Ubu *Aderesi igezweho: Cologne 50931, Ubudage, Ubushakashatsi ku itsinda ry’abahanga mu by’ubuhanga rya Cologne ku bijyanye no guhangana n’ihungabana ry’uturemangingo mu ndwara zijyanye n’izabukuru (CECAD).
Kugabanuka k'ubwonko ku ndwara za mitochondrial bifatwa nk'ibidasubirwaho kuko imikorere mibi ya neuron iri muke, ariko ingaruka z'imikorere mibi ya mitochondrial ku bushobozi bw'uturemangingo bwo gukora imikorere y'imitsi mu mubiri ntizumvikana neza. Aha, turagaragaza proteome yihariye ya neuron ya Purkinje hamwe n'ibura rya OXPHOS rikomeza kwiyongera bitewe n'imikorere mibi ya mitochondrial. Twasanze imikorere mibi ya mitochondrial yateje impinduka zikomeye mu rwego rwa proteomics, amaherezo byatumye gahunda nyazo za metabolic zitangira gukora mbere y'uko uturemangingo dupfa. Mu buryo butunguranye, twasanze ubwiyongere bugaragara bwa pyruvate carboxylase (PCx) n'izindi enzymes zirwanya gusaza zuzuza intermediates ya TCA cycle. Kubuza PCx kongera imbaraga za oxidative stress no kwangirika kw'ubwonko, bigaragaza ko atherosclerosis ifite ingaruka zo kurinda neurons zidafite OXPHOS. Kugarura imikorere ya mitochondrial mu neurons zangiritse burundu bihindura burundu iyi miterere ya metabolic, bityo bikarinda urupfu rw'uturemangingo. Ibyo twabonye bigaragaza inzira zitazwi mbere zitanga ubushobozi bwo kwihanganira imikorere mibi ya mitochondrial kandi bigaragaza ko kwangirika kw'ubwonko gushobora gusubizwa inyuma ndetse no mu byiciro bya nyuma by'indwara.
Uruhare rw'ingenzi rwa mitochondria mu kubungabunga ingufu z'imitsi y'ubwonko rugaragazwa n'ibimenyetso byinshi by'imitsi bifitanye isano n'indwara za mitochondria ku bantu. Inyinshi muri izi ndwara ziterwa n'impinduka za gene zigenga igaragara rya gene ya mitochondria (1, 2) cyangwa kwangirika kwa gene bifitanye isano n'imikorere ya mitochondria, bigira ingaruka ku buryo butaziguye ku ituze rya ADN ya mitochondria (mtDNA) (3, 4). Akazi mu nyamaswa kagaragaje ko mu rwego rwo gusubiza imikorere mibi ya mitochondria mu ngingo zikikije, inzira za metabolic zihamye (5-7) zishobora gukoreshwa, ibyo bikaba bitanga amakuru y'ingenzi yo gusobanukirwa neza imiterere y'izi ndwara zikomeye. Mu buryo bunyuranye cyane, gusobanukirwa kwacu impinduka za metabolic z'ubwoko bwihariye bw'uturemangingo ziterwa no kunanirwa rusange kw'umusaruro wa adenosine triphosphate (ATP) mu bwonko ni ingenzi (8), bigashimangira ko ari ngombwa kumenya intego zo kuvura zishobora gukoreshwa mu gukumira cyangwa gukumira indwara. Kurinda kwangirika kw'ubwonko (9). Kubura amakuru ni uko uturemangingo tw'imitsi dufatwa cyane nk'aho dufite ubushobozi buke bwo guhindura imikorere y'imitsi ugereranije n'ubwoko bw'uturemangingo tw'uturemangingo tuyikikije (10). Bitewe nuko izi selile zigira uruhare runini mu guhuza uburyo selile zitanga metabolite mu mitsi kugira ngo ziteze imbere ihererekanya rya synaptic no gusubiza ibibazo by’imvune n’indwara, ubushobozi bwo guhuza imikorere y’ingirabuzimafatizo n’imimerere igoye y’ingirabuzimafatizo z’ubwonko bugarukira hafi ku selile za glial (11-14). Byongeye kandi, itandukaniro riri hagati y’ingirabuzimafatizo z’ingirabuzimafatizo z’ubwonko ribangamira cyane inyigo y’impinduka mu mikorere y’ingirabuzimafatizo ziba mu matsinda yihariye y’imitsi. Kubera iyo mpamvu, nta byinshi bizwi ku ngaruka nyazo z’ingirabuzimafatizo n’imikorere y’ingirabuzimafatizo z’imitsi.
Kugira ngo dusobanukirwe ingaruka z’imikorere mibi ya mitochondrial, twatoranyije neurons za Purkinje (PNs) mu byiciro bitandukanye byo kwangirika kw’imitsi biterwa no kwangirika kwa mitochondrial outer membrane fusion (Mfn2). Nubwo impinduka za Mfn2 mu bantu zifitanye isano n’ubwoko bw’indwara yo mu bwonko iterwa n’uruhererekane rw’imyanya y’umubiri izwi nka Charcot-Marie-Tooth type 2A (15), kwangirika kwa Mfn2 mu mbeba ni uburyo buzwi bwo gutera oxidation​​ Phosphorylation (OXPHOS) kudakora neza. Ubwoko butandukanye bw’imitsi (16-19) hamwe n’uburyo bwo kwangirika kw’imitsi buvamo biherekezwa n’ibimenyetso by’imitsi bigenda bikura, nko kutagira imikorere myiza (18, 19) cyangwa serebela ataxia (16). Dukoresheje uburyo bwo guhuza proteomics zidafite label quantitative (LFQ), metabolomics, imaging, na virological, twerekanye ko kwangirika kw’imitsi bigenda bikura cyane pyruvate carboxylase (PCx) n’ibindi bintu bigira uruhare mu gutera arteriosclerosis ya PNs in vivo The expression of enzymes. Kugira ngo tumenye akamaro k'iki gitekerezo, twagabanyije cyane cyane uburyo PCx igaragara muri PN zidafite Mfn2, kandi twasanze iki gikorwa cyongereye stress ya oxidative ndetse kikanatuma ubwonko bwihuta, bityo bigaragaza ko azoospermia ituma uturemangingo dupfa. Kugaragara cyane kwa MFN2 bishobora gukiza burundu kwangirika kwa PN hamwe n'ibura rikomeye rya OXPHOS, gukoresha ADN ya mitochondrial cyane, ndetse n'urusobe rwa mitochondrial rusa n'aho rwangiritse, ibi bikaba bishimangira ko ubu buryo bwo kwangirika kw'ubwonko bushobora no gukira mu gihe cy'indwara mbere y'uko uturemangingo dupfa.
Kugira ngo turebe mitochondria muri PN za Mfn2, twakoresheje ubwoko bw'imbeba butuma mitochondria ishingiye kuri Cre yibasira poroteyine y'umuhondo (YFP) (mtYFP) (20) Cre expression kandi twagenzuye imiterere ya mitochondria mu mubiri. Twasanze kwangirika kwa gene ya Mfn2 muri PN byazatuma habaho igabanyuka ry'urusobe rwa mitochondria buhoro buhoro (Ishusho S1A), kandi impinduka ya mbere yabonetse ku byumweru 3 by'amavuko. Mu buryo bunyuranye, kwangirika gukomeye k'urwego rw'uturemangingo twa PN, nk'uko bigaragazwa no gutakaza immunostaining ya Calbindin, ntibyatangiye kugeza ku byumweru 12 by'amavuko (Ishusho ya 1, A na B). Kutamenyana kw'igihe hagati y'impinduka za mbere mu miterere ya mitochondria n'itangira rigaragara ry'urupfu rw'imitsi byatumye dukora ubushakashatsi ku mpinduka za metabolisme zatewe no kudakora neza kwa mitochondria mbere y'uko uturemangingo dupfa. Twakoze ingamba zo gutondeka uturemangingo dukoresha fluorescence (FACS) kugira ngo dutandukanye PN ikoresha YFP (YFP+) (Ishusho ya 1C), no mu mbeba zigenzura (Mfn2 + / loxP :: mtYFP loxP- stop-loxP: : L7-cre), hano hifashishijwe CTRL (Ishusho ya S1B). Gutunganya ingamba zo gufunga bishingiye ku bukana bw'ikimenyetso cya YFP bidufasha gusukura umubiri wa YFP+ (YFPhigh) wa PN uhereye ku tutari PN (YFPneg) (Ishusho ya S1B) cyangwa ibice bya axon/dendritic fluorescent (YFPlow; Ishusho ya S1D, ibumoso), byemejwe na microscope ya confocal (Ishusho ya S1D, iburyo). Kugira ngo twemeze umwirondoro w'abantu bashyizwe mu byiciro, twakoze LFQ proteomics hanyuma dusesengura ibice by'ingenzi, maze dusanga hari itandukaniro rigaragara hagati y'uturemangingo twa YFPhigh na YFPneg (Ishusho ya S1C). Uturemangingo twa YFPhigh twagaragaje ubwiyongere bw’ibimenyetso bya PN bizwi (urugero: Calb1, Pcp2, Grid2 na Itpr3) (21, 22), ariko nta bwiyongere bwa poroteyine bukunze kugaragara mu tunyangingo cyangwa mu tundi duce tw’uturemangingo (Ishusho ya 1D)). Igereranya hagati y’ibipimo mu tunyangingo twa YFPhigh twakusanyijwe mu bushakashatsi bwigenga ryagaragaje ko coefficient y’isano> 0.9, igaragaza ko ikwirakwira neza hagati y’utunyangingo tw’ibinyabuzima (Ishusho ya S1E). Muri make, aya makuru yemeje gahunda yacu yo kwitandukanya PN ishoboka mu buryo bwihuse kandi bwihariye. Kubera ko sisitemu ya L7-cre ikoreshwa ituma habaho kwihuza kwa mosaic mu cyumweru cya mbere nyuma yo kubyara (23), twatangiye gukuramo imbeba muri CTRL na conditional (Mfn2 loxP / loxP :: mtYFP loxP-stop-loxP :: L7-cre) Gukusanya neurons. Nyuma yo kwihuza kurangiye, yitwa Mfn2cKO ku byumweru 4 by’amavuko. Nk’iherezo, twahisemo ibyumweru 8 by’amavuko igihe urwego rwa PN rwari rumeze neza nubwo hari ibice bya mitochondrial bigaragara (Ishusho ya 1B n’Ishusho ya S1A). Muri rusange, twapimye poroteyine 3013, muri zo hafi 22% byari bishingiye ku nyandiko za MitoCarta 2.0 zishingiye kuri proteome ya mitochondrial nka mitochondria (Ishusho ya 1E) (Ishusho ya 1E) (24). Isesengura ry’itandukaniro ry’imisemburo ya gene ryakozwe mu cyumweru cya 8 ryagaragaje ko 10.5% gusa bya poroteyine zose ari byo byagize impinduka zikomeye (Ishusho ya 1F n’Ishusho ya S1F), muri zo poroteyine 195 zagabanutseho naho poroteyine 120 zongereweho (Ishusho ya 1F). Ni ngombwa kumenya ko "isesengura ry’inzira nshya" ry’iyi seti y’amakuru rigaragaza ko gene zigaragara mu buryo butandukanye ahanini ziri mu itsinda ry’inzira runaka zikoreshwa mu mikorere (Ishusho ya 1G). Igishimishije ni uko nubwo kugabanya inzira zijyanye na OXPHOS na calcium signaling byemeza ko mitochondrial idakora neza mu mitochondrial PN zidafite fusion, ibindi byiciro byiganjemo aside amine metabolism byiyongera cyane, ibi bikaba bihuye n'ihinduka ry'imikorere ribaho muri mitochondrial PN.
(A) Amafoto y’inyuma y’imbeba za CTRL na Mfn2cKO agaragaza ko PNs zigenda zitakaza buhoro buhoro (calbindin, imvi); imiterere y’inyuma yafashwe na DAPI. (B) Isuzuma rya (A) (isesengura ry’uburyo bumwe bw’impinduka, ***P<0.001; n = uruziga 4 kugeza kuri 6 rw’imbeba eshatu). (C) Imikorere y’igerageza. (D) Ishusho y’ubushyuhe bw’ibimenyetso byihariye kuri Purkinje (hejuru) n’ubundi bwoko bw’uturemangingo (hagati). (E) Ishusho ya Venn yerekana umubare wa poroteyine za mitochondrial zabonetse muri PN yashyizwe mu byiciro. (F) Igishushanyo cy’ibirunga cya poroteyine zitandukanye muri neurons za Mfn2cKO mu byumweru 8 (agaciro k’ingenzi ka 1.3). (G) Isesengura ry’inzira y’ubuhanga rigaragaza inzira eshanu z’ingenzi zo kongera (umutuku) no kugabanya (ubururu) muri Mfn2cKO PN yashyizwe mu byiciro 8. Igipimo mpuzandengo cy’igaragazwa rya buri poroteyine yabonetse kiragaragazwa. Ishusho y’ubushyuhe bwa Grayscale: agaciro ka P kahinduwe. ns, ntabwo ari ingenzi.
Amakuru ya Proteomics yagaragaje ko imiterere ya poroteyine ya complexes I, III, na IV yagabanutse buhoro buhoro. Complexes I, III, na IV zose zari zirimo utunyangingo tw’ingenzi twa mtDNA, mu gihe complex II, yari ifite nucleus gusa, nta ngaruka yari ifite (Ishusho ya 2A n’Ishusho ya S2A). . Bijyanye n’ibyavuye muri proteomics, immunohistochemistry y’ibice by’utunyangingo twa cerebellar yagaragaje ko urwego rwa MTCO1 (mitochondrial cytochrome C oxidase subunit 1) rwa complex IV muri PN rwagabanutse buhoro buhoro (Ishusho ya 2B). Utunyangingo twa mtDNA twa Mtatp8 twagabanutse cyane (Ishusho ya S2A), mu gihe urwego rwa static-state rwa ATP synthase subunit rwa nucleus rwagumyeho, ibyo bikaba bihuye n’utunyangingo twa ATP synthase subassembly F1 tuzwiho igihe imiterere ya mtDNA ihamye. Imiterere irahamye. Interrupt (7). Isuzuma ry’urugero rwa mtDNA muri Mfn2cKO PNs zatoranyijwe hakoreshejwe reaction ya polymerase chain reaction (qPCR) ryemeje ko umubare wa kopi ya mtDNA wagabanutse buhoro buhoro. Ugereranyije n’itsinda ry’abagenzuye, ku byumweru 8 by’amavuko, hafi 20% by’urugero rwa mtDNA ni byo byagumanye (Ishusho ya 2C). Dukurikije ibi byavuye muri ubu bushakashatsi, hakoreshejwe mikroskopi ya confocal ya Mfn2cKO PNs mu gupima ADN, bigaragaza ikoreshwa rya nucleotides za mitochondrial rishingiye ku gihe (Ishusho ya 2D). Twasanze hari abakandida bamwe gusa bagize uruhare mu kwangirika kwa poroteyine ya mitochondrial no gusubiza stress ari bo bongerewe, harimo Lonp1, Afg3l2 na Clpx, na OXPHOS complex assembly factors. Nta mpinduka zikomeye ku rugero rwa poroteyine zigira uruhare mu ihindagurika ry’uturemangingo (Ishusho ya S2B). Mu buryo nk’ubwo, twasanze mitochondria na endoplasmic reticulum channels zigira uruhare mu gutwara kalisiyumu gusa (Ishusho ya S2C). Byongeye kandi, isuzuma rya poroteyine zifitanye isano na autophagy nta mpinduka zikomeye ryabonye, ​​bihuye n'uburyo autophagosomes zigaragara mu mubiri zibonwa mu buryo bw'umubiri hakoreshejwe immunohistochemistry na electron microscopy (Ishusho S3). Ariko, imikorere mibi ya OXPHOS muri PN iherekezwa n'impinduka zigaragara za mitochondrial. Amatsinda ya mitochondrial ashobora kugaragara mu ngingo z'uturemangingo no mu biti bya Mfn2cKO PNs zifite ibyumweru 5 na 8, kandi imiterere y'imbere mu ruhu yahindutse cyane (Ishusho S4, A na B). Bijyanye n'izi mpinduka za ultrastructural ndetse no kugabanuka gukomeye kwa mtDNA, isesengura rya cerebellar cerebellum slices hamwe na tetramethylrhodamine methyl ester (TMRM) ryagaragaje ko ubushobozi bwa mitochondrial membrane muri Mfn2cKO PNs bwagabanutse cyane (Ishusho S4C).
