Present *Endagiriro eriwo kati: Cologne 50931, Germany, Cologne Excellence Cluster Okunoonyereza ku kuddamu kw’obutoffaali ku situleesi mu ndwadde ezeekuusa ku kukaddiwa (CECAD).
Okuvunda kw’obusimu bw’endwadde z’obusimu kutwalibwa ng’okutasobola kuddamu kubanga obuveera bw’enkyukakyuka y’obusimu bukoma, naye ekikolwa ky’obutakola bulungi bwa mitochondrial ku bwetwaze bw’obutoffaali bw’enkyukakyuka y’obusimu mu mubiri tekitegeerekeka bulungi. Wano, twanjula proteome ey’obutoffaali ey’obusimu bwa Purkinje n’obutaba na OXPHOS obugenda mu maaso obuva ku nkyukakyuka y’okuyungibwa kw’emitochondria okutaataaganyizibwa. Twakizuula nti obutakola bulungi bwa mitochondrial bwaleetawo enkyukakyuka ey’amaanyi mu kisaawe kya proteomics, okukkakkana nga kivuddeko okukola mu mutendera gwa pulogulaamu entuufu ez’enkyukakyuka mu mubiri nga obutoffaali tebunnafa. Nga tetusuubira, twasalawo okuyingiza okweyoleka okwa pyruvate carboxylase (PCx) ne peroxidases endala ezikwatagana n’okugatta ku TCA cycle intermediates. Okuziyiza PCx kwayongera ku situleesi y’okwokya n’okuvunda kw’obusimu, ekiraga nti okuzimba emisuwa kulina ekikolwa eky’obukuumi mu busimu obutaliimu OXPHOS. Okuzzaawo okuyungibwa kwa mitochondrial mu busimu obukendedde mu nkomerero kukyusa ddala engeri zino ez’enkyukakyuka mu mubiri, bwe kityo ne kiziyiza okufa kw’obutoffaali. Ebizuuliddwa byaffe bizuula amakubo agaali tegamanyiddwa emabegako agawa obugumiikiriza eri obutakola bulungi bwa mitochondria era biraga nti okuvunda kw’obusimu kuyinza okukyusibwa ne mu mitendera egy’oluvannyuma egy’obulwadde.
Omulimu omukulu ogwa mitochondria mu kukuuma enkyukakyuka y’amasoboza g’obusimu guggumiza obubonero obw’obusimu obw’amaanyi obukwatagana n’endwadde z’obusimu bw’omuntu. Endwadde zino ezisinga ziva ku nkyukakyuka mu buzaale ezifuga okulaga kw’obuzaale bwa mitochondrial (1, 2) oba okusaanawo kw’obuzaale obukwatagana n’enkyukakyuka ya mitochondrial, ezikosa mu ngeri etali butereevu nnywevu ya DNA ya mitochondrial (mtDNA) (3, 4). Omulimu mu bikolwa by’ebisolo gulaze nti mu kuddamu obutakola bulungi bwa mitochondrial mu bitundu ebigyetoolodde, amakubo g’enkyukakyuka y’emmere agakuuma (5-7) gasobola okukolebwa, bwe kityo ne kiwa amagezi amakulu ku ntandikwa y’endwadde zino enzibu. Okwawukana ennyo, okutegeera kwaffe ku nkyukakyuka mu mubiri gw’ebika by’obutoffaali ebitongole ebiva ku kulemererwa okwa bulijjo okw’okukola mitochondrial adenosine triphosphate (ATP) mu bwongo kya musingi (8), nga kiggumiza obwetaavu bw’okuzuula ebigendererwa by’obujjanjabi ebiyinza okukozesebwa okuziyiza oba okuziyiza endwadde. Okuziyiza okuvunda kw’obusimu (9). Obutabeera na mawulire kwe kuba nti obutoffaali bw’obusimu butwalibwa nnyo okuba n’obusobozi obutono ennyo mu nkyukakyuka y’emmere bw’ogeraageranya n’ebika by’obutoffaali eby’ebitundu ebibyetoolodde ( 10 ). Okusinziira ku kuba nti obutoffaali buno bukola kinene mu kukwasaganya okugabibwa kw’ebirungo ebikyusa ebiriisa eri obusimu okutumbula okutambuza kw’obusimu n’okuddamu embeera z’obuvune n’endwadde, obusobozi okukyusa enkyukakyuka y’obutoffaali n’embeera ezisoomoozebwa ez’ebitundu by’obwongo kumpi bukoma ku butoffaali bwa glial ( 11-14 ). Okugatta ku ekyo, obutafaanagana bw’obutoffaali obuzaaliranwa obw’ebitundu by’obwongo okusinga bulemesa okunoonyereza ku nkyukakyuka mu nkyukakyuka mu mubiri ezibeerawo mu bibinja by’obusimu ebitongole. N’ekyavaamu, kitono ekimanyiddwa ku biva mu butoffaali n’enkyukakyuka y’emmere entuufu olw’obutakola bulungi bwa mitochondria mu busimu obuyitibwa neurons.
Okusobola okutegeera ebiva mu nkyukakyuka y’emmere olw’obutakola bulungi bwa mitochondrial, twawula obusimu bwa Purkinje (PNs) mu mitendera egy’enjawulo egy’okuvunda kw’obusimu obuva ku kusaanyizibwawo kw’okuyungibwa kw’olususu olw’ebweru olwa mitochondrial (Mfn2). Newankubadde enkyukakyuka za Mfn2 mu bantu zikwatagana n’engeri y’obulwadde bw’obusimu obuyitibwa heditary motor sensory neuropathy obumanyiddwa nga Charcot-Marie-Tooth type 2A (15), okusaanyaawo Mfn2 mu bukwakkulizo mu mbeba nkola emanyiddwa bulungi ey’okuleeta oxidation Phosphorylation (OXPHOS) dysfunction method. Ebika by’obusimu eby’enjawulo (16-19) n’enkula y’obusimu evuddemu biwerekerwako obubonero bw’obusimu obugenda mu maaso, gamba ng’obuzibu mu ntambula ( 18 , 19 ) oba obutafaali bwa bwongo ( 16 ). Nga tukozesa omugatte gwa label-free quantitative (LFQ) proteomics, metabolomics, imaging, ne virological enkola, tulaga nti progressive neurodegeneration strongly induces pyruvate carboxylase (PCx) n’ensonga endala ezikwatibwako arteriosclerosis of PNs in vivo Okwolesebwa kwa enzymes. Okukakasa obukulu bw’ekizuuliddwa kino, twassa wansi mu ngeri ey’enjawulo okwolesebwa kwa PCx mu PNs ezibulamu Mfn2, era ne tuzuula nti okulongoosebwa kuno kwayongera okunyigirizibwa okw’okwokya n’okwanguyiza okuvunda kw’obusimu, bwe tutyo ne tukakasa nti azoospermia egaba okufa kw’obutoffaali Metabolic adaptability. Okwolesebwa okw’amaanyi okwa MFN2 kuyinza okununula ddala PN ey’okuvunda okusembayo n’obutaba na OXPHOS okw’amaanyi, okukozesa ennyo DNA ya mitochondrial, era nga kirabika omukutu gwa mitochondrial gumenyese, ekyongera okuggumiza nti engeri eno ey’okuvunda kw’obusimu esobola n’okudda engulu mu mutendera ogw’oku ntikko ogw’obulwadde nga obutoffaali tebunnafa.
Okusobola okulaba mu birowoozo mitochondria mu Mfn2 knockout PNs, twakozesa ekika ky’ebibe ekisobozesa Cre-dependent mitochondria okutunuulira yellow fluorescent protein (YFP) (mtYFP) (20) Cre expression era ne tukebera mitochondria morphology mu vivo. Twakizuula nti okusaanawo kw’obuzaale bwa Mfn2 mu PNs kwandiviiriddeko okugabanya mpolampola kw’omukutu gwa mitochondrial (Figure S1A), era enkyukakyuka eyasooka yazuulibwa ku wiiki 3 ez’emyaka. Okwawukana ku ekyo, okuvunda okw’amaanyi okw’oluwuzi lw’obutoffaali bwa PN, nga bwe kiragibwa okufiirwa Calbindin immunostaining, tekwatandika okutuusa nga wayise wiiki 12 (Ekifaananyi 1, A ne B). Obutakwatagana mu biseera wakati w’enkyukakyuka ezaasooka mu nkula ya mitochondria n’okutandika okulabika okw’okufa kw’obusimu bwatuleetera okunoonyereza ku nkyukakyuka mu nkyukakyuka mu mubiri ezireetebwa obutakola bulungi bwa mitochondria nga obutoffaali tebunnafa. Twakola enkola eyesigamiziddwa ku fluorescence-activated cell sorting (FACS) okwawula YFP (YFP +)-okwolesa PN (Figure 1C), ne mu mbeba ezifuga (Mfn2 + / loxP :: mtYFP loxP- stop-loxP: : L7-cre), wano oluvannyuma eyitibwa CTRL (Figure S1B). Okulongoosa enkola ya gating okusinziira ku maanyi agakwatagana ag’akabonero ka YFP kitusobozesa okulongoosa omubiri gwa YFP + (YFPhigh) ogwa PNs okuva mu non-PNs (YFPneg) (Figure S1B) oba putative fluorescent axon/dendritic fragments (YFPlow; Ekifaananyi S1D, kkono), ekikakasibwa confocal microscope (Figure S1D, ku ddyo). Okusobola okukakasa endagamuntu y’abantu abasengekeddwa, twakola LFQ proteomics n’oluvannyuma okwekenneenya ebitundu ebikulu, era ne tuzuula nti waliwo okwawukana okutegeerekeka wakati w’obutoffaali bwa YFPhigh ne YFPneg (Figure S1C). YFPhigh cells yalaga okugaggawala okutuufu okw’obubonero bwa PNs obumanyiddwa (kwe kugamba Calb1, Pcp2, Grid2 ne Itpr3) (21, 22), naye tewali kugaggawaza kwa puloteyina ezitera okulagibwa mu busimu oba ebika by’obutoffaali ebirala (Figure 1D) ). Okugerageranya wakati wa sampuli mu butoffaali bwa YFPhigh obusengekeddwa obukung’aanyiziddwa mu kugezesa okwetongodde kwalaga omugerageranyo gw’enkolagana> 0.9, nga kiraga okuddamu okulungi wakati w’ebiramu ebikoppiddwa (Figure S1E). Mu bufunze, data zino zakakasa enteekateeka yaffe ey’okwawula okw’amangu n’okw’enjawulo okwa PN esoboka. Olw’okuba enkola ya L7-cre driver ekozesebwa ereeta mosaic recombination mu wiiki esooka oluvannyuma lw’okuzaala (23), twatandika okusala ebibe okuva mu CTRL n’obukwakkulizo (Mfn2 loxP / loxP :: mtYFP loxP-stop-loxP :: L7-cre) Okukung’aanya obusimu. Oluvannyuma lw’okumaliriza okugatta, kiyitibwa Mfn2cKO ku wiiki 4 ez’obukulu. Nga enkomerero, twalonda wiiki 8 ez’emyaka nga PN layer ekyali mu mbeera wadde nga ebitundu bya mitochondrial byeyoleka (Figure 1B and Figure S1A). Okutwalira awamu, twapima obungi bwa puloteyina 3013, nga ku zino nga 22% zaali zeesigamiziddwa ku MitoCarta 2.0 annotations nga zeesigamiziddwa ku mitochondrial proteome nga mitochondria (Figure 1E) (Figure 1E) (24). Okwekenenya okw’enjawulo okwakolebwa mu wiiki 8 kwalaga nti ebitundu 10.5% byokka ku puloteyina zonna bye byalina enkyukakyuka ez’amaanyi (Figure 1F ne Figure S1F), nga ku zino puloteyina 195 zaali zifugibwa wansi ate puloteyina 120 zaali zitereezeddwa waggulu (Ekifaananyi 1F). Kinajjukirwa nti “okwekenneenya amakubo ag’obuyiiya” kw’ekibiina kino eky’amawulire kulaga nti obuzaale obulaga mu ngeri ey’enjawulo okusinga buba mu kibinja ekikugirwa eky’amakubo ag’enjawulo ag’enkyukakyuka mu mubiri (Figure 1G). Ekyewuunyisa, wadde ng’okukka wansi kw’amakubo agakwatagana ne OXPHOS ne calcium signaling kukakasa okuleetebwa kw’obutakola bulungi bwa mitochondrial mu PNs ezibulamu fusion, ebika ebirala ebisinga okuzingiramu okukyusakyusa amino acid biba bikulu nnyo waggulu-regulated, ekikwatagana n’enkyukakyuka ebeerawo mu mitochondrial PNs. Okuddamu okukuba waya (rewiring) kukwatagana. obutakola bulungi.
(A) Ebifaananyi ebikiikirira confocal ebitundu by’obwongo eby’ebibe CTRL ne Mfn2cKO ebiraga okufiirwa okugenda mu maaso kwa PNs (calbindin, enzirugavu); nuclei zaali za counterstained ne DAPI. (B) Okugera obungi bwa (A) (okwekenneenya enjawulo mu ngeri emu, ***P <0.001; n = enzirugavu 4 ku 6 okuva mu mbeba ssatu). (C) Enkola y’emirimu ey’okugezesa. (D) Ensaasaanya ya maapu y’ebbugumu ey’obubonero obukwata ku Purkinje (waggulu) n’ebika by’obutoffaali ebirala (wakati). (E) Ekifaananyi kya Venn ekiraga omuwendo gwa puloteyina za mitochondrial ezizuuliddwa mu PN eyasengekeddwa. (F) Volcano plot of differentially expressed proteins mu busimu bwa Mfn2cKO ku wiiki 8 (omuwendo gw’okusalako ogw’amakulu ogwa 1.3). (G) Okwekenenya ekkubo ly’obuyiiya kulaga amakubo ataano agasinga obukulu ag’okulinnya (emmyufu) n’okukka wansi (bbululu) mu Mfn2cKO PN agasengekeddwa nga wiiki 8. Omutindo gw’okwolesebwa ogwa wakati ogwa buli puloteyina ezuuliddwa gulagibwa. Maapu y’ebbugumu ery’enzirugavu: omuwendo gwa P ogutereezeddwa. ns, si kikulu.
