Kwanyalo *Likheli lanyalo: Cologne 50931, eJalimane, Cologne Lucwaningo Lwelicembu Lekusebenta Kahle Ngekuphendvula Kwekucindzeteleka Kwemaseli Etifweni Letihlobene Nekuguga (CECAD).
Kubola kwetinzwa kwetifo te-mitochondrial kutsatfwa njengalokungabuyiselwa emuva ngoba ipulasitiki yekugaya ema-neurons ilinganiselwe, kepha umphumela wekungasebenti kahle kwe-mitochondrial ekutimeleni kwemaseli ekugaya ema-neuronal emtimbeni awucondzakali kahle. Lapha, setfula i-proteome lekhetsekile yemaseli e-Purkinje neurons ngekushoda kwe-OXPHOS lokuchubekako lokubangelwa kuphazamiseka kwe-mitochondrial fusion. Sitfole kutsi kungasebenti kahle kwe-mitochondrial kwabangela lushintjo lolukhulu emkhakheni we-proteomics, lokwagcina kuholele ekusebentiseni lokulandzelanako kwetinhlelo leticondzile tekugaya kudla ngaphambi kwekufa kwemaseli. Ngekungalindzeleki, sancuma kufaka lokusobala kwe-pyruvate carboxylase (PCx) kanye naletinye tintfo letilwa nekuguga letingeta tintfo letisemkhatsini temjikeleto we-TCA. Kuvinjelwa kwe PCx kwakhulisa kucindzeteleka kwe-oxidative kanye nekubola kwemitsambo, lokukhombisa kutsi i-atherosclerosis inemphumela wekuvikela kuma-neurons lashoda nge-OXPHOS. Kubuyiselwa kwe-mitochondrial fusion kuma-neurons laphelile kubuyisela emuva ngalokuphelele letimphawu tekugaya kudla, ngaleyo ndlela kuvikele kufa kwemaseli. Lokutfolwe ngitsi kukhomba tindlela lebetingatiwa ngaphambilini letiniketa kucinisela ekungasebenti kahle kwe-mitochondrial futsi kukhombisa kutsi i-neurodegeneration ingabuyiselwa emuva ngisho nasetigaba tekugcina talesifo.
Indima lesemkhatsini ye-mitochondria ekugcineni umjikeleto wemandla e-neuronal igcizelelwa timphawu letibanti te-neurological letihambisana netifo te-mitochondrial tebantfu. Linyenti laletifo libangelwa kugucuka kwemajini lalawula kuveta kwemajini e-mitochondrial (1, 2) noma kubhujiswa kwemajini lokuhlobene nemandla e-mitochondrial, lokutsintsa ngalokungacondzi kusimama kwe-DNA ye-mitochondrial (mtDNA) (3, 4). Umsebenti kumamodeli etilwane ukhombise kutsi ngekuphendvula kungasebenti kahle kwe mitochondrial eticubini letitungeletile, tindlela tekugaya letilondvolotako (5-7) tingasebenta, letiniketa lwati lolubalulekile lwekuvisisa lokujulile kwe pathogenesis yaletifo letiyinkimbinkimbi. Ngalokwehlukile kakhulu, kuvisisa kwetfu tingucuko tekugaya kudla kwetinhlobo letitsite temaseli letibangelwa kwehluleka jikelele kwekukhicita i-mitochondrial adenosine triphosphate (ATP) yengcondvo kusisekelo (8), kugcizelela sidzingo sekubona tintfo letihlosiwe tekwelapha letingasetjentiswa kuvikela noma kuvikela sifo. Vikela kugula kwemiva (9). Kushoda kwelwati liciniso lekutsi emaseli etinzwa atsatfwa kakhulu kutsi anekugucugucuka lokuncane kakhulu kwekugaya uma kucatsaniswa netinhlobo temaseli eticubu letitungeletile (10). Njengobe kuniketwe kutsi lamaseli adlala indzima lesemkhatsini ekuhlanganiseni kuniketwa kwe-metabolites kuma-neurons kukhutsata kudluliselwa kwe-synaptic kanye nekuphendvula ekulimaleni kanye netimo tesifo, likhono lekuvumelanisa i-metabolism yemaseli netimo letiyinselele teticubu tebuchopho licishe lilinganiselwe kumaseli e-glial (11-14). Ngetulu kwaloko, i-heterogeneity yemaseli lekhonako yeticubu tebuchopho ivimbela kakhulu kufundza tingucuko tekugaya kudla letenteka emacenjini lakhetsekile e-neuronal. Ngenca yaloko, kuncane lokwatiwako ngemiphumela lecondzile yemaseli kanye nekugaya kudla kwekungasebenti kahle kwe-mitochondrial kuma-neurons.
Kute sivisise imiphumela yekugaya kudla kwekungasebenti kahle kwe-mitochondrial, sahlukanisa ema-neurons e-Purkinje (ema-PN) etigaba letehlukene tekubola kwemizwa lokubangelwa kubhujiswa kwekuhlanganiswa kwe-mitochondrial yangaphandle (Mfn2). Noma nje kuguculwa kwe-Mfn2 kubantfu kuhlobene nendlela yekugula kwemoto lokutsatfwako lokwatiwa ngekutsi yi-Charcot-Marie-Tooth luhlobo 2A (15), kubhujiswa kwe-Mfn2 lokunemibandzela kumagundvwane kuyintfo leyatiwa kahle yekungasebenti kahle kwe-oxidation Phosphorylation (OXthoPHOS). Letinhlobo letehlukene tetinzwa (16-19) kanye ne-phenotype lephumako yetinzwa ihambisana netimphawu tetinzwa letichubekako, njengekuphatfwa kabi kwekuhamba (18, 19) noma i-ataxia ye-cerebellar (16). Ngekusebentisa inhlanganisela ye-proteomics lengakabaluleki (LFQ), i-metabolomics, imifanekiso, kanye netindlela te-virological, sikhombisa kutsi kugucuka kwemiva lokuchubekako kukhutsata kakhulu i-pyruvate carboxylase (PCx) kanye naletinye tintfo letitsintsekako ekugucukeni kwemitsambo ye-PNs ku-vivo Kuveta ema-enzyme. Kucinisekisa kuhambisana kwalokutfolakele, sacondzisa ngalokukhetsekile kuveta kwe-PCx ku-PNs lenesifo se-Mfn2, futsi satfola kutsi lokusebenta kwakhulisa kucindzeteleka kwe-oxidative kanye nekusheshisa kwe-neurodegeneration, ngaleyo ndlela kufakazela kutsi i-azoospermia iniketa kufa kwemaseli Kuvumelana nesimo se-Metabolic. Kuveta lokukhulu kwe-MFN2 kungakhulula ngalokuphelele i-PN lephelile yekubola ngekushoda lokukhulu kwe-OXPHOS, kusetjentiswa lokukhulu kwe-DNA ye-mitochondrial, kanye nenethiwekhi ye-mitochondrial lephukile lebonakalako, lokuphindze kugcizelele kutsi lendlela yekubola kwe-neurodegeneath ingaphindze ilulame esigabeni lesiphakeme sesifo se-cell.
Kute sibone ngemehlo engcondvo i-mitochondria ku-Mfn2 knockout PNs, sasebentisa luhlobo lwegundvwane loluvumela i-mitochondria lencike ku-Cre kutsi ihlose iphrotheni lephuti lekhanyako (YFP) (mtYFP) (20) Kuveta kwe-Cre futsi sahlola indlela ye-mitochondrial ku-vivo. Sitfole kutsi kubhujiswa kwe-Mfn2 gene kuma-PNs kutawuholela ekuhlukanisweni kancane kancane kwenethiwekhi ye-mitochondrial (Umfanekiso S1A), futsi lushintjo lwekucala lwatfolakala emavikini lama-3 budzala. Ngalokwehlukile, kubola lokukhulu kwelucwencwe lwemaseli e-PN, njengoba kufakazelwa kulahlekelwa kwe-Calbindin immunostaining, akuzange kucale kwaze kwaba ngemaviki langu-12 budzala (Umfanekiso 1, A na B). Kungahambisani kwesikhatsi emkhatsini wetingucuko tekucala te-mitochondrial morphology kanye nekucala lokubonakalako kwekufa kwe-neuronal kwasikhutsata kutsi siphenye tingucuko tekugaya kudla letibangwa kungasebenti kahle kwe-mitochondrial ngaphambi kwekufa kwemaseli. Sakha isu lekuhlunga emaseli lasebenta nge-fluorescence (FACS) lelisekelwe ekuhlukaniseni i-YFP (YFP +)-leveta i-PN (Umfanekiso 1C), kanye nasemagundvwaneni ekulawula (Mfn2 + / loxP :: mtYFP loxP- stop-loxP: : L7-cre), lapha ibitwa ngekutsi yi-CT1BRL (Umfanekiso). Kusebenta kahle kwesu lekuvula lisango lokusekelwe emandla lahlobene esiginali ye-YFP kusivumela kutsi sihlante umtimba we-YFP + (YFPhigh) we-PNs kusuka ku-non-PNs (YFPneg) (Umfanekiso S1B) noma tincetu te-axon / dendritic letikhanyako (YFPlow; Umfanekiso S1D, confoscopecal, kwesokunxele). Kute sicinisekise bunjalo bebantfu labahlukanisiwe, senta i-LFQ proteomics bese sihlatiya tincenye letiyinhloko, futsi satfola kutsi kunekuhlukaniswa lokucacile emkhatsini wemangcamuzane e-YFPhigh kanye ne-YFPneg (Umfanekiso S1C). Emaseli e-YFPhigh akhombisa kuceceshwa kwenethi kwemamaki e-PNs latiwako (lokusho Calb1, Pcp2, Grid2 kanye ne-Itpr3) (21, 22), kepha kute kuceceshwa kwemaprotheni lavame kuvetwa kuma-neurons noma letinye tinhlobo temaseli (Umfanekiso 1D) ). Kucatsanisa emkhatsini wemasampula kumaseli eYFPhigh lahlukanisiwe labutfwe ekuhlolweni lokutimele kwakhombisa i-coefficient yekuhambisana> 0.9, lekhombisa kukhicita lokuhle emkhatsini wekuphindzaphindza kwetilwane (Umfanekiso S1E). Ngalokufishane, lemininingwane yacinisekisa luhlelo lwetfu lwekuhlukaniswa lokubukhali nalokucondzile kwe-PN lekhonakalako. Ngenca yekutsi luhlelo lwemshayeli we-L7-cre lolusetjentisiwe lukhutsata kuhlanganiswa kabusha kwe-mosaic evikini lekucala ngemuva kwekuletsa (23), sacala kubulala emagundvwane kusuka ku-CTRL kanye nemibandela (Mfn2 loxP / loxP :: mtYFP loxP-stop-loxP :: L7-cre) Butsa ema-neurons. Ngemuva kwekuhlanganiswa kabusha sekuphelile, kubitwa ngekutsi yi Mfn2cKO ngemaviki lamane budzala. Njengephuzu lekugcina, sakhetsa emaviki langu-8 eminyaka lapho ungqimba lwe-PN lwaluphelele naphezu kwekuhlukaniswa kwe-mitochondrial lokusobala (Umfanekiso 1B kanye neMfanekiso S1A). Sekukonkhe, salinganisa emaprotheni langu-3013, lapho khona cishe 22% bekasekelwe ku-MitoCarta 2.0 tichasiselo letisekelwe ku-proteome ye-mitochondrial njenge-mitochondria (Umfanekiso 1E) (Umfanekiso 1E) (24). Kuhlatiya kwekuveta emajini lahlukile lokwentiwe evikini lesi-8 kwakhombisa kutsi ngu-10.5% kuphela wawo onkhe emaprotein abe netingucuko letibalulekile (Umfanekiso 1F kanye neMfanekiso S1F), lapho khona emaprotein langu-195 alawulwa phansi kantsi emaprotein langu-120 alawulwa ngetulu (Umfanekiso 1F). Kufanele kucashelwe kutsi “kuhlatiya indlela lensha” yalesethi yedatha kukhombisa kutsi emajini lavetwe ngekwehlukana ikakhulukati avela kusethi levinjelwe yetindlela letitsite tekugaya (Umfanekiso 1G). Kuyajabulisa kutsi, noma ngabe kwehliswa kwetindlela letihlobene ne-OXPHOS kanye nekukhombisa kwe-calcium kucinisekisa kungeniswa kwekungasebenti kahle kwe-mitochondrial kuma-PN lashodako e-fusion, letinye tigaba letifaka ekhatsi kakhulu kusebenta kwe-amino acid tilawulwa kakhulu, lokuhambisana nekugaya kwe-mitochondrial lokwenteka ku-P condrion. kungasebenti kahle.
