Hona Joale *Aterese ea Hona Joale: Cologne 50931, Jeremane, Patlisiso ea Sehlopha sa Bokhabane sa Cologne mabapi le Karabelo ea Khatello ea Lisele Mafung a Amanang le Botsofali (CECAD).
Ho senyeha ha methapo ea kutlo ha mafu a mitochondrial ho nkoa e sa fetohe hobane ho se sebetse hantle ha methapo ea kutlo hoa fokola, empa phello ea ho se sebetse hantle ha mitochondrial ho ikemela ha sele ha metabolism ea methapo ea kutlo 'meleng ha e utloisisoe hantle. Mona, re hlahisa proteome e ikhethileng ea lisele tsa li-neurone tsa Purkinje tse nang le khaello ea OXPHOS e tsoelang pele e bakoang ke ho senyeha ha matla a ho kopanya ha mitochondrial. Re fumane hore ho se sebetse hantle ha mitochondrial ho bakile phetoho e kholo tšimong ea li-proteomics, e leng se ileng sa qetella se lebisitse ho ts'ebetsong e latellanang ea mananeo a nepahetseng a metabolism pele ho lefu la sele. Ka tšohanyetso, re ile ra fumana tlhahiso e totobetseng ea pyruvate carboxylase (PCx) le li-enzyme tse ling tse loantšang botsofali tse tlatsetsang lipakeng tsa potoloho ea TCA. Thibelo ea PCx e mpefalitse khatello ea oxidative le ho senyeha ha methapo ea kutlo, e bontšang hore atherosclerosis e na le phello e sireletsang li-neurone tse haellang OXPHOS. Ho tsosolosoa ha ho kopana ha mitochondrial li-neurone tse senyehileng ka ho feletseng ho fetola litšobotsi tsena tsa metabolism ka botlalo, ka hona ho thibela lefu la sele. Liphuputso tsa rona li khetholla litsela tse neng li sa tsejoe pele tse fanang ka matla a ho se sebetse hantle ha mitochondrial 'me li bontša hore ho senyeha ha methapo ea kutlo ho ka khutlisoa esita le mehatong ea morao ea lefu lena.
Karolo ea bohlokoa ea mitochondria ho bolokeng metabolism ea matla a methapo ea kutlo e hatisoa ke matšoao a mangata a methapo a amanang le mafu a mitochondrial a batho. Bongata ba mafu ana a bakoa ke liphetoho tsa liphatsa tsa lefutso tse laolang polelo ea liphatsa tsa lefutso tsa mitochondrial (1, 2) kapa timetso ea liphatsa tsa lefutso tse amanang le matla a mitochondrial, tse amang botsitso ba DNA ea mitochondrial (mtDNA) ka tsela e sa tobang (3, 4). Mosebetsi mehlaleng ea liphoofolo o bontšitse hore ho arabela ho se sebetse hantle ha mitochondrial liseleng tse potolohileng, litsela tsa metabolism tse bolokang (5-7) li ka kenngoa tšebetsong, e leng se fanang ka tlhahisoleseling ea bohlokoa bakeng sa kutloisiso e tebileng ea pathogenesis ea mafu ana a rarahaneng. Ka lehlakoreng le leng, kutloisiso ea rona ea liphetoho tsa metabolism tsa mefuta e itseng ea lisele tse bakoang ke ho hloleha ka kakaretso ha tlhahiso ea adenosine triphosphate (ATP) ea boko ke ea bohlokoa (8), e hatisang tlhoko ea ho tsebahatsa lipheo tsa kalafo tse ka sebelisoang ho thibela kapa ho thibela mafu. Thibela ho senyeha ha methapo ea kutlo (9). Ho hloka tlhahisoleseling ke taba ea hore lisele tsa methapo li nkoa haholo li na le ho tenyetseha ho fokolang ha metabolism ha li bapisoa le mefuta ea lisele tsa lisele tse potolohileng (10). Kaha lisele tsena li bapala karolo ea bohlokoa ho hokahanya phepelo ea li-metabolite ho li-neuron ho khothaletsa phetiso ea synaptic le ho arabela likotsing le maemong a lefu, bokhoni ba ho fetola metabolism ea sele ho latela maemo a thata a lisele tsa boko bo batla bo lekanyelitsoe ho lisele tsa glial (11-14). Ho phaella moo, ho se tšoane ha lisele tsa tlhaho tsa lisele tsa boko ho sitisa haholo thuto ea liphetoho tsa metabolism tse etsahalang lihlopheng tse itseng tsa neuronal. Ka lebaka leo, ho tsejoa hanyane ka liphello tse nepahetseng tsa sele le metabolism tsa ho se sebetse hantle ha mitochondrial li-neuron.
E le ho utloisisa liphello tsa metabolism tsa ho se sebetse hantle ha mitochondrial, re khethile li-neuron tsa Purkinje (PNs) mehatong e fapaneng ea ho senyeha ha methapo ea kutlo e bakoang ke timetso ea ho kopana ha lera la kantle la mitochondrial (Mfn2). Le hoja liphetoho tsa Mfn2 bathong li amahanngoa le mofuta oa lefu la methapo ea kutlo ea lefutso la lefutso le tsejoang e le Charcot-Marie-Tooth type 2A (15), timetso e nang le maemo ea Mfn2 ho litoeba ke mokhoa o tsebahalang oa ho kenngoa ha oxidation​​ Phosphorylation (OXPHOS) dysfunction. Mefuta e fapaneng ea neuronal (16-19) le phenotype e hlahang ea neurodegenerative li tsamaea le matšoao a tsoelang pele a methapo ea kutlo, joalo ka mathata a ho sisinyeha (18, 19) kapa cerebellar ataxia (16). Ka ho sebelisa motsoako oa li-proteomics tse se nang label-quantitative (LFQ), metabolomics, imaging, le mekhoa ea virological, re bontša hore progressive neurodegeneration e baka ka matla pyruvate carboxylase (PCx) le lintlha tse ling tse amehang ho arteriosclerosis ea PNs in vivo The polelo ea li-enzyme. Ho netefatsa bohlokoa ba phumano ena, re ile ra fokotsa ka ho khetheha tlhahiso ea PCx ho li-PN tse haelloang ke Mfn2, 'me ra fumana hore ts'ebetso ena e mpefalitse khatello ea oxidative le ho potlakisa ho bola ha methapo ea kutlo, ka hona ra paka hore azoospermia e fa lisele lefu la ho ikamahanya le maemo a metabolism. Ponahalo e matla ea MFN2 e ka pholosa ka botlalo ho bola ha PN ka lebaka la khaello e matla ea OXPHOS, tšebeliso e kholo ea DNA ea mitochondrial, le marang-rang a mitochondrial a bonahalang a robehile, e leng se totobatsang hore mofuta ona oa ho bola ha methapo ea kutlo o ka boela oa hlaphoheloa mohatong o tsoetseng pele oa lefu pele sele e shoa.
E le ho bona ka mahlo a kelello mitochondria ho Mfn2 knockout PNs, re sebelisitse mofuta oa toeba o lumellang mitochondria e itšetlehileng ka Cre ho shebana le protheine e mosehla ea fluorescent (YFP) (mtYFP) (20) Cre expression mme ra hlahloba sebopeho sa mitochondrial in vivo. Re fumane hore timetso ea gene ea Mfn2 ho PNs e tla lebisa karohanong e butle-butle ea marang-rang a mitochondrial (Setšoantšo sa S1A), 'me phetoho ea pele e fumanoe ha motho a le libeke tse 3. Ka lehlakoreng le leng, ho senyeha ho hoholo ha lera la sele ea PN, joalo ka ha ho pakoa ke tahlehelo ea immunostaining ea Calbindin, ha hoa ka ha qala ho fihlela a le libeke tse 12 (Setšoantšo sa 1, A le B). Ho se lumellane ha nako pakeng tsa liphetoho tsa pele ho sebopeho sa mitochondrial le ho qala ho bonahalang ha lefu la neuronal ho re susumelitse ho batlisisa liphetoho tsa metabolism tse bakoang ke ho se sebetse hantle ha mitochondrial pele ho lefu la sele. Re qapile leano le thehiloeng ho fluorescence-activated cell sorting (FACS) ho arola YFP (YFP+)-expressing PN (Setšoantšo sa 1C), le ho litoeba tse laolang (Mfn2 + / loxP :: mtYFP loxP- stop-loxP: : L7-cre), eo ho tla thoe ke CTRL (Setšoantšo sa S1B). Ho ntlafatsa leano la ho thibela ho ipapisitse le matla a amanang le lets'oao la YFP ho re lumella ho hloekisa 'mele oa YFP+ (YFPhigh) oa li-PN ho tsoa ho li-non-PN (YFPneg) (Setšoantšo sa S1B) kapa likaroloana tsa fluorescent axon/dendritic (YFPlow; Setšoantšo sa S1D, ka letsohong le letšehali), tse tiisitsoeng ke microscope ea confocal (Setšoantšo sa S1D, ka ho le letona). E le ho netefatsa boitsebiso ba baahi ba arotsoeng, re entse LFQ proteomics ebe re etsa tlhahlobo ea likarolo tse kholo, 'me ra fumana hore ho na le karohano e hlakileng lipakeng tsa lisele tsa YFPhigh le YFPneg (Setšoantšo sa S1C). Lisele tse phahameng tsa YFP li bontšitse ntlafatso ea netweke ea matšoao a tsejoang a PN (ke hore Calb1, Pcp2, Grid2 le Itpr3) (21, 22), empa ha ho ntlafatso ea liprotheine tse atisang ho hlahisoa li-neuron kapa mefuta e meng ea lisele (Setšoantšo sa 1D)). Papiso pakeng tsa lisampole liseleng tse phahameng tsa YFP tse bokelletsoeng litekong tse ikemetseng e bontšitse coefficient ea kamano> 0.9, e bonts'ang ho ikatisa ho hotle pakeng tsa likopi tsa baeloji (Setšoantšo sa S1E). Ka bokhutšoanyane, lintlha tsena li netefalitse morero oa rona oa ho arohana ka tšohanyetso le ka ho khetheha ha PN e ka khonehang. Hobane sistimi ea mokhanni oa L7-cre e sebelisitsoeng e baka ho kopana hape ha mosaic bekeng ea pele kamora ho pepa (23), re qalile ho bolaea litoeba ho tsoa ho CTRL le conditional (Mfn2 loxP / loxP :: mtYFP loxP-stop-loxP :: L7-cre) Bokella li-neuron. Kamora hore ho kopana hape ho phetheloe, e bitsoa Mfn2cKO ha e le libeke tse 4. E le ntlha ea ho qetela, re khethile libeke tse 8 tsa lilemo ha lera la PN le ne le sa senyeha ho sa tsotellehe karohano e totobetseng ea mitochondrial (Setšoantšo sa 1B le Setšoantšo sa S1A). Ka kakaretso, re lekantse kakaretso ea liprotheine tse 3013, tseo hoo e ka bang 22% ea tsona li neng li thehiloe ho litlhaloso tsa MitoCarta 2.0 tse thehiloeng ho proteome ea mitochondrial e le mitochondria (Setšoantšo sa 1E) (Setšoantšo sa 1E) (24). Tlhahlobo ea phapang ea polelo ea liphatsa tsa lefutso e entsoeng bekeng ea 8 e bontšitse hore ke 10.5% feela ea liprotheine tsohle tse neng li e-na le liphetoho tse kholo (Setšoantšo sa 1F le Setšoantšo sa S1F), tseo liprotheine tse 195 tsa tsona li neng li theohile 'me liprotheine tse 120 li ne li phahamisitsoe (Setšoantšo sa 1F). Hoa hlokomeleha hore "tlhahlobo ea tsela e ncha" ea sete ena ea data e bontša hore liphatsa tsa lefutso tse hlahisoang ka mokhoa o fapaneng haholo-holo ke tsa sete e thibetsoeng ea litsela tse itseng tsa metabolism (Setšoantšo sa 1G). Hoa thahasellisa hore, leha ho fokotseha ha litsela tse amanang le OXPHOS le calcium signaling ho tiisa ho kenngoa ha ho se sebetse hantle ha mitochondrial ho li-PN tse haelloang ke fusion, mekhahlelo e meng e kenyeletsang haholo metabolism ea amino acid e eketsehile haholo, e leng se tsamaellanang le metabolism e etsahalang ho li-PN tsa mitochondrial.
(A) Litšoantšo tse emelang tsa confocal tsa likarolo tsa cerebellar tsa litoeba tsa CTRL le Mfn2cKO tse bontšang tahlehelo e tsoelang pele ea li-PN (calbindin, gray); li-nuclei li ile tsa haneloa ka DAPI. (B) Tekanyo ea (A) (tlhahlobo ea tsela e le 'ngoe ea phapang, ***P<0.001; n = didikadikwe tse 4 ho isa ho tse 6 ho tsoa ho litoeba tse tharo). (C) Mosebetsi oa liteko. (D) Kabo ea 'mapa oa mocheso oa matšoao a ikhethileng ho Purkinje (holimo) le mefuta e meng ea lisele (bohareng). (E) Setšoantšo sa Venn se bontšang palo ea liprotheine tsa mitochondrial tse khethiloeng ho PN e khethiloeng. (F) Polane ea seretse se chesang ea liprotheine tse hlahisitsoeng ka mokhoa o fapaneng ho li-neurons tsa Mfn2cKO libekeng tse 8 (boleng ba ho khaola bohlokoa ba 1.3). (G) Tlhahlobo ea tsela ea boqapi e bontša litsela tse hlano tsa bohlokoa ka ho fetisisa tsa ho phahamisa (khubelu) le ho theola (putsoa) ho Mfn2cKO PN e khethiloeng e le libeke tse 8. Boemo ba karolelano ba polelo ea protheine ka 'ngoe e fumanoeng bo bontšoa. 'Mapa oa mocheso oa Grayscale: boleng ba P bo fetotsoeng. ns, ha bo bohlokoa.
