Amrɔ nɛɛ *Adrɛs ni yɔɔ amrɔ nɛɛ: Cologne 50931, Germany, Cologne Kuu ni Fe Fɛɛ Nibii Amli Taomɔ ni kɔɔ bɔ ni gbɔmɔtso lɛ mli nibii ni haa mɔ tsui nyɔɔ emli lɛ tsuɔ nii ehaa yɛ helai ni kɛ dalɛ baa lɛ ahe (CECAD).
Asusuɔ akɛ mitochondrial helai ni haa mɔ tsui nyɔɔ emli lɛ nyɛɛɛ aku sɛɛ aba ejaakɛ tsɔnei ni haa mɔ tsui nyɔɔ emli lɛ amli nibii ni haa mɔ tsui nyɔɔ emli lɛ faaa, shi anuuu nɔ ni mitochondrial nitsumɔ ni ejaaa lɛ tsuɔ yɛ tsɔnei ni haa mɔ tsui nyɔɔ emli lɛ amli lɛ shishi jogbaŋŋ. Yɛ biɛ lɛ, wɔkɛ Purkinje tsɔnei ni haa mɔ tsui nyɔɔ emli lɛ amli wala yibii pɔtɛɛ ni yɔɔ OXPHOS ni faaa ni mitochondrial fusion dynamics ni efite lɛ kɛbaa lɛ jieɔ kpo. Wɔna akɛ mitochondrial nitsumɔ ni ejaaa lɛ ha tsakemɔ kpele ko ba yɛ proteomics he nitsumɔ mli, ni yɛ naagbee lɛ ekɛ metabolic gbɛjianɔtoi pɔtɛɛ ni nyiɛɔ sɛɛ baa dani wala yibii lɛ gboi. Yɛ gbɛ ni wɔkpaaa gbɛ nɔ lɛ, wɔyoo pyruvate carboxylase (PCx) kɛ peroxidases krokomɛi ni kɔɔ TCA sɛɛkuu lɛ teŋgbɛ nibii lɛ ahe lɛ mlibotemɔ ni yɔɔ faŋŋ lɛ he. PCx ni tsiɔ naa lɛ ha oxidative stress kɛ jwɛŋmɔŋ helai ni haa mɔ he jɔ̃ɔ ehe lɛ mli bawo wu, ni tsɔɔ akɛ atherosclerosis lɛ yɛ hewalɛ ni buɔ mɔ he yɛ jwɛŋmɔŋ helai ni bɛ OXPHOS lɛ amli. Mitochondrial fusion ni akuɔ sɛɛ akɛbaa yɛ neurons ni efite kwraa lɛ mli lɛ tsakeɔ nɛkɛ metabolic sui nɛɛ kwraa, ni no tsiɔ wala yibii lɛ agbele naa. Nibii ni wɔna lɛ tsɔɔ gbɛi ni aleee tsutsu ni haa mɔ nyɛɔ eshiɔ mitochondrial nitsumɔ ni esaaa lɛ, ni etsɔɔ akɛ abaanyɛ aku sɛɛ atsake jwɛŋmɔŋ helai ni haa mɔ tsui nyɔɔ emli lɛ yɛ hela lɛ naagbee gbɛ po.
Nɔ ni maa gbɛfaŋnɔ titri ni mitochondria tsuɔ yɛ jwɛŋmɔŋ hewalɛ ni tsakeɔ lɛ mli lɛ nɔ mi kɛtsɔ jwɛŋmɔŋ helai ni fa babaoo ni kɛ gbɔmɔ mitochondria helai baa lɛ nɔ mi. Helai nɛɛ ateŋ babaoo jɛɔ tsɛŋemɔ helai amli tsakemɔi ni kudɔɔ tsɛŋemɔ helai ni haa mɔ mli wala yibii lɛ jieɔ amɛhe kpo lɛ mli (1, 2) loo tsɛŋemɔ helai ni haa mɔ mli wala yibii lɛ fiteɔ ni kɔɔ tsɛŋemɔ helai ahe nibii ahe lɛ mli, ni enɛ saa tsɛŋemɔ helai amli DNA (mtDNA) lɛ shifimɔ he yɛ gbɛ ni ejaaa nɔ (3, 4). Nitsumɔ yɛ kooloi ahe nɔkwɛmɔnii amli lɛ etsɔɔ akɛ yɛ mitochondrial ni tsuuu nii jogbaŋŋ yɛ fãji ni ebɔle amɛhe lɛ amli lɛ hetoohamɔ mli lɛ, abaanyɛ atsu nibii ni haa mɔ he jɔɔ lɛ (5-7) lɛ ahe nii, ni no haa anáa nibii ni he hiaa ni kɔɔ bɔ ni helai ni mli wawai nɛɛ baa lɛ he. Nɔ ni tamɔɔɔ enɛ lɛ, wɔ shishinumɔ ni wɔyɔɔ yɛ tsakemɔi ni baa yɛ wala yibii pɔtɛɛ komɛi amli ni jɛɔ jwɛŋmɔŋ mitochondrial adenosine triphosphate (ATP) feemɔ ni nyɛɛɛ atsu nii jogbaŋŋ lɛ mli baa lɛ he lɛ ji nɔ ni he hiaa waa (8), ni emaa bɔ ni ehe hiaa ni ana tsofafeemɔ mli otii ni abaanyɛ akɛtsu nii kɛtsi hela naa loo atsĩ naa lɛ nɔ mi. Tsi jwɛŋmɔŋ helai ni haa mɔ he jɔ̃ɔ ehe lɛ naa (9). Saji ni bɛ lɛ ji anɔkwale ni eji akɛ, asusuɔ akɛ tsitsi mli wala yibii lɛ bɛ hewalɛ ko kwraa kɛ akɛto wala yibii ni yɔɔ fãji ni ebɔle amɛhe lɛ amli lɛ ahe (10). Kɛ akɛto he akɛ nɛkɛ wala yibii nɛɛ tsuɔ gbɛfaŋnɔ titri ko he nii yɛ nibii ni haa mɔ tsui nyɔɔ emli lɛ ahe gbɛjianɔtoo mli koni ekɛwo synaptic transmission lɛ he hewalɛ ni ekɛha hetoo kɛha pilamɔi kɛ helai shihilɛi lɛ, nyɛmɔ ni ayɔɔ ni akɛtsakeɔ wala yibii lɛ amli wala yibii lɛ kɛyaa shihilɛi ni wa ni yɔɔ jwɛŋmɔŋ fãji lɛ amli lɛ ji nɔ ni miihe ashɛ glial wala yibii lɛ pɛ he (11-14). Kɛfata he lɛ, bɔ ni jwɛŋmɔ mli wala yibii lɛ yɔɔ ha lɛ tsiɔ tsakemɔi ni baa yɛ jwɛŋmɔŋ fãji akui pɔtɛɛ komɛi amli lɛ akasemɔ naa waa. Nɔ ni jɛ mli ba ji akɛ, nɔ ko fioo pɛ ale yɛ nɔ pɔtɛɛ ni jɛɔ mitochondrial ni tsuuu nii jogbaŋŋ yɛ tsitsii amli lɛ mli baa lɛ he.
Bɔni afee ni wɔnu nɔ ni jɛɔ mitochondrial nitsumɔ ni ejaaa mli baa lɛ shishi lɛ, wɔjie Purkinje neurons (PNs) lɛ kɛjɛ tsitsii ni fiteɔ ni mitochondrial kponɔgbɛ membrane fusion (Mfn2) lɛ hiɛkpatamɔ kɛbaa lɛ mli. Eyɛ mli akɛ Mfn2 tsakemɔi ni baa adesai amli lɛ kɛ gboshinii ko ni haa mɔ tsui nyɔɔ emli ni ale lɛ akɛ Charcot-Marie-Tooth type 2A (15) lɛ yɔɔ tsakpaa moŋ, shi Mfn2 hiɛkpatamɔ yɛ shihilɛ mli yɛ ŋmeii amli lɛ ji nɔ ko ni ale jogbaŋŋ ni haa mɔ tsui nyɔɔ emli (OXthoPHOS) lɛ. 16-19) kɛ nɔ ni jɛɔ mli baa lɛ mli lɛ, jwɛŋmɔŋ helai ni yaa hiɛ, tamɔ gbɔmɔtsoŋ helai ni haa mɔ he jɔ̃ɔ ehe (18, 19) loo cerebellar ataxia (16) fataa he. Kɛtsɔ tsɔne ni akɛfeɔ proteomics, metabolomics, mfonirifeemɔ, kɛ helatsamɔ gbɛi ni akɛtsuɔ nii lɛ nɔ lɛ, wɔtsɔɔ akɛ, tsɔne ni haa mɔ tsui nyɔɔ emli ni yaa hiɛ lɛ haa pyruvate carboxylase (PCx) kɛ nibii krokomɛi ni fata PNs ni yɔɔ gbɔmɔtso lɛ mli lɛ ahe lɛ tsuɔ nii waa. Kɛha nɛkɛ niiamlipɛimɔ nɛɛ he sɛɛnamɔ lɛ he odaseyeli lɛ, wɔkɛ PCx kpojiemɔ lɛ ba shi yɛ Mfn2-deficient PNs lɛ mli, ni wɔna akɛ nɛkɛ nitsumɔ nɛɛ haa oxidative haomɔ lɛ mli woɔ wu ni ehaa jwɛŋmɔŋ helai ni haa mɔ he jɔ̃ɔ ehe lɛ yaa hiɛ oya, ni no tsɔɔ akɛ azoospermia haa wala yibii lɛ gboiɔ Metabolic adaptability. MFN2 kpojiemɔ ni naa wa baanyɛ ajie PN ni fiteɔ lɛ kwraa kɛ OXPHOS ni faaa waa, mitochondrial DNA ni ayeɔ babaoo, kɛ etamɔ nɔ ni mitochondrial network ni ekumɔ lɛ, ni maa nɔ mi ekoŋŋ akɛ nɛkɛ neurodegeneration nɛɛ baanyɛ aya hiɛ po yɛ hela ni etee hiɛ lɛ mli.
Bɔni afee ni wɔna mitochondria ni yɔɔ Mfn2 knockout PNs lɛ mli lɛ he mfoniri yɛ wɔjwɛŋmɔ mli lɛ, wɔkɛ ŋmei ko ni haa mitochondria ni damɔ Cre nɔ lɛ tsɔɔ yellow fluorescent protein (YFP) (mtYFP) (20) Cre kpojiemɔ lɛ nɔ ni wɔkwɛɔ bɔ ni mitochondria lɛ yɔɔ ha yɛ vivo mli. Wɔna akɛ Mfn2 tsɛŋemɔ hela lɛ hiɛkpatamɔ lɛ baaha mitochondrial network lɛ mli agbala fiofio (Mfoniri S1A), ni ana tsakemɔ ni ba mra fe fɛɛ lɛ yɛ otsii 3 mli. Nɔ ni tamɔɔɔ enɛ lɛ, PN tsɛŋemɔ hela lɛ mli nibii ni fiteɔ waa lɛ, taakɛ Calbindin immunostaining lɛ ni laajeɔ lɛ yeɔ he odase lɛ, ejeee shishi kɛyashi be mli ni eye otsii 12 (Mfoniri 1, A kɛ B). Be ni kɛ mra be mli tsakemɔi ni baa yɛ mitochondrial morphology mli kɛ neuron gbele ni anaa lɛ shishijee ni anaa lɛ kpãaa gbee lɛ tsirɛ wɔ ni wɔpɛi tsakemɔi ni mitochondrial nitsumɔ ni ejaaa lɛ kɛbaa lɛ mli dani wala yibii lɛ gboi. 1C), kɛ yɛ ŋmeii ni kudɔɔ (Mfn2 + / loxP :: mtYFP loxP- stop-loxP: : L7-cre), ni sɛɛ mli lɛ atsɛɔ lɛ CT1BRL (Mfoniri) lɛ mli. Nibii ni afeɔ lɛ jogbaŋŋ ni damɔ YFP okadi lɛ mli hewalɛ nɔ lɛ haa wɔtsuuɔ YFP + gbɔmɔtso lɛ (YFPhigh) ni ji PNs lɛ kɛjɛɔ mɛi ni jeee PNs (YFPneg) (Mfoniri S1B) loo putative fluorescent axon / dendritic fãi (YFPlow; Mfoniri S1D, confoscopecal kɛtsɔ abɛkugbɛ), abɛkugbɛ). Bɔni afee ni wɔkwɛ bɔ ni gbɔmɛi ni ato amɛhe gbɛjianɔ lɛ ji lɛ, wɔfee LFQ proteomics kɛ no sɛɛ lɛ, wɔfee nibii titrii ni yɔɔ mli lɛ amlipɛimɔ, ni wɔna akɛ mligbalamɔ ko ni yɔɔ faŋŋ yɛ YFPhigh kɛ YFPneg wala yibii lɛ ateŋ (Mfoniri S1C). YFPhigh tsɛŋemɔ helai tsɔɔ PNs okadii ni ale lɛ (ni ji Calb1, Pcp2, Grid2 kɛ Itpr3) (21, 22), shi tsɛŋemɔ helai ni afɔɔ feemɔ yɛ tsɛŋemɔ helai loo tsɛŋemɔ helai krokomɛi amli lɛ shweremɔ ko bɛ (Mfoniri 1D) ). Nɔkwɛmɔnii ni yɔɔ YFPhigh wala yibii ni ato he gbɛjianɔ ni abua naa yɛ amɛ diɛŋtsɛ amɛhe shikpamɔi amli lɛ ateŋ nɔkwɛmɔ nɔ tsɔɔ tsakpaa ni yɔɔ > 0.9, ni tsɔɔ fɔmɔ kpakpa ni yɔɔ wala yibii ni afee ekoŋŋ lɛ ateŋ (Mfoniri S1E). Yɛ kukufoo mli lɛ, saji nɛɛ ma wɔ gbɛjianɔtoo kɛha PN ni baanyɛ aba lɛ mligbalamɔ ni mli wa kɛ nɔ pɔtɛɛ lɛ nɔ mi. Akɛni L7-cre tsɔne kudɔmɔ gbɛjianɔtoo ni akɛtsuɔ nii lɛ haa mosaic sɛɛkuu baa yɛ klɛŋklɛŋ otsi lɛ mli yɛ fɔmɔ sɛɛ (23) hewɔ lɛ, wɔbɔi ŋmeii ni jɛ CTRL kɛ shihilɛ mli (Mfn2 loxP / loxP :: mtYFP loxP-stop-loxP :: L7-cre) Buamɔ tsɔnei ni haa mɔ tsui nyɔɔ emli lɛ anaa. Kɛji akɛ agbe sɛɛkuu lɛ naa lɛ, atsɛɔ lɛ Mfn2cKO beni eye otsii 4 lɛ. Akɛ naagbee lɛ, wɔhala otsii 8 ni eye beni PN fã lɛ kã shi lolo yɛ mitochondrial mli fãi ni yɔɔ faŋŋ lɛ fɛɛ sɛɛ (Mfoniri 1B kɛ Mfoniri S1A). Yɛ fɛɛ mli lɛ, wɔbu protein 3013 ni fa fe fɛɛ, ni aaafee 22% damɔ MitoCarta 2.0 niŋmaai ni damɔ mitochondrial proteome lɛ nɔ lɛ nɔ akɛ mitochondria (Mfoniri 1E) (Mfoniri 1E) (24). Srɔtofeemɔ ni yɔɔ tsɛŋemɔ hela mli lɛ mlipɛimɔ ni afee yɛ otsi ni ji 8 lɛ mli lɛ tsɔɔ akɛ, 10.5% pɛ yɛ protein fɛɛ mli ni tsakemɔi ni sa kadimɔ ba (Mfoniri 1F kɛ Mfoniri S1F), ni protein 195 ji nɔ ni akudɔ shi ni protein 120 ji nɔ ni akudɔ ŋwɛi (Mfoniri 1F). Ehe miihia ni wɔkadi akɛ “gbɛi heei ni atsɔɔ nɔ akwɛɔ nibii amli” ni yɔɔ saji nɛɛ amli lɛ tsɔɔ akɛ, tsɛŋemɔ helai ni ajieɔ lɛ kpo yɛ gbɛi srɔtoi anɔ lɛ titri ji gbɛi pɔtɛɛ ni atsɔɔ nɔ atsakeɔ nii lɛ (Mfoniri 1G). Nɔ ni yɔɔ miishɛɛ ji akɛ, eyɛ mli akɛ gbɛi ni kɔɔ OXPHOS kɛ calcium he lɛ ahe mlai ni baa shi lɛ maa mitochondrial nitsumɔ ni bɛ fusion-deficient PNs lɛ nɔ mi moŋ, shi kui krokomɛi ni amino acid tsakemɔ fata he titri lɛ ji nɔ ni ato he gbɛjianɔ jogbaŋŋ, ni kɛ metabolism ni yɔɔ mitochdrion P mli lɛ kpãa gbee. Rewiring lɛ yaa nɔ daa. nitsumɔ ni ehiii.
(A) Mfonirii ni damɔ shi kɛha cerebellar fãi ni yɔɔ CTRL kɛ Mfn2cKO ŋmeii lɛ amli lɛ ni tsɔɔ PNs (calbindin, gray) ni laajeɔ lɛ yaa hiɛ; nuclei lɛ ji nɔ ni akɛ DAPI wo mli. (B) (A) lɛ falɛ (gbɛ kome ni atsɔɔ nɔ akwɛɔ srɔtofeemɔ lɛ mli, ***P<0.001; n = 4 kɛyashi 6 kooyigbɛ kɛjɛ ŋmeii etɛ mli). (C) Nitsumɔ mli kaa. (D) Heat map mlijaa ni kɔɔ kadimɔi pɔtɛɛ ni kɔɔ Purkinje he (ŋwɛi) kɛ wala yibii krokomɛi ahe (teŋgbɛ). (E) Venn mfoniri ni tsɔɔ mitochondrial proteins ni ayɔse yɛ PN ni ato he gbɛjianɔ lɛ mli lɛ ayibɔ. (F) Volcano mfoniri ni tsɔɔ protein ni ajieɔ lɛ kpo yɛ gbɛi srɔtoi anɔ yɛ Mfn2cKO neurons lɛ mli yɛ otsii 8 mli (sɛɛnamɔ ni afo mli ni ji 1.3). (G) Nibɔɔ gbɛ lɛ mlipɛimɔ lɛ tsɔɔ gbɛi enumɔ ni he hiaa fe fɛɛ ni ji ŋwɛi-mla (red) kɛ shishi-mla (blue) ni yɔɔ Mfn2cKO PN ni ato he gbɛjianɔ akɛ otsii 8 lɛ mli. Atsɔɔ bɔ ni protein fɛɛ protein ni ana lɛ jieɔ ehe kpo ehaa lɛ mlijaa. Grayscale dɔlɛ shikpɔŋ he mfoniri: P sɛɛnamɔ ni atsake. ns, ehe ehiaaa.
