Okwamanje *Ikheli lamanje: Cologne 50931, eJalimane, iCologne Excellence Cluster Research on Cellular Stress Response in Aging-related Diseases (CECAD).
Ukuwohloka kwemizwa kwezifo ze-mitochondrial kubhekwa njengokungenakulungiseka ngoba ukuguquguquka kwe-metabolic kwama-neurons kunqunyelwe, kodwa umphumela wokungasebenzi kahle kwe-mitochondrial ekuzimeleni kweseli kwe-neuronal metabolism emzimbeni awuqondakali kahle. Lapha, sethula i-proteome yama-neurons e-Purkinje aqondene neseli enokuntuleka kwe-OXPHOS okuqhubekayo okubangelwa ukuphazamiseka kwe-mitochondrial fusion dynamics. Sithole ukuthi ukungasebenzi kahle kwe-mitochondrial kwabangela ushintsho olukhulu emkhakheni we-proteomics, okwagcina kuholele ekusebenzeni okulandelanayo kwezinhlelo ze-metabolic eziqondile ngaphambi kokufa kweseli. Ngokungalindelekile, sithole ukungeniswa okusobala kwe-pyruvate carboxylase (PCx) kanye namanye ama-enzyme okulwa nokuguga anezela ama-intermediate omjikelezo we-TCA. Ukuvinjelwa kwe-PCx kwandise ukucindezeleka kwe-oxidative kanye ne-neurodegeneration, okubonisa ukuthi i-atherosclerosis inomphumela wokuvikela kuma-neurons angenayo i-OXPHOS. Ukubuyiselwa kokuhlanganiswa kwe-mitochondrial kuma-neurons awohloka ngokuphelele kuguqula ngokuphelele lezi zici ze-metabolic, ngaleyo ndlela kuvimbele ukufa kweseli. Okutholakele kwethu kukhomba izindlela ezazingaziwa ngaphambili ezinikeza ukuqina kokungasebenzi kahle kwe-mitochondrial futhi kukhombisa ukuthi ukonakala kwemizwa kungaguqulwa ngisho nasezigabeni zokugcina zesifo.
Indima ebalulekile ye-mitochondria ekugcineni i-metabolism yamandla e-neuronal igcizelelwa yizimpawu eziningi ze-neurological ezihlobene nezifo ze-mitochondrial zabantu. Iningi lalezi zifo libangelwa ukuguqulwa kwezakhi zofuzo okulawula ukubonakaliswa kwezakhi zofuzo ze-mitochondrial (1, 2) noma ukubhujiswa kwezakhi zofuzo okuhlobene ne-mitochondrial dynamics, okuthinta ngokungaqondile ukuzinza kwe-DNA ye-mitochondrial (mtDNA) (3, 4). Umsebenzi kumamodeli ezilwane ukhombisile ukuthi ekuphenduleni ukungasebenzi kahle kwe-mitochondrial ezicutshini ezizungezile, izindlela ze-metabolism ezilondolozayo (5-7) zingasebenza, okunikeza ulwazi olubalulekile lokuqonda ngokujulile i-pathogenesis yalezi zifo eziyinkimbinkimbi. Ngokuphambene kakhulu, ukuqonda kwethu izinguquko ze-metabolism zezinhlobo ezithile zamaseli ezibangelwa ukwehluleka okujwayelekile kokukhiqizwa kwe-brain mitochondrial adenosine triphosphate (ATP) kubalulekile (8), kugcizelela isidingo sokuhlonza izinhloso zokwelapha ezingasetshenziswa ukuvimbela noma ukuvimbela izifo. Vimbela ukuwohloka kwe-neuro (9). Ukuntuleka kolwazi iqiniso lokuthi amaseli ezinzwa abhekwa kabanzi ukuthi anokuguquguquka okulinganiselwe kwe-metabolism uma kuqhathaniswa nezinhlobo zamaseli zezicubu ezizungezile (10). Njengoba la maseli edlala indima ebalulekile ekuhlanganiseni ukuhlinzekwa kwama-metabolites kuma-neurons ukukhuthaza ukudluliselwa kwe-synaptic nokusabela ezimweni zokulimala nezifo, ikhono lokuvumelanisa i-metabolism yamaseli nezimo eziyinselele zezicubu zobuchopho cishe lilinganiselwe kumaseli e-glial (11-14). Ngaphezu kwalokho, ukungafani kwamangqamuzana kwezicubu zobuchopho kuvimbela kakhulu ukutadisha izinguquko ze-metabolic ezenzeka emaqenjini athile e-neuronal. Ngenxa yalokho, kuncane okwaziwayo ngemiphumela eqondile yamaseli kanye ne-metabolic yokungasebenzi kahle kwe-mitochondrial kuma-neurons.
Ukuze siqonde imiphumela ye-metabolic yokungasebenzi kahle kwe-mitochondrial, sihlukanise ama-neuron e-Purkinje (ama-PN) ezigabeni ezahlukene zokuwohloka kwemizwa okubangelwa ukubhujiswa kwe-mitochondrial outer membrane fusion (i-Mfn2). Nakuba ukuguqulwa kwe-Mfn2 kubantu kuhlotshaniswa nohlobo lwe-hereditary motor sensory neuropathy eyaziwa ngokuthi i-Charcot-Marie-Tooth type 2A (15), ukubhujiswa okunemibandela kwe-Mfn2 emagundwini kuyindlela eqashelwa kahle yokwenziwa kwe-oxidation​​ Phosphorylation (OXPHOS) dysfunction method. Izinhlobo ezahlukene ze-neuronal (16-19) kanye ne-neurodegenerative phenotype ephumelayo kuhambisana nezimpawu ze-neurological eziqhubekayo, njengokuphazamiseka kokunyakaza (18, 19) noma i-cerebellar ataxia (16). Ngokusebenzisa inhlanganisela ye-proteomics ye-quantitative (LFQ) engenalebula, i-metabolomics, i-imaging, kanye ne-virological methods, sibonisa ukuthi i-progressive neurodegeneration ibangela kakhulu i-pyruvate carboxylase (PCx) nezinye izinto ezihilelekile ku-arteriosclerosis yama-PNs in vivo The expression of enzymes. Ukuze siqinisekise ukubaluleka kwalokhu okutholakele, sinciphise ngokukhethekile ukuvezwa kwe-PCx kuma-PN angenayo i-Mfn2, futhi sathola ukuthi lokhu kusebenza kwandise ukucindezeleka kwe-oxidative kanye nokusheshisa ukuwohloka kwemizwa, ngaleyo ndlela kufakazela ukuthi i-azoospermia inika ukufa kweseli Ukuzivumelanisa nezimo kwe-Metabolic. Ukubonakaliswa okukhulu kwe-MFN2 kungasindisa ngokuphelele ukuwohloka kwe-PN okugcina ngokuntuleka okukhulu kwe-OXPHOS, ukusetshenziswa okukhulu kwe-DNA ye-mitochondrial, kanye nenethiwekhi ye-mitochondrial ebonakala iphukile, okugcizelela ukuthi lolu hlobo lokuwohloka kwemizwa lungalulama ngisho nasesigabeni esithuthukile sesifo ngaphambi kokufa kweseli.
Ukuze sibone ngeso lengqondo i-mitochondria kuma-PN e-Mfn2 knockout, sisebenzise uhlobo lwegundane oluvumela i-mitochondria encike ku-Cre ukuthi ihlose iphrotheni ekhanyayo ephuzi (i-YFP) (mtYFP) (20) Ukubonakaliswa kwe-Cre futhi sahlola isimo se-mitochondrial in vivo. Sithole ukuthi ukubhujiswa kwe-gene ye-Mfn2 kuma-PN kuzoholela ekuhlukanisweni kancane kancane kwenethiwekhi ye-mitochondrial (Isithombe S1A), futhi ushintsho lokuqala lwatholakala emavikini ama-3 ubudala. Ngokuphambene nalokho, ukuwohloka okukhulu kwengqimba yeseli le-PN, njengoba kufakazelwa ukulahleka kwe-Calbindin immunostaining, akuzange kuqale kuze kube amasonto ayi-12 ubudala (Isithombe 1, A no-B). Ukungafani kwesikhathi phakathi kwezinguquko zokuqala esimweni se-mitochondrial kanye nokuqala okubonakalayo kokufa kwe-neuronal kwasishukumisela ukuthi sihlole izinguquko ze-metabolic ezibangelwa ukungasebenzi kahle kwe-mitochondrial ngaphambi kokufa kweseli. Sakhe isu elisekelwe ku-fluorescence-activated cell sorting (FACS) ukuze sihlukanise i-YFP (YFP+)-expressing PN (Isithombe 1C), kanye namagundane alawulayo (Mfn2 + / loxP :: mtYFP loxP- stop-loxP: : L7-cre), okuzobizwa ngokuthi i-CTRL (Isithombe S1B). Ukwenziwa ngcono kwesu lokubopha ngokusekelwe ekuqineni okuhlobene kwesignali ye-YFP kusenza sikwazi ukuhlanza umzimba we-YFP+ (YFPhigh) wama-PN kusuka kuma-non-PN (YFPneg) (Isithombe S1B) noma izingcezu ze-axon/dendritic ezifakazelwa yi-fluorescent fluorescent (YFPlow; Isithombe S1D, kwesobunxele), okuqinisekiswe yi-confocal microscope (Isithombe S1D, kwesokudla). Ukuze siqinisekise ukuthi ungubani wabantu abahlukanisiwe, senze i-LFQ proteomics bese kuba ukuhlaziywa kwezingxenye eziyinhloko, futhi sathola ukuthi kukhona ukuhlukaniswa okucacile phakathi kwamaseli e-YFPhigh kanye ne-YFPneg (Isithombe S1C). Amaseli aphezulu e-YFP abonise ukucebisa okunetha kwama-PN markers aziwayo (okungukuthi i-Calb1, i-Pcp2, i-Grid2 kanye ne-Itpr3) (21, 22), kodwa akukho ukucebisa amaprotheni avame ukuvezwa kuma-neurons noma kwezinye izinhlobo zamaseli (Isithombe 1D)). Ukuqhathaniswa phakathi kwamasampula kumaseli aphezulu e-YFP aqoqwe ekuhlolweni okuzimele kubonise i-coefficient yokuxhumana> 0.9, okubonisa ukuphindaphindeka okuhle phakathi kokuphindaphindeka kwezinto eziphilayo (Isithombe S1E). Ngamafuphi, le datha iqinisekisile uhlelo lwethu lokuhlukaniswa okubukhali nokuqondile kwe-PN engenzeka. Ngenxa yokuthi uhlelo lomshayeli we-L7-cre olusetshenziswayo lubangela ukuphindaphindeka kwe-mosaic ngesonto lokuqala ngemuva kokubeletha (23), saqala ukubulala amagundane ku-CTRL kanye ne-conditional (Mfn2 loxP / loxP :: mtYFP loxP-stop-loxP :: L7-cre) Qoqa ama-neurons. Ngemva kokuthi ukuphindaphindeka kuqediwe, kubizwa ngokuthi i-Mfn2cKO emavikini ama-4 ubudala. Njengesiphetho, sikhethe amasonto ayi-8 ubudala lapho ungqimba lwe-PN lwaluphelele naphezu kokuqhekeka okusobala kwe-mitochondrial (Isithombe 1B kanye nesithombe S1A). Sekukonke, silinganise inani lamaprotheni angu-3013, cishe angu-22% awo ayesekelwe ku-MitoCarta 2.0 izichasiselo ezisekelwe ku-mitochondrial proteome njenge-mitochondria (Isithombe 1E) (Isithombe 1E) (24). Ukuhlaziywa kokubonakaliswa kwezakhi zofuzo ezihlukile okwenziwe ngesonto lesi-8 kubonise ukuthi yi-10.5% kuphela yawo wonke amaprotheni ayenezinguquko ezibalulekile (Isithombe 1F kanye nesithombe S1F), lapho amaprotheni angu-195 ayephansi khona kanti amaprotheni angu-120 ayephezulu (Isithombe 1F). Kubalulekile ukuqaphela ukuthi "ukuhlaziywa kwendlela entsha" kwale sethi yedatha kukhombisa ukuthi izakhi zofuzo ezivezwe ngokuhlukile ikakhulukazi zingesethi ekhawulelwe yezindlela ezithile ze-metabolic (Isithombe 1G). Ngokuthakazelisayo, nakuba ukwehla kwezindlela ezihlobene ne-OXPHOS kanye ne-calcium signaling kuqinisekisa ukuqaliswa kokungasebenzi kahle kwe-mitochondrial kuma-PN angenayo i-fusion, ezinye izigaba ezihilela kakhulu i-metabolism yama-amino acid zikhushulwa kakhulu, okuhambisana ne-metabolism eyenzeka kuma-PN e-mitochondrial.
(A) Izithombe ezimele eziyimfihlo zezingxenye ze-cerebellar zamagundane e-CTRL kanye ne-Mfn2cKO ezibonisa ukulahleka okuqhubekayo kwama-PN (calbindin, grey); ama-nuclei aphikiswana ne-DAPI. (B) Ukulinganiswa kwe-(A) (ukuhlaziywa kwendlela eyodwa kokuhlukahluka, ***P<0.001; n = imibuthano engu-4 kuya ku-6 evela kumagundane amathathu). (C) Ukuhamba komsebenzi kokuhlola. (D) Ukusatshalaliswa kwemephu yokushisa kwamamaki athile ku-Purkinje (phezulu) nakwezinye izinhlobo zamaseli (phakathi). (E) Umdwebo we-Venn okhombisa inani lamaprotheni e-mitochondrial atholakale ku-PN ehlukanisiwe. (F) Isakhiwo se-volcano samaprotheni achazwe ngokuhlukile kuma-neurons e-Mfn2cKO emavikini angu-8 (inani lokunqunywa kokubaluleka elingu-1.3). (G) Ukuhlaziywa kwendlela yokudala kukhombisa izindlela ezinhlanu ezibaluleke kakhulu zokuphakamisa (okubomvu) kanye nokwehlisa (okuluhlaza okwesibhakabhaka) ku-Mfn2cKO PN ehlukaniswe njengamaviki angu-8. Izinga lokubonakaliswa elimaphakathi leprotheni ngayinye etholakele liyaboniswa. Imephu yokushisa ye-Grayscale: inani le-P elilungisiwe. ns, alibalulekile.