Lintlha tsa Proteomics li bontšitse hore tlhahiso ea protheine ea li-complexes tsa I, III, le IV e ile ea fokotseha butle-butle. Li-complexes tsa I, III, le IV kaofela li ne li e-na le li-subunit tsa bohlokoa tse kentsoeng ke mtDNA, ha complex II, e neng e ngotsoe ke nuclear-code feela, e ne e sa amehe (Setšoantšo sa 2A le Setšoantšo sa S2A). . Ho latela liphetho tsa proteomics, immunohistochemistry ea likarolo tsa lisele tsa cerebellar e bontšitse hore boemo ba subunit ea MTCO1 (mitochondrial cytochrome C oxidase subunit 1) ea complex IV ho PN bo ile ba fokotseha butle-butle (Setšoantšo sa 2B). Subunit e kentsoeng ke mtDNA Mtatp8 e ile ea fokotseha haholo (Setšoantšo sa S2A), ha boemo ba steady-state ba subunit ea ATP synthase e kentsoeng ke nuclear-coded bo ile ba lula bo sa fetohe, e leng se lumellanang le complex e tsejoang ea ATP synthase subassembly F1 ha tlhahiso ea mtDNA e tsitsitse. Sebopeho se tsitsitse. Interrupt (7). Tlhahlobo ea boemo ba mtDNA ho li-PN tsa Mfn2cKO tse hlophisitsoeng ka karabelo ea ketane ea polymerase ea nako ea sebele (qPCR) e tiisitse phokotseho e fokolang ea palo ea kopi ea mtDNA. Ha ho bapisoa le sehlopha sa taolo, ha ba le libeke tse 8, ke hoo e ka bang 20% ​​feela ea boemo ba mtDNA e ileng ea bolokoa (Setšoantšo sa 2C). Ho latela liphetho tsena, ho daea ha confocal microscopy ea li-PN tsa Mfn2cKO ho sebelisitsoe ho lemoha DNA, ho bontša tšebeliso ea li-nucleotide tsa mitochondrial e itšetlehileng ka nako (Setšoantšo sa 2D). Re fumane hore ke bakhethoa ba bang feela ba amehang ho senyeheng ha protheine ea mitochondrial le karabelo ea khatello ea maikutlo ba ileng ba eketseha, ho kenyeletsoa Lonp1, Afg3l2 le Clpx, le lintlha tsa kopano ea OXPHOS complex. Ha ho liphetoho tse kholo maemong a liprotheine tse amehang ho apoptosis tse ileng tsa fumanoa (Setšoantšo sa S2B). Ka mokhoa o ts'oanang, re fumane hore mitochondria le likanale tsa endoplasmic reticulum tse amehang ho tsamaiseng calcium li na le liphetoho tse nyane feela (Setšoantšo sa S2C). Ho phaella moo, tlhahlobo ea liprotheine tse amanang le autophagy ha ea ka ea fumana liphetoho tse kholo, e leng se lumellanang le tlhahiso e bonahalang ea li-autophagosomes tse bonoang ka har'a 'mele ka immunohistochemistry le microscopy ea elektrone (Setšoantšo sa S3). Leha ho le joalo, ho se sebetse hantle ha OXPHOS ho tsoelang pele ho li-PN ho tsamaea le liphetoho tse totobetseng tsa mitochondrial tsa ultrastructural. Lihlopha tsa mitochondrial li ka bonoa 'meleng ea sele le lifateng tsa dendritic tsa Mfn2cKO PNs tse lilemo li 5 le 8,' me sebopeho sa lera la ka hare se bile le liphetoho tse tebileng (Setšoantšo sa S4, A le B). Ho latela liphetoho tsena tsa ultrastructural le phokotso e kholo ea mtDNA, tlhahlobo ea likarolo tsa cerebellar cerebral cerebral tse nang le tetramethylrhodamine methyl ester (TMRM) e bontšitse hore bokhoni ba lera la mitochondrial ho li-PN tsa Mfn2cKO bo fokotsehile haholo (Setšoantšo sa S4C).
(A) Tlhahlobo ea nako ea boemo ba tlhahiso ea motsoako oa OXPHOS. Nahana feela ka liprotheine tse nang le P<0.05 libekeng tse 8 (ANOVA ea litsela tse peli). Mohala o nang le matheba: Ha ho na phetoho ha e bapisoa le CTRL. (B) Ka letsohong le letšehali: Mohlala oa karolo ea cerebellar e ngotsoeng ka antibody ea anti-MTCO1 (bar ea sekala, 20 μm). Sebaka se nkiloeng ke 'mele ea lisele tsa Purkinje se koahetsoe ke 'mala o mosehla. Ka letsohong le letona: Tekanyo ea maemo a MTCO1 (tlhahlobo ea tsela e le 'ngoe ea phapang; n = lisele tse 7 ho isa ho tse 20 tse hlahlobiloeng ho tsoa ho litoeba tse tharo). (C) Tlhahlobo ea qPCR ea nomoro ea kopi ea mtDNA ho PN e hlophisitsoeng (tlhahlobo ea tsela e le 'ngoe ea phapang; n = litoeba tse 3 ho isa ho tse 7). (D) Ka letsohong le letšehali: Mohlala oa selae sa cerebellar se ngotsoeng ka antibody ea anti-DNA (bar ea sekala, 20 μm). Sebaka se nkiloeng ke 'mele ea lisele tsa Purkinje se koahetsoe ke 'mala o mosehla. Ka letsohong le letona: Tekanyo ea liso tsa mtDNA (tlhahlobo ea tsela e le 'ngoe ea phapang; n = lisele tse 5 ho isa ho tse 9 ho tsoa ho litoeba tse tharo). (E) Mohlala oa karolo e matla ea cerebellar e bonts'ang lisele tsa mitoYFP + Purkinje (motsu) ho rekoto ea clamp ea patch ea sele eohle. (F) Tekanyo ea mothapo oa IV. (G) Lirekoto tse emelang tsa ente ea hona joale ea depolarizing liseleng tsa CTRL le Mfn2cKO Purkinje. Tekanyo e kaholimo: Pulse ea pele e qalileng AP. Tekanyo e ka tlase: Maqhubu a AP a phahameng ka ho fetisisa. (H) Tekanyo ea li-input tsa postsynaptic tse sa reroang (sPSPs). Tekanyo ea rekoto e emelang le karolelano ea eona ea zoom li bontšoa ho (I). Tlhahlobo ea tsela e le 'ngoe ea phapang e hlahlobiloe n = lisele tse 5 ho isa ho tse 20 ho tsoa ho litoeba tse tharo. Lintlha li hlahisoa e le karolelano±SEM; *P<0.05; **P<0.01; ***P<0.001. (J) Mehlala e emelang ea AP e sa reroang e tlalehiloeng ho sebelisoa mokhoa oa clamp ea patch e nang le masoba. Tekanyo e kaholimo: Maqhubu a AP a phahameng ka ho fetisisa. Tekanyo e ka tlase: zoom ea AP e le 'ngoe. (K) Tekanyo ea maqhubu a tloaelehileng le a phahameng ka ho fetisisa a AP ho latela (J). Teko ea Mann-Whitney; n = lisele tse 5 li hlahlobiloe ho tsoa ho litoeba tse 'ne. Lintlha li hlahisoa e le karolelano ea ± SEM; ha li bohlokoa.
Tšenyo e totobetseng ea OXPHOS e fumanoe ho Mfn2cKO PN ea libeke tse 8, e leng se bontšang hore ts'ebetso ea 'mele ea li-neuron e sa tloaeleha haholo. Ka hona, re hlahlobile litšobotsi tsa motlakase tse sa sebetseng tsa li-neuron tse haelloang ke OXPHOS libekeng tse 4 ho isa ho tse 5 le libeke tse 7 ho isa ho tse 8 ka ho etsa lirekoto tsa clamp ea patch ea sele eohle ka har'a li-slices tsa cerebellar tse matla (Setšoantšo sa 2E). Ka mokhoa o sa lebelloang, bokhoni bo tloaelehileng ba lera la ho phomola le khanyetso ea ho kenya ea li-neuron tsa Mfn2cKO li ne li tšoana le taolo, leha ho ne ho e-na le liphapang tse poteletseng lipakeng tsa lisele (Tafole ea 1). Ka mokhoa o ts'oanang, libekeng tse 4 ho isa ho tse 5 tsa lilemo, ha ho liphetoho tse kholo kamanong ea motlakase oa hona joale (mokhahlelo oa IV) tse fumanoeng (Setšoantšo sa 2F). Leha ho le joalo, ha ho li-neuron tsa Mfn2cKO tse libeke tse 7 ho isa ho tse 8 tse ileng tsa pholoha mokhoa oa IV (mohato oa hyperpolarization), tse bontšang hore ho na le kutloisiso e hlakileng ea bokhoni ba hyperpolarization mohatong ona oa morao. Ka lehlakoreng le leng, ho li-neuron tsa Mfn2cKO, maqhubu a depolarizing a bakang ho ntšoa ha matla a ketso e pheta-phetoang (AP) a mamelleha hantle, ho bontšang hore mekhoa ea bona ea ho ntšoa ha e fapane haholo le ea li-neuron tsa taolo tsa libeke tse 8 (Tafole ea 1 le Setšoantšo sa 2G). Ka mokhoa o ts'oanang, maqhubu le boholo ba maqhubu a postsynaptic a sa reroang (sPSCs) li ne li tšoana le tsa sehlopha sa taolo, 'me makhetlo a liketsahalo a eketsehile ho tloha libekeng tse 4 ho isa libekeng tse 5 ho isa libekeng tse 7 ho isa libekeng tse 8 ka keketseho e ts'oanang (Setšoantšo sa 2, H le I). Nako ea ho hola ha synaptic ho li-PN (25). Liphetho tse ts'oanang li fumanoe kamora li-patches tsa PN tse phunyehileng. Sebopeho sena se thibela puseletso e ka bang teng ea liphoso tsa ATP tsa sele, joalo ka ha ho ka etsahala ho rekotong ea clamp ea patch ea sele eohle. Haholo-holo, bokhoni ba lera la phomolo le maqhubu a ho thunya a sa reroang a li-neuron tsa Mfn2cKO ha lia ka tsa ameha (Setšoantšo sa 2, J le K). Ka bokhutšoanyane, liphetho tsena li bontša hore li-PN tse nang le ho se sebetse hantle ha OXPHOS li ka sebetsana hantle le mekhoa ea ho ntša metsi khafetsa, e leng se bontšang hore ho na le mokhoa oa puseletso o li lumellang ho boloka likarabo tsa electrophysiological tse batlang li tloaelehile.
Lintlha li hlahisoa e le karolelano ea ± SEM (tlhahlobo ea tsela e le 'ngoe ea phapang, teko ea papiso e mengata ea Holm-Sidak; *P<0.05). Nomoro ea yuniti e bontšoa ka li-bracket.
Re qalile ho batlisisa hore na sehlopha sefe kapa sefe sebokeng sa data sa proteomics (Setšoantšo sa 1G) se kenyelletsa litsela tse ka loantšang khaello e matla ea OXPHOS, ka hona re hlalosa hore na ke hobane'ng ha PN e amehileng e ka boloka electrophysiology e batlang e tloaelehile (Setšoantšo sa 2, E ho isa ho K). . Tlhahlobo ea proteomics e bontšitse hore li-enzyme tse amehang ho catabolism ea li-amino acid tsa ketane e nang le makala (BCAA) li ne li ntlafalitsoe haholo (Setšoantšo sa 3A le Setšoantšo sa S5A), 'me sehlahisoa sa ho qetela acetyl-CoA (CoA) kapa succinyl CoA li ka tlatsa li-tricarboxylates potolohong ea arteriosclerosis Acid (TCA). Re fumane hore litaba tsa BCAA transaminase 1 (BCAT1) le BCAT2 ka bobeli li eketsehile. Li susumetsa mohato oa pele oa catabolism ea BCAA ka ho hlahisa glutamate ho tsoa ho α-ketoglutarate (26). Diyuniti tsohle tse etsang motsoako wa keto acid dehydrogenase (BCKD) wa ketane e nang le makala di a eketseha (motswako ona o susumetsa decarboxylation e latelang le e ke keng ya fetolwa ya masapo a carbon a BCAA a hlahang) (Setshwantsho sa 3A le Setshwantsho sa S5A). Leha ho le jwalo, ha ho diphetoho tse totobetseng ho BCAA ka boyona tse fumanweng ho PN e hlophisitsweng, e ka bang ka lebaka la ho amohelwa ha di-amino acid tsena tsa bohlokwa ka bongata kapa tshebediso ya mehlodi e meng (glucose kapa lactic acid) ho tlatsetsa potoloho ya TCA (Setshwantsho sa S5B). Di-PN tse se nang OXPHOS le tsona di bontshitse keketseho ya ho bola ha glutamine le mesebetsi ya transamination ha di le dibeke tse 8, e leng se ka bontshwang ke keketseho ya dienzyme tsa mitochondrial glutaminase (GLS) le glutamine pyruvate transaminase 2 (GPT2) (Setshwantsho sa 3, A le C). Ho bohlokoa ho hlokomela hore keketseho ea GLS e lekanyelitsoe ho spliced ​​​​isoform glutaminase C (GLS-GAC) (phetoho ea Mfn2cKO/CTRL e ka ba makhetlo a 4.5, P = 0.05), 'me keketseho ea eona e ikhethang liseleng tsa mofetše E ka tšehetsa bioenergy ea mitochondrial. (27).
(A) 'Mapa oa mocheso o bontša phetoho ea ho mena ha boemo ba protheine bakeng sa tsela e boletsoeng libekeng tse 8. (B) Mohlala oa selae sa cerebellar se ngotsoeng ka anti-PCx antibody (sekala sa bar, 20 μm). Motsu o mosehla o supa 'meleng oa sele ea Purkinje. (C) Tlhahlobo ea polelo ea protheine ea nako e khetholloang e le mokhethoa oa bohlokoa oa atherosclerosis (tlhahlobo ea t e ngata, *FDR <5%; n = litoeba tse 3-5). (D) Ka holimo: Setšoantšo sa schematic se bontšang litsela tse fapaneng tsa ho kena ka har'a carbon e ngotsoeng e fumanehang ho [1-13C]pyruvate tracer (ke hore, ka PDH kapa tsela ea trans-arterial). Tlase: Chate ea violin e bontša peresente ea carbon e nang le leibole e le 'ngoe (M1) e fetotsoeng ho aspartic acid, citric acid le malic acid kamora ho ngola lilae tse matla tsa cerebellar ka [1-13C]pyruvate (tlhahlobo ea t e kopantsoeng; ** P <0.01). (E) Tlhahlobo e felletseng ea nalane ea nako ea tsela e bontšitsoeng. Nahana feela ka liprotheine tse nang le P<0.05 libekeng tse 8. Mohala o nang le matheba: ha ho na boleng ba tokiso (tlhahlobo ea mahlakore a mabeli ea phapang; * P <0.05; *** P <0.001). Lintlha li hlahisoa e le karolelano±SEM.
Tlhahlobong ea rona, catabolism ea BCAA e fetohile e 'ngoe ea litsela tsa bohlokoa tsa ho ntlafatsa taolo. Ntlha ena e fana ka maikutlo a matla a hore bophahamo ba moea bo kenang potolohong ea TCA bo ka fetoloa ha PN e haelloa ke OXPHOS. Sena se ka emela mofuta o moholo oa ho kopanya methapo ea methapo ea kutlo, e ka bang le tšusumetso e tobileng ho fisioloji ea methapo le ho phela nakong ea ho boloka ho se sebetse hantle ha OXPHOS. Tumellanong le khopolo-taba ena, re fumane hore enzyme e kholo ea anti-atherosclerotic PCx e laoloa ka ho eketsehileng (Mfn2cKO/CTRL e fetoha ka makhetlo a ka bang 1.5; Setšoantšo sa 3A), se susumetsang phetoho ea pyruvate ho oxaloacetate (28), eo ho lumeloang hore e teng liseleng tsa boko. Polelo ea in e lekanyelitsoe ho astrocytes (29, 30). Tumellanong le liphetho tsa proteomics, microscopy ea confocal e bontšitse hore polelo ea PCx e eketsehile ka ho khetheha le ka mokhoa o makatsang ho li-PN tse haelloang ke OXPHOS, ha reactivity ea PCx e ne e lekanyelitsoe haholo liseleng tsa glial tsa Bergmann tse haufi tsa taolo (Setšoantšo sa 3B). Ho leka ka mokhoa o sebetsang keketseho e bonoang ea PCx, re ile ra phekola likaroloana tsa cerebellar tse matla ka [1-13C]pyruvate tracer. Ha pyruvate e ne e oxidized ke pyruvate dehydrogenase (PDH), leibole ea eona ea isotope e ile ea nyamela, Empa e kenyelletsoa lipakeng tsa potoloho ea TCA ha pyruvate e fetoloa ke liketso tsa methapo ea mali (Setšoantšo sa 3D). Ho tšehetsa lintlha tsa rona tsa proteomics, re bone palo e kholo ea matšoao a tsoang ho tracer ena ho aspartic acid ea likaroloana tsa Mfn2cKO, ha citric acid le malic acid le tsona li ne li e-na le mokhoa o itekanetseng, leha li sa bohlokoa (Setšoantšo sa 3D).
