Adireshin da ke akwai a yanzu: Cologne 50931, Jamus, Cologne Excellence Cluster Research on Cellular Response to Stress Response in Cellular Diseases Related Tsofaffi (CECAD).
Ana ɗaukar cewa rashin daidaituwar cututtukan mitochondrial ba zai iya canzawa ba saboda ƙarfin metabolism na neurons yana da iyaka, amma tasirin rashin daidaituwar mitochondrial akan ikon mallakar ƙwayoyin halitta na metabolism na neuronal a cikin jiki ba a fahimta sosai. A nan, mun gabatar da furotin na musamman na ƙwayoyin Purkinje tare da ƙarancin OXPHOS mai ci gaba wanda ya haifar da raguwar haɗin mitochondrial. Mun gano cewa rashin daidaituwar mitochondrial ya haifar da babban canji a fannin proteomics, wanda a ƙarshe ya haifar da kunna shirye-shiryen metabolism na daidai kafin mutuwar ƙwayoyin halitta. Ba zato ba tsammani, mun gano bayyanar haifar da pyruvate carboxylase (PCx) da sauran enzymes masu hana tsufa waɗanda ke ƙara tsaka-tsakin zagayowar TCA. Hana PCx ya ƙara tsananta damuwa da neurodegeneration, yana nuna cewa atherosclerosis yana da tasirin kariya a cikin ƙwayoyin halitta waɗanda ba su da OXPHOS. Maido da haɗin mitochondrial a cikin ƙwayoyin halitta da suka lalace gaba ɗaya yana juya waɗannan halayen metabolism gaba ɗaya, don haka yana hana mutuwar ƙwayoyin halitta. Bincikenmu ya gano hanyoyin da ba a sani ba a baya waɗanda ke ba da juriya ga rashin daidaituwar mitochondrial kuma yana nuna cewa ana iya mayar da neurodegeneration ko da a ƙarshen matakan cutar.
Babban rawar da mitochondria ke takawa wajen kula da tsarin samar da makamashin jijiyoyi yana da matuƙar muhimmanci ta hanyar yawan alamomin jijiyoyi da ke da alaƙa da cututtukan mitochondrial na ɗan adam. Yawancin waɗannan cututtukan suna faruwa ne sakamakon maye gurbi na kwayoyin halitta waɗanda ke daidaita bayyanar kwayoyin halittar mitochondrial (1, 2) ko lalata kwayoyin halitta da ke da alaƙa da yanayin mitochondrial, wanda ke shafar kwanciyar hankalin DNA na mitochondrial (mtDNA) kai tsaye (3, 4). Aiki a cikin samfuran dabbobi ya nuna cewa a martanin rashin aikin mitochondrial a cikin kyallen da ke kewaye, hanyoyin rayuwa masu ra'ayin mazan jiya (5-7) za a iya kunna su, wanda ke ba da mahimman bayanai don fahimtar tushen waɗannan cututtuka masu rikitarwa. Sabanin haka, fahimtarmu game da canje-canjen rayuwa na takamaiman nau'ikan ƙwayoyin halitta da gazawar gabaɗaya na samar da adenosine triphosphate na kwakwalwa (ATP) abu ne mai mahimmanci (8), yana mai jaddada buƙatar gano manufofin magani waɗanda za a iya amfani da su don hana ko hana cututtuka. Hana neurodegeneration (9). Rashin bayanai shine gaskiyar cewa ana ɗaukar ƙwayoyin jijiyoyi a matsayin suna da ƙarancin sassaucin rayuwa idan aka kwatanta da nau'ikan ƙwayoyin halitta na kyallen da ke kewaye (10). Ganin cewa waɗannan ƙwayoyin suna taka muhimmiyar rawa wajen daidaita samar da metabolites ga ƙwayoyin jijiyoyi don haɓaka watsawar synaptic da kuma mayar da martani ga yanayin rauni da cututtuka, ikon daidaita metabolism na ƙwayoyin halitta zuwa ga yanayin ƙalubalen ƙwayoyin kwakwalwa kusan yana iyakance ga ƙwayoyin glial (11-14). Bugu da ƙari, bambancin ƙwayoyin halitta na ƙwayoyin kwakwalwa galibi yana hana nazarin canje-canjen metabolism da ke faruwa a cikin takamaiman ƙananan ƙungiyoyin neuronal. Sakamakon haka, ba a san komai game da ainihin sakamakon ƙwayoyin halitta da metabolism na rashin aikin mitochondrial a cikin ƙwayoyin jijiyoyi ba.
Domin fahimtar sakamakon metabolism na rashin aikin mitochondrial, mun ware ƙwayoyin halittar Purkinje (PNs) a matakai daban-daban na neurodegeneration wanda ya haifar da lalata haɗin membrane na waje na mitochondrial (Mfn2). Duk da cewa maye gurbi na Mfn2 a cikin mutane yana da alaƙa da wani nau'in neuropathy na jijiyoyi na gado wanda aka sani da Charcot-Marie-Tooth type 2A (15), lalata yanayin Mfn2 a cikin beraye wata hanya ce da aka sani ta haifar da oxidation Phosphorylation (OXPHOS). Nau'ikan ƙananan ƙwayoyin halitta daban-daban (16-19) da kuma yanayin neurodegenerative da ya haifar suna tare da alamun ci gaba na jijiyoyi, kamar rikicewar motsi (18, 19) ko cerebellar ataxia (16). Ta hanyar amfani da haɗin proteomics, metabolomics, hoto, da hanyoyin virological marasa lakabi, mun nuna cewa ci gaba da neurodegeneration yana haifar da pyruvate carboxylase (PCx) da sauran abubuwan da ke da hannu a cikin arteriosclerosis na PNs a cikin vivo Bayyanar enzymes. Domin tabbatar da muhimmancin wannan binciken, mun rage girman bayyanar PCx ​​a cikin PNs marasa Mfn2, kuma mun gano cewa wannan aikin ya ƙara tsananta damuwa ta oxidative da kuma hanzarta neurodegeneration, don haka ya tabbatar da cewa azoospermia yana ba da mutuwar ƙwayoyin halitta daidaitawar metabolism. Bayyanar MFN2 mai tsanani na iya ceton gaba ɗaya lalacewar PN tare da ƙarancin OXPHOS mai tsanani, yawan amfani da DNA na mitochondrial, da kuma karyewar hanyar sadarwa ta mitochondrial, wanda ya ƙara jaddada cewa wannan nau'in neurodegeneration na iya murmurewa a matakin ci gaba na cuta kafin mutuwar ƙwayoyin halitta.
Domin mu hango mitochondria a cikin PNs na Mfn2 knockout, mun yi amfani da nau'in linzamin kwamfuta wanda ke ba da damar mitochondria mai dogaro da Cre-dependent don kai hari ga furotin mai launin rawaya (YFP) (mtYFP) (20) Bayyanar Cre kuma mun duba yanayin mitochondrial a cikin vivo. Mun gano cewa lalata kwayar halittar Mfn2 a cikin PNs zai haifar da rarrabuwa a hankali na hanyar sadarwar mitochondrial (Hoto na S1A), kuma an sami canjin farko a makonni 3 na haihuwa. Sabanin haka, mummunan lalacewar layin kwayar halittar PN, kamar yadda aka nuna ta hanyar asarar Calbindin immunostaining, bai fara ba har sai makonni 12 na haihuwa (Hoto na 1, A da B). Rashin daidaiton lokaci tsakanin canje-canje na farko a cikin yanayin mitochondrial da farkon mutuwar jijiyoyi ya sa mu bincika canje-canjen rayuwa da rashin aikin mitochondrial ya haifar kafin mutuwar tantanin halitta. Mun ƙirƙiro wata dabarar tantance ƙwayoyin halitta (FACS) da aka kunna ta hanyar hasken rana (YFP+) don ware PN mai bayyana YFP (YFP+) (Hoto na 1C), da kuma a cikin beraye masu sarrafawa (Mfn2 + / loxP :: mtYFP loxP-stop-loxP: : L7-cre), daga nan aka kira shi CTRL (Hoto na S1B). Inganta dabarun gating bisa ga ƙarfin siginar YFP yana ba mu damar tsarkake jikin YFP+ (YFPhigh) na PNs daga waɗanda ba PN ba (YFPneg) (Hoto na S1B) ko gutsuttsuran axon/dendritic masu haske (YFPlow; Hoto na S1D, hagu), wanda aka tabbatar ta hanyar na'urar hangen nesa ta confocal (Hoto na S1D, dama). Domin tabbatar da asalin mutanen da aka ware, mun gudanar da proteomics na LFQ sannan muka gudanar da babban binciken sassan, kuma muka gano cewa akwai rabuwa bayyananne tsakanin ƙwayoyin YFPhigh da YFPneg (Hoto na S1C). Kwayoyin YFP masu girma sun nuna wadatar da aka samu daga alamun PN da aka sani (misali Calb1, Pcp2, Grid2 da Itpr3) (21, 22), amma babu wadatar sunadarai da aka fi bayyanawa a cikin jijiyoyi ko wasu nau'ikan tantanin halitta (Hoto na 1D)). Kwatanta tsakanin samfuran a cikin manyan ƙwayoyin YFP da aka tattara a cikin gwaje-gwaje masu zaman kansu ya nuna ma'aunin haɗin kai > 0.9, yana nuna kyakkyawan sake haifuwa tsakanin kwafi na halittu (Hoto na S1E). A taƙaice, waɗannan bayanan sun tabbatar da shirinmu na ware PN mai yiwuwa cikin gaggawa da takamaiman. Saboda tsarin direban L7-cre da aka yi amfani da shi yana haifar da sake haɗuwa da mosaic a makon farko bayan bayarwa (23), mun fara cire beraye daga CTRL da yanayin (Mfn2 loxP / loxP :: mtYFP loxP-stop-loxP :: L7-cre) Tattara ƙwayoyin jijiyoyi. Bayan an kammala sake haɗawa, ana kiransa Mfn2cKO yana da makonni 4 na haihuwa. A matsayin ƙarshen lokacin, mun zaɓi makonni 8 na shekaru lokacin da layin PN ya kasance cikakke duk da rarrabuwar mitochondrial da aka bayyana (Hoto na 1B da Hoto na S1A). A jimilla, mun ƙididdige jimlar sunadaran 3013, waɗanda kusan 22% sun dogara ne akan bayanin MitoCarta 2.0 bisa ga furotin na mitochondrial a matsayin mitochondria (Hoto na 1E) (Hoto na 1E) (24). Binciken bambancin bayyanar kwayoyin halitta da aka yi a mako na 8 ya nuna cewa kashi 10.5% ne kawai na dukkan sunadaran suka sami manyan canje-canje (Hoto na 1F da Hoto na S1F), wanda daga cikinsu an rage yawan sunadaran kuma an ƙara yawan sunadaran 120 (Hoto na 1F). Ya kamata a lura cewa "binciken hanyoyin kirkire-kirkire" na wannan saitin bayanai ya nuna cewa kwayoyin halittar da aka bayyana daban-daban galibi suna cikin takamaiman tsarin hanyoyin rayuwa (Hoto na 1G). Abin sha'awa, kodayake raguwar hanyoyin da suka shafi OXPHOS da siginar calcium sun tabbatar da haifar da rashin aiki na mitochondrial a cikin PNs marasa haɗin kai, sauran nau'ikan da galibi suka shafi metabolism na amino acid suna da ƙarfi sosai, wanda ya yi daidai da metabolism ɗin da ke faruwa a cikin PNs na mitochondrial. Rewiring yana da daidaito. rashin aiki.
(A) Hotunan da aka ɗauka a matsayin confocal na sassan cerebellar na berayen CTRL da Mfn2cKO waɗanda ke nuna asarar ci gaba na PNs (calbindin, launin toka); an yi wa ƙwayoyin halittar da DAPI. (B) Ƙididdigewa na (A) (binciken bambanci na hanya ɗaya, ***P<0.001; n = da'ira 4 zuwa 6 daga berayen uku). (C) Aikin gwaji. (D) Rarraba taswirar zafi na alamomi na musamman ga Purkinje (sama) da sauran nau'ikan tantanin halitta (tsakiya). (E) Zane na Venn wanda ke nuna adadin sunadaran mitochondrial da aka gano a cikin PN da aka ware. (F) Tsarin aman wuta na sunadaran da aka bayyana daban-daban a cikin ƙwayoyin Mfn2cKO a makonni 8 (ƙimar yankewa mai mahimmanci na 1.3). (G) Binciken hanyar kerawa yana nuna mahimman hanyoyi guda biyar mafi mahimmanci na haɓaka (ja) da ragewa (shuɗi) a cikin PN na Mfn2cKO da aka ware a matsayin makonni 8. An nuna matsakaicin matakin bayyana kowane furotin da aka gano. Taswirar zafi mai launin toka: ƙimar P da aka gyara. ns, ba mahimmanci ba.
