Tunah hian *Tun dinhmunah chuan: Cologne 50931, Germany, Cologne Excellence Cluster Research on Cellular Stress Response in Aging-related Diseases (CECAD) a awm mek a ni.
Mitochondrial natna neurodegeneration hi tihdanglam theih loh anga ngaih a ni a, a chhan chu neurons te metabolic plasticity a tlem a, mahse mitochondrial dysfunction hian taksaa neuronal metabolism cell autonomy a nghawng dan hi hriat chian a la ni lo. Hetah hian mitochondrial fusion dynamics tihbuai avanga OXPHOS tlakchhamna zual zel nei Purkinje neurons cell-specific proteome kan rawn tarlang a ni. Mitochondrial dysfunction hian proteomics field-ah inthlak danglamna thuk tak a thlen tih kan hmuchhuak a, chu chuan a tawpah chuan cell thih hmaa precise metabolic programs sequential activation a thlen ta a ni. Beisei loh takin TCA cycle-a intermediate-te supplement tu pyruvate carboxylase (PCx) leh anti-aging enzyme dangte induction chiang tak kan hmuchhuak a. PCx tihkhawtlai hian oxidative stress leh neurodegeneration a tizual a, hei hian OXPHOS nei lo neurons-ah atherosclerosis hian venhimna a nei tih a tilang. Terminally degenerated neurons-a mitochondrial fusion siam thar leh hian heng metabolic characteristics te hi a tidanglam vek a, chu chuan cell thihna a veng thei a ni. Kan thil hmuhchhuahte hian mitochondrial dysfunction laka invenna pe thei kawng hriat lohte a hmuchhuak a, natna hnuhnung berah pawh neurodegeneration chu tihdanglam theih a nih thu a tarlang bawk.
Neuronal energy metabolism vawn thatna kawnga mitochondria-te chanvo pawimawh tak chu mihring mitochondrial natna nena inzawm neurological symptoms zau tak takte hian a uar hle. Heng natna tam zawk hi mitochondrial gene expression (1, 2) tidanglamtu gene mutations emaw, mitochondrial dynamics nena inzawm gene tihchhiat emaw avanga lo awm a ni a, chu chuan mitochondrial DNA (mtDNA) stability chu indirect takin a nghawng a ni (3, 4). Animal model-a hnathawh chuan a chhehvel tissue-a mitochondrial dysfunction chhanna atan conservative metabolic pathways (5-7) te chu activate theih a ni tih a tarlang a, hei hian heng natna khirh tak takte pathogenesis thuk taka hriatthiamna atan information pawimawh tak a pe a ni. Chumi danglamna chu, thluaka mitochondrial adenosine triphosphate (ATP) siam chhuah hlawhchhamna tlangpui avanga cell chi hrang hrangte metabolic changes kan hriatthiamna hi a bulpui ber a ni (8), natna laka invenna emaw, venna emaw atana hman theih tur therapeutic target hriatchhuah a tulzia a sawi uar hle. Neurodegeneration tih loh nan (9). Thu hriat tur awm lohna chhan chu nerve cell te hi a chhehvel tissue cell chi hrang hrang nena khaikhin chuan metabolic flexibility nei tlem hle nia ngaih a ni (10). Heng cell te hian neurons te hnena metabolites supply inrualremna kawngah hian central role an nei a, chu chuan synaptic transmission a tichak a, hliam leh natna dinhmun a chhang let thei a, thluak tissue dinhmun harsa tak takah cell metabolism insiamrem theihna chu glial cells ah chauh a awm tawh mai (11-14). Chu bakah, thluak tissue-a inherent cellular heterogeneity hian neuronal subgroup bik hrang hranga metabolic changes thlengte zirchianna chu nasa takin a tikhawlo a ni. Chuvang chuan neurons-a mitochondrial dysfunction avanga cellular leh metabolic consequence dik tak hi hriat tur a tlem hle.
Mitochondrial dysfunction avanga metabolic consequences hriatthiam theih nan mitochondrial outer membrane fusion (Mfn2) tihchhiat avanga neurodegeneration stage hrang hranga awm Purkinje neurons (PNs) te kan isolated a. Mihringah Mfn2 mutations hi hereditary motor sensory neuropathy chi khat Charcot-Marie-Tooth type 2A tia hriat nen inzawm ni mahse, mice-a Mfn2 conditional destruction hi oxidation Phosphorylation (OXPHOS) dysfunction method induction hriat lar tak a ni. Neuronal subtype hrang hrang (16-19) leh a chhuak neurodegenerative phenotype te hi neurological symptoms progressive tak tak, movement disorders (18, 19) emaw cerebellar ataxia (16) te nen a inzawm a ni. Label-free quantitative (LFQ) proteomics, metabolomics, imaging, leh virological methods te inzawmkhawm hmangin, progressive neurodegeneration hian pyruvate carboxylase (PCx) leh PNs in vivo-a arteriosclerosis-a inrawlh thil dangte chu nasa takin a tichhuak tih kan lantir a ni The expression of enzymes. He thil hmuhchhuah hi a inzawm tlat tih finfiah nan Mfn2 tlachham PN-a PCx expression chu a bik takin kan down-regulate a, he operation hian oxidative stress a tizual a, neurodegeneration a ti chak tih kan hmu a, chu chuan azoospermia hian cell thihna a pe tih kan finfiah Metabolic adaptability a pe a ni. MFN2 expression na tak chuan OXPHOS tlakchhamna nasa tak nei, mitochondrial DNA tam tak ei tamna, leh mitochondrial network chhe tawh anga lang, terminal degeneration PN chu a chhanchhuak vek thei a, hei hian he neurodegeneration chi hi cell thih hmaa natna advanced stage-ah pawh a lo dam leh thei tih a tichiang lehzual a ni.
Mfn2 knockout PNs-a mitochondria awmte hmuh theih nan Cre-dependent mitochondria-te chu yellow fluorescent protein (YFP) (mtYFP) (20) Cre expression target-a siam thei mouse strain kan hmang a, in vivo-ah mitochondrial morphology kan check bawk. PNs-a Mfn2 gene tihchhiat hian mitochondrial network chu a inthen zauh zauh dawn tih kan hmuchhuak a (Figure S1A), a danglamna hmasa ber chu kum 3 mi lek a nih laiin hmuhchhuah a ni. Chumi danglamna chu, Calbindin immunostaining hloh atanga a lan danin, PN cell layer degeneration nasa tak chu kar 12 an tlin thlengin a intan lo (Figure 1, A leh B). Mitochondrial morphology inthlak danglamna hmasa ber leh neuronal thihna hmuh theiha lo awm tan hun inang lo tak chuan cell thih hmaa mitochondrial dysfunction avanga metabolic changes lo awmte chhui turin min fuih a ni. YFP (YFP+)-expressing PN (Figure 1C) isolate turin fluorescence-activated cell sorting (FACS)-based strategy kan siam a, control mice (Mfn2 + / loxP :: mtYFP loxP- stop-loxP: : L7-cre), hetah hian CTRL tia sawi a ni ang (Figure S1B). YFP signal relative intensity atanga gating strategy optimization hian non-PNs (YFPneg) (Figure S1B) emaw putative fluorescent axon/dendritic fragments (YFPlow; Figure S1D, veilam) atanga PNs YFP+ body (YFPhigh) chu kan tithianghlim thei a, hei hi confocal microscope hmanga nemngheh a ni (Figure S1D, dinglam). Classified population te identity finfiah nan LFQ proteomics kan nei a, chutah chuan principal component analysis kan nei a, YFPhigh leh YFPneg cell te inkarah hian inthenna chiang tak a awm tih kan hmu chhuak a (Figure S1C). YFPhigh cells hian PNs marker hriat tawh (ie Calb1, Pcp2, Grid2 leh Itpr3) te net enrichment an nei a (21, 22), mahse neurons emaw cell chi dang emaw-a proteins express tlangpui enrichment an nei lo (Figure 1D) ). Independent experiment-a classified YFPhigh cells-a sample lakkhawmte khaikhin chuan correlation coefficient> 0.9 a awm a, hei hian biological replicate inkara reproducibility tha tak a lantir a ni (Figure S1E). A tawi zawngin, heng data te hian feasible PN acute leh specific isolation atana kan ruahmanna chu a nemnghet a ni. L7-cre driver system hman hian nau neih hnu kar hmasa berah mosaic recombination a thlen avangin (23), CTRL atanga mice te chu kan culling tan a, conditional (Mfn2 loxP / loxP :: mtYFP loxP-stop-loxP :: L7-cre) Collect neurons kan nei tan a. Recombination zawh hnuah kar 4 a tlin chuan Mfn2cKO an ti a. End point atan chuan mitochondrial fragmentation chiang tak awm mahse PN layer chu a awm reng laiin kum 8 mi kan thlang a (Figure 1B leh Figure S1A). A vaiin protein zawng zawng 3013 kan quantified a, chung zinga 22% vel chu mitochondrial proteome hmanga MitoCarta 2.0 annotations hmanga mitochondria anga siam a ni (Figure 1E) (Figure 1E) (24). Kar 8-naa differential gene expression analysis an neihah chuan protein zawng zawng zinga 10.5% chauhvin danglamna nasa tak an nei tih hmuhchhuah a ni (Figure 1F leh Figure S1F), heng zinga protein 195 chu down-regulated niin, protein 120 chu up-regulated a ni (Figure 1F). Hriat tur chu he data set-a “innovative pathway analysis” atanga a lan dan chuan differentially expressed genes te hi a bik takin restricted set of specific metabolic pathways-a tel an ni (Figure 1G). Ngaihven awm tak chu OXPHOS leh calcium signaling nena inzawm pathways downregulation hian fusion-deficient PNs-a mitochondrial dysfunction induction a awm tih a nemnghet a, mahse amino acid metabolism inrawlh ber category dangte chu nasa takin upregulated an ni a, hei hi mitochondrial metabolism-a metabolism thleng nen a inmil a ni Rewiring is consistent. hnathawh lohna (disfunction) a ni.
(A) CTRL leh Mfn2cKO mice-a cerebellar section-a PNs (calbindin, gray) hloh zual zel dan entir (representative confocal photographs) nuclei te chu DAPI hmangin counterstain an ni. (B) (A) quantification (one-way analysis of variance, ***P<0.001; n = mice pathum atanga circle 4 atanga 6). (C) Experimental hnathawh dan tur. (D) Purkinje (chunglam) leh cell chi dang (middle) tana marker bikte heat map-a insem dan. (E) Venn diagram-ah hian classified PN-a mitochondrial protein hmuhchhuah zat tarlan a ni. (F) Kar 8-ah Mfn2cKO neurons-a protein hrang hrang expressed volcano plot (significance cut-off value 1.3). (G) Creativity pathway analysis hian Mfn2cKO PN-a up-regulation (red) leh down-regulation (blue) pathway pawimawh ber panga chu kar 8 chhunga classified a ni. Protein hmuhchhuah tinte expression level average chu tarlan a ni. Grayscale heat map: P value siamrem a ni. ns, a pawimawh lo.
Proteomics data atanga a lan dan chuan complex I, III, leh IV te protein expression chu a tlahniam zauh zauh a. Complex I, III, leh IV-ah te hian mtDNA-encoded subunit pawimawh tak tak an nei vek a, complex II, nuclear-coded chauh awm chu a bul berah chuan a nghawng lo (Figure 2A leh Figure S2A). . Proteomics result nena inmil takin cerebellar tissue section immunohistochemistry hmangin PN-a complex IV-a MTCO1 (mitochondrial cytochrome C oxidase subunit 1) subunit level chu a tlahniam zauh zauh tih hmuhchhuah a ni (Figure 2B). mtDNA-encoded subunit Mtatp8 chu nasa takin a tlahniam a (Figure S2A), chutih laiin nuclear-encoded ATP synthase subunit-a steady-state level chu a danglam lo a, hei hi mtDNA expression a stable laiin stable ATP synthase subassembly F1 complex hriat tawh nen a inmil hle. Formation pawh a inmil hle. Interrupt (7) a ni. Mfn2cKO PNs sorted-a mtDNA level chu real-time polymerase chain reaction (qPCR) hmanga endik a nih chuan mtDNA copy number a tlahniam zauh zauh tih a nemnghet. Control group nen khaikhin chuan kum 8 an tlin chuan mtDNA level 20% vel chauh Was retained (Figure 2C). Heng result nena inmil takin Mfn2cKO PNs confocal microscopy staining hmangin DNA hmuhchhuah a ni a, hei hian mitochondrial nucleotide hman dan hun a innghat tih a tarlang (Figure 2D). Mitochondrial protein tihchhiatna leh stress response-a inrawlh candidate thenkhat chauh chu up-regulated an ni tih kan hmuchhuak a, chung zingah chuan Lonp1, Afg3l2 leh Clpx, leh OXPHOS complex assembly factors te pawh an tel. Apoptosis-a inrawlh protein level-ah hian danglamna lian tham a awm lo (Figure S2B). Chutiang bawkin, calcium transport-a inrawlh mitochondria leh endoplasmic reticulum channel-ah pawh inthlak danglamna tlemte chauh a awm tih kan hmuchhuak bawk (Figure S2C). Chu bakah, autophagy-related proteins evaluation-ah pawh danglamna lian tham a awm lo a, hei hi immunohistochemistry leh electron microscopy hmanga in vivo-a autophagosomes hmuh theih induction hmuhchhuah nen a inmil hle (Figure S3). Mahse, PNs-a OXPHOS dysfunction lo zual zel hian ultrastructural mitochondrial changes langsar tak a keng tel a ni. Mfn2cKO PNs kar 5 leh kar 8 inkar cell taksa leh dendritic tree-ah mitochondrial cluster hmuh tur a awm a, inner membrane structure-ah pawh inthlak danglamna thuk tak a awm a (Figure S4, A leh B). Heng ultrastructural changes leh mtDNA tlahniam nasa tak nen hian inmil takin, acute cerebral cerebellar slices tetramethylrhodamine methyl ester (TMRM) hmanga an zirchiannaah chuan Mfn2cKO PNs-a mitochondrial membrane potential chu nasa takin a tlahniam tih hmuhchhuah a ni (Figure S4C).
