I kare ni *Kabedo me kare ni: Cologne 50931, Germany, Cologne Excellence Cluster Research on Cellular Stress Response in Aging-related Diseases (CECAD).
Rweny pa two ma mako dul kom ma ki lwongo ni neurodegeneration ni ki ngeyo ni pe kitwero dwoko cen pien kit ma dul kom ma tiyo kwede i kom dano nok, ento adwogi pa tic pa dul kom ma pe tiyo maber i kom kit ma kom dano tiyo kwede i kom dano pe ki ngeyo maber. Kany, wa kelo cell-specific proteome pa Purkinje neurons ki OXPHOS ma tye ka mede ma oa ki i tic pa mitochondrial fusion ma obale. Wanongo ni tic pa mitochondrial okelo alokaloka madit i dul pa proteomics, ma okelo tic pa jami ma kwako kom ma peya cell oto. Labongo akalakala, wa nongo ni jami ma ki lwongo ni pyruvate carboxylase (PCx) ki peroxidases mukene ma mako medo i kom TCA cycle intermediates. Gengo PCx okelo peko me oxidative ki neurodegeneration, ma nyutu ni atherosclerosis tye ki adwogi me gwoko neurons ma pe ki OXPHOS. Dwogo pa dul kom ma ki lwongo ni mitochondrial fusion i dul kom ma dong obale loko kit ma kom dano tiyo kwede, ma gengo to pa cell. Gin ma waniano nyutu yoo ma onongo pe ki ngeyo ma miyo teko i kom tic pa mitochondrial ki nyutu ni goro pa wic twero dwogo cen kadi wa i agiki me two ni.
Tic ma pire tek pa mitochondria me gwoko teko pa dul kom ma ki lwongo ni neuronal metabolism ki nyutu ki lanyut mapol ma mako wic ma mako two ma mako mitochondria pa dano. Pol pa two magi oa ki i alokaloka me gene ma doro tic pa gene me mitochondria (1, 2) onyo balo gene ma mako kit ma mitochondrial tiyo kwede, ma weko mitochondrial DNA (mtDNA) bedo maber (3, 4). Tic ma kitimo i kom ler onyutu ni me gamo peko me tic pa mitochondrial i kom ma orumowa, yoo ma gwoko kom (5-7) twero tic, ma miyo ngec ma pire tek i kom kit ma two magi dongo kwede. I apoka poka, niang wa i kom alokaloka me kom pa dul kom ma pat pat ma oa ki i rwenyo tic pa dul kom ma ki lwongo ni adenosine triphosphate (ATP) tye gin ma pire tek (8), ma nyutu miti me nongo yoo me cang ma twero tic me gengo nyo gengo two. Gengo two ma mako wic (9). Pek pa ngec tye ni dul kom ma ki lwongo ni nerve cells ki ngeyo ni gitye ki kero me loko jami ma mako kom ka kiporo ki cell ma tye i kom dul kom ma orumowa (10). Ki miino ni cell magi gi tiyo tic ma pire tek me yubu jami ma kelo alokaloka bot neurons me kelo cwalo jami ki gamo peko ki two, kero me loko kit ma cell tiyo kwede me rwate ki peko me dul kom ma i wic tye cok ki glial cells (11-14). Medo ikom meno, kit ma dul kom ma pat pat ma tye i dul kom ma i wic gengo kwan i kom alokaloka ma time i dul kom ma pat pat. Kit ma adugi ne, jami manok aye ki ngeyo i kom adwogi pa cell ki metabolic ma bino pi tic pa mitochondrial i neurons.
Me niang adwogi pa tic pa mitochondrial, wa poko Purkinje neurons (PNs) i rwom ma pat pat me rweny pa neurodegeneration ma oa ki i balo mitochondrial outer membrane fusion (Mfn2). Kadi bed ni Mfn2 ma ki loko i kom dano rwate ki kit two ma ki lwongo ni Hereditary motor sensory neuropathy ma ki ngeyo ni Charcot-Marie-Tooth type 2A (15), balo Mfn2 i kom oyoo obedo gin ma ki ngeyo maber ma kelo peko me tic pa oxidation Phosphorylation (OXPHOS). Kit dul kom ma pat pat (16-19) ki kit ma dul kom ma balo wic gi bino kwede ki lanyut ma mako wic, ma calo peko me ngwec (18, 19) onyo ataxia me wic (16). Nono tic ki dul ma ki lwongo ni label-free quantitative (LFQ) proteomics, metabolomics, imaging, ki yoo me virological, wanyutu ni dongo pa neurodegeneration kelo pyruvate carboxylase (PCx) ki jami mukene ma kwako arteriosclerosis pa PNs in vivo Nyutu pa enzymes. Me moko ni gin ma kinongo ni tye kakare, wa dwoko piny rwom me nyutu pa PCx i Mfn2-deficient PNs, ki wanongo ni tic man omedo peko me oxidative ki ojingo goro pa wic, ma nyutu ni azoospermia miyo to pa cell Metabolic adaptability. Nyutu matek pa MFN2 twero laro PN ma tye ka rweny woko ki peko me OXPHOS, tic ki DNA me mitochondrial madwong, ki nen calo mitochondrial network ma otur, ma medo nyutu ni kit me rweny pa wic ni twero cang i kare ma two dong okato ma peya cell otoo.
Me neno mitochondria i Mfn2 knockout PNs, watiyo ki dul ler ma weko Cre-dependent mitochondria me keto yellow fluorescent protein (YFP) (mtYFP) (20) Cre expression ki neno kit ma mitochondrial nen kwede i kom dano. Wanongo ni balo Mfn2 gene i PNs obi kelo poko kin mitochondrial network (lanyut S1A), ki alokaloka ma okwongo ni kinongo i cabit 3 me mwaka. Ka i poro, rweny pa PN cell layer, ma ki nyutu ki rwenyo pa Calbindin immunostaining, pe oyabe nio wa i cabit 12 me mwaka (lanyut 1, A ki B). Kare ma pe rwate i kin alokaloka macon i kit ma mitochondrial nen kwede ki acaki me to pa neurons oweko wa kwedo alokaloka ma bino pi tic pa mitochondrial ma peya cell oto. Wa yubu yoo me poko cell ma ki lwongo ni fluorescence-activated cell sorting (FACS) me poko YFP (YFP+)-ma nyutu PN (lanyut 1C), ki i kom oyot ma ki lwongo ni control mice (Mfn2 + / loxP :: mtYFP loxP- stop-loxP: : L7-cre), ma ki lwongo ni CTRL (lanyut S1BRL). Kit ma ki yubu kwede yoo me donyo ma lubu rwom me tic pa YFP miyo wa twero me lonyo kom YFP+ (YFPhigh) pa PNs ki i kom non-PNs (YFPneg) (lanyut S1B) onyo putative fluorescent axon/dendritic fragments (YFPlow; cal S1D, tung lacuc), ma ki moko ki confocal microscope (lanyut S1D tung acuc). Me moko ni dul ma ki poko ni tye, wa timo LFQ proteomics ki dong wa timo analysis i kom dul ma pire tek, ki wa nongo ni tye apoka poka ma nen maber i kin YFPhigh ki YFPneg cells (lanyut S1C). YFPhigh cells onyutu rwom me dongo pa PNs ma ki ngeyo (ie Calb1, Pcp2, Grid2 ki Itpr3) (21, 22), ento pe dongo pa proteins ma pol kare ki nyutu i neurons onyo cell mukene (lanyut 1D) ). Apoka poka ma tye i kin jami ma ki kwanyo ki i YFPhigh cells ma ki kwanyo ki i yub ma pat pat onyutu ni rwom me rwate> 0.9, ma nyutu ni twero bedo maber i kin jami ma ki dwoko cen (lanyut S1E). I agiki ne, ngec eni moko yub wa pi poko kin PN ma twero time. Pien L7-cre driver system ma ki tiyo kwede kelo rwate i cabit mukwongo inge nyodo (23), wa cako neko oyot ki i CTRL ki conditional (Mfn2 loxP / loxP :: mtYFP loxP-stop-loxP :: L7-cre) Coko neurons. Iyonge tyeko rwate, ki lwongo ni Mfn2cKO i cabit me 4 me mwaka. Ma calo agiki, wa yero cabit 8 me mwaka ma dul kom PN onongo pud tye maber kede ma onongo tye ki dul kom ma nen (lanyut 1B ki cal S1A). I kom weny, wa kwano wel 3013 pa proteins, ma romo 22% onongo jenge i kom MitoCarta 2.0 ma jenge i kom mitochondrial proteome calo mitochondria (lanyut 1E) (lanyut 1E) (24). Kwedo gin ma ki lwongo ni differential gene expression analysis ma kitimo i cabit me 8 onyutu ni 10.5% keken pa proteins weng onongo tye ki alokaloka madit (lanyut 1F ki cal S1F), ma i kine 195 proteins onongo tye piny ki 120 proteins onongo tye up-regulated (lanyut 1F). Ber me poyo ni "kwano yoo manyen" pa jami ma ki nongo ni nyutu ni gene ma ki nyutu ma pat pat ni tye i dul ma ki gengo me tic ki jami ma kwako kom (lanyut 1G). I yo me miti, kadi bed ni dwoko piny yoo ma mako OXPHOS ki calcium nyutu ni mitochondrial dysfunction i PNs ma pe ki rwate, dul mukene ma kwako amino acid metabolism gi medo rwom gi, ma rwate ki metabolism ma time i mitochondrial PNs. loko waya tye maber. tic ma pe tiyo.
(A) cal ma ki kwanyo ma nyutu dul kom ma ki lwongo ni cerebellar pa CTRL ki Mfn2cKO ma nyutu rweny pa PNs (calbindin, gray); nuclei ni ki keto i DAPI. (B) Kwan me (A) (one-way analysis of variance, ***P<0.001; n = 4 to 6 circles from three mice). (C) Kit me tic ma ki temo. (D) Poko pa lanyut ma mako Purkinje (i wi) ki dul cell mukene (i kin). (E) cal pa Venn ma nyutu wel jami ma ki lwongo ni mitochondrial proteins ma ki nongo i PN ma ki poko. (F) Volcano plot pa jami ma ki nyutu ma pat pat i Mfn2cKO neurons i cabit 8 (gin ma pire tek ma ki keto me 1.3). (G) Kwedo yoo me yubu jami ma ki yubu nyutu yoo abic ma pire tek ma ki lwongo ni up-regulation (red) ki down-regulation (blue) pathways i Mfn2cKO PN ma ki poko ni cabit 8. Ki nyutu rwom me nyutu pa protein acel acel ma kinongo. Grayscale heat map: ki loko P value. ns, pe pire tek.
Lok ma ki nongo ki i kom jami ma ki lwongo ni Proteomics onyutu ni rwom me tic pa jami ma ki lwongo ni complexes I, III, ki IV odok piny mot mot. Complex I, III, ki IV weng tye ki mtDNA-encoded subunits, kun complex II, ma onongo tye ki nuclear-coded keken, onongo pe ki balo (lanyut 2A ki cal S2A). . Ma rwate ki adwogi me proteomics, immunohistochemistry pa dul kom ma ki lwongo ni cerebellar tissue onyutu ni MTCO1 (mitochondrial cytochrome C oxidase subunit 1) subunit level me complex IV i PN odok piny mot mot (lanyut 2B). Gin ma ki lwongo ni mtDNA-encoded subunit Mtatp8 ni odok piny tutwal (lanyut S2A), ma nongo rwom me nuclear-encoded ATP synthase subunit odong ma pe oloko, ma rwate ki rwom ma ki ngeyo ni ATP synthase subassembly F1 complex ka mtDNA expression tye maber. Kit ma ki yubu kwede tye ma rwate. Turo lok (7). Temo rwom me mtDNA i Mfn2cKO PNs ma ki poko ki real-time polymerase chain reaction (qPCR) moko ni wel mtDNA odok piny mot mot. Ka kiporo ki gurup ma ki temo, i cabit 8 me mwaka, 20% keken me rwom me mtDNA aye ki gwoko (lanyut 2C). Ma rwate ki adwogi eni, jami ma ki lwongo ni confocal microscopy staining pa Mfn2cKO PNs me nongo DNA nyutu kare ma ki tiyo kwede me mitochondrial nucleotides (lanyut 2D). Wanongo ni jami mogo ma tye i mitochondrial protein degradation ki stress response aye ki dwoko piny, medo ki Lonp1, Afg3l2 ki Clpx, ki OXPHOS complex assembly factors. Pe ki nongo alokaloka mo ma pire tek i rwom me protein ma tye i apoptosis (lanyut S2B). I yo acel-lu, wanongo ni mitochondria ki endoplasmic reticulum channels ma tye i tic me tero calcium tye ki alokaloka manok keken (lanyut S2C). Medo ikom meno, kwedo jami ma mako autophagy-related proteins pe onongo alokaloka mo ma pire tek, ma rwate ki neno ma ki neno i kom autophagosomes ma ki neno i kom dano ki immunohistochemistry ki electron microscopy (lanyut S3). Ento, tic pa OXPHOS ma tye ka mede i PNs bino ki alokaloka ma nen i kom dul kom ma ki lwongo ni ultrastructural mitochondrial. Mitochondrial clusters ki twero neno gi i kom cell ki yadi ma ki lwongo ni dendritic trees pa Mfn2cKO PNs ma mwaka gi tye 5 ki 8 cabit, ki dul kom ma i iye ni okato ki i alokaloka madit (lanyut S4, A ki B). Ma rwate ki alokaloka magi ki dwoko piny ma pire tek i mtDNA, kwedo dul kom ma ki lwongo ni acute cerebral cerebellar slices ki tetramethylrhodamine methyl ester (TMRM) onyutu ni teko pa dul kom ma ki lwongo ni mitochondrial membrane i Mfn2cKO PNs onongo tye ma piny tutwal (lanyut S4C).
