Obwire obu *Endagiriro y‘obwire obu: Cologne 50931, Germany, Cologne Ekibiina ky‘okucondooza aha kugarukamu kw‘okushoberwa kw‘obutafaari omu ndwara ezirikureetwaho okukura (CECAD).
Okusheeshasheesha kw‘obutafaari bw‘omubiri (neurodegeneration) kw‘endwara z‘omubiri nikutwarwaho nk‘okutarikubaasa kugaruka enyima ahabw‘okuba obutafaari bw‘omubiri oburikuhinduka (metabolic plasticity) nibukye, kwonka ebirikuruga omu kusheeshasheesha kw‘obutafaari bw‘omubiri aha butafaari bw‘omubiri (cell autonomy) bw‘okuhindura eby‘okurya omu mubiri tibirikwetegyerezibwa gye. Aha, nitutandikaho puroteyini y‘obutafaari bw‘omubiri obw‘obutafaari bwa Purkinje oburimu okubura kwa OXPHOS okurikutunguuka okurikureetwaho okutabura enkora y‘okuteerana kw‘obutafaari bw‘omubiri. Twashanga ngu okuremwa kukora gye kwa mitochondrial kukareetaho empindahinduka mpango omu by’obuhangwa bw’omubiri, aha muheru kyareetera puroguraamu z’okuhindura eby’okurya ezihikire omu nkora y’okukuratana obutafaari butakafiire. Omu muringo ogutarikuteekateekwaho, tukamanya okutandika kw‘okureebeka kwa pyruvate carboxylase (PCx) n‘ebindi bihingwa ebiine akakwate n‘okugaita ebiri ahagati y‘enzindo ya TCA. Okuzibira PCx kukayongyera aha kushoberwa kw‘okwosya n‘okusheeshasheesha kw‘obutafaari bw‘omubiri, ekirikworeka ngu okusheeshasheesha kw‘emitsi y‘omubiri kwine omugasho gw‘okwerinda omu butafaari bw‘omubiri obutaine OXPHOS. Okugarukaho kw’okuteerana kw’ebicweka by’omubiri ebirikwetwa mitochondrial fusion omu bicweka by’omubiri ebihweireho nibihindura kimwe emitwarize egi ey’okuhindura eby’okurya, obwo nibuzibira obutafaari kufa. Ebi twashanga nibyoreka emihanda eyaabaire etamanyirwe ira erikuhereza obugumisiriza aha kutakora gye kw‘emitsi y‘omubiri kandi neeyoreka ngu okusheeshasheesha kw‘obutafaari bw‘omubiri nikubaasa kugaruka enyima n‘omu mitwarize y‘aha muheru y‘endwara.
Omurimo mukuru gwa mitochondria omu kuguma n‘okuhindura kw‘amaani g‘ebihaha niguhamibwa obumanyiso bw‘ebihaha obuhango oburikukwatagana n‘endwara z‘ebihaha z‘abantu. Endwara ezi nyingi nizireetwa okuhinduka kw‘ebicweka by‘omubiri ebirikutegyeka okureebeka kw‘ebicweka by‘omubiri (1, 2) nari okucwekyerera kw‘ebicweka by‘omubiri ebiine akakwate n‘entunguuka y‘ebicweka by‘omubiri, ebirikuteganisa omu muringo ogutarikuhikaana n‘okuhama kwa DNA y‘omubiri (mtDNA) (3, 4). Okukora omu bishushani by‘enyamaishwa kworekire ngu omu kugarukamu okuremwa kukora kwa mitochondria omu bicweka by‘omubiri ebyetooroire, emihanda y‘okuhindura eby‘okurya (5-7) neebaasa kutandika kukora, n‘ahabw‘ekyo neeha okumanya okukuru aha nkora y‘endwara ezi ezigumire. Omu kugyeragyeranisa n‘ekyo, okwetegyereza kwaitu aha mpindahinduka z‘okuhindura eby‘okurya omu miringo y‘obutafaari emwe ezirikureetwaho okuremwa kw‘okukora kw‘obwongo okwa mitochondrial adenosine triphosphate (ATP) omu bwijwire n‘eky‘omugasho munonga (8), kuhamya ekyetengo ky‘okumanya ebigyendererwa by‘obujanjabi ebirikubaasa kukoresibwa kwetantara nari kutangira endwara. Zibira okusheeshasheesha kw’obutafaari bw’omubiri (9). Okubura amakuru n‘okugira ngu obutafaari bw‘omubiri nibuteekateekwaho munonga kuba bwine obushoboorozi bukye munonga bw‘okuhindura eby‘okurya waagyeragyeranisa n‘ebika by‘obutafaari bw‘ebicweka by‘omubiri ebyetooroire (10). Okumanya ngu obutafaari obu nibukora omurimo muhango omu kukwatisa okuha ebiriisa omu nkora y‘obutafaari bw‘omubiri kutunguura okujanjaaza kwa synaptic n‘okugarukamu embeera z‘obuhuta n‘endwara, obushoboorozi bw‘okuhindura obutafaari bw‘omubiri kuhikaana n‘embeera ezigumire z‘ebicweka by‘obwongo nibuhika haihi n‘obutafaari bwa glial (11-14). Okwongyera ahari ekyo, obutashushana bw‘obutafaari bw‘omubiri oburi omu bwongo niburemesa munonga okwega aha mpindahinduka z‘omubiri ezirikubaho omu guruupu z‘obutafaari bw‘omubiri ezitari zimwe na zimwe. Ekirugiremu, tihariho bingi ebirikumanywa aha birugire omu butafaari n‘okuhindura eby‘okurya ebihikire ahabw‘okuremwa kukora gye kw‘obutafaari bw‘omubiri omu nkora y‘obutafaari bw‘omubiri.
Okubaasa kushoboorora ebirikuruga omu kuhindura eby‘okurya omu nkora y‘okuremwa kukora gye kw‘ebicweka by‘omubiri, tukataanisaho obutafaari bwa Purkinje (PNs) omu mitwarize etari emwe n‘emwe y‘okusheeshasheesha kw‘obutafaari oburikureetwaho okucwekyerera kw‘okuteerana kw‘ebicweka by‘omubiri eby‘aheeru (Mfn2). N‘obu okuhinduka kwa Mfn2 omu bantu kuraabe nikukwatagana n‘omuringo gw‘oburwaire bw‘okuhurira oburikumanyirwa nka Charcot-Marie-Tooth type 2A (15), okucwekyerera kw‘embeera kwa Mfn2 omu mbeba n‘okureetaho okuremwa kukora kw‘okwotsya (OXthoPHOS) okumanyirwe gye. Ebika by‘obutafaari bw‘omubiri ebitari bimwe na bimwe (16-19) n‘ekirikurugamu ekirikureetaho okusheeshasheesha kw‘obutafaari bw‘omubiri nibihererukayo n‘obumanyiso bw‘obutafaari bw‘omubiri oburikutunguuka, nk‘oburemeezi bw‘okutambura (18, 19) nari cerebellar ataxia (16). Omu kukoresa omugaati gw‘eby‘okurya ebitariho akamanyiso (LFQ) eby‘okureeberaho by‘eby‘okurya, eby‘okurya, eby‘okureeberaho, n‘eby‘okureeberaho by‘obukooko, nitworeka ngu okutunguuka kw‘obutafaari bw‘omubiri nikureetaho munonga pyruvate carboxylase (PCx) n‘ebindi ebirikwejumba omu kusheeshasheesha kw‘emitsi ya PNs omu nkora y‘amagara Okworeka kw‘enzigye. Okuhamya obukuru bw‘ekirugire omu kucondooza oku, tukakyendeeza aha kushoboorora kwa PCx omu PNs ezitarimu Mfn2, kandi twashanga ngu okukora oku kukayongyera aha kushoberwa kw‘okwosya n‘okutunguura okusheeshasheesha kw‘obutafaari bw‘omubiri, n‘ahabw‘ekyo nikihamya ngu azoospermia neeha okufa kw‘obutafaari Metabolic adaptability. Okworeka munonga kwa MFN2 nikubaasa kucungura kimwe PN y‘okusheeshasheesha kw‘aha muheru n‘okubura OXPHOS kw‘amaani, okukoresa munonga DNA y‘omubiri, n‘okureebeka ngu omukura gwa mitochondrial guhendagurikire, ekirikwongyera kuhamya ngu omuringo ogu gw‘okusheeshasheesha kw‘obutafaari bw‘omubiri nigubaasa n‘okugaruka omu buteeka omu bwire bw‘okusheeshasheesha kw‘obutafaari bw‘omubiri oburwaire butakahikire.
Okubaasa kureeba omu maisho emitochondria omu Mfn2 knockout PNs, tukakoresa oruganda rw’embeba orurikubaasisa mitochondria erikwegamira ahari Cre kugyendera aha puroteyini erikwetwa yellow fluorescent puroteyini (YFP) (mtYFP) (20) Cre expression kandi twakyebera enkora y’emitochondria omu nkora y’amagara. Twashanga ngu okucwekyerera kw‘omubazi gwa Mfn2 omu PNs kukaba nikwija kureetera okugabanisamu mporampora kw‘omutimbagano gw‘emibiri (ekishushani S1A), kandi empindahinduka ey‘okubanza ekashangwa aha myaka 3 y‘obukuru. Okutaana n‘ekyo, okukyendeera omu nkora y‘obutafaari bwa PN, nk‘oku kirikworekwa omu kufeerwa kw‘obutafaari bwa Calbindin immunostaining, tikwatandikire kuhitsya omu wiiki 12 z‘obukuru (ekishushani 1, A na B). Obwire obutarikushushana ahagati y‘empindahinduka z‘okubanza omu nkora y‘obutafaari bw‘omubiri n‘okutandika okurikureebwa kw‘okufa kw‘obutafaari bw‘omubiri kukatureetera kucondooza aha mpindahinduka z‘omubiri ezirikureetwaho okuremwa kukora gye kw‘obutafaari bw‘omubiri obutafaari butakafiire. Twakora enkora erikwegamira aha kushoroora obutafaari oburikukoresibwa omushana (FACS) kubaasa kutaanisa YFP (YFP+)-erikworeka PN (ekishushani 1C), n‘omu mbeba ezirikutegyekwa (Mfn2 + / loxP :: mtYFP loxP- stop-loxP: : L7-cre), ahaifo nizimanyirwa nka CT1BRL (ekishushani). Okutunguura enkora y‘okukinga omuryango kurugiirira aha amaani g‘okugyeragyeranisa g‘akamanyiso ka YFP nikutubaasisa kushemeza omubiri gwa YFP+ (YFPhigh) gwa PNs kuruga omu bitari bya PNs (YFPneg) (ekishushani S1B) nari ebicweka by‘omubiri ebirikuteekateekwaho kuba eby‘omubiri (YFPlow; ekishushani S1D, confopecal by‘aha rubaju rwa bumosho). Okubaasa kuhamya okumanyisa kw‘abantu abashorooziibwe, tukakora LFQ proteomics bwanyima twakora okucondooza aha bicweka ebikuru, kandi twashanga ngu hariho okwahukana okurikworeka gye ahagati y‘obutafaari bwa YFPhigh na YFPneg (Ekishushani S1C). Obutafaari bwa YFPhigh bukayoreka okugaita kw‘obutimba bw‘obumanyiso bwa PNs obumanyirwe (nikwo kugira ngu Calb1, Pcp2, Grid2 na Itpr3) (21, 22), kwonka tihaine kugaita kwa puroteyini ezirikukira kworekwa omu nkora y‘obutafaari bw‘omubiri nari ebindi bika by‘obutafaari (Ekishushani 1D) ). Okugyeragyeranisa ahagati y‘eby‘okureeberaho omu butafaari bwa YFPhigh obutebeekanisiibwe obushorooziibwe omu kucondooza okwetongoire kukooreka omubaro gw‘enkoragana> 0.9, kworeka okuzaara kurungi ahagati y‘ebihangirwe ebiine amagara (ekishushani S1E). Omu bugufu, ebihandiiko ebi bikahamya enteekateeka yaitu ey‘okwetaanisa aha PN erikubaasika. Ahabw‘okugira ngu enkora y‘okuvuga L7-cre erikukoresibwa neereetaho okugaruka kw‘okuteerana kwa mosaic omu wiiki y‘okubanza bwanyima y‘okuzaara (23), tukatandika kwita embeba kuruga omu CTRL n‘embeera (Mfn2 loxP / loxP :: mtYFP loxP-stop-loxP :: L7-cre) Kuteerana obutafaari bw‘omubiri. Bwanyima y‘okugaruka kujwanzya, nikyetwa Mfn2cKO aha myaka 4 y‘obukuru. Nk‘enshonga y‘okuhendera, tukatoorana wiiki 8 z‘emyaka obu PN layer yaabaire ekiriho n‘obu ebicweka bya mitochondrial biraabe byabaire nibireebeka gye (ekishushani 1B n‘ekishushani S1A). Omu bwijwire, tukabara omubaro gw‘ebiriisa 3013, ahari ebyo nka 22% bikaba biri aha bihandiiko bya MitoCarta 2.0 ebirikwegamira aha puroteyini y‘omubiri nk‘ebiriisa by‘omubiri (ekishushani 1E) (ekishushani 1E) (24). Okushwijuma okurikworeka ebiri omu nkora y‘obuzaarwa (differential gene expression analysis) okwakozirwe omu wiiki 8 kukareebeka ngu 10.5% zonka ez‘ebirungo byona zikaba ziine empindahinduka z‘amaani (ekishushani 1F n‘ekishushani S1F), ahari ebyo puroteyini 195 zikaba ziri ahansi kandi 120 zikaba ziri ahaiguru (ekishushani 1F). Nikirungi kumanya ngu “okushwijuma emihanda y‘okukora ebintu bisya” eby‘ekicweka ky‘amakuru eki nikyoreka ngu ebiri omu nkora y‘obuzaarwa ebirikworekwa omu miringo etarikushushana okukira munonga nibiruga omu nkora y‘okuzibira ey‘emihanda y‘okuhindura eby‘okurya (ekishushani 1G). Ekirikushemeza, n‘obu okukyendeeza aha nkora y‘emihanda erikukwatagana na OXPHOS n‘okumanyisa kwa calcium kuraabe nikuhamya okutandika kw‘okuremwa kukora kwa mitochondrial omu PNs ezitaine fusion, ebindi biika ebirikukirayo kwejumba omu kuhindura kw‘ebiriisa bya amino acid nibihindurwa munonga, ekirikuhikaana n‘okuhindura kw‘ebiriisa omu mitochN P okurikubaho. Okugaruka kutaho waya nikugumaho. obutakora gye.
(A) Ebishushani by‘okujwekyera eby‘ebicweka by‘omubiri eby‘omubiri eby‘embeba za CTRL na Mfn2cKO ebirikworeka okufeerwa kw‘okutunguuka kw‘ebicweka bya PNs (calbindin, gray); nuclei zikaba ziine erangi ya DAPI. (B) Okupima (A) (okushwijuma kw‘omuringo gumwe kw‘okuhinduka, ***P<0.001; n = 4 kuhika 6 enzindo kuruga omu mbeba ishatu). (C) Okugyezesa okukora emirimo. (D) Okugabwa kwa maapu y‘okwotsya kw‘obumanyiso oburikworeka Purkinje (ahaiguru) n‘ebindi bika by‘obutafaari (ahagati). (E) Ekishushani kya Venn ekirikworeka omubaro gw‘ebiriisa by‘omubiri ebimanyirwe omu PN erikushoroorwa. (F) Volcano plot y‘ebirungo ebirikworekwa omu miringo etarikushushana omu nkora ya Mfn2cKO aha wiiki 8 (omuhendo gw‘okusharaho ogw‘omugasho gwa 1.3). (G) Okushwijuma omuhanda gw‘okuhangahanga nikworeka emihanda etaano erikukirayo obukuru ey‘okutunguuka (etukutuku) n‘okugaruka ahansi (bururu) omu Mfn2cKO PN erikuteebwa omu kicweka kya wiiki 8. Omutindo gw‘okumanyisa ogwa buri puroteyini erikushangwa nigworeka. Maapu y‘okwotsya ey‘enzindo: omuhendo gwa P oguhindukire. ns, ti kikuru.
