Present *Current address: Cologne 50931, Germany, Cologne Excellence Cluster Research on Cellular Stress Response in Aging-related Diseases (CECAD).
Ing neurodegeneration da reng sakit a mitochondrial tuturing deng e malyaring ibalik uling limitadu ya ing metabolic plasticity da reng neuron, dapot e masyadung aintindian ing epektu ning mitochondrial dysfunction king cell autonomy ning neuronal metabolism king katawan. Keni, ipakilala mi ing cell-specific proteome da reng Purkinje neurons a maki progresibong kakulangan king OXPHOS a sangkan ning mesira nang mitochondrial fusion dynamics. Ikit mi na ing mitochondrial dysfunction meging sangkan ning maragul a pamagbayu keng larangan ning proteomics, na meging sangkan ning sequential activation da reng precise metabolic programs bayu la mete deng cell. E mi asahan, abalu mi ing malino induction ning pyruvate carboxylase (PCx) ampo reng aliwang anti-aging enzymes na magdagdag kareng intermediates ning TCA cycle. Ing pamag-inhibit ning PCx pepalala ne ing oxidative stress ampo ing neurodegeneration, a magpakit na ing atherosclerosis atin yang proteksiun a epektu kareng neurons a alang OXPHOS. Ing pamagbalik ning mitochondrial fusion kareng terminally degenerated neurons ganap nang babaligtad deng metabolic characteristics a reni, inya pin makaiwas ing kamatayan da reng cell. Ing kekaming akit a makikilala kareng e pa balu a dalan na mamye resilience keng mitochondrial dysfunction ampo papakit na ing neurodegeneration malyari yang mibalik agyang keng tauling yugtu ning sakit.
Ing sentru ning mitochondria keng pamag-maintain ning neuronal energy metabolism mipapansin ya kareng malawak a sintomas ning neurological a makasuglung kareng sakit ning mitochondria ning tau. keraklan kareng sakit a reni manibatan la kareng gene mutations na magregulate keng mitochondrial gene expression (1, 2) o gene destruction a maki kaugnayan keng mitochondrial dynamics, na e direktang makaapektu keng katatagan ning mitochondrial DNA (mtDNA) (3, 4). Ing obra kareng modelung animal pepakit na na bilang pakibat keng mitochondrial dysfunction kareng makapadurut a tissue, deng conservative metabolic pathways (5-7) malyari lang ma-activate, na mamye importanting impormasyun para keng malalam a pamanintindi keng pathogenesis da reng komplikadung sakit a reni. Ing pamiyaliwa, ing kekatamung pamanintindi kareng pamagbayu ning metabolic da reng partikular a uri ning cell a sangkan ning pangkabilugan a pangabagsak ning pamaglalang ning mitochondrial adenosine triphosphate (ATP) ning utak metung yang pundasyunal (8), a didinan pansin ing pamangailangan a akilala la reng therapeutic targets a malyaring gamitan para aiwasan o aiwasan ing sakit. Iwasan ing neurodegeneration (9). Ing kakulangan keng impormasyun yapin ing katutwan na deng nerve cells maralas lang tuturing a atin lang limitadu a metabolic flexibility kumpara kareng cell a uri da reng makapadurut a tissue (10). Uling deng cell a reni atin lang sentrung papil keng pamag-coordinate keng supply da reng metabolites kareng neurons para i-promote ing synaptic transmission ampo pakibatan ing injury ampo sakit, ing abilidad na para i-adapt ing cell metabolism kareng masakit a kundisyun ning brain tissue halus limitadu ya kareng glial cells (11-14). Dagdag pa, ing inherent cellular heterogeneity ning tissue ning utak maragul yang makasabat keng pamagaral kareng metabolic changes a malilyari kareng specific neuronal subgroups. Uli na niti, ditak mu ing balu tamu tungkul keng eksaktung cellular ampo metabolic a bunga ning mitochondrial dysfunction kareng neurons.
Para aintindian ing metabolic a bunga ning mitochondrial dysfunction, me-isolate kami kareng Purkinje neurons (PNs) kareng miyayaliwang yugtu ning neurodegeneration a sangkan ning pangasira ning mitochondrial outer membrane fusion (Mfn2). Agyang deng Mfn2 mutations kareng tau makasuglung la keng metung a uri ning hereditary motor sensory neuropathy a kilala bilang Charcot-Marie-Tooth type 2A (15), ing conditional a pangasira ning Mfn2 kareng mice metung yang makikilalang induction ning oxidation Phosphorylation (OXPHOS) dysfunction method. Deng miyayaliwang neuronal subtypes (16-19) ampo ing resulta ning neurodegenerative phenotype kayabe la reng progresibong sintomas ning neurological, antimo reng movement disorders (18, 19) o cerebellar ataxia (16). Gamit ing kombinasyun ning label-free quantitative (LFQ) proteomics, metabolomics, imaging, ampo virological a paralan, papakit mi na ing progresibong neurodegeneration masikan yang mag-induce pyruvate carboxylase (PCx) ampo reng aliwang sangkan a maki kaugnayan keng arteriosclerosis da reng PNs in vivo Ing pamagpahayag da reng enzymes. Para abalu ing kaugnayan na niting akit, partikular ming pepababa ing expression ning PCx kareng Mfn2-deficient PNs, at ikit mi na ing operasyun ayni peparagul ne ing oxidative stress at pepabilis ne ing neurodegeneration, inya pin patunayan na ing azoospermia mamye yang cell death Metabolic adaptability. Ing matinding pamagpahayag ning MFN2 malyari yang lubus a iligtas ing terminal degeneration PN a maki matinding kakulangan king OXPHOS, maragul a pamangailangan king mitochondrial DNA, at malino mesira a mitochondrial network, a mas magpalwal king ing pormang ini ning neurodegeneration malyari yang mibawi king matas a yugtu ning sakit bayu ya mate ing cell.
Para akit mi ing mitochondria keng Mfn2 knockout PNs, ginamit kaming mouse strain na magpaintulut kareng Cre-dependent mitochondria para i-target ing yellow fluorescent protein (YFP) (mtYFP) (20) Cre expression ampo linawe mi ing mitochondrial morphology in vivo. Ikit mi na ing pangasira ning Mfn2 gene kareng PNs magdala yang gradual a pamikawani ning mitochondrial network (Figure S1A), at ing mumunang pamagbayu ikit ya keng 3 duminggu ning edad. Ing pamiyaliwa, ing maragul a pangasira ning PN cell layer, antimo ing pepakit ning pangawala ning Calbindin immunostaining, e ya megumpisa anggang 12 duminggu ing edad (Figure 1, A ampo B). Ing time mismatch pilatan da reng mumunang pamagbayu keng mitochondrial morphology ampo ing akakit a pamagumpisa ning neuronal death ing meging sangkan para manimbistiga kami kareng metabolic changes a me-trigger keng mitochondrial dysfunction bayu ing cell death. Mekagawa kaming fluorescence-activated cell sorting (FACS)-based strategy para i-isolate ing YFP (YFP+)-expressing PN (Figure 1C), ampo kareng control mice (Mfn2 + / loxP :: mtYFP loxP- stop-loxP: : L7-cre), kaybat ausan deng CTRL (Figure SBRL). Ing pamag-optimize ning gating strategy base keng relative intensity ning YFP signal paintulutan na katamung linisan ing YFP+ body (YFPhigh) da reng PNs ibat kareng non-PNs (YFPneg) (Figure S1B) o putative fluorescent axon/dendritic fragments (YFPlow; Figure S1D, wanan), a mekumpirma kapamilatan ning confocal microscope (Figure S1D, wanan). Para abalu mi ing pangatau da reng me-classify a populasyun, ginawa kaming LFQ proteomics at kaybat ing principal component analysis, at ikit mi na atin yang malino pamiyaliwa ing YFPhigh ampo YFPneg cells (Figure S1C). YFPhigh cells pepakit na ing net enrichment da reng balu dang PNs markers (ie Calb1, Pcp2, Grid2 ampo Itpr3) (21, 22), pero alang enrichment da reng protina a keraklan mipalabas kareng neurons o aliwang uri da reng cell (Figure 1D) ). Ing pamagkumpara kareng sample kareng classified YFPhigh cells a tinipun kareng independenting experiment pepakit na ing correlation coefficient> 0.9, papakit na ing masanting a reproducibility kareng biological replicates (Figure S1E). keng pamamutut, deng data a reni pepatutuan da ing kekaming planu para keng acute ampo specific a pamag-isolate kareng feasible PN. Uling ing L7-cre driver system a ginamit mag induce yang mosaic recombination keng mumunang duminggu kaybat ning pangabait (23), megumpisa kaming mekamate kareng mice ibat keng CTRL ampo conditional (Mfn2 loxP / loxP :: mtYFP loxP-stop-loxP :: L7-cre) Collect neurons. Kaybat nang meyari ing recombination, ausan deng Mfn2cKO keng 4 a duminggu ning edad. Bilang ing wakas, pinili mi ing 8 duminggu ning edad nung kapilan ing PN layer intact ya agyang malino ing mitochondrial fragmentation (Figure 1B ampo Figure S1A). Ing kabilugan, me-quantify kami keng kabilugan a 3013 a protina, nung nukarin mga 22% ing makabasi keng MitoCarta 2.0 annotations base keng mitochondrial proteome bilang mitochondria (Figure 1E) (Figure 1E) (24). Ing differential gene expression analysis a megawa keng duminggu 8 pepakit na na 10.5% mu kareng eganaganang protina ing atin makabaldugan a pamagbayu (Figure 1F ampo Figure S1F), nung nukarin 195 a protina ing me-down-regulate ampo 120 a protina ing me-up-regulate (Figure 1F). Dapat papansinan na ing "innovative pathway analysis" na niting data set papakit na ing differentially expressed genes keraklan kayabe la keng metung a restricted set da reng specific metabolic pathways (Figure 1G). Ing makawili, agyang ing pamag-downregulate kareng pathways a maki kaugnayan keng OXPHOS ampo calcium signaling kukumpirma na ing induction ning mitochondrial dysfunction kareng fusion-deficient PNs, deng aliwang kategorya na keraklan maki kaugnayan keng amino acid metabolism makabaldugan lang mi-upregulate, na makalinya keng metabolism a malilyari keng mitochondrial PNs Rewiring consistent ya. disfunction.
(A) Representative confocal photographs da reng cerebellar sections da reng CTRL ampo Mfn2cKO a mice a papakit da reng progresibong pangawala da reng PNs (calbindin, gray); nuclei me counterstain la keng DAPI. (B) Quantification ning (A) (one-way analysis ning variance, ***P<0.001; n = 4 anggang 6 a gulis ibat kareng atlung mice). (C) Experimental workflow. (D) Heat map distribution da reng marker a partikular keng Purkinje ( babo) ampo reng aliwang uri da reng cell (libutad). (E) Venn diagram papakit na ing bilang da reng mitochondrial proteins a mekilala keng classified PN. (F) Volcano plot da reng differentially expressed proteins keng Mfn2cKO neurons keng 8 a duminggu (significance cut-off value ning 1.3). (G) Ing creativity pathway analysis papakit na ing limang pekamaulaga nang up-regulation (pula) ampo down-regulation (asul) a dalan keng Mfn2cKO PN a me-classify bilang 8 duminggu. Ing average a expression level ning balang meyakit a protina mipalabas ya. Grayscale heat map: me-adjust ing P value. ns, ali importanti.
