Kawoni *Kabedo ma kawoni: Cologne 50931, Germany, Cologne Excellence Cluster Research on Cellular Stress Response in Ageing-related Diseases (CECAD).
Nyoth pa two pa mitochondrial nen calu piny ma copo dwoko ngo, kum metabolic plasticity pa neurons utiye nok, ento adwogi pa mitochondrial dysfunction ikum cell autonomy pa neuronal metabolism i kum ungeyere ngo maber. Kae, wabe ketho cell-specific proteome pa Purkinje neurons ku progressive OXPHOS deficiency ma wok kud i disrupted mitochondrial fusion dynamics. Wanwango ni mitochondrial dysfunction ukelo aloka loka madit in thenge mi proteomics, ma ukelo tic pa yub pa metabolic ma rwinyore ma podi cell utho ngo. Ma dhanu ungiyu ngo, wanwango nya pyruvate carboxylase (PCx) man enzymes mukende ma jwigo dongo ma medo ikum TCA cycle. Gengo pa PCx umedire peko mi oxidative man neurodegeneration, ma nyutho nia atherosclerosis utiye ku tic mi gwoko neurons ma epe ku OXPHOS. Dwoko pa mitochondrial fusion in neurons ma dong unyai ungo loko kit pa metabolic eni, man eketho cell tho. Lembe ma wanwango nyutho yore ma con ma jungeyo ngo ma mio tego pa mitochondrial dysfunction man nyutho nia neurodegeneration copo dwoko cen kadi i thum pa two.
Tic ma pire tek pa mitochondria i gwoko kero pa ler mi wic nenre ku lanyuth ma lee pa wic ma mako two pa mitochondria pa dhanu. Pol pa two eni wok kud i aloka loka pa gene ma doro tic pa mitochondrial (1, 2) nyo nyoth pa gene ma mako mitochondrial dynamics, ma nyayu aloka loka pa mitochondrial DNA (mtDNA) (3, 4). Tic i kum lei unyutho nia i dwokowang lembe pa mitochondrial dysfunction i dul kum ma ceng kudu, yore mi metabolic (5-7) copo caku tic, ma mio ngec ma pire tek pi nyang matuc pa pathogenesis pa two ma tek eni. I tung ocele, nyang mwa ikum aloka loka pa dul kom moko ma wok kud i timo pa mitochondrial adenosine triphosphate (ATP) ikum wic ubedo piny ma pire tek (8), ma nyutho yeny mi nwangu yore mi yath ma juromo tiyo kudu pi juku two. Gwok two ma mako wic (9). Bedo mbe pa lembe utiye lemandha ni nerve cells nen ni gitiye ku metabolic flexibility ma nok ka juporo ku kit cells mi tissues ma ucungo kudu (10). Ni lubre ku nia cells eni timo tic ma pire tek i yub pa metabolites ni neurons pi ketho synaptic transmission man dwoko wang ret man two, kero mi loko cell metabolism ni lembe ma tek mi wic utiye kende kende i glial cells (11-14). Mi medo, tung tung pa ler mi wic nyayu peko i somo pa aloka loka ma timere i dul pa wic moko. Macalu adwogi, lembe manok kende ungeye ikum adwogi pa cell man metabolic pa mitochondrial dysfunction in neurons.
Pi ni nyang adwogi pa tic pa mitochondrial, wapoko Purkinje neurons (PNs) i rwom matung tung mi neurodegeneration ma wok kud i nyoth pa mitochondrial outer membrane fusion (Mfn2). Kadi bende aloka loka pa Mfn2 i dhanu utiye ku kit two moko ma julwongo ni Charcot-Marie-Tooth type 2A (15), nyoth pa Mfn2 i oyo ubedo yore maber mi kelo oxidation Phosphorylation (OXPHOS) dysfunction method. Kit neuronal matung tung (16-19) man kit ma neurodegenerative phenotype ma wok kud i iye bino ku lanyuth ma dongo dongo mi neurological, calu ve peko mi ngwec (18, 19) nyo cerebellar ataxia (16). Ni tiyo ku dul pa label-free quantitative (LFQ) proteomics, metabolomics, imaging, man virological methods, wanyutho nia progressive neurodegeneration nyayu pyruvate carboxylase (PCx) man piny mukende ma mako arteriosclerosis pa PNs in vivo The expression of enzymes. Mi ketho cing ikum lembe ma wanwango, wa dwoko piny rwom pa PCx in Mfn2-deficient PNs, man wanwango ni tic eni umedire peko mi oxidative man umedire rwom pa neurodegeneration, eno nyutho nia azoospermia mio tho pa cell Metabolic adaptability. Nyuthu ma tek pa MFN2 copo laru PN ma rwinyo ku OXPHOS ma tek, camu DNA pa mitochondrial madwong, man mitochondrial network ma rwinyo, ma medo nyutho nia kit neurodegeneration eni copo dwogo i rwom ma malu pa two ma podi cell utho ngo.
Pi ni neno mitochondria in Mfn2 knockout PNs, watiyo ku mouse strain ma weko Cre-dependent mitochondria ni ketho yellow fluorescent protein (YFP) (mtYFP) (20) Cre expression man waneno kit ma mitochondrial morphology in vivo. Wanwango ni nyoth pa gene pa Mfn2 in PNs bi telu poko pa mitochondrial network (lanyut S1A), man aloka loka ma kwong eca junwange i yenga adek mi oro. I ayi pa tung tung, nyoth pa PN cell layer, calu ma junyutho ku rweny pa Calbindin immunostaining, ucaku ungo nitundu juma 12 (lanyut 1, A ku B). Saa ma rwinyo ngo ikind aloka loka ma kwong'a i kite ma mitochondrial nen kudu man acaki pa tho pa neurons uketho wa lubo tok aloka loka ma wok kud i tic pa mitochondrial ma podi cell utho ngo. Wa yubu yore mi poko cell ma tiyo ku ler (FACS) pi poko YFP (YFP+)-ma nyuthu PN (lanyut 1C), man i oyo ma juporo (Mfn2 + / loxP :: mtYFP loxP- stop-loxP: : L7-cre), ma kawoni julwongo ni CT (Lanyut S1BRL). Medu pa yore mi dhugola malubiri ku tego pa YFP signal konyowa ni yweyu YFP+ body (YFPhigh) pa PNs kud i non-PNs (YFPneg) (Figure S1B) nyo putative fluorescent axon/dendritic fragments (YFPlow; Figure S1D, yo piny), ma jumoko ku confocal microscope (Figure S1D, yo cing yor acam). Pi ni ketho cing ikum dul pa dhanu ma jupoko, watimo LFQ proteomics man i ngeye watimo analysis pa dul madongo, man wanwango nya apoka poka utiye ikind YFPhigh ku YFPneg cells (Figure S1C). YFPhigh cells unyutho rwom ma malu pa PNs markers (niwacu Calb1, Pcp2, Grid2 man Itpr3) (21, 22), ento rwom pa proteins ma nwangre in neurons nyo kit cell mange (Figure 1D) ). Poro ikind lanyuth ma jupoko i YFPhigh cells ma jucoko i lembe ma jutimo giri unyutho correlation coefficient> 0.9, ma nyutho bedo ber pa dwoko ikind biological replicates (Figure S1E). I ayi ma kicoko, lembe eni uketho yub mwa pi poko PN ma copo timere. Pien L7-cre driver system ma jutiyo kudu nyayu mosaic recombination i yenga mir acel ingey nyol (23), wacaku neko oyo kud i CTRL man conditional (Mfn2 loxP / loxP :: mtYFP loxP-stop-loxP :: L7-cre) Collect neurons. I ngei thum pa dwoko piny, julwonge Mfn2cKO i ngei yenga angwen. Calu thum pa lembe, wayero yenga 8 mi oro ma PN layer ubedo maber kadi nang mitochondrial fragmentation nen (lanyut 1B man cal S1A). I wel, wa kwanu wel pa 3013 pa proteins, ma romo 22% ubedo ikum MitoCarta 2.0 annotations ma ucungo ikum mitochondrial proteome calu mitochondria (Figure 1E) (Figure 1E) (24). Lembe ma tung tung ma jutimo i yenga mir 8 unyutho nia 10.5% kende pa proteins ceke re ma ubedu ku aloka loka madit (lanyut 1F man cal S1F), ma 195 pa proteins ubedu piny man 120 ubedu up-regulated (lanyut 1F). Ber ni ngeyo ni "innovative pathway analysis" pa lembe eni nyutho nia genes ma juweco pigi tung tung gibedo ikind yore moko ma juketho (lanyut 1G). Ber pa lembe, kadi nya dwoko piny pa yore ma mako OXPHOS man calcium signaling nyutho nyayu pa mitochondrial dysfunction in fusion-deficient PNs, dul mange ma mako amino acid metabolism utiye ku rwom ma malu, ma rwate ku metabolism ma timere i mitochondrial PNs rewiring utiye marom. tic marac.
(A) Cal ma nyutho dul pa wic pa CTRL man Mfn2cKO ma nyutho rwinyo pa PNs (calbindin, gray); nuclei juyike ku DAPI. (B) Kwantify pa (A) (one-way analysis of variance, ***P<0.001; n = 4 to 6 circles from three mice). (C) Tic mi temo. (D) Heat map poko pa lanyuth ma mako Purkinje (i wi) man kit cell mange (ikind). (E) Venn diagram ma nyutho wel pa mitochondrial proteins ma junwango i PN ma jupoko. (F) Volcano plot pa proteins ma tung tung in Mfn2cKO neurons i yenga 8 (significant cut-off value of 1.3). (G) Lapor pa yore mi cwio piny nyutho yore abic ma pire tek mi dongo (makwar) man yore mi dwoko piny (macol) i Mfn2cKO PN ma jupoko ni yenga 8. Kite ma rwom pa protein acel acel ma junwango nyutho kudu. Grayscale heat map: P value ma juloko. ns, piretek ungo.
Lembe pa proteomics unyutho nia nyuthu pa protein pa complexes I, III, man IV udok piny moth moth. Complex I, III, man IV zo ubedu ku mtDNA-encoded subunits, man complex II, ma ubedu nuclear-coded kende, ubedu ungo ku adwogi marac (lanyut 2A man cal S2A). . Malube ku adwogi pa proteomics, immunohistochemistry pa cerebellar tissue sections unyutho ni MTCO1 (mitochondrial cytochrome C oxidase subunit 1) subunit level pa complex IV in PN udok piny moth moth (Figure 2B). MtDNA-encoded subunit Mtatp8 ubedo piny ma pire tek (Figure S2A), man steady-state level pa nuclear-encoded ATP synthase subunit udong ungo, ma rwate ku ATP synthase subassembly F1 complex ma jung'eyo ka mtDNA expression ubedo stable. Kit ma juyubu kudu utiye marom. Tur (7). Pima pa mtDNA in Mfn2cKO PNs ma jupoko ku real-time polymerase chain reaction (qPCR) unyutho nya wel pa mtDNA udok piny. Ka juporo ku dul ma jutimo i iye lembe, i yenga 8 mi oro, 20% kende pa mtDNA ma jugwoko (lanyut 2C). Malube ku adwogi eni, confocal microscopy staining pa Mfn2cKO PNs jutiyo kudu pi nwangu DNA, ma nyutho tic pa mitochondrial nucleotides (lanyut 2D). Wanwango ni jumoko kende ma gitiye i mitochondrial protein degradation man stress response re ma judwokogi malu, ikinde utiye Lonp1, Afg3l2 man Clpx, man OXPHOS complex assembly factors. Aloka loka madit ope i kadhiri pa proteins ma tiye i apoptosis (lanyut S2B). Kumeno bende, wanwango ni mitochondria man endoplasmic reticulum channels ma timo tic i calcium transport utiye ku aloka loka manok kende (Figure S2C). Mi medo, pimo pa autophagy-related proteins unwangu ngo aloka loka madit, ma rwate ku nen pa autophagosomes ma juneno in vivo ku immunohistochemistry man electron microscopy (Figure S3). Ento, timo marac pa OXPHOS in PNs bino ku aloka loka madit mi mitochondrial. Mitochondrial clusters copo nen i kum cell man dendritic trees pa Mfn2cKO PNs ma oro migi 5 ku 8, man dul pa membrane ma iye ukadhu kud i aloka loka madit (lanyut S4, A ku B). Malube ku aloka loka eni man dwoko piny pa mtDNA, nyang ikum dul pa wic ku tetramethylrhodamine methyl ester (TMRM) unyutho nia mitochondrial membrane potential in Mfn2cKO PNs ubedo piny madit (Figure S4C).