(A) Kwano kare me nyutu rwom me nyutu pa OXPHOS complex. Tam i kom protein ma tye ki P<0.05 i cabit 8 (two-way ANOVA). Lain ma ki keto: Pe ki loko ka kiporo ki CTRL. (B) Tung cem: Labol pa dul kom ma ki keto iye anti-MTCO1 antibody (scale bar, 20 μm). Kabedo ma kom ler ma ki lwongo ni Purkinje tye iye ki umo ki yellow. Tung acuc: Kwano rwom me MTCO1 (kwano yoo acel me apoka poka; n = 7 onyo 20 cell ma ki kwano ki i kom oyoo adek). (C) qPCR nyamo namba pa mtDNA i PN ma ki yero (nyamo ma pat pat; n = 3 onyo 7 oyoo). (D) Tung lacuc: Labol pa dul kom ma ki lwongo ni cerebellar slice ma ki keto iye anti-DNA antibody (scale bar, 20 μm). Kabedo ma kom ler ma ki lwongo ni Purkinje tye iye ki umo ki yellow. Tung acuc: Kwano wel mtDNA ma obale (kwano yoo acel me apoka poka; n = 5 onyo 9 cell ki i kom oyoo adek). (E) Labol pa dul kom ma ki lwongo ni acute cerebellar ma nyutu mitoYFP + Purkinje cells (arrow) i dul kom ma ki lwongo ni whole-cell patch clamp recording. (F) Kwanyo wel pa IV curve. (G) Gin ma ki coyo ma nyutu kit ma ki keto kwede depolarizing current i CTRL ki Mfn2cKO Purkinje cells. Labol ma malo: gin ma ki lwongo ni pulse mukwongo ma okelo AP. Labol ma piny: Wel AP ma malo. (H) Kwano wel jami ma bino pi tic ma pe ki ngeyo (sPSPs). Gin ma ki mako ma ki nyutu ki rwom me dongo ne ki nyutu i (I). Kwedo yoo acel me apoka poka ma ki kwano n = 5 onyo 20 cell ki i kom oyoo adek. Ngec ni ki nyutu calo mean±SEM; *P<0.05; **P<0.01; ***P<0.001. (J) Gin ma nyutu AP ma ki coyo kun kitiyo ki yoo me mako gin ma ki lwongo ni perforated patch clamp. Labol ma malo: rwom me AP ma malo. Labol ma piny: medo AP acel. (K) Kwan wel ma rwate ki ma malo pa AP ma lubu (J). Temo pa Mann-Whitney; n = 5 cell ma ki kwano ki i kom oyoo angwen. Ngec ni ki nyutu calo mean±SEM; pe pire tek.
Balo pa OXPHOS ma nen i Mfn2cKO PN ma tye cabit 8, ma nyutu ni tic pa dul kom ma ki lwongo ni neurons ni tye ma pi opore. Pi meno, wa kwano kit ma teko pa OXPHOS-deficient neurons tiyo kwede i cabit 4 onyo 5 ki cabit 7 onyo 8 kun wa timo jami ma ki mako i cell ma ki lwongo ni acute cerebellar slices (lanyut 2E). Labongo akalakala, rwom me yweyo pa dul kom ma ki lwongo ni Mfn2cKO neurons onongo rom ki ma ki temo, kadi bed ni onongo tye apokapoka manok i kin cell (Table 1). I yo acel-lu, i cabit me 4 onyo 5 me mwaka, pe ki nongo alokaloka mo ma pire tek i kin rwom me teko (IV curve) (lanyut 2F). Ento, pe tye Mfn2cKO neurons ma mwaka gi tye 7 onyo 8 ma okato ki i IV regimen (hyperpolarization step), ma nyutu ni tye rwom me hyperpolarization i kare me agiki ni. Ka i poro, i Mfn2cKO neurons, depolarizing currents ma kelo repetitive action potential (AP) ma ki cwalo maber, ma nyutu ni kit ma gi cwalo kwede pe pat ki pa 8-week-old control neurons (Table 1 ki Figure 2G). I yo acel-lu, pol pa jami ma time i nge kom (sPSCs) onongo rom ki pa gurup ma ki temo, ki pol pa jami ma otimme omede ki i cabit 4 nio wa i cabit 5 nio wa i cabit 7 nio wa i cabit 8 ki medo ma rom (lanyut 2, H ki I). Kare me dongo pa jami ma ki lwongo ni synaptic i PNs (25). Adwogi ma rom ki man kinongo inge keto PNs ma ki buru. Kit ma ki yubu kwede ni gengo cul ma twero bedo tye pi peko me ATP ma tye i cell, ma twero time i kom rekod pa cell ducu. I kom lok, teko pa dul kom ma ki lwongo ni resting membrane potential ki spontaneous firing frequency pa Mfn2cKO neurons pe ki balo (lanyut 2, J ki K). I agiki ne, adwogi eni nyutu ni PNs ma tye ki peko me OXPHOS twero tic maber ki rwom me ngwec ma malo, ma nyutu ni tye yoo me culo kwor ma weko gi gwoko rwom me tic pa mac ma rwate.
Ngec ni ki nyutu calo rwom me ± SEM (one-way analysis of variance, Holm-Sidak's multiple comparison test; *P<0.05). Namba me dul ni ki nyutu i kin braket.
Wa keto yub me kwedo ka ce dul mo kiken i kin jami ma ki nongo ki ikom jami makwo (lanyut 1G) tye ki yoo ma twero juku peko me OXPHOS, ma weko wa tito pingo PN ma obale twero gwoko electrophysiology ma rwate (lanyut 2, E to K). . Kwedo jami ma ki lwongo ni Proteomics onyutu ni enzymes ma tye i catabolism pa branched chain amino acids (BCAA) onongo tye ka mede malo (lanyut 3A ki cal S5A), ki gin ma ki yubu me acetyl-CoA (CoA) onyo succinyl CoA twero medo tricarboxylates i arteriosclerosis Acid (TCA) cycle. Wanongo ni wel BCAA transaminase 1 (BCAT1) ki BCAT2 weng omede. Gin gu kelo rwom me acel me BCAA catabolism kun gi kelo glutamate ki i α-ketoglutarate (26). Jami weny ma gi yubu dul ma ki lwongo ni branched chain keto acid dehydrogenase (BCKD) complex ni ki medo gi (complex ni kelo decarboxylation ma lubo ki ma pe kitwero dwoko cen pa BCAA carbon skeleton) (lanyut 3A ki cal S5A). Ento, pe ki nongo alokaloka mo ma nen i BCAA kikome i PN ma ki poko, ma twero bedo pi medo rwom me tic pa amino acid magi ma pire tek onyo tic ki jami mukene (glucose onyo lactic acid) me medo TCA cycle (lanyut S5B). PNs ma pe ki OXPHOS bene onyutu dongo pa glutamine decomposition ki transamination i cabit 8 me mwaka, ma twero nen ki i rwom ma malo pa mitochondrial enzymes glutaminase (GLS) ki glutamine pyruvate transaminase 2 (GPT2) (lanyut 3, A ki C). Ber me poyo ni rwom me dongo pa GLS tye ma ki keto i kom gin ma ki lwongo ni spliced ​​fisoform glutaminase C (GLS-GAC) (alokaloka pa Mfn2cKO/CTRL tye ma romo 4.5-fold, P = 0.05), ki rwom me dongo ne i kom ler ma tye ki two kanca twero konyo mitochondrial bioenergy. (27).
(A) Map me lyeto nyutu alokaloka i rwom me protein pi yoo ma ki moko i cabit 8. (B) Labol pa dul kom ma ki lwongo ni anti-PCx antibody (scale bar, 20 μm). Lanyut ma yellow ni nyutu kom cell me Purkinje. (C) Kwedo kit ma protein nyute kwede i kare ma kinongo ni obedo gin ma pire tek pi two ma mako remo (multiple t-test, *FDR <5%; n = 3-5 mice). (D) Malo: cal ma nyutu yoo ma pat pat me donyo i [1-13C]pyruvate tracer (ma calo, ki i PDH onyo trans-arterial route). Piny: Karatac me violin nyutu wel pa single-labeled carbon (M1) ma ki loko doko aspartic acid, citric acid ki malic acid ingee keto lanyut i kom dul kom ma ki lwongo ni acute cerebellar slices ki [1-13C]pyruvate (paired t-test; ** P <0.01). (E) Kwano lok ma mako kare ma ki nyutu ni. Tam i kom proteins ma tye ki P<0.05 i cabit 8 keken. Dashed line: pe tye wel ma ki loko (kwedo yoo aryo me apoka poka; * P <0.05; *** P <0.001). Ngec ni ki nyutu calo mean±SEM.
I kwedo wa, BCAA catabolism odoko yoo acel ma pire tek me dongo. Lok man nyutu ni yamo ma donyo i TCA twero loko i PN ma pe ki OXPHOS. Man twero nyutu kit ma pire tek me dwoko rwom me tic pa ler ma i wic, ma twero bedo ki adwogi i kom kit ma ler tiyo kwede ki kwo i kare me gwoko peko me OXPHOS. Ma rwate ki tam man, wanongo ni enzyme ma gengo two ma mako ler ma ki lwongo ni PCx tye ma ki medo (Mfn2cKO/CTRL loko tyen 1.5; cal 3A), ma weko pyruvate loko doko oxaloacetate (28), ma ki geno ni tye i kom ler ma ki lwongo ni brainsue. (29, 30). Ma rwate ki adwogi me proteomics, confocal microscopy onyutu ni PCx expression onongo tye ka mede i OXPHOS-deficient PNs, kun PCx reactivity onongo tye ka cung matek i kom Bergmann glial cells ma tye cok ki labol (lanyut 3B). Me temo tic pa PCx, wa tiyo ki dul kom ma ki lwongo ni acute cerebellar slices ki [1-13C]pyruvate tracer. I kare ma pyruvate onongo oxidized ki pyruvate dehydrogenase (PDH), isotope label ne orweny woko, Ento ki medo i TCA cycle intermediates ka pyruvate ki loko ki vascular reactions (lanyut 3D). Me miiyo kony bot ngec ma mako jami ma ki lwongo ni proteomics, waneno wel lanyut mapol ki i kom gin ma ki lwongo ni aspartic acid pa Mfn2cKO slices, kun citric acid ki malic acid bene onongo tye ki rwom ma lapiny, kadi bed ni pe pire tek (lanyut 3D).
I kom dul kom ma ki lwongo ni dopamine neurons pa MitoPark mice ma tye ki goro pa mitochondrial ma oa ki i dopamine neurons ma tye ka balo mitochondrial transcription factor A gene (Tfam) (lanyut S6B), nyutu pa PCx bene onongo tye ka mede (31), ma nyutu ni acetone acid arteriosclerosis. OXPHOS i kom. Ber me poyo ni kinongo ni enzymes ma pat pat (32-34) ma twero bedo ni ki nyutu i neurons ma twero bedo ki wat ki arteriosclerosis gi tye ka mede i PNs ma pe ki OXPHOS, calo propionyl-CoA carboxylase (PCC-A), Malonyl-CoA loko propionyl me doko propionyl-CoA ki succilchondrial enzyme 3 (ME3), ma tic ne madit tye me nongo pyruvate ki i malate (lanyut 3, A ki C) (33, 35). Medo ikom meno, wanongo ni Pdk3 enzyme omede, ma phosphorylates ki dong inactivate PDH (36), kun nongo alokaloka mo pe ki nongo i Pdp1 enzyme ma weko PDH tic onyo PDH enzyme complex kikome (lanyut 3A). Labongo akalakala, i Mern2cKO PNs, phosphorylation pa α1 subunit α (PDHE1α) subunit pa pyruvate dehydrogenase E1 ma tye i PDH complex i Ser293 (ma ki ngeyo ni gengo tic pa enzyme pa PDH) omede ( cal S6C) ( cal S6C). Pyruvate pe ki yoo me donyo i remo.