Ing data ning proteomics pepakit na ing protein expression da reng complex I, III, ampo IV bagya bagya yang mibaba. Deng complex I, III, ampo IV atin lang essential mtDNA-encoded subunits, kabang ing complex II, na nuclear-coded mu, ala yang apektadu (Figure 2A ampo Figure S2A). . Agpang kareng resulta ning proteomics, ing immunohistochemistry da reng cerebellar tissue sections pepakit na ing MTCO1 (mitochondrial cytochrome C oxidase subunit 1) subunit level ning complex IV keng PN bagya bagya yang mebaba (Figure 2B). Ing mtDNA-encoded subunit Mtatp8 metung yang makabaldugan a pamagbawas (Figure S2A), kabang ing steady-state level ning nuclear-encoded ATP synthase subunit menatili yang e mibayu, a maki kaugnayan king balu nang stable ATP synthase subassembly F1 complex nung ing mtDNA expression matatag ya. Ing formation consistent ya. Putulan (7). Ing pamagsuri king mtDNA level kareng me-sort a Mfn2cKO PNs kapamilatan ning real-time polymerase chain reaction (qPCR) kinumpirma na ing bagya-bagyang pamagbaba king mtDNA copy number. Nung ikumpara me keng control group, keng 8 a duminggu ning edad, mga 20% mu ning mtDNA level ing mitagan (Figure 2C). Agpang kareng resultang deni, ing confocal microscopy staining ning Mfn2cKO PNs ginamit ya para akit ing DNA, papakit na ing time-dependent a pamangailangan da reng mitochondrial nucleotides (Figure 2D). Ikit mi na mapilan mung kandidatu ing makiabe keng mitochondrial protein degradation ampo ing stress response ing me-up-regulate, kayabe na ing Lonp1, Afg3l2 ampo ing Clpx, ampo reng OXPHOS complex assembly factors. Ala yang ikit a makabaldugan a pamagbayu keng lebel da reng protina a maki kaugnayan keng apoptosis (Figure S2B). makanyan mu naman, ikit mi na ing mitochondria ampo ing endoplasmic reticulum channels a makiabe keng calcium transport atin lang ditak a pamagbayu (Figure S2C). Dagdag pa, ing pamagsuri kareng protina a maki kaugnayan king autophagy ala yang ikit a makabaldugan a pamagbayu, a makayagpang king akakit a induction da reng autophagosomes a mayayakit king vivo kapamilatan ning immunohistochemistry ampong electron microscopy (Figure S3). Makanyan man, ing progresibong OXPHOS dysfunction kareng PNs kayabe ne ing malino ultrastructural mitochondrial a pamagbayu. Deng mitochondrial clusters akakit la kareng cell bodies ampo kareng dendritic trees da reng Mfn2cKO PNs a maki edad 5 ampo 8 a duminggu, ampo ing inner membrane structure dinalan yang maragul a pamagbayu (Figure S4, A ampo B). Agpang kareng ultrastructural a pamagbayu ampo ing makabaldugan a pamagbaba keng mtDNA, ing pamagsuri kareng acute cerebral cerebellar slices gamit ing tetramethylrhodamine methyl ester (TMRM) pepakit na ing mitochondrial membrane potential keng Mfn2cKO PNs makabaldugan yang mebaba (Figure S4C).
(A) Time course analysis ning expression level ning OXPHOS complex. Isipan mu la reng protina na ating P<0.05 keng 8 a duminggu (two-way ANOVA). Dotted line: Ala yang adjustment kumpara keng CTRL. (B) Kaili: Metung a alimbawa ning cerebellar section a makalabel keng anti-MTCO1 antibody (scale bar, 20 μm). Ing lugal a sasakupan da reng katawan da reng cell ning Purkinje tatakpan ne ning dilaw. Wanan: Quantification ning MTCO1 levels (one-way analysis of variance; n = 7 anggang 20 cells a me-analyze ibat kareng atlung mice). (C) qPCR analysis ning mtDNA copy number keng me-sort a PN (one-way analysis ning variance; n = 3 anggang 7 a mice). (D) Kaili: Metung a alimbawa ning cerebellar slice a makalabel keng anti-DNA antibody (scale bar, 20 μm). Ing lugal a sasakupan da reng katawan da reng cell ning Purkinje tatakpan ne ning dilaw. Wanan: Quantification da reng mtDNA lesions (one-way analysis of variance; n = 5 anggang 9 cells ibat kareng atlung mice). (E) Metung a alimbawa ning acute cerebellar section a papakit mitoYFP + Purkinje cells (pana) keng metung a whole-cell patch clamp recording. (F) Quantification ning IV curve. (G) Representative recordings ning depolarizing current injection keng CTRL ampo Mfn2cKO Purkinje cells. Top trace: Ing mumunang pulse na mekapag trigger keng AP. Bottom trace: Maximum AP frequency. (H) Quantification da reng postsynaptic spontaneous inputs (sPSPs). Ing representative recording trace ampo ing zoom ratio na mayayakit ya keng (I). One-way analysis of variance me-analyze ya n = 5 anggang 20 cells ibat kareng atlung mice. Ing data mipalabas ya bilang mean±SEM; *P<0.05; **P<0.01; ***P<0.001. (J) Representative traces ning spontaneous AP a mitala gamit ing perforated patch clamp mode. Top trace: Maximum AP frequency. Bottom trace: zoom ning metung a AP. (K) I-quantify ing average ampo maximum AP frequency agpang keng (J). Mann-Whitney test; n = 5 cells ing me-analisa ibat kareng apat a mice. Ing data mipalabas ya bilang mean±SEM; e importanti.
Ing malino OXPHOS damage meyakit ya keng 8-week-old Mfn2cKO PN, papakit na ing physiological function da reng neurons sobrang abnormal ya. Uli na niti, sinuri mi la reng passive electrical characteristics da reng OXPHOS-deficient neurons keng 4 anggang 5 a duminggu ampo 7 anggang 8 a duminggu kapamilatan ning pamag-perform kareng whole-cell patch clamp recordings kareng acute cerebellar slices (Figure 2E). E asahan, ing average a resting membrane potential ampo ing input resistance da reng Mfn2cKO neurons parehu la keng control, agyang atin lang subtle a pamiyaliwa kareng cell (Table 1). makanyan mu naman, keng 4 anggang 5 a duminggu ning edad, alang makabaldugan a pamagbayu keng current-voltage relationship (IV curve) ing ikit (Figure 2F). Makanyan man, alang Mfn2cKO neurons 7 anggang 8 a duminggu ing makaligtas keng IV regimen (hyperpolarization step), a magpakit na atin yang malino sensitivity keng hyperpolarization potential keng tauling yugtu. Ing pamiyaliwa, kareng Mfn2cKO neurons, deng depolarizing currents na magdalang repetitive action potential (AP) discharges masanting lang a-tolerate, a papakit na ing karelang kabilugan a discharge patterns ali la makabaldugan a pamiyaliwa kareng 8-week-old control neurons (Table 1 and Figure 2G). makanyan mu naman, ing frequency ampo ing amplitude da reng spontaneous postsynaptic currents (sPSCs) parehu la kareng control group, ampo ing frequency da reng events meragdagan ya manibat 4 duminggu angga 5 duminggu angga 7 duminggu angga 8 duminggu a maki parehung pamagtas (Figure 2, H ampo I). Ing panaun ning synaptic maturation kareng PNs (25). Parehu lang resulta deng mekwa kaybat da reng perforated PNs patches. Ing configuration ayni pipigilan na ing posibling pamamayad kareng cellular ATP defects, antimo ing malyari keng whole-cell patch clamp recording. Ing partikular, ing resting membrane potential ampo ing spontaneous firing frequency da reng Mfn2cKO neurons e la meapektuan (Figure 2, J ampo K). keng pamamutut, deng resulta a reni papakit da na deng PNs na ating malino OXPHOS dysfunction malyari lang makipaglaban kareng high-frequency discharge patterns, papakit na atin yang mekanismo ning pamamayad na magpaintulut karela na manatili lang malapit-normal a electrophysiological a pakibat.
Ing data mipalabas ya bilang mean ± SEM (one-way analysis of variance, Holm-Sidak's multiple comparison test; *P<0.05). Ing unit number makasulat ya keng brackets.
Migmula kami para manimbistiga nung nanu mang kategorya keng proteomics dataset (Figure 1G) kayabe la reng dalan na malyaring makakontra keng grabing kakulangan ning OXPHOS, inya sa't mipaliwanag nung bakit ing apektadung PN malyari yang manatili keng malapit-normal a electrophysiology (Figure 2, E anggang K). . Ing pamagsuri king proteomics pepakit na ing enzymes a maki kaugnayan king catabolism da reng branched chain amino acids (BCAA) makabaldugan lang mi-up-regulate (Figure 3A ampo Figure S5A), at ing tauling produktu a acetyl-CoA (CoA) o succinyl CoA malyari yang magdagdag kareng tricarboxylates king arteriosclerosis Acid (TCA) cycle. ikit mi na ing laman ning BCAA transaminase 1 (BCAT1) ampo BCAT2 parehu lang meragdagan. Ila reng mag-catalyze keng mumunang step ning BCAA catabolism kapamilatan ning pamaglalang glutamate ibat keng α-ketoglutarate (26). Deng eganaganang subunits a gagawang branched chain keto acid dehydrogenase (BCKD) complex maka-upregulate la (ing complex catalyze ne ing tutuking at e malyaring ibalik a decarboxylation ning resulta ning BCAA carbon skeleton) (Figure 3A ampo Figure S5A). Makanyan man, alang malino pamagbayu keng BCAA mismu ing ikit da keng me-sort a PN, na malyaring maging sangkan ning pamagtas ning cellular uptake da reng essential amino acids o ing pamangamit kareng aliwang panibatan (glucose o lactic acid) para dagdagan ing TCA cycle (Figure S5B). Deng PNs a alang OXPHOS pepakit da ing mas maragul a glutamine decomposition ampo transamination activities keng 8 duminggu ning edad, na malyaring mipalabas keng up-regulation da reng mitochondrial enzymes glutaminase (GLS) ampo glutamine pyruvate transaminase 2 (GPT2) (Figure 3, A ampo C). Dapat papansinan na ing up-regulation ning GLS limitadu ya keng spliced ​​isoform glutaminase C (GLS-GAC) (ing pamagbayu ning Mfn2cKO/CTRL manga 4.5-fold ya, P = 0.05), ampo ing kayang specific a up-regulation kareng cancer tissues Malyari yang suportan ing mitochondrial bioenergy bioenergy. (27).
(A) Ing heat map papakit na ing pamagbayu ning protina para keng metung a ruta keng 8 a duminggu. (B) Alimbawa ning cerebellar slice a makalabel keng anti-PCx antibody (scale bar, 20 μm). Ing dilaw a pana makaturu ya keng Purkinje cell body. (C) Time course protein expression analysis a mekilala bilang metung a importanting kandidatu para keng atherosclerosis (multiple t-test, *FDR <5%; n = 3-5 mice). (D) babo: metung a schematic diagram a papakit kareng miyayaliwang paralan ning pamaglub keng makalabel a carbon a atyu keng [1-13C]pyruvate tracer (ie, kapamilatan ning PDH o trans-arterial route). Lalam: Ing violin chart papakit ne ing porsyentu ning single-labeled carbon (M1) a mibayu keng aspartic acid, citric acid ampo malic acid kaybat dang me-label deng acute cerebellar slices gamit ing [1-13C]pyruvate (paired t-test; ** P <0.01). (E) Komprehensibung pamagsuri keng kasaysayan ning panaun ning makatuldung dalan. Isipan mu la reng protina na ating P<0.05 keng 8 a duminggu. Dashed line: alang adjustment value (two-way analysis of variance; * P <0.05; *** P <0.001). Ing data mipalabas ya bilang mean±SEM.
keng kekaming pamagsuri, ing BCAA catabolism meging yang metung kareng susi ning up-regulation pathways. Ing katutwan ayni masikan yang magsuggest na ing ventilation volume a lulub keng TCA cycle malyari yang mibayu keng PN a alang OXPHOS. Iti malyari yang magrepresenta keng maragul a porma ning neuronal metabolic rewiring, na malyaring mika direktang epektu keng neuronal physiology ampo survival kabang mag maintain ya ing grabing OXPHOS dysfunction. Agpang keng hypothesis ayni, ikit mi na ing main anti-atherosclerotic enzyme PCx up-regulated ya (Mfn2cKO/CTRL magbayu yang 1.5 times; Figure 3A), na mag-catalyze keng pamagbayu ning pyruvate keng oxaloacetate (28), na paniwalan da keng brainsutes ing astrocy expression makabawal ya (29, 30). Agpang kareng resulta ning proteomics, ing confocal microscopy pepakit na ing PCx expression metung yang partikular at makabaldugan a pamagtas kareng OXPHOS-deficient PNs, kabang ing PCx reactivity keraklan makabawal ya kareng makalapit a Bergmann glial cells ning control (Figure 3B). Para asuri ing upregulation ning PCx, ginamit mi la reng acute cerebellar slices gamit ing [1-13C]pyruvate tracer. Inyang ing pyruvate me-oxidize ya kapamilatan ning pyruvate dehydrogenase (PDH), ing kayang isotope label mewala ya, dapot miyabe ya kareng TCA cycle intermediates nung ing pyruvate me-metabolize ya kapamilatan da reng vascular reactions (Figure 3D). Bilang suporta keng kekaming proteomics data, ikit mi ing maragul a bilang da reng marker ibat kening tracer keng aspartic acid da reng Mfn2cKO slices, kabang ing citric acid ampo ing malic acid atin lang katamtaman a trend, agyang e makabaldugan (Figure 3D).
