Fifia *Adrɛs si li fifia: Cologne 50931, Germany, Cologne Excellence Cluster Numekuku le Lãmenugbagbeviwo ƒe Nuteɖeamedzi ƒe Ŋuɖoɖo le Tsitsi ƒe Dɔlélewo me (CECAD).
Wobua mitochondrial dɔlélewo ƒe ahɔhɔ̃mekawo ƒe gbegblẽ be womate ŋu atrɔe o elabena ahɔhɔ̃mekawo ƒe lãmenugbagbeviwo ƒe dɔwɔwɔ ƒe asitɔtrɔ le wo ŋu mesɔ gbɔ o, gake womese ŋusẽ si mitochondria ƒe dɔmawɔmawɔ nyuie kpɔna ɖe ahɔhɔ̃mekawo ƒe lãmenugbagbeviwo ƒe dɔwɔwɔ le ŋutilã me ƒe lãmenugbagbeviwo ƒe ɖokuisinɔnɔ dzi gɔme nyuie o. Le afisia la, míeto Purkinje ahɔhɔ̃mekawo ƒe lãmenugbagbevi tɔxɛ proteome si me OXPHOS ƒe anyimanɔmanɔ vivivi si tso mitochondrial fusion dynamics si tɔtɔ gbɔ la vɛ. Míekpɔe be mitochondria ƒe dɔmawɔmawɔ nyuie he tɔtrɔ gã aɖe vɛ le proteomics ƒe akpa dzi, si na mlɔeba be lãmenugbagbeviwo ƒe dɔwɔwɔ ƒe ɖoɖo siwo sɔ pɛpɛpɛ ƒe dɔwɔwɔ ɖe wo nɔewo yome hafi lãmenugbagbeviwo ku. Le vome la, míekpɔ alesi pyruvate carboxylase (PCx) kple enzyme bubu siwo tsia tsitsi nu siwo kpena ɖe TCA ƒe tsatsam ƒe domedomenuwo ŋu ƒe dɔwɔwɔ si dze ƒã. PCx ƒe mɔxexe ɖe enu na oxidative stress kple ahɔhɔ̃mekawo ƒe dɔmawɔmawɔ nyuie gasẽ ɖe edzi, si ɖee fia be ʋukawo ƒe ʋuʋu kpɔa ame ta le ahɔhɔ̃meka siwo me OXPHOS mele o me. Mitochondrial fusion gbugbɔgaɖoanyi le ahɔhɔ̃meka siwo gblẽ le nuwuwu me trɔa asi le lãmenugbagbeviwo ƒe dɔwɔwɔ ƒe nɔnɔme siawo ŋu keŋkeŋ, si wɔnɛ be lãmenugbagbeviwo ƒe kuku xea mɔ na wo. Nusiwo ŋu míeke ɖo la de dzesi mɔ siwo míenya tsã o siwo naa ame te ŋu nɔa te ɖe mitochondria ƒe dɔmawɔmawɔ nyuie nu eye wòɖenɛ fiana be woate ŋu atrɔ ahɔhɔ̃mekawo ƒe dɔmawɔmawɔ nyuie le dɔlélea ƒe nuwuwu gɔ̃ hã.
Wote gbe ɖe akpa vevi si mitochondria wɔna le ahɔhɔ̃mekawo ƒe ŋusẽ ƒe asitɔtrɔ me léle ɖe asi me dzi to ahɔhɔ̃mekawo ƒe dzesi gbogbo siwo do ƒome kple amegbetɔ ƒe mitochondria dɔlélewo me. Dɔléle siawo ƒe akpa gãtɔ tsoa domenyiŋusẽfianu ƒe tɔtrɔ siwo kpɔa mitochondrial domenyiŋusẽfianu ƒe ɖeɖefia dzi (1, 2) alo domenyiŋusẽfianu ƒe tsɔtsrɔ̃ si do ƒome kple mitochondrial dynamics, si kpɔa ŋusẽ ɖe mitochondrial DNA (mtDNA) ƒe liƒo dzi le mɔ si mele tẽ o nu (3, 4). Dɔwɔwɔ le lãwo ƒe kpɔɖeŋuwo me ɖee fia be le mitochondria ƒe dɔmawɔmawɔ nyuie le lãkusi siwo ƒo xlãe me ta la, woate ŋu awɔ dɔ na lãmenugbagbeviwo ƒe dɔwɔwɔ ƒe mɔnu siwo me wodzraa nu ɖo (5-7), si naa nyatakaka veviwo hena dɔléle sesẽ siawo ƒe dɔléle dzɔdzɔ gɔmesese deto. To vovo kura la, míaƒe gɔmesese le lãmenugbagbevi ƒomevi aɖewo koŋ ƒe lãmenugbagbeviwo ƒe dɔwɔwɔ ƒe tɔtrɔ siwo ahɔhɔ̃ me mitochondrial adenosine triphosphate (ATP) wɔwɔ ƒe kpododonu le goawo katã me hena vɛ ŋu nye nu vevi aɖe (8), si te gbe ɖe alesi wòhiã be woade dzesi atikewɔwɔ ƒe taɖodzinu siwo woate ŋu azã atsɔ axe mɔ ɖe dɔléle nu alo axe mɔ ɖe enu dzi. Xɔ mɔ ɖe ahɔhɔ̃mekawo ƒe dɔmawɔmawɔ nyuie nu (9). Nyatakaka ƒe anyimanɔmanɔe nye nyateƒe si wònye be ame geɖe bua lãmekawo ƒe lãmenugbagbeviwo be woƒe lãmenugbagbeviwo ƒe dɔwɔwɔ mete ŋu trɔna ɖe nɔnɔmewo ŋu boo o ne wotsɔe sɔ kple lãmenugbagbevi ƒomevi siwo le lãkusi siwo ƒo xlã wo me (10). Esi wònye be lãmenugbagbevi siawo wɔa akpa vevi aɖe le ɖoɖowɔwɔ ɖe lãmenugbagbeviwo ƒe dɔwɔwɔ ŋu na ahɔhɔ̃mekawo be woado lãmenugbagbeviwo ƒe dɔwɔwɔ ɖe ŋgɔ eye woawɔ nu ɖe ​​abi kple dɔléle ƒe nɔnɔmewo ŋu ta la, ŋutete si le wo si be woatrɔ asi le lãmenugbagbeviwo ƒe dɔwɔwɔ ŋu wòasɔ ɖe ahɔhɔ̃mekawo ƒe nɔnɔme sesẽwo nu la se ɖe glial lãmenugbagbeviwo ko kloe (11-14). Tsɔ kpe ɖe eŋu la, ahɔhɔ̃mekawo ƒe lãmenugbagbeviwo ƒe vovototo si le dzɔdzɔme nu xea mɔ na nusɔsrɔ̃ tso lãmenugbagbeviwo ƒe tɔtrɔ siwo dzɔna le ahɔhɔ̃mekawo ƒe hatsotso sue aɖewo koŋ me ŋu vevie. Esia wɔe be womenya nu boo aɖeke tso lãmenugbagbeviwo kple lãmenugbagbeviwo ƒe dɔwɔwɔ me tsonu siwo tututu dona tso mitochondria ƒe dɔmawɔmawɔ nyuie le ahɔhɔ̃mekawo me ŋu o.
Be míase nusiwo dona tso mitochondrial ƒe dɔmawɔmawɔ nyuie me gɔme la, míeɖe Purkinje ahɔhɔ̃mekawo (PNs) ɖe vovo le ahɔhɔ̃mekawo ƒe dɔmawɔmawɔ nyuie ƒe akpa vovovo siwo mitochondrial gotagome lãkusi ƒe fusion (Mfn2) ƒe tsɔtsrɔ̃ hena vɛ. Togbɔ be Mfn2 ƒe tɔtrɔ le amegbetɔwo me do ƒome kple domenyiŋusẽfianu ƒe seselelãme ƒe ahɔhɔ̃mekawo ƒe dɔléle ƒomevi aɖe si woyɔna be Charcot-Marie-Tooth type 2A (15) hã la, Mfn2 ƒe tsɔtsrɔ̃ le nɔnɔme aɖe me le sisiblisiwo me nye oxidation Phosphorylation (OXPHOS) ƒe dɔmawɔmawɔ nyuie ƒe mɔnu si wonya nyuie. Ahɔhɔ̃mekawo ƒe hatsotso sue vovovoawo (16-19) kple ahɔhɔ̃mekawo ƒe dɔmawɔmawɔ nyuie ƒe nɔnɔme si dona tso eme la kpena ɖe ahɔhɔ̃mekawo ƒe dzesi siwo yia ŋgɔ, abe ʋuʋu ƒe kuxiwo (18, 19) alo ahɔhɔ̃mekawo ƒe dɔmawɔmawɔ nyuie (16) ene ŋu. To label-free quantitative (LFQ) proteomics, metabolomics, imaging, kple virological mɔnuwo zazã me la, míeɖee fia be ahɔhɔ̃mekawo ƒe gbegblẽ vivivi hea pyruvate carboxylase (PCx) kple nu bubu siwo kpɔa gome le PNs ƒe ʋukawo ƒe ʋuʋu me le vivo Enzymewo ƒe ɖeɖefia vɛ vevie. Be míaɖo kpe nusi ŋu woke ɖo sia ƒe vevienyenye dzi la, míeɖe PCx ƒe ɖeɖefia dzi kpɔtɔ etɔxɛe le PN siwo me Mfn2 mele o me, eye míekpɔe be dɔwɔwɔ sia na oxidative stress nu sẽ eye wòna ahɔhɔ̃mekawo ƒe gbegblẽ kabakaba, si wɔe be míeɖo kpe edzi be azoospermia naa lãmenugbagbeviwo ƒe ku Metabolic adaptability. MFN2 ƒe ɖeɖefia sesẽ ate ŋu aɖe PN si gblẽ le nuwuƒe si me OXPHOS ƒe anyimanɔmanɔ vevie, mitochondrial DNA ɖuɖu fũ, kple mitochondrial network si dze abe ɖe wògbã ene, si gate gbe ɖe edzi be ahɔhɔ̃mekawo ƒe dɔmawɔmawɔ nyuie ƒomevi sia ate ŋu ahaya gɔ̃ hã le dɔlélea ƒe akpa si de ŋgɔ hafi lãmenugbagbeviwo naku.
Be míate ŋu akpɔ mitochondria le susu me le Mfn2 knockout PNs me la, míezã sisiblisi ƒomevi aɖe si ɖea mɔ na mitochondria siwo nɔa te ɖe Cre dzi be woaɖo taɖodzinu na yellow fluorescent protein (YFP) (mtYFP) (20) Cre ƒe ɖeɖefia eye míelé ŋku ɖe mitochondria ƒe nɔnɔme ŋu le vivo. Míekpɔe be Mfn2 domenyiŋusẽfianu ƒe tsɔtsrɔ̃ le PNwo me ana mitochondrial network la nama vivivi (Nɔnɔmetata S1A), eye wokpɔ tɔtrɔ gbãtɔ le kwasiɖa 3 xɔxɔ me. To vovo na ema la, PN lãmenugbagbeviwo ƒe ƒuƒoƒoa ƒe gbegblẽ vevie, abe alesi Calbindin ƒe dɔlélenutsiŋutete ƒe amadede si bu ɖee fia ene la, medze egɔme o vaseɖe esime wòxɔ kwasiɖa 12 (Nɔnɔmetata 1, A kple B). Ɣeyiɣi si mesɔ o le tɔtrɔ gbãtɔ siwo va le mitochondria ƒe nɔnɔme me kple ahɔhɔ̃mekawo ƒe ku ƒe gɔmedzedze si wokpɔna dome na míeku nu me le lãmenugbagbeviwo ƒe tɔtrɔ siwo mitochondria ƒe dɔmawɔmawɔ nyuie hena vɛ hafi lãmenugbagbeviwo ku ŋu. Míewɔ mɔnu si wotu ɖe fluorescence-activated cell sorting (FACS) dzi tsɔ ɖe YFP (YFP +)-ɖea PN fiana (Nɔnɔmetata 1C), kple le dziɖuɖu mice (Mfn2 + / loxP :: mtYFP loxP- stop-loxP: : L7-cre), si woyɔna le afisia be CTRL (Nɔnɔmetata S1B). Gating ƒe aɖaŋu ƒe nyonyome si wotu ɖe YFP ƒe dzesi ƒe sesẽme si sɔ dzi na míete ŋu kɔ YFP + ŋutilã (YFPhigh) si le PNs me tso non-PNs (YFPneg) (Nɔnɔmetata S1B) alo putative fluorescent axon/dendritic fragments (YFPlow; Figure S1D, miame), si ŋu woɖo kpee to confocal microscope (Nɔnɔmetata S1D, ɖusime). Be míate ŋu aɖo kpe ameha si woɖo ɖe hatsotsowo me ƒe dzedzeme dzi la, míewɔ LFQ proteomics eye emegbe míewɔ akpa veviwo me dzodzro, eye míekpɔe be mama si dze ƒã le YFPhigh kple YFPneg lãmenugbagbeviwo dome (Nɔnɔmetata S1C). YFPhigh lãmenugbagbeviwo ɖe PNs dzesi siwo wonya (si nye Calb1, Pcp2, Grid2 kple Itpr3) ƒe dzidziɖedzi ŋutɔŋutɔ fia (21, 22), gake protein siwo woɖena fiana zi geɖe le ahɔhɔ̃mekawo alo lãmenugbagbevi ƒomevi bubuwo me ƒe dzidziɖedzi aɖeke menɔ anyi o (Nɔnɔmetata 1D) ). Kpɔɖeŋu siwo le YFPhigh lãmenugbagbevi siwo woda ɖe hatsotsowo me siwo woƒo ƒu le dodokpɔ siwo wowɔ le wo ɖokui si me tsɔtsɔ sɔ kple wo nɔewo ɖee fia be ƒomedodo ƒe xexlẽdzesi> 0.9, si ɖee fia be woate ŋu agbugbɔ nu gbagbe siwo wogbugbɔ wɔ dome nyuie (Nɔnɔmetata S1E). Kpuie ko la, nyatakaka siawo ɖo kpe míaƒe ɖoɖo si míewɔ ɖe PN si woate ŋu awɔ la ɖeɖe ɖe aga kpata kple esi le tẽ ŋu dzi. Esi wònye be L7-cre ʋukulawo ƒe ɖoɖo si wozã la hea mosaic recombination vɛ le kwasiɖa gbãtɔ me le vidzidzi megbe ( 23 ) ta la, míedze mice wuwu gɔme tso CTRL kple conditional (Mfn2 loxP / loxP :: mtYFP loxP-stop-loxP :: L7-cre) Collect neurons. Ne wowu ƒoƒo gbugbɔgaƒoƒo nu vɔ la, woyɔnɛ be Mfn2cKO le kwasiɖa 4 xɔxɔ me. Abe nuwuƒe ene la, míetia kwasiɖa 8 ƒe ƒexɔxɔ esime PN layer la me gblẽ o togbɔ be mitochondrial ƒe mama dze ƒã hã (Nɔnɔmetata 1B kple Nɔnɔmetata S1A). Le wo katã me la, míebu protein 3013 ƒe agbɔsɔsɔme, siwo dometɔ siwo ade 22% nɔ te ɖe MitoCarta 2.0 ƒe nuŋlɔɖi siwo wotu ɖe mitochondrial proteome dzi abe mitochondria ene (Nɔnɔmetata 1E) (Nɔnɔmetata 1E) (24). Domenyiŋusẽfianu ƒe vovototodedeameme ƒe numekuku si wowɔ le kwasiɖa 8 lia me ɖee fia be proteinwo katã ƒe 10.5% koe tɔtrɔ ɖedzesiwo le (Nɔnɔmetata 1F kple Nɔnɔmetata S1F), siwo dometɔ 195 ƒe dɔwɔwɔ ɖiɖi eye protein 120 ƒe dziyiyi (Nɔnɔmetata 1F). Enyo be míade dzesii be nyatakaka sia ƒe “mɔ yeyewo me dzodzro” ɖee fia be domenyiŋusẽfianu siwo woɖena fiana le mɔ vovovowo nu la nye lãmenugbagbeviwo ƒe dɔwɔwɔ ƒe mɔnu tɔxɛ aɖewo siwo ŋu seɖoƒe li na la tɔ koŋ (Nɔnɔmetata 1G). Enyo be míade dzesii be togbɔ be mɔ siwo do ƒome kple OXPHOS kple calcium ƒe dzesiwo ƒe ɖiɖi ɖo kpe mitochondrial ƒe dɔmawɔmawɔ nyuie ƒe dzɔdzɔ dzi le PN siwo me fusion mele o me hã la, hatsotso bubu siwo me amino acid ƒe asitɔtrɔ koŋ le la dzi ɖe edzi ŋutɔ, si sɔ kple lãmenugbagbeviwo ƒe dɔwɔwɔ si dzɔna le mitochondrial metabolis me Rewiring is consistent. dɔmawɔmawɔ nyuie.
(A) CTRL kple Mfn2cKO mice ƒe ahɔhɔ̃mekawo ƒe akpa siwo le teƒenɔlawo ƒe confocal foto siwo ɖe PNs (calbindin, grey) ƒe bu vivivi fia; wotsɔ DAPI tsɔ tsi tre ɖe nuclei ŋu. (B) Agbɔsɔsɔme (A) (mɔ ɖeka dzi numekuku le vovototo ŋu, ***P <0.001; n = 4 va ɖo 6 nutome tso sisiblisi etɔ̃ me). (C) Dodokpɔ ƒe dɔwɔwɔ ƒe ɖoɖo. (D) Dzoxɔxɔ ƒe nɔnɔmetata ƒe mama na dzesi siwo ku ɖe Purkinje koŋ ŋu (tame) kple lãmenugbagbevi ƒomevi bubuwo (titina). (E) Venn ƒe nɔnɔmetata si ɖe mitochondrial protein ƒe xexlẽme si wokpɔ le PN si woda ɖe hatsotso me me fia. (F) Dzoto ƒe nɔnɔmetata si me protein siwo woɖe fia le mɔ vovovowo nu le Mfn2cKO ahɔhɔ̃mekawo me le kwasiɖa 8 me (vevienyenye ƒe cut-off value of 1.3). (G) Nuwɔwɔ ƒe mɔnu ƒe numekuku ɖe up-regulation (dzẽ) kple down-regulation (blue) mɔ vevitɔ atɔ̃ siwo le Mfn2cKO PN si woda ɖe hatsotso 8 me la fia. Woɖe protein ɖesiaɖe si wokpɔ ƒe ɖeɖefia ƒe agbɔsɔsɔme le mama dedie nu fia. Dzoxɔxɔ ƒe nɔnɔmetata si le ɣie: P ƒe asixɔxɔ si woɖɔli. ns, mele vevie o.