Bayanan Proteomics sun nuna cewa bayyanar furotin na hadaddun I, III, da IV a hankali ya ragu. Complexes I, III, da IV duk sun ƙunshi ƙananan sassan mtDNA masu mahimmanci, yayin da complex II, wanda aka yi masa code na nukiliya kawai, ba shi da wani tasiri (Hoto na 2A da Hoto na S2A). . Daidai da sakamakon proteomics, immunohistochemistry na sassan ƙwayoyin cerebellar ya nuna cewa matakin ƙananan sassan MTCO1 (mitochondrial cytochrome C oxidase subunit 1) na hadaddun IV a cikin PN ya ragu a hankali (Hoto na 2B). An rage girman ƙaramin sashin mtDNA mai code na Mtatp8 sosai (Hoto na S2A), yayin da matakin tsayayyen matakin sashin ATP synthase mai encoded na nukiliya bai canza ba, wanda ya yi daidai da sanannen rukunin ATP synthase mai karko F1 lokacin da bayyanar mtDNA ta tabbata. Samuwar ta kasance daidai. Katsewa (7). Kimanta matakin mtDNA a cikin PNs ɗin Mfn2cKO da aka tsara ta hanyar amsawar sarkar polymerase ta ainihin lokaci (qPCR) ya tabbatar da raguwar a hankali a cikin lambar kwafin mtDNA. Idan aka kwatanta da ƙungiyar kulawa, a makonni 8 na haihuwa, kusan kashi 20% ne kawai na matakin mtDNA An riƙe (Hoto na 2C). Daidai da waɗannan sakamakon, an yi amfani da tabon microscopy na confocal na Mfn2cKO PNs don gano DNA, yana nuna yawan amfani da nucleotides na mitochondrial wanda ya dogara da lokaci (Hoto na 2D). Mun gano cewa wasu 'yan takara ne kawai da ke da hannu a cikin lalacewar furotin na mitochondrial da amsawar damuwa aka ƙara daidaita su, gami da Lonp1, Afg3l2 da Clpx, da abubuwan haɗuwa masu rikitarwa na OXPHOS. Ba a gano manyan canje-canje a cikin matakan furotin da ke da hannu a cikin apoptosis ba (Hoto na S2B). Hakazalika, mun gano cewa tashoshin mitochondria da endoplasmic reticulum da ke da hannu a cikin jigilar calcium suna da ƙananan canje-canje kawai (Hoto na S2C). Bugu da ƙari, kimantawar sunadaran da suka shafi autophagy ba ta sami wani muhimmin canji ba, wanda ya yi daidai da yadda ake iya ganin autophagosomes da aka gani a cikin jiki ta hanyar immunohistochemistry da electron microscopy (Hoto na S3). Duk da haka, rashin aikin OXPHOS mai ci gaba a cikin PNs yana tare da canje-canje a bayyane na mitochondrial. Ana iya ganin tarin mitochondrial a cikin jikin tantanin halitta da bishiyoyin dendritic na Mfn2cKO PNs masu makonni 5 da 8, kuma tsarin membrane na ciki ya sami manyan canje-canje (Hoto na S4, A da B). Daidai da waɗannan canje-canje na ultrastructural da raguwa mai yawa a cikin mtDNA, nazarin yanka cerebellar na kwakwalwa mai tsanani tare da tetramethylrhodamine methyl ester (TMRM) ya nuna cewa yuwuwar membrane na mitochondrial a cikin Mfn2cKO PNs ya ragu sosai (Hoto na S4C).
(A) Binciken lokaci na matakin bayyanar hadaddun OXPHOS. Yi la'akari da sunadaran da ke da P<0.05 kawai a makonni 8 (ANOVA mai hanyoyi biyu). Layin da aka nuna: Babu daidaitawa idan aka kwatanta da CTRL. (B) Hagu: Misali na sashin cerebellar mai lakabi da antibody anti-MTCO1 (mashigin sikelin, 20 μm). Yankin da jikin ƙwayoyin Purkinje ke mamaye ya rufe da rawaya. Dama: Adadin matakan MTCO1 (binciken bambanci na hanya ɗaya; n = ƙwayoyin 7 zuwa 20 da aka bincika daga beraye uku). (C) Binciken qPCR na lambar kwafin mtDNA a cikin PN da aka tsara (binciken bambanci na hanya ɗaya; n = beraye 3 zuwa 7). (D) Hagu: Misali na yanki na cerebellar mai lakabi da antibody anti-DNA (mashigin sikelin, 20 μm). Yankin da jikin ƙwayoyin Purkinje ke mamaye ya rufe da rawaya. Dama: Adadin raunuka na mtDNA (binciken bambanci na hanya ɗaya; n = ƙwayoyin 5 zuwa 9 daga beraye uku). (E) Misali na sashin kwakwalwa mai tsanani wanda ke nuna ƙwayoyin mitoYFP + Purkinje (kibiya) a cikin rikodin maƙallin faci na tantanin halitta gaba ɗaya. (F) Ƙididdige lanƙwasa na IV. (G) Rikodin wakilci na allurar depolarizing current a cikin ƙwayoyin CTRL da Mfn2cKO Purkinje. Babban alama: Bugawa ta farko da ta haifar da AP. Alamar ƙasa: Matsakaicin mitar AP. (H) Ƙididdige shigarwar postsynaptic spontant (sPSPs). An nuna alamar rikodi mai wakiltar da rabon zuƙowa a cikin (I). Binciken bambanci na hanya ɗaya da aka bincika n = 5 zuwa 20 ƙwayoyin daga beraye uku. An bayyana bayanai a matsayin matsakaicin ± SEM; *P<0.05; **P<0.01; ***P<0.001. (J) Alamomin AP na bazata da aka yi rikodin su ta amfani da yanayin maƙallin faci mai huda. Alamun sama: Matsakaicin mitar AP. Alamun ƙasa: zuƙowa na AP guda ɗaya. (K) Ƙididdige matsakaicin mitar AP bisa ga (J). Gwajin Mann-Whitney; an yi nazarin n = ƙwayoyin 5 daga beraye huɗu. Ana bayyana bayanai a matsayin matsakaicin ± SEM; ba mahimmanci ba ne.
An gano lalacewar OXPHOS a fili a cikin Mfn2cKO PN mai makonni 8, wanda ke nuna cewa aikin ilimin halittar jijiyoyi yana da matukar rashin daidaituwa. Saboda haka, mun yi nazarin halayen lantarki marasa aiki na jijiyoyi marasa OXPHOS a makonni 4 zuwa 5 da makonni 7 zuwa 8 ta hanyar yin rikodin manne na faci na tantanin halitta gaba ɗaya a cikin yanka na cerebellar mai tsanani (Hoto na 2E). Ba zato ba tsammani, matsakaicin ƙarfin membrane na hutawa da juriyar shigarwa na jijiyoyi na Mfn2cKO sun yi kama da na sarrafawa, kodayake akwai bambance-bambance masu zurfi tsakanin ƙwayoyin halitta (Tebur na 1). Hakazalika, a makonni 4 zuwa 5 na haihuwa, ba a sami manyan canje-canje a cikin dangantakar wutar lantarki ta yanzu (layin IV) ba (Hoto na 2F). Duk da haka, babu jijiyoyi na Mfn2cKO masu makonni 7 zuwa 8 da suka tsira daga tsarin IV (matakin hyperpolarization), wanda ke nuna cewa akwai bayyanannen jin daɗin yuwuwar hyperpolarization a wannan matakin ƙarshe. Sabanin haka, a cikin ƙwayoyin Mfn2cKO, kwararar depolarizing waɗanda ke haifar da fitowar ƙarfin aiki mai maimaitawa (AP) an yarda da su sosai, wanda ke nuna cewa tsarin fitarwa gabaɗaya ba su da bambanci sosai da na ƙwayoyin sarrafawa masu makonni 8 (Tebur 1 da Hoto na 2G). Hakazalika, mita da girman kwararar postsynaptic mai saurin canzawa (sPSCs) sun yi daidai da na ƙungiyar kulawa, kuma yawan abubuwan da suka faru ya ƙaru daga makonni 4 zuwa makonni 5 zuwa makonni 7 zuwa makonni 8 tare da ƙaruwa iri ɗaya (Hoto na 2, H da I). ​​Lokacin balaga na synaptic a cikin PNs (25). An sami irin wannan sakamako bayan facin PNs da aka huda. Wannan tsari yana hana yiwuwar diyya ga lahani na ATP na tantanin halitta, kamar yadda zai iya faruwa a cikin rikodin manne na facin tantanin halitta gaba ɗaya. Musamman ma, ƙarfin membrane na hutawa da mitar harbi na bazata na ƙwayoyin Mfn2cKO ba su shafi ba (Hoto na 2, J da K). A taƙaice, waɗannan sakamakon sun nuna cewa PNs waɗanda ke da matsalar OXPHOS a bayyane za su iya jure wa yanayin fitar da iska mai yawan gaske, wanda ke nuna cewa akwai hanyar diyya da ke ba su damar kiyaye martanin lantarki na kusan al'ada.
An bayyana bayanai a matsayin matsakaicin ± SEM (binciken bambanci na hanya ɗaya, gwajin kwatantawa da yawa na Holm-Sidak; *P<0.05). An nuna lambar naúrar ta hanyar maƙallan baka.
Mun fara bincike ko wani rukuni a cikin bayanan proteomics (Hoto na 1G) ya haɗa da hanyoyin da za su iya magance matsalar ƙarancin OXPHOS mai tsanani, don haka ya bayyana dalilin da yasa PN da abin ya shafa zai iya ci gaba da kusan al'ada electrophysiology (Hoto na 2, E zuwa K). . Binciken Proteomics ya nuna cewa enzymes da ke cikin catabolism na amino acid masu reshe (BCAA) an inganta su sosai (Hoto na 3A da Hoto na S5A), kuma samfurin ƙarshe na acetyl-CoA (CoA) ko succinyl CoA na iya ƙara tricarboxylates a cikin zagayowar arteriosclerosis Acid (TCA). Mun gano cewa abubuwan da ke cikin BCAA transaminase 1 (BCAT1) da BCAT2 duka sun ƙaru. Suna haɓaka matakin farko na catabolism na BCAA ta hanyar samar da glutamate daga α-ketoglutarate (26). Duk ƙananan sassan da suka ƙunshi hadaddun keto acid dehydrogenase (BCKD) mai reshe an daidaita su (haɗarin yana haɓaka rabuwar da ke faruwa da kuma wanda ba za a iya canzawa ba na kwarangwal ɗin carbon BCAA da ya haifar) (Hoto na 3A da Hoto na S5A). Duk da haka, babu wani canji a bayyane a cikin BCAA da kanta da aka samu a cikin PN da aka tsara, wanda wataƙila ya faru ne saboda ƙaruwar shan waɗannan amino acid masu mahimmanci ko amfani da wasu tushe (glucose ko lactic acid) don ƙara yawan zagayowar TCA (Hoto na S5B). PNs da ba su da OXPHOS sun kuma nuna ƙaruwar rushewar glutamine da ayyukan transamination a makonni 8 na haihuwa, wanda za a iya nuna shi ta hanyar haɓaka tsarin enzymes na mitochondrial glutaminase (GLS) da glutamine pyruvate transaminase 2 (GPT2) (Hoto na 3, A da C). Ya kamata a lura cewa haɓaka GLS yana iyakance ga isoform glutaminase C (GLS-GAC) mai haɗin gwiwa (canjin Mfn2cKO/CTRL yana kusan ninki 4.5, P = 0.05), kuma takamaiman haɓaka shi a cikin kyallen kansa na iya tallafawa makamashin mitochondrial. (27).
(A) Taswirar zafi tana nuna canjin ninki a matakin furotin don hanyar da aka ƙayyade a makonni 8. (B) Misali na yanki na cerebellar mai lakabi da anti-PCx antibody (ma'aunin sikelin, 20 μm). Kibiyar rawaya tana nuna jikin ƙwayar Purkinje. (C) Binciken bayyanar furotin na lokaci wanda aka gano a matsayin muhimmin ɗan takara don atherosclerosis (gwajin t da yawa, *FDR <5%; n = beraye 3-5). (D) A sama: Zane mai zane wanda ke nuna hanyoyi daban-daban na shigar da carbon mai lakabin da ke cikin [1-13C]pyruvate tracer (watau, ta hanyar PDH ko hanyar trans-arterial). Ƙasa: Taswirar violin tana nuna kashi na carbon mai lakabin guda ɗaya (M1) wanda aka canza zuwa aspartic acid, citric acid da malic acid bayan an yiwa yanka cerebellar mai tsanani lakabi da [1-13C]pyruvate (gwajin t-haɗi; ** P <0.01). (E) Cikakken nazarin tarihin lokaci na hanyar da aka nuna. Yi la'akari da sunadaran da ke da P<0.05 a makonni 8. Layi mai lanƙwasa: babu ƙimar daidaitawa (biyu nazarin bambance-bambance; * P <0.05; *** P <0.001). Ana bayyana bayanai a matsayin matsakaicin ± SEM.
A cikin bincikenmu, tasirin BCAA ya zama ɗaya daga cikin manyan hanyoyin haɓaka tsari. Wannan gaskiyar ta nuna cewa yawan iska da ke shiga cikin zagayowar TCA na iya canzawa a cikin PN ba tare da OXPHOS ba. Wannan na iya wakiltar babban nau'in sake haɗa hanyoyin metabolism na neuronal, wanda zai iya yin tasiri kai tsaye ga ilimin halittar jijiyoyi da rayuwa yayin kula da mummunan aikin OXPHOS. Dangane da wannan hasashe, mun gano cewa babban enzyme na anti-atherosclerotic PCx yana ƙaruwa (Mfn2cKO/CTRL yana canzawa kusan sau 1.5; Hoto na 3A), wanda ke haɓaka juyawar pyruvate zuwa oxaloacetate (28), wanda ake kyautata zaton yana cikin kyallen kwakwalwa. Furucin in an iyakance shi ga astrocytes (29, 30). Daidai da sakamakon proteomics, na'urar microscopy ta confocal ta nuna cewa bayyanar PCx ​​ta ƙaru musamman kuma sosai a cikin PNs marasa OXPHOS, yayin da amsawar PCx ​​galibi an iyakance shi ga ƙwayoyin glial Bergmann da ke kusa da su na sarrafawa (Hoto na 3B). Domin gwada yadda aka lura da ƙaruwar PCx ​​a cikin aiki, mun yi maganin yanka na cerebellar mai tsanani da [1-13C]pyruvate tracer. Lokacin da pyruvate dehydrogenase (PDH) ya yi oxidize da pyruvate, alamar isotope ɗinsa ta ɓace, Amma an haɗa ta cikin tsaka-tsakin zagayowar TCA lokacin da pyruvate ya metabolize ta hanyar halayen jijiyoyin jini (Hoto na 3D). Don tallafawa bayanan proteomics ɗinmu, mun lura da adadi mai yawa na alamomi daga wannan tracer a cikin aspartic acid na yanka Mfn2cKO, yayin da citric acid da malic acid suma suna da yanayin matsakaici, kodayake ba su da mahimmanci (Hoto na 3D).
A cikin ƙwayoyin dopamine na beraye na MitoPark waɗanda ke da matsalar mitochondrial wanda ƙwayoyin dopamine ke haifarwa musamman lalata kwayar halittar mitochondrial transcription factor A (Tfam) (Hoto na S6B), an kuma ƙara yawan bayyanar PCx ​​sosai (31), wanda ke nuna cewa acetone acid arteriosclerosis Ana daidaita faruwar cutar yayin rashin aikin neuronal OXPHOS a jiki. Ya kamata a lura cewa an gano cewa enzymes na musamman (32-34) waɗanda za a iya bayyana a cikin ƙwayoyin neurons waɗanda za a iya dangantawa da arteriosclerosis suna da ƙarfi sosai a cikin PNs marasa OXPHOS, kamar propionyl-CoA carboxylase (PCC-A), Malonyl-CoA yana canza propionyl-CoA zuwa succinyl-CoA da mitochondrial malic enzyme 3 (ME3), wanda babban aikinsa shine dawo da pyruvate daga malate (Hoto na 3, A da C) (33, 35). Bugu da ƙari, mun sami ƙaruwa mai yawa a cikin enzyme na Pdk3, wanda ke haifar da phosphorylation kuma ta haka yana kashe PDH (36), yayin da ba a gano wani canji a cikin enzyme na Pdp1 wanda ke kunna PDH ko hadaddun enzyme na PDH da kansa ba (Hoto na 3A). A koyaushe, a cikin Mern2cKO PNs, an inganta phosphorylation na sashin α1 subunit α (PDHE1α) na sashin pyruvate dehydrogenase E1 na hadaddun PDH a cikin Ser293 (wanda aka sani da hana ayyukan enzyme na PDH) (Hoto na S6C) (Hoto na S6C). Pyruvate ba shi da hanyar shiga jijiyoyin jini.
