Atu *A tu umnak: Cologne 50931, Germany, Cologne Excellence Cluster Research on Cellular Stress Response in Aging-related Diseases (CECAD).
Mitochondrial zawtnak hna i an thluak rawhralnak cu thlen khawh lomi ah ruah a si, zeicahtiah neurons hna i an metabolic plasticity cu a tlawm, asinain mitochondrial riantuan ningcang lo nih pum chung i neuronal metabolism cell autonomy cungah a chuahpimi thil cu ttha tein theihthiam a si lo. Hika ah, mitochondrial fusion dynamics a rawkmi ruangah a chuakmi OXPHOS tlamtlin lonak a ngeimi Purkinje neurons cell-specific proteome kha kan langhter. Mitochondrial riantuan ningcang lo nih proteomics lei ah thlennak nganpi a chuahter ti kan hmuh, a donghnak ah cell thih hlan ah a dikmi metabolic program hna kha a zulmi riantuannak ah a hruai. Ruah lo piin, TCA cycle karlak thilri pawl chapnak he aa pehtlaimi pyruvate carboxylase (PCx) le a dang peroxidases pawl a fiangmi chuahternak kha kan hmuh. PCx khamnak nih oxidative stress le neurodegeneration a zualter, cu nih cun atherosclerosis nih OXPHOS a ngei lomi neurons ah huhphenhnak a ngeih ti a langhter. A donghnak ah a rawkmi neurons ah mitochondrial fusion remh ṭhannak nih hi metabolic sining hna kha a thlen dih, cucaah cell thihnak kha a kham. Kan hmuhmi nih a hlan ah theih lomi lam hna kha a langhter i cu nih cun mitochondrial riantuan ningcang lonak kha a tuar khawh i zawtnak a donghnak lei hmanh ah thluak rawhnak cu a thlen khawh ti kha a langhter.
Minung mitochondrial zawtnak he aa pehtlaimi a kau ngaimi thluak lei zawtnak hmelchunhnak nih neuronal thazaang thlennak kilvennak ah mitochondria nih a biapi bikmi rian a ttuanmi kha a langhter. Hi zawtnak tam deuh cu mitochondrial gene langhternak (1, 2) a ukmi gene thlennak asiloah mitochondrial thlennak he aa pehtlaimi gene hrawhnak nih a chuahtermi a si, cu nih cun mitochondrial DNA (mtDNA) dirhmun kha a hnorsuan (3, 4). Saram zohchunhnak ah riantuannak nih a langhter mi cu a pawngkam tissue hna ah mitochondrial riantuan ningcang lo ruangah, conservative metabolic lam (5-7) cu a cawlcang kho, cucaah hi a harmi zawtnak hna i an chuahkehnak kongah a biapi mi hmuhnak a pek. Cu he aa dang ngai in, thluak mitochondrial adenosine triphosphate (ATP) chuahternak a tlamtlin lonak ruangah a chuakmi cell phun cheukhat hna i an thlennak kong kan hngalhthiamnak cu a hrampi a si (8), zawtnak khamnak asiloah khamnak caah hman khawhmi thlopnak tinhmi hngalh a herhnak kha a langhter. Thluak rawhnak kha kham (9). Theihhngalhnak a tlawmmi cu nerve cells pawl cu a pawngkam tissues cells phun pawl he tahchunh tikah a tlawm tukmi metabolic flexibility an ngei tiah mi tampi nih an ruah hna caah a si (10). Hi cells pawl nih hin synaptic transmission thanchoternak le hliamhma le zawtnak dirhmun ah lehrulhnak caah neurons sinah metabolites peknak ah a biapi tukmi rian an ttuan caah, thluak tissue a harmi dirhmun ah cell metabolism remh khawhnak cu glial cells lawng ah a tlawm (11-14). Cun, thluak tissue chung i a ummi cellular heterogeneity nih hin neuronal phu hme tete ah a cangmi metabolic thlennak kong hlathlainak kha a kham ngaingai. Cu ruangah, neurons chung i mitochondrial riantuan ningcang lo nih a chuahpimi cellular le metabolic thil sining kong kha tlawmte lawng theih a si.
Mitochondrial riantuan ningcang lo nih a chuahpimi thil thlennak hngalhthiam khawhnak ding caah, mitochondrial lenglei membrane fonhnak (Mfn2) rawhralnak ruangah a chuakmi thluak rawhnak a phunphun ah Purkinje neurons (PNs) kha kan thleidan hna. Minung chung i Mfn2 thlennak cu Charcot-Marie-Tooth phun 2A (15) tiah theihmi hrinsor in a chuakmi thluak lei zawtnak phunkhat he aa pehtlai ko nain, vok chung i Mfn2 a rawhralnak cu oxidation Phosphorylation (OXPHOS) riantuan ningcang lonak phunglam a chuahtertu a si tiah theihhngalhmi a si. Neuronal phun dangdang (16-19) le a chuakmi neurodegenerative phenotype cu a karh thluahmahmi thluak lei zawtnak hmelchunhnak, cawlcanghnak zawtnak (18, 19) asiloah cerebellar ataxia (16) tibantuk he an i pehtlai. Label-free quantitative (LFQ) proteomics, metabolomics, hmanthlaknak le virusological lam hna fonh in hmannak thawngin, a karh cuahmahmi thluak rawhnak nih pyruvate carboxylase (PCx) le PNs arteriosclerosis ah aa telmi thil dangdang hna kha fakpi in a chuahter ti kha kan langhter. Hi hmuhmi he aa pehtlaimi a si le si lo hngalh awkah, Mfn2-tlawmmi PNs ah PCx langhternak kha a hleiin kan tlawmter, cun hi operation nih hin oxidative stress a zualter i thluak rawhnak a rangter ti kha kan hmuh, cucaah azoospermia nih cell thihnak a pek ti kha a langhter. MFN2 a fakmi langhternak nih a donghnak rawhralnak PN kha a fakmi OXPHOS tlamtlin lonak, mitochondrial DNA tam tuk hmannak, le a langhning in a rawkmi mitochondrial network he tling tein a khamh khawh, cu nih cun hi bantuk neurodegeneration phun hi cell thih hlan ah zawtnak a sang deuhmi dirhmun hmanh ah a dam kho ti kha a langhter chin.
Mfn2 knockout PNs chung i mitochondria hmuh khawhnak ding caah, Cre-dependent mitochondria nih yellow fluorescent protein (YFP) (mtYFP) (20) Cre langhternak kha a target khawh i mitochondrial morphology kha in vivo ah kan zoh. PNs chung i Mfn2 gene hrawhnak nih mitochondrial network kha tlawmpal in a tthencheu lai ti kan hmuh (Figure S1A), cun a hmasa bik thlennak cu zarh 3 a si ah hmuh a si. Cu he aa dang in, PN cell tthennak a tlawm tukmi cu, Calbindin immunostaining a tlaunak nih a langhter bantukin, zarh 12 a tlin hlan tiang aa thawk lo (Figure 1, A le B). Mitochondrial morphology ah a hmasa bik thlennak le hmuh khawhmi neuronal thihnak aa thawknak karlak caan aa tlak lonak nih cell thih hlan ah mitochondrial riantuan ningcang lo nih a chuahtermi metabolic thlennak hna kha hlathlai dingin a kan forh. YFP (YFP+)-langhtermi PN (Figure 1C) thleidannak caah fluorescence-activated cell sorting (FACS)-based strategy kan ser, cun control mice (Mfn2 + / loxP :: mtYFP loxP- stop-loxP: : L7-cre) ah, a hnu ah CTRL (Figure S1BRL) tiah auh a si lai. YFP signal i aa pehtlaimi thazaang cung ah hram bunh in gating strategy tthaternak nih PNs a si lomi (YFPneg) (Figure S1B) asiloah putative fluorescent axon/dendritic fragments (YFPlow; Figure S1D, kehlei) in confocal microscope in fehtermi (Figure S1D orhlei) in YFP+ pum (YFPhigh) kha thianh khawhnak a kan pek. Thleidanmi milu zat hngalh khawhnak ding caah, LFQ proteomics le cu hnu ah a hrampi thilri hlathlainak kan tuah, cun YFPhigh le YFPneg cells karlak ah fiang tein thleidannak a um ti kan hmuh (Figure S1C). YFPhigh cells nih theihmi PNs hmelchunhnak (cucu Calb1, Pcp2, Grid2 le Itpr3) (21, 22) a tthanchoter, asinain neurons asiloah cell phun dang ah a lang tawnmi proteins a tthawnter lo (Figure 1D) ). Independent hneksaknak ah lakmi thleidanmi YFPhigh cells chung i sample pawl tahchunhnak nih pehtlaihnak coefficient> 0.9 a langhter, cu nih cun biological replicates karlak ah ttha tein chuah khawhnak a langhter (Figure S1E). A tawinak in chim ahcun, hi data nih hin a cang khomi PN a rannak le a bik in thleidannak caah kan timhtuahnak kha a fehter. Zeicatiah L7-cre driver system hmanmi nih hrin hnu zarh khatnak ah mosaic recombination a chuahter caah (23), CTRL le conditional (Mfn2 loxP / loxP :: mtYFP loxP-stop-loxP :: L7-cre) Collect neurons in vok thah kan thawk. Recombination a dih hnu ah, zarh 4 a si ah Mfn2cKO tiah auh a si. A donghnak ah, a fiangmi mitochondrial tthencheunak a um ko nain PN tthen cu a rawk rih lo lio zarh 8 kum kha kan thim (Figure 1B le Figure S1A). A dihlak ah, protein 3013 kan tuak, cu chung ah 22% hrawng cu MitoCarta 2.0 annotations cung ah hram bunh in mitochondria proteome cu mitochondria (Figure 1E) (Figure 1E) (24) ah hram bunh in an si. Zarh 8 ah tuahmi aa dangmi gene langhternak hlathlainak nih a langhter mi cu protein vialte chungin 10.5% lawng nih thlennak nganpi an ngei (Figure 1F le Figure S1F), cu chung ah protein 195 cu a niam deuhmi an si i protein 120 cu a sang deuhmi an si (Figure 1F). Hi data set i “thil thar sernak lamthluan hlathlainak” nih a langhtermi cu aa dang tein langhtermi genes pawl cu a bik in a hleiin metabolic lamthluan tlawmte ah aa telmi an si (Figure 1G). A hngalh a nuammi cu, OXPHOS le calcium signaling he aa pehtlaimi lamthluan hna i an tlawmternak nih fusion-deficient PNs ah mitochondrial riantuan ningcang lo a chuahter ti a fehter ko nain, amino acid metabolism aa telmi phun dang hna cu a tlawm deuhmi an si, cucu mitochondrial PNs ah a cangmi metabolism he aa tlak. Rewiring cu aa khat. riantuan khawh lonak.
(A) CTRL le Mfn2cKO vok hna i an thluak cheukhat hna i PNs (calbindin, gray) a tlau thluahmahmi a langhtermi aiawhtu confocal hmanthlak hna; nuclei cu DAPI in an rak thlen hna. (B) (A) zat tahnak (thleidannak lam khatlei hlathlainak, ***P<0.001; n = 4 in 6 tiang vok pathum chungin circle). (C) Hneksaknak riantuan ning. (D) Purkinje (cunglei) le a dang cell phun (laifang ah) caah a hleiin hmelchunhnak hna i lumnak map phawtzamhnak. (E) Venn diagram nih tthencheuhmi PN ah hmuhmi mitochondrial protein zat a langhter. (F) Zarh 8 ah Mfn2cKO neurons ah aa dang tein langhtermi protein pawl meitlang hmanthlak (1.3 a biapitnak tlawmternak man). (G) Sersiam khawhnak lam hlathlainak nih zarh 8 tiah tthenmi Mfn2cKO PN ah a biapi bikmi tthanchonak (sen) le tanglei (sen) lam panga a langhter. Hmuhmi protein pakhat cio i a tlangpi langhternak tlukruannak kha langhter a si. Grayscale heat map: remhmi P man. ns, a biapi lo.