Proteomics he saji tsɔɔ akɛ protein ni yɔɔ complex I, III, kɛ IV lɛ mli lɛ baa shi fiofio. Complexes I, III, kɛ IV lɛ fɛɛ hiɛ mtDNA-encoded subunits ni he hiaa waa, yɛ be mli ni complex II, ni ji nuklea-kode pɛ lɛ, nɔ titri ni enaaa nɔ ko nɔ ko (Mfoniri 2A kɛ Mfoniri S2A). . Kɛha nɔ ni jɛ mli ba lɛ, immunohistochemistry ni kɔɔ cerebellar fãji lɛ ahe lɛ tsɔɔ akɛ MTCO1 (mitochondrial cytochrome C oxidase subunit 1) subunit ni ji IV ni yɔɔ PN lɛ mli lɛ baa shi fiofio (Mfoniri 2B). mtDNA-encoded subunit Mtatp8 lɛ ba shi kwraa (Mfoniri S2A), yɛ be mli ni nuklea-encoded ATP synthase subunit lɛ mli shihilɛ ni yɔɔ shiŋŋ lɛ tee nɔ ehi shi shiŋŋ, ni kɛ ATP synthase subassembly F1 ni ale ni yɔɔ shiŋŋ lɛ kpãa gbee beni mtDNA kpojiemɔ lɛ damɔ shi shiŋŋ lɛ. Gbɛjianɔtoo lɛ yaa nɔ daa. Gbalamɔ mli (7). Nibii ni akwɛɔ yɛ mtDNA lɛ mli yɛ Mfn2cKO PNs ni ahala lɛ mli lɛ mli kɛtsɔ be-mli polymerase chain reaction (qPCR) nɔ lɛ ma nɔ mi akɛ mtDNA lɛ mlijaa lɛ ayibɔ baa shi fiofio. Kɛ akɛto kuu ni kudɔɔ lɛ he lɛ, yɛ otsii 8 mli lɛ, mtDNA lɛ mlijaa 20 pɛ ji nɔ ni ahiɛ (Mfoniri 2C). Nɔ ni kɛ nɔ ni jɛ mli ba nɛɛ kpãa gbee lɛ, Mfn2cKO PNs ni akɛkwɛɔ nibii bibii amli lɛ mli nibii bibii ni akɛkwɛɔ nii amli lɛ tsɔɔ bɔ ni mitochondrial nucleotides lɛ yeɔ be ni damɔ shi (Mfoniri 2D). Wɔna akɛ mɛi komɛi pɛ ni kɛ amɛhe woɔ mitochondrial protein fitemɔ kɛ nɔnyɛɛ hetoo mli lɛ ji mɛi ni ato amɛhe gbɛjianɔ, ni Lonp1, Afg3l2 kɛ Clpx, kɛ OXPHOS nibii ni haa mɔ feɔ ekome lɛ fata he. Ayooo tsakemɔi ni sa kadimɔ yɛ protein ni fata apoptosis he lɛ mli (Mfoniri S2B). Nakai nɔŋŋ hu, wɔna akɛ mitochondria kɛ endoplasmic reticulum gbɛi ni kɛ amɛhe woɔ calcium tsɔne mli lɛ yɛ tsakemɔi fioo pɛ (Mfoniri S2C). Kɛfata he lɛ, tsɔne ni haa mɔ he jɔɔ lɛ he niiamlipɛimɔ lɛ enaaa tsakemɔ ko ni sa kadimɔ, ni kɛ tsɔne ni haa mɔ he jɔɔ lɛ ni anaa yɛ wala mli kɛtsɔ immunohistochemistry kɛ electron microscopy nɔ lɛ kpãa gbee (Mfoniri S3). Shi kɛlɛ, OXPHOS nitsumɔ ni yaa hiɛ yɛ PNs lɛ mli lɛ fataa tsakemɔi ni yɔɔ faŋŋ ni baa yɛ mitochondrial mli lɛ he. Mitochondrial kui baanyɛ ana yɛ Mfn2cKO PNs ni eye otsii 5 kɛ 8 lɛ amli wala yibii kɛ dendritic tsei lɛ amli, ni mligbɛ tsɔne lɛ mli tsɔne lɛ etsɔ tsakemɔi ni mli kwɔ mli (Mfoniri S4, A kɛ B). Kɛha nɛkɛ tsakemɔi ni baa trukaa nɛɛ kɛ mtDNA lɛ nɔ ni baa shi waa lɛ, tsɔne ni akɛ tetramethylrhodamine methyl ester (TMRM) tsuɔ nii yɛ mli lɛ mlipɛimɔ tsɔɔ akɛ, tsɔne ni haa mɔ tsui nyɔɔ emli lɛ mli hewalɛ ni yɔɔ Mfn2cKO PNs lɛ mli lɛ ba shi waa (Mfoniri S4C).
(A) Be mli nibii amlipɛimɔ ni kɔɔ OXPHOS complex lɛ kpojiemɔ lɛ he. Susumɔ protein ni yɔɔ P<0.05 yɛ otsii 8 mli pɛ he (gbɛi enyɔ ANOVA). Dotted line: Tsakemɔ ko bɛ kɛ akɛto CTRL he. (B) Abɛkugbɛ: Nɔkwɛmɔnɔ ni kɔɔ cerebellar fã ko ni akɛ tsofa ni gbeɔ MTCO1 he lɛ ewo nɔ (skala bar, 20 μm). He ni Purkinje wala yibii lɛ yɔɔ lɛ ji he ni ŋmɔŋ ehà nɔ. Ninejurɔgbɛ: MTCO1 lɛ mlijaa (gbɛ kome nɔ ni akɛpɛiɔ srɔtofeemɔ lɛ mli; n = 7 kɛyashi 20 ni akwɛ mli kɛjɛ ŋmeii etɛ mli). (C) qPCR mlipɛimɔ ni kɔɔ mtDNA nɔkwɛmɔ nɔ yibɔ ni yɔɔ PN ni ahala lɛ mli lɛ he (gbɛ kome mlipɛimɔ ni kɔɔ srɔtofeemɔ he; n = 3 kɛyashi 7 mice). (D) Abɛkugbɛ: Nɔkwɛmɔnɔ ni kɔɔ cerebellar fã ko ni akɛ nɔ ko ni gbeɔ DNA he lɛ ewo nɔ (skala bar, 20 μm). He ni Purkinje wala yibii lɛ yɔɔ lɛ ji he ni ŋmɔŋ ehà nɔ. Ninejurɔgbɛ: mtDNA helai lɛ ayibɔ (gbɛ kome ni atsɔɔ nɔ akwɛɔ srɔtofeemɔ lɛ mli; n = 5 kɛyashi 9 wala yibii ni jɛ ŋmeii etɛ mli). (E) Nɔkwɛmɔnɔ ko ni kɔɔ cerebellar fã ko ni mli wa ni tsɔɔ mitoYFP + Purkinje wala yibii (akpɔlɔ) yɛ wala yibii muu lɛ fɛɛ ni akɛwoɔ klamp mli lɛ he. (F) IV curve lɛ falɛ. (G) Najiaŋdamɔlɔi ni aŋmala ashwie shi ni kɔɔ ŋmɛnɛŋmɛnɛ beaŋ tsofa ni akɛwoɔ mɔ mli lɛ he yɛ CTRL kɛ Mfn2cKO Purkinje wala yibii lɛ amli. Top trace: Klɛŋklɛŋ pulse ni tsirɛ AP. Nɔ ni yɔɔ shishigbɛ: AP shii abɔ ni fa fe fɛɛ. (H) Nibii ni baa trukaa lɛ ayibɔ (sPSPs). Atsɔɔ najiaŋdamɔlɔ ni aŋmalaa nɔ nii lɛ kɛ e-zoom ratio lɛ yɛ (I). Gbɛ kome ni atsɔɔ nɔ akwɛɔ srɔtofeemɔ mli lɛ kwɛɔ n = 5 kɛyashi 20 mli wala yibii ni jɛ ŋmeii etɛ mli lɛ mli. Ajieɔ saji lɛ akpo akɛ mean±SEM; *P<0.05; **P<0.01; ***p<0.001. (J) AP ni baa trukaa lɛ he najiaŋdamɔlɔi ni aŋmala ashwie shi kɛtsɔ tsɔne ni akɛfoɔ tsɔne ni akɛwoɔ tsɔne mli lɛ nɔ. Nɔ ni yɔɔ ŋwɛi: AP shii abɔ ni fa fe fɛɛ. Nɔ ni yɔɔ shishigbɛ: AP kome pɛ. (K) Tsɔɔmɔ AP ni afɔɔ feemɔ lɛ mlijaa kɛ nɔ ni fe fɛɛ lɛ falɛ taakɛ (J) tsɔɔ lɛ. Mann-Whitney kaa lɛ; n = 5 cells ni akwɛ mli kɛjɛ ŋmeii ejwɛ mli. Ajieɔ saji lɛ akpo akɛ mean±SEM; ehe ehiaaa.
Ana OXPHOS fitemɔ ni yɔɔ faŋŋ yɛ Mfn2cKO PN ni eye otsii 8 lɛ mli, ni tsɔɔ akɛ gbɔmɔtsoŋ nitsumɔ ni kɔɔ tsɔnei ni haa mɔ tsui nyɔɔ emli lɛ ahe lɛ ejaaa kwraa. No hewɔ lɛ, wɔpɛi tsɔnei ni haa mɔ tsui nyɔɔ emli ni haa mɔ tsui nyɔɔ emli lɛ amli yɛ otsii 4 kɛyashi 5 kɛ otsii 7 kɛyashi 8 mli kɛtsɔ tsɔne ni akɛwoɔ tsɔne mli ni haa mɔ tsui nyɔɔ emli lɛ mli lɛ nɔ (Mfoniri 2E). Yɛ gbɛ ni akpaaa gbɛ nɔ lɛ, sɛɛnamɔ ni yɔɔ Mfn2cKO tsɔnei ni haa mɔ tsui nyɔɔ emli lɛ ahe lɛ tamɔ nɔ ni kudɔɔ lɛ, eyɛ mli akɛ srɔtofeemɔi ni yɔɔ mlɛo yɛ wala yibii lɛ ateŋ moŋ (Table 1). Nakai nɔŋŋ hu, yɛ otsii 4 kɛyashi 5 mli lɛ, anaaa tsakemɔ ko ni sa kadimɔ yɛ ŋmɛnɛŋmɛnɛ-voltage wekukpaa lɛ mli (IV curve) (Mfoniri 2F). Shi kɛlɛ, Mfn2cKO tsɔnei ni eye otsii 7 kɛyashi 8 lɛ eko kwraa yeee wala yɛ IV gbɛjianɔtoo lɛ mli (hyperpolarization step), ni tsɔɔ akɛ henumɔ ko ni yɔɔ faŋŋ ni kɔɔ hyperpolarization he lɛ yɛ nɛkɛ naagbee be nɛɛ mli. Nɔ ni tamɔɔɔ enɛ lɛ, yɛ Mfn2cKO tsɔnei lɛ amli lɛ, depolarizing currents ni kɛ repetitive action potential (AP) tsɔnei lɛ baa lɛ heɔ amɛyeɔ jogbaŋŋ, ni tsɔɔ akɛ amɛ tsɔnei ni ajieɔ lɛ fɛɛ lɛ jeee nɔ ni yɔɔ srɔto kwraa yɛ tsɔnei ni kudɔɔ otsii 8 lɛ ahe (Table 1 kɛ Figure 2G). Nakai nɔŋŋ hu, bɔ ni spontaneous postsynaptic currents (sPSCs) lɛ fɔɔ baa kɛ bɔ ni egbɛɔ eshwaa lɛ ji nɔ ni akɛtoɔ kuu ni kudɔɔ lɛ he, ni nibii ni baa lɛ tee hiɛ kɛjɛ otsii 4 kɛtee otsii 5 kɛtee otsii 7 kɛtee otsii 8 kɛ shweremɔ ni tamɔ nakai nɔŋŋ (Mfoniri 2, H kɛ I). Be ni akɛfeɔ synaptic shweremɔ yɛ PNs (25). Ana nɔ ni tamɔ nakai nɔŋŋ yɛ PNs ni afo mli lɛ sɛɛ. Nɛkɛ gbɛjianɔtoo nɛɛ tsiɔ ATP ni ehiii ni yɔɔ wala yibii lɛ amli lɛ he nyɔmɔwoo naa, taakɛ ebaanyɛ eba yɛ wala yibii muu lɛ fɛɛ mli klamp ni aŋmalaa lɛ mli lɛ. Yɛ gbɛ titri ko nɔ lɛ, hejɔɔmɔ membrane hewalɛ kɛ Mfn2cKO neurons lɛ ni tswaa lɛ shii abɔ lɛ enaaa nɔ ko nɔ ko (Mfoniri 2, J kɛ K). Yɛ kukufoo mli lɛ, nɔ ni jɛ mli ba nɛɛ tsɔɔ akɛ PNs ni yɔɔ OXPHOS ni tsuuu nii jogbaŋŋ lɛ baanyɛ akpee sɛɛkuu ni yaa hiɛ waa lɛ naa jogbaŋŋ, ni tsɔɔ akɛ gbɛ ko yɛ ni atsɔɔ nɔ awoɔ nyɔmɔ ni haa amɛhiɛɔ electrophysiological hetooi ni bɛŋkɛɔ bɔ ni sa lɛ mli.
Ajieɔ saji lɛ akpo akɛ nɔ ni tsɔɔ ± SEM (gbɛ kome mlipɛimɔ ni kɔɔ srɔtofeemɔ he, Holm-Sidak srɔtofeemɔi babaoo kaa; * P <0.05). Akɛ brakɛti tsɔɔ unit yibɔ lɛ.
Wɔto gbɛjianɔ koni wɔpɛi mli kɛji akɛ gbɛi ni baanyɛ aku OXPHOS ni faaa waa lɛ naa lɛ fata kuu ko he (Mfoniri 1G) lɛ, ni no tsɔɔ nɔ hewɔ ni PN ni ena naagba lɛ baanyɛ ahi electrophysiology ni bɛŋkɛɔ bɔ ni sa lɛ mli (Mfoniri 2, E kɛyashi K). . Nibii ni akwɛɔ mli lɛ tsɔɔ akɛ, nibii ni haa mɔ tsui nyɔɔ emli lɛ ateŋ nɔ ni fe fɛɛ ji nɔ ni haa mɔ tsui nyɔɔ emli (Mfoniri 3A kɛ Mfoniri S5A), ni naagbee nɔ ni jɛɔ mli baa lɛ baanyɛ aye abua tricarboxylates ni yɔɔ lá mli lɛ. Wɔna akɛ nɔ ni yɔɔ BCAA transaminase 1 (BCAT1) kɛ BCAT2 lɛ fɛɛ mli lɛ tee hiɛ. Amɛha BCAA mli nibii ni tsakeɔ lɛ klɛŋklɛŋ gbɛ lɛ tee hiɛ kɛtsɔ glutamate ni jɛ α-ketoglutarate mli lɛ nɔ (26). Akɛ fãi bibii fɛɛ ni feɔ branched chain keto acid dehydrogenase (BCKD) lɛ tsuɔ nii (nifeemɔ nɛɛ haa BCAA carbon skeleton ni jɛɔ mli baa lɛ mli nibii ni nyiɛɔ sɛɛ ni anyɛɛɛ aku sɛɛ atsuɔ he nii lɛ yaa hiɛ) (Mfoniri 3A kɛ Mfoniri S5A). Shi kɛlɛ, anaaa tsakemɔ ko ni yɔɔ faŋŋ yɛ BCAA lɛ diɛŋtsɛ mli yɛ PN ni ahala lɛ mli, ni ekolɛ ejɛ amino acids ni he hiaa nɛɛ ni tsɛŋemɔ helai amli lɛ yaa hiɛ loo nibii krokomɛi ni akɛtsuɔ nii (glucose loo lactic acid) ni akɛtsuɔ nii koni akɛfata TCA sɛɛkuu lɛ he lɛ hewɔ (Mfoniri S5B). PNs ni bɛ OXPHOS lɛ hu jie glutamine fitemɔ kɛ transamination nitsumɔi ni yaa hiɛ yɛ otsii 8 mli, ni baanyɛ ajie lɛ kpo kɛtsɔ mitochondrial enzymes glutaminase (GLS) kɛ glutamine pyruvate transaminase 2 (GPT2) lɛ nɔyaa nɔ (Mfoniri 3, A kɛ C). Ehe miihia ni wɔkadi akɛ, GLS lɛ nɔyaa lɛ ji isoform glutaminase C (GLS-GAC) ni ajie lɛ kpo lɛ pɛ (Mfn2cKO/CTRL tsakemɔ lɛ ji nɔ ni miihe ashɛ 4.5-fold, P = 0.05), ni egbɛjianɔtoo pɔtɛɛ ni yɔɔ kansa fãji amli lɛ baanyɛ aye abua mitochnerghondrial. (27).
(A) Heat map lɛ tsɔɔ tsakemɔ ni baa yɛ protein lɛ mli kɛha gbɛ pɔtɛɛ ni atsɔɔ nɔ lɛ yɛ otsii 8 mli. (B) Nɔkwɛmɔnɔ ni kɔɔ cerebellar fã ko ni akɛ tsofa ni gbeɔ PCx he lɛ ewo nɔ (skala bar, 20 μm). Akpɔlɔi ni yɔɔ ŋmɔŋ lɛ tsɔɔ Purkinje wala yibii lɛ gbɔmɔtso lɛ. (C) Be mli ni protein lɛ jieɔ ehe kpo lɛ mlipɛimɔ ni ayɔse akɛ eji mɔ ko ni he hiaa waa kɛha lá ni hoɔ yɛ gbɔmɔtso lɛ mli lɛ (t-test babaoo, *FDR <5%; n = 3-5 mice). (D) Yɛ ŋwɛi: Mfoniri ni tsɔɔ gbɛi srɔtoi ni atsɔɔ nɔ aboteɔ carbon ni aŋmala nɔ nii ni yɔɔ [1-13C]pyruvate tracer lɛ mli lɛ mli (ni ji, kɛtsɔ PDH loo trans-arterial gbɛ nɔ). 13C]pyruvate (paired t-test; ** P <0.01) ni akɛwo cerebellar mli fãi ni mli wa lɛ amli lɛ mlijaa oha mlijaa 100. (E) Be mli yinɔsane mlipɛimɔ ni mli kwɔ ni kɔɔ gbɛ ni atsɔɔ lɛ he. Susumɔ protein ni yɔɔ P<0.05 yɛ otsii kpaanyɔ mli pɛ he. Line ni yɔɔ ŋmɔlɔŋmɔlɔ: tsakemɔ sɛɛnamɔ ko bɛ (gbɛi enyɔ mlipɛimɔ ni kɔɔ srɔtofeemɔ he; * P <0.05; *** P <0.001). Ajieɔ saji lɛ akpo akɛ tsɔɔ ± SEM.
Yɛ wɔpɛimɔ lɛ mli lɛ, BCAA catabolism ebatsɔ gbɛi titrii ni atsɔɔ nɔ akwɛɔ nibii anɔ lɛ ateŋ ekome. Nɛkɛ anɔkwale nɛɛ tsɔɔ waa akɛ kɔɔyɔɔ ni boteɔ TCA sɛɛkuu lɛ mli lɛ baanyɛ atsake yɛ PN ni OXPHOS bɛ mli lɛ mli. Enɛ baanyɛ adamɔ shi aha tsɔne ni haa mɔ tsui nyɔɔ emli lɛ mli nibii ni haa mɔ tsui nyɔɔ emli lɛ ahe nitsumɔ, ni baanyɛ aná tsɔne lɛ mli nibii ni haa mɔ tsui nyɔɔ emli lɛ anɔ hewalɛ tɛ̃ɛ yɛ OXPHOS nitsumɔ ni naa wa lɛ nɔkwɛmɔ be mli. Yɛ nɛkɛ susumɔ nɛɛ naa lɛ, wɔna akɛ, nɔ titri ni teɔ shi woɔ lá ni hoɔ yɛ gbɔmɔtso lɛ mli lɛ PCx lɛ ji nɔ ni ato he gbɛjianɔ (Mfn2cKO/CTRL tsakeɔ aaafee shii 1.5; Mfoniri 3A), ni haa pyruvate lɛ tsakeɔ kɛyaa oxaloacetate (28), ni aheɔ ayeɔ akɛ eyɛ brain mli lɛ mli. 30). Kɛha proteomics hetooi lɛ, confocal microscopy tsɔɔ akɛ PCx kpojiemɔ lɛ tee hiɛ waa yɛ OXPHOS-deficient PNs lɛ mli, yɛ be mli ni PCx nifeemɔ lɛ ji nɔ titri ni tsiɔ Bergmann glial cells ni bɛŋkɛ lɛ ni kudɔɔ lɛ lɛ naa (Mfoniri 3B). Kɛha nitsumɔ he kaa ni ana lɛ, wɔkɛ [1-13C]pyruvate tracer tsu cerebellar fãji ni mli wa lɛ ahe nii. Beni pyruvate lɛ ji nɔ ni pyruvate dehydrogenase (PDH) lɛ ha lɛ, e-isotope kadimɔ lɛ laaje , Shi akɛwoɔ TCA sɛɛkuu lɛ teŋgbɛ nibii lɛ amli beni pyruvate lɛ tsakeɔ kɛtsɔɔ lá mli nibii ni yaa nɔ lɛ anɔ (Mfoniri 3D). Yɛ wɔ proteomics saji lɛ asɛɛfimɔ mli lɛ, wɔkwɛ kadimɔi babaoo ni jɛ nɛkɛ tracer nɛɛ mli yɛ aspartic acid ni yɔɔ Mfn2cKO fãi lɛ amli lɛ mli, yɛ be mli ni citric acid kɛ malic acid hu yɛ nɔ ko ni yaa nɔ yɛ gbɛ ni sa nɔ, eyɛ mli akɛ jeee nɔ ni he hiaa waa moŋ (Mfoniri 3D).
