Ikheli lamanje *Ikheli lamanje: Cologne 50931, e-Germany, i-Cologne Excellence Cluster Research on Cellular Stress Response in Aging-related Diseases (CECAD).
Ukuwohloka kwemizwa yamalwelwe we-mitochondrial kuthathwa njengokungabuyiseleki emuva ngombana iplastiki ye-metabolic yama-neuron ilinganiselwe, kodwana umphumela wokungasebenzi kuhle kwe-mitochondrial ekuzimeleni kwamaseli we-metabolism ye-neuron emzimbeni awuzwisiseki kuhle. Lapha, sethula i-proteome ekhethekileko yamaseli we-Purkinje neurons ngokutlhogeka kwe-OXPHOS okubangelwa kuphazamiseka kokuhlangana kwe-mitochondrial. Sithole bona ukungasebenzi kuhle kwe-mitochondrial kwabangela itjhuguluko elikhulu emkhakheni we-proteomics, ekugcineni kwaholela ekusebenzeni okulandelanako kwamahlelo we-metabolic ngaphambi kokufa kwamaseli. Ngokungakalindeleki, sathola ukungeniswa okusobala kwe-pyruvate carboxylase (PCx) namanye ama-peroxidases ahlobene nokungezelela ama-TCA cycle intermediates. Ukuvinjelwa kwe-PCx kwakhuphula ukugandeleleka kwe-oxidative kanye nokuwohloka kwemithambo yeengazi, okutjengisa bona i-atherosclerosis inomphumela wokuvikela eenzwaneni ezingenayo i-OXPHOS. Ukubuyiselwa kwe-mitochondrial fusion kuma-neurons aphelelwe sikhathi kuguqula ngokupheleleko ubujamo bokugaya ukudla, ngaleyo ndlela kuvimbele ukufa kwamaseli. Okutholwe sikhomba iindlela ebezingaziwa ngaphambilini ezinikela ukujamelana nokungasebenzi kuhle kwe-mitochondrial begodu zitjengisa bona ukuwohloka kwemizwa kungabuyiselwa emuva ngitjho nasesigabeni sokugcina sesifo.
Indima eqakathekileko ye-mitochondria ekugcineni ukusebenza kwamandla we-neuron igandelelwa ziimpawu ezinengi ze-neurological ezihlobene namalwelwe we-mitochondria yabantu. Inengi lamalwelwe la abangelwa kutjhuguluka kwamajini alawula ukuvezwa kwamajini we-mitochondrial (1, 2) nofana ukubhujiswa kwamajini okuhlobene nokuguquguquka kwe-mitochondrial, okuthinta ngokungakaqondi ukuzinza kwe-DNA ye-mitochondrial (mtDNA) (3, 4). Umsebenzi emamodelini weenlwana utjengise bona ngokuphendula ukungasebenzi kuhle kwe-mitochondrial eencutjhweni ezizungezeko, iindlela zokugaya ukudla ezilondolozako (5-7) zingasebenza, lokho kunikela ilwazi eliqakathekileko nge-pathogenesis yalezi zifo eziyinkimbinkimbi. Ngokuphikisako, ukuzwisisa kwethu amatjhuguluko wokugaya ukudla kwemihlobo ethileko yamaseli abangelwa kukubhalelwa kokukhiqizwa kwe-brain mitochondrial adenosine triphosphate (ATP) kuqakathekile (8), kugandelela isidingo sokukhomba amathagethi wezokwelapha angasetjenziselwa ukukhandela nofana ukukhandela amalwelwe. Vimbela ukuwohloka kwemizwa (9). Ukutlhogeka kwelwazi kuliqiniso lokuthi amaseli wezinzwa athathwa njengalawo anokuguquguquka okuncani khulu nakuqathaniswa nemihlobo yamaseli wezicubu ezizungezeko (10). Njengombana amaseli la adlala indima eqakathekileko ekuhlanganiseni ukunikelwa kwama-metabolites kuma-neurons ukukhuthaza ukudluliselwa kwe-synaptic nokuphendula ebujameni bokulimala nezifo, ikghono lokutjhugulula ukugaya ukudla kwamaseli ebujameni obubudisi bezicubu zobuchopho pheze lilinganiselwe kumaseli we-glial (11-14). Ukungezelela, ukuhlukahluka kwamaseli wezicubu zobuchopho kuvimbela khulu ukufundwa kwamatjhuguluko we-metabolic enzeka emaqenjini athileko we-neuronal. Njengomphumela, kuncani okwaziwako ngemiphumela yamaseli neyokugaya ukudla yokungasebenzi kuhle kwe-mitochondrial kuma-neuron.
Ukuze sizwisise imiphumela yokugaya ukudla yokungasebenzi kuhle kwe-mitochondrial, sihlukanise ama-Purkinje neurons (ama-PN) emazingeni ahlukileko wokuwohloka kwe-neuro okubangelwa kutjhabalaliswa kwe-mitochondrial outer membrane fusion (Mfn2). Nanyana ukutjhuguluka kwe-Mfn2 ebantwini kuhlotjaniswa nohlobo lwe-hereditary motor sensory neuropathy eyaziwa nge-Charcot-Marie-Tooth type 2A (15), ukubhujiswa okunemibandela kwe-Mfn2 emagundeni kuyindlela eyaziwako yokungeniswa kwe-oxidation Phosphorylation (OXPHOS) dysfunction method. Iinhlobo ezihlukahlukeneko ze-neuronal (16-19) kanye nomphumela we-neurodegenerative phenotype zihambisana namatshwayo we-neurological, afana nokuphazamiseka kokunyakazisa (18, 19) nofana i-taxia ye-cerebellar (16). Ngokusebenzisa inhlanganisela ye-label-free quantitative (LFQ) proteomics, metabolomics, imaging, kanye neendlela ze-virological, sitjengisa bona ukuwohloka kwe-neurodegeneration okuragela phambili kubangela khulu i-pyruvate carboxylase (PCx) nezinye izinto ezithintekako e-arteriosclerosis yama-PNs in vivo Ukuvezwa kwama-enzayimu. Ukuqinisekisa ukufaneleka kwalokhu okutholakeleko, silawule phasi ukuvezwa kwe-PCx kuma-PN atlhoga i-Mfn2, begodu sathola bona ukusebenza lokhu kwakhuphula ukugandeleleka kwe-oxidative begodu kwakhawulezisa ukuwohloka kwemizwa, lokho kufakazela bona i-azoospermia inikela ukufa kwamaseli ukuvumelana nokutjhuguluka kwe-Metabolic. Ukuvezwa okukhulu kwe-MFN2 kungasindisa ngokupheleleko ukuwohloka kwe-PN ngokutlhogeka okukhulu kwe-OXPHOS, ukusetjenziswa okukhulu kwe-DNA ye-mitochondrial, kanye nenethiwekhi ye-mitochondrial ephukileko, okuragela phambili nokugandelela bona indlela le yokuwohloka kwemizwa ingalulama esigabeni esiphambili sesifo ngaphambi kokufa kwamaseli.
Ukuze sibone ngelihlo lengqondo i-mitochondria ku-Mfn2 knockout PNs, sisebenzise umhlobo wegundane ovumela i-Cre-dependent mitochondria bona ihlose i-yellow fluorescent protein (YFP) (mtYFP) (20) Cre expression begodu sihlole i-mitochondrial morphology ku-vivo. Sithole bona ukubhujiswa kwejini le-Mfn2 kuma-PN kuzokurholela ekuhlukanisweni kancanikancani kwenethiwekhi ye-mitochondrial (Umfanekiso S1A), begodu ukutjhuguluka kokuthoma kwatholakala emavikini ama-3 ubudala. Ngokuphikisako, ukuwohloka okukhulu kwesendlalelo seseli le-PN, njengombana kufakazelwa kulahleka kwe-Calbindin immunostaining, azange kuthome bekwaba ziimveke ezili-12 (Umfanekiso 1, A no-B). Ukungafani kwesikhathi hlangana namatjhuguluko wokuthoma we-mitochondrial morphology nokuthoma okubonakalako kokufa kwe-neuron kwasenza bona siphenye amatjhuguluko we-metabolic abangelwa kukungasebenzi kuhle kwe-mitochondrial ngaphambi kokufa kwamaseli. Sakha iqhinga lokuhlunga amaseli elisebenza nge-fluorescence (FACS) ukuhlukanisa i-YFP (YFP+)-expressing PN (Umfanekiso 1C), kanye namagundane wokulawula (Mfn2 + / loxP :: mtYFP loxP- stop-loxP: : L7-cre), lapha okubizwa ngokuthi yi-CTRL (Umfanekiso S1BRL). Ukuthuthukiswa kweqhinga lokungena ngokuya ngokuqina kwesiginali ye-YFP kusivumela bona sihlanze umzimba we-YFP+ (YFPhigh) wama-PN ukusuka kuma-PN (YFPneg) (Umfanekiso S1B) nofana ama-axon/dendritic fragments we-fluorescent (YFPlow; Umfanekiso S1D, kwesokunxele), okuqinisekiswe yi-confocal microscope (Umfanekiso S1D, kwesokudla). Ukuqinisekisa ubunikazi besibalo esihlukanisiweko, senze i-LFQ proteomics bese senza ukuhlaziywa kweengcenye eziphambili, begodu sathola bona kunokuhlukana okucacileko hlangana namaseli we-YFPhigh ne-YFPneg (Umfanekiso S1C). Amaseli we-YFPhigh atjengisa ukunothiswa okupheleleko kwamamaki we-PNs aziwako (okutjho i-Calb1, Pcp2, Grid2 kanye ne-Itpr3) (21, 22), kodwana akukho ukunothiswa kwamaphrotheyini avamise ukuvezwa kuma-neurons nofana eminye imihlobo yamaseli (Umfanekiso 1D) ). Ukumadanisa hlangana namasampula wamaseli we-YFPhigh ahlukanisiweko abuthelelwe ekuhlolweni okuzijameleko kutjengise i-correlation coefficient> 0.9, okutjengisa ukukhiqiza okuhle hlangana nokuphindaphinda kwezinto eziphilako (Umfanekiso S1E). Ngokufingqa, idatha le iqinisekise ihlelo lethu lokuhlukaniswa okubukhali nokuthileko kwe-PN engenzeka. Ngombana ihlelo lomtjhayeli we-L7-cre elisetjenzisiweko likhuthaza ukuhlanganiswa kabutjha kwe-mosaic ngeveke yokuthoma ngemva kokubeletha (23), sathoma ukubulala amagundane avela ku-CTRL begodu sibuthelele ama-neurons. Ngemva kokuthi ukuhlanganiswa kabutjha kuqedwe, kubizwa ngokuthi yi-Mfn2cKO emavikini ama-4 ubudala. Njengesiphetho, sikhethe amaviki abunane wobudala lokha isendlalelo se-PN nasisesekhona nanyana bekuneengcezu ze-mitochondrial ezibonakalako (Umfanekiso 1B noMfanekiso S1A). Sekukonke, salinganisa inani lamaphrotheyini ama-3013, lapho amaphesenti angaba ma-22 bekasekelwe kuma-MitoCarta 2.0 annotations asekelwe ku-mitochondrial proteome njenge-mitochondria (Umfanekiso 1E) (Umfanekiso 1E) (24). Ukuhlaziywa kokuvezwa kwezakhi zofuzo okuhlukileko okwenziwe ngeveke yesi-8 kutjengise bona yi-10.5% kwaphela yawo woke amaphrotheyini anamatjhuguluko aqakathekileko (Umfanekiso 1F nomfanekiso S1F), lapho amaphrotheyini ali-195 alawulwa phasi kuthi amaphrotheyini ali-120 alawulwa phezulu (Umfanekiso 1F). Kuqakathekile ukutjheja bona "ukuhlaziywa kweendlela ezitjha" zesethi yedatha le kutjengisa bona amajini avezwe ngokuhlukileko ikakhulukazi angawesethi ekhawulelweko yeendlela ezithileko zokugaya ukudla (Umfanekiso 1G). Ngokuthakazelisako, nanyana ukwehliswa kweendlela ezihlobene ne-OXPHOS kanye ne-calcium signaling kuqinisekisa ukuthonywa kokungasebenzi kuhle kwe-mitochondrial kuma-PN angabi nokuhlanganiswa, ezinye iingaba ezibandakanya khulu ukugaya ukudla kwe-amino acid zilawulwa khulu, okukhambisana nokusebenza kwe-mitochondrial PN. Ukufaka iintambo kabutjha kuyafana. ukungasebenzi kuhle.
(A) Iinthombe ezijameleko zeengcenye ze-cerebellar ze-CTRL ne-Mfn2cKO ezitjengisa ukulahlekelwa okuragela phambili kwama-PNs (i-calbindin, grey); iinyukliya zafakwa nge-DAPI. (B) Ukulinganisa kwe-(A) (ukuhlaziywa kwendlela eyodwa yokuhluka, ***P<0.001; n = 4 ukuya ku-6 imibuthano evela emagundeni amathathu). (C) Ukusebenza kokuhlola. (D) Ukusabalalisa kwemephu yokutjhisa kwamamaki aqondene ne-Purkinje (phezulu) neminye imihlobo yamaseli (phakathi). (E) Umdwebo we-Venn otjengisa inani lamaphrotheyini we-mitochondrial akhonjwe ku-PN ehlukanisiweko. (F) Iplothi ye-volcano yamaphrotheyini avezwe ngokuhlukileko kuma-neurons we-Mfn2cKO emavikini abunane (inani lokusika eliqakathekileko le-1.3). (G) Ukuhlaziywa kwendlela yokutlama kutjengisa iindlela ezihlanu eziqakatheke khulu zokukhuphuka (ezibomvu) nezokwehla (ezihlaza satjani) ku-Mfn2cKO PN ehlukaniswe njengamaviki ayi-8. Izinga eliphakathi lokuvezwa kwephrotheyini ngayinye etholakeleko liyatjengiswa. Imephu yokutjhisa ye-greyscale: inani le-P elilungisiweko. ns, akukaqakatheki.
