Lino *Kkeyala yamazubaano: Cologne 50931, Germany, Cologne Buvwuntauzi Bwakuchita Kabunga Kabotu Kujatikizya Malwazi Aajatikizya Kuchembaala (CECAD).
Kubola kwabongo kwamalwazi aamubulowa bwamumubili kulabonwa kuti takukonzyi kujokolosyegwa nkaambo kuchincha kwatunsingo mumubili nkusyoonto, asi mpindu yakubula nguzu kwamizeezo mumubili takuzyibidwe kabotu pe. Aawa, twatalisya maseelo aa Purkinje neurons aabulwazi bwa OXPHOS buboola akaambo kakunyongana kwa mitochondrial fusion dynamics. Twakajana kuti kutabeleka kabotu kwa mitochondrial kwakapa kuti kube kucinca kupati mumulimo wa proteomics, kumamanino kwakapa kuti kube kubeleka kwazibeela zya metabolic kakutanafwa maseelo. Kakunyina kulangila, twakajana kuti kuli kuyungizigwa kwa pyruvate carboxylase (PCx) azimwi peroxidases zijatikizyidwe akuyungizya TCA cycle intermediates. Kulesyegwa kwa PCx kwakayungizya kukatazyigwa kwa oxidative antoomwe akubola kwabongo, kutondezya kuti bulwazi bwakuyuma kwamitsinga bulaanguzu zyakukwabilila mubongo butakwe OXPHOS. Kubukulusya kuswaangana kwa mitochondrial muma neurons aamana kumana kuchincha bube oobu bwa metabolic, aboobo kulesya lufu lwamaseelo. Zyintu nzyotwajana zitondezya nzila zitazyibidwe kaindi zipa kuti mitochondrial itabeleki kabotu alimwi zitondezya kuti kubola kwabongo kulakonzya kujokolosyegwa nokuba kumamanino aabulwazi.
Mulimu mupati wa mitochondria mukubamba manguzu aamubongo ulakankaizyigwa azitondezyo zyiingi zyabongo zijatikizyidwe amalwazi aa mitochondria yabantu. Bunji bwamalwazi aaya aletwa akusanduka kwamajini aayeendelezya zitondezyo zyamajini aamubulowa (1, 2) naa kunyonyoonwa kwamajini aajatikizya mbozibeleka zintu mubulowa, eezyo zijatikizya kukkalikana kwa DNA (mtDNA) (3, 4). Mulimo muzikozyanyo zyabanyama wakatondezya kuti mukwiingula kutabeleka kabotu kwa mitochondrial muzibeela zyanyama zizingulukide, nzila zya metabolic zikwabilila (5-7) zilakonzya kubelesegwa, aboobo kupa luzibo lupati kujatikizya nzila zyakutalisya malwazi aaya mayumu. Mukwiimpana, kumvwisisisya kwesu kwakusanduka kwamaseelo amwi akaambo kakulezya kwakupanga adenosine triphosphate (ATP) mubongo nchintu chipati (8), kukankaizya mbokuyandika kujana nzila zyakusilikwa zikonzya kubelesegwa kukwabilila naa kukwabilila malwazi. Kulesya kubola kwabongo (9). Kubula makani nkakasimpe kakuti maselu aamunsinga alabonwa kuti taakwe nguzu zyakusanduka zyakulya kweelanisigwa amaseelo aazingulukide (10). Twaambilizya kuti maselu aaya alabeleka mulimo mupati mukubamba nzila zyakutola ma metabolites ku neurons kuchitila kuti asumpule kusanduka kwa synaptic akusandula kukulichisa amalwazi, inguzu zyakucinca metabolism yamaseelo kuti zibelesyegwe mubukkale bukatazya bwazibeela zyabongo zili buyo aamaselu aa glial (11-14). Mukuyungizya, kusiyana kwamaseelo aamubongo kulalesya chiiyo chakusanduka kwazyakulya kuchitika muzibeela zimwi zyabongo. Mukujana mpindu, mbichani chizibinkene aatala azintu zikonzya kuchitika akaambo kakutabeleka kabotu kwama mitochondrial muma neurons.
Kuti tumvwisisisye zyintu zikonzya kuchitika akaambo kakutabeleka kabotu kwamaseelo, twakaandaanya ma Purkinje neurons (PNs) mumasena aasiyene-siyene aakunyonyooka kwamizeezo akaambo kakunyonyooka kwamaseelo aanze (Mfn2). Nikuba kuti kusanduka kwa Mfn2 mubantu kulaswaangene abulwazi bwamubili buzyibidwe kuti Charcot-Marie-Tooth type 2A (15), kunyonyoonwa kwa Mfn2 mu mbeba nchintu chizyibidwe loko chakutalisya nzila ya oxidation Phosphorylation (OXPHOS) dysfunction method. Misyobo yiindene-indene yabongo (16-19) antoomwe abukkale bwakubula nguzu bwabongo buboola akaambo kazeezi zilatobelwa azitondezyo zyabongo ziyaambele, mbuli kubula nguzu kweenda (18, 19) naa kutabeleka kabotu kwabongo (16). Kwiinda mukubelesya kusanganya kwa label-free quantitative (LFQ) proteomics, metabolomics, imaging, anzila zya virological, tutondezya kuti kuyaambele kwakubola kwabongo kulasungwaazya pyruvate carboxylase (PCx) azintu zimwi zijatikizyidwe mu arteriosclerosis ya PNs in vivo Kutondezya ma enzymes. Kusinizya kweendelana kwakujana ooku, twakacheesya kutondezya kwa PCx mu Mfn2-deficient PNs, mpawo twakajana kuti ooku kuchita kwakayungizya kukatazyigwa kwa oxidative akufwambaanya kubola kwabongo, aboobo kutondezya kuti azoospermia ilapa lufu lwamaseelo Metabolic adaptability. Kuyungizya kwa MFN2 kulakonzya kuvwuna PN iimana akubula OXPHOS, kubelesya DNA ya mitochondrial mpati, antoomwe akutyoka kwa mitochondrial network, eezyo zikankaizya kuti nzila eeyi yakubola kwabongo ilakonzya kupona muchiimo chipati chabulwazi kakutanafwa maseelo.
Kuti tubone mitochondria mu Mfn2 knockout PNs, twakabelesya musyobo wa mbeba uuzumizya Cre-dependent mitochondria kuti ichite ma protein aasalala pyu (YFP) (mtYFP) (20) Cre expression mpawo twakalanga mbuli mitochondrial morphology in vivo. Twakajana kuti kunyonyoonwa kwa Mfn2 gene mu PNs inga kwasololela kukuandaana kwa mitochondrial network (Chitondeezyo S1A), mpawo kusanduka kutaanzi kwakajanwa amviki zyotatwe zyakuzyalwa. Mukwiimpana, kubola kupati kwa PN cell layer, mbuli mbokutondezegwa akusweekelwa kwa Calbindin immunostaining, tiikwakatalika kusikila kumviki zili kkumi azibili zyakuzyalwa (Chitondeezyo 1, A a B). Kutamvwana kwachiindi aakati kakusanduka kwakusaanguna kwabupange bwama mitochondria akutalika kwalufu lwa neuron kwakatukulwaizya kuti tuvwuntauzye kuchincha kwa metabolic kwakachitwa akutabeleka kabotu kwa mitochondria kaitanafwa maseelo. Twakabamba nzila ya fluorescence-activated cell sorting (FACS)-based isolate YFP (YFP+)-expressing PN (Figure 1C), alimwi amumbeba zyakweendelezya (Mfn2 + / loxP :: mtYFP loxP- stop-loxP: : L7-cre), lino zyaambwa kuti CT CT (Figure S1BRL). Kubambulula nzila yakujala kweendelanya anguzu zya YFP kutuzumizya kusalazya mubili wa YFP+ (YFPhigh) wa PNs kuzwa kuli baabo batali ba PNs (YFPneg) (Chitondeezyo S1B) naakuti zibeela zya axon/dendritic ziyeeyelwa kuti zili mumuni (YFPlow; Cifwanikiso S1D, kulumwensyi), zyakasinizyigwa a confocal microscope (Cifwanikiso S1D, kululyo). Kuti tusinizye mweelwe wabantu bakabikkwa muzibeela, twakachita LFQ proteomics mpawo twakachita kuvwuntauzya kwazibeela zipati, mpawo twakajana kuti kuli kwaandaana kulibonya aakati ka YFPhigh a YFPneg maselu (Chitondeezyo S1C). Maseelo aa YFPhigh akatondezya kuvwuba kwama PNs aazyibidwe (nkokuti Calb1, Pcp2, Grid2 a Itpr3) (21, 22), pesi taakwe kuvwuzya kwama protein aajanika muma neurons naakuti mumaseelo aambi (Chitondeezyo 1D) ). Kweezyanisya aakati kamasampu mumaseelo aa YFPhigh aakabungikwa mumasunko aalikke kwakatondezya kuti kuli kweendelana> 0.9, kutondezya kuzyalana kubotu aakati kazyintu zipona (Chitondeezyo S1E). Muchi chifwiifwi, data eeyi yakasinizya bubambe bwesu bwakugwisya PN iikonzya kuchitwa. Nkaambo kakuti L7-cre driver system yakabelesegwa ilasungwaazya kuswaangana kwa mosaic mumviki yakusaanguna kuzwa nikwakazyalwa (23), twakatalika kujaya mbeba kuzwa ku CTRL antoomwe a conditional (Mfn2 loxP / loxP :: mtYFP loxP-stop-loxP :: L7-cre) Collect neurons. Kuswaanganya kwamana, kwiitwa kuti Mfn2cKO amviki zyone zyakuzyalwa. Mbuli mamanino, twakasala nsondo zili 8 zyakuzyalwa aawo PN niyakachili kabotu nikuba kuti kwakali zibeela zya mitochondrial (Chitondeezyo 1B aChitondeezyo S1A). Mukuvwula, twakabala mweelwe wa ma protein aali 3013, aali 22% akali kweendelana a MitoCarta 2.0 annotations kweendelana a mitochondrial proteome mbuli mitochondria (Chitondeezyo 1E) (Chitondeezyo 1E) (24). Kuvwuntauzya kwakusiyana kwama gene kwakachitwa mumviki ya 8 kwakatondezya kuti buyo 10.5% yamapuloteni ngaakajisi kusanduka kupati (Mufanikisyo 1F aMufanikisyo S1F), oomo mapulotini ali 195 akacheya mpawo ali 120 akasumpulwa (Chitondeezyo 1F). Nchintu chibotu kuzyiba kuti "kuvwuntauzya kwanzila mpya" ya data eeyi itondezya kuti ma genes aatondezyedwe munzila ziindene-indene ngaajanika munzila zimwi zya metabolic (Chitondeezyo 1G). Chakugambya, nikuba kuti kucesya kwanzila zijatikizyidwe a OXPHOS a calcium signaling kusinizya kutalika kwakubula kubeleka kwa mitochondrial mu PNs zitakwe fusion, zimwi zibeela zijatikizya amino acid metabolism zilasumpulwa kapati, eezyo zyeendelana a metabolism iichitika mu mitochondrial PNs. Kujokezya ntambo nchintu chizumanana. kutabeleka kabotu.
(A) Zifwoto zitondezya zibeela zyabongo zya CTRL antoomwe a Mfn2cKO mbeba zitondezya kusweekelwa kwa PNs (calbindin, gray); nuclei zyakabikkwa DAPI. (B) Kuvwuntauzya kwa (A) (kuvwuntauzya kwakusiyana kwanzila yomwe, ***P<0.001; n = 4 kusikila ku 6 zikobela kuzwa ku mbeba zyotatwe). (C) Nzila yakubeleka yakusola. (D) Kwaabila kwamaapu yakupya kwazitondezyo zya Purkinje (atala) amisyobo imwi yamaseelo (akati). (E) Cifwanikiso cha Venn chitondeezya mweelwe wama puloteni aa mitochondrial aajanika mu PN yakabikkidwe. (F) Chitondeezyo cha volcano cha ma protein aatondezyedwe munzila yiindene mu Mfn2cKO neurons mumviki zili 8 (mpindu ya 1.3). (G) Buvwuntauzi bwanzila zyakulenga butondezya nzila zili musanu ziyandikana loko zyakusumpula (mubala uusalala pyu) azyaansi (mubala wa bbuluu) mu Mfn2cKO PN yakabikkwa mumviki zili 8. Mweelwe waakati-kati wakutondezya kwa protein imwi aimwi yakajanwa utondezyedwe. Maapu yakupya kwa grayscale: mweelwe wa P wakabambululwa. ns, taziyandiki pe.