(A) Titfombe letimele te-confocal tetincenye te-cerebellar temagundvwane e-CTRL kanye ne-Mfn2cKO letikhombisa kulahleka lokuchubekako kwe-PNs (calbindin, grey); nuclei bebaphikisana ne-DAPI. (B) Kulinganisa kwe (A) (kuhlatiya kwendlela yinye yekuhluka, *** P <0.001; n = 4 kuya ku 6 imibutsano kusuka kumagundvwane lamatsatfu). (C) Kusebenta kwekuhlola. (D) Kusabalaliswa kwemephu yekushisa kwemamaki lacondzile ku Purkinje (ngetulu) kanye naletinye tinhlobo temaseli (emkhatsini). (E) Umdvwebo weVenn lokhombisa linani lemaprotheni e-mitochondrial lakhonjwe ku-PN lehlukanisiwe. (F) Volcano plot yemaprotheni lavetwe ngekwehlukana kuma-neurons e-Mfn2cKO emavikini langu-8 (kubaluleka kwekusika kwe-1.3). (G) Kuhlatiya indlela yekudala kukhombisa tindlela letisihlanu letibaluleke kakhulu tekulawula phezulu (bovu) kanye nekulawula phansi (luhlata sasibhakabhaka) ku-Mfn2cKO PN lehlukaniswe njengemaviki langu-8. Lizinga lekuveta lelisemkhatsini lephrotheni ngayinye letfolakele liyakhonjiswa. Imephu yekushisa ye-Grayscale: inani le-P lelilungisiwe. ns, akubalulekile.
Idatha ye-proteomics yakhombisa kutsi kuveta emaprotheni etinkimbinkimbi I, III, na-IV kwehla kancane kancane. Tinkimbinkimbi I, III, kanye ne IV tonkhe betinema-subunits labalulekile lakhodiwe nge-mtDNA, kantsi i-complex II, lebeyikhodiwe ngenuzi kuphela, beyingakatsintseki ngalokuyinhloko (Umfanekiso 2A kanye neMfanekiso S2A). . Kuhambisana nemiphumela ye-proteomics, i-immunohistochemistry yetincenye teticubu tebuchopho yakhombisa kutsi i-MTCO1 (i-mitochondrial C oxidase subunit 1) lizinga le-subunit le-IV leyinkimbinkimbi ku-PN lehla kancane kancane (Umfanekiso 2B). I-mtDNA-lekhodiwe subunit Mtatp8 yancishiswa kakhulu (Umfanekiso S2A), kantsi lizinga le-simo lesimile le-ATP synthase subunit lekhodiwe ngenuzi lahlala lingashintjwa, lokuhambisana ne-ATP synthase subassembly F1 leyatiwako lecinile lapho kuveta kwe-mtDNA kuzinzile. Kubunjwa kwakhe kuyahambisana. Phazamisa (7). Kuhlolwa kwelizinga le-mtDNA kuma-PNs e-Mfn2cKO lahlukanisiwe ngekuphendvula kwemaketanga e-polymerase ngesikhatsi sangempela (qPCR) kucinisekisile kwehla kancane kancane kwenombolo yekhophi ye-mtDNA. Nawucatsanisa nelicembu lekulawula, emavikini langu-8 budzala, kuphela cishe 20% wezinga le-mtDNA Lagcinwa (Umfanekiso 2C). Kuhambisana nalemiphumela, kugcotjwa kwesibonakhulu se-confocal kwe-Mfn2cKO PNs kwasetjentiswa kutfola i-DNA, kukhombisa kusetjentiswa lokuncike esikhatsini se-nucleotides ye-mitochondrial (Umfanekiso 2D). Sitfole kutsi labanye kuphela labakhetsiwe labatsintseka ekucedzeni emaprotheni e-mitochondrial kanye nekuphendvula kwekucindzeteleka bekulawulwa, kufaka ekhatsi i-Lonp1, Afg3l2 ne-Clpx, kanye ne-OXPHOS tici tekuhlangana letiyinkimbinkimbi. Kute tingucuko letibalulekile emazingeni emaprotheni latsintsekako apoptosis kwatfolakala (Umfanekiso S2B). Ngendlela lefanako, satfola kutsi imigudu ye-mitochondria kanye ne-endoplasmic reticulum lehilelekile ekuhambiseni i-calcium inetingucuko letincane kuphela (Umfanekiso S2C). Ngetulu kwaloko, kuhlolwa kwemaprotheni lahlobene ne-autophagy akutfolanga tingucuko letibalulekile, lokuhambisana nekufakwa lokubonakalako kwema-autophagosomes labonwa ku-vivo nge-immunohistochemistry kanye ne-electron microscopy (Umfanekiso S3). Kepha, kusebenta kabi kwe-OXPHOS lokuchubekako kuma-PN kuhambisana netingucuko letisobala te-mitochondrial. Emacembu eMitochondrial angabonakala emitimbeni yemaseli kanye netihlahla te dendritic te Mfn2cKO PNs letineminyaka lengu 5 kanye na 8 emaviki, futsi sakhiwo se membrane lengekhatsi sesibe netingucuko letijulile (Umfanekiso S4, A na B). Kuhambisana naletingucuko tesakhiwo lesikhulu kanye nekwehla lokukhulu kwe-mtDNA, kuhlatiya tincetu tebuchopho lobubukhali nge-tetramethylrhodamine methyl ester (TMRM) kwakhombisa kutsi emandla elucwencwe lwe-mitochondrial ku-Mfn2cKO PNs anciphe kakhulu (Umfanekiso S4C).
(A) Sikhatsi sifundvo kuhlatiya izinga incazelo OXPHOS inkimbinkimbi. Cabanga kuphela emaprotheni nge P <0.05 emavikini langu-8 (ANOVA yetindlela letimbili). Umugca lonemachashazi: Kute kulungiswa uma kucatsaniswa ne-CTRL. (B) Ngesencele: Sibonelo sesigaba se-cerebellar lesibhalwe nge-anti-MTCO1 (ibha yesikali, 20 μm). Indzawo lehlalwa yimitimba yemaseli ePurkinje imbonywe ngumtfubi. Ngasekudla: Kulinganisa emazinga eMTCO1 (kuhlatiya kwendlela yinye yekuhluka; n = 7 kuya ku 20 emaseli lahlatiya kusuka kumagundvwane lamatsatfu). (C) qPCR kuhlatiya mtDNA ikhophi inombolo ku PN ehlukanisiwe (kuhlatiya indlela yinye yekuhluka; n = 3 kuya ku-7 emagundvwane). (D) Ngesencele: Sibonelo sesicephu se-cerebellar lesibhalwe nge-anti-DNA (ibha yesikali, 20 μm). Indzawo lehlalwa yimitimba yemaseli ePurkinje imbonywe ngumtfubi. Ngasekudla: Kulinganisa tilonda te mtDNA (kuhlatiya kwendlela yinye yekuhluka; n = 5 kuya ku 9 emaseli kusuka kumagundvwane lamatsatfu). (E) Sibonelo sesigaba se-cerebellar lesibucayi lesikhombisa emaseli e-mitoYFP + Purkinje (umcibisholo) ekubhalweni kwe-clamp ye-patch yeseli yonkhe. (F) Kulinganisa kwe-IV curve. (G) Kubhalwa lokumele kwekujova kwanyalo lokukhipha umfutfo kumaseli e-CTRL kanye ne-Mfn2cKO Purkinje. Top trace: I-pulse yekucala leyabangela i-AP. Umkhondo longentasi: Imvamisa lephakeme ye-AP. (H) Kulinganisa lokufakwako lokutentakalelako kwe-postsynaptic (sPSPs). Umkhondo wekubhala lommeleli kanye nesilinganiso sayo sekusondzeta kukhonjisiwe ku (I). Kuhlatiya kwendlela yinye kwekuhluka kuhlatiya n = 5 kuya ku 20 emaseli kusuka emagundvwane lamatsatfu. Idatha ivezwa njengendlela ± SEM; *P<0.05; **P<0.01; ***P<0.001. (J) Imilandvo lemele AP letentekelako lebhalwe kusetjentiswa imodi ye-patch clamp lebhobokile. Umkhondo lophezulu: Imvamisa lephakeme ye-AP. Umkhondo longentasi: zoom ye-AP yinye. (K) Lindza imvamisa ye-AP lesemkhatsini nalephakeme ngekuya kwe (J). Kuhlolwa kwaMann-Whitney; n = 5 amaseli ahlaziywa kusuka emagundvwane amane. Idatha ivezwa njengendlela ± SEM; akukabaluleki.
Umonakalo we-OXPHOS losobala watfolakala ku-Mfn2cKO PN lenemaviki langu-8, lokukhombisa kutsi umsebenti wemtimba we-neurons awukavami kakhulu. Ngako-ke, sahlatiya timphawu tagezi letingasebenti te-OXPHOS-neurons letinesifo emavikini langu-4 kuya ku-5 kanye nemaviki langu-7 kuya ku-8 ngekwenta lokubhalwe phansi kwe-patch clamp ye-cell yonkhe etincenyeni te-cerebellar letibukhali (Umfanekiso 2E). Ngekungalindzeleki, silinganiso sekuphumula kwe-membrane emandla kanye nekumelana kwekufaka kwe-Mfn2cKO neurons bekufana nekulawula, noma bekunemehluko longabonakali emkhatsini wemangcamuzane (Lithebula 1). Ngendlela lefanako, emavikini langu-4 kuya kulangu-5 budzala, kute tingucuko letibalulekile ebudlelwaneni be-voltage yamanje (IV curve) letatfolakala (Umfanekiso 2F). Kepha, kute ema-neurons e-Mfn2cKO lanemaviki langu-7 kuya ku-8 lasindza ku-IV regimen (sinyatselo se-hyperpolarization), lokukhombisa kutsi kunekuzwela lokucacile kwemandla e-hyperpolarization kulesigaba sekugcina. Ngalokwehlukile, ku-Mfn2cKO neurons, imisinga ye-depolarizing lebangela kukhishwa kwesento lesiphindzaphindvwako (AP) ibeketelelwa kahle, lokukhombisa kutsi emaphethini abo ekukhishwa jikelele akafani kakhulu nalawo e-neurons yekulawula emaviki langu-8 (Lithebula 1 kanye neMfanekiso 2G). Ngendlela lefanako, imvamisa kanye ne-amplitude ye-postsynaptic currents letentekelako (sPSCs) beyifanana naleyo yelicembu lekulawula, futsi imvamisa yetintfo letenteka yakhula kusuka kumaviki langu-4 kuya kumaviki langu-5 kuya kumaviki langu-7 kuya kumaviki langu-8 ngekukhula lokufanako (Umfanekiso 2, H na-I). Sikhatsi sekuvutfwa kwe-synaptic ku-PNs (25). Imiphumela lefanako yatfolakala ngemuva kwemabala e-PNs labhobokile. Lokucushwa kuvimbela sincephetelo lesingahle sibe khona setikweleti te-ATP yemaseli, njengobe kungenteka ekubhaleni kwe-clamp ye-patch yeseli yonkhe. Ikakhulukati, emandla e-membrane ekuphumula kanye nemvamisa yekudubula lokutentakalelako kwe-Mfn2cKO neurons akuzange kuthinteke (Umfanekiso 2, J na K). Ngalokufishane, lemiphumela ikhombisa kutsi ema-PNs lanekungasebenti kahle kwe-OXPHOS lokusobala angabhekana nemaphethini ekukhishwa kwemvamisa lesetulu kahle, lokukhombisa kutsi kunendlela yekunxephetela lebavumela kutsi bagcine timphendvulo letisedvute-nekuvamile te-electrophysiological.
