale mua o ka synapse i ka lolo (51). Hiki ke hōʻiliʻili kēia mea synaptic i ka CSF, ʻo ia ke kumu e ʻike ai mākou i ka piʻi ʻana o ka CSF ma ka panel neuron. Hiki ke wehewehe i ka hoʻonui ʻia o nā protein glial a mākou e ʻike ai i loko o ka lolo a me ka wai cerebrospinal i loko o ke kaʻina hana maʻi. Ma waho aʻe o ka synaptic pruning, hiki ke alakaʻi i nā ʻano like ʻole o ke ala exocytic i nā ʻano lolo like ʻole a me nā hōʻike CSF o nā māka neuronal. Ua hōʻike ʻia kekahi mau haʻawina ua loli ka ʻike o nā exosome i ka pathogenesis o ka lolo AD (52). Hoʻopili pū ʻia ke ala extracellular i ka hoʻonui ʻana o Aβ (53, 54). He mea pono e hoʻomaopopo i ka hoʻopau ʻana i ka exosomal secretion hiki ke hōʻemi i ka pathology like me AD i nā hiʻohiʻona kiʻi transgenic AD (55).
I ka manawa like, ua hōʻike ka protein i ka panel vascular i ka piʻi haʻahaʻa o ka lolo AD, akā ua emi nui i ka CSF. Hiki i ka pale koko-lolo (BBB) ​​ke wehewehe i kēia mau ʻike. Nui nā haʻawina kanaka postmortem kūʻokoʻa i hōʻike i ka haki ʻana o BBB i AD (56, 57). Ua hōʻoia kēia mau haʻawina i nā hana like ʻole e hoʻopuni ana i kēia papa paʻa paʻa o nā cell endothelial, me ka leakage capillary lolo a me ka hōʻiliʻili ʻana o nā protein i lawe ʻia i ke koko (57). Hiki i kēia ke hāʻawi i kahi wehewehe maʻalahi no nā protein vascular kiʻekiʻe i ka lolo, akā ʻaʻole hiki ke wehewehe piha i ka pau ʻana o kēia mau protein i loko o ka wai cerebrospinal. ʻO kekahi mea hiki ke hoʻokaʻawale ikaika ka ʻōnaehana nerve waena i kēia mau mole e hoʻoponopono i ka pilikia o ka hoʻonui ʻana i ka mumū a me ke kaumaha oxidative. ʻO ka hoʻemi ʻana i kekahi o nā protein CSF koʻikoʻi loa i kēia papa, ʻoi aku ka poʻe i pili i ka hoʻoponopono lipoprotein, pili i ka pale ʻana i nā pae ʻino o ka mumū a me ke kaʻina neuroprotective o nā ʻano oxygen reactive. He ʻoiaʻiʻo kēia no Paroxonase 1 (PON1), he enzyme hoʻopaʻa lipoprotein i kuleana no ka hoʻohaʻahaʻa ʻana i nā pae koʻikoʻi oxidative i ke kahe (58, 59). ʻO Alpha-1-microglobulin/bikunin precursor (AMBP) kekahi mea hōʻailona hoʻohaʻahaʻa i lalo o ka pūʻulu vascular. ʻO ia ka mua o ka bikunin transporter lipid, kahi i komo pū i ka hoʻopau ʻana a me ka pale neurological (60, 61).
ʻOiai ʻo nā kuhiakau hoihoi like ʻole, ʻo ka hiki ʻole ke ʻike pololei i nā mīkini maʻi biochemical kahi palena kaulana o ka nānā ʻana i nā proteomics ʻike. No laila, pono ka noiʻi hou ʻana e wehewehe pono i nā hana ma hope o kēia mau panela biomarker. I mea e neʻe ai i ka hoʻomohala ʻana i ka ʻimi noiʻi maʻi MS-based, pono nō ka ʻaoʻao o ka wā e hiki mai ana i ka hoʻohana ʻana i nā ʻano hana quantitative i manaʻo ʻia no ka hōʻoia biomarker nui, e like me ka nānā ʻana a i ʻole ka nānā ʻana i ka hopena (62). Ua hoʻohana hou mākou i ka nānā ʻana i ka hopena like (63) e hōʻoia i ka nui o nā hoʻololi protein CSF i wehewehe ʻia ma aneʻi. Ua helu ʻia kekahi mau pahuhopu papa mua me ka pololei nui, me YWHAZ, ALDOA, a me SMOC1, ka palapala ʻāina i kā mākou synapse, metabolism, a me nā panela ʻulaʻula, kēlā me kēia (63). Hiki ke hoʻohana ʻia ka ʻIke ʻIke Kūʻokoʻa (DIA) a me nā hoʻolālā ʻē aʻe e pili ana i ka MS no ka hōʻoia ʻana. ʻO Bud et al. (64) Ua hōʻike ʻia i kēia manawa aia kahi ʻano nui ma waena o nā biomarkers AD i ʻike ʻia i kā mākou CSF discovery data set a me ka DIA-MS data set kūʻokoʻa, aia ma kahi o 200 CSF samples mai ʻekolu mau ʻāpana ʻEulopa. Ke kākoʻo nei kēia mau haʻawina hou i ka hiki o kā mākou mau panela e hoʻololi i ka ʻike ʻike ʻia ʻo MS. He mea koʻikoʻi nō hoʻi ka antibody kuʻuna a me ka ʻike aptamer no ka hoʻomohala hou ʻana i nā biomarkers AD koʻikoʻi. Ma muli o ka haʻahaʻa haʻahaʻa o CSF, ʻoi aku ka paʻakikī o ka ʻike ʻana i kēia mau biomarkers me ka hoʻohana ʻana i nā ala MS kiʻekiʻe. ʻO NEFL a me NRGN ʻelua mau hiʻohiʻona o nā biomarkers CSF haʻahaʻa haʻahaʻa, i hoʻopaʻa ʻia i ka panel i kā mākou loiloi piha, akā ʻaʻole hiki ke ʻike ʻia me ka hoʻohana ʻana i kā mākou hoʻolālā MS hoʻokahi. ʻO ka hoʻolālā ʻana i nā hoʻolālā e pili ana i nā antibodies he nui, e like me ka PEA, hiki ke hāpai i ka hoʻololi ʻana o kēia mau māka.
