Cutar cutar Alzheimer (AD) ba ta da ma'auni na furotin wanda ke nuna nau'in ilimin halittar jiki da yawa, yana hana ci gaban ganewar asali da magani. Anan, muna amfani da cikakkun bayanan kariya don gano alamun ƙwayoyin cuta na cerebrospinal (CSF) waɗanda ke wakiltar kewayon kewayon cututtukan cututtukan AD. Multiplex mass spectrometry gano kusan 3,500 da kusan sunadaran 12,000 a cikin AD CSF da kwakwalwa, bi da bi. Binciken cibiyar sadarwa na proteome na kwakwalwa ya warware nau'ikan nau'ikan nau'ikan halittu guda 44, 15 daga cikinsu sun mamaye proteome ruwan cerebrospinal. Alamar CSF AD a cikin waɗannan rukunoni masu haɗaka an naɗe su zuwa ƙungiyoyin furotin guda biyar, waɗanda ke wakiltar matakai daban-daban na pathophysiological. Synapses da metabolites a cikin kwakwalwar AD suna raguwa, amma CSF yana ƙaruwa, yayin da glial-rich myelination da ƙungiyoyin rigakafi a cikin kwakwalwa da CSF suna ƙaruwa. An tabbatar da daidaito da ƙayyadaddun cututtuka na canje-canjen panel a cikin ƙarin samfuran CSF fiye da 500. Waɗannan ƙungiyoyin kuma sun gano ƙananan ƙungiyoyin halittu a cikin asymptomatic AD. Gabaɗaya, waɗannan sakamakon mataki ne mai ban sha'awa ga kayan aikin biomarker na tushen yanar gizo don aikace-aikacen asibiti a AD.
Cutar Alzheimer (AD) ita ce mafi yawan abin da ke haifar da lalata neurodegenerative a duk duniya kuma yana da nau'i mai yawa na rashin aikin tsarin halitta, ciki har da watsa synaptic, glial-mediated rigakafi, da kuma mitochondrial metabolism (1-3). Duk da haka, kafaffen furotin biomarkers har yanzu suna mayar da hankali kan gano amyloid da furotin tau, sabili da haka ba za su iya yin la'akari da wannan nau'in pathophysiology ba. Wadannan abubuwan gina jiki na "core" wadanda aka fi dogara da su a cikin ruwa na cerebrospinal (CSF) sun hada da (i) amyloid beta peptide 1-42 (Aβ1-42), wanda ke nuna samuwar amyloid plaques na cortical; (ii) jimlar tau, alamar lalacewar axon; (iii) phospho-tau (p-tau), wakilin pathological tau hyperphosphorylation (4-7). Ko da yake waɗannan ma'aunin ruwa na cerebrospinal sun taimaka mana sosai wajen gano cututtukan furotin AD "alama" (4-7), suna wakiltar ɗan ƙaramin sashi na hadadden ilimin halitta a bayan cutar.
Rashin bambance-bambancen pathophysiological na AD biomarkers ya haifar da ƙalubale da yawa, ciki har da (i) rashin iya ganowa da ƙididdige nau'o'in halittu na marasa lafiya AD, (ii) rashin isasshen ma'auni na rashin lafiya da ci gaba, musamman a cikin matakin farko, Kuma (ii) iii) haɓakar magungunan warkewa waɗanda suka kasa magance gaba ɗaya duk abubuwan da suka shafi lalacewar jijiyoyin jiki. Dogaro da mu ga alamomin cututtukan cututtuka don siffanta AD daga cututtukan da ke da alaƙa yana ƙara tsananta waɗannan matsalolin. Shaidu da yawa sun nuna cewa yawancin tsofaffi waɗanda ke fama da cutar hauka suna da halayen cututtukan cuta fiye da ɗaya na raguwar fahimi (8). Kimanin kashi 90% ko fiye na mutanen da ke da cututtukan cututtukan AD suma suna da cututtukan jijiyoyin jini, haɗawar TDP-43, ko wasu cututtukan lalacewa (9). Waɗannan ɗimbin yawa na rikice-rikice na cututtukan cututtuka sun rushe tsarin binciken mu na yanzu don cutar hauka, kuma ana buƙatar ƙarin cikakkiyar ma'anar cutar ta jiki.
Bisa la'akari da buƙatar gaggawa don nau'ikan alamun halittu na AD, filin yana ƙara ɗaukar hanyar "omics" dangane da tsarin gabaɗaya don gano alamun halittu. The Accelerated Pharmaceutical Partnership (AMP) -AD Alliance an ƙaddamar da shi a cikin 2014 kuma yana kan gaba a cikin shirin. Wannan ƙoƙarce-ƙoƙarce da yawa ta Cibiyar Kiwon Lafiya ta ƙasa, ilimi, da masana'antu na nufin yin amfani da dabarun tushen tsarin don mafi kyawun ma'anar pathophysiology na AD da haɓaka ƙididdigar bincike na biodiversity da dabarun jiyya (10). A matsayin wani ɓangare na wannan aikin, hanyoyin sadarwa na cibiyar sadarwa ya zama kayan aiki mai ban sha'awa don ci gaban tsarin tushen halittu a cikin AD. Wannan hanyar da ba ta dace da bayanan da aka yi amfani da ita ba tana tsara hadaddun bayanan proteomics saiti zuwa ƙungiyoyi ko "modules" na sunadaran da aka bayyana tare da ke da alaƙa da takamaiman nau'ikan tantanin halitta, gabobin jiki, da ayyukan nazarin halittu (11-13). Kusan 12 bayanai-arziƙi nazarin hanyoyin sadarwa proteomics an gudanar a kan kwakwalwa AD (13-23). Gabaɗaya, waɗannan nazarin suna nuna cewa cibiyar sadarwar kwakwalwa ta AD proteome tana kula da ƙungiya mai ƙima sosai a cikin ƙungiyoyi masu zaman kansu da yankuna masu yawa. Bugu da ƙari, wasu daga cikin waɗannan nau'ikan suna nuna canje-canjen da za a iya maimaitawa a cikin yalwar da ke da alaƙa da AD a cikin tsarin bayanai, suna nuna ilimin ilimin cututtuka na cututtuka masu yawa. Gabaɗaya, waɗannan binciken suna nuna maƙasudin ƙwaƙƙwaran ƙwanƙwasa don gano ƙwayar ƙwayar cuta ta hanyar sadarwa a matsayin tushen tsarin halittu a cikin AD.
Domin canza hanyar sadarwar kwakwalwar AD zuwa masu amfani da tsarin rayuwa masu amfani da tsarin, mun haɗu da hanyar sadarwar da aka samu ta kwakwalwa tare da nazarin proteomic na AD CSF. Wannan tsarin haɗin gwiwar ya haifar da gano nau'o'i biyar masu ban sha'awa na CSF biomarkers waɗanda ke da alaƙa da nau'o'in ilimin cututtuka na kwakwalwa, ciki har da synapses, tasoshin jini, myelination, kumburi, da kuma rashin aiki na hanyoyin rayuwa. Mun sami nasarar inganta waɗannan bangarori na biomarker ta hanyar nazarin maimaitawa da yawa, gami da samfuran CSF sama da 500 daga cututtukan neurodegenerative daban-daban. Waɗannan ƙididdigar tabbatarwa sun haɗa da nazarin maƙasudin rukuni a cikin CSF na marasa lafiya tare da asymptomatic AD (AsymAD) ko kuma nuna shaidar tarin amyloid mara kyau a cikin yanayin fahimi na yau da kullun. Wadannan nazarin suna nuna mahimmancin bambancin halittu a cikin yawan AsymAD da kuma gano alamun panel waɗanda za su iya ƙaddamar da mutane a farkon matakan cutar. Gabaɗaya, waɗannan sakamakon suna wakiltar wani muhimmin mataki a cikin haɓaka kayan aikin furotin biomarker bisa tsarin da yawa waɗanda zasu iya samun nasarar magance yawancin ƙalubalen asibiti da AD ke fuskanta.
Babban makasudin wannan binciken shine don gano sabbin ƙwayoyin ƙwayoyin cuta na cerebrospinal wanda ke nuna nau'ikan cututtukan cututtukan ƙwayoyin cuta waɗanda ke haifar da AD. Hoto S1 yana zayyana hanyoyin binciken mu, wanda ya haɗa da (i) cikakken bincike wanda aka gudanar ta hanyar binciken farko na AD CSF da kuma tsarin kwakwalwar cibiyar sadarwa don gano alamun cututtukan CSF da ke da alaƙa da kwakwalwa da yawa, da (ii) maimaitawa na gaba Wadannan alamun halittu suna cikin cerebrospinal masu zaman kansu da yawa. ƙungiyoyin ruwa. Binciken da aka samo asali ya fara ne tare da nazarin bambancin bayanin CSF a cikin mutane 20 na al'ada na al'ada da kuma 20 AD marasa lafiya a Emory Goizueta Alzheimer's Disease Research Center (ADRC). An bayyana ganewar asali na AD a matsayin babban rashin fahimta a gaban ƙananan Aβ1-42 da matakan girma na jimlar tau da p-tau a cikin ruwa na cerebrospinal [Ma'anar Ƙididdigar Ƙwararrun Ƙwararrun Montreal (MoCA), 13.8 ± 7.0] [ELISA (ELISA) )]] (Table S1A). Gudanarwa (ma'anar MoCA, 26.7 ± 2.2) yana da matakan al'ada na CSF biomarkers.
CSF ɗan adam yana da alaƙa da kewayon yawan furotin mai ƙarfi, wanda albumin da sauran sunadaran sunadaran da yawa zasu iya hana gano sunadaran sha'awa (24). Don ƙara zurfin binciken sunadaran, mun cire furotin 14 na farko masu yawa daga kowane samfurin CSF kafin bincike na spectrometry (MS) (24). An gano jimlar peptides 39,805 ta MS, waɗanda aka tsara su zuwa 3691 proteomes a cikin samfuran 40. Ana yin ƙididdige adadin furotin ta hanyar lakabin tandem mass tag (TMT) da yawa (18, 25). Don warware bayanan da suka ɓace, mun haɗa kawai waɗannan sunadaran da aka ƙididdige su a cikin aƙalla 50% na samfuran a cikin bincike na gaba, don haka a ƙarshe ƙididdige proteomes 2875. Saboda babban bambanci a cikin jimlar yawan adadin furotin, samfurin sarrafawa an yi la'akari da ƙididdiga a matsayin mafifici (13) kuma ba a haɗa shi cikin bincike na gaba ba. An daidaita yawan ƙimar sauran samfuran 39 bisa ga shekaru, jinsi, da haɗin kai (13-15, 17, 18, 20, 26).