Me agiki ne, wanongo ni yoo ma malo pa serine ki glycine biosynthesis, ma rwate ki mitochondrial folate (1C) cycle ki proline biosynthesis (lanyut 1G ki cal S5C) weng gi tye ka mede, malube ki ripot, i kare me tic. Gin ma orumo kom ni gi tye ka tic ki mitochondrial dysfunction (5-7). Kwedo lok ma mako jami ma ki lwongo ni proteomics onyutu ni i PN ma OXPHOS pe tye, dul ler ma ki lwongo ni cerebellar slices pa oyot ma mwaka gi tye 8 ki keto gi i serine hydroxymethyltransferase 2 (SHMT2), enzyme ma pire tek i mitochondrial folate cycle. Lagam pa kom ma pire tek (lanyut S5D). I 13 CU-glucose-incubated acute cerebellar slices, metabolic tracing experiments omedo ki moko ni serine ki proline biosynthesis omede, ma nyutu ni rwom me carbon isoforms i serine ki proline omede (lanyut S5E). Kit ma jami ma GLS ki GPT2 kelo gi tye ki twero i kom yubu glutamate ki i glutamine ki transamination i kin glutamate ki α-ketoglutarate, rwom gi me dongo nyutu ni OXPHOS-deficient neurons tye ki miti madwong pi glutamate, Man twero bedo me gwoko biosynthesis ma omede pa proline (Figure S5). Ka iporo ki alokaloka magi, kwan ma kitimo i kom dul kom ma ki lwongo ni cerebellar astrocytes ki i PN-specific Mfn2cKO mice onyutu ni yoo magi (ma tye iye antiperoxidases weny) pe oloko tutwal i kit ma ki nyutu kwede, man nyutu ni metabolic redirection ni tye ma kiyero me PN ma obale (Fig. S6, D to G).
I agiki ne, jami ma ki kwano ni onyutu kit ma pat pat me tic pa yoo ma pat pat i PNs. Kadi bed ni tic pa ler ma ki lwongo ni neuronal mitochondrial ma pe tye kakare twero kelo atherosclerosis ki 1C remodeling (lanyut 3E ki cal S5C), ki bene alokaloka ma ki twero byeko i nyutu pa I ki IV complexes, alokaloka i serine de novo synthesis tye keken. En onongo nen i kare me agiki. OXPHOS ma pe tiyo maber (lanyut 3E ki cal S5C). Gin ma kinongo ni nyutu kit ma dul kom ma ki lwongo ni stress-induced mitochondrial (1C cycle) ki cytoplasmic (serine biosynthesis) gi gamo kacel ki dongo pa atherosclerosis i TCA cycle me loko kit ma kom dano tiyo kwede.
8-week-old OXPHOS-deficient PNs twero gwoko high-frequency excitation activity ki kato ki i metabolic reconnection ma pire tek me culo pi mitochondrial dysfunction. Gin ma kinongo ni okelo lok ma mako tam ni kadi wa i kare ni, cell magi bene twero nongo kony me cango two me juku onyo gengo two ma mako wic. Gal. Wa tyeko lok man kun wok ki i yub aryo ma pat pat. I yoo me acel, wa yubu Cre-dependent adeno-associated virus (AAV) vector wek MFN2 ki twero nyute i OXPHOS-deficient PNs in vivo (Figure S7A). AAV ma coyo MFN2 ki fluorescent reporter gene mCherry (Mfn2-AAV) ki moko i primary neuron cultures in vitro, ma oweko MFN2 onyutu i yoo ma jenge i kom Cre ki laro kit ma mitochondrial nen kwede, ma oweko ki gengo neuromutation i Mfn2cKO neurons ( cal S7, B, D ki B). Ma lubo, wa timo jami ma ki temo i kom dano me miyo Mfn2-AAV ma mwaka ne tye 8 bot cerebellar cortex pa Mfn2cKO ki oyot ma ki temo, ki wa kwano oyot ma mwaka ne tye 12 (lanyut 4A). Twoni ma ki miyo yat Mfn2cKO ni otoo (lanyut 1, A ki B) (16). Lok pa kwidi i kom dano okelo nyutu pa PN i dul kom mogo (lanyut S7, G ki H). keto AAV ma nyutu mCherry keken (Ctrl-AAV) onongo pe ki adwogi mo i kom rwom me rweny pa ler ma ki lwongo ni Mfn2cKO. Ka i poro, kwedo Mfn2cKOs ma ki loko ki Mfn2-AAV onyutu ni tye ki gwok ma pire tek i kom PN cell layer (lanyut 4, B ki C). I kom lok, dwong pa ler ma ki lwongo ni neuron nen calo pe kitwero poko ne ki ler ma ki temo (lanyut 4, B ki C, ki cal S7, H ki I). Nyutu pa MFN1 ento pi MFN2 tye maber me gwoko to pa ler (lanyut 4C ki cal S7, C ki F), ma nyutu ni nyutu pa ectopic MFN1 twero medo peko me MFN2. Medo kwedo lok i kom rwom me PN acel onyutu ni Mfn2-AAV okonyo tutwal me laro rwom me mitochondria, odwoko rwom me mtDNA piny, ki oloko rwom me nyutu ma malo pa lanyut me anti-angiogenesis marker PCx ​​(lanyut 4, C to E ). Neno ki wang i kom oyot ma ki laro ma ki lwongo ni Mfn2cKO ma onongo gitye ka yweyo onyutu ni kit ma gi cung kwede ki kit ma gi tiyo kwede (woto S1 onyo S3) onongo ber. Me agiki ne, jami ma ki temo ni nyutu ni dwoko cen MFN2 i PNs ma tye ki peko madwong i OXPHOS romo dwoko cen tic ki mtDNA ki kelo two ma mako remo, ma gengo rweny pa axon ki too pa neuronal i kom dano.
(A) Yub ma nyutu yub me temo keto AAV ma coyo MFN2 ka yoo ma ki nyutu ni tye ka tic. (B) Cal ma ki kwanyo ma nyutu dul kom ma mwaka ne 12 ma ki keto i Mfn2cKO mice i cabit 8 ki ki keto lanyut i kom anti-Calbindin antibody. Tung acuc: rwom me axon fibers. Kit ma axon zoom tye kwede tye 450 ki 75 μm. (C) Tung cem: Kwano wel cell ma ki lwongo ni Purkinje i AAV transduction loop (AAV+) (one-way analysis of variance; n = 3 mice). Tung acuc: mtDNA ma ki keto i PN ma ki loko i cabit me 12 (t-test ma pe ki rwate; n = 6 cells ki i kom oyoo adek). * P <0.05; ** P <0.01. (D) Gin ma ki nyutu ma ki lwongo ni transmission electron micrographs pa PNs pa Mfn2cKO cerebellar sections ma ki loko ki viral vectors ma ki nyutu. Gin ma ki lwongo ni pink mask nyutu kabedo ma dendrites tye iye, ki yellow dotted square nyutu zoom ma kimiyo tung acuc; n nyutu nucleus. Lapim, 1μm. (E) nyutu labol pa PCx ma ki keto i PN ma ki loko i cabit 12. Lapim, 20μm. OE, nyutu ma okato kare; FC, loko dul.
Me agiki ne, wa kwedo kit ma peroxidase-induced cell survival kwede i PNs ma gubedo ki peko me OXPHOS. Wa yubu mCherry ma tye ki AAV-shRNA (short hairpin RNA) ma tye ka tic ki mouse PCx mRNA (AAV-shPCx), ki wa keto kwidi ni onyo gin ma ki lwongo ni scrambled control (AAV-scr) i cerebellum pa Mfn2cKO mice. Ki keto yat i cabit me angwen me mwaka (lanyut 5A) me nongo PCx ma tiyo maber i kare ma PCx omede (lanyut 3C) ki PN cell layer onongo pud tye maber (lanyut 1A). Ber me ngeyo ni turo PCx (lanyut S8A) kelo rwom ma malo me too pa PN, ma en aye gin acel ma gengo two (lanyut 5, B ki C). Me niang kit ma tic pa kom ma ki lwongo ni PCx up-regulation okelo kwede, wa kwano redox status pa PNs inge PCx knockdown ki AAV-mediated optical biosensor Grx1-roGFP2 ma ki nyutu i kare acel (lanyut S8, B to D) me temo glutathione alokaloka ma rwate ki redox peptide (383). I ngeye, wa otimo gin ma ki lwongo ni two-photon fluorescence lifetime imaging microscopy (FLIM) i dul kom ma ki lwongo ni acute brain slices pa Mfn2cKO ma mwaka ne 7 onyo dul kom ma ki lwongo ni control littermates me nongo alokaloka ma twero bedo tye i cytoplasmic redox status ingee moko kit ma FLIM tye kwede (lanyut S8, E to G). Kwedo ni onyutu ni rwom me oxidation pa Mfn2cKO PNs acel ma pe ki PCx expression, ma pat ki control neurons onyo Mfn2cKO PNs ma nyutu scrambled shRNA keken (lanyut 5, D ki E). Ka PCx nyutu ni ki dwoko piny, wel pa Mfn2cKO PNs ma nyutu ni tye ki oxidized ma malo omede tyen adek kulu (lanyut 5E), ma nyutu ni PCx up-regulation ogwoko kero me redox pa degenerated neurons.
(A) Yub ma nyutu yub me temo keto AAV ma nyutu shPCx ka yoo ma ki nyutu ni tye ka tic. (B) Cal ma ki kwanyo ma nyutu dul kom ma mwaka ne tye 8 i Mfn2cKO ma ki keto i kom ler ma ki lwongo ni anti-calcineurin antibody i cabit 4. Lapim, 450μm. (C) Kwano wel cell ma ki lwongo ni Purkinje i AAV-transduced loops (one-way analysis of variance; n = 3 to 4 mice). Ngec ni ki nyutu calo mean±SEM; ***P<0.001. (D) Cal ma nyutu FLIM nyutu kwo pa PN ma tye cabit 7 ma nyutu glutathione redox sensor Grx1-roGFP2 i te jami ma ki temo. LUT (look-up table) ratio: kare me kwo (i picoseconds). Lapim, 25μm. (E) Histogram nyutu poko pa Grx1-roGFP2 ma tye i kare me kwo pa (D) (n=158 onyo 368 cells i kom oyot aryo i te kit acel acel). Labol ma malo i kom histogram acel acel: nyutu wel cell ma tye ki kwo mabor (makwar, ma oxidized) onyo ma nok (ma bulu, ma ki dwoko piny) ma kato 1 SD me kwo ma rwate i CTRL-AAV-scr. (F) Kit ma ki yubu ni nyutu adwogi me gwoko pa rwom me dongo pa PCx.
I kom lok, ngec ma wamiyo kany nyutu ni dwoko rwom me MFN2 twero laro PN ma odongo ki peko me OXPHOS, rweny pa mtDNA, ki kit ma nen kwede ma pe rwate, ma miyo dongo kadi wa i two ma odongo. Neurodegeneration miyo caden ma twero dwoko cen i kom kare ma cell peya oto. Kit me loko kit ma kom dano tiyo kwede ki medo ki teko pa neurons me kelo atherosclerosis (rewiring me TCA cycle), ma gengo PCx i PNs ma pe ki OXPHOS ki medo to pa cell, ma weko bedo ki tic me gwoko (lanyut 5F).
I kwan man, wamiyo caden ni lagam pa PNs i kom OXPHOS ma pe tiyo maber tye me cito mot mot i TCA cycle atherosclerosis kun wok ki i yoo ma pat pat ma ki tiyo kwede ki yub me metabolic. Wa moko ni jami ma ki lwongo ni proteomic analysis tye ki yoo mapol ma rwate ki waniang ni ka ki keto peko i kom tic pa mitochondrial, neurons tye ki kit ma onongo pe ki ngeyo kwede me metabolic elasticity. Ki awura wa, yub me dwoko rwom me tic pa ler ni weny pe nyutu kit ma kom dano tye ka wot kwede mot mot ki labongo dwoko cen, ento ngec wa nyutu ni twero bedo gin ma gwoko ler kadi wa i kare ma cell pwod peya oto. Gin ma kinongo ni nyutu ni dul kom ma ki lwongo ni neurons tye ki rwom me tic ma lamal i kom. Lok man nyutu ni dwoko cen MFN2 lacen twero loko kit ma lanyut ma pire tek me tic ki kom ki gengo rweny pa PN. I kom lok adaa, en gengo two ma mako remo ki weko dul kom ma tiyo i kom dano oyot. ngat ma loko rangi del kome.