Ho li-neuron tsa dopamine tsa litoeba tsa MitoPark tse nang le ho se sebetse hantle ha mitochondrial ho bakoang ke li-neuron tsa dopamine tse senyang ka ho khetheha gene ea transcription factor A ea mitochondrial (Tfam) (Setšoantšo sa S6B), polelo ea PCx le eona e ile ea ntlafatsoa haholo (31), e leng se bontšang hore acetone acid arteriosclerosis Ho hlaha ha lefu lena ho laoloa nakong ea ho se sebetse hantle ha OXPHOS ea neuronal 'meleng. Hoa hlokomeleha hore ho fumanoe hore li-enzyme tse ikhethang (32-34) tse ka hlahisoang ho li-neuron tse ka amanang le arteriosclerosis li ntlafalitsoe haholo ho li-PN tse haellang ho OXPHOS, joalo ka propionyl-CoA carboxylase (PCC-A), Malonyl-CoA e fetola propionyl-CoA ho succinyl-CoA le mitochondrial malic enzyme 3 (ME3), eo karolo ea eona e ka sehloohong e leng ho khutlisa pyruvate ho tsoa ho malate (Setšoantšo sa 3, A le C) (33, 35). Ho phaella moo, re fumane keketseho e kgolo ho enzyme ya Pdk3, e etsang phosphoryl mme ka hona e etsa hore PDH (36) e se sebetse, athe ha ho diphetoho tse ileng tsa fumanwa ho enzyme ya Pdp1 e kenyang PDH tshebetsong kapa enzyme ya PDH ka boyona (Setshwantsho sa 3A). Ka ho tswela pele, ho Mern2cKO PNs, phosphorylation ya α1 subunit α (PDHE1α) subunit ya karolo ya pyruvate dehydrogenase E1 ya PDH complex ho Ser293 (e tsejwang ka ho thibela mosebetsi wa enzyme wa PDH) e ile ya ntlafatswa (Setshwantsho sa S6C) (Setshwantsho sa S6C). Pyruvate ha e na phihlello ya methapo ya madi.
Qetellong, re fumane hore tsela e ntle ea serine le glycine biosynthesis, potoloho e amanang ea mitochondrial folate (1C) le proline biosynthesis (Setšoantšo sa 1G le Setšoantšo sa S5C) kaofela li ntlafalitsoe haholo, ho ea ka litlaleho, nakong ea ts'ebetso ea ts'ebetso. Lisele tse potolohileng lia kenngoa tšebetsong ka ho se sebetse hantle ha mitochondrial (5-7). Tlhahlobo ea Confocal e tšehetsang lintlha tsena tsa proteomics e bontšitse hore ho PN e nang le OXPHOS e sieo, lilae tsa cerebellar tsa litoeba tse libeke tse 8 li ile tsa kenngoa serine hydroxymethyltransferase 2 (SHMT2), e leng enzyme ea bohlokoa ea potoloho ea mitochondrial folate. Karabelo e kholo ea 'mele ea ho itšireletsa mafung (Setšoantšo sa S5D). Lilaeeng tse 13 tsa cerebellar tse hlahisitsoeng ke CU-glucose, liteko tsa ho latela metabolism li tiisitse ho eketseha ha serine le proline biosynthesis, ho bontšang hore phallo ea carbon isoforms ho ea serine le proline e eketsehile (Setšoantšo sa S5E). Kaha dikarabelo tse kgothalletswang ke GLS le GPT2 di ikarabella bakeng sa tlhahiso ya glutamate ho tswa ho glutamine le transamination pakeng tsa glutamate le α-ketoglutarate, ho eketseha ha tsona ho bontsha hore di-neuron tse haellang OXPHOS di na le tlhoko e eketsehileng ya glutamate, Sena se ka rerelwa ho boloka biosynthesis e eketsehileng ya proline (Setshwantsho sa S5C). Ho fapana le diphetoho tsena, tlhahlobo ya proteomic ya di-astrocyte tsa cerebellar ho tswa ho ditoeba tsa Mfn2cKO tse ikgethang tsa PN di bontshitse hore ditsela tsena (ho kenyeletswa le di-antiperoxidases tsohle) ha di a fetoha haholo tlhahisong, ka hona di bontsha hore phetisetso ena ya metabolism e kgetha PN e senyehileng (Setshwantsho sa S6, D ho ya ho G).
Ka bokhutšoanyane, litlhahlobo tsena li senotse mekhoa e fapaneng haholo ea ts'ebetso ea nakoana ea litsela tse itseng tsa metabolism ho li-PN. Le hoja ts'ebetso e sa tloaelehang ea mitochondrial ea methapo ea kutlo e ka lebisa ho atherosclerosis ea pele le ho nchafatsoa ha 1C (Setšoantšo sa 3E le Setšoantšo sa S5C), esita le liphetoho tse ka boleloang esale pele polelong ea metsoako ea I le IV, liphetoho tlhahisong ea serine de novo li bonahala feela mekhahlelong ea morao. Ho se sebetse hantle ha OXPHOS (Setšoantšo sa 3E le Setšoantšo sa S5C). Liphuputso tsena li hlalosa ts'ebetso e latellanang eo ho eona mitochondrial e bakoang ke khatello ea maikutlo (potoloho ea 1C) le cytoplasmic (serine biosynthesis) li arabelang ka tsela e lumellanang le keketseho ea atherosclerosis potolohong ea TCA ho bopa bocha metabolism ea methapo.
Li-PN tse haelloang ke OXPHOS tse libeke tse 8 li ka boloka ts'ebetso ea ho hlasimolla khafetsa 'me tsa feta ho hokahanngoa ho hoholo ha metabolism ho lefella ho se sebetse hantle ha mitochondrial. Ho sibolloa hona ho hlahisa monyetla o khahlisang oa hore esita le motsotsong ona, lisele tsena li ka boela tsa fumana thuso ea kalafo ho liehisa kapa ho thibela ho senyeha ha methapo. Morao tjena. Re rarollotse monyetla ona ka mekhoa e 'meli e ikemetseng. Mokhoeng oa pele, re qapile vector ea adeno-associated virus (AAV) e itšetlehileng ka Cre e le hore MFN2 e ka hlahisoa ka mokhoa o khethiloeng ho li-PN tse haelloang ke OXPHOS ka har'a vivo (Setšoantšo sa S7A). AAV e kenyang MFN2 le gene ea moqolotsi oa litaba ea fluorescent mCherry (Mfn2-AAV) li netefalitsoe litsong tsa mantlha tsa li-neuron ka har'a vitro, e leng se ileng sa etsa hore MFN2 e hlahisoe ka mokhoa o itšetlehileng ka Cre 'me ea pholosa sebopeho sa mitochondrial, ka hona ea thibela neuromutation ho li-neurons tsa Mfn2cKO (Setšoantšo sa S7, B, D le E). Ka mor'a moo, re entse liteko tsa in vivo ho isa Mfn2-AAV ea libeke tse 8 ka mokhoa oa stereotactic ho cerebellar cortex ea Mfn2cKO le litoeba tsa taolo, 'me ra sekaseka litoeba tsa libeke tse 12 (Setšoantšo sa 4A). Litoeba tsa Mfn2cKO tse phekotsoeng li ile tsa shoa (Setšoantšo sa 1, A le B) (16). Phetisetso ea vaerase in vivo e ile ea fella ka tlhahiso e khethiloeng ea PN li-circles tse ling tsa cerebellar (Setšoantšo sa S7, G le H). Ente ea AAV ea taolo e hlahisang mCherry feela (Ctrl-AAV) ha ea ka ea ba le phello e kholo tekanyong ea ho senyeha ha methapo ea kutlo liphoofolong tsa Mfn2cKO. Ka lehlakoreng le leng, tlhahlobo ea Mfn2cKOs e fetisitsoeng ka Mfn2-AAV e bontšitse phello e kholo ea tšireletso ea lera la sele ea PN (Setšoantšo sa 4, B le C). Haholo-holo, bongata ba neuron bo bonahala bo batla bo sa khethoe ho liphoofolo tse laolang (Setšoantšo sa 4, B le C, le Setšoantšo sa S7, H le I). Pontšo ea MFN1 empa eseng MFN2 e sebetsa hantle ka ho lekana ho pholoseng lefu la methapo ea kutlo (Setšoantšo sa 4C le Setšoantšo sa S7, C le F), e leng se bontšang hore polelo ea ectopic MFN1 e ka tlatsetsa ka katleho khaello ea MFN2. Tlhahlobo e eketsehileng boemong ba PN e le 'ngoe e bontšitse hore Mfn2-AAV e pholositse haholo sebopeho sa mitochondria, ea etsa maemo a mtDNA a tloaelehileng, 'me ea fetola polelo e phahameng ea letšoao la anti-angiogenesis PCx ​​(Setšoantšo sa 4, C ho isa ho E). Tlhahlobo ea pono ea litoeba tse pholositsoeng tsa Mfn2cKO li le boemong ba ho phomola e bontšitse hore boemo ba tsona le matšoao a ho sisinyeha (motsamao oa S1 ho ea ho S3) li ntlafalitsoe. Qetellong, liteko tsena li bontša hore ho liehisa ho kenngoa hape ha MFN2 ho li-PN tse haellang haholo ho OXPHOS ho lekane ho khutlisetsa morao tšebeliso ea mtDNA le ho baka atherosclerosis, ka hona ho thibela ho senyeha ha axon le lefu la methapo ea kutlo ka har'a 'mele.
(A) Sekema se bontshang kemiso ya teko ya ho kenya AAV encoding MFN2 ha tsela ya metabolism e bontshitsweng e kentswe tshebetsong. (B) Ditshwantsho tse emelang tse ka hare tsa di-cerebellar tse dibeke tse 12 tse fetisitsweng dibekeng tse 8 ho dipeba tsa Mfn2cKO mme di ngotswe ka antibody ya anti-Calbindin. Ka ho le letona: Ho lekanya difaeba tsa axon. Sekala sa axon zoom ke 450 le 75 μm. (C) Ka ho le letshehadi: Tekanyo ya bongata ba sele ya Purkinje ho AAV transduction loop (AAV+) (tlhahlobo ya tsela e le nngwe ya phapang; n = dipeba tse 3). Ka ho le letona: tlhahlobo ya ho tsepamisa maikutlo ya mtDNA ho PN e fetisitsweng bekeng ya 12 (tlhahlobo ya t e sa pakwang; n = disele tse 6 ho tswa ho dipeba tse tharo). * P <0.05; ** P <0.01. (D) Di-micrograph tsa elektrone tse emelang phetiso ya di-PN tsa dikarolo tsa cerebellar tsa Mfn2cKO tse fetisitsweng ka di-vector tsa vaerase tse bontshitsweng. Maske e pinki e bontša sebaka se nkiloeng ke li-dendrite, 'me sekwere se sesehla se nang le matheba se bontša zoom e fanoeng ka ho le letona; n e emela nucleus. Sekala sa scale, 1μm. (E) se bontša mohlala oa ho taka ha PCx ho PN e fetotsoeng libekeng tse 12. Sekala sa scale, 20μm. OE, ho pepeseha ho feteletseng; FC, phetoho ea ho mena.
Qetellong, re batlisisitse bohlokoa ba ho pholoha ha sele e bakoang ke peroxidase ho li-PN tse bileng le bothata ba OXPHOS. Re hlahisitse mCherry encoding AAV-shRNA (moriri o mokhutšoanyane oa RNA) e shebaneng ka ho khetheha le PCx mRNA ea toeba (AAV-shPCx), 'me ra kenya vaerase kapa taolo ea eona e qhekelletsoeng (AAV-scr) ka har'a cerebellum ea litoeba tsa Mfn2cKO. Ente e entsoe bekeng ea bone ea lilemo (Setšoantšo sa 5A) ho fihlela PCx knockdown e sebetsang nakong eo tlhahiso ea PCx e neng e eketseha (Setšoantšo sa 3C) 'me lera la sele ea PN le ne le ntse le le teng (Setšoantšo sa 1A). Hoa bohlokoa ho hlokomela hore ho qhaqha PCx (Setšoantšo sa S8A) ho lebisa ho potlakisong e kholo ea lefu la PN, e leng se lekanyelitsoeng ho lesale le tšoaelitsoeng (Setšoantšo sa 5, B le C). E le ho utloisisa mokhoa oa liphello tsa metabolism tse bakoang ke ho phahamisoa ha PCx, re ithutile boemo ba redox ba li-PN kamora ho emisoa ha PCx ​​le AAV-mediated optical biosensor Grx1-roGFP2 li hlahisitsoe ka nako e le 'ngoe (Setšoantšo sa S8, B ho isa ho D) ho lekola phetoho e amanang le glutathione ea peptide redox potential (38). Eaba, re etsa microscopy ea imaging ea bophelo bohle ea photon fluorescence (FLIM) ka har'a li-slices tsa boko bo matla ba Mfn2cKO ea libeke tse 7 kapa li-littermate tsa taolo ho lemoha liphetoho tse ka bang teng boemong ba redox ea cytoplasmic kamora ho netefatsa maemo a FLIM (Setšoantšo sa S8, E ho isa ho G). Tlhahlobo e bontšitse keketseho e kholo boemong ba oxidation ba li-PN tse le 'ngoe tsa Mfn2cKO tse se nang tlhahiso ea PCx, tse fapaneng le li-neurons tsa taolo kapa li-PN tsa Mfn2cKO tse hlahisang shRNA e qhekelletsoeng feela (Setšoantšo sa 5, D le E). Ha tlhahiso ea PCx e ne e theohile, peresente ea li-PN tsa Mfn2cKO tse bonts'ang boemo bo nang le oxidized e ile ea eketseha ka makhetlo a fetang a mararo (Setšoantšo sa 5E), e leng se bontšang hore ho phahama ha taolo ea PCx ho bolokile bokhoni ba redox ba li-neurone tse senyehileng.
(A) Sekema se bontshang kemiso ya teko ya ho kenya AAV encoding shPCx ha tsela e bontshitsweng ya metabolism e kentswe tshebetsong. (B) Difoto tse emelang tse ka hare tsa dikarolo tsa cerebellar tsa dibeke tse 8 ho dipeba tsa Mfn2cKO tse fetisitsweng le ho ngolwa ka antibody ya anti-calcineurin dibekeng tse 4. Sekala sa tekanyo, 450μm. (C) Tekanyo ya bongata ba sele ya Purkinje ho di-loop tse fetisitsweng ke AAV (tlhahlobo ya tsela e le nngwe ya phapang; n = dipeba tse 3 ho isa ho tse 4). Lintlha di hlahiswa e le mekgahlelo±SEM; ***P<0.001. (D) Setshwantsho se emelang sa FLIM se bontsha nako e tloaelehileng ya bophelo ba sensor ya glutathione redox ya dibeke tse 7 ya PN e hlahisang glutathione redox Grx1-roGFP2 tlasa maemo a boletsweng a teko. Karolelano ya LUT (tafole ya ho sheba): nako ya ho phela (ka di-picosecond). Sekala sa tekanyo, 25μm. (E) Histogram e bonts'a kabo ea boleng ba bophelo ba Grx1-roGFP2 ho tloha ho (D) (n=158 ho isa ho lisele tse 368 ho litoeba tse peli tlas'a boemo ka bong). Chate ea phae ka holim'a histogram ka 'ngoe: e bonts'a palo ea lisele tse nang le boleng ba bophelo bo bolelele haholo (bofubelu, bo nang le oxidized) kapa bo bokhutšoanyane (boputsoa, ​​bo fokotsehileng), bo fetang 1 SD ea boleng ba bophelo bo tloaelehileng ho CTRL-AAV-scr. (F) Mohlala o sisintsoeng o bonts'a phello e sireletsang ea ho phahama ha PCx ea methapo.