Data ya Proteomics yalaga nti protein expression ya complexes I, III, ne IV yakendeera mpolampola. Complexes I, III, ne IV zonna zaali zirimu subunits enkulu eziriko mtDNA, ate complex II, eyali eriko enkodi ya nuclear yokka, okusinga teyaliikosebwa (Figure 2A ne Figure S2A). . Okukwatagana n’ebivudde mu proteomics, immunohistochemistry y’ebitundu by’ebitundu by’obwongo yalaga nti MTCO1 (mitochondrial cytochrome C oxidase subunit 1) subunit level ya complex IV mu PN yakendeera mpolampola (Figure 2B). Subunit Mtatp8 eriko enkodi ya mtDNA yakendeera nnyo (Figure S2A), ate nga level ya steady-state ya nuclear-encoded ATP synthase subunit yasigala nga tekyuse, ekikwatagana n’ekimanyiddwa stable ATP synthase subassembly F1 complex nga mtDNA expression enywevu. Ensengeka eno ekwatagana. Okusalako (7). Okwekenenya omutindo gwa mtDNA mu Mfn2cKO PNs ezisunsuddwa nga tuyita mu kiseera ekituufu ekya polymerase chain reaction (qPCR) kyakakasa okukendeera mpolampola mu muwendo gwa mtDNA copy number. Bw’ogeraageranya n’ekibinja ekifuga, ku wiiki 8 ez’emyaka, ebitundu nga 20% byokka ku mutindo gwa mtDNA bye byasigala (Figure 2C). Okukwatagana n’ebivuddemu bino, okusiiga kwa confocal microscopy okwa Mfn2cKO PNs okuzuula DNA kulaga enkozesa eyesigamiziddwa ku kiseera eya mitochondrial nucleotides (Figure 2D). Twakizuula nti abamu bokka abesimbyewo abeenyigira mu mitochondrial protein degradation ne stress response baali up-regulated, omuli Lonp1, Afg3l2 ne Clpx, ne OXPHOS complex assembly factors. Tewali nkyukakyuka nnene mu miwendo gya puloteyina ezenyigira mu apoptosis yazuulibwa (Figure S2B). Mu ngeri y’emu, twakizuula nti emikutu gya mitochondria ne endoplasmic reticulum egyenyigira mu ntambula ya calcium girina enkyukakyuka entonotono zokka (Figure S2C). Okugatta ku ekyo, okwekenneenya obutoffaali obukwata ku autophagy tekwazudde nkyukakyuka nnene, ekikwatagana n’okuyingiza okulabika kwa autophagosomes okwalabiddwa mu vivo nga bakozesa immunohistochemistry ne electron microscopy (Figure S3). Naye, obutakola bulungi bwa OXPHOS obugenda mu maaso mu PNs buwerekerwako enkyukakyuka ezeeyolese mu mitochondrial ez’ensengekera y’obutonde (ultrastructural mitochondrial changes). Ebibinja by’obutoffaali (mitochondrial clusters) bisobola okulabibwa mu mibiri gy’obutoffaali n’emiti gya dendritic egya Mfn2cKO PNs ez’emyaka 5 ne 8, era ensengekera y’olususu olw’omunda efunye enkyukakyuka ez’amaanyi (Ekifaananyi S4, A ne B). Okukwatagana n’enkyukakyuka zino ez’ensengekera y’obutonde (ultrastructural changes) n’okukendeera okw’amaanyi mu mtDNA, okwekenneenya ebitundu by’obwongo eby’amaanyi (acute cerebral cerebellar slices) ne tetramethylrhodamine methyl ester (TMRM) kwalaga nti obusobozi bw’olususu lw’emitochondrial mu Mfn2cKO PNs bwakendeera nnyo (Figure S4C).
(A) Okwekenenya ekkubo ly’ebiseera ku ddaala ly’okwolesebwa kw’ekizibu kya OXPHOS. Lowooza ku puloteyina zokka ezirina P<0.05 ku wiiki 8 (two-way ANOVA). Layini eriko ennukuta: Tewali nnongoosereza bw’ogeraageranya ne CTRL. (B) Ku kkono: Ekyokulabirako ky’ekitundu ky’obwongo ekiwandiikiddwako anti-MTCO1 antibody (scale bar, 20 μm). Ekitundu ekirimu emirambo gy’obutoffaali bwa Purkinje kibikkiddwako langi ya kyenvu. Ku ddyo: Okugera obungi bw’emiwendo gya MTCO1 (okwekenneenya enjawulo mu ngeri emu; n = obutoffaali 7 ku 20 obwekenneenyeddwa okuva mu mbeba ssatu). (C) okwekenneenya qPCR okw’omuwendo gwa kkopi ya mtDNA mu PN eyasunsulwa (okwekenneenya enjawulo okw’engeri emu; n = mbeba 3 ku 7). (D) Ku kkono: Ekyokulabirako ky’ekitundu ky’obwongo ekiwandiikiddwako ekirungo ekiziyiza DNA (scale bar, 20 μm). Ekitundu ekirimu emirambo gy’obutoffaali bwa Purkinje kibikkiddwako langi ya kyenvu. Ku ddyo: Okugera obungi bw’ebiwundu bya mtDNA (okwekenneenya enjawulo mu ngeri emu; n = obutoffaali 5 ku 9 okuva mu mbeba ssatu). (E) Ekyokulabirako ky’ekitundu ky’obwongo ekizibu ekiraga obutoffaali bwa mitoYFP + Purkinje (akasaale) mu kuwandiika kwa patch clamp ey’obutoffaali bwonna. (F) Okugera obungi bwa IV curve. (G) Ebiwandiiko ebikiikirira eby’empiso ya kasasiro eya depolarizing mu butoffaali bwa CTRL ne Mfn2cKO Purkinje. Top trace: Pules eyasooka eyasitula AP. Wansi okulondoola: Frequency ya AP esinga obunene. (H) Okugera obungi bw’ebiyingizibwa mu ngeri ey’okwekolako (sPSPs). Omulondo gw’okukwata ogukiikirira n’omugerageranyo gwayo ogw’okuzimba biragiddwa mu (I). Okwekenenya enjawulo mu ngeri emu kwekenneenya obutoffaali n = 5 ku 20 okuva mu mbeba ssatu. Data ziragibwa nga mean±SEM; *P<0.05 nga bwe kiri; **P <0.01 nga bwe kiri; ***P<0.001 nga bano. (J) Ebifo ebikiikirira AP eyeetongodde ebiwandiikiddwa nga tukozesa enkola ya perforated patch clamp mode. Top trace: Frequency ya AP esinga obunene. Wansi okulondoola: zoom ya AP emu. (K) Omuwendo gwa frequency ya AP eya wakati n’esinga obunene okusinziira ku (J). Okugezesebwa kwa Mann-Whitney; n = obutoffaali 5 bwakeberebwa okuva mu mbeba nnya. Data ziragibwa nga mean±SEM; si kikulu.
Okwonooneka kwa OXPHOS okweyoleka kwazuulibwa mu Mfn2cKO PN eya wiiki 8, ekiraga nti omulimu gw’omubiri gw’obusimu si gwa bulijjo nnyo. Nolwekyo, twekenneenya engeri z’amasannyalaze ezitakola (passive electrical characteristics) ez’obusimu obutaliimu OXPHOS ku wiiki 4 ku 5 ne wiiki 7 ku 8 nga tukola ebiwandiiko by’obutoffaali bwonna (whole-cell patch clamp recordings) mu bitundu by’obwongo ebizibu (Figure 2E). Mu ngeri etasuubirwa, obusobozi bw’olususu oluwummula wakati n’okuziyiza okuyingira kw’obusimu bwa Mfn2cKO byali bifaanagana n’eby’okufuga, wadde nga waaliwo enjawulo entonotono wakati w’obutoffaali (Table 1). Mu ngeri y’emu, ku wiiki 4 ku 5 ez’obukulu, tewali nkyukakyuka nnene mu nkolagana ya kasasiro ne vvulovumenti (IV curve) yazuulibwa (Ekifaananyi 2F). Wabula, tewali busimu bwa Mfn2cKO obwa wiiki 7 ku 8 bwawona enkola ya IV (omutendera gwa hyperpolarization), ekiraga nti waliwo okutegeera okutegeerekeka ku busobozi bwa hyperpolarization ku mutendera guno ogw’oluvannyuma. Okwawukana ku ekyo, mu busimu bwa Mfn2cKO, amasannyalaze aga depolarizing agavaako okufuluma kw’obusobozi bw’okukola okuddiŋŋana (AP) gagumiikiriza bulungi, ekiraga nti enkola zaabwe ez’okufulumya okutwalira awamu tezaawukana nnyo ku z’obusimu obufuga obwa wiiki 8 (Table 1 and Figure 2G). Mu ngeri y’emu, emirundi n’obunene bwa spontaneous postsynaptic currents (sPSCs) byali bigeraageranyizibwa ku by’ekibinja ekifuga, era emirundi gy’ebintu ebibaawo gyeyongera okuva ku wiiki 4 okutuuka ku wiiki 5 okutuuka ku wiiki 7 okutuuka ku wiiki 8 nga kweyongera okufaananako (Figure 2, H and I). Ekiseera ky’okukula kw’obusimu mu PNs (25). Ebivuddemu ebifaananako bwe bityo byafunibwa oluvannyuma lw’okussaamu ebitundu bya PNs ebituli. Ensengeka eno eremesa okuliyirira okusoboka okw’obulema bwa ATP obw’obutoffaali, nga bwe kiyinza okubaawo mu kuwandiika kwa patch clamp ey’obutoffaali bwonna. Okusingira ddala, obusobozi bw’olususu oluwummula n’emirundi gy’okukuba amasasi mu ngeri ey’okwekolako ey’obusimu bwa Mfn2cKO tebyakosebwa (Ekifaananyi 2, J ne K). Mu bufunze, ebivuddemu bino biraga nti PNs ezirina obuzibu obweyoleka obwa OXPHOS zisobola okugumira obulungi enkola z’okufulumya amazzi aga frequency enkulu, ekiraga nti waliwo enkola y’okuliyirira ezibasobozesa okukuuma eby’okuddamu eby’amasannyalaze kumpi ebya bulijjo.
Data ziragibwa nga mean ± SEM (one-way analysis of variance, Holm-Sidak’s multiple comparison test; *P<0.05). Ennamba ya yuniti eragiddwa n’obukwakulizo.
Twatandika okunoonyereza oba ekika kyonna mu proteomics dataset (Figure 1G) kirimu amakubo agayinza okuziyiza OXPHOS ebbula ery’amaanyi, bwe kityo ne tunnyonnyola lwaki PN ekoseddwa esobola okukuuma kumpi-bulijjo electrophysiology (Figure 2, E to K). . Okwekenenya proteomics kwalaga nti enzymes ezikwatibwako mu catabolism ya branched chain amino acids (BCAA) zaali nnyo up-regulated (Figure 3A ne Figure S5A), era ekintu ekisembayo acetyl-CoA (CoA) oba succinyl CoA kisobola okwongera ku tricarboxylates mu arteriosclerosis Acid (TCA) cycle. Twakizuula nti ebirimu BCAA transaminase 1 (BCAT1) ne BCAT2 byombi byeyongera. Ba catalyzed omutendera ogusooka ogwa BCAA catabolism nga bakola glutamate okuva mu α-ketoglutarate ( 26 ). Subunits zonna ezikola branched chain keto acid dehydrogenase (BCKD) complex zibeera upregulated (ekizibu kino kikola decarboxylation eddirira era etakyukakyuka ey’amagumba ga kaboni ga BCAA agavaamu) (Ekifaananyi 3A n’Ekifaananyi S5A). Naye, tewali nkyukakyuka yeeyoleka mu BCAA yennyini yasangibwa mu PN eyasunsulwa, ekiyinza okuba nga kiva ku kweyongera kw’obutoffaali okutwalibwa kwa amino asidi zino enkulu oba okukozesa ensibuko endala (glucose oba lactic acid) okugatta ku nsengekera ya TCA (Figure S5B). PNs ezibulamu OXPHOS nazo zalaga okweyongera kw’okuvunda kwa glutamine n’emirimu gy’okutambuza omusaayi ku wiiki 8 ez’obukulu, ekiyinza okweyolekera mu kulongoosa enziyiza za mitochondrial glutaminase (GLS) ne glutamine pyruvate transaminase 2 (GPT2) (Ekifaananyi 3, A ne C). Kikulu okumanya nti up-regulation ya GLS ekoma ku spliced ​​isoform glutaminase C (GLS-GAC) (enkyukakyuka ya Mfn2cKO/CTRL eri nga 4.5-fold, P = 0.05), era up-regulation yaayo specific mu bitundu bya kookolo Asobola okuwagira mitochondrial bioenergy. (27) (27).
(A) Maapu y’ebbugumu eraga enkyukakyuka y’emirundi mu ddaala lya puloteyina ku kkubo eriragiddwa ku wiiki 8. (B) Ekyokulabirako ky’ekitundu ky’obwongo ekiwandiikiddwako ekiziyiza ekiziyiza PCx (scale bar, 20 μm). Akasaale aka kyenvu kalaga omubiri gw’obutoffaali bwa Purkinje. (C) Okwekenenya okwolesebwa kwa puloteyina mu kkoosi y’obudde okwazuulibwa ng’omuntu omukulu eyeesimbyewo ku kuzimba emisuwa (multiple t-test, *FDR <5%; n = 3-5 mice). (D) Waggulu: Ekifaananyi ekiraga engeri ez’enjawulo ez’okuyingira mu kaboni eyawandiikibwako ali mu [1-13C]pyruvate tracer (kwe kugamba, okuyita mu PDH oba trans-arterial route). Wansi: Ekipande kya violin kiraga ebitundu ku kikumi ebya kaboni (M1) alina akabonero kamu akyusiddwa okufuuka asidi wa aspartic, asidi wa citric ne asidi malic oluvannyuma lw’okuwandiika ebitundu by’obwongo eby’amaanyi ne [1-13C]pyruvate (paired t-test; ** P <0.01). (E) Okwekenenya ebyafaayo by’ebiseera ebijjuvu eby’ekkubo eriragiddwa. Lowooza ku puloteyina ezirina P<0.05 ku wiiki 8 zokka . Layini eriko enkokola: tewali muwendo gwa kulongoosa (okwekenneenya enjawulo mu ngeri bbiri; * P <0.05; *** P <0.001). Data ziragibwa nga mean±SEM.
Mu kwekenneenya kwaffe, BCAA catabolism efuuse emu ku makubo amakulu aga up-regulation. Ensonga eno eraga nnyo nti obuzito bw’empewo obuyingira mu nsengekera ya TCA buyinza okukyusibwa mu PN etaliimu OXPHOS. Kino kiyinza okukiikirira engeri enkulu ey’okusiba waya mu nkyukakyuka y’obusimu, ekiyinza okuba n’akakwate obutereevu ku nkola y’obusimu n’okuwangaala mu kiseera ky’okukuuma obutakola bulungi bwa OXPHOS. Okukwatagana n’endowooza eno, twakizuula nti enziyiza enkulu anti-atherosclerotic enzyme PCx is up-regulated (Mfn2cKO / CTRL enkyukakyuka nga 1.5 emirundi; Ekifaananyi 3A), ekitabula okukyusa pyruvate okudda mu oxaloacetate (28), ekiteeberezebwa okuba mu bwongo ebitundu The expression in is restricted to astrocytes (29, 30). Okukwatagana n’ebivudde mu proteomics, confocal microscopy yalaga nti PCx expression yali specifically era significantly okweyongera mu OXPHOS-deficient PNs, so PCx reactivity okusinga yali ekoma ku adjacent Bergmann glial cells of the control (Figure 3B). Okugezesa emirimu okugezesa okutunuulirwa upregulation ya PCx, twajjanjaba acute cerebellar slices ne [1-13C]pyruvate tracer. Pyruvate bwe yafuulibwa oxidized ne pyruvate dehydrogenase (PDH), isotope label yaayo yabula , Naye eyingizibwa mu TCA cycle intermediates nga pyruvate ekyusibwakyusibwa okuyita mu nkola z’emisuwa (Figure 3D). Mu kuwagira data yaffe eya proteomics, twalaba omuwendo omunene ogw’obubonero okuva mu tracer eno mu aspartic acid ya Mfn2cKO slices, ate nga citric acid ne malic acid nazo zaali zirina omuze ogw’ekigero, wadde nga si gwa maanyi (Figure 3D).
