Parizvino *Kero yazvino: Cologne 50931, Germany, Cologne Excellence Cluster Research on Cellular Stress Response in Aging-related Diseases (CECAD).
Kuora kwetsinga dzemitochondrial kunoonekwa sekusingadzoreki nekuti metabolic plasticity yema neuron yakaganhurirwa, asi mhedzisiro yekusashanda zvakanaka kwe mitochondrial pakuzvitonga kwemasero kwe neuron metabolism mumuviri hainzwisiswi zvakanaka. Pano, tinoisa proteome yemasero ePurkinje neurons ine kushomeka kweOXPHOS kunoenderera mberi kunokonzerwa nekukanganiswa kwe mitochondrial fusion dynamics. Takaona kuti kusashanda zvakanaka kwe mitochondrial kwakakonzera shanduko huru mumunda we proteomics, izvo zvakazoita kuti zvirongwa zve metabolic chaiyo zviitwe sequentially cell before death. Tisingatarisiri, takaona kupinza kuri pachena kwe pyruvate carboxylase (PCx) nemamwe ma enzymes ekudzivirira kukwegura anowedzera intermediates yeTCA cycle. Kudziviswa kwePCx kwakawedzera oxidative stress uye neurodegeneration, zvichiratidza kuti atherosclerosis ine mhedzisiro yekudzivirira muma neuron asina OXPHOS. Kudzorerwa kwe mitochondrial fusion muma neuron apera simba kunoshandura zvachose hunhu uhwu hwe metabolic, nokudaro zvichidzivirira kufa kwema cell. Zvatakawana zvinoratidza nzira dzisati dzambozivikanwa dzinopa simba rekusashanda zvakanaka kwe mitochondrial uye zvinoratidza kuti neurodegeneration inogona kudzoserwa kunyangwe muchikamu chekupedzisira chechirwere.
Basa guru remitochondria mukuchengetedza masero esimba remitsipa rinosimbiswa nezviratidzo zvakawanda zvetsinga zvine chekuita nezvirwere zvemitochondrial zvevanhu. Zvizhinji zvezvirwere izvi zvinokonzerwa nekuchinja kwemajini kunodzora kutaura kwemajini emitochondrial (1, 2) kana kuparadzwa kwemajini kune chekuita nekuchinja kwemitochondrial, izvo zvinokanganisa kusagadzikana kweDNA yemitochondrial (mtDNA) (3, 4). Basa mumhando dzemhuka rakaratidza kuti mukupindura kusashanda zvakanaka kwemitochondrial mumatishu akakomberedza, nzira dzekuchengetedza metabolic (5-7) dzinogona kuvhurwa, izvo zvinopa ruzivo rwakakosha rwekunzwisisa kwakadzama kwepathogenesis yezvirwere izvi zvakaoma. Kusiyana neizvi, kunzwisisa kwedu shanduko dzemetabolism dzemhando dzakasiyana dzemasero dzinokonzerwa nekukundikana kukuru kwekugadzirwa kweadenosine triphosphate (ATP) muuropi kwakakosha (8), zvichisimbisa kudiwa kwekuona zvinangwa zvekurapa zvinogona kushandiswa kudzivirira kana kudzivirira chirwere. Kudzivirira kuora kwetsinga (9). Kushaikwa kweruzivo ndechekuti masero etsinga anofungidzirwa zvakanyanya kuva nekushanduka kushoma kwemetabolism zvichienzaniswa nemarudzi emasero ematishu akakomberedza (10). Zvichienderana nekuti masero aya anoita basa guru mukuronga kugoverwa kwemametabolites kuma neuron kuti akurudzire kutapurirana kwesynaptic uye kupindura kumatambudziko ekukuvara uye zvirwere, kugona kugadzirisa metabolism yema cell kune mamiriro akaoma e brain tissue kwakangogumira ku glial cells (11-14). Pamusoro pezvo, kusiyana kwemasero aripo mu brain tissue kunonyanya kukanganisa kudzidza shanduko dze metabolic dzinoitika mu specific neuron subgroups. Nekuda kweizvozvo, zvishoma zvinozivikanwa nezvemhedzisiro chaiyo ye cellular uye metabolic ye mitochondrial dysfunction mu neuron.
Kuti tinzwisise mhedzisiro yekushanda zvisina kunaka kwemitochondrial, takaparadzanisa maPurkinje neurons (PNs) muzvikamu zvakasiyana zvekuora kwetsinga kunokonzerwa nekuparadzwa kwemitochondrial outer membrane fusion (Mfn2). Kunyangwe Mfn2 mutations muvanhu ichibatanidzwa nerudzi rwe neuropathy yemota inozivikanwa seCharcot-Marie-Tooth type 2A (15), kuparadzwa kweMfn2 mumakonzo inzira inozivikanwa ye oxidation​​ Phosphorylation (OXPHOS) dysfunction method. Mhando dzakasiyana dze neuronal (16-19) uye neurodegenerative phenotype inokonzereswa ne progressive neurological symptoms, dzakadai se movement disorders (18, 19) kana cerebellar ataxia (16). Nekushandisa musanganiswa we label-free quantitative (LFQ) proteomics, metabolomics, imaging, uye virological methods, tinoratidza kuti progressive neurodegeneration inokonzera zvakanyanya pyruvate carboxylase (PCx) nezvimwe zvinhu zvine chekuita ne arteriosclerosis yePNs in vivo The expression ye enzymes. Kuti tione kukosha kwekuwana uku, takaderedza zvakanyanya kutaurwa kwePCx muMfn2-deficient PNs, uye takaona kuti kuvhiyiwa uku kwakawedzera oxidative stress uye kwakakurumidzisa neurodegeneration, zvichiratidza kuti azoospermia inopa kufa kwemasero. Kuratidzwa kwakanyanya kweMFN2 kunogona kuponesa zvachose degeneration yePN ine kushomeka kwakanyanya kweOXPHOS, kushandiswa kwakanyanya kweDNA yemitochondrial, uye network yemitochondrial yakatyoka, izvo zvinosimbisa zvakare kuti rudzi urwu rwe degeneration yemitochondrial runogona kutopora padanho repamusoro rechirwere masero asati afa.
Kuti tikwanise kuona mitochondria muMfn2 knockout PNs, takashandisa mhando yembeva inobvumira mitochondria inoenderana neCre kuti inange protein yellow fluorescent (YFP) (mtYFP) (20) Cre expression uye takatarisa chimiro chemitochondrial in vivo. Takaona kuti kuparadzwa kwegene reMfn2 muPNs kwaizotungamira mukukamurwa zvishoma nezvishoma kwenetwork yemitochondrial (Mufananidzo S1A), uye shanduko yekutanga yakawanikwa pazera remavhiki matatu. Kusiyana neizvi, kuora kwakanyanya kwePN cell layer, sezvakaratidzwa nekurasikirwa kweCalbindin immunostaining, hakuna kutanga kusvika mavhiki gumi nemaviri ekuberekwa (Mufananidzo 1, A naB). Kusawirirana kwenguva pakati pekuchinja kwekutanga muchimiro chemitochondrial uye kutanga kunoonekwa kwekufa kweneuronal kwakaita kuti tiongorore shanduko dzemetabolism dzinokonzereswa nekusashanda zvakanaka kwemitochondrial cell isati yafa. Takagadzira nzira yekusarudza ma cell anoshandiswa ne fluorescence (FACS) kuti tibvise YFP (YFP+)-expressing PN (Mufananidzo 1C), uye mu control mice (Mfn2 + / loxP :: mtYFP loxP- stop-loxP: : L7-cre), pano inonzi CTRL (Mufananidzo S1B). Kugadziriswa kwenzira yekuisa gating zvichibva pahuwandu hwechiratidzo cheYFP kunotibvumira kuchenesa muviri weYFP+ (YFPhigh) wePN kubva kune vasiri PN (YFPneg) (Mufananidzo S1B) kana putative fluorescent axon/dendritic fragments (YFPlow; Mufananidzo S1D, kuruboshwe), zvakasimbiswa ne confocal microscope (Mufananidzo S1D, kurudyi). Kuti tisimbise hunhu hwevanhu vakarongwa, takaita LFQ proteomics uye tobva taongorora principal component, uye takaona kuti pane musiyano wakajeka pakati peYFPhigh neYFPneg cells (Mufananidzo S1C). Masero eYFPhigh akaratidza kuwedzera kwezviratidzo zvePN zvinozivikanwa (kureva Calb1, Pcp2, Grid2 naItpr3) (21, 22), asi hapana kuwedzera kwemapuroteni anowanzoonekwa muma neuron kana mamwe marudzi emasero (Mufananidzo 1D)). Kuenzanisa pakati pemasampuli ari mumasero eYFPhigh akaunganidzwa mukuyedza kwakazvimirira kwakaratidza kuwirirana kwe correlation coefficient> 0.9, zvichiratidza kuberekana kwakanaka pakati pezvakaunganidzwa zve biological (Mufananidzo S1E). Muchidimbu, data iri rakasimbisa hurongwa hwedu hwekupatsanurwa kwePN inogoneka uye chaiyo. Nekuti sisitimu yekutyaira yeL7-cre inoshandiswa inokonzera kupatsanurwa kwemosaic muvhiki yekutanga mushure mekusununguka (23), takatanga kuuraya mbeva kubva kuCTRL uye conditional (Mfn2 loxP / loxP :: mtYFP loxP-stop-loxP :: L7-cre) Kuunganidza ma neuron. Mushure mekunge kupatsanurwa kwapera, inonzi Mfn2cKO pamavhiki mana ekuberekwa. Semugumo, takasarudza mavhiki masere ezera apo PN layer yakanga isina kupararira pasinei nekupatsanuka kuri pachena kwe mitochondrial (Mufananidzo 1B neMufananidzo S1A). Pakazara, takayera huwandu hwemapuroteni 3013, ayo angangoita 22% akavakirwa paMitoCarta 2.0 annotations zvichibva pa mitochondrial proteome se mitochondria (Mufananidzo 1E) (Mufananidzo 1E) (24). Kuongororwa kwe differential gene expression analysis kwakaitwa pavhiki rechisere kwakaratidza kuti 10.5% chete yemapuroteni ese aiva nekuchinja kukuru (Mufananidzo 1F neMufananidzo S1F), ayo mapuroteni 195 akanga aderedzwa uye mapuroteni 120 akanga awedzerwa (Mufananidzo 1F). Zvakakosha kuziva kuti "innovative pathway analysis" yedata iri inoratidza kuti majini akaratidzwa zvakasiyana-siyana anonyanya kuve eboka rakaganhurirwa renzira dzakati wandei dze metabolic (Mufananidzo 1G). Zvinofadza kuti, kunyangwe kudzikiswa kwenzira dzine chekuita neOXPHOS uye calcium signaling kuchisimbisa kutangwa kwekusashanda zvakanaka kwemitochondrial muPN dzisina fusion, mamwe mapoka anonyanya kusanganisira metabolism yeamino acid akawedzerwa zvakanyanya, zvinoenderana nemetabolism inoitika muPN dzemitochondrial.
(A) Mifananidzo inomiririra yezvikamu zve cerebellar zve CTRL ne Mfn2cKO mbeva ichiratidza kurasikirwa kuri kuramba kuchiitika kwePNs (calbindin, grey); nuclei dzakakanganiswa neDAPI. (B) Kuyera (A) (kuongorora kwega kwega kwevariance, ***P<0.001; n = 4 kusvika 6 denderedzwa kubva kumakonzo matatu). (C) Kushanda kwekuyedza. (D) Kugoverwa kwemepu yekupisa kwemamaki chaiwo kuPurkinje (kumusoro) nedzimwe mhando dzemasero (pakati). (E) Venn diagram inoratidza huwandu hwemapuroteni e mitochondrial akaonekwa muPN yakatsanangurwa. (F) Volcano plot yemapuroteni anoratidzwa zvakasiyana muMfn2cKO neurons pamavhiki masere (kukosha kwekucheka kwe1.3). (G) Kuongorora nzira yekugadzira kunoratidza nzira shanu dzakakosha dzekusimudzira (tsvuku) uye kudzikisa (bhuruu) muMfn2cKO PN yakatsanangurwa semavhiki masere. Avhareji yehuwandu hweproteni yega yega yakawanikwa inoratidzwa. Grayscale heat map: yakagadziriswa P value. ns, haina kukosha.
