Tam sim no *Chaw nyob tam sim no: Cologne 50931, Lub Tebchaws Yelemees, Cologne Excellence Cluster Research on Cellular Stress Response in Aging-related Diseases (CECAD).
Qhov kev puas tsuaj ntawm cov kab mob mitochondrial yog suav tias tsis rov qab tau vim tias qhov kev hloov pauv ntawm cov hlwb neurons yog txwv, tab sis qhov cuam tshuam ntawm mitochondrial dysfunction ntawm lub cell autonomy ntawm neuronal metabolism hauv lub cev tsis nkag siab zoo. Ntawm no, peb qhia txog cov proteome tshwj xeeb ntawm Purkinje neurons nrog kev tsis txaus OXPHOS uas tshwm sim los ntawm kev cuam tshuam mitochondrial fusion dynamics. Peb pom tias mitochondrial dysfunction ua rau muaj kev hloov pauv loj heev hauv thaj chaw ntawm proteomics, uas thaum kawg ua rau muaj kev ua haujlwm ntawm cov kev pab cuam metabolic ua ntej kev tuag ntawm lub hlwb. Tsis tau xav txog, peb tau txiav txim siab qhov kev tshwm sim pom tseeb ntawm pyruvate carboxylase (PCx) thiab lwm yam enzymes tiv thaiv kev laus uas ntxiv cov intermediates ntawm lub voj voog TCA. Kev txwv ntawm PCx ua rau muaj kev ntxhov siab oxidative thiab neurodegeneration, qhia tias atherosclerosis muaj kev tiv thaiv hauv neurons tsis muaj OXPHOS. Kev kho dua tshiab ntawm mitochondrial fusion hauv cov neurons degenerated terminally thim rov qab cov yam ntxwv metabolic no, yog li tiv thaiv kev tuag ntawm lub hlwb. Peb cov kev tshawb pom txheeb xyuas cov kev tsis paub yav dhau los uas muab kev ywj pheej rau mitochondrial dysfunction thiab qhia tias neurodegeneration tuaj yeem thim rov qab txawm tias nyob rau theem kawg ntawm tus kab mob.
Lub luag haujlwm tseem ceeb ntawm mitochondria hauv kev tswj hwm lub zog metabolism neuronal yog hais txog los ntawm cov tsos mob neurological dav dav uas cuam tshuam nrog tib neeg mitochondrial kab mob. Feem ntau ntawm cov kab mob no yog tshwm sim los ntawm kev hloov pauv gene uas tswj hwm mitochondrial gene expression (1, 2) lossis gene destruction cuam tshuam nrog mitochondrial dynamics, uas cuam tshuam tsis ncaj qha rau kev ruaj khov ntawm mitochondrial DNA (mtDNA) (3, 4). Kev ua haujlwm hauv cov qauv tsiaj tau qhia tias hauv kev teb rau mitochondrial dysfunction hauv cov ntaub so ntswg ib puag ncig, cov txheej txheem metabolic conservative (5-7) tuaj yeem ua haujlwm, uas muab cov ntaub ntawv tseem ceeb rau kev nkag siab tob txog pathogenesis ntawm cov kab mob nyuaj no. Hauv kev sib piv, peb qhov kev nkag siab txog kev hloov pauv metabolic ntawm cov hom cell tshwj xeeb uas tshwm sim los ntawm kev ua tsis tiav ntawm lub hlwb mitochondrial adenosine triphosphate (ATP) tsim yog qhov tseem ceeb (8), hais txog qhov xav tau los txheeb xyuas cov hom phiaj kho mob uas tuaj yeem siv los tiv thaiv lossis tiv thaiv kab mob. Tiv thaiv neurodegeneration (9). Qhov tsis muaj cov ntaub ntawv yog qhov tseeb tias cov hlwb paj hlwb tau suav tias muaj kev ywj pheej metabolic tsawg heev piv rau cov hom cell ntawm cov ntaub so ntswg ib puag ncig (10). Vim tias cov hlwb no ua lub luag haujlwm tseem ceeb hauv kev sib koom tes muab cov metabolites rau cov neurons los txhawb kev sib kis synaptic thiab teb rau kev raug mob thiab kab mob, lub peev xwm los hloov kho cov metabolism ntawm cov hlwb rau cov xwm txheej nyuaj ntawm cov ntaub so ntswg hauv lub hlwb yuav luag txwv rau cov hlwb glial (11-14). Tsis tas li ntawd, qhov sib txawv ntawm cov hlwb ntawm cov ntaub so ntswg hauv lub hlwb feem ntau cuam tshuam kev kawm txog kev hloov pauv metabolic uas tshwm sim hauv cov pab pawg neuronal tshwj xeeb. Yog li ntawd, me ntsis paub txog qhov tseeb ntawm cov cellular thiab metabolic tshwm sim ntawm mitochondrial dysfunction hauv neurons.
Yuav kom nkag siab txog cov teeb meem metabolic ntawm mitochondrial dysfunction, peb tau cais Purkinje neurons (PNs) hauv ntau theem ntawm neurodegeneration los ntawm kev puas tsuaj ntawm mitochondrial sab nraud membrane fusion (Mfn2). Txawm hais tias Mfn2 mutations hauv tib neeg yog txuam nrog ib hom ntawm hereditary motor sensory neuropathy hu ua Charcot-Marie-Tooth type 2A (15), qhov kev puas tsuaj ntawm Mfn2 hauv nas yog qhov paub zoo induction ntawm oxidation Phosphorylation (OXPHOS) dysfunction method. Ntau yam neuronal subtypes (16-19) thiab cov neurodegenerative phenotype tshwm sim nrog cov tsos mob neurological nce qib, xws li kev txav mus los tsis zoo (18, 19) lossis cerebellar ataxia (16). Los ntawm kev siv kev sib xyaw ua ke ntawm cov ntawv lo tsis muaj ntau yam (LFQ) proteomics, metabolomics, imaging, thiab virological txoj kev, peb qhia tau hais tias kev nce qib ntawm neurodegeneration ua rau muaj zog pyruvate carboxylase (PCx) thiab lwm yam uas cuam tshuam nrog arteriosclerosis ntawm PNs hauv vivo Kev qhia tawm ntawm cov enzymes. Txhawm rau txheeb xyuas qhov tseeb ntawm qhov kev tshawb pom no, peb tau txo qis qhov kev qhia tawm ntawm PCx hauv Mfn2-deficient PNs, thiab pom tias qhov kev ua haujlwm no ua rau muaj kev ntxhov siab oxidative thiab ua kom neurodegeneration sai dua, yog li ua pov thawj tias azoospermia muab kev tuag ntawm tes Metabolic adaptability. Kev qhia tawm hnyav ntawm MFN2 tuaj yeem cawm tag nrho qhov kev puas tsuaj ntawm PN nrog OXPHOS tsis txaus, kev noj ntau ntawm mitochondrial DNA, thiab pom tseeb tias tawg mitochondrial network, uas ntxiv qhov tseeb tias daim ntawv no ntawm neurodegeneration tuaj yeem rov zoo hauv theem siab ntawm tus kab mob ua ntej cell tuag.
Yuav kom pom tau cov mitochondria hauv Mfn2 knockout PNs, peb siv cov nas uas tso cai rau Cre-dependent mitochondria los tsom rau cov protein fluorescent daj (YFP) (mtYFP) (20) Cre kev qhia tawm thiab kuaj xyuas cov mitochondrial morphology hauv vivo. Peb pom tias kev puas tsuaj ntawm Mfn2 gene hauv PNs yuav ua rau muaj kev faib maj mam ntawm lub network mitochondrial (Daim Duab S1A), thiab qhov kev hloov pauv thaum ntxov tshaj plaws tau pom thaum muaj hnub nyoog 3 lub lis piam. Qhov sib piv, qhov kev puas tsuaj loj heev ntawm cov txheej cell PN, raws li pov thawj los ntawm kev poob ntawm Calbindin immunostaining, tsis tau pib txog thaum muaj hnub nyoog 12 lub lis piam (Daim Duab 1, A thiab B). Lub sijhawm tsis sib xws ntawm qhov kev hloov pauv thaum ntxov tshaj plaws hauv mitochondrial morphology thiab qhov pom kev pib ntawm kev tuag neuronal tau ua rau peb tshawb xyuas cov kev hloov pauv metabolic uas tshwm sim los ntawm mitochondrial dysfunction ua ntej kev tuag ntawm tes. Peb tau tsim ib txoj kev xaiv cell fluorescence-activated (FACS)-raws li txoj kev los cais YFP (YFP+)-expressing PN (Daim Duab 1C), thiab hauv cov nas tswj (Mfn2 + / loxP :: mtYFP loxP- nres-loxP: : L7-cre), tom qab no hu ua CTRL (Daim Duab S1B). Kev ua kom zoo dua ntawm txoj kev gating raws li qhov sib piv ntawm lub teeb liab YFP tso cai rau peb kom ntxuav YFP+ lub cev (YFPhigh) ntawm PNs los ntawm cov tsis yog PNs (YFPneg) (Daim Duab S1B) lossis cov khoom tawg fluorescent axon/dendritic (YFPlow; Daim Duab S1D, sab laug), lees paub los ntawm confocal microscope (Daim Duab S1D, sab xis). Txhawm rau txheeb xyuas tus kheej ntawm cov pej xeem raug cais tawm, peb tau ua LFQ proteomics thiab tom qab ntawd kev tshuaj xyuas cov khoom tseem ceeb, thiab pom tias muaj kev sib cais meej ntawm YFPhigh thiab YFPneg hlwb (Daim Duab S1C). Cov hlwb YFPhigh tau qhia txog kev nplua nuj ntawm cov cim PNs paub (piv txwv li Calb1, Pcp2, Grid2 thiab Itpr3) (21, 22), tab sis tsis muaj kev nplua nuj ntawm cov protein feem ntau qhia hauv cov neurons lossis lwm hom cell (Daim Duab 1D)). Kev sib piv ntawm cov qauv hauv cov hlwb YFPhigh uas tau muab cais ua pawg hauv kev sim ywj pheej tau qhia txog tus lej sib raug zoo> 0.9, qhia txog kev rov ua dua zoo ntawm cov kab mob rov ua dua (Daim Duab S1E). Hauv kev xaus, cov ntaub ntawv no tau lees paub peb txoj kev npaj rau kev rho tawm sai thiab tshwj xeeb ntawm PN ua tau. Vim tias lub kaw lus tsav tsheb L7-cre siv ua rau muaj kev sib xyaw ua ke hauv thawj lub lim tiam tom qab yug me nyuam (23), peb tau pib tua cov nas los ntawm CTRL thiab cov xwm txheej (Mfn2 loxP / loxP :: mtYFP loxP-stop-loxP :: L7-cre) Sau cov neurons. Tom qab kev sib xyaw ua ke tiav, nws hu ua Mfn2cKO thaum muaj hnub nyoog 4 lub lis piam. Ua qhov kawg, peb xaiv 8 lub lis piam hnub nyoog thaum lub txheej PN tseem zoo li qub txawm tias muaj kev sib cais ntawm mitochondrial (Daim Duab 1B thiab Daim Duab S1A). Tag nrho, peb tau suav tag nrho 3013 cov protein, uas kwv yees li 22% raws li MitoCarta 2.0 cov lus piav qhia raws li mitochondrial proteome ua mitochondria (Daim Duab 1E) (Daim Duab 1E) (24). Kev tshuaj xyuas kev qhia txog cov noob caj noob ces sib txawv ua rau lub lim tiam 8 qhia tau tias tsuas yog 10.5% ntawm tag nrho cov protein muaj kev hloov pauv tseem ceeb (Daim Duab 1F thiab Daim Duab S1F), uas 195 cov protein tau down-regulated thiab 120 cov protein tau up-regulated (Daim Duab 1F). Nws tsim nyog sau cia tias "kev tshuaj xyuas txoj hauv kev tshiab" ntawm cov ntaub ntawv teeb tsa no qhia tau hais tias cov noob caj noob ces sib txawv feem ntau yog cov txheej txheem txwv ntawm cov txheej txheem metabolic tshwj xeeb (Daim Duab 1G). Qhov txaus siab yog, txawm hais tias qhov kev txo qis ntawm txoj hauv kev cuam tshuam nrog OXPHOS thiab calcium signaling lees paub qhov kev ua haujlwm ntawm mitochondrial dysfunction hauv fusion-deficient PNs, lwm pawg uas feem ntau cuam tshuam nrog amino acid metabolism yog upregulated tseem ceeb, uas yog nyob rau hauv kab nrog metabolism uas tshwm sim hauv mitochondrial PNs Rewiring yog sib xws. disfunction.
(A) Cov duab sawv cev ntawm cov ntu cerebellar ntawm CTRL thiab Mfn2cKO nas uas qhia txog kev poob ntawm PNs (calbindin, grey); nuclei tau counterstained nrog DAPI. (B) Kev ntsuas ntawm (A) (ib txoj kev tshuaj xyuas ntawm qhov sib txawv, ***P <0.001; n = 4 txog 6 lub voj voog los ntawm peb tus nas). (C) Kev ua haujlwm sim. (D) Daim ntawv qhia cua sov faib ntawm cov cim tshwj xeeb rau Purkinje (sab saum toj) thiab lwm hom cell (nruab nrab). (E) Daim duab Venn qhia tus lej ntawm cov protein mitochondrial uas tau txheeb xyuas hauv PN uas tau cais tawm. (F) Daim duab roob hluav taws ntawm cov protein sib txawv hauv Mfn2cKO neurons ntawm 8 lub lis piam (qhov tseem ceeb txiav-tawm tus nqi ntawm 1.3). (G) Kev tshuaj xyuas txoj kev muaj tswv yim qhia txog tsib txoj kev tseem ceeb tshaj plaws up-regulation (liab) thiab down-regulation (xiav) hauv Mfn2cKO PN uas tau cais tawm ua 8 lub lis piam. Qib kev qhia nruab nrab ntawm txhua cov protein uas tau kuaj pom tau qhia. Daim ntawv qhia cua sov grayscale: kho tus nqi P. ns, tsis tseem ceeb.