(A) OXPHOS complex expression level atanga a hun kal dan thlirletna. Kar 8-a P<0.05 nei protein chauh ngaihtuah tur (two-way ANOVA). Dotted line: CTRL nen khaikhin chuan adjustment a awm lo. (B) Veilam: Cerebellar section entirnan anti-MTCO1 antibody (scale bar, 20 μm) hmanga label a ni. Purkinje cell body-te luahna hmun chu yellow-in a khuh a ni. Right: MTCO1 level quantification (one-way analysis of variance; n = mice pathum atanga cell 7 atanga 20 thlirlet). (C) PN sorted-a mtDNA copy number qPCR hmanga zirchianna (one-way analysis of variance; n = 3 to 7 mice). (D) Veilam: Cerebellar slice entir nan anti-DNA antibody (scale bar, 20 μm) hmanga label a ni. Purkinje cell body-te luahna hmun chu yellow-in a khuh a ni. Right: mtDNA lesions quantification (one-way analysis of variance; n = mice pathum atanga cell 5 atanga 9 thleng). (E) Acute cerebellar section entirnan, whole-cell patch clamp recording-a mitoYFP + Purkinje cells (arrow) te a lan dan. (F) IV curve zat chhiar chhuah. (G) CTRL leh Mfn2cKO Purkinje cell-a depolarizing current injection aiawhtu recording. Top trace: AP tichhuaktu pulse hmasa ber. Bottom trace: AP frequency sang ber. (H) Postsynaptic spontaneous inputs (sPSPs) awm zat tehna. Representative recording trace leh a zoom ratio chu (I) ah hian tarlan a ni. One-way analysis of variance hmangin mice pathum atanga cell n = 5 atanga 20 vel an zirchiang a. Data chu mean±SEM anga tarlan a ni a; *P<0.05 a ni a; **P<0.01 a ni a; ***P<0.001 a ni. (J) Perforated patch clamp mode hmanga AP recorded spontaneous traces aiawhtu. Top trace: AP frequency sang ber. Bottom trace: AP pakhat zoom a ni. (K) (J) angin AP frequency average leh maximum chu quantified rawh. Mann-Whitney-a test a ni a; n = 5 cells chu mice pali atangin an zirchiang a. Data chu mean±SEM anga tarlan a ni a; pawimawh lo.
Kar 8 mi Mfn2cKO PN-ah hian OXPHOS chhiatna chiang tak hmuh a ni a, hei hian neurons-te physiological function chu a dik lo hle tih a tilang a ni. Chuvangin, OXPHOS tlachham neurons te passive electrical characteristics chu kar 4 atanga kar 5 leh kar 7 atanga kar 8 chhungin acute cerebellar slices-a whole-cell patch clamp recording hmangin kan zirchiang a (Figure 2E). Beisei loh takin Mfn2cKO neurons te average resting membrane potential leh input resistance chu control nen a inang a, mahse cell hrang hrangah danglamna fiah lo tak tak a awm (Table 1). Chutiang bawkin kar 4 atanga kar 5 inkar ah pawh current-voltage relationship (IV curve) ah pawh danglamna lian tham a awm lo (Figure 2F). Mahse, IV regimen (hyperpolarization step) hmang hian kar 7 atanga kar 8 inkar Mfn2cKO neuron pakhat mah an damchhuak lo a, hei hian he stage hnuhnung berah hian hyperpolarization potential-ah sensitivity chiang tak a awm tih a tilang a ni. Chumi danglamna chu Mfn2cKO neurons-ah chuan repetitive action potential (AP) discharge siamtu depolarizing currents te chu an tuar tha hle a, hei hian an overall discharge pattern chu kar 8 mi control neurons te nen hian a danglam vak lo tih a tilang (Table 1 leh Figure 2G). Chutiang bawkin, spontaneous postsynaptic currents (sPSCs) awm zat leh amplitude chu control group-a awmte nen tehkhin theih a ni a, thil thleng tamna pawh kar 4 atanga kar 5 atanga kar 7 atanga kar 8 thleng a pung a, chutiang bawkin a pung bawk (Figure 2, H leh I). PNs-a synaptic maturation hun chhung (25). Perforated PNs patch hnuah pawh hetiang chiah hian result a awm bawk. He configuration hian cellular ATP chhiatna compensation awm thei chu a veng a, chu chu whole-cell patch clamp recording-ah pawh a thleng thei a ni. A bik takin Mfn2cKO neurons te resting membrane potential leh spontaneous firing frequency te chu a nghawng lo (Figure 2, J leh K). A tawi zawngin, heng results te hian OXPHOS dysfunction langsar tak nei PN te hian high-frequency discharge pattern te chu an tuar tha thei tih a tilang a, hei hian compensation mechanism a awm tih a tilang a, chu chuan near-normal electrophysiological responses te chu a vawng reng thei a ni.
Data chu mean ± SEM (one-way analysis of variance, Holm-Sidak-a multiple comparison test; *P<0.05) anga tarlan a ni. Unit number chu bracket hmanga tarlan a ni.
Proteomics dataset-a category eng pawh (Figure 1G)-ah hian OXPHOS tlakchhamna nasa tak do thei tur kawng a awm em tih chhui turin kan insiam a, chu chuan a nghawng PN-in near-normal electrophysiology a neih theih chhan kan sawifiah a ni (Figure 2, E to K). . Proteomics analysis atanga a lan dan chuan branched chain amino acids (BCAA) catabolism-a inrawlh enzyme te chu nasa takin up-regulated an ni (Figure 3A leh Figure S5A), final product acetyl-CoA (CoA) emaw succinyl CoA emaw hian arteriosclerosis Acid (TCA) cycle-a tricarboxylates te chu a tipung thei a ni. BCAA transaminase 1 (BCAT1) leh BCAT2 te hi a pung vek tih kan hmuchhuak. Anni hian α-ketoglutarate atanga glutamate siamin BCAA catabolism step hmasa ber chu an catalyze a ( 26 ). Branched chain keto acid dehydrogenase (BCKD) complex siamtu subunit zawng zawng chu upregulated an ni vek (complex hian a hnu lama BCAA carbon skeleton lo chhuak decarboxylation lo awm tur leh irreversible decarboxylation chu a tichak a ni) (Figure 3A leh Figure S5A). Mahse, BCAA ngei pawh hi sorted PN-ah hian danglamna chiang tak hmuh tur a awm lo a, hei hi heng amino acid pawimawh tak takte cellular uptake tihpun vang emaw, TCA cycle tihpunna atana source dang (glucose emaw lactic acid) hman vang emaw a ni thei (Figure S5B). OXPHOS nei lo PNs te pawhin kar 8 an tlin chuan glutamine decomposition leh transamination activity a tipung tih an hmuchhuak a, hei hi mitochondrial enzymes glutaminase (GLS) leh glutamine pyruvate transaminase 2 (GPT2) up-regulation atanga lang thei a ni (Figure 3, A leh C). Hriat tur chu GLS up-regulation hi spliced ​​isoform glutaminase C (GLS-GAC) (Mfn2cKO/CTRL inthlak danglamna hi a let 4.5 vel a ni a, P = 0.05) chauh a ni a, cancer tissue-a a specific up-regulation hian mitochondrial bioenergy a support thei bawk. (27) a ni.
(A) Heat map-ah hian kar 8 chhunga kawng ruat sa atana protein level fold inthlak dan a lang. (B) Anti-PCx antibody (scale bar, 20 μm) hmanga label cerebellar slice entir nan. Arrow sen hian Purkinje cell taksa chu a kawk a. (C) Time course protein expression analysis chu atherosclerosis atana candidate pawimawh tak nia hriat a ni (multiple t-test, *FDR <5%; n = 3-5 mice). (D) A chungah: [1-13C]pyruvate tracer-a awm labeled carbon luh dan hrang hrang (chu chu PDH emaw trans-arterial route hmanga) schematic diagram a ni. A hnuai lam: Violin chart-ah hian acute cerebellar slice-te chu [1-13C]pyruvate hmanga label hnua aspartic acid, citric acid leh malic acid-a inthlak, single-labeled carbon (M1) percentage tarlan a ni (paired t-test; ** P <0.01). (E) Kawng tarlanna hun chanchin kimchang taka thlirletna. Kar 8-ah P<0.05 nei protein chauh ngaihtuah tur a ni . Dashed line: siamthatna value a awm lo (two-way analysis of variance; * P <0.05; *** P <0.001). Data chu mean±SEM anga tarlan a ni.
Kan zirchiannaah chuan BCAA catabolism hi up-regulation pathway pawimawh ber pakhat a lo ni ta a ni. He thudik hian TCA cycle chhunga ventilation volume lut chu OXPHOS nei lo PN-ah a danglam thei tih a tilang chiang hle. Hei hian neuronal metabolic rewiring chi khat lian tak a entir thei a, hei hian OXPHOS dysfunction na tak a awm reng laiin neuronal physiology leh dam khawchhuahnaah nghawng direct a nei thei a ni. He hypothesis nena inmil takin, anti-atherosclerotic enzyme ber PCx ​​chu up-regulated (Mfn2cKO/CTRL hi vawi 1.5 vel a inthlak a; Figure 3A) a ni tih kan hmuchhuak a, hei hian pyruvate chu oxaloacetate-ah a chantir a (28), chu chu thluak tissue-ah a awm nia rin a ni The expression in is restricted to astrocytes (29, 30). Proteomics result nena inmil takin confocal microscopy hmangin OXPHOS tlachham PN-ah PCx expression chu a bik takin leh nasa takin a pung tih hmuhchhuah a ni a, PCx reactivity erawh chu control-a Bergmann glial cell kianga awmte chauh a ni ber thung (Figure 3B). PCx upregulation hmuhchhuah chu functionally test turin acute cerebellar slices te chu [1-13C]pyruvate tracer hmangin kan enkawl a. Pyruvate chu pyruvate dehydrogenase (PDH) hmanga oxidized a nih chuan a isotope label chu a bo , But chu pyruvate chu vascular reaction hmanga metabolized a nih chuan TCA cycle intermediates-ah a lut a ni (Figure 3D). Kan proteomics data thlawpna atan hian he tracer atang hian Mfn2cKO slices-a aspartic acid-ah hian marker tam tak kan hmu a, citric acid leh malic acid te pawhin trend hniam tak an nei a, mahse a langsar lo hle (Figure 3D).
