Natan *Kaplesan ya address: Cologne 50931, Germany, Cologne Excellence Cluster Research on Cellular Stress Response ed Aging-related Diseases (CECAD).
Say neurodegeneration na saray sakit ed mitochondria et ipapasen ya agnauman lapud limitado so metabolic plasticity na saray neuron, balet say epekto na mitochondrial dysfunction ed cell autonomy na neuronal metabolism ed laman et ag tanton natalosan. Dia, ipaparungtal mi so cell-specific proteome na Purkinje neurons ya walay progresibon kakulangan na OXPHOS ya sengegan na ag-inkayadyari na mitochondrial fusion dynamics. Nalmoan mi ya say mitochondrial dysfunction so sengegan na baleg a pananguman ed lawak na proteomics, ya diad kaunoran et angitonton ed nantutumbokan ya pakaaktibo na eksakton metabolic programs antis ya ompatey so selula. Diad ag inilaloan, nadetermina mi so mapatnag ya induction na pyruvate carboxylase (PCx) tan arum niran peroxidases ya misiglaotan ed pangiter na TCA cycle intermediates. Say pangisebel ed PCx so amaloor ed oxidative stress tan neurodegeneration, ya mangipapanengneng ya say atherosclerosis et walay proteksion to ed saray neuron ya anggapoy OXPHOS. Say kipawil na mitochondrial fusion ed saray neurons ya aderal la et sigpot ya mamasimbalo ed sarayan metabolic characteristics, kanian ag-onpatey so selula. Say naromog mi et mangipapanengneng na saray dalan ya ag ni kabat nensaman ya mangiiter na resistensya ed mitochondrial dysfunction tan mangipapanengneng ya say neurodegeneration et nayarin nauman anggan diad unor lan yugtan na sakit.
Say manunan betang na mitochondria ed pamasiansia ed metabolismo na enerhiya na neuron et napapabitar na malaknab iran sintomas na neurological ya misiglaotan ed saray sakit ed mitochondria na too. Maslak ed sarayan sakit et lapud gene mutations ya mangireregula ed mitochondrial gene expression (1, 2) odino say gene destruction ya misiglaotan ed mitochondrial dynamics, ya agdirektan mangaapekta ed inkapekder na mitochondrial DNA (mtDNA) (3, 4). Say kimey ed saray modelo na ayayep so angipatnag ya bilang ebat ed mitochondrial dysfunction ed kaliberliber iran tisyu, saray konserbatibon metabolic pathways (5-7) so nayarin na-activate, kanian mangiter na importantin pakatalos ed pathogenesis na sarayan komplikadon sakit. Diad pidumaan, say pakatalos tayo ed saray pananguman ed metabolismo na espisipikon klase na selula ya sengegan na inkalapagan ya ag-inkayadyari na produksyon na adenosine triphosphate (ATP) ed utek et manuna (8), ya mangipapabitar ed pankaukolan ya nabirbir iray target na panagtambal ya nayarin usaren pian naprebentaan odino naprebentaan so sakit. Ipaliis so neurodegeneration (9). Say kakulangan na impormasyon et say katuaan ya saray selula na nerbiyos et ipapasen ya walaan na limitadon metabolic flexibility no ikompara ed saray klase na selula ed kaliberliber iran tisyu (10). Lapud sarayan selula so walaan na manunan betang ed pangikoordina na suplay na metabolites ed saray neuron pian napaaligwas so synaptic transmission tan onkiwas ed saray kondisyon na injury tan sakit, say abilidad ya iyadapta so metabolismo na selula ed saray makapaangat a kondisyon na tisyu na utek et ngalngalin limitado ed saray glial cells (11-14). Niarum ni, say inherent cellular heterogeneity na tissue na utek so baleg ya mangaamper ed panaral ed saray metabolic changes ya nagagawa ed espisipikon neuronal subgroups. Lapud saya, daiset so amta nipaakar ed eksakton epekto na selula tan metabolismo na mitochondrial dysfunction ed saray neuron.
Pian natalosan so epekto na metabolismo na mitochondrial dysfunction, insian mi ray Purkinje neurons (PNs) ed nanduruman yugtan na neurodegeneration ya sengegan na inkaderal na mitochondrial outer membrane fusion (Mfn2). Anggaman saray mutation na Mfn2 ed totoo et walay koneksion to ed sakey a klase na hereditary motor sensory neuropathy ya tatawagen ya Charcot-Marie-Tooth type 2A (15), say kondisyonal ya pakaderal na Mfn2 ed saray ayep et sakey a kabkabat ya induction of oxidation Phosphorylation (OXPHOS) dysfunction method. Saray nanduruman neuronal subtypes (16-19) tan say resulta na neurodegenerative phenotype et walay kaiba ton progresibon sintomas na neurological, a singa say movement disorders (18, 19) odino cerebellar ataxia (16). Diad pangusar na kombinasyon na label-free quantitative (LFQ) proteomics, metabolomics, imaging, tan virological iran paraan, ipapanengneng mi ya say progresibon neurodegeneration et mabiskeg ya mamapawala na pyruvate carboxylase (PCx) tan arum niran bengatla ya nalalanor ed arteriosclerosis na PNs ed vivo. Pian naseguro so kaimportantian na sayan adiskobre, espisipikon in-down-regulate mi so expression na PCx ed Mfn2-deficient PNs, tan aromog min sayan operasyon so amaloor na oxidative stress tan amapeles na neurodegeneration, kanian apaneknekan ya say azoospermia et mangiiter na cell death Metabolic adaptability. Say graben panangibalikas na MFN2 et sigpot ya makapangiliktar ed terminal degeneration PN ya walay graben kakulangan na OXPHOS, baleg ya konsumo na mitochondrial DNA, tan ompatnag ya abuyak so mitochondrial network, ya lalon mangipapabitar ya sayan klase na neurodegeneration et nayarin onabig ni ingen ed atagey lan yugtan na sakit antis ya ompatey so selula.
Pian nanengneng so mitochondria ed Mfn2 knockout PNs, angusar kami na mouse strain ya mangiiter na Cre-dependent mitochondria ya man-target na yellow fluorescent protein (YFP) (mtYFP) (20) Cre expression tan in-check so mitochondrial morphology ed vivo. Nalmoan mi ya say pakaderal na Mfn2 gene ed PNs et manresulta ed kalkalnan inkaapag na mitochondrial network (Figure S1A), tan say sankaunaan ya pananguman et aromog ed 3 simba na edad. Diad pidumaan, say baleg ya inkagba na PN cell layer, ya napatnagan ed inkaandi na Calbindin immunostaining, et ag ginmapo anggad 12 simba so edad to (Figure 1, A tan B). Say agpantatalosan na panaon ed baetan na sankaunaan ya pananguman ed morpolohiya na mitochondrial tan say nanengneng ya gapoan na impatey na neuron so amakiwas ed sikami ya imbestigaan iray pananguman ed metabolismo ya sengegan na ag-inkayadyari na mitochondrial sakbay na ipatey na selula. Nanggawa kami na fluorescence-activated cell sorting (FACS)-based ya estratehiya pian nai-isolate so YFP (YFP+)-expressing PN (Figure 1C), tan diad saray control mice (Mfn2 + / loxP :: mtYFP loxP- stop-loxP: : L7-cre), ya tatawagen ya CTRL (Figure S1BRL). Say pangi-optimize ed estratehiya na gating base ed relatibon inka-intensibo na YFP signal et mamaabuloy ed sikatayo pian nalinisan so YFP+ body (YFPhigh) na PNs manlapud saray aliwan PNs (YFPneg) (Figure S1B) odino putative fluorescent axon/dendritic fragments (YFPlow; Figure S1D, kawigi), ya akumpirma na confocal microscope (Figure S1D, kawanan). Pian na-verify so pakabidbiran na saray niklasipikan populasyon, nanggawa kami na LFQ proteomics insan principal component analysis, tan aromog mi ya walay malinew ya pandumaan na YFPhigh tan YFPneg cells (Figure S1C). Saray YFPhigh cells so angipatnag na net enrichment na saray kabkabat ya PNs markers (salanti, Calb1, Pcp2, Grid2 tan Itpr3) (21, 22), balet anggapoy enrichment na saray protina ya kaslakan ya napatnagan ed saray neuron odino arum nin klase na selula (Figure 1D) ). Say panangikompara ed baetan na saray sample ed saray klasipikadon YFPhigh cells ya naala ed saray independentin eksperimento et angipatnag na correlation coefficient> 0.9, ya mangipapanengneng na maong ya reproducibility ed baetan na saray biological replicates (Figure S1E). Diad sumaryo, sarayan datos so angipabitar ed plano mi parad maples tan espisipikon pangibiig ed posiblin PN. Lapud say L7-cre driver system ya inusar et mangiiter na mosaic recombination ed unonan simba kayari na pangiyanak (23), ginapoan min pinatey iray ayep manlapud CTRL tan conditional (Mfn2 loxP / loxP :: mtYFP loxP-stop-loxP :: L7-cre) Collect neurons. Kayari na recombination, tatawagen itan ya Mfn2cKO no 4 simba so edad to. Bilang sampot, pinili mi so 8 simba na edad nen say PN layer et intakto ni anggaman wala ray mapatnag ya mitochondrial fragments (Figure 1B tan Figure S1A). Diad inkalapagan, nabilang mi so dagup na 3013 a protina, a manga 22% ed saratan et nibase ed MitoCarta 2.0 ya anotasyon base ed mitochondrial proteome bilang mitochondria (Figure 1E) (Figure 1E) (24). Say differential gene expression analysis ya ginawa nen simba 8 et angipatnag ya 10.5% labat ed amin ya protina so walaan na signipikantin pananguman (Figure 1F tan Figure S1F), ya 195 ya protina so inmabeba tan 120 ya protina so inmatagey (Figure 1F). Nepeg ya imanoen ya say “innovative pathway analysis” na sayan data set et mangipapanengneng ya saray differentially expressed genes et manunan kabiangan na limitadon set na espisipikon metabolic pathways (Figure 1G). Makapainteres, anggaman say down-regulation na saray pathway ya misiglaotan ed OXPHOS tan calcium signaling so mangikukumpirma ed induction na mitochondrial dysfunction ed fusion-deficient PNs, arum niran kategorya ya manunan mangilalanor ed amino acid metabolism et signipikantin inmatagey, ya mipara ed metabolismo ya nagagawa ed mitochondrial PNs. Say pangi-rewire et tuloytuloy. ag-inkayadyari.
(A) Representative confocal photographs na cerebellar sections na CTRL tan Mfn2cKO ya ayep ya mangipapanengneng na progresibon pakaandi na PNs (calbindin, gray); saray nuclei et inkontra na DAPI. (B) Say bilang na (A) (one-way analysis of variance, ***P<0.001; n = 4 anggad 6 ya sirkulo manlapud taloran ayep). (C) Experimental workflow. (D) Heat map distribution na saray marker ya espisipiko ed Purkinje (tagey) tan arum niran klase na selula (pegley). (E) Venn diagram ya mangipapanengneng na bilang na mitochondrial proteins ya abitla ed niklasipika ya PN. (F) Volcano plot na saray protina ya nandurumay nipatnag da ed Mfn2cKO neurons ed loob na 8 simba (significance cut-off value ya 1.3). (G) Say creativity pathway analysis et mangipapanengneng na limaran sankaimportantian ya up-regulation (ambalanga) tan down-regulation (asul) ya dalan ed Mfn2cKO PN ya niklasipika bilang 8 simba. Nipanengneng so average ya lebel na ekspresyon na kada naimano ya protina. Grayscale heat map: inuman so P value. ns, aliwan importante.