Idatha ivezwa njengekusho ± SEM (kuhlatiya kwendlela yinye yekuhluka, luhlolo lwekucatsanisa lolunyenti lwaHolm-Sidak; * P <0.05). Inombolo yeyunithi ikhonjiswa kubakaki.
Sibeka kuphenya kutsi ngabe ngusiphi sigaba ku-dataset ye-proteomics (Umfanekiso 1G) sifaka ekhatsi tindlela letingalwa nekushoda lokukhulu kwe-OXPHOS, ngaleyo ndlela sichaze kutsi kungani i-PN letsintsekile ingagcina i-electrophysiology lesedvute-nekujwayelekile (Umfanekiso 2, E kuya ku-K). . Kuhlatiya kwema-proteomics kwakhombisa kutsi ema-enzyme latsintseka ekuguculeni ema-amino acid lanemagatja (BCAA) alawulwa kakhulu (Umfanekiso 3A kanye neMfanekiso S5A), futsi umkhicito wekugcina we-acetyl-CoA (CoA) noma i-succinyl CoA ungafaka ema-tricarboxylates ku-Acteriosclerosis. Sitfole kutsi lokucuketfwe yi-BCAA 1 (BCAT1) ne-BCAT2 kokubili kwandza. Bakhutsata sinyatselo sekucala se-BCAA ngekutsi bakhicite i-glutamate kusuka ku-α-ketoglutarate (26). Onkhe emayunithi lamancane lakha umjikeleto lonemagatja we-keto acid dehydrogenase (BCKD) leyinkimbinkimbi ilawulwa (leyinkimbinkimbi ikhutsata kukhishwa kwe-carboxylation lokulandzelako nalokungabuyiselwa emuva kwe-BCAA carbon skeleton) (Umfanekiso 3A kanye neMfanekiso S5A). Kepha, kute tingucuko letibonakalako ku BCAA yona ngekwayo letatfolakala ku PN lehlukanisiwe, lokungenteka kutsi kungenca yekukhula kwekutsatfwa kwemaseli kwalama amino acid labalulekile noma kusetjentiswa kwaletinye tintfo (glucose noma lactic acid) kwentela kufaka umjikeleto we TCA (Umfanekiso S5B). Ema PN lashoda nge OXPHOS aphindze akhombisa kukhula kwekubola kwe glutamine kanye nemisebenti yekukhicita emavikini langu 8 budzala, lokungabonakala ngekulawulwa kwe-enzymes ye-mitochondrial glutaminase (GLS) kanye ne-glutamine pyruvate transaminase 2 (GPT2) (Umfanekiso 3, A na C). Kufanele kucashelwe kutsi kulawulwa kwe-GLS kukhawulelwe ku-isoform glutaminase C (GLS-GAC) lehlanganisiwe (kushintja kwe-Mfn2cKO / CTRL kucishe kube tikhatsi letingu-4.5, P = 0.05), kanye nekulawulwa kwayo lokutsite eticubini temdlavuza. (27).
(A) Imephu yekushisa ikhombisa kushintja kwekugoqa esigabeni seprotein yendlela lechaziwe emavikini langu-8. (B) Sibonelo sesicephu se-cerebellar lesibhalwe nge-anti-PCx antibody (ibha yesikali, 20 μm). Umcibisholo lomtfubi ukhomba emtimbeni weseli lePurkinje. (C) Kuhlatiya kwekuveta emaprotheni esikhatsini lokukhonjwe njengemmeleli lobalulekile we-atherosclerosis (t-test leminyenti, * FDR <5%; n = 3-5 emagundvwane). (D) Ngetulu: Umdvwebo lokhombisa tindlela letehlukene tekufaka ikhabhoni lebhalwe lecuketfwe ku [1-13C] pyruvate tracer (lokusho kutsi, nge PDH noma umzila we-trans-arterial). Ngezansi: Lishadi le-violin likhombisa liphesenti lekhabhoni lebhalwe yinye (M1) leguculwe yaba yi-aspartic esidi, esidi ye-citric kanye ne-malic esidi ngemuva kwekulebula tincetu te-cerebellar letibukhali nge [1-13C]pyruvate (kuhlolwa kwe-t lokubhanjiwe; ** P <0.01). (E) Kuhlatiya umlandvo wesikhatsi lesiphelele sendlela lekhonjisiwe. Cabanga kuphela emaprotheyini lane-P<0.05 emavikini langu-8 . Umugca lonemabala: kute linani lekulungisa (kuhlatiya kwetindlela letimbili tekuhluka; * P <0.05; *** P <0.001). Idatha ivezwa njengendlela ± SEM.
Ekuhlaziyeni kwetfu, i-BCAA catabolism seyibe ngulenye yetindlela letibalulekile tekulawula. Leliciniso likhombisa kakhulu kutsi umthamo wemoya longena emjikelezweni we-TCA ungashintjwa ku-PN lete i-OXPHOS. Loku kungamelela luhlobo lolukhulu lwekuphindze kufakwe ema-neuronal metabolic, lokungaba nemtselela locondzile ku-neuronal physiology kanye nekuphila ngesikhatsi sekugcinwa kwekungasebenti kahle kwe-OXPHOS lokukhulu. Kuhambisana nalomcabango, satfola kutsi i-enzyme leyinhloko yekulwa nekugula kwemitsambo PCx ilawulwa ngetulu (Mfn2cKO / CTRL ishintsha cishe tikhatsi letingu-1.5; Umfanekiso 3A), lekhutsata kuguculwa kwe-pyruvate ibe yi-oxaloacetate (28), lokukholelwa kutsi ku-brain, 30). Kuhambisana nemiphumela ye-proteomics, i-microscopy ye-confocal yakhombisa kutsi kuveta kwe-PCx kwakhuphuka ngalokukhetsekile futsi kwakhuphuka kakhulu kuma-PNs lashoda nge-OXPHOS, kantsi kusebenta kwe-PCx bekuvinjelwe kakhulu kumaseli e-Bergmann glial lasedvute ekulawula (Umfanekiso 3B). Kuhlola ngendlela lesebentako lokubonwako kwe-PCx, saphatsa tincetu te-cerebellar letibukhali nge- [1-13C] pyruvate tracer. Ngesikhatsi i-pyruvate i-oxidized yi-pyruvate dehydrogenase (PDH), ilebuli yayo ye-isotope yanyamalala , Kepha ifakwa etintfweni letisemkhatsini temjikeleto we-TCA lapho i-pyruvate iguculwa ngekuphendvula kwemitsambo (Umfanekiso 3D). Ekusekeleni idatha yetfu ye-proteomics, sabona linani lelikhulu letimphawu letivela kulomlandzeli ku-aspartic acid yetincetu te-Mfn2cKO, kantsi i-citric acid ne-malic acid nayo beyinemkhuba lolinganiselwe, noma ingabalulekile (Umfanekiso 3D).
Kule-neurons ye-dopamine yemagundvwane aseMitoPark lanekungasebenti kahle kwe-mitochondrial lokubangelwa tinhlayiya te-dopamine letibhubhisa ngalokukhetsekile sici sekubhala se-mitochondrial A gene (Tfam) (Umfanekiso S6B), kuveta kwe-PCx kwaphindze kulawulwa kakhulu (31), lokukhombisa kutsi i-acetonal acid leyentekako ye- OXPHOS emtimbeni. Kufanele kucashelwe kutsi kutfolakele kutsi ema-enzyme lahlukile (32-34) langavetwa kuma-neurons langahle ahlobane nekugula kwemitsambo yemithambo alawulwa kakhulu kuma-PN lashoda nge-OXPHOS, njenge-propionyl-CoA carboxylase (PCC-A), i-Malonyl-CoA-mi-CoA igucula ibe yi- i-enzyme 3 (ME3), indzima yayo lenkhulu kutsi itfole i-pyruvate kusuka ku-malate (Umfanekiso 3, A na-C) (33, 35). Ngetulu kwaloko, satfola kwandza lokukhulu kwe-enzyme ye-Pdk3, le-phosphorylates futsi ngaleyo ndlela yenta kutsi i-PDH ingasebenti (36), kantsi kute tingucuko letatfolakala ku-enzyme ye-Pdp1 lesebenta i-PDH noma i-PDH enzyme complex yona (Umfanekiso 3A). Ngekungagucugucuki, ku-Mern2cKO PNs, i-phosphorylation ye-α1 α (PDHE1α) ye-pyruvate dehydrogenase E1 yengcenye ye-PDH leyinkimbinkimbi ku-Ser293 (leyatiwa ngekuvimbela umsebenti we-enzyme ye-PDH) yakhuliswa ( Umfanekiso S6C ) (Umfanekiso S6C). I-Pyruvate ayinayo indlela yekufinyelela kwemitsambo.
Ekugcineni, satfola kutsi indlela lephakeme ye-serine ne-glycine, umjikeleto lohlobene we-mitochondrial folate (1C) kanye ne-proline biosynthesis (Umfanekiso 1G kanye neMfanekiso S5C) konkhe kulawulwa kakhulu, ngekuya kwemibiko, ngesikhatsi senkambo yekusebenta. Leticubu letitungeletile tisebenta ngekungasebenti kahle kwe-mitochondrial (5-7). Kuhlatiya kwe-confocal lokusekela lemininingwane ye-proteomics kwakhombisa kutsi ku-PN lene-OXPHOS lelahlekile, tincetu te-cerebellar temagundvwane lanemaviki langu-8 abekwa ngaphansi kwe-serine hydroxymethyltransferase 2 (SHMT2), i-enzyme lebalulekile yemjikeleto we-mitochondrial folate. Kuphendvula lokubalulekile kwemasotja emtimba (Umfanekiso S5D). Ku-13 CU-glucose-incubated cerebellar tincetu letibucayi, kuhlolwa kwekulandzelela kwemetabolic kwaphindze kwacinisekisa kulawulwa kwe-serine kanye ne-proline biosynthesis, lokukhombisa kutsi kuhamba kwema-isoforms e-carbon abe yi-serine kanye ne-proline kwandza (Umfanekiso S5E). Njengobe kuphendvula lokukhutsatwa yi-GLS ne-GPT2 kunesibopho sekuhlanganiswa kwe-glutamate kusuka ku-glutamine kanye nekuguculwa emkhatsini we-glutamate ne-α-ketoglutarate, kulawulwa kwabo kukhombisa kutsi ema-neurons lashoda nge-OXPHOS anesidzingo lesikhulu se-glutamate , Loku kungaba kuhloswe ngako ekugcineni i-biosynthesis lekhulile ye-proFig5). Ngalokwehlukile kuletingucuko, kuhlatiya kwe-proteomic kwe-astrocytes ye-cerebellar kusuka kumagundvwane e-Mfn2cKO lakhetsekile e-PN kwakhombisa kutsi letindlela (kufaka ekhatsi tonkhe ti-antiperoxidases) atizange tishintje kakhulu ekuboniseni, ngaleyo ndlela kukhombisa Loku kucondziswa kabusha kwe-metabolic kukhetsa ku-PN leyehlisiwe (Fig. S6).