Gin acel ma oyomo cwinya ma kinongo i kwan wa tye ni PNs ma pe ki OXPHOS twero loko kit ma TCA tiyo kwede kun gi medo enzymes ma weko two ma mako remo. Metabolic rearrangement obedo gin ma pol pa cell me two kanca, mogo ma gi jenge i kom glutamine me medo TCA cycle intermediates me kelo reducing equivalents, ma doro respiratory chain ki gwoko yubu lipid ki nucleotide biosynthesis precursors (39 , 40). Kwan ma ki tiyo cok cok ni onyutu ni i kom dul kom ma tye ka wok ki peko me OXPHOS, rwate pa glutamine/glutamate bene tye gin ma pire tek (5, 41), ma yoo ma glutamine donyo kwede i TCA cycle cung i kom Pien peko me OXPHOS (41). ). Ento, pe tye lanyut ma nen maber i kom kit ma kom dano rwate kwede ki kit ma twero bedo kwede i kom two. I kwan ma ki timo i kom gin ma ki lwongo ni primary cortical neurons ki nyutu ni gi tiyo ki glutamate pools pi neurotransmission, ma weko oxidative metabolism ki atherosclerosis i te peko me metabolic (42). Ber me poyo ni ite yat ma ki gengo ki TCA cycle enzyme succinate dehydrogenase, pyruvate carboxylation ki geno ni gwoko yub pa oxaloacetate i cerebellar granule neurons ma ki dongo (34). Ento, kit ma jami magi rwate kwede ki dul kom ma i wic (ma ki geno ni two ma mako ler ma mako remo pol kare bedo i astrocytes) pud tye ki tyen lok ma pire tek (43). I kom lok man, ngec wa nyutu ni PNs ma ki balo ki OXPHOS i kom dano twero loko gi me doko BCAA ma balo ki pyruvate carboxylation, ma obedo yoo aryo ma ki medo iye TCA pool intermediates. Kadi bed ni kony pa BCAA i kom teko pa ler ma ki lwongo ni neuronal metabolism dong ki waco, medo i kom tic pa glutamate ki GABA pi neurontransmission (44), pud pe tye gin mo ma nyutu ni jami eni tiime i kom dano. Pi meno, yot me tamo ni PNs ma pe tiyo maber twero culo pi tic ki jami ma ki lwongo ni TCA ma ki kelo ki i yub me rwate ki medo two ma mako remo. I kom lok, medo PCx twero bedo gin ma mite me gwoko miti ma omede pi aspartic acid, ma ki nyutu i cell ma tye ka nya ki mitochondrial dysfunction (45). Ento, kwan wa me metabolomics pe onyutu alokaloka mo ma pire tek i rwom me aspartic acid i Mfn2cKO PNs (lanyut S6A), ma nyutu kit ma tic ki aspartic acid i kin cell ma tye ka nya ki post-mitotic neurons. Kadi bed ni kit ma PCx mede kwede i kom dul kom ma pe tiyo maber i kom dano pud peya ki ngeyo, wanyutu ni lagam man ma peya kare oromo ni tye ki tic ma pire tek me gwoko kit ma dul kom ma ki lwongo ni redox tye kwede, ma ki nyutu i FLIM ma ki temo i kom dul kom ma ki lwongo ni cerebellar slices. I kom lok, gengo PNs me medo rwom me tic pa PCx twero kelo rwom me oxidized ki weko cell too oyotoyot. Tic pa BCAA ma balo ki carboxylation pa pyruvate pe obedo yoo me nyutu kit ma kom ma tye i nget mitochondrial tye ka tic kwede marac (7). Pi meno, nen calo gin ma pire tek bot dul kom ma pe ki OXPHOS, kadi bed ni pe obedo gin acel keken, ma pire tek pi rweny pa dul kom. .
Two ma mako wic obedo two ma mako wic ma pol kare nyute calo ataxia ki pol kare balo PNs (46). Dul pa dul kom ma ki lwongo ni neuron ni tye ki peko me tic pa mitochondrial pien rweny pa gi i kom oyot romo kelo lanyut mapol ma nyutu spinocerebellar ataxia pa dano (16, 47, 48). Malube ki ripot, gin ma ki lwongo ni transgenic mouse model ki mutant gene tye ki rwate ki human spinocerebellar ataxia ki tye ki mitochondrial dysfunction (49, 50), ma nyutu teko me kwano adwogi me OXPHOS deficiency i PNPH. Pi meno, ber tutwal me poko ki kwano dul kom ma pat pat ni. Ento, kit ma PNs gi winyo kwede i kom teko ki gi tye ki dul manok pa cell me cerebellar, pi kwan mapol ma jenge ikom omics, poko kin gi calo cell weny pud tye gin ma tek. Kadi bed ni tek tutwal me nongo peke me balo dul kom mukene (tutwale dul kom pa dano madongo), wa keto rwom me poko kin jami ma tiyo maber ki FACS me nongo wel ma oromo pa dul kom ma twero kwo pi kwan ma ki lwongo ni downstream proteomics, ki tye ki rwom me protein ma malo (ma romo 3000 proteins) ka kiporo ki ngec ma tye i cerebellum set (15). Ki gwoko kwo pa cell weny, yoo ma wa miyo kany miyo kare botwa me neno alokaloka i yoo me tic pa cell i mitochondria keken, ento bene me neno alokaloka i cytoplasmic counterparts, ma medo tic ki mitochondrial membrane label enrichment ma rwate ki cell types Yo manyen me wel mitochondria i tisu (255, 25). Yoo ma wa tito ni pe kubbe ki kwan pa cell me Purkinje keken, ento twero bedo yot me tic kwede i kom cell mo keken me cobo alokaloka me metabolic i obwono ma tye ki two, medo ki kit mukene me tic pa mitochondrial.
Me agiki ne, wa nongo yoo me cang i kare me yub man ma twero loko lanyut ma pire tek me peko me cell ki gengo rweny pa neuronal. Pi meno, niang tic pa tic pa rewiring ma kiloko iye kany twero miyo ngec ma pire tek i kom kit me cango two ma twero gwoko kwo pa dul kom i kare ma mitochondrial tye ka tic marac. Kwedo ngec me anyim ma tye ka temo poko alokaloka i kit ma teko tiyo kwede i kom dul kom mukene ma i wic mitte me nyutu kit ma cik man twero tic kwede i kom two mukene ma mako wic.
MitoPark mice dong ki waco gii con (31). C57BL/6N mice ki loxP flanking Mfn2 genes dong ki waco con (18) ki ki rwate ki L7-Cre mice (23). Gin ma ki nongo ni tye ki ler ma ki lwongo ni double heterozygous ni ki rwate ki homozygous Mfn2loxP/Mfn2loxP mice me kelo Purkinje-specific gene knockouts pi Mfn2 (Mfn2loxP/Mfn2loxP; L7-cre). I dul ma ki poko me nyom, Gt (ROSA26) SorStop-mito-YFP allele (stop-mtYFP) ki keto i kin rwate mukene (20). Kit me tic ki ler ni ki tiimo malube ki cik me lobo Europe, lobo ki dul ma ki ngeyo ki LandesamtfürNatur me Umwelt ki Verbraucherschutz, North Rhine-Westphalia, Germany. Tic i kom ler bene lubu tela pa dul pa European Federation of Laboratory Animal Sciences Associations.
Iyonge keto arem i kom dako ma tye ki ic, ki kwanyo latin oyoo ni (E13). Ki poko dul kom ma ki lwongo ni Cortex i Hanks' Balanced Salt Solution (HBSS) ma ki medo ki 10 mM Hepes ki ki keto i kom Dulbecco's Modified Eagle's Medium ma tye ki papain (20 U/ml) ki cysteine ​​(1μg/ml). Ket kom ni i DMEM) ki poko ne ki enzymatic digestion. Ml) i 37°C pi dakika 20, ki dong ki juku ne i DMEM ma ki medo ki 10% remo pa dyang ma tye i ic. Gin ma ki lwongo ni cells ni ki keto gi i kom gin ma ki lwongo ni glass coverslips ma ki umu ki polylysine i rwom me 2×106 pi 6 cm culture dish onyo i rwom me 0.5×105 cells/cm2 pi neno cal. Iyonge cawa 4, ki loko gin ma ki tiyo kwede ki gin ma ki lwongo ni Neurobasal serum-free medium ma tye ki 1% B27 supplement ki 0.5 mM GlutaMax. Gin ma ki lwongo ni neurons ni ki gwoko gi i 37°C ki 5% CO2 i kare me temo ni, ki kimiyo cam bot gi acel i cabit acel acel. Me kelo dwoko rwom me tic i vitro, 3μl (24-well culture dish) onyo 0.5μl (24-well plate) pa AAV9 virus vector ma lubo ni ki tiyo kwede me cano neurons i nino me aryo i vitro: AAV9.CMV.PI.eGFP. WPRE.bGH (Addgene, namba me catalog 105530-AAV9) ki AAV9.CMV.HI.eGFP-Cre.WPRE.SV40 (Addgene, namba me catalog 105545-AAV9).
Mouse Mfn1 ki Mfn2 ma rwate ki DNA (ma ki nongo ki i Addgene plasmid #23212 ki #23213, kit meno) ki keto lanyut i kom V5 sequence (GKPIPNPLLGLDST) i C-terminus, ki ki rubu ki mCherry i frame kun wok ki i T2A sequence. Grx1-roGFP2 obedo mic ma aa ki bot Heidelberg TP Dick DFKZ (Deutsches Krebsforschungszentrum). Ki loko tdTomato cassette kun kitiyo ki yoo me cloning macon, cassette ni ki keto i pAAV-CAG-FLEX-tdTomato backbone (Addgene reference namba 28306) me yubu pAAV-CAG-FLEX-mCherry-T2A-MFN2-V5, pAAV-CAG-G FLEX-mCherry-T2A-MFN1-V5 ki pAAV-CAG-FLEX-Grx-roGFP2 vectors. Kit me tic ma kit meno ki tiyo kwede me yubu gin ma ki lwongo ni control vector pAAV-CAG-FLEX-mCherry. Me yubu AAV-shPCx construct, plasmid AAV vector (VectorBuilder, pAAV [shRNA] -CMV-mCherry-U6-mPcx- [shRNA#1]) mite, ma tye ki DNA sequence ma nyutu shRNA ma tye ka tic ki mouse PCx (5′CTTTCGCTAGCTAGGCTAAGGCTAAGGCTAGACTTAGA 3′) I te loc pa U6 promoter, mCherry ki tiyo kwede i te loc pa CMV promoter. Yub me AAV vectors ma konyo ni kitimo kit ma luyub ne owaco kwede (Cell Biolabs). Ki lok macek, tii ki gin ma ki lwongo ni transfer plasmid ma tye ki mCherry-T2A-MFN2-V5 (pAAV-CAG-FLEX-mCherry-T2A-MFN2-V5), mCherry-T2A-MFN1-V5 (pAAV-CAG-FLEX-mCherry) Transfection of 23A-MFN2-V5-MFN2-V5 cell mCherry (pAAV-CAG-FLEX-mCherry) onyo Grx-roGFP2 (pAAV-CAG-FLEX-Grx-roGFP2) ma coyo gene, kacel ki coyo AAV1 capsid protein ki accessory protein Packaging plasmid plasmid, kun kitiyo ki yoo me calcium phosphate. Gin ma ki lwongo ni crude virus supernatant ni ki nongo ki i freeze-thaw cycles i dry ice/ethanol bath ki lysed cells i phosphate buffered saline (PBS). Gin ma ki lwongo ni AAV vector ni ki lonyo ki iodixanol gradient ultracentrifugation (cawa 24 i 32,000 rpm ki 4°C) ki ki keto i tic ki Amicon ultra-15 centrifugal filter. Gin ma ki lwongo ni Genome titer pa 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 onongo kit ma ki waco kwede con (PCR) -MFN1-V5 (1.9×1013 GC/ml) ki AAV1-CAG-FLEX-Grx-roGFP2 (8.9×1012 GC/ml).
Ki kwanyo dul kom ma ki lwongo ni Primary neurons ki i ice-cold 1x PBS, ki poko, ki dong ki keto i 0.5% Triton X-100 / 0.5% sodium deoxycholate/PBS lysis buffer ma tye ki phosphatase ki protease inhibitor (Roche). Kwano wel protein ni ki tiyo ki bicinchoninic acid assay (Thermo Fisher Scientific). Gin ma ki lwongo ni proteins ni ki poko gii ki SDS-polyacrylamide gel electrophoresis, ki dong ki keto gi i kom polyvinylidene fluoride membrane (GE Healthcare). Gengo kabedo ma pe ki ngeyo ki iket ki antibody ma pire tek (nen Table S1 pi ngec mukene) i cak 5% i TBST (Tris-buffered saline ki Tween), rwom me lwoko ki antibody me aryo i TBST Ket. Ket ki gin ma gengo two pi dyewor i +4°C. Iyonge lwoko, ket gin ma ki lwongo ni secondary antibody pi cawa 2 i rwom me lyeto me ot. I ngeye, ki keto gin ma ki lwongo ni blot acel-lu ki anti-β-actin antibody, ki moko ni ki keto gin acel-lu. Nongo ne ki loko ne me doko chemiluminescence ki medo chemiluminescence (GE Healthcare).