Idatha ye-proteomics itjengise bona ukuvezwa kwamaphrotheyini wama-complexes I, III, kanye ne-IV kwehla kancanikancani. Iinhlanganisela I, III, kanye ne-IV zoke bezinama-subunits aqakathekileko we-mtDNA, kuthi i-Complex II, ebegade ikhodiwe nge-nuclear kwaphela, beyingakathinteki (Umfanekiso 2A noMfanekiso S2A). . Ngokuya ngemiphumela ye-proteomics, i-immunohistochemistry yeengcenye zezicubu ze-cerebellar itjengise bona i-MTCO1 (mitochondrial cytochrome C oxidase subunit 1) izinga le-subunit ye-complex IV ku-PN lehla kancanikancani (Umfanekiso 2B). I-mtDNA-encoded subunit Mtatp8 yehliswe khulu (Umfanekiso S2A), lokha izinga elizinzileko le-nuclear-encoded ATP synthase subunit lihlala lingatjhuguluki, okuhambisana ne-ATP synthase subassembly F1 complex eyaziwako lokha i-mtDNA expression izinzile. Ukwakheka kuyafana. Phakamisa (7). Ukuhlolwa kwezinga le-mtDNA kuma-PNs we-Mfn2cKO ahlelwe nge-real-time polymerase chain reaction (qPCR) kwaqinisekisa ukwehla kancanikancani kwenani lamakhophi we-mtDNA. Nawuqathanisa nesiqhema sokulawula, emavikini abunane, kwaba yi-20% kwaphela yezinga le-mtDNA (Umfanekiso 2C). Ngokuvumelana nemiphumela le, ukufakwa kwe-confocal microscopy kwe-Mfn2cKO PNs ukuthola i-DNA kutjengisa ukusetjenziswa kwesikhathi okuthembele ema-mitochondrial nucleotides (Umfanekiso 2D). Sithole bona abanye abakhethiweko abazibandakanya ekuwohlokeni kwamaphrotheyini we-mitochondrial nokuphendula kokugandeleleka abalawulwa phezulu, kufaka hlangana i-Lonp1, i-Afg3l2 kanye ne-Clpx, kanye nama-OXPHOS complex assembly factors. Akukho amatjhuguluko aqakathekileko emazingeni wamaphrotheyini abandakanyeka ekufeni kwawo (Umfanekiso S2B). Ngokufanako, sithole bona ama-mitochondria kanye nama-endoplasmic reticulum channels abandakanyeka ekuthuthweni kwe-calcium anamatjhuguluko amancani (Umfanekiso S2C). Ukungezelela, ukuhlolwa kwamaphrotheyini ahlobene ne-autophagy akutholanga amatjhuguluko aqakathekileko, okuhambisana nokufakwa okubonakalako kwama-autophagosomes okubonwe ku-vivo nge-immunohistochemistry kanye ne-electron microscopy (Umfanekiso S3). Kodwana, ukungasebenzi kuhle kwe-OXPHOS kuma-PN kukhambisana namatjhuguluko abonakalako we-ultrastructural mitochondrial. Amaqoqo we-mitochondrial angabonakala emizimbeni yamaseli kanye nemithi ye-dendritic ye-Mfn2cKO PNs eneminyaka emi-5 ukuya kweyi-8, begodu isakhiwo sangaphakathi se-membrane sitjhuguluke khulu (Umfanekiso S4, A no-B). Ngokuvumelana namatjhuguluko we-ultrastructural kanye nokwehla okukhulu kwe-mtDNA, ukuhlaziywa kweengcezu ze-cerebral cerebellar ezibukhali nge-tetramethylrhodamine methyl ester (TMRM) kutjengise bona amandla we-mitochondrial membrane ku-Mfn2cKO PNs wehlile khulu (Umfanekiso S4C).
(A) Ukuhlaziywa kwesikhathi sezinga lokuvezwa kwe-OXPHOS. Cabanga kwaphela amaphrotheyini ane-P<0.05 emavikini ayi-8 (i-ANOVA yeendlela ezimbili). Umugqa onamachashazi: Akukho ukulungiswa nayiqathaniswa ne-CTRL. (B) Kwesokunxele: Isibonelo sesigaba se-cerebellar esifakwe i-anti-MTCO1 antibody (isikali, 20 μm). Indawo ehlala imizimba yamaseli we-Purkinje imbozwe mthubi. Ngakwesokudla: Ukulinganisa amazinga we-MTCO1 (ukuhlaziywa kwendlela eyodwa yokuhluka; n = 7 ukuya ku-20 amaseli ahlaziywe ukusuka emagundeni amathathu). (C) Ukuhlaziywa kwe-qPCR kwenomboro yekhophi ye-mtDNA ku-PN ehlelweko (ukuhlaziywa kwendlela eyodwa yokuhluka; n = 3 ukuya ku-7 amagundane). (D) Kwesokunxele: Isibonelo sesilayi se-cerebellar esifakwe i-anti-DNA antibody (isikali, 20 μm). Indawo ehlala imizimba yamaseli we-Purkinje imbozwe mthubi. Ngakwesokudla: Ukulinganisa amabala we-mtDNA (ukuhlaziywa kwendlela eyodwa yokuhluka; n = 5 ukuya ku-9 amaseli avela emakhondweni amathathu). (E) Isibonelo sesigaba se-cerebellar esibukhali esitjengisa amaseli we-mitoYFP + Purkinje (umcibisholo) ekurekhodeni kwe-whole-cell patch clamp. (F) Ukulinganisa kwejika le-IV. (G) Ukurekhoda okujameleko kokujova kwamanje okukhipha i-polarizing kumaseli we-CTRL kanye ne-Mfn2cKO Purkinje. Umtlhala ophezulu: I-pulse yokuthoma eyabangela i-AP. Umtlhala ongaphasi: Ifreyikhwensi ephezulu ye-AP. (H) Ukulinganisa ukufakwa okuzenzakalelako kwe-postsynaptic (sPSPs). Umtlhala wokurekhoda omeleko kanye nesilinganiso sawo sokusondeza kutjengiswa ku-(I). Ukuhlaziywa kwendlela eyodwa yokuhluka kuhlaziywe n = 5 ukuya ku-20 amaseli avela emagundeni amathathu. Idatha ivezwa njenge-mean±SEM; *P<0.05; **P<0.01; ***P<0.001. (J) Imikhondo ejameleko ye-AP ezenzekelayo etlolwe ngokusebenzisa imodi yokubopha isiqetjhana esineembobo. Umkhondo ophezulu: Imvamisa ephezulu ye-AP. Umkhondo ongezansi: ukusondeza kwe-AP eyodwa. (K) Qala isilinganiso nesilinganiso esiphezulu se-AP ngokuya ngo-(J). Ukuhlolwa kwe-Mann-Whitney; n = 5 amaseli ahlaziywa ukusuka emagundeni amane. Idatha ivezwa njenge-mean±SEM; akukaqakatheki.
Umonakalo obonakalako we-OXPHOS watholakala ku-Mfn2cKO PN enamaviki abunane, okutjengisa bona ukusebenza komzimba kwama-neuron akukajayeleki khulu. Ngalokho-ke, sihlaziye ubujamo begezi obungenzi litho be-OXPHOS-deficient neurons emavikini ama-4 ukuya kwayi-5 kanye namaviki ama-7 ukuya kwayi-8 ngokwenza ukurekhoda kwe-whole-cell patch clamp eencenyeni ze-cerebellar ezibukhali (Umfanekiso 2E). Ngokungakalindeleki, isilinganiso se-membrane yokuphumula nokujamelana kokufaka kwama-neuron we-Mfn2cKO bekufana nokulawula, nanyana bekunomehluko ongabonakaliko hlangana namaseli (Ithebula 1). Ngokufanako, emavikini ama-4 ukuya kwama-5 ubudala, akukho amatjhuguluko aqakathekileko ebudlelwaneni bevoltheji yamanje (i-IV curve) eyatholakala (Umfanekiso 2F). Kodwana, ayikho i-Mfn2cKO ye-neurons eneemveke ezi-7 ukuya kwezi-8 ezisinde emthethweni we-IV (igadango lokukhula), okutjengisa bona kunokuzwela okucacileko emandleni wokukhula khulu esigabeni sokugcina. Ngokuphikisako, kuma-neurons we-Mfn2cKO, imisinga yokukhipha i-depolarizing ebanga ukukhishwa kwe-action potential (AP) ibekezelelwe kuhle, okutjengisa bona amaphetheni wokukhipha kwawo woke awafani khulu nalawo wama-neurons wokulawula aneveke ezi-8 (Ithebula 1 noMfanekiso 2G). Ngokufanako, ukuvama nobukhulu bama-postsynaptic currents (ama-sPSC) bekufana nalawo wesiqhema sokulawula, begodu ukuvama kwezehlakalo kwanda ukusuka emavikini ama-4 ukuya emavikini amahlanu ukuya emavikini ayi-7 ukuya emavikini abunane ngokunyuka okufanako (Umfanekiso 2, H no-I). Isikhathi sokuvuthwa kwe-synaptic kuma-PN (25). Imiphumela efanako yatholakala ngemva kwamaphetjhana we-PNs abhoboziweko. Ukuhlela lokhu kuvimbela ukubhadelwa okungenzekako kwamaphutha we-ATP yamaseli, njengombana kungenzeka ekurekhodeni kwe-clamp ye-patch yamaseli woke. Ngokukhethekileko, amandla we-membrane yokuphumula kanye nokudubula okuzenzakalelako kwama-neuron we-Mfn2cKO azange kuthinteke (Umfanekiso 2, J no-K). Ngokufingqa, imiphumela le itjengisa bona ama-PN anokungasebenzi kuhle kwe-OXPHOS angajamelana namaphetheni wokukhipha amavama aphezulu kuhle, okutjengisa bona kunendlela yokubhadela ebavumela bona bagcine iimpendulo ze-electrophysiological eziseduze nezijayelekileko.
Idatha ivezwa njenge-mean ± SEM (ukuhlaziywa kwendlela eyodwa yokuhluka, ukuhlolwa kokumadanisa okunengi kuka-Holm-Sidak; *P<0.05). Inomboro yeyunithi itjengiswa ngabakaki.
Sizimisele ukuphenya bona ingabe nanyana ngiyiphi isigaba ku-dataset ye-proteomics (Umfanekiso 1G) ifaka hlangana iindlela ezingalwisana nokutlhayela okukhulu kwe-OXPHOS, ngaleyindlela sihlathulula bona kubayini i-PN ethintekako ingagcina i-electrophysiology eseduze nejwayelekileko (Umfanekiso 2, E ukuya ku-K). . Ukuhlaziywa kwe-proteomics kutjengise bona ama-enzayimu abandakanyeka ekuguqulweni kwama-amino acid we-branched chain (BCAA) bekakhuphuke khulu (Umfanekiso 3A noMfanekiso S5A), begodu umkhiqizo wokugcina we-acetyl-CoA (CoA) nofana i-succinyl CoA ungangezelela ama-tricarboxylates ku-arteriosclerosis Acid (TCA) cycle. Sithole bona okuqukethwe kwe-BCAA transaminase 1 (BCAT1) kanye ne-BCAT2 kokubili kwanda. Bakhuthaza igadango lokuthoma le-BCAA catabolism ngokukhiqiza i-glutamate ukusuka ku-α-ketoglutarate (26). Woke ama-subunits akha i-branched chain keto acid dehydrogenase (BCKD) complex alawulwa phezulu (i-complex ikhuthaza ukukhipha i-carboxylation elandelako nengabuyiselwa emuva ye-BCAA carbon skeleton) (Umfanekiso 3A noMfanekiso S5A). Kodwana, akukho amatjhuguluko abonakalako ku-BCAA ngokwawo atholakala ku-PN ehleliweko, okungenzeka kube ngebanga lokukhuphuka kokuthathwa kwamaseli wama-amino acid aqakathekileko nofana ukusetjenziswa kweminye imithombo (iglukhozi nofana i-lactic acid) ukungezelela umjikelezo we-TCA (Umfanekiso S5B). Ama-PN angenayo i-OXPHOS nawo atjengisa ukwanda kokubola kwe-glutamine kanye nemisebenzi yokutjhugululwa emavikini ayi-8 ubudala, okungabonakala ngokulawulwa phezulu kwama-enzyme we-mitochondrial glutaminase (GLS) kanye ne-glutamine pyruvate transaminase 2 (GPT2) (Umfanekiso 3, A no-C). Kuqakathekile ukutjheja bona ukulawulwa kwe-GLS kukhawulelwe ku-isoform glutaminase C (GLS-GAC) (ukutjhuguluka kwe-Mfn2cKO/CTRL kungaba 4.5-fold, P = 0.05), begodu ukulawulwa kwayo okukhethekileko emathitjhu womdlavuza Kungasekela i-mitochondrial bioenergy. (27).
(A) Imephu yokutjhisa itjengisa ukutjhuguluka okuphindaphindiweko kwezinga lamaphrotheyini ngendlela ebekiweko emavikini ayi-8. (B) Isibonelo sesilayi se-cerebellar esifakwe i-anti-PCx antibody (isikali, 20 μm). Umcibisholo ophuzi ukhomba emzimbeni weseli le-Purkinje. (C) Ukuhlaziywa kokuvezwa kwamaphrotheyini kwesikhathi okukhonjwe njengekhetho eliqakathekileko le-atherosclerosis (i-t-test eminengi, *FDR <5%; n = 3-5 amagundane). (D) Ngehla: Umdwebo otjengisa iindlela ezihlukileko zokungena kwekhabhoni ebhaliweko equkethwe ku-[1-13C]pyruvate tracer (okutjho, nge-PDH nofana indlela yokudlula kwemithambo). Ngezansi: Itjhadi le-violin litjengisa iphesenteji ye-single-labeled carbon (M1) etjhugululwe yaba yi-aspartic acid, i-citric acid kanye ne-malic acid ngemva kokulebula iinlayi ze-cerebellar ezibukhali nge-[1-13C]pyruvate (paired t-test; ** P <0.01). (E) Ukuhlaziywa okupheleleko komlando wesikhathi wendlela etjengisiweko. Cabanga ngamaphrotheyini ane-P<0.05 emavikini ayi-8 kwaphela. Umugqa ophukileko: ayikho inani lokulungiswa (ukuhlaziywa kweendlela ezimbili zokuhluka; * P <0.05; *** P <0.001). Idatha ivezwa njenge-mean±SEM.
Ekuhlaziyweni kwethu, i-BCAA catabolism sele ibe ngenye yeendlela eziqakathekileko zokulawula. Iqiniso leli lisikisela khulu bona ivolumu yokungenisa umoya engena emjikelezweni we-TCA ingatjhuguluka ku-PN engenayo i-OXPHOS. Lokhu kungaba yindlela ekulu yokuvuselelwa kwe-metabolic ye-neuronal, okungaba nomthelela oqondileko emzimbeni we-neuronal nokusinda ngesikhathi sokugcinwa kokungasebenzi kuhle kwe-OXPHOS. Ngokuvumelana nalokhu kucabanga, sithole bona i-enzayimu ekulu yokulwa ne-atherosclerotic i-PCx ilawulwa phezulu (i-Mfn2cKO/CTRL itjhuguluka pheze isikhathi esiyi-1.5; Umfanekiso 3A), okukhuthaza ukutjhuguluka kwe-pyruvate ibe yi-oxaloacetate (28), okukholelwa bona kuvezwa ebuchosheni (29, 30). Ngokuvumelana nemiphumela ye-proteomics, i-confocal microscopy itjengise bona ukuvezwa kwe-PCx kwakhuphuka khulukhulu kuma-PN atlhoga i-OXPHOS, ngesikhathi ukuphendula kwe-PCx bekukhawulelwe khulu kumaseli we-Bergmann glial aseduze wokulawula (Umfanekiso 3B). Ukuhlola ukusebenza kokulawulwa kwe-PCx, siphathe iinlayi ze-cerebellar ezibukhali nge-[1-13C]pyruvate tracer. Lokha i-pyruvate nayi-oxidized nge-pyruvate dehydrogenase (PDH), ilebula yayo ye-isotope yanyamalala, Kodwana ifakwa hlangana ne-TCA cycle intermediates lokha i-pyruvate nayitjhugululwa ngokusabela kwemithambo (Umfanekiso 3D). Ukusekela idatha yethu ye-proteomics, sibone inani elikhulu lamatshwayo avela ku-tracer le ku-aspartic acid yeengcezu ze-Mfn2cKO, ngesikhathi i-citric acid ne-malic acid nayo gade inomkhuba olinganiselwe, nanyana ingakaqakatheki (Umfanekiso 3D).