Proteomics data nih a langhter mi cu complex I, III, le IV hna i an protein langhternak cu a zor thluahmah. Complex I, III, le IV vialte nih a herhmi mtDNA-encoded subunits an ngei dih, cu lio ah complex II, nuclear-coded lawng a simi cu a hrampi in a hnorsuan lo (Figure 2A le Figure S2A). . Proteomics hmuhmi he aa tlak in, cerebellar tissue tthencheunak immunohistochemistry nih PN chung i complex IV i MTCO1 (mitochondrial cytochrome C oxidase subunit 1) subunit level cu a zor thluahmah ti a langhter (Figure 2B). mtDNA-encoded subunit Mtatp8 cu a zor ngaingai (Figure S2A), cu lio ah nuclear-encoded ATP synthase subunit i a fekmi dirhmun cu aa thleng lo, cucu mtDNA langhternak a fek tikah theihmi a fekmi ATP synthase subassembly F1 complex he aa tlak. A dirhmun cu aa khat. (7) kha kham. A caan hmaan tein polymerase chain reaction (qPCR) hmangin thleidanmi Mfn2cKO PNs chung i mtDNA level zohfelnak nih mtDNA copy zat a zor thluahmahmi kha a fehter. Control phu he tahchunh ahcun, zarh 8 an si ah, mtDNA level 20% hrawng lawng a tang (Figure 2C). Hi hmuhmi he aa tlak in, DNA hmuh khawhnak ding caah Mfn2cKO PNs i confocal microscopy staining nih mitochondrial nucleotides caan-a hngatmi hmannak a langhter (Figure 2D). Mitochondrial protein rawhnak le lungretheihnak lehrulhnak ah aa telmi candidate cheukhat lawng kha Lonp1, Afg3l2 le Clpx, le OXPHOS complex assembly factors telh in an tthanchoter ti kan hmuh. Apoptosis ah aa telmi protein hna an tlawmtam ah thlennak nganpi hmuh a si lo (Figure S2B). Cu bantuk cun, calcium phurhnak ah aa telmi mitochondria le endoplasmic reticulum lam hna cu thlennak hmete lawng an ngei ti kan hmuh (Figure S2C). Cun, autophagy he aa pehtlaimi protein pawl zohfelnak nih a biapi mi thlennak a hmu lo, cucu immunohistochemistry le electron microscopy (Figure S3) in vivo ah hmuhmi autophagosomes hmuh khawhmi induction he aa tlak. Sihmanhsehlaw, PNs ah a karh cuahmahmi OXPHOS riantuan lonak cu a fiangmi ultrastructural mitochondrial thlennak he aa pehtlai. Zarh 5 le 8 a simi Mfn2cKO PNs cell pum le dendritic thingkung ah mitochondrial bu pawl hmuh khawh an si, cun a chunglei membrane sining cu thlennak nganpi a tong (Figure S4, A le B). Hi ultrastructural thlennak le mtDNA a zornak he aa tlak in, tetramethylrhodamine methyl ester (TMRM) he acute cerebral cerebellar slices hlathlainak nih Mfn2cKO PNs ah mitochondrial membrane potential cu a zor ngaingai ti a langhter (Figure S4C).
(A) OXPHOS complex langhternak tlukruannak caan kalning hlathlainak. Zarh 8 ah P<0.05 a ngeimi protein lawng kha ruat (two-way ANOVA). Dotted line: CTRL he tahchunh ahcun thlennak a um lo. (B) Orhlei: Anti-MTCO1 antibody (scale bar, 20 μm) in min bunhmi thluak tthen tahchunhnak. Purkinje cell pum nih a lakmi hmun cu a senmi nih a khuh. Orhlei: MTCO1 tlukruannak (thleidannak lam pakhat lawng in hlathlainak; n = 7 in 20 tiang cells cu vok pathum in hlathlainak tuahmi). (C) qPCR hlathlainak mtDNA copy zat kha thleidanmi PN ah (aa dangmi lam khatlei hlathlainak; n = 3 in 7 mice). (D) Kehlei: Anti-DNA antibody (scale bar, 20 μm) in min bunhmi thluak tlawmpal tahchunhnak. Purkinje cell pum nih a lakmi hmun cu a senmi nih a khuh. Orhlei: mtDNA hma zat tahnak (thleidannak lam khatlei hlathlainak; n = 5 in 9 tiang vok pathum chungin cells). (E) Cell dihlak patch clamp recording ah mitoYFP + Purkinje cells (thal) a langhtermi a fakmi thluak tthen tahchunhnak. (F) IV curve tahnak. (G) CTRL le Mfn2cKO Purkinje cells ah depolarizing current luhternak aiawhtu cazin. A cunglei hmelchunhnak: AP a chuahtertu a hmasa bik tthawnnak. A tanglei hmelchunhnak: A tam bik AP frequency. (H) Postsynaptic spontaneous inputs (sPSPs) zat tahnak. Aiawhtu hmanthlaknak hmelchunhnak le a kauh ning cu (I) ah langhter a si. Lam khatlei in aa dangmi hlathlainak nih vok pathum chungin n = 5 in 20 tiang cells a hlathlai. Data cu a tlangpi±SEM in langhter a si; *P<0.05; **P<0.01; ***P<0.001. (J) Perforated patch clamp mode hmangin ttialmi mah tein a cangmi AP aiawhtu hmelchunhnak hna. A cunglei hmelchunhnak: A tam bik AP frequency. A tanglei hmelchunhnak: AP pakhat lawng zoom. (K) (J) ningin a tlangpi le a tam bik AP frequency kha tuak. Mann-Whitney hneksaknak; n = 5 cells cu vok pali chungin an hlathlai hna. Data cu a tlangpi±SEM in langhter a si; a biapi lo.
Zarh 8 a simi Mfn2cKO PN ah OXPHOS rawhnak a fiangmi hmuh a si, cu nih cun neurons hna an pumsa riantuan ning cu a ttha lo ngai ti a langhter. Cucaah, zarh 4 in 5 le zarh 7 in 8 karlak ah OXPHOS-a tlawmmi neurons hna i a cawlcang lomi electric sining kha acute cerebellar slices ah whole-cell patch clamp recordings tuahnak in kan hlathlai (Figure 2E). Ruah lo piin, Mfn2cKO neurons hna i a tlangpi in dinhnak membrane tthawnnak le luhternak doh khawhnak cu control he aa lo, asinain cells karlak ah a fiang lomi thleidannak a um (Table 1). Cu bantuk cun, zarh 4 in 5 karlak ah, a tu-tthawnnak pehtlaihnak (IV curve) ah a biapi mi thlennak a um lo (Figure 2F). Sihmanhsehlaw, zarh 7 in 8 kar a simi Mfn2cKO neurons pakhat hmanh IV regimen (hyperpolarization step) in an luat lo, cucu a donghnak lei ah hyperpolarization tuah khawhnak ah fiang tein theih khawhnak a um ti a langhter. Cu he aa dang in, Mfn2cKO neurons ah, aa peh tthanmi thil tuah khawhnak (AP) chuahternak a chuahtertu depolarizing currents cu ttha tein an tuar khawh, cu nih cun an dihlak in chuahternak pattern cu zarh 8 a simi control neurons he aa dang tuk lo ti a langhter (Table 1 le Figure 2G). Cu bantuk cun, a chuakmi postsynaptic currents (sPSCs) a tam le a tam cu control phu he tahchunh khawh an si, cun thil cangmi a tam ning cu zarh 4 in zarh 5 tiang zarh 7 in zarh 8 tiang aa khatmi karhternak he a karh (Figure 2, H le I). PNs (25) ah synaptic tthanchonak caan. Aa khatmi phichuak cu PNs tthencheunak hnu ah hmuh a si. Hi dirhmun nih hin cell ATP tlamtlin lonak a um khomi remh khawhnak kha a kham, cucu cell dihlak patch clamp recording ah a cang khomi a si. A bik in, Mfn2cKO neurons hna i an i dinhnak membrane thilti khawhnak le anmah tein an kah tawnmi kha a hnorsuan lo (Figure 2, J le K). A tawinak in chim ahcun, hi hmuhmi nih hin OXPHOS riantuan ningcang lo a fiangmi PN pawl nih a sangmi chuahnak pattern pawl kha ttha tein an tei khawh ti a langhter, cu nih cun a ttha ngaimi electrophysiological lehrulhnak ngeih khawhnak ding caah tlukruannak lam a um ti a langhter.
Data cu a tlangpi in ± SEM (thleidannak pakhat lei hlathlainak, Holm-Sidak's multiple comparison test; *P<0.05) in langhter a si. Unit nambar cu tthencheu in langhter a si.
Proteomics dataset (Figure 1G) chung i zeibantuk phun hmanh nih OXPHOS tlamtlin lonak fakpi a doh khomi lam aa tel maw tel lo ti hlathlainak tuah dingin kan i tim, cucaah a tlunhmi PN nih a ttha ngaimi electrophysiology (Figure 2, E in K tiang) a ngeih khawhnak a ruang kha a fianter. . Proteomics hlathlainak nih a langhter mi cu branched chain amino acids (BCAA) catabolism ah aa telmi enzymes pawl cu an tthangcho ngaingai (Figure 3A le Figure S5A), cun a donghnak thilchuak acetyl-CoA (CoA) asiloah succinyl CoA nih arteriosclerosis Acid (TCA) cycle chung i tricarboxylates pawl kha a bawmh khawh hna. BCAA transaminase 1 (BCAT1) le BCAT2 an pahnih in an karh ti kan hmuh. α-ketoglutarate (26) in glutamate sernak in BCAA catabolism a hmasa bik kehlan kha an thawk. Branched chain keto acid dehydrogenase (BCKD) complex a sertu subunits vialte cu an tthancho (complex nih a chuakmi BCAA carbon ruhro a hnu i a kir khawh lomi decarboxylation kha a chuahter) (Figure 3A le Figure S5A). Sihmanhsehlaw, BCAA hrimhrim ah a fiangmi thlennak pakhat hmanh thleidanmi PN ah hmuh a si lo, cucu hi a herhmi amino acid pawl cell nih a lakmi a karh caah asiloah TCA cycle bawmhchanhnak caah a dang hrampi (glucose asiloah lactic acid) hmannak ruangah a si kho men (Figure S5B). OXPHOS a ngei lomi PNs zong nih zarh 8 an tlin tikah glutamine rawhnak le transamination cawlcanghnak a karhter, cucu mitochondrial enzymes glutaminase (GLS) le glutamine pyruvate transaminase 2 (GPT2) (Figure 3, A le C) a karhmi nih a langhter khawh. GLS a tthanchonak cu spliced ​​isoform glutaminase C (GLS-GAC) (Mfn2cKO/CTRL thlennak cu 4.5-fold hrawng a si, P = 0.05) lawng ah a si ti kha hngalh awk tlak a si, cun cancer tissues ah a hleiin a tthanchonak nih mitochondrial bioenergy a bawmh khawh. (27).
(A) Heat map nih zarh 8 ah a langhtermi lam caah protein level thlennak a langhter. (B) Anti-PCx antibody (scale bar, 20 μm) in min bunhmi thluak tlawmpal tahchunhnak. A senmi thal nih Purkinje cell pum kha a langhter. (C) Caan karlak protein langhternak hlathlainak cu atherosclerosis caah a biapi mi candidate pakhat in hmuh a si (multiple t-test, *FDR <5%; n = 3-5 mice). (D) A cunglei: [1-13C]pyruvate tracer chung i aa telmi min bunhmi carbon luh khawhnak lam phunphun a langhtermi hmanthlak (cucu, PDH asiloah trans-arterial lam in). A tanglei: Violin chart nih [1-13C]pyruvate (paired t-test; ** P <0.01) in a fakmi thluak tthencheu pawl kha aspartic acid, citric acid le malic acid ah aa thlengmi single-labeled carbon (M1) zat kha a langhter. (E) A langhtermi lam i a tlingmi caan tuanbia hlathlainak. Zarh 8 ah P<0.05 a ngeimi protein lawng kha ruat. A tthenmi hri: remh khawhnak man a um lo (two-way-way analysis of variance; * P <0.05; *** P <0.001). Data cu a tlangpi±SEM in langhter a si.
Kan hlathlainak ah, BCAA catabolism cu a biapi bikmi tthanchonak lam pakhat ah a cang. Hi thil nih hin TCA cycle chung a lutmi thli luhchuahnak zat cu OXPHOS a ngei lomi PN ah aa thleng kho ti kha fakpi in a langhter. Mah nih hin neuronal metabolic rewiring phun nganpi pakhat a aiawh kho, mah nih hin OXPHOS riantuan ningcang lo fakpi a ngeih lio ah neuronal physiology le nunnak cungah direct in hnahnawhnak a pek khawh. Hi ruahnak he aa tlak in, kan hmuhmi cu a tlangpi in thi kalnak lam dohnak enzyme PCx cu a tthangcho (Mfn2cKO/CTRL cu voi 1.5 hrawng aa thleng; Figure 3A), cu nih cun pyruvate kha oxaloacetate (28) ah a thlenter, cucu thluak ah a ummi a si lai tiah zumh a si. (29, 30). Proteomics hmuhmi he aa tlak in, confocal microscopy nih PCx langhternak cu OXPHOS-tlawmmi PNs ah a hleiin le a biapi in a karh ti a langhter, cu lio ah PCx reactivity cu control i a pawngkam Bergmann glial cells ah a tlawm deuh (Figure 3B). PCx a karhnak hmuhmi kha riantuan ning in hneksaknak tuah awkah, [1-13C]pyruvate tracer in a fakmi thluak tthen pawl kha kan thlop hna. Pyruvate cu pyruvate dehydrogenase (PDH) nih a thlen tikah, a isotope hmelchunhnak cu a tlau, asinain pyruvate cu thihri thlennak in a thlen tikah TCA cycle karlak thilri ah aa tel (Figure 3D). Kan proteomics data bawmhchanhnak ah, Mfn2cKO slices aspartic acid ah hi tracer in hmelchunhnak tampi kan hmuh, cu lio ah citric acid le malic acid zong nih a tlawm deuhmi sining an ngei, asinain a biapi lo (Figure 3D).