Ruzivo rweProteomics rwakaratidza kuti kuratidzwa kweprotein kwemacomplexes I, III, uye IV kwakadzikira zvishoma nezvishoma. MaComplexes I, III, uye IV ese aive nemasubunits akakosha e mtDNA-encoded, nepo complex II, iyo yaive nuclear-coded chete, yakanga isina kukanganiswa (Mufananidzo 2A neMufananidzo S2A). . Zvichienderana nemhedzisiro yeproteomics, immunohistochemistry yezvikamu zve cerebellar tissue yakaratidza kuti MTCO1 (mitochondrial cytochrome C oxidase subunit 1) subunit level ye complex IV muPN yakaderera zvishoma nezvishoma (Mufananidzo 2B). Subunit ye ​​mtDNA-encoded Mtatp8 yakaderedzwa zvakanyanya (Mufananidzo S2A), nepo steady-state level ye nuclear-encoded ATP synthase subunit yakaramba isina kuchinja, izvo zvinoenderana ne stable ATP synthase subassembly F1 complex inozivikanwa kana mtDNA expression yakagadzikana. Kuumbwa kwayo kwakafanana. Interrupt (7). Kuongororwa kwehuwandu hwemtDNA muMfn2cKO PNs dzakarongwa ne real-time polymerase chain reaction (qPCR) kwakaratidza kudzikira zvishoma nezvishoma kwenhamba yekopi yemtDNA. Zvichienzaniswa neboka rekutonga, pazera remavhiki masere, inenge 20% chete yehuwandu hwemtDNA ndiyo yakachengetedzwa (Mufananidzo 2C). Zvichienderana nemhedzisiro iyi, kudhirowa kwe confocal microscopy kweMfn2cKO PNs kwakashandiswa kuona DNA, zvichiratidza kushandiswa kwe mitochondrial nucleotides kunoenderana nenguva (Mufananidzo 2D). Takaona kuti vamwe chete vanobatanidzwa mukuora kwemapuroteni e mitochondrial uye kupindura kwekushushikana ndivo vakakwidziridzwa, kusanganisira Lonp1, Afg3l2 naClpx, uye OXPHOS complex assembly factors. Hapana shanduko dzakakosha mumazinga emapuroteni anobatanidzwa muapoptosis akaonekwa (Mufananidzo S2B). Saizvozvowo, takaona kuti mitochondria uye endoplasmic reticulum channels dzinobatanidzwa mukufambisa calcium dzine shanduko diki chete (Mufananidzo S2C). Pamusoro pezvo, kuongororwa kwemapuroteni ane chekuita neautophagy hakuna kuwana shanduko dzakakosha, izvo zvinoenderana nekupinza kunoonekwa kweautophagosomes kunoonekwa muhupenyu hwemuviri ne immunohistochemistry uye electron microscopy (Mufananidzo S3). Zvisinei, kusashanda zvakanaka kweOXPHOS muPNs kunoperekedzwa nekuchinja kuri pachena kwe ultrastructural mitochondrial. Mitochondrial clusters inogona kuonekwa mumasero uye dendritic trees dzeMfn2cKO PNs dzine mavhiki mashanu nemasere, uye chimiro chemukati che membrane chakachinja zvakanyanya (Mufananidzo S4, A na B). Zvichienderana nekuchinja uku kwe ultrastructural uye kudzikira kukuru kwe mtDNA, kuongororwa kwe acute cerebral cerebellar slices ne tetramethylrhodamine methyl ester (TMRM) kwakaratidza kuti mitochondrial membrane potential muMfn2cKO PNs yakaderera zvakanyanya (Mufananidzo S4C).
(A) Kuongororwa kwenguva kwehuwandu hwekutaura kweOXPHOS complex. Funga nezvemapuroteni ane P<0.05 chete pamavhiki masere (two-way ANOVA). Mutsetse une madotsi: Hapana kugadziriswa kana tichienzanisa neCTRL. (B) Kuruboshwe: Muenzaniso wechikamu checerebellar chakanyorwa ne anti-MTCO1 antibody (scale bar, 20 μm). Nzvimbo inogarwa nePurkinje cell bodies yakafukidzwa neyero. Kurudyi: Kuyera MTCO1 levels (one-way analysis of variance; n = 7 kusvika 20 cells studies from three mouses). (C) Kuongororwa kweqPCR kwe mtDNA copy number muPN yakarongeka (one-way analysis of variance; n = 3 kusvika 7 mouses). (D) Kuruboshwe: Muenzaniso wecerebellar slice yakanyorwa ne anti-DNA antibody (scale bar, 20 μm). Nzvimbo inogarwa nePurkinje cell bodies yakafukidzwa yeyero. Kurudyi: Kuyera mtDNA loses (one-way analysis of variance; n = 5 kusvika 9 cells from three mouses). (E) Muenzaniso wechikamu che cerebellar chinoratidza mitoYFP + Purkinje masero (museve) mukurekodha kwe clamp ye whole-cell patch. (F) Kuyera kwe IV curve. (G) Kurekodha kunomiririra depolarizing current injection mu CTRL ne Mfn2cKO masero e Purkinje. Kuyera kwekumusoro: Kurova kwekutanga kwakakonzera AP. Kuyera kwezasi: Kuyera kwe AP frequency. (H) Kuyera kwe postsynaptic spontaneous inputs (sPSPs). Kuyera kwe representative recording trace uye zoom ratio yayo zvinoratidzwa mu (I). One-way analysis ye variance yakaongororwa n = 5 kusvika 20 masero kubva kumakonzo matatu. Data rinoratidzwa se mean±SEM; *P<0.05; **P<0.01; ***P<0.001. (J) Kuyera kwe spontaneous AP yakarekodhwa uchishandisa perforated patch clamp mode. Kuyera kwekumusoro: Kuyera kwe AP frequency. Kuyera kwezasi: zoom ye single AP. (K) Kuyera avhareji uye maximum AP frequency zvichienderana ne (J). Mann-Whitney test; n = 5 masero akaongororwa kubva kumakonzo mana. Data rinoratidzwa seavhareji ±SEM; harina kukosha.
Kukuvadzwa kuri pachena kweOXPHOS kwakaonekwa muMfn2cKO PN yemavhiki masere, zvichiratidza kuti mashandiro emuviri emaneuron haana kujairika zvakanyanya. Nokudaro, takaongorora hunhu hwemagetsi husina simba hwemaneuron asina kukwana eOXPHOS pamavhiki mana kusvika mashanu uye mavhiki manomwe kusvika masere nekuita zvinyorwa zveclamp ye whole-cell patch muzvikamu zve acute cerebellar (Mufananidzo 2E). Zvisingatarisirwi, avhareji ye resting membrane potential uye input resistance yema neuron eMfn2cKO zvakafanana ne control, kunyangwe paive nemisiyano midiki pakati pemasero (Tafura 1). Saizvozvowo, pamavhiki mana kusvika mashanu ekuberekwa, hapana shanduko dzakakosha mu current-voltage relationship (IV curve) dzakawanikwa (Mufananidzo 2F). Zvisinei, hapana ma neuron eMfn2cKO ane mavhiki manomwe kusvika masere ekuberekwa akapona IV regimen (hyperpolarization step), zvichiratidza kuti pane kunzwisisika kwakajeka kune hyperpolarization potential padanho iri rekupedzisira. Kusiyana neizvi, muma neuron eMfn2cKO, ma currents e depolarizing anokonzera repetitive action potential (AP) anobvumirwa zvakanaka, zvichiratidza kuti maitiro avo ese ekubuda haana kusiyana zvakanyanya neaya ema neurons ekudzora ane mavhiki masere (Tafura 1 neMufananidzo 2G). Saizvozvowo, frequency uye amplitude yema spontaneous postsynaptic currents (sPSCs) akafanana neaya eboka rekudzora, uye frequency yezviitiko yakawedzera kubva pamavhiki mana kusvika kumavhiki mashanu kusvika kumavhiki manomwe kusvika kumavhiki masere nekuwedzera kwakafanana (Mufananidzo 2, H naI). Nguva yekukura kwe synaptic muPNs (25). Mhedzisiro yakafanana yakawanikwa mushure mePNs patches dzakaboorwa. Kugadziriswa uku kunodzivirira kubhadharwa kwezvikanganiso zveATP zvemasero, sezvinogona kuitika mukurekodha clamp ye whole-cell patch. Kunyanya, kugona kwe membrane resting uye frequency yekuputika kwe spontaneous yeMfn2cKO neurons hazvina kukanganiswa (Mufananidzo 2, J naK). Muchidimbu, mhedzisiro iyi inoratidza kuti maPN ane kutadza kushanda zvakanaka kweOXPHOS anogona kubata nemapatani ekubuda kweropa kakawanda, zvichiratidza kuti pane nzira yekudzorera ropa inovabvumira kuchengetedza mhinduro dzemagetsi dziri pedyo nedzakajairika.
Data rinoratidzwa seavhareji ± SEM (kuongorora musiyano wenzira imwe chete, bvunzo yekuenzanisa kwakawanda yaHolm-Sidak; *P<0.05). Nhamba yeyuniti inoratidzwa nemabhuraketi.
Takatanga kuongorora kana paine chero chikamu chiri mudata reproteomics (Mufananidzo 1G) chinosanganisira nzira dzinogona kurwisa kushomeka kwakanyanya kweOXPHOS, nokudaro tichitsanangura kuti nei PN yakakanganiswa ichigona kuchengetedza electrophysiology iri pedyo neyakajairika (Mufananidzo 2, E kusvika K). . Ongororo yeProteomics yakaratidza kuti ma enzymes anobatanidzwa mu catabolism ye branched chain amino acids (BCAA) akawedzerwa zvakanyanya (Mufananidzo 3A neMufananidzo S5A), uye chigadzirwa chekupedzisira acetyl-CoA (CoA) kana succinyl CoA inogona kuwedzera ma tricarboxylates mu arteriosclerosis Acid (TCA) cycle. Takaona kuti huwandu hweBCAA transaminase 1 (BCAT1) neBCAT2 zvese zvakawedzera. Zvinobatsira danho rekutanga re BCAA catabolism nekugadzira glutamate kubva ku α-ketoglutarate (26). Ma subunits ese anoumba branched chain keto acid dehydrogenase (BCKD) complex anowedzerwa (complex iyi inokonzeresa decarboxylation inotevera uye isingadzoreki yeBCAA carbon skeleton inobuda) (Mufananidzo 3A neMufananidzo S5A). Zvisinei, hapana shanduko dziri pachena muBCAA pachayo dzakawanikwa muPN yakarongwa, izvo zvinogona kunge zvichikonzerwa nekuwedzera kwekutora maamino acid aya akakosha kana kushandiswa kwezvimwe zvinhu (glucose kana lactic acid) kuwedzera TCA cycle (Mufananidzo S5B). MaPN asina OXPHOS akaratidzawo kuwedzera kwe glutamine decomposition uye transamination activity pamavhiki masere ekuberekwa, izvo zvinogona kuratidzwa nekukwira kwe mitochondrial enzymes glutaminase (GLS) uye glutamine pyruvate transaminase 2 (GPT2) (Mufananidzo 3, A na C). Zvakakosha kuziva kuti kuwedzera kweGLS kunogumira kune spliced ​​​​isoform glutaminase C (GLS-GAC) (shanduko yeMfn2cKO/CTRL inenge 4.5-fold, P = 0.05), uye kuwedzera kwayo kwakananga mumatishu ekenza Kunogona kutsigira mitochondrial bioenergy. (27).
(A) Mepu yekupisa inoratidza kuchinja kwehuwandu hweprotein munzira yakatarwa pamavhiki masere. (B) Muenzaniso wechikamu checerebellar chakanyorwa ne anti-PCx antibody (scale bar, 20 μm). Museve weyero unonongedzera kumuviri wesero rePurkinje. (C) Kuongorora kweprotein expression ye time course kwakacherechedzwa senzira yakakosha ye atherosclerosis (multiple t-test, *FDR <5%; n = mbeva 3-5). (D) Pamusoro: Dhiyagiramu inoratidza nzira dzakasiyana dzekupinda mu carbon yakanyorwa iri mu [1-13C]pyruvate tracer (kureva, kuburikidza nePDH kana trans-arterial route). Pazasi: Chati yeviolin inoratidza chikamu checarbon ine label imwe (M1) yakachinjwa kuita aspartic acid, citric acid uye malic acid mushure mekunyora acute cerebellar slices ne [1-13C]pyruvate (paired t-test; ** P <0.01). (E) Kuongorora kwakazara kwenhoroondo yenguva yenzira yakaratidzwa. Funga nezvemapuroteni ane P<0.05 pamavhiki masere. Mutsetse wakakamurwa: hapana kukosha kwekugadzirisa (kuongorora musiyano wemativi maviri; * P <0.05; *** P <0.001). Data rinoratidzwa seavhareji±SEM.
Mukuongorora kwedu, BCAA catabolism yave imwe yenzira huru dzekusimudzira mitemo. Izvi zvinoratidza zvakasimba kuti huwandu hwemhepo hunopinda muTCA cycle hunogona kuchinjwa muPN isina OXPHOS. Izvi zvinogona kureva rudzi rukuru rwe neuronal metabolic rewiring, iyo inogona kukanganisa zvakananga physiology ye neuronal uye kurarama panguva yekuchengetedza OXPHOS dysfunction yakanyanya. Zvichienderana nefungidziro iyi, takaona kuti PCx enzyme huru inodzivirira atherosclerotic inowedzera (Mfn2cKO/CTRL inochinja kanenge ka1.5; Mufananidzo 3A), iyo inokonzeresa kushandurwa kwe pyruvate kuita oxaloacetate (28), iyo inofungidzirwa kuti iri muuropi. Kutaurwa kwe in kwakaganhurirwa kune astrocytes (29, 30). Zvichienderana nemhedzisiro yeproteomics, confocal microscopy yakaratidza kuti kutaura kwePCx kwakawedzera zvakanyanya muOXPHOS-deficient PNs, nepo PCx reactivity yakaganhurirwa zvakanyanya kuBergmann glial cells dziri pedyo dze control (Mufananidzo 3B). Kuti tiedze kukwira kwakaonekwa kwePCx, takarapa zvidimbu zve cerebellar zvine acute ne [1-13C]pyruvate tracer. Apo pyruvate yakasanganiswa ne pyruvate dehydrogenase (PDH), isotope label yayo yakanyangarika , Asi inosanganiswa muTCA cycle intermediates apo pyruvate inogadziriswa ne vascular reactions (Mufananidzo 3D). Mukutsigira data redu reproteomics, takaona nhamba huru yezviratidzo kubva ku tracer iyi mu aspartic acid yeMfn2cKO slices, nepo citric acid ne malic acid zvaivewo netsika iri pakati nepakati, kunyangwe zvisina kukosha (Mufananidzo 3D).
