Sisan *Sisan ladɛrɛsi: Kɔlɔn 50931, Alimanjamana, Kɔlɔn Excellence Cluster Research on Cellular Stress Response in Aging-related Diseases (CECAD).
Mitokondriyaw ka banaw ka sɔgɔsɔgɔninjɛ bɛ jate ko a tɛ se ka segin kɔ barisa sɔgɔsɔgɔninjɛw ka farikoloɲɛnajɛ plastiki dan ye, nka mitokondriyaw ka baarakɛbaliya nɔ min bɛ senfagabana farikoloɲɛnajɛ selilɛri yɛrɛmahɔrɔnya la farikolo la, o ma faamuya kosɛbɛ. Yan, an bɛ Purkinje senfagabana ka selilɛri kɛrɛnkɛrɛnnen proteome jira ni OXPHOS dɛsɛ bɛ taa ɲɛ min bɛ sɔrɔ mitokondriyaw ka fusion dynamics tiɲɛni fɛ. An y’a ye ko mitokondriyaw ka baarakɛbaliya ye fɛnba dɔ lawuli proteomiki siratigɛ la, o min labanna ka kɛ sababu ye ka farikoloɲɛnajɛ porogaramu tigitigiw baara ɲɔgɔn kɔ ka kɔn selilɛriw ka saya ɲɛ. An ma miiri min na, an ye piruvate carboxylase (PCx) ni kɔrɔya kɛlɛli anzimu wɛrɛw bilali jɛlen dɔn minnu bɛ TCA sɛrɛkili cɛmancɛw fara ɲɔgɔn kan. PCx balili ye oksidan degun ni farikolojidɛsɛ juguya, o b’a jira ko joli siraw gɛlɛya bɛ mɔgɔ tanga farikolojidɛsɛ ma OXPHOS tɛ minnu na. Mitokondriyaw ka ɲɔgɔn sɔrɔli seginni senkɔniw na minnu tiɲɛna laban na, o bɛ o farikoloɲɛnajɛ kɛcogo ninnu kɔsegin pewu, o la, a bɛ seliluw ka saya bali. An ka sɔrɔw bɛ siraw jira minnu tun ma dɔn fɔlɔ, minnu bɛ se ka muɲu di mitokondriyaw ka baarakɛbaliya ma, wa u b’a jira ko farikolojidɛsɛ bɛ se ka kɔsegin hali bana in labanw na.
Mitokondriyaw jɔyɔrɔba min bɛ senfagabana fanga sɛnɛcogo sabatili la, o sinsinnen bɛ hakililata taamasiɲɛ caman kan minnu bɛ tali kɛ hadamadenw ka mitokondriyaw banaw la. Nin bana ninnu fanba bɛ sɔrɔ jamu caman cili fɛ minnu bɛ mitokondriyaw ka jamu jiracogo ɲɛnabɔ (1, 2) walima jamu tiɲɛni min bɛ tali kɛ mitokondriyaw ka fanga la, o bɛ nɔ bila mitokondriyaw ka ADN sabatili la cogo la min tɛ ɲɛ (ADNm) (3, 4). Baara kɛlen baganw ka misaliw la, o y’a jira ko mitokondriyaw ka baarakɛbaliya jaabi la farikolo yɔrɔw la, farikoloɲɛnajɛ sira kɔrɔlenw (5-7) bɛ se ka baara kɛ, o bɛ kunnafoni nafamaw di walasa ka nin bana gɛlɛn ninnu banakisɛ sɔrɔcogo faamuya kosɛbɛ. O ni ɲɔgɔn cɛ, an ka faamuyali min bɛ farikolokisɛ suguya kɛrɛnkɛrɛnnenw ka farikoloɲɛnajɛ yeli kan, o min bɛ sɔrɔ kunsɛmɛ mitokondriyaw adenosini trifosfati (ATP) dilanni dɛsɛba fɛ, o ye fɛnba ye (8), o b’a sinsin a kan ko a ka kan ka furakɛli laɲiniw dɔn minnu bɛ se ka kɛ ka banaw bali walima ka banaw bali. Aw bɛ farikolojidɛsɛ bali (9). Kunnafoni dɛsɛ ye a ye ko farikoloɲɛnajɛw bɛ jate kosɛbɛ ko u ka farikoloɲɛnajɛ kɛcogo ka dɔgɔ kosɛbɛ ni i y’a suma ni farikolo yɔrɔw selilɛri suguyaw ye (10). Ni an y’a jateminɛ ko nin farikolokisɛ ninnu jɔyɔrɔ ka bon farikolojɔlifɛnw dicogo labɛnni na senfagabana la walasa ka sinankunya jɛnsɛnni sabati ani ka joginda ni bana cogoyaw jaabi, se min bɛ se ka farikolokisɛw ladilan ka kɛɲɛ ni kunsɛmɛnasumaya gɛlɛyaw ye, o bɛ se ka dan glial selilɛriw ma (11-14). Ka fara o kan, kunsɛmɛnasumaya selilɛriw ka ɲɔgɔndan danbe bɛ farikoloɲɛnajɛ fɛn caman sɛgɛsɛgɛli bali kosɛbɛ, fɛn minnu bɛ kɛ senfagabana kulu fitinin kɛrɛnkɛrɛnnenw na. O de kosɔn, fɛn caman tɛ dɔn mitokondriyaw ka baarakɛbaliya kɔlɔlɔ tigitigiw kan selilɛriw ni farikoloɲɛnajɛw la senfagabana kɔnɔ.
Walasa ka mitokondriyaw ka baarakɛbaliya kɔlɔlɔw faamuya, an ye Purkinje senfagabana (PN) bɔ ɲɔgɔn na farikolojidɛsɛ taabolo danfaralenw na min bɛ sɔrɔ mitokondriyaw kɔkanna fɛnɲɛnamafagalanw (Mfn2) tiɲɛni fɛ. Hali ni Mfn2 jiginni hadamadenw na, o bɛ tali kɛ farikolo-ɲɛnajɛ-banakisɛ suguya dɔ la min bɛ wele ko Charcot-Marie-Tooth suguya 2A (15), Mfn2 tiɲɛni cogoya la sosow la, o ye oksidan Phosphorylation (OXPHOS) baarakɛbaliya fɛɛrɛ ye min dɔnna kosɛbɛ. Senfagabana suguya fitinin suguyaw (16-19) ani farikolojidɛsɛbanakisɛ min bɛ sɔrɔ o la, o bɛ taa ni senfagabana taamasiɲɛw ye minnu bɛ taa ɲɛ, i n’a fɔ lamaga-lamagalibaliya (18, 19) walima kunsɛmɛnasumaya (16). Ni an bɛ baara kɛ ni protéomique quantitative (LFQ) minnu tɛ label ye, metabolomiki, ja lajɛ ani banakisɛfagalanw fɛɛrɛw faralen ɲɔgɔn kan, an b’a jira ko farikolojidɛsɛ min bɛ taa ɲɛ, o bɛ piruvate carboxylase (PCx) ni fɛn wɛrɛw bila kosɛbɛ minnu bɛ PNw ka joli siraw sɔgɔsɔgɔ in vivo The expression of enzymes. Walasa ka nin sɔrɔ in nafa sɛgɛsɛgɛ, an ye PCx jirali dɔgɔya kɛrɛnkɛrɛnnenya la PNw la minnu tɛ Mfn2 ye, wa an y’a ye ko nin opereli in ye oksidan degun juguya ani ka farikolojidɛsɛ teliya, o la, a y’a jira ko azoospermia bɛ selilɛri saya di Metabolic adaptability ma. MFN2 jirali jugu bɛ se ka PN laban kisi pewu ni OXPHOS dɛsɛ jugumanba ye, ni mitokondriyaw ka ADN dunni ye kosɛbɛ, ani a bɛ iko mitokondriyaw ka rezo tiɲɛna, o bɛ sinsin a kan ka taa a fɛ ko nin farikolojidɛsɛ suguya in bɛ se ka kɛnɛya yɛrɛ bana jugumanba la sani selilɛri ka sa.
Walasa ka mitokondriyaw yecogo ye Mfn2 knockout PNw kɔnɔ, an ye soso suguya dɔ kɛ min b’a to mitokondriyaw minnu bɛ Cre-dependant (Cre-dependant mitokondriyaw) bila u ka se ka poroteyini jɛman fluorescent (YFP) (mtYFP) (mtYFP) Cre jiracogo laɲini ani ka mitokondriyaw cogoya lajɛ in vivo. An y’a ye ko Mfn2 jamu tiɲɛni PNw kɔnɔ, o bɛna kɛ sababu ye ka mitokondriyaw ka rezo tila dɔɔnin dɔɔnin (Ja S1A), wa fɛn caman Changement fɔlɔ sɔrɔla dɔgɔkun 3 la. O ni PN selilɛri lamini tiɲɛniba, i n’a fɔ a jiralen bɛ cogo min na ni Calbindin farikolo tangacogo bɔnɛni ye, o ma daminɛ fo dɔgɔkun 12 (Jaa 1, A ani B). Waati bɛnbaliya min bɛ mitokondriyaw cogoya caman yeli ni senfagabana saya daminɛ yelen cɛ, o y’an bila ka farikoloɲɛnajɛ fɛn caman sɛgɛsɛgɛ minnu bɛ sɔrɔ mitokondriyaw ka baarakɛbaliya fɛ ka kɔn selilɛriw ka saya ɲɛ. An ye fɛɛrɛ dɔ labɛn min sinsinnen bɛ fluorescence-activated cell sorting (FACS) kan walasa ka YFP (YFP+)-expressing PN (Ja 1C) bɔ ɲɔgɔn na, ani control sogow la (Mfn2 + / loxP :: mtYFP loxP- stop-loxP: : L7-cre), n’a bɛ fɔ o ma kɔfɛ ko CTRL (Ja S1B). Gating fɛɛrɛ ɲuman min sinsinnen bɛ YFP taamasiɲɛ fanga danfaralen kan, o b’a to an bɛ se ka PNw ka YFP+ farikolo (YFPhigh) saniya ka bɔ PNw tɛ minnu na (YFPneg) (Ja S1B) walima ka bɔ axon/dendritic fragments fluorescentes putatives (YFPlow; Jaa S1D, numan fɛ), o tiɲɛna ni mikroskɔpu konfokali ye (Ja S1D, kinin fɛ). Walasa ka jamanaden danfaralenw ka danfara sɛgɛsɛgɛ, an ye LFQ proteomics kɛ, o kɔfɛ, principal component analysis, ka sɔrɔ k’a ye ko danfara jɛlen bɛ YFPhigh ni YFPneg selilɛriw cɛ (Ja S1C). YFPhigh cellules ye PNs taamasiyɛn dɔntaw nafabɔli jɛlen jira (i n’a fɔ Calb1, Pcp2, Grid2 ani Itpr3) (21, 22), nka farikolojɔlifɛnw nafabɔli ma sɔrɔ minnu bɛ jira ka caya senfagabana walima selilu suguya wɛrɛw la (Ja 1D) ). YFPhigh cellules classifiés (YFPhigh cellules classés) kɔnɔfɛnw ni ɲɔgɔn cɛ minnu lajɛra yɛrɛmahɔrɔnya sɛgɛsɛgɛliw la, o y’a jira ko jɛɲɔgɔnya coefficient> 0,9, o b’a jira ko se ka lasegincogo ɲuman jira fɛnɲɛnamafagalanw ni ɲɔgɔn cɛ (Jaa S1E). Kuma surun na, nin kunnafoniw y’an ka laɲini sinsin PN seko ni dɔnko isolation acute et spécifique kan. Ikomi L7-cre driver system min bɛ kɛ, o bɛ mozayika recombination bila dɔgɔkun fɔlɔ la jiginni kɔfɛ (23), an y’a daminɛ ka sosow faga ka bɔ CTRL la ani ka conditionnel (Mfn2 loxP / loxP :: mtYFP loxP-stop-loxP :: L7-cre) Collect neurons. Ni ɲɔgɔndan kɛra ka ban, a bɛ wele ko Mfn2cKO dɔgɔkun 4 la. I n’a fɔ labanyɔrɔ, an ye dɔgɔkun 8 sugandi waati min na PN layini tun ma tiɲɛ hali ni mitokondriyaw tilacogo jɛlen don (Jaa 1B ani ja S1A). K’a bɛɛ lajɛlen ta, an ye poroteyini 3013 bɛɛ lajɛlen hakɛ jateminɛ, minnu na 22% ɲɔgɔn tun sinsinnen bɛ MitoCarta 2.0 sɛbɛnniw kan minnu sinsinnen bɛ mitokondriyaw ka poroteyome kan i n’a fɔ mitokondriyaw (Ja 1E) (Ja 1E) (24). Diferential gene expression analysis min kɛra dɔgɔkun 8nan na, o y’a jira ko 10,5% dɔrɔn de ye fɛn caman Changements significatifs sɔrɔ farikolojɔlifɛnw bɛɛ la (Ja 1F ani Jaa S1F), o la, poroteyini 195 tun bɛ jigin, ka 120 kɛ ka wuli (Jaa 1F). A ka kan ka kɔlɔsi ko nin kunnafonidilan in “sira kura sɛgɛsɛgɛli” b’a jira ko jamu minnu jiralen don danfara la, olu bɛ farikoloɲɛnajɛ sira kɛrɛnkɛrɛnnenw kulu dantigɛlen dɔ de la kosɛbɛ (Ja 1G). A nafa ka bon kosɛbɛ, hali ni sira minnu bɛ tali kɛ OXPHOS ni kalisiyɔmu taamasiɲɛw la, olu jigincogo bɛ mitokondriyaw ka baarakɛbaliya bilali dafa PNw la minnu tɛ fusion sɔrɔ, kulu wɛrɛ minnu bɛ asidi aminikiw sɛnɛcogo de kan kosɛbɛ, olu bɛ wuli kosɛbɛ, o bɛ bɛn farikoloɲɛnajɛ ma min bɛ kɛ mitokondriyaw ka PNw la Rewiring is consistent. baarakɛbaliya (disfunction) (baarakɛbaliya).
(A) CTRL ni Mfn2cKO sogow ka ɲɛgɛnɛsira yɔrɔw jiracogo ja minnu bɛ PNw (calbindin, gris) bɔnɛni jira ka taa a fɛ; nukiliw tun bɛ counterstained ni DAPI ye. (B) (A) hakɛ jateminɛ (sɛgɛsɛgɛli kɛcogo kelen fɛ, ***P<0,001; n = sɛrɛkili 4 fo 6 ka bɔ soso saba la). (C) Baarakɛcogo min bɛ kɛ ni kɔrɔbɔli ye. (D) Taamaʃyɛn minnu kɛrɛnkɛrɛnnen don Purkinje (sanfɛ) ni selilɛri suguya wɛrɛw ma (cɛmancɛ) olu ka funteni karti tilatilali. (E) Venn ja min bɛ mitokondriyaw ka poroteyini hakɛ jira minnu dɔnna PN danfaralen kɔnɔ. (F) Tasuma-fanga ja min bɛ farikolojɔlifɛnw jira cogoya wɛrɛw la Mfn2cKO senfagabana kɔnɔ dɔgɔkun 8 kɔnɔ (nafa tigɛlen nafa ye 1,3 ye). (G) Daɲɛgafe sira sɛgɛsɛgɛli bɛ sanfɛla (bilen) ni jigin (bulu) sira duuru jira minnu nafa ka bon kosɛbɛ Mfn2cKO PN kɔnɔ minnu jatera dɔgɔkun 8 ye. Protéine kelen-kelen minnu kɔlɔsira, olu jiracogo hakɛ danmadɔ jiralen don. Funteni karti gris: P hakɛ ladilanen. ns, nafa tɛ.