Yɛ MitoPark ŋmeii ni yɔɔ mitochondrial nitsumɔ ni ehiii ni jɛɔ dopamine ŋmeii ni fiteɔ mitochondrial niŋmaa factor A gene (Tfam) (Mfoniri S6B) lɛ mli lɛ, PCx kpojiemɔ lɛ hu ji nɔ ni ato he gbɛjianɔ jogbaŋŋ (31), ni tsɔɔ akɛ acetone acid ni haa mɔ tsui nyɔɔ emli lɛ ji hela ni haa mɔ tsui nyɔɔ emli lɛ. OXPHOS ni yɔɔ gbɔmɔtso lɛ mli lɛ. Ehe miihia ni wɔkadi akɛ, ana akɛ enzymes ni yɔɔ srɔto (32-34) ni baanyɛ ajie ehe kpo yɛ tsɔnei ni haa mɔ tsui nyɔɔ emli ni baanyɛ aha lá ni hoɔ yɛ gbɔmɔtso lɛ mli lɛ aba lɛ, akwɛɔ nɔ jogbaŋŋ yɛ PNs ni bɛ OXPHOS lɛ amli, tamɔ propionyl-CoA carboxylase (PCC-A), Malonyl-CoA mitochylCoA tsakeɔ propionyl-CoA kɛyaa propionyl-CoA mitochyl-CoA . (ME3), ni egbɛfaŋnɔ titri ji ni ejie pyruvate kɛjɛ malate mli (Mfoniri 3, A kɛ C) (33, 35). Kɛfata he lɛ, wɔna akɛ Pdk3 enzyme, ni haa PDH lɛ feɔ phosphorylates ni no haaa etsu nii lɛ mliyaa ni sa kadimɔ waa (36), yɛ be mli ni anaaa tsakemɔ ko yɛ Pdp1 enzyme ni haa PDH loo PDH enzyme complex lɛ diɛŋtsɛ tsuɔ nii lɛ mli (Mfoniri 3A). Yɛ be fɛɛ mli lɛ, yɛ Mern2cKO PNs lɛ mli lɛ, α1 fã bibioo α (PDHE1α) fã bibioo ni ji pyruvate dehydrogenase E1 fã ni ji PDH fã ni yɔɔ Ser293 (ni ale akɛ etsĩɔ PDH enzyme nitsumɔ lɛ naa) lɛ mli lɛ tee hiɛ ( Mfoniri S6C ) (Mfoniri S6). Pyruvate bɛ gbɛ ni tsɔɔ lá mli.
Yɛ naagbee lɛ, wɔna akɛ gbɛ ni nɔ kwɔ ni akɛfeɔ serine kɛ glycine nibii ni wala yɔɔ mli lɛ, mitochondrial folate (1C) ni kɔɔ he lɛ kɛ proline nibii ni wala yɔɔ mli lɛ (Mfoniri 1G kɛ Mfoniri S5C) lɛ fɛɛ ji nɔ ni akwɛɔ nɔ jogbaŋŋ, taakɛ amaniɛbɔi tsɔɔ lɛ, yɛ nitsumɔ lɛ mli. Akɛ mitochondrial ni tsuuu nii jogbaŋŋ lɛ tsuɔ nii (5-7). Nibii amlipɛimɔ ni fiɔ nɛkɛ proteomics saji nɛɛ asɛɛ lɛ tsɔɔ akɛ yɛ PN ni OXPHOS bɛ mli lɛ mli lɛ, akɛ cerebellar fãi ni eye otsii 8 lɛ wo serine hydroxymethyltransferase 2 (SHMT2), ni ji enzyme titri ko ni haa mitochondrial folate sɛɛkuu lɛ tsuɔ nii lɛ mli. Hewalɛ ni tsiɔ hela naa lɛ hetoo ni he hiaa waa (Mfoniri S5D). Yɛ 13 CU-glucose-ni akɛwoɔ cerebellar slices mli lɛ, metabolic tracing shikpamɔi lɛ ma nɔ mi ekoŋŋ akɛ serine kɛ proline wala yibii ahe nitsumɔ lɛ yaa hiɛ, ni tsɔɔ akɛ carbon isoforms ni hoɔ kɛyaa serine kɛ proline mli lɛ tee hiɛ (Mfoniri S5E). Akɛni nibii ni GLS kɛ GPT2 woɔ he hewalɛ lɛ ji nɔ ni haa glutamate kɛjɛɔ glutamine mli kɛ transamination ni yɔɔ glutamate kɛ α-ketoglutarate teŋ lɛ hewɔ lɛ, amɛ upregulation tsɔɔ akɛ OXPHOS-ni bɛ tsɔnei ni haa mɔ tsui nyɔɔ emli lɛ yɛ glutamate he taomɔ ni yaa hiɛ , Enɛ baanyɛ afee nɔ ni akɛtoɔ proureline StaureC ni yaa hiɛ lɛ he (Fig5). Nɔ ni tamɔɔɔ tsakemɔi nɛɛ, proteomic mlipɛimɔ ko ni kɔɔ cerebellar astrocytes ni jɛ PN-specific Mfn2cKO ŋmeii amli lɛ tsɔɔ akɛ gbɛi nɛɛ (ni antiperoxidases fɛɛ fata he) tsakeee kwraa yɛ wiemɔ mli, ni no tsɔɔ akɛ Nɛkɛ metabolic redirection nɛɛ ji nɔ ni halaa PN ni efite lɛ (Fig. S6).
Yɛ kukufoo mli lɛ, nɛkɛ mlipɛimɔi nɛɛ jie gbɛi srɔtoi ni yɔɔ PNs lɛ amli lɛ kpo. Eyɛ mli akɛ tsɔne ni haa mɔ tsui nyɔɔ emli lɛ nitsumɔ ni ejaaa lɛ baanyɛ aha mɔ he ajɔ lɛ mra kɛ 1C tsakemɔ (Mfoniri 3E kɛ Mfoniri S5C), kɛ tsakemɔi ni abaanyɛ atsɔɔ hiɛ atsɔɔ po yɛ I kɛ IV complexes lɛ kpojiemɔ mli moŋ, shi tsakemɔi ni baa yɛ serine de novo synthesis lɛ mli lɛ pɛ Ebafee faŋŋ yɛ naagbee sta lɛ mli pɛ. OXPHOS nitsumɔ ni ejaaa (Mfoniri 3E kɛ Mfoniri S5C). Nibii ni ana nɛɛ tsɔɔ gbɛ ni atsɔɔ nɔ atsuɔ nii ni mitochondrial (1C sɛɛkuu) kɛ cytoplasmic (serine biosynthesis) ni haa mɔ tsui nyɔɔ emli lɛ tsuɔ nii yɛ ekomefeemɔ mli kɛ lá ni hoɔ yɛ gbɔmɔtso lɛ mli lɛ mliyaa yɛ TCA sɛɛkuu lɛ mli koni ekɛtsake tsɔne ni haa mɔ tsui nyɔɔ emli lɛ mli nibii ni haa mɔ tsui nyɔɔ emli lɛ.
8-otsii OXPHOS-deficient PNs baanyɛ ahi shii abɔ ni haa mɔ tsui nyɔɔ emli lɛ nɔ ni etsɔ metabolic reconnection ni he hiaa waa mli koni ekɛwo mitochondrial nitsumɔ ni ehiii lɛ he nyɔmɔ. Nɛkɛ nii ni ana nɛɛ teɔ nɔ ko ni yɔɔ miishɛɛ shi akɛ yɛ nɛkɛ be nɛɛ mli po lɛ, nɛkɛ wala yibii nɛɛ hu baanyɛ Aná tsofafeemɔ mli nifeemɔi ni baaha jwɛŋmɔŋ helai ni haa mɔ tsui nyɔɔ emli lɛ asɛɛ afo loo atsĩ naa. Otsɛ́. Wɔtsu nɛkɛ gbɛ ni baanyɛ aba nɛɛ he nii kɛtsɔ mɛi enyɔ ni kɛ amɛhe wo mli lɛ anɔ. Yɛ gbɛ klɛŋklɛŋ nɔ lɛ, wɔfee Cre-ni damɔ shi kɛha adeno-ni kɛ hela (AAV) vector koni MFN2 lɛ anyɛ ajie lɛ kpo yɛ OXPHOS-ni bɛ PNs mli yɛ vivo mli (Mfoniri S7A). AAV ni kɛ MFN2 lɛ woɔ mli lɛ kɛ fluorescent adafitswaa wolo mCherry (Mfn2-AAV) lɛ ji nɔ ni akwɛ mli yɛ shishijee tsɔne ni haa mɔ tsui nyɔɔ emli lɛ mli yɛ vitro mli, ni no ha ajie MFN2 kpo yɛ gbɛ ni damɔ Cre nɔ ni ejie mitochondrial morphology lɛ yiwala, ni no ha tsɔne ni haa mɔ tsui nyɔɔ emli lɛ tsakeɔ yɛ Mfns, B Figure S7). No sɛɛ lɛ, wɔfee vivo mli kaai koni wɔkɛ stereotactically aha otsii 8 Mfn2-AAV kɛya cerebellar cortex ni ji Mfn2cKO kɛ ŋmeii ni kudɔɔ amɛ lɛ, ni wɔpɛi ŋmeii ni eye otsii 12 lɛ amli (Mfoniri 4A). Mfn2cKO ŋmeii ni atsa amɛ lɛ gboi (Mfoniri 1, A kɛ B) (16). Viral tsakemɔ yɛ vivo mli lɛ ha PN kpojiemɔ ni ahala yɛ cerebellar kpokpai komɛi amli (Mfoniri S7, G kɛ H). AAV ni kudɔɔ lɛ ni jieɔ mCherry (Ctrl-AAV) pɛ kpo lɛ ni akɛwoɔ mɔ mli lɛ bɛ hewalɛ ko kwraa yɛ bɔ ni jwɛŋmɔŋ helai ni haa mɔ tsui nyɔɔ emli lɛ yaa hiɛ ehaa yɛ Mfn2cKO kooloi lɛ amli lɛ nɔ. Nɔ ni tamɔɔɔ enɛ lɛ, Mfn2cKOs ni akɛ Mfn2-AAV tsake lɛ mlipɛimɔ lɛ tsɔɔ akɛ PN cell layer lɛ buɔ mɔ he waa (Mfoniri 4, B kɛ C). Yɛ gbɛ titri ko nɔ lɛ, etamɔ nɔ ni srɔtofeemɔ ko bɛ tsɔne ni haa mɔ tsui nyɔɔ emli lɛ mli kɛjɛ kooloi ni akudɔ amɛ lɛ ateŋ (Mfoniri 4, B kɛ C, kɛ Mfoniri S7, H kɛ I). MFN1 kpojiemɔ shi jeee MFN2 lɛ tsuɔ nii jogbaŋŋ yɛ tsɔne ni haa mɔ tsui nyɔɔ emli lɛ gbele yiwalaheremɔ mli (Mfoniri 4C kɛ Mfoniri S7, C kɛ F), ni tsɔɔ akɛ ectopic MFN1 kpojiemɔ lɛ baanyɛ aye abua MFN2 ni bɛ lɛ jogbaŋŋ. Nibii krokomɛi ni akwɛ mli yɛ PN kome lɛ nɔ lɛ tsɔɔ akɛ Mfn2-AAV lɛ jie mitochondria lɛ mli ultrastructure lɛ babaoo, eha mtDNA lɛ mli nibii lɛ bafee nɔ ni sa, ni ekɛ PCx ni ji anti-angiogenesis marker lɛ kpojiemɔ ni nɔ kwɔ lɛ ku sɛɛ (Mfoniri 4, C kɛyashi E ). Nibii ni akɛ hiŋmɛii kwɛɔ Mfn2cKO ŋmeii ni ajie amɛ lɛ yɛ hejɔɔmɔ shihilɛ mli lɛ tsɔɔ akɛ amɛhe ni amɛdamɔ kɛ amɛhe ni amɛtsɔɔ (nyiɛmɔ S1 kɛyashi S3) lɛ etee hiɛ. Yɛ naagbee lɛ, nɛkɛ kaai nɛɛ tsɔɔ akɛ, sɛɛkuu ni akɛwoɔ MFN2 mli ekoŋŋ kɛyaa PNs ni OXPHOS bɛ mli waa lɛ mli lɛ fa bɔ ni sa ni baaha mtDNA ni akɛtsuɔ nii lɛ aku sɛɛ aba ni ekɛ atherosclerosis aba, ni no baatsi axon fitemɔ kɛ neuronal gbele naa yɛ vivo mli.
(A) Gbɛjianɔtoo ko ni tsɔɔ kaa gbɛjianɔtoo ni akɛbaatsu nii kɛwo AAV ni kɛ MFN2 woɔ mli lɛ mli beni atsɔɔ gbɛ ni atsɔɔ nɔ atsakeɔ nii lɛ mli lɛ. (B) Najiaŋdamɔlɔi ni damɔ shi kɛha cerebellar fãi ni eye otsii 12 ni akɛ Mfn2cKO ŋmeii tsake yɛ otsii 8 mli ni akɛ anti-Calbindin antibody wo nɔ lɛ. Ninejurɔgbɛ: Axon fibers lɛ akwɛmɔ. Axon zoom lɛ sɛkɛl ji 450 kɛ 75 μm. (C) Abɛkugbɛ: Purkinje wala yibii lɛ ayibɔ ni yɔɔ AAV shishitsɔɔmɔ loop (AAV +) (gbɛ kome mlipɛimɔ ni kɔɔ srɔtofeemɔ he; n = 3 mice). Ninejurɔgbɛ: mtDNA hiɛdɔɔ mlipɛimɔ yɛ PN ni atsake lɛ mli yɛ otsi 12 (t-test ni bɛ enyɔ; n = 6 cells kɛjɛ ŋmeii etɛ mli). * P <0.05; ** P <0.01. (D) Najiaŋdamɔlɔ ni tsɔɔ bɔ ni PNs ni ji Mfn2cKO cerebellar fãi lɛ tsɔɔ mli kɛ tsɔnei ni akɛtsɔɔ helai ni baa mɔ mli lɛ. Mask ni ji pinki lɛ tsɔɔ he ni dendrites lɛ hiɔ lɛ, ni shwɛmɔ ni yɔɔ ŋmɔlɔŋmɔlɔ ni yɔɔ ŋmɔlɔŋmɔlɔ lɛ tsɔɔ bɔ ni akɛ zoom ha yɛ ninejurɔgbɛ lɛ; n damɔ shi kɛha nukleus lɛ. Skel bar, 1μm. (E) tsɔɔ PCx staining yɛ PN ni atsake yɛ otsii 12 mli lɛ he nɔkwɛmɔnɔ. Skel bar, 20μm. OE, wiemɔ ni ajieɔ lɛ kpo fe nine; FC, tsakemɔ.
Yɛ naagbee lɛ, wɔtao bɔ ni ehe hiaa ni peroxidase-ni haa wala yibii lɛ ahi shi yɛ PNs ni ena OXPHOS nitsumɔ ni ejaaa lɛ mli lɛ mli. Wɔfee mCherry ni kɛ AAV-shRNA (hairpin RNA kuku) ni tsɔɔ ŋmei PCx mRNA (AAV-shPCx) lɛ tsuɔ nii tɛ̃ɛ, ni wɔkɛ hela lɛ loo e-scrambled control (AAV-scr) lɛ wo Mfn2cKO ŋmei lɛ atsitsi mli. Akɛ tsofa lɛ wo mɔ lɛ mli yɛ otsi ni ji ejwɛ lɛ mli (Mfoniri 5A) koni anyɛ ana PCx knockdown ni tsuɔ nii jogbaŋŋ yɛ be mli ni PCx kpojiemɔ lɛ yaa hiɛ (Mfoniri 3C) ni PN cell layer lɛ kã he ehi shi lolo lɛ (Mfoniri 1A). E sa nɛ wa kai kaa PCx (Mfoniri S8A) nɛ a maa ngɔ lɛ ɔ haa nɛ PN gbenɔ ɔ yaa oya wawɛɛ, nɛ lɔ ɔ ji nɔ́ pɛ nɛ a tsiɔ tsɛŋemɔ hela ɔ he (Mfoniri 5, B kɛ C). Bɔni afee ni wɔnu bɔ ni PCx ŋwɛi-mla lɛ tsirɛɔ lɛ lɛ shishi lɛ, wɔkase PNs lɛ redox shihilɛ lɛ yɛ PCx gbee shi lɛ sɛɛ, ni AAV-mediated optical biosensor Grx1-roGFP2 lɛ jie ehe kpo yɛ be kome mli (Mfoniri S8, B kɛyashi D) koni wɔkɛpɛi glutathione ni ji potentix3 lɛ mli. No sɛɛ lɛ, wɔfee enyɔ-foton fluorescence wala be mli mfonirifeemɔ bibioo (FLIM) yɛ jwɛŋmɔŋ fãi ni mli wa ni ji Mfn2cKO ni eye otsii 7 loo littermates ni kudɔɔ lɛ mli koni wɔna tsakemɔi ni baanyɛ aba yɛ cytoplasmic redox shihilɛ mli yɛ FLIM shihilɛi lɛ amlipɛimɔ sɛɛ (Mfoniri S8, E kɛyashi G). Nibii amlipɛimɔ lɛ tsɔɔ akɛ, Mfn2cKO PNs kome ni bɛ PCx kpojiemɔ lɛ mli oksideshin shihilɛ lɛ etee hiɛ waa, ni esoro no kwraa yɛ tsɔnei ni kudɔɔ tsɔnei ni haa mɔ tsui nyɔɔ emli loo Mfn2cKO PNs ni jieɔ shRNA ni atswa pɛ kpo lɛ he (Mfoniri 5, D kɛ E). Beni PCx kpojiemɔ lɛ ba shi lɛ, Mfn2cKO PNs ni tsɔɔ shihilɛ ni mli wa waa lɛ mli oha mlijaa lɛ tee hiɛ fe shii etɛ (Mfoniri 5E), ni tsɔɔ akɛ PCx nɔyaa lɛ haa redox nyɛmɔ ni yɔɔ tsitsii ni efite lɛ amli lɛ hiɔ shi.
(A) Gbɛjianɔtoo ko ni tsɔɔ kaa gbɛjianɔtoo ni akɛbaatsu nii kɛwo AAV ni kɛ shPCx woɔ mli lɛ mli beni atsɔɔ gbɛ ni atsɔɔ nɔ atsakeɔ nii lɛ mli lɛ. (B) Najiaŋdamɔlɔi ni damɔ shi kɛha otsii 8 cerebellar fãi ni yɔɔ Mfn2cKO ŋmeii amli ni atsake ni akɛ anti-calcineurin antibody wo nɔ yɛ otsii 4 mli. Skel tso, 450μm. (C) Purkinje wala yibii lɛ ayibɔ ni yɔɔ AAV-ni atsɔɔ mli lɛ mli (gbɛ kome ni akɛpɛiɔ srɔtofeemɔ mli; n = 3 kɛyashi 4 mice). Ajieɔ saji lɛ akpo akɛ mean±SEM; ***p<0.001. (D) Najiaŋdamɔlɔ FLIM mfoniri tsɔɔ wala be ni PN ni eye otsii kpawo lɛ jieɔ glutathione redox sensor Grx1-roGFP2 kpo yɛ shikpamɔ shihilɛi ni atsɔɔ lɛ shishi. LUT (kwɛmɔ okpɔlɔ) mlijaa: wala be teŋ (yɛ picoseconds mli). Skel bar, 25μm. (E) Histogram lɛ tsɔɔ bɔ ni Grx1-roGFP2 wala be mli sɛɛnamɔi lɛ mlijaa jɛ (D) (n=158 kɛyashi 368 tsɛŋemɔ helai enyɔ mli yɛ shihilɛ fɛɛ shihilɛ shishi). Pie chart ni yɔɔ histogram fɛɛ histogram nɔ lɛ: tsɔɔ wala yibii ayibɔ ni yɔɔ wala be mli jara ni sɛɛ tsɛɔ waa (tsuru, oxidized) loo kuku (bluu, eba shi), ni feɔ 1 SD ni ji wala be mli jara ni yɔɔ CTRL-AAV-scr lɛ mli lɛ. (F) Nɔkwɛmɔnɔ ni atsɔɔ lɛ tsɔɔ bɔ ni PCx ni yɔɔ tsitsi lɛ nɔ lɛ buɔ mɔ he ehaa.