Cov ntaub ntawv Proteomics qhia tau hais tias qhov kev qhia tawm ntawm cov protein ntawm cov complexes I, III, thiab IV maj mam txo qis. Cov Complexes I, III, thiab IV tag nrho muaj cov subunits tseem ceeb mtDNA-encoded, thaum complex II, uas tsuas yog nuclear-coded, yeej tsis cuam tshuam (Daim Duab 2A thiab Daim Duab S2A). . Sib xws nrog cov txiaj ntsig proteomics, immunohistochemistry ntawm cov ntaub so ntswg cerebellar qhia tau hais tias MTCO1 (mitochondrial cytochrome C oxidase subunit 1) subunit theem ntawm complex IV hauv PN maj mam txo qis (Daim Duab 2B). Lub mtDNA-encoded subunit Mtatp8 tau txo qis heev (Daim Duab S2A), thaum lub xeev ruaj khov ntawm lub nuclear-encoded ATP synthase subunit tseem tsis hloov pauv, uas yog sib xws nrog qhov paub ruaj khov ATP synthase subassembly F1 complex thaum mtDNA kev qhia ruaj khov. Kev tsim yog sib xws. Kev cuam tshuam (7). Kev ntsuam xyuas ntawm qib mtDNA hauv Mfn2cKO PNs uas tau txheeb los ntawm kev tshuaj xyuas polymerase chain reaction (qPCR) tau lees paub qhov kev txo qis maj mam ntawm tus lej mtDNA. Piv nrog rau pawg tswj hwm, thaum muaj hnub nyoog 8 lub lis piam, tsuas yog kwv yees li 20% ntawm qib mtDNA tau khaws cia (Daim Duab 2C). Sib xws nrog cov txiaj ntsig no, kev siv tshuaj confocal microscopy ntawm Mfn2cKO PNs tau siv los ntes DNA, qhia txog kev siv mitochondrial nucleotides raws sijhawm (Daim Duab 2D). Peb pom tias tsuas yog qee tus neeg sib tw koom nrog hauv kev puas tsuaj ntawm mitochondrial protein thiab kev teb rau kev ntxhov siab tau nce siab, suav nrog Lonp1, Afg3l2 thiab Clpx, thiab OXPHOS cov yam ntxwv sib dhos. Tsis muaj kev hloov pauv tseem ceeb hauv cov qib ntawm cov protein koom nrog hauv apoptosis tau pom (Daim Duab S2B). Ib yam li ntawd, peb pom tias mitochondria thiab endoplasmic reticulum channels koom nrog hauv kev thauj calcium tsuas yog muaj kev hloov pauv me me xwb (Daim Duab S2C). Ntxiv mus, kev ntsuam xyuas cov protein ntsig txog autophagy tsis pom muaj kev hloov pauv tseem ceeb, uas yog sib xws nrog qhov pom kev ntawm autophagosomes pom hauv vivo los ntawm immunohistochemistry thiab electron microscopy (Daim Duab S3). Txawm li cas los xij, qhov kev ua haujlwm OXPHOS tsis zoo hauv PNs yog nrog los ntawm kev hloov pauv ultrastructural mitochondrial pom tseeb. Mitochondrial clusters tuaj yeem pom hauv cov cell lub cev thiab cov ntoo dendritic ntawm Mfn2cKO PNs hnub nyoog 5 thiab 8 lub lis piam, thiab cov qauv membrane sab hauv tau dhau los ua kev hloov pauv loj heev (Daim Duab S4, A thiab B). Sib xws nrog cov kev hloov pauv ultrastructural no thiab kev txo qis tseem ceeb hauv mtDNA, kev tshuaj xyuas ntawm cov kab mob cerebral cerebellar nrog tetramethylrhodamine methyl ester (TMRM) qhia tau hais tias lub peev xwm ntawm mitochondrial membrane hauv Mfn2cKO PNs tau txo qis tseem ceeb (Daim Duab S4C).
(A) Kev tshuaj xyuas lub sijhawm ntawm qib kev qhia tawm ntawm OXPHOS complex. Tsuas yog xav txog cov protein nrog P <0.05 ntawm 8 lub lis piam (ob-txoj kev ANOVA). Kab dotted: Tsis muaj kev hloov kho piv rau CTRL. (B) Sab laug: Ib qho piv txwv ntawm ib ntu cerebellar uas muaj cov tshuaj tiv thaiv anti-MTCO1 (scale bar, 20 μm). Thaj chaw uas Purkinje cell lub cev nyob yog npog los ntawm daj. Sab xis: Kev ntsuas ntawm MTCO1 theem (ib-txoj kev tshuaj xyuas ntawm variance; n = 7 txog 20 cell soj ntsuam los ntawm peb tus nas). (C) qPCR kev tshuaj xyuas ntawm mtDNA daim ntawv theej hauv PN uas tau txheeb xyuas (ib-txoj kev tshuaj xyuas ntawm variance; n = 3 txog 7 tus nas). (D) Sab laug: Ib qho piv txwv ntawm ib daim cerebellar uas muaj cov tshuaj tiv thaiv anti-DNA (scale bar, 20 μm). Thaj chaw uas Purkinje cell lub cev nyob yog npog los ntawm daj. Sab xis: Kev ntsuas ntawm mtDNA lesions (ib-txoj kev tshuaj xyuas ntawm variance; n = 5 txog 9 cell los ntawm peb tus nas). (E) Ib qho piv txwv ntawm ib ntu cerebellar mob hnyav uas qhia txog mitoYFP + Purkinje hlwb (xub) hauv kev kaw cov ntaub ntawv tag nrho ntawm lub hlwb. (F) Kev ntsuas ntawm IV nkhaus. (G) Cov ntaub ntawv sawv cev ntawm kev txhaj tshuaj tam sim no hauv CTRL thiab Mfn2cKO Purkinje hlwb. Cov kab saum toj kawg nkaus: Lub zog thawj zaug uas ua rau AP. Cov kab hauv qab: Qhov siab tshaj plaws AP zaus. (H) Kev ntsuas ntawm cov tswv yim postsynaptic spontaneous (sPSPs). Cov kab sau ntawv sawv cev thiab nws qhov zoom piv tau qhia hauv (I). Ib txoj kev tshuaj xyuas ntawm qhov sib txawv tau tshuaj xyuas n = 5 txog 20 lub hlwb los ntawm peb tus nas. Cov ntaub ntawv tau qhia ua qhov nruab nrab ± SEM; *P <0.05; **P <0.01; ***P <0.001. (J) Cov kab sawv cev ntawm spontaneous AP kaw siv hom perforated patch clamp. Cov kab saum toj kawg nkaus: Qhov siab tshaj plaws AP zaus. Cov kab hauv qab: zoom ntawm ib qho AP. (K) Ntsuas qhov nruab nrab thiab siab tshaj plaws AP zaus raws li (J). Mann-Whitney xeem; n = 5 lub hlwb tau tshuaj xyuas los ntawm plaub tus nas. Cov ntaub ntawv raug qhia ua qhov nruab nrab ± SEM; tsis tseem ceeb.
Pom tseeb tias OXPHOS puas tsuaj tau pom tseeb hauv Mfn2cKO PN uas muaj 8 lub lis piam, qhia tias lub cev ua haujlwm ntawm cov neurons tsis zoo li qub. Yog li ntawd, peb tau tshuaj xyuas cov yam ntxwv hluav taws xob tsis zoo ntawm OXPHOS-deficient neurons thaum 4 txog 5 lub lis piam thiab 7 txog 8 lub lis piam los ntawm kev ua cov ntaub ntawv kaw tag nrho cov hlwb hauv cov kab mob cerebellar (Daim Duab 2E). Tsis tau xav txog, qhov nruab nrab ntawm lub peev xwm so thiab kev tawm tsam ntawm Mfn2cKO neurons zoo ib yam li kev tswj hwm, txawm hais tias muaj qhov sib txawv me ntsis ntawm cov hlwb (Rooj 1). Ib yam li ntawd, thaum muaj hnub nyoog 4 txog 5 lub lis piam, tsis muaj kev hloov pauv tseem ceeb hauv kev sib raug zoo tam sim no-voltage (IV nkhaus) tau pom (Daim Duab 2F). Txawm li cas los xij, tsis muaj Mfn2cKO neurons uas muaj 7 txog 8 lub lis piam tau dim ntawm IV regimen (kauj ruam hyperpolarization), qhia tias muaj kev nkag siab meej rau lub peev xwm hyperpolarization ntawm theem lig no. Qhov sib piv, hauv Mfn2cKO neurons, cov dej ntws depolarizing uas ua rau muaj kev rov ua dua (AP) tso tawm tau zoo, qhia tias lawv cov qauv tso tawm tag nrho tsis txawv ntawm cov neurons tswj 8-lub lis piam (Rooj 1 thiab Daim Duab 2G). Ib yam li ntawd, qhov zaus thiab qhov loj ntawm cov dej ntws postsynaptic spontaneous (sPSCs) tau sib piv rau cov ntawm pawg tswj, thiab qhov zaus ntawm cov xwm txheej nce ntxiv los ntawm 4 lub lis piam mus rau 5 lub lis piam mus rau 7 lub lis piam mus rau 8 lub lis piam nrog qhov nce zoo sib xws (Daim Duab 2, H thiab I). Lub sijhawm ntawm synaptic maturation hauv PNs (25). Cov txiaj ntsig zoo sib xws tau txais tom qab perforated PNs patches. Qhov kev teeb tsa no tiv thaiv qhov ua tau ntawm cov teeb meem cellular ATP, zoo li yuav tshwm sim hauv tag nrho-cell patch clamp sau. Tshwj xeeb, lub peev xwm so membrane thiab qhov zaus tua hluav taws ntawm Mfn2cKO neurons tsis raug cuam tshuam (Daim Duab 2, J thiab K). Hauv kev xaus, cov txiaj ntsig no qhia tau tias PNs nrog OXPHOS dysfunction pom tseeb tuaj yeem tiv taus cov qauv tso tawm ntau zaus zoo, qhia tias muaj cov txheej txheem them nyiaj uas tso cai rau lawv kom tswj tau cov lus teb electrophysiological ze li qub.
Cov ntaub ntawv raug qhia ua qhov nruab nrab ± SEM (kev tshuaj xyuas ib txoj kev ntawm qhov sib txawv, Holm-Sidak qhov kev xeem sib piv ntau yam; *P<0.05). Tus lej ntawm chav tsev raug qhia los ntawm cov ntawv cim.
Peb tau teem sijhawm los tshawb nrhiav seb puas muaj pawg twg hauv cov ntaub ntawv proteomics (Daim Duab 1G) suav nrog cov kev uas tuaj yeem tiv thaiv qhov tsis txaus OXPHOS hnyav, yog li piav qhia vim li cas PN cuam tshuam tuaj yeem tswj tau ze li qub electrophysiology (Daim Duab 2, E rau K). . Kev tshuaj xyuas Proteomics qhia tau hais tias cov enzymes koom nrog hauv catabolism ntawm branched chain amino acids (BCAA) tau tswj hwm ntau heev (Daim Duab 3A thiab Daim Duab S5A), thiab cov khoom kawg acetyl-CoA (CoA) lossis succinyl CoA tuaj yeem ntxiv cov tricarboxylates hauv arteriosclerosis Acid (TCA) voj voog. Peb pom tias cov ntsiab lus ntawm BCAA transaminase 1 (BCAT1) thiab BCAT2 ob qho tib si nce ntxiv. Lawv catalyze thawj kauj ruam ntawm BCAA catabolism los ntawm kev tsim glutamate los ntawm α-ketoglutarate (26). Txhua lub subunits uas ua rau cov branched chain keto acid dehydrogenase (BCKD) complex raug upregulated (cov complex ua rau muaj qhov decarboxylation tom qab thiab tsis rov qab tau ntawm cov BCAA carbon skeleton) (Daim Duab 3A thiab Daim Duab S5A). Txawm li cas los xij, tsis muaj kev hloov pauv pom tseeb hauv BCAA nws tus kheej tau pom hauv sorted PN, uas tej zaum yog vim muaj kev nce ntxiv ntawm cov amino acids tseem ceeb no lossis siv lwm qhov chaw (glucose lossis lactic acid) los ntxiv rau TCA lub voj voog (Daim Duab S5B). PNs uas tsis muaj OXPHOS kuj tau qhia txog kev nce ntxiv ntawm glutamine decomposition thiab transamination cov haujlwm thaum muaj hnub nyoog 8 lub lis piam, uas tuaj yeem pom los ntawm kev nce ntxiv ntawm cov mitochondrial enzymes glutaminase (GLS) thiab glutamine pyruvate transaminase 2 (GPT2) (Daim Duab 3, A thiab C). Nws yog ib qho tsim nyog sau tseg tias qhov kev nce qib ntawm GLS tsuas yog txwv rau qhov spliced ​​​​isoform glutaminase C (GLS-GAC) (qhov kev hloov pauv ntawm Mfn2cKO / CTRL yog kwv yees li 4.5-npaug, P = 0.05), thiab nws qhov kev nce qib tshwj xeeb hauv cov ntaub so ntswg mob qog noj ntshav tuaj yeem txhawb nqa mitochondrial bioenergy. (27).
(A) Daim ntawv qhia cua sov qhia txog qhov hloov pauv ntawm cov protein rau txoj kev teev tseg ntawm 8 lub lis piam. (B) Piv txwv ntawm ib daim cerebellar uas muaj cov tshuaj anti-PCx antibody (scale bar, 20 μm). Tus xub daj taw rau Purkinje cell body. (C) Kev tshuaj xyuas cov protein qhia txog lub sijhawm tau txheeb xyuas tias yog tus neeg sib tw tseem ceeb rau atherosclerosis (ntau t-test, *FDR <5%; n = 3-5 nas). (D) Saum toj no: Daim duab qhia txog ntau txoj hauv kev nkag mus rau cov pa roj carbon uas muaj nyob hauv [1-13C] pyruvate tracer (piv txwv li, ntawm PDH lossis trans-arterial route). Hauv qab: Daim ntawv qhia violin qhia txog feem pua ​​​​​​ntawm cov pa roj carbon ib leeg (M1) hloov mus rau aspartic acid, citric acid thiab malic acid tom qab sau cov cerebellar slices nrog [1-13C] pyruvate (paired t-test; ** P <0.01). (E) Kev tshuaj xyuas keeb kwm lub sijhawm ntawm txoj kev qhia. Tsuas yog xav txog cov protein nrog P <0.05 ntawm 8 lub lis piam. Kab txiav: tsis muaj tus nqi hloov kho (kev tshuaj xyuas ob txoj kev ntawm qhov sib txawv; * P <0.05; *** P <0.001). Cov ntaub ntawv tau qhia ua qhov nruab nrab ± SEM.
Hauv peb qhov kev tshuaj xyuas, BCAA catabolism tau dhau los ua ib qho ntawm cov kev tswj hwm tseem ceeb. Qhov tseeb no qhia tau hais tias qhov ntim cua nkag mus rau hauv lub voj voog TCA yuav raug hloov pauv hauv PN tsis muaj OXPHOS. Qhov no yuav sawv cev rau ib daim ntawv tseem ceeb ntawm kev hloov pauv neuronal metabolic, uas yuav muaj kev cuam tshuam ncaj qha rau neuronal physiology thiab kev ciaj sia nyob thaum lub sijhawm tswj hwm OXPHOS dysfunction hnyav. Sib xws nrog qhov kev xav no, peb pom tias lub ntsiab anti-atherosclerotic enzyme PCx yog up-regulated (Mfn2cKO / CTRL hloov pauv kwv yees li 1.5 zaug; Daim Duab 3A), uas catalyzes qhov hloov pauv ntawm pyruvate rau oxaloacetate (28), uas ntseeg tias nyob hauv cov ntaub so ntswg hauv lub hlwb. Kev qhia hauv yog txwv rau astrocytes (29, 30). Sib xws nrog cov txiaj ntsig proteomics, confocal microscopy qhia tau hais tias PCx kev qhia tau tshwj xeeb thiab tseem ceeb nce hauv OXPHOS-deficient PNs, thaum PCx reactivity feem ntau txwv rau Bergmann glial hlwb ntawm kev tswj hwm (Daim Duab 3B). Yuav kom kuaj tau qhov kev nce siab ntawm PCx, peb tau kho cov kab mob cerebellar nrog [1-13C] pyruvate tracer. Thaum pyruvate raug oxidized los ntawm pyruvate dehydrogenase (PDH), nws cov ntawv cim isotope ploj mus, Tab sis tau koom ua ke rau hauv TCA lub voj voog intermediates thaum pyruvate raug metabolized los ntawm cov tshuaj tiv thaiv vascular (Daim Duab 3D). Los txhawb peb cov ntaub ntawv proteomics, peb tau pom ntau tus cim los ntawm cov tracer no hauv cov kua qaub aspartic ntawm Mfn2cKO slices, thaum citric acid thiab malic acid kuj muaj qhov sib txawv nruab nrab, txawm tias tsis tseem ceeb (Daim Duab 3D).