Mu busimu bwa dopamine obw’ebibe bya MitoPark n’obutakola bulungi bwa mitochondrial obuva ku busimu bwa dopamine obusaanyaawo mu ngeri ey’enjawulo obusimu bwa mitochondrial transcription factor A (Tfam) (Figure S6B), PCx expression nayo yali significantly up-regulated (31), ekiraga nti acetone acid arteriosclerosis Okubeerawo kw’obulwadde kulung’amibwa mu kiseera ky’obutakola bulungi bw’obusimu OXPHOS mu mubiri. Kinajjukirwa nti kizuuliddwa nti enziyiza ez’enjawulo (32-34) eziyinza okulagibwa mu busimu obuyitibwa neurons eziyinza okuba nga zikwatagana n’okuzimba emisuwa zitereera nnyo mu PNs ezibulamu OXPHOS, nga propionyl-CoA carboxylase (PCC-A), Malonyl-CoA ekyusa propionyl-CoA okufuuka succinyl-CoA ne mitochondrial malic 3 (ME3), . omulimu gwe omukulu kwe kuzzaawo pyruvate okuva mu malate (Ekifaananyi 3, A ne C) (33, 35). Okugatta ku ekyo, twazuula okweyongera okw’amaanyi mu enzyme ya Pdk3, ekola phosphorylates era bwetyo ne inactivate PDH ( 36 ), so nga tewali nkyukakyuka yazuulibwa mu enzyme ya Pdp1 ekola PDH oba PDH enzyme complex yennyini (Figure 3A). Mu ngeri ekwatagana, mu Mern2cKO PNs, phosphorylation ya α1 subunit α (PDHE1α) subunit ya pyruvate dehydrogenase E1 ekitundu kya PDH complex mu Ser293 (ekimanyiddwa okuziyiza enzyme activity ya PDH) yayongerwako ( Ekifaananyi S6C) (Figure S6C). Pyruvate terina misuwa gye giyingira.
Mu kusembayo, twakizuula nti ekkubo erya super erya serine ne glycine biosynthesis, enzirukanya ya mitochondrial folate (1C) ekwatagana ne proline biosynthesis (Figure 1G ne Figure S5C) byonna bikulu nnyo up-regulated, okusinziira ku lipoota, mu kiseera ky’enkola y’okukola. Ebitundu ebigyetoolodde bikola nga mitochondrial dysfunction (5-7). Okwekenenya kwa confocal okuwagira data zino proteomics kwalaga nti mu PN nga OXPHOS abuze, cerebellar slices z’ebibe ebya wiiki 8 zakolebwako serine hydroxymethyltransferase 2 (SHMT2), enzyme enkulu mu mitochondrial folate cycle. Okuddamu kw’abaserikale b’omubiri okw’amaanyi (Figure S5D). Mu 13 CU-glucose-incubated acute cerebellar slices, okugezesa okulondoola metabolic yayongera okukakasa up-regulation ya serine ne proline biosynthesis, ekiraga nti flux ya carbon isoforms mu serine ne proline okweyongera (Figure S5E). Okuva enkola ezitumbulwa GLS ne GPT2 bwe zivunaanyizibwa ku kusengejja glutamate okuva mu glutamine n’okutambuza wakati wa glutamate ne α-ketoglutarate, upregulation yaabwe eraga nti obusimu obutaliimu OXPHOS bulina obwetaavu obweyongedde obwa glutamate , Kino kiyinza okuba nga kigendereddwamu okukuuma biosynthesis eyeyongedde eya proline (Figure S5C). Okwawukana ku nkyukakyuka zino, okwekenneenya proteomic ya cerebellar astrocytes okuva mu PN-specific Mfn2cKO mice kwalaga nti amakubo gano (nga mw’otwalidde antiperoxidases zonna) tegaakyuka nnyo mu kwolesebwa, bwe kityo kiraga This metabolic redirection is selective to degraded PN (Fig. S6, D to G).
Mu bufunze, okwekenneenya kuno kwalaga enkola ez’enjawulo ennyo ez’okukola okw’ekiseera okw’amakubo ag’enjawulo ag’enkyukakyuka mu PNs. Newankubadde nga enkola y’obusimu obuyitibwa mitochondrial etali ya bulijjo eyinza okuvaako okuzimba emisuwa nga bukyali n’okuddamu okukola 1C (Figure 3E ne Figure S5C), era n’enkyukakyuka eziteeberezebwa mu kwolesebwa kwa I ne IV complexes, enkyukakyuka mu serine de novo synthesis zokka It only became apparent in the late stages. OXPHOS obutakola bulungi (Ekifaananyi 3E n’Ekifaananyi S5C). Ebizuuliddwa bino bitegeeza enkola ey’omuddiring’anwa nga situleesi ereetebwa mitochondrial (1C cycle) ne cytoplasmic (serine biosynthesis) ziddamu mu ngeri ey’okukwatagana n’okweyongera kw’okuzimba emisuwa mu TCA cycle okuddamu okubumba enkyukakyuka y’obusimu.
PNs ezibulamu OXPHOS eza wiiki 8 zisobola okukuuma omulimu gw’okusikirizibwa ogw’emirundi mingi era ne ziyita mu kuddamu okuyungibwa kw’enkyukakyuka y’emmere okw’amaanyi okusasula obutakola bulungi bwa mitochondria. Okuzuula kuno kuleeta ekisoboka ekinyuvu nti ne mu kiseera kino, obutoffaali buno era buyinza Okufuna okuyingira mu nsonga z’obujjanjabi okulwawo oba okuziyiza okuvunda kw’obusimu. Kikeerezi. Kino twakigonjoola nga tuyita mu nkola bbiri ezeetongodde. Mu nkola esooka, twakola dizayini ya Cre-dependent adeno-associated virus (AAV) vector MFN2 esobole okulonda okulagibwa mu OXPHOS-deficient PNs mu vivo (Figure S7A). AAV encoding MFN2 ne fluorescent reporter gene mCherry (Mfn2-AAV) zakakasibwa mu buwangwa bw’obusimu obusookerwako mu vitro, ekyaleetera MFN2 okulaga mu ngeri eyeesigama ku Cre era ne zitaasa enkula y’emitochondria, bwe kityo ne kiziyiza enkyukakyuka y’obusimu mu busimu bwa Mfn2cKO ( Ekifaananyi S7, B, D ne E). Ekiddako, twakola okugezesa mu vivo okutuusa mu ngeri ya stereotactically Mfn2-AAV eya wiiki 8 ku cerebellar cortex ya Mfn2cKO n’ebibe ebifuga, era ne twekenneenya ebibe ebya wiiki 12 ( Ekifaananyi 4A ). Ebibe ebya Mfn2cKO ebyajjanjabwa byafa (Ekifaananyi 1, A ne B) (16). Okukyusa akawuka mu vivo kwavaamu okulonda okwolesebwa kwa PN mu nkulungo ezimu ez’obwongo (Figure S7, G and H). Empiso ya control AAV eraga mCherry yokka (Ctrl-AAV) teyaliiko kinene kye yakola ku ddaala ly’okuvunda kw’obusimu mu bisolo bya Mfn2cKO. Okwawukana ku ekyo, okwekenneenya Mfn2cKOs ezikyusiddwa ne Mfn2-AAV kwalaga ekikolwa eky’obukuumi ekinene eky’oluwuzi lw’obutoffaali bwa PN (Ekifaananyi 4, B ne C). Okusingira ddala, obungi bw’obusimu obuyitibwa neuron density bulabika nga kumpi tebwawukana ku bisolo ebifuga (Ekifaananyi 4, B ne C, n’Ekifaananyi S7, H ne I). Okwolesebwa kwa MFN1 naye si MFN2 kukola kyenkanyi mu kutaasa okufa kw’obusimu (Ekifaananyi 4C n’ekifaananyi S7, C ne F), ekiraga nti okwolesebwa kwa MFN1 okw’ebweru w’olubuto kuyinza bulungi okwongera ku bbula lya MFN2. Okwongera okwekenneenya ku ddaala lya PN omu kwalaga nti Mfn2-AAV okusinga yanunula ultrastructure ya mitochondria, normalized mtDNA levels, era n’ezzaawo okwolesebwa okw’amaanyi okw’akabonero akalwanyisa angiogenesis PCx (Figure 4, C to E ). Okukebera n’amaaso ebibe bya Mfn2cKO ebyanunuddwa mu mbeera y’okuwummula kwalaga nti enyimirira yaabwe n’obubonero bw’enkola y’emirimu (okutambula S1 okutuuka ku S3) byalongooka. Mu kumaliriza, okugezesa kuno kulaga nti okulwawo okuddamu okuyingiza MFN2 mu PNs ezibula ennyo OXPHOS kimala okuzzaawo okukozesa mtDNA n’okuleeta okuzimba emisuwa, bwe kityo ne kiziyiza okuvunda kw’emisuwa n’okufa kw’obusimu mu vivo.
(A) Enteekateeka eraga enteekateeka y’okugezesa ey’okukuba empiso ya AAV eriko enkodi ya MFN2 ng’ekkubo ly’okukyusakyusa emmere eriragiddwa likoleddwa. (B) Ebifaananyi ebikiikirira confocal eby’ebitundu by’obwongo eby’emyaka 12 ebyakyusibwa n’ebibe bya Mfn2cKO ku wiiki 8 ne biwandiikibwako anti-Calbindin antibody. Ku ddyo: Okusika kw’obuwuzi bwa axon. Ekipimo kya axon zoom kiri 450 ne 75 μm. (C) Kkono: Okubala obungi bw’obutoffaali bwa Purkinje mu AAV transduction loop (AAV+) (okwekenneenya enjawulo mu ngeri emu; n = 3 mice). Ku ddyo: okwekenneenya okussa essira ku mtDNA mu PN eyakyusibwa ku wiiki 12 (unpaired t-test; n = 6 cells okuva mu mbeba ssatu). * P <0.05 nga bwe kiri; ** P <0.01. (D) Ebifaananyi ebitonotono eby’obusannyalazo eby’okutambuza ebikiikirira PNs z’ebitundu by’obwongo ebya Mfn2cKO ebikyusiddwa n’ebirungo ebiraga akawuka. Masiki eya pinki eraga ekitundu ekituuliddwamu dendrites, ate square eriko ennyiriri eza kyenvu eraga zoom eweereddwa ku ddyo; n ekiikirira ekitundu ekiyitibwa nucleus. Ebbaala ya minzaani, 1μm. (E) eraga ekyokulabirako ky’okusiiga kwa PCx mu PN eyakyusibwa ku wiiki 12. Ebbaala ya minzaani, 20μm. OE, okulaga ekisukkiridde; FC, okukyusa emirundi.
Mu kusembayo, twanoonyereza ku bukulu bw’okuwangaala kw’obutoffaali obuleetebwa peroxidase mu PNs ezifunye obuzibu mu kukola kwa OXPHOS. Twakola mCherry encoding AAV-shRNA (short hairpin RNA) specifically targeting mouse PCx mRNA (AAV-shPCx), era ne tufuyira akawuka oba scrambled control yaako (AAV-scr) mu cerebellum y’ebibe Mfn2cKO. Empiso yakolebwa mu wiiki ey’okuna ey’emyaka (Ekifaananyi 5A) okutuuka ku PCx knockdown ennungi mu kiseera nga PCx expression yeeyongedde (Figure 3C) era nga PN cell layer ekyali intact (Figure 1A). Kinajjukirwa nti okukoona wansi PCx (Ekifaananyi S8A) kiviirako okwanguya okw’amaanyi okw’okufa kwa PN, nga eno ye mpeta yokka ekugirwa ey’okukwatibwa (Ekifaananyi 5, B ne C). Okusobola okutegeera enkola y’ebikolwa by’okukyusakyusa ebiriisa ebireetebwa PCx up-regulation, twasoma embeera ya redox eya PNs oluvannyuma lwa PCx knockdown ne AAV-mediated optical biosensor Grx1-roGFP2 byalagibwa omulundi gumu (Figure S8, B to D) okwekenneenya glutathione Enkyukakyuka ey’enjawulo ey’obusobozi bwa peptide redox (38). Awo, twakola two-photon fluorescence lifetime imaging microscopy (FLIM) mu acute obwongo slices 7-week-old Mfn2cKO oba control littermates okuzuula enkyukakyuka eziyinza okubaawo mu cytoplasmic redox status oluvannyuma lw’okukakasa FLIM embeera (Figure S8, E to G). Okwekenenya kwalaga okweyongera okw’amaanyi mu mbeera y’okwokya (oxidation state) ya Mfn2cKO PNs emu ezibulamu PCx expression, eky’enjawulo ku control neurons oba Mfn2cKO PNs eziraga scrambled shRNA yokka (Figure 5, D ne E). Bwe PCx expression yali down-regulated, ebitundu ku kikumi ebya Mfn2cKO PNs okulaga embeera oxidized ennyo byeyongera emirundi egisukka mu esatu (Figure 5E), ekiraga nti PCx up-regulation yakuuma obusobozi redox obwa degenerated neurons.
(A) Enteekateeka eraga enteekateeka y’okugezesa ey’okukuba empiso ya AAV eriko enkodi ya shPCx ng’ekkubo ly’okukyusakyusa emmere eriragiddwa likoleddwa. (B) Ebifaananyi ebikiikirira confocal ebitundu by’obwongo ebya wiiki 8 mu mbeba za Mfn2cKO ezikyusiddwa era ne ziwandiikibwako anti-calcineurin antibody ku wiiki 4. Ebbaala ya minzaani, 450μm. (C) Okugera obungi bw’obutoffaali bwa Purkinje mu loopu ezikyusiddwa AAV (okwekenneenya enjawulo okw’engeri emu; n = mbeba 3 ku 4). Data ziragibwa nga mean±SEM; ***P<0.001 nga bano. (D) Ekifaananyi ekikiikirira FLIM kiraga obulamu bwa wakati obwa PN ow’emyaka 7 alaga glutathione redox sensor Grx1-roGFP2 wansi w’embeera z’okugezesa eziragiddwa. LUT (emmeeza etunula waggulu) omugerageranyo: ekiseera ky’okuwangaala (mu picoseconds). Ebbaala ya minzaani, 25μm. (E) Histogram eraga ensaasaanya y’emiwendo gy’obulamu bwa Grx1-roGFP2 okuva ku (D) (n=158 okutuuka ku 368 obutoffaali mu mbeba bbiri wansi wa buli mbeera). Ekipande kya paayi waggulu wa buli histogram: kiraga omuwendo gw’obutoffaali obulina emiwendo gy’obulamu obuwanvu ennyo (emmyufu, oxidized) oba obumpi (bbululu, obukendeezeddwa), obusukka 1 SD ku muwendo gw’obulamu obwa wakati mu CTRL-AAV-scr. (F) Omuze oguteeseddwa gulaga ekikolwa eky’obukuumi eky’okulinnyisa omutindo gw’obusimu PCx.
Byonna awamu, data gye tuwa wano eraga nti okuddamu okwolesebwa kwa MFN2 kusobola okununula ddala PN ey’omulembe n’obutaba na OXPHOS okw’amaanyi, okukendeera kwa mtDNA okw’amaanyi, n’enkula eringa ista etali ya bulijjo ennyo, bwe kityo ne kiwa enkulaakulana egenda mu maaso ne mu ndwadde ezikuze. Okuvunda kw’obusimu kuwa obujulizi obukyusibwakyusibwa ku mutendera nga obutoffaali tebunnafa. Diguli eno ey’okukyukakyuka mu nkyukakyuka y’emmere eyongera okuggumiza obusobozi bw’obusimu obuyitibwa neurons okuleeta atherosclerosis (a rewiring of the TCA cycle), ekiziyiza PCx expression mu PNs ezibulamu OXPHOS era ne kyongera ku kufa kw’obutoffaali, bwe kityo ne kizannya ekifo eky’obukuumi (Figure 5F).