A ƙarshe, mun gano cewa babban hanyar serine da glycine biosynthesis, da zagayowar mitochondrial folate (1C) da kuma proline biosynthesis (Hoto na 1G da Hoto na S5C) duk an daidaita su sosai, a cewar rahotanni, yayin aikin kunnawa. Ana kunna kyallen da ke kewaye da su tare da rashin aikin mitochondrial (5-7). Binciken Confocal wanda ke goyan bayan waɗannan bayanan proteomics ya nuna cewa a cikin PN tare da ɓacewar OXPHOS, yanka cerebellar na berayen makonni 8 an yi musu serine hydroxymethyltransferase 2 (SHMT2), wani muhimmin enzyme na zagayowar folate na mitochondrial. Babban martanin garkuwar jiki (Hoto na S5D). A cikin yanka cerebellar guda 13 da aka haɗa da CU-glucose, gwaje-gwajen bin diddigin metabolism sun ƙara tabbatar da haɓaka tsarin serine da proline biosynthesis, yana nuna cewa kwararar carbon isoforms zuwa serine da proline ya ƙaru (Hoto na S5E). Tunda halayen da GLS da GPT2 ke haɓaka suna da alhakin haɗa glutamine daga glutamine da kuma transamination tsakanin glutamate da α-ketoglutarate, haɓaka su yana nuna cewa ƙwayoyin jijiyoyi marasa OXPHOS suna da ƙaruwar buƙatar glutamate. Wannan na iya nufin ci gaba da ƙaruwar biosynthesis na proline (Hoto na S5C). Sabanin waɗannan canje-canjen, wani bincike na proteomic na ƙwayoyin halittar cerebellar daga beraye Mfn2cKO na musamman na PN ya nuna cewa waɗannan hanyoyin (gami da duk antiperoxidases) ba su canza sosai a cikin magana ba, don haka yana nuna cewa wannan juyawar metabolism zaɓi ne ga lalacewar PN (Hoto na S6, D zuwa G).
A taƙaice, waɗannan nazarin sun nuna bambance-bambancen da ke tsakanin tsarin kunnawa na ɗan lokaci na takamaiman hanyoyin rayuwa a cikin PNs. Duk da cewa aikin mitochondrial na neuronal mara kyau na iya haifar da farkon atherosclerosis da sake fasalin 1C (Hoto na 3E da Hoto na S5C), har ma da canje-canjen da ake iya faɗi a cikin bayyanar hadaddun I da IV, canje-canje a cikin haɗin serine de novo kawai Ya bayyana ne kawai a cikin matakai na ƙarshe. Rashin aiki na OXPHOS (Hoto na 3E da Hoto na S5C). Waɗannan binciken sun bayyana tsari mai zuwa wanda mitochondrial (zagaye na 1C) da cytoplasmic (serine biosynthesis) suka mayar da martani tare da ƙaruwar atherosclerosis a cikin zagayowar TCA don sake fasalin metabolism na neuronal.
Kwayoyin halittar OXPHOS masu tsawon makonni 8 na iya ci gaba da ayyukan motsa jiki masu yawa kuma suna fuskantar babban haɗin metabolism don rama rashin aikin mitochondrial. Wannan binciken ya haifar da wata dama mai ban sha'awa cewa ko da a wannan lokacin, waɗannan ƙwayoyin suna iya samun maganin warkewa don jinkirta ko hana neurodegeneration. A makare. Mun warware wannan yiwuwar ta hanyar shiga tsakani guda biyu masu zaman kansu. A cikin hanyar farko, mun tsara ƙwayar cuta mai alaƙa da adeno (AAV) ta Cre-dependent don a iya bayyana MFN2 a cikin PNs masu ƙarancin OXPHOS a cikin vivo (Hoto na S7A). An tabbatar da AAV mai ɓoye MFN2 da kwayar halittar mai ba da rahoto mai haske mCherry (Mfn2-AAV) a cikin al'adun jijiyoyi na farko a cikin vitro, wanda ya sa aka bayyana MFN2 ta hanyar da ta dogara da Cre kuma ya ceci yanayin mitochondrial, ta haka yana hana neurodegeneration a cikin ƙwayoyin halittar Mfn2cKO (Hoto na S7, B, D da E). Na gaba, mun gudanar da gwaje-gwaje a cikin jiki don isar da Mfn2-AAV mai makonni 8 zuwa ga ƙwayar kwakwalwa ta Mfn2cKO da kuma sarrafa beraye, sannan muka yi nazarin beraye masu makonni 12 (Hoto na 4A). Berayen Mfn2cKO da aka yi wa magani sun mutu (Hoto na 1, A da B) (16). Juyawar ƙwayoyin cuta a cikin jiki ya haifar da zaɓin bayyanar PN a wasu da'irori na cerebellar (Hoto na S7, G da H). Allurar AAV mai sarrafa kansa da ke bayyana mCherry kawai (Ctrl-AAV) ba ta da wani tasiri mai mahimmanci kan matakin neurodegeneration a cikin dabbobin Mfn2cKO. Sabanin haka, nazarin Mfn2cKOs da aka yi wa Mfn2-AAV ya nuna babban tasirin kariya na layin ƙwayoyin PN (Hoto na 4, B da C). Musamman ma, yawan ƙwayoyin jijiyoyi kusan ba a iya bambance su da dabbobin da ke sarrafawa ba (Hoto na 4, B da C, da Hoto na S7, H da I). Bayyanar MFN1 amma ba MFN2 ba yana da tasiri daidai gwargwado wajen ceton mutuwar jijiyoyi (Hoto na 4C da Hoto na S7, C da F), wanda ke nuna cewa bayyanar MFN1 mai kama da ƙashi zai iya ƙarawa rashin MFN2 yadda ya kamata. Ƙarin bincike a matakin PN guda ɗaya ya nuna cewa Mfn2-AAV ya ceto tsarin mitochondria, daidaita matakan mtDNA, kuma ya juya babban bayyanar alamar anti-angiogenesis PCx ​​(Hoto na 4, C zuwa E). Duba gani na berayen Mfn2cKO da aka ceto a yanayin hutawa ya nuna cewa an inganta yanayinsu da alamun motsinsu (motsi na S1 zuwa S3). A ƙarshe, waɗannan gwaje-gwajen sun nuna cewa jinkirin sake dawo da MFN2 cikin PNs wanda ke da ƙarancin OXPHOS ya isa ya mayar da amfani da mtDNA da haifar da atherosclerosis, ta haka yana hana lalacewar axon da mutuwar jijiyoyi a cikin jiki.
(A) Tsarin da ke nuna jadawalin gwaji don allurar AAV mai ɓoye MFN2 lokacin da aka kunna hanyar metabolism da aka nuna. (B) Hotunan confocal na yanka cerebellar na makonni 12 da aka ɗauka a makonni 8 a cikin beraye Mfn2cKO kuma aka yi musu lakabi da anti-Calbindin antibody. Dama: Ƙara zaruruwan axon. Girman axon zoom shine 450 da 75 μm. (C) Hagu: Adadin yawan ƙwayoyin Purkinje a cikin madauki na transduction AAV (AAV+) (binciken bambanci na hanya ɗaya; n = beraye 3). Dama: nazarin mayar da hankali na mtDNA a cikin PN da aka ɗauka a mako na 12 (gwajin t-ba tare da haɗin gwiwa ba; n = ƙwayoyin 6 daga beraye uku). * P <0.05; ** P <0.01. (D) Wakilan micrographs na watsa lantarki na PNs na sassan cerebellar Mfn2cKO da aka ɗauka tare da vectors na ƙwayoyin cuta da aka nuna. Abin rufe fuska mai ruwan hoda yana nuna yankin da dendrites ke zaune, kuma murabba'in rawaya mai dige-dige yana nuna zuƙowar da aka bayar a dama; n yana wakiltar tsakiya. Sandar sikelin, 1μm. (E) yana nuna misalin tabon PCx a cikin PN da aka canza a makonni 12. Sandar sikelin, 20μm. OE, yawan magana; FC, canjin ninkawa.
A ƙarshe, mun bincika mahimmancin rayuwar ƙwayoyin halitta da peroxidase ke haifarwa a cikin PNs waɗanda suka fuskanci matsalar OXPHOS. Mun samar da mCherry mai rubuta AAV-shRNA (gajeren RNA mai gashi) wanda ke nufin PCx mRNA na linzamin kwamfuta (AAV-shPCx), kuma mun yi allurar ƙwayar cuta ko kuma maganinta mai ƙarfi (AAV-scr) a cikin cerebellum na beraye Mfn2cKO. An yi allurar a mako na huɗu na haihuwa (Hoto na 5A) don cimma ingantaccen bugun PCx a lokacin da bayyanar PCx ​​ta ƙaru (Hoto na 3C) kuma layin ƙwayar PN har yanzu yana nan (Hoto na 1A). Ya kamata a lura cewa bugun PCx (Hoto na S8A) yana haifar da ƙaruwar mutuwar PN, wanda aka iyakance ga zoben da ya kamu da cutar (Hoto na 5, B da C). Domin fahimtar tsarin tasirin metabolism wanda haɓakawar PCx ​​ke haifarwa, mun yi nazarin matsayin redox na PNs bayan bugun PCx da kuma na'urar hangen nesa ta AAV Grx1-roGFP2 an bayyana su a lokaci guda (Hoto na S8, B zuwa D) don kimanta glutathione. Canjin dangi na yuwuwar peptide redox (38). Sannan, mun yi amfani da na'urar hangen nesa ta hasken rana ta photon biyu (FLIM) a cikin sassan kwakwalwa masu tsanani na Mfn2cKO mai makonni 7 ko abokan hulɗar sarrafawa don gano canje-canje masu yuwuwa a cikin yanayin redox na cytoplasmic bayan tabbatar da yanayin FLIM (Hoto na S8, E zuwa G). Binciken ya nuna ƙaruwa mai yawa a cikin yanayin oxidation na PN guda ɗaya na Mfn2cKO waɗanda ba su da bayyanar PCx, wanda ya bambanta da ƙwayoyin sarrafawa ko PN na Mfn2cKO waɗanda ke bayyana shRNA kawai da aka lalata (Hoto na 5, D da E). Lokacin da aka rage yawan bayyanar PCx, kashi na Mfn2cKO PNs da ke nuna yanayin oxidized ya ƙaru da fiye da sau uku (Hoto na 5E), wanda ke nuna cewa haɓaka PCx yana kiyaye ƙarfin redox na ƙwayoyin jijiyoyi da suka lalace.
(A) Tsarin da ke nuna jadawalin gwaji don allurar AAV mai ɓoye shPCx lokacin da aka kunna hanyar metabolism da aka nuna. (B) Hotunan confocal na sassan cerebellar na makonni 8 a cikin beraye Mfn2cKO da aka ɗauka aka kuma yi musu lakabi da anti-calcineurin antibody a makonni 4. Madaurin sikelin, 450μm. (C) Adadin yawan ƙwayoyin Purkinje a cikin madaukai da aka ɗauka ta hanyar AAV (nazarin bambancin hanya ɗaya; n = beraye 3 zuwa 4). An bayyana bayanai a matsayin matsakaicin ±SEM; ***P<0.001. (D) Hoton wakilin FLIM yana nuna matsakaicin tsawon rayuwar PN mai makonni 7 mai bayyana glutathione redox firikwensin Grx1-roGFP2 a ƙarƙashin yanayin gwaji da aka ƙayyade. Rabon LUT (teburin duba sama): tazara lokacin tsira (a cikin picoseconds). Madaurin sikelin, 25μm. (E) Histogram ɗin yana nuna rarrabawar ƙimar rayuwar Grx1-roGFP2 daga (D) (n=158 zuwa ƙwayoyin halitta 368 a cikin beraye biyu a ƙarƙashin kowane yanayi). Jadawalin kek da ke sama da kowane histogram: yana nuna adadin ƙwayoyin da ke da ƙimar tsawon rai mai tsawo (ja, oxidized) ko gajere (shuɗi, raguwa), waɗanda suka wuce 1 SD na matsakaicin ƙimar tsawon rai a cikin CTRL-AAV-scr. (F) Tsarin da aka gabatar yana nuna tasirin kariya na haɓaka PCx na jijiyoyi.
Gabaɗaya, bayanan da muka bayar a nan sun nuna cewa sake bayyana MFN2 zai iya ceton ci gaba gaba ɗaya na PN tare da ƙarancin OXPHOS mai tsanani, raguwar mtDNA mai tsanani, da kuma yanayin ista mai kama da na ista, ta haka yana samar da ci gaba mai ɗorewa ko da a cikin cututtuka masu tasowa. Lalacewar jijiyoyi yana ba da shaidar da za a iya canzawa game da matakin kafin mutuwar ƙwayoyin halitta. Wannan matakin sassaucin metabolism an ƙara jaddada shi ta hanyar ikon neurons na haifar da atherosclerosis (sake haɗawa da zagayowar TCA), wanda ke hana bayyanar PCx ​​a cikin PNs marasa OXPHOS kuma yana haɓaka mutuwar ƙwayoyin halitta, don haka yana taka rawa ta kariya (Hoto na 5F).