Muma neuron edopamine emakonzo eMitoPark ane kusashanda zvakanaka kwemitochondrial kunokonzerwa nema neuron edopamine achiparadza mitochondrial transcription factor A gene (Tfam) (Mufananidzo S6B), PCx expression yakavandudzwawo zvakanyanya (31), zvichiratidza kuti acetone acid arteriosclerosis Kuitika kwechirwere ichi kunodzorwa panguva yekusashanda zvakanaka kwe neuronal OXPHOS mumuviri. Zvakakosha kuziva kuti zvakawanikwa kuti ma enzymes akasiyana (32-34) anogona kuratidzwa muma neuron anogona kunge akabatana ne arteriosclerosis akadzorwa zvakanyanya muPNs dzisina OXPHOS, dzakadai sepropionyl-CoA carboxylase (PCC-A), Malonyl-CoA inoshandura propionyl-CoA kuita succinyl-CoA uye mitochondrial malic enzyme 3 (ME3), iyo basa rayo guru nderekudzosa pyruvate kubva ku malate (Mufananidzo 3, A na C) (33, 35). Pamusoro pezvo, takawana kuwedzera kukuru kwePdk3 enzyme, iyo inoisa phosphoryls uye nokudaro ichiisa PDH (36), nepo pasina shanduko dzakaonekwa muPdp1 enzyme iyo inoisa PDH kana PDH enzyme complex pachayo (Mufananidzo 3A). Nguva dzose, muMern2cKO PNs, phosphorylation yeα1 subunit α (PDHE1α) subunit yepyruvate dehydrogenase E1 chikamu chePDH complex muSer293 (inozivikanwa nekudzivisa mashandiro eenzyme ePDH) yakawedzerwa (Mufananidzo S6C) (Mufananidzo S6C). Pyruvate haina mukana wekupinda mutsinga dzeropa.
Pakupedzisira, takaona kuti nzira yepamusoro ye serine ne glycine biosynthesis, iyo yakabatana mitochondrial folate (1C) cycle uye proline biosynthesis (Mufananidzo 1G neMufananidzo S5C) zvese zvakavandudzwa zvakanyanya, zvichienderana nemishumo, panguva ye activation process. Nyama dzakatenderedza dzinoshanda ne mitochondrial dysfunction (5-7). Confocal analysis inotsigira iyi proteomics data yakaratidza kuti muPN isina OXPHOS, cerebellar slices dzembeva dzine mavhiki masere dzakaiswa serine hydroxymethyltransferase 2 (SHMT2), iyo key enzyme ye mitochondrial folate cycle. Kupindura kwakakosha kwekudzivirira muviri (Mufananidzo S5D). Mu 13 CU-glucose-incubated acute cerebellar slices, metabolic tracing experiments yakasimbisa kuwedzera kwe serine ne proline biosynthesis, zvichiratidza kuti flux ye carbon isoforms mu serine ne proline yakawedzera (Mufananidzo S5E). Sezvo maitiro anokurudzirwa neGLS neGPT2 ari iwo anokonzera kugadzirwa kweglutamate kubva kuglutamine uye transamination pakati peglutamate neα-ketoglutarate, kukwidziridzwa kwavo kunoratidza kuti ma neuron asina OXPHOS ane kudiwa kwakawedzerwa kweglutamate, Izvi zvinogona kunge zvichivavarira kuchengetedza kuwedzera kwe biosynthesis yeproline (Mufananidzo S5C). Kusiyana nekuchinja uku, ongororo yeproteomic ye cerebellar astrocytes kubva kumakonzo ePN-specific Mfn2cKO yakaratidza kuti nzira idzi (kusanganisira ese antiperoxidases) hadzina kuchinja zvakanyanya mukutaura, zvichiratidza kuti metabolic redirection iyi inosarudza PN yakaora (Mufananidzo S6, D kusvika G).
Muchidimbu, ongororo idzi dzakaratidza maitiro akasiyana-siyana ekushanda kwenguva pfupi kwenzira dzakati dze metabolic muPNs. Kunyangwe kushanda kwe neuronal mitochondrial kusingawanzoitiki kunogona kutungamira ku atherosclerosis yekutanga uye kugadziriswa kwe1C (Mufananidzo 3E neMufananidzo S5C), uye kunyange shanduko dzinofungidzirwa mukutaura kweI neIV complexes, shanduko mu serine de novo synthesis dzinongoitika chete. Zvakangoonekwa chete muzvikamu zvekupedzisira. Kusashanda zvakanaka kweOXPHOS (Mufananidzo 3E neMufananidzo S5C). Izvi zvakawanikwa zvinotsanangura maitiro akatevedzana umo mitochondrial inokonzerwa nekushushikana (1C cycle) uye cytoplasmic (serine biosynthesis) zvinopindura pamwe chete nekuwedzera kwe atherosclerosis muTCA cycle kuti pavezve nekuchinja kwe neuronal metabolism.
MaPN eOXPHOS ane mavhiki masere ekuberekwa anogona kuchengetedza chiitiko chekukurumidzira zvakanyanya uye kupinda mukubatanidzwa kwakakosha kwemetabolism kuti atsive kusashanda zvakanaka kwemitochondrial. Kuwanikwa uku kunomutsa mukana unonakidza wekuti kunyangwe panguva ino, masero aya anogonawo kugamuchira rubatsiro rwekurapa kuti anonoke kana kudzivirira kuora kwetsinga. Tanonoka. Takagadzirisa mukana uyu kuburikidza nekupindira kuviri kwakazvimirira. Munzira yekutanga, takagadzira vector yeCre-dependent adeno-associated virus (AAV) kuitira kuti MFN2 igone kuratidzwa nenzira yakasarudzwa muOXPHOS-deficient PNs in vivo (Mufananidzo S7A). AAV encoding MFN2 uye fluorescent reporter gene mCherry (Mfn2-AAV) zvakaongororwa mu primary neuron cultures in vitro, izvo zvakaita kuti MFN2 iratidzwe nenzira inoenderana neCre uye yakaponesa mitochondrial morphology, nokudaro ichidzivirira neuromutation muMfn2cKO neurons (Mufananidzo S7, B, D naE). Tevere, takaita bvunzo dzemuviri kuti tiendese Mfn2-AAV yemavhiki masere ku cerebellar cortex yeMfn2cKO ne mbeva dzinodzora, uye takaongorora mbeva dzemavhiki gumi nemaviri (Mufananidzo 4A). Mbeva dzakarapwa dzeMfn2cKO dzakafa (Mufananidzo 1, A na B) (16). Kuendeswa kwehutachiona mumuviri kwakaguma nekutaura kwePN mune mamwe ma cerebellar circles (Mufananidzo S7, G na H). Kuiswa kweAAV yekudzora ichitaura mCherry chete (Ctrl-AAV) hakuna kukanganisa zvakanyanya padanho rekuora kwetsinga mumhuka dzeMfn2cKO. Kusiyana neizvi, kuongororwa kweMfn2cKOs dzakaendeswa neMfn2-AAV kwakaratidza kudzivirira kwakakosha kwePN cell layer (Mufananidzo 4, B na C, uye Mufananidzo S7, H na I). Kuonekwa kweMFN1 asi kwete MFN2 kunoshanda zvakaenzana mukuchengetedza kufa kwetsinga dzeropa (Mufananidzo 4C neMufananidzo S7, C naF), izvo zvinoratidza kuti kuonekwa kweMFN1 mudumbu kunogona kuwedzera kushaikwa kweMFN2. Ongororo yakawedzerwa padanho rimwe chete rePN yakaratidza kuti Mfn2-AAV yakabatsira zvikuru pakugadzirisa marongerwo emasero eropa, yakagadzirisa mazinga emtDNA, uye yakadzora kuratidzwa kwakanyanya kwechiratidzo cheanti-angiogenesis PCx ​​(Mufananidzo 4, C kusvika E). Kuongororwa kwembeva dzeMfn2cKO dzakaponeswa dziri mumamiriro ekuzorora kwakaratidza kuti chimiro chadzo uye zviratidzo zvekufamba (kufamba kweS1 kuenda kuS3) zvakavandudzika. Mukupedzisa, kuedza uku kunoratidza kuti kunonoka kudzoserwa kweMFN2 muPNs dzisina kukwana muOXPHOS kwakakwana kuderedza kushandiswa kwemtDNA uye kukonzera atherosclerosis, nokudaro kudzivirira kuora kweaxon uye kufa kwetsinga dzeropa mumuviri.
(A) Chirongwa chinoratidza purogiramu yekuedza yekuisa AAV encoding MFN2 kana nzira yemetabolic yaratidzwa yatanga kushanda. (B) Mifananidzo inomiririra confocal yezvidimbu zvecerebellar zvemavhiki gumi nemaviri zvakatorwa pamavhiki masere mumbeva dzeMfn2cKO uye zvakanyorwa ne anti-Calbindin antibody. Kurudyi: Kuyera axon fibers. Chikero che axon zoom i450 ne75 μm. (C) Kuruboshwe: Kuyera kwePurkinje cell density muAAV transduction loop (AAV+) (one-way analysis of variance; n = 3 mbeva). Kurudyi: mtDNA focus analysis mu transduced PN pavhiki 12 (unpaired t-test; n = 6 masero kubva kumambeva matatu). * P <0.05; ** P <0.01. (D) Mienzaniso ye transmission electron micrographs yePNs dzeMfn2cKO cerebellar sections dzakatorwa nemavirus vectors akaratidzwa. Chifukidzo chepingi chinoratidza nzvimbo inogarwa nedendrites, uye sikweya yero ine madotsi inoratidza zoom yakapihwa kurudyi; n inomiririra nucleus. Scale bar, 1μm. (E) inoratidza muenzaniso wePCx staining muPN transduced pamavhiki gumi nemaviri. Scale bar, 20μm. OE, overexpression; FC, fold change.
Pakupedzisira, takaongorora kukosha kwekurarama kwemasero anokonzerwa neperoxidase mumasero ePNs akasangana neOXPHOS disadvantage. Takagadzira mCherry encoding AAV-shRNA (short hairpin RNA) yakanangana negonzo PCx mRNA (AAV-shPCx), uye takabaya hutachiona kana scrambled control (AAV-scr) mucerebellum yemakonzo eMfn2cKO. Jekiseni iri rakaitwa muvhiki yechina yekuberekwa (Mufananidzo 5A) kuti PCx ibudirire panguva iyo PCx expression yakawedzera (Mufananidzo 3C) uye PN cell layer yakanga ichiripo (Mufananidzo 1A). Zvakakosha kuziva kuti kudonha PCx (Mufananidzo S8A) kunotungamira mukukurumidzira kukuru kwekufa kwePN, izvo zvinongogumira kune ring ine hutachiona (Mufananidzo 5, B naC). Kuti tinzwisise mashandiro emhedzisiro yemetabolic inokonzerwa nePCx up-regulation, takadzidza mamiriro e redox ePNs mushure mekunge PCx ​​knockdown yaitwa uye AAV-mediated optical biosensor Grx1-roGFP2 yakaratidzwa panguva imwe chete (Mufananidzo S8, B kusvika D) kuti tiongorore glutathione The relative change yepeptide redox potential (38). Zvadaro, takaita two-photon fluorescence lifetime imaging microscopy (FLIM) mu acute brain slices dzeMfn2cKO yemavhiki manomwe kana kudzora littermates kuti tione shanduko dzinogona kuitika mu cytoplasmic redox status mushure mekusimbisa mamiriro eFLIM (Mufananidzo S8, E kusvika G). Ongororo iyi yakaratidza kuwedzera kukuru mu oxidation state yeMfn2cKO PNs imwe chete isina PCx expression, iyo yakasiyana ne control neurons kana Mfn2cKO PNs dzinongoburitsa scrambled shRNA (Mufananidzo 5, D naE). Pakaderedzwa kuratidzwa kwePCx, chikamu cheMfn2cKO PNs chinoratidza mamiriro e oxidized zvakanyanya chakawedzera kanopfuura katatu (Mufananidzo 5E), zvichiratidza kuti PCx yakawedzera kugona kwayo kuita redox kwema neuron akaora.
(A) Chirongwa chinoratidza purogiramu yekuedza yekuisa AAV encoding shPCx kana nzira yemetabolic yakaratidzwa yavhurwa. (B) Mifananidzo ye confocal inoratidza zvikamu zve cerebellar zvemavhiki masere mumbeva dzeMfn2cKO dzakatorwa uye dzakanyorwa ne anti-calcineurin antibody pamavhiki mana. Scale bar, 450μm. (C) Kuyera huwandu hwemasero ePurkinje muAAV-transduced loops (kuongorora kwega kwega kwevariance; n = mbeva 3 kusvika 4). Data rinoratidzwa seavhareji±SEM; ***P<0.001. (D) Mufananidzo weFLIM unoratidza avhareji yehupenyu hwePN yemavhiki manomwe inoratidza glutathione redox sensor Grx1-roGFP2 pasi pemamiriro ezvinhu ekuedza akatarwa. LUT (tarisa-up table) ratio: nguva yekupona (mu picoseconds). Scale bar, 25μm. (E) Histogram inoratidza kugoverwa kweGrx1-roGFP2 lifetime values ​​​​kubva pa (D) (n=158 kusvika 368 masero mumakonzo maviri pasi pemamiriro ese). Chati yepie iri pamusoro pehistogram yega yega: inoratidza huwandu hwemasero ane marefu zvakanyanya (tsvuku, oxidized) kana mapfupi (bhuruu, akaderedzwa) lifetime values, ayo anopfuura 1 SD yeavhareji lifetime value muCTRL-AAV-scr. (F) Muenzaniso unoratidzwa unoratidza kudzivirira kwekukwidziridzwa kweneuronal PCx.