Ngalokufishane, lokuhlatiya kwaveta emaphethini lehluke kakhulu ekusebenta kwesikhashana kwetindlela letitsite tekugaya kudla ku-PNs. Noma umsebenti longakavami we-neuronal mitochondrial ungaholela ekucaleni kwe-atherosclerosis kanye nekuvuselela kwe-1C (Umfanekiso 3E kanye neMfanekiso S5C), ngisho netingucuko leticagelako ekuveteni kwetinkimbinkimbi te-I ne-IV, tingucuko tekuhlanganiswa kwe-serine de novo kuphela Kwabonakala kuphela ngesikhatsi sekugcina. OXPHOS kungasebenti kahle (Umfanekiso 3E kanye Umfanekiso S5C). Lokutfolwe kuchaza inchubo lelandzelanako lapho khona i-mitochondrial (umjikeleto we-1C) kanye ne-cytoplasmic (i-biosynthesis ye-serine) lebangelwa kucindzeteleka iphendvula ngekusebentisana nekwandziswa kwekugula kwemitsambo emjikelezweni we-TCA kute ibumbe kabusha umsebenti we-neuronal.
Ema-PNs lanesifo se-OXPHOS-emaviki langu-8 angagcina umsebenti wekujabula wemvamisa lesetulu futsi ahambe nekuhlanganiswa kabusha lokubalulekile kwe-metabolic kute anxephetele kungasebenti kahle kwe-mitochondrial. Lokutfolakala kuphakamisa kungenteka lokujabulisako kwekutsi ngisho nakulesikhatsi, lamaseli angaphindze Atfole kungenelela kwekwelapha kubambezeleka noma kuvimbela neurodegeneration. Kwephuta. Silungise loku kungenteka ngekungenelela lokubili lokutimele. Ngendlela yekucala, saklama i-vector yegciwane lelihambisana ne-adeno lencike ku-Cre (AAV) kute i-MFN2 ingavetwa ngekukhetsa ku-PNs lenesifo se-OXPHOS ku-vivo (Umfanekiso S7A). I-AAV lekhoda i-MFN2 kanye ne-mCherry ye-fluorescent gene gene (Mfn2-AAV) yacinisekiswa emasikweni e-neuron layinhloko ku-vitro, leyabangela kutsi i-MFN2 ivetwe ngendlela lencike ku-Cre futsi yakhulula i-mitochondrial morphology, ngaleyo ndlela yavimbela kugucuka kwe-neuron ku-Mfns D, B Umfanekiso S7). Ngemuva kwaloko, sachuba kuhlolwa kwe-vivo kute siletse ngendlela le-stereotactically i-Mfn2-AAV lenemaviki langu-8 ku-cerebellar cortex ye-Mfn2cKO kanye nekulawula emagundvwane, futsi sahlatiya emagundvwane lanemaviki langu-12 (Umfanekiso 4A). Emagundvwane eMfn2cKO lalashwa afa (Umfanekiso 1, A na B) (16). Kuguculwa kwegciwane ku-vivo kwaholela ekuboniseni lokukhetsiwe kwe-PN kuleminye imibutsano ye-cerebellar (Umfanekiso S7, G na-H). Umjovo wekulawula i-AAV leveta kuphela i-mCherry (Ctrl-AAV) bewute umphumela lomkhulu esigabeni sekubola kwemiva etilwaneni te-Mfn2cKO. Ngalokwehlukile, kuhlatiya kwe-Mfn2cKOs leguculwe nge-Mfn2-AAV kwakhombisa umphumela lomkhulu wekuvikela we-PN cell layer (Umfanekiso 4, B na C). Ikakhulu, le neuron density ibonakala icishe ingahlukaniswa netilwane letilawulako (Umfanekiso 4, B na C, kanye ne Umfanekiso S7, H na I). Kuveta i-MFN1 kepha hhayi i-MFN2 kusebenta ngendlela lefanako ekusindiseni kufa kwe-neuronal (Umfanekiso 4C kanye neMfanekiso S7, C na-F), lokukhombisa kutsi kuveta i-ectopic MFN1 kungangeta ngemphumelelo kushoda kwe-MFN2. Kuhlatiya lokunyenti ezingeni le-PN linye kwakhombisa kutsi i-Mfn2-AAV yakhulula kakhulu i-ultrastructure ye-mitochondria, emazinga e-mtDNA lajwayelekile, futsi yabuyisela emuva kuveta lokusezingeni leliphakeme kwe-PCx ye-anti-angiogenesis (Umfanekiso 4, C kuya ku-E ). Kuhlolwa lokubonwako kwemagundvwane eMfn2cKO lakhululiwe asesimeni sekuphumula kwakhombisa kutsi kuma kwawo kanye netimphawu temoto (kuhamba S1 kuya ku S3) kwaba ncono. Ekuphetseni, lokuhlolwa kukhombisa kutsi kubambezeleka kwekuphindze kwetfulwe kwe-MFN2 kuma-PNs lashoda kakhulu ku-OXPHOS kwanele kubuyisela emuva kusetjentiswa kwe-mtDNA kanye nekubangela i-atherosclerosis, ngaleyo ndlela kuvikeleke kubola kwe-axon kanye nekufa kwe-neuronal ku-vivo.
(A) Luhlelo lolukhombisa luhlelo lwekuhlola lwekujova i-AAV lekhoda i-MFN2 lapho indlela lekhonjisiwe yekugaya isebenta. (B) Imifanekiso lemele ye-confocal yetincetu te-cerebellar letinemaviki langu-12 letiguculwe emavikini langu-8 kumagundvwane e-Mfn2cKO futsi letibhalwe nge-anti-Calbindin antibody. Ngasekudla: Kukala imicu ye-axon. Sikali se-axon zoom ngu-450 na-75 μm. (C) Kwesencele: Kulinganisa kwebuningi bemaseli ePurkinje ku-loop yekuhumusha ye-AAV (AAV +) (kuhlatiya kwendlela yinye yekuhluka; n = 3 emagundvwane). Ngakwesokudla: mtDNA kugcila kuhlatiya ku PN transduced evikini 12 (unpaired t-test; n = 6 amaseli kusuka emagundvwane lamatsatfu). * P <0.05; ** P <0.01. (D) Ema-micrographs e-electron ekudluliswa lamele ema-PNs etincenye te-cerebellar te-Mfn2cKO letiguculwe ngetintfo letikhonjisiwe teligciwane. Le mask lepinki ikhombisa indzawo lehlalwa ngema dendrites, bese sikwele lesimtfubi lesinemachashazi sikhombisa kusondzeta lokuniketwe ngasekudla; n imelela i-nucleus. Ibha yesikali, 1μm. (E) ukhombisa sibonelo se-PCx staining ku-PN leguculwe emavikini langu-12. Ibha yesikali, 20μm. OE, kuveta kakhulu; FC, goca kushintja.
Ekugcineni, saphenya kubaluleka kwekusindza kwemaseli labangelwa yi-peroxidase kuma-PNs lahlangabetana nekungasebenti kahle kwe-OXPHOS. Sikhicite i-mCherry lekhoda i-AAV-shRNA (i-RNA lemfisha yetinwele) lecondziswe ngco ku-mRNA ye-PCx yegundane (i-AAV-shPCx), futsi sajova leligciwane noma kulawula kwalo lokuhlanganisiwe (i-AAV-scr) ebuchosheni bemagundvwane e-Mfn2cKO. Lomjovo wentiwa evikini lesine leminyaka (Umfanekiso 5A) kuze kuzuzwe kushaywa kwe-PCx lokuphumelelako ngesikhatsi lapho kuveta kwe-PCx kwandza khona (Umfanekiso 3C) futsi lucwencwe lweseli le-PN belusolo lukhona (Umfanekiso 1A). Kufanele kucashelwe kutsi kushaya phansi i-PCx (Umfanekiso S8A) kuholela ekusheshiseni lokukhulu kwekufa kwe-PN, lokulinganiselwe endandathoni leneligciwane (Umfanekiso 5, B na C). Kute sicondze indlela yemiphumela yekugaya kudla lebangelwa yi-PCx up-umtsetfo, safundza simo se-redox se-PNs ngemuva kwekushaywa kwe-PCx kanye ne-AAV-lelamulako ye-biosensor ye-optical Grx1-roGFP2 yavetwa ngaso sonkhe sikhatsi (Umfanekiso S8, B kuya ku-D) kuhlola i-glutax3one I-potential). Ngemuva kwaloko, senta sibonakaliso sekutsatsa titfombe temphilo yonkhe ye-photon lembili (FLIM) etincenyeni tebuchopho letibukhali te-Mfn2cKO lenemaviki langu-7 noma kulawula littermates kutfola tingucuko letingaba khona esimeni se-cytoplasmic redox ngemuva kwekucinisekisa timo te-FLIM (Umfanekiso S8, E kuya ku-G). Lokuhlatiya kwakhombisa kukhula lokukhulu kwesimo se-oxidation se-Mfn2cKO PNs yinye lete kuveta kwe-PCx, lokuhlukile kuma-neurons ekulawula noma ema-Mfn2cKO PNs laveta kuphela i-shRNA le-scrambled (Umfanekiso 5, D na-E). Ngesikhatsi kuveta kwe-PCx bekukulawulwa phansi, liphesenti le-Mfn2cKO PNs lekhombisa simo se-oxidized kakhulu lakhula ngetikhatsi letingetulu kwaletintsatfu (Umfanekiso 5E), lokukhombisa kutsi kulawulwa kwe-PCx phezulu kwagcina likhono le-redox le-neurons lewohlokile.
(A) Luhlelo lolukhombisa luhlelo lwekuhlola lwekujova i-AAV encoding shPCx lapho indlela lekhonjisiwe yekugaya isebenta. (B) Titfombe letimele te-confocal tetincenye te-cerebellar letinemaviki langu-8 kumagundvwane e-Mfn2cKO laguculwe futsi labhalwe nge-anti-calcineurin antibody emavikini langu-4. Ibha yesikali, 450μm. (C) Kulinganisa kwebuningi bemaseli ePurkinje kuma-loop laguculwe nge-AAV (kuhlatiya kwendlela yinye yekuhluka; n = 3 kuya ku-4 emagundvwane). Idatha ivezwa njengendlela ± SEM; ***P<0.001. (D) Sitfombe se-FLIM lesimele sikhombisa sikhatsi sekuphila lesisemkhatsini se-PN lenemaviki langu-7 leveta inzwa ye-glutathione redox Grx1-roGFP2 ngaphansi kwetimo tekuhlola letichaziwe. LUT (litafula lekubuka) silinganiso: sikhatsi sekuphila (nge-picoseconds). Ibha yesikali, 25μm. (E) I-histogram ikhombisa kusabalaliswa kwemanani ekuphila kwe-Grx1-roGFP2 kusuka ku-(D) (n = 158 kuya ku-368 emaseli emagundvwane lamabili ngaphansi kwesimo ngasinye). Lishadi le pie lelingetulu kwe histogram ngayinye: likhombisa linani lemangcamuzane lanemanani ekuphila lamadze kakhulu (bovu, la-oxidized) noma lamafishane (luhlata sasibhakabhaka, lancishisiwe), langetulu kwe 1 SD yelinani lekuphila lelisemkhatsini ku CTRL-AAV-scr. (F) Imodeli lephakanyisiwe ikhombisa umphumela wekuvikela upregulation PCx neuronal.
