Matenda a Alzheimer's (AD) alibe ma protein biomarkers omwe amawonetsa ma pathophysiology ake angapo, zomwe zimalepheretsa kupita patsogolo kwa matenda ndi chithandizo. Apa, timagwiritsa ntchito ma proteomics okwanira kuti tizindikire ma biomarker a cerebrospinal fluid (CSF) omwe amayimira mitundu yambiri ya AD pathophysiology. Multiplex mass spectrometry adazindikira pafupifupi 3,500 ndi pafupifupi 12,000 mapuloteni mu AD CSF ndi ubongo, motsatana. Kusanthula kwa netiweki kwa proteome yaubongo kunathetsa ma module 44 amitundu yosiyanasiyana, 15 omwe adalumikizana ndi cerebrospinal fluid proteome. Zolemba za CSF AD m'magawo ophatikizika awa amapindika m'magulu asanu a mapuloteni, kuyimira njira zosiyanasiyana zapathophysiological. Ma synapses ndi metabolites mu ubongo wa AD amachepetsa, koma CSF imawonjezeka, pamene glial-rich miyelination ndi magulu a chitetezo cha mthupi mu ubongo ndi CSF akuwonjezeka. Kusasinthika ndi kutsimikizika kwa matenda a kusintha kwa gululi kunatsimikiziridwa mu zitsanzo zowonjezera za 500 za CSF. Maguluwa adazindikiranso timagulu tachilengedwe tating'onoting'ono ta asymptomatic AD. Ponseponse, zotsatirazi ndi gawo lopatsa chiyembekezo ku zida zapaintaneti za biomarker pazogwiritsa ntchito zamankhwala mu AD.
Matenda a Alzheimer's (AD) ndi omwe amayambitsa matenda a neurodegenerative padziko lonse lapansi ndipo amadziwika ndi kusokonekera kosiyanasiyana kwa biological system, kuphatikiza kufalikira kwa ma synaptic, chitetezo chamthupi cha glial, ndi metabolism ya mitochondrial (1-3). Komabe, ma biomarkers ake okhazikika a protein amayang'anabe pakuzindikira mapuloteni a amyloid ndi tau, motero sangathe kuwonetsa mitundu yosiyanasiyana ya matenda. Ma "core" a protein biomarkers omwe amayezedwa modalirika mu cerebrospinal fluid (CSF) akuphatikizapo (i) amyloid beta peptide 1-42 (Aβ1-42), yomwe imasonyeza mapangidwe a cortical amyloid plaques; (ii) tau yathunthu, chizindikiro cha kuwonongeka kwa axon; (iii) phospho-tau (p-tau), woimira pathological tau hyperphosphorylation (4-7). Ngakhale kuti cerebrospinal fluid biomarkers izi zatithandizira kwambiri kuzindikira matenda a mapuloteni a AD "odziwika" (4-7), amangoimira gawo laling'ono la biology yovuta kuseri kwa matendawa.
Kuperewera kwa ma pathophysiological diversity of AD biomarkers kwadzetsa zovuta zambiri, kuphatikiza (i) kulephera kuzindikira ndi kuwerengera kuchuluka kwachilengedwe kwa odwala AD, (ii) kuyeza kosakwanira kwa kuuma kwa matenda ndi kupita patsogolo, makamaka mu gawo la preclinical, Ndipo ( iii) chitukuko cha mankhwala ochiritsira omwe analephera kuthetsa kwathunthu mbali zonse za kuwonongeka kwa mitsempha. Kudalira kwathu pa matenda odziwika bwino pofotokoza AD kuchokera ku matenda okhudzana nawo kumangowonjezera mavutowa. Umboni wochulukirachulukira ukuwonetsa kuti okalamba ambiri omwe ali ndi vuto la dementia amakhala ndi mawonekedwe opitilira chidziwitso chimodzi (8). Pafupifupi 90% kapena kupitilira apo omwe ali ndi matenda a AD alinso ndi matenda amitsempha, TDP-43 inclusions, kapena matenda ena osokonekera (9). Kuchulukana kwakukulu kwa kuphatikizika kwa matenda kwasokoneza njira yathu yodziwira matenda a dementia, ndipo kutanthauzira kokwanira kwa matendawa ndikofunikira.
Poganizira kufunikira kwachangu kwamitundu yosiyanasiyana ya AD biomarkers, gawoli likutengera njira ya "omics" kutengera dongosolo lonse lodziwikiratu ma biomarker. Mgwirizano wa Accelerated Pharmaceutical Partnership (AMP)-AD unakhazikitsidwa mu 2014 ndipo uli patsogolo pa pulogalamuyi. Khama losiyanasiyana lopangidwa ndi National Institutes of Health, maphunziro, ndi mafakitale likufuna kugwiritsa ntchito njira zokhazikitsidwa ndi dongosolo kuti lifotokoze bwino za matenda a AD ndikupanga kusanthula kwamitundu yosiyanasiyana yazachilengedwe ndi njira zamankhwala (10). Monga gawo la polojekitiyi, network proteomics yakhala chida chothandizira kupititsa patsogolo ma biomarkers mu AD. Njira yosakondera iyi yoyendetsedwa ndi data imakonza ma data a proteomics ovuta kukhala m'magulu kapena "ma module" a mapuloteni omwe amalumikizidwa ndi ma cell ena, organelles, ndi ntchito zachilengedwe (11-13). Pafupifupi maphunziro 12 olemera a network proteomics achitika pa ubongo wa AD (13-23). Ponseponse, kusanthula uku kukuwonetsa kuti ma proteome a ubongo a AD amakhala ndi gulu lotetezedwa kwambiri m'magulu odziyimira pawokha komanso zigawo zingapo za cortical. Kuphatikiza apo, ena mwa ma modulewa akuwonetsa kusintha kobwerezabwereza kwa kuchuluka kwa AD-zokhudzana ndi kuchuluka kwa ma data, kuwonetsa ma pathophysiology a matenda angapo. Pazonse, zomwe zapezazi zikuwonetsa chiyembekezo chotsimikizika cha kupezeka kwa netiweki yaubongo proteome ngati biomarker yochokera mu AD.
Kuti tisinthe ma proteome a AD brain network kukhala ma biomarker othandiza azachipatala, tidaphatikiza maukonde otengedwa muubongo ndi kusanthula kwa proteinomic kwa AD CSF. Njira yophatikizikayi idatsogolera kuzindikirika kwa magawo asanu olonjeza a CSF biomarkers omwe amalumikizidwa ndi mitundu ingapo yokhudzana ndi ubongo, kuphatikiza ma synapses, mitsempha yamagazi, myelination, kutupa, ndi kusokonekera kwa njira zama metabolic. Tidatsimikizira bwino mapanelo a biomarkerwa kudzera mu kusanthula kangapo, kuphatikiza zitsanzo zopitilira 500 CSF kuchokera ku matenda osiyanasiyana a neurodegenerative. Kufufuza kovomerezeka kumeneku kumaphatikizapo kufufuza zolinga zamagulu mu CSF ya odwala omwe ali ndi AD asymptomatic (AsymAD) kapena kusonyeza umboni wa kudzikundikira kwachilendo kwa amyloid m'malo ozindikira bwino. Kusanthula uku kukuwonetsa kusiyanasiyana kwachilengedwe kwachiwerengero cha anthu a AsymAD ndikuzindikira zolembera zomwe zitha kutsitsa anthu m'magawo oyambilira a matendawa. Ponseponse, zotsatirazi zikuyimira gawo lofunikira pakupanga zida zama protein biomarker zochokera pamakina angapo omwe amatha kuthetsa mavuto ambiri azachipatala omwe AD amakumana nawo.
Cholinga chachikulu cha phunziroli ndikuzindikira ma biomarker atsopano a cerebrospinal fluid omwe amawonetsa ma pathophysiology osiyanasiyana a muubongo omwe amatsogolera ku AD. Chithunzi S1 ikufotokoza njira yathu yofufuzira, yomwe imaphatikizapo (i) kusanthula kwathunthu koyendetsedwa ndi zomwe adapeza mu AD CSF ndi netiweki yaubongo proteome kuti azindikire ma biomarker okhudzana ndi matenda a CSF okhudzana ndi ubongo, ndi (ii) kubwereza kotsatira magulu a madzi. Kafukufuku wokhudzana ndi zopezekazo adayamba ndikuwunika kusiyanitsa kwa CSF mwa anthu 20 odziwika bwino komanso odwala 20 AD ku Emory Goizueta Alzheimer's Research Research Center (ADRC). Kuzindikira kwa AD kumatanthauzidwa kuti ndi vuto lalikulu lachidziwitso pamaso pa Aβ1-42 yotsika komanso kuchuluka kwa tau ndi p-tau mu cerebrospinal fluid [Mean Montreal Cognitive Assessment (MoCA), 13.8 ± 7.0] [ELISA (ELISA) )]] (Table S1A). Kuwongolera (kutanthauza MoCA, 26.7 ± 2.2) kunali ndi magawo abwinobwino a CSF biomarkers.
CSF yaumunthu imadziwika ndi kuchuluka kwa mapuloteni ambiri, momwe albumin ndi mapuloteni ena ochuluka kwambiri amatha kulepheretsa kupezeka kwa mapuloteni omwe amawakonda (24). Kuti tiwonjezere kuzama kwa kupezeka kwa mapuloteni, tidachotsa mapuloteni 14 ochuluka kwambiri pachitsanzo chilichonse cha CSF tisanayambe kusanthula kwa mass spectrometry (MS) (24). Ma peptides okwana 39,805 adadziwika ndi MS, omwe adajambulidwa kukhala ma proteome 3691 mu zitsanzo 40. Kuchulukitsidwa kwa mapuloteni kumapangidwa ndi zilembo zambiri za tandem mass tag (TMT) (18, 25). Kuti tithetse deta yomwe ikusowa, tinaphatikizapo mapuloteni okhawo omwe anawerengedwa osachepera 50% ya zitsanzo pakuwunika kotsatira, motero potsirizira pake timawerengera mapuloteni a 2875. Chifukwa cha kusiyana kwakukulu kwa kuchuluka kwa mapuloteni okwanira, chitsanzo chowongolera chimawonedwa ngati chachilendo (13) ndipo sichinaphatikizidwe pakuwunika kotsatira. Kuchuluka kwa zitsanzo 39 zotsalazo zidasinthidwa malinga ndi zaka, jenda, komanso ma batch covariance (13-15, 17, 18, 20, 26).