(A) Lembe ma jutimo i saa mi timo lembe pa OXPHOS complex. Nen kende proteins ma P<0.05 i yenga 8 (two-way ANOVA). Dotted line: Pe timo aloka loka ka juporo ku CTRL. (B) Yor acam: Lanyuth pa dul wic ma jugoro ku anti-MTCO1 antibody (scale bar, 20 μm). Kabedo ma Purkinje cell bodies ubedo i iye umo ku yellow. Ku cing cing: Kwan pa MTCO1 (ngiyo acel mi tung tung; n = 7 ku 20 cells ma juporo kud i oyo adek). (C) qPCR analysis pa mtDNA copy number in the sorted PN (one-way analysis of variance; n = 3 to 7 mice). (D) Yor acam: Lanyuth pa cerebellar slice ma jugoro ku anti-DNA antibody (scale bar, 20 μm). Kabedo ma Purkinje cell bodies ubedo i iye umo ku yellow. Ku cing: Kwan pa mtDNA lesions (one-way analysis of variance; n = 5 to 9 cells from three mice). (E) Lanyuth pa dul wic ma nyai ma nyutho mitoYFP + Purkinje cells (arrow) in whole-cell patch clamp recording. (F) Kwantif pa IV curve. (G) Lembe ma jugoro ma nyutho depolarizing current injection in CTRL man Mfn2cKO Purkinje cells. Top trace: Pulse mir acel ma nyayu AP. Lanyuth ma piny: Frequency ma malu pa AP. (H) Kwantify pa postsynaptic spontaneous inputs (sPSPs). Lanyuth pa lembe ma jugoro man zoom ratio pare nen i (I). Yo acel mi nyang ikum tung tung uketho n = 5 ku 20 cells kud i oyo adek. Lembe majunyutho calu mean±SEM; *P<0.05; **P<0.01; ***P<0.001. (J) Lembe ma nyutho AP ma wok kende ma jugoro ku tic pa perforated patch clamp mode. Lanyuth ma malu: Frequency ma malu pa AP. Lembe ma piny: zoom pa AP acel. (K) Kwan wel ma romo man ma malu pa AP frequency nimakere ku (J). Mann-Whitney test; n = 5 cells juporo kud i oyo angwen. Lembe majunyutho calu mean±SEM; piretek ungo.
Peko pa OXPHOS unwangre i Mfn2cKO PN ma oro pare 8, ma nyutho nia tic pa neurons utiye marac akeca. Pieno, wa ngiyo kit ma OXPHOS-deficient neurons tiyo kudu ikind yenga 4 ku 5 man yenga 7 ku 8 ni kadhu kud i timo patch clamp pa cell i acute cerebellar slices (lanyut 2E). Ma dhanu ungiyu ngo, rwom pa membrane ma ling man kero pa Mfn2cKO neurons ubedo marom ku ma juporo, kadi nang ubedu ku tung tung manok ikind cells (Table 1). Kumeno bende, i kind yenga 4 ku 5 mi oro, aloka loka madit ope i kind current-voltage (IV curve) (Figure 2F). Ento, Mfn2cKO neurons moko mbe ma oro migi 7 ku 8 ma ukwo i IV regimen (hyperpolarization step), ma nyutho nya etiye ku rwom ma malu mi hyperpolarization i kare ma tok cen. I ayi pa tung tung, i Mfn2cKO neurons, depolarizing currents ma nyayu repetitive action potential (AP) discharges juyio maber, ma nyutho nia discharge patterns migi zo epe ku tung tung ku pa 8-week-old control neurons (Table 1 man Figure 2G). Kumeno bende, wel man dwong pa lembe ma wok kende kende (sPSCs) ubedo marom ku pa ju ma jutimo igi sayusac, man wel pa lembe umedire nicaku i yenga 4 nitundo i yenga 5 nitundo i yenga 7 nitundo i yenga 8 ku mediri marom (lanyut 2, H man I). Kare mi dongo pa synaptic i PNs (25). Adwogi marom ju nwango ingey ketho PNs ma juyiko. Kit yub eni juku dwoko piny ma rac pa ATP mi cell, calu ma copo timere i whole-cell patch clamp recording. I andha, kero pa membrane ma ling man kero pa Mfn2cKO neurons ma wok kende kende ubedu ngo ku matoke (lanyut 2, J man K). I ayi ma kicoko, adwogi eni nyutho nia PNs ku OXPHOS dysfunction ma nen kamaleng copo timo tic maber ku high-frequency discharge patterns, ma nyutho nia yore moko nuti ma copo ketho gi bedo ku dwokowang ma rwinyo.
Lembe jukwanu calu rwom ± SEM (one-way analysis of variance, Holm-Sidak's multiple comparison test; *P<0.05). Namba pa unit ju nyuthu ku brackets.
Wa caku timo sayusac ka nyo dul moku ci ikind lembe mi proteomics (lanyut 1G) uketho i iye yore ma copo juku peko pa OXPHOS, ma uketho waweco ikum pirango ma PN ma rac copo gwoko electrophysiology ma ceng ikum kare (lanyut 2, E ku K). . Proteomics analysis unyutho nia enzymes ma timo tic i catabolism pa branched chain amino acids (BCAA) ubedo ma malu (lanyut 3A man cal S5A), man adwogi ma tok cen acetyl-CoA (CoA) nyo succinyl CoA copo medo tricarboxylates in arteriosclerosis Acid (TCA) cycle. Wanwango ni BCAA transaminase 1 (BCAT1) man BCAT2 zo umedire. Gi ketho rwom mir acel pa BCAA catabolism ni kelo glutamate kud i α-ketoglutarate (26). The subunits ceke ma gi timo branched chain keto acid dehydrogenase (BCKD) complex gi upregulated (complex catalyzes the subsequent and irreversible decarboxylation of resulting BCAA carbon skeleton) (Figure 3A and Figure S5A). Ento, aloka loka moko ope ma nwangre i BCAA gire i PN ma jupoko, ma copo bedu pi medo pa rwom pa amino acids ma pigi tek eni nyo tic ku piny mukende (glucose nyo lactic acid) pi medo TCA cycle (Figure S5B). PNs ma ope ku OXPHOS de unyutho dongo pa glutamine decomposition man transamination activities i yenga 8 mi oro, ma copo nenre ku up-regulation pa mitochondrial enzymes glutaminase (GLS) man glutamine pyruvate transaminase 2 (GPT2) (Figure 3, A ku C). Ber ni ngeyo ni rwom ma malu pa GLS utiye kende kende i spliced ​​isoform glutaminase C (GLS-GAC) (lok pa Mfn2cKO/CTRL utiye 4.5-fold, P = 0.05), man rwom pare ma malu i dul pa two kanca copo konyo mitochondrial bioenergy. (27).
(A) Heat map nyutho aloka loka pa rwom pa protein pi yo ma jukoro i yenga mir 8. (B) Lanyuth pa dul wic ma jugoro ku anti-PCx antibody (scale bar, 20 μm). Lanyuth ma yellow nyuthu kum pa cell pa Purkinje. (C) Time course protein expression analysis ungiyure ni piny ma pire tek pi atherosclerosis (multiple t-test, *FDR <5%; n = 3-5 mice). (D) Malu: Cal ma nyutho yore matung tung mi mondo i [1-13C]pyruvate tracer (niwacu, nikadhu kud i PDH nyo trans-arterial route). Piny: Violin chart nyutho wel pa single-labeled carbon (M1) ma juloko ni aspartic acid, citric acid man malic acid ingey ketho nying ikum cerebellar slices ku [1-13C]pyruvate (paired t-test; ** P <0.01). (E) Ponji mi lembe mi kare ma ukadhu pa yo ma junyutho. Nen kende kende proteins ma P<0.05 i yenga mir 8. Dashed line: no adjustment value (two-way analysis of variance; * P <0.05; *** P <0.001). Lembe jukwanu calu mean±SEM.
I lembe mu'a, BCAA catabolism udoko acel ikin yore madongo mi medo rwom. Lembe eni nyutho kamaleng nia rwom pa yamu ma mondo i TCA cycle copo lokere i PN ma OXPHOS epe. Eni copo bedu kit yore madit mi dwoko rwom pa tic pa ler mi wic, ma copo bedu ku adwogi ikum tic pa ler mi wic man kwo pare i saa mi gwoko OXPHOS ma rac. Malube ku paru eni, wanwango ni enzyme madit mi juku remo PCx utiye malu (Mfn2cKO/CTRL loko wang 1.5; cal 3A), ma ketho pyruvate doko oxaloacetate (28), ma juyio ni utiye i brainsutes. (29, 30). Malube ku adwogi pa proteomics, confocal microscopy unyutho nia PCx expression ubedo ma pire tek man ma pire tek i OXPHOS-deficient PNs, ma PCx reactivity ubedo ma pire tek i adjacent Bergmann glial cells mi control (Figure 3B). Mi temo tic pa PCx, wa timo tic ku [1-13C]pyruvate tracer. Kinde ma pyruvate ubedo oxidized ku pyruvate dehydrogenase (PDH), isotope label pare rwinyo, ento edaru ketho i TCA cycle intermediates kinde ma pyruvate ubedo metabolized ku vascular reactions (Figure 3D). Ni konyo lembe mwa mi proteomics, waneno wel madit pa lanyuth kud i tracer eni i aspartic acid pa Mfn2cKO slices, ma citric acid ku malic acid de ubedu ku rwom ma lageng, kadi nang epe (lanyut 3D).
I dopamine neurons pa MitoPark mice ku mitochondrial dysfunction ma wok kud i dopamine neurons ma nyayu peko i mitochondrial transcription factor A gene (Tfam) (Figure S6B), PCx expression de ubedo ma malu (31), ma nyutho nia acetone acid arteriosclerosis. OXPHOS i kum. Ber ni ngeyo ni junwango ni enzymes ma tung tung (32-34) ma copo bedu in neurons ma copo bedu ku ribri ku arteriosclerosis utiye ma malu in PNs ma epe ku OXPHOS, calu ve propionyl-CoA carboxylase (PCC-A), Malonyl-CoA loko propionyl-CoA ni propio-CoA succil-Acondrial enzyme 3 (ME3), ma tic pare madit utiye mi dwoko pyruvate kud i malate (lanyut 3, A ku C) (33, 35). Mi medo, wanwango mediri madit pa Pdk3 enzyme, ma phosphorylates man inactivates PDH (36), man aloka loka moko ope in Pdp1 enzyme ma activates PDH nyo PDH enzyme complex kende (lanyut 3A). Ku lapor, i Mern2cKO PNs, phosphorylation pa α1 subunit α (PDHE1α) subunit pa pyruvate dehydrogenase E1 component pa PDH complex i Ser293 (ma jung'eyo ni juku tic pa enzyme pa PDH) ubedo maber ( cal S6C) ( cal S6C). Pyruvate epe ku yore mi remo.