Konkhe loku, idatha lesiyiniketa lapha ikhombisa kutsi kuphindze kuvetwe i-MFN2 kungakhulula ngalokuphelele i-PN lesetulu ngekushoda lokukhulu kwe-OXPHOS, kucinywa lokukhulu kwe-mtDNA, kanye ne-morphology lefana ne-ista lengakavami kakhulu, ngaleyo ndlela kuniketa inchubekelembili lechubekako ngisho nasetifweni letisetulu. Neurodegeneration iniketa bufakazi lobubuyela emuva besigaba ngaphambi kwekufa kwemaseli. Lelizinga lekugucugucuka kwemetabolic liphindze ligcizelelwa likhono le-neurons lekutsi lifake i-atherosclerosis (kuphindze kufakwe umjikeleto we-TCA), lokuvimbela kuveta kwe-PCx kuma-PN lashoda nge-OXPHOS futsi kutfutfukisa kufa kwemaseli, ngaleyo ndlela kudlala indzima yekuvikela (Umfanekiso 5F).
Kulesifundvo, sinikete bufakazi bekutsi impendulo PNs OXPHOS dysfunction kutsi kancane kancane converge TCA umjikelezo atherosclerosis ngendlela umehluko kusebenta kusebenta tinhlelo metabolic. Sicinisekisile kuhlatiya kwe-proteomic ngetindlela letinyenti letiphelele futsi saveta kutsi uma inselele yekungasebenti kahle kwe-mitochondrial lokukhulu, ema-neurons anendlela lebeyingatiwa ngaphambilini yekugucuka kwe-metabolic. Kuyamangala kutsi yonkhe inchubo yekuphindze ufake tintambo ayiphawuli simo se-metabolic sekugcina lesihambisana ne-neurodegeneration kancane kancane futsi ngendlela lengabuyiselwa emuva, kepha idatha yetfu iphakamisa kutsi ingahle ibe yi-neuron yekunakekelwa ngisho nasesigabeni ngaphambi kwekufa kwemaseli Indlela yekunxephetela lesebentako. Loku lokutfolakele kukhombisa kutsi ema-neurons anelizinga lelikhulu le-plasticity yekugaya emtimbeni. Leliciniso lifakazela kutsi kuphindze kwetfulwe kamuva kwe-MFN2 kungabuyisela emuva kuveta kwemamaki labalulekile ekugaya kudla futsi kuvikele kubola kwe-PN. Ngalokwehlukile, kuvimbela i-atherosclerosis futsi kusheshisa imizwa. bantfu labagucukela ebulilini.
Lokunye lokutfolakele lokutsakatelisa kakhulu elucwaningweni letfu kutsi ema-PN lashoda nge-OXPHOS angagucula umjikeleto we-TCA ngekutsi alawule ema-enzyme lakhutsata ngalokukhetsekile i-arteriosclerosis. Kuhlelwa kabusha kwemetabolic kuyintfo levamile yemangcamuzane emdlavuza, lamanye awo atsembele ku-glutamine kufaka tintfo letisemkhatsini temjikeleto we-TCA kute akhicite tintfo letilinganako letinciphisako, letichuba umjikeleto wekuphefumula futsi tigcine kukhicitwa kwetintfo letihamba embili te-lipid kanye ne-nucleotide biosynthesis (39 , 40). Lucwaningo lwamuva lukhombise kutsi eticubini letiseceleni letihlangabetana nekungasebenti kahle kwe-OXPHOS, kuhlanganiswa kabusha kwe-glutamine / glutamate metabolism nako kuyintfo lebonakalako (5, 41), lapho khona sicondziso sekungena kwe-glutamine emjikelezweni we-TCA sincike ku Ngenca yebukhulu bekulimala kwe-OXPHOS (41). ). Kepha, kukhona kushoda kwebufakazi lobucacile nganoma ngukuphi kufana kwe-neuronal metabolic plasticity emtimbeni kanye nekuhambisana kwayo lokungenteka esimeni sesifo. Esifundvweni sakamuva se-vitro, ema-neurons layinhloko e-cortical akhonjiswa kutsi ahlanganise emachibi e-glutamate ekudluliselwa kwemizwa, ngaleyo ndlela akhutsate kusebenta kwe-oxidative kanye ne-atherosclerosis ngaphansi kwetimo tekucindzeteleka kwe-metabolic (42). Kuyaphawuleka kutsi ngaphansi kwekuvimbela kwemitsi kwe-TCA umjikeleto enzyme succinate dehydrogenase, i-pyruvate carboxylation kukholelwa kutsi igcina kuhlanganiswa kwe-oxaloacetate kuma-neurons e-cerebellar granule lakhulisiwe (34). Kepha, kuhambisana kwemtimba kwaletindlela neticubu tebuchopho (lapho kukholelwa kutsi i-atherosclerosis ikakhulukati ivalelwe kuma-astrocytes) kusanekubaluleka lokubalulekile kwemtimba (43). Kulesi simo, idatha yetfu ikhombisa kutsi ema PNs lalimatwa yi OXPHOS emtimbeni angashintjwa abe nekuwohloka kwe BCAA kanye ne pyruvate carboxylation, lokuyimitfombo lemibili leyinhloko yekungeta tintfo letisemkhatsini te TCA pool. Noma umnikelo lotsatfwako we-BCAA catabolism ekusebentiseni emandla e-neuronal sewuphakanyisiwe, ngaphezu kwendzima ye-glutamate ne-GABA ye-neurotransmission (44), kusekhona bufakazi baletindlela ku-vivo. Ngako-ke, kulula kucabanga kutsi ema-PN langasebenti angancephetela ngekuzenzakalela kudliwa kwetintfo letisemkhatsini te-TCA leticondziswa yinchubo yekuhlanganiswa ngekukhulisa i-atherosclerosis. Ikakhulukati, upregulation PCx kungadzingeka kutsi kugcinwe sidzingo lesikhulu se-aspartic acid, lephakanyiswa ekwandziseni emaseli ngekungasebenti kahle kwe-mitochondrial (45). Kepha, kuhlatiya kwetfu kwe-metabolomics akuzange kuvete tingucuko letibalulekile esigabeni lesicinile se-aspartic acid ku-Mfn2cKO PNs (Umfanekiso S6A), lokucabanga kutsi kukhombisa kusetjentiswa lokuhlukile kwe-aspartic acid emkhatsini wemangcamuzane landzako kanye ne-neurons ye-post-mitotic . Noma indlela lecondzile ye-PCx upregulation kuma-neurons langasebenti ku-vivo isafanele kutsi ikhonjiswe, sakhombisa kutsi lempendulo lengakafiki idlala indzima lebalulekile ekugcineni simo se-redox se-neurons, leyakhonjiswa ekuhlolweni kwe-FLIM kuma-cerebellar slices. Ikakhulukati, kuvimbela ema PNs kusuka ekulawuleni i-PCx kungaholela esimeni se-oxidized kakhulu futsi kusheshise kufa kwemaseli. Kusebenta kwekubola kwe-BCAA kanye ne-carboxylation ye-pyruvate akusito tindlela tekuveta ticubu letiseceleni tekungasebenti kahle kwe-mitochondrial (7). Ngako-ke, kubonakala ngatsi sici lesibalulekile se-OXPHOS-nesifo se-neurons, noma kungasiso sici kuphela, lokubalulekile ekugucukeni kwe-neurodegeneration. .
Sifo se-Cerebellar luhlobo lolwehlukene lwesifo se-neurodegenerative lesivame kubonakala njenge-ataxia futsi sivame kulimata ema-PN (46). Lelinani le-neuron lisengotini kakhulu yekungasebenti kahle kwe-mitochondrial ngoba kubola kwabo lokukhetsiwe emagundvwane kwanele kukhicita timphawu letinyenti temoto letichaza i-ataxia ye-spinocerebellar yebantfu (16, 47, 48). Ngekuya kwemibiko, imodeli yegundvwane le-transgenic lene-gene legucukako ihlobene ne-spinocerebellar ataxia yebantfu futsi inekungasebenti kahle kwe-mitochondrial (49, 50), kugcizelela kubaluleka kwekufundza imiphumela yekushoda kwe-OXPHOS ku-PNPH. Ngako-ke, kufanelekile ikakhulukati kuhlukanisa ngemphumelelo kanye nekufundza lelinani lelikhetsekile le-neuron. Kepha, kuniketwa kutsi ema PNs ayatsintseka kakhulu ekucindzetelekeni futsi alandzisa ngesilinganiso lesiphansi salo lonkhe linani lemaseli e-cerebellar, etifundvweni letinyenti letisekelwe ku-omics, kuhlukaniswa lokukhetsiwe kwawo njengemangcamuzane laphelele kuseseyintfo leyinselele. Noma kucishe kungenteki kutsi kuzuzwe kushoda lokuphelele kwekungcoliswa kwaletinye tinhlobo temaseli (ikakhulukati ticubu tebantfu labadzala), sahlanganisa sinyatselo sekuhlukana lesiphumelelako ne-FACS kutfola inombolo leyanele ye-neurons lephilako yekuhlatiya kwe-proteomics lesezansi, futsi sibe nekuhlanganiswa kwemaprotheni laphakeme kakhulu (cishe 3000 le-databel le-e) (51). Ngekugcina kusebenta kwemangcamuzane onkhe, indlela lesiyiniketa lapha isivumela kutsi singagcini nje ngekuhlola tingucuko letikhona etindleleni tekugaya kudla ku-mitochondria, kepha futsi sihlole tingucuko letikhona kubalingani bayo be-cytoplasmic, lokuphelele kusetjentiswa kwemathegi elucwencwe lwe-mitochondrial ku-mitochondria kucebisa luhlobo lwemaseli le-tichomber le-mithox missund (52, 53). Indlela lesiyichazako ayihlobene nekufundza emaseli ePurkinje kuphela, kepha ingasetjentiswa kalula kunobe nguyiphi inhlobo yemaseli kubhekana netingucuko tekugaya kudla ebuchosheni lobugulako, kufaka ekhatsi letinye timodeli tekungasebenti kahle kwe-mitochondrial.
Ekugcineni, sikhonje lifasitelo lekwelapha ngesikhatsi salenchubo yekuhlela kabusha kwe-metabolic lengabuyisela emuva ngalokuphelele timphawu letibalulekile tekucindzeteleka kwemaseli futsi ivikele kubola kwe-neuronal. Ngako-ke, kuvisisa imiphumela lesebentako ye-rewiring lechazwe lapha kunganiketa lwati lolusisekelo ngetindlela tekwelapha letingenteka tekugcina kusebenta kwe-neuronal ngesikhatsi sekungasebenti kahle kwe-mitochondrial. Lucwaningo lwesikhatsi lesitako loluhlose kuhlukanisa tingucuko ekusebentiseni emandla kuletinye tinhlobo temaseli ebuchosheni luyadzingeka kute kuvetwe ngalokugcwele kusebenta kwalesimiso kuletinye tifo tetinzwa.
MitoPark emagundvwane sekuchazwe ngaphambilini (31). C57BL / 6N emagundvwane nge loxP flanking Mfn2 ufuzo sekuchazwe ngaphambilini (18) futsi awela L7-Cre emagundvwane (23). Lomphumela lophindvwe kabili we-heterozygous wabese uyawela nemagundvwane e-Mfn2loxP/Mfn2loxP lafanako kwentela kukhicita ema-knockouts e-Purkinje-lakhetsekile e-Mfn2 (Mfn2loxP/Mfn2loxP; L7-cre). Ku-subset yekuhlangana, i-Gt (ROSA26) SorStop-mito-YFP allele (yema-mtYFP) yetfulwa ngekwewela lokungetiwe (20). Tonkhe tindlela tetilwane tentiwa ngekulandzela imihlahlandlela yase Europe, yavelonkhe kanye neyetikhungo futsi yavunywa yi LandesamtfürNatur yase Umwelt kanye ne Verbraucherschutz, eNorth Rhine-Westphalia, eJalimane. Umsebenti wetilwane uphindze ulandzele sicondziso seNhlangano Yetilwane Tesayensi Yelabhorethri yase Europe.