A cikin wannan binciken, mun bayar da shaida cewa martanin PNs ga rashin aikin OXPHOS shine a hankali su haɗu zuwa zagayowar TCA atherosclerosis ta hanyar hanyar kunnawa daban-daban da shirye-shiryen metabolism ke kunnawa. Mun tabbatar da nazarin proteomic tare da hanyoyi da yawa masu dacewa kuma mun bayyana cewa lokacin da aka ƙalubalanci matsalar mitochondrial mai tsanani, ƙwayoyin jijiyoyi suna da nau'in elasticity na metabolism wanda ba a san shi ba a da. Abin mamaki, duk tsarin sake haɗawa ba lallai bane ya nuna yanayin metabolism na ƙarshe wanda ke tare da neurodegeneration a hankali kuma ba za a iya jurewa ba, amma bayananmu sun nuna cewa yana iya zama neuron mai kulawa ko da a matakin kafin mutuwar ƙwayoyin halitta Tsarin diyya na aiki. Wannan binciken ya nuna cewa ƙwayoyin jijiyoyi suna da babban matakin elasticity na metabolism a cikin jiki. Wannan gaskiyar ta tabbatar da cewa sake farawa na MFN2 daga baya zai iya juya bayyanar manyan alamomin metabolism kuma ya hana lalacewar PN. Akasin haka, yana hana atherosclerosis kuma yana hanzarta jijiyoyi. transsexual.
Ɗaya daga cikin abubuwan da suka fi ban sha'awa a bincikenmu shine cewa PNs marasa OXPHOS na iya canza tsarin metabolism na zagayowar TCA ta hanyar haɓaka enzymes waɗanda ke motsa arteriosclerosis musamman. Sake fasalin metabolism abu ne da aka saba gani a cikin ƙwayoyin cutar kansa, waɗanda wasu daga cikinsu sun dogara da glutamine don ƙara wa tsaka-tsakin zagayowar TCA don samar da daidaitattun abubuwa, waɗanda ke jagorantar sarkar numfashi da kuma kula da samar da abubuwan da suka fara samar da lipid da nucleotide biosynthesis (39, 40). Wani bincike na baya-bayan nan ya nuna cewa a cikin kyallen da ke kewaye da ke fuskantar matsalar OXPHOS, sake haɗa metabolism na glutamine/glutamate shima babban fasali ne (5, 41), inda alkiblar shigar glutamine cikin zagayowar TCA ta dogara ne akan Saboda tsananin raunin OXPHOS (41). Duk da haka, akwai rashin wata hujja bayyananniya kan duk wani kamanceceniya na yanayin metabolism na neuronal a cikin jiki da yuwuwar dacewarsa a cikin mahallin cutar. A cikin wani bincike na in vitro na baya-bayan nan, an nuna cewa manyan ƙwayoyin cortical suna motsa wuraren glutamate don watsawar jijiyoyi, ta haka suna haɓaka metabolism na oxidative da atherosclerosis a ƙarƙashin yanayin damuwa na metabolism (42). Ya kamata a lura cewa a ƙarƙashin hana maganin enzyme na zagayowar TCA succinate dehydrogenase, ana kyautata zaton pyruvate carboxylation yana kiyaye haɗakar oxaloacetate a cikin ƙwayoyin sel masu haɓaka (34). Duk da haka, mahimmancin waɗannan hanyoyin ga kyallen kwakwalwa (inda ake tsammanin atherosclerosis galibi yana iyakance ga ƙwayoyin halitta) har yanzu yana da mahimmancin ilimin halittar jiki (43). A wannan yanayin, bayananmu sun nuna cewa PNs da OXPHOS ya lalata a jiki za a iya canzawa zuwa lalata BCAA da pyruvate carboxylation, waɗanda sune manyan hanyoyin biyu na ƙarin tsaka-tsakin TCA. Kodayake an gabatar da gudummawar da aka bayar ta hanyar amfani da BCAA catabolism ga metabolism na makamashin jijiyoyi, ban da rawar da glutamate da GABA ke takawa wajen isar da sakonnin jijiyoyi (44), har yanzu babu wata shaida ga waɗannan hanyoyin a cikin jiki. Saboda haka, yana da sauƙi a yi hasashe cewa PNs marasa aiki na iya ramawa ta atomatik don amfani da tsaka-tsakin TCA wanda tsarin haɗuwa ke haifarwa ta hanyar ƙara atherosclerosis. Musamman ma, ana iya buƙatar haɓaka PCx don ci gaba da buƙatar aspartic acid, wanda aka ba da shawara a cikin ƙwayoyin da ke yaɗuwa tare da rashin aikin mitochondrial (45). Duk da haka, bincikenmu na metabolomics bai bayyana wani muhimmin canji a cikin matakin aspartic acid mai ɗorewa a cikin Mfn2cKO PNs ba (Hoto na S6A), wanda wataƙila yana nuna bambancin amfani da aspartic acid tsakanin ƙwayoyin da ke yaɗuwa da ƙwayoyin bayan mitotic. Kodayake ainihin hanyar haɓaka PCx a cikin ƙwayoyin da ba su da aiki a cikin jiki har yanzu ba a san ta ba, mun nuna cewa wannan martanin da ya yi kafin lokaci yana taka muhimmiyar rawa wajen kiyaye yanayin redox na ƙwayoyin halitta, wanda aka nuna a cikin gwaje-gwajen FLIM akan yanka cerebellar. Musamman ma, hana PNs haɓaka PCx na iya haifar da yanayin oxidized da haɓaka mutuwar ƙwayoyin halitta. Kunna lalacewar BCAA da carboxylation na pyruvate ba hanyoyi ne na siffanta kyallen jikin da ke gefe na rashin aikin mitochondrial ba (7). Saboda haka, da alama su ne babban fasalin ƙwayoyin halitta marasa OXPHOS, koda kuwa ba shine kawai fasalin ba, wanda yake da mahimmanci ga neurodegeneration. .
Cutar kwakwalwa nau'i ne na cututtukan jijiyoyi daban-daban wanda yawanci yakan bayyana a matsayin ataxia kuma yakan lalata PNs (46). Wannan yawan ƙwayoyin jijiyoyi suna da matuƙar rauni ga aikin mitochondrial saboda lalacewar zaɓin su a cikin beraye ya isa ya haifar da yawancin alamun motsa jiki waɗanda ke nuna spinocerebellar ataxia na ɗan adam (16, 47, 48). A cewar rahotanni, samfurin linzamin kwamfuta mai canza halitta tare da kwayar halittar maye gurbi yana da alaƙa da spinocerebellar ataxia na ɗan adam kuma yana da matsalar mitochondrial (49, 50), yana jaddada mahimmancin nazarin sakamakon rashin OXPHOS a cikin PNPH. Saboda haka, ya dace musamman don ware da kuma nazarin wannan ƙwayar jijiyoyi ta musamman yadda ya kamata. Duk da haka, idan aka yi la'akari da cewa PNs suna da matukar damuwa ga matsin lamba kuma suna lissafin ƙarancin adadin dukkan ƙwayoyin cerebellar, ga yawancin nazarin da aka yi bisa ga omics, rabuwar su a matsayin ƙwayoyin halitta gaba ɗaya har yanzu babban al'amari ne mai ƙalubale. Ko da yake kusan ba zai yiwu a cimma cikakkiyar rashin gurɓata wasu nau'ikan ƙwayoyin halitta ba (musamman kyallen manya), mun haɗu da ingantaccen matakin rabuwa da FACS don samun isassun adadin ƙwayoyin halitta masu aiki don nazarin proteomics na ƙasa, kuma muna da cikakken ɗaukar furotin (kimanin sunadaran 3000) idan aka kwatanta da saitin bayanai na yanzu na cerebellum gaba ɗaya (51). Ta hanyar kiyaye wanzuwar ƙwayoyin halitta gaba ɗaya, hanyar da muka bayar a nan ba wai kawai tana ba mu damar duba canje-canje a cikin hanyoyin rayuwa a cikin mitochondria ba, har ma da duba canje-canje a cikin takwarorinta na cytoplasmic, wanda ke ƙara amfani da alamun membrane na mitochondrial don wadatar da nau'in ƙwayoyin halitta Sabuwar hanyar don adadin mitochondria a cikin kyallen halitta masu rikitarwa (52, 53). Hanyar da muka bayyana ba wai kawai tana da alaƙa da nazarin ƙwayoyin Purkinje ba, har ma ana iya amfani da ita cikin sauƙi ga kowace irin ƙwayar halitta don magance canje-canjen rayuwa a cikin kwakwalwar da ke fama da rashin lafiya, gami da wasu samfuran rashin aikin mitochondrial.
A ƙarshe, mun gano wata hanya ta magani a yayin wannan tsarin sake fasalin metabolism wanda zai iya juya manyan alamun damuwa na ƙwayoyin halitta gaba ɗaya da kuma hana lalacewar jijiyoyi. Saboda haka, fahimtar tasirin aikin sake fasalin da aka bayyana a nan na iya samar da fahimta mai mahimmanci game da hanyoyin magancewa don kiyaye dorewar jijiyoyi yayin rashin aikin mitochondrial. Ana buƙatar bincike na gaba da nufin gano canje-canje a cikin metabolism na makamashi a cikin wasu nau'ikan ƙwayoyin kwakwalwa don bayyana cikakken amfani da wannan ka'ida ga sauran cututtukan jijiyoyi.
An riga an bayyana beraye na MitoPark a baya (31). An riga an bayyana beraye na C57BL/6N tare da kwayoyin halittar Mfn2 masu gefen loxP (18) kuma an haɗa su da beraye na L7-Cre (23). Daga nan aka haɗa beraye masu heterozygous guda biyu da suka haifar da beraye masu homozygous Mfn2loxP/Mfn2loxP don samar da ƙwayoyin halittar Purkinje na musamman ga Mfn2 (Mfn2loxP/Mfn2loxP; L7-cre). A cikin wani ɓangare na haɗuwa, an gabatar da Gt (ROSA26) SorStop-mito-YFP allele (stop-mtYFP) ta hanyar ƙarin haɗuwa (20). An gudanar da duk hanyoyin dabbobi daidai da jagororin Turai, na ƙasa da na hukumomi kuma LandesamtfürNatur na Umwelt da Verbraucherschutz, North Rhine-Westphalia, Jamus sun amince da su. Aikin dabbobi kuma yana bin jagorancin Tarayyar Turai ta Ƙungiyoyin Kimiyyar Dabbobi.
Bayan an yi wa mace mai juna biyu maganin sa barci a mahaifa, sai a ware tayin linzamin kwamfuta (E13). An cire kwayar halittar a cikin maganin Hanks' Balanced Salt Solution (HBSS) wanda aka ƙara masa 10 mM Hepes sannan aka wuce da Dulbecco's Modified Eagle's Medium mai ɗauke da papain (20 U/ml) da cysteine ​​​​(1μg/ml). A saka nama a cikin DMEM) sannan a raba ta ta hanyar narkewar enzymatic. Ml) a zafin jiki na 37°C na minti 20, sannan a niƙa ta hanyar injiniya a cikin DMEM tare da ƙarin 10% na serum na shanu na tayi. An shuka ƙwayoyin halitta a kan murfin gilashi da aka shafa da polylysine a yawan 2×106 a kowace kwano mai girman 6 cm ko kuma a yawan 0.5×105 ƙwayoyin halitta/cm2 don nazarin hoto. Bayan awanni 4, an maye gurbin maganin da maganin Neurobasal wanda ba shi da serum wanda ya ƙunshi ƙarin B27 1% da 0.5 mM GlutaMax. Daga nan aka kiyaye ƙwayoyin jijiyoyi a zafin 37°C da 5% na CO2 a duk tsawon gwajin, sannan aka ciyar da su sau ɗaya a mako. Domin a samar da sake haɗa su a cikin vitro, an yi amfani da 3μl (abincin rijiyoyi 24) ko 0.5μl (faranti 24) na wannan ƙwayar cuta ta AAV9 don magance ƙwayoyin jijiyoyi a rana ta biyu a cikin vitro: AAV9.CMV.PI.eGFP. WPRE.bGH (Addgene, lambar kasida 105530-AAV9) da AAV9.CMV.HI.eGFP-Cre.WPRE.SV40 (Addgene, lambar kasida 105545-AAV9).
An yiwa linzamin kwamfuta Mfn1 da Mfn2 ƙarin DNA (wanda aka samo daga plasmid na Addgene #23212 da #23213, bi da bi) alama da jerin V5 (GKPIPNPLLGLDST) a C-terminus, kuma an haɗa su da mCherry a cikin firam ta hanyar jerin T2A. Grx1-roGFP2 kyauta ce daga Heidelberg TP Dick DFKZ (Deutsches Krebsforschungszentrum). Ta hanyar maye gurbin kaset ɗin tdTomato ta amfani da hanyoyin cloning na gargajiya, an haɗa kaset ɗin cikin kashin baya na pAAV-CAG-FLEX-tdTomato (lambar ambaton Addgene 28306) don samar da vectors na pAAV-CAG-FLEX-mCherry-T2A-MFN2-V5, pAAV-CAG-FLEX-mCherry-T2A-MFN1-V5 da pAAV-CAG-FLEX-Grx-roGFP2. An yi amfani da irin wannan dabarar don samar da na'urar sarrafawa ta pAAV-CAG-FLEX-mCherry. Domin samar da ginin AAV-shPCx, ana buƙatar na'urar plasmid AAV (VectorBuilder, pAAV [shRNA] -CMV-mCherry-U6-mPcx- [shRNA#1]), wacce ke ɗauke da jerin DNA wanda ke ƙunshe da linzamin kwamfuta mai niyya ta shRNA PCx (5′CTTTCGCTCTAAGGTGCTAACTCGAGTTTAGCACCTTAGAGCGAAAG 3′) A ƙarƙashin ikon mai haɓaka U6, ana amfani da mCherry a ƙarƙashin ikon mai haɓaka CMV. An gudanar da samar da na'urorin AAV masu taimako bisa ga umarnin masana'anta (Cell Biolabs). A takaice, yi amfani da plasmid mai canja wuri wanda ke ɗauke da mCherry-T2A-MFN2-V5 (pAAV-CAG-FLEX-mCherry-T2A-MFN2-V5), mCherry-T2A-MFN1-V5 (pAAV-CAG-FLEX-mCherry) a cikin ɗan lokaci Canja wurin ƙwayoyin 293AAV-T2A-MFN1-V5), mCherry (pAAV-CAG-FLEX-mCherry) ko Grx-roGFP2 (pAAV-CAG-FLEX-Grx-roGFP2) na kwayar halittar da ke ɗauke da sunadaran AAV1, da kuma rubuta sunadaran capsid da furotin na haɗin gwiwa. Marufi plasmid na plasmid, ta amfani da hanyar calcium phosphate. An samo ɗanyen ƙwayar cuta ta hanyar daskare-narkewa a cikin busasshen wanka na kankara/ethanol da ƙwayoyin da aka lysed a cikin saline mai ɗauke da sinadarin phosphate (PBS). An tsarkake na'urar AAV ta hanyar amfani da na'urar rage gudu ta iodixanol (awanni 24 a 32,000 rpm da 4°C) sannan aka mayar da hankali kan matattarar centrifugal ta Amicon ultra-15. Matsayin kwayoyin halitta na AAV1-CAG-FLEX-mCherry-T2A-MFN2-V5 [2.9×1013 kwafin kwayoyin halitta (GC)/ml], AAV1-CAG-FLEX-mCherry (6.1×1012 GC/ml), an auna AAV1-CAG-FLEX kamar yadda aka bayyana a baya (54), an auna ta hanyar ainihin PCR mai ƙima (qPCR) -MFN1-V5 (1.9×1013 GC/ml) da AAV1-CAG-FLEX-Grx-roGFP2 (8.9×1012 GC/ml).