Data ya Proteomics yakatondeezya kuti zitondezyo zya ma protein zya complexes I, III, a IV zyakaselemuka asyoonto-asyoonto. Zibeela I, III, antoomwe a IV zyoonse zyakalijisi zibeela ziyandikana zya mtDNA, mpawo zibeela II, zyakali buyo a nuclear-coded, tiizyakali kujatikizyigwa pe (Chitondeezyo 2A aChitondeezyo S2A). . Kweendelana azyakazwa mu proteomics, immunohistochemistry yazibeela zyanyama zyabongo yakatondezya kuti MTCO1 (mitochondrial cytochrome C oxidase subunit 1) subunit level of complex IV mu PN yakaselemuka asyoonto-syoonto (Chitondeezyo 2B). MtDNA-encoded subunit Mtatp8 yakacheya loko (Chitondeezyo S2A), mpawo steady-state level ya nuclear-encoded ATP synthase subunit tiiyakachincha pe, eezi zyeendelana a stable ATP synthase subassembly F1 complex aawo mtDNA noili kabotu. Bupange bulazumanana. Konyonganya (7). Kulangisisya mweelwe wa mtDNA mu Mfn2cKO PNs zyakasalwa kwiinda mukubelesya nzila ya real-time polymerase chain reaction (qPCR) kwakasinizya kucheya kwamweelwe wa mtDNA. Kweezyanisya akabunga kakweendelezya, amviki zili 8 zyakuzyalwa, buyo 20% ya mtDNA level yakazumanana (Chitondeezyo 2C). Kweendelana azintu eezyi zyajanwa, kuvwuntauzya kwa Mfn2cKO PNs kwiinda mukubelesya nzila ya confocal microscopy kuti ibone DNA kutondezya kubelesya kwa mitochondrial nucleotides kweendelana achiindi (Chitondeezyo 2D). Twakajana kuti buyo bamwi bakali kujatikizyidwe mukunyonyoona ma protein aamu mitochondrial akusanduka kwakukataazikana bakayungizigwa, kubikkilizya a Lonp1, Afg3l2 a Clpx, antoomwe a OXPHOS complex assembly factors. Taakwe kucinca kupati kwakajanwa muma protein aajatikizyidwe mu apoptosis (Chitondeezyo S2B). Mbubwenya buyo, twakajana kuti mitochondria anzila zya endoplasmic reticulum zijatikizyidwe mukutola calcium zili buyo akucinca kusyoonto (Chitondeezyo S2C). Mukuyungizya, kulanga-langa ma protein aajatikizya kulya kwamubili tiikwakajana kucinca kupati pe, ooko kweendelana akuboneka kwa autophagosomes kwakabonwa mumubili kwiinda mukubelesya immunohistochemistry antoomwe a electron microscopy (Chitondeezyo S3). Nokuba boobo, kutabeleka kabotu kwa OXPHOS mu PNs kweendelana akuchincha kwa mitochondrial. Tubunga twama mitochondrial tulakonzya kubonwa mumibili yamaseelo amusamu wa dendritic wa Mfn2cKO PNs uujisi myaka yakuzyalwa iili 5 a 8, mpawo bupange bwa membrane bwamukati bwakachincha kapati (Chitondeezyo S4, A a B). Kweendelana akuchincha ooku kwa ultrastructural akuseluka kupati kwa mtDNA, kuvwuntauzya kwa acute cerebral cerebellar slices a tetramethylrhodamine methyl ester (TMRM) kwakatondezya kuti nguzu zya mitochondrial membrane mu Mfn2cKO PNs zyakali kuseluka kapati (Chitondeezyo S4C).
(A) Kuvwuntauzya kwachiindi kwakutondezya kwa OXPHOS complex. Koyeeya buyo ma protein ajisi P<0.05 mumviki zili 8 (two-way ANOVA). Muzila uulaa madoti: Kunyina kuchincha kweelanisigwa a CTRL. (B) Kulumwensyi: Chikozyano chacibeela chabongo chakalembedwe anti-MTCO1 antibody (chipimyo, 20 μm). Busena buzwide maselu aa Purkinje bulivwumbidwe aamubala wamubala wamubala wa yellow. Kululyo: Kupima mweelwe wa MTCO1 (kuvwuntauzya kwakusiyana kwanzila yomwe; n = 7 kusikila ku 20 maselu aakavwuntauzyigwa kuzwa ku mbeba zyotatwe). (C) qPCR kuvwuntauzya kwanamba ya mtDNA mu PN yakasalwa (kuvwuntauzya kwakusiyana kwanzila yomwe; n = 3 kusikila ku 7 mbeba). (D) Kulumwensyi: Chikozyano chachibeela cha boongo chilembedwe a anti-DNA antibody (chipimyo, 20 μm). Busena buzwide maselu aa Purkinje bulivwumbidwe aamubala wamubala wamubala wa yellow. Kululyo: Kupima zilonda zya mtDNA (kuvwuntauzya kwakusiyana kwanzila yomwe; n = 5 kusikila ku 9 maselu kuzwa ku mbeba zyotatwe). (E) Chikozyano chacibeela chabongo chakufwambaana chitondezya maseelo aa mitoYFP + Purkinje (musemo) mukulekkooda kwamaseelo woonse. (F) Kupima kwa IV curve. (G) Kulekkooda kwakugwisya manguzu mumaseelo aa CTRL antoomwe a Mfn2cKO Purkinje. Chitondeezyo chakujulu: Kuzungaana kutaanzi kwakatalisya AP. Chitondeezyo chaansi: Mweelwe mupati wa AP. (H) Kupima mweelwe wazyintu zitalibonyi (sPSPs). Chitondeezyo chakulekkooda chiiminina antoomwe akweezyanisya kwacho zitondezyedwe muli (I). Kuvwuntauzya kwakusiyana kwanzila yomwe kwakavwuntauzya n = 5 kusikila ku 20 maselu kuzwa ku mbeba zyotatwe. Data itondezyedwe mbuli mweelwe±SEM; *P<0.05; **P<0.01; ***P<0.001. (J) Zitondeezyo zya AP zyakachitika biyo zyakalembwa kubelesya nzila ya perforated patch clamp mode. Mulongo wakujulu: Mweelwe mupati wa AP. Chitondeezyo chaansi: kuvwungulula kwa AP yomwe. (K) Bala mweelwe waakati aakati amweelwe mupati wa AP kweendelanya a (J). Musunko wa Mann-Whitney; n = 5 maselu akavwuntauzyigwa kuzwa ku mbeba zyone. Data itondezyedwe mbuli mweelwe±SEM; taziyandiki pe.
Kunyonyooka kwa OXPHOS kwakajanwa mu 8-week-old Mfn2cKO PN, kutondezya kuti mulimo wa physiological wa neurons tauli kabotu pe. Aboobo, twakavwuntauzya bube bwamagesi butabeleki bwa OXPHOS-deficient neurons mumviki zili 4 kusikila kuli 5 alimwi amvwiki zili 7 kusikila kuli 8 kwiinda mukulekkooda maseelo woonse muzibeela zya cerebellar (Chitondeezyo 2E). Chakutalangilwi, mweelwe waakati-kati wakulyookezya kwa membrane akukazyigwa kwa Mfn2cKO neurons kwakalikozyene akweendelezya, nikuba kuti kwakali kwiimpana kusyoonto aakati kamaseelo (Table 1). Mbubwenya buyo, kumviki zili 4 kusikila kuli 5 zyakuzyalwa, taakwe kucinca kupati kwakajanwa muchilongwe chamagesi-anguzu (IV curve) (Chitondeezyo 2F). Nokuba boobo, taakwe Mfn2cKO neurons zya 7 kusikila ku 8 nsondo zyakapona kuzwa ku IV regimen (ntaamu ya hyperpolarization), kutondezya kuti kuli kumvwisisisya ku hyperpolarization muchiindi eechi. Mukwiimpana, mu Mfn2cKO neurons, i depolarizing currents iipa kuti kube kuzwa kwanguzu zyakubeleka (AP) zilazuminwa obotu, kutondezya kuti nzila zyabo zyoonse zyakuzwa taziindene kapati azyeezyo zya 8-week-old control neurons (Table 1 and Figure 2G). Mbubwenya buyo, mweelwe abwaanda bwakuzwa mumubili (sPSCs) zyakali kwelanisigwa azyeezyo zyakabunga kakweendelezya, mpawo mweelwe wazintu zyakali kuchitika wakatanta kuzwa kumviki zyone kusika kumviki zili musanu kusikila kumviki zili musanu atubili kusikila kumviki zili musanu atutatu kakuli kuyungizigwa kukozyenye (Mufanikisyo 2, H a I). Chiindi chakukomena kwa synaptic mu PNs (25). Izyazwa zikozyania zyakajanwa musule aa PNs zyakabikkwa mabwe. Ooku kubamba kulesya kubambululwa kwatumpenda twa ATP mumaseelo, mbuli mbokukonzya kuchitika mukulekkooda kwamaseelo woonse. Mubwini, nguzu zya membrane yakulyookezya antoomwe akufwambaana kwakuzwa kwa Mfn2cKO neurons tiizyakajatikizyigwa pe (Chitondeezyo 2, J a K). Muchi chifwiifwi, eezyi mpindu zitondezya kuti ma PNs ajisi kutabeleka kabotu kwa OXPHOS alakonzya kuliyumya kabotu anzila zyakuzwa kwachiindi chilamfu, kutondeezya kuti kuli nzila yakugwasya iikonzya kubagwasya kuti kabazumanana kuchita zintu zili afwaafwi.
Data itondezyedwe mbuli mweelwe ± SEM (kulanga-langa kwakusiyana kwanzila yomwe, Holm-Sidak's multiple comparison test; *P<0.05). Inamba ya yuniti itondezyedwe mumabala.
Twakatalika kuyanduulisisya kuti naa kuli chibaka chili mu dataset ya proteomics (Chitondeezyo 1G) chibikkilizya nzila zikonzya kulwana kubula OXPHOS, aboobo kupandulula kaambo PN iijatikizyidwe ncoikonzya kubamba electrophysiology iili afwaafwi (Chitondeezyo 2, E kusikila ku K). . Kuvwuntauzya kwa Proteomics kwakatondezya kuti ma enzymes aajatikizyidwe mukusanduka kwa branched chain amino acids (BCAA) akasumpulwa kapati (Chitondeezyo 3A a Cifwanikiso S5A), mpawo acetyl-CoA (CoA) naakuti succinyl CoA ilakonzya kuyungizya ma tricarboxylates mu arteriosclerosis Acid (TCA) cycle. Twakajana kuti BCAA transaminase 1 (BCAT1) antoomwe a BCAT2 zyoonse zyakatanta. Bakasumpula ntaamu yakusaanguna ya BCAA catabolism kwiinda mukupanga glutamate kuzwa ku α-ketoglutarate (26). Tubeela toonse tupangwa aama branched chain keto acid dehydrogenase (BCKD) complex tulasumpulwa (eeyi complex ilagwasya kugwisigwa kwa BCAA carbon skeleton) (Chitondeezyo 3A aChitondeezyo S5A). Nokuba boobo, taakwe kucinca kulibonya mu BCAA kwakajanika mu PN yakasalwa, eecho chikonzya kuba akaambo kakuyungizya kutambula ma amino acid aaya aayandika naakuti kubelesya zimwi zintu (glucose naa lactic acid) kuyungizya TCA cycle (Chitondeezyo S5B). PNs zitakwe OXPHOS azyalo zyakatondezya kuyungizigwa kwakubola kwa glutamine akubeleka kwa transamination kumviki zili 8 zyakuzyalwa, eezyo zinga zyatondezyegwa akutanta kwa mitochondrial enzymes glutaminase (GLS) antoomwe a glutamine pyruvate transaminase 2 (GPT2) (Chitondeezyo 3, A a C). Nchintu chibotu kuzyiba kuti kusumpuka kwa GLS kuli buyo a isoform glutaminase C (GLS-GAC) (kusanduka kwa Mfn2cKO/CTRL kuli afwaafwi ziindi zili 4.5, P = 0.05), mpawo kusumpuka kwayo muzibeela zya kkansa kulakonzya kugwasya mitochondrial bioenergy. (27).
(A) Maapu yakupya itondezya kuchincha kwama protein munzila yakaambwa mumviki zili 8. (B) Chikozyano chachibeela cha boongo chilembedwe a anti-PCx antibody (chipimyo, 20 μm). Musemu uusalala pyu utondeezya kumubili waseelo lya Purkinje. (C) Kuvwuntauzya kwakutondezya ma protein kwachiindi kwakajanwa kuti nchintu chiyandikana loko mukulwana bulwazi bwakuzimba kwamitsinga (multiple t-test, *FDR <5%; n = 3-5 mbeba). (D) Atala: Cifwanikiso chitondeezya nzila zisiyene zyakunjizya kaboni uulembedwe uuli mu [1-13C]pyruvate tracer (nkokuti, kwiinda mu PDH naakuti nzila ya trans-arterial). Munsi: Chaati ya violin itondezya mweelwe wa single-labeled carbon (M1) yakasandulwa kuba aspartic acid, citric acid antoomwe a malic acid nikwakamana kulembwa zibeela zya cerebellar a [1-13C]pyruvate (paired t-test; ** P <0.01). (E) Kuvwuntauzya kwachiindi chilamfu kwanzila itondezyedwe. Koyeeya buyo ma protein ajisi P<0.05 mumviki zili 8. Muzila uukkwene: taakwe mpindu yakucinca (kuvwuntauzya kwakusiyana kwanzila zyobile; * P <0.05; *** P <0.001). Data itondezyedwe mbuli mweelwe ± SEM.
Mukuvwuntauzya kwesu, BCAA catabolism yaba nzila imwi mpati yakusumpula. Kaambo aaka katondezya kuti mweelwe wamuwo uunjila mumulongo wa TCA ulakonzya kuchincha mu PN iitakwe OXPHOS. Eezi zilakonzya kwiiminina nzila mpati yakubambulula zyintu zili mubongo, zikonzya kuba ampindu mpati kubuumi bwabongo akupona kwabo muchiindi chakutabeleka kabotu kwa OXPHOS. Kweendelana a muzeezo ooyu, twakajana kuti enzyme mpati iilwana bulwazi bwakuyuma kwamitsinga ya PCx ilisumpukide (Mfn2cKO/CTRL ilasanduka ziindi zili 1.5; Cifwanikiso 3A), eeyo iipa kuti pyruvate isanduke kuba oxaloacetate (28), eeyo iisyomwa kuti ilajanika mubongo. (29, 30). Kweendelana azyakajanwa mu proteomics, confocal microscopy yakatondezya kuti PCx yakayungizigwa kapati mu OXPHOS-deficient PNs, kakuli PCx reactivity yakabikkilizyigwa kapati kumaseelo aali afwaafwi aa Bergmann glial aakweendelezya (Chitondeezyo 3B). Kuti tusunke kweendelana kwa PCx, twakasilika zibeela zyabongo zipati a [1-13C]pyruvate tracer. Eelyo pyruvate niyakasandulwa a pyruvate dehydrogenase (PDH), zyina lyayo lya isotope lyakamana, pesi lyakabikkilizyigwa mu TCA cycle intermediates eelyo pyruvate niyakasanduka kwiinda mukusanduka kwamitsinga (Chitondeezyo 3D). Mukugwasyilizya data yesu ya proteomics, twakabona mweelwe mupati wazitondezyo kuzwa kuli eechi chitondezyo mu aspartic acid ya Mfn2cKO slices, mpawo citric acid a malic acid azyalo zyakaba achiimo chili akati-akati, nikuba kuti tiizyakali kuyandika pe (Chitondeezyo 3D).