Mu kunoonyereza kuno, twawa obujulizi nti okuddamu kwa PNs eri OXPHOS dysfunction kwe kukwatagana mpolampola ku TCA cycle atherosclerosis okuyita mu differential activation pathway activated by metabolic programs. Twakakasa okwekenneenya kwa proteomic n’enkola nnyingi ezijjuliza era ne tuzuula nti bwe tusoomoozebwa obutakola bulungi bwa mitochondrial, obusimu obuyitibwa neurons bulina engeri etamanyiddwa emabegako eya metabolic elasticity. Ekyewuunyisa, enkola yonna ey’okusiba waya tekitegeeza nti embeera y’okukyusakyusa emmere ey’enkomerero ewerekera okuvunda kw’obusimu mpolampola era mu ngeri etakyuka, naye data yaffe eraga nti eyinza okukola obusimu obuddaabiriza ne mu mutendera nga obutoffaali tebunnafa Enkola y’okuliyirira emirimu. Kino ekizuuliddwa kiraga nti obusimu obuyitibwa neurons bulina ekigero ekinene eky’obuveera bw’enkyukakyuka mu mubiri. Ensonga eno ekakasa nti okuddamu okuyingiza MFN2 oluvannyuma kuyinza okuzzaawo okwolesebwa kw’obubonero obukulu obw’enkyukakyuka y’emmere n’okuziyiza okuvunda kwa PN. Okwawukana ku ekyo, kiziyiza okuzimba emisuwa n’okwanguyiza obusimu. transsexual.
Ekimu ku bisinga okusikiriza mu kunoonyereza kwaffe kwe kuba nti PNs ezibulamu OXPHOS zisobola okukyusa TCA cycle metabolism nga up-regulating enzymes that specifically stimulate arteriosclerosis. Okuddamu okusengeka metabolic kintu kya bulijjo mu butoffaali bwa kookolo, ebimu ku byo byesigamye ku glutamine okugatta ku TCA cycle intermediates okukola reducing equivalents, ezivuga olujegere lw’okussa n’okukuuma okukola lipid ne nucleotide biosynthesis precursors (39 , 40). Okunoonyereza okwakakolebwa kwalaga nti mu bitundu by’omubiri eby’okumpi ebifuna obutakola bulungi bwa OXPHOS, okuddamu okuyungibwa kw’enkyukakyuka ya glutamine/glutamate nakyo kintu kimanyiddwa nnyo ( 5 , 41 ), ng’obulagirizi bw’okuyingira kwa glutamine mu nsengekera ya TCA kwesigamye ku Olw’obuzibu bw’obuvune bwa OXPHOS ( 41 ). ). Naye, tewali bujulizi bulambulukufu ku kufaanagana kwonna okw’obuveera bw’enkyukakyuka y’obusimu mu mubiri n’obukulu bwayo obusoboka mu mbeera y’obulwadde. Mu kunoonyereza okwakakolebwa mu vitro, obusimu obusookerwako obw’omubiri (primary cortical neurons) bwalagibwa okukungaanya ebidiba bya glutamate okutambuza obusimu, bwe kityo ne kitumbula oxidative metabolism ne atherosclerosis wansi w’embeera z’okunyigirizibwa kw’enkyukakyuka mu mubiri ( 42 ). Kikulu okumanya nti wansi w’okuziyiza eddagala kwa TCA cycle enzyme succinate dehydrogenase, pyruvate carboxylation kirowoozebwa nti ekuuma okusengejja oxaloacetate mu cultured cerebellar granule neurons ( 34 ). Naye, obukulu bw’enkola zino mu mubiri ku bitundu by’obwongo (nga obulwadde bw’emisuwa bulowoozebwa nti okusinga bukoma ku astrocytes) bukyalina amakulu amakulu mu mubiri ( 43 ). Mu mbeera eno, data yaffe eraga nti PNs ezonoonese OXPHOS mu mubiri zisobola okukyusibwa okudda mu BCAA degradation ne pyruvate carboxylation, nga zino ze nsibuko ebbiri enkulu ez’okugatta TCA pool intermediates. Newankubadde nga ekigambibwa nti BCAA catabolism ekikola ku neuronal energy metabolism kiteeseddwa, ng’oggyeeko omulimu gwa glutamate ne GABA ku neurotransmission ( 44 ), n’okutuusa kati tewali bujulizi bwonna ku nkola zino mu vivo. N’olwekyo, kyangu okuteebereza nti PN ezitakola bulungi zisobola okusasula mu ngeri ey’otoma olw’okukozesa ebirungo eby’omu makkati ebya TCA ebivugibwa enkola y’okugatta nga zongera ku kuzimba emisuwa. Okusingira ddala, upregulation ya PCx eyinza okwetaagisa okukuuma obwetaavu obweyongedde obwa aspartic acid, ekiragiddwa mu butoffaali obweyongera n’obutakola bulungi bwa mitochondrial ( 45 ). Wabula, okwekenneenya kwaffe metabolomics tekwalaga nkyukakyuka yonna nnene mu steady-state level ya aspartic acid mu Mfn2cKO PNs (Figure S6A), ekiteeberezebwa nti kiraga enjawulo metabolic enkozesa ya aspartic acid wakati proliferating cells ne post-mitotic neurons . Newankubadde enkola entuufu eya PCx upregulation mu dysfunctional neurons mu vivo ekyalina okumanyibwa, twalaga nti kino premature response ekola kinene mu kukuuma redox embeera ya neurons, ekyalagibwa mu FLIM experiments ku cerebellar slices. Okusingira ddala, okuziyiza PNs oku up-regulating PCx kiyinza okuvaako embeera esingako oxidized n’okwanguyiza okufa kw’obutoffaali. Okukola kw’okuvunda kwa BCAA n’okufuuka carboxylation ya pyruvate si ngeri ya kulaga bitundu by’omubiri eby’okumpi eby’obutakola bulungi bwa mitochondrial ( 7 ). N’olwekyo, zirabika nga kye kintu ekikulu eky’obusimu obutaliimu OXPHOS, ne bwe kiba nga si kye kintu kyokka, ekikulu mu kuvunda kw’obusimu. .
Obulwadde bwa cerebellar kika kya bulwadde bwa neurodegenerative ekitera okweyoleka nga ataxia era nga kitera okwonoona PNs ( 46 ). Omuwendo guno ogw’obusimu naddala guli mu bulabe bw’obutakola bulungi bwa mitochondrial kubanga okuvunda kwazo okulonda mu mbeba kumala okuzaala obubonero bungi obw’enkola y’omubiri obulaga obulwadde bwa spinocerebellar ataxia bw’omuntu ( 16 , 47 , 48 ). Okusinziira ku lipoota, ekifaananyi ky’ebibe ekikyusiddwa obuzaale n’obuzaale obukyusiddwa kikwatagana n’okutaataaganyizibwa kw’omugongo gw’omuntu era kirina obutakola bulungi bwa mitochondrial ( 49 , 50 ), nga kiggumiza obukulu bw’okunoonyereza ku biva mu bbula lya OXPHOS mu PNPH. N’olwekyo, kirungi nnyo okwawula obulungi n’okunoonyereza ku bungi bw’obusimu buno obw’enjawulo. Naye, okusinziira ku kuba nti PNs ziwulikika nnyo ku puleesa era nga zikola ekitundu ekitono ku bungi bw’obutoffaali bw’obwongo bwonna, ku kunoonyereza kungi okwesigamiziddwa ku omics, okwawula okulondako kwazo ng’obutoffaali bwonna kukyali nsonga esoomooza. Newankubadde nga kumpi tekisoboka kutuuka ku butabeera na bucaafu bwonna obw’ebika by’obutoffaali ebirala (naddala ebitundu by’omubiri eby’abantu abakulu), twagatta omutendera omulungi ogw’okukutuka ne FACS okufuna omuwendo ogumala ogw’obusimu obusobola okukola wansi okwekenneenya proteomics, era nga tulina Quite high protein coverage (nga 3000 proteins) bw’ogeraageranya ne data set eriwo ey’obwongo bwonna (51). Nga tukuuma obulamu bw’obutoffaali bwonna, enkola gye tuwa wano tetusobozesa kukebera nkyukakyuka mu makubo g’enkyukakyuka mu mitochondria zokka, naye era n’okukebera enkyukakyuka mu bannaabwe aba cytoplasmic, ekijjuliza enkozesa y’okugaggawaza akabonero k’oluwuzi lw’emitochondrial specific to cell types Enkola empya ey’omuwendo gwa mitochondria mu bitundu ebizibu (52, 53). Enkola gye twogerako tekwatagana na kunoonyereza ku butoffaali bwa Purkinje bwokka, naye esobola bulungi okukozesebwa ku kika kyonna eky’obutoffaali okukola ku nkyukakyuka mu nkyukakyuka mu bwongo obulwadde, omuli n’ebikolwa ebirala eby’obutakola bulungi bwa mitochondria.
Mu kusembayo, tuzudde eddirisa ly’obujjanjabi mu kiseera kino eky’okuddamu okutegeka enkyukakyuka y’emmere eriyinza okuzzaawo ddala obubonero obukulu obw’okunyigirizibwa kw’obutoffaali n’okuziyiza okuvunda kw’obusimu. Nolwekyo, okutegeera ebiva mu nkola y’okusiba waya ebyogeddwako wano kiyinza okuwa amagezi amakulu ku bujjanjabi obusoboka obw’okukuuma obulamu bw’obusimu mu kiseera ky’obutakola bulungi bwa mitochondria. Okunoonyereza mu biseera eby’omu maaso okugendereddwamu okusalasala enkyukakyuka mu nkyukakyuka y’amasoboza mu bika by’obutoffaali bw’obwongo ebirala kwetaagibwa okulaga mu bujjuvu okukozesebwa kw’omusingi guno ku ndwadde endala ez’obusimu.
MitoPark mice zaayogerwako emabegako ( 31 ). C57BL/6N mice with loxP flanking Mfn2 genes zaayogerwako emabegako ( 18 ) era ne zisalagana n’ebibe L7-Cre ( 23 ). Olwo ezzadde eryavaamu ery’emirundi ebiri erya heterozygous lyasalasala n’ebibe bya homozygous Mfn2loxP/Mfn2loxP okukola Purkinje-specific gene knockouts for Mfn2 (Mfn2loxP/Mfn2loxP; L7-cre). Mu kibinja ekitono eky’okugatta, Gt (ROSA26) SorStop-mito-YFP allele (stop-mtYFP) yayingizibwa okuyita mu kusalaba okw’enjawulo ( 20 ). Enkola zonna ez’ebisolo zakolebwa nga zigoberera ebiragiro by’Abazungu, eby’eggwanga n’ebitongole era nga zikkirizibwa LandesamtfürNatur ow’e Umwelt ne Verbraucherschutz, North Rhine-Westphalia, Germany. Omulimu gw’ebisolo nagwo gugoberera obulagirizi bw’ekibiina kya European Federation of Laboratory Animal Sciences Associations.
Oluvannyuma lw’okubudamya omukyala ow’olubuto okusesema kw’omumwa gwa nnabaana, embuto y’ekibe yawulwamu (E13). Ekitundu ky’omubiri kyasalibwamu mu Hanks’ Balanced Salt Solution (HBSS) nga kigattibwako 10 mM Hepes ne kiyisibwa ku Dulbecco’s Modified Eagle’s Medium nga mulimu papain (20 U/ml) ne cysteine ​​(1μg/ml). Fumbira ekitundu mu DMEM) era okikutule nga oyita mu kugaaya enziyiza. Ml) ku 37°C okumala eddakiika 20, n’oluvannyuma n’asiigibwa mu ngeri ey’ebyuma mu DMEM ng’eyongeddeko 10% fetal bovine serum. Obutoffaali bwasimbibwa ku bibikka endabirwamu ebisiigiddwako polylysine ku density ya 2×106 buli 6 cm culture dish oba ku density ya 0.5×105 cells/cm2 okusobola okwekenneenya ebifaananyi. Oluvannyuma lw’essaawa 4, ekirungo kino kyakyusibwa ne kifuulibwa ekirungo ekitaliimu serum ekya Neurobasal nga kirimu 1% B27 supplement ne 0.5 mM GlutaMax. Olwo obusimu obuyitibwa neurons bwakuumibwa ku 37°C ne 5% CO2 mu kugezesa kwonna, era ne buliisibwa omulundi gumu mu wiiki. Okusobola okuleeta okugatta okuddamu mu vitro, 3μl (24-well culture dish) oba 0.5μl (24-well plate) ya AAV9 virus vector eno wammanga yakozesebwa okujjanjaba obusimu ku lunaku olwokubiri mu vitro: AAV9.CMV.PI.eGFP. WPRE.bGH (Addgene, nnamba ya katalogu 105530-AAV9) ne AAV9.CMV.HI.eGFP-Cre.WPRE.SV40 (Addgene, nnamba ya katalogu 105545-AAV9).
Mouse Mfn1 ne Mfn2 complementary DNA (efunibwa okuva mu Addgene plasmid #23212 ne #23213, respectively) ziteekebwako akabonero n’omutendera gwa V5 (GKPIPNPLLGLDST) ku C-terminus, era ziyungibwa ne mCherry mu frame okuyita mu T2A sequence. Grx1-roGFP2 kirabo okuva mu kkampuni ya Heidelberg T. P. Dick DFKZ (Deutsches Krebsforschungszentrum). Nga tukyusa kaseti ya tdTomato nga tukozesa enkola eza bulijjo ez’okukoppa, kaseti yakyusibwa mu mugongo gwa pAAV-CAG-FLEX-tdTomato (Addgene reference number 28306) okukola pAAV-CAG-FLEX-mCherry-T2A-MFN2-V5, pAAV-CAG- FLEX-mCherry-T2A-MFN1-V5 ne pAAV-CAG-FLEX-Grx-roGFP2 ebiwuka ebiziyiza endwadde. Enkola efaananako bwetyo yakozesebwa okukola ekirungo ekifuga ekirungo kya pAAV-CAG-FLEX-mCherry. Okusobola okukola AAV-shPCx construct, plasmid AAV vector (VectorBuilder, pAAV [shRNA] -CMV-mCherry-U6-mPcx- [shRNA#1]) yeetaagibwa, erimu omutendera gwa DNA oguwandiika enkodi ya shRNA egenderera PCx y’ebibe (5′CTTTCGCTCTAAGGTGCTAAACTCGAGTTTAGCACCTTAGAGCGAAAG 3′) Wansi w’okufugibwa promoter ya U6, mCherry ekozesebwa wansi w’okufugibwa promoter ya CMV. Okukola ebirungo ebiyamba AAV vectors kwakolebwa okusinziira ku biragiro by’abakola (Cell Biolabs). Mu bufunze, kozesa plasmid y’okukyusa etisse mCherry-T2A-MFN2-V5 (pAAV-CAG-FLEX-mCherry-T2A-MFN2-V5), mCherry-T2A-MFN1-V5 (pAAV-CAG-FLEX-mCherry) mu kiseera ekitono Okukyusa obutoffaali 293AAV-T2A-MFN1-V5), mCherry (pAAV-CAG-FLEX-mCherry) oba Grx-roGFP2 (pAAV-CAG-FLEX-Grx-roGFP2) ekola enkoodi y’obuzaale, wamu n’okuwandiika enkoodi ya puloteyina ya AAV1 capsid ne puloteyina ey’obuyambi Packaging plasmid plasmid, nga tukozesa enkola ya calcium phosphate. Ekisengejjero ky’akawuka ekitali kirongoofu kyafunibwa nga tuyita mu nsengekera za freeze-thaw mu kinaabiro kya ice/ethanol omukalu n’obutoffaali obusengekeddwa mu phosphate buffered saline (PBS). Ekirungo kya AAV kyalongoosebwa nga kiyita mu nkola ya discontinuous iodixanol gradient ultracentrifugation (essaawa 24 ku 32,000 rpm ne 4°C) ne kisengejebwa nga tukozesa Amicon ultra-15 centrifugal filter. Genome titer ya AAV1-CAG-FLEX-mCherry-T2A-MFN2-V5 [2.9×1013 genome copy (GC)/ml], AAV1-CAG-FLEX-mCherry (6.1×1012 GC/ml), AAV1-CAG- FLEX yali nga bwe kyayogerwako emabegako (54), eyapimibwa mu kiseera ekituufu quantitative PCR (qPCR) -MFN1-V5 (1.9×1013 GC/ml) ne AAV1-CAG-FLEX-Grx-roGFP2 (8.9×1012 GC/ml).