Yin amfani da ƙididdigar t-gwajin ƙididdiga don kimanta bambancin magana akan saitin bayanan koma baya, wannan bincike ya gano sunadaran da yawan adadin su ya canza sosai (P <0.05) tsakanin masu sarrafawa da AD (Table S2A). Kamar yadda aka nuna a cikin hoto na 1A, yawan adadin sunadaran sunadaran 225 a AD ya ragu sosai, kuma yawan sunadaran 303 ya karu sosai. Waɗannan sunadaran da aka bayyana daban-daban sun haɗa da alamomin alamar cerebrospinal ruwa AD da yawa a baya, irin su furotin mai alaƙa da microtubule tau (MAPT; P = 3.52 × 10-8), neurofilament (NEFL; P = 6.56 × 10-3), Protein mai alaƙa da haɓaka 43. (GAP43; P = 1.46 × 10−5), Fatty Acid Binding Protein 3 (FABP3; P = 2.00 × 10-5), Chitinase 3 kamar 1 (CHI3L1; P = 4.44 × 10-6), Neural Granulin (NRGN; P = 3.43 × 10-4) da kuma haɓakar haɓakar jijiya na VGF (VGF; P = 4.83 × 10-3) (4-6). Duk da haka, mun kuma gano wasu maƙasudai masu mahimmanci, irin su GDP dissociation inhibitor 1 (GDI1; P = 1.54 × 10-10) da SPARC da ke da alaƙa da ƙwayar calcium mai ɗaure 1 (SMOC1; P = 6.93 × 10-9). Binciken Gene Ontology (GO) na 225 ya rage yawan sunadaran sunadaran sun nuna kusancin haɗin kai tare da hanyoyin ruwa na jiki kamar su metabolism na steroid, haɗin jini, da ayyukan hormone (Hoto 1B da Table S2B). Sabanin haka, haɓakar furotin mai mahimmanci na 303 yana da alaƙa da tsarin tantanin halitta da kuzarin kuzari.
(A) Matsakaicin dutsen mai aman wuta yana nuna canjin log2 ninka (x-axis) dangane da ƙimar -log10 kididdigar P (y-axis) da aka samu ta hanyar t-gwajin, wanda ake amfani da shi don gano bambancin magana tsakanin sarrafawa (CT) da Abubuwan AD na furotin CSF Na duk sunadaran. Sunadaran da ke da matakan raguwa sosai (P <0.05) a cikin AD ana nuna su cikin shuɗi, yayin da sunadaran da ke da haɓakar matakan cuta suna nunawa a ja. Sunan furotin da aka zaɓa. (B) Manyan kalmomin GO masu alaƙa da furotin sun ragu sosai (blue) kuma suna ƙaruwa (ja) a cikin AD. Yana nuna sharuɗɗan GO guda uku tare da mafi girman z-maki a cikin fagagen tafiyar matakai na rayuwa, ayyuka na ƙwayoyin cuta, da sassan salula. (C) MS ya auna matakin MAPT a cikin samfurin CSF (hagu) da alaƙarta da samfurin ELISA tau matakin (dama). Ana nuna ƙimar haɗin Pearson tare da ƙimar P mai dacewa. Saboda rashin bayanan ELISA na shari'ar AD ɗaya, waɗannan alkalumman sun haɗa da ƙima don 38 daga cikin 39 da aka bincika. (D) Binciken cluster mai kulawa (P <0.0001, Benjamini-Hochberg (BH) gyara P <0.01) akan sarrafawa da AD CSF sun sami samfurori ta amfani da 65 mafi mahimmancin sunadarai sun canza a cikin bayanan bayanan. Daidaita, daidaita.
Matsayin proteomic na MAPT yana da alaƙa da kusanci da matakin ELISA tau wanda aka auna kansa (r = 0.78, P = 7.8 × 10-9; Hoto 1C), yana goyan bayan ingancin ma'aunin MS ɗin mu. Bayan narkewar trypsin a matakin furotin amyloid precursor (APP), takamaiman peptides na musamman da aka tsara zuwa C-terminus na Aβ1-40 da Aβ1-42 ba za a iya ionized da kyau ba (27, 28). Don haka, peptides na APP da muka gano ba su da alaƙa da matakan ELISA Aβ1-42. Don yin la'akari da bambance-bambancen maganganun kowane hali, mun yi amfani da sunadaran da aka bayyana daban-daban tare da P <0.0001 (FDR) da aka gyara P <0.01) don yin nazarin gungu mai kulawa na samfurori (Table S2A). Kamar yadda aka nuna a cikin Hoto na 1D, waɗannan sunadaran sunadaran 65 masu matuƙar mahimmanci na iya haɗa samfuran daidai gwargwado bisa ga yanayin cuta, sai dai yanayin AD guda ɗaya tare da halaye masu kama da sarrafawa. Daga cikin waɗannan sunadaran 65, 63 sun karu a AD, yayin da biyu kawai (CD74 da ISLR) suka ragu. Gabaɗaya, waɗannan nazarin ruwa na cerebrospinal sun gano ɗaruruwan sunadaran sunadaran a cikin AD waɗanda zasu iya zama alamomin cutar.
Sa'an nan kuma mun yi wani bincike mai zaman kansa na hanyar sadarwa na kwakwalwar AD. Ƙungiyar kwakwalwa na wannan binciken sun haɗa da dorsolateral prefrontal cortex (DLPFC) daga sarrafawa (n = 10), cutar Parkinson (PD; n = 10), gauraye AD/PD (n = 10) da AD (n = 10). ) Misali. Emery Goizueta ADRC. An yi bayanin ƙididdiga na waɗannan shari'o'i 40 a baya (25) kuma an taƙaita su a cikin Tebur S1B. Mun yi amfani da TMT-MS don nazarin waɗannan kyallen jikin kwakwalwa guda 40 da ƙungiyar kwafi na lokuta 27. Gabaɗaya, waɗannan bayanan bayanan kwakwalwa guda biyu sun samar da peptides na musamman guda 227,121, waɗanda aka tsara su zuwa 12,943 proteomes (25). Waɗannan sunadaran ne kawai waɗanda aka ƙididdige su a cikin aƙalla kashi 50% na lokuta an haɗa su cikin bincike na gaba. Saitin bayanan binciken ƙarshe ya ƙunshi sunadaran ƙididdiga 8817. Daidaita yawan adadin furotin dangane da shekaru, jinsi, da tazarar mutuwa (PMI). Binciken bambance-bambancen bayanan bayanan da aka saita bayan sake dawowa ya nuna cewa> 2000 matakan furotin sun canza sosai [P <0.05, nazarin bambance-bambancen (ANOVA)] a cikin ƙungiyoyi biyu ko fiye. Sa'an nan kuma, mun yi nazarin gungu mai kulawa bisa la'akari da sunadaran da aka bayyana daban-daban, da P <0.0001 a cikin AD / sarrafawa da / ko AD / PD kwatancen (Hoto S2, A da B, Table S2C). Waɗannan sunadaran sunadaran 165 da aka canza sosai suna kwatanta lokuta tare da ilimin halittar AD daga sarrafawa da samfuran PD, suna mai tabbatar da sauye-sauye na musamman AD a cikin dukkan proteome.
Daga nan sai muka yi amfani da wani algorithm mai suna Weighted Gene Co-expression Network Analysis (WGCNA) don yin nazarin hanyar sadarwa a kan ƙwayar ƙwayar cuta da aka gano, wanda ke tsara bayanan da aka saita zuwa nau'ikan furotin mai kama da yanayin magana (11-13). Binciken ya gano nau'o'in 44 (M) sunadaran da aka bayyana, an tsara su kuma an ƙidaya su daga mafi girma (M1, n = 1821 sunadaran) zuwa mafi ƙanƙanta (M44, n = 34 sunadaran) (Figure 2A da Table S2D) ). Kamar yadda aka ambata a sama (13) Yi ƙididdige bayanin bayanin wakilci ko furotin halayen kowane nau'in, kuma daidaita shi tare da yanayin cutar da ilimin halittar AD, wato, kafa ƙawancen Registry Disease Registry (CERAD) da Braak Score (Figure 2B). Gabaɗaya, samfuran 17 suna da alaƙa da alaƙa da neuropathology AD (P <0.05). Yawancin waɗannan nau'ikan abubuwan da ke da alaƙa da cuta kuma suna da wadatar alamomin takamaiman nau'in tantanin halitta (Hoto na 2B). Kamar yadda aka ambata a sama (13), ana ƙayyade nau'in tantanin halitta ta hanyar nazarin abin da ke tattare da juna da jerin abubuwan da suka shafi takamaiman nau'in tantanin halitta. Wadannan kwayoyin halitta an samo su ne daga bayanan da aka buga a cikin keɓaɓɓen ƙwayoyin linzamin kwamfuta, endothelial da glial sel. RNA sequencing (RNA-seq) gwaji (29).
(A) Gano WGCNA na proteome na kwakwalwa. (B) Matsakaicin matsakaicin nauyi (BiCor) nazarin furotin sa hannu na zamani (babban ɓangaren farko na furotin furotin) tare da halayen neuropathological AD (saman), gami da CERAD (Aβ plaque) da maki Braak (tau tangles). Ana nuna ƙarfin ma'amala mai kyau (ja) da korau (blue) ta taswirar zafi mai launi biyu, kuma asterisks suna nuna mahimmancin ƙididdiga (P <0.05). Yi amfani da Gwajin Haɗin Fisher na Hypergeometric (FET) (ƙasa) don tantance ƙungiyar nau'in tantanin halitta na kowane nau'in furotin. Ƙarfin inuwar ja yana nuna matakin haɓaka nau'in tantanin halitta, kuma alamar alama tana nuna mahimmancin ƙididdiga (P <0.05). Yi amfani da hanyar BH don gyara ƙimar P da aka samu daga FET. (C) GO bincike na sunadaran sunadaran. Ana nuna mafi kusantar hanyoyin nazarin halittu ga kowane nau'i ko rukuni mai alaƙa. oligo, oligodendrocyte.