Hauv cov dopamine neurons ntawm MitoPark nas nrog mitochondrial dysfunction los ntawm dopamine neurons tshwj xeeb rhuav tshem mitochondrial transcription factor A gene (Tfam) (Daim Duab S6B), PCx kev qhia kuj tau nce siab heev (31), qhia tias acetone acid arteriosclerosis Qhov tshwm sim ntawm tus kab mob yog tswj hwm thaum lub sijhawm ua haujlwm tsis zoo ntawm neuronal OXPHOS hauv lub cev. Nws yog ib qho tsim nyog sau cia tias nws tau pom tias cov enzymes tshwj xeeb (32-34) uas yuav qhia tawm hauv neurons uas yuav cuam tshuam nrog arteriosclerosis tau nce siab heev hauv PNs tsis muaj OXPHOS, xws li propionyl-CoA carboxylase (PCC-A), Malonyl-CoA hloov propionyl-CoA rau succinyl-CoA thiab mitochondrial malic enzyme 3 (ME3), uas nws lub luag haujlwm tseem ceeb yog rov qab pyruvate los ntawm malate (Daim Duab 3, A thiab C) (33, 35). Ntxiv rau, peb pom muaj kev nce ntxiv ntawm Pdk3 enzyme, uas phosphorylates thiab yog li ntawd inactivates PDH (36), thaum tsis muaj kev hloov pauv tau pom hauv Pdp1 enzyme uas ua kom PDH lossis PDH enzyme complex nws tus kheej (Daim Duab 3A). Tsis tu ncua, hauv Mern2cKO PNs, phosphorylation ntawm α1 subunit α (PDHE1α) subunit ntawm pyruvate dehydrogenase E1 Cheebtsam ntawm PDH complex hauv Ser293 (paub tias inhibit qhov enzyme ua haujlwm ntawm PDH) tau txhim kho (Daim Duab S6C) (Daim Duab S6C). Pyruvate tsis muaj kev nkag mus rau hauv cov hlab ntsha.
Thaum kawg, peb pom tias txoj kev super ntawm serine thiab glycine biosynthesis, qhov cuam tshuam mitochondrial folate (1C) voj voog thiab proline biosynthesis (Daim Duab 1G thiab Daim Duab S5C) yog txhua yam up-regulated, raws li cov ntawv ceeb toom, thaum lub sijhawm ua kom muaj zog. Cov ntaub so ntswg nyob ib puag ncig raug ua kom muaj zog nrog mitochondrial dysfunction (5-7). Kev tshuaj xyuas confocal txhawb nqa cov ntaub ntawv proteomics no qhia tau tias hauv PN nrog OXPHOS ploj lawm, cerebellar slices ntawm 8-lub lis piam-laus nas tau raug rau serine hydroxymethyltransferase 2 (SHMT2), ib qho enzyme tseem ceeb ntawm mitochondrial folate voj voog. Kev teb tiv thaiv kab mob tseem ceeb (Daim Duab S5D). Hauv 13 CU-glucose-incubated acute cerebellar slices, kev sim metabolic tracing ntxiv lees paub qhov up-regulation ntawm serine thiab proline biosynthesis, qhia tias cov flux ntawm carbon isoforms rau hauv serine thiab proline nce (Daim Duab S5E). Vim tias cov tshuaj tiv thaiv uas GLS thiab GPT2 txhawb nqa yog lub luag haujlwm rau kev tsim cov glutamate los ntawm glutamine thiab kev hloov pauv ntawm glutamate thiab α-ketoglutarate, lawv qhov kev nce qib qhia tau hais tias OXPHOS-deficient neurons muaj kev thov ntau ntxiv rau glutamate, Qhov no yuav yog lub hom phiaj ntawm kev tswj hwm qhov kev tsim cov proline ntau ntxiv (Daim Duab S5C). Hauv kev sib piv rau cov kev hloov pauv no, kev tshuaj xyuas proteomic ntawm cerebellar astrocytes los ntawm PN-specific Mfn2cKO nas tau qhia tias cov kev no (suav nrog txhua yam antiperoxidases) tsis tau hloov pauv ntau hauv kev qhia tawm, yog li qhia tau tias qhov kev hloov pauv metabolic no yog xaiv rau PN uas puas tsuaj (Daim Duab S6, D rau G).
Hauv kev xaus, cov kev tshuaj xyuas no tau qhia txog cov qauv sib txawv ntawm kev ua haujlwm ntawm lub sijhawm ntawm cov txheej txheem metabolic tshwj xeeb hauv PNs. Txawm hais tias kev ua haujlwm tsis zoo ntawm neuronal mitochondrial tuaj yeem ua rau atherosclerosis thaum ntxov thiab 1C remodeling (Daim Duab 3E thiab Daim Duab S5C), thiab txawm tias qhov kev hloov pauv kwv yees tau hauv kev qhia tawm ntawm I thiab IV complexes, qhov kev hloov pauv hauv serine de novo synthesis tsuas yog Nws tsuas yog pom tseeb hauv cov theem kawg. OXPHOS dysfunction (Daim Duab 3E thiab Daim Duab S5C). Cov kev tshawb pom no txhais cov txheej txheem sib law liag uas kev ntxhov siab ua rau mitochondrial (1C voj voog) thiab cytoplasmic (serine biosynthesis) teb synergistically nrog kev nce hauv atherosclerosis hauv TCA voj voog los hloov kho neuronal metabolism.
Cov PNs uas tsis muaj OXPHOS uas muaj 8 lub lis piam tuaj yeem tswj tau qhov kev ua haujlwm siab thiab rov ua dua qhov kev sib txuas lus tseem ceeb los them rau qhov tsis ua haujlwm ntawm mitochondrial. Qhov kev tshawb pom no ua rau muaj qhov ua tau zoo uas txawm tias lub sijhawm no, cov hlwb no kuj tseem yuav tau txais kev kho mob kom ncua lossis tiv thaiv kev puas tsuaj ntawm lub hlwb. Lig. Peb tau daws qhov ua tau no los ntawm ob qho kev cuam tshuam ywj pheej. Hauv thawj txoj kev, peb tau tsim Cre-dependent adeno-associated virus (AAV) vector kom MFN2 tuaj yeem xaiv tau hauv OXPHOS-deficient PNs hauv vivo (Daim Duab S7A). AAV encoding MFN2 thiab fluorescent reporter gene mCherry (Mfn2-AAV) tau txheeb xyuas hauv cov kab lis kev cai neuron thawj zaug hauv vitro, uas ua rau MFN2 raug qhia tawm hauv Cre-dependent thiab cawm mitochondrial morphology, yog li tiv thaiv kev hloov pauv ntawm lub hlwb hauv Mfn2cKO neurons (Daim Duab S7, B, D thiab E). Tom ntej no, peb tau ua qhov kev sim hauv vivo kom xa Mfn2-AAV uas muaj 8 lub lis piam mus rau lub hlwb cerebellar ntawm Mfn2cKO thiab cov nas tswj, thiab tau tshuaj xyuas cov nas uas muaj 12 lub lis piam (Daim Duab 4A). Cov nas Mfn2cKO uas tau kho tau tuag lawm (Daim Duab 1, A thiab B) (16). Kev hloov pauv ntawm cov kab mob hauv vivo ua rau muaj kev xaiv ntawm PN hauv qee lub voj voog cerebellar (Daim Duab S7, G thiab H). Kev txhaj tshuaj ntawm AAV tswj uas qhia tsuas yog mCherry (Ctrl-AAV) tsis muaj qhov cuam tshuam loj rau qib ntawm kev puas tsuaj ntawm cov hlwb hauv Mfn2cKO cov tsiaj. Qhov sib txawv, kev tshuaj xyuas ntawm Mfn2cKOs hloov pauv nrog Mfn2-AAV tau qhia txog qhov cuam tshuam loj ntawm cov txheej PN cell (Daim Duab 4, B thiab C). Tshwj xeeb, qhov ceev ntawm cov hlwb neuron zoo li yuav luag tsis sib txawv ntawm cov tsiaj tswj (Daim Duab 4, B thiab C, thiab Daim Duab S7, H thiab I). Qhov kev qhia tawm ntawm MFN1 tab sis tsis yog MFN2 muaj txiaj ntsig zoo ib yam hauv kev cawm txoj kev tuag ntawm cov hlab ntsha (Daim Duab 4C thiab Daim Duab S7, C thiab F), uas qhia tau tias qhov kev qhia tawm ntawm ectopic MFN1 tuaj yeem pab txhawb qhov tsis muaj MFN2. Kev tshuaj xyuas ntxiv ntawm ib qib PN qhia tau tias Mfn2-AAV feem ntau cawm tau ultrastructure ntawm mitochondria, ua kom cov qib mtDNA zoo li qub, thiab thim rov qab qhov kev qhia tawm siab ntawm cov cim tiv thaiv angiogenesis PCx ​​(Daim Duab 4, C rau E). Kev tshuaj xyuas pom ntawm cov nas Mfn2cKO uas tau cawm dim hauv lub xeev so tau qhia tias lawv cov tsos mob thiab cov tsos mob ntawm lub cev (kev txav mus los S1 rau S3) tau zoo dua. Hauv kev xaus, cov kev sim no qhia tau tias kev ncua rov qab ntawm MFN2 rau hauv PNs uas tsis muaj zog hauv OXPHOS txaus los thim rov qab mtDNA noj thiab ua rau atherosclerosis, yog li tiv thaiv axon degeneration thiab neuronal tuag hauv vivo.
(A) Ib daim phiaj xwm qhia txog lub sijhawm sim rau kev txhaj tshuaj AAV encoding MFN2 thaum txoj kev metabolic tau qhia. (B) Cov duab confocal sawv cev ntawm 12-lub lis piam-laus cerebellar slices transduced ntawm 8 lub lis piam hauv Mfn2cKO nas thiab cim nrog anti-Calbindin antibody. Sab xis: Scaling ntawm axon fibers. Qhov ntsuas ntawm axon zoom yog 450 thiab 75 μm. (C) Sab laug: Quantification ntawm Purkinje cell density hauv AAV transduction loop (AAV +) (ib txoj kev tshuaj xyuas ntawm variance; n = 3 nas). Sab xis: mtDNA tsom xam hauv transduced PN ntawm lub lim tiam 12 (unpaired t-test; n = 6 hlwb los ntawm peb nas). * P <0.05; ** P <0.01. (D) Tus sawv cev kis electron micrographs ntawm PNs ntawm Mfn2cKO cerebellar seem transduced nrog cov kab mob qhia vectors. Lub ntsej muag liab qhia txog thaj chaw uas dendrites nyob, thiab lub xwmfab daj qhia txog qhov zoom uas muab rau sab xis; n sawv cev rau lub nucleus. Scale bar, 1μm. (E) qhia txog ib qho piv txwv ntawm PCx staining hauv PN transduced ntawm 12 lub lis piam. Scale bar, 20μm. OE, overexpression; FC, quav hloov.
Thaum kawg, peb tau tshawb nrhiav qhov tseem ceeb ntawm kev ciaj sia ntawm cov hlwb peroxidase hauv PNs uas tau ntsib OXPHOS dysfunction. Peb tau tsim mCherry encoding AAV-shRNA (luv luv hairpin RNA) tshwj xeeb rau nas PCx mRNA (AAV-shPCx), thiab txhaj tshuaj tus kab mob lossis nws cov kev tswj hwm scrambled (AAV-scr) rau hauv cerebellum ntawm Mfn2cKO nas. Kev txhaj tshuaj tau ua tiav hauv lub lim tiam plaub ntawm hnub nyoog (Daim Duab 5A) kom ua tiav PCx knockdown zoo thaum lub sijhawm thaum PCx kev qhia nce ntxiv (Daim Duab 3C) thiab PN cell txheej tseem nyob ruaj khov (Daim Duab 1A). Nws yog ib qho tsim nyog sau tseg tias kev khob PCx (Daim Duab S8A) ua rau muaj kev nrawm ntawm PN tuag, uas tsuas yog txwv rau lub nplhaib kis kab mob (Daim Duab 5, B thiab C). Yuav kom nkag siab txog cov txheej txheem ntawm cov teebmeem metabolic uas tshwm sim los ntawm PCx up-regulation, peb tau kawm txog qhov xwm txheej redox ntawm PNs tom qab PCx knockdown thiab AAV-mediated optical biosensor Grx1-roGFP2 tau qhia tib lub sijhawm (Daim Duab S8, B txog D) los ntsuas glutathione Qhov kev hloov pauv ntawm peptide redox peev xwm (38). Tom qab ntawd, peb tau ua ob-photon fluorescence lifetime imaging microscopy (FLIM) hauv cov hlwb ntse ntawm 7-lub lis piam-laus Mfn2cKO lossis cov neeg tswj hwm kom pom cov kev hloov pauv hauv cytoplasmic redox xwm txheej tom qab txheeb xyuas FLIM cov xwm txheej (Daim Duab S8, E txog G). Qhov kev tshuaj xyuas tau qhia txog kev nce ntxiv hauv lub xeev oxidation ntawm ib qho Mfn2cKO PNs uas tsis muaj PCx kev qhia, uas txawv ntawm kev tswj neurons lossis Mfn2cKO PNs qhia tsuas yog scrambled shRNA (Daim Duab 5, D thiab E). Thaum PCx kev qhia tawm raug txo qis, feem pua ​​​​​​ntawm Mfn2cKO PNs uas qhia txog lub xeev oxidized heev tau nce ntau dua peb zaug (Daim Duab 5E), qhia tias PCx kev tswj hwm tau tswj hwm lub peev xwm redox ntawm cov neurons degenerated.
(A) Ib daim duab qhia txog lub sijhawm sim rau kev txhaj tshuaj AAV encoding shPCx thaum txoj kev metabolic tau qhia. (B) Cov duab confocal sawv cev ntawm 8-lub lis piam-laus cerebellar seem hauv Mfn2cKO nas transduced thiab labeled nrog anti-calcineurin antibody ntawm 4 lub lis piam. Scale bar, 450μm. (C) Kev ntsuas ntawm Purkinje cell ceev hauv AAV-transduced loops (ib txoj kev tshuaj xyuas ntawm variance; n = 3 txog 4 nas). Cov ntaub ntawv tau qhia ua mean ± SEM; ***P <0.001. (D) Daim duab sawv cev FLIM qhia txog lub neej nruab nrab ntawm 7-lub lis piam-laus PN qhia glutathione redox sensor Grx1-roGFP2 nyob rau hauv cov xwm txheej sim tshwj xeeb. LUT (saib-up rooj) piv: lub sijhawm ciaj sia (hauv picoseconds). Scale bar, 25μm. (E) Daim histogram qhia txog kev faib tawm ntawm Grx1-roGFP2 lub neej tus nqi los ntawm (D) (n = 158 txog 368 lub hlwb hauv ob lub nas nyob rau hauv txhua qhov xwm txheej). Daim duab qhia txog lub ncuav qab zib saum toj no txhua daim histogram: qhia tus lej ntawm cov hlwb nrog lub neej ntev dua (liab, oxidized) lossis luv dua (xiav, txo qis), uas tshaj 1 SD ntawm qhov nruab nrab lub neej tus nqi hauv CTRL-AAV-scr. (F) Tus qauv pom zoo qhia txog kev tiv thaiv ntawm kev nce qib ntawm neuronal PCx.