Obusimu obusookerwako bwasekulwa mu 1x PBS ennyogovu nga ice, ne bufuulibwa obukuta, n’oluvannyuma ne bufuuka homogenized mu 0.5% Triton X-100 / 0.5% sodium deoxycholate/PBS lysis buffer erimu phosphatase ne protease inhibitor (Roche). Okugera obungi bwa puloteyina kwakolebwa nga tukozesa enkola ya bicinchoninic acid assay (Thermo Fisher Scientific). Oluvannyuma puloteyina zaawulwamu nga zikozesa enkola ya SDS-polyacrylamide gel electrophoresis, oluvannyuma ne zifuumuulwa ku luwuzi lwa polyvinylidene fluoride (GE Healthcare). Zibira ebifo ebitali bitongole era ofumbe n’ekiziyiza ekisookerwako (laba ekipande S1 okumanya ebisingawo) mu mata 5% mu TBST (Tris-buffered saline with Tween), emitendera gy’okunaaba n’ekiziyiza eky’okubiri mu TBST Incubate. Fumbira n’ekiziyiza ekisookerwako okumala ekiro ku +4°C. Oluvannyuma lw’okunaaba, ssaako ekiziyiza eky’okubiri okumala essaawa 2 ku bbugumu erya bulijjo. Oluvannyuma, nga bafumbiza blot y’emu n’ekirungo ekiziyiza β-actin, okutikka kwe kumu kwakakasibwa. Okuzuula nga tukyusa okudda mu chemiluminescence n’okutumbula chemiluminescence (GE Healthcare).
Obusimu obuyitibwa neurons obwasooka okusimbibwa ku bibikka ku ndabirwamu bwassibwa ne 4% paraformaldehyde (PFA)/PBS mu kiseera ekiragiddwa ku bbugumu erya bulijjo okumala eddakiika 10. Ebibikka bisooka kuyingizibwamu 0.1% Triton X-100/PBS okumala eddakiika 5 ku bbugumu erya bulijjo, n’oluvannyuma mu blocking buffer [3% bovine serum albumin (BSA)/PBS]. Ku lunaku olwokubiri, ebibikka byanaazibwa ne blocking buffer ne bifumbirwa n’ekirungo ekituufu ekiziyiza obulwadde bwa fluorophore-conjugated secondary antibody okumala essaawa 2 ku bbugumu erya bulijjo; ku nkomerero, sampuli zaanaazibwa bulungi mu PBS nga 4′,6-diamidino-2 -Phenylindole (DAPI) is counterstained n’oluvannyuma n’enyweza ku microscope slide ne Aqua-Poly/Mount.
Ebibe (ebisajja n’ebikazi) byabuzibwabuzibwa nga bifuyira ketamine (130 mg/kg) ne xylazine (10 mg/kg) mu lubuto ne biweebwa wansi w’olususu n’eddagala eriweweeza ku bulumi erya carprofen (5 mg/kg) , Ne biteekebwa mu kyuma ekiziyiza obulumi (Kopf) nga kirimu paadi ebuguma. Okwolese ekiwanga era okozese ekyuma ekisima amannyo okugonza ekitundu ky’ekitundu ky’omubiri ekiyitibwa cerebellar cortex ekikwatagana n’eggumba erya mis (okuva mu lambda: omukira 1.8, lateral 1, ogukwatagana ne lobules IV ne V). Kozesa empiso y’empiso eriko enkokola okukola n’obwegendereza ekituli ekitono mu kiwanga okwewala okutaataaganya emisuwa wansi. Olwo omusuwa omugonvu ogw’endabirwamu ogukubiddwa guyingizibwa mpola mu kinnya ekitono (okuva ku -1.3 okutuuka ku -1 ku ludda lw’omu lubuto olwa dura mater), era AAV 200 okutuuka ku 300 nl efukibwa mu micro-injector (Narishige) n’empiso z’omu ngalo (Narishige) emirundi egiwerako ku puleesa entono mu bbanga ery’eddirisa ery’eddakiika 10 ku 20. Oluvannyuma lw’okufuyira, teeka emisuwa okumala eddakiika endala 10 okusobozesa akawuka okusaasaana ddala. Oluvannyuma lw’okuggyibwako emisuwa, olususu lutungibwa n’obwegendereza okukendeeza ku kuzimba kw’ebiwundu n’okusobozesa ekisolo okudda engulu. Ebisolo byajjanjabwa n’eddagala eriweweeza ku bulumi (caspofen) okumala ennaku eziwera oluvannyuma lw’okulongoosebwa, mu kiseera ekyo embeera y’omubiri gwabyo yalondoolebwa n’obwegendereza oluvannyuma ne bittibwa mu kiseera ekyalagirwa. Emitendera gyonna gyakolebwa nga gigoberera ebiragiro by’Abazungu, eby’eggwanga n’ebitongole era nga byakkirizibwa LandesamtfürNatur ow’e Umwelt ne Verbraucherschutz, North Rhine-Westphalia, Germany.
Ebisolo byabuzibwabuzibwa ne ketamine (100 mg/kg) ne xylazine (10 mg/kg), era omutima ne gusooka kufukibwako 0.1 M PBS, n’oluvannyuma ne 4% PFA mu PBS. Ekitundu kyasalibwamu ne kiteekebwa mu 4% PFA/PBS okumala ekiro ku 4°C. Ekiso ekikankana (Leica Microsystems GmbH, Vienna, Austria) kyakozesebwa okuteekateeka ebitundu bya sagittal (50 μm obuwanvu) okuva mu bwongo obutakyukakyuka mu PBS. Okuggyako nga kirambikiddwa bulala, okusiiga ebitundu ebitengejja mu ddembe kwakolebwa nga bwe kyayogerwako waggulu (13) ku bbugumu erya bulijjo n’okutabula. Mu bufunze, okusooka, ebitundu ebifunibwa byayisibwa ne 0.5% Triton X-100/PBS okumala eddakiika 15 ku bbugumu erya bulijjo; ku epitopes ezimu (Pcx ne Shmt2), nga mu tris-EDTA buffer ku 80°C (PH 9) bbugumya slices okumala eddakiika 25 mu kifo ky’omutendera guno. Ekiddako, ebitundu byafumbirwa n’ekiziyiza ekisookerwako (laba Table S1) mu buffer eziyiza (3% BSA/PBS) ku 4°C okumala ekiro nga bitabuddwa. Enkeera, ebitundu byanaazibwa ne blocking buffer ne bifumbirwa n’ekirungo ekituufu ekiziyiza obulwadde bwa fluorophore-conjugated secondary antibody okumala essaawa 2 ku bbugumu erya bulijjo; ku nkomerero, ebitundu byanaazibwa bulungi mu PBS, ne biddamu okusiiga langi ya DAPI, oluvannyuma ne bitereezebwa ne AquaPolymount Ku slayidi ya microscope.
Laser scanning confocal microscope (TCS SP8-X oba TCS Digital Light Sheet, Leica Microsystems) nga eriko layisi y’ekitangaala ekyeru ne layisi ya 405 diode ultraviolet yakozesebwa okukuba ekifaananyi kya sampuli. Nga bacamula fluorophore n’okukung’aanya siginiini ne Hybrid Detector (HyDs), pulogulaamu ya LAS-X yakozesebwa okukung’aanya ebifaananyi ebitumbiddwa ebikwatagana n’okutwala sampuli za Nyquist mu ngeri ey’omuddiring’anwa: ku bipande ebitali bya bungi, biba siginiini ezikyukakyuka ennyo (okugeza, mu butoffaali bw’omubiri ne dendrites) mtYFP) Kozesa HyD okuzuula omuwendo gwa PNs mu mbeera ya BrightR). Gating ya 0.3 ku 6 ns ekozesebwa okukendeeza ku background.
Okukuba ebifaananyi mu kiseera ekituufu ku butoffaali obusunsuddwa. Oluvannyuma lw’okusunsula mu Neurobasal-A medium erimu 1% B27 supplement ne 0.5 mM GlutaMax, obutoffaali bwasimbibwa mangu ku poly-l-lysine-coated glass slides (μ-Slide8 Well, Ibidi, catalog number 80826) , N’oluvannyuma ne bagikuuma ku 37°C ne 5% CO2 okumala essaawa 1 okusobozesa obutoffaali okusenga. Okukuba ebifaananyi mu kiseera ekituufu kwakolebwa ku Leica SP8 laser scanning confocal microscope nga eriko layisi enjeru, HyD, 63×[1.4 numerical aperture (NA)] oil objective lens ne heating stage.
Ekibe kyabuzibwabuzibwa mangu ne kaboni dayokisayidi ne kisalibwako omutwe, obwongo ne buggyibwa mangu ku kiwanga, ne busalibwamu ekitundu kya sagittal obuwanvu 200μm (ku 13C labeling experiment) oba 275μm obuwanvu (ku kugezesa kwa photon okubiri) nga kijjudde ebintu bino wammanga The ice cream (HM-650 V, Thermo Fisher Scientific, Walldorf, Germany) ejjula ebintu bino wammanga: 125 mM ebinyogovu nga ice, ebijjudde kaboni (95% O2 ne 5% CO2) Ca2 + entono + amazzi ag’ekikugu ag’omu bwongo (ACSF) NaCl, 2.5 mM KCl, 1.25 mM sodium phosphate buffer, 25 mM NaHCO3, 25 mM glucose, 0.5 mM CaCl2 ne 3.5 mM MgCl2 (puleesa y’okuwuuma eya 310 okutuuka ku 330 mmol). Teeka ebitundu by’obwongo ebifunibwa mu kisenge nga tonnafumbirwa nga mulimu Ca2 + ACSF esingako (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 ne 2.0 mM MgCl2) Medium) pH 7.4 ne 310 okutuuka ku 320 mmol).
Mu nkola y’okukuba ebifaananyi, ebitundu byasengulwa mu kisenge ekyetongodde eky’okukuba ebifaananyi, era okugezesa kwakolebwa wansi w’okufuyira kwa ACSF okutambula obutasalako ku bbugumu eritakyukakyuka erya 32° okutuuka ku 33°C. Microscope ya multiphoton laser scanning microscope (TCS SP8 MP-OPO, Leica Microsystems) eriko Leica 25x objective lens (NA 0.95, amazzi), Ti: Sapphire laser (Chameleon Vision II, Coherent) yakozesebwa okukuba ebifaananyi eby’ebitundutundu. Module ya FLIM (PicoHarp300, PicoQuant) nga bwe kiri.
FLIM ya Grx1-roGFP2. Enkyukakyuka mu mbeera ya cytoplasmic redox eya PNs zapimibwa n’ebirungo bibiri-photon FLIM mu sagittal brain slices, nga Grx1-roGFP2 biosensor egenderera PNs. Mu layeri ya PN, ekifo ky’okufuna kirondebwa nga 50 ku 80 μm wansi w’olutimbe lw’ekitundu okukakasa nti waliwo PN esobola okukolebwa (kwe kugamba, obutaba na nsengekera ya bimuli oba enkyukakyuka mu nkula y’obusimu ku dendrites) ne sensa ya roGFP2 ey’emirundi ebiri ennungi ne AAV encoding shRNA PCx oba omutendera gwayo ogw’okufuga (buli wamu alaga mCherry). Kuŋŋaanya ebifaananyi eby’omutambi gumu nga olina 2x digital zoom [obuwanvu bw’amayengo ag’okusikirizibwa: 890 nm; 512 nm 512 ppikisi]. Okuzuula: HyD ey’omunda, ekibinja ky’omusengejja ekya fluorescein isothiocyanate (FITC)] n’okugerageranya ebifaananyi mu ddakiika 2 ku 3 bikozesebwa okukakasa nti obutangaavu obumala bukuŋŋaanyizibwa (obutangaavu 1000 bwonna awamu) okusobola okutuuka ku kikoona. Obuwulize bwa Grx1-roGFP2 probe n’okukakasa embeera za FLIM byakolebwa nga balondoola omuwendo gw’obulamu bwa roGFP2 nga bongerako exogenous 10 mM H2O2 ku perfusion ACSF (okulinnyisa oxidation, ekivaamu okweyongera obulamu), n’oluvannyuma okugattako 2 mM dithiothreitol (okukendeeza ku ddaala ly’okukendeeza, ekivaamu okukendeera mu bulamu) (Ekifaananyi S8, D okutuuka ku G). Kozesa pulogulaamu ya FLIMfit 5.1.1 okwekenneenya ebivuddemu ebifunibwa, okutuukanya ekipimo ky’okuvunda okw’ekigerageranyo ekimu eky’ekifaananyi kyonna ku IRF epimiddwa (omulimu gw’okuddamu kw’ebikozesebwa), era χ2 eri nga 1. Okubala obulamu bwa PN emu, masiki eyeetoolodde omubiri gw’obusimu yakubiddwa mu ngalo, era obulamu obwa wakati mu buli masiki bwakozesebwa okupima obungi.
Okwekenenya obusobozi bwa mitochondrial. Oluvannyuma lw’ekitundu eky’amangu okufumbirwa ne 100 nM TMRM eyagattibwa butereevu mu ACSF eyafukibwa okumala eddakiika 30, enkyukakyuka z’obusobozi bwa mitochondrial eza PNs zapimibwa n’ekipima eky’obutafaali obubiri. Okukuba ebifaananyi mu TMRM kwakolebwa nga bacamula probe ku 920 nm era nga bakozesa HyD ey’omunda (tetramethylrhodamine isothiocyanate: 585/40 nm) okukung’aanya obubonero; nga tukozesa obuwanvu bw’amayengo ag’okusikirizibwa bwe bumu naye nga tukozesa HyD ey’omunda ey’enjawulo (FITC :525/50) okukuba ekifaananyi kya mtYFP. Kozesa ImageJ's Image Calculator plug-in okwekenneenya obusobozi bwa mitochondrial ku ddaala ly'obutoffaali obumu. Mu bufunze, ensengekera ya plug-in: signal = min (mtYFP, TMRM) ekozesebwa okuzuula ekitundu kya mitochondrial ekiraga akabonero ka TMRM mu Purkinje Somali mu kifaananyi kya confocal ekya single-stack confocal eky’omukutu ogukwatagana. Olwo ekitundu kya pikseli mu masiki evuddemu kipimibwa mu bungi, n’oluvannyuma ne kifuuka kya bulijjo ku kifaananyi ekikwatagana eky’omusingi ogw’omutendera gumu ogw’omukutu gwa mtYFP okufuna ekitundu kya mitochondrial ekiraga obusobozi bwa mitochondrial.
Ekifaananyi kino kyalongoosebwamu nga bakozesa pulogulaamu ya Huygens Pro (Scientific Volume Imaging). Ku bifaananyi ebya sikaani ebya tile, montage ya tile emu ekolebwa nga tukozesa enkola ya automatic stitching algorithm ewereddwa pulogulaamu ya LAS-X. Oluvannyuma lw’okupima ekifaananyi, kozesa ImageJ ne Adobe Photoshop okwongera okukola ku kifaananyi n’okutereeza mu ngeri y’emu okwakaayakana n’enjawulo. Kozesa Adobe Illustrator okuteekateeka ebifaananyi.