Protéomique kunnafoniw y’a jira ko complexes I, III ani IV ka proteine ​​jirali dɔgɔyara dɔɔni dɔɔni. Komplekisi I, III ani IV bɛɛ tun bɛ ni fɛn fitininw ye minnu bɛ ADNm kodeli la, k’a sɔrɔ complexe II, min tun ye nukliyɛri kode dɔrɔn ye, o tun tɛ nɔ bila a jɔyɔrɔba la (Jaa 2A ani Jaa S2A). . Ka bɛn ni protéomique jaabiw ye, immunohistochimie de cerebellar tissues sections y’a jira ko MTCO1 (mitochondrial cytochrome C oxidase subunit 1) subunit hakɛ min bɛ complex IV la PN kɔnɔ, o dɔgɔyara dɔɔni dɔɔni (Jaa 2B). ADNmt-kodelen yɔrɔ fitinin Mtatp8 dɔgɔyara kosɛbɛ (Ja S2A), k’a sɔrɔ ATP sintazi yɔrɔ fitinin min bɛ nukilila kodɔn, o hakɛ jɔlen tora a cogo la, o min bɛ bɛn ATP sintazi subassembly F1 complexe sabatilen dɔnta ma ni mtDNA jirali sabatilen don. A dabɔcogo bɛ Kɛ cogo kelen na. A ka kuma tigɛ (7). ADNmt hakɛ jateminɛni Mfn2cKO PN suguyaw la ni polimerazi cakɛda ye waati yɛrɛ la (qPCR) ye ADNmt kopi hakɛ dɔgɔyali dɔɔni dɔɔni jira. Ni an ye a suma ni kɔrɔsili kulu ye, dɔgɔkun 8 la, ADNmt hakɛ 20% ɲɔgɔn dɔrɔn de tun bɛ to a la (Jaa 2C). Ka bɛn ni nin jaabi ninnu ye, Mfn2cKO PNw ka mikroskɔpi konfokali nɔgɔli kɛra ka ADN dɔn, o b’a jira ko mitokondriyaw nukilitɔnw dunni bɛ bɔ waati la (Jaa 2D). An y’a ye ko kandida dɔw dɔrɔn de sen bɛ mitokondriyaw ka poroteyini tiɲɛni na ani degun jaabi la, olu de bɛ wuli ka taa sanfɛ, i n’a fɔ Lonp1, Afg3l2 ani Clpx, ani OXPHOS complex assembly factors. Yɛlɛmaba si ma sɔrɔ farikolojɔli dumuniw hakɛ la minnu bɛ apoptose la (Jaa S2B). O cogo kelen na, an y’a ye ko mitokondriyaw ni endoplasmique reticulum channels minnu bɛ kalisiyɔmu tali la, olu bɛ fɛn misɛnninw dɔrɔn de Changer (Jaa S2C). Ka fara o kan, otofaji ni farikolojɔli dumuniw jateminɛni ma fɛn caman Changements significatifs sɔrɔ, o bɛ bɛn otofagosomes induction yelen ma min kɔlɔsira in vivo ni immunohistochimie ni électron microscopie ye (Ja S3). Nka, OXPHOS baarakɛbaliya min bɛ taa ɲɛ PNw la, o bɛ taa ni fɛn caman yeli ye min bɛ ye ka ɲɛ mitokondriyaw la. Mitokondriyaw kuluw bɛ se ka ye Mfn2cKO PNw ka selilɛri farikolow ni dendɛrɛsi jiriw la minnu si bɛ dɔgɔkun 5 ni 8 la, wa kɔnɔnatumuw jɔcogo yeli caman kɛra (Jaa S4, A ani B). Ka kɛɲɛ ni nin fɛn caman yeli ye min bɛ kɛ ni ultrastructural ye ani mtDNA dɔgɔyaliba ye, kunsɛmɛnasumaya sɔgɔsɔgɔninjɛ jugumanw sɛgɛsɛgɛli ni tetramethylrhodamine methyl ester (TMRM) ye, o y’a jira ko mitokondriyaw ka seko min bɛ Mfn2cKO PNw kɔnɔ, o dɔgɔyara kosɛbɛ (Jaa S4C).
(A) Waati taabolo sɛgɛsɛgɛli OXPHOS complexe jiracogo hakɛ kan. Aw bɛ farikolojɔli dumuniw dɔrɔn jateminɛ minnu ka P<0,05 dɔgɔkun 8 kɔnɔ (ANOVA sira fila). Layini dotted: Ladilanni si ma kɛ ni i y’a suma ni CTRL ye. (B) Nɛgɛbere : Misali dɔ ye ɲɛgɛnɛsira yɔrɔ dɔ ye min sɛbɛnnen don ni anti-MTCO1 anticorps ye (scale bar, 20 μm). Purkinje selilɛri farikolow bɛ yɔrɔ min minɛ, o bɛ datugu ni jɛman ye. Nimanbolo fɛ : MTCO1 hakɛ jateminɛ (sɛgɛsɛgɛli kɛcogo kelen fɛ; n = selilu 7 fo 20 minnu sɛgɛsɛgɛra ka bɔ soso saba la). (C) ADNmt kopi hakɛ sɛgɛsɛgɛli qPCR fɛ PN suguya la (sɛgɛsɛgɛli kɛcogo kelen fɛ; n = soso 3 fo 7). (D) Nɛgɛbere : Misali dɔ ye ɲɛgɛnɛsira sɔgɔlen ye min sɛbɛnnen don ni ADN kɛlɛlan ye (skala bar, 20 μm). Purkinje selilɛri farikolow bɛ yɔrɔ min minɛ, o bɛ datugu ni jɛman ye. Nimanbolo fɛ : ADNmt joginda hakɛ jateminɛ (sɛgɛsɛgɛli kɛcogo kelen fɛ; n = selilu 5 fo 9 minnu bɔra soso saba la). (E) Misali dɔ ye cerebellar section acute ye min bɛ mitoYFP + Purkinje selilɛriw jira (tɛgɛ) selilɛri bɛɛ ka patch clamp sɛbɛnni na. (F) IV kurulen hakɛ jateminɛni. (G) Kuran pikiri depolarizelen jirali sɛbɛnw CTRL ni Mfn2cKO Purkinje selilɛriw kɔnɔ. Top trace: Pulse fɔlɔ min ye AP daminɛ. Jiginni taamasiyɛn: AP frequency maksimali. (H) Postsynaptique spontané inputs (sPSPs) hakɛ jateminɛni. Representative recording trace ani a zoom ratio jiralen bɛ (I) kɔnɔ. Sɛgɛsɛgɛli min kɛra sira kelen fɛ, o ye n = 5 fo 20 sɛgɛsɛgɛ ka bɔ soso saba la. Kunnafoni bɛ jira ni cɛmancɛ±SEM ye; *P<0,05 ye; **P<0,01 ye; ***P<0,001 ye. (J) AP yɛrɛmabila taamasiɲɛw jiralenw minnu sɛbɛnna ni perforated patch clamp mode ye. Sanfɛla taamasiyɛn: AP frequency maksimali. Jiginni taamasiyɛn: AP kelen ka zoom. (K) Aw bɛ AP hakɛ danmadɔ ni a caya hakɛ jateminɛ ka kɛɲɛ ni (J) ye. Mann-Whitney ka sɛgɛsɛgɛli; n = 5 selilu sɛgɛsɛgɛra ka bɔ soso naani na. Kunnafoni bɛ jira ni cɛmancɛ±SEM ye; nafa tɛ o la.
OXPHOS tiɲɛni jɛlen sɔrɔla dɔgɔkun 8 Mfn2cKO PN la, o b’a jira ko senfagabana farikolo baara tɛ cogo la kosɛbɛ. O la, an ye OXPHOS dɛsɛbagatɔw ka kuran cogoya pasifw sɛgɛsɛgɛ dɔgɔkun 4 fo 5 ani dɔgɔkun 7 fo 8 kɔnɔ, an kɛtɔ ka selilu bɛɛ ka patch clamp recordings kɛ cerebellar slices acute (Jaa 2E). K’a sɔrɔ a ma labɛn, Mfn2cKO senfagabana lafiɲɛbɔlan seko ni dɔnko hakɛ danmadɔ tun bɛ tali kɛ kɔrɔsili la, hali ni danfara misɛnniw tun bɛ seliluw ni ɲɔgɔn cɛ (Taabolo 1). O cogo kelen na, dɔgɔkun 4 fo dɔgɔkun 5 la, fɛn caman ma yeli kɛ sisan ni tension cɛsira la (IV curve) (Jaa 2F). Nka, Mfn2cKO senfagabana si ma se ka kisi IV furakɛli ma (hyperpolarisation step), o b’a jira ko sɛgɛsɛgɛli jɛlen bɛ hyperpolarisation seko la nin waati laban in na. O ni Mfn2cKO senfagabana ta tɛ kelen ye, depolarizing currents minnu bɛ kɛ sababu ye ka repetitive action potential (AP) bɔli kɛ, olu bɛ muɲu kosɛbɛ, o b’a jira k’u bɔli cogoya bɛɛ lajɛlen tɛ danfaraba don ni dɔgɔkun 8 kɔrɔsili senkɔniw ta ye (Taabolo 1 ani Jaa 2G). O cogo kelen na, spontané postsynaptic currents (sPSCs) ka caya ni u bonya tun bɛ se ka suma ni kɔrɔsili kulu ta ye, wa ko kɛlenw caya cayara ka bɔ dɔgɔkun 4 na ka se dɔgɔkun 5 ma ka se dɔgɔkun 7 ma ka se dɔgɔkun 8 ma ni dɔ farali ɲɔgɔn kan (Jaa 2, H ani I). Sinankunya kɔgɔli waati PNw la (25). O jaabi kelenw sɔrɔla PNs patches perforés kɔfɛ. O cogoya in bɛ selilɛri ATP nafamafɛnw musakabɔcogo bali, i n’a fɔ a bɛ se ka kɛ cogo min na selilu bɛɛ ka patch clamp sɛbɛnni na. Kɛrɛnkɛrɛnnenya la, Mfn2cKO senkɔniw ka lafiɲɛbɔ membrane seko ni u yɛrɛma tasuma kɛcogo ma nɔ bila o la (Jaa 2, J ani K). Kuma surun na, nin jaabi ninnu b’a jira ko PN minnu ka OXPHOS baarakɛbaliya jɛlen don, olu bɛ se ka se ka jibɔcogo caman kunbɛn ka ɲɛ, o b’a jira ko musakabɔ fɛɛrɛ dɔ bɛ yen min b’a to u bɛ se ka kuran fiziyɔmu jaabiw mara minnu bɛ se ka surunya.
Kunnafoni bɛ jira ni hakɛ danmadɔ ye ± SEM (sɛgɛsɛgɛli min bɛ kɛ sira kelen fɛ, Holm-Sidak ka sɛgɛsɛgɛli caman kɛcogo; *P<0.05). Unit nimɔrɔ bɛ jira ni brackets ye.
An y’a daminɛ ka sɛgɛsɛgɛli kɛ ni suguya o suguya bɛ proteomiki kunnafonidilan kɔnɔ (Ja 1G) siraw bɛ yen minnu bɛ se ka OXPHOS dɛsɛ jugumanba kɛlɛ, o la, an b’a ɲɛfɔ mun na PN min tɔɔrɔla, o bɛ se ka kuran fiziyɔmu min bɛ kɛrɛfɛ, o mara (Jaa 2, E ka se K ma). . Protéomique sɛgɛsɛgɛli y’a jira ko anzimu minnu bɛ cakɛda amino acide branchés (BCAA) ka katabolisme la, olu tun bɛ wuli kosɛbɛ (Ja 3A ani Jaa S5A), wa a laban na, acetyl-CoA (CoA) walima succinyl CoA bɛ se ka tricarboxylates fara a kan arteriosclerose Acid (TCA) cycle kɔnɔ. An y’a ye ko BCAA transaminase 1 (BCAT1) ni BCAT2 fila bɛɛ hakɛ cayara. U bɛ BCAA katabolisme (banakisɛfagalan) taabolo fɔlɔ kɛ ka glutamate (glutamate) bɔ α-ketoglutarate la (26). Subunits bɛɛ minnu bɛ branched chain keto acid dehydrogenase (BCKD) complexe kɛ, olu bɛ upregulated (o complexe bɛ BCAA carbone skeleton sɔrɔlen decarboxylation kɔfɛ ani min tɛ se ka wuli) catalyse (Jaa 3A ani Jaa S5A). Nka, fɛn jɛlen si ma sɔrɔ BCAA yɛrɛ la PN suguya la, o bɛ se ka kɛ sababu ye ka nin asidi aminiki nafama ninnu tali caya selilɛriw la walima ka bɔ sɔrɔyɔrɔ wɛrɛw (glucose walima asidi laktiki) tali la walasa ka TCA sɛrɛkili dafa (Jaa S5B). PN minnu tɛ OXPHOS sɔrɔ, olu fana ye dɔ fara glutamine tiɲɛni ni transamination baara kan dɔgɔkun 8 kɔnɔ, o bɛ se ka jira ni mitokondriyaw ka enzymes glutaminase (GLS) ani glutamine pyruvate transaminase 2 (GPT2) ka wuliliko ye (Jaa 3, A ani C). A ka kan ka kɔlɔsi ko GLS ka wuli-ka-bɔli dan ye isoform glutaminase C (GLS-GAC) spliced ​​spliced ​​isoform glutaminase C (GLS-GAC) ye (Mfn2cKO/CTRL caman cili bɛ se siɲɛ 4,5 ma, P = 0,05), wa a wulicogo kɛrɛnkɛrɛnnen kansɛri farikolo yɔrɔw la Se bɛ mitokondriyaw ka biyoenerji dɛmɛ. (27) la.
(A) Funteni karti bɛ foli caman yeli jira farikolojɔli dumuniw hakɛ la sira kofɔlen na dɔgɔkun 8 kɔnɔ. (B) Misali la, ɲɛgɛnɛsira sɔgɔlen min sɛbɛnnen don ni PCx kɛlɛlan ye (skala bar, 20 μm). Tile jɛman bɛ Purkinje selilɛri farikolo jira. (C) Waati taabolo poroteyini jiracogo sɛgɛsɛgɛli min jirala ko a ye kandida nafama ye joli siraw la (t-test caman, *FDR <5%; n = 3-5 sosow). (D) Sanfɛ: Ja min bɛ kɛ kabon taamasiyɛnlen doncogo danfaralenw jira min bɛ [1-13C]piruvate tracer kɔnɔ (o kɔrɔ ye ko PDH walima trans-arterial route fɛ). Jiginni : Viyolin jatebɔsɛbɛn bɛ karɔbɔni taamasiɲɛ kelen (M1) kɛmɛsarada la jira min jiginna ka kɛ asidi aspartitiki, asidi sitriki ani asidi maliki ye, ka sɔrɔ ka ɲɛgɛnɛsira sɔgɔlenw taamasiɲɛ kɛ ni [1-13C]piruvati ye (t-test fila-fila; ** P <0.01). (E) Sira jiralen in waati tariku sɛgɛsɛgɛli bɛɛjɛfanga. aw bɛ farikolojɔli dumuniw dɔrɔn jateminɛ minnu bɛ P<0,05 dɔgɔkun 8 kɔnɔ . Layini tigɛlen : ladilanni nafa tɛ (sɛgɛsɛgɛli fila-fila fɛ; * P <0,05; *** P <0,001). Kunnafoni bɛ jira ni cɛmancɛ±SEM ye.
An ka sɛgɛsɛgɛli la, BCAA katabolisme kɛra up-regulation sira jɔnjɔn dɔ ye. O ko in b’a jira kosɛbɛ ko fiɲɛbɔ hakɛ min bɛ don TCA sɛrɛkili la, o bɛ se ka Changé PN la min tɛ OXPHOS sɔrɔ. O bɛ se ka kɛ senfagabana farikololabɔli siraba dɔ ye, min bɛ se ka nɔ bila senfagabana farikoloɲɛnajɛ ni ɲɛnamaya la OXPHOS baarakɛbaliya jugumanba marali waati. Ka bɛn ni nin hakilina in ye, an y’a ye ko joli siraw kɛlɛli anzimu kunba PCx bɛ wuli ka taa sanfɛ (Mfn2cKO/CTRL bɛ Changé siɲɛ 1,5 ɲɔgɔn; Jaa 3A), o bɛ piruvate jiginni kɛ ka kɛ oxaloacetate ye (28), o min dalen bɛ a la ko a bɛ hakili la The expression in is restricted to astrocytes (29, 30). Ka bɛn ni poroteyomiki jaabiw ye, mikroskɔpi konfokali y’a jira ko PCx jirali cayara kɛrɛnkɛrɛnnenya la ani kosɛbɛ OXPHOS dɛsɛbagatɔ PNw la, k’a sɔrɔ PCx waleyali dantigɛlen don kosɛbɛ kɔrɔsilikɛla ka Bergmann glial selilɛriw kɛrɛfɛ (Jaa 3B). Walasa ka PCx ka wulicogo kɔlɔsilen sɛgɛsɛgɛ baarakɛcogo la, an ye ɲɛgɛnɛsira sɔgɔlenw furakɛ ni [1-13C]pyruvate tracer ye. Ni piruvati kɛra oksidan ye piruvate dehydrogenase (PDH) fɛ, a isotope taamasiɲɛ tununna , Nka a bɛ don TCA cycle intermediates kɔnɔ ni piruvate bɛ metabolize ni joli siraw ye (Ja 3D). An ka proteomics kunnafoniw dɛmɛni kama, an ye taamasiyɛn caman kɔlɔsi ka bɔ nin tracer in na Mfn2cKO slices ka asidi aspartiki la, ka sɔrɔ asidi sitriki ni asidi maliki fana tun bɛ ni ŋaniya damadɔ ye, hali n’a ma kɛ ko nafama ye (Ja 3D).
MitoPark sogow ka dopamine senkɔniw na minnu ka mitokondriyaw ka baarakɛbaliya bɛ sɔrɔ dopamine senkɔniw fɛ minnu bɛ mitokondriyaw ka sɛbɛnnikɛlan A jamu (Tfam) tiɲɛ kɛrɛnkɛrɛnnenya la (Ja S6B), PCx jirali fana tun bɛ wuli kosɛbɛ (31), o b’a jira ko asidi asetɔni joli siraw sɔgɔsɔgɔninjɛ Bana in sɔrɔli bɛ labɛn senfagabana baarakɛbaliya waati OXPHOS bɛ farikolo la. A ka kan ka kɔlɔsi ko a jirala ko anzimu kɛrɛnkɛrɛnnenw (32-34) minnu bɛ se ka jira senfagabana la minnu bɛ se ka kɛ sababu ye ka joli siraw sɔgɔsɔgɔ, olu bɛ wuli kosɛbɛ PNw la minnu tɛ OXPHOS sɔrɔ, i n’a fɔ propionyl-CoA carboxylase (PCC-A), Malonyl-CoA bɛ propionyl-CoA sɛmɛntiya ka kɛ succinyl-CoA ye ani mitokondriyali malic enzyme 3 (ME3), min jɔyɔrɔba ye ka piruvati sɔrɔ malate la (Jaa 3, A ani C) (33, 35). Ka fara o kan, an ye dɔ fara kosɛbɛ Pdk3 anzimu kan, o min bɛ fosforilasi kɛ, o cogo la a bɛ PDH kɛ baarakɛbali ye (36), k’a sɔrɔ fɛn si ma ye Pdp1 anzimu la min bɛ PDH baara walima PDH anzimu complexe yɛrɛ (Jaa 3A). O cogo kelen na, Mern2cKO PNw kɔnɔ, PDH complexe ka piruvate dehydrogenase E1 yɔrɔ α1 subunit α (PDHE1α) subunit ka fosforilasi cayara Ser293 kɔnɔ (a dɔnna ko a bɛ PDH ka anzimu baara bali) ( Jaa S6C) (Ja S6C). Piruvate tɛ se ka don joli sira fɛ.