Tag nrho hauv txhua qhov, cov ntaub ntawv peb muab ntawm no qhia tau hais tias qhov rov qhia tawm ntawm MFN2 tuaj yeem cawm tau tag nrho cov PN siab heev nrog OXPHOS tsis txaus, mtDNA poob qis heev, thiab morphology zoo li ista tsis zoo, yog li muab kev nce qib txuas ntxiv txawm tias nyob rau hauv cov kab mob siab heev. Neurodegeneration muab pov thawj rov qab tau ntawm theem ua ntej cell tuag. Qib ntawm kev hloov pauv metabolic no tau hais ntxiv los ntawm lub peev xwm ntawm neurons los ua rau atherosclerosis (kev rov ua dua ntawm lub voj voog TCA), uas inhibits PCx kev qhia tawm hauv PNs tsis muaj OXPHOS thiab ua rau cell tuag, yog li ua lub luag haujlwm tiv thaiv (Daim Duab 5F).
Hauv kev tshawb fawb no, peb tau muab pov thawj tias cov lus teb ntawm PNs rau OXPHOS dysfunction yog maj mam sib sau ua ke rau TCA lub voj voog atherosclerosis los ntawm txoj kev sib txawv ua haujlwm ua los ntawm cov kev pab cuam metabolic. Peb tau lees paub qhov kev tshuaj xyuas proteomic nrog ntau txoj kev sib txuas thiab qhia tias thaum muaj kev sib tw los ntawm kev ua haujlwm tsis zoo mitochondrial, neurons muaj daim ntawv tsis paub yav dhau los ntawm metabolic elasticity. Rau peb qhov xav tsis thoob, tag nrho cov txheej txheem rewiring tsis tas yuav cim lub xeev kawg metabolic uas nrog neurodegeneration maj mam thiab tsis rov qab, tab sis peb cov ntaub ntawv qhia tias nws yuav tsim ib qho neuron txij nkawm txawm tias nyob rau theem ua ntej cell tuag Functional compensation mechanism. Qhov kev tshawb pom no qhia tau hais tias neurons muaj qib siab ntawm metabolic plasticity hauv lub cev. Qhov tseeb no ua pov thawj tias qhov rov qab los ntawm MFN2 tom qab tuaj yeem thim rov qab qhov kev qhia ntawm cov cim tseem ceeb metabolic thiab tiv thaiv PN degeneration. Ntawm qhov tsis sib xws, nws inhibits atherosclerosis thiab ua kom cov hlab ntsha ceev. transsexual.
Ib qho ntawm cov kev tshawb pom zoo tshaj plaws hauv peb txoj kev tshawb fawb yog tias PNs uas tsis muaj OXPHOS tuaj yeem hloov kho TCA lub voj voog metabolism los ntawm kev nce-tswj cov enzymes uas tshwj xeeb txhawb arteriosclerosis. Metabolic rearrangement yog ib qho feature ntawm cov qog nqaij hlav, qee qhov uas vam khom glutamine los ntxiv TCA lub voj voog intermediates los tsim cov khoom sib npaug, uas tsav cov saw hlau ua pa thiab tswj kev tsim cov lipid thiab nucleotide biosynthesis precursors (39, 40). Ib txoj kev tshawb fawb tsis ntev los no tau qhia tias hauv cov ntaub so ntswg peripheral uas muaj OXPHOS dysfunction, qhov kev sib txuas ntawm glutamine / glutamate metabolism kuj yog ib qho feature tseem ceeb (5, 41), qhov twg qhov kev taw qhia ntawm glutamine nkag mus rau hauv TCA lub voj voog nyob ntawm Vim yog qhov hnyav ntawm OXPHOS raug mob (41). Txawm li cas los xij, tsis muaj pov thawj meej meej txog kev zoo sib xws ntawm neuronal metabolic plasticity hauv lub cev thiab nws qhov cuam tshuam rau hauv cov kab mob. Hauv kev tshawb fawb tsis ntev los no hauv vitro, thawj cortical neurons tau pom tias yuav mobilize glutamate pas dej rau neurotransmission, yog li txhawb nqa oxidative metabolism thiab atherosclerosis nyob rau hauv metabolic kev ntxhov siab mob (42). Nws yog ib qho tsim nyog sau tseg tias nyob rau hauv kev txwv tsis pub siv tshuaj ntawm TCA lub voj voog enzyme succinate dehydrogenase, pyruvate carboxylation ntseeg tau tias yuav tswj tau qhov kev tsim cov oxaloacetate hauv cov kab lis kev cai cerebellar granule neurons (34). Txawm li cas los xij, qhov tseem ceeb ntawm cov txheej txheem no rau cov ntaub so ntswg hauv lub hlwb (qhov twg atherosclerosis ntseeg tau tias feem ntau yog txwv rau astrocytes) tseem muaj qhov tseem ceeb ntawm lub cev (43). Hauv qhov no, peb cov ntaub ntawv qhia tau hais tias PNs puas tsuaj los ntawm OXPHOS hauv lub cev tuaj yeem hloov mus rau BCAA degradation thiab pyruvate carboxylation, uas yog ob qhov chaw tseem ceeb ntawm kev ntxiv ntawm TCA pas dej nruab nrab. Txawm hais tias qhov kev koom tes ntawm BCAA catabolism rau neuronal lub zog metabolism tau raug pom zoo, ntxiv rau lub luag haujlwm ntawm glutamate thiab GABA rau neurotransmission (44), tseem tsis muaj pov thawj rau cov txheej txheem no hauv vivo. Yog li ntawd, nws yooj yim los xav tias dysfunctional PNs tuaj yeem them rau kev noj ntawm TCA intermediates tsav los ntawm cov txheej txheem assimilation los ntawm kev nce atherosclerosis. Tshwj xeeb, kev nce qib ntawm PCx yuav tsum tau ua kom muaj kev thov ntau ntxiv rau aspartic acid, uas tau hais hauv cov hlwb proliferating nrog mitochondrial dysfunction (45). Txawm li cas los xij, peb qhov kev tshuaj xyuas metabolimomics tsis tau qhia txog kev hloov pauv tseem ceeb hauv qib ruaj khov ntawm aspartic acid hauv Mfn2cKO PNs (Daim Duab S6A), uas tej zaum yuav qhia txog kev siv metabolic sib txawv ntawm aspartic acid ntawm cov hlwb proliferating thiab post-mitotic neurons. Txawm hais tias qhov tseeb ntawm PCx upregulation hauv dysfunctional neurons hauv vivo tseem yuav tsum tau piav qhia, peb tau qhia tias qhov kev teb ua ntej no ua lub luag haujlwm tseem ceeb hauv kev tswj hwm lub xeev redox ntawm neurons, uas tau pom hauv FLIM kev sim ntawm cerebellar slices. Tshwj xeeb, kev tiv thaiv PNs los ntawm kev nce qib PCx tuaj yeem ua rau lub xeev oxidized ntau dua thiab ua kom lub hlwb tuag sai dua. Kev ua haujlwm ntawm BCAA degradation thiab carboxylation ntawm pyruvate tsis yog txoj hauv kev los piav qhia cov ntaub so ntswg ntawm mitochondrial dysfunction (7). Yog li ntawd, lawv zoo li yog qhov tseem ceeb ntawm cov neurons uas tsis muaj OXPHOS, txawm tias tsis yog tib qho tseem ceeb, uas yog qhov tseem ceeb rau kev puas tsuaj ntawm lub paj hlwb.
Kab mob Cerebellar yog ib hom kab mob neurodegenerative heterogeneous uas feem ntau tshwm sim ua ataxia thiab feem ntau ua rau PNs puas tsuaj (46). Cov pej xeem neuron no tshwj xeeb tshaj yog muaj kev phom sij rau mitochondrial dysfunction vim tias lawv qhov kev xaiv degeneration hauv nas txaus los rov ua ntau yam ntawm cov tsos mob ntawm lub cev uas piav qhia txog tib neeg spinocerebellar ataxia (16, 47, 48). Raws li cov lus ceeb toom, tus qauv nas transgenic nrog lub noob hloov pauv yog cuam tshuam nrog tib neeg spinocerebellar ataxia thiab muaj mitochondrial dysfunction (49, 50), hais txog qhov tseem ceeb ntawm kev kawm txog cov txiaj ntsig ntawm OXPHOS tsis txaus hauv PNPH. Yog li ntawd, nws yog qhov tsim nyog tshwj xeeb los cais tawm thiab kawm txog cov pej xeem neuron tshwj xeeb no. Txawm li cas los xij, muab hais tias PNs muaj kev nkag siab heev rau qhov siab thiab suav nrog qhov feem pua ​​​​​​tsawg ntawm tag nrho cov pej xeem cerebellar cell, rau ntau qhov kev tshawb fawb raws li omics, kev sib cais xaiv ntawm lawv ua tag nrho cov hlwb tseem yog qhov nyuaj. Txawm hais tias nws yuav luag tsis yooj yim sua kom tsis muaj kev sib kis ntawm lwm hom cell (tshwj xeeb tshaj yog cov ntaub so ntswg laus), peb tau ua ke ib kauj ruam dissociation zoo nrog FACS kom tau txais cov neurons txaus rau kev tshuaj xyuas proteomics downstream, thiab muaj cov protein ntau heev (kwv yees li 3000 protein) piv rau cov ntaub ntawv teeb tsa uas twb muaj lawm ntawm tag nrho cerebellum (51). Los ntawm kev khaws cia qhov muaj sia nyob ntawm tag nrho cov cell, txoj kev uas peb muab ntawm no tso cai rau peb tsis yog tsuas yog xyuas cov kev hloov pauv hauv cov txheej txheem metabolic hauv mitochondria, tab sis kuj xyuas cov kev hloov pauv hauv nws cov cytoplasmic counterparts, uas ua tiav kev siv cov cim mitochondrial membrane los nplua nuj hom cell Txoj kev tshiab rau tus lej ntawm mitochondria hauv cov ntaub so ntswg nyuaj (52, 53). Txoj kev uas peb piav qhia tsis yog tsuas yog cuam tshuam nrog kev kawm ntawm Purkinje hlwb, tab sis tuaj yeem siv tau yooj yim rau txhua hom cell los daws cov kev hloov pauv metabolic hauv cov hlwb mob, suav nrog lwm cov qauv ntawm mitochondrial dysfunction.
Thaum kawg, peb tau txheeb xyuas lub qhov rais kho mob thaum lub sijhawm hloov pauv metabolic no uas tuaj yeem thim rov qab cov cim tseem ceeb ntawm kev ntxhov siab ntawm tes thiab tiv thaiv kev puas tsuaj ntawm cov hlab ntsha. Yog li ntawd, kev nkag siab txog cov txiaj ntsig ntawm kev hloov pauv uas tau piav qhia ntawm no yuav muab cov kev nkag siab tseem ceeb rau kev kho mob rau kev tswj hwm lub zog ntawm cov hlab ntsha thaum lub sijhawm ua haujlwm tsis zoo ntawm mitochondrial. Kev tshawb fawb yav tom ntej uas tsom mus rau kev txiav txim siab cov kev hloov pauv hauv kev hloov pauv zog hauv lwm hom hlwb hlwb yog qhov xav tau los qhia tag nrho qhov kev siv tau ntawm lub hauv paus ntsiab lus no rau lwm yam kab mob neurological.
Cov nas MitoPark tau piav qhia ua ntej lawm (31). Cov nas C57BL/6N nrog cov noob caj noob ces loxP flanking Mfn2 tau piav qhia ua ntej lawm (18) thiab hla nrog cov nas L7-Cre (23). Cov noob caj noob ces ob chav heterozygous tau hla nrog cov nas homozygous Mfn2loxP/Mfn2loxP los tsim Purkinje-specific gene knockouts rau Mfn2 (Mfn2loxP/Mfn2loxP; L7-cre). Hauv ib pawg ntawm kev sib deev, Gt (ROSA26) SorStop-mito-YFP allele (nres-mtYFP) tau qhia los ntawm kev hla ntxiv (20). Txhua txoj kev ua haujlwm ntawm tsiaj tau ua raws li cov lus qhia ntawm European, lub tebchaws thiab cov koom haum thiab tau pom zoo los ntawm LandesamtfürNatur ntawm Umwelt thiab Verbraucherschutz, North Rhine-Westphalia, Lub Tebchaws Yelemees. Kev ua haujlwm ntawm tsiaj kuj ua raws li kev taw qhia ntawm European Federation of Laboratory Animal Sciences Associations.
Tom qab muab tshuaj loog rau tus poj niam cev xeeb tub lub caj dab, tus nas embryo raug rho tawm (E13). Lub cortex tau txiav hauv Hanks' Balanced Salt Solution (HBSS) ntxiv nrog 10 mM Hepes thiab dhau mus rau Dulbecco's Modified Eagle's Medium uas muaj papain (20 U / ml) thiab cysteine ​​​​(1μg / ml). Incubate cov ntaub so ntswg hauv DMEM) thiab dissociate nws los ntawm enzymatic digestion. Ml) ntawm 37 ° C rau 20 feeb, thiab tom qab ntawd mechanically milled hauv DMEM ntxiv nrog 10% fetal bovine serum. Cov hlwb tau cog rau ntawm iav npog coated nrog polylysine ntawm qhov ceev ntawm 2 × 106 ib 6 cm kab lis kev cai tais lossis ntawm qhov ceev ntawm 0.5 × 105 hlwb / cm2 rau kev tshuaj ntsuam duab. Tom qab 4 teev, cov nruab nrab tau hloov nrog Neurobasal serum-free nruab nrab uas muaj 1% B27 ntxiv thiab 0.5 mM GlutaMax. Cov neurons ces tau khaws cia ntawm 37 ° C thiab 5% CO2 thoob plaws hauv kev sim, thiab pub ib zaug ib lub lim tiam. Txhawm rau kom muaj kev sib xyaw ua ke hauv vitro, 3μl (24-qhov tais kab lis kev cai) lossis 0.5μl (24-qhov tais) ntawm cov kab mob AAV9 hauv qab no tau siv los kho cov neurons rau hnub thib ob hauv vitro: AAV9.CMV.PI.eGFP. WPRE.bGH (Addgene, tus lej catalog 105530-AAV9) thiab AAV9.CMV.HI.eGFP-Cre.WPRE.SV40 (Addgene, tus lej catalog 105545-AAV9).
Cov DNA sib txuas ntawm nas Mfn1 thiab Mfn2 (tau los ntawm Addgene plasmid #23212 thiab #23213, feem) yog cim nrog V5 kab lus (GKPIPNPLLGLDST) ntawm C-terminus, thiab raug sib xyaw nrog mCherry hauv thav duab los ntawm T2A kab lus. Grx1-roGFP2 yog khoom plig los ntawm Heidelberg TP Dick DFKZ (Deutsches Krebsforschungszentrum). Los ntawm kev hloov tdTomato cassette siv cov txheej txheem cloning ib txwm muaj, lub cassette tau subcloned rau hauv pAAV-CAG-FLEX-tdTomato backbone (Addgene tus lej siv 28306) los tsim pAAV-CAG-FLEX-mCherry-T2A-MFN2-V5, pAAV-CAG-FLEX-mCherry-T2A-MFN1-V5 thiab pAAV-CAG-FLEX-Grx-roGFP2 vectors. Ib txoj kev zoo sib xws tau siv los tsim cov vector tswj pAAV-CAG-FLEX-mCherry. Yuav kom tsim tau AAV-shPCx construct, yuav tsum muaj plasmid AAV vector (VectorBuilder, pAAV [shRNA] -CMV-mCherry-U6-mPcx- [shRNA#1]) uas muaj cov DNA sequence encoding lub shRNA targeting nas PCx (5′CTTTCGCTCTAAGGTGCTAAACTCGAGTTTAGCACCTTAGAGCGAAAG 3′) Nyob rau hauv kev tswj hwm ntawm U6 promoter, mCherry yog siv nyob rau hauv kev tswj hwm ntawm CMV promoter. Kev tsim cov auxiliary AAV vectors tau ua raws li cov lus qhia ntawm cov chaw tsim khoom (Cell Biolabs). Hauv ntej, siv lub plasmid hloov pauv uas nqa mCherry-T2A-MFN2-V5 (pAAV-CAG-FLEX-mCherry-T2A-MFN2-V5), mCherry-T2A-MFN1-V5 (pAAV-CAG-FLEX-mCherry) ib ntus Transfection ntawm 293AAV hlwb-T2A-MFN1-V5), mCherry (pAAV-CAG-FLEX-mCherry) lossis Grx-roGFP2 (pAAV-CAG-FLEX-Grx-roGFP2) coding gene, nrog rau coding AAV1 capsid protein thiab accessory protein Packaging plasmid plasmid, siv txoj kev calcium phosphate. Cov kab mob nyoos supernatant tau txais los ntawm kev khov-thaw voj voog hauv cov dej khov qhuav/ethanol da dej thiab cov hlwb lysed hauv phosphate buffered saline (PBS). Tus AAV vector tau purified los ntawm discontinuous iodixanol gradient ultracentrifugation (24 teev ntawm 32,000 rpm thiab 4 ° C) thiab concentrated siv lub lim dej Amicon ultra-15 centrifugal. Genome titer ntawm 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 yog raws li tau piav qhia ua ntej (54), ntsuas los ntawm lub sijhawm tiag tiag quantitative PCR (qPCR) -MFN1-V5 (1.9 × 1013 GC / ml) thiab AAV1-CAG-FLEX-Grx-roGFP2 (8.9 × 1012 GC / ml).