Muma dopamine neurons aa MitoPark mbeba ajisi mitochondrial dysfunction akaambo kakuti dopamine neurons zyakanyonyoona mitochondrial transcription factor A gene (Tfam) (Chitondeezyo S6B), PCx expression yakayungizigwa kapati (31), kutondezya kuti acetone acid arteriosclerosis Kuchitika kwabulwazi oobu kweendelezyegwa muchiindi chakutabeleka kabotu kwa neurons OXPHOS mumubili. Nchintu chibotu kuzyiba kuti kwajanwa kuti ma enzyme aalibedelede (32-34) aakonzya kutondezyegwa muma neurons aakonzya kuswaangene a arteriosclerosis alasumpulwa kapati muma PNs aatakwe OXPHOS, mbuli propionyl-CoA carboxylase (PCC-A), Malonyl-CoA isandula propionyl-CoA kuba mito-ACoA succil-condrial enzyme 3 (ME3), ooyo mulimo mupati ngwakujana pyruvate kuzwa ku malate (Chitondeezyo 3, A a C) (33, 35). Mukuyungizya, twakajana kuyungizigwa kupati kwa Pdk3 enzyme, eeyo iipa kuti PDH itabeleki (36), kakuli taakwe kucinca kwakajanwa mu Pdp1 enzyme iipa kuti PDH ibeleke naakuti PDH enzyme complex (Chitondeezyo 3A). Mbubwenya, mu Mern2cKO PNs, phosphorylation ya α1 subunit α (PDHE1α) subunit ya pyruvate dehydrogenase E1 component ya PDH complex mu Ser293 (iizyibidwe kuti ilalesyela enzyme activity ya PDH) yakayungizigwa (Chitondeezyo S6C) (Chitondeezyo S6C). Pyruvate taikwe nzila yakunjilila mumitsinga.
Kumamanino, twakajana kuti nzila mpati ya serine a glycine biosynthesis, mitochondrial folate (1C) cycle iiswaangene antoomwe a proline biosynthesis (Chitondeezyo 1G a Cifwanikiso S5C) zyoonse zyakasumpulwa, kweendelanya amalipooti, ​​muchiindi chakutalisya. Zibeela zizingulukide zilabeleka akaambo kakutabeleka kabotu kwa mitochondrial (5-7). Kuvwuntauzya kwakugwasyilizya data eeyi kwakatondeezya kuti mu PN kakunyina OXPHOS, zibeela zyabongo zyambeba zyamviki zili 8 zyakabikkwa serine hydroxymethyltransferase 2 (SHMT2), enzyme iiyandika kapati mu mitochondrial folate cycle. Ikwiingula kwamubili kupati (Chitondeezyo S5D). Mu 13 CU-glucose-incubated acute cerebellar slices, metabolic tracing experiments yakasinizya kusumpuka kwa serine a proline biosynthesis, kutondezya kuti kunjila kwa carbon isoforms mu serine a proline kwakayungizyika (Chitondeezyo S5E). Mbwaanga nsandulo zisungwaazyigwa a GLS a GPT2 nzizyo zipa kuti kube kupanga glutamate kuzwa ku glutamine akusanduka aakati ka glutamate a α-ketoglutarate, kuyungizigwa kwazyo kutondeezya kuti OXPHOS-deficient neurons zilijisi kuyandika kupati kwa glutamate, Eezi zilakonzya kuchitwa kuti zizumanane kubamba biosynthesis ya proline (Figure S5). Kwiimpana akusanduka ooku, buvwuntauzi bwa proteomic bwa cerebellar astrocytes kuzwa ku PN-specific Mfn2cKO mice bwakatondezya kuti eezyi nzila (kubikkilizya ama antiperoxidases oonse) tiizyakacinca pe mukutondezya, aboobo kutondezya kuti Ooku kucinca kwa metabolic kulasala ku degraded PN (Fig. S6, D to G).
Muchi chifwiifwi, ooku kuvwuntauzya kwakatondeezya nzila zisiyene zyakutalisya nzila zya metabolic mu PNs. Nikuba kuti kutabeleka kabotu kwa mitochondrial yabongo kulakonzya kuleta kubola kwamitsinga ya 1C (Chitondeezyo 3E a Cifwanikiso S5C), mpawo akuchincha kwa I a IV complexes, kuchincha kwa serine de novo synthesis kwakalibonya buyo kumamanino. OXPHOS kutabeleka kabotu (Chitondeezyo 3E aChitondeezyo S5C). Eezyi zyajanwa zipandulula nzila iitobelana oomo mitochondrial (1C cycle) antoomwe a cytoplasmic (serine biosynthesis) zipa kuti kube kukatazyigwa antoomwe akuyungizigwa kwa atherosclerosis mu TCA cycle kuchitila kuti kube kubambulula metabolism yabongo.
8-week-old OXPHOS-deficient PNs ilakonzya kubamba mulimo wakukankaminwa kwachiindi chilamfu mpawo ilakonzya kunjila mukuswaangana kwa metabolic kuchitila kuti ibwezelezye kutabeleka kabotu kwa mitochondrial. Ooku kujana kwabusya kaambo kakondelezya kakuti nikuba achiindi eechi, maselu aaya alakonzya kutambula lugwasyo lwabusilisi kuchitila kuti achede naakuti alesye kubola kwabongo. Kuchedwa. Twakamana penzi eeli kwiinda mukubelesya nzila zyobile zyakuliimya. Munzila yakusaanguna, twakabamba Cre-dependent adeno-associated virus (AAV) vector kuchitila kuti MFN2 ikonzye kusalwa mu OXPHOS-deficient PNs in vivo (Figure S7A). AAV iibamba MFN2 antoomwe a fluorescent reporter gene mCherry (Mfn2-AAV) zyakasinizyigwa mu primary neuron cultures in vitro, eezyo zyakapa kuti MFN2 itondezyegwe munzila iiyeeme a Cre mpawo yakavwuna mitochondrial morphology, aboobo yakalesya kuchincha kwa neurons mu Mfn2cKO neurons ( Cifwanikiso S7, D, D, B, D, B). Kuzwa waawo, twakachita zyintu zyakusunka mubuumi bwamuntu kuti tupe Mfn2-AAV yamviki zili 8 ku cerebellar cortex ya Mfn2cKO mbeba zyakweendelezya, mpawo twakavwuntauzya mbeba zyamviki zili kkumi azibili (Chitondeezyo 4A). Mbeba zya Mfn2cKO zyakasilikwa zyakafwa (Chitondeezyo 1, A a B) (16). Kusanduka kwa kazunda mumubili kwakapa kuti kube kusala kwa PN muzibeela zimwi zyabongo (Chitondeezyo S7, G a H). Kubikkwa kwa AAV yeendelezya iitondezya buyo mCherry (Ctrl-AAV) tiikwakajisi mpindu mpati aatala akubola kwabongo mubanyama ba Mfn2cKO. Mukwiimpana, kuvwuntauzya kwa Mfn2cKOs kwakasandulwa a Mfn2-AAV kwakatondezya kukwabilila kupati kwa PN cell layer (Chitondeezyo 4, B a C). Mubwini, bunji bwama neuron bulibonya kuti tabukonzyi kwaandaanisigwa abanyama batakwe mulimo (Chitondeezyo 4, B a C, a Cifwanikiso S7, H a I). Kutondezya kwa MFN1 pesi kutali kwa MFN2 kulagwasya mbweenya buyo mukuvwuna lufu lwabongo (Chitondeezyo 4C aChitondeezyo S7, C a F), eezi zitondezya kuti kutondezya kwa MFN1 kunze lya MFN2 kulakonzya kugwasya kubula kwa MFN2. Kuvwuntauzya kumwi a PN yomwe kwakatondeezya kuti Mfn2-AAV yakavwuna kapati bupange bwa mitochondria, yakapa kuti mtDNA ibe kabotu, mpawo yakapilusya chitondezyo cha anti-angiogenesis marker PCx ​​(Chitondeezyo 4, C kusikila E). Kulangisisya kwameso kwa mbeba zya Mfn2cKO zyakavwunwa kazili mukulyookezya kwakatondeezya kuti mbobakali kwiima azitondezyo zyabo (kweenda S1 kusikila S3) zyakaba kabotu. Mukumanizya, aaya masunko atondezya kuti kujokezyegwa kwa MFN2 mu PNs zitakwe OXPHOS zilazula kujokolosya kubelesya mtDNA akupa kuti kube kuciswa kwamitsinga, aboobo kulesya kubola kwa axon akufwa kwa neuronal in vivo.
(A) Chitondeezyo chitondeezya mabambe aakusunka aakunjizya AAV iilembedwe MFN2 aawo nzila ya metabolic yakabikkwa. (B) Zifwoto zitondezya zibeela zyabongo zyamviki zili kkumi azibili zyakasandulwa a Mfn2cKO mbeba mumviki zili cisambomwe mpawo zyakabikkwa ma anti-Calbindin antibody. Kululyo: Kuyungizya nsinga zya axon. Chiimo cha axon zoom 450 a 75 μm. (C) Kulumwensyi: Kupima bunji bwamaseelo aa Purkinje mu AAV transduction loop (AAV+) (kuvwuntauzya kwakusiyana kwanzila yomwe; n = 3 mbeba). Kululyo: kulanga-langa kwa mtDNA mu PN yakasandulwa mumviki ya 12 (t-test iitakwe mweenzinyina; n = maseelo aali 6 kuzwa ku mbeba zyotatwe). * P <0.05; ** P <0.01. (D) Zitondezyo zyakutola ma electron micrographs zya PNs zya Mfn2cKO zibeela zya cerebellar zyakasandulwa ama virus vectors aatondezyedwe. Chivwumbyo cha pinki chitondeezya busena buzwide ma dendrites, mpawo sikweya uujisi madoti aamubala wa yellow utondeezya busena buli kululyo; n wiiminina nucleus. Chipimyo, 1μm. (E) itondezya zikozyanyo zya PCx staining mu PN yakasandulwa mumviki zili kkumi azibili. Chipimyo, 20μm. OE, kuyungizya; FC, kusanduka kwakupeta.
Kumamanino, twakavwuntauzya mbociyandika kupona kwamaseelo aakaletwa a peroxidase muma PNs aakajana OXPHOS dysfunction. Twakapanga mCherry iijisi AAV-shRNA (short hairpin RNA) iilanganya kapati mouse PCx mRNA (AAV-shPCx), mpawo twakanjizya kazunda naakuti scrambled control (AAV-scr) mubongo bwa Mfn2cKO mice. Injection yakachitwa mumviki yone yakuzyalwa (Chitondeezyo 5A) kuchitila kuti PCx ichite obotu muchiindi eecho PCx niyakayungizya (Chitondeezyo 3C) mpawo PN cell layer niyakachili kabotu (Chitondeezyo 1A). Nchintu chibotu kuzyiba kuti kugonka PCx (Mufanikisyo S8A) kuletela kufwambana kupati kwalufu lwa PN, oolo ndoluli lulikke lulesyedwe lwabulwazi (Mufanikisyo 5, B a C). Kuti tumvwisisisye nzila zyakusanduka kwamubili ziletwa a PCx up-regulation, twakaiya chiimo cha redox cha PNs nikwakainda PCx knockdown antoomwe a AAV-mediated optical biosensor Grx1-roGFP2 zyakatondezyegwa achiindi chomwe (Chitondeezyo S8, B kusikila ku D) kuti tubone glutathione Kuchincha kwa redox peptide (388). Mpawo, twakachita zibelesyo zyakupima buumi bwamafotoni obile (FLIM) muzibeela zyabongo zya Mfn2cKO zyamviki zili musanu ababili naakuti zyeendelezya littermates kuti tubone kucinca kukonzya kuchitika muchiimo cha cytoplasmic redox twamana kusinizya bukkale bwa FLIM (Chitondeezyo S8, E kusikila ku G). Kuvwuntauzya kwakatondeezya kuyungizigwa kupati kwachiimo chakusanduka kwa Mfn2cKO PNs yomwe iitakwe PCx expression, eeyo yiindene a control neurons naakuti Mfn2cKO PNs iitondezya buyo shRNA iisanganizidwe (Chitondeezyo 5, D a E). Eelyo PCx niyakagwisigwa, mweelwe wa Mfn2cKO PNs wakatondezya chiimo cha oxidized wakatanta ziindi ziinda kuli zyotatwe (Chitondeezyo 5E), kutondezya kuti PCx up-regulation yakabamba nguzu zya redox zya neurons zyakabola.
(A) Chitondeezyo chitondeezya mabambe aakusunka aakunjizya AAV iilembedwe shPCx aawo nzila ya metabolic niyabelesegwa. (B) Zifwoto zitondezya zibeela zyabongo zyamviki zili 8 mu mbeba zya Mfn2cKO zyakasandulwa akubikkwa ma anti-calcineurin antibody mumviki zyone. Chipimyo, 450μm. (C) Kupima bunji bwamaseelo aa Purkinje mu AAV-transduced loops (kulanga-langa kwakusiyana kwanzila yomwe; n = 3 kusikila ku 4 mbeba). Data itondezyedwe mbuli mweelwe±SEM; ***P<0.001. (D) Cifwanikiso cha FLIM chitondezya buumi bwa 7-week-old PN itondezya glutathione redox sensor Grx1-roGFP2 mubukkale bwakusola bwakaambwa. LUT (look-up table) mweelwe: chiindi chakupona (mu picoseconds). Chipimyo, 25μm. (E) Ihistogilamu itondezya kwaabwa kwa Grx1-roGFP2 kwabuumi kuzwa ku (D) (n=158 kusika ku 368 maselu mu mbeba zyobile mubukkale buli boonse). Ichaati ya pie atala a histogram imwi aimwi: itondezya mweelwe wamaseelo ajisi buumi bulamfwu (mubala uusalala pyu, uujisi busani) naa bufwaafwi (bbuluu, buceya) bwabuumi, oobo buinda 1 SD yabuumi bwaakati-kati mu CTRL-AAV-scr. (F) Inzila yakayeeyelwa itondezya bukwabilizi bwakusumpula PCx ya neuronal.
Zyoonse antoomwe, data njotupede aawa itondezya kuti kujokolosya kwa MFN2 kulakonzya kuvwuna PN iisumpukide akubula OXPHOS, kumana kwa mtDNA, antoomwe a ista-like morphology iitaluleme, aboobo kupa kuyaambele kuzumanana nokuba mumalwazi aasumpukide. Kubola kwabongo kupa bumboni bukonzya kujokoloka bwachiindi maseelo katanafwa. Ooku kucinca kwa metabolic kulayungizyigwa akukonzya kwama neurons kuleta atherosclerosis (kucinca kwa TCA cycle), ooko kulesya PCx kutondezya mu PNs zitakwe OXPHOS akusumpula lufu lwamaseelo, aboobo kuchita mulimo wakukwabilila (Chitondeezyo 5F).