Saitin astrocyte guda biyar da ke da alaƙa da microglia-rich modules (M30, M29, M18, M24, da M5) sun nuna kyakkyawar alaƙa mai kyau tare da AD neuropathology (Hoto 2B). Binciken Ontology yana haɗa waɗannan samfuran glial tare da haɓakar tantanin halitta, haɓakawa, da rigakafi (Hoto 2C da Table S2E). Ƙarin nau'ikan glial guda biyu, M8 da M22, suma suna da ƙarfi sosai a cikin cuta. M8 yana da alaƙa sosai da hanyar karɓa kamar Toll-kamar mai karɓa, kasidar sigina wacce ke taka muhimmiyar rawa a cikin amsawar rigakafi ta asali (30). A lokaci guda, M22 yana da alaƙa ta kut da kut da gyare-gyaren bayan fassarar. M2, wanda ke da wadata a cikin oligodendrocytes, yana nuna kyakkyawar dangantaka mai kyau tare da ilimin cututtuka na AD da kuma haɗin kai na ontological tare da haɗin nucleoside da kwafin DNA, yana nuna haɓakar haɓakar ƙwayoyin cuta a cikin cututtuka. Gabaɗaya, waɗannan binciken suna tallafawa haɓaka samfuran glial waɗanda muka lura a baya a cikin hanyar sadarwar AD (13, 17). A halin yanzu an gano cewa yawancin nau'ikan glial masu alaƙa da AD a cikin hanyar sadarwa suna nuna ƙananan matakan magana a cikin sarrafawa da kuma lamuran PD, suna nuna ƙayyadaddun cututtukan su wanda aka ɗaukaka a cikin AD (Figure S2C).
Nau'o'i huɗu ne kawai a cikin proteome na cibiyar sadarwar mu (M1, M3, M10, da M32) suna da alaƙa da mummunan alaƙa tare da ilimin halittar AD (P <0.05) (Hoto 2, B da C). Dukansu M1 da M3 suna da wadata a alamomin neuronal. M1 yana da alaƙa sosai da siginar synaptic, yayin da M3 ke da alaƙa da aikin mitochondrial. Babu wata shaida na wadatar nau'in tantanin halitta don M10 da M32. M32 yana nuna haɗin kai tsakanin M3 da ƙwayar sel, yayin da M10 yana da alaƙa da haɓakar tantanin halitta da aikin microtubule. Idan aka kwatanta da AD, duk nau'ikan nau'ikan guda huɗu suna haɓaka cikin sarrafawa da PD, suna ba su takamaiman canje-canjen AD (Hoto S2C). Gabaɗaya, waɗannan sakamakon suna goyan bayan raguwar wadatattun kayayyaki masu wadatar neuron waɗanda muka riga muka lura a cikin AD (13, 17). A taƙaice, nazarin hanyar sadarwa na proteome na kwakwalwa wanda muka gano ya samar da wasu sauye-sauye na musamman na AD daidai da binciken mu na baya.
AD yana da alamar farkon asymptomatic matakin (AsymAD), wanda mutane ke nuna tarin amyloid ba tare da raguwar fahimi na asibiti ba (5, 31). Wannan matakin asymptomatic yana wakiltar taga mai mahimmanci don ganowa da wuri da sa baki. Mun riga mun nuna ingantaccen tsarin kiyayewa na AsymAD da AD kwakwalwar cibiyar sadarwar proteome a cikin saitin bayanai masu zaman kansu (13, 17). Domin tabbatar da cewa cibiyar sadarwar kwakwalwa da muka gano a halin yanzu ta yi daidai da waɗannan binciken da aka yi a baya, mun yi nazarin adana nau'o'i 44 a cikin bayanan da aka kwafi daga ƙungiyoyin DLPFC 27. Waɗannan ƙungiyoyi sun haɗa da iko (n = 10), AsymAD (n = 8) Da AD (n = 9). Samfuran sarrafawa da AD an haɗa su a cikin nazarin ƙungiyar kwakwalwar binciken mu (Table S1B), yayin da shari'o'in AsymAD suka kasance na musamman kawai a cikin ƙungiyar kwafi. Waɗannan shari'o'in AsymAD kuma sun fito ne daga bankin kwakwalwar Emory Goizueta ADRC. Kodayake cognition ya kasance al'ada a lokacin mutuwa, matakan amyloid sun kasance da yawa (ma'anar CERAD, 2.8 ± 0.5) (Table S1B).
Binciken TMT-MS na waɗannan ƙwayoyin kwakwalwa 27 ya haifar da ƙididdige adadin 11,244 proteomes. Wannan ƙidayar ƙarshe ta ƙunshi waɗannan sunadaran da aka ƙididdige su a cikin aƙalla kashi 50% na samfuran. Wannan saitin bayanan da aka kwafi ya ƙunshi 8638 (98.0%) na sunadaran 8817 da aka gano a cikin binciken kwakwalwar binciken mu, kuma kusan 3000 sun canza sunadaran sosai tsakanin ƙungiyoyin sarrafawa da AD (P <0.05, bayan Tukey's paired t test for analysis of variance) Table S2F). Daga cikin waɗannan sunadaran da aka bayyana daban-daban, 910 kuma ya nuna mahimman canje-canje tsakanin AD da lamuran sarrafa proteome na kwakwalwa (P <0.05, bayan ANOVA Tukey sun haɗa t-gwajin). Ya kamata a lura cewa waɗannan alamomin 910 suna da daidaituwa sosai a cikin jagorancin canji tsakanin proteomes (r = 0.94, P <1.0 × 10-200) (Hoto S3A). Daga cikin haɓakar sunadaran, sunadaran da ke da mafi daidaito canje-canje tsakanin saitin bayanai galibi membobi ne na glial-rich M5 da M18 modules (MDK, COL25A1, MAPT, NTN1, SMOC1, da GFAP). Daga cikin raguwar sunadaran, waɗanda ke da mafi daidaito canje-canje sun kasance kusan mambobi ne na tsarin M1 (NPTX2, VGF, da RPH3A) masu alaƙa da synapse. Mun ƙara tabbatar da canje-canje masu alaƙa da AD na midkine (MDK), CD44, ɓoyayyiyar furotin mai alaƙa da frizzled 1 (SFRP1) da VGF ta hanyar lalatar yamma (Hoto S3B). Binciken adana kayan aiki ya nuna cewa game da 80% na nau'ikan furotin (34/44) a cikin proteome na kwakwalwa an kiyaye su sosai a cikin saitin bayanan kwafi (z-score> 1.96, FDR ya gyara P <0.05) (Figure S3C). Goma sha huɗu daga cikin waɗannan ƙa'idodi an kebe su ne musamman tsakanin abubuwan kariya biyu (z-score> 10, FDR ta gyara P <1.0 × 10-23). Gabaɗaya, ganowa da kwafi na babban matakin daidaito a cikin furci daban-daban da tsarin daidaitawa tsakanin proteome na kwakwalwa yana nuna sake fasalin canje-canje a cikin sunadaran cortex na gaba na AD. Bugu da ƙari, ya kuma tabbatar da cewa AsymAD da ƙarin cututtukan da suka ci gaba suna da tsarin cibiyar sadarwar kwakwalwa mai kama da juna.
Ƙarin cikakken bayani game da bambance-bambance a cikin saitin bayanan kwafi na kwakwalwa yana nuna mahimman digiri na canje-canjen furotin AsymAD, ciki har da jimlar 151 da suka canza sunadaran sunadaran tsakanin AsymAD da sarrafawa (P <0.05) (Figure S3D). Daidai da nauyin amyloid, APP a cikin kwakwalwar AsymAD da AD ya karu sosai. MAPT kawai yana canzawa sosai a cikin AD, wanda yayi daidai da haɓaka matakan tangles da sanannen alaƙarta tare da raguwar fahimi (5, 7). Abubuwan glial-rich modules (M5 da M18) suna nunawa sosai a cikin haɓakar sunadaran a cikin AsymAD, yayin da tsarin M1 da ke da alaƙa da neuron shine mafi wakilcin raguwar sunadarai a cikin AsymAD. Yawancin waɗannan alamomin AsymAD suna nuna manyan canje-canje a cikin cututtukan cututtuka. Daga cikin waɗannan alamomin akwai SMOC1, furotin glial na M18, wanda ke da alaƙa da ciwace-ciwacen ƙwaƙwalwa da haɓaka idanu da gaɓoɓi (32). MDK shine haɓakar haɓakar heparin da ke da alaƙa da haɓakar tantanin halitta da angiogenesis (33), wani memba na M18. Idan aka kwatanta da ƙungiyar kulawa, AsymAD ya karu sosai, ya biyo baya mafi girma a cikin AD. Sabanin haka, furotin synaptic neuropentraxin 2 (NPTX2) ya ragu sosai a cikin kwakwalwar AsymAD. NPTX2 an haɗa shi a baya tare da neurodegeneration kuma yana da rawar da aka sani a cikin tsaka-tsaki na synapses mai ban sha'awa (34). Gabaɗaya, waɗannan sakamakon suna bayyana sauye-sauye na furotin na asali iri-iri a cikin AD waɗanda da alama suna ci gaba tare da tsananin cutar.
Ganin cewa mun sami babban zurfin ɗaukar furotin a cikin gano ƙwayoyin proteome na kwakwalwa, muna ƙoƙarin fahimtar dalla-dalla yadda ya zo tare da bayanan matakin AD na cibiyar sadarwa. Saboda haka, mun kwatanta proteome na kwakwalwa da muka gano tare da tsarin da muka ƙirƙira a baya daga ma'aunin microarray na kwayoyin 18,204 a cikin AD (n = 308) da sarrafawa (n = 157) kyallen takarda DLPFC (13). jerawa. Gabaɗaya, mun gano nau'ikan nau'ikan RNA daban-daban guda 20, waɗanda yawancinsu sun nuna haɓaka takamaiman nau'ikan tantanin halitta, gami da neurons, oligodendrocytes, astrocytes, da microglia (Hoto 3A). Canje-canje da yawa na waɗannan kayayyaki a cikin AD ana nuna su a hoto na 3B. Daidai da binciken da muka gabata na haɗin gwiwar furotin-RNA ta amfani da mafi zurfi mara lahani na proteome MS (kimanin sunadaran 3000) (13), yawancin nau'ikan 44 a cikin cibiyar sadarwar proteome na kwakwalwa da muka samu suna cikin cibiyar sadarwa ta rubutu. Bincikenmu da kwafi na nau'ikan sunadaran sunadaran 34 waɗanda aka adana sosai a cikin proteome na kwakwalwa, kawai 14 (~ 40%) sun wuce ainihin gwajin Fisher (FET) ya tabbatar da cewa yana da ƙima mai mahimmanci tare da rubutun rubutu (Figure 3A). Mai jituwa tare da gyaran lalacewar DNA (P-M25 da P-M19), fassarar furotin (P-M7 da P-M20), RNA dauri / splicing (P-M16 da P-M21) da furotin da aka yi niyya (P-M13 da P- M23) baya zoba tare da kayayyaki a cikin rubutun. Saboda haka, ko da yake ana amfani da saitin bayanan proteome mai zurfi a cikin bincike mai zurfi na yanzu (13), yawancin proteome na cibiyar sadarwa na AD ba a tsara su zuwa cibiyar sadarwar da aka rubuta ba.