I ajiki, wanwango ni yo madit pa serine ku glycine, dul pa mitochondrial folate (1C) man proline biosynthesis (lanyut 1G man cal S5C) zo gi ubedo malu, nimakere ku lapor, i kare mi tic. Jam piny ma ucungu kudu timo tic ku mitochondrial dysfunction (5-7). Confocal analysis ma cwaku lembe maeni unyutho nia i PN ma OXPHOS upe, cerebellar slices pa 8-week-old mice jutimo igi serine hydroxymethyltransferase 2 (SHMT2), enzyme ma pire tek mi mitochondrial folate cycle. Adwogi ma pire tek pa kum (lanyut S5D). I 13 CU-glucose-incubated acute cerebellar slices, metabolic tracing experiments umedire ni ketho serine ku proline biosynthesis umedire, ma nyutho nia flux pa carbon isoforms i serine ku proline umedire (Figure S5E). Calu ma lembe ma GLS ku GPT2 gi kelo gitiye ku twero mi yubu glutamate kud i glutamine man transamination ikind glutamate ku α-ketoglutarate, rwom migi nyutho nia OXPHOS-deficient neurons utiye ku yeny madit pi glutamate, Eni copo bedu pi gwoko dongo pa biosynthesis pa proline (Figure S5). I tung tung ku aloka loka eni, nyang pa proteomic pa cerebellar astrocytes kud i PN-specific Mfn2cKO mice unyutho ni yore eni (ma uketho i iye antiperoxidases zo) uloko ngo lee i nyuthu, eno nyutho nia metabolic redirection eni utiye selective ni degraded PN (Fig. S6, D to G).
I ayi ma kicoko, lembe maeni unyutho kit ma tung tung mi tic pa yore moko mi tic pa piny i PNs. Kadi bende tic pa neuronal mitochondrial ma rac copo telu atherosclerosis man 1C remodeling (lanyut 3E ku cal S5C), man kadi aloka loka ma jular jukoro i nyuthu pa I ku IV complexes, aloka loka i serine de novo synthesis ubedo kende. Ebedo nen kende i thum pa stages. OXPHOS timo marac (lanyut 3E man cal S5C). Lembe ma junwango eni nyuthu yore ma rwom pa mitochondrial (1C cycle) man cytoplasmic (serine biosynthesis) dwoko wang lembe karacelu ku medu pa atherosclerosis i TCA cycle mi loko metabolism pa neuronal.
8-week-old OXPHOS-deficient PNs copo bedo ku tic ma tek man timo tic ma pire tek mi dwoko rwom pa tic pa mitochondrial. Lembe ma junwango eni ukelo lembe ma nyangu dhanu nia kadi i kare eni de, dul kom eni copo nwangu kony mi yath pi jwigo nyo jwigo two pa wic. Galo. Wadaru lembe eni nikadhu kud i timo lembe aryo matung tung. I yore mir acel, wayubu Cre-dependent adeno-associated virus (AAV) vector kara MFN2 ubed selectively expressed in OXPHOS-deficient PNs in vivo (Figure S7A). AAV ma ketho MFN2 man fluorescent reporter gene mCherry (Mfn2-AAV) juporo gi i primary neuron cultures in vitro, ma uketho MFN2 ubedo expressed in a Cre-dependent manner man ekonyo mitochondrial morphology, ma uketho jugwoko neuromutation i Mfn2cKO neurons ( cal S7, B, D man B). I ngeye, watimo in vivo experiments pi tero 8-week-old Mfn2-AAV i cerebellar cortex pa Mfn2cKO man control mice, man waporo 12-week-old mice (lanyut 4A). Mfn2cKO ma jutimo igi yath utho (lanyut 1, A ku B) (16). Viral transduction in vivo ukelo nyuthu pa PN i cerebellar circles moko (lanyut S7, G man H). Cwalu pa AAV ma nyuthu mCherry kende (Ctrl-AAV) ubedu ungo ku adwogi madit ikum kadhiri pa neurodegeneration i lei Mfn2cKO. I ayi pa tung tung, nyang ikum Mfn2cKOs ma juloko ku Mfn2-AAV unyutho bedo maber pa PN cell layer (lanyut 4, B ku C). I andha, dwong pa neuron nen ni epe ku lei ma jutiyo kudu (lanyut 4, B ku C, man cal S7, H ku I). Nyuthu pa MFN1 ento ungo MFN2 utiye ber marom i laru tho pa neuronal (lanyut 4C man cal S7, C ku F), ma nyutho ni nyuthu pa ectopic MFN1 copo medo kero pa MFN2. Lembe mukende ma jutimo i PN acel unyutho nia Mfn2-AAV ukonyo ultrastructure pa mitochondria, uketho mtDNA ubedo maber, man udwoko rwom ma malu pa anti-angiogenesis marker PCx ​​(lanyut 4, C ku E ). Neno ku wang pa oyo Mfn2cKO ma jular jukonyo gi i kare mi yom cwiny unyutho nia kite ma gicungu kudu man kit ma giwotho kudu (wotho S1 nitundo S3) udoko ber. I thum pa lembe, lembe maeni nyutho nia dwoko cen pa MFN2 i PNs ma OXPHOS upe i iye romo dwoko mtDNA man nyayu atherosclerosis, man eketho axon degeneration man tho pa neurons in vivo.
(A) Lembe ma nyutho yub mi temo pi ketho AAV ma ketho MFN2 ka yore mi tic pa piny ma juweco pire ubedo ka tic. (B) Cal ma nyutho 12-week-old cerebellar slices ma juloko i 8 weeks i Mfn2cKO mice man ma jugoro ku anti-Calbindin antibody. Ku cing: Medu pa axon fibers. Kit pa axon zoom utiye 450 ku 75 μm. (C) Yor acam: Kwan pa Purkinje cell density in AAV transduction loop (AAV+) (one-way analysis of variance; n = 3 mice). Ku cing cing: mtDNA nen i PN ma juloko i yenga mir 12 (unpaired t-test; n = 6 cells from three mice). * P <0.05; ** P <0.01. (D) Representative transmission electron micrographs of PNs of Mfn2cKO cerebellar sections transduced with the indicated viral vectors. Mask ma pink nyutho kabedo ma dendrites ubedo i iye, man square ma yellow ma juketho i iye lanyuth nyutho zoom ma juketho i cing' ma yor acam; n nyuthu nucleus. Scale bar, 1μm. (E) nyutho lanyuth pa rangi pa PCx i PN ma juloko i yenga 12. Scale bar, 20μm. OE, nyuthu lembe madwong; FC, loko loko.
I ajiki, walubo tok beru pa kwo pa cell ma peroxidase uketho i PNs ma ukadhu kud i peko pa OXPHOS. Wacwio mCherry ma uketho AAV-shRNA (short hairpin RNA) ma ucungo iwi PCx mRNA pa oyo (AAV-shPCx), man wabolo two ne nyoke ma jucwalo (AAV-scr) i cerebellum pa oyo Mfn2cKO. Yath ne jutimo i yenga mir angwen mi oro (lanyut 5A) mi nwangu PCx knockdown maber i kare ma PCx umedire (lanyut 3C) man PN cell layer podi utiye maber (lanyut 1A). Ber ni ngeyo ni turo PCx (lanyut S8A) ucwalu tho pa PN pio pio, ma utiye kende kende i ring ma two (lanyut 5, B ku C). Pi ni nyang ikum kit ma tic pa PCx up-regulation wok kudu, wa somo redox status pa PNs ingey PCx knockdown man AAV-mediated optical biosensor Grx1-roGFP2 jubedu ka nyuthu gi marom (lanyut S8, B ku D) pi ni ngiyo glutathione aloka loka pa redox peptide (3838). I ngeye, watimo two-photon fluorescence lifetime imaging microscopy (FLIM) i acute brain slices pa 7-week-old Mfn2cKO nyo control littermates ni neno aloka loka ma copo timere i cytoplasmic redox status ingey ketho FLIM (Figure S8, E to G). Lembe ma jutimo unyutho mediri madit pa rwom pa oxidation pa Mfn2cKO PNs acel ma epe ku PCx expression, ma tung tung ku control neurons nyo Mfn2cKO PNs ma nyutho scrambled shRNA kende (lanyut 5, D man E). Kinde ma PCx ucungo piny, wel pa Mfn2cKO PNs ma nyutho bedo ma oxidized umedire wang adek (lanyut 5E), ma nyutho nia PCx up-regulation umedire redox capacity pa degenerated neurons.
(A) Lembe ma nyutho yub mi temo pi ketho AAV ma ketho shPCx ka yore mi tic pa piny ma juweco pire ubedo ka tic. (B) Cal ma nyutho dul pa cerebellar ma oro migi 8 i Mfn2cKO mice ma juloko man juketho i iye anti-calcineurin antibody i yenga 4. Scale bar, 450μm. (C) Kwantify pa Purkinje cell density in AAV-transduced loops (one-way analysis of variance; n = 3 to 4 mice). Lembe majunyutho calu mean±SEM; ***P<0.001. (D) Cal pa FLIM nyutho kwo pa PN ma oro pare 7 ma nyuthu glutathione redox sensor Grx1-roGFP2 i thenge mi lembe ma jukoro. LUT (look-up table) ratio: kare mi kwo (i picoseconds). Scale bar, 25μm. (E) Histogram nyutho poko pa wel pa Grx1-roGFP2 ma kwo kud i (D) (n=158 nicidho 368 cells i oyo ario i thenge acel acel). Pie chart ma malu pa histogram acel acel: nyutho wel pa cell ma kwo migi bor (makwar, oxidized) nyo ma nok (macol, ma nok) ma kadhu 1 SD pa kwo migi i CTRL-AAV-scr. (F) Model ma juyiko nyuthu bero pa upregulation pa neuronal PCx.
I lembe zo, lembe ma wabe mio kany nyutho nia dwoko MFN2 copo laru PN ma ukadhu ku peko mi OXPHOS, mtDNA ma nok, man ista-like morphology ma rwinyo, ma copo mio adwogi maber kadi i two ma ukadhu. Neurodegeneration mio lanyuth ma copo dwoko wange pa kare ma podi cell utho ngo. Kite ma metabolic flexibility eni nen kudu ikum kero pa neurons mi kelo atherosclerosis (rewiring pa TCA cycle), ma juku PCx expression in PNs ma epe ku OXPHOS man medo tho pa cell, ma etimo tic mi gwoko (lanyut 5F).
I lembe maeni, wabe mio lanyuth nia dwokowang pa PNs ni OXPHOS utiye ni rwinyo nyathoth ni TCA cycle atherosclerosis nikadhu kud i yore mi tic ma tung tung ma jutiyo kudu ku program mi metabolic. Wamoko lembe mi proteomic ku yore madwong man wanyutho nia ka jutimo igi peko mi mitochondrial dysfunction, neurons gitiye ku kit metabolic elasticity ma jung'eyo ongo con. Ku iworo mwa, yub mi dwoko waya eca uketho ngo rwom pa piny ma bino ku neurodegeneration moth moth man ma copo dwoko ngo, ento lembe mu'a nyutho nya ecopu bedu ni neuron ma gwoko kadi i kare ma podi cell utho ngo Functional compensation mechanism. Lembe ma junwango nyuthu nia neurons utiye ku rwom ma malu mi metabolic plasticity i kum. Lembe eni nyutho nia dwoko MFN2 i ngeye ecopu loko nyuthu pa lanyuth ma pire tek mi metabolic man juku PN degeneration. Ento, ejuku atherosclerosis man eketho nerves doko tek. transsexual.