Pogwiritsa ntchito kusanthula kwa t-test kuti muwone kusiyana kwa kusiyana kwa deta yobwereranso, kusanthula uku kunazindikiritsa mapuloteni omwe kuchuluka kwake kunasinthidwa kwambiri (P <0.05) pakati pa kulamulira ndi milandu ya AD (Table S2A). Monga momwe tawonetsera pa Chithunzi 1A, kuchuluka kwa mapuloteni a 225 mu AD kunachepetsedwa kwambiri, ndipo kuchuluka kwa mapuloteni a 303 kunawonjezeka kwambiri. Mapuloteni owonetsedwa mosiyanasiyanawa akuphatikizapo zizindikiro zingapo za AD za cerebrospinal fluid, monga microtubule-associated protein tau (MAPT; P = 3.52 × 10−8), neurofilament (NEFL; P = 6.56 × 10−3), Mapuloteni okhudzana ndi kukula 43 (GAP43; P = 1.46 × 10−5), Fatty Acid Binding Protein 3 (FABP3; P = 2.00 × 10−5), Chitinase 3 ngati 1 (CHI3L1; P = 4.44 × 10−6), Neural Granulin (NRGN; P = 3.43 × 10-4) ndi VGF mitsempha kukula factor (VGF; P = 4.83 × 10-3) (4-6). Komabe, tinazindikiranso zolinga zina zofunika kwambiri, monga GDP dissociation inhibitor 1 (GDI1; P = 1.54 × 10-10) ndi SPARC yokhudzana ndi calcium modular kumanga 1 (SMOC1; P = 6.93 × 10-9) . Kusanthula kwa Gene Ontology (GO) kwa mapuloteni 225 ochepetsedwa kwambiri kunawonetsa kugwirizana kwambiri ndi njira zamadzimadzi am'thupi monga steroid metabolism, magazi coagulation, ndi zochita za mahomoni (Chithunzi 1B ndi Table S2B). Mosiyana ndi izi, mapuloteni ochuluka kwambiri a 303 amagwirizana kwambiri ndi mapangidwe a maselo ndi kagayidwe ka mphamvu.
(A) Chiwembu cha volcano chikuwonetsa kusintha kwa log2 (x-axis) poyerekeza ndi -log10 statistical P value (y-axis) yopezedwa ndi t-test, yomwe imagwiritsidwa ntchito kuzindikira kusiyana pakati pa kuwongolera (CT) ndi Matenda a AD a CSF proteinome Yamapuloteni onse. Mapuloteni okhala ndi milingo yocheperako kwambiri (P <0.05) mu AD amawonetsedwa mu buluu, pomwe mapuloteni omwe ali ndi milingo yowonjezereka ya matenda amawonetsedwa mofiira. Mapuloteni osankhidwa amalembedwa. (B) Mawu apamwamba a GO okhudzana ndi mapuloteni amachepetsedwa kwambiri (buluu) ndikuwonjezeka (ofiira) mu AD. Imawonetsa mawu atatu a GO omwe ali ndi z-scores apamwamba kwambiri pazachilengedwe, magwiridwe antchito a ma cell, ndi zida zam'manja. (C) MS anayeza mlingo wa MAPT mu chitsanzo cha CSF (kumanzere) ndi kugwirizanitsa kwake ndi chitsanzo cha ELISA tau mlingo (kumanja). Pearson coefficient coefficient yokhala ndi P yofunikira ikuwonetsedwa. Chifukwa chakusowa kwa data ya ELISA pamilandu imodzi ya AD, ziwerengerozi zikuphatikizanso mikhalidwe 38 mwa milandu 39 yomwe yawunikidwa. (D) Kuwunika kwamagulu oyang'anira (P <0.0001, Benjamini-Hochberg (BH) adasintha P <0.01) pa ulamuliro ndipo AD CSF inapeza zitsanzo pogwiritsa ntchito mapuloteni a 65 osinthidwa kwambiri mu deta. Standardize, normalize.
Mlingo wa proteinomic wa MAPT umagwirizana kwambiri ndi mulingo wa ELISA tau wodziyimira pawokha (r = 0.78, P = 7.8 × 10-9; Chithunzi 1C), kuthandizira kutsimikizika kwa kuyeza kwathu kwa MS. Pambuyo pa kugaya kwa trypsin pamlingo wa amyloid precursor protein (APP), ma peptides enieni a isoform omwe amajambulidwa ku C-terminus ya Aβ1-40 ndi Aβ1-42 sangathe kupangidwa bwino (27, 28). Chifukwa chake, ma peptides a APP omwe tidazindikira alibe chochita ndi ELISA Aβ1-42 milingo. Pofuna kuyesa kusiyanasiyana kwa nkhani iliyonse, tidagwiritsa ntchito mapuloteni opangidwa mosiyanasiyana ndi P <0.0001 [false discovery rate (FDR) yokonzedwa P <0.01] kuti tichite kusanthula kwamagulu koyang'aniridwa kwa zitsanzo (Table S2A). Monga momwe tawonetsera pa Chithunzi 1D, mapuloteni ofunikira kwambiri a 65 amatha kusonkhanitsa zitsanzo molingana ndi momwe matenda amakhalira, kupatulapo vuto limodzi la AD lomwe lili ndi machitidwe olamulira. Mwa mapuloteni 65 awa, 63 adawonjezeka mu AD, pomwe awiri okha (CD74 ndi ISLR) adatsika. Pazonse, kusanthula kwa cerebrospinal fluid iyi kwapeza mapuloteni mazana ambiri mu AD omwe amatha kukhala ngati ma biomarkers a matenda.
Kenako tidachita kafukufuku wodziyimira pawokha wa AD brain proteome. Gulu laubongo lomwe lapezekali linaphatikizapo dorsolateral prefrontal cortex (DLPFC) kuchokera ku control (n = 10), matenda a Parkinson (PD; n = 10), osakaniza AD / PD (n = 10) ndi AD (n = 10) milandu. ) Chitsanzo. Emery Goizueta ADRC. Ziwerengero za milandu 40 iyi zidafotokozedwa kale (25) ndipo zafotokozedwa mwachidule mu Table S1B. Tidagwiritsa ntchito TMT-MS kusanthula minyewa 40 yaubongo ndi gulu lobwereza la milandu 27. Pazonse, ma seti awiri aubongo awa adatulutsa ma peptide apadera a 227,121, omwe adajambulidwa kukhala ma protein 12,943 (25). Mapuloteni okhawo omwe amawerengedwa osachepera 50% a milandu adaphatikizidwa muzofufuza zotsatila. Deta yomaliza yopezeka ili ndi mapuloteni 8817 owerengeka. Sinthani kuchuluka kwa mapuloteni potengera zaka, jenda, ndi nthawi ya post-mortem (PMI). Kusanthula kwamafotokozedwe osiyanitsa a data yomwe idakhazikitsidwa pambuyo pobwerera kukuwonetsa kuti> 2000 mapuloteni adasinthidwa kwambiri [P <0.05, kusanthula kusiyanasiyana (ANOVA)] m'magulu awiri kapena angapo a matenda. Kenaka, tinachita kafukufuku wamagulu oyang'aniridwa pogwiritsa ntchito mapuloteni owonetsera mosiyana, ndi P <0.0001 mu AD / control ndi / kapena AD / PD kufananitsa (Chithunzi S2, A ndi B, Table S2C). Mapuloteni osinthidwa kwambiri a 165 akuwonetseratu milandu yokhala ndi AD pathology kuchokera ku ulamuliro ndi zitsanzo za PD, kutsimikizira kusintha kwakukulu kwa AD mu proteome yonse.
Kenako tinagwiritsa ntchito algorithm yotchedwa Weighted Gene Co-expression Network Analysis (WGCNA) kuti tifufuze maukonde pa proteome yaubongo yomwe idapezeka, yomwe imalinganiza zomwe zimayikidwa m'mapuloteni okhala ndi mawonekedwe ofanana (11-13). Kusanthula kunazindikiritsa ma modules a 44 (M) omwe amawonetsa mapuloteni, osankhidwa ndi owerengedwa kuchokera ku zazikulu (M1, n = 1821 mapuloteni) mpaka ochepa kwambiri (M44, n = 34 mapuloteni) (Chithunzi 2A ndi Table S2D)). Monga tafotokozera pamwambapa (13) Kuwerengera woimira mawu mbiri kapena khalidwe mapuloteni gawo lililonse, ndi kugwirizana ndi matenda boma ndi AD matenda, ndiye kukhazikitsa mgwirizano wa Alzheimer's Disease Registry (CERAD) ndi Braak Score (Chithunzi 2B). Ponseponse, ma module a 17 anali ogwirizana kwambiri ndi AD neuropathology (P <0.05). Ambiri mwa ma module okhudzana ndi matendawa alinso ndi zolembera zamtundu wa cell (Chithunzi 2B). Monga tafotokozera pamwambapa (13), kulemeretsa kwamtundu wa cell kumatsimikiziridwa ndikuwunika kuchulukana kwa ma module ndi mndandanda wamawu amtundu wamtundu wa cell. Majini awa amachokera ku deta yosindikizidwa mu ma neuroni a mbewa, endothelial ndi ma cell a glial. RNA sequencing (RNA-seq) kuyesa (29).
(A) Dziwani za WGCNA ya proteome ya ubongo. (B) Biweight midcorrelation (BiCor) kusanthula kwa mapuloteni a siginecha modular (chinthu choyamba chachikulu cha modular protein expression) ndi AD neuropathological properties (pamwamba), kuphatikizapo CERAD (Aβ plaque) ndi Braak (tau tangles) zambiri. Kulimba kwa kulumikizana kwabwino (kofiira) ndi koyipa (buluu) kumawonetsedwa ndi mapu amitundu iwiri kutentha, ndipo nyenyezi zimawonetsa kufunikira kwa ziwerengero (P <0.05). Gwiritsani Ntchito Mayeso Enieni a Hypergeometric Fisher's (FET) (pansi) kuti muwone kuyanjana kwamtundu wa ma cell a protein iliyonse. Kuchuluka kwa mthunzi wofiira kumasonyeza kukula kwa mtundu wa maselo, ndipo nyenyezi imasonyeza kufunikira kwa chiwerengero (P <0.05). Gwiritsani ntchito njira ya BH kukonza mtengo wa P wochokera ku FET. (C) GO kusanthula kwa mapuloteni okhazikika. Njira zofananira kwambiri zachilengedwe zimawonetsedwa pagawo lililonse kapena gulu lofananira. oligo, oligodendrocyte.