(A) Hypergeometric FET yana nuna wadatar takamaiman alamomin tantanin halitta a cikin RNA module na AD transcriptome (saman) da matakin haɗuwa tsakanin RNA (x-axis) da furotin (y-axis) na kwakwalwar AD. (kasa) . Ƙarfin jajayen shading yana nuna ƙimar haɓaka nau'ikan tantanin halitta a cikin babban panel da kuma ƙarfin haɗuwa na kayayyaki a cikin ɓangaren ƙasa. Asterisks suna nuna mahimmancin ƙididdiga (P <0.05). (B) Matsayin daidaitawa tsakanin halayen halayen kowane nau'in juzu'i da matsayin AD. Abubuwan da ke gefen hagu sun fi dacewa da AD (blue), kuma waɗanda ke hannun dama sun fi dacewa da AD (ja). Ƙimar P mai gyara BH da aka canza log yana nuna ƙimar mahimmancin ƙididdiga na kowane alaƙa. (C) Mahimman nau'ikan jeri tare da wadatar nau'in tantanin halitta. (D) Binciken daidaitawa na canjin log2 ninka na furotin da aka lakafta (x-axis) da RNA (y-axis) a cikin madaidaicin tsarin. Ana nuna ƙimar haɗin Pearson tare da ƙimar P mai dacewa. Micro, microglia; jikin sama, astrocytes. CT, sarrafawa.
Yawancin sunadaran sunadaran haɗe-haɗe da na'urorin RNA suna raba nau'in bayanan haɓaka nau'in tantanin halitta iri ɗaya da daidaitattun kwatancen AD (Hoto na 3, B da C). A wasu kalmomi, tsarin M1 mai alaka da synapse na proteome na kwakwalwa (PM1) an tsara shi zuwa nau'o'in RNA na homologous guda uku (R-M1, R-M9 da RM16), waɗanda ke cikin AD Dukansu sun nuna. matakin ragewa. Hakazalika, glial-rich M5 da M18 sunadaran sunadaran suna haɗuwa tare da samfuran RNA masu wadatar astrocytes da alamomin microglial (R-M3, RM-M7, da RM-M10) kuma suna da hannu sosai cikin haɓakar cututtuka. Waɗannan fasalulluka na yau da kullun da aka raba tsakanin saitin bayanai guda biyu suna ƙara tallafawa nau'in tantanin halitta da sauye-sauye masu alaƙa da cuta da muka lura a cikin proteome na kwakwalwa. Koyaya, mun lura da bambance-bambance masu yawa tsakanin matakan RNA da furotin na alamomin daidaikun mutane a cikin waɗannan samfuran da aka raba. Binciken daidaitawa na banbamcin bayanin proteomics da kwafi na kwayoyin halitta a cikin waɗannan nau'ikan da suka mamaye (Hoto 3D) yana haskaka wannan rashin daidaituwa. Misali, APP da wasu sunadaran sunadaran glial da yawa (NTN1, MDK, COL25A1, ICAM1, da SFRP1) sun nuna karuwa mai yawa a cikin furotin AD, amma kusan babu wani canji a cikin kwafin AD. Waɗannan sauye-sauye na ƙayyadaddun furotin na iya kasancewa da alaƙa da alaƙa da amyloid plaques (23, 35), suna nuna proteome a matsayin tushen sauye-sauyen cututtukan cututtuka, kuma waɗannan canje-canjen bazai iya nunawa a cikin rubutun ba.
Bayan yin nazarin kwakwalwa da kansa da ƙwayoyin kariya na CSF da muka gano, mun gudanar da cikakken bincike game da saitin bayanai guda biyu don gano alamun AD CSF masu alaƙa da ilimin cututtukan cututtukan ƙwayar cuta na cibiyar sadarwar kwakwalwa. Dole ne mu fara ayyana abin da ke tattare da proteome biyu. Kodayake an yarda da cewa CSF tana nuna canje-canjen neurochemical a cikin kwakwalwar AD (4), ainihin matakin haɗuwa tsakanin kwakwalwar AD da proteome CSF ba a sani ba. Ta hanyar kwatanta adadin samfuran kwayoyin halittar da aka gano a cikin sinadarai guda biyu, mun gano cewa kusan 70% (n = 1936) na sunadaran da aka gano a cikin ruwan cerebrospinal kuma an ƙididdige su a cikin kwakwalwa (Hoto 4A). Yawancin waɗannan sunadaran da suka mamaye (n = 1721) an tsara su zuwa ɗaya daga cikin nau'ikan bayanin haɗin gwiwa guda 44 daga saitin bayanan kwakwalwar ganowa (Hoto 4B). Kamar yadda aka zata, manyan na'urori guda shida na kwakwalwa (M1 zuwa M6) sun nuna mafi girman adadin ruɗewar CSF. Koyaya, akwai ƙananan na'urori na kwakwalwa (misali, M15 da M29) waɗanda ke samun babban mataki ba zato ba tsammani, wanda ya fi girma fiye da tsarin kwakwalwa sau biyu girmansa. Wannan yana motsa mu mu ɗauki ƙarin daki-daki, hanyar ƙwaƙƙwaran ƙididdiga don ƙididdige abin da ke tsakanin kwakwalwa da ruwa na cerebrospinal.
(A da B) Sunadaran da aka gano a cikin kwakwalwar ganowa da saitin bayanai na CSF sun zo juna. Yawancin waɗannan sunadaran da suka mamaye suna da alaƙa da ɗayan nau'ikan 44 na haɗin kai na cibiyar sadarwar haɗin gwiwar kwakwalwa. (C) Gano haɗuwa tsakanin proteome ruwa na cerebrospinal da proteome cibiyar sadarwar kwakwalwa. Kowane jeri na taswirar zafin rana yana wakiltar wani keɓantaccen nazarin jeri na hypergeometric FET. Jeri na sama yana kwatanta abin da ya faru (farin launin toka/baƙar shading) tsakanin tsarin kwakwalwa da gabaɗayan proteome na CSF. Layi na biyu yana nuna cewa haɗuwa tsakanin sassan kwakwalwa da furotin CSF (mai inuwa a ja) an daidaita shi sosai a cikin AD (P <0.05). Jeri na uku yana nuna cewa haɗuwa tsakanin sassan kwakwalwa da furotin CSF (shading blue) yana da mahimmanci a cikin AD (P <0.05). Yi amfani da hanyar BH don gyara ƙimar P da aka samu daga FET. (D) Kwamitin nadawa bisa tushen ƙungiyar nau'in tantanin halitta da sharuddan GO masu alaƙa. Wadannan bangarori sun ƙunshi jimillar sunadaran da ke da alaƙa da ƙwaƙwalwa 271, waɗanda ke da ma'anar ma'anar ma'anar su a cikin proteome na CSF.
Yin amfani da FETs masu wutsiya guda ɗaya, mun tantance mahimmancin haɗin sunadaran sunadaran tsakanin proteome na CSF da kowane nau'ikan kwakwalwa. Binciken ya nuna cewa jimillar nau'ikan kwakwalwar kwakwalwa na 14 a cikin saitin bayanan CSF suna da ƙididdiga masu mahimmanci (FDR da aka gyara P <0.05), da kuma ƙarin ƙirar (M18) wanda haɗin gwiwa ya kusa da mahimmanci (FDR gyara P = 0.06) (Hoto 4C). , layi na sama). Har ila yau, muna sha'awar kayayyaki waɗanda ke da ƙarfi sosai tare da sunadaran CSF da aka bayyana daban-daban. Saboda haka, mun yi amfani da ƙarin ƙarin nazarin FET guda biyu don sanin wane na (i) furotin CSF ya karu sosai a cikin AD kuma (ii) furotin CSF ya ragu sosai a AD (P <0.05, an haɗa t gwajin AD / sarrafawa) Na'urorin kwakwalwa tare da ma'ana mai ma'ana. tsakanin su. Kamar yadda aka nuna a cikin layuka na tsakiya da ƙasa na Hoto 4C, waɗannan ƙarin bincike sun nuna cewa 8 na 44 na kwakwalwar kwakwalwa suna da mahimmanci tare da furotin da aka kara a AD CSF (M12, M1, M2, M18, M5, M44, M33, da M38). . ), yayin da kawai nau'i biyu (M6 da M15) sun nuna ma'ana mai ma'ana tare da rage yawan furotin a AD CSF. Kamar yadda aka zata, duk nau'ikan nau'ikan 10 suna cikin samfuran 15 tare da mafi girma tare da proteome CSF. Sabili da haka, muna ɗauka cewa waɗannan nau'ikan 15 sune tushen samar da albarkatu masu girma na AD masu amfani da ƙwayoyin halitta na CSF.
Mun naɗe waɗannan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan 'ya'yan itace da kuma nau'ikan kwayoyin halitta (Hoto na 4D). Rukunin farko yana ƙunshe da kayayyaki masu wadata a alamomin neuron da sunadarai masu alaƙa da synapse (M1 da M12). Ƙungiyar synaptic ta ƙunshi jimillar sunadaran 94, kuma matakan da ke cikin proteome na CSF sun canza sosai, yana mai da shi mafi girma tushen alamun CSF masu alaka da kwakwalwa a cikin bangarori biyar. Rukuni na biyu (M6 da M15) sun nuna kusancin haɗin gwiwa tare da alamomin sel na endothelial da jikin jijiyoyin jini, irin su "warkar da rauni" (M6) da "ka'idar amsawar rashin jin daɗi" (M15). M15 kuma yana da alaƙa da haɓakar lipoprotein metabolism, wanda ke da alaƙa da endothelium (36). Ƙungiyar jijiyoyin jini ta ƙunshi alamun 34 CSF masu alaƙa da kwakwalwa. Ƙungiya ta uku ta haɗa da kayayyaki (M2 da M4) waɗanda ke da alaƙa da mahimmancin alamomin oligodendrocyte da yaduwar kwayar halitta. Misali, manyan sharuddan ontology na M2 sun haɗa da "tabbatacciyar ƙa'ida ta kwafin DNA" da "tsarin biosynthesis na purine". A halin yanzu, na M4 sun haɗa da "bambance-bambancen cell glial" da "rarrabuwar chromosome". Ƙungiyar myelination ta ƙunshi alamomin CSF 49 masu alaƙa da kwakwalwa.