Cov hlwb tseem ceeb tau raug khawb tawm hauv cov dej khov 1x PBS, pelleted, thiab tom qab ntawd homogenized hauv 0.5% Triton X-100 / 0.5% sodium deoxycholate/PBS lysis buffer uas muaj phosphatase thiab protease inhibitor (Roche). Kev ntsuas protein tau ua los ntawm kev siv bicinchoninic acid assay (Thermo Fisher Scientific). Cov protein tau raug cais los ntawm SDS-polyacrylamide gel electrophoresis, thiab tom qab ntawd blotted rau ntawm polyvinylidene fluoride membrane (GE Healthcare). Thaiv cov chaw tsis tshwj xeeb thiab incubate nrog cov tshuaj tiv thaiv thawj zaug (saib Rooj S1 rau cov ntsiab lus) hauv 5% mis nyuj hauv TBST (Tris-buffered saline nrog Tween), cov kauj ruam ntxuav thiab cov tshuaj tiv thaiv thib ob hauv TBST Incubate. Incubate nrog cov tshuaj tiv thaiv thawj zaug ib hmos ntawm +4 ° C. Tom qab ntxuav, siv cov tshuaj tiv thaiv thib ob rau 2 teev ntawm chav sov. Tom qab ntawd, los ntawm kev incubating tib lub blot nrog cov tshuaj tiv thaiv anti-β-actin, tib qhov kev thauj khoom tau lees paub. Kev kuaj pom los ntawm kev hloov mus rau chemiluminescence thiab txhim kho chemiluminescence (GE Healthcare).
Cov neurons uas tau cog rau ntawm cov iav npog tau kho nrog 4% paraformaldehyde (PFA) / PBS ntawm lub sijhawm teev tseg ntawm chav sov li 10 feeb. Cov npog tau nkag mus rau hauv thawj zaug nrog 0.1% Triton X-100 / PBS rau 5 feeb ntawm chav sov, thiab tom qab ntawd hauv blocking buffer [3% bovine serum albumin (BSA) / PBS]. Nyob rau hnub thib ob, cov npog tau ntxuav nrog blocking buffer thiab incubated nrog cov tshuaj fluorophore-conjugated secondary antibody tsim nyog rau 2 teev ntawm chav sov; thaum kawg, cov qauv tau ntxuav kom huv si hauv PBS nrog 4′, 6-diamidino-2 -Phenylindole (DAPI) yog counterstained thiab tom qab ntawd kho rau ntawm lub microscope slide nrog Aqua-Poly / Mount.
Cov nas (txiv neej thiab poj niam) tau raug tshuaj loog los ntawm kev txhaj tshuaj ketamine (130 mg/kg) thiab xylazine (10 mg/kg) rau hauv plab thiab muab tshuaj carprofen analgesic (5 mg/kg) rau hauv qab daim tawv nqaij, Thiab muab tso rau hauv lub cuab yeej stereotactic (Kopf) uas muaj lub ncoo sov so. Qhib lub pob txha taub hau thiab siv lub tshuab xuas hniav kom nyias nyias ib feem ntawm lub hlwb cerebellar cortex uas sib raug rau lub pob txha mis (los ntawm lambda: tus Tsov tus tw 1.8, sab 1, sib raug rau lobules IV thiab V). Siv lub koob txhaj tshuaj nkhaus kom ua tib zoo tsim ib lub qhov me me hauv lub pob txha taub hau kom tsis txhob cuam tshuam cov hlab ntsha hauv qab no. Tom qab ntawd cov capillary iav nyias nyias maj mam ntxig rau hauv lub qhov me me (los ntawm -1.3 txog -1 ntawm sab ventral ntawm dura mater), thiab 200 txog 300 nl AAV raug txhaj rau hauv lub koob txhaj tshuaj me me (Narishige) nrog cov koob txhaj tshuaj tes (Narishige) ntau zaus ntawm qhov siab qis dhau lub sijhawm 10 txog 20 feeb qhov rai. Tom qab txhaj tshuaj lawm, muab cov hlab ntsha tso rau lwm 10 feeb kom tus kab mob kis tau tag nrho. Tom qab cov hlab ntsha raug rho tawm, daim tawv nqaij raug xaws kom zoo kom txo qhov mob o thiab cia tus tsiaj rov zoo. Cov tsiaj tau kho nrog tshuaj analgesics (caspofen) rau ob peb hnub tom qab kev phais, thaum lub sijhawm ntawd lawv lub cev tau saib xyuas zoo thiab tom qab ntawd lawv tau raug tua pov tseg ntawm lub sijhawm teev tseg. Txhua txoj kev kho mob tau ua raws li cov lus qhia ntawm European, lub tebchaws thiab cov koom haum thiab tau pom zoo los ntawm LandesamtfürNatur ntawm Umwelt thiab Verbraucherschutz, North Rhine-Westphalia, Lub Tebchaws Yelemees.
Cov tsiaj tau raug tshuaj loog nrog ketamine (100 mg / kg) thiab xylazine (10 mg / kg), thiab lub plawv tau perfused nrog 0.1 M PBS ua ntej, thiab tom qab ntawd nrog 4% PFA hauv PBS. Cov ntaub so ntswg tau txiav thiab kho hauv 4% PFA / PBS hmo ntuj ntawm 4 ° C. Ib rab riam vibrating (Leica Microsystems GmbH, Vienna, Austria) tau siv los npaj cov ntu sagittal (50 μm tuab) los ntawm lub hlwb kho hauv PBS. Tshwj tsis yog tias tau teev tseg lwm yam, staining ntawm cov ntu dawb-floating tau ua raws li tau piav qhia saum toj no (13) ntawm chav tsev kub thiab do. Hauv ntej, ua ntej, cov slices tau txais tau permeabilized nrog 0.5% Triton X-100 / PBS rau 15 feeb ntawm chav tsev kub; rau qee qhov epitopes (Pcx thiab Shmt2), los ntawm hauv tris-EDTA buffer ntawm 80 ° C (PH 9) cua sov cov slices rau 25 feeb es tsis txhob ua cov kauj ruam no. Tom ntej no, cov seem tau muab tso rau hauv lub thawv nrog cov tshuaj tiv thaiv thawj zaug (saib Rooj S1) hauv lub thawv thaiv (3% BSA/PBS) ntawm 4°C ib hmos nrog kev sib tov. Hnub tom qab, cov seem tau ntxuav nrog lub thawv thaiv thiab muab tso rau hauv lub thawv nrog cov tshuaj tiv thaiv thib ob uas muaj fluorophore-conjugated rau 2 teev ntawm chav sov; thaum kawg, cov seem tau ntxuav kom huv si hauv PBS, pleev xim rau DAPI, thiab tom qab ntawd kho nrog AquaPolymount Ntawm lub tshuab microscope.
Ib lub tshuab tsom iav laser scanning confocal (TCS SP8-X lossis TCS Digital Light Sheet, Leica Microsystems) uas muaj lub teeb dawb laser thiab lub teeb ultraviolet 405 diode tau siv los thaij duab cov qauv. Los ntawm kev ua kom lub fluorophore zoo siab thiab sau cov teeb liab nrog Hybrid Detector (HyDs), LAS-X software tau siv los sau cov duab sib dhos uas ua raws li Nyquist sampling hauv hom sib lawv liag: rau cov vaj huam sib luag tsis muaj ntau, nws yog cov teeb liab dynamic heev (piv txwv li, hauv somatic cell thiab dendrites) mtYFP) Siv HyD los ntes tus lej ntawm PNs hauv hom BrightR). Gating los ntawm 0.3 txog 6 ns yog siv los txo keeb kwm yav dhau.
Kev thaij duab tiag tiag ntawm cov hlwb uas tau xaiv. Tom qab kev xaiv hauv Neurobasal-A nruab nrab uas muaj 1% B27 ntxiv thiab 0.5 mM GlutaMax, cov hlwb tau raug cog rau ntawm cov iav poly-l-lysine-coated (μ-Slide8 Well, Ibidi, tus lej catalog 80826), Thiab tom qab ntawd khaws cia rau ntawm 37 ° C thiab 5% CO2 rau 1 teev kom cov hlwb tau daws. Kev thaij duab tiag tiag tau ua tiav ntawm Leica SP8 laser scanning confocal microscope uas muaj lub laser dawb, HyD, 63 × [1.4 numerical aperture (NA)] roj lub lens thiab theem cua sov.
Tus nas tau raug tshuaj loog sai sai nrog cov pa roj carbon dioxide thiab txiav lub taub hau, lub hlwb tau raug tshem tawm sai sai ntawm lub pob txha taub hau, thiab txiav ua 200μm tuab (rau 13C labeling experiment) lossis 275μm tuab (rau ob qhov kev sim photon) sagittal seem puv nrog cov khoom siv hauv qab no Cov dej khov nab kuab (HM-650 V, Thermo Fisher Scientific, Walldorf, Lub Tebchaws Yelemees) tau puv nrog cov tshuaj hauv qab no: 125 mM dej khov-txias, carbon-saturated (95% O2 thiab 5% CO2) qis Ca2 + artificial cerebrospinal fluid (ACSF) NaCl, 2.5 mM KCl, 1.25 mM sodium phosphate buffer, 25 mM NaHCO3, 25 mM glucose, 0.5 mM CaCl2 thiab 3.5 mM MgCl2 (osmotic pressure ntawm 310 txog 330 mmol). Hloov cov hlais hlwb uas tau txais mus rau hauv chav ua ntej uas muaj Ca2 + ACSF siab dua (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 thiab 2.0 mM MgCl2) Nruab Nrab) pH 7.4 thiab 310 txog 320 mmol).
Thaum lub sijhawm ua cov duab, cov hlais tau raug tsiv mus rau hauv chav thaij duab tshwj xeeb, thiab qhov kev sim tau ua tiav hauv qab ACSF perfusion tas mus li ntawm qhov kub tas li ntawm 32 ° txog 33 ° C. Lub tshuab ntsuas laser multiphoton (TCS SP8 MP-OPO, Leica Microsystems) nruab nrog Leica 25x lub lens (NA 0.95, dej), Ti: Sapphire laser (Chameleon Vision II, Coherent) tau siv rau kev thaij duab hlais. FLIM module (PicoHarp300, PicoQuant).
FLIM ntawm Grx1-roGFP2. Cov kev hloov pauv hauv lub xeev cytoplasmic redox ntawm PNs tau ntsuas los ntawm ob-photon FLIM hauv sagittal lub hlwb slices, qhov twg Grx1-roGFP2 biosensor tsom PNs. Hauv PN txheej, qhov chaw tau txais raug xaiv txog 50 txog 80 μm hauv qab qhov chaw txiav kom ntseeg tau tias muaj PN muaj sia nyob (uas yog, tsis muaj cov qauv beaded lossis kev hloov pauv neuronal morphological raws li dendrites) thiab ob npaug zoo roGFP2 sensor thiab AAV encoding shRNA PCx lossis nws cov kab ke tswj (txhua tus co-expressing mCherry). Sau cov duab ib-stack nrog 2x digital zoom [excitation wavelength: 890 nm; 512 nm 512 pixels]. Kev tshawb pom: sab hauv HyD, fluorescein isothiocyanate (FITC) lim pawg] thiab duab nruab nrab hauv 2 txog 3 feeb raug siv los xyuas kom meej tias muaj cov photons txaus tau sau (1000 photons tag nrho) rau kev sib phim nkhaus. Qhov rhiab heev ntawm Grx1-roGFP2 probe thiab kev txheeb xyuas cov xwm txheej FLIM tau ua los ntawm kev saib xyuas lub neej ntawm roGFP2 thaum ntxiv exogenous 10 mM H2O2 rau hauv perfusion ACSF (kom ua kom oxidation ntau tshaj plaws, ua rau lub neej ntev dua), thiab tom qab ntawd ntxiv 2 mM dithiothreitol (ua kom txo qis qib ntawm kev txo qis, ua rau lub neej luv dua) (Daim Duab S8, D rau G). Siv FLIMfit 5.1.1 software los tshuaj xyuas cov txiaj ntsig tau txais, haum rau ib qho exponential decay curve ntawm tag nrho cov duab rau IRF ntsuas (kev ua haujlwm teb ntawm cov cuab yeej), thiab χ2 yog kwv yees li 1. Txhawm rau xam lub neej ntawm ib qho PN, lub ntsej muag nyob ib puag ncig lub cev paj hlwb tau kos tes, thiab lub neej nruab nrab hauv txhua lub ntsej muag tau siv rau kev ntsuas.
Kev tshuaj xyuas lub peev xwm mitochondrial. Tom qab qhov seem mob hnyav raug incubated nrog 100 nM TMRM ncaj qha ntxiv rau ACSF perfused rau 30 feeb, cov kev hloov pauv mitochondrial ntawm PNs tau ntsuas los ntawm lub tshuab microscope ob-photon. TMRM imaging tau ua los ntawm kev ua kom lub probe zoo siab ntawm 920 nm thiab siv sab hauv HyD (tetramethylrhodamine isothiocyanate: 585/40 nm) los sau cov teeb liab; los ntawm kev siv tib lub wavelength excitation tab sis siv lwm yam sab hauv HyD (FITC: 525/50) los thaij duab mtYFP. Siv ImageJ's Image Calculator plug-in los ntsuas lub peev xwm mitochondrial ntawm theem ib lub cell. Hauv ntej, cov kab zauv plug-in: teeb liab = min (mtYFP, TMRM) yog siv los txheeb xyuas thaj chaw mitochondrial uas qhia lub teeb liab TMRM hauv Purkinje Somali hauv daim duab confocal ib leeg ntawm cov channel sib xws. Tom qab ntawd thaj chaw pixel hauv lub ntsej muag tshwm sim raug ntsuas, thiab tom qab ntawd normalized ntawm qhov sib xws threshold ib-stack duab ntawm mtYFP channel kom tau txais cov mitochondrial fraction uas qhia txog mitochondrial potential.