Impanengneng na datos na proteomics ya say protein expression na complex I, III, tan IV et kalkalnan dinmaak. Saray complex I, III, tan IV et walaan na amin ya importantin mtDNA-encoded subunits, bangta say complex II, ya nuclear-coded labat, et ag-apektado (Figure 2A tan Figure S2A). . Mitunosan ed resulta na proteomics, say immunohistochemistry na saray seksion na tisyu na cerebellar so angipatnag ya say MTCO1 (mitochondrial cytochrome C oxidase subunit 1) subunit level na complex IV ed PN et kalkalnan inmabeba (Figure 2B). Say mtDNA-encoded subunit Mtatp8 et signipikantin abawasan (Figure S2A), bangta say steady-state level na nuclear-encoded ATP synthase subunit et ag anguman, ya mipara ed kabkabat ya stable ya ATP synthase subassembly F1 complex no say mtDNA expression et stable. Say inkaporma et mantutunosan. Itunda (7). Say impangusisa ed lebel na mtDNA ed saray inurnos ya Mfn2cKO PNs panamegley na real-time polymerase chain reaction (qPCR) so angikumpirma ed kalkalnan ibabawas na bilang na kopya na mtDNA. No ikompara ed grupon kontrol, diad edad ya 8 simba, manga 20% labat na mtDNA so natilak (Figure 2C). Mitunosan ed sarayan resulta, say confocal microscopy staining na Mfn2cKO PNs pian nanengneng so DNA et mangipapanengneng na panaon-dependent ya konsumo na mitochondrial nucleotides (Figure 2D). Nalmoan mi ya pigara labat ed saray kandidato ya nalalanor ed mitochondrial protein degradation tan stress response so na-up-regulate, kaiba lay Lonp1, Afg3l2 tan Clpx, tan saray OXPHOS complex assembly factors. Anggapoy naimano ya signipikantin pananguman ed saray protina ya nalalanor ed apoptosis (Figure S2B). Ontan met, aromog mi ya say mitochondria tan endoplasmic reticulum channels ya nalalanor ed transportasyon na calcium et walaan labat na daiset iran pananguman (Figure S2C). Niarum ni, say impanaral ed saray protina ya misiglaotan ed autophagy et anggapoy aromog dan signipikantin pananguman, ya mipara ed nanengneng ya induction na autophagosomes ya naobserbaan ed vivo panamegley na immunohistochemistry tan electron microscopy (Figure S3). Balet, say progresibon OXPHOS dysfunction ed PNs et kaiba na mapatnag iran pananguman ed ultrastructural mitochondrial. Saray mitochondrial clusters et nanengneng ed saray cell bodies tan dendritic trees na Mfn2cKO PNs ya manedad na 5 tan 8 simba, tan say istruktura na loob na membrane et walaan na baleg iran pananguman (Figure S4, A tan B). Mitunosan ed sarayan pananguman ed ultrastructural tan say signipikantin ibawas na mtDNA, say impanaral ed saray acute cerebral cerebellar slices ya walay tetramethylrhodamine methyl ester (TMRM) so angipatnag ya say potensyal na mitochondrial membrane ed Mfn2cKO PNs et signipikantin inmabeba (Figure S4C).
(A) Panaon ya panagsukimat ed lebel na panangibalikas na OXPHOS complex. Ikonsidera labat iray protina ya walay P<0.05 ed 8 simba (two-way ANOVA). Dotted line: Anggapoy pananguman no ikompara ed CTRL. (B) Kawigi: Sakey ya alimbawa na seksion na cerebellar ya walay marka ton anti-MTCO1 antibody (scale bar, 20 μm). Say lugar ya okupaan na Purkinje cell bodies et asakbongan na duyaw. Kawanan: Say bilang na MTCO1 levels (one-way analysis of variance; n = 7 anggad 20 ya selula ya inanalisa manlapud taloran ayep). (C) qPCR analysis na bilang na kopya na mtDNA ed in-sort ya PN (one-way analysis of variance; n = 3 anggad 7 ya ayep). (D) Kawigi: Sakey ya alimbawa na cerebellar slice ya walay marka ton anti-DNA antibody (scale bar, 20 μm). Say lugar ya okupaan na Purkinje cell bodies et asakbongan na duyaw. Kawanan: Say bilang na saray sugat ed mtDNA (one-way analysis of variance; n = 5 anggad 9 ya selula manlapud taloran ayep). (E) Sakey ya alimbawa na acute cerebellar section ya mangipapanengneng na mitoYFP + Purkinje cells (pana) diad sakey a whole-cell patch clamp recording. (F) Say bilang na IV curve. (G) Saray representatibon rekording na depolarizing current injection ed CTRL tan Mfn2cKO Purkinje cells. Sankatageyan a bakas: Say inmunan pulso ya amawala na AP. Bottom trace: Maximum AP frequency. (H) Say bilang na postsynaptic spontaneous inputs (sPSPs). Say representative recording trace tan say zoom ratio to et nanengneng ed (I). Say one-way analysis of variance et nan-analisa na n = 5 anggad 20 ya selula manlapud taloran ayep. Saray datos et nipatnag bilang mean±SEM; *P<0.05; **P<0.01; ***P<0.001. (J) Saray representatibon bakat na spontaneous AP ya inrekord ya inusar so perforated patch clamp mode. Sankatageyan a bakas: Sankatageyan a frequency na AP. Bottom trace: zoom na sakey ya AP. (K) Bilangen so average tan sankatageyan ya AP frequency unong ed (J). Mann-Whitney test; n = 5 ya selula so inanalisa manlapud apatiran ayep. Saray datos et nipatnag bilang mean±SEM; aliwan importante.
Say mapatnag ya depekto na OXPHOS et anengneng ed 8-simban Mfn2cKO PN, ya mangipapanengneng ya say physiological function na saray neurons et graben abnormal. Kanian, inanalisa mi so passive electrical characteristics na OXPHOS-deficient neurons ed 4 anggad 5 simba tan 7 anggad 8 simba diad panggawa na whole-cell patch clamp recordings ed saray acute cerebellar slices (Figure 2E). Ag-inilaloan, say average ya resting membrane potential tan input resistance na Mfn2cKO neurons et mipara ed kontrol, anggaman wala ray ag tanton pandumaan na saray selula (Table 1). Ontan met, diad edad ya 4 anggad 5 simba, anggapoy anengneng ya signipikantin pananguman ed relasyon na current-voltage (IV curve) (Figure 2F). Balet, anggapoy Mfn2cKO neurons ya 7 anggad 8 simba so akaliktar ed IV regimen (hyperpolarization step), ya mangipapanengneng ya walay malinew ya sensitivity ed hyperpolarization potential ed sayan unor lan yugtan. Diad pidumaan, diad saray Mfn2cKO neurons, saray depolarizing currents ya pansengegan na repetitive action potential (AP) discharges et maong ya itepel, ya mangipapanengneng ya say inkalapagan ya discharge patterns da et aliwan signipikantin midumaan ed saramay 8-simba lan control neurons (Table 1 tan Figure 2G). Ontan met, say frequency tan amplitude na spontaneous postsynaptic currents (sPSCs) et mipara ed saray control group, tan say frequency na saray ebento et dinmaak manlapud 4 a simba anggad 5 simba anggad 7 simba anggad 8 simba ya walaan na mipadparan indaak (Figure 2, H tan I). Say panaon na synaptic maturation ed PNs (25). Mipadparan resulta so naala kayari na saray perforated PNs patches. Sayan configuration et mangaamper ed posiblin pakabawi na saray depekto na cellular ATP, a singa nayarin nagawa ed whole-cell patch clamp recording. Diad partikular, say resting membrane potential tan say spontaneous firing frequency na Mfn2cKO neurons et ag-apektado (Figure 2, J tan K). Diad sumaryo, ipapanengneng na sarayan resulta ya saray PN ya walaan na mapatnag ya OXPHOS dysfunction et makayagel a maong ed saray high-frequency discharge patterns, ya mangipapanengneng ya walay mekanismo na kompensasyon ya mamaabuloy ed sikaran mantinien so ngalngalin normal iran electrophysiological ya ebat.
Saray datos et nipatnag bilang mean ± SEM (one-way analysis of variance, Holm-Sidak's multiple comparison test; *P<0.05). Say numero na yunit et nipanengneng ed saray bracket.
Inisip mi no kasin walay kategorya ed proteomics dataset (Figure 1G) ya walaan na saray dalan ya makakontra ed graben kakulangan na OXPHOS, diad ontan et nipaliwawa no akin ya say apektadon PN et makapansiansia na ngalngalin normal ya electrophysiology (Figure 2, E anggad K). . Impanengneng na proteomics analysis ya saray enzymes ya nalalanor ed catabolism na branched chain amino acids (BCAA) et signipikantin inmatagey (Figure 3A tan Figure S5A), tan say pinal ya produkto ya acetyl-CoA (CoA) odino succinyl CoA et nayarin makatulong ed saray tricarboxylates ed arteriosclerosis Acid (TCA) cycle. Nalmoan mi ya say karga na BCAA transaminase 1 (BCAT1) tan BCAT2 et parehon dinmaak. In-catalyze da so unonan kundang na BCAA catabolism diad impamawala na glutamate manlapud α-ketoglutarate (26). Amin ya subunits ya manitibukel ed branched chain keto acid dehydrogenase (BCKD) complex et na-upregulate (say complex so man-catalyze ed ontumbok tan agnauman ya decarboxylation na resulta na BCAA carbon skeleton) (Figure 3A tan Figure S5A). Balet, anggapoy naimano ya pananguman ed BCAA a mismo ed in-sort ya PN, ya nayarin lapud indakel na cellular uptake na sarayan importantin amino acid odino say pangusar na arum a lapuan (glucose odino lactic acid) pian nadagdag so TCA cycle (Figure S5B). Saray PN ya anggapoy OXPHOS et angipatnag met na indaak na glutamine decomposition tan transamination activities nen 8 simba so edad da, ya nayarin nanengneng ed up-regulation na mitochondrial enzymes glutaminase (GLS) tan glutamine pyruvate transaminase 2 (GPT2) (Figure 3, A tan C). Nepeg ya imanoen ya say up-regulation na GLS et limitado ed spliced ​​fisoform glutaminase C (GLS-GAC) (say pananguman na Mfn2cKO/CTRL et ngalngalin 4.5-fold, P = 0.05), tan say espisipikon up-regulation to ed saray tisyu na kanser et nayarin onsuporta ed mitochondrial bioenergy. (27).
(A) Ipapanengneng na heat map so fold change na protein level parad samay espisipikon rota ed loob na 8 simba. (B) Alimbawa na sakey a slice na cerebellar ya walay marka ton anti-PCx antibody (scale bar, 20 μm). Say duyaw ya pana et ontuturo ed laman na selula na Purkinje. (C) Time course protein expression analysis ya abitla ya importantin kandidato parad atherosclerosis (multiple t-test, *FDR <5%; n = 3-5 mice). (D) Diad tagey: Sakey ya schematic diagram ya mangipapanengneng na nanduruman paraan na iloob na carbon ya walad [1-13C]pyruvate tracer (salanti, diad panamegley na PDH odino trans-arterial route). Diad leksab: Ipapanengneng na violin chart so porsiento na single-labeled carbon (M1) ya nagmaliw ya aspartic acid, citric acid tan malic acid kayari na impangi-label ed saray acute cerebellar slices na [1-13C]pyruvate (paired t-test; ** P <0.01). (E) Komprehensibon panagsukimat ed awaran na panaon na nipanengneng a dalan. Ikonsidera labat iray protina ya walay P<0.05 ed 8 simba. Dashed line: anggapoy adjustment value (two-way analysis of variance; * P <0.05; *** P <0.001). Saray datos et nipatnag bilang mean±SEM.
Diad impanaral mi, say BCAA catabolism et nagmaliw ya sakey ed saray manunan dalan na up-regulation. Sayan katuaan et mabiskeg ya mangisusuheri ya say karakel na bentilasyon ya onloloob ed TCA cycle et nayarin manguman ed PN ya anggapoy OXPHOS. Saya so nayarin mangirerepresenta na manunan nengneng na neuronal metabolic rewiring, ya nayarin walaan na direktan epekto ed neuronal physiology tan survival legan na pamasiansia na graben OXPHOS dysfunction. Mitunosan ed sayan hypothesis, aromog mi ya say manunan anti-atherosclerotic enzyme PCx et up-regulated (Mfn2cKO/CTRL et manguuman na ngalngalin 1.5 beses; Figure 3A), ya mamapawala na pyruvate ed oxaloacetate (28), ya papanisiaan ya walad brainsutes et limitado ed astrocyte expression (29, 30). Mitunosan ed resulta na proteomics, impanengneng na confocal microscopy ya say PCx expression et espisipikon tan signipikantin inmatagey ed OXPHOS-deficient PNs, bangta say PCx reactivity et manunan limitado ed saray asingger ya Bergmann glial cells na kontrol (Figure 3B). Pian nasubok so naobserbaan ya indaak na PCx, inikdan mi na [1-13C]pyruvate tracer iray acute cerebellar slices. Nen say pyruvate et na-oxidize na pyruvate dehydrogenase (PDH), naandi so isotope label to, balet ta inlaktip ed TCA cycle intermediates no say pyruvate et na-metabolize na vascular reactions (Figure 3D). Bilang suporta ed datos mi nipaakar ed proteomics, anengneng mi so baleg a bilang na saray marker manlapud sayan tracer ed aspartic acid na Mfn2cKO slices, bangta say citric acid tan malic acid et walaan met na moderate ya trend, anggaman aliwan makabkabaliksan (Figure 3D).