MitoPark vok dopamine neurons ah dopamine neurons nih mitochondrial transcription factor A gene (Tfam) (Figure S6B) a hrawh ruangah a chuakmi mitochondrial riantuan lonak a ngeimi dopamine neurons ah, PCx langhternak zong a sang ngai in a tthangcho (31), cu nih cun acetone acid arteriosclerosis Zawtnak a chuahkehnak cu neuron riantuan ning a ttha lo lio ah a ukmi a si ti a langhter pum chung i OXPHOS. Arteriosclerosis he aa pehtlaimi neurons ah a lang khomi aa dangmi enzymes (32-34) cu OXPHOS a ngei lomi PNs ah a tthangcho ngaingai ti hmuh a si ti kha hngalh awk tlak a si, cu hna cu propionyl-CoA carboxylase (PCC-A), Malonyl-CoA nih propionyl-CoA carboxylase (PCC-A) tibantuk a si. enzyme 3 (ME3), a rian nganbik cu malate chungin pyruvate hmuh ṭhan a si (Figure 3, A le C) (33, 35). Cun, Pdk3 enzyme ah a karh tukmi kan hmuh, cu nih cun PDH kha a phosphorylate i cucaah PDH kha a rian a ttuan ti lo (36), cu lio ah PDH a rian a ttuantertu Pdp1 enzyme asiloah PDH enzyme complex hrimhrim ah thlennak pakhat hmanh hmuh a si lo (Figure 3A). Aa khat tein, Mern2cKO PNs ah, Ser293 (PDH enzyme cawlcanghnak a kham tiah theihmi) chung i PDH complex i pyruvate dehydrogenase E1 component α1 subunit α (PDHE1α) subunit phosphorylation cu a tthancho (Figure S6C) (Figure S6C). Pyruvate nih thi kalnak lam a ngei lo.
A donghnak ah, serine le glycine biosynthesis super pathway, aa pehtlaimi mitochondrial folate (1C) cycle le proline biosynthesis (Figure 1G le Figure S5C) hna cu report ning in, riantuannak kalning chungah a ttha ngaimi an si ti kan hmuh. A pawngkam ummi tissue pawl cu mitochondrial riantuan lonak he an cawlcang (5-7). Hi proteomics data a dirkamhtu confocal hlathlainak nih OXPHOS a um lomi PN ah, zarh 8 a simi vok hna i an thluak tthencheu hna cu mitochondrial folate cycle ah a biapi bikmi enzyme serine hydroxymethyltransferase 2 (SHMT2) ah an chiah hna ti a langhter. A biapi mi ralvennak lehrulhnak (Figure S5D). CU-glucose-incubated acute cerebellar slices 13 ah, metabolic tracing hneksaknak nih serine le proline biosynthesis a tthanchonak kha a fehter chin, cu nih cun serine le proline ah carbon isoforms a luh ning a karh ti a langhter (Figure S5E). GLS le GPT2 nih a chuahtermi lehrulhnak hna cu glutamine in glutamate sernak le glutamate le α-ketoglutarate karlak transamination caah tuanvo an lak caah, an tthanchonak nih OXPHOS-a tlawmmi neurons nih glutamate an herhnak a karh ti a langhter, Mah cu proline sernak a karhmi kilvennak caah a si kho men (Figure S5). Hi thlennak hna he aa dang in, PN-specific Mfn2cKO vok hna chungin cerebellar astrocytes hna i proteomic hlathlainak nih hi lam hna (antiperoxidases vialte telh chih in) hna cu langhternak ah a biapi tukmi thlennak an ngei lo ti a langhter, cucaah Hi metabolic thlennak cu a rawkmi PN (Fig. S6, D in G tiang) caah a thimmi a si ti a langhter.
A tawinak in chim ahcun, hi hlathlainak hna nih PNs chung i a hleiin metabolic lamthluan hna i caan tawite chung riantuannak aa dang tukmi an langhter. A ttha lomi neuronal mitochondrial riantuannak nih a hlan deuh ah atherosclerosis le 1C remhnak (Figure 3E le Figure S5C) a chuahter khawh ko nain, cun I le IV complexes langhternak ah chimchung khawhmi thlennak hmanh a chuahter khawh, serine de novo sernak ah thlennak cu a hnu lei lawng ah a lang. OXPHOS riantuan ningcang lo (Hmanthlak 3E le Hmanthlak S5C). Hi hmuhmi nih hin lungretheihnak nih a chuahtermi mitochondrial (1C cycle) le cytoplasmic (serine biosynthesis) nih TCA cycle ah atherosclerosis a karhmi he aa pehtlaimi a zulmi thil kalning kha a fianter.
Zarh 8 a simi OXPHOS-a tlawmmi PNs nih a sangmi lungthawhnak cawlcanghnak kha an kilven khawh i mitochondrial riantuan ningcang lo caah a biapi mi metabolic pehtlaihnak an tuah khawh. Hi hmuhmi nih hin a tu lio caan hmanh ah, hi cells pawl nih hin thluak rawhnak tlaiter asiloah kham khawhnak ding caah thlopnak bawmhnak an hmuh ve men lai timi thil sining hngalh a nuammi a chuahter. Khua hnut. Hi thil cang khomi hi aa hngat lomi thlennak pahnih in kan tawlrel. A hmasa bik lam ah, Cre-dependent adeno-associated virus (AAV) vector kan suai i cucaah MFN2 cu OXPHOS-a tlawmmi PNs ah in vivo ah thim tein langhter khawh a si (Figure S7A). MFN2 a codemi AAV le a ceumi thawngthanhtu gene mCherry (Mfn2-AAV) cu in vitro ah a hrampi neuron cinnak ah fehter an si, cu nih cun MFN2 cu Cre-a hngatmi lam in a langhter i mitochondrial morphology a khamh, cucaah Mfn2cKO neurons ah neuromutation a kham ( Figure S7, D le B, B). A hnu ah, zarh 8 a simi Mfn2-AAV kha Mfn2cKO le control vok hna i an cerebellar cortex ah stereotactically in kuat khawhnak ding caah in vivo hneksaknak kan tuah, cun zarh 12 a simi vok pawl kha kan hlathlai hna (Figure 4A). Thlopmi Mfn2cKO vok cu an thi (Figure 1, A le B) (16). In vivo ah virus thlennak nih cerebellar circle cheukhat ah PN thimnak langhternak a chuahter (Figure S7, G le H). mCherry (Ctrl-AAV) lawng a langhtermi control AAV thunnak nih Mfn2cKO saram hna ah thluak rawhnak tlukruannak ah zeihmanh thlennak a chuahpi lo. Cu he aa dang in, Mfn2-AAV in thlenmi Mfn2cKOs hlathlainak nih PN cell tthennak nih a biapi tukmi huhphenhnak a langhter (Figure 4, B le C). A bik in, neuron density cu control saram hna he thleidan khawh a si lo (Figure 4, B le C, le Figure S7, H le I). MFN1 langhternak nain MFN2 langhternak cu neuronal thihnak khamhnak ah aa tluk tein a ttha (Figure 4C le Figure S7, C le F), cucu ectopic MFN1 langhternak nih MFN2 a um lonak kha ttha tein a bawmh khawh ti a langhter. PN level pakhat lawng ah hlathlainak tuah chapnak nih Mfn2-AAV nih mitochondria ultrastructure kha a tam deuh in a khamh, mtDNA level kha a tthatter, cun anti-angiogenesis marker PCx ​​a sangmi langhternak kha a thlen (Figure 4, C in E ). A din lio ah khamhmi Mfn2cKO vok pawl mit in zohfelnak nih an dirhmun le an cawlcanghnak hmelchunhnak (S1 in S3 tiang cawlcanghnak) a ttha deuh ti a langhter. A donghnak ah, hi hneksaknak hna nih hin OXPHOS a tlawm tukmi PNs ah MFN2 luhter tthannak nih mtDNA hmannak a thlen khawh i atherosclerosis a chuahter khawh, cucaah axon rawhnak le neuronal thihnak kha in vivo ah a kham khawh ti kha a langhter.
(A) A langhtermi metabolic lam a cawlcangh tikah MFN2 a codemi AAV thunnak caah hneksaknak caan suaimi a langhtermi timhtuahnak. (B) Zarh 12 a simi thluak tthencheunak hna cu zarh 8 ah Mfn2cKO vok he thlenmi le anti-Calbindin antibody in min bunhmi aiawhtu confocal hmanthlak hna. Orhlei: Axon hri hna i an i tluknak. Axon zoom tahfung cu 450 le 75 μm a si. (C) Kehlei: AAV thlennak loop (AAV+) chung i Purkinje cell tlawmtam tahnak (thleidannak lam khatlei hlathlainak; n = 3 vok). Orhlei: zarh 12 ah thlenmi PN ah mtDNA focus hlathlainak (aa pehtlaimi t-test; n = vok pathum chungin cell 6). * P <0.05; ** P <0.01. (D) A langhtermi virus vectors he thlenmi Mfn2cKO thluak tthencheunak PNs aiawhtu thlennak electron micrographs. A senmi hmaikhuh nih dendrites nih an lakmi hmun kha a langhter, cun a senmi dot square nih orhlei ah pekmi zoom kha a langhter; n nih nucleus a aiawh. Tahnak hri, 1μm. (E) nih zarh 12 ah thlenmi PN ah PCx rong thlennak tahchunhnak a langhter. Tahnak hri, 20μm. OE, a hlei in langhternak; FC, thlennak.
A donghnak ah, OXPHOS riantuan lonak a tongmi PNs ah peroxidase-induced cell nunnak a biapitnak kha kan hlathlai. Mouse PCx mRNA (AAV-shPCx) kha a bik in aa tinhmi AAV-shRNA (short hairpin RNA) a langhtermi mCherry kan ser, cun Mfn2cKO vok hna i an thluak chung ah virus asiloah a scrambled control (AAV-scr) kha kan thun. PCx langhternak a karh (Figure 3C) le PN cell layer a rawk rih lo (Figure 1A) chung ah ttha tein PCx knockdown hmuh khawhnak ding caah kum linak zarh (Figure 5A) ah thunnak cu tuah a si. PCx (Figure S8A) hrawhnak nih PN thihnak a rangter ngaingai ti kha hngalh awk tlak a si, cucu zawtnak hrik a khammi pakhat lawng a si (Figure 5, B le C). PCx up-regulation nih a chuahtermi metabolic effects hna an kalning hngalhthiam khawhnak ding caah, PCx knockdown hnu ah PNs redox dirhmun le AAV-mediated optical biosensor Grx1-roGFP2 cu caankhatte ah an langhter (Figure S8, B in D) glutathione Redox peptide thlennak (383) tuaktannak caah kan hlathlai. Cun, FLIM dirhmun kan hngalh hnu ah cytoplasmic redox dirhmun ah thlennak a um khomi hmuh khawhnak ding caah zarh 7 a simi Mfn2cKO asiloah control littermates hna i an thluak tlawmpal ah two-photon fluorescence lifetime imaging microscopy (FLIM) kan tuah (Figure S8, E in G). Hlathlainak nih PCx langhternak a ngei lomi Mfn2cKO PNs pakhat lawng i oxidation dirhmun ah a karh ngaingai ti a langhter, cucu control neurons asiloah Mfn2cKO PNs nih scrambled shRNA lawng a langhtermi he aa dang (Figure 5, D le E). PCx langhternak kha a niamter tikah, Mfn2cKO PNs nih a sang tukmi oxidized dirhmun a langhtermi zatuak cu a let thum nak tam in a karh (Figure 5E), PCx a sangmi nih a rawkralmi neurons redox thilti khawhnak kha a kilven ti a langhter.
(A) A langhtermi metabolic lam a cawlcangh tikah shPCx a codemi AAV thunnak caah hneksaknak caan suaimi a langhtermi timhtuahnak. (B) Zarh 4 ah anti-calcineurin antibody in thlenmi le min bunhmi Mfn2cKO vok hna i zarh 8 a simi thluak tthencheu hna i aiawhtu confocal hmanthlak hna. Tahnak hri, 450μm. (C) AAV-transduced loops ah Purkinje cell density tahnak (variance pakhat lei hlathlainak; n = 3 in 4 mice). Data cu a tlangpi±SEM in langhter a si; ***P<0.001. (D) Aiawhtu FLIM hmanthlak nih a langhtermi hneksaknak thil sining tangah zarh 7 a simi PN nih a langhtermi glutathione redox sensor Grx1-roGFP2 nunchung caan sauhnak kha a langhter. LUT (zoh khawhnak cabuai) zat: nunnak caan karlak (picoseconds in). Tahnak hri, 25μm. (E) Histogram nih (D) in Grx1-roGFP2 nunchung man hna i phawtzamhnak a langhter (n=158 in 368 cells tiang vok pahnih ah thil sining pakhat cio tangah). Histogram pakhat cio cunglei pie chart nih: CTRL-AAV-scr ah a tlangpi nunchung man 1 SD a lonhmi, a sau deuhmi (sen, oxidized) asiloah a tawi deuhmi (sen, a zormi) nunchung man a ngeimi cells zat kha a langhter. (F) Ruahnak cheuhnak model nih hin neuronal PCx tthanchonak nih a huhphenh khawhnak kha a langhter.