Kareng dopamine neurons da reng MitoPark a mice a maki mitochondrial dysfunction a sangkan da reng dopamine neurons a partikular a sisira king mitochondrial transcription factor A gene (Tfam) (Figure S6B), ing PCx expression metung yang makabaldugan a up-regulated (31), a papakit na ing acetone acid arteriosclerosis Ing pangapalyari ning sakit makaregulate ya kabang dysfunction ning neuron OXPHOS keng katawan. Dapat papansinan na ing ikit da na deng unique enzymes (32-34) na malyaring mipalabas kareng neurons na malyaring mikaugne keng arteriosclerosis makabaldugan lang mi-up-regulate kareng PNs a alang OXPHOS, antimo ing propionyl-CoA carboxylase (PCC-A), Malonyl-CoA converts ne ing propionyl-CoA carboxylase ampo ing succil-Coa succilchondrial enzyme 3 (ME3), na ing peka-papel na ing makabawi keng pyruvate ibat keng malate (Figure 3, A ampo C) (33, 35). Dagdag pa, menakit kaming maragul a pamagtas king Pdk3 enzyme, a mag-phosphorylate at makanyan yang mag-inactivate king PDH (36), kabang alang ikit a pamagbayu king Pdp1 enzyme a mag-activate king PDH o king PDH enzyme complex mismu (Figure 3A). Parati, keng Mern2cKO PNs, ing phosphorylation ning α1 subunit α (PDHE1α) subunit ning pyruvate dehydrogenase E1 component ning PDH complex keng Ser293 (balu na maka-inhibit keng enzyme activity ning PDH) me-enhance ya ( Figure S6C) (Figure S6C). Ing pyruvate ala yang access keng vascular.
Katataulian, ikit mi na ing super pathway ning serine ampo glycine biosynthesis, ing kaugnayan a mitochondrial folate (1C) cycle ampo proline biosynthesis (Figure 1G ampo Figure S5C) ila ngan makabaldugan a mi-up-regulate, agpang kareng ulat, keng prosesu ning pamag-activate. Deng makapadurut a tissue me-activate la keng mitochondrial dysfunction (5-7). Ing confocal analysis a susuporta kareng proteomics data pepakit na ing PN a alang OXPHOS, deng cerebellar slices da reng 8-week-old a mice mipailalam la keng serine hydroxymethyltransferase 2 (SHMT2), metung a susi a enzyme ning mitochondrial folate cycle. Makabaldugan a immune response (Figure S5D). keng 13 CU-glucose-incubated acute cerebellar slices, ing metabolic tracing experiments lalu yang mekumpirma ing up-regulation ning serine ampo proline biosynthesis, a magpakit na ing flux da reng carbon isoforms keng serine ampo proline meragdagan ya (Figure S5E). Uling deng reaksyun a susulung ning GLS ampo GPT2 ila reng maki pakibatan keng synthesis ning glutamate ibat keng glutamine ampo ing transamination pilatan ning glutamate ampo α-ketoglutarate, ing karelang upregulation papakit na ing OXPHOS-deficient neurons atin lang mas maragul a pamangailangan para keng glutamate, Iti malyari yang maglalang para panatilyan ing mas maragul a biosynthesis ning proline (Figure S5). Ing pamiyaliwa da reng pamagbayu a reni, ing proteomic analysis da reng cerebellar astrocytes ibat kareng PN-specific Mfn2cKO mice pepakit na na deng pathways a reni (kayabe nala reng eganaganang antiperoxidases) e la maragul a pamagbayu keng expression, inya pepakit na ing metabolic redirection ayni selective ya keng degraded PN (Fig. S6, D to G).
keng pamamutut, deng pamagsuri a reni pepakit da ing makabaldugan a miyayaliwang pattern ning temporal activation da reng specific a metabolic pathways kareng PNs. Agyang ing abnormal a neuronal mitochondrial function malyari yang magdalang early atherosclerosis ampo 1C remodeling (Figure 3E ampo Figure S5C), ampo pati na ing predictable a pamagbayu keng expression da reng I ampo IV complexes, ing pamagbayu keng serine de novo synthesis ila mung meging malino keng late stages. OXPHOS dysfunction (Figure 3E ampo Figure S5C). Deting akit magdefine lang metung a sequential a prosesu nung nukarin ing stress-induced mitochondrial (1C cycle) ampo ing cytoplasmic (serine biosynthesis) pakibatan da ing synergistically keng pamagtas ning atherosclerosis keng TCA cycle para i-reshape ing neuronal metabolism.
8-week-old OXPHOS-deficient PNs malyari lang manatili keng high-frequency excitation activity ampo dumalan lang makabaldugan a metabolic reconnection para makabawi keng mitochondrial dysfunction. Ing tuklas ayni magdalang metung a makawili a posibilidad na agyang ngeni, deng cell a reni malyari la namang tanggap therapeutic intervention para ma-delay o ma-prevent ing neurodegeneration. Metalwi. Ayari mi ing posibilidad ayni kapamilatan da reng adwang independenting pamakialam. Ing mumunang paralan, dinisenyu mi ing Cre-dependent adeno-associated virus (AAV) vector ban ing MFN2 malyari yang mapiling mipalabas kareng OXPHOS-deficient PNs in vivo (Figure S7A). Ing AAV a mag-encode king MFN2 ampo ing fluorescent reporter gene mCherry (Mfn2-AAV) me-verify la kareng primary neuron cultures in vitro, a meging sangkan ning MFN2 a mipalabas king Cre-dependent a paralan at miligtas ya ing mitochondrial morphology, inya sa’t me-prevent ing neuromutation kareng Mfn2cKO neurons ( Figure S7, D, B and D E). Kaybat, ginawa kaming in vivo experiments para stereotactically ideliver ing 8-week-old Mfn2-AAV keng cerebellar cortex ning Mfn2cKO ampo control mice, ampo inanalisa mi la reng 12-week-old mice (Figure 4A). Ing meulu a Mfn2cKO a mice mete ya (Figure 1, A ampo B) (16). Ing viral transduction in vivo meging sangkan ning selective expression ning PN kareng mapilan a cerebellar circles (Figure S7, G ampo H). Ing pamag-inject ning control AAV a mag-express mung mCherry (Ctrl-AAV) ala yang makabaldugan a epektu keng degree ning neurodegeneration kareng animal a Mfn2cKO. Ing pamiyaliwa, ing pamagsuri kareng Mfn2cKOs a mi-transduce keng Mfn2-AAV pepakit na ing makabaldugan a proteksiun a epektu ning PN cell layer (Figure 4, B ampo C). Ing partikular, ing neuron density balamu e ya malyaring akilala kareng control a animal (Figure 4, B ampo C, ampo Figure S7, H ampo I). Ing pamagpakilala ning MFN1 pero ali ing MFN2 parehu yang epektibo keng pamagligtas keng neuronal death (Figure 4C ampo Figure S7, C ampo F), na magpakit na ing pamagpakilala ning ectopic MFN1 malyari yang epektibung magdagdag keng kakulangan ning MFN2. Ing karagdagang pamagsuri keng metung a PN level pepakit na ing Mfn2-AAV maragul yang pamagligtas keng ultrastructure ning mitochondria, pepa-normalize na ing mtDNA levels, ampo pepabalik ne ing matas a expression ning anti-angiogenesis marker PCx ​​(Figure 4, C to E ). Ing pamagsuri kareng meligtas a Mfn2cKO mice keng pamagpainawa pepakit na ing karelang postura ampo motor symptoms (movement S1 to S3) meging masanting la. keng pangwakas, deng experiment a reni papakit da na ing delayed reintroduction ning MFN2 kareng PNs a grabing kulang keng OXPHOS sapat na para ibalik ing mtDNA consumption ampo mag induce atherosclerosis, para aiwasan ing axon degeneration ampo neuronal death in vivo.
(A) Metung a scheme a papakit keng experimental schedule para keng pamag-inject AAV encoding MFN2 nung ing me-activate a metabolic pathway me-activate ya. (B) Representative confocal images ning 12-week-old cerebellar slices a mi-transduce keng 8 weeks kareng Mfn2cKO mice ampo mi-label keng anti-Calbindin antibody. Wanan: Scaling da reng axon fibers. Ing sukad ning axon zoom 450 ya ampo 75 μm. (C) Kaliwa: Quantification ning Purkinje cell density keng AAV transduction loop (AAV+) (one-way analysis of variance; n = 3 mice). Wanan: mtDNA focus analysis keng transduced PN keng duminggu 12 (unpaired t-test; n = 6 cells ibat kareng atlung mice). * P <0.05; ** P <0.01. (D) Representative transmission electron micrographs da reng PNs ning Mfn2cKO cerebellar sections a mi-transduce kareng mipalabas a viral vectors. Ing pink mask papakit ne ing lugal a sasakupan da reng dendrites, at ing yellow dotted square papakit ne ing zoom a atyu keng wanan; n ing magrepresenta keng nucleus. Scale bar, 1μm. (E) papakit ne ing alimbawa ning PCx staining keng PN a mipalitan keng 12 a duminggu. Scale bar, 20μm. OE, overexpression; FC, lipat pamagbayu.
katataulian, siniasat mi ing importansya ning peroxidase-induced cell survival kareng PNs na mika OXPHOS dysfunction. Mekagawa kaming mCherry encoding AAV-shRNA (makuyad a hairpin RNA) a partikular a magtarget keng mouse PCx mRNA (AAV-shPCx), at inject mi ing virus o ing kayang scrambled control (AAV-scr) keng cerebellum da reng Mfn2cKO a mice. Ing injection megawa ya keng kapat a duminggu ning edad (Figure 5A) para akwa ing epektibung PCx knockdown keng panaun nung kapilan ing PCx expression meragul ya (Figure 3C) ampo ing PN cell layer intact ya pa (Figure 1A). Dapat papansinan na ing pamag-knock down keng PCx (Figure S8A) magdalang maragul a pamibilis ning PN death, na limitadu ya keng infected ring (Figure 5, B ampo C). Para aintindian ing mekanismo da reng metabolic effects a sangkan ning PCx up-regulation, inaral mi ing redox status da reng PNs kaybat ning PCx knockdown ampo ing AAV-mediated optical biosensor Grx1-roGFP2 a sabay-sabay mipalabas (Figure S8, B to D) para abalu ing glutathione Ing relatibung pamagbayu ning redox peptide potential (383). Kaybat, ginawa mi ing two-photon fluorescence lifetime imaging microscopy (FLIM) kareng acute brain slices ning 7-week-old Mfn2cKO o control littermates para akit mi ing posibling pamagbayu keng cytoplasmic redox status kaybat ming me-verify ing FLIM conditions (Figure S8, E to G). Ing pamagsuri pepakit na ing maragul a pamagtas king oxidation state ning metung a Mfn2cKO PNs a alang PCx expression, a miyayaliwa kareng control neurons o Mfn2cKO PNs a magpalwal mung scrambled shRNA (Figure 5, D ampong E). Nung ing PCx expression me-down-regulate ya, ing porsyentu da reng Mfn2cKO PNs a magpakit king matas a oxidized state meragdagan yang maigit atlung besis (Figure 5E), a papakit na ing PCx up-regulation menatili ya king redox capacity da reng degenerated neurons.
(A) Metung a scheme a papakit keng experimental schedule para keng pamag-inject AAV encoding shPCx nung ing me-activate a metabolic pathway me-activate ya. (B) Representative confocal photographs da reng 8-week-old cerebellar sections kareng Mfn2cKO mice a mi-transduce ampo mi-label keng anti-calcineurin antibody keng 4 a duminggu. Scale bar, 450μm. (C) Quantification ning Purkinje cell density keng AAV-transduced loops (one-way analysis ning variance; n = 3 anggang 4 a mice). Ing data mipalabas ya bilang mean±SEM; ***P<0.001. (D) Ing larawan ning FLIM papakit ne ing average a bie ning 7-week-old PN a magpalwal glutathione redox sensor Grx1-roGFP2 lalam ning makatuldung kundisyun ning eksperimentu. LUT (look-up table) ratio: survival time interval (king picoseconds). Scale bar, 25μm. (E) Ing histogram papakit na ing pamikalat da reng Grx1-roGFP2 lifetime values ​​manibat (D) (n=158 anggang 368 cells kareng adwang mice lalam ning balang kundisyun). Ing pie chart babo ning balang histogram: papakit na ing bilang da reng cell a maki makabaldugan a mas makaba (pula, oxidized) o mas makuyad (asul, mabawas) a ulaga ning bie, a lalagpus king 1 SD ning average a ulaga ning bie king CTRL-AAV-scr. (F) Ing mipanukalang model papakit na ing proteksiun a epektu ning pamagtas ning neuronal PCx.
Ing kabilugan, ing data a binie mi keni papakit na ing pamagbalik ning MFN2 malyari yang lubus a magligtas king advanced PN a maki grabing kakulangan king OXPHOS, grabing mtDNA depletion, at sobrang abnormal a ista-like morphology, inya sa’t mamye yang patuloy a pamagprogresu agyang kareng advanced a sakit. Ing neurodegeneration mamye yang reversible a ebidensya ning yugtu bayu ya mete ing cell. Ing degree ning metabolic flexibility ayni mas mipakilala ya keng abilidad da reng neurons na mag induce atherosclerosis (metung a rewiring ning TCA cycle), na mag inhibit keng PCx expression kareng PNs a alang OXPHOS ampo magpasyag keng cell death, inya maglarin yang protective role (Figure 5F).