Piny acel ma pire tek ma wanwango i sayu lembe mwa nya PNs ma epe ku OXPHOS copo loko TCA cycle metabolism ni medo enzymes ma nyayu two arteriosclerosis. Metabolic rearrangement ubedo piny ma rwinyo pa two kanca, moko ikum gi geno glutamine ni medo TCA cycle intermediates ni kelo reducing equivalents, ma telo respiratory chain man gwoko yik pa lipid man nucleotide biosynthesis precursors (39 , 40). Somo moko ma kawoni unyutho nia i dul kum ma OXPHOS timo ngo tic maber, dwoko rwom pa glutamine/glutamate de ubedo piny ma pire tek (5, 41), kama yo ma glutamine mondo i TCA cycle ucungo iwi pi peko pa ret pa OXPHOS (41). ). Ento, lembe moko mbe ma nyutho kamaleng pa neuronal metabolic plasticity i kum man kite ma ecopo bedu kudu i two. I lembe manyen ma jutimo in vitro, junyutho nia primary cortical neurons copo ketho glutamate pools pi neurotransmission, ma uketho oxidative metabolism man atherosclerosis i thenge mi metabolic stress (42). Ber ni ngeyo ni i the loc pa yath pa TCA cycle enzyme succinate dehydrogenase, pyruvate carboxylation juyio ni gwoko synthesis pa oxaloacetate in cultured cerebellar granule neurons (34). Ento, beru pa lembe eni ikum ler mi wic (ma atherosclerosis juyio ni utiye ikum astrocytes) podi utiye ku tic ma pire tek (43). I lembe maeni, lembe mwa nyutho nia PNs ma OXPHOS unyothu i kum dhanu copo lokore ni BCAA degradation man pyruvate carboxylation, ma gitiye yore ario madongo mi medo pa TCA pool intermediates. Kadi bende kony pa BCAA catabolism i neuronal energy metabolism juweco pire, ikum tic pa glutamate ku GABA pi neurotransmission (44), podi lembe moko mbe ma nyutho nia yore eni tiye in vivo. Pieno, e yot ni paru nia PNs ma timo ngo tic maber copo culo kwor pa TCA ma wok kud i tic mi assimilation ni medo atherosclerosis. I andha, medo rwom pa PCx copo bedu piny ma mitere pi gwoko yeny pa aspartic acid, ma juyero i proliferating cells ku mitochondrial dysfunction (45). Ento, sayusac mwa mi metabolomics unyutho ngo aloka loka moku madit i rwom pa aspartic acid in Mfn2cKO PNs (lanyut S6A), ma nyutho tic matung tung pa aspartic acid ikind proliferating cells ku post-mitotic neurons. Kadi bende yore ma PCx timo kudu tic i dysfunctional neurons in vivo podi ungeyere ngo, wanyutho nia dwokowang ma rwinyo eni timo tic ma pire tek i gwoko redox state pa neurons, ma junyutho i FLIM experiments iwi cerebellar slices. I andha, juku PNs kud i up-regulate PCx ecopu telu ikum oxidized state man nyayu tho pa cell. Tic pa BCAA man carboxylation pa pyruvate epe yore mi nyuthu kit ma mitochondrial tiyu kudu marac (7). Pieno, nen calu gibedo piny ma pire tek pa OXPHOS-deficient neurons, kadi nang epe kende, ma pire tek pi neurodegeneration. .
Twoyo mi wic ubedo kit two ma tung tung ma nyayu peko i wic ma pol kare nyutre calu ataxia man pol kare nyayu peko i kum PNs (46). Dul pa neuron eni utiye ku peko madit ikum mitochondrial dysfunction pi en nyothre migi i oyo romo nyayu lanyuth mapol ma nyutho spinocerebellar ataxia pa dhanu (16, 47, 48). Ni lubre ku lapor, lanyuth pa oyo ma juloko ku gene ma juloko utiye ku ataxia pa spinocerebellar pa dhanu man etiye ku mitochondrial dysfunction (49, 50), ma nyutho beru pa somo adwogi pa OXPHOS ma nok in PNPH. Pieno, eber mandha ni poko man ni ponjo dul pa neuron ma tung tung eni. Ento, ni lubre ku PNs ma giwinjo lembe ma tek man gibedo ku dul ma nok pa dul pa dul pa cerebellar cells, pi somo madwong ma omics-based, poko gi calu cells zo podi utiye lembe ma tek. Kadok ecopu bedu tek ni nwangu bedo ma rwinyo pa kit cell mange (mandha ne dul pa dhanu madongo), wa ketho rwom mi poko piny maber ku FACS ni nwangu wel ma romo pa neurons ma copo kwo pi timo sayusac mi proteomics, man bedu ku rwom ma malu pa protein (maromo 3000 proteins) ka juporo ku data ma con pa cerebellum (15). Ni gwoko kwo pa cell zo, yore ma wabe miyo keni konyowa ni neno aloka loka mi yore mi metabolic i mitochondria kende, ento bende ni neno aloka loka mi cytoplasmic counterparts, ma medo tic ku mitochondrial membrane tags pi medo cell type The new method for the number of mitochondria in complex tissues (555). Yore ma wabeweco pire epe kende ku somo pa Purkinje cells, ento ecopu bedu yot ni tiyo kudu i kit cell moko ci pi cobo aloka loka mi metabolic i wic ma two, ikinde utiye kit tic mange pa mitochondrial dysfunction.
I ajiki, wanwango yore mi keyo i saa mi dwoko piny ma nyayu remo ma copo loko lanyuth ma pire tek pa peko mi dul kom man juku nyoth pa dul kum. Pieno, nyang ikum tic pa rewiring ma juloko keni copo mio ngec ma pire tek ikum yore mi gwoko kwo pa neurons i saa mi tic pa mitochondrial. Lembe manyen ma jutimo pi nyang ikum aloka loka mi tic pa kero i kit cell mi wic mange mitere pi nyutho tic pa cik eni ikum two mukende mi wic.
MitoPark mice juweco pire con (31). C57BL/6N oyo ku loxP flanking Mfn2 genes juweco pire con (18) man juporo ku L7-Cre oyo (23). Adwogi pa nyithindho ma rangi migi ario ma tung tung juporo ku Mfn2loxP/Mfn2loxP mice pi niketho Purkinje-specific gene knockouts pi Mfn2 (Mfn2loxP/Mfn2loxP; L7-cre). I kind dul ma nok mi nyom, Gt (ROSA26) SorStop-mito-YFP allele (stop-mtYFP) jukelo nikadhu kud i rwate mange (20). Lembe ceke ma jutimo ikum lei jutimo nimakere ku cik mi Europe, mi ngom man mi kanisa man juyio ku LandesamtfürNatur mi Umwelt man Verbraucherschutz, North Rhine-Westphalia, Germany. Tic mi lei de lubu telowic pa European Federation of Laboratory Animal Sciences Associations.
I ngei timo anesthesia i kum dhaku ma ic, juwodho nyathin pa oyo (E13). Cortex jupoko i Hanks' Balanced Salt Solution (HBSS) ma ju medo ku 10 mM Hepes man ju medo i Dulbecco's Modified Eagle's Medium ma tye ku papain (20 U/ml) man cysteine ​​(1μg/ml). Keth adila i DMEM) man poko ku enzymatic digestion. Ml) i 37°C pi dakika 20, man i ngeye juyiko ku cuma i DMEM ma jumedo ku 10% pa rimo pa dhyeng. Jupodho cells iwi glass coverslips ma juketho i iye polylysine i rwom pa 2×106 pi 6 cm culture dish nyo i rwom pa 0.5×105 cells/cm2 pi neno cal. I ngei sawa angwen, judwoko piny ma jutiyo kudu ku Neurobasal serum-free medium ma tye ku 1% B27 supplement man 0.5 mM GlutaMax. I ngeye jugwoko neurons i 37°C man 5% CO2 i kare mi temo, man jumiyo igi cam wang acel i yenga acel. Pi ni ketho recombination in vitro, 3μl (24-well culture dish) nyo 0.5μl (24-well plate) pa AAV9 virus vector ma e piny eni jutiyo kudu pi timo yath pa neurons i ceng mir aryo in vitro: AAV9.CMV.PI.eGFP. WPRE.bGH (Addgene, namba mi catalog 105530-AAV9) man AAV9.CMV.HI.eGFP-Cre.WPRE.SV40 (Addgene, namba mi catalog 105545-AAV9).
Mouse Mfn1 man Mfn2 complementary DNA (ma ju nwango kud i Addgene plasmid #23212 man #23213, kubang wadi) ju goyo ku V5 sequence (GKPIPNPLLGLDST) i C-terminus, man ju ketho gi ku mCherry in frame nikadhu kud i T2A sequence. Grx1-roGFP2 ubedo mic pa Heidelberg TP Dick DFKZ (Deutsches Krebsforschungszentrum). Ni loko tdTomato cassette ni tiyo ku yore macon mi cloning, cassette ne judwoko i pAAV-CAG-FLEX-tdTomato backbone (Addgene reference number 28306) mi yubu pAAV-CAG-FLEX-mCherry-T2A-MFN2-V5, pAAV-CAG-G FLEX-mCherry-T2A-MFN1-V5 man pAAV-CAG-FLEX-Grx-roGFP2 vectors. Yore marom jutiyo kudu pi yubu vector mi pAAV-CAG-FLEX-mCherry. Pi niketho AAV-shPCx construct, plasmid AAV vector (VectorBuilder, pAAV [shRNA] -CMV-mCherry-U6-mPcx- [shRNA#1]) mitere, ma tingo DNA sequence ma ketho shRNA ma ucungo iwi mouse PCx (5′CTTTCGCTAGCTAGGCTAAGGCTAAGGCTAGACTTAGA 3′) I the loc pa U6 promoter, mCherry jutiyo kudu ithe loc pa CMV promoter. Yubu pa AAV vectors jutimo nimakere ku cik pa ju ma yiku (Cell Biolabs). I lembe ma nok, ti ​​ku plasmid ma tingo mCherry-T2A-MFN2-V5 (pAAV-CAG-FLEX-mCherry-T2A-MFN2-V5), mCherry-T2A-MFN1-V5 (pAAV-CAG-FLEX-mCherry) pi kare manok. mCherry (pAAV-CAG-FLEX-mCherry) nyo Grx-roGFP2 (pAAV-CAG-FLEX-Grx-roGFP2) coding gene, man bende coding AAV1 capsid protein man accessory protein Packaging plasmid plasmid, using calcium phosphate method. Jam piny ma nyai pa two ne ju nwango ku yub mi ngico-yweyo i pii ma ngic/ethanol man jupoko cells i phosphate buffered saline (PBS). AAV vector juyike ku discontinuous iodixanol gradient ultracentrifugation (sawa 24 i 32,000 rpm man 4°C) man juyike ku Amicon ultra-15 centrifugal filter. 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 ubedo calu ma jukoro con (jupimo ku PCR) -MFN1-V5 (1.9×1013 GC/ml) man AAV1-CAG-FLEX-Grx-roGFP2 (8.9×1012 GC/ml).