An cire ƙwayoyin jijiyoyi na farko a cikin PBS mai sanyi 1x, an cire su, sannan aka haɗa su da wani sinadari mai kama da na sodium deoxycholate/PBS mai 0.5% Triton X-100 / 0.5% sodium deoxycholate/PBS lysis buffer wanda ke ɗauke da phosphatase da protease inhibitor (Roche). An yi amfani da gwajin bicinchoninic acid (Thermo Fisher Scientific). Daga nan aka raba sunadaran ta hanyar electrophoresis na gel na SDS-polyacrylamide, sannan aka goge su a kan membrane na polyvinylidene fluoride (GE Healthcare). Toshe wuraren da ba na musamman ba kuma a saka su da babban antibody (duba Tebur S1 don cikakkun bayanai) a cikin madarar 5% a cikin TBST (Tris-buffered saline tare da Tween), matakan wankewa da kuma antibody na biyu a cikin TBST Incubate. A saka su da babban antibody na dare a +4°C. Bayan wankewa, a shafa antibody na biyu na tsawon awanni 2 a zafin ɗaki. Daga baya, ta hanyar sanya wannan blot ɗin tare da antibody na anti-β-actin, an tabbatar da ɗaukar nauyin iri ɗaya. Ganowa ta hanyar canzawa zuwa chemiluminescence da haɓaka chemiluminescence (GE Healthcare).
An gyara ƙwayoyin jijiyoyi da aka riga aka shuka a kan gilashin murfin da aka rufe da 4% paraformaldehyde (PFA)/PBS a lokacin da aka ƙayyade a zafin ɗaki na tsawon minti 10. Da farko ana zuba murfin da 0.1% Triton X-100/PBS na tsawon minti 5 a zafin ɗaki, sannan a sanya shi a cikin toshewar buffer [3% bovine serum albumin (BSA)/PBS]. A rana ta biyu, an wanke murfin da toshewar buffer kuma an saka shi da maganin rigakafi na biyu mai dacewa da fluorophore-conjugated na tsawon awanni 2 a zafin ɗaki; a ƙarshe, an wanke samfuran sosai a cikin PBS tare da 4′,6-diamidino-2 -Phenylindole (DAPI) an shafa shi a kan maƙallin microscope tare da Aqua-Poly/Mount.
An yi wa beraye (maza da mata) sabulun sabulu ta hanyar allurar ketamine (130 mg/kg) da xylazine (10 mg/kg) a cikin ciki sannan a ba su maganin rage zafi (5 mg/kg). Sannan a sanya su a cikin wani kayan aiki mai kama da na'urar rage zafi (Kopf) wanda aka sanya masa wani abin ɗumi. A fallasa kwanyar sannan a yi amfani da abin haƙori don rage sashin cerebellar cortex wanda ya yi daidai da ƙashin ƙugu (daga lambda: wutsiya 1.8, gefe 1, wanda ya yi daidai da lobules IV da V). Yi amfani da allurar sirinji mai lanƙwasa don ƙirƙirar ƙaramin rami a cikin kwanyar a hankali don guje wa lalata jijiyoyin jini da ke ƙasa. Sannan a saka siririn murfin gilashi a hankali a cikin ƙaramin rami (daga -1.3 zuwa -1 a gefen ventral na dura mater), kuma a saka 200 zuwa 300 nl AAV a cikin ƙaramin allurar (Narishige) tare da sirinji na hannu (Narishige) sau da yawa a ƙaramin matsin lamba na tsawon lokaci na mintuna 10 zuwa 20. Bayan an yi jiko, a sanya murfin na tsawon mintuna 10 domin kwayar cutar ta yaɗu gaba ɗaya. Bayan an cire murfin, sai a dinka fatar a hankali don rage kumburin rauni da kuma ba wa dabbar damar murmurewa. An yi wa dabbobin magani da maganin rage radadi (caspofen) na tsawon kwanaki da dama bayan tiyatar, a lokacin kuma aka sa ido sosai kan yanayin jikinsu sannan aka kashe su a daidai lokacin da aka ƙayyade. An gudanar da dukkan hanyoyin bisa ga ƙa'idojin Turai, na ƙasa da na hukumomi kuma LandesamtfürNatur na Umwelt da Verbraucherschutz, North Rhine-Westphalia, Jamus sun amince da su.
An yi wa dabbobin maganin sa barci da ketamine (100 mg/kg) da xylazine (10 mg/kg), sannan aka zuba wa zuciya 0.1 M PBS da farko, sannan aka zuba 4% PFA a cikin PBS. An cire nama sannan aka gyara shi da 4% PFA/PBS cikin dare a 4°C. An yi amfani da wuka mai girgiza (Leica Microsystems GmbH, Vienna, Austria) don shirya sassan sagittal (kauri 50 μm) daga kwakwalwar da aka gyara a cikin PBS. Sai dai idan an ƙayyade akasin haka, an yi tabo na sassan da ke iyo kyauta kamar yadda aka bayyana a sama (13) a zafin ɗaki kuma ana juyawa. A takaice, da farko, an yi tabo na sassan da aka samu da 0.5% Triton X-100/PBS na minti 15 a zafin ɗaki; ga wasu epitopes (Pcx da Shmt2), ta hanyar tris-EDTA buffer a 80°C (PH9) a zafafa yanka na minti 25 maimakon wannan matakin. Na gaba, an saka sassan da babban maganin rigakafi (duba Tebur S1) a cikin toshewar buffer (3% BSA/PBS) a 4°C da dare tare da juyawa. Washegari, an wanke sassan da toshewar buffer sannan aka saka su da maganin rigakafi na biyu mai dacewa da fluorophore-conjugated na tsawon awanni 2 a zafin ɗaki; a ƙarshe, an wanke sassan sosai a cikin PBS, an shafa musu DAPI, sannan aka gyara su da AquaPolymount On a microscope slide.
An yi amfani da na'urar hangen nesa ta laser confocal scanning microscope (TCS SP8-X ko TCS Digital Light Sheet, Leica Microsystems) wacce aka sanye da farin laser da kuma diode ultraviolet laser mai ƙarfin 405 don ɗaukar hoton samfurin. Ta hanyar kunna fluorophore da kuma tattara siginar tare da Hybrid Detector (HyDs), an yi amfani da software na LAS-X don tattara hotuna da suka dace da samfurin Nyquist a cikin yanayin jere: ga bangarorin da ba na adadi ba, sigina ne masu ƙarfi (misali, a cikin ƙwayoyin somatic da dendrites) mtYFP) Yi amfani da HyD don gano adadin PNs a cikin yanayin BrightR). Ana amfani da ƙofar daga 0.3 zuwa 6 ns don rage bango.
Hoton ƙwayoyin da aka ware a ainihin lokaci. Bayan an rarraba su a cikin Neurobasal-A medium wanda ke ɗauke da ƙarin B27 1% da 0.5 mM GlutaMax, nan da nan aka shuka ƙwayoyin a kan gilashin da aka rufe da poly-l-lysine (μ-Slide8 Well, Ibidi, lambar kasida 80826), Sannan a ajiye shi a 37°C da 5% CO2 na tsawon awa 1 don ba da damar ƙwayoyin su zauna. An yi hoton ainihin lokaci akan na'urar daukar hoto ta Leica SP8 wacce aka sanye da farin laser, HyD, ruwan tabarau mai girman 63×[1.4 numerical aperture (NA)] da kuma matakin dumama.
An yi wa linzamin kwamfuta sabulun da sauri da carbon dioxide sannan aka yanke masa kai, aka cire kwakwalwa da sauri daga kwanyar, aka yanka ta zuwa kauri 200μm (don gwajin lakabin 13C) ko kauri 275μm (don gwaje-gwajen photon guda biyu) sashen sagittal cike da kayan da ke ƙasa. An cika ice cream (HM-650 V, Thermo Fisher Scientific, Walldorf, Jamus) da abubuwa masu zuwa: 125 mM kankara mai sanyi, mai cike da carbon (95% O2 da 5% CO2) ƙarancin Ca2 + ruwan cerebrospinal na wucin gadi (ACSF) NaCl, 2.5 mM KCl, 1.25 mM sodium phosphate buffer, 25 mM NaHCO3, 25 mM glucose, 0.5 mM CaCl2 da 3.5 mM MgCl2 (matsin osmotic na 310 zuwa 330 mmol). Canja wurin sassan kwakwalwar da aka samu zuwa ɗakin da aka riga aka shirya wanda ke ɗauke da mafi girman Ca2 + ACSF (125.0 mM NaCl, 2.5 mM KCl, 1.25 mM sodium phosphate buffer, 25.0 mM NaHCO3, 25.0 mM d-glucose, 1.0 mM CaCl2 da 2.0 mM MgCl2) Matsakaici) pH 7.4 da 310 zuwa 320 mmol).
A lokacin aikin ɗaukar hoton, an mayar da sassan zuwa wani ɗaki na musamman na ɗaukar hoton, kuma an yi gwajin a ƙarƙashin ci gaba da tura ACSF a yanayin zafi na 32° zuwa 33°C. An yi amfani da na'urar hangen nesa ta laser multiphoton scanning microscope (TCS SP8 MP-OPO, Leica Microsystems) wacce aka sanye da ruwan tabarau na Leica 25x (NA 0.95, ruwa), Ti: Sapphire laser (Chameleon Vision II, Coherent) don ɗaukar hoton sassan. Module na FLIM (PicoHarp300, PicoQuant).
FLIM na Grx1-roGFP2. An auna canje-canje a yanayin redox na cytoplasmic na PNs ta hanyar amfani da FLIM mai photon biyu a cikin sassan kwakwalwa na sagittal, inda na'urar gano abubuwa ta Grx1-roGFP2 ta yi niyya ga PNs. A cikin layin PN, an zaɓi filin siye kusan 50 zuwa 80 μm a ƙasa da saman yanki don tabbatar da cewa akwai PN mai dorewa (wato, rashin tsarin beads ko canje-canje na yanayin jijiyoyi tare da dendrites) da firikwensin roGFP2 mai kyau sau biyu da AAV mai ɓoye shRNA PCx ko jerin sarrafawarsa (kowane mCherry mai bayyana tare). Tattara hotuna masu tari ɗaya tare da zuƙowa na dijital 2x [tsawo mai motsawa: 890 nm; 512 nm 512 pixels]. Ganowa: HyD na ciki, ƙungiyar matattarar fluorescein isothiocyanate (FITC)] da matsakaicin hoto cikin mintuna 2 zuwa 3 ana amfani da su don tabbatar da cewa an tattara isasshen photons (jimlar photons 1000) don daidaita lanƙwasa. An yi amfani da na'urar bincike ta Grx1-roGFP2 da kuma tabbatar da yanayin FLIM ta hanyar sa ido kan darajar tsawon rayuwar roGFP2 lokacin da aka ƙara 10 mM H2O2 na waje zuwa ACSF (don haɓaka iskar shaka, wanda ke haifar da ƙaruwar tsawon rai), sannan aka ƙara 2 mM dithiothreitol (yana rage matakin raguwa, wanda ke haifar da raguwar tsawon rai) (Hoto na S8, D zuwa G). Yi amfani da software na FLIMfit 5.1.1 don nazarin sakamakon da aka samu, daidaita lanƙwasa ɗaya ta ruɓewar hoto gaba ɗaya zuwa ga IRF da aka auna (aikin amsawar kayan aiki), kuma χ2 yana kusan 1. Don ƙididdige tsawon rayuwar PN guda ɗaya, an zana abin rufe fuska da ke kewaye da jikin jijiya da hannu, kuma an yi amfani da matsakaicin tsawon rai a cikin kowane abin rufe fuska don ƙididdigewa.
Binciken yuwuwar Mitochondrial. Bayan an saka sashin da ke da rauni tare da ƙara 100 nM TMRM kai tsaye zuwa ACSF mai perfused na tsawon mintuna 30, an auna canje-canjen yuwuwar mitochondrial na PNs ta hanyar amfani da na'urar hangen nesa ta photon biyu. An yi hoton TMRM ta hanyar kunna binciken a 920 nm da kuma amfani da HyD na ciki (tetramethylrhodamine isothiocyanate: 585/40 nm) don tattara sigina; ta hanyar amfani da irin wannan tsayin motsawa amma ta amfani da HyD na ciki daban (FITC :525/50) don ɗaukar hoton mtYFP. Yi amfani da plugin ɗin ImageJ's Image Calculator don kimanta yuwuwar mitochondrial a matakin tantanin halitta guda ɗaya. A takaice, ana amfani da lissafin plug-in: signal = min (mtYFP, TMRM) don gano yankin mitochondrial wanda ke nuna siginar TMRM a cikin Purkinje Somali a cikin hoton confocal guda ɗaya na tashar da ta dace. Sannan a auna yankin pixel a cikin abin rufe fuska da aka samu, sannan a daidaita shi akan hoton da ya dace da tashar mtYFP guda ɗaya don samun ɓangaren mitochondrial wanda ke nuna ƙarfin mitochondrial.
An cire hoton da software na Huygens Pro (Scientific Volume Imaging). Don hotunan tayal da aka duba, ana yin saitin tayal ɗaya ta amfani da algorithm ɗin ɗinki ta atomatik da software na LAS-X ya bayar. Bayan daidaita hoto, yi amfani da ImageJ da Adobe Photoshop don ƙara sarrafa hoton kuma daidaita haske da bambanci daidai gwargwado. Yi amfani da Adobe Illustrator don shirya zane.