Ƙungiya ta huɗu ta ƙunshi mafi yawan kayayyaki (M30, M29, M18, M24, da M5), kuma kusan dukkanin kayayyaki suna da wadata a cikin microglia da alamomin astrocyte. Hakazalika da faifan myelination, panel na huɗu kuma ya ƙunshi kayayyaki (M30, M29, da M18) waɗanda ke da alaƙa da haɓakar tantanin halitta. Sauran nau'ikan da ke cikin wannan rukunin suna da alaƙa sosai da sharuɗɗan rigakafi, kamar "tsarin tasiri na rigakafi" (M5) da "ka'idojin amsawa na rigakafi" (M24). Ƙungiyar rigakafi ta glial ta ƙunshi alamomin CSF 42 masu alaƙa da kwakwalwa. A ƙarshe, rukunin na ƙarshe ya haɗa da alamomin da ke da alaƙa da ƙwaƙwalwa na 52 akan nau'ikan nau'ikan guda huɗu (M44, M3, M33, da M38), waɗanda duk suna cikin jikin da ke da alaƙa da ajiyar kuzari da haɓaka. Mafi girma daga cikin waɗannan kayayyaki (M3) yana da alaƙa ta kud da kud da mitochondria kuma yana da wadatar alamomin takamaiman neuron. M38 yana ɗaya daga cikin ƙananan mambobi a cikin wannan metabolome kuma yana nuna matsakaicin ƙayyadaddun neuron.
Gabaɗaya, waɗannan bangarori guda biyar suna nuna nau'ikan tantanin halitta da ayyuka iri-iri a cikin kogin AD, kuma tare sun ƙunshi alamomin CSF masu alaƙa da kwakwalwa guda 271 (Table S2G). Don kimanta ingancin waɗannan sakamakon MS, mun yi amfani da ƙimar kusancin kusanci (PEA), fasaha ce ta tushen antibody mai ƙarfi tare da iyawar juzu'i, babban hankali da ƙayyadaddun bayanai, kuma mun sake nazarin samfuran ruwan cerebrospinal da muka samo wani yanki na waɗannan 271 biomarkers. (n = 36). Wadannan makasudin 36 suna nuna canji a cikin yawan AD na PEA, wanda ke da alaƙa da alaƙa da binciken mu na MS (r = 0.87, P = 5.6 × 10-12) , Wanda ya tabbatar da ingantaccen sakamakon binciken mu na MS (Hoto S4). ).
Jigogi na nazarin halittu da ƙungiyoyin mu biyar suka jaddada, daga siginar synaptic zuwa makamashi metabolism, duk suna da alaƙa da pathogenesis na AD (1-3). Sabili da haka, duk nau'ikan nau'ikan 15 da ke ɗauke da waɗannan bangarorin suna da alaƙa da ilimin halittar AD a cikin proteome na kwakwalwa wanda muka gano (Hoto 2B). Babban abin lura shine babban ingantacciyar alaƙar cututtukan cututtukan cututtukan ƙwayar cuta tsakanin samfuran glial ɗinmu da ƙaƙƙarfan alaƙar cututtukan cututtukan cuta tsakanin manyan samfuran neuronal ɗinmu (M1 da M3). Binciken bambance-bambancen bayanin proteome na kwakwalwarmu da aka kwafi (Hoto S3D) shima yana haskaka M5 da M18 sunadaran glial da aka samu. A cikin AsymAD da alamar alamar AD, mafi yawan sunadaran glial da ke da alaƙa da synapses na M1 An rage yawan furotin. Wadannan abubuwan lura sun nuna cewa alamomin ruwa na cerebrospinal 271 da muka gano a cikin ƙungiyoyi biyar suna da alaƙa da tsarin cututtuka a cikin AD cortex, ciki har da waɗanda ke faruwa a farkon matakan asymptomatic.
Domin ingantacciyar nazarin canjin canjin sunadaran panel a cikin kwakwalwa da ruwan kashin baya, mun zana abubuwan da ke biyowa ga kowane nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan 15: (i) sami matakin yalwar module a saitin bayanan kwakwalwa da (ii) tsarin. furotin An bayyana bambanci a cikin ruwan cerebrospinal (Hoto S5). Kamar yadda aka ambata a baya, WGCNA ana amfani da ita don tantance yawan adadin ko ƙimar furotin a cikin kwakwalwa (13). Ana amfani da taswirar dutsen mai aman wuta don bayyana bambancin kalaman sunadaran sunadaran a cikin ruwan cerebrospinal (AD/control). Wadannan alkaluma sun nuna cewa uku daga cikin bangarori biyar suna nuna yanayin magana daban-daban a cikin kwakwalwa da ruwan kashin baya. Nau'o'i biyu na panel synapse (M1 da M12) suna nuna raguwa a cikin yawan adadin da ke cikin kwakwalwar AD, amma suna da mahimmanci tare da karuwar furotin a cikin AD CSF (Hoto S5A). Abubuwan da ke da alaƙa da neuron da ke ɗauke da metabolome (M3 da M38) sun nuna kwakwalwar kwakwalwa iri ɗaya da tsarin maganganun ruwa na cerebrospinal ba daidai ba (Hoto S5E). Ƙungiyar jijiyoyin jini ta kuma nuna nau'o'in maganganu daban-daban, kodayake nau'ikansa (M6 da M15) sun karu da yawa a cikin kwakwalwar AD kuma sun ragu a cikin CSF marasa lafiya (Figure S5B). Ragowar bangarorin biyu sun ƙunshi manyan hanyoyin sadarwa na glial waɗanda sunadaran sunadaran suna da tsari akai-akai a cikin sassan biyu (Hoto S5, C da D).
Lura cewa waɗannan dabi'un ba kowa bane ga duk alamomi a cikin waɗannan bangarorin. Misali, kwamitin synaptic ya haɗa da sunadaran sunadarai da yawa waɗanda aka rage su sosai a cikin kwakwalwar AD da CSF (Hoto S5A). Daga cikin waɗannan alamomin ruwa na cerebrospinal da aka tsara sune NPTX2 da VGF na M1, da chromogranin B na M12. Koyaya, duk da waɗannan keɓancewa, yawancin alamun mu na synaptic suna haɓaka cikin ruwan kashin baya na AD. Gabaɗaya, waɗannan nazarin sun sami damar bambance mahimman abubuwan ƙididdiga a cikin kwakwalwa da matakan ruwa na cerebrospinal a cikin kowane fanni na mu guda biyar. Wadannan dabi'un suna haskaka hadaddun dangantaka da sau da yawa daban-daban tsakanin kwakwalwa da bayanin furotin CSF a cikin AD.
Sa'an nan, mun yi amfani da high-throughput MS kwafi bincike (CSF kwafi 1) don kunkuntar mu 271 saitin biomarkers zuwa mafi alƙawari da reproducible hari (Hoto 5A). Kwafin CSF 1 ya ƙunshi jimlar samfuran 96 daga Emory Goizueta ADRC, gami da sarrafawa, AsymAD, da ƙungiyar AD (Table S1A). Waɗannan shari'o'in AD suna da alaƙa da raguwar fahimi mai sauƙi (ma'anar MoCA, 20.0 ± 3.8), da canje-canje a cikin alamun halittun AD waɗanda aka tabbatar a cikin ruwan cerebrospinal (Table S1A). Sabanin nazarin CSF da muka samo, ana yin wannan kwafin ta amfani da mafi inganci da ingantaccen hanyar "harbi ɗaya" MS (ba tare da ɓangarorin layi ba), gami da ƙayyadaddun tsari na shirye-shiryen samfuri wanda ke kawar da buƙatar rigakafi na samfuran mutum ɗaya. . Madadin haka, ana amfani da “tashar haɓakawa” guda ɗaya da ta rage garkuwar jiki don haɓaka siginar sunadaran da ba su da yawa (37). Ko da yake yana rage yawan ɗaukar hoto na proteome, wannan hanyar harbi guda ɗaya yana rage yawan lokacin inji kuma yana ƙara yawan samfuran da aka yiwa lakabi da TMT waɗanda za'a iya tantance su (17, 38). Gabaɗaya, binciken ya gano peptides 6,487, waɗanda aka tsara zuwa 1,183 proteomes a cikin shari'o'i 96. Kamar yadda binciken CSF da muka samu, kawai waɗannan sunadaran da aka ƙididdige su a cikin aƙalla 50% na samfuran an haɗa su a cikin ƙididdiga masu zuwa, kuma bayanan sun koma baya don tasirin shekaru da jinsi. Wannan ya haifar da ƙididdige adadin ƙwayoyin proteome 792, 95% kuma an gano su a cikin saitin bayanan CSF da aka samu.
(A) Maƙasudin furotin CSF masu alaƙa da ƙwaƙwalwa an tabbatar da su a cikin rukunin CSF na farko da aka kwafi kuma an haɗa su cikin rukunin ƙarshe (n = 60). (B zuwa E) Matakan ƙirar halittu (composite z-scores) da aka auna a cikin ƙungiyoyin kwafi na CSF guda huɗu. An yi amfani da haɗe-haɗe t-tests ko ANOVA tare da Tukey's bayan gyara an yi amfani da su don kimanta mahimmancin ƙididdiga na canje-canjen da yawa a cikin kowane bincike mai kwafi. CT, sarrafawa.
Tunda muna da sha'awar tabbatar da maƙasudin CSF ɗinmu na 271 masu alaƙa da ƙwaƙwalwa ta hanyar cikakken bincike, za mu iyakance ƙarin gwajin wannan sinadari mai kwafi ga waɗannan alamomi. Daga cikin waɗannan sunadaran 271, an gano 100 a cikin kwafin CSF 1. Hoto S6A yana nuna bambancin maganganun waɗannan alamomin 100 masu haɗuwa tsakanin sarrafawa da samfurori na AD. Synaptic da metabolite histones suna ƙaruwa mafi yawa a cikin AD, yayin da sunadaran jijiyoyin jini suna rage mafi yawan cututtuka. Yawancin alamomin 100 masu haɗuwa (n = 70) sun kiyaye alƙawarin canji guda ɗaya a cikin saitin bayanai guda biyu (Hoto S6B). Waɗannan 70 ingantattun alamomin CSF masu alaƙa da kwakwalwa (Table S2H) galibi suna nuna yanayin yanayin magana da aka lura a baya, wato, ƙa'idodin ƙa'idodin sunadaran jijiyoyin jini da haɓakar duk sauran bangarorin. Kashi 10 ne kawai daga cikin waɗannan ingantattun sunadaran 70 sun nuna canje-canje a cikin yawan AD wanda ya saba wa waɗannan yanayin tsarin. Domin samar da wani kwamiti wanda ya fi dacewa yana nuna yanayin gaba ɗaya na kwakwalwa da ruwa na cerebrospinal, mun cire waɗannan sunadaran 10 daga rukunin sha'awa wanda a ƙarshe muka tabbatar (Hoto 5A). Don haka, rukuninmu a ƙarshe ya haɗa da jimlar sunadaran 60 waɗanda aka tabbatar a cikin ƙungiyoyin CSF AD biyu masu zaman kansu ta amfani da shirye-shiryen samfuri daban-daban da kuma nazarin dandamali na MS. Makircin maganganun z-score na waɗannan bangarori na ƙarshe a cikin kwafin kwafin 1 na CSF da shari'o'in AD sun tabbatar da yanayin kwamitin da aka lura a cikin ƙungiyar CSF da muka samo (Hoto 5B).