Yɛ fɛɛ mli lɛ, saji ni wɔkɛhaa yɛ biɛ lɛ tsɔɔ akɛ MFN2 lɛ kpojiemɔ ekoŋŋ lɛ baanyɛ ajie PN ni etee hiɛ lɛ kwraa kɛ OXPHOS ni faaa waa, mtDNA ni faaa waa, kɛ ista-like morphology ni bɛ mlɛo kwraa, ni no haa hiɛyaa yaa nɔ yɛ helai ni etee hiɛ po amli. Neurodegeneration haa odaseyeli ni anyɛɔ atsakeɔ yɛ shihilɛ ni yɔɔ dani wala yibii lɛ gboɔ lɛ he. Nɛkɛ bɔ ni gbɔmɔtso lɛ mli nibii ni haa mɔ tsui nyɔɔ emli lɛ tsakeɔ ehaa lɛ maa nɔ mi babaoo kɛtsɔ nyɛmɔ ni tsɔnei ni haa mɔ tsui nyɔɔ emli lɛ yɔɔ ni amɛkɛbaa atherosclerosis (TCA sɛɛkuu lɛ sɛɛkuu), ni tsiɔ PCx kpojiemɔ yɛ PNs ni bɛ OXPHOS lɛ mli lɛ naa ni ehaa wala yibii gboiɔ lɛ yaa hiɛ, ni no haa etsuɔ ​​hebuu gbɛfaŋnɔ ko he nii (Mfoniri 5F).
Yɛ nikasemɔ nɛɛ mli lɛ, wɔkɛ odaseyeli ha akɛ PNs lɛ hetoo ni amɛhaa OXPHOS nitsumɔ ni ejaaa lɛ ji ni amɛyashɛ TCA sɛɛkuu atherosclerosis he fiofio kɛtsɔ srɔtofeemɔ nitsumɔ gbɛ ni metabolic gbɛjianɔtoi lɛ tsuɔ he nii lɛ nɔ. Wɔkɛ gbɛi babaoo ni yeɔ ebuaa lɛ ma proteomic mlipɛimɔ lɛ nɔ mi ni wɔjie lɛ kpo akɛ, kɛ́ mitochondrial nitsumɔ ni naa wa waa lɛ tsi enaa lɛ, tsɔnei ni haa mɔ tsui nyɔɔ emli lɛ yɛ bɔ ni tsɔne lɛ mli nibii ni haa mɔ tsui nyɔɔ emli lɛ tsuɔ nii ni aleee tsutsu lɛ. Nɔ ni haa wɔnaa kpɛɔ wɔhe ji akɛ, rewiring gbɛjianɔtoo lɛ fɛɛ ekadiii naagbee metabolic shihilɛ ni fataa neurodegeneration he fiofio ni anyɛɛɛ aku sɛɛ aba lɛ, shi wɔ saji lɛ tsɔɔ akɛ ebaanyɛ efee neuron ni kwɛɔ nɔ yɛ be mli ni wala yibii lɛ gboɔ lɛ po mli. Nɛkɛ niiamlipɛimɔ nɛɛ tsɔɔ akɛ, tsɔnei ni haa mɔ tsui nyɔɔ emli lɛ yɛ tsɔne ni haa mɔ he jɔɔ lɛ babaoo yɛ gbɔmɔtso lɛ mli. Nɛkɛ anɔkwale nɛɛ maa nɔ mi akɛ, sɛɛ mli lɛ, MFN2 ni akɛba ekoŋŋ lɛ baanyɛ aku sɛɛ aha nibii ni he hiaa ni haa mɔ he jɔɔ lɛ ajieɔ amɛhe kpo, ni etsĩ PN fitemɔ naa. Nɔ ni tamɔɔɔ enɛ lɛ, etsĩɔ lá ni hoɔ yɛ gbɔmɔtso lɛ mli lɛ naa, ni ehaa tsitsii lɛ yaa oya. hii kɛ yei ni kɛ amɛhe feɔ yakayaka nii.
Nɔ kome ni yɔɔ miishɛɛ waa ni wɔna yɛ wɔ niiamlitaomɔ lɛ mli ji akɛ, PNs ni bɛ OXPHOS lɛ baanyɛ atsake TCA sɛɛkuu lɛ mli nibii ni tsakeɔ lɛ kɛtsɔ enzymes ni haa lá tsɔɔ mli lɛ nɔ. Metabolic rearrangement ji nɔ ko ni afɔɔ naa yɛ kansa mli wala yibii lɛ amli, ni ekomɛi kɛ amɛhe fɔ̃ɔ glutamine nɔ koni amɛkɛfata TCA sɛɛkuu mli nibii ni baa shi lɛ ahe koni amɛfee nɔ ni tamɔ nakai ni baa shi, ni tsirɛɔ mumɔ mli tsɔne lɛ ni ekwɛɔ lipid kɛ nucleotide biosynthesis precursors lɛ afeemɔ nɔ (39 , 40). Nikasemɔ ko ni afee nyɛsɛɛ nɛɛ tsɔɔ akɛ yɛ fãji ni yɔɔ sɛɛgbɛ ni kɛ OXPHOS tsuuu nii jogbaŋŋ lɛ amli lɛ, glutamine/glutamate metabolism lɛ ni akɛtsakeɔ amɛhe lɛ hu ji nɔ ko ni he hiaa waa (5, 41), he ni gbɛ ni glutamine lɛ tsɔɔ nɔ eboteɔ TCA sɛɛkuu lɛ mli lɛ damɔ OXPHOS pilamɔ lɛ mli wa ha lɛ nɔ (41). ). Shi kɛlɛ, odaseyeli ni yɔɔ faŋŋ bɛ ni tsɔɔ bɔ ni tsɔne ni haa mɔ tsui nyɔɔ emli lɛ mli nibii ni haa mɔ tsui nyɔɔ emli lɛ yeɔ egbɔ yɛ gbɔmɔtso lɛ mli kɛ bɔ ni ebaanyɛ ekɛ ehe awo hela lɛ mli lɛ he. Yɛ nyɛsɛɛ nɛɛ mli nikasemɔ ko ni afee yɛ vitro mli lɛ, atsɔɔ akɛ shishijee cortical neurons lɛ buaa glutamate pools lɛ anaa kɛha tsɔne ni haa mɔ tsui nyɔɔ emli lɛ, ni no haa tsɔne ni haa mɔ he jɔɔ lɛ kɛ tsɔne ni haa mɔ he jɔ̃ɔ ehe lɛ yaa hiɛ yɛ tsɔne ni haa mɔ tsui nyɔɔ emli lɛ mli (42). Ehe miihia ni wɔkadi akɛ, yɛ tsofafeemɔ mli nibii ni tsiɔ TCA sɛɛkuu lɛ naa lɛ shishi lɛ, aheɔ ayeɔ akɛ pyruvate carboxylation lɛ haa oxaloacetate lɛ feɔ ekome yɛ cerebellar granule neurons ni akwɛɔ nɔ lɛ mli (34). Shi kɛlɛ, bɔ ni nɛkɛ gbɛjianɔtoi nɛɛ kɔɔ jwɛŋmɔŋ fãji ahe (he ni aheɔ ayeɔ akɛ atherosclerosis lɛ ji astrocytes lɛ pɛ nɔ titri) lɛ yɛ gbɔmɔtsoŋ shishinumɔ ni he hiaa lolo (43). Yɛ shihilɛ nɛɛ mli lɛ, wɔ saji lɛ tsɔɔ akɛ PNs ni OXPHOS efite yɛ gbɔmɔtso lɛ mli lɛ baanyɛ atsake kɛya BCAA fitemɔ kɛ pyruvate carboxylation, ni ji jɛɛhei titrii enyɔ ni akɛfataa TCA pool mli nibii lɛ ahe lɛ nɔ. Eyɛ mli akɛ awie yelikɛbuamɔ ni asusuɔ akɛ BCAA catabolism kɛhaa neuronal hewalɛ tsakemɔ lɛ he moŋ, shi kɛfata glutamate kɛ GABA nitsumɔ kɛha neurotransmission (44) lɛ he lɛ, odaseyeli ko bɛ lolo kɛha nɛkɛ gbɛi nɛɛ yɛ vivo mli. No hewɔ lɛ, ewaaa akɛ aaasusu akɛ PNs ni tsuuu nii jogbaŋŋ lɛ baanyɛ awo TCA teŋgbɛ nibii ni assimilation gbɛjianɔtoo lɛ tsirɛɔ lɛ lɛ ni ayeɔ lɛ he nyɔmɔ kɛtsɔ atherosclerosis ni yaa hiɛ lɛ nɔ. Yɛ gbɛ titri ko nɔ lɛ, ekolɛ ehe baahia ni akwɛ PCx lɛ nɔ koni akɛkwɛ aspartic acid ni ataoɔ lɛ nɔ, ni ajie lɛ kpo yɛ wala yibii ni yaa hiɛ ni mitochondrial tsuuu nii jogbaŋŋ lɛ amli (45). Shi kɛlɛ, wɔ metabolomics mlipɛimɔ lɛ ejieee tsakemɔ ko ni he hiaa yɛ aspartic acid ni yɔɔ Mfn2cKO PNs (Mfoniri S6A) lɛ mli lɛ kpo, ni eeenyɛ efee akɛ etsɔɔ bɔ ni aspartic acid lɛ tsuɔ nii ehaa yɛ wala yibii ni yaa hiɛ kɛ post-mitotic neurons lɛ ateŋ. Eyɛ mli akɛ gbɛ pɔtɛɛ ni PCx upregulation lɛ tsuɔ nii yɛ dysfunctional neurons mli yɛ vivo mli lɛ ji nɔ ni esa akɛ atsɔɔ mli moŋ, shi wɔtsɔɔ akɛ nɛkɛ hetoo ni baa oya nɛɛ tsuɔ gbɛfaŋnɔ ko ni he hiaa he nii yɛ redox shihilɛ ni yɔɔ neurons lɛ mli lɛ mlihilɛ mli, ni ajie lɛ kpo yɛ FLIM shikpamɔi ni afee yɛ cerebellar slices lɛ anɔ lɛ mli. Yɛ gbɛ titri ko nɔ lɛ, PNs ni atsiɔ naa kɛjɛɔ PCx ni kudɔɔ nii lɛ nɔ lɛ baanyɛ aha shihilɛ ni mli wa aba ni eha wala yibii lɛ agbo oya. BCAA fitemɔ lɛ nitsumɔ kɛ pyruvate lɛ carboxylation lɛ jeee gbɛi ni atsɔɔ nɔ ajieɔ fãji ni yɔɔ sɛɛgbɛ ni haa mɔ tsui nyɔɔ emli lɛ kpo (7). No hewɔ lɛ, etamɔ nɔ ni amɛji nɔ ko ni he hiaa fe fɛɛ yɛ OXPHOS-deficient neurons lɛ amli, kɛji akɛ jeee amɛ pɛ po, ni he hiaa kɛha tsitsii ni fiteɔ lɛ. .
Cerebellar hela ji hela ko ni haa mɔ tsui nyɔɔ emli ni bei pii lɛ ejieɔ ehe kpo akɛ ataxia ni bei pii lɛ efiteɔ PNs (46). Nɛkɛ tsɔne ni haa mɔ tsui nyɔɔ emli nɛɛ ji nɔ ni yɔɔ oshara waa kɛha mitochondrial nitsumɔ ni ejaaa ejaakɛ amɛhalamɔ ni fiteɔ yɛ ŋmeii amli lɛ fa bɔ ni sa ni ekɛ tsɔne mli okadii babaoo ni jieɔ gbɔmɔ spinocerebellar ataxia kpo lɛ baa ekoŋŋ (16, 47, 48). Taakɛ amaniɛbɔi tsɔɔ lɛ, tsɛŋemɔ hela ko ni akɛtsakeɔ tsɛŋemɔ hela ni haa mɔ he jɔ̃ɔ ehe lɛ kɛ gbɔmɔ spinocerebellar ataxia lɛ yɔɔ tsakpaa ni eyɛ mitochondrial nitsumɔ ni ehiii (49, 50), ni emaa bɔ ni ehe hiaa ni akase nɔ ni jɛɔ OXPHOS ni faaa yɛ PNPH mli lɛ mli baa lɛ nɔ mi. No hewɔ lɛ, ehi jogbaŋŋ akɛ abaajie nɛkɛ tsɔnei ni haa mɔ tsui nyɔɔ emli nɛɛ ayibɔ ni yɔɔ srɔto nɛɛ kɛjɛ mli jogbaŋŋ ni akase amɛ. Shi, akɛni PNs lɛ nuɔ nɔnyɛɛ he waa ni amɛhaa cerebellar cell muu lɛ fɛɛ mlijaa bibioo ko hewɔ lɛ, kɛha nikasemɔi babaoo ni damɔ omics nɔ lɛ, amɛmligbalamɔ ni ahala akɛ cells muu lɛ ji nɔ ko ni wa lolo. Eyɛ mli akɛ enyɛŋ eba akɛ aaaná mujiwoo ni bɛ kwraa yɛ wala yibii krokomɛi ahe (titri lɛ, fãji ni eda lɛ), shi wɔkɛ FACS fata he koni wɔnine ashɛ tsɔnei ni haa mɔ tsui nyɔɔ emli lɛ ayibɔ ni fa bɔ ni sa kɛha proteomics mlipɛimɔ ni yɔɔ shishigbɛ lɛ nɔ, ni wɔyɛ protein ni fa babaoo ni akɛtoɔ cerxibel the data (51). Kɛtsɔ wala yibii muu lɛ fɛɛ ni wɔɔhi shi lɛ nɔ lɛ, gbɛ ni wɔtsɔɔ nɔ wɔkɛhaa yɛ biɛ lɛ haaa wɔkwɛ tsakemɔi ni baa yɛ wala yibii lɛ amli lɛ pɛ, shi moŋ wɔkwɛɔ tsakemɔi ni baa yɛ e-cytoplasmic compounds lɛ amli lɛ hu, ni no yeɔ ebuaa mitochondrial membrane label enrichment pɔtɛɛ ni kɔɔ wala yibii lɛ ahe lɛ he. (52 kɛ 53). Gbɛ ni wɔtsɔɔ mli lɛ kɔɔɔ Purkinje wala yibii lɛ ahe nikasemɔ pɛ he, shi moŋ abaanyɛ akɛtsu nii yɛ wala yibii srɔtoi fɛɛ amli yɛ gbɛ ni waaa nɔ koni akɛtsu tsakemɔi ni baa yɛ jwɛŋmɔ ni hela mɔ amɛ lɛ mli lɛ ahe nii, ni nɔ ni fata he ji mitochondrial dysfunction he nɔkwɛmɔnii krokomɛi.
Yɛ naagbee lɛ, wɔna tsofafeemɔ samflɛ ko yɛ nɛkɛ nibii ni haa mɔ he jɔɔ lɛ ahe gbɛjianɔtoo nɛɛ mli ni baanyɛ aku sɛɛ aha okadii titrii ni tsɔɔ akɛ tsɛŋemɔ hela lɛ mli fãji lɛ miigba enaa lɛ kwraa, ni etsĩ tsɛŋemɔ helai ahe nibii ni haa mɔ tsui nyɔɔ emli lɛ anaa. No hewɔ lɛ, shishinumɔ ni kɔɔ nitsumɔ ni kɔɔ rewiring ni atsɔɔ mli yɛ biɛ lɛ he lɛ baanyɛ aha shishitoo mlai ni kɔɔ helatsamɔi ni baanyɛ aba koni akɛkwɛ tsɔne ni haa mɔ tsui nyɔɔ emli lɛ nɔ yɛ be mli ni mitochondrial tsuuu nii jogbaŋŋ lɛ he. Wɔsɛɛ be mli niiamlitaomɔ ni yiŋtoo ji ni akwɛ tsakemɔi ni baa hewalɛ ni tsakeɔ yɛ jwɛŋmɔŋ wala yibii krokomɛi amli lɛ mli lɛ he miihia koni ajie bɔ ni shishitoo mla nɛɛ tsuɔ nii ahaa yɛ jwɛŋmɔŋ helai krokomɛi ahe lɛ kpo kwraa.
Atsɔɔ MitoPark ŋmeii lɛ amli momo (31). Atsɔɔ C57BL/6N ŋmeii ni loxP flanking Mfn2 ŋmeii lɛ yɔɔ mli lɛ amli momo (18) ni akɛ L7-Cre ŋmeii (23) efo mli. No sɛɛ lɛ, akɛ seshibii ni ji heterozygous enyɔ ni jɛ mli ba lɛ fo mli kɛ homozygous Mfn2loxP/Mfn2loxP ŋmeii koni akɛfee Purkinje-sɔɔmɔ tsɛŋemɔ hela ni ji Mfn2 (Mfn2loxP/Mfn2loxP; L7-cre) lɛ. Yɛ gbalashihilɛ mli hefatalɔi akuu bibioo ko mli lɛ, ajie Gt (ROSA26) SorStop-mito-YFP allele (stop-mtYFP) lɛ kpo kɛtsɔ krɔs krokomɛi ni akɛfata he lɛ nɔ (20). Afee kooloi ahe nifeemɔi fɛɛ yɛ Europa, maŋ kɛ nitsumɔhe gbɛtsɔɔmɔi anaa, ni LandesamtfürNatur ni yɔɔ Umwelt kɛ Verbraucherschutz, North Rhine-Westphalia, Germany lɛ kpɛlɛ nɔ. Koolooi ahe nitsumɔ hu nyiɛɔ gbɛtsɔɔmɔ ni European Laboratory Koolooi Ahe Saji Akui lɛ kɛhaa lɛ sɛɛ.
Beni awo yoo ni hiɛ musu lɛ fɔmɔ kotoku lɛ mli tsofa ni haa mɔ he jɔɔ lɛ sɛɛ lɛ, ajieɔ tsina fɔmɔ kotoku lɛ kɛjɛɔ mli (E13). Akɛ 10 mM Hepes fata he lɛ mli, ni akɛwo Dulbecco Modified Eagle Medium ni papain (20 U/ml) kɛ cysteine ​​(1μg/ml) yɔɔ mli lɛ mli. Akɛ fãji lɛ awo DMEM mli) ni ajie lɛ kɛtsɔ enzymatic digestion nɔ. Ml) yɛ 37°C mli minitii 20, ni no sɛɛ lɛ, akɛ tsɔne shwieɔ DMEM ni akɛ 10% tsina fɔmɔ kotoku mli lá fataa he lɛ mli. Akɛ tsɛŋemɔ helai wo glase coverslips ni akɛ polylysine ehà nɔ lɛ nɔ yɛ 2×106 yɛ 6 cm kusum niyenii mli loo yɛ 0.5×105 tsɛŋemɔ helai/cm2 mli kɛha mfonirifeemɔ mlipɛimɔ. Yɛ ŋmɛlɛtswai 4 sɛɛ lɛ, akɛ tsofa ni ji Neurobasal ni lá bɛ mli ni 1% B27 ni akɛfataa he kɛ 0.5 mM GlutaMax yɔɔ mli lɛ tsake tsofa lɛ. No sɛɛ lɛ, akwɛ tsɔnei ni haa mɔ tsui nyɔɔ emli lɛ anɔ yɛ 37°C kɛ 5% CO2 mli yɛ kaa lɛ mli fɛɛ, ni aha amɛ niyenii shikome yɛ otsi mli. Bɔni afee ni afee nɔ ko ni akɛfee ekome ekoŋŋ yɛ vitro mli lɛ, akɛ 3μl (24-well culture dish) loo 0.5μl (24-well plate) ni ji AAV9 hela ni nyiɛ sɛɛ nɛɛ tsu nii kɛtsa tsɔnei ni haa mɔ tsui nyɔɔ emli lɛ yɛ gbi ni ji enyɔ lɛ nɔ yɛ vitro mli: AAV9.CMV.PI.eGFP. 105530-AAV9) kɛ AAV9.CMV.HI.eGFP-Cre.WPRE.SV40 (Addgene, wolo lɛ yibɔ 105545-AAV9).