Idatha ye-Proteomics ibonise ukuthi ukubonakaliswa kwamaprotheni kwama-complex I, III, kanye ne-IV kwehla kancane kancane. Ama-Complex I, III, kanye ne-IV wonke aqukethe ama-subunit abalulekile e-mtDNA, kuyilapho i-complex II, eyayine-nuclear-coded kuphela, yayingathinteki (Isithombe 2A kanye nesithombe S2A). . Ngokuhambisana nemiphumela ye-proteomics, i-immunohistochemistry yezingxenye zezicubu ze-cerebellar ibonise ukuthi izinga le-subunit le-MTCO1 (mitochondrial cytochrome C oxidase subunit 1) le-complex IV ku-PN lehla kancane kancane (Isithombe 2B). I-subunit e-mtDNA-encoded Mtatp8 yehliswe kakhulu (Isithombe S2A), kuyilapho izinga le-steady-state le-subunit ye-ATP synthase e-nuclear-encoded ATP synthase lihlala lingashintshi, okuhambisana ne-complex eyaziwayo ye-ATP synthase subassembly F1 lapho ukubonakaliswa kwe-mtDNA kuzinzile. Ukwakheka kuyavumelana. I-Interrupt (7). Ukuhlolwa kwezinga le-mtDNA kuma-PN e-Mfn2cKO ahlungiwe yi-real-time polymerase chain reaction (qPCR) kuqinisekisile ukwehla kancane kancane kwenombolo yekhophi ye-mtDNA. Uma kuqhathaniswa neqembu lokulawula, emavikini angu-8 ubudala, cishe ama-20% kuphela wezinga le-mtDNA agcinwe (Isithombe 2C). Ngokuhambisana nale miphumela, ukudaya kwe-confocal microscopy kwama-PN e-Mfn2cKO kwasetshenziswa ukuthola i-DNA, okubonisa ukusetshenziswa kwe-nucleotide ye-mitochondrial okuncike esikhathini (Isithombe 2D). Sithole ukuthi abanye kuphela abantu abathintekayo ekuwohlokeni kwamaprotheni e-mitochondrial kanye nokusabela kokucindezeleka abakhushuliwe, okuhlanganisa i-Lonp1, i-Afg3l2 kanye ne-Clpx, kanye nezici zokuhlanganisa eziyinkimbinkimbi ze-OXPHOS. Azikho izinguquko ezibalulekile emazingeni amaprotheni ahilelekile ku-apoptosis ezitholiwe (Isithombe S2B). Ngokufanayo, sithole ukuthi iziteshi ze-mitochondria kanye ne-endoplasmic reticulum ezihilelekile ekuthuthweni kwe-calcium zinezinguquko ezincane kuphela (Isithombe S2C). Ngaphezu kwalokho, ukuhlolwa kwamaprotheni ahlobene ne-autophagy akutholanga izinguquko ezibalulekile, okuhambisana nokufakwa okubonakalayo kwama-autophagosomes abonwe emzimbeni nge-immunohistochemistry kanye ne-electron microscopy (Isithombe S3). Kodwa-ke, ukungasebenzi kahle kwe-OXPHOS okuqhubekayo kuma-PN kuhambisana nezinguquko ezisobala ze-ultrastructural mitochondrial. Amaqoqo e-Mitochondrial angabonakala emizimbeni yamaseli kanye nasezihlahleni ze-dendritic zama-Mfn2cKO PNs aneminyaka engu-5 no-8, futhi isakhiwo se-membrane yangaphakathi siye sashintsha kakhulu (Isithombe S4, A no-B). Ngokuhambisana nalezi zinguquko ze-ultrastructural kanye nokwehla okukhulu kwe-mtDNA, ukuhlaziywa kwezingcezu ze-cerebral cerebellar ezibukhali ezine-tetramethylrhodamine methyl ester (TMRM) kubonise ukuthi amandla e-mitochondrial membrane kuma-Mfn2cKO PNs anciphe kakhulu (Isithombe S4C).
(A) Ukuhlaziywa kwesikhathi sezinga lokubonakaliswa kwe-OXPHOS complex. Cabanga kuphela ngamaprotheni ane-P<0.05 emavikini angu-8 (i-ANOVA enezindlela ezimbili). Umugqa onamachashazi: Akukho ukulungiswa uma kuqhathaniswa ne-CTRL. (B) Kwesobunxele: Isibonelo sesigaba se-cerebellar esibhalwe nge-anti-MTCO1 antibody (ibha yesikali, 20 μm). Indawo ehlalwa yimizimba yamaseli e-Purkinje imbozwe ngophuzi. Kwesokudla: Ukulinganiswa kwamazinga e-MTCO1 (ukuhlaziywa kwendlela eyodwa kokuhlukahluka; n = amaseli angu-7 kuya kwangu-20 ahlaziywe kusuka kumagundane amathathu). (C) Ukuhlaziywa kwe-qPCR kwenombolo yekhophi ye-mtDNA ku-PN ehlungiwe (ukuhlaziywa kwendlela eyodwa kokuhlukahluka; n = amagundane angu-3 kuya kwangu-7). (D) Kwesobunxele: Isibonelo sesilayi se-cerebellar esibhalwe nge-anti-DNA antibody (ibha yesikali, 20 μm). Indawo ehlalwa yimizimba yamaseli e-Purkinje imbozwe ngophuzi. Kwesokudla: Ukulinganiswa kwezilonda ze-mtDNA (ukuhlaziywa kwendlela eyodwa kokuhlukahluka; n = amaseli angu-5 kuya kwangu-9 avela kumagundane amathathu). (E) Isibonelo sesigaba se-cerebellar esibukhali esibonisa amaseli e-mitoYFP + Purkinje (umcibisholo) ekurekhodweni kwe-clamp ye-patch yeseli lonke. (F) Ukulinganiswa kwejika le-IV. (G) Ukuqoshwa okumele kokufakwa kwe-depolarizing current kumaseli e-CTRL kanye ne-Mfn2cKO Purkinje. Ukulandelela okuphezulu: Ukushaya kokuqala okubangele i-AP. Ukulandelela okuphansi: Imvamisa ephezulu ye-AP. (H) Ukulinganisa kokufakwayo okuzenzakalelayo kwe-postsynaptic (sPSPs). Ukulandelela okuzimele kokurekhoda kanye nesilinganiso sawo sokusondeza kuboniswe ku-(I). Ukuhlaziywa kwendlela eyodwa kokuhlukahluka kuhlaziywe n = amaseli angu-5 kuya kwangu-20 avela kumagundane amathathu. Idatha ivezwa njenge-mean±SEM; *P<0.05; **P<0.01; ***P<0.001. (J) Ukulandelela okumele kwe-AP ezenzakalelayo kuqoshwe kusetshenziswa imodi ye-clamp ye-patch enezimbobo. Ukulandelela okuphezulu: Imvamisa ephezulu ye-AP. Ukulandelela okuphansi: ukusondeza kwe-AP eyodwa. (K) Ukulinganisa imvamisa ephakathi nendawo kanye nephezulu ye-AP ngokusho kwe-(J). Ukuhlolwa kwe-Mann-Whitney; n = amaseli angu-5 ahlaziywe kumagundane amane. Idatha ivezwa njenge-average±SEM; ayibalulekile.
Umonakalo osobala we-OXPHOS utholakale ku-Mfn2cKO PN enamasonto ayi-8 ubudala, okubonisa ukuthi umsebenzi womzimba wama-neurons awujwayelekile kakhulu. Ngakho-ke, sihlaziye izici zikagesi ezingasebenzi zama-neurons angenawo ama-OXPHOS emavikini ama-4 kuya kwayi-5 kanye namasonto ayi-7 kuya kwayi-8 ngokwenza ukuqoshwa kwe-clamp ye-patch yeseli lonke ezingcezu ze-cerebellar ezibukhali (Isithombe 2E). Ngokungalindelekile, amandla okuphumula ajwayelekile kanye nokumelana kokufaka kwama-neurons e-Mfn2cKO kwakufana nokulawula, yize kwakukhona umehluko omncane phakathi kwamaseli (Ithebula 1). Ngokufanayo, emavikini ama-4 kuya kwayi-5 ubudala, akukho zinguquko ezibalulekile ebuhlotsheni bamanje-voltage (ijika le-IV) ezitholakele (Isithombe 2F). Kodwa-ke, awekho ama-neurons e-Mfn2cKO anamasonto ayi-7 kuya kwayi-8 ubudala asinda ohlelweni lwe-IV (isinyathelo se-hyperpolarization), okubonisa ukuthi kukhona ukuzwela okucacile kwamandla e-hyperpolarization kulesi sigaba sokugcina. Ngokuphambene nalokho, kuma-neurons e-Mfn2cKO, ama-current depolarizing abangela ukukhishwa kwe-repetitive action potential (AP) abekezelelwa kahle, okubonisa ukuthi amaphethini awo okukhishwa ahlukile kakhulu kulawo ama-neurons okulawula amasonto ayi-8 (Ithebula 1 kanye nesithombe 2G). Ngokufanayo, imvamisa kanye nobukhulu be-sponsive postsynaptic currents (sPSCs) kwakufaniswa nokweqembu lokulawula, futhi imvamisa yezehlakalo yanda kusukela emavikini ama-4 kuya emavikini ama-5 kuya emavikini ayi-7 kuya emavikini ayi-8 ngokukhula okufanayo (Isithombe 2, H kanye no-I). Isikhathi sokuvuthwa kwe-synaptic kuma-PN (25). Imiphumela efanayo yatholakala ngemuva kwama-patches e-PN anezimbobo. Lokhu kulungiselelwa kuvimbela isinxephezelo esingaba khona samaphutha e-ATP eseli, njengoba kungenzeka ekurekhodweni kwe-clamp ye-patch yeseli lonke. Ikakhulukazi, amandla okuphumula kwe-membrane kanye nemvamisa yokudubula okuzenzakalelayo kwama-neurons e-Mfn2cKO akuzange kuthinteke (Isithombe 2, J kanye no-K). Ngamafuphi, le miphumela ikhombisa ukuthi ama-PN anokungasebenzi kahle kwe-OXPHOS okusobala angabhekana kahle namaphethini okukhishwa kwe-frequency ephezulu, okubonisa ukuthi kunendlela yokubuyisela evumela ukuthi alondoloze izimpendulo ze-electrophysiological ezicishe zibe zijwayelekile.
Idatha ivezwa njenge-mean ± SEM (ukuhlaziywa kwe-one-way of variance, ukuhlolwa kokuqhathanisa okuningi kukaHolm-Sidak; *P<0.05). Inombolo yeyunithi iboniswa ngamabakaki.
Siqale ukuphenya ukuthi ngabe yisiphi isigaba kusethi yedatha ye-proteomics (Isithombe 1G) sihlanganisa izindlela ezingamelana nokuntuleka okukhulu kwe-OXPHOS, ngaleyo ndlela sichaza ukuthi kungani i-PN ethintekile ingagcina i-electrophysiology ecishe ibe yinto evamile (Isithombe 2, E kuya ku-K). . Ukuhlaziywa kwe-Proteomics kubonise ukuthi ama-enzyme ahilelekile ku-catabolism yama-amino acids ahlanganisiwe (BCAA) akhuliswe kakhulu (Isithombe 3A kanye nesithombe S5A), kanti umkhiqizo wokugcina i-acetyl-CoA (CoA) noma i-succinyl CoA ingangezelela ama-tricarboxylates kumjikelezo we-arteriosclerosis Acid (TCA). Sithole ukuthi okuqukethwe kwe-BCAA transaminase 1 (BCAT1) kanye ne-BCAT2 kokubili kwanda. Zivuselela isinyathelo sokuqala se-catabolism ye-BCAA ngokukhiqiza i-glutamate kusuka ku-α-ketoglutarate (26). Zonke izingxenye ezincane ezakha i-branched chain keto acid dehydrogenase (BCKD) complex ziyanda (i-complex ivuselela i-decarboxylation elandelayo futhi engaguquki ye-BCAA carbon skeleton ephumela) (Isithombe 3A kanye nesithombe S5A). Kodwa-ke, akukho zinguquko ezicacile ku-BCAA ngokwayo ezitholakale ku-PN ehlungiwe, okungenzeka ukuthi kungenxa yokwanda kokuthathwa kwalawa ma-amino acid abalulekile noma ukusetshenziswa kweminye imithombo (i-glucose noma i-lactic acid) ukuze kuhambisane nomjikelezo we-TCA (Isithombe S5B). Ama-PN angenayo i-OXPHOS nawo abonise ukwanda kwemisebenzi yokubola kwe-glutamine kanye ne-transamination emavikini angu-8 ubudala, okungabonakala ngokukhushulwa kwama-enzyme e-mitochondrial glutaminase (GLS) kanye ne-glutamine pyruvate transaminase 2 (GPT2) (Isithombe 3, A kanye no-C). Kuyaphawuleka ukuthi ukukhushulwa kwe-GLS kukhawulelwe ku-isoform glutaminase C (GLS-GAC) ehlanganisiwe (ushintsho lwe-Mfn2cKO/CTRL lucishe lube yi-4.5-fold, P = 0.05), kanye nokukhushulwa kwayo okuqondile ezicutshini zomdlavuza Kungasekela i-bioenergy ye-mitochondrial. (27).
(A) Imephu yokushisa ikhombisa ushintsho lokugoba ezingeni leprotheyini lomzila ocacisiwe emavikini angu-8. (B) Isibonelo sesilayi se-cerebellar esibhalwe nge-anti-PCx antibody (ibha yesikali, 20 μm). Umcibisholo ophuzi ukhomba umzimba weseli le-Purkinje. (C) Ukuhlaziywa kokubonakaliswa kwephrotheyini yenkambo yesikhathi okukhonjwe njengomuntu obalulekile we-atherosclerosis (ukuhlolwa kwe-t okuningi, *i-FDR <5%; n = amagundane angu-3-5). (D) Ngenhla: Umdwebo weskimu okhombisa izindlela ezahlukene zokungena ku-carbon ebhalwe nge-[1-13C]pyruvate tracer (okungukuthi, nge-PDH noma umzila we-trans-arterial). Ngezansi: Ishadi le-violin likhombisa iphesenti le-carbon ene-single-labeled (M1) eguqulwe yaba yi-aspartic acid, i-citric acid kanye ne-malic acid ngemuva kokufaka ilebula izingcezu ze-cerebellar ezibukhali nge-[1-13C]pyruvate (ukuhlolwa kwe-t okubhangqiwe; ** P <0.01). (E) Ukuhlaziywa komlando wesikhathi okuphelele kwendlela ebonisiwe. Cabanga kuphela ngamaprotheni ane-P<0.05 emavikini angu-8. Umugqa onamachashazi: akukho xabiso lokulungisa (ukuhlaziywa kwezindlela ezimbili kokuhlukahluka; * P <0.05; *** P <0.001). Idatha ivezwa njengesilinganiso±SEM.
Ekuhlaziyeni kwethu, i-BCAA catabolism isibe enye yezindlela ezibalulekile zokukhulisa ukulawulwa. Leli qiniso lisikisela ngokuqinile ukuthi ivolumu yokungenisa umoya engena emjikelezweni we-TCA ingashintshwa ku-PN engenawo i-OXPHOS. Lokhu kungase kufanekisele uhlobo olukhulu lokushintsha kabusha i-neuronal metabolic, okungaba nomthelela oqondile ku-physiology ye-neuronal kanye nokusinda ngesikhathi sokugcinwa kokungasebenzi kahle kwe-OXPHOS okukhulu. Ngokuhambisana nale mbono, sithole ukuthi i-enzyme eyinhloko yokulwa ne-atherosclerotic PCx ilawulwa kakhulu (i-Mfn2cKO/CTRL ishintsha cishe izikhathi ezingu-1.5; Umfanekiso 3A), okhuthaza ukuguqulwa kwe-pyruvate ibe yi-oxaloacetate (28), okukholakala ukuthi isezicutshini zobuchopho. Ukubonakaliswa kwe-in kukhawulelwe kuma-astrocyte (29, 30). Ngokuhambisana nemiphumela ye-proteomics, i-confocal microscopy ibonise ukuthi ukubonakaliswa kwe-PCx kwanda ngokukhethekile nangokuphawulekayo kuma-PN angenayo i-OXPHOS, kuyilapho ukusabela kwe-PCx kwakukhawulelwe kakhulu kumaseli e-glial aseBergmann aseduze okulawula (Umfanekiso 3B). Ukuze sihlole ngokusebenzayo ukwanda okubonwe kwe-PCx, selaphe izingcezu ze-cerebellar ezibukhali nge-[1-13C]pyruvate tracer. Lapho i-pyruvate i-oxidized yi-pyruvate dehydrogenase (PDH), ilebula layo le-isotope lanyamalala, Kodwa lifakwa kuma-intermediate omjikelezo we-TCA lapho i-pyruvate iguqulwa yi-vascular reactions (Isithombe 3D). Ukusekela idatha yethu ye-proteomics, sibone inani elikhulu lezimpawu ezivela kule tracer ku-aspartic acid yezingcezu ze-Mfn2cKO, kuyilapho i-citric acid ne-malic acid nazo zazinomkhuba ophakathi, nakuba zingabalulekile (Isithombe 3D).