Zvose pamwe chete, data ratinopa pano rinoratidza kuti kudzokororwa kweMFN2 kunogona kubatsira zvachose PN yakafambira mberi ine kushomeka kwakanyanya kweOXPHOS, kudzikira kwakanyanya kwemtDNA, uye chimiro chisina kujairika chakafanana ne ista, nokudaro zvichipa kufambira mberi kunoramba kuripo kunyangwe muzvirwere zvakanyanya. Kudzikira kwetsinga dzeropa kunopa humbowo hunodzokororwa hwedanho risati rasvika rekufa kwemasero. Kuchinjika uku kwemetabolism kunosimbiswazve nekukwanisa kwema neuron kukonzera atherosclerosis (kudzokororwa kweTCA cycle), izvo zvinodzivisa kuratidzwa kwePCx muPN dzisina OXPHOS uye zvinowedzera kufa kwemasero, nokudaro zvichiita basa rekudzivirira (Mufananidzo 5F).
Muchidzidzo ichi, takapa humbowo hwekuti mhinduro yePNs kuOXPHOS dysfunction ndeyekubatana zvishoma nezvishoma kuTCA cycle atherosclerosis kuburikidza nenzira ye differential activation pathway inoitiswa nema metabolic programs. Takasimbisa ongororo yeproteomic nenzira dzakawanda dzinoenderana uye takaratidza kuti kana paine dambudziko nekusashanda zvakanaka kwe mitochondrial, ma neuron ane chimiro che metabolic elasticity chisati chambozivikanwa. Zvakatishamisa kuti, maitiro ese e rewiring haafanire kuratidza mamiriro ekupedzisira e metabolic anoenderana ne neurodegeneration zvishoma nezvishoma uye zvisingadzoreki, asi data redu rinoratidza kuti inogona kugadzira maintainance neuron kunyangwe padanho risati rasvika rekufa kwemasero. Functional compensation mechanism. Izvi zvinoratidza kuti ma neuron ane metabolic plasticity yakakura mumuviri. Izvi zvinoratidza kuti kudzoserwa kweMFN2 gare gare kunogona kushandura kutaura kwe key metabolic markers uye kudzivirira PN degeneration. Kusiyana neizvi, inodzivirira atherosclerosis uye inokurumidza tsinga.
Chimwe chezvakawanikwa zvinonakidza muongororo yedu ndechekuti maPN asina OXPHOS anogona kugadzirisa metabolism yeTCA cycle nekuwedzera ma enzymes anokurudzira arteriosclerosis. Kugadziriswazve kwemetabolic chinhu chinowanzoitika mumasero ekenza, mamwe acho anovimba ne glutamine kuwedzera ma TCA cycle intermediates kuti agadzire zvinhu zvinoderedza simba rekufema uye kuchengetedza kugadzirwa kwe lipid ne nucleotide biosynthesis precursors (39, 40). Ongororo ichangobva kuitwa yakaratidza kuti mu peripheral tissues dzine OXPHOS dysfunction, kubatanazve kwe glutamine/glutamate metabolism chinhu chakakoshawo (5, 41), uko nzira yekupinda kwe glutamine muTCA cycle inoenderana Nekuda kwekuoma kwekukuvara kweOXPHOS (41). Zvisinei, hapana humbowo hwakajeka pane chero kufanana kwe neuronal metabolic plasticity mumuviri uye kukosha kwayo muchirwere. Mukudzidza kwakaitwa munguva pfupi yapfuura mu vitro, ma primary cortical neurons akaratidzwa kuti anounganidza madziva e glutamate kuti awane neurotransmission, nokudaro achikurudzira oxidative metabolism uye atherosclerosis pasi pemamiriro ekumanikidzwa kwemetabolic (42). Zvakakosha kuziva kuti pasi pekudziviswa kweTCA cycle enzyme succinate dehydrogenase, pyruvate carboxylation inofungidzirwa kuti inochengetedza kugadzirwa kweoxaloacetate muma neurons e cerebellar granule akakura (34). Zvisinei, kukosha kwemuviri kwemaitiro aya kumatishu europi (uko atherosclerosis inofungidzirwa kuti inonyanya kuganhurirwa kuma astrocytes) kuchiri kukosha kwemuviri (43). Muchiitiko ichi, data redu rinoratidza kuti maPN akakuvadzwa neOXPHOS mumuviri anogona kuchinjwa kuita BCAA degradation uye pyruvate carboxylation, ayo ari manyuko maviri makuru ekuwedzera maTCA pool intermediates. Kunyangwe mupiro wekufungidzira weBCAA catabolism kune neuronal energy metabolism wakakurudzirwa, kuwedzera kune basa re glutamate neGABA ye neurotransmission (44), hapana humbowo hwemaitiro aya mumuviri. Nokudaro, zviri nyore kufungidzira kuti maPN asina kushanda anogona kutsiva otomatiki kushandiswa kweTCA intermediates kunokonzerwa ne assimilation process nekuwedzera atherosclerosis. Kunyanya, kuwedzera kwePCx kunogona kudiwa kuti kudiwa kweaspartic acid kurambe kuripo, izvo zvinokurudzirwa mumasero ari kuwedzera ane kusashanda zvakanaka kwemitochondrial (45). Zvisinei, ongororo yedu yemetabolomics haina kuratidza shanduko dzakakosha muhuwandu hweaspartic acid muMfn2cKO PNs (Mufananidzo S6A), izvo zvinofungidzirwa kuti zvinoratidza kushandiswa kwakasiyana kweaspartic acid pakati pemasero ari kuwedzera uye maneuron epashure pemitotic. Kunyangwe nzira chaiyo yekuwedzera kwePCx mumasero asina kushanda zvakanaka muvivo ichiri kutsanangurwa, takaratidza kuti iyi mhinduro isati yasvika inoita basa rakakosha mukuchengetedza mamiriro e redox emaneuron, ayo akaratidzwa mukuyedza kweFLIM pazvidimbu zvecerebellar. Kunyanya, kudzivirira maPN kubva pakuwedzera PCx kunogona kutungamira kune mamiriro e oxidized uye kukurumidzisa kufa kwemasero. Kushanda kweBCAA degradation uye carboxylation ye pyruvate hazvisi nzira dzekuratidza peripheral tissues ekusashanda zvakanaka kwemitochondrial (7). Saka, zvinoita sekuti ndiwo masero anonyanya kukosha emasero etsinga asina OXPHOS, kunyangwe asiri iwo ega, ayo akakosha pakukura kwetsinga.
Chirwere cheCerebellar chirwere che neurodegenerative chakasiyana-siyana chinowanzoonekwa se ataxia uye chinowanzo kuvadza maPN (46). Huwandu hwema neuron uhwu hunonyanya kukanganiswa nekushanda zvisina kunaka kwe mitochondrial nekuti kusashanda zvakanaka kwemasero mumasero emunhu (16, 47, 48). Sekureva kwemishumo, modhi yembeva ye transgenic ine gene rakachinja inobatanidzwa ne human spinocerebellar ataxia uye ine kusashanda zvakanaka kwe mitochondrial (49, 50), zvichisimbisa kukosha kwekudzidza mhedzisiro yekushaikwa kweOXPHOS muPNPH. Saka, zvakakodzera kuparadzanisa uye kudzidza huwandu uhwu hwema neuron hwakasiyana zvinobudirira. Zvisinei, tichifunga kuti maPN anonyanya kunzwa kumanikidzwa uye anotsanangura chikamu chidiki chehuwandu hwemasero ese ecerebellar, kune dzakawanda zvidzidzo zvinobva ku omics, kuparadzaniswa kwavo semasero akazara kuchiri chinhu chakaoma. Kunyangwe zvisingaite kuti tisawane kusvibiswa kwemamwe marudzi emasero (kunyanya nyama dzevakuru), takabatanidza danho rinoshanda rekuparadzanisa neFACS kuti tiwane huwandu hwakakwana hwema neuron anoshanda ekuongorora proteomics, uye tiwane huwandu hwakawanda hweprotein (anenge mapuroteni 3000) zvichienzaniswa nedata riripo recerebellum yese (51). Nekuchengetedza kurarama kwemasero ese, nzira yatinopa pano inotibvumira kwete kungotarisa shanduko munzira dzemetabolism mumitochondria, asiwo kutarisa shanduko mune cytoplasmic counterparts dzayo, izvo zvinozadzisa kushandiswa kwematagi emitochondrial membrane kupfumisa mhando yemasero Nzira itsva yehuwandu hwemitochondria munyama dzakaoma (52, 53). Nzira yatinotsanangura haina kungoenderana nekudzidza kwemasero ePurkinje chete, asi inogona kushandiswa nyore nyore kune chero rudzi rwesero kugadzirisa shanduko dzemetabolism muuropi hwakarwara, kusanganisira mamwe mamodheru ekusashanda zvakanaka kwemitochondrial.
Pakupedzisira, takaona nzira yekurapa panguva iyi yekugadzirisa metabolic process iyo inogona kubvisa zvachose zviratidzo zvikuru zvekushushikana kwemasero uye kudzivirira kuora kwetsinga dzetsinga. Nokudaro, kunzwisisa mashandiro ekubatanidza tambo dzetsinga kunotsanangurwa pano kunogona kupa ruzivo rwakakosha mukurapa kunogona kuchengetedza tsinga dzetsinga panguva yekusashanda zvakanaka kwemitochondrial. Tsvagiridzo yeramangwana yakanangana nekutsanangura shanduko mukushandiswa kwesimba mune mamwe marudzi emasero europi inodiwa kuti iratidze zvizere kushanda kwenheyo iyi kune zvimwe zvirwere zvetsinga.
Mbeva dzeMitoPark dzakambotsanangurwa kare (31). Mbeva dzeC57BL/6N dzine majini eMfn2 akabatana neloxP dzakambotsanangurwa kare (18) uye dzakasanganiswa nembeva dzeL7-Cre (23). Mbeva mbiri dzakabuda dzakazosanganiswa nembeva dzeMfn2loxP/Mfn2loxP dzakafanana kuti dzigadzire magene ePurkinje-specific knockouts eMfn2 (Mfn2loxP/Mfn2loxP; L7-cre). Muchikamu chekubatana, Gt (ROSA26) SorStop-mito-YFP allele (stop-mtYFP) yakaunzwa kuburikidza nekupatsanurwa kwekuwedzera (20). Maitiro ese emhuka akaitwa zvichienderana nemirairo yeEurope, yenyika uye yemasangano uye akatenderwa neLandesamtfürNatur yeUmwelt neVerbraucherschutz, North Rhine-Westphalia, Germany. Mabasa emhuka anoteverawo nhungamiro yeEuropean Federation of Laboratory Animal Sciences Associations.
Mushure mekupedza kurara kwemukadzi ane pamuviri, mwana wembeva anobviswa (E13). Korekti yakachekwa muHanks' Balanced Salt Solution (HBSS) yakawedzeredzwa ne10 mM Hepes ndokupfuudzwa Dulbecco's Modified Eagle's Medium ine papain (20 U/ml) uye cysteine ​​​​(1μg/ml). Isa nyama muDMEM) woiparadzanisa nekugaya enzyme. Ml) pa37°C kwemaminitsi makumi maviri, wozoikuya ne DMEM yakawedzeredzwa ne 10% fetal bovine serum. Masero akadyarwa pamagirazi akaputirwa ne polylysine pa density ye 2×106 pa 6 cm culture dish kana pa density ye 0.5×105 cells/cm2 kuti iongororwe. Mushure memaawa mana, medium yakatsiviwa ne Neurobasal serum-free medium ine 1% B27 supplement uye 0.5 mM GlutaMax. Ma neuron akazochengetwa pa37°C uye 5% CO2 mukati mekuedza, uye akadyiswa kamwe chete pasvondo. Kuti zvikonzere kusanganiswa kwemasero mu vitro, 3μl (ndiro yekurima ine 24-well) kana 0.5μl (ndiro ine 24-well) yeAAV9 virus vector inotevera yakashandiswa kurapa ma neuron pazuva rechipiri mu vitro: AAV9.CMV.PI.eGFP. WPRE.bGH (Addgene, nhamba yekatalogu 105530-AAV9) uye AAV9.CMV.HI.eGFP-Cre.WPRE.SV40 (Addgene, nhamba yekatalogu 105545-AAV9).