A dihlak in, hika ah kan pekmi data nih a langhter mi cu MFN2 langhternak nih a sangmi PN kha a fakmi OXPHOS tlamtlin lonak, a fakmi mtDNA tlawmternak, le a ttha lomi ista-bantuk morphology he tling tein a khamh khawh, cucaah a sangmi zawtnak hmanh ah pehzulh tein tthanchonak a pek khawh. Neurodegeneration nih cell thih hlan dirhmun kha a thlen khawhmi tehte a pek. Hi tluk in metabolic thlen khawhnak cu neurons nih atherosclerosis (TCA cycle thlennak) a chuahter khawhnak nih a langhter chin, mah nih hin OXPHOS a ngei lomi PNs ah PCx langhternak a kham i cell thihnak a karhter, cucaah kilvennak rian a ttuan (Figure 5F).
Hi hlathlainak ah, OXPHOS riantuan lonak ah PNs nih an lehrulhnak cu metabolic program nih a cawlcangtermi aa dangmi riantuannak lam hmangin TCA cycle atherosclerosis ah zaangfah tein aa fonh ti kha tehte kan pek. Proteomic hlathlainak cu aa pehtlaimi lam tampi in kan fehter i mitochondrial riantuan ningcang lo fakpi nih a kan doh tikah, neurons nih a hlan ah theih lomi metabolic elasticity phun an ngei ti kha kan langhter. Kan khuaruah a har, rewiring tuahnak dihlak nih hin neurodegeneration he aa pehtlaimi a donghnak metabolic dirhmun kha zaangfah tein le thlen khawh lomi in a langhter hrimhrim lo, asinain kan data nih a langhter mi cu cell thih hlan dirhmun hmanh ah zohkhenhnak neuron a si kho men. Hi hmuhmi nih hin neurons nih hin pum chung ah metabolic plasticity tlawmpal an ngei ti a langhter. Hi thil nih hin a hnu ah MFN2 luhter tthannak nih a biapi mi metabolic hmelchunhnak pawl langhternak kha a thlen khawh i PN rawhnak a kham khawh ti kha a langhter. Cu he aa ralchanh in, thihri zawtnak a kham i thluak a rangter. nu le pa sualnak a tuahmi.
Kan hlathlainak ah a hngalh a nuammi hmuhmi pakhat cu OXPHOS a ngei lomi PNs nih TCA cycle metabolism kha a hleiin arteriosclerosis a chuahtertu enzyme pawl kha a tthanchoter khawh hna. Metabolic rearrangement cu cancer cells ah a um tawnmi thil pakhat a si, cheukhat nih cun TCA cycle karlak thilri pawl bawmhchanhnak caah glutamine kha an i bochan i a zortertu tlukruannak an chuahter, cu nih cun thawchuahnak lam kha a hruai i lipid le nucleotide biosynthesis precursors chuahternak kha a kilven (39 , 40). Nai ah tuahmi hlathlainak nih a langhter ning ahcun OXPHOS riantuan ningcang lo a tongmi pawngkam vun hna ah, glutamine/glutamate thlennak pehtlaihnak zong a langmi thil pakhat a si (5, 41), cuka ahcun TCA cycle chung i glutamine luh khawhnak lam cu OXPHOS hliamhma a fah tuk caah (41) cungah aa hngat. ). Sihmanhsehlaw, pum chung i neuronal metabolic plasticity aa lawhnak le zawtnak kong he aa pehtlaimi a si khomi kongah fiang tein tehte a um lo. Nai ah tuahmi in vitro hlathlainak ah, a hrampi cortical neurons nih neurotransmission caah glutamate pools kha an cawlcangter khawh ti an hmuh, cu nih cun metabolic stress dirhmun tangah oxidative metabolism le atherosclerosis a thanchoter (42). TCA cycle enzyme succinate dehydrogenase sii lei khamnak tangah, pyruvate carboxylation nih cinmi cerebellar granule neurons ah oxaloacetate sernak kha a kilven tiah zumh a si ti kha hngalh awk tlak a si (34). Sihmanhsehlaw, hi thilri hna i thluak tissue (atherosclerosis cu astrocytes lawng ah aa hngat tiah zumhmi) he an i pehtlaihnak cu a biapi mi pumsa lei sullam a ngei rih (43). Hi kong ah, kan data nih a langhter mi cu pum chung i OXPHOS nih a hrawhmi PNs cu BCAA rawhnak le pyruvate carboxylation ah thlen khawh a si, cu hna cu TCA pool karlak thilri pawl chapnak hrampi pahnih an si. BCAA catabolism nih neuronal thazaang thlennak ah a chuahpi tiah ruahmi bawmhnak cu ruahnak cheuh a si ko nain, thluak thlennak caah glutamate le GABA riantuannak lengah (44), hi thilri hna hi in vivo ah tehte a um rih lo. Cucaah, rian a ttuan kho lomi PNs nih atherosclerosis karhternak in assimilation process nih a hruaimi TCA karlak thilri hmannak kha amah tein a tlawmter khawh tiah ruah a fawi. A bik in, PCx upregulation cu aspartic acid caah a karhmi herhnak kilvennak caah a herh men lai, cucu mitochondrial riantuan lonak a ngeimi a karhmi cells ah hmuh khawh a si (45). Sihmanhsehlaw, kan metabolomics hlathlainak nih Mfn2cKO PNs (Figure S6A) ah aspartic acid a fekmi dirhmun ah thlennak nganpi a langhter lo, cucu a karh cuahmahmi cells le post-mitotic neurons karlak ah aspartic acid metabolic hmannak aa dangmi a langhter tiah zumh a si. In vivo ah rian a ttuan kho lomi neurons ah PCx upregulation a dikthliar in a kalning cu hngalh khawh a si rih lo nain, hi a caan hlan ah lehnak nih hin neurons redox dirhmun kilvennak ah a biapi tukmi rian a ttuan ti kha kan langhter, cucu cerebellar slices cung i FLIM hneksaknak ah langhter a si. A bik in, PNs nih PCx a tthanchoter lonak ding caah khamnak nih hin oxidized dirhmun a chuahter khawh i cell thihnak a rangter khawh. BCAA rawhnak le pyruvate carboxylation riantuannak cu mitochondrial riantuan ningcang lo (7) i a pawngkam vun sining langhternak lam a si lo. Cucaah, annih cu OXPHOS-tlawmmi neurons hna i biapi bikmi sining an si ko rua tiah a lang, cu sining pakhat lawng a si lo hmanh ah, cu cu thluak rawhnak caah a biapi. .
Cerebellar zawtnak cu aa khat lomi thluak lei zawtnak phunkhat a si i a tlangpi in ataxia in a lang i PNs a hrawh tawn (46). Hi neuron milu hi mitochondrial riantuan ningcang lo ah a hlei in a fawimi an si, zeicahtiah vok ah an thimmi rawhnak nih minung spinocerebellar ataxia a langhtermi motor zawtnak hmelchunhnak tampi chuahter khawhnak a za (16, 47, 48). Report ning in, aa thlengmi gene a ngeimi transgenic mouse model cu minung spinocerebellar ataxia he aa pehtlai i mitochondrial riantuan ningcang lo a ngei (49, 50), PNPH ah OXPHOS tlamtlin lonak nih a chuahpimi thil kong hlathlainak a biapitnak kha a langhter. Cucaah, hi aa dangmi neuron milu kha ttha tein thleidan le hlathlai awkah aa tlak khun. Sihmanhsehlaw, PNs cu hneknak ah an tuar khawh tuk i cerebellar cell dihlak ah tlawmte lawng an si caah, omics-based hlathlainak tampi caah, cell dihlak in thim in tthen cu a har rih. Cell phun dang (a bik in upa tissue) hna i thurhnawmhnak a um lonak ding cu a si kho lo nain, FACS he ttha tein tthennak step kan fonh i a tanglei proteomics hlathlainak caah a ttha mi neurons zat kan hmuh, cun a um cangmi cerebel data he tahchunh tikah protein coverage (protein 3000 hrawng) kan ngei (155). Cell dihlak nun khawhnak kilvennak thawngin, hika ah kan pekmi lam nih hin mitochondria chung i thlennak lamthluan thlennak lawng si loin a cytoplasmic counterparts thlennak zong zoh khawhnak a kan pek, cucu cell phun he aa pehtlaimi mitochondrial membrane label enrichment hmannak kha a bawmh. Kan langhtermi lam cu Purkinje cells hlathlainak he aa pehtlaimi lawng si loin, zawtnak a ngeimi thluak chung i metabolic thlennak tawlrelnak caah cell phun paoh ah fawi tein hman khawh a si, cu chungah mitochondrial riantuan ningcang lonak model dang zong aa tel.
A donghnak ah, hi metabolic remhthannak kalning chungah hin cellular stress hmelchunhnak biapi pawl kha tling tein a thlen khawhmi le neuronal rawhnak a kham khomi thlopnak window pakhat kan hmuh. Cucaah, hika ah langhtermi rewiring riantuannak sullam hngalhthiamnak nih mitochondrial riantuan ningcang lo lio ah neuronal nun khawhnak kilvennak caah a si khomi thlopnak kongah a hrampi hmuhnak a pek khawh. Hmailei ah thluak cell phun dang hna i thazaang thlennak thlennak hna kha thleidan khawhnak ding caah hi phunglam hi thluak lei zawtnak dang hna ah hman khawhnak tling tein langhter khawhnak ding caah hlathlainak tuah a herh.
MitoPark vok cu a hlan ah an rak langhter cang (31). C57BL/6N vok pawl cu loxP he aa pehtlaimi Mfn2 genes hna cu a hlan ah an rak langhter cang (18) i L7-Cre vok pawl he an rak cawh cang hna (23). A chuakmi heterozygous pahnih cu homozygous Mfn2loxP/Mfn2loxP vok he an cawh hna i Mfn2 (Mfn2loxP/Mfn2loxP; L7-cre) caah Purkinje-specific gene knockouts an ser. Nu le pa sualnak tuahnak ah, Gt (ROSA26) SorStop-mito-YFP allele (stop-mtYFP) cu a dang cross (20) hmangin an rak luhpi. Saram tuahsernak vialte cu European, ram pumpi le bupi lamhruainak ningin tuah an si i Umwelt le Verbraucherschutz, North Rhine-Westphalia, Germany i LandesamtfürNatur nih an cohlan. Saram riantuannak zong nih European Federation of Laboratory Animal Sciences Associations lamhruainak kha a zulh.
Nau a pawi liomi nu a hngawng a tlongmi kha hngilhternak sii an pek hnu ah, vok hrinmi cu an thleidan (E13). Cortex cu Hanks' Balanced Salt Solution (HBSS) ah 10 mM Hepes he an cawh i Dulbecco's Modified Eagle's Medium ah papain (20 U/ml) le cysteine ​​(1μg/ml) aa telmi ah an pek. DMEM ah tissue kha chiah) law enzymatic rawl ei in tthen. Ml) cu 37°C ah minutes 20 chung, cun DMEM ah 10% nau caw thi he cawhmi ah seh in cawh. Cell pawl cu hmanthlak hlathlainak caah 6 cm cinnak kuang ah 2×106 asiloah 0.5×105 cells/cm2 tluk in polylysine in thuhmi thlalang khuhnak cung ah an cin hna. Suimilam 4 hnu ah, 1% B27 supplement le 0.5 mM GlutaMax aa telmi Neurobasal serum-free medium in an thlen. Cu hnu ah neurons pawl cu hneksaknak chung vialte 37°C le 5% CO2 ah an chiah hna, cun zarhkhat ah voikhat rawl an pek hna. In vitro ah fonh tthan khawhnak ding caah, a tanglei AAV9 virus vector 3μl (24-well culture dish) asiloah 0.5μl (24-well plate) cu in vitro ah ni hnihnak ah neurons thlopnak caah hman a si: AAV9.CMV.PI.eGFP. WPRE.bGH (Addgene, catalog number 105530-AAV9) le AAV9.CMV.HI.eGFP-Cre.WPRE.SV40 (Addgene, catalog number 105545-AAV9).
Mouse Mfn1 le Mfn2 complementary DNA (Addgene plasmid #23212 le #23213 in hmuhmi) cu C-terminus ah V5 sequence (GKPIPNPLLGLDST) in hmelchunhnak an tuah, cun T2A sequence hmangin mCherry he an i fonh. Grx1-roGFP2 cu Heidelberg TP Dick DFKZ (Deutsches Krebsforschungszentrum) nih a kan pekmi laksawng a si. tdTomato cassette kha a hlanlio cloning tuahnak lam hmangin thlennak thawngin, cassette cu pAAV-CAG-FLEX-tdTomato backbone (Addgene reference number 28306) ah pAAV-CAG-FLEX-mCherry-T2A-MFN2-V5, pAAV-CAG-CA chuah khawhnak ding caah subclone tuah a si FLEX-mCherry-T2A-MFN1-V5 le pAAV-CAG-FLEX-Grx-roGFP2 vectors. Cu bantuk strategy cu control vector pAAV-CAG-FLEX-mCherry sernak caah hman a si. AAV-shPCx sermi ser khawhnak ding caah, plasmid AAV vector (VectorBuilder, pAAV [shRNA] -CMV-mCherry-U6-mPcx- [shRNA#1]) a herh, cu nih cun shRNA targeting mouse PCx (5′CTTTCGCTAGCTAGGCTAAGGCTAAGGCTAGACTTAGACTAGACTAGA) a codemi DNA sequence aa tel 3′) U6 promoter uknak tangah, mCherry cu CMV promoter uknak tangah hman a si. Bawmtu AAV vector sernak cu sertu nih a chimmi ningin (Cell Biolabs) tuah a si. A tawinak in, mCherry-T2A-MFN2-V5 (pAAV-CAG-FLEX-mCherry-T2A-MFN2-V5), mCherry-T2A-MFN1-V5 (pAAV-CAG-FLEX-mCherry) aa putmi thlennak plasmid kha hmang. mCherry (pAAV-CAG-FLEX-mCherry) asiloah Grx-roGFP2 (pAAV-CAG-FLEX-Grx-roGFP2) code gene, cun code AAV1 capsid protein le a dang protein code Packaging plasmid plasmid, calcium phosphate phunglam hmangin. A thiang lomi virus supernatant cu a ro cangmi tikhor/ethanol ti kholhnak ah a lum-a linmi thlennak in hmuh a si i phosphate buffered saline (PBS) ah lysed cells in hmuh a si. AAV vector cu a pehzulh lomi iodixanol gradient ultracentrifugation (32,000 rpm le 4°C ah suimilam 24) in thianh a si i Amicon ultra-15 centrifugal filter hmangin a tlawmter. 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 hna i an genome titer cu a hlan ah langhtermi bantuk in a si (PCR) in quantitative tahnak -MFN1-V5 (1.9×1013 GC/ml) le AAV1-CAG-FLEX-Grx-roGFP2 (8.9×1012 GC/ml).