A laban na, an y’a ye ko serini ni glycine biyosintɛzi ka super sira, mitokondriyali folate (1C) cycle min bɛ tali kɛ o la ani proline biyosintɛzi (Ja 1G ani ja S5C) bɛɛ bɛ wuli kosɛbɛ, ka kɛɲɛ ni kunnafoniw ye, baarakɛcogo senfɛ. Lamini yɔrɔw bɛ baara kɛ ni mitokondriyaw ka baarakɛbaliya ye (5-7). Konfokali sɛgɛsɛgɛli min bɛ nin poroteyomiki kunnafoniw dɛmɛ, o y’a jira ko PN kɔnɔ ni OXPHOS tununna, dɔgɔkun 8 sogow kunkolo sɔgɔlenw kɛra serine hydroxymethyltransferase 2 (SHMT2) ye, o ye mitokondriyaw folate cycle ka anzimu jɔnjɔn ye. Banakisɛfagalanw ka jaabi nafama (Jaa S5D). CU-glucose-incubés acute cerebellar slices 13 kɔnɔ, farikoloɲɛnajɛ ɲininiw ye serine ni proline biyosintɛzi wulicogo sinsin ka taa a fɛ, o b’a jira ko karɔbɔni isofɔmuw ka don serine ni proline kɔnɔ, o cayara (Ja S5E). Ikomi GLS ni GPT2 bɛ wale minnu lawuli, olu de bɛ glutamate sɔrɔli sababu ye ka bɔ glutamine la ani ka transamination kɛ glutamate ni α-ketoglutarate cɛ, u ka wulicogo b’a jira ko OXPHOS dɛsɛbagatɔw bɛ glutamate ɲinini caya , O bɛ se ka kɛ sababu ye ka proline ka biyosintɛzi cayalenba sabati (Ja S5C). Nin fɛn caman ɲɔgɔnna yeli ni ɲɔgɔn cɛ, ɲɛgɛnɛsirala sɔgɔsɔgɔninjɛw sɛgɛsɛgɛli min kɛra ni PN kɛrɛnkɛrɛnnen Mfn2cKO sogow ye, o y’a jira ko nin sira ninnu (antiperoxidases bɛɛ sen bɛ o la) ma Changé kosɛbɛ u jiracogo la, o la, a b’a jira ko Nin farikoloɲɛnajɛ sinsinni in bɛ sugandi ka kɛ PN tiɲɛnenw ye (Ja S6, D ka taa G la).
Kuma surun na, nin sɛgɛsɛgɛliw y’a jira ko farikoloɲɛnajɛ sira kɛrɛnkɛrɛnnenw baarakɛcogo waatilama baarakɛcogo tɛ kelen ye kosɛbɛ PNw kɔnɔ. Hali ni senfagabana mitokondriyaw ka baara kɛcogo jugu bɛ se ka kɛ sababu ye ka joli siraw sɔgɔsɔgɔ joona ani ka 1C labɛn kokura (Ja 3E ani ja S5C), ani hali fɛn minnu bɛ se ka fɔ ka ɲɛ I ni IV complexes jiracogo la, fɛn minnu bɛ se ka fɔ ka ɲɛ, olu ye serine de novo sintezi ye dɔrɔn A kɛra ka jɛya dɔrɔn a labanw na. OXPHOS baarakɛbaliya (Jaa 3E ani ja S5C). Nin sɔrɔlen ninnu bɛ taabolo dɔ ɲɛfɔ min bɛ tugu ɲɔgɔn na, min kɔnɔ, degun bɛ sɔrɔ mitokondriyaw (1C cycle) ni sitoplasma (serine biosynthèse) jaabi la ni jɛɲɔgɔnya ye ni joli siraw sɔgɔsɔgɔli caya ye TCA cycle kɔnɔ walasa ka senfagabana farikoloɲɛnajɛ kɛcogo kura.
Dɔgɔkun 8 PN minnu tɛ OXPHOS sɔrɔ, olu bɛ se ka wulicogo caman mara ani ka farikoloɲɛnajɛ caman kɛ walasa ka mitokondriyaw ka baarakɛbaliya kunbɛn. Nin sɔrɔli in bɛ seko nafama dɔ lawuli ko hali nin waati in na, o farikolokisɛ ninnu fana bɛ se ka furakɛli fɛɛrɛw sɔrɔ walasa ka farikolojidɛsɛ bila kɔfɛ walima k’a bali. Lɛrɛ kɔfɛ. An ye o seko in ɲɛnabɔ ni yɛrɛmahɔrɔnya wale fila ye. Fɛɛrɛ fɔlɔ la, an ye Cre-dependent adeno-associated virus (AAV) fɛnɲɛnamafagalan dɔ labɛn walasa MFN2 ka se ka jira cogo sugandilen na OXPHOS dɛsɛbagatɔ PNw la in vivo (Jaa S7A). AAV min bɛ MFN2 kofɔ ani fluorescent reporter gene mCherry (Mfn2-AAV) sɛgɛsɛgɛra senfagabana fɔlɔw la in vitro, o kɛra sababu ye ka MFN2 jira cogo la min bɛ tali kɛ Cre la ani ka mitokondriyaw cogoya kisi, o la, a ye senfagabana jiginni bali Mfn2cKO senfagabana kɔnɔ ( Jaa S7, B, D ani E). O kɔfɛ, an ye sɛgɛsɛgɛliw kɛ in vivo walasa ka dɔgɔkun 8 Mfn2-AAV lase Mfn2cKO ni kɔrɔsili sogow ka ɲɛgɛnɛsira la ni stɛriyɔmu ye, ka dɔgɔkun 12 sogow sɛgɛsɛgɛ (Jaa 4A). Mfn2cKO sogo furakɛlenw sara (Jaa 1, A ani B) (16). Virus transduction in vivo kɛra sababu ye ka PN jiracogo sugandilen kɛ kunsɛmɛnasumaya lamini dɔw la (Jaa S7, G ani H). Kɔlɔsili AAV pikiri min bɛ mCherry (Ctrl-AAV) dɔrɔn jira, o ma nɔba bila Mfn2cKO baganw ka farikolojidɛsɛ hakɛ la. O ni Mfn2cKOw ta tɛ kelen ye, minnu jiginna ni Mfn2-AAV ye, olu sɛgɛsɛgɛli y’a jira ko PN selilɛri lakanani nafa ka bon (Jaa 4, B ani C). Kɛrɛnkɛrɛnnenya la, a bɛ iko senfagabana hakɛ tɛ se ka danfara bɔ ni baganw ye minnu bɛ kɔrɔsi (Jaa 4, B ani C, ani ja S7, H ani I). MFN1 jirali nka MFN2 tɛ, o bɛ se ka kɛ sababu ye ka senfagabana saya kisi cogo kelen na (Ja 4C ani ja S7, C ani F), o b’a jira ko MFN1 bɔli kɔnɔbara kɔkan, o bɛ se ka dɔ fara MFN2 dɛsɛ kan kosɛbɛ. Sɛgɛsɛgɛli wɛrɛw kɛra PN hakɛ kelen na, o y’a jira ko Mfn2-AAV ye mitokondriyaw ka ultrastructure kisi kosɛbɛ, ka mtDNA hakɛw normalize, ani ka anti-angiogenesis marker PCx ​​jiracogo caman kɔsegin (Ja 4, C ka taa E la). Mfn2cKO sogo kisilenw sɛgɛsɛgɛli ni ɲɛ ye lafiɲɛbɔ cogo la, o y’a jira k’u jɔcogo n’u ka farikoloɲɛnajɛ taamasiɲɛw (taamacogo S1 ka taa S3 la) ɲɛnabɔra. Kuncɛli la, nin kɔrɔbɔli ninnu b’a jira ko MFN2 seginni kɔfɛ PNw kɔnɔ minnu dɛsɛlen don kosɛbɛ OXPHOS la, o bɛ se ka ADNmt dunni kɔsegin ani ka joli siraw gɛlɛyali bila mɔgɔ la, o la, a bɛ axonw tiɲɛni ni senfagabana saya bali in vivo.
(A) Fɛɛrɛ min bɛ AAV pikiri kɛcogo jira min bɛ MFN2 kofɔ ni farikoloɲɛnajɛ sira jiralen bɛ baara kɛ. (B) Dɔgɔkun 12 kunsɛmɛnasumaya sɔgɔsɔgɔninjɛ minnu kɛra dɔgɔkun 8 kɔnɔ Mfn2cKO sogow la, olu ja confocal jiralenw, minnu taamasiɲɛw kɛra ni anti-Calbindin anticorps ye. Nimanbolo fɛ: Axon fibres (axon fibres) sɔgɔli. Axon zoom (axon zoom) hakɛ ye 450 ni 75 μm ye. (C) Nɛgɛbere : Purkinje selilɛri density hakɛ jateminɛni AAV transduction loop (AAV+) kɔnɔ (sɛgɛsɛgɛli sira kelen fɛ; n = soso 3). Nimanbolo fɛ : ADNmt sinsinni sɛgɛsɛgɛli PN jiginni na dɔgɔkun 12nan na (t-test unpaired; n = 6 cellules ka bɔ soso saba la). * P <0,05 ye; ** P <0,01 ye. (D) Mfn2cKO cerebellar sections PNw ka transmission électronique micrographes représentants transduites ni banakisɛfagalanw ye minnu jiralen don. Maskɛ pinki bɛ yɔrɔ jira min bɛ dendɛrɛsiw bolo, ani kɛnɛ jɛman min bɛ ni yɔrɔ jɛman ye, o bɛ zoom jira min dira kinin fɛ; n bɛ nucleus (nucleus) jira. Skala bara, 1μm. (E) bɛ PCx nɔgɔli misali jira PN la min jiginna dɔgɔkun 12 kɔnɔ. Skala bara, 20μm. OE, a jirali kojugu; FC, foli caman caman cili.
A laban na, an ye sɛgɛsɛgɛli kɛ perokisidazi ka selilɛriw ka ɲɛnamaya nafa kan PNw la minnu ye OXPHOS baarakɛbaliya sɔrɔ. An ye mCherry dilan min bɛ AAV-shRNA (ARN kunsigi surun) kofɔ min bɛ sosow ka PCx mRNA (AAV-shPCx) laɲini kɛrɛnkɛrɛnnenya la, ka banakisɛ in walima a sɛgɛsɛgɛlikɛlan sɔgɔlen (AAV-scr) pikiri kɛ Mfn2cKO sogow kunsɛmɛ na. Pikiri kɛra dɔgɔkun naaninan na (Jaa 5A) walasa ka PCx knockdown nafama sɔrɔ waati min na PCx jirali cayara (Jaa 3C) ani PN selilɛri lamini tun bɛ a cogo la hali bi (Ja 1A). A ka kan ka kɔlɔsi ko PCx (Jaa S8A) gosili bɛ na ni PN saya teliyaba ye, o min bɛ dan banakisɛfagalan ma (Jaa 5, B ani C). Walasa ka farikoloɲɛnajɛ nɔ minnu bɛ sɔrɔ PCx wulicogo fɛ, olu kɛcogo faamuya, an ye PNw ka redox cogoya sɛgɛsɛgɛ PCx knockdown kɔfɛ ani AAV-mediated optical biosensor Grx1-roGFP2 jirala waati kelen na (Ja S8, B ka taa D) walasa ka glutathione jateminɛ The relative change of peptide redox potential (38). O kɔfɛ, an ye foton fila fluorescence lifetime imaging microscopy (FLIM) kɛ dɔgɔkun 7 Mfn2cKO walima control littermates ka hakili sɔgɔlenw na walasa ka fɛn dɔw dɔn minnu bɛ se ka Changements potentiels de cytoplasmique redox status (Jiracogo S8, E ka taa G la). Sɛgɛsɛgɛli y’a jira ko dɔ farala kosɛbɛ Mfn2cKO PN kelen ka oksidan cogoya la min tɛ PCx jira, o ni kɔrɔsili senfagabana walima Mfn2cKO PN minnu bɛ shRNA sɔgɔlen dɔrɔn jira, olu tɛ kelen ye (Jaa 5, D ani E). Ni PCx jirali tun bɛ jigin, Mfn2cKO PN minnu bɛ oksidan cogoya jira, olu kɛmɛsarada la, o cayara ka tɛmɛ siɲɛ saba kan (Jaa 5E), o b’a jira ko PCx jiginni jiginni ye senfagabana tiɲɛnenw ka redox seko mara.
(A) Fɛɛrɛ min bɛ AAV pikiri kɛcogo jira min bɛ shPCx kofɔ ni farikoloɲɛnajɛ sira jiralen bɛ baara kɛ. (B) Dɔgɔkun 8 ɲɛgɛnɛsira yɔrɔw jiracogo jiralenw Mfn2cKO sogow la minnu jiginna ka u taamasiɲɛ kɛ ni anti-calcineurin anticorps ye dɔgɔkun 4 kɔnɔ. Skala bara, 450μm. (C) Purkinje selilɛri hakɛ jateminɛni AAV-transduced loops kɔnɔ (sɛgɛsɛgɛli sira kelen fɛ; n = 3 ka se 4 ma). Kunnafoni bɛ jira ni cɛmancɛ±SEM ye; ***P<0,001 ye. (D) FLIM ja min ye ciden ye, o bɛ dɔgɔkun 7 PN min bɛ glutathione redox sensor Grx1-roGFP2 jira, o ka ɲɛnamaya kɛcogo cɛmancɛ jira, k’a sɔrɔ a kɛcogo ɲɛfɔlenw kɔnɔ. LUT (tabali lajɛ) hakɛ : ɲɛnamaya waati cɛmancɛ (pikosekɔndi kɔnɔ). Skala bara, 25μm. (E) Histogramme bɛ Grx1-roGFP2 ɲɛnamaya hakɛw tilatilali jira ka bɔ (D) la (n=158 ka se 368 ma soso fila kɔnɔ cogoya kelen-kelen bɛɛ la). Tablo min bɛ histogramme kelen-kelen bɛɛ sanfɛ : o bɛ selilu hakɛ jira minnu si janya ka jan kosɛbɛ (bilen, oksizɛni) walima minnu ka surun (bulu, dɔgɔyalen) ye, minnu bɛ tɛmɛ 1 SD kan ɲɛnamaya hakɛ danmadɔ la CTRL-AAV-scr kɔnɔ. (F) Modeli sugandilen bɛ PCx senfagabana wulicogo lakanani nɔ jira.
O bɛɛ lajɛlen na, an bɛ kunnafoni minnu di yan, olu b’a jira ko MFN2 jirali seginni bɛ se ka PN kɔrɔlenw kisi pewu ni OXPHOS dɛsɛ jugumanba ye, ADNmt dɔgɔyali jugumanba, ani ista-like morphologie juguba, o la, ɲɛtaa bɛ to senna hali bana jugumanw na. Neurodegeneration bɛ dalilu caman di minnu bɛ se ka segin kɔ, minnu b’a jira ko a bɛ kɛ cogo min na sani selilɛri ka sa. Nin farikoloɲɛnajɛ sɛgɛsɛgɛli hakɛ in bɛ sinsin ka taa a fɛ ni senfagabana seko ye ka joli siraw sɔgɔsɔgɔ (TCA sɛrɛkili ka seginkanni kɛ), o min bɛ PCx jirali bali PNw la minnu tɛ OXPHOS sɔrɔ ani ka selilɛriw ka saya bonya, o la, a bɛ jɔyɔrɔba ta lakanani na (Jaa 5F).
Nin kalan in kɔnɔ, an ye daliluw di minnu b’a jira ko PNw ka jaabi OXPHOS baarakɛbaliya la, o ye ka ɲɔgɔn sɔrɔ dɔɔnin dɔɔnin ka taa TCA cycle atherosclerose la, diferential activation sira fɛ min bɛ baara kɛ ni farikoloɲɛnajɛ porogaramuw ye. An ye poroteyomi sɛgɛsɛgɛli sinsin ni fɛɛrɛ caman ye minnu bɛ ɲɔgɔn dafa, wa an y’a jira ko ni gɛlɛya donna mitokondriyaw ka baarakɛbaliya juguman fɛ, sɔgɔsɔgɔninjɛw bɛ ni farikoloɲɛnajɛ kɛcogo ye min tun ma dɔn fɔlɔ. An kabakoyara kosɛbɛ k’a ye ko rewiring kɛcogo bɛɛ tɛ farikoloɲɛnajɛ laban taamasiɲɛ ye min bɛ taa ni farikolojidɛsɛ ye dɔɔni dɔɔni ani cogo min tɛ se ka segin kɔ, nka an ka kunnafoniw b’a jira ko a bɛ se ka kɛ ladonni senfagabana ye hali ni a kɛra waati la sani selilɛri ka sa Functional compensation mechanism. Nin sɔrɔ in b’a Jira ko farikolo-ɲɛnajɛ-minɛnw bɛ ni farikolo-ɲɛnajɛ-plastiki hakɛ caman ye. Nin ko in b’a jira ko MFN2 seginni kɔfɛ, o bɛ se ka farikoloɲɛnajɛ taamasiɲɛ jɔnjɔnw jirali kɔsegin ani ka PN tiɲɛni bali. O kɔfɛ, a bɛ joli siraw gɛlɛya bali ani ka farikolojidɛsɛ teliya. cɛnimusoya kɛli.