Daim duab tau raug tshem tawm nrog Huygens Pro (Scientific Volume Imaging) software. Rau cov duab scanned ntawm cov vuas, qhov montage ntawm ib lub vuas yog ua los ntawm kev siv cov algorithm stitching tsis siv neeg uas muab los ntawm LAS-X software. Tom qab calibration duab, siv ImageJ thiab Adobe Photoshop los ua cov duab ntxiv thiab kho qhov ci thiab qhov sib piv sib npaug. Siv Adobe Illustrator rau kev npaj duab.
Kev tshuaj xyuas qhov tsom mus rau mtDNA. Tus naj npawb ntawm cov qhov txhab mtDNA tau ntsuas rau ntawm cov ntu cerebellar uas muaj cov tshuaj tiv thaiv DNA los ntawm lub tshuab tsom iav confocal. Txhua thaj chaw tsom tau tsim rau lub cev cell thiab lub nucleus ntawm txhua lub cell, thiab thaj chaw ntawd tau suav siv Multi Measure plug-in (ImageJ software). Rho thaj chaw nuclear ntawm thaj chaw cell body kom tau thaj chaw cytoplasmic. Thaum kawg, Analyze Particles plug-in (ImageJ software) tau siv los ntsuas cov ntsiab lus cytoplasmic DNA uas qhia txog mtDNA ntawm daim duab threshold, thiab cov txiaj ntsig tau txais tau raug kho kom zoo rau PN nruab nrab ntawm CTRL nas. Cov txiaj ntsig tau qhia ua tus naj npawb nruab nrab ntawm nucleosides ib lub cell.
Kev tshuaj xyuas kev qhia tawm ntawm cov protein. Siv ImageJ's Image Calculator plug-in los ntsuas kev qhia tawm ntawm cov protein hauv PN ntawm theem ib lub cell. Hauv ntej, hauv daim duab confocal ib txheej ntawm cov channel sib xws, los ntawm qhov sib npaug: teeb liab = min (mtYFP, antibody), thaj tsam mitochondrial uas qhia txog immunoreactivity rau qee yam antibody hauv Purkina tau txheeb xyuas. Tom qab ntawd thaj chaw pixel hauv lub ntsej muag tshwm sim tau ntsuas, thiab tom qab ntawd normalized ntawm daim duab threshold ib-stack ntawm mtYFP channel kom tau txais cov feem mitochondrial ntawm cov protein uas tau tso tawm.
Kev tshuaj xyuas qhov ceev ntawm cov hlwb Purkinje. Lub Cell Counter plug-in ntawm ImageJ tau siv los ntsuas qhov ceev ntawm Purkinje los ntawm kev faib tus naj npawb ntawm cov hlwb Purkinje suav los ntawm qhov ntev ntawm lub nplhaib cerebellar uas cov hlwb suav tau nyob.
Kev npaj thiab sau cov qauv. Cov hlwb los ntawm pawg tswj thiab Mfn2cKO nas tau kho rau hauv 2% PFA/2.5% glutaraldehyde hauv 0.1 M phosphate buffer (PB), thiab tom qab ntawd cov ntu coronal tau npaj siv ciliates (Leica Mikrosysteme GmbH, Vienna, Austria) (Tuab 50 txog 60 μm). Tom qab ntawd kho rau hauv PB buffer hauv 1% os tetraoxide thiab 1.5% potassium ferrocyanide ntawm chav tsev kub rau 1 teev. Cov ntu tau ntxuav peb zaug nrog dej distilled, thiab tom qab ntawd stained nrog 70% ethanol uas muaj 1% uranyl acetate rau 20 feeb. Cov ntu tom qab ntawd tau qhuav hauv cawv graded thiab muab tso rau hauv Durcupan ACM (Araldite casting resin M) epoxy resin (Electron Microscopy Sciences, catalog number 14040) ntawm cov iav silicon-coated, thiab thaum kawg ntawm 60°C Polymerize hauv qhov cub rau 48 teev. Thaj chaw cerebellar cortex tau raug xaiv thiab 50 nm ultrathin seem tau txiav rau ntawm Leica Ultracut (Leica Mikrosysteme GmbH, Vienna, Austria) thiab khaws rau ntawm 2 × 1 hli tooj liab slit grid coated nrog polystyrene zaj duab xis. Cov seem tau stained nrog cov tshuaj ntawm 4% uranyl acetate hauv H2O rau 10 feeb, ntxuav nrog H2O ntau zaus, tom qab ntawd nrog Reynolds lead citrate hauv H2O rau 10 feeb, thiab tom qab ntawd ntxuav nrog H2O ntau zaus. Micrographs tau coj nrog lub tshuab hluav taws xob kis tau Philips CM100 (Thermo Fisher Scientific, Waltham, MA, USA) siv TVIPS (Tietz Video thiab Image Processing System) TemCam-F416 digital koob yees duab (TVIPS GmbH, Gauting, USA). Lub teb chaws Yelemees).
Rau cov nas uas muaj tus kab mob AAV, lub hlwb raug cais thiab txiav ua ntu 1 hli tuab, thiab lub hlwb cerebellum raug kuaj xyuas siv lub tshuab tsom iav fluorescence los txheeb xyuas lub nplhaib uas muaj tus kab mob AAV (uas yog, mCherry expressing). Tsuas yog kev sim uas AAV txhaj tshuaj ua rau muaj kev ua haujlwm siab heev ntawm Purkinje cell txheej (piv txwv li yuav luag tag nrho txheej) hauv tsawg kawg yog ob lub nplhaib cerebellar sib law liag. Lub voj voog AAV-transduced tau microdissected rau hmo ntuj tom qab kho (4% PFA thiab 2.5% glutaraldehyde hauv 0.1 M cocoate buffer) thiab ua tiav ntxiv. Rau EPON embedding, cov ntaub so ntswg kho tau ntxuav nrog 0.1 M sodium cocoate buffer (Applichem), thiab incubated nrog 2% OsO4 (os, Science Services; Caco) hauv 0.1 M sodium cocoate buffer (Applichem) 4 Teev, tom qab ntawd ntxuav rau 2 teev. Rov ua dua 3 zaug nrog 0.1 M cocamide buffer. Tom qab ntawd, cov koob tshuaj ethanol nce mus rau saum toj tau siv los ua kom txhua cov tshuaj ethanol ntawm 4°C rau 15 feeb kom qhuav cov ntaub so ntswg. Cov ntaub so ntswg tau hloov mus rau propylene oxide thiab ua kom sov ib hmos hauv EPON (Sigma-Aldrich) ntawm 4°C. Muab cov ntaub so ntswg tso rau hauv EPON tshiab ntawm chav tsev kub rau 2 teev, thiab tom qab ntawd muab tso rau ntawm 62°C rau 72 teev. Siv ultramicrotome (Leica Microsystems, UC6) thiab rab riam pob zeb diamond (Diatome, Biel, Switzerland) los txiav 70 nm ultrathin seem, thiab pleev xim nrog 1.5% uranyl acetate rau 15 feeb ntawm 37°C, thiab pleev xim nrog cov kua citrate lead 4 feeb. Cov duab hluav taws xob micrographs tau coj los siv JEM-2100 Plus transmission electron microscope (JEOL) nruab nrog Camera OneView 4K 16-bit (Gatan) thiab DigitalMicrograph software (Gatan). Rau kev tsom xam, cov duab hluav taws xob micrographs tau txais nrog 5000 × lossis 10,000 × digital zoom.
Kev tshuaj xyuas morphological ntawm mitochondria. Rau txhua qhov kev tshuaj xyuas, cov duab ntawm cov mitochondria ib leeg tau raug piav qhia hauv cov duab digital siv ImageJ software. Cov kev ntsuas morphological sib txawv raug tshuaj xyuas. Qhov ceev ntawm mitochondrial yog qhia ua feem pua ​​​​​​tau los ntawm kev faib tag nrho thaj chaw mitochondrial ntawm txhua lub cell los ntawm thaj chaw cytoplasm (cheeb tsam cytoplasm = thaj chaw cell-cheeb tsam nucleus cell) × 100. Qhov puag ncig ntawm mitochondria yog xam nrog cov mis [4π∙(cheeb tsam/perimeter 2)]. Lub morphology ntawm mitochondria tau raug tshuaj xyuas thiab muab faib ua ob pawg ("tubular" thiab "blister") raws li lawv cov duab tseem ceeb.
Tus lej thiab kev tshuaj xyuas qhov ceev ntawm Autophagosome/lysosome. Siv ImageJ software los kos cov duab ntawm txhua lub autophagosome/lysosome hauv daim duab digital. Thaj chaw Autophagosome/lysosome yog qhia ua feem pua ​​​​​​uas suav los ntawm kev faib tag nrho thaj chaw qauv autophagosome/lysosome ntawm txhua lub cell los ntawm thaj chaw cytoplasm (thaj chaw cytoplasm = thaj chaw cell-cheeb tsam nucleus) × 100. Qhov ceev ntawm autophagosomes/lysosomes yog suav los ntawm kev faib tag nrho tus lej los ntawm tus lej ntawm cov qauv autophagosome/lysosome ib lub cell (hauv thaj chaw cytoplasmic) (cheeb chaw cytoplasmic = thaj chaw cell-cheeb tsam nuclear).
Kev sau ntawv rau kev txiav cov hlwb thiab kev npaj cov qauv. Rau cov kev sim uas xav tau kev sau ntawv glucose, hloov cov hlwb hlwb mus rau chav ua ntej-incubation, uas muaj cov pa roj carbon saturated (95% O2 thiab 5% CO2), Ca2 + ACSF siab (125.0 mM NaCl, 2.5 mM KCl, 1.25 mM sodium phosphate buffer, 25.0 mM NaHCO 3, 25.0 mM d-glucose, 1.0 mM CaCl 2 thiab 2.0 mM MgCl 2, kho rau pH 7.4 thiab 310 txog 320 mOsm), uas glucose yog 13 C 6- Glucose hloov (Eurisotop, tus lej catalog CLM-1396). Rau cov kev sim uas xav tau pyruvate labeling, hloov cov hlais hlwb mus rau Ca2 + ACSF siab dua (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 thiab Ntxiv 2.0mM MgCl2, kho kom pH 7.4 thiab 310 txog 320mOsm), thiab ntxiv 1mM 1-[1-13C]pyruvate (Eurisotop, tus lej catalog CLM-1082). Incubate cov seem rau 90 feeb ntawm 37 ° C. Thaum kawg ntawm kev sim, cov seem tau ntxuav sai sai nrog cov kua dej (pH 7.4) uas muaj 75 mM ammonium carbonate, thiab tom qab ntawd homogenized hauv 40:40:20 (v:v:v) acetonitrile (ACN): methanol: dej. Tom qab cov seem raug muab tso rau hauv dej khov rau 30 feeb, cov qauv raug centrifuged ntawm 21,000 g rau 10 feeb ntawm 4°C, thiab cov supernatant ntshiab raug ziab hauv SpeedVac concentrator. Cov metabolite pellet qhuav tau khaws cia ntawm -80°C kom txog thaum kuaj xyuas.
Kev tshuaj xyuas kua chromatography-mass spectrometry ntawm 13 C-labeled amino acids. Rau kev tshuaj xyuas kua chromatography-mass spectrometry (LC-MS), cov metabolite pellet tau rov ua dua hauv 75μl ntawm LC-MS qib dej (Honeywell). Tom qab centrifugation ntawm 21,000 g rau 5 feeb ntawm 4 ° C, 20 μl ntawm cov supernatant clarified tau siv rau kev tshuaj xyuas amino acid flux, thaum cov extract seem tau siv tam sim ntawd rau kev tshuaj xyuas anion (saib hauv qab no). Kev tshuaj xyuas amino acid tau ua tiav siv cov txheej txheem derivatization benzoyl chloride uas tau piav qhia ua ntej (55, 56). Hauv thawj kauj ruam, 10μl ntawm 100 mM sodium carbonate (Sigma-Aldrich) tau ntxiv rau 20μl ntawm metabolite extract, thiab tom qab ntawd 10μl ntawm 2% benzoyl chloride (Sigma-Aldrich) tau ntxiv rau LC qib ACN. Cov qauv tau raug vortexed luv luv thiab tom qab ntawd centrifuged ntawm 21,000 g rau 5 feeb ntawm 20 ° C. Hloov cov supernatant uas tau ntxuav mus rau hauv lub raj mis 2 ml autosampler nrog lub khob iav conical (200 μl ntim). Cov qauv tau soj ntsuam siv Acquity iClass ultra-high performance LC system (Waters) txuas nrog Q-Exactive (QE)-HF (Ultra High Field Orbitrap) high-resolution precision mass spectrometer (Thermo Fisher Scientific). Rau kev soj ntsuam, 2μl ntawm cov qauv derivatized tau txhaj rau hauv 100 × 1.0 mm high-strength silica T3 kem (Waters) uas muaj 1.8μm particles. Tus nqi ntws yog 100μl / min, thiab lub kaw lus buffer muaj buffer A (10 mM ammonium formate thiab 0.15% formic acid hauv dej) thiab buffer B (ACN). Qhov gradient yog raws li nram no: 0%B ntawm 0 feeb; 0%B. 0 txog 15% B thaum 0 txog 0.1 feeb; 15 txog 17% B thaum 0.1 txog 0.5 feeb; B thaum 17 txog 55% thaum 0.5 txog 14 feeb; B thaum 55 txog 70% thaum 14 txog 14.5 feeb; thaum 14.5 txog 70 txog 100% B thaum 18 feeb; 100% B thaum 18 txog 19 feeb; 100 txog 0% B thaum 19 txog 19.1 feeb; 0% B thaum 19.1 txog 28 feeb (55, 56). Lub QE-HF mass spectrometer ua haujlwm hauv hom positive ionization nrog qhov ntau ntawm m/z (mass/charge ratio) ntawm 50 txog 750. Qhov kev daws teeb meem siv yog 60,000, thiab lub hom phiaj gain control (AGC) ion tau txais yog 3 × 106, thiab lub sijhawm ion siab tshaj plaws yog 100 milliseconds. Lub tshuab hluav taws xob electrospray ionization (ESI) ua haujlwm ntawm lub zog hluav taws xob ntawm 3.5 kV, qhov kub ntawm capillary ntawm 250 ° C, lub cua ntsawj ntawm 60 AU (arbitrary units), thiab lub cua ntsawj ntawm 20 AU. 250 ° C. Lub lens S tau teeb tsa rau 60 AU.
Kev tshuaj xyuas Anion chromatography-MS ntawm 13C labeled organic acids. Cov metabolite precipitate seem (55μl) tau tshuaj xyuas siv Dionex ion chromatography system (ICS 5000+, Thermo Fisher Scientific) txuas nrog QE-HF mass spectrometer (Thermo Fisher Scientific). Hauv ntej, 5μl ntawm metabolite extract tau txhaj rau hauv Dionex IonPac AS11-HC kem uas muaj HPLC (2 mm × 250 mm, qhov loj me ntawm cov khoom me me 4μm, Thermo Fisher Scientific) hauv hom thawb-hauv ib nrab voj voog nrog qhov sib piv ntawm 1.) Dionex IonPac AG11-HC guard kem (2 mm x 50 mm, 4μm, Thermo Fisher Scientific). Qhov kub ntawm kem raug tswj ntawm 30°C, thiab lub autosampler raug teeb tsa rau 6°C. Siv lub potassium hydroxide cartridge uas muab nrog dej deionized los tsim cov potassium hydroxide gradient los ntawm lub eluent generator. Kev sib cais cov metabolites ntawm tus nqi ntws ntawm 380μl/min, siv cov gradient hauv qab no: 0 txog 3 feeb, 10 mM KOH; 3 txog 12 feeb, 10 txog 50 mM KOH; 12 txog 19 feeb, 50 txog 100 mM KOH; 19 txog 21 feeb, 100 mM KOH; 21 txog 21.5 feeb, 100 txog 10 mM KOH. Lub kem tau rov sib npaug dua hauv qab 10 mM KOH rau 8.5 feeb.