DNA yeMfn1 neMfn2 (yakawanikwa kubva kuAddgene plasmid #23212 uye #23213, zvichiteerana) dzakanyorwa neV5 sequence (GKPIPNPLLGLDST) paC-terminus, uye dzakasanganiswa nemCherry mufuremu kuburikidza neT2A sequence. Grx1-roGFP2 chipo kubva kuna Heidelberg TP Dick DFKZ (Deutsches Krebsforschungszentrum). Nekutsiva tdTomato cassette uchishandisa nzira dzekare dze cloning, tapesti yakaiswa pasi pe pAAV-CAG-FLEX-tdTomato backbone (Addgene reference number 28306) kugadzira pAAV-CAG-FLEX-mCherry-T2A-MFN2-V5, pAAV-CAG-FLEX-mCherry-T2A-MFN1-V5 uye pAAV-CAG-FLEX-Grx-roGFP2 vectors. Nzira yakafanana yakashandiswa kugadzira vector yekudzora pAAV-CAG-FLEX-mCherry. Kuti pagadzirwe AAV-shPCx construct, plasmid AAV vector (VectorBuilder, pAAV [shRNA] -CMV-mCherry-U6-mPcx- [shRNA#1]) inodiwa, ine DNA sequence inovhara shRNA targeting mouse PCx (5′CTTTCGCTCTAAGGTGCTAAACTCGAGTTTAGCACCCTTAGAGCGAAAG 3′) Pasi pekutonga kweU6 promoter, mCherry inoshandiswa pasi pekutonga kweCMV promoter. Kugadzirwa kweAAV vectors dzekubatsira kwakaitwa zvichienderana nemirairo yemugadziri (Cell Biolabs). Muchidimbu, shandisa plasmid inotapurirana inotakura mCherry-T2A-MFN2-V5 (pAAV-CAG-FLEX-mCherry-T2A-MFN2-V5), mCherry-T2A-MFN1-V5 (pAAV-CAG-FLEX-mCherry) Transfection yemasero 293AAV-T2A-MFN1-V5), mCherry (pAAV-CAG-FLEX-mCherry) kana Grx-roGFP2 (pAAV-CAG-FLEX-Grx-roGFP2) coding gene, pamwe necoding AAV1 capsid protein uye accessory protein Packaging plasmid plasmid, uchishandisa calcium phosphate method. crude virus supernatant yakawanikwa ne freeze-thaw cycles mu dry ice/ethanol bath uye lysed cells mu phosphate buffered saline (PBS). Vector yeAAV yakacheneswa ne discontinuous iodixanol gradient ultracentrifugation (maawa makumi maviri nemana pa32,000 rpm uye 4°C) uye yakasimbiswa uchishandisa Amicon ultra-15 centrifugal filter. Genome titer yeAAV1-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 yakaita sezvakatsanangurwa kare (54), yakayerwa ne real-time quantitative PCR (qPCR) -MFN1-V5 (1.9×1013 GC/ml) uye AAV1-CAG-FLEX-Grx-roGFP2 (8.9×1012 GC/ml).
Ma neurons ekutanga akakweshwa mu 1x PBS inotonhora zvikuru, obva aiswa mu 0.5% Triton X-100 / 0.5% sodium deoxycholate/PBS lysis buffer ine phosphatase uye protease inhibitor (Roche). Kuyera mapuroteni kwakaitwa nekushandisa bicinchoninic acid assay (Thermo Fisher Scientific). Mapuroteni acho akabva aparadzaniswa neSDS-polyacrylamide gel electrophoresis, obva abviswa pa polyvinylidene fluoride membrane (GE Healthcare). Vhara nzvimbo dzisiri dzechokwadi wobva waisa mu incubate ne primary antibody (ona Table S1 kuti uwane rumwe ruzivo) mu 5% mukaka mu TBST (Tris-buffered saline neTween), washing steps uye secondary antibody mu TBST Incubate. Isa mu incubate ne primary antibody husiku hwese pa +4°C. Mushure mekusuka, isa secondary antibody kwemaawa maviri patembiricha yemumba. Zvadaro, nekuisa mu incubate blot rimwechete ne anti-β-actin antibody, mutoro wakafanana wakasimbiswa. Kuonekwa kwemasero nekushandura kuita chemiluminescence uye kuwedzera chemiluminescence (GE Healthcare).
Ma neuron akambodyarwa pamagirazi akavharirwa akaiswa 4% paraformaldehyde (PFA)/PBS panguva yakatarwa patembiricha yemukamuri kwemaminitsi gumi. Magirazi akavharirwa anotanga anyudzwa ne 0.1% Triton X-100/PBS kwemaminitsi mashanu patembiricha yemukamuri, uye ozoiswa mu blocking buffer [3% bovine serum albumin (BSA)/PBS]. Pazuva rechipiri, magirazi akavharirwa akagezwa ne blocking buffer ndokuiswa mu incubator yakakodzera fluorophore-conjugated secondary antibody kwemaawa maviri patembiricha yemukamuri; pakupedzisira, sampuro dzakagezwa zvakanaka muPBS ne 4′,6-diamidino-2-Phenylindole (DAPI) yakamiswa uye yozoiswa pa microscope slide ne Aqua-Poly/Mount.
Mbeva (dzechirume nedzechikadzi) dzakabayiwa ketamine (130 mg/kg) uye xylazine (10 mg/kg) mukati medumbu uye dzakapihwa pasi peganda necarprofen analgesic (5 mg/kg), Uye dzakaiswa muchinhu chinonzi stereotactic (Kopf) chakashongedzerwa nepedhi rinodziya. Fukura dehenya woshandisa dental drill kuti utetepese chikamu checerebellar cortex chinoenderana nepfupa remis (kubva ku lambda: tail 1.8, lateral 1, inoenderana ne lobules IV na V). Shandisa tsono yesirinji yakakombama kuti ugadzire buri diki mudehenya kuti usakanganisa mitsipa iri pazasi. Zvadaro capillary yegirazi yakatetepa inopinzwa zvishoma nezvishoma mu micro-hole (kubva -1.3 kusvika -1 parutivi rwe dura mater), uye 200 kusvika 300 nl AAV inopinzwa mu micro-injector (Narishige) nemasirinji emaoko (Narishige) kakawanda nekumanikidzwa kwakaderera kwenguva yakareba kwemaminitsi gumi kusvika makumi maviri. Mushure mekuisirwa mushonga, isa capillary kwemaminitsi gumi kuti hutachiona hupararire zvachose. Mushure mekunge capillaries dzabviswa, ganda rinokwenywa nokungwarira kuderedza kuzvimba kweronda uye kubvumira mhuka kuti ipore. Mhuka dzakarapwa nemishonga inonyaradza marwadzo (caspofen) kwemazuva akati wandei mushure mekuvhiyiwa, panguva iyo mamiriro adzo emuviri ainyatsotariswa uye ipapo dzakazourayiwa panguva yakatarwa. Maitiro ese akaitwa zvichienderana nemirairo yeEurope, yenyika uye yemasangano uye akatenderwa neLandesamtfürNatur yeUmwelt neVerbraucherschutz, North Rhine-Westphalia, Germany.
Mhuka dzakapihwa mushonga wekutindivadza neketamine (100 mg/kg) uye xylazine (10 mg/kg), uye mwoyo wakazorwa 0.1 M PBS kutanga, uye wozoiswa 4% PFA muPBS. Nyama yacho yakachekwa-chekwa ndokuiswa mu4% PFA/PBS husiku hwese pa4°C. Banga rinodedera (Leica Microsystems GmbH, Vienna, Austria) rakashandiswa kugadzira zvikamu zve sagittal (50 μm gobvu) kubva muuropi hwakagadzika muPBS. Kunze kwekunge zvataurwa neimwe nzira, kudhirowa kwezvikamu zvinoyangarara zvakasununguka kwakaitwa sezvakatsanangurwa pamusoro apa (13) patembiricha yemukamuri uye kukurunga. Muchidimbu, kutanga, zvidimbu zvakawanikwa zvakanyungudutswa ne 0.5% Triton X-100/PBS kwemaminitsi gumi nemashanu patembiricha yemukamuri; kune mamwe ma epitopes (Pcx naShmt2), nekuisa mu tris-EDTA buffer pa80°C (PH 9) pisa zvidimbu kwemaminitsi makumi maviri nemashanu panzvimbo pedanho iri. Zvadaro, zvikamu zvakaiswa muchimiro cheprimary antibody (ona Tafura S1) mu blocking buffer (3% BSA/PBS) pa4°C husiku hwese nekukurunga. Zuva rakatevera, zvikamu zvakagezwa ne blocking buffer ndokuiswa muchimiro che fluorophore-conjugated secondary antibody kwemaawa maviri patembiricha yemumba; pakupedzisira, zvikamu zvakagezwa zvakanaka muPBS, zvakaiswa counter-stained neDAPI, uye zvakazogadziriswa neAquaPolymount On a microscope slide.
Maikorosikopu yelaser scanning confocal (TCS SP8-X kana TCS Digital Light Sheet, Leica Microsystems) yakashongedzerwa nelaser chena yechiedza uye 405 diode ultraviolet laser yakashandiswa kutora mufananidzo wemuenzaniso. Nekunakisa fluorophore uye kuunganidza chiratidzo neHybrid Detector (HyDs), LAS-X software yakashandiswa kuunganidza mifananidzo yakaunganidzwa inoenderana neNyquist sampling mu sequential mode: kune non-quantitative panels, i highly dynamic signals (semuenzaniso, mu somatic cells uye dendrites) mtYFP) Shandisa HyD kuona huwandu hwePNs muBrightR mode). Gating kubva pa0.3 kusvika 6 ns inoshandiswa kuderedza background.
Kufungidzira masero akarongwa panguva chaiyo. Mushure mekuronga muNeurobasal-A medium ine 1% B27 supplement uye 0.5 mM GlutaMax, masero akabva angodyarwa pamasiraidhi egirazi ane poly-l-lysine (μ-Slide8 Well, Ibidi, catalog number 80826), Wobva waichengeta pa37°C uye 5% CO2 kweawa imwe chete kuti masero agadzikane. Kufungidzira kwenguva chaiyo kwakaitwa paLeica SP8 laser scanning confocal microscope ine laser chena, HyD, 63×[1.4 numeral aperture (NA)] oil objective lens uye nzvimbo yekupisa.
Mbeva yakakurumidza kupiswa necarbon dioxide ndokugurwa musoro, uropi hwakakurumidza kubviswa kubva mudehenya, ndokuchekwa kuita 200μm ukobvu (yekuyedza 13C labeling) kana 275μm ukobvu (yekuyedza kaviri photon) chikamu che sagittal chakazadzwa nezvinhu zvinotevera. Ice cream (HM-650 V, Thermo Fisher Scientific, Walldorf, Germany) yakazadzwa nezvinhu zvinotevera: 125 mM ice-cold, carbon-saturated (95% O2 uye 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 uye 3.5 mM MgCl2 (osmotic pressure ye310 kusvika 330 mmol). Tumira zvidimbu zveuropi zvawanikwa mukamuri risati raiswa mudumbu rine Ca2 + ACSF yakakwira (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 uye 2.0 mM MgCl2) Medium) pH 7.4 uye 310 kusvika 320 mmol).
Munguva yekutora mifananidzo, zvidimbu zvakaendeswa muimba yekutora mifananidzo yakatsaurirwa, uye kuyedza kwakaitwa pasi peACSF perfusion inoenderera mberi patembiricha inogara iri pakati pe32° kusvika 33°C. Microscope ine multiphoton laser scanning (TCS SP8 MP-OPO, Leica Microsystems) yakashongedzerwa neLeica 25x objective lens (NA 0.95, mvura), Ti: Sapphire laser (Chameleon Vision II, Coherent) yakashandiswa pakutora mifananidzo yezvidimbu. FLIM module (PicoHarp300, PicoQuant).
FLIM yeGrx1-roGFP2. Shanduko mu cytoplasmic redox state yePNs dzakayerwa ne two-photon FLIM mu sagittal brain slices, uko Grx1-roGFP2 biosensor yakanangana nePNs. MuPN layer, nzvimbo yekuwana inosarudzwa inenge 50 kusvika 80 μm pasi pe slice surface kuti ive nechokwadi chekuti pane PN inoshanda (ndiko kuti, kushaikwa kwe beaded structure kana neuronal morphological changes pamwe chete ne dendrites) uye double positive roGFP2 sensor uye AAV encoding shRNA PCx kana control sequence yayo (imwe neimwe inobatanidza mCherry). Unganidza mifananidzo ye single-stack ine 2x digital zoom [excitation wavelength: 890 nm; 512 nm 512 pixels]. Kuona: internal HyD, fluorescein isothiocyanate (FITC) filter group] uye averaging yemufananidzo mukati memaminitsi maviri kusvika matatu zvinoshandiswa kuona kuti ma photon akakwana aunganidzwa (1000 photons in total) kuti curve fitting. Kunzwa kweGrx1-roGFP2 probe uye kusimbiswa kwemamiriro eFLIM kwakaitwa nekutarisa kukosha kwehupenyu hweroGFP2 pakuwedzera exogenous 10 mM H2O2 ku ACSF inoyerera (kuwedzera oxidation, zvichikonzera kuwedzera kwehupenyu), uye wobva wawedzera 2 mM dithiothreitol (inoderedza mwero wekudzikira, zvichikonzera kudzikira kwehupenyu) (Mufananidzo S8, D kusvika G). Shandisa FLIMfit 5.1.1 software kuongorora mhedzisiro yakawanikwa, batanidza single exponential decay curve yemufananidzo wese neIRF yakayerwa (instrument response function), uye χ2 inenge 1. Kuti uverenge hupenyu hwePN imwe chete, mask yakatenderedza tsinga yemuviri yakadhirowewa nemaoko, uye avhareji yehupenyu hwese mumaski yega yega yakashandiswa pakuyera.
Kuongororwa kweMitochondrial potential. Mushure mekunge chikamu cheacute chaiswa 100 nM TMRM yakawedzerwa zvakananga kuACSF yakapiswa kwemaminitsi makumi matatu, shanduko dzemitochondrial potential dzePN dzakayerwa ne microscope ine ma photon maviri. TMRM imaging yakaitwa nekunakisa probe pa920 nm uye kushandisa internal HyD (tetramethylrhodamine isothiocyanate: 585/40 nm) kuunganidza zviratidzo; nekushandisa wavelength imwechete ye excitation asi uchishandisa internal HyD yakasiyana (FITC :525/50) kumufananidzo we mtYFP. Shandisa ImageJ's Image Calculator plug-in kuti uongorore mitochondrial potential padanho re single cell. Muchidimbu, plug-in equation: signal = min (mtYFP, TMRM) inoshandiswa kuona nzvimbo yemitochondrial inoratidza TMRM signal muPurkinje Somali mumufananidzo we confocal we single-stack we channel inoenderana. Zvadaro nzvimbo yepikisi iri mumasiki inobuda inoyerwa, uye yozoongororwa pamufananidzo wechikamu chimwe chete chemuganho unoenderana we mtYFP channel kuti pawanikwe chikamu che mitochondrial chinoratidza kugona kwe mitochondrial.