Kening pamagaral ayni, dininan ming ebidensya na ing pakibat da reng PNs keng OXPHOS dysfunction yapin ing bagya-bagyang pamag-converge keng TCA cycle atherosclerosis kapamilatan ning differential activation pathway a mipa-activate kareng metabolic programs. Kinumpirma mi ing proteomic analysis kareng dakal a complementary a paralan at pepakit mi na nung mipapag-challenge la keng grabing mitochondrial dysfunction, deng neurons atin lang e pa balu a uri ning metabolic elasticity. Ing kekaming pamagmulala, ing mabilug a prosesu ning pamag-rewiring e na kailangang markahan ing terminal metabolic state a kayabe ning neurodegeneration bagya-bagya at e malyaring ibalik, dapot ing kekaming data sasabyan na malyari yang maging maintenance neuron agyang king yugtu bayu ya mete ing cell Functional compensation mechanism. Ing akit a ini papakit na ing neurons atin yang maragul a degree ning metabolic plasticity keng katawan. Ing katutwan ayni patunayan na ing tauli nang pamagbalik ning MFN2 malyari yang ibalik ing pamagpahayag da reng susi a metabolic markers at pigilan ing pangasira ning PN. Ing kabaligtaran, pipigilan na ing atherosclerosis ampo papabilis ne ing nerbyus. transsexual.
Metung kareng peka-makabaldugan a akit mi keng kekaming pamanyaliksik yapin ing PNs a alang OXPHOS malyari deng i-modify ing TCA cycle metabolism kapamilatan ning pamag-up-regulate kareng enzymes na partikular a mag-stimulate arteriosclerosis. Ing metabolic rearrangement metung yang karaniwan a katangian da reng cancer cells, deng mapilan kareti magdependi la keng glutamine para dagdagan ing TCA cycle intermediates para makagawa lang reducing equivalents, na mag drive keng respiratory chain ampo magpanatili keng pamaglalang da reng lipid ampo nucleotide biosynthesis precursors (39 , 40). Ing bayung pamagaral pepakit na kareng peripheral tissues a maki OXPHOS dysfunction, ing reconnection ning glutamine/glutamate metabolism metung ya namang prominenting feature (5, 41), nung nukarin ing direksyun ning pamaglub ning glutamine keng TCA cycle makayagpang ya keng uling keng severity ning OXPHOS injury (41). ). Makanyan man, ala pang malino ebidensya keng nanu mang pamikakatulad ning neuronal metabolic plasticity keng katawan ampo ing kayang posibling kaugnayan keng kontekstu ning sakit. King metung a bayung in vitro a pamagaral, ding mumunang cortical neurons pepakit da na magmobilize lang glutamate pools para keng neurotransmission, inya sa’t magpromote lang oxidative metabolism ampo atherosclerosis lalam ning metabolic stress a kundisyun (42). Dapat papansinan na ing lalam ning pharmacological inhibition ning TCA cycle enzyme succinate dehydrogenase, ing pyruvate carboxylation paniwalan dang manatili ing synthesis ning oxaloacetate kareng cultured cerebellar granule neurons (34). Makanyan man, ing physiological a kaugnayan da reng mekanismo a reni keng tissue ning utak (nung nukarin ing atherosclerosis paniwalan da na keraklan makakulong ya kareng astrocytes) atin ya pa muring importanting physiological a kabaldugan (43). Kening kasu, ing kekaming data papakit na ing PNs a mesira keng OXPHOS keng katawan malyari yang mipalitan keng BCAA degradation ampo pyruvate carboxylation, na ila reng adwang manimunang panibatan ning supplementation da reng TCA pool intermediates. Agyang ing putative a ambag ning BCAA catabolism keng neuronal energy metabolism mipanukala ya, dagdag pa keng papil ning glutamate ampo GABA para keng neurotransmission (44), ala pa muring ebidensya para kareng mekanismo a reni in vivo. Uli na niti, madali mung isipan na deng dysfunctional PNs malyari lang automatic a magbayad keng pamangan da reng TCA intermediates a ibat keng prosesu ning pamag-asimila kapamilatan ning pamagdake ning atherosclerosis. Ing partikular, ing pamag-upregulate ning PCx malyari yang kailangan para manatili ing mas maragul a pamangailangan para keng aspartic acid, na mumusbung kareng proliferating cells a maki mitochondrial dysfunction (45). Makanyan man, ing kekaming metabolomics analysis ala yang pepakit a makabaldugan a pamagbayu keng steady-state level ning aspartic acid keng Mfn2cKO PNs (Figure S6A), na siguru papakit na ing miyayaliwang metabolic a pamangamit ning aspartic acid pilatan da reng proliferating cells ampo reng post-mitotic neurons. Agyang ing eksaktung mekanismo ning PCx upregulation kareng dysfunctional neurons in vivo e pa abalu, pepakit mi na ing e pa panaun a pakibat ayni atin yang importanting papil keng pamagpanatili keng redox state da reng neurons, a pepakit mi keng FLIM experiments kareng cerebellar slices. Ing partikular, ing pamag-iwas kareng PNs keng pamag-up-regulate ning PCx malyari yang magdalang mas oxidized a estado at magpabilis king pangamate da reng cell. Ing pamag-activate ning BCAA degradation ampo ing carboxylation ning pyruvate ali la paralan para abalu ing peripheral tissues ning mitochondrial dysfunction (7). Uli na niti, balamu ila ing priority feature da reng OXPHOS-deficient neurons, agyang ali mu kabud feature, na importanti para keng neurodegeneration. .
Ing sakit a cerebellar metung yang heterogeneous a uri ning sakit a neurodegenerative a keraklan mayayakit bilang ataxia at maralas makasira kareng PNs (46). Ing populasyun da reng neuron ayni partikular yang vulnerable keng mitochondrial dysfunction uling ing karelang selective degeneration kareng mice sapat na para magreproduce kareng dakal a motor symptoms a maki katangian ning human spinocerebellar ataxia (16, 47, 48). Agpang kareng ulat, ing metung a transgenic a modelung mouse a maki mutant a gene makasuglung ya king human spinocerebellar ataxia at atin yang mitochondrial dysfunction (49, 50), a magpalwal king importansya ning pamagaral kareng bunga ning kakulangan king OXPHOS king PNPH. Uli na niti, partikular yang masanting ing epektibung pamag-isolate at pamag-aral kaniting makatagulaling a populasyun ning neuron. Makanyan man, uling deng PNs sensitibo la keng pressure ampo mababa la mu keng mabilug a populasyun ning cerebellar cell, para kareng dakal a omics-based a pamagaral, ing selective separation da bilang mabilug a cell metung ya pa muring masakit a aspetu. Agyang halus imposibli ing akwa ing absolute a kakulangan ning kontaminasyun da reng aliwang uri ning cell (lalu na reng tissue da reng matwa), pisamut mi ing epektibung step ning dissociation kareng FACS para mika sapat a bilang da reng viable neurons para keng downstream proteomics analysis, at atin lang matas a protein coverage (mga 3000 proteins) kumpara keng existing data set ning cerebellum set (515). King pamagpreserba king viability da reng mabilug a cell, ing paralan a bibie mi keni paintulutan na katamung e mu akit ing pamagbayu kareng metabolic pathways kareng mitochondria, nune pati na ing pamagbayu kareng kayang cytoplasmic counterparts, a magdagdag king pamangamit kareng mitochondrial membrane tags para pasikanan ing cell type Ing bayung paralan para king bilang da reng mitochondria kareng complex tissues (2535). Ing paralan a ilarawan mi ali yamu makaugne keng pamagaral kareng Purkinje cells, pero malyari yang gamitan keng nanu mang uri ning cell para a-address ing metabolic changes kareng sakit a utak, kayabe nala reng aliwang model ning mitochondrial dysfunction.
Katataulian, akilala mi ing metung a therapeutic window keng prosesu ning metabolic rearrangement na malyaring lubus a magbalik kareng susi ning cellular stress ampo makaiwas keng neuronal degeneration. Uli na niti, ing pamanintindi kareng functional implikasyon ning rewiring a milarawan keni malyari yang mamye pundasyunal a pamanalastas kareng posibling panulu para king pamagpanatili king neuronal viability kabang mitochondrial dysfunction. Ing daratang a pamanyaliksik a maglalang king pamag-dissect kareng pamagbayu king metabolismu ning enerhiya kareng aliwang uri ning cell ning utak kailangan ya bang lubus a mibulalag ing panga-applicable na niting prinsipyu kareng aliwang sakit king neurological.
MitoPark mice me describe na la kanitang minuna (31). C57BL/6N mice a maki loxP flanking Mfn2 genes me describe na la (18) ampo me cross la kareng L7-Cre mice (23). Ing resulta ning double heterozygous progeny mipapag-cross la kareng homozygous Mfn2loxP/Mfn2loxP mice para makagawa lang Purkinje-specific gene knockouts para keng Mfn2 (Mfn2loxP/Mfn2loxP; L7-cre). Ing metung a subset ning pamiyabe, ing Gt (ROSA26) SorStop-mito-YFP allele (stop-mtYFP) mipakilala ya kapamilatan da reng karagdagang crossings (20). Deng eganaganang animal procedures megawa la agpang kareng European, national ampo institutional guidelines ampo me-aprubahan la keng LandesamtfürNatur ning Umwelt ampo Verbraucherschutz, North Rhine-Westphalia, Germany. Ing obra kareng animal tutuki ya keng tuntunan ning European Federation of Laboratory Animal Sciences Associations.
Kaybat dang me-anesthetize ing cervical dislocation ning mabuktut, ing embryo ning mouse me-isolate ya (E13). Ing cortex me-dissect ya keng Hanks' Balanced Salt Solution (HBSS) a midagdag 10 mM Hepes ampo mipasa ya keng Dulbecco's Modified Eagle's Medium a maki papain (20 U/ml) ampo cysteine ​​(1μg/ml). I-incubate me ing tissue keng DMEM) ampo i-dissociate me kapamilatan ning enzymatic digestion. Ml) keng 37°C kilub ning 20 minutu, at kaybat mechanically milled ya keng DMEM a mika 10% fetal bovine serum. Deng cells mitanam la keng glass coverslips a makabalut keng polylysine keng density a 2×106 balang 6 cm culture dish o keng density a 0.5×105 cells/cm2 para keng imaging analysis. Kaybat ning 4 oras, ing medium mipalitan ya keng Neurobasal serum-free medium a maki 1% B27 supplement ampo 0.5 mM GlutaMax. Deng neurons me-maintain la keng 37°C ampo 5% CO2 keng mabilug a experiment, ampo pepakanan da la misan keng paruminggu. Para mika recombination in vitro, 3μl (24-well culture dish) o 0.5μl (24-well plate) ning tutuking AAV9 virus vector ing ginamit para gamutan la reng neurons keng kaduang aldo in vitro: AAV9.CMV.PI.eGFP. WPRE.bGH (Addgene, catalog number 105530-AAV9) ampo AAV9.CMV.HI.eGFP-Cre.WPRE.SV40 (Addgene, catalog number 105545-AAV9).
Ing mouse Mfn1 ampo Mfn2 complementary DNA (mekwa la keng Addgene plasmid #23212 ampo #23213, respectively) makamarka la keng V5 sequence (GKPIPNPLLGLDST) keng C-terminus, ampo me-fuse la keng mCherry keng frame kapamilatan ning T2A sequence. Ing Grx1-roGFP2 metung yang regalu ibat keng Heidelberg TP Dick DFKZ (Deutsches Krebsforschungszentrum). King pamagpalit king tdTomato cassette gamit ing conventional a paralan ning pamag-clone, ing cassette me-subclone ya king pAAV-CAG-FLEX-tdTomato backbone (Addgene reference number 28306) para gawang pAAV-CAG-FLEX-mCherry-T2A-MFN2-V5, pAAV-CAG-G FLEX-mCherry-T2A-MFN1-V5 ampo pAAV-CAG-FLEX-Grx-roGFP2 vectors. Metung a kalupa nang diskarti ing ginamit para gawan ing control vector pAAV-CAG-FLEX-mCherry. Para makagawa yang AAV-shPCx construct, kailangan ing plasmid AAV vector (VectorBuilder, pAAV [shRNA] -CMV-mCherry-U6-mPcx- [shRNA#1]), na maglaman keng DNA sequence a mag-encode keng shRNA a mag-target keng mouse PCx (5′CTTTCGCTAGCTAGGCTAAGGCTAAGGCTAGACTTAGA 3′) King lalam ning kontrol ning U6 promoter, ing mCherry magagamit ya lalam ning kontrol ning CMV promoter. Ing pamaglalang kareng auxiliary AAV vectors megawa ya agpang kareng instruksyun ning manufacturer (Cell Biolabs). king makuyad a salita, gamitan me ing transfer plasmid a magdalang mCherry-T2A-MFN2-V5 (pAAV-CAG-FLEX-mCherry-T2A-MFN2-V5), mCherry-T2A-MFN1-V5 (pAAV-CAG-FLEX-mCherry) transiently Transfection of 23A-MFN2-MFN2-V5 cell mCherry (pAAV-CAG-FLEX-mCherry) o Grx-roGFP2 (pAAV-CAG-FLEX-Grx-roGFP2) coding gene, ampo coding AAV1 capsid protein ampo accessory protein Packaging plasmid plasmid, gamit ing calcium phosphate method. Ing crude virus supernatant mekwa ya kapamilatan ning freeze-thaw cycles keng dry ice/ethanol bath ampo lysed cells keng phosphate buffered saline (PBS). Ing AAV vector me purify ya kapamilatan ning discontinuous iodixanol gradient ultracentrifugation (24 oras keng 32,000 rpm ampo 4°C) ampo me concentrate ya gamit ing Amicon ultra-15 centrifugal filter. Ing genome titer ning 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 antimo ing minunang misulat (5), quantitative PCR (d PCR) -MFN1-V5 (1.9×1013 GC/ml) ampo AAV1-CAG-FLEX-Grx-roGFP2 (8.9×1012 GC/ml).