Akɛ V5 sɛɛkuu (GKPIPNPLLGLDST) kadiɔ tsina Mfn1 kɛ Mfn2 ni yeɔ ebuaa DNA (ni anáa kɛjɛɔ Addgene plasmid #23212 kɛ #23213, yɛ amɛteŋ lɛ) yɛ C-naagbee lɛ, ni akɛ mCherry ebɔ he yɛ freim mli kɛtsɔɔ T2A sɛɛkuu lɛ nɔ. Grx1-roGFP2 ji nikeenii ni jɛ Heidelberg TP Dick DFKZ (Glemanbii anitsumɔhe lɛ). Kɛtsɔ tdTomato kasɛt lɛ ni akɛto gbɛi ni atsɔɔ nɔ afee nɔ ko lɛ nɔ lɛ, ajie kasɛt lɛ awo pAAV-CAG-FLEX-tdTomato sɛɛkuu lɛ mli (Addgene reference yibɔ 28306) koni ekɛfee pAAV-CAG-FLEX-mCherry-T2A-MFGAV2-V5, pAAV-CAG-FLEX-mCherry-T2A-MFNGAV2-V5, Flɛks-mCɛri-T2A-MFN1-V5 kɛ pAAV-CAG-Flɛks-Grx-roGFP2 tsɔnei. Akɛ gbɛ ni tamɔ nakai nɔŋŋ tsu nii ni akɛfee pAAV-CAG-FLEX-mCherry ni kudɔɔ tsɔne lɛ. Bɔni afee ni afee AAV-shPCx lɛ, ehe miihia ni afee plasmid AAV tsɔne (VectorBuilder, pAAV [shRNA] -CMV-mCherry-U6-mPcx- [shRNA#1]), ni DNA sɛɛkuu ni tsɔɔ shRNA ni tsɔɔ ŋmei PCx (5′GTAGCTAGGTGTGTGACTAACTA) lɛ yɔɔ mli. 3′) Yɛ U6 sɛɛnyiɛlɔ lɛ kudɔmɔ shishi lɛ, akɛ mCherry tsuɔ nii yɛ CMV sɛɛnyiɛlɔ lɛ kudɔmɔ shishi. Afee AAV vectors ni yeɔ ebuaa lɛ yɛ bɔ ni mɔ ni fee lɛ tsɔɔ lɛ naa (Cell Biolabs). Yɛ kukufoo mli lɛ, kɛ plasmid ni akɛyaa he kroko ni hiɛ mCherry-T2A-MFN2-V5 (pAAV-CAG-FLEX-mCherry-MFN2-V5), mCherry-T2A-MFN1-V5 (pAAV-CAG-FLEX-mCherry) lɛ atsu nii be kukuoo mli. (pAAV-CAG-FLEX-mCherry) loo Grx-roGFP2 (pAAV-CAG-FLEX-Grx-roGFP2) ni haa mɔ he jɔɔ lɛ, kɛ agbɛnɛ hu, AAV1 capsid protein kɛ nibii ni fata he lɛ ahe kood ni akɛwoɔ plasmid plasmid mli, kɛtsɔ calcium phosphate gbɛ nɔ. Akɛ tsɛŋemɔ hela ni haa mɔ he jɔ̃ɔ ehe lɛ tsu nii kɛtsɔ ais ni egbi/ethanol hejuu mli kɛ lysed cells ni yɔɔ phosphate buffered saline (PBS) mli lɛ nɔ. Akɛ AAV vector lɛ tsu nii kɛtsɔ iodixanol ni sɛɛ tsɛɔ ni ajieɔ kɛjɛɔ mli lɛ nɔ (ŋmɛlɛtswai 24 yɛ 32,000 rpm kɛ 4°C) ni akɛ Amicon ultra-15 tsɔne ni haa hulutsoo lɛ tsu nii ni akɛwo mli. 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 lɛ falɛ ji nɔ ni asusu he yɛ be ni eho lɛ mli lɛ mli. 1013 GC/ml) kɛ AAV1-CAG-FLEX-Grx-roGFP2 (8.9×1012 GC/ml).
100 / 0.5% sodium deoxycholate/PBS lysis buffer ni phosphatase kɛ protease inhibitor (Roche) yɔɔ mli lɛ ji nɔ ni ajie shishijee tsɔnei ni haa mɔ tsui nyɔɔ emli lɛ. Akɛ tsofa ni ji bicinchoninic acid assay (Thermo Fisher Scientific) lɛ tsu nii kɛtao niyenii ni haa mɔ he waa lɛ mli. No sɛɛ lɛ, akɛ SDS-polyacrylamide gel electrophoresis gbala protein lɛ mli, ni no sɛɛ lɛ akɛwo polyvinylidene fluoride membrane (GE Healthcare) nɔ. Blɔ hei ni jeee pɔtɛɛ ni okɛ shishijee tsofa ni gbeɔ hela lɛ awo mli (kwɛmɔ Table S1 kɛha mlitsɔɔmɔ) yɛ 5% mliki mli yɛ TBST (Tris-buffered saline kɛ Tween), tsɔnei ni akɛfɔɔ he kɛ tsofa ni gbeɔ hela ni ji enyɔ lɛ mli yɛ TBST Incubate mli. Kɛ tsofa ni gbeɔ hela lɛ awo mli nyɔɔŋ kome yɛ +4°C. Kɛji akɛ afɔ he lɛ, okɛ tsofa ni gbeɔ hela ni ji enyɔ lɛ awo mli ŋmɛlɛtswai 2 yɛ shĩa mli latsaa mli. No sɛɛ lɛ, kɛtsɔ tsofa ko ni gbeɔ hela ni ji anti-β-actin lɛ ni akɛwoɔ mli lɛ nɔ lɛ, ama nɔ mi akɛ tsɔne lɛ nɔŋŋ ji nɔ ni akɛwoɔ mli lɛ. Nibii ni ana kɛtsɔ tsakemɔ kɛmiiya tsofa ni haa hulu kpɛɔ kɛ tsofa ni haa hulu kpɛɔ lɛ nɔyaa nɔ (GE Healthcare).
Akɛ 4% paraformaldehyde (PFA)/PBS fĩ tsɔnei ni haa mɔ tsui nyɔɔ emli ni awo tsutsu lɛ awo glase nɔ lɛ mli yɛ be pɔtɛɛ ni ato lɛ mli yɛ tsu mli dɔlɛ mli minitii 10. Akɛ 0.1% Triton X-100/PBS woɔ tsɔne lɛ mli klɛŋklɛŋ minitii enumɔ yɛ tsu mli latsaa mli, ni no sɛɛ lɛ akɛwoɔ tsɔne ni tsiɔ nii anaa lɛ mli [3% tsina lá mli tsofa (BSA)/PBS]. Yɛ gbi ni ji enyɔ lɛ nɔ lɛ, akɛ tsofa ni tsiɔ nii anaa lɛ tsuɔ coverslips lɛ ahe, ni akɛ fluorophore-conjugated secondary antibody ni sa lɛ wo mli ŋmɛlɛtswai 2 yɛ tsu mli latsaa mli; Yɛ naagbee lɛ, afɔ nɔkwɛmɔnii lɛ ahe jogbaŋŋ yɛ PBS mli kɛ 4′,6-diamidino-2 -Phenylindole (DAPI) ni akɛwoɔ mli lɛ mli, ni no sɛɛ lɛ akɛ Aqua-Poly/Mount fĩɔ tsɔne bibioo ni akɛkwɛɔ nii lɛ nɔ.
130 mg/kg) kɛ xylazine (10 mg/kg) ni akɛwoɔ amɛmli lɛ nɔ, ni akɛ tsofa ni haa mɔ he jɔ̃ɔ ehe ni ji carprofen (5 mg/kg) wo amɛhewolo shishi, ni akɛwo tsɔne ko ni haa mɔ he jɔ̃ɔ ehe (Kopd a pamf) mli. Ajie tsitsi lɛ kpo ni akɛ tsɔne ni akɛfoɔ ŋmeii lɛ atsu nii ni akɛfo cerebellar cortex lɛ fã ni kɛ mis wu lɛ kpãa gbee lɛ mli (kɛjɛ lambda: tsitsi 1.8, lateral 1, ni kɛ lobules IV kɛ V lɛ kpãa gbee lɛ mli). Kɛ tsɔne ni akɛwoɔ mɔ mli ni ebɔle ehe lɛ afee bu bibioo ko yɛ tsitsi lɛ mli jogbaŋŋ koni akafite lá ni hoɔ yɛ shishigbɛ lɛ. No sɛɛ lɛ, akɛ glase ni ajie lɛ kpo lɛ woɔ tsɔne bibioo lɛ mli fiofio (kɛjɛ -1.3 kɛyashi -1 yɛ dura mater lɛ sɛɛgbɛ), ni akɛ 200 kɛyashi 300 nl AAV woɔ tsɔne bibioo ni akɛwoɔ tsɔne mli lɛ mli (Narishige) kɛ tsɔne ni akɛ niji tsuɔ nii (Narishige tisure) yɛ be mli ni shɛɔ 100 ml. minitii samflɛ. Kɛ́ okɛ tsofa lɛ wo mli ogbe naa lɛ, okɛ kapilɛri lɛ awo mli minitii 10 kroko koni hela lɛ agbɛ ashwã kwraa. Beni ajie lá fãji lɛ sɛɛ lɛ, akpɛɔ hewolo lɛ jogbaŋŋ koni pilamɔi lɛ amli fuumɔ aba shi ni eha kooloo lɛ aná hewalɛ. Akɛ tsofai ni haa mɔ he jɔ̃ɔ ehe (caspofen) tsu koolooi lɛ ahe nii gbii babaoo yɛ opireshɛn lɛ sɛɛ, ni yɛ nakai beaŋ lɛ, akwɛ amɛgbɔmɔtsoŋ shihilɛ lɛ jogbaŋŋ, ni no sɛɛ lɛ agbe amɛ yɛ be ni ato lɛ mli. Afee gbɛjianɔtoi lɛ fɛɛ yɛ Europa, maŋ kɛ nitsumɔhe gbɛtsɔɔmɔi anaa, ni LandesamtfürNatur ni yɔɔ Umwelt kɛ Verbraucherschutz, North Rhine-Westphalia, Germany lɛ kpɛlɛ nɔ.
Akɛ tsofa ni ji ketamine (100 mg/kg) kɛ xylazine (10 mg/kg) wo kooloi lɛ amli, ni akɛ 0.1 M PBS wo tsui lɛ mli klɛŋklɛŋ, ni no sɛɛ lɛ akɛ 4% PFA wo PBS mli. Afo fãji lɛ amli ni akɛwo 4% PFA/PBS mli nyɔɔŋ kome yɛ 4°C. Akɛ tsɔne ko ni hosoɔ (Leica Microsystems GmbH, Vienna, Austria) tsu nii ni akɛsaa fãi ni yɔɔ tsitsi ni yɔɔ PBS mli lɛ mli (ni kɛlɛ ji 50 μm) kɛjɛ jwɛŋmɔ ni yɔɔ shiŋŋ lɛ mli. Kɛji akɛ atsɔɔɔ gbɛ kroko nɔ lɛ, afee fãi ni fɔ̃ɔ amɛhe lɛ ahe muji tamɔ bɔ ni atsɔɔ mli yɛ yiteŋgbɛ lɛ (13) yɛ tsu mli latsaa kɛ ŋmɔlɔ mli. Yɛ kukufoo mli lɛ, klɛŋklɛŋ lɛ, akɛ 0.5% Triton X-100/PBS wo ŋmeii ni ana lɛ amli minitii 15 yɛ shĩa mli latsaa mli; kɛha epitopes komɛi (Pcx kɛ Shmt2), kɛtsɔ tris-EDTA buffer ni yɔɔ 80°C (PH 9) mli lɛ nɔ lɛ, shãa fãi lɛ minitii 25 yɛ nɛkɛ gbɛ nɛɛ najiaŋ. No sɛɛ lɛ, akɛ fãi lɛ wo shishijee tsofa ni gbeɔ hela (kwɛmɔ Table S1) mli yɛ buffer ni tsiɔ nii anaa (3% BSA/PBS) mli yɛ 4°C mli gbɛkɛ muu fɛɛ kɛ ŋmɔlɔ. Enɔ jetsɛremɔ lɛ, akɛ tsofa ni tsiɔ nii anaa lɛ tsuɔ fãi lɛ ahe nii, ni akɛ tsofa ni sa ni ji fluorophore-conjugated secondary antibody lɛ wo mli ŋmɛlɛtswai 2 yɛ tsu mli latsaa mli; yɛ naagbee lɛ, afɔ fãi lɛ ahe jogbaŋŋ yɛ PBS mli, ni akɛ DAPI wo mli, ni no sɛɛ lɛ akɛ AquaPolymount On a microscope slide fiɔ he.
Akɛ laser ni akɛkwɛɔ nibii bibii ni yɔɔ ŋwɛi lɛ amli lɛ tsu nii ni akɛfee nɔkwɛmɔnɔ lɛ he mfoniri. Kɛtsɔ fluorophore lɛ miishɛɛ kɛ okadi lɛ naa kɛ Hybrid Detector (HyDs) nɔ lɛ, akɛ LAS-X tsɔne lɛ tsu nii ni akɛbua mfonirii ni ato he gbɛjianɔ ni kɛ Nyquist nɔkwɛmɔnɔ lɛ kpãa gbee lɛ anaa yɛ gbɛ ni nyiɛɔ sɛɛ nɔ: kɛha nɔ ni jeee nɔ ni falɛ yɔɔ mli lɛ, eji okadi ni yɔɔ hewalɛ waa (nɔkwɛmɔnɔ, yɛ gbɔmɔtso mli wala yibii kɛ numberites ni ji Hybrid) mt BrightR gbɛ nɔ). Akɛ Gating ni ji 0.3 kɛyashi 6 ns tsuɔ nii koni akɛba sɛɛgbɛ nibii shi.
Be-mli mfonirifeemɔ ni kɔɔ wala yibii ni ato he gbɛjianɔ lɛ ahe. Beni ajie lɛ kpo yɛ Neurobasal-A mli ni 1% B27 ni akɛfataa he kɛ 0.5 mM GlutaMax yɔɔ mli lɛ, amrɔ nɔŋŋ lɛ, adu wala yibii lɛ awo glase slide ni akɛ poly-l-lysine ewo mli lɛ nɔ (μ-Slide8 Jogbaŋŋ, Ibidi, katalog yibɔ 80826) , Ni no sɛɛ lɛ, aŋmɛ lɛ yɛ a1% CO2 lɛ nɔ. koni amɛhi shi. Akɛ mfonirifeemɔ ni yɔɔ be mli diɛŋtsɛ lɛ tsu nii yɛ Leica SP8 laser skanning confocal microscope ni akɛ laser yɛŋ, HyD, 63×[1.4 yibɔi ni ajieɔ lɛ kpo (NA)] mu ni akɛkwɛɔ nibii amli kɛ tsɔne ni haa hulu tsoɔ lɛ ewo mli lɛ nɔ.
Akɛ tsofa ni haa mɔ he jɔɔ lɛ wo tsina lɛ mli oya ni afo eyitso, ajie jwɛŋmɔ lɛ kɛjɛ eyitso lɛ mli oya, ni afo mli awo 200μm ŋmɔlɔ (kɛha 13C kadimɔ kaa) loo 275μm ŋmɔlɔ (kɛha foton shikpamɔi enyɔ) sagittal fã ni eyi kɛ nibii ni nyiɛ sɛɛ nɛɛ (VHr50). 125 mM ni mli ejɔ tamɔ ais, ni kɔɔyɔɔ ni mli eyi obɔ kɛ kɔɔyɔɔ (95% O2 kɛ 5% CO2) ni Ca2 ni faaa + ni ji jwɛŋmɔŋ nu ni akɛfee lɛ (ACSF) NaCl, 2.5 mM KCl, 1.25 mM sodium m2, 1.25 mM sodium pho2. glucose, 0.5 mM CaCl2 kɛ 3.5 mM MgCl2 (osmotic hewalɛ ni ji 310 kɛyashi 330 mmol). 125.0 mM NaCl, 2.5 mM KCl, 1.25 mM sodium phosphate buffer, 25.0 mM NaHCO3, 25.0 mM d-glucose. 7.4 kɛ 310 kɛyashi 320 mmol).
Yɛ mfonirifeemɔ nitsumɔ lɛ mli lɛ, ajie fãi lɛ kɛtee mfonirifeemɔ tsu ko ni ajɔɔ nɔ aha lɛ mli, ni afee kaa lɛ yɛ ACSF ni akɛwoɔ mli ni yaa nɔ lɛ shishi yɛ hulutsoo be ni ji 32° kɛyashi 33°C mli daa. Akɛ tsɔne ko ni akɛkwɛɔ nibii bibii ni yɔɔ ŋwɛi lɛ amli lɛ tsu nii kɛfee mfoniri ni akɛkwɛɔ nibii bibii ni yɔɔ ŋwɛi lɛ amli lɛ. FLIM nɔkwɛmɔnɔ (PikoHarp300, PikoKuant).
FLIM ni ji Grx1-roGFP2. Akɛ foton enyɔ FLIM ni yɔɔ sagittal jwɛŋmɔŋ fãi amli lɛ susu tsakemɔi ni baa PNs lɛ amli lɛ he, he ni Grx1-roGFP2 biosensor lɛ tsɔɔ PNs lɛ nɔ lɛ. Yɛ PN fã lɛ mli lɛ, ahalaa nibii ni anáa lɛ aaafee 50 kɛyashi 80 μm yɛ fã lɛ shishi koni ana nɔmimaa akɛ PN ni baanyɛ atsu nii (ni ji, ŋtayaai ahe gbɛjianɔtoo loo ŋtayaai ahe tsakemɔi ni bɛ dendrites lɛ ahe lɛ bɛ) kɛ ŋtayaai enyɔ ni ji roGFP2 sensor kɛ AAVx incee encoding sh mThɛri). Buamɔ mfoniri ni ato he kome ni akɛ 2x dijitaal zoom [miishɛɛ wavelength: 890 nm; 512 nm 512 piksɛli]. Nibii ni ana: mligbɛ HyD, fluorescein isothiocyanate (FITC) filter kuu] kɛ mfoniri ni akɛtoɔ minitii 2 kɛyashi 3 mli lɛ tsuɔ nii koni ana akɛ abua foton ni fa bɔ ni sa naa (foton 1000 fɛɛ) kɛha curve fitting. Akɛ Grx1-roGFP2 niiamlipɛimɔ lɛ henumɔi kɛ FLIM shihilɛi lɛ ahe odaseyeli lɛ tsu nii kɛtsɔ roGFP2 wala be mli sɛɛnamɔ lɛ nɔkwɛmɔ nɔ beni akɛ exogenous 10 mM H2O2 fataa ACSF ni akɛwoɔ nu mli lɛ he (koni ekɛha deoxidation aya hiɛ, ni no haa wala be mli baa lɛ yaa hiɛ), ni no sɛɛ lɛ akɛ mrethimithol of shishijee, ni no haa wala be lɛ baa shi) (Mfoniri S8, D kɛyashi G). Kɛ FLIMfit 5.1.1 tsɔne lɛ atsu nii ni okɛpɛi nɔ ni jɛ mli ba lɛ mli, ni okɛ mfoniri muu lɛ fɛɛ mli fitemɔ ni yɔɔ ŋwɛi lɛ mli lɛ ato IRF ni asusu lɛ he (nibii ni akɛtsuɔ nii lɛ ahetoo nitsumɔ), ni χ2 ji nɔ ni miihe ashɛ 1. ni akɛtsuɔ nii kɛtsɔɔ yibɔi amli.