Kuma-neuron e-dopamine amagundane aseMitoPark anokungasebenzi kahle kwe-mitochondrial okubangelwa ama-neurons e-dopamine abhubhisa ngqo i-mitochondrial transcription factor A gene (Tfam) (Isithombe S6B), ukuvezwa kwe-PCx nakho kwathuthukiswa kakhulu (31), okubonisa ukuthi i-acetone acid arteriosclerosis Ukuvela kwalesi sifo kulawulwa ngesikhathi sokungasebenzi kahle kwe-neuronal OXPHOS emzimbeni. Kubalulekile ukuqaphela ukuthi kutholakale ukuthi ama-enzyme ahlukile (32-34) angavezwa kuma-neurons angase ahlotshaniswe ne-arteriosclerosis athuthukiswa kakhulu kuma-PN antula i-OXPHOS, njenge-propionyl-CoA carboxylase (PCC-A), i-Malonyl-CoA iguqula i-propionyl-CoA ibe yi-succinyl-CoA kanye ne-mitochondrial malic enzyme 3 (ME3), enendima eyinhloko yokuthola i-pyruvate kusuka ku-malate (Isithombe 3, A kanye no-C) (33, 35). Ngaphezu kwalokho, sithole ukwanda okukhulu kwe-enzyme ye-Pdk3, efaka i-phosphorylates bese ivala i-PDH (36), kuyilapho kungekho zinguquko ezitholakele ku-enzyme ye-Pdp1 esebenza ku-PDH noma i-enzyme complex ye-PDH ngokwayo (Isithombe 3A). Ngokuqhubekayo, kuma-PN e-Mern2cKO, i-phosphorylation ye-α1 subunit α (PDHE1α) subunit yengxenye ye-pyruvate dehydrogenase E1 ye-PDH complex ku-Ser293 (eyaziwa ngokuvimbela umsebenzi we-enzyme we-PDH) yathuthukiswa (Isithombe S6C) (Isithombe S6C). I-Pyruvate ayinayo indlela yokufinyelela imithambo yegazi.
Ekugcineni, sithole ukuthi indlela ephezulu ye-serine kanye ne-glycine biosynthesis, umjikelezo we-mitochondrial folate (1C) ohlobene kanye ne-proline biosynthesis (Isithombe 1G kanye nesithombe S5C) konke kukhuliswe kakhulu, ngokusho kwemibiko, ngesikhathi senqubo yokusebenza. Izicubu ezizungezile ziyavuselelwa ngokungasebenzi kahle kwe-mitochondrial (5-7). Ukuhlaziywa kwe-Confocal okusekela le datha ye-proteomics kubonise ukuthi ku-PN lapho i-OXPHOS ingekho, izingcezu ze-cerebellar zamagundane aneminyaka engu-8 ubudala zafakwa ku-serine hydroxymethyltransferase 2 (SHMT2), i-enzyme eyinhloko yomjikelezo we-mitochondrial folate. Impendulo ebalulekile yomzimba (Isithombe S5D). Kuzingcezu ze-cerebellar ezi-acute ze-CU-glucose-incubated eziyi-13, izivivinyo zokulandelela i-metabolic zaqinisekisa ukukhushulwa kwe-serine kanye ne-proline biosynthesis, okubonisa ukuthi ukugeleza kwe-carbon isoforms ku-serine kanye ne-proline kwanda (Isithombe S5E). Njengoba ukusabela okukhuthazwa yi-GLS kanye ne-GPT2 kuyimbangela yokwenziwa kwe-glutamate kusuka ku-glutamine kanye nokuguqulwa kwe-transamination phakathi kwe-glutamate kanye ne-α-ketoglutarate, ukukhushulwa kwazo kubonisa ukuthi ama-neurons angenayo i-OXPHOS anesidingo esikhulu se-glutamate, Lokhu kungase kuhloswe ekugcineni ukwanda kwe-biosynthesis ye-proline (Isithombe S5C). Ngokuphambene nalezi zinguquko, ukuhlaziywa kwe-proteomic kwama-astrocyte e-cerebellar avela kumagundane e-Mfn2cKO athile e-PN kubonise ukuthi lezi zindlela (kufaka phakathi wonke ama-antiperoxidases) azishintshanga kakhulu ekubonakalisweni, ngaleyo ndlela kukhombisa ukuthi lokhu kuguqulwa kwe-metabolic kukhetha i-PN eyonakele (Isithombe S6, D kuya ku-G).
Ngamafuphi, lokhu kuhlaziya kwembule amaphethini ahlukene kakhulu okusebenza kwesikhashana kwezindlela ezithile ze-metabolic kuma-PN. Nakuba ukusebenza okungajwayelekile kwe-neuronal mitochondrial kungaholela ekushayweni kwe-atherosclerosis kwasekuqaleni kanye nokuguqulwa kwe-1C (Isithombe 3E kanye nesithombe S5C), kanye nezinguquko ezibikezelwayo ekubonakalisweni kwe-I kanye ne-IV complexes, izinguquko ekuhlanganisweni kwe-serine de novo kuphela. Kwabonakala kuphela ezigabeni zokugcina. Ukungasebenzi kahle kwe-OXPHOS (Isithombe 3E kanye nesithombe S5C). Lokhu okutholakele kuchaza inqubo elandelanayo lapho i-mitochondrial ebangelwa ukucindezeleka (umjikelezo we-1C) kanye ne-cytoplasmic (serine biosynthesis) kusabela ngokubambisana nokwanda kwe-atherosclerosis emjikelezweni we-TCA ukuze kuphinde kuguqulwe i-metabolism ye-neuronal.
Ama-PN angenawo amaviki ayi-8 e-OXPHOS angagcina umsebenzi wokuvusa imvamisa ephezulu futhi abhekane nokuxhumeka okuphawulekayo kwe-metabolic ukuze kulungiswe ukungasebenzi kahle kwe-mitochondrial. Lokhu kutholwa kuphakamisa ithuba elithakazelisayo lokuthi ngisho nangalesi sikhathi, la maseli angathola nokungenelela kokwelapha ukuze kubambezeleke noma kuvinjelwe ukonakala kwemizwa. Sekwephuzile. Sixazulule leli thuba ngokungenelela okubili okuzimele. Endleleni yokuqala, saklama i-vector ye-adeno-associated virus (AAV) encike ku-Cre ukuze i-MFN2 ivezwe ngokukhetha kuma-PN angenawo ama-OXPHOS ku-vivo (Isithombe S7A). I-AAV ebhala i-MFN2 kanye ne-fluorescent reporter gene mCherry (Mfn2-AAV) kwaqinisekiswa kumasiko e-primary neuron in vitro, okwabangela ukuthi i-MFN2 ivezwe ngendlela encike ku-Cre futhi kwasindisa i-mitochondrial morphology, ngaleyo ndlela kuvinjelwe ukuguqulwa kwemizwa ku-Mfn2cKO neurons (Isithombe S7, B, D kanye no-E). Okulandelayo, senze izivivinyo ze-in vivo ukuze sihambise i-Mfn2-AAV enamasonto ayi-8 ubudala ku-cerebellar cortex ye-Mfn2cKO kanye namagundane okulawula, futhi sahlaziya amagundane anamasonto ayi-12 ubudala (Isithombe 4A). Amagundane e-Mfn2cKO aphathwe afa (Isithombe 1, A kanye no-B) (16). Ukudluliselwa kwegciwane kwi-vivo kwaholela ekuvezweni okukhethiwe kwe-PN kwezinye iziyingi ze-cerebellar (Isithombe S7, G kanye no-H). Ukujova kwe-AAV yokulawula eveza i-mCherry (Ctrl-AAV) kuphela akuzange kube nomthelela omkhulu ezingeni lokuwohloka kwemizwa ezilwaneni ze-Mfn2cKO. Ngokuphambene nalokho, ukuhlaziywa kwama-Mfn2cKO adluliselwe nge-Mfn2-AAV kubonise umphumela obalulekile wokuvikela wesendlalelo seseli le-PN (Isithombe 4, B kanye no-C). Ikakhulukazi, ubuningi be-neuron bubonakala bucishe bungafani nezilwane ezilawulayo (Isithombe 4, B kanye no-C, kanye nesithombe S7, H kanye no-I). Ukuvezwa kwe-MFN1 kodwa hhayi i-MFN2 kusebenza ngokulinganayo ekusindiseni ukufa kwe-neuronal (Isithombe 4C kanye nesithombe S7, C kanye no-F), okubonisa ukuthi ukuvezwa kwe-ectopic MFN1 kunganezela ngempumelelo ukuntuleka kwe-MFN2. Ukuhlaziywa okwengeziwe ezingeni elilodwa le-PN kubonise ukuthi i-Mfn2-AAV isindise kakhulu isakhiwo se-mitochondria, yalinganisa amazinga e-mtDNA, futhi yaguqula ukuvezwa okuphezulu kwe-anti-angiogenesis marker PCx ​​​​(Isithombe 4, C kuya ku-E). Ukuhlolwa okubonakalayo kwamagundane e-Mfn2cKO asindisiwe esimweni sokuphumula kubonise ukuthi ukuma kwawo kanye nezimpawu zokunyakaza (ukunyakaza kwe-S1 kuya ku-S3) kuthuthukisiwe. Ekuphetheni, lezi zivivinyo zibonisa ukuthi ukubuyiselwa kwe-MFN2 okubambezelekile kuma-PN antula kakhulu i-OXPHOS kwanele ukuguqula ukusetshenziswa kwe-mtDNA futhi kubangele i-atherosclerosis, ngaleyo ndlela kuvimbele ukuwohloka kwe-axon kanye nokufa kwe-neuronal emzimbeni.
(A) Uhlelo olubonisa isimiso sokuhlola sokufaka ikhodi ye-AAV ku-MFN2 lapho indlela ye-metabolic ekhonjisiwe ivuliwe. (B) Izithombe ezimele eziyimfihlo zezingcezu ze-cerebellar ezinamaviki ayi-12 ezidluliselwe emavikini ayi-8 kumagundane e-Mfn2cKO futhi zilebula nge-anti-Calbindin antibody. Kwesokudla: Ukulinganiswa kwemicu ye-axon. Isikali se-axon zoom singama-450 no-75 μm. (C) Kwesobunxele: Ukulinganiswa kobuningi bamaseli e-Purkinje ku-AAV transduction loop (AAV+) (ukuhlaziywa kwendlela eyodwa kokuhlukahluka; n = amagundane ama-3). Kwesokudla: Ukuhlaziywa kokugxila kwe-mtDNA ku-PN edluliselwe esontweni le-12 (ukuhlolwa kwe-t okungabhangqiwe; n = amaseli ayi-6 avela kumagundane amathathu). * P <0.05; ** P <0.01. (D) Ama-micrograph e-electron okudlulisela amele ama-PN ezingxenyeni ze-cerebellar ze-Mfn2cKO ezidluliselwe ngama-vector egciwane abonisiwe. Imaski epinki ibonisa indawo ehlalwa ama-dendrite, kanti isikwele esiphuzi esinamachashazi sibonisa i-zoom enikeziwe ngakwesokudla; ​​u-n umelela i-nucleus. Ibha yesikali, 1μm. (E) ikhombisa isibonelo sokufakwa kwe-PCx ku-PN okudluliselwe emavikini ayi-12. Ibha yesikali, 20μm. OE, ukuvezwa ngokweqile; i-FC, ushintsho lokugoqa.
Ekugcineni, siphenye ukubaluleka kokusinda kwamaseli abangelwa yi-peroxidase kuma-PN abhekane nokungasebenzi kahle kwe-OXPHOS. Sikhiqize i-mCherry encoding AAV-shRNA (i-short hairpin RNA) eqondise ngqo igundane i-PCx mRNA (AAV-shPCx), futhi safaka igciwane noma i-scrambled control (AAV-scr) ku-cerebellum yamagundane e-Mfn2cKO. Umjovo wenziwa esontweni lesine leminyaka (Isithombe 5A) ukuze kufezwe i-PCx knockdown esebenzayo ngesikhathi lapho ukubonakaliswa kwe-PCx kwanda khona (Isithombe 3C) futhi ungqimba lweseli le-PN lwalusaphelele (Isithombe 1A). Kubalulekile ukuqaphela ukuthi ukushayisa i-PCx (Isithombe S8A) kuholela ekusheshisweni okukhulu kokufa kwe-PN, okukhawulelwe ku-ring ethelelekile (Isithombe 5, B kanye no-C). Ukuze siqonde indlela esebenza ngayo imiphumela ye-metabolic ebangelwa ukukhushulwa kwe-PCx, sifunde isimo se-redox sama-PN ngemva kokukhishelwa phansi kwe-PCx kanye ne-AAV-mediated optical biosensor Grx1-roGFP2 ivezwe ngasikhathi sinye (Isithombe S8, B kuya ku-D) ukuhlola ushintsho oluhlobene lwe-peptide redox potential (38). Ngemuva kwalokho, senze i-two-photon fluorescence lifetime imaging microscopy (FLIM) ezingcezu zobuchopho ezibukhali ze-Mfn2cKO enamasonto ayi-7 noma ama-littermates okulawula ukuze sithole izinguquko ezingaba khona esimweni se-cytoplasmic redox ngemuva kokuqinisekisa izimo ze-FLIM (Isithombe S8, E kuya ku-G). Ukuhlaziywa kubonise ukwanda okukhulu kwesimo se-oxidation se-Mfn2cKO PN eyodwa engenakho ukubonakaliswa kwe-PCx, okuhlukile kuma-neurons okulawula noma ama-Mfn2cKO PN aveza i-shRNA eqhekekile kuphela (Isithombe 5, D no-E). Lapho ukubonakaliswa kwe-PCx kwehliswa, iphesenti lama-Mfn2cKO PN abonisa isimo esinomoya omningi landa ngokuphindwe kathathu (Isithombe 5E), okubonisa ukuthi ukulawulwa kwe-PCx okwandisiwe kwagcina amandla e-redox ama-neurons agugile.
(A) Uhlelo olubonisa ishejuli yokuhlola yokufaka ikhodi ye-AAV e-shPCx lapho indlela ye-metabolic ekhonjisiwe ivuliwe. (B) Izithombe ezimele ze-confocal zezingxenye ze-cerebellar ezinamaviki angu-8 kumagundane e-Mfn2cKO adluliselwe futhi alebula nge-anti-calcineurin antibody emavikini angu-4. Ibha yesikali, 450μm. (C) Ukulinganiswa kobuningi bamaseli e-Purkinje kuma-loops adluliselwe e-AAV (ukuhlaziywa kwendlela eyodwa kokuhlukahluka; n = amagundane angu-3 kuya kwangu-4). Idatha ivezwa njenge-mean±SEM; ***P<0.001. (D) Isithombe esimele se-FLIM sibonisa isikhathi sokuphila esimaphakathi senzwa ye-glutathione redox ye-PN enamasonto angu-7 ubudala i-Grx1-roGFP2 ngaphansi kwezimo zokuhlola ezichaziwe. Isilinganiso se-LUT (ithebula lokuhlola): isikhathi sokusinda (kuma-picosecond). Ibha yesikali, 25μm. (E) I-histogram ikhombisa ukusatshalaliswa kwamanani empilo ye-Grx1-roGFP2 kusukela ku-(D) (n=158 kuya kumaseli angu-368 kumagundane amabili ngaphansi kwesimo ngasinye). Ishadi lephayi ngaphezu kwe-histogram ngayinye: libonisa inani lamaseli anamanani empilo amade kakhulu (abomvu, axutshwe) noma amafushane (aluhlaza okwesibhakabhaka, ancishisiwe), adlula i-1 SD yenani elimaphakathi lempilo ku-CTRL-AAV-scr. (F) Imodeli ephakanyisiwe ikhombisa umphumela wokuvikela wokwenyuka kwe-neuronal PCx.
Sekukonke, idatha esiyinikezayo lapha ikhombisa ukuthi ukuvezwa kabusha kwe-MFN2 kungasindisa ngokuphelele i-PN ethuthukile ngokuntuleka okukhulu kwe-OXPHOS, ukuncipha okukhulu kwe-mtDNA, kanye nokwakheka okungavamile kakhulu okufana ne-ista, ngaleyo ndlela kuhlinzeke ngentuthuko eqhubekayo ngisho nasezifweni ezithuthukile. Ukonakala kwemizwa kunikeza ubufakazi obuguqukayo besigaba ngaphambi kokufa kweseli. Leli zinga lokuguquguquka kwe-metabolic ligcizelelwa kakhulu yikhono lama-neurons lokudala i-atherosclerosis (ukuhlanganiswa kabusha komjikelezo we-TCA), okuvimbela ukubonakaliswa kwe-PCx kuma-PN angenayo i-OXPHOS futhi kuthuthukisa ukufa kweseli, ngaleyo ndlela kudlala indima yokuvikela (Isithombe 5F).