MitoPark mice-a mitochondrial dysfunction nei, dopamine neurons-in mitochondrial transcription factor A gene (Tfam) a bik taka a tihchhiat avanga dopamine neurons-ah pawh PCx expression chu nasa takin up-regulated a ni bawk (31), hei hian acetone acid arteriosclerosis Neuronal dysfunction laiin natna lo awm dan chu a regulate tih a tilang a ni OXPHOS taksaah a awm. Hriat tur chu, arteriosclerosis nena inzawm thei neurons-a express theih enzyme danglam bik (32-34) te chu OXPHOS nei lo PN-ah nasa takin up-regulated an ni tih hmuhchhuah a ni a, chungte chu propionyl-CoA carboxylase (PCC-A) te hi an ni a, Malonyl-CoA hian propionyl-CoA chu succinyl-CoA ah a chantir a, mitochondrial malic 3 (ME3), a hna ber chu malate atanga pyruvate lakchhuah a ni (Figure 3, A leh C) (33, 35). Chu bakah, PDH phosphorylate leh inactivate tu Pdk3 enzyme-ah nasa takin a pung tih kan hmu bawk ( 36 ), chutih laiin PDH activate tu Pdp1 enzyme emaw PDH enzyme complex ngeiah pawh danglamna hmuh tur a awm lo (Figure 3A). Chutiang bawkin, Mern2cKO PNs-ah chuan Ser293-a PDH complex-a pyruvate dehydrogenase E1 component-a α1 subunit α (PDHE1α) subunit phosphorylation chu a tichak a ( Figure S6C) (Figure S6C). Pyruvate hian vascular access a nei lo.
A tawp berah chuan serine leh glycine biosynthesis super pathway, a kaihhnawih mitochondrial folate (1C) cycle leh proline biosynthesis (Figure 1G leh Figure S5C) te chu activation process chhung hian nasa takin up-regulated vek an ni tih kan hmuchhuak a, report-te chuan. A chhehvel tissue te hi mitochondrial dysfunction nen an activate thin (5-7). Heng proteomics data thlawptu confocal analysis atanga a lan dan chuan OXPHOS bo nei PN-ah chuan kar 8 mi mice cerebellar slices chu mitochondrial folate cycle-a enzyme pawimawh tak serine hydroxymethyltransferase 2 (SHMT2) hmangin an enfiah a ni. Immune response pawimawh tak (Figure S5D). CU-glucose-incubated acute cerebellar slice 13-ah metabolic tracing experiment-ah serine leh proline biosynthesis up-regulation a awm tih a nemnghet lehzual a, hei hian carbon isoforms serine leh proline-a flux a pung tih a tilang (Figure S5E). GLS leh GPT2-in a tihchak reaction te hian glutamine atanga glutamate siamna leh glutamate leh α-ketoglutarate inkara transamination siamtu a nih avangin an upregulation hian OXPHOS-deficient neurons te hian glutamate mamawhna an nei sang zawk tih a tilang a , Hei hi proline biosynthesis tihpunna vawn reng tumna a ni thei (Figure S5C). Heng inthlak danglamna te nen a danglamna chu PN-specific Mfn2cKO mice atanga cerebellar astrocytes proteomic analysis an neihah chuan heng pathways (antiperoxidases zawng zawng telin) te hi expression ah a danglam vak lo tih hmuhchhuah a ni a, chu chuan This metabolic redirection is selective to degraded PN (Fig. S6, D to G) tih a lantir a ni.
A tawi zawngin, heng analysis te hian PNs-a specific metabolic pathways temporal activation pattern hrang hrang a awm tih a tarlang. Neuronal mitochondrial function pangngai lo chuan atherosclerosis hmasa ber leh 1C remodeling (Figure 3E leh Figure S5C) a thlen thei a, I leh IV complex expression-a inthlak danglamna pawh sawi lawk theih ni mahse, serine de novo synthesis-a inthlak danglamna chu a tawp lam chauh a ni. OXPHOS hnathawh that lohna (Figure 3E leh Figure S5C). Heng thil hmuhchhuah te hian stress-induced mitochondrial (1C cycle) leh cytoplasmic (serine biosynthesis) te hian TCA cycle-a atherosclerosis tihpunna nen synergistically-in neuronal metabolism a siam thar lehna tur process inzawmkhawm a sawifiah a ni.
Kar 8 mi OXPHOS tlachham PN te hian high-frequency excitation activity an vawng reng thei a, mitochondrial dysfunction compensate turin metabolic reconnection nasa tak an nei thei bawk. He thil hmuhchhuah hian thil awmdan ngaihnawm tak a siam a, tun dinhmunah pawh hian heng cell te hian neurodegeneration tihkhawtlai emaw, venna emaw atan therapeutic intervention an dawng thei bawk. Tlai. He thil awm thei hi independent intervention pahnih hmangin kan chinfel sak a ni. A hmasa berah chuan Cre-dependent adeno-associated virus (AAV) vector kan design a, chutiang chuan MFN2 chu OXPHOS tlachham PN-ah in vivo-ah thlan bik takin a lang thei a ni (Figure S7A). MFN2 encoding AAV leh fluorescent reporter gene mCherry (Mfn2-AAV) te chu primary neuron culture-ah in vitro-ah finfiah a ni a, hei hian MFN2 chu Cre-dependent takin a express a, mitochondrial morphology a chhanchhuak a, chu chuan Mfn2cKO neurons-a neuromutation a veng thei a ni ( Figure S7, B, D leh E). A dawtah chuan in vivo experiment kan nei a, kar 8 mi Mfn2-AAV chu Mfn2cKO leh control mice-te cerebellar cortex-ah stereotactically-in kan thlen a, kar 12 mi mice-te chu kan zirchiang bawk (Figure 4A). Mfn2cKO mice enkawl tawhte chu an thi ta a ni (Figure 1, A leh B) (16). Viral transduction in vivo hian cerebellar circle thenkhatah PN chu selective expression a siam a (Figure S7, G leh H). mCherry (Ctrl-AAV) chauh expressing control AAV injection hian Mfn2cKO rannung neurodegeneration degree-ah nghawng lian tham a nei lo. Chumi danglamna chu Mfn2-AAV hmanga transduced Mfn2cKOs te zirchiannaah chuan PN cell layer hian venhimna nasa tak a nei tih hmuhchhuah a ni (Figure 4, B leh C). A bik takin neuron density hi control animals te nen chuan a danglam thei lo tluk niin a lang (Figure 4, B leh C, leh Figure S7, H leh I). MFN1 expression ni lovin MFN2 expression hian neuronal death chhanchhuahna kawngah a thawk tha ve ve a (Figure 4C leh Figure S7, C leh F), hei hian ectopic MFN1 expression hian MFN2 tlakchhamna chu a tichak thei tih a tilang a ni. Single PN level-a zirchianna dang neih belh leh chuan Mfn2-AAV hian mitochondria ultrastructure chu nasa takin a chhanchhuak a, mtDNA level a normal a, anti-angiogenesis marker PCx ​​expression sang tak chu a tidanglam tih hmuhchhuah a ni (Figure 4, C to E ). Mfn2cKO mice chhanchhuah te chu chawlh hahdamna hmuna mit hmuha enfiah a nih chuan an dinhmun leh an motor symptoms (movement S1 to S3) a tha zawk tih hmuhchhuah a ni. Thutawp atan chuan heng experiment-te hian OXPHOS tlakchhamna nasa tak nei PN-a MFN2 dah leh hun sawn chu mtDNA ei leh in tihdanglam nan a tawk a, atherosclerosis a thlen a, chu chuan axon degeneration leh neuronal death in vivo-ah a veng thei tih a tarlang.
(A) Metabolic pathway tarlan chu activate a nih chuan MFN2 encoding AAV inject dan tur experimental schedule tarlanna scheme. (B) Mfn2cKO mice-a kar 8-a transduced leh anti-Calbindin antibody hmanga label-a kar 12 mi cerebellar slices representative confocal images. Dinglam: Axon fibers te scaling. Axon zoom scale hi 450 leh 75 μm a ni. (C) Veilam: AAV transduction loop (AAV+)-a Purkinje cell density zat chhiar (one-way analysis of variance; n = 3 mice). Right: kar 12-naah transduced PN-a mtDNA focus analysis (unpaired t-test; n = mice pathum atanga cell 6). * P <0.05 a ni a; ** P <0.01 a ni. (D) Viral vector tarlan hmanga transduced Mfn2cKO cerebellar section-a PN-te aiawhtu transmission electron micrographs. Pink mask hian dendrite-te luahna hmun a entir a, yellow dotted square hian dinglama zoom pek chu a entir bawk a; n hian nucleus a entir a ni. Scale bar, 1μm a ni. (E)-ah hian kar 12-a PN transduced-a PCx staining entir a ni. Scale bar, 20μm a ni. OE, sawi chhuah tam lutuk; FC, fold thlak danglam.
A tawp berah chuan OXPHOS dysfunction tawk PN-a peroxidase-induced cell survival pawimawhzia kan zirchiang a. Mouse PCx mRNA (AAV-shPCx) bik target-tu AAV-shRNA (short hairpin RNA) encoding mCherry kan siam a, virus emaw a scrambled control (AAV-scr) emaw chu Mfn2cKO mice cerebellum-ah kan inject a ni. PCx expression a san lai (Figure 3C) leh PN cell layer a la awm reng lai (Figure 1A) chhunga PCx knockdown tha tak neih theih nan kum li a nih laiin injection hi an ti a (Figure 1A). Hriat tur chu PCx (Figure S8A) tihchhiat hian PN thihna nasa takin a ti chak a, chu chu infected ring-ah chauh a awm (Figure 5, B leh C). PCx up-regulation-in a thlen metabolic effects mechanism hriatthiam nan PCx knockdown hnua PNs redox status leh AAV-mediated optical biosensor Grx1-roGFP2 chu a ruala express a nih hnuah (Figure S8, B to D) glutathione The relative change of peptide redox potential (38) tehna kan zirchiang a. Tichuan, kar 7 mi Mfn2cKO emaw control littermates emaw acute brain slices-ah two-photon fluorescence lifetime imaging microscopy (FLIM) kan hmang a, FLIM conditions kan finfiah hnua cytoplasmic redox status-a inthlak danglamna awm thei te kan hmuchhuak a (Figure S8, E to G). Analysis-ah hian PCx expression nei lo Mfn2cKO PN pakhat chauh oxidation state a sang hle tih hmuhchhuah a ni a, hei hi control neurons emaw scrambled shRNA chauh express Mfn2cKO PNs emaw nen a danglam a ni (Figure 5, D leh E). PCx expression chu down-regulated a nih chuan Mfn2cKO PNs oxidized state sang tak nei percentage chu a let thum aia tam a pung a (Figure 5E), hei hian PCx up-regulation hian degenerated neurons te redox capacity a vawng reng tih a tilang a ni.
(A) Metabolic pathway tarlan chu activate a nih chuan shPCx encoding AAV inject dan tur experimental schedule tarlanna scheme. (B) Mfn2cKO mice-a kar 8 mi cerebellar section transduced leh kar 4-a anti-calcineurin antibody hmanga label-a representative confocal photographs. Scale bar, 450μm a ni. (C) AAV-transduced loop-a Purkinje cell density zat chhiar (one-way analysis of variance; n = 3 to 4 mice). Data chu mean±SEM anga tarlan a ni a; ***P<0.001 a ni. (D) Representative FLIM picture hian kar 7 mi PN expressing glutathione redox sensor Grx1-roGFP2 chu experimental condition tarlan hnuaia a dam chhung zatve a tarlang a. LUT (look-up table) ratio: dam rei hun chhung (picoseconds-ah). Scale bar, 25μm a ni. (E) Histogram hian (D) atanga Grx1-roGFP2 lifetime value insem dan a tarlang a (n=158 atanga 368 cells in mice pahnih condition tin hnuaiah). Histogram tin chunga pie chart: hian cell dam rei zawk (red, oxidized) emaw, a tawi zawk (blue, reduced) emaw zat a tarlang a, hei hi CTRL-AAV-scr-a dam rei zawng value average 1 SD aia tam a ni. (F) Model ruahman hian neuronal PCx upregulation-in a humhimna a lantir a.
A vaiin, hetia kan data kan pek hian MFN2 re-expression hian OXPHOS tlakchhamna nasa tak, mtDNA tlakchhamna nasa tak, leh ista-like morphology danglam tak tak nei advanced PN chu a chhanchhuak vek thei tih a tarlang a, chu chuan natna zual takah pawh hmasawnna chhunzawm zel a pe thei a ni. Neurodegeneration hian cell thih hmaa stage awm dan reversible evidence a pe a ni. Hetiang degree of metabolic flexibility hi neurons te hian atherosclerosis (TCA cycle rewiring) an thlen theihna hian a tilang lehzual a, hei hian OXPHOS nei lo PNs-a PCx expression a titawp a, cell thihna a tipung a, chu chuan venhimna hna a thawk a ni (Figure 5F).