Ebihandiiko bya puroteyini nibyoreka ngu okureebeka kwa puroteyini z’ebicweka I, III, na IV kukaba nikukyendeera mporampora. Ebizibu I, III, na IV byona bikaba birimu ebicweka ebikuru ebiine koodi ya mtDNA, obwo ekizibu II, ekyabaire kiine koodi ya nukiriya yonka, omu kubanza kikaba kitaine kabi koona (ekishushani 2A n‘ekishushani S2A). . Okurugiirira aha byarugamu omu kucondooza kwa puroteyini, okucondooza aha nkora y‘okurwanisa endwara z‘omubiri (immunohistochemistry) kw‘ebicweka by‘enyama z‘omubiri z‘omu bwongo kukooreka ngu MTCO1 (mitochondrial cytochrome C oxidase subunit 1) omutindo gw‘ekicweka ky‘omubiri ekigumire IV omu PN gukeyongyera mporampora (ekishushani 2B). Ekicweka kya mtDNA-enkoded Mtatp8 kikakyendeera munonga (ekishushani S2A), obwo omutindo gw‘okugumaho ogw‘ekicweka kya ATP synthase ekiine enkokora ya nukiriya gukaguma gutahindukire, ekirikuhikaana n‘ekicweka ky‘omubiri ekimanyirwe ekigumire ekya ATP synthase subassembly F1 complex obu mtDNA expression erikugumaho. Okuhangwa nikugumaho. Okutabura (7). Okushwijuma omutindo gwa mtDNA omu PNs za Mfn2cKO ezitoorainwe kurabira omu nkora ya polymerase chain reaction (qPCR) omu bwire obwabuzima (qPCR) kukahamya okukyendeera mporampora kw’omubaro gw’okukopa kwa mtDNA. Kugyeragyeranisa n‘ekibiina ky‘okukyebera, aha myaka 8 y‘obukuru, ebicweka nka 20% byonka eby‘omutindo gwa mtDNA bikagumaho (ekishushani 2C). Okurugiirira aha byarugamu ebi, okucondooza kwa confocal microscopy okwa Mfn2cKO PNs kubaasa kumanya DNA nikworeka okukoresa kw‘obwire okurikurugiirira aha nkora y‘okukoresa ebiriisa by‘omubiri (ekishushani 2D). Twashanga ngu abamwe bonka abarikwenda kwejumba omu kusheenya puroteyini y‘omubiri n‘okugarukamu kw‘okushoberwa nibo baabaire nibateebwaho ebiragiro, otwariiremu Lonp1, Afg3l2 na Clpx, n‘enshonga z‘okuteerana ezigumire eza OXPHOS. Tihariho mpindahinduka z‘amaani omu mitwarize ya puroteyini ezirikwejumba omu kusheeshasheesha ezaashangirwe (ekishushani S2B). Omu muringo nigwo gumwe, tukashanga ngu emihanda ya mitochondria na endoplasmic reticulum erikwejumba omu kutwara ekiriisa kya calcium eine empindahinduka nkye (ekishushani S2C). Okwongyera ahari ekyo, okushwijuma puroteyini eziine akakwate n‘okurya eby‘okurya by‘omubiri tikwashangireho mpindahinduka z‘amaani, ekirikuhikaana n‘okureebeka kw‘okutandika kw‘okurya eby‘okurya by‘omubiri (autophagosomes) okureebirwe omu nkora y‘omubiri (vivo) kurabira omu immunohistochemistry na electron microscopy (ekishushani S3). Kwonka, okuremwa kukora kwa OXPHOS okurikugyenda omu maisho omu PNs nikugyenda n‘empindahinduka z‘omubiri ezirikureebwa butunu. Ebirundo bya mitochondrial nibibaasa kureebwa omu mibiri y‘obutafaari n‘emiti ya dendritic ya Mfn2cKO PNs ey‘emyaka 5 na 8, kandi omubumbi w‘omubiri gw‘omunda guhindukire munonga (ekishushani S4, A na B). Okuhikaana n‘empindahinduka ezi z‘omubiri n‘okukyendeera kw‘amaani omu mtDNA, okweshwijuma kw‘ebicweka by‘omubiri eby‘omubiri ebiine tetramethylrhodamine methyl ester (TMRM) kukooreka ngu obushoboorozi bw‘omubiri gw‘omubiri omu Mfn2cKO PNs bukakyendeera munonga (ekishushani S4C).
(A) Okushwijuma obwire bw‘okushwijuma omutindo gw‘okworeka OXPHOS complex. Teekateeka aha puroteyini zonka eziine P<0.05 aha wiiki 8 (ANOVA y‘emiringo ebiri). Omuringo gw’obutonyezi: Tihariho kuhindura waagyeragyeranisa na CTRL. (B) Aha rubaju rwa bumosho: Eky‘okureeberaho ky‘ekicweka ky‘obwongo ekihandiikirweho obutafaari oburikurwanisa MTCO1 (omubaro gw‘okupima, 20 μm). Omwanya ogurimu emibiri y’obutafaari bwa Purkinje gushwekirwe ekirungo kya kyenju. Aha rubaju rwa buryo: Okupima emiringo ya MTCO1 (okushwijuma kw‘omuringo gumwe kw‘okuhinduka; n = obutafaari 7 kuhika 20 obushwijumirwe kuruga omu mbeba ishatu). (C) qPCR okushwijuma omubaro gwa kopi ya mtDNA omu PN etoorainwe (okushwijuma kw‘omuringo gumwe okw‘okuhinduka; n = 3 kuhika ahari 7 empunu). (D) Aha rubaju rwa bumosho: Eky‘okureeberaho ky‘ekicweka ky‘obwongo ekihandiikirweho ekirikurwanisa DNA (omubaro gw‘okupima, 20 μm). Omwanya ogurimu emibiri y’obutafaari bwa Purkinje gushwekirwe ekirungo kya kyenju. Aha rubaju rwa buryo: Okupima obuhango bw‘ebizimba bya mtDNA (okushwijuma kw‘omuringo gumwe kw‘okuhinduka; n = obutafaari 5 kuhika 9 kuruga omu mbeba ishatu). (E) Eky‘okureeberaho ky‘ekicweka ky‘obwongo ekirikworeka obutafaari bwa mitoYFP + Purkinje (omusyo) omu kuhandiika kw‘obutafaari bwona. (F) Okupima omubaro gw‘ekicweka kya IV. (G) Ebihandiiko ebirikujwekyera eby‘okutaho omubazi gw‘okukyendeeza aha nkora y‘okuteerana omu butafaari bwa CTRL na Mfn2cKO Purkinje. Top trace: Pulse y‘okubanza eyaareeteire AP. Okukuratira ahansi: Emirundi ya AP erikukirayo. (H) Okupima omubaro gw‘ebintu ebirikutaaha omu nkora bwanyima y‘okuhurira (sPSPs). Omuringo gw‘okuhandiika ogurikujwekyera n‘omubaro gwagwo gw‘okutunguura nigworeka omu (I). Okushwijuma kw‘omuringo gumwe okw‘okuhinduka kukashwijuma obutafaari n = 5 kuhika 20 kuruga omu mbeba ishatu. Ebihandiiko nibyorekwa nk‘ebiri ahagati±SEM; *P<0.05; **Ekicweka<0.01; ***P<0.001. (J) Ebishushani ebirikujwekyera AP y‘okwetunguura ehandiikirwe barikukoresa omuringo gw‘okukwatiraho ekipande ekirimu ebitobo. Okukuratira ahaiguru: Emirundi ya AP erikukirayo. Okukuratira ahansi: zoom ya AP emwe. (K) Baza omubaro gw‘ebicweka bya AP eby‘ahagati n‘eby‘ahaiguru kurugiirira ahari (J). Ekigyezo kya Mann-Whitney; n = obutafaari 5 bukakyeberwa kuruga omu mbeba ina. Ebihandiiko nibyorekwa nk‘ebiri ahagati±SEM; ti kikuru.
Okushiisha kwa OXPHOS okurikworekwa kukashangwa omu Mfn2cKO PN ey‘emyaka 8, ekirikworeka ngu emirimo y‘omubiri y‘obutafaari bw‘omubiri n‘ey‘omutaano munonga. Nahabwekyo, tukashwijuma emitwarize y‘amashanyarazi etarikukora (passive electrical neurons) ey‘obutafaari obutaine OXPHOS aha wiiki 4 kuhika ahari 5 na wiiki 7 kuhika ahari 8 turikukora okuhandiika kw‘obutafaari bwona obw‘okuteerana omu bicweka by‘omubiri ebiine obuhuta bw‘omubiri (ekishushani 2E). Tikirikuteekateekwaho, obushoboorozi bw‘okuhuumura obw‘omubaro gw‘ahaiguru n‘okuzibira okutaaha kw‘obutafaari bwa Mfn2cKO bikaba nibishushana n‘okutegyeka, n‘obu haraabe haabaireho entaaniso nkye ahagati y‘obutafaari (Ekipande 1). Omu muringo nigwo gumwe, aha myaka 4 kuhika ahari 5, tihaine mpindahinduka z‘amaani omu nkoragana y‘obwire obu n‘amaani (IV curve) ezaashangirwe (ekishushani 2F). Kwonka, tihaine nshonga yoona eya Mfn2cKO ey‘emyaka 7 kuhika 8 eyahonokireho omu nkora ya IV (eitembezo ry‘okutambura munonga), ekirikworeka ngu hariho okuhurira okurikworeka gye obushoboorozi bw‘okutambura munonga aha rurengo oru orurikuhererukayo. Okutaana n‘ekyo, omu nkora ya Mfn2cKO, amashanyarazi g‘okukyendeeza aha nkora (depolarizing currents) agarikureetaho okushohoza kw‘okugarukamu kw‘obushoboorozi bw‘okukora (AP) nigugumisiriza gye, ekirikworeka ngu enkora zaabo z‘okushohoza zoona tizirikutaana munonga n‘ez‘obutafaari bw‘okutegyeka obw‘emyaka 8 (Ekipande 1 n‘ekishushani 2G). Omu muringo nigwo gumwe, emirundi n‘obuhango bw‘ebintu ebirikubaho bwanyima y‘okuhurira (sPSCs) bikagyeragyeranisibwa n‘eby‘ekibiina ky‘okukyebera, kandi emirundi y‘ebintu ekaba neeyeyongyera kuruga aha wiiki 4 kuhika ahari wiiki 5 kuhika ahari wiiki 7 kuhika ahari wiiki 8 n‘okwongyezibwaho kumwe (ekishushani 2, H na I). Obwire bw‘okukura kw‘ebicweka by‘omubiri omu PNs (25). Ebirugiremu nk‘ebi bikashangwa bwanyima y‘okutaho ebipande bya PNs ebiine ebitobo. Entebekanisa egi neezibira okurihiirira okurikubaasa kubaho ahabw‘oburemeezi bwa ATP omu butafaari, nk‘oku kirikubaasa kubaho omu kuhandiika kw‘obutafaari bwona. Okukira munonga, obushoboorozi bw‘okuhuumura kw‘omubiri n‘okuteera kw‘okwetunguura kw‘obutafaari bwa Mfn2cKO tikukwatsirweho (ekishushani 2, J na K). Omu bugufu, ebyarugamu ebi nibyoreka ngu PNs eziine oburemeezi bw‘okukora kwa OXPHOS oburikworekwa nizibaasa kugumira gye enkora z‘okushohoza ebiri omu nkora y‘okushohoza ebiriisa by‘emirundi mingi, ekirikworeka ngu hariho enkora y‘okurihiirira erikubabaasisa kuguma baine okugarukamu kw‘amashanyarazi okuri haihi n‘okwa buriijo.
Ebihandiiko nibyorekwa nk‘ebiri ahagati ± SEM (okushwijuma kw‘omuringo gumwe kw‘okuhinduka, ekigyezo ky‘okugyeragyeranisa emirundi mingi ekya Holm-Sidak; *P<0.05). Enamba y’ekicweka neeyorekwa omu bubonero.
Tukataho okucondooza yaaba ekicweka kyona omu nkora ya proteomics dataset (ekishushani 1G) kirimu emihanda erikubaasa kurwanisa okubura OXPHOS kw‘amaani, obwo turikushoboorora ahabwenki PN erikukwatwaho neebaasa kuguma n‘eby‘amashanyarazi ebiri haihi n‘eby‘obutoosha (ekishushani 2, E kuhika K). . Okushwijuma kwa puroteyini kukooreka ngu enzymes ezirikwejumba omu kuhindura ebiriisa by‘omubiri (BCAA) zikaba zitebeekanisiibwe munonga (ekishushani 3A n‘ekishushani S5A), kandi ekirugiremu eky‘aha muheru acetyl-CoA (CoA) nari succinyl CoA nikibaasa kwongyera aha tricarboxylates omu kusheeshasheesha kw‘emitsi. Twashanga ngu ebiri omu BCAA 1 (BCAT1) na BCAT2 byombi nibyeyongyera. Bakareetaho eitembezo ry’okubanza ry’okuhindura BCAA barikukora ebiriisa kuruga omu α-ketoglutarate (26). Ebicweka byona ebirikukora ekicweka ky‘omubiri ekiine amataagi ga keto acid dehydrogenase (BCKD) nibiteebwaho (ekicweka eki nikireetaho okukyendeeza okurikukuratiraho kandi okutarikugaruka enyima kw‘omubiri gw‘omubiri gwa BCAA carbon skeleton) (ekishushani 3A n‘ekishushani S5A). Kwonka, tihaine mpindahinduka ezirikureebwa omu BCAA ahabwayo ezashangirwe omu PN eyashorooziibwe, ekirikubaasa kuba nikireetwaho okweyongyera kw‘obutafaari bw‘okutwara amino acids ezi ezikuru nari okukoresa ebindi bihingwa (shukari nari lactic acid) kwongyera aha nkora ya TCA (ekishushani S5B). PNs ezitaine OXPHOS nazo zikooreka okweyongyera kw‘okuvunda kwa glutamine n‘emirimo y‘okujanjaaza aha myaka 8 y‘obukuru, ekirikubaasa kworekwa omu kutunguuka kw‘okuhabura kw‘eshagama y‘omubiri (GLS) na glutamine pyruvate transaminase 2 (GPT2) (ekishushani 3, A na C). Nikirungi kumanya ngu okutunguuka kwa GLS nikugarukira aha nkora ya glutaminase C (GLS-GAC) (empindahinduka ya Mfn2cKO/CTRL neehika emirundi 4.5, P = 0.05), kandi okutunguuka kwayo okw‘omutaano omu bicweka by‘omubiri eby‘amagara Nikubaasa kushagika mitochnergyondrial. (27).
(A) Maapu y‘okwotsya neeyoreka empindahinduka y‘emirundi omu rurengo rwa puroteyini ahabw‘omuhanda ogumanyirwe aha wiiki 8. (B) Eky‘okureeberaho ky‘ekicweka ky‘obwongo ekihandiikirweho obutafaari oburikurwanisa PCx (omubaro gw‘okupima, 20 μm). Omushaari gwa kyenju nigworeka omubiri gw’obutafaari bwa Purkinje. (C) Okushwijuma okurikworeka puroteyini omu bwire okwamanyirwe nk‘ekintu kikuru ekirikureetaho okusharara kw‘emitsi (okukyebera kwa t kwingi, *FDR <5%; n = 3-5 embeba). (D) Aharuguru: Ekishushani ky‘enteekateeka ekirikworeka emiringo etari emwe n‘emwe y‘okutaaha omu kaboni ehandiikirweho erimu omu [1-13C]pyruvate tracer (nikwo kugira ngu, kurabira omu PDH nari omuhanda gw‘okuraba omu mubiri). Ahansi: Ekipande kya violin nikyoreka ebicweka 10 ahari 100 bya kaboni (M1) erimu akabonero kamwe akahindukire asidi ya aspartic, asidi ya citric na asidi ya malic bwanyima y‘okuhandiika ebicweka by‘omubiri ebiine akamanyiso ka [1-13C]pyruvate (paired t-test; ** P <0.01). (E) Okushwijuma ebyafaayo by‘obwire omu bwijwire eby‘omuhanda ogurikworekwa. Teekateeka aha puroteyini eziine P<0.05 aha wiiki 8 zonka. Omuringo gw‘okuhindura: tihaine muheendo gw‘okuhindura (okushwijuma kw‘emiringo ebiri kw‘okuhinduka; * P <0.05; *** P <0.001). Ebihandiiko nibyorekwa nk‘ebiri ahagati±SEM.
Omu kweshwijuma kwaitu, BCAA catabolism ebaire emwe aha mihanda mikuru y‘okutunguura. Enshonga egi neeyoreka munonga ngu omubaro gw‘okutaho omuyaga ogurikutaaha omu nkora ya TCA nigubaasa kuhindurwa omu PN etaine OXPHOS. Eki nikibaasa kujwekyera omuringo muhango gw‘okugaruzaho eby‘okurya omu nkora y‘obutafaari bw‘omubiri, ekirikubaasa kugira eki kyakora butoosha aha nkora y‘obutafaari bw‘omubiri n‘okugumaho omu bwire bw‘okurinda oburemeezi bw‘amaani bwa OXPHOS. Okurugiirira aha nteekateeka egi, tukashanga ngu enzyme mpango erikurwanisa okusharara kw‘emitsi PCx neehindurwa ahaiguru (Mfn2cKO/CTRL neehinduka emirundi nka 1.5; ekishushani 3A), ekirikureetaho okuhindura pyruvate kuba oxaloacetate (28), ekirikuteekateekwaho kuba kiri omu brain, to9. 30). Okurugiirira aha byarugamu omu kucondooza kwa proteomics, confocal microscopy ekooreka ngu okureebeka kwa PCx kukaba kweyongyeire omu muringo gw‘omutaano kandi gw‘amaani omu PNs ezitaine OXPHOS, obwo okugarukamu kwa PCx kwabaire nikukirayo kugarukira aha butafaari bwa Bergmann glial oburi haihi obw‘okutegyeka (ekishushani 3B). Okugyezaho omu muringo gw‘okukora okutunguuka okureebirwe okwa PCx, tukajanjaba ebicweka by‘omubiri ebiine obuhuta bw‘amaani n‘omubazi gwa [1-13C]pyruvate tracer. Pyruvate ku yaabaire neehindurwa okwitsya ahabw‘okukoresa pyruvate dehydrogenase (PDH), akabonero kaayo aka isotope kakabura , Kwonka nikataaha omu nkora ya TCA ey‘omugati obu pyruvate erikuhindurwa ahabw‘okugarukamu kw‘emitsi (Ekishushani 3D). Omu kushagika ebihandiiko byaitu ebya proteomics, tukareeba omubaro muhango gw’obumanyiso kuruga omu mubazi ogu omu aspartic acid y’ebicweka bya Mfn2cKO, obwo citric acid na malic acid nabyo biine omuze gw’omutindo gw’ahaiguru, n’obu biraabe bitari bikuru (Ekishushani 3D).