Gulu la ma modules asanu ogwirizana kwambiri a astrocyte ndi microglia-rich (M30, M29, M18, M24, ndi M5) adawonetsa mgwirizano wabwino kwambiri ndi AD neuropathology (Chithunzi 2B). Kusanthula kwa ontology kumalumikiza ma module a glial ndi kukula kwa maselo, kuchulukana, komanso chitetezo chokwanira (Chithunzi 2C ndi Table S2E). Ma module awiri owonjezera a glial, M8 ndi M22, amawongoleranso kwambiri matenda. M8 imagwirizana kwambiri ndi njira ya Toll-like receptor, njira yolumikizira yomwe imatenga gawo lalikulu pakuyankhidwa kwachilengedwe kwa chitetezo chamthupi (30). Panthawi imodzimodziyo, M22 ikugwirizana kwambiri ndi kusinthidwa pambuyo pomasulira. M2, yomwe ili ndi oligodendrocyte, imasonyeza mgwirizano wamphamvu ndi AD pathology ndi kugwirizana kwa ontological ndi nucleoside synthesis ndi DNA replication, kusonyeza kuwonjezeka kwa maselo mu matenda. Ponseponse, zotsatirazi zimathandizira kukwera kwa ma module a glial omwe tidawonapo kale mu AD network proteome (13, 17). Pakalipano akupezeka kuti ma module ambiri okhudzana ndi AD okhudzana ndi glial pamaneti amawonetsa milingo yocheperako pakuwongolera ndi milandu ya PD, kuwonetsa momwe matenda awo alili omwe amakwezedwa mu AD (Chithunzi S2C).
Ma module anayi okha mu network yathu ya proteome (M1, M3, M10, ndi M32) amalumikizana moyipa kwambiri ndi AD pathology (P <0.05) (Chithunzi 2, B ndi C). Onse M1 ndi M3 ali olemera mu zolembera za neuronal. M1 imagwirizana kwambiri ndi zizindikiro za synaptic, pamene M3 imagwirizana kwambiri ndi ntchito ya mitochondrial. Palibe umboni wowonjezera mtundu wa cell wa M10 ndi M32. M32 imasonyeza kugwirizana pakati pa M3 ndi maselo a metabolism, pamene M10 imagwirizana kwambiri ndi kukula kwa maselo ndi ntchito ya microtubule. Poyerekeza ndi AD, ma modules anayi onse amawonjezedwa ndikuwongolera ndi PD, kuwapatsa kusintha kwa AD kwa matenda (Chithunzi S2C). Ponseponse, zotsatirazi zimathandizira kuchepa kwa ma module olemera a neuron omwe tidawonapo kale mu AD (13, 17). Mwachidule, kusanthula kwa netiweki kwa proteome yaubongo komwe tidapeza kunapanga ma module osinthidwa a AD omwe amagwirizana ndi zomwe tapeza m'mbuyomu.
AD imadziwika ndi gawo loyambirira la asymptomatic (AsymAD), momwe anthu amawonetsa kudzikundikira kwa amyloid popanda kuchepa kwa chidziwitso chachipatala (5, 31). Gawo la asymptomatic iyi likuyimira zenera lofunikira pakuzindikira koyambirira komanso kuchitapo kanthu. Tawonetsa kale kutetezedwa kolimba kwa AsymAD ndi AD ubongo network proteome kudutsa ma data odziyimira pawokha (13, 17). Pofuna kuonetsetsa kuti maukonde aubongo omwe tapeza pano akugwirizana ndi zomwe tapeza m'mbuyomu, tidasanthula kusungidwa kwa ma module a 44 muzotengera zomwe zidapangidwanso kuchokera ku mabungwe a 27 DLPFC. Mabungwewa akuphatikizapo kulamulira (n = 10), AsymAD (n = 8) Ndi AD (n = 9) milandu. Zitsanzo zowongolera ndi AD zidaphatikizidwa pakuwunikira gulu lathu laubongo (Table S1B), pomwe milandu ya AsymAD inali yapadera pagulu lobwerezabwereza. Milandu ya AsymAD iyi idachokeranso ku banki yaubongo ya Emory Goizueta ADRC. Ngakhale kuzindikira kunali kwachilendo pa nthawi ya imfa, ma amyloid anali okwera kwambiri (kutanthauza CERAD, 2.8 ± 0.5) (Table S1B).
Kusanthula kwa TMT-MS kwa minyewa yaubongo ya 27 kudapangitsa kuti ma protein a 11,244 achuluke. Kuwerengera komalizaku kumaphatikizapo mapuloteni okhawo omwe amawerengedwa osachepera 50% ya zitsanzo. Deta yotsatiridwayi ili ndi 8638 (98.0%) ya mapuloteni a 8817 omwe apezeka mu kufufuza kwathu kwa ubongo, ndipo pafupifupi 3000 yasintha kwambiri mapuloteni pakati pa olamulira ndi ma AD cohorts (P <0.05, pambuyo pa Tukey's paired t test for analysis of kusiyana) ( Gulu S2F). Pakati pa mapuloteni owonetsedwa mosiyanasiyana, 910 idawonetsanso kusintha kwakukulu pakati pa AD ndi ma proteome control kesi (P <0.05, pambuyo pa ANOVA Tukey paired t-test). Ndizofunikira kudziwa kuti zizindikiro za 910 zimagwirizana kwambiri pakusintha pakati pa ma proteomes (r = 0.94, P <1.0 × 10-200) (Chithunzi S3A). Pakati pa mapuloteni owonjezereka, mapuloteni omwe ali ndi kusintha kosasintha pakati pa seti ya deta makamaka mamembala a glial-rich M5 ndi M18 modules (MDK, COL25A1, MAPT, NTN1, SMOC1, ndi GFAP). Pakati pa mapuloteni ochepetsedwa, omwe ali ndi kusintha kosasinthasintha anali pafupifupi mamembala a M1 module (NPTX2, VGF, ndi RPH3A) yogwirizana ndi synapse. Tidatsimikiziranso kusintha kokhudzana ndi AD kwa midkine (MDK), CD44, protein 1 yokhudzana ndi frizzled (SFRP1) ndi VGF ndi Western blotting (Chithunzi S3B). Kusanthula kosungirako ma module kunawonetsa kuti pafupifupi 80% ya ma module a protein (34/44) mu proteome yaubongo adasungidwa kwambiri mu seti ya data yobwereza (z-score> 1.96, FDR yokonzedwa P <0.05) (Chithunzi S3C). Makumi ndi anayi mwa ma module awa adasungidwa mwapadera pakati pa ma proteome awiri (z-score> 10, FDR yokonzedwa P <1.0 × 10-23). Ponseponse, kupezedwa ndi kubwereza kwa kuchuluka kwa kusasinthika kwamafotokozedwe osiyanitsidwa ndi kapangidwe kake pakati pa proteome yaubongo kumawunikira kupangidwanso kwa kusintha kwa mapuloteni a AD frontal cortex. Kuphatikiza apo, idatsimikiziranso kuti AsymAD ndi matenda otsogola kwambiri ali ndi mawonekedwe ofanana kwambiri muubongo.
Kusanthula mwatsatanetsatane kwa kusiyanasiyana kwa mafotokozedwe muubongo wobwereza deta kumawonetsa kuchuluka kwakukulu kwa kusintha kwa mapuloteni a AsymAD, kuphatikiza ma protein a 151 osintha kwambiri pakati pa AsymAD ndi control (P <0.05) (Chithunzi S3D). Mogwirizana ndi katundu wa amyloid, APP mu ubongo wa AsymAD ndi AD inakula kwambiri. MAPT imangosintha kwambiri mu AD, yomwe imagwirizana ndi kuchuluka kwa ma tangles komanso kulumikizana kwake kodziwika ndi kuchepa kwa chidziwitso (5, 7). Ma modules olemera a glial (M5 ndi M18) amawonekera kwambiri mu mapuloteni owonjezereka ku AsymAD, pamene gawo la M1 la neuron ndilomwe limayimira kwambiri mapuloteni omwe amachepetsa ku AsymAD. Zambiri mwa zolembera za AsymAD zikuwonetsa kusintha kwakukulu kwa matenda azizindikiro. Zina mwa zolemberazi ndi SMOC1, mapuloteni a glial a M18, omwe amalumikizidwa ndi zotupa muubongo komanso kukula kwa maso ndi miyendo (32). MDK ndi chinthu chomangika cha heparin chokhudzana ndi kukula kwa maselo ndi angiogenesis (33), membala wina wa M18. Poyerekeza ndi gulu lolamulira, AsymAD inakula kwambiri, kutsatiridwa ndi kuwonjezeka kwakukulu kwa AD. Mosiyana ndi izi, synaptic protein neuropentraxin 2 (NPTX2) idachepetsedwa kwambiri muubongo wa AsymAD. NPTX2 idalumikizidwa kale ndi neurodegeneration ndipo ili ndi gawo lodziwika pakuyanjanitsa ma synapses osangalatsa (34). Ponseponse, zotsatirazi zikuwonetsa kusintha kosiyanasiyana kwa mapuloteni oyambira mu AD omwe akuwoneka kuti akupita patsogolo ndi kuopsa kwa matendawa.
Poganizira kuti tapeza kuzama kwakukulu kwa kufalikira kwa mapuloteni pakutulukira kwa proteome ya muubongo, tikuyesera kumvetsetsa bwino kuphatikizika kwake ndi netiweki ya AD transcriptome. Chifukwa chake, tidafanizira proteome yaubongo yomwe tidapeza ndi gawo lomwe tidapangapo kale kuchokera muyeso la microarray la jini 18,204 mu AD (n = 308) ndikuwongolera (n = 157) minofu ya DLPFC (13). kudutsana. Ponseponse, tidazindikira ma module a 20 osiyanasiyana a RNA, ambiri omwe adawonetsa kulemerera kwamitundu ina ya maselo, kuphatikiza ma neuroni, oligodendrocytes, astrocytes, ndi microglia (Chithunzi 3A). Kusintha kangapo kwa ma module awa mu AD kukuwonetsedwa mu Chithunzi 3B. Mogwirizana ndi kafukufuku wathu wam'mbuyomu wa protein-RNA overlap pogwiritsa ntchito zakuya zosalemba za MS proteome (pafupifupi 3000 mapuloteni) (13), ma modules ambiri a 44 mu network ya proteome network omwe tapeza ali mu network ya transcriptome Palibe kuphatikizika kwakukulu. Kupeza kwathu ndi kubwereza kwa ma modules a 34 a mapuloteni omwe amasungidwa kwambiri mu proteome ya ubongo, 14 (~ 40%) okha omwe adapambana mayeso enieni a Fisher (FET) adatsimikizira kuti ali ndi chiwerengero chachikulu chophatikizira ndi transcriptome (Chithunzi 3A). Kugwirizana ndi kukonza zowonongeka kwa DNA (P-M25 ndi P-M19), kumasulira kwa mapuloteni (P-M7 ndi P-M20), RNA binding/splicing (P-M16 ndi P-M21) ndi kutsata mapuloteni (P-M13 ndi P- M23) sichiphatikizana ndi ma module mu transcriptome. Choncho, ngakhale deta yozama ya proteome imagwiritsidwa ntchito pakuwunika kwaposachedwa (13), ambiri a AD network proteome samajambulidwa ku netiweki ya transcriptome.