Diad dopamine neurons na MitoPark ya ayep ya walay mitochondrial dysfunction ya sengegan na dopamine neurons ya espisipikon maneral ed mitochondrial transcription factor A gene (Tfam) (Figure S6B), say PCx expression et signipikantin inmatagey met (31), ya mangipapanengneng ya say acetone acid arteriosclerosis Say inkagawa na sakit et naregula legan na dysfunction na neuron OXPHOS ed laman. Nepeg ya imanoen ya nalmoan ya saray nikadkaduman enzyme (32-34) ya nayarin napatnagan ed saray neuron ya nayarin misiglaotan ed arteriosclerosis et signipikantin inmatagey ed saray PN ya anggapoy OXPHOS, a singa say propionyl-CoA carboxylase (PCC-A), say Malonyl-CoA et manguuman ed propionyl-CoA mitoconyl-CoA tan succic-Acondrial enzyme 3 (ME3), ya say manunan papel to et say pakabawi na pyruvate manlapud malate (Figure 3, A tan C) (33, 35). Niarum ni, aromog mi so signipikantin indaak na Pdk3 enzyme, ya man-phosphorylate tan diad ontan et man-inactivate na PDH (36), bangbalet anggapoy anengneng ya pananguman ed Pdp1 enzyme ya mama-activate ed PDH odino say PDH enzyme complex a mismo (Figure 3A). Diad Mern2cKO PNs, say phosphorylation na α1 subunit α (PDHE1α) subunit na pyruvate dehydrogenase E1 component na PDH complex ed Ser293 (ya kabkabat ya mangaamper ed aktibidad na enzyme na PDH) et inmaligwas ( Figure S6C) (Figure S6C). Say pyruvate et anggapoy vascular access to.
Diad kaunoran, aromog mi ya say super pathway na serine tan glycine biosynthesis, say misiglaotan a mitochondrial folate (1C) cycle tan proline biosynthesis (Figure 1G tan Figure S5C) et amin et signipikantin inmatagey, unong ed saray report, legan na proseso na panag-aktibo. Saray kaliberliber ya tisyu et na-activate lapud mitochondrial dysfunction (5-7). Say confocal analysis ya onsusuporta ed sarayan datos na proteomics et angipatnag ya diad PN ya anggapoy OXPHOS, saray cerebellar slices na 8-simban ayep so inpaarap ed serine hydroxymethyltransferase 2 (SHMT2), sakey a manunan enzyme na mitochondrial folate cycle. Makatantandan ebat na immune (Figure S5D). Diad 13 ya CU-glucose-incubated acute cerebellar slices, saray eksperimento ed metabolic tracing so lalon angikumpirma ed up-regulation na serine tan proline biosynthesis, ya mangipapanengneng ya say flux na carbon isoforms ed serine tan proline et dinmaak (Figure S5E). Lapud saray reaksion ya ipapaseseg na GLS tan GPT2 so responsable ed panaggawa na glutamate manlapud glutamine tan say transamination ed baetan na glutamate tan α-ketoglutarate, say itagey da et mangipapanengneng ya saray OXPHOS-deficient neurons et walaan na mas baleg a pankaukolan parad glutamate, Nayarin saya so gagala pian napansiansia so mas dakel a biosynthesis na proline (Figure S5). Diad pidumaan ed sarayan pananguman, say proteomic analysis na cerebellar astrocytes manlapud PN-specific Mfn2cKO ya ayep so angipatnag ya sarayan dalan (kaiba lay amin ya antiperoxidases) et ag-anguman a maong ed panangibalikas, kanian ipapanengneng na sayan metabolic redirection et mapili ed degraded PN (Fig. S6, D anggad G).
Diad sumaryo, sarayan panagsukimat so angipatnag na signipikantin nanduruman padron na temporal ya aktibasyon na espisipikon metabolic pathways ed PNs. Anggaman say abnormal ya neuronal mitochondrial function et nayarin pansengegan na masakbay ya atherosclerosis tan 1C remodeling (Figure 3E tan Figure S5C), tan anggan saray naisip ya pananguman ed panangibalikas na I tan IV complexes, saray pananguman ed serine de novo synthesis et napatnagan labat ed saray unor lan yugtan. OXPHOS dysfunction (Figure 3E tan Figure S5C). Sarayan naromog et mangiter na kabaliksan na mantutumbokan a proseso ya say stress-induced mitochondrial (1C cycle) tan cytoplasmic (serine biosynthesis) et mankakasakey ed itatagey na atherosclerosis ed TCA cycle pian nauman so neuronal metabolism.
Saray 8-simban OXPHOS-deficient PNs et makapansiansia na high-frequency excitation activity tan makadalan na signipikantin metabolic reconnection pian nabawi so mitochondrial dysfunction. Sayan adiskobre so mangiiter na makapainteres a posibilidad ya anggan diad sayan bekta, sarayan selula et nayarin makaawat met na tambal pian naatrasado odino naprebentaan so neurodegeneration. Atrasado. Naresolbe mi yan posibilidad panamegley na duaran independentin interbensyon. Diad inmunan paraan, nandesinyo kami na Cre-dependent adeno-associated virus (AAV) vector pian say MFN2 et nayarin napili ya ibalikas ed OXPHOS-deficient PNs ed vivo (Figure S7A). Say AAV ya man-encode na MFN2 tan say fluorescent reporter gene ya mCherry (Mfn2-AAV) et na-verify ed primary neuron cultures in vitro, ya sengegan na pakapatnagan na MFN2 ed Cre-dependent ya paraan tan nisalba so mitochondrial morphology, kanian naprebentaan so neuromutation ed Mfn2cKO neurons ( Figure S7, B, B tan D). Kayari to, nanggawa kami na in vivo iran eksperimento pian stereotactically niter so 8-simba ya Mfn2-AAV ed cerebellar cortex na Mfn2cKO tan control mice, tan inanalisa mi so 12-simba ya ayep (Figure 4A). Inatey so tinambal ya Mfn2cKO ya ayep (Figure 1, A tan B) (16). Say viral transduction ed vivo et nanresulta ed selective expression na PN ed pigaran cerebellar circles (Figure S7, G tan H). Say pangi-ineksion na control AAV ya walaan labat na mCherry (Ctrl-AAV) et anggapoy signipikantin epekto to ed laknab na neurodegeneration ed Mfn2cKO ya ayayep. Diad pidumaan, say impanaral ed Mfn2cKOs ya in-transduce na Mfn2-AAV et angipatnag na signipikantin proteksion ya epekto na PN cell layer (Figure 4, B tan C). Diad partikular, say karakel na neuron et singa ngalngali ag nabidbir ed saray ayep ya kontrol (Figure 4, B tan C, tan Figure S7, H tan I). Say pangibalikas na MFN1 balet aliwan MFN2 et parehon epektibo ed pangisalba ed ipapatey na neuron (Figure 4C tan Figure S7, C tan F), ya mangipapanengneng ya say pangibalikas na ectopic MFN1 et epektibon makatulong ed kakulangan na MFN2. Say kaaruman ya impanaral ed sakey a PN level et angipatnag ya say Mfn2-AAV so angiligtas ed ultrastructure na mitochondria, angi-normalize na mtDNA levels, tan angipawil ed atagey ya expression na anti-angiogenesis marker PCx ​​(Figure 4, C to E ). Say pakanengneng ed saray nisalba ya Mfn2cKO ya ayep ya manpapainawa et angipatnag ya inmaligwas so postura tan saray sintomas na motor da (galaw S1 anggad S3). Diad konklusyon, ipapanengneng na sarayan eksperimento ya say atrasadon pangiloob lamet na MFN2 ed saray PN ya kulang na OXPHOS et magenap la pian nabawi so konsumo na mtDNA tan nawalaan na atherosclerosis, diad ontan et naprebentaan so axon degeneration tan neuronal death ed vivo.
(A) Sakey ya iskema ya mangipapanengneng na eskedyul na eksperimento parad pangi-inject na AAV ya man-encode na MFN2 no say nipanengneng ya metabolic pathway et na-activate. (B) Representative confocal photographs na 12-simba ya seksion na cerebellar ya in-transduce na Mfn2cKO ya ayep ed 8 simba tan in-label na anti-Calbindin antibody. Kawanan: Pan-scale na axon fibers. Say sukat na axon zoom et 450 tan 75 μm. (C) Kawigi: Say bilang na Purkinje cell density ed AAV transduction loop (AAV+) (one-way analysis of variance; n = 3 mice). Kawanan: mtDNA focus analysis ed transduced PN nen simba 12 (unpaired t-test; n = 6 cells manlapud taloran ayep). * P <0.05; ** P <0.01. (D) Representative transmission electron micrographs na PNs na Mfn2cKO cerebellar sections ya in-transduce ed saray nipanengneng ya viral vectors. Say pink ya maskara et mangipapatnag na lugar ya okupaan na saray dendrite, tan say duyaw ya tuldok ya kuadrado et mangipapatnag na zoom ya walad nikawanan; say n et mangirerepresenta ed nucleus. Scale bar, 1μm. (E) so mangipapanengneng na alimbawa na PCx staining ed PN ya in-transduce ed 12 simba. Scale bar, 20μm. OE, sobran panangibalikas; FC, fold change.
Diad kaunoran, inimbestigaan mi so kaimportantian na peroxidase-induced cell survival ed saray PN ya akaeksperiensya na OXPHOS dysfunction. Nanggawa kami na mCherry ya man-encode na AAV-shRNA (antikey ya hairpin RNA) ya espisipikon man-target ed mouse PCx mRNA (AAV-shPCx), tan in-inject mi so virus odino say scrambled control to (AAV-scr) ed cerebellum na Mfn2cKO ya ayep. Say ineksion et ginawa nen komapat a simba na edad (Figure 5A) pian nadampot so epektibon PCx knockdown legan na panaon ya dinmaak so PCx expression (Figure 3C) tan say PN cell layer et wala ni (Figure 1A). Nepeg ya imanoen ya say pangitumba ed PCx (Figure S8A) et mangitonton ed signipikantin kapeles na ipatey na PN, ya sikato labat so limitadon singsing na impeksion (Figure 5, B tan C). Pian natalosan so mekanismo na saray epekto na metabolismo ya sengegan na PCx up-regulation, inaral mi so redox status na PNs kayari na PCx knockdown tan say AAV-mediated optical biosensor Grx1-roGFP2 so nankakasakey ya inpaway (Figure S8, B anggad D) pian nasuri so relatibon pananguman na redox peptide potential (383). Insan, nanggawa kami na two-photon fluorescence lifetime imaging microscopy (FLIM) ed saray acute brain slices na 7-simba ya Mfn2cKO odino control littermates pian nanengneng iray posiblin pananguman ed cytoplasmic redox status kayari na impan-verify ed saray kondisyon na FLIM (Figure S8, E anggad G). Say analysis so angipatnag na signipikantin indaak na oxidation state na sakey a Mfn2cKO PNs ya anggapoy PCx expression, ya duma ed saray control neurons odino Mfn2cKO PNs ya walaan labat na scrambled shRNA (Figure 5, D tan E). Nen say PCx expression et in-down-regulate, say porsiento na Mfn2cKO PNs ya mangipapanengneng na highly oxidized state et dinmaak na masulok ya taloran danay (Figure 5E), ya mangipapanengneng ya say PCx up-regulation et nansiansia so redox capacity na saray degenerated neurons.
(A) Sakey ya iskema ya mangipapanengneng na eskedyul na eksperimento parad pangi-inject na AAV ya man-encode na shPCx no say nipanengneng ya metabolic pathway et na-activate. (B) Representatibon litrato na 8-simban seksion na cerebellar ed Mfn2cKO ya ayep ya in-transduce tan in-label na anti-calcineurin antibody ed loob na 4 a simba. Scale bar, 450μm. (C) Say bilang na Purkinje cell density ed AAV-transduced loops (one-way analysis of variance; n = 3 anggad 4 ya ayep). Saray datos et nipatnag bilang mean±SEM; ***P<0.001. (D) Say representative ya litrato na FLIM et mangipapanengneng na average ya karukey na bilay na 7-simban PN ya mangipapanengneng na glutathione redox sensor Grx1-roGFP2 diad silong na saray nibaga ya kondisyon na eksperimento. LUT (look-up table) ratio: survival time interval (diad picoseconds). Scale bar, 25μm. (E) Ipapanengneng na histogram so inkaapag-apag na Grx1-roGFP2 ya balor na bilay manlapud (D) (n=158 anggad 368 ya selula ed duaran ayep ed kada kondisyon). Say pie chart ed tagey na kada histogram: et mangipapanengneng na bilang na saray selula ya walaan na mas andukey (ambalanga, oxidized) odino mas antikey (asul, nabawasan) ya balor na bilay, ya onlampas ed 1 SD na average ya bilay ed CTRL-AAV-scr. (F) Say iproproponin modelo et mangipapanengneng na proteksion ya epekto na upregulation na neuronal PCx.
Diad amin, say datos ya inter mi dia et mangipapanengneng ya say pangibalikas lamet na MFN2 et sigpot ya makapangisalba ed advanced PN ya walay graben kakulangan na OXPHOS, graben mtDNA depletion, tan alablabas ya abnormal ya ista-like morphology, kanian makapangiter na tuloytuloy ya iyaaligwas anggan diad saray advanced ya sakit. Say neurodegeneration et mangiiter na nauman ya ebidensya na yugtan antis na ipatey na selula. Sayan grado na inka-flexible na metabolismo et lalon napapabitar na abilidad na saray neuron ya mamawala na atherosclerosis (sakey a rewiring na TCA cycle), ya mangaamper ed PCx expression ed PNs ya anggapoy OXPHOS tan mamaaligwas ed ipatey na selula, kanian walay proteksion to (Figure 5F).