Omu nkora ya dopamine ey‘embeba za MitoPark eziine obutakora gye oburikureetwaho obutafaari bwa dopamine oburikucwekyereza omu bwijwire ekicweka ky‘omubiri ekirikuhandiika A gene (Tfam) (ekishushani S6B), okureebeka kwa PCx nakwo kukaba nikuteebwaho omu nkora (31), ekirikworeka ngu acetone acid harring dysfunction of the neuron arteriosclerosis of The OXPHOS omu mubiri. Nikirungi kumanya ngu kishangirwe ngu enzymes z‘omutaano (32-34) ezirikubaasa kworekwa omu nkora y‘obutafaari bw‘omubiri ezirikubaasa kuba ziine akakwate n‘okusharara kw‘emitsi nizitunguuka munonga omu PN ezitarimu OXPHOS, nk‘ebiriisa ebirikukora nk‘ebiriisa (PCC-A), Malonyl-CoA mitochylCoA ebirikuhindura propionyl-CoA carboxylase. enzyme 3 (ME3), omurimo gwayo omukuru n‘okugaruraho pyruvate kuruga omu malate (ekishushani 3, A na C) (33, 35). Okwongyera ahari ekyo, tukashanga okweyongyera kw‘amaani omu nkora ya Pdk3, erikukora phosphorylates kandi obwo erikureetera PDH kutakora (36), obwo hatariho mpindahinduka ezaashangirwe omu nkora ya Pdp1 erikukora PDH nari omu nkora ya PDH ahabwayo (ekishushani 3A). Omu muringo ogurikushushana, omu Mern2cKO PNs, okuhindura kw‘ekicweka kya α1 α (PDHE1α) eky‘ekicweka kya pyruvate dehydrogenase E1 eky‘ekicweka kya PDH omu Ser293 (ekimanyirwe okuzibira emirimo y‘enzigye ya PDH) kukaba nikutunguuka ( Ekishushani S6C) (Ekishushani S6C). Pyruvate teine ​​nkora y’okutunga emitsi.
Eky‘aha muheru, tukashanga ngu omuhanda gw‘ahaiguru gw‘okukora ebiriisa bya serine na glycine, enzindo y‘okukora ebiriisa bya mitochondrial folate (1C) erikukwatagana n‘okukora ebiriisa bya proline (ekishushani 1G n‘ekishushani S5C) byona nibitunguuka munonga, kurugiirira ahari ripoota, omu bwire bw‘okukora. Emisi eyehinguririize neekora n‘okuremwa kukora gye kw‘ebicweka by‘omubiri (5-7). Okushwijuma okwa confocal okurikuhagira ebihandiiko ebi ebya puroteyini kukooreka ngu omu PN erimu OXPHOS etarimu, ebicweka by‘omubiri eby‘embeba z‘emyaka 8 bikateebwa omu nkora ya serine hydroxymethyltransferase 2 (SHMT2), enzyme y‘omugasho omu nkora ya folate y‘omubiri. Okugarukamu kw‘amaani kw‘obutafaari bw‘omubiri (ekishushani S5D). Omu bicweka 13 eby‘omubiri ebiine CU-glucose-incubated acute cerebellar slices, okucondooza okurikukuratira eby‘okurya omu mubiri kukagaruka kwahamya okutunguuka kw‘eby‘okurya ebirimu ebiriisa bya serine na proline, ekirikworeka ngu okuhinduka kw‘ebicweka bya carbon isoforms omu serine na proline kukaba kweyongyera (ekishushani S5E). Ahabw‘okugira ngu ebikorwa ebirikutunguuka GLS na GPT2 nibikora aha kuhanga glutamate kuruga omu glutamine n‘okuhindura ahagati ya glutamate na α-ketoglutarate, okutunguuka kwabo nikworeka ngu obutafaari bw‘omubiri obutaine OXPHOS bwine okweyongyera kw‘okwenda glutamate , Eki nikibaasa kuba kigyendereire kuguma n‘okwongyera aha proureline Staureline (Fig5). Okutaana n‘empindahinduka ezi, okucondooza kwa proteomic okwa cerebellar astrocytes kuruga omu mbeba za Mfn2cKO eziine PN-specific kworekire ngu emihanda egi (obariiremu antiperoxidases zoona) tehindukire munonga omu kumanyisa, n‘ahabw‘ekyo nikworeka ngu Okuhindura oku kw‘eby‘okurya nikutoorana PN erikusheenya (Fig. S6, G).
Omu bugufu, okweshwijuma oku kukashohoza enkora zitari zimwe na zimwe z‘okukora kw‘obwire bukye kw‘emihanda y‘okuhindura eby‘okurya omu PNs. N‘obu okukora kw‘obutafaari bw‘omubiri okutari kwa buriijo kuraabe nikubaasa kureetera okusharara kw‘emitsi omu bwire bukye n‘okugaruzaho 1C (ekishushani 3E n‘ekishushani S5C), nangwa n‘empindahinduka ezirikuteberezibwa omu kumanyisa kw‘ebicweka bya I na IV, empindahinduka omu kuhanga kwa serine de novo nizo zonka Ekatandika kureebeka omu sta y‘aha muheru. OXPHOS obutakora gye (ekishushani 3E n‘ekishushani S5C). Ebirugire omu kucondooza oku nibishoboorora omuringo gw‘okukuratana ogu emitsi y‘omubiri erikureetwaho okushoberwa (1C cycle) n‘ebiri omu nkora y‘omubiri (serine biosynthesis) birikugarukamu omu muringo gw‘okukoragana n‘okwongyera aha kusheeshasheesha kw‘emitsi omu nkora ya TCA kugaruka kuhindura omubiri gw‘omubiri.
PNs eziine OXPHOS ezitarikubura OXPHOS ez‘emyaka 8 nizibaasa kuguma ziine emirimo y‘okusiimura ey‘emirundi mingi kandi zihikye omu kugarukaho kw‘eby‘okurya kw‘amaani kubaasa kugaruzaho okuremwa kukora gye kw‘emibiri. Okuzoora oku nikureetaho okubaasa okurikushemeza ngu n‘omu bwire obu, obutafaari obu nibubaasa n‘okutunga obujanjabi bw‘okukyerererwa nari okuzibira okusheeshasheesha kw‘obutafaari bw‘omubiri. Kucerererwa. Twashoboorora okubaasa oku kurabira omu kuhaajaana kw‘abantu babiri ab‘obwegyese. Omu muringo gw‘okubanza, tukakora omubazi gw‘okutwara akakoko akarikwegamira ahari Cre (AAV) kugira ngu MFN2 ebaasa kutooranwa kworekwa omu PNs ezitaine OXPHOS omu nkora y‘amagara (Ekishushani S7A). AAV erikuhandiika MFN2 n‘omubazi gw‘okumanyisa ebiri omu mushana mCherry (Mfn2-AAV) bikahamibwa omu buhangwa bw‘obutafaari bw‘omubiri obukuru omu nkora ya vitro, ekyareeteire MFN2 kworekwa omu muringo ogurikurugiirira ahari Cre kandi kyacungura enkora y‘obutafaari bw‘omubiri, obwo kikazibira okuhinduka kw‘obutafaari omu Mfns, B Ekishushani S2c7). Ekindi, tukakora okucondooza omu vivo kubaasa kuhitsya omu muringo gw‘okureeberaho Mfn2-AAV ey‘emyaka 8 aha cerebellar cortex ya Mfn2cKO n‘embeba z‘okutegyeka, kandi twashwijuma embeba z‘emyaka 12 (ekishushani 4A). Embeba za Mfn2cKO ezaabaire ziine obujanjabi zikafa (Ekishushani 1, A na B) (16). Okuhindura kwa virus omu nkora y‘amagara (vivo) kukarugamu okutoorana PN omu nkora ezimwe z‘obutafaari bw‘omubiri (ekishushani S7, G na H). Okuteebwamu omubazi gw‘okutegyeka AAV ogurikworeka mCherry yonka (Ctrl-AAV) tiguine ki gwakora aha rurengo rw‘okusheeshasheesha kw‘obutafaari bw‘omubiri omu nyamaishwa za Mfn2cKO. Okutaana n‘ekyo, okweshwijuma kwa Mfn2cKOs ezaabaire zihindurwa na Mfn2-AAV kukooreka ngu hariho oburinzi bw‘amaani aha rubaju rw‘obutafaari bwa PN (ekishushani 4, B na C). Okukira munonga, oburemeezi bw‘obutafaari bw‘omubiri nibushusha nk‘obutarikutaanisibwa haihi n‘enyamaishwa ezirikutegyekwa (ekishushani 4, B na C, n‘ekishushani S7, H na I). Okworeka MFN1 kwonka hatariho MFN2 nikukora gye nk‘oku kiri omu kujuna okufa kw‘obutafaari bw‘omubiri (ekishushani 4C n‘ekishushani S7, C na F), ekirikworeka ngu okweyoreka kwa ectopic MFN1 nikubaasa kwongyera gye aha kubura MFN2. Okweyongyera kweshwijuma aha rurengo rwa PN rumwe kukooreka ngu Mfn2-AAV ekacungura munonga ultrastructure ya mitochondria, ekahindura omutindo gwa mtDNA, kandi ekagarura enyima okureebeka kw‘ahaiguru kw‘akamanyiso k‘okurwanisa okusheeshasheesha PCx (Ekishushani 4, C kuhika E ). Okukyebera okurikureebwa kw‘embeba za Mfn2cKO ezacungwirwe omu mbeera y‘okuhuumura kworekire ngu oku zirikuguma n‘obumanyiso bw‘okuvuga (okutambura S1 kuhika S3) zikaba ziri nungi. Omu kuhendera, okucondooza oku nikworeka ngu okukyerererwa kugaruka kuta MFN2 omu PNs eziine oburemeezi bw‘amaani omu OXPHOS nikimara kugaruza enyima okukoresa mtDNA n‘okureetaho okusharara kw‘emitsi, obwo nikuzibira okusheeshasheesha kw‘emitsi n‘okufa kw‘obutafaari bw‘omubiri omu nkora y‘amagara.
(A) Enteekateeka erikworeka entebekanisa y‘okugyezesa ey‘okuta AAV encoding MFN2 obu omuhanda gw‘okuhindura eby‘okurya ogurikworekwa gurikukora. (B) Ebishushani by‘okujwekyera confocal eby‘ebicweka by‘obwongo eby‘emyaka 12 ebyabaire bihindurwa n‘embeba za Mfn2cKO aha wiiki 8 kandi bikahandiikwaho obutafaari oburikurwanisa Calbindin. Aha rubaju rwa buryo: Okupima ebicweka by’omubiri ebirikwetwa axon. Ekipimo ky’okutunguura axon ni 450 na 75 μm. (C) Aha rubaju rwa bumosho: Okupima obuhango bw‘obutafaari bwa Purkinje omu nkora y‘okuhindura AAV (AAV+) (okushwijuma kw‘omuringo gumwe kw‘okuhinduka; n = 3 empunu). Aha rubaju rwa buryo: mtDNA focus okucondooza omu PN ehindwirwemu aha wiiki 12 (t-test etaine babiri; n = obutafaari 6 kuruga omu mbeba ishatu). * P <0.05; ** P <0.01. (D) Ebishushani by‘okujanjaaza eby‘amashanyarazi ebirikujwekyera PNs z‘ebicweka by‘obwongo bya Mfn2cKO ebihindwirwemu n‘ebintu ebirikutwara obukooko oburikworekwa. Ekijwaro kya pinka nikyoreka omwanya ogurimu ebihimba, kandi ekikwato ekirimu obutonyezi bwa kyenju nikyoreka zoom erikuheebwa aha rubaju rwa buryo; n nikijwekyera ekicweka ky’omubiri. Omubaro gw’okupima, 1μm. (E) nikyoreka eky‘okureeberaho ky‘okutaho erangi ya PCx omu PN erikuhindurwa aha wiiki 12. Omubaro gw’okupima, 20μm. OE, okugira eki waagamba munonga; FC, okuhindura.
Eky‘aha muheru, tukacondooza aha bukuru bw‘okugumaho kw‘obutafaari oburikureetwa peroxidase omu PNs ezitungire oburemeezi bwa OXPHOS. Twakora mCherry erikuhandiika AAV-shRNA (RNA y’eishokye rigufu) erikuta omutima munonga aha mRNA ya PCx y’embeba (AAV-shPCx), kandi twata akakoko aka nari okutegyeka kwako okurikuteganisa (AAV-scr) omu bwongo bw’embeba za Mfn2cKO. Enjegyere ekakorwa omu wiiki ya kana y‘emyaka (ekishushani 5A) kubaasa kutunga okuteera PCx okuhikire omu bwire obu PCx expression yayeyongyeire (ekishushani 3C) kandi PN cell layer ekaba ekiriho (ekishushani 1A). Nikirungi kumanya ngu okuteera PCx ahansi (ekishushani S8A) nikireetaho okutunguuka kw‘amaani kw‘okufa kwa PN, niyo mpeta yonka erikuzibirwa y‘okukwatwa endwara (ekishushani 5, B na C). Okubaasa kushoboorora omuringo gw‘ebirikuruga omu kuhindura eby‘okurya ebirikureetwa okutunguuka kwa PCx, tukacondooza aha mbeera ya redox ya PNs bwanyima y‘okuteera PCx kandi AAV-mediated optical biosensor Grx1-roGFP2 ekaba neeyorekwa omu bwire bumwe (ekishushani S8, B kuhika D) kukyebera glutathione potential of3). Bwanyima, tukakora okucondooza kw‘ebishushani by‘amagara g‘abantu (FLIM) omu bicweka by‘obwongo ebigumire eby‘emyaka 7 eby‘emyaka 7 bya Mfn2cKO nari okutegyeka littermates kureeba empindahinduka ezirikubaasa kubaho omu mbeera ya cytoplasmic redox bwanyima y‘okuhamya embeera za FLIM (ekishushani S8, E kuhika G). Okweshwijuma kukooreka okweyongyera kw‘amaani omu mbeera y‘okuhindura ebiriisa (oxidation state) ya Mfn2cKO PNs emwe etaine PCx expression, ekirikutaana n‘obutafaari bw‘omubiri oburikutegyeka nari Mfn2cKO PNs ezirikworeka shRNA erimu scrambled yonka (ekishushani 5, D na E). PCx expression ku yaabaire neeteebwa ahansi, ebicweka 10 ahari 100 bya Mfn2cKO PNs ebirikworeka embeera y‘okugira okwijuzibwa kw‘amaani bikeyongyera emirundi erikurenga ahari eshatu (ekishushani 5E), ekirikworeka ngu PCx up-regulation ekaguma n‘obushoboorozi bwa redox obw‘obutafaari bw‘omubiri obuhweireho.
(A) Enteekateeka erikworeka entebeekanisa y‘okugyezesa ey‘okuta AAV encoding shPCx obu omuhanda gw‘okuhindura eby‘okurya ogurikworekwa gurikukora. (B) Ebishushani by‘okujwekyera confocal eby‘ebicweka by‘obwongo eby‘emyaka 8 omu mbeba za Mfn2cKO ezaabaire zihindurwa kandi zihandiikirweho obutafaari oburikurwanisa calcineurin aha wiiki 4. Omubaro gw’okupima, 450μm. (C) Okupima obuhango bw‘obutafaari bwa Purkinje omu nkora ya AAV-transduced loops (okushwijuma kw‘omuringo gumwe kw‘okuhinduka; n = 3 kuhika 4 empunu). Ebihandiiko nibyorekwa nk‘ebiri ahagati±SEM; ***P<0.001. (D) Ekishushani kya FLIM ekirikujwekyera nikyoreka emyaka y‘amagara eya PN ey‘emyaka 7 erikworeka glutathione redox sensor Grx1-roGFP2 ahansi y‘embeera z‘okugyezesa ezimanyirwe. LUT (emeeza y‘okureeba) omubaro: obwire bw‘okugumaho (omu picoseconds). Omubaro gw’okupima, 25μm. (E) Histogram neeyoreka okugabwa kw‘emihendo y‘amagara ga Grx1-roGFP2 kuruga ahari (D) (n=158 kuhika 368 obutafaari omu mbeba ibiri ahansi ya buri mbeera). Ekipande kya pie ahaiguru ya buri histogram: nikyoreka omubaro gw‘obutafaari obuine emihendo y‘amagara erikukirayo oburaingwa (etukutuku, oxidized) nari mikye (bururu, okukyendeera), erikurenga 1 SD y‘omuhendo gw‘amagara ogwagati omu CTRL-AAV-scr. (F) Eky‘okureeberaho ekiteirweho nikyoreka oburinzi bw‘okutunguura PCx y‘obutafaari bw‘omubiri.