Okwekenenya essira ku mtDNA. Omuwendo gw’ebiwundu bya mtDNA gwapimibwa ku bitundu by’obwongo ebyawandiikibwako obutoffaali obuziyiza DNA nga bakozesa confocal microscope. Buli kitundu ekigendererwa kyatondebwawo ku mubiri gw’obutoffaali ne nucleus ya buli katoffaali, era ekitundu ekikwatagana kyabalirirwa nga tukozesa Multi Measure plug-in (ImageJ software). Ggyako ekitundu kya nyukiliya okuva mu kitundu ky’omubiri gw’obutoffaali okufuna ekitundu kya cytoplasmic. Mu kusembayo, Analyze Particles plug-in (ImageJ software) yakozesebwa okugera otomatika omuwendo gwa cytoplasmic DNA points eziraga mtDNA ku threshold image, era ebivuddemu ebyafunibwa bya normalized ku PN average ya CTRL mice. Ebivuddemu biragibwa nga omuwendo gwa nucleosides ogwa wakati buli katoffaali.
Okwekenenya okwolesebwa kwa puloteyina. Kozesa ImageJ’s Image Calculator plug-in okwekenneenya okulaga kwa puloteyina mu PN ku ddaala ly’obutoffaali obumu. Mu bufunze, mu kifaananyi kya confocal ekya layeri emu eky’omukutu ogukwatagana, okuyita mu nsengekera: signal = min (mtYFP, antibody), ekitundu kya mitochondrial ekiraga immunoreactivity eri antibody ezimu mu Purkina kizuulibwa. Olwo ekitundu kya pikseli mu masiki evuddemu kipimibwa mu bungi, n’oluvannyuma ne kifuuka kya bulijjo ku kifaananyi ekikwatagana ekya threshold single-stack ekifaananyi eky’omukutu gwa mtYFP okufuna ekitundu kya mitochondrial ekya puloteyina eragiddwa.
Okwekenenya obungi bw’obutoffaali bwa Purkinje. Plug-in ya Cell Counter eya ImageJ yakozesebwa okwekenneenya density ya Purkinje nga egabanya omuwendo gw’obutoffaali bwa Purkinje obubalibwa n’obuwanvu bw’empeta y’obwongo etudde obutoffaali obubalibwa.
Okuteekateeka sampuli n’okukung’aanya. Obwongo okuva mu kibinja ekifuga n’ebibe bya Mfn2cKO byassibwa mu 2% PFA/2.5% glutaraldehyde mu 0.1 M phosphate buffer (PB), n’oluvannyuma ebitundu bya coronal ne bitegekebwa nga tukozesa ciliates (Leica Mikrosysteme GmbH, Vienna, Austria) (Obugumu 50 ku 60 μm) Oluvannyuma ne bitereezebwa mu PB buffer mu 1% os tetraoxide ne 1.5% potassium ferrocyanide ku bbugumu erya bulijjo okumala essaawa emu. Ebitundu byanaazibwa emirundi esatu n’amazzi agafumbiddwa, oluvannyuma ne bifukibwako ethanol 70% erimu 1% uranyl acetate okumala eddakiika 20. Olwo ebitundu byaggyibwamu amazzi mu mwenge ogwa graded ne biteekebwa mu Durcupan ACM (Araldite casting resin M) epoxy resin (Electron Microscopy Sciences, catalog number 14040) wakati wa silicon-coated glass slides, era okukkakkana nga ku 60°C Polymerize mu oven okumala essaawa 48. Ekitundu ky’omutwe gw’obwongo kyalondebwa era ebitundu ebigonvu ennyo ebya 50 nm ne bisalibwa ku Leica Ultracut (Leica Mikrosysteme GmbH, Vienna, Austria) ne bilondebwa ku 2×1 mm copper slit grid eyasiigibwako firimu ya polystyrene. Ebitundu byasiigibwa langi ya solution ya 4% uranyl acetate mu H2O okumala eddakiika 10, ne binaazibwa ne H2O emirundi egiwerako, oluvannyuma ne Reynolds lead citrate mu H2O okumala eddakiika 10, oluvannyuma ne binaazibwa ne H2O emirundi egiwerako. Ebifaananyi ebitonotono byakwatibwa n’ekyuma ekikebera obusannyalazo obutambuza Philips CM100 (Thermo Fisher Scientific, Waltham, MA, USA) nga tukozesa TVIPS (Tietz Video and Image Processing System) TemCam-F416 digital camera (TVIPS GmbH, Gauting, USA). Girimaani).
Ku mbeba ezaakwatibwa AAV, obwongo bwawulwamu ne busalibwa mu kitundu kya sagittal ekiwanvu mm 1, era cerebellum yakeberebwa nga tukozesa fluorescence microscope okuzuula empeta erimu AAV (kwe kugamba, mCherry expressing). Okugezesa kwokka nga empiso ya AAV evaamu obulungi ennyo obw’okukyusakyusa obw’oluwuzi lw’obutoffaali bwa Purkinje (kwe kugamba kumpi oluwuzi lwonna) mu mpeta z’obwongo ezitakka wansi wa bbiri eziddiring’ana ze zikozesebwa. Loopu eyakyusibwa AAV yasalibwamu microdissected okumala ekiro kyonna oluvannyuma lw’okunyweza (4% PFA ne 2.5% glutaraldehyde mu 0.1 M cocoate buffer) era n’eyongera okulongoosebwa. Ku EPON embedding, ekitundu ekinywevu kyanaazibwa ne 0.1 M sodium cocoate buffer (Applichem), ne kifumbirwa ne 2% OsO4 (os, Science Services; Caco) mu 0.1 M sodium cocoate buffer (Applichem) essaawa 4, oluvannyuma ne kinaazibwa okumala essaawa 2. Ddamu emirundi 3 ne 0.1 M cocamide buffer. Oluvannyuma, omuddirirwa gwa ethanol ogulinnya gwakozesebwa okufumbiza buli solution ya ethanol ku 4°C okumala eddakiika 15 okuggyamu amazzi mu bitundu by’omubiri. Ekitundu kino kyakyusibwa ne kifuulibwa propylene oxide ne kifumbirwa ekiro kyonna mu EPON (Sigma-Aldrich) ku 4°C. Teeka ekitundu kino mu EPON empya ku bbugumu erya bulijjo okumala essaawa 2, n’oluvannyuma okiteeke ku 62°C okumala essaawa 72. Kozesa ultramicrotome (Leica Microsystems, UC6) n’akambe ka dayimanda (Diatome, Biel, Switzerland) okusala ebitundu ebigonvu ennyo ebya 70 nm, era osiige ne 1.5% uranyl acetate okumala eddakiika 15 ku 37°C, era osiige n’omusulo gwa lead citrate okumala eddakiika 4. Ebifaananyi ebitonotono eby’obusannyalazo byakwatibwa nga bakozesa ekintu ekirabika obulungi ekya JEM-2100 Plus transmission electron microscope (JEOL) nga kirimu Camera OneView 4K 16-bit (Gatan) ne pulogulaamu ya DigitalMicrograph (Gatan). Okusobola okwekenneenya, ebifaananyi ebitonotono eby’obusannyalazo byafunibwa nga bikozesa 5000× oba 10,000× digital zoom.
Okwekenenya enkula y’ebirungo ebiyitibwa mitochondria. Ku kwekenneenya kwonna, enkula za mitochondria ssekinnoomu zaalambikibwa mu ngalo mu bifaananyi bya digito nga tukozesa pulogulaamu ya ImageJ. Ebipimo eby’enjawulo eby’enkula (morphological parameters) byekenneenyezebwa. Densite ya mitochondria eraga nga ebitundu ku kikumi ebifunibwa nga tugabanya ekitundu kya mitochondrial kyonna ekya buli katoffaali n’ekitundu kya cytoplasm (ekitundu kya cytoplasm = ekitundu ky’obutoffaali-ekitundu kya nucleus y’obutoffaali) × 100. Obuzito bwa mitochondria bubalirirwa n’ensengekera [4π∙(ekitundu/perimeter 2)]. Enkula ya ista eya mitochondria yeekenneenyezebwa era n’egabanyizibwamu ebika bibiri (“tubular” ne “blister”) okusinziira ku nkula zazo enkulu.
Okwekenenya omuwendo gwa autophagosome/lysosome ne density. Kozesa pulogulaamu ya ImageJ okulaga mu ngalo enkula za buli autophagosome/lysosome mu kifaananyi kya digito. Ekitundu kya autophagosome/lysosome kiragibwa nga ebitundu ku kikumi ebibalirirwa nga tugabanya ekitundu kyonna eky’ensengekera ya autophagosome/lysosome eya buli katoffaali n’ekitundu kya cytoplasm (ekitundu kya cytoplasm=ekitundu ky’obutoffaali-ekitundu kya nucleus)×100. Densite ya autophagosomes/lysosomes ebalibwa nga tugabanya omuwendo gwonna n’omuwendo gw’ensengekera za autophagosome/lysosome buli katoffaali (mu kitundu kya cytoplasmic) (cytoplasmic area = cell area-nuclear area).
Okuwandiika ebiwandiiko okusobola okugabanyaamu ebitundu eby’amangu n’okuteekateeka sampuli. Ku kugezesa okwetaaga okuwandiika ku glucose, kyusa ebitundu by’obwongo eby’amangu mu kisenge ekisooka okufumbirwa, ekirimu kaboni omujjuvu (95% O2 ne 5% CO2), Ca2 + ACSF omungi (125.0 mM NaCl, 2.5 mM KCl, 1.25 mM sodium phosphate buffer, 25.0 mM NaHCO 3, 25.0 mM d-glucose, 1.0 mM CaCl 2 ne 2.0 mM MgCl 2, nga zitereezeddwa ku pH 7.4 ne 310 okutuuka ku 320 mOsm), nga mu kino glucose eri 13 C 6- Okukyusa glucose (Eurisotop, katalogu nnamba CLM-1396). Ku kugezesa okwetaaga okuwandiika ku pyruvate, kyusa ebitundu by’obwongo eby’amangu mu Ca2 + ACSF eya waggulu (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 era oteekemu 2.0mM MgCl2, tereeza ku pH 7.4 ne 310 okutuuka ku 320mOsm), era osseeko 1mM 1-[1-13C]pyruvate (Eurisotop, ennamba ya katalogu CLM-1082). Ebitundu bifumbe okumala eddakiika 90 ku 37°C. Ku nkomerero y’okugezesa, ebitundu byanaazibwa mangu n’ekisengejjero eky’amazzi (pH 7.4) ekirimu 75 mM ammonium carbonate, n’oluvannyuma ne bifuuka homogenized mu 40:40:20 (v:v:v) acetonitrile (ACN): methanol: amazzi. Oluvannyuma lw’ebitundu okufumbirwa ku ice okumala eddakiika 30, sampuli zasengejebwa ku 21,000 g okumala eddakiika 10 ku 4°C, era amazzi amayonjo agafumbiddwa waggulu ne gakalira mu SpeedVac concentrator. Ekikuta kya metabolite ekikalu ekyavaamu kyaterekebwa ku -80°C okutuusa nga kyekenneenyezebwa.
Okwekenenya mu ngeri y’amazzi (liquid chromatography-mass spectrometry analysis) ku amino asidi 13 eziwandiikiddwako C. Ku lw’okwekenneenya amazzi mu ngeri ya liquid chromatography-mass spectrometry (LC-MS), ekikuta ky’ebirungo ebikyusa ebiriisa kyaddamu okusibwa mu 75μl z’amazzi aga LC-MS grade (Honeywell). Oluvannyuma lw’okufuumuula ku 21,000 g okumala eddakiika 5 ku 4°C, 20 μl z’amazzi agatangaaziddwa gaakozesebwa okwekenneenya amino acid flux, ate ekitundu ekisigadde ekyaggibwamu kyakozesebwa mangu okwekenneenya anion (laba wansi). Okwekenenya amino asidi kwakolebwa nga tukozesa enkola ya benzoyl chloride derivatization eyayogerwako emabegako ( 55 , 56 ). Mu mutendera ogusooka, 10μl za 100 mM sodium carbonate (Sigma-Aldrich) zateekebwa mu 20μl eza metabolite extract, n’oluvannyuma 10μl za 2% benzoyl chloride (Sigma-Aldrich) ne ziteekebwa mu LC grade ACN. Sampuli yafuumuulwa mu bufunze n’oluvannyuma n’efuumuulwa ku 21,000 g okumala eddakiika 5 ku 20°C. Teeka amazzi agasukkulumye ku mazzi agalongooseddwa mu kibya kya autosampler ekya 2 ml nga kiriko ekintu ekiyingizibwamu endabirwamu eya kkooni (200 μl volume). Sampuli zino zeekenneenyezebwa nga tukozesa enkola ya Acquity iClass ultra-high performance LC system (Waters) eyungibwa ku Q-Exactive (QE)-HF (Ultra High Field Orbitrap) high-resolution precision mass spectrometer (Thermo Fisher Scientific). Okusobola okwekenneenya, 2μl za sampuli eyava mu nkola yafukibwa mu 100×1.0 mm high-strength silica T3 column (Waters) erimu obutundutundu bwa 1.8μm. Omuwendo gw’amazzi agakulukuta guli 100μl/min, era enkola ya buffer erimu buffer A (10 mM ammonium formate ne 0.15% formic acid mu mazzi) ne buffer B (ACN). Enkyukakyuka eri bweti: 0%B ku ddakiika 0; 0%B. 0 ku 15% B ku ddakiika 0 ku 0.1; 15 ku 17% B ku ddakiika 0.1 ku 0.5; B ku bitundu 17 ku 55% ku ddakiika 0.5 ku 14; B ku bitundu 55 ku 70% ku ddakiika 14 ku 14.5; ku 14.5 ku 70 ku 100% B ku ddakiika 18; 100% B ku ddakiika 18 okutuuka ku 19; 100 ku 0% B ku ddakiika 19 ku 19.1; 0% B ku ddakiika 19.1 okutuuka ku 28 (55, 56). QE-HF mass spectrometer ekola mu mbeera ya ionization ennungi nga mass range ya m/z (mass/charge ratio) ya 50 okutuuka ku 750. Resolution ekozesebwa eri 60,000, era gain control (AGC) ion target efunibwa ye 3×106, era obudde bwa ion obusinga obunene buli 100 milliseconds. Ensibuko y’amasannyalaze agabuguma agafuuyira ionization (ESI) ekola ku vvulovumenti y’okufuuyira eya 3.5 kV, ebbugumu ly’emisuwa erya 250°C, empewo y’omu kikuta eya 60 AU (yuniti ezitali za bulijjo), n’empewo ey’obuyambi eya 20 AU. 250°C. Lenzi ya S eteekeddwa ku 60 AU.
Okwekenenya kwa anion chromatography-MS okwa asidi z’obutonde eziwandiikiddwako 13C. Ekisengejjero ky’ebirungo ebisigaddewo (55μl) kyakeberebwa nga tukozesa enkola ya Dionex ion chromatography system (ICS 5000+, Thermo Fisher Scientific) eyungibwa ku QE-HF mass spectrometer (Thermo Fisher Scientific). Mu bufunze, 5μl za metabolite extract zafukibwa mu Dionex IonPac AS11-HC column nga erimu HPLC (2 mm×250 mm, particle size 4μm, Thermo Fisher Scientific) mu push-in partial loop mode nga erimu omugerageranyo gw’okujjuza ogwa 1. )on. Empagi y’omukuumi eya Dionex IonPac AG11-HC (mm 2 x mm 50, 4μm, Thermo Fisher Scientific). Ebbugumu ly’empagi likuumibwa ku 30°C, era autosampler eteekebwa ku 6°C. Kozesa ekirungo kya potassium hydroxide ekiweereddwa n’amazzi agataliimu ayoni okukola ekigerageranyo kya potassium hydroxide okuyita mu jenereta y’ekiwujjo. Okwawula ebirungo ebikyusa ebiriisa ku sipiidi y’okukulukuta eya 380μl/min, nga osiiga ekigerageranyo kino wammanga: eddakiika 0 ku 3, 10 mM KOH; Eddakiika 3 ku 12, 10 ku 50 mM KOH; Eddakiika 12 ku 19, 50 ku 100 mM KOH; eddakiika 19 okutuuka ku 21 , 100 mM KOH; Eddakiika 21 ku 21.5, 100 ku 10 mM KOH. Empagi yaddamu okutebenkera wansi wa 10 mM KOH okumala eddakiika 8.5.