Ngemuva kwekubulala buhlungu kwekuphuka kwesibeletho semfati lokhulelwe, umbungu weligundvwane uyahlukaniswa (E13). Le cortex yahlukaniswa ku Hanks’ Sisombululo Seluswayi Lelilinganiselwe (HBSS) lengetiwe nge Hepes lengu 10 mM futsi yadluliselwa ku Dulbecco’s Loguculwe Lusweti Lolusemkhatsini lolucuketse ipapain (20 U/ml) kanye ne cysteine ​​(1μg/ml). Fukamela leticubu ku DMEM) bese uyatihlukanisa ngekugaya kwe enzymatic. Ml) ku 37°C imizuzu lengemashumi lamabili, bese kuyagaywa ngemshini ku DMEM lengetiwe nge 10% we serum yetinkhomo letingakazalwa. Emaseli abehlanyelwa kuma coverslips engilazi lagcotjwe nge polylysine ngesilinganiso se 2×106 ngesitja selisiko le 6 cm noma ngesilinganiso se 0.5×105 emaseli/cm2 kwentela kuhlatiya titfombe. Ngemuva kwemahora langu 4, lendzawo yatsatfwa esikhundleni sayo lesemkhatsini lete serum ye Neurobasal lecuketse 1% we B27 kanye ne 0.5 mM GlutaMax. Ema neurons abese agcinwa ku 37°C kanye na 5% CO2 kulo lonkhe lokuhlolwa, futsi adliwa kanye ngeliviki. Kute kukhutsatwe kuhlanganiswa kabusha ku vitro, 3μl (sitja selisiko lelinemgodzi longu 24) noma 0.5μl (ipuleti lelinemigodzi lengu 24) yaleligciwane le-AAV9 lelilandzelako lasetjentiswa kwelapha ema-neurons ngelilanga lesibili ku-vitro: AAV9.CMV.PI.eGFP. WPRE.bGH (I-Addgene, inombolo yekhathalogu 105530-AAV9) kanye ne-AAV9.
Igundvwane Mfn1 kanye ne Mfn2 DNA lephelele (letfolakala ku Addgene plasmid #23212 kanye #23213, ngekulandzelana) imakwe ngekulandzelana kwe V5 (GKPIPNPLLGLDST) ku C-terminus, futsi ihlanganiswa ne mCherry kufreyimu ngekulandzelana kwe T2A. I-Grx1-roGFP2 sipho lesivela ku-Heidelberg TP Dick DFKZ. Ngekufaka esikhundleni sekhasethi ye-tdTomato kusetjentiswa tindlela tekukhicita letivamile, lekhasethi yakhicitwa ngaphansi kwemgogodla we-pAAV-CAG-FLEX-tdTomato (inombolo yekubhekisa ye-Addgene 28306) kute kukhicitwe i-pAAV-CAG-FLEX-mCherry-T2A-MFNGAV-V5, pAAV-CAG-FLEX-tdTomato. I-FLEX-mCherry-T2A-MFN1-V5 kanye ne-pAAV-CAG-FLEX-Grx-roGFP2 emavectors. Lisu lelifanako lasetjentiswa kukhicita i-vector yekulawula pAAV-CAG-FLEX-mCherry. Kute kukhicitwe kwakha kwe-AAV-shPCx, kudzingeka i-plasmid AAV vector (i-VectorBuilder, i-pAAV [shRNA] -CMV-mCherry-U6-mPcx- [shRNA # 1]), lecuketse kulandzelana kwe-DNA lokukhoda i-shRNA lecondziswe ku-PCx yegundvwane (5′ 3′) Ngaphansi kwekulawula kwemgcugcuteli we-U6, i-mCherry isetjentiswa ngaphansi kwekulawula kwemgcugcuteli we-CMV. Kukhicitwa kwema vectors e AAV lasitako kwentiwa ngekuya kweticondziso temkhiciti (Cell Biolabs). Ngamafuphi, sebentisa i-plasmid yekudlulisa lethwele i-mCherry-T2A-MFN2-V5 (i-pAAV-CAG-FLEX-mCherry-MFN2-V5), i-mCherry-T2A-MFN1-V5 (i-pAAV-CAG-FLEX-mCherry) ngesikhashana (i-pAAV-CAG-FLEX-mCherry) noma i-Grx-roGFP2 (i-pAAV-CAG-FLEX-Grx-roGFP2) yekukhoda lifutsi, kanye nekukhoda iphrotheni ye-AAV1 capsid kanye nephrotheni lesekelako Kupakisha i-plasmid plasmid, kusetjentiswa indlela ye-calcium phosphate. Leligciwane leliluhlata lelisetulu latfolakala ngemijikeleto yekubandza-kuncibilika ekugezeni kwe ayisi/ethanol leyomile kanye nemaseli lahlanyelwe ku phosphate buffered saline (PBS). Le vector ye AAV yahlanjululwa ngekungachubeki kwe iodixanol gradient (emahora langu 24 ku 32,000 rpm na 4°C) futsi yagcizelelwa kusetjentiswa luhlu lwe Amicon ultra-15 lwekuhlunga. I-Genome titer ye-AAV1-CAG-FLEX-mCherry-T2A-MFN2-V5 [2.9 × 1013 ikhophi ye-genome (GC) / ml], i-AAV1-CAG-FLEX-mCherry (6.1 × 1012 GC / ml), i-AAV1-CAG-FLEX-mCherry (6.1 × 1012 GC / ml), i-AAV1-CAG-FLEX beyilinganiswa njengaphambilini, (qPCR) -MFN1-V5 (1.9 × 1013 GC / ml) kanye ne-AAV1-CAG-FLEX-Grx-roGFP2 (8.9 × 1012 GC / ml).
Ema neurons layinhloko aklwebhelwa ku 1x PBS lebandza njengeqhwa, ahlanganiswa, bese ahlanganiswa ku 0.5% Triton X-100 / 0.5% sodium deoxycholate / PBS lysis buffer lecuketse i-phosphatase kanye ne-protease inhibitor (Roche). Kulinganisa emaprotein kwentiwa ngekusebentisa kuhlolwa kwe esidi ye bicinchoninic (iThermo Fisher yesayensi). Lamaprotheyini abese ahlukaniswa nge-SDS-polyacrylamide gel electrophoresis, bese ayabhalwa elucwencweni lwe-polyvinylidene fluoride (i-GE Healthcare). Vimba tindzawo letingakakhetsi bese uyafukamela nge antibody leyinhloko (buka Lithebula S1 ngemininingwane) ku 5% welubisi ku TBST (Tris-buffered saline nge Tween), tinyatselo tekugeza kanye ne antibody yesibili ku TBST Incubate. Fukamela nge antibody leyinhloko busuku bonkhe ku +4°C. Ngemuva kwekugeza, faka i-antibody yesibili emahora lamabili ngekushisa kweligumbi. Ngemuva kwaloko, ngekufukamela le-blot lefanako nge-anti-β-actin antibody, kulayishwa lokufanako kwacinisekiswa. Kutfolakala ngekutsi kuguculwe kube kukhanya kwemakhemikhali kanye nekutfutfukisa kukhanya kwemakhemikhali (GE Healthcare).
Leti neurons lebetihlanyelwe ngaphambilini kuma coverslips engilazi talungiswa nge 4% paraformaldehyde (PFA)/PBS ngesikhatsi lesibekiwe ngesikhatsi sekushisa ekamelweni imizuzu lelishumi. Letimbonyo ticala kugcwaliswa nge 0.1% Triton X-100/PBS imizuzu lesihlanu ngekushisa kweligumbi, bese kuvimbela [3% albumin ye serum yenkhomo (BSA)/PBS]. Ngelilanga lesibili, lama coverslips agezwa nge buffer yekuvimba bese afutfunyelwa nge antibody yesibili lefanele ye fluorophore-conjugated emahora lamabili ngekushisa kweligumbi; ekugcineni, lamasampula agezwa kahle ku PBS nge 4 ',6-diamidino-2 -Phenylindole (DAPI) iyaphikiswa bese iyalungiswa kusilayidi sesibonakhulu nge Aqua-Poly / Intsaba.
Emagundvwane (lamadvodza nalamasikati) abulawa buhlungu ngekutsi ajovwe nge-ketamine (130 mg/kg) kanye ne-xylazine (10 mg/kg) futsi aniketwa ngaphansi kwesikhumba nge-carprofen yekubulala buhlungu (5 mg/kg) , Futsi abekwa esitfweni sekubulala buhlungu (Kopd a). Veta luhlaka lwekhanda bese usebentisa ibhobo lematinyo kwentela kucedza incenye ye cerebellar cortex lehambisana nelitsambo le mis (kusuka ku lambda: umsila 1.8, lateral 1, lehambisana ne lobules IV na V). Sebentisa inyalisi yesirinji legobile kwenta ngekucophelela imbobo lencane kule skull kwentela kugwema kuphazamisa imitsambo lengephansi. Ngemuva kwaloko le-capillary yengilazi lencane ledonswe ifakwa kancane kancane emgodzini lomncane (kusuka ku -1.3 kuya ku -1 eceleni kwe-ventral ye-dura mater), bese 200 kuya ku-300 nl AAV ijovwa ku-micro-injector (Narishige) ngemijovo yesandla (Narishige tisure) ngesikhatsi sekucindzeteleka kwe-2. lifasitelo lemizuzu. Ngemuva kwekufakwa, beka le capillary leminye imizuzu lelishumi kwentela kuvumela leligciwane kutsi lisakateke ngalokuphelele. Ngemuva kwekutsi ema capillaries akhishiwe, sikhumba siyathungwa kahle kwentela kunciphisa kuvuvuka kwesilonda bese kuvumela lesilwane kutsi silulame. Letilwane talashwa ngetindlela tekucedza buhlungu (caspofen) emalanga lamaningi ngemuva kwekuhlindvwa, ngaleso sikhatsi simo sato semtimba besigadvwa kahle bese tiyabulawa ngesikhatsi lesibekiwe. Tonkhe tindlela tekusebenta tentiwa ngekuhambisana nemihlahlandlela yaseYurophu, yavelonkhe kanye neyetikhungo futsi tavunywa yi LandesamtfürNatur yase Umwelt kanye nase Verbraucherschutz, eNorth Rhine-Westphalia, eJalimane.
Letilwane tabulawa buhlungu nge ketamine (100 mg/kg) kanye ne xylazine (10 mg/kg), bese inhlitiyo yafakwa nge 0.1 M PBS kucala, bese nge 4% PFA ku PBS. Leticubu tahlukaniswa futsi talungiswa ku 4% PFA/PBS busuku bonkhe ku 4°C. Ummese lodvudvutako (Leica Microsystems GmbH, eVienna, e-Austria) wasetjentiswa kulungisa tincenye te-sagittal (50 μm ngebukhulu) kusuka engcondvweni lecinile ku-PBS. Ngaphandle uma kucaciswe ngalenye indlela, kufaka emabala etincenye letintantako letikhululekile kwentiwa njengoba kuchaziwe ngenhla (13) ngekushisa kweligumbi kanye nekuvuselela. Ngamafuphi, kwekucala, letincetu letitfolakele tatsatfwa nge 0.5% Triton X-100 / PBS imizuzu lengu 15 ngesikhatsi sekushisa ekamelweni; kulamanye ema epitopes (Pcx na Shmt2), ngekutsi ku tris-EDTA buffer ku 80°C (PH 9) kushisa letincetu imizuzu lengemashumi lamabili nesihlanu esikhundleni salesinyatselo. Ngemuva kwaloko, letincenye tafutfunyelwa nge antibody leyinhloko (bona Lithebula S1) ku buffer yekuvimba (3% BSA/PBS) ku 4°C busuku bonkhe ngekuvuselela. Ngelilanga lelilandzelako, letincenye tagezwa nge buffer yekuvimba futsi tafutfunyelwa nge antibody yesibili lefanele ye fluorophore-conjugated emahora lamabili ngekushisa kweligumbi; ekugcineni, letincenye tagezwa kahle ku PBS, taphindze tatsatfwa nge DAPI, bese tiyalungiswa nge AquaPolymount Ku silayidi sesibonakhulu.