Ka kakaretso, lintlha tseo re fanang ka tsona mona li bontša hore ho hlaha hape ha MFN2 ho ka pholosa PN e tsoetseng pele ka botlalo ka khaello e matla ea OXPHOS, ho fokotseha ho matla ha mtDNA, le sebopeho se sa tloaelehang haholo se kang ista, ka hona ho fana ka tsoelo-pele e tsoelang pele esita le mafung a tsoetseng pele. Ho senyeha ha methapo ea kutlo ho fana ka bopaki bo ka fetoloang ba mohato pele ho lefu la sele. Tekanyo ena ea ho tenyetseha ha metabolism e hatisoa hape ke bokhoni ba li-neuron ba ho baka atherosclerosis (ho kopanya hape potoloho ea TCA), e thibelang tlhahiso ea PCx ho li-PN tse haellang OXPHOS le ho eketsa lefu la sele, ka hona e bapala karolo ea tšireletso (Setšoantšo sa 5F).
Thutong ena, re fane ka bopaki ba hore karabelo ea li-PN ho ho se sebetse hantle ha OXPHOS ke ho kopana butle-butle le atherosclerosis ea potoloho ea TCA ka tsela e fapaneng ea ts'ebetso e hlahisoang ke mananeo a metabolism. Re tiisitse tlhahlobo ea proteomic ka mekhoa e mengata e tlatsanang 'me ra senola hore ha li phephetsoa ke ho se sebetse hantle ho matla ha mitochondrial, li-neuron li na le mofuta o sa tsejoeng oa ho tenyetseha ha metabolism pele. Ho makatsang ke hore ts'ebetso eohle ea ho kopanya hape ha e hlile ha e tšoaee boemo ba metabolic bo qetellang bo tsamaeang le neurodegeneration butle-butle le ka mokhoa o ke keng oa fetoloa, empa lintlha tsa rona li bontša hore e ka 'na ea e-ba neuron ea tlhokomelo esita le mohatong oa pele ho lefu la sele Mokhoa oa puseletso o sebetsang. Ho fumanoa hona ho bontša hore li-neuron li na le tekanyo e kholo ea metabolic plasticity 'meleng. Ntlha ena e paka hore ho khutlisetsoa hape ha MFN2 ho ka fetola polelo ea matšoao a bohlokoa a metabolism le ho thibela ho senyeha ha PN. Ho fapana le hoo, e thibela atherosclerosis le ho potlakisa methapo.
E 'ngoe ea lintho tse khahlisang ka ho fetisisa lipatlisisong tsa rona ke hore li-PN tse se nang OXPHOS li ka fetola metabolism ea potoloho ea TCA ka ho eketsa li-enzyme tse laolang arteriosclerosis ka ho khetheha. Ho hlophisoa bocha ha metabolism ke tšobotsi e tloaelehileng ea lisele tsa mofetše, tseo tse ling tsa tsona li itšetlehileng ka glutamine ho tlatsa li-intermediate tsa potoloho ea TCA ho hlahisa lintho tse fokotsang tse lekanang, tse tsamaisang ketane ea ho hema le ho boloka tlhahiso ea li-precursors tsa biosynthesis tsa lipid le nucleotide (39, 40). Phuputso ea morao tjena e bontšitse hore liseleng tse ka ntle tse nang le bothata ba OXPHOS, ho hokahanngoa hape ha metabolism ea glutamine/glutamate le hona ke tšobotsi e hlahelletseng (5, 41), moo tataiso ea ho kena ha glutamine potolohong ea TCA e itšetlehileng ka Ka lebaka la boima ba kotsi ea OXPHOS (41). Leha ho le joalo, ho na le khaello ea bopaki bo hlakileng mabapi le ho tšoana leha e le hofe ha plasticity ea metabolism ea neuronal 'meleng le bohlokoa ba eona boemong ba lefu lena. Phuputsong ea morao tjena ea in vitro, li-neuron tsa mantlha tsa cortical li bontšitsoe li hlohlelletsa matamo a glutamate bakeng sa phetisetso ea methapo, ka hona li khothalletsa metabolism ea oxidative le atherosclerosis tlas'a maemo a khatello ea metabolism (42). Hoa hlokomeleha hore tlas'a thibelo ea meriana ea enzyme ea potoloho ea TCA succinate dehydrogenase, pyruvate carboxylation ho lumeloa hore e boloka tlhahiso ea oxaloacetate ka har'a li-neurons tsa cerebellar granule tse lengoang (34). Leha ho le joalo, bohlokoa ba 'mele ba mekhoa ena ho lisele tsa boko (moo ho lumeloang hore atherosclerosis e lekanyelitsoe haholo ho li-astrocyte) bo ntse bo na le bohlokoa ba 'mele (43). Tabeng ena, lintlha tsa rona li bontša hore li-PN tse senyehileng ke OXPHOS 'meleng li ka fetoloa ho senyeha ha BCAA le pyruvate carboxylation, e leng mehloli e 'meli e meholo ea tlatsetso ea li-intermediate tsa letamo la TCA. Leha monehelo o belaelloang oa BCAA catabolism ho metabolism ea matla a neuronal o se o sisintsoe, ho phaella ho karolo ea glutamate le GABA bakeng sa phetisetso ea methapo (44), ha ho na bopaki ba mekhoa ena ka har'a 'mele. Ka hona, ho bonolo ho nahana hore li-PN tse sa sebetseng li ka lefella ka bohona tšebeliso ea li-intermediate tsa TCA tse susumetsoang ke ts'ebetso ea ho monya ka ho eketsa atherosclerosis. Haholo-holo, ho ka hlokahala keketseho ea PCx ho boloka tlhoko e eketsehileng ea aspartic acid, e leng se khothalletsoang ho ata ha lisele tse nang le bothata ba mitochondrial (45). Leha ho le joalo, tlhahlobo ea rona ea metabolomics ha ea ka ea senola liphetoho tse kholo boemong bo tsitsitseng ba aspartic acid ho Mfn2cKO PNs (Setšoantšo sa S6A), seo ho ka etsahalang hore se bonts'a ts'ebeliso e fapaneng ea metabolism ea aspartic acid lipakeng tsa lisele tse atang le li-neurone tsa post-mitotic. Leha mokhoa o nepahetseng oa keketseho ea PCx ho li-neurone tse sa sebetseng hantle ka har'a vivo o ntse o lokela ho hlalosoa, re bontšitse hore karabelo ena ea pele ho nako e bapala karolo ea bohlokoa ho bolokeng boemo ba redox ba li-neurone, bo bontšitsoeng litekong tsa FLIM holim'a li-cerebellar slices. Haholo-holo, ho thibela li-PN ho phahamisa PCx ho ka lebisa boemong ba oxidized haholoanyane le ho potlakisa lefu la sele. Ho kengoa tšebetsong ha BCAA degeneration le carboxylation ea pyruvate ha se litsela tsa ho khetholla lisele tse ka ntle tsa ho se sebetse hantle ha mitochondrial (7). Ka hona, ho bonahala e le tšobotsi ea bohlokoa ea li-neurone tse haelloang ke OXPHOS, leha e se eona feela tšobotsi, e leng ea bohlokoa bakeng sa ho senyeha ha methapo ea kutlo.
Lefu la Cerebellar ke mofuta o fapaneng oa lefu la methapo le atisang ho iponahatsa e le ataxia 'me hangata le senya li-PN (46). Palo ena ea li-neuron e kotsing haholo ea ho se sebetse hantle ha mitochondrial hobane ho senyeha ha tsona ho khethollang litoeba ho lekane ho hlahisa matšoao a mangata a tsamaiso a khethollang ataxia ea spinocerebellar ea motho (16, 47, 48). Ho ea ka litlaleho, mohlala oa toeba ea transgenic o nang le gene e fetotsoeng o amahanngoa le ataxia ea spinocerebellar ea motho 'me o na le ho se sebetse hantle ha mitochondrial (49, 50), ho hatisa bohlokoa ba ho ithuta liphello tsa khaello ea OXPHOS ho PNPH. Ka hona, ho loketse haholo ho arola le ho ithuta palo ena e ikhethang ea li-neuron ka katleho. Leha ho le joalo, kaha li-PN li na le kutloelo-bohloko haholo khatellong 'me li ikarabella bakeng sa karolo e tlase ea palo eohle ea lisele tsa cerebellar, bakeng sa lithuto tse ngata tse thehiloeng ho omics, karohano e khethollang ea tsona e le lisele tse felletseng e ntse e le karolo e phephetsang. Leha ho batla ho le thata ho fihlella khaello e felletseng ea tšilafalo ea mefuta e meng ea lisele (haholo-holo lisele tsa batho ba baholo), re kopantse mohato o atlehang oa ho arohana le FACS ho fumana palo e lekaneng ea li-neurone tse sebetsang bakeng sa tlhahlobo ea proteomics e theohelang, 'me re na le tšireletso e phahameng haholo ea protheine (liprotheine tse ka bang 3000) ha li bapisoa le sete ea data e teng ea cerebellum eohle (51). Ka ho boloka matla a lisele tsohle, mokhoa oo re o fanang ka mona o re lumella eseng feela ho hlahloba liphetoho litseleng tsa metabolism mitochondria, empa hape le ho hlahloba liphetoho ho li-sytoplasmic counterparts tsa eona, tse tlatsang tšebeliso ea li-tag tsa mitochondrial membrane ho ntlafatsa mofuta oa sele Mokhoa o mocha oa palo ea mitochondria liseleng tse rarahaneng (52, 53). Mokhoa oo re o hlalosang ha o amane feela le thuto ea lisele tsa Purkinje, empa o ka sebelisoa habonolo ho mofuta ofe kapa ofe oa sele ho sebetsana le liphetoho tsa metabolism bokong bo kulang, ho kenyeletsoa le mehlala e meng ea ho se sebetse hantle ha mitochondrial.
Qetellong, re hlwaile fensetere ea phekolo nakong ea ts'ebetso ena ea ho hlophisa bocha metabolism e ka fetolang ka botlalo matšoao a bohlokoa a khatello ea sele le ho thibela ho senyeha ha methapo ea kutlo. Ka hona, ho utloisisa litlamorao tsa ts'ebetso ea ho kopanya likhoele tse hlalositsoeng mona ho ka fana ka temohisiso ea motheo mabapi le mekhoa ea kalafo e ka bang teng bakeng sa ho boloka bophelo ba methapo ea kutlo nakong ea ho se sebetse hantle ha mitochondrial. Lipatlisiso tsa nakong e tlang tse reretsoeng ho sekaseka liphetoho metabolism ea matla mefuteng e meng ea lisele tsa boko lia hlokahala ho senola ka botlalo hore na molao-motheo ona o sebetsa joang mafung a mang a methapo ea kutlo.
Litoeba tsa MitoPark li se li hlalositsoe pele (31). Litoeba tsa C57BL/6N tse nang le liphatsa tsa lefutso tsa Mfn2 tse nang le loxP li hlalositsoe pele (18) 'me tsa kopanngoa le litoeba tsa L7-Cre (23). Litholoana tse peli tse hlahang tse nang le heterozygous li ile tsa kopanngoa le litoeba tsa Mfn2loxP/Mfn2loxP tse nang le homozygous ho hlahisa litoeba tse ikhethileng tsa liphatsa tsa lefutso tsa Purkinje bakeng sa Mfn2 (Mfn2loxP/Mfn2loxP; L7-cre). Karolong e nyenyane ea ho kopana, allele ea Gt (ROSA26) SorStop-mito-YFP (stop-mtYFP) e ile ea hlahisoa ka ho kopanya ho eketsehileng (20). Mekhoa eohle ea liphoofolo e entsoe ho latela litataiso tsa Europe, tsa naha le tsa setheo 'me ea amoheloa ke LandesamtfürNatur ea Umwelt le Verbraucherschutz, North Rhine-Westphalia, Jeremane. Mosebetsi oa liphoofolo o boetse o latela tataiso ea Mokhatlo oa Europe oa Li-Laboratory Animal Sciences Associations.
Kamora ho robatsa molomo oa popelo oa mosali oa moimana, lesea le sa tsoa emoloa lea aroloa (E13). cortex e ile ea senngoa ka Hanks' Balanced Salt Solution (HBSS) e tlatselitsoeng ka 10 mM Hepes 'me ea fetisetsoa ho Dulbecco's Modified Eagle's Medium e nang le papain (20 U/ml) le cysteine ​​​​(1μg/ml). Kenya lisele ho DMEM) 'me u li arole ka tšilo ea lik'hemik'hale. Ml) mochesong oa 37°C metsotso e 20, ebe u siloa ka mechine ho DMEM e tlatselitsoeng ka 10% ea serum ea likhomo tsa fetal. Lisele li ile tsa jaloa holim'a likhalase tse koahetsoeng ka polylysine ka bongata ba 2 × 106 ka sejana sa tlhahiso sa 6 cm kapa ka bongata ba lisele tsa 0.5 × 105 / cm2 bakeng sa tlhahlobo ea litšoantšo. Kamora lihora tse 4, setshelo se ile sa nkeloa sebaka ke setshelo se se nang serum sa Neurobasal se nang le 1% B27 supplement le 0.5 mM GlutaMax. Di-neurone di ile tsa bolokwa mochesong wa 37°C le 5% CO2 nakong yohle ya teko, mme tsa fepuwa hang ka beke. E le ho susumetsa ho kopana hape ka hara vitro, 3μl (sejana sa temo sa di-well tse 24) kapa 0.5μl (poleiti ya di-well tse 24) ya vector e latelang ya vaerase ya AAV9 e ile ya sebediswa ho phekola di-neurone letsatsing la bobedi ka hara vitro: AAV9.CMV.PI.eGFP. WPRE.bGH (Addgene, nomoro ya khataloganyo 105530-AAV9) le AAV9.CMV.HI.eGFP-Cre.WPRE.SV40 (Addgene, nomoro ya khataloganyo 105545-AAV9).
DNA e tlatsetsang ea Mouse Mfn1 le Mfn2 (e fumanoeng ho tsoa ho Addgene plasmid #23212 le #23213, ka ho latellana) li tšoailoe ka tatellano ea V5 (GKPIPNPLLGLDST) ho C-terminus, 'me li kopantsoe le mCherry ka foreimi ho pholletsa le tatellano ea T2A. Grx1-roGFP2 ke mpho e tsoang ho Heidelberg TP Dick DFKZ (Deutsches Krebsforschungszentrum). Ka ho nkela khasete ea tdTomato sebaka ho sebelisoa mekhoa e tloaelehileng ea ho kopanya, khasete e ile ea kenngoa mokokotlong oa pAAV-CAG-FLEX-tdTomato (nomoro ea referense ea Addgene 28306) ho hlahisa li-vector tsa pAAV-CAG-FLEX-mCherry-T2A-MFN2-V5, pAAV-CAG-FLEX-mCherry-T2A-MFN1-V5 le pAAV-CAG-FLEX-Grx-roGFP2. Leano le tšoanang le sebelisitsoe ho hlahisa vekthara ea taolo ea pAAV-CAG-FLEX-mCherry. E le ho hlahisa sebopeho sa AAV-shPCx, vekthara ea plasmid AAV (VectorBuilder, pAAV [shRNA] -CMV-mCherry-U6-mPcx- [shRNA#1]) ea hlokahala, e nang le tatellano ea DNA e kenyang PCx ea toeba e shebaneng le shRNA (5′CTTTCGCTCTAAGGTGCTAAACTCGAGTTTAGCACCCTTAGAGCGAAAG 3′) Tlas'a taolo ea promoter ea U6, mCherry e sebelisoa tlas'a taolo ea promoter ea CMV. Tlhahiso ea li-vekthara tse thusang tsa AAV e entsoe ho latela litaelo tsa moetsi (Cell Biolabs). Ka bokhutšoanyane, sebelisa plasmid e fetisang e jereng mCherry-T2A-MFN2-V5 (pAAV-CAG-FLEX-mCherry-T2A-MFN2-V5), mCherry-T2A-MFN1-V5 (pAAV-CAG-FLEX-mCherry) ka nakoana Phetisetso ea lisele tse 293AAV-T2A-MFN1-V5), mCherry (pAAV-CAG-FLEX-mCherry) kapa gene ea ho ngola Grx-roGFP2 (pAAV-CAG-FLEX-Grx-roGFP2), hammoho le ho ngola protheine ea capsid ea AAV1 le protheine ea lisebelisoa Packaging plasmid plasmid, ho sebelisoa mokhoa oa calcium phosphate. Supernatant ea kokoana-hloko e tala e fumanoe ka lipotoloho tsa ho qhibilihisa leqhoa le omileng/ethanol bath le lisele tse lysed ka har'a saline ea phosphate buffered (PBS). Vekthara ea AAV e hloekisitsoe ka ho se tsoe ho iodixanol gradient ultracentrifugation (lihora tse 24 ka lebelo la 32,000 rpm le 4°C) 'me ea hokahanngoa ho sebelisoa sefe sa Amicon ultra-15 centrifugal. Tekanyo ea genome ea 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 e ne e le joalo ka ha ho hlalositsoe pele (54), e lekantsoe ka real-time quantitative PCR (qPCR) -MFN1-V5 (1.9×1013 GC/ml) le AAV1-CAG-FLEX-Grx-roGFP2 (8.9×1012 GC/ml).