Deng mumunang neuron me-scrape la keng ice-cold 1x PBS, me-pellet, at kaybat me-homogenize la keng 0.5% Triton X-100 / 0.5% sodium deoxycholate/PBS lysis buffer a maki phosphatase ampo protease inhibitor (Roche). Ing pamag-quantify ning protina megawa ya kapamilatan ning bicinchoninic acid assay (Thermo Fisher Scientific). Deng protina mekawani la kapamilatan ning SDS-polyacrylamide gel electrophoresis, at kaybat me-blot la keng polyvinylidene fluoride membrane (GE Healthcare). I-block la reng e partikular a lugal at i-incubate la reng mumunang antibody (lawen ya ing Table S1 para kareng detalye) keng 5% gatas keng TBST (Tris-buffered saline with Tween), washing steps ampo ing secondary antibody keng TBST Incubate. I-incubate me ing primary antibody magdamag keng +4°C. Kaybat meng me hugas, gamitan me ing secondary antibody kilub ning adwang oras keng room temperature. Kaybat, kapamilatan ning pamag-incubate king pareung blot king anti-β-actin antibody, ing pareung loading mekumpirma ya. Detection kapamilatan ning pamagbayu keng chemiluminescence ampo pamagpabuti keng chemiluminescence (GE Healthcare).
Deng neurons a minunang mitanam kareng glass coverslips me-fix la keng 4% paraformaldehyde (PFA)/PBS keng metung a oras keng temperatura ning silid kilub ning 10 minutu. Deng coverslips mumuna lang mipapalwal keng 0.1% Triton X-100/PBS kilub ning 5 minutu keng temperatura ning silid, at kaybat keng blocking buffer [3% bovine serum albumin (BSA)/PBS]. keng kaduang aldo, ding coverslips me hugas la keng blocking buffer ampo me incubate la keng ustung fluorophore-conjugated secondary antibody kilub ning adwang oras keng room temperature; katataulian, ding sample me hugas lang masalese keng PBS gamit ing 4′,6-diamidino-2 -Phenylindole (DAPI) a me counterstain at kaybat me fix ya keng microscope slide gamit ing Aqua-Poly/Mount.
Deng mice (lalaki ampo babai) me-anesthesia la kapamilatan ning intraperitoneal injection ning ketamine (130 mg/kg) ampo xylazine (10 mg/kg) ampo midinan lang subcutaneously carprofen analgesic (5 mg/kg) , at mibili la keng stereotactic instrument (Kopf) a maki warm. I-expose me ing bungu at gamitan me ing dental drill para manipis ya ing bage ning cerebellar cortex a maki kaugnayan keng mis bone (ibat keng lambda: tail 1.8, lateral 1, makatuglung keng lobules IV ampo V). Gamitan me ing makakurbang syringe needle para gawan meng malating butul ing bungu para aiwasan ing pangasira ning vasculature king lalam. Kaybat ing manipis a glass capillary mabagal yang milub keng micro-hole (ibat -1.3 anggang -1 keng ventral side ning dura mater), ampo 200 anggang 300 nl AAV mi-inject ya keng micro-injector (Narishige) gamit ing manual syringes (Narishige) marakal a besis keng mababang pressure kilub ning 20 anggang 20 minutu. Kaybat ning pamag-infuse, ibili me ing capillary kilub ning 10 minutu para lubus yang mikalat ing virus. Kaybat dang mewala deng capillary, ing balat mitahi yang masalese para mabawas ing pamangalbag ning sugat at paintulutan neng kumayap ing animal. Deng animal meulu la kareng analgesics (caspofen) kilub ning mapilan a aldo kaybat ning operasyun, nung nukarin ing karelang kondisyun ning katawan maingat yang me-monitor at kaybat me-euthanize la keng sinabi nang oras. Deng eganaganang pamamaralan megawa la agpang kareng European, national ampo institutional guidelines ampo me-aprubahan la keng LandesamtfürNatur ning Umwelt ampo Verbraucherschutz, North Rhine-Westphalia, Germany.
Deng animal me-anesthesia la keng ketamine (100 mg/kg) ampo xylazine (10 mg/kg), ampo ing pusu me-perfuse ya keng 0.1 M PBS mumuna, at kaybat 4% PFA keng PBS. Ing tissue me-dissect ya at me-fix ya keng 4% PFA/PBS magdamag keng 4°C. Ing vibrating knife (Leica Microsystems GmbH, Vienna, Austria) ginamit ya para isadya ing sagittal sections (50 μm kapal) ibat keng fixed a utak keng PBS. nung ali ya makasulat, ing pamag-stain kareng free-floating sections megawa ya antimo ing makasulat babo (13) keng room temperature ampo pamag-stir. king makuyad a salita, mumuna, ding mekwa a hiwa me-permeabilize la keng 0.5% Triton X-100/PBS kilub ning 15 minutu keng room temperature; para kareng mapilan a epitopes (Pcx ampo Shmt2), kapamilatan ning tris-EDTA buffer keng 80°C (PH 9) painitan la reng hiwa kilub ning 25 minutu imbes na ining step. Kaybat, ding section mi-incubate la kareng primary antibody (lawen ya ing Table S1) keng blocking buffer (3% BSA/PBS) keng 4°C magdamag a maki stirring. keng tutuking aldo, ding section me hugas la keng blocking buffer ampo me incubate la keng ustung fluorophore-conjugated secondary antibody kilub ning adwang oras keng room temperature; katataulian, ding section me hugas lang masalese keng PBS, me counter-stain la keng DAPI, at kaybat me fix la keng AquaPolymount keng microscope slide.
Ing laser scanning confocal microscope (TCS SP8-X o TCS Digital Light Sheet, Leica Microsystems) a ating white light laser ampo ing 405 diode ultraviolet laser ing ginamit para i-image ing sample. King pamag-excite king fluorophore ampo pamagkolekta king signal gamit ing Hybrid Detector (HyDs), ing LAS-X software ginamit ya para makolekta kareng stacked a larawan a makayagpang king Nyquist sampling king sequential mode: para kareng non-quantitative a panel, iti matas yang dynamic a signal (alimbawa, kareng somatic cells ampong dendrites) mtY UFP to detect ing bilang da reng BPNR mode). Ing gating manibat 0.3 anggang 6 ns ing gagamitan para mabawas ing background.
Real-time imaging da reng me-sort a cell. Kaybat ning pamag-sort keng Neurobasal-A medium a maki 1% B27 supplement ampo 0.5 mM GlutaMax, deng cells agad lang mitanam keng poly-l-lysine-coated glass slides (μ-Slide8 Well, Ibidi, catalog number 80826) , at kaybat itabi de keng 37°C ampo 12% CO kilub ning metung a oras para keng cell ibalakad. Ing real-time imaging megawa ya keng Leica SP8 laser scanning confocal microscope a ating white laser, HyD, 63×[1.4 numerical aperture (NA)] oil objective lens ampo heating stage.
Ing mouse mabilis yang me-anesthesia keng carbon dioxide ampo meputut ya buntuk, ing utak mabilis yang mewala keng bungu, ampo meputut ya keng 200μm kapal (para keng 13C labeling experiment) o 275μm kapal (para keng adwang photon experiments) sagittal section a mitmu kareng makatuking materyales Ing ice cream (HM-605, Vher, Scientific, Scientific Walldorf, Germany) mitmu ya kareng makatuking sangkap: 125 mM ice-cold, carbon-saturated (95% O2 ampo 5% CO2) mababang Ca2 + artificial cerebrospinal fluid (ACSF) NaCl, 2.5 mM KCl, 1.25 mM sodium phosphate buffer, NaCl, 2.5 mM, gluse 0.5 mM CaCl2 ampo 3.5 mM MgCl2 (osmotic pressure ning 310 anggang 330 mmol). Ilipat la reng mekwa nang utak keng pre-incubation chamber a maki mas matas a Ca2 + ACSF (125.0 mM NaCl, 2.5 mM KCl, 1.25 mM sodium phosphate buffer, 25.0 mM NaHCO3, 25.0 mM d-glucose, 10 mM Ca. MgCl2) Medium) pH 7.4 ampo 310 anggang 320 mmol).
keng prosesu ning pamag-imaging, ding slices mipalitan la keng metung a dedicated imaging room, at ing experiment megawa ya lalam ning patuloy a ACSF perfusion keng pare-pareung temperatura a 32° anggang 33°C. Ing multiphoton laser scanning microscope (TCS SP8 MP-OPO, Leica Microsystems) a ating Leica 25x objective lens (NA 0.95, danum), Ti: Sapphire laser (Chameleon Vision II, Coherent) ing ginamit para keng slice imaging. FLIM module (PicoHarp300, PicoQuant).
FLIM ning Grx1-roGFP2. Ing pamagbayu keng cytoplasmic redox state da reng PNs mesukad ya kapamilatan ning two-photon FLIM keng sagittal brain slices, nung nukarin ing Grx1-roGFP2 biosensor target no reng PNs. Ing PN layer, ing acquisition field mepili ya mga 50 anggang 80 μm lalam ning slice surface para makasiguradung atin yang viable PN (yapin ing alang beaded structure o neuronal morphological changes kareng dendrites) ampo ing double positive roGFP2 sensor ampo AAV encoding shRNA PCx o ing kayang control sequence (meach copressing mCherry). Manipun kang single-stack a larawan gamit ing 2x digital zoom [excitation wavelength: 890 nm; 512 nm 512 pixels]. Detection: internal HyD, fluorescein isothiocyanate (FITC) filter group] ampo ing image averaging kilub ning 2 anggang 3 minutu ing gagamitan para makasiguradung sapat a photons ing makakolekta (1000 photons keng kabilugan) para keng curve fitting. Ing sensitivity ning Grx1-roGFP2 probe ampo ing pamag-verify kareng FLIM a kundisyun megawa ya kapamilatan ning pamag-monitor king lifespan value ning roGFP2 nung dagdagan ing exogenous 10 mM H2O2 king perfusion ACSF (para mas maragul ing oxidation, a magresulta king mas maragul a lifespan), at kaybat dagdagan ing 2 mM dithiothreit (magbawas ing degree ning reduction, a magresulta king pamagbawas mabawas ing bie) (Figure S8, D anggang G). Gamitan me ing FLIMfit 5.1.1 software para asuri la reng resulta, i-fit me ing metung a exponential decay curve ning mabilug a larawan keng mesukad a IRF (instrument response function), at ing χ2 manga 1 ya. Para makalkula ing lifetime ning metung a PN, ing mask makapadurut keng nerve body manual yang ginuhit, ing average lifetime ing ginamit para keng balang masktime.
Mitochondrial potential analysis. Kaybat ning acute section me-incubate ya keng 100 nM TMRM a diretsung miyabe keng perfused ACSF kilub ning 30 minutu, ing mitochondrial potential changes da reng PNs mesukad ya kapamilatan ning two-photon microscope. Ing TMRM imaging megawa ya kapamilatan ning pamag-excite keng probe keng 920 nm ampo gamit ing internal HyD (tetramethylrhodamine isothiocyanate: 585/40 nm) para makakolekta signal; gamit ing pareung excitation wavelength pero gamit ing aliwang internal HyD (FITC :525/50) para i-image ing mtYFP. Gamitan me ing ImageJ's Image Calculator plug-in para abalu me ing mitochondrial potential keng metung a cell level. Ing makuyad a salita, ing plug-in equation: signal = min (mtYFP, TMRM) magagamit ya para akilala ing mitochondrial region na papakit ing TMRM signal keng Purkinje Somali keng single-stack confocal image ning katumbas a channel. Kaybat ing pixel area keng resulta ning mask me quantify ya, at kaybat me normalize ya keng corresponding threshold single-stack image ning mtYFP channel para akwa ing mitochondrial fraction a papakit keng mitochondrial potential.