Proteomics nyatakakawo ɖee fia be protein ƒe ɖeɖefia le complex I, III, kple IV me dzi ɖe kpɔtɔ vivivi. MtDNA-si woŋlɔ ɖe mɔ̃memi me ƒe akpa sue veviwo nɔ complex I, III, kple IV katã me, gake le gɔmedzedzea me la, mekpɔ ŋusẽ ɖe complex II, si ŋu nuklia-kɔda koe le dzi o (Nɔnɔmetata 2A kple Nɔnɔmetata S2A). . Le ɖekawɔwɔ me kple proteomics me tsonu la, dɔlélenutsiŋutete ƒe lãmenugbagbeviwo ƒe dɔwɔwɔ le ahɔhɔ̃mekawo ƒe akpawo me ɖee fia be MTCO1 (mitochondrial cytochrome C oxidase subunit 1) ƒe subunit ƒe agbɔsɔsɔ si le complex IV me le PN me dzi ɖe kpɔtɔ vivivi (Nɔnɔmetata 2B). Woɖe mtDNA-si woŋlɔ ɖe Mtatp8 ƒe akpa suea dzi kpɔtɔ ŋutɔ (Nɔnɔmetata S2A), esime nuklia-si woŋlɔ ɖe ATP synthase ƒe akpa suea ƒe nɔnɔme si li ke metrɔ o, si sɔ kple ATP synthase subassembly F1 complex si li ke si wonya ne mtDNA ƒe ɖeɖefia li ke. Nuwɔwɔa sɔ ɖe enu. Dzudzɔ (7). MtDNA ƒe agbɔsɔsɔme ƒe dodokpɔ le Mfn2cKO PN siwo woɖo ɖe hatsotsowo me to ɣeyiɣi ŋutɔŋutɔ me polymerase chain reaction (qPCR) me ɖo kpe mtDNA ƒe kɔpi xexlẽme dzi ɖeɖe kpɔtɔ vivivi dzi. Ne wotsɔe sɔ kple ƒuƒoƒo si me wokpɔa wo gbɔ le la, le kwasiɖa 8 xɔxɔ me la, mtDNA ƒe agbɔsɔsɔ si ade 20% koe Wolé ɖe asi (Nɔnɔmetata 2C). Le ɖekawɔwɔ me kple emetsonu siawo la, wozã confocal microscopy staining of Mfn2cKO PNs tsɔ de dzesi DNA, si ɖe mitochondrial nucleotides zazã si nɔ te ɖe ɣeyiɣi dzi fia (Nɔnɔmetata 2D). Míekpɔe be ame aɖewo siwo kpɔa gome le mitochondrial protein ƒe gbegblẽ kple nuteɖeamedzi ƒe ŋuɖoɖo me koe wodo ɖe dzi, siwo dometɔ aɖewoe nye Lonp1, Afg3l2 kple Clpx, kple OXPHOS complex assembly factors. Womekpɔ tɔtrɔ ɖedzesi aɖeke le protein siwo kpɔa gome le apoptosis me ƒe agbɔsɔsɔ me o (Nɔnɔmetata S2B). Nenema ke míekpɔe be tɔtrɔ sue aɖewo koe le mitochondria kple endoplasmic reticulum channels siwo kpɔa gome le calcium tsɔtsɔ yi teƒe bubuwo me (Nɔnɔmetata S2C). Tsɔ kpe ɖe eŋu la, protein siwo do ƒome kple autophagy ƒe dodokpɔ mekpɔ tɔtrɔ ɖedzesi aɖeke o, si sɔ kple autophagosomes ƒe ʋuʋu si wokpɔna si wokpɔ le vivo to immunohistochemistry kple electron microscopy me (Nɔnɔmetata S3). Gake OXPHOS ƒe dɔmawɔmawɔ nyuie si yia edzi le PNwo me kpena ɖe tɔtrɔ siwo dze ƒã le ultrastructural mitochondrial ŋu. Woateŋu akpɔ mitochondrial ƒuƒoƒo le lãmenugbagbeviwo ƒe ŋutilã kple dendritic atiwo me le Mfn2cKO PN siwo xɔ kwasiɖa 5 kple 8 me, eye tɔtrɔ gãwo va le lãkusi ememetɔ ƒe wɔwɔme me (Nɔnɔmetata S4, A kple B). Le ɖekawɔwɔ me kple ultrastructural ƒe tɔtrɔ siawo kple mtDNA ƒe ɖiɖi ŋutɔ la, numekuku le acute cerebral cerebellar slices ŋu kple tetramethylrhodamine methyl ester (TMRM) ɖee fia be mitochondrial membrane potential le Mfn2cKO PNs me dzi ɖe kpɔtɔ ŋutɔ (Nɔnɔmetata S4C).
(A) Ɣeyiɣi ƒe zɔzɔme me dzodzro le OXPHOS complex ƒe nyagbɔgblɔ ƒe seƒe ŋu. Bu protein siwo ƒe P<0.05 le kwasiɖa 8 me ko ŋu (mɔ eve dzi ANOVA). Fli si dzi woŋlɔ nu ɖo: Asitɔtrɔ aɖeke meli ne wotsɔe sɔ kple CTRL o. (B) Miame: Ahɔhɔ̃ ƒe akpa aɖe si wotsɔ anti-MTCO1 dɔlékuiwutike (scale bar, 20 μm) de dzesii ƒe kpɔɖeŋu. Teƒe si Purkinje lãmenugbagbeviwo ƒe ŋutilãwo xɔ la nye ɣie. Nuɖusi: MTCO1 ƒe agbɔsɔsɔ ƒe agbɔsɔsɔme (mɔ ɖeka dzi numekuku le vovototo ŋu; n = lãmenugbagbevi 7 va ɖo 20 siwo woda kpɔ tso sisiblisi etɔ̃ me). (C) qPCR numekuku le mtDNA kɔpi xexlẽme le PN si woɖo ɖe hatsotsowo me (mɔ ɖeka dzi numekuku le vovototo; n = 3 va ɖo 7 mice). (D) Miame: Ahɔhɔ̃meka si wotsɔ dɔlékuiwutike si tsi tre ɖe DNA ŋu (scale bar, 20 μm) de dzesii ƒe kpɔɖeŋu. Teƒe si Purkinje lãmenugbagbeviwo ƒe ŋutilãwo xɔ la nye ɣie. Nuɖusi: mtDNA ƒe abiwo ƒe agbɔsɔsɔme nyanya (mɔ ɖeka dzi numekuku le vovototo ŋu; n = lãmenugbagbevi 5 va ɖo 9 tso sisiblisi etɔ̃ me). (E) Ahɔhɔ̃meka ƒe akpa si le sesẽm ƒe kpɔɖeŋu si ɖe mitoYFP + Purkinje lãmenugbagbeviwo fia (aŋutsrɔe) le lãmenugbagbevi blibo ƒe patch clamp nuŋlɔɖi me. (F) IV curve ƒe agbɔsɔsɔme nyanya. (G) Teƒenɔla ƒe nuŋlɔɖiwo le depolarizing fifia dodo le CTRL kple Mfn2cKO Purkinje lãmenugbagbeviwo me. Top trace: Pulse gbãtɔ si ʋã AP. Bottom trace: AP ƒe ɣeyiɣi si sɔ gbɔ wu. (H) Agbɔsɔsɔme si le postsynaptic spontaneous inputs (sPSPs) ŋu. Woɖe nuŋlɔɖi ƒe dzesi si le eteƒe kple eƒe lolome ƒe sɔsɔme fia le (I) me. Mɔ ɖeka dzi numekuku le vovototo ŋu da lãmenugbagbevi n = 5 va ɖo 20 kpɔ tso sisiblisi etɔ̃ me. Woɖea nyatakakawo fiana abe mama dedie±SEM ene; *P <0.05 ƒe; **P <0.01 ƒe xexlẽme; ***P <0.001 ƒe xexlẽme. (J) AP si dzɔ le eɖokui si ƒe dzesi siwo woŋlɔ ɖi to perforated patch clamp mode zazã me ƒe dzesiwo teƒenɔlawo. Top trace: AP ƒe ɣeyiɣi si sɔ gbɔ wu. Bottom trace: AP ɖeka ƒe lolome. (K) Bu AP ƒe ɣeyiɣi ƒe didime le mama dedie nu kple esi sɔ gbɔ wu ƒe agbɔsɔsɔme le (J) nu. Mann-Whitney ƒe dodokpɔ; Woda lãmenugbagbevi n = 5 kpɔ tso sisiblisi ene me. Woɖea nyatakakawo fiana abe mama dedie±SEM ene; mele vevie o.
Wokpɔ OXPHOS ƒe nugbegblẽ si dze ƒã le kwasiɖa 8 ƒe Mfn2cKO PN me, si ɖee fia be ahɔhɔ̃mekawo ƒe ŋutilã ƒe dɔwɔwɔ mesɔ kura o. Eyata míebu elektrikŋusẽ ƒe nɔnɔme siwo mewɔa dɔ o siwo le ahɔhɔ̃meka siwo me OXPHOS mele o ŋu le kwasiɖa 4 va ɖo 5 kple kwasiɖa 7 va ɖo 8 me to lãmenugbagbevi blibo ƒe patch clamp recordings wɔwɔ le acute cerebellar slices me (Nɔnɔmetata 2E). Le mɔ si womekpɔ mɔ na o nu la, Mfn2cKO ahɔhɔ̃mekawo ƒe gbɔɖeme ƒe lãkusi ƒe ŋutete kple nusiwo wotsɔ dea eme ƒe tsitretsitsi le mama dedie nu sɔ kple esi dzi woɖu, togbɔ be vovototo siwo menya kpɔna dzea sii bɔbɔe o nɔ lãmenugbagbeviwo dome hã (Tabla 1). Nenema ke le kwasiɖa 4 va ɖo 5 xɔxɔ me la, womekpɔ tɔtrɔ ɖedzesi aɖeke le elektrikŋusẽ kple elektrikŋusẽ ƒe ƒomedodo (IV curve) me o (Nɔnɔmetata 2F). Gake Mfn2cKO ahɔhɔ̃meka aɖeke si xɔ kwasiɖa 7 va ɖo 8 metsi agbe le IV-dɔdamɔnu (afɔɖeɖe si nye hyperpolarization) me o, si ɖee fia be seselelãme si dze ƒã le hyperpolarization ƒe ŋutete ŋu le afɔɖeɖe sia si tsi megbe. To vovo na esia la, le Mfn2cKO ahɔhɔ̃mekawo me la, woɖea mɔ ɖe depolarizing currents siwo hea repetitive action potential (AP) discharges ŋu nyuie, si ɖee fia be woƒe discharge ƒe ɖoɖo bliboa meto vovo boo tso kwasiɖa 8 vi control neurons tɔ gbɔ o (Tabla 1 kple Figure 2G). Nenema ke spontaneous postsynaptic currents (sPSCs) ƒe dzidziɖedzi kple eƒe sesẽme sɔ kple esiwo le ƒuƒoƒo si me wokpɔa wo gbɔ le, eye nudzɔdzɔawo ƒe dzidziɖedzi tso kwasiɖa 4 va ɖo kwasiɖa 5 va ɖo kwasiɖa 7 va ɖo kwasiɖa 8 kple dzidziɖedzi ma tɔgbe (Nɔnɔmetata 2, H kple I). Ɣeyiɣi si me synaptic tsitsi le PNs (25). Wokpɔ nu mawo tɔgbe le PNs ƒe akpa siwo me do le megbe. Ðoɖo sia xea mɔ na lãmenugbagbeviwo ƒe ATP ƒe gbɔdzɔgbɔdzɔwo ƒe fexexe si ate ŋu adzɔ, abe alesi wòate ŋu adzɔ le lãmenugbagbevi bliboa ƒe patch clamp nuŋlɔɖi me ene. Vevietɔ la, mekpɔ ŋusẽ ɖe Mfn2cKO ahɔhɔ̃mekawo ƒe ɖiɖiɖeme ƒe lãkusi ƒe ŋutete kple tudada le wo ɖokui si ƒe zi gbɔ zi geɖe dzi o (Nɔnɔmetata 2, J kple K). Kpuie ko la, emetsonu siawo ɖee fia be PN siwo si OXPHOS ƒe dɔmawɔmawɔ nyuie si dze ƒã le ate ŋu anɔ te ɖe tsi si dona le ɣeyiɣi geɖe me ƒe nɔnɔmewo nu nyuie, si ɖee fia be fexexemɔnu aɖe li si wɔnɛ be woate ŋu alé elektrikŋusẽ ƒe ŋuɖoɖo siwo sɔ kloe me ɖe asi.
Woɖea nyatakakawo fiana abe mama dedie ± SEM (mɔ ɖeka dzi numekuku le vovototo ŋu, Holm-Sidak ƒe dodokpɔ si wotsɔ sɔ kple wo nɔewo zi geɖe; *P<0.05). Wotsɔa axadzinuŋɔŋlɔwo ɖea unit ƒe xexlẽdzesi fiana.
Míeɖoe be míaku nu me nenye be hatsotso aɖe si le proteomics dataset (Nɔnɔmetata 1G) me la lɔ mɔ siwo ate ŋu atsi OXPHOS ƒe anyimanɔmanɔ sesẽ nu ɖe ​​eme, si to esia me ɖe nusita PN si ŋu wògblẽ nu le ate ŋu alé elektrikŋusẽ ƒe dɔwɔwɔ si sɔ kloe me ɖe asi (Nɔnɔmetata 2, E vaseɖe K). . Proteomics numekuku ɖee fia be enzyme siwo kpɔa gome le branched chain amino acids (BCAA) ƒe catabolism me la dzi ɖe edzi ŋutɔ (Nɔnɔmetata 3A kple Nɔnɔmetata S5A), eye nusi wowɔ mamlɛtɔ acetyl-CoA (CoA) alo succinyl CoA ate ŋu akpe ɖe tricarboxylates ŋu le arteriosclerosis Acid (TCA) ƒe tsatsam me. Míekpɔe be BCAA transaminase 1 (BCAT1) kple BCAT2 siaa ƒe agbɔsɔsɔ dzi ɖe edzi. Wodoa ŋusẽ BCAA ƒe ʋuʋu ƒe afɔɖeɖe gbãtɔ to glutamate wɔwɔ tso α-ketoglutarate me ( 26 ). Wodoa subunits siwo katã wɔ branched chain keto acid dehydrogenase (BCKD) complex la ɖe dzi (complex la ʋãa BCAA carbon skeleton si do tso eme ƒe decarboxylation si kplɔe ɖo eye womate ŋu atrɔ o) (Nɔnɔmetata 3A kple Nɔnɔmetata S5A). Gake womekpɔ tɔtrɔ aɖeke si dze ƒã le BCAA ŋutɔ me le PN si woɖo ɖe hatsotsowo me o, si ate ŋu atso amino acid vevi siawo ƒe lãmenugbagbeviwo ƒe dzidziɖedzi alo dzɔtsoƒe bubuwo (glucose alo lactic acid) zazã atsɔ akpe ɖe TCA ƒe tsatsam ŋu gbɔ (Nɔnɔmetata S5B). PN siwo me OXPHOS mele o hã ɖe glutamine ƒe gbegblẽ kple transamination dɔwɔnawo ƒe dzidziɖedzi fia le kwasiɖa 8 xɔxɔ me, si ateŋu adze to mitochondrial enzymes glutaminase (GLS) kple glutamine pyruvate transaminase 2 (GPT2) ƒe dziyiyi me (Nɔnɔmetata 3, A kple C). Enyo be míade dzesii be GLS ƒe dziyiyi seɖoƒe li na spliced ​​isoform glutaminase C (GLS-GAC) (Mfn2cKO/CTRL ƒe tɔtrɔ anɔ abe zi gbɔ zi 4.5 ene, P = 0.05), eye eƒe dziyiyi tɔxɛ le kansa ƒe lãkusiwo me Ate ŋu ado alɔ mitochondrial bioenergy. (27) .
(A) Dzoxɔxɔ ƒe nɔnɔmetata la fia protein ƒe agbɔsɔsɔ ƒe tɔtrɔ zi gbɔ zi geɖe na mɔ si woɖo ɖi le kwasiɖa 8 me. (B) Ahɔhɔ̃meka si wotsɔ anti-PCx dɔlékui de dzesii ƒe kpɔɖeŋu (scale bar, 20 μm). Aŋutrɔ ɣia fia asi Purkinje lãmenugbagbevi ƒe ŋutilã. (C) Ɣeyiɣi ƒe zɔzɔme protein ƒe ɖeɖefia me dzodzro si wokpɔ be enye ame vevi aɖe si woate ŋu azã na ʋukawo ƒe ʋuʋu (multiple t-test, *FDR <5%; n = 3-5 mice). (D) Le etame: Nɔnɔmetata si ɖe mɔ vovovo siwo dzi woato age ɖe carbon si woŋlɔ ŋkɔ na si le [1-13C]pyruvate tracer me (si nye to PDH alo trans-arterial route dzi) fia. Ete: Violin ƒe nɔnɔmetata la ɖe carbon si woŋlɔ ɖe dzesi ɖeka (M1) si wotrɔ zu aspartic acid, citric acid kple malic acid ƒe alafa memamã fia le esi wotsɔ [1-13C]pyruvate de dzesi ahɔhɔ̃meka siwo le sesẽm la vɔ megbe (paired t-test; ** P <0.01). (E) Mɔ si woɖe fia ƒe ɣeyiɣi ŋutinya me dzodzro bliboe. Bu protein siwo ƒe P<0.05 le kwasiɖa 8 me ko ŋu . Fli si woɖo ɖe fli me: asitɔtrɔ ƒe asixɔxɔ aɖeke meli o (mɔ eve dzi numekuku le vovototo ŋu; * P <0.05; *** P <0.001). Woɖea nyatakakawo fiana abe mama dedie±SEM ene.
Le míaƒe numekuku me la, BCAA catabolism va zu up-regulation mɔ veviwo dometɔ ɖeka. Nyateƒenya sia ɖee fia vevie be yaƒoƒo ƒe agbɔsɔsɔme si gena ɖe TCA ƒe tsatsam me ate ŋu atrɔ le PN si me OXPHOS mele o me. Esia ate ŋu atsi tre ɖi na ahɔhɔ̃mekawo ƒe lãmenugbagbeviwo ƒe dɔwɔwɔ ƒe kaƒoƒo ƒomevi vevi aɖe, si ate ŋu akpɔ ŋusẽ ɖe ahɔhɔ̃mekawo ƒe dɔwɔwɔ kple agbetsitsi dzi tẽ le OXPHOS ƒe dɔmawɔmawɔ nyuie sesẽa dzi kpɔkpɔ me. Le ɖekawɔwɔ me kple nukpɔsusu sia la, míekpɔe be anti-atherosclerotic enzyme vevitɔ PCx nye up-regulated (Mfn2cKO/CTRL trɔna abe zi 1.5 ene; Nɔnɔmetata 3A), si doa ŋusẽ pyruvate ƒe tɔtrɔ wòzua oxaloacetate (28), si woxɔ se be ele ahɔhɔ̃mekawo me The expression in is restricted to astrocytes (29, 30). Le ɖekawɔwɔ me kple proteomics ƒe emetsonuwo la, confocal microscopy ɖee fia be PCx ƒe ɖeɖefia dzi ɖe edzi koŋ eye wòdzi ɖe edzi ŋutɔ le PN siwo me OXPHOS mele o me, esime PCx ƒe dɔwɔwɔ ya se ɖe Bergmann glial lãmenugbagbevi siwo te ɖe wo nɔewo ŋu koŋ le dziɖuɖua me (Nɔnɔmetata 3B). Be míado PCx ƒe dziyiyi si wokpɔ la akpɔ le dɔwɔwɔ me la, míetsɔ [1-13C]pyruvate tracer da dɔ na ahɔhɔ̃mekawo ƒe akpa siwo le sesẽm. Esi wotsɔ pyruvate dehydrogenase (PDH) wɔ oxidized la, eƒe isotope ƒe dzesi bu , Gake wotsɔe de TCA ƒe tsatsam ƒe domedomenuwo me ne pyruvate le metabolized to ʋukawo ƒe dɔwɔwɔ me (Nɔnɔmetata 3D). Le kpeɖodzi na míaƒe proteomics nyatakakawo la, míekpɔ dzesi gbogbo aɖewo tso tracer sia me le aspartic acid si le Mfn2cKO slices me, esime citric acid kple malic acid hã ƒe nɔnɔme le titina, togbɔ be meɖe dzesi o hã (Nɔnɔmetata 3D).