He zirchiannaah hian OXPHOS dysfunction-a PN-te chhanna chu metabolic program-in a tihchak differential activation pathway hmanga TCA cycle atherosclerosis-ah zawi zawiin a inzawm khawm tih finfiahna kan pe a ni. Proteomic analysis hi complementary method tam tak hmangin kan nemnghet a, mitochondrial dysfunction na tak avanga challenge a nih chuan neurons te hian a hmaa hriat ngai loh metabolic elasticity form an nei tih kan hmuchhuak a ni. Kan mak tak mai chu, rewiring process pumpui hian neurodegeneration nena inzawm, zawi zawiin leh tihdanglam theih loh khawpa terminal metabolic state chu a chhinchhiahna a ni hauh lo va, mahse kan data chuan cell thih hma stage-ah pawh maintenance neuron a siam thei tih a tarlang Functional compensation mechanism. He thil hmuhchhuah hian neurons te hian taksaah metabolic plasticity nasa tak an nei tih a tilang a ni. He thudik hian a hnu lama MFN2 rawn luh leh hian key metabolic marker expression a tidanglam thei a, PN degeneration a veng thei tih a tilang chiang hle. Chu ai chuan atherosclerosis a titawp a, nerve a ti chak zawk bawk. transsexual tih hi a ni.
Kan zirchianna atanga kan hmuh chhuah ngaihnawm ber pakhat chu OXPHOS nei lo PN te hian TCA cycle metabolism chu a tidanglam thei a, chu chu arteriosclerosis bik tichaktu enzyme te up-regulate in a tidanglam thei a ni. Metabolic rearrangement hi cancer cell-te thil langsar tak a ni a, a then chu glutamine-ah an innghat a, TCA cycle intermediates supplement-in reducing equivalents an siam a, hei hian thawkna chain a khalh a, lipid leh nucleotide biosynthesis precursors siam chhuahna a vawng reng bawk (39 , 40). Tun hnaia zirchianna pakhatah chuan OXPHOS dysfunction tawk peripheral tissue-ah chuan glutamine/glutamate metabolism inzawm lehna pawh hi thil langsar tak a ni tih a tarlang (5, 41), hetah hian TCA cycle-a glutamine luhna lam chu Because of the severity of OXPHOS injury (41) ah a innghat a ni. ). Mahse, taksaa neuronal metabolic plasticity inang lo tak tak leh natna context-a a inzawmna awm thei chungchangah hian finfiahna chiang tak a awm lo. Tun hnaia in vitro study-ah chuan primary cortical neurons te hian neurotransmission atan glutamate pools an mobilise tih hmuhchhuah a ni a, chu chuan metabolic stress condition hnuaiah oxidative metabolism leh atherosclerosis a tichak a ni (42). Hriat tur chu TCA cycle enzyme succinate dehydrogenase pharmacological inhibition hnuaiah hian pyruvate carboxylation hian cultured cerebellar granule neurons-a oxaloacetate synthesis a vawng reng niin an ngai (34). Mahse, heng mechanism-te hi thluak tissue (atherosclerosis hi astrocyte-ah chauh a awm nia rinna hmun) nena physiological relevance hian physiological significance pawimawh tak a la nei tho (43). Hetiang a nih chuan kan data atanga a lan dan chuan taksaa OXPHOS-in a tihchhiat PNs te chu BCAA degradation leh pyruvate carboxylation-ah an thlak thei a, chungte chu TCA pool intermediates supplementation source lian ber pahnih an ni. BCAA catabolism hian neuronal energy metabolism-a a thawhhlawk nia sawi ni mah se, neurotransmission atana glutamate leh GABA te chanvo bakah hian ( 44 ), in vivo-ah heng mechanism te hi finfiahna a la awm lo. Chuvangin, dysfunctional PNs hian atherosclerosis tihpunna hmangin assimilation process-in a khalh TCA intermediates ei zat chu automatic-in a compensate thei tih ngaihtuah a awlsam hle. A bik takin, aspartic acid mamawhna sang zel vawng reng turin PCx upregulation a ngai mai thei a, hei hi mitochondrial dysfunction nei cell proliferating-ah chuan an rawt a ni ( 45 ). Mahse, kan metabolomics analysis-ah chuan Mfn2cKO PNs-a aspartic acid steady-state level-ah danglamna lian tham a awm lo (Figure S6A), hei hian proliferating cells leh post-mitotic neurons inkara aspartic acid metabolic utilization hrang hrang a lantir nia ngaih a ni. In vivo-a dysfunctional neurons-a PCx upregulation mechanism dik tak chu hriat chian a la ni lo nain, he premature response hian neurons-te redox state vawn thatna kawngah hmun pawimawh tak a chang tih kan hmuchhuak a, hei hi cerebellar slice-a FLIM experiment-ah hmuhchhuah a ni. A bik takin PNs ten PCx up-regulate lo tura ven hian oxidized state a thlen thei a, cell thihna a ti chak thei bawk. BCAA degradation activation leh pyruvate carboxylation te hi mitochondrial dysfunction peripheral tissue te characterize dan tur a ni lo (7). Chuvangin, OXPHOS tlachham neurons-te thil pawimawh ber niin a lang a, chu chu anmahni chauh ni lo mah se, chu chu neurodegeneration atana pawimawh tak a ni. .
Cerebellar disease hi neurodegenerative disease chi hrang hrang heterogeneous a ni a, ataxia angin a lang tlangpui a, PNs a tichhe fo bawk (46). He neuron population hi mitochondrial dysfunction-ah a hlauhawm hle a, a chhan chu mice-a an selective degeneration hian mihring spinocerebellar ataxia-a langsar motor symptom tam tak a siam chhuah theih nan a tawk a ni (16, 47, 48). Report-te tarlan danin, mutant gene nei transgenic mouse model chu mihring spinocerebellar ataxia nen a inzawm a, mitochondrial dysfunction a nei a ( 49 , 50 ), PNPH-a OXPHOS tlakchhamna avanga thil thleng zirchian a pawimawhzia a sawi uar hle. Chuvangin, he neuron population danglam tak hi \ha taka isolate leh zirchian hi a \ha hle. Mahse, PN-te hi pressure-ah an sensitive hle a, cerebellar cell population zawng zawngah a tlem hle tih ngaihtuah chuan omics-based study tam takah chuan cell pum anga selective separation hi thil harsa tak a la ni reng a ni. Cell chi dang (a bik takin puitling tissue)-a contamination awm lohna tak tak chu thil theih loh tluk zet ni mah se, FACS nen dissociation step \ha tak kan hmang dun a, downstream proteomics analysis atan neurons nung tling tak kan hmu a, cerebellum pumpui data set awmsa nen khaikhin chuan Quite high protein coverage (protein 3000 vel) kan nei (51). Cell pum pui nunna humhimna hmang hian hetia kan pek dan hian mitochondria-a metabolic pathways inthlak danglamna te enfiah mai bakah a cytoplasmic counterparts-a inthlak danglamna te pawh kan enfiah thei a, hei hian cell type enrich turin mitochondrial membrane tags hmanna a tichak a ni The new method for the number of mitochondria in complex tissues (52, 53). Kan sawi dan hi Purkinje cells zirchianna nen chauh a inzawm lo va, cell chi hrang hrangah awlsam takin hman theih a ni a, chu chuan thluak natna neia metabolic changes te chu a hmachhawn thei a, mitochondrial dysfunction model dangte pawh a huam tel bawk.
A tawp berah chuan he metabolic rearrangement process chhung hian therapeutic window kan hmuchhuak a, chu chuan cellular stress chhinchhiahna pawimawh tak takte chu a tidanglam vek thei a, neuronal degeneration a veng thei bawk. Chuvangin, hetia kan sawi tawh rewiring-in a hnathawh dan hriatthiamna hian mitochondrial dysfunction laia neuronal viability vawng reng tura enkawlna awm thei chungchangah fundamental insights a pe thei a ni. Nakin lawka thluak cell chi dangtea energy metabolism inthlak danglamna dissecting tumna zirchianna neih a ngai a, chu chuan he principle hi neurological disease dangte pawha hman theih a nih dan kimchang takin a pholang thei ang.
MitoPark mice hi a hmain kan lo sawi tawh a ( 31 ). C57BL/6N mice loxP flanking Mfn2 genes nei hi a hmain an lo sawi tawh a ( 18 ) L7-Cre mice nen an cross bawk ( 23 ). Chumi hnuah double heterozygous progeny lo chhuak chu homozygous Mfn2loxP/Mfn2loxP mice nen crossed in Mfn2 (Mfn2loxP/Mfn2loxP; L7-cre) tan Purkinje-specific gene knockouts siam a ni. Mating subset pakhatah chuan Gt (ROSA26) SorStop-mito-YFP allele (stop-mtYFP) chu crossing dang hmangin an rawn luhtir a ( 20 ). Rannung tih dan zawng zawng hi European, national leh institutional guidelines angin tih a ni a, LandesamtfürNatur of Umwelt and Verbraucherschutz, North Rhine-Westphalia, Germany-in a pawmpui a ni. Ran hnathawh pawh hi European Federation of Laboratory Animal Sciences Associations kaihhruaina zawm a ni.
Hmeichhe naupai serh (cervical dislocation) chu anesthetize hnuah mouse embryo chu isolated (E13) a ni. Cortex chu Hanks’ Balanced Salt Solution (HBSS) 10 mM Hepes supplemented-ah dissect a ni a, Dulbecco’s Modified Eagle’s Medium papain (20 U/ml) leh cysteine ​​(1μg/ml) awmnaah pass a ni. Tissue chu DMEM) ah incubate la, enzymatic digestion hmangin dissociate rawh. Ml) 37°C-ah minute 20 chhung dah a ni a, chutah chuan DMEM-ah 10% fetal bovine serum supplemented-ah mechanically milled a ni. Cell te chu glass coverslips polylysine coated-ah 6 cm culture dish-ah 2×106 density-ah emaw, 0.5×105 cells/cm2 density-ah emaw imaging analysis atan seeded an ni. Darkar 4 hnuah medium chu Neurobasal serum-free medium 1% B27 supplement leh 0.5 mM GlutaMax awmna hmangin thlak a ni. Chumi hnuah neurons te chu experiment chhung zawngin 37°C leh 5% CO2 ah an dah a, kar khatah vawi khat chaw pek an ni. In vitro-a recombination induce turin a hnuaia AAV9 virus vector 3μl (24-well culture dish) emaw 0.5μl (24-well plate) emaw hmangin ni hnihnaah in vitro-ah neurons enkawl a ni: AAV9.CMV.PI.eGFP. WPRE.bGH (Addgene, catalog number 105530-AAV9) leh AAV9.CMV.HI.eGFP-Cre.WPRE.SV40 (Addgene, catalog number 105545-AAV9) te a ni.
Mouse Mfn1 leh Mfn2 complementary DNA (Addgene plasmid #23212 leh #23213 atanga lak chhuah) te chu C-terminus-ah V5 sequence (GKPIPNPLLGLDST) hmanga chhinchhiah a ni a, T2A sequence kaltlangin frame-ah mCherry nen fuse an ni. Grx1-roGFP2 hi Heidelberg TP Dick DFKZ (Deutsches Krebsforschungszentrum) hnen atanga thilpek a ni. Cloning method pangngai hmanga tdTomato cassette thlak danglamin, cassette chu pAAV-CAG-FLEX-tdTomato backbone (Addgene reference number 28306)-ah subclone-in pAAV-CAG-FLEX-mCherry-T2A-MFN2-V5, pAAV-CAG- FLEX-mCherry-T2A-MFN1-V5 leh pAAV-CAG-FLEX-Grx-roGFP2 vectors te hi a ni. Hetiang bawk hian control vector pAAV-CAG-FLEX-mCherry siam nan pawh hman a ni. AAV-shPCx construct siam tur chuan plasmid AAV vector (VectorBuilder, pAAV [shRNA] -CMV-mCherry-U6-mPcx- [shRNA#1]) a ngai a, chutah chuan mouse PCx targeting shRNA encode tu DNA sequence (5′CTTTCGCTCTAAGGTGCTAAACTCGAGTTTAGCACCTTAGAGCGAAAG) a awm a ni 3′) U6 promoter control hnuaiah mCherry hi CMV promoter control hnuaiah hman a ni. Auxiliary AAV vector siamna chu siamtute thupek (Cell Biolabs) angin an ti a ni. A tawi zawngin, transfer plasmid hmanga mCherry-T2A-MFN2-V5 (pAAV-CAG-FLEX-mCherry-T2A-MFN2-V5), mCherry-T2A-MFN1-V5 (pAAV-CAG-FLEX-mCherry) transiently Transfection of 293AAV cells-T2A-MFN1-V5), mCherry (pAAV-CAG-FLEX-mCherry) emaw Grx-roGFP2 (pAAV-CAG-FLEX-Grx-roGFP2) coding gene, chubakah AAV1 capsid protein leh accessory protein Coding Packaging plasmid plasmid, calcium phosphate method hmanga siam a ni. Crude virus supernatant chu dry ice/ethanol bath-a freeze-thaw cycle hmanga hmuh a ni a, phosphate buffered saline (PBS)-a lysed cells hmanga hmuh a ni bawk. AAV vector chu discontinuous iodixanol gradient ultracentrifugation (32,000 rpm leh 4°C-a darkar 24) hmangin tihthianghlim a ni a, Amicon ultra-15 centrifugal filter hmangin concentrate a ni. 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 te genome titer chu a hmaa kan sawi tawh ang khan (54) a ni a, real-time quantitative PCR (qPCR) hmanga teh a ni. -MFN1-V5 (1.9×1013 GC/ml) leh AAV1-CAG-FLEX-Grx-roGFP2 (8.9×1012 GC/ml) te a ni.