Primary neurons cu tikhal bantuk a lummi 1x PBS ah an hlonh hna, an tthen hna, cun 0.5% Triton X-100 / 0.5% sodium deoxycholate/PBS lysis buffer ah phosphatase le protease inhibitor (Roche) aa telmi ah an i fonh hna. Protein zat tuaktannak cu bicinchoninic acid hneksaknak (Thermo Fisher Scientific) hmangin tuah a si. Protein pawl cu SDS-polyacrylamide gel electrophoresis in an tthen hna, cun polyvinylidene fluoride membrane (GE Healthcare) ah an thlet hna. A hleiin a si lomi hmun pawl kha phih law TBST (Tris-buffered saline with Tween) ah 5% cawhnuk ah a hrampi antibody (a tawinak caah Table S1 zoh) he chiah, ṭawlnak step le TBST chung i a pahnihnak antibody kha chiah. +4°C ah zankhuadei primary antibody he chiah. Na tawl hnu ah, a pahnihnak antibody kha innchung lumnak ah suimilam 2 chung thuh. Cu hnu ah, cu blot cu anti-β-actin antibody he cawh in, cu bantuk loading cu fehter a si. Chemiluminescence ah thlennak le chemiluminescence ṭhanchoternak in hmuh khawhnak (GE Healthcare).
A hlan ah thlalang khuhnak cung ah an rak cinmi neurons pawl cu 4% paraformaldehyde (PFA)/PBS in a caan khiahmi ah khaan lumnak ah minutes 10 chung an rak chiah hna. A khuhmi hna cu 0.1% Triton X-100/PBS in innchung lumnak ah minutes 5 chung an hnim hmasa hna, cun khamnak buffer [3% bovine serum albumin (BSA)/PBS] ah an hnim hna. Ni hnihnak ah, coverslips cu blocking buffer in an tawl hna i aa tlakmi fluorophore-conjugated secondary antibody he innchung lumnak ah suimilam 2 chung an chiah hna; a donghnak ah, sample pawl cu PBS ah 4′,6-diamidino-2 -Phenylindole (DAPI) he ttha tein an tawl hna i cu hnu ah Aqua-Poly/Mount he microscope slide ah an chiah hna.
Zu (pa le nu) cu ketamine (130 mg/kg) le xylazine (10 mg/kg) in an paw chung ah an thun hna i carprofen analgesic (5 mg/kg) in an vun tang ah an pek hna, cun stereotactic thilri (Kopf) ah an chiah hna i a lumter. Luruh kha langhter law ha thlopnak seh hmangin mis ruh he aa tlakmi cerebellar cortex tthen kha tlawmter (lambda in: tail 1.8, lateral 1, lobules IV le V he aa tlakmi). A tanglei thi kalnak lam hna kha hrawh lo awkah luruh ah ralring tein khur hmete ser awkah a tlikmi syringe hri hmang. Cun a tlawmmi hnuhmi thlalang capillary cu micro-hole chungah zaangfah tein an khumh (dura mater ventral lei ah -1.3 in -1 tiang), cun 200 in 300 nl AAV cu micro-injector (Narishige) chungah kut in tuahmi syringe (Narishige) hmangin minutes 20 in 20 chung caan tlawmpal chung ah voi zeimawzat an thun. Thil thlet hnu ah, thihri kha minutes 10 chung chiah tthan law virus cu a karh dih lai. Thi kalnak lam pawl an chuah hnu ah, hma a fahtermi a zorter i saram cu a dam khawh nakhnga vun cu ralring tein an tah. Saram pawl cu operation an tuah hnu ni zeimawzat chung fahnak zorternak sii (caspofen) in an thlop hna, cu caan chung ah an pumsa dirhmun kha ralring tein an zohfel hna i cu hnuah an chimmi caan ah an thah hna. Tuahsernak vialte cu European, ram pumpi le bupi lamhruainak ningin tuah a si i Umwelt le Verbraucherschutz, North Rhine-Westphalia, Germany i LandesamtfürNatur nih an cohlan.
Saram pawl cu ketamine (100 mg/kg) le xylazine (10 mg/kg) in an hngilhter hna, cun lungthin cu 0.1 M PBS in an thlet hmasa, cun PBS chung i 4% PFA in an thlet. Tissue cu an thleh i 4% PFA/PBS ah 4°C ah zankhuadei an chiah. PBS chung i a fekmi thluak chungin sagittal sections (50 μm thick) timhtuahnak caah a hninhmi namte (Leica Microsystems GmbH, Vienna, Austria) kha hman a si. A dang tein langhter a si lo ahcun, a cunglei (13) ah langhtermi bantuk in a zalongmi tthen pawl kha khaan lumternak le cawhnak ah tuah a si. A tawinak in, pakhatnak ah, hmuhmi tthen pawl cu 0.5% Triton X-100/PBS in innchung lumnak ah minutes 15 chung an luhter hna; epitopes cheukhat (Pcx le Shmt2) caah, hi step si loin 80°C (PH 9) ah tris-EDTA buffer ah minutes 25 chung lumter. A hnu ah, tthencheunak hna cu a hrampi antibody (Table S1 zoh) he khamnak buffer (3% BSA/PBS) ah 4°C ah zankhuadei cawh in an chiah hna. A thaizing ah, tthen pawl cu khamnak buffer in an tawl hna i aa tlakmi fluorophore-conjugated secondary antibody he innchung lumnak ah suimilam 2 chung an chiah hna; a donghnak ah, tthen pawl cu PBS ah ttha tein an tawl hna, DAPI in an thlen hna, cun AquaPolymount in an remh hna On a microscope slide.
Laser scanning confocal microscope (TCS SP8-X asiloah TCS Digital Light Sheet, Leica Microsystems) cu ceunak rang laser le 405 diode ultraviolet laser he aa pehtlaimi kha sample hmanthlaknak caah hman a si. Fluorophore kha a thawhter i Hybrid Detector (HyDs) in signal kha laknak thawngin, LAS-X software cu Nyquist sampling he aa tlakmi hmanthlak pawl kha a zulmi lam in laknak caah hman a si: quantitative a si lomi panel caah, a tthawng tukmi signals (tahchunhnak ah, somatic cells le dendrites ah) mty UFPD in Hybrid Detector zat ah Hybrid detect ah hmuh khawh a si mode). Hnulei tlawmternak caah 0.3 in 6 ns tiang kharnak hman a si.
A caan hmaan tein thleidanmi cells hmanthlaknak. Neurobasal-A medium ah 1% B27 supplement le 0.5 mM GlutaMax aa telmi ah thleidan hnu ah, cells pawl cu poly-l-lysine-coated glass slides (μ-Slide8 Well, Ibidi, catalog number 80826) ah an cin colh hna, cun 37°C ah 37°C le 12% chung chiah in cell kha suimilam pakhat chung an chiah hna um. Caan hmaan tein hmanthlaknak cu Leica SP8 laser scanning confocal microscope ah tuah a si i, cu ah cun white laser, HyD, 63×[1.4 numerical aperture (NA)] oil objective lens le heating stage he an i tlum.
Zu cu carbon dioxide in rang tein an hngilhter i a lu an tan, a thluak cu a luruh chungin rang tein an chuah, cun 200μm a thukmi (13C min bunhnak hneksaknak caah) asiloah 275μm a thukmi (photon hneksaknak pahnih caah) a tanglei thilri hna he a khatmi sagittal section ah an tan. Walldorf, Germany) cu a tanglei thilri hna in a khat: 125 mM tikhor-a lummi, carbon-a khatmi (95% O2 le 5% CO2) tlawmte Ca2 + artificial cerebrospinal fluid (ACSF) NaCl, 2.5 mM KCl, 1.25 mM sodium phosphate buffer, NaCl 2.5 mM, gluse 25 mM 0.5 mM CaCl2 le 3.5 mM MgCl2 (osmotic pressure of 310 in 330 mmol). Hmuhmi thluak tthen pawl kha a sang deuhmi 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 le 20 mM Ca2) aa telmi pre-incubation khaan ah thlen. MgCl2) Medium) pH 7.4 le 310 in 320 mmol).
Hmanthlaknak tuah lio ah, tthencheu pawl cu a hleiin hmanthlaknak khaan ah an ṭhial hna, cun hneksaknak cu 32° in 33°C tiang aa thleng lomi lumnak ah pehzulh tein ACSF thletnak tangah tuah a si. Leica 25x objective lens (NA 0.95, ti), Ti: Sapphire laser (Chameleon Vision II, Coherent) he aa pehtlaimi multiphoton laser scanning microscope (TCS SP8 MP-OPO, Leica Microsystems) cu slice hmanthlaknak caah hman a si. FLIM module (PicoHarp300, PicoQuant).
Grx1-roGFP2 i FLIM. PNs cytoplasmic redox dirhmun thlennak cu sagittal thluak tthen ah two-photon FLIM in tah a si, cuka ahcun Grx1-roGFP2 biosensor nih PNs a target hna. PN tthen ah, hmuh khawhnak hmun cu slice cunglei tang ah 50 in 80 μm hrawng ah thim a si i, a ttha mi PN (cucu, dendrites pawng ah beaded structure asiloah neuronal morphological thlennak a um lo) le shRNA PCx asiloah a control sequence a ukmi AAV pahnih a um khawhnak hnga caah thim a si. 2x digital zoom [excitation wavelength: 890 nm; 512 nm 512 pixels]. Hmuh khawhnak: chunglei HyD, fluorescein isothiocyanate (FITC) filter phu] le minutes 2 in 3 chung ah hmanthlak tlukruannak cu curve fitting caah a za tawk in photons (a dihlak ah photons 1000) lak khawhnak ding caah hman a si. Grx1-roGFP2 hlathlainak i a tuar khawhnak le FLIM dirhmun hngalhternak cu perfusion ACSF ah exogenous 10 mM H2O2 chap tikah roGFP2 nunchung man kha zohfelnak in tuah a si (oxidation tamter awkah, nunchung sauternak a chuahter), cun 2 mM dithiothreit chapnak nih a tlawmter, a zorter nunchung caan a zor) (Figure S8, D in G tiang). FLIMfit 5.1.1 software hmangin hmuhmi thil sining hna kha hlathlai, hmanthlak dihlak i a pakhatnak exponential decay curve kha tahmi IRF (instrument response function) he i tlakter, cun χ2 cu 1 hrawng a si. PN pakhat nunchung tuak awkah, nerve pum pawngkam i hmaikhuh kha kut in suai a si, hmaikhuh pakhat cio caah nunnak caan tahnak kha hman a si.
Mitochondrial thilti khawhnak hlathlainak. A fakmi tthen cu 100 nM TMRM he direct in perfused ACSF ah minutes 30 chung an cawh hnu ah, PNs mitochondrial potential thlennak cu two-photon microscope in tah a si. TMRM hmanthlaknak cu 920 nm ah probe kha lungthawhter in le signal laknak caah chunglei HyD (tetramethylrhodamine isothiocyanate: 585/40 nm) hmangin tuah a si; mtYFP hmanthlaknak caah aa khatmi lungthawhnak wavelength hmannak in asinain a dang chunglei HyD (FITC :525/50) hmannak in. ImageJ's Image Calculator plug-in kha cell pakhat level ah mitochondrial thilti khawhnak tuaktan awkah hmang. A tawinak in, plug-in equation: signal = min (mtYFP, TMRM) cu aa pehtlaimi channel i single-stack confocal hmanthlak ah Purkinje Somali ah TMRM signal a langhtermi mitochondrial region hngalh khawhnak caah hman a si. Cun a chuakmi hmaikhuh chung i pixel area cu zat in tuak a si, cun mtYFP channel i aa pehtlaimi threshold single-stack hmanthlak ah mitochondrial thilti khawhnak a langhtermi mitochondrial tthencheunak hmuh khawhnak ding caah a tlarimi ah chiah a si.