Omuri byona, ebihandiiko ebi turikuha aha nibyoreka ngu okugaruka kworeka MFN2 nikubaasa kucungura kimwe PN etunguukire n‘okubura OXPHOS kw‘amaani, okubura mtDNA kw‘amaani, n‘okugira enkora y‘omubiri erikushushana n‘eya ista etari ya buriijo munonga, obwo nikiha entunguuka etarikuhwaho nangwa n‘omu ndwara ezitunguukire. Okusheeshasheesha kw‘obutafaari bw‘omubiri nikuha obuhame oburikugaruka enyima obw‘omutindo gw‘obutafaari butakafiire. Omutindo ogu gw‘okuhindurahindura eby‘okurya nigweyongyera kuhamibwa obushoboorozi bw‘obutafaari bw‘omubiri kureetaho okusharara kw‘emitsi (okugaruka kuhindura omu nkora ya TCA), ekirikuzibira PCx kworeka omu PNs ezitaine OXPHOS kandi kyongyera aha kufa kw‘obutafaari, obwo nibukora omurimo gw‘okwerinda (ekishushani 5F).
Omu kucondooza oku, tukaha obuhame ngu okugarukamu kwa PNs aha kutakora gye kwa OXPHOS n‘okuhindura mporampora kuhika aha TCA cycle atherosclerosis kurabira omu muhanda gw‘okukora ogw‘entaaniso ogurikukoresibwa puroguraamu z‘okuhindura eby‘okurya. Twahamya okucondooza kwa proteomic n‘emiringo mingi y‘okuhagira kandi twashohoza ngu ku barikuhakanisibwa okuremwa kukora gye kw‘emibiri, obutafaari bw‘omubiri nibugira omuringo gw‘okuhindura eby‘okurya ogwabaire gutamanyirwe ira. Ekirikututangaaza, omuringo gwona ogw‘okugaruzaho waya tigurikumanyisa embeera y‘okuhindura eby‘okurya ey‘aha muheru erikuhererukayo n‘okusheeshasheesha kw‘obutafaari bw‘omubiri mporampora kandi okutarikugaruka enyima, kwonka ebihandiiko byaitu nibyoreka ngu nibibaasa kukora obutafaari bw‘omubiri oburikurinda n‘obu bwakuba omu rurengo oru obutafaari butakafiire Functional compensation mechanism. Ekirugire omu kucondooza oku nikyoreka ngu obutafaari bw‘omubiri oburikwetwa neurons bwine omutindo muhango gw‘okuhindura eby‘okurya omu mubiri. Amazima aga nigahamya ngu okugaruka kutaho MFN2 bwanyima nikubaasa kuhindura okureebeka kw‘obumanyiso bw‘okuhindura eby‘okurya ebikuru n‘okuzibira okusheeshasheesha kwa PN. Kureka, nikizibira okusharara kw’emitsi kandi nikitunguura emitsi. okuhindura abashaija n’abakazi.
Ekimwe aha byashangwa omu kucondooza kwaitu okurikushemeza munonga n‘okugira ngu PNs ezitaine OXPHOS nizibaasa kuhindura omu nkora ya TCA cycle metabolism barikuhindura enzymes ezirikureetaho okusharara kw‘emitsi. Okugaruka omu ntebeekanisa y‘eby‘okurya n‘ekintu ekirikukirayo omu butafaari bwa kansa, obumwe aharibwo nibwesiga glutamine kwongyera aha biri ahagati y‘enzindo ya TCA kureetaho ebirikushushana ebirikukyendeeza, ebirikuvuga omurunga gw‘okuhuuta n‘okurinda okukora ebiriisa by‘omubiri n‘eby‘okurya ebirikubanza (39 , 40). Okucondooza okukozirwe juba kworekire ngu omu bicweka by‘omubiri ebiri aha rubaju ebirikutunga oburemeezi bwa OXPHOS, okugaruka kukwatiraine n‘okuhindura glutamine/glutamate nakyo n‘ekintu kikuru (5, 41), ahu orubaju rw‘okutaaha kwa glutamine omu nkora ya TCA rurikurugiirira aha Ahabw‘oburemeezi bw‘okuhutaara kwa OXPHOS (41). ). Kwonka, tihaine buhame oburikworeka gye aha kushushana kwona kw‘obutafaari bw‘omubiri oburikuhindura eby‘okurya omu mubiri n‘okubaasa kuba kuriho omu mbeera y‘endwara. Omu kucondooza okuherize kukorwa omu vitro, obutafaari bw‘omubiri oburikubanza bukareebeka ngu nibuteerana ebiziba bya glutamate ahabw‘okujanjaaza obutafaari bw‘omubiri, obwo nibutunguura okuhindura kw‘omubiri n‘okusharara kw‘emitsi ahansi y‘embeera z‘okushoberwa kw‘omubiri (42). Nikirungi kumanya ngu ahansi y‘okuzibira kw‘eby‘emibazi kw‘enzigye y‘omuringo gwa TCA succinate dehydrogenase, pyruvate carboxylation neeteekateekwaho kuguma n‘okuteerana kwa oxaloacetate omu nkora y‘obuhangwa bw‘omubiri (34). Kwonka, obukuru bw‘omubiri bw‘enkora ezi n‘ebicweka by‘obwongo (ahu oburwaire bw‘okusharara kw‘emitsi burikwikirizibwa ngu nibukira kuba omu nkora z‘omubiri) bukyaine omugasho gw‘omubiri gw‘amaani (43). Omu muringo ogu, ebihandiiko byaitu nibyoreka ngu PNs ezirikushiisha OXPHOS omu mubiri nizibaasa kuhindurwa zibe omu kusheenya BCAA na pyruvate carboxylation, eziri enshonga ibiri nkuru z‘okugaita eby‘okurya by‘omugati bya TCA pool. N‘obu okuteekateeka ngu BCAA catabolism neehwera omu kuhindura amaani g‘obutafaari bw‘omubiri kyateebwaho, okwiha aha murimo gwa glutamate na GABA omu kuhindura obutafaari bw‘omubiri (44), hakiriho buhame bw‘emiringo egi omu nkora y‘amagara. Nahabwekyo, kyorobi kuteekateeka ngu PNs ezitarikukora gye nizibaasa kwerihiirira ahabw‘okukoresa eby‘omugati bya TCA ebirikugyenderwaho omu nkora y‘okushoroora (assimilation process) ahabw‘okwongyera aha kusheeshasheesha kw‘emitsi. Okukira munonga, okutunguura PCx nikubaasa kwetengyesa kubaasa kuguma n‘okweyongyera kw‘okwenda aspartic acid, ekirikworekwa omu kukanyisa obutafaari oburimu oburemeezi bw‘okukora gye (45). Kwonka, okucondooza kwaitu okwa metabolomics tikwayorekire empindahinduka yoona ey’amaani omu rurengo rw’okugumaho rwa aspartic acid omu Mfn2cKO PNs (ekishushani S6A), ekirikuteekateekwaho kworeka okukoresa kw’eby’okurya okutarikushushana ahagati y’obutafaari oburikweyongyera n’obutafaari oburikukura bwanyima y’okuhinduka. N‘obu omuringo gwenyini ogw‘okutunguura PCx omu nkora y‘obutafaari bw‘omubiri obutarikukora gye omu nkora y‘amagara guraabe niguguma nigumanyirwa, tukooreka ngu okugarukamu oku okutakahikire nikukora omurimo muhango omu kuguma n‘embeera ya redox y‘obutafaari bw‘omubiri, ekyayorekwa omu kucondooza kwa FLIM aha bicweka by‘omubiri eby‘omubiri. Okukira munonga, okuzibira PNs kuruga omu kutunguura PCx nikibaasa kureetera embeera y‘okugira okwijuzibwa kwingi n‘okutunguura okufa kw‘obutafaari. Okukora kw‘okusheenya kwa BCAA n‘okuhindura kwa carboxylation kwa pyruvate ti miringo y‘okumanyisa enyama z‘aheeru y‘omubiri ezitarikukora gye (7). Nahabwekyo, nibireebeka nk‘ekintu ky‘okubanza omu nkora y‘obutafaari bw‘omubiri obutaine OXPHOS, n‘obu kiraabe kitari kyonka, ekikuru omu kusheeshasheesha kw‘obutafaari bw‘omubiri. .
Endwara ya cerebellar n‘omuringo gw‘endwara z‘obutafaari bw‘omubiri ezitarikushushana ezirikukira kweyoreka nka ataxia kandi ezirikukira kushiisha PNs (46). Omubaro gw‘obutafaari obu nigubaasa kukwatwaho munonga ahabw‘okuremwa kukora gye kw‘ebicweka by‘omubiri ahabw‘okuba okukyendeera kwabyo okutoorainwe omu mbeba nikumara kuzaara obumanyiso bwingi bw‘okuvuga oburikworeka obuhuta bw‘omubiri bw‘omuntu (16, 47, 48). Okurugirira ahari ripoota, omuringo gw‘embeba oguhindwirwemu n‘omubazi ogurikuhinduka nigukwatagana n‘omuntu spinocerebellar ataxia kandi gwine obutakora gye bw‘emibiri (49, 50), ekirikuhamya obukuru bw‘okwega ebirikuruga omu kubura OXPHOS omu PNPH. Nahabwekyo, nikiba kiri kirungi munonga kwetaanisa gye n’okwega aha nkora egi ey’omutaano ey’obutafaari bw’omubiri. Kwonka, ahabw‘okugira ngu PNs nizihurira munonga okugyemeserezibwa kandi nizitwara ekicweka kikye ky‘obutafaari bwona obw‘omu bwongo, ahabw‘okucondooza kwingi okurikugyendera aha omics, okuzitaanisa omu kutoorana nk‘obutafaari bwona n‘ekintu ekigumire. N‘obu kiraabe kitarikubaasika kuhikiiriza okubura obukooko obuhikire obw‘ebindi biika by‘obutafaari (na munonga ebicweka by‘omubiri eby‘abantu bakuru), tukagaita eitembezo ry‘okutaana eririkukorera gye na FACS kutunga omubaro gw‘obutafaari oburikumara obw‘okushwijuma ebiri omu nkora y‘okukyebera ebiriisa by‘omubiri, kandi twine ebiriisa by‘omubiri ebiri ahaiguru munonga (nk‘ebicweka 300 the cerebel the proteins) (51) Omu kurinda obushoboorozi bw’obutafaari bwona, omuringo ogu turikuha aha nigutubaasisa kutakyebera empindahinduka omu mihanda y’okuhindura eby’okurya omu mitochondria yonka, kureka n’okukyebera empindahinduka omu banywani baayo aba cytoplasmic, ekirikuhagira okukoresa obutafaari bw’omubiri oburikugambwaho omu miringo y’obutafaari bw’omubiri the new methox number the (52, 53). Omuringo ogu turikushoboorora tigurikukwatagana n‘okwega obutafaari bwa Purkinje bwonka, kureka nigubaasa kukoresibwa omu muringo gw‘obutafaari gwona omu muringo gworobi kushoboorora empindahinduka z‘okuhindura eby‘okurya omu bwongo obuine endwara, otwariiremu n‘ebindi bishushani by‘okutakora gye kw‘obutafaari bw‘omubiri.
Eky‘aha muheru, tukamanya edirisa y‘okujanjaba omu bwire bw‘omuringo ogu gw‘okugaruka kutebeekanisa eby‘okurya ebirikubaasa kuhindura kimwe obumanyiso obukuru obw‘okushoberwa kw‘obutafaari n‘okuzibira okusheeshasheesha kw‘obutafaari bw‘omubiri. Nahabwekyo, okwetegyereza ebirikuruga omu kukora kw‘okugaruzaho waya okushobooroirwe aha nikubaasa kuha okumanya kw‘omutaano aha bujanjabi oburikubaasa kubaho ahabw‘okurinda obushoboorozi bw‘obutafaari bw‘omubiri omu bwire bw‘okuremwa kukora gye kw‘obutafaari bw‘omubiri. Okucondooza okw‘omumaisho okugyendereire kucondooza empindahinduka omu kuhindura kw‘amaani omu miringo endiijo y‘obutafaari bw‘obwongo nikwetengyesa kworeka omu bwijwire oku omusingye ogu gurikubaasa kukoresibwa aha ndwara ezindi z‘obwongo.
Embeba za MitoPark zikaba zishobooroirwe enyima (31). Embeba za C57BL/6N eziine loxP flanking Mfn2 genes zikaba zishobooroirwe enyima (18) kandi zikaba zirimu embeba za L7-Cre (23). Ebirugiremu, abaana b’emiringo ebiri abatari bamwe na bamwe bakaheza baazaana n’embeba eziine emiringo emwe eya Mfn2loxP/Mfn2loxP kubaasa kureetaho okuteerana kw’ebicweka by’omubiri ebya Purkinje ebya Mfn2 (Mfn2loxP/Mfn2loxP; L7-cre). Omu kicweka ky’okuteerana, Gt (ROSA26) SorStop-mito-YFP allele (okurekyera aho-mtYFP) ekatandikwa kurabira omu kusharaho okundi (20). Enkora zoona z’okucondooza aha nyamaishwa zikakorwa kurugiirira aha buhabuzi bwa Buraaya, obw’eihanga n’obw’ebitongore kandi zikaikirizibwa LandesamtfürNatur eya Umwelt na Verbraucherschutz, North Rhine-Westphalia, Bugirimaani. Omurimo gw’okukora aha nyamaishwa nagwo nigukuratira obuhabuzi bw’ekibiina kya Buraaya eky’ebibiina by’enyamaishwa z’omu laboratory.
Bwanyima y‘okutaho obuhuta bw‘okusheeshasheesha kw‘omukazi oine enda, akazaano k‘embeba nikataanisibwa (E13). Ekicweka ky‘omubiri eky‘omubiri kikasharamu omu nkora ya Hanks ey‘omunyugunyu (HBSS) eyongyeirwemu Hepes 10 mM kandi kyahingura aha nkora ya Dulbecco ehindwirwemu ey‘enkongoro erimu papain (20 U/ml) na cysteine ​​(1μg/ml). Incubate enyama omu DMEM) kandi ogitaanise orikukoresa enzymatic okugigabura. Ml) aha 37°C kumara edakiika 20, reero bwanyima bakagisenda omu DMEM erimu 10% y’eshagama y’ente ey’omunda. Obutafaari bukabyarwa aha bipande by’okukingiraho endabirwamu ebiteirweho polylysine aha nkora ya 2×106 ahari buri dishi y’obuhangwa ya cm 6 nari aha nkora ya 0.5×105 obutafaari/cm2 ahabw’okushwijuma ebishushani. Bwanyima y‘eshaaha 4, omubazi gukahindurwa omubazi ogutarimu serum ya Neurobasal ogurimu 1% B27 y‘okugaita hamwe na 0.5 mM GlutaMax. Bwanyima obutafaari obu bukaguma buri aha 37°C na 5% CO2 omu kucondooza kwona, kandi bwariisibwa omurundi gumwe omu wiiki. Okubaasa kureetaho okugaruka omu nkora omu nkora y‘okukyebera, 3μl (ekikopo ky‘obuhangwa bw‘ebiina 24) nari 0.5μl (ekikopo ky‘ebiina 24) ky‘omubazi gw‘akakoko ka AAV9 ogurikukurataho gukakoresibwa kujanjaba obutafaari bw‘omubiri aha kiro kya kabiri omu nkora y‘okukyebera: AAV9.CMV.PI.eGFP. WPRE.bGH (Eky’okureeberaho, enamba y’akatabo 105530-AAV9) na AAV9.CMV.HI.eGFP-Cre.