An ka ɲininiw na, fɛn minnu ye mɔgɔw ɲɛnajɛ kosɛbɛ, olu dɔ ye ko PN minnu tɛ ni OXPHOS ye, olu bɛ se ka TCA sɛrɛkili farikoloɲɛnajɛ caman sɛmɛntiya ni anzimuw wulicogo ye minnu bɛ joli siraw sɔgɔsɔgɔ kɛrɛnkɛrɛnnenya la. Fɛnɲɛnɛmafagalanw labɛncogo kura ye kansɛribanakisɛw ka ko ye min bɛ kɛ tuma caman na, dɔw bɛ u jigi da glutamine kan walasa ka TCA cycle intermédiaires fara u kan walasa ka fɛnw bɔ minnu bɛ dɔ bɔ ɲɔgɔn na, minnu bɛ ninakili cakɛda bila ani ka lipid ni nucléotide biyosintɛzi ɲɛbilafɛnw dilanni sabati (39 , 40). Kɔsa in na, sɛgɛsɛgɛli dɔ y’a jira ko farikolo yɔrɔw la minnu bɛ OXPHOS baarakɛbaliya sɔrɔ, glutamine/glutamate farikoloɲɛnajɛ jɛɲɔgɔnya seginni fana ye fɛn ye min bɛ ye kosɛbɛ (5, 41), yɔrɔ min na glutamine doncogo sira bɛ TCA sɛrɛkili la, o bɛ bɔ OXPHOS jogin juguya kosɔn (41). ). Nka, dalilu jɛlenw tɛ yen minnu bɛ tali kɛ senfagabana farikoloɲɛnajɛ plastiki ɲɔgɔn na farikolo la ani a nafa bɛ se ka kɛ bana in kɔnɔ. Kɔsa in na, sɛgɛsɛgɛli dɔ la min kɛra in vitro kɔnɔ, a jirala ko ɲɛgɛnɛsira fɔlɔw bɛ glutamate pools lajɛ walasa ka farikoloɲɛnajɛ kɛ, o bɛ oksidan farikolojɔli ni joli siraw gɛlɛyali sabati farikoloɲɛnajɛ degun cogoyaw la (42). A ka kan ka kɔlɔsi ko TCA cycle enzyme succinate dehydrogenase ka furakɛli balili kɔnɔ, a dalen b’a la ko piruvate carboxylation bɛ oxaloacetate sɔrɔli sabati cerebellar granule neurons sɛnɛlenw na (34). Nka, nin fɛɛrɛ ninnu nafa ka bon farikolo ta fan fɛ ni kunsɛmɛnasumaya ye (yɔrɔ min na a dalen b’a la ko joli siraw sɔgɔsɔgɔli bɛ dan astrocytes de la kosɛbɛ) nafa ka bon farikolo ta fan fɛ hali bi (43). O cogo la, an ka kunnafoniw b’a jira ko PN minnu tiɲɛna OXPHOS fɛ farikolo la, olu bɛ se ka wuli ka taa BCAA tiɲɛni ni piruvate karɔbɔli la, olu minnu ye TCA pooli cɛmancɛfɛnw farali sababuba fila ye. Hali ni a fɔra ko BCAA ka katabolisme bɛ se ka kɛ sababu ye ka senfagabana fanga sɛnɛcogo la, ka fara glutamate ni GABA jɔyɔrɔ kan farikolojidɛsɛ la (44), dalilu si tɛ yen hali bi o fɛɛrɛw kan in vivo. O la, a ka nɔgɔn ka miiri ko PN minnu tɛ baara kɛ, olu bɛ se ka TCA cɛmancɛfɛnw dunni musaka bɔ u yɛrɛma, minnu bɛ ɲɛminɛ ni asimilasiyɔn taabolo ye, ni dɔ farali ye joli siraw la. Kɛrɛnkɛrɛnnenya la, PCx jiginni bɛ se ka kɛ wajibi ye walasa ka asidi aspartitiki ɲinini cayalenba mara, o min bɛ fɔ farikolokisɛw la minnu bɛ caya ni mitokondriyaw ka baarakɛbaliya ye (45). Nka, an ka fɛnɲɛnamafagalanw sɛgɛsɛgɛli ma fɛn caman jira asidi asidi aspartiki hakɛ jɔlen na Mfn2cKO PNw kɔnɔ (Ja S6A), o min bɛ miiri ko asidi aspartitiki baarakɛcogo danfaralen jira farikolo la selilu camanba ni mitozi kɔfɛ senfagabana cɛ Hali n’a y’a sɔrɔ PCx jiginni fɛɛrɛ tigitigi min bɛ sɔrɔ senfagabana baarakɛbaliw la in vivo, o ma dɔn fɔlɔ, an y’a jira ko nin jaabi min bɛ kɛ ka kɔn a waati ɲɛ, o jɔyɔrɔ ka bon senfagabana redox cogoya sabatili la, o min jirala FLIM ka kɔlɔsiliw la kunsɛmɛnasumaya sɔgɔsɔgɔninjɛw kan. Kɛrɛnkɛrɛnnenya la, ni PNw bali ka PCx kɔrɔta, o bɛ se ka kɛ sababu ye ka oksidan sɔrɔ kosɛbɛ ani ka selilɛriw ka saya teliya. BCAA tiɲɛni baarakɛli ni piruvati karɔbɔli tɛ fɛɛrɛw ye minnu bɛ se ka mitokondriyaw ka baarakɛbaliya lamini yɔrɔw jira (7). O la, a bɛ iko u ye OXPHOS dɛsɛbagatɔw ka fɔlɔfɔlɔko ye, hali n’o tɛ u kelenpe ye, min nafa ka bon farikolojidɛsɛ la. .
Kunsɛmɛnasumaya ye farikolojidɛsɛbana suguya ye min tɛ kelen ye, a ka ca a la a bɛ ye i n’a fɔ ataxia, wa a ka teli ka PNw tiɲɛ (46). Nin senfagabana jamana in ka teli ka kɛ kɛrɛnkɛrɛnnenya la mitokondriyaw ka baarakɛbaliya la bawo u ka sɛgɛn sugandilen sosow la, o bɛ se ka kɛ sababu ye ka farikoloɲɛnajɛ taamasiɲɛ caman lasegin minnu bɛ hadamadenw ka kɔkolo-sɔgɔsɔgɔninjɛ ataxie (16, 47, 48). Ka kɛɲɛ ni kunnafoniw ye, soso modɛli min jiginna ni jamu caman ye, o bɛ tali kɛ hadamadenw ka kɔkolo-sɔgɔsɔgɔninjɛ ataxie la, wa a bɛ ni mitokondriyaw baarakɛbaliya ye (49, 50), o b’a sinsin a kan ko OXPHOS dɛsɛ kɔlɔlɔw sɛgɛsɛgɛli nafa ka bon PNPH la. O la, a bɛnnen don kɛrɛnkɛrɛnnenya la ka nin sɔgɔsɔgɔninjɛ suguya kɛrɛnkɛrɛnnen in faranfasi ka ɲɛ ani k’a sɛgɛsɛgɛ. Nka, k’a da a kan PNw bɛ degun kosɛbɛ, wa u bɛ kɛ sababu ye ka ɲɛgɛnɛsiralabanaw bɛɛ lajɛlen hakɛ dɔgɔya, omiki kalan caman na, u farali sugandilen na i n’a fɔ selilu kuuru, o ye gɛlɛya ye hali bi. Hali n’a bɛ se ka kɛ ko a tɛ se ka kɛ ka nɔgɔ dɛsɛ pewu selilu suguya wɛrɛw la (kɛrɛnkɛrɛnnenya la balikuw ka farikolo yɔrɔw), an ye faranfasi fɛɛrɛ ɲuman dɔ fara ɲɔgɔn kan ni FACS ye walasa ka senfagabana ɲɛnama hakɛ bɛrɛ sɔrɔ walasa ka farikolojɔlifɛnw sɛgɛsɛgɛli kɛ duguma, ani ka farikolojɔlifɛnw datugucogo caman sɔrɔ kosɛbɛ (poroteyini 3000 ɲɔgɔn) ni i y’a suma ni kunnafonidilan bɛɛ ye min bɛ yen (51). Ni an ye selilu kuuru ka ɲɛnamaya lakana, an bɛ fɛɛrɛ min di yan, o t’a to an tɛ se ka farikoloɲɛnajɛ siraw caman yeli dɔrɔn lajɛ mitokondriyaw kɔnɔ, nka an bɛ se ka fɛn caman sɛgɛsɛgɛli fana kɛ a sitoplasma ɲɔgɔnna fɛnw na, o min bɛ mitokondriyaw ka membrane tags tali dafa walasa ka selilu suguya nafa Fɛɛrɛ kura min bɛ mitokondriyaw hakɛ la farikolo yɔrɔ gɛlɛnw na (52, 53). An bɛ fɛɛrɛ min ɲɛfɔ, o tɛ tali kɛ Purkinje selilɛriw kalanni dɔrɔn na, nka a bɛ se ka kɛ nɔgɔya la selilu suguya bɛɛ la walasa ka farikoloɲɛnajɛ caman yeli ɲɛnabɔ banabagatɔw ka hakili la, hali mitokondriyaw ka baarakɛbaliya misali wɛrɛw.
A laban na, an ye furakɛli finɛtiri dɔ jira nin farikoloɲɛnajɛ labɛncogo kura in senfɛ min bɛ se ka selilɛriw ka degun taamasiɲɛ jɔnjɔnw kɔsegin pewu ani ka senfagabana tiɲɛni bali. O la, ni an ye seginkanni kɛcogo faamuya min ɲɛfɔlen don yan, o bɛ se ka hakilina jɔnjɔnw di furakɛlicogo dɔw kan minnu bɛ se ka kɛ walasa ka senfagabana ɲɛnamaya sabati mitokondriyaw baarakɛbaliya waati. Sɛgɛsɛgɛli nataw minnu kun ye ka fanga sɛnɛcogo yeli fɛn caman tigɛli ye kunsɛmɛnasumaya suguya wɛrɛw la, olu ka kan ka kɛ walasa ka nin sariyakolo in waleyacogo jira kosɛbɛ hakilibana wɛrɛw la.
MitoPark sogow ɲɛfɔra ka tɛmɛ (31). C57BL/6N sogo minnu bɛ ni loxP flanking Mfn2 jamu ye, olu ɲɛfɔra ka tɛmɛ (18) ani u ni L7-Cre sogow cɛtigɛ (23). O kɔfɛ, den fila hɛtɛrozigɔti minnu sɔrɔla, olu ni Mfn2loxP/Mfn2loxP sogo homozigɔtiw tigɛra walasa ka Purkinje kɛrɛnkɛrɛnnenw ka jɛnɛya knockouts sɔrɔ Mfn2 kama (Mfn2loxP/Mfn2loxP; L7-cre). Jɛɲɔgɔnya kulu fitinin dɔ kɔnɔ, Gt (ROSA26) SorStop-mito-YFP alelɛ (stop-mtYFP) donna a kɔnɔ ni tɛmɛsira wɛrɛw ye (20). Baganw ka taabolo bɛɛ kɛra ka kɛɲɛ ni Erɔpu, jamana ani sigidamɔgɔw ka laadilikanw ye, wa u sɔnna LandesamtfürNatur fɛ min bɛ Umwelt ani Verbraucherschutz, Rhine-Westphalie woroduguyanfan fɛ, Alimanjamana na. Baganw ka baara fana bɛ tugu Erɔpu laboratuwari bagan dɔnniya tɔnba ka laadilikanw kɔ.
Musokɔnɔma ka kɔnɔbara basigilen lafiyalen kɔfɛ, soso den bɛ bɔ a la (E13). Kɔnɔbara tigɛlen don Hanks ka kɔgɔ balansi (HBSS) la min farala Hepes milimɛtɛrɛ 10 kan, ka tɛmɛ Dulbecco ka Modified Eagle’s Medium kan, papayini (20 U/ml) ni sitɛyini (1μg/ml) bɛ min na. Aw bɛ farikolo yɔrɔ in bila DMEM kɔnɔ) ka a faranfasi ni anzimu ye. Ml) la 37°C la miniti 20 kɔnɔ, ka sɔrɔ ka a sɔgɔ ni masin ye DMEM la min farala denso kɔnɔ misiden 10% kan. Bugunw tun bɛ kisɛ kɛ galasi datugulanw kan minnu datugulen don ni polilisini ye ni u hakɛ ye 2×106 ye santimɛtɛrɛ 6 sɛnɛkɛminɛn kɔnɔ walima ni u hakɛ ye 0,5×105 ye selilu/santimɛtɛrɛ 2 walasa ka ja sɛgɛsɛgɛli kɛ. Lɛrɛ 4 kɔfɛ, aw bɛ furakisɛ bila a nɔ na ni Neurobasal serum-free medium ye, B27 faralen 1% ani GlutaMax 0,5 mM bɛ min na. O kɔfɛ, sɔgɔsɔgɔninjɛw tun bɛ mara 37°C ni 5% CO2 la k’u kɛ k’a lajɛ bɛɛ kɔnɔ, ka dumuni di u ma siɲɛ kelen dɔgɔkun kɔnɔ. Walasa ka se ka ɲɔgɔn sɔrɔ kokura in vitro, nin AAV9 banakisɛfagalan in 3μl (kɔlɔn 24 bɛ min kɔnɔ) walima 0,5μl (kɔlɔn 24 plati) kɛra ka senfagabana furakɛ tile filanan na in vitro: AAV9.CMV.PI.eGFP. WPRE.bGH (Addgene, gafe nimɔrɔ 105530-AAV9) ani AAV9.CMV.HI.eGFP-Cre.WPRE.SV40 (Addgene, gafe nimɔrɔ 105545-AAV9).
Sogo Mfn1 ni Mfn2 ADN dafalen (minnu sɔrɔla Addgene plasmide #23212 ani #23213 fɛ), olu taamasiyɛn bɛ kɛ ni V5 sinsinni ye (GKPIPNPLLGLDST) C-terminus la, wa u bɛ fara ɲɔgɔn kan ni mCherry ye frame kɔnɔ T2A sinsinni fɛ. Grx1-roGFP2 ye nilifɛn ye min bɔra Heidelberg T. P. Dick DFKZ (Deutsches Krebsforschungszentrum) fɛ. Ni tdTomato kasɛti bilala a nɔ na ni cloning fɛɛrɛ kɔrɔw ye, kasɛti kɛra subcloned ye pAAV-CAG-FLEX-tdTomato kɔkolo la (Addgene reference number 28306) walasa ka pAAV-CAG-FLEX-mCherry-T2A-MFN2-V5, pAAV-CAG- FLEX-mCherry-T2A-MFN1-V5 ani pAAV-CAG-FLEX-Grx-roGFP2 banakisɛfagalanw. O fɛɛrɛ kelen in kɛra ka kɔrɔsili vektɔri pAAV-CAG-FLEX-mCherry dilan. Walasa ka AAV-shPCx dilancogo dilan, plasmide AAV vecteur (VectorBuilder, pAAV [shRNA] -CMV-mCherry-U6-mPcx- [shRNA#1]) de ka kan, min kɔnɔ ADN sinsinni bɛ sɔrɔ min bɛ shRNA laɲini soso PCx (5′CTTTCGCTCTCTAAGGTGCTAAACTCGAGTTTAGCACCTTAGAGCGAAAG) sɔrɔ 3′) U6 promotɛri ka kɔlɔsili kɔnɔ, mCherry bɛ kɛ CMV promotɛri ka kɔlɔsili kɔnɔ. AAV fɛnɲɛnamafagalan dɛmɛnanw dilanni kɛra ka kɛɲɛ ni a dilannikɛla ka laadilikanw ye (Cell Biolabs). Kuma surun na, aw bɛ baara kɛ ni plasmide transfert ye min bɛ mCherry-T2A-MFN2-V5 (pAAV-CAG-FLEX-mCherry-T2A-MFN2-V5), mCherry-T2A-MFN1-V5 (pAAV-CAG-FLEX-mCherry) ta waati dɔɔnin kɔnɔ Transfection of 293AAV cells-T2A-MFN1-V5), mCherry (pAAV-CAG-FLEX-mCherry) walima Grx-roGFP2 (pAAV-CAG-FLEX-Grx-roGFP2) kodeli jɛnɛya, ka fara AAV1 kapisidi poroteyini kodeli ni a dɛmɛnan poroteyini Packaging plasmid plasmid, ni kalisiyɔmu fositɛri fɛɛrɛ ye. Virus (banakisɛfagalan) min bɛ sanfɛ, o sɔrɔla ni freeze-thaw cycles (sɔgɔsɔgɔninjɛ-sɔgɔsɔgɔninjɛ) ye jisuma jalen/ethanol bath kɔnɔ ani ka selilu lysed (lysed cells) kɛ phosphate buffered saline (PBS) kɔnɔ. AAV fɛnɲɛnamafagalan saniyalen don ni iodixanol gradient ultracentrifugation discontinuous ye (lɛrɛ 24 32.000 rpm ani 4°C) ka sɔrɔ ka a caman kɛ ni Amicon ultra-15 centrifugal filter ye. 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 ka genome titer tun bɛ i n’a fɔ a ɲɛfɔra cogo min ka tɛmɛ (54), o suman ni real-time quantitative PCR (qPCR) ye -MFN1-V5 (1,9×1013 GC/ml) ani AAV1-CAG-FLEX-Grx-roGFP2 (8,9×1012 GC/ml).