Mukuvwuntauzya ooku, twakapa bumboni bwakuti nsandulo ya PNs kukutabeleka kabotu kwa OXPHOS nkwiinda asyoonto-asyoonto kusika ku TCA cycle atherosclerosis kwiinda munzila yakusiyana iibelesyegwa amapulogilamu aa metabolic. Twakasinizya buvwuntauzi bwa proteomic anzila zyiingi zigwasya mpawo twakayubununa kuti nibakasunkwa akutabeleka kabotu kwa mitochondrial, ma neurons alaa nzila iitazyibidwe ya metabolic elasticity. Kugambwa kwesu, nzila yoonse yakubambulula ntambo tiikonzyi kwaamba kuti ntambo zyakumaninizya zyeendelanya akubola kwabongo asyoonto-asyoonto alimwi kakunyina kujokoloka, pesi data yesu itondezya kuti ilakonzya kubamba ntambo zyabongo nikuba muchiindi chakufwa kwamaseelo. Nzila yakugwasya. Eezyi zyajanwa zitondezya kuti ma neurons alakonzya kuchita obotu mumubili. Kaambo aaka katondezya kuti kujokezyegwa kwa MFN2 kulakonzya kuchincha zitondezyo zipati zya metabolic akukwabilila kubola kwa PN. Kakuchili boobo, chilesya kubola kwamitsinga mpawo chifwambaanya nsinga. sikucinca koonana.
Chimwi chazintu zikondelezya loko nzyotwajana mukuvwuntauzya kwesu nchakuti ma PNs aatakwe OXPHOS alakonzya kuchincha TCA cycle metabolism kwiinda mukusumpula ma enzymes aayo asungwaazya arteriosclerosis. Kubambulula kwamubili nchintu chidumide mumaseelo aakkansa, amwi aayo asyoma glutamine kuti ayungizye TCA cycle intermediates kuti azwe zintu zikozyenye, eezyo zyeendelezya nzila yakuyoya akubamba kupanga ma lipid a nucleotide biosynthesis precursors (39 , 40). Buvwuntauzi bwakachitika ino-ino bwakatondezya kuti muzibeela zyaatala zili mumapenzi aa OXPHOS, kuswaangana alimwi kwa glutamine/glutamate metabolism akwalo nchintu chipati (5, 41), aawo nzila ya glutamine kunjila mu TCA cycle njiiyeeme a Akaambo kabupati bwakuchisa kwa OXPHOS (41). ). Nokuba boobo, taakwe bukamboni busalala bwakukozyanya kwakusanduka kwatunsinga twabongo mumubili antoomwe ambokukonzya kugwasya mubukkale bwamalwazi. Mukuvwuntauzya kwalino-lino kwa in vitro, ma neurons aakusaanguna akatondezyegwa kuti alakonzya kuyobolola glutamate pools kuti ibelesye neurotransmission, aboobo akusumpula oxidative metabolism antoomwe a atherosclerosis mubukkale bwakukatazyigwa kwa metabolic (42). Nchintu chibotu kuzyiba kuti munsi lyakulesyegwa kwa misamu ya TCA cycle enzyme succinate dehydrogenase, pyruvate carboxylation ilasyomwa kuti ilabamba kupanga kwa oxaloacetate mu cerebellar granule neurons (34). Nokuba boobo, kweendelana kwamubili kwanzila eezyi kuzibeela zyabongo (oomo atherosclerosis nkokusyomwa kuti kujanika buyo kuma astrocytes) kuchili amulimu mupati (43). Muchiimo eechi, data yesu itondezya kuti ma PNs akanyonyoonwa a OXPHOS mumubili alakonzya kusanduka kuya kukunyonyooka kwa BCAA antoomwe a pyruvate carboxylation, eezyo nzintu zyobile zipati zipa kuti kube TCA pool intermediates. Nikuba kuti lugwasyo lwa BCAA catabolism mukusanduka kwanguzu zyabongo lwakayeeyelwa, kunze lyamulimo wa glutamate a GABA mukutumizya boongo (44), kunyina bumboni bwanzila eezyi mumubili. Aboobo, nchuubauba kweezyeezya kuti ma PN aatabeleki kabotu alakonzya kuligwasya kulya kwa TCA aakati-akati kweendelezyegwa anzila yakusanganya kwiinda mukuyungizya bulwazi bwakuzimba kwamitsinga. Mubwini, kuyungizya PCx kulakonzya kuyandika kuti kuzumanane kuyandika kwa aspartic acid, ooko kwakatondezyegwa mumaseelo aayungizyika ajisi mitochondrial dysfunction (45). Nokuba boobo, buvwuntauzi bwesu bwa metabolomics tiibwakayubununa kucinca kuli koonse kupati muchiimo cha aspartic acid mu Mfn2cKO PNs (Chitondeezyo S6A), eecho chikonzya kutondezya kusiyana kwakubelesya kwa aspartic acid aakati kamaseelo aayungizyika antoomwe a post-mitotic neurons. Nikuba kuti nzila ncobeni yakuyungizya PCx muma neurons aatabeleki kabotu mumubili tiiyakazyibwa pe, twakatondezya kuti kwiingula ooku kakutanasika chiindi kulagwasya kapati mukubamba redox state ya neurons, eeyo yakatondezyegwa mumasunko aa FLIM aazibeela zya cerebellar. Mubwini, kulesya ma PN kuti atabi aanguzu zyakusumpula PCx kulakonzya kuleta chiimo cha oxidized akufwambaanya lufu lwamaseelo. Kuchita kuti BCAA inyonyoonwe a carboxylation ya pyruvate tazili nzila zyakuziba nyama zili kunze lya mitochondrial dysfunction (7). Aboobo, zilibonya kuba chintu chiyandikana loko ku OXPHOS-deficient neurons, nikuba kuti tazili zyeezyo zilikke, eezyo ziyandikana kukubula nguzu kwabongo. .
Bulwazi bwa cerebellar mbulwazi bwiindene-indene bwakubula nguzu kwabongo oobo kanjikanji bulibonya mbuli ataxia alimwi kanjikanji bulanyonganya PNs (46). Eeyi nkamu yama neuron ilakonzya kuchiswa kapati akaambo kakutabeleka kabotu kwa mitochondrial nkaambo kusaala kwayo mu mbeba kulakonzya kuzyala zitondezyo zyiingi zya spinocerebellar ataxia (16, 47, 48). Kweelana amalipooti, ​​mbeba yakasandulwa ilijisi gene yakasanduka ilaswaangene a spinocerebellar ataxia yabantu alimwi ilaa mitochondrial dysfunction (49, 50), kukankaizya mbociyandika kwiiya ntenda zyakubula OXPHOS mu PNPH. Aboobo, nchintu chibotu loko kwaandaanya akwiiya nkamu eeyi iilibedelede. Nokuba boobo, nkaambo kakuti ma PN alamvwa loko kukusinikizyigwa mpawo ngaajisi mweelwe musyoonto wamaseelo woonse aamubongo, muziiyo zyiingi zyeendelana a omics, kwaandaanya kwawo mbuli maseelo woonse kuchili chintu chiyumu. Nikuba kuti nchintu chiyumu kuzuzikizya kubula kusofwaazyigwa kwamaseelo amwi (ikapati nyama yabantu bapati), twakaswaanganya ntaamu yakugwisya maseelo a FACS kuti tujane mweelwe uuzulide wama neurons aakonzya kubelesegwa mukuvwuntauzya kwa proteomics, mpawo tube aama protein miingi (aasika ku 3000 ma protein) kweelanisigwa a data yoonse ya cerebel (155). Kwiinda mukukwabilila buumi bwamaseelo woonse, nzila njotupa aawa itupa kuti tubone kuchincha kwanzila zyakusanduka kwamaseelo muma mitochondria, pesi akulangisisya kuchincha kwazibeela zyamaseelo, eezyo zigwasilizya kubelesya nzila yakuyungizya maseelo aamaseelo. Nzila njotupandulula tayeendelani akwiiya maselu aa Purkinje luzutu pe, pesi ilakonzya kubelesegwa kuli kufumbwa maseelo kuchitila kuti kube kuchincha kwa metabolic mubongo buchiswa, kubikkilizya azimwi nzila zya mitochondrial dysfunction.
Kumamanino, twakajana nzila yakusilisya muchiindi eechi chakubambulula chakulya chikonzya kuchincha zitondezyo zipati zyakukataazikana kwamaseelo akukwabilila kumana kwamaseelo. Aboobo, kumvwisisisya mulimo wakucinca ntambo zyaambwa aawa kulakonzya kupa luzibo lupati kujatikizya nzila zikonzya kugwasya kubamba buumi bwabongo muchiindi chakutabeleka kabotu kwamaseelo. Buvwuntauzi bwakumbele bujisi makanze aakulanga-langa kucinca kwanguzu zyakubelesya mumaseelo aambi aabongo bulayandika kutegwa kuyubununwe cakumaninina mboikonzya kubelesyegwa njiisyo eeyi kumalwazi aambi aajatikizya bongo.
Mbeba zya MitoPark zyakapandululwa musule (31). C57BL/6N mbeba zili a loxP zili kumbali a Mfn2 genes zyakapandululwa musule (18) mpawo zyakaswaanganizyigwa a L7-Cre mbeba (23). Lunyungu lwa heterozygous zyobile lwakazwa mpawo lwakasanganizigwa a homozygous Mfn2loxP/Mfn2loxP mbeba kuchitila kuti kube Purkinje-specific gene knockouts ya Mfn2 (Mfn2loxP/Mfn2loxP; L7-cre). Muchibeela chakuzyalana, Gt (ROSA26) SorStop-mito-YFP allele (stop-mtYFP) yakanjizigwa kwiinda mukuyungizigwa (20). Nzila zyoonse zyakusilikwa kwabanyama zyakachitilwa kweendelana amalailile aakuYulopu, aacisi alimwi aambunga alimwi zyakazumizyigwa a LandesamtfürNatur waku Umwelt a Verbraucherschutz, North Rhine-Westphalia, Germany. Mulimo wabanyama awalo utobelezya busolozi bwa European Federation of Laboratory Animal Sciences Associations.
Musule akulesya kuchisa kwamukaintu uumitide, mwana wa mbeba wakagwisigwa (E13). Icikanda chakakosolwa mu Hanks' Balanced Salt Solution (HBSS) yakayungizigwa a 10 mM Hepes mpawo yakabikkwa a Dulbecco's Modified Eagle's Medium iijisi papain (20 U/ml) antoomwe a cysteine ​​(1μg/ml). Bikka nyama mu DMEM) mpawo uyigwisye kwiinda mukugaya. Ml) a 37°C kwamaminiti aali makumi abili, mpawo yakagayigwa mu DMEM yakayungizigwa a 10% yabulowa bwa ng'ombe. Maseelo akabikkwa aama gilazi aakavwumbidwe a polylysine abulemu bwa 2×106 a 6 cm yakukomezya naakuti abulemu bwa 0.5×105 maselu/cm2 kuti kube kulanga-langa. Nikwakainda mahola one, musamu wakachinchwa a musamu uutakwe bulowa bwa Neurobasal uujisi 1% B27 supplement a 0.5 mM GlutaMax. Ma neurons mpawo akabikkwa a 37°C a 5% CO2 muchiindi choonse chakusola, mpawo akapegwa chakulya chiindi chomwe amviki. Kuti kube kusanganya alimwi mu vitro, 3μl (24-well culture dish) naa 0.5μl (24-well plate) ya AAV9 virus vector yakabelesegwa kusilika ma neurons mubuzuba bwabili mu vitro: AAV9.CMV.PI.eGFP. WPRE.bGH (Addgene, namba ya 105530-AAV9) antoomwe a AAV9.CMV.HI.eGFP-Cre.WPRE.SV40 (Addgene, namba ya 105545-AAV9).
Mfn1 a Mfn2 zya mbeba (zyakajanwa kuzwa ku Addgene plasmid #23212 antoomwe a #23213, kweendelana) zililembedwe a V5 sequence (GKPIPNPLLGLDST) ku C-terminus, mpawo zyakaswaanganizyigwa a mCherry muchifwanikiso kwiinda mu T2A sequence. Grx1-roGFP2 nchipego kuzwa kuli Heidelberg TP Dick DFKZ (Deutsches Krebsforschungszentrum). Kwiinda mukuchincha tdTomato kaseti kubelesya nzila zyakukozyanisya zyakale, kaseti yakabikkwa mu pAAV-CAG-FLEX-tdTomato backbone (Addgene reference number 28306) kuti ipange pAAV-CAG-FLEX-mCherry-T2A-MFN2-V5, pAAV-CAG-G FLEX-mCherry-T2A-MFN1-V5 antoomwe a pAAV-CAG-FLEX-Grx-roGFP2 vectors. Nzila iikozyenye yakabelesegwa kupanga pAAV-CAG-FLEX-mCherry. Kuti kube kupanga kwa AAV-shPCx, plasmid AAV vector (VectorBuilder, pAAV [shRNA] -CMV-mCherry-U6-mPcx- [shRNA#1]) ilayandika, eeyo iijisi DNA sequence iibamba shRNA iilanganya mouse PCx (5′CTTTCGCTAGCTAGGCTAAGGCTAAGGCTAGACTTAGACTTAGA 3′) Mubweendelezi bwa U6 promoter, mCherry ibelesyegwa mubweendelezi bwa CMV promoter. Kupanga kwa AAV vectors kwakachitwa kweendelana amalayilile aabasikupanga (Cell Biolabs). Mubufwiifwi, belesya plasmid iijisi mCherry-T2A-MFN2-V5 (pAAV-CAG-FLEX-mCherry-T2A-MFN2-V5), mCherry-T2A-MFN1-V5 (pAAV-CAG-FLEX-mCherry) muchiindi chifwiifwi. mCherry (pAAV-CAG-FLEX-mCherry) naa Grx-roGFP2 (pAAV-CAG-FLEX-Grx-roGFP2) iibamba zyintu, antoomwe a AAV1 capsid protein antoomwe a accessory protein Packaging plasmid plasmid, using calcium phosphate method. Kazunda katakwe busani kakajanwa kwiinda mukutontola-kutontola muchisambilo cha aisi/ethanol mpawo maselu akakosolwa mu phosphate buffered saline (PBS). I AAV vector yaka salazyigwa a iodixanol gradient ultracentrifugation (mawoola aali makumi obilwe atune a 32,000 rpm a 4°C) mpawo yakabikkwa antoomwe kubelesya Amicon ultra-15 centrifugal filter. Genome titer ya 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 yakali mbuli mbukwakapandululwa musule (pccr) -MFN1-V5 (1.9×1013 GC/ml) antoomwe a AAV1-CAG-FLEX-Grx-roGFP2 (8.9×1012 GC/ml).