Ema-neuron we-dopamine we-MitoPark amagundane anokungasebenzi kuhle kwe-mitochondrial okubangelwa ma-neuron we-dopamine abhubhisa ngokukhethekileko i-mitochondrial transcription factor A (Tfam) (Umfanekiso S6B), ukuvezwa kwe-PCx nakho kwalawulwa khulu (31), okutjengisa bona i-acetone acid arteriosclerosis Ukwenzeka kwesifo kulawulwa ngesikhathi sokusebenza kwe-neuron I-OXPHOS emzimbeni. Kuqakathekile ukutjheja bona kutholakele bona ama-enzyme ahlukileko (32-34) angavezwa ema-neurons angahlotjaniswa ne-arteriosclerosis alawulwa khulu kuma-PN angenayo i-OXPHOS, njenge-propionyl-CoA carboxylase (PCC-A), i-Malonyl-CoA itjhugulula i-propionyl-CoA carboxylase kanye ne-malonyl-CoA i-enzyme 3 (ME3), indima yayo ekulu kukubuyisa i-pyruvate ku-malate (Umfanekiso 3, A no-C) (33, 35). Ukungezelela, sithole ukwanda okukhulu kwe-enzyme ye-Pdk3, eyenza i-phosphorylate begodu yenza bona i-PDH ingasebenzi (36), ngesikhathi kungekho amatjhuguluko atholakele ku-enzyme ye-Pdp1 eyenza bona i-PDH isebenze nofana i-enzyme ye-PDH ngokwayo (Umfanekiso 3A). Ngokungaguquki, ku-Mern2cKO PNs, i-phosphorylation ye-α1 subunit α (PDHE1α) subunit ye-pyruvate dehydrogenase E1 ye-PDH complex ku-Ser293 (eyaziwa ngokuvimbela ukusebenza kwe-enzyme ye-PDH) kwathuthukiswa (Umfanekiso S6C) (Umfanekiso S6C). I-Pyruvate ayikwazi ukungena kwemithambo yeengazi.
Ekugcineni, sithole bona indlela ephezulu ye-serine ne-glycine biosynthesis, umjikelezo ohlobene ne-mitochondrial folate (1C) kanye ne-proline biosynthesis (Umfanekiso 1G noMfanekiso S5C) koke kulawulwa khulu, ngokuya kwemibiko, ngesikhathi senqubo yokusebenza. Iincubu ezizungezeko zisebenza ngokungasebenzi kuhle kwe-mitochondrial (5-7). Ukuhlaziywa kwe-confocal okusekela idatha ye-proteomics kutjengise bona ku-PN ene-OXPHOS engekho, iinlayi ze-cerebellar zamagundane aneveke ezi-8 zafakwa i-serine hydroxymethyltransferase 2 (SHMT2), i-enzayimu eqakathekileko yomjikelezo we-mitochondrial folate. Ukusabela okuqakathekileko kwamasotja womzimba (Umfanekiso S5D). Ku-13 CU-glucose-incubated acute cerebellar slices, ukuhlolwa kokulandelela umzimba kwaqinisekisa ukulawulwa kwe-serine ne-proline biosynthesis, okutjengisa bona ukugeleza kwama-isoform we-carbon ku-serine ne-proline kwanda (Umfanekiso S5E). Njengombana ukusabela okukhuthazwa yi-GLS ne-GPT2 kunomthwalo wokwenziwa kwe-glutamate ukusuka ku-glutamine kanye nokutjhugululwa hlangana ne-glutamate ne-α-ketoglutarate, ukulawulwa kwawo kutjengisa bona ama-neuron atlhoga i-OXPHOS anesidingo esikhulu se-glutamate, Lokhu kungenzeka kuhloswe ngakho ukugcina ukwanda kwe-biosynthesis ye-proline (Umfanekiso S5). Ngokungafani namatjhuguluko la, ukuhlaziywa kwe-proteomic yama-astrocyte we-cerebellar avela emagundeni we-PN-specific Mfn2cKO kutjengise bona iindlela lezi (okufaka hlangana woke ama-antiperoxidases) azange zitjhuguluke khulu ekuvezweni, lokho kutjengisa bona ukuguqulwa kwe-metabolic kukhetha i-PN ewohlokileko (Umfanekiso S6, D ukuya ku-G).
Ngokufingqa, ukuhlaziywa lokhu kuveze amaphethini ahluke khulu wokusebenza kwesikhatjhana kweendlela ezithileko zokugaya ukudla kuma-PN. Nanyana ukusebenza okungakajayeleki kwe-neuronal mitochondrial kungadosela ekuthomeni kwe-atherosclerosis kanye nokuvuselelwa kwe-1C (Umfanekiso 3E noMfanekiso S5C), kanye nokutjhuguluka okubikezelwako ekuvezweni kwe-I ne-IV complexes, ukutjhuguluka kwe-serine de novo synthesis kuvele kwabonakala kwaphela emazingeni wokugcina. Ukungasebenzi kuhle kwe-OXPHOS (Umfanekiso 3E noMfanekiso S5C). Iindaba ezitholweko zihlathulula ikambiso elandelanako lapho i-mitochondrial (umjikelezo we-1C) kanye ne-cytoplasmic (i-serine biosynthesis) ebangelwa kugandeleleka iphendula ngokubambisana nokwanda kwe-atherosclerosis emjikelezweni we-TCA ukubumba kabutjha ukusebenza kwe-neuronal.
Ama-PN we-OXPHOS anamaviki abunane angagcina umsebenzi wokuvuselela okuphezulu begodu enze ukuhlanganiswa kabutjha okukhulu kwe-metabolic ukunxephezela ukungasebenzi kuhle kwe-mitochondrial. Lokhu okutholakeleko kuphakamisa ithuba elikarisako lokuthi ngitjho nanje, amaseli la angathola ukungenelela kwezokwelapha ukulibazisa nofana ukukhandela ukuwohloka kwemizwa. Ngemva kwesikhathi. Sirarulule lokhu ngokusebenzisa ukungenelela okubili okuzijameleko. Ngendlela yokuthoma, saklama i-Cre-dependent adeno-associated virus (AAV) vector ukwenzela bona i-MFN2 ingavezwa ngokukhethekileko ku-OXPHOS-deficient PNs ku-vivo (Umfanekiso S7A). I-AAV ekhoda i-MFN2 kanye ne-fluorescent reporter gene mCherry (Mfn2-AAV) yaqinisekiswa emasikweni we-neuron aphambili ku-vitro, okwenza bona i-MFN2 ivezwe ngendlela ethembele ku-Cre begodu yasindisa i-mitochondrial morphology, ngaleyo ndlela ivimbele ukuguquka kwe-neuromutation kuma-neuron we-Mfn2cKO ( Umfanekiso D, D, S7 no B). Okulandelako, senza ukuhlolwa kwe-vivo ukuletha i-Mfn2-AAV enamaviki ayi-8 ku-cerebellar cortex ye-Mfn2cKO namagundane wokulawula, begodu sahlaziya amagundane amaviki ali-12 (Umfanekiso 4A). Amagundane we-Mfn2cKO alatjhwako ahlongakala (Umfanekiso 1, A no-B) (16). Ukudluliselwa kwevayirasi ku-vivo kwabangela ukuvezwa okukhethiweko kwe-PN kweminye imibuthano ye-cerebellar (Umfanekiso S7, G no-H). Ukujova kwe-AAV yokulawula eveza i-mCherry kwaphela (Ctrl-AAV) bekunganamthelela omkhulu ezingeni lokuwohloka kwemizwa eenlwaneni ze-Mfn2cKO. Ngokuphikisako, ukuhlaziywa kwe-Mfn2cKOs etjhugululwe nge-Mfn2-AAV kutjengise umphumela oqakathekileko wokuvikela we-PN cell layer (Umfanekiso 4, B no-C). Ngokukhethekileko, ukuminyana kwe-neuron kubonakala kungahlukaniswa neenlwana ezilawulwako (Umfanekiso 4, B no-C, kanye noMfanekiso S7, H no-I). Ukuvezwa kwe-MFN1 kodwana ingasi i-MFN2 kusebenza ngokufanako ekusindiseni ukufa kwe-neuronal (Umfanekiso 4C nomfanekiso S7, C no-F), okutjengisa bona ukuvezwa kwe-ectopic MFN1 kungangezelela kuhle ukutlhogeka kwe-MFN2. Ukuhlaziywa okuragela phambili ezingeni le-PN elilodwa kutjengise bona i-Mfn2-AAV yasindisa khulu i-ultrastructure ye-mitochondria, yenza amazinga we-mtDNA ajwayelekileko, begodu yabuyisela emuva ukuvezwa okuphezulu kwe-anti-angiogenesis marker PCx ​​(Umfanekiso 4, C ukuya ku-E ). Ukuhlolwa kwamehlo kwamagundane we-Mfn2cKO ahlengiweko asebujameni bokuphumula kutjengise bona ukuma kwawo kanye namatshwayo wokunyakaza (ukunyakaza kwe-S1 ukuya ku-S3) kwathuthuka. Ekuphetheni, iimhloliso lezi zitjengisa bona ukubuyiselwa emuva kwe-MFN2 kuma-PNs atlhoga khulu i-OXPHOS kwanele ukubuyisela emuva ukusetjenziswa kwe-mtDNA begodu kubangele i-atherosclerosis, ngaleyo ndlela kuvimbele ukuwohloka kwe-axon nokuhlongakala kwe-neuronal ku-vivo.
(A) Isikimu esitjengisa ihlelo lokuhlola lokujova i-AAV ekhoda i-MFN2 lokha indlela etjengisiweko yokugaya ukudla nayisebenzako. (B) Iinthombe ezijameleko zeengcenye ze-cerebellar ezineemveke ezili-12 ezitjhugululwe ngamagundane we-Mfn2cKO emavikini ayi-8 begodu zibhalwe nge-anti-Calbindin antibody. Ngakwesokudla: Ukukhula kwemicu ye-axon. Isikali se-axon zoom ngu-450 no-75 μm. (C) Kwesokunxele: Ukulinganisa ukuminyana kwamaseli we-Purkinje ku-AAV transduction loop (AAV+) (ukuhlaziywa kwendlela eyodwa yokuhluka; n = 3 amagundane). Ngakwesokudla: ukuhlaziywa kokugxila kwe-mtDNA ku-PN etjhugululiweko ngeveke ye-12 (i-t-test engabhangqwanga; n = amaseli ayi-6 avela emakhondweni amathathu). * P <0.05; ** P <0.01. (D) Iinthombe ezijameleko zokudluliselwa kwe-elekthronikhi zama-PNs weengaba ze-Mfn2cKO ze-cerebellar ezitjhugululwe ngama-vector wegciwane atjengisiweko. Imaski epinki itjengisa indawo ethathwe ma-dendrite, begodu isikwere esiphuzi esinamachashaza sitjengisa ukusondeza okunikelwe ngesidleni; n imele inyukliya. Ibha yesikali, 1μm. (E) itjengisa isibonelo sokugcotshwa kwe-PCx ku-PN etjhugululwe emavikini ali-12. Ibha yesikali, 20μm. OE, ukuvezwa ngokweqileko; FC, ukugoqa ukutjhuguluka.
Ekugcineni, sihlole ukuqakatheka kokuphila kwamaseli okubangelwa yi-peroxidase kuma-PN ahlangabezane nokungasebenzi kuhle kwe-OXPHOS. Sakhiqiza i-mCherry ekhoda i-AAV-shRNA (i-RNA emfitjhani yeenhluthu) eqalise khulukhulu i-mouse PCx mRNA (AAV-shPCx), begodu sajova igciwane nofana ukulawula kwayo okuhlangeneko (AAV-scr) ngaphakathi kwe-cerebellum yamagundane we-Mfn2cKO. Ukujova kwenziwa ngeveke yesine yobudala (Umfanekiso 5A) ukufezekisa ukuqedwa okuphumelelako kwe-PCx ngesikhathi lapho ukuvezwa kwe-PCx kwanda khona (Umfanekiso 3C) begodu isendlalelo seseli se-PN besisesekhona (Umfanekiso 1A). Kuqakathekile ukutjheja bona ukuwisa i-PCx (Umfanekiso S8A) kurholela ekutheni kube nokurhaba okukhulu kokufa kwe-PN, okukuphela kwendandatho evinjelweko yokutheleleka (Umfanekiso 5, B no-C). Ukuze sizwisise indlela imiphumela yokugaya ukudla ebangelwa kulawulwa kwe-PCx, sifunde ubujamo be-redox yama-PN ngemva kokuqedwa kwe-PCx begodu i-AAV-mediated optical biosensor Grx1-roGFP2 yavezwa ngasikhathi sinye (Umfanekiso S8, B ukuya ku-D) ukuhlola i-glutathione Ukutjhuguluka okuhlobeneko kwe-redox peptide (383). Bese, senza i-two-photon fluorescence lifetime imaging microscopy (FLIM) eengcenyeni zobuchopho ezibukhali ze-Mfn2cKO enamaviki ali-7 nofana ama-littermates wokulawula ukuthola amatjhuguluko angaba khona ebujameni be-cytoplasmic redox ngemva kokuqinisekisa ubujamo be-FLIM (Umfanekiso S8, E ukuya ku-G). Ukuhlaziywa kutjengise ukwanda okukhulu kwesimo se-oksijini se-Mfn2cKO PNs eyodwa engenakho ukuvezwa kwe-PCx, okuhlukileko kuma-neurons wokulawula nofana ama-Mfn2cKO PNs aveza i-shRNA ehlanjululweko kwaphela (Umfanekiso 5, D no-E). Lokha ukuvezwa kwe-PCx nakulawulwa phasi, iphesenteji ye-Mfn2cKO PNs etjengisa ubujamo obune-oksijini ephezulu ikhuphuke ngaphezu kweenkhathi ezintathu (Umfanekiso 5E), okutjengisa bona ukulawulwa kwe-PCx kugcine amandla we-redox wama-neurons awohlokileko.
(A) Isikimu esitjengisa ihlelo lokuhlola lokujova i-AAV ekhoda i-shPCx lokha indlela etjengisiweko yokugaya ukudla nayisebenzako. (B) Iinthombe ezijameleko zeengcenye ze-cerebellar ezineemveke ezi-8 emagundeni we-Mfn2cKO atjhugululwe begodu abhalwe nge-anti-calcineurin antibody emavikini ama-4. Ibha yesikali, 450μm. (C) Ukulinganisa ukuminyana kwamaseli we-Purkinje kuma-loop atjhugululwe yi-AAV (ukuhlaziywa kwendlela eyodwa yokuhluka; n = 3 ukuya ku-4 amagundane). Idatha ivezwa njenge-mean±SEM; ***P<0.001. (D) Isithombe esijameleko se-FLIM sitjengisa isikhathi sokuphila esiphakathi se-PN enamaviki ali-7 eveza i-glutathione redox sensor Grx1-roGFP2 ngaphasi kweemeko ezibekiweko zokuhlola. I-LUT (itafula lokubuka) isilinganiso: isikhathi sokusinda (ngama-picosecond). Ibha yesikali, 25μm. (E) I-histogram itjengisa ukusabalala kwamanani wesikhathi sokuphila se-Grx1-roGFP2 ukusuka ku-(D) (n=158 ukuya ku-368 amaseli emagundeni amabili ngaphasi kobujamo ngabunye). Itjhadi lephayi elingaphezulu kwe-histogram ngayinye: litjengisa inani lamaseli anamagugu wesikhathi sokuphila eside khulu (ebomvu, esine-oksijini) nofana esifitjhani (esihlaza satjani, esinciphileko), edlula i-1 SD yesilinganiso sesikhathi sokuphila ku-CTRL-AAV-scr. (F) Imodeli ehlongozwayo itjengisa umphumela wokuvikela wokulawulwa kwe-PCx ye-neuronal.
Sekukonke, idatha esiyinikelako lapha itjengisa bona ukuvezwa kabutjha kwe-MFN2 kungasindisa ngokupheleleko i-PN ethuthukileko ngokutlhogeka okukhulu kwe-OXPHOS, ukuphelelwa mamandla kwe-mtDNA, kanye nesakhiwo esifana ne-ista esingakajayeleki khulu, ngaleyo ndlela kunikelwe ituthuko eragela phambili ngitjho nasemalwelweni athuthukileko. Ukuwohloka kwemizwa kunikela ubufakazi obubuyiselwa emuva besigaba sangaphambi kokufa kwamaseli. Izinga lokuguquguquka kwe-metabolic liphinde ligcizelelwa yikghono lama-neuron lokubangela i-atherosclerosis (ukuvuselelwa komjikelezo we-TCA), okuvimbela ukuvezwa kwe-PCx kuma-PN angenayo i-OXPHOS begodu kukhuphule ukufa kwamaseli, ngaleyo ndlela kudlala indima yokuvikela (Umfanekiso 5F).