Le dopamine ahɔhɔ̃meka siwo le MitoPark mice siwo me mitochondrial ƒe dɔmawɔmawɔ nyuie si dopamine ahɔhɔ̃meka siwo gblẽa mitochondrial transcription factor A domenyiŋusẽfianu (Tfam) koŋ gbɔ (Nɔnɔmetata S6B) me la, PCx ƒe ɖeɖefia hã dzi ɖe edzi ŋutɔ (31), si fia be acetone acid arteriosclerosis Wokpɔa dɔlélea ƒe dzɔdzɔ dzi le ahɔhɔ̃mekawo ƒe dɔmawɔmawɔ nyuie me OXPHOS le ŋutilã me. Edze be míade dzesii be wokpɔe be enzyme tɔxɛwo (32-34) siwo ate ŋu adze le ahɔhɔ̃meka siwo ate ŋu ado ƒome kple ʋukawo ƒe ʋuʋu me la dzina ɖe edzi ŋutɔ le PN siwo me OXPHOS mele o me, abe propionyl-CoA carboxylase (PCC-A), Malonyl-CoA trɔa propionyl-CoA wòzua succinyl-CoA kple mitochondrial malic enzyme 3 (ME3), . si ƒe dɔ vevitɔe nye be wòaxɔ pyruvate tso malate me (Nɔnɔmetata 3, A kple C) (33, 35). Tsɔ kpe ɖe eŋu la, míekpɔ dzidziɖedzi gã aɖe le Pdk3 enzyme, si wɔa phosphorylate eye wòtoa esia me naa PDH mewɔa dɔ o ( 36 ), evɔ womekpɔ tɔtrɔ aɖeke le Pdp1 enzyme si naa PDH alo PDH enzyme complex ŋutɔ me o (Nɔnɔmetata 3A). Ɣesiaɣi la, le Mern2cKO PNwo me la, wodo ŋusẽ α1 subunit α (PDHE1α) subunit si le pyruvate dehydrogenase E1 ƒe akpa si le PDH complex me le Ser293 (si wonya be exea mɔ na PDH ƒe enzyme ƒe dɔwɔwɔ) ƒe phosphorylation ( Nɔnɔmetata S6C) (Nɔnɔmetata S6C). Ʋuka aɖeke mele pyruvate ŋu o.
Mlɔeba la, míekpɔe be serine kple glycine biosynthesis ƒe super mɔ, mitochondrial folate (1C) ƒe tsatsam si do ƒome kplii kple proline biosynthesis (Nɔnɔmetata 1G kple Nɔnɔmetata S5C) katã dzi ɖe edzi ŋutɔ, le nyatakakawo nu, le dɔwɔwɔ ƒe ɖoɖoa me. Wowɔa lãmenugbagbevi siwo ƒo xlãe la dɔ kple mitochondria ƒe dɔmawɔmawɔ nyuie (5-7). Confocal numekuku si ɖo kpe proteomics nyatakaka siawo dzi ɖee fia be le PN si me OXPHOS bu le me la, wotsɔ serine hydroxymethyltransferase 2 (SHMT2), si nye mitochondrial folate ƒe tsatsam ƒe enzyme vevi aɖe na mice siwo xɔ kwasiɖa 8 ƒe ahɔhɔ̃mekawo. Dɔlélenutsiŋutete ƒe ŋuɖoɖo ɖedzesi aɖe (Nɔnɔmetata S5D). Le 13 CU-glucose-incubated acute cerebellar slices me la, metabolic tracing dodokpɔwo gaɖo kpe serine kple proline biosynthesis ƒe dziyiyi dzi, si ɖee fia be carbon isoforms ƒe sisi yi serine kple proline me dzi ɖe edzi (Nɔnɔmetata S5E). Esi wònye be nuwɔna siwo GLS kple GPT2 do ɖe ŋgɔe nye nusi gbɔ glutamate ƒe wɔwɔme tso glutamine me kple transamination le glutamate kple α-ketoglutarate dome gbɔ ta la, woƒe upregulation ɖee fia be OXPHOS-manɔsitɔwo ƒe ahɔhɔ̃mekawo ƒe glutamate didi dzi ɖe edzi , Esia ate ŋu anye taɖodzinu be woalé proline ƒe biosynthesis si dzi ɖe edzi la me ɖe asi (Nɔnɔmetata S5C). To vovo na tɔtrɔ siawo la, proteomic numekuku le cerebellar astrocytes tso PN-koŋ Mfn2cKO mice me ɖee fia be mɔ siawo (si me antiperoxidases katã hã le) metrɔ boo le eɖeɖefia me o, si ɖee fia be This metabolic redirection is selective to degraded PN (Fig. S6, D to G).
Kpuie ko la, numekuku siawo ɖe ɣeyiɣi aɖe ƒe dɔwɔwɔ le lãmenugbagbeviwo ƒe dɔwɔwɔ ƒe mɔnu tɔxɛwo me le PNwo me fia. Togbɔ be ahɔhɔ̃mekawo ƒe mitochondrial ƒe dɔwɔwɔ si mesɔ o ate ŋu ahe ʋukawo ƒe ʋuʋu kabakaba kple 1C ƒe tɔtrɔ vɛ (Nɔnɔmetata 3E kple Nɔnɔmetata S5C), eye tɔtrɔ siwo woate ŋu agblɔ ɖi gɔ̃ hã le I kple IV ƒe ƒuƒoƒo ƒe ɖeɖefia me hã la, tɔtrɔ siwo va le serine de novo ƒe wɔwɔme me koe nye It only became apparent in the late stages. OXPHOS ƒe dɔmawɔmawɔ nyuie (Nɔnɔmetata 3E kple Nɔnɔmetata S5C). Nusiwo ŋu woke ɖo siawo ɖe ɖoɖo si kplɔ wo nɔewo ɖo si me mitochondrial (1C cycle) kple cytoplasmic (serine biosynthesis) si nuteɖeamedzi hena vɛ la wɔa nu aduadu kple ʋukawo ƒe ʋuʋu ƒe dzidziɖedzi le TCA ƒe tsatsam me be woagbugbɔ atrɔ asi le ahɔhɔ̃mekawo ƒe lãmenugbagbeviwo ƒe dɔwɔwɔ ŋu.
Kwasiɖa 8 ƒe PN siwo me OXPHOS mele o ate ŋu alé dzidzɔdonamedɔ si dzɔna zi geɖe me ɖe asi eye woato lãmenugbagbeviwo ƒe dɔwɔwɔ ƒe kadodo vevi aɖe me be woatsɔ axe fe ɖe mitochondrial ƒe dɔmawɔmawɔ nyuie ta. Nusi ŋu woke ɖo sia fɔ nu dodzidzɔname aɖe ɖe te be le ɣeyiɣi sia me gɔ̃ hã la, lãmenugbagbevi siawo ate ŋu Axɔ atikewɔwɔ ƒe nudede eme hã be woatsɔ ahe ahɔhɔ̃mekawo ƒe dɔmawɔmawɔ nyuie ɖe megbe alo axe mɔ ɖe enu. Tsi megbe. Míekpɔ nusi ate ŋu adzɔ sia gbɔ to nudede nyawo me le mía ɖokui si me. Le mɔnu gbãtɔ me la, míewɔ Cre-dependent adeno-associated virus (AAV) vector ale be woate ŋu atia MFN2 le PN siwo me OXPHOS mele o me le vivo (Nɔnɔmetata S7A). Woɖo kpe AAV si ŋlɔa MFN2 kple fluorescent reporter gene mCherry (Mfn2-AAV) dzi le ahɔhɔ̃mekawo ƒe dekɔnu gbãtɔwo me le vitro, si na be MFN2 dze le mɔ si nɔ te ɖe Cre dzi nu eye wòɖe mitochondrial ƒe nɔnɔme, si to esia me xe mɔ na ahɔhɔ̃mekawo ƒe tɔtrɔ le Mfn2cKO ahɔhɔ̃mekawo me ( Nɔnɔmetata S7, B, D kple E ). Eyome, míewɔ dodokpɔwo le vivo me be míatsɔ stereotactically atsɔ kwasiɖa 8 Mfn2-AAV ayi cerebellar cortex na Mfn2cKO kple control mice, eye míeda kwasiɖa 12 vi mice (Nɔnɔmetata 4A). Mfn2cKO mice siwo ŋu wowɔ atike ɖo la ku (Nɔnɔmetata 1, A kple B) (16). Dɔlékui ƒe tɔtrɔ le agbe me na PN ƒe ɖeɖefia tiatia le ahɔhɔ̃meka aɖewo me (Nɔnɔmetata S7, G kple H). AAV si wotsɔ ɖua wo dzi si ɖea mCherry (Ctrl-AAV) ɖeɖeko fiana la dodo mekpɔ ŋusẽ ɖe ahɔhɔ̃mekawo ƒe dɔmawɔmawɔ nyuie ƒe agbɔsɔsɔme dzi le Mfn2cKO lãwo me o. To vovo na ema la, Mfn2cKO siwo wotsɔ Mfn2-AAV trɔ asi le ƒe numekuku ɖe PN lãmenugbagbeviwo ƒe ƒuƒoƒoa ƒe ametakpɔkpɔ ƒe ŋusẽkpɔɖeamedzi ɖedzesi aɖe fia (Nɔnɔmetata 4, B kple C). Vevietɔ la, edze abe womate ŋu ade vovototo ahɔhɔ̃mekawo ƒe kpekpeme kple lã siwo dzi woɖu la dome kloe o ene (Nɔnɔmetata 4, B kple C, kple Nɔnɔmetata S7, H kple I). MFN1 ƒe ɖeɖefia gake menye MFN2 o wɔa dɔ nyuie nenema ke le ahɔhɔ̃mekawo ƒe ku ɖeɖe me (Nɔnɔmetata 4C kple Nɔnɔmetata S7, C kple F), si ɖee fia be MFN1 si le vidzidɔa godo ƒe ɖeɖefia ate ŋu akpe ɖe MFN2 ƒe anyimanɔmanɔ ŋu nyuie. Kukuɖenuŋu bubuwo le PN ƒe ɖoɖo ɖeka me ɖee fia be Mfn2-AAV ɖe mitochondria ƒe ultrastructure akpa gãtɔ, na mtDNA ƒe agbɔsɔsɔme sɔ, eye wòtrɔ anti-angiogenesis marker PCx ​​ƒe ɖeɖefia gã la (Nɔnɔmetata 4, C vaseɖe E ). Mfn2cKO mice siwo woɖe la me dzodzro kple ŋku le ɖiɖiɖeme ƒe nɔnɔme me ɖee fia be woƒe nɔnɔme kple woƒe dɔwɔwɔ ƒe dzesiwo (ʋuʋu S1 yi S3) nyo ɖe edzi. Le nyataƒoƒo me la, dodokpɔ siawo ɖee fia be MFN2 gbugbɔgade PN siwo me OXPHOS mele vevie o me megbedede sɔ gbɔ be wòatrɔ mtDNA zazã eye wòahe ʋukawo ƒe ʋuʋu vɛ, si ana woaxe mɔ ɖe axon ƒe gbegblẽ kple ahɔhɔ̃mekawo ƒe ku nu le vivo.
(A) Ðoɖo si ɖe dodokpɔ ƒe ɖoɖowɔɖi si woatsɔ ado AAV si ŋlɔa MFN2 ne wowɔ dɔ le lãmenugbagbeviwo ƒe dɔwɔwɔ ƒe mɔnu si woɖe fia ŋu. (B) Kwasiɖa 12 vi ƒe ahɔhɔ̃meka siwo woɖɔli le kwasiɖa 8 me le Mfn2cKO mice me eye wotsɔ anti-Calbindin antibody de dzesi wo ƒe nɔnɔmetata siwo le tsitre ɖi na confocal. Nuɖusi: Axon kawo ƒe ʋuʋu. Axon zoom ƒe lolome nye 450 kple 75 μm. (C) Miame: Purkinje lãmenugbagbeviwo ƒe kpekpeme ƒe agbɔsɔsɔme le AAV transduction loop (AAV+) (mɔ ɖeka dzi numekuku le vovototo ŋu; n = 3 mice). Nuɖusi: mtDNA ƒe susu ƒe numekuku le PN si wogbugbɔ trɔ me le kwasiɖa 12 lia me (unpaired t-test; n = 6 cells from three mice). * P <0.05, kple 100; ** P <0.01 ƒe xexlẽme. (D) Teƒenɔla ƒe kaka elektrɔnik nusuekpɔmɔ̃wo ƒe PN siwo le Mfn2cKO ahɔhɔ̃mekawo ƒe akpa siwo wotsɔ dɔlékui ƒe dɔlékuiwutike siwo woɖe fia la trɔ. Ametakpɔnu pink la ɖe teƒe si dendritewo xɔ la fia, eye dzogoe ene me ɣi si dzi woŋlɔ nu ɖo la ɖe alesi wowɔe le ɖusime la fia; n tsi tre ɖi na nucleus la. Nudzidzenu ƒe ʋuƒo, 1μm. (E) ɖe PCx ƒe amadede ƒe kpɔɖeŋu aɖe fia le PN si wogbugbɔ trɔ le kwasiɖa 12 me. Nudzidzenu ƒe ʋuƒo, 20μm. OE, nyagbɔgblɔ si gbɔ eme; FC, agbalẽ xatsaxatsa ƒe tɔtrɔ.
Mlɔeba la, míeku nu me le lãmenugbagbeviwo ƒe agbetsitsi si peroxidase hena vɛ ƒe vevienyenye ŋu le PN siwo to OXPHOS ƒe dɔmawɔmawɔ nyuie me. Míewɔ mCherry si ŋlɔa AAV-shRNA (short hairpin RNA) si tɔa ŋku mouse PCx mRNA (AAV-shPCx) koŋ, eye míedo dɔlékuia alo eƒe scrambled control (AAV-scr) ɖe Mfn2cKO mice ƒe ahɔhɔ̃mekawo me. Wodo atike la le ƒe enelia me (Nɔnɔmetata 5A) be woakpɔ PCx ƒe tsɔtsrɔ̃ nyuie le ɣeyiɣi si me PCx ƒe ɖeɖefia dzi ɖe edzi (Nɔnɔmetata 3C) eye PN lãmenugbagbeviwo ƒe ƒuƒoƒoa gakpɔtɔ nɔ anyi (Nɔnɔmetata 1A). Enyo be míade dzesii be PCx (Nɔnɔmetata S8A) ƒoƒo ɖe anyi hea PN ƒe ku kabakaba ŋutɔ, si nye asigɛ si me dɔlélea le ko (Nɔnɔmetata 5, B kple C). Be míase mɔnu si lãmenugbagbeviwo ƒe dɔwɔwɔ ƒe ŋusẽkpɔɖeamedzi siwo PCx ƒe dziyiyi hena vɛ gɔme la, míesrɔ̃ nu tso redox nɔnɔme si le PNs ŋu le PCx ƒe tsɔtsrɔ̃ kple AAV-tome ŋkume biosensor Grx1-roGFP2 si woɖe fia le ɣeyiɣi ɖeka me (Nɔnɔmetata S8, B vaseɖe D) be míada glutathione The relative change of peptide redox potential (38) kpɔ. Emegbe, míewɔ eve-photon fluorescence agbenɔƒe nɔnɔmetata nusuekpɔmɔ̃ (FLIM) le acute ahɔhɔ̃ slices kwasiɖa 7 Mfn2cKO alo dziɖuɖu littermates be míade dzesi tɔtrɔ siwo ate ŋu ava le cytoplasmic redox nɔnɔme me le FLIM nɔnɔmewo me toto vɔ megbe (Nɔnɔmetata S8, E vaseɖe G). Kukuɖenuŋua ɖee fia be dzidziɖedzi gã aɖe le oxidation nɔnɔme me le Mfn2cKO PN ɖeka si me PCx ƒe ɖeɖefia mele o, si to vovo na control neurons alo Mfn2cKO PNs siwo ɖea shRNA si wotsɔ ƒo ƒu ko fiana (Nɔnɔmetata 5, D kple E). Ne woɖe PCx ƒe ɖeɖefia dzi kpɔtɔ la, Mfn2cKO PN siwo ɖea nɔnɔme si me oxidized le ŋutɔ fiana ƒe alafa memamã dzi ɖe edzi wu zi gbɔ zi etɔ̃ (Nɔnɔmetata 5E), si ɖee fia be PCx ƒe dziyiyi lé ahɔhɔ̃meka siwo gblẽ ƒe redox ŋutete me ɖe asi.
(A) Ðoɖo si ɖe dodokpɔ ƒe ɖoɖowɔɖi si woatsɔ ado AAV si ŋlɔa shPCx ne wowɔ dɔ le lãmenugbagbeviwo ƒe dɔwɔwɔ ƒe mɔnu si woɖe fia ŋu. (B) Teƒenɔla confocal fotowo ƒe kwasiɖa 8 ƒe ahɔhɔ̃mekawo ƒe akpawo le Mfn2cKO mice siwo wotrɔ eye wotsɔ anti-calcineurin antibody de dzesi wo le kwasiɖa 4 me. Nudzidzenu ƒe ʋuƒo, 450μm. (C) Purkinje lãmenugbagbeviwo ƒe kpekpeme ƒe agbɔsɔsɔme le AAV-transduced loops me (mɔ ɖeka dzi numekuku le vovototo ŋu; n = 3 va ɖo 4 mice). Woɖea nyatakakawo fiana abe mama dedie±SEM ene; ***P <0.001 ƒe xexlẽme. (D) Teƒenɔla FLIM nɔnɔmetata ɖe kwasiɖa 7 vi PN si ɖea glutathione redox sensor Grx1-roGFP2 fiana le dodokpɔ ƒe nɔnɔme siwo wogblɔ me ƒe agbenɔƒe le mama dedie nu fia. LUT (kpɔkpɔ ƒe kplɔ̃) ƒe xexlẽme: agbetsitsi ƒe ɣeyiɣi ƒe dometsotso (le picoseconds me). Nudzidzenu ƒe ʋuƒo, 25μm. (E) Histogram la fia Grx1-roGFP2 ƒe agbenɔƒe ƒe asixɔxɔwo ƒe mama tso (D) (n=158 va ɖo 368 lãmenugbagbevi le mice eve me le nɔnɔme ɖesiaɖe me). Pie chart si le histogram ɖesiaɖe tame: fia lãmenugbagbevi siwo ƒe agbenɔƒe didi ŋutɔ (dzẽ, oxidized) alo kpuie (blɔ, dzi ɖe kpɔtɔ) ƒe xexlẽme, si wu 1 SD le agbenɔƒe ƒe asixɔxɔ le mama dedie nu le CTRL-AAV-scr. (F) Kpɔɖeŋu si wodo ɖa la ɖe ahɔhɔ̃mekawo ƒe PCx ƒe dziyiyi ƒe ametakpɔkpɔ ƒe ŋusẽkpɔɖeamedzi fia.