DNA y’embeba Mfn1 na Mfn2 erikuhwera (etungirwe kuruga omu Addgene plasmid #23212 na #23213, buri kimwe) neehandiikwa omu nkora ya V5 (GKPIPNPLLGLDST) aha muheru gwa C, kandi neekwatanisibwa na mCherry omu fureemu kurabira omu nkora ya T2A. Grx1-roGFP2 n’ekiconco kuruga omu kitongore ky’eby’amagara ekya Heidelberg. Omu kugaruzaho akatambi ka tdTomato barikukoresa emiringo y’okukora ebishushani eya butoosha, kaasi ekahindurwa omu mugongo gwa pAAV-CAG-FLEX-tdTomato (Addgene reference number 28306) kubaasa kureetaho pAAV-CAG-FLEX-mCherry-T2A-MFNGAV-V5, pAAV-CAG-FLEX-tdTomato. FLEX-mCherry-T2A-MFN1-V5 na pAAV-CAG-FLEX-Grx-roGFP2 ebiri omu nkora. Enkora nk‘egyo ekakoresibwa kureetaho omubazi gw‘okutegyeka pAAV-CAG-FLEX-mCherry. Okubaasa kureetaho ekyombeko kya AAV-shPCx, omubazi gwa plasmid AAV (VectorBuilder, pAAV [shRNA] -CMV-mCherry-U6-mPcx- [shRNA#1]) nigwetengyesa, ogurimu omukuratana gwa DNA ogurikuhandiika shRNA erikuta omutima aha mbeba PCx (5′GTAGCTAGGTGTGCTACTACTA 3′) Ahansi y’obushoboorozi bw’omuhi w’obuhwezi wa U6, mCherry neekoresibwa ahansi y’obushoboorozi bw’omuhi w’obuhwezi wa CMV. Okukora ebikwato by’okuhwera AAV vectors kukakorwa kurugiirira aha biragiro by’abakozire (Cell Biolabs). Omu bugufu, kozesa plasmid y‘okuhingisa erikutwara mCherry-T2A-MFN2-V5 (pAAV-CAG-FLEX-mCherry-MFN2-V5), mCherry-T2A-MFN1-V5 (pAAV-CAG-FLEX-mCherry) omu bwire bukye Okuhingisa kw‘obutafaari bwa 29MFCAV2, (pAAV-CAG-FLEX-mCherry) nari Grx-roGFP2 (pAAV-CAG-FLEX-Grx-roGFP2) ekirikuhandiika ebiri omu nkora y‘obuzaarwa, hamwe n‘okuhandiika ebiri omu puroteyini ya AAV1 n‘ebiri omu puroteyini y‘okuhweraho Okupakira puroteyini ya plasmid, orikukoresa omuringo gwa calcium phosphate. Ekirungo ky’akakoko akatari kakye kikatunga omu nkora y’okufuka-okufuka omu kyombeko ky’orubaare/ethanol eyomire kandi obutafaari bwasheesheka omu saline erimu phosphate buffered (PBS). Omubazi gwa AAV gukashemezibwa omu kukyebera okurikukoresibwa iodixanol gradient okutarikugumizamu (eshaaha 24 ahari 32,000 rpm na 4°C) kandi gwateebwa omu nkora barikukoresa ekicweka ky’okukyebera ekya Amicon ultra-15. 2.9×1013 kopi y‘omubiri (GC)/ml], AAV1-CAG-FLEX-mCherry (6.1×1012 GC/ml), AAV1-CAG-FLEX-mCherry (6.1×1012 GC/ml), AAV1-CAG-FLEX ey‘omubaro gw‘amazima ekaba neepimwa nk‘oku kyabaire kiri enyima. (qPCR) -MFN1-V5 (1.9×1013 GC/ml) na AAV1-CAG-FLEX-Grx-roGFP2 (8.9×1012 GC/ml).
Obutafaari bw‘omubiri obukuru bukasharaho omu 1x PBS erikwotsya nk‘orubaare, bwateebwa omu nshaho, bwanyima bwahindurwa omu 0.5% Triton X-100 / 0.5% sodium deoxycholate/PBS lysis buffer erimu phosphatase n‘ekirungo ky‘okuzibira puroteyini (Roche). Okupima obuhango bwa puroteyini kukakorwa barikukoresa okukyebera kwa asidi ya bicinchoninic (Thermo Fisher Scientific). Ebirungo ebyo bikarugaho byataanisibwa omu nkora ya SDS-polyacrylamide gel, reero byateebwa aha rubaju rw’omubiri orurikwetwa GE Healthcare. Zibira emyanya etari y‘omutaano kandi oshukyeere n‘omubazi gw‘okubanza (reeba ekipande S1 ahabw‘ebirikukiraho) omu mate 5% omu TBST (Tris-buffered saline na Tween), amatembezo g‘okwozya n‘omubazi gw‘okubanza omu TBST Incubate. Ote omu nkungu n’ebirungo by’okubanza ekiro kyona ahari +4°C. Bwanyima y‘okwozya, teekaho omubazi gw‘okurwanisa endwara ogwa kabiri kumara eshaaha 2 aha butagasi bw‘omu kishengye. Okurugirira ahari ekyo, okutaho ekicweka ekyo kimwe n’ekirungo ekirikurwanisa β-actin, okuremeera kumwe kukahamibwa. Okukyebera orikuhindura omu nkora y‘okukyebera n‘okutunguura okukyebera (GE Healthcare).
Obutafaari bw‘omubiri obwabaire buteirwe enyima aha bipande by‘okukingira endabirwamu bukahamibwa na 4% paraformaldehyde (PFA)/PBS omu bwire obuteirweho aha butagasi bw‘omu kishengye kumara edakiika 10. Ebipande by‘okukingira nibibanza byateebwamu 0.1% Triton X-100/PBS kumara edakiika 5 aha butagasi bw‘omu kishengye, reero bwanyima omu buffer y‘okuzibira [3% y‘eshagama y‘ente (BSA)/PBS]. Aha kiro kya kabiri, ebipande by’okukingiraho bikanaabwa n’ekirungo ky’okuzibira kandi byateebwa omu nkora n’ekirungo ky’okurwanisa endwara ekihikire ekya fluorophore-conjugated secondary antibody kumara eshaaha 2 aha butagasi bw’omu kishengye; aha muheru, eshampu zikanaabwa gye omu PBS na 4′,6-diamidino-2 -Phenylindole (DAPI) nizikyeberwa reero zihamibwa aha rupapura rw’okukyeberaho na Aqua-Poly/Mount.
Embeba (ez‘omushaija n‘ez‘omukazi) zikakyeberwa barikuzita omunda y‘ekibunu omubazi gw‘okukyebera (130 mg/kg) na xylazine (10 mg/kg) kandi zikateebwa ahansi y‘oruhu n‘omubazi gw‘okukyendeeza obusaasi ogwa carprofen (5 mg/kg) , Kandi ziteebwa omu kikwato ky‘okukyebera obusaasi (Kopd a pamf) equi. Okworeka ekibunu kandi okozese ekikwato ky‘okuteera ameino kukyendeeza aha kicweka ky‘omubiri ekirikuhikaana n‘eigufa rya mis (kuruga omu lambda: omukira 1.8, aha rubaju 1, ekirikuhikaana na lobules IV na V). Koresa empiira y‘okuteera empiso erikugoma kuhangaho akaina kakye omu kifuba n‘obwegyendesereza okwetantara okuteganisa emitsi eri ahansi. Bwanyima akapiira k‘endabirwamu akakye akarikuteebwaho nikateebwa mpora mpora omu kiina kikye (kuruga ahari -1.3 kuhika ahari -1 aha rubaju rw‘omunda rw‘ekibunu ky‘omubiri), kandi 200 kuhika 300 nl AAV neeteebwa omu kiina ky‘omubiri ekikye (Narishige) n‘empiso z‘omu ngaro (Narishige tisure) aha bwire bwa sever a2. edirisa y’edakiika. Bwanyima y'okugiteekaho, taho akacupa aka akapiira kumara edakiika ezindi 10 kubaasa kuha akakoko aka kujanjaara kimwe. Bwanyima y‘okuihaho obutafaari oburikwetwa capillaries, oruhu nirutunganirwa n‘obwegyendesereza kukyendeeza aha kuzimba kw‘ebihuta n‘okureetera enyamaishwa kugaruka omu buteeka. Ebinyonyi bikajanjabwa n’emibazi y’okukyendeeza obusaasi (caspofen) kumara ebiro bingi bwanyima y’okushemezibwa, omu bwire obwo embeera y’omubiri gwabyo ekaba neekyeberwa gye reero bwanyima byaitwa omu bwire obugambirweho. Enkora zoona zikakorwa kurugiirira aha buhabuzi bwa Buraaya, obw‘eihanga n‘obw‘ebitongore kandi zikaikirizibwa LandesamtfürNatur eya Umwelt na Verbraucherschutz, North Rhine-Westphalia, Bugirimaani.
Ebinyonyi bikakyeberwa n’omubazi gwa ketamine (100 mg/kg) na xylazine (10 mg/kg), kandi omutima gukabanza gwateebwamu 0.1 M PBS, bwanyima gwateebwamu 4% PFA omu PBS. Emisi ekasharaho kandi yaateebwa omu 4% PFA/PBS ekiro kyona ahari 4°C. Omusyo ogurikutengeeta (Leica Microsystems GmbH, Vienna, Austria) gukakozesibwa kutebeekanisa ebicweka by‘omubiri ebirikwetwa sagittal (oburaingwa bwa 50 μm) kuruga omu bwongo obutaine mpinduka omu PBS. Okwihaho kyaba kitarikworekwa, okutaho erangi aha bicweka ebirikwetoorora busha kukakorwa nk’oku kyashoboororwa ahaiguru (13) aha butagasi bw’omu kishengye n’okubiteera. Omu bugufu, eky’okubanza, ebicweka ebyatungirwe bikakyeberwa 0.5% Triton X-100/PBS kumara edakiika 15 aha butagasi bw’omu kishengye; aha epitope ezimwe (Pcx na Shmt2), kurabira omu tris-EDTA buffer ahari 80°C (PH 9) kwotsya ebicweka by‘omubiri kumara edakiika 25 omu mwanya gw‘eidaara eri. Ekindi, ebicweka bikateebwa omu nkora y‘okurwanisa endwara y‘okubanza (reeba ekipande S1) omu buffer y‘okuzibira (3% BSA/PBS) aha 4°C ekiro kyona n‘okugiteera. Ekiro ekyakuratsireho, ebicweka ebyo bikayozibwa n’ekirungo ky’okuzibira kandi byateebwa omu nkora n’ekirungo ky’akabiri ekihikire ekya fluorophore-conjugated kumara eshaaha 2 aha butagasi bw’omu kishengye; aha muheru, ebicweka bikayozibwa gye omu PBS, byakyeberwa na DAPI, reero byahamibwa na AquaPolymount Aha slide ya microscope.
Ekikwato ky‘okukyebera ebiri omu mushana (TCS SP8-X nari TCS Digital Light Sheet, Leica Microsystems) ekirimu omushana gw‘omushana ogurikwera n‘omushana gw‘omushana ogwa 405 diode kikakoresibwa kureeba ekishushani ky‘eky‘okureeberaho. Omu kushemeza fluorophore n‘okuteerana akamanyiso na Hybrid Detector (HyDs), puroguraamu ya LAS-X ekakoresibwa okuteerana ebishushani ebiteirwe omu nshaho ebirikuhikaana n‘okutoorana kwa Nyquist omu muringo gw‘okukuratana: ahabw‘ebipande ebitari by‘omubaro, n‘obumanyiso bw‘amaani maingi (nk‘eky‘okureeberaho, omu butafaari bw‘omubiri n‘ebicweka by‘omubiri) mt . Omuringo gwa BrightR). Gating ya 0.3 kuhika 6 ns neekoresibwa kukyendeeza aha nshonga z‘enyima.
Okushushanisa obutafaari obutoorainwe omu bwire obwabuzima. Bwanyima y’okushoroora omu nkora ya Neurobasal-A erimu 1% B27 y’okugaita hamwe na 0.5 mM GlutaMax, obutafaari bukabyarwa ahonaaho aha bipande by’endabirwamu ebiine poly-l-lysine (μ-Slide8 Eiziina, Ibidi, enamba ya katalogi 80826) , Kandi bwanyima bukaguma aha ho°C kuhika ahari 3% CO2. okutuurayo. Okuteera ebishushani omu bwire obwabuzima kukakorwa aha kikwato ky‘okucondooza ekya Leica SP8 ekiine laser erikwera, HyD, 63×[1.4 omubaro gw‘okukinga (NA)] amajuta g‘okureeba n‘omutindo gw‘okuhemba.
Embeba ekakyeberwa ahonaaho n‘omuyaga gwa carbon dioxide kandi yasharaho omutwe, obwongo bwaruga omu kifuba ahonaaho, kandi bwasharaho omu 200μm oburaingwa (ahabw‘okugyezesa okuhandiika Fi 13C) nari 275μm oburaingwa (ahabw‘okugyezesa kwa fotoni ibiri) ekicweka kya sagittal ekiijwire ebintu ebi ahaifo cremoam The VHr5. Scientific, Walldorf, Germany) eijwiire ebintu ebi: 125 mM ebirikufuka nk’orubaare, ebirimu kaboni (95% O2 na 5% CO2) Ca2 nkye + amaizi g’omubiri agakozirwe abantu (ACSF) NaCl, 2.5 mM KCl, 1.25 mM bufferte m2, Na2. shukaari, 0.5 mM CaCl2 na 3.5 mM MgCl2 (omuhimbo gw‘okusheeshasheesha ogwa 310 kuhika 330 mmol). Hindurira ebicweka by‘obwongo ebitungirwe omu kishengye ky‘okubanza ekirimu Ca2 + ACSF y‘ahaiguru (125.0 mM NaCl, 2.5 mM KCl, 1.25 mM sodium phosphate buffer, 25.0 mM NaHCO3, 25.0 mM d-glucose, 25.0 mM d-glucose. MgCl2) Omugati) pH 7.4 na 310 kuhika 320 mmol).
Omu bwire bw’okuteera ebishushani, ebicweka bikatwarwa omu kishengye ekihairweyo eky’okuteera ebishushani, kandi okucondooza kukakorwa ahansi y’okuguma nibasheeshasheesha ACSF aha bwire bw’obutagatsi obutarikuhinduka bwa 32° kuhika 33°C. Ekikwato ky‘okukyebera ebiri omu itaka (TCS SP8 MP-OPO, Leica Microsystems) ekirimu ekikwato ky‘okureeba ebiri omu itaka (NA 0.95, amaizi), Ti: Sapphire laser (Chameleon Vision II, Coherent) kikakoresibwa omu kukyebera ebicweka. Omushomo gwa FLIM (Ekicweka ky’Ekicweka 300, Ekicweka ky’Ekicweka).
FLIM ya Grx1-roGFP2. Empindahinduka omu mbeera y‘okuhindura ebiri omu nkora ya PNs zikapimwa FLIM y‘ebishushani bibiri omu bicweka by‘obwongo ebya sagittal, ahu Grx1-roGFP2 biosensor yaabaire neegyenderera PNs. Omu nkora ya PN, omusiri gw‘okutunga nigutooranwa nka 50 kuhika 80 μm ahansi y‘eitaka ry‘ekicweka kureeba ngu hariho PN erikubaasa kukora (nikwo kugira ngu, okubura omutindo gw‘ebirungo nari empindahinduka omu nkora y‘obutafaari bw‘omubiri aha rubaju rw‘ebicweka by‘omubiri) n‘okutegyeka roGFP2 sensor na AAVxexe iRNA sh mCheri). Okuteerana ebishushani by‘omutindo gumwe n‘okutunguura kwa dijitali 2x [oburaingwa bw‘omuyaga gw‘okushemererwa: 890 nm; 512 nm 512 ebishushani]. Okucondooza: HyD y‘omunda, ekibiina ky‘okushwijuma ekya fluorescein isothiocyanate (FITC)] n‘okugyeragyeranisa ebishushani omu dakiika 2 kuhika ahari 3 nibikoresibwa kureeba ngu ebishushani ebirikumara byarundaanwa (ebishushani 1000 omu bwijwire) ahabw‘okuhikaana n‘ebishushani by‘okugoma. Okuhurira kw‘okucondooza kwa Grx1-roGFP2 n‘okuhamya embeera za FLIM kukakorwa barikureeba omuhendo gw‘amagara ga roGFP2 baaba nibongyera exogenous 10 mM H2O2 aha ACSF y‘okusheeshasheesha (okutunguura deoxidation, ekirikureetaho okweyongyera kw‘amagara ga roGFP2), reero bwanyima bakayongyeraho mremizitzo of2). okukyendeeza, ekirikureetaho okukyendeera omu myaka y‘amagara) (ekishushani S8, D kuhika G). Koresa puroguraamu ya FLIMfit 5.1.1 kucondooza ebyarugamu ebitungirwe, hikaana n‘omubaro gw‘okusheeshasheesha kw‘ekishushani kyona aha IRF eyapimirwe (omurimo gw‘okugarukamu kw‘ekikwato), kandi χ2 ni nk‘oku 1. Kubara obwire bw‘amagara ga PN emwe, ekijwaro ky‘okukingira amaisho kikaba kiri omu ngaro obwire bw‘okusheeshasheesha okwehinguririize omutsi . ekirikukoresibwa omu kupima omubaro.
Okushwijuma obushoboorozi bw’omubiri. Bwanyima y‘ekicweka ky‘amaani kukyeberwa na 100 nM TMRM eyongyeirwe butunu aha ACSF erimu okusheeshasheesha kumara edakiika 30, empindahinduka z‘obushoboorozi bw‘emitochondria eza PNs zikapimwa n‘ekikwato ky‘okukyebera ebiri omu itaka (2-photon microscope). Okushushanisa kwa TMRM kukakorwa omu kushemeza ekikwato ky‘okucondooza aha 920 nm kandi barikukoresa HyD y‘omunda (tetramethylrhodamine isothiocyanate: 585/40 nm) kushorooza obumanyiso; omu kukozesa oburaingwa bw‘omuyaga bw‘okusiimura nibwo bumwe kwonka orikukoresa HyD y‘omunda etari emwe n‘emwe (FITC :525/50) kushushanisa mtYFP. Koresa ekikwato kya ImageJ eky‘okubara ebishushani kukyebera obushoboorozi bw‘obutafaari bw‘omubiri aha rurengo rw‘obutafaari bumwe. Omu bugufu, ekibaro ky‘okutaho: akamanyiso = min (mtYFP, TMRM) nikikoresibwa kumanyisa ekicweka ky‘omubiri ekirikworeka akamanyiso ka TMRM omu Purkinje Somali omu kishushani ky‘omutindo gumwe eky‘omuhanda ogurikuhikaana. Bwanyima omwanya gwa pixel omu mask erikurugamu nigubarwa, reero guhindurwa ogwa butoosha aha kishushani ky‘omutindo gumwe ekirikuhikaana n‘omuhanda gwa mtYFP kutunga ekicweka kya mitochondrial ekirikworeka obushoboorozi bwa mitochondrial.