Ing larawan me-deconvolute ya keng Huygens Pro (Scientific Volume Imaging) software. Para kareng me-scan a larawan da reng tile, ing montage ning metung a tile gagawan de gamit ing automatic stitching algorithm a binie ning LAS-X software. Kaybat ning pamag-calibrate ning larawan, gamitan me ing ImageJ ampo ing Adobe Photoshop para mas iprosesu me ing larawan ampo para a-adjust me ing brightness ampo ing contrast. Gamitan me ing Adobe Illustrator para keng pamagsadya keng graphic.
mtDNA focus analysis. Ing bilang da reng mtDNA lesions me-quantify ya kareng cerebellar sections a maka-label kareng antibodies laban keng DNA kapamilatan ning confocal microscope. Balang target area megawa ya para keng katawan ning cell ampo ing nucleus ning balang cell, ampo ing balang area mekwa ya gamit ing Multi Measure plug-in (ImageJ software). Ibawas ing nuclear area keng cell body area para akwa ing cytoplasmic area. Katataulian, ing Analyze Particles plug-in (ImageJ software) ginamit ya para automatic yang ma-quantify ing cytoplasmic DNA points a magpakit mtDNA keng threshold image, at ing mekwa a resulta me-normalize ya keng PN average da reng CTRL mice. Ing resulta mipalabas ya bilang ing average a bilang da reng nucleosides balang cell.
Pamagsuri king pamagpakilala ning protina. Gamitan me ing ImageJ's Image Calculator plug-in para abalu me ing protein expression keng PN keng single cell level. king makuyad a salita, keng single-layer confocal image ning corresponding channel, kapamilatan ning equation: signal = min (mtYFP, antibody), ing mitochondrial region na magpakit immunoreactivity keng metung a antibody keng Purkina mekilala ya. Kaybat ing pixel area keng resulta ning mask me-quantify ya, at kaybat me-normalize ya keng corresponding threshold single-stack image ning mtYFP channel para akwa ing mitochondrial fraction ning mipalabas a protina.
Purkinje cell density analysis. Ing Cell Counter plug-in ning ImageJ ginamit ya para abalu ing Purkinje density kapamilatan ning pamikawani da reng bilang da reng Purkinje cells a mebilang king kaba ning cerebellar ring a sasakupan da reng mebilang a cell.
Sample preparation at collection. Deng utak ibat keng control group ampo reng Mfn2cKO a mice me-fix la keng 2% PFA/2.5% glutaraldehyde keng 0.1 M phosphate buffer (PB), at kaybat ding coronal sections me-fix la gamit ing ciliates (Leica Mikrosysteme GmbH, Vienna, Austria) (Kapal 50 anggang 60) Ing fix buffer keng P1% μm tetraoxide ampo 1.5% potassium ferrocyanide keng temperatura ning silid kilub ning metung oras. Deng section me hugas lang atlung besis keng distilled water, at kaybat me stain la keng 70% ethanol a maki 1% uranyl acetate kilub ning 20 minutus. Deng section kaybat me-dehydrate la keng graded alcohol ampo mi-embed la keng Durcupan ACM (Araldite casting resin M) epoxy resin (Electron Microscopy Sciences, catalog number 14040) pilatan da reng silicon-coated glass slides, at katataulian keng 60°C mi-polymerize la keng oven kilub ning 48 oras. Ing cerebellar cortex area mepili ya at 50 nm ultrathin sections meputut la keng Leica Ultracut (Leica Mikrosysteme GmbH, Vienna, Austria) ampo mepili la keng 2×1 mm copper slit grid a makabalut keng polystyrene film. Deng section me-stain la keng solusyun ning 4% uranyl acetate keng H2O kilub ning 10 minutu, me hugas la keng H2O marakal a besis, kaybat keng Reynolds lead citrate keng H2O kilub ning 10 minutu, at kaybat me hugas la keng H2O marakal a besis. Deng micrographs mekwa la gamit ing transmission electron microscope Philips CM100 (Thermo Fisher Scientific, Waltham, MA, USA) gamit ing TVIPS (Tietz Video and Image Processing System) TemCam-F416 digital camera (TVIPS GmbH, Gauting, USA). Germany).
Para kareng mice a ating AAV, ing utak me-separate ya at me-slice ya keng 1 mm a kapal a sagittal section, at ing cerebellum me-eksamin ya gamit ing fluorescence microscope para akilala ya ing AAV-infected ring (yapin ing mCherry expressing). Deng mung eksperimentu nung nukarin ing AAV injection magresulta ya keng matas a transduction efficiency ning Purkinje cell layer (ie halus ing mabilug a layer) keng at least adwang sunod-sunod a cerebellar rings ila reng gagamitan. Ing AAV-transduced loop me-microdissect ya para keng bengi kaybat ning pamag-fix (4% PFA ampo 2.5% glutaraldehyde keng 0.1 M cocoate buffer) ampo meprosesu ya pa. Para keng EPON embedding, ing fixed tissue me hugas ya keng 0.1 M sodium cocoate buffer (Applichem), ampo me incubate ya keng 2% OsO4 (os, Science Services; Caco) keng 0.1 M sodium cocoate buffer (Applichem) 4 oras, kaybat me hugas ya kilub ning 2 oras. Ulitan me atlung besis gamit ing 0.1 M cocamide buffer. Kaybat, ing ascending series ning ethanol ginamit ya para i-incubate ing balang ethanol solution keng 4°C kilub ning 15 minutus para ma-dehydrate ing tissue. Ing tissue mipalitan ya keng propylene oxide at mi-incubate ya keng EPON (Sigma-Aldrich) keng 4°C. Ibili me ing tissue keng bayung EPON keng room temperature kilub ning 2 oras, at kaybat i-embed me keng 62°C kilub ning 72 oras. Gamitan me ing ultramicrotome (Leica Microsystems, UC6) ampo ing diamond knife (Diatome, Biel, Switzerland) para pututan me ing 70 nm a ultrathin a section, ampo i-stain me ing 1.5% uranyl acetate kilub ning 15 minutu keng 37°C, ampo i-stain me ing lead citrate solution kilub ning 4 minutu. Deng electron micrographs mekwa la gamit ing JEM-2100 Plus transmission electron microscope (JEOL) a ating Camera OneView 4K 16-bit (Gatan) ampo DigitalMicrograph software (Gatan). Para keng pamagsuri, deng electron micrographs mekwa la gamit ing 5000× o 10,000× digital zoom.
Morphological analysis ning mitochondria. Para kareng eganaganang pamagsuri, ding contours da reng indibidwal a mitochondria manual lang mibalangkas kareng digital a larawan gamit ing ImageJ software. Deng miyayaliwang morphological parameters me-analisa la. Ing densidad ning mitochondria mipalabas ya bilang porsyentu a ikua kapamilatan ning pamikawani ning kabilugan a mitochondrial area ning balang cell king cytoplasm area (cytoplasm area = cell area-cell nucleus area) × 100. Ing pangabilug ning mitochondria makalkula ya kapamilatan ning formula [4π∙(area/perimeter 2)]. Ing ista morphology da reng mitochondria me-analisa ya at me-divide ya kareng adwang kategorya ("tubular" ampo "blister") agpang kareng karelang manimunang porma.
Autophagosome/lysosome number ampo density analysis. Gamitan me ing ImageJ software para manual meng i-outline ing contours ning balang autophagosome/lysosome keng digital a larawan. Ing autophagosome/lysosome area mipalabas ya bilang porsyentu a makalkula kapamilatan ning pamikawani ning kabilugan a autophagosome/lysosome structure area ning balang cell king cytoplasm area (cytoplasm area=cell area-nucleus area)×100. Ing densidad da reng autophagosome/lysosomes makalkula ya kapamilatan ning pamikawani da reng kabilugan a bilang king bilang da reng autophagosome/lysosome structures balang cell (keng paralan ning cytoplasmic area) (cytoplasmic area = cell area-nuclear area).
Pamaglabel para keng acute sectioning ampo pamagsadya keng sample. Para kareng eksperimentu na mangailangan glucose labeling, ilipat la reng acute brain slices keng pre-incubation chamber, na atin saturated carbon (95% O2 ampo 5% CO2), matas a Ca2 + ACSF (125.0 mM NaCl, 2.5 mM KCl, 1.25 mM sodium phosphate buffer, 2.5 mM NaCl, 2.5 mM, 205 mM. d-glucose, 1.0 mM CaCl 2 ampo 2.0 mM MgCl 2, mi-adjust ya keng pH 7.4 ampo 310 anggang 320 mOsm), nung nukarin ing glucose 13 C 6- Glucose substitution (Eurisotop, catalog number CLM-1396). Para kareng experiment na mangailangan pyruvate labeling, ilipat la reng acute brain slices keng mas matas a Ca2 + ACSF (125.0 mM NaCl, 2.5 mM KCl, 1.25 mM sodium phosphate buffer, 25.0 mM NaHCO3, 25.0 mM d-glucose, 10.0 mM Ca20 Ml Ca. MgCl2, i-adjust me ing pH 7.4 ampo 310 anggang 320mOsm), ampo idagdag me ing 1mM 1-[1-13C]pyruvate (Eurisotop, catalog number CLM-1082). I-incubate la reng section kilub ning 90 minutus keng 37°C. keng wakas ning experiment, deng section mabilis lang me hugas keng aqueous solution (pH 7.4) a maki 75 mM ammonium carbonate, at kaybat me homogenize la keng 40:40:20 (v:v:v) acetonitrile (ACN): methanol: danum. Kaybat deng section me-incubate la keng yelo kilub ning 30 minutu, ding sample me-centrifuge la keng 21,000 g kilub ning 10 minutu keng 4°C, at ing malino a supernatant me-dry ya keng SpeedVac concentrator. Ing resulta ning metuling a metabolite pellet mitagan ya keng -80°C angga na keng pamagsuri.
Liquid chromatography-mass spectrometry analysis da reng 13 C-labeled amino acids. Para keng liquid chromatography-mass spectrometry (LC-MS) analysis, ing metabolite pellet mibalik ya keng 75μl ning LC-MS grade water (Honeywell). Kaybat ning centrifugation keng 21,000 g kilub ning 5 minutu keng 4°C, 20 μl ning me-clear a supernatant ing ginamit para keng amino acid flux analysis, kabang ing mitagan a extract agad yang ginamit para keng anion analysis (lawen ya ing lalam). Ing amino acid analysis megawa ya gamit ing minunang misulat a benzoyl chloride derivatization protocol (55, 56). keng mumunang lakbang, 10μl ning 100 mM sodium carbonate (Sigma-Aldrich) ing miyabe keng 20μl ning metabolite extract, at kaybat 10μl ning 2% benzoyl chloride (Sigma-Aldrich) ing miyabe keng LC grade ACN. Ing sample me vortex ya saglit at kaybat me centrifuge ya keng 21,000 g kilub ning 5 minutu keng 20°C. Ilipat me ing melinis a supernatant keng 2 ml autosampler vial a maki conical glass insert (200 μl volume). Deng sample me-analisa la gamit ing Acquity iClass ultra-high performance LC system (Waters) a makakonekta keng Q-Exactive (QE)-HF (Ultra High Field Orbitrap) high-resolution precision mass spectrometer (Thermo Fisher Scientific). Para keng pamagsuri, 2μl ning derivatized sample mi-inject ya keng 100×1.0 mm high-strength silica T3 column (Waters) a maki 1.8μm a particles. Ing flow rate 100μl/min ya, at ing buffer system atin yang buffer A (10 mM ammonium formate ampo 0.15% formic acid keng danum) ampo buffer B (ACN). Ing gradient makanini ya: 0%B keng 0 minutu; 0%B. 0 anggang 15% B keng 0 anggang 0.1 minutu; 15 anggang 17% B keng 0.1 anggang 0.5 minutu; B keng 17 anggang 55% keng 0.5 anggang 14 minutu; B keng 55 anggang 70% keng 14 anggang 14.5 minutus; keng 14.5 anggang 70 anggang 100% B keng 18 minutu; 100% B keng 18 anggang 19 minutu; 100 anggang 0% B keng 19 anggang 19.1 minutu; 0% B keng 19.1 anggang 28 minutu (55, 56). Ing QE-HF mass spectrometer mag-operate ya keng positibong ionization mode a maki mass range a m/z (mass/charge ratio) a 50 anggang 750. Ing resolution a migamit 60,000 ya, at ing gain control (AGC) ion target a ikua 3×106 ya, at ing maximum a ion time 10 milliseconds ya. Ing heated electrospray ionization (ESI) source mag operate ya keng spray voltage a 3.5 kV, ing capillary temperature a 250°C, ing sheath airflow a 60 AU (arbitrary units), ampo ing auxiliary airflow a 20 AU. 250°C. Ing S lens maka set ya keng 60 AU.