Primary neurons te chu ice-cold 1x PBS-ah scraped off a ni a, pelleted a ni a, chutah chuan 0.5% Triton X-100 / 0.5% sodium deoxycholate/PBS lysis buffer phosphatase leh protease inhibitor (Roche) awmnaah homogenized a ni. Protein quantification chu bicinchoninic acid assay (Thermo Fisher Scientific) hmangin an ti a. Chumi hnuah protein te chu SDS-polyacrylamide gel electrophoresis hmangin an inthen a, polyvinylidene fluoride membrane (GE Healthcare)-ah blot a ni. Non-specific sites block la, primary antibody (a chipchiar zawkna chu Table S1 en rawh) nen TBST (Tris-buffered saline with Tween)-a 5% milk-ah incubate la, washing steps leh secondary antibody chu TBST Incubate-ah incubate rawh. Primary antibody nen zan khat +4°C ah incubate tur a ni. Silfai zawhah secondary antibody chu darkar 2 chhung room temperature-ah hnawih tur a ni. Chumi hnuah chuan blot ang chiah chu anti-β-actin antibody hmanga incubate-in loading inang chiah chu a dik tih finfiah a ni. Chemiluminescence-a inthlak leh chemiluminescence tihchakna hmanga hriatchhuah (GE Healthcare).
Glass coverslip-a a hmaa seeded neurons te chu 4% paraformaldehyde (PFA)/PBS hmangin room temperature-ah minute 10 chhung an fix a. Coverslips te hi 0.1% Triton X-100/PBS hmangin room temperature-ah minute 5 chhung an permeate hmasa a, chutah chuan blocking buffer [3% bovine serum albumin (BSA)/PBS]-ah an permeate leh bawk. Ni hnihnaah chuan coverslips te chu blocking buffer hmangin silfai a ni a, a remchan dan angin fluorophore-conjugated secondary antibody nen room temperature-ah darkar 2 chhung an incubate a a tawpah chuan sample te chu PBS-ah 4′,6-diamidino-2 -Phenylindole (DAPI) hmangin uluk takin silfai a ni a, chutah chuan microscope slide-ah Aqua-Poly/Mount hmangin fix a ni.
Mice (mipa leh hmeichhia) te chu ketamine (130 mg/kg) leh xylazine (10 mg/kg) intraperitoneal injection hmangin anesthetized an ni a, carprofen analgesic (5 mg/kg) hmangin vun hnuaiah an pe a, Steretactic instrument (Kopf)-ah pad lum tak dahin an dah bawk. Luruh chu pholang la, ha drill hmangin mis ruh nena inmil cerebellar cortex hmun (lambda atanga: tail 1.8, lateral 1, lobules IV leh V nena inmil) chu thin turin. Curved syringe needle hmangin uluk takin luruh chhungah khur te tak te siam la, a hnuaia vasculature tihbuai loh nan. Tichuan, glass capillary te tak te chu micro-hole-ah zawi zawiin (dura mater ventral lam -1.3 atanga -1 thleng) dah a ni a, AAV 200 atanga 300 nl chu micro-injector (Narishige)-ah manual syringe (Narishige) hmangin vawi tam tak pressure hniam takah minute 10 atanga 20 chhung window-ah inject a ni. Infusion zawhah capillary chu minute 10 dang dah leh la, chu chuan virus chu a darh vek theih nan. Capillaries te chu lakchhuah a nih hnuah vun chu uluk takin an suture a, chu chuan hliam natna a tihziaawm theih nan leh rannung chu a dam leh theih nan a pui a ni. Rannungte hi operation an neih hnu ni engemaw zat chu analgesics (caspofen) hmanga enkawl an ni a, chutih chhung chuan an taksa dinhmun uluk takin an enfiah a, chumi hnuah hun bituk angin an that leh a ni. Procedure zawng zawng hi European, national leh institutional guidelines angin tih a ni a, Umwelt leh Verbraucherschutz, North Rhine-Westphalia, Germany-a LandesamtfürNatur-in a pawmpui a ni.
Rannungte chu ketamine (100 mg/kg) leh xylazine (10 mg/kg) hmangin anesthetized an ni a, thinlung chu 0.1 M PBS hmangin an perfuse hmasa a, chutah chuan PBS-ah 4% PFA hmangin an perfuse leh a ni. Tissue chu dissected niin 4% PFA/PBS-ah 4°C-ah zan khat chhung fix a ni. PBS-a thluak nghet tak atanga sagittal section (50 μm thick) siam nan vibrating knife (Leica Microsystems GmbH, Vienna, Austria) hman a ni. A danglamna a awm loh chuan, free-floating section-te chu a chunga kan sawi tawh ang khan (13) room temperature-ah staining leh stirring-in an ti a. A tawi zawngin, a hmasa berah chuan slice hmuhte chu 0.5% Triton X-100/PBS hmangin room temperature-ah minute 15 chhung permeabilized a ni a; epitopes thenkhat (Pcx leh Shmt2) tan chuan, by in tris-EDTA buffer at 80°C (PH 9) he step ai chuan slices te hi minute 25 chhung tihlum tur a ni. A dawtah chuan section te chu primary antibody (Table S1 en la) nen blocking buffer (3% BSA/PBS) ah 4°C ah zan khat chhung stirring nen incubate a ni. A tuk zingah chuan section te chu blocking buffer hmangin silfai a ni a, a remchan dan angin fluorophore-conjugated secondary antibody nen darkar 2 chhung room temperature-ah an incubate a a tawpah chuan section te chu PBS-ah uluk takin silfai a ni a, DAPI hmangin counter-stained a ni a, chutah chuan AquaPolymount On microscope slide hmangin fix a ni.
Sample hi laser scanning confocal microscope (TCS SP8-X emaw TCS Digital Light Sheet, Leica Microsystems) hmangin white light laser leh 405 diode ultraviolet laser hmanga thuam a ni. Fluorophore exciting leh Hybrid Detector (HyDs) hmanga signal lakkhawm hmangin LAS-X software hmangin Nyquist sampling nena inmil stacked images chu sequential mode-ah lakkhawm a ni: non-quantitative panel tan chuan highly dynamic signals (entir nan, somatic cell leh dendrites-ah) mtYFP) BrightR mode-a PNs awm zat hriat nan HyD hmang rawh). Background tihtlem nan 0.3 atanga 6 ns thleng gating hman a ni.
Cell sorted te chu real-time imaging a ni. 1% B27 supplement leh 0.5 mM GlutaMax awmna Neurobasal-A medium-a sorting hnuah, cell-te chu poly-l-lysine-coated glass slide (μ-Slide8 Well, Ibidi, catalog number 80826)-ah seeded nghal a ni a, chutah chuan 37°C leh 5% CO2-ah darkar 1 chhung dah a ni a, cell-te chu a awm theih nan. Real-time imaging hi Leica SP8 laser scanning confocal microscope hmangin laser dum, HyD, 63×[1.4 numerical aperture (NA)] oil objective lens leh heating stage hmanga thuam a ni.
Mouse chu rang takin carbon dioxide hmangin anesthetized a ni a, lu tansak a ni a, thluak chu rang takin luruh atanga lakchhuah a ni a, 200μm thick (13C labeling experiment atan) emaw 275μm thick (photon experiment pahnih atan) emaw sagittal section-ah a hnuaia thil awmte nen a khat The ice cream (HM-650 V, Thermo Fisher Scientific, Walldorf, Germany) chu a hnuaia thilte hian a khat a ni: 125 mM ice-cold, carbon-saturated (95% O2 leh 5% CO2) low 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 leh 3.5 mM MgCl2 (osmotic pressure 310 atanga 330 mmol) a ni. Brain slices hmuhte chu pre-incubation chamber-ah Ca2 + ACSF sang zawk (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 leh 2.0 mM MgCl2) awmnaah dah la. Medium) pH 7.4 leh 310 atanga 320 mmol).
Imaging process chhung hian slices te chu dedicated imaging room ah an sawn a, experiment chu continuous ACSF perfusion hnuaiah constant temperature 32° atanga 33°C ah an ti a. Slice imaging atan hian multiphoton laser scanning microscope (TCS SP8 MP-OPO, Leica Microsystems) Leica 25x objective lens (NA 0.95, tui), Ti: Sapphire laser (Chameleon Vision II, Coherent) hmanga thuam hman a ni. FLIM module (PicoHarp300, PicoQuant) hmanga siam a ni.
Grx1-roGFP2 FLIM hmanga siam a ni. PNs cytoplasmic redox state inthlak danglamna hi sagittal brain slices-a two-photon FLIM hmanga teh a ni a, Grx1-roGFP2 biosensor hian PNs a target a ni. PN layer-ah chuan acquisition field chu slice surface hnuai 50 atanga 80 μm vel a thlan a ni a, chu chu viable PN (chu chu dendrites-a beaded structure emaw neuronal morphological changes awm lohna) leh double positive roGFP2 sensor leh AAV encoding shRNA PCx emaw a control sequence (each co-expressing mCherry) a awm theih nan a ni. 2x digital zoom [excitation wavelength: 890 nm; 512 nm 512 pixels a ni]. Detection: internal HyD, fluorescein isothiocyanate (FITC) filter group] leh minute 2 atanga 3 chhunga image averaging hmangin curve fitting atan photon tling (a vaiin photon 1000) a khawlkhawm theih nan hman a ni. Grx1-roGFP2 probe sensitivity leh FLIM conditions verification chu perfusion ACSF-a exogenous 10 mM H2O2 dah a nih hunah (oxidation tihpun nan, dam rei theihna tur) 2 mM dithiothreitol dah belh (reduction degree tih tlem a, tlahniam a awm) chuan roGFP2 lifespan value enfiahin an ti a ni dam chhungin) (Figure S8, D atanga G thleng). FLIMfit 5.1.1 software hmangin result hmuh tawhte chu zirchiang la, image pumpui exponential decay curve pakhat chu IRF tehna (instrument response function)-ah fit rawh, χ2 chu 1 vel a ni a, PN pakhat dam chhung chhiar tur chuan nerve body vel mask chu kut hmangin an siam a, mask tina dam chhung zatve chu quantification atan hman a ni.
Mitochondrial thiltihtheihna thlirletna. Acute section chu 100 nM TMRM direct-a perfused ACSF-a dahin minute 30 chhung an incubate hnuah PNs-te mitochondrial potential inthlak danglamna chu two-photon microscope hmangin an teh a ni. TMRM imaging chu probe chu 920 nm-ah exciting-in internal HyD (tetramethylrhodamine isothiocyanate: 585/40 nm) hmangin signals lakkhawm a ni a; excitation wavelength inang hmang mahse mtYFP image turin internal HyD danglam (FITC :525/50) hmangin. ImageJ-a Image Calculator plug-in hmangin mitochondrial potential chu single cell level-ah teh theih a ni. A tawi zawngin, plug-in equation: signal = min (mtYFP, TMRM) hmang hian Purkinje Somali-a TMRM signal chu a inmil channel-a single-stack confocal image-a mitochondrial region a lantir a ni. Tichuan, mask lo chhuak chhunga pixel area chu quantified a ni a, chutah chuan mtYFP channel-a threshold single-stack image inmil takah normalized a ni a, mitochondrial potential lantir thei mitochondrial fraction chu hmuh theih a ni.