Ekishushani eki kikahindurwa puroguraamu ya Huygens Pro (Ekishushani ky’Ebishushani by’Omubaro gwa Sayansi). Aha bishushani by’ebipande ebikozirwe omu simu, okuteerana kw’ebipande bimwe nikukorwa barikukoresa enkora y’okutunga ebitafaari ebirikwekorera ebirikuheebwa puroguraamu ya LAS-X. Bwanyima y’okugyeragyeranisa ebishushani, kozesa ImageJ na Adobe Photoshop kwongyera kukora aha kishushani kandi ohindure omu muringo gumwe okujwara gye n’okutaana. Koresa Adobe Illustrator kutebeekanisa ebishushani.
mtDNA okuta omutima aha kweshwijuma. Omubaro gw‘ebizimba bya mtDNA gukakyeberwa aha bicweka by‘obwongo ebihandiikirweho obutafaari oburikurwanisa DNA kurabira omu confocal microscope. Buri mwanya ogurikugyenderwaho gukakorwa ahabw‘omubiri gw‘obutafaari n‘omubiri gwa buri butafaari, kandi omwanya ogurikuhikaana gukabarwa barikukoresa ekikwato kya Multi Measure plug-in (ImageJ software). Ihaho omwanya gw’enyukiriya kuruga omu mwanya gw’omubiri gw’obutafaari kutunga omwanya gw’obutafaari bw’omubiri. Eky’aha muheru, ekikwato kya Analyze Particles plug-in (ImageJ software) kikakozesebwa kugyeragyeranisa enshonga za DNA z’omubiri ezirikworeka mtDNA aha kishushani ky’okutandikiraho, kandi ebyarugamu bikahindurwa byaba ebya butoosha kuhika aha PN average y’embeba za CTRL. Ebirugiremu nibyorekwa nk’omubaro gw’ebicweka by’omubiri ebirikwetwa nucleosides ahari buri kahuri.
Okushwijuma oku puroteyini zirikworeka. Koresa ekikwato kya ImageJ eky‘okubara ebishushani kukyebera oku puroteyini zirikworekwa omu PN aha rurengo rw‘obutafaari bumwe. Omu bugufu, omu kishushani ky‘omutindo gumwe eky‘omuhanda ogurikuhikaana, kurabira omu nkora: akamanyiso = min (mtYFP, antibody), ekicweka kya mitochondrial ekirikworeka obutafaari bw‘omubiri oburikurwanisa endwara obumwe omu Purkina nikimanyirwa. Bwanyima omwanya gwa pixel omu mask erikurugamu nigubarwa, reero guhindurwa ogwa butoosha aha kishushani ky‘omutindo gumwe ekirikuhikaana n‘omuhanda gwa mtYFP kutunga ekicweka kya mitochondrial eky‘ekiriisa ekirikworekwa.
Okushwijuma obutafaari bwa Purkinje. Ekikwato kya Cell Counter plug-in ekya ImageJ kikakoresibwa kukyebera oburemeezi bwa Purkinje barikugabanisa omubaro gw‘obutafaari bwa Purkinje obubariirwe n‘oburaingwa bw‘empeta y‘obwongo erikutuurwamu obutafaari obubariirwe.
Okutebeekanisa n’okushorooza eby’okureeberaho. Obwongo kuruga omu guruupu y‘okukyebera n‘embeba za Mfn2cKO bukahamibwa omu 2% PFA/2.5% glutaraldehyde omu 0.1 M phosphate buffer (PB), reero ebicweka by‘omubiri eby‘omubiri bikatebeekanisibwa barikukoresa ebihimba by‘omubiri (Leica Mikrosysteme GmbH, Vienna, Austria) (Obuhango bw‘omubiri 50 kuhika ahari The60). tetraoxide na 1.5% y’ekiriisa kya potasiyumu aha bwire bw’obutagatsi bw’omu kishengye kumara eshaaha 1. Ebicweka ebyo bikayozibwa emirundi eshatu n’amaizi agarimu ebiriisa, bwanyima byateebwaho erangi 70% ethanol erimu 1% uranyl acetate kumara edakiika 20. Ebicweka ebyo bikarugaho byarugaho amaizi omu by’omwonyo ebitebeekanisiibwe kandi byateebwa omu nshaho ya Durcupan ACM (Araldite casting resin M) epoxy resin (Electron Microscopy Sciences, enamba y’akatabo 14040) ahagati y’ebipande by’endabirwamu ebiine silikoni, kandi aha muheru aha 60°C Polymerize48 oven. Ekicweka ky‘omubiri gw‘omutwe kikatooranwa kandi ebicweka by‘omubiri ebikye munonga ebya 50 nm bikasharaho aha Leica Ultracut (Leica Mikrosysteme GmbH, Vienna, Austria) kandi byatooranwa aha rubaaho rw‘omuringa rwa mm 2×1 orurimu ebiriisa by‘omuringa ebiteirweho firimu ya polystyrene. Ebicweka bikateebwaho erangi n’omubazi gwa 4% uranyl acetate omu H2O kumara edakiika 10, byayozibwa na H2O emirundi mingi, bwanyima byayozibwa na Reynolds lead citrate omu H2O kumara edakiika 10, bwanyima byanaabibwa na H2O emirundi mingi. Ebishushani ebikye bikakwatwa n‘ekikwato ky‘okujanjaaza ebishushani ekya Philips CM100 (Thermo Fisher Scientific, Waltham, MA, USA) barikukoresa kamera ya dijitali eya TVIPS (Tietz vidiyo n‘ebishushani) TemCam-F416 (TVIPS GmbH, Gauting, USA). Bugirimaani).
Aha mbeba ezaabaire ziine AAV, obwongo bukataanisibwa kandi bwasharamu ekicweka kya sagittal ekiine oburaingwa bwa mm 1, kandi cerebellum ekakyeberwa barikukoresa fluorescence microscope kubaasa kumanya empeta erimu AAV (nikwo kugira ngu, mCherry expressing). Okugyezesa kwonka oku okutaho AAV kurikurugamu okuhindura kw‘ahaiguru munonga kw‘ekicweka ky‘obutafaari bwa Purkinje (nikwo kugira ngu haihi ekicweka kyona) omu mpeta z‘obwongo haakiri ibiri ezirikukuratana nizo zirikukoresibwa. Orubaju orurikuhindurwa AAV rukakyeberwa omu bwire bw‘ekiro bwanyima y‘okukihamya (4% PFA na 2.5% glutaraldehyde omu 0.1 M cocoate buffer) kandi rwagaruka rwakorwaho. Aha kutaho EPON, enyama egumire ekanaabwa n’omubazi gw’okukyebera 0.1 M (Applichem), kandi yaateebwa omu 2% OsO4 (os, Obuheereza bwa sayansi; Caco) omu 0.1 M y’omubazi gw’okukyebera (Applichem) Eshaha 4, bwanyima yaanaabwa kumara eshaaha 2. Garuka okore emirundi 3 n‘ekirungo kya cocamide 0.1 M. Okurugirira ahari ekyo, omubaro gw’okutemba ogwa ethanol gukakoresibwa kuteeka buri nshonga ya ethanol aha 4°C kumara edakiika 15 kubaasa kukyendeeza amaizi omu nyama. Enyama egi ekatwarwa omu nshaho y’okukyebera kandi yaateebwa omu EPON (Sigma-Aldrich) ekiro kyona ahari 4°C. Teeka enyama omu EPON ensya aha butagasi bw‘omu kishengye kumara eshaaha 2, reero ogite aha 62°C kumara eshaaha 72. Koresa ekikwato ky’okukyebera ebiri omu itaka (Leica Microsystems, UC6) n’omusyo gw’eibaare ry’omuhendo (Diatome, Biel, Switzerland) kutema ebicweka by’omubiri ebikye munonga eby’oburaingwa bwa 70 nm, kandi oshukyeho erangi n’ekiriisa kya uranyl 1.5% kumara edakiika 15 ahari 37°C, kandi oshukyeho erangi n’omubazi gw’ekiriisa kya lead edakiika 4. Ebishushani by’eby’amashanyarazi bikakwatwa barikukoresa ekikwato kya JEM-2100 Plus ekirimu eby’okureeberaho by’eby’amashanyarazi (JEOL) ebiine kamera OneView 4K 16-bit (Gatan) hamwe na puroguraamu ya DigitalMicrograph (Gatan). Okushwijuma, ebishushani by’eby’amashanyarazi bikatunga 5000× nari 10,000× zoom ya dijitali.
Okushwijuma enkora y’ebicweka by’omubiri ebirikwetwa mitochondria. Aha kweshwijuma kwona, enkora za mitochondria z‘omuntu ahabwe zikaba zihandiikirwe omu ngaro omu bishushani bya dijitali barikukoresa puroguraamu ya ImageJ. Ebintu ebitarikushushana eby’enkora y’ebigambo nibishwijumwa. Oburemeezi bwa mitochondria nibumanyisibwa nk’ebicweka 10 ahari igana ebirikutunga okugabanisa omwanya gwona ogwa mitochondria ogwa buri butafaari n’omwanya gwa cytoplasm (omwanya gwa cytoplasm = omwanya gw’obutafaari-omwanya gw’obutafaari bw’omubiri) × 100. Okwetoorora kwa mitochondria nikubarwa n’omuringo [4π∙2(area)]./perimeter. Enkora ya ista eya mitochondria ekakyeberwa kandi yaabaganisibwamu ebicweka bibiri (“tubular” na “blister”) kurugiirira aha nkora zaabo enkuru.
Omubaro gw‘eby‘okurya/eby‘okurya by‘omubiri n‘okushwijuma oburemeezi. Koresa puroguraamu ya ImageJ kuhandiika omu ngaro enkora za buri kimwe ekirikukora aha nshonga z‘omubiri (autophagosome/lysosome) omu kishushani kya dijitali. Ekicweka kya autophagosome/lysosome nikyorekwa nk‘ebicweka 10 ahari 100 ebirikubarwa orikugabanisa ekicweka kyona eky‘omutindo gwa autophagosome/lysosome ekya buri butafaari n‘ekicweka kya cytoplasm (ekicweka kya cytoplasm=ekicweka ky‘obutafaari-ekicweka ky‘omubiri)×100. Oburemeezi bw‘ebicweka by‘omubiri ebirikurya ebiriisa/ebicweka by‘omubiri ebirikukora nk‘ebiriisa nibubarwa orikugabanisa omubaro gwona n‘omubaro gw‘ebicweka by‘omubiri ebirikurya ebiriisa/ebicweka by‘omubiri ahari buri butafaari (omu kureeba omwanya gw‘obutafaari) (omwanya gw‘obutafaari = omwanya gw‘obutafaari-omwanya gw‘enyukiriya).
Okuhandiika aha kusharaho kw‘amaani n‘okutebeekanisa sampo. Aha kugyezesa okurikwetengyesa okuhandiika shukaari, hindura ebicweka by‘obwongo ebiine obuhuta omu kishengye ky‘okubanza ky‘okukyebera, ekirimu kaboni eijwiire (95% O2 na 5% CO2), Ca2 + ACSF y‘ahaiguru (125.0 mM NaCl, 2.5 mM KCl, 1.25 mM sodium m3O phosphate, 1.25 mM sodium phosphate. 25.0 mM d-shukari, 1.0 mM CaCl 2 na 2.0 mM MgCl 2, ebihindwirwemu pH 7.4 na 310 kuhika 320 mOsm), omu shukaari eri 13 C 6- Okuhindura shukaari (Eurisotop, enamba y‘akatabo69-1). Aha kugyezesa okurikwetengyesa okuhandiika kwa pyruvate, hindura ebicweka by‘obwongo ebiine obuhuta bw‘amaani kuza omu Ca2 + ACSF y‘ahaiguru (125.0 mM NaCl, 2.5 mM KCl, 1.25 mM sodium phosphate buffer, 25.0 mM NaHCO3, 25.0 mM Ad2M d-glucose, Ca00). MgCl2, hindura aha pH 7.4 na 310 kuhika ahari 320mOsm), kandi otemu 1mM 1-[1-13C]pyruvate (Eurisotop, enamba y‘akatabo CLM-1082). Ebicweka ebyo obite omu nkungu kumara edakiika 90 aha 37°C. Aha muheru gw’okugyezesa, ebicweka bikayozibwa ahonaaho n’omubazi gw’amaizi (pH 7.4) ogurimu 75 mM ammonium carbonate, reero byahindurwa omu 40:40:20 (v:v:v) asetonitiri (ACN): methanol: amaizi. Bwanyima y’ebicweka ebyo kuteebwa aha ice kumara edakiika 30, eshampu zikaba ziteirwe omu centrifuge aha 21,000 g kumara edakiika 10 ahari 4°C, kandi ebiri aha ice ebirikwera bikayomezibwa omu SpeedVac concentrator. Ekirugiremu ekirungo kya metabolite ekyomire kikabiikwa aha -80°C kuhitsya obu kyakyeberwa.
Okushwijuma okuri omu maizi-okushwijuma okuri omu nkora ya amino acid 13 eziine akamanyiso ka C. Aha kucondooza kw’amaizi (LC-MS), ekirungo ky’eby’okurya kikagaruka kyateebwa omu 75μl y’amaizi g’omutindo gwa LC-MS (Honeywell). Bwanyima y’okukyebera aha 21,000 g kumara edakiika 5 ahari 4°C, 20 μl y’ekirungo ekishobooroirwe kikakozesebwa omu kucondooza aha amino acid flux, obwo ekicweka ekisigaireho eky’okushohoza kyakoresibwa ahonaaho omu kucondooza aha anion (reeba ahaifo). Okushwijuma kwa amino acid kukakorwa barikukoresa enkora y‘okushohoza benzoyl chloride eyashobooroirwe enyima (55, 56). Omu nkora y‘okubanza, 10 μl y‘ekiriisa kya 100 mM (Sigma-Aldrich) ekateebwa omu 20 μl y‘ekirungo ky‘eby‘okurya, reero 10 μl y‘ekiriisa kya 2% (Sigma-Aldrich) ekateebwa omu ACN y‘omutindo gwa LC. Sampuli ekakyeberwa omu bwire bukye reero bwanyima yakyeberwa aha 21,000 g kumara edakiika 5 ahari 20°C. Hindurira ebiri aha mutwe ebishemeziibwe omu kacupa ka miriyoni 2 aka autosampler akarimu ekikwato ky‘okutaho endabirwamu y‘ekibunu (200 μl volume). Sampuli zikeeberwa barikukoresa enkora ya Acquity iClass ey’amaani munonga (Amaizi) erikukwatanisa n’ekikwato kya Q-Exactive (QE)-HF (Ultra High Field Orbitrap) eky’omutaano eky’omutaano (Thermo Fisher Scientific). Okweshwijuma, 2μl y’eky’okureeberaho ekyarugiremu ekateebwa omu 100×1.0 mm ey’amaani maingi eya silika T3 column (Amaizi) erimu obucweka bwa 1.8μm. Omubaro gw‘okusheeshasheesha ni 100μl/edakiika, kandi enkora y‘okukyebera erimu okukyebera A (10 mM ammonium formate na 0.15% formic acid omu maizi) n‘okukyebera B (ACN). Omubaro gw‘okutunguuka nigushushana n‘ogu: 0%B aha dakiika 0; 0%B. 0 kuhika 15% B aha dakiika 0 kuhika 0.1; 15 kuhika 17% B aha dakiika 0.1 kuhika 0.5; B aha 17 kuhika 55% aha dakiika 0.5 kuhika 14; B aha 55 kuhika 70% aha dakiika 14 kuhika 14.5; aha 14.5 kuhika 70 kuhika 100% B aha dakiika 18; 100% B omu dakiika 18 kuhika 19; 100 kuhika 0% B aha dakiika 19 kuhika 19.1; 0% B aha dakiika 19.1 kuhika 28 (55, 56). Ekipimo ky’oburemeezi bwa QE-HF nikikora omu muringo gw’okuhindura ionization nungi n’omubaro gw’oburemeezi bwa m/z (omubaro gw’oburemeezi/okushashura) gwa 50 kuhika 750. Okushoboorora okurikukoresibwa ni 60,000, kandi ekigyendererwa ky’okutegyeka okutunga (AGC) ion ekirikutunga ni 3×106, kandi obwire bwa ion oburikukirayo ni millise1. Enshonga y‘okufuuhirira ey‘okwotsya (ESI) neekora aha amaani g‘okufuuhirira ga 3.5 kV, obutagatsi bw‘omubiri oburikwetwa capillary 250°C, omuyaga gw‘omu sheath ogwa 60 AU (ebicweka ebitarikushushana), n‘omuyaga gw‘okuhwera ogwa 20 AU. 250°C. S lensi eteirwe aha 60 AU.