Gin ma ki lwongo ni neurons ma onongo ki keto i kom gin ma ki lwongo ni glass coverslips onongo ki keto gi ki 4% paraformaldehyde (PFA)/PBS i kare ma ki moko i rwom me lyeto me ot pi dakika 10. Gin ma ki lwongo ni coverslips ni ki keto i 0.1% Triton X-100/PBS pi dakika 5 i rwom me lyeto me ot, ki dong i blocking buffer [3% bovine serum albumin (BSA)/PBS]. I nino me aryo, ki lwoko bongo ma ki lwongo ni coverslips ki blocking buffer ki ki gwoko ki fluorophore-conjugated secondary antibody pi cawa 2 i rwom me lyeto me ot; me agiki, jami ma ki nyutu ni ki lwoko maber i PBS ki 4′,6-diamidino-2 -Phenylindole (DAPI) ma ki keto i kom gin ma ki lwongo ni microscope ki Aqua-Poly/Mount.
Twoni (co ki mon) ki keto gi i kom ler ma ki lwongo ni intraperitoneal injection me ketamine (130 mg/kg) ki xylazine (10 mg/kg) ki ki miyo gi i te del kom ki carprofen analgesic (5 mg/kg) , ki ki keto gi i gin ma ki lwongo ni stereotactic instrument (Kopf) ma tye ki warm. Nyut wic ki tic ki gin ma ki lwongo ni dental drill me dwoko piny dul kom ma rwate ki mis bone (cake i lambda: yibe 1.8, lateral 1, ma rwate ki lobules IV ki V). Tii ki gin ma ki lwongo ni syringe needle me yubu bur matidi i wic me gengo balo yoo ma tye piny. Ki dong ki keto gin ma ki lwongo ni glass capillary mot mot i micro-hole (cake i -1.3 nio wa i -1 i tung lacuc me dura mater), ki 200 onyo 300 nl AAV ki keto i micro-injector (Narishige) ki manual syringes (Narishige) tyen mapol i rwom ma piny pi kare ma romo dakika 20 onyo 20. Iyonge keto remo, ket capillary pi dakika 10 mukene me weko kwidi ni onya woko. Inge kwanyo ler ma ki lwongo ni capillaries ni, ki tweyo del kom ni maber me dwoko piny pong pa buru ki weko ler ni cang. Ki miyo kony bot ler ni ki yat me juku arem (caspofen) pi nino mapol ingee tic, i kare meno ki neno kit ma kom gi tye kwede maber ki dong ki neko gi i kare ma ki waco ni. Jami weny ma kitimo ni ki tiimo malube ki cik me lobo Europe, lobo ki dul ma ki ngeyo ki LandesamtfürNatur me Umwelt ki Verbraucherschutz, North Rhine-Westphalia, Germany.
Lee ni ki miyo gi yat ma ki lwongo ni ketamine (100 mg/kg) ki xylazine (10 mg/kg), ki cwiny gi ki keto iye 0.1 M PBS mukwongo, ki dong ki keto 4% PFA i PBS. Ki kwanyo del kom ni ki ki keto i 4% PFA/PBS pi dyewor i 4°C. Pala ma lyel (Leica Microsystems GmbH, Vienna, Austria) ki tiyo kwede me yubu dul ma ki lwongo ni sagittal sections (50 μm thick) ki i kom wic ma ki keto i PBS. Ka pi ki waco mukene, keto rangi i kom dul kom ma tye ka mol maber ni kitimo kit ma ki waco kwede malo ni (13) i rwom me lyeto me ot ki ki buru ne. Ki lok macek, mukwongo, dul ma ki nongo ni ki keto iye 0.5% Triton X-100/PBS pi dakika 15 i rwom me lyeto me ot; pi epitopes mogo (Pcx ki Shmt2), ki i tris-EDTA buffer i 80°C (PH 9) lyeto dul kom ni pi dakika 25 ma ka timo gin man. Ma lubo, dul ni ki gwoko ki antibody ma pire tek (nen Table S1) i blocking buffer (3% BSA/PBS) i 4°C pi dyewor ki buru. I nino ma lubo, dul ni ki lwoko ki jami ma gengo ki ki keto i pii ma rwate ki fluorophore-conjugated secondary antibody pi cawa 2 i rwom me lyeto me ot; me agiki, dul ni ki lwoko maber i PBS, ki keto rangi i kom DAPI, ki dong ki keto ki AquaPolymount i kom gin ma ki lwongo ni microscope slide.
Gin ma ki lwongo ni laser scanning confocal microscope (TCS SP8-X onyo TCS Digital Light Sheet, Leica Microsystems) ma ki keto iye laser ma ler ki 405 diode ultraviolet laser ki tiyo kwede me mako cal ma ki nyutu ni. Ki tic ki fluorophore ki coko lanyut ki Hybrid Detector (HyDs), LAS-X software ni ki tiyo kwede me coko cal ma ki keto kacel ma rwate ki Nyquist sampling i kit ma lubo: pi non-quantitative panels, obedo lanyut ma tye ki teko madwong (me labolle, i somatic cells ki dendrites) mtY UFP me nongo namba me Hybrid Detector i Hybrids kit). Ki keto rwom me 0.3 onyo 6 ns me dwoko piny gin ma tye angec.
Cal ma ki kwanyo i kare kikome pa cell ma ki poko. Iyonge poko ne i Neurobasal-A ma tye ki 1% B27 supplement ki 0.5 mM GlutaMax, ki keto cell ni cut cut i kom poly-l-lysine-coated glass slides (μ-Slide8 Well, Ibidi, catalog number 80826) , ki dong ki gwoko i 37°C ki 5% CO pi cawa acel me weko cell ni bedo piny winye. Neno cal i kare kikome kitimo i kom Leica SP8 laser scanning confocal microscope ma tye ki laser makwar, HyD, 63×[1.4 numerical aperture (NA)] oil objective lens ki heating stage.
Ki keto yat ma ki lwongo ni mouse ni ki yat ma ki lwongo ni carbon dioxide ki dong ki turo wic ne, ki kwanyo wic ne oyotoyot ki i wiye, ki ki turo i 200μm (pi 13C labeling experiments) onyo 275μm thick (pi photon aryo experiments) sagittal section ma ki pongo ki jami ma lubo ni Ice cream (HM-605, VHer, Scientific, Scientific Walldorf, Germany) ki pongo ki jami ma lubo ni: 125 mM ice-cold, carbon-saturated (95% O2 ki 5% CO2) low Ca2 + artificial cerebrospinal fluid (ACSF) NaCl, 2.5 mM KCl, 1.25 mM sodium phosphate buffer, NaCl mM, 2.25 mM 0.5 mM CaCl2 ki 3.5 mM MgCl2 (tek pa 310 onyo 330 mmol). Lok dul kom ma ki nongo ni i ot ma ki keto iye Ca2 + ACSF ma malo (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 ki 310 onyo 320 mmol).
I kare me kwanyo cal, ki tero dul kom ni i ot ma ki yubu me kwanyo cal, ki temo ni kitimo i te ACSF ma ki keto i rwom me lyeto ma rwate ki 32° onyo 33°C. Gin ma ki lwongo ni multiphoton laser scanning microscope (TCS SP8 MP-OPO, Leica Microsystems) ma ki keto iye Leica 25x objective lens (NA 0.95, pii), Ti: Sapphire laser (Chameleon Vision II, Coherent) ki tiyo kwede me kwanyo cal. FLIM module (PicoHarp300, PicoQuant).
FLIM pa Grx1-roGFP2. Aloka loka ma tye i cytoplasmic redox state pa PNs ki pimo ki two-photon FLIM i sagittal brain slices, ma Grx1-roGFP2 biosensor ni otemo PNs. I dul PN, ka ma ki nongo iye jami ni ki yero ma romo 50 onyo 80 μm piny i kom dul ma ki poko ni me neno ni tye PN ma twero tic (ma en aye, peke pa dul kom ma ki lwongo ni beaded structure onyo neuronal morphological changes i kom dendrites) ki gin ma ki lwongo ni double positive roGFP2 sensor ki AAV ma nyutu shRNA PCx onyo kit ma ki lwongo ni control sequence (coache-expressing mCherry). Kwany cal ma ki keto kacel ki 2x digital zoom [excitation wavelength: 890 nm; 512 nm 512 pixels]. Nongo: HyD ma i iye, dul ma ki lwongo ni fluorescein isothiocyanate (FITC) filter group] ki cal ma ki nongo i kin dakika 2 onyo 3 ki tiyo kwede me neno ni photons ma oromo ki nongo (1000 photons weny) pi rwate ki curve. Kit ma Grx1-roGFP2 probe ni winyo kwede ki moko pa FLIM ni kitimo kun ki neno kwo pa roGFP2 ka ki medo exogenous 10 mM H2O2 i perfusion ACSF (me medo oxidation, ma kelo medo kwo), ki dong medo 2 mM dithiothreit (ma weko dwoko piny rwom me rection, dwoko piny kare me kwo) (lanyut S8, D onyo G). Tii ki jami ma ki lwongo ni FLIMfit 5.1.1 me nyamo adwogi ma kinongo, med gin ma ki lwongo ni single exponential decay curve me cal ni i kom IRF ma ki pimo (gin ma ki tiyo kwede me gamo), ki χ2 tye ma romo 1. Me kwano kwo pa PN acel, ki goyo gin ma ki lwongo ni mask ma orumo kom nerve ki cing, ki kwano kwo pa mask acel acel.
Kwedo teko pa Mitochondrial. Iyonge dul ma ki lwongo ni acute dul ma ki keto i 100 nM TMRM ma ki medo i ACSF ma ki keto i pii pi dakika 30, alokaloka ma twero time i mitochondrial pa PNs ki pimo ki microscope ma tye ki ler aryo. TMRM imaging ki otimo kun ki keto gin ma ki lwongo ni probe i 920 nm ki tic ki HyD ma tye iye (tetramethylrhodamine isothiocyanate: 585/40 nm) me nongo ngec; kun kitiyo ki excitation wavelength acel ento kitiyo ki HyD ma pat (FITC :525/50) me nyutu mtYFP. Tii ki ImageJ's Image Calculator plug-in me temo teko pa mitochondrial i rwom me cell acel. Ki lok macek, gin ma ki lwongo ni plug-in equation: signal = min (mtYFP, TMRM) ki tiyo kwede me nongo mitochondrial region ma nyutu TMRM signal i Purkinje Somali i single-stack confocal image me channel ma rwate. Ki dong kabedo pa pixel ma tye i gin ma ki lwongo ni mask ni ki kwano, ki dong ki dwoko ne i kom cal ma ki keto i kom mtYFP channel me nongo dul pa mitochondrial ma nyutu teko pa mitochondrial.
Cal ni ki kwanyo ki Huygens Pro (Scientific Volume Imaging) software. Pi cal ma ki kwanyo ki ikom tile, ki yubu tile acel kun kitiyo ki yoo me tweyo ma ki miyo ki LAS-X software. Iyonge rwom me cal, tii ki ImageJ ki Adobe Photoshop me medo tic ki cal ki loko rwom me ryeny ki apoka poka. Tii ki Adobe Illustrator me yubu cal.
mtDNA ma ki keto iye tam. Wel mtDNA ma obale ki kwano i kom dul kom ma ki keto iye antibodies i kom DNA ki confocal microscope. Kabedo acel acel ma ki yubu pi kom cell ki nucleus pa cell acel acel, ki kabedo ni ki kwano kun kitiyo ki Multi Measure plug-in (ImageJ software). Kwany kabedo ma tye i kom cell ki i kom cell me nongo kabedo ma tye i cytoplasm. Me agiki ne, gin ma ki lwongo ni Analyze Particles plug-in (ImageJ software) onwongo ki tiyo kwede me kwano wel DNA ma nyutu mtDNA i kom cal ma ki nyutu, ki adwogi ma kinongo ni ki rwate ki PN average pa CTRL mice. Adwogi ne ki nyutu calo wel nucleosides ma tye i kin cell acel acel.
Kwano kit ma protein nyute kwede. Tii ki ImageJ's Image Calculator plug-in me temo kit ma protein nyute kwede i PN i rwom me cell acel. Ki lok macek, i cal ma ki lwongo ni single-layer confocal me channel ma rwate, kun wok ki i equation: signal = min (mtYFP, antibody), dul kom ma nyutu immunoreactivity i kom antibody mo i Purkina ki ngeyo. Ki dong kabedo pa pixel ma tye i mask ma ki nongo ni ki kwano, ki dong ki dwoko ne i kom cal ma ki keto i kom mtYFP channel me nongo dul pa mitochondrial pa protein ma ki nyutu ni.