Mitokondrial hewalɛ mlipɛimɔ. 100 nM TMRM ni akɛfata ACSF ni awo mli lɛ he tɛ̃ɛ minitii 30 sɛɛ lɛ, akɛ mitochondrial potential tsakemɔi ni yɔɔ PNs lɛ mli lɛ tsu nii kɛtsɔ foton enyɔ ni akɛkwɛɔ nibii bibii anɔ lɛ nɔ. Akɛ TMRM mfonirifeemɔ tsu nii kɛtsɔ nɔ ni akɛtaoɔ nii lɛ mli lɛ ni ajieɔ lɛ kpo yɛ 920 nm lɛ nɔ, ni akɛ HyD ni yɔɔ mli lɛ tsuɔ nii koni akɛbua okadii anaa; kɛtsɔ ŋwɛi ŋta ni haa mɔ tsui nyɔɔ emli lɛ nɔŋŋ ni akɛtsuɔ nii shi akɛ HyD ni yɔɔ mli ni yɔɔ srɔto (FITC :525/50) tsuɔ nii kɛfeɔ mtYFP mfoniri lɛ nɔ. Kɛ ImageJ mfoniri he akɔntaabuu tsɔne lɛ atsu nii ni okɛpɛi mitochondrial hewalɛ mli yɛ wala yibii kome mli. Yɛ kukufoo mli lɛ, akɛ plug-in equation: okadi = min (mtYFP, TMRM) lɛ tsuɔ nii koni akɛyoo mitochondrial kpokpaa ni tsɔɔ TMRM okadi lɛ yɛ Purkinje Somali mli yɛ mfoniri ni yɔɔ ŋwɛigbɛ ni kɛ lɛ kpãa gbee lɛ mli. No sɛɛ lɛ, asusuɔ pixel he ni yɔɔ mask ni jɛɔ mli baa lɛ mli lɛ he, ni no sɛɛ lɛ, afeɔ lɛ bɔ ni sa yɛ mtYFP gbɛ lɛ nɔ mfoniri kome ni kɛ lɛ kpãa gbee lɛ nɔ koni ana mitochondrial fã ni tsɔɔ mitochondrial nyɛmɔ lɛ.
Akɛ Huygens Pro (Jeŋ Nilee Mfonirifeemɔ) tsɔne lɛ jie mfoniri lɛ mli. Kɛha tile mfoniri ni ajie lɛ kpo lɛ, akɛ tsɔne ni akɛkpɛɔ nii ni LAS-X tsɔne lɛ kɛhaa lɛ tsuɔ nii kɛfeɔ tile kome pɛ ni akɛwoɔ mli lɛ. Kɛji akɛ okɛ mfoniri lɛ to he lɛ, okɛ ImageJ kɛ Adobe Photoshop atsu nii ni okɛtsu mfoniri lɛ he nii babaoo ni okɛtsake bɔ ni ekpɛɔ kɛ bɔ ni eyɔɔ srɔto ha lɛ yɛ gbɛ kome nɔ. Kɛ Adobe Illustrator atsu nii kɛha mfonirifeemɔ he saamɔ.
mtDNA hiɛdɔɔ mlipɛimɔ. Akɛ mtDNA helai lɛ ayibɔ to cerebellar fãi ni aŋmala nɔ nibii ni teɔ shi woɔ DNA lɛ ahe kɛtsɔ confocal microscope nɔ. Afee he fɛɛ he ni akɛmamɔ hiɛ lɛ aha wala yibii lɛ gbɔmɔtso lɛ kɛ wala yibii lɛ eko fɛɛ eko mli fã ni yɔɔ mli lɛ, ni akɛ Multi Measure plug-in (ImageJ software) lɛ bu he fɛɛ he lɛ he akɔntaa. Jiemɔ nuklea he lɛ kɛjɛ wala yibii lɛ gbɔmɔtso lɛ he lɛ mli koni ona cytoplasmic he lɛ. Yɛ naagbee lɛ, akɛ Analyze Particles plug-in (ImageJ software) lɛ tsu nii koni akɛtsɔɔ mtDNA ni yɔɔ threshold mfoniri lɛ nɔ lɛ mli lɛ, ni akɛ nɔ ni jɛ mli ba lɛ to PN mlijaa ni ji CTRL ŋmeii lɛ ahe. Ajieɔ nɔ ni jɛɔ mli baa lɛ kpo akɛ nucleosides ni yɔɔ wala yibii kome mli lɛ mlijaa mlijaa.
Protein kpojiemɔ mlipɛimɔ. Kɛ ImageJ mfoniri he akɔntaabuu plug-in lɛ atsu nii ni okɛpɛi protein kpojiemɔ yɛ PN mli yɛ wala yibii kome mli. Yɛ kukufoo mli lɛ, yɛ mfoniri ni yɔɔ ŋwɛigbɛ ni kɔɔ gbɛ ni kɛ lɛ kpãa gbee lɛ he lɛ mli lɛ, kɛtsɔ equation lɛ nɔ: okadi = min (mtYFP, antibody), ayɔseɔ mitochondrial kpokpaa ni tsɔɔ bɔ ni gbɔmɔtso lɛ mli hewalɛ ni gbeɔ helai komɛi ni yɔɔ Purkina lɛ tsuɔ nii eha lɛ. No sɛɛ lɛ, asusuɔ pixel he ni yɔɔ mask ni jɛɔ mli baa lɛ mli lɛ he, ni no sɛɛ lɛ, afeɔ lɛ bɔ ni sa yɛ mtYFP gbɛ lɛ nɔ mfoniri kome ni kɛ lɛ kpãa gbee lɛ nɔ koni ana mitochondrial fã ni ji protein ni ajieɔ lɛ kpo lɛ.
Purkinje wala yibii amlipɛimɔ. Akɛ ImageJ lɛ mli Cell Counter plug-in lɛ tsu nii ni akɛpɛi Purkinje mli tsɔne lɛ mli kɛtsɔ Purkinje mli wala yibii ni akane lɛ ayibɔ ni akɛ cerebellar ring ni wala yibii ni akane lɛ hiɔ mli lɛ mlijaa nɔ.
Nɔkwɛmɔnɔ he saamɔ kɛ nibii ni abua naa. Akɛ jwɛŋmɔi ni jɛ kuu ni kudɔɔ amɛ lɛ kɛ Mfn2cKO ŋmeii lɛ amli lɛ to 2% PFA/2.5% glutaraldehyde mli yɛ 0.1 M phosphate buffer (PB) mli, ni no sɛɛ lɛ, akɛ ciliates (Leica Mikrosysteme GmbH, Vienna, Austria) tsu nii kɛsaa coronal fãi lɛ. tetraoxide kɛ 1.5% ni akɛwoɔ ŋwɛi fɛ̃i mli ŋmɛlɛtswaa kome. Akɛ nu ni ajie mli nu lɛ tsu fãi lɛ ahe shii etɛ, ni no sɛɛ lɛ akɛ ethanol 70% ni uranyl acetate 1% yɔɔ mli lɛ wo mli minitii 20 sɔŋŋ. No sɛɛ lɛ, ajie nu kɛjɛ fãi lɛ amli yɛ dãa ni ato he gbɛjianɔ mli ni akɛwo Durcupan ACM (Araldite sũ mli sũ M) epoxy sũ (Electron Microscopy Sciences, wolokpo yibɔ 14040) mli yɛ glase ni akɛ silikon ehà nɔ lɛ ateŋ, ni naagbee lɛ yɛ 60°C Polymerize yɛ flɔnɔɔ mli. Ahala tsitsi lɛ mli fãi ni yɔɔ tsitsi lɛ mli lɛ ni afo 50 nm fãi ni yɔɔ mlɛo waa lɛ anɔ yɛ Leica Ultracut (Leica Mikrosysteme GmbH, Vienna, Austria) nɔ ni ajie yɛ 2×1 mm akɔɔble ŋta ni akɛ polystyrene sini ehà nɔ lɛ nɔ. Akɛ 4% uranyl acetate ni yɔɔ H2O mli lɛ mli nu lɛ wo fãi lɛ amli minitii 10, ni akɛ H2O tsu he shii abɔ, no sɛɛ lɛ akɛ Reynolds lead citrate ni yɔɔ H2O mli lɛ tsu nii minitii 10, ni no sɛɛ lɛ akɛ H2O tsu he shii abɔ. Akɛ TVIPS (Tietz Vidio kɛ Mfoniri He Nitsumɔ He Gbɛjianɔtoo) TemCam-F416 dijitaal kamera (TVIPS GmbH, Gauting, USA) tsu nii kɛfo mfoniri bibii. Germany).
Yɛ ŋmeii ni AAV emɔ amɛ lɛ agbɛfaŋ lɛ, aja jwɛŋmɔ lɛ mli ni afolɔ mli awo sagittal fã ni kɛlɛ ji 1 mm mli, ni akɛ fluorescence microscope tsu nii kɛkwɛ cerebellum lɛ mli koni ana AAV hela lɛ ring (ni ji, mCherry ni jieɔ ehe kpo). Shikpamɔi ni AAV ni akɛwoɔ mɔ mli lɛ haa Purkinje cell layer (ni ji nɔ ni miihe ashɛ layer muu lɛ fɛɛ) lɛ tsuɔ nii jogbaŋŋ waa yɛ kɛ hoo lɛ cerebellar rings enyɔ ni nyiɛɔ amɛsɛɛ lɛ amli lɛ pɛ ji nɔ ni akɛtsuɔ nii. Akɛ AAV-ni atsake lɛ mli fã bibioo ko tsu nii kɛha shwane mli sɛɛkuu (4% PFA kɛ 2.5% glutaraldehyde yɛ 0.1 M kookoo buffer mli) ni atsu he nii ekoŋŋ. Kɛha EPON ni akɛwoɔ mli lɛ, akɛ 0.1 M sodium kookoo buffer (Applichem) tsuɔ fãji ni yɔɔ shiŋŋ lɛ ahe nii, ni akɛ 2% OsO4 (os, Science Services; Caco) woɔ 0.1 M sodium kookoo buffer (Applichem) mli ŋmɛlɛtswai 4 mli, no sɛɛ lɛ afɔɔ he ŋmɛlɛtswai 2. Tĩi mli shii etɛ kɛ 0.1 M cocamide buffer. No sɛɛ lɛ, akɛ ethanol ni yaa ŋwɛi lɛ tsu nii ni akɛwo ethanol mli nu lɛ eko fɛɛ eko mli yɛ 4°C mli minitii 15 koni ekɛjie nu ni yɔɔ fãji lɛ amli lɛ. Akɛ fãji lɛ tee tsɔne ni akɛwoɔ tsɔne mli lɛ mli ni akɛwo EPON (Sigma-Aldrich) mli nyɔɔŋ kome yɛ 4°C. Okɛ fãji lɛ awo EPON hee mli yɛ ŋwɛi fɛ̃i mli ŋmɛlɛtswai 2, ni no sɛɛ lɛ okɛwo mli yɛ 62°C mli ŋmɛlɛtswai 72. Kɛ tsɔne ni akɛkwɛɔ nibii bibii amli lɛ (Leica Microsystems, UC6) kɛ ojarawatɛi ni akɛfeɔ nibii bibii (Diatome, Biel, Switzerland) atsu nii ni okɛfolɔ fãi ni mli tseee ni kɛlɛ ji 70 nm lɛ, ni okɛ 1.5% uranyl acetate awo mli minitii 15 yɛ 37°C, ni okɛ lead citrate solution awo mli minitii 4. Akɛ tsɔne ko ni akɛ Camera OneView 4K 16-bit (Gatan) kɛ DigitalMicrograph tsɔne (Gatan) wo mli lɛ tsu nii kɛfee tsɔne ni akɛkwɛɔ nibii bibii ni yɔɔ ŋwɛi lɛ amli lɛ. Kɛha mlipɛimɔ lɛ, ana electron mfoniri bibii ni akɛ 5000× loo 10,000× dijitaal zoom tsuɔ nii.
Mitochondria mlipɛimɔ. Kɛha mlipɛimɔi fɛɛ lɛ, akɛ niji tsɔɔ mitochondria aŋkroaŋkroi lɛ amli yɛ mfonirii ni akɛ ImageJ tsɔne lɛ tsu nii. Akwɛɔ bɔ ni gbɔmɔtso lɛ yɔɔ ha lɛ mli. Ajieɔ mitochondrial density lɛ kpo akɛ oha mlijaa ni anáa kɛtsɔ mitochondrial kpokpaa fɛɛ ni yɔɔ wala yibii lɛ eko fɛɛ eko mli lɛ mlijaa kɛ cytoplasm kpokpaa lɛ nɔ (cytoplasm kpokpaa = wala yibii lɛ kpokpaa-wala yibii lɛ amli nucleus kpokpaa) × 100. Akɛ fomula [4π∙2(area)/perimeter lɛ buɔ mitochondria lɛ kpokpaa lɛ he akɔntaa. Akwɛ bɔ ni mitochondria lɛ yɔɔ ista lɛ mli ni aja mli awo kui enyɔ mli (“tubular” kɛ “blister”) yɛ bɔ ni amɛhe yɔɔ ha lɛ naa.
Otofagozom/laisozom yibɔ kɛ emli shweremɔ mlipɛimɔ. Kɛ ImageJ tsɔne lɛ atsu nii ni okɛ onine atsɔɔ bɔ ni autophagosome/lysosome lɛ eko fɛɛ eko yɔɔ yɛ mfoniri ni yɔɔ dijitaal lɛ mli lɛ mli. Ajieɔ autophagosome/lysosome kpokpaa lɛ kpo akɛ oha mlijaa ni abuɔ he akɔntaa kɛtsɔ autophagosome/lysosome gbɛjianɔtoo he fɛɛ ni yɔɔ wala yibii lɛ eko fɛɛ eko mli lɛ mlijaa kɛ cytoplasm kpokpaa lɛ nɔ (cytoplasm he=wala yibii he-nucleus he)×100. Akɛ autophagosomes/lysosomes lɛ ayibɔ buɔ he akɔntaa kɛtsɔ yibɔ fɛɛ ni akɛ autophagosome/lysosomes shishitoo mlai lɛ ayibɔ ni yɔɔ wala yibii lɛ eko mli lɛ mlijaa nɔ (yɛ cytoplasmic kpokpaa lɛ he) (cytoplasmic kpokpaa = wala yibii kpokpaa-nuklea kpokpaa).
Nibii ni akɛwoɔ mli kɛha fãi ni mli wa kɛ nɔkwɛmɔnɔ he saamɔ. Kɛha shikpamɔi ni biɔ ni aŋmala glucose he okadi lɛ, ajie jwɛŋmɔŋ fãi ni mli wa lɛ kɛya tsu ko ni akɛwoɔ mli lɛ mli, ni kɔɔyɔɔ ni mli eyi obɔ (95% O2 kɛ 5% CO2), Ca2 + ACSF ni nɔ kwɔ (125.0 mM NaCl, 2.5 mM KCl, 1.25 mM sodium m3O phos05te, 1.25 mM NaCl. 25.0 mM d-glucose, 1.0 mM CaCl 2 kɛ 2.0 mM MgCl 2, ni atsake kɛtee pH 7.4 kɛ 310 kɛyashi 320 mOsm), ni mli lɛ glucose ji 13 C 6- glucose najiaŋdamɔlɔ (Eurisotop, katalog yibɔ CLM93-1). 125.0 mM NaCl, 2.5 mM KCl, 1.25 mM sodium phosphate buffer, 25.0 mM NaHCO3, 25.0 mM d-glucose, Ca1 1082) ni akɛ 1-[1-13C]pyruvate (Eurisotop, wolokpo lɛ yibɔ ni ji CLM-1082) lɛ fata he. Okɛ fãi lɛ awo mli minitii 90 yɛ 37°C. Yɛ kaa lɛ naagbee lɛ, akɛ nu mli nu (pH 7.4) ni ammonium carbonate 75 mM yɔɔ mli lɛ tsu fãi lɛ ahe oya, ni no sɛɛ lɛ, afee lɛ ekome yɛ 40:40:20 (v:v:v) acetonitrile (ACN): methanol: nu mli. Beni akɛ fãi lɛ wo ais nɔ minitii 30 sɛɛ lɛ, akɛ nɔkwɛmɔnii lɛ wo 21,000 g mli minitii 10 yɛ 4°C, ni agbi nu ni mli tse ni yɔɔ mli lɛ yɛ SpeedVac concentrator mli. Akɛ metabolite pellet ni egbi ni jɛ mli ba lɛ to -80°C kɛyashi be mli ni akwɛ mli.
Nu mli kromatografi-mass spektrometri mlipɛimɔ ni kɔɔ amino acids 13 ni akadi C lɛ he. Kɛha nu mli tsɔne ni akɛkwɛɔ nibii amli lɛ mlipɛimɔ (LC-MS) lɛ, atsake tsɔne ni haa mɔ he jɔɔ lɛ lɛ awo 75μl ni ji LC-MS grade nu (Honeywell) mli. Yɛ 21,000 g mli minitii 5 yɛ 4°C sɛɛ lɛ, akɛ 20 μl ni ji supernatant ni ajie lɛ kpo lɛ tsu nii kɛpɛi amino acid flux mli, yɛ be mli ni akɛ nɔ ni eshwɛ ni ajie lɛ mli lɛ tsu nii amrɔ nɔŋŋ kɛpɛi anion mli (kwɛmɔ shishigbɛ). Akɛ benzoyl chloride ni atsɔɔ mli tsutsu lɛ tsu nii kɛpɛi amino acid mli (55, 56). Yɛ klɛŋklɛŋ gbɛ nɔ lɛ, akɛ 10μl ni ji 100 mM sodium carbonate (Sigma-Aldrich) fata 20μl ni ji metabolite mli nu lɛ he, ni no sɛɛ lɛ akɛ 2% benzoyl chloride (Sigma-Aldrich) ni ji 10μl fata LC grade ACN lɛ he. Akɛ nɔkwɛmɔnɔ lɛ wo vortex mli kuku ni no sɛɛ lɛ akɛwo centrifuge mli yɛ 21,000 g mli minitii enumɔ yɛ 20°C. Okɛ nu ni ajie lɛ awo 2 ml autosampler tsinsii ni akɛ glase ni yɔɔ ŋwɛi ni akɛwoɔ mli (200 μl volume) mli. Akɛ Acquity iClass nitsumɔ ni nɔ kwɔ waa ni ji LC gbɛjianɔtoo (Nu) ni akɛtsa Q-Exactive (QE)-HF (Ultra High Field Orbitrap) ni ji ŋwɛi ŋtayaa ni nɔ kwɔ waa (Thermo Fisher Scientific) lɛ tsu nii kɛpɛi nɔkwɛmɔnii lɛ amli. Kɛha mlipɛimɔ lɛ, akɛ 2μl ni ji nɔkwɛmɔnɔ ni ajie lɛ kpo lɛ wo 100×1.0 mm hewalɛ ni nɔ kwɔ ni ji silica T3 column (Waters) ni 1.8μm nibii bibii yɔɔ mli lɛ mli. 100μl/min, ni nɔ ni yɔɔ tsɔne ni akɛbuɔ nu mli lɛ mli ji tsɔne ni akɛbuɔ nu he lɛ A (10 mM ammonium formate kɛ 0.15% formic acid ni yɔɔ nu mli) kɛ tsɔne ni akɛbuɔ nu mli B (ACN). Nɔ ni tsɔɔ mli lɛ ji nɔ ni nyiɛ sɛɛ nɛɛ: 0%B yɛ minitii 0 mli; 0%B. 0 kɛyashi 15% B yɛ minitii 0 kɛyashi 0.1 mli; 15 kɛyashi 17% B yɛ minitii 0.1 kɛyashi 0.5 mli; B yɛ 17 kɛyashi 55% yɛ minitii 0.5 kɛyashi 14 mli; B yɛ 55 kɛyashi 70% yɛ minitii 14 kɛyashi 14.5 mli; yɛ 14.5 kɛyashi 70 kɛyashi 100% B yɛ minitii 18 mli; 100% B yɛ minitii 18 kɛyashi 19 mli; 100 kɛyashi 0% B yɛ minitii 19 kɛyashi 19.1 mli; 0% B yɛ minitii 19.1 kɛyashi 28 mli (55, 56). QE-HF mass spectrometer lɛ tsuɔ nii yɛ ionization gbɛ ni hi jogbaŋŋ nɔ kɛ m/z (mass/charge ratio) ni ji 50 kɛyashi 750. Yiŋkpɛɛ ni akɛtsuɔ nii lɛ ji 60,000, ni sɛɛnamɔ ni anáa lɛ nɔkwɛmɔ (AGC) ion target ni anáa lɛ ji 3×106, ni ion be ni fe fɛɛ ji milliconds. Electrospray ionization (ESI) ni mli edɔ lɛ jɛɛhe lɛ tsuɔ nii yɛ 3.5 kV ni shwieɔ shi, hulutsoo ni ji 250°C, kɔɔyɔɔ ni hoɔ yɛ shwiili mli ni ji 60 AU (nibii ni asumɔɔɔ), kɛ kɔɔyɔɔ ni yeɔ ebuaa ni hoɔ yɛ 20 AU mli. 250°C. S lens lɛ ji 60 AU.