Cov metabolites eluted raug muab sib xyaw nrog 150μl/min isopropanol ntxiv dej tom qab lub kem thiab tom qab ntawd coj mus rau lub tshuab ntsuas qhov siab ua haujlwm hauv hom ionization tsis zoo. MS tab tom saib xyuas qhov ntau ntawm m/z 50 txog 750 nrog qhov kev daws teeb meem ntawm 60,000. AGC tau teeb tsa rau 1 × 106, thiab lub sijhawm ion siab tshaj plaws khaws cia ntawm 100 ms. Lub qhov cua sov ESI tau ua haujlwm ntawm lub zog tsuag ntawm 3.5 kV. Lwm qhov chaw ntawm lub qhov cua sov yog raws li nram no: capillary kub 275 ° C; sheath roj ntws, 60 AU; auxiliary roj ntws, 20 AU ntawm 300 ° C, thiab S lens teeb tsa rau 60 AU.
Kev tshuaj xyuas cov ntaub ntawv ntawm 13C cov metabolites uas tau cim. Siv TraceFinder software (version 4.2, Thermo Fisher Scientific) rau kev tshuaj xyuas cov ntaub ntawv ntawm isotope piv. Tus kheej ntawm txhua lub compound tau txheeb xyuas los ntawm ib qho kev siv tau zoo thiab tau tshuaj xyuas ywj pheej. Txhawm rau ua qhov kev tshuaj xyuas isotope enrichment, thaj chaw ntawm cov ion chromatogram (XIC) uas tau rho tawm ntawm txhua 13C isotope (Mn) tau rho tawm ntawm [M + H] +, qhov twg n yog tus lej carbon ntawm lub hom phiaj compound, siv los tshuaj xyuas cov amino acids lossis [MH] + yog siv los tshuaj xyuas cov anions. Qhov tseeb ntawm XIC yog tsawg dua tsib feem ib lab, thiab qhov tseeb ntawm RT yog 0.05 feeb. Kev tshuaj xyuas enrichment yog ua los ntawm kev xam qhov sib piv ntawm txhua lub isotope uas tau kuaj pom rau qhov sib npaug ntawm txhua lub isotopes ntawm cov compound sib xws. Cov piv no tau muab ua feem pua ​​rau txhua lub isotope, thiab cov txiaj ntsig tau qhia ua molar feem pua ​​enrichment (MPE), raws li tau piav qhia ua ntej (42).
Cov pellet neuron khov tau homogenized hauv dej khov-txias 80% methanol (v/v), vortexed, thiab incubated ntawm -20°C rau 30 feeb. Vortex cov qauv dua thiab do ntawm +4°C rau 30 feeb. Cov qauv tau centrifuged ntawm 21,000 g rau 5 feeb ntawm 4°C, thiab tom qab ntawd cov supernatant tau sau thiab ziab siv SpeedVac concentrator ntawm 25°C rau kev tshuaj xyuas tom ntej. Raws li tau piav qhia saum toj no, LC-MS kev tshuaj xyuas tau ua tiav ntawm cov amino acids ntawm cov hlwb xaiv. Siv TraceFinder (version 4.2, Thermo Fisher Scientific), kev tshuaj xyuas cov ntaub ntawv tau ua tiav siv monoisotopic pawg ntawm txhua lub compound. Quantile normalization ntawm cov ntaub ntawv metabolite tau ua tiav siv cov pob software preprocessCore (57).
Npaj daim. Tus nas tau raug tshuaj loog sai sai nrog cov pa roj carbon dioxide thiab txiav lub taub hau, lub hlwb tau raug tshem tawm sai sai ntawm lub pob txha taub hau, thiab rab riam uas muaj dej khov puv (HM-650 V, Thermo Fisher Scientific, Walldorf, Lub Tebchaws Yelemees) tau siv los txiav nws ua 300 txog 375 μm sagittal seem Kev ua pa roj carbon txias (95% O2 thiab 5% CO2) Ca2 + ACSF qis (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 thiab 6.0 mM MgCl2 Kho kom haum rau pH 7.4 thiab 310 txog 330 mOsm). Hloov cov hlais hlwb uas tau txais mus rau hauv lub chamber uas muaj Ca2 + ACSF siab dua (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 CaCl2 thiab mM 3.5 MgCl2) pH 7.4 thiab 310 txog 320 mOsm). Khaws cov hlais rau 20 txog 30 feeb kom lawv rov qab tau ua ntej sau cia.
kev kaw suab. Ib lub tshuab microscope theem nruab nrog lub chamber kaw suab ruaj khov thiab lub lens 20x immersion dej (Scientifica) tau siv rau txhua qhov kev kaw suab. Cov hlwb Purkinje uas tau muab tso rau hauv qhov chaw tau txheeb xyuas los ntawm (i) qhov loj ntawm lub cev, (ii) qhov chaw ntawm lub cerebellum, thiab (iii) kev qhia tawm ntawm cov noob fluorescent mtYFP reporter. Lub pipette patch nrog lub taub hau tsis kam ntawm 5 txog 11 megohms tau rub tawm los ntawm lub capillary iav borosilicate (GB150-10, 0.86 mm × 1.5 mm × 100 mm, Science Products, Hofheim, Lub Tebchaws Yelemees) thiab lub pipette kab rov tav (P-1000, Sutter), Novato, CA). Txhua qhov kev kaw suab tau ua los ntawm ELC-03XS npi patch clamp amplifier (npi electronic GmbH, Tam, Lub Tebchaws Yelemees), uas tau tswj hwm los ntawm software Signal (version 6.0, Cambridge Electronic, Cambridge, UK). Qhov kev sim tau kaw ntawm tus nqi kuaj ntawm 12.5 kHz. Lub teeb liab raug lim nrog ob lub lim dej luv luv Bessel nrog cov zaus txiav ntawm 1.3 thiab 10 kHz feem. Lub peev xwm ntawm daim nyias nyias thiab lub pipette raug them los ntawm lub voj voog them nyiaj siv lub amplifier. Tag nrho cov kev sim tau ua nyob rau hauv kev tswj hwm ntawm Orca-Flash 4.0 lub koob yees duab (Hamamatsu, Gerden, Lub Tebchaws Yelemees), uas tau tswj hwm los ntawm Hokawo software (version 2.8, Hamamatsu, Gerden, Lub Tebchaws Yelemees).
Kev teeb tsa thiab kev tshuaj xyuas tag nrho cov hlwb. Ua ntej sau cia, muab cov kua dej sab hauv uas muaj cov tshuaj hauv qab no rau hauv lub pipette: 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 (Mg), 0.5 mM Guanosine triphosphate (GTP) (Na) thiab 10.0 mM creatinine phosphate tau kho rau pH 7.25, thiab lub siab osmotic yog 290 mOsm (sucrose). Tom qab siv lub zog ntawm 0 pA los rhuav lub membrane, lub peev xwm so ntawm lub membrane tau ntsuas. Qhov tsis kam ntawm cov tswv yim raug ntsuas los ntawm kev siv cov hluav taws xob hyperpolarized ntawm -40, -30, -20, thiab -10 pA. Ntsuas qhov loj ntawm qhov teb voltage thiab siv Ohm txoj cai los xam qhov tsis kam ntawm cov tswv yim. Kev ua ub ua no tau raug kaw cia rau hauv lub clamp voltage rau 5 feeb, thiab sPSC tau txheeb xyuas thiab ntsuas hauv Igor Pro (version 32 7.01, WaveMetrics, Lake Oswego, Oregon, USA) siv daim ntawv sau semi-automatic. Cov kab nkhaus IV thiab cov hluav taws xob ruaj khov raug ntsuas los ntawm kev clamp lub roj teeb ntawm ntau lub peev xwm sib txawv (pib ntawm -110 mV) thiab nce qhov hluav taws xob hauv 5 mV kauj ruam. Kev tsim tawm ntawm AP tau raug sim los ntawm kev siv cov hluav taws xob depolarizing. Clamp lub cell ntawm -70 mV thaum siv cov hluav taws xob depolarizing. Kho qhov loj me ntawm txhua chav kaw suab sib cais (10 txog 60 pA). Xam qhov siab tshaj plaws AP zaus los ntawm kev suav cov pulse spikes uas ua rau qhov siab tshaj plaws AP zaus. Qhov AP threshold raug soj ntsuam los ntawm kev siv qhov thib ob ntawm cov depolarization pulse uas ua rau ib lossis ntau dua APs.
Kev teeb tsa thiab kev tshuaj xyuas cov khoom uas muaj qhov perforated. Ua cov ntaub ntawv perforated patch siv cov txheej txheem txheem. Siv lub pipette ATP- thiab GTP-free uas tsis muaj cov khoom xyaw hauv qab no: 128 mM gluconate K, 10 mM KCl, 10 mM Hepes, 0.1 mM EGTA thiab 2 mM MgCl2, thiab kho kom haum pH 7.2 (siv KOH). ATP thiab GTP raug tshem tawm ntawm cov tshuaj intracellular kom tiv thaiv kev nkag mus tsis tau ntawm lub cell membrane. Lub pipette patch yog puv nrog cov tshuaj amphotericin uas muaj sab hauv (kwv yees li 200 txog 250μg/ml; G4888, Sigma-Aldrich) kom tau txais cov ntaub ntawv punched patch. Amphotericin tau yaj rau hauv dimethyl sulfoxide (qhov kawg concentration: 0.1 txog 0.3%; DMSO; D8418, Sigma-Aldrich). Qhov concentration ntawm DMSO siv tsis muaj qhov cuam tshuam loj rau cov neurons uas tau kawm. Thaum lub sijhawm ua haujlwm xuas nrig, qhov kev tiv thaiv ntawm cov channel (Ra) tau raug saib xyuas tas li, thiab qhov kev sim tau pib tom qab qhov amplitude ntawm Ra thiab AP ruaj khov (20-40 feeb). Kev ua haujlwm spontaneous raug ntsuas hauv lub voltage thiab / lossis tam sim no clamp rau 2 txog 5 feeb. Kev tshuaj xyuas cov ntaub ntawv tau ua tiav siv Igor Pro (version 7.05.2, WaveMetrics, USA), Excel (version 2010, Microsoft Corporation, Redmond, USA) thiab GraphPad Prism (version 8.1.2, GraphPad Software Inc., La Jolla, CA). Tebchaws Meskas). Txhawm rau txheeb xyuas cov APs spontaneous, IgorPro's NeuroMatic v3.0c plug-in siv. Txheeb xyuas cov APs siv qhov threshold, uas tau kho ib tus zuj zus rau txhua cov ntaub ntawv. Siv lub sijhawm spike, txiav txim siab qhov zaus spike nrog qhov siab tshaj plaws instantaneous spike zaus thiab qhov nruab nrab spike zaus.
Kev rho tawm PN. Los ntawm kev hloov kho rau cov txheej txheem luam tawm ua ntej, PNs tau ntxuav los ntawm cov nas cerebellum ntawm theem teev tseg (58). Hauv ntej, cov cerebellum tau txiav thiab minced hauv cov dej khov-txias dissociation medium [tsis muaj HBSS Ca2 + thiab Mg2 +, ntxiv nrog 20 mM glucose, penicillin (50 U / ml) thiab streptomycin (0.05 mg / ml)], thiab tom qab ntawd zom cov nruab nrab hauv papain [HBSS, ntxiv nrog 1-cysteine ​​​​HCl (1 mg / ml), papain (16 U / ml) thiab deoxyribonuclease I (DNase I; 0.1 mg / ml)] Kho rau 30 feeb ntawm 30 ° C. Ua ntej, ntxuav cov ntaub so ntswg hauv HBSS nruab nrab uas muaj cov qe hnoos qeev (10 mg/ml), BSA (10 mg/ml) thiab DNase (0.1 mg/ml) ntawm chav tsev kub kom tiv thaiv kev zom zaub mov enzymatic, thiab tom qab ntawd hauv HBSS nruab nrab uas muaj 20 mM glucose. Maj mam sib tsoo hauv HBSS, penicillin (50 U/ml), streptomycin (0.05 mg/ml) thiab DNase (0.1 mg/ml) tso tawm ib lub cell. Cov cell suspension tau raug lim los ntawm lub cell strainer 70μm, tom qab ntawd cov cell tau pelleted los ntawm centrifugation (1110 rpm, 5 feeb, 4°C) thiab rov ua dua hauv sorting nruab nrab [HBSS, ntxiv nrog 20 mM glucose, 20% fetal bovine) Serum, penicillin (50 U/ml) thiab streptomycin (0.05 mg/ml)]; soj ntsuam qhov muaj sia nyob ntawm cell nrog propidium iodide thiab kho qhov ceev ntawm cell rau 1 × 106 txog 2 × 106 cell/ml. Ua ntej flow cytometry, cov tshuaj raug lim los ntawm lub cell strainer 50 μm.
Flow cytometer. Kev cais cov cell tau ua tiav ntawm 4 ° C siv lub tshuab FACSAria III (BD Biosciences) thiab FACSDiva software (BD Biosciences, version 8.0.1). Lub cell ncua tau raug cais siv 100 μm nozzle nyob rau hauv lub siab ntawm 20 psi ntawm tus nqi ntawm ~ 2800 qhov xwm txheej / sec. Txij li thaum cov qauv gating ib txwm muaj (qhov loj ntawm cell, bimodal kev faib tawm, thiab cov yam ntxwv scattering) tsis tuaj yeem ua kom muaj kev sib cais ntawm PN los ntawm lwm hom cell, lub tswv yim gating tau teeb tsa raws li kev sib piv ncaj qha ntawm YFP qhov muaj zog thiab autofluorescence hauv mitoYFP + ​​thiab tswj mitoYFP − Nas. YFP zoo siab los ntawm kev irradiating cov qauv nrog 488 nm laser kab, thiab lub teeb liab tau kuaj pom siv 530/30 nm band pass lim. Hauv mitoYFP + ​​nas, lub zog sib piv ntawm Rosa26-mitoYFP reporter gene kuj tseem siv los cais cov neuronal lub cev thiab axon fragments. 7-AAD raug ua kom zoo siab nrog lub laser daj 561 nm thiab ntes tau nrog lub lim bandpass 675/20 nm kom tshem tawm cov hlwb tuag. Yuav kom cais cov astrocytes tib lub sijhawm, lub cell suspension tau pleev xim nrog ACSA-2-APC, tom qab ntawd cov qauv tau irradiated nrog kab laser 640 nm, thiab lub lim bandpass 660/20 nm tau siv los ntes cov teeb liab.
Cov hlwb uas tau sau los tau raug muab zom los ntawm centrifugation (1110 rpm, 5 feeb, 4 ° C) thiab khaws cia rau -80 ° C kom txog thaum siv. Cov nas Mfn2cKO thiab lawv cov menyuam dev raug cais tawm tib hnub kom txo qis qhov sib txawv ntawm cov txheej txheem. Kev nthuav qhia cov ntaub ntawv FACS thiab kev tshuaj xyuas tau ua tiav siv FlowJo software (FlowJo LLC, Ashland, Oregon, USA).