Purkinje cell density analysis. Gin ma ki lwongo ni Cell Counter plug-in me ImageJ ni ki tiyo kwede me pimo Purkinje density kun ki poko wel Purkinje cells ma ki kwano ki bor pa cerebellar ring ma cell ma ki kwano ni tye iye.
Yub ki coko labol. Wic ma aa ki i dul ma ki temo ki Mfn2cKO mice ki keto i 2% PFA/2.5% glutaraldehyde i 0.1 M phosphate buffer (PB), ki dong ki yubu dul kom ma ki lwongo ni coronal sections kun kitiyo ki ciliates (Leica Mikrosysteme GmbH, Vienna, Austria) (Thickness 50 to 60 μm) Buffer ma ki keto i 1% PB tetraoxide ki 1.5% potassium ferrocyanide i rwom me lyeto me ot pi cawa 1. Ki lwoko dul ni tyen adek ki pii ma ki yubu maber, ki dong ki keto rangi ki 70% ethanol ma tye ki 1% uranyl acetate pi dakika 20. Ki dong ki kwanyo pii i kom dul ni ki kongo ma ki poko ki medo i Durcupan ACM (Araldite casting resin M) epoxy resin (Electron Microscopy Sciences, namba 14040) i kin jami ma ki lwongo ni silicon-coated glass slides, ki dong i 60°C Polymerize i keno pi cawa 48. Kabedo ma ki lwongo ni cerebellar cortex ki yero ki 50 nm ultrathin sections ki kwanyo ki i Leica Ultracut (Leica Mikrosysteme GmbH, Vienna, Austria) ki ki kwanyo ki i kom 2×1 mm copper slit grid ma ki umo ki polystyrene film. Ki keto rangi i kom dul ni ki 4% uranyl acetate i H2O pi dakika 10, ki lwoko ki H2O tyen mapol, ki dong ki Reynolds lead citrate i H2O pi dakika 10, ki dong ki lwoko ki H2O tyen mapol. Ki kwanyo cal ma ki neno ki microscope ma ki lwongo ni transmission electron microscope Philips CM100 (Thermo Fisher Scientific, Waltham, MA, USA) kun kitiyo ki TVIPS (Tietz Video and Image Processing System) TemCam-F416 digital camera (TVIPS GmbH, Gauting, USA). Germany).
Pi oyot ma tye ki two AAV, ki poko wic gi ki ki poko i dul ma lac 1 mm, ki cerebellum ki temo kun kitiyo ki microscope me nongo ring ma tye ki two AAV (ma en aye, mCherry ma nyutu). Temo keken ma AAV injection okelo adwogi maber i kom Purkinje cell layer (ma en aye layer weny) i cerebellar rings ma romo aryo ma lubo gii aye ki tiyo kwede. Gin ma ki lwongo ni AAV-transduced loop ni ki poko ne pi dyewor ma okato (4% PFA ki 2.5% glutaraldehyde i 0.1 M cocoate buffer) ki ki medo tic kwede. Pi keto EPON, ki lwoko kom ma ki keto ni ki 0.1 M sodium cocoate buffer (Applichem), ki ki keto i 2% OsO4 (os, Science Services; Caco) i 0.1 M sodium cocoate buffer (Applichem) pi cawa 4, ki dong ki lwoko pi cawa 2. Dok cen tyen 3 ki 0.1 M cocamide buffer. I ngeye, ki tiyo ki ethanol ma tye ka mede me gwoko ethanol acel acel i 4°C pi dakika 15 me kwanyo pii i kom. Gin ma ki lwongo ni tissue ni ki keto i propylene oxide ki ki gwoko pi dyewor i EPON (Sigma-Aldrich) i 4°C. Ket del kom ni i EPON ma nyen i rwom me lyeto me ot pi cawa 2, ki dong i ket i 62°C pi cawa 72. Tii ki ultramicrotome (Leica Microsystems, UC6) ki pala ma ki lwongo ni diamond knife (Diatome, Biel, Switzerland) me ngolo dul ma tidi ma romo 70 nm, ki ruk ki 1.5% uranyl acetate pi dakika 15 i 37°C, ki ruk ki lead citrate solution pi dakika 4. Gin ma ki lwongo ni electron micrographs ni ki kwanyo ki tic ki JEM-2100 Plus transmission electron microscope (JEOL) ma tye ki Camera OneView 4K 16-bit (Gatan) ki DigitalMicrograph software (Gatan). Pi nyamo ne, ki nongo cal ma ki lwongo ni electron micrographs ki 5000× onyo 10,000× digital zoom.
Kwano kit ma mitochondria nen kwede. Pi jami weny ma ki kwano, dul kom pa mitochondria acel acel ki nyutu ki cing i cal ma ki tiyo kwede ki ImageJ software. Ki kwano jami ma pat pat ma nyutu kit ma kome nen kwede. Mitochondrial density ki nyutu calo pacenti ma ki nongo ki poko wel mitochondrial ma tye i cell acel acel ki cytoplasm area (cytoplasm area = cell area-cell nucleus area) × 100. Ki kwano rwom me mitochondria ki formula [4π∙(area/perimeter 2)]. Kit ma mitochondria nen kwede ki poko ne i dul aryo ("tubular" ki "blister") malube ki kit ma gi nen kwede.
Namba pa autophagosome/lysosome ki pek pa gin moni. Tii ki jami ma ki lwongo ni ImageJ me nyutu kit ma autophagosome/lysosome acel acel tye kwede i cal ma ki coyo ni. Kabedo pa Autophagosome/lysosome ki nyutu calo pacenti ma ki kwano ki poko kabedo pa autophagosome/lysosome ducu pa cell acel acel ki kabedo me cytoplasm (kabedo me cytoplasm=kabedo me cell-kabedo me nucleus)×100. Pek pa autophagosomes/lysosomes ki kwano ki poko wel ni ki wel pa autophagosome/lysosome ma tye i cell acel acel (i kit ma cytoplasmic area) (cytoplasmic area = cell area-nuclear area).
Coyo nying pi poko kin jami ma otiime ki yubu kamu kene. Pi temo ma mito keto lanyut i kom glucose, lok dul kom ma ki lwongo ni acute brain slices i ot ma ki keto iye jami ma ki lwongo ni saturated carbon (95% O2 ki 5% CO2), Ca2 + ACSF ma malo (125.0 mM NaCl, 2.5 mM KCl, 1.25 mM sodium phosphate buffer, 2.5 mM NaCl, 2.5 mM NaCl. d-glucose, 1.0 mM CaCl 2 ki 2.0 mM MgCl 2, ma ki medo i pH 7.4 ki 310 me 320 mOsm), ma iye glucose tye 13 C 6- Glucose substitution (Eurisotop, namba me catalog CLM-1396). Pi temo ma mito pyruvate labeling, lok dul kom ma ki lwongo ni acute brain slices ni i Ca2 + ACSF ma malo (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.Cml ki Ad20 MM. MgCl2, lok pH 7.4 ki 310 me 320mOsm), ki medo 1mM 1-[1-13C]pyruvate (Eurisotop, namba me catalog CLM-1082). Ket dul ni pi dakika 90 i 37°C. I agiki me temo ni, dul ni ki lwoko oyoto ki pii (pH 7.4) ma tye ki 75 mM ammonium carbonate, ki dong ki keto i 40:40:20 (v:v:v) acetonitrile (ACN): methanol: pii. Iyonge ma ki gwoko dul ni i kom pee pi dakika 30, ki keto kamu kene ni i 21,000 g pi dakika 10 i 4°C, ki gin ma ki kwanyo ni ki juku i SpeedVac concentrator. Gin ma ki nongo ni ki gwoko i -80°C nio ka ki temo.
Liquid chromatography-mass spectrometry analysis pa 13 C-labeled amino acids. Pi jami ma ki lwongo ni liquid chromatography-mass spectrometry (LC-MS) analysis, ki dwoko gin ma ki lwongo ni metabolite pellet ni i 75μl me pii ma ki lwongo ni LC-MS grade (Honeywell). Iyonge centrifugation i 21,000 g pi dakika 5 i 4°C, 20 μl me pii ma ki kwanyo ni ki tiyo kwede me nyamo amino acid flux, kun jami mukene ma ki kwanyo ni ki tiyo kwede cut cut me nyamo anion (nen piny). Kwano amino acid ki tiimo kun kitiyo ki yoo ma ki waco con ni me kwanyo benzoyl chloride (55, 56). I tyelo mukwongo, 10μl me 100 mM sodium carbonate (Sigma-Aldrich) ki medo i 20μl me metabolite extract, ki dong 10μl me 2% benzoyl chloride (Sigma-Aldrich) ki medo i LC grade ACN. Gin ma ki nyutu ni ki loko ne manok ki dong ki keto i 21,000 g pi dakika 5 i 20°C. Lok gin ma ki kwanyo ni i 2 ml me autosampler vial ki gin ma ki lwongo ni conical glass insert (200 μl volume). Kamu kene ni ki kwano kun kitiyo ki Acquity iClass ultra-high performance LC system (Waters) ma ki keto i Q-Exactive (QE)-HF (Ultra High Field Orbitrap) high-resolution precision mass spectrometer (Thermo Fisher Scientific). Pi kwedo, 2μl me gin ma ki kwanyo ki ikom gin ma ki kwanyo ni ki keto i 100×1.0 mm high-strength silica T3 column (Waters) ma tye ki 1.8μm particles. Kit ma mol kwede tye 100μl/min, ki gin ma ki lwongo ni buffer system tye ki buffer A (10 mM ammonium formate ki 0.15% formic acid i pii) ki buffer B (ACN). Kit ma rwom ne nen kwede tye kit man: 0%B i dakika 0; 0%B. 0 onyo 15% B i 0 onyo 0.1 dakika; 15 onyo 17% B i 0.1 onyo 0.5 dakika; B i 17 onyo 55% i 0.5 onyo 14 dakika; B i 55 onyo 70% i dakika 14 onyo 14.5; i 14.5 onyo 70 onyo 100% B i dakika 18; 100% B i dakika 18 onyo 19; 100 onyo 0% B i dakika 19 onyo 19.1; 0% B i 19.1 onyo 28 dakika (55, 56). Gin ma ki lwongo ni QE-HF mass spectrometer tiyo i kit me ionization maber ki mass range me m/z (mass/charge ratio) me 50 onyo 750. Kit ma ki tiyo kwede me neno tye 60,000, ki gain control (AGC) ion target ma kinongo tye 3×106, ki ion time ma malo loyo tye 10 milliseconds. Gin ma ki lwongo ni heated electrospray ionization (ESI) source tiyo ki kero me 3.5 kV, rwom me lyeto me capillary me 250°C, yamo ma mol ki 60 AU (arbitrary units), ki yamo ma konyo 20 AU. 250°C. Gin ma ki lwongo ni S lens ni ki keto i 60 AU.
Anion chromatography-MS analysis pa 13C ma ki keto i kom jami ma kwo. Gin ma odong ma ki lwongo ni metabolite precipitate (55μl) ki kwano kun kitiyo ki Dionex ion chromatography system (ICS 5000+, Thermo Fisher Scientific) ma ki keto i QE-HF mass spectrometer (Thermo Fisher Scientific). Ki lok macek, 5μl me jami ma ki kwanyo ki i kom dano ki keto i Dionex IonPac AS11-HC column ma ki keto iye HPLC (2 mm×250 mm, dit pa gin ni 4μm, Thermo Fisher Scientific) i kit ma ki lwongo ni push-in partial loop mode ki filling ratio me 1. )on. Dionex IonPac AG11-HC guard column (2 mm x 50 mm, 4μm, Thermo Fisher Scientific). Ki gwoko rwom me lyeto pa gin ni i 30°C, ki gin ma ki lwongo ni autosampler ni ki keto i 6°C. Tii ki gin ma ki lwongo ni potassium hydroxide cartridge ma ki miino ki pii ma ki kwanyo iye ion me yubu potassium hydroxide gradient ki i eluent generator. Poko jami ma ki lwongo ni metabolites i rwom me mol pa 380μl/min, kun kitiyo ki rwom ma lubo ni: 0 onyo 3 dakika, 10 mM KOH; 3 onyo 12 dakika, 10 onyo 50 mM KOH; 12 onyo 19 dakika, 50 onyo 100 mM KOH; 19 onyo 21 dakika, 100 mM KOH; 21 onyo 21.5 dakika, 100 onyo 10 mM KOH. Gin ma ki lwongo ni column ni ki dwoko rwom ne piny i te 10 mM KOH pi dakika 8.5.