(A) Hypergeometric FET imawonetsa kulemetsedwa kwa zolembera zamtundu wa cell mu gawo la RNA la AD transcriptome (pamwamba) ndi kuchuluka kwa kuphatikizika pakati pa ma module a RNA (x-axis) ndi protein (y-axis) a ubongo wa AD. (pansi). Kuchuluka kwa shading yofiira kumawonetsa kuchuluka kwa kuchuluka kwa ma cell mu gulu lapamwamba komanso kuchuluka kwa kuphatikizika kwa ma modules pansi. Nyenyezi zimasonyeza kufunikira kwa chiwerengero (P <0.05). (B) Mlingo wa kulumikizana pakati pamtundu wamtundu uliwonse wa transcriptome module ndi AD. Ma modules omwe ali kumanzere ndi omwe amatsutsana kwambiri ndi AD (buluu), ndipo omwe ali kumanja ndi omwe amagwirizana kwambiri ndi AD (ofiira). Mtengo wosinthidwa wa chipika wa BH-wowongolera P ukuwonetsa kuchuluka kwa kufunikira kwa kulumikizana kulikonse. (C) Ma module ofunikira omwe ali ndi ma cell omwe amagawana nawo. (D) Kusanthula kwa kulumikizana kwa kusintha kwa log2 fold kwa protein yolembedwa (x-axis) ndi RNA (y-axis) mugawo lopitilira. Pearson coefficient coefficient yokhala ndi P yofunikira ikuwonetsedwa. Microglia; matupi akuthambo, astrocyte. CT, control.
Mapuloteni ambiri ophatikizika ndi ma module a RNA amagawana mbiri yofananira yamtundu wa cell komanso njira zosinthira za AD (Chithunzi 3, B ndi C). Mwanjira ina, gawo la M1 lokhudzana ndi synapse la ubongo proteome (PM1) limapangidwa kukhala ma module atatu a neuronal-rich homologous RNA (R-M1, R-M9 ndi R-M16), omwe ali mu AD mlingo wochepetsedwa. Mofananamo, ma modules a glial-rich M5 ndi M18 amaphatikizana ndi ma module a RNA olemera mu astrocytes ndi ma microglial markers (R-M3, R-M7, ndi R-M10) ndipo amakhudzidwa kwambiri ndi matenda Kuwonjezeka. Izi zomwe zimagawidwa modulira pakati pa ma data awiriwa zimathandizira kukulitsa kwamtundu wa cell komanso kusintha kokhudzana ndi matenda komwe tawona mu proteome yaubongo. Komabe, tidawona kusiyana kwakukulu pakati pa RNA ndi kuchuluka kwa mapuloteni a zolembera zapayekha m'magawo ogawana nawo. Kusanthula kwa kulumikizana kwa kusiyanasiyana kwa ma proteomics ndi ma transcriptomics a mamolekyu omwe ali mkati mwa ma module ophatikizika awa (Chithunzi 3D) akuwonetsa kusagwirizana uku. Mwachitsanzo, APP ndi mapuloteni ena angapo a glial module (NTN1, MDK, COL25A1, ICAM1, ndi SFRP1) adawonetsa kuwonjezeka kwakukulu kwa AD proteome, koma panalibe pafupifupi kusintha kwa AD transcriptome. Kusintha kwa mapuloteniwa kumatha kukhala kogwirizana kwambiri ndi zolembera za amyloid (23, 35), kuwonetsa proteome ngati gwero la kusintha kwa ma pathological, ndipo zosinthazi sizingawonekere muzolemba.
Titafufuza mozama zaubongo ndi ma CSF ma proteomes omwe tidapeza, tidasanthula mwatsatanetsatane ma seti awiriwa kuti tizindikire ma biomarker a AD CSF okhudzana ndi pathophysiology ya netiweki yaubongo. Choyamba tiyenera kutanthauzira kuphatikizika kwa ma proteome awiri. Ngakhale zimavomerezedwa kuti CSF imawonetsa kusintha kwa neurochemical mu ubongo wa AD (4), kuchuluka kwenikweni kwapang'onopang'ono pakati pa ubongo wa AD ndi CSF proteome sikudziwika bwino. Poyerekeza kuchuluka kwa ma jini omwe amagawidwa m'maproteome athu awiri, tidapeza kuti pafupifupi 70% (n = 1936) ya mapuloteni odziwika mu cerebrospinal fluid adawerengedwanso muubongo (Chithunzi 4A). Ambiri mwa mapuloteni ophatikizikawa (n = 1721) amajambulidwa ku imodzi mwama module 44 ofotokozera kuchokera kuzinthu zopezeka muubongo (Chithunzi 4B). Monga zikuyembekezeredwa, ma module asanu ndi limodzi akuluakulu a ubongo (M1 mpaka M6) adawonetsa kuchuluka kwakukulu kwa CSF. Komabe, pali ma modules ang'onoang'ono a ubongo (mwachitsanzo, M15 ndi M29) omwe amakwaniritsa mosayembekezereka kuchulukana kwakukulu, kwakukulu kuposa gawo la ubongo kawiri kukula kwake. Izi zimatilimbikitsa kutengera njira yatsatanetsatane, yoyendetsedwa ndi ziwerengero yowerengera kuchulukana pakati pa ubongo ndi cerebrospinal fluid.
(A ndi B) Mapuloteni omwe amapezeka muubongo wopezeka ndi ma data a CSF amalumikizana. Ambiri mwa mapuloteni ophatikizikawa amalumikizidwa ndi imodzi mwama 44 ma co-expression module a network co-expression network. (C) Dziwani kuphatikizika pakati pa cerebrospinal fluid proteome ndi proteome network network. Mzere uliwonse wa mapu otentha umayimira kusanthula kosiyana kwa hypergeometric FET. Mzere wapamwamba umasonyeza kuphatikizika (mthunzi wotuwa / wakuda) pakati pa gawo la ubongo ndi proteome yonse ya CSF. Mzere wachiwiri ukuwonetsa kuti kuphatikizika pakati pa ma module a ubongo ndi mapuloteni a CSF (ofiira ofiira) kumayendetsedwa kwambiri mu AD (P <0.05). Mzere wachitatu umasonyeza kuti kuphatikizika pakati pa ma modules a ubongo ndi mapuloteni a CSF (blue shading) kumakhala pansi kwambiri mu AD (P <0.05). Gwiritsani ntchito njira ya BH kukonza mtengo wa P wochokera ku FET. (D) Pindani gawo la gawo lotengera mayanjano amtundu wa cell ndi mawu okhudzana ndi GO. Mapulogalamuwa ali ndi mapuloteni okhudzana ndi ubongo a 271, omwe ali ndi tanthauzo losiyana mu CSF proteome.
Pogwiritsa ntchito ma FET okhala ndi mchira umodzi, tinawona kufunikira kwa mapuloteni pakati pa CSF proteome ndi ma module a ubongo. Kufufuzaku kunawonetsa kuti ma modules a ubongo a 14 mu data ya CSF ali ndi zowonjezereka zowonjezereka (FDR yosinthidwa P <0.05), ndi gawo lina (M18) lomwe kuphatikizika kwake kuli pafupi ndi kufunikira (FDR yosinthidwa P = 0.06) (Chithunzi 4C , mzere wapamwamba). Tilinso ndi chidwi ndi ma module omwe amalumikizana kwambiri ndi mapuloteni a CSF owonetsedwa mosiyanasiyana. Chifukwa chake, tidagwiritsa ntchito kuwunika kuwiri kowonjezera kwa FET kuti tidziwe (i) mapuloteni a CSF omwe adachulukitsidwa kwambiri mu AD ndipo (ii) mapuloteni a CSF adatsika kwambiri mu AD (P <0.05, paired t test AD/control) ma module aubongo okhala ndi kuphatikizika koyenera. pakati pawo. Monga momwe mizere yapakati ndi yapansi ya Chithunzi 4C, kusanthula kowonjezera kumeneku kumasonyeza kuti 8 ya ma modules a ubongo a 44 amagwirizana kwambiri ndi mapuloteni owonjezeredwa mu AD CSF (M12, M1, M2, M18, M5, M44, M33, ndi M38) . ), pamene ma modules awiri okha (M6 ndi M15) adawonetsa kugwirizanitsa kopindulitsa ndi mapuloteni ochepetsedwa mu AD CSF. Monga zikuyembekezeredwa, ma module onse a 10 ali m'mamodule 15 omwe amalumikizana kwambiri ndi CSF proteome. Choncho, tikuganiza kuti ma modules a 15 ndi magwero apamwamba a AD omwe amachokera ku ubongo wa CSF biomarkers.
Tidapinda ma modules 15 odumphadumphawa kukhala mapanelo akuluakulu asanu a protein kutengera kuyandikira kwawo pachithunzi cha mtengo wa WGCNA komanso kulumikizana kwawo ndi mitundu ya ma cell ndi gene ontology (Chithunzi 4D). Gulu loyamba lili ndi ma module okhala ndi zolembera za neuron ndi mapuloteni okhudzana ndi synapse (M1 ndi M12). Gulu la synaptic lili ndi mapuloteni onse a 94, ndipo milingo ya CSF proteome yasintha kwambiri, ndikupangitsa kuti ikhale gwero lalikulu la zolembera za CSF zokhudzana ndi ubongo pakati pa mapanelo asanu. Gulu lachiwiri (M6 ndi M15) linasonyeza kugwirizana kwapafupi ndi zizindikiro za endothelial cell ndi mitsempha ya mitsempha, monga "machiritso a mabala" (M6) ndi "kuwongolera kuyankha kwa humoral immune" (M15). M15 imagwirizananso kwambiri ndi lipoprotein metabolism, yomwe imagwirizana kwambiri ndi endothelium (36). Gulu la mitsempha lili ndi zolembera za 34 CSF zokhudzana ndi ubongo. Gulu lachitatu limaphatikizapo ma modules (M2 ndi M4) omwe amagwirizana kwambiri ndi zizindikiro za oligodendrocyte ndi kuchuluka kwa maselo. Mwachitsanzo, mawu apamwamba a ontology a M2 akuphatikizapo "kuwongolera kwabwino kwa DNA replication" ndi "purine biosynthesis process". Pakadali pano, a M4 akuphatikizapo "kusiyana kwa ma cell a glial" ndi "kusiyana kwa chromosome". Gulu la myelination lili ndi zolembera za 49 CSF zokhudzana ndi ubongo.
Gulu lachinayi lili ndi ma modules ambiri (M30, M29, M18, M24, ndi M5), ndipo pafupifupi ma modules onse ali olemera kwambiri mu microglia ndi astrocyte markers. Mofanana ndi gulu la myelination, gulu lachinayi limakhalanso ndi ma modules (M30, M29, ndi M18) omwe amagwirizana kwambiri ndi kuchuluka kwa maselo. Ma modules ena omwe ali m'gululi amagwirizana kwambiri ndi mawu a immunological, monga "chitetezo cha chitetezo cha mthupi" (M5) ndi "chitetezo cha chitetezo cha mthupi" (M24). Gulu la chitetezo cha glial lili ndi zolembera za 42 CSF zokhudzana ndi ubongo. Potsirizira pake, gulu lomaliza limaphatikizapo zizindikiro za 52 zokhudzana ndi ubongo pa ma modules anayi (M44, M3, M33, ndi M38), zonse zomwe zili m'thupi zokhudzana ndi kusungirako mphamvu ndi metabolism. Yaikulu kwambiri mwa ma module awa (M3) imagwirizana kwambiri ndi mitochondria ndipo imakhala ndi zolembera za neuron. M38 ndi amodzi mwamagawo ang'onoang'ono mu metabolome iyi komanso amawonetsa kutsimikizika kwa neuron.