Binciken mayar da hankali na mtDNA. An ƙididdige adadin raunukan mtDNA akan sassan cerebellar da aka yiwa lakabi da ƙwayoyin rigakafi akan DNA ta hanyar na'urar hangen nesa ta confocal. An ƙirƙiri kowane yanki da aka nufa don jikin tantanin halitta da kuma nucleus na kowace tantanin halitta, kuma an ƙididdige yankin da ya dace ta amfani da plugin ɗin Multi Measure (software na ImageJ). Rage yankin nukiliya daga yankin jikin tantanin halitta don samun yankin cytoplasmic. A ƙarshe, an yi amfani da plugin ɗin Analyse Particles (software na ImageJ) don ƙididdige maki na DNA na cytoplasmic ta atomatik wanda ke nuna mtDNA akan hoton ƙofar, kuma an daidaita sakamakon da aka samu zuwa matsakaicin PN na berayen CTRL. Ana bayyana sakamakon a matsayin matsakaicin adadin nucleosides a kowace tantanin halitta.
Binciken bayyanar furotin. Yi amfani da kayan haɗin ImageJ's Image Calculator don kimanta bayyanar furotin a cikin PN a matakin ƙwayar halitta guda ɗaya. A takaice, a cikin hoton confocal mai layi ɗaya na tashar da ta dace, ta hanyar lissafi: sigina = min (mtYFP, antibody), an gano yankin mitochondrial wanda ke nuna aikin rigakafi ga wani takamaiman antibody a Purkina. Sannan ana auna yankin pixel a cikin abin rufe fuska da ya haifar, sannan a daidaita shi akan hoton tari ɗaya daidai na tashar mtYFP don samun ɓangaren mitochondrial na furotin da aka nuna.
Binciken yawan ƙwayoyin Purkinje. An yi amfani da ƙarin na'urar tantance yawan ƙwayoyin halitta ta ImageJ don kimanta yawan ƙwayoyin Purkinje ta hanyar raba adadin ƙwayoyin Purkinje da aka ƙidaya da tsawon zoben cerebellar da ƙwayoyin da aka ƙidaya suka mamaye.
Shirya samfurin da tattarawa. An gyara kwakwalwar daga ƙungiyar sarrafawa da beraye Mfn2cKO a cikin 2% PFA/2.5% glutaraldehyde a cikin 0.1 M phosphate buffer (PB), sannan aka shirya sassan coronal ta amfani da ciliates (Leica Mikrosysteme GmbH, Vienna, Austria) (Kauri 50 zuwa 60 μm). Sannan a saka a cikin PB buffer a cikin 1% os tetraoxide da 1.5% potassium ferrocyanide a zafin ɗaki na tsawon awa 1. An wanke sassan sau uku da ruwa mai narkewa, sannan a shafa musu fenti da 70% ethanol wanda ke ɗauke da 1% uranyl acetate na tsawon mintuna 20. Sannan an cire sassan a cikin barasa mai daraja sannan aka saka su a cikin Durcupan ACM (Araldite casting resin M) epoxy resin (Electron Microscopy Sciences, lambar kasida 14040) tsakanin gilashin da aka rufe da silicon, kuma a ƙarshe a 60°C Polymerize a cikin tanda na tsawon awanni 48. An zaɓi yankin cerebellar cortex kuma an yanke sassan 50 nm masu siriri sosai a kan Leica Ultracut (Leica Mikrosysteme GmbH, Vienna, Austria) sannan aka ɗauki su a kan grid ɗin jan ƙarfe mai girman 2 × 1 mm wanda aka shafa da fim ɗin polystyrene. An yi wa sassan fenti da maganin uranyl acetate na 4% a cikin H2O na tsawon mintuna 10, aka wanke su da H2O sau da yawa, sannan aka wanke su da citrate na Reynolds a cikin H2O na tsawon mintuna 10, sannan aka wanke su da H2O sau da yawa. An ɗauki ƙananan hotuna da na'urar hangen nesa ta lantarki Philips CM100 (Thermo Fisher Scientific, Waltham, MA, Amurka) ta amfani da kyamarar dijital ta TVIPS (Tietz Video and Image Processing System) TemCam-F416 (TVIPS GmbH, Gauting, Amurka). Jamus).
Ga beraye da suka kamu da AAV, an raba kwakwalwa aka kuma yanke ta zuwa wani sashe mai kauri mm 1, sannan aka duba cerebellum ta amfani da na'urar hangen nesa ta fluorescence don gano zoben da ya kamu da AAV (wato, mCherry expressing). Gwaje-gwaje ne kawai da allurar AAV ke haifar da ingantaccen transduction na layin ƙwayoyin Purkinje (watau kusan dukkan layin) a cikin aƙalla zoben cerebellar guda biyu a jere aka yi amfani da su. An yi amfani da madauki mai transduced AAV don microdissecting na dare ɗaya bayan an gyara shi (4% PFA da 2.5% glutaraldehyde a cikin 0.1 M cocoate buffer) sannan aka ƙara sarrafa shi. Don saka EPON, an wanke kyallen da aka gyara da 0.1 M sodium cocoate buffer (Applichem), sannan aka saka 2% OsO4 (os, Science Services; Caco) a cikin 0.1 M sodium cocoate buffer (Applichem) awanni 4, sannan a wanke na tsawon awanni 2. A maimaita sau 3 da 0.1 M cocamide buffer. Daga baya, an yi amfani da jerin ethanol masu hawa sama don saka kowace maganin ethanol a zafin 4°C na tsawon mintuna 15 don fitar da ruwa daga kyallen. An mayar da kyallen zuwa propylene oxide sannan aka saka shi cikin EPON (Sigma-Aldrich) a zafin 4°C. Sanya kyallen a cikin sabon EPON a zafin ɗaki na tsawon awanni 2, sannan a saka shi a zafin 62°C na tsawon awanni 72. Yi amfani da ultramicrotome (Leica Microsystems, UC6) da wuka mai lu'u-lu'u (Diatome, Biel, Switzerland) don yanke sassan 70 nm masu siriri, sannan a yi masa tabo da 1.5% uranyl acetate na tsawon mintuna 15 a zafin 37°C, sannan a yi masa tabo da maganin citrate na tsawon mintuna 4. An ɗauki ƙananan ƙwayoyin lantarki ta amfani da na'urar hangen nesa ta JEM-2100 Plus (JEOL) wacce aka sanye da manhajar Camera OneView 4K 16-bit (Gatan) da DigitalMicrograph (Gatan). Don yin nazari, an samo na'urorin auna haske na lantarki ta amfani da zuƙowa ta dijital ta 5000× ko 10,000×.
Binciken yanayin mitochondria. Ga dukkan nazarin, an tsara yanayin mitochondria na mutum ɗaya da hannu a cikin hotunan dijital ta amfani da software na ImageJ. Ana nazarin sigogi daban-daban na yanayin halitta. Ana bayyana yawan mitochondria a matsayin kashi da aka samu ta hanyar raba jimlar yankin mitochondria na kowace tantanin halitta ta hanyar yankin cytoplasm (yankin cytoplasm = yankin ƙwayar halitta-ƙwayar halitta) × 100. Ana ƙididdige zagayen mitochondria tare da dabarar [4π∙(yanki/gefe 2)]. An yi nazarin yanayin mitochondria kuma an raba shi zuwa rukuni biyu ("tubular" da "blister") bisa ga manyan siffofinsu.
Binciken lambobi da yawa na Autophagosome/lysosome. Yi amfani da manhajar ImageJ don zana siffofi na kowane autophagosome/lysosome da hannu a cikin hoton dijital. Ana bayyana yankin Autophagosome/lysosome a matsayin kashi da aka ƙididdige ta hanyar raba jimlar yankin tsarin autophagosome/lysosome na kowace tantanin halitta ta hanyar yankin cytoplasm (yankin cytoplasm=yankin tantanin halitta-yankin nucleus) × 100. Ana ƙididdige yawan autophagosomes/lysosomes ta hanyar raba jimlar adadin da adadin tsarin autophagosome/lysosome a kowace tantanin halitta (dangane da yankin cytoplasmic) (yankin cytoplasmic = yankin tantanin halitta-yankin nukiliya).
Lakabi don sassaka mai tsanani da shirya samfuri. Don gwaje-gwajen da ke buƙatar lakabin glucose, a canja wurin yankawar kwakwalwa mai tsanani zuwa ɗakin da aka riga aka shirya, wanda ke ɗauke da cikakken carbon (95% O2 da 5% CO2), babban Ca2 + ACSF (125.0 mM NaCl, 2.5 mM KCl, 1.25 mM sodium phosphate buffer, 25.0 mM NaHCO3, 25.0 mM d-glucose, 1.0 mM CaCl2 da 2.0 mM MgCl2, wanda aka daidaita zuwa pH 7.4 da 310 zuwa 320 mOsm), inda glucose yake 13 C 6- maye gurbin glucose (Eurisotop, lambar kasida CLM-1396). Don gwaje-gwajen da ke buƙatar lakabin pyruvate, a canja wurin yanka kwakwalwa mai tsanani zuwa Ca2 + ACSF mafi girma (125.0 mM NaCl, 2.5 mM KCl, 1.25 mM sodium phosphate buffer, 25.0 mM NaHCO3, 25.0 mM d-glucose, 1.0 mM CaCl2 kuma a ƙara 2.0mM MgCl2, a daidaita zuwa pH 7.4 da 310 zuwa 320mOsm), sannan a ƙara 1mM 1-[1-13C]pyruvate (Eurisotop, lambar kasida CLM-1082). A saka sassan na tsawon mintuna 90 a 37°C. A ƙarshen gwajin, an wanke sassan da sauri da ruwan magani (pH 7.4) wanda ke ɗauke da 75 mM ammonium carbonate, sannan a daidaita su a cikin 40:40:20 (v:v:v) acetonitrile (ACN): methanol: ruwa. Bayan an saka sassan a kan kankara na tsawon mintuna 30, an sanya samfuran a cikin centrifuge a 21,000 g na tsawon mintuna 10 a zafin jiki na 4°C, sannan aka busar da ruwan da ke cikin iska mai haske a cikin na'urar tattara bayanai ta SpeedVac. An adana busasshen ƙwayar metabolite da aka samu a -80°C har sai an yi bincike.
Binciken sinadarin chromatography-mass na ruwa mai dauke da amino acid 13 masu lakabin C. Don nazarin sinadarin chromatography-mass spectrometry (LC-MS), an sake dakatar da sinadarin metabolite a cikin 75μl na ruwan LC-MS (Honeywell). Bayan an yi amfani da centrifugation a 21,000 g na tsawon mintuna 5 a zafin jiki na 4°C, an yi amfani da 20 μl na sinadarin da aka bayyana don nazarin sinadarin amino acid, yayin da aka yi amfani da sauran sinadarin nan take don nazarin anion (duba ƙasa). An yi nazarin sinadarin amino acid ta amfani da tsarin derivatization na benzoyl chloride da aka bayyana a baya (55, 56). A mataki na farko, an ƙara 10μl na sodium carbonate 100 mM (Sigma-Aldrich) zuwa 20μl na sinadarin metabolite, sannan aka ƙara 10μl na 2% benzoyl chloride (Sigma-Aldrich) zuwa ACN na LC. An yi amfani da na'urar jujjuyawar samfurin na ɗan lokaci sannan aka sanya shi a cikin centrifuge a 21,000 g na tsawon mintuna 5 a zafin jiki na 20°C. A mayar da ruwan da aka share zuwa kwalbar autosampler mai girman 2 ml tare da gilashin conical insert (ƙarfin 200 μl). An yi nazarin samfuran ta amfani da tsarin Acquity iClass ultra-high performance LC (Waters) wanda aka haɗa da Q-Exactive (QE)-HF (Ultra High Field Orbitrap) high-resolution precision mass spectrometer (Thermo Fisher Scientific). Don yin bincike, an yi allurar 2μl na samfurin da aka samo a cikin ginshiƙin silica T3 mai ƙarfi 100 × 1.0 mm (Waters) wanda ke ɗauke da ƙwayoyin cuta 1.8μm. Yawan kwarara shine 100μl/min, kuma tsarin buffer ya ƙunshi buffer A (10 mM ammonium formate da 0.15% formic acid a cikin ruwa) da buffer B (ACN). Gradient ɗin shine kamar haka: 0%B a mintuna 0; 0%B. 0 zuwa 15% B a minti 0 zuwa 0.1; 15 zuwa 17% B a minti 0.1 zuwa 0.5; B a minti 17 zuwa 55% a minti 0.5 zuwa 14; B a minti 55 zuwa 70% a minti 14 zuwa 14.5; a minti 14.5 zuwa 70 zuwa 100% B a minti 18; 100% B a minti 18 zuwa 19; 100 zuwa 0% B a minti 19 zuwa 19.1; 0% B a minti 19.1 zuwa 28 (55, 56). Na'urar auna ma'aunin QE-HF tana aiki a yanayin ionization mai kyau tare da kewayon taro na m/z (rabo na taro/caji) na 50 zuwa 750. Matsakaicin ƙudurin da aka yi amfani da shi shine 60,000, kuma maƙasudin ion na iko (AGC) da aka samu shine 3×106, kuma matsakaicin lokacin ion shine milise 100. Tushen ionization na electrospray (ESI) mai zafi yana aiki a ƙarfin feshi na 3.5 kV, zafin jiki na capillary na 250°C, iskar sheath na AU 60 (raka'o'i marasa tsari), da kuma iskar iska mai taimako na AU 20. 250°C. An saita ruwan tabarau na S zuwa AU 60.
Binciken chromatography na Anion-MS na acid mai suna 13C. An yi nazarin ragowar metabolite precipitate (55μl) ta amfani da tsarin chromatography na Dionex ion (ICS 5000+, Thermo Fisher Scientific) wanda aka haɗa da na'urar auna ma'aunin QE-HF (Thermo Fisher Scientific). A takaice, an allurar da 5μl na cire metabolite a cikin ginshiƙin Dionex IonPac AS11-HC wanda aka sanye da HPLC (2 mm × 250 mm, girman barbashi 4μm, Thermo Fisher Scientific) a cikin yanayin madauki na turawa tare da rabon cikawa na 1.) Ginshiƙin tsaro na Dionex IonPac AG11-HC (2 mm x 50 mm, 4μm, Thermo Fisher Scientific). Ana kiyaye zafin ginshiƙin a 30°C, kuma an saita samfurin atomatik zuwa 6°C. Yi amfani da harsashin potassium hydroxide da aka bayar tare da ruwan da aka cire don samar da gradient na potassium hydroxide ta hanyar janareta mai ƙarfin lantarki. Raba metabolites a cikin saurin kwarara na 380μl/min, ana amfani da wannan juzu'i: minti 0 zuwa 3, 10 mM KOH; minti 3 zuwa 12, minti 10 zuwa 50 mM KOH; minti 12 zuwa 19, 50 zuwa 100 mM KOH; minti 19 zuwa 21, 100 mM KOH; minti 21 zuwa 21.5, minti 100 zuwa 10 mM KOH. An sake daidaita ginshiƙin a ƙarƙashin 10 mM KOH na minti 8.5.