Li-neurone tsa mantlha li ile tsa hohloa ka har'a 1x PBS e batang haholo, tsa pelletsoa, ​​​​'me tsa fetoloa homogenized ka har'a 0.5% Triton X-100 / 0.5% sodium deoxycholate/PBS lysis buffer e nang le phosphatase le protease inhibitor (Roche). Ho lekanngoa ha protheine ho entsoe ka ho sebelisa bicinchoninic acid assay (Thermo Fisher Scientific). Liprotheine li ile tsa aroloa ka SDS-polyacrylamide gel electrophoresis, 'me tsa tlosoa holim'a polyvinylidene fluoride membrane (GE Healthcare). Thibela libaka tse seng tse ikhethileng 'me u li fuame ka antibody ea mantlha (sheba Tafole S1 bakeng sa lintlha tse qaqileng) ka lebese la 5% ho TBST (Tris-buffered saline with Tween), mehato ea ho hlatsoa le antibody ea bobeli ho TBST Incubate. Fuame ka antibody ea mantlha bosiu bohle ho +4°C. Kamora ho hlatsoa, ​​​​sebelisa antibody ea bobeli lihora tse 2 mochesong oa kamore. Ka mor'a moo, ka ho fuame blot e tšoanang ka antibody ea anti-β-actin, mojaro o tšoanang o ile oa tiisoa. Ho lemoha ka ho fetohela ho chemiluminescence le ho ntlafatsa chemiluminescence (GE Healthcare).
Li-neuron tse neng li peoe pele holim'a li-covers tsa khalase li ile tsa lokisoa ka 4% paraformaldehyde (PFA)/PBS nakong e boletsoeng mochesong oa kamore metsotso e 10. Li-covers li qala ka ho kenngoa ka 0.1% Triton X-100/PBS metsotso e 5 mochesong oa kamore, ebe li kenngoa ka har'a blocking buffer [3% bovine serum albumin (BSA)/PBS]. Ka letsatsi la bobeli, li-covers li ile tsa hlatsuoa ka blocking buffer 'me tsa kenngoa ka antibody ea secondary e kopantsoeng le fluorophore ka lihora tse 2 mochesong oa kamore; qetellong, lisampole li ile tsa hlatsuoa hantle ka har'a PBS ka 4′,6-diamidino-2 -Phenylindole (DAPI) e hanyetsoang ebe e kenngoa ka har'a microscope slide ka Aqua-Poly/Mount.
Litoeba (tse tona le tse tšehali) li ile tsa robatsoa ka ente ea ketamine (130 mg/kg) le xylazine (10 mg/kg) ka hare ho peritoneal 'me tsa fuoa ka tlas'a mpa ka carprofen analgesic (5 mg/kg), 'me tsa kenngoa ka har'a sesebelisoa sa stereotactic (Kopf) se nang le pad e mofuthu. Pepesa lehata 'me u sebelise drill ea meno ho tšesaane karolo ea cerebellar cortex e tsamaellanang le mis bone (ho tloha lambda: mohatla 1.8, lateral 1, e tsamaellanang le lobules IV le V). Sebelisa nale ea syringe e kobehileng ho etsa lesoba le lenyenyane ka hloko lehateng ho qoba ho sitisa methapo ea mali ka tlase. Ebe capillary e tšesaane ea khalase e huloang e kenngoa butle-butle ka har'a lesoba le lenyenyane (ho tloha -1.3 ho isa ho -1 ka lehlakoreng la ventral la dura mater), 'me 200 ho isa ho 300 nl AAV e kenngoa ka har'a micro-injector (Narishige) ka li-syringe tsa letsoho (Narishige) ka makhetlo a 'maloa ka khatello e tlase ka nako e telele metsotso e 10 ho isa ho e 20. Kamora ho kenngoa ha moriana, beha capillary metsotso e meng e 10 ho lumella vaerase ho ata ka botlalo. Kamora hore methapo ea mali e ntšoe, letlalo le hohoa ka hloko ho fokotsa ho ruruha ha maqeba le ho lumella phoofolo ho fola. Liphoofolo li ile tsa phekoloa ka meriana e kokobetsang bohloko (caspofen) matsatsi a 'maloa kamora opereishene, eo ka nako eo boemo ba tsona ba' mele bo ileng ba hlahlojoa ka hloko ebe li bolaoa ka nako e boletsoeng. Mekhoa eohle e ile ea etsoa ho latela litataiso tsa Europe, tsa naha le tsa mekhatlo 'me ea amoheloa ke LandesamtfürNatur ea Umwelt le Verbraucherschutz, North Rhine-Westphalia, Jeremane.
Liphoofolo li ile tsa fuoa ketamine (100 mg/kg) le xylazine (10 mg/kg), 'me pelo ea tšeloa ka 0.1 M PBS pele, eaba ka 4% PFA ho PBS. Linama li ile tsa senngoa 'me tsa lokisoa ka 4% PFA/PBS bosiu bohle ho 4°C. Thipa e thothomelang (Leica Microsystems GmbH, Vienna, Austria) e ile ea sebelisoa ho lokisa likarolo tsa sagittal (tse teteaneng ka 50 μm) ho tloha bokong bo tsitsitseng ho PBS. Ntle le haeba ho boletsoe ka tsela e 'ngoe, ho silafatsa likarolo tse phaphametseng ka bolokolohi ho entsoe joalo ka ha ho hlalositsoe kaholimo (13) mochesong oa kamore le ho hlohlelletsa. Ka bokhutšoanyane, pele, likotoana tse fumanoeng li ile tsa koaheloa ka 0.5% Triton X-100/PBS metsotso e 15 mochesong oa kamore; bakeng sa li-epitopes tse ling (Pcx le Shmt2), ka ho kenya tris-EDTA buffer ho 80°C (PH 9) futhumatsa likotoana metsotso e 25 ho fapana le mohato ona. Ka mor'a moo, likarolo li ile tsa kenngoa ka antibody ea mantlha (sheba Tafole S1) ka har'a buffer e thibelang (3% BSA/PBS) ho 4°C bosiu ka ho hlohlelletsa. Letsatsing le hlahlamang, likarolo li ile tsa hlatsuoa ka buffer e thibelang 'me tsa kenngoa ka antibody ea bobeli e loketseng e kopantsoeng le fluorophore ka lihora tse 2 mochesong oa kamore; qetellong, likarolo li ile tsa hlatsuoa hantle ka PBS, tsa silafatsoa ka DAPI, 'me tsa lokisoa ka AquaPolymount On microscope slide.
Ho sebedisitsoe microscope ya confocal e hlahlobang ka laser (TCS SP8-X kapa TCS Digital Light Sheet, Leica Microsystems) e nang le laser e tshweu ya lesedi le laser ya 405 diode ultraviolet ho etsa setshwantsho sa sampole. Ka ho hlasimolla fluorophore le ho bokella letshwao ka Hybrid Detector (HyDs), software ya LAS-X e sebedisitsweng ho bokella ditshwantsho tse pakilweng tse tsamaellanang le sampling ya Nyquist ka mokgwa o latellanang: bakeng sa diphanele tse seng tsa bongata, ke matshwao a matla haholo (mohlala, diseleng tsa somatic le di-dendrites) mtYFP) Sebedisa HyD ho lemoha palo ya di-PN ka mokgwa wa BrightR). Gating ho tloha ho 0.3 ho isa ho 6 ns e sebediswa ho fokotsa bokamorao.
Ho nka ditshwantsho tsa nako ya sebele tsa disele tse hlophisitsweng. Kamora ho hlopha ka hara Neurobasal-A medium e nang le 1% B27 supplement le 0.5 mM GlutaMax, disele di ile tsa mela hang-hang hodima di-slide tsa khalase tse manehilweng ka poly-l-lysine (μ-Slide8 Well, Ibidi, nomoro ya khataloganyo 80826), Mme ebe di di boloka mochesong wa 37°C le 5% CO2 ka hora e le nngwe ho dumella disele ho dula. Ho nka ditshwantsho tsa nako ya sebele ho entswe hodima microscope ya confocal ya Leica SP8 scanning laser e nang le laser e tshweu, HyD, lense ya oli ya 63×[1.4 numeral aperture (NA)] le mohato wa ho futhumatsa.
Toeba e ile ea robatsoa ka potlako ka carbon dioxide 'me ea khaoloa hlooho, boko bo ile ba tlosoa ka potlako lehateng, 'me ba seha ka botenya ba 200μm (bakeng sa teko ea ho ngola mabitso ea 13C) kapa botenya ba 275μm (bakeng sa liteko tse peli tsa photon) karolo ea sagittal e tlatsitsoeng ka lisebelisoa tse latelang. Ice cream (HM-650 V, Thermo Fisher Scientific, Walldorf, Jeremane) e tletse lintho tse latelang: 125 mM e batang ka leqhoa, e tletseng khabone (95% O2 le 5% CO2) Ca2 e tlase + mokelikeli oa maiketsetso oa cerebrospinal (ACSF) NaCl, 2.5 mM KCl, 1.25 mM sodium phosphate buffer, 25 mM NaHCO3, 25 mM glucose, 0.5 mM CaCl2 le 3.5 mM MgCl2 (khatello ea osmotic ea 310 ho isa ho 330 mmol). Fetisetsa dikotwana tsa boko tse fumanweng ka phaposing ya pele ho ho ikatisa e nang le Ca2 + ACSF e phahameng (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 le 2.0 mM MgCl2) Medium) pH 7.4 le 310 ho isa ho 320 mmol).
Nakong ea ts'ebetso ea ho nka litšoantšo, likotoana li ile tsa isoa ka phapusing e khethehileng ea ho nka litšoantšo, 'me teko e entsoe tlas'a phallo e tsoelang pele ea ACSF mochesong o sa fetoheng oa 32° ho isa ho 33°C. Microscope ea ho hlahloba laser e nang le li-photon tse ngata (TCS SP8 MP-OPO, Leica Microsystems) e nang le lense ea Leica 25x objective (NA 0.95, metsi), Ti: Sapphire laser (Chameleon Vision II, Coherent) e sebelisitsoe bakeng sa ho nka litšoantšo tsa likotoana. FLIM module (PicoHarp300, PicoQuant).
FLIM ea Grx1-roGFP2. Liphetoho boemong ba redox ea cytoplasmic ea li-PN li lekantsoe ka FLIM ea li-photon tse peli ka har'a li-slices tsa boko ba sagittal, moo biosensor ea Grx1-roGFP2 e neng e shebane le li-PN. Lera la PN, tšimo ea ho fumana e khethoa hoo e ka bang 50 ho isa ho 80 μm ka tlase ho bokaholimo ba selae ho netefatsa hore ho na le PN e sebetsang (ke hore, khaello ea sebopeho se nang le lifaha kapa liphetoho tsa sebopeho sa neuronal ho potoloha li-dendrites) le sensor ea roGFP2 e nang le double positive le AAV e kenyang shRNA PCx kapa tatellano ea eona ea taolo (mCherry e 'ngoe le e 'ngoe e bonts'ang hammoho). Bokella litšoantšo tsa stack e le 'ngoe ka zoom ea dijithale ea 2x [bolelele ba bolelele ba thabo: 890 nm; 512 nm 512 pixels]. Ho lemoha: HyD ea ka hare, sehlopha sa sefe sa fluorescein isothiocyanate (FITC)] le karolelano ea setšoantšo ka har'a metsotso e 2 ho isa ho e 3 li sebelisoa ho netefatsa hore li-photon tse lekaneng li bokelloa (li-photon tse 1000 ka kakaretso) bakeng sa ho lokisa curve. Kutloisiso ea probe ea Grx1-roGFP2 le netefatso ea maemo a FLIM li entsoe ka ho beha leihlo boleng ba bophelo ba roGFP2 ha ho eketsoa 10 mM H2O2 ea kantle ho ACSF ea phallo ea mali (ho eketsa oxidation, e fellang ka keketseho ea bophelo), ebe ho eketsoa 2 mM dithiothreitol (e fokotsa tekanyo ea phokotso, e fellang ka phokotso ea bophelo) (Setšoantšo sa S8, D ho isa ho G). Sebelisa software ea FLIMfit 5.1.1 ho sekaseka liphetho tse fumanoeng, ho lekana le lekhalo le le leng la ho bola ha exponential la setšoantšo sohle ho IRF e lekantsoeng (mosebetsi oa karabelo ea sesebelisoa), 'me χ2 e batla e le 1. Ho bala bophelo ba PN e le 'ngoe, maske e potolohileng 'mele oa methapo e ile ea huloa ka letsoho, 'me bophelo bo tloaelehileng ba maske ka 'ngoe bo ile ba sebelisoa bakeng sa ho lekanya.
Tlhahlobo ea bokhoni ba Mitochondrial. Kamora hore karolo e bohale e kenngoe ka 100 nM TMRM e eketsoe ka ho toba ho ACSF e hloekisitsoeng metsotso e 30, liphetoho tsa bokhoni ba mitochondrial tsa li-PN li ile tsa lekanngoa ka microscope ea li-photon tse peli. Setšoantšo sa TMRM se entsoe ka ho hlasimolla probe ho 920 nm le ho sebelisa HyD ea kahare (tetramethylrhodamine isothiocyanate: 585/40 nm) ho bokella matšoao; ka ho sebelisa bolelele bo tšoanang ba ho hlasimolla empa ho sebelisoa HyD ea kahare e fapaneng (FITC :525/50) ho etsa setšoantšo sa mtYFP. Sebedisa plug-in ea ImageJ's Image Calculator ho lekola bokhoni ba mitochondrial boemong ba sele e le 'ngoe. Ka bokhutšoanyane, equation ea plug-in: signal = min (mtYFP, TMRM) e sebelisoa ho khetholla sebaka sa mitochondrial se bontšang letšoao la TMRM ho Purkinje Somali setšoantšong sa confocal se nang le stack e le 'ngoe sa kanale e tsamaellanang. Ebe sebaka sa pixel ka har'a maske e hlahang sea lekanngoa, ebe se etsoa hore se be boemong bo tloaelehileng setšoantšong se tsamaellanang sa moeli o le mong oa kanale ea mtYFP ho fumana karolo ea mitochondrial e bonts'ang bokhoni ba mitochondrial.
Setšoantšo se ile sa tlosoa ka software ea Huygens Pro (Scientific Volume Imaging). Bakeng sa litšoantšo tse skenoeng tsa lithaele, montage ea thaele e le 'ngoe e etsoa ho sebelisoa mokhoa oa ho roka o iketsang o fanoeng ke software ea LAS-X. Kamora ho lekanya setšoantšo, sebelisa ImageJ le Adobe Photoshop ho sebetsana le setšoantšo haholoanyane le ho fetola khanya le phapang ka mokhoa o ts'oanang. Sebelisa Adobe Illustrator bakeng sa ho lokisa litšoantšo.