He thlalak hi Huygens Pro (Scientific Volume Imaging) software hmanga deconvoluted a ni. Tiles scanned pictures atan chuan tile pakhat montage chu LAS-X software-in automatic stitching algorithm a pek hmangin siam a ni. Image calibration zawhah ImageJ leh Adobe Photoshop hmangin image chu process lehzual la, brightness leh contrast chu uniform takin adjust rawh. Graphic buatsaih nan Adobe Illustrator hmang ang che.
mtDNA focus hmanga zirchianna. mtDNA lesions awm zat chu confocal microscope hmangin DNA laka antibodies hmanga label cerebellar section-ah quantified a ni. Target area tinte chu cell taksa leh cell tin nucleus atan siam a ni a, Multi Measure plug-in (ImageJ software) hmangin area hrang hrang chu chhut a ni. Cell body area atanga nuclear area chu paih la, cytoplasmic area chu i hmu ang. A tawpah chuan Analyze Particles plug-in (ImageJ software) hmangin threshold image-a mtDNA tarlanna cytoplasmic DNA point te chu automatic-in quantified a ni a, result hmuhte chu CTRL mice PN average-ah normalized a ni. Results chu cell khata nucleoside awm zat average anga tarlan a ni.
Protein expression thlirletna a ni. ImageJ-a Image Calculator plug-in hmangin PN-a protein expression chu single cell level-ah evaluate rawh. A tawi zawngin, a inmil channel-a single-layer confocal image-ah chuan, equation: signal = min (mtYFP, antibody) hmangin, Purkina-a antibody engemaw zat immunoreactivity lantir thei mitochondrial region chu hriatchhuah a ni. Tichuan, a chhuak mask-a pixel area chu quantified a ni a, chutah chuan mtYFP channel-a threshold single-stack image inmil takah normalized a ni a, chu chuan protein tarlan mitochondrial fraction chu a hmu chhuak a ni.
Purkinje cell density thlirletna a ni. ImageJ-a Cell Counter plug-in hmangin Purkinje density chu Purkinje cell chhiar zat chu chhiar chhuah cell-te’n an luah cerebellar ring sei zawng nen an zat a ni.
Sample buatsaih leh lakkhawm. Control group leh Mfn2cKO mice atanga thluak te chu 0.1 M phosphate buffer (PB) ah 2% PFA/2.5% glutaraldehyde ah fix a ni a, chutah chuan coronal section te chu ciliates (Leica Mikrosysteme GmbH, Vienna, Austria) (Thickness 50 to 60 μm) hmangin siam a ni a, Chumi hnuah PB buffer in 1% os tetraoxide ah fix leh 1.5% potassium ferrocyanide chu room temperature-ah darkar 1 chhung dah tur a ni. Section te chu distilled water hmangin vawi thum silfai a ni a, chutah chuan 70% ethanol 1% uranyl acetate awmna hmangin minute 20 chhung stain a ni. Chumi hnuah section te chu graded alcohol-ah dehydrate a ni a, Durcupan ACM (Araldite casting resin M) epoxy resin (Electron Microscopy Sciences, catalog number 14040)-ah silicon-coated glass slide inkarah embedded a ni a, a tawpah 60°C-ah oven-ah darkar 48 chhung Polymerize a ni. Cerebellar cortex area chu thlan a ni a, 50 nm ultrathin section chu Leica Ultracut (Leica Mikrosysteme GmbH, Vienna, Austria)-ah cut a ni a, 2×1 mm copper slit grid-ah polystyrene film-a khuh-ah picked a ni. Section te chu H2O-a 4% uranyl acetate solution hmangin minute 10 chhung stain a ni a, H2O hmangin vawi tam tak silfai a ni a, chutah chuan Reynolds lead citrate hmangin H2O-ah minute 10 chhung silfai a ni a, chutah chuan H2O hmangin vawi tam tak silfai a ni. Micrograph te hi transmission electron microscope Philips CM100 (Thermo Fisher Scientific, Waltham, MA, USA) hmangin TVIPS (Tietz Video and Image Processing System) TemCam-F416 digital camera (TVIPS GmbH, Gauting, USA) hmangin lak a ni. Germany) a ni).
AAV vei mice te tan chuan thluak chu then hranin 1 mm thick sagittal section-ah sliced ​​a ni a, cerebellum chu fluorescence microscope hmangin AAV kai ring (chu chu mCherry expressing) hriat theih nan an enfiah a ni. AAV injection hmanga Purkinje cell layer (chu chu layer pumpui deuhthaw) chu cerebellar ring pahnih tal a zawna transduction efficiency sang tak a neih theihna experiment chauh hman a ni. AAV-transduced loop chu zan khat post-fixation atan microdissected (0.1 M cocoate buffer-ah 4% PFA leh 2.5% glutaraldehyde) a ni a, process leh a ni. EPON embedding atan chuan fixed tissue chu 0.1 M sodium cocoate buffer (Applichem) hmangin silfai a ni a, 2% OsO4 (os, Science Services; Caco) nen 0.1 M sodium cocoate buffer (Applichem)-ah Darkar 4 chhung incubate a ni a, chutah chuan darkar 2 chhung silfai a ni. 0.1 M cocamide buffer hmangin vawi 3 tih leh tur a ni. Chumi hnuah chuan ascending series of ethanol hmangin ethanol solution tinte chu 4°C-ah minute 15 chhung an incubate a, tissue chu a dehydrate a ni. Tissue chu propylene oxide-ah dahin EPON (Sigma-Aldrich)-ah 4°C-ah zan khat chhung an incubate a. Tissue chu EPON thar ah room temperature ah darkar 2 chhung dah la, chutah chuan 62°C ah darkar 72 chhung embed rawh. Ultramicrotome (Leica Microsystems, UC6) leh diamond knife (Diatome, Biel, Switzerland) hmangin 70 nm ultrathin section te chu tan la, 1.5% uranyl acetate hmangin 37°C ah minute 15 chhung stain la, lead citrate solution hmangin minute 4 stain rawh. Electron micrograph te hi JEM-2100 Plus transmission electron microscope (JEOL) hmanga lak niin, Camera OneView 4K 16-bit (Gatan) leh DigitalMicrograph software (Gatan) hmanga thuam a ni. Analysis atan chuan electron micrographs chu 5000× emaw 10,000× digital zoom hmangin an la a.
Mitochondria te hi a pianphung (morphological analysis) a ni. Analysis zawng zawngah chuan mitochondria pakhat zel contour chu ImageJ software hmangin digital image-ah manual-in an outline a. Morphological parameter hrang hrang an zirchiang a. Mitochondrial density hi cell tinte mitochondrial area zawng zawng chu cytoplasm area (cytoplasm area = cell area-cell nucleus area) × 100-a then atanga percentage hmuh chhuah anga tarlan a ni a, mitochondria roundness chu formula [4π∙(area/perimeter 2)] hmangin chhut a ni. Mitochondria ista morphology chu an zirchiang a, an pianzia ber angin category hnih (“tubular” leh “blister”) ah an then a.
Autophagosome/lysosome awm zat leh density thlirletna. ImageJ software hmangin digital image-a autophagosome/lysosome tinte contour chu manual-in outline rawh. Autophagosome/lysosome area hi cell tinte autophagosome/lysosome structure area zawng zawng chu cytoplasm area (cytoplasm area=cell area-nucleus area)×100 hmanga then a percentage anga chhut a ni. Autophagosomes/lysosomes density hi cell khata autophagosome/lysosome structure awm zat (cytoplasmic area atanga thlirin) (cytoplasmic area = cell area-nuclear area) atanga a zatve zat atanga chhut a ni.
Acute sectioning leh sample buatsaihna atana labeling siam. Glucose labeling mamawh experiment atan chuan acute brain slices te chu pre-incubation chamber-ah transfer tur a ni a, chutah chuan saturated carbon (95% O2 leh 5% CO2), Ca2 + ACSF sang (125.0 mM NaCl, 2.5 mM KCl, 1.25 mM sodium phosphate buffer, 25.0 mM NaHCO 3, 25.0 mM) a awm a ni d-glucose, 1.0 mM CaCl 2 leh 2.0 mM MgCl 2, pH 7.4 leh 310 atanga 320 mOsm-a siamrem), chutah chuan glucose chu 13 C 6- Glucose substitution (Eurisotop, catalog number CLM-1396) a ni. Pyruvate labeling mamawh experiment atan chuan acute brain slices te chu Ca2 + ACSF sang zawk (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 leh Add 2.0mM ah transfer tur a ni MgCl2, pH 7.4 leh 310 atanga 320mOsm-ah siamrem la, 1mM 1-[1-13C]pyruvate (Eurisotop, catalog number CLM-1082) dah bawk ang che. Section te chu 37°C ah minute 90 chhung incubate tur a ni. Experiment tawp lamah chuan section te chu 75 mM ammonium carbonate awmna aqueous solution (pH 7.4) hmangin rang takin an silfai a, chutah chuan 40:40:20 (v:v:v) acetonitrile (ACN): methanol: tuiah homogenized an ni. Section te chu ice-ah minute 30 chhung an incubate hnuah sample te chu 21,000 g-ah minute 10 chhung 4°C-ah centrifuge-in, clear supernatant chu SpeedVac concentrator-ah an vawt a. Chuta chhuak dried metabolite pellet chu -80°C-ah analysis thlengin dah a ni.
C-labeled amino acid 13 chu liquid chromatography-mass spectrometry hmanga enfiah a ni. Liquid chromatography-mass spectrometry (LC-MS) analysis atan chuan metabolite pellet chu LC-MS grade water (Honeywell) 75μl-ah resuspended a ni. 21,000 g-a 4°C-a minute 5 chhung centrifugation hnuah clarified supernatant 20 μl chu amino acid flux analysis atan hman a ni a, a bak zawng chu anion analysis atan hman nghal a ni (a hnuaia mi hi en la). Amino acid analysis hi a hmaa kan sawi tawh angin benzoyl chloride derivatization protocol hmangin an ti a (55, 56). Step hmasa berah chuan metabolite extract 20μl-ah 100 mM sodium carbonate (Sigma-Aldrich) 10μl dah a ni a, chutah chuan LC grade ACN-ah 2% benzoyl chloride (Sigma-Aldrich) 10μl dah a ni. Sample chu rei vak lo vortex-in 21,000 g-ah 20°C-ah minute 5 chhung centrifuged a ni. Supernatant clear tawh chu 2 ml autosampler vial-ah conical glass insert (200 μl volume) hmangin dah la. Sample te hi Acquity iClass ultra-high performance LC system (Waters) hmangin Q-Exactive (QE)-HF (Ultra High Field Orbitrap) high-resolution precision mass spectrometer (Thermo Fisher Scientific) nena inzawm hmangin an zirchiang a ni. Analysis atan chuan derivatized sample 2μl chu 100×1.0 mm high-strength silica T3 column (Waters)-ah 1.8μm particles awmnaah inject a ni. Flow rate hi 100μl/min a ni a, buffer system hi buffer A (10 mM ammonium formate leh tuiah 0.15% formic acid) leh buffer B (ACN) te a ni. Gradient chu hetiang hi a ni: minute 0-ah 0%B; 0%B a ni. 0 atanga 15% B chu minute 0 atanga 0.1 chhungin; 15 atanga 17% B chu minute 0.1 atanga 0.5 chhungin; B chu minute 0.5 atanga 14 chhungin 17 atanga 55% a ni a; B chu minute 14 atanga 14.5 chhungin 55 atanga 70% a ni a; minute 18-ah 14.5 atanga 70 atanga 100% B-ah; Minute 18 atanga 19 chhungin 100% B; Minute 19 atanga 19.1 chhungin 100 atanga 0% B; 0% B chu minute 19.1 atanga 28 chhung (55, 56) a ni. QE-HF mass spectrometer hi positive ionization mode-ah a thawk a, mass range m/z (mass/charge ratio) 50 atanga 750 a ni a, applied resolution chu 60,000 a ni a, gain control (AGC) ion target hmuh chu 3×106 a ni a, ion time sang ber chu 100 milliseconds a ni. Heated electrospray ionization (ESI) source hi spray voltage 3.5 kV, capillary temperature 250°C, sheath airflow 60 AU (arbitrary units), leh auxiliary airflow 20 AU-ah a thawk a ni. 250°C a ni. S lens hi 60 AU ah dah a ni.
Anion chromatography-MS hmanga 13C labeled organic acid te chu an enfiah a. Metabolite precipitate la awm (55μl) chu QE-HF mass spectrometer (Thermo Fisher Scientific) nena inzawm Dionex ion chromatography system (ICS 5000+, Thermo Fisher Scientific) hmangin an zirchiang a. A tawi zawngin, metabolite extract 5μl chu HPLC (2 mm×250 mm, particle size 4μm, Thermo Fisher Scientific) hmanga thuam Dionex IonPac AS11-HC column-ah push-in partial loop mode-ah filling ratio 1. ) Dionex IonPac AG11-HC guard column (2 mm x 50 mm, 4μm, Thermo Fisher Scientific lam a ni). Column temperature chu 30°C-ah dah a ni a, autosampler chu 6°C-ah dah a ni. Eluent generator kaltlangin potassium hydroxide gradient siam turin deionized water awmna potassium hydroxide cartridge hmang ang che. Metabolite te chu flow rate 380μl/min-a then hran, a hnuaia gradient hmang hian: minute 0 atanga 3, 10 mM KOH; Minute 3 atanga 12 chhung, KOH 10 atanga 50 mM; Minute 12 atanga 19 chhung, 50 atanga 100 mM KOH; Minute 19 atanga 21 chhung , 100 mM KOH; Minute 21 atanga 21.5 chhung, 100 atanga 10 mM KOH. Column chu 10 mM KOH hnuaiah minute 8.5 chhung equilibrate leh a ni.