Diad sayan impanaral, angiter kami na ebidensya ya say ebat na PNs ed OXPHOS dysfunction et kalkalnan onsasabi ed TCA cycle atherosclerosis diad panamegley na differential activation pathway ya na-activate na saray metabolic programs. Kinompirma mi so proteomic analysis panamegley na dakel a komplementaryon paraan tan imparungtal mi ya no nasubok ed graben mitochondrial dysfunction, saray neuron et walaan na ag kabat ya nengneng na metabolic elasticity. Diad pankelaw mi, say interon proseso na rewiring et agmet kaukolan ya markaan so terminal metabolic state ya kaiba na neurodegeneration ya kalkalna tan agnauman, balet isusuheri na datos mi ya nayarin saya so manitibukel na maintenance neuron anggan diad yugtan antis na ipatey na selula Functional compensation mechanism. Ipapanengneng na sayan adiskobre ya saray neuron et walaan na baleg a grado na metabolic plasticity ed laman. Sayan katuaan so mamaneknek ya say saginonor ya pangipawil na MFN2 et makapangibalikas ed panangibalikas na saray manunan metabolic markers tan makaamper ed pakaandi na PN. Diad kasunian, saya so mangaamper ed atherosclerosis tan mamapeles ed saray nerbiyos. transsexual.
Sakey ed saray sankainteresan ya adiskobre ed impansukimat mi et saray PN ya anggapoy OXPHOS et nayarin manguman ed TCA cycle metabolism diad panamegley na pangi-up-regulate ed saray enzyme ya espisipikon mamapakiwas ed arteriosclerosis. Say metabolic rearrangement et kaslakan a kabiangan na saray selula na kanser, ya arum ed saratan et mandedependi ed glutamine pian nadagdag iray TCA cycle intermediates pian makapawala na saray reducing equivalents, ya mamapaarap ed respiratory chain tan mamasiansia ed produksyon na lipid tan nucleotide biosynthesis precursors (39 , 40). Impanengneng na sakey ya agano nin impanaral ya diad saray peripheral tissues ya walay OXPHOS dysfunction, say pankokonekta lamet na glutamine/glutamate metabolism et sakey met ya prominentin feature (5, 41), ya say direksion na iloob na glutamine ed TCA cycle et mandedependi ed Lapud inkagrabe na OXPHOS injury (41). ). Balet, anggapoy malinew ya ebidensya ed antokaman a pampareho na neuronal metabolic plasticity ed laman tan say posiblin kaimportantian to ed konteksto na sakit. Diad sakey ya in vitro ya impanaral, saray manunan cortical neurons et nipanengneng ya makapangigalaw na glutamate pools parad neurotransmission, kanian napaaligwas so oxidative metabolism tan atherosclerosis diad silong na metabolic stress conditions (42). Nepeg ya imanoen ya diad silong na pharmacological inhibition na TCA cycle enzyme succinate dehydrogenase, say pyruvate carboxylation et papanisiaan ya mamasiansia ed synthesis na oxaloacetate ed saray cultured cerebellar granule neurons (34). Balet, say physiological ya kabaliksan na sarayan mekanismo ed tisyu na utek (ya say atherosclerosis et papanisiaan ya manunan walad saray astrocytes) et walaan ni na importantin physiological ya kabaliksan (43). Diad sayan kaso, ipapanengneng na datos mi ya saray PN ya aderal na OXPHOS ed laman et nayarin iyalis ed BCAA degradation tan pyruvate carboxylation, ya duaran manunan lapuan na suplemento na TCA pool intermediates. Anggaman niproponin say kontribusyon na BCAA catabolism ed neuronal energy metabolism, nilikud ed papel na glutamate tan GABA parad neurotransmission (44), anggapo ni so ebidensya parad sarayan mekanismo ed vivo. Kanian, mainomay ya isipen ya saray dysfunctional PNs et automatikon makabawi ed konsumo na TCA intermediates ya ipapaarap na proseso na assimilation diad pamabaleg na atherosclerosis. Diad partikular, say upregulation na PCx et nayarin nakaukolan pian napansiansia so indakel na pankaukolan parad aspartic acid, ya napatnagan ed saray ondarakel ya selula ya walay mitochondrial dysfunction (45). Balet, say metabolomics analysis mi et anggapoy angipatnag na antokaman ya signipikantin pananguman ed steady-state level na aspartic acid ed Mfn2cKO PNs (Figure S6A), ya posiblin mangipapatnag na pandumaan na metabolic ya panguusar na aspartic acid ed baetan na saray proliferating cells tan post-mitotic neurons. Anggaman agni ni nabidbir so eksakton mekanismo na PCx upregulation ed saray dysfunctional neurons ed vivo, impanengneng mi ya sayan masakbay ya ebat et walay importantin betang to ed pamasiansia ed redox state na neurons, ya impanengneng ed saray eksperimento na FLIM ed saray cerebellar slices. Diad partikular, say pangisebel ed saray PN ya mangi-up-regulate ed PCx et nayarin manresulta ed mas oxidized ya estado tan mamapeles ed ipatey na selula. Say pakaaktibo na BCAA degradation tan say carboxylation na pyruvate et aliwan paraan pian nabidbir iray peripheral tissues na mitochondrial dysfunction (7). Kanian, singa ra prayuridad a kabiangan na saray neuron ya kulang na OXPHOS, anggano aliwan satan labat so kabiangan, ya importante parad neurodegeneration. .
Say sakit ed utek et sakey a klase na sakit ya manlalapud utek ya kaslakan ya ompapatnag bilang ataxia tan mabetbet ya makaderal ed saray PN (46). Sayan populasyon na neuron et mainomay ya naapektoan na mitochondrial dysfunction lapud say selective degeneration da ed saray ayep et magenap lan mamawala na dakel ed saray sintomas na motor ya pakabidbiran na human spinocerebellar ataxia (16, 47, 48). Unong ed saray report, say transgenic ya mouse model ya walay mutant gene to et walay koneksion tod human spinocerebellar ataxia tan walay mitochondrial dysfunction to (49, 50), ya mangipapabitar ed kaimportantian na panaral ed saray resulta na kakulangan na OXPHOS ed PNPH. Kanian, mas matukoy so epektibon pangibiig tan panaral ed sayan nikadkaduman populasyon na neuron. Balet, lapud saray PN et sensitibo ed pressure tan abeban proporsyon na interon populasyon na selula na cerebellar, parad dakel ya impanaral ya akabase ed omics, say mapilin pangibiig ed sikara bilang interon selula et sakey nin mairap ya aspekto. Anggaman ngalngali imposiblin nadampot so absoluton kakulangan na kontaminasyon ed arum a klase na selula (lalo la ed saray tisyu na adulto), intipon mi so epektibon dissociation step ed FACS pian nawalaan na magenap a bilang na viable neurons parad downstream proteomics analysis, tan walaan kami na medyo atagey a protein coverage (manga 3000 proteins) no ikompara ed walan datos na interon cerebel set (15). Diad pangipreserba ed inkabilay na interon selula, say paraan ya itatarya mi dia et aglabat mangiter na pankanawnawan nanengneng iray pananguman ed saray metabolic pathways ed mitochondria, noagta nanengneng met iray pananguman ed saray cytoplasmic counterparts to, ya ontutulong ed pangusar na mitochondrial membrane label enrichment ya espisipiko ed saray klase na selula. Say paraan ya indeskribe mi et aliwan misiglaotan labat ed panaral ed saray selula na Purkinje, noagta mainomay ya iyaplika ed antokaman a klase na selula pian naresolbe iray pananguman ed metabolismo na saray mansasakit ya utek, kaiba lay arum nin modelo na mitochondrial dysfunction.
Diad kaunoran, walay naimano min therapeutic window legan na sayan proseso na metabolic rearrangement ya sigpot ya makabawi ed saray manunan tanda na cellular stress tan makaamper ed neuronal degeneration. Kanian, say pakatalos ed saray implikasyon na rewiring ya adeskribe dia et nayarin mangiter na manunan pakatalos ed saray posiblin panagtambal pian napansiansia so neuronal viability legan na mitochondrial dysfunction. Say arapen ya panagsukimat ya say getma to et say pangusisa ed saray pananguman ed metabolismo na enerhiya ed arum ya klase na selula na utek et nakaukolan pian sigpot ya niparungtal so inkaaplika na sayan prinsipyo ed arum ya sakit ed utek.
Saray MitoPark mice et adeskribe la nensaman (31). Saray C57BL/6N ya ayep ya walay loxP ya akaabay ed Mfn2 genes et adeskribe la nensaman (18) tan in-cross ed L7-Cre ya ayep (23). Say resulta na doble heterozygous ya anak et in-cross ed homozygous Mfn2loxP/Mfn2loxP ya ayep pian makagawa na Purkinje-specific gene knockouts parad Mfn2 (Mfn2loxP/Mfn2loxP; L7-cre). Diad sakey a subset na panag-asawa, say Gt (ROSA26) SorStop-mito-YFP allele (stop-mtYFP) so inloob panamegley na kaaruman iran cross (20). Amin ya pamaakaran ed ayayep et ginawa unong ed saray totontonen na Europa, nasyonal tan institusyon tan inaprobaan na LandesamtfürNatur na Umwelt tan Verbraucherschutz, North Rhine-Westphalia, Germany. Say kimey ed ayayep et ontutumbok met ed panangigiya na European Federation of Laboratory Animal Sciences Associations.
Kayari na impan-anesthesia ed cervical dislocation na malukon a bii, say embryo na mouse et nibiig (E13). Say cortex et in-dissect ed Hanks' Balanced Salt Solution (HBSS) ya inaruman na 10 mM Hepes tan inpasa ed Dulbecco's Modified Eagle's Medium ya walay papain (20 U/ml) tan cysteine ​​(1μg/ml). I-incubate so tissue ed DMEM) tan i-dissociate itan panamegley na enzymatic digestion. Ml) ed 37°C ed loob na 20 minutos, insan mekanikal ya giniling ed DMEM ya inaruman na 10% fetal bovine serum. Saray selula so inseed ed saray glass coverslips ya pinaltog na polylysine ed densidad ya 2×106 kada 6 cm ya culture dish odino diad densidad ya 0.5×105 cells/cm2 parad imaging analysis. Kayari na 4 oras, say medium et sinalatan na Neurobasal serum-free medium ya walay 1% B27 supplement tan 0.5 mM GlutaMax. Saray neurons et pinansiansia ed 37°C tan 5% CO2 legan na eksperimento, tan pinakan da maminsan ed sakey simba. Pian na-induce so recombination ed vitro, 3μl (24-well culture dish) odino 0.5μl (24-well plate) na onggendan ya AAV9 virus vector so inusar pian tambalen iray neurons ed komaduan agew ed vitro: AAV9.CMV.PI.eGFP. WPRE.bGH (Addgene, catalog number 105530-AAV9) tan AAV9.CMV.HI.eGFP-Cre.WPRE.SV40 (Addgene, catalog number 105545-AAV9).
Say Mouse Mfn1 tan Mfn2 ya komplementaryon DNA (ya naala ed Addgene plasmid #23212 tan #23213, unong ed inkauksoy da) et akamarka na V5 sequence (GKPIPNPLLGLDST) diad C-terminus, tan aka-fuse ed mCherry ed frame panamegley na T2A sequence. Say Grx1-roGFP2 et regalo manlapud Heidelberg TP Dick DFKZ (Deutsches Krebsforschungszentrum). Diad impangisalat ed tdTomato cassette ya inusar so tradisyonal iran paraan na panag-clone, say cassette et in-subclone ed pAAV-CAG-FLEX-tdTomato backbone (Addgene reference number 28306) pian makagawa na pAAV-CAG-FLEX-mCherry-T2A-MFN2-V5, pAAV-CAG-G FLEX-mCherry-T2A-MFN1-V5 tan pAAV-CAG-FLEX-Grx-roGFP2 vectors. Say mipadparan estratehiya so inusar pian nagawa so control vector ya pAAV-CAG-FLEX-mCherry. Pian nagawaan so AAV-shPCx construct, nakaukolan so plasmid AAV vector (VectorBuilder, pAAV [shRNA] -CMV-mCherry-U6-mPcx- [shRNA#1]) ya walaan na DNA sequence ya mangi-encode ed shRNA ya manpupuntirya ed mouse PCx (5′CTTTCGGTAGCTAGGCTAAGGCTAAGGCTAGACTAGCTAGCTAGCTAGCT 3′) Diad silong na kontrol na U6 promoter, say mCherry et uusaren ed silong na kontrol na CMV promoter. Say produksyon na auxiliary AAV vectors et ginawa unong ed instruksion na manggagawa (Cell Biolabs). Diad antikey, mangusar na transfer plasmid ya walaan na mCherry-T2A-MFN2-V5 (pAAV-CAG-FLEX-mCherry-T2A-MFN2-V5), mCherry-T2A-MFN1-V5 (pAAV-CAG-FLEX-mCherry) ya temporaryon Transfection na 29AV-MFN2-V5-MFN2-V5, mCherry (pAAV-CAG-FLEX-mCherry) odino Grx-roGFP2 (pAAV-CAG-FLEX-Grx-roGFP2) ya coding gene, ontan met ed coding ya AAV1 capsid protein tan accessory protein Packaging plasmid plasmid, ya uusaren so calcium phosphate method. Say krudon supernatant na virus et naala panamegley na freeze-thaw cycles ed dry ice/ethanol bath tan lysed cells ed phosphate buffered saline (PBS). Say AAV vector et alinisan panamegley na discontinuous iodixanol gradient ultracentrifugation (24 oras ed 32,000 rpm tan 4°C) tan inkonsentrat ya inusar so Amicon ultra-15 centrifugal filter. Say genome titer na 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 et unong ed akauna lan indeskribe (45), say quantitative ya PCR (PCR) -MFN1-V5 (1.9×1013 GC/ml) tan AAV1-CAG-FLEX-Grx-roGFP2 (8.9×1012 GC/ml).