Hmanthlak cu Huygens Pro (Scientific Volume Imaging) software hmangin a tthencheu. Tile hmanthlak scanmi hna caah, LAS-X software nih a pekmi amah tein thlennak algorithm hmangin tile pakhat lawng i fonhnak tuah a si. Hmanthlak tahfung na thlen hnu ah, ImageJ le Adobe Photoshop hmangin hmanthlak kha ttha tein thlen khawh a si i a ceunak le aa dannak kha aa khat tein remh khawh a si. Hmanthlak timhtuahnak caah Adobe Illustrator hmang.
mtDNA zohfelnak. mtDNA hma zat cu confocal microscope hmangin DNA dohnak antibodies he min bunhmi thluak tthencheunak ah tah a si. Target hmun pakhat cio cu cell pum le cell pakhat cio i a hrampi caah ser a si, cun aa pehtlaimi hmun cu Multi Measure plug-in (ImageJ software) hmangin tuak a si. Cytoplasmic area hmuh khawhnak ding caah cell pum area in nuclear area kha tlawmter. A donghnak ah, Analyze Particles plug-in (ImageJ software) cu threshold hmanthlak cung i mtDNA a langhtermi cytoplasmic DNA points kha amah tein zat tuaktannak caah hman a si, cun hmuhmi phichuak cu CTRL vok PN average he aa tlakmi ah ser a si. A phichuak cu cell pakhat ah nucleosides zat tlawmpal in langhter a si.
Protein langhternak hlathlainak. ImageJ's Image Calculator plug-in kha PN ah cell pakhat lawng ah protein langhternak kha tuaktan awkah hmang. A tawinak in, aa pehtlaimi channel i a tthen pakhat lawng a ummi hmanthlak ah, equation hmangin: signal = min (mtYFP, antibody), Purkina chung i antibody pakhatkhat cungah immunoreactivity a langhtermi mitochondrial region kha hmuh khawh a si. Cun a chuakmi hmaikhuh chung i pixel area kha zat in tuak a si, cun langhtermi protein i mitochondrial tthencheu hmuh khawhnak dingah mtYFP channel i aa pehtlaimi threshold single-stack hmanthlak ah a tlarimi ah chiah a si.
Purkinje cell tlawmtam hlathlainak. ImageJ i Cell Counter plug-in cu Purkinje density tuaktannak caah hman a si i, relmi cells nih an lakmi cerebellar ring saunak in relmi Purkinje cells zat kha tthen a si.
Sample timhtuahnak le laknak. Control phu le Mfn2cKO vok hna thluak cu 0.1 M phosphate buffer (PB) ah 2% PFA/2.5% glutaraldehyde ah an chiah hna, cun ciliates (Leica Mikrosysteme GmbH, Vienna, Austria) (Thickness 50 in 60 μm) hmangin coronal sections an timhtuah hna. tetraoxide le 1.5% potassium ferrocyanide kha innchung lumnak ah suimilam 1 chung chiah. Cu tthen pawl cu ti thiang in voithum an tawl hna, cun 1% uranyl acetate aa telmi 70% ethanol in minutes 20 chung an thuh hna. Cu hnu ah tthencheunak hna cu zu tlawmpal ah an thlet hna i Durcupan ACM (Araldite casting resin M) epoxy resin (Electron Microscopy Sciences, catalog number 14040) ah silicon-coated glass slides karlak ah an khumh hna, cun a donghnak ah 60°C ah suimilam 48 chung oven chungah Polymerize an tuah hna. Cerebellar cortex area cu thim a si i 50 nm ultrathin sections cu Leica Ultracut (Leica Mikrosysteme GmbH, Vienna, Austria) ah an tan i polystyrene film in thuhmi 2×1 mm copper slit grid ah an lak. Cu tthen pawl cu H2O ah 4% uranyl acetate cawhmi in minutes 10 chung an thuh hna, H2O in voi zeimawzat an tawl hna, cun H2O ah Reynolds lead citrate in minutes 10 chung an tawl hna, cun H2O in voi zeimawzat an tawl hna. Micrographs cu TVIPS (Tietz Video le Image Processing System) TemCam-F416 digital camera (TVIPS GmbH, Gauting, USA) hmangin transmission electron microscope Philips CM100 (Thermo Fisher Scientific, Waltham, MA, USA) in an lak hna. Germany).
AAV zawtnak a ngeimi vok caah, thluak cu tthen a si i 1 mm a thukmi sagittal section ah tthen a si, cun cerebellum cu fluorescence microscope hmangin AAV zawtnak a ngeimi kuang (cucu, mCherry langhternak) hngalh khawhnak ding caah zohfel a si. AAV thunnak nih Purkinje cell tthen (cucu tthen dihlak tluk) a tlawmbik ah aa pehtlaimi cerebellar ring pahnih ah a sang tukmi thlennak tthatnak a chuahtermi hneksaknak lawng kha hman a si. AAV-transduced loop cu zankhuadei post-fixation (4% PFA le 2.5% glutaraldehyde in 0.1 M cocoate buffer) caah microdissected a si i a pehzulh in an tuah. EPON hrennak caah, a fekmi vun cu 0.1 M sodium cocoate buffer (Applichem) in tawl a si, cun 2% OsO4 (os, Science Services; Caco) he 0.1 M sodium cocoate buffer (Applichem) ah Suimilam 4 chung chiah a si, cun suimilam 2 chung tawl a si. 0.1 M cocamide buffer he voi 3 tuah tthan. Cu hnu ah, a kai thluahmahmi ethanol cu ​​ethanol cawhmi pakhat cio kha 4°C ah minutes 15 chung chiah in tissue kha ti a tlawmter. Tissue cu propylene oxide ah thlen a si i EPON (Sigma-Aldrich) ah 4°C ah zankhuadei chiah a si. Tissue cu EPON thar ah innchung lumnak ah suimilam 2 chung chiah, cun 62°C ah suimilam 72 chung chiah. Ultramicrotome (Leica Microsystems, UC6) le diamond namte (Diatome, Biel, Switzerland) hmangin 70 nm ultrathin sections kha tan law, 1.5% uranyl acetate in 37°C ah minutes 15 chung hnih, cun lead citrate cawhmi in minutes 4 chung hnih. Electron micrographs cu JEM-2100 Plus transmission electron microscope (JEOL) hmangin an lak hna i Camera OneView 4K 16-bit (Gatan) le DigitalMicrograph software (Gatan) he an i tlum. Hlathlainak caah, electron micrographs cu 5000× asiloah 10,000× digital zoom in an lak.
Mitochondria sining zohfelnak. Hlathlainak vialte caah, ImageJ software hmangin digital hmanthlak ah mitochondria pakhat cio an sining cu kut in suai an si. Aa dangmi sining sining hna kha an hlathlai. Mitochondrial density cu cell pakhat cio i mitochondrial area dihlak kha cytoplasm area (cytoplasm area = cell area-cell nucleus area) × 100 in tthennak in hmuhmi zatuak in langhter a si. Mitochondria a tlongmi cu [4π∙(area/perimeter 2)] timi formula in tuak a si. Mitochondria ista morphology cu hlathlainak tuah a si i an muisam bik ning in phun hnih (“tubular” le “blister”) ah ṭhen a si.
Autophagosome/lysosome zat le tlawmtam hlathlainak. Digital hmanthlak chung i autophagosome/lysosome pakhat cio i an muisam kha kut in suai awkah ImageJ software kha hmang. Autophagosome/lysosome area cu cell pakhat cio i autophagosome/lysosome sining dihlak kha cytoplasm area (cytoplasm area=cell area-nucleus area)×100 in tthennak in tuakmi zatuak in langhter a si. Autophagosomes/lysosomes an tlawm le tam cu a dihlak zat kha cell pakhat ah autophagosome/lysosome sining zat in tthen in tuak a si (cytoplasmic area in) (cytoplasmic area = cell area-nuclear area).
A fakmi tthencheunak le sample timhtuahnak caah min bunh. Glucose min bunh a herhmi hneksaknak caah, a fakmi thluak tthen pawl kha a tlingmi carbon (95% O2 le 5% CO2), a sangmi Ca2 + ACSF (125.0 mM NaCl, 2.5 mM KCl, 1.25 mM sodium phosphate buffer, 2.5 mM NaCl, 25 mM MH) aa telmi pre-incubation chamber ah thlen. d-glucose, 1.0 mM CaCl 2 le 2.0 mM MgCl 2, pH 7.4 le 310 in 320 mOsm tiang remhmi), cu chungah glucose cu 13 C 6- Glucose thlennak (Eurisotop, catalog number CLM-1396) a si. Pyruvate min bunh a herhmi hneksaknak caah, a fakmi thluak tthen pawl kha a sang deuhmi 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 Ca2 le Ad. MgCl2, pH 7.4 le 310 in 320mOsm tiang remh), cun 1mM 1-[1-13C]pyruvate (Eurisotop, catalog number CLM-1082) chap. Cu tthen pawl cu 37°C ah minutes 90 chung chiah hna. Hneksaknak a donghnak ah, tthen pawl cu 75 mM ammonium carbonate aa telmi ti (pH 7.4) in rang tein an tawl hna, cun 40:40:20 (v:v:v) acetonitrile (ACN): methanol: ti ah an i fonh. Section pawl cu minutes 30 chung tikhor cung ah an chiah hna hnu ah, sample pawl cu 21,000 g ah minutes 10 chung 4°C ah an thlet hna, cun a thiangmi a cunglei ti cu SpeedVac concentrator ah an roter. A chuakmi a ro cangmi metabolite pellet cu hlathlainak tuah hlan tiang -80°C ah chiah a si.
C-labeled amino acid 13 hna i ti rong thlennak-mass spectrometry hlathlainak. Ti chromatography-mass spectrometry (LC-MS) hlathlainak caah, metabolite pellet cu LC-MS grade ti (Honeywell) 75μl ah an hnim ṭhan. 21,000 g ah 4°C ah minutes 5 chung centrifugation tuah hnu ah, 20 μl a fiangmi supernatant cu amino acid flux hlathlainak caah hman a si, a tangmi chuahmi cu anion hlathlainak caah hman colh a si (a tanglei ah zoh). Amino acid hlathlainak cu a hlan ah langhtermi benzoyl chloride derivatization protocol (55, 56) hmangin tuah a si. A hmasa bik ah, 100 mM sodium carbonate (Sigma-Aldrich) 10μl cu metabolite extract 20μl ah an cawh, cun LC grade ACN ah 2% benzoyl chloride (Sigma-Aldrich) 10μl an cawh. Sample cu caan tawite chung vortex tuah a si i 21,000 g ah 20°C ah minutes 5 chung centrifuge tuah a si. A thiangmi a cunglei ti kha 2 ml autosampler vial ah conical glass insert (200 μl volume) he thlen. Sample pawl cu Q-Exactive (QE)-HF (Ultra High Field Orbitrap) high-resolution precision mass spectrometer (Thermo Fisher Scientific) he aa pehtlaimi Acquity iClass ultra-high performance LC system (Waters) hmangin hlathlainak an tuah hna. Hlathlainak caah, 2μl chuahmi sample cu 100×1.0 mm a fekmi silica T3 column (Waters) ah 1.8μm particles aa telmi ah an thun. A luan ning cu 100μl/min a si, cun buffer system cu buffer A (10 mM ammonium formate le ti chung i 0.15% formic acid) le buffer B (ACN) in a um. A tluknak cu a tanglei bantuk in a si: 0%B at 0 minutes; 0%B. 0 in 15% B ah 0 in 0.1 minutes ah; 15 in 17% B 0.1 in 0.5 minutes ah; B cu 17 in 55% tiang 0.5 in 14 minutes ah; B cu 55 in 70% tiang minutes 14 in 14.5 karlak ah; minutes 18 ah 14.5 in 70 in 100% B ah; minutes 18 in 19 karlak ah 100% B; 100 in 0% B cu minutes 19 in 19.1 karlak ah; 0% B cu 19.1 in 28 minutes (55, 56) ah a si. QE-HF mass spectrometer cu m/z (mass/charge ratio) 50 in 750 karlak mass range he positive ionization mode in a rian a ttuan. A hmanmi resolution cu 60,000 a si, cun gain control (AGC) ion target hmuhmi cu 3×106 a si, cun a tam bik ion caan cu 10 milliseconds a si. A lummi electrospray ionization (ESI) hrampi cu 3.5 kV thletnak voltage, 250°C capillary lumnak, 60 AU (a duh lomi unit) thli luhchuahnak le 20 AU bawmtu thli luhchuahnak ah a rian a ttuan. 250°C. S lens cu 60 AU ah chiah a si.