Ma neurons mataanzi akagwisigwa mu ice-cold 1x PBS, akabikwa, mpawo akabikkwa antoomwe mu 0.5% Triton X-100 / 0.5% sodium deoxycholate/PBS lysis buffer iijisi phosphatase antoomwe a protease inhibitor (Roche). Kupima ma protein kwakachitwa kwiinda mukubelesya bicinchoninic acid assay (Thermo Fisher Scientific). Ma protein mpawo akaandaanizyigwa a SDS-polyacrylamide gel electrophoresis, mpawo akabikkwa a polyvinylidene fluoride membrane (GE Healthcare). Jala masena aatali aampindu mpawo ubike antoomwe a antibody yakusaanguna (Langa Table S1 kuti ujane makani woonse) mu 5% yamalili mu TBST (Tris-buffered saline a Tween), ntaamu zyakusanzya antoomwe a antibody yabili mu TBST. Bikka antoomwe a antibody yakusaanguna masiku woonse a +4°C. Mwamana kusamba, bikka musamu wabili wakulwana malwazi kwamahola obile muchiimo chakutontola. Kuzwa waawo, kwiinda mukubikka bbala ndyoonya eelyo a anti-β-actin antibody, kuvwumbilila mbweenya kwakasinizyigwa. Kuzyiba kwiinda mukusandula kuya ku chemiluminescence akuyungizya chemiluminescence (GE Healthcare).
Ma neurons aakabikkidwe kale aamagilazi akabikkwa a 4% paraformaldehyde (PFA)/PBS achiindi chakabikkidwe aawo mpaakali kupya loko kwamaminiti aali kkumi. Zivwunisyo zyakasaanguna kubikkwa a 0.1% Triton X-100/PBS kwamaminiti aali 5 aawo mpokutontola, mpawo zyakabikkwa muchikwabilizyo [3% bovine serum albumin (BSA)/PBS]. Mubuzuba bwabili, zivwunisyo zyakasanzigwa achisinkilizyo chakukwabilila mpawo zyakabikkwa antoomwe a fluorophore-conjugated secondary antibody kwamahola obile muchiimo chakutontola; kumamanino, zitondezyo zyakasanzigwa kabotu-kabotu mu PBS a 4′,6-diamidino-2 -Phenylindole (DAPI) yakabikkwa mubala mpawo yakabikkwa aa microscope slide a Aqua-Poly/Mount.
Mbeba (baalumi abanakazi) bakabikkwa mumubili kwiinda mukubikkwa musamu wa ketamine (130 mg/kg) a xylazine (10 mg/kg) mpawo bakapegwa musamu wa carprofen analgesic (5 mg/kg), mpawo bakabikkwa muchibelesyo cha stereotactic (Kopf) chili a warm. Amugwisye mutwe wamuntu mpawo ubelesye chibelesyo chakugela meno kuchesya cibeela cha cerebellar cortex ceendelana a mis bone (kuzwa ku lambda: mucila 1.8, lateral 1, ceendelana a lobules IV a V). Belesya nyeleti ya syringe iigonkede kuti upange kabotu-kabotu mpako nsyoonto mumutwe kutegwa utanyonyooni nsinga zili munsi. Mpawo nsinga zyagilazi ziteteete zilabikkwa asyoonto-asyoonto mumulyango muniini (kuzwa ku -1.3 kusika ku -1 kulubazu lwamukati lwa dura mater), mpawo 200 kusikila ku 300 nl AAV ilanjizyigwa mumulyango muniini (Narishige) ama syringe aabelesyegwa amaanza (Narishige) ziindi zyiingi aansi muchiindi chamaminiti aali 20 kusikila ku 10. Musule akubikkwa, bikka nsinga zyabulowa kwamaminiti aambi aali kkumi kuchitila kuti kazunda kamwaike koonse. Nokwamana kugwisigwa nsinga zyabulowa, chikumba chilasontwa kabotu-kabotu kuchitila kuti chilonda chitazimbi mpawo munyama apone. Banyama bakapegwa misamu iikola (caspofen) kwamazuba manji kuzwa nobakasilikwa, muchiindi eecho bukkale bwabo bwakali kulangilwa oobotu mpawo bakajaigwa muchiindi cheelede. Nzila zyoonse zyakachitilwa kweendelana amalailile aakuYulopu, aacisi alimwi aambunga alimwi zyakazumizyigwa a LandesamtfürNatur waku Umwelt a Verbraucherschutz, North Rhine-Westphalia, Germany.
Banyama bakapegwa musamu wa ketamine (100 mg/kg) antoomwe a xylazine (10 mg/kg), mpawo moyo wakabikkwa 0.1 M PBS kusaanguna, mpawo 4% PFA mu PBS. Nyama yakakosolwa mpawo yabikkwa mu 4% PFA/PBS masiku woonse a 4°C. Peni lizungaana (Leica Microsystems GmbH, Vienna, Austria) lyakabelesegwa kubamba zibeela zya sagittal (50 μm mubufwaafwi) kuzwa kubongo butazungaani mu PBS. Kusiya kwakuti kwaambwa bumbi, kupenta zibeela zitazungaani kwakachitwa mbuli mbokwaambidwe atala aawa (13) aawo mpokutontola mpawo kakuvwungululwa. Mubufwiifwi, chakusanguna, zibeela zyakajanwa zyakabikwa a 0.5% Triton X-100/PBS kwamaminiti ali kkumi amusanu muchiimo chakutontola; kuli zimwi zibeela (Pcx a Shmt2), kwiinda mu tris-EDTA buffer a 80°C (PH 9) kupya zibeela kwamaminiti aali 25 muciindi chakuchita ntaamu eeyi. Kuzwa waawo, zibeela zyakabikkwa antoomwe a antibody yakusaanguna (Langa Table S1) mubbuloking buffer (3% BSA/PBS) a 4°C masiku woonse kakuli kuzungaanizyigwa. Buzuba bwakatobela, zibeela zyakasanzigwa achikwabilizyo chakusinkilila mpawo zyakabikkwa antoomwe a fluorophore-conjugated secondary antibody kwamahola obile muchiimo chakutontola; kumamanino, zibeela zyakasanzigwa kabotu-kabotu mu PBS, zyakasanzyigwa a DAPI, mpawo zyakabambululwa a AquaPolymount aa microscope slide.
I laser scanning confocal microscope (TCS SP8-X naa TCS Digital Light Sheet, Leica Microsystems) iijisi laser yamumuni uutuba antoomwe a 405 diode ultraviolet laser yakabelesegwa kutondezya musamu. Kwiinda mukukkomanisya fluorophore akuyobolola citondezyo a Hybrid Detector (HyDs), pulogilamu ya LAS-X yakabelesegwa kuyobolola zifwanikiso zibikkidwe antoomwe kweendelana a Nyquist sampling munzila iitobelana: kuzibeela zitali zya mweelwe, nzitondezyo zijisi nguzu kapati (mbuli, mumaseelo aamubili alimwi ama dendrites) mtYFPD kuya kunamba ya Hybrid ya BPN nzila). Kujala kwa 0.3 kusika ku 6 ns kubelesegwa kuchesya zyintu zili musule.
Kufwotola kwachiindi cheenzu kwamaseelo aasalidwe. Nikwakamana kusalazyigwa mu Neurobasal-A yakajisi 1% B27 supplement antoomwe a 0.5 mM GlutaMax, maselu akabikkwa mpoonya-mpoonya a poly-l-lysine-coated glass slides (μ-Slide8 Well, Ibidi, catalog number 80826) , mpawo wakabamba a 37°C a 12% CO kwawoola lyomwe kuti seelo lileke kkalinkana. Kufotozya muchiindi cheenzu kwakachitwa a Leica SP8 laser scanning confocal microscope iijisi laser iituba, HyD, 63×[1.4 numerical aperture (NA)] oil objective lens antoomwe a heating stage.
Mbeba yakafwambaanisigwa a carbon dioxide mpawo yakosolwa mutwe, boongo bwakagwisigwa kuzwa kumutwe, mpawo yakakosolwa kuba 200μm mubufwaafwi (kutegwa 13C labeling experiments) naa 275μm bupati (muma photon experiments obile) sagittal section yakazwide zintu zitobela The ice cream (HM-605, Themorific, The Scientifics, Scientific Walldorf, Germany) ilizwide zintu zitobela: 125 mM ice-cold, carbon-saturated (95% O2 and 5% CO2) low Ca2 + artificial cerebrospinal fluid (ACSF) NaCl, 2.5 mM KCl, 1.25 mM sodium phosphate buffer, NaCl 25 MM, 25 MM gluse 0.5 mM CaCl2 antoomwe a 3.5 mM MgCl2 (kusinikizyigwa kwa 310 kusika ku 330 mmol). Bweza zibeela zyabongo zyajanwa muchibaka chakusaanguna chijisi Ca2 + ACSF (125.0 mM NaCl, 2.5 mM KCl, 1.25 mM sodium phosphate buffer, 25.0 mM NaHCO3, 25.0 mM d-glucose, 10 mM Ca. MgCl2) Aakati) pH 7.4 a 310 kusika ku 320 mmol).
Muchiindi chakutola zifwanikiso, zibeela zyakatolwa mukaanda kakutola zifwanikiso, mpawo kusunka kwakachitwa a ACSF perfusion iizumanana a 32° kusika ku 33°C. I multiphoton laser scanning microscope (TCS SP8 MP-OPO, Leica Microsystems) iijisi Leica 25x objective lens (NA 0.95, maanzi), Ti: Sapphire laser (Chameleon Vision II, Coherent) yakabelesegwa mukupanga zifwanikiso. FLIM module (PicoHarp300, PicoQuant).
FLIM ya Grx1-roGFP2. Kuchincha kwachiimo cha PNs kwakapimwa a two-photon FLIM mu sagittal brain slices, ooko Grx1-roGFP2 biosensor nkoyakalanga PNs. Muchibaka cha PN, busena bwakujana bulasalwa aali 50 kusikila ku 80 μm munsi lyachibeela chachibeela kuchitila kuti kube PN iikonzya kubeleka (nkokuti, kubula bupange bwamabwe naa kucinca kwabupange bwabongo kumbali aama dendrites) antoomwe a double positive roGFP2 sensor antoomwe a AAV iibamba shRNA PCx naa kutobelana kwayo (mCherry coexpressing). Bweza zifwanikiso zyaatala a 2x digital zoom [excitation wavelength: 890 nm; 512 nm 512 pixels]. Kuzyiba: HyD yamukati, fluorescein isothiocyanate (FITC) filter group] antoomwe akulanga-langa zifwanikiso mukati kamaminiti obile kusikila kuli otatwe zilabelesyegwa kusinizya kuti mafotoni aakkwana ayobololwa (mafotoni aali 1000 woonse antoomwe) kuti akonzye kweendelana. Kumvwisisisya kwa Grx1-roGFP2 probe akusinizya bukkale bwa FLIM kwakachitwa kwiinda mukulanga buumi bwa roGFP2 aawo nokwakayungizyigwa 10 mM H2O2 ku perfusion ACSF (kuyungizya oxidation, kupa kuti buumi buyungizigwe), mpawo akuyungizya 2 mM dithiothreit (kucheya kwa rection, kucheya kwabuumi) (Chitondeezyo S8, D kusikila ku G). Belesya FLIMfit 5.1.1 software kulanga-langa mpindu zyajanwa, kobamba nzila yomwe yakubola kwachifanikisyo choonse ku IRF (instrument response function) yakapimwa, mpawo χ2 ili afwaafwi a 1. Kubala buumi bwa PN yomwe, chivwumbyo chakazyungulukide nsinga chakajanwa amaanza, mpawo mweelwe waakati-kati wabuumi bwachivwumbyo wakabelesegwa.
Kuvwuntauzya nguzu zya mitochondrial. Nikwakamana kubikkwa 100 nM TMRM mu ACSF kwamaminiti aali 30, kuchincha kwa mitochondrial kwa PNs kwakapimwa a microscope yama photon obile. Kufotozya kwa TMRM kwakachitwa akubotezya chitondezyo a 920 nm akubelesya HyD yamukati (tetramethylrhodamine isothiocyanate: 585/40 nm) kuyobolola zitondezyo; kwiinda mukubelesya bulamfwu bwakuzungaanya mbubwenya pele kubelesya HyD yamukati iindene (FITC :525/50) kutondezya mtYFP. Belesya ImageJ's Image Calculator plug-in kuti ubone nguzu zya mitochondrial a cell yomwe. Mubufwiifwi, equation ya plug-in: signal = min (mtYFP, TMRM) ilabelesyegwa kuzyiba busena bwa mitochondrial butondezya TMRM signal mu Purkinje Somali muchifwanikiso cha single-stack confocal cha channel yeendelana. Mpawo busena bwa pixel muchikozyano chakajanwa bulabalilwa, mpawo bwabikkwa mbuli mbukweelede achifanikisyo cha mtYFP channel kuchitila kuti tujane chibeela cha mitochondrial chitondezya nguzu zya mitochondrial.