(A) Isesengura ry'igihe cy'igaragazwa ry'urugero rw'ishusho ya OXPHOS complex. Tekereza gusa poroteyine zifite P<0.05 mu byumweru 8 (ANOVA y'inzira ebyiri). Umurongo w'utudomo: Nta gihinduka ugereranije na CTRL. (B) Ibumoso: Urugero rw'igice cya cerebellar cyanditseho anti-MTCO1 antibody (umurongo w'ubugari, 20 μm). Agace kari mu turemangingo twa Purkinje gatwikiriwe n'umuhondo. Iburyo: Gupima urugero rwa MTCO1 (isesengura ry'uburyo bumwe bw'itandukaniro; n = uturemangingo 7 kugeza kuri 20 twasesenguwe n'imbeba eshatu). (C) Isesengura rya qPCR ry'umubare wa kopi ya mtDNA muri PN yatoranyijwe (isesengura ry'uburyo bumwe bw'itandukaniro; n = imbeba 3 kugeza kuri 7). (D) Ibumoso: Urugero rw'agace ka cerebellar kanditseho anti-DNA antibody (umurongo w'ubugari, 20 μm). Agace kari mu turemangingo twa Purkinje gatwikiriwe n'umuhondo. Iburyo: Gupima ibisebe bya mtDNA (isesengura ry'uburyo bumwe bw'itandukaniro; n = uturemangingo 5 kugeza kuri 9 twavuye ku mbeba eshatu). (E) Urugero rw'igice cy'ubuhumekero cy'inyuma kigaragaza utunyangingo twa mitoYFP + Purkinje (umwambi) mu ifatwa ry'imashini ifata ingirangingo yose. (F) Ingano y'umuyoboro wa IV. (G) Incamake y'ishyirwa ry'umuyoboro wa depolarizing current muri CTRL na Mfn2cKO Purkinje. Incamake yo hejuru: Ingufu ya mbere yateye AP. Incamake yo hasi: Ingano ntarengwa ya AP. (H) Ingano y'ibyinjira by'inyuma ya synaptic (sPSPs). Incamake y'inyuguti ihagarariye hamwe n'igipimo cya zoom yayo bigaragazwa muri (I). Isesengura ry'uburyo bumwe ry'impinduka ryasesenguwe n = utunyangingo 5 kugeza kuri 20 bivuye ku mbeba eshatu. Amakuru agaragazwa nka mean ±SEM; *P <0.05; **P <0.01; ***P <0.001. (J) Ingano y'inyuguti za AP yihariye yafashwe hakoreshejwe uburyo bwa patch clamp ifite imyobo. Incamake yo hejuru: Ingano ntarengwa ya AP. Incamake yo hasi: Ingano y'inyuguti imwe. (K) Ingano mpuzandengo n'ingano ntarengwa ya AP hakurikijwe (J). Ikizamini cya Mann-Whitney; n = utunyangingo 5 twasesenguwe mu mbeba enye. Amakuru agaragazwa nk'ikigereranyo ± SEM; ntabwo ari ngombwa.
Kwangirika kugaragara kwa OXPHOS kwagaragaye muri Mfn2cKO PN y'ibyumweru 8, bigaragaza ko imikorere y'imitsi y'ubwonko idasanzwe cyane. Kubwibyo, twasesenguye imiterere y'amashanyarazi idakora ya neuron zidafite OXPHOS mu byumweru 4 kugeza kuri 5 n'ibyumweru 7 kugeza kuri 8 dukoresha uburyo bwo gufata amashusho ya patch patch mu turemangingo twose mu bice by'ubwonko bya acute cerebellar (Ishusho ya 2E). Mu buryo butunguranye, ubushobozi busanzwe bwo kuruhuka no kwirinda kwinjira kwa neuron za Mfn2cKO byari bisa n'uburyo bwo kugenzura, nubwo hari itandukaniro rito hagati y'uturemangingo (Imbonerahamwe ya 1). Mu buryo nk'ubwo, ku byumweru 4 kugeza kuri 5 by'amavuko, nta mpinduka zikomeye mu mibanire y'amashanyarazi n'amashanyarazi (IV curve) zabonetse (Ishusho ya 2F). Ariko, nta neuron za Mfn2cKO zifite ibyumweru 7 kugeza kuri 8 zarokotse IV regimen (intambwe ya hyperpolarization), bigaragaza ko hari uburyo bwo kumva neza ubushobozi bwa hyperpolarization muri iki cyiciro cya nyuma. Mu buryo bunyuranye, muri neurone za Mfn2cKO, imiyoboro ya depolarizating itera repetitive action potential (AP) irahanganirwa neza, bigaragaza ko imiterere yayo muri rusange idatandukanye cyane n'iya neurone zo mu rwego rwo kugenzura zifite ibyumweru 8 (Imbonerahamwe ya 1 n'Ishusho ya 2G). Mu buryo nk'ubwo, inshuro n'ubwinshi bw'imiyoboro ya possinapike (sPSCs) byari bingana n'iby'itsinda ryo kugenzura, kandi inshuro z'ibintu byagiye byiyongera kuva ku byumweru 4 kugeza ku byumweru 5 kugeza ku byumweru 7 kugeza ku byumweru 8 hamwe n'ubwiyongere busa (Ishusho ya 2, H na I). ​​Igihe cyo gukura kwa synaptic muri PN (25). Ibisubizo nk'ibyo byabonetse nyuma y'uduce twa PN twatobowe. Iyi miterere ibuza kwishyura ubusembwa bwa ATP mu turemangingo, nk'uko bishobora kubaho mu kwandika clamp y'uturemangingo twose. By'umwihariko, ubushobozi bwo kuruhuka bwa membrane n'ubwinshi bwo kurasa bwa neurone za Mfn2cKO ntibyigeze bigerwaho (Ishusho ya 2, J na K). Muri make, ibi bisubizo bigaragaza ko PN zifite ikibazo cya OXPHOS gishobora guhangana neza n’imiterere y’ibisohoka mu maraso cyane, bigaragaza ko hari uburyo bwo kubikemura butuma zigumana uburyo busanzwe bwo gusubiza amashanyarazi.
Amakuru agaragazwa nk'ikigereranyo cya ± SEM (isesengura ry'uburyo bumwe bw'impinduka, ikizamini cya Holm-Sidak cyo kugereranya inshuro nyinshi; *P<0.05). Umubare w'igice ugaragazwa n'udukingirizo.
Twatangiye gukora ubushakashatsi ku icyiciro icyo ari cyo cyose mu makuru ya proteomics (Ishusho ya 1G) kirimo inzira zishobora kurwanya ibura rikomeye rya OXPHOS, bityo tugasobanura impamvu PN yagizweho ingaruka ishobora kugumana electrophysiology isanzwe (Ishusho ya 2, E kugeza K). Isesengura rya proteomics ryagaragaje ko enzymes zigize uruhare mu gukatabolisma kwa aside amine z’uruhererekane rw’amashami (BCAA) zari zariyongereye cyane (Ishusho ya 3A n’Ishusho ya S5A), kandi umusaruro wa nyuma acetyl-CoA (CoA) cyangwa succinyl CoA bishobora kongera tricarboxylates mu mizunguko ya arteriosclerosis Acid (TCA). Twasanze ingano ya BCAA transaminase 1 (BCAT1) na BCAT2 byombi byiyongereye. Bituma intambwe ya mbere ya catabolisma ya BCAA ikora glutamate iva kuri α-ketoglutarate (26). Uduce duto twose tugize uruhererekane rw'amashami rwa keto aside dehydrogenase (BCKD) turazamuka (uru ruvange rutuma habaho ihinduka ry'imisemburo ya BCAA karubone) (Ishusho ya 3A n'Ishusho ya S5A). Ariko, nta mpinduka zigaragara muri BCAA ubwayo zabonetse muri PN yatoranyijwe, ishobora guterwa no kwiyongera kw'iyi aside amine y'ingenzi cyangwa ikoreshwa ry'andi masoko (glucose cyangwa aside lactic) kugira ngo yongere imikorere ya TCA (Ishusho ya S5B). PN zidafite OXPHOS nazo zagaragaje ko glutamine yagabanutse kandi igahindura imikorere ku byumweru 8 by'amavuko, ibi bishobora kugaragazwa no kwiyongera kwa enzymes za mitochondrial glutaminase (GLS) na glutamine pyruvate transaminase 2 (GPT2) (Ishusho ya 3, A na C). Ni ngombwa kumenya ko kwiyongera kwa GLS bigarukira kuri isoform glutaminase C (GLS-GAC) (impinduka ya Mfn2cKO/CTRL ni hafi inshuro 4.5, P = 0.05), kandi kwiyongera kwayo mu buryo bwihariye mu turemangingo twa kanseri Bishobora gushyigikira bioenergy ya mitochondrial. (27).
(A) Ikarita y'ubushyuhe igaragaza impinduka mu rugero rwa poroteyine ku nzira yagenwe mu byumweru 8. (B) Urugero rw'agace ka cerebellar kanditseho anti-PCx antibody (umurongo w'ubugari, 20 μm). Akambi k'umuhondo kerekana umubiri wa selile ya Purkinje. (C) Isesengura ry'igihe poroteyine igaragaramo ryagaragaye nk'ikintu cy'ingenzi cya atherosclerosis (ikizamini cya t-multiple, *FDR <5%; n = imbeba 3-5). (D) Hejuru: Ishusho yerekana uburyo butandukanye bwo kwinjira muri karuboni ifite ikimenyetso kiri muri [1-13C]pyruvate tracer (ni ukuvuga, binyuze muri PDH cyangwa inzira ya trans-arterial). Hasi: Imbonerahamwe ya violon yerekana ijanisha rya karuboni ifite ikimenyetso kimwe (M1) yahinduwe aside aspartic, aside citric na aside malic nyuma yo gushyiramo uduce twa cerebellar tw’ubushyuhe hamwe na [1-13C]pyruvate (ikizamini cya t-paired; ** P <0.01). (E) Isesengura ryuzuye ry'amateka y'inzira yagaragajwe. Tekereza gusa poroteyine zifite P <0.05 mu byumweru 8. Umurongo uhanamye: nta gaciro ko guhindura (isesengura ry'impinduka mu buryo bubiri; * P <0.05; *** P <0.001). Amakuru agaragazwa nk'ikigereranyo ± SEM.
Mu isesengura ryacu, BCAA catabolism yabaye imwe mu nzira z'ingenzi zo kongera ubushobozi bwo guhumeka. Ibi bigaragaza cyane ko ingano y'umwuka winjira mu ruziga rwa TCA ishobora guhinduka mu gihe PN idafite OXPHOS. Ibi bishobora kuba ari ubwoko bukomeye bwo kongera imbaraga mu mikorere y'imitsi y'ubwonko, bishobora kugira ingaruka zitaziguye ku mikorere y'imitsi y'ubwonko no kubaho kwayo mu gihe cyo gukomeza gukora nabi cyane kwa OXPHOS. Dukurikije iki gitekerezo, twasanze enzyme ikomeye irwanya atherosclerotic PCx iriyongera (Mfn2cKO/CTRL ihinduka inshuro zigera kuri 1.5; Ishusho ya 3A), ituma pyruvate ihinduka oxaloacetate (28), bikekwa ko iri mu bwonko. Imvugo ya in igarukira ku nyenyeri (29, 30). Dukurikije ibyavuye mu bushakashatsi bwa proteomics, microscopy ya confocal yagaragaje ko imiterere ya PCx yiyongereye cyane kandi cyane mu misemburo ya PN idafite OXPHOS, mu gihe imikorere ya PCx yari igarukira cyane ku turemangingo twa Bergmann glial twegeranye (Ishusho ya 3B). Kugira ngo tugerageze uburyo PCx yagaragaye yiyongera, twavuje uduce twa cerebellar dukoresheje [1-13C]pyruvate tracer. Ubwo pyruvate yakurwagamo oxidized na pyruvate dehydrogenase (PDH), ikirango cyayo cya isotope cyarazimye, Ariko yinjizwa mu bice bya TCA cycle iyo pyruvate ihinduwemo metabolize bitewe n'imikorere y'imitsi (Ishusho ya 3D). Mu gushyigikira amakuru yacu ya proteomics, twabonye umubare munini w'ibimenyetso byavuye muri iyi tracer muri aside aspartic ya Mfn2cKO slices, mu gihe aside citric na aside malic nabyo byari bifite icyerekezo kiringaniye, nubwo bitari binini (Ishusho ya 3D).
Mu mitsi ya dopamine y’imbeba za MitoPark ifite ikibazo cya mitochondrial giterwa na dopamine neurons zangiza cyane cyane gene ya mitochondrial transcription factor A (Tfam) (Ishusho ya S6B), PCx nayo yazamuwe cyane (31), bigaragaza ko aside acetone arteriosclerosis Ivuka ry’iyi ndwara rigenzurwa mu gihe cy’imikorere mibi ya OXPHOS y’imitsi mu mubiri. Ni ngombwa kumenya ko byagaragaye ko enzymes zidasanzwe (32-34) zishobora kugaragara mu mitsi ishobora kuba ifitanye isano na arteriosclerosis zigenzurwa cyane mu mitsi ya PN ibura muri OXPHOS, nka propionyl-CoA carboxylase (PCC-A), Malonyl-CoA ihindura propionyl-CoA muri succinyl-CoA na mitochondrial malic enzyme 3 (ME3), uruhare rwayo runini ari ukugarura pyruvate muri malate (Ishusho ya 3, A na C) (33, 35). Byongeye kandi, twabonye ukwiyongera gukomeye kwa enzyme ya Pdk3, ikora phosphoryl bityo ikabuza PDH (36), mu gihe nta mpinduka zagaragaye muri enzyme ya Pdp1 ikora PDH cyangwa enzyme ya PDH ubwayo (Ishusho ya 3A). Mu buryo buhoraho, muri Mern2cKO PNs, phosphorylation ya α1 subunit α (PDHE1α) subunit y'igice cya pyruvate dehydrogenase E1 cy'ihuriro rya PDH muri Ser293 (kizwiho kubuza imikorere ya enzyme ya PDH) yariyongereye (Ishusho ya S6C) (Ishusho ya S6C). Pyruvate nta mitsi ishobora kugeramo.