Ana haɗa metabolites ɗin da aka fitar tare da kwararar isopropanol 150μl/min bayan ginshiƙi sannan a tura su zuwa na'urar auna taro mai ƙuduri mai girma wacce ke aiki a yanayin ionization mara kyau. MS tana sa ido kan kewayon taro daga m/z 50 zuwa 750 tare da ƙudurin 60,000. An saita AGC zuwa 1×106, kuma matsakaicin lokacin ion ana kiyaye shi a 100 ms. An sarrafa tushen ESI mai zafi a ƙarfin feshi na 3.5 kV. Sauran saitunan tushen ion sune kamar haka: zafin capillary 275°C; kwararar iskar gas ta sheath, 60 AU; kwararar iskar gas mai taimako, 20 AU a 300°C, da saitin ruwan tabarau na S zuwa 60 AU.
Binciken bayanai na metabolites masu lakabin 13C. Yi amfani da manhajar TraceFinder (sigar 4.2, Thermo Fisher Scientific) don nazarin bayanai na rabon isotope. An tabbatar da asalin kowane mahaɗi ta hanyar ingantaccen mahaɗin tunani kuma an yi nazari kansa. Domin yin nazarin wadatar isotope, an cire yankin chromatogram na ion da aka cire (XIC) na kowane isotope na 13C (Mn) daga [M + H] +, inda n shine adadin carbon na mahaɗin da aka yi niyya, wanda aka yi amfani da shi don nazarin amino acid ko [MH] + ana amfani da shi don nazarin anions. Daidaiton taro na XIC bai wuce sassa biyar a kowace miliyan ba, kuma daidaiton RT shine mintuna 0.05. Ana yin nazarin wadatarwa ta hanyar ƙididdige rabon kowane isotope da aka gano zuwa jimlar duk isotopes na mahaɗin da ya dace. Ana ba da waɗannan rabo azaman ƙimar kashi ga kowane isotope, kuma ana bayyana sakamakon a matsayin wadatar yawan molar percentage (MPE), kamar yadda aka bayyana a baya (42).
An haɗa ƙwayar neuron da aka daskare a cikin methanol mai sanyi 80% (v/v), aka yi tururi, sannan aka saka a cikin -20°C na minti 30. A sake juya samfurin a juya a +4°C na minti 30. An sanya centrifuge a 21,000 g na minti 5 a 4°C, sannan aka tattara supernatant ɗin da ya fito aka busar da shi ta amfani da na'urar tattara SpeedVac a 25°C don bincike na gaba. Kamar yadda aka bayyana a sama, an yi nazarin LC-MS akan amino acid na ƙwayoyin da aka ware. Ta amfani da TraceFinder (sigar 4.2, Thermo Fisher Scientific), an yi nazarin bayanai ta amfani da nauyin monoisotopic na kowane mahaɗi. An yi daidaita adadin bayanai na metabolite ta amfani da fakitin software na preprocessCore (57).
Shirya yanka. An yi wa linzamin kwamfuta sabulu da sauri da carbon dioxide sannan aka yanke masa kai, aka cire kwakwalwa da sauri daga kwanyar, sannan aka yi amfani da wuka mai girgiza da aka cika da kankara (HM-650 V, Thermo Fisher Scientific, Walldorf, Jamus) don yanke ta zuwa sassan sagittal 300 zuwa 375 μm na iskar gas mai sanyi (95% O2 da 5% CO2) Ƙaramin Ca2 + ACSF (125.0 mM NaCl, 2.5 mM KCl, 1.25 mM sodium phosphate buffer, 25.0 mM NaHCO3, 25.0 mM d-glucose, 1.0 mM CaCl2 da 6.0 mM MgCl2 Daidaita zuwa pH 7.4 da 310 zuwa 330 mOsm). A mayar da sassan kwakwalwar da aka samu zuwa wani ɗaki mai ɗauke da mafi girman Ca2 + ACSF (125.0 mM NaCl, 2.5 mM KCl, 1.25 mM sodium phosphate buffer, 25.0 mM NaHCO3, 25.0 mM d-glucose, 4.0 mM CaCl2 da mM 3.5 MgCl2) pH 7.4 da 310 zuwa 320 mOsm). A ajiye sassan na tsawon mintuna 20 zuwa 30 domin a iya mayar da su kafin a yi rikodi.
rikodi. An yi amfani da wani ma'aunin microscope wanda aka sanye shi da ɗakin rikodi mai tsayayyen tsari da kuma ruwan tabarau mai nutsewa cikin ruwa sau 20 (Scientifica) don duk rikodin. An gano ƙwayoyin Purkinje masu kama da juna ta (i) girman jiki, (ii) wurin da cerebellum yake, da kuma (iii) bayyanar kwayar halittar mai ba da rahoto mai haske ta mtYFP. An fitar da bututun faci mai juriya daga gefuna na megohms 5 zuwa 11 ta hanyar amfani da murfin gilashin borosilicate (GB150-10, 0.86 mm×1.5 mm×100 mm, Science Products, Hofheim, Jamus) da kuma Kayan Aiki na Pipette na kwance (P-1000, Sutter), Novato, CA. An yi duk rikodin ta hanyar amplifier na ELC-03XS npi patch clamp amplifier (npi electronic GmbH, Tam, Jamus), wanda software Signal (sigar 6.0, Cambridge Electronic, Cambridge, UK) ke sarrafawa. An yi gwajin a cikin ƙimar samfurin 12.5 kHz. Ana tace siginar da matattarar Bessel guda biyu masu gajeren wucewa tare da mitoci na yankewa na 1.3 da 10 kHz bi da bi. Ana rama ƙarfin membrane da pipette ta hanyar da'irar diyya ta amfani da amplifier. An yi duk gwaje-gwajen a ƙarƙashin ikon kyamarar Orca-Flash 4.0 (Hamamatsu, Gerden, Jamus), wacce software na Hokawo ke sarrafawa (sigar 2.8, Hamamatsu, Gerden, Jamus).
Tsarin ƙwayoyin halitta da bincike na yau da kullun. Nan da nan kafin yin rikodi, cika pipette da maganin ciki wanda ke ɗauke da waɗannan abubuwa: 4.0 mM KCl, 2.0 mM NaCl, 0.2 mM EGTA, 135.0 mM potassium gluconate, 10.0 mM Hepes, 4.0 mM ATP (Mg), 0.5 mM Guanosine triphosphate (GTP) (Na) da 10.0 mM creatinine phosphate an daidaita su zuwa pH 7.25, kuma matsin lambar osmotic shine 290 mOsm (sucrose). Nan da nan bayan amfani da ƙarfin 0 pA don karya membrane, an auna ƙarfin membrane mai hutawa. Ana auna juriyar shigarwa ta hanyar amfani da kwararar iska mai yawan polarized na -40, -30, -20, da -10 pA. Auna girman martanin ƙarfin lantarki kuma yi amfani da dokar Ohm don ƙididdige juriyar shigarwa. An yi rikodin ayyukan da ba zato ba tsammani a cikin maƙallin ƙarfin lantarki na tsawon mintuna 5, kuma an gano sPSC kuma an auna ta a cikin Igor Pro (sigar 32 7.01, WaveMetrics, Lake Oswego, Oregon, Amurka) ta amfani da rubutun gane atomatik na semi-atomatik. Ana auna lanƙwasa na IV da kuma yanayin tsayayyen yanayi ta hanyar matse batirin a wurare daban-daban (farawa daga -110 mV) da kuma ƙara ƙarfin lantarki a matakai 5 mV. An gwada samar da AP ta hanyar amfani da hasken depolarizing. Matse tantanin halitta a -70 mV yayin amfani da bugun depolarizing current. Daidaita girman matakin kowane na'urar rikodi daban (10 zuwa 60 pA). Lissafa matsakaicin mitar AP ta hanyar ƙidaya ƙwanƙwasa bugun jini da hannu wanda ke haifar da mafi girman mitar AP. Ana nazarin matakin AP ta amfani da na biyu na bugun depolarization wanda da farko ke haifar da ɗaya ko fiye APs.
Tsarin facin da aka huda da kuma nazarinsa. Yi rikodin facin da aka huda ta amfani da ƙa'idodi na yau da kullun. Yi amfani da pipette mara ATP da GTP wanda ba ya ƙunshi waɗannan sinadaran: 128 mM gluconate K, 10 mM KCl, 10 mM Hepes, 0.1 mM EGTA da 2 mM MgCl2, kuma a daidaita shi zuwa pH 7.2 (ta amfani da KOH). An cire ATP da GTP daga maganin cikin jiki don hana shigar membrane na tantanin halitta ba tare da kulawa ba. An cika patch pipette da maganin ciki mai ɗauke da amphotericin (kimanin 200 zuwa 250μg/ml; G4888, Sigma-Aldrich) don samun rikodin facin da aka huda. An narkar da Amphotericin a cikin dimethyl sulfoxide (ƙarshen taro: 0.1 zuwa 0.3%; DMSO; D8418, Sigma-Aldrich). Yawan DMSO da aka yi amfani da shi bai yi wani tasiri mai mahimmanci ga ƙwayoyin jijiyoyi da aka yi nazari a kansu ba. A lokacin aikin huda, ana ci gaba da sa ido kan juriyar tashar (Ra), kuma an fara gwajin bayan girman Ra da AP sun daidaita (minti 20-40). Ana auna ayyukan da ba zato ba tsammani a cikin matsewar wutar lantarki da/ko na yanzu na tsawon mintuna 2 zuwa 5. An yi nazarin bayanai ta amfani da Igor Pro (sigar 7.05.2, WaveMetrics, Amurka), Excel (sigar 2010, Microsoft Corporation, Redmond, Amurka) da GraphPad Prism (sigar 8.1.2, GraphPad Software Inc., La Jolla, CA). Amurka). Domin gano APs na bazata, ana amfani da plugin na NeuroMatic v3.0c na IgorPro. Gano APs ta atomatik ta amfani da iyaka da aka bayar, wanda aka daidaita daban-daban ga kowane rikodi. Ta amfani da tazara tazara tazara, ƙayyade mitar karuwa tare da matsakaicin mitar karuwa nan take da matsakaicin mitar karuwa.
Warewar PN. Ta hanyar daidaitawa da ka'idar da aka buga a baya, an tsarkake PNs daga cerebellum na linzamin kwamfuta a wani mataki da aka ƙayyade (58). A takaice, an yanke cerebellum ɗin kuma an niƙa shi a cikin ruwan sanyi mai sanyi [ba tare da HBSS Ca2+ da Mg2+ ba, an ƙara masa 20 mM glucose, penicillin (50 U/ml) da streptomycin (0.05 mg/ml)], sannan a narke maganin a cikin papain [HBSS, wanda aka ƙara masa 1-cysteine·HCl (1 mg/ml), papain (16 U/ml) da deoxyribonuclease I (DNase I; 0.1 mg/ml)] A sha na minti 30 a zafin jiki na 30°C. Da farko a wanke kyallen takarda a cikin maganin HBSS wanda ke ɗauke da majina na ƙwai (10 mg/ml), BSA (10 mg/ml) da DNase (0.1 mg/ml) a zafin ɗaki don hana narkewar enzymatic, sannan a cikin maganin HBSS wanda ke ɗauke da glucose 20 mM. A niƙa a hankali a cikin HBSS, penicillin (50 U/ml), streptomycin (0.05 mg/ml) da DNase (0.1 mg/ml) ƙwayoyin da suka saki. An tace dakatarwar da aka samu ta hanyar tace ƙwayar tantanin halitta ta 70μm, sannan aka cire ƙwayoyin ta hanyar centrifugation (1110 rpm, mintuna 5, 4°C) sannan a sake dakatar da su a cikin maganin rarrabawa [HBSS, wanda aka ƙara masa 20 mM glucose, 20% na naman sa na tayi), penicillin (50 U/ml) da streptomycin (0.05 mg/ml)]; a kimanta yuwuwar ƙwayoyin tantanin halitta tare da propidium iodide kuma a daidaita yawan ƙwayoyin zuwa 1×106 zuwa 2×106 ƙwayoyin/ml. Kafin a fara amfani da cytometry, an tace dakatarwar ta hanyar tace ƙwayoyin halitta mai girman μm 50.
Mai auna kwararar ƙwayoyin halitta. An yi rarraba ƙwayoyin halitta a zafin 4°C ta amfani da na'urar FACSAria III (BD Biosciences) da software na FACSDiva (BD Biosciences, sigar 8.0.1). An rarraba dakatarwar ƙwayoyin halitta ta amfani da bututun ƙarfe 100 μm a ƙarƙashin matsin lamba na 20 psi a ƙimar ~2800 abubuwan da suka faru/sec. Tunda ka'idojin gating na gargajiya (girman ƙwayoyin halitta, wariyar bimodal, da halayen warwatsewa) ba za su iya tabbatar da warewar PN daidai daga sauran nau'ikan ƙwayoyin ba, an saita dabarun gating bisa ga kwatanta kai tsaye na ƙarfin YFP da autofluorescence a cikin mitoYFP+ ​​​​da mitoYFP − Mice. YFP yana jin daɗi ta hanyar haskaka samfurin da layin laser 488 nm, kuma ana gano siginar ta amfani da matatar wucewa ta band 530/30 nm. A cikin mice na mitoYFP+, ana amfani da ƙarfin dangin kwayar halittar Rosa26-mitoYFP don bambance jikin jijiyoyi da gutsuttsuran axon. Ana amfani da na'urar laser mai launin rawaya ta 561 nm kuma an gano ta da matattarar bandpass ta 675/20 nm don cire ƙwayoyin da suka mutu. Domin raba ƙwayoyin astrocytes a lokaci guda, an yi wa dakatarwar tantanin halitta fenti da ACSA-2-APC, sannan aka yi wa samfurin haske da layin laser na 640 nm, sannan aka yi amfani da matattarar bandpass ta 660/20 nm don gano siginar.
An yi wa ƙwayoyin da aka tattara pellet ta hanyar amfani da centrifugation (1110 rpm, mintuna 5, 4°C) sannan aka adana su a -80°C har sai an yi amfani da su. Ana rarraba beraye Mfn2cKO da 'yan kwikwiyon su a rana ɗaya don rage bambancin tsari. An gudanar da gabatar da bayanai da kuma nazarin FACS ta amfani da software na FlowJo (FlowJo LLC, Ashland, Oregon, Amurka).