Ponseponse, mapanelo asanuwa amawonetsa mitundu yosiyanasiyana ya maselo ndi ntchito mu AD cortex, ndipo palimodzi ali ndi 271 zolembera za CSF zokhudzana ndi ubongo (Table S2G). Kuti tiwone ngati zotsatira za MS izi ndi zowona, tidagwiritsa ntchito proximity extension assay (PEA), ukadaulo wa orthogonal antibody wokhala ndi kuthekera kochulukira, kukhudzika kwakukulu komanso kutsimikizika, ndikuwunikanso zitsanzo zamadzimadzi a muubongo omwe tapeza Kagawo kakang'ono ka 271 biomarkers. (n = 36). Zolinga za 36 izi zikuwonetsa kusintha kwa AD angapo a PEA, omwe amagwirizana kwambiri ndi zomwe tapeza kuchokera ku MS (r = 0.87, P = 5.6 × 10-12), Zomwe zinatsimikizira mwamphamvu zotsatira za kusanthula kwathu kwakukulu kwa MS (Chithunzi S4 ).
Mitu yazachilengedwe yomwe imagogomezeredwa ndi magulu athu asanu, kuyambira pa synaptic signing to energy metabolism, zonse zimagwirizana ndi matenda a AD (1-3). Choncho, ma modules onse a 15 omwe ali ndi mapanelowa amagwirizana ndi AD pathology mu proteome ya ubongo yomwe tidapeza (Chithunzi 2B). Chodziwika kwambiri ndi kulumikizana kwabwino kwapathological pakati pa ma module athu a glial ndi kulumikizana kwamphamvu kwapathological pakati pa ma module athu akuluakulu a neuronal (M1 ndi M3). Kusanthula kwamafotokozedwe osiyanasiyana a proteome yathu yaubongo (Chithunzi S3D) ikuwonetsanso mapuloteni a glial a M5 ndi M18. Mu AsymAD ndi chizindikiro cha AD, mapuloteni ochuluka kwambiri a glial ndi ma synapses okhudzana ndi M1 Puloteni imachepetsedwa kwambiri. Zomwe taziwonazi zikuwonetsa kuti zizindikiro za 271 cerebrospinal fluid zomwe tazizindikira m'magulu asanu zimagwirizana ndi njira za matenda mu AD cortex, kuphatikizapo zomwe zimachitika kumayambiriro kwa asymptomatic.
Pofuna kusanthula bwino momwe mapuloteni amapangidwira muubongo ndi madzi am'mimba, tidajambula zotsatirazi pagawo lililonse la 15 lomwe limadutsamo: (i) adapeza kuchuluka kwa gawo mu seti ya data yaubongo ndi (ii) gawo. mapuloteni Kusiyana kumawonetsedwa mu cerebrospinal fluid (Chithunzi S5). Monga tanena kale, WGCNA imagwiritsidwa ntchito kudziwa kuchuluka kwa gawo kapena kuchuluka kwa mapuloteni muubongo (13). Mapu a phiri lamoto amagwiritsidwa ntchito kufotokoza kusiyana kwa mapuloteni amtundu wa cerebrospinal fluid (AD/control). Ziwerengerozi zikuwonetsa kuti mapanelo atatu mwa asanu amawonetsa machitidwe osiyanasiyana muubongo ndi msana. Ma module awiri a gulu la synapse (M1 ndi M12) akuwonetsa kuchepa kwa kuchuluka kwa kuchuluka kwa ubongo wa AD, koma amalumikizana kwambiri ndi mapuloteni ochulukirapo mu AD CSF (Chithunzi S5A). Ma module okhudzana ndi ma neuron omwe ali ndi metabolome (M3 ndi M38) adawonetsa mawonekedwe ofanana a ubongo ndi cerebrospinal fluid zosagwirizana (Chithunzi S5E). Gulu la mitsempha linawonetsanso machitidwe osiyanasiyana, ngakhale kuti ma modules (M6 ndi M15) adawonjezeka pang'onopang'ono mu ubongo wa AD ndipo anachepa mu CSF odwala (Chithunzi S5B). Mapanelo awiri otsalawo ali ndi maukonde akulu a glial omwe mapuloteni ake amakhala osakhazikika m'zipinda zonse ziwiri (Chithunzi S5, C ndi D).
Chonde dziwani kuti izi sizodziwika kwa zolembera zonse pamapanelo awa. Mwachitsanzo, gulu la synaptic limaphatikizapo mapuloteni angapo omwe amachepetsedwa kwambiri mu ubongo wa AD ndi CSF (Chithunzi S5A). Zina mwa zolembera za cerebrospinal fluid zotsika ndi NPTX2 ndi VGF ya M1, ndi chromogranin B ya M12. Komabe, ngakhale izi ndizosiyana, zolembera zathu zambiri za synaptic zimakwezedwa mu AD spinal fluid. Ponseponse, kusanthula uku kunatha kusiyanitsa momwe zinthu zimayendera muubongo ndi cerebrospinal fluid m'magulu athu asanu. Izi zikuwonetsa mgwirizano wovuta komanso wosiyana pakati pa ubongo ndi CSF protein expression mu AD.
Kenako, tidagwiritsa ntchito kusanthula kwapamwamba kwa MS replication (CSF replication 1) kuti tichepetse seti yathu ya 271 ya biomarkers ku mipherezero yodalirika komanso yobwereketsa (Chithunzi 5A). CSF kope 1 lili ndi chiwerengero cha zitsanzo 96 kuchokera Emory Goizueta ADRC, kuphatikizapo control, AsymAD, ndi AD gulu (Table S1A). Milandu ya AD iyi imadziwika ndi kuchepa kwachidziwitso pang'ono (kutanthauza MoCA, 20.0 ± 3.8), ndi kusintha kwa ma biomarkers a AD omwe amatsimikiziridwa mu cerebrospinal fluid (Table S1A). Mosiyana ndi kusanthula kwa CSF komwe tapeza, kubwereza uku kumachitika pogwiritsa ntchito njira yabwino kwambiri komanso yapamwamba kwambiri "yowombera imodzi" ya MS (popanda kugawa kwapaintaneti), kuphatikiza ndondomeko yophweka yokonzekera yomwe imathetsa kufunika kwa immunodepletion ya zitsanzo za munthu aliyense. . M'malo mwake, "njira yowonjezera" yomwe ili ndi chitetezo chamthupi imodzi imagwiritsidwa ntchito kukulitsa chizindikiro cha mapuloteni ocheperako (37). Ngakhale amachepetsa kufalikira kwa proteome, njira yowombera imodziyi imachepetsa kwambiri nthawi yamakina ndikuwonjezera kuchuluka kwa zitsanzo zolembedwa ndi TMT zomwe zitha kuyesedwa zotheka (17, 38). Pazonse, kuwunikaku kudapeza ma peptides 6,487, omwe adawonetsa ma protein 1,183 pamilandu 96. Mofanana ndi kusanthula kwa CSF komwe tidapeza, mapuloteni okhawo omwe amawerengedwa osachepera 50% mwa zitsanzo adaphatikizidwa muzowerengera zotsatizana, ndipo deta idasinthidwa chifukwa cha zaka ndi jenda. Izi zinapangitsa kuti chiwerengero chomaliza cha ma proteome 792, 95% omwe adadziwikanso mu data ya CSF yomwe yapezeka.
(A) Zolinga zama protein za CSF zokhudzana ndi ubongo zomwe zidatsimikiziridwa mu gulu loyamba lofananizidwa la CSF ndikuphatikizidwa mu gulu lomaliza (n = 60). (B mpaka E) Magulu a biomarker (composite z-scores) amayezedwa m'magulu anayi obwereza a CSF. Mayeso a t-pawiri kapena ANOVA yokhala ndi kuwongolera pambuyo pa Tukey adagwiritsidwa ntchito kuwunika tanthauzo la kuchuluka kwa kusintha kochulukira pakuwunika kulikonse. CT, control.
Popeza tili ndi chidwi chofuna kutsimikizira zolinga zathu za 271 zokhudzana ndi ubongo za CSF kudzera mu kusanthula mwatsatanetsatane, tichepetsa kuwunikanso kwa proteome yobwerezedwa iyi kwa zolembera izi. Pakati pa mapuloteni a 271, 100 anapezeka mu kubwereza kwa CSF 1. Chithunzi cha S6A chimasonyeza kusiyana kwa zizindikiro za 100 zomwe zikudutsana pakati pa kulamulira ndi AD kubwereza zitsanzo. Synaptic ndi metabolite histones amachulukitsa kwambiri mu AD, pomwe mapuloteni am'mitsempha amachepetsa kwambiri matenda. Zambiri mwa zolembera za 100 zomwe zikudutsana (n = 70) zinasunga njira yofanana yosinthira ma data awiri (Chithunzi S6B). Izi 70 zovomerezeka za CSF zokhudzana ndi ubongo (Table S2H) zimawonetsa kwambiri machitidwe omwe adawonedwa kale, ndiko kuti, kutsika kwa mapuloteni am'mitsempha komanso kuwongolera kwa mapanelo ena onse. 10 yokha mwa mapuloteni 70 ovomerezekawa adawonetsa kusintha kwa kuchuluka kwa AD komwe kumatsutsana ndi machitidwe awa. Kuti tipange gulu lomwe likuwonetsa bwino momwe ubongo ndi cerebrospinal fluid zimayendera, sitinaphatikizepo mapuloteni 10 awa pagulu lachidwi lomwe tidatsimikizira pomaliza pake (Chithunzi 5A). Chifukwa chake, gulu lathu pamapeto pake limaphatikizapo mapuloteni okwana 60 otsimikiziridwa m'magulu awiri odziyimira pawokha a CSF AD pogwiritsa ntchito kukonzekera kwa zitsanzo ndi kusanthula kwa nsanja ya MS. Mafotokozedwe a z-score a mapanelo omalizawa mu CSF kuwongolera 1 ndi milandu ya AD adatsimikizira zomwe zidawonedwa mu gulu la CSF lomwe tidapeza (Chithunzi 5B).