Cifwanikiso chakagwisigwa a Huygens Pro (Scientific Volume Imaging) software. Kumifanikisyo yamatayilisi yakaskenwa, kusanganya tile lyomwe kuchitwa kubelesya nzila yakusona iizwa kuli pulogilamu ya LAS-X. Musule akubamba mufanikisyo, kobelesya ImageJ antoomwe a Adobe Photoshop kuti uzumanane kubamba mufanikisyo mpawo ubambulule kumweka akusiyana. Belesya Adobe Illustrator kulibambila zifwanikiso.
mtDNA kulanga-langa. Mweelwe wazilonda zya mtDNA wakabalilwa kuzibeela zyabongo zyakalembwa ama antibodies aalwana DNA a confocal microscope. Busena buli boonse bwakabambilwa mubili wa selo antoomwe a nucleus ya selo imwi aimwi, mpawo busena oobo bwakabalilwa kubelesya Multi Measure plug-in (ImageJ software). Gwisya busena bwa nyukiliya kuzwa kubusena bwamubili wamaseelo kuti ujane busena bwa cytoplasm. Kumamanino, nzila ya Analyze Particles plug-in (ImageJ software) yakabelesegwa kujana mweelwe wa DNA ya cytoplasmic kutondezya mtDNA achifanikisyo, mpawo mpindu zyakajanwa zyakabikkwa antoomwe a PN average ya CTRL mice. Izyazwa zilatondezyegwa mbuli mweelwe waakati-kati wama nucleosides mu selo lyomwe.
Kuvwuntauzya kwakutondezya ma protein. Belesya ImageJ's Image Calculator plug-in kuti ubone mboizwa ma protein mu PN aa selo lyomwe. Mubufwiifwi, mumufanikisyo wachibaka chomwe, kwiinda mu equation: signal = min (mtYFP, antibody), busena bwa mitochondrial butondezya immunoreactivity ku antibody imwi mu Purkina bulajanwa. Mpawo busena bwa pixel muchikozyano chakajanwa bulabalilwa, mpawo bwabikkwa mbuli mbukweelede achifanikisyo cha mtYFP channel kuchitila kuti tujane cibeela cha mitochondrial cha protein itondezyedwe.
Kuvwuntauzya bunji bwamaseelo aa Purkinje. I Cell Counter plug-in ya ImageJ yakabelesegwa kulanga-langa bunji bwa Purkinje kwiinda mukwaandaanya mweelwe wamaseelo aa Purkinje aakabalilwa abulamfu bwa ringi ya cerebellar yakakkede maseelo aakabalilwa.
Kulibambila akuyobolola zikozyanyo. Boongo kuzwa mukabunga kakweendelezya antoomwe a Mfn2cKO mbeba bakabikkwa mu 2% PFA/2.5% glutaraldehyde mu 0.1 M phosphate buffer (PB), mpawo zibeela zya coronal zyakabambwa kubelesya ciliates (Leica Mikrosysteme GmbH, Vienna, Austria) (Bupati bwa 50 kusikila ku 60 μm) Buffer buffer mu 1% PB tetraoxide antoomwe a 1.5% potassium ferrocyanide aawo mpokupya loko kwawoola lyomwe. Zibeela zyakasanzigwa ziindi zyotatwe amaanzi aasalala, mpawo zyakasanzyigwa a 70% ethanol iijisi 1% uranyl acetate kwamaminiti aali makumi abili. Zibeela eezyo mpawo zyakagwisigwa maanzi mubukoko bwakabikkidwe kabotu mpawo zyakabikkwa mu Durcupan ACM (Araldite casting resin M) epoxy resin (Electron Microscopy Sciences, catalog number 14040) aakati kamagilazi aajisi silicon, mpawo kumamanino a 60°C zyakabikkwa mu oveni kwamahola aali makumi one atutatwe. Busena bwa cerebellar cortex bwakasalwa mpawo 50 nm zibeela ziteteete zyakakosolwa a Leica Ultracut (Leica Mikrosysteme GmbH, Vienna, Austria) mpawo zyakakosolwa a 2×1 mm copper slit grid yakavwumbidwe a polystyrene film. Zibeela zyakasanzigwa amusamu wa 4% uranyl acetate mu H2O kwamaminiti aali kkumi, zyakasanzyigwa a H2O ziindi zyiingi, mpawo a Reynolds lead citrate mu H2O kwamaminiti aali kkumi, mpawo zyakasanzyigwa a H2O ziindi zyiingi. Micrographs yakatolwa a transmission electron microscope Philips CM100 (Thermo Fisher Scientific, Waltham, MA, USA) kubelesya TVIPS (Tietz Video and Image Processing System) TemCam-F416 digital camera (TVIPS GmbH, Gauting, USA). Germany).
Kumbeba zyakajatwa bulwazi bwa AAV, boongo bwakaandaanizyigwa mpawo bwakakosolwa muchibeela cha 1 mm, mpawo bongo bwakalangwa-langwa kubelesya chipekupeku cha fluorescence kuti kuzyibe ringi yakajisi bulwazi bwa AAV (nkokuti, mCherry expressing). Kusola buyo ooko AAV injection nkokupa kuti kube kuchincha kwa Purkinje cell layer (nkokuti afwaafwi a layer yoonse) muzibeela zyobile zitobelana zya cerebellar nkokubelesegwa. I AAV-transduced loop yakakosolwa mubusiku boonse (4% PFA antoomwe a 2.5% glutaraldehyde mu 0.1 M cocoate buffer) mpawo yakayungizigwa. Kuti EPON ibikkwe, nyama yakasanzyigwa a 0.1 M sodium cocoate buffer (Applichem), mpawo yakabikkwa a 2% OsO4 (os, Science Services; Caco) mu 0.1 M sodium cocoate buffer (Applichem) 4 Hours, mpawo yakasanzyigwa kwa 2 hours. Kozumanana ziindi zyotatwe a 0.1 M cocamide buffer. Kuzwa waawo, mulongo wa ethanol uutanta wakabelesegwa kubamba musamu wa ethanol a 4°C kwamaminiti ali kkumi amusanu kuchitila kuti nyama imane maanzi. Nyama yakabikkwa mu propylene oxide mpawo yakabikkwa masiku woonse mu EPON (Sigma-Aldrich) a 4°C. Bika nyama mu EPON mpya aawo mpokutontola kwamahola obile, mpawo uyibike a 62°C kwamahola aali 72. Belesya ultramicrotome (Leica Microsystems, UC6) antoomwe achipeni cha daimondi (Diatome, Biel, Switzerland) kugela 70 nm zibeela ziteteete, mpawo uzibike a 1.5% uranyl acetate kwamaminiti aali kkumi amusanu a 37°C, mpawo uzibike a lead citrate solution kwamaminiti aali 4. Ma electron micrographs akatolwa kubelesya JEM-2100 Plus transmission electron microscope (JEOL) iijisi Camera OneView 4K 16-bit (Gatan) antoomwe a DigitalMicrograph software (Gatan). Mukuvwuntauzya, ma electron micrographs akajanwa a 5000× naa 10,000× digital zoom.
Kuvwuntauzya kwabupange bwama mitochondria. Kukuvwuntauzya koonse, mibala ya mitochondria yomwe-yomwe yakabikkwa amaanza muzifwanikiso zya dijitali kubelesya pulogilamu ya ImageJ. Zibeela zyaandeene zyabupange zilalingulwa. Bunji bwama mitochondria bulatondezyegwa mbuli mweelwe uujanwa kwiinda mukwaandaanya busena boonse bwama mitochondria bwa selo lyomwe lyomwe abusena bwa cytoplasm (busena bwa cytoplasm = busena bwa selo-busena bwa selo nucleus) × 100. Buzunguluki bwa mitochondria bulabalilwa anzila ya [4π∙(area/perimeter 2)]. Bupange bwa ista bwa mitochondria bwakavwuntauzyigwa mpawo bwakaandaanizyigwa muzibeela zyobile ("tubular" a "blister") kweendelanya abubambe bwazyo bupati.
Mweelwe wa autophagosome/lysosome akuvwuntauzya bunji bwawo. Belesya pulogilamu ya ImageJ kuti ulembe omwini nzila zya autophagosome/lysosome muchifwanikiso. Busena bwa Autophagosome/lysosome butondezyegwa mbuli mweelwe uubalilwa kwiinda mukwaandaanya busena boonse bwa autophagosome/lysosome bwa selo lyomwe lyomwe abusena bwa cytoplasm (busena bwa cytoplasm=busena bwa selo-busena bwa nucleus)×100. Bunji bwama autophagosomes/lysosomes bulabalilwa kwiinda mukwaandaanya mweelwe woonse amweelwe wa autophagosome/lysosome structures mu selo lyomwe (kweendelana abusena bwa cytoplasm) (busena bwa cytoplasm = busena bwa selo-busena bwa nyukiliya).
Kulemba mabala aakubelesya kuchitila kuti kube kugwisigwa kwachiindi chifwiifwi akubamba sampulu. Kumasunko ayanda kulemba glucose, bikka zibeela zyabongo zibikkidwe muchibaka chakusaanguna, chili a saturated carbon (95% O2 a 5% CO2), Ca2 + ACSF (125.0 mM NaCl, 2.5 mM KCl, 1.25 mM sodium phosphate buffer, 2.5 mM NaCl, 205 mM. d-glucose, 1.0 mM CaCl 2 a 2.0 mM MgCl 2, yakacincwa kusika ku pH 7.4 a 310 kusika ku 320 mOsm), oomo glucose moili 13 C 6- Glucose substitution (Eurisotop, catalog number CLM-1396). Kumasunko aayandika kulembwa a pyruvate, bikka zibeela zyabongo zipati ku Ca2 + ACSF (125.0 mM NaCl, 2.5 mM KCl, 1.25 mM sodium phosphate buffer, 25.0 mM NaHCO3, 25.0 mM d-glucose, 10 mM Ca.Cm a Add 20 MM. MgCl2, bambulula pH 7.4 a 310 kusika ku 320mOsm), mpawo ubike 1mM 1-[1-13C]pyruvate (Eurisotop, namba ya CLM-1082). Bika zibeela eezyo kwamaminiti aali 90 a 37°C. Kumamanino aakusola, zibeela zyakasanzyigwa aamaanzi (pH 7.4) aajisi 75 mM ammonium carbonate, mpawo zyakabikkwa antoomwe mu 40:40:20 (v:v:v) acetonitrile (ACN): methanol: maanzi. Nikwakainda zibeela zyakabikkwa mu aisi kwamaminiti aali 30, zitondezyo zyakabikkwa mu 21,000 g kwamaminiti aali 10 a 4°C, mpawo maanzi aasalala akayuminizyigwa mu SpeedVac concentrator. Ibbwe lya metabolite lyakayuma lyakabambwa a -80°C kusikila lyakavwuntauzyigwa.
Kuvwuntauzya kwama amino acid aali kkumi atutatu aalembedwe kuti C. Mukuvwuntauzya kwa liquid chromatography-mass spectrometry (LC-MS), musamu wa metabolite wakabikkwa alimwi mu 75μl yamaanzi aa LC-MS (Honeywell). Musule akusanzya a 21,000 g kwamaminiti aali 5 a 4°C, 20 μl ya supernatant yakasalala yakabelesegwa kulanga-langa amino acid flux, mpawo zyoonse zyakagwisigwa zyakabelesegwa kulanga-langa anion (langa munsi). Kuvwuntauzya kwa amino acid kwakachitwa kubelesya nzila ya benzoyl chloride derivatization yakaambwa musule (55, 56). Munzila yakusaanguna, 10μl ya 100 mM sodium carbonate (Sigma-Aldrich) yakabikkwa ku 20μl ya metabolite extract, mpawo 10μl ya 2% benzoyl chloride (Sigma-Aldrich) yakabikkwa ku LC grade ACN. Isampulu yakazungaanizyigwa kwakaindi kaniini mpawo yakabikkwa mu 21,000 g kwamaminiti aali 5 a 20°C. Bweza maanzi aasalala mubbokesi lya 2 ml lya autosampler lijisi gilazi lya conical (200 μl volume). Isampulu zyakavwuntauzyigwa kubelesya Acquity iClass ultra-high performance LC system (Waters) yakaswaangene a Q-Exactive (QE)-HF (Ultra High Field Orbitrap) high-resolution precision mass spectrometer (Thermo Fisher Scientific). Mukulanga-langa, 2μl ya musamu wakabikkwa mu 100×1.0 mm high-strength silica T3 column (Waters) iijisi 1.8μm particles. Mweelwe wakuzuza ngu 100μl/min, mpawo nzila yakukwabilila ilijisi kukwabilila A (10 mM ammonium formate a 0.15% asidi yafoomu mumaanzi) antoomwe akukwabilila B (ACN). Ikusanduka kuli boobu: 0%B a 0 maminiti; 0%B. 0 kusika ku 15% B a 0 kusikila ku 0.1 maminiti; 15 kusika ku 17% B a 0.1 kusikila ku 0.5 maminiti; B a 17 kusika ku 55% a 0.5 kusikila ku 14 maminiti; B a 55 kusikila ku 70% a 14 kusikila ku 14.5 maminiti; a 14.5 kusika ku 70 kusika ku 100% B kumaminiti ali kkumi atutatwe; 100% B mumaminiti aali 18 kusikila ku 19; 100 kusika ku 0% B kumaminiti aali 19 kusikila ku 19.1; 0% B a 19.1 kusikila ku 28 maminiti (55, 56). I QE-HF mass spectrometer ibelekela munzila ya positive ionization a mass range ya m/z (mass/charge ratio) ya 50 kusikila ku 750. Kusalazya kwakabelesegwa nku 60,000, mpawo gain control (AGC) ion target yakajanwa nja 3×106, mpawo ion time mpati 100 milliseconds. Inzila yakusansaila magesi iipya (ESI) ibelekela a 3.5 kV, kupya kwa 250°C, muya uuzwa mumubili wa 60 AU (mayuniti aatali kabotu), antoomwe amuuya uugwasilizya wa 20 AU. 250°C. I lensi ya S ilibikkidwe a 60 AU.