Kulolu cwaningo, sinikeze ubufakazi bokuthi impendulo yama-PN ekungasebenzi kahle kwe-OXPHOS iwukuhlangana kancane kancane ne-TCA cycle atherosclerosis ngendlela yokuhlukanisa ukusebenza evuselelwa yizinhlelo ze-metabolic. Siqinisekisile ukuhlaziywa kwe-proteomic ngezindlela eziningi ezihambisanayo futhi sembule ukuthi lapho inselele ngenxa yokungasebenzi kahle okukhulu kwe-mitochondrial, ama-neurons anesimo esingaziwa ngaphambili sokunwebeka kwe-metabolic. Okumangazayo ukuthi yonke inqubo yokushintsha izintambo ayisho ngempela isimo sokugcina se-metabolic esihambisana nokuwohloka kwe-neuro kancane kancane nangokungaguquki, kodwa idatha yethu iphakamisa ukuthi ingaba yi-neuron yokugcina ngisho nasesigabeni sangaphambi kokufa kweseli. Indlela yokubuyisela ukusebenza. Lokhu okutholakele kubonisa ukuthi ama-neurons anezinga elikhulu le-metabolic plasticity emzimbeni. Leli qiniso lifakazela ukuthi ukubuyiselwa kabusha kwe-MFN2 kamuva kungaguqula ukubonakaliswa kwezimpawu ezibalulekile ze-metabolic futhi kuvimbele ukuwohloka kwe-PN. Ngokuphambene nalokho, kuvimbela i-atherosclerosis futhi kusheshise imizwa. transsexual.
Enye yezinto ezitholwe ezithakazelisayo kakhulu ocwaningweni lwethu ukuthi ama-PN angenayo i-OXPHOS angashintsha imetabolism yomjikelezo we-TCA ngokukhuphula ama-enzyme alawula i-arteriosclerosis. Ukuhlelwa kabusha kwe-metabolic kuyisici esivamile samaseli omdlavuza, amanye awo athembele ku-glutamine ukuze angezelele ama-intermediate e-TCA cycle ukuze akhiqize ama-reducing equivalent, aqhuba uchungechunge lokuphefumula futhi agcine ukukhiqizwa kwama-lipid kanye ne-nucleotide biosynthesis precursors (39, 40). Ucwaningo lwamuva lubonise ukuthi ezicutshini zangaphandle ezibhekene nokungasebenzi kahle kwe-OXPHOS, ukuxhumeka kabusha kwe-glutamine/glutamate metabolism nakho kuyisici esivelele (5, 41), lapho isiqondiso sokungena kwe-glutamine emjikelezweni we-TCA sincike khona Ngenxa yobunzima bokulimala kwe-OXPHOS (41). Kodwa-ke, kukhona ukuntuleka kobufakazi obucacile kunoma yikuphi ukufana kwe-neuronal metabolic plasticity emzimbeni kanye nokufaneleka kwayo okungenzeka kumongo wesifo. Esifundweni samuva nje se-in vitro, ama-cortical neurons ayinhloko aboniswe ukuthi ahlanganisa amachibi e-glutamate ukuze adlulisele i-neurotransmission, ngaleyo ndlela akhuthaze imetabolism ye-oxidative kanye ne-atherosclerosis ngaphansi kwezimo zokucindezeleka kwe-metabolic (42). Kuyaphawuleka ukuthi ngaphansi kokuvinjelwa kwe-pharmacological kwe-enzyme yomjikelezo we-TCA i-succinate dehydrogenase, i-pyruvate carboxylation kukholakala ukuthi igcina ukwakheka kwe-oxaloacetate kuma-neurons e-cerebellar granule akhuliswe (34). Kodwa-ke, ukubaluleka kwe-physiological kwalezi zindlela ezicutshini zobuchopho (lapho i-atherosclerosis kukholakala ukuthi ivalwe kakhulu kuma-astrocyte) kusenokubaluleka okubalulekile kwe-physiological (43). Kulesi simo, idatha yethu ikhombisa ukuthi ama-PN alimele yi-OXPHOS emzimbeni angashintshelwa ekuwohlokeni kwe-BCAA kanye ne-pyruvate carboxylation, okuyimithombo emibili eyinhloko yokwengezwa kwama-intermediates e-TCA pool. Nakuba kuphakanyiswe umnikelo we-BCAA catabolism ku-metabolism yamandla e-neuronal, ngaphezu kwendima ye-glutamate ne-GABA yokudluliselwa kwe-neurotransmission (44), abukho ubufakazi balezi zindlela emzimbeni. Ngakho-ke, kulula ukuqagela ukuthi ama-PN angasebenzi kahle angakhokhela ngokuzenzakalelayo ukusetshenziswa kwama-intermediates e-TCA aqhutshwa yinqubo yokuhlanganisa ngokwandisa i-atherosclerosis. Ikakhulukazi, ukukhushulwa kwe-PCx kungadingeka ukuze kugcinwe isidingo esikhulu se-aspartic acid, okuphakanyiswayo kumaseli akhula ngokungasebenzi kahle kwe-mitochondrial (45). Kodwa-ke, ukuhlaziywa kwethu kwe-metabolomics akuzange kwembule noma yiziphi izinguquko ezibalulekile ezingeni elizinzile le-aspartic acid kuma-Mfn2cKO PNs (Isithombe S6A), okungenzeka ukuthi sibonisa ukusetshenziswa okuhlukile kwe-metabolic ye-aspartic acid phakathi kwamaseli akhula ngokuqina kanye nama-neuron e-post-mitotic. Nakuba indlela eqondile yokwanda kwe-PCx kuma-neurons angasebenzi kahle emzimbeni isazochazwa, sibonise ukuthi le mpendulo yangaphambi kwesikhathi idlala indima ebalulekile ekugcineni isimo se-redox sama-neurons, esaboniswa ekuhlolweni kwe-FLIM kuma-cerebellar slices. Ikakhulukazi, ukuvimbela ama-PN ukuthi angakhushulwa i-PCx kungaholela esimweni esincishisiwe futhi kusheshiswe ukufa kwamaseli. Ukusebenza kokuwohloka kwe-BCAA kanye ne-carboxylation ye-pyruvate akuzona izindlela zokubonisa izicubu zangaphandle zokungasebenzi kahle kwe-mitochondrial (7). Ngakho-ke, kubonakala sengathi ziyisici esibalulekile sama-neuron antula i-OXPHOS, noma ngabe akuyona yodwa isici, esibalulekile ekuwohlokeni kwemizwa.
Isifo se-cerebellar uhlobo oluhlukile lwesifo se-neurodegenerative esivame ukubonakala njenge-ataxia futhi sivame ukulimaza ama-PN (46). Leli nani lama-neuron lisengozini enkulu yokungasebenzi kahle kwe-mitochondrial ngoba ukuwohloka kwalo okukhethayo kumagundane kwanele ukukhiqiza izimpawu eziningi zokunyakaza ezichaza i-human spinocerebellar ataxia (16, 47, 48). Ngokusho kwemibiko, imodeli yegundane eguqulwe nge-genic ene-gene mutant ihlotshaniswa ne-human spinocerebellar ataxia futhi inokungasebenzi kahle kwe-mitochondrial (49, 50), okugcizelela ukubaluleka kokutadisha imiphumela yokuntuleka kwe-OXPHOS ku-PNPH. Ngakho-ke, kufanelekile kakhulu ukuhlukanisa nokufunda leli nani lama-neuron elihlukile ngempumelelo. Kodwa-ke, njengoba ama-PN ezwela kakhulu ekucindezelweni futhi abangela ingxenye ephansi yalo lonke inani lamaseli e-cerebellar, ezifundweni eziningi ezisekelwe kuma-omics, ukuhlukaniswa kwawo okukhethayo njengamaseli aphelele kuseyisici esiyinselele. Nakuba cishe kungenakwenzeka ukufeza ukuntuleka okuphelele kokungcola kwezinye izinhlobo zamaseli (ikakhulukazi izicubu zabantu abadala), sihlanganise isinyathelo sokuhlukana esiphumelelayo ne-FACS ukuze sithole inani elanele lama-neurons asebenzayo okuhlaziywa kwe-proteomics engezansi, futhi sibe nokumbozwa okuphezulu kwamaprotheni (cishe amaprotheni angu-3000) uma kuqhathaniswa nesethi yedatha ekhona ye-cerebellum yonke (51). Ngokugcina ukusebenza kwamaseli aphelele, indlela esiyinikezayo lapha isivumela ukuthi singagcini nje ngokubheka izinguquko ezindleleni ze-metabolic ku-mitochondria, kodwa futhi nokubheka izinguquko kontanga bayo be-cytoplasmic, okuhambisana nokusetshenziswa kwamathegi e-mitochondrial membrane ukuze kucebiswe uhlobo lweseli Indlela entsha yenani le-mitochondria ezicutshini eziyinkimbinkimbi (52, 53). Indlela esiyichazayo ayihlobene nje kuphela nokutadisha amaseli e-Purkinje, kodwa ingasetshenziswa kalula kunoma yiluphi uhlobo lweseli ukubhekana nezinguquko ze-metabolic ebuchosheni obugulayo, kufaka phakathi amanye amamodeli okungasebenzi kahle kwe-mitochondrial.
Okokugcina, sithole ifasitela lokwelapha phakathi nale nqubo yokuhlelwa kabusha kwe-metabolic engaguqula ngokuphelele izimpawu ezibalulekile zokucindezeleka kwamaseli futhi ivimbele ukuwohloka kwe-neuronal. Ngakho-ke, ukuqonda imiphumela yokusebenza kwe-rewiring echazwe lapha kunganikeza ukuqonda okuyisisekelo mayelana nokwelashwa okungenzeka kokugcina ukusebenza kwe-neuronal ngesikhathi sokungasebenzi kahle kwe-mitochondrial. Ucwaningo lwesikhathi esizayo oluhlose ukuhlaziya izinguquko ku-metabolism yamandla kwezinye izinhlobo zamaseli obuchopho luyadingeka ukuze kwembulwe ngokugcwele ukusebenza kwalesi simiso kwezinye izifo ze-neurological.
Amagundane aseMitoPark achazwe ngaphambilini (31). Amagundane e-C57BL/6N anezakhi zofuzo ze-Mfn2 ezihambisana ne-loxP achazwe ngaphambilini (18) futhi ahlanganiswa namagundane e-L7-Cre (23). Inzalo ephindwe kabili ye-heterozygous eyalandela yahlanganiswa namagundane e-Mfn2loxP/Mfn2loxP ahambisanayo ukuze kukhiqizwe ama-knockouts e-Purkinje aqondene nezakhi zofuzo ze-Mfn2 (Mfn2loxP/Mfn2loxP; L7-cre). Esigabeni esincane sokuhlangana, i-allele ye-Gt (ROSA26) SorStop-mito-YFP (stop-mtYFP) yethulwa ngokuhlanganisa okwengeziwe (20). Zonke izinqubo zezilwane zenziwa ngokuhambisana neziqondiso zaseYurophu, zezwe kanye nezezikhungo futhi zavunywa yi-LandesamtfürNatur yase-Umwelt naseVerbraucherschutz, eNyakatho Rhine-Westphalia, eJalimane. Umsebenzi wezilwane uphinde ulandele isiqondiso se-European Federation of Laboratory Animal Sciences Associations.
Ngemva kokubulala i-anesthesia ekuphumeni komlomo wesibeletho kowesifazane okhulelwe, umbungu wegundane uyahlukaniswa (E13). I-cortex yasikwa ku-Hanks' Balanced Salt Solution (HBSS) engezwe nge-10 mM Hepes yadluliselwa ku-Dulbecco's Modified Eagle's Medium equkethe i-papain (20 U/ml) kanye ne-cysteine ​​​​(1μg/ml). Faka izicubu ku-DMEM bese uzihlukanisa ngokugaya kwe-enzyme. Ml) ku-37°C imizuzu engama-20, bese zigaywa ngomshini ku-DMEM engezwe nge-10% ye-fetal bovine serum. Amaseli ahlwanyelwa ezimbozweni zengilazi ezimbozwe nge-polylysine ngobuningi obungu-2×106 ngesidlo sokukhuliswa esingu-6 cm noma ngobuningi obungu-0.5×105 cells/cm2 ukuze kuhlaziywe izithombe. Ngemva kwamahora ama-4, lesi sitshalo sathathelwa indawo yi-Neurobasal serum-free medium equkethe i-1% B27 supplement kanye ne-0.5 mM GlutaMax. Ama-neurons abe esegcinwa ku-37°C kanye no-5% CO2 kulo lonke ucwaningo, futhi anikwa kanye ngesonto. Ukuze kukhuthazwe ukuhlanganiswa kabusha kwe-in vitro, i-3μl (isidlo sokukhulisa ama-24-well) noma i-0.5μl (ipuleti lama-24-well) ye-vector yegciwane le-AAV9 elandelayo yasetshenziswa ukwelapha ama-neurons ngosuku lwesibili lwe-in vitro: AAV9.CMV.PI.eGFP. WPRE.bGH (Addgene, inombolo yekhathalogi 105530-AAV9) kanye ne-AAV9.CMV.HI.eGFP-Cre.WPRE.SV40 (Addgene, inombolo yekhathalogi 105545-AAV9).
I-DNA ye-Mfn1 yegundane kanye ne-Mfn2 ehambisanayo (etholakale ku-Addgene plasmid #23212 kanye no-#23213, ngokulandelana) imakwe ngochungechunge lwe-V5 (GKPIPNPLLGLDST) ku-C-terminus, futhi ihlanganiswe ne-mCherry ohlakeni ngokusebenzisa uhlu lwe-T2A. I-Grx1-roGFP2 iyisipho esivela ku-Heidelberg TP Dick DFKZ (Deutsches Krebsforschungszentrum). Ngokushintsha ikhasethi ye-tdTomato kusetshenziswa izindlela ezivamile zokuhlanganisa, ikhasethi yafakwa ngaphansi kwe-pAAV-CAG-FLEX-tdTomato backbone (inombolo yereferensi ye-Addgene 28306) ukuze kukhiqizwe amavekhtha e-pAAV-CAG-FLEX-mCherry-T2A-MFN2-V5, i-pAAV-CAG-FLEX-mCherry-T2A-MFN1-V5 kanye ne-pAAV-CAG-FLEX-Grx-roGFP2. Isu elifanayo lisetshenziswe ukukhiqiza i-control vector pAAV-CAG-FLEX-mCherry. Ukuze kukhiqizwe ukwakheka kwe-AAV-shPCx, kudingeka i-plasmid AAV vector (i-VectorBuilder, i-pAAV [shRNA] -CMV-mCherry-U6-mPcx- [shRNA#1]), equkethe ukulandelana kwe-DNA okuqopha igundane eliqondisa i-shRNA PCx (5′CTTTCGCTCTAAGGTGCTAAACTCGAGTTTAGCACCCTTAGAGCGAAAG 3′) Ngaphansi kokulawulwa kwe-U6 promoter, i-mCherry isetshenziswa ngaphansi kokulawulwa kwe-CMV promoter. Ukukhiqizwa kwama-AAV vector asizayo kwenziwa ngokwemiyalelo yomenzi (ama-Cell Biolabs). Ngamafuphi, sebenzisa i-plasmid yokudlulisa ephethe i-mCherry-T2A-MFN2-V5 (pAAV-CAG-FLEX-mCherry-T2A-MFN2-V5), mCherry-T2A-MFN1-V5 (pAAV-CAG-FLEX-mCherry) okwesikhashana Ukudluliselwa kwamaseli angu-293AAV-T2A-MFN1-V5), mCherry (pAAV-CAG-FLEX-mCherry) noma i-Grx-roGFP2 (pAAV-CAG-FLEX-Grx-roGFP2) ikhodi yejini, kanye nokubhala ikhodi ye-AAV1 capsid protein kanye ne-accessory protein Packaging plasmid plasmid, kusetshenziswa indlela ye-calcium phosphate. I-crude virus supernatant itholwe ngemijikelezo yokuqandisa ebhavini elomile leqhwa/i-ethanol kanye namaseli afakwe i-lysed ku-phosphate buffered saline (PBS). I-AAV vector ihlanzwe nge-discontinuous iodixanol gradient ultracentrifugation (amahora angu-24 ku-32,000 rpm kanye no-4°C) futhi yagxiliswa kusetshenziswa isihlungi se-Amicon ultra-15 centrifugal. I-Genome titer ye-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 yayinjengoba kuchaziwe ngaphambilini (54), ilinganiswa nge-real-time quantitative PCR (qPCR) -MFN1-V5 (1.9×1013 GC/ml) kanye ne-AAV1-CAG-FLEX-Grx-roGFP2 (8.9×1012 GC/ml).