Sibonakhulu sekukhanya se-laser (i-TCS SP8-X noma i-TCS Lishidi Lekukhanya Ledijithali, Leica Microsystems) lefakwe i-laser yekukhanya lemhlophe kanye ne-laser ye-ultraviolet ye-405 diode yasetjentiswa kutsatsa sitfombe sesampula. Ngekujabulisa i-fluorophore kanye nekubutsela ndzawonye luphawu nge-Hybrid Detector (HyDs), isoftware ye-LAS-X yasetjentiswa kubutsela ndzawonye titfombe letihlanganisiwe letihambisana nekutsatsa sampula kwe-Nyquist ngendlela yekulandzelana: kumaphaneli langalinganiswa, timphawu letishintjashintjako kakhulu (sibonelo, kumaseli e-somatic kanye ne-dendrites) ye-PFPY) mt . Imodi ye-BrightR). Gating kusuka ku 0.3 kuya ku 6 ns kusetjentiswa kunciphisa imvelaphi.
Kutsatsa titfombe ngesikhatsi sangempela semangcamuzane lahlungiwe. Ngemuva kwekuhlunga ku Neurobasal-A lesemkhatsini lecuketse 1% B27 wesengeto kanye ne 0.5 mM GlutaMax, emaseli asheshe abekwa imbewu kumaslayidi engilazi lambonywe nge poly-l-lysine (μ-Slide8 Kahle, Ibidi, inombolo yekhathalogu 80826) , Bese ayigcina ku 1%s kuya ku 3000. kuhlala. Kutsatfwa kwemifanekiso ngesikhatsi sangempela kwentiwa ngesibonakhulu se-Leica SP8 lesiskena nge-laser lefakwe i-laser lemhlophe, i-HyD, 63 × [1.4 kuvula kwetinombolo (NA)] lens lenhloso yemafutsa kanye nesigaba sekushisa.
Leligundvwane lasheshe labulawa buhlungu nge carbon dioxide futsi lancunywa inhloko, ingcondvo yasuswa ngekushesha ekhanda, futsi yasikwa yaba ngu 200μm ngebukhulu (kwekuhlolwa kwekulebula kwe Fi3C) noma 275μm ngebukhulu (kwekuhlolwa kwe photon lembili) sigaba se sagittal lesigcwele letintfo letilandzelako te crece I-VH-50. Isayensi, eWalldorf, eJalimane) igcwele letintfo letilandzelako: 125 mM lebandza njengeqhwa, legcwele ikhabhoni (95% O2 kanye na 5% CO2) iCa2 lephansi + lutfuli lwebuchopho lolwentiwe (ACSF) NaCl, 2.5 mM KCl, 1.25 mM sodium pho2, 1.25 mM sodium pho2. glucose, 0.5 mM CaCl2 kanye na 3.5 mM MgCl2 (umfutfo we-osmotic we-310 kuya ku-330 mmol). Dlulisela letincetu tebuchopho letitfolakele egumbini lekucala lekufukamela lelicuketse i-Ca2 + ACSF lephakeme (125.0 mM NaCl, 2.5 mM KCl, 1.25 mM sodium phosphate buffer, 25.0 mM NaHCO3, 25.0 mM d-glucose, 25.0 mM d-glucose. MgCl2) Lesemkhatsini) pH 7.4 kanye na 310 kuya ku 320 mmol).
Ngesikhatsi senchubo yekutsatsa titfombe, letincetu tatfutfwa taya ekamelweni lekutsatsa titfombe letinikele, futsi lokuhlolwa kwentiwa ngaphansi kwekuchubeka nekufutfumala kwe ACSF ngesikhatsi sekushisa lokuhlala njalo kwa 32° kuya ku 33°C. Sibonakhulu sekuskena se-laser se-multiphoton (i-TCS SP8 MP-OPO, i-Leica Microsystems) lefakwe lens lehlosiwe ye-Leica 25x (i-NA 0.95, emanti), i-Ti: I-laser ye-Sapphire (i-Chameleon Vision II, I-Coherent) yasetjentiswa kutsatsa titfombe tetincetu. Imojuli ye-FLIM (i-PicoHarp300, i-PicoQuant).
I-FLIM ye-Grx1-roGFP2. Tingucuko esimeni se-cytoplasmic redox se-PNs talinganiswa nge-FLIM lene-photon lemibili etincetu tebuchopho be-sagittal, lapho khona i-biosensor ye-Grx1-roGFP2 ihlose ema-PN. Kulesendlalelo se-PN, insimu yekutfola ikhetfwa cishe 50 kuya ku-80 μm ngaphansi kwemhlabatsi wesicephu kucinisekisa kutsi kune-PN lephilako (lokusho kutsi, kungabi khona kwesakhiwo sebuhlalu noma tingucuko tesimo se-neuronal eceleni kwema-dendrites) kanye ne-roGFPque lephindvwe kabili lenhle ye-roGFP2 inzwa kanye ne-AAVx-incoding sh mCheri). Hlanganisa titfombe letine-stack yinye nge-2x zoom yedijithali [budze begagasi lekujabula: 890 nm; 512 nm 512 emaphikseli]. Kutfola: iHyD lengekhatsi, licembu lekuhlunga le fluorescein isothiocyanate (FITC)] kanye nekulinganisa sitfombe kungakapheli imizuzu lemibili kuya kulemitsatfu kusetjentiswa kucinisekisa kutsi emaphoton lanele ayabutfwa (emaphoton langu 1000 sekukonkhe) kwentela kufaka emajika. Kuzwela kwe-Grx1-roGFP2 probe kanye nekucinisekiswa kwetimo te-FLIM kwentiwa ngekugadza linani lekuphila kwe-roGFP2 uma kungetwa i-exogenous 10 mM H2O2 ku-ACSF yekufutsa (kukhulisa i-oxidation, lokuholela ekukhuleni kwekuphila kwe-mrethizol of2). kuncishiswa, lokuholela ekuncipheni kwesikhatsi sekuphila) (Umfanekiso S8, D kuya ku G). Sebentisa isoftware ye-FLIMfit 5.1.1 kuhlatiya imiphumela letfoliwe, linganisa i-curve yekubola kwe-exponential yinye yaso sonkhe sitfombe ku-IRF lelinganisiwe (umsebenti wekuphendvula wetintfo tekusebenta), futsi χ2 cishe ngu-1. isetjentiselwa kulinganisa.
Kuhlatiya emandla e-mitochondrial. Ngemuva kwekutsi lesigaba lesibucayi sifuywe nge-100 nM TMRM lengetwe ngco ku-ACSF lephefumulelwe imizuzu lengu-30, tingucuko letingaba khona te-mitochondrial te-PNs talinganiswa ngesibonakhulu se-photon lembili. Kutsatfwa kwetitfombe kwe-TMRM kwentiwa ngekutsi kujabulise le-probe ku-920 nm kanye nekusebentisa i-HyD lengekhatsi (i-sothiocyanate ye-tetramethylrhodamine: 585/40 nm) kuhlanganisa timphawu; ngekusebentisa budze begagasi lelifanako lekujabula kepha kusebentisa i-HyD lehlukile lengekhatsi (FITC :525/50) ku-mtYFP. Sebentisa ipulaki ye-ImageJ ye-Image Calculator kuhlola emandla e-mitochondrial ezingeni leseli linye. Ngamafuphi, isilinganiso se-plug-in: luphawu = min (mtYFP, TMRM) sisetjentiselwa kukhomba sifundza se-mitochondrial lesikhombisa luphawu lwe-TMRM ePurkinje Somali esitfombeni se-confocal se-stack sinye sesiteshi lesihambisanako. Ngemuva kwaloko indzawo ye-pixel ku-mask lephumelelako iyalinganiswa, bese iyajwayelekile esitfombeni lesihambisanako se-single-stack sesiteshi se-mtYFP kute kutfolakale ingcenye ye-mitochondrial lekhombisa emandla e-mitochondrial.
Lesitfombe sakhishwa nge-software ye-Huygens Pro (Yesayensi Yekutsatsa Titfombe). Kuletitfombe letiskeniwe temathayela, kuhlanganiswa kwethayela linye kwentiwa kusetjentiswa i-algorithm yekutfunga letentekelako leniketwa yi-software ye-LAS-X. Ngemuva kwekulinganisa sitfombe, sebentisa i-ImageJ ne-Adobe Photoshop kute uchubeke ucubungule sitfombe futsi ulungise ngendlela lefanako kukhanya kanye nekucatsanisa. Sebentisa i-Adobe Illustrator kute ulungiselele imifanekiso.
mtDNA kuhlatiya lokugcila. Inani letilonda te-mtDNA lalinganiswa etincenyeni te-cerebellar letibhalwe ngema-antibodies lalwa ne-DNA ngesibonakhulu se-confocal. Indzawo ngayinye lehlosiwe yadalelwa umtimba weseli kanye ne-nucleus yeseli ngalinye, futsi indzawo lelandzelanako yabalwa kusetjentiswa i-plug-in ye-Multi Measure (software ye-ImageJ). Susa indzawo yenuzi endzaweni yemtimba weseli kute utfole indzawo ye-cytoplasmic. Ekugcineni, i-plug-in yekuhlatiya tinhlayiya (isofthiwe ye-ImageJ) yasetjentiswa kulinganisa ngekuzenzakalela emaphuzu e-cytoplasmic DNA lakhombisa i-mtDNA esitfombeni sekucala, futsi imiphumela letfolakele yajwayelekile ku-PN isilinganiso semagundvwane e-CTRL. Imiphumela ivetwa njengenani lelisemkhatsini le-nucleosides ngeseli ngalinye.
Kuhlatiya kuveta emaprotheyini. Sebentisa i-plug-in ye-ImageJ ye-Image Calculator kuhlola kuveta kwemaprotheni ku-PN ezingeni leseli linye. Ngamafuphi, esitfombeni se-confocal selucwencwe lunye lwesiteshi lesihambisanako, ngesilinganiso: luphawu = min (mtYFP, umtimba), sifundza se-mitochondrial lesikhombisa kuvikeleka kwemtimba ku-antibody letsite ePurkina siyakhonjwa. Ngemuva kwaloko indzawo ye-pixel ku-mask lephumelelako iyalinganiswa, bese iyajwayelekile esitfombeni lesihambisanako se-single-stack sesiteshi se-mtYFP kute kutfolakale ingcenye ye-mitochondrial yephrotheni lekhonjisiwe.
Kuhlatiya buningi bemaseli ePurkinje. I-plug-in ye-Cell Counter ye-ImageJ yasetjentiswa kuhlola bukhulu be-Purkinje ngekuhlukanisa linani lemangcamuzane e-Purkinje labalwa ngebudze bendandatho ye-cerebellar lehlalwa ngulamaseli labalwe.