Kuvwuntauzya kwa anion chromatography-MS kwa 13C iilembedwe kuti organic acids. Ma metabolite aakasyaala (55μl) akavwuntauzyigwa kubelesya nzila ya Dionex ion chromatography (ICS 5000+, Thermo Fisher Scientific) yakaswaangene a QE-HF mass spectrometer (Thermo Fisher Scientific). Mubufwiifwi, 5μl ya metabolite yakabikkwa mu Dionex IonPac AS11-HC column iijisi HPLC (2 mm×250 mm, bupati bwatubeela 4μm, Thermo Fisher Scientific) munzila yakusunka mukati kachintu chibeela cha 1. )on. Dionex IonPac AG11-HC guard column (2 mm x 50 mm, 4μm, Thermo Fisher Scientific). Ikupya kwamulongo kulazumanana a 30°C, mpawo autosampler yakabikkwa a 6°C. Belesya bbokesi lya potassium hydroxide lipegwa amaanzi aatakwe ayoni kuti upange potassium hydroxide gradient kwiinda mumuncini uuzwa maanzi. Kwaandaanya zintu zipangwa mumubili amweelwe wa 380μl/min, kubelesya nzila eeyi: 0 kusikila ku 3 maminiti, 10 mM KOH; 3 kusikila ku 12 maminiti, 10 kusikila ku 50 mM KOH; 12 kusikila ku 19 maminiti, 50 kusikila ku 100 mM KOH; 19 kusikila ku 21 maminiti, 100 mM KOH; 21 kusikila ku 21.5 maminiti, 100 kusikila ku 10 mM KOH. Mulongo wakabambululwa aansi aa 10 mM KOH kwamaminiti aali 8.5.
Ma metabolites azwa aswaanganizigwa a 150μl/min isopropanol supplement stream musule lya column mpawo asololelwa ku high-resolution mass spectrometer iibeleka munzila ya negative ionization. MS ili mukulanga bupati kuzwa ku m/z 50 kusika ku 750 a resolution ya 60,000. AGC yakabikkwa a 1×106, mpawo chiindi chilamfu cha ion chakabikkwa a 100 ms. I ESI yakapya yakali kubelesegwa a 3.5 kV. Amwi mabambe aa ion source ngaaya: kupya kwa capillary 275°C; kweenda kwamuwo, 60 AU; kweenda kwamuya uugwasya, 20 AU a 300°C, antoomwe a S lens kubikkwa a 60 AU.
Kuvwuntauzya data ya 13C iilembedwe kuti metabolites. Belesya pulogilamu ya TraceFinder (version 4.2, Thermo Fisher Scientific) mukulanga-langa data ya isotope ratio. Kuzyibwa kwa musamu umwi aumwi kwakasinizyigwa amusamu uusyomeka mpawo wakavwuntauzyigwa. Kuti tuchite buvwuntauzi bwakuvwuzya isotope, busena bwa ion chromatogram (XIC) yakagwisigwa ya 13C isotope (Mn) yakagwisigwa kuzwa ku [M + H] +, ooko n nkoili namba ya kaboni ya musamu uuyanda kubelesegwa, yakabelesegwa kulanga amino acids naakuti [ MH] + yakabelesegwa kulanga ma anions. Kulondoka kwabunji bwa XIC kuli munsi aazibeela zyosanwe muli miliyoni, mpawo kulondoka kwa RT nku 0.05 maminiti. Kuvwuntauzya kwakuvwuzya kuchitwa kwiinda mukubala mweelwe wa isotope imwi aimwi yakajanika kumweelwe wa isotope zyoonse zya musamu uukozyenye. Eezyi zibeela zipegwa mbuli mweelwe wamapesenti kuli isotope imwi aimwi, mpawo mpindu zilatondezyegwa mbuli molar percent enrichment (MPE), mbuli mbokwaambidwe musule (42).
Ibbwe lya neuron lyakayuminina lyakabikkwa antoomwe mu 80% methanol (v/v) iitontola mbuli aisi, lyakavwungululwa, mpawo lyakabikkwa mu -20°C kwamaminiti aali 30. Kozungaanya musamu alimwi mpawo uvwungulule a +4°C kwamaminiti aali 30. Isampulu yakabikkwa a 21,000 g kwamaminiti aali 5 a 4°C, mpawo maanzi aakazwa akabungikwa akuyuminizyigwa kubelesya SpeedVac concentrator a 25°C kuti chibelesyegwe mukulanga-langa. Mbuli mbokwaambidwe atala aawa, kuvwuntauzya kwa LC-MS kwakachitwa kuma amino acid aamaseelo aakasalwa. Kubelesya TraceFinder (version 4.2, Thermo Fisher Scientific), kuvwuntauzya kwa data kwakachitwa kubelesya monoisotopic mass ya compound imwi aimwi. Kubambulula mweelwe wa data ya metabolite kwakachitwa kubelesya pulogilamu ya preprocessCore (57).
Kulibambila zibeela. Mbeba yakafwambaanisigwa a carbon dioxide mpawo yakosolwa mutwe, boongo bwakagwisigwa kuzwa kumutwe, mpawo peni lyakazwide aayizi (HM-650 V, Thermo Fisher Scientific, Walldorf, Germany) lyakabelesegwa kutebula muzibeela zya 300 kusikila ku 375 μm sagittal gas carbon gas (25% O) Ca2 + ACSF nsyoonto (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 a 6.0 mM MgCl2 Kuchincha kusika ku pH 37 a 30 mO). Bweza zibeela zyabongo zyajanwa muchibaka chili a Ca2 + ACSF (125.0 mM NaCl, 2.5 mM KCl, 1.25 mM sodium phosphate buffer, 25.0 mM NaHCO3, 25.0 mM d-glucose, 4.0 mM Ca 2.2 mM a mM 2 mC 7.4 a 310 kusika ku 320 mOsm). Bikka zibeela kwamaminiti aali 20 kusikila ku 30 kuchitila kuti zikonzye kujokolosyegwa kakutana lekkoodwa.
kulekkooda. Chibeela cha microscope chijisi busena bwakulekkooda butazungaani antoomwe a 20x water immersion objective lens (Scientifica) chakabelesegwa mukulekkooda koonse. Maseelo aa Purkinje ayeeyelwa akajanwa a (i) bupati bwamubili, (ii) busena bwabongo, antoomwe a (iii) kutondezya kwa fluorescent mtYFP reporter gene. Ipipeti yachikwabilizyo iijisi nguzu zyakukwabilila kuzwa ku 5 kusikila ku 11 megohms ilagwisigwa a borosilicate glass capillary (GB150-10, 0.86 mm×1.5 mm×100 mm, Science Products, Hofheim, Germany) antoomwe a pipette iilangene Instruments (P-10000), Notova, CA). Kulekkooda koonse kwakachitwa a ELC-03XS npi patch clamp amplifier (npi electronic GmbH, Tam, Germany), eeyo yakali kweendelezyegwa a software Signal (version 6.0, Cambridge Electronic, Cambridge, UK). Kusola ooku kwakalembwa amweelwe wa 12.5 kHz. Chitondeezyo chisefwa amasefa obile aa Bessel aajisi 1.3 a 10 kHz kweendelana. Inguzu zya membrane a pipette zilabbadelwa a compensation circuit kubelesya amplifier. Zyoonse zyakusola zyakachitilwa kweendelezyegwa a Orca-Flash 4.0 camera (Hamamatsu, Gerden, Germany), eeyo yakali kweendelezyegwa a Hokawo software (version 2.8, Hamamatsu, Gerden, Germany).
Kubamba akulanga-langa maseelo woonse. Kakutanaba kulekkooda, zuzya pipette amusamu wamukati uulaa zintu eezyi: 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 (MMM), 0.5 mM Gunophosiphate. (GTP) (Na) antoomwe a 10.0 mM creatinine phosphate zyakabambululwa kusika ku pH 7.25, mpawo osmotic pressure yakali 290 mOsm (sucrose). Mpawo-mpawo nikwakamana kubelesya manguzu aa 0 pA kutyolaula chikumba, nguzu zyachikumba zyakali kulyookezya zyakapimwa. Ikukazyigwa kwakunjila kulapimwa akubelesya nguzu zya -40, -30, -20, antoomwe a -10 pA. Pima bupati bwakuvwiila kwanguzu zyamagesi mpawo ubelesye mulawu wa Ohm kuti ubale kukazyigwa kwakunjilila. Kuchita kwakutayeeyela kwakalembwa muchikwabilizyo chamagesi kwamaminiti aali 5, mpawo sPSC yakajanwa akupimwa mu Igor Pro (version 32 7.01, WaveMetrics, Lake Oswego, Oregon, USA) kubelesya semi-automatic recognition script. IV curve antoomwe a steady-state current zilapimwa akujatilila bbattery aamanguzu aasiyene (kutalikila ku -110 mV) akuyungizya nguzu zya magesi mu 5 mV ntaamu. Kupanga kwa AP kwakasunkwa akubelesya nguzu zya depolarizing current. Jama selo a -70 mV mpawo kobelesya nguzu zyakugwisya nguzu. Bambulula bupati bwantaamu yachintu chimwi achimwi chakulekkooda (10 kusika ku 60 pA). Bala AP frequency mpati kwiinda mukubala amaanza ma pulse spikes aaleta AP frequency mpati. I AP threshold ilalingulwa kubelesya nzila yabili ya depolarization pulse eeyo iisaanguna kutalisya AP yomwe naa zyiingi.
Kubamba kwachibaka chakavwumbidwe akuvwuntauzya. Chita kulekkooda kwamabala aajisi zilonda kobelesya nzila zizibilikide. Belesya ATP- a GTP-free pipette iitakwe zintu eezyi: 128 mM gluconate K, 10 mM KCl, 10 mM Hepes, 0.1 mM EGTA antoomwe a 2 mM MgCl2, mpawo ubweze pH 7.2 (kubelesya KOH). ATP a GTP zilagwisigwa mumusamu uuli mukati kamaseelo kuchitila kutabilila kunjila kutali kabotu kwachikumba chamaseelo. Ibbokesi lyachikwabilizyo lilazuzyigwa amusamu wamukati uujisi amphotericin (aasika ku 200 kusikila ku 250 μg/ml; G4888, Sigma-Aldrich) kuchitila kuti kujane lugwalo lwachikwabilizyo chakavwumbidwe. Amphotericin yakanyonyoonwa mu dimethyl sulfoxide (kumamanino: 0.1 kusika ku 0.3%; DMSO; D8418, Sigma-Aldrich). Bunji bwa DMSO yakabelesegwa tiibwakajisi mpindu mpati kuma neurons aakavwuntauzyigwa. Muchiindi chakuuma, kukazyigwa kwanzila (Ra) kwakali kulangilwa lyoonse, mpawo kusunka kwakatalisyigwa nikwakamana kukkalwa kwa Ra a AP (maminiti aali 20-40). Kuchita kwakutayeeyelwa kulapimwa muchikwabilizyo chamagesi a/naakuti nguzu zyamagesi kwamaminiti aali 2 kusikila kuli 5. Kuvwuntauzya data kwakachitwa kubelesya Igor Pro (version 7.05.2, WaveMetrics, USA), Excel (version 2010, Microsoft Corporation, Redmond, USA) antoomwe a GraphPad Prism (version 8.1.2, GraphPad Software Inc., La Jolla, CA). I Amelika). Kuti tuzyibe ma AP aatalibonyi, IgorPro's NeuroMatic v3.0c plug-in ilabelesyegwa. Kozyiba ma APs kolikke kubelesya mweelwe uubikkidwe, ooyo uucincwa umwi awumwi kuli zyoonse zilembedwe. Kubelesya chiindi chama spike, ziba mweelwe wa spike antoomwe a frequency ya spike mpati loko antoomwe a mweelwe waakati aakati wa spike frequency.
PN isolation. Kwiinda mukuzyibila nzila yakamwaigwa musule, PNs yakasalazyigwa kuzwa kubongo bwa mbeba achiindi cheelede (58). Mubufwiifwi, cerebellum yakakosolwa akukosolwa mu ice-cold dissociation medium [kakwiina HBSS Ca2+ a Mg2+, yakayungizigwa a 20 mM glucose, penicillin (50 U/ml) a streptomycin (0.05 mg/ ml)], mpawo yakagaya mu medium ya papain [HBSS, HC2+ a steine (1 mg / ml), papain (16 U / ml) antoomwe a deoxyribonuclease I (DNase I; 0.1 mg/ml)] Kosilika kwamaminiti aali 30 a 30°C. Kusaanguna kosanzya nyama mu HBSS iijisi mafwuta aamazai (10 mg/ml), BSA (10 mg/ml) a DNase (0.1 mg/ml) aawo mpokupya kutegwa kutabi kugaya kwa enzymatic, mpawo mu HBSS iijisi 20 mM glucose Kugaya kabotu-kabotu mu HBSS peilin, peillin/ml/ml, strep (0.05 mg/ml) antoomwe a DNase (0.1 mg/ml) zigwisya maseelo aalikke. Maselu aakazwa akasefwa kwiinda mu 70μm cell strainer, mpawo maselu akakosolwa kwiinda mukubelesya nzila ya centrifugation (1110 rpm, 5 minutes, 4°C) mpawo akabikkwa mubusena bwakusalazya [HBSS, yakayungizigwa a 20 mM glucose, 20% fetal bovine ) Serum, pelinic (50) U/5 (0.05 mg/ml)]; kulanga-langa buumi bwamaseelo a propidium iodide mpawo uchinche bunji bwamaseelo kusika ku 1×106 kusika ku 2×106 maseelo/ml. Kakutanaba flow cytometry, musamu wakasefwa kwiinda mu 50 μm cell strainer.
Flow cytometer. Kusalazya maseelo kwakachitwa a 4°C kubelesya FACSAria III muchini (BD Biosciences) antoomwe a FACSDiva software (BD Biosciences, version 8.0.1). Ikujatikizigwa kwamaseelo kwakasalwa kubelesya 100 μm nozzle munsi lya 20 psi amweelwe wa ~2800 events/sec. Mbwaanga nzila zyakujala zyakale (bupati bwamaseelo, kusalulula kwazibeela zyobile, abube bwakumwaika) tazikonzyi kusinizya kwaandaanya PN kuzwa kumisyobo imwi yamaseelo, nzila yakujana nzila yabikkwa kweendelana akweezyanisya kwa YFP intensity a autofluorescence mu mitoYFP+ ​​alimwi a control mitoYFP − Mice. YFP ilabotelwa akubelesya mumuni wa 488 nm laser line, mpawo chitondezyo chajanwa kubelesya 530/30 nm band pass filter. Mumbeba zya mitoYFP+, manguzu aa Rosa26-mitoYFP reporter gene alabelesyegwa kwaandaanya mubili wa neuronal azibeela zya axon. 7-AAD ilabotelwa a 561 nm laser yellow mpawo yakajanwa a 675/20 nm bandpass filter kuti igwisye maselu aafwide. Kuti kwaandaanye ma astrocytes achiindi chomwe, maseelo akabikkwa ACSA-2-APC, mpawo musamu wakabikkwa mumumuni wa 640 nm laser line, mpawo 660/20 nm bandpass filter yakabelesegwa kujana citondezyo.