Kulesi sifundo, sinikele ubufakazi bokuthi ukuphendula kwama-PNs ekungasebenzini kwe-OXPHOS kuzokuhlangana kancanikancani ku-TCA cycle atherosclerosis ngendlela yokusebenza ehlukileko eyenziwa ziinhlelo ze-metabolic. Siqinisekise ukuhlaziywa kwe-proteomic ngeendlela ezinengi ezihambisanako begodu saveza bona lokha nayitlhoriswa kukungasebenzi kuhle kwe-mitochondrial, ama-neurons anendlela engakaziwa ngaphambilini yokuthamba kwe-metabolic. Ngokumangalisako, ikambiso yoke yokubuyisela iintambo ayitshwayi ubujamo bokuphela kwe-metabolic obukhambisana nokuwohloka kwemizwa kancanikancani begodu ngokungabuyiseleki emuva, kodwana idatha yethu iphakamisa bona ingaba yi-neuron yokugcina ngitjho nasesigabeni sangaphambi kokufa kwamaseli Indlela yokubhadela esebenzako. Lokhu okutholakeleko kutjengisa bona ama-neuron anezinga elikhulu lokutjhuguluka komzimba emzimbeni. Iqiniso leli lifakazela bona ukubuyiselwa kwe-MFN2 kamuva kungabuyisela emuva ukuvezwa kwamatshwayo aqakathekileko we-metabolic begodu kuvimbele ukuwohloka kwe-PN. Ngokuphikisako, ivimbela ukuvuvuka kwemithambo yegazi begodu irhabisa imizwa. otjhugulula ubulili.
Okhunye okutholwe kuhle khulu erhubhululweni lethu kukuthi ama-PN angenayo i-OXPHOS angatjhugulula ukusebenza komjikelezo we-TCA ngokulawula ama-enzyme akhuthaza khulukhulu ukuvuvuka kwemithambo yeengazi. Ukuhlelwa kabutjha kwe-metabolic yinto ejayelekileko yamaseli womdlavuza, amanye wawo athembele ku-glutamine ukungezelela ama-TCA cycle intermediates ukukhiqiza ukulingana okunciphisako, okuqhuba iketango lokuphefumula begodu kugcine ukukhiqizwa kwama-lipid nama-nucleotide biosynthesis precursors (39 , 40). Irhubhululo lakamuva litjengise bona eencutjhweni ezingaphandle ezihlangabezana nokungasebenzi kuhle kwe-OXPHOS, ukuhlanganiswa kabutjha kwe-glutamine/glutamate nakho kuyinto eqakathekileko (5, 41), lapho indlela yokungena kwe-glutamine emjikelezweni we-TCA incike ngebanga lobukhulu bokulimala kwe-OXPHOS (41). ). Kodwana, kunobufakazi obucacileko bokufana kokusebenza kwe-neuronal emzimbeni kanye nokuhlobana kwayo emzimbeni wesifo. Esifundweni sakamuva se-in vitro, ama-neurons we-cortical aphambili atjengiswe ukuhlanganisa amadamu we-glutamate ukudluliselwa kwe-neuro, ngaleyo ndlela akhuthaze ukugaya ukudla kwe-oxidative kanye ne-atherosclerosis ngaphasi kweemeko zokugandeleleka kwe-metabolic (42). Kuqakathekile ukutjheja bona ngaphasi kokuvinjelwa kwe-TCA cycle enzyme succinate dehydrogenase, i-pyruvate carboxylation kukholelwa bona igcina ukwakheka kwe-oxaloacetate kuma-neuron we-cerebellar granule (34). Kodwana, ukuqakatheka komzimba kwalezindlela emathitjhu wengqondo (lapho kukholelwa bona i-atherosclerosis ivame khulu kuma-astrocytes) kusese nokuqakatheka okuqakathekileko komzimba (43). Kulokhu, idatha yethu itjengisa bona ama-PN alinyazwe yi-OXPHOS emzimbeni angatjhugululwa abe yi-BCAA ukuwohloka kanye ne-pyruvate carboxylation, okuyimithombo emibili eqakathekileko yokungezelelwa kwama-TCA pool intermediates. Nanyana igalelo elicatshangelwako le-BCAA catabolism ekusebenzeni kwamandla we-neuron sele liphakanyisiwe, ngaphezu kwendima ye-glutamate ne-GABA yokudluliswa kwe-neuro (44), akukabi nobufakazi beendlela lezi ku-vivo. Ngalokho-ke, kulula ukuqagela bona ama-PN angasebenziko angazibuyisela ngokuzenzakalelayo ukusetjenziswa kwe-TCA ephakathi eqhutshwa yindlela yokuhlanganiswa ngokukhuphula i-atherosclerosis. Ngokukhethekileko, ukulawulwa kwe-PCx kungatlhogeka ukugcina isidingo esikhuphukileko se-aspartic acid, okutjengiswe emaseli andako anokungasebenzi kuhle kwe-mitochondrial (45). Kodwana, ukuhlaziywa kwethu kwe-metabolomics azange kuveze amatjhuguluko aqakathekileko ezingeni elizinzileko le-aspartic acid ku-Mfn2cKO PNs (Umfanekiso S6A), okucatshangelwa bona kutjengisa ukusetjenziswa okuhlukileko kwe-aspartic acid hlangana namaseli akhulako kanye nama-neurons we-post-mitotic. Nanyana indlela eqondileko yokukhuphulwa kwe-PCx ema-neuron angasebenzi kuhle ku-vivo isazokuvezwa, sitjengise bona ukuphendula kwangaphambi kwesikhathi kudlala indima eqakathekileko ekugcineni ubujamo be-redox be-neuron, okwatjengiswa ekuhlolweni kwe-FLIM eenlayini ze-cerebellar. Khulukhulu, ukukhandela ama-PN bona angalawuli i-PCx kungarholela ekutheni ibe ne-oksijini begodu kurhabisa ukufa kwamaseli. Ukusebenza kokuwohloka kwe-BCAA kanye nokukhiqizwa kwe-pyruvate akusizo iindlela zokuveza amathitjhu angaphandle wokungasebenzi kuhle kwe-mitochondrial (7). Ngalokho-ke, kubonakala ngasuthi ziyinto eqakathekileko ye-OXPHOS-deficient neurons, ngitjho nanyana kungasi yiyo kwaphela, eqakathekileko ekuwohlokeni kwemizwa. .
Ubulwelwe be-cerebellar mhlobo ohlukileko wobulwelwe be-neurodegenerative obuvamise ukubonakala njenge-ataxia begodu buvame ukulimaza ama-PN (46). Inani lama-neuron lisengozini khulu yokungasebenzi kuhle kwe-mitochondrial ngombana ukuwohloka kwawo okukhethiweko emagundeni kwanele ukukhiqiza amatshwayo amanengi wemoto atjengisa i-taxia ye-spinocerebellar yabantu (16, 47, 48). Ngokuya kwemibiko, imodeli yegundwane etjhugululweko ene-mutant gene ihlotjaniswa ne-taxia ye-spinocerebellar yabantu begodu inokungasebenzi kuhle kwe-mitochondrial (49, 50), okugandelela ukuqakatheka kokufunda imiphumela yokutlhogeka kwe-OXPHOS ku-PNPH. Ngalokho-ke, kufanele khulu ukuhlukanisa nokufunda inani le-neuron elihlukileko. Kodwana, njengombana ama-PN azwela khulu ukugandeleleka begodu athatha ingcenye ephasi yawo woke amaseli we-cerebellar, eemfundweni ezinengi ezisekelwe ku-omics, ukuhlukaniswa kwawo njengamaseli woke kusese yinto ebudisi. Nanyana kungakghonakali ukufikelela ukutlhogeka okupheleleko kokusilaphazeka kweminye imihlobo yamaseli (khulukhulu amathitjhu wabantu abadala), sihlanganise igadango lokuhlukanisa eliphumelelako ne-FACS ukuthola inani elaneleko lama-neurons asebenzako wokuhlaziywa kwe-proteomics, begodu sibe nokuhlanganiswa okuphezulu kwamaphrotheyini (amaphrotheyini angaba ma-3000) nayimadaniswa nedatha ekhona ye-cerebel (155). Ngokulondoloza ukuphila kwamaseli woke, indlela esiyinikela lapha ayivumeli ukuhlola amatjhuguluko weendlela zokugaya ukudla ku-mitochondria kwaphela, kodwana nokuhlola amatjhuguluko kuma-cytoplasmic counterparts wayo, okuhambisana nokusetjenziswa kwe-mitochondrial membrane label enrichment eqondene nemihlobo yamaseli. Indlela esiyichazako ayihlobene nokufunda kwamaseli we-Purkinje kwaphela, kodwana ingasetjenziswa lula kiyo nanyana ngimuphi umhlobo wamaseli ukutjheja amatjhuguluko we-metabolic ebuchosheni obugulako, kufaka hlangana amanye amamodeli wokungasebenzi kuhle kwe-mitochondrial.
Ekugcineni, sithole ifasitela lokwelapha ngesikhathi senqubo yokuhlelwa kabutjha kwe-metabolic engabuyisela emuva ngokupheleleko amatshwayo aqakathekileko wokugandeleleka kwamaseli begodu ivimbele ukuwohloka kwe-neuronal. Ngalokho-ke, ukuzwisisa imiphumela yokusebenza kokubuyiselwa kwentambo okuchazwe lapha kunganikela ilwazi eliyisisekelo ngeendlela zokwelapha ezingaba khona zokugcina ukusebenza kwe-neuron ngesikhathi sokungasebenzi kuhle kwe-mitochondrial. Irhubhululo lesikhathi esizako elihlose ukuhlukanisa amatjhuguluko ekusebenzeni kwamandla kezinye iinhlobo zamaseli wengqondo liyatlhogeka ukuveza ngokuzeleko ukusetjenziswa kwalomthetho kezinye iinlwelwe zemizwa.
Amagundane we-MitoPark achazwe ngaphambilini (31). Amagundane we-C57BL/6N anamajini we-loxP akhambisana ne-Mfn2 sele ahlathululwe ngaphambilini (18) begodu ahlanganiswa namagundane we-L7-Cre (23). Iinzukulwana eziphindwe kabili ezihlukileko zabe sezihlanganiswa namagundane we-Mfn2loxP/Mfn2loxP afanako ukukhiqiza amajini we-Purkinje-specific we-Mfn2 (Mfn2loxP/Mfn2loxP; L7-cre). Esigabeni esincani sokuhlangana, i-Gt (ROSA26) SorStop-mito-YFP allele (stop-mtYFP) yethulwa ngokusebenzisa ezinye iinqhema (20). Zoke iinkambiso zeenlwana zenziwa ngokuya kwemihlahlandlela ye-Europe, yeentjhabatjhaba neyeenkhungo begodu yavunywa yi-LandesamtfürNatur ye-Umwelt ne-Verbraucherschutz, e-North Rhine-Westphalia, e-Germany. Umsebenzi weenlwana godu ulandela isiqondiso se-European Federation of Laboratory Animal Sciences Associations.
Ngemva kokubulala ubuhlungu ukuphuka komlomo wesibeletho womfazi okhulelweko, umbungu wegundane uyahlukaniswa (E13). I-cortex yahlukaniswa nge-Hanks' Balanced Salt Solution (HBSS) efakwe i-10 mM Hepes begodu yadluliselwa ku-Dulbecco's Modified Eagle's Medium ene-papain (20 U/ml) kanye ne-cysteine ​​(1μg/ml). Fukamela amathitjhu ku-DMEM) bese uwahlukanisa ngokugayeka kwe-enzymatic. Ml) ku-37°C imizuzu ema-20, bese igaywa nge-DMEM ene-10% ye-fetal bovine serum. Amaseli atjalwa phezu kweengilazi ezigcotshwe nge-polylysine nge-density engu-2×106 ngesitja se-6 cm sokulima nofana nge-density engu-0.5×105 amaseli/cm2 ukuhlaziywa kwemifanekiso. Ngemva kwama-awa ama-4, umphakathi wathathelwa indawo yi-Neurobasal serum-free medium equkethe i-1% B27 supplement kanye ne-0.5 mM GlutaMax. Ama-neurons abese agcinwa ku-37°C kanye ne-5% CO2 kiyo yoke isilingo, begodu adliswa kanye ngeveke. Ukuze kukhuthazwe ukuhlanganiswa kabutjha nge-vitro, i-3μl (24-well culture dish) nofana i-0.5μl (24-well plate) ye-AAV9 virus vector elandelako yasetjenziselwa ukwelapha ama-neurons ngelanga lesibili in vitro: AAV9.CMV.PI.eGFP. I-WPRE.bGH (Addgene, inombolo yekhathalogu 105530-AAV9) kanye ne-AAV9.CMV.HI.eGFP-Cre.WPRE.SV40 (Addgene, inombolo yekhathalogu 105545-AAV9).
I-Mouse Mfn1 ne-Mfn2 i-DNA (etholwe ku-Addgene plasmid #23212 kanye ne-#23213, ngokulandelana) itshwaywe nge-V5 sequence (GKPIPNPLLGLDST) ku-C-terminus, begodu ihlanganiswe ne-mCherry kufreyimu ngokusebenzisa i-T2A sequence. I-Grx1-roGFP2 sisipho esivela ku-Heidelberg TP Dick DFKZ (Deutsches Krebsforschungszentrum). Ngokubuyisela ikhasethi ye-tdTomato kusetjenziswa iindlela ezijayelekileko zokukhiqiza, ikhasethi yafakwa ngaphasi komgogodla we-pAAV-CAG-FLEX-tdTomato (inombolo yokureferensi ye-Addgene 28306) ukwenza i-pAAV-CAG-FLEX-mCherry-T2A-MFN2-V5, pAAV-CAG-G I-FLEX-mCherry-T2A-MFN1-V5 kanye ne-pAAV-CAG-FLEX-Grx-roGFP2 amavektha. Iqhinga elifanako lasetjenziselwa ukwenza i-vector yokulawula i-pAAV-CAG-FLEX-mCherry. Ukuze kwenziwe ukwakhiwa kwe-AAV-shPCx, i-plasmid AAV vector (VectorBuilder, pAAV [shRNA] -CMV-mCherry-U6-mPcx- [shRNA#1]) iyatlhogeka, equkethe ukulandelana kwe-DNA okukhowuda i-shRNA ehloselwe i-mouse PCx (5′CTTTCGCTAGCTAGGCTAAGGCTAAGGCTAGACTTAGA 3′) Ngaphasi kokulawulwa komkhuthazi we-U6, i-mCherry isetjenziswa ngaphasi kokulawulwa komkhuthazi we-CMV. Ukukhiqizwa kwama-AAV vectors asizako kwenziwa ngokuya kwemiyalo yomkhiqizi (Cell Biolabs). Ngamafuphi, sebenzisa iplasmidi yokudlulisa ephethe i-mCherry-T2A-MFN2-V5 (pAAV-CAG-FLEX-mCherry-T2A-MFN2-V5), i-mCherry-T2A-MFN1-V5 (pAAV-CAG-FLEX-mCherry) ngokudluliswa kwamaseli we-29A-MFN2-V5), I-mCherry (pAAV-CAG-FLEX-mCherry) nofana i-Grx-roGFP2 (pAAV-CAG-FLEX-Grx-roGFP2) ikhoda isakhi sofuzo, kanye nekhowudi yeprotheyini ye-AAV1 capsid kanye nephrotheyini esekelako Ukupakisha iplasmidi yeplasmidi, kusetjenziswa indlela ye-calcium phosphate. Igciwane elingakahlwengiswa eliphezulu latholwa ngokusebenzisa imijikelezo yokuqanda-ukuncibilikisa nge-ayisi eyomileko/ye-ethanol begodu amaseli ahlanjululwe nge-phosphate buffered saline (PBS). I-AAV vector yahlanjululwa nge-iodixanol gradient ultracentrifugation (ama-awa ama-24 nge-32,000 rpm kanye ne-4°C) begodu yahlanganiswa kusetjenziswa i-Amicon ultra-15 centrifugal filter. I-genome titer ye-AAV1-CAG-FLEX-mCherry-T2A-MFN2-V5 [2.9×1013 ikhophi ye-genome (GC)/ml], i-AAV1-CAG-FLEX-mCherry (6.1×1012 GC/ml), i-AAV1-CAG-FLEX beyihlathululwe ngaphambilini (i-PCR-5d) -MFN1-V5 (1.9×1013 GC/ml) kanye ne-AAV1-CAG-FLEX-Grx-roGFP2 (8.9×1012 GC/ml).