Neurons ma pire tek juwodho gi i ice-cold 1x PBS, jupoko gi, man judwokogi maromrom i 0.5% Triton X-100 / 0.5% sodium deoxycholate/PBS lysis buffer ma tye ku phosphatase man protease inhibitor (Roche). Kwan pa protein jutimo ku tic ku bicinchoninic acid assay (Thermo Fisher Scientific). Jam piny ne jupoko ku SDS-polyacrylamide gel electrophoresis, man jupoko iwi polyvinylidene fluoride membrane (GE Healthcare). Juk kabedo ma nen ungo man iket ku antibody ma pire tek (nen Table S1 pi lembe mukende) i 5% mi cak i TBST (Tris-buffered saline ku Tween), rwom mi lwoko man antibody mir aryo i TBST Keth. Keth ku antibody ma pire tek dyewor i +4°C. I ngei lwoko, med antibody mir aryo pi sawa 2 i rwom ma piny. I ngeye, ku ketho blot marom ku anti-β-actin antibody, rwom marom jumoko. Nwangu ku loko ni chemiluminescence man medo chemiluminescence (GE Healthcare).
Neurons ma jular juketho iwi glass coverslips juyike ku 4% paraformaldehyde (PFA)/PBS i kare ma juketho i ot pi dakika apar. Coverslips jucaku ku 0.1% Triton X-100/PBS pi dakika 5 i rwom ma piny, man i blocking buffer [3% bovine serum albumin (BSA)/PBS]. I ceng mir aryo, juyiko coverslips ku blocking buffer man juyiko ku fluorophore-conjugated secondary antibody pi sawa 2 i ot; i ajiki, ju lwoko lanyuth ne maber i PBS ku 4′,6-diamidino-2 -Phenylindole (DAPI) ma juyiko man juyike iwi microscope slide ku Aqua-Poly/Mount.
Oyo (co ku mon) jutimo igi anesthesia ku injection mi ketamine (130 mg/kg) man xylazine (10 mg/kg) i iye man jumiyo igi piny ku carprofen analgesic (5 mg/kg) , man juketho gi i stereotactic instrument (Kopf) ma juketho i iye warm. Nyuth wic man iti ku jamb tic mi lak pi jwigo thenge mi cerebellar cortex ma rwate ku mis bone (nicaku i lambda: yibe 1.8, lateral 1, ma rwate ku lobules IV ku V). Ti ku syringe needle ma ucungo pi yubu vur ma nok i wic kara kud unyoth vasculature ma piny. I ngeye, jubolo capillary ma nyathotho i micro-hole (nicaku i -1.3 nitundo i -1 i thenge ma yo piny pa dura mater), man 200 nitundo 300 nl AAV jubolo i micro-injector (Narishige) ku manual syringes (Narishige) wang mapol i pressure ma piny pi kare ma romo dakika 20 nitundo i windows. I ngei mi ketho rimo, keth capillary pi dakika 10 mukende wek two ne unyai zo. I ngei ma juwodho capillaries, juyiko adila maber mi jwigo lyethu pa ret man weko lei ne udok maber. Jutimo yath ni lei ne ku yath mi kwiyu remu (caspofen) pi nindu madwong ingey timo yath, ikind kare eni jubedu ka lubo tok kum migi maber man ingeye junego gi i kare ma jukwanu. Lembe ceke jutimo nimakere ku cik mi Europe, mi ngom man mi kanisa man juyio ku LandesamtfürNatur mi Umwelt man Verbraucherschutz, North Rhine-Westphalia, Germany.
Lei ne juyike ku ketamine (100 mg/kg) man xylazine (10 mg/kg), man juyike i adunde ku 0.1 M PBS mir acel, man i ngeye ku 4% PFA i PBS. Jupoko adila man juyike i 4% PFA/PBS dyewor i 4°C. Pala ma yeng yeng (Leica Microsystems GmbH, Vienna, Austria) jutiyo kudu pi yiko sagittal sections (50 μm thick) kud i wic ma juketho i PBS. Ka jukwanu ngo yo mukende, rangi pa dul ma mol iwi pii jutimo calu ma jukoro malu (13) i rwom ma piny pa ot man juyike. I lembe ma nok, mir acel, jupoko dul ma jucaku ku 0.5% Triton X-100/PBS pi dakika 15 i lyethu mi ot; pi epitopes moko (Pcx ku Shmt2), ku tris-EDTA buffer i 80°C (PH 9) lyeth dul ne pi dakika 25 kakare mi timo lembe eni. I ngeye, jupoko dul ne ku antibody ma pire tek (nen Table S1) i blocking buffer (3% BSA/PBS) i 4°C dyewor ku ngwec. Iceng ma lubo, juyike dul ne ku blocking buffer man juyike ku fluorophore-conjugated secondary antibody pi sawa 2 i ot; i ajiki, juyike dul ne maber i PBS, juyike ku DAPI, man juyike ku AquaPolymount iwi microscope slide.
Laser scanning confocal microscope (TCS SP8-X nyo TCS Digital Light Sheet, Leica Microsystems) ma juketho i iye laser ma ler man 405 diode ultraviolet laser jutiyo kudu pi goyo lanyuth. Ni ketho fluorophore man ni coko signal ku Hybrid Detector (HyDs), LAS-X software jutiyo kudu pi coko cal ma jucoko ma lubu Nyquist sampling in sequential mode: pi non-quantitative panels, etiye signals ma tek (lapor, i somatic cells man dendrites) mtYFPD ni Hybrid Detector in the right detect numbers kit). Gating nicaku 0.3 nicidho 6 ns jutiyo kudu pi jwigo piny ma ngeye.
Cal pa cell ma jupoko i kare mandha. I ngei poko i Neurobasal-A ma tye ku 1% B27 supplement man 0.5 mM GlutaMax, jupoko cells iwi poly-l-lysine-coated glass slides (μ-Slide8 Well, Ibidi, catalog number 80826) , man i ngeye jugwoke i 37°C man 12% pi sawa acel ni cell bedi. Cal mi kare mandha jutimo iwi Leica SP8 laser scanning confocal microscope ma juketho i iye laser matar, HyD, 63×[1.4 numerical aperture (NA)] oil objective lens man heating stage.
Jutimo anesthesia pio pio ku carbon dioxide man jupoko wiye, juwodhe wic pio pio kud i wiye, man jupoko i 200μm (pi 13C labeling experiment) nyo 275μm thick (pi photon aryo) sagittal section ma jupong ku piny ma e. Walldorf, Germany) upong ku piny ma e: 125 mM ice-cold, carbon-saturated (95% O2 and 5% CO2) low Ca2 + artificial cerebrospinal fluid (ACSF) NaCl, 2.5 mM KCl, 1.25 mM sodium phosphate buffer, NaCl 2.5 mM, gluse 3 MM 0.5 mM CaCl2 man 3.5 mM MgCl2 (osmotic pressure pa 310 ku 330 mmol). Lok dul wic ma inwango i ot ma juketho i iye 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 man Ca.20 mM. MgCl2) Medium) pH 7.4 man 310 ku 320 mmol).
I kare mi timo cal, juwodho dul ne i ot moko ma juyiko pi goyo cal, man jutimo sayusac i the ACSF perfusion ma rwinyo i lyethu ma rwinyo 32° ku 33°C. Multiphoton laser scanning microscope (TCS SP8 MP-OPO, Leica Microsystems) ma juketho ku Leica 25x objective lens (NA 0.95, pii), Ti: Sapphire laser (Chameleon Vision II, Coherent) jutiyo kudu pi goyo cal. FLIM module (PicoHarp300, PicoQuant).
FLIM pa Grx1-roGFP2. Aloka loka mi cytoplasmic redox state pa PNs jupimu ku two-photon FLIM in sagittal brain slices, kama Grx1-roGFP2 biosensor uketho PNs. I PN layer, kabedo mi nwangu piny juyeru 50 ku 80 μm piny pa slice surface mi neno nia PN maber utiye (niwacu, bedo mbe pa beaded structure nyo neuronal morphological changes along the dendrites) man double positive roGFP2 sensor man AAV encoding shRNA PCx nyo control sequence pare (coaching mCherry copressing). Cok cal acel acel ku 2x digital zoom [excitation wavelength: 890 nm; 512 nm 512 pixels]. Nwangu: HyD ma iye, fluorescein isothiocyanate (FITC) filter group] man cal ma juporo ikind dakika 2 ku 3 jutiyo kudu pi neno nia photons ma romo (1000 photons i wel) pi curve fitting. Ngec pa Grx1-roGFP2 probe man nyang pa FLIM jutimo ku lubo tok kwo pa roGFP2 kinde ma ju medo exogenous 10 mM H2O2 i perfusion ACSF (pi medo oxidation, ma kelo kwo madwong), man i ngeye medo 2 mM dithiothreit (ma dwoko piny rwom pa kwo pa roGFP2) dwoko piny kwo) (lanyut S8, D ni G). Ti ku FLIMfit 5.1.1 software mi ngiyo adwogi ma junwango, med single exponential decay curve pa cal ceke ni IRF ma jupimu (tic pa jam tic), man χ2 utiye 1. Pi kwanu kwo pa PN acel, mask ma ucungu ikum nerve body ju goyo ku cing, man wel pa kwo pa mask acel acel ju tiyo kudu.
Mitochondrial potential analysis. I ngei ma jupoko dul ma nyai ku 100 nM TMRM maju medo i ACSF pi dakika 30, aloka loka pa mitochondrial pa PNs jupimu ku two-photon microscope. TMRM imaging jutimo ku nyayu probe i 920 nm man tic ku HyD (tetramethylrhodamine isothiocyanate: 585/40 nm) mi coko lembe; ni tiyo ku wavelength marom ento nitio ku HyD ma iye matung tung (FITC :525/50) ni goyo cal pa mtYFP. Ti ku ImageJ's Image Calculator plug-in pi pimo kero pa mitochondrial i rwom pa cell acel. I lembe ma nok, equation mi plug-in: signal = min (mtYFP, TMRM) jutiyo kudu pi nyutho kabedo mi mitochondrial ma nyutho TMRM signal in Purkinje Somali in single-stack confocal image of the corresponding channel. I ngeye, kabedo pa pixel in mask ma wok jupimu, man juyike iwi cal acel acel pa mtYFP channel pi nwangu dul pa mitochondrial ma nyutho kero pa mitochondrial.
Cal ne juyike ku Huygens Pro (Scientific Volume Imaging) software. Pi cal ma juwodho pa tiles, montage pa tile acel jutimo ku kony pa automatic stitching algorithm ma LAS-X software umio. I ngei ketho cal, ti ku ImageJ man Adobe Photoshop mi medo tic ku cal man mi dwoko ler man tung tung pa cal. Ti ku Adobe Illustrator pi yiko cal.
mtDNA nen lembe. Wel pa ret pa mtDNA jupimu iwi dul pa cerebellar ma jugoro ku antibodies ikum DNA ku confocal microscope. Kabedo acel acel juyike pi kum pa cell man nucleus pa cell acel acel, man kabedo acel acel ju kwanu ku Multi Measure plug-in (ImageJ software). Kwany kabedo pa nuclear kud i kabedo pa cell body pi nwangu kabedo pa cytoplasm. I ajiki, Analyze Particles plug-in (ImageJ software) jutiyo kudu pi pimo cytoplasmic DNA points ma nyutho mtDNA iwi threshold image, man adwogi ma junwango judwoko ni PN average pa CTRL mice. Adwogi ne nen calu wel pa nucleosides i cell acel acel.