Amaherezo, twasanze inzira ikomeye ya serine na glycine biosynthesis, cycle ya mitochondrial folate (1C) ifitanye isano na proline biosynthesis (Ishusho ya 1G n'Ishusho ya S5C) byose byazamutse cyane, nk'uko raporo zibivuga, mu gihe cyo gukora. Ingirangingo zikikije zikora neza hamwe n'imikorere mibi ya mitochondrial (5-7). Isesengura rya Confocal rishyigikira aya makuru ya proteomics ryagaragaje ko muri PN hamwe na OXPHOS ibura, ibice bya cerebellar by'imbeba z'ibyumweru 8 byakorewe serine hydroxymethyltransferase 2 (SHMT2), enzyme y'ingenzi ya mitochondrial folate cycle. Ubudahangarwa bw'umubiri bugaragara cyane (Ishusho ya S5D). Mu bice 13 bya CU-glucose-incubated acute cerebellar slices, igerageza ryo gukurikirana metabolic ryemeje ko serine na proline biosynthesis byiyongera, bigaragaza ko urujya n'uruza rwa carbon isoforms muri serine na proline rwiyongereye (Ishusho ya S5E). Kubera ko ibikorwa byatewe inkunga na GLS na GPT2 ari byo bituma glutamate iva muri glutamine ihinduka ndetse n’uburyo glutamate na α-ketoglutarate bihinduka, kwiyongera kwabyo bigaragaza ko neurons zidafite OXPHOS zikeneye glutamate nyinshi, Ibi bishobora kuba bigamije gukomeza kwiyongera kwa biosynthesis ya proline (Ishusho ya S5C). Bitandukanye n’izi mpinduka, isesengura rya proteomic rya astrocytes zo mu bwoko bwa cerebellar ziva ku mbeba za Mfn2cKO zihariye za PN ryagaragaje ko izi nzira (harimo na antiperoxidases zose) zitahindutse cyane mu mikorere, bityo bigaragaza ko iyi redirection ya metabolic ihitamo PN yangiritse (Ishusho ya S6, D kugeza kuri G).
Muri make, ubu bushakashatsi bwagaragaje imiterere itandukanye cyane y’imikorere y’igihe runaka y’inzira runaka za metabolic muri PN. Nubwo imikorere idasanzwe ya mitochondrial y’imitsi ishobora gutuma habaho atherosclerosis hakiri kare no kuvugurura 1C (Ishusho ya 3E n’Ishusho ya S5C), ndetse n’impinduka zitezwe mu mikorere ya complexes za I na IV, impinduka mu ikorwa rya serine de novo ni zo gusa. Byagaragaye gusa mu byiciro bya nyuma. Imikorere mibi ya OXPHOS (Ishusho ya 3E n’Ishusho ya S5C). Ibi byavuye mu bushakashatsi bisobanura inzira ikurikirana aho mitochondrial iterwa n’stress (1C cycle) na cytoplasmic (serine biosynthesis) bisubiza hamwe n’ubwiyongere bwa atherosclerosis mu ikorwa rya TCA kugira ngo bihindure imiterere ya neurons.
PN zo mu bwoko bwa OXPHOS zifite ibyumweru 8 zishobora gukomeza gukora ibikorwa byo gushyushya cyane no guhura n’ibibazo bikomeye byo guhuza imikorere y’umubiri kugira ngo zisubize ikibazo cy’imikorere mibi ya mitochondrial. Ubu buvumbuzi bugaragaza ko hari amahirwe menshi ko no muri iki gihe, izi selile zishobora no kubona ubufasha bwo kuvura kugira ngo zitinde cyangwa zikumire kwangirika kw’imitsi. Bitinze. Twakemuye iki kibazo binyuze mu buryo bubiri bwigenga. Mu buryo bwa mbere, twakoze vector ya virusi iterwa na adeno-associated virus (AAV) kugira ngo MFN2 ibashe kugaragara neza muri PN zo mu bwoko bwa OXPHOS zidafite OXPHOS (Ishusho ya S7A). AAV ikora MFN2 hamwe na gene ya fluorescent reporter mCherry (Mfn2-AAV) byemejwe mu miterere y’imitsi y’ibanze muri vitro, byatumye MFN2 igaragara mu buryo buterwa na Cre kandi ikiza imiterere ya mitochondrial, bityo bikarinda ihinduka ry’imitsi muri neurons za Mfn2cKO (Ishusho ya S7, B, D na E). Hanyuma, twakoze igerageza ryakozwe mu buryo bwa "in vivo" kugira ngo twohereze Mfn2-AAV y'ibyumweru 8 kuri cortex ya Mfn2cKO n'imbeba zigenzura, tunasesengura imbeba z'ibyumweru 12 (Ishusho ya 4A). Imbeba zavuwe za Mfn2cKO zapfuye (Ishusho ya 1, A na B) (16). Gutera virusi mu buryo bwa "in vivo" byatumye PN igaragara neza mu duce tumwe na tumwe twa "cerebellar" (Ishusho ya S7, G na H). Gutera AAV igenzura gusa (Ctrl-AAV) nta ngaruka nini byagize ku rugero rwo kwangirika kw'imitsi mu nyamaswa za Mfn2cKO. Ibinyuranye n'ibyo, isesengura rya Mfn2cKOs ryakozwe na Mfn2-AAV ryagaragaje ingaruka zikomeye zo kurinda urwego rw'uturemangingo twa PN (Ishusho ya 4, B na C, na Ishusho ya S7, H na I). Kugaragara kwa MFN1 ariko atari MFN2 bifite akamaro kanini mu kurokora urupfu rw'imitsi (Ishusho ya 4C n'Ishusho ya S7, C na F), bigaragaza ko kugaragara kwa MFN1 yo mu nda bishobora kongera kubura kwa MFN2. Isesengura ryinshi ku rwego rumwe rwa PN ryagaragaje ko Mfn2-AAV yarokoye cyane imiterere ya mitochondria, ihindura urugero rwa mtDNA, kandi ihindura imiterere ya PCx ​​yo kurwanya angiogenesis (Ishusho ya 4, C kugeza kuri E). Isuzuma ry'imbeba za Mfn2cKO zarokowe mu gihe cyo kuruhuka ryagaragaje ko imiterere yazo n'ibimenyetso by'imikorere yazo (kuva kuri S1 kugera kuri S3) byarushijeho kuba byiza. Mu gusoza, ubu bushakashatsi bwerekana ko gutinda kongera kwinjiza MFN2 muri PN zidafite OXPHOS bihagije kugira ngo zisubize inyuma ikoreshwa rya mtDNA no gutera indwara z'imitsi, bityo birinde kwangirika kwa axon no gupfa kw'imitsi mu mubiri.
(A) Igishushanyo kigaragaza gahunda y'igerageza ryo gutera AAV encoding MFN2 iyo inzira ya metabolic yagaragajwe ikora. (B) Amashusho y'inyuma y'ibice bya cerebellar by'ibyumweru 12 byashyizwe mu byumweru 8 mu mbeba za Mfn2cKO kandi byanditseho anti-Calbindin antibody. Iburyo: Gupima fibres za axon. Igipimo cya axon zoom ni 450 na 75 μm. (C) Ibumoso: Isuzuma ry'ubucucike bw'uturemangingo twa Purkinje muri AAV transduction loop (AAV+) (isesengura ry'uburyo bumwe bw'impinduka; n = 3 mbeba). Iburyo: Isesengura ry'icyerekezo cya mtDNA muri transduced PN mu cyumweru cya 12 (isuzuma rya t ritavanze; n = 6 selile zivuye ku mbeba eshatu). * P <0.05; ** P <0.01. (D) Imikorografi ya electron ihagarariye transmission ya PNs za Mfn2cKO selile zashyizwe mu turemangingo twa virusi twagaragajwe. Agapfukamunwa k'umutuku kerekana agace gatuwemo na dendrites, naho kare k'umuhondo karimo utudomo kagaragaza imiterere y'ishusho iboneka iburyo; n ihagarariye nucleus. Umurongo wa scale, 1μm. (E) yerekana urugero rw'irangi rya PCx muri PN ryahinduwe mu byumweru 12. Umurongo wa scale, 20μm. OE, ubwiyongere bw'ishusho; FC, impinduka mu mikurire.
Amaherezo, twasesenguye akamaro ko kubaho kw'uturemangingo duterwa na peroxidase muri PN zagize ikibazo cyo kudakora neza kwa OXPHOS. Twakoze mCherry ikora AAV-shRNA (agace gato k'umusatsi) kigamije cyane cyane imbeba PCx mRNA (AAV-shPCx), maze dutera virusi cyangwa uburyo bwayo bwo kuyigenzura (AAV-scr) mu bwonko bw'imbeba za Mfn2cKO. Urushinge rwakozwe mu cyumweru cya kane cy'amavuko (Ishusho ya 5A) kugira ngo PCx ikore neza mu gihe PCx yagendaga yiyongera (Ishusho ya 3C) kandi urwego rw'uturemangingo twa PN rwari rukiri rwose (Ishusho ya 1A). Ni ngombwa kumenya ko gukubita PCx (Ishusho ya S8A) bitera kwihuta cyane kw'urupfu rwa PN, ibyo bikaba bigarukira ku mpeta yanduye (Ishusho ya 5, B na C). Kugira ngo dusobanukirwe uburyo ingaruka za metabolic ziterwa no kwiyongera kwa PCx, twize ku miterere ya redox ya PNs nyuma yo gutsindwa kwa PCx na AAV-mediated optical biosensor Grx1-roGFP2 byagaragajwe icyarimwe (Ishusho ya S8, B kugeza kuri D) kugira ngo dusuzume impinduka ya glutathione ya peptide redox potential (38). Hanyuma, twakoze microscopy ya fluorescence lifetime imaging microscopy (FLIM) ebyiri mu bice by'ubwonko bya Mfn2cKO y'ibyumweru 7 cyangwa uduce duto two kugenzura kugira ngo tumenye impinduka zishobora kubaho mu miterere ya cytoplasmic redox nyuma yo kugenzura imiterere ya FLIM (Ishusho ya S8, E kugeza kuri G). Isesengura ryagaragaje ubwiyongere bunini mu miterere ya oxidation ya Mfn2cKO PNs imwe idafite PCx expression, bitandukanye na neurons zo kugenzura cyangwa Mfn2cKO PNs zigaragaza shRNA gusa (Ishusho ya 5, D na E). Ubwo PCx yagabanywaga, igipimo cya Mfn2cKO PNs cyagaragazaga imiterere ya oxidation nyinshi cyariyongereyeho inshuro zirenga eshatu (Ishusho ya 5E), bigaragaza ko PCx yazamuye ubushobozi bwa redox bwa neuron zangiritse.
(A) Igishushanyo kigaragaza gahunda y'igerageza ryo gutera AAV encoding shPCx iyo inzira ya metabolic yagaragajwe ikora. (B) Amafoto y'inyuma y'ibice by'ubugimbi by'ibyumweru 8 mu mbeba za Mfn2cKO yahinduwe kandi yashyizweho ikimenyetso cya anti-calcineurin antibody mu byumweru 4. Umugozi w'isuzuma, 450μm. (C) Isuzuma ry'ubucucike bw'uturemangingo twa Purkinje mu duce twahinduwe na AAV (isesengura ry'uburyo bumwe bw'impinduka; n = imbeba 3 kugeza kuri 4). Amakuru agaragazwa nka ±SEM; ***P<0.001. (D) Ishusho ya FLIM igaragaza igihe mpuzandengo cy'ubuzima bwa sensor ya glutathione redox ya PN y'ibyumweru 7 ikora Grx1-roGFP2 mu bihe byagenwe by'igerageza. Igipimo cya LUT (imbonerahamwe yo kureba): igihe cyo kubaho (mu masegonda ya pico). Umugozi w'isuzuma, 25μm. (E) Histogramu yerekana uko agaciro k'ubuzima bwa Grx1-roGFP2 gakwirakwizwa kuva kuri (D) (n=158 kugeza kuri 368 mu mbeba ebyiri muri buri kibazo). Imbonerahamwe iri hejuru ya buri histogramu: igaragaza umubare w'uturemangingo dufite agaciro k'ubuzima burebure cyane (umutuku, oxidized) cyangwa bugufi (ubururu, bwagabanutse), birenga 1 SD y'agaciro k'ubuzima mpuzandengo muri CTRL-AAV-scr. (F) Icyitegererezo cyatanzwe kigaragaza ingaruka zo kurinda kwiyongera kwa PCx y'imitsi.
Muri rusange, amakuru dutanga hano agaragaza ko kongera kwigaragaza kwa MFN2 bishobora gukiza burundu PN iteye imbere ifite ikibazo gikomeye cya OXPHOS, igabanuka rikomeye rya mtDNA, ndetse n'imiterere idasanzwe isa na ista, bityo bigatanga iterambere rihoraho ndetse no mu ndwara zikomeye. Gusenyuka kw'imitsi bitanga ibimenyetso by'intambwe mbere y'uko ingirabuzimafatizo zipfa. Iki cyiciro cy'ubworoherane mu mikorere y'umubiri gishimangirwa cyane n'ubushobozi bw'imitsi yo gutera indwara z'imitsi (guhindura uruziga rwa TCA), ibi bikabuza PCx kwigaragaza muri PN zidafite OXPHOS kandi bikongera urupfu rw'ingirabuzimafatizo, bityo bikagira uruhare mu kurinda (Ishusho ya 5F).
Muri ubu bushakashatsi, twatanze ibimenyetso byerekana ko igisubizo cya PNs ku mikorere mibi ya OXPHOS ari ukwihuza buhoro buhoro na atherosclerosis ya TCA cycle binyuze mu nzira inyuranye yo gukora ikorwa na gahunda za metabolic. Twemeje isesengura rya proteomic hakoreshejwe uburyo bwinshi bwuzuzanya kandi twagaragaje ko iyo ihanganye n'imikorere mibi ikomeye ya mitochondrial, neurons zifite uburyo butazwi bwo guhindagurika kwa metabolic. Igitangaje ni uko inzira yose yo kongera gukoresha ibyuma ntabwo igaragaza imiterere ya metabolic igendana no kwangirika k'ubwonko buhoro buhoro kandi mu buryo budasubirwaho, ariko amakuru yacu agaragaza ko ishobora kuba neuron yo kubungabunga ndetse no mu cyiciro cya mbere cy'urupfu rw'uturemangingo. Iki cyavumbuwe kigaragaza ko neurons zifite urugero runini rwa metabolic plastike mu mubiri. Ibi bigaragaza ko kongera gukoresha MFN2 nyuma bishobora guhindura imyitwarire y'ibimenyetso by'ingenzi bya metabolic no gukumira kwangirika kwa PN. Ibinyuranye n'ibyo, bibuza atherosclerosis kandi bikihutisha imitsi.