Ama-neuron aphambili aklwetjhwa nge-1x PBS ebandako, abekwa ama-pellet, bese ahlanganiswa nge-0.5% Triton X-100 / 0.5% sodium deoxycholate/PBS lysis buffer ene-phosphatase ne-protease inhibitor (Roche). Ukulinganiswa kwamaphrotheyini kwenziwa ngokusebenzisa i-bicinchoninic acid assay (Thermo Fisher Scientific). Amaphrotheyini la ahlukaniswa nge-SDS-polyacrylamide gel electrophoresis, bese afakwa ku-polyvinylidene fluoride membrane (GE Healthcare). Vimba iindawo ezingakaqakatheki bese ufukamela nge-antibody yokuthoma (qala iThebula S1 malungana nemininingwana) ngebisi elingama-5% ku-TBST (i-Tris-buffered saline nge-Tween), amagadango wokuhlanza kanye ne-antibody yesibili ku-TBST Ukufukamela. Fukamela nge-antibody yokuthoma ubusuku boke ku-+4°C. Ngemva kokugeza, faka i-antibody yesibili ama-awa amabili emazingeni womtjhiso wegumbi. Ngemva kwalokho, ngokufukamela i-blot efanako nge-anti-β-actin antibody, ukulayisha okufanako kwaqinisekiswa. Ukutholakala ngokutjhugulula kube yi-chemiluminescence nokuthuthukisa i-chemiluminescence (i-GE Healthcare).
Ama-neurons ebegade atjalwe ngaphambilini ema-glass coverslips alungiswa nge-4% paraformaldehyde (PFA)/PBS ngesikhathi esibekiweko emazingeni womtjhiso wegumbi imizuzu eli-10. Ama-coverslips athoma ngokufakwa nge-0.1% Triton X-100/PBS imizuzu emi-5 emazingeni womtjhiso wegumbi, bese kuthi ngemva kwalokho kube yi-blocking buffer [3% bovine serum albumin (BSA)/PBS]. Ngelanga lesibili, ama-coverslips ahlanzwa nge-blocking buffer begodu afukamela nge-fluorophore-conjugated secondary antibody ama-awa amabili emazingeni womtjhiso wegumbi; ekugcineni, amasampula ahlanzwa kuhle ku-PBS nge-4′,6-diamidino-2 -Phenylindole (DAPI) efakwe i-counterstain bese ilungiswa esilayidini se-microscope nge-Aqua-Poly/Mount.
Amagundane (amaduna nawesifazana) ahlatjhwa ngokujova ngaphakathi kwesisu se-ketamine (130 mg/kg) kanye ne-xylazine (10 mg/kg) begodu anikelwa ngaphasi kwesikhumba nge-carprofen analgesic (5 mg/kg), begodu afakwa esitjeni se-stereotactic (Kopf) esine-warm. Vula ikhanda bese usebenzisa i-dental drill ukunciphisa ingcenye ye-cerebellar cortex ehambelana nethambo le-mis (kusuka ku-lambda: umsila 1.8, lateral 1, ohambelana nama-lobules IV no-V). Sebenzisa inaliti yesirinji egobileko ukwenza umgodi omncani ekhanda ukugwema ukuphazamisa imithambo yeengazi engezansi. Bese i-capillary yengilazi edonswe kancanikancani ifakwa kancanikancani ngaphakathi kwe-micro-hole (kusuka ku-1.3 ukuya ku--1 ngehlangothini le-ventral le-dura mater), bese kuthi i-200 ukuya ku-300 nl AAV ijovwe ngaphakathi kwe-micro-injector (i-Narishige) ngama-manual syringes (i-Narishige) kanengi ngegandelelo eliphasi esikhathini esingaba mizuzu ema-20 ukuya kweli-10. Ngemva kokufakwa, faka i-capillary eminye imizuzu eli-10 ukuvumela bona ivayirasi irhatjheke ngokupheleleko. Ngemva kobana imithambo yeengazi ikhutjhwe, isikhumba sithungwa ngokutjheja ukwenzela bona kunciphiswe ukuvuvuka kwesilonda begodu kuvumele isilwana bona silulame. Iinlwana zaphathwa ngemithi yokuqeda ubuhlungu (i-caspofen) amalanga ambalwa ngemva kokuhlinzwa, phakathi nesikhathi lapho ubujamo bazo bomzimba bebuqalwa ngokutjheja bese zibulawa ngesikhathi esibekiweko. Zoke iinkambiso zenziwa ngokuya kwemihlahlandlela ye-Europe, yeentjhabatjhaba neyeenkhungo begodu zavunywa yi-LandesamtfürNatur ye-Umwelt ne-Verbraucherschutz, e-North Rhine-Westphalia, e-Germany.
Iinlwana zafakwa i-ketamine (100 mg/kg) kanye ne-xylazine (10 mg/kg), begodu ihliziyo yafakwa i-0.1 M PBS kokuthoma, bese ifakwa i-4% PFA ku-PBS. Ithitjhu yahlukaniswa begodu yalungiswa ku-4% PFA/PBS ubusuku boke ku-4°C. Ummese odlidlizako (i-Leica Microsystems GmbH, Vienna, Austria) wasetjenziselwa ukulungiselela iingaba ze-sagittal (50 μm ubukhulu) ukusuka ebuchosheni obujameleko ku-PBS. Ngaphandle kobana kuvezwe ngenye indlela, ukutjhwaba kweengaba ezithambileko kwenziwa njengombana kuchaziwe ngehla (13) emazingeni womtjhiso wegumbi nokuvuselelwa. Ngamafuphi, kokuthoma, iinlayi ezitholakeleko zafakwa nge-0.5% Triton X-100/PBS imizuzu eli-15 emazingeni womtjhiso wegumbi; kwamanye ama-epitope (i-Pcx ne-Shmt2), nge-tris-EDTA buffer ku-80°C (PH 9) ukutjhisa iinlayi imizuzu ema-25 esikhundleni segadango leli. Okulandelako, iingaba zafukanywa nge-antibody yokuthoma (qala iThebula S1) ku-block buffer (3% BSA/PBS) ku-4°C ubusuku boke ngokuvuselela. Ngelanga elilandelako, iingaba zahlanzwa nge-block buffer begodu zafakwa nge-fluorophore-conjugated secondary antibody ama-awa ama-2 emazingeni womtjhiso wegumbi; ekugcineni, iingaba zahlanzwa kuhle nge-PBS, zafakwa ibala nge-DAPI, bese zalungiswa nge-AquaPolymount Isilayidi se-microscope.
I-laser scanning confocal microscope (i-TCS SP8-X nofana i-TCS Digital Light Sheet, i-Leica Microsystems) ene-laser emhlophe kanye ne-405 diode ultraviolet laser yasetjenziselwa ukuthatha isithombe sesampula. Ngokuthabisa i-fluorophore nokubuthelela isiginali nge-Hybrid Detector (HyDs), isofthiwe ye-LAS-X yasetjenziselwa ukubuthelela iinthombe ezihlanganisiweko ezihambisana ne-Nyquist sampling ngendlela elandelanako: kumaphaneli angalinganiko, ziinthombe ezinamandla khulu (ngokwesibonelo, kumaseli we-somatic kanye nama-dendrite) mty-UPD YFP ukuya kunombolo ye-Hybrid Detector imodi). Ukuvala okungu-0.3 ukuya ku-6 ns kusetjenziswa ukwehlisa isizinda.
Ukuthathwa kwemifanekiso yesikhathi sangempela yamaseli ahlelweko. Ngemva kokuhlunga ku-Neurobasal-A medium ene-1% B27 supplement kanye ne-0.5 mM GlutaMax, amaseli atjalwa khonokho kuma-slide we-poly-l-lysine-coated glass (μ-Slide8 Kulungile, Ibidi, inombolo yekhathalogu 80826) , Bese wayigcina ku-37°C kanye ne-awa ye-CO hlala. Ukuthatha iinthombe ngesikhathi sangempela kwenziwa nge-Leica SP8 laser scanning confocal microscope ene-laser emhlophe, i-HyD, i-63×[1.4 numerical aperture (NA)] ilensi ye-oyile kanye ne-heating stage.
Igundane labulawa msinyana nge-carbon dioxide begodu laqunywa ikhanda, ubuchopho basuswa msinyana ekhanda, begodu yasikwa yaba yi-200μm ubukhulu (ngokulinga ukulebula i-13C) nofana i-275μm ubukhulu (ukuhlolwa kwama-photon amabili) isigaba se-sagittal esizele izinto ezilandelako. I-Walldorf, e-Germany) izele izinto ezilandelako: 125 mM ice-cold, i-carbon-saturated (95% O2 kanye ne-5% CO2) i-Ca2 ephasi + artificial cerebrospinal fluid (ACSF) NaCl, 2.5 mM KCl, 1.25 mM sodium phosphate buffer, 2.5 mM, mM, gluse 0.5 mM CaCl2 kanye ne-3.5 mM MgCl2 (umfutho we-osmotic we-310 ukuya ku-330 mmol). Dlulisela iinlayi zobuchopho ezitholiweko egumbini lokufukamela eline-Ca2 + ACSF ephezulu (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) Okuphakathi) pH 7.4 kanye no-310 ukuya ku-320 mmol).
Ngesikhathi senqubo yokuthatha iinthombe, iinlayi zathuthelwa egumbini lokuthatha iinthombe, begodu ukuhlolwa kwenziwa ngaphasi kokufuthelwa kwe-ACSF okuragela phambili emazingeni womtjhiso angaguqukiko ama-32° ukuya ku-33°C. I-multiphoton laser scanning microscope (TCS SP8 MP-OPO, Leica Microsystems) ene-Leica 25x objective lens (NA 0.95, amanzi), i-Ti: Sapphire laser (Chameleon Vision II, Coherent) yasetjenziselwa ukuthatha iinthombe. imojuli ye-FLIM (PicoHarp300, PicoQuant).
I-FLIM ye-Grx1-roGFP2. Ukutjhuguluka kwesimo se-cytoplasmic redox sama-PNs kwalinganiswa nge-FLIM yama-photon amabili eengqeni zobuchopho be-sagittal, lapho i-Grx1-roGFP2 biosensor iqothe khona ama-PNs. Ngaphakathi kwesendlalelo se-PN, indawo yokuthola ikhethwa ngama-50 ukuya ku-80 μm ngaphasi kwendawo yesilayi ukuqinisekisa bona kune-PN esebenzako (okutjho bona, ukutlhogeka kwesakhiwo esinama-beaded nofana amatjhuguluko we-neuronal morphological eceleni kwama-dendrite) kanye nesensa ye-roGFP2 ephindwe kabili kanye ne-AAV ekhoda i-shRNA PCx nofana ukulandelana kwayo kokulawula (ukuvezwa kwe-mChery). Buthelela iinthombe ezine-single-stack nge-2x digital zoom [excitation wavelength: 890 nm; 512 nm 512 amaphikseli]. Ukutholwa: i-HyD yangaphakathi, i-fluorescein isothiocyanate (FITC) filter group] kanye nokulinganisa isithombe ngaphakathi kwemizuzu emi-2 ukuya kwemi-3 kusetjenziselwa ukuqinisekisa bona ama-photon aneleko abuthelelwa (ama-photon ayi-1000 woke) ukulinganisa i-curve. Ukuzwela kwe-Grx1-roGFP2 probe nokuqinisekiswa kwemibandela ye-FLIM kwenziwa ngokutjheja inani lesikhathi sokuphila kwe-roGFP2 lokha nakufakwa i-exogenous 10 mM H2O2 ku-perfusion ACSF (ukukhulisa i-oksijini, okurholela ekukhuphukeni kwesikhathi sokuphila), bese kufakwa i-2 mM dithiothreit (ukunciphisa izinga lokuphila, okurholela ekunciphiseni ukwehla kwesikhathi sokuphila) (Umfanekiso S8, D ukuya ku-G). Sebenzisa isofthiwe ye-FLIMfit 5.1.1 ukuhlaziya imiphumela etholakeleko, ufanise ijika lokubola elilodwa lesithombe soke ku-IRF elinganisiweko (umsebenzi wokuphendula wethulusi), begodu i-χ2 ilinganiselwa ku-1. Ukubala isikhathi sokuphila se-PN eyodwa, imaski ezungeze umzimba wezinzwa yadwetshwa ngesandla, begodu kwasetjenziswa ukulinganisa kwesikhathi ngasinye semaski.
Ukuhlaziywa kwamandla we-mitochondrial. Ngemva kokuthi isigaba esibukhali sifukanywe nge-100 nM TMRM efakwe ngqo ku-ACSF ephuziweko imizuzu ema-30, amatjhuguluko we-mitochondrial we-PNs alinganiswa nge-microscope ye-photon ezimbili. Ukuthathwa kwemifanekiso ye-TMRM kwenziwa ngokuvusa i-probe ku-920 nm begodu kusetjenziswa i-HyD yangaphakathi (tetramethylrhodamine isothiocyanate: 585/40 nm) ukubuthelela amatshwayo; ngokusebenzisa i-wavelength efanako yokuvuselela kodwana ngokusebenzisa i-HyD yangaphakathi ehlukileko (FITC :525/50) ukuthatha isithombe se-mtYFP. Sebenzisa i-ImageJ's Image Calculator plug-in ukuhlola amandla we-mitochondrial ezingeni leseli elilodwa. Ngamafuphi, i-plug-in equation: signal = min (mtYFP, TMRM) isetjenziselwa ukukhomba indawo ye-mitochondrial etjengisa isignali ye-TMRM ku-Purkinje Somali esithombeni se-single-stack confocal sesiteshi esihambisanako. Bese kuthi indawo yamaphikseli emaskini ephumelako iyalinganiswa, bese yenziwa ibe yinto ejayelekileko esithombeni esifanako se-mtYFP ukuthola ingcenye ye-mitochondrial etjengisa amandla we-mitochondrial.
Isithombe sahlukaniswa ngesofthiwe ye-Huygens Pro (Scientific Volume Imaging). Eenthombeni eziskeniweko zamathayela, ukuhlanganiswa kwethayela elilodwa kwenziwa kusetjenziswa i-algorithm yokuthunga ezenzakalelayo enikelwa yisofthiwe ye-LAS-X. Ngemva kokulinganisa isithombe, sebenzisa i-ImageJ ne-Adobe Photoshop ukuragela phambili nokucubungula isithombe begodu ulungise ngokufanako ukukhanya nokuphambene. Sebenzisa i-Adobe Illustrator ukulungiselela iinthombe.