Anion kromatograf-MS mlipɛimɔ ni kɔɔ 13C ni aŋmala nibii ni wala yɔɔ mli lɛ ahe. Akɛ Dionex ion chromatography gbɛjianɔtoo (ICS 5000+, Thermo Fisher Scientific) ni akɛtsa QE-HF mass spectrometer (Thermo Fisher Scientific) lɛ tsu nii kɛpɛi ŋmɔtɔ ni eshwɛ lɛ mli. Yɛ kukufoo mli lɛ, akɛ 5μl ni ji metabolite mli nu lɛ wo Dionex IonPac AS11-HC ŋwɛitsu ni akɛ HPLC (2 mm×250 mm, fãji lɛ dalɛ 4μm, Thermo Fisher Scientific) wo mli yɛ push-in fã ko ni akɛwoɔ mli lɛ mli kɛ 1. )on. Dionex IonPac AG11-HC bulɔ akutso (2 mm x 50 mm, 4μm, Thermo Fisher jeŋ nilee). Akwɛɔ ŋwɛi hulutsoo be lɛ nɔ yɛ 30°C, ni akɛ autosampler lɛ toɔ 6°C. Kɛ potassium hydroxide katrid ni akɛ nu ni mli ewo muji lɛ ewo mli lɛ atsu nii ni okɛfee potassium hydroxide ni tsɔɔ eluent generator lɛ nɔ. 380μl/min, ni akɛ ŋwɛi ŋta ni nyiɛ sɛɛ nɛɛ tsuɔ nii: minitii 0 kɛyashi 3, 10 mM KOH; minitii 3 kɛyashi 12, 10 kɛyashi 50 mM KOH; 12 kɛyashi 19 minitii, 50 kɛyashi 100 mM KOH; 19 kɛyashi 21 minitii , 100 mM KOH; 21 kɛyashi minitii 21.5, 100 kɛyashi 10 mM KOH. Akɛ ŋwɛi ŋta lɛ to 10 mM KOH shishi ekoŋŋ minitii 8.5.
150μl/min isopropanol ni akɛfataa he lɛ fataa he yɛ ŋwɛi ŋta lɛ sɛɛ, ni no sɛɛ lɛ, atsɔɔ ŋwɛi ŋwɛi ŋta ni tsuɔ nii yɛ ionization gbɛ ni ehiii nɔ lɛ nɔ. MS miikwɛ mass lɛ nɔ kɛjɛ m/z 50 kɛyashi 750 kɛ 60,000 yiŋkpɛɛ. AGC lɛ ji 1×106, ni ion be ni fa fe fɛɛ lɛ toɔ 100 ms. Akɛ ESI jɛɛhe ni mli edɔ lɛ tsu nii yɛ 3.5 kV ni akɛshwieɔ shi lɛ nɔ. Nibii krokomɛi ni ato he gbɛjianɔ yɛ ion jɛɛhe lɛ ji: kapilɛri mli latsaa 275°C; shwiili mli la ni hoɔ, 60 AU; yelikɛbuamɔ ni haa kɔɔyɔɔ lɛ hoɔ, 20 AU yɛ 300°C, kɛ S lens ni atoɔ 60 AU.
13C ni aŋmala nɔ nibii ni haa mɔ he jɔɔ lɛ mlipɛimɔ. Kɛ TraceFinder tsɔne (sɛɛ 4.2, Thermo Fisher jeŋ nilee) atsu nii kɛha saji amlipɛimɔ ni kɔɔ isotope mlijaa he. Akɛ tsofa ko ni anyɛɔ akɛ he fɔ̃ɔ nɔ lɛ tsu nii kɛkwɛ bɔ ni tsofa lɛ eko fɛɛ eko ji lɛ mli, ni akwɛ mli jogbaŋŋ. Bɔni afee ni afee isotope nibii babaoo mlipɛimɔ lɛ, ajie he ni ajie ion chromatogram (XIC) ni yɔɔ 13C isotope (Mn) lɛ eko fɛɛ eko mli lɛ kɛjɛ [M + H] + mli, he ni n ji carbon yibɔ ni yɔɔ tsɔne ni akɛtaoɔ nii lɛ mli lɛ mli, ni akɛtsuɔ nii kɛpɛiɔ amino acids mli loo akɛ [ MH] + tsuɔ nii kɛpɛiɔ anions mli. XIC lɛ mass accuracy lɛ faaa fe fãi enumɔ yɛ akpekpe kome mli, ni RT lɛ jalɛsaneyeli lɛ ji minitii 0.05. Afeɔ nibii ni haa mɔ tsui nyɔɔ emli lɛ amlipɛimɔ lɛ kɛtsɔ isotope fɛɛ isotope ni ana lɛ mlijaa kɛ isotope fɛɛ ni yɔɔ compound ni kɛ lɛ kpãa gbee lɛ mli lɛ mlijaa nɔ. Akɛ nɛkɛ sɛɛnamɔi nɛɛ haa akɛ oha mlijaa mlijaa kɛha isotope fɛɛ isotope, ni ajieɔ nɔ ni jɛɔ mli baa lɛ kpo akɛ molar oha mlijaa mli nibii ni akɛwoɔ mli (MPE), taakɛ atsɔɔ mli kɛtsɔ hiɛ lɛ (42).
Akɛ tsɔne ni haa mɔ tsui nyɔɔ emli lɛ fee ekome yɛ 80% methanol (v/v) ni mli ejɔ tamɔ ais mli, ni akɛwo mli, ni akɛwo -20°C mli minitii 30. Vortex nɔkwɛmɔnɔ lɛ ekoŋŋ ni okɛfutu yɛ +4°C minitii 30. Akɛ nɔkwɛmɔnɔ lɛ wo 21,000 g mli minitii enumɔ yɛ 4°C, ni no sɛɛ lɛ abua nɔ ni jɛ mli ba lɛ naa ni agbi kɛtsɔ SpeedVac concentrator ni yɔɔ 25°C nɔ koni akɛpɛi mli yɛ sɛɛ mli. Taakɛ atsɔɔ mli yɛ yiteŋgbɛ lɛ, afee LC-MS mlipɛimɔ yɛ amino acids ni yɔɔ wala yibii ni ahala lɛ amli lɛ anɔ. Kɛtsɔ TraceFinder (ni ji 4.2, Thermo Fisher Scientific) lɛ nɔ lɛ, akɛ nibii ni yɔɔ mli lɛ eko fɛɛ eko mli nibii ni yɔɔ mli lɛ mlipɛimɔ tsu nii. Akɛ preprocessCore tsɔne ni akɛtsuɔ nii (57) lɛ tsu nii kɛfee nibii ni haa mɔ he jɔɔ lɛ ahe saji lɛ.
Slice he saamɔ. Akɛ kɔɔyɔɔ ni haa mɔ he jɔ̃ɔ ehe lɛ wo tsina lɛ mli oya ni afo eyitso, ajie jwɛŋmɔ lɛ kɛjɛ etsitsi lɛ mli oya, ni akɛ tsɔne ni hosoɔ ni ais eyi mli obɔ (HM-650 V, Thermo Fisher Scientific, Walldorf, Germany) lɛ tsu nii ni akɛfo mli ni efee 300 kɛyashi 375% carbon gas O5d. 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 kɛ 6.0 mM MgCl372. msm). 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 pHC2l2l. kɛ 310 kɛyashi 320 mOsm). Okɛ fãi lɛ ato minitii 20 kɛyashi 30 bɔni afee ni anyɛ aku sɛɛ akɛwo mli dani aŋmala.
ni aŋmala ashwie shi. Akɛ tsɔne bibioo ko ni akɛkwɛɔ nibii bibii amli ni akɛ tsɔne ko ni akɛtswaa nibii ni yɔɔ shiŋŋ kɛ 20x nu mli nibii ni akɛkwɛɔ nibii amli (Scientifica) tsu nii kɛha nibii fɛɛ ni aŋmalaa ashwieɔ shi lɛ. Ayoo Purkinje wala yibii ni asusuɔ akɛ amɛji lɛ kɛtsɔ (i) gbɔmɔtso lɛ dalɛ, (ii) he ni cerebellum lɛ yɔɔ, kɛ (iii) mtYFP adafitswaa wolo ni kpɛɔ lɛ kpojiemɔ nɔ. 150-10, 0.86 mm×1.5 mm×100 mm, Science Products, Hofheim, Germany) kɛ pipet ni yɔɔ ŋwɛigbɛ ni akɛtsuɔ nii (P-1000, CA) lɛ ji nɔ ni ajieɔ kɛjɛɔ mli. ELC-03XS npi patch clamp amplifier (npi tsɔne ni akɛtswaa lala, Tam, Germany) lɛ ji nɔ ni akɛtswa nibii fɛɛ ni aŋmala ashwie shi lɛ, ni tsɔne ni atsɛɔ lɛ Signal (ni ji 6.0, Cambridge Electronic, Cambridge, UK) lɛ kudɔɔ. Akɛ kaa lɛ wo 12.5 kHz mli. Akɛ Bessel tsɔnei enyɔ ni tsɔɔ kuku ni sɛɛ tsɛɔ ni ji 1.3 kɛ 10 kHz lɛ tsuɔ okadi lɛ he nii. Akɛ amplifier lɛ tsuɔ nii kɛwoɔ tsɔne ni akɛwoɔ tsɔne mli lɛ mli hewalɛ ni yɔɔ membrane lɛ kɛ pipette lɛ mli lɛ he nyɔmɔ. Afee kaai lɛ fɛɛ yɛ Orca-Flash 4.0 mfonirifeemɔ tsɔne ko (Hamamatsu, Gerden, Germany) kudɔmɔ shishi, ni Hokawo tsɔne ni akɛtsuɔ nii yɛ tsɔne mli lɛ kudɔɔ.
Cell muu lɛ fɛɛ he gbɛjianɔtoo kɛ mlipɛimɔ ni afeɔ daa. Amrɔ nɔŋŋ dani obaaŋmala nɔ ko lɛ, okɛ mligbɛ tsofa ni nibii ni nyiɛ sɛɛ nɛɛ yɔɔ mli lɛ awo pipɛt lɛ mli: 4.0 mM KCl, 2.0 mM NaCl, 0.2 mM EGTA, 135.0 mM potassium gluconate, 10.0 mM Hepes, 4.0 mM Guphano.TPGsine (4.0 mM ATP.5g) (Na) kɛ 10.0 mM kreatinin fɔsfɛt lɛ tsake pH 7.25, ni osmotic hewalɛ ji 290 mOsm (sukrose). Beni akɛ hewalɛ ni ji 0 pA tsu nii ni ekɛgbala ŋmei lɛ mli amrɔ nɔŋŋ lɛ, asusu ŋmei ni heɔ ehe lɛ he hewalɛ ni eyɔɔ lɛ he. Asusuɔ hewalɛ ni akɛwoɔ mli lɛ he kɛtsɔ -40, -30, -20, kɛ -10 pA ni akɛtsuɔ nii lɛ nɔ. Susumɔ bɔ ni voltage lɛ hetoo lɛ da ha lɛ he ni okɛ Ohm mla lɛ abu nɔ ni akɛwoɔ mli lɛ he akɔntaa. Akɛ nibii ni baa trukaa lɛ wo voltage klamp mli minitii enumɔ, ni ayoo sPSC ni asusu yɛ Igor Pro (version 32 7.01, WaveMetrics, Lake Oswego, Oregon, USA) mli kɛtsɔ niŋmaa ni haa mɔ leɔ nii ni ekɛ ehe woɔ mli lɛ nɔ. Asusuɔ IV curve kɛ steady-state current lɛ he kɛtsɔ batri lɛ ni akɛwoɔ mli yɛ hewalɛi srɔtoi anɔ (ni jeɔ shishi kɛjɛɔ -110 mV) kɛ voltage ni yaa hiɛ yɛ 5 mV steps mli lɛ nɔ. Akɛ depolarizing current tsu nii kɛka AP lɛ feemɔ akwɛ. Kpɛtɛɔ tsɔne lɛ he yɛ -70 mV be mli ni okɛ depolarizing ŋmɛnɛŋmɛnɛ beaŋ pulse tsuɔ nii. Tsakemɔ step size ni yɔɔ lalai ni aŋmalaa lɛ eko fɛɛ eko mli lɛ mli srɔtoi (10 kɛyashi 60 pA). Buu AP ni fa fe fɛɛ lɛ he akɔntaa kɛtsɔ pulse spikes ni kɛ AP ni fa fe fɛɛ baa lɛ ni okɛ onine akaneɔ lɛ nɔ. Akwɛɔ AP shishitoo mla lɛ mli kɛtsɔ depolarization pulse ni ji enyɔ ni jɛɔ AP kome loo nɔ ni fe nakai mli klɛŋklɛŋ lɛ ni akɛtsuɔ nii lɛ nɔ.
Patch ni afo mli lɛ he gbɛjianɔtoo kɛ mlipɛimɔ. Feemɔ perforated patch niŋmaa kɛtsɔ protocol ni yɔɔ ŋmɛnɛ lɛ nɔ. 128 mM gluconate K, 10 mM KCl, 10 mM Hepes, 0.1 mM EGTA kɛ 2 mM MgCl2, ni okɛto pH 7.2 he (kɛtsɔ KOH nɔ). Ajieɔ ATP kɛ GTP kɛjɛɔ wala yibii lɛ amli tsofa lɛ mli koni akɛtsi wala yibii lɛ amli tsɔne ni anyɛɛɛ akudɔ lɛ naa. Akɛ amphotericin ni yɔɔ mli lɛ woɔ tsɔne ni akɛwoɔ tsɔne mli lɛ mli (aaafee 200 kɛyashi 250 μg/ml; G4888, Sigma-Aldrich) koni anyɛ ana tsɔne ni akɛwoɔ tsɔne mli lɛ he saji ni aŋmala ashwie shi. Akɛ amphotericin wo dimethyl sulfoxide mli (naagbee shweremɔ: 0.1 kɛyashi 0.3%; DMSO; D8418, Sigma-Aldrich). DMSO ni akɛtsu nii lɛ mli falɛ lɛ bɛ hewalɛ ko kwraa yɛ tsitsii ni akaseɔ lɛ anɔ. Yɛ be mli ni atswaa tsɔne lɛ, akwɛɔ gbɛ ni tsiɔ mɔ naa (Ra) lɛ nɔ be fɛɛ be, ni ajeɔ kaa lɛ shishi beni Ra kɛ AP lɛ amplitude lɛ ehi shi shiŋŋ sɛɛ (minitii 20-40). Asusuɔ nifeemɔ ni baa trukaa lɛ he yɛ voltage kɛ/loo ŋmɛnɛŋmɛnɛ klamp mli minitii 2 kɛyashi 5. Akɛ Igor Pro (ni ji 7.05.2, WaveMetrics, USA), Excel (ni ji 2010, Microsoft Corporation, Redmond, USA) kɛ GraphPad Prism (ni ji 8.1.2, GraphPad Software Inc., La Jolla, CA) tsu nii kɛpɛi saji amli. Amerika). Bɔni afee ni ayoo APs ni baa trukaa lɛ, akɛ IgorPro NeuroMatic v3.0c plug-in lɛ tsuɔ nii. Etsɔɔ APs lɛ diɛŋtsɛ kɛtsɔ shishitoo mla ko ni aha lɛ nɔ, ni atsakeɔ lɛ eko fɛɛ eko kɛha niŋmaa lɛ eko fɛɛ eko. Kɛtsɔ spike teŋgbɛ lɛ nɔ lɛ, tsɔɔmɔ spike frekuensi kɛ spike frekuensi ni fe fɛɛ amrɔ nɔŋŋ kɛ spike frekuensi ni yɔɔ teŋgbɛ.
PN hetsemɔ. Kɛtsɔ gbɛjianɔtoo ni akala tsutsu lɛ nɔ lɛ, atsuu PNs lɛ he kɛjɛ ŋmɔtɔ cerebellum lɛ mli yɛ be pɔtɛɛ ko mli (58). Yɛ kukufoo mli lɛ, atse cerebellum lɛ mli ni atsɔ mli yɛ ais ni mli ejɔ mli [ni HBSS Ca2+ kɛ Mg2+ bɛ mli, ni akɛ glucose 20 mM, penicillin (50 U/ml) kɛ streptomycin (0.05 mg/ ml) fata he], ni no sɛɛ lɛ akɛ mgSSHB-1steine ​​fata he. / ml), papain (16 U / ml) kɛ deoksiribonuklease I (DNase I; 0.1 mg/ml)] Akɛ minitii 30 atsu nii yɛ 30°C nɔ. Klɛŋklɛŋ lɛ, afɔ fãji lɛ ahe yɛ HBSS mli ni ŋmei mli muji (10 mg/ml), BSA (10 mg/ml) kɛ DNase (0.1 mg/ml) yɔɔ mli lɛ mli yɛ shĩa mli latsaa mli koni ekɛtsi enzymatic mli nibii ni shwieɔ shi lɛ anaa, ni no sɛɛ lɛ, okɛ HBSS mli nibii ni mli eyi obɔ kɛ 20 mM glucose lɛ atsu nii. (0.05 mg/ml) kɛ DNase (0.1 mg/ml) lɛ jieɔ wala yibii komekomei. 1110 rpm, minitii 5, 4°C) ni akɛwo mli ekoŋŋ yɛ tsɔne ni akɛhalaa nii lɛ mli [HBSS, ni akɛ 20 mM glucose, 20% fetal bovinic U.50) Serum, 5 (0.05 mg/ml)]; kwɛɔ bɔ ni wala yibii lɛ baanyɛ ahi shi aha lɛ mli kɛtsɔ propidium iodide nɔ ni atsake wala yibii lɛ ayibɔ ni ji 1×106 kɛyashi 2×106 wala yibii/ml. Dani abaafee flow cytometry lɛ, atsɔ 50 μm cell strainer nɔ ajie nu ni akɛwoɔ mli lɛ mli.
Saitɔmɛta ni hoɔ. Akɛ FACSAria III tsɔne (BD Biosciences) kɛ FACSDiva tsɔne (BD Biosciences, 8.0.1) tsu nii kɛhala wala yibii lɛ amli yɛ 4°C. 100 μm nozzle yɛ 20 psi nɔnyɛɛ shishi yɛ ~2800 events/sec mli. Akɛni blema shinaa gbɛjianɔtoo (wala yibii lɛ adalɛ, bimodal mligbalamɔ, kɛ gbɛfaŋnɔi ni gbɛɔ eshwaa) nyɛŋ ana nɔmimaa akɛ PN lɛ jeɔ kpo kɛjɛɔ wala yibii krokomɛi ahe hewɔ lɛ, shinaa gbɛjianɔtoo lɛ damɔ YFP hewalɛ kɛ autofluorescence ni yɔɔ mitoYFP + kɛ MitoYFP ni kudɔɔ lɛ nɔ. YFP miishɛɛ ji kɛtsɔ 488 nm laser line ni ekɛjieɔ nɔkwɛmɔnɔ lɛ nɔ lɛ nɔ, ni akɛ 530/30 nm band pass filter tsuɔ nii kɛnaa okadi lɛ. Yɛ mitoYFP+ ​​ŋmeii amli lɛ, akɛ Rosa26-mitoYFP adafitswaa wolo lɛ mli hewalɛ ni kɔɔ he lɛ hu tsuɔ nii koni akɛyoo srɔtofeemɔ ni yɔɔ tsɔne ni haa mɔ tsui nyɔɔ emli lɛ kɛ axon mli fãi lɛ ateŋ. 7-AAD lɛ kɛ laser ni ji 561 nm ni ji ŋmɔŋ lɛ haa mɔ tsui nyɔɔ emli ni akɛ 675/20 nm bandpass filter lɛ naa koni ejie wala yibii ni egboi lɛ kɛjɛ mli. Bɔni afee ni ajie astrocytes lɛ mli yɛ be kome mli lɛ, akɛ ACSA-2-APC wo wala yibii lɛ amli, no sɛɛ lɛ akɛ 640 nm laser line shwie nɔkwɛmɔnɔ lɛ nɔ, ni akɛ 660/20 nm bandpass filter tsu nii koni akɛna okadi lɛ.