Daga cikin wadannan sunadaran 60, akwai kwayoyin da aka sani suna da alaƙa da AD, irin su osteopontin (SPP1), wanda shine cytokine pro-inflammatory wanda aka danganta da AD a yawancin binciken (39-41), da GAP43, A synaptic protein. wanda ke da alaƙa a fili zuwa neurodegeneration (42). Mafi yawan sunadaran da aka tabbatar da su sune alamun da ke da alaƙa da wasu cututtuka na neurodegenerative, irin su amyotrophic lateral sclerosis (ALS) superoxide dismutase 1 (SOD1) da cutar Parkinson da ke da alaka da desaccharase (PARK7). Mun kuma tabbatar da cewa yawancin sauran alamomi, irin su SMOC1 da siginar siginar abun da ke ciki na kwakwalwa 1 (BASP1), sun iyakance hanyoyin haɗin da suka gabata zuwa neurodegeneration. Yana da kyau a lura cewa saboda ƙarancin wadatar su gaba ɗaya a cikin furotin CSF, yana da wahala a gare mu mu yi amfani da wannan babban hanyar gano harbi guda ɗaya don dogaro da gano MAPT da wasu sunadaran da ke da alaƙa da AD (misali, NEFL da NRGN). ) ( 43, 44).
Daga nan mun duba waɗannan alamomin fifiko guda 60 a cikin ƙarin bincike guda uku. A cikin CSF Kwafi 2, mun yi amfani da TMT-MS guda ɗaya don nazarin ƙungiyoyi masu zaman kansu na kulawar 297 da samfuran AD daga Emory Goizueta ADRC (17). Kwafin CSF 3 ya haɗa da sake nazarin bayanan TMT-MS da ke samuwa daga kulawar 120 da marasa lafiya AD daga Lausanne, Switzerland (45). Mun gano fiye da kashi biyu bisa uku na alamomin fifiko 60 a cikin kowane saitin bayanai. Kodayake binciken na Swiss ya yi amfani da dandamali daban-daban na MS da hanyoyin ƙididdige TMT (45, 46), mun yi karfi da sake sake fasalin tsarin mu a cikin maimaitawa guda biyu (Hoto 5, C da D, da Tables S2, I, da J). Don kimanta ƙayyadaddun cutar ta ƙungiyarmu, mun yi amfani da TMT-MS don bincika saitin bayanan kwafi na huɗu (CSF replication 4), wanda ya haɗa ba kawai iko (n = 18) da AD (n = 17) lokuta ba, har ma PD ( n = 14)), ALS (n = 18) da kuma gaban dementia na gaba (FTD) samfurori (n = 11) (Table S1A). Mun sami nasarar ƙididdige kusan kashi biyu bisa uku na furotin a cikin wannan rukunin (38 cikin 60). Waɗannan sakamakon suna nuna ƙayyadaddun sauye-sauye na AD a cikin dukkan fa'idodin biomarker guda biyar (Hoto 5E da Table S2K). Haɓakawa a cikin rukunin metabolite ya nuna ƙayyadaddun ƙaƙƙarfan AD, sannan ƙungiyar myelination da glial ta biyo baya. Har ila yau, FTD yana nuna karuwa tsakanin waɗannan bangarori, wanda zai iya nuna irin canje-canjen hanyoyin sadarwa (17). Sabanin haka, ALS da PD sun nuna kusan myelination, glial, da bayanan martaba iri ɗaya kamar ƙungiyar kulawa. Gabaɗaya, duk da bambance-bambance a cikin shirye-shiryen samfurin, dandamali na MS, da hanyoyin ƙididdigewa na TMT, waɗannan ƙididdigar maimaitawa sun nuna cewa alamun kwamitinmu na fifiko suna da daidaitattun sauye-sauye na takamaiman AD a cikin samfuran CSF na musamman na 500.
AD neurodegeneration an san shi da yawa shekaru da yawa kafin farawar alamun fahimi, don haka akwai buƙatar gaggawa ga masu binciken halittu na AsymAD (5, 31). Duk da haka, ƙarin shaidu sun nuna cewa ilimin halitta na AsymAD ya yi nisa daga kamanni, kuma hadaddun hulɗar haɗari da juriya yana haifar da babban bambance-bambancen mutum a cikin ci gaban cututtuka na gaba (47). Ko da yake an yi amfani da su don gano lokuta na AsymAD, matakan core CSF biomarkers (Aβ1-42, total tau da p-tau) ba su tabbatar da cewa za su iya dogara ga wanda zai ci gaba zuwa dementia (4, 7), yana nuna ƙarin Yana iya zama. ya zama dole don haɗa da cikakkun kayan aikin biomarker dangane da bangarori da yawa na ilimin halittar kwakwalwa don daidaita haɗarin wannan yawan. Sabili da haka, daga baya mun yi nazarin panel ɗin mu na biomarker na AD a cikin adadin AsymAD na CSF kwafin 1. Waɗannan lokuta na 31 AsymAD sun nuna matakan da ba su dace ba (Aβ1-42 / jimlar tau ELISA rabo, <5.5) da cikakkiyar fahimta (ma'anar MoCA, 27.1). ± 2.2) (Table S1A). Bugu da kari, duk mutanen da ke tare da AsymAD suna da makin cutar dementia na asibiti na 0, wanda ke nuna cewa babu wata shaida ta raguwar fahimi ko aiki na yau da kullun.
Mun fara bincikar matakan ingantattun bangarorin a cikin duk 96 CSF kwafi na 1, gami da ƙungiyar AsymAD. Mun gano cewa bangarori da yawa a cikin ƙungiyar AsymAD suna da manyan canje-canje masu yawa kamar AD, ƙungiyar jijiyoyi sun nuna yanayin ƙasa a cikin AsymAD, yayin da sauran bangarorin suka nuna haɓakar haɓaka (Hoto 6A). Sabili da haka, duk bangarorin sun nuna alaƙa mai mahimmanci tare da ELISA Aβ1-42 da jimlar matakan tau (Hoto 6B). Sabanin haka, alaƙar da ke tsakanin ƙungiyar da ƙimar MoCA ba ta da kyau. Ɗayan ƙarin binciken da ya fi dacewa daga waɗannan nazarin shine babban kewayon ɗimbin panel a cikin ƙungiyar AsymAD. Kamar yadda aka nuna a cikin Hoto 6A, matakin panel na ƙungiyar AsymAD yakan ƙetare matakin panel na ƙungiyar kulawa da kuma ƙungiyar AD, yana nuna babban bambanci. Don ci gaba da gano wannan nau'in nau'in AsymAD, mun yi amfani da bincike na Multidimensional Scaling (MDS) zuwa 96 CSF kwafi 1 lokuta. Binciken MDS yana ba da damar ganin kamanni tsakanin shari'o'i dangane da wasu masu canji a cikin saitin bayanai. Don wannan binciken tari, muna amfani da waɗancan ingantattun alamomin panel waɗanda ke da babban canji na ƙididdiga (P <0.05, AD/control) a cikin binciken CSF da maimaita matakin 1 proteome (n = 29) (Table S2L). Wannan bincike ya haifar da fayyace tari tsakanin ikonmu da shari'ar AD (Hoto 6C). Sabanin haka, wasu shari'o'in AsymAD sun taru a fili a cikin rukunin kulawa, yayin da wasu suna cikin shari'o'in AD. Don ƙarin bincika wannan nau'in AsymAD, mun yi amfani da taswirar MDS don ayyana ƙungiyoyi biyu na waɗannan shari'o'in AsymAD. Ƙungiya ta farko ta haɗa da shari'o'in AsymAD da aka taru kusa da sarrafawa (n = 19), yayin da rukuni na biyu yana da alamun AsymAD tare da bayanin martaba kusa da AD (n = 12).
(A) Matsayin magana (z-score) na ƙungiyar biomarker na CSF a cikin duk samfuran 96 a cikin ƙungiyar CSF replica 1 cohort, gami da AsymAD. An yi amfani da nazarin bambance-bambance tare da Tukey's bayan gyara don kimanta mahimmancin ƙididdiga na yawan canje-canjen panel. (B) Binciken daidaituwa na matakin yawan furotin panel (z-score) tare da maki MoCA da jimlar tau matakin a cikin ELISA Aβ1-42 da CSF kwafin 1 samfurori. Ana nuna ƙimar haɗin Pearson tare da ƙimar P mai dacewa. (C) MDS na 96 CSF kwafin 1 lokuta ya dogara ne akan yawan matakan 29 ingantattun alamomin panel, waɗanda aka canza su sosai a duka binciken da CSF kwafin 1 bayanan bayanan [P <0.05 AD / sarrafawa (CT)]. Anyi amfani da wannan bincike don rarraba ƙungiyar AsymAD zuwa iko (n = 19) da AD (n = 12) ƙananan ƙungiyoyi. (D) Makircin volcano yana nuna bambancin bayanin duk sunadaran CSF kwafi 1 tare da canjin log2 (x-axis) dangane da ƙimar P-log10 na ƙididdigewa tsakanin ƙungiyoyin AsymAD guda biyu. Alamar kwamfutoci masu launi. (E) CSF kwafi 1 yawan matakin ɗimbin matakin ƙungiyar zaɓaɓɓun alamun halittu an bayyana su daban-daban tsakanin ƙungiyoyin AsymAD. An yi amfani da nazarin bambance-bambancen da Tukey ya yi don tantance mahimmancin ƙididdiga.