Mufananidzo wacho wakabviswa muchimiro cheHuygens Pro (Scientific Volume Imaging). Pamifananidzo yemataira yakaskeniwa, montage yetaira imwe chete inogadzirwa uchishandisa automatic stitching algorithm yakapihwa neLAS-X software. Mushure mekugadzirisa mufananidzo, shandisa ImageJ neAdobe Photoshop kuti uwedzere kugadzirisa mufananidzo uye kugadzirisa kupenya nekusiyana kwawo zvakaenzana. Shandisa Adobe Illustrator pakugadzirira mifananidzo.
Kuongorora kwekuongorora kwemtDNA. Huwandu hwemaronda emtDNA hwakayerwa pazvikamu zvecerebellar zvakanyorwa nema antibodies anorwisa DNA ne confocal microscope. Nzvimbo yega yega yakanangwa yakagadzirwa yemuviri wesero nenucleus yesero rega rega, uye nzvimbo yacho yakaverengerwa uchishandisa Multi Measure plug-in (ImageJ software). Bvisa nzvimbo yenyukireya kubva munzvimbo yemuviri wesero kuti uwane nzvimbo ye cytoplasmic. Pakupedzisira, Analyse Particles plug-in (ImageJ software) yakashandiswa kuyera otomatiki mapoinzi e cytoplasmic DNA anoratidza mtDNA pamufananidzo wepakati, uye mhedzisiro yakawanikwa yakajairwa kusvika paavhareji yePN yemakonzo eCTRL. Mhedzisiro yacho inoratidzwa seavhareji yenhamba ye nucleosides pasero rega rega.
Kuongororwa kwemaitiro emapuroteni. Shandisa plug-in yeImageJ's Image Calculator kuti uongorore maitiro emapuroteni muPN padanho remasero rimwe chete. Muchidimbu, mumufananidzo we single-layer confocal wechiteshi chinoenderana, kuburikidza ne equation: signal = min (mtYFP, antibody), nzvimbo ye mitochondrial inoratidza immunoreactivity kune imwe antibody muPurkina inoonekwa. Zvadaro nzvimbo ye pixel iri mu mask inobuda inoyerwa, uye yozogadziriswa pamufananidzo we single-stack unoenderana we mtYFP channel kuti uwane mitochondrial fraction yeprotein inoratidzwa.
Kuongororwa kwehuwandu hwemasero ePurkinje. Iyo Cell Counter plug-in yeImageJ yakashandiswa kuongorora huwandu hwePurkinje nekukamura huwandu hwemasero ePurkinje akaverengwa nehurefu hwe cerebellar ring inobatwa nemasero akaverengwa.
Kugadzirira uye kuunganidzwa kwemuenzaniso. Uropi hweboka rekutonga neMfn2cKO mbeva dzakagadziriswa mu2% PFA/2.5% glutaraldehyde mu0.1 M phosphate buffer (PB), uye zvikamu zve coronal zvakagadziriswa uchishandisa ciliates (Leica Mikrosysteme GmbH, Vienna, Austria) (Kukora 50 kusvika 60 μm). Wobva waisa muPB buffer mu1% os tetraoxide uye 1.5% potassium ferrocyanide patembiricha yemumba kweawa imwe chete. Zvikamu zvakagezwa katatu nemvura yakasanganiswa, uye wozozoiswa 70% ethanol ine 1% uranyl acetate kwemaminitsi makumi maviri. Zvikamu zvakazobviswa mvura mualcohol yakarongwa ndokuiswa muDurcupan ACM (Araldite casting resin M) epoxy resin (Electron Microscopy Sciences, catalog number 14040) pakati pemasiraidhi egirazi akaputirwa nesilicon, uye pakupedzisira pa60°C Polymerize muovheni kwemaawa makumi mana nemasere. Nzvimbo yecerebellar cortex yakasarudzwa uye zvikamu zvitete zve50 nm zvakachekwa paLeica Ultracut (Leica Mikrosysteme GmbH, Vienna, Austria) ndokutorwa pagridi yemhangura ye2×1 mm yakafukidzwa nepolystyrene film. Zvikamu zvakaiswa ruvara rwe4% uranyl acetate muH2O kwemaminitsi gumi, zvakagezwa neH2O kakawanda, zvadaro neReynolds lead citrate muH2O kwemaminitsi gumi, uye zvadaro zvakagezwa neH2O kakawanda. Micrographs dzakatorwa ne transmission electron microscope Philips CM100 (Thermo Fisher Scientific, Waltham, MA, USA) uchishandisa TVIPS (Tietz Video and Image Processing System) TemCam-F416 digital camera (TVIPS GmbH, Gauting, USA). Germany).
Kune mbeva dzine AAV, uropi hwakaparadzaniswa ndokuchekwa kuita chikamu che sagittal chine gobvu re 1 mm, uye cerebellum yakaongororwa uchishandisa maikorosikopu ye fluorescence kuti ione ring ine AAV (kureva, mCherry expressing). Kuedza chete uko jekiseni reAAV rinoita kuti jira rePurkinje cell rishande zvakanaka (kureva kuti jira rese) muma cerebellar rings maviri akatevedzana ndiko kunoshandiswa. AAV-transduced loop yakachekwa microscope kuti igadziriswe husiku hwese (4% PFA uye 2.5% glutaraldehyde mu 0.1 M cocoate buffer) uye yakazogadziriswazve. Pakuisa EPON, tissue yakagadzika yakagezwa ne 0.1 M sodium cocoate buffer (Applichem), uye yakaputirwa ne 2% OsO4 (os, Science Services; Caco) mu 0.1 M sodium cocoate buffer (Applichem) Maawa mana, wozogeza kwemaawa maviri. Dzokorora katatu ne 0.1 M cocamide buffer. Zvadaro, ethanol yakasimudzwa yakashandiswa kufukidza mhinduro yega yega yeethanol pa4°C kwemaminitsi gumi nemashanu kuti ibvise mvura mumuviri. Nyama yacho yakaendeswa kupropylene oxide ndokuiswa muincubation husiku hwese muEPON (Sigma-Aldrich) pa4°C. Isa nyama yacho muEPON itsva patembiricha yemumba kwemaawa maviri, wobva waiisa pa62°C kwemaawa makumi manomwe nemaviri. Shandisa ultramicrotome (Leica Microsystems, UC6) nebanga redhaimani (Diatome, Biel, Switzerland) kucheka zvikamu zvitete zve70 nm, wozoisa ruvara ne1.5% uranyl acetate kwemaminitsi gumi nemashanu pa37°C, wozoisa ruvara ne lead citrate solution kwemaminitsi mana. Ma electron micrographs akatorwa uchishandisa JEM-2100 Plus transmission electron microscope (JEOL) ine Camera OneView 4K 16-bit (Gatan) uye DigitalMicrograph software (Gatan). Kuti zviongororwe, maelektron micrographs akawanikwa achishandisa 5000× kana 10,000× digital zoom.
Kuongororwa kwechimiro chemitochondria. Pakuongorora kwese, marongerwo emitochondria yega yega akatsanangurwa nemaoko mumifananidzo yedhijitari uchishandisa software yeImageJ. Maparamita akasiyana echimiro anoongororwa. Kuwanda kwemitochondria kunoratidzwa sechikamu chinowanikwa nekukamura nzvimbo yese yemitochondria yesero rega rega nenzvimbo yecytoplasm (nzvimbo yecytoplasm = nzvimbo yesero-nucleus yesero) × 100. Kutenderera kwemitochondria kunoverengerwa nefomula [4π∙(nzvimbo/perimeter 2)]. Ista morphology yemitochondria yakaongororwa ikakamurwa kuita mapoka maviri ("tubular" uye "blister") zvichienderana nemaumbirwo azvo makuru.
Nhamba yeAutophagosome/lysosome uye kuongorora density. Shandisa software yeImageJ kuti urondedzere nemaoko marongerwo e autophagosome/lysosome yega yega mumufananidzo wedhijitari. Nzvimbo yeAutophagosome/lysosome inoratidzwa sechikamu chinoverengerwa nekukamura nzvimbo yese ye autophagosome/lysosome yesero rega rega nenzvimbo ye cytoplasm (nzvimbo ye cytoplasm=nzvimbo yesero-nucleus)×100. Density ye autophagosomes/lysosomes inoverengerwa nekukamura nhamba yese nenhamba ye autophagosome/lysosome structures pasero rega rega (maererano nenzvimbo ye cytoplasmic) (nzvimbo ye cytoplasmic = nzvimbo yesero-nucleus).
Kunyora mazita ekugadzirisa zvikamu zvemuviri uye kugadzirira sampuro. Kune bvunzo dzinoda kunyora mazita eglucose, tumira zvidimbu zveuropi zveacute mukamuri risati raiswa mushonga, rine kabhoni yakaguta (95% O2 ne5% CO2), Ca2 + ACSF yakakwira (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 ne2.0 mM MgCl2, yakagadziriswa ku pH 7.4 ne310 kusvika 320 mOsm), umo glucose iri 13 C 6- Glucose substitution (Eurisotop, catalog number CLM-1396). Kune zviedzo zvinoda pyruvate label, fambisa zvidimbu zveuropi zvakanyanya kuCa2 + ACSF yakakwira (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 uye Wedzera 2.0mM MgCl2, gadzirisa pH 7.4 uye 310 kusvika 320mOsm), uye wedzera 1mM 1-[1-13C]pyruvate (Eurisotop, catalog number CLM-1082). Isa zvikamu kwemaminitsi makumi mapfumbamwe pa37°C. Pakupera kwekuyedza, zvikamu zvakakurumidza kugezwa nemvura ine mvura (pH 7.4) ine 75 mM ammonium carbonate, uye zvakazosanganiswa mu 40:40:20 (v:v:v) acetonitrile (ACN): methanol: mvura. Mushure mekunge zvikamu zvaiswa muchando kwemaminitsi makumi matatu, sampuro dzakaiswa mu centrifuge pa 21,000 g kwemaminitsi gumi pa 4°C, uye supernatant yakaomeswa mu SpeedVac concentrator. Metabolite pellet yakaomeswa yakachengetwa pa -80°C kusvika yaongororwa.
Kuongororwa kweLiquid chromatography-mass spectrometry ye13 C-labeled amino acids. Pakuongorora kweliquid chromatography-mass spectrometry (LC-MS), metabolite pellet yakadzorerwa mu75μl yeLC-MS grade water (Honeywell). Mushure mekutenderera pa21,000 g kwemaminitsi mashanu pa4°C, 20 μl ye clearened supernatant yakashandiswa pakuongorora amino acid flux, nepo zvimwe zvakabviswa zvakashandiswa pakarepo pakuongorora anion (ona pazasi). Amino acid analysis yakaitwa uchishandisa benzoyl chloride derivatization protocol yakatsanangurwa kare (55, 56). Padanho rekutanga, 10μl ye100 mM sodium carbonate (Sigma-Aldrich) yakawedzerwa ku20μl ye metabolite extract, uye ipapo 10μl ye2% benzoyl chloride (Sigma-Aldrich) yakawedzerwa kuLC grade ACN. Sampuro yakabviswa mvura kwenguva pfupi yozoiswa mu centrifuge pa 21,000 g kwemaminitsi mashanu pa 20°C. Isa supernatant yakacheneswa mu 2 ml autosampler vial ine conical glass insert (200 μl volume). Sampuro dzakaongororwa uchishandisa Acquity iClass ultra-high performance LC system (Waters) yakabatana neQ-Exactive (QE)-HF (Ultra High Field Orbitrap) high-resolution precision mass spectrometer (Thermo Fisher Scientific). Kuti iongororwe, 2μl yesample yakabviswa yakapinzwa mu 100×1.0 mm high-strength silica T3 column (Waters) ine 1.8μm particles. Mwero wekuyerera kwemvura i100μl/min, uye buffer system ine buffer A (10 mM ammonium formate uye 0.15% formic acid mumvura) uye buffer B (ACN). Gradient ndeiyi inotevera: 0%B pa 0 maminitsi; 0%B. 0 kusvika 15% B pamaminitsi 0 kusvika 0.1; 15 kusvika 17% B pamaminitsi 0.1 kusvika 0.5; B pa17 kusvika 55% pamaminitsi 0.5 kusvika 14; B pa55 kusvika 70% pamaminitsi 14 kusvika 14.5; pa14.5 kusvika 70 kusvika 100% B pamaminitsi 18; 100% B pamaminitsi 18 kusvika 19; 100 kusvika 0% B pamaminitsi 19 kusvika 19.1; 0% B pamaminitsi 19.1 kusvika 28 (55, 56). Iyo QE-HF mass spectrometer inoshanda mu positive ionization mode ine huwandu hwe m/z (mass/charge ratio) ye50 kusvika 750. Iyo applied resolution i60,000, uye gain control (AGC) ion target yakawanikwa i3×106, uye nguva yepamusoro ye ion i100 milliseconds. Iyo heated electrospray ionization (ESI) source inoshanda pa spray voltage ye3.5 kV, capillary tembiricha ye250°C, sheath airflow ye60 AU (arbitrary units), uye assistant airflow ye20 AU. 250°C. Iyo S lens yakaiswa ku60 AU.