Senfagabana fɔlɔw tun bɛ sɔgɔsɔgɔ 1x PBS kɔnɔ, ka u kɛ bɔgɔlan ye, ka sɔrɔ ka u kɛ kelen ye 0,5% Triton X-100 / 0,5% sodium deoxycholate/PBS lysis buffer kɔnɔ, fosifatazi ni protease inhibiteur (Roche) bɛ min kɔnɔ. Protéines hakɛ jateminɛ kɛra ni asidi bicinchoninic assay (Thermo Fisher Scientific) ye. O kɔfɛ, farikolojɔlifɛnw farala ɲɔgɔn kan ni SDS-polyacrylamide gel electrophoresis ye, ka sɔrɔ ka u ko ka kɛ polivinilideni fluoride membrane (GE Healthcare) kan. Aw bɛ yɔrɔ kɛrɛnkɛrɛnnenw bali ani ka u bila ni banakisɛfagalan fɔlɔ ye (aw ye sɛbɛn ɲɛ Tablo S1 lajɛ walasa ka kunnafoni wɛrɛw sɔrɔ) nɔnɔ 5% la TBST la (Tris-buffered saline with Tween), kocogo taabolo ani banakisɛfagalan filanan TBST Incubate kɔnɔ. Aw bɛ a tobi ni banakisɛfagalan fɔlɔ ye su fɛ +4°C la. Koli kɔfɛ, aw bɛ banakisɛfagalan filanan kɛ fo lɛri 2 so funteni na. O kɔfɛ, ni o blot kelen in kɛra ni anti-β-actin anticorps ye, o doni kelen in dafara. Kɔlɔsili ni fɛn caman tigɛli ye ka kɛ kemiluminisɛnsi ye ani ka kemiluminisɛnsi fanga bonya (GE Healthcare).
Senfagabana minnu tun kisɛra gilasi datugulanw kan ka kɔrɔ, olu sirilen don ni parafɔrimulidihidi (PFA)/PBS 4% ye waati latigɛlen na so funteni na miniti 10 kɔnɔ. Aw bɛ fɔlɔ ka datugulanw don ni Triton X-100/PBS 0,1% ye miniti 5 kɔnɔ so funteni na, ka sɔrɔ ka u don tampon bloquant [3% bovine serum albumin (BSA)/PBS) kɔnɔ. Tile filanan na, datugulanw ko ni tampon bloquant ye ka u bila ni anticorps secondaire conjugué fluorophore bɛnnen ye sanga 2 kɔnɔ so funteni na a laban na, u ko ka ɲɛ PBS la ni 4′,6-diamidino-2 -Phenylindole (DAPI) bɛ counterstained ye ka sɔrɔ ka u siri mikroskɔpu slide kan ni Aqua-Poly/Mount ye.
Sogow (cɛ ni muso) ye lafiya sɔrɔ ni ketamini (miligaramu 130/kg) ni zilazini (miligaramu 10/kg) pikiri ye kɔnɔbara la ani ka u kɛ fari jukɔrɔ ni dimimadafura ye karɔporofɛni (miligaramu 5/kg) , Ka u bila stɛriyɔmu minɛn kɔnɔ (Kopf) min bɛ ni fɛn sumalen ye. Aw bɛ kungolo bɔ kɛnɛ kan ani ka ɲinw furakɛcogo kɛ walasa ka ɲɛgɛnɛsira yɔrɔ min bɛ bɛn mis kolo ma (ka bɔ lambda la: kɔ 1,8, kɛrɛfɛ 1, min bɛ bɛn lobules IV ni V ma) fin. Aw bɛ pikirijikɛlan kurulen kɛ ka dingɛ fitini dɔ da kungolo la ka ɲɛ walasa aw kana duguma joli siraw tiɲɛ. O kɔ, gilasi ɲɛgɛnɛsira finman min jalen don, o bɛ don dɔɔni dɔɔni mikro-dingɛ kɔnɔ (ka bɔ -1,3 la ka se -1 ma dura mater kɔnɔbara fan fɛ), ka sɔrɔ ka AAV 200 fo 300 nl pikiri kɛ mikro-pikirikɛlan (Narishige) la ni bolola pikirijikɛlanw ye (Narishige) siɲɛ caman ni tansiyɔn dɔgɔman ye waati dɔ kɔnɔ miniti 10 fo miniti 20 finɛtiri kɔnɔ. Pikiri kɛlen kɔfɛ, aw bɛ ɲɛgɛnɛsira bila miniti 10 wɛrɛ kɔnɔ walasa banakisɛ ka jɛnsɛn pewu. Kapilɛriw bɔlen kɔ, u bɛ fari siri ka ɲɛ walasa ka joginda funu dɔgɔya ani ka bagan bila a ka kɛnɛya. Baganw furakɛra ni dimimadafura ye (kaspofen) tile damadɔ kɔnɔ opereli kɔfɛ, o waati kɔnɔna na, u farikolo cogoya tun bɛ kɔlɔsi kosɛbɛ, o kɔfɛ, u fagara waati fɔlen na. Taabolo bɛɛ kɛra ka kɛɲɛ ni Erɔpu, jamana ani sigidamɔgɔw ka laadilikanw ye, wa u sɔnna LandesamtfürNatur fɛ min bɛ Umwelt ani Verbraucherschutz, Rhin Worodugu-Westfalen, Alimanjamana na.
Baganw lafiyalen don ni ketamini (miligaramu 100/kg) ni zilazini (miligaramu 10/kg) ye, ka sɔrɔ ka dusukun pikiri ni 0,1 M PBS ye fɔlɔ, ka sɔrɔ ka 4% PFA kɛ PBS kɔnɔ. Aw bɛ farikolo yɔrɔ tigɛtigɛ ka a siri 4% PFA/PBS la su fɛ 4°C la. Muru min bɛ yɛrɛyɛrɛ (Leica Microsystems GmbH, Vienne, Ɔtirisi) kɛra ka sagittal yɔrɔw labɛn (50 μm janya) ka bɔ kunkolo jɔlen na PBS kɔnɔ. Fo ni a fɔra cogo wɛrɛ la, yɔrɔw nɔgɔli kɛra i n’a fɔ a ɲɛfɔlen don cogo min na sanfɛ (13) so funteni na ani ka u lamaga. Kuma surun na, fɔlɔ, sɔgɔsɔgɔninjɛ sɔrɔlenw kɛra permeabiliser ye ni 0,5% Triton X-100/PBS ye miniti 15 kɔnɔ so funteni na; ka ɲɛsin epitɔpi dɔw ma (Pcx ni Shmt2), ni aw bɛ tris-EDTA tampon na 80°C (PH 9) la, aw bɛ sɔgɔsɔgɔninjɛw sumaya miniti 25 kɔnɔ nin taamasiɲɛ in nɔ na. O kɔfɛ, tilayɔrɔw tun bɛ tobi ni banakisɛfagalan fɔlɔ ye (aw ye sɛbɛn ɲɛ Tablo S1 lajɛ) tampon bloquant (3% BSA/PBS) kɔnɔ 4°C la su fɛ ni u wulilen ye. O dugujɛ, yɔrɔw ko ni tampon bloquant ye ka u bila ni anticorps secondaire conjugué fluorophore bɛnnen ye sanga 2 kɔnɔ so funteni na; a laban na, yɔrɔw ko ka ɲɛ PBS la, ka u nɔgɔ ni DAPI ye, ka sɔrɔ ka u siri ni AquaPolymount On a microscope slide ye.
Laser scanning confocal microscope (TCS SP8-X walima TCS Digital Light Sheet, Leica Microsystems) min bɛ ni laser yeelen finman ye ani laser ultraviolet 405 diode ye, o kɛra ka ja in ja. Ni fluorophore (fluorophore) lawulili ye ani ka taamasiyɛn lajɛ ni Hybrid Detector (HyDs) ye, LAS-X porogaramu kɛra ka ja caman lajɛ minnu bɛ bɛn Nyquist ka misali ta ma cogo la min bɛ tugu ɲɔgɔn na : panɛriw la minnu tɛ hakɛ ye, o ye taamasiyɛnw ye minnu bɛ fanga sɔrɔ kosɛbɛ (misali la, somatiki selilɛriw ni dendɛrɛsiw kɔnɔ) mtYFP) Baara kɛ ni HyD ye walasa ka PN hakɛ dɔn BrightR cogo la). Gating min bɛ Bɔ 0,3 na ka Se 6 ns ma, o bɛ Kɛ walasa ka kɔkanna Dɔgɔya.
Selilu minnu tilalen don, olu jatebɔ waati yɛrɛ la. Tilalen kɔfɛ Neurobasal-A kɔnɔ, B27 faralen 1% ni GlutaMax 0,5 mM bɛ min kɔnɔ, o yɔrɔnin bɛɛ la, seliluw kisɛra gilasi slayidiw kan minnu datugulen don ni poli-l-lisini ye (μ-Slide8 Well, Ibidi, katalogu nimɔrɔ 80826) , Ka sɔrɔ k’a bila 37°C ani 5% CO2 la sanga 1 kɔnɔ walasa seliluw ka sigi. Ja minnu bɛ kɛ waati yɛrɛ la, olu kɛra Leica SP8 laser scanning confocal microscope kan, min bɛ ni laser finman ye, HyD, 63×[1.4 numerical aperture (NA)] tulu laɲini lens ani tasuma-yɔrɔ.
Sogo in lafiyala joona ni gazi ɲuman ye ka a kunkolo tigɛ, ka a kunkolo bɔ joona a kungolo la, ka tila ka kɛ 200μm ye (ka kɛɲɛ ni 13C taamasiyɛn kɛcogo ye) walima ka kɛ 275μm ye (ka ɲɛsin foton sɛgɛsɛgɛli fila ma) sagittal section min falen bɛ nin fɛn ninnu na The ice cream (HM-650 V, Thermo Fisher Scientific, Walldorf, Germany) bɛ fa ni nin fɛn ninnu ye : 125 mM jisuma na, karɔbɔni caman bɛ min na (95% O2 ani 5% CO2) Ca2 dɔgɔyalen + kunsɛmɛnasumaya jilama dilannen (ACSF) NaCl, 2,5 mM KCl, 1,25 mM fosifate sodiyɔmu tampon, 25 mM NaHCO3, 25 mM glucose, 0,5 mM CaCl2 ani 3,5 mM MgCl2 (osmotique tansiyɔn bɛ se miligaramu 310 fo 330 ma). Aw bɛ kunsɛmɛnasumaya sɔrɔlenw bila yɔrɔ la sani u ka wuli ka taa a bila yɔrɔ la, Ca2 + ACSF caman bɛ min kɔnɔ (NaCl 125,0 mM, KCl 2,5 mM, tampon fosifate sodiyɔmu 1,25 mM, NaHCO3 25,0 mM, d-glucose 25,0 mM, CaCl2 mililitiri 1,0 ani MgCl2 mililitiri 2,0) bɛ sɔrɔ min kɔnɔ. Medium) pH 7,4 ani miligaramu 310 fo 320).
Jalabɔ senfɛ, sɔgɔsɔgɔninjɛw bilala ja lajɛyɔrɔ kɛrɛnkɛrɛnnen dɔ kɔnɔ, wa o kɔlɔsili kɛra ACSF pikirijikɛlan basigilen kɔnɔ, funteni basigilen na min bɛ se 32° fo 33°C ma. Laser scanning microscope multiphoton (TCS SP8 MP-OPO, Leica Microsystems) min bɛ ni Leica 25x objective lens (NA 0.95, ji) ye, Ti: Sapphire laser (Chameleon Vision II, Coherent) kɛra ka slice ja lajɛ. Module FLIM (PicoHarp300, PicoQuant) ye baara kɛ ni min ye.
Grx1-roGFP2 ka FLIM. Yɛlɛma minnu kɛra PNw ka sitoplasma redox cogoya la, olu suman ni foton fila FLIM ye sagittal brain slices kɔnɔ, yɔrɔ min na Grx1-roGFP2 biosensor tun bɛ PNw laɲini. PN layɛrɛ kɔnɔ, sɔrɔyɔrɔ bɛ sugandi 50 fo 80 μm ɲɔgɔn na sɔgɔsɔgɔninjɛ sanfɛla jukɔrɔ walasa ka a jira ko PN bɛ se ka kɛ (o kɔrɔ ye ko biɲɛw jɔcogo dɛsɛ walima senfagabana cogoya caman yeli dendɛrɛsiw kɛrɛfɛ) ani roGFP2 sensɛri ɲuman fila ni AAV min bɛ shRNA PCx kode walima a kɔlɔsili sinsinni (u kelen-kelen bɛɛ bɛ mCherry jira ɲɔgɔn fɛ). Ja minnu bɛ kɛ kulu kelen na, olu lajɛ ni 2x zoom numérique ye [excitation wavelength: 890 nm; 512 nm 512 ye pixel ye]. Kɔlɔsili : kɔnɔna HyD, fluorescein isothiocyanate (FITC) filɛri kulu] ani ja cɛmancɛw kɛli miniti 2 fo 3 kɔnɔ, olu bɛ kɛ walasa ka foton caman lajɛ (foton 1000 bɛɛ lajɛlen) walasa ka kurufew don. Grx1-roGFP2 sonda ka sensitivite ani FLIM cogoyaw sɛgɛsɛgɛli kɛra ni roGFP2 ka ɲɛnamaya nafa kɔlɔsili ye ni a bɛ H2O2 10 mM kɛnɛma fara perfusion ACSF kan (walasa ka oksidan caya, o bɛ na ni si hakɛ caya ye), o kɔfɛ, ka dithiothreitol 2 mM fara a kan (o bɛ dɔgɔyali hakɛ dɔgɔya, o bɛ na ni a dɔgɔyali ye ɲɛnamaya kɔnɔ) (Ja S8, D ka taa G la). Aw bɛ baara kɛ ni FLIMfit 5.1.1 porogaramu ye walasa ka jaabi sɔrɔlenw sɛgɛsɛgɛ, ka ja bɛɛ ka tiɲɛni kuru kelen bɛ bɛn IRF sumanlen ma (minɛn jaabi baarakɛcogo), ani χ2 bɛ bɛn 1 ma. Walasa ka PN kelen ka ɲɛnamaya jateminɛ, nɛrɛmuguma farikolo lamini masike ja kɛra bolo la, wa masɔrɔ kelen-kelen bɛɛ si hakɛ danmadɔ kɛra hakɛ jateminɛni ye.
Mitokondriyaw seko ni dɔnko sɛgɛsɛgɛli. Dakun jugumanba in bilalen kɔ ni TMRM 100 nM ye min farala ACSF perfusé kan k’a ɲɛsin miniti 30 ma, PNw ka mitokondriyaw seko caman yeli suman ni foton fila mikroskɔpu ye. TMRM ja lajɛ kɛra ni sonda lawulili ye 920 nm la ani ka baara kɛ ni kɔnɔna HyD (tetramethylrhodamine isothiocyanate: 585/40 nm) ye walasa ka taamasiɲɛw lajɛ; ni baara kɛli ye ni wulicogo kelen ye nka ka baara kɛ ni kɔnɔna HyD wɛrɛ ye (FITC :525/50) walasa ka mtYFP ja. Baara kɛ ni ImageJ ka Image Calculator plugin ye walasa ka mitokondriyaw seko jateminɛ selilɛri kelen ta fan fɛ. Kuma surun na, plug-in equation : signal = min (mtYFP, TMRM) bɛ kɛ ka mitokondriyaw yɔrɔ dɔn min bɛ TMRM taamasiyɛn jira Purkinje Somalikan na, o bɛ kɛ ni kanal min bɛ bɛn o ma, o ja confocal single-stack kɔnɔ. O kɔfɛ, pixel yɔrɔ min bɛ mask sɔrɔlen kɔnɔ, o bɛ jate, o kɔfɛ, a bɛ normalize kɛ mtYFP kanal ka threshold single-stack image kan min bɛ bɛn o ma walasa ka mitokondriyaw tilayɔrɔba sɔrɔ min bɛ mitokondriyaw seko jira.
Ja in deconvoluté ni Huygens Pro (Scientific Volume Imaging) porogaramu ye. Tile ja minnu sɛgɛsɛgɛra, tile kelen montage bɛ kɛ ni automatique stitching algorithm ye min bɛ di LAS-X porogaramu fɛ. Ja jateminɛni kɔfɛ, baara kɛ ni ImageJ ni Adobe Photoshop ye walasa ka ja in baara ka taa a fɛ ani ka yeelen ni danfara ladilan cogo kelen na. Baara kɛ ni Adobe Illustrator ye walasa ka ja labɛn.
mtDNA sinsinnan sɛgɛsɛgɛli. ADNmt joginda hakɛ jateminɛna kunsɛmɛ yɔrɔw kan minnu taamasiyɛn kɛra ni ADN kɛlɛli furaw ye ni mikroskɔpu konfokali ye. Laɲini yɔrɔ kelen-kelen bɛɛ dabɔra seli farikolo ni seli kelen-kelen bɛɛ nucleus kama, wa yɔrɔ kelen-kelen bɛɛ jatebɔ kɛra ni Multi Measure plug-in (ImageJ porogaramu) ye. Aw bɛ nukili yɔrɔ bɔ selilɛri farikolo yɔrɔ la walasa ka sitoplasma yɔrɔ sɔrɔ. A laban na, Analyze Particles plug-in (ImageJ porogaramu) kɛra ka ADN sitoplasma yɔrɔw hakɛ jateminɛ otomatiki la minnu bɛ mtDNA jira ja dakun na, ani jaabi sɔrɔlenw kɛra normal ye ka kɛɲɛ ni CTRL sogow ka PN moyenne ye. O jaabiw bɛ jira ni nukilisi hakɛ cɛmancɛ ye seli kelen kɔnɔ.