Ebirungo ebikyusa ebiriisa ebifukiddwa bigattibwa wamu n’omugga ogw’okugatta isopropanol ogwa 150μl/min oluvannyuma lw’empagi n’oluvannyuma ne bilagirwa ku kipima ekipima ekinene eky’obunene (high-resolution mass spectrometer) ekikola mu mbeera ya negativu ionization. MS erondoola obuzito okuva ku m/z 50 okutuuka ku 750 nga erina okusalawo kwa 60,000. AGC eteekebwa ku 1×106, era obudde bwa ion obusinga obunene bukuumibwa ku 100 ms. Ensibuko ya ESI eyabuguma yakolebwa ku vvulovumenti y’okufuuyira eya kV 3.5. Ensengeka endala ez’ensibuko ya ion ze zino wammanga: ebbugumu ly’emisuwa 275°C; okutambula kwa ggaasi mu kikuta, 60 AU; okutambula kwa ggaasi omuyambi, 20 AU ku 300°C, ne S lenzi okuteekebwa ku 60 AU.
Okwekenenya data ku 13C labeled metabolites. Kozesa pulogulaamu ya TraceFinder (version 4.2, Thermo Fisher Scientific) okwekenneenya data y’omugerageranyo gwa isotope. Obumanyirivu bwa buli kirungo kyakakasibwa ekirungo ekyesigika eky’okujuliza era ne kyekenneenyezebwa mu ngeri eyeetongodde. Okusobola okukola okwekenneenya okugaggawaza isotopu, ekitundu kya chromatogram ya ion eggiddwamu (XIC) eya buli isotope ya 13C (Mn) kyaggyibwa mu [M + H] +, nga n ye namba ya kaboni y’ekirungo ekigendererwa, ekozesebwa okwekenneenya amino asidi oba [ MH] + ekozesebwa okwekenneenya aniyoni. Obutuufu bw’obuzito bwa XIC buba wansi w’ebitundu bitaano buli bukadde, ate obutuufu bwa RT buli ddakiika 0.05. Okwekenenya okugaggawaza kukolebwa nga tubalirira omugerageranyo gwa buli isotopu ezuuliddwa ku mugatte gwa isotopu zonna ez’ekirungo ekikwatagana. Emigerageranyo gino giweebwa nga emiwendo gy’ebitundu ku kikumi ku buli isotopu, era ebivuddemu biragibwa nga okugaggawala kw’ebitundu ku kikumi (MPE), nga bwe kyayogerwako emabegako (42).
Ekikuta ky’obusimu obufuuse bbugumu kyafuulibwa mu ngeri ya ice-cold 80% methanol (v/v), ne kifuumuulwa, ne kifumbirwa ku -20°C okumala eddakiika 30. Ddamu okufuumuula sampuli onyige ku +4°C okumala eddakiika 30. Sampuli yafuumuulwa ku 21,000 g okumala eddakiika 5 ku 4°C, n’oluvannyuma amazzi agavaamu ne gakuŋŋaanyizibwa ne gakala nga tukozesa ekirungo kya SpeedVac concentrator ku 25°C okusobola okwekenneenya oluvannyuma. Nga bwe kyayogeddwa waggulu, okwekenneenya kwa LC-MS kwakolebwa ku amino asidi z’obutoffaali obwalondebwa. Nga tukozesa TraceFinder (version 4.2, Thermo Fisher Scientific), okwekenneenya data kwakolebwa nga tukozesa obuzito bwa monoisotopic mass bwa buli kirungo. Quantile normalization ya metabolite data yakolebwa nga tukozesa preprocessCore software package ( 57 ).
Okuteekateeka slice. Ekibe kyabuzibwabuzibwa mangu ne kaboni dayokisayidi ne kisalibwako omutwe, obwongo ne buggyibwa mangu ku kiwanga, era ekiso ekikankana ekijjudde ice (HM-650 V, Thermo Fisher Scientific, Walldorf, Germany) kyakozesebwa okukisala mu bitundu bya sagittal ebya 300 ku 375 μm Cold carbon gasification (95% O2 ne 5% CO2) Low Ca2 + ACSF (125.0 mM NaCl, 2.5 mM KCl, 1.25 mM sodium phosphate buffer, 25.0 mM NaHCO3, 25.0 mM d-glucose, 1.0 mM CaCl2 ne 6.0 mM MgCl2 Teekateeka ku pH 7.4 ne 310 okutuuka ku 330 mOsm). Kyuusa ebitundu by’obwongo ebifunibwa mu kisenge ekirimu Ca2 + ACSF esingako (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 ne mM 3.5 MgCl2) pH 7.4 ne 310 okutuuka ku 320 mOsm). Ebitundu bitereke okumala eddakiika 20 ku 30 bisobole okuddamu okubikwata nga tonnakwata.
okukwata ebifaananyi. Omutendera gwa microscope ogulimu ekisenge ekikwata ebifaananyi ekinywevu n’ekiruubirirwa ky’okunnyika mu mazzi ekya 20x (Scientifica) kyakozesebwa mu kukwata kwonna. Obutoffaali bwa Purkinje obuteeberezebwa bwazuulibwa nga (i) obunene bw’omubiri, (ii) ekifo ky’omubiri gw’obwongo, ne (iii) okwolesebwa kw’obuzaale bw’omusasi wa mtYFP obutangaavu. Pipeti ya patch eriko obuziyiza bw’ensonga okuva ku megohms 5 ku 11 eggyibwamu omusuwa gwa borosilicate glass capillary (GB150-10, 0.86 mm×1.5 mm×100 mm, Science Products, Hofheim, Germany) ne pipette horizontal Instruments (P-1000, Sutter), Novato, CA). Ebikwata byonna byakolebwa mu ngeri ya ELC-03XS npi patch clamp amplifier (npi electronic GmbH, Tam, Germany), eyafugibwa pulogulaamu ya Signal (version 6.0, Cambridge Electronic, Cambridge, UK). Okugezesa kuno kwawandiikibwa ku sipiidi ya sampling eya 12.5 kHz. Siginini eno esengejebwa n’ebisengejja bibiri ebya Bessel ebiyita mu bumpimpi nga biriko firikwensi z’okusalako eza 1.3 ne 10 kHz. Capacitance ya membrane ne pipette eliyirirwa circuit y’okuliyirira nga ekozesa amplifier. Okugezesa kwonna kwakolebwa wansi w’okufugibwa kkamera ya Orca-Flash 4.0 (Hamamatsu, Gerden, Germany), eyafugibwa pulogulaamu ya Hokawo (version 2.8, Hamamatsu, Gerden, Germany).
Ensengeka ya bulijjo ey’obutoffaali bwonna n’okwekenneenya. Amangu ddala nga tonnaba kuwandiika, jjuza pipette n’ekisengejjero eky’omunda ekirimu ebintu bino wammanga: 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) ne 10.0 mM creatinine phosphate zatereezebwa okutuuka ku pH 7.25, era puleesa ya osmotic yali 290 mOsm (sucrose). Amangu ddala nga tumaze okussaako empalirizo ya 0 pA okukutula oluwuzi, obusobozi bw’oluwuzi oluwummula bwapimibwa. Obuziyiza obuyingizibwa bupimibwa nga tukozesa amasannyalaze aga hyperpolarized aga -40, -30, -20, ne -10 pA. Pima obunene bw’okuddamu kwa vvulovumenti era okozese etteeka lya Ohm okubala obuziyiza bw’okuyingiza. Emirimu egy’okwekolako gyawandiikibwa mu vvulovumenti clamp okumala eddakiika 5, era sPSC yazuulibwa era n’epimibwa mu Igor Pro (version 32 7.01, WaveMetrics, Lake Oswego, Oregon, USA) nga tukozesa ekiwandiiko ekitegeera ekitali kya otomatiki. IV curve ne steady-state current zipimibwa nga zisiba bbaatule ku potentials ez’enjawulo (okutandika okuva ku -110 mV) n’okwongera ku voltage mu mitendera gya 5 mV. Okukola AP kwagezesebwa nga bakozesa akasannyalazo aka depolarizing. Siba akatoffaali ku -70 mV ng’ossaako ‘depolarizing current pulse’. Teekateeka obunene bw’omutendera gwa buli kitundu ekikwata ebifaananyi okwawukana (10 ku 60 pA). Bala frequency ya AP esinga obunene ng’obala mu ngalo ebiwujjo bya pulse ebireeta frequency ya AP esinga obunene. Omusingi gwa AP gwekenneenyezebwa nga tukozesa ekivaamu ekyokubiri ekya depolarization pulse ekisooka okusitula AP emu oba eziwera.
Ensengeka y’ebitundu ebituli n’okwekenneenya. Kola perforated patch recording nga okozesa enkola ya standard. Kozesa pipette etaliimu ATP ne GTP etaliimu birungo bino wammanga: 128 mM gluconate K, 10 mM KCl, 10 mM Hepes, 0.1 mM EGTA ne 2 mM MgCl2, era otereeze ku pH 7.2 (ng’okozesa KOH). ATP ne GTP zirekeddwa mu kisengejjero ekiri mu butoffaali okuziyiza okuyita okutafugibwa kw’oluwuzi lw’obutoffaali. Pipette ya patch ejjula n’ekizimbulukusa eky’omunda ekirimu amphotericin (nga 200 ku 250 μg/ml; G4888, Sigma-Aldrich) okufuna likodi y’ekikuta ekirimu ebituli. Amphotericin yasaanuusibwa mu dimethyl sulfoxide (ekigero ekisembayo: 0.1 okutuuka ku 0.3%; DMSO; D8418, Sigma-Aldrich). Ekirungo kya DMSO ekyakozesebwa tekyalina kinene kye kyakola ku busimu obuyitibwa neurons obwasomesebwa. Mu nkola y’okukuba ebikonde, obuziyiza bw’omukutu (Ra) bwalondoolebwa obutasalako, era okugezesa kwatandikibwawo oluvannyuma lwa amplitude ya Ra ne AP okutebenkera (eddakiika 20-40). Emirimu egy’okwekolako gipimibwa mu vvulovumenti ne/oba ekikwaso kya kasasiro okumala eddakiika 2 ku 5. Okwekenenya amawulire kwakolebwa nga tukozesa Igor Pro (enkyusa 7.05.2, WaveMetrics, Amerika), Excel (enkyusa 2010, Microsoft Corporation, Redmond, Amerika) ne GraphPad Prism (enkyusa 8.1.2, GraphPad Software Inc., La Jolla, CA). Amerika). Okusobola okuzuula APs ezeetongodde, plug-in ya IgorPro eya NeuroMatic v3.0c ekozesebwa. Laba APs mu ngeri ey’otoma ng’okozesa omusingi oguweereddwa, ogutereezebwa kinnoomu ku buli likodi. Ng’okozesa ekiseera kya spike, manya frequency ya spike nga erina frequency ya spike esinga obunene ey’akaseera katono ne frequency ya spike eya wakati.
PN okweyawula ku balala. Nga tukwatagana n’enkola eyafulumizibwa emabegako, PNs zalongoosebwa okuva mu bwongo bw’ebibe ku mutendera ogulagirwa ( 58 ). Mu bufunze, ekisenge ky’obwongo kyasalibwamu ne kisalibwa mu kifo eky’okukutula ekinyogovu nga ice [nga tekirina HBSS Ca2+ ne Mg2+, nga kigattibwako 20 mM glucose, penicillin (50 U/ml) ne streptomycin (0.05 mg/ ml)], n’oluvannyuma ne kigaaya ekifo mu papain [HBSS, nga kigattibwako 1-cysteine·HCl (1 mg / ml), papain (16 U / ml) ne deoxyribonuclease I (DNase I; 0.1 mg/ml)] Zijjanjaba okumala eddakiika 30 ku 30°C. Sooka onaabe ebitundu by’omubiri mu HBSS medium erimu omusulo gw’amagi (10 mg/ml), BSA (10 mg/ml) ne DNase (0.1 mg/ml) ku bbugumu erya bulijjo okuziyiza enziyiza okugaaya, n’oluvannyuma mu HBSS medium erimu 20 mM glucose Gently grinding in HBSS, penicillin (50 U/ml), streptomycin (0.05 mg/ml) ne DNase (0.1 mg/ml) efulumya obutoffaali obumu. Ekisengejjero ky’obutoffaali ekyavaamu kyasengejebwa okuyita mu kisengejja ky’obutoffaali ekya 70μm, olwo obutoffaali ne bufuulibwa obukuta nga buyita mu kusengejja (1110 rpm, eddakiika 5, 4°C) ne buddamu okusibwa mu kifo eky’okusunsulamu [HBSS, nga kigattibwako 20 mM glucose, 20% fetal bovine ) Serum, penicillin (50 U/ml) ne streptomycin (0.05 mg/ml)]; okwekenneenya obulamu bw’obutoffaali ne propidium iodide era otereeze density y’obutoffaali okutuuka ku 1×106 okutuuka ku 2×106 cells/ml. Nga tebannaba kukola flow cytometry, ekiyimiriziddwa kyasengejebwa okuyita mu 50 μm cell strainer.
Ekipima obutoffaali obukulukuta. Okusunsula obutoffaali kwakolebwa ku 4°C nga tukozesa ekyuma kya FACSAria III (BD Biosciences) ne pulogulaamu ya FACSDiva (BD Biosciences, version 8.0.1). Okuyimirizaawo obutoffaali kwasunsulwa nga tukozesa entuuyo ya 100 μm wansi wa puleesa ya 20 psi ku sipiidi ya ~2800 events/sec. Okuva emisingi gy’okuyingiza enzigi egy’ennono (obunene bw’obutoffaali, okusosola mu bimodal, n’engeri y’okusaasaana) tegitasobola kukakasa kwawula kutuufu kwa PN okuva mu bika by’obutoffaali ebirala, enkola y’okuggala eteekebwawo okusinziira ku kugeraageranya obutereevu amaanyi ga YFP n’okumasamasa mu mitoYFP + n’ebibe ebifuga. YFP ecamuka nga etangaaza sampuli ne layini ya layisi eya 488 nm, era siginiini ezuulibwa nga ekozesa 530/30 nm band pass filter. Mu mbeba za mitoYFP+, amaanyi agakwatagana ag’obuzaale bw’omusasi wa Rosa26-mitoYFP era gakozesebwa okwawula ebitundu by’omubiri gw’obusimu n’obusimu obuyitibwa axon. 7-AAD ecamulwa ne layisi eya kyenvu eya 561 nm era n’ezuulibwa n’omusengejja gwa 675/20 nm bandpass okuggyamu obutoffaali obufu. Okusobola okwawula astrocytes mu kiseera kye kimu, ekisengejjo ky’obutoffaali kyasiigibwa ACSA-2-APC, olwo sampuli n’efukibwako layini ya layisi eya 640 nm, era ekyuma ekisengejja 660/20 nm bandpass kyakozesebwa okuzuula akabonero.