Kuongororwa kweAnion chromatography-MS kwe13C labeled organic acids. Yasara metabolite precipitate (55μl) yakaongororwa uchishandisa Dionex ion chromatography system (ICS 5000+, Thermo Fisher Scientific) yakabatana neQE-HF mass spectrometer (Thermo Fisher Scientific). Muchidimbu, 5μl yemetabolite extract yakapinzwa muDionex IonPac AS11-HC column yakagadzirwa neHPLC (2 mm×250 mm, particle size 4μm, Thermo Fisher Scientific) mu push-in partial loop mode ine filling ratio ye1. ) Dionex IonPac AG11-HC guard column (2 mm x 50 mm, 4μm, Thermo Fisher Scientific). Tembiricha yecolumn inochengetwa pa30°C, uye autosampler yakaiswa pa6°C. Shandisa potassium hydroxide cartridge ine deionized water kugadzira potassium hydroxide gradient kuburikidza ne eleent generator. Kupatsanurwa kwemametabolites pachiyero chekuyerera kwe380μl/min, uchishandisa gradient inotevera: 0 kusvika 3 maminitsi, 10 mM KOH; 3 kusvika 12 maminitsi, 10 kusvika 50 mM KOH; 12 kusvika 19 maminitsi, 50 kusvika 100 mM KOH; 19 kusvika 21 maminitsi, 100 mM KOH; 21 kusvika 21.5 maminitsi, 100 kusvika 10 mM KOH. Koramu yakadzokororwa pasi pe10 mM KOH kwemaminitsi 8.5.
Ma metabolites aya anosanganiswa ne 150μl/min isopropanol supplement stream mushure me column uye obva aendeswa kune high-resolution mass spectrometer inoshanda mu negative ionization mode. MS iri kutarisa huwandu hwemasasi kubva m/z 50 kusvika 750 ine resolution ye60,000. AGC yakaiswa pa 1×106, uye nguva yepamusoro ye ion inochengetwa pa 100 ms. Sosi yeESI inopisa yakashandiswa pa spray voltage ye 3.5 kV. Mamwe marongero e sosi ye ion ndeaya anotevera: capillary temperature 275°C; sheath gas flow, 60 AU; assistant gas flow, 20 AU pa 300°C, uye S lens setting kusvika 60 AU.
Kuongororwa kwedata remetabolites dzakanyorwa 13C. Shandisa software yeTraceFinder (vhezheni 4.2, Thermo Fisher Scientific) kuti uongorore data re isotope ratio. Kuzivikanwa kwekombuta yega yega kwakasimbiswa nekombuta yakavimbika uye yakaongororwa yakazvimirira. Kuti uite ongororo ye isotope enrichment, nzvimbo ye ion chromatogram (XIC) yakabviswa ye 13C isotope (Mn) yega yega yakatorwa kubva ku [M + H]+, uko n iri nhamba yekabhoni yekombuta yakatarwa, inoshandiswa kuongorora amino acids kana [MH] + inoshandiswa kuongorora anions. Kururama kwehuwandu hweXIC kuri pasi pezvikamu zvishanu pamiriyoni, uye kururama kweRT maminetsi 0.05. Kuongororwa kwe enrichment kunoitwa nekuverenga chiyero che isotope yega yega yakawanikwa nehuwandu hwe isotopes dzese dzekombuta inoenderana. Aya ma ratios anopiwa sehuwandu hwe percentage ye isotope yega yega, uye mhedzisiro yacho inoratidzwa se molar percentage enrichment (MPE), sezvakatsanangurwa kare (42).
Pellet ye neuron yakachengetwa mu 80% methanol (v/v) yakatonhora zvakanyanya, yakasanganiswa nemhepo, uye yakaiswa mu -20°C kwemaminitsi makumi matatu. Vhortex sampuro zvakare woikurunga pa +4°C kwemaminitsi makumi matatu. Sampuro yakaiswa mu centrifuge pa 21,000 g kwemaminitsi mashanu pa 4°C, uye supernatant yakabuda yakaunganidzwa yoomeswa uchishandisa SpeedVac concentrator pa 25°C kuti iongororwezve. Sezvakatsanangurwa pamusoro apa, LC-MS analysis yakaitwa pama amino acids emasero akarongwa. Uchishandisa TraceFinder (vhezheni 4.2, Thermo Fisher Scientific), ongororo yedata yakaitwa uchishandisa monoisotopic mass ye compound yega yega. Quantile normalization ye metabolite data yakaitwa uchishandisa preprocessCore software package (57).
Kugadzirira zvidimbu. Mbeva yakakurumidza kupiswa necarbon dioxide ndokugurwa musoro, uropi hwakakurumidza kubviswa mudehenya, uye banga rakazadzwa nechando (HM-650 V, Thermo Fisher Scientific, Walldorf, Germany) rakashandiswa kuricheka kuita zvikamu zve300 kusvika 375 μm sagittal. Cold carbon gasification (95% O2 uye 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 uye 6.0 mM MgCl2. Chinja kune pH 7.4 uye 310 kusvika 330 mOsm). Tumira zvidimbu zveuropi zvawanikwa mukamuri rine Ca2 + ACSF yakakwira (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 uye mM 3.5 MgCl2) pH 7.4 uye 310 kusvika 320 mOsm). Chengeta zvidimbu kwemaminitsi makumi maviri kusvika makumi matatu kuitira kuti zvigone kudzoreredzwa usati wanyora.
kurekodha. Danho remaikorosikopu rakagadzirwa nekamuri rekurekodha rakagadzikwa uye lenzi ye20x immersion mumvura (Scientifica) rakashandiswa pakurekodha kwese. Masero ePurkinje anofungidzirwa kuti akaonekwa ne (i) saizi yemuviri, (ii) nzvimbo yeanatomiki yecerebellum, uye (iii) kuratidzwa kwejini re reporter reflux mtYFP. Pipette yepatch ine resistance yetip ye5 kusvika 11 megohms inoburitswa ne borosilicate glass capillary (GB150-10, 0.86 mm×1.5 mm×100 mm, Science Products, Hofheim, Germany) uye horizontal pipette Instruments (P-1000, Sutter), Novato, CA). Kurekodha kwese kwakaitwa neELC-03XS npi patch clamp amplifier (npi electronic GmbH, Tam, Germany), iyo yaidzorwa nesoftware Signal (vhezheni 6.0, Cambridge Electronic, Cambridge, UK). Kuedza uku kwakarekodhwa pachiyero che12.5 kHz. Chiratidzo chinoseferwa nemafirita maviri eBessel ane mafrequencies e1.3 ne10 kHz zvichiteerana. Kukwanisa kwemembrane nepipette kunodzoserwa necircuit yekupa simba uchishandisa amplifier. Kuedza kwese kwakaitwa pasi pekutungamirirwa kwekamera yeOrca-Flash 4.0 (Hamamatsu, Gerden, Germany), iyo yaidzorwa nesoftware yeHokawo (vhezheni 2.8, Hamamatsu, Gerden, Germany).
Rongai magadzirirwo emasero ese uye kuongorora. Nguva pfupi usati wanyora, zadzai pipette nemhinduro yemukati ine zvinhu zvinotevera: 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) uye 10.0 mM creatinine phosphate zvakagadziriswa kuita pH 7.25, uye kumanikidzwa kweosmotic kwaive 290 mOsm (sucrose). Mushure mekushandisa simba re0 pA kuti membrane iparare, mukana we membrane yakasara wakayerwa. Kuramba kwekupinda kunoyerwa nekushandisa hyperpolarized currents ye -40, -30, -20, uye -10 pA. Yera hukuru hwemhinduro yevoltage uye shandisa mutemo weOhm kuverenga kuramba kwekupinda. Basa rekuzvidira rakanyorerwa mu voltage clamp kwemaminitsi mashanu, uye sPSC yakawanikwa ndokuyerwa muIgor Pro (vhezheni 32 7.01, WaveMetrics, Lake Oswego, Oregon, USA) uchishandisa semi-automatic recognition script. IV curve uye steady-state current zvinoyerwa nekubatanidza bhatiri pamatanho akasiyana (kutanga kubva -110 mV) uye kuwedzera voltage mumatanho mashanu mV. Kugadzirwa kweAP kwakaedzwa nekushandisa depolarizing current. Bata sero pa -70 mV uchishandisa depolarizing current pulse. Gadzirisa saizi yedanho reyuniti yega yega yekurekodha zvakasiyana (10 kusvika 60 pA). Verenga ma frequency epamusoro eAP nekuverenga nemaoko ma pulse spikes anokonzera AP frequency yakakwira. Muganho weAP unoongororwa nekushandisa derivative yechipiri ye depolarization pulse inotanga kukonzera imwe kana kupfuura APs.
Kugadziriswa uye kuongororwa kwechigamba chakaboorwa. Ita kunyorwa kwechigamba chakaboorwa uchishandisa maprotocol akajairwa. Shandisa pipette isina ATP neGTP isina zvinhu zvinotevera: 128 mM gluconate K, 10 mM KCl, 10 mM Hepes, 0.1 mM EGTA uye 2 mM MgCl2, uye gadzirisa ku pH 7.2 (uchishandisa KOH). ATP neGTP zvinobviswa mu intracellular solution kudzivirira kupindira kusingadzoreki kwe cell membrane. Patch pipette yakazadzwa ne amphotericin-containing internal solution (inenge 200 kusvika 250μg/ml; G4888, Sigma-Aldrich) kuti uwane punched patch record. Amphotericin yakanyungudutswa mu dimethyl sulfoxide (kuwanda kwekupedzisira: 0.1 kusvika 0.3%; DMSO; D8418, Sigma-Aldrich). Kuwanda kweDMSO yakashandiswa hakuna kukanganisa zvakanyanya ma neuron akadzidzwa. Munguva yekubaya, kuramba kwemugero (Ra) kwairamba kuchiongororwa, uye kuyedza kwacho kwakatangwa mushure mekunge amplitude yeRa neAP yagadzikana (maminetsi 20-40). Basa rinoitwa nega rega rinoyerwa muvoltage uye/kana current clamp kwemaminitsi 2 kusvika ku5. Kuongororwa kwedata kwakaitwa uchishandisa Igor Pro (vhezheni 7.05.2, WaveMetrics, USA), Excel (vhezheni 2010, Microsoft Corporation, Redmond, USA) uye GraphPad Prism (vhezheni 8.1.2, GraphPad Software Inc., La Jolla, CA). United States). Kuti uzive maAP anongoerekana aitika, plug-in yeIgorPro's NeuroMatic v3.0c inoshandiswa. Ziva maAP otomatiki uchishandisa threshold yakapihwa, iyo inogadziriswa yega yega kune rekodhi yega yega. Uchishandisa spike interval, sarudza spike frequency ine max instantaneous spike frequency uye avhareji spike frequency.
Kupatsanurwa kwePN. Nekuchinjika kuprotocol yakamboburitswa, maPN akacheneswa kubva kucerebellum yembeva padanho rakatarwa (58). Muchidimbu, cerebellum yakachekwa-chekwa ndokuchekwa muchimiro chechando-chando chekuparadzanisa [isina HBSS Ca2+ neMg2+, yakawedzerwa 20 mM glucose, penicillin (50 U/ml) uye streptomycin (0.05 mg/ml)], uye yozogaya chimiro mupapain [HBSS, yakawedzerwa 1-cysteine·HCl (1 mg/ml), papain (16 U / ml) uye deoxyribonuclease I (DNase I; 0.1 mg/ml)] Rapa kwemaminitsi makumi matatu pa30°C. Kutanga gezai matishu ari muchikamu cheHBSS chine manyoka emazai (10 mg/ml), BSA (10 mg/ml) uye DNase (0.1 mg/ml) patembiricha yemukamuri kudzivirira kugaya enzyme, uye wozopinda muchikamu cheHBSS chine 20 mM glucose. Zvinyoronyoro muHBSS, penicillin (50 U/ml), streptomycin (0.05 mg/ml) uye DNase (0.1 mg/ml) inoburitsa masero mamwechete. Kumiswa kwemasero kwakakonzerwa kwakaseferwa kuburikidza ne 70μm cell strainer, ipapo masero acho akaputirwa ne centrifugation (1110 rpm, 5 mins, 4°C) uye akadzorerwa muchikamu chekusarudza [HBSS, yakawedzerwa ne 20 mM glucose, 20% fetal bovine ) Serum, penicillin (50 U/ml) uye streptomycin (0.05 mg/ml)]; ongororai kushanda kwemasero ne propidium iodide uye gadzirisai density yemasero kusvika pa 1×106 kusvika pa 2×106 masero/ml. Pamberi pekuongorora flow cytometry, suspension yacho yaisefetwa kuburikidza ne 50 μm cell strainer.
Flow cytometer. Kuronga masero kwakaitwa pa4°C uchishandisa muchina weFACSAria III (BD Biosciences) uye FACSDiva software (BD Biosciences, vhezheni 8.0.1). Kumiswa kwemasero kwakarongwa uchishandisa 100 μm nozzle pasi pekumanikidzwa kwe20 psi pachiyero che ~2800 events/sec. Sezvo nzira dzechinyakare dzegating (hukuru hwemasero, bimodal discrimination, uye scattering characteristics) dzisingakwanise kuve nechokwadi chekuparadzaniswa kwakakodzera kwePN kubva kune mamwe marudzi emasero, nzira yegating inoiswa zvichibva pakuenzanisa zvakananga kweYFP intensity uye autofluorescence mu mitoYFP+ ​​​​uye control mitoYFP − Mice. YFP inofara nekuisa sample ne 488 nm laser line, uye chiratidzo chinowanikwa uchishandisa 530/30 nm band pass filter. Mu mitoYFP+ ​​​​mbeva, simba reRosa26-mitoYFP reporter gene rinoshandiswawo kusiyanisa zvidimbu zvemuviri we neuronal ne axon. 7-AAD inofadzwa ne laser yero ye561 nm uye yakaonekwa ne 675/20 nm bandpass filter kuti isaburitse masero akafa. Kuti iparadzanise ma astrocytes panguva imwe chete, kumiswa kwemasero kwacho kwakaiswa ACSA-2-APC, uye sampuro yakapiswa ne 640 nm laser line, uye 660/20 nm bandpass filter yakashandiswa kuona chiratidzo.