Protéinew jiracogo sɛgɛsɛgɛli. Baara kɛ ni ImageJ ka Image Calculator plugin ye walasa ka farikolojɔlifɛnw jiracogo jateminɛ PN kɔnɔ selilu kelen hakɛ la. Kuma surun na, kanal min bɛ bɛn o ma, o ja min bɛ kɛ ni fɛn kelen ye, o kɔnɔ, o bɛ sɔrɔ ni ɲɔgɔndan ye: taamasiɲɛ = min (mtYFP, anticorps), mitokondriyaw yɔrɔ min bɛ immunoreactivité jira anticorps dɔ la Purkina kɔnɔ, o bɛ dɔn. O kɔfɛ, pixel yɔrɔ min bɛ mask sɔrɔlen kɔnɔ, o bɛ jate, o kɔfɛ, a bɛ normalize kɛ mtYFP kanal ka threshold single-stack image correspondant kan walasa ka protéine jiralen ka mitokondriyaw tilayɔrɔba sɔrɔ.
Purkinje selilɛri density sɛgɛsɛgɛli. ImageJ ka Cell Counter plug-in kɛra ka Purkinje density jateminɛ ni Purkinje selilu jatelen hakɛ tilali ye ni cerebellar ring janya ye min minɛna selilu jatelenw fɛ.
Misaliw labɛnni ni u lajɛli. Kɔlɔsili kulu ni Mfn2cKO sogow ka hakili minnu bɔra PFA/2,5% glutaraldehyde la fositɛri tampon (PB) 0,1 M kɔnɔ, o kɔfɛ, koronawirisi yɔrɔw labɛnna ni ciliatiw ye (Leica Mikrosysteme GmbH, Vienne, Ɔtirisi) (a girinya bɛ se 50 fo 60 μm ma) O kɔfɛ, u sirilen don PB tampon na os tetraoxide 1% kɔnɔ ani Aw bɛ potasiyɔmu ferosiyanidi 1,5% kɛ so funteni na fo lɛri 1. Aw bɛ yɔrɔw ko siɲɛ saba ni ji wulilen ye, ka sɔrɔ ka u nɔgɔ ni etanɔli 70% ye, uranili asetati 1% bɛ min na miniti 20 kɔnɔ. O kɔfɛ, yɔrɔw ji bɔra dɔlɔ hakɛ la, ka u don Durcupan ACM (Araldite casting resin M) epoxy resin (Electron Microscopy Sciences, katalogu nimɔrɔ 14040) kɔnɔ, gilasi sɔgɔlenw ni ɲɔgɔn cɛ, minnu datugulen don ni siliyɔmu ye, ka laban ka kɛ 60°C la, u bɛ Polimerize kɛ foro kɔnɔ sanga 48 kɔnɔ. Cerebellar cortex yɔrɔ sugandira ka 50 nm ultrathin yɔrɔw tigɛ Leica Ultracut (Leica Mikrosysteme GmbH, Vienne, Austria) kan ka u ta 2×1 mm nɛgɛ sɔgɔlen grid kan min datugulen don ni polisitiri filimu ye. Tilayɔrɔw nɔgɔlen don ni uranili asetati 4% jilama ye H2O la miniti 10 kɔnɔ, ka u ko ni H2O ye siɲɛ caman, o kɔfɛ ni Reynolds plɔmu sitɛrɛti ye H2O kɔnɔ miniti 10 kɔnɔ, ka sɔrɔ ka u ko ni H2O ye siɲɛ caman. Mikorografuw kɛra ni transmission électronique microscope Philips CM100 (Thermo Fisher Scientific, Waltham, MA, Etazuni) ye ni TVIPS (Tietz Video and Image Processing System) TemCam-F416 kamera numérique ye (TVIPS GmbH, Gauting, Etazuni). Alimanjamana na).
AAV banakisɛ bɛ sogo minnu na, u kunkolo farala ɲɔgɔn kan ka u tigɛtigɛ ka kɛ sagittal yɔrɔ ye min janya ye milimɛtɛrɛ 1 ye, ka ɲɛgɛnɛsira sɛgɛsɛgɛ ni fluorescence microscope ye walasa ka AAV banakisɛ sɔrɔlen bololanɛgɛ dɔn (o kɔrɔ ye ko mCherry expressing). Kɔrɔbɔli minnu na AAV pikiri bɛ na ni Purkinje selilɛri (i n’a fɔ a bɛ se ka fɔ ko a bɛɛ lajɛlen) jiginni nafa ka bon kosɛbɛ, olu dɔrɔn de bɛ kɛ ni kunsɛmɛnasumaya bololanɛgɛ fila tugu-tugulen ye a dɔgɔyalenba la. AAV-transduite loop (AV-transduire loop) kɛra microdissected (mikɔrɔdisection) ye walasa ka su kɛ fixation kɔfɛ (4% PFA ani 2,5% glutaraldehyde 0,1 M cocoate buffer kɔnɔ) ka sɔrɔ ka baara kɛ ka taa a fɛ. Walasa ka EPON don, farikolo yɔrɔ jɔlen ko ni 0,1 M sodium cocoate buffer (Applichem) ye, ka sɔrɔ ka a tobi ni 2% OsO4 (os, Science Services; Caco) ye 0,1 M sodium cocoate buffer (Applichem) kɔnɔ Lɛrɛ 4, o kɔ aw bɛ a ko sanga 2 kɔnɔ. Aw bɛ segin o kan siɲɛ 3 ni kokamidi tampon 0,1 M ye. O kɔfɛ, etanɔli jiginni sinsinni kɛra ka etanɔli jilama kelen-kelen bɛɛ bila 4°C la miniti 15 kɔnɔ walasa ka farikolojidɛsɛ bɔ a la. Aw bɛ farikolo yɔrɔ in bila poropilini ɔkisidi la ka a bila su fɛ EPON (Sigma-Aldrich) kɔnɔ 4°C la. Aw bɛ a fɛnɲɛnamafagalan bila EPON kura kɔnɔ so funteni na fo lɛri 2, o kɔ aw bɛ a don 62°C la fo lɛri 72. Aw bɛ ultramicrotome (Leica Microsystems, UC6) ni diamant muru (Diatome, Biel, Switzerland) kɛ ka yɔrɔw tigɛ minnu ka fin kosɛbɛ 70 nm, ka u nɔgɔ ni uranili asetati 1,5% ye miniti 15 kɔnɔ 37°C la, ka u nɔgɔ ni sitɛrɛti plɔmu jilama ye miniti 4 kɔnɔ. Elektɔron mikorografu ninnu kɛra ni JEM-2100 Plus transmission electron microscope (JEOL) ye min bɛ ni Camera OneView 4K 16-bit (Gatan) ani DigitalMicrograph porogaramu (Gatan) ye. Walasa ka sɛgɛsɛgɛli kɛ, ɛlektrɔniki mikrografuw sɔrɔla ni 5000× walima 10.000× zoom numérique ye.
Mitokondriyaw cogoya sɛgɛsɛgɛli. Sɛgɛsɛgɛli bɛɛ kama, mitokondriyaw kelen-kelen bɛɛ ka kontoronw ɲɛfɔra bolo la ja digitɛriw kɔnɔ ni ImageJ porogaramu ye. Fɛnɲɛnɛma-ko-paramɛtiriw danfaralenw bɛ Sɛgɛsɛgɛ. Mitokondriyaw density bɛ jira ni kɛmɛsarada la min bɛ sɔrɔ ni selilu kelen-kelen bɛɛ ka mitokondriyaw yɔrɔ bɛɛ tilalen don ni sitoplasma yɔrɔ ye (sitoplasma yɔrɔ = selilɛri yɔrɔ-seli nucléaire yɔrɔ) × 100. Mitokondriyaw lamini hakɛ bɛ jate ni formula ye [4π∙(yɔrɔ/perimɛtiri 2) Mitokondriyaw ka ista cogoya sɛgɛsɛgɛra ka tila tila fila ye (“tubular” ani “blister”) ka kɛɲɛ n’u cogoya kunbabaw ye.
Otofagozomu/lizozomu hakɛ ni a density sɛgɛsɛgɛli. Baara kɛ ni ImageJ porogaramu ye walasa ka otofagozomu/lizozomu kelen-kelen bɛɛ cogoya jira ni bolo ye ja digitɛri kɔnɔ. Otofagozomu/lizozomu yɔrɔ bɛ jira ni kɛmɛsarada la jatebɔ ye ni selilu kelen-kelen bɛɛ ka otofagozomu/lizozomu jɔcogo yɔrɔ bɛɛ tilalen ye sitoplasma yɔrɔ la (sitoplasma yɔrɔ=seli yɔrɔ-nucleus yɔrɔ)×100. Otofagozomu/lizozomu density jate bɛ kɛ ni otofagozomu/lizozomu jɔcogo hakɛ bɛɛ tilalen ye seli kelen kɔnɔ (sitoplasma yɔrɔ siratigɛ la) (sitoplasma yɔrɔ = selilɛri yɔrɔ-nukilila yɔrɔ).
Labeling for acute sectioning ani sample labɛnni. Ni aw bɛ sɛgɛsɛgɛliw kɛ minnu bɛ sukaro taamasiɲɛ kɛ, aw bɛ kunsɛmɛnasumaya sɔgɔsɔgɔninjɛw bila yɔrɔ la min bɛ kɛ sani u ka wuli, o kɔnɔ, karɔbɔli saturated (95% O2 ani 5% CO2) bɛ sɔrɔ min kɔnɔ, Ca2 + ACSF ka ca (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 ani 2,0 mM MgCl 2, ladilanna ka kɛ pH 7,4 ye ani 310 ka se 320 mOsm ma), o la glucose ye 13 C 6- Glucose substitution (Eurisotop, katalogu nimɔrɔ CLM-1396). Ni aw bɛ k’a lajɛ minnu bɛ piruvate taamasiɲɛ kɛ, aw bɛ kunsɛmɛnasumaya sɔgɔlenw bila Ca2 + ACSF caman na (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 ani ka 2,0mM fara a kan MgCl2, aw bɛ a ladilan ka kɛ pH 7,4 ye ani 310 fo ka se 320mOsm ma), ka sɔrɔ ka 1mM 1-[1-13C]piruvati (Eurisotop, katalogu nimɔrɔ CLM-1082) fara a kan. Aw bɛ tilayɔrɔw tobi miniti 90 kɔnɔ 37°C la. Kɔrɔbɔli laban na, yɔrɔw ko joona ni jilama ye (pH 7,4) min kɔnɔ, amoniyɔmu karɔbɔni 75 mM bɛ sɔrɔ, o kɔfɛ, u kɛra kelen ye asetonitirili (ACN) 40:40:20 (v:v:v) la: mɛtanɔli: ji. Tilayɔrɔw bilalen kɔ jisuma kan miniti 30 kɔnɔ, u bɛ centrifugage kɛ garamu 21.000 la miniti 10 kɔnɔ 4°C la, ka sɔrɔ ka ji jɛlen min bɛ sanfɛ, o jalen don SpeedVac concentrateur kɔnɔ. Metabolitɛri jalen min sɔrɔla, o maralen bɛ -80°C la fo ka se a sɛgɛsɛgɛli ma.
Liquid chromatography-mass spectrometry analysis (sɛgɛsɛgɛli min bɛ kɛ ni amino acide 13 ye minnu taamasiyɛn ye C ye. Walasa ka ji sɛgɛsɛgɛli kɛ ni sɛgɛsɛgɛli ye min bɛ wele ko liquid chromatography-mass spectrometry (LC-MS), farikolojidɛsɛ furakisɛw bɛ segin ka kɛ LC-MS grade ji 75μl la (Honeywell). Santirifugare kɔfɛ garamu 21.000 la miniti 5 kɔnɔ 4°C la, a sanfɛla jɛlen 20 μl kɛra ka asidi aminiki jiginni sɛgɛsɛgɛ, ka sɔrɔ a bɔlen tɔ kɛra o yɔrɔnin bɛɛ ka kɛ aniyoni sɛgɛsɛgɛli ye (aw ye sɛbɛn ɲɛ jukɔrɔ lajɛ). Asidi aminiki sɛgɛsɛgɛli kɛra ni kuloridi bɛnzoyili bɔli fɛɛrɛ ye min ɲɛfɔra ka tɛmɛ (55, 56). Fɔlɔ la, karɔbɔni sodiyɔmu 100 mM (Sigma-Aldrich) 10μl farala fɛnɲɛnamafagalan 20μl kan, o kɔfɛ, bɛnzoyi kuloridi 2% (Sigma-Aldrich) 10μl farala LC grade ACN kan. Aw bɛ sɛgɛsɛgɛli kɛ ka vortex kɛ waati kunkurunnin kɔnɔ, ka sɔrɔ ka centrifuged kɛ garamu 21.000 la miniti 5 kɔnɔ 20°C la. Aw bɛ jisuma min saniyalen don, aw bɛ o kɛ mililitiri 2 otosamplɛri fiyɛlilan kɔnɔ ni galasi konikɛlen ye (a hakɛ 200 μl). O misaliw sɛgɛsɛgɛra ni Acquity iClass ultra-high performance LC system (Waters) ye min sirilen don Q-Exactive (QE)-HF (Ultra High Field Orbitrap) mas spectromètre (Thermo Fisher Scientific) la, min bɛ se ka jateminɛ kosɛbɛ. Walasa ka sɛgɛsɛgɛli kɛ, fɛn min bɔra a la, o pikiri kɛra milimɛtɛrɛ 100×1,0 ye, min fanga ka bon silika T3 koloma (Jiw) la, 1,8μm fɛnɲɛnamafagalanw bɛ min kɔnɔ. Jibɔ hakɛ ye 100μl/min ye, wa tampon sitɛmu bɛ kɛ ni tampon A (10 mM amoniyɔmu foromati ani 0,15% asidi foromiki ji la) ani tampon B (ACN). Gradient bɛ kɛ nin cogo la: 0%B miniti 0 kɔnɔ; 0%B ye. 0 ka se 15% B ma miniti 0 ka se 0,1 ma; 15 ka se 17% B ma miniti 0,1 fo 0,5 la; B ni 17 fo 55% ye miniti 0,5 fo miniti 14; B ni 55 fo 70% ye miniti 14 fo 14,5 la; ni 14,5 fo 70 fo 100% B ye miniti 18 kɔnɔ; 100% B miniti 18 fo miniti 19 kɔnɔ; 100 ka se 0% B ma miniti 19 fo 19,1 kɔnɔ; 0% B la miniti 19,1 fo miniti 28 (55, 56). QE-HF mass spectrometer bɛ baara kɛ ionizasiyɔn ɲuman cogo la ni mass range ye m/z (mass/charge ratio) ye 50 fo 750. Resolution min bɛ kɛ, o ye 60.000 ye, ani gain control (AGC) ion target sɔrɔlen ye 3×106 ye, ani ion waati hakɛ cayalenba ye milisekɔndi 100 ye. Elektrofiyɛli ionizasiyɔn (ESI) sɔrɔyɔrɔ min bɛ funteni na, o bɛ baara kɛ ni fiyɛli fanga ye 3,5 kV, kapilɛri funteni ye 250°C ye, ka fiɲɛbɔlan 60 AU (unités arbitraires), ani fiɲɛbɔ dɛmɛnan ye 20 AU ye. 250°C ye. S lensɛri bɛ bila 60 AU la.
Aniyɔni chromatography-MS sɛgɛsɛgɛli asidi organique taamasiɲɛ 13C kan. Metabolite precipitate tɔ (55μl) sɛgɛsɛgɛra ni Dionex ion chromatography system (ICS 5000+, Thermo Fisher Scientific) ye min ni QE-HF mass spectrometer (Thermo Fisher Scientific) sirilen don. Kuma surun na, farikolojɔlifɛn 5μl pikiri kɛra Dionex IonPac AS11-HC koloma kɔnɔ min bɛ ni HPLC ye (2 mm×250 mm, particule size 4μm, Thermo Fisher Scientific) ka kɛɲɛ ni push-in partial loop mode ye ni fasa hakɛ ye 1. ) Dionex IonPac AG11-HC garadi koloni (2 mm x 50 mm, 4μm, Thermo Fisher Scientifique ka baara). Kolon funteni bɛ mara 30°C la, ani autosampler bɛ bila 6°C la. Aw bɛ baara kɛ ni potasiyɔmu idɔrɔkisidi kariti ye min dilen don ni ji ye min ma ionize, walasa ka potasiyɔmu idɔrɔkisidi gradient sɔrɔ eluent generator fɛ. Fɛnɲɛnɛmafagalanw farali ni jibɔ hakɛ ye min ye 380μl/min ye, ka nin sɛgɛsɛgɛli in kɛ: miniti 0 fo miniti 3, KOH milimɛtɛrɛ 10; miniti 3 fo miniti 12, KOH mililitiri 10 fo 50; miniti 12 fo miniti 19, KOH milimɛtɛrɛ 50 fo 100; miniti 19 fo miniti 21 , 100 mM KOH; miniti 21 fo miniti 21,5, KOH milimɛtɛrɛ 100 fo 10. Kolon in bɛ segin ka balansi kɛ KOH milimɛtɛrɛ 10 kɔnɔ miniti 8,5 kɔnɔ.