Ukuhlaziywa kokugxila kwe-mtDNA. Inani leenlonda ze-mtDNA lalinganiswa eengcenyeni ze-cerebellar ezifakwe ama-antibodies aphikisana ne-DNA nge-confocal microscope. Indawo ngayinye ehlosiweko yenzelwe umzimba weseli kanye ne-nucleus yeseli ngayinye, begodu indawo ehlukileko yabalwa kusetjenziswa iplagi ye-Multi Measure (isofthiwe ye-ImageJ). Susa indawo yenyukliya endaweni yomzimba weseli ukuthola indawo ye-cytoplasmic. Ekugcineni, i-Analyze Particles plug-in (isofthiwe ye-ImageJ) yasetjenziselwa ukulinganisa ngokuzenzakalelako amaphuzu we-DNA we-cytoplasmic atjengisa i-mtDNA esithombeni se-threshold, begodu imiphumela etholakeleko yenziwa ibe yinto ejwayelekileko ye-PN yamagundane we-CTRL. Imiphumela ivezwa njengenani eliphakathi lama-nucleosides ngeseli ngalinye.
Ukuhlaziywa kokuvezwa kwamaphrotheyini. Sebenzisa i-ImageJ's Image Calculator plug-in ukuhlola ukuvezwa kwamaphrotheyini ku-PN ezingeni leseli elilodwa. Ngamafuphi, esithombeni esine-single-layer confocal sesiteshi esihambelanako, ngokusebenzisa i-equation: isignali = umzuzu (mtYFP, i-antibody), indawo ye-mitochondrial etjengisa ukuzivikela kwe-antibody ethileko e-Purkina iyakhonjwa. Bese kuthi indawo yephikseli emaskini ephumelako iyalinganiswa, bese yenziwa ibe yinto ejayelekileko esithombeni esifanako sesikhwama esisodwa sesiteshi se-mtYFP ukuthola ingcenye ye-mitochondrial yephrotheyini etjengisiweko.
Ukuhlaziywa kobukhulu bamaseli we-Purkinje. I-Cell Counter plug-in ye-ImageJ yasetjenziselwa ukuhlola ukuminyana kwe-Purkinje ngokuhlukanisa inani lamaseli we-Purkinje abalweko ngobude bendandatho ye-cerebellar ehlala amaseli abalweko.
Ukulungiselela nokubuthelela isampula. Ubuchopho obuvela esiqhemeni sokulawula kanye namagundane we-Mfn2cKO kwalungiswa ku-2% PFA/2.5% glutaraldehyde ku-0.1 M phosphate buffer (PB), bese kuthi iingaba ze-coronal zalungiswa kusetjenziswa ama-ciliates (Leica Mikrosysteme GmbH, Vienna, Austria) (Ubukhulu 50 ukuya ku-60) Ukulungiswa ku-B buffer ku-P1% i-tetraoxide kanye ne-1.5% ye-potassium ferrocyanide emazingeni womtjhiso wegumbi i-awa elilodwa. Iingcenye zahlanzwa kathathu ngamanzi ahlanjululweko, bese zafakwa ibala nge-70% ye-ethanol ene-1% ye-uranyl acetate imizuzu ema-20. Iingcenye zabe seziphelelwa mamanzi nge-alkhoholi ehlelweko begodu zafakwa ngaphakathi kwe-Durcupan ACM (Araldite casting resin M) epoxy resin (Electron Microscopy Sciences, inomboro yekhathalogu 14040) hlangana nama-slide weklasi agcotjhwe nge-silicon, begodu ekugcineni nge-60°C zafakwa ngaphakathi kwe-oveni ama-awa ama-48. Indawo ye-cerebellar cortex yakhethwa begodu iingaba ezi-50 nm ezincani zasikwa ku-Leica Ultracut (Leica Mikrosysteme GmbH, Vienna, Austria) begodu zakhethwa ku-2×1 mm copper slit grid ehlanganiswe nge-polystyrene film. Iingaba zafakwa ibala ngesisombululo se-4% ye-uranyl acetate ku-H2O imizuzu eli-10, zahlanzwa nge-H2O kanengi, bese zahlanzwa nge-Reynolds lead citrate ku-H2O imizuzu eli-10, bese zahlanzwa nge-H2O kanengi. Iinthombe ezincani zathathwa nge-transmission electron microscope Philips CM100 (Thermo Fisher Scientific, Waltham, MA, USA) kusetjenziswa i-TVIPS (Tietz Video and Image Processing System) TemCam-F416 digital camera (TVIPS GmbH, Gauting, USA). I-Germany).
Emagundaneni atheleleke nge-AAV, ubuchopho bebuhlukaniswa begodu bube yingcenye ye-sagittal ebukhulu obungu-1 mm, begodu i-cerebellum yahlolwa kusetjenziswa i-fluorescence microscope ukukhomba indandatho ene-AAV (okutjho, ukuveza i-mCherry). Iimhloliso kwaphela lapho ukujova kwe-AAV kuphumela ekusebenzeni okuphezulu khulu kwesendlalelo seseli le-Purkinje (okutjho pheze isendlalelo soke) okungenani emaringini amabili alandelanako we-cerebellar asetjenziswa. I-AAV-transduced loop yahlukaniswa ngobusuku boke ngemva kokulungiswa (4% PFA kanye ne-2.5% glutaraldehyde ku-0.1 M cocoate buffer) begodu yaragela phambili nokusetjenzwa. Ukufaka i-EPON, isicubu esiqinileko sahlanzwa nge-0.1 M ye-sodium cocoate buffer (Applichem), begodu safukanywa nge-2% OsO4 (os, Science Services; Caco) ku-0.1 M ye-sodium cocoate buffer (Applichem) ama-awa ama-4, bese sihlanzwa ama-awa ama-2. Phinda kathathu nge-0.1 M ye-cocamide buffer. Ngemva kwalokho, uchungechunge olukhuphukako lwe-ethanol lwasetjenziselwa ukufukamela isisombululo ngasinye se-ethanol ku-4°C imizuzu eli-15 ukuqeda amanzi esicutshini. Ithitjhu yadluliselwa ku-propylene oxide begodu yafukamela ubusuku boke ku-EPON (Sigma-Aldrich) ku-4°C. Faka isicubu ku-EPON etja emazingeni womtjhiso wegumbi ama-awa amabili, bese uyifaka ku-62°C ama-awa ama-72. Sebenzisa i-ultramicrotome (Leica Microsystems, UC6) kanye nommese wedayimani (Diatome, Biel, Switzerland) ukusika iingaba ezi-70 nm ezincani khulu, bese ufaka ibala nge-1.5% ye-uranyl acetate imizuzu eli-15 ku-37°C, bese ufaka ibala ngesisombululo se-lead citrate imizuzu emi-4. Iinthombe ze-elekthronikhi zithathwe kusetjenziswa i-JEM-2100 Plus transmission electron microscope (JEOL) ene-Camera OneView 4K 16-bit (Gatan) kanye nesofthiwe ye-DigitalMicrograph (Gatan). Ukuhlaziywa, ama-electron micrographs atholwa nge-5000× nofana i-10,000× yedijithali zoom.
Ukuhlaziywa kwesakhiwo sama-mitochondria. Kikho koke ukuhlaziywa, amakhontu we-mitochondria ngayinye avezwe ngesandla eenthombeni zedijithali kusetjenziswa isofthiwe ye-ImageJ. Amapharamitha ahlukileko wesakhiwo ayahlaziywa. Ukuminyana kwe-mitochondria kuvezwa njengephesenteji etholwe ngokuhlukanisa indawo ye-mitochondrial yoke yeseli ngayinye ngendawo ye-cytoplasm (indawo ye-cytoplasm = indawo yeseli-indawo ye-nucleus yeseli) × 100. Ubukhulu be-mitochondria bubalwa ngefomula [4π∙(area/perimeter 2)]. I-ista morphology yama-mitochondria yahlaziywa begodu yahlukaniswa yaba ziinkoro ezimbili ("tubular" kanye "ne-blister") ngokuya ngejamo layo elikhulu.
Inomboro ye-autophagosome/lysosome nokuhlaziywa kobukhulu. Sebenzisa isofthiwe ye-ImageJ ukuveza ngesandla amakhontu we-autophagosome/lysosome ngayinye esithombeni sedijithali. Indawo ye-autophagosome/lysosome ivezwa njengephesenteji ebalwa ngokuhlukanisa indawo yesakhiwo se-autophagosome/lysosome yeseli ngayinye ngendawo ye-cytoplasm (indawo ye-cytoplasm=indawo yeseli-indawo ye-nucleus)×100. Ukuminyana kwama-autophagosomes/ama-lysosomes kubalwa ngokuhlukanisa inani elipheleleko ngenani lezakhiwo ze-autophagosome/lysosome ngeseli ngalinye (ngokuya ngendawo ye-cytoplasmic) (indawo ye-cytoplasmic = indawo yeseli-indawo yenyukliya).
Ukulebula ukuhlukaniswa okubukhali nokulungiselela isampula. Ukuhlolwa okutlhoga ukulebula kwe-glucose, dlulisela iinqetjhana zobuchopho ezibukhali ekamelweni lokufukamela, eliqukethe ikhabhoni egcweleko (95% O2 kanye ne-5% CO2), i-Ca2 + ACSF ephezulu (125.0 mM NaCl, 2.5 mM KCl, 1.25 mM sodium phosphate buffer, 2.5 mM NaCl, 2.5 mM. d-glucose, 1.0 mM CaCl 2 kanye ne-2.0 mM MgCl 2, elungiselelwe ku-pH 7.4 kanye ne-310 ukuya ku-320 mOsm), lapho i-glucose iyi-13 C 6- Glucose substitution (Eurisotop, inombolo yekhathalogu CLM-1396). Ukuhlolwa okutlhoga ukulebula nge-pyruvate, dlulisa iinlayi zobuchopho ezibukhali zibe yi-Ca2 + ACSF ephezulu (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. I-MgCl2, yenza ibe yi-pH 7.4 kanye ne-310 ukuya ku-320mOsm), bese ufaka i-1mM 1-[1-13C]pyruvate (Eurisotop, inombolo yekhathalogu CLM-1082). Fukamela iingcenye imizuzu ema-90 ku-37°C. Ekupheleni kokuhlola, iingaba zahlanzwa msinyana ngesisombululo samanzi (pH 7.4) esine-75 mM ammonium carbonate, bese zihlanganiswa ku-40:40:20 (v:v:v) acetonitrile (ACN): methanol: amanzi. Ngemva kobana iingaba zifakwe phezu kweqhwa imizuzu ema-30, amasampula ahlanganiswa ku-21,000 g imizuzu eli-10 ku-4°C, begodu i-supernatant ecacileko yomiswe ku-SpeedVac concentrator. I-pellet ye-metabolite eyomileko ebegade igcinwe ku--80°C bekwaba kulapho ihlaziywa khona.
Ukuhlaziywa kwe-liquid chromatography-mass spectrometry yama-amino acid ali-13 ane-C. Ukuhlaziywa kwe-liquid chromatography-mass spectrometry (LC-MS), i-metabolite pellet yabuyiselwa emuva ku-75μl yamanzi wezinga le-LC-MS (Honeywell). Ngemva kokukhiqizwa nge-21,000 g imizuzu emi-5 ku-4°C, i-20 μl ye-supernatant ehlanjululweko yasetjenziselwa ukuhlaziywa kwe-amino acid flux, kuthi okuseleko kwasetjenziselwa ukuhlaziywa kwe-anion (qala ngenzasi). Ukuhlaziywa kwe-amino acid kwenziwa kusetjenziswa iphrothokholi yokuthola i-benzoyl chloride echazwe ngaphambilini (55, 56). Esigabeni sokuthoma, i-10μl ye-100 mM ye-sodium carbonate (Sigma-Aldrich) yafakwa ku-20μl ye-metabolite extract, bese i-10μl ye-2% benzoyl chloride (Sigma-Aldrich) yafakwa ku-LC grade ACN. Isampula yatjhugululwa kancani bese yatjhugululwa ku-21,000 g imizuzu emi-5 ku-20°C. Dlulisela i-supernatant ehlanjululweko ebhodini le-2 ml le-autosampler eline-insert ye-conical glass (200 μl yevolumu). Amasampula ahlaziywa kusetjenziswa i-Acquity iClass ultra-high performance LC system (Waters) exhunywe ku-Q-Exactive (QE)-HF (Ultra High Field Orbitrap) high-resolution precision mass spectrometer (Thermo Fisher Scientific). Ukuhlaziywa, i-2μl yesampula ethathweko yafakwa ngaphakathi kwe-100×1.0 mm ephezulu ye-silica T3 kholomu (Waters) eneenhlayiya eziyi-1.8μm. Izinga lokugeleza lingu-100μl/min, begodu ihlelo lokuvikela linebhafa A (10 mM ammonium formate kanye ne-0.15% formic acid emanzini) kanye nebhafa B (ACN). I-gradient ilandelako: 0%B ngemizuzu eyi-0; 0%B. 0 ukuya ku-15% B ngemizuzu engu-0 ukuya ku-0.1; 15 ukuya ku-17% B ngemizuzu engu-0.1 ukuya ku-0.5; B ku-17 ukuya ku-55% ngemizuzu engu-0.5 ukuya ku-14; B ku-55 ukuya ku-70% ngemizuzu eli-14 ukuya kweli-14.5; ku-14.5 ukuya ku-70 ukuya ku-100% B ngemizuzu eli-18; 100% B emizuzwini eli-18 ukuya keli-19; 100 ukuya ku-0% B emizuzwini eli-19 ukuya kweli-19.1; 0% B emizuzwini eli-19.1 ukuya kweli-28 (55, 56). I-QE-HF mass spectrometer isebenza ngendlela ye-ionization ehle ene-mass range ye-m/z (mass/charge ratio) yama-50 ukuya ku-750. Isisombululo esisetjenzisiweko ngu-60,000, begodu i-gain control (AGC) ion target etholweko ngu-3×106, begodu isikhathi esiphezulu se-ion siyi-100 yamamilimitha. Umthombo we-electrospray ionization (ESI) otjhisako usebenza nge-voltage yokufafaza engu-3.5 kV, izinga lokutjhisa le-capillary elingu-250°C, ukugeleza komoya kwe-sheath okungu-60 AU (amayunithi angakahleleki), kanye nokugeleza komoya okusizako okungu-20 AU. 250°C. Ilensi ye-S isethwe ku-60 AU.
Ukuhlaziywa kwe-anion chromatography-MS yama-organic acid alebhulwe nge-13C. I-metabolite eseleko (55μl) yahlaziywa kusetjenziswa i-Dionex ion chromatography system (ICS 5000+, Thermo Fisher Scientific) exhunywe ku-QE-HF mass spectrometer (Thermo Fisher Scientific). Ngamafuphi, i-5μl ye-metabolite extract yafakwa ngaphakathi kwekholomu ye-Dionex IonPac AS11-HC ene-HPLC (2 mm×250 mm, ubukhulu bezinhlayiya 4μm, i-Thermo Fisher Scientific) ngemodi ye-push-in partial loop nge-filling ratio ye-1. )on. Ikholomu yokuvikela i-Dionex IonPac AG11-HC (2 mm x 50 mm, 4μm, Thermo Fisher Scientific). Izinga lokutjhisa lekholomu ligcinwa ku-30°C, begodu i-autosampler isethwe ku-6°C. Sebenzisa ikhatriji ye-potassium hydroxide enikelwe ngamanzi ahlanjululweko ukwenza i-potassium hydroxide gradient nge-eluent generator. Ukuhlukaniswa kwama-metabolites ngezinga lokugeleza elingu-380μl/min, kusetjenziswa i-gradient elandelako: 0 ukuya ku-3 imizuzu, 10 mM KOH; 3 ukuya ku-12 imizuzu, 10 ukuya ku-50 mM KOH; 12 ukuya ku-19 imizuzu, 50 ukuya ku-100 mM KOH; 19 ukuya ku-21 imizuzu, 100 mM KOH; 21 ukuya ku-21.5 imizuzu, 100 ukuya ku-10 mM KOH. Ikholomu yaphinde yalinganiswa ngaphasi kwe-10 mM KOH imizuzu eyi-8.5.