Eluted metabolites te chu column hnuah 150μl/min isopropanol supplement stream nen an inzawm khawm a, chu chu negative ionization mode-a thawk high-resolution mass spectrometer-ah an hruai a ni. MS hian m/z 50 atanga 750 thlenga mass range chu resolution 60,000 neiin a enfiah mek a ni. AGC chu 1×106 ah dah a ni a, ion time sang ber chu 100 ms ah dah a ni. ESI source lum tak chu spray voltage 3.5 kV-ah an thawk a ni. Ion source setting dangte chu hetiang hi a ni: capillary temperature 275°C; sheath gas luang chhuak, 60 AU; auxiliary gas flow, 300°C-ah 20 AU, leh S lens chu 60 AU-a dah a ni.
13C labeled metabolites te data thlirletna a ni. Isotope ratio data thlirletna atan TraceFinder software (version 4.2, Thermo Fisher Scientific) hmang ang che. Compound tinte identity chu reference compound rintlak takin an finfiah a, independent takin an zirchiang a ni. Isotope enrichment analysis tih nan hian 13C isotope (Mn) tinte extracted ion chromatogram (XIC) area chu [M + H] + atangin lakchhuah a ni a, chutah chuan n chu target compound carbon number a ni a, amino acids analysis atan emaw [ MH] + hmangin anions analysis atan hman a ni. XIC mass accuracy hi part nga aia tlem a ni a, RT accuracy chu minute 0.05 a ni. Enrichment analysis hi isotope hmuhchhuah tinte leh a inmil compound isotope zawng zawng zat zat ratio chhut chhuah a ni. Heng ratio te hi isotope tin tan percentage value anga pek a ni a, a result chu molar percent enrichment (MPE) anga tarlan a ni a, a hmaa kan sawi tawh ang khan (42).
Frozen neuron pellet chu ice-cold 80% methanol (v/v)-ah homogenized a ni a, vortex-ah dah a ni a, -20°C-ah minute 30 chhung incubate a ni. Sample chu vortex leh la, +4°C ah minute 30 chhung stir leh rawh. Sample chu 21,000 g-ah minute 5 chhung 4°C-ah centrifuged a ni a, chutah chuan supernatant lo chhuak chu lakkhawm niin, SpeedVac concentrator hmangin 25°C-ah a hnua analysis atan an vawt a ni. A chunga kan sawi tawh ang khan LC-MS analysis hmangin sorted cells amino acids te chu an ti a. TraceFinder (version 4.2, Thermo Fisher Scientific) hmangin compound tinte monoisotopic mass hmangin data analysis an nei a. Metabolite data quantile normalization chu preprocessCore software package hmangin an ti a (57).
Slice buatsaih dan tur. Mouse chu rang takin carbon dioxide hmangin anesthetized a ni a, lu tansak a ni a, thluak chu rang takin a luruh atanga lakchhuah a ni a, ice-filled vibrating knife (HM-650 V, Thermo Fisher Scientific, Walldorf, Germany) hmangin 300 atanga 375 μm sagittal section-ah an tan a, Cold carbon gasification (95% O2 leh 5% CO2) Low Ca2 + ACSF (125.0 mM NaCl, 2.5 mM KCl, 1.25 mM sodium phosphate buffer, 25.0 mM NaHCO3, 25.0 mM d-glucose, 1.0 mM CaCl2 leh 6.0 mM MgCl2 pH 7.4 leh 310 atanga 330 mOsm thlenga siamrem). Brain slices hmuhte chu Ca2 + ACSF sang zawk (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 leh mM 3.5 MgCl2) pH 7.4 leh 310 atanga 320 mOsm thleng a ni). Slices te hi minute 20 atanga 30 vel dah la, chutiang chuan record hmain a lo awm leh thei ang.
recording tih a ni. Recording zawng zawng atan hian microscope stage, fixed recording chamber leh 20x water immersion objective lens (Scientifica) hmanga thuam hman a ni. Purkinje cells nia sawite hi (i) taksa lian leh te, (ii) cerebellum anatomical location, leh (iii) fluorescent mtYFP reporter gene expression hmanga hriat theih a ni. Tip resistance megohms 5 atanga 11 nei patch pipette hi borosilicate glass capillary (GB150-10, 0.86 mm×1.5 mm×100 mm, Science Products, Hofheim, Germany) leh horizontal pipette Instruments (P-1000, Sutter), Novato, CA hmanga lakchhuah a ni. Recording zawng zawng hi ELC-03XS npi patch clamp amplifier (npi electronic GmbH, Tam, Germany) hmanga tih a ni a, chu chu software Signal (version 6.0, Cambridge Electronic, Cambridge, UK) hmanga control a ni. Experiment hi sampling rate 12.5 kHz-a record a ni. Signal hi short-pass Bessel filter pahnih hmanga filter a ni a, cutoff frequency 1.3 leh 10 kHz a ni. Membrane leh pipette capacitance chu amplifier hmanga compensation circuit hmangin a compensate a ni. Experiment zawng zawng hi Orca-Flash 4.0 camera (Hamamatsu, Gerden, Germany) control hnuaia tih a ni a, chu chu Hokawo software (version 2.8, Hamamatsu, Gerden, Germany) hmanga control a ni.
Cell pum pui configuration leh analysis tih dan pangngai. Recording hma lawk hian pipette chhungah hian a chhunga solution awm chu a hnuaia thil awmte hi dah la: 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) leh 10.0 mM creatinine phosphate chu pH 7.25-ah an siam a, osmotic pressure chu 290 mOsm (sucrose) a ni. Membrane rupture turin force 0 pA an pek hnu lawkah resting membrane potential chu an teh nghal a. Input resistance hi hyperpolarized current -40, -30, -20, leh -10 pA hmanga teh a ni. Voltage response magnitude teh la, Ohm’s law hmangin input resistance chu chhut rawh. Voltage clamp-ah minute 5 chhung spontaneous activity record a ni a, semi-automatic recognition script hmangin Igor Pro (version 32 7.01, WaveMetrics, Lake Oswego, Oregon, USA)-ah sPSC chu hriatchhuah leh teh a ni. IV curve leh steady-state current hi battery chu potential hrang hrang (-110 mV atanga tan)-a clamp a, 5 mV step-a voltage tihpun hmanga teh a ni. AP siam chhuah dan chu depolarizing current hmanga test a ni. Depolarizing current pulse hmangin cell chu -70 mV ah clamp rawh. Recording unit tinte step size chu a hranin (10 to 60 pA) siamrem rawh. AP frequency sang ber thlentu pulse spike te chu manual-a chhiarin AP frequency sang ber chu chhut rawh. AP threshold hi AP pakhat emaw a aia tam emaw trigger hmasa ber depolarization pulse atanga derivative pahnihna hmangin an zirchiang a ni.
Perforated patch configuration leh analysis te pawh a awm bawk. Standard protocol hmangin perforated patch recording tih tur a ni. ATP- leh GTP-free pipette hmang la, a hnuaia thil awmte hi a awm lo: 128 mM gluconate K, 10 mM KCl, 10 mM Hepes, 0.1 mM EGTA leh 2 mM MgCl2, pH 7.2 (KOH hmangin)-ah siamrem rawh. Cell membrane-a uncontrolled permeability awm loh nan intracellular solution atang hian ATP leh GTP te hi paih chhuah a ni. Patch pipette chu amphotericin awmna internal solution (200 atanga 250μg/ml vel; G4888, Sigma-Aldrich) hmanga khat a ni a, punched patch record hmuh theih a ni. Amphotericin chu dimethyl sulfoxide (a tawp ber: 0.1 atanga 0.3%; DMSO; D8418, Sigma-Aldrich)-ah a hmin a. DMSO hman zat hian neurons zirchian tawhte chungah nghawng lian tham a nei lo. Punching process chhung hian channel resistance (Ra) chu enfiah chhunzawm zel a ni a, Ra leh AP amplitude a stabilized hnuah (minute 20-40) experiment tan a ni. Spontaneous activity chu voltage leh/ emaw current clamp hmangin minute 2 atanga 5 chhung teh a ni. Data thlirletna hi Igor Pro (version 7.05.2, WaveMetrics, USA), Excel (version 2010, Microsoft Corporation, Redmond, USA) leh GraphPad Prism (version 8.1.2, GraphPad Software Inc., La Jolla, CA) hmangin an ti a ni. United States a ni). Spontaneous APs hriat theih nan IgorPro-a NeuroMatic v3.0c plug-in hman a ni. Threshold pek hmangin AP te chu automatic-in hriat theih a ni a, chu chu record tin atan a hranpaa siamrem a ni. Spike interval hmangin instantaneous spike frequency sang ber leh spike frequency average nei spike frequency chu teh rawh.
PN isolation a ni. A hmaa protocol tihchhuah tawh angin insiamrem a, PNs chu mouse cerebellum atang hian stage ruat saah tihthianghlim a ni ( 58 ). A tawi zawngin, cerebellum chu ice-cold dissociation medium [HBSS Ca2+ leh Mg2+ tel lo, 20 mM glucose, penicillin (50 U/ml) leh streptomycin (0.05 mg/ ml) nena supplemented]-ah dissected leh minced a ni a, chutah chuan papain [HBSS, 1-cysteine·HCl supplemented)-ah digest a ni (1 mg / ml), papain (16 U / ml) leh deoxyribonuclease I (DNase I; 0.1 mg/ml)] 30°C-ah minute 30 chhung enkawl tur a ni. Enzymatic digestion awm loh nan egg mucus (10 mg/ml), BSA (10 mg/ml) leh DNase (0.1 mg/ml) awmna HBSS medium-ah silfai hmasa la, chutah chuan 20 mM glucose awmna HBSS medium-ah Gently grinding in HBSS, penicillin (50 U/ml), streptomycin (0.05 mg/ml) leh DNase (0.1 mg/ml) hian cell pakhat chauh a chhuah tir thin. Cell suspension lo chhuak chu 70μm cell strainer hmangin filter a ni a, chutah chuan cell te chu centrifugation (1110 rpm, minute 5, 4°C) hmangin pellet a ni a, sorting medium [HBSS, 20 mM glucose, 20% fetal bovine ) Serum, penicillin (50 U/ml) leh streptomycin te nen supplemented-ah resuspended a ni (0.05 mg/ml)] a ni a; propidium iodide hmanga cell viability endik a, cell density chu 1×106 atanga 2×106 cells/ml thlenga siamrem. Flow cytometry hmain suspension chu 50 μm cell strainer hmangin an filter a.
Flow cytometer hmanga chhut a ni. Cell sorting chu 4°C-ah FACSAria III machine (BD Biosciences) leh FACSDiva software (BD Biosciences, version 8.0.1) hmangin an ti a. Cell suspension chu 100 μm nozzle hmangin 20 psi pressure hnuaiah ~2800 events/sec rate-in an thliar hrang a. Traditional gating criteria (cell size, bimodal discrimination, leh scattering characteristics) hmangin cell chi dang atanga PN isolation dik tak a awm theih loh avangin, gating strategy hi mitoYFP+ ​​leh control mitoYFP − Mice-a YFP intensity leh autofluorescence direct comparison atanga siam a ni. YFP hi 488 nm laser line hmanga sample irradiate-in a excited a, 530/30 nm band pass filter hmangin signal chu detect a ni. MitoYFP+ ​​mice-ah chuan Rosa26-mitoYFP reporter gene relative strength hmangin neuronal body leh axon fragment te pawh an thliar hrang thin. 7-AAD hi 561 nm yellow laser hmangin excited a ni a, 675/20 nm bandpass filter hmangin detected niin cell thi te chu paih chhuah a ni. Astrocyte te chu a ruala then hran theih nan cell suspension chu ACSA-2-APC hmangin stain a ni a, chutah chuan sample chu 640 nm laser line hmangin irradiate a ni a, 660/20 nm bandpass filter hmangin signal chu hmuhchhuah a ni.