Obutoffaali obukung’aanyiziddwa bwafuulibwa obukuta nga buyita mu kusengejja (1110 rpm, eddakiika 5, 4°C) ne buterekebwa ku -80°C okutuusa lwe bukozesebwa. Ebibe bya Mfn2cKO n’abaana baabwe abafuuse kasasiro basengekebwa ku lunaku lwe lumu okukendeeza ku nkyukakyuka mu nkola. Okwanjula n’okwekenneenya ebikwata ku FACS byakolebwa nga tukozesa pulogulaamu ya FlowJo (FlowJo LLC, Ashland, Oregon, USA).
Nga bwe kyayogeddwa waggulu (59), real-time PCR ekozesebwa okwawula DNA okuva mu busimu obusunsuddwa okusobola okupima mtDNA oluvannyuma. Linearity ne threshold sensitivity byasooka kukeberebwa nga bakola qPCR ku muwendo gw’obutoffaali ogw’enjawulo. Mu bufunze, kukungaanya PN 300 mu lysis buffer erimu 50 mM tris-HCl (pH 8.5), 1 mM EDTA, 0.5% Tween 20 ne proteinase K (200 ng/ml) ofumbe ku 55°C okumala eddakiika 120. Obutoffaali bwayongera okufumbirwa ku 95°C okumala eddakiika 10 okukakasa nti proteinase K tekola mu bujjuvu.Nga tukozesa TaqMan probe (Thermo Fisher) eyeetongodde ku mt-Nd1, mtDNA yapimibwa mu semi-quantitative PCR mu nkola ya 7900HT Real-Time PCR system (Thermo Fisher Scientific). Sayansi, ennamba ya katalogu Mm04225274_s1), mt-Nd6 (Thermo Fisher Scientific, nnamba ya katalogu AIVI3E8) ne 18S (Thermo Fisher Scientific, nnamba ya katalogu Hs99999901_s1) obuzaale.
Okuteekateeka sampuli ya proteome. Nga obugumya ekisengejjero ku 95°C okumala eddakiika 10 n’okola sonicating, mu lysis buffer [6 M guanidine chloride, 10 mM tris (2-carboxyethyl) phosphine hydrochloride, 10 mM chloroacetamide ne 100 mM tris- Lyse frozen neuron pellets mu HCl]. Ku Bioruptor (Diagenode) okumala eddakiika 10 (sekondi 30 pulse / sekondi 30 pause period). Sampuli yafukibwa 1:10 mu 20 mM tris-HCl (pH 8.0), n’etabulwa ne 300 ng trypsin gold (Promega), n’efumbirwa ekiro kyonna ku 37°C okutuuka ku kugaaya okujjuvu. Ku lunaku olwokubiri, sampuli yafuumuulwa ku g 20,000 okumala eddakiika 20. Ekisengejjero ekyafukibwako asidi wa formic 0.1%, era ekisengejjero ne kiggyibwamu omunnyo ne StageTips eyeekoledde. Sampuli yakalizibwa mu kyuma kya SpeedVac (Eppendorf concentrator plus 5305) ku 45°C, n’oluvannyuma peptide n’eyimirizibwa mu 0.1% formic acid. Sampuli zonna zaategekebwa mu kiseera kye kimu omuntu omu. Okusobola okwekenneenya sampuli za astrocyte, 4 μg desalted peptides zawandiikibwako tandem mass tag (TMT10plex, catalog number 90110, Thermo Fisher Scientific) nga peptide ku TMT reagent ratio ya 1:20. Ku kussaako akabonero ka TMT, 0.8 mg za TMT reagent zaddamu okusibwa mu 70 μl za ACN ezitaliimu mazzi, era peptide enkalu n’eddamu okukolebwa mu 9 μl za 0.1 M TEAB (triethylammonium bicarbonate), nga ku zino 7 μl za TMT reagent mu ACN zaateekebwamu. Ebirungo bino byali 43.75%. Oluvannyuma lw’eddakiika 60 nga zifumbiddwa, ensengekera yazikizibwa ne 2 μl za 5% hydroxylamine. Peptides eziwandiikiddwa zaakuŋŋaanyizibwa, ne zikalira, ne ziddamu okusibwa mu 200μl za 0.1% formic acid (FA), ne zigabanyizibwamu bibiri, oluvannyuma ne ziggyibwamu omunnyo nga tukozesa StageTips ezeekoledde. Nga tukozesa UltiMate 3000 ultra high performance liquid chromatograph (UltiMate 3000 ultra high performance liquid chromatograph), ekimu ku bitundu ebibiri kyakutulwamu ku mpagi ya Acquity chromatographic eya mm 1 x mm 150 nga ejjudde obutundutundu bwa C18 obwa 130Å1.7μm (Waters, catalog No. SKU: 186006935). Thermo Fisher Omuvubi wa Sayansi). Yawula peptides ku sipiidi y’okukulukuta eya 30μl/min, yawula okuva ku 1% okutuuka ku 50% buffer B okumala eddakiika 85 nga olina stepwise gradient ya eddakiika 96, okuva ku 50% okutuuka ku 95% buffer B okumala eddakiika 3, olwo eddakiika 8 ku 95 % Buffer B; Buffer A eri 5% ACN ne 10 mM ammonium bicarbonate (ABC), ate buffer B eri 80% ACN ne 10 mM ABC. Kuŋŋaanya obutundutundu buli luvannyuma lwa ddakiika 3 obugatte mu bibinja bibiri (1 + 17, 2 + 18, n’ebirala) obikalize mu vacuum centrifuge.
Okwekenenya kwa LC-MS/MS. Ku mass spectrometry, peptides (ennamba r119.aq) zaawulwa ku 25 cm, 75 μm inner diameter PicoFrit analytical column (new objective lens, part number PF7508250) nga zirina 1.9 μm ReproSil-Pur 120 C18-AQ medium (Dr. Maisch, mat), Use EASY-nLC 1200 (Ekitongole kya Thermo Fisher Scientific, Bugirimaani). Empagi yakuumibwa ku 50°C. Buffers A ne B ziri 0.1% formic acid mu mazzi ne 0.1% formic acid mu 80% ACN, mu kulondako. Peptides zaawulwamu ne stepwise gradient ya 200 nl/min okuva ku 6% okutuuka ku 31% buffer B okumala eddakiika 65, ate okuva ku 31% okutuuka ku 50% buffer B okumala eddakiika 5. Peptides ezaalongoosebwa zaakeberebwa ku Orbitrap Fusion mass spectrometer (Thermo Fisher Scientific). Okupima peptide precursor m/z kukolebwa nga kulina okusalawo kwa 120,000 mu bbanga lya 350 ne 1500 m/z. Nga tukozesa amasoboza g’okutomeragana aga bulijjo aga 27%, ekisookerwako ekisinga amaanyi nga kirina embeera ya chajingi okuva ku 2 ku 6 kirondebwa okusobola okukutuka kw’amasoboza amangi C trap dissociation (HCD). Obudde bw’enzirukanya buteekebwa ku s 1. Omuwendo gwa m/z ogw’ekitundu kya peptide gwapimibwa mu mutego gwa ion nga tukozesa ekiruubirirwa kya AGC ekisinga obutono ekya 5×104 n’obudde obusinga obunene obw’okukuba empiso bwa 86 ms. Oluvannyuma lw’okukutulakutula, ekisookerwako kyateekebwa ku lukalala lw’okuggyibwako okukyukakyuka okumala s 45. Peptides eziwandiikiddwako TMT zaawulwa ku 50 cm, 75 μm Acclaim PepMap column (Thermo Fisher Scientific, catalog number 164942), era spectra z’okusenguka zaakeberebwa ku Orbitrap Lumos Tribrid mass spectrometer (Thermo Fisher Scientific) nga zirina ebyuma ebiyitibwa high-field asymmetric waveform ions (FAIMS). (Thermo Fisher Scientific) ekola ku vvulovumenti bbiri ez’okuliyirira eza −50 ne −70 V. MS3 eyalondebwa okusinziira ku kisookerwako eky’okukwatagana ekozesebwa okupima obubonero bwa ion lipoota ya TMT. Okwawula peptide kwakolebwa ku EASY-nLC 1200, nga tukozesa 90% linear gradient elution, nga buffer concentration ya 6% okutuuka ku 31%; buffer A yali 0.1% FA, ate buffer B yali 0.1% FA ne 80% ACN. Empagi eyeekenneenya ekolebwa ku 50°C. Kozesa FreeStyle (version 1.6, Thermo Fisher Scientific) okugabanya fayiro eyasooka okusinziira ku vvulovumenti y’okuliyirira eya FAIMS.
Okuzuula ebirungo ebizimba omubiri n’okubigera obungi. Nga tukozesa enkola y’okunoonya eya Andromeda ekwataganye, data eyasooka yakeberebwa nga tukozesa MaxQuant version 1.5.2.8 (https://maxquant.org/). Ng’oggyeeko ensengekera za Cre recombinase ne YFP ezafunibwa okuva mu Aequorea victoria, spektira z’obutundutundu bwa peptide zanoonyezebwa ensengekera y’ennono n’ensengekera ya isoform eya proteome y’ebibe ey’okujuliza (Proteome ID UP000000589, eyawanulibwa okuva mu UniProt mu May 2017) . Methionine oxidation ne protein N-terminal acetylation byateekebwawo nga enkyukakyuka ezikyukakyuka; cysteine ​​carbamoyl methylation yateekebwawo nga enkyukakyuka ezitakyukakyuka. Ebipimo by’okugaaya emmere biteekebwa ku “specificity” ne “trypsin/P”. Omuwendo omutono ogwa peptides ne razor peptides ezikozesebwa okuzuula puloteyina guli 1; omuwendo omutono ogwa peptides ez’enjawulo guli 0. Mu mbeera z’okukwatagana kwa maapu ya peptide, omuwendo gw’okuzuula puloteyina gwali 0.01. Enkola ya “Second Peptide” ekoleddwa. Kozesa enkola ya “match between runs” okukyusa okuzuula okuwangudde wakati wa fayiro ez’enjawulo ez’olubereberye. Kozesa LFQ omugerageranyo omutono count 1 ku label-free quantification (LFQ) (60). Amaanyi ga LFQ gasengejebwa waakiri emiwendo ebiri emituufu mu kibinja kya genotype waakiri kimu ku buli kiseera, era ne gafulumizibwa okuva mu nsasaanya eya bulijjo n’obugazi bwa. 0.3 n’ogenda wansi 1.8. Kozesa omukutu gwa kompyuta ogwa Perseus (https://maxquant.net/perseus/) ne R (https://r-project.org/) okwekenneenya ebivudde mu LFQ. Okugezesebwa kwa t okw’ekigero okw’engeri bbiri okuva mu limma software package kwakozesebwa okwekenneenya okwolesebwa okw’enjawulo ( 61 ). Okwekenenya ebikwata ku bantu mu kunoonyereza kukolebwa nga tukozesa ggplot, FactoMineR, factoextra, GGally ne pheatmap. Data ya proteomics eyesigamiziddwa ku TMT yakeberebwa nga tukozesa MaxQuant version 1.6.10.43. Noonya data ya raw proteomics okuva mu UniProt’s human proteomics database, eyawanulibwa mu September 2018. Okwekenenya kuno kuliko ensonga y’okutereeza obulongoofu bwa isotope eyaweebwa abakola. Kozesa limma mu R okwekenneenya okwolesebwa okw’enjawulo. Data eyasooka, ebivudde mu kunoonya database, n’enkola y’emirimu mu kwekenneenya data n’ebivuddemu byonna biterekebwa mu mukago gwa ProteomeXchange okuyita mu tterekero ly’omukwanaganya wa PRIDE n’ekimanyisa data set PXD019690.
Ennyiriri eziraga emirimu zigaggawaza okwekenneenya. Ekintu ekiyitibwa Ingenuity Pathway Analysis (QIAGEN) kyakozesebwa okuzuula obugagga bw’ebigambo by’obugambo ebikola eby’ebiwandiiko ebiteekeddwawo ku wiiki 8 (Ekifaananyi 1). Mu bufunze, olukalala lwa puloteyina olw’omuwendo olufunibwa okuva mu kwekenneenya amawulire ga LC-MS/MS (tandem mass spectrometry) lukozesebwa n’emisingi gino egy’okusengejja: Mus musculus elondebwa ng’ekika n’ensibuko, era ekika kiraga omuwendo gwa P ogwatereezebwa Benjamini olw’okugaggawaza 0.05 oba wansi gutwalibwa ng’ogw’amaanyi. Ku giraafu eno, ebika ebisukkiridde ebitaano eby’oku ntikko mu buli kibinja okusinziira ku muwendo gwa P ogutereezeddwa biragiddwa. Nga tukozesa multiple t-test, nga tukozesa enkola ya linear boost program ey’emitendera ebiri eya Benjamini, Krieger, ne Yekutieli (Q = 5%), okwekenneenya okwolesebwa kwa puloteyina mu kiseera ky’omusomo kukolebwa ku beesimbyewo abakulu abazuuliddwa mu buli mutendera, era buli lunyiriri lwekenneenyezebwa okwawukana. Tekyetaagisa kwettanira SD ekwatagana.
Okusobola okugeraageranya ebivudde mu kunoonyereza kuno ne databases ezifulumiziddwa n’okukola ekifaananyi kya Venn mu kifaananyi 1, twagatta olukalala lwa puloteyina ez’omuwendo ne MitoCarta 2.0 annotations ( 24 ). Kozesa ekintu ekiri ku mutimbagano Draw Venn Diagram (http://bioinformatics.psb.ugent.be/webtools/Venn/) okukola ekifaananyi.
Okumanya ebisingawo ku nkola z’ebibalo ezikozesebwa okwekenneenya proteomics, laba ekitundu ekikwatagana ekya Materials and Methods. Ku kugezesa okulala kwonna, amawulire amajjuvu gasobola okusangibwa mu lugero olukwatagana. Okuggyako nga kirambikiddwa bulala, data zonna ziragibwa nga mean ± SEM, era okwekenneenya kwonna okw’ebibalo kwakolebwa nga tukozesa pulogulaamu ya GraphPad Prism 8.1.2.
Okumanya ebikozesebwa eby’okugatta ku kiwandiiko kino, laba http://advances.sciencemag.org/cgi/content/full/6/35/eaba8271/DC1
Kino kiwandiiko ekiggule ekisaasaanyizibwa wansi w’ebiragiro bya Creative Commons Attribution-Non-Commercial License, ekikkiriza okukozesa, okusaasaanya n’okuzzaawo mu ngeri yonna, kasita enkozesa esembayo si ya kuganyulwa mu by’obusuubuzi era nga n’ensonga eri nti omulimu ogwasooka mutuufu. Okwebuuza ewalala.
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Ebyawandiikibwa E. Motori, I. Atanassov, S. M. V. Kochan, K. Folz-Donahue, V. Sakthivelu, P. Giavalisco, N. Toni, J. Puyal, N.-G. Larson, omuwandiisi w’ebitabo
Okwekenenya obusimu obutakola bulungi mu by’obutonde (proteomics analysis) kwalaga nti pulogulaamu z’enkyukakyuka mu mubiri zikolebwa okusobola okulwanyisa okuvunda kw’obusimu.
Ebyawandiikibwa E. Motori, I. Atanassov, S. M. V. Kochan, K. Folz-Donahue, V. Sakthivelu, P. Giavalisco, N. Toni, J. Puyal, N.-G. Larson, omuwandiisi w’ebitabo
Okwekenenya obusimu obutakola bulungi mu by’obutonde (proteomics analysis) kwalaga nti pulogulaamu z’enkyukakyuka mu mubiri zikolebwa okusobola okulwanyisa okuvunda kw’obusimu.
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