Saray manunan neuron et inekal ed ambetel ya 1x PBS, in-pellet, insan in-homogenize ed 0.5% Triton X-100 / 0.5% sodium deoxycholate/PBS lysis buffer ya walay phosphatase tan protease inhibitor (Roche). Say bilang na protina et ginawa diad impangusar na bicinchoninic acid assay (Thermo Fisher Scientific). Saray protina et insian panamegley na SDS-polyacrylamide gel electrophoresis, insan in-blot ed polyvinylidene fluoride membrane (GE Healthcare). I-block iray aliwan espisipikon pasen tan i-incubate ed manunan antibody (nengnengen so Table S1 parad detalye) ed 5% a gatas ed TBST (Tris-buffered saline ya walay Tween), saray kundang na panag-uras tan say segundaryon antibody ed TBST I-incubate. I-incubate ed primary antibody ed sanlabi ed +4°C. Kayari na panag-uras, iyaplika so segundaryon antibody ed loob na 2 oras diad temperatura na kuarto. Kayari to, diad pangi-incubate ed parehon blot ya walay anti-β-actin antibody, akumpirma so parehon loading. Say pakadetektaan diad pangi-convert ed chemiluminescence tan pamaaligwas ed chemiluminescence (GE Healthcare).
Saray neurons ya datin intanem ed saray glass coverslips et inayos na 4% paraformaldehyde (PFA)/PBS diad aturon panaon diad temperatura na kuarto ed loob na 10 minutos. Saray coverslips et unaan ya inlooban na 0.1% Triton X-100/PBS ed loob na 5 minuto ed temperatura na kuarto, insan inyan ed blocking buffer [3% bovine serum albumin (BSA)/PBS]. Diad komaduan agew, saray coverslips et inurasan na blocking buffer tan in-incubate na matukoy ya fluorophore-conjugated secondary antibody ed loob na 2 oras ed temperatura na kuarto; diad kaunoran, saray sample et inurasan a maong ed PBS ya walay 4′,6-diamidino-2 -Phenylindole (DAPI) ya in-counterstain insan inyan ed microscope slide ya walay Aqua-Poly/Mount.
Saray ayep (lalaki tan bii) et in-anesthesia panamegley na intraperitoneal injection na ketamine (130 mg/kg) tan xylazine (10 mg/kg) tan inikdan na carprofen analgesic (5 mg/kg) ed silong na baog, tan inyan ed stereotactic instrument (Kopf) ya walay warm. Iparungtal so bungo tan mangusar na dental drill pian manipis so parte na cerebellar cortex ya mipara ed mis bone (manlapud lambda: tail 1.8, lateral 1, mipara ed lobules IV tan V). Mangusar na kurbadon hiringgilya pian maalwar ya manggawa na melag ya abut ed bungo pian ag nadisturbo so ugat ed leksab. Insan say manipis ya ginuyod a salming a capillary et kalkalnan isuksok ed micro-hole (manlapud -1.3 anggad -1 diad ventral side na dura mater), tan 200 anggad 300 nl AAV so itusok ed micro-injector (Narishige) ya walay manual syringes (Narishige) ya aminpiga ed abeban presyon ed loob na 10 anggad 20 minuto. Kayari na infusion, iyan so capillary ed loob na 10 minutos pian sigpot ya onkayat so virus. Kayari na pangekal ed saray capillary, say baog et tahien a maong pian nabawasan so impeksion na sugat tan onabig so ayep. Saray ayayep et inikdan na analgesics (caspofen) ed loob na pigaran agew kayari na operasyon, legan na satan a panaon et maalwar ya inbantayan so kondisyon na laman da insan ira pinatey ed nibaga ya oras. Amin ya proseso et ginawa unong ed saray totontonen na Europa, nasyonal tan institusyonal tan inaprobaan na LandesamtfürNatur na Umwelt tan Verbraucherschutz, North Rhine-Westphalia, Germany.
Saray ayayep et in-anesthesia na ketamine (100 mg/kg) tan xylazine (10 mg/kg), tan say puso et in-perfuse na 0.1 M PBS ya unona, insan in-perfuse na 4% PFA ed PBS. Say tissue et in-dissect tan in-fix ed 4% PFA/PBS ed sanlabi ed 4°C. Say vibrating knife (Leica Microsystems GmbH, Vienna, Austria) so inusar pian iparaan so sagittal sections (50 μm kapal) manlapud akapeket ya utek ed PBS. Likud no walay arum ya nibaga, say panagmantsa ed saray free-floating sections et ginawa unong ed adeskribe ed tagey (13) diad temperatura na kuarto tan in-stir. Diad antikey, unona, saray naala ya slice et inpermeabilize na 0.5% Triton X-100/PBS ed loob na 15 minutos ed temperatura na kuarto; parad pigaran epitopes (Pcx tan Shmt2), diad panamegley na tris-EDTA buffer ed 80°C (PH 9) painitan iray slice ed loob na 25 minutos imbes ya sayan kundang. Insan, saray seksion so in-incubate ed primary antibody (nengnengen so Table S1) diad blocking buffer (3% BSA/PBS) ed 4°C ed sanlabi ya in-stir. Diad tinmumbok ya agew, saray seksion et inurasan na blocking buffer tan in-incubate na matukoy ya fluorophore-conjugated secondary antibody ed loob na 2 oras ed temperatura na kuarto; diad kaunoran, saray seksion et inurasan a maong ed PBS, in-counter-stain na DAPI, insan inayos na AquaPolymount Diad sakey a microscope slide.
Say laser scanning confocal microscope (TCS SP8-X o TCS Digital Light Sheet, Leica Microsystems) ya walay white light laser tan 405 diode ultraviolet laser so inusar pian nanengneng so sample. Diad pangi-excite ed fluorophore tan pangikolekta na signal panamegley na Hybrid Detector (HyDs), say LAS-X software so inusar pian mankolekta na saray aka-stack ya imahen ya ontutumbok ed Nyquist sampling diad sequential mode: parad saray non-quantitative panel, saray signal ya dinamikon tuloy (alimbawa, diad somatic cells tan dendrites) mtY UFP ed Hybrid Detector ya nanengneng so Hybrid Detector mode). Say gating ya 0.3 anggad 6 ns et inyaplika pian nabawasan so background.
Real-time ya panag-imadyin na saray na-sort ya selula. Kayari na impan-sort ed Neurobasal-A medium ya walay 1% B27 supplement tan 0.5 mM GlutaMax, saray selula so tampol ya intanem ed poly-l-lysine-coated glass slides (μ-Slide8 Well, Ibidi, catalog number 80826) , insan inyan ed 37°C tan 12% ya CO ed loob na sakey oras pian naabuloyan so selula iresolbe. Say real-time imaging et ginawa ed Leica SP8 laser scanning confocal microscope ya walay white laser, HyD, 63×[1.4 numerical aperture (NA)] oil objective lens tan heating stage.
Say mouse et maples ya in-anesthesia na carbon dioxide tan pinuter so ulo to, say utek to et maples ya inekal ed bungo to, tan pinuter ed 200μm kapal (parad 13C labeling experiment) odino 275μm kapal (parad duaran photon experiments) sagittal section ya pinano na sarayan materyales. Say Walldorf, Germany) et napno na sarayan substansia: 125 mM ice-cold, carbon-saturated (95% O2 tan 5% CO2) abeban Ca2 + artificial cerebrospinal fluid (ACSF) NaCl, 2.5 mM KCl, 1.25 mM sodium phosphate buffer, NaCl, 2.5 mM MCO2 0.5 mM CaCl2 tan 3.5 mM MgCl2 (osmotic pressure ya 310 anggad 330 mmol). Iyalis iray naala ya brain slices ed pre-incubation chamber ya walay mas atagey ya Ca2 + ACSF (125.0 mM NaCl, 2.5 mM KCl, 1.25 mM sodium phosphate buffer, 25.0 mM NaHCO3, 25.0 mM d-glucose, 10 mM Ca. MgCl2) Medium) pH 7.4 tan 310 anggad 320 mmol).
Legan na proseso na panag-imadyin, saray slice et inyalis ed sakey a kuarton panag-imahe, tan say eksperimento et ginawa ed silong na tuloytuloy ya ACSF perfusion diad pirmin temperatura ya 32° anggad 33°C. Say multiphoton laser scanning microscope (TCS SP8 MP-OPO, Leica Microsystems) ya walay Leica 25x objective lens (NA 0.95, danum), Ti: Sapphire laser (Chameleon Vision II, Coherent) so inusar parad slice imaging. FLIM module (PicoHarp300, PicoQuant).
FLIM na Grx1-roGFP2. Say pananguman ed cytoplasmic redox state na PNs et sinukat na two-photon FLIM ed sagittal brain slices, ya ditan et say Grx1-roGFP2 biosensor et nan-target ed PNs. Diad PN layer, say acquisition field et napili ed manga 50 anggad 80 μm ed leksab na tapew na slice pian naseguro ya walay viable PN (salanti, say kakulangan na beaded structure odino neuronal morphological changes ed saray dendrites) tan say double positive roGFP2 sensor tan AAV ya mangi-encode na shRNA PCx odino say sequence to ya mankokontrol ed kada sakey a mCherry expressing. Mankolekta na single-stack iran imahen ya walay 2x digital zoom [excitation wavelength: 890 nm; 512 nm 512 pixels]. Detection: internal HyD, fluorescein isothiocyanate (FITC) filter group] tan image averaging ed loob na 2 anggad 3 minuto so nausar pian naseguro ya magenap so nakolekta ya photons (1000 photons ed amin) parad curve fitting. Say inkasensitibo na Grx1-roGFP2 probe tan say panag-verify na FLIM conditions et ginawa diad panamegley na panag-monitor ed lifespan value na roGFP2 no inyarum so exogenous 10 mM H2O2 ed perfusion ACSF (pian napalaknab so oxidation, ya manresulta ed idakel na lifespan), insan inyarum so 2 mM dithiothreit (manresulta ed pambawas na degree na reduction ibawas na bilay) (Figure S8, D anggad G). Usaren so FLIMfit 5.1.1 software pian naanalisa iray naala ya resulta, i-fit so single exponential decay curve na interon imahen ed sinukat ya IRF (instrument response function), tan say χ2 et ngalngalin 1. Pian nakalkula so bilay na sakey ya PN, say mask ya akapaliber ed laman na nerbiyos et mano-mano ya indrowing, tan say average ya bilay ed kada mask et inusar.
Panagsukimat ed potensyal na mitochondria. Kayari na acute section ya in-incubate ed 100 nM TMRM ya direktan inyarum ed perfused ACSF ed loob na 30 minutos, say mitochondrial potential changes na PNs et sinukat panamegley na two-photon microscope. Say TMRM imaging et ginawa diad pangi-excite ed probe ed 920 nm tan inusar so internal HyD (tetramethylrhodamine isothiocyanate: 585/40 nm) pian makala na signal; diad pangusar na parehon excitation wavelength balet ta pangusar na sananey ya internal HyD (FITC :525/50) pian nanengneng so mtYFP. Usaren so ImageJ's Image Calculator plug-in pian nasuri so potensyal na mitochondrial ed sakey a selula. Diad antikey, say plug-in equation: signal = min (mtYFP, TMRM) et uusaren pian nabirbir so mitochondrial region ya mangipapanengneng na TMRM signal ed Purkinje Somali diad single-stack confocal image na mipetekan a channel. Insan say pixel area ed resulta na mask et nabilang, insan na-normalize ed mipetekan ya threshold single-stack image na mtYFP channel pian naala so mitochondrial fraction ya mangipapanengneng na mitochondrial potential.