Mun bincika bambance-bambancen bayanin furotin tsakanin waɗannan iko da AD-kamar maganganun AsymAD (Hoto 6D da Table S2L). Taswirar dutsen mai aman wuta da aka samu ya nuna cewa alamomi 14 sun canza sosai tsakanin ƙungiyoyin biyu. Yawancin waɗannan alamomin mambobi ne na synapse da metabolome. Duk da haka, SOD1 da myristoylated alanine-rich protein kinase C substrate (MARCKS), waɗanda suke mambobi ne na myelin da glial rigakafi kungiyoyin, bi da bi, kuma suna cikin wannan rukuni (Hoto 6, D da E). Ƙungiyar jijiyoyin jini kuma ta ba da gudummawar alamomi guda biyu waɗanda aka rage su sosai a cikin rukunin AD-kamar AsymAD, gami da furotin mai ɗaure AE 1 (AEBP1) da kuma cikakken memba na C9. Babu wani bambanci mai mahimmanci tsakanin ƙungiyoyin sarrafawa da AD-kamar AsymAD subgroups a cikin ELISA AB1-42 (P = 0.38) da p-tau (P = 0.28), amma akwai babban bambanci a cikin jimlar tau matakin (P = 0.0031). ) (Hoto S7). Akwai alamomi da yawa waɗanda ke nuna cewa canje-canje tsakanin ƙungiyoyin AsymAD biyu sun fi mahimmanci fiye da jimlar matakan tau (misali, YWHAZ, SOD1, da MDH1) (Hoto 6E). Gabaɗaya, waɗannan sakamakon suna nuna cewa ingantaccen kwamitinmu na iya ƙunsar alamomin halittu waɗanda za su iya jujjuyawar haɗarin marasa lafiya da cutar asymptomatic.
Akwai buƙatar gaggawa don kayan aikin tushen tsarin halittu don ingantacciyar ma'auni da manufa daban-daban na ilimin halittar jiki a bayan AD. Ana sa ran waɗannan kayan aikin ba wai kawai za su canza tsarin binciken mu na AD ba, har ma suna haɓaka ɗaukar tasiri, takamaiman dabarun jiyya na haƙuri (1, 2). Don wannan ƙarshen, mun yi amfani da cikakkiyar tsarin kariya na rashin son rai ga kwakwalwar AD da CSF don gano alamun abubuwan rayuwa na CSF na yanar gizo waɗanda ke nuna nau'ikan cututtukan cututtukan ƙwayoyin cuta na kwakwalwa. Bincikenmu ya samar da bangarori biyar na CSF biomarker, wanda (i) yana nuna synapses, tasoshin jini, myelin, rigakafi da rashin aiki na rayuwa; (ii) nuna karfi reproducibility a kan daban-daban MS dandamali; (iii) Nuna takamaiman canje-canjen cututtuka na ci gaba a cikin farkon da ƙarshen matakan AD. Gabaɗaya, waɗannan binciken suna wakiltar mataki mai ban sha'awa ga haɓaka iri-iri, abin dogaro, kayan aikin biomarker na yanar gizo don bincike na AD da aikace-aikacen asibiti.
Sakamakonmu yana nuna ƙungiyar da aka tanada sosai ta hanyar sadarwar kwakwalwar AD kuma tana goyan bayan amfani da ita azaman anka don haɓaka tushen tsarin halittu. Binciken mu ya nuna cewa bayanan TMT-MS masu zaman kansu guda biyu da suka ƙunshi AD da kwakwalwar AsymAD suna da ƙarfi mai ƙarfi. Waɗannan binciken sun haɓaka aikinmu na baya, yana nuna adana kayan aiki masu ƙarfi na ƙwayoyin kwakwalwa fiye da 2,000 daga ƙungiyoyi masu zaman kansu da yawa a cikin gaba, parietal, da cortex na ɗan lokaci (17). Wannan haɗin gwiwar haɗin gwiwar yana nuna canje-canje daban-daban masu alaƙa da cututtuka da aka gani a cikin bincike na yanzu, gami da haɓakar glial-rich modules inflammatory modules da kuma raguwar kayan aikin neuron. Kamar bincike na yanzu, wannan babbar hanyar sadarwar kuma tana da sauye-sauye masu mahimmanci a cikin AsymAD, yana nuna nau'ikan cututtukan cututtuka daban-daban (17).
Koyaya, a cikin wannan tsarin tushen tsarin mai ra'ayin mazan jiya, akwai mafi kyawun nau'ikan nau'ikan halitta, musamman tsakanin daidaikun mutane a farkon matakan AD. Ƙungiyar mu ta biomarker tana iya nuna ƙungiyoyi biyu a cikin AsymAD, waɗanda ke nuna mahimmancin bambance-bambancen alamomin CSF da yawa. Ƙungiyarmu ta sami damar haskaka bambance-bambancen nazarin halittu tsakanin waɗannan ƙananan ƙungiyoyi biyu, waɗanda ba a bayyane suke ba a matakin ainihin AD biomarkers. Idan aka kwatanta da ƙungiyar kulawa, adadin Aβ1-42/ jimlar tau na waɗannan mutanen AsymAD sun yi ƙasa sosai. Koyaya, jimlar matakan tau kawai sun bambanta sosai tsakanin ƙungiyoyin AsymAD guda biyu, yayin da matakan Aβ1-42 da p-tau sun kasance masu kama da juna. Tun da babban CSF tau alama ya zama mafi kyawun hangen nesa na alamun fahimi fiye da matakan Aβ1-42 (7), muna tsammanin cewa ƙungiyoyin AsymAD guda biyu na iya samun haɗari daban-daban na ci gaban cuta. Idan aka ba da ƙayyadaddun girman samfurin mu na AsymAD da kuma rashin bayanan dogon lokaci, ana buƙatar ƙarin bincike don zana waɗannan yanke shawara. Koyaya, waɗannan sakamakon suna nuna cewa rukunin CSF na tushen tsarin zai iya haɓaka ikonmu na daidaita daidaitattun mutane yayin matakin asymptomatic na cutar.
Gabaɗaya, bincikenmu yana goyan bayan rawar ayyuka na halitta da yawa a cikin pathogenesis na AD. Koyaya, ƙarancin kuzarin kuzari ya zama fitaccen jigon duk ingantattun bangarori biyar ɗin mu. Sunadaran gina jiki, irin su hypoxanthine-guanine phosphoribosyltransferase 1 (HPRT1) da lactate dehydrogenase A (LDHA), sune mafi inganci ingantattun magungunan biomarkers, wanda ke nuna cewa karuwa a cikin AD CSF shine jima'i mai girma. Tasoshin jinin mu da kuma ginshiƙan glial suma sun ƙunshi alamomi da yawa da ke da hannu a cikin metabolism na abubuwan da ke da iskar oxygen. Wadannan binciken sun yi daidai da mahimmin rawar da tsarin rayuwa ke takawa a cikin kwakwalwa gaba daya, ba kawai don saduwa da buƙatun makamashi mai ƙarfi na neurons ba, har ma don saduwa da buƙatun makamashi na astrocytes da sauran ƙwayoyin glial (17, 48). Sakamakonmu yana goyan bayan ƙarar shaidar cewa canje-canje a cikin yuwuwar redox da katsewar hanyoyin makamashi na iya zama babban hanyar haɗin kai tsakanin manyan hanyoyin aiwatarwa da ke cikin pathogenesis na AD, gami da rikicewar mitochondrial, kumburin tsaka-tsaki na glial, da lalacewar Vascular (49). Bugu da ƙari, masu samar da ruwa na cerebrospinal na rayuwa suna dauke da adadi mai yawa na sunadaran sunadaran masu arziki a tsakanin ikonmu da kuma AD-like AsymAD subgroups, suna ba da shawarar cewa rushewar waɗannan hanyoyin makamashi da redox na iya zama mahimmanci a cikin matakin farko na cutar.
Daban-daban nau'ikan kwakwalwa da yanayin yanayin ruwan cerebrospinal da muka lura kuma suna da tasirin ilimin halitta masu ban sha'awa. Synapses da metabolomes masu arziki a cikin neurons suna nuna raguwar matakan a cikin kwakwalwar AD da karuwar yawan ruwa a cikin cerebrospinal. Ganin cewa neurons suna da wadata a cikin mitochondria masu samar da makamashi a synapses don samar da makamashi don yawan siginar su na musamman (50), ana sa ran kamanni na bayanan bayanan waɗannan ƙungiyoyin neuron guda biyu. Asarar neurons da extrusion na sel masu lalacewa na iya yin bayanin waɗannan kwakwalwar kwakwalwa da yanayin panel na CSF a cikin cututtuka na baya, amma ba za su iya bayyana canje-canje na farko da muke lura da su ba (13). Ɗaya daga cikin bayanin da zai yiwu ga waɗannan binciken a farkon cutar asymptomatic shine pruning synaptic mara kyau. Sabbin shaida a cikin ƙirar linzamin kwamfuta suna nuna cewa ana iya kunna phagocytosis mai tsaka-tsaki na microglia a cikin AD kuma ya haifar da asarar synapse na farko a cikin kwakwalwa (51). Wannan kayan aikin synaptic da aka jefar na iya tarawa a cikin CSF, wanda shine dalilin da ya sa muke lura da karuwar CSF a cikin sashin neuron. Tsarewar synaptic mai tsaka-tsaki na rigakafi na iya yin bayanin wani bangare na karuwa a cikin sunadaran glial da muke gani a cikin kwakwalwa da ruwan cerebrospinal a cikin tsarin cutar. Baya ga pruning synaptic, gabaɗayan rashin daidaituwa a cikin hanyar exocytic na iya haifar da kwakwalwa daban-daban da maganganun CSF na alamomin neuronal. Yawancin karatu sun nuna cewa abun ciki na exosomes a cikin pathogenesis na kwakwalwar AD ya canza (52). Hanya ta waje kuma tana cikin haɓakar Aβ (53, 54). Yana da kyau a lura cewa ƙunshewar ɓoyewar exosomal na iya rage cututtukan cututtukan AD a cikin nau'ikan linzamin kwamfuta na AD transgenic (55).
A lokaci guda, sunadaran da ke cikin rukunin jijiyoyin jini sun nuna matsakaicin haɓaka a cikin kwakwalwar AD, amma ya ragu sosai a cikin CSF. Rashin aiki na jini-kwakwalwa (BBB) na iya yin bayanin waɗannan binciken. Yawancin binciken ɗan adam na bayan mutuwa sun nuna raunin BBB a AD (56, 57). Wadannan binciken sun tabbatar da wasu ayyuka marasa kyau da ke kewaye da wannan shingen shinge na sel na endothelial, gami da zubar jini na kwakwalwa da tarin jijiyoyin jini na sunadaran da ke haifar da jini (57). Wannan zai iya ba da bayani mai sauƙi don haɓakar sunadaran jijiyoyi a cikin kwakwalwa, amma ba zai iya yin cikakken bayani game da raguwar waɗannan sunadarai guda ɗaya a cikin ruwa na cerebrospinal ba. Ɗaya daga cikin yuwuwar ita ce tsarin kulawa na tsakiya yana ware waɗannan kwayoyin halitta don magance matsalar ƙara yawan kumburi da damuwa na oxidative. Ragewar wasu sunadaran CSF mafi tsanani a cikin wannan kwamiti, musamman ma waɗanda ke cikin tsarin lipoprotein, yana da alaƙa da hana matakan cutarwa na kumburi da tsarin neuroprotective na nau'in oxygen mai amsawa. Wannan gaskiya ne ga Paroxonase 1 (PON1), enzyme mai ɗaure lipoprotein da ke da alhakin rage matakan damuwa na oxidative a cikin wurare dabam dabam (58, 59). Alpha-1-microglobulin/bikunin precursor (AMBP) wata alama ce mai mahimmanci da aka tsara ta ƙungiyar jijiyoyin jini. Ita ce mafarin mai jigilar lipid bikunin, wanda kuma ke da hannu wajen hana kumburi da Kariyar jijiyoyin jiki (60, 61).