Anion chromatography-MS 13C tiah min an bunhmi organic acid pawl hlathlainak. A tangmi metabolite precipitate (55μl) cu Dionex ion chromatography system (ICS 5000+, Thermo Fisher Scientific) hmangin QE-HF mass spectrometer (Thermo Fisher Scientific) he pehtlai in hlathlainak tuah a si. A tawinak in, 5μl metabolite chuahmi cu HPLC (2 mm×250 mm, particle size 4μm, Thermo Fisher Scientific) he aa pehtlaimi Dionex IonPac AS11-HC column chungah 1. )on a tlingmi ratio he push-in partial loop mode in an thun. Dionex IonPac AG11-HC ralvengtu tung (2 mm x 50 mm, 4μm, Thermo Fisher Scientific). Column lumnak cu 30°C ah chiah a si, cun autosampler cu 6°C ah chiah a si. Eluent generator hmangin potassium hydroxide gradient chuah khawhnak ding caah deionized ti he pekmi potassium hydroxide cartridge kha hmang. A tanglei gradient hmangin 380μl/min a luangmi thilri pawl tthennak: 0 in 3 minutes, 10 mM KOH; minutes 3 in 12, 10 in 50 mM KOH; minutes 12 in 19, 50 in 100 mM KOH; 19 in 21 minutes, 100 mM KOH; 21 in 21.5 minutes, 100 in 10 mM KOH. Column cu 10 mM KOH tangah minutes 8.5 chung an i tlukter ṭhan.
A chuakmi thilri pawl cu column hnu ah 150μl/min isopropanol supplement stream he an fonh hna i cu hnu ah a ttha lomi ionization mode in rian a ttuanmi high-resolution mass spectrometer ah an hruai hna. MS nih m/z 50 in 750 tiang a ritmi kha 60,000 tluk in a zoh. AGC cu 1×106 ah chiah a si, cun a tam bik ion caan cu 100 ms ah chiah a si. A linmi ESI hrampi cu 3.5 kV thletnak voltage in rian a ttuan. Ion source a dang chiahmi hna cu a tanglei bantuk in an si: capillary lumnak 275°C; sheath gas flow, 60 AU; bawmtu thli luannak, 300°C ah 20 AU, le S lens 60 AU ah chiah.
13C tiah min an bunhmi thilri hna kong hlathlainak. Isotope ratio data hlathlainak caah TraceFinder software (version 4.2, Thermo Fisher Scientific) kha hmang. Compound pakhat cio an sining cu zumhawktlak zohchunhnak compound nih a fehter i mah tein hlathlainak tuah a si. Isotope rumternak hlathlainak tuah khawhnak ding caah, 13C isotope (Mn) pakhat cio i chuahmi ion chromatogram (XIC) area cu [M + H] + chungin chuah a si, cuka ah n cu target compound i carbon nambar a si, amino acid hlathlainak caah hmanmi asiloah [ MH] + cu anions hlathlainak caah hmanmi a si. XIC i a ritmi a dikthliarnak cu million pakhat ah cheu panga nak tlawm deuh a si, cun RT i a dikthliarnak cu minutes 0.05 a si. Ruahnak hlathlainak cu hmuhmi isotope pakhat cio le aa pehtlaimi compound i isotope vialte fonhmi zat tuaknak in tuah a si. Hi zat hna cu isotope pakhat cio caah zatuak man in pek an si, cun a phichuak cu a hlan ah langhtermi (42) bantuk in molar zatuak tthanchonak (MPE) in langhter a si.
A kikmi neuron pellet cu tikhor-a lummi 80% methanol (v/v) ah an cawh, an cawh, cun -20°C ah minutes 30 chung an chiah. Sample kha vortex tuah tthan law +4°C ah minutes 30 chung cawh. Sample cu 21,000 g ah 4°C ah minutes 5 chung centrifuge a si, cun a chuakmi a cunglei ti cu lak a si i SpeedVac concentrator hmangin 25°C ah a hnu ah hlathlainak tuah a si. A cunglei ah langhtermi bantuk in, LC-MS hlathlainak cu thleidanmi cells amino acids cungah tuah a si. TraceFinder (version 4.2, Thermo Fisher Scientific) hmangin, data hlathlainak cu compound pakhat cio i monoisotopic mass hmangin tuah a si. Metabolite data zat tlukruannak cu preprocessCore software package (57) hmangin tuah a si.
Slice timhtuahnak. Zu cu carbon dioxide in rang tein an hngilhter i a lu an tan, a thluak cu a luruh chungin rang tein an chuah, cun tikhor in a khatmi a hninhmi namte (HM-650 V, Thermo Fisher Scientific, Walldorf, Germany) hmangin 300 in 375 μm sagittal sections carbon gas le carbon gas (25% O2) ah an tan Ca2 + ACSF tlawm (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 le 6.0 mM MgCl2 pH 37 le 30 mO ah remh). Hmuhchuahmi thluak tthen pawl kha a sang deuhmi 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 Ca2 mM le 2.5 mM pH) aa telmi khaan ah thlen 7.4 le 310 in 320 mOsm tiang). Record tuah hlanah an remh khawh nakhnga tlawmpal tete kha minutes 20 in 30 tiang chiah hna.
record tuah. A fekmi ttialnak khaan le 20x ti hnimhnak thilri lens (Scientifica) he aa pehtlaimi microscope stage cu ttialnak vialte caah hman a si. Purkinje cells tiah ruahmi hna cu (i) pum ngan le hme, (ii) cerebellum dirhmun, le (iii) a ceumi mtYFP reporter gene langhternak in hngalh khawh an si. Megohms tiang a tthawnnak a ngeimi patch pipette cu borosilicate glass capillary (GB150-10, 0.86 mm×1.5 mm×100 mm, Science Products, Hofheim, Germany) le a tlarimi pipette Instruments (P-1000, Sutter, Sutter), CA) nih a chuah. Record vialte cu ELC-03XS npi patch clamp amplifier (npi electronic GmbH, Tam, Germany) nih a tuah, cucu software Signal (version 6.0, Cambridge Electronic, Cambridge, UK) nih a uk. Hneksaknak cu 12.5 kHz sampling rate in an khumh. Signal cu 1.3 le 10 kHz tthencheunak a ngeimi short-pass Bessel filter pahnih in thleidan a si. Membrane le pipette i an capacitance cu amplifier hmangin compensation circuit nih a tlawmter. Hneksaknak vialte cu Orca-Flash 4.0 camera (Hamamatsu, Gerden, Germany) uknak tangah tuah a si, cucu Hokawo software (version 2.8, Hamamatsu, Gerden, Germany) nih a uk.
Cell dihlak dirhmun le hlathlainak tuah tawnmi. Record tuah hlan deuh te ah, a tanglei thilri hna aa telmi chunglei cawhnuk in pipette kha khahter: 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), Gunophosiphate. (GTP) (Na) le 10.0 mM creatinine phosphate cu pH 7.25 ah an remh hna, cun osmotic pressure cu 290 mOsm (sucrose) a si. Membrane thleh awkah 0 pA thazaang hman hnu tlawmpal ah, a dinmi membrane tthawnnak kha tah a si. A luhtermi ralchanhnak cu -40, -30, -20, le -10 pA a simi hyperpolarized current hmannak in tah a si. Voltage lehnak a ngan le hme kha tah law Ohm phunglam hmangin luhtermi ralchanhnak kha tuak. Mah tein a cangmi thil cu minutes 5 chung voltage clamp ah an khumh, cun sPSC cu Igor Pro (version 32 7.01, WaveMetrics, Lake Oswego, Oregon, USA) ah semi-automatic recognition script hmangin an hngalh i an tah. IV curve le steady-state current cu battery kha a dangdang potentials (-110 mV in thawk) ah hren in le 5 mV step in voltage karhternak in tah a si. AP chuah khawhnak cu depolarizing current hmannak in hneksak a si. Depolarizing current pulse hman lio ah -70 mV ah cell kha hren. Recording unit pakhat cio i a dang tein (10 in 60 pA tiang) step size kha remh. A sang bik AP frequency a chuahtertu pulse spikes kha kut in relnak in a sang bik AP frequency kha tuak. AP threshold cu AP pakhat asiloah tam deuh a chuahtertu depolarization pulse i a pahnihnak derivative hmangin hlathlainak tuah a si.
A kuaimi patch dirhmun le hlathlainak. A tlarimi phunglam hmangin a kuaimi patch hmanthlaknak tuah. A tanglei thilri pawl aa tel lomi ATP- le GTP-um lomi pipette hmang: 128 mM gluconate K, 10 mM KCl, 10 mM Hepes, 0.1 mM EGTA le 2 mM MgCl2, cun pH 7.2 (KOH hmangin) ah remh. ATP le GTP cu cell membrane chung i uk khawh lomi luh khawhnak khamnak caah intracellular cawhnuk chungin chuah an si. Patch pipette cu amphotericin aa telmi chunglei cawhnuk (200 in 250 μg/ml hrawng; G4888, Sigma-Aldrich) in a khat i a kuaimi patch cazin hmuh khawh a si. Amphotericin cu dimethyl sulfoxide (a donghnak tlawmtam: 0.1 in 0.3%; DMSO; D8418, Sigma-Aldrich) ah a cawh. DMSO hmanmi tlawmtam nih hlathlainak tuahmi neurons cungah zeihmanh thlennak a chuahpi lo. Hnawmhnak tuah lio ah, channel dohnak (Ra) cu pehzulh tein zohkhenh a si, cun Ra le AP amplitude a ttha (20-40 minutes) hnu ah hneksaknak cu thawk a si. Mah tein a cangmi cawlcanghnak cu voltage le/asiloah current clamp ah minutes 2 in 5 tiang tah a si. Data hlathlainak cu Igor Pro (version 7.05.2, WaveMetrics, USA), Excel (version 2010, Microsoft Corporation, Redmond, USA) le GraphPad Prism (version 8.1.2, GraphPad Software Inc., La Jolla, CA) hmangin tuah a si. United States). Mah tein a chuakmi AP pawl hngalh khawhnak dingah, IgorPro's NeuroMatic v3.0c plug-in kha hman a si. Pekmi threshold hmangin APs kha amah tein hngalh khawh a si, cucu record pakhat cio caah pakhat cio in remh a si. Spike karlak hmangin, a sang bik a rannak in a chuakmi spike frequency le a tlangpi spike frequency he spike frequency kha tuak.
PN thleidannak. A hlan ah chuahmi phunglam he aa tlakmi in, PNs cu vok thluak chungin a hleiin a ummi dirhmun ah thianh a si (58). A tawinak in, cerebellum cu vur-a kikmi tthencheunak thilri [HBSS Ca2+ le Mg2+ tel loin, 20 mM glucose, penicillin (50 U/ml) le streptomycin (0.05 mg/ ml) he chapmi] ah an thleh i an cawh, cun cu hnuah papa in [HBSS, HCS-steine ​​chapmi] ah an cawh. (1 mg / ml), papain (16 U / ml) le deoxyribonuclease I (DNase I; 0.1 mg/ml)] 30°C ah minutes 30 chung thlop. HBSS medium ah egg mucus (10 mg/ml), BSA (10 mg/ml) le DNase (0.1 mg/ml) aa telmi ah tissue pawl kha khaan lumternak ah tawl hmasa hna law enzymatic digestion khamnak caah, cun HBSS medium ah 20 mM glucose aa telmi HBSS medium ah nem tein hnimh, peillin, U/ml/ml, strep (0.05 mg/ml) le DNase (0.1 mg/ml) nih cell pakhat lawng an chuahter. A chuakmi cell hnahnawhnak cu 70μm cell thletnak in thlet a si, cun cells pawl cu centrifugation (1110 rpm, minutes 5, 4°C) in an thlet hna i thleidannak thilri ah an thlet ṭhan hna [HBSS, 20 mM glucose, 20% fetal bovine he chapmi) Serum, pelinic (50/0/5) le strepcyn (0.05 mg/ml)]; propidium iodide hmangin cell nun khawhnak kha tuaktan law cell tlawmtam kha 1×106 in 2×106 cells/ml tiang remh. Flow cytometry tuah hlan ah, 50 μm cell strainer in a thletmi cu a thlet.
Flow cytometer. Cell thleidannak cu FACSAria III seh (BD Biosciences) le FACSDiva software (BD Biosciences, version 8.0.1) hmangin 4°C ah tuah a si. Cell tthencheunak cu 100 μm nozzle hmangin 20 psi hneknak tangah ~2800 thil cangmi/sec tluk in tthen a si. Hlanlio kharnak phunglam (cell ngan, bimodal thleidannak, le tthencheunak sining) nih cell phun dang hna he PN kha a dikmi thleidannak a tuah khawh lo caah, kharnak strategy cu mitoYFP+ ​​le control mitoYFP − Mice ah YFP thazaang le autofluorescence direct tahchunhnak cungah hram bunh in chiah a si. YFP cu 488 nm laser line in sample kha ceunak in a thawhter, cun signal cu 530/30 nm band pass filter hmangin hmuh khawh a si. MitoYFP+ ​​vok ah, Rosa26-mitoYFP reporter gene i aa pehtlaimi thazaang zong kha neuronal pum le axon tthencheu thleidan khawhnak caah hman a si. 7-AAD cu 561 nm a senmi laser in a thawhter i a thimi cell pawl chuahter awkah 675/20 nm bandpass filter in hmuh a si. Astrocytes pawl kha caankhat te ah thleidan khawhnak ding caah, cell suspension cu ACSA-2-APC in an thuh, cun sample cu 640 nm laser line in an ceuter, cun 660/20 nm bandpass filter hmangin signal kha an hmuh.