Kimwe mu byavumbuwe mu bushakashatsi bwacu ni uko PN zidafite OXPHOS zishobora guhindura imikorere ya TCA cycle metabolism binyuze mu kongera enzymes zituma arteriosclerosis itera imbere cyane cyane. Guhindura imikorere ya metabolism ni ikintu gikunze kugaragara ku turemangingo twa kanseri, zimwe muri zo zishingikiriza kuri glutamine kugira ngo zongere imikorere ya TCA cycle intermediates kugira ngo zikore ibintu bigabanya, biyobora urunigi rw'ubuhumekero kandi bigakomeza gukora lipid na nucleotide biosynthesis precursors (39, 40). Ubushakashatsi buherutse gukorwa bwagaragaje ko mu ngingo z'umubiri zihura n'ikibazo cya OXPHOS, kongera guhuza imikorere ya glutamine/glutamate nabyo ari ikintu kigaragara cyane (5, 41), aho icyerekezo cya glutamine mu imikorere ya TCA giterwa n'ubukana bw'ingaruka za OXPHOS (41). Ariko, nta bimenyetso bifatika bigaragaza isano iri hagati ya neural metabolic plasticity mu mubiri n'akamaro kayo mu ndwara. Mu bushakashatsi buherutse gukorwa muri vitro, neurons zo mu mutwe za mbere zagaragaye ko zishishikariza glutamate transmissions mu miyoboro y'ubwonko, bityo zigateza imbere imikorere ya oxidative na atherosclerosis mu bihe by'ihungabana rya metabolism (42). Ni ngombwa kumenya ko mu gihe habayeho gukumira imiti ya enzyme ya TCA cycle succinate dehydrogenase, carboxylation ya pyruvate yizewe ko ikomeza gukora oxaloacetate mu mitsi ya cerebellar granule neurons (34). Ariko, akamaro k'iyi mikorere ku ngingo z'ubwonko (aho atherosclerosis ifatwa cyane cyane n'abantu bareba mu kirere) karacyafite akamaro kanini mu mikorere (43). Muri iki kibazo, amakuru yacu agaragaza ko PN zangiritse na OXPHOS mu mubiri zishobora guhinduka BCAA degradation na pyruvate carboxylation, ari zo soko ebyiri z'ingenzi zo kongeramo TCA pool intermediates. Nubwo hagaragajwe ko uruhare rwa BCAA catabolism mu mikorere y'ingufu z'imitsi rwatanzwe, uretse uruhare rwa glutamate na GABA mu gutuma habaho uburyo bwo kohereza mu mitsi (44), nta bimenyetso birahari by'iyi mikorere mu mubiri. Kubwibyo, biroroshye gutekereza ko PN zidakora neza zishobora kwishyura ikoreshwa rya TCA intermediates bitewe n'uburyo bwo guhuza mu mitsi binyuze mu kongera atherosclerosis. By’umwihariko, kongera umubare wa PCx bishobora kuba ngombwa kugira ngo hakomeze kwiyongera kwa aside aspartic, nk’uko bivugwa mu turemangingo twiyongera dufite ikibazo cya mitochondrial (45). Ariko, isesengura ryacu rya metabolomics ntiryagaragaje impinduka zikomeye mu rugero ruhamye rwa aside aspartic muri Mfn2cKO PNs (Ishusho S6A), bigaragara ko bigaragaza uburyo butandukanye bwo gukoresha aside aspartic hagati y’uturemangingo twiyongera n’uturemangingo twa nyuma ya mitochondrial. Nubwo uburyo nyabwo bwo kongera umubare wa PCx muri neurons zidakora neza mu mubiri butaramenyekana, twagaragaje ko iyi ngaruka itaratangira igira uruhare runini mu kubungabunga imiterere ya redox ya neurons, nk’uko byagaragajwe mu bushakashatsi bwa FLIM ku bice bya cerebellar. By’umwihariko, gukumira PNs kongera umubare wa PCx bishobora gutuma habaho imiterere ya oxidized kandi byihutishe urupfu rw’uturemangingo. Gukora kwangirika kwa BCAA no gushyira pyruvate mu mubiri si uburyo bwo kuranga ingirabuzimafatizo z’imikorere mibi ya mitochondrial (7). Bityo rero, bisa nkaho ari ikintu cy'ingenzi ku mitsi idafite OXPHOS, nubwo atari yo yonyine, ari ingenzi mu kwangirika kw'imitsi.
Indwara ya Cerebellar ni ubwoko butandukanye bw'indwara ya mitsi itera indwara ikunze kugaragara nka ataxia kandi akenshi yangiza PN (46). Ubu bwoko bw'imitsi bukunze kwibasirwa cyane no kudakora neza kwa mitochondrial kuko kwangirika kwayo mu mbeba bihagije kugira ngo hagaragare ibimenyetso byinshi by'imikorere biranga spinocerebellar ataxia y'umuntu (16, 47, 48). Nk'uko raporo zibivuga, icyitegererezo cy'imbeba ya transgenic ifite gene ya mutant ifitanye isano na spinocerebellar ataxia y'umuntu kandi ifite imikorere mibi ya mitochondrial (49, 50), bishimangira akamaro ko kwiga ingaruka zo kubura OXPHOS muri PNPH. Kubwibyo, birakwiye cyane gutandukanya no kwiga neza ubu bwoko bwihariye bw'imitsi. Ariko, bitewe nuko PN zigira ikibazo gikomeye ku muvuduko w'amaraso kandi zikaba zifite igice gito cy'uturemangingo two mu cerebellar, ku bushakashatsi bwinshi bushingiye ku bumenyi bw'umubiri, gutandukanya uturemangingo nk'uturemangingo twose biracyari ikibazo gikomeye. Nubwo bigoye cyane kugera ku kutagira ubwandu bw’izindi selile (cyane cyane ingirabuzimafatizo z’abantu bakuru), twahuje intambwe nziza yo kwitandukanya na FACS kugira ngo tubone umubare uhagije w’uturemangingo dushobora gusesengura proteomics, kandi tukagira poroteyine nyinshi cyane (poroteyine zigera kuri 3000) ugereranije n’amakuru ariho kuri cerebellum yose (51). Mu kubungabunga ubuzima bw’ingirabuzimafatizo zose, uburyo dutanga hano ntibudufasha gusa kugenzura impinduka mu nzira za metabolike muri mitochondria, ahubwo bunatuma tugenzura impinduka mu miterere ya cytoplasmic, byuzuza ikoreshwa rya tagi za mitochondria mu kunoza ubwoko bw’ingirabuzimafatizo. Uburyo bushya bwo kugereranya umubare wa mitochondria mu ngirabuzimafatizo zikomeye (52, 53). Ubu buryo dusobanura ntibufitanye isano gusa n’inyigo y’ingirabuzimafatizo za Purkinje, ahubwo bushobora gukoreshwa byoroshye ku bwoko ubwo aribwo bwose bw’ingirabuzimafatizo kugira ngo hakemurwe impinduka mu mikorere y’ubwonko burwaye, harimo n’izindi ngero z’imikorere mibi ya mitochondrial.
Amaherezo, twabonye uburyo bwo kuvura muri iki gikorwa cyo guhindura imikorere y’uturemangingo bushobora gusubiza inyuma burundu ibimenyetso by’ingenzi by’imihangayiko y’uturemangingo no gukumira kwangirika kw’imitsi. Kubwibyo, gusobanukirwa ingaruka z’imikorere y’imitsi ivugwa hano bishobora gutanga ubumenyi bw’ibanze ku buryo bushoboka bwo kuvura imitsi ikomeza kubaho mu gihe cy’imikorere mibi ya mitochondrial. Ubushakashatsi bw’ejo hazaza bugamije gusesengura impinduka mu mikorere y’ingufu mu zindi ngirabuzima fatizo z’ubwonko burakenewe kugira ngo hagaragazwe neza uburyo iri hame rikoreshwa ku zindi ndwara z’imitsi.
Imbeba za MitoPark zavuzwe mbere (31). Imbeba za C57BL/6N zifite genes za loxP zifatanye na Mfn2 zavuzwe mbere (18) kandi zavanzwe n'imbeba za L7-Cre (23). Imbeba ebyiri zavuyemo heterozygous zavanzwe n'imbeba za Mfn2loxP/Mfn2loxP zimeze nka homozygous kugira ngo hakorwe gene knockouts za Purkinje kuri Mfn2 (Mfn2loxP/Mfn2loxP; L7-cre). Mu itsinda ry'imibonano mpuzabitsina, allele ya Gt (ROSA26) SorStop-mito-YFP (stop-mtYFP) yashyizwemo binyuze mu buryo bwo kongeramo (20). Uburyo bwose bwo gukora inyamaswa bwakozwe hakurikijwe amabwiriza y'i Burayi, igihugu n'ibigo kandi byemejwe na LandesamtfürNatur ya Umwelt na Verbraucherschutz, Rhine-Westphalia y'Amajyaruguru, mu Budage. Umurimo w'inyamaswa nawo ukurikiza ubuyobozi bw'Ishyirahamwe ry'Uburayi ry'Ubumenyi bw'Inyamaswa rya Laboratwari.
Nyuma yo gusinzira umugore utwite yavuye mu nda, urusoro rw'imbeba rurakurwamo (E13). Inyama z'imbeba zaciwe muri Hanks' Balanced Salt Solution (HBSS) yongewemo 10 mM Hepes hanyuma zoherezwa kuri Dulbecco's Modified Eagle's Medium irimo papain (20 U/ml) na cysteine ​​​​(1μg/ml). Shyira inyama muri DMEM) hanyuma uzitandukanye binyuze mu igogora rya enzymatic. Ml) kuri 37°C mu gihe cy'iminota 20, hanyuma zigasigwa muri DMEM zongewemo 10% by'amaraso y'inka yo mu nda. Uturemangingo twatewe ku birahuri byasizwe na polylysine ku bucucike bwa 2 × 106 kuri buri sahani ya cm 6 cyangwa ku bucucike bwa 0.5 × 105 selile / cm2 kugira ngo hakoreshwe isuzuma. Nyuma y'amasaha 4, ubwo bucucike bwasimbuwe na Neurobasal serum-free medium irimo 1% B27 supplement na 0.5 mM GlutaMax. Hanyuma uturemangingo twa neurons twakomeje kubungabungwa kuri 37°C na 5% bya CO2 mu gihe cyose cy'igerageza, kandi tugaburirwa rimwe mu cyumweru. Kugira ngo twongere imbaraga zo kongera guhurirana muri vitro, 3μl (isahani yo gukuriramo ibiryo 24) cyangwa 0.5μl (isahani y'ibiryo 24) bya vector ya virusi ya AAV9 ikurikira yakoreshejwe mu kuvura uturemangingo ku munsi wa kabiri muri vitro: AAV9.CMV.PI.eGFP. WPRE.bGH (Addgene, nimero ya catalog 105530-AAV9) na AAV9.CMV.HI.eGFP-Cre.WPRE.SV40 (Addgene, nimero ya catalog 105545-AAV9).
ADN yuzuzanya ya Mfn1 na Mfn2 (yabonetse muri plasmid ya Addgene #23212 na #23213, uko bikurikirana) irangwa n'uruhererekane rwa V5 (GKPIPNPLLGLDST) kuri C-terminus, kandi ihuzwa na mCherry mu ishusho binyuze muri uruhererekane rwa T2A. Grx1-roGFP2 ni impano ya Heidelberg TP Dick DFKZ (Deutsches Krebsforschungszentrum). Mu gusimbuza kaseti ya tdTomato hakoreshejwe uburyo busanzwe bwo gushushanya, kaseti yashyizwe mu mugongo wa pAAV-CAG-FLEX-tdTomato (nimero y'ifatizo ya Addgene 28306) kugira ngo hakorwe vekteri za pAAV-CAG-FLEX-mCherry-T2A-MFN2-V5, pAAV-CAG-FLEX-mCherry-T2A-MFN1-V5 na pAAV-CAG-FLEX-Grx-roGFP2. Ingamba nk'iyi yakoreshejwe mu gukora vekteri igenzura pAAV-CAG-FLEX-mCherry. Kugira ngo hakorwe imiterere ya AAV-shPCx, hakenewe vekteri ya plasmid AAV (VectorBuilder, pAAV [shRNA] -CMV-mCherry-U6-mPcx- [shRNA#1]), irimo uruhererekane rwa ADN rukubiyemo shRNA targeting mouse PCx (5′CTTTCGCTCTAAGGTGCTAAACTCGAGTTTAGCACCCTTAGAGCGAAAG 3′) Iyobowe na promoter ya U6, mCherry ikoreshwa iyobowe na promoter ya CMV. Gukora vekteri z'inyongera za AAV byakozwe hakurikijwe amabwiriza y'uwabikoze (Cell Biolabs). Muri make, koresha plasmide itwara mCherry-T2A-MFN2-V5 (pAAV-CAG-FLEX-mCherry-T2A-MFN2-V5), mCherry-T2A-MFN1-V5 (pAAV-CAG-FLEX-mCherry) by'agateganyo Guhindura uturemangingo 293AAV-T2A-MFN1-V5), mCherry (pAAV-CAG-FLEX-mCherry) cyangwa Grx-roGFP2 (pAAV-CAG-FLEX-Grx-roGFP2) coding gene, ndetse no kwandika poroteyine ya capsid ya AAV1 na poroteyine y'inyongera Gushyira plasmid plasmid, hakoreshejwe uburyo bwa calcium phosphate. Supernatant ya virusi yabonetse hakoreshejwe uburyo bwo gukonjesha mu mwobo wumye w'urubura/ethanol hamwe n'uturemangingo twa lysed muri phosphate buffered saline (PBS). Vektori ya AAV yejejwe hakoreshejwe iodixanol gradient ultracentrifugation (amasaha 24 kuri 32.000 rpm na 4°C) kandi yinjijwemo hakoreshejwe filter ya Amicon ultra-15 centrifugal. Titer ya genome ya AAV1-CAG-FLEX-mCherry-T2A-MFN2-V5 [2.9×1013 genome copy (GC)/ml], AAV1-CAG-FLEX-mCherry (6.1×1012 GC/ml), AAV1-CAG-FLEX yari nkuko byasobanuwe mbere (54), yapimwe hakoreshejwe real-time quantitative PCR (qPCR) -MFN1-V5 (1.9×1013 GC/ml) na AAV1-CAG-FLEX-Grx-roGFP2 (8.9×1012 GC/ml).