Ama-metabolites ahlanjululweko ahlanganiswa ne-150μl/min isopropanol supplement stream ngemva kwekholomu bese aqondiswa ku-mass spectrometer ene-resolution ephezulu esebenza ngendlela ye-ionization embi. I-MS igada ububanzi obusuka ku-m/z 50 ukuya ku-750 ngesisombululo esingu-60,000. I-AGC ibekwe ku-1×106, begodu isikhathi esiphezulu se-ion sigcinwa ku-100 ms. Umthombo we-ESI otjhisako bewusebenza ngevoltheji yokufafaza engu-3.5 kV. Amanye amasethingi womthombo we-ayoni alandelako: izinga lokutjhisa le-capillary 275°C; ukugeleza kwegesi, 60 AU; ukugeleza kwegesi, 20 AU ku-300°C, kanye ne-S lens ukubekwa ku-60 AU.
Ukuhlaziywa kwedatha yama-metabolites abhalwe nge-13C. Sebenzisa isofthiwe ye-TraceFinder (inguqulo 4.2, Thermo Fisher Scientific) ukuhlaziywa kwedatha ye-isotope ratio. Ubunjalo bekhompawundi ngayinye kwaqinisekiswa yikhompawundi ethembekileko begodu yahlaziywa ngokuzijamela. Ukuze kwenziwe ukuhlaziywa kokucebisa i-isotope, indawo ye-extracted ion chromatogram (XIC) ye-13C isotope ngayinye (Mn) yakhishwa ku-[M + H] +, lapho n kuyinombolo yekhabhoni ye-compound ehlosiweko, esetjenziselwa ukuhlaziya ama-amino acid nofana [ MH] + isetjenziselwa ukuhlaziya ama-anion. Ukunemba kwesisindo se-XIC kungaphasi kwamalunga amahlanu ngesigidi, begodu ukunemba kwe-RT kungu-0.05 wemizuzu. Ukuhlaziywa kokunothisa kwenziwa ngokubala isilinganiso se-isotope ngayinye etholakeleko ukuya ekuhlanganisweni kwawo woke ama-isotopu wekhompawundi ehambelana nayo. Ama-ratio la anikelwa njengamanani wamaphesente we-isotope ngayinye, begodu imiphumela ivezwa njenge-molar percent enrichment (MPE), njengombana kuchaziwe ngaphambilini (42).
I-neuron pellet efriziweko yahlanganiswa nge-80% ye-methanol (v/v) ebanda njengeqhwa, yahlanganiswa, begodu yafukanywa ku--20°C imizuzu ema-30. Hlanganisa isampula godu bese uvuselela ku-+4°C imizuzu ema-30. Isampula yahlanganiswa ku-21,000 g imizuzu emi-5 ku-4°C, bese kuthi i-supernatant eyaba khona yaqoqwa yayomiswa kusetjenziswa i-SpeedVac concentrator ku-25°C ukuhlaziywa okulandelako. Njengombana kuchaziwe ngehla, ukuhlaziywa kwe-LC-MS kwenziwa kuma-amino acid wamaseli ahlukanisiweko. Ngokusebenzisa i-TraceFinder (inguqulo 4.2, Thermo Fisher Scientific), ukuhlaziywa kwedatha kwenziwa kusetjenziswa ubukhulu be-monoisotopic ye-compound ngayinye. Ukulinganisa idatha ye-metabolite kwenziwa kusetjenziswa iphakheji yesofthiwe ye-preprocessCore (57).
Ukulungiselela isilayi. Igundwane labulawa msinyana nge-carbon dioxide begodu laqunywa ihloko, ubuchopho basuswa msinyana ekhanda, begodu umukhwa wokuthuthumela ogcwele iqhwa (HM-650 V, Thermo Fisher Scientific, Walldorf, Germany) wasetjenziselwa ukuwusika ube ziingcenye ezingu-300 ukuya ku-375 μm ze-sagittal carbon gas (25% CO) I-Ca2 + ACSF ephasi (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 kanye ne-6.0 mM MgCl2 Lungisa ibe ku-pH ukuya ku-pH 430 mm Dlulisela iinqetjhana zobuchopho ezitholiweko uye ekamelweni eline-Ca2 + ACSF ephezulu (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 ml Ca 2 mM kanye ne-pHC mg 7.4 kanye no-310 ukuya ku-320 mOsm). Gcina iinlayi imizuzu ema-20 ukuya kwema-30 ukwenzela bona zibuyiselwe ngaphambi kokurekhoda.
ukurekhoda. Isiteji se-microscope esifakwe ikamelo lokurekhoda elingatjhugulukiko kanye ne-20x water immersion objective lens (i-Scientifica) sasetjenziselwa koke ukurekhoda. Amaseli we-Purkinje akhonjwa (i) ngobukhulu bomzimba, (ii) indawo yomzimba ye-cerebellum, kanye (iii) nokuvezwa kwe-fluorescent mtYFP reporter gene. I-patch pipette ene-tip resistance yama-5 ukuya ku-11 megohms idonswa yi-borosilicate glass capillary (GB150-10, 0.86 mm×1.5 mm×100 mm, Science Products, Hofheim, Germany) kanye ne-horntal pipette Instruments (P-10000), Notova, CA). Zoke iinrekhodi zenziwa nge-ELC-03XS npi patch clamp amplifier (npi electronic GmbH, Tam, Germany), ebeyilawulwa yi-software Signal (inguqulo 6.0, Cambridge Electronic, Cambridge, UK). Ukuhlolwa kwarekhodwa ngezinga lokusampula le-12.5 kHz. Isiginali ihlungwa ngezihlungi ezimbili ze-Bessel ezifitjhani ezinamafrikhwensi wokusika we-1.3 kanye ne-10 kHz ngokulandelana. I-capacitance ye-membrane ne-pipette inxephezelwa yi-compensation circuit ngokusebenzisa i-amplifier. Zoke iimhloliso zenziwa ngaphasi kokulawulwa kwekhamera ye-Orca-Flash 4.0 (Hamamatsu, Gerden, Germany), ebegade ilawulwa yisofthiwe ye-Hokawo (inguqulo 2.8, Hamamatsu, Gerden, Germany).
Ukuhlelwa nokuhlaziywa kwamaseli woke. Ngaphambi kokurekhoda, gcwalisa i-pipette ngesisombululo sangaphakathi esinezinto ezilandelako: 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 (M0 mM), Gunophosiphate. (GTP) (Na) kanye ne-10.0 mM creatinine phosphate zalungiswa zaba yi-pH 7.25, begodu umfutho we-osmotic bewungu-290 mOsm (i-sucrose). Msinyana ngemva kokusebenzisa amandla we-0 pA ukudabula ulwelwesi, kwalinganiswa amandla welwelwesi lokuphumula. Ukujamelana okungenako kulinganiswa ngokusebenzisa ama-hyperpolarized currents we--40, -30, -20, kanye ne--10 pA. Linganisa ubukhulu bokuphendula kwevoltheji bese usebenzisa umthetho ka-Ohm ukubala ukujamelana kokufaka. Ukusebenza ngokuzenzakalelako kwarekhodwa nge-voltage clamp imizuzu emi-5, begodu i-sPSC yahlonzwa begodu yalinganiswa ku-Igor Pro (inguqulo 32 7.01, WaveMetrics, Lake Oswego, Oregon, USA) kusetjenziswa iskripthi sokuqaphela esizenzakalelayo. I-IV curve kanye ne-steady-state current kulinganiswa ngokubophelela ibhetri ngamandla ahlukileko (ukuthoma ku--110 mV) begodu kukhuphulwe ivoltheji ngamagadango we-5 mV. Ukukhiqizwa kwe-AP kwahlolwa ngokusebenzisa umtjhini wokukhiqiza. Bopha iseli ku--70 mV ngesikhathi usebenzisa i-pulse yamanje. Lungisa ubukhulu besinyathelo seyunithi ngayinye yokurekhoda ngokwehlukana (10 ukuya ku-60 pA). Bala imvamisa ephezulu ye-AP ngokubala ngesandla ama-pulse spikes abangela imvamisa ephezulu ye-AP. I-AP threshold ihlaziywa ngokusebenzisa i-derivative yesibili ye-depolarization pulse eqala ukuvusa i-AP eyodwa nofana ngaphezulu.
Ukuhlelwa nokuhlaziywa kwephetjhi eneembobo. Yenza ukurekhoda kwe-patch enebhoboza ngokusebenzisa amaphrothokholi ajwayelekileko. Sebenzisa i-ATP- kanye ne-GTP-free pipette engenazo iinthako ezilandelako: 128 mM gluconate K, 10 mM KCl, 10 mM Hepes, 0.1 mM EGTA kanye ne-2 mM MgCl2, bese uzilungiselela ku-pH 7.2 (usebenzisa i-KOH). I-ATP ne-GTP azifakwa esixazululweni sangaphakathi kwamaseli ukukhandela ukungena okungalawulekiko kwe-membrane yeseli. I-patch pipette izaliswa ngesisombululo sangaphakathi esine-amphotericin (cishe ku-200 ukuya ku-250 μg/ml; G4888, Sigma-Aldrich) ukuthola irekhodi le-patch enembobo. I-amphotericin yancibilikiswa ku-dimethyl sulfoxide (ukugxila kokugcina: 0.1 ukuya ku-0.3%; DMSO; D8418, Sigma-Aldrich). Ukugxila kwe-DMSO esetjenzisiweko akuzange kube nomthelela omkhulu kuma-neurons afundwako. Ngesikhathi senqubo yokutjhwaba, ukujamelana kwetjhaneli (i-Ra) bekuhlala kugadwa, begodu ukuhlolwa kwathonywa ngemva kokuthi i-amplitude ye-Ra ne-AP zizinzile (imizuzu ema-20-40). Umsebenzi ozenzekelayo ulinganiswa ngevoltheji kanye/nofana nge-clamp yamanje imizuzu emibili ukuya kwemihlanu. Ukuhlaziywa kwedatha kwenziwa kusetjenziswa i-Igor Pro (inguqulo 7.05.2, WaveMetrics, USA), Excel (inguqulo 2010, Microsoft Corporation, Redmond, USA) kanye ne-GraphPad Prism (inguqulo 8.1.2, GraphPad Software Inc., La Jolla, CA). I-United States). Ukuze kutholakale ama-AP azenzekelayo, i-IgorPro's NeuroMatic v3.0c plug-in iyasetjenziswa. Khomba ama-AP ngokuzenzakalelako ngokusebenzisa umkhawulo onikelweko, olungiswa ngamunye ngamunye kurekhodi ngalinye. Ngokusebenzisa isikhala se-spike, hlola imvamisa ye-spike ngefrikhwensi ephezulu ye-spike kanye nefrikhwensi ye-spike ephakathi.
Ukuzihlukanisa kwe-PN. Ngokuvumelana nephrothokholi egadangisiweko ngaphambilini, ama-PN ahlanjululwa ebuchosheni begundane esigabeni esithileko (58). Ngamafuphi, i-cerebellum yahlukaniswa begodu yagaywa ngaphakathi kwe-ice-cold dissociation medium [ngaphandle kwe-HBSS Ca2+ ne-Mg2+, enezelelwe nge-20 mM glucose, i-penicillin (50 U/ml) kanye ne-streptomycin (0.05 mg/ ml)], bese igaywa i-medium ku-papa [HBSS, ene-HBSS, ene-1 (1 mg / ml), i-papain (16 U / ml) kanye ne-deoxyribonuclease I (DNase I; 0.1 mg/ml)] Lapha imizuzu ema-30 ku-30°C. Kokuthoma hlamba amathitjhu nge-HBSS ene-egg mucus (10 mg/ml), i-BSA (10 mg/ml) kanye ne-DNase (0.1 mg/ml) emazingeni womtjhiso wegumbi ukukhandela ukugayeka kwe-enzymatic, bese kuthi nge-HBSS ene-20 mM ye-glucose Ukugaya ngobumnene ku-HBSS, i-peillin (10 mg/ml) (0.05 mg/ml) kanye ne-DNase (0.1 mg/ml) zikhupha amaseli awodwa. Ukumiswa kwamaseli okuphumelako kwahlungwa ngesihlungi samaseli esingu-70μm, bese amaseli ahlungwa nge-centrifugation (1110 rpm, imizuzu emi-5, 4°C) begodu abuyiselwa emkhathini wokuhlunga [HBSS, enezwe nge-20 mM glucose, 20% fetal bovine ) Serum, pelinic (50/5) (0.05 mg/ml)]; hlola ukusebenza kwamaseli nge-propidium iodide bese ulungisa ukuminyana kwamaseli kube yi-1×106 ukuya ku-2×106 amaseli/ml. Ngaphambi kwe-flow cytometry, ukumiswa kwahlungwa nge-50 μm cell strainer.
I-flow cytometer. Ukuhlunga amaseli kwenziwa ku-4°C kusetjenziswa umtjhini we-FACSAria III (BD Biosciences) kanye nesofthiwe ye-FACSDiva (BD Biosciences, inguqulo 8.0.1). Ukumiswa kwamaseli kwahlukaniswa kusetjenziswa umlomo we-100 μm ngaphasi komfutho we-20 psi ngezinga le-~2800 yezehlakalo/sekhondi. Njengombana indlela yokuvala yendabuko (ubukhulu bamaseli, ukubandlulula okubili, kanye nobujamo bokusakazeka) ingakghoni ukuqinisekisa ukuhlukaniswa okufaneleko kwe-PN kweminye imihlobo yamaseli, isu lokuvala libekwa ngokuya ngokumadanisa okunqophileko kwamandla we-YFP nokuzibonakalisa ku-mitoYFP+ ​​kanye ne-mitoYFP − Mice yokulawula. I-YFP ithabile ngokufaka isampula ngelayini ye-488 nm ye-laser, begodu isiginali itholwa ngokusebenzisa isihlungi se-530/30 nm se-band pass. Emagundeni we-mitoYFP+, amandla ahlobeneko we-Rosa26-mitoYFP reporter gene nawo asetjenziselwa ukuhlukanisa umzimba we-neuron kanye nama-axon fragments. I-7-AAD ithabile nge-561 nm ye-laser ephuzi begodu itholakala nge-675/20 nm bandpass filter ukukhipha amaseli ahlongakeleko. Ukuze kuhlukaniswe ama-astrocytes ngesikhathi sinye, ukumiswa kwamaseli kwafakwa ibala nge-ACSA-2-APC, bese isampula yafakwa nge-640 nm laser line, bese kuthi i-660/20 nm bandpass filter yasetjenziselwa ukuthola isignali.
Amaseli abuthelelweko ahlanganiswa nge-centrifugation (1110 rpm, imizuzu emi-5, 4°C) begodu agcinwa ku--80°C bekwaba kulapho asetjenziswa khona. Amagundane we-Mfn2cKO kanye namadzinyana wawo ahlukaniswa ngelanga elifanako ukunciphisa ukuhlukahluka kwendlela yokusebenza. Ukwethulwa kwedatha ye-FACS nokuhlaziywa kwenziwa kusetjenziswa isofthiwe ye-FlowJo (FlowJo LLC, Ashland, Oregon, USA).