Pakati pa mapuloteni 60 awa, pali mamolekyu omwe amadziwika kuti amagwirizana ndi AD, monga osteopontin (SPP1), yomwe ndi pro-inflammatory cytokine yomwe yakhala ikugwirizana ndi AD m'maphunziro ambiri (39-41), ndi GAP43, A synaptic protein. zomwe zimagwirizana bwino ndi neurodegeneration (42). Mapuloteni otsimikizika kwambiri ndi zolembera zokhudzana ndi matenda ena a neurodegenerative, monga amyotrophic lateral sclerosis (ALS) yokhudzana ndi superoxide dismutase 1 (SOD1) ndi matenda a Parkinson desaccharase (PARK7). Tatsimikiziranso kuti zolembera zina zambiri, monga SMOC1 ndi ma membrane olemera muubongo omwe amasainira protein 1 (BASP1), ali ndi maulalo am'mbuyomu a neurodegeneration. Ndizofunikira kudziwa kuti chifukwa chochepa kwambiri mu CSF proteome, n'kovuta kuti tigwiritse ntchito njira yodziwikiratu yamtundu umodzi kuti tizindikire bwino MAPT ndi mapuloteni ena okhudzana ndi AD (mwachitsanzo, NEFL ndi NRGN). ( 43, 44 ).
Kenako tidayang'ana zolembera zoyambira 60 pakuwunikanso katatu kobwerezabwereza. Mu CSF Copy 2, tidagwiritsa ntchito TMT-MS imodzi kusanthula gulu loyima palokha la 297 control ndi AD zitsanzo kuchokera ku Emory Goizueta ADRC (17). Kubwereza kwa CSF 3 kumaphatikizapo kusanthulanso kwa data ya TMT-MS yomwe ilipo kuchokera ku 120 control ndi AD odwala ochokera ku Lausanne, Switzerland (45). Tapeza zoposa magawo awiri pa atatu a zolembera zofunika 60 pagulu lililonse. Ngakhale kuti kafukufuku wa ku Switzerland adagwiritsa ntchito mapulatifomu osiyanasiyana a MS ndi njira zowerengera za TMT (45, 46), tinapanganso mwamphamvu machitidwe athu amagulu mu kusanthula kuwiri kobwerezabwereza (Chithunzi 5, C ndi D, ndi Matebulo S2, I, ndi J) . Kuti tiwone momwe matenda a gulu lathu lakhalira, tinagwiritsa ntchito TMT-MS kusanthula deta yachinayi yobwerezabwereza (CSF replication 4), yomwe inaphatikizapo osati kulamulira (n = 18) ndi AD (n = 17) milandu, komanso PD ( n = 14)), ALS (n = 18) ndi zitsanzo za frontotemporal dementia (FTD) (n = 11) (Table S1A). Tidawerengera bwino pafupifupi magawo awiri pa atatu a mapuloteni amgululi (38 mwa 60). Zotsatirazi zikuwonetsa kusintha kwa AD-enieni pamagulu onse asanu a biomarker (Chithunzi 5E ndi Table S2K). Kuwonjezeka kwa gulu la metabolite kunawonetsa kutsimikizika kwamphamvu kwa AD, kutsatiridwa ndi gulu la myelination ndi glial. Pang'ono pang'ono, FTD ikuwonetsanso kuwonjezeka pakati pa mapanelo awa, omwe angasonyeze kusintha kofanana kwa maukonde (17). Mosiyana ndi izi, ALS ndi PD adawonetsa pafupifupi mbiri yofananira, glial, ndi metabolome monga gulu lolamulira. Ponseponse, ngakhale pali kusiyana kokonzekera zitsanzo, nsanja ya MS, ndi njira zowerengera za TMT, kusanthula kobwerezabwerezaku kukuwonetsa kuti zolembera zathu zamagulu otsogola zimakhala ndi zosintha zofananira za AD pazopitilira 500 zapadera za CSF.
AD neurodegeneration yadziwika kwambiri zaka zingapo zisanachitike zizindikiro zachidziwitso, kotero pakufunika mwachangu ma biomarkers a AsymAD (5, 31). Komabe, maumboni ochulukirachulukira akuwonetsa kuti biology ya AsymAD ili kutali kwambiri, ndipo kuyanjana kovutirapo pachiwopsezo ndi kulimba mtima kumabweretsa kusiyana kwakukulu pakukula kwa matenda (47). Ngakhale kuti amagwiritsidwa ntchito pozindikira milandu ya AsymAD, milingo ya ma CSF biomarkers (Aβ1-42, tau yonse ndi p-tau) sanatsimikizire kuti athe kulosera modalirika yemwe angapite patsogolo ku dementia (4, 7), kuwonetsa zambiri. zofunikira kuphatikiza zida zonse za biomarker zochokera kuzinthu zingapo zaubongo kuti zitsimikizire molondola chiwopsezo cha anthuwa. Choncho, pambuyo pake tidasanthula gulu lathu la AD lovomerezeka la biomarker mu chiwerengero cha AsymAD cha CSF kopi 1. Milandu iyi ya 31 AsymAD inasonyeza milingo yosadziwika bwino ya biomarker (Aβ1-42 / chiwerengero cha tau ELISA, <5.5) ndi kuzindikira kwathunthu (kutanthauza MoCA, 27.1) ± 2.2) (Table S1A). Kuonjezera apo, anthu onse omwe ali ndi AsymAD ali ndi chiwerengero cha dementia cha 0, kusonyeza kuti palibe umboni wa kuchepa kwa chidziwitso cha tsiku ndi tsiku kapena kugwira ntchito.
Tidasanthula kaye kuchuluka kwa mapanelo ovomerezeka muzolemba zonse za 96 CSF 1, kuphatikiza gulu la AsymAD. Tidapeza kuti mapanelo angapo mu gulu la AsymAD anali ndi kusintha kwakukulu kwa AD-ngati kuchuluka, gulu la mitsempha likuwonetsa kutsika kwa AsymAD, pomwe mapanelo ena onse adawonetsa kukwera pamwamba (Chithunzi 6A). Chifukwa chake, mapanelo onse adawonetsa kulumikizana kofunikira kwambiri ndi ELISA Aβ1-42 ndi milingo yonse ya tau (Chithunzi 6B). Mosiyana ndi izi, kulumikizana pakati pa gululo ndi chiwopsezo cha MoCA ndikocheperako. Chimodzi mwazochititsa chidwi kwambiri pakuwunikaku ndi kuchuluka kwamagulu ambiri mu gulu la AsymAD. Monga momwe tawonetsera pa Chithunzi 6A, gulu la gulu la AsymAD nthawi zambiri limadutsa gulu la gulu lolamulira ndi gulu la AD, kusonyeza kusiyana kwakukulu. Kuti tipitirize kufufuza kusiyanasiyana kwa AsymAD, tinagwiritsa ntchito kusanthula kwa Multidimensional Scaling (MDS) ku 96 CSF replication 1 milandu. Kusanthula kwa MDS kumathandizira kuwona kufanana pakati pamilandu kutengera zosintha zina mu seti ya data. Pakuwunika masangowa, timangogwiritsa ntchito zolembera zovomerezeka zomwe zili ndi kusintha kwakukulu (P <0.05, AD/control) pakupeza CSF ndi replication 1 proteome (n = 29) (Table S2L). Kusanthula uku kunapanga kusanja bwino kwapakati pakati pa ulamuliro wathu ndi milandu ya AD (Chithunzi 6C). Mosiyana ndi izi, milandu ina ya AsymAD imasonkhanitsidwa bwino mugulu lowongolera, pomwe ena amapezeka mumilandu ya AD. Kuti tifufuzenso kusiyanasiyana kwa AsymAD, tidagwiritsa ntchito mapu athu a MDS kutanthauzira magulu awiri amilandu iyi ya AsymAD. Gulu loyamba lidaphatikizapo milandu ya AsymAD yolumikizidwa pafupi ndi kuwongolera (n = 19), pomwe gulu lachiwiri limadziwika ndi milandu ya AsymAD yokhala ndi mbiri yoyandikira pafupi ndi AD (n = 12).
(A) Mulingo wa mawu (z-score) wa gulu la CSF biomarker mu zitsanzo zonse 96 mu CSF replication 1 cohort, kuphatikiza AsymAD. Kuwunika kwa kusiyana ndi kuwongolera pambuyo pa Tukey kunagwiritsidwa ntchito kuwunika kufunikira kwa kusintha kwa kuchuluka kwa gulu. (B) Kusanthula kwa kulumikizana kwa mulingo wa kuchuluka kwa mapuloteni (z-score) ndi mphambu ya MoCA ndi kuchuluka kwa tau mu ELISA Aβ1-42 ndi CSF kukopera zitsanzo 1. Pearson coefficient coefficient yokhala ndi P yofunikira ikuwonetsedwa. (C) MDS ya 96 CSF kopi ya 1 milandu inakhazikitsidwa pa kuchuluka kwa zizindikiro zovomerezeka za 29 zovomerezeka, zomwe zinasinthidwa kwambiri pakupeza ndi CSF copy 1 data sets [P <0.05 AD/control (CT)]. Kusanthula uku kunagwiritsidwa ntchito kugawa gulu la AsymAD kuti lizilamulira (n = 19) ndi AD (n = 12) timagulu tating'ono. (D) Chiwembu cha phirili chikuwonetsa kusiyanitsa kwa mapuloteni onse a CSF obwereza 1 okhala ndi kusintha kwa log2 (x-axis) mogwirizana ndi -log10 statistical P mtengo pakati pamagulu awiri a AsymAD. Ma biomarkers apagulu ndi amitundu. (E) CSF replication 1 kuchuluka kwa zolembera zamagulu osankhidwa zimawonetsedwa mosiyana pakati pamagulu ang'onoang'ono a AsymAD. Kusanthula kosinthidwa kwa Tukey kwa kusiyana kunagwiritsidwa ntchito poyesa kufunikira kwa ziwerengero.
Tidawunikanso kusiyana kwa mapuloteni pakati pa kuwongolera uku ndi milandu ya AD-ngati AsymAD (Chithunzi 6D ndi Table S2L). Mapu a phirili akuwonetsa kuti zolembera 14 zasintha kwambiri pakati pa magulu awiriwa. Zambiri mwa zolemberazi ndi mamembala a synapse ndi metabolome. Komabe, SOD1 ndi myristoylated alanine-rich protein kinase C substrate (MARCKS), omwe ndi mamembala a myelin ndi magulu a chitetezo cha glial, motero, amakhalanso a gulu ili (Chithunzi 6, D ndi E) . Gulu la mitsempha la mitsempha linaperekanso zizindikiro ziwiri zomwe zinachepetsedwa kwambiri mu AD-monga AsymAD gulu, kuphatikizapo AE kumanga mapuloteni 1 (AEBP1) ndikuthandizira wachibale C9. Panalibe kusiyana kwakukulu pakati pa magulu ang'onoang'ono a AD-ngati AsymAD mu ELISA AB1-42 (P = 0.38) ndi p-tau (P = 0.28), koma panalidi kusiyana kwakukulu mu mlingo wa tau wonse (P = 0.0031 ) (Mkuyu S7). Pali zolembera zingapo zomwe zikuwonetsa kuti kusintha pakati pa magulu awiri a AsymAD ndi ofunika kwambiri kuposa kuchuluka kwa tau (mwachitsanzo, YWHAZ, SOD1, ndi MDH1) (Chithunzi 6E). Ponseponse, zotsatirazi zikuwonetsa kuti gulu lathu lovomerezeka litha kukhala ndi ma biomarkers omwe amatha kutsitsa ndikuyika chiwopsezo cha odwala omwe ali ndi matenda asymptomatic.