Akɛ tsɛŋemɔ helai ni abua naa lɛ tsu nii kɛtsɔ tsɔne ni akɛwoɔ mli lɛ nɔ (1110 rpm, minitii 5, 4°C) ni akɛto -80°C kɛyashi akɛtsu nii. Akɛ Mfn2cKO ŋmeii kɛ amɛbii bibii lɛ toɔ gbi kome nɔ koni akɛba gbɛjianɔtoo lɛ mli tsakemɔi lɛ shi. Akɛ FlowJo tsɔne ni akɛtsuɔ nii (FlowJo LLC, Ashland, Oregon, USA) lɛ tsu nii kɛtsɔɔ FACS saji amli kɛ mlipɛimɔ.
Taakɛ atsĩ tã yɛ yiteŋgbɛ lɛ (59), akɛ real-time PCR tsuɔ nii koni akɛjie DNA kɛjɛ tsɔnei ni ahala lɛ amli kɛha mtDNA yibɔ ni nyiɛ sɛɛ lɛ. Shishijee lɛ, aka linearity kɛ threshold sensitivity lɛ akwɛ kɛtsɔ qPCR ni akɛtsuɔ nii yɛ tsɛŋemɔ helai srɔtoi anɔ lɛ nɔ. Yɛ kukufoo mli lɛ, abua PN 300 naa yɛ lysis buffer ni 50 mM tris-HCl (pH 8.5), 1 mM EDTA, 0.5% Tween 20 kɛ proteinase K (200 ng/ml) yɔɔ mli lɛ mli ni okɛwo 55°C mli minitii 120. Akɛ TaqMan probe (Thermo Fisher) ni kɔɔ mt-Nd1 he lɛ tsu nii kɛsusu mtDNA lɛ he kɛtsɔ PCR ni ji fã-kɛ-fã nɔ yɛ 7900HT Real-Time Ficientmosher PCR gbɛjianɔtoo (Therificmosher PCR system (Therificmosher) lɛ mli). Jeŋ nilee, wolokpo yibɔ Mm04225274_s1), mt-Nd6 (Thermo Fisher jeŋ nilee, wolokpo yibɔ AIVI3E8) kɛ 18S (Thermo Fisher jeŋ nilee, wolokpo yibɔ Hs99999901_s1) tsɛŋemɔ helai.
Proteome nɔkwɛmɔnɔ he saamɔ. Kɛtsɔ la ni tsoɔ yɛ 95°C nɔ minitii 10 kɛ sonicating, yɛ lysis buffer [6 M guanidine kloraid, 10 mM tris (2-carboxyethyl) phosphine hydrochloride, 10 mM kloroacetamide kɛ 100 mM tris- Lyse fɛ̃i tsɔne mli]. Yɛ Bioruptor (Diagenode) lɛ nɔ minitii 10 (sɛkɛndii 30 pulse / sɛkɛndii 30 hejɔɔmɔ be). Akɛ nɔkwɛmɔnɔ lɛ wo 20 mM tris-HCl (pH 8.0) mli 1:10, ni akɛ trypsin shika tsuru (Promega) 300 ng futu mli, ni akɛwo mli nyɔɔŋ kome yɛ 37°C koni enyɛ eshɛ niyenii ni shwieɔ shi lɛ he kwraa. Yɛ gbi ni ji enyɔ lɛ nɔ lɛ, akɛ nɔkwɛmɔnɔ lɛ wo 20,000 g mli minitii 20. Akɛ 0.1% formic acid wo nu ni hoɔ lɛ mli, ni akɛ StageTips ni mɔ diɛŋtsɛ fee lɛ jie nu lɛ mli nu lɛ. Akɛ nɔkwɛmɔnɔ lɛ gĩ yɛ SpeedVac dɛŋdade ko mli (Eppendorf tsɔne ni akɛwoɔ mli lɛ kɛ 5305) yɛ 45°C, ni no sɛɛ lɛ, akɛ peptide lɛ wo 0.1% formic acid mli. Mɔ kome too lɛ nɔŋŋ ji mɔ ni fee nɔkwɛmɔnii lɛ fɛɛ yɛ be kome mli. Bɔni afee ni akwɛ ŋulamiiaŋkwɛmɔi lɛ amli lɛ, akɛ 4 μg ni mli edɔ lɛ wo nɔ ni ji 1:20 (TMT10plex, yibɔ ni ji 90110, Thermo Fisher Scientific) kɛ nɔ ni ji 1:20. Kɛha TMT kadimɔ lɛ, atsake TMT nibii ni akɛtsuɔ nii lɛ 0.8 mg awo ACN ni nu bɛ mli lɛ 70 μl mli, ni atsake peptide ni egbi lɛ awo 9 μl ni ji 0.1 M TEAB (triethylammonium bicarbonate) mli, ni akɛ TMT nibii ni akɛtsuɔ nii yɛ ACN mli lɛ 7 μl fata he. Nibii ni yɔɔ mli lɛ ji 43.75%. Yɛ minitii 60 ni akɛwo mli lɛ sɛɛ lɛ, akɛ 2 μl ni ji 5% hydroxylamine lɛ gbe nɔ ni tee nɔ lɛ. Akɛ peptides ni aŋmala nɔ nii lɛ bua naa, agbi, ni akɛwo 200μl ni ji 0.1% formic acid (FA) mli ekoŋŋ, ni aja mli enyɔ, ni no sɛɛ lɛ akɛ StageTips ni mɔ diɛŋtsɛ fee lɛ tsu nii ni ekɛjie ŋmei kɛjɛ mli. 3000 ultra nu kromatograf (UltiMate 3000 ultra nu kromatograf) lɛ, ajara fãi enyɔ lɛ ateŋ ekome mli yɛ 1mm x 150mm Acquity kromatograf kolum ni eyi kɛ 130Å1.7μm C18 nibii bibii (NoWaters, catalog1850) lɛ nɔ. Thermo Fisher jeŋ nilee). Gbalamɔ peptides lɛ mli yɛ 30μl/min mli, gbalamɔ kɛjɛ 1% kɛyashi 50% buffer B mli minitii 85 kɛ stepwise gradient ni ji minitii 96, kɛjɛ 50% kɛyashi 95% buffer B mli minitii 3, no sɛɛ lɛ minitii 8 kɛha 95 % Buffer B; Nibii ni akɛwoɔ mli lɛ ji 5% ACN kɛ 10 mM ammonium bicarbonate (ABC), ni nɔ ni akɛwoɔ mli lɛ ji 80% ACN kɛ 10 mM ABC. Buamɔ fãi lɛ anaa daa minitii 3 ni okɛ amɛ afee kui enyɔ (1 + 17, 2 + 18, kɛ ekrokomɛi) ni okɛgbi yɛ vacuum centrifuge mli.
LC-MS/MS mlipɛimɔ. Kɛha ŋwɛi ŋtayaa lɛ, ajie peptides lɛ (yibɔ ni ji r119.aq) lɛ mli yɛ 25 cm, 75 μm mligbɛ ŋtayaa ni ji PicoFrit mlipɛimɔ ŋwɛi ŋta (yiŋtoo hee, fã yibɔ ni ji PF7508250) ni akɛ 1.9 μm ReproSil-Pur-Pur-Purse, 1200 (Tɛrmo Fisher Jeŋ Nilee, Germany). Akwɛ ŋwɛitsu lɛ nɔ yɛ 50°C. Buffers A kɛ B ji 0.1% fɔmik asid ni yɔɔ nu mli kɛ 0.1% fɔmik asid ni yɔɔ 80% ACN mli, yɛ gbɛ kome nɔ. Akɛ 200 nl/min ni ji 6% kɛyashi 31% buffer B lɛ mli minitii 65, kɛjɛ 31% kɛyashi 50% buffer B lɛ mli minitii 5 mli lɛ gbala peptides lɛ amli. Akwɛ nibii ni ajie lɛ mli lɛ amli yɛ Orbitrap Fusion mass spectrometer (Thermo Fisher Scientific) nɔ. 120,000 ni yɔɔ 350 kɛyashi 1500 m/z lɛ mli lɛ ji peptide precursor m/z susumɔ. Kɛtsɔ 27% ni ji gbeekpamɔ hewalɛ ni akɛtsuɔ nii daa lɛ nɔ lɛ, ahalaa nɔ ni tsɔɔ hiɛ ni he wa fe fɛɛ ni yɔɔ charge shihilɛ ni ji 2 kɛyashi 6 kɛha hewalɛ ni nɔ kwɔ C trap dissociation (HCD) cleavage. Akɛ sɛɛkuu be lɛ eto 1 s. Asusu m/z jara ni yɔɔ peptide fã lɛ mli lɛ he yɛ ion trap lɛ mli kɛtsɔ AGC oti bibioo fe fɛɛ ni ji 5×104 kɛ 86 ms ni akɛwoɔ mɔ mli lɛ be ni fe fɛɛ lɛ nɔ. Yɛ mligbalamɔ sɛɛ lɛ, akɛ nɔ ni tsɔɔ hiɛ lɛ wo dynamic exclusion list lɛ nɔ kɛha 45 s. Ajara peptides ni akɛkadiɔ TMT lɛ mli yɛ 50 cm, 75 μm Acclaim PepMap ŋwɛi ŋta (Thermo Fisher jeŋ nilee, wolokpo yibɔ 164942), ni akwɛ shifãa lɛ mli yɛ Orbitrap Lumos Tribrid mass spectrometer (Thermo Fisher jeŋ nilee ni nɔ kwɔ) nɔ. (FAIMS) nibii (Thermo Fisher Scientific) tsuɔ nii yɛ nyɔmɔwoo voltaji enyɔ ni ji −50 kɛ −70 V. MS3 ni ahala yɛ sɛɛkuu-kɛsaramɔ ni tsɔɔ hiɛ lɛ nɔ lɛ ji nɔ ni akɛtsuɔ nii kɛhaa TMT amaniɛbɔɔ ion okadi susumɔ. 1200 lɛ nɔ, ni akɛ 90% linear gradient elution tsu nii, ni buffer ni mli eyi obɔ kɛ 6% kɛyashi 31%; FA ni ji 0.1%, ni FA ni ji 0.1% kɛ ACN ni ji 80%. Akɛ analytic column lɛ tsuɔ nii yɛ 50°C. Kɛ FreeStyle (shishitsɔɔmɔ 1.6, Thermo Fisher Scientific) atsu nii ni okɛja shishijee faili lɛ mli yɛ FAIMS nyɔmɔwoo voltage lɛ naa.
Protein ayɔsemɔ kɛ bɔ ni eyɔɔ ha. Kɛtsɔ Andromeda taomɔ tsɔne ni akɛfee ekome lɛ nɔ lɛ, akɛ MaxQuant 1.5.2.8 (https://maxquant.org/) lɛ tsu nii kɛpɛi shishijee saji lɛ amli. Kɛfata Cre recombinase kɛ YFP sɛɛkuu ni ana kɛjɛ Aequorea victoria lɛ he lɛ, atao peptide mli fãi ni yɔɔ srɔtoi lɛ amli kɛha sɛɛkuu ni yɔɔ mla naa kɛ isoform sɛɛkuu ni kɔɔ ŋmei mli nibii ni akɛtsuɔ nii lɛ ahe lɛ (Proteome ID UP000000589, ni ajie kɛjɛ UniProt)777 lɛ mli yɛ May 2010 mli. Akɛ methionine oksideshin kɛ protein N-naagbee asetilɛshin to akɛ tsakemɔi ni tsakeɔ; Akɛ cysteine ​​carbamoyl methylation lɛ to akɛ tsakemɔi ni yɔɔ shiŋŋ. Akɛ niyenii ni shwieɔ shi lɛ he nibii lɛ toɔ “pɔtɛɛ” kɛ “trypsin/P” he. Peptides kɛ razor peptides ni akɛtsuɔ nii kɛyooɔ protein lɛ ayibɔ ni baa shi fe fɛɛ ji 1; peptides pɔtɛɛi ayibɔ ni baa shi fe fɛɛ ji 0. Yɛ peptide shikpɔŋ he mfoniri ni kɛ amɛhe kpãa gbee lɛ shishi lɛ, protein lɛ ayɔsemɔ mlijaa ji 0.01. Akɛ “Peptide ni Ji Enyɔ” lɛ tsuɔ nii. Kɛ “match entre runs” lɛ atsu nii ni okɛ omanyeyeli nibii ni tsɔɔ shishijee faili srɔtoi lɛ ateŋ lɛ atsu nii. Kɛ LFQ mlijaa bibioo fe fɛɛ lɛ akane 1 kɛha yibɔ ni bɛ yibɔ (LFQ) (60). Akɛ LFQ hewalɛ lɛ tsuɔ nii kɛha kɛ hoo lɛ sɛɛnamɔi enyɔ ni sa yɛ kɛ hoo lɛ genotype kuu kome mli yɛ be fɛɛ mli, ni ajieɔ kɛjɛɔ mlijaa ni sa ni mlilɛɛmɔ ji. 0.3 ni oya shi 1.8. Kɛ Perseus kɔmpiuta gbɛjianɔtoo (https://maxquant.net/perseus/) kɛ R (https://r-project.org/) atsu nii ni okɛpɛi LFQ hetooi lɛ amli. Akɛ gbɛi enyɔ ni yɔɔ mlɛo t kaa ni jɛ limma tsɔne lɛ mli lɛ tsu nii kɛha srɔtofeemɔ wiemɔi amlipɛimɔ (61). Akɛ ggplot, FactoMineR, Factoekstra, GGally kɛ pheatmap tsuɔ nii kɛpɛiɔ saji amli. Akɛ MaxQuant henɔ 1.6.10.43 tsu nii kɛpɛi TMT mli nibii ni haa mɔ he waa lɛ amli. Taomɔ proteomics he saji ni afeee nɔ ko kɛjɛ UniProt gbɔmɔtso mli proteomics he saji atoohe lɛ, ni ajie yɛ September 2018. Nɔ ni fata niiamlipɛimɔ lɛ he ji isotope hetsemɔ tsakemɔ nibii ni mɔ ni fee lɛ kɛha lɛ. Kɛ limma yɛ R mli kɛha srɔtofeemɔ wiemɔ mlipɛimɔ. Data shishijee, databeis taomɔ hetooi, kɛ data mlipɛimɔ nitsumɔ kɛ hetooi lɛ fɛɛ akɛtoɔ ProteomeXchange kpaŋmɔ lɛ mli kɛtsɔ PRIDE hefatalɔi nitoohe kɛ data kuu ni tsɔɔ PXD019690.
Nitsumɔ he saji ni aŋmala ashwie shi lɛ haa mlipɛimɔ lɛ yaa hiɛ. Akɛ Ingenuity Pathway Analysis (QIAGEN) dɛŋdade lɛ tsu nii koni akɛna bɔ ni nitsumɔ mli saji ni aŋmala ashwie shi lɛ mli saji ni ato he gbɛjianɔ lɛ mli nibii babaoo yɔɔ ha yɛ otsii 8 mli (Mfoniri 1). Yɛ kukufoo mli lɛ, akɛ protein ni falɛ ni anáa kɛjɛɔ LC-MS/MS (tandem mass spectrometry) mli lɛ mlipɛimɔ mli lɛ tsuɔ nii kɛ filta shishitoo mlai ni nyiɛ sɛɛ nɛɛ: Ahala Mus musculus akɛ bɔɔ nii kɛ sɛɛgbɛ, ni fã lɛ tsɔɔ P sɛɛnamɔ ni Benjamini tsake kɛha nibii ni haa mɔ tsui nyɔɔ emli 0.05 loo nɔ ni baa shi lɛ akɛ nɔ ni he hiaa waa. Kɛha nɛkɛ mfoniri nɛɛ, atsɔɔ ŋwɛi kui enumɔ ni fa fe nine yɛ kuu fɛɛ kuu mli ni damɔ P sɛɛnamɔ ni atsake lɛ nɔ lɛ. Kɛtsɔ t-test babaoo ni akɛtsuɔ nii, ni akɛ Benjamini, Krieger, kɛ Yekutieli (Q = 5%) linear boost program ni yɔɔ fãi enyɔ lɛ tsuɔ nii lɛ nɔ lɛ, afeɔ be-kɛ-be mli protein kpojiemɔ mlipɛimɔ yɛ mɛi ni he hiaa ni ayɔse amɛ yɛ kui lɛ eko fɛɛ eko mli lɛ anɔ, ni akwɛɔ line fɛɛ line mli srɔtoi. Ehe ehiaaa ni akɛ SD ni yaa nɔ daa lɛ atsu nii.
Bɔni afee ni wɔkɛ nɔ ni jɛ nikasemɔ nɛɛ mli ba lɛ ato saji ni akala lɛ ahe ni wɔfee Venn mfoniri ni yɔɔ mfoniri 1 lɛ mli lɛ, wɔkɛ protein ni falɛ ni aŋmala ashwie shi lɛ fata MitoCarta 2.0 niŋmaai ahe (24). Kɛ intanɛt nɔ dɛŋdade ni ji Draw Venn Mfoniri lɛ atsu nii ni okɛfee mfoniri lɛ.
Kɛji ootao saji fitsofitso ni kɔɔ akɔntaabuu gbɛjianɔtoi ni akɛtsuɔ nii kɛpɛiɔ proteomics mli lɛ he lɛ, ofainɛ kwɛmɔ nibii kɛ gbɛi ni atsɔɔ nɔ atsuɔ nii lɛ fã ni kɛ lɛ kpãa gbee lɛ. Kɛha kaai krokomɛi fɛɛ lɛ, abaanyɛ ana saji fitsofitso yɛ abɛbua ni kɛ lɛ kpãa gbee lɛ mli. Kɛji akɛ atsɔɔɔ gbɛ kroko nɔ lɛ, ajieɔ saji fɛɛ akpo akɛ ± SEM, ni akɛ GraphPad Prism 8.1.2 tsɔne lɛ tsu nii kɛpɛi akɔntaabuu mli.
Kɛji ootao nibii krokomɛi ni fata he kɛha niŋmaa nɛɛ, ofainɛ kwɛmɔ http://advances.sciencemag.org/cgi/content/full/6/35/eaba8271/DC1.
Enɛ ji niŋmaa ko ni anyɛɔ ashɛɔ he ni ajaa yɛ Creative Commons Attribution-Non-Commercial License lɛ mlai lɛ ashishi, ni ŋmɛɔ gbɛ akɛ akɛtsuɔ nii, ajaa ni afee ekoŋŋ yɛ gbɛ fɛɛ gbɛ nɔ, kɛji akɛ naagbee nitsumɔ lɛ jeee kɛha jarayeli sɛɛnamɔ ni nɔ ni yɔɔ mli ji akɛ shishijee nitsumɔ lɛ ja. Kaimɔ nii.
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E. Motori, I. Atanassov, SMV Kochan, K. Folz-Donahue, V. Sakthivelu, P. Giavalisco, N. Toni, J. Puyal, N.-G. Larson
Proteomics mlipɛimɔ ni kɔɔ tsɔnei ni tsuuu nii jogbaŋŋ lɛ ahe lɛ jie lɛ kpo akɛ, tsɔnei ni haa mɔ tsui nyɔɔ emli lɛ tsuɔ nii koni ekɛtsi tsɔnei ni haa mɔ tsui nyɔɔ emli lɛ anaa.
E. Motori, I. Atanassov, SMV Kochan, K. Folz-Donahue, V. Sakthivelu, P. Giavalisco, N. Toni, J. Puyal, N.-G. Larson
Proteomics mlipɛimɔ ni kɔɔ tsɔnei ni tsuuu nii jogbaŋŋ lɛ ahe lɛ jie lɛ kpo akɛ, tsɔnei ni haa mɔ tsui nyɔɔ emli lɛ tsuɔ nii koni ekɛtsi tsɔnei ni haa mɔ tsui nyɔɔ emli lɛ anaa.
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