Fɛnɲɛnɛmafagalan minnu kolen don, olu bɛ fara ɲɔgɔn kan ni isoporopanɔli faralen ji ye min bɛ se 150μl/min ma koloni kɔfɛ, o kɔfɛ, u bɛ ɲɛsin masɔrɔ spektromɛtiri ma min bɛ baara kɛ ionizasiyɔn juguman na. MS bɛ ka masala hakɛ kɔlɔsi k’a ta m/z 50 na ka se 750 ma ni 60.000 ye. AGC bɛ sigi 1×106 kan, wa ion waati hakɛ maksimali bɛ mara 100 ms. ESI sɔrɔyɔrɔ min sumana, o tun bɛ baara kɛ ni fiyɛli fanga ye min ye 3,5 kV ye. Iyɔn sɔrɔyɔrɔ sigicogo tɔw ye ninnu ye: kapilɛri funteni 275°C; gazi min bɛ kɛ ka bɔ a kɔnɔ, o ye 60 AU ye; gazi dɛmɛnan bɔli, 20 AU 300°C la, ani S lens sigili 60 AU la.
Kunnafoni sɛgɛsɛgɛli 13C taamasiɲɛ kɛlenw farikolojɔlifɛnw kan. Baara kɛ ni TraceFinder porogaramu ye (version 4.2, Thermo Fisher Scientific) walasa ka kunnafoniw sɛgɛsɛgɛ isotopu hakɛ kan. Fɛnɲɛnɛma kelen-kelen bɛɛ danfara sɛgɛsɛgɛra ni fɛnɲɛnamafagalan ye min bɛ se ka da a kan, wa a sɛgɛsɛgɛra a yɛrɛma. Walasa ka isotopu nafama sɛgɛsɛgɛli kɛ, 13C isotopu (Mn) kelen-kelen bɛɛ ka ion chromatogramme (XIC) bɔlen yɔrɔ bɔra [M + H] + la, n ye laɲini fɛnɲɛnamafagalan ka karɔbɔli hakɛ ye, min bɛ kɛ ka asidi aminikiw sɛgɛsɛgɛ walima [ MH] + bɛ kɛ ka aniyoni sɛgɛsɛgɛ. XIC ka masala tiɲɛni bɛ dɔgɔya ni tilayɔrɔ duuru ye miliyɔn kelen na, wa RT ka tiɲɛni ye miniti 0,05 ye. Nafolosɔrɔ-sɛgɛsɛgɛli bɛ Kɛ ni isotopu kelen-kelen minnu Sɔrɔla, olu ni ɲɔgɔn cɛ isotopu bɛɛ lajɛlen jatebɔ ye. O hakɛw bɛ di i n’a fɔ kɛmɛsarada la izotopu kelen-kelen bɛɛ la, wa a jaabiw bɛ jira ni kɛmɛsarada la molari nafa ye (MPE), i n’a fɔ a ɲɛfɔra cogo min ka tɛmɛ (42).
Senfagabana bɔgɔlan min sumana, o kɛra kelen ye mɛtanɔli 80% (v/v) la min nɛnɛna ni jisuma ye, ka a kɛ vortex ye, ka a bila -20°C la miniti 30 kɔnɔ. Aw bɛ segin ka a wuli ka a wuli ni +4°C ye miniti 30 kɔnɔ. Aw bɛ sɛgɛsɛgɛli kɛ ni 21.000 g ye miniti 5 kɔnɔ 4°C la, o kɔfɛ, ji min bɛ sɔrɔ a kan, o bɛ lajɛ ka ja ni SpeedVac fɛnɲɛnamafagalan ye 25°C la walasa ka sɛgɛsɛgɛli kɛ kɔfɛ. I n’a fɔ a ɲɛfɔra cogo min na sanfɛ, LC-MS sɛgɛsɛgɛli kɛra farikolokisɛ suguyaw ka asidi aminikiw kan. Ni baara kɛra ni TraceFinder (version 4.2, Thermo Fisher Scientific) ye, kunnafoniw sɛgɛsɛgɛli kɛra ni fɛnɲɛnamafagalan kelen-kelen bɛɛ ka mass monoisotopique ye. Metabolite kunnafoniw ka normalisation quantile kɛra ni preprocessCore porogaramu ye (57).
Slice labɛnni. Sogo in lafiyala joona ni gazi ɲuman ye, ka a kunkolo tigɛ, ka a kunkolo bɔ joona a kunkolo la, ka muru yɛrɛyɛrɛlen min falen bɛ jikuruw la (HM-650 V, Thermo Fisher Scientific, Walldorf, Allemagne) kɛra ka a tigɛ ka kɛ sagittal sections 300 fo 375 μm ye Cold carbon gasification (95% O2 ani 5% CO2) Dɔgɔya Ca2 + ACSF (NaCl 125,0 mM, KCl 2,5 mM, fosifati sodiyɔmu tampon 1,25 mM, NaHCO3 25,0 mM, d-glucose 25,0 mM, CaCl2 1,0 mM ani 6,0 mM MgCl2 Aw bɛ a ladilan ka kɛɲɛ ni pH 7,4 ye ani 310 fo 330 mOsm). Aw bɛ kunsɛmɛnasumaya sɔrɔlenw bila so kɔnɔ min kɔnɔ Ca2 + ACSF ka ca (125,0 mM NaCl, 2,5 mM KCl, 1,25 mM fositɛri fositɛri tampon, 25,0 mM NaHCO3, 25,0 mM d-glucose, 4,0 mM CaCl2 ani mM 3,5 MgCl2) pH 7,4 ani 310 fo 320 mOsm). Aw bɛ o sɔgɔsɔgɔninjɛw mara miniti 20 fo miniti 30 walasa u ka se ka segin u cogo kɔrɔ la sani aw ka sɛbɛnni kɛ.
fɔlisenw sɛbɛnni. Mikɔrɔskɔpu dakun min bɛ ni sɛbɛnnikɛlan jɔlen ye ani ji su ji la 20x objective lens (Scientifica) de kɛra sɛbɛnni bɛɛ la. Purkinje selilɛri minnu bɛ jate, olu dɔnna ni (i) farikolo bonya ye, (ii) kunsɛmɛnasumaya sigiyɔrɔ farikolo la, ani (iii) mtYFP kunnafonidila jɛnɛya fluorescent jirali fɛ. Patɔrɔn pipɛti min kuncɛ bɛ se megomɛ 5 fo 11 ma, o bɛ sama ka bɔ ni borosilikati gilasi kapilɛri ye (GB150-10, 0,86 mm×1,5 mm×100 mm, Science Products, Hofheim, Alimanjamana) ani pipɛti horizontal Instruments (P-1000, Sutter), Novato, CA). O fɔlisenw bɛɛ Kɛra ELC-03XS npi patch clamp amplifier (npi electronic GmbH, Tam, Allemagne) fɛ, o min tùn bɛ Kɔrɔsi ni porogaramu Signal ye (version 6.0, Cambridge Electronic, Cambridge, UK). Kɔrɔbɔli in sɛbɛnna ni misali ta hakɛ ye min ye 12,5 kHz ye. Siginiden bɛ filɛ ni Bessel filɛri surun fila ye minnu tigɛcogo ye 1,3 ani 10 kHz ye. Membrane ni pipette ka capacitance bɛ musaka kɛ ni compensation circuit ye ​​ni amplificateur ye. Kɔrɔbɔliw bɛɛ kɛra Orca-Flash 4.0 kamera (Hamamatsu, Gerden, Alimanjamana) ka bolo kan, o min tun bɛ Hokawo porogaramu fɛ (version 2.8, Hamamatsu, Gerden, Alimanjamana).
Kɔnɔnatumu bɛɛ lajɛlen labɛncogo ni a sɛgɛsɛgɛli kɛcogo kɔrɔlen. Sani aw ka sɛbɛnni kɛ dɔrɔn, aw bɛ pipɛti fa ni kɔnɔnaji ye min kɔnɔ nin fɛn ninnu bɛ sɔrɔ: KCl mililitiri 4,0, NaCl mililitiri 2,0, EGTA mililitiri 0,2, Glukonati potasiyɔmu mililitiri 135,0, Hepes mililitiri 10,0, ATP (Mg) mililitiri 4,0, Guanosine triphosphate (GTP) (Na) mililitiri 0,5 ani Kɛrɛyatinini fositɛri 10,0 mM ladilanna ka se pH 7,25 ma, ani osmotique tansiyɔn kɛra 290 mOsm (sucrose) ye. O yɔrɔnin bɛɛ fanga 0 pA kɛli kɔfɛ walasa ka ɲɛgɛnɛsira kari, lafiɲɛbɔlan seko jateminɛna. Donanw ka rezisti bɛ suman ni -40, -30, -20, ani -10 pA ka kuran hyperpolarisé (kurun polarizelenw) kɛli ye. Aw bɛ tension jaabi bonya suman ani ka Ohm ka sariya kɛ ka donna rezisti jate. Yɛrɛlabaara kɛcogo sɛbɛnna tension clamp kɔnɔ miniti 5 kɔnɔ, ani sPSC dɔnna ka suman Igor Pro (version 32 7.01, WaveMetrics, Lake Oswego, Oregon, USA) kɔnɔ ni semi-automatique recognition script ye. IV curve ni steady-state current bɛ suman ni batiri sirili ye seko ni dɔnko danfaralenw na (a bɛ daminɛ -110 mV la) ani ka dɔ fara tension kan 5 mV senfɛ. AP dilanni kɔrɔbɔra ni depolarizing current (kurun depolarize) kɛli ye. Aw bɛ selilabana siri ni -70 mV ye ka a sɔrɔ aw bɛ depolarizing current pulse kɛ. Aw bɛ sɛbɛnnikɛlan kelen-kelen bɛɛ senfɛta hakɛ ladilan u danma (10 fo 60 pA). Aw bɛ AP hakɛ cayalenba jateminɛ ni aw ye bololabaara kɛ ni pulse spikes ye minnu bɛ kɛ sababu ye ka AP frequency camanba sɔrɔ. AP dakun bɛ sɛgɛsɛgɛ ni depolarize pulse bɔlen filanan ye min bɛ fɔlɔ ka AP kelen walima caman lawuli.
Perforés patch configuration ani a sɛgɛsɛgɛli. Aw bɛ perforated patch recording kɛ ni protocoles standards ye. Aw bɛ pipɛti kɛ min tɛ ATP ni GTP ye, ni nin fɛn ninnu tɛ a la: Gluconate K 128 mM, KCl 10 mM, Hepes 10 mM, EGTA 0,1 mM ani 2 mM MgCl2, ani ka a ladilan ka kɛ pH 7,2 ye (aw bɛ baara kɛ ni KOH ye). ATP ni GTP bɛ bɔ selilu kɔnɔ furaji la walasa ka selilɛriw ka doncogo bali min tɛ kɔlɔsi. Paki pipɛti bɛ fa ni kɔnɔnaji ye min kɔnɔ anfoterisini bɛ sɔrɔ (a bɛ se ka kɛ 200 fo 250μg/ml ye; G4888, Sigma-Aldrich) walasa ka patɔrɔn sɛbɛn punched sɔrɔ. Anfoterisini tun bɛ wuli dimetil sulfokisidi la (a hakɛ laban: 0,1 fo 0,3%; DMSO; D8418, Sigma-Aldrich). DMSO hakɛ min kɛra, o ma nɔba bila senfagabana sɛgɛsɛgɛlenw na. Punching kɛcogo la, kanal resistance (Ra) tun bɛ kɔlɔsi tuma bɛɛ, wa k’a daminɛ ka Ra ni AP ka amplitude sabatilen kɔfɛ (miniti 20-40). Yɛrɛlabaara bɛ suman ni tension ni/walima current clamp ye miniti 2 fo miniti 5 kɔnɔ. Kunnafoni sɛgɛsɛgɛli kɛra ni Igor Pro (7.05.2 bɔko ye, WaveMetrics, Ameriki), Excel (2010 bɔko, Microsoft Corporation, Redmond, Ameriki) ani GraphPad Prism (8.1.2 bɔko, GraphPad Software Inc., La Jolla, CA). Etazuni). Walasa ka AP yɛrɛmabilalenw dɔn, IgorPro ka NeuroMatic v3.0c plug-in bɛ kɛ. APw dɔn otomatiki la ni dakun dilen ye, min bɛ ladilan kelen-kelen sɛbɛn kelen-kelen bɛɛ kama. Ni aw bɛ baara kɛ ni spike interval ye, ka spike frequency (spike frequency) jateminɛ ni spike frequency (spike frequency) ye min bɛ kɛ yɔrɔnin kelen na ani spike frequency (spike frequency) hakɛ danmadɔ.
PN ka danfara. Ni u bɛ ladamu ka kɛɲɛ ni sariyasun ye min bɔra ka tɛmɛ, PNw saniyalen don ka bɔ sosow ka kunsɛmɛ na waati latigɛlen dɔ la (58). Kuma surun na, kunsɛmɛnasumaya tigɛra ka a tigɛtigɛ ka a tigɛtigɛ ni jisuma ye [HBSS Ca2+ ni Mg2+ tɛ min na, ka fara sukaro 20 mM kan, penisilini (50 U/ml) ani sitɛrɛpitɔmisini (0,05 mg/ ml)], ka sɔrɔ ka furaji sɔgɔ papayini na [HBSS, min farala 1-cysteine·HCl kan (miligaramu 1 / mililitiri), papayini (16 U / mililitiri) ani deoxyribonuclease I (DNase I; miligaramu 0,1/ml)] Aw bɛ a furakɛ miniti 30 kɔnɔ 30°C la. Aw bɛ fɔlɔ ka farikolo yɔrɔw ko HBSS furakisɛ la, kɔnɔbara nɔgɔ bɛ min kɔnɔ (miligaramu 10/ml), BSA (miligaramu 10/ml) ani DNase (miligaramu 0,1/ml) bɛ sɔrɔ so funteni na walasa ka anzimu ka dumuni toli bali, o kɔ aw bɛ HBSS furakisɛ la min kɔnɔ sukaro miligaramu 20 bɛ sɔrɔ ka a sɔgɔ nɔgɔya la HBSS, penisilini (50 U/ml), sitɛrɛpitɔmisini (miligaramu 0,05/ml) ani DNase (0,1 mg/ml) bɛ farikolokisɛ kelenw bɔ. Kɔnɔnatumuw wulicogo min sɔrɔla, o filɛra ni selilɛri 70μm ye, o kɔfɛ, seliluw kɛra bɔgɔdaga ye ni santirifugare ye (1110 rpm, miniti 5, 4°C) ka segin ka u wuli ka u wuli kokura sɛgɛsɛgɛlikɛlan kɔnɔ [HBSS, ka fara a kan ni 20 mM glucose ye, 20% fetal bovine ) Serum, penicilline (50 U/ml) ani streptomycine (miligaramu 0,05/ml)]; aw bɛ seliluw ka ɲɛnamaya jateminɛ ni iyodi poropidiyɔmu ye ani ka selilu hakɛ ladilan ka se 1×106 fo 2×106 selilu/ml ma. Sani flow cytometry ka kɛ, suspension tun bɛ filɛri kɛ ni 50 μm cell strainer ye.
Flow cytomètre (banakisɛfagalan) min bɛ wele ko cytomètre. Kɔnɔnatumuw tilatilali kɛra 4°C la ni FACSAria III masin ye (BD Biosciences) ani FACSDiva porogaramu (BD Biosciences, version 8.0.1). Kɔnɔnatumuw jɔcogo tun bɛ tila ni 100 μm fiɲɛbɔlan ye ni tansiyɔn 20 psi ye ni hakɛ ye ~2800 ko kɛlenw/sekɔndi kɔnɔ. Ikomi laadala gating sariyaw (seli hakɛ, bimodali danfara, ani jɛnsɛnni jogo) tɛ se ka PN danfara ka ɲɛ ka bɔ selilu suguya wɛrɛw la, gating fɛɛrɛ bɛ sigi sen kan ka da YFP fanga ni otofluorescence sumalen tilennen kan mitoYFP+ ​​ni control mitoYFP − Sogow la. YFP bɛ nisɔndiya ni sɛgɛsɛgɛli ye ni 488 nm laser layini ye, wa taamasiyɛn bɛ dɔn ni 530/30 nm band pass filter ye. MitoYFP+ ​​sogow la, Rosa26-mitoYFP kunnafonidila jamu fanga danfaralen fana bɛ kɛ ka senfagabana farikolo ni axon yɔrɔw danfara. 7-AAD bɛ nisɔndiya ni 561 nm laser jɛman ye ani ka sɔrɔ ni 675/20 nm bandpass filter ye walasa ka seli salenw bɔ kɛnɛ kan. Walasa ka astrocytes fara ɲɔgɔn kan waati kelen na, selilu suspension nɔgɔra ni ACSA-2-APC ye, o kɔfɛ, a ye sɛgɛsɛgɛli kɛ ni 640 nm laser line ye, ka 660/20 nm bandpass filter kɛ ka taamasiɲɛ in dɔn.
Bugun minnu lajɛra, olu kɛra bɔgɔlan ye ni santirifugare ye (1110 rpm, miniti 5, 4°C) ka u mara -80°C la fo ka se u kɛli ma. Mfn2cKO sogow n’u ka nɔgɔw bɛ tila don kelen na walasa ka taabolo caman ɲɔgɔnna dɔgɔya. FACS kunnafoniw jirali ni u sɛgɛsɛgɛli kɛra ni FlowJo porogaramu ye (FlowJo LLC, Ashland, Oregon, Etazuni).