Okushwijuma kwa anion-MS okwa 13C okuhandiikirweho asidi z‘ebihangirwe. Ekicweka ky’omubiri ekyasigaireho (55μl) kikakyeberwa barikukoresa enkora ya Dionex ion chromatography (ICS 5000+, Thermo Fisher Scientific) erikukwatanisa n’ekikwato ky’okupima obuhango bw’ebintu (QE-HF). Omu bugufu, 5μl y‘ebirungo by‘omubiri bikateebwa omu nkora ya Dionex IonPac AS11-HC erimu HPLC (2 mm×250 mm, obuhango bw‘ebicweka 4μm, Thermo Fisher Scientific) omu muringo gw‘okuteera omu nkora y‘ekicweka ky‘omubiri n‘omubaro gw‘okujwisa 1. )aha. Enyomyo y‘okurinda eya Dionex IonPac AG11-HC (mm 2 x mm 50, mm 4, ey‘omujubi wa sayansi ey‘okwotsya). Ebiriisa by’omu nkora nibiguma biri ahari 30°C, kandi autosampler neeteebwa ahari 6°C. Kozesa akacupa ka potassium hydroxide akaheebwa amaizi agarimu deionized kureetaho potassium hydroxide gradient kurabira omu nkora y‘okureetaho ebiriisa. Okwahukana kw‘ebintu ebirikuhindura omubiri aha sipiidi y‘okusheeshasheesha kwa 380μl/min, orikukoresa enkora egi: edakiika 0 kuhika ahari 3, 10 mM KOH; Edakkika 3 kuhika 12, 10 kuhika 50 mM KOH; Edakkika 12 kuhika 19, 50 kuhika 100 mM KOH; Edakkika 19 kuhika 21 , 100 mM KOH; 21 kuhika 21.5 edakiika, 100 kuhika 10 mM KOH. Enyomyo ekagaruka yagyeragyeranisibwa ahansi ya 10 mM KOH kumara edakiika 8.5.
Ebirungo by‘omubiri ebishohoziibwe nibigambwaho n‘omugyera gw‘okugaita isopropanol ogwa 150μl/min bwanyima y‘okuteera enyomyo reero bwanyima bigyendererwa aha nkora y‘okupima obuhango bw‘ebintu ey‘omutindo gw‘ahaiguru erikukora omu muringo gw‘okukyendeeza aha ionization. MS eriyo neereeberera oburemeezi kuruga ahari m/z 50 kuhika ahari 750 n’okushoboorora kwa 60,000. AGC neeteebwa ahari 1×106, kandi obwire bw’ayoni oburikukirayo nibuguma ahari 100 ms. Enshonga ya ESI erikwotsya ekaba neekora aha amaani g‘okufuuhirira ga 3.5 kV. Ebindi ebitebeekanisiibwe eby’entandikwa ya ion ni nk’ebi: obutagatsi bwa capillary 275°C; okutambura kwa gaasi y’omu kifuba, 60 AU; okutambura kwa gaasi okurikuhwera, 20 AU ahari 300°C, n‘okutaho S lensi ahari 60 AU.
Okushwijuma amakuru g‘ebintu ebihandiikirweho 13C. Kozesa puroguraamu ya TraceFinder (omuringo 4.2, Thermo Fisher Scientific) ahabw‘okushwijuma amakuru g‘omubaro gw‘ebicweka by‘omubiri. Okumanya buri kirungo kukahamibwa ekirungo ky‘okureeberaho ekirikwesigwa kandi kyashwijumwa kyonka. Okubaasa kukora okucondooza kw’okugaita isotope, omwanya gw’okushohoza ion chromatogram (XIC) ya buri isotope ya 13C (Mn) gukaihwaho kuruga omu [M + H] +, ahu n eri namba ya kaboni y’ekirungo ekigyendereirwe, erikukoresibwa kucondooza amino acids nari [ MH] + erikukoresibwa kucondooza anions. Obuhikire bwa mass bwa XIC buri ahansi y‘ebicweka bitaano ahari buri miriyoni, kandi obuhikire bwa RT buri edakiika 0.05. Okushwijuma okugaigahaza nikukorwa omu kubara omubaro gwa buri isotope erikushangwa n’omubaro gwa isotope zoona ez’ekirungo ekirikuhikaana. Ebipimo ebi nibiheebwa nk’ebicweka bya buri isotopu, kandi ebyarugamu nibyorekwa nk’okugaita ebicweka bya molar (MPE), nk’oku kyashoboororwa enyima (42).
Ekirungo ky’obutafaari bw’omubiri ekyabaire kifukiire kikahindurwa kimwe omu 80% methanol (v/v) erikwotsya nk’orubaare, kyahindurwa, kandi kyateebwa omu -20°C kumara edakiika 30. Garuka ohindure sampo kandi ogite aha +4°C kumara edakiika 30. Sampuli ekateebwa omu centrifuge aha 21,000 g kumara edakiika 5 ahari 4°C, reero ebyarugiremu bikatorotoorwaho kandi byayomezibwa barikukoresa ekikwato kya SpeedVac concentrator ahari 25°C kubaasa kukyebera bwanyima. Nkoku kyashoboororwa ahaiguru, okucondooza kwa LC-MS kukakorwa aha amino acids z’obutafaari obutoorainwe. Okukozesa TraceFinder (omuringo 4.2, Thermo Fisher Scientific), okucondooza kw‘ebihandiiko kukakorwa barikukoresa oburemeezi bw‘ekicweka kimwe kya buri kirungo. Okuhindura ebiri omu nkora y‘okuhindura ebiri omu mubiri kukakorwa barikukoresa puroguraamu ya preprocessCore (57).
Okutebeekanisa ebicweka. Embeba ekakyeberwa ahonaaho n’omuyaga gwa kaboni kandi yaasharaho omutwe, obwongo bwaihwaho ahonaaho kuruga omu kifuba, kandi omusyo ogurikutengeeta oguijwire orubaare (HM-650 V, Thermo Fisher Scientific, Walldorf, Germany) gukakoresibwa kugisharaho omu bicweka 300 kuhika 375 μ% eby’omuyaga O9 sa5d. CO2) Ca2 + ACSF nkye (NaCl 125.0 mM, KCl 2.5 mM, ekirungo kya sodium phosphate 1.25 mM, NaHCO3 25.0 mM, d-shukari 25.0 mM, CaCl2 1.0 mM na Adju 6.0 mM kuhika ahari pH3702. mOsm). Hindurira ebicweka by‘obwongo ebitungirwe omu kishengye ekirimu Ca2 + ACSF y‘ahaiguru (NaCl 125.0 mM, KCl 2.5 mM, ekirungo kya sodium phosphate 1.25 mM, NaHCO3 25.0 mM, d-glucose 25.0 mM, pH2 mM 4.0 mM Ca3l5C. na 310 kuhika ahari 320 mOsm). Biika ebicweka ebyo kumara edakiika 20 kuhika ahari 30 kugira ngu bigarurweho otakabihandiikire.
okuteera akatambi. Eitembezo rya microscope eririmu ekishengye ky’okuhandiikamu ekigumire n’ekishushani ky’okuta omu maizi 20x (Scientifica) kikakoresibwa omu kuhandiika kwona. Obutafaari bwa Purkinje oburikuteekateekwaho bukamanyirwa aha (i) obuhango bw‘omubiri, (ii) omwanya gw‘omubiri gw‘obutafaari bw‘omubiri obukye, na (iii) okureebeka kw‘omubazi gw‘okumanyisa mtYFP ogurikujwara omushana. Pipeti y‘ekipande erimu obuzibu bw‘okuzibira 5 kuhika ahari 11 megohms neekururwaho n‘ekipande ky‘endabirwamu y‘ekyoma (GB150-10, 0.86 mm×1.5 mm×100 mm, Ebintu bya sayansi, Hofheim, Bugirimaani) n‘ekipande ky‘okupiira ekirikuhikaana n‘ebikwato (P-1000, No). Ebihandiiko byona bikakorwa omu nkora ya ELC-03XS npi patch clamp amplifier (npi eby‘amashanyarazi GmbH, Tam, Germany), ebyabaire nibitegyekwa puroguraamu ya Signal (omuringo 6.0, eby‘amashanyarazi, Cambridge, Bungyereza). Okugyezesa kukahandiikwa aha nkora y‘okutoorana 12.5 kHz. Ekimanyiso nikishwijumwa n‘ebishwijumwa bibiri bya Bessel ebirikuraba omu bwire bukye kandi ebiine emirundi y‘okusharaho 1.3 na 10 kHz. Obushoboorozi bw’ekibunu n’ekipiira niburihirwa omu nkora y’okurihiirira erikukoresa ekirikutunguura. Okucondooza kwona kukakorwa ahansi y’obushoboorozi bwa kamera ya Orca-Flash 4.0 (Hamamatsu, Gerden, Bugirimaani), eyabaire neetegyekwa puroguraamu ya Hokawo (omuringo 2.8, Hamamatsu, Gerden, Bugirimaani).
Okutebeekanisa n’okushwijuma obutafaari bwona okwa buriijo. Ahonaaho otakahandiikire, ijuza ekipimo ky’omubazi gw’omunda ogurimu ebintu ebi: 4.0 mM KCl, 2.0 mM NaCl, 0.2 mM EGTA, 135.0 mM potassium gluconate, 10.0 mM Hepes, 4.0 mM Guphano.TPGsine mM) (Na) na 10.0 mM ebiriisa by’omubiri bikahindurwa byahika ahari pH 7.25, kandi omuhimbo gw’okusheeshasheesha gukaba guri 290 mOsm (sukrose). Ahonaaho bwanyima y‘okutaho amaani ga 0 pA kuhendagura omubiri, obushoboorozi bw‘omubiri oburikuhuumura bukapimwa. Okuzibira kw‘okuta omu nkora nikupimwa barikukoresa amashanyarazi g‘okukyendeeza munonga -40, -30, -20, na -10 pA. Pima obuhango bw’okugarukamu kw’amaani kandi okozese ekiragiro kya Ohm kubara okuzibira kw’okutaaha. Emirimo y‘okwemanyiza ekahandiikwa omu nkora y‘okukwatiraho amashanyarazi kumara edakiika 5, kandi sPSC ekamanyirwa kandi yapimwa omu Igor Pro (omuringo 32 7.01, WaveMetrics, enyanja ya Oswego, Oregon, USA) barikukoresa ekihandiiko ky‘okumanya ekirikukoresibwa omu bwire bw‘okwemanyiza. IV curve na steady-state current nibipimwa barikukwaata battery aha bushoborozi butari bumwe na bumwe (okutandika na -110 mV) n‘okwongyera aha amaani omu matembezo ga 5 mV. Okukora AP kukagyezibwa barikukoresa amashanyarazi g’okukyendeeza aha nkora ya depolarizing. Kwata akacweka ka -70 mV obwo orikukoresa omuvuyo gw‘okukyendeeza aha nkora. Tebeekanisa obuhango bw‘eitembezo rya buri kicweka ky‘okuhandiika kyonka (10 kuhika 60 pA). Barira emirundi ya AP erikukirayo orikubara omu ngaro ebipimo by‘okuteera ebirikureetaho emirundi ya AP erikukirayo. Ekipimo kya AP nikikyeberwa barikukoresa ekirugire omu nkora ya kabiri ey‘okuteerana (depolarization pulse) ekirikubanza kureetaho AP emwe nari nyingi.
Okutebekanisa n’okushwijuma ebipande ebiine ebitobo. Kora okuhandiika kw’ebicweka ebiine ebitobo orikukoresa enkora z’omutindo. Koresa ekikopo ekitarimu ATP- na GTP ekitarimu ebirungo ebi: 128 mM shukaari K, 10 mM KCl, 10 mM Hepes, 0.1 mM EGTA na 2 mM MgCl2, kandi ohindure pH 7.2 (orikukoresa KOH). ATP na GTP niziruga omu nkora y’omunda y’obutafaari kubaasa kuzibira okutaaha omu nkora y’obutafaari obutarikutegyekwa. Pipeti y‘okutaho ekipande neeijuzibwa omubazi gw‘omunda ogurimu amphotericin (enkumi 200 kuhika 250 μg/ml; G4888, Sigma-Aldrich) kutunga ekihandiiko ky‘ekipande ekirimu ebitobo. Amphotericin ekaba erimu omubazi gw’okukyebera (omubaro gw’aha muheru: 0.1 kuhika 0.3%; DMSO; D8418, Sigma-Aldrich). Okuteerana kwa DMSO okwakozesiibwe kukaba kutaine ki kwakozire aha nkora y‘obutafaari bw‘omubiri obucondooziibwe. Omu bwire bw‘okuteera ebikoni, okuzibira kw‘omuhanda (Ra) kukaguma nikukyeberwa, kandi okugyezesa kukatandika bwanyima y‘okuhamira aha nkora ya Ra na AP (edakiika 20-40). Emirimo y‘okwetunguura neepimwa omu nkora y‘okuteera amashanyarazi n‘/nari ekikwato ky‘okuteera amashanyarazi kumara edakiika 2 kuhika ahari 5. Okushwijuma amakuru kukakorwa barikukoresa Igor Pro (omuringo gwa 7.05.2, WaveMetrics, USA), Excel (omuringo gwa 2010, ekitongore kya Microsoft, Redmond, USA) na GraphPad Prism (omuringo gwa 8.1.2, puroguraamu ya GraphPad Inc., La Jolla, CA). Amerika). Okubaasa kumanya APs ezirikwetunguura, ekikwato kya IgorPro ekya NeuroMatic v3.0c nikikoresibwa. Okumanya APs omu muringo gw‘okwemanyiza orikukoresa omutindo oguhairwe, ogurikuhindurwa buri omwe ahari buri rikodi. Okwejunisa obwire bw‘okusiika, manya emirundi y‘okusiika n‘emirundi y‘okusiika erikukirayo omu bwire bukye n‘emirundi y‘okusiika eri ahagati.
PN okwetaanisa aha bandi. Omu kuhindura omu nkora eyaabaire eshohoziibwe enyima, PNs zikashemezibwa kuruga omu cerebellum y‘embeba aha rurengo orumanyirwe (58). Omu bugufu, obutafaari bw‘omubiri obukye bukasharaho kandi bwasharara omu nkora y‘okutaana erikwotsya nk‘orubaare [obutarimu HBSS Ca2+ na Mg2+, bwayongyerwamu shukaari 20 mM, penisilini (50 U/ml) na streptomycin (0.05 mg/ ml)], reero bwanyima bwaganywa· omubazi ogwo na mgHC-cysteine ​​omu papal1. / miriyoni), papaini (16 U / ml) na DNase I (DNase I; 0.1 mg/ml)] Tambira kumara edakiika 30 aha 30°C. Okubanza oyoze enyama omu nkora ya HBSS erimu omubazi gw‘amahuri (10 mg/ml), BSA (10 mg/ml) na DNase (0.1 mg/ml) aha bwire bw‘obutagatsi bw‘omu kishengye okuzibira okugaaya kw‘enzigye, reero omu nkora ya HBSS erimu shukaari 20 mM), okushenda mpora mpora omu HBmylm0. (0.05 mg/miriyoni) na DNase (0.1 mg/miriyoni) nizirekura obutafaari bumwe. Obutafaari oburugiremu bukakyeberwa kurabira omu nkora y‘okukyebera obutafaari bwa 70μm, bwanyima obutafaari bukakyeberwa omu nkora y‘okukyebera (1110 rpm, edakiika 5, 4°C) kandi bwagaruka bwakyeberwa omu nkora y‘okushoroora [HBSS, eyongyeirwemu shukaari 20 mM, 20% fetal stremylin/peptomylin ) Serum, 1110 rpm, edakiika 5, 4°C). (0.05 mg/miriyoni)]; okushwijuma okubaasa kw‘obutafaari n‘okukoresa propidium iodide kandi ohindure oburemeezi bw‘obutafaari kuhika ahari 1×106 kuhika ahari 2×106 obutafaari/ml. Flow cytometry etakatandikire, ekirungo kikaba kishwijumwa kurabira omu nkora y‘okushwijuma obutafaari bwa 50 μm.