Tlhahlobo ea ho tsepamisa maikutlo ho mtDNA. Palo ea liso tsa mtDNA e lekantsoe likarolong tsa cerebellar tse ngotsoeng ka li-antibodies khahlanong le DNA ka microscope ea confocal. Sebaka se seng le se seng se reretsoeng se entsoe bakeng sa 'mele oa sele le nucleus ea sele ka 'ngoe, 'me sebaka se amehang se baliloe ho sebelisoa Multi Measure plug-in (software ea ImageJ). Tlosa sebaka sa nyutlelie sebakeng sa 'mele oa sele ho fumana sebaka sa cytoplasmic. Qetellong, plug-in ea Analyse Particles (software ea ImageJ) e sebelisitsoe ho lekanya ka bohona lintlha tsa cytoplasmic DNA tse bontšang mtDNA setšoantšong sa moeli, 'me liphetho tse fumanoeng li ile tsa tloaeleha ho ea ho karolelano ea PN ea litoeba tsa CTRL. Liphetho li hlahisoa e le palo e tloaelehileng ea li-nucleoside ka sele.
Tlhahlobo ea tlhahiso ea protheine. Sebelisa plug-in ea ImageJ's Image Calculator ho lekola tlhahiso ea protheine ho PN boemong ba sele e le 'ngoe. Ka bokhutšoanyane, setšoantšong sa confocal sa lera le le leng sa kanale e tsamaellanang, ka equation: signal = min (mtYFP, antibody), sebaka sa mitochondrial se bontšang ho itšireletsa mafung ho antibody e itseng ho Purkina sea khetholloa. Ebe sebaka sa pixel maskeng e hlahang sea lekanngoa, ebe se etsoa hore se be boemong bo tloaelehileng setšoantšong se tsamaellanang sa moeli o le mong oa kanale ea mtYFP ho fumana karolo ea mitochondrial ea protheine e bontšitsoeng.
Tlhahlobo ea bongata ba lisele tsa Purkinje. Plug-in ea Cell Counter ea ImageJ e sebelisitsoe ho lekola bongata ba Purkinje ka ho arola palo ea lisele tsa Purkinje tse baloang ka bolelele ba lesale la cerebellar le nkiloeng ke lisele tse baliloeng.
Tokiso le pokello ea mehlala. Boko bo tsoang sehlopheng sa taolo le litoeba tsa Mfn2cKO bo ile ba lokisoa ka har'a 2% PFA/2.5% glutaraldehyde ho 0.1 M phosphate buffer (PB), ebe likarolo tsa coronal li lokisoa ho sebelisoa ciliates (Leica Mikrosysteme GmbH, Vienna, Austria) (Botenya ba 50 ho isa ho 60 μm) Ebe u lokisa ka har'a PB buffer ho 1% os tetraoxide le 1.5% potassium ferrocyanide mochesong oa kamore ka hora e le 'ngoe. Likarolo li ile tsa hlatsuoa ka makhetlo a mararo ka metsi a hloekisitsoeng, ebe li dauoa ka 70% ethanol e nang le 1% uranyl acetate metsotso e 20. Likarolo li ile tsa omisoa ka har'a joala bo nang le maemo 'me tsa kenngoa ka har'a Durcupan ACM (Araldite casting resin M) epoxy resin (Electron Microscopy Sciences, catalog number 14040) pakeng tsa li-slide tsa khalase tse koahetsoeng ka silicon, 'me qetellong ho 60°C Polymerize ka ontong ka lihora tse 48. Sebaka sa cerebellar cortex se ile sa khethoa 'me likarolo tse tšesaane tsa 50 nm li ile tsa sehoa ho Leica Ultracut (Leica Mikrosysteme GmbH, Vienna, Austria) 'me tsa khethoa holim'a gridi ea koporo ea 2×1 mm e koahetsoeng ka filimi ea polystyrene. Likarolo li ile tsa dauoa ka tharollo ea 4% uranyl acetate ho H2O metsotso e 10, tsa hlatsuoa ka H2O makhetlo a 'maloa, eaba li hlatsuoa ka Reynolds lead citrate ho H2O metsotso e 10, 'me tsa hlatsuoa ka H2O makhetlo a 'maloa. Li-micrograph li ile tsa nkoa ka microscope ea elektrone ea phetisetso Philips CM100 (Thermo Fisher Scientific, Waltham, MA, USA) ho sebelisoa khamera ea dijithale ea TVIPS (Tietz Video and Image Processing System) TemCam-F416 (TVIPS GmbH, Gauting, USA). Jeremane).
Bakeng sa litoeba tse tšoaelitsoeng ke AAV, boko bo ile ba aroloa 'me ba sehoa ka karolo ea sagittal e teteaneng ea 1 mm, 'me cerebellum e ile ea hlahlojoa ka microscope ea fluorescence ho khetholla lesale le tšoaelitsoeng ke AAV (ke hore, mCherry expressing). Ke liteko feela tseo ho tsona ente ea AAV e fellang ka katleho e phahameng haholo ea transduction ea lera la sele la Purkinje (ke hore hoo e ka bang lera lohle) bonyane masaleng a mabeli a latellanang a cerebellar a sebelisoang. Sekoahelo se fetotsoeng ke AAV se ile sa aroloa ka microscope bakeng sa ho lokisoa bosiu bohle (4% PFA le 2.5% glutaraldehyde ho 0.1 M cocoate buffer) 'me sa sebetsoa hape. Bakeng sa ho kenngoa ha EPON, lisele tse tsitsitseng li ile tsa hlatsuoa ka 0.1 M sodium cocoate buffer (Applichem), 'me tsa kenngoa ka 2% OsO4 (os, Science Services; Caco) ho 0.1 M sodium cocoate buffer (Applichem) Lihora tse 4, ebe li hlatsuoa lihora tse 2. Pheta makhetlo a 3 ka 0.1 M cocamide buffer. Ka mora moo, letoto la ethanol le nyolohang le ile la sebediswa ho futhumatsa tharollo ka nngwe ya ethanol ho 4°C metsotso e 15 ho ntsha metsi dinama. Dinama di ile tsa fetisetswa ho propylene oxide mme tsa futhumatswa bosiu bohle ho EPON (Sigma-Aldrich) ho 4°C. Beha dinama ka hara EPON e foreshe mochesong wa kamore ka dihora tse 2, ebe o di kenya ho 62°C ka dihora tse 72. Sebedisa ultramicrotome (Leica Microsystems, UC6) le thipa ya daemane (Diatome, Biel, Switzerland) ho seha dikarolo tse tshesane tsa 70 nm, mme o di futhumatse ka 1.5% uranyl acetate metsotso e 15 ho 37°C, mme o di futhumatse ka tharollo ya lead citrate metsotso e 4. Di-micrograph tsa di-electron di ile tsa nkuwa ho sebediswa microscope ya JEM-2100 Plus transmission electron (JEOL) e nang le software ya Camera OneView 4K 16-bit (Gatan) le DigitalMicrograph (Gatan). Bakeng sa tlhahlobo, di-micrograph tsa dielektrone di ile tsa fumanwa ka zoom ya dijithale ya 5000× kapa 10,000×.
Tlhahlobo ea sebopeho sa mitochondria. Bakeng sa litlhahlobo tsohle, libopeho tsa mitochondria ka bomong li hlalositsoe ka letsoho litšoantšong tsa dijithale ho sebelisoa software ea ImageJ. Litekanyetso tse fapaneng tsa sebopeho lia hlahlojoa. Bongata ba mitochondria bo hlahisoa e le peresente e fumanoeng ka ho arola sebaka sohle sa mitochondrial sa sele ka 'ngoe ka sebaka sa cytoplasm (sebaka sa cytoplasm = sebaka sa sele-nucleus ea sele) × 100. Bophara ba mitochondria bo baloa ka foromo [4π∙ (sebaka/perimeter 2)]. Sebopeho sa ista sa mitochondria se ile sa hlahlojoa 'me sa aroloa ka lihlopha tse peli ("tubular" le "blister") ho latela libopeho tsa tsona tse ka sehloohong.
Tlhahlobo ea palo ea Autophagosome/lysosome le bongata. Sebelisa software ea ImageJ ho hlalosa ka letsoho sebopeho sa autophagosome/lysosome ka 'ngoe setšoantšong sa dijithale. Sebaka sa Autophagosome/lysosome se hlahisoa e le peresente e baloang ka ho arola sebaka sohle sa sebopeho sa autophagosome/lysosome sa sele ka 'ngoe ka sebaka sa cytoplasm (sebaka sa cytoplasm=sebaka sa sele-nucleus)×100. Bongata ba li-autophagosome/lysosome bo baloa ka ho arola palo eohle ka palo ea libopeho tsa autophagosome/lysosome ka sele (ho latela sebaka sa cytoplasm) (sebaka sa cytoplasm = sebaka sa sele-nucleus).
Ho ngola mabitso bakeng sa ho arola ka potlako le ho lokisa sampole. Bakeng sa liteko tse hlokang ho ngola mabitso a tsoekere, fetisetsa likarolo tsa boko bo matla ka phaposing ea pele ho ho ikatisa, e nang le khabone e tletseng (95% O2 le 5% CO2), Ca2 + ACSF e phahameng (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 le 2.0 mM MgCl 2, e fetotsoeng ho pH 7.4 le 310 ho isa ho 320 mOsm), moo tsoekere e leng 13 C 6- Phetolo ea tsoekere (Eurisotop, nomoro ea khataloganyo CLM-1396). Bakeng sa liteko tse hlokang ho ngolwa ha pyruvate, fetisetsa dikotwana tsa boko bo matla ho Ca2 + ACSF e phahameng (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 le Eketsa 2.0mM MgCl2, lokisa ho pH 7.4 le 310 ho isa ho 320mOsm), mme o kenye 1mM 1-[1-13C]pyruvate (Eurisotop, nomoro ya khataloganyo CLM-1082). Kenya dikarolo metsotso e 90 mochesong wa 37°C. Qetellong ea teko, likarolo li ile tsa hlatsuoa ka potlako ka tharollo ea metsi (pH 7.4) e nang le 75 mM ammonium carbonate, 'me tsa fetoloa homogenized ka 40:40:20 (v:v:v) acetonitrile (ACN): methanol: metsi. Kamora hore likarolo li behoe leqhoeng metsotso e 30, lisampole li ile tsa tšeloa ka centrifuge ho 21,000 g metsotso e 10 ho 4°C, 'me supernatant e hlakileng e ile ea omisoa ka har'a concentrator ea SpeedVac. Pellet ea metabolite e omisitsoeng e hlahisitsoeng e ile ea bolokoa ho -80°C ho fihlela tlhahlobo.
Tlhahlobo ea chromatography ea metsi-boima ba spectrometry ea li-amino acid tse 13 tse nang le label ea C. Bakeng sa tlhahlobo ea chromatography ea metsi-boima ba spectrometry (LC-MS), pellet ea metabolite e ile ea emisoa hape ka har'a 75μl ea metsi a sehlopha sa LC-MS (Honeywell). Kamora ho centrifugation ho 21,000 g metsotso e 5 ho 4°C, 20 μl ea supernatant e hlakisitsoeng e ile ea sebelisoa bakeng sa tlhahlobo ea amino acid flux, ha karolo e setseng ea extract e ile ea sebelisoa hang-hang bakeng sa tlhahlobo ea anion (sheba ka tlase). Tlhahlobo ea amino acid e entsoe ho sebelisoa protocol e hlalositsoeng pele ea benzoyl chloride derivatization (55, 56). Mohatong oa pele, 10μl ea 100 mM sodium carbonate (Sigma-Aldrich) e ile ea eketsoa ho 20μl ea metabolite extract, ebe 10μl ea 2% benzoyl chloride (Sigma-Aldrich) e eketsoa ho LC grade ACN. Sampole e ile ea ntšoa ka mouoane ka nakoana ebe e tšeloa ka seterifung ho 21,000 g metsotso e 5 mochesong oa 20°C. Fetisetsa supernatant e hloekisitsoeng ka har'a vial ea 2 ml ea autosampler e nang le khalase e kobehileng (bophahamo ba 200 μl). Lisampole li ile tsa hlahlojoa ho sebelisoa sistimi ea Acquity iClass ultra-high performance LC (Metsi) e hokahantsoeng le Q-Exactive (QE)-HF (Ultra High Field Orbitrap) high-resolution precision mass spectrometer (Thermo Fisher Scientific). Bakeng sa tlhahlobo, 2μl ea sampole e nkiloeng e ile ea kenngoa ka har'a kholomo ea silica T3 ea 100×1.0 mm e matla ka ho fetisisa e nang le likaroloana tsa 1.8μm. Sekhahla sa phallo ke 100μl/min, 'me sistimi ea buffer e na le buffer A (10 mM ammonium formate le 0.15% formic acid ka metsing) le buffer B (ACN). Gradient ke e latelang: 0%B ka metsotso e 0; 0%B. 0 ho isa ho 15% B ka metsotso e 0 ho isa ho 0.1; 15 ho isa ho 17% B ka metsotso e 0.1 ho isa ho 0.5; B ka 17 ho isa ho 55% ka metsotso e 0.5 ho isa ho e 14; B ka 55 ho isa ho 70% ka metsotso e 14 ho isa ho e 14.5; ka 14.5 ho isa ho 70 ho isa ho 100% B ka metsotso e 18; 100% B ka metsotso e 18 ho isa ho e 19; 100 ho isa ho 0% B ka metsotso e 19 ho isa ho e 19.1; 0% B ka metsotso e 19.1 ho isa ho e 28 (55, 56). Seteishene sa boima sa QE-HF se sebetsa ka mokhoa o motle oa ionization ka mefuta e fapaneng ea boima ba m/z (boima/karolelano ea tjhaja) ea 50 ho isa ho 750. Qeto e sebelisitsoeng ke 60,000, 'me sepheo sa ion sa taolo ea gain (AGC) se fumanoeng ke 3×106, 'me nako e phahameng ka ho fetisisa ea ion ke li-millisecond tse 100. Mohloli oa ionization ea electrospray e futhumetseng (ESI) o sebetsa ka motlakase oa spray oa 3.5 kV, mocheso oa capillary oa 250°C, moea o phallang oa sheath oa 60 AU (li-unit tse sa reroang), le moea o thusang oa 20 AU. 250°C. Lense ea S e behiloe ho 60 AU.
Tlhahlobo ea Anion chromatography-MS ea li-organic acid tse nang le mabitso a 13C. Methapo e setseng ea metabolite (55μl) e hlahlobiloe ho sebelisoa sistimi ea Dionex ion chromatography (ICS 5000+, Thermo Fisher Scientific) e hokahantsoeng le QE-HF mass spectrometer (Thermo Fisher Scientific). Ka bokhutšoanyane, 5μl ea metabolite extract e ile ea kenngoa ka har'a kholomo ea Dionex IonPac AS11-HC e nang le HPLC (2 mm×250 mm, boholo ba likaroloana 4μm, Thermo Fisher Scientific) ka mokhoa oa push-in partial loop ka karolelano ea ho tlatsa ea 1. ) Kholomo ea tšireletso ea Dionex IonPac AG11-HC (2 mm x 50 mm, 4μm, Thermo Fisher Scientific). Mocheso oa kholomo o bolokoa ho 30°C, 'me autosampler e behiloe ho 6°C. Sebelisa cartridge ea potassium hydroxide e fanoeng ka metsi a deionized ho hlahisa gradient ea potassium hydroxide ka jenereithara ea eluent. Karohano ea li-metabolite ka sekhahla sa phallo ea 380μl/min, ho sebelisoa gradient e latelang: metsotso e 0 ho isa ho e 3, 10 mM KOH; metsotso e 3 ho isa ho e 12, 10 ho isa ho e 50 mM KOH; metsotso e 12 ho isa ho e 19, 50 ho isa ho e 100 mM KOH; metsotso e 19 ho isa ho e 21, 100 mM KOH; metsotso e 21 ho isa ho e 21.5, 100 ho isa ho e 10 mM KOH. Kholomo e ile ea lekanngoa hape tlas'a 10 mM KOH ka metsotso e 8.5.