Le wo katã me la, nyatakaka siwo míena le afisia ɖee fia be MFN2 ƒe gbugbɔgaɖeɖe ate ŋu aɖe PN deŋgɔ si me OXPHOS ƒe anyimanɔmanɔ vevie, mtDNA ƒe vɔvɔ sesẽ, kple nɔnɔme si le abe ista ene si mesɔ kura o la keŋkeŋ, si ana ŋgɔyiyi si yia edzi le dɔléle siwo de ŋgɔ gɔ̃ hã me. Ahɔhɔ̃mekawo ƒe dɔmawɔmawɔ nyuie naa kpeɖodzi si woate ŋu atrɔ le afɔɖeɖe si do ŋgɔ na lãmenugbagbeviwo ƒe ku ŋu. Wogate gbe ɖe lãmenugbagbeviwo ƒe asitɔtrɔ le nɔnɔmewo ŋu ƒe seƒe sia dzi to ŋutete si le ahɔhɔ̃mekawo si be woahe ʋukawo ƒe ʋuʋu (TCA ƒe tsatsam ƒe kaƒoƒo) vɛ, si xea mɔ na PCx ƒe ɖeɖefia le PN siwo me OXPHOS mele o me eye wòdoa lãmenugbagbeviwo ƒe ku ɖe ŋgɔ, si wɔnɛ be wòwɔa akpa si kpɔa ame ta (Nɔnɔmetata 5F).
Le numekuku sia me la, míena kpeɖodzi be PNwo ƒe ŋuɖoɖo ɖe OXPHOS ƒe dɔmawɔmawɔ nyuie ŋue nye be woaƒo ƒu vivivi ɖe TCA ƒe tsatsam ƒe ʋukawo ƒe ʋuʋu ŋu to vovototodedeameme ƒe dɔwɔwɔ ƒe mɔnu si lãmenugbagbeviwo ƒe dɔwɔwɔ ƒe ɖoɖowo wɔa dɔ le dzi. Míetsɔ mɔnu geɖe siwo kpe ɖe eŋu ɖo kpe proteomic numekukua dzi eye míeɖee fia be ne mitochondrial ƒe dɔmawɔmawɔ nyuie sesẽ do mo ɖa la, ahɔhɔ̃mekawo ƒe lãmenugbagbeviwo ƒe dɔwɔwɔ ƒe ʋuʋu ƒe nɔnɔme si womenya tsã o la nɔa wo ŋu. Ewɔ nuku na mí be kaƒoƒo ƒe ɖoɖo bliboa medea dzesi lãmenugbagbeviwo ƒe dɔwɔwɔ ƒe nɔnɔme mamlɛtɔ si kpena ɖe ahɔhɔ̃mekawo ƒe dɔmawɔmawɔ nyuie ŋu vivivi eye womate ŋu atrɔe o, gake míaƒe nyatakakawo ɖee fia be ate ŋu anye beléle na ahɔhɔ̃meka le afɔɖeɖe si do ŋgɔ na lãmenugbagbeviwo ƒe ku gɔ̃ hã me Functional compensation mechanism. Nusi ŋu woke ɖo sia ɖee fia be ahɔhɔ̃mekawo ƒe lãmenugbagbeviwo ƒe dɔwɔwɔ ƒe tɔtrɔ le ŋutilãa me sɔ gbɔ ŋutɔ. Nyateƒenya sia ɖo kpe edzi be MFN2 gbugbɔgava emegbe ate ŋu atrɔ lãmenugbagbeviwo ƒe dzesi veviwo ƒe ɖeɖefia eye wòaxe mɔ ɖe PN ƒe gbegblẽ nu. Kura o, exea mɔ na ʋukawo ƒe ʋuʋu eye wònaa lãmekawo wɔa dɔ kabakaba. transsexual.
Nusiwo do dzidzɔ na ame wu le míaƒe numekukua me dometɔ ɖekae nye be PN siwo me OXPHOS mele o ate ŋu atrɔ asi le TCA ƒe tsatsam ƒe lãmenugbagbeviwo ƒe dɔwɔwɔ ŋu to enzyme siwo doa ŋusẽ ʋukawo ƒe ʋuʋu koŋ dzi ɖeɖe kpɔtɔ me. Lãmetsiŋusẽ ƒe tɔtrɔ nye nusi bɔ ɖe kansa lãmenugbagbeviwo me, eye wo dometɔ aɖewo ɖoa ŋu ɖe glutamine ŋu be wòatsɔ akpe ɖe TCA ƒe tsatsam ƒe domedomenuwo ŋu be woawɔ nusiwo sɔ kple wo nɔewo siwo dzi ɖena kpɔtɔna, siwo ʋãa gbɔgbɔtsixe ƒe kɔsɔkɔsɔa eye woléa lipid kple nucleotide biosynthesis precursors ƒe wɔwɔ me ɖe asi (39 , 40). Numekuku aɖe si wowɔ nyitsɔ laa ɖee fia be le lãmeka siwo le gota siwo me OXPHOS ƒe dɔmawɔmawɔ nyuie le me la, glutamine/glutamate ƒe lãmenugbagbeviwo ƒe dɔwɔwɔ ƒe kadodo ake hã nye nu vevi aɖe ( 5 , 41 ), afisi mɔfiame si glutamine gena ɖe TCA ƒe tsatsam la nɔ te ɖe Le esi OXPHOS ƒe abixɔxɔ ƒe sesẽ ta ( 41 ). ). Gake kpeɖodzi si me kɔ aɖeke meli ku ɖe ahɔhɔ̃mekawo ƒe lãmenugbagbeviwo ƒe dɔwɔwɔ ƒe tɔtrɔ ɖesiaɖe si ɖi wo nɔewo le ŋutilã me kple alesi wòate ŋu asɔ le dɔlélea ƒe nɔnɔme me ŋu o. Le numekuku aɖe si wowɔ nyitsɔ laa le vitro me la, woɖee fia be ahɔhɔ̃meka gbãtɔwo ƒoa glutamate-tawo nu ƒu hena ahɔhɔ̃mekawo ƒe kaka, si wɔnɛ be wodoa oxidative metabolism kple atherosclerosis ɖe ŋgɔ le metabolic stress nɔnɔmewo me ( 42 ). Edze be míade dzesii be le atikeŋutinunya ƒe mɔxexe ɖe TCA ƒe tsatsam ƒe enzyme succinate dehydrogenase nu te la, woxɔe se be pyruvate carboxylation léa oxaloacetate ƒe wɔwɔme ɖe te le ahɔhɔ̃meka siwo wode agble me le ahɔhɔ̃mekawo me ( 34 ). Gake vevienyenye vevi aɖe gakpɔtɔ le mɔ̃ siawo ƒe ŋutilã me kadodo kple ahɔhɔ̃mekawo ŋu (afisi woxɔe se be ʋukawo me ʋu ƒe sisi koŋue nye ɣletivimefakaka) ( 43 ). Le go sia me la, míaƒe nyatakakawo ɖee fia be woate ŋu atrɔ PN siwo OXPHOS gblẽ le ŋutilã me la ɖe BCAA ƒe gbegblẽ kple pyruvate carboxylation, siwo nye TCA pool intermediates ƒe kpeɖeŋutɔtsoƒe vevi eve. Togbɔ be wodo alesi wosusu be BCAA ƒe ʋuʋudedi wɔna le ahɔhɔ̃mekawo ƒe ŋusẽ ƒe asitɔtrɔ me hã la, tsɔ kpe ɖe akpa si glutamate kple GABA wɔna le ahɔhɔ̃mekawo ƒe dɔwɔwɔ me ŋu ( 44 ), kpeɖodzi aɖeke megali na mɔnu siawo le agbe me o. Eyata ele bɔbɔe be woasusui be PN siwo mewɔa dɔ nyuie o ate ŋu axe fe ɖe TCA domedomenuwo zazã si assimilation ƒe ɖoɖoa ʋãna ta le wo ɖokui si to ʋukawo ƒe ʋuʋu dzi ɖeɖe kpɔtɔ me. Vevietɔ la, PCx ƒe dziyiyi ate ŋu ahiã be woalé aspartic acid ƒe didi si dzi ɖe edzi me ɖe asi, si wodo ɖa le lãmenugbagbevi siwo le dzidzim ɖe edzi kple mitochondrial ƒe dɔmawɔmawɔ nyuie me ( 45 ). Gake míaƒe lãmenugbagbeviwo ƒe dɔwɔwɔ ŋuti numekuku meɖe tɔtrɔ gã aɖeke fia le aspartic acid ƒe agbɔsɔsɔ si le nɔnɔme si li ke me le Mfn2cKO PNs me o (Nɔnɔmetata S6A), si wosusu be eɖe aspartic acid ƒe lãmenugbagbeviwo ƒe dɔwɔwɔ vovovo siwo le lãmenugbagbevi siwo le dzidzim ɖe edzi kple ahɔhɔ̃meka siwo le mitotic megbe dome fiana . Togbɔ be womekpɔ de dzesi mɔnu si tututu PCx ƒe dziyiyi le ahɔhɔ̃meka siwo mewɔa dɔ nyuie o me le vivo me hã la, míeɖee fia be ŋuɖoɖo sia si do ŋgɔ na ɣeyiɣia wɔa akpa vevi aɖe le ahɔhɔ̃mekawo ƒe redox nɔnɔme me léle ɖe asi me, si woɖe fia le FLIM dodokpɔwo me le ahɔhɔ̃mekawo ƒe kakɛwo ŋu. Vevietɔ la, mɔxexe ɖe PNwo nu be woagawɔ PCx ƒe dziyiyi o ate ŋu ana lãmenugbagbeviwo ƒe nɔnɔme nanye oxidized wu eye wòana lãmenugbagbeviwo naku kabakaba. BCAA ƒe gbegblẽ ƒe dɔwɔwɔ kple pyruvate ƒe carboxylation menye mɔ siwo dzi woato ade dzesi mitochondrial ƒe dɔmawɔmawɔ nyuie ƒe lãmeka siwo le gota gome o (7). Eyata edze abe woawoe nye nusi wotsɔ ɖo nɔƒe gbãtɔ na ahɔhɔ̃meka siwo me OXPHOS mele o ene, ne menye woawo koe o gɔ̃ hã, si le vevie na ahɔhɔ̃mekawo ƒe dɔmawɔmawɔ nyuie. .
Ahɔhɔ̃mekawo ƒe dɔléle nye ahɔhɔ̃mekawo ƒe dɔléle ƒomevi vovovowo si dzena zi geɖe abe ataxia ene eye zi geɖe la, egblẽa nu le PNwo ŋu ( 46 ). Ahɔhɔ̃mekawo ƒe hatsotso sia nyea mitochondrial ƒe dɔmawɔmawɔ nyuie bɔbɔe elabena woƒe tiatiawɔblɔɖe ƒe gbegblẽ le sisiblisiwo me sɔ gbɔ be woagbugbɔ lãmeka ƒe dzesi geɖe siwo ɖe dzesi le amegbetɔ ƒe akɔta ƒe ahɔhɔ̃mekawo ƒe ataxia ŋu ( 16 , 47 , 48 ). Nyatakakawo ɖee fia be sisiblisi ƒe kpɔɖeŋu si wotsɔ domenyiŋusẽfianu trɔ si me domenyiŋusẽfianu si trɔ la do ƒome kple amegbetɔ ƒe ahɔhɔ̃mekawo ƒe ataxia eye mitochondrial ƒe dɔmawɔmawɔ nyuie le eŋu ( 49 , 50 ), si te gbe ɖe alesi wòle vevie be woasrɔ̃ nu tso OXPHOS ƒe anyimanɔmanɔ me tsonu ŋu le PNPH me dzi. Eyata esɔ ŋutɔ be woaɖe ahɔhɔ̃mekawo ƒe hatsotso tɔxɛ sia ɖe vovo nyuie ahasrɔ̃ nu tso eŋu. Gake esi wònye be PN-wo sea veve ɖe nyaƒoɖeamenu nu ŋutɔ eye woxɔa ahɔhɔ̃mekawo ƒe lãmenugbagbeviwo ƒe xexlẽme bliboa ƒe akpa sue aɖe ta la, le numekuku geɖe siwo wotu ɖe omics dzi gome la, wo mama tiatia abe lãmenugbagbevi blibowo ene gakpɔtɔ nye akpa sesẽ aɖe. Togbɔ be manya wɔ kloe be woakpɔ ɖiƒoƒo ƒe anyimanɔmanɔ kura gbɔ le lãmenugbagbevi ƒomevi bubuwo (vevietɔ ame tsitsiwo ƒe lãkusiwo) me o hã la, míetsɔ afɔɖeɖe nyui aɖe si nye vovototodedeameme ƒo ƒu kple FACS be míakpɔ ahɔhɔ̃meka siwo ate ŋu anɔ agbe ƒe xexlẽme si sɔ hena proteomics numekuku le anyime, eye protein ƒe xɔxlɔ̃ si lolo ŋutɔ (si ade protein 3000) nanɔ mía si ne wotsɔe sɔ kple ahɔhɔ̃meka bliboa ƒe nyatakaka siwo li xoxo (51). To lãmenugbagbevi blibowo ƒe agbenɔnɔ ta kpɔkpɔ me la, mɔnu si míena le afisia ɖe mɔ na mí be menye ɖeko míate ŋu alé ŋku ɖe tɔtrɔ siwo va le lãmenugbagbeviwo ƒe dɔwɔwɔ ƒe mɔnuwo me le mitochondria me ŋu ko o, ke boŋ be míalé ŋku ɖe tɔtrɔ siwo le eƒe cytoplasmic hatiwo me hã ŋu, si kpe ɖe mitochondrial membrane tags zazã ŋu be woatsɔ ado lãmenugbagbeviwo ƒe ƒomevi ɖe ŋgɔ Mɔnu yeye si wozãna na mitochondria ƒe xexlẽme le lãmeka sesẽwo me (52, 53). Menye ɖeko mɔnu si míeɖɔ la do ƒome kple Purkinje lãmenugbagbeviwo ŋuti nusɔsrɔ̃ ko o, ke woate ŋu azãe bɔbɔe ɖe lãmenugbagbevi ƒomevi ɖesiaɖe ŋu atsɔ akpɔ lãmenugbagbeviwo ƒe dɔwɔwɔ ƒe tɔtrɔwo gbɔ le ahɔhɔ̃ siwo ŋu dɔléle le me, siwo dome mitochondrial ƒe dɔmawɔmawɔ nyuie ƒe kpɔɖeŋu bubuwo hã le.
Mlɔeba la, míede dzesi atikewɔwɔ ƒe fesre aɖe le lãmenugbagbeviwo ƒe tɔtrɔ ƒe ɖoɖo sia me si ate ŋu atrɔ lãmenugbagbeviwo ƒe nuteɖeamedzi ƒe dzesi veviwo keŋkeŋ eye wòaxe mɔ ɖe ahɔhɔ̃mekawo ƒe gbegblẽ nu. Eyata dɔwɔwɔ ƒe gɔmesese si le kaƒoƒo si ŋu woƒo nu tsoe le afisia ŋu gɔmesese ate ŋu ana gɔmesese veviwo nasu mía si ku ɖe atikewɔwɔ siwo woate ŋu azã atsɔ alé ahɔhɔ̃mekawo ƒe agbenɔnɔ me ɖe asi le mitochondria ƒe dɔmawɔmawɔ nyuie me ŋu. Numekuku siwo woawɔ le etsɔme si ƒe taɖodzinue nye be woatso tɔtrɔ siwo vaa ŋusẽ ƒe asitɔtrɔ le ahɔhɔ̃mekawo ƒe lãmenugbagbevi ƒomevi bubuwo me ŋu be woaɖe gɔmeɖose sia zazã le ahɔhɔ̃medɔ bubuwo me afia bliboe.
Woƒo nu tso MitoPark mice ŋu va yi ( 31 ). Woƒo nu tso C57BL/6N mice siwo si loxP flanking Mfn2 domenyiŋusẽfianuwo le ŋu va yi ( 18 ) eye wotso wo kple L7-Cre mice ( 23 ). Emegbe wotsɔ dzidzimevi eve siwo nye heterozygous eve siwo do tso eme la tsaka kple homozygous Mfn2loxP/Mfn2loxP mice be woawɔ Purkinje-koŋ domenyiŋusẽfianu ƒe knockouts na Mfn2 (Mfn2loxP/Mfn2loxP; L7-cre). Le srɔ̃ɖeɖe ƒe hatsotso sue aɖe me la, woto Gt (ROSA26) SorStop-mito-YFP allele (stop-mtYFP) vɛ to crossing bubuwo me ( 20 ). Wowɔ lãwo ƒe dɔwɔwɔwo katã le Europa, dukɔa kple dɔwɔƒewo ƒe mɔfiamewo nu eye LandesamtfürNatur si le Umwelt kple Verbraucherschutz, North Rhine-Westphalia, Germany da asi ɖe edzi. Lãwo ƒe dɔwɔwɔ hã zɔna ɖe Europa Lãwo Ŋuti Nusrɔ̃ƒe ƒe Habɔbɔ ƒe mɔfiafia dzi.
Ne wodo atike na funɔa ƒe vidzidɔ ƒe ʋuʋu vɔ la, woɖea sisiblisi ƒe fugboea ɖe vovo (E13). Wotso lãkusia le Hanks ƒe Dzetsi si Da sɔ (HBSS) si wotsɔ Hepes 10 mM kpe ɖe eŋu me eye wotsɔ Dulbecco ƒe Modified Eagle’s Medium si me papain (20 U/ml) kple cysteine ​​(1μg/ml) le la na. Tsɔ lãkusia de DMEM me) eye nàmae to enzyme ƒe nuɖuɖumeŋusẽ me. Ml) le 37°C me hena miniti 20, eye emegbe wotsɔ mɔ̃ ƒoe le DMEM si me wotsɔ fugboe ƒe nyitsu ƒe ʋumenugbagbeviɣi 10% kpe ɖe eŋu me. Woƒã lãmenugbagbeviwo ɖe ahuhɔ̃e ƒe nutsyɔnu siwo wotsɔ polylysine tsyɔ dzi le eƒe kpekpeme si nye 2×106 le 6 cm dekɔnu nuɖugba ɖesiaɖe me alo le kpekpeme si nye 0.5×105 cells/cm2 me hena nɔnɔmetatawo me dzodzro. Le gaƒoƒo 4 megbe la, wotsɔ Neurobasal serum-free medium si me 1% B27 kpeɖeŋutɔ kple 0.5 mM GlutaMax le ɖɔli atike la. Emegbe wolé ahɔhɔ̃mekaawo ɖe dzoxɔxɔ 37°C kple 5% CO2 me le dodokpɔa katã me, eye wona nuɖuɖu wo zi ɖeka le kwasiɖa me. Be woate ŋu ana woagaƒo ƒu ake le vitro la, wozã AAV9 dɔlékui ƒe dɔlékuiwutike si gbɔna ƒe 3μl (vudo 24 ƒe dekɔnu nuɖugba) alo 0.5μl (do 24 ƒe agba) tsɔ da dɔ na ahɔhɔ̃mekawo le ŋkeke evelia dzi le vitro: AAV9.CMV.PI.eGFP. WPRE.bGH (Addgene, agbalẽdzraɖoƒe ƒe xexlẽdzesi 105530-AAV9) kple AAV9.CMV.HI.eGFP-Cre.WPRE.SV40 (Addgene, agbalẽdzraɖoƒe ƒe xexlẽdzesi 105545-AAV9).