Nyamu lembe pa protein. Ti ku ImageJ's Image Calculator plug-in mi pimo kite ma protein tiyu kudu i PN i rwom pa cell acel. I lembe ma nok, i cal acel mi dul acel, nikadhu kud i equation: signal = min (mtYFP, antibody), kabedo mi mitochondrial ma nyutho immunoreactivity ni antibody moko i Purkina ungiyure. I ngeye, kabedo pa pixel i mask ma wok jupimo, man i ngeye juyike iwi cal acel acel pa mtYFP channel pi nwangu mitochondrial fraction pa protein ma ju nyuthu.
Purkinje cell density analysis. Cell Counter plug-in pa ImageJ jutiyo kudu pi pimo Purkinje density ni poko wel pa Purkinje cells ma jukwanu ku bor pa cerebellar ring ma cells ma jukwanu ubedo i iye.
Yub man coko lanyuth. Obwongo pa dul ma jucaku man Mfn2cKO mice juyike i 2% PFA/2.5% glutaraldehyde i 0.1 M phosphate buffer (PB), man i ngeye juyiko coronal sections ku tic ku ciliates (Leica Mikrosysteme GmbH, Vienna, Austria) (Thickness 50 to 60 μm) Buffer in 1% PB tetraoxide man 1.5% potassium ferrocyanide i lyethu mi ot pi sawa acel. Juyiko dul ne wang adek ku pii ma leng, man juyike ku 70% ethanol ma tye ku 1% uranyl acetate pi dakika 20. Thenge eca juwodho pii kud i kongo ma juyiko man juyiko i Durcupan ACM (Araldite casting resin M) epoxy resin (Electron Microscopy Sciences, namba 14040) ikind silicon-coated glass slides, man i ajiki i 60°C Polymerize i oven pi sawa 48. Kabedo pa cerebellar cortex juyeru man 50 nm ultrathin sections jupoko iwi Leica Ultracut (Leica Mikrosysteme GmbH, Vienna, Austria) man jukwanyo iwi 2×1 mm copper slit grid ma juketho ku polystyrene film. Thenge ne juyike ku 4% uranyl acetate in H2O pi dakika 10, ju lwoko ku H2O wang mapol, man ju lwoko ku Reynolds lead citrate i H2O pi dakika 10, man ju lwoko ku H2O wang mapol. Micrographs juwodho ku transmission electron microscope Philips CM100 (Thermo Fisher Scientific, Waltham, MA, USA) ma jutiyo kudu ku TVIPS (Tietz Video and Image Processing System) TemCam-F416 digital camera (TVIPS GmbH, Gauting, USA). Germany).
Pi oyo ma AAV unwangu, wic jupoko man jupoko i 1 mm thick sagittal section, man cerebellum juporo ku tiyo ku fluorescence microscope pi ni ngeyo ring ma AAV unwangu (niwacu, mCherry expressing). Lembe ma jutimo kende ma AAV injection kelo adwogi maber pa Purkinje cell layer (niwacu, layer zo) i cerebellar rings ario ma lubo. AAV-transduced loop ubedo microdissected pi overnight post-fixation (4% PFA man 2.5% glutaraldehyde in 0.1 M cocoate buffer) man ju medo tic kudu. Pi EPON embedding, juyike ku 0.1 M sodium cocoate buffer (Applichem), man juyike ku 2% OsO4 (os, Science Services; Caco) i 0.1 M sodium cocoate buffer (Applichem) pi sawa angwen, man juyike pi sawa 2. Med kendo wang 3 ku 0.1 M cocamide buffer. I ngeye, jutiyo ku ethanol ma ube medere malu pi ketho ethanol acel acel i 4°C pi dakika 15 mi jwigo pii kud i kum. Juloko adila ne i propylene oxide man juyike dyewor i EPON (Sigma-Aldrich) i 4°C. Keth adila ne i EPON manyen i lyethu mi ot pi sawa 2, man idok i 62°C pi sawa 72. Ti ku ultramicrotome (Leica Microsystems, UC6) man diamond knife (Diatome, Biel, Switzerland) mi thum 70 nm ultrathin sections, man iket rangi ku 1.5% uranyl acetate pi dakika 15 i 37°C, man iket rangi ku lead citrate solution pi dakika 4. Electron micrographs juwodho ku tic ku JEM-2100 Plus transmission electron microscope (JEOL) ma juketho i iye Camera OneView 4K 16-bit (Gatan) man DigitalMicrograph software (Gatan). Pi nyang ikum lembe, electron micrographs ju kwanu ku 5000× nyo 10,000× digital zoom.
Kwano kit ma mitochondria nen kudu. Pi lembe ceke ma jutimo, nen pa mitochondria acel acel ju goyo ku cing gi i cal ma jutiyo kudu ku jam tic mi ImageJ. Lembe matung tung mi nen pa piny juporo. Mitochondrial density jukwanu calu pacent ma jupoko ku poko mitochondrial area pa cell acel acel ku cytoplasm area (cytoplasm area = cell area-cell nucleus area) × 100. Roundness pa mitochondria ju kwanu ku formula [4π∙(area/perimeter 2)]. Ista morphology pa mitochondria juporo man jupoko i dul aryo ("tubular" man "blister") nimakere ku kit migi.
wel pa autophagosome/lysosome man pek pa piny. Ti ku ImageJ software ni goyo nen pa autophagosome/lysosome acel acel i cal. Kabedo pa autophagosome/lysosome jukwanu ni wel ma jupoko ku poko kabedo pa autophagosome/lysosome pa cell acel acel ku kabedo pa cytoplasm (kabedo pa cytoplasm=kabedo pa cell-kabedo pa nucleus)×100. Pek pa autophagosomes/lysosomes ju kwanu ku poko wel ceke ku wel pa autophagosome/lysosome structures pa cell acel acel (i thenge pa cytoplasmic area) (cytoplasmic area = cell area-nuclear area).
Kethu nying pi poko poko man yiko lanyuth. Pi lembe ma jutimo ma mitu ketho lanyuth pa glucose, lok dul pa wic ma nyai i ot ma podi juyiko ngo, ma utiye ku saturated carbon (95% O2 man 5% CO2), Ca2 + ACSF (125.0 mM NaCl, 2.5 mM KCl, 1.25 mM sodium phosphate buffer, 2.5 mM NaCl, 2.5 mM. d-glucose, 1.0 mM CaCl 2 man 2.0 mM MgCl 2, ma juloko ni pH 7.4 man 310 ni 320 mOsm), ma glucose utiye 13 C 6- Glucose substitution (Eurisotop, namba mi catalog CLM-1396). Pi lembe ma jutimo ma mitu pyruvate labeling, lok acute brain slices i Ca2 + ACSF ma malu (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 Ca2 man Ad. MgCl2, lok pH 7.4 man 310 ni 320mOsm), man medu 1mM 1-[1-13C]pyruvate (Eurisotop, namba mi catalog CLM-1082). Keth dul ne pi dakika 90 i 37°C. I thum pa lembe ma jutimo, juyiko dul ne pio pio ku pii (pH 7.4) ma tye ku 75 mM ammonium carbonate, man juyike i 40:40:20 (v:v:v) acetonitrile (ACN): methanol: pii. I ngei ma juketho dul ne iwi pee pi dakika 30, juyike i 21,000 g pi dakika 10 i 4°C, man juyike pii maleng i SpeedVac concentrator. Adwogi pa metabolite ma muthwo jugwoko i -80°C nitundu jutimo sayusac.
Liquid chromatography-mass spectrometry analysis pa 13 C-labeled amino acids. Pi liquid chromatography-mass spectrometry (LC-MS) analysis, metabolite pellet judwoko i 75μl pa LC-MS grade water (Honeywell). I ngei centrifugation i 21,000 g pi dakika 5 i 4°C, 20 μl pa supernatant ma juyiko jutiyo kudu pi amino acid flux analysis, man extract mukende jutiyo kudu pi anion analysis (nen piny). Amino acid analysis jutimo ku tic ku benzoyl chloride derivatization protocol (55, 56). I acaki, 10μl pa 100 mM sodium carbonate (Sigma-Aldrich) jumedo i 20μl pa metabolite extract, man 10μl pa 2% benzoyl chloride (Sigma-Aldrich) jumedo i LC grade ACN. Lanyuth ne juyike nyanok man juyike i 21,000 g pi dakika abic i 20°C. Lok pii ma juyiko i 2 ml autosampler vial ku conical glass insert (200 μl volume). Lanyuth juporo ku kony pa Acquity iClass ultra-high performance LC system (Waters) ma jukubo ku Q-Exactive (QE)-HF (Ultra High Field Orbitrap) high-resolution precision mass spectrometer (Thermo Fisher Scientific). Pi nyang ikum lembe, 2μl pa lanyuth ma juwodho jubolo i 100×1.0 mm high-strength silica T3 column (Waters) ma tye ku 1.8μm particles. Kite ma pii mol kudu utiye 100μl/min, man buffer system utiye ku buffer A (10 mM ammonium formate man 0.15% formic acid i pii) man buffer B (ACN). Gradient utiye ma e: 0%B i dakika 0; 0%B. 0 ku 15% B i 0 ku 0.1 dakika; 15 ku 17% B i 0.1 ku 0.5 dakika; B i 17 ku 55% i 0.5 ku 14 dakika; B i 55 ku 70% i 14 ku 14.5 dakika; i 14.5 ku 70 ku 100% B i dakika 18; 100% B i dakika 18 ku 19; 100 ku 0% B i dakika 19 ku 19.1; 0% B i dakika 19.1 ku 28 (55, 56). QE-HF mass spectrometer timo tic i positive ionization mode ku mass range pa m/z (mass/charge ratio) pa 50 ku 750. Resolution ma jutiyo kudu utiye 60,000, man gain control (AGC) ion target ma junwango utiye 3×106, man ion time ma dit utiye 100 milliseconds. The heated electrospray ionization (ESI) source operations at spray voltage of 3.5 kV, the capillary temperature of 250°C, a sheath airflow of 60 AU (arbitrary units), and the auxiliary airflow of 20 AU. 250°C. S lens juketho i 60 AU.
Anion chromatography-MS analysis pa 13C ma jukiewo ikum organic acids. Piny ma udong (55μl) juporo ku kony pa Dionex ion chromatography system (ICS 5000+, Thermo Fisher Scientific) ma jukubo ku QE-HF mass spectrometer (Thermo Fisher Scientific). I lembe ma nok, 5μl pa metabolite extract jubolo i Dionex IonPac AS11-HC column ma juketho i iye HPLC (2 mm×250 mm, particle size 4μm, Thermo Fisher Scientific) i push-in partial loop mode ku filling ratio pa 1. ) Dionex IonPac AG11-HC mm column (x250 mm guard column, 50 4μm, Thermo Fisher Scientific). Lyethu pa column ubedo 30°C, man autosampler ubedo 6°C. Ti ku potassium hydroxide cartridge ma juketho i iye pii ma deionized pi niketho potassium hydroxide gradient nikadhu kud i eluent generator. Poko pa metabolites i rwom pa mol pa 380μl/min, ni tiyo ku gradient ma e: 0 ku 3 dakika, 10 mM KOH; 3 ku 12 dakika, 10 ku 50 mM KOH; 12 ku 19 dakika, 50 ku 100 mM KOH; 19 ku 21 dakika, 100 mM KOH; 21 ku 21.5 dakika, 100 ku 10 mM KOH. Column ne ju dwoko piny i 10 mM KOH pi dakika 8.5.