Masero akaunganidzwa akaputirwa ne centrifugation (1110 rpm, maminetsi mashanu, 4°C) ndokuchengetwa pa -80°C kusvika ashandiswa. Mbeva dzeMfn2cKO nevana vadzo vanoiswa muzvikamu pazuva rimwe chete kuderedza kusiyana kwemaitiro. Kuratidzwa kwedata reFACS nekuongorora kwakaitwa uchishandisa FlowJo software (FlowJo LLC, Ashland, Oregon, USA).
Sezvambotaurwa pamusoro apa (59), real-time PCR inoshandiswa kupatsanura DNA kubva kuma neuron akarongwa kuti pave nekuongororwa kwe mtDNA. Kugona kwemutsara uye kunzwisiswa kwemasero kwakaedzwa pakutanga nekushandisa qPCR pamasero akasiyana. Muchidimbu, unganidza 300 PN mu lysis buffer ine 50 mM tris-HCl (pH 8.5), 1 mM EDTA, 0.5% Tween 20 uye proteinase K (200 ng/ml) uye isa proteinase K (200 ng/ml) uye isa proteinase K (200 ng/ml) kwemaminitsi 120. Masero aya akaiswazve pa95°C kwemaminitsi gumi kuti ave nechokwadi chekuti proteinase K haishande zvachose. Uchishandisa TaqMan probe (Thermo Fisher) chaiyo kune mt-Nd1, mtDNA yakayerwa ne semi-quantitative PCR mu 7900HT Real-Time PCR system (Thermo Fisher Scientific). Sainzi, nhamba yekatarogu Mm04225274_s1), mt-Nd6 (Thermo Fisher Scientific, nhamba yekatarogu AIVI3E8) uye 18S (Thermo Fisher Scientific, nhamba yekatarogu Hs99999901_s1) majini.
Kugadzirira sampuro yeProteome. Nekudziisa mhinduro pa95°C kwemaminitsi gumi uye kuisa sonicating, mu lysis buffer [6 M guanidine chloride, 10 mM tris(2-carboxyethyl) phosphine hydrochloride, 10 mM chloroacetamide uye 100 mM tris-Lyse frozen neuron pellets muHCl]. PaBioruptor (Diagenode) kwemaminitsi gumi (30 seconds pulse / 30 seconds pause period). Sampuro yakaderedzwa 1:10 mu 20 mM tris-HCl (pH 8.0), yakasanganiswa ne 300 ng trypsin gold (Promega), uye yakachengetwa husiku hwese pa37°C kuti iwane kugaya kwakakwana. Pazuva rechipiri, sampuro yakaderedzwa centrifuge pa 20,000 g kwemaminitsi makumi maviri. Supernatant yakaderedzwa ne 0.1% formic acid, uye mhinduro yakabviswa munyu neStageTips yakagadzirwa nega. Sampuro yakaomeswa muSpeedVac instrument (Eppendorf concentrator plus 5305) pa45°C, uye peptide yakasungirirwa mu 0.1% formic acid. Sampuro dzese dzakagadzirwa panguva imwe chete nemunhu mumwe chete. Kuti vaongorore sampuro dze astrocyte, 4 μg desalted peptides dzakanyorwa ne tandem mass tag (TMT10plex, catalog number 90110, Thermo Fisher Scientific) ine peptide to TMT reagent ratio ye1:20. Pakunyora TMT, 0.8 mg ye TMT reagent yakasimudzwa mu 70 μl ye anhydrous ACN, uye peptide yakaomeswa yakasanganiswa kuita 9 μl ye 0.1 M TEAB (triethylammonium bicarbonate), iyo 7 μl ye TMT reagent mu ACN yakawedzerwa. Kuwanda kwacho kwaive 43.75%. Mushure memaminitsi makumi matanhatu ekuiswa muchikamu chemvura, mhinduro yacho yakamiswa ne2 μl ye5% hydroxylamine. Mapeptides ane mazita akaunganidzwa, akaomeswa, akasungirirwazve mu200μl ye0.1% formic acid (FA), akakamurwa kuita maviri, uye akazobviswa munyu uchishandisa StageTips yakagadzirwa nemaoko. Uchishandisa UltiMate 3000 ultra high performance liquid chromatograph (UltiMate 3000 ultra high performance liquid chromatograph), imwe yehafu mbiri yakakamurwa pachikamu che1mm x 150mm Acquity chromatographic column yakazadzwa ne130Å1.7μm C18 particles (Waters, catalog No. SKU: 186006935). Thermo Fisher Scientific). Paradzanisa mapeptides padanho rekuyerera kwe30μl/min, patsanura kubva pa1% kusvika 50% buffer B kwemaminitsi 85 ne stepwise gradient yemaminitsi 96, kubva pa50% kusvika 95% buffer B kwemaminitsi 3, wozotevera maminitsi 8 e 95% Buffer B; Buffer A i5% ACN uye 10 mM ammonium bicarbonate (ABC), uye buffer B i80% ACN uye 10 mM ABC. Unganidza zvikamu mumaminitsi matatu ega ega wozvisanganisa mumapoka maviri (1 + 17, 2 + 18, nezvimwewo) wozviomesa mu vacuum centrifuge.
Kuongororwa kweLC-MS/MS. Pakuongorora mass spectrometry, mapeptide (nhamba r119.aq) akaparadzaniswa pachikamu chekuongorora chePicoFrit che 25 cm, 75 μm dhayamita yemukati (lenzi itsva, nhamba yechikamu PF7508250) chakashongedzerwa ne 1.9 μm ReproSil-Pur 120 C18-AQ medium (Dr. Maisch, mat), Shandisa EASY-nLC 1200 (Thermo Fisher Scientific, Germany). Chikamu chacho chakachengetedzwa pa 50°C. Mabuffers A naB 0.1% formic acid mumvura uye 0.1% formic acid mu 80% ACN, zvichiteerana. Mapeptides akaparadzaniswa kubva pa 6% kusvika 31% buffer B kwemaminitsi makumi matanhatu nemashanu uye kubva pa 31% kusvika 50% buffer B kwemaminitsi mashanu ne gradient ye 200 nl/min. Mapeptides aya akaongororwa paOrbitrap Fusion mass spectrometer (Thermo Fisher Scientific). Kuyerwa kwePeptide precursor m/z kunoitwa ne resolution ye120,000 iri pakati pe350 kusvika 1500 m/z. Uchishandisa 27% normalized collision energy, precursor yakasimba ine charge state ye2 kusvika 6 inosarudzwa kuti pave ne high energy C trap dissociation (HCD) cleavage. Nguva yekutenderera yakaiswa pa 1 s. Kukosha kwe m/z kwe peptide fragment kwakayerwa mu ion trap uchishandisa diki AGC target ye 5×104 uye nguva yepamusoro ye injection ye 86 ms. Mushure mekupatsanurwa, precursor yakaiswa pane dynamic exclusion list kwe 45 s. Mapeptide ane TMT-labeled akaparadzaniswa pa 50 cm, 75 μm Acclaim PepMap column (Thermo Fisher Scientific, catalog number 164942), uye migration spectra yakaongororwa pa Orbitrap Lumos Tribrid mass spectrometer (Thermo Fisher Scientific) yakashongedzerwa ne high-field asymmetric waveform ions (FAIMS) equipment (Thermo Fisher Scientific) inoshanda pa consolidation voltages mbiri dze −50 uye −70 V. MS3 yakasarudzwa zvichibva pane synchronization precursor inoshandiswa pakuyera TMT report ion signal. Kupatsanurwa kwepeptide kwakaitwa paEASY-nLC 1200, uchishandisa 90% linear gradient elution, ine buffer concentration ye 6% kusvika 31%; buffer A yaive 0.1% FA, uye buffer B yaive 0.1% FA uye 80% ACN. Analytical column inoshandiswa pa50°C. Shandisa FreeStyle (vhezheni 1.6, Thermo Fisher Scientific) kupatsanura faira rekutanga zvichienderana nemagetsi ekubhadhara eFAIMS.
Kuzivikanwa kwemapuroteni uye kuverengwa kwehuwandu. Uchishandisa injini yekutsvaga yeAndromeda yakabatanidzwa, data rekutanga rakaongororwa uchishandisa MaxQuant version 1.5.2.8 (https://maxquant.org/). Pamusoro peCre recombinase uye YFP sequences dzakawanikwa kubva kuAequorea victoria, peptide fragment spectra dzakatsvakwa canonical sequence uye isoform sequence ye mouse reference proteome (Proteome ID UP000000589, yakatorwa kubva kuUniProt muna Chivabvu 2017). Methionine oxidation uye protein N-terminal acetylation zvakaiswa se variable modifications; cysteine ​​​​carbamoyl methylation yakaiswa se fixed modifications. Ma digestion parameters akaiswa ku "specificity" uye "trypsin/P". Nhamba shoma yepeptides uye razor peptides dzinoshandiswa pakuziva mapuroteni i1; nhamba shoma yepeptides dzakasiyana i0. Pasi pemamiriro ekufananidza peptide map, chiyero chekuzivikanwa kwemapuroteni chaive 0.01. Sarudzo ye "Second Peptide" inogoneswa. Shandisa sarudzo ye "match between runs" kuti utamise ruzivo rwakabudirira pakati pemafaira epakutanga akasiyana. Shandisa LFQ minimum ratio count 1 yekuenzanisa kusina label (LFQ) (60). Kusimba kweLFQ kunoseferwa kune kanenge ma values ​​maviri anoshanda muboka rimwe chete re genotype panguva imwe neimwe, uye kunotorwa kubva pakugoverwa kwakajairika kune upamhi hwe. 0.3 uye kudzika pasi 1.8. Shandisa Perseus computing platform (https://maxquant.net/perseus/) uye R (https://r-project.org/) kuongorora mhinduro dzeLFQ. Bvunzo ye two-way moderate t kubva ku limma software package yakashandiswa pakuongorora kwe differential expression (61). Kuongorora data rekutsvaga kunoitwa uchishandisa ggplot, FactoMineR, factoextra, GGally uye pheatmap. Data re TMT-based proteomics rakaongororwa uchishandisa MaxQuant version 1.6.10.43. Tsvaga data reproteomics kubva kuUniProt's human proteomics database, iro rakatorwa muGunyana 2018. Ongororo iyi inosanganisira isotope purity correction factor yakapihwa nemugadziri. Shandisa limma muR kuti uongorore differential expression. Data rekutanga, mhedzisiro yekutsvaga database, uye workflow yekuongorora data nemigumisiro zvese zvakachengetwa muProteomeXchange alliance kuburikidza nePRIDE partner repository ine data set identifier PXD019690.
Kutsanangura kwekushanda kunovandudza ongororo. Chishandiso cheIngenuity Pathway Analysis (QIAGEN) chakashandiswa kuona kupfuma kwemashoko ekutsvaga kwekushanda kwedata rakaiswa pamavhiki masere (Mufananidzo 1). Muchidimbu, runyorwa rweprotein quantitative rwakawanikwa kubva kuLC-MS/MS (tandem mass spectrometry) data analysis rinoshandiswa nematanho anotevera efilter: Mus musculus inosarudzwa semhando uye background, uye chikamu chinoratidza kuti P value yakagadziriswa naBenjamini yekuwedzera 0.05 kana pasi inoonekwa seyakakosha. Pagirafu iyi, mapoka mashanu epamusoro akawandisa muboka rega rega zvichibva paP value yakagadziriswa anoratidzwa. Uchishandisa multiple t-test, uchishandisa two-stage linear boost program yeBenjamini, Krieger, naYekutieli (Q = 5%), time-course protein expression analysis inoitwa pavanhu vakakosha vakawanikwa muchikamu chimwe nechimwe, uye mutsetse wega wega unoongororwa zvakasiyana. Hapana chikonzero chekushandisa SD inoenderana.
Kuti tienzanise mhedzisiro yechidzidzo ichi nemadhatabhesi akabudiswa uye kugadzira dhayagiramu yeVenn muMufananidzo 1, takabatanidza runyorwa rweprotein yehuwandu neMitoCarta 2.0 annotations (24). Shandisa chishandiso chepamhepo chinonzi Draw Venn Diagram (http://bioinformatics.psb.ugent.be/webtools/Venn/) kugadzira dhayagiramu.
Kuti uwane rumwe ruzivo rwakadzama nezvemaitiro ehuwandu hwe ...
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NaE. Motori, I. Atanassov, SMV Kochan, K. Folz-Donahue, V. Sakthivelu, P. Giavalisco, N. Toni, J. Puyal, N.-G. Larson
Kuongororwa kweproteomics kwema neuron asina kushanda zvakanaka kwakaratidza kuti mapurogiramu ekugadzirisa mashandiro emuviri anoshandiswa kurwisa kuora kwetsinga.
NaE. Motori, I. Atanassov, SMV Kochan, K. Folz-Donahue, V. Sakthivelu, P. Giavalisco, N. Toni, J. Puyal, N.-G. Larson
Kuongororwa kweproteomics kwema neuron asina kushanda zvakanaka kwakaratidza kuti mapurogiramu ekugadzirisa mashandiro emuviri anoshandiswa kurwisa kuora kwetsinga.
©2020 American Association for the Advancement of Science. kodzero dzose dzakachengetedzwa. AAAS ishamwari yeHINARI, AGORA, OARE, CHORUS, CLOCKSS, CrossRef uye COUNTER. ScienceAdvans ISSN 2375-2548.