Wotsɔ V5 ƒe ɖoɖo (GKPIPNPLLGLDST) de dzesi sisiblisi Mfn1 kple Mfn2 kpeɖeŋutɔ DNA (si wokpɔ tso Addgene plasmid #23212 kple #23213, le wo nɔewo yome) le C-nuwuƒe, eye wotsɔa mCherry tsakana le frame me to T2A ƒe ɖoɖoa me. Grx1-roGFP2 nye nunana tso Heidelberg T. P. Dick DFKZ (Deutsches Krebsforschungszentrum) gbɔ. To tdTomato kasɛt la ɖɔliɖɔli to nɔnɔmetatawo wɔwɔ ƒe mɔnu bɔbɔewo zazã me la, woɖe kasɛt la ɖe pAAV-CAG-FLEX-tdTomato ƒe megbeƒu me (Addgene ƒe xexlẽdzesi 28306) be woawɔ pAAV-CAG-FLEX-mCherry-T2A-MFN2-V5, pAAV-CAG- FLEX-mCherry-T2A-MFN1-V5 kple pAAV-CAG-FLEX-Grx-roGFP2 dɔlékuiwutikewo. Wozã mɔnu ma tɔgbe tsɔ wɔ dɔlékuiwutike si nye pAAV-CAG-FLEX-mCherry. Be woateŋu awɔ AAV-shPCx ƒe wɔwɔme la, wohiã plasmid AAV vector (VectorBuilder, pAAV [shRNA] -CMV-mCherry-U6-mPcx- [shRNA#1]), si me DNA ƒe ɖoɖo si ŋlɔa shRNA si ɖo taɖodzinu na sisiblisi PCx (5′CTTTCGCTCTAAGGTGCTAAACTCGAGTTTAGCACCTTAGAGCGAAAG) le 3′) Le U6 ƒe ŋgɔdola ƒe dzikpɔkpɔ te la, wozãa mCherry le CMV ƒe ŋgɔdola ƒe dzikpɔkpɔ te. Wowɔ kpekpeɖeŋu AAV dɔlékuiwutikewo wɔwɔ le atikewɔƒea ƒe mɔfiamewo nu (Cell Biolabs). Kpuie ko la, zã transfer plasmid si tsɔ mCherry-T2A-MFN2-V5 (pAAV-CAG-FLEX-mCherry-T2A-MFN2-V5), mCherry-T2A-MFN1-V5 (pAAV-CAG-FLEX-mCherry) hena ɣeyiɣi kpui aɖe ƒe Transfection of 293AAV cells-T2A-MFN1-V5), mCherry (pAAV-CAG-FLEX-mCherry) alo Grx-roGFP2 (pAAV-CAG-FLEX-Grx-roGFP2) si ŋlɔa domenyiŋusẽfianu, kpakple AAV1 capsid protein kple kpeɖeŋutɔ protein si ŋlɔa nu Packaging plasmid plasmid, to calcium phosphate mɔnu zazã me. Wokpɔ dɔlékui ƒuƒu si le tsia dzi to tsikpe ƒuƒu ƒe tsikpe/ethanol tsilele me kple lãmenugbagbevi siwo woɖe le phosphate buffered saline (PBS) me. Wokɔ AAV dɔlékuiwutikea ŋu to iodixanol gradient ultracentrifugation si metso o (gaƒoƒo 24 le 32,000 rpm kple 4°C) me eye wozã Amicon ultra-15 centrifugal filter tsɔ ƒoe nu ƒu. AAV1-CAG-FLEX-mCherry-T2A-MFN2-V5 [2.9×1013 domenyiŋusẽfianu ƒe kɔpi (GC)/ml], AAV1-CAG-FLEX-mCherry (6.1×1012 GC/ml), AAV1-CAG- FLEX ƒe domenyiŋusẽfianu ƒe agbɔsɔsɔme nɔ abe alesi woɖɔe va yi ene (54), si wodzidze to ɣeyiɣi ŋutɔŋutɔ ƒe agbɔsɔsɔme ƒe PCR (qPCR) . -MFN1-V5 (1.9×1013 GC/ml) kple AAV1-CAG-FLEX-Grx-roGFP2 (8.9×1012 GC/ml).
Woɖe ahɔhɔ̃meka gbãtɔwo ɖa le 1x PBS si fa miamiamia abe tsikpe ene me, wotsɔ wo ƒo ƒui, eye emegbe wowɔ wo ɖekae le 0.5% Triton X-100 / 0.5% sodium deoxycholate/PBS lysis buffer si me phosphatase kple protease inhibitor (Roche) le me. Wowɔ protein ƒe agbɔsɔsɔme to bicinchoninic acid dodokpɔ (Thermo Fisher Scientific) zazã me. Emegbe wotsɔ SDS-polyacrylamide gel electrophoresis ɖe proteinawo ɖa, eye emegbe wotu wo ɖe polyvinylidene fluoride membrane (GE Healthcare) dzi. Xlẽ teƒe siwo menye teƒe tɔxɛwo o eye nàtsɔ dɔlékuiwutike gbãtɔ (kpɔ Tabla S1 hena numeɖeɖewo) de notsi 5% me le TBST (Tris-buffered saline with Tween), tsilele ƒe afɔɖeɖewo kple dɔlékuiwutike evelia le TBST Incubate me. Tsɔ dɔlékuiwutike gbãtɔ de eme zã bliboa le +4°C. Ne èklɔe vɔ la, nàtsɔ dɔlékuiwutike evelia asisi ɖe eŋu gaƒoƒo 2 le xɔme ƒe dzoxɔxɔ nu. Emegbe to blot ma ke tsɔtsɔ de anti-β-actin dɔlékuiwutike me me la, woɖo kpe agba ma ke dzi. Deteksi to tɔtrɔ ɖe chemiluminescence ŋu kple chemiluminescence (GE Healthcare) dodo ɖe ŋgɔ me.
Wotsɔ 4% paraformaldehyde (PFA)/PBS ɖɔ ahɔhɔ̃meka siwo woƒã ɖe glass nutsyɔnu dzi va yi la ɖo le ɣeyiɣi si woɖo ɖi me le xɔme ƒe dzoxɔxɔ me hena miniti 10. Wotsɔa 0.1% Triton X-100/PBS dea nutsyɔnuawo me gbã hena miniti 5 le xɔme ƒe dzoxɔxɔ nu, eye emegbe wotsɔa blocking buffer [3% bovine serum albumin (BSA)/PBS] dea wo me. Le ŋkeke evelia dzi la, woklɔ nutsyɔnuawo kple blocking buffer eye wotsɔ fluorophore-conjugated secondary antibody si sɔ la de wo me gaƒoƒo 2 le xɔme ƒe dzoxɔxɔ nu; mlɔeba la, woklɔ kpɔɖeŋuawo nyuie le PBS me kple 4′,6-diamidino-2 -Phenylindole (DAPI) nye counterstained eye emegbe wotsɔ Aqua-Poly/Mount ɖo wo ɖe nusuekpɔmɔ̃ ƒe slide la dzi.
Wotsɔ ketamine (130 mg/kg) kple xylazine (10 mg/kg) dodo ɖe dɔgboa me na alɔ̃dɔdɔ sisiblisiwo (atsu kple nyɔnu) eye wotsɔ carprofen vevesesenutsitike (5 mg/kg) do wo ɖe ŋutigbalẽ te , Eye wotsɔ wo de stereotactic dɔwɔnu (Kopf) si me wotsɔ aŋe si me dzo le me. Ðe taɖua ɖe go eye nàzã aɖutike atsɔ awɔ ahɔhɔ̃mekawo ƒe akpa si sɔ kple mis ƒu (tso lambda: asike 1.8, axadzi 1, si sɔ kple lobules IV kple V) la tsɛ. Zã atikekui si woƒo ƒu nàtsɔ awɔ do sue aɖe ɖe taɖua me nyuie be nàgagblẽ ʋuka siwo le ete la me o. Emegbe wotsɔa ahuhɔ̃e me ʋuka tsɛ si woɖe la dea do suea me vivivi (tso -1.3 va ɖo -1 le dura mater ƒe akɔta), eye wodoa AAV 200 va ɖo 300 nl ɖe micro-injector (Narishige) me kple asi ƒe atikekui (Narishige) zi geɖe le nyaƒoɖeamenu sue aɖe me le ɣeyiɣi aɖe si xɔa miniti 10 va ɖo 20 ƒe fesre me. Ne èdoe vɔ la, da ʋukaa me aɖabaƒoƒo 10 bubu be dɔlékuia nakaka keŋkeŋ. Ne woɖe ʋukaawo ɖa vɔ la, woblaa ŋutigbalẽa nyuie be abi nagaxɔ abi o eye wòana lãa nahaya. Wotsɔ vevesesenutsitike (caspofen) da dɔ na lãawo ŋkeke geɖe le dɔwɔwɔ nɛ vɔ megbe, eye le ɣeyiɣi ma me la, wolé ŋku ɖe woƒe ŋutilã ƒe nɔnɔme ŋu nyuie eye emegbe wowu wo le ɣeyiɣi si wogblɔ me. Wowɔ dɔawo katã le Europa, dukɔa kple dɔwɔƒewo ƒe mɔfiamewo nu eye LandesamtfürNatur si le Umwelt kple Verbraucherschutz, North Rhine-Westphalia, Germany da asi ɖe wo dzi.
Wotsɔ ketamine (100 mg/kg) kple xylazine (10 mg/kg) fa alɔ̃ na lãawo, eye wotsɔ 0.1 M PBS de dzia me gbã, eye emegbe wotsɔ 4% PFA de PBS me. Wotso lãkusia eye wotsɔe ɖo 4% PFA/PBS me zã bliboa le 4°C. Wozã hɛ si ʋuʋuna (Leica Microsystems GmbH, Vienna, Austria) tsɔ dzra sagittal sections (50 μm titrime) ɖo tso ahɔhɔ̃ si wotu ɖe teƒe ɖeka me le PBS me. Negbe ɖe wogblɔe bubui ko la, wowɔa amadede na akpa siwo le tsia dzi faa abe alesi míegblɔe ɖe etame ene (13) le xɔme ƒe dzoxɔxɔ nu eye wotsɔa wo ʋuʋuna. Kpuie ko la, gbã la, wotsɔ 0.5% Triton X-100/PBS de kakɛ siwo wokpɔ la me hena miniti 15 le xɔme ƒe dzoxɔxɔ nu; na epitopes aɖewo (Pcx kple Shmt2), to le tris-EDTA buffer le 80°C (PH 9) me do dzo kakɛawo hena miniti 25 ɖe afɔɖeɖe sia teƒe. Eyome, wotsɔ dɔlékuiwutike gbãtɔ (kpɔ Tabla S1) de mɔxenu (3% BSA/PBS) me le 4°C me zã bliboa kple ʋuʋu. Le ŋufɔke la, woklɔ akpaawo kple blocking buffer eye wotsɔ fluorophore-conjugated secondary antibody si sɔ la de wo me gaƒoƒo 2 le xɔme ƒe dzoxɔxɔ nu; mlɔeba la, woklɔ akpaawo nyuie le PBS me, wotsɔ DAPI tsɔ tsi tre ɖe wo ŋu, eye emegbe wotsɔ AquaPolymount On a microscope slide ɖɔ wo ɖo.
Wozã laser scanning confocal microscope (TCS SP8-X alo TCS Digital Light Sheet, Leica Microsystems) si me wotsɔ kekeli ɣi laser kple 405 diode ultraviolet laser ɖo tsɔ wɔ kpɔɖeŋua ƒe nɔnɔmetata. To fluorophore ƒe dzidzɔkpɔkpɔ kple dzesia nuƒoƒoƒu kple Hybrid Detector (HyDs) me la, wozã LAS-X kɔmpiuta dɔwɔɖoɖo tsɔ ƒo nɔnɔmetata siwo woƒo ƒu siwo sɔ kple Nyquist ƒe kpɔɖeŋuwo nu ƒu le ɖoɖo si wowɔ ɖe wo nɔewo yome ƒe nɔnɔme me: le non-quantitative panels gome la, enye dzesi siwo ŋu ŋusẽ le ŋutɔ (le kpɔɖeŋu me, le somatic cells kple dendrites me) mtYFP) Zã HyD nàtsɔ ade dzesi PNs ƒe xexlẽme le BrightR mode me). Wozãa gating tso 0.3 va ɖo 6 ns tsɔ ɖea megbenyawo dzi kpɔtɔna.
Lãmenugbagbevi siwo woɖo ɖe hatsotsowo me ƒe nɔnɔmetatawo ɖeɖe le ɣeyiɣi ŋutɔŋutɔ me. Esi woda lãmenugbagbeviwo ɖe hatsotsowo me le Neurobasal-A medium si me 1% B27 kpeɖeŋutɔ kple 0.5 mM GlutaMax le me vɔ la, woƒã lãmenugbagbeviwo enumake ɖe poly-l-lysine-coated glass slides (μ-Slide8 Well, Ibidi, catalog number 80826) , Eye emegbe wotsɔe de 37°C kple 5% CO2 me gaƒoƒo 1 be lãmenugbagbeviawo nate ŋu anɔ anyi. Wowɔ nɔnɔmetatawo le ɣeyiɣi ŋutɔŋutɔ me le Leica SP8 laser scanning confocal microscope si me wotsɔ laser ɣi, HyD, 63×[1.4 numerical aperture (NA)] ami ƒe taɖodzinu ƒe lens kple dzoxɔxɔ ƒe akpa aɖe ɖo.
Wotsɔ carbon dioxide fa alɔ̃ na sisiblisi la kaba eye wotso ta, woɖe ahɔhɔ̃a le tagbɔ kaba, eye wotsoe ɖe sagittal ƒe akpa si ƒe titrime nye 200μm (hena 13C ƒe dzesidede dodokpɔ) alo 275μm (na foton dodokpɔ eve) si me wotsɔ nu siwo gbɔna yɔ fũu The ice cream (HM-650 V, Thermo Fisher Scientific, Walldorf, Germany) . me yɔ fũ kple nusiwo gbɔna: 125 mM tsikpe fafɛ, carbon-saturated (95% O2 kple 5% CO2) si me Ca2 + si mebɔ o + ahɔhɔ̃meka me tsi si wowɔ (ACSF) NaCl, 2.5 mM KCl, 1.25 mM sodium phosphate buffer, 25 mM NaHCO3, 25 mM glucose, 0.5 mM CaCl2 kple 3.5 mM MgCl2 (osmotic ƒe nyaƒoɖeamenu si nye 310 va ɖo 330 mmol). Tsɔ ahɔhɔ̃meka siwo nèkpɔ la yi xɔ si me Ca2 + ACSF si lolo wu (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 kple 2.0 mM MgCl2) le do ŋgɔ na vidzidzi me. Medium) pH 7.4 kple 310 va ɖo 320 mmol).
Le nɔnɔmetatawo ɖeɖe ƒe ɖoɖoa me la, woɖe kakɛawo yi nɔnɔmetataxɔƒe si woɖo ɖi koŋ na wo, eye wowɔ dodokpɔa le ACSF ƒe tsi si wodona ɖaa me le dzoxɔxɔ si nɔa anyi ɖaa si nye 32° va ɖo 33°C me. Wozã fotoɖemɔ̃ geɖe ƒe laser scanning microscope (TCS SP8 MP-OPO, Leica Microsystems) si me Leica 25x objective lens (NA 0.95, tsi), Ti: Sapphire laser (Chameleon Vision II, Coherent) le hena slice nɔnɔmetatawo ɖeɖe. FLIM ƒe mɔ̃ (PicoHarp300, PicoQuant).
FLIM si nye Grx1-roGFP2 ƒe FLIM. Wotsɔ foton eve FLIM dzidze tɔtrɔ siwo va le PNwo ƒe cytoplasmic redox nɔnɔme me le sagittal ahɔhɔ̃ ƒe kakɛwo me, afisi Grx1-roGFP2 biosensor la tɔ ŋku PNwo le. Le PN ƒe ƒuƒoƒoa me la, wotiaa xɔxlɔ̃ ƒe agblea abe 50 va ɖo 80 μm le slice ƒe anyigba te be woakpɔ egbɔ be PN si ate ŋu anɔ anyi (si nye beaded structure alo neuronal morphological changes ƒe anyimanɔmanɔ le dendrites) kple double positive roGFP2 sensor kple AAV encoding shRNA PCx alo eƒe control sequence (each co-expressing mCherry) li. Ƒo nɔnɔmetata siwo wotsɔ ƒo ƒu ɖekae nu ƒu kple 2x digital zoom [excitation wavelength: 890 nm; 512 nm 512 piksel]. Deteksi: wozãa HyD ememetɔ, fluorescein isothiocyanate (FITC) filter group] kple nɔnɔmetata ƒe mamã dedie le miniti 2 va ɖo 3 me tsɔ kpɔa egbɔ be woƒo foton siwo sɔ nu ƒu (foton 1000 le wo katã me) hena curve fitting. Wowɔ Grx1-roGFP2 probe ƒe seselelãme kple FLIM nɔnɔmewo ƒe kpeɖodzi to ŋkuléle ɖe roGFP2 ƒe agbenɔƒe ƒe asixɔxɔ ŋu ne wotsɔ exogenous 10 mM H2O2 kpe ɖe perfusion ACSF ŋu (be woatsɔ adzi oxidation ɖe edzi, si ana agbenɔƒe nadzi ɖe edzi), eye emegbe wotsɔ 2 mM dithiothreitol kpe ɖe eŋu (eɖea alesi gbegbe edzi ɖena kpɔtɔnae dzi kpɔtɔna, si wɔnɛ be edzi ɖe kpɔtɔ le agbenɔƒe me) (Nɔnɔmetata S8, D vaseɖe G). Zã FLIMfit 5.1.1 kɔmpiuta dɔwɔɖoɖo nàtsɔ adzro emetsonu siwo wokpɔ me, atsɔ nɔnɔmetata bliboa ƒe gbegblẽ ƒe ʋuʋudedi ɖeka si wotsɔ ƒo ƒui la sɔ kple IRF si wodzidze (dɔwɔnu ƒe ŋuɖoɖo ƒe dɔwɔwɔ), eye χ2 anɔ abe 1. Be woabu PN ɖeka ƒe agbenɔƒe la, wotsɔ asi ta ametakpɔnu si ƒo xlã lãmeka ƒe ŋutilã, eye wozã agbenɔƒe si le mamã dedie nu le ametakpɔnu ɖesiaɖe me tsɔ bu agbɔsɔsɔme.