Njengombana kutjhiwo ngehla (59), i-PCR yesikhathi sangempela isetjenziselwa ukuhlukanisa i-DNA ema-neurons ahlukanisiweko ukulinganisa i-mtDNA. Ukulandelana nokuzwela komkhawulo kwahlolwa ekuthomeni ngokusebenzisa i-qPCR eenombolweni ezihlukileko zamaseli. Ngamafuphi, buthelela i-300 PN ngaphakathi kwe-lysis buffer ene-50 mM tris-HCl (pH 8.5), i-1 mM EDTA, i-0.5% Tween 20 kanye ne-proteinase K (200 ng/ml) bese uyifukamela ku-55°C imizuzu eli-120. Amaseli aragela phambili nokufukamela ku-95°C imizuzu eli-10 ukuqinisekisa ukungasebenzi okupheleleko kwe-proteinase K. Ngokusebenzisa i-TaqMan probe (Thermo Fisher) eqondene ne-mt-Nd1, i-mtDNA yalinganiswa nge-semi-quantitative PCR ku-7900HT Real-Time PCR system (Thermoherific Scientifics). Isayensi, inombolo yekhathalogu Mm04225274_s1), mt-Nd6 (Thermo Fisher Scientific, inombolo yekhathalogu AIVI3E8) kanye ne-18S (Thermo Fisher Scientific, inombolo yekhathalogu Hs99999901_s1) amajini.
Ukulungiselela isampula yamaphrotheyimu. Ngokutjhisa isisombululo ku-95°C imizuzu eli-10 bese u-sonicate, ngaphakathi kwe-lysis buffer [6 M guanidine chloride, 10 mM tris(2-carboxyethyl) phosphine hydrochloride, 10 mM chloroacetamide kanye ne-100 mM tris-Lyse frozen neuron pellets in HC]. Ku-Bioruptor (Diagenode) imizuzu eli-10 (i-pulse yemizuzwana ema-30 / isikhathi sokujama semizuzwana ema-30). Isampula yahlanjululwa ngo-1:10 ku-20 mM tris-HCl (pH 8.0), yahlanganiswa ne-300 ng ye-trypsin gold (Promega), begodu yafukamela ubusuku boke ku-37°C ukufikelela ukugayeka okupheleleko. Ngelanga lesibili, isampula yatjhugululwa ku-20,000 g imizuzu ema-20. I-pernatant yahlanjululwa nge-0.1% ye-formic acid, begodu isisombululo sasuswa itswayi nge-StageTips ezenziwe ngokwayo. Isampula yomiswa nge-SpeedVac (i-Eppendorf concentrator plus 5305) ku-45°C, bese i-peptide yalengiswa ku-0.1% formic acid. Woke amasampula alungiselelwe ngasikhathi sinye mumuntu munye. Ukuze kuhlaziywe amasampula we-astrocyte, ama-peptide ama-4 μg we-desalted abhalwe nge-tandem mass tag (TMT10plex, inombolo yekhathalogu 90110, Thermo Fisher Scientific) nge-peptide ukuya ku-TMT reagent ratio ye-1:20. Ukulebula i-TMT, i-0.8 mg ye-TMT reagent yafakwa kabutjha ku-70 μl ye-ACN enganamanzi, begodu i-peptide eyomileko yabuyiselwa yaba yi-9 μl ye-0.1 M TEAB (triethylammonium bicarbonate), lapho i-7 μl ye-TMT reagent ku-ACN yengezwa khona. Ukugxila bekungu-43.75%. Ngemva kwemizuzu ema-60 yokufukamela, ukusabela kwacinywa nge-2 μl ye-5% hydroxylamine. Ama-peptide alebuliweko abuthelelwa, omiswa, abuyiselwa emuva ku-200μl ye-0.1% ye-formic acid (FA), ahlukaniswe aba mabili, bese asuswa itswayi kusetjenziswa i-StageTips eyenziwe ngokwayo. Ngokusebenzisa i-UltiMate 3000 ultra high performance liquid chromatograph (i-UltiMate 3000 ultra high performance liquid chromatograph), enye yeengcenye ezimbili yahlukaniswa kukholomu ye-1mm x 150mm Acquity chromatographic ezele iinhlayiya ze-130Å1.7μm C18 (Waters, ikhathalogu Nombolo SKU: 196366). Thermo Fisher Scientific). Hlukanisa ama-peptide ngezinga lokugeleza elingama-30μl/min, hlukanisa ukusuka ku-1% ukuya ku-50% we-buffer B imizuzu ema-85 nge-gradient ye-96 yemizuzu, ukusuka ku-50% ukuya ku-95% ye-buffer B imizuzu emi-3, bese kuba yimizuzu emi-8 ukuya ku-95 % ye-buffer B; Ibhafa A yi-5% ACN kanye ne-10 mM ammonium bicarbonate (ABC), begodu ibhafa B yi-80% ACN kanye ne-10 mM ABC. Buthelela amafraktjhini qobe ngemva kwemizuzu emi-3 bese uwahlanganisa abe maqembu amabili (1 + 17, 2 + 18, njll.) bese uwomisa nge-vacuum centrifuge.
Ukuhlaziywa kwe-LC-MS/MS. Ukuze i-mass spectrometry, ama-peptide (inomboro r119.aq) ahlukaniswe ku-25 cm, 75 μm ububanzi bangaphakathi be-PicoFrit ikholomu yokuhlaziya (ilensi etja yomnqopho, inomboro yengcenye PF7508250) efakwe i-1.9 μm ReproSil-Pur 120 C18-AQ medium (inombolo r119.aq) i-1.9 μm ye-ReproSil-Pur 120 C18-AQ medium (Maischr, Maisch, EY-AS. 1200 (Thermo Fisher Scientific, e-Germany). Ikholomu beyigcinwe ku-50°C. Amabhafa A no-B ziyi-0.1% ye-formic acid emanzini kanye ne-0.1% ye-formic acid ku-80% ye-ACN, ngokulandelana. Ama-peptide ahlukaniswe nge-gradient ye-200 nl/min ukusuka ku-6% ukuya ku-31% ye-buffer B imizuzu ema-65, begodu ukusuka ku-31% ukuya ku-50% ye-buffer B imizuzu emi-5. Ama-peptide ahlanjululweko ahlaziywa ku-Orbitrap Fusion mass spectrometer (Thermo Fisher Scientific). Ukulinganisa kwe-peptide precursor m/z kwenziwa ngesisombululo esingu-120,000 phakathi kwe-350 ukuya ku-1500 m/z. Ukusebenzisa amandla wokutjhayisana ajwayelekileko angama-27%, isandulela esinamandla khulu esinesimo sokutjhaja esingu-2 ukuya ku-6 sikhethelwa ukuhlukaniswa kwe-C trap dissociation (HCD) yamandla aphezulu. Isikhathi somjikelezo sibekwe ku-1 s. Inani le-m/z lesiqephu se-peptide lalinganiswa emgibeni we-ayoni kusetjenziswa ithagethi encani ye-AGC ye-5×104 kanye nesikhathi esiphezulu sokujova se-86 ms. Ngemva kokuhlukaniswa, isandulela safakwa erhelweni lokukhipha eliguquguqukako ama-45 s. Ama-peptide anelebula nge-TMT ahlukaniswe ku-50 cm, 75 μm Acclaim PepMap kholomu (i-Thermo Fisher Scientific, inombolo yekhathalogu 164942), begodu ama-spectra wokufuduka ahlaziywa ku-Orbitrap Lumos Tribrid mass spectrometer (Thermo Fisher Scientific) efakwe ama-aveons we-high-symmetric aveform (IMS) imitjhini (i-Thermo Fisher Scientific) isebenza ngamavoltheji amabili wokubhadela we-−50 no-−70 V. I-MS3 ekhethwe ngokuya ngesandulela sokuvumelanisa isetjenziselwa ukulinganisa isignali ye-ion yombiko we-TMT. Ukuhlukaniswa kwe-peptide kwenziwa ku-EASY-nLC 1200, kusetjenziswa i-90% ye-linear gradient elution, nge-buffer concentration ye-6% ukuya ku-31%; ibhafa A beyiyi-0.1% FA, begodu ibhafa B beyiyi-0.1% FA kanye ne-80% ACN. Ikholomu yokuhlaziya isetjenziswa ku-50°C. Sebenzisa i-FreeStyle (inguqulo 1.6, Thermo Fisher Scientific) ukuhlukanisa ifayela lokuthoma ngokuya nge-FAIMS compensation voltage.
Ukukhonjwa kwamaphrotheyini nokulinganisa. Ngokusebenzisa injini yokusesha ye-Andromeda, idatha yasekuthomeni yahlaziywa kusetjenziswa i-MaxQuant version 1.5.2.8 (https://maxquant.org/). Ngaphezu kwe-Cre recombinase kanye ne-YFP sequences etholwe ku-Aequorea victoria, ama-peptide fragment spectra afunwa ukulandelana kwe-canonical kanye ne-isoform sequence ye-mouse reference proteome (Proteome ID UP000000589, elandwe ku-UniProt ngoMeyi 2017). I-methionine oxidation kanye ne-protein N-terminal acetylation kwabekwa njengeenguquko ezihlukahlukeneko; i-cysteine ​​  carbamoyl methylation yabekwa njengeentjhuguluko ezingaguqukiko. Imingcele yokugaya isethwe ku-"specificity" kanye "ne-trypsin/P". Inani eliphasi lama-peptide nama-peptide we-razor asetjenziselwa ukuhlonza amaphrotheyini ngu-1; inani eliphasi lama-peptide ahlukileko ngu-0. Ngaphasi kweemeko zokufanisa imephu yama-peptide, izinga lokukhonjwa kwamaphrotheyini belingu-0.01. Inketho ethi "I-Peptide Yesibili" inikwe amandla. Sebenzisa inketho "yokufanisa hlangana nokugijima" ukudlulisa ukuhlonza okuphumeleleko hlangana namafayela wekuthomeni ahlukileko. Sebenzisa i-LFQ ubuncani be-ratio count 1 ukulinganisa ngaphandle kwamalebula (LFQ) (60). Ukuqina kwe-LFQ kuhlungwa okungenani ngamanani amabili avumelekileko okungenani esiqhemeni sinye se-genotype ngesikhathi ngasinye, begodu kukhishwa ukusuka ekusabalalisweni okujayelekileko ngobubanzi be. 0.3 bese wehlela phasi ngo-1.8. Sebenzisa iplatifomu yekhomphyutha ye-Perseus (https://maxquant.net/perseus/) kanye ne-R (https://r-project.org/) ukuhlaziya imiphumela ye-LFQ. Ukuhlolwa kwe-t okulinganiselweko okuvela kuphakheji yesofthiwe ye-limma kwasetjenziselwa ukuhlaziywa kokuvezwa okuhlukileko (61). Ukuhlaziywa kwedatha yokuhlola kwenziwa kusetjenziswa i-ggplot, FactoMineR, factoextra, GGally kanye ne-pheatmap. Idatha ye-TMT-based proteomics yahlaziywa kusetjenziswa i-MaxQuant version 1.6.10.43. Sesha idatha ye-proteomics engakalungiswa evela ku-UniProt's human proteomics database, eyalandwa ngoSeptemba 2018. Ukuhlaziywa kufaka hlangana i-isotope purity correction factor enikelwe mkhiqizi. Sebenzisa i-limma ku-R ukuhlaziywa kokuvezwa okuhlukileko. Idatha yasekuthomeni, imiphumela yokusesha yedathabheyisi, kanye nokusebenza kokuhlaziywa kwedatha nemiphumela yoke igcinwa ku-ProteomeXchange alliance nge-PRIDE partner repository ene-data set identifier PXD019690.
Iinhlathululo ezisebenzako zinothisa ukuhlaziywa. Ithulusi lokuhlaziywa kwendlela yobuhlakani (i-QIAGEN) lasetjenziselwa ukuthola ukunotha kwamagama wokuhlathulula okusebenzako wedatha ebekwe emavikini abunane (Umfanekiso 1). Ngamafuphi, irhelo lamaphrotheyini atholwe ku-LC-MS/MS (tandem mass spectrometry) ukuhlaziywa kwedatha lisetjenziswa ngendlela elandelako yokuhlunga: I-Mus musculus ikhethwa njengomhlobo nesizinda, begodu isigaba sitjengisa inani le-P elilungiswe ngu-Benjamini ukunothisa okungu-0.05 nofana ngaphasi lithathwa njengeliqakathekileko. Ngegrafu le, iinkoro ezihlanu eziphezulu eziphezulu esiqhemeni ngasinye ngokuya ngenani le-P elilungisiweko zitjengiswa. Ngokusebenzisa i-t-test eminengi, ngokusebenzisa ihlelo lokukhuthaza i-linear boost enezigaba ezimbili ze-Benjamini, Krieger, no-Yekutieli (Q = 5%), ukuhlaziywa kokuvezwa kwamaphrotheyini kwesikhathi kwenziwa ebantwini abaqakathekileko abakhonjwe esigabeni ngasinye, begodu umugqa ngamunye uhlaziywa ngokwehlukana. Asikho isidingo sokwamukela i-SD engaguqukiko.
Ukuze simadanise imiphumela yesifundo lesi nama-database agadangisiweko begodu senze umdwebo we-Venn kuMfanekiso 1, sihlanganise irhelo lamaphrotheyini we-quantitative nama-annotations we-MitoCarta 2.0 (24). Sebenzisa ithulusi le-inthanethi elithi Draw Venn Diagram (http://bioinformatics.psb.ugent.be/webtools/Venn/) ukwenza umdwebo.
Ukuthola ilwazi elinabileko ngeenkambiso zezibalo ezisetjenziselwa ukuhlaziywa kwe-proteomics, sibawa uqale isigaba esihambelanako se-Materials and Methods. Kizo zoke ezinye iimhloliso, ilwazi elinemininingwana lingatholakala enganekwaneni ehambelana nayo. Ngaphandle kobana kuvezwe ngenye indlela, yoke idatha ivezwa njenge-mean ± SEM, begodu koke ukuhlaziywa kwezibalo kwenziwa kusetjenziswa isofthiwe ye-GraphPad Prism 8.1.2.
Ukuthola izinto ezingezelelweko ze-atikili le, sibawa uqale i-http://advances.sciencemag.org/cgi/content/full/6/35/eaba8271/DC1
Le yi-atikili evulekileko esabalaliswa ngaphasi kwemibandela ye-Creative Commons Attribution-Non-Commercial License, evumela ukusetjenziswa, ukusabalalisa nokukhiqizwa kabutjha nganoma ngiyiphi indlela, inqobo nje nangabe ukusetjenziswa kokugcina kungasi kwezokurhweba begodu umqondo kukuthi umsebenzi wokuthoma ulungile. Ireferensi.
Tjheja: Sikubawa bona unikele ngekheli lakho le-imeyili ukwenzela bona umuntu omncomako ekhasini azi bona ufuna bona abone i-imeyili begodu akusiyo i-spam. Angeze sathatha amakheli we-imeyili.
Umbuzo lo usetjenziselwa ukuhlola bona usivakatjhi begodu uvimbele ukuthunyelwa kwe-spam okuzenzakalelayo.
Ngu-E. Motori, I. Atanassov, SMV Kochan, K. Folz-Donahue, V. Sakthivelu, P. Giavalisco, N. Toni, J. Puyal, N.-G. U-Larson
Ukuhlaziywa kwe-proteomics yama-neuron angasebenziko kuveze bona amahlelo we-metabolic asebenza ukulwisana nokuwohloka kwe-neuron.
Ngu-E. Motori, I. Atanassov, SMV Kochan, K. Folz-Donahue, V. Sakthivelu, P. Giavalisco, N. Toni, J. Puyal, N.-G. U-Larson
Ukuhlaziywa kwe-proteomics yama-neuron angasebenziko kuveze bona amahlelo we-metabolic asebenza ukulwisana nokuwohloka kwe-neuron.
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