Cell lakkhawmte chu centrifugation (1110 rpm, 5 minutes, 4°C) hmangin pellet a ni a, hman thlengin -80°C-ah dah a ni. Mfn2cKO mice leh an litter pups te chu ni khatah classified an ni a, hei hian procedural variability a tihtlem phah a ni. FACS data presentation leh analysis hi FlowJo software (FlowJo LLC, Ashland, Oregon, USA) hmangin an ti a ni.
A chunga kan sawi tawh ang khan (59) real-time PCR hmangin sorted neurons atanga DNA chu a hnu lama mtDNA quantification atan isolate nan hman a ni. Linearity leh threshold sensitivity hi a tirah chuan cell number hrang hrangah qPCR run hmangin an test a. A tawi zawngin, lysis buffer 50 mM tris-HCl (pH 8.5), 1 mM EDTA, 0.5% Tween 20 leh proteinase K (200 ng/ml) awmnaah PN 300 la khawm la, 55°C-ah minute 120 chhung incubate rawh. Cell te chu 95°C ah minute 10 chhung an incubate leh a, proteinase K chu a inactivation kim vek a, mt-Nd1 specific TaqMan probe (Thermo Fisher) hmangin mtDNA chu 7900HT Real-Time PCR system (Thermo Fisher Scientific) ah semi-quantitative PCR hmangin an teh a ni. Science, catalog number Mm04225274_s1), mt-Nd6 (Thermo Fisher Scientific, catalog number AIVI3E8) leh 18S (Thermo Fisher Scientific, catalog number Hs99999901_s1) te genes te hi a ni.
Proteome sample buatsaih dan tur. Solution chu 95°C-ah minute 10 chhung tihlum a, sonicate-in, lysis buffer-ah [6 M guanidine chloride, 10 mM tris(2-carboxyethyl) phosphine hydrochloride, 10 mM chloroacetamide leh 100 mM tris- Lyse frozen neuron pellets in HCl Bioruptor (Diagenode) ah minute 10 (second 30 pulse / second 30 pause period) chhung dah a ni. Sample chu 20 mM tris-HCl (pH 8.0)-ah 1:10-in diluted a ni a, 300 ng trypsin gold (Promega) nen chawhpawlh a ni a, 37°C-ah zan khat chhung incubate a ni a, a hmin vek a ni. Ni hnihnaah chuan sample chu 20,000 g-ah minute 20 chhung centrifuged a ni. Supernatant chu 0.1% formic acid hmangin diluted a ni a, chu solution chu self-made StageTips hmangin desalted a ni. Sample chu SpeedVac instrument (Eppendorf concentrator plus 5305)-ah 45°C-ah an vawt a, chutah chuan peptide chu 0.1% formic acid-ah an suspended a ni. Sample zawng zawng hi mi pakhatin a rualin a siam vek a ni. Astrocyte sample enfiah nan desalted peptide 4 μg chu tandem mass tag (TMT10plex, catalog number 90110, Thermo Fisher Scientific) hmangin peptide to TMT reagent ratio 1:20 hmangin an label a. TMT labeling atan chuan TMT reagent 0.8 mg chu anhydrous ACN 70 μl-ah resuspended a ni a, peptide vawt chu 0.1 M TEAB (triethylammonium bicarbonate) 9 μl-ah siam thar leh a ni a, chutah chuan ACN-a TMT reagent 7 μl dah a ni. A concentration chu 43.75% a ni. Minute 60 vel an incubation hnuah 5% hydroxylamine 2 μl hmangin an quench leh a. Labeled peptide te chu an la khawm a, an vawt a, 0.1% formic acid (FA) 200μl-ah resuspended an ni a, pahnih ah then a ni a, chutah chuan self-made StageTips hmangin desalted an ni. UltiMate 3000 ultra high performance liquid chromatograph (UltiMate 3000 ultra high performance liquid chromatograph) hmangin a chanve pahnih zinga pakhat chu 1mm x 150mm Acquity chromatographic column-ah 130Å1.7μm C18 particles (Waters, catalog No. SKU: 186006935)-a khatah fractionated a ni. Thermo Fisher Scientific lam atanga lo chhuak a ni). Peptide te chu flow rate 30μl/min-ah then la, 1% atanga 50% buffer B minute 85 chhung stepwise gradient minute 96, 50% atanga 95% buffer B minute 3, chutah minute 8 chhung 95 % Buffer B Buffer A hi 5% ACN leh 10 mM ammonium bicarbonate (ABC) a ni a, buffer B hi 80% ACN leh 10 mM ABC a ni. Minute 3 danah fraction te chu la khawm la, group hnih (1 + 17, 2 + 18, etc.) ah dah khawm la, vacuum centrifuge ah vawt rawh.
LC-MS/MS hmanga zirchian a ni. Mass spectrometry atan chuan peptide (number r119.aq) te chu 25 cm, 75 μm inner diameter PicoFrit analytical column (new objective lens, part number PF7508250) 1.9 μm ReproSil-Pur 120 C18-AQ medium (Dr. Maisch, mat) hmanga thuam, EASY-nLC hmangin an inthen a 1200 (Thermo Fisher Scientific, Germany) ah a lo piang tawh a ni. Column chu 50°C ah an dah a. Buffer A leh B te hi tuiah 0.1% formic acid leh 80% ACN ah 0.1% formic acid an ni. Peptide te chu 6% atanga 31% buffer B ah minute 65 chhung leh 31% atanga 50% buffer B ah minute 5 chhung gradient 200 nl/min ah then a ni. Peptide eluted te chu Orbitrap Fusion mass spectrometer (Thermo Fisher Scientific) hmangin an zirchiang a. Peptide precursor m/z tehna hi 350 atanga 1500 m/z inkar resolution 120,000 hmanga tih a ni. 27% normalized collision energy hmangin charge state 2 atanga 6 nei precursor chak ber chu high energy C trap dissociation (HCD) cleavage atan thlan a ni. Cycle hun chhung chu 1 s ah dah a ni. Peptide fragment m/z value chu ion trap-ah AGC target tenau ber 5×104 leh injection hun rei ber 86 ms hmangin an teh a ni. Fragmentation hnuah precursor chu dynamic exclusion list-ah 45 s chhung dah a ni. TMT-labeled peptide te chu 50 cm, 75 μm Acclaim PepMap column (Thermo Fisher Scientific, catalog number 164942)-ah an thliar hrang a, migration spectra te chu Orbitrap Lumos Tribrid mass spectrometer (Thermo Fisher Scientific) hmangin high-field asymmetric waveform ions (FAIMS) equipment hmanga thuam hmangin an zirchiang a ni (Thermo Fisher Scientific) hian compensation voltage pahnih −50 leh −70 V-ah hna a thawk a, synchronization precursor atanga thlan chhuah MS3 chu TMT report ion signal tehna atan hman a ni. Peptide separation hi EASY-nLC 1200 hmangin an ti a, 90% linear gradient elution hmangin, buffer concentration 6% atanga 31% inkar a ni a; buffer A chu 0.1% FA a ni a, buffer B chu 0.1% FA leh 80% ACN a ni. Analytical column hi 50°C-ah an thawk a ni. FreeStyle (version 1.6, Thermo Fisher Scientific) hmangin FAIMS compensation voltage angin original file chu then rawh.
Protein hriatchhuah leh quantification. Integrated Andromeda search engine hmangin original data chu MaxQuant version 1.5.2.8 (https://maxquant.org/) hmangin an zirchiang a ni. Aequorea victoria atanga Cre recombinase leh YFP sequence hmuhchhuah bakah hian peptide fragment spectra-ah mouse reference proteome (Proteome ID UP000000589, May 2017-a UniProt atanga download) canonical sequence leh isoform sequence te chu an zawng a ni. Methionine oxidation leh protein N-terminal acetylation te chu variable modification atan an dah a; cysteine ​​carbamoyl methylation chu fixed modification atan an dah a ni. Digestion parameters chu “specificity” leh “trypsin/P” ah dah a ni. Protein hriat theihna atana peptide leh razor peptide hman tlem ber chu 1 a ni a; the minimum number of unique peptides is 0. Peptide map matching condition hnuaiah chuan protein identification rate chu 0.01 a ni. “Second Peptide” tih kha enable a ni. Original file hrang hrang inkara identification hlawhtling tak tak transfer nan “match between runs” option hmang la. Label-free quantification (LFQ) atan LFQ minimum ratio count 1 hmang rawh (60). LFQ intensity hi time point tinah genotype group pakhat tal valid value pahnih tal filter a ni a, normal distribution atanga extrapolate a ni a, a zau zawng chu. 0.3 leh 1.8 hnuai lam pan rawh. LFQ result thlirletna atan Perseus computing platform (https://maxquant.net/perseus/) leh R (https://r-project.org/) te hmang la. Differential expression analysis atan limma software package atanga two-way moderate t test hman a ni ( 61 ). Exploratory data analysis hi ggplot, FactoMineR, factoextra, GGally leh pheatmap hmanga tih a ni. TMT-based proteomics data chu MaxQuant version 1.6.10.43 hmangin an zirchiang a ni. UniProt-a human proteomics database aṭanga raw proteomics data zawng rawh, September 2018 khan download a ni a, he analysis-ah hian siamtuin isotope purity correction factor a pek chu a tel a ni. Differential expression analysis atan R ah limma hmang rawh. Data original, database search result, leh data analysis workflow leh result te chu PRIDE partner repository kaltlangin ProteomeXchange alliance ah data set identifier PXD019690 hmangin dah vek a ni.
Functional annotations hian analysis chu a tihausa hle. Kar 8 chhunga data set-a functional annotation terms hausakna hriat nan Ingenuity Pathway Analysis (QIAGEN) tool hman a ni (Figure 1). A tawi zawngin, LC-MS/MS (tandem mass spectrometry) data analysis atanga quantitative protein list hmuh chu a hnuaia filter criteria hmang hian hman a ni: Mus musculus chu species leh background atan thlan a ni a, category-ah hian Benjamini-a’n enrichment 0.05 emaw a aia hniam emaw atana P value a siamrem chu a pawimawh hle nia ngaih a ni. He graph atan hian cluster tina excess category sang ber panga chu adjusted P value atanga thlirin tarlan a ni. Multiple t-test hmangin Benjamini, Krieger, leh Yekutieli (Q = 5%) te two-stage linear boost program hmangin category tina candidate pawimawh hmuhchhuah te chu time-course protein expression analysis an nei a, row tin chu a hranin an zirchiang a ni. SD mumal tak hman a ngai lo.
He zirchianna result hi database chhuah tawhte nena khaikhin a, Figure 1-a Venn diagram siam nan hian quantitative protein list chu MitoCarta 2.0 annotations nen kan inzawm khawm a ( 24 ). Online tool Draw Venn Diagram (http://bioinformatics.psb.ugent.be/webtools/Venn/) hmangin diagram siam rawh.
Proteomics analysis atana statistical procedure hman dan chipchiar zawka hriat duh chuan Materials and Methods tih section inmil hi en la. Experiment dang zawng zawng tan chuan, a kaihhnawih legend-ah hian thu chipchiar tak hmuh theih a ni. A danglamna a awm loh chuan data zawng zawng chu mean ± SEM anga tarlan a ni a, statistical analysis zawng zawng chu GraphPad Prism 8.1.2 software hmanga tih a ni.
He thuziak atana supplementary materials hriat duh chuan http://advances.sciencemag.org/cgi/content/full/6/35/eaba8271/DC1 ah hian en theih a ni
Hei hi Creative Commons Attribution-Non-Commercial License thuthlung hnuaia sem chhuah, open access article a ni a, chu chuan eng medium pawha hman, sem leh tihchhuah phalsak a ni a, a tawp bera hmanna chu sumdawnna atana hlawkna tur a nih loh phawt chuan leh a bulpui ber chu a hnathawh hmasa ber chu a dik tih a nih phawt chuan. Kawhhmuh.
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E. Motori, I. Atanassov, S. M. V. Kochan, K. Folz-Donahue, V. Sakthivelu, P. Giavalisco, N. Toni, J. Puyal, N.-G te ziah a ni. Larson chuan a rawn ti a
Dysfunctional neurons te proteomics analysis an neihah chuan neurodegeneration do turin metabolic program te chu activate a ni tih hmuhchhuah a ni.
E. Motori, I. Atanassov, S. M. V. Kochan, K. Folz-Donahue, V. Sakthivelu, P. Giavalisco, N. Toni, J. Puyal, N.-G te ziah a ni. Larson chuan a rawn ti a
Dysfunctional neurons te proteomics analysis an neihah chuan neurodegeneration do turin metabolic program te chu activate a ni tih hmuhchhuah a ni.
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