Mitochondrial ƒe ŋutete me dzodzro. Esi wotsɔ TMRM 100 nM si wotsɔ de ACSF si wodo ɖa me tẽe de akpa si nu sẽ la me aɖabaƒoƒo 30 vɔ la, wotsɔ foton eve ƒe nusuekpɔmɔ̃ dzidze PNwo ƒe mitochondrial ŋutete ƒe tɔtrɔwo. Wowɔ TMRM nɔnɔmetatawo to dzodzo le nusi wotsɔna dzroa nu me le 920 nm me eye wozã HyD ememetɔ (tetramethylrhodamine isothiocyanate: 585/40 nm) tsɔ ƒo dzesiwo nu ƒu; to dzidzɔdoname ƒe ƒutsotsoe ƒe didime ɖeka zazã me gake wozã ememe HyD vovovo (FITC :525/50) tsɔ wɔ mtYFP ƒe nɔnɔmetata. Zã ImageJ ƒe Image Calculator plug-in nàtsɔ ada mitochondrial ƒe ŋutete kpɔ le lãmenugbagbevi ɖeka ƒe ɖoɖo nu. Kpuie ko la, wozãa plug-in equation: signal = min (mtYFP, TMRM) tsɔ dea dzesi mitochondrial nuto si ɖea TMRM dzesi fiana le Purkinje Somali me le single-stack confocal image si le channel si sɔ la me. Emegbe wobua pixel ƒe teƒe si le mask si do tso eme la ƒe agbɔsɔsɔme, eye emegbe wowɔa esia wòsɔna ɖe mtYFP mɔ̃a ƒe threshold single-stack image si sɔ dzi be woakpɔ mitochondrial ƒe akpa si ɖe mitochondrial ƒe ŋutete fia.
Wotsɔ Huygens Pro (Scientific Volume Imaging) kɔmpiuta dɔwɔɖoɖo aɖe nɔnɔmetata la ɖa. Le tile ƒe nɔnɔmetata siwo woɖe le scan me gome la, wozãa automatic stitching algorithm si LAS-X kɔmpiuta dɔwɔɖoɖo na tsɔ wɔa tile ɖeka ƒe montage. Le nɔnɔmetata ƒe dzidzedze vɔ megbe la, zã ImageJ kple Adobe Photoshop nàtsɔ awɔ nɔnɔmetata la ŋudɔ geɖe wu eye nàtrɔ asi le eƒe keklẽ kple vovototoa ŋu ɖekae. Zã Adobe Illustrator hena nɔnɔmetatawo dzadzraɖo.
mtDNA ƒe susu ƒe numekuku. Wotsɔ confocal microscope bu mtDNA ƒe abi ƒe xexlẽme le ahɔhɔ̃mekawo ƒe akpa siwo wotsɔ dɔlékuiwutike siwo tsi tre ɖe DNA ŋu de dzesii la dzi. Wowɔ teƒe ɖesiaɖe si woɖo taɖodzinu na na lãmenugbagbevi ƒe ŋutilã kple lãmenugbagbevi ɖesiaɖe ƒe nucleus, eye wozã Multi Measure plug-in (ImageJ kɔmpiuta dɔwɔɖoɖo) tsɔ bu teƒe si woɖo la. Ðe nuklia ƒe akpa si le lãmenugbagbevi ƒe ŋutilã ƒe akpa si le lãmenugbagbeviwo me be nàkpɔ lãmenugbagbeviwo ƒe teƒea. Mlɔeba la, wozã Analyze Particles plug-in (ImageJ software) tsɔ bu cytoplasmic DNA teƒe siwo fia mtDNA le threshold image la dzi le wo ɖokui si, eye wowɔ emetsonu siwo wokpɔ la wòsɔ ɖe CTRL mice ƒe PN mamã dedie nu. Woɖea emetsonuwo fiana abe nucleoside ƒe xexlẽme si le lãmenugbagbevi ɖeka me le mama dedie nu ene.
Protein ƒe ɖeɖefia me dzodzro. Zã ImageJ ƒe Image Calculator plug-in nàtsɔ ada protein ƒe ɖeɖefia kpɔ le PN me le lãmenugbagbevi ɖeka ƒe ɖoɖo nu. Kpuie ko la, le mɔ̃ si sɔ ƒe nɔnɔmetata si wotsɔ ƒo ƒu ɖekae me la, to sɔsɔŋusẽ si nye: dzesi = min (mtYFP, dɔlékuiwutike) dzi la, wode dzesi mitochondrial nuto si ɖea dɔlélenutsiŋutete ƒe dɔwɔwɔ ɖe dɔlékuiwutike aɖe ŋu le Purkina me. Emegbe wobua pixel ƒe teƒe si le mask si do tso eme la ƒe agbɔsɔsɔme, eye emegbe wowɔa esia wòsɔna ɖe mtYFP mɔ̃a ƒe threshold single-stack image si sɔ dzi be woakpɔ protein si woɖe fia ƒe mitochondrial fraction.
Purkinje lãmenugbagbeviwo ƒe kpekpeme me dzodzro. Wozã ImageJ ƒe Cell Counter plug-in tsɔ da Purkinje ƒe kpekpeme kpɔ to Purkinje lãmenugbagbevi siwo woxlẽ ƒe xexlẽme mama ɖe ahɔhɔ̃meka ƒe asigɛ si me lãmenugbagbevi siwo woxlẽ la xɔ la ƒe didime nu.
Kpɔɖeŋuwo dzadzraɖo kple wo nu ƒoƒo ƒu. Woɖo ahɔhɔ̃ siwo tso ƒuƒoƒo si me wokpɔa wo gbɔ le kple Mfn2cKO mice me ɖe 2% PFA/2.5% glutaraldehyde me le 0.1 M phosphate buffer (PB) me, eye emegbe wotsɔ ciliates (Leica Mikrosysteme GmbH, Vienna, Austria) (Titrime 50 va ɖo 60 μm) dzra coronal ƒe akpawo ɖo Emegbe wotsɔe ɖo PB buffer me le 1% os tetraoxide kple 1.5% potassium ferrocyanide le xɔme ƒe dzoxɔxɔ me hena gaƒoƒo 1. Wotsɔ tsi si woɖe tso tsia me klɔ akpaawo zi etɔ̃, eye emegbe wotsɔ ethanol 70% si me uranyl acetate 1% le de ama wo hena miniti 20. Emegbe woɖe tsi le akpaawo me le aha si woɖo ɖe ɖoɖo nu me eye wotsɔ wo de Durcupan ACM (Araldite casting resin M) epoxy resin (Electron Microscopy Sciences, catalog number 14040) me le glass slide siwo ŋu silicon-fa ɖo dome, eye mlɔeba le 60°C me la, wotsɔ Polymerize de dzodoƒe gaƒoƒo 48. Wotia ahɔhɔ̃mekawo ƒe akpa si le lãkusia me eye wotso akpa siwo le sue ŋutɔ siwo ƒe didime nye 50 nm le Leica Ultracut (Leica Mikrosysteme GmbH, Vienna, Austria) dzi eye woɖe wo le akɔbli si ƒe lolome nye 2×1 mm ƒe ʋuƒo si wotsɔ polystyrene film tsyɔ la dzi. Wotsɔ 4% uranyl acetate ƒe tsi si le H2O me de ama akpaawo hena miniti 10, woklɔ wo kple H2O zi geɖe, emegbe wotsɔ Reynolds lead citrate le H2O me hena miniti 10, eye emegbe woklɔ wo kple H2O zi geɖe. Wotsɔ TVIPS (Tietz Video kple Nɔnɔmetatawo Ŋuti Dɔwɔɖoɖo) TemCam-F416 dijitaal fotoɖemɔ̃ (TVIPS GmbH, Gauting, U.S.A.) tsɔ elektrɔnik nusuekpɔmɔ̃ si wotsɔna ɖoa nyatakakawo ɖa si nye Philips CM100 (Thermo Fisher Scientific, Waltham, MA, U.S.A.) tsɔ nu suesuesuewo. Germany).
Le sisiblisi siwo ŋu AAV le gome la, woɖe ahɔhɔ̃a ɖe vovo eye wotsoe ɖe sagittal ƒe akpa si ƒe titrime nye milimeta 1 me, eye wozã fluorescence microscope tsɔ do ahɔhɔ̃a kpɔ be woatsɔ ade dzesi asigɛ si ŋu AAV le (si fia be mCherry ɖee fia). Dodokpɔ siwo me AAV dodo na Purkinje lãmenugbagbeviwo ƒe ƒuƒoƒo (si nye ƒuƒoƒo bliboa kloe) ƒe dɔwɔwɔ nyuie ŋutɔ le ahɔhɔ̃meka eve ya teti siwo kplɔ wo nɔewo ɖo me koe wozãna. Woɖe AAV-trɔtrɔ ƒe loop la ƒe microdissected hena zã ɖeka le fixation megbe (4% PFA kple 2.5% glutaraldehyde le 0.1 M cocoate buffer me) eye wogatrɔ asi le eŋu. Le EPON ƒe tsɔtsɔ de eme gome la, woklɔ lãkusi si woɖo ɖi la kple 0.1 M sodium cocoate buffer (Applichem), eye wotsɔ 2% OsO4 (os, Science Services; Caco) de 0.1 M sodium cocoate buffer (Applichem) me Gaƒoƒo 4, emegbe woklɔe gaƒoƒo 2. Gbugbɔ wɔe zi 3 kple 0.1 M cocamide buffer. Emegbe la, wozã ethanol si kplɔ wo nɔewo ɖo si le dzi yim tsɔ tsɔ ethanol-tsi ɖesiaɖe de dzoxɔxɔ 4°C me aɖabaƒoƒo 15 be tsi naɖe le lãkusia me. Wotsɔ lãkusia de propylene oxide me eye wotsɔe de EPON (Sigma-Aldrich) me zã bliboa le dzoxɔxɔ 4°C me. De lãkusia EPON yeye me le xɔme ƒe dzoxɔxɔ nu gaƒoƒo 2, eye emegbe nàtsɔe ade dzoxɔxɔ 62°C me gaƒoƒo 72. Zã ultramicrotome (Leica Microsystems, UC6) kple diamond hɛ (Diatome, Biel, Switzerland) nàtsɔ atso 70 nm ƒe akpa siwo le sue ŋutɔ, eye nàtsɔ 1.5% uranyl acetate aƒo ɖi wo aɖabaƒoƒo 15 le 37°C, eye nàtsɔ lead citrate solution aƒo ɖi wo aɖabaƒoƒo 4. Wozã JEM-2100 Plus elektrɔnik nusuekpɔmɔ̃ (JEOL) si me Camera OneView 4K 16-bit (Gatan) kple DigitalMicrograph kɔmpiuta dɔwɔɖoɖo (Gatan) le tsɔ ɖe elektrɔnik nusuekpɔmɔ̃awo. Le numekuku ta la, wotsɔ 5000× alo 10,000× digital zoom xɔ elektrɔnik nusuekpɔmɔ̃wo.
Mitochondria ƒe nɔnɔme me dzodzro. Le numekukuawo katã gome la, wotsɔ asi ŋlɔ mitochondria ɖekaɖekawo ƒe nɔnɔmewo le dijitaal nɔnɔmetatawo me to ImageJ kɔmpiuta dɔwɔɖoɖo zazã me. Wodzroa nɔnɔme ƒe nɔnɔme vovovowo me. Woɖea mitochondria ƒe kpekpeme fiana abe alafa memamã si wokpɔ to lãmenugbagbevi ɖesiaɖe ƒe mitochondrial ƒe teƒe bliboa mama ɖe cytoplasm ƒe teƒe nu (cytoplasm area = cell area-cell nucleus area) × 100. Wobu mitochondria ƒe goglome kple mɔfiame [4π∙(area/perimeter 2)]. Woda mitochondria ƒe ista nɔnɔme kpɔ eye womae ɖe hatsotso eve me (“tubular” kple “blister”) le woƒe nɔnɔme veviwo nu.
Autophagosome/lysosome ƒe xexlẽme kple eƒe kpekpeme me dzodzro. Zã ImageJ kɔmpiuta dɔwɔɖoɖo nàtsɔ asi agblɔ autophagosome/lysosome ɖesiaɖe ƒe nɔnɔme siwo le dijitaal nɔnɔmetata la me. Woɖea autophagosome/lysosome ƒe teƒe fiana abe alafa memamã si wobu to autophagosome/lysosome ƒe wɔwɔme ƒe teƒe bliboa mama le lãmenugbagbevi ɖesiaɖe me ɖe cytoplasm ƒe teƒe (cytoplasm area=cell area-nucleus area)×100 nu. Wobua autophagosomes/lysosomes ƒe kpekpeme to xexlẽme bliboa mama ɖe autophagosome/lysosome ƒe wɔwɔme ƒe xexlẽme le lãmenugbagbevi ɖeka me (le cytoplasmic area gome) (cytoplasmic area = cell area-nuclear area).
Dzesidede na acute sectioning kple kpɔɖeŋu dzadzraɖo. Le dodokpɔ siwo hiã be woade dzesi suklidɔ la, tsɔ ahɔhɔ̃ ƒe kakɛ siwo le sesẽm la yi xɔ si me wodo ŋgɔ na vidzidzi, si me saturated carbon (95% O2 kple 5% CO2), Ca2 + ACSF si sɔ gbɔ (125.0 mM NaCl, 2.5 mM KCl, 1.25 mM sodium phosphate buffer, 25.0 mM NaHCO 3, 25.0 mM) le d-glucose, 1.0 mM CaCl 2 kple 2.0 mM MgCl 2, si woɖɔli ɖe pH 7.4 kple 310 va ɖo 320 mOsm), si me sukli nye 13 C 6- Glucose ɖɔliɖɔli (Eurisotop, agbalẽdzraɖoƒe ƒe xexlẽdzesi CLM-1396). Le dodokpɔ siwo hiã be woade dzesi pyruvate la, tsɔ ahɔhɔ̃ ƒe kakɛ siwo le sesẽm la yi Ca2 + ACSF si kɔkɔ wu (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 eye nàtsɔ 2.0mM akpe ɖe eŋu MgCl2, trɔ asi le pH 7.4 kple 310 va ɖo 320mOsm), eye nàtsɔ 1mM 1-[1-13C]pyruvate (Eurisotop, agbalẽdzraɖoƒe ƒe xexlẽdzesi CLM-1082) akpe ɖe eŋu. Tsɔ akpaawo de dzoxɔxɔ 37°C me aɖabaƒoƒo 90. Le dodokpɔa ƒe nuwuwu la, woklɔ akpaawo kabakaba kple tsi (pH 7.4) si me ammonium carbonate 75 mM le, eye emegbe wotsɔ 40:40:20 (v:v:v) acetonitrile (ACN): methanol: tsi wɔ wo ɖekae. Esi wotsɔ akpaawo de tsikpe dzi aɖabaƒoƒo 30 vɔ la, wotsɔa kpɔɖeŋuawo dea tsikpe me le 21,000 g me hena miniti 10 le dzoxɔxɔ 4°C me, eye wotsɔa tsi si le wo dzi si me kɔ la ƒua gbe le SpeedVac concentrator me. Wodzra metabolite pellet ƒuƒu si do tso eme la ɖo ɖe -80°C vaseɖe esime wodzro eme.
Liquid chromatography-mass spectrometry numekuku le amino acid 13 siwo wotsɔ C-de dzesii ŋu. Le liquid chromatography-mass spectrometry (LC-MS) numekuku me la, wogbugbɔ tsɔ metabolite pellet la de LC-MS grade tsi 75μl me (Honeywell). Le centrifugation le 21,000 g me aɖabaƒoƒo 5 le 4°C megbe la, wozã tsi si le edzi si me kɔ la ƒe 20 μl hena amino acid flux numekuku, gake wozã nusi woɖe tso eme ƒe akpa mamlɛa enumake hena anion numekuku (kpɔ ete). Wozã benzoyl chloride derivatization protocol si ŋu míeƒo nu tsoe va yi ( 55 , 56 ). Le afɔɖeɖe gbãtɔ me la, wotsɔ 10μl 100 mM sodium carbonate (Sigma-Aldrich) de 20μl metabolite extract me, eye emegbe wotsɔ 10μl 2% benzoyl chloride (Sigma-Aldrich) de LC grade ACN me. Wotsɔ vortex ƒo kpɔɖeŋua kpuie eye emegbe wotsɔ centrifuged le 21,000 g me aɖabaƒoƒo 5 le 20°C. Tsɔ tsi si le edzi si woɖe ɖa la de 2 ml autosampler vial si me conical glass insert (200 μl volume) le me. Wozã Acquity iClass ultra-high performance LC system (Waters) si do ƒome kple Q-Exactive (QE)-HF (Ultra High Field Orbitrap) high-resolution precision mass spectrometer (Thermo Fisher Scientific) tsɔ da kpɔɖeŋuawo kpɔ. Le numekuku me la, wodo kpɔɖeŋu si woɖe tso eme la ƒe 2μl ɖe 100×1.0 mm si ƒe ŋusẽ lolo silica T3 sɔti (Tsiwo) si me 1.8μm ƒe nusuewo le la me. Sisi ƒe agbɔsɔsɔme nye 100μl/min, eye buffer system la nye buffer A (10 mM ammonium formate kple 0.15% formic acid le tsi me) kple buffer B (ACN). Nusiwo le ʋuʋum la le ale: 0%B le miniti 0 me; 0%B. 0 va ɖo 15% B le miniti 0 va ɖo 0.1 me; 15 va ɖo 17% B le miniti 0.1 va ɖo 0.5 me; B le 17 va ɖo 55% le miniti 0.5 va ɖo 14 me; B le 55 va ɖo 70% le miniti 14 va ɖo 14.5 me; le 14.5 va ɖo 70 va ɖo 100% B le miniti 18 me; 100% B le miniti 18 va ɖo 19 me; 100 va ɖo 0% B le miniti 19 va ɖo 19.1 me; 0% B le miniti 19.1 va ɖo 28 me (55, 56). QE-HF mass spectrometer wɔa dɔ le ionization mode nyuitɔ me kple mass ƒe didime si nye m/z (mass/charge ratio) si nye 50 va ɖo 750. Resolution si wozã la nye 60,000, eye gain control (AGC) ion target si wokpɔ la nye 3×106, eye ion ƒe ɣeyiɣi si sɔ gbɔ wu nye miliseconds 100. Heated electrospray ionization (ESI) dzɔtsoƒea wɔa dɔ le spray voltage si nye 3.5 kV, capillary temperature si nye 250°C, sheath airflow si nye 60 AU (arbitrary units), kple auxiliary ya si nye 20 AU. 250°C ƒe dzoxɔxɔ. Woɖo S lens la ɖe 60 AU dzi.