Pakufunika mwachangu zida zogwiritsira ntchito biomarker kuti muyeze bwino ndikuwongolera ma pathophysiology omwe ali kumbuyo kwa AD. Zidazi zikuyembekezeredwa kuti zisasinthe ndondomeko yathu yowonetsera AD, komanso kulimbikitsa kukhazikitsidwa kwa njira zothandizira, zothandizira odwala (1, 2). Kuti izi zitheke, tidagwiritsa ntchito njira yosakondera yokhudzana ndi ma proteomics ku ubongo wa AD ndi CSF kuti tidziwe zolembera za CSF zapaintaneti zomwe zimawonetsa mitundu ingapo yamatenda okhudza ubongo. Kusanthula kwathu kunatulutsa mapanelo asanu a CSF biomarker, omwe (i) amawonetsa ma synapses, mitsempha yamagazi, myelin, kuwonongeka kwa chitetezo chamthupi ndi kagayidwe kachakudya; (ii) kuwonetsa kuberekana mwamphamvu pamapulatifomu osiyanasiyana a MS; ( iii) Onetsani kusintha kwapang'onopang'ono kwa matenda m'magawo oyambirira ndi kumapeto kwa AD. Ponseponse, zomwe zapezazi zikuyimira gawo lopatsa chiyembekezo pakupanga zida zosiyanasiyana, zodalirika, zokhazikika pa intaneti pa kafukufuku wa AD ndi ntchito zamankhwala.
Zotsatira zathu zikuwonetsa gulu lotetezedwa kwambiri la AD brain network proteome ndikuthandizira kugwiritsidwa ntchito kwake ngati nangula wa chitukuko chokhazikitsidwa ndi biomarker. Kusanthula kwathu kukuwonetsa kuti ma dataset awiri odziyimira pawokha a TMT-MS okhala ndi ubongo wa AD ndi AsymAD ali ndi modularity wamphamvu. Zomwe tapezazi zimakulitsa ntchito yathu yam'mbuyomu, kuwonetsa kusungidwa kwa ma module amphamvu opitilira 2,000 minyewa yaubongo kuchokera kumagulu angapo odziyimira pawokha kutsogolo, parietal, ndi temporal cortex (17). Maukonde ogwirizanawa akuwonetsa kusintha kosiyanasiyana kokhudzana ndi matenda komwe kumawonedwa mu kafukufuku wapano, kuphatikiza kuchuluka kwa ma module otumphukira a glial komanso kuchepa kwa ma module olemera a neuron. Monga kafukufuku waposachedwa, netiweki yayikuluyi imakhalanso ndi zosintha zazikulu mu AsymAD, kuwonetsa mitundu yosiyanasiyana yamatenda a preclinical pathophysiology (17).
Komabe, mkati mwa dongosolo lokhazikika lokhazikikali, pali kusiyanasiyana kwachilengedwe kwachilengedwe, makamaka pakati pa anthu omwe ali koyambirira kwa AD. Gulu lathu la biomarker limatha kuwonetsa timagulu ting'onoting'ono ta AsymAD, zomwe zikuwonetsa kusiyanitsa kwakukulu kwa zolembera zingapo za CSF. Gulu lathu lidatha kuwonetsa kusiyana kwachilengedwe pakati pa magulu awiriwa, omwe sanawonekere pamlingo wa AD biomarkers. Poyerekeza ndi gulu lolamulira, ma Aβ1-42/total tau ratios a anthu a AsymAD awa anali otsika kwambiri. Komabe, kuchuluka kwa tau kokha kunali kosiyana kwambiri pakati pa magulu awiri a AsymAD, pomwe ma Aβ1-42 ndi p-tau adakhalabe ofanana. Popeza tau yapamwamba ya CSF ikuwoneka ngati yolosera bwino zazizindikiro kuposa milingo ya Aβ1-42 (7), tikukayikira kuti magulu awiriwa a AsymAD atha kukhala ndi ziwopsezo zosiyanasiyana zakukula kwa matenda. Poganizira kukula kwachitsanzo chochepa cha AsymAD yathu komanso kusowa kwa data yotalikirapo, kufufuza kwina kumafunika kuti tipeze mfundo izi molimba mtima. Komabe, zotsatirazi zikuwonetsa kuti gulu lokhazikika la CSF litha kupititsa patsogolo luso lathu lokhazikitsa bwino anthu panthawi yomwe matendawa ali asymptomatic.
Ponseponse, zomwe tapeza zimathandizira gawo la magwiridwe antchito angapo achilengedwe mu pathogenesis ya AD. Komabe, dysregulated energy metabolism idakhala mutu wotsogola wamagulu athu onse asanu ovomerezeka. Mapuloteni a metabolism, monga hypoxanthine-guanine phosphoribosyltransferase 1 (HPRT1) ndi lactate dehydrogenase A (LDHA), ndizomwe zimatsimikiziridwa mwamphamvu kwambiri za synaptic biomarkers, zomwe zimasonyeza kuti kuwonjezeka kwa AD CSF ndiko kugonana kwambiri. Mitsempha yathu yamagazi ndi mapanelo a glial alinso ndi zolembera zingapo zomwe zimakhudzidwa ndi metabolism ya zinthu zotulutsa okosijeni. Zomwe zapezazi zikugwirizana ndi gawo lofunikira lomwe njira za metabolic zimagwira muubongo wonse, osati kungokwaniritsa kuchuluka kwamphamvu kwa ma neuron, komanso kukwaniritsa kufunikira kwamphamvu kwa ma astrocyte ndi ma cell ena a glial (17, 48). Zotsatira zathu zimathandizira umboni womwe ukukula wosonyeza kuti kusintha kwa redox komanso kusokonezeka kwa njira zamagetsi kungakhale kulumikizana kwakukulu pakati pa njira zingapo zofunika zomwe zimakhudzidwa ndi pathogenesis ya AD, kuphatikiza kusokonezeka kwa mitochondrial, kutupa kwa glial, ndi kuwonongeka kwa Mitsempha (49). Kuphatikiza apo, ma metabolic cerebrospinal fluid biomarkers ali ndi mapuloteni ambiri olemera mosiyanasiyana pakati pa zowongolera zathu ndi magulu a AD-monga AsymAD, zomwe zikuwonetsa kuti kusokonezeka kwa mphamvu izi ndi njira za redox kungakhale kofunika kwambiri mu gawo loyambirira la matendawa.
Mitundu yosiyanasiyana yaubongo ndi cerebrospinal fluid panel yomwe tawona ilinso ndi chidwi chokhudza chilengedwe. Ma Synapses ndi metabolomes olemera mu ma neuron amawonetsa kuchepa kwa ubongo wa AD ndikuchulukirachulukira mu cerebrospinal fluid. Popeza kuti ma neuron ali olemera mu mitochondria yopanga mphamvu pa ma synapses kuti apereke mphamvu pazizindikiro zawo zingapo zapadera (50), kufanana kwa mbiri yamagulu awiriwa amayembekezeredwa. Kutayika kwa ma neuroni ndi kutulutsa kwa maselo owonongeka kumatha kufotokozera zomwe zimachitika muubongo ndi CSF pamatenda am'tsogolo, koma sangathe kufotokozera zosintha zoyambira zomwe timawona (13). Kufotokozera kumodzi kwazomwe zapezedwa m'matenda oyambilira asymptomatic ndikudulira kwachilendo kwa synaptic. Umboni watsopano pamawonekedwe a mbewa ukuwonetsa kuti ma microglia-mediated synaptic phagocytosis atha kutsegulidwa modabwitsa mu AD ndikupangitsa kuti ubongo uwonongeke muubongo (51). Zinthu zotayidwa za synaptic zitha kudziunjikira mu CSF, ndichifukwa chake timawona kuwonjezeka kwa CSF mu gulu la neuron. Kudulira kolumikizana ndi chitetezo chamthupi kumatha kufotokozeranso pang'ono kuchuluka kwa mapuloteni a glial omwe timawona muubongo ndi cerebrospinal fluid panthawi yonse ya matendawa. Kuphatikiza pa kudulira kwa synaptic, zovuta zonse munjira ya exocytic zitha kubweretsanso ku ubongo ndi ma CSF mafotokozedwe a neuronal markers. Kafukufuku wambiri wasonyeza kuti zomwe zili mu exosomes mu pathogenesis ya ubongo wa AD zasintha (52). Njira ya extracellular imakhudzidwanso ndi kufalikira kwa Aβ (53, 54). Ndizofunikira kudziwa kuti kuponderezedwa kwa katulutsidwe ka exosomal kumatha kuchepetsa matenda ngati AD mumitundu ya AD transgenic mbewa (55).
Panthawi imodzimodziyo, mapuloteni mu gulu la mitsempha amasonyeza kuwonjezeka kwapakati mu ubongo wa AD, koma kunachepa kwambiri mu CSF. Kulephera kugwira ntchito kwa magazi-ubongo (BBB) kumatha kufotokozera zomwe zapezazi. Maphunziro ambiri odziyimira pawokha a postmortem awonetsa kuwonongeka kwa BBB mu AD (56, 57). Kafukufukuyu adatsimikizira zochitika zosiyanasiyana zachilendo zozungulira ma cell otsekedwa bwino a endothelial, kuphatikiza kutayikira kwa capillary muubongo komanso kudzikundikira kwa mapuloteni obwera m'magazi (57). Izi zingapereke kufotokozera kosavuta kwa mapuloteni okwera a mitsempha mu ubongo, koma sangathe kufotokoza bwino za kuchepa kwa mapuloteni omwewa mu cerebrospinal fluid. Chotheka chimodzi ndi chakuti dongosolo lapakati la mitsempha likulekanitsa mamolekyuwa kuti athetse vuto la kuwonjezeka kwa kutupa ndi kupsinjika kwa okosijeni. Kuchepa kwa mapuloteni ena owopsa kwambiri a CSF pagululi, makamaka omwe akukhudzidwa ndi malamulo a lipoprotein, amagwirizana ndi kuletsa kwamphamvu yotupa yotupa komanso njira ya neuroprotective yamitundu ya okosijeni yokhazikika. Izi ndi zoona kwa Paroxonase 1 (PON1), enzyme yomanga lipoprotein yomwe imathandizira kuchepetsa kupsinjika kwa okosijeni m'magazi (58, 59). Alpha-1-microglobulin/bikunin precursor (AMBP) ndi chizindikiro china chotsika kwambiri cha gulu la mitsempha. Ndiye kalambulabwalo wa lipid transporter bikunin, yomwe imakhudzidwanso ndi kuponderezana kwa kutupa komanso Chitetezo cha minyewa (60, 61).