Ma keʻano holoʻokoʻa, hāʻawi kēia haʻawina i kahi ala proteomic kūʻokoʻa no ka ʻike a me ka hōʻoia ʻana o nā biomarkers CSF AD e pili ana i nā ʻōnaehana like ʻole. ʻO ka hoʻomaikaʻi ʻana i kēia mau panela marker ma waena o nā cohorts AD hou a me nā kahua MS hiki ke hōʻoia i ka hoʻohiki ʻana e holomua i ka stratification a me ka mālama ʻana i ka AD. He mea koʻikoʻi nō hoʻi nā haʻawina e loiloi ana i ka pae lōʻihi o kēia mau panela i ka wā e hoʻoholo ai i ka hui ʻana o nā māka e hoʻopaʻa maikaʻi i ka pilikia o ka maʻi mua a me nā loli i ka nui o ka maʻi.
Koe no na laana 3 i kopeia e CSF, ua ohiia na laana CSF a pau i hoohana ia ma keia a'o ana ma lalo o ka mana o Emory ADRC a i ole na keena noii pili pili. Ua hoʻohana ʻia he ʻehā pūʻulu o nā laʻana Emory CSF i kēia mau haʻawina proteomics. Ua ʻike ʻia ka cohort CSF i nā laʻana mai 20 mau mana olakino a me 20 mau maʻi maʻi AD. Hoʻokomo ʻia ka CSF kope 1 i nā laʻana mai 32 mau mana olakino, 31 poʻe AsymAD, a me 33 mau kānaka AD. Aia ka CSF kope 2 he 147 mana a me 150 AD laana. ʻO ka nui o nā maʻi CSF replication 4 cohort i loaʻa i nā mana 18, 17 AD, 19 ALS, 13 PD, a me 11 FTD nā mea hoʻohālike. Wahi a ka ʻaelike i ʻae ʻia e ka Emory University Institutional Review Board, ua loaʻa i nā mea aʻo Emory a pau ka ʻae ʻike. Wahi a ka 2014 National Institute of Aging Best Practice Guidelines for Alzheimer's Centers (https://alz.washington.edu/BiospecimenTaskForce.html), ua hōʻiliʻili ʻia ka wai cerebrospinal a mālama ʻia e ka lumbar puncture. Ua loaʻa i nā maʻi Control a me AsymAD a me AD ka loiloi cognitive maʻamau ma Emory Cognitive Neurology Clinic a i ʻole Goizueta ADRC. Ua ho'āʻoʻia kā lākou mau'ōpana wai cerebrospinal e INNO-BIA AlzBio3 Luminex no ELISA Aβ1-42, ka nui o ka tau a me ka helu p-tau (65). Hoʻohana ʻia nā waiwai ELISA e kākoʻo i ka hoʻokaʻawale diagnostic o nā kumuhana i hoʻokumu ʻia i nā koina ʻokiʻoki biomarker AD (66, 67). Loaʻa ʻia ka ʻikepili demographic a me ka diagnostic no nā maʻi CSF ʻē aʻe (FTD, ALS, a me PD) mai Emory ADRC a i ʻole nā ​​hui noiʻi pili. Hiki ke loaʻa ka metadata hihia hōʻuluʻulu no kēia mau hihia Emory CSF ma ka Papa S1A. Ua paʻi mua ʻia nā ʻano o ka Swiss CSF replication 3 cohort (45).