Ama-neuron ayinhloko akhuhlwe ku-1x PBS ebandayo, ahlungwe, bese ehlanganiswa ku-0.5% Triton X-100 / 0.5% sodium deoxycholate/PBS lysis buffer equkethe i-phosphatase kanye ne-protease inhibitor (Roche). Ukulinganiswa kwamaphrotheni kwenziwa ngokusebenzisa i-bicinchoninic acid assay (Thermo Fisher Scientific). Amaphrotheni abe esehlukaniswa yi-SDS-polyacrylamide gel electrophoresis, abese ecishwa ku-polyvinylidene fluoride membrane (GE Healthcare). Vimba izindawo ezingacacisiwe bese ufaka i-incubate ne-primary antibody (bheka Ithebula S1 ukuthola imininingwane) kubisi lwe-5% ku-TBST (Tris-buffered saline with Tween), izinyathelo zokuwasha kanye ne-secondary antibody ku-TBST Incubate. Faka i-incubate ne-primary antibody ubusuku bonke ku-+4°C. Ngemva kokugeza, faka i-secondary antibody amahora ama-2 ekushiseni kwegumbi. Ngemuva kwalokho, ngokufaka i-blot efanayo ne-antibody ye-anti-β-actin, umthwalo ofanayo waqinisekiswa. Ukutholwa ngokuguqula i-chemiluminescence nokuthuthukisa i-chemiluminescence (GE Healthcare).
Ama-neurons atshalwe ngaphambili kuma-coverlips engilazi aqiniswa nge-4% paraformaldehyde (PFA)/PBS ngesikhathi esibekiwe ekushiseni kwegumbi imizuzu eyi-10. Ama-coverlips aqala agcwala nge-0.1% Triton X-100/PBS imizuzu emi-5 ekushiseni kwegumbi, bese kuba ku-blocking buffer [3% bovine serum albumin (BSA)/PBS]. Ngosuku lwesibili, ama-coverlips ahlanzwa nge-blocking buffer futhi afakwa nge-antibody yesibili ehlanganisiwe ne-fluorophore amahora ama-2 ekushiseni kwegumbi; ekugcineni, amasampula ahlanzwa kahle ku-PBS nge-4′,6-diamidino-2-Phenylindole (DAPI) eqiniswa bese iqiniswa ku-microscope slide nge-Aqua-Poly/Mount.
Amagundane (abesilisa nabesifazane) atholwa i-anesthesia ngokujova i-ketamine (130 mg/kg) kanye ne-xylazine (10 mg/kg) ngaphakathi kwesisu futhi anikezwa ngaphansi kwesikhumba nge-carprofen analgesic (5 mg/kg), futhi afakwa kuthuluzi le-stereotactic (Kopf) elifakwe iphedi efudumele. Veza ugebhezi bese usebenzisa i-dental drill ukuze unciphise ingxenye ye-cerebellar cortex ehambisana nethambo le-mis (kusukela ku-lambda: umsila 1.8, ohlangothini 1, oluhambisana nama-lobule IV no-V). Sebenzisa inaliti yesirinji egobile ukuze udale ngokucophelela imbobo encane ekhanda ukuze ugweme ukuphazamisa imithambo yegazi engezansi. Ngemuva kwalokho i-capillary yengilazi encane edonswe ngaphakathi ifakwa kancane kancane emgodini omncane (kusukela ku--1.3 kuya ku--1 ohlangothini lwe-ventral lwe-dura mater), bese kuthi i-200 kuya ku-300 nl AAV ifakwe ku-micro-injector (Narishige) ngama-syringes esandla (Narishige) izikhathi eziningana ngokucindezela okuphansi esikhathini esithile imizuzu eyi-10 kuya kwengama-20 iwindi. Ngemva kokufakwa, beka i-capillary eminye imizuzu eyi-10 ukuze igciwane lisabalale ngokuphelele. Ngemva kokukhishwa kwama-capillary, isikhumba siyathungwa ngokucophelela ukuze kuncishiswe ukuvuvukala kwenxeba futhi kuvunyelwe isilwane ukuba silulame. Izilwane zelashwe ngemithi yokunciphisa ubuhlungu (caspofen) izinsuku eziningana ngemva kokuhlinzwa, lapho isimo sazo somzimba sasiqashwe ngokucophelela bese zibulawa ngesikhathi esibekiwe. Zonke izinqubo zenziwa ngokuhambisana neziqondiso zaseYurophu, zezwe kanye nezezikhungo futhi zavunyelwa yiLandesamtfürNatur yase-Umwelt naseVerbraucherschutz, eNyakatho Rhine-Westphalia, eJalimane.
Izilwane zahlanzwa nge-ketamine (100 mg/kg) kanye ne-xylazine (10 mg/kg), kwathi inhliziyo yagcotshwa nge-0.1 M PBS kuqala, kwabe sekufakwa i-4% PFA ku-PBS. Izicubu zasikwa futhi zaqiniswa ku-4% PFA/PBS ubusuku bonke ku-4°C. Ummese odlidlizayo (Leica Microsystems GmbH, eVienna, e-Austria) wasetshenziswa ukulungiselela izingxenye ze-sagittal (ubukhulu obungu-50 μm) ezivela ebuchosheni obuqinile ku-PBS. Ngaphandle kokuthi kuchazwe ngenye indlela, ukudaya izingxenye ezintantayo ngokukhululekile kwenziwa njengoba kuchaziwe ngenhla (13) ekushiseni kwegumbi kanye nokuxuba. Ngamafuphi, okokuqala, izingcezu ezitholiwe zafakwa nge-0.5% Triton X-100/PBS imizuzu eyi-15 ekushiseni kwegumbi; kwamanye ama-epitopes (Pcx kanye ne-Shmt2), ngokufaka i-tris-EDTA buffer ku-80°C (PH 9) shisa izingcezu imizuzu engama-25 esikhundleni salesi sinyathelo. Okulandelayo, izingxenye zafakwa i-antibody eyinhloko (bheka Ithebula S1) ku-blocking buffer (3% BSA/PBS) ku-4°C ubusuku bonke ngokuxubha. Ngosuku olulandelayo, izingxenye zagezwa nge-blocking buffer futhi zafakwa i-incubator efanelekile ye-fluorophore-conjugated secondary antibody amahora ama-2 ekushiseni kwegumbi; ekugcineni, izingxenye zagezwa kahle ku-PBS, zagcotshwa nge-DAPI, zabe seziqiniswa nge-AquaPolymount On a microscope slide.
I-laser scanning confocal microscope (TCS SP8-X noma i-TCS Digital Light Sheet, i-Leica Microsystems) ifakwe i-laser yokukhanya okumhlophe kanye ne-405 diode ultraviolet laser yasetshenziswa ukuthatha isithombe sesampula. Ngokushukumisa i-fluorophore nokuqoqa isignali nge-Hybrid Detector (HyDs), isofthiwe ye-LAS-X yasetshenziswa ukuqoqa izithombe ezihlanganisiwe ezihambisana ne-Nyquist sampling kwimodi elandelanayo: kumaphaneli angewona awenani, kuyizimpawu ezinamandla kakhulu (isibonelo, kumaseli e-somatic nama-dendrites) mtYFP) Sebenzisa i-HyD ukuthola inani lama-PN kwimodi ye-BrightR). Ukulinganisa kusuka ku-0.3 kuya ku-6 ns kusetshenziswa ukunciphisa ingemuva.
Ukuthwebula izithombe ngesikhathi sangempela kwamaseli ahlungiwe. Ngemva kokuhlunga ku-Neurobasal-A medium equkethe i-1% B27 supplement kanye ne-0.5 mM GlutaMax, amaseli ahlwanyelwa ngokushesha kumaslayidi engilazi ambozwe nge-poly-l-lysine (μ-Slide8 Well, Ibidi, inombolo yekhathalogi 80826), Bese uyigcina ku-37°C kanye ne-5% CO2 ihora eli-1 ukuvumela amaseli ukuthi ahlale. Ukuthwebula izithombe ngesikhathi sangempela kwenziwa kuma-microscope e-confocal e-Leica SP8 laser scanning confocal ahlonyiswe nge-laser emhlophe, i-HyD, ilensi ye-oil objective engu-63×[1.4 numeral aperture (NA)] kanye nesigaba sokushisa.
Igundane laphuzwa ngokushesha nge-carbon dioxide futhi lanqunywa ikhanda, ubuchopho basuswa ngokushesha ekhanda, banqunywa baba ugqinsi olungama-200μm (kokuhlolwa kokulebula okungu-13C) noma ugqinsi olungama-275μm (kokuhlolwa okubili kwe-photon) isigaba se-sagittal esigcwaliswe ngezinto ezilandelayo. I-ayisikhilimu (HM-650 V, Thermo Fisher Scientific, Walldorf, Germany) igcwele izinto ezilandelayo: 125 mM ice-cold, carbon-saturated (95% O2 kanye no-5% CO2) i-Ca2 ephansi + i-cerebrospinal fluid yokwenziwa (ACSF) NaCl, 2.5 mM KCl, 1.25 mM sodium phosphate buffer, 25 mM NaHCO3, 25 mM glucose, 0.5 mM CaCl2 kanye no-3.5 mM MgCl2 (umfutho we-osmotic ongu-310 kuya ku-330 mmol). Dlulisa izingcezu zobuchopho ezitholiwe ekamelweni lokufuthwa eliqukethe i-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, 1.0 mM CaCl2 kanye no-2.0 mM MgCl2) Medium) pH 7.4 kanye no-310 kuya ku-320 mmol).
Ngesikhathi senqubo yokuthwebula izithombe, izingcezu zayiswa ekamelweni elikhethekile lokuthwebula izithombe, futhi ukuhlolwa kwenziwa ngaphansi kokugeleza okuqhubekayo kwe-ACSF ekushiseni okungaguquki okungu-32° kuya ku-33°C. I-microscope yokuskena nge-laser ene-multiphoton (TCS SP8 MP-OPO, Leica Microsystems) efakwe ilensi ye-Leica 25x objective (NA 0.95, amanzi), i-Ti: Sapphire laser (Chameleon Vision II, Coherent) yasetshenziswa ekuthwebuleni izithombe ngezingcezu. I-FLIM module (PicoHarp300, PicoQuant).
I-FLIM ye-Grx1-roGFP2. Izinguquko esimweni se-cytoplasmic redox sama-PN zilinganiswe yi-FLIM enama-photon amabili ezingcezu zobuchopho be-sagittal, lapho i-Grx1-roGFP2 biosensor ihlose khona ama-PN. Esigabeni se-PN, insimu yokuthola ikhethwa cishe ngama-50 kuya ku-80 μm ngaphansi kobuso besilayidi ukuqinisekisa ukuthi kukhona i-PN esebenzayo (okungukuthi, ukuntuleka kwesakhiwo esinama-beaded noma izinguquko ze-neuronal morphological eceleni kwama-dendrites) kanye nenzwa ye-roGFP2 ephindwe kabili kanye ne-AAV encoding shRNA PCx noma ukulandelana kwayo kokulawula (i-mCherry ngayinye eveza ngokubambisana). Qoqa izithombe ze-single-stack nge-2x digital zoom [ububanzi be-excitation: 890 nm; 512 nm ama-pixel angu-512]. Ukutholwa: i-HyD yangaphakathi, iqembu lesihlungi se-fluorescein isothiocyanate (FITC)] kanye nesilinganiso sesithombe ngaphakathi kwemizuzu emi-2 kuya kwemi-3 kusetshenziswa ukuqinisekisa ukuthi kuqoqwe ama-photon anele (ama-photon ayi-1000 esewonke) ukuze kufakwe ijika. Ukuzwela kweprobe ye-Grx1-roGFP2 kanye nokuqinisekiswa kwezimo ze-FLIM kwenziwa ngokuqapha inani lempilo ye-roGFP2 lapho kungezwa i-10 mM H2O2 yangaphandle ku-ACSF yokugeleza kwegazi (ukukhulisa i-oxidation, okuholela ekwandeni kwempilo), bese kungezwa i-2 mM dithiothreitol (kunciphisa izinga lokunciphisa, okuholela ekwehleni kwempilo) (Isithombe S8, D kuya ku-G). Sebenzisa isofthiwe ye-FLIMfit 5.1.1 ukuhlaziya imiphumela etholiwe, ukulinganisa ijika elilodwa lokubola kwe-exponential lesithombe sonke ku-IRF elinganisiwe (umsebenzi wokuphendula kwensimbi), kanti i-χ2 cishe ingu-1. Ukuze kubalwe isikhathi sokuphila se-PN eyodwa, imaski ezungeze umzimba wezinzwa yadwetshwa ngesandla, futhi isikhathi sokuphila esimaphakathi kumaski ngayinye sasetshenziselwa ukulinganisa.
Ukuhlaziywa kwamandla e-Mitochondrial. Ngemva kokuba ingxenye ebukhali ifakwe i-100 nM TMRM engezwe ngqo ku-ACSF efakwe amanzi imizuzu engama-30, izinguquko zamandla e-mitochondrial zama-PN zalinganiswa nge-microscope ene-photon ezimbili. Ukuthwebula izithombe ze-TMRM kwenziwa ngokushukumisa i-probe ku-920 nm nokusebenzisa i-HyD yangaphakathi (tetramethylrhodamine isothiocyanate: 585/40 nm) ukuqoqa izimpawu; ngokusebenzisa ubude be-excitation obufanayo kodwa kusetshenziswa i-HyD yangaphakathi ehlukile (FITC :525/50) ukuze kuthathwe isithombe se-mtYFP. Sebenzisa i-plug-in ye-ImageJ's Image Calculator ukuhlola amandla e-mitochondrial ezingeni leseli elilodwa. Ngamafuphi, i-plug-in equation: signal = min (mtYFP, TMRM) isetshenziselwa ukuhlonza isifunda se-mitochondrial esibonisa isignali ye-TMRM ku-Purkinje Somali esithombeni se-single-stack confocal sesiteshi esihambisanayo. Bese kubalwa indawo yephikseli kumaski ophumayo, bese ibekwa esimweni esijwayelekile esithombeni esihambisanayo se-threshold single-stack sesiteshi se-mtYFP ukuze kutholakale ingxenye ye-mitochondrial ekhombisa amandla e-mitochondrial.
Isithombe sasuswa ngesofthiwe ye-Huygens Pro (Scientific Volume Imaging). Ezithombeni eziskeniwe zamathayili, ukuhlanganiswa kwethayili elilodwa kwenziwa kusetshenziswa i-algorithm yokuthunga ezenzakalelayo enikezwe yisofthiwe ye-LAS-X. Ngemva kokulinganiswa kwesithombe, sebenzisa i-ImageJ ne-Adobe Photoshop ukuze uqhubeke nokucubungula isithombe futhi ulungise ukukhanya nokuqhathanisa ngokulinganayo. Sebenzisa i-Adobe Illustrator ukulungiselela imidwebo.