Uturemangingo tw’ibanze twakuwe mu ifu y’urubura 1x PBS, dushyirwamo pellet, hanyuma dushyirwa mu buryo bumwe muri 0.5% Triton X-100 / 0.5% sodium deoxycholate / PBS lysis buffer irimo phosphatase na protease inhibitor (Roche). Gupima poroteyine byakozwe hakoreshejwe isuzuma rya aside bicinchoninic (Thermo Fisher Scientific). Hanyuma poroteyine zatandukanyijwe na SDS-polyacrylamide gel electrophoresis, hanyuma zishyirwa ku membrane ya polyvinylidene fluoride (GE Healthcare). Zingira ahantu hatari hazwi hanyuma ushyiremo antibody y’ibanze (reba Imbonerahamwe S1 ku bisobanuro birambuye) mu mata ya 5% muri TBST (Tris-buffered saline with Tween), intambwe zo gukaraba n’antibody y’inyongera muri TBST Incubate. Shyiramo antibody y’inyongera ijoro ryose kuri +4°C. Nyuma yo gukaraba, shyira antibody y’inyongera mu gihe cy’amasaha 2 ku bushyuhe bw’icyumba. Nyuma yaho, ushyizemo blot imwe ukoresheje antibody ya anti-β-actin, umutwaro umwe wemejwe. Kumenya hakoreshejwe uburyo bwa chemiluminescence no kongera imiterere ya chemiluminescence (GE Healthcare).
Uturemangingo twatewe mbere ku dupfundikizo tw’ibirahure twashyizwemo 4% ya paraformaldehyde (PFA) / PBS ku gihe cyagenwe ku bushyuhe bw’icyumba mu gihe cy’iminota 10. Udupfundikizo twabanje gushyirwamo 0.1% ya Triton X-100 / PBS mu gihe cy’iminota 5 ku bushyuhe bw’icyumba, hanyuma tugashyirwa mu gice cyo gufunga [3% ya albumin y’inka (BSA) / PBS]. Ku munsi wa kabiri, udupfundikizo twogejwe n’igice cyo gufunga hanyuma dushyirwamo antibody ya kabiri ikoreshwa na fluorophore mu gihe cy’amasaha 2 ku bushyuhe bw’icyumba; amaherezo, ingero zogejwe neza muri PBS hamwe na 4′,6-diamidino-2 -Phenylindole (DAPI) irashyirwa ku gipimo cya 4′,6-diamidino-2 -Phenylindole (DAPI) hanyuma ishyirwa ku gice cyo gufunga cya mikorosikopi hakoreshejwe Aqua-Poly / Mount.
Imbeba (iz'abagabo n'iz'ingore) zatewe ikinya binyuze mu gutera ketamine (130 mg/kg) na xylazine (10 mg/kg) mu nda y'umura, hanyuma zigaterwa mu buryo bworoshye hifashishijwe carprofen analgesic (5 mg/kg), hanyuma zigashyirwa mu gikoresho cya stereotactic (Kopf) gifite agakoresho gashyushye. Shyira agahanga mu mwanya wacyo hanyuma ukoreshe imashini yo gutobora amenyo kugira ngo ugabanye igice cya cerebellar cortex gihuye n'igufwa rya mis (kuva kuri lambda: umurizo wa 1.8, uruhande rwa 1, rujyanye na lobules IV na V). Koresha urushinge rwa syringe rufite umugozi kugira ngo ukore umwobo muto mu gahanga kugira ngo wirinde kwangiza imitsi y'amaraso iri hepfo. Hanyuma umuyoboro muto w'ikirahure ushyirwa buhoro buhoro mu mwobo muto (kuva kuri -1.3 kugeza kuri -1 ku ruhande rw'imbere rw'umuyoboro wa dura), hanyuma 200 kugeza kuri 300 nl AAV igashyirwa mu cyuma gitera imitsi mito (Narishige) hakoreshejwe intoki (Narishige) inshuro nyinshi ku muvuduko muto mu gihe cy'iminota 10 kugeza kuri 20. Nyuma yo guterwa, shyira agace k'imitsi mu minota 10 kugira ngo virusi ikwirakwire burundu. Nyuma yo gukuramo imitsi, uruhu rushyirwamo umuti witonze kugira ngo rugabanye ububabare bw'ibikomere kandi rureke gukira. Inyamaswa zavuwe imiti igabanya ububabare (caspofen) iminsi myinshi nyuma yo kubagwa, muri icyo gihe imiterere yazo y'umubiri yakurikiranwaga neza hanyuma zigahita zicwa mu gihe cyagenwe. Uburyo bwose bwakozwe hakurikijwe amabwiriza y'i Burayi, igihugu n'ibigo kandi byemejwe na LandesamtfürNatur yo muri Umwelt na Verbraucherschutz, Rhine-Westphalia y'Amajyaruguru, mu Budage.
Inyamaswa zahawe ikinyabutabire hakoreshejwe ketamine (100 mg/kg) na xylazine (10 mg/kg), maze umutima utangira gutogoshwa na 0.1 M PBS, hanyuma hagashyirwamo 4% PFA muri PBS. Uduce twakuwemo uduce duto dushyirwamo 4% PFA/PBS mu ijoro ryose kuri 4°C. Imbugita inyeganyega (Leica Microsystems GmbH, Vienna, Otirishiya) yakoreshejwe mu gutegura ibice bya sagittal (ubugari bwa 50 μm) bivuye mu bwonko buhamye muri PBS. Keretse byavuzwe ukundi, gusiga irangi ku bice bireremba nk'uko byasobanuwe haruguru (13) ku bushyuhe bw'icyumba no kuvanga. Muri make, mbere na mbere, uduce twabonetse twashyizwemo 0.5% Triton X-100/PBS mu minota 15 ku bushyuhe bw'icyumba; kuri epitopes zimwe na zimwe (Pcx na Shmt2), hakoreshejwe tris-EDTA buffer kuri 80°C (PH 9) shyushya uduce mu minota 25 aho kuba iyi ntambwe. Hanyuma, ibice byashyizwemo antibody y’ibanze (reba Imbonerahamwe S1) muri buffer yo kuziba (3% BSA / PBS) kuri 4°C nijoro bivanze. Umunsi wakurikiyeho, ibice byogejwemo buffer yo kuzibamo hanyuma bishyirwamo antibody ya kabiri ijyanye na fluorophore mu gihe cy’amasaha 2 ku bushyuhe bw’icyumba; amaherezo, ibice byogejwe neza muri PBS, bishyirwamo DAPI, hanyuma bikosorwa na AquaPolymount On a microscope slide.
Mikorosikopi ya laser scanning confocal (TCS SP8-X cyangwa TCS Digital Light Sheet, Leica Microsystems) ifite laser y'umweru hamwe na laser ya ultraviolet ya diode 405 yakoreshejwe mu gushushanya icyitegererezo. Mu gukurura fluorophore no gukusanya ikimenyetso hifashishijwe Hybrid Detector (HyDs), porogaramu ya LAS-X yakoreshejwe mu gukusanya amashusho ajyanye na Nyquist sampling mu buryo bukurikiranye: ku ma paneli atari ingano, ni ibimenyetso bihinduka cyane (urugero, mu turemangingo two mu mubiri na dendrites) mtYFP) Koresha HyD kugira ngo umenye umubare wa PN mu buryo bwa BrightR). Gate kuva kuri 0.3 kugeza kuri 6 ns ikoreshwa kugira ngo ugabanye inyuma.
Gufata amashusho y’uturemangingo mu buryo bw’igihe nyacyo. Nyuma yo gutondeka mu buryo bwa Neurobasal-A burimo 1% B27 supplement na 0.5 mM GlutaMax, uturemangingo twahise duterwa ku birahuri bya poly-l-lysine (μ-Slide8 Well, Ibidi, catalog nomero 80826), hanyuma tukabigumana kuri 37°C na 5% CO2 mu gihe cy’isaha 1 kugira ngo uturemangingo dushobore gutuza. Gufata amashusho mu buryo bw’igihe nyacyo byakorewe kuri mikorosikopi ya Leica SP8 scanning confocal ifite laser yera, HyD, lenzi ya 63×[1.4 numeral aperture (NA)] oil objective lens hamwe n’urwego rwo gushyushya.
Imbeba yahise iterwa ikinyabutabire na dioxyde de carbone hanyuma icibwa umutwe, ubwonko buhita bukurwa mu gahanga, bucibwamo ubugari bwa 200μm (ku igerageza rya 13C) cyangwa ubugari bwa 275μm (ku igerageza rya fotoni ebyiri) igice cya sagittal cyuzuyemo ibi bikurikira. Ice cream (HM-650 V, Thermo Fisher Scientific, Walldorf, Ubudage) yuzuyemo ibi bikurikira: 125 mM ikonje, ifunze neza (95% O2 na 5% CO2) Ca2 nkeya + amazi y’ubwonko y’ubukorano (ACSF) NaCl, 2.5 mM KCl, 1.25 mM sodium phosphate buffer, 25 mM NaHCO3, 25 mM glucose, 0.5 mM CaCl2 na 3.5 mM MgCl2 (umuvuduko wa osmotic wa 310 kugeza 330 mmol). Subiza ibice by'ubwonko byabonetse mu cyumba cyo kwinjiriramo kirimo Ca2 + ACSF iri hejuru (125.0 mM NaCl, 2.5 mM KCl, 1.25 mM sodium phosphate buffer, 25.0 mM NaHCO3, 25.0 mM d-glucose, 1.0 mM CaCl2 na 2.0 mM MgCl2) Medium) pH 7.4 na 310 kugeza 320 mmol).
Mu gihe cyo gufata amashusho, uduce twimuwe mu cyumba cyagenewe gufata amashusho, maze igerageza rikorwa hifashishijwe ACSF ikomeza gusohora ku bushyuhe buhoraho bwa 32° kugeza 33°C. Mikorosikopi ya laser scanning multiphoton (TCS SP8 MP-OPO, Leica Microsystems) ifite lens ya Leica 25x objective (NA 0.95, amazi), Ti: Sapphire laser (Chameleon Vision II, Coherent) yakoreshejwe mu gufata amashusho y'uduce. FLIM module (PicoHarp300, PicoQuant).
FLIM ya Grx1-roGFP2. Impinduka mu miterere ya redox ya cytoplasmic ya PN zapimwe hakoreshejwe FLIM ifite photon ebyiri mu duce tw'ubwonko twa sagittal, aho biosensor ya Grx1-roGFP2 yibandaga kuri PN. Mu cyiciro cya PN, agace ko gufata katoranywa nko muri μm 50 kugeza kuri 80 munsi y'ubuso bw'agace kugira ngo harebwe ko hari PN ifatika (ni ukuvuga kutagira imiterere y'udupira cyangwa impinduka mu miterere y'ubwonko ku duce twa dendrites) hamwe na sensor ya roGFP2 ifite double positive na AAV encoding shRNA PCx cyangwa umurongo wayo wo kugenzura (buri mCherry ihuza). Gukusanya amashusho ya single-stack hamwe na 2x digital zoom [ubugari bw'umuvuduko: 890 nm; 512 nm 512 pixels]. Gutahura: HyD y'imbere, itsinda ry'urufunguzo rwa fluorescein isothiocyanate (FITC)] n'ikigereranyo cy'ishusho mu minota 2 kugeza kuri 3 birakoreshwa kugira ngo harebwe ko photon zihagije zakusanyijwe (photon 1000 zose hamwe) kugira ngo zihuzwe. Uburyo bwo kumenya neza imiterere ya probe ya Grx1-roGFP2 no kugenzura imiterere ya FLIM byakozwe hagenzurwa agaciro k'ubuzima bwa roGFP2 ubwo hongerwagaho 10 mM H2O2 yo hanze muri ACSF (kugira ngo hongerwe oxidation, bigatuma ubuzima bwiyongera), hanyuma hongerwamo 2 mM dithiothreitol (bigabanya urugero rwo kugabanuka, bigatuma ubuzima bugabanuka) (Ishusho S8, D kugeza kuri G). Koresha porogaramu ya FLIMfit 5.1.1 kugira ngo usesengure ibyavuye mu bushakashatsi, ushyire umurongo umwe wa exponential decay w'ishusho yose kuri IRF yapimwe (imikorere yo gusubiza igikoresho), kandi χ2 ni hafi 1. Kugira ngo ubare ubuzima bwa PN imwe, agapfukamunwa gakikije umubiri w'imitsi kashushanyijwe n'intoki, kandi impuzandengo y'ubuzima muri buri gapfukamunwa yakoreshejwe mu gupima.
Isesengura ry’ubushobozi bwa Mitochondrial. Nyuma y’uko igice cy’ingufu gishyizwemo 100 nM TMRM yongewemo mu buryo butaziguye kuri ACSF yakozwe mu gihe cy’iminota 30, impinduka za mitochondrial potential za PN zapimwe hakoreshejwe mikorosikopi ebyiri. Isuzuma rya TMRM ryakozwe hakoreshejwe gukurura probe kuri 920 nm no gukoresha HyD y’imbere (tetramethylrhodamine isothiocyanate: 585/40 nm) kugira ngo hakusanywe ibimenyetso; hakoreshejwe uburebure bumwe bwo gushyushya ariko hakoreshejwe HyD y’imbere itandukanye (FITC :525/50) kugira ngo hamenyekane mtYFP. Koresha porogaramu ya ImageJ's Image Calculator kugira ngo usuzume ubushobozi bwa mitochondrial ku rwego rw’akaremangingo kamwe. Muri make, ikigereranyo cya plug-in: signal = min (mtYFP, TMRM) gikoreshwa mu kumenya agace ka mitochondrial kerekana ikimenyetso cya TMRM muri Purkinje Somali mu ishusho imwe ya confocal y’umuyoboro ujyanye nayo. Hanyuma agace ka pixel kari muri mask ivuyemo karapimwa, hanyuma kagashyirwa ku ishusho ijyanye n'umupaka umwe w'umuyoboro wa mtYFP kugira ngo haboneke igice cya mitochondrial kigaragaza ubushobozi bwa mitochondrial.
Ishusho yakuwemo ihurizo hakoreshejwe porogaramu ya Huygens Pro (Scientific Volume Imaging). Ku mashusho yasuzumwe y'amatafari, ihurizo ry'amatafari rimwe rikorwa hakoreshejwe uburyo bwo kudoda bwikora bwatanzwe na porogaramu ya LAS-X. Nyuma yo gupima amashusho, koresha ImageJ na Adobe Photoshop kugira ngo urusheho gutunganya ishusho no guhindura ubwiza n'itandukaniro ryayo mu buryo bumwe. Koresha Adobe Illustrator mu gutegura amashusho.