Ua loaʻa iā CSF ka laʻana. I mea e hoʻonui ai i ka hohonu o kā mākou ʻike ʻana i ka hoʻonohonoho ʻikepili CSF, ua hana ʻia ka hoʻohana ʻana o nā protein kiʻekiʻe ma mua o ka trypsinization. I ka pōkole, 130 μl o CSF ​​mai 40 mau mea hoʻohālike CSF a me ka nui like (130 μl) o High Select Top14 Abundance Protein Depletion Resin (Thermo Fisher Scientific, A36372) i waiho ʻia i loko o kahi kolamu milo (Thermo Fisher Scientific, A89868) ma ka lumi. wela Incubate). Ma hope o ka milo ʻana no 15 mau minuke, e centrifuge ka hāpana ma 1000g no 2 mau minuke. Ua hoʻohana ʻia kahi mea kānana ultracentrifugal 3K (Millipore, UFC500396) e noʻonoʻo i ka hāpana effluent ma ka centrifuging ma 14,000g no 30 mau minuke. E hoʻoheheʻe i nā mea laʻana a pau i 75 μl me ka paʻakai phosphate buffered. Ua loiloi ʻia ka ʻike protein e ka bicinchoninic acid (BCA) e like me ka protocol a ka mea hana (Thermo Fisher Scientific). ʻO ka immunodepleted CSF (60 μl) mai nā laʻana 40 a pau i digested me lysyl endopeptidase (LysC) a me trypsin. I pōkole, ka hāpana ua ho'ēmi a alkylated me 1.2 μl 0.5 M tris-2 (-carboxyethyl) -phosphine a me 3 μl 0.8 M chloroacetamide ma 90 ° C no 10 minuke, a laila sonicated i loko o ka wai auau no 15 minuke. Ua hoʻoheheʻe ʻia ka hāpana me 193 μl 8 M urea buffer [8 M urea a me 100 mM NaHPO4 (pH 8.5)] i ka hopena hope o 6 M urea. Hoʻohana ʻia ʻo LysC (4.5 μg; Wako) no ka ʻai ʻana i ka pō ma ka wela o ka lumi. Ua hoʻoheheʻe ʻia ka hāpana i 1 M urea me 50 mM ammonium bicarbonate (ABC) (68). E hoʻohui i ka nui like (4.5 μg) o trypsin (Promega), a laila e hoʻoulu i ka hāpana no 12 mau hola. E hoʻokaʻina i ka solution peptide i hoʻoheheʻe ʻia i ka hopena hope loa o 1% formic acid (FA) a me 0.1% trifluoroacetic acid (TFA) (66), a laila hoʻopau i ka wai me kahi kolamu 50 mg Sep-Pak C18 (Waters) e like me ka mea i hōʻike ʻia ma luna (25) . A laila hoʻokaʻawale ʻia ka peptide i 1 ml o 50% acetonitrile (ACN). No ka hoʻohālikelike ʻana i ka helu ʻana o ka protein ma waena o nā pūʻulu (25), ua hui pū ʻia nā 100 μl aliquots mai nā 40 CSF samples e hana i kahi laʻana i hui ʻia, a laila māhele ʻia i ʻelima mau hōʻailona kūloko kūloko (GIS) (48). Hoʻomaloʻo ʻia nā mea hoʻohālike a pau a me nā maʻamau i hui ʻia e ka māmā holo wikiwiki (Labconco).
Hoʻopili ʻo CSF ​​i ka laʻana. Ua wehewehe mua ʻo Dayon a me nā hoa hana i ka hoʻopau ʻana o ka immune a me ka hoʻoheheʻe ʻana o ka CSF kope 3 samples (45, 46). ʻAʻole i hoʻopau pono ʻia nā mea hoʻohālike i koe. E ʻeli i kēia mau laʻana i wehe ʻole ʻia ma trypsin e like me ka mea i wehewehe mua ʻia (17). No kēlā me kēia loiloi hou, 120 μl aliquots o ka peptide eluted mai kēlā me kēia hāpana i hui pū ʻia a māhele ʻia i nā aliquots volume like e hoʻohana ʻia e like me ka TMT-labeled global internal standard (48). Hoʻomaloʻo ʻia nā mea hoʻohālike a pau a me nā maʻamau i hui ʻia e ka māmā holo wikiwiki (Labconco). I mea e hoʻonui ai i ka hōʻailona o ka protein CSF haʻahaʻa haʻahaʻa, ma ka hoʻohui ʻana i ka 125 μl mai kēlā me kēia hāpana, ua hoʻomākaukau ʻia kahi laʻana "hoʻonui" no kēlā me kēia hoʻopili hou ʻana [ʻo ia hoʻi, kahi laʻana olaola e hoʻohālike i ka hāpana noiʻi, akā ʻo ka nui i loaʻa. ʻoi aku ka nui (37, 69)] i hoʻohui ʻia i kahi laʻana CSF hui pū ʻia (17). Ua hoʻoneʻe ʻia ka laʻana i hui ʻia me ka hoʻohana ʻana i ka 12 ml o High Select Top14 Abundance Protein Removal Resin (Thermo Fisher Scientific, A36372), i ʻeli ʻia e like me ka mea i hōʻike ʻia ma luna, a hoʻokomo ʻia i loko o ka lepili TMT lehulehu.