Anion chromatography-MS analysis da reng 13C a makalabel a organic acids. Ing mitagan a metabolite precipitate (55μl) me-analisa ya gamit ing Dionex ion chromatography system (ICS 5000+, Thermo Fisher Scientific) a makakonekta keng QE-HF mass spectrometer (Thermo Fisher Scientific). king makuyad a salita, 5μl ning metabolite extract ing mi-inject keng Dionex IonPac AS11-HC column a maki HPLC (2 mm×250 mm, particle size 4μm, Thermo Fisher Scientific) keng push-in partial loop mode a maki filling ratio a 1. ) Dionex IonPac AG11-HC mm guard column ( x 4μm, Thermo Fisher Scientific). Ing temperatura ning column manatili ya keng 30°C, at ing autosampler maka set ya keng 6°C. Gamitan me ing potassium hydroxide cartridge a ating deionized a danum para makagawa kang potassium hydroxide gradient kapamilatan ning eluent generator. Ing pamikawani da reng metabolites keng flow rate a 380μl/min, gamit ing makatuking gradient: 0 anggang 3 minutu, 10 mM KOH; 3 anggang 12 minutu, 10 anggang 50 mM KOH; 12 anggang 19 minutu, 50 anggang 100 mM KOH; 19 anggang 21 minutu, 100 mM KOH; 21 anggang 21.5 minutu, 100 anggang 10 mM KOH. Ing column mibalik ya keng 10 mM KOH kilub ning 8.5 minutus.
Deng eluted metabolites mipagkalub la keng 150μl/min isopropanol supplement stream kaybat ning column at kaybat midirekta la keng high-resolution mass spectrometer na mag-operate keng negative ionization mode. Ing MS babanten ne ing mass range manibat m/z 50 anggang 750 a maki resolution a 60,000. Ing AGC maka-set ya keng 1×106, at ing maximum a ion time maka-set ya keng 100 ms. Ing me-init a ESI source me-operate ya keng spray voltage a 3.5 kV. Deng aliwang setting ning ion source ilapin deng makatuki: capillary temperature 275°C; sheath gas flow, 60 AU; auxiliary gas flow, 20 AU keng 300°C, ampo ing S lens setting keng 60 AU.
Data analysis da reng 13C a maki label a metabolites. Gamitan me ing TraceFinder software (version 4.2, Thermo Fisher Scientific) para keng data analysis ning isotope ratio. Ing pangatau ning balang compound me-verify ya kapamilatan ning metung a mapanalig a reference compound at me-analisa yang independenti. Para gawan ing isotope enrichment analysis, ing area ning me-extract ion chromatogram (XIC) ning balang 13C isotope (Mn) me-extract ya ibat keng [M + H] +, nung nukarin ing n ya ing carbon number ning target compound, gagamitan ya para mag-analisa kareng amino acids o [ MH] + ing gagamitan para mag-analisa kareng anions. Ing mass accuracy ning XIC mas ditak ya keng limang bage balang milyun, at ing accuracy ning RT 0.05 minutes ya. Ing enrichment analysis gagawan de kapamilatan ning pamagkwenta king ratio ning balang meyakit a isotope king kabilugan da reng eganaganang isotope ning katumbas a compound. Deting ratio mibye la bilang porsyentu para kareng balang isotope, at ding resulta mipalabas la bilang molar percent enrichment (MPE), antimo ing misulat na (42).
Ing frozen neuron pellet me-homogenize ya keng ice-cold 80% methanol (v/v), me-vortex ya, ampo me-incubate ya keng -20°C kilub ning 30 minutus. I-vortex me ing sample pasibayu at i-stir keng +4°C kilub ning 30 minutus. Ing sample me-centrifuge ya keng 21,000 g kilub ning 5 minutu keng 4°C, at kaybat ing resulta ning supernatant mekolekta ya at me-dry ya gamit ing SpeedVac concentrator keng 25°C para keng tutuking pamagsuri. Antimo ing makasulat babo, ing LC-MS analysis megawa ya kareng amino acids da reng me-sort a cell. Gamit ing TraceFinder (version 4.2, Thermo Fisher Scientific), ing data analysis megawa ya gamit ing monoisotopic mass ning balang compound. Ing quantile normalization ning metabolite data megawa ya gamit ing preprocessCore software package (57).
Pamagsadya keng slice. Ing mouse mabilis yang me-anesthesia keng carbon dioxide ampo meputut ya buntuk, ing utak mabilis yang mewala keng bungu, ampo ing ice-filled vibrating knife (HM-650 V, Thermo Fisher Scientific, Walldorf, Germany) ginamit de para pututan ya keng 300 anggang 375 μm sagittal sections carbon gasification (25% O25) Mababang Ca2 + ACSF (125.0 mM NaCl, 2.5 mM KCl, 1.25 mM sodium phosphate buffer, 25.0 mM NaHCO3, 25.0 mM d-glucose, 1.0 mM CaCl2 ampo 6.0 mM MgCl2 I-adjust keng pH 40 mM to 3030 mO). Ilipat la reng mekwa nang slice ning utak keng metung a silid a maki mas matas a Ca2 + ACSF (125.0 mM NaCl, 2.5 mM KCl, 1.25 mM sodium phosphate buffer, 25.0 mM NaHCO3, 25.0 mM d-glucose, 4.0 ml MM Ca ampo 5 MM pH 7.4 ampo 310 anggang 320 mOsm). Ibili la reng hiwa kilub ning 20 anggang 30 minutu ban mibalik la bayu la i-record.
pamagrekord. Ing microscope stage a ating fixed recording chamber ampo ing 20x water immersion objective lens (Scientifica) ing ginamit para kareng eganaganang recording. Deng putative Purkinje cells mekilala la kapamilatan ning (i) sukad ning katawan, (ii) anatomical a lugal ning cerebellum, ampo (iii) expression ning fluorescent mtYFP reporter gene. Ing patch pipette a maki tip resistance a 5 anggang 11 megohms mibubu ya kapamilatan ning borosilicate glass capillary (GB150-10, 0.86 mm×1.5 mm×100 mm, Science Products, Hofheim, Germany) ampo ing horizontal pipette Instruments (P-10000), Sutterva, CA). Deng eganaganang pamagrekord megawa la kapamilatan ning ELC-03XS npi patch clamp amplifier (npi electronic GmbH, Tam, Germany), a mekontrol king software Signal (version 6.0, Cambridge Electronic, Cambridge, UK). Ing experiment merecord ya keng sampling rate a 12.5 kHz. Ing signal me-filter ya kareng adwang short-pass Bessel filters a maki cutoff frequencies a 1.3 ampo 10 kHz. Ing capacitance ning membrane ampo ing pipette mibayaran ya keng compensation circuit gamit ing amplifier. Deng eganaganang eksperimentu megawa la lalam ning kontrol ning Orca-Flash 4.0 camera (Hamamatsu, Gerden, Germany), a kontroladu ning Hokawo software (version 2.8, Hamamatsu, Gerden, Germany).
Routine whole-cell configuration ampo analysis. Bayu ya pa mu rin i-record, katmuan me ing pipette keng internal a solusyun a maki makatuking sangkap: 4.0 mM KCl, 2.0 mM NaCl, 0.2 mM EGTA, 135.0 mM potassium gluconate, 10.0 mM Hepes, 4.0 mM ATP (MM), 5 MM triphone. (GTP) (Na) ampo 10.0 mM creatinine phosphate me-adjust la keng pH 7.25, ampo ing osmotic pressure 290 mOsm (sucrose). Agad kaybat nang megamit ing pwersa a 0 pA para masira ya ing membrane, ing resting membrane potential mesukad ya. Ing input resistance sukad ya kapamilatan ning pamag apply kareng hyperpolarized currents a -40, -30, -20, ampo -10 pA. Sukat me ing magnitude ning voltage response at gamitan me ing Ohm's law para makalkula me ing input resistance. Ing spontaneous activity mitala ya keng voltage clamp kilub ning 5 minutu, at ing sPSC mekilala ya at mesukad ya keng Igor Pro (version 32 7.01, WaveMetrics, Lake Oswego, Oregon, USA) gamit ing semi-automatic recognition script. Ing IV curve ampo ing steady-state current susukat ya kapamilatan ning pamag-clamp keng battery kareng miyayaliwang potentials (magumpisa keng -110 mV) ampo pamagdake keng voltage keng 5 mV steps. Ing pamaglalang ning AP me-test ya kapamilatan ning pamag-apply king depolarizing current. I-clamp me ing cell keng -70 mV kabang mag apply kang depolarizing current pulse. I-adjust ing step size ning balang recording unit (10 anggang 60 pA). Karkulan me ing maximum AP frequency kapamilatan ning pamamilang kareng pulse spikes na magdalang pekamatas a AP frequency. Ing AP threshold me-analisa ya kapamilatan ning pamangamit king kaduang derivative ning depolarization pulse a mumunang mag-trigger king metung o maigit pang APs.
Perforated patch configuration ampo analysis. Gawa kang perforated patch recording gamit ing standard protocols. Gamitan me ing ATP- ampo GTP-free pipette na ala kareng makatuking sangkap: 128 mM gluconate K, 10 mM KCl, 10 mM Hepes, 0.1 mM EGTA ampo 2 mM MgCl2, ampo i-adjust me keng pH 7.2 (gamit ing KOH). ATP ampo GTP e la milub keng intracellular solution para aiwasan ing e makontrol a permeability ning cell membrane. Ing patch pipette mitmu ya keng amphotericin-containing internal solution (mga 200 anggang 250μg/ml; G4888, Sigma-Aldrich) para mika punched patch record. Ing amphotericin metunaw ya keng dimethyl sulfoxide (katataulian a konsentrasyun: 0.1 anggang 0.3%; DMSO; D8418, Sigma-Aldrich). Ing konsentrasyun ning DMSO a ginamit ala yang makabaldugan a epektu kareng neurons a me-aral. keng prosesu ning pamanuktuk, ing channel resistance (Ra) patuloy yang mamonitor, at ing experiment megumpisa ya kaybat ning amplitude ning Ra ampo AP me-stabilize (20-40 minutus). Ing spontaneous activity susukat ya keng voltage ampo/o current clamp kilub ning 2 anggang 5 minutu. Ing pamagsuri keng data megawa ya gamit ing Igor Pro (version 7.05.2, WaveMetrics, USA), Excel (version 2010, Microsoft Corporation, Redmond, USA) ampo ing GraphPad Prism (version 8.1.2, GraphPad Software Inc., La Jolla, CA). United States). Para akilala la reng spontaneous APs, ing IgorPro's NeuroMatic v3.0c plug-in ing gagamitan. Automatic lang akilala deng APs gamit ing metung a threshold, na mi-adjust balang metung para kareng balang record. Gamit ing spike interval, abalu me ing spike frequency keng maximum a instantaneous spike frequency ampo ing average spike frequency.
PN isolation. King pamakibagay king minunang mibulalag a protocol, ing PNs melinis ya ibat king cerebellum ning mouse king metung a metung a yugtu (58). king makuyad a salita, ing cerebellum me-dissect ya at me-mince ya keng ice-cold dissociation medium [alang HBSS Ca2+ ampo Mg2+, midagdag ya keng 20 mM glucose, penicillin (50 U/ml) ampo streptomycin (0.05 mg/ ml)], at kaybat me-digest ya ing medium keng papain [HBSS, HCS, 1·1 (1 mg / ml), papain (16 U / ml) ampo deoxyribonuclease I (DNase I; 0.1 mg/ml)] Gamutan kilub ning 30 minutu keng 30°C. Mumuna, hugasan la reng tissue keng HBSS medium a maki egg mucus (10 mg/ml), BSA (10 mg/ml) ampo DNase (0.1 mg/ml) keng room temperature para aiwasan ing enzymatic digestion, at kaybat keng HBSS medium a maki 20 mM glucose. (0.05 mg/ml) ampo DNase (0.1 mg/ml) magpalwal lang single cells. Ing resulta ning cell suspension me-filter ya kapamilatan ning 70μm cell strainer, kaybat ding cell me-pellet la kapamilatan ning centrifugation (1110 rpm, 5 minutes, 4°C) at mi-resuspended la king sorting medium [HBSS, supplemented with 20 mM glucose, 20% fetal bovine ) Serum, pelinic (50/5/5) (0.05 mg/ml)]; evaluate ing cell viability gamit ing propidium iodide ampo i-adjust ing cell density keng 1×106 anggang 2×106 cells/ml. Bayu ing flow cytometry, ing suspension me filter ya keng 50 μm cell strainer.