Isesengura rya mtDNA. Umubare w'ibisebe bya mtDNA wapimwe ku bice bya cerebellar byanditseho antibody antibodies militants ADN hakoreshejwe mikorosikopi ya confocal. Buri gace k'ingenzi karemewe umubiri w'akaremangingo n'ingirabuzimafatizo za buri karemangingo, maze agace kabigenewe kabarwa hakoreshejwe porogaramu ya Multi Measure plug-in (ImageJ software). Kuramo agace ka nuclear mu gace k'umubiri w'akaremangingo kugira ngo ubone agace ka cytoplasmic. Amaherezo, porogaramu ya Analyse Particles plug-in (ImageJ software) yakoreshejwe mu gupima mu buryo bwikora ingingo za ADN za cytoplasmic zigaragaza mtDNA ku ishusho y'umupaka, kandi ibisubizo byabonetse byagereranyijwe ku mpuzandengo ya PN y'imbeba za CTRL. Ibisubizo bigaragazwa nk'umubare mpuzandengo wa nucleosides kuri buri karemangingo.
Isesengura ry’imikorere ya poroteyine. Koresha porogaramu ya ImageJ yo kubara amashusho kugira ngo usuzume imikorere ya poroteyine muri PN ku rwego rw’akaremangingo kamwe. Muri make, mu ishusho y’icyiciro kimwe cy’umuyoboro ujyanye, binyuze mu kigereranyo: ikimenyetso = min (mtYFP, antibody), agace ka mitochondrial kagaragaza uburyo bwo kwirinda indwara ku muyoboro runaka muri Purkina karamenyekana. Hanyuma agace ka pixel kari muri mask ivuyemo karapimwa, hanyuma kagashyirwa ku ishusho ijyanye n’umuyoboro umwe w’umuyoboro wa mtYFP kugira ngo ubone igice cya mitochondrial cya poroteyine yagaragajwe.
Isesengura ry'ubucucike bw'uturemangingo twa Purkinje. Porogaramu yo gupima ubucucike bw'uturemangingo ya ImageJ yakoreshejwe mu gusuzuma ubucucike bwa Purkinje igabanya umubare w'uturemangingo twa Purkinje twabazwe n'uburebure bw'impeta y'ubwonko yari ifitwe n'uturemangingo twabazwe.
Gutegura no gukusanya ingero. Ubwonko bw’itsinda ry’abagenzuzi n’imbeba za Mfn2cKO bwashyizwe muri 2% PFA/2.5% glutaraldehyde muri 0.1 M phosphate buffer (PB), hanyuma ibice bya coronal bitegurwa hakoreshejwe ciliates (Leica Mikrosysteme GmbH, Vienna, Otirishiya) (Ubunini bwa 50 kugeza kuri 60 μm). Hanyuma shyira muri PB buffer muri 1% os tetraoxide na 1.5% potasiyumu ferrocyanide ku bushyuhe bw’icyumba mu gihe cy’isaha 1. Ibice byogejwe inshuro eshatu n’amazi yaciwe, hanyuma bisiga irangi rya 70% ethanol irimo 1% uranyl acetate mu gihe cy’iminota 20. Ibice byashyizwemo alcohol yagenwe hanyuma bishyirwa muri Durcupan ACM (Araldite casting resin M) epoxy resin (Electron Microscopy Sciences, catalog nomero 14040) hagati y’ibirahuri bya silikoni, hanyuma birangira kuri 60°C Polymerize mu ifuru mu gihe cy’amasaha 48. Agace k’ubwonko bw’inyuma katoranyijwe maze ibice bito cyane bya nanometero 50 byacishwa kuri Leica Ultracut (Leica Mikrosysteme GmbH, Vienne, Otirishiya) hanyuma bitoranywa ku mugozi w’umuringa wa 2 × 1 mm usizwe na polystyrene film. Ibice byasizwe irangi rya 4% uranyl acetate muri H2O mu minota 10, byozwa na H2O inshuro nyinshi, hanyuma bikoreshwa na Reynolds lead citrate muri H2O mu minota 10, hanyuma byozwa na H2O inshuro nyinshi. Mikorografi zafashwe hakoreshejwe mikorosikopi ya elegitoroniki ya Philips CM100 (Thermo Fisher Scientific, Waltham, MA, USA) hakoreshejwe kamera ya digitale ya TVIPS (Tietz Video and Image Processing System) TemCam-F416 (TVIPS GmbH, Gauting, USA). Ubudage.
Ku mbeba zanduye AAV, ubwonko bwaciwemo ibice bibiri by'ubugari bwa sagittal, maze ubwonko busuzumwa hakoreshejwe mikorosikopi ya fluorescence kugira ngo hamenyekane impeta yanduye AAV (ni ukuvuga mCherry expressing). Hakoreshwa gusa igerageza aho inshinge za AAV zituma urwego rwa selile ya Purkinje rukora neza cyane (ni ukuvuga hafi urwego rwose) byibuze mu mpeta ebyiri zikurikiranye za cerebellar. Umuzunguruko wa AAV wahinduwe waciwemo mikorosi kugira ngo ushyirwemo mu ijoro ryose (4% PFA na 2.5% glutaraldehyde muri 0.1 M cocoate buffer) hanyuma ugakomeza gutunganywa. Kugira ngo EPON ishyirwemo, imiterere y'umubiri wogejwe na 0.1 M sodium cocoate buffer (Applichem), hanyuma ishyirwamo 2% OsO4 (os, Science Services; Caco) muri 0.1 M sodium cocoate buffer (Applichem) amasaha 4, hanyuma woge mu gihe cy'amasaha 2. Subiramo inshuro 3 ukoresheje 0.1 M cocamide buffer. Nyuma y’ibyo, urukurikirane rw’amavuta ya ethanoli rwakoreshejwe mu gukamura buri gikoresho cya ethanoli kuri dogere selisiyusi 4 mu gihe cy’iminota 15 kugira ngo inyure amazi mu mubiri. Uduce twashyizwe muri propylene oxide hanyuma dushyirwa muri EPON (Sigma-Aldrich) mu gihe cy’amasaha 2. Shyira utwo duce muri EPON nshya ku bushyuhe bw’icyumba mu gihe cy’amasaha 2, hanyuma utwo duce dushire kuri dogere selisiyusi 62 mu gihe cy’amasaha 72. Koresha ultramicrotome (Leica Microsystems, UC6) n’icyuma cya diyama (Diatome, Biel, Switzerland) kugira ngo ukate ibice bya 70 nm bito cyane, hanyuma ushyireho ibara rya 1.5% uranyl acetate mu gihe cy’iminota 15 kuri dogere selisiyusi 37, hanyuma ushyireho ibara rya citrate y’icyitegererezo mu gihe cy’iminota 4. Imikorografi ya ethanoli yafashwe hakoreshejwe mikorosikopi ya JEM-2100 Plus transmission electron (JEOL) ifite Camera OneView 4K 16-bit (Gatan) na porogaramu ya DigitalMicrograph (Gatan). Mu gusesengura, mikorografi za elegitoroniki zabonetse hakoreshejwe zoom ya dijitali ya 5000× cyangwa 10.000×.
Isesengura ry’imiterere ya mitochondria. Ku isesengura ryose, imiterere ya mitochondria ku giti cyayo yagaragajwe n’intoki mu mashusho ya digitale hakoreshejwe porogaramu ya ImageJ. Ibipimo bitandukanye by’imiterere birasesengurwa. Ubucucike bwa mitochondria bugaragazwa nk'ijanisha ryabonetse hagabanywa agace kose ka mitochondria ka buri selile n’agace ka cytoplasm (agace ka cytoplasm = agace k’agace k’uturemangingo-agace k’ingirabuzimafatizo) × 100. Ubuzenguruke bwa mitochondria bubarwa hakoreshejwe formula [4π∙ (agace/uruziga rwa 2)]. Imiterere ya mitochondria yasesenguwe kandi igabanywamo ibyiciro bibiri ("tubular" na "blister") hakurikijwe imiterere yabyo y'ingenzi.
Isesengura ry'umubare wa Autophagosome/lysosome n'ubucucike. Koresha porogaramu ya ImageJ kugira ngo ugaragaze imiterere ya buri autophagosome/lysosome mu ishusho ya digitale. Agace ka Autophagosome/lysosome kagaragazwa nk'ijanisha ryabazwe hagabanywa agace kose k'imiterere ya autophagosome/lysosome ka buri selile n'agace ka cytoplasm (agace ka cytoplasm = agace k'agace k'ingirabuzimafatizo) × 100. Ubucucike bwa autophagosomes/lysosomes bubarwa hagabanywa umubare wose n'umubare w'imiterere ya autophagosome/lysosome kuri buri selile (mu bijyanye n'agace ka cytoplasm) (agace ka cytoplasmi = agace k'agace k'ingirabuzimafatizo).
Gushyiramo ibirango byo gutandukanya ibice by’umubiri no gutegura icyitegererezo. Ku bipimo bisaba gushyiramo ibirango bya glucose, shyira ibice by’ubwonko by’umubiri mu cyumba cyo kwinjiriramo mbere yo guteramo, kirimo karubone yuzuye (95% O2 na 5% CO2), Ca2 + ACSF nyinshi (125.0 mM NaCl, 2.5 mM KCl, 1.25 mM sodium phosphate buffer, 25.0 mM NaHCO 3, 25.0 mM d-glucose, 1.0 mM CaCl 2 na 2.0 mM MgCl 2, yahinduwe kuri pH 7.4 na 310 kugeza 320 mOsm), aho glucose ari 13 C 6- Isimbura glucose (Eurisotop, nimero ya kataloge CLM-1396). Ku bipimo bisaba gushyiramo pyruvate, shyira ibice by'ubwonko by'ako kanya kuri Ca2 + ACSF iri hejuru (125.0 mM NaCl, 2.5 mM KCl, 1.25 mM sodium phosphate buffer, 25.0 mM NaHCO3, 25.0 mM d-glucose, 1.0 mM CaCl2 na Ongeraho 2.0mM MgCl2, hindura pH 7.4 na 310 kugeza kuri 320mOsm), hanyuma wongereho 1mM 1-[1-13C]pyruvate (Eurisotop, nimero ya kataloge CLM-1082). Shyira ibice mu gihe cy'iminota 90 kuri 37°C. Mu mpera z'igerageza, ibice byasutswe vuba hakoreshejwe umuti w'amazi (pH 7.4) urimo 75 mM ammonium carbonate, hanyuma bihuzwa muri 40:40:20 (v:v:v) acetonitrile (ACN): methanol: amazi. Nyuma y'uko ibice bishyizwe ku rubura mu gihe cy'iminota 30, ingero zashyizwe kuri centrifuge kuri 21.000 g mu gihe cy'iminota 10 kuri 4°C, hanyuma supernatant isobanutse yumishwa muri SpeedVac concentrator. Agace k'ibinyabutabire byumye kavuyemo kabikwa kuri -80°C kugeza igihe isesengura ryakorewe.
Isesengura rya chromatography y'amazi-mass spectrometry rya aside amine 13 zifite label ya C. Ku isesengura rya chromatography y'amazi-mass spectrometry (LC-MS), agace ka metabolite kasubiwemo muri 75μl y'amazi ya LC-MS (Honeywell). Nyuma yo gupima kuri 21.000 g mu gihe cy'iminota 5 kuri 4°C, 20μl y'umusemburo wasobanuwe wakoreshejwe mu isesengura rya aside amine flux, mu gihe andi mashanyarazi yakoreshejwe mu isesengura rya anion (reba hepfo). Isesengura rya aside amine ryakozwe hakoreshejwe protocole ya benzoyl chloride derivatization yavuzwe mbere (55, 56). Mu ntambwe ya mbere, 10μl ya 100 mM sodium carbonate (Sigma-Aldrich) yongewe kuri 20μl y'isesengura rya metabolite, hanyuma 10μl ya 2% benzoyl chloride (Sigma-Aldrich) yongewe kuri LC grade ACN. Icyitegererezo cyasuzumwe mu buryo bw'igihe gito hanyuma gishyirwa kuri centrifuge kuri 21.000 g mu gihe cy'iminota 5 kuri 20°C. Subiza supernatant yasukuwe mu kabari ka 2 ml autosampler gafite ikirahure cy'inyuguti (200 μl volume). Icyitegererezo cyasesenguwe hakoreshejwe sisitemu ya Acquity iClass ultra-high performance LC (Amazi) ihujwe na Q-Exactive (QE)-HF (Ultra High Field Orbitrap) high-resolution precision mass spectrometer (Thermo Fisher Scientific). Mu gusesengura, 2μl y'icyitegererezo cyavuyemo cyashyizwe mu gikoresho cya silica T3 (Amazi) gifite uburebure bwa 100×1.0 mm gifite uduce twa 1.8μm. Igipimo cy'amazi ni 100μl/min, kandi sisitemu ya buffer igizwe na buffer A (10 mM ammonium formate na 0.15% formic acid mu mazi) na buffer B (ACN). Uburemere ni ubu bukurikira: 0%B mu minota 0; 0%B. 0 kugeza kuri 15% B ku munota wa 0 kugeza kuri 0.1; 15 kugeza kuri 17% B ku munota wa 0.1 kugeza kuri 0.5; B ku munota wa 17 kugeza kuri 55% ku munota wa 0.5 kugeza kuri 14; B ku munota wa 55 kugeza kuri 70% ku munota wa 14 kugeza kuri 14.5; ku munota wa 14.5 kugeza kuri 70 kugeza kuri 100% B ku munota wa 18; 100% B ku munota wa 18 kugeza kuri 19; 100 kugeza kuri 0% B ku munota wa 19 kugeza kuri 19.1; 0% B ku munota wa 19.1 kugeza kuri 28 (55, 56). Spektrometero y'uburemere ya QE-HF ikora mu buryo bwa ionization nziza ifite uburebure bwa m/z (igipimo cy'uburemere/charge) kuva kuri 50 kugeza kuri 750. Ubushobozi bukoreshwa ni 60.000, naho intego ya ion yo kugenzura (AGC) yabonetse ni 3 × 106, kandi igihe ntarengwa cya ion ni milisegonda 100. Isoko ya ionization ya electrospray (ESI) ishyushye ikora kuri voltage ya spray ya 3.5 kV, ubushyuhe bwa capillary bwa 250 ° C, sheath airflow ya 60 AU (ibice by'ibanga), n'umwuka w'inyongera wa 20 AU. 250 ° C. Lens ya S ishyirwa kuri 60 AU.
Isesengura rya Anion chromatography-MS rya aside organic ya 13C. Ingano ya metabolite isigaye (55μl) yasesenguwe hakoreshejwe sisitemu ya Dionex ion chromatography (ICS 5000+, Thermo Fisher Scientific) ihujwe na QE-HF mass spectrometer (Thermo Fisher Scientific). Muri make, 5μl y'ibikomoka kuri metabolite yashyizwe mu nkingi ya Dionex IonPac AS11-HC ifite HPLC (2 mm × 250 mm, ingano y'uduce 4μm, Thermo Fisher Scientific) mu buryo bwo gusunika igice cy'urukiramende hamwe n'igipimo cyo kuzuza cya 1.) Dionex IonPac AG11-HC guard column (2 mm x 50 mm, 4μm, Thermo Fisher Scientific). Ubushyuhe bw'inkingi buguma kuri 30°C, kandi autosampler ishyirwa kuri 6°C. Koresha cartridge ya potasiyumu hydroxide ifite amazi ya deionized kugira ngo ukore gradient ya potasiyumu hydroxide binyuze muri generator ya eluent. Gutandukanya metabolites ku gipimo cya 380μl/min, hakoreshejwe ubu buryo bukurikira: iminota 0 kugeza kuri 3, 10 mM KOH; iminota 3 kugeza kuri 12, 10 kugeza kuri 50 mM KOH; iminota 12 kugeza kuri 19, 50 kugeza kuri 100 mM KOH; iminota 19 kugeza kuri 21, 100 mM KOH; iminota 21 kugeza kuri 21.5, 100 kugeza kuri 10 mM KOH. Inkingi yongeye kuringanizwa munsi ya 10 mM KOH mu gihe cy'iminota 8.5.