Kamar yadda aka ambata a sama (59), ana amfani da PCR na ainihin lokaci don ware DNA daga ƙwayoyin jijiyoyi da aka tsara don ƙididdige mtDNA na gaba. An fara gwada daidaiton layi da ƙarfin ma'auni ta hanyar gudanar da qPCR akan lambobi daban-daban na ƙwayoyin halitta. A takaice, tattara PN 300 a cikin ma'aunin lysis wanda ya ƙunshi 50 mM tris-HCl (pH 8.5), 1 mM EDTA, 0.5% Tween 20 da proteinase K (200 ng/ml) sannan a saka a cikin 55°C na mintuna 120. An ƙara sanya ƙwayoyin a cikin 95°C na mintuna 10 don tabbatar da cikakken dakatar da proteinase K. Ta amfani da binciken TaqMan (Thermo Fisher) wanda ya keɓance ga mt-Nd1, an auna mtDNA ta hanyar semi-quantitative PCR a cikin tsarin 7900HT Real-Time PCR (Thermo Fisher Scientific). Kimiyya, lambar kasida Mm04225274_s1), mt-Nd6 (Thermo Fisher Scientific, lambar kasida AIVI3E8) da 18S (Thermo Fisher Scientific, lambar kasida Hs99999901_s1) kwayoyin halitta.
Shirya samfurin Proteome. Ta hanyar dumama maganin a zafin 95°C na minti 10 da kuma sonicating, a cikin lysis buffer [6 M guanidine chloride, 10 mM tris(2-carboxyethyl) phosphine hydrochloride, 10 mM chloroacetamide da 100 mM tris-Lyse daskararrun ƙwayoyin neuron a cikin HCl]. A kan Bioruptor (Diagenode) na minti 10 (daƙiƙa 30 bugun jini / daƙiƙa 30 na dakatarwa). An narkar da samfurin 1:10 a cikin 20 mM tris-HCl (pH 8.0), an haɗa shi da 300 ng trypsin gold (Promega), sannan aka sanya shi cikin dare a zafin 37°C don cimma cikakken narkewar abinci. A rana ta biyu, an narkar da samfurin a 20,000 g na minti 20. An narkar da ruwan da ke cikinsa da 0.1% formic acid, kuma an narkar da ruwan da kansa da StageTips. An busar da samfurin a cikin kayan aikin SpeedVac (Eppendorf concentrator plus 5305) a zafin jiki na 45°C, sannan aka dakatar da peptide ɗin a cikin 0.1% formic acid. An shirya dukkan samfuran a lokaci guda ta mutum ɗaya. Domin yin nazarin samfuran astrocyte, an yiwa peptides masu narkewa 4 μg lakabi da tandem mass tag (TMT10plex, lambar kasida 90110, Thermo Fisher Scientific) tare da rabon peptide zuwa TMT reagent na 1:20. Don lakabin TMT, an sake dakatar da 0.8 mg na TMT reagent a cikin 70 μl na anhydrous ACN, kuma an sake haɗa peptide ɗin busasshen zuwa 9 μl na 0.1 M TEAB (triethylammonium bicarbonate), wanda aka ƙara 7 μl na TMT reagent a cikin ACN. Yawan ya kasance 43.75%. Bayan mintuna 60 na haɗuwa, an kashe amsawar da 2 μl na 5% hydroxylamine. An tattara peptides ɗin da aka yiwa lakabi, an busar da su, an sake haɗa su a cikin 200μl na 0.1% formic acid (FA), an raba su biyu, sannan aka tace gishirin ta amfani da StageTips da aka yi da kanka. Ta amfani da UltiMate 3000 ultra high performance liquid chromatograph (UltiMate 3000 ultra high performance liquid chromatograph), an raba ɗaya daga cikin rabi biyun a kan ginshiƙin chromatographic na Acquity 1mm x 150mm wanda aka cika da ƙwayoyin C18 130Å1.7μm (Waters, catalog No. SKU: 186006935). Thermo Fisher Scientific). Raba peptides a cikin ƙimar kwarara na 30μl/min, an raba daga 1% zuwa 50% buffer B na minti 85 tare da gradient na mataki-mataki na minti 96, daga 50% zuwa 95% buffer B na minti 3, sannan mintuna 8 don 95% Buffer B; Buffer A shine kashi 5% na ACN da 10 mM na ammonium bicarbonate (ABC), kuma buffer B shine kashi 80% na ACN da 10 mM na ABC. A tattara sassan a kowane minti 3 sannan a haɗa su zuwa ƙungiyoyi biyu (1 + 17, 2 + 18, da sauransu) sannan a busar da su a cikin injin centrifuge mai amfani da injin tsabtace iska.
Binciken LC-MS/MS. Don nazarin mass spectrometry, an raba peptides (lambar r119.aq) a kan ginshiƙin bincike na ciki na PicoFrit mai girman cm 25, 75 μm (sabon ruwan tabarau mai ma'ana, lambar sashi PF7508250) wanda aka sanye shi da matsakaicin ReproSil-Pur 120 C18-AQ 1.9 μm (Dr. Maisch, mat), Yi amfani da EASY-nLC 1200 (Thermo Fisher Scientific, Jamus). An kiyaye ginshiƙin a 50°C. Buffers A da B suna da 0.1% formic acid a cikin ruwa da 0.1% formic acid a cikin 80% ACN, bi da bi. An raba Peptides daga 6% zuwa 31% buffer B na tsawon mintuna 65 da kuma daga 31% zuwa 50% buffer B na tsawon mintuna 5 tare da gradient na 200 nl/min. An yi nazarin peptides ɗin da aka fitar a kan na'urar auna yawan Orbitrap Fusion (Thermo Fisher Scientific). Ana yin ma'aunin peptide precursor m/z tare da ƙuduri na 120,000 a cikin kewayon 350 zuwa 1500 m/z. Ta amfani da makamashin karo na yau da kullun 27%, an zaɓi mafi ƙarfin precursor tare da yanayin caji na 2 zuwa 6 don yankewar tarko mai ƙarfi na C (HCD). An saita lokacin zagayowar zuwa 1 s. An auna ƙimar m/z na ɓangaren peptide a cikin tarkon ion ta amfani da ƙaramin maƙasudin AGC na 5 × 104 da matsakaicin lokacin allura na 86 ms. Bayan rarrabuwa, an sanya precursor akan jerin keɓancewa masu ƙarfi na daƙiƙa 45. An raba peptides masu alamar TMT akan ginshiƙin Acclaim PepMap mai tsawon cm 50, 75 μm (Thermo Fisher Scientific, lambar kasida 164942), kuma an yi nazarin spectra na ƙaura akan na'urar auna ma'aunin Orbitrap Lumos Tribrid (Thermo Fisher Scientific) wacce aka sanye da kayan aikin ions masu girman filin asymmetric waveform (FAIMS) (Thermo Fisher Scientific) tana aiki a ƙarfin lantarki guda biyu na −50 da −70 V. Ana amfani da MS3 da aka zaɓa bisa ga precursor na daidaitawa don auna siginar ion rahoton TMT. An gudanar da rabuwar peptide akan EASY-nLC 1200, ta amfani da 90% linear gradient exution, tare da yawan buffer na 6% zuwa 31%; buffer A shine 0.1% FA, kuma buffer B shine 0.1% FA da 80% ACN. Ana gudanar da ginshiƙin nazari a 50°C. Yi amfani da FreeStyle (sigar 1.6, Thermo Fisher Scientific) don raba fayil ɗin asali bisa ga ƙarfin diyya na FAIMS.
Gano furotin da ƙididdigewa. Ta amfani da injin binciken Andromeda da aka haɗa, an yi nazarin asalin bayanan ta amfani da MaxQuant sigar 1.5.2.8 (https://maxquant.org/). Baya ga jerin Cre recombinase da YFP da aka samu daga Aequorea victoria, an nemi spectra na peptide fragment don jerin canonical da jerin isoform na linzamin kwamfuta proteome (Proteome ID UP000000589, wanda aka sauke daga UniProt a watan Mayu 2017). An saita oxidation na methionine da furotin N-terminal acetylation azaman gyare-gyare masu canzawa; an saita cysteine ​​​​carbamoyl methylation azaman gyare-gyare masu tsayayye. An saita sigogin narkewar abinci zuwa "takamaiman" da "trypsin/P". Mafi ƙarancin adadin peptides da peptides na razor da ake amfani da su don gano furotin shine 1; mafi ƙarancin adadin peptides na musamman shine 0. A ƙarƙashin yanayin daidaitawar taswirar peptide, ƙimar gano furotin shine 0.01. An kunna zaɓin "Peptide na Biyu". Yi amfani da zaɓin "daidaitawa tsakanin gudu" don canja wurin ganowa mai nasara tsakanin fayiloli na asali daban-daban. Yi amfani da ƙimar rabo mafi ƙaranci na LFQ 1 don ƙididdigewa mara lakabi (LFQ) (60). Ana tace ƙarfin LFQ don aƙalla ƙima biyu masu inganci a cikin aƙalla rukunin genotype ɗaya a kowane lokaci, kuma ana cire shi daga rarrabawa na yau da kullun tare da faɗin . 0.3 kuma a matsa ƙasa 1.8. Yi amfani da dandamalin kwamfuta na Perseus (https://maxquant.net/perseus/) da R (https://r-project.org/) don nazarin sakamakon LFQ. An yi amfani da gwajin t mai matsakaici na hanyoyi biyu daga fakitin software na limma don nazarin bayyanar bambance-bambancen (61). Ana gudanar da nazarin bayanai ta amfani da ggplot, FactoMineR, factoextra, GGally da pheatmap. An yi nazarin bayanan proteomics na tushen TMT ta amfani da MaxQuant sigar 1.6.10.43. Nemi bayanai na proteomics daga bayanan UniProt na ɗan adam na proteomics, wanda aka sauke a watan Satumba na 2018. Binciken ya haɗa da ma'aunin gyara tsarkin isotope da masana'anta suka bayar. Yi amfani da limma a cikin R don nazarin bayyanar bambance-bambance. Bayanan asali, sakamakon binciken bayanai, da aikin nazarin bayanai da sakamako duk an adana su a cikin haɗin gwiwar ProteomeXchange ta hanyar ma'ajiyar abokin hulɗar PRIDE tare da mai gano saitin bayanai PXD019690.
Bayanan aiki suna ƙara wa binciken ƙarfi. An yi amfani da kayan aikin Binciken Hanyar Ingenuity (QIAGEN) don tantance wadatar sharuɗɗan bayanin aiki na bayanan da aka saita a makonni 8 (Hoto na 1). A takaice, ana amfani da jerin sunadaran adadi da aka samu daga nazarin bayanai na LC-MS/MS (tandem mass spectrometry) tare da waɗannan sharuɗɗan tacewa: An zaɓi Musculus a matsayin nau'in da asalinsa, kuma rukunin yana nuna ƙimar P da Benjamini ya daidaita don wadatarwa 0.05 ko ƙasa da haka ana ɗaukarsa da mahimmanci. Ga wannan jadawalin, an nuna manyan rukunoni biyar da suka wuce gona da iri a cikin kowane rukuni bisa ga ƙimar P da aka gyara. Ta amfani da gwajin t da yawa, ta amfani da shirin haɓaka layi biyu na Benjamini, Krieger, da Yekutieli (Q = 5%), ana yin nazarin bayyanar furotin na lokaci-lokaci akan mahimman 'yan takara da aka gano a cikin kowane rukuni, kuma ana nazarin kowane layi daban. Babu buƙatar ɗaukar SD mai daidaito.
Domin kwatanta sakamakon wannan binciken da bayanan da aka buga da kuma samar da zane na Venn a Hoto na 1, mun haɗa jerin sunadaran adadi tare da bayanin MitoCarta 2.0 (24). Yi amfani da kayan aikin kan layi Zana Venn Diagram (http://bioinformatics.psb.ugent.be/webtools/Venn/) don samar da zane.
Don cikakken bayani game da hanyoyin ƙididdiga da ake amfani da su don nazarin proteomics, da fatan za a duba sashin da ya dace na Kayan Aiki da Hanyoyi. Ga duk sauran gwaje-gwaje, ana iya samun cikakken bayani a cikin tatsuniyar da ta dace. Sai dai idan aka ƙayyade akasin haka, duk bayanan ana bayyana su a matsayin matsakaicin ± SEM, kuma duk nazarin ƙididdiga an yi su ta amfani da software na GraphPad Prism 8.1.2.
Don ƙarin kayan aiki don wannan labarin, da fatan za a duba http://advances.sciencemag.org/cgi/content/full/6/35/eaba8271/DC1
Wannan wani labari ne da aka rarraba a ƙarƙashin sharuɗɗan Lasisin Creative Commons Attribution-Non-Commercial, wanda ke ba da damar amfani, rarrabawa da sake bugawa a kowace hanya, matuƙar amfani na ƙarshe ba don ribar kasuwanci ba ne kuma hujjar ita ce ainihin aikin daidai ne. Nassoshi.
Lura: Muna roƙon ka ne kawai ka bayar da adireshin imel ɗinka domin mutumin da ka ba da shawarar zuwa shafin ya san kana son ya ga imel ɗin kuma ba spam ba ne. Ba za mu kama duk wani adireshin imel ba.
Ana amfani da wannan tambayar don gwada ko kai baƙo ne kuma hana aika saƙonnin banza ta atomatik.
By E. Motori, I. Atanassov, SMV Kochan, K. Folz-Donahue, V. Sakthivelu, P. Giavalisco, N. Toni, J. Puyal, N.-G. Larson
Binciken proteomics na neurons marasa aiki ya nuna cewa shirye-shiryen metabolism suna aiki don magance neurodegeneration.
By E. Motori, I. Atanassov, SMV Kochan, K. Folz-Donahue, V. Sakthivelu, P. Giavalisco, N. Toni, J. Puyal, N.-G. Larson
Binciken proteomics na neurons marasa aiki ya nuna cewa shirye-shiryen metabolism suna aiki don magance neurodegeneration.
©2020 Ƙungiyar Amirka don Ci gaban Kimiyya. duk haƙƙin mallaka. AAAS abokin tarayya ne na HINARI, AGORA, OARE, CHORUS, CLOCKSS, CrossRef da COUNTER. KimiyyaCi gaban ISSN 2375-2548.