Gin ma ki lwongo ni eluted metabolites ni ki rubu ki 150μl/min isopropanol supplement stream inge column ki dong ki cwalo gi i high-resolution mass spectrometer ma tiyo i negative ionization mode. MS tye ka neno pek pa gin ni ki i m/z 50 nio wa i 750 ki rwom me 60,000. AGC ki keto i 1×106, ki kare me ion ma malo ki gwoko i 100 ms. Gin ma ki lwongo ni ESI source ni ki tiyo kwede ki kero me 3.5 kV. Kit ma ki keto kwede ion source ni tye kit man: rwom me lyeto pa capillary 275°C; mol pa yamo, 60 AU; yamo ma konyo, 20 AU i 300°C, ki S lens ma ki keto i 60 AU.
Kwano ngec pa 13C ma ki keto i kom jami ma ki lwongo ni metabolites. Tii ki jami ma ki lwongo ni TraceFinder (version 4.2, Thermo Fisher Scientific) me nyamo ngec ma mako rwom me isotope. Kit ma gin acel acel tye kwede ki moko ki gin ma ki twero geno ki ki kwano ne kene. Me timo kwedo rwom me dongo pa isotope, kabedo pa ion chromatogram (XIC) ma ki kwanyo ki i 13C isotope (Mn) acel acel ki kwanyo ki [M + H] +, ma n obedo namba me carbon pa gin ma ki yubu, ma kitiyo kwede me kwedo amino acids onyo [ MH] + kitiyo kwede me kwedo anions. Kit ma pek pa XIC tye kwede tye piny ki dul abic pi million, ki kit ma RT tye kwede tye dakika 0.05. Kwedo rwom me dongo ni ki tiimo kun ki kwano ratio pa isotope acel acel ma ki nongo ki wel isotope weny ma tye i kom gin ni. Ratio magi kimiyo calo wel pacenti pi isotope acel acel, ki adwogi ne ki nyutu calo molar percent enrichment (MPE), kit ma kiloko kwede con (42).
Gin ma ki lwongo ni frozen neuron pellet ni ki keto i ice-cold 80% methanol (v/v), ki loko, ki ki keto i -20°C pi dakika 30. Wir gin ma ki nyutu ni odoco ki rub ne i +4°C pi dakika 30. Gin ma ki nyutu ni ki keto i 21,000 g pi dakika 5 i 4°C, ki dong ki kwanyo gin ma okati ki iye ki ki juku kun kitiyo ki gin ma ki lwongo ni SpeedVac concentrator i 25°C pi kwedo ne. Kit ma kiloko kwede malo ni, LC-MS analysis otime i kom amino acids pa cell ma ki poko. Nono TraceFinder (version 4.2, Thermo Fisher Scientific), kwedo ngec ni kitimo kun kitiyo ki pek pa gin acel acel. Kwantile normalization me metabolite data ni kitimo kun kitiyo ki preprocessCore software package (57).
Yub me poko ne. Ki keto yat ma ki lwongo ni mouse ni ki yat ma ki lwongo ni carbon dioxide ki dong ki kwanyo wiye woko, ki kwanyo wic ne oyotoyot ki i wic, ki pala ma ki lwongo ni ice-filled vibrating knife (HM-650 V, Thermo Fisher Scientific, Walldorf, Germany) ki tiyo kwede me ngolo ne i 300 onyo 375 μm sagittal carbon gas ki carbon gas (25% O) Ca2 + ACSF ma lapiny (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 ki 6.0 mM MgCl2 Lok i pH me 7 ki 30 mO3). Cwal dul kom ma ki nongo ni i ot ma tye ki Ca2 + ACSF ma malo (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 Ca2 mM ki pH 2 MM 7.4 ki 310 onyo 320 mOsm). Gwok jami ma ki ngolo ni pi dakika 20 onyo 30 wek ki twero dwogo cen ma pwod pi ki coyo ne.
mako lok. Gin ma ki lwongo ni microscope ma ki keto iye kama ki mako iye jami ki 20x water immersion objective lens (Scientifica) ki tiyo kwede pi mako jami weny. Gin ma ki lwongo ni Purkinje cells ni ki ngeyo ki (i) dit pa kom, (ii) kabedo pa cerebellum, ki (iii) nyutu pa fluorescent mtYFP reporter gene. Gin ma ki lwongo ni patch pipette ki tip resistance me 5 onyo 11 megohms ki kwanyo ki borosilicate glass capillary (GB150-10, 0.86 mm×1.5 mm×100 mm, Science Products, Hofheim, Germany) ki horizontal pipette Instruments (P-10000), Sutter Notova, CA). Jami ma ki mako ni ki tiimo ki ELC-03XS npi patch clamp amplifier (npi electronic GmbH, Tam, Germany), ma onwongo ki doro ki software Signal (version 6.0, Cambridge Electronic, Cambridge, UK). Gin ma ki temo ni ki mako i rwom me 12.5 kHz. Gin ma ki cwalo ni ki kwanyo ki jami aryo ma ki lwongo ni short-pass Bessel ma tye ki cutoff frequency me 1.3 ki 10 kHz kit meno. Kit ma dul kom ma ki lwongo ni membrane ki pipette ni ki medo kwede ki gin ma ki lwongo ni compensation circuit ma tiyo ki amplifier. Temo weng ki otimo i te loc pa Orca-Flash 4.0 camera (Hamamatsu, Gerden, Germany), ma onongo ki doro ki Hokawo software (version 2.8, Hamamatsu, Gerden, Germany).
Kit me yubu cell weny ki nyamo ne. Cut cut ma peya ki coyo, pong pipette ki gin ma tye iye ma tye ki jami ma lubo ni: 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 (MMMM), Gunophone. (GTP) (Na) ki 10.0 mM creatinine phosphate ki dwoko pH 7.25, ki osmotic pressure onwongo tye 290 mOsm (sucrose). Cut cut inge keto teko me 0 pA me turo dul kom, ki pimo teko pa dul kom ma tye ka yweyo. Gin ma ki keto i iye ki pimo ki keto teko ma ki keto i kom -40, -30, -20, ki -10 pA. Pim dit pa lagam pa teko ki tic ki cik pa Ohm me kwano teko ma ki keto iye. Tic ma otiime piire kene ki coyo i gin ma ki lwongo ni voltage clamp pi dakika 5, ki sPSC ki nongo ki kipimo i Igor Pro (version 32 7.01, WaveMetrics, Lake Oswego, Oregon, USA) kun kitiyo ki gin ma ki lwongo ni semi-automatic recognition script. IV curve ki steady-state current ki pimo kun ki keto battery i rwom ma pat pat (cake ki -110 mV) ki medo voltage i 5 mV steps. Kit ma ki yubu kwede AP ni ki temo kun ki keto depolarizing current. Ket cell ni i -70 mV nongo itye ka tic ki depolarizing current pulse. Lok dit pa gin ma ki mako ni acel acel ma pat pat (10 onyo 60 pA). Kwan wel AP ma malo loyo kun i kwano ki cingi wel ma ki lwongo ni pulse spikes ma kelo AP frequency ma malo. AP threshold ki kwano kun kitiyo ki derivative me aryo me depolarization pulse ma okwongo kelo AP acel onyo mapol.
Kit ma ki yubu kwede ki nyamo ne. Tim jami ma ki mako ma ki lwongo ni perforated patch kun itiyo ki yoo ma ki ngeyo. Tii ki pipette ma pe ki ATP- ki GTP ma pe tye ki jami ma lubo ni: 128 mM gluconate K, 10 mM KCl, 10 mM Hepes, 0.1 mM EGTA ki 2 mM MgCl2, ki lok pH 7.2 (kun itiyo ki KOH). ATP ki GTP ki kwanyo gi ki i gin ma ki lwongo ni intracellular me gengo donyo ma pe ki twero i kom cell membrane. Gin ma ki lwongo ni patch pipette ni ki pongo ki amphotericin-containing internal solution (ma romo 200 onyo 250 μg/ml; G4888, Sigma-Aldrich) me nongo rekod pa gin ma ki lwongo ni perforated patch. Amphotericin onongo ki rubu i dimethyl sulfoxide (rwom me agiki: 0.1 onyo 0.3%; DMSO; D8418, Sigma-Aldrich). Kit ma DMSO ma ki tiyo kwede ni pe ki adwogi mo i kom dul kom ma ki kwano. I kare me tic ki jami ma ki lwongo ni punching, kibedo ka neno rwom me tic pa channel (Ra) kare ki kare, ki temo ni oyabe inge amplitude pa Ra ki AP ma ocung matek (dakika 20-40). Tic ma time kene ki pimo i voltage ki/onyo current clamp pi dakika 2 onyo 5. Kwano ngec ni kitimo kun kitiyo ki Igor Pro (version 7.05.2, WaveMetrics, USA), Excel (version 2010, Microsoft Corporation, Redmond, USA) ki GraphPad Prism (version 8.1.2, GraphPad Software Inc., La Jolla, CA). Lobo Amerika). Me nongo APs ma bino piire kene, IgorPro's NeuroMatic v3.0c plug-in ki tiyo kwede. Ngeyo APs labongo tic ki rwom ma kimiyo, ma ki loko acel acel pi rekod acel acel. Nono kare me dongo, nongo rwom me dongo ki rwom me dongo ma malo ki rwom me dongo ma tye i kin piny.
PN isolation. Ki loko kit ma ki coyo kwede con, PNs ki kwanyo ki i kom ler ma ki lwongo ni mouse cerebellum i kare ma ki moko (58). Ki lok macek, dul kom ma ki lwongo ni cerebellum ni ki turo ki ki turo i ice-cold dissociation medium [labongo HBSS Ca2+ ki Mg2+, ma ki medo ki 20 mM glucose, penicillin (50 U/ml) ki streptomycin (0.05 mg/ ml)], ki dong ki digest medium ni i papa [HBSs ma ki medo ki steine (1 mg / ml), papain (16 U / ml) ki deoxyribonuclease I (DNase I; 0.1 mg/ml)] Tii pi dakika 30 i 30°C. Mukwongo lwoko kom ni i HBSS ma tye ki egg mucus (10 mg/ml), BSA (10 mg/ml) ki DNase (0.1 mg/ml) i rwom me lyeto me ot me gengo enzymatic digestion, ki dong i HBSS ma tye ki 20 mM glucose Gently grinding in HBSS, peillinic (0.5/ml), strep (0.05 mg/ml) ki DNase (0.1 mg/ml) gi gonyo cell acel acel. Gin ma ki nongo ni ki kwanyo ki i 70μm cell strainer, ki dong ki kwanyo cell ni ki centrifugation (1110 rpm, 5 minutes, 4°C) ki ki dwoko cen i sorting medium [HBSS, supplemented with 20 mM glucose, 20% fetal bovine ) Serum, pelinic (50) (0.05 mg/ml)]; temo kit ma cell twero kwo kwede ki propidium iodide ki lok dwong pa cell ni 1×106 onyo 2×106 cells/ml. Ma peya kitimo flow cytometry, gin ma ki keto ni ki kwanyo ki i 50 μm cell strainer.
Flow cytometer. Ki poko cell i 4°C kun kitiyo ki cuma ma ki lwongo ni FACSAria III (BD Biosciences) ki FACSDiva software (BD Biosciences, version 8.0.1). Gin ma ki lwongo ni cell suspension ni ki poko kun kitiyo ki 100 μm nozzle ma tye ite rwom me 20 psi i rwom me ~2800 events/sec. Kit ma rwom me gating macon (dit pa cell, poko kin jami aryo, ki kit ma ki poko kwede) pe twero moko ni ki poko PN ki cell mukene, yoo me gating ni ki keto malube ki poro pa YFP intensity ki autofluorescence i mitoYFP+ ​​ki mitoYFP − Mice. YFP ki nongo ki i keto mac i kom gin ma ki nyutu ni ki 488 nm laser line, ki lanyut ni ki nongo ki tic ki 530/30 nm band pass filter. I mitoYFP+ ​​fimice, teko ma rwate pa Rosa26-mitoYFP reporter gene bene kitiyo kwede me poko kin dul kom ma ki lwongo ni neuronal ki dul kom ma ki lwongo ni axon. 7-AAD ki miiyo ki 561 nm yellow laser ki kinongo ki 675/20 nm bandpass filter me kwanyo cell ma otoo. Me poko astrocytes i kare acel, cell suspension ni ki keto ACSA-2-APC, ki dong ki keto lanyut ni ki 640 nm laser line, ki 660/20 nm bandpass filter ki tiyo kwede me nongo lanyut ni.
Gin ma ki nongo ni ki poko ne ki centrifugation (1110 rpm, dakika 5, 4°C) ki ki gwoko i -80°C nio ka ki tiyo kwede. Twoni ma ki lwongo ni Mfn2cKO ki lutino gi ma ki nywalo gi ki poko gi i nino acel me dwoko piny apoka poka me kit ma ki tiimo kwede. Nyutu ki nyamo ngec pa FACS ki tiimo kun kitiyo ki jami ma ki lwongo ni FlowJo (FlowJo LLC, Ashland, Oregon, USA).