I n’a fɔ a fɔra cogo min na sanfɛ (59), PCR bɛ kɛ waati yɛrɛ la walasa ka ADN bɔ senfagabana suguyaw la walasa ka ADNmt hakɛ jateminɛ kɔfɛ. Linearité ni threshold sensitivity sɛgɛsɛgɛra fɔlɔ ni qPCR boli ye selilu hakɛ danfaralenw kan. Kuma surun na, aw bɛ PN 300 lajɛ lysis tampon kɔnɔ min kɔnɔ, tris-HCl mililitiri 50 (pH 8,5), EDTA mililitiri 1, Tween 20 0,5% ani poroteyini K (200 ng/ml) bɛ sɔrɔ ka a bila 55°C la miniti 120 kɔnɔ. Bugunw bilala ka taa a fɛ 95°C la miniti 10 kɔnɔ walasa ka a jira ko poroteyini K ma baara kɛ pewu.Ni baara kɛra ni TaqMan sonda (Thermo Fisher) ye min kɛrɛnkɛrɛnnen don mt-Nd1 ma, ADNmt suman ni semi-quantitative PCR ye 7900HT Real-Time PCR system (Thermo Fisher Scientific) kɔnɔ. Science, katalogu nimɔrɔ Mm04225274_s1), mt-Nd6 (Thermo Fisher Scientific, katalogu nimɔrɔ AIVI3E8) ani 18S (Thermo Fisher Scientific, katalogu nimɔrɔ Hs99999901_s1) jamu.
Protéome (poroteyɔmu) misali labɛnni. Ni aw ye furaji sumaya 95°C la miniti 10 kɔnɔ ani ka sonika kɛ, ka a kɛ lysis buffer kɔnɔ [6 M guanidine chloride, 10 mM tris(2-carboxyethyl) phosphine hydrochloride, 10 mM chloroacetamide ani 100 mM tris- Lyse frozen neuron pellets in HCl Bioruptor (Diagenode) kan miniti 10 kɔnɔ (segin 30 pulse / segin 30 lafiɲɛbɔ waati). Aw bɛ a lajɛ 1:10 tris-HCl milimɛtiri 20 (pH 8,0) la, ka a ɲagami ni tripsin sanu ng 300 ye (Promega), ka sɔrɔ ka a bila su fɛ 37°C la walasa ka dumuni dafalen sɔrɔ. Tile filanan na, aw bɛ a lajɛ ni santirifugu ye garamu 20 000 na miniti 20 kɔnɔ. Aw bɛ a sanfɛji kɛ ni asidi formiki 0,1% ye, ka sɔrɔ ka a jilama kɔgɔ bɔ ni StageTips ye min dilannen don u yɛrɛ ye. O sɛgɛsɛgɛli kɛra SpeedVac minɛn kɔnɔ (Eppendorf concentrator plus 5305) 45°C la, o kɔfɛ, pepitiri in bɛ wuli asidi formiki 0,1% la. Sɛgɛsɛgɛli bɛɛ labɛnna waati kelen na mɔgɔ kelen fɛ. Walasa ka astrocyte tacogo sɛgɛsɛgɛ, pepitiri kɔgɔlen 4 μg taamasiyɛn kɛra ni tandem mass tag ye (TMT10plex, katalogu nimɔrɔ 90110, Thermo Fisher Scientific) ni pepitiri ni TMT reagent hakɛ ye 1:20 ye. Walasa ka TMT taamasiɲɛ kɛ, TMT fɛnɲɛnamafagalan miligaramu 0,8 bɛ segin ka kɛ ACN jilama 70 μl kɔnɔ, ka pepitiri jalen kɛ ka segin ka kɛ TEAB 0,1 M (triethylammonium bicarbonate) 9 μl ye, TMT fɛnɲɛnamafagalan 7 μl faralen a kan ACN kɔnɔ. A hakɛ tun ye 43,75% ye. Miniti 60 kɛlen kɔ ka a wuli, aw bɛ a furakɛ ni 2 μl 5% hidroksilamini ye. Pepitiri minnu sɛbɛnnen don, olu lajɛra, ka u ja, ka u lasegin asidi formiki 0,1% (FA) 200μl kɔnɔ, ka u tila fila ye, ka sɔrɔ ka kɔgɔ bɔ u la ni StageTips ye minnu dilannen don u yɛrɛ ye. Ni UltiMate 3000 ultra high performance liquid chromatograph (UltiMate 3000 ultra high performance liquid chromatograph) ye, tilayɔrɔ fila la kelen tilalen don Acquity chromatographie koloma 1mm x 150mm kan min falen bɛ 130Å1.7μm C18 fɛnɲɛnamafagalanw na (Waters, catalogue No. SKU: 186006935). Thermo Fisher ka dɔnniyakalan). Aw bɛ pepitiriw fara ɲɔgɔn kan ni jibɔ hakɛ ye min ye 30μl/min ye, ka u faranfasi ka bɔ 1% la ka se 50% ma tampon B la miniti 85 kɔnɔ ni taamasiɲɛw ye miniti 96 kɔnɔ, ka bɔ 50% na ka se 95% ma tampon B la miniti 3 kɔnɔ, o kɔ miniti 8 ka bɔ 95 % tampon B la Tampon A ye 5% ACN ye ani 10 mM amoniyɔmu bikarbonati (ABC), ani tampon B ye 80% ACN ni 10 mM ABC ye. Aw bɛ tilayɔrɔw lajɛ miniti 3 o miniti 3 ka u fara ɲɔgɔn kan ka kɛ kulu fila ye (1 + 17, 2 + 18, a ɲɔgɔnnaw) ka u ja centrifuge (santirifuge) kɔnɔ min bɛ kɛ ni fɛnɲɛnamafagalan ye.
LC-MS/MS sɛgɛsɛgɛli. Walasa ka mass spectrometry kɛ, pepitiriw (jate r119.aq) farala ɲɔgɔn kan PicoFrit analytical column kan, min kɔnɔna janya ye santimɛtɛrɛ 25 ye, min janya ye 75 μm ye (laɲini lensɛri kura, yɔrɔ nimɔrɔ PF7508250) min bɛ ni 1,9 μm ReproSil-Pur 120 C18-AQ medium (Dr. Maisch, mat) ye, ka baara kɛ ni EASY-nLC ye 1200 (Thermo Fisher Scientific, Alimanjamana na). Kolon in tun bɛ mara 50°C la. Tampon A ni B ye asidi foromiki 0,1% ye ji la ani asidi foromiki 0,1% ye ACN 80% la. Pepitidiw farala ɲɔgɔn kan ka bɔ tampon B 6% la ka se 31% ma miniti 65 kɔnɔ ani ka bɔ tampon B 31% la ka se 50% ma miniti 5 kɔnɔ ni jiginni ye 200 nl/min. Pepitiri minnu bɔra, olu sɛgɛsɛgɛra Orbitrap Fusion mass spectrometer (Thermo Fisher Scientific) kan. Peptide ɲɛbila m/z sumani bɛ kɛ ni 120.000 ye min bɛ se 350 fo 1500 m/z ma. Ni baara kɛra ni 27% normalisé collision energy ye, precurseur barikama min ka charge state bɛ 2 fo 6 la, o bɛ sugandi high energy C trap dissociation (HCD) cleavage kama. Sikili waati bɛ sigi 1 s kan. Pepitiri yɔrɔ in m/z hakɛ jateminɛna ion trap kɔnɔ ni AGC laɲini fitinin ye min ye 5×104 ye ani pikiri waati min ka ca ni 86 ms ye. Tila-tilalen kɔfɛ, ɲɛbila in bilala lisi kan min bɛ wele ko dynamique exclusion list fo s 45. Pepitiri minnu taamasiyɛn ye TMT ye, olu farala ɲɔgɔn kan santimɛtɛrɛ 50, 75 μm Acclaim PepMap kulu kan (Thermo Fisher Scientific, katalogu nimɔrɔ 164942), ka jiginni spekterw sɛgɛsɛgɛ Orbitrap Lumos Tribrid mass spectrometer (Thermo Fisher Scientific) kan, min bɛ ni high-field asymmetric waveform ions (FAIMS) minɛnw ye (Thermo Fisher Scientific) bɛ baara kɛ ni musakabɔlan fila ye minnu ye −50 ani −70 V. MS3 min sugandira ka da sinsinnan ɲɛbila kan, o bɛ kɛ TMT ka kunnafoni ion taamasiɲɛ sumani kama. Pepitiri farali kɛra EASY-nLC 1200 kan, ni 90% linear gradient elution ye, ni tampon hakɛ ye 6% fo 31% ye; tampon A tun ye 0,1% FA ye, tampon B tun ye 0,1% FA ye ani 80% ACN. Sɛgɛsɛgɛli koloma bɛ baara kɛ ni 50°C ye. Baara kɛ ni FreeStyle (version 1.6, Thermo Fisher Scientific) ye walasa ka dosiye fɔlɔ tila ka kɛɲɛ ni FAIMS ka musaka hakɛ ye.
Protéines (banakisɛfagalanw) dɔnni ni u hakɛ jateminɛni. Baara kɛlen ni Andromeda ɲininikɛlan jɛlen ye, kunnafoni fɔlɔw sɛgɛsɛgɛra ni MaxQuant bɔko 1.5.2.8 ye (https://maxquant.org/). Ka fara Cre recombinase ni YFP sinsinni kan minnu sɔrɔla Aequorea victoria fɛ, pepitiri yɔrɔw spekterw ɲinina ka sosow ka poroteyomu (Proteome ID UP000000589, telesarse ka bɔ UniProt la san 2017 mɛkalo la) ka sinsinni kanon ni isoform sinsinni kan Metiyoni oksidan ni poroteyini N-terminal asetilasi bilala sen kan i n’a fɔ fɛn caman b’a la ka fɛn caman sɛmɛntiya; sitɛyini karɔbamoyili metilasi bilala sen kan i n’a fɔ fɛn caman sɛmɛntiyalenw. Dumunikɛcogo paramɛtiriw bɛ sigi “kɛrɛnkɛrɛnnenya” ani “tripsin/P” la. Pepitiriw ni razɔri pepitiri minnu bɛ kɛ ka farikolojɔli dumuniw dɔn, olu hakɛ fitinin ye 1 ye; pepitiri kɛrɛnkɛrɛnnenw hakɛ fitinin ye 0. Pepitiri karti bɛnkan cogoyaw la, farikolojɔlifɛnw dɔnni hakɛ tun ye 0,01 ye. “Péptide filanan” sugandilen bɛ baara la. Baara kɛ ni “match between runs” sugandi ye walasa ka dantigɛli ɲɛnabɔlenw bila dosiye fɔlɔ suguya wɛrɛw cɛ. Aw bɛ LFQ hakɛ dɔgɔyalen jate 1 kɛ walasa ka hakɛ jateminɛ min tɛ labeli ye (LFQ) (60). LFQ fanga bɛ filɛri kɛ ka kɛɲɛ ni nafa bɛnnen fila ye a dɔgɔyalenba la jamu kulu kelen kɔnɔ waati kelen-kelen bɛɛ la, wa a bɛ bɔ kɛnɛ kan ka bɔ jatebɔcogo nɔgɔman na ni a bonya ye. 0,3 ani ka jigin 1,8. Baara kɛ ni Perseus jatebɔyɔrɔ (https://maxquant.net/perseus/) ani R (https://r-project.org/) ye walasa ka LFQ jaabiw sɛgɛsɛgɛ. T test moderate fila-fila min bɔra limma porogaramu pake la, o kɛra ka diferential expression analysis (61) kɛ. Kunnafoni sɛgɛsɛgɛli ɲinini bɛ kɛ ni ggplot, FactoMineR, factoextra, GGally ani pheatmap ye. TMT-based proteomics kunnafoniw sɛgɛsɛgɛra ni MaxQuant version 1.6.10.43 ye. Protéomique raw kunnafoniw ɲini ka bɔ UniProt ka hadamadenw ka proteomiki kunnafonidilan na, o min telesarsera sɛtanburukalo san 2018. Sɛgɛsɛgɛli in kɔnɔ, isotopu saniya latilenni factor min dira a dilabaga fɛ. Baara kɛ ni limma ye R kɔnɔ walasa ka danfara jiracogo sɛgɛsɛgɛli kɛ. Donan fɔlɔw, kunnafonidilanw ɲinini jaabiw, ani kunnafonidilanw sɛgɛsɛgɛli baarakɛcogo n’u jaabiw bɛɛ bɛ mara ProteomeXchange jɛkulu kɔnɔ PRIDE jɛɲɔgɔnw ka marayɔrɔ fɛ ni kunnafonidilanw taamasiyɛn ye PXD019690.
Baarakɛcogo ɲɛfɔliw bɛ sɛgɛsɛgɛli nafa bonya. Ingenuity Pathway Analysis (QIAGEN) baarakɛminɛn kɛra sababu ye ka kunnafonidilanw ka baarakɛcogo ɲɛfɔli daɲɛw nafa dɔn dɔgɔkun 8 kɔnɔ (Ja 1). Kuma surun na, poroteyini hakɛlama lisɛli min sɔrɔla LC-MS/MS (tandem mass spectrometry) kunnafonidilanw fɛ, o bɛ kɛ ni nin filɛri sariyaw ye: Mus musculus bɛ sugandi ka kɛ suguya ni kɔkanna ye, wa o kulu bɛ P hakɛ jira min ladilanna Benjamini fɛ walasa ka nafolo sɔrɔ 0,05 walima a dɔgɔyalenba la, o jatera ko a nafa ka bon. Nin ja in kama, sanfɛla duuru tɛmɛnenw jiralen don kulu kelen-kelen bɛɛ kɔnɔ ka da P hakɛ ladilanni kan. Ni baara kɛra ni t-test caman ye, ka baara kɛ ni Benjamini, Krieger ani Yekutieli ka porogaramu ye min bɛ kɛ ka fɛnw bonya, minnu bɛ kɛ ni taabolo fila ye (Q = 5%), waati-dafalen poroteyini jiracogo sɛgɛsɛgɛli bɛ kɛ kandida nafamaw kan minnu dɔnna kulu kelen-kelen bɛɛ kɔnɔ, wa jirisun kelen-kelen bɛɛ bɛ sɛgɛsɛgɛ u danma. Wajibi tɛ ka SD basigilen ta.
Walasa ka nin kalan in jaabiw suma ni kunnafonidilanw ye minnu bɔra kɛnɛ kan ani ka Venn ja dɔ labɛn ja 1 kɔnɔ, an ye poroteyini hakɛlama lisɛli ni MitoCarta 2.0 ɲɛfɔliw fara ɲɔgɔn kan (24). Baara kɛ ni ɛntɛrinɛti baarakɛminɛn ye min tɔgɔ ye ko Draw Venn Diagram (http://bioinformatics.psb.ugent.be/webtools/Venn/) walasa ka ja dilan.
Walasa ka kunnafoni caman sɔrɔ jateminɛ kɛcogo minnu bɛ kɛ ka poroteyomiki sɛgɛsɛgɛli kɛ, aw bɛ se ka sɛbɛn ɲɛ Fɛnw ni fɛɛrɛw yɔrɔ lajɛ min bɛ bɛn o ma. Kɔrɔbɔli tɔw bɛɛ la, kunnafoni caman bɛ se ka sɔrɔ nsiirin min bɛ bɛn o ma. Fo n’a fɔra cogo wɛrɛ la, kunnafoniw bɛɛ bɛ jira iko cɛmancɛ ± SEM, wa jateminɛw bɛɛ kɛra ni porogaramu GraphPad Prism 8.1.2 ye.
Walasa ka kunnafoni wɛrɛw sɔrɔ nin barokun in kan, aw bɛ se ka http://advances.sciencemag.org/cgi/content/full/6/35/eaba8271/DC1 lajɛ
Nin ye dakun dafalen ye min bɛ tilatila Creative Commons Attribution-Non-Commercial License sariyaw kɔnɔ, min bɛ sira di baara ma, a tilatilali ani a lasegincogo ma fɛɛrɛ suguya bɛɛ la, ni baara laban tɛ jago tɔnɔ kama ani ni a dabɔra ko baara fɔlɔ ye tiɲɛ ye. Tàyɔrɔ.
Kɔlɔsili: An b’a ɲini i fɛ dɔrɔn i k’i ka e-mail ladɛrɛsi di walasa i bɛ mɔgɔ min laadi ɲɛ kan, o k’a dɔn ko i b’a fɛ u ka o e-mail ye, wa ko o tɛ spam ye. An tɛna imɛri ladɛrɛsi si minɛ.
Nin ɲininkali in bɛ kɛ walasa k’a lajɛ n’i ye taamakɛla ye ani ka spam ci otomatiki bali.
E. Motori fɛ, I. Atanassov fɛ, S. M. V. Kochan, K. Folz-Donahue, V. Sakthivelu fɛ, P. Giavalisco fɛ, N. Toni fɛ, J. Puyal fɛ, N.-G. Larson ye baara kɛ
Protéomique sɛgɛsɛgɛli kɛra senfagabana baarakɛbaliya kan, o y’a jira ko farikoloɲɛnajɛ porogaramuw bɛ baara kɛ walasa ka senfagabana kɛlɛ.
E. Motori fɛ, I. Atanassov fɛ, S. M. V. Kochan, K. Folz-Donahue, V. Sakthivelu fɛ, P. Giavalisco fɛ, N. Toni fɛ, J. Puyal fɛ, N.-G. Larson ye baara kɛ
Protéomique sɛgɛsɛgɛli kɛra senfagabana baarakɛbaliya kan, o y’a jira ko farikoloɲɛnajɛ porogaramuw bɛ baara kɛ walasa ka senfagabana kɛlɛ.
©2020 Ameriki ka dɔnniya yiriwali tɔnba. jo bɛɛ bɛ a bolo. AAAS ye HINARI, AGORA, OARE, CHORUS, CLOCKSS, CrossRef ani COUNTER ka jɛɲɔgɔn ye. Dɔnniya ɲɛtaa ISSN 2375-2548.