Say imahen et in-deconvolute panamegley na Huygens Pro (Scientific Volume Imaging) software. Parad saray in-scan ya litrato na tile, say montage na sakey a tile et gagawaen ya uusaren so automatic stitching algorithm ya inter na LAS-X software. Kayari na panag-calibrate na imahen, usaren so ImageJ tan Adobe Photoshop pian lalon naproseso so imahen tan pareparehon na-adjust so liwawa tan pandumaan. Usaren so Adobe Illustrator parad panagparaan na graphic.
mtDNA focus analysis. Say bilang na mtDNA lesions et nabilang ed saray seksion na cerebellar ya walay antibodies sumpad DNA panamegley na confocal microscope. Kada target area et ginawa parad laman na selula tan say nucleus na kada selula, tan say nanduruman area et kinalkula ya inusar so Multi Measure plug-in (ImageJ software). Ibawas so nuclear area ed cell body area pian naala so cytoplasmic area. Diad kaunoran, say Analyze Particles plug-in (ImageJ software) so inusar pian automatikon nabilang so cytoplasmic DNA points ya mangipapanengneng na mtDNA ed threshold image, tan saray naala ya resulta et in-normalize ed PN average na CTRL ya ayep. Saray resulta et nipatnag bilang say average ya bilang na nucleosides ed kada selula.
Panagsukimat ed panangibalikas na protina. Usaren so ImageJ's Image Calculator plug-in pian na-evaluate so protein expression ed PN diad single cell level. Diad antikey, diad single-layer confocal image na mipetekan a channel, diad panamegley na equation: signal = min (mtYFP, antibody), say mitochondrial region ya mangipapanengneng na immunoreactivity ed sakey a antibody ed Purkina et nabirbir. Insan say pixel area ed resulta na mask et nabilang, insan na-normalize ed mipetekan ya threshold single-stack image na mtYFP channel pian naala so mitochondrial fraction na nipanengneng ya protina.
Purkinje cell density analysis. Say Cell Counter plug-in na ImageJ so inusar pian nasuri so Purkinje density diad pangibiig ed bilang na Purkinje cells ya abilang ed karukey na cerebellar ring ya okupaan na saray abilang ya selula.
Panagparaan tan panagkolekta na sample. Saray utek na control group tan Mfn2cKO ya ayep et in-fix ed 2% PFA/2.5% glutaraldehyde ed 0.1 M phosphate buffer (PB), insan saray coronal section et imparaan ya inusar iray ciliates (Leica Mikrosysteme GmbH, Vienna, Austria) (Kapal 50 anggad 60) Say in-fix buffer ed P1% μm tetraoxide tan 1.5% potassium ferrocyanide ed temperatura na kuarto ed loob na 1 oras. Saray seksion et inurasan na mamitlo na distilled water, insan in-stain na 70% ya ethanol ya walay 1% ya uranyl acetate ed loob na 20 minutos. Saray seksion et in-dehydrate ed graded alcohol tan inyan ed Durcupan ACM (Araldite casting resin M) epoxy resin (Electron Microscopy Sciences, catalog number 14040) ed baetan na saray silicon-coated glass slides, tan diad kaunoran et diad 60°C et in-Polymerize ed oven ed loob na 48 oras. Say cerebellar cortex area so pinili tan 50 nm ultrathin sections so pinuter ed Leica Ultracut (Leica Mikrosysteme GmbH, Vienna, Austria) tan pinuter ed 2×1 mm copper slit grid ya pinaltog na polystyrene film. Saray seksion et inkoloran na solusyon na 4% uranyl acetate ed H2O ed loob na 10 minutos, inurasan na H2O na pigaran danay, insan inurasan na Reynolds lead citrate ed H2O ed loob na 10 minutos, insan inurasan na H2O na pigaran danay. Saray mikrograpo et inala panamegley na transmission electron microscope Philips CM100 (Thermo Fisher Scientific, Waltham, MA, USA) ya inusar so TVIPS (Tietz Video and Image Processing System) TemCam-F416 digital camera (TVIPS GmbH, Gauting, USA). Alemanya).
Parad saray ayep ya walay AAV, say utek da et insian tan pinuter ed 1 mm ya kapal na sagittal section, tan say cerebellum et ineksamin ya inusar so fluorescence microscope pian nabirbir so AAV-infected ring (salanti, mCherry expressing). Saray eksperimento labat ya say AAV injection et manresulta ed atagey ya transduction efficiency na Purkinje cell layer (salanti, ngalngalin interon layer) ed ag-onkulang ed duaran mantutumbokan ya cerebellar rings so nausar. Say AAV-transduced loop et in-microdissect parad sanlabi ya post-fixation (4% PFA tan 2.5% glutaraldehyde ed 0.1 M cocoate buffer) tan inproseso ni. Parad EPON embedding, say akapeket ya tissue et inurasan na 0.1 M sodium cocoate buffer (Applichem), tan in-incubate na 2% OsO4 (os, Science Services; Caco) ed 0.1 M sodium cocoate buffer (Applichem) ed loob na 4 oras, insan inurasan ed loob na 2 oras. Uliten ya mamitlo ya walay 0.1 M cocamide buffer. Kayari to, say ontatagey ya serye na ethanol so inusar pian na-incubate so kada solusyon na ethanol ed 4°C ed loob na 15 minutos pian na-dehydrate so tissue. Say tissue et inyalis ed propylene oxide tan in-incubate ed EPON (Sigma-Aldrich) ed 4°C ed loob na sanlabi. Iyan so tissue ed balon EPON diad temperatura na kuarto ed loob na 2 oras, insan i-embed ed 62°C ed loob na 72 oras. Mangusar na ultramicrotome (Leica Microsystems, UC6) tan diamond knife (Diatome, Biel, Switzerland) pian putoten so 70 nm ya ultrathin ya seksion, tan mantsa na 1.5% uranyl acetate ed loob na 15 minutos ed 37°C, tan mantsa na lead citrate solution ed loob na 4 minutos. Saray electron micrograph et inala ya inusar so JEM-2100 Plus transmission electron microscope (JEOL) ya walay Camera OneView 4K 16-bit (Gatan) tan DigitalMicrograph software (Gatan). Parad panagsukimat, saray electron micrographs et naala ya walay 5000× o 10,000× ya digital zoom.
Morpolohiya ya panagsukimat ed mitochondria. Parad amin ya panagsukimat, saray kurti na indibidual a mitochondria so manual ya insulat ed digital iran imahen ya inusar so ImageJ software. Naanalisa so nanduruman parametro na morpolohiya. Say densidad na mitochondria et nipatnag bilang porsiento ya naala diad pangibiig ed dagup na mitochondrial area na kada selula ed cytoplasm area (cytoplasm area = cell area-cell nucleus area) × 100. Say inkabilog na mitochondria et nakalkula ed pormula ya [4π∙(area/perimeter 2)]. Say ista morphology na mitochondria et inaral tan inapag ed duaran kategorya ("tubular" tan "blister") unong ed manunan kurti ra.
Autophagosome/lysosome number tan density analysis. Usaren so ImageJ software pian manual ya i-outline so kurti na kada autophagosome/lysosome ed digital ya imahen. Say autophagosome/lysosome area et nipatnag bilang porsiento ya nakalkula diad pangibiig ed dagup na autophagosome/lysosome structure area na kada selula ed cytoplasm area (cytoplasm area=cell area-nucleus area)×100. Say karakel na autophagosome/lysosomes et nakalkula diad pangibiig ed dagup na bilang ed bilang na autophagosome/lysosome ya istruktura ed kada selula (unong ed cytoplasmic area) (cytoplasmic area = cell area-nuclear area).
Say pangi-label parad acute sectioning tan panagparaan na sample. Parad saray eksperimento ya mankaukolan na glucose labeling, iyalis iray acute brain slices ed pre-incubation chamber, ya walaan na saturated carbon (95% O2 tan 5% CO2), atagey a Ca2 + ACSF (125.0 mM NaCl, 2.5 mM KCl, 1.25 mM sodium phosphate buffer, 2.5 mM NaCl, 2.5 mM NaCl, 2.5 mM. d-glucose, 1.0 mM CaCl 2 tan 2.0 mM MgCl 2, ya in-adjust ed pH 7.4 tan 310 anggad 320 mOsm), ya say glucose et 13 C 6- Glucose substitution (Eurisotop, catalog number CLM-1396). Parad saray eksperimento ya mankaukolan na pyruvate labeling, iyalis iray acute brain slices ed mas atagey ya Ca2 + ACSF (125.0 mM NaCl, 2.5 mM KCl, 1.25 mM sodium phosphate buffer, 25.0 mM NaHCO3, 25.0 mM d-glucose, 10 mM Ca.Cl tan 20 MM Ad. MgCl2, i-adjust ed pH 7.4 tan 310 anggad 320mOsm), tan idagdag so 1mM 1-[1-13C]pyruvate (Eurisotop, catalog number CLM-1082). I-incubate iray seksion ed loob na 90 minutos ed 37°C. Diad sampot na eksperimento, saray seksion et maples ya inurasan na danum ya solusyon (pH 7.4) ya walay 75 mM ammonium carbonate, insan in-homogenize ed 40:40:20 (v:v:v) acetonitrile (ACN): methanol: danum. Kayari na impan-incubate na saray seksion ed yelo ed loob na 30 minutos, saray sample et in-centrifuge ed 21,000 g ed loob na 10 minutos ed 4°C, tan say malinew ya supernatant et pinamagaan ed SpeedVac concentrator. Say resulta na pinamaga ya metabolite pellet et insimpen ed -80°C anggad naanalisa.
Liquid chromatography-mass spectrometry analysis na 13 C-labeled amino acids. Parad liquid chromatography-mass spectrometry (LC-MS) ya panagsukimat, say metabolite pellet et insuspinde lamet ed 75μl na LC-MS grade water (Honeywell). Kayari na centrifugation ed 21,000 g ed loob na 5 minuto ed 4°C, 20 μl na pinalinew ya supernatant so inusar parad amino acid flux analysis, bangta say arum nin extract et tampol ya inusar parad anion analysis (nengnengen ed leksab). Say amino acid analysis et ginawa ya inusar so akauna lan adeskribe ya benzoyl chloride derivatization protocol (55, 56). Diad unonan kundang, 10μl na 100 mM sodium carbonate (Sigma-Aldrich) so inyarum ed 20μl na metabolite extract, insan 10μl na 2% benzoyl chloride (Sigma-Aldrich) so inyarum ed LC grade ACN. Say sample et in-vortex na antikey insan in-centrifuge ed 21,000 g ed loob na 5 minuto ed 20°C. Iyalis so alinisan ya supernatant ed 2 ml ya autosampler vial ya walay conical glass insert to (200 μl volume). Saray sample et inanalisa ya inusar so Acquity iClass ultra-high performance LC system (Waters) ya akakonekta ed Q-Exactive (QE)-HF (Ultra High Field Orbitrap) high-resolution precision mass spectrometer (Thermo Fisher Scientific). Parad panagsukimat, 2μl na derivatized sample so inyan ed 100×1.0 mm high-strength silica T3 column (Waters) ya walaan na 1.8μm particles. Say flow rate et 100μl/min, tan say buffer system et tugyopen na buffer A (10 mM ammonium formate tan 0.15% formic acid ed danum) tan buffer B (ACN). Say gradient et onia: 0%B ed 0 minuto; 0%B. 0 anggad 15% B ed 0 anggad 0.1 minuto; 15 anggad 17% B ed 0.1 anggad 0.5 minutos; B ed 17 anggad 55% ed 0.5 anggad 14 minutos; B ed 55 anggad 70% ed 14 anggad 14.5 minutos; diad 14.5 anggad 70 anggad 100% B ed 18 minutos; 100% B ed 18 anggad 19 minutos; 100 anggad 0% B ed 19 anggad 19.1 minutos; 0% B ed 19.1 anggad 28 minutos (55, 56). Say QE-HF mass spectrometer et mankikimey ed positibon ionization mode ya walay mass range ya m/z (mass/charge ratio) ya 50 anggad 750. Say inyaplikan resolusyon et 60,000, tan say gain control (AGC) ion target ya naala et 3×106, tan say sankatageyan ya ion time et 10 milliseconds. Say heated electrospray ionization (ESI) source et mankikimey ed spray voltage ya 3.5 kV, temperatura na capillary ya 250°C, sheath airflow ya 60 AU (arbitrary units), tan auxiliary airflow ya 20 AU. 250°C. Say S lens et aka-set ed 60 AU.
Anion chromatography-MS ya panagsukimat ed 13C ya walay marka ton organikon asido. Say nakekeraan ya metabolite precipitate (55μl) et inaral ya inusar so Dionex ion chromatography system (ICS 5000+, Thermo Fisher Scientific) ya inkonekta ed QE-HF mass spectrometer (Thermo Fisher Scientific). Diad antikey, 5μl na metabolite extract so in-inject ed Dionex IonPac AS11-HC column ya walay HPLC (2 mm×250 mm, particle size 4μm, Thermo Fisher Scientific) diad push-in partial loop mode ya walay filling ratio ya 1. )on. Dionex IonPac AG11-HC guard column (2 mm x 50 mm, 4μm, Thermo Fisher Scientific). Say temperatura na kolum et mansiansian 30°C, tan say autosampler et aka-set ed 6°C. Mangusar na potassium hydroxide cartridge ya walay deionized ya danum pian makagawa na potassium hydroxide gradient panamegley na eluent generator. Say pangibiig ed saray metabolites ed flow rate ya 380μl/min, ya iyaplika so onggendan ya gradient: 0 anggad 3 minuto, 10 mM KOH; 3 anggad 12 minutos, 10 anggad 50 mM KOH; 12 anggad 19 minutos, 50 anggad 100 mM KOH; 19 anggad 21 minutos, 100 mM KOH; 21 anggad 21.5 minutos, 100 anggad 10 mM KOH. Say kolum et in-equilibrate lamet ed silong na 10 mM KOH ed loob na 8.5 minutos.