Raws li tau hais los saum toj no (59), PCR tiag tiag yog siv los cais DNA los ntawm cov neurons uas tau txheeb xyuas rau kev ntsuas mtDNA tom qab. Qhov linearity thiab threshold sensitivity tau sim thawj zaug los ntawm kev khiav qPCR ntawm ntau tus lej ntawm cov hlwb. Hauv ntej, sau 300 PN hauv lub lysis buffer uas muaj 50 mM tris-HCl (pH 8.5), 1 mM EDTA, 0.5% Tween 20 thiab proteinase K (200 ng / ml) thiab incubate ntawm 55 ° C 120 feeb. Cov hlwb tau ntxiv incubated ntawm 95 ° C rau 10 feeb kom ntseeg tau tias tag nrho inactivation ntawm proteinase K. Siv TaqMan probe (Thermo Fisher) tshwj xeeb rau mt-Nd1, mtDNA tau ntsuas los ntawm semi-quantitative PCR hauv 7900HT Real-Time PCR system (Thermo Fisher Scientific). Science, daim ntawv teev npe tus lej Mm04225274_s1), mt-Nd6 (Thermo Fisher Scientific, daim ntawv teev npe tus lej AIVI3E8) thiab 18S (Thermo Fisher Scientific, daim ntawv teev npe tus lej Hs99999901_s1) cov noob caj noob ces.
Kev npaj cov qauv Proteome. Los ntawm kev ua kom sov cov tshuaj ntawm 95 ° C rau 10 feeb thiab sonicating, hauv lysis buffer [6 M guanidine chloride, 10 mM tris (2-carboxyethyl) phosphine hydrochloride, 10 mM chloroacetamide thiab 100 mM tris-Lyse frozen neuron pellets hauv HCl]. Ntawm Bioruptor (Diagenode) rau 10 feeb (30 vib nas this pulse / 30 vib nas this ncua sijhawm). Cov qauv tau diluted 1:10 hauv 20 mM tris-HCl (pH 8.0), sib xyaw nrog 300 ng trypsin kub (Promega), thiab incubated hmo ntuj ntawm 37 ° C kom ua tiav kev zom zaub mov tiav. Nyob rau hnub thib ob, cov qauv tau centrifuged ntawm 20,000 g rau 20 feeb. Cov supernatant tau diluted nrog 0.1% formic acid, thiab cov tshuaj tau desalted nrog tus kheej ua StageTips. Cov qauv tau qhuav hauv lub cuab yeej SpeedVac (Eppendorf concentrator ntxiv rau 5305) ntawm 45°C, thiab tom qab ntawd cov peptide tau muab tso rau hauv 0.1% formic acid. Tag nrho cov qauv tau npaj tib lub sijhawm los ntawm tib tus neeg. Txhawm rau txheeb xyuas cov qauv astrocyte, 4 μg desalted peptides tau muab cim nrog lub cim tandem mass (TMT10plex, catalog number 90110, Thermo Fisher Scientific) nrog rau peptide rau TMT reagent piv ntawm 1:20. Rau TMT labeling, 0.8 mg ntawm TMT reagent tau rov ua dua hauv 70 μl ntawm anhydrous ACN, thiab cov peptide qhuav tau rov ua dua rau hauv 9 μl ntawm 0.1 M TEAB (triethylammonium bicarbonate), uas ntxiv 7 μl ntawm TMT reagent hauv ACN. Qhov concentration yog 43.75%. Tom qab 60 feeb ntawm kev sib xyaw, qhov tshuaj tiv thaiv tau quenched nrog 2 μl ntawm 5% hydroxylamine. Cov peptides uas muaj daim ntawv lo tau sau, ziab, rov ua dua hauv 200μl ntawm 0.1% formic acid (FA), faib ua ob, thiab tom qab ntawd desalted siv tus kheej ua StageTips. Siv UltiMate 3000 ultra high performance liquid chromatograph (UltiMate 3000 ultra high performance liquid chromatograph), ib qho ntawm ob lub halves tau fractionated ntawm 1mm x 150mm Acquity chromatographic kem uas muaj 130Å1.7μm C18 particles (Waters, catalog No. SKU: 186006935). Thermo Fisher Scientific). Cais cov peptides ntawm tus nqi ntws ntawm 30μl / feeb, cais los ntawm 1% txog 50% buffer B rau 85 feeb nrog stepwise gradient ntawm 96 feeb, los ntawm 50% txog 95% buffer B rau 3 feeb, tom qab ntawd 8 feeb rau 95% Buffer B; Buffer A yog 5% ACN thiab 10 mM ammonium bicarbonate (ABC), thiab buffer B yog 80% ACN thiab 10 mM ABC. Sau cov feem pua ​​txhua 3 feeb thiab muab lawv ua ob pawg (1 + 17, 2 + 18, thiab lwm yam) thiab ziab lawv hauv lub tshuab centrifuge nqus tsev.
Kev tshuaj xyuas LC-MS/MS. Rau kev ntsuas pawg, cov peptides (tus lej r119.aq) tau sib cais rau ntawm 25 cm, 75 μm sab hauv txoj kab uas hla PicoFrit analytical kem (lub lens tshiab, tus lej ib feem PF7508250) nruab nrog 1.9 μm ReproSil-Pur 120 C18-AQ nruab nrab (Dr. Maisch, lev), Siv EASY-nLC 1200 (Thermo Fisher Scientific, Lub Tebchaws Yelemees). Lub kem tau khaws cia ntawm 50°C. Buffers A thiab B yog 0.1% formic acid hauv dej thiab 0.1% formic acid hauv 80% ACN, raws li. Peptides tau sib cais los ntawm 6% txog 31% buffer B rau 65 feeb thiab los ntawm 31% txog 50% buffer B rau 5 feeb nrog qhov gradient ntawm 200 nl/min. Cov peptides eluted tau tshuaj xyuas rau ntawm Orbitrap Fusion mass spectrometer (Thermo Fisher Scientific). Kev ntsuas peptide precursor m/z yog ua tiav nrog kev daws teeb meem ntawm 120,000 hauv qhov ntau ntawm 350 txog 1500 m/z. Siv 27% lub zog sib tsoo normalized, tus precursor muaj zog tshaj plaws nrog lub xeev them ntawm 2 txog 6 yog xaiv rau lub zog siab C trap dissociation (HCD) cleavage. Lub sijhawm voj voog yog teeb tsa rau 1 s. Tus nqi m/z ntawm cov peptide fragment tau ntsuas hauv lub ion trap siv lub hom phiaj AGC me tshaj plaws ntawm 5 × 104 thiab lub sijhawm txhaj tshuaj siab tshaj plaws ntawm 86 ms. Tom qab fragmentation, tus precursor tau muab tso rau ntawm daim ntawv teev npe dynamic exclusion rau 45 s. Cov peptides uas muaj daim ntawv lo TMT tau raug cais tawm ntawm ib kab Acclaim PepMap uas ntev li 50 cm, 75 μm (Thermo Fisher Scientific, daim ntawv teev npe lej 164942), thiab cov spectra migration tau raug soj ntsuam ntawm Orbitrap Lumos Tribrid mass spectrometer (Thermo Fisher Scientific) uas muaj cov khoom siv high-field asymmetric waveform ions (FAIMS) (Thermo Fisher Scientific) ua haujlwm ntawm ob lub zog them rov qab ntawm −50 thiab −70 V. MS3 xaiv raws li qhov ua ntej synchronization siv rau kev ntsuas TMT daim ntawv qhia ion teeb liab. Kev sib cais peptide tau ua tiav ntawm EASY-nLC 1200, siv 90% linear gradient elution, nrog rau qhov concentration ntawm buffer ntawm 6% txog 31%; buffer A yog 0.1% FA, thiab buffer B yog 0.1% FA thiab 80% ACN. Kab analytical tau ua haujlwm ntawm 50°C. Siv FreeStyle (version 1.6, Thermo Fisher Scientific) los faib cov ntaub ntawv qub raws li lub zog them rov qab FAIMS.
Kev txheeb xyuas thiab ntsuas cov protein. Siv lub tshuab tshawb nrhiav Andromeda, cov ntaub ntawv qub tau raug tshuaj xyuas siv MaxQuant version 1.5.2.8 (https://maxquant.org/). Ntxiv rau Cre recombinase thiab YFP cov kab ke tau los ntawm Aequorea victoria, peptide fragment spectra tau raug tshawb nrhiav rau cov kab ke canonical thiab isoform kab ke ntawm nas siv proteome (Proteome ID UP000000589, rub tawm los ntawm UniProt thaum Lub Tsib Hlis 2017). Methionine oxidation thiab protein N-terminal acetylation tau teeb tsa ua kev hloov pauv hloov pauv; cysteine ​​​​carbamoyl methylation tau teeb tsa ua kev hloov pauv ruaj khov. Cov txheej txheem zom zaub mov tau teeb tsa rau "specificity" thiab "trypsin / P". Tus lej tsawg kawg nkaus ntawm peptides thiab razor peptides siv rau kev txheeb xyuas protein yog 1; tus lej tsawg kawg nkaus ntawm cov peptides tshwj xeeb yog 0. Nyob rau hauv cov xwm txheej ntawm peptide daim ntawv qhia sib phim, tus nqi txheeb xyuas protein yog 0.01. Qhov kev xaiv "Second Peptide" tau qhib. Siv qhov kev xaiv "sib phim ntawm kev khiav" los hloov cov kev txheeb xyuas zoo ntawm cov ntaub ntawv qub sib txawv. Siv LFQ qhov sib piv tsawg kawg nkaus suav 1 rau kev ntsuas tsis muaj daim ntawv lo (LFQ) (60). Qhov muaj zog LFQ raug lim rau tsawg kawg yog ob qhov txiaj ntsig raug cai hauv tsawg kawg yog ib pawg genotype ntawm txhua lub sijhawm, thiab raug extrapolated los ntawm kev faib tawm ib txwm muaj nrog qhov dav ntawm. 0.3 thiab txav mus rau 1.8. Siv Perseus computing platform (https://maxquant.net/perseus/) thiab R (https://r-project.org/) los tshuaj xyuas cov txiaj ntsig LFQ. Ob txoj kev sim t nruab nrab los ntawm limma software pob tau siv rau kev tshuaj xyuas kev qhia sib txawv (61). Kev tshuaj xyuas cov ntaub ntawv tshawb nrhiav tau ua tiav siv ggplot, FactoMineR, factoextra, GGally thiab pheatmap. Cov ntaub ntawv proteomics raws li TMT tau tshuaj xyuas siv MaxQuant version 1.6.10.43. Tshawb nrhiav cov ntaub ntawv raw proteomics los ntawm UniProt lub database tib neeg proteomics, uas tau rub tawm thaum lub Cuaj Hli 2018. Qhov kev tshuaj xyuas suav nrog qhov kev kho kom huv si isotope uas muab los ntawm cov chaw tsim khoom. Siv limma hauv R rau kev tshuaj xyuas qhov sib txawv. Cov ntaub ntawv qub, cov txiaj ntsig tshawb nrhiav database, thiab cov txheej txheem tshuaj xyuas cov ntaub ntawv thiab cov txiaj ntsig tau khaws cia rau hauv ProteomeXchange alliance los ntawm PRIDE tus khub repository nrog cov ntaub ntawv teeb tsa tus lej PXD019690.
Cov lus piav qhia txog kev ua haujlwm ua rau kev tshuaj xyuas nplua nuj. Lub cuab yeej Ingenuity Pathway Analysis (QIAGEN) tau siv los txiav txim siab qhov nplua nuj ntawm cov lus piav qhia txog kev ua haujlwm ntawm cov ntaub ntawv teeb tsa ntawm 8 lub lis piam (Daim Duab 1). Hauv ntej, cov npe protein ntau tau los ntawm LC-MS / MS (tandem mass spectrometry) kev tshuaj xyuas cov ntaub ntawv siv nrog cov txheej txheem lim dej hauv qab no: Mus musculus raug xaiv ua hom tsiaj thiab keeb kwm yav dhau los, thiab pawg qhia tus nqi P kho los ntawm Benjamini rau kev nplua nuj 0.05 lossis qis dua yog suav tias yog qhov tseem ceeb. Rau daim duab no, tsib pawg sab saum toj tshaj plaws hauv txhua pawg raws li tus nqi P kho tau pom. Siv ntau t-xeem, siv ob-theem linear boost program ntawm Benjamini, Krieger, thiab Yekutieli (Q = 5%), lub sijhawm-chav kawm protein kev qhia tawm tau ua tiav rau cov neeg sib tw tseem ceeb uas tau txheeb xyuas hauv txhua pawg, thiab txhua kab raug tshuaj xyuas sib cais. Tsis tas yuav siv SD sib xws.
Yuav kom piv cov txiaj ntsig ntawm txoj kev tshawb fawb no nrog cov ntaub ntawv luam tawm thiab tsim daim duab Venn hauv Daim Duab 1, peb tau muab cov npe protein ntau nrog MitoCarta 2.0 cov lus piav qhia (24). Siv cov cuab yeej online Kos Venn Diagram (http://bioinformatics.psb.ugent.be/webtools/Venn/) los tsim daim duab.
Yog xav paub ntxiv txog cov txheej txheem suav lej siv rau kev tshuaj xyuas proteomics, thov saib ntu ntawm Cov Khoom Siv thiab Cov Txheej Txheem. Rau txhua qhov kev sim lwm yam, cov ntaub ntawv qhia ntxaws tuaj yeem pom hauv cov lus piav qhia sib xws. Tshwj tsis yog tias tau teev tseg lwm yam, tag nrho cov ntaub ntawv tau qhia ua qhov nruab nrab ± SEM, thiab tag nrho cov kev tshuaj xyuas suav lej tau ua tiav siv GraphPad Prism 8.1.2 software.
Yog xav paub ntxiv txog tsab xov xwm no, thov mus saib http://advances.sciencemag.org/cgi/content/full/6/35/eaba8271/DC1
Qhov no yog ib tsab xov xwm qhib rau kev nkag mus tau faib raws li cov nqe lus ntawm Creative Commons Attribution-Non-Commercial License, uas tso cai rau kev siv, kev faib tawm thiab kev tsim dua tshiab hauv txhua qhov chaw, tsuav yog qhov kev siv zaum kawg tsis yog rau kev lag luam thiab lub hauv paus yog tias cov haujlwm thawj yog qhov tseeb. Kev siv.
Lus Cim: Peb tsuas thov kom koj muab koj qhov chaw nyob email kom tus neeg uas koj pom zoo rau nplooj ntawv paub tias koj xav kom lawv pom email thiab nws tsis yog spam. Peb yuav tsis ntes cov chaw nyob email twg li.
Lo lus nug no yog siv los sim seb koj puas yog tus qhua thiab tiv thaiv kev xa tawm spam tsis siv neeg.
Los ntawm E. Motori, I. Atanassov, SMV Kochan, K. Folz-Donahue, V. Sakthivelu, P. Giavalisco, N. Toni, J. Puyal, N.-G. Larson
Kev tshuaj xyuas cov protein ntawm cov neurons uas tsis ua haujlwm tau qhia tias cov kev pab cuam metabolic tau qhib los tiv thaiv kev puas tsuaj ntawm cov neurodegeneration.
Los ntawm E. Motori, I. Atanassov, SMV Kochan, K. Folz-Donahue, V. Sakthivelu, P. Giavalisco, N. Toni, J. Puyal, N.-G. Larson
Kev tshuaj xyuas cov protein ntawm cov neurons uas tsis ua haujlwm tau qhia tias cov kev pab cuam metabolic tau qhib los tiv thaiv kev puas tsuaj ntawm cov neurodegeneration.
© 2020 American Association rau Advancement of Science. tag nrho cov cai. AAAS yog tus khub ntawm HINARI, AGORA, OARE, CHORUS, CLOCKSS, CrossRef thiab COUNTER. ScienceAdvances ISSN 2375-2548.