Maseelo aakabungikwa akakosolwa kwiinda mukubelesya nzila ya centrifugation (1110 rpm, 5 minutes, 4°C) mpawo akabikkwa a -80°C kusikila akabelesegwe. Mfn2cKO mbeba abana babo balabikkwa muzibeela mubuzuba bomwe kuchitila kuti kutabi kusiyana kwanzila. Kutondezya akuvwuntauzya kwa FACS kwakachitwa kubelesya pulogilamu ya FlowJo (FlowJo LLC, Ashland, Oregon, USA).
Mbuli mbukwaambidwe atala aawa (59), PCR yachiindi cheenzu ilabelesyegwa kwaandaanya DNA kuzwa kuma neurons aasalidwe kuti mtDNA ibelesyegwe. Bwiime bwakutobelezya antoomwe akumvwisisisya kwakusaanguna kwakasunkwa kwiinda mukubelesya qPCR kunamba zyamaseelo zisiyene. Mubufwiifwi, bweza 300 PN mu lysis buffer iijisi 50 mM tris-HCl (pH 8.5), 1 mM EDTA, 0.5% Tween 20 antoomwe a proteinase K (200 ng/ml) mpawo ubike a 55°C 120 maminiti. Maseelo akayungizyigwa kuyobolwa a 95°C kwamaminiti aali kkumi kuchitila kuti proteinase K itabeleki pe. Kubelesya TaqMan probe (Thermo Fisher) iijatikizya mt-Nd1, mtDNA yakapimwa a semi-quantitative PCR mu 7900HT Real-Time PCR system (Thermoherific Scientifics). Sayensi, namba yamulongo Mm04225274_s1), mt-Nd6 (Thermo Fisher Scientific, namba yamulongo AIVI3E8) antoomwe a 18S (Thermo Fisher Scientific, namba yamulongo Hs99999901_s1) ma genes.
Kubamba sampulu ya proteome. Kwiinda mukupya musamu a 95°C kwamaminiti aali kkumi akuumpa, mu lysis buffer [6 M guanidine chloride, 10 mM tris(2-carboxyethyl) phosphine hydrochloride, 10 mM chloroacetamide a 100 mM tris- Lyse frozen neuron pellets in HC pellets]. A Bioruptor (Diagenode) kwamaminiti aali kkumi (masekondi aali makumi otatwe aakuuma / masekondi aali makumi otatwe aakulyookezya). Isampulu yakasanzyigwa 1:10 mu 20 mM tris-HCl (pH 8.0), yakasanganyigwa a 300 ng ya trypsin gold (Promega), mpawo yakabikkwa mubusiku boonse a 37°C kuti izuzikizye kugaya. Mubuzuba bwabili, musamu wakabikwa a 20,000 g kwamaminiti aali 20. Maanzi aakasyaala akasanzyigwa a 0.1% ya asidi yafoomu, mpawo musamu wakagwisigwa munyo a StageTips yakapangwa. Isampulu yakayuminizyigwa muchibelesyo cha SpeedVac (Eppendorf concentrator plus 5305) a 45°C, mpawo peptide yakabikkwa mu 0.1% formic acid. Zitondezyo zyoonse zyakabambwa achiindi chomwe amuntu omwe. Mukulanga-langa zitondezyo zya astrocyte, 4 μg yama peptide aatakwe munyo akalembedwe a tandem mass tag (TMT10plex, catalog number 90110, Thermo Fisher Scientific) a peptide ku TMT reagent ratio ya 1:20. Mukulemba TMT, 0.8 mg ya TMT reagent yakabikkwa alimwi mu 70 μl ya ACN iitakwe maanzi, mpawo peptide yakayumide yakabikkwa alimwi mu 9 μl ya 0.1 M TEAB (triethylammonium bicarbonate), ooko 7 μl ya TMT reagent mu ACN nkoyakayungizyigwa. Bunji bwazintu bwakali 43.75%. Nikwakainda maminiti aali makumi musanu aabili aakukkazika, kusanduka kwakazimwa a 2 μl ya 5% hydroxylamine. Ma peptide aakalembwa akabungikwa, akayuminizyigwa, akabikkwa mu 200μl ya 0.1% formic acid (FA), akaandaanizyigwa muzibeela zyobile, mpawo akagwisigwa munyo kubelesya StageTips yakapangwa. Kubelesya UltiMate 3000 ultra high performance liquid chromatograph (UltiMate 3000 ultra high performance liquid chromatograph), chimwi chazibeela zyobile chakakosolwa a 1mm x 150mm Acquity chromatographic column yakazwide 130Å1.7μm C18 particles (Waters, catalog No SKU: 196366). Thermo Fisher Scientific). Aandaanya ma peptide aamweelwe wa 30μl/min, aandaanye kuzwa ku 1% kusika ku 50% buffer B kwamaminiti aali 85 anzila ya 96 maminiti, kuzwa ku 50% kusika ku 95% buffer B kwamaminiti otatwe, mpawo maminiti aali 8 kuya ku 95 % Buffer B; Buffer A ngu 5% ACN a 10 mM ammonium bicarbonate (ABC), mpawo buffer B ngu 80% ACN a 10 mM ABC. Kobweza zibeela mumaminiti otatwe mpawo uzisanganye mutubunga tobile (1 + 17, 2 + 18, azimwi) mpawo uziyuminizye mumuncini uuzwa mumulengalenga.
Kuvwuntauzya kwa LC-MS/MS. Mukupima bunji bwazintu, ma peptide (namba r119.aq) akaandaanizyigwa a 25 cm, 75 μm mukati ka PicoFrit analytical column (new objective lens, part number PF7508250) iijisi 1.9 μm ReproSil-Pur 120 C18-AQ medium (Maisch, ELC-D), Maisch-YAS. 1200 (Thermo Fisher Scientific, Germany). Mulongo wakabamba a 50°C. Zikwabilizyo A a B zili 0.1% asidi yafoomu mumaanzi a 0.1% asidi yafoomu mu 80% ACN, kweendelana. Ma peptide akaandaanizyigwa a 200 nl/min kuzwa ku 6% kusika ku 31% buffer B kwamaminiti aali 65, mpawo kuzwa ku 31% kusika ku 50% buffer B kwamaminiti aali 5. Ma peptide aakagwisigwa akavwuntauzyigwa a Orbitrap Fusion mass spectrometer (Thermo Fisher Scientific). Kupima kwa peptide precursor m/z kuchitwa akusalazya kwa 120,000 aakati ka 350 kusika ku 1500 m/z. Kubelesya 27% yanguzu zyakudonkolana, chintu chiyumu loko chilaanguzu zya 2 kusikila ku 6 chasalwa kuti chibelesyegwe munguzu zya C trap dissociation (HCD) cleavage. Chiindi chakuzyunguluka chabikkwa a 1 s. M/z value ya peptide fragment yakapimwa mu ion trap kubelesya AGC target nsyoonto ya 5×104 antoomwe achiindi chilamfu chakunjizya cha 86 ms. Nikwakamana kutyompokelwa, chitondezyo chakabikkwa mumulongo wakugwisya kwachiindi chamasekondi aali makumi aane amusanu. Ma peptide aalembedwe TMT akaandaanizyigwa a 50 cm, 75 μm Acclaim PepMap column (Thermo Fisher Scientific, catalog namba 164942), mpawo kweendeenda kwakavwuntauzyigwa a Orbitrap Lumos Tribrid mass spectrometer (Thermo Fisher Scientific) iijisi high-ammetric iform iMS (IMS) zibelesyo (Thermo Fisher Scientific) zibeleka aama voltage obile aa −50 a −70 V. MS3 yasalwa kweendelana a synchronization precursor ibelesyegwa mu TMT report ion signal measurement. Kwaandaanya ma peptide kwakachitwa a EASY-nLC 1200, kubelesya 90% ya linear gradient elution, a buffer concentration ya 6% kusika ku 31%; chikwabilizyo A chakali 0.1% FA, mpawo chikwabilizyo B chakali 0.1% FA a 80% ACN. Mulongo wakulanga-langa ubelekela a 50°C. Belesya FreeStyle (version 1.6, Thermo Fisher Scientific) kwaandaanya fayela lyakusaanguna kweendelanya a FAIMS compensation voltage.
Kuziba ma protein akupima mweelwe. Kubelesya injini yakuyandaula ya Andromeda, data yakusaanguna yakavwuntauzyigwa kubelesya MaxQuant version 1.5.2.8 (https://maxquant.org/). Mukuyungizya ku Cre recombinase antoomwe a YFP zyakajanwa kuzwa ku Aequorea victoria, peptide fragment spectra zyakavwuntauzyigwa kuti zibe zya canonical sequence antoomwe a isoform sequence ya mouse reference proteome (Proteome ID UP000000589, yakagwisigwa kuzwa ku UniProt mu May 2017). Methionine oxidation antoomwe a protein N-terminal acetylation zyakabikkwa mbuli zintu zikonzya kuchincha; cysteine ​​ficarbamoyl methylation yakabikkwa mbuli zintu zitacinci. Zibeela zyakugaya zyakulya zyabikkwa a "kusalala" antoomwe a "trypsin/P". Mweelwe musyoonto wama peptide ama peptide aabelesyegwa kuzyiba ma protein ngu 1; mweelwe musyoonto wama peptide aalibedelede ngu 0. Mubukkale bwakukozyanisya maapu yama peptide, mweelwe wakuzyibwa kwama protein wakali 0.01. Inzila ya "Peptide yabili" ilizumizyidwe. Belesya nzila ya "kweezyanisya aakati kakuchijaana" kuti ubweze zitondezyo zyazwidilila aakati kama fayela aasiyene-siyene. Belesya LFQ mweelwe muniini wakubala 1 mukupima kakunyina kulemba (LFQ) (60). Bupati bwa LFQ bulasefwa kuti butasiki kuli zyobile zili kabotu mukabunga komwe ka genotype achiindi choonse, mpawo bulagwisigwa kuzwa kukumwaika kuli kabotu abwamba bwa. 0.3 mpawo uselemuke 1.8. Belesya Perseus computing platform (https://maxquant.net/perseus/) antoomwe a R (https://r-project.org/) kulanga-langa mpindu zya LFQ. Nzila zyobile zya t test kuzwa ku limma software package yakabelesegwa mukuvwuntauzya kwakusiyana (61). Kuvwuntauzya kwa data kwachitwa kubelesya ggplot, FactoMineR, factoextra, GGally antoomwe a pheatmap. Data ya TMT-based proteomics yakavwuntauzyigwa kubelesya MaxQuant version 1.6.10.43. Yanduula data yama proteomics kuzwa ku UniProt's human proteomics database, eeyo yakagwisigwa mu September 2018. Kuvwuntauzya ooku kubikkilizya a isotope purity correction factor yakapegwa abasikupanga. Belesya limma mu R mukulanga-langa kwiimpana kwamajwi. Data yakusaanguna, mpindu zyakuvwuntauzya, anzila zyakuvwuntauzya data zyoonse zilabikkwa mu ProteomeXchange alliance kwiinda mu PRIDE partner repository antoomwe a data set identifier PXD019690.
Malembe aabeleka alasumpula buvwuntauzi. Chibelesyo cha Ingenuity Pathway Analysis (QIAGEN) chakabelesegwa kujana buvubi bwamabala aabelesyegwa a data yakabikkwa mumviki zili 8 (Chitondeezyo 1). Mubufwiifwi, mulongo wamapulotini aajanwa kuzwa ku LC-MS/MS (tandem mass spectrometry) data analysis ubelesyegwa anzila zyakusefa zitobela: Mus musculus wasalwa mbuli musyobo alubono, mpawo chibaka chitondezya kuti P value yakabambululwa a Benjamini kuti ibe 0.05 naa munsi ilabonwa kuti ilayandika. Ku graph eeyi, zibeela zyosanwe zyaatala zyaatala mukabunga kamwi akamwi kweendelanya a P value yakacincwa zilatondezyegwa. Kubelesya t-test myiingi, kubelesya pulogilamu ya Benjamini, Krieger, a Yekutieli (Q = 5%), kuvwuntauzya kwakutondezya ma protein kwachiindi chilachitwa kuli baabo bayandikana bakajanwa mumulongo umwi aumwi, mpawo mulongo umwi aumwi ulavwuntauzyigwa alikke. Takuyandiki pe kutambula SD iitacinci.
Mukweezyanisya mpindu zya chiiyo eechi ama databezi aakamwaigwa akupanga mufanikisyo wa Venn muchifanikisyo 1, twakaswaanganya mulongo wamapulotini a MitoCarta 2.0 annotations (24). Belesya chibelesyo chaa intaneti cha Draw Venn Diagram (http://bioinformatics.psb.ugent.be/webtools/Venn/) kuti upange mufanikisyo.
Kuti ujane makani manji aatala anzila zyakubala zibelesyegwa mukuvwuntauzya ma proteomics, chonde kolanga chibeela cha Materials and Methods. Kumasunko aambi woonse, makani aasalala alakonzya kujanwa mumakani aakozyenye. Kusikila kwaambwa bumbi, data yoonse itondezyedwe mbuli mweelwe ± SEM, mpawo zyoonse zyakuvwuntauzya zyakachitilwa kubelesya pulogilamu ya GraphPad Prism 8.1.2.
Kuti ujane zimwi zilembedwe muchibalo eechi, ndakulomba kuti ubone http://advances.sciencemag.org/cgi/content/full/6/35/eaba8271/DC1
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Kuli E. Motori, I. Atanassov, SMV Kochan, K. Folz-Donahue, V. Sakthivelu, P. Giavalisco, N. Toni, J. Puyal, N.-G. Larson
Kuvwuntauzya kwa Proteomics kwama neurons aatabeleki kabotu kwakatondeezya kuti mapulogilamu aa metabolic alabeleka kulwana kubola kwabongo.
Kuli E. Motori, I. Atanassov, SMV Kochan, K. Folz-Donahue, V. Sakthivelu, P. Giavalisco, N. Toni, J. Puyal, N.-G. Larson
Kuvwuntauzya kwa Proteomics kwama neurons aatabeleki kabotu kwakatondeezya kuti mapulogilamu aa metabolic alabeleka kulwana kubola kwabongo.
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