Saray eluted metabolites et ipila ed 150μl/min ya isopropanol supplement stream kayari na column insan ipaarap ed high-resolution mass spectrometer ya mankikimey ed negatibon ionization mode. Say MS et manbabantay ed mass range manlapud m/z 50 anggad 750 ya walay resolusyon ya 60,000. Say AGC et aka-set ed 1×106, tan say sankatageyan ya ion time et 100 ms. Say pinainit ya ESI source et pinaandar ed spray voltage ya 3.5 kV. Saray arum ya setting na ion source et singa onggendan: temperatura na capillary 275°C; sheath gas flow, 60 AU; say auxiliary gas flow, 20 AU ed 300°C, tan say S lens setting ed 60 AU.
Say data analysis na 13C ya walay label ya metabolites. Usaren so TraceFinder software (version 4.2, Thermo Fisher Scientific) parad data analysis na isotope ratio. Say pakabidbiran na kada compound et na-verify na sakey a napanmatalkan ya reperensya ya compound tan independentin inaral. Pian nagawaan so isotope enrichment analysis, say area na extracted ion chromatogram (XIC) na kada 13C isotope (Mn) et inala manlapud [M + H] +, ya say n et say carbon number na target compound, ya inusar ed panagsukimat na amino acids odino [ MH] + so inusar ed panagsukimat na anions. Say inkasusto na masa na XIC et kulang ed limaran parte kada milyon, tan say inkasusto na RT et 0.05 minutos. Say enrichment analysis et gagawaen diad pankuenta na ratio na kada naimano ya isotope ed dagup na amin ya isotope na mipetekan a compound. Sarayan ratio et niiter bilang porsiento na kada isotope, tan saray resulta et nibalikas bilang molar percent enrichment (MPE), unong ya adeskribe la (42).
Say frozen neuron pellet et in-homogenize ed ice-cold 80% methanol (v/v), in-vortex, tan in-incubate ed -20°C ed loob na 30 minutos. I-vortex lamet so sample tan i-stir ed +4°C ed loob na 30 minutos. Say sample et in-centrifuge ed 21,000 g ed loob na 5 minuto ed 4°C, insan say resulta na supernatant et inala tan pinamagaan ya inusar so SpeedVac concentrator ed 25°C parad ontumbok ya panagsukimat. Unong ed adeskribe ed tagey, ginawa so LC-MS ya panagsukimat ed saray amino acid na saray inurnos ya selula. Diad pangusar na TraceFinder (version 4.2, Thermo Fisher Scientific), say data analysis et ginawa ya inusar so monoisotopic mass na kada compound. Say quantile normalization na metabolite data et ginawa ya inusar so preprocessCore software package (57).
Panagparaan na pispis. Say mouse et maples ya in-anesthesia na carbon dioxide tan pinuter so ulo to, say utek to et maples ya inekal ed bungo to, tan say ice-filled vibrating knife (HM-650 V, Thermo Fisher Scientific, Walldorf, Germany) so inusar pian putoten itan ed 300 anggad 375 μm sagittal sections carbon dioxide tan carbon gas (25% O) Abeban Ca2 + ACSF (125.0 mM NaCl, 2.5 mM KCl, 1.25 mM sodium phosphate buffer, 25.0 mM NaHCO3, 25.0 mM d-glucose, 1.0 mM CaCl2 tan 6.0 mM MgCl2 I-adjust ed pH 7 tan 30 mO30). Iyalis iray naala ya piraso na utek ed sakey a silir ya walaan na mas atagey a Ca2 + ACSF (125.0 mM NaCl, 2.5 mM KCl, 1.25 mM sodium phosphate buffer, 25.0 mM NaHCO3, 25.0 mM d-glucose, 4.0 ml Ca 2.2 mM tan 2.3 mM pH 7.4 tan 310 anggad 320 mOsm). Isimpen iray slice ed loob na 20 anggad 30 minutos pian napawil iratan antis ya irekord.
panagrekord. Say microscope stage ya walay fixed recording chamber to tan 20x water immersion objective lens (Scientifica) so inusar ed amin ya rekording. Saray ipapasen ya Purkinje cells et nabirbir ed (i) kabaleg na laman, (ii) kawalaan na cerebellum, tan (iii) panangibalikas na fluorescent mtYFP reporter gene. Say patch pipette ya walay resistensya na tip ya 5 anggad 11 megohms et ipapaway na borosilicate glass capillary (GB150-10, 0.86 mm×1.5 mm×100 mm, Science Products, Hofheim, Germany) tan sakey ya horizontal pipette Instruments (P-10000), Sutter, CA). Amin ya rekording et ginawa na ELC-03XS npi patch clamp amplifier (npi electronic GmbH, Tam, Germany), ya kinontrol na software Signal (version 6.0, Cambridge Electronic, Cambridge, UK). Say eksperimento et nirekord ed sampling rate ya 12.5 kHz. Say signal et na-filter na duaran short-pass Bessel filter ya walay cutoff frequency ya 1.3 tan 10 kHz. Say kapasidad na membrane tan say pipette et nabayaran na compensation circuit ya uusaren so amplifier. Amin ya eksperimento et ginawa ed silong na kontrol na Orca-Flash 4.0 camera (Hamamatsu, Gerden, Germany), ya inkontrol na Hokawo software (version 2.8, Hamamatsu, Gerden, Germany).
Rotina ya panag-configure tan panagsukimat ed interon selula. Antis ya irekord, panoen so pipette na internal solution ya walaan na sarayan substansia: 4.0 mM KCl, 2.0 mM NaCl, 0.2 mM EGTA, 135.0 mM potassium gluconate, 10.0 mM Hepes, 4.0 mM ATP (MM MM), Gunophone. (GTP) (Na) tan 10.0 mM creatinine phosphate so in-adjust ed pH 7.25, tan say osmotic pressure et 290 mOsm (sucrose). Kayari na pangiyaplika na puersa ya 0 pA pian nabuyak so membrane, say resting membrane potential et sinukat. Say input resistance et nasukat diad pangiyaplika na hyperpolarized currents ya -40, -30, -20, tan -10 pA. Sukaten so kabaleg na ebat na boltahe tan usaren so ganggan nen Ohm pian nakalkula so resistensya na input. Say spontaneous ya aktibidad et nirekord ed sakey a voltage clamp ed loob na 5 minuto, tan say sPSC et abitla tan sinukat ed Igor Pro (version 32 7.01, WaveMetrics, Lake Oswego, Oregon, USA) ya inusar so semi-automatic recognition script. Say IV curve tan steady-state current et nasukat diad pangi-clamp ed baterya ed nanduruman potensyal (manlapud -110 mV) tan pamarakep ed boltahe ed 5 mV steps. Say produksyon na AP et sinubok diad pangiyaplika na depolarizing current. I-clamp so cell ed -70 mV legan ya iyaaplika so depolarizing current pulse. I-adjust so step size na kada recording unit ya manbibiigan (10 anggad 60 pA). Kalkulaen so sankatageyan ya AP frequency diad pangibilang na mano-mano ed saray pulse spike ya sengegan na sankatageyan ya AP frequency. Say AP threshold et naanalisa diad pangusar na komaduan derivative na depolarization pulse ya unonan mamapawala na sakey odino masulok nin APs.
Konpigurasyon tan panagsukimat na perforated patch. Manggawa na perforated patch recording ya uusaren iray estandarten protocol. Mangusar na ATP- tan GTP-free pipette ya anggapoy sarayan ingredients: 128 mM gluconate K, 10 mM KCl, 10 mM Hepes, 0.1 mM EGTA tan 2 mM MgCl2, tan i-adjust ed pH 7.2 (usar so KOH). Say ATP tan GTP et inekal ed solusyon ed loob na selula pian ag nakontrol so pakaloob na selula. Say patch pipette et panoen na amphotericin-containing internal solution (manga 200 anggad 250 μg/ml; G4888, Sigma-Aldrich) pian naala so rekord na perforated patch. Say amphotericin et tinunaw ed dimethyl sulfoxide (kaunoran ya konsentrasyon: 0.1 anggad 0.3%; DMSO; D8418, Sigma-Aldrich). Say konsentrasyon na DMSO ya inusar et anggapoy signipikantin epekto to ed saray neuron ya inaral. Legan na proseso na panagsuntok, say channel resistance (Ra) et tuloytuloy ya nabantayan, tan say eksperimento et ginmapo kayari na inka-estabilisa na amplitude na Ra tan AP (20-40 minutos). Say spontaneous ya aktibidad et nasukat ed boltahe tan/odino current clamp ed loob na 2 anggad 5 minuto. Say data analysis et ginawa ya inusar so Igor Pro (version 7.05.2, WaveMetrics, USA), Excel (version 2010, Microsoft Corporation, Redmond, USA) tan GraphPad Prism (version 8.1.2, GraphPad Software Inc., La Jolla, CA). Estados Unidos). Pian nabirbir iray spontaneous APs, say NeuroMatic v3.0c plug-in na IgorPro so uusaren. Automatikon nabidbir iray AP ya uusaren so sakey a threshold, ya i-adjust ya indibidual parad kada rekord. Diad pangusar ed spike interval, amtaen so spike frequency ya walay sankatageyan ya instantaneous spike frequency tan say average ya spike frequency.
PN isolation. Diad pangiyadapta ed akaunan nipalapag ya protocol, saray PNs et nalinisan manlapud mouse cerebellum diad sakey ya espisipikon yugtan (58). Diad antikey, say cerebellum et in-dissect tan in-mince ed ice-cold dissociation medium [ya anggapoy HBSS Ca2+ tan Mg2+, ya inaruman na 20 mM glucose, penicillin (50 U/ml) tan streptomycin (0.05 mg/ ml)], insan in-digest so medium ed papain [HBSS, HCS, ya initdan na steine (1 mg / ml), papain (16 U / ml) tan deoxyribonuclease I (DNase I; 0.1 mg/ml)] Itambal ed loob na 30 minutos ed 30°C. Unona et urasan iray tisyu ed HBSS medium ya walay egg mucus (10 mg/ml), BSA (10 mg/ml) tan DNase (0.1 mg/ml) ed temperatura na kuarto pian ag-ontunaw so enzymatic, insan diad HBSS medium ya walay 20 mM glucose. (0.05 mg/ml) tan DNase (0.1 mg/ml) so mangibubuga na saksakey a selula. Say resulta na cell suspension et sinala panamegley na 70μm cell strainer, insan saray selula et in-pellet panamegley na centrifugation (1110 rpm, 5 minutos, 4°C) tan insuspension lamet ed sorting medium [HBSS, ya inaruman na 20 mM glucose, 20% fetal bovine ) Serum, pelinic (50) (0.05 mg/ml)]; usisaen so cell viability panamegley na propidium iodide tan i-adjust so cell density ed 1×106 anggad 2×106 cells/ml. Antis na flow cytometry, say suspension et sinala ed 50 μm cell strainer.
Flow cytometer. Say panag-sort na selula et ginawa ed 4°C ya inusar so FACSAria III machine (BD Biosciences) tan FACSDiva software (BD Biosciences, version 8.0.1). Say cell suspension et inurnos ya inusar so 100 μm nozzle diad pressure ya 20 psi diad rate ya ~2800 events/sec. Lapud saray tradisyonal a gating criteria (kabaleg na selula, bimodal discrimination, tan scattering characteristics) et agmakaseguro na suston pangibiig ed PN manlapud arum a klase na selula, say estratehiya na gating et niletneg base ed direktan panangikompara na YFP intensity tan autofluorescence ed mitoYFP+ ​​tan say control mitoYFP − Mice. Say YFP et na-excite diad pangi-irradiate ed sample panamegley na 488 nm laser line, tan say signal et nanengneng panamegley na 530/30 nm band pass filter. Diad mitoYFP+ ​​ya ayep, say relatibon biskeg na Rosa26-mitoYFP reporter gene et uusaren met pian nibiig so neuronal body tan axon fragments. Say 7-AAD et na-excite na 561 nm ya yellow laser tan na-detect na 675/20 nm bandpass filter pian naekal iray inatey ya selula. Pian naisian iray astrocytes ed parehon panaon, say cell suspension et in-stain na ACSA-2-APC, insan say sample et in-irradiate na 640 nm laser line, tan inusar so 660/20 nm bandpass filter pian nanengneng so signal.