Ngakhale pali malingaliro osiyanasiyana ochititsa chidwi, kulephera kuzindikira mwachindunji njira zamatenda am'thupi ndi njira yodziwika bwino yowunikira ma proteinomics. Chifukwa chake, kufufuza kwina ndikofunikira kuti mufotokozere molimba mtima njira zomwe zimathandizira mapanelo a biomarker awa. Kuti tipitirire ku chitukuko cha kusanthula kwachipatala kochokera ku MS, mayendedwe amtsogolo amafunikiranso kugwiritsa ntchito njira zowunikira pakutsimikizira kwakukulu kwa biomarker, monga kuwunika kosankha kapena kufanana (62). Posachedwa tagwiritsa ntchito kuwunika kofananira (63) kutsimikizira kusintha kwa mapuloteni ambiri a CSF omwe akufotokozedwa pano. Zolinga zingapo zofunika kwambiri zimawerengedwa molondola kwambiri, kuphatikiza YWHAZ, ALDOA, ndi SMOC1, zomwe zimayendera ma synapse, metabolism, ndi mapanelo otupa, motsatana (63). Independent Data Acquisition (DIA) ndi njira zina zochokera ku MS zitha kukhala zothandiza pakutsimikizira zomwe mukufuna. Bud ndi al. (64) Posachedwapa zawonetsedwa kuti pali kuphatikizika kwakukulu pakati pa ma biomarker a AD omwe azindikirika mu seti yathu ya CSF yopezeka ndi data yodziyimira payokha ya DIA-MS, yomwe ili ndi pafupifupi 200 CSF zitsanzo kuchokera kumagulu atatu osiyanasiyana aku Europe. Maphunziro aposachedwawa amathandizira kuthekera kwa mapanelo athu kuti asinthe kukhala odalirika ozindikira a MS. Kuzindikira kwachikhalidwe ndi ma aptamer ndikofunikanso pakupititsa patsogolo ma biomarker ofunika kwambiri a AD. Chifukwa cha kuchepa kwa CSF, ndizovuta kwambiri kuzindikira zolembera izi pogwiritsa ntchito njira zapamwamba za MS. NEFL ndi NRGN ndi zitsanzo ziwiri zotere za CSF biomarkers zotsika kwambiri, zomwe zimayikidwa pagulu pakuwunika kwathu mwatsatanetsatane, koma sizingadziwike modalirika pogwiritsa ntchito njira yathu imodzi ya MS. Njira zowunikira zotengera ma antibodies angapo, monga PEA, zitha kulimbikitsa kusintha kwachipatala kwa zolemberazi.
Ponseponse, kafukufukuyu akupereka njira yapadera ya proteinomics pozindikiritsa ndi kutsimikizira za CSF AD biomarkers kutengera machitidwe osiyanasiyana. Kuwongolera mapanelo awa pamagawo owonjezera a AD ndi nsanja za MS zitha kutsimikizira kupititsa patsogolo kusamvana kwachiwopsezo cha AD ndi chithandizo. Maphunziro omwe amayesa kutalika kwa mapanelowa pakapita nthawi ndi ofunikiranso kuti adziwe kuti ndi mitundu iti ya zolembera zomwe zimatsimikizira kuopsa kwa matenda oyamba komanso kusintha kwa kuopsa kwa matenda.
Kupatula zitsanzo za 3 zojambulidwa ndi CSF, zitsanzo zonse za CSF zomwe zinagwiritsidwa ntchito mu phunziroli zinasonkhanitsidwa mothandizidwa ndi Emory ADRC kapena mabungwe ofufuza ogwirizana kwambiri. Ma seti anayi a zitsanzo za Emory CSF adagwiritsidwa ntchito mu maphunziro a proteinomics. Gulu la CSF lidapezeka kuti lili ndi zitsanzo kuchokera ku maulamuliro athanzi 20 ndi odwala 20 AD. Kopi ya CSF 1 imaphatikizapo zitsanzo kuchokera ku maulamuliro athanzi 32, anthu 31 a AsymAD, ndi anthu 33 AD. CSF kope 2 lili 147 amazilamulira ndi 150 AD zitsanzo. Gulu la CSF replication 4 lamitundu yambiri limaphatikizapo zowongolera 18, 17 AD, 19 ALS, 13 PD, ndi zitsanzo 11 za FTD. Malinga ndi mgwirizano womwe wavomerezedwa ndi Emory University Institutional Review Board, onse omwe adachita nawo kafukufuku wa Emory adalandira chilolezo chodziwitsidwa. Malinga ndi 2014 National Institute of Aging Best Practice Guidelines for Alzheimer's Centers (https://alz.washington.edu/BiospecimenTaskForce.html), cerebrospinal fluid inasonkhanitsidwa ndikusungidwa ndi lumbar puncture. Odwala a Control and AsymAD ndi AD adalandira kuwunika kokhazikika kwachidziwitso ku Emory Cognitive Neurology Clinic kapena Goizueta ADRC. Zitsanzo zawo za cerebrospinal fluid zinayesedwa ndi INNO-BIA AlzBio3 Luminex kwa ELISA Aβ1-42, kusanthula kwathunthu kwa tau ndi p-tau (65). Makhalidwe a ELISA amagwiritsidwa ntchito kuthandizira kugawika kwa maphunziro potengera njira zodulira za AD biomarker (66, 67). Deta yachiwerengero ya anthu komanso yowunikira matenda ena a CSF (FTD, ALS, ndi PD) imapezekanso kuchokera ku Emory ADRC kapena mabungwe ogwirizana nawo. Metadata yachidule yamilandu iyi ya Emory CSF ikupezeka mu Table S1A. Makhalidwe a Swiss CSF replication 3 cohort adasindikizidwa kale (45).
CSF idapeza chitsanzo. Kuti tiwonjezere kuya kwazomwe tapeza pa data ya CSF, kugwiritsa ntchito chitetezo chamthupi kwa mapuloteni ochuluka kwambiri kunachitika musanayambe trypsinization. Mwachidule, 130 μl ya CSF kuchokera ku zitsanzo 40 za CSF ndi voliyumu yofanana (130 μl) ya High Select Top14 Abundance Protein Depletion Resin (Thermo Fisher Scientific, A36372) anayikidwa mu mzere wa spin (Thermo Fisher Scientific, A89868) Kutentha kwa kutentha). Pambuyo kupota kwa mphindi 15, centrifuge chitsanzo pa 1000g kwa mphindi ziwiri. Chipangizo cha 3K ultracentrifugal filter (Millipore, UFC500396) chinagwiritsidwa ntchito kuyika zitsanzo za utsi ndi centrifuging pa 14,000g kwa mphindi 30. Sungunulani zitsanzo zonse za 75 μl ndi phosphate buffer saline. Kuchuluka kwa mapuloteni kunayesedwa ndi njira ya bicinchoninic acid (BCA) molingana ndi ndondomeko ya opanga (Thermo Fisher Scientific). The immunodepleted CSF (60 μl) kuchokera ku zitsanzo zonse 40 idagayidwa ndi lysyl endopeptidase (LysC) ndi trypsin. Mwachidule, chitsanzo anali yafupika ndi alkylated ndi 1.2 μl 0.5 M tris-2 (-carboxyethyl) -phosphine ndi 3 μl 0,8 M chloroacetamide pa 90 ° C kwa mphindi 10, ndiyeno sonicated mu osamba madzi kwa mphindi 15. Chitsanzocho chinachepetsedwa ndi 193 μl 8 M urea buffer [8 M urea ndi 100 mM NaHPO4 (pH 8.5)] mpaka ndende yomaliza ya 6 M urea. LysC (4.5 μg; Wako) imagwiritsidwa ntchito pogaya usiku kutentha kutentha. Chitsanzocho chinachepetsedwa kukhala 1 M urea ndi 50 mM ammonium bicarbonate (ABC) (68). Onjezani kuchuluka kofanana (4.5 μg) kwa trypsin (Promega), ndiyeno muyimire chitsanzocho kwa maola 12. Tsitsani njira ya peptide yogayidwa mpaka 1% formic acid (FA) ndi 0.1% trifluoroacetic acid (TFA) (66), kenako mchere ndi 50 mg Sep-Pak C18 column (Madzi) monga tafotokozera pamwambapa (25) . Kenako peptide idatulutsidwa mu 1 ml ya 50% acetonitrile (ACN). Kuti akhazikitse kuchuluka kwa mapuloteni m'magulu (25), ma aliquots 100 μl kuchokera ku zitsanzo zonse 40 za CSF adaphatikizidwa kuti apange zitsanzo zosakanikirana, zomwe zidagawidwa kukhala zitsanzo zisanu zapadziko lonse lapansi (GIS) (48). Zitsanzo zonse za munthu payekha ndi miyezo yophatikizidwa imawumitsidwa ndi vacuum yothamanga kwambiri (Labconco).
CSF imakopera chitsanzocho. Dayon ndi anzawo adafotokoza kale za kuchepa kwa chitetezo chamthupi komanso kugayidwa kwa ma CSF kopi 3 zitsanzo (45, 46). Zitsanzo zotsalira zotsalira sizinali payekhapayekha immunodepleted. Gawani zitsanzo zomwe sizinachotsedwe mu trypsin monga tafotokozera kale (17). Pakuwunika kobwerezabwereza kulikonse, 120 μl aliquots wa peptide eluted kuchokera ku chitsanzo chilichonse adasonkhanitsidwa pamodzi ndikugawidwa kukhala ma aliquots ofanana kuti agwiritsidwe ntchito ngati muyezo wapadziko lonse wa TMT (48). Zitsanzo zonse za munthu payekha ndi miyezo yophatikizidwa imawumitsidwa ndi vacuum yothamanga kwambiri (Labconco). Pofuna kupititsa patsogolo chidziwitso cha mapuloteni otsika kwambiri a CSF, pophatikiza 125 μl kuchokera ku chitsanzo chilichonse, chitsanzo "chowonjezera" chinakonzedwa kuti chisanthulenso chofanana [ie, chitsanzo chachilengedwe chomwe chimatsanzira chitsanzo cha kafukufuku, koma kuchuluka komwe kulipo ndi chokulirapo (37, 69)] chophatikizidwa kukhala chitsanzo cha CSF chosakanikirana (17). Zitsanzo zosakanikiranazo zidachotsedwanso pogwiritsa ntchito 12 ml ya High Select Top14 Abundance Protein Removal Resin (Thermo Fisher Scientific, A36372), idagayidwa monga tafotokozera pamwambapa, ndikuphatikizidwa muzolemba zambiri za TMT.
Nthawi yotumiza: Aug-27-2021