An lakmi cells pawl cu centrifugation (1110 rpm, 5 minutes, 4°C) in an tthen hna i hman hlan tiang -80°C ah an chiah hna. Mfn2cKO vok le an fale cu nikhat te ah tthencheu an si i cu nih cun phunglam aa dangmi a zorter. FACS data langhternak le hlathlainak cu FlowJo software (FlowJo LLC, Ashland, Oregon, USA) hmangin tuah a si.
A cunglei ah kan chim cang bantuk in (59), real-time PCR cu a hnu mtDNA zat tuaktannak caah thleidanmi neurons chungin DNA thleidannak caah hman a si. Linearity le threshold sensitivity cu a hramthawk ah cell zat aa dangmi ah qPCR tuahnak in hneksaknak an tuah. A tawinak in, 50 mM tris-HCl (pH 8.5), 1 mM EDTA, 0.5% Tween 20 le proteinase K (200 ng/ml) aa telmi lysis buffer ah 300 PN kha lak law 55°C ah minutes 120 chung chiah. Cell pawl cu 95°C ah minutes 10 chung an chiah hna i proteinase K kha a tling tein a ngolter. mt-Nd1 he aa pehtlaimi TaqMan probe (Thermo Fisher) hmangin, mtDNA cu 7900HT Real-Time PCR system (Thermoherific Fiscients) ah semi-quantitative PCR in tah a si. Science, catalog number Mm04225274_s1), mt-Nd6 (Thermo Fisher Scientific, catalog number AIVI3E8) le 18S (Thermo Fisher Scientific, catalog number Hs99999901_s1) genes.
Proteome zohchunhnak timhtuahnak. Minute 10 chung 95°C ah lumternak le sonication tuahnak in, lysis buffer [6 M guanidine chloride, 10 mM tris(2-carboxyethyl) phosphine hydrochloride, 10 mM chloroacetamide le 100 mM tris- Lyse frozen neuron pellets in HC]. Bioruptor (Diagenode) kha minutes 10 chung (second 30 tthawnnak / second 30 dinh caan) on. Sample cu 20 mM tris-HCl (pH 8.0) ah 1:10 in cawh a si, 300 ng trypsin gold (Promega) he cawh a si, cun 37°C ah zankhuadei chiah a si i a tlingmi rawl ei khawh a si. Nihnihnak ah, sample cu 20,000 g ah minutes 20 chung an cawh. A cunglei ti cu 0.1% formic acid in cawh a si, cun a cawhmi cu mah tein sermi StageTips in chiti hloh a si. Sample cu SpeedVac thilri (Eppendorf concentrator plus 5305) ah 45°C ah an roter, cun peptide cu 0.1% formic acid ah an chiah. Sample vialte cu mi pakhat nih hmunkhat te ah a timhtuah dih hna. Astrocyte sample pawl hlathlainak tuah khawhnak ding caah, 4 μg desalteted peptides cu tandem mass tag (TMT10plex, catalog number 90110, Thermo Fisher Scientific) in peptide le TMT reagent ratio 1:20 he min an bunh hna. TMT min bunhnak caah, TMT reagent 0.8 mg cu ti um lomi ACN 70 μl ah an hnim ṭhan, cun a ro cangmi peptide cu 0.1 M TEAB (triethylammonium bicarbonate) 9 μl ah an ser ṭhan, cu ah cun ACN chung i TMT reagent 7 μl an chap. A tlawmtam cu 43.75% a si. Minute 60 chung an chiah hnu ah, 2 μl 5% hydroxylamine in an hmit. Min bunhmi peptide pawl cu an lak hna, an roter hna, 0.1% formic acid (FA) 200μl ah an hnim ṭhan hna, pahnih ah an ṭhen hna, cun mah tein sermi StageTips hmangin chiti an hloh hna. UltiMate 3000 ultra high performance liquid chromatograph (UltiMate 3000 ultra high performance liquid chromatograph) hmangin, a cheu hnih lakah pakhat cu 130Å1.7μm C18 particles in a khatmi 1mm x 150mm Acquity chromatographic column ah tthen a si (Waters, catalog No: SKU:1966. Thermo Fisher Scientific). Peptide pawl kha 30μl/min a luannak in thleidan hna, 1% in 50% tiang buffer B kha minutes 85 chung minutes 96 chung stepwise gradient in thleidan hna, 50% in 95% tiang buffer B kha minutes 3 chung, cun 95 % Buffer B caah minutes 8; Buffer A cu 5% ACN le 10 mM ammonium bicarbonate (ABC) a si, cun buffer B cu 80% ACN le 10 mM ABC a si. Minute 3 dan ah tthencheu pawl kha lak hna law phu hnih (1 + 17, 2 + 18, tbk.) ah fonh hna law vacuum centrifuge ah roter hna.
LC-MS/MS hlathlainak. Mass spectrometry caah, peptides (number r119.aq) cu 25 cm, 75 μm chunglei diameter PicoFrit analytical column (new objective lens, part number PF7508250) ah 1.9 μm ReproSil-Pur 120 C18-AQ medium (Maisch, Maisch-YAS) he an i tthen. 1200 (Thermo Fisher Scientific, Germany). Column cu 50°C ah chiah a si. Buffer A le B cu ti chung i 0.1% formic acid le 80% ACN chung i 0.1% formic acid an si. Peptide pawl cu 6% in 31% buffer B in minutes 65 chung, cun 31% in 50% buffer B tiang minutes 5 chung 200 nl/min tluk in tthen an si. A chuakmi peptide pawl cu Orbitrap Fusion mass spectrometer (Thermo Fisher Scientific) ah an hlathlai hna. Peptide precursor m/z tahnak cu 350 in 1500 m/z karlak ah 120,000 resolution in tuah a si. 27% a tlarimi i pahnak thazaang hmangin, 2 in 6 tiang a tthawnnak dirhmun a ngeimi a tthawng bikmi hramthawknak cu thazaang sang C trap dissociation (HCD) tthennak caah thim a si. Cycle caan cu 1 s ah chiah a si. Peptide tthencheunak m/z man cu ion trap ah a hme bik AGC target 5×104 le a tam bik thunnak caan 86 ms hmangin tah a si. A cheu hnu ah, a hramthawknak cu 45 s chung aa thleng lengmangmi hrialnak cazin ah chiah a si. TMT-labeled peptides cu 50 cm, 75 μm Acclaim PepMap column (Thermo Fisher Scientific, catalog number 164942) ah an tthen hna, cun an i ṭhialnak spectra cu Orbitrap Lumos Tribrid mass spectrometer (Thermo Fisher Scientific) ah high-field aveform iMS (IMS) he aa pehtlaimi ah an hlathlai hna thilri (Thermo Fisher Scientific) cu −50 le −70 V tlukruannak voltage pahnih ah a rian a ttuan. MS3 cu synchronization precursor cung ah hram bunh in thim a si i TMT report ion signal tahnak caah hman a si. Peptide tthennak cu EASY-nLC 1200 ah tuah a si, 90% linear gradient elution hmangin, buffer concentration 6% in 31% tiang; buffer A cu 0.1% FA a si, cun buffer B cu 0.1% FA le 80% ACN a si. Hlathlainak column cu 50°C ah rian a ttuan. FreeStyle (version 1.6, Thermo Fisher Scientific) kha hmang law FAIMS tlukruannak voltage ningin a hramthawk file kha ṭhen.
Protein hngalh khawhnak le zat tuak khawhnak. Andromeda kawlnak seh fonhmi hmangin, a hramthawk data cu MaxQuant version 1.5.2.8 (https://maxquant.org/) hmangin hlathlainak tuah a si. Aequorea victoria in hmuhmi Cre recombinase le YFP sequences lengah, peptide fragment spectra cu mouse reference proteome (Proteome ID UP000000589, May 2017 ah UniProt in download tuahmi) i canonical sequence le isoform sequence caah kawl a si. Methionine oxidation le protein N-terminal acetylation cu aa thleng khomi remhnak ah chiah an si; cysteine ​​carbamoyl methylation cu a hmunmi remhnak ah chiah a si. Rawl ei ningcang hna cu “a hlei in” le “trypsin/P” ah chiah an si. Protein hngalh khawhnak caah hmanmi peptide le razor peptide tlawm bik cu 1 a si; a tlawmbik aa dangmi peptide zat cu 0 a si. Peptide map i tluknak dirhmun tangah, protein hngalh khawhnak rate cu 0.01 a si. “Peptide Pahnihnak” timi thimnak cu on a si. “Match between runs” timi kha hmang law a dang original file karlak ah hlawhtlinnak a hmumi hngalh khawhnak kha thlen. Cafang tel loin tahnak (LFQ) (60) caah LFQ a niam bik zat relnak 1 kha hmang. LFQ thazaang cu caan pakhat cio ah a tlawmbik genotype phu pakhat ah a tlawmbik a hmanmi man pahnih caah thleidan a si, cun a kauhnak he a tlarimi phawtzamhnak in chuah a si. 0.3 le 1.8 in ttum. LFQ hmuhmi hlathlainak caah Perseus computer platform (https://maxquant.net/perseus/) le R (https://r-project.org/) kha hmang. Limma software package chungin lam pahnih in a tlawm deuhmi t hneksaknak cu aa dangmi langhternak hlathlainak caah hman a si (61). Hlathlainak data hlathlainak cu ggplot, FactoMineR, factoextra, GGally le pheatmap hmangin tuah a si. TMT-based proteomics data cu MaxQuant version 1.6.10.43 hmangin hlathlainak tuah a si. UniProt's human proteomics database chungin raw proteomics data kawl, cucu September 2018 ah download tuahmi a si. Hlathlainak ah sertu nih a pekmi isotope thianhlimnak remhnak factor aa tel. Aa dangmi langhternak hlathlainak caah R ah limma hmang. A hramthawk data, database kawlnak hmuhmi, le data hlathlainak riantuan ning le hmuhmi vialte cu PRIDE partner repository hmangin ProteomeXchange alliance ah data set identifier PXD019690 he chiah an si.
Riantuannak fianternak nih hlathlainak kha a ṭhanchoter. Ingenuity Pathway Analysis (QIAGEN) thilri cu zarh 8 ah data set i riantuannak fianternak biafang hna a rumnak hngalh khawhnak caah hman a si (Figure 1). A tawinak in, LC-MS/MS (tandem mass spectrometry) data hlathlainak in hmuhmi quantitative protein cazin cu a tanglei filter tahfung he hman a si: Mus musculus cu phun le hnulei ah thim a si, cun phun nih Benjamini nih 0.05 asiloah a niam deuhmi rumternak caah a remhmi P man kha a langhter i a biapi tiah ruah a si. Hi graph caah, remhmi P man cung ah hram bunh in cluster pakhat cio ah a sang bik a hleimi phun panga kha langhter an si. T-test tampi hmangin, Benjamini, Krieger, le Yekutieli (Q = 5%) i a tthen hnih linear boost program hmangin, caan-caan chung protein langhternak hlathlainak cu phun pakhat cio ah hmuhmi a biapi mi candidate hna cungah tuah a si, cun tlar pakhat cio cu a dang tein hlathlainak tuah a si. Aa khatmi SD hman a hau lo.
Hi hlathlainak hmuhmi kha chuahmi cazin hna he tahchunh awk le hmanthlak 1 ah Venn suaisam ser awk ah, zatlang protein cazin kha MitoCarta 2.0 fianternak he kan fonh (24). Diagram sernak caah online thilri Draw Venn Diagram (http://bioinformatics.psb.ugent.be/webtools/Venn/) kha hmang.
Proteomics hlathlainak caah hmanmi zatlang phunglam kong he pehtlai in a tawinak in theih na duh ahcun, thilri le lamthluan he aa pehtlaimi ṭhen kha zoh. A dang hneksaknak vialte caah, a tlingmi thawngpang cu aa pehtlaimi tuanbia ah hmuh khawh a si. A dang tein langhter a si lo ahcun, data vialte cu a tlangpi ± SEM in langhter an si, cun zatlang hlathlainak vialte cu GraphPad Prism 8.1.2 software hmangin tuah an si.
Hi capar caah bawmtu thilri caah, http://advances.sciencemag.org/cgi/content/full/6/35/eaba8271/DC1 zoh
Hihi Creative Commons Attribution-Non-Commercial License phunglam ningin phawtzamhmi a ongmi capar a si, mah nih hin zeibantuk lam hmanh in hmannak, phawtzamhnak le chuahnak nawl a pek, a donghnak hmannak cu chawlehnak caah a si lo ahcun le a hramthawk riantuannak cu a dikmi a si ahcun. Namuna.
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Hi biahalnak hi khual na si le si lo hneksaknak caah le mah tein spam kuatnak khamnak caah hmanmi a si.
E. Motori, I. Atanassov, SMV Kochan, K. Folz-Donahue, V. Sakthivelu, P. Giavalisco, N. Toni, J. Puyal, N.-G. Larson
Rian a ttuan lomi thluak hna i proteomics hlathlainak nih thluak rawhnak doh awkah thlennak program hna cu an cawlcang ti a langhter.
E. Motori, I. Atanassov, SMV Kochan, K. Folz-Donahue, V. Sakthivelu, P. Giavalisco, N. Toni, J. Puyal, N.-G. Larson
Rian a ttuan lomi thluak hna i proteomics hlathlainak nih thluak rawhnak doh awkah thlennak program hna cu an cawlcang ti a langhter.
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