Ibinyabutabire bya eluted bihuzwa n'umuyoboro w'inyongera wa isopropanol wa 150μl/min nyuma y'inkingi hanyuma bigashyirwa kuri spectrometer y'uburemere ifite ubushobozi bwo hejuru ikora mu buryo bwa ionization ya negative. MS ikurikirana ingano y'uburemere kuva kuri m/z 50 kugeza 750 hamwe n'umusaruro wa 60.000. AGC ishyirwa kuri 1 × 106, kandi igihe ntarengwa cya iyoni gikomeza kuri 100 ms. Isoko rya ESI rishyushye ryakoreshejwe kuri voltage ya spray ya 3.5 kV. Izindi genamiterere ry'isoko rya iyoni ni izi zikurikira: ubushyuhe bwa capillary 275°C; sheath gaz flow, 60 AU; support gaz flow, 20 AU kuri 300°C, na S lens setting kuri 60 AU.
Isesengura ry'amakuru ya metabolites ya 13C ifite ikirango. Koresha porogaramu ya TraceFinder (version 4.2, Thermo Fisher Scientific) mu isesengura ry'amakuru ku gipimo cya isotope. Indangamuntu ya buri kintu yemejwe n'icyitegererezo cyizewe kandi yasesenguwe ku giti cye. Kugira ngo hakorwe isesengura ry'inyongera ya isotope, agace ka chromatogram ya ion yakuwemo (XIC) ya buri isotope ya 13C (Mn) kakuwe muri [M + H] +, aho n ari umubare wa karuboni w'icyigize kigamije, gikoreshwa mu gusesengura aside amine cyangwa [MH] + gikoreshwa mu gusesengura anions. Ubuziranenge bwa XIC buri munsi y'ibice bitanu kuri miliyoni, naho ubuziranenge bwa RT ni iminota 0.05. Isesengura ry'inyongera rikorwa hakorwa ikigereranyo cya buri isotope yabonetse n'igiteranyo cya isotope zose z'icyigize kijyanye nayo. Ibi bigereranyo bitangwa nk'agaciro k'ijanisha kuri buri isotope, kandi ibisubizo bigaragazwa nka molar percentage enrichment (MPE), nk'uko byasobanuwe mbere (42).
Agace k'imitsi y'ubwonko kakonjeshejwe kahinduwe mu buryo bumwe mu buryo bukonje cyane bwa 80% methanol (v/v), gashyirwamo umwuka, hanyuma gashyirwa kuri -20°C mu gihe cy'iminota 30. Ongera uvange icyitegererezo hanyuma uvange kuri +4°C mu gihe cy'iminota 30. Icyitegererezo cyashyizwemo centrifuge kuri 21.000 g mu gihe cy'iminota 5 kuri 4°C, hanyuma supernatant yavuyemo irakusanywa ikaniyumisha hakoreshejwe SpeedVac concentrator kuri 25°C kugira ngo hakorwe isesengura rikurikiraho. Nkuko byasobanuwe haruguru, isesengura rya LC-MS ryakozwe kuri aside amine zo mu turemangingo twatoranijwe. Hakoreshejwe TraceFinder (version 4.2, Thermo Fisher Scientific), isesengura ry'amakuru ryakozwe hakoreshejwe uburemere bwa monoisotopic bwa buri kintu. Gutunganya amakuru ya metabolite hakoreshejwe porogaramu ya preprocessCore (57).
Gutegura ibice. Imbeba yahise iterwa ikinyabutabire na dioxyde de carbone hanyuma icibwa umutwe, ubwonko buhita bukurwa mu gahanga, maze icyuma cyuzuye urubura gishyuha (HM-650 V, Thermo Fisher Scientific, Walldorf, Ubudage) gikoreshwa mu kuyikatamo ibice bya sagittal bya 300 kugeza kuri 375 μm. Gasification ya karubone ikonje (95% O2 na 5% CO2) Ca2 nkeya + ACSF (125.0 mM NaCl, 2.5 mM KCl, 1.25 mM sodium phosphate buffer, 25.0 mM NaHCO3, 25.0 mM d-glucose, 1.0 mM CaCl2 na 6.0 mM MgCl2. Hindura kuri pH 7.4 na 310 kugeza 330 mOsm). Subiza ibice by'ubwonko byabonetse mu cyumba kirimo Ca2 + ACSF iri hejuru (125.0 mM NaCl, 2.5 mM KCl, 1.25 mM sodium phosphate buffer, 25.0 mM NaHCO3, 25.0 mM d-glucose, 4.0 mM CaCl2 na mM 3.5 MgCl2) pH 7.4 na 310 kugeza 320 mOsm). Bika ibice mu gihe cy'iminota 20 kugeza 30 kugira ngo bibashe kugarurwa mbere yo kwandika.
gufata amashusho. Icyiciro cya mikorosikopi gifite icyumba cyo gufata amashusho gihamye hamwe n'indorerwamo y'amazi ya 20x (Scientifica) cyakoreshejwe mu gufata amashusho yose. Uturemangingo twa Purkinje twagaragajwe hifashishijwe (i) ingano y'umubiri, (ii) aho cerebellum iherereye, na (iii) uburyo gene ya mtYFP igaragara. Umuyoboro wa patch ufite ubushobozi bwo guhangana n'impande za megohms 5 kugeza kuri 11 ukurwamo na kapilari y'ikirahure ya borosilicate (GB150-10, 0.86 mm × 1.5 mm × 100 mm, Science Products, Hofheim, Germany) na pipette Instruments (P-1000, Sutter), Novato, CA). Amajwi yose yafashwe hifashishijwe ELC-03XS npi patch clamp amplifier (npi electronic GmbH, Tam, Germany), yagenzurwaga na software Signal (version 6.0, Cambridge Electronic, Cambridge, UK). Igerageza ryafashwe ku gipimo cya 12.5 kHz. Ikimenyetso gishungurwa hakoreshejwe filter ebyiri za Bessel zigenda gahoro zifite umurongo wa cutoff wa 1.3 na 10 kHz. Capacitance ya membrane na pipette yishyurwa na circuit compensation ikoresheje amplifier. Igerageza ryose ryakozwe hifashishijwe kamera ya Orca-Flash 4.0 (Hamamatsu, Gerden, Ubudage), yagenzurwaga na software ya Hokawo (version 2.8, Hamamatsu, Gerden, Ubudage).
Gutegura no gusesengura buri gihe ingirabuzimafatizo zose. Mbere yo kwandika, uzuza pipette umuti w’imbere urimo ibintu bikurikira: 4.0 mM KCl, 2.0 mM NaCl, 0.2 mM EGTA, 135.0 mM potassium gluconate, 10.0 mM Hepes, 4.0 mM ATP (Mg), 0.5 mM Guanosine triphosphate (GTP) (Na) na 10.0 mM creatinine phosphate byahinduwe kuri pH 7.25, kandi umuvuduko wa osmotic wari 290 mOsm (sucrose). Nyuma yo gukoresha imbaraga za 0 pA kugira ngo membrane ivunike, ubushobozi bwa membrane busigaye bwapimwe. Ubudahangarwa bw’injira bupimwa hakoreshejwe imiyoboro ya hyperpolarized ya -40, -30, -20, na -10 pA. Pima ingano y’umuvuduko wa voltage hanyuma ukoreshe itegeko rya Ohm kugira ngo ubare ubudahangarwa bw’injira. Igikorwa cyakozwe ku buryo bwikora cyafashwe mu gice cy’amashanyarazi mu gihe cy’iminota 5, maze sPSC iramenyekana kandi ipimwa muri Igor Pro (version 32 7.01, WaveMetrics, Lake Oswego, Oregon, USA) hakoreshejwe script yo kumenya ibintu byikora. Umuyoboro wa IV n’umuyoboro uhoraho bipimwa hakoreshejwe gufunga bateri ku buryo butandukanye (kuva kuri -110 mV) no kongera voltage mu ntambwe 5 mV. Umusaruro wa AP wageragejwe hakoreshejwe umuyoboro ugabanya amashanyarazi. Shyira akagari kuri -70 mV mu gihe ukoresha umuyoboro ugabanya amashanyarazi. Hindura ingano y’intambwe ya buri gice cyo gufata amajwi ukwacyo (10 kugeza 60 pA). Bara umuyoboro ntarengwa wa AP ubara intoki imivuduko y’amaraso itera umuyoboro mwinshi wa AP. Umuyoboro wa AP usesengurwa hakoreshejwe derivative ya kabiri ya depolarization pulse itangira gutera AP imwe cyangwa nyinshi.
Imiterere n'isesengura ry'agace gafite imyobo. Kora ifatwa ry'agace gafite imyobo ukoresheje protocole zisanzwe. Koresha pipette idafite ATP na GTP idafite ibi bikurikira: 128 mM gluconate K, 10 mM KCl, 10 mM Hepes, 0.1 mM EGTA na 2 mM MgCl2, hanyuma uhindure pH 7.2 (ukoresheje KOH). ATP na GTP bikurwa mu gisubizo cy'imbere mu ngirabuzimafatizo kugira ngo hirindwe ko membrane y'uturemangingo idashobora kwinjira neza. Pipette y'agace gafite imyobo y'imbere irimo amphotericin (hafi 200 kugeza 250μg/ml; G4888, Sigma-Aldrich) kugira ngo ubone agace gafite imyobo. Amphotericin yashongeshejwe muri dimethyl sulfoxide (igipimo cya nyuma: 0.1 kugeza 0.3%; DMSO; D8418, Sigma-Aldrich). Igipimo cya DMSO cyakoreshejwe nta ngaruka nini cyagize ku mitsi yakozweho ubushakashatsi. Mu gihe cyo gukubita, uburyo bwo guhangana n'umuyoboro (Ra) bwakurikiranwaga buri gihe, maze igerageza ritangira nyuma y'uko uburebure bwa Ra na AP buhagaze neza (iminota 20-40). Igikorwa cyo kwihitiramo gipimwa mu buryo bwa voltage na/cyangwa icyuma cy'amashanyarazi mu gihe cy'iminota 2 kugeza kuri 5. Isesengura ry'amakuru ryakozwe hakoreshejwe Igor Pro (version 7.05.2, WaveMetrics, USA), Excel (version 2010, Microsoft Corporation, Redmond, USA) na GraphPad Prism (version 8.1.2, GraphPad Software Inc., La Jolla, CA). Leta Zunze Ubumwe za Amerika). Kugira ngo hamenyekane AP zitunguranye, hakoreshwa plug-in ya IgorPro ya NeuroMatic v3.0c. Hitamo AP mu buryo bwikora ukoresheje umupaka runaka, uhindurwa ukwawo kuri buri nyandiko. Ukoresheje interval ya spike, menya frequency ya spike hamwe na frequency ntarengwa ya spike hamwe na frequency mpuzandengo ya spike.
Gutandukanya PN. Mu guhuza na protocole yasohotse mbere, PNs zakuwe mu cyuma cy’imbeba ku ntera runaka (58). Muri make, cy’imbeba cyaciwemo ibice hanyuma gicukurwa mu buryo bukonje cyane [budafite HBSS Ca2+ na Mg2+, cyongerwamo glucose ya 20 mM, penisiline (50 U/ml) na streptomycin (0.05 mg/ml)], hanyuma gicukurwa mu cyuma cy’imbeba [HBSS, cyongerwamo 1-cysteine·HCl (1 mg/ml), papain (16 U / ml) na deoxyribonuclease I (DNase I; 0.1 mg/ml)] Vura iminota 30 kuri 30°C. Banza woze utwo dutsi mu buryo bwa HBSS burimo urumogi rw'igi (10 mg/ml), BSA (10 mg/ml) na DNase (0.1 mg/ml) ku bushyuhe bw'icyumba kugira ngo wirinde igogorwa rya enzyme, hanyuma mu buryo bwa HBSS burimo glucose ya 20 mM. Ugasya buhoro buhoro muri HBSS, penisiline (50 U/ml), streptomycin (0.05 mg/ml) na DNase (0.1 mg/ml) uturekure uturemangingo. Uduce twavuyemo twasuzumwe binyuze mu gikoresho cya 70μm, hanyuma uturemangingo dushyirwamo centrifugation (1110 rpm, iminota 5, 4°C) hanyuma twongera gushyirwa mu buryo bwo gutondeka [HBSS, hiyongereyeho glucose ya 20 mM, 20% y'inka y'umwana) Serum, penisiline (50 U/ml) na streptomycin (0.05 mg/ml)]; suzuma ubushobozi bw'uturemangingo ukoresheje propidium iodide hanyuma uhindure ubucucike bw'uturemangingo kuri 1 × 106 kugeza kuri 2 × 106 uturemangingo / ml. Mbere yo gusuzuma flow cytometry, suspension yashungurwaga binyuze mu cyuma cya 50 μm cell strainer.
Isuzuma ry’urumuri rw’amazi. Gusesengura utunyangingo byakozwe kuri dogere selisiyusi 4 hakoreshejwe imashini ya FACSAria III (BD Biosciences) na porogaramu ya FACSDiva (BD Biosciences, verisiyo ya 8.0.1). Guhagarika utunyangingo byashyizwe mu byiciro hakoreshejwe umunwa wa μm 100 munsi y’umuvuduko wa psi 20 ku gipimo cya ~2800 events/sec. Kubera ko ibipimo gakondo byo gutandukanya utunyangingo (ingano y’utunyangingo, itandukaniro ry’utunyangingo, n’imiterere yo gukwirakwira) bidashobora kwemeza ko PN itandukaniye neza n’izindi selisi, ingamba zo gutandukanya zishyirwaho hashingiwe ku kugereranya neza imbaraga za YFP na autofluorescence muri mitoYFP+ ​​​​na mitoYFP yo kugenzura - Imbeba. YFP ishishikajwe no gushyira icyitegererezo ku murongo wa laser wa 488 nm, kandi ikimenyetso kiboneka hakoreshejwe akayunguruzo ka 530/30 nm. Mu mbeba za mitoYFP+, imbaraga zigereranywa za gene ya Rosa26-mitoYFP reporter nazo zikoreshwa mu gutandukanya ibice by’umubiri w’imitsi n’ibice bya axon. 7-AAD yatewe inkunga na laser y'umuhondo ya 561 nm kandi yabonetse hakoreshejwe akayunguruzo ka bandpass ka 675/20 nm kugira ngo hatabaho uturemangingo twapfuye. Kugira ngo hatandukanye astrocytes icyarimwe, suspension y'uturemangingo yasizwe ibara rya ACSA-2-APC, hanyuma icyitegererezo gishyirwamo umurongo wa laser wa 640 nm, hanyuma akayunguruzo ka bandpass ka 660/20 nm gakoreshwa mu kumenya ikimenyetso.