Ekipimo ky’okusheeshasheesha. Okushoroora obutafaari kukakorwa aha 4°C barikukoresa ekyoma kya FACSAria III (BD Biosciences) na puroguraamu ya FACSDiva (BD Biosciences, omuringo 8.0.1). Obutafaari oburikwemerera bukashoroorwa barikukoresa ekikwato kya 100 μm ahansi y‘omuhimbo gwa 20 psi aha sipiidi ya ~2800 events/sec. Ahabw‘okugira ngu enkora y‘obuhangwa ey‘okutaho omuryango (obuhango bw‘obutafaari, okushoroora kw‘emiringo ebiri, n‘emitwarize y‘okujanjaara) terikubaasa kuhamya okwetaanisa okuhikire okwa PN kuruga omu miringo endiijo y‘obutafaari, enkora y‘okutaho omuryango neeteebwaho kurugiirira aha kugyeragyeranisa okuhikire kw‘amaani ga YFP n‘okweshushaniriza omu mitoYFP+ ​​n‘okutegyeka mitoYFP −ce. YFP neeshemererwa ahabw’okujanjaaza sampo n’omurongooti gwa laser ogwa 488 nm, kandi akamanyiso nikamanyirwa aha kukoresa ekicweka ky’okushwijuma band pass ekya 530/30 nm. Omu mbeba za mitoYFP+, amaani g‘okugyeragyeranisa ag‘omubazi gw‘okumanyisa Rosa26-mitoYFP nago nigakoresibwa kutaanisa omubiri gw‘obutafaari bw‘omubiri n‘ebicweka bya axon. 7-AAD neeshemererwa na laser ya kyenju eya 561 nm kandi neekyeberwa n’ekicweka ky’okushwijuma bandpass ekya 675/20 nm kubaasa kubinga obutafaari obufiire. Okubaasa kutaanisa obutafaari bw’enyonyoozi omu bwire nibwo bumwe, ekicweka ky’obutafaari kikateebwaho erangi ya ACSA-2-APC, bwanyima sampo ekakyeberwa n’omurongooti gwa laser ogwa 640 nm, kandi ekicweka ky’okushwijuma ekya 660/20 nm ekya bandpass kikakoresibwa kureeba akamanyiso.
Obutafaari obwarundainwe bukateebwa omu nshaho (1110 rpm, edakiika 5, 4°C) kandi bwabiikwa aha -80°C kuhitsya obu bwakoresiibwe. Embeba za Mfn2cKO n‘embeba zaazo z‘okuzaara nizishoroorwa aha izooba rimwe kukyendeeza aha kuhinduka kw‘enkora. Okworeka n‘okushwijuma amakuru ga FACS kukakorwa barikukoresa puroguraamu ya FlowJo (FlowJo LLC, Ashland, Oregon, USA).
Nkoku kyagambirweho ahaiguru (59), PCR y‘obwire obwabuzima neekoresibwa kutaanisa DNA kuruga omu nkora y‘obutafaari obutoorainwe ahabw‘okupima mtDNA okurikukuratiraho. Okuhurira kw’omutindo n’okuhurira kw’omutindo bikabanza byakyeberwa barikukoresa qPCR aha mibaro y’obutafaari etari emwe n’emwe. Omu bugufu, teerana PN 300 omu nkora y‘okusheeshasheesha erimu tris-HCl 50 mM (pH 8.5), EDTA 1 mM, Tween 20 0.5% na puroteyini K (200 ng/ml) kandi obite aha 55°C edakiika 120. Obutafaari bukagaruka bwateebwa omu 95°C kumara edakiika 10 kureeba ngu puroteyini K yaakora kimwe. Sayansi, enamba y’ekitabo Mm04225274_s1), mt-Nd6 (Ekitabo ky’eby’obuhangwa, enamba y’ekitabo AIVI3E8) na 18S (Eby’obuhangwa Hs99999901_s1) ebiri omu nkora y’obuzaarwa.
Okutebeekanisa sampo ya puroteyini. Omu kuhemba omubazi aha 95°C kumara edakiika 10 n’okuteera akaari, omu nkora y’okusheeshasheesha [6 M, 10 mM tris(2-carboxyethyl) phosphine hydrochloride, 10 mM chloroacetamide na 100 mM tris-HC Lyse pellet omu nkora y’okusheeshasheesha]. Aha Bioruptor (Diagenode) kumara edakiika 10 (obwire bw‘okuhuumuraho bwa sekondi 30 / obwire bw‘okuhuumuraho bwa sekondi 30). Eky’okureeberaho kikakyeberwa 1:10 omu 20 mM tris-HCl (pH 8.0), kyataburwamu 300 ng zaabu ya trypsin (Promega), kandi kyateebwa omu nkungu ekiro kyona ahari 37°C kubaasa kutunga okugabura okuhikire. Aha kiro kya kabiri, sampo ekakyeberwa aha 20,000 g kumara edakiika 20. Ekirungo ky’ahaiguru kikaba kiine 0.1% ya formic acid, kandi omubazi gukakyeberwa n’ebintu bya StageTips ebyayekoreire. Sampuli ekayomezibwa omu kikwato kya SpeedVac (ekikwato kya Eppendorf ekirikuteerana hamwe na 5305) ahari 45°C, reero bwanyima peptide ekahanikwa omu 0.1% ya formic acid. Sampuli zoona zikatebeekanisibwa omurundi gumwe n’omuntu omwe. Okubaasa kukyebera eby’okureeberaho by’enyonyoozi, 4 μg y’ebicweka by’omubiri ebitarimu omunyugunyu bikahandiikwaho akamanyiso k’oburemeezi bw’omubiri (TMT10plex, enamba ya katalogu 90110, Thermo Fisher Scientific) n’omubaro gw’ebicweka by’omubiri ebiine omunyugunyu gwa 1:20. Aha kuhandiika TMT, 0.8 mg y’ekirungo kya TMT ekagaruka yaateebwa omu 70 μl ya ACN etaine maizi, kandi peptide eyomire ekagaruka yaateebwa omu 9 μl ya 0.1 M TEAB (triethylammonium bicarbonate), ahu 7 μl y’ekirungo kya TMT omu ACN yayongyerwamu. Okuta omutima aha nshonga kukaba kuri 43.75%. Bwanyima y’okumara edakiika 60 z’okukyebera, ekirungo eki kikazimwa na 2 μl ya 5% hydroxylamine. Ebirungo ebihandiikirweho akamanyiso bikatorotoorwaho, byayomibwa, byagaruka byateebwa omu 200μl ya 0.1% formic acid (FA), byabaganisibwamu bibiri, reero byarugaho omunyugunyu barikukoresa StageTips ezaakozirwe ahabwabo. Okukozesa ekikwato ky’okupima amaizi g’omubiri ekya UltiMate 3000 eky’amaani maingi munonga (ekikwato ky’okupima amaizi g’omubiri eky’amaani munonga ekya UltiMate 3000), kimwe aha bicweka bibiri kikabaganisibwa aha nkora y’okupima amaizi eya Acquity eya 1mm x 150mm eijwiire obucweka bwa C18 130Å1.7μm (NoWaters, cataKU6050). Omujubi w’eby’obuhangwa). Taana peptides aha nkora y‘okusheeshasheesha ya 30μl/min, taanisa kuruga ahari 1% kuhika ahari 50% buffer B kumara edakiika 85 n‘okutambura kw‘eitembezo ry‘edakiika 96, kuruga ahari 50% kuhika ahari 95% buffer B kumara edakiika 3, bwanyima edakiika 8 ahabwa 95 % Buffer B; Ekirungo A ni 5% ACN na 10 mM ABC, kandi ekirungo B ni 80% ACN na 10 mM ABC. Kurundaana obucweka buri dakiika 3 kandi obite omu guruupu ibiri (1 + 17, 2 + 18, n’ebindi) kandi obyome omu nkora ya vacuum centrifuge.
Okushwijuma kwa LC-MS/MS. Aha bikwatiraine n’okupima obuhango bw’ebintu, ebicweka by’omubiri (enamba r119.aq) bikataanisibwa aha nkora y’okucondooza eya PicoFrit ey’omunda ey’oburaingwa bwa sentimita 25, 75 μm (ekishushani kisya ky’ekigyendererwa, enamba y’ekicweka PF7508250) erimu 1.9 μm ReproSil-Pur C. EASY-nLC 1200 (Ekitongore ky’eby’obuhangwa, Bugirimaani). Enyomyo ekaba eguma eri aha 50°C. Buffers A na B ni 0.1% omu maizi na 0.1% omu 80% ACN, nk’oku biri. Peptides zikaba zitaana n‘omutindo gw‘okutunguuka ogwa 200 nl/min kuruga ahari 6% kuhika ahari 31% buffer B kumara edakiika 65, kandi kuruga ahari 31% kuhika ahari 50% buffer B kumara edakiika 5. Ebirungo ebyabaire bikyebeirwe bikakyeberwa aha kikwato ky’okupima obuhango bw’ebintu (Thermo Fisher Scientific). Okupima m/z y‘okubanza kwa peptide nikukorwa n‘okushoboorora kwa 120,000 omu nkora ya 350 kuhika 1500 m/z. Okukozesa 27% y‘amaani g‘okuteerana agatebeekanisiibwe, ekirikubanza ekirikukirayo amaani ekiine embeera y‘okushashura 2 kuhika ahari 6 nikitooranwa ahabw‘amaani maingi C trap dissociation (HCD) cleavage. Obwire bw‘okwetoroora nibuteebwa ahari 1 s. Omuhendo gwa m/z gw‘ekicweka kya peptide gukapimwa omu mutego gwa ion barikukoresa ekigyendererwa kya AGC ekikye munonga ekya 5×104 n‘obwire bw‘okutaho enjegyere oburikukirayo obuhango bwa 86 ms. Bwanyima y‘okucwanyagura, omubazi gw‘okubanza gukateebwa aha runyiriri rw‘okutaho amaani kumara s 45. Ebicweka by‘omubiri ebiine akamanyiso ka TMT bikataanisibwa aha nkora ya Acclaim PepMap eya sentimita 50, 75 μm (Thermo Fisher Scientific, enamba y‘akatabo 164942), kandi ebiri omu nkora y‘okufuruka bikakyeberwa aha nkora y‘okupima obuhango bwa Orbitrap Lumos Tribrid nk‘ebiri omu nkora y‘okufuruka. (FAIMS) eby‘okukoresa (Thermo Fisher Scientific) nibikora aha nkora ibiri z‘okurihiirira eza −50 na −70 V. MS3 etoorainwe kurugiirira aha nkora y‘okubanza y‘okuteerana neekoresibwa omu kupima obumanyiso bwa ion ripooti ya TMT. Okwahukana kwa peptide kukakorwa aha EASY-nLC 1200, barikukoresa 90% y’okushohoza kw’omuringo gumwe, n’okushohoza kwa buffer okwa 6% kuhika 31%; obutafaari A bukaba buri 0.1% FA, kandi obutafaari B bukaba buri 0.1% FA na 80% ACN. Enyomyo y’okushwijuma neekora aha 50°C. Kozesa FreeStyle (omuringo 1.6, Thermo Fisher Scientific) kugabanisamu fairo y‘okubanza kurugiirira aha nkora y‘okurihiirira eya FAIMS.
Okumanya puroteyini n’okuzipima. Okukozesa omukura gw’okusherura ogwa Andromeda ogurimu, ebihandiiko by’okubanza bikakyeberwa barikukoresa omuringo gwa MaxQuant 1.5.2.8 (https://maxquant.org/). Oyihireho okukuratana kwa Cre recombinase na YFP okwatungirwe kuruga omu Aequorea victoria, ebicweka bya peptide ebyabaire nibikuratana bikarondwa ahabw’okukuratana kw’ebiragiro n’okukuratana kwa isoform kw’ekirungo ky’okureeberaho eky’embeba (Proteome ID UP000000589, ekyatungirwe kuruga omu UniProt)77 omu Kwakataano2020. Okuhindura kwa methionine n’okuhindura kwa puroteyini N-terminal kukateebwaho nk’okuhindura okurikuhinduka; okuhindura ebiri omu mubiri kukateebwaho nk’okuhindura okutaine mpinduka. Ebirikukwata aha kugaaya eby’okurya nibiteebwa aha “bumanyiso” na “trypsin/P”. Omubaro mukye gw’ebicweka by’omubiri ebirikwetwa peptides na razor peptides ebirikukoresibwa omu kumanyisa puroteyini ni 1; omubaro mukye gw’ebicweka by’omubiri eby’omutaano ni 0. Omu mbeera z’okuhikaana kwa maapu y’ebicweka by’omubiri, omubaro gw’okumanyisa puroteyini gukaba guri 0.01. “Peptide ya kabiri” neekora. Koresa “okuhikaana ahagati y’emisinde” okuhingisa okumanyisa okusingire ahagati ya fairo z’oburugo zitari zimwe na zimwe. Koresa LFQ omubaro gw‘ahansi gw‘omubaro 1 ahabw‘okupima okutaine nkora (LFQ) (60). Obuhango bwa LFQ nibushwijumwa haakiri emihendo ebiri ehikire omu guruupu emwe y‘omuringo gw‘obuzaarwa ahari buri bwire, kandi nibukyeberwa kuruga omu kugabwa okwa buriijo n‘obugazi bwa. 0.3 kandi oze ahansi 1.8. Koresa omukura gwa kompyuta ogwa Perseus () na R (purojekiti.org/) kucondooza ebyarugamu bya LFQ. Ekigyezo kya t eky‘emiringo ebiri eky‘omutindo kuruga omu nkora ya limma software kikakoresibwa omu kucondooza aha kushoboorora kw‘entaaniso (61). Okushwijuma ebihandiiko by‘okucondooza nikukorwa barikukoresa ggplot, FactoMineR, factoextra, GGally na pheatmap. Ebihandiiko bya puroteyini ebirikwegamira ahari TMT bikakyeberwa barikukoresa omuringo gwa MaxQuant 1.6.10.43. Ronda amakuru g’eby’okurya ebitari birungi kuruga omu nkora ya UniProt ey’eby’obuhangwa bw’abantu, eyatungirwe omu Kwamwenda 2018. Okweshwijuma oku kurimu enshonga y’okutereeza obushemeire bwa isotope erikuheebwa omukozi. Koresa limma omu R ahabw‘okushwijuma okushoboorora okurikutaana. Ebihandiiko by‘okubanza, ebyarugamu omu kusherura omu nkora y‘okucondooza, n‘okukora kw‘okushwijuma ebihandiiko n‘ebyarugamu byona nibibiikwa omu mukago gwa ProteomeXchange kurabira omu kibiiko ky‘omukago gwa PRIDE n‘ekimanyiso ky‘ebihandiiko PXD019690.
Ebihandiiko ebirikukora nibigaita okweshwijuma. Ekikwato ky‘okushwijuma omuhanda gw‘obwengye (QIAGEN) kikakoresibwa kureeba obugaiga bw‘ebigambo by‘okushoboorora ebirikukora eby‘ebihandiiko ebyateirweho aha wiiki 8 (ekishushani 1). Omu bugufu, orunyiriri rwa puroteyini z‘omubaro ezitungirwe kuruga omu kucondooza kwa LC-MS/MS (tandem mass spectrometry) amakuru nirukoresibwa n‘enkora z‘okushwijuma ezi: Mus musculus neetooranwa nk‘ekika n‘enyima, kandi ekicweka nikyoreka omuhendo gwa P ogwahinduirwe Benjamini ahabw‘okugaita 0.05 nari ahansi nigutwarwa nk‘ogw‘omugasho. Aha giraafu egi, ebicweka bitaano eby‘ahaiguru eby‘okurengaho omu buri kicweka kurugiirira aha muheendo gwa P oguhindwirwemu nibyorekwa. Okukozesa t-test nyingi, okukoresa puroguraamu y‘okutunguura ebiri omu nkora y‘emitendekyere ebiri eya Benjamini, Krieger, na Yekutieli (Q = 5%), okucondooza kw‘okumanyisa puroteyini omu bwire nikukorwa aha bantu abakuru abamanyirwe omu buri kicweka, kandi buri rubaju nirushwijumwa omu muringo gw‘okwetaanisa. Tihariho kwetenga kutwara SD etarikuhinduka.
Okubaasa kugyeragyeranisa ebyarugamu omu kucondooza oku n‘ebihandiiko ebihandiikirwe n‘okureetaho ekishushani kya Venn omu kishushani 1, tukagaita orunyiriri rwa puroteyini z‘omubaro n‘ebihandiiko bya MitoCarta 2.0 (24). Koresa ekikwato ky‘aha Intaneeti Teera ekishushani kya Venn (http://eby‘amagara.psb.ugent.be/webtools/Venn/) kureetaho ekishushani.
Okumanya ebirikukiraho aha nkora z‘embariirira ezirikukoresibwa omu kucondooza kwa puroteyini, reeba ekicweka ekirikuhikaana n‘ebintu n‘emiringo y‘okukora. Aha kugyezesa okundi kwona, amakuru agahikire nigabaasa kushangwa omu nfumu erikuhikaana. Okwihaho kyaba kitarikworekwa, ebihandiiko byona nibyorekwa nk‘ebiri ahagati ± SEM, kandi okucondooza kwona okw‘embariirira kukakorwa barikukoresa puroguraamu ya GraphPad Prism 8.1.2.
Okumanya ebihandiiko by’okwongeraho aha kihandiiko eki, reeba aha http://advances.sciencemag.org/cgi/ebirimu/omu bwijwire/6/35/eaba8271/DC1.
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Omuringo gw’okuhandiika ebiri omu nkora ya E. Motori, I. Atanassov, SMV Kochan, K. Folz-Donahue, V. Sakthivelu, P. Giavalisco, N. Toni, J. Puyal, N.-G. Larson
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