Duk da hasashe masu ban sha'awa iri-iri, rashin iya gano hanyoyin cututtukan ƙwayoyin cuta kai tsaye sanannen iyakance ne na bincike-binciken ƙwayoyin cuta. Sabili da haka, ƙarin bincike ya zama dole don amintacce da ƙayyadaddun hanyoyin da ke bayan waɗannan bangarorin biomarker. Domin matsawa zuwa ci gaban nazarin asibiti na tushen MS, jagorar gaba kuma tana buƙatar amfani da hanyoyin ƙididdigewa da aka yi niyya don tabbatar da babban sikelin biomarker, kamar zaɓi ko saka idanu na daidaitaccen amsa (62). Kwanan nan mun yi amfani da saka idanu na daidaitawa (63) don inganta yawancin canje-canjen furotin CSF da aka kwatanta a nan. An ƙididdige maƙasudin kwamitin fifiko da yawa tare da mahimmin daidaito, gami da YWHAZ, ALDOA, da SMOC1, waɗanda ke taswirar synapse ɗinmu, metabolism, da bangarorin kumburi, bi da bi (63). Samun Bayanai mai zaman kansa (DIA) da sauran dabarun tushen MS na iya zama da amfani don tabbatar da manufa. Bud et al. (64) Kwanan nan an nuna cewa akwai gagarumin cikowa tsakanin masu nazarin halittu na AD da aka gano a cikin saitin bayanan binciken mu na CSF da saitin bayanan DIA-MS mai zaman kansa, wanda ya ƙunshi kusan samfuran CSF 200 daga ƙungiyoyin Turai daban-daban guda uku. Waɗannan karatun na baya-bayan nan suna goyan bayan yuwuwar bangarorin mu don canzawa zuwa gano tushen MS abin dogaro. Maganin rigakafi na gargajiya da gano tushen aptamer shima yana da mahimmanci don haɓaka ci gaban maɓalli na AD biomarkers. Saboda ƙarancin wadatar CSF, yana da wahala a gano waɗannan alamomin halittu ta amfani da manyan hanyoyin MS. NEFL da NRGN su ne misalai guda biyu na ƙananan ƙananan ƙwayoyin cuta na CSF, waɗanda aka yi taswira zuwa kwamitin a cikin cikakken bincikenmu, amma ba za a iya dogara da su ta amfani da dabarun MS ɗinmu ɗaya ba. Dabarun da aka yi niyya bisa ga ƙwayoyin rigakafi da yawa, kamar PEA, na iya haɓaka canjin asibiti na waɗannan alamomin.
Gabaɗaya, wannan binciken yana ba da wata hanyar kariya ta musamman don ganowa da kuma tabbatar da masu amfani da halittu na CSF AD bisa tsarin daban-daban. Haɓaka waɗannan bangarorin masu alama a cikin ƙarin ƙungiyoyin AD da dandamali na MS na iya tabbatar da alƙawarin haɓaka haɗarin AD da jiyya. Nazarin da ke kimanta matakin tsayin daka na waɗannan bangarori na tsawon lokaci suma suna da mahimmanci don tantance waɗanne haɗakar alamomin da suka fi ƙayyadaddun haɗarin kamuwa da cuta na farko da canje-canje a cikin tsananin cutar.
Sai dai samfuran 3 da CSF ta kwafa, duk samfuran CSF da aka yi amfani da su a cikin wannan binciken an tattara su a ƙarƙashin kulawar Emory ADRC ko cibiyoyin bincike masu alaƙa. An yi amfani da jimillar nau'ikan samfuran Emory CSF guda huɗu a cikin waɗannan nazarin ilimin halittu. An gano ƙungiyar CSF tana ɗauke da samfurori daga kulawar lafiya 20 da marasa lafiya 20 AD. Kwafin CSF 1 ya haɗa da samfurori daga kulawar lafiya 32, daidaikun AsymAD 31, da daidaikun 33 AD. Kwafin CSF 2 ya ƙunshi sarrafawa 147 da samfuran AD 150. Ƙungiyoyin 4 na CSF masu cutar da yawa sun haɗa da sarrafawa 18, 17 AD, 19 ALS, 13 PD, da samfuran FTD 11. Dangane da yarjejeniyar da Hukumar Binciken Cibiyoyin Jami'ar Emory ta amince da ita, duk mahalarta binciken Emory sun sami izini na sanarwa. Bisa ga 2014 National Institute of Aging Best Practice Guidelines for Alzheimer's Centers (https://alz.washington.edu/BiospecimenTaskForce.html), an tattara ruwan cerebrospinal kuma an adana shi ta hanyar huda lumbar. Sarrafa da AsymAD da AD marasa lafiya sun sami daidaitattun ƙima a Emory Cognitive Neurology Clinic ko Goizueta ADRC. INNO-BIA AlzBio3 Luminex ya gwada samfuran ruwan su na cerebrospinal don ELISA Aβ1-42, jimlar tau da p-tau bincike (65). Ana amfani da ƙimar ELISA don tallafawa rarrabuwa na batutuwa dangane da ƙayyadaddun ka'idodin yanke biomarker AD (66, 67). Asalin bayanan alƙaluman jama'a da bincike don sauran cututtukan CSF (FTD, ALS, da PD) ana samun su daga Emory ADRC ko cibiyoyin bincike masu alaƙa. Za'a iya samun takamaiman metadata na waɗannan shari'o'in Emory CSF a cikin Tebur S1A. An buga halayen ƙungiyar 3 na Swiss CSF maimaitawa a baya (45).
CSF ta sami samfurin. Don ƙara zurfin binciken mu na saitin bayanan CSF, an yi amfani da rigakafi na furotin masu yawa kafin trypsinization. A takaice, 130 μl na CSF daga samfuran CSF guda 40 da daidaitaccen girma (130 μl) na Babban Zaɓin Babban Zaɓuɓɓuka Top14 Abundance Protein Depletion Resin (Thermo Fisher Scientific, A36372) an sanya shi a cikin juzu'in juzu'i (Thermo Fisher Scientific, A89868) a cikin ɗaki. zafin jiki Incubate). Bayan yin juyawa na mintina 15, sanya samfurin a 1000g na minti 2. An yi amfani da na'urar tacewa ta 3K ultracentrifugal (Millipore, UFC500396) don mayar da hankali samfurin datti ta hanyar sakawa a 14,000g na mintuna 30. Tsarma duk samfurin samfurin zuwa 75 μl tare da phosphate buffered saline. An kimanta yawan furotin ta hanyar bicinchoninic acid (BCA) bisa ga ka'idar masana'anta (Thermo Fisher Scientific). CSF mai rauni (60 μl) daga duk samfuran 40 an narkar da shi tare da lysyl endopeptidase (LysC) da trypsin. A takaice, samfurin da aka rage da kuma alkylated da 1.2 μl 0.5 M tris-2 (-carboxyethyl) -phosphine da 3 μl 0.8 M chloroacetamide a 90 ° C na minti 10, sa'an nan sonicated a cikin wani ruwa wanka na 15 minutes. An narkar da samfurin tare da 193 μl 8 M urea buffer [8 M urea da 100 mM NaHPO4 (pH 8.5)] zuwa taro na ƙarshe na 6M urea. Ana amfani da LysC (4.5 μg; Wako) don narkewar dare a zafin jiki. Sa'an nan kuma an diluted samfurin zuwa 1 M urea tare da 50 mM ammonium bicarbonate (ABC) (68). Ƙara daidai adadin (4.5 μg) na trypsin (Promega), sa'an nan kuma kunsa samfurin na 12 hours. Acidify maganin peptide da aka narkar da shi zuwa matakin ƙarshe na 1% formic acid (FA) da 0.1% trifluoroacetic acid (TFA) (66), sannan a desalt tare da 50 MG Sep-Pak C18 shafi (Ruwa) kamar yadda aka bayyana a sama (25) . An cire peptide a cikin 1 ml na 50% acetonitrile (ACN). Don daidaita ƙididdiga na furotin a cikin batches (25), 100 μl aliquots daga duk samfuran 40 CSF an haɗa su don samar da samfurin gauraye, wanda aka raba zuwa samfuran ciki biyar na duniya (GIS) (48). Duk samfuran ɗaiɗaikun ɗaiɗaikun ɗaiɗaikun ɗaiɗai da ƙa'idodin haɗe an bushe su ta hanyar injin mai saurin sauri (Labconco).
CSF tana kwafin samfurin. Dayon da abokan aiki a baya sun bayyana raunin rigakafi da narkewar samfuran 3 na CSF (45, 46). Ragowar samfuran kwafin ba su kasance daidaiku ba. Narke waɗannan samfuran da ba a cire su a cikin trypsin kamar yadda aka bayyana a baya (17). Don kowane bincike mai maimaitawa, 120 μl aliquots na peptide eluted daga kowane samfurin an haɗa su tare kuma an raba su zuwa daidaitattun ƙididdiga masu girma da za a yi amfani da su azaman ma'aunin ciki na duniya mai alamar TMT (48). Duk samfuran ɗaiɗaikun ɗaiɗaikun ɗaiɗaikun ɗaiɗai da ƙa'idodin haɗe an bushe su ta hanyar injin mai saurin sauri (Labconco). Don haɓaka siginar ƙarancin furotin CSF mai ƙarancin yawa, ta hanyar haɗa 125 μl daga kowane samfurin, an shirya samfurin “inganta” don kowane bincike na kwafi [watau samfurin nazarin halittu wanda ke kwaikwayon samfurin bincike, amma adadin da ake samu shine. ya fi girma (37, 69)] haɗe cikin samfurin CSF gauraye (17). Sa'an nan kuma an cire samfurin gauraye ta hanyar amfani da 12 ml na Babban Zaɓan Babban Zaɓuɓɓuka na Top14 Abundance Protein Removal Resin (Thermo Fisher Scientific, A36372), wanda aka narkar da shi kamar yadda aka bayyana a sama, kuma an haɗa shi a cikin lakabin TMT da yawa na gaba.
Lokacin aikawa: Agusta-27-2021