Kit ma kiloko kwede malo ni (59), PCR me kare kikome ki tiyo kwede me poko DNA ki ikom neurons ma ki yero pi kwano mtDNA ma lubo. Kit ma ki rwate kwede ki kit ma ki ngeyo kwede ki temo ne ki tic ki qPCR i kom wel cell ma pat pat. Ki lok macek, coko 300 PN i lysis buffer ma tye ki 50 mM tris-HCl (pH 8.5), 1 mM EDTA, 0.5% Tween 20 ki proteinase K (200 ng/ml) ki iket i 55°C pi dakika 120. Gin ma ki lwongo ni cells ni ki medo gi i 95°C pi dakika 10 me neno ni proteinase K otum woko. Ki tic ki TaqMan probe (Thermo Fisher) ma rwate ki mt-Nd1, mtDNA ki pimo ki semi-quantitative PCR i 7900HT Real-Time PCR system (Thermoherific Scientifics). Cayan, namba me catalog Mm04225274_s1), mt-Nd6 (Thermo Fisher Scientific, namba me catalog AIVI3E8) ki 18S (Thermo Fisher Scientific, namba me catalog Hs99999901_s1) genes.
Yub me lanyut me Proteome. Ki lyeto gin ma ki lwongo ni 95°C pi dakika 10 ki keto dwan, i lysis buffer [6 M guanidine chloride, 10 mM tris(2-carboxyethyl) phosphine hydrochloride, 10 mM chloroacetamide ki 100 mM tris- Lyse frozen neuron pellets in HC pellets]. I kom Bioruptor (Diagenode) pi dakika 10 (ceken 30 me ngwec / kare me ling pi ceken 30). Gin ma ki nyutu ni ki dilute 1:10 i 20 mM tris-HCl (pH 8.0), ki rubu ki 300 ng trypsin gold (Promega), ki ki gwoko pi dyewor i 37°C me nongo digestion ma otum. I nino me aryo, gin ma ki nyutu ni ki keto i 20,000 g pi dakika 20. Gin ma odong ni ki rubu ki 0.1% formic acid, ki gin ma ki rubu ni ki kwanyo kadu ki StageTips ma ki yubu piri keni. Gin ma ki nyutu ni ki juku i gin ma ki lwongo ni SpeedVac (Eppendorf concentrator plus 5305) i 45°C, ki dong ki keto peptide ni i 0.1% formic acid. Kamu kene weny ki yubu i kare acel ki ngat acel. Me temo jami ma ki kwanyo ki i kom ler ma ki lwongo ni astrocyte, 4 μg me desated peptides ki keto i kom tandem mass tag (TMT10plex, namba me catalog 90110, Thermo Fisher Scientific) ki peptide ki TMT reagent ratio me 1:20. Pi keto TMT, 0.8 mg me TMT reagent ki dwoko i 70 μl me ACN ma pe ki pii, ki peptide ma otwo ni ki dwoko cen i 9 μl me 0.1 M TEAB (triethylammonium bicarbonate), ma ki medo iye 7 μl me TMT reagent i ACN. Kit ma ki keto kwede onongo tye 43.75%. Iyonge dakika 60 me gwoko ne, ki neko gin ni ki 2 μl me 5% hydroxylamine. Peptides ma ki coyo ni ki kwanyo, ki juku, ki dwoko i 200μl me 0.1% formic acid (FA), ki poko i aryo, ki dong ki kwanyo kadu kun ki tiyo ki StageTips ma ki yubu piri keni. Nono UltiMate 3000 ultra high performance liquid chromatograph (UltiMate 3000 ultra high performance liquid chromatograph), acel i kin nucu aryo ni ki poko i kom 1mm x 150mm Acquity chromatographic column ma opong ki 130Å1.7μm C18 particles (Waters, catalog No SKU: 196366). Thermo Fisher Scientific). Pok peptides ki rwom me mol pa 30μl/min, poko ki 1% onyo 50% buffer B pi dakika 85 ki rwom me 96 dakika, ki 50% onyo 95% buffer B pi dakika 3, ki dong dakika 8 pi 95% Buffer B; Buffer A tye 5% ACN ki 10 mM ammonium bicarbonate (ABC), ki buffer B tye 80% ACN ki 10 mM ABC. Cok dul ma ki poko ni i nge dakika 3 ki rubu gi i dul aryo (1 + 17, 2 + 18, ki mukene) ki juku gi i vacuum centrifuge.
LC-MS/MS analysis. Pi mass spectrometry, peptides (namba r119.aq) ki poko gi i kom 25 cm, 75 μm inner diameter PicoFrit analytical column (lens manyen, namba me dul PF7508250) ma ki keto iye 1.9 μm ReproSil-Pur 120 C18-AQ medium medium (Maisch, Maisch-YAS). 1200 (Thermo Fisher Scientific, Germany). Gin ma ki lwongo ni column ni ki gwoko i 50°C. Gin ma ki lwongo ni Buffers A ki B tye 0.1% formic acid i pii ki 0.1% formic acid i 80% ACN, kit meno. Ki poko jami ma ki lwongo ni peptides ki rwom me 200 nl/min ki i 6% onyo 31% buffer B pi dakika 65, ki ki i 31% onyo 50% buffer B pi dakika 5. Gin ma ki lwongo ni eluted peptides ni ki kwano i kom Orbitrap Fusion mass spectrometer (Thermo Fisher Scientific). Pim ma ki lwongo ni peptide precursor m/z ki tiimo ki rwom me 120,000 i kin 350 onyo 1500 m/z. Nono 27% me teko me rwate, gin ma tek loyo ma tye ki kero me 2 onyo 6 ki yero pi teko ma malo C trap dissociation (HCD) cleavage. Cawa me tic ni ki keto i 1 s. wel m/z pa dul kom ma ki lwongo ni peptide ni ki pimo i ion trap kun kitiyo ki AGC ma nok loyo ma tye 5×104 ki kare me keto ma malo loyo ma tye 86 ms. Iyonge poko ne, gin ma okelo ne ki keto i kom gin ma ki kwanyo woko pi 45 s. TMT-labeled peptides ki poko gii i kom 50 cm, 75 μm Acclaim PepMap column (Thermo Fisher Scientific, namba me catalog 164942), ki migration spectra ni ki kwano i kom Orbitrap Lumos Tribrid mass spectrometer (Thermo Fisher Scientific) ma tye ki high-field ammetric waveform iMS (IMS) jami tic (Thermo Fisher Scientific) tiyo ki voltage aryo me −50 ki −70 V. MS3 ma ki yero malube ki gin ma ki lwongo ni synchronization precursor ki tiyo kwede pi TMT report ion signal measurement. poko peptide ni kitimo i EASY-nLC 1200, kun kitiyo ki 90% linear gradient elution, ki buffer concentration ma tye 6% onyo 31%; buffer A onwongo tye 0.1% FA, ki buffer B onwongo tye 0.1% FA ki 80% ACN. Gin ma ki lwongo ni analytical column ni ki tiyo kwede i 50°C. Tii ki FreeStyle (version 1.6, Thermo Fisher Scientific) me poko gin ma ki coyo ni malube ki rwom me FAIMS.
Ngeyo ki kwano wel jami ma konyo kom. Nono Andromeda search engine, ngec macon ni ki kwano kun kitiyo ki MaxQuant version 1.5.2.8 (https://maxquant.org/). Medo i kom Cre recombinase ki YFP ma kinongo ki i Aequorea victoria, jami ma ki lwongo ni peptide fragment spectra ki yenyo pi canonical sequence ki isoform sequence pa mouse reference proteome (Proteome ID UP000000589, ma ki kwanyo ki i UniProt i dwe me abic mwaka 2017). Methionine oxidation ki protein N-terminal acetylation ki keto calo alokaloka ma pat pat; cysteine ​​  carbamoyl methylation onwongo ki keto calo gin ma ki loko. Kit ma ki tiyo kwede me juku cam ki keto i "kit ma pat pat" ki "trypsin/P". Wel ma nok loyo pa peptides ki razor peptides ma ki tiyo kwede me ngeyo protein tye 1; wel ma nok loyo pa peptide ma pat pat tye 0. I te cik me rwate pa peptide map, wel ma ki nongo ki ikom protein onwongo tye 0.01. Gin ma ki lwongo ni "Peptide me aryo" ki yabo. Tii ki "rwate i kin ngwec" me keto gin ma ki ngeyo maber i kin jami ma ki coyo ma pat pat. Tii ki LFQ wel ma piny loyo 1 pi label-free quantification (LFQ) (60). Kit ma LFQ tek kwede ki kwanyo pi wel ma romo aryo ma tye kakare i dul pa genotype acel i kare acel acel, ki ki kwanyo ki i rwom ma opore ki lac pa. 0.3 ki cito piny 1.8. Tii ki Perseus computing platform (https://maxquant.net/perseus/) ki R (https://r-project.org/) me nyamo adwogi me LFQ. T test ma ki lwongo ni two-way moderate t test ki i kom jami ma ki lwongo ni limma software package ni ki tiyo kwede me nyamo lok ma pat pat (61). Kwedo ngec ma ki nongo ki tiimo kun kitiyo ki ggplot, FactoMineR, factoextra, GGally ki pheatmap. TMT-based proteomics data ni ki kwano kun kitiyo ki MaxQuant version 1.6.10.43. Yeny pi ngec ma ki nongo ki i kom jami ma ki lwongo ni human proteomics database pa UniProt, ma ki kwanyo i dwe me Abugwen mwaka 2018. Kwedo ne tye ki gin ma ki lwongo ni isotope purity correction factor ma ki miino ki bot luyub ne. Tii ki limma i R pi nyamo lok ma pat pat. Lok macon, adwogi me yenyo i database, ki kit ma ki tiyo kwede ki adwogi ne ki gwoko i ProteomeXchange alliance kun wok ki i PRIDE partner repository ki data set identifier PXD019690.
Coc ma ki coyo maber medo rwom me nyamo lok. Gin ma ki lwongo ni Ingenuity Pathway Analysis (QIAGEN) ki tiyo kwede me moko rwom me tic pa jami ma ki nongo i cabit 8 (lanyut 1). Ki lok macek, wel protein ma ki nongo ki i LC-MS/MS (tandem mass spectrometry) data analysis ni ki tiyo kwede ki jami ma lubo ni: Mus musculus ki yero calo dul ki rwom, ki dul ni nyutu P value ma ki loko ki Benjamini pi medo 0.05 onyo ma piny ni ki ngeyo ni pire tek. Pi karatac man, dul ma malo abic ma okato i dul acel acel ma lubu wel P ma ki loko ki nyutu. Nono t-test mapol, no tiyo ki yub me rwom aryo pa Benjamini, Krieger, ki Yekutieli (Q = 5%), time-course protein expression analysis ki tiimo i kom jami ma pire tek ma ki nongo i dul acel acel, ki dul acel acel ki kwano pat pat. Pe tye gin mo ma miite me tic ki SD ma rwate.
Me poro adwogi me kwan man ki database ma ki coyo ki me yubu cal me Venn i cal 1, wa keto wel protein ma ki kwano ki MitoCarta 2.0 annotations (24). Tii ki gin ma ki tiyo kwede i intanet me goyo cal pa Venn (http://bioinformatics.psb.ugent.be/webtools/Venn/) me yubu cal ni.
Pi ngec matut ikom kit me kwano wel ma kitiyo kwede me nyamo jami ma kwako kom, nen dul ma rwate ki jami ma kitiyo kwede ki yoo me tic kwede. Pi jami mukene ma ki temo, ngec matut ki twero nongo ne i lok ma ki coyo ni. Ka pi ki waco mukene, ngec weny ki nyutu calo rwom me ± SEM, ki jami weny ma ki kwano ki tiimo no tic ki jami ma ki lwongo ni GraphPad Prism 8.1.2.
Pi jami ma ki medo i kom pwony man, nen http://advances.sciencemag.org/cgi/content/full/6/35/eaba8271/DC1
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Ki bot E. Motori, I. Atanassov, SMV Kochan, K. Folz-Donahue, V. Sakthivelu, P. Giavalisco, N. Toni, J. Puyal, N.-G. Larson
Kwano jami ma ki lwongo ni Proteomics i kom dul kom ma pe tiyo maber onyutu ni yub me tic pa kom dano tye ka tic me gengo rweny pa dul kom.
Ki bot E. Motori, I. Atanassov, SMV Kochan, K. Folz-Donahue, V. Sakthivelu, P. Giavalisco, N. Toni, J. Puyal, N.-G. Larson
Kwano jami ma ki lwongo ni Proteomics i kom dul kom ma pe tiyo maber onyutu ni yub me tic pa kom dano tye ka tic me gengo rweny pa dul kom.
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