Ukuhlaziywa kokugxila kwe-mtDNA. Inani lezilonda ze-mtDNA lalinganiswa ezingxenyeni ze-cerebellar ezibhalwe ngama-antibodies ngokumelene ne-DNA nge-microscope ye-confocal. Indawo ngayinye eqondiwe yadalelwa umzimba weseli kanye ne-nucleus yeseli ngalinye, futhi indawo efanele yabalwa kusetshenziswa i-Multi Measure plug-in (isofthiwe ye-ImageJ). Susa indawo yenuzi endaweni yomzimba weseli ukuze uthole indawo ye-cytoplasmic. Ekugcineni, i-Analyze Particles plug-in (isofthiwe ye-ImageJ) yasetshenziswa ukulinganisa ngokuzenzakalelayo amaphuzu e-cytoplasmic DNA abonisa i-mtDNA esithombeni esinqunyiwe, futhi imiphumela etholakele yalinganiswa ngokwesilinganiso se-PN samagundane e-CTRL. Imiphumela ivezwa njengenani elimaphakathi lama-nucleoside ngeseli ngalinye.
Ukuhlaziywa kokubonakaliswa kwamaprotheni. Sebenzisa i-plug-in ye-ImageJ's Image Calculator ukuhlola ukubonakaliswa kwamaprotheni ku-PN ezingeni leseli elilodwa. Ngamafuphi, esithombeni esiyimfihlo se-single-layer sesiteshi esihambisanayo, ngokusebenzisa i-equation: isignali = min (mtYFP, i-antibody), isifunda se-mitochondrial esibonisa ukusabela komzimba ku-antibody ethile ePurkina siyahlonzwa. Bese indawo ye-pixel kumaski ephumayo iyalinganiswa, bese ibekwa esimweni esijwayelekile esithombeni esihambisanayo se-threshold single-stack yesiteshi se-mtYFP ukuze kutholakale ingxenye ye-mitochondrial yeprotheni ebonisiwe.
Ukuhlaziywa kobuningi bamaseli e-Purkinje. I-plug-in ye-Cell Counter ye-ImageJ isetshenziswe ukuhlola ubuningi be-Purkinje ngokuhlukanisa inani lamaseli e-Purkinje abalwe ngobude bendandatho ye-cerebellar ehlalwa amaseli abaliwe.
Ukulungiswa nokuqoqwa kwesampula. Ubuchopho obuvela eqenjini lokulawula kanye namagundane e-Mfn2cKO buqiniswe ku-2% PFA/2.5% glutaraldehyde ku-0.1 M phosphate buffer (PB), bese kulungiswa izingxenye ze-coronal kusetshenziswa ama-ciliates (Leica Mikrosysteme GmbH, Vienna, Austria) (Ubukhulu 50 kuya ku-60 μm). Bese kufakwa ku-PB buffer ku-1% os tetraoxide kanye ne-1.5% potassium ferrocyanide ekushiseni kwegumbi ihora eli-1. Izingxenye zagezwa kathathu ngamanzi acwengekile, bese zigcotshwa nge-ethanol engu-70% equkethe i-1% uranyl acetate imizuzu engama-20. Izingxenye zabe sezicwiliswa ku-alcohol ehlungiwe futhi zafakwa ku-Durcupan ACM (Araldite casting resin M) epoxy resin (Electron Microscopy Sciences, inombolo yekhathalogi 14040) phakathi kwamaslayidi engilazi ambozwe nge-silicon, futhi ekugcineni ku-60°C Polymerize kuhhavini amahora angama-48. Indawo ye-cerebellar cortex yakhethwa kwathi izingxenye ezincane kakhulu ezingama-50 nm zasikwa ku-Leica Ultracut (Leica Mikrosysteme GmbH, eVienna, e-Austria) zathathwa kugridi yethusi engu-2×1 mm embozwe ngefilimu ye-polystyrene. Izingxenye zagcotshwa ngesisombululo se-4% uranyl acetate ku-H2O imizuzu eyi-10, zagezwa nge-H2O izikhathi eziningana, kwabe sekusetshenziswa i-lead citrate kaReynolds ku-H2O imizuzu eyi-10, kwabe sekusetshenziswa i-H2O izikhathi eziningana. Ama-Micrograph athathwe nge-transmission electron microscope i-Philips CM100 (Thermo Fisher Scientific, Waltham, MA, USA) kusetshenziswa ikhamera yedijithali ye-TVIPS (Tietz Video and Image Processing System) i-TemCam-F416 (TVIPS GmbH, Gauting, USA). Germany).
Kumagundane atheleleke nge-AAV, ubuchopho bahlukaniswa futhi banqunywa baba yisigaba se-sagittal esingu-1 mm ubukhulu, kwathi i-cerebellum yahlolwa kusetshenziswa i-microscope ye-fluorescence ukuze kutholakale indandatho etheleleke nge-AAV (okungukuthi, i-mCherry expressing). Kusetshenziswa kuphela izivivinyo lapho umjovo we-AAV uphumela ekusebenzeni kahle kakhulu kokudluliselwa kwengqimba yeseli le-Purkinje (okungukuthi cishe ungqimba lonke) okungenani ezindandatho ezimbili ezilandelanayo ze-cerebellar. I-loop edluliselwe yi-AAV yahlukaniswa nge-micro ukuze ilungiswe ubusuku bonke (4% PFA kanye ne-2.5% glutaraldehyde ku-0.1 M cocoate buffer) futhi yaqhubeka nokucutshungulwa. Ukuze kufakwe i-EPON, izicubu ezilungisiwe zagezwa nge-0.1 M sodium cocoate buffer (Applichem), futhi zafakwa nge-2% OsO4 (os, Science Services; Caco) ku-0.1 M sodium cocoate buffer (Applichem) Amahora ama-4, bese ugeza amahora ama-2. Phinda izikhathi ezi-3 nge-0.1 M cocamide buffer. Ngemva kwalokho, uchungechunge lwe-ethanol olukhuphukayo lwasetshenziswa ukufulela isisombululo ngasinye se-ethanol ku-4°C imizuzu eyi-15 ukuze kuncibilikiswe izicubu. Izicubu zadluliselwa ku-propylene oxide futhi zafulelwa ubusuku bonke ku-EPON (Sigma-Aldrich) ku-4°C. Beka izicubu ku-EPON entsha ekushiseni kwegumbi amahora ama-2, bese uzifaka ku-62°C amahora angama-72. Sebenzisa i-ultramicrotome (Leica Microsystems, UC6) kanye nommese wedayimane (Diatome, Biel, Switzerland) ukusika izingxenye ezincane kakhulu ezingama-70 nm, bese ufaka i-stain nge-1.5% uranyl acetate imizuzu eyi-15 ku-37°C, bese ufaka i-stain ngesisombululo se-lead citrate imizuzu emi-4. Ama-micrograph e-electron athathwe kusetshenziswa i-JEM-2100 Plus transmission electron microscope (JEOL) efakwe i-Camera OneView 4K 16-bit (Gatan) kanye ne-DigitalMicrograph software (Gatan). Ukuze kuhlaziywe, ama-micrograph e-electron atholakale nge-zoom yedijithali engu-5000× noma engu-10,000×.
Ukuhlaziywa kwesimo se-mitochondria. Kuzo zonke izihlaziyi, imigqa ye-mitochondria ngayinye ichazwe ngesandla ezithombeni zedijithali kusetshenziswa isofthiwe ye-ImageJ. Kuhlaziywa amapharamitha ahlukene esimo se-mitochondria. Ubuningi be-mitochondria buvezwa njengephesenti elitholwe ngokuhlukanisa indawo yonke ye-mitochondrial yeseli ngalinye ngendawo ye-cytoplasm (indawo ye-cytoplasm = indawo yeseli-indawo ye-nucleus yeseli) × 100. Ubuyindilinga be-mitochondria bubalwa ngefomula [4π∙(indawo/ipherimitha 2)]. I-ista morphology ye-mitochondria ihlaziywe futhi yahlukaniswa ngezigaba ezimbili ("i-tubular" kanye "ne-blister") ngokwezimo zazo eziyinhloko.
Ukuhlaziywa kwenombolo ye-Autophagosome/lysosome kanye nobuningi. Sebenzisa isofthiwe ye-ImageJ ukuze uchaze ngesandla imidwebo ye-autophagosome/lysosome ngayinye esithombeni sedijithali. Indawo ye-Autophagosome/lysosome ivezwa njengephesenti elibalwa ngokuhlukanisa indawo yesakhiwo se-autophagosome/lysosome iyonke yeseli ngalinye ngendawo ye-cytoplasm (indawo ye-cytoplasm=indawo yeseli-nucleus)×100. Ubuningi be-autophagosomes/lysosomes bubalwa ngokuhlukanisa inombolo iyonke ngenani lezakhiwo ze-autophagosome/lysosome ngeseli ngalinye (ngokwendawo ye-cytoplasmic) (indawo ye-cytoplasmic = indawo yeseli-nuclear).
Ukulebula kokuhlukanisa okubukhali kanye nokulungiselela isampula. Kokuhlolwa okudinga ukulebula kwe-glucose, dlulisela izingcezu zobuchopho obubukhali ekamelweni langaphambi kokufukamela, eliqukethe ikhabhoni egcwele (95% O2 kanye ne-5% CO2), i-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, 1.0 mM CaCl2 kanye ne-2.0 mM MgCl2, elungiswe ku-pH 7.4 kanye ne-310 kuya ku-320 mOsm), lapho i-glucose ingu-13 C 6- Ukufakwa esikhundleni kwe-glucose (i-Eurisotop, inombolo yekhathalogi CLM-1396). Ngezivivinyo ezidinga ukulebula kwe-pyruvate, dlulisela izingcezu zobuchopho ezibukhali ku-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, 1.0 mM CaCl2 kanye ne-Add 2.0mM MgCl2, lungisa ku-pH 7.4 kanye no-310 kuya ku-320mOsm), bese ufaka i-1mM 1-[1-13C]pyruvate (i-Eurisotop, inombolo yekhathalogi CLM-1082). Faka i-incubate imizuzu engama-90 ku-37°C. Ekupheleni kokuhlolwa, izingxenye zagezwa ngokushesha ngesisombululo samanzi (pH 7.4) esiqukethe i-ammonium carbonate engu-75 mM, bese zihlanganiswa zibe yi-homogenized ku-40:40:20 (v:v:v) i-acetonitrile (ACN): i-methanol: amanzi. Ngemva kokuba izingxenye zifakwe eqhweni imizuzu engama-30, amasampula afakwa ku-centrifuge ku-21,000 g imizuzu eyi-10 ku-4°C, kanti i-supernatant ecacile yomiswa ku-SpeedVac concentrator. I-metabolite pellet eyomile eyaphuma yagcinwa ku--80°C kuze kube yilapho ihlaziywa.
Ukuhlaziywa kwe-chromatography-mass spectrometry yama-amino acid angu-13 anelebula le-C. Ukuhlaziywa kwe-chromatography-mass spectrometry (LC-MS) okumanzi, i-metabolite pellet yaphinde yamiswa ku-75μl wamanzi e-LC-MS grade (Honeywell). Ngemva kokufaka i-centrifugation ku-21,000 g imizuzu emi-5 ku-4°C, ama-20 μl e-supernatant ecacisiwe asetshenziswa ekuhlaziyweni kwe-amino acid flux, kuyilapho okusele kokukhishwa kwasetshenziswa ngokushesha ekuhlaziyweni kwe-anion (bheka ngezansi). Ukuhlaziywa kwe-amino acid kwenziwa kusetshenziswa iphrothokholi yokukhishwa kwe-benzoyl chloride echazwe ngaphambilini (55, 56). Esinyathelweni sokuqala, ama-10μl e-100 mM sodium carbonate (Sigma-Aldrich) afakwe ku-20μl yokukhishwa kwe-metabolite, bese kuthi ama-10μl e-2% benzoyl chloride (Sigma-Aldrich) afakwe ku-LC grade ACN. Isampula yahlungwa isikhashana yabe isifakwa ku-centrifuge ku-21,000 g imizuzu emi-5 ku-20°C. Dlulisa i-supernatant esusiwe ku-2 ml autosampler vial ene-conical glass insert (200 μl volume). Amasampula ahlaziywe kusetshenziswa uhlelo lwe-Acquity iClass ultra-high performance LC (Waters) oluxhunywe ku-Q-Exactive (QE)-HF (Ultra High Field Orbitrap) high-resolution precision mass spectrometer (Thermo Fisher Scientific). Ukuze kuhlaziywe, i-2μl yesampula esuselwe kufakwe kukholomu ye-silica T3 (Waters) engu-100×1.0 mm high-strength equkethe izinhlayiya ezingu-1.8μm. Izinga lokugeleza lingu-100μl/min, kanti uhlelo lwe-buffer luqukethe i-buffer A (10 mM ammonium formate kanye ne-0.15% formic acid emanzini) kanye ne-buffer B (ACN). I-gradient ilandelayo: 0%B ngemizuzu engu-0; 0%B. 0 kuya ku-15% B ngemizuzu engu-0 kuya ku-0.1; 15 kuya ku-17% B ngemizuzu engu-0.1 kuya ku-0.5; B ngemizuzu engu-17 kuya ku-55% ngemizuzu engu-0.5 kuya ku-14; B ngemizuzu engu-55 kuya ku-70% ngemizuzu engu-14 kuya ku-14.5; 14.5 kuya ku-70 kuya ku-100% B ngemizuzu engu-18; 100% B ngemizuzu engu-18 kuya ku-19; 100 kuya ku-0% B ngemizuzu engu-19 kuya ku-19.1; 0% B ngemizuzu engu-19.1 kuya ku-28 (55, 56). I-QE-HF mass spectrometer isebenza kumodi ye-ionization enhle enobubanzi besisindo esingu-m/z (isilinganiso sesisindo/seshaja) esingu-50 kuya ku-750. Isixazululo esisetshenzisiwe singu-60,000, kanti ithagethi ye-ion yokulawula i-gain control (AGC) etholiwe ingu-3×106, kanti isikhathi esiphezulu se-ion singu-100 milliseconds. Umthombo we-ionization ye-electrospray eshisayo (ESI) usebenza ku-voltage ye-spray engu-3.5 kV, izinga lokushisa le-capillary elingu-250°C, ukuhamba komoya kwe-sheath okungu-60 AU (amayunithi angahleliwe), kanye nokuhamba komoya okusizayo okungu-20 AU. 250°C. Ilensi ye-S isethwe ku-60 AU.
Ukuhlaziywa kwe-anion chromatography-MS kwama-organic acids angu-13C anelebula. I-metabolite precipitate esele (55μl) ihlaziywe kusetshenziswa uhlelo lwe-Dionex ion chromatography (ICS 5000+, Thermo Fisher Scientific) oluxhunywe ku-QE-HF mass spectrometer (Thermo Fisher Scientific). Ngamafuphi, i-5μl ye-metabolite extract ifakwe kukholomu ye-Dionex IonPac AS11-HC efakwe i-HPLC (2 mm×250 mm, usayizi wezinhlayiya ongu-4μm, Thermo Fisher Scientific) kumodi ye-push-in partial loop enesilinganiso sokugcwalisa esingu-1. ) Ikholomu ye-Dionex IonPac AG11-HC guard (2 mm x 50 mm, 4μm, Thermo Fisher Scientific). Izinga lokushisa lekholomu ligcinwa ku-30°C, kanti i-autosampler isethwe ku-6°C. Sebenzisa i-cartridge ye-potassium hydroxide enikezwe amanzi ahlanzekile ukuze ukhiqize i-gradient ye-potassium hydroxide nge-generator eluent. Ukuhlukaniswa kwama-metabolites ngesilinganiso sokugeleza esingu-380μl/min, kusetshenziswa i-gradient elandelayo: imizuzu engu-0 kuya kwemi-3, i-10 mM KOH; imizuzu emi-3 kuya kweyi-12, i-10 kuya kweyi-50 mM KOH; imizuzu eyi-12 kuya kweyi-19, i-50 kuya kweyi-100 mM KOH; imizuzu eyi-19 kuya kweyi-21, i-100 mM KOH; imizuzu engama-21 kuya kweyi-21.5, i-100 kuya kweyi-10 mM KOH. Ikholomu yaphinde yalinganiswa ngaphansi kwe-10 mM KOH imizuzu engu-8.5.