Anion chromatography-MS ƒe numekuku le 13C ƒe dzesi organic acidwo ŋu. Wozã Dionex ion chromatography system (ICS 5000+, Thermo Fisher Scientific) si do ƒome kple QE-HF mass spectrometer (Thermo Fisher Scientific) tsɔ da metabolite ƒe tsidzadza susɔea (55μl) kpɔ. Kpuie ko la, wodo 5μl metabolite extract ɖe Dionex IonPac AS11-HC sɔti si me HPLC (2 mm×250 mm, nusuewo ƒe lolome 4μm, Thermo Fisher Scientific) le me le push-in partial loop mode me kple yɔyɔ ƒe xexlẽme si nye 1. ) Dionex IonPac AG11-HC dzɔla sɔti (2 mm x 50 mm, 4μm, Thermo Fisher Dzɔdzɔmeŋutinunya). Woléa sɔti ƒe dzoxɔxɔ ɖe te le 30°C, eye woɖoa autosampler la ɖe 6°C. Zã potassium hydroxide ƒe ʋuƒo si me tsi si me ion mele o le nàtsɔ awɔ potassium hydroxide ƒe ʋuʋudedi to eluent generator la dzi. Nusiwo wotsɔ ɖɔa li lãmenugbagbeviwoe ƒe mama le sisi ƒe agbɔsɔsɔme si nye 380μl/min me, wozãa ʋuʋudedi si gbɔna: miniti 0 va ɖo 3, 10 mM KOH; miniti 3 va ɖo 12, 10 va ɖo 50 mM KOH; miniti 12 va ɖo 19, 50 va ɖo 100 mM KOH; miniti 19 va ɖo 21 , 100 mM KOH; miniti 21 va ɖo 21.5, 100 va ɖo 10 mM KOH. Wogatsɔ sɔti la da sɔ le 10 mM KOH te hena miniti 8.5.
Wotsɔa metabolite siwo woɖe ɖa la ƒoa ƒu kple isopropanol kpeɖeŋutɔ ƒe sisi si ƒe didime nye 150μl/min le sɔti la megbe eye emegbe woɖoa wo ɖe mass spectrometer si ƒe nɔnɔme deŋgɔ si le dɔ wɔm le ionization mode manyomanyo me. MS le ŋku lém ɖe kpekpeme ƒe didime ŋu tso m/z 50 va ɖo 750 kple 60,000 ƒe vovototodedeameme. Woɖo AGC ɖe 1×106, eye wotsɔ ion ƒe ɣeyiɣi si sɔ gbɔ wu la ɖo 100 ms. Wowɔ dɔ le ESI dzɔtsoƒe si wodo dzoe la ŋu le atike si wotsɔna ƒua gbe ƒe ŋusẽ si nye 3.5 kV me. Ion dzɔtsoƒea ƒe ɖoɖo bubuawoe nye esiawo: ʋukawo ƒe dzoxɔxɔ 275°C; gas si le kotoku me, 60 AU; gas kpeɖeŋutɔ ƒe sisi, 20 AU le 300°C, kple S lens ɖoɖo ɖe 60 AU.
Nyatakakawo me dzodzro le 13C ƒe dzesidenu siwo wotsɔ ɖɔa lãmenugbagbeviwoe ŋu. Zã TraceFinder kɔmpiuta dɔwɔɖoɖo (trɔtrɔ 4.2, Thermo Fisher Scientific) hena nyatakakawo me dzodzro le isotope ƒe xexlẽme ŋu. Wotsɔ atike si ŋu kakaɖedzi le si dzi woate ŋu aƒo nu tsoe ɖo kpe atike ɖesiaɖe ƒe dzedzeme dzi eye woda wo kpɔ le wo ɖokui si. Be woateŋu awɔ isotope ƒe dzidziɖedzi ŋuti numekuku la, woɖe 13C isotope (Mn) ɖesiaɖe ƒe ion chromatogram (XIC) si woɖe tso eme ƒe teƒe tso [M + H] + me, afisi n nye carbon xexlẽme si le atike si woɖo taɖodzinu na me, si wozãna tsɔ daa amino acidwo kpɔ alo wozãa [ MH] + tsɔ kua anionwo me. XIC ƒe agbɔsɔsɔme ƒe nyateƒetoto mede akpa atɔ̃ le miliɔn ɖeka me o, eye RT ƒe nyateƒetoto nye miniti 0.05. Wowɔa nusiwo wotsɔ doa ŋusẽe ƒe numekukua to akɔntabubu le isotope ɖesiaɖe si wokpɔ ƒe sɔsɔme kple isotopewo katã ƒe ƒuƒoƒo si le atike si sɔ kplii me. Wotsɔ xexlẽme siawo na abe alafa memamã ƒe asixɔxɔwo na isotope ɖesiaɖe ene, eye woɖea emetsonuwo fiana abe molar percent enrichment (MPE), abe alesi míegblɔe va yi ene (42).
Wowɔ ahɔhɔ̃mekawo ƒe ƒuƒoƒo si wofa tsikpe la wòzu ɖeka le methanol 80% (v/v) si fa miamiamia me, wotsɔe ƒo ƒui, eye wotsɔe de -20°C me aɖabaƒoƒo 30. Gaʋuʋu kpɔɖeŋua ake eye nàʋuʋui le +4°C aɖabaƒoƒo 30. Wotsɔa centrifuged na kpɔɖeŋua le 21,000 g me hena miniti 5 le 4°C, eye emegbe woƒoa tsi si do tso eme la nu ƒu heƒua gbe to SpeedVac concentrator si le 25°C zazã me hena numekuku emegbe. Abe alesi míegblɔe le etame ene la, wowɔ LC-MS numekuku le lãmenugbagbevi siwo woɖo ɖe hatsotsowo me ƒe amino acidwo ŋu. Wozã TraceFinder (version 4.2, Thermo Fisher Scientific) tsɔ wɔ nyatakakawo me dzodzro to atike ɖesiaɖe ƒe monoisotopic mass zazã me. Wowɔ metabolite nyatakakawo ƒe agbɔsɔsɔme si sɔ pɛpɛpɛ to preprocessCore kɔmpiuta dɔwɔɖoɖo (57) zazã me.
Slice dzadzraɖo. Wotsɔ carbon dioxide fa alɔ̃ na sisiblisi la kaba eye wotso ta nɛ, woɖe ahɔhɔ̃a le tagbɔ kaba, eye wozã hɛ si ʋuʋuna si me tsikpe yɔ fũu (HM-650 V, Thermo Fisher Scientific, Walldorf, Germany) tsɔ lãe ɖe 300 va ɖo 375 μm sagittal sections me Cold carbon gasification (95% O2 kple 5% CO2) Low 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 kple 6.0 mM MgCl2 Trɔ asi le pH 7.4 ŋu eye 310 va ɖo 330 mOsm). Tsɔ ahɔhɔ̃meka siwo nèkpɔ la yi xɔ si me Ca2 + ACSF si lolo wu (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 kple mM 3.5 MgCl2) pH 7.4 kple 310 va ɖo 320 mOsm). Dzra kakɛawo ɖo aɖabaƒoƒo 20 va ɖo 30 ale be woate ŋu agbugbɔ wo aɖo wo te hafi alé wo ɖe mɔ̃ dzi.
nuŋɔŋlɔ si wolé ɖe mɔ̃ dzi. Wozã nusuekpɔmɔ̃ si me wotsɔa nuléƒe si wotu ɖe teƒe ɖeka kple tsi me nyɔnyrɔ̃ ƒe taɖodzinu ƒe ŋkumeɖonu si ƒe didime nye 20x (Scientifica) ɖo la tsɔ léa nu ɖe ​​mɔ̃ dzi katã. Wode dzesi Purkinje lãmenugbagbevi siwo wosusu be woto (i) ŋutilã ƒe lolome, (ii) ahɔhɔ̃mekawo ƒe ŋutilã ƒe teƒe si wòle, kple (iii) fluorescent mtYFP reporter gene ƒe ɖeɖefia. Wohea patch pipette si ƒe nugbɔ ƒe tsitretsitsi nye megohms 5 va ɖo 11 la doa goe to borosilicate glass capillary (GB150-10, 0.86 mm×1.5 mm×100 mm, Science Products, Hofheim, Germany) kple pipette si le tsia dzi Instruments (P-1000, Sutter), Novato, CA). Wotsɔ ELC-03XS npi patch clamp amplifier (npi electronic GmbH, Tam, Germany), si dzi kɔmpiutadziɖoɖo si nye Signal (version 6.0, Cambridge Electronic, Cambridge, UK) kpɔ ŋusẽ ɖo lae wɔ nuŋlɔɖiawo katã. Wolé dodokpɔa ɖe mɔ̃ dzi le kpɔɖeŋu ƒe tsɔtsɔme si nye 12.5 kHz me. Wotsɔa Bessel filter eve siwo toa kpuie siwo ƒe cutoff frequencies nye 1.3 kple 10 kHz tsia dzesia me. Wotsɔa compensation circuit si zãa amplifier la xea fe na membrane kple pipette ƒe capacitance. Wowɔ dodokpɔawo katã le Orca-Flash 4.0 fotoɖemɔ̃ (Hamamatsu, Gerden, Germany) ƒe dzikpɔkpɔ te, si dzi Hokawo kɔmpiuta dɔwɔɖoɖo (version 2.8, Hamamatsu, Gerden, Germany) kpɔ ŋusẽ ɖo.
Lãmenugbagbevi blibowo ƒe ɖoɖowɔwɔ kple wo me dzodzro edziedzi. Hafi nàlée ɖe mɔ̃ dzi teti la, tsɔ ememe tsi si me nusiwo gbɔna le la yɔ pipette la me: 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) kple Wotrɔ asi le creatinine phosphate 10.0 mM ŋu wòɖo pH 7.25, eye osmotic ƒe nyaƒoɖeamenu nye 290 mOsm (sucrose). Esi wozã ŋusẽ si nye 0 pA tsɔ gbã lãkusia vɔ teti la, wodzidze lãkusia ƒe ŋutete si le gbɔɖemeƒe. Wodzidzea tsitretsitsi si wotsɔ dea eme la to elektrikŋusẽ si me ʋuʋudedi le si nye -40, -30, -20, kple -10 pA zazã me. Dzidze elektrikŋusẽ ƒe ŋuɖoɖo ƒe lolome eye nàzã Ohm ƒe se atsɔ abu tsitretsitsi si wotsɔ de eme. Woŋlɔ dɔwɔwɔ si dzɔna le eɖokui si ɖe voltage clamp me hena miniti 5, eye wode dzesi sPSC eye wodzidzee le Igor Pro (version 32 7.01, WaveMetrics, Lake Oswego, Oregon, USA) me to semi-automatic recognition script zazã me. Wodzidzea IV curve kple steady-state current to batri la ƒoƒo ɖe ŋusẽ vovovowo me (edzea egɔme tso -110 mV) kple voltage dzi ɖeɖe kpɔtɔ le 5 mV afɔɖeɖewo me. Wodo AP ƒe wɔwɔ kpɔ to depolarizing current zazã me. Kpla lãmenugbagbevi la le -70 mV esime nèle depolarizing current pulse zãm. Trɔ asi le nuƒomɔ̃ ɖesiaɖe ƒe afɔɖeɖe ƒe lolome ŋu ɖe vovo (10 va ɖo 60 pA). Bu AP ƒe tsɔtsɔme si sɔ gbɔ wu to asi xexlẽ le pulse spikes siwo hea AP frequency si lolo wu vɛ me. Wodzroa AP ƒe dzidzenu me to depolarization pulse ƒe derivative evelia si ʋãa AP ɖeka alo esi wu nenema gbã la zazã me.
Perforated patch ɖoɖowɔwɔ kple numekuku. Wɔ perforated patch recording to ɖoɖowɔɖi siwo wozãna ɖaa zazã me. Zã pipette si me ATP kple GTP mele o si me nusiwo gbɔna mele o: 128 mM gluconate K, 10 mM KCl, 10 mM Hepes, 0.1 mM EGTA kple 2 mM MgCl2, eye nàtrɔ asi le eŋu wòaɖo pH 7.2 (zã KOH). Woɖea ATP kple GTP ɖa le lãmenugbagbeviwo me tsia me be woaxe mɔ ɖe lãmenugbagbeviwo ƒe lãkusi ƒe gege ɖe eme si dzi womeɖu o nu. Wotsɔa ememe tsi si me amphotericin le (si ade 200 va ɖo 250μg/ml; G4888, Sigma-Aldrich) yɔa patch pipette la me be woakpɔ patch record si woƒo. Wotsɔ amphotericin ƒo dimethyl sulfoxide (agbɔsɔsɔ mamlɛtɔ: 0.1 va ɖo 0.3%; DMSO; D8418, Sigma-Aldrich). DMSO ƒe agbɔsɔsɔ si wozã mekpɔ ŋusẽ gã aɖeke ɖe ahɔhɔ̃meka siwo ŋu wosrɔ̃ nu tsoe dzi o. Le punching ƒe ɖoɖoa me la, woléa ŋku ɖe channel resistance (Ra) ŋu ɣesiaɣi, eye wodze dodokpɔa gɔme le Ra kple AP ƒe amplitude ƒe liƒo megbe (aɖabaƒoƒo 20-40). Wodzidzea dɔwɔwɔ le eɖokui si le elektrikŋusẽ kple/alo elektrikŋusẽ ƒe nulénu me hena miniti 2 va ɖo 5. Wozã Igor Pro (tɔtrɔ 7.05.2, WaveMetrics, USA), Excel (tɔtrɔ 2010, Microsoft Corporation, Redmond, USA) kple GraphPad Prism (tɔtrɔ 8.1.2, GraphPad Software Inc., La Jolla, CA) tsɔ wɔ nyatakakawo me dzodzro. United States). Be woate ŋu ade dzesi AP siwo dzɔna le wo ɖokui si la, wozãa IgorPro ƒe NeuroMatic v3.0c plug-in. De dzesi APwo le ɖokuiwò si to dzidzenu si wona zazã me, si wotrɔ asi le ɖekaɖeka na nuŋlɔɖi ɖesiaɖe. Zã spike dometsotsoa nàtsɔ anya spike frequency si me spike frequency si sɔ gbɔ wu enumake le kple spike frequency le mama dedie nu.
PN ƒe ame ɖokui ɖeɖe ɖe aga. To tɔtrɔ ɖe ɖoɖowɔɖi si wota va yi ŋu me la, wokɔ PN-wo ŋu tso sisiblisi ƒe ahɔhɔ̃meka me le afɔɖeɖe aɖe koŋ me ( 58 ). Kpuie ko la, wotso ahɔhɔ̃mekaa eye wotsoe le tsikpe fafɛ ƒe vovototodedeameme ƒe tsi me [si me HBSS Ca2+ kple Mg2+ mele o, wotsɔ sukli 20 mM, penicillin (50 U/ml) kple streptomycin (0.05 mg/ ml) kpe ɖe eŋu], eye emegbe woɖu nusi le papain me [HBSS, si wotsɔ 1-cysteine·HCl kpe ɖe eŋu (1 mg / ml), papain (16 U / ml) kple deoxyribonuclease I (DNase I; 0.1 mg/ml)] Tsɔe da dɔe aɖabaƒoƒo 30 le dzoxɔxɔ 30°C me. Gbã la, klɔ lãkusiawo le HBSS tsi si me azi ƒe ʋuʋudedi (10 mg/ml), BSA (10 mg/ml) kple DNase (0.1 mg/ml) le me le xɔme ƒe dzoxɔxɔ nu be wòaxe mɔ ɖe enzymatic nuɖuɖumeŋusẽ nu, eye emegbe nàklɔ lãkusiawo le HBSS nusi me sukli 20 mM le me blewuu le HBSS, penicillin (50 U/ml), streptomycin (0.05 mg/ml) kple DNase (0.1 mg/ml) ɖea lãmenugbagbevi ɖekaɖekawo ɖa. Woɖe lãmenugbagbevi siwo do tso eme la to lãmenugbagbeviwo ƒe ʋuʋudedi si ƒe lolome nye 70μm me, emegbe wotsɔ centrifugation (1110 rpm, miniti 5, 4°C) ƒo lãmenugbagbeviawo ƒu eye wogatsɔ wo ƒo ƒu ɖe vovototodedeameme me [HBSS, si wotsɔ 20 mM glucose, 20% fetal bovine ) Serum, penicillin (50 U/ml) kple streptomycin kpe ɖe eŋu (0.05 mg/ml)]; tsɔ propidium iodide da lãmenugbagbeviwo ƒe agbenɔnɔ kpɔ eye woatrɔ asi le lãmenugbagbeviwo ƒe kpekpeme ŋu wòanye 1×106 va ɖo 2×106 cells/ml. Hafi woawɔ flow cytometry la, woɖea tsi si wotsɔ ƒoe la to 50 μm cell strainer me.
Sisi ƒe lãmenugbagbeviwodzidzenu. Wowɔ lãmenugbagbeviwo ƒe vovototodedeameme le 4°C to FACSAria III mɔ̃ (BD Biosciences) kple FACSDiva kɔmpiuta dɔwɔɖoɖo (BD Biosciences, version 8.0.1) zazã me. Wozã 100 μm ƒe nugbɔ tsɔ ƒo lãmenugbagbevi ƒe ʋuʋu ɖe hatsotsowo me le nyaƒoɖeamenu si nye 20 psi me le ~2800 nudzɔdzɔwo/sekɛnd ƒe agbɔsɔsɔ me. Esi wònye be gating dzidzenu xoxowo (lãmenugbagbeviwo ƒe lolome, bimodal vovototodedeameme, kple kaka ƒe nɔnɔmewo) mate ŋu akpɔ egbɔ be woɖe PN ɖe vovo nyuie tso lãmenugbagbevi ƒomevi bubuwo gbɔ o ta la, wotu gating ƒe aɖaŋua ɖe YFP ƒe sesẽme kple autofluorescence tsɔtsɔ sɔ kple wo nɔewo tẽ le mitoYFP+ ​​kple control mitoYFP − Mice me dzi. Wodoa dzidzɔ na YFP to keklẽŋusẽ si wotsɔ 488 nm laser-ka ƒoa kpɔɖeŋuae me, eye wozãa 530/30 nm band pass filter tsɔ dea dzesi dzesia. Le mitoYFP+ ​​mice me la, wozãa Rosa26-mitoYFP nyadzɔdzɔgblɔla domenyiŋusẽfianu ƒe ŋusẽ si sɔ hã tsɔ dea vovototo ahɔhɔ̃mekawo ƒe ŋutilã kple axon kakɛwo dome. Wotsɔa 561 nm yellow laser doa dzidzɔ na 7-AAD eye wotsɔa 675/20 nm bandpass filter dea dzesii be woaɖe lãmenugbagbevi kukuwo ɖa. Be woate ŋu ama ɣletivimefakaka le ɣeyiɣi ɖeka me la, wotsɔ ACSA-2-APC de ama lãmenugbagbevi ƒe ʋuʋudedi la, emegbe wotsɔ 640 nm laser line klẽ ɖe kpɔɖeŋua dzi, eye wozã 660/20 nm bandpass filter tsɔ de dzesi dzesia.