Jam piny ma juwodho juporo ku 150μl/min isopropanol supplement stream ingey column man jucwalo gi i high-resolution mass spectrometer ma tiyo i negative ionization mode. MS ube lubo tok pek pa m/z 50 nitundo 750 ku rwom pa 60,000. AGC juketho i 1×106, man kare ma malu pa ion juketho i 100 ms. Jam piny ma lyeth ma julwongo ni ESI ubedo ka tiyo ku voltage pa 3.5 kV. Kit mukende pa ion source utiye ma e: lyethu pa capillary 275°C; sheath gas flow, 60 AU; mol pa yamo ma konyo, 20 AU i 300°C, man S lens ma juketho i 60 AU.
Kwano lembe pa 13C ma jugoro ku metabolites. Ti ku TraceFinder software (version 4.2, Thermo Fisher Scientific) pi ngiyo lembe pa isotope ratio. Kit pa piny acel acel juyike ku piny ma jucopu geno man jutimo sayusac iwie giri. Pi timo sayusac mi dongo pa isotope, kabedo pa ion chromatogram (XIC) pa 13C isotope acel acel (Mn) juwodho kud i [M + H] +, kama n utiye namba pa carbon pa piny ma juyenyo, ma jutiyo kudu pi ngiyo amino acids nyo [ MH] + jutiyo kudu pi ngiyo anions. Ber pa XIC utiye piny pa dul abic pi million, man ber pa RT utiye dakika 0.05. Enrichment analysis jutimo ku kwanu ratio pa isotope acel acel ma junwango ku wel pa isotope ceke mi compound ma rwate. Ratio eni jumiyo calu wel pa acel acel pa isotope, man adwogi ne junyutho calu molar percent enrichment (MPE), calu ma juloko con (42).
Pellet pa neuron ma jukutho juyike i ice-cold 80% methanol (v/v), juyike, man juyike i -20°C pi dakika 30. Vortex sample kendu man irub i +4°C pi dakika 30. Lanyuth ne ju centrifuge i 21,000 g pi dakika 5 i 4°C, man i ngeye ju kwalo man ju juku ku SpeedVac concentrator i 25°C pi timo sayusac mukende. Calu ma jukoro malu, LC-MS jutimo ikum amino acids pa cell ma jupoko. Ku tic ku TraceFinder (version 4.2, Thermo Fisher Scientific), jutimo sayusac ikum lembe ma jutiyo kudu ku monoisotopic mass pa compound acel acel. Quantile normalization pa metabolite data utimere ku kony pa preprocessCore software package (57).
Yub pa dul. Jutimo anesthesia pio pio ku carbon dioxide man jupoko wiye, juwodho wic pare pio pio kud i wic, man jutiyo ku pala ma upong ku pee (HM-650 V, Thermo Fisher Scientific, Walldorf, Germany) pi poko 300 ku 375 μm sagittal sections COd carbon gas man CO (25% 25%) Ca2 + ACSF ma nok (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 man 6.0 mM MgCl2 Lok ikum pH ni 37 mM man 30 mO). Lok dul wic ma inwango i ot ma tye ku 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 mM Ca 2.2 mM man pH 2 MM 7.4 man 310 ku 320 mOsm). Gwok dul ne pi dakika 20 ku 30 kara gicop dwoko gi maber mapodi i goyo wer.
mako wer. Microscope stage ma juketho i iye kabedo mi goyo cal man 20x water immersion objective lens (Scientifica) jutiyo kudu pi goyo cal zo. Purkinje cells ma juporo ku (i) dit pa adi, (ii) kabedo pa cerebellum, man (iii) nyuthu pa fluorescent mtYFP reporter gene. Patch pipette ku tip resistance pa 5 ku 11 megohms juwodho woko ku borosilicate glass capillary (GB150-10, 0.86 mm×1.5 mm×100 mm, Science Products, Hofheim, Germany) man horizontal pipette Instruments (P-1000, Sutter, Notova, CA). Lembe ceke ma jugoro jutimo ku ELC-03XS npi patch clamp amplifier (npi electronic GmbH, Tam, Germany), ma jutiyo kudu ku software Signal (version 6.0, Cambridge Electronic, Cambridge, UK). Temo ne ju goyo i sampling rate pa 12.5 kHz. Lanyuth ne juyike ku filter ario ma short-pass Bessel ku cutoff frequency pa 1.3 ku 10 kHz kubang acel acel. Kero pa membrane man pipette judwoko ku circuit mi dwoko piny ma jutiyo kudu ku amplifier. Lembe ceke ma jutimo jutimo ku Orca-Flash 4.0 camera (Hamamatsu, Gerden, Germany), ma jutiyo kudu ku Hokawo software (version 2.8, Hamamatsu, Gerden, Germany).
Yub pa cell ceke man nyang ikum gi. Mapodi i goyo wer, pong pipette ku pii ma iiye ma tye ku piny ma e: 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 (MMM), 5.0 mM Gunophosine. (GTP) (Na) man 10.0 mM creatinine phosphate juyike ni pH 7.25, man osmotic pressure ubedo 290 mOsm (sucrose). Ndhu ndhu ingey tiyo ku tego pa 0 pA mi turo membrane, jupimu kero pa membrane. Pek pa piny ma jucwalo jupimu ku tiyo ku kero ma lee pa -40, -30, -20, man -10 pA. Pim dit pa dwokowang pa voltage man ti ku cik pa Ohm ni kwanu resistance pa input. Tic ma timo kende kende jukyewo i voltage clamp pi dakika abic, man sPSC junwango man jupimu i Igor Pro (version 32 7.01, WaveMetrics, Lake Oswego, Oregon, USA) ku tic ku semi-automatic recognition script. IV curve man steady-state current jupimu ku mako battery i rwom matung tung (nicaku i -110 mV) man medo voltage i 5 mV steps. Tic pa AP jutemo ku tic ku depolarizing current. Keth cell i -70 mV podi itiyo ku depolarizing current pulse. Lok dit pa step acel acel mi rekod acel acel (10 ku 60 pA). Kwan AP frequency ma malo ni kwanu pulse spikes ma kelo AP frequency ma malu. AP threshold juporo ku tic ku derivative mir aryo pa depolarization pulse ma kwong ma nyayu AP acel nyo madwong.
Kit ma juyubu kudu man nyang kudu. Tim rekod pa patch ma jugoro ku tiyo ku cik ma rwom pare lapiny. Ti ku ATP- man GTP-free pipette ma epe ku piny ma e: 128 mM gluconate K, 10 mM KCl, 10 mM Hepes, 0.1 mM EGTA man 2 mM MgCl2, man ilok pH 7.2 (ku tic ku KOH). ATP ku GTP juweko gi kud i pii ma iye pi juku pii ma rwinyo ngo pa cell membrane. Patch pipette upong ku amphotericin-containing internal solution (maromo 200 ku 250μg/ml; G4888, Sigma-Aldrich) mi nwangu rekod pa patch. Amphotericin juyike i dimethyl sulfoxide (dwong pa piny: 0.1 ku 0.3%; DMSO; D8418, Sigma-Aldrich). Kit pa DMSO ma jutiyo kudu ubedu ungo ku adwogi madit ikum neurons ma jutimo ikumgi sayusac. I kare mi timo tic mi goyo, rwom pa channel (Ra) jubedu ka lubo tok jeni, man jucaku timo ne ingey amplitude pa Ra ku AP ubedo maber (dakika 20-40). Tic ma timo kende kende jupimu i voltage man/nyo current clamp pi dakika 2 ku 5. Kwano lembe jutimo ku kony pa Igor Pro (version 7.05.2, WaveMetrics, USA), Excel (version 2010, Microsoft Corporation, Redmond, USA) man GraphPad Prism (version 8.1.2, GraphPad Software Inc., La Jolla, CA). Amerika). Pi ni nyang APs ma wok kende, IgorPro's NeuroMatic v3.0c plug-in jutiyu kudu. Nwangu APs kende kende ni tiyo ku threshold moko, ma juloko acel acel pi rekod acel acel. Ni tiyo ku spike interval, ngeyo spike frequency ku maximum instantaneous spike frequency man average spike frequency.
PN isolation. Ni lubo cik ma jugoro con, PNs juyike kud i cerebellum pa oyo i kare moko (58). I lembe ma nok, cerebellum jupoko man jupoko i ice-cold dissociation medium [labongo HBSS Ca2+ man Mg2+, jumedo ku 20 mM glucose, penicillin (50 U/ml) man streptomycin (0.05 mg/ ml)], man juyike i medium i papain [HBSS, 1·1 (1 mg / ml), papain (16 U / ml) man deoxyribonuclease I (DNase I; 0.1 mg/ml)] Tim pi dakika 30 i 30°C. Mir acel, lwok adila i HBSS ma tye ku egg mucus (10 mg/ml), BSA (10 mg/ml) man DNase (0.1 mg/ml) i rwom ma piny mi ot pi juku lyethu pa enzymatic, man i ngeye i HBSS ma tye ku 20 mM glucose Gently grinding in HBSS peillinic, peillin (0.5/ml), strep (0.05 mg/ml) man DNase (0.1 mg/ml) wodhu cell acel acel. Adwogi pa cell suspension juwodho kud i 70μm cell strainer, man cells jupoko ku centrifugation (1110 rpm, 5 minutes, 4°C) man judwoko cen i sorting medium [HBSS, supplemented with 20 mM glucose, 20% fetal bovine ) Serum, pelinic (50) man strep (0.05 mg/ml)]; pimo kwo pa cell ku propidium iodide man ilok pek pa cell ni 1×106 ku 2×106 cells/ml. Mapodi flow cytometry utimere, juyike piny ma juciko ku 50 μm cell strainer.
Flow cytometer. Poko cell jutimo i 4°C ku tic ku FACSAria III machine (BD Biosciences) man FACSDiva software (BD Biosciences, version 8.0.1). Cell suspension jupoko ku tic ku 100 μm nozzle ithe tegu pa 20 psi i rwom pa ~2800 events/sec. Calu ma lembe macon mi gating (dit pa cell, bimodal discrimination, man scattering characteristics) copo ketho ngo poko PN kud i cell mange, yore mi gating juketho malubiri ku poro pa YFP intensity man autofluorescence i mitoYFP+ ​​man control mitoYFP − Mice. YFP ubedo kud anyonga ni nyayu lero pa lanyuth ku 488 nm laser line, man lanyuth ne junwango ku tic ku 530/30 nm band pass filter. I mitoYFP+ ​​oyo, tego pa Rosa26-mitoYFP reporter gene de jutiyo kudu pi poko kind dul kum pa ler man dul pa axon. 7-AAD juyike ku 561 nm laser ma yellow man junwango ku 675/20 nm bandpass filter mi kwanyu cell ma tho. Pi poko astrocytes i sawa marom, cell suspension juyike ku ACSA-2-APC, man juyike ku 640 nm laser line, man 660/20 nm bandpass filter jutiyo kudu pi nwangu signal.
Cells ma jucoko jupoko ku centrifugation (1110 rpm, dakika 5, 4°C) man jugwoko i -80°C nitundu jutiyo kudu. Mfn2cKO mice ku nyithindgi ma gipodho jupoko gi in ceng acel pi jwigu aloka loka mi timo lembe. Nyuthu pa lembe pa FACS man nyang kudu jutimo ku kony pa FlowJo software (FlowJo LLC, Ashland, Oregon, USA).