Ama-metabolite aluted ahlanganiswa nomsinga wokwengeza we-isopropanol ongu-150μl/min ngemuva kwekholomu bese eqondiswa ku-spectrometer yesisindo esinesinqumo esiphezulu esebenza kwimodi ye-ionization engemihle. I-MS iqapha ububanzi bobuningi kusukela ku-m/z 50 kuya ku-750 ngesinqumo esingu-60,000. I-AGC isethwe ku-1×106, kanti isikhathi esiphezulu se-ion sigcinwa ku-100 ms. Umthombo we-ESI oshisayo wasebenza nge-voltage yokufafaza engu-3.5 kV. Ezinye izilungiselelo zomthombo we-ion zimi kanje: izinga lokushisa le-capillary 275°C; ukugeleza kwegesi ye-sheath, 60 AU; ukugeleza kwegesi okusizayo, 20 AU ku-300°C, kanye nokusetha ilensi ye-S ku-60 AU.
Ukuhlaziywa kwedatha kwama-metabolites anelebula le-13C. Sebenzisa isofthiwe ye-TraceFinder (inguqulo 4.2, i-Thermo Fisher Scientific) ukuze kuhlaziywe idatha yesilinganiso se-isotope. Ubunikazi bekhompawundi ngayinye buqinisekiswe yikhompawundi ethembekile futhi buhlaziywe ngokuzimela. Ukuze kwenziwe ukuhlaziywa kokunongwa kwe-isotope, indawo ye-ion chromatogram ekhishwe (XIC) ye-isotope ngayinye ye-13C (Mn) ikhishwe ku-[M + H]+, lapho i-n iyinombolo yekhabhoni yekhompawundi eqondiwe, esetshenziselwa ukuhlaziya ama-amino acid noma i-[MH] + isetshenziselwa ukuhlaziya ama-anion. Ukunemba kwesisindo se-XIC kungaphansi kwezingxenye ezinhlanu ngesigidi, kanti ukunemba kwe-RT kungama-0.05 imizuzu. Ukuhlaziywa kokunongwa kwenziwa ngokubala isilinganiso se-isotope ngayinye etholakele nesamba sawo wonke ama-isotope ekhompawundi ehambisanayo. Lezi zilinganiso zinikezwa njengamanani amaphesenti e-isotope ngayinye, futhi imiphumela ivezwa njenge-molar percentage enrichment (MPE), njengoba kuchaziwe ngaphambilini (42).
I-pellet ye-neuron eqandisiwe yahlanganiswa yaba yi-homogenized ku-80% methanol (v/v) ebandayo njengeqhwa, yafakwa i-vortex, yabe isifakwa ku--20°C imizuzu engama-30. Phinda ujikeleze isampula bese uvuselela ku-+4°C imizuzu engama-30. Isampula yafakwa ku-centrifuge ku-21,000 g imizuzu emi-5 ku-4°C, bese kuthi i-supernatant ephumayo iqoqwe futhi yomiswe kusetshenziswa i-SpeedVac concentrator ku-25°C ukuze kuhlaziywe okulandelayo. Njengoba kuchaziwe ngenhla, ukuhlaziywa kwe-LC-MS kwenziwa kuma-amino acid amaseli ahlungiwe. Kusetshenziswa i-TraceFinder (inguqulo 4.2, i-Thermo Fisher Scientific), ukuhlaziywa kwedatha kwenziwa kusetshenziswa isisindo se-monoisotopic se-compound ngayinye. Ukulungiswa kwe-quantile kwedatha ye-metabolite kwenziwa kusetshenziswa iphakheji yesofthiwe ye-preprocessCore (57).
Ukulungiswa kwezingcezu. Igundane laphuzwa ngokushesha nge-carbon dioxide futhi lanqunywa ikhanda, ubuchopho basuswa ngokushesha ekhanda, kwathi ummese odlidlizayo ogcwele iqhwa (HM-650 V, Thermo Fisher Scientific, Walldorf, Germany) wasetshenziswa ukuwusika ube yizingxenye ezingama-300 kuya ku-375 μm sagittal. I-Cold carbon gasification (95% O2 kanye ne-5% CO2) I-Ca2 ephansi + i-ACSF (125.0 mM NaCl, 2.5 mM KCl, 1.25 mM sodium phosphate buffer, 25.0 mM NaHCO3, 25.0 mM d-glucose, 1.0 mM CaCl2 kanye ne-6.0 mM MgCl2 Lungisa ku-pH 7.4 kanye ne-310 kuya ku-330 mOsm). Dlulisa izingcezu zobuchopho ezitholiwe ekamelweni eliqukethe i-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 mM CaCl2 kanye ne-mM 3.5 MgCl2) pH 7.4 kanye no-310 kuya ku-320 mOsm). Gcina izingcezu imizuzu engama-20 kuya kwengama-30 ukuze zikwazi ukubuyiselwa ngaphambi kokurekhoda.
ukuqopha. Isigaba se-microscope esihlonyiswe ngegumbi lokuqopha elihleliwe kanye nelensi yokucwiliswa emanzini engu-20x (i-Scientifica) yasetshenziswa kuzo zonke iziqopha. Amaseli e-Purkinje acatshangelwa (i) ubukhulu bomzimba, (ii) indawo ye-anatomical ye-cerebellum, kanye (iii) ukubonakaliswa kwe-fluorescent mtYFP reporter gene. I-patch pipette enokumelana kwe-tip engu-5 kuya ku-11 megohms ikhishwa yi-borosilicate glass capillary (GB150-10, 0.86 mm×1.5 mm×100 mm, Science Products, Hofheim, Germany) kanye ne-horizontal pipette Instruments (P-1000, Sutter), Novato, CA). Konke ukuqopha kwenziwe yi-ELC-03XS npi patch clamp amplifier (npi electronic GmbH, Tam, Germany), eyayilawulwa yi-software Signal (version 6.0, Cambridge Electronic, Cambridge, UK). Ukuhlolwa kwaqoshwa ngesilinganiso sokusampula esingu-12.5 kHz. Isignali ihlungwa ngezihlungi ezimbili zeBessel ezidlula ngokushesha ezinemvamisa yokusika engu-1.3 no-10 kHz ngokulandelana. Amandla e-membrane kanye ne-pipette akhokhelwa yisekethe yokunciphisa umsindo kusetshenziswa i-amplifier. Zonke izivivinyo zenziwe ngaphansi kokulawulwa kwekhamera ye-Orca-Flash 4.0 (eHamamatsu, eGerden, eJalimane), eyayilawulwa yisoftware yeHokawo (inguqulo 2.8, eHamamatsu, eGerden, eJalimane).
Yenza ukumiswa nokuhlaziywa kweseli lonke njalo. Ngaphambi nje kokurekhoda, gcwalisa i-pipette ngesisombululo sangaphakathi esiqukethe izinto ezilandelayo: 4.0 mM KCl, 2.0 mM NaCl, 0.2 mM EGTA, 135.0 mM potassium gluconate, 10.0 mM Hepes, 4.0 mM ATP (Mg), 0.5 mM Guanosine triphosphate (GTP) (Na) kanye ne-10.0 mM creatinine phosphate kwalungiswa kwaba yi-pH 7.25, kanti ingcindezi ye-osmotic yayingu-290 mOsm (sucrose). Ngokushesha ngemva kokusebenzisa amandla angu-0 pA ukudabula i-membrane, amandla e-membrane aphumulile alinganiswa. Ukumelana kokufaka kulinganiswa ngokusebenzisa ama-currents e-hyperpolarized angu--40, -30, -20, kanye no--10 pA. Linganisa ubukhulu bempendulo ye-voltage bese usebenzisa umthetho we-Ohm ukuze ubale ukumelana kokufaka. Umsebenzi ozenzakalelayo waqoshwa ku-voltage clamp imizuzu emi-5, kwathi i-sPSC yatholakala futhi yalinganiswa ku-Igor Pro (inguqulo 32 7.01, i-WaveMetrics, iLake Oswego, i-Oregon, e-USA) kusetshenziswa iskripthi sokuqashelwa okuzenzakalelayo. Ijika le-IV kanye nogesi ozinzile kulinganiswa ngokufaka ibhethri kuma-potential ahlukene (kusukela ku--110 mV) nokwandisa i-voltage ngezinyathelo ezi-5 mV. Ukukhiqizwa kwe-AP kwahlolwa ngokusebenzisa i-depolarizing current. Faka iseli ku--70 mV ngenkathi usebenzisa i-depolarizing current pulse. Lungisa usayizi wesinyathelo seyunithi ngayinye yokurekhoda ngokwehlukana (10 kuya ku-60 pA). Bala imvamisa ephezulu ye-AP ngokubala ngesandla ama-pulse spikes abangela imvamisa ephezulu ye-AP. Umkhawulo we-AP uhlaziywa ngokusebenzisa i-derivative yesibili ye-depolarizing pulse eqala ngokuqala i-AP eyodwa noma ngaphezulu.
Ukucushwa kanye nokuhlaziywa kwe-patch enezimbobo. Yenza ukuqoshwa kwe-patch enezimbobo usebenzisa amaphrothokholi ajwayelekile. Sebenzisa i-pipette engenazo i-ATP ne-GTP engenazo izithako ezilandelayo: i-128 mM gluconate K, i-10 mM KCl, i-10 mM Hepes, i-0.1 mM EGTA kanye ne-2 mM MgCl2, bese ulungisa ku-pH 7.2 (usebenzisa i-KOH). I-ATP kanye ne-GTP zisusiwe esixazululweni sangaphakathi kweseli ukuvimbela ukungena okungalawuleki kwe-membrane yeseli. I-patch pipette igcwele isixazululo sangaphakathi esiqukethe i-amphotericin (cishe i-200 kuya ku-250μg/ml; G4888, Sigma-Aldrich) ukuthola irekhodi le-patch elibhoboziwe. I-Amphotericin yancibilikiswa ku-dimethyl sulfoxide (ukuhlushwa kokugcina: 0.1 kuya ku-0.3%; DMSO; D8418, Sigma-Aldrich). Ukuhlushwa kwe-DMSO esetshenzisiwe akuzange kube nomthelela omkhulu kuma-neurons afundwe. Ngesikhathi senqubo yokubhoboza, ukumelana kwesiteshi (i-Ra) kwaqalwa njalo, futhi ukuhlolwa kwaqalwa ngemva kokuba ubukhulu be-Ra ne-AP buzinzile (imizuzu engama-20-40). Umsebenzi ozenzakalelayo ulinganiswa nge-voltage kanye/noma i-current clamp imizuzu emi-2 kuya kwemi-5. Ukuhlaziywa kwedatha kwenziwa kusetshenziswa i-Igor Pro (inguqulo 7.05.2, i-WaveMetrics, i-USA), i-Excel (inguqulo 2010, i-Microsoft Corporation, i-Redmond, i-USA) kanye ne-GraphPad Prism (inguqulo 8.1.2, i-GraphPad Software Inc., i-La Jolla, i-CA). I-United States). Ukuze kutholakale ama-AP angahleliwe, kusetshenziswa i-plug-in ye-IgorPro's NeuroMatic v3.0c. Khomba ngokuzenzakalelayo ama-AP kusetshenziswa umkhawulo onikeziwe, olungiswa ngawodwana kwirekhodi ngalinye. Usebenzisa isikhawu se-spike, nquma imvamisa ye-spike ngemvamisa ye-spike ephezulu kakhulu kanye nemvamisa ye-spike evamile.
Ukuhlukaniswa kwe-PN. Ngokuzivumelanisa nephrothokholi eyanyatheliswa ngaphambilini, ama-PN ahlanzwa ku-cerebellum yegundane esigabeni esithile (58). Ngamafuphi, i-cerebellum yasikwa futhi yagaywa endaweni yokuhlukanisa ebandayo [ngaphandle kwe-HBSS Ca2+ kanye ne-Mg2+, yanezelwa nge-glucose engu-20 mM, i-penicillin (50 U/ml) kanye ne-streptomycin (0.05 mg/ml)], bese igaywa indawo ku-papain [HBSS, enezelwe nge-1-cysteine·HCl (1 mg/ml), i-papain (16 U/ml) kanye ne-deoxyribonuclease I (DNase I; 0.1 mg/ml)] Phatha imizuzu engama-30 ku-30°C. Okokuqala geza izicubu endaweni ye-HBSS equkethe i-mucus yeqanda (10 mg/ml), i-BSA (10 mg/ml) kanye ne-DNase (0.1 mg/ml) ekamelweni lokushisa ukuvimbela ukugaya kwe-enzyme, bese kuba endaweni ye-HBSS equkethe i-glucose engu-20 mM. Gaya kancane ku-HBSS, i-penicillin (50 U/ml), i-streptomycin (0.05 mg/ml) kanye ne-DNase (0.1 mg/ml) ekhipha amaseli angawodwa. Ukumiswa kweseli okuholele kwahlungwa nge-strainer yeseli engu-70μm, bese amaseli ahlungwa nge-centrifugation (1110 rpm, imizuzu emi-5, 4°C) futhi aphinde amiswe endaweni yokuhlunga [HBSS, engezwe nge-glucose engu-20 mM, i-20% yenkomo yengane) i-Serum, i-penicillin (50 U/ml) kanye ne-streptomycin (0.05 mg/ml)]; hlola ukusebenza kweseli nge-propidium iodide bese ulungisa ubuningi beseli ku-1×106 kuya ku-2×106 amaseli/ml. Ngaphambi kokuhlolwa kwe-flow cytometry, i-suspension yayihlungwa nge-strainer yamaseli angu-50 μm.
I-Flow cytometer. Ukuhlunga amaseli kwenziwe ku-4°C kusetshenziswa umshini we-FACSAria III (BD Biosciences) kanye nesofthiwe ye-FACSDiva (BD Biosciences, inguqulo 8.0.1). Ukumiswa kwamaseli kuhlelwe kusetshenziswa i-nozzle engu-100 μm ngaphansi kwengcindezi engu-20 psi ngesilinganiso semicimbi engu-~2800/sec. Njengoba izindlela zokulinganisa zendabuko (usayizi wamaseli, ukuhlukaniswa kwe-bimodal, kanye nezici zokuhlakazeka) zingakwazi ukuqinisekisa ukuhlukaniswa okufanele kwe-PN kwezinye izinhlobo zamaseli, isu lokulinganisa lisethwe ngokusekelwe ekuqhathanisweni okuqondile kokuqina kwe-YFP kanye ne-autofluorescence ku-mitoYFP+ ​​​​kanye ne-control mitoYFP − Amagundane. I-YFP iyajabula ngokukhanyisa isampula ngomugqa we-laser ongu-488 nm, futhi isignali itholakala kusetshenziswa isihlungi se-band pass esingu-530/30 nm. Kumagundane e-mitoYFP+, amandla ahlobene ne-gene reporter ye-Rosa26-mitoYFP nawo asetshenziselwa ukuhlukanisa izingcezu zomzimba we-neuronal kanye ne-axon. I-7-AAD ivuselelwe nge-laser ephuzi engu-561 nm futhi itholwe ngesihlungi se-bandpass esingu-675/20 nm ukuze kukhishwe amaseli afile. Ukuze kuhlukaniswe ama-astrocyte ngesikhathi esifanayo, ukumiswa kweseli kwagcotshwa nge-ACSA-2-APC, bese isampula yashiswa ngomugqa we-laser ongu-640 nm, kwase kusetshenziswa isihlungi se-bandpass esingu-660/20 nm ukuthola isignali.
Amaseli aqoqwe ahlungwa nge-centrifugation (1110 rpm, imizuzu emi-5, 4°C) futhi agcinwa ku--80°C kuze kube yilapho esetshenziswa. Amagundane e-Mfn2cKO kanye namantshontsho awo ahlukaniswa ngosuku olufanayo ukuze kuncishiswe ukuguquguquka kwenqubo. Ukwethulwa kanye nokuhlaziywa kwedatha ye-FACS kwenziwa kusetshenziswa isofthiwe ye-FlowJo (FlowJo LLC, Ashland, Oregon, USA).