Li-metabolite tse lerootho li kopantsoe le phallo ea tlatsetso ea isopropanol ea 150μl/min ka mor'a kholomo ebe li lebisoa ho spectrometer ea boima bo phahameng e sebetsang ka mokhoa oa negative ionization. MS e shebile mefuta ea boima ho tloha ho m/z 50 ho isa ho 750 ka qeto ea 60,000. AGC e behiloe ho 1×106, 'me nako e phahameng ka ho fetisisa ea ion e bolokoa ho 100 ms. Mohloli oa ESI o futhumetseng o sebelitsoe ka motlakase oa spray oa 3.5 kV. Litlhophiso tse ling tsa mohloli oa ion ke tse latelang: mocheso oa capillary 275°C; phallo ea khase ea sheath, 60 AU; phallo ea khase e thusang, 20 AU ho 300°C, le ho beha lense ea S ho 60 AU.
Tlhahlobo ea data ea li-metabolite tse nang le mabitso a 13C. Sebelisa software ea TraceFinder (mofuta oa 4.2, Thermo Fisher Scientific) bakeng sa tlhahlobo ea data ea karolelano ea isotope. Boitsebiso ba motsoako ka mong bo netefalitsoe ke motsoako o tšepahalang oa litšupiso 'me bo hlahlobiloe ka boikemelo. E le ho etsa tlhahlobo ea ntlafatso ea isotope, sebaka sa chromatogram ea ion e ntšitsoeng (XIC) ea isotope ka 'ngoe ea 13C (Mn) se ntšitsoe ho [M + H]+, moo n e leng nomoro ea carbon ea motsoako o reriloeng, o sebelisetsoang ho sekaseka li-amino acid kapa [MH] + e sebelisetsoang ho sekaseka li-anion. Ho nepahala ha boima ba XIC ho ka tlase ho likarolo tse hlano ka milione, 'me ho nepahala ha RT ke metsotso e 0.05. Tlhahlobo ea ntlafatso e etsoa ka ho bala karolelano ea isotope ka 'ngoe e fumanoeng ho kakaretso ea li-isotope tsohle tsa motsoako o tsamaellanang. Li-ratios tsena li fanoa e le boleng ba peresente bakeng sa isotope ka 'ngoe, 'me liphetho li hlahisoa e le ntlafatso ea peresente ea molar (MPE), joalo ka ha ho hlalositsoe pele (42).
Pellet ea neuron e hatselitsoeng e ile ea fetoloa homogenized ka har'a 80% methanol (v/v) e batang joaloka leqhoa, ea koaheloa ka moea, 'me ea beoa ka har'a -20°C metsotso e 30. Fokotsa sampole hape 'me u hlohlelletse ho +4°C metsotso e 30. Sampole e ile ea tšeloa ka setering ho 21,000 g metsotso e 5 ho 4°C, ebe supernatant e hlahang e bokelloa 'me ea omisoa ho sebelisoa SpeedVac concentrator ho 25°C bakeng sa tlhahlobo e latelang. Joalokaha ho hlalositsoe kaholimo, tlhahlobo ea LC-MS e entsoe holim'a li-amino acid tsa lisele tse hlophisitsoeng. Ho sebelisoa TraceFinder (mofuta oa 4.2, Thermo Fisher Scientific), tlhahlobo ea data e entsoe ho sebelisoa boima ba monoisotopic ba motsoako o mong le o mong. Ho normalize data ea metabolite ka bongata ho entsoe ho sebelisoa sephutheloana sa software sa preprocessCore (57).
Ho lokisa selae. Toeba e ile ea robatsoa ka potlako ka carbon dioxide 'me ea khaoloa hlooho, boko ba tlosoa kapele lehateng, 'me thipa e thothomelang e tletseng leqhoa (HM-650 V, Thermo Fisher Scientific, Walldorf, Jeremane) e ile ea sebelisoa ho e seha likarolo tsa sagittal tsa 300 ho isa ho 375 μm Cold carbon gasification (95% O2 le 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 le 6.0 mM MgCl2 Fetola ho pH 7.4 le 310 ho isa ho 330 mOsm). Fetisetsa dikotwana tsa boko tse fumanweng ka phapusing e nang le Ca2 + ACSF e phahameng (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 le mM 3.5 MgCl2) pH 7.4 le 310 ho isa ho 320 mOsm). Boloka dikotwana metsotso e 20 ho isa ho e 30 e le hore di ka tsosolosoa pele di rekotwa.
ho rekota. Mokhahlelo oa microscope o nang le kamore e tsitsitseng ea ho rekota le lense ea 20x ea ho qoelisoa ka metsing (Scientifica) o ile oa sebelisoa bakeng sa lirekoto tsohle. Lisele tsa Purkinje tse ka bang teng li ile tsa khetholloa ka (i) boholo ba 'mele, (ii) sebaka sa anatomical sa cerebellum, le (iii) polelo ea gene ea reporter ea fluorescent mtYFP. Pipette ea patch e nang le khanyetso ea ntlha ea megohms tse 5 ho isa ho tse 11 e huloa ke capillary ea khalase ea borosilicate (GB150-10, 0.86 mm×1.5 mm×100 mm, Science Products, Hofheim, Jeremane) le pipette Instruments e otlolohileng (P-1000, Sutter), Novato, CA). Lirekoto tsohle li entsoe ke ELC-03XS npi patch clamp amplifier (npi electronic GmbH, Tam, Jeremane), e neng e laoloa ke software Signal (mofuta oa 6.0, Cambridge Electronic, Cambridge, UK). Teko e tlalehiloe ka sekhahla sa sampole sa 12.5 kHz. Letšoao le sefshoa ka li-filter tse peli tsa Bessel tse fetang ka nako e khuts'oane tse nang le maqhubu a ho khaola a 1.3 le 10 kHz ka ho latellana. Bokhoni ba lera le pipette bo lefelloa ke potoloho ea puseletso ho sebelisoa amplifier. Liteko tsohle li entsoe tlas'a taolo ea khamera ea Orca-Flash 4.0 (Hamamatsu, Gerden, Jeremane), e neng e laoloa ke software ea Hokawo (mofuta oa 2.8, Hamamatsu, Gerden, Jeremane).
Tlhophiso le tlhahlobo ya sele yohle kamehla. Hang pele o rekota, tlatsa pipette ka tharollo ya kahare e nang le dintho tse latelang: 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) le 10.0 mM creatinine phosphate di ile tsa lokiswa ho pH 7.25, mme kgatello ya osmotic e ne e le 290 mOsm (sucrose). Hang ka mora ho sebedisa matla a 0 pA ho taboha lera, bokgoni ba lera le phomotseng bo ile ba lekanngwa. Khanyetso ya ho kenya e lekanngwa ka ho sebedisa maqhubu a hyperpolarized a -40, -30, -20, le -10 pA. Lekanya boholo ba karabelo ya motlakase mme o sebedise molao wa Ohm ho bala khanyetso ya ho kenya. Mosebetsi o iketsahalletseng o ile oa tlalehoa ka har'a clamp ea motlakase ka metsotso e 5, 'me sPSC e ile ea fumanoa le ho lekanngoa ho Igor Pro (mofuta oa 32 7.01, WaveMetrics, Lake Oswego, Oregon, USA) ho sebelisoa sengoloa sa ho lemoha semi-automatic. Curve ea IV le steady-state current li lekanngoa ka ho tiisa betri ka matla a fapaneng (ho qala ho tloha ho -110 mV) le ho eketsa voltage ka mehato e 5 mV. Tlhahiso ea AP e ile ea lekoa ka ho sebelisa depolarizing current. Tlama sele ho -70 mV ha u ntse u sebelisa depolarizing current pulse. Fetola boholo ba mohato oa yuniti ka 'ngoe ea ho rekota ka thoko (10 ho isa ho 60 pA). Bala maqhubu a holimo a AP ka ho bala ka letsoho li-spikes tsa pulse tse bakang maqhubu a phahameng ka ho fetisisa a AP. Moeli oa AP o hlahlojoa ka ho sebelisa derivative ea bobeli ea depolarization pulse e qalang AP e le 'ngoe kapa ho feta.
Tlhophiso le tlhahlobo ea patch e nang le masoba. Etsa rekoto ea patch e nang le masoba u sebelisa liprothokholi tse tloaelehileng. Sebelisa pipette e se nang ATP le GTP e se nang metsoako e latelang: 128 mM gluconate K, 10 mM KCl, 10 mM Hepes, 0.1 mM EGTA le 2 mM MgCl2, 'me u ikamahanye le pH 7.2 (u sebelisa KOH). ATP le GTP li tlosoa tharollong ea ka hare ho sele ho thibela ho kenella ho sa laoleheng ha lera la sele. Pipette ea patch e tlatsitsoe ka tharollo ea ka hare e nang le amphotericin (hoo e ka bang 200 ho isa ho 250μg/ml; G4888, Sigma-Aldrich) ho fumana rekoto ea patch e phunngoeng. Amphotericin e qhibilihisitsoe ka dimethyl sulfoxide (khohlano ea ho qetela: 0.1 ho isa ho 0.3%; DMSO; D8418, Sigma-Aldrich). Khohlano ea DMSO e sebelisitsoeng ha ea ka ea ba le phello e kholo ho li-neuron tse ithutiloeng. Nakong ea ts'ebetso ea ho phunya, khanyetso ea kanale (Ra) e ile ea hlahlojoa khafetsa, 'me teko e ile ea qalisoa ka mor'a hore boholo ba Ra le AP bo tsitse (metsotso e 20-40). Mosebetsi o iketsahallang o lekanngoa ka motlakase le/kapa clamp ea hona joale metsotso e 2 ho isa ho e 5. Tlhahlobo ea data e entsoe ho sebelisoa Igor Pro (mofuta oa 7.05.2, WaveMetrics, USA), Excel (mofuta oa 2010, Microsoft Corporation, Redmond, USA) le GraphPad Prism (mofuta oa 8.1.2, GraphPad Software Inc., La Jolla, CA). United States). E le ho khetholla li-AP tse sa reroang, ho sebelisoa plug-in ea IgorPro ea NeuroMatic v3.0c. Khetholla li-AP ka boiketsetso ho sebelisoa moeli o fanoeng, o fetoloang ka bonngoe bakeng sa rekoto ka 'ngoe. U sebelisa nako ea spike, fumana maqhubu a spike ka maqhubu a phahameng a spike a hang-hang le maqhubu a tloaelehileng a spike.
Ho ikarola ha PN. Ka ho ikamahanya le protocol e phatlalalitsoeng pele, li-PN li ile tsa hloekisoa ho tloha ho cerebellum ea mouse mohatong o boletsoeng (58). Ka bokhutšoanyane, cerebellum e ile ea senngoa 'me ea siloa ka har'a mokelikeli o batang oa ho arohana [ntle le HBSS Ca2+ le Mg2+, ea tlatsetsoa ka glucose ea 20 mM, penicillin (50 U/ml) le streptomycin (0.05 mg/ ml)], ebe e sila mokelikeli ka har'a papain [HBSS, e tlatselitsoeng ka 1-cysteine·HCl (1 mg/ ml), papain (16 U / ml) le deoxyribonuclease I (DNase I; 0.1 mg/ml)] Phekola metsotso e 30 mochesong oa 30°C. Hlatsoa dinama tse ka hare ho HBSS e nang le mucus ya lehe (10 mg/ml), BSA (10 mg/ml) le DNase (0.1 mg/ml) mochesong wa kamore ho thibela tshilo ya dijo ya enzymatic, ebe o di kenya ka hare ho HBSS e nang le glucose ya 20 mM. Sila ka bonolo ho HBSS, penicillin (50 U/ml), streptomycin (0.05 mg/ml) le DNase (0.1 mg/ml) ho lokolla disele tse le nngwe. Sele e hlahisitsweng e ile ya sefshwa ka sehatsetsi sa sele sa 70μm, ebe disele di tjhesetswa ka centrifugation (1110 rpm, metsotso e 5, 4°C) mme tsa tjhesetswa hape ka hare ho selaete [HBSS, e tlatselletswa ka glucose ya 20 mM, 20% ya kgomo ya fetal) Serum, penicillin (50 U/ml) le streptomycin (0.05 mg/ml)]; hlahloba ho phela ha sele ka propidium iodide mme o fetole bongata ba sele ho disele tse 1 × 106 ho isa ho tse 2 × 106/ml. Pele ho potoloho ea mali, ho emisoa ho ne ho sefshoa ka har'a sesefa sa sele sa 50 μm.
Sesebediswa sa ho lekanya phallo ya disele. Ho hlopha disele ho entswe ka mocheso wa 4°C ho sebediswa mochini wa FACSAria III (BD Biosciences) le software ya FACSDiva (BD Biosciences, mofuta wa 8.0.1). Ho emiswa ha sele ho hlophisitswe ka ho sebedisa nozzle ya 100 μm tlasa kgatello ya 20 psi ka sekgahla sa ~2800 diketsahalo/motsotswana. Kaha mekgwa ya setso ya ho kgema (boholo ba sele, kgethollo ya bimodal, le dibopeho tsa ho hasana) e ke ke ya netefatsa karohano e nepahetseng ya PN ho mefuta e meng ya disele, leano la ho kgema le behilwe ho latela papiso e tobileng ya matla a YFP le autofluorescence ho mitoYFP+ ​​​​le mitoYFP ya taolo − Ditoeba. YFP e thabisitswe ka ho bonesa sampole ka mola wa laser wa 488 nm, mme letshwao le fumanwa ho sebediswa sefe ya ho fetisa sehlopha sa 530/30 nm. Ho mitoYFP+, matla a amanang a gene ya moqolotsi wa Rosa26-mitoYFP le ona a sebediswa ho kgetholla dikarolo tsa mmele wa neuronal le axon. 7-AAD e thabisitswe ka laser e tshehla ya 561 nm mme e fumanwe ka sefe ya bandpass ya 675/20 nm ho ntsha disele tse shweleng. E le ho arola di-astrocyte ka nako e le nngwe, sele e emisitsweng e ne e tshwaeditswe ka ACSA-2-APC, ebe sampole e tjhajwa ka mola wa laser wa 640 nm, mme sefe ya bandpass ya 660/20 nm e sebediseditswe ho lemoha letshwao.
Lisele tse bokelletsoeng li ile tsa tšeloa ka ho centrifugation (1110 rpm, metsotso e 5, 4°C) 'me tsa bolokoa ho -80°C ho fihlela li sebelisoa. Litoeba tsa Mfn2cKO le malinyane a tsona a lithōle li aroloa ka letsatsi le le leng ho fokotsa ho feto-fetoha ha mekhoa. Tlhahiso le tlhahlobo ea data ea FACS li entsoe ho sebelisoa software ea FlowJo (FlowJo LLC, Ashland, Oregon, USA).