Saray nakolekta ya selula et in-pellet panamegley na centrifugation (1110 rpm, 5 minutos, 4°C) tan insimpen ed -80°C anggad nausar. Saray Mfn2cKO mice tan saray litter pups da et niklasipika ed parehon agew pian nabawasan so pandurumaan na proseso. Say FACS data presentation tan analysis et ginawa ya inusar so FlowJo software (FlowJo LLC, Ashland, Oregon, USA).
Unong ya asalambit ed tagey (59), say real-time PCR et uusaren pian nibiig so DNA manlapud saray na-sort ya neurons parad ontumbok ya mtDNA quantification. Say linearity tan threshold sensitivity et inmuna ya sinubok diad pangipaakseb na qPCR ed nanduruman bilang na selula. Diad antikey, kolektaen so 300 PN ed lysis buffer ya walay 50 mM tris-HCl (pH 8.5), 1 mM EDTA, 0.5% Tween 20 tan proteinase K (200 ng/ml) tan i-incubate ed 55°C ed loob na 120 minutos. Saray selula so lalon in-incubate ed 95°C ed loob na 10 minutos pian naseguro so sigpot ya inkaandi na proteinase K. Diad impangusar na TaqMan probe (Thermo Fisher) ya espisipiko ed mt-Nd1, say mtDNA et sinukat panamegley na semi-quantitative PCR diad 7900HT Real-Time PCR system (Thermoherific Scientifics). Science, catalog number Mm04225274_s1), mt-Nd6 (Thermo Fisher Scientific, catalog number AIVI3E8) tan 18S (Thermo Fisher Scientific, catalog number Hs99999901_s1) ya genes.
Panagparaan na sample na proteome. Diad pangipainit ed solusyon ed 95°C ed loob na 10 minuto tan pangi-sonicate, diad lysis buffer [6 M guanidine chloride, 10 mM tris(2-carboxyethyl) phosphine hydrochloride, 10 mM chloroacetamide tan 100 mM tris-Lyse frozen neuron pellets ed HC pellets]. Diad Bioruptor (Diagenode) ed loob na 10 minutos (30 segundon pulso / 30 segundon pause period). Say sample et inlaok na 1:10 ed 20 mM tris-HCl (pH 8.0), inlaok ed 300 ng trypsin gold (Promega), tan inkulongan ed sanlabi ed 37°C pian nadampot so sigpot a panagtunaw. Diad komaduan agew, say sample et in-centrifuge ed 20,000 g ed loob na 20 minutos. Say supernatant et inlaok na 0.1% ya formic acid, tan say solusyon et inekal na asin na self-made StageTips. Say sample et pinamaga ed SpeedVac ya instrumento (Eppendorf concentrator plus 5305) ed 45°C, insan say peptide et insuspinde ed 0.1% formic acid. Amin ya sample et nankakasakey ya imparaan na saksakey a too. Pian naanalisa iray sample na astrocyte, 4 μg ya desalted peptides so inmarkaan na tandem mass tag (TMT10plex, catalog number 90110, Thermo Fisher Scientific) ya walay peptide to TMT reagent ratio ya 1:20. Parad TMT labeling, 0.8 mg na TMT reagent so insuspinde lamet ed 70 μl na anhydrous ACN, tan say pinamaga ya peptide et inreconstitute ed 9 μl na 0.1 M TEAB (triethylammonium bicarbonate), ya inyarum so 7 μl na TMT reagent ed ACN. Say konsentrasyon et 43.75%. Kayari na 60 minutos ya inkubasyon, say reaksion et inekal na 2 μl na 5% hydroxylamine. Saray peptide ya walay label to et tinipon, pinamaga, insuspinde lamet ed 200μl na 0.1% formic acid (FA), inapag ed duara, insan in-desalt ya inusar so self-made StageTips. Diad impangusar na UltiMate 3000 ultra high performance liquid chromatograph (UltiMate 3000 ultra high performance liquid chromatograph), sakey ed duaran kapaldua so in-fraction ed 1mm x 150mm Acquity chromatographic column ya napno na 130Å1.7μm C18 particles (Waters, catalog No: 1966366). Thermo Fisher Scientific). Ibiig iray peptide ed flow rate ya 30μl/min, ibiig so 1% anggad 50% buffer B ed loob na 85 minutos ya walay stepwise gradient ya 96 minutos, manlapud 50% anggad 95% buffer B ed loob na 3 minuto, insan 8 minuto parad 95% Buffer B; Say buffer A et 5% ACN tan 10 mM ammonium bicarbonate (ABC), tan say buffer B et 80% ACN tan 10 mM ABC. Mantipon na saray piraso kada 3 minuto tan pankasakeyen iratan ed duaran grupo (1 + 17, 2 + 18, tan arum nira) tan pamaraan iratan ed vacuum centrifuge.
LC-MS/MS ya panagsukimat. Parad mass spectrometry, saray peptide (numero r119.aq) so insian ed 25 cm, 75 μm ya inner diameter ya PicoFrit analytical column (balon objective lens, part number PF7508250) ya walaan na 1.9 μm ReproSil-Pur 120 C18-AQ medium (Maischr, Maisch-AQ), Maischr. 1200 (Thermo Fisher Scientific, Germany). Say kolum et nansiansia ed 50°C. Say buffer A tan B et 0.1% formic acid ed danum tan 0.1% formic acid ed 80% ACN, unong ed inkauksoy da. Saray peptide et insian ed stepwise gradient ya 200 nl/min manlapud 6% anggad 31% buffer B ed loob na 65 minutos, tan manlapud 31% anggad 50% buffer B ed loob na 5 minutos. Saray eluted peptides et inaral ed Orbitrap Fusion mass spectrometer (Thermo Fisher Scientific). Say panagsukat na peptide precursor m/z et gagawaen ed resolusyon ya 120,000 diad baetan na 350 anggad 1500 m/z. Diad pangusar na 27% ya normalized collision energy, say sankabiskegan ya precursor ya walay charge state ya 2 anggad 6 so napili parad high energy C trap dissociation (HCD) cleavage. Say panaon na siklo et aka-set ed 1 s. Say m/z value na peptide fragment et sinukat ed ion trap ya inusar so sankamelagan ya AGC target ya 5×104 tan say sankatageyan ya oras na injection ya 86 ms. Kayari na inkaapag-apag, say precursor et inyan ed listaan ​​na dinamikon ag-inlaktip ed loob na 45 s. Saray TMT-labeled peptides et insian ed 50 cm, 75 μm ya Acclaim PepMap column (Thermo Fisher Scientific, catalog number 164942), tan saray migration spectra et inanalisa ed Orbitrap Lumos Tribrid mass spectrometer (Thermo Fisher Scientific) ya walaan na high-field a ammetric iMS ions (IMS) say kagawaan (Thermo Fisher Scientific) et mankikimey ed duaran boltahe na −50 tan −70 V. Say MS3 ya pinili base ed synchronization precursor et uusaren parad TMT report ion signal measurement. Say peptide separation et ginawa ed EASY-nLC 1200, ya inusar so 90% linear gradient elution, ya walay buffer concentration ya 6% anggad 31%; say buffer A et 0.1% FA, tan say buffer B et 0.1% FA tan 80% ACN. Say analytical column et uusaren ed 50°C. Usaren so FreeStyle (version 1.6, Thermo Fisher Scientific) pian naapag so orihinal ya file unong ed FAIMS compensation voltage.
Say pakabidbiran tan pakabilang na protina. Diad impangusar ed integrated Andromeda search engine, say orihinal ya datos et inaral ya inusar so MaxQuant version 1.5.2.8 (https://maxquant.org/). Nilikud ed Cre recombinase tan YFP sequences ya naala ed Aequorea victoria, saray peptide fragment spectra so inanap parad canonical sequence tan isoform sequence na mouse reference proteome (Proteome ID UP000000589, ya in-download manlapud UniProt nen Mayo 2017). Say methionine oxidation tan protein N-terminal acetylation so inletneg bilang nanduruman pananguman; say cysteine ​​ficarbamoyl methylation et inletneg bilang pihon pananguman. Saray parametro na panagtunaw et aka-set ed “specificity” tan “trypsin/P”. Say sankaabebaan ya bilang na peptides tan razor peptides ya uusaren parad pakabidbiran na protina et 1; say sankaabebaan ya bilang na nikadkaduman peptide et 0. Diad silong na kondisyon na peptide map matching, say protein identification rate et 0.01. Say “Second Peptide” ya opsyon et aka-enable. Usaren so “match between runs” ya opsyon pian nipaliis iray matalonan pakabidbiran ed baetan na nanduruman orihinal iran file. Usaren so LFQ minimum ratio count 1 parad label-free quantification (LFQ) (60). Say LFQ intensity et na-filter parad ag-onkulang ed duaran balidon balor ed ag-onkulang ed sakey a grupo na genotype ed kada punto na panaon, tan na-extrapolate manlapud normal ya distribusyon ya walay kalapar na. 0.3 tan onleksab na 1.8. Usaren so Perseus computing platform (https://maxquant.net/perseus/) tan R (https://r-project.org/) pian naanalisa iray resulta na LFQ. Say two-way moderate t test manlapud limma software package so inusar parad differential expression analysis (61). Say exploratory data analysis et gagawaen ya uusaren so ggplot, FactoMineR, factoextra, GGally tan pheatmap. Say TMT-based proteomics data et inanalisa ya inusar so MaxQuant version 1.6.10.43. Anapen so raw proteomics data manlapud human proteomics database na UniProt, ya in-download nen Setyembre 2018. Say analysis et kaiba so isotope purity correction factor ya inter na manufacturer. Usaren so limma ed R parad differential expression analysis. Say orihinal ya data, resulta na panagsukimat ed database, tan data analysis workflow tan resulta et amin et aka-imbantak ed ProteomeXchange alliance panamegley na PRIDE partner repository ya walay data set identifier ya PXD019690.
Saray functional annotations so mamaaligwas ed panagsukimat. Say Ingenuity Pathway Analysis (QIAGEN) ya tool so inusar pian naamtaan so karakel na functional annotation terms na data set ed 8 simba (Figure 1). Diad antikey, say quantitative protein list ya naala ed LC-MS/MS (tandem mass spectrometry) data analysis et inusar ed sarayan filter criteria: Say Mus musculus so pinili bilang species tan background, tan say kategorya so mangipapanengneng na P value ya in-adjust nen Benjamini parad enrichment 0.05 odino abeba ni et ipapasen ya makabkabaliksan. Parad sayan graph, nipanengneng so sankatageyan ya limaran sobran kategorya ed kada cluster base ed inuman ya P value. Diad pangusar na multiple t-test, ya uusaren so two-stage linear boost program di Benjamini, Krieger, tan Yekutieli (Q = 5%), say time-course protein expression analysis et ginawa ed saray importantin kandidato ya abitla ed kada kategorya, tan kada hilera et inaral ya manbibiigan. Anggapoy kaukolan ya mangagamil na tuloytuloy ya SD.
Pian nakompara so resulta na sayan impanaral ed saray nipalapag ya database tan makagawa na Venn diagram ed Figure 1, intipon mi so quantitative protein list ed MitoCarta 2.0 annotations (24). Usaren so online tool ya Draw Venn Diagram (http://bioinformatics.psb.ugent.be/webtools/Venn/) pian nagawaan so diagram.
Parad detalyadon impormasyon nipaakar ed saray statistical procedures ya inusar parad proteomics analysis, nengnengen so mipetekan a seksion na Materials and Methods. Parad amin ya arum ya eksperimento, say detalyadon impormasyon et naromog ed mipetekan ya alamat. Likud no walay arum ya nibaga, amin ya datos et nibalikas bilang mean ± SEM, tan amin ya statistical analyses et ginawa ya inusar so GraphPad Prism 8.1.2 software.
Parad kaaruman iran materyales parad sayan artikulo, nengnengen so http://advances.sciencemag.org/cgi/content/full/6/35/eaba8271/DC1
Saya so open access ya artikulo ya ibubunog unong ed saray kondisyon na Creative Commons Attribution-Non-Commercial License, ya mangiiter na pankanawnawan mangusar, mangibunog tan manpawala lamet ed antokaman a medium, basta say unor ya pangusar et aliwan parad komersyal a gunggona tan say premis et duga so orihinal ya kimey. Reperensya.
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Insulat nen E. Motori, I. Atanassov, SMV Kochan, K. Folz-Donahue, V. Sakthivelu, P. Giavalisco, N. Toni, J. Puyal, N.-G. Larson
Say proteomics analysis na dysfunctional neurons et angipatnag ya saray metabolic programs et na-activate pian nasumpal so neurodegeneration.
Insulat nen E. Motori, I. Atanassov, SMV Kochan, K. Folz-Donahue, V. Sakthivelu, P. Giavalisco, N. Toni, J. Puyal, N.-G. Larson
Say proteomics analysis na dysfunctional neurons et angipatnag ya saray metabolic programs et na-activate pian nasumpal so neurodegeneration.
©2020 American Association for the Advancement of Science. akareserba so amin a kanepegan. Say AAAS et partner na HINARI, AGORA, OARE, CHORUS, CLOCKSS, CrossRef tan COUNTER. ScienceAdvances ISSN 2375-2548.