Njengoba kushiwo ngenhla (59), i-PCR yesikhathi sangempela isetshenziselwa ukuhlukanisa i-DNA kuma-neurons ahlungiwe ukuze kuhlolwe i-mtDNA elandelayo. Ukuzwela komugqa kanye nokuzwela komkhawulo kwahlolwa ekuqaleni ngokusebenzisa i-qPCR ezinambeni ezahlukene zamaseli. Ngamafuphi, qoqa ama-PN angu-300 ku-lysis buffer equkethe i-50 mM tris-HCl (pH 8.5), i-1 mM EDTA, i-0.5% Tween 20 kanye ne-proteinase K (200 ng/ml) bese uyibeka ku-55°C imizuzu eyi-120. Amaseli abekwa ku-95°C imizuzu eyi-10 ukuqinisekisa ukungasebenzi ngokuphelele kwe-proteinase K. Kusetshenziswa i-probe ye-TaqMan (Thermo Fisher) eqondene ne-mt-Nd1, i-mtDNA ilinganiswe nge-semi-quantitative PCR ohlelweni lwe-7900HT Real-Time PCR (Thermo Fisher Scientific). Isayensi, inombolo yekhathalogi Mm04225274_s1), mt-Nd6 (Thermo Fisher Scientific, inombolo yekhathalogi AIVI3E8) kanye ne-18S (Thermo Fisher Scientific, inombolo yekhathalogi Hs99999901_s1) izakhi zofuzo.
Ukulungiswa kwesampula ye-Proteome. Ngokufudumeza isixazululo ku-95°C imizuzu eyi-10 kanye nokufaka i-sonicating, ku-lysis buffer [6 M guanidine chloride, 10 mM tris(2-carboxyethyl) phosphine hydrochloride, 10 mM chloroacetamide kanye ne-100 mM tris-Lyse ama-neuron pellets aqandisiwe ku-HCl]. Ku-Bioruptor (Diagenode) imizuzu eyi-10 (imizuzwana engama-30 yokushaya kwenhliziyo / imizuzwana engama-30 isikhathi sokuphumula). Isampula yancishiswa ngo-1:10 ku-20 mM tris-HCl (pH 8.0), ixutshwe ne-300 ng trypsin gold (Promega), futhi yafakwa ubusuku bonke ku-37°C ukuze kufezwe ukugaya okuphelele. Ngosuku lwesibili, isampula yafakwa ku-centrifuge ku-20,000 g imizuzu engama-20. I-supernatant yancishiswa nge-0.1% formic acid, kanti isixazululo sasuswa usawoti nge-StageTips ezenziwe ngumuntu. Isampula yomiswe kuthuluzi le-SpeedVac (i-Eppendorf concentrator plus 5305) ku-45°C, bese i-peptide ilenga ku-0.1% formic acid. Wonke amasampula alungiswa ngesikhathi esisodwa ngumuntu ofanayo. Ukuze kuhlaziywe amasampula e-astrocyte, ama-peptide angu-4 μg akhishwe usawoti abhalwe nge-tandem mass tag (TMT10plex, inombolo yekhathalogi 90110, Thermo Fisher Scientific) enesilinganiso se-peptide kuya ku-TMT reagent esingu-1:20. Ekulebeni kwe-TMT, i-0.8 mg ye-reagent ye-TMT yaphinde yamiswa ku-70 μl ye-ACN engenamanzi, kanti i-peptide eyomisiwe yaphinde yahlanganiswa yaba yi-9 μl ye-0.1 M TEAB (triethylammonium bicarbonate), lapho kwanezelwa khona i-7 μl ye-reagent ye-TMT ku-ACN. Ukuhlushwa kwakungu-43.75%. Ngemva kwemizuzu engama-60 yokufukamela, ukusabela kwacinywa nge-2 μl ye-5% hydroxylamine. Ama-peptide anelebula aqoqwa, omiswa, aphinde axutshwe ku-200μl ye-0.1% formic acid (FA), ahlukaniswa kabili, abese ekhishwa usawoti kusetshenziswa i-StageTips eyenziwe ngumuntu ngokwakhe. Kusetshenziswa i-UltiMate 3000 ultra high performance liquid chromatograph (UltiMate 3000 ultra high performance liquid chromatograph), enye yezingxenye ezimbili yahlukaniswa kukholomu ye-1mm x 150mm Acquity chromatographic egcwele izinhlayiya ze-130Å1.7μm C18 (Amanzi, ikhathalogi No. SKU: 186006935). Thermo Fisher Scientific). Hlukanisa ama-peptide ngesilinganiso sokugeleza esingu-30μl/min, uhlukanise kusuka ku-1% kuya ku-50% we-buffer B imizuzu engu-85 nge-gradient ejikelezayo yemizuzu engu-96, kusukela ku-50% kuya ku-95% we-buffer B imizuzu emi-3, bese kuba imizuzu engu-8 ye-95% ye-Buffer B; i-Buffer A ingu-5% we-ACN kanye ne-10 mM ammonium bicarbonate (ABC), kanti i-buffer B ingu-80% we-ACN kanye ne-10 mM ABC. Qoqa izingxenyana njalo ngemizuzu emi-3 bese uzihlanganisa zibe amaqembu amabili (1 + 17, 2 + 18, njll.) bese uzomisa ku-vacuum centrifuge.
Ukuhlaziywa kwe-LC-MS/MS. Kokuhlolwa kwe-mass spectrometry, ama-peptide (inombolo r119.aq) ahlukaniswe kukholomu yokuhlaziya ye-PicoFrit engama-25 cm, 75 μm ububanzi bangaphakathi (ilensi entsha yenhloso, inombolo yengxenye PF7508250) efakwe i-1.9 μm ReproSil-Pur 120 C18-AQ medium (Dr. Maisch, mat), Sebenzisa i-EASY-nLC 1200 (Thermo Fisher Scientific, Germany). Ikholomu igcinwe ku-50°C. Ama-Buffers A no-B angama-0.1% formic acid emanzini kanye nama-0.1% formic acid ku-80% ACN, ngokulandelana. Ama-Peptides ahlukaniswe kusuka ku-6% kuya ku-31% buffer B imizuzu engama-65 kanye nama-31% buffer B imizuzu emi-5 nge-gradient engu-200 nl/min. Ama-peptides aluted ahlaziywe ku-Orbitrap Fusion mass spectrometer (Thermo Fisher Scientific). Ukulinganiswa kwe-Peptide precursor m/z kwenziwa ngesisombululo esingu-120,000 ebangeni eliphakathi kuka-350 no-1500 m/z. Kusetshenziswa amandla okushayisana ajwayelekile angu-27%, i-precursor enamandla kakhulu enesimo sokushaja esingu-2 kuya ku-6 ikhethwa ukuhlukana kwe-C trap dissociation (HCD) yamandla aphezulu. Isikhathi somjikelezo sisethwe ku-1 s. Inani le-m/z le-peptide fragment lilinganiswe ku-ion trap kusetshenziswa i-AGC target encane kakhulu engu-5×104 kanye nesikhathi esiphezulu sokufaka esingu-86 ms. Ngemva kokuqhekeka, i-precursor yabekwa ohlwini lwe-dynamic exclusion imizuzwana engu-45. Ama-peptide anelebula le-TMT ahlukaniswe kukholomu engu-50 cm, 75 μm ye-Acclaim PepMap (i-Thermo Fisher Scientific, inombolo yekhathalogi 164942), kanti ama-spectra okufuduka ahlaziywe ku-Orbitrap Lumos Tribrid mass spectrometer (i-Thermo Fisher Scientific) ehlonyiswe ngemishini ye-high-field asymmetric waveform ions (FAIMS) (i-Thermo Fisher Scientific) esebenza kuma-voltage amabili okubuyisela angu-−50 kanye no-−70 V. I-MS3 ekhethiwe ngokusekelwe ku-precursor yokuvumelanisa esetshenziselwa ukulinganisa isignali ye-ion yokubika ye-TMT. Ukuhlukaniswa kwe-peptide kwenziwa ku-EASY-nLC 1200, kusetshenziswa i-90% linear gradient elution, ene-buffer concentration engu-6% kuya ku-31%; i-buffer A yayingu-0.1% FA, kanti i-buffer B yayingu-0.1% FA kanye no-80% ACN. Ikholomu yokuhlaziya isebenza ku-50°C. Sebenzisa i-FreeStyle (inguqulo 1.6, i-Thermo Fisher Scientific) ukuze uhlukanise ifayela lokuqala ngokwe-voltage yesinxephezelo se-FAIMS.
Ukuhlonza amaprotheni kanye nokulinganisa. Kusetshenziswa injini yokusesha ye-Andromeda ehlanganisiwe, idatha yokuqala yahlaziywa kusetshenziswa inguqulo ye-MaxQuant 1.5.2.8 (https://maxquant.org/). Ngaphezu kokulandelana kwe-Cre recombinase kanye ne-YFP okutholwe ku-Aequorea victoria, ama-spectra e-peptide fragment ahlolwe ukulandelana kwe-canonical kanye nokulandelana kwe-isoform kwe-mouse reference proteome (Proteome ID UP000000589, elandiwe ku-UniProt ngoMeyi 2017). I-Methionine oxidation kanye ne-protein N-terminal acetylation kwasethwa njengokuguqulwa okuguquguqukayo; i-cysteine ​​​​carbamoyl methylation yasethwa njengokuguqulwa okuhleliwe. Amapharamitha okugaya abekwe ku-"specificity" kanye ne-"trypsin/P". Inani elincane lama-peptide kanye nama-razor peptide asetshenziselwa ukuhlonza amaprotheni lingu-1; inani elincane lama-peptide ahlukile lingu-0. Ngaphansi kwezimo zokufanisa imephu ye-peptide, izinga lokuhlonza amaprotheni lalingu-0.01. Inketho ethi "Second Peptide" iyasebenza. Sebenzisa inketho ethi “match between run” ukuze udlulise ukuhlonza okuphumelelayo phakathi kwamafayela okuqala ahlukene. Sebenzisa inani elincane le-LFQ elingu-1 ukuze uthole ukukala okungenalebula (LFQ) (60). Ukuqina kwe-LFQ kuhlungwa okungenani amanani amabili avumelekile okungenani eqenjini elilodwa le-genotype ngesikhathi ngasinye, futhi kuthathwe kusukela ekusabalalisweni okujwayelekile okunobubanzi obungu-0.3 bese wehla ngo-1.8. Sebenzisa ipulatifomu yokubala ye-Perseus (https://maxquant.net/perseus/) kanye ne-R (https://r-project.org/) ukuze uhlaziye imiphumela ye-LFQ. Ukuhlolwa kwe-t okulinganiselwe okubili okuvela kuphakheji yesofthiwe ye-limma kusetshenziswe ekuhlaziyweni kokubonakaliswa okuhlukile (61). Ukuhlaziywa kwedatha yokuhlola kwenziwa kusetshenziswa i-ggplot, i-FactoMineR, i-factoextra, i-GGally kanye ne-pheatmap. Idatha ye-proteomics esekelwe ku-TMT ihlaziywe kusetshenziswa inguqulo ye-MaxQuant 1.6.10.43. Sesha idatha ye-proteomics eluhlaza kusuka kusizindalwazi se-proteomics somuntu se-UniProt, esalandwa ngoSepthemba 2018. Ukuhlaziywa kufaka phakathi isici sokulungisa ubumsulwa be-isotope esinikezwe umenzi. Sebenzisa i-limma ku-R ukuze uhlaziye ukubonakaliswa kokungafani. Idatha yokuqala, imiphumela yokusesha yesizindalwazi, kanye nomsebenzi wokuhlaziya idatha kanye nemiphumela konke kugcinwa ku-ProteomeXchange alliance ngokusebenzisa indawo yokugcina yabalingani be-PRIDE ene-data set identifier PXD019690.
Izichasiselo ezisebenzayo zicebisa ukuhlaziywa. Ithuluzi le-Ingenuity Pathway Analysis (QIAGEN) lisetshenziswe ukunquma ukuceba kwemigomo yesichasiselo esisebenzayo sedatha ebekwe emavikini ayi-8 (Isithombe 1). Ngamafuphi, uhlu lwamaprotheni olutholakala ku-LC-MS/MS (tandem mass spectrometry) ukuhlaziywa kwedatha lusetshenziswa ngezinqubo zokuhlunga ezilandelayo: I-Mus musculus ikhethwa njengohlobo kanye nesizinda, futhi isigaba sibonisa inani le-P elilungiswe yi-Benjamini ukuze licebise ngo-0.05 noma ngaphansi libhekwa njengelibalulekile. Kule grafu, kuboniswa izigaba ezinhlanu eziphezulu kakhulu kuqoqo ngalinye ngokusekelwe enanini le-P elilungisiwe. Kusetshenziswa ukuhlolwa kwe-t okuningi, kusetshenziswa uhlelo lwe-two-stage linear boost lwe-Benjamini, Krieger, kanye ne-Yekutieli (Q = 5%), ukuhlaziywa kokubonakaliswa kwe-protein yesikhathi kwenziwa kubantu ababalulekile abakhonjwe esigabeni ngasinye, futhi umugqa ngamunye uhlaziywa ngokwehlukana. Asikho isidingo sokwamukela i-SD ehambisanayo.
Ukuze siqhathanise imiphumela yalolu cwaningo nezizindalwazi ezishicilelwe futhi sikhiqize umdwebo we-Venn ku-Figure 1, sihlanganise uhlu lwamaprotheni olunenani nezichasiselo ze-MitoCarta 2.0 (24). Sebenzisa ithuluzi eliku-inthanethi elithi Draw Venn Diagram (http://bioinformatics.psb.ugent.be/webtools/Venn/) ukuze sikhiqize umdwebo.
Ukuze uthole ulwazi oluningiliziwe ngezinqubo zezibalo ezisetshenziselwa ukuhlaziywa kwe-proteomics, sicela ubheke isigaba esihambisanayo se-Materials and Methods. Kuzo zonke ezinye izivivinyo, ulwazi oluningiliziwe lungatholakala ku-legend ehambisanayo. Ngaphandle kokuthi kuchazwe ngenye indlela, yonke idatha ivezwa njenge-mean ± SEM, futhi konke ukuhlaziywa kwezibalo kwenziwe kusetshenziswa isofthiwe ye-GraphPad Prism 8.1.2.
Ukuze uthole izinto ezengeziwe zalesi sihloko, sicela ubheke ku-http://advances.sciencemag.org/cgi/content/full/6/35/eaba8271/DC1
Lesi yisihloko sokufinyelela okuvulekile esisatshalaliswa ngaphansi kwemigomo ye-Creative Commons Attribution-Non-Commercial License, evumela ukusetshenziswa, ukusatshalaliswa kanye nokukhiqizwa kabusha kunoma iyiphi indlela, inqobo nje uma ukusetshenziswa kokugcina kungengenxa yenzuzo yezentengiselwano futhi isisekelo siwukuthi umsebenzi wokuqala ulungile. Ireferensi.
Qaphela: Sicela kuphela ukuthi unikeze ikheli lakho le-imeyili ukuze umuntu omncomayo ekhasini azi ukuthi ufuna abone i-imeyili nokuthi akuyona ugaxekile. Ngeke sithathe noma yimaphi amakheli e-imeyili.
Lo mbuzo usetshenziselwa ukuhlola ukuthi uyisivakashi yini futhi uvimbele ukuthunyelwa kogaxekile okuzenzakalelayo.
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Ukuhlaziywa kwe-Proteomics kwama-neurons angasebenzi kahle kwembule ukuthi izinhlelo ze-metabolic ziyasebenza ukulwa nokuwohloka kwemizwa.
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Ukuhlaziywa kwe-Proteomics kwama-neurons angasebenzi kahle kwembule ukuthi izinhlelo ze-metabolic ziyasebenza ukulwa nokuwohloka kwemizwa.
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