Jwalo ka ha ho boletswe ka hodimo (59), PCR ya nako ya sebele e sebedisetswa ho arola DNA ho tswa ho di-neuron tse hlophisitsweng bakeng sa ho lekanya mtDNA ho latelang. Ho ba le kutlwisiso ya ho lekalekana le ho utlwahala ha moedi ho ile ha lekwa qalong ka ho sebedisa qPCR dipalong tse fapaneng tsa disele. Ka bokhutshwane, bokella 300 PN ka hara lysis buffer e nang le 50 mM tris-HCl (pH 8.5), 1 mM EDTA, 0.5% Tween 20 le proteinase K (200 ng/ml) mme o e futhumale ka 55°C metsotso e 120. Disele di ile tsa futhumala ka ho eketsehileng ka 95°C metsotso e 10 ho netefatsa ho se sebetse ka botlalo ha proteinase K. Ho sebediswa probe ya TaqMan (Thermo Fisher) e ikgethang ho mt-Nd1, mtDNA e ile ya lekanngwa ka semi-quantitative PCR tsamaisong ya 7900HT Real-Time PCR (Thermo Fisher Scientific). Saense, nomoro ea khataloge Mm04225274_s1), mt-Nd6 (Thermo Fisher Scientific, nomoro ea khataloge AIVI3E8) le 18S (Thermo Fisher Scientific, nomoro ea khataloge Hs99999901_s1) liphatsa tsa lefutso.
Tokisetso ea sampole ea Proteome. Ka ho futhumatsa tharollo ka 95°C metsotso e 10 le ho e hlaba ka sonicating, ka har'a lysis buffer [6 M guanidine chloride, 10 mM tris(2-carboxyethyl) phosphine hydrochloride, 10 mM chloroacetamide le 100 mM tris-Lyse li-pellets tsa neuron tse hoamisitsoeng ho HCl]. Ho Bioruptor (Diagenode) metsotso e 10 (metsotsoana e 30 ea pulse / metsotsoana e 30 ea ho emisa). Sampole e ile ea hlapolloa ka 1:10 ho 20 mM tris-HCl (pH 8.0), e kopantsoe le 300 ng trypsin gold (Promega), 'me ea beoa bosiu bohle ho 37°C ho fihlela tšilo ea lijo e felletseng. Ka letsatsi la bobeli, sampole e ile ea tšeloa ka centrifuge ho 20,000 g metsotso e 20. Supernatant e ile ea hlapolloa ka 0.1% formic acid, 'me tharollo ea tlosoa letsoai ka StageTips e entsoeng ke motho ka boeena. Sampole e omisitsoe ka har'a sesebelisoa sa SpeedVac (Eppendorf concentrator plus 5305) mochesong oa 45°C, ebe peptide e emisoa ka har'a 0.1% formic acid. Lisampole tsohle li lokisitsoe ka nako e le 'ngoe ke motho a le mong. E le ho sekaseka lisampole tsa astrocyte, li-peptide tse 4 μg tse se nang letsoai li ile tsa ngoloa ka tandem mass tag (TMT10plex, catalog number 90110, Thermo Fisher Scientific) ka karolelano ea peptide ho TMT reagent ea 1:20. Bakeng sa ho ngola TMT, 0.8 mg ea reagent ea TMT e ile ea emisoa hape ka 70 μl ea ACN e sa nang le metsi, 'me peptide e omisitsoeng e ile ea kopanngoa hape ho 9 μl ea 0.1 M TEAB (triethylammonium bicarbonate), eo ho eona ho ileng ha eketsoa 7 μl ea reagent ea TMT ho ACN. Khakanyo e ne e le 43.75%. Kamora metsotso e 60 ea ho ikatisa, karabelo e ile ea tima ka 2 μl ea 5% hydroxylamine. Li-peptide tse ngotsoeng li ile tsa bokelloa, tsa omisoa, tsa emisoa hape ka har'a 200μl ea 0.1% formic acid (FA), tsa aroloa ka tse peli, eaba li tlosoa letsoai ho sebelisoa StageTips e entsoeng ke motho ka boeena. Ho sebelisoa UltiMate 3000 ultra high performance liquid chromatograph (UltiMate 3000 ultra high performance liquid chromatograph), e 'ngoe ea likarolo tse peli e ile ea aroloa ka har'a kholomo ea 1mm x 150mm Acquity chromatographic e tletseng likaroloana tsa 130Å1.7μm C18 (Metsi, catalog No. SKU: 186006935). Thermo Fisher Scientific). Arola li-peptide ka sekhahla sa phallo sa 30μl/min, u arohane le 1% ho isa ho 50% buffer B metsotso e 85 ka gradient e otlolohileng ea metsotso e 96, ho tloha ho 50% ho isa ho 95% buffer B metsotso e 3, ebe metsotso e 8 bakeng sa 95% Buffer B; Buffer A ke 5% ACN le 10 mM ammonium bicarbonate (ABC), 'me buffer B ke 80% ACN le 10 mM ABC. Bokella likaroloana metsotso e meng le e meng e 3 'me u li kopanye ka lihlopha tse peli (1 + 17, 2 + 18, jj.) 'me u li omise ka har'a vacuum centrifuge.
Tlhahlobo ea LC-MS/MS. Bakeng sa tlhahlobo ea boima ba 'mele, li-peptide (nomoro ea r119.aq) li ile tsa aroloa kholomong ea tlhahlobo ea PicoFrit ea bophara ba 25 cm, 75 μm (lense e ncha ea sepheo, nomoro ea karolo ea PF7508250) e nang le 1.9 μm ReproSil-Pur 120 C18-AQ medium (Dr. Maisch, mat), Sebelisa EASY-nLC 1200 (Thermo Fisher Scientific, Jeremane). Kholomo e ile ea bolokoa mochesong oa 50°C. Li-buffers A le B ke 0.1% formic acid metsing le 0.1% formic acid ho 80% ACN, ka ho latellana. Li-peptide li ile tsa aroloa ho tloha ho 6% ho isa ho 31% buffer B metsotso e 65 le ho tloha ho 31% ho isa ho 50% buffer B metsotso e 5 ka gradient ea 200 nl/min. Li-peptide tse lerootho li ile tsa hlahlojoa ho Orbitrap Fusion mass spectrometer (Thermo Fisher Scientific). Tekanyo ea peptide precursor m/z e etsoa ka qeto ea 120,000 pakeng tsa 350 le 1500 m/z. Ho sebelisoa matla a 27% a ho thulana a tloaelehileng, precursor e matla ka ho fetisisa e nang le boemo ba tjhaja ba 2 ho isa ho 6 e khethoa bakeng sa karohano ea C trap dissociation (HCD) ea matla a phahameng. Nako ea potoloho e behiloe ho 1 s. Boleng ba m/z ba peptide fragment bo lekantsoe ka har'a ion trap ho sebelisoa sepheo se senyenyane ka ho fetisisa sa AGC sa 5×104 le nako e phahameng ka ho fetisisa ea ente ea 86 ms. Kamora ho arohana, precursor e ile ea beoa lenaneng la ho tlosoa ka matla ka metsotsoana e 45. Li-peptide tse nang le mabitso a TMT li arotsoe kholomong ea Acclaim PepMap ea 50 cm, 75 μm (Thermo Fisher Scientific, nomoro ea khataloge 164942), 'me li-spectra tsa ho falla li ile tsa hlahlojoa ho Orbitrap Lumos Tribrid mass spectrometer (Thermo Fisher Scientific) e nang le lisebelisoa tsa high-field asymmetric waveform ions (FAIMS) (Thermo Fisher Scientific) e sebetsa ka li-voltage tse peli tsa puseletso tsa −50 le −70 V. MS3 e khethiloeng ho latela selelekela sa synchronization se sebelisetsoang ho lekanya lets'oao la ion la tlaleho ea TMT. Karohano ea peptide e entsoe ho EASY-nLC 1200, ho sebelisoa 90% linear gradient elution, ka khatello ea buffer ea 6% ho isa ho 31%; buffer A e ne e le 0.1% FA, 'me buffer B e ne e le 0.1% FA le 80% ACN. Kholomo ea tlhahlobo e sebelisoa ho 50°C. Sebelisa FreeStyle (mofuta oa 1.6, Thermo Fisher Scientific) ho arola faele ea pele ho latela motlakase oa puseletso oa FAIMS.
Tlhahlobo le ho lekanya protheine. Ka ho sebedisa enjene ya ho batla ya Andromeda e kopantsweng, tlhahisoleseding ya mantlha e ile ya sekasekwa ho sebediswa mofuta wa MaxQuant 1.5.2.8 (https://maxquant.org/). Ntle le tatellano ya Cre recombinase le YFP e fumanweng ho Aequorea victoria, di-spectra tsa peptide fragment di ile tsa batlisiswa bakeng sa tatellano ya canonical le tatellano ya isoform ya proteome ya referense ya mouse (Proteome ID UP000000589, e jarollotsweng ho UniProt ka Motsheanong 2017). Oxidation ya Methionine le acetylation ya protheine ya N-terminal di ile tsa bewa e le diphetoho tse fetofetohang; cysteine ​​​​carbamoyl methylation e ile ya bewa e le diphetoho tse tsitsitseng. Diparamitha tsa tshilo ya dijo di bewa ho "boikgethelo" le "trypsin/P". Palo e fokolang ya di-peptide le di-peptide tsa razor tse sebedisetswang ho tsebahatsa protheine ke 1; palo e fokolang ya di-peptide tse ikgethang ke 0. Tlas'a maemo a ho bapisa 'mapa wa peptide, sekgahla sa ho tsebahatsa protheine e ne e le 0.01. Khetho ya "Peptide ya Bobedi" e a buletswe. Sebelisa khetho ea "papali pakeng tsa ho matha" ho fetisetsa boitsebiso bo atlehileng pakeng tsa lifaele tse fapaneng tsa mantlha. Sebelisa palo ea karolelano ea bonyane ea LFQ 1 bakeng sa ho lekanya ntle le leiboleng (LFQ) (60). Matla a LFQ a sefshoa bakeng sa bonyane boleng bo peli bo nepahetseng bonyane sehlopheng se le seng sa genotype ntlheng e 'ngoe le e 'ngoe ea nako, 'me a ntšoa ho tsoa kabong e tloaelehileng e nang le bophara ba. 0.3 'me a theohele tlase ho 1.8. Sebelisa sethala sa k'homphieutha sa Perseus (https://maxquant.net/perseus/) le R (https://r-project.org/) ho sekaseka liphetho tsa LFQ. Teko ea t e itekanetseng ea litsela tse peli ho tsoa sephutheloana sa software sa limma e sebelisitsoe bakeng sa tlhahlobo ea polelo e fapaneng (61). Tlhahlobo ea data e batlisisang e etsoa ho sebelisoa ggplot, FactoMineR, factoextra, GGally le pheatmap. Lintlha tsa proteomics tse thehiloeng ho TMT li se li sekasekiloe ho sebelisoa mofuta oa MaxQuant 1.6.10.43. Batla data ya proteomics e tala ho tswa ho database ya proteomics ya motho ya UniProt, e ileng ya jarollwa ka Loetse 2018. Tlhahlobo ena e kenyelletsa ntlha ya tokiso ya bohloeki ba isotope e fanweng ke moetsi. Sebedisa limma ho R bakeng sa tlhahlobo ya polelo e fapaneng. Data ya pele, diphetho tsa patlo ya database, le mosebetsi wa tlhahlobo ya data le diphetho kaofela di bolokilwe kopanong ya ProteomeXchange ka polokelo ya molekane wa PRIDE e nang le sesupo sa sete ya data PXD019690.
Litlhaloso tse sebetsang li ntlafatsa tlhahlobo. Sesebelisoa sa Ingenuity Pathway Analysis (QIAGEN) se sebelisitsoe ho fumana leruo la mantsoe a ts'ebetso a data e behiloeng libekeng tse 8 (Setšoantšo sa 1). Ka bokhutšoanyane, lethathamo la protheine ea bongata le fumanoeng ho tsoa tlhahlobong ea data ea LC-MS/MS (tandem mass spectrometry) le sebelisoa ka litekanyetso tse latelang tsa sefa: Mus musculus e khethiloe e le mofuta le nalane, 'me sehlopha se bontša boleng ba P bo fetotsoeng ke Benjamini bakeng sa ntlafatso ea 0.05 kapa ka tlase bo nkoa bo le bohlokoa. Bakeng sa kerafo ena, lihlopha tse hlano tse holimo tse feteletseng sehlopheng ka seng ho latela boleng ba P bo fetotsoeng lia bontšoa. Ho sebelisoa teko ea t-teko e mengata, ho sebelisoa lenaneo la ho phahamisa linear la mekhahlelo e 'meli la Benjamini, Krieger, le Yekutieli (Q = 5%), tlhahlobo ea polelo ea protheine ea nako e etsoa ho bakhethoa ba bohlokoa ba khethiloeng sehlopheng ka seng, 'me mola o mong le o mong o hlahlojoa ka thoko. Ha ho hlokahale ho amohela SD e tsitsitseng.
E le ho bapisa liphetho tsa phuputso ena le li-database tse phatlalalitsoeng le ho hlahisa setšoantšo sa Venn ho Setšoantšo sa 1, re kopantse lethathamo la protheine ea bongata le litlhaloso tsa MitoCarta 2.0 (24). Sebelisa sesebelisoa sa inthanete sa Draw Venn Diagram (http://bioinformatics.psb.ugent.be/webtools/Venn/) ho hlahisa setšoantšo.
Bakeng sa tlhahisoleseding e qaqileng mabapi le mekhoa ea lipalo-palo e sebelisoang bakeng sa tlhahlobo ea proteomics, ka kopo sheba karolo e tsamaellanang ea Lisebelisoa le Mekhoa. Bakeng sa liteko tse ling tsohle, tlhahisoleseling e qaqileng e ka fumanoa tšōmong e tsamaellanang. Ntle le haeba ho boletsoe ka tsela e 'ngoe, lintlha tsohle li hlahisoa e le karolelano ea ± SEM, 'me litlhahlobo tsohle tsa lipalo-palo li entsoe ho sebelisoa software ea GraphPad Prism 8.1.2.
Bakeng sa lisebelisoa tse ling bakeng sa sengoloa sena, ka kopo sheba http://advances.sciencemag.org/cgi/content/full/6/35/eaba8271/DC1
Ena ke sengoloa se bulehileng se ajoang tlas'a lipehelo tsa Creative Commons Attribution-Non-Commercial License, e lumellang tšebeliso, kabo le tlhahiso-leseling ka mokhoa ofe kapa ofe, ha feela tšebeliso ea ho qetela e se bakeng sa phaello ea khoebo 'me motheo ke hore mosebetsi oa pele o nepahetse. Referense.
Tlhokomeliso: Re kopa feela hore o fane ka aterese ea hau ea lengolo-tsoibila e le hore motho eo u mo khothaletsang leqepheng a tsebe hore u batla hore a bone lengolo-tsoibila le hore ha se spam. Re ke ke ra hapa liaterese life kapa life tsa lengolo-tsoibila.
Potso ena e sebelisetsoa ho leka hore na u moeti le ho thibela ho romelloa ha spam ka bohona.
Ka E. Motori, I. Atanassov, SMV Kochan, K. Folz-Donahue, V. Sakthivelu, P. Giavalisco, N. Toni, J. Puyal, N.-G. Larson
Tlhahlobo ea li-proteomics tsa li-neurone tse sa sebetseng hantle e senotse hore mananeo a metabolism aa kenngoa tšebetsong ho loantša ho senyeha ha methapo ea kutlo.
Ka E. Motori, I. Atanassov, SMV Kochan, K. Folz-Donahue, V. Sakthivelu, P. Giavalisco, N. Toni, J. Puyal, N.-G. Larson
Tlhahlobo ea li-proteomics tsa li-neurone tse sa sebetseng hantle e senotse hore mananeo a metabolism aa kenngoa tšebetsong ho loantša ho senyeha ha methapo ea kutlo.
©2020 Mokhatlo oa Amerika oa Tsoelo-pele ea Saense. litokelo tsohle li sirelelitsoe. AAAS ke molekane oa HINARI, AGORA, OARE, CHORUS, CLOCKSS, CrossRef le COUNTER. ScienceAdvances ISSN 2375-2548.