Calu ma juweco pire malu (59), PCR mi kare mandha jutiyo kudu pi poko DNA kud i kind neurons ma jupoko pi mtDNA ma lubo. Linearity man threshold sensitivity ju temo ku qPCR iwi wel pa cell matung tung. I lembe ma nok, coko 300 PN i lysis buffer ma tye ku 50 mM tris-HCl (pH 8.5), 1 mM EDTA, 0.5% Tween 20 man proteinase K (200 ng/ml) man iket i 55°C pi dakika 120. Judwoko cell ne i 95°C pi dakika 10 mi neno nia proteinase K uthum ungo. Ku tic ku TaqMan probe (Thermo Fisher) ma pire tek ni mt-Nd1, mtDNA jupimu ku semi-quantitative PCR i 7900HT Real-Time PCR system (Thermoherific Scientifics). Sayans, namba mi catalog Mm04225274_s1), mt-Nd6 (Thermo Fisher Scientific, namba mi catalog AIVI3E8) man 18S (Thermo Fisher Scientific, namba mi catalog Hs99999901_s1) genes.
Yub pa lanyuth pa proteome. Ku lyethu pii i 95°C pi dakika 10 man sonicating, i lysis buffer [6 M guanidine chloride, 10 mM tris(2-carboxyethyl) phosphine hydrochloride, 10 mM chloroacetamide man 100 mM tris- Lyse frozen neuron pellets in HC]. I Bioruptor (Diagenode) pi dakika 10 (30 seconds pulse / 30 seconds pause period). Lanyuth ne juyike 1:10 in 20 mM tris-HCl (pH 8.0), jurubu ku 300 ng trypsin gold (Promega), man juyike dyewor i 37°C mi nwangu digestion ma oromo. I ceng mir aryo, juyiko lanyuth ne i 20,000 g pi dakika 20. Piny ma ucungo iwi pii ne juyike ku 0.1% formic acid, man juyike ku StageTips ma juyubu giri. Lanyuth ne juyike i SpeedVac instrument (Eppendorf concentrator plus 5305) i 45°C, man peptide ne juyike i 0.1% formic acid. Sampol zo juyike i saa acel ku ngati acel. Pi ni ngiyo lanyuth pa astrocyte, 4 μg desated peptides ju goyo ku tandem mass tag (TMT10plex, catalog number 90110, Thermo Fisher Scientific) ku peptide ku TMT reagent ratio pa 1:20. Pi ketho TMT, 0.8 mg pa TMT reagent ju dwoko i 70 μl pa anhydrous ACN, man peptide ma muthwo ju dwoko i 9 μl pa 0.1 M TEAB (triethylammonium bicarbonate), ma 7 μl pa TMT reagent i ACN ju medo i iye. Kit ma piny ubedo kudu ubedo 43.75%. I ngei dakika 60 mi gwoko, judwoko piny ne ku 2 μl pa 5% hydroxylamine. Peptides ma jugoro nying gi jucoko, jutwo, judwoko gi i 200μl pa 0.1% formic acid (FA), jupoko i ario, man judwokogi ku kadu ku StageTips ma juyubu giri. Ku tic ku UltiMate 3000 ultra high performance liquid chromatograph (UltiMate 3000 ultra high performance liquid chromatograph), acel ikin nusu aryo ne jupoko iwi 1mm x 150mm Acquity chromatographic column ma upong ku 130Å1.7μm C18 particles (Waters, catalog No SKU: 196366). Thermo Fisher Scientific). Pok peptides ku rwom mi mol pa 30μl/min, poko kud i 1% ku 50% buffer B pi dakika 85 ku stepwise gradient pa dakika 96, kud i 50% ku 95% buffer B pi dakika 3, man dakika 8 pi 95 % Buffer B; Buffer A utiye 5% ACN man 10 mM ammonium bicarbonate (ABC), man buffer B utiye 80% ACN man 10 mM ABC. Cok dul ma nok kubang dakika 3 man iket gi i ungu aryo (1 + 17, 2 + 18, man mukende mapol) man iket gi i vacuum centrifuge.
LC-MS/MS analysis. Pi mass spectrometry, peptides (namba r119.aq) jupoko iwi 25 cm, 75 μm inner diameter PicoFrit analytical column (lens manyen, namba mi thenge PF7508250) ma juketho i iye 1.9 μm ReproSil-Pur 120 C18-AQD medium (Maisch, UAS-YAS). 1200 (Thermo Fisher Scientific, Germany). Column ne jugwoko i 50°C. Buffers A ku B utiye 0.1% formic acid i pii man 0.1% formic acid i 80% ACN, kubang wadi. Peptides jupoko kud i 6% ku 31% buffer B pi dakika 65 man kud i 31% ku 50% buffer B pi dakika 5 ku gradient pa 200 nl/min. Peptides ma juwodho juporo ku Orbitrap Fusion mass spectrometer (Thermo Fisher Scientific). Peptide precursor m/z measurement is performed with resolution of 120,000 in the range of 350 to 1500 m/z. Ni tiyo ku 27% pa kero mi rwinyo, piny ma tek uloyu ku kero pa 2 ku 6 juyeru pi kero madit pa C trap dissociation (HCD) cleavage. Saa mi ngwec juyike ni 1 s. Wel m/z pa dul pa peptide jupimu i ion trap ku tic ku AGC ma nok loyo pa 5×104 man kare ma dit pa 86 ms. I ngei poko, piny ma kwong eca juketho iwi piny ma juwodho woko pi 45 s. TMT-labeled peptides jupoko iwi 50 cm, 75 μm Acclaim PepMap column (Thermo Fisher Scientific, namba mi catalog 164942), man migration spectra juporo iwi Orbitrap Lumos Tribrid mass spectrometer (Thermo Fisher Scientific) ma juketho ku high-field ammetric aveform iMS (IMS) jam tic (Thermo Fisher Scientific) tiyo ku voltage aryo mi −50 ku −70 V. MS3 ma juyeru malubiri ku synchronization precursor jutiyo kudu pi TMT report ion signal measurement. Poko peptide utimere iwi EASY-nLC 1200, ku tic ku 90% linear gradient elution, ku buffer concentration pa 6% ku 31%; buffer A ubedo 0.1% FA, man buffer B ubedo 0.1% FA man 80% ACN. Analytical column tiyu kudu i 50°C. Ti ku FreeStyle (version 1.6, Thermo Fisher Scientific) mi poko fail macon nimakere ku FAIMS compensation voltage.
Nyuthu man kwanu pa protein. Ni tiyo ku Andromeda search engine, lembe macon juporo ku MaxQuant version 1.5.2.8 (https://maxquant.org/). Mi medo ikum Cre recombinase man YFP ma junwango kud i Aequorea victoria, peptide fragment spectra juyenyo pi canonical sequence man isoform sequence pa mouse reference proteome (Proteome ID UP000000589, ma juwodho kud i UniProt i Dwi mir Abic 2017). Methionine oxidation man protein N-terminal acetylation juyike calu aloka loka matung tung; cysteine ​​  carbamoyl methylation juyike calu aloka loka ma ucungo. Lembe ma mako nyayu cam juyike ni "specificity" man "trypsin/P". Wel ma nok pa peptides man razor peptides ma jutiyo kudu pi ngeyo protein utiye 1; wel ma nok pa peptides ma tung tung utiye 0. I thenge mi peptide map matching, wel mi ngec pa protein ubedo 0.01. "Peptide mir aryo" utiye tic. Ti ku "match between runs" pi cwalo lembe maber ikind fail matung tung. Ti ku LFQ ratio ma nok 1 pi label-free quantification (LFQ) (60). LFQ intensity juyike pi wel ario ma ber i dul acel acel mi genotype i kare acel acel, man juwodho kud i rwom ma lageng ku lac pa. 0.3 man idok piny 1.8. Ti ku Perseus computing platform (https://maxquant.net/perseus/) man R (https://r-project.org/) pi nyang ikum adwogi pa LFQ. T test ma rwom pare ario kud i limma software package jutiyo kudu pi nyang ikum lembe ma tung tung (61). Lembe ma jutimo ku ggplot, FactoMineR, factoextra, GGally man pheatmap. TMT-based proteomics data juporo ku MaxQuant version 1.6.10.43. Sayu lembe ma mako proteomics kud i UniProt's human proteomics database, ma juwodho i Dwi mir Abora 2018. Lembe ma jutimo uketho i iye isotope purity correction factor ma juyiko ku ju ma yiku. Ti ku limma in R pi nyang ikum lembe ma tung tung. Lembe macon, adwogi mi sayu lembe, man tic mi nyang ikum lembe man adwogi zo jugwoko i ProteomeXchange alliance nikadhu kud i PRIDE partner repository ku data set identifier PXD019690.
Lembe ma jugoro maber konyowa ni nyang ikum lembe. Jam tic mi Ingenuity Pathway Analysis (QIAGEN) jutiyo kudu pi nyang ikum rwom pa lembe ma jugoro i lembe ma jucoko i yenga mir 8 (lanyut 1). I lembe ma nok, wel pa protein ma ju nwango kud i LC-MS/MS (tandem mass spectrometry) data analysis jutiyo kudu ku filter criteria ma e: Mus musculus juyeru calu kit lei man thenge ma piny, man dul ne nyutho P value ma judwoko ku Benjamini pi medo 0.05 nyo piny ma jukwanu ni piny ma pire tek. Pi graph eni, dul abic ma malu pa dul acel acel malubiri ku wel pa P ma juloko ju nyuthu. Ni tiyo ku t-test madwong, nitio ku two-stage linear boost program pa Benjamini, Krieger, man Yekutieli (Q = 5%), time-course protein expression analysis jutimo ikum ju ma pire tek ma junwango i dul acel acel, man dul acel acel jutimo sayusac iwie. Pe tiye ku yeny mi lubo SD ma rwate.
Pi poro adwogi pa somo eni ku lembe ma juwodho man niketho Venn diagram i cal mir acel, wa ketho wel pa protein ku MitoCarta 2.0 annotations (24). Ti ku jam tic mi intanet Draw Venn Diagram (http://bioinformatics.psb.ugent.be/webtools/Venn/) mi yubu cal.
Pi ngec matuc ikum yore mi kwan ma jutiyo kudu pi nyang ikum proteomics, nen dul ma rwate ku jam tic man yore. Pi lembe mukende ma jutimo, lembe matung tung copo nwangire i lembe ma rwate. Ka jukwanu ngo yo mukende, lembe zo jukwanu calu mean ± SEM, man lembe ceke ma jukwanu jutimo ku tic pa GraphPad Prism 8.1.2 software.
Pi lembe mukende ma medo ikum lembe maeni, nen http://advances.sciencemag.org/cgi/content/full/6/35/eaba8271/DC1
Eni ubedo lembe ma dhanu ceke copo tiyo kudu ma jupoko i the cik pa Creative Commons Attribution-Non-Commercial License, ma yio tic kudu, poko man dwoko piny i yore moku ci, tek tic ma tok cen epe pi kelo lim man lembe ma pire tek utiye nia tic ma kwong eca utiye kakare. Mamako kume.
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Ku E. Motori, I. Atanassov, SMV Kochan, K. Folz-Donahue, V. Sakthivelu, P. Giavalisco, N. Toni, J. Puyal, N.-G. Larson
Proteomics analysis pa dysfunctional neurons unyutho nia tic pa metabolic programs timo tic pi juku neurodegeneration.
Ku E. Motori, I. Atanassov, SMV Kochan, K. Folz-Donahue, V. Sakthivelu, P. Giavalisco, N. Toni, J. Puyal, N.-G. Larson
Proteomics analysis pa dysfunctional neurons unyutho nia tic pa metabolic programs timo tic pi juku neurodegeneration.
©2020 Kanica mi Amerika pi dongo ngec mi ngec. twero ceke jugwoko. AAAS ubedo wadi pa HINARI, AGORA, OARE, CHORUS, CLOCKSS, CrossRef man COUNTER. ScienceAdvances ISSN 2375-2548.