Flow cytometer. Ing pamag-sort kareng cell megawa ya keng 4°C gamit ing FACSAria III machine (BD Biosciences) ampo ing FACSDiva software (BD Biosciences, version 8.0.1). Ing cell suspension me-sort ya gamit ing 100 μm nozzle lalam ning pressure a 20 psi keng rate a ~2800 events/sec. Uling deng tradisyunal a gating criteria (cell size, bimodal discrimination, ampo scattering characteristics) e la makasiguradung ustu ing pamag-isolate ning PN kareng aliwang uri ning cell, ing gating strategy maka-set ya base keng direktang pamagkumpara ning YFP intensity ampo autofluorescence keng mitoYFP+ ​​ampo ing control mitoYFP − Mice. Ing YFP me-excite ya kapamilatan ning pamag-irradiate king sample gamit ing 488 nm laser line, at ing signal mayayakit ya gamit ing 530/30 nm band pass filter. kareng mitoYFP+ ​​a mice, ing relatibung sikanan ning Rosa26-mitoYFP reporter gene magagamit ya para akilala ing neuronal body ampo reng axon fragments. Ing 7-AAD me-excite ya keng 561 nm yellow laser ampo me-detect ya keng 675/20 nm bandpass filter para i-exclude la reng mete cells. Para mikawani la reng astrocytes keng parehung oras, ing cell suspension me-stain ya keng ACSA-2-APC, kaybat ing sample me-irradiate ya keng 640 nm laser line, ampo ing 660/20 nm bandpass filter ginamit ya para akit ing signal.
Deng tinipun a cell me-pellet la kapamilatan ning centrifugation (1110 rpm, 5 minutus, 4°C) at mitagan la king -80°C anggang gamitan la. Deng Mfn2cKO mice ampo reng karelang litter pups me-classify la keng parehung aldo para mabawas ing procedural variability. Ing FACS data presentation ampo analysis megawa ya gamit ing FlowJo software (FlowJo LLC, Ashland, Oregon, USA).
Antimo ing sinabi ku babo (59), ing real-time PCR magagamit ya para i-isolate ing DNA kareng me-sort a neurons para keng tutuking mtDNA quantification. Ing linearity ampo ing threshold sensitivity me-test la kapamilatan ning pamag-run qPCR kareng miyayaliwang bilang da reng cell. king makuyad a salita, tipunan me ing 300 PN keng lysis buffer a maki 50 mM tris-HCl (pH 8.5), 1 mM EDTA, 0.5% Tween 20 ampo proteinase K (200 ng/ml) ampo i-incubate me keng 55°C 120 minutus. Deng cells mi-incubate la pa keng 95°C kilub ning 10 minutu para siguraduan ing kumpletung inactivation ning proteinase K. Gamit ing TaqMan probe (Thermo Fisher) a specific keng mt-Nd1, ing mtDNA mesukad ya kapamilatan ning semi-quantitative PCR keng 7900HT Real-Time PCR system (Thermoherific Scientifics). Science, catalog number Mm04225274_s1), mt-Nd6 (Thermo Fisher Scientific, catalog number AIVI3E8) ampo 18S (Thermo Fisher Scientific, catalog number Hs99999901_s1) genes.
Pamagsadya keng sample ning proteome. King pamangisnawa king solusyun king 95°C kilub ning 10 minutu at pamag-sonicate, king lysis buffer [6 M guanidine chloride, 10 mM tris(2-carboxyethyl) phosphine hydrochloride, 10 mM chloroacetamide ampo 100 mM tris- Lyse frozen neuron pellets king HC pellets]. keng Bioruptor (Diagenode) kilub ning 10 minutu (30 segundo pulse / 30 segundo pause period). Ing sample me dilute yang 1:10 keng 20 mM tris-HCl (pH 8.0), me timpla ya keng 300 ng trypsin gold (Promega), ampo me incubate yang magdamag keng 37°C para akwa ing kumpletung digestion. keng kaduang aldo, ing sample me centrifuge ya keng 20,000 g kilub ning 20 minutus. Ing supernatant me dilute ya keng 0.1% formic acid, at ing solusyun me desalt ya keng self-made StageTips. Ing sample me-dry ya keng SpeedVac instrument (Eppendorf concentrator plus 5305) keng 45°C, at kaybat ing peptide me-suspende ya keng 0.1% formic acid. Deng eganaganang sample mesadia lang sabay-sabay ning metung a tau. Para a-analisa la reng sample da reng astrocyte, 4 μg desalted peptides ing mi-label keng tandem mass tag (TMT10plex, catalog number 90110, Thermo Fisher Scientific) a maki peptide to TMT reagent ratio a 1:20. Para keng TMT labeling, 0.8 mg ning TMT reagent ing mibalik keng 70 μl ning anhydrous ACN, at ing dried peptide mibalik ya keng 9 μl ning 0.1 M TEAB (triethylammonium bicarbonate), nung nukarin 7 μl ning TMT reagent keng ACN ing midagdag. Ing konsentrasyun 43.75% ya. Kaybat ning 60 minutung pamag-incubate, ing reaksyun me-quench ya keng 2 μl ning 5% hydroxylamine. Deng makalabel a peptides tinipun la, pepatuyu la, mibalik la keng 200μl ning 0.1% formic acid (FA), mepitna la keng adwa, at kaybat me-desalt la gamit ing self-made StageTips. Gamit ing UltiMate 3000 ultra high performance liquid chromatograph (UltiMate 3000 ultra high performance liquid chromatograph), metung kareng adwang kapitna ing me-fractionate keng 1mm x 150mm Acquity chromatographic column a mitmung 130Å1.7μm C18 particles (Waters, catalog No SKU: 196366). Thermo Fisher Scientific). Mikawani la reng peptides keng flow rate a 30μl/min, mikawani la keng 1% anggang 50% buffer B kilub ning 85 minutu a maki stepwise gradient a 96 minutu, manibat 50% anggang 95% buffer B kilub ning 3 minutu, kaybat 8 ​​minutu para keng 95 % Buffer B; Ing buffer A 5% ACN ya ampo 10 mM ammonium bicarbonate (ABC), ampo ing buffer B 80% ACN ya ampo 10 mM ABC. Tipunan la reng fractions balang 3 minutu at isamut la kareng adwang grupu (1 + 17, 2 + 18, atbp.) at patuyan la keng vacuum centrifuge.
LC-MS/MS analysis. Para keng mass spectrometry, ding peptides (number r119.aq) mikawani la keng 25 cm, 75 μm inner diameter PicoFrit analytical column (bayung objective lens, part number PF7508250) a ating 1.9 μm ReproSil-Pur 120 C18-AQ medium (Maisch, Maisch-AQ), E. 1200 (Thermo Fisher Scientific, Germany). Ing column menatili ya keng 50°C. Deng buffer A ampo B 0.1% formic acid la keng danum ampo 0.1% formic acid keng 80% ACN, agpang kareti. Deng peptides mikakawani la ibat 6% anggang 31% buffer B kilub ning 65 minutu ampo ibat 31% anggang 50% buffer B kilub ning 5 minutu a maki gradient a 200 nl/min. Deng eluted peptides me-analisa la keng Orbitrap Fusion mass spectrometer (Thermo Fisher Scientific). Ing peptide precursor m/z a sukad gagawan de keng resolution a 120,000 keng range ning 350 anggang 1500 m/z. Gamit ing 27% normalized collision energy, ing pekamasikan a precursor a maki charge state a 2 anggang 6 ing mepili para keng matas a enerhiya C trap dissociation (HCD) cleavage. Ing oras ning siklo makatakda ya keng 1 s. Ing m/z value ning peptide fragment mesukad ya keng ion trap gamit ing pekamalating AGC target a 5×104 ampo ing maximum injection time a 86 ms. Kaybat ning fragmentation, ing precursor mibili ya keng dynamic exclusion list kilub ning 45 s. Deng TMT-labeled peptides mekawani la keng 50 cm, 75 μm Acclaim PepMap column (Thermo Fisher Scientific, catalog number 164942), at ding migration spectra me-analisa la keng Orbitrap Lumos Tribrid mass spectrometer (Thermo Fisher Scientific) a maki high-field a ammetric iMS iveons (IMS) ing kasangkapan (Thermo Fisher Scientific) mag-operate ya keng adwang compensation voltages a −50 ampo −70 V. Ing MS3 a mepili base keng synchronization precursor ing gagamitan para keng TMT report ion signal measurement. Ing peptide separation megawa ya keng EASY-nLC 1200, gamit ing 90% linear gradient elution, a maki buffer concentration a 6% anggang 31%; ing buffer A 0.1% FA ya, at ing buffer B 0.1% FA ya ampo 80% ACN. Ing analytical column gagawan de keng 50°C. Gamitan me ing FreeStyle (version 1.6, Thermo Fisher Scientific) para pisabyan me ing orihinal a file agpang keng FAIMS compensation voltage.
Pamikilala at pamag-quantify kareng protina. Gamit ing integrated Andromeda search engine, ing orihinal a data me-analisa ya gamit ing MaxQuant version 1.5.2.8 (https://maxquant.org/). Dagdag pa kareng Cre recombinase ampo YFP sequences a ibat keng Aequorea victoria, ing peptide fragment spectra me-search ya para keng canonical sequence ampo isoform sequence ning mouse reference proteome (Proteome ID UP000000589, me-download ibat keng UniProt inyang Mayu 2017). Ing methionine oxidation ampo ing protein N-terminal acetylation mitakda la bilang miyayaliwang pamagbayu; cysteine ​​ficarbamoyl methylation me set ya bilang fixed modifications. Ing digestion parameters maka set ya keng "specificity" ampo "trypsin/P". Ing pekamalating bilang da reng peptides ampo razor peptides a gagamitan para keng pamangilala kareng protina 1 ya; ing minimum a bilang da reng unique peptides 0 ya. keng lalam ning kundisyun ning peptide map matching, ing protein identification rate 0.01 ya. Ing "Second Peptide" option maka-enable ya. Gamitan me ing "match between runs" a opsyun para ilipat ing matagumpeng pamikilala kareng miyayaliwang orihinal a file. Gamitan me ing LFQ minimum ratio count 1 para keng label-free quantification (LFQ) (60). Ing LFQ intensity me-filter ya para kareng at least adwang valid values ​​keng at least metung a genotype group keng balang time point, at me-extrapolate ya ibat keng normal a distribution a maki lapad a. 0.3 at mibaba 1.8. Gamitan ing Perseus computing platform (https://maxquant.net/perseus/) ampo ing R (https://r-project.org/) para abalu ing resulta ning LFQ. Ing two-way moderate t test ibat keng limma software package ing ginamit para keng differential expression analysis (61). Ing exploratory data analysis gagawan de gamit ing ggplot, FactoMineR, factoextra, GGally ampo pheatmap. Ing TMT-based proteomics data me-analisa ya gamit ing MaxQuant version 1.6.10.43. Manintun kang raw proteomics data ibat keng UniProt's human proteomics database, na me-download ya inyang September 2018. Ing pamagsuri kayabe ya ing isotope purity correction factor a binie ning manufacturer. Gamitan me ing limma keng R para keng differential expression analysis. Ing orihinal a data, ing resulta ning pamanintun keng database, ampo ing data analysis workflow ampo ing resulta makalage la ngan keng ProteomeXchange alliance kapamilatan ning PRIDE partner repository a maki data set identifier PXD019690.
Deng functional annotations papayaman da ing pamagsuri. Ing Ingenuity Pathway Analysis (QIAGEN) tool ginamit ya para abalu ing kayamanan da reng functional annotation terms ning data set keng 8 duminggu (Figure 1). Ing makuyad a salita, ing quantitative protein list a ibat keng LC-MS/MS (tandem mass spectrometry) data analysis magagamit ya kareng makatuking filter criteria: Ing Mus musculus mepili ya bilang species ampo background, at ing kategorya papakit na ing P value a me-adjust nang Benjamini para keng enrichment 0.05 o mas mababa metung yang makabaldugan. Para keng graph ayni, ding limang pekamatas a kategorya keng balang cluster base keng me-adjust a P value ila ring mipalabas. Gamit ing multiple t-test, gamit ing two-stage linear boost program da ri Benjamini, Krieger, ampo Yekutieli (Q = 5%), ing time-course protein expression analysis gagawan de kareng importanting kandidatu a mekilala keng balang kategorya, ampo ing balang row me-analisa yang miyayaliwa. E na kailangan ing gamitan ing consistent a SD.
Para akumpara la reng resulta na niting pamagaral kareng mibulalag a database at makagawa yang Venn diagram king Figure 1, pisamut mi ing quantitative protein list kareng MitoCarta 2.0 annotations (24). Gamitan me ing online tool Draw Venn Diagram (http://bioinformatics.psb.ugent.be/webtools/Venn/) para gawan me ing diagram.
Para keng detalyadung impormasyun kareng statistical procedures a gagamitan para keng proteomics analysis, paki-refer me ing makatuglung a section ning Materials and Methods. Para kareng aliwang experiment, deng detalyadung impormasyun mayayakit la keng makatuglung a alamat. Nung ali ya makasulat, ing eganaganang data mipalabas ya bilang mean ± SEM, at eganaganang statistical analyses megawa la gamit ing GraphPad Prism 8.1.2 software.
Para kareng karagdagang materyales para kening artikulu, lawan me ing http://advances.sciencemag.org/cgi/content/full/6/35/eaba8271/DC1
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