Wotsɔ centrifugation (1110 rpm, miniti 5, 4°C) ƒo lãmenugbagbevi siwo woƒo ƒu la ƒu eye wodzra wo ɖo ɖe -80°C vaseɖe esime woazã wo. Woda Mfn2cKO mice kple woƒe gbeɖuɖɔ viwo ɖe hatsotsowo me le ŋkeke ɖeka dzi be woaɖe dɔwɔwɔ ƒe tɔtrɔ dzi akpɔtɔ. Wozã FlowJo kɔmpiuta dɔwɔɖoɖo (FlowJo LLC, Ashland, Oregon, USA) tsɔ wɔ FACS nyatakakawo ɖeɖefia kple wo me dzodzro.
Abe alesi míegblɔe le etame ene (59) la, wozãa ɣeyiɣi ŋutɔŋutɔ me PCR tsɔ ɖea DNA ɖe vovo tso ahɔhɔ̃meka siwo woɖo ɖe hatsotsowo me hena mtDNA ƒe agbɔsɔsɔme didi emegbe. Wodo linearity kple threshold sensitivity kpɔ gbã to qPCR wɔwɔ ɖe lãmenugbagbevi ƒe xexlẽme vovovowo dzi me. Kpuie ko la, ƒo PN 300 nu ƒu ɖe lysis buffer si me 50 mM tris-HCl (pH 8.5), 1 mM EDTA, 0.5% Tween 20 kple proteinase K (200 ng/ml) le me eye nàtsɔe ade 55°C me aɖabaƒoƒo 120. Wogatsɔ lãmenugbagbeviawo de dzoxɔxɔ 95°C me hena miniti 10 be woakpɔ egbɔ be proteinase K megawɔa dɔ keŋkeŋ o. Wozã TaqMan probe (Thermo Fisher) si ku ɖe mt-Nd1 koŋ ŋu tsɔ dzidze mtDNA to semi-quantitative PCR me le 7900HT Real-Time PCR system (Thermo Fisher Scientific) me. Dzɔdzɔmeŋutinunya, agbalẽdzraɖoƒe ƒe xexlẽdzesi Mm04225274_s1), mt-Nd6 (Thermo Fisher Scientific, agbalẽdzraɖoƒe ƒe xexlẽdzesi AIVI3E8) kple 18S (Thermo Fisher Scientific, agbalẽdzraɖoƒe ƒe xexlẽdzesi Hs99999901_s1) domenyiŋusẽfianuwo.
Proteome ƒe kpɔɖeŋu dzadzraɖo. To dzoxɔxɔ na tsi si le 95°C hena miniti 10 kple sonicating, le lysis buffer [6 M guanidine chloride, 10 mM tris (2-carboxyethyl) phosphine hydrochloride, 10 mM chloroacetamide kple 100 mM tris- Lyse frozen neuron pellets le HCl me]. Le Bioruptor (Diagenode) dzi hena miniti 10 (sɛkɛnd 30 ƒe ʋu ƒe sisi / sɛkɛnd 30 ƒe ɖiɖiɖemeɣi). Wotsɔ 1:10 ƒo kpɔɖeŋua le 20 mM tris-HCl (pH 8.0) me, wotsɔe tsaka kple 300 ng trypsin gold (Promega), eye wotsɔe de dzo me zã bliboa le 37°C me be wòaɖu nu bliboe. Le ŋkeke evelia dzi la, wotsɔa centrifuge tsɔa kpɔɖeŋua dea g 20,000 me aɖabaƒoƒo 20. Wotsɔ formic acid 0.1% tsi tsi si le edzi la me, eye wotsɔ StageTips siwo wowɔ wo ɖokui la ɖe dze le tsi si le tsia dzi la ɖa. Wotsɔa SpeedVac dɔwɔnu (Eppendorf concentrator plus 5305) ƒua gbe le dzoxɔxɔ 45°C me, eye emegbe wotsɔa peptide la ƒua gbe ɖe 0.1% formic acid me. Ame ɖeka ma ke dzra kpɔɖeŋuawo katã ɖo le ɣeyiɣi ɖeka me. Be woate ŋu ada astrocyte ƒe kpɔɖeŋuwo kpɔ la, wotsɔ tandem mass tag (TMT10plex, catalog number 90110, Thermo Fisher Scientific) de dzesi peptide siwo me dze mele o 4 μg kple peptide kple TMT reagent ƒe xexlẽme si nye 1:20. Le TMT ƒe dzesidede gome la, wogbugbɔ tsɔ TMT reagent 0.8 mg de ACN si me tsi mele o 70 μl me, eye wogbugbɔ ƒo peptide si ƒu la wòzu 9 μl 0.1 M TEAB (triethylammonium bicarbonate), si me wotsɔ TMT reagent 7 μl si le ACN me kpe ɖe eŋu. Nusiwo le emee nye 43.75%. Le aɖabaƒoƒo 60 ƒe vidzidzi megbe la, wotsɔ 2 μl 5% hydroxylamine tsi nuwɔna la nu. Woƒo peptide siwo woŋlɔ ŋkɔ na la nu ƒu, wotsɔ wo ƒu gbe, wogatsɔ wo de 0.1% formic acid (FA) 200μl me, woma wo ɖe eve me, eye emegbe woɖe dze le wo me to StageTips siwo wowɔ na wo ɖokui zazã me. Wozã UltiMate 3000 ultra high performance liquid chromatograph (UltiMate 3000 ultra high performance liquid chromatograph) tsɔ ma afã eveawo dometɔ ɖeka ɖe 1mm x 150mm Acquity chromatographic sɔti si yɔ fũ kple 130Å1.7μm C18 ƒe nusuewo (Waters, catalog No. SKU: 186006935) dzi. Thermo Fisher Dzɔdzɔmeŋutinunya). Ma peptidewo ɖe vovo le sisi ƒe agbɔsɔsɔme si nye 30μl/min me, mãe tso 1% va ɖo 50% buffer B hena miniti 85 kple afɔɖeɖe ɖesiaɖe ƒe tsɔtsɔme si nye miniti 96, tso 50% va ɖo 95% buffer B hena miniti 3, emegbe miniti 8 na 95 % Buffer B; Buffer A nye 5% ACN kple 10 mM ammonium bicarbonate (ABC), eye buffer B nye 80% ACN kple 10 mM ABC. Ƒo nu suesuesuewo nu ƒu le miniti 3 ɖesiaɖe me eye nàƒo wo nu ƒu ɖe hatsotso eve me (1 + 17, 2 + 18, kple bubuawo) eye nàƒu wo le vacuum centrifuge me.
LC-MS/MS ƒe numekuku. Le mass spectrometry gome la, woɖe peptideawo (xexlẽdzesi r119.aq) ɖe vovo le PicoFrit numekuku sɔti si ƒe didime nye sentimeta 25, si ƒe didime nye 75 μm (taɖodzinu ƒe lens yeye, akpa ƒe xexlẽdzesi PF7508250) si me wotsɔ 1.9 μm ReproSil-Pur 120 C18-AQ medium (Dr. Maisch, mat) ɖo, zã EASY-nLC 1200 (Thermo Fisher Dzɔdzɔmeŋutinunya, Germany). Wolé sɔti la ɖe te le dzoxɔxɔ 50°C me. Buffer A kple B nye 0.1% formic acid le tsi me kple 0.1% formic acid le 80% ACN me, le wo nɔewo yome. Woɖe peptidewo ɖa tso 6% va ɖo 31% buffer B hena miniti 65 eye tso 31% va ɖo 50% buffer B hena miniti 5 kple gradient si nye 200 nl/min. Woda peptide siwo woɖe ɖa la kpɔ le Orbitrap Fusion mass spectrometer (Thermo Fisher Scientific) dzi. Wowɔa peptide precursor m/z dzidzedze kple 120,000 ƒe vovototo le 350 va ɖo 1500 m/z dome. Le 27% normalized collision energy zazã me la, wotiaa ŋgɔdola sesẽtɔ kekeake si ƒe charge nɔnɔme tso 2 va ɖo 6 hena ŋusẽ gã C trap dissociation (HCD) cleavage. Woɖo ɣeyiɣi si woatsɔ awɔ tsatsam la ɖe s 1 dzi. Wodzidze peptide kakɛa ƒe m/z ƒe asixɔxɔ le ion mɔ̃a me to AGC ƒe taɖodzinu suetɔ kekeake si nye 5×104 kple ɣeyiɣi si woatsɔ adoe si sɔ gbɔ wu si nye 86 ms zazã me. Le mama megbe la, wotsɔ ŋgɔdonya la de dynamic exclusion list me hena s 45. Woɖe peptide siwo wotsɔ dzesi TMT de dzesii la ɖe vovo le Acclaim PepMap sɔti si ƒe didime nye sentimeta 50, si ƒe didime nye 75 μm (Thermo Fisher Scientific, agbalẽdzraɖoƒe ƒe xexlẽdzesi 164942), eye woda ʋuʋu ƒe ɣeyiɣi ƒe didime kpɔ le Orbitrap Lumos Tribrid mass spectrometer (Thermo Fisher Scientific) si me wotsɔ high-field asymmetric waveform ions (FAIMS) dɔwɔnuwo ɖo la dzi (Thermo Fisher Scientific) wɔa dɔ le fexexe ƒe ʋuʋudedi eve siwo nye −50 kple −70 V. Wozãa MS3 si wotia ɖe synchronization precursor dzi hena TMT nyatakaka ion dzesi dzidzedze. Wowɔ peptide ƒe mama le EASY-nLC 1200 dzi, wozã 90% linear gradient elution, kple buffer ƒe agbɔsɔsɔme si nye 6% va ɖo 31%; buffer A nye 0.1% FA, eye buffer B nye 0.1% FA kple 80% ACN. Wowɔa numekuku ƒe sɔti la le dzoxɔxɔ 50°C me. Zã FreeStyle (version 1.6, Thermo Fisher Scientific) nàtsɔ ama faɛl gbãtɔa ɖe FAIMS ƒe fexexe ƒe elektrikŋusẽ nu.
Protein ƒe dzesidede kple eƒe agbɔsɔsɔme. Wozã Andromeda ƒe nyatakakadimɔ̃ si wotsɔ wɔ ɖekae tsɔ ku nyatakaka gbãtɔawo me to MaxQuant ƒe tɔtrɔ 1.5.2.8 (https://maxquant.org/) zazã me. Tsɔ kpe ɖe Cre recombinase kple YFP ƒe ɖoɖo siwo wokpɔ tso Aequorea victoria gbɔ ŋu la, wodi peptide kakɛ ƒe spektrumwo hena canonical sequence kple isoform sequence si le mouse reference proteome (Proteome ID UP000000589, si woɖe tso UniProt le May 2017 me) . Woɖo methionine oxidation kple protein N-terminal acetylation be woanye tɔtrɔ siwo trɔna; woɖo cysteine ​​carbamoyl methylation be wòanye tɔtrɔ siwo woɖo ɖi. Woɖo nuɖuɖumeŋusẽ ƒe nɔnɔmewo ɖe “nu tɔxɛ” kple “trypsin/P” dzi. Peptide kple razor peptide siwo wozãna tsɔ dea dzesi protein ƒe xexlẽme suetɔ kekeakee nye 1; peptide tɔxɛwo ƒe xexlẽme suetɔ kekeake nye 0. Le peptide ƒe anyigbatata ƒe sɔsɔ ƒe nɔnɔmewo nu la, protein ƒe dzesidede ƒe agbɔsɔsɔme nye 0.01. Wowɔ “Second Peptide” ƒe tiatia la ŋudɔ. Zã “match between runs” tiatia be nàtsɔ dzesideŋkɔ siwo kpɔ dzidzedze la ayi faɛl gbãtɔ vovovowo dome. Zã LFQ ƒe xexlẽme suetɔ kekeake ƒe xexlẽme 1 hena agbɔsɔsɔme si womede dzesii o (LFQ) (60). Woɖea LFQ ƒe sesẽme hena asixɔxɔ eve ya teti siwo sɔ le domenyiŋusẽfianu ƒe hatsotso ɖeka ya teti me le ɣeyiɣi ɖesiaɖe me, eye woɖenɛ tsoa mama si sɔ me si ƒe kekeme nye. 0.3 eye nàɖiɖi 1.8. Zã Perseus kɔmpiutadziɖoɖo (https://maxquant.net/perseus/) kple R (https://r-project.org/) nàtsɔ adzro LFQ ƒe emetsonuwo me. Wozã mɔ eve ƒe t dodokpɔ si sɔ gbɔ tso limma kɔmpiuta dɔwɔɖoɖoa me hena vovototodedeameme ƒe nyagbɔgblɔ me dzodzro ( 61 ). Wowɔa nyatakakawo me dzodzro le numekuku me to ggplot, FactoMineR, factoextra, GGally kple pheatmap zazã me. Wozã MaxQuant ƒe tɔtrɔ 1.6.10.43 tsɔ ku proteomics nyatakaka siwo wotu ɖe TMT dzi la me. Di proteomics nyatakaka xoxowo tso UniProt ƒe amegbetɔ ƒe proteomics nyatakakadzraɖoƒe, si woɖe le September 2018. Kukuɖenuŋua lɔ isotope dzadzɛnyenye ɖɔɖɔɖo factor si adzɔha la na ɖe eme. Zã limma le R me hena vovototodedeameme ƒe nyagbɔgblɔ me dzodzro. Wodzraa nyatakaka gbãtɔwo, nyatakakadzraɖoƒe didi me tsonu, kple nyatakakawo me dzodzro ƒe dɔwɔwɔ kple emetsonuwo katã ɖo ɖe ProteomeXchange ƒe nubabla me to PRIDE hadɔwɔla ƒe nudzraɖoƒe dzi kple nyatakakawo ƒe ƒuƒoƒo ƒe dzesi PXD019690.
Dɔwɔwɔ ŋuti nuŋlɔɖiwo naa numekukua nyona ɖe edzi. Wozã Ingenuity Pathway Analysis (QIAGEN) dɔwɔnu tsɔ kpɔ nyatakaka siwo woɖo ɖe kwasiɖa 8 me ƒe dɔwɔwɔ ŋuti nuŋlɔɖinyawo ƒe kesinɔnuwo (Nɔnɔmetata 1). Kpuie ko la, wozãa protein ƒe agbɔsɔsɔme ƒe xexlẽdzesi si wokpɔ tso LC-MS/MS (tandem mass spectrometry) nyatakakawo me dzodzro me kple filter dzidzenu siwo gbɔna: Wotia mus musculus abe ƒomevi kple megbenya ene, eye hatsotso la ɖe P ƒe asixɔxɔ si Benjamini trɔ asi le hena kesinɔnuwo 0.05 alo esi mede nenema o la fia be wobu be eɖe dzesi. Le nɔnɔmetata sia gome la, woɖe hatsotso atɔ̃ siwo sɔ gbɔ wu siwo le ƒuƒoƒo ɖesiaɖe me siwo wotu ɖe P ƒe asixɔxɔ si woɖɔli dzi la fia. To multiple t-test zazã me, to Benjamini, Krieger, kple Yekutieli ƒe afɔɖeɖe eve ƒe linear boost program (Q = 5%) zazã me, wowɔa protein ƒe ɖeɖefia ŋuti numekuku le ɣeyiɣi ƒe didime nu le ame vevi siwo wokpɔ le hatsotso ɖesiaɖe me ŋu, eye wodaa fli ɖesiaɖe kpɔna ɖe vovo. Mehiã be woaxɔ SD si mewɔa tɔtrɔ o.
Be míatsɔ numekuku sia me tsonu asɔ kple nyatakakadzraɖoƒe siwo wota eye míawɔ Venn ƒe nɔnɔmetata le Nɔnɔmetata 1 me la, míetsɔ protein ƒe agbɔsɔsɔme ƒe xexlẽdzesiwo ƒo ƒu kple MitoCarta 2.0 ƒe nuŋlɔɖiwo ( 24 ). Zã dɔwɔnu si le Internet dzi si nye Draw Venn Diagram (http://bioinformatics.psb.ugent.be/webtools/Venn/) nàtsɔ awɔ nɔnɔmetata la.
Ne èdi nyatakaka tsitotsito tso akɔntabubu ƒe mɔnu siwo wozãna hena proteomics numekuku ŋu la, taflatse kpɔ Nuwo Kple Mɔnuwo ƒe akpa si sɔ kplii. Le dodokpɔ bubuawo katã gome la, woate ŋu akpɔ nyatakaka tsitotsito le xotutu si sɔ kplii me. Negbe ɖe wogblɔe bubui ko la, woɖea nyatakakawo katã fiana abe mama dedie ± SEM ene, eye wozã GraphPad Prism 8.1.2 kɔmpiuta dɔwɔɖoɖo tsɔ wɔ akɔntabubuwo me dzodzrowo katã.
Ne èdi nyatakaka kpeɖeŋutɔ na nyati sia la, taflatse kpɔ http://advances.sciencemag.org/cgi/content/full/6/35/eaba8271/DC1
Esia nye nyati si woateŋu akpɔ le mɔ gbadza nu si woma le Creative Commons Attribution-Non-Commercial License ƒe sewo nu, si ɖe mɔ ɖe eŋudɔwɔwɔ, mama kple egbugbɔgawɔ le mɔnu ɖesiaɖe me, nenye be zazã mamlɛtɔ menye asitsatsa ƒe viɖe ta o eye gɔmeɖoanyia nye be dɔ gbãtɔa sɔ. Ɖeɖe fiã.
De dzesii: Ðeko míebia tso asiwò be nàtsɔ wò e-mail adrɛs ana ale be amesi nèkafu na axaa nanya be yedi be yeakpɔ e-mail la eye be menye nyatakaka gbegblẽe wònye o. Mímalé e-mail adrɛs aɖeke ɖe mɔ̃ dzi o.
Wozãa biabia sia tsɔ doa amedzro kpɔ nenye be amedzroe nènye eye woxea mɔ na nyatakaka gbegblẽwo ɖoɖo ɖe amewo le wo ɖokui si.
E. Motori, I. Atanassov, S. M. V. Kochan, K. Folz-Donahue, V. Sakthivelu, P. Giavalisco, N. Toni, J. Puyal, N.-G., ye ŋlɔe. Larson ye nye esia
Proteomics numekuku le ahɔhɔ̃meka siwo mewɔa dɔ nyuie o ŋu ɖee fia be wowɔa lãmenugbagbeviwo ƒe dɔwɔwɔ ƒe ɖoɖowo be woatsɔ atsi ahɔhɔ̃mekawo ƒe dɔmawɔmawɔ nyuie nu.
E. Motori, I. Atanassov, S. M. V. Kochan, K. Folz-Donahue, V. Sakthivelu, P. Giavalisco, N. Toni, J. Puyal, N.-G., ye ŋlɔe. Larson ye nye esia
Proteomics numekuku le ahɔhɔ̃meka siwo mewɔa dɔ nyuie o ŋu ɖee fia be wowɔa lãmenugbagbeviwo ƒe dɔwɔwɔ ƒe ɖoɖowo be woatsɔ atsi ahɔhɔ̃mekawo ƒe dɔmawɔmawɔ nyuie nu.
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