Uturemangingo twakusanyijwe twashyizwe mu buryo bwo gupima ubushyuhe (1110 rpm, iminota 5, 4°C) hanyuma tubikwa kuri -80°C kugeza igihe bizakoreshwa. Imbeba za Mfn2cKO n'ibyana byazo bishyirwa mu byiciro umunsi umwe kugira ngo bigabanye itandukaniro ry'imikorere. Kwerekana no gusesengura amakuru ya FACS byakozwe hakoreshejwe porogaramu ya FlowJo (FlowJo LLC, Ashland, Oregon, muri Amerika).
Nkuko byavuzwe haruguru (59), PCR y'igihe nyacyo ikoreshwa mu gutandukanya ADN mu mitsi yatoranijwe kugira ngo hamenyekane neza mtDNA. Uburyo bwo gupima umurongo n'uburyo bwo kumenya ingano byabanje gupimwa hakoreshejwe qPCR ku mibare itandukanye y'uturemangingo. Muri make, kusanya PN 300 mu gikoresho cya lysis kigizwe na 50 mM tris-HCl (pH 8.5), 1 mM EDTA, 0.5% Tween 20 na proteinase K (200 ng / ml) hanyuma ushyire kuri 55°C mu minota 120. Uturemangingo twongeye gushyirwa kuri 95°C mu gihe cy'iminota 10 kugira ngo proteinase K idakora neza. Hakoreshejwe probe ya TaqMan (Thermo Fisher) yihariye kuri mt-Nd1, mtDNA yapimwe hakoreshejwe PCR y'ingano nto muri sisitemu ya PCR ya 7900HT Real-Time (Thermo Fisher Scientific). Ubumenyi, nimero ya katalogi Mm04225274_s1), mt-Nd6 (Thermo Fisher Scientific, nimero ya katalogi AIVI3E8) na 18S (Thermo Fisher Scientific, nimero ya katalogi Hs99999901_s1) genes.
Gutegura icyitegererezo cya Proteome. Mu gushyushya umuti kuri 95°C mu gihe cy'iminota 10 no gukoresha sonicating, muri lysis buffer [6 M guanidine chloride, 10 mM tris (2-carboxyethyl) phosphine hydrochloride, 10 mM chloroacetamide na 100 mM tris-Lyse neurons frozen pellets muri HCl]. Kuri Bioruptor (Diagenode) mu gihe cy'iminota 10 (amasegonda 30 pulse / amasegonda 30 igihe cyo guhagarara). Icyitegererezo cyavanzwe 1:10 muri 20 mM tris-HCl (pH 8.0), kivanze na 300 ng trypsin gold (Promega), hanyuma gishyirwa mu bushyuhe ijoro ryose kuri 37°C kugira ngo kigere ku igogora ryuzuye. Ku munsi wa kabiri, icyitegererezo cyashyizwe muri centrifuge kuri 20.000 g mu gihe cy'iminota 20. Supernatant yavanzwe na 0.1% formic acid, maze umuti uvanwamo umunyu na StageTips wikoreye. Icyitegererezo cyumishijwe mu gikoresho cya SpeedVac (Eppendorf concentrator plus 5305) kuri 45°C, hanyuma peptide ishyirwa muri aside formic ya 0.1%. Ingero zose zateguwe icyarimwe n'umuntu umwe. Kugira ngo basesengure ingero za astrocyte, peptide 4 μg zakuwemo umunyu zanditseho tandem mass tag (TMT10plex, catalog number 90110, Thermo Fisher Scientific) hamwe na peptide na TMT reagent ratio ya 1:20. Ku bijyanye no kwandika TMT, 0.8 mg ya TMT reagent yongeye gushyirwa muri 70 μl ya ACN ya anhydrous, maze peptide yumye yongera gushyirwamo 9 μl ya 0.1 M TEAB (triethylammonium bicarbonate), aho hongewemo 7 μl ya TMT reagent muri ACN. Igipimo cyari 43.75%. Nyuma y'iminota 60 yo gushyushya, igisubizo cyarazimijwe na 2 μl ya 5% ya hydroxylamine. Peptide zanditseho amazina zakusanyijwe, zirumishwa, zongera gushyirwa muri 200μl ya aside formic ya 0.1% (FA), zigaburwamo kabiri, hanyuma zikurwamo umunyu hakoreshejwe StageTips zakozwe n'umuntu ubwe. Hakoreshejwe UltiMate 3000 ultra high performance liquid chromatograph (UltiMate 3000 ultra high performance liquid chromatograph), kimwe muri bibiri cyagabanyijwe ku gipande cya 1mm x 150mm Acquity chromatographic cyuzuyemo uduce twa 130Å1.7μm C18 (Amazi, catalog No. SKU: 186006935). Thermo Fisher Scientific). Gutandukanya peptides ku muvuduko wa 30μl/min, utandukanye na 1% kugeza kuri 50% ya buffer B mu minota 85 hamwe n'umurongo ugenda buhoro buhoro w'iminota 96, kuva kuri 50% kugeza kuri 95% ya buffer B mu minota 3, hanyuma iminota 8 kuri 95% ya Buffer B; Buffer A ni 5% ACN na 10 mM ammonium bicarbonate (ABC), naho buffer B ni 80% ACN na 10 mM ABC. Kusanya ibice buri minota 3 ubivange mu matsinda abiri (1 + 17, 2 + 18, nibindi) hanyuma ubimye mu cyuma gitanga umwuka.
Isesengura rya LC-MS/MS. Ku bijyanye na spectrometry y'uburemere, peptides (umubare r119.aq) zatandukanyijwe ku gipimo cy'imbere cya PicoFrit gifite umurambararo wa cm 25, 75 μm (lens nshya y'icyitegererezo, umubare w'igice PF7508250) gifite 1.9 μm ReproSil-Pur 120 C18-AQ medium (Dr. Maisch, mat), Koresha EASY-nLC 1200 (Thermo Fisher Scientific, Ubudage). Igipimo cyagumye kuri 50°C. Buffers A na B ni 0.1% aside formic mu mazi na 0.1% aside formic muri 80% ACN, uko bikurikirana. Peptides zatandukanyijwe kuva kuri 6% kugeza kuri 31% buffer B mu minota 65 na kuva kuri 31% kugeza kuri 50% buffer B mu minota 5 hamwe n'umuvuduko wa 200 nl/min. Peptides zavuyemo zasesenguwe kuri Orbitrap Fusion mass spectrometer (Thermo Fisher Scientific). Igipimo cya m/z gipima peptide precursor kigaragazwa n’ubushobozi bwa 120.000 hagati ya 350 na 1500 m/z. Hakoreshejwe ingufu za 27% zisanzwe zo gukubitana, precursor ikomeye cyane ifite imiterere ya charge ya 2 kugeza kuri 6 itoranywa mu gucika kw’imitego ya C (HCD) ifite ingufu nyinshi. Igihe cyo kuzenguruka gishyirwa kuri 1 sec. Agaciro ka m/z k’agace ka peptide kapimwe mu mutego wa ion hakoreshejwe intego ntoya ya AGC ya 5 × 104 n’igihe ntarengwa cyo guteramo 86 ms. Nyuma yo gucikamo ibice, precursor yashyizwe ku rutonde rw’ibintu bishobora gukurwaho mu gihe cy’amasegonda 45. Peptide zanditseho TMT zatandukanyijwe ku nkingi ya Acclaim PepMap ya cm 50, 75 μm (Thermo Fisher Scientific, nimero ya kataloge 164942), maze spectra yo kwimuka yasesenguwe kuri Orbitrap Lumos Tribrid mass spectrometer (Thermo Fisher Scientific) ifite ibikoresho bya high-field asymmetric waveform ions (FAIMS) (Thermo Fisher Scientific) ikora kuri voltage ebyiri za −50 na −70 V. MS3 yatoranijwe hashingiwe ku buryo bwo guhuza bukoreshwa mu gupima ikimenyetso cya ion cya TMT. Gutandukanya peptide byakozwe kuri EASY-nLC 1200, hakoreshejwe 90% linear gradient elution, hamwe n'ubunini bwa buffer kuva kuri 6% kugeza kuri 31%; buffer A yari 0.1% FA, naho buffer B yari 0.1% FA na 80% ACN. Inkingi yo gusesengura ikoreshwa kuri 50°C. Koresha FreeStyle (verisiyo ya 1.6, Thermo Fisher Scientific) kugira ngo ugabanye dosiye y'umwimerere ukurikije voltage ya FAIMS.
Kumenya poroteyine no gupima. Hakoreshejwe moteri y’ishakisha ya Andromeda, amakuru y’umwimerere yasesenguwe hakoreshejwe verisiyo ya MaxQuant 1.5.2.8 (https://maxquant.org/). Uretse Cre recombinase na YFP sequences byakuwe muri Aequorea victoria, peptide fragment spectra zashakishijwe sequence ya canonical na isoform sequence ya proteome y’imbeba (Proteome ID UP000000589, yakuwe muri UniProt muri Gicurasi 2017). Oxidation ya Methionine na protein N-terminal acetylation byashyizweho nk'impinduka zihinduka; cysteine ​​​​carbamoyl methylation yashyizweho nk'impinduka zihoraho. Ibipimo by'igogora bishyirwa kuri "specificiality" na "trypsin / P". Umubare muto wa peptide na peptide za razor zikoreshwa mu kumenya poroteyine ni 1; umubare muto wa peptide zidasanzwe ni 0. Mu buryo bwo guhuza peptide map, igipimo cyo kumenya poroteyine cyari 0.01. Amahitamo ya "Second Peptide" arafunguwe. Koresha uburyo bwa "match between runs" kugira ngo wohereze ibimenyetso byatsinze hagati y’amadosiye atandukanye y’umwimerere. Koresha LFQ minimum ratio count 1 kuri quantification idafite label (LFQ) (60). Ubukana bwa LFQ bushungurwa nibura ku gaciro kabiri kemewe muri nibura itsinda rimwe rya genotype kuri buri gihe, kandi buvanwa mu igabanywa risanzwe rifite ubugari bwa. 0.3 hanyuma umanuke 1.8. Koresha platform ya Perseus computing (https://maxquant.net/perseus/) na R (https://r-project.org/) kugira ngo usesengure ibyavuye muri LFQ. Ikizamini cya t giciriritse cy’uburyo bubiri cyakoreshejwe muri limma software package cyakoreshejwe mu gusesengura differential expression (61). Isesengura ry’amakuru rikorwa hakoreshejwe ggplot, FactoMineR, factoextra, GGally na pheatmap. Amakuru ya proteomics ashingiye kuri TMT yasesenguwe hakoreshejwe MaxQuant verisiyo 1.6.10.43. Shakisha amakuru ya proteomics asanzwe muri database ya proteomics y’abantu ya UniProt, yakuwe muri Nzeri 2018. Isesengura ririmo ikintu cyakozwe n’uwakoze isotope purity correction factor. Koresha limma muri R kugira ngo usesengura differential expression. Amakuru y’umwimerere, ibisubizo by’ishakisha rya database, n’imikorere y’isesengura ry’amakuru n’ibisubizo byose bibikwa muri ProteomeXchange alliance binyuze mu bubiko bw’abafatanyabikorwa ba PRIDE hamwe n’ikimenyetso cy’amakuru PXD019690.
Ibisobanuro by'imikorere byongera isesengura. Igikoresho cya Ingenuity Pathway Analysis (QIAGEN) cyakoreshejwe kugira ngo hamenyekane ubwinshi bw'amagambo y'imikorere y'amakuru yashyizweho mu byumweru 8 (Ishusho ya 1). Muri make, urutonde rwa poroteyine z'ingano rwavuye mu isesengura ry'amakuru rya LC-MS/MS (tandem mass spectrometry) rukoreshwa hifashishijwe ibi bikurikira: Mus musculus yatoranijwe nk'ubwoko n'inkomoko, kandi icyiciro kigaragaza agaciro ka P kahinduwe na Benjamini kugira ngo gakomeze 0.05 cyangwa munsi yacyo gafatwa nk'akamaro. Kuri iyi mbonerahamwe, hagaragazwa ibyiciro bitanu by'ikirenga muri buri cluster hashingiwe ku gaciro ka P kahinduwe. Hakoreshejwe ikizamini cya t-multiple, hakoreshejwe gahunda ya kabiri ya linear boost ya Benjamini, Krieger, na Yekutieli (Q = 5%), isesengura ry'imvugo ya poroteyine y'igihe rikorwa ku bakandida b'ingenzi bagaragajwe muri buri cyiciro, kandi buri murongo usesengurwa ukwawo. Nta mpamvu yo gukoresha SD ihoraho.
Kugira ngo tugereranye ibyavuye muri ubu bushakashatsi n'amakuru yasohotse kandi tugakora imbonerahamwe ya Venn iri ku Ishusho ya 1, twahuje urutonde rwa poroteyine z'ingano n'inyandiko za MitoCarta 2.0 (24). Koresha igikoresho cyo kuri interineti cyitwa Draw Venn Diagram (http://bioinformatics.psb.ugent.be/webtools/Venn/) kugira ngo ukore imbonerahamwe.
Kugira ngo ubone amakuru arambuye ku buryo bw'ibarurishamibare bukoreshwa mu gusesengura proteomics, nyamuneka reba igice gifitanye isano n'Ibikoresho n'Uburyo. Ku bindi bigeragezo byose, amakuru arambuye aboneka mu nkuru ijyanye nabyo. Keretse iyo byavuzwe ukundi, amakuru yose agaragazwa nk'ikigereranyo cya ± SEM, kandi isesengura ryose ry'ibarurishamibare ryakozwe hakoreshejwe porogaramu ya GraphPad Prism 8.1.2.
Ku bindi bikoresho by'inyongera kuri iyi nkuru, reba http://advances.sciencemag.org/cgi/content/full/6/35/eaba8271/DC1
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Bya E. Motori, I. Atanassov, SMV Kochan, K. Folz-Donahue, V. Sakthivelu, P. Giavalisco, N. Toni, J. Puyal, N.-G. Larson
Isesengura rya poroteyomike ku mikorere mibi y’ubwonko ryagaragaje ko gahunda za metabolic zikora kugira ngo zirwanye kwangirika kw’ubwonko.
Bya E. Motori, I. Atanassov, SMV Kochan, K. Folz-Donahue, V. Sakthivelu, P. Giavalisco, N. Toni, J. Puyal, N.-G. Larson
Isesengura rya poroteyomike ku mikorere mibi y’ubwonko ryagaragaje ko gahunda za metabolic zikora kugira ngo zirwanye kwangirika kw’ubwonko.
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