Kulungiselela isampula kanye nekubutsa. Lengcondvo levela ecenjini lekulawula kanye nemagundvwane eMfn2cKO yalungiswa ku 2% PFA / 2.5% glutaraldehyde ku 0.1 M phosphate buffer (PB), bese tincenye te coronal talungiswa kusetjentiswa ema ciliates (Leica Mikrosysteme GmbH, Vienna, Austria) (Kugcwala 50 kuya ku The60 tetraoxide kanye na 1.5% we potassium ferrocyanide ngesikhatsi sekushisa ekamelweni lihora linye. Letigaba tagezwa katsatfu ngemanti lahlungiwe, bese tigcotjwa nge 70% ethanol lecuketse 1% uranyl acetate imizuzu lengemashumi lamabili. Letigaba tase tibese tiphelelwa ngemanti etjwale lobulinganisiwe bese tifakwa ku Durcupan ACM (i-Araldite casting resin M) i-epoxy resin (Isayensi ye-Electron Microscopy, inombolo yekhathalogu 14040) emkhatsini wema-slide engilazi lagcotjwe nge-silicon, bese ekugcineni ku-60°C Polymerize48 ehhavini. Indzawo ye cerebellar cortex yakhetfwa futsi tincenye letincane kakhulu letingu 50 nm tasikwa ku Leica Ultracut (Leica Mikrosysteme GmbH, eVienna, e-Austria) bese tiyakhetfwa ku gridi ye 2 × 1 mm yelitfusi lelinemgodzi lefakwe ifilimu ye polystyrene. Letigaba tahlanganiswa ngesisombululo se 4% uranyl acetate ku H2O imizuzu lelishumi, tagezwa nge H2O tikhatsi letiningi, bese nge Reynolds lead citrate ku H2O imizuzu lelishumi, bese tiyagezwa nge H2O tikhatsi letiningi. Ema-micrographs atsatfwa ngesibonakhulu se-elekthronikhi sekudlulisela i-Philips CM100 (i-Thermo Fisher Scientific, Waltham, MA, eMelika) kusetjentiswa ikhamera yedijithali ye-TVIPS (Luhlelo Lwekucubungula Ividiyo Netitfombe LwaseTietz) TemCam-F416 (i-TVIPS GmbH, Gauting, eMelika). eJalimane).
Kumagundvwane latseleleke nge AAV, ingcondvo yahlukaniswa yase iyasikwa yaba sigaba se sagittal lesinebukhulu lobungaba ngu 1 mm, bese i cerebellum yahlolwa kusetjentiswa sibonakhulu se fluorescence kwentela kukhomba indandatho letseleleke nge AAV (lokusho kutsi, i-mCherry leveta). Kuhlolwa kuphela lapho khona umjovo we-AAV uphumela ekusebenteni kahle kakhulu kwekugucula kwelucwencwe lwemaseli ePurkinje (lokusho kutsi cishe lonkhe lucwencwe) lokungenani emaringi lamabili lalandzelanako e-cerebellar ayasetjentiswa. Le loop leguculwe yi AAV yahlukaniswa kancane kwentela kutsi ibekwe ebusuku ngemuva kwekucinisa (4% PFA kanye na 2.5% glutaraldehyde ku 0.1 M cocoate buffer) bese iyacubungulwa. Kufaka EPON, leticubu letimile tagezwa nge 0.1 M ye sodium cocoate buffer (Applichem), bese tiyafutfunyelwa nge 2% OsO4 (os, Tinsita Tesayensi; Caco) ku 0.1 M sodium cocoate buffer (Applichem) Emahora lamane, bese tiyagezwa emahora lamabili. Phindza tikhatsi letingu-3 nge-0.1 M cocamide buffer. Ngemuva kwaloko, loluchungechunge lwe ethanol lolukhuphukako lwasetjentiswa kufukamela sisombululo ngasinye se ethanol ku 4°C imizuzu lelishumi nesihlanu kwentela kukhipha emanti kuleticubu. Leticubu tadluliselwa ku propylene oxide futsi tafutfunyelwa busuku bonkhe ku EPON (Sigma-Aldrich) ku 4°C. Faka leticubu ku EPON lensha ngekushisa kweligumbi emahora lamabili, bese uyayifaka ku 62°C emahora lamashumi lasikhombisa nakubili. Sebentisa i-ultramicrotome (i-Leica Microsystems, UC6) kanye nemukhwa welidayimane (i-Diatome, i-Biel, eSwitzerland) kusika tincenye letincane kakhulu letingu-70 nm, bese ufaka libala nge-1.5% ye-uranyl acetate imizuzu lelishumi nesihlanu ku-37°C, bese ufaka libala ngesisombululo se-lead minutes citrate imizuzu lengu-4. Letitfombe letincane te-elekthronikhi tatsatfwa kusetjentiswa sibonakhulu se-elekthronikhi sekudluliswa kwe-JEM-2100 Plus (i-JEOL) lesifakwe i-Camera OneView 4K 16-bit (i-Gatan) kanye nesofthiwe ye-DigitalMicrograph (i-Gatan). Kute kuhlaziywe, ema-micrographs e-electron atfolakala nge-5000 × noma 10,000 × yekusondzeta kwedijithali.
Kuhlatiya kwesimo se-mitochondria. Kuko konkhe kuhlatiya, ema-contours e-mitochondria ngamunye abekwa ngesandla ngetitfombe letidijithali kusetjentiswa isoftware ye-ImageJ. Kuhlaziywa imikhawulo lehlukene ye-morphological. Kucina kwe-mitochondrial kuvetwa njengeliphesenti lelitfolakala ngekuhlukanisa indzawo ye-mitochondrial lephelele yeseli ngalinye ngendzawo ye-cytoplasm (indzawo ye-cytoplasm = indzawo yeseli-indzawo ye-nucleus yeseli) × 100. Kuyindingilizi kwe-mitochondria kubalwa ngefomula [4π∙2(indzawo)]. Le ista morphology ye mitochondria yahlaziywa futsi yahlukaniswa yaba tigaba letimbili (“emashubhu” kanye “nemabhubhu”) ngekuya ngetindlela tabo letiyinhloko.
Inombolo ye-autophagosome / ye-lysosome kanye nekuhlatiya buningi. Sebentisa isoftware ye-ImageJ kuveta ngesandla imigca ye-autophagosome/lysosome ngayinye esitfombeni sedijithali. Indzawo ye-autophagosome/lysosome ivetwa njengeliphesenti lelibalwa ngekuhlukanisa indzawo yesakhiwo se-autophagosome/lysosome lephelele yeseli ngalinye ngendzawo ye-cytoplasm (indzawo ye-cytoplasm=indzawo yeseli-indzawo ye-nucleus)×100. Kucina kwema autophagosomes/lysosomes kubalwa ngekuhlukanisa inombolo lephelele ngelinani letakhiwo te autophagosome/lysosome ngeseli ngalinye (ngekuya ngendzawo ye cytoplasmic) (indzawo ye cytoplasmic = indzawo yeseli-indzawo yenuzi).
Kulebula kwekuhlukanisa lokubucayi kanye nekulungiselela isampula. Kute kuhlolelwe lokudzinga kulebula glucose, dlulisela letincetu tebuchopho letibukhali egumbini lekucala lekufukamela, lelicuketse ikhabhoni legcwele (95% O2 kanye na 5% CO2), iCa2 + ACSF lesetulu (125.0 mM NaCl, 2.5 mM KCl, 1.25 mM sodium phosphate, 1.25 mM sodium phosphate. 25.0 mM d-shukela, 1.0 mM CaCl 2 kanye na 2.0 mM MgCl 2, lelungiswe ku pH 7.4 kanye na 310 kuya ku 320 mOsm), lapho khona shukela 13 C 6- Kufaka esikhundleni sashukela (Eurisotop, inombolo yekhathalogu CLM9-1). Kuhlolwa lokudzinga kulebula kwe-pyruvate, dlulisela tincetu tebuchopho letibukhali ku-Ca2 + ACSF lephakeme (125.0 mM NaCl, 2.5 mM KCl, 1.25 mM sodium phosphate buffer, 25.0 mM NaHCO3, 25.0 mM d-glucose, 25.0 mM d-glucose. MgCl2, hlela ku pH 7.4 kanye na 310 kuya ku 320mOsm), bese wengeta 1mM 1-[1-13C]pyruvate (Eurisotop, inombolo yekhathalogu CLM-1082). Fukamela letincenye imizuzu lengu 90 ku 37°C. Ekupheleni kwekuhlola, letincenye tasheshe tagezwa ngesisombululo lesinemanti (pH 7.4) lesine 75 mM ammonium carbonate, bese tiyahlanganiswa ku 40:40:20 (v:v:v) i-acetonitrile (ACN): i-methanol: emanti. Ngemuva kwekutsi letincenye tifutfumele ku ice imizuzu lengemashumi lamatsatfu, lamasampula abekwa endzaweni lesetulu ku 21,000 g imizuzu lelishumi ku 4°C, bese le supernatant lecacile yomiswa ku SpeedVac concentrator. Le pellet ye metabolite leyomile lephumelelako yagcinwa ku -80°C kwaze kwaba ngulapho ihlatiya.
Kuhlatiya kwe-chromatography-yebuningi be-amino esidi le-13 lebhalwe nge-C. Kute kuhlaziywe ngendlela lesemantini (LC-MS), le pellet ye metabolite yaphindze yafakwa ku 75μl wemanti elizinga le LC-MS (Honeywell). Ngemuva kwekucindzetelwa ku 21,000 g imizuzu lesihlanu ku 4°C, 20 μl we supernatant lecacisiwe yasetjentiswa kuhlatiya i-amino acid flux, kantsi lokunye lokukhishwako kwasetjentiswa ngekushesha kuhlatiya anion (buka ngephansi). Kuhlatiya kwe-amino acid kwentiwa kusetjentiswa indlela yekukhishwa kwe-benzoyl chloride lechazwe ngaphambilini (55, 56). Esinyatselweni sekucala, 10 μl we 100 mM we sodium carbonate (Sigma-Aldrich) wangetwa ku 20 μl wekukhishwa kwe metabolite, kwase kutsi 10 μl we 2% we benzoyl chloride (Sigma-Aldrich) wangetwa ku LC grade ACN. Lesampula yavutsiwe kafishane bese iya centrifuged ku 21,000 g imizuzu lesihlanu ku 20°C. Dlulisela le supernatant lehlanjululwe ku vial ye autosampler lengu 2 ml ngekufaka ingilazi ye conical (200 μl ivolumu). Lamasampula ahlaziywa kusetjentiswa luhlelo lwe-LC lwe-Acquity iClass lolusebenta kakhulu (Emanti) loluhlanganiswe ne-Q-Exactive (QE)-HF (I-Orbitrap Yensimu Lephakeme kakhulu) lenesisombululo lesiphakeme se-mass spectrometer (i-Thermo Fisher Scientific). Kuhlatiya, 2μl yesampula lekhishwako yajovwa ku 100 × 1.0 mm silica T3 column lenemandla lamakhulu (Emanti) lecuketse tinhlayiya letingu 1.8μm. Lizinga lekugeleta lingu 100μl/min, futsi lendlela yekuvikela ifaka sivikelo A (10 mM ammonium formate kanye na 0.15% we formic acid emantini) kanye nekuvikela B (ACN). I gradient injalo: 0%B ngemizuzu lengu 0; 0%B. 0 kuya ku 15% B ngemizuzu lengu 0 kuya ku 0.1; 15 kuya ku 17% B ngemizuzu lengu 0.1 kuya ku 0.5; B ku 17 kuya ku 55% ku 0.5 kuya ku 14 imizuzu; B ku 55 kuya ku 70% ku 14 kuya ku 14.5 imizuzu; ku 14.5 kuya ku 70 kuya ku 100% B ngemizuzu lengu 18; 100% B ngemizuzu lengu 18 kuya ku 19; 100 kuya ku 0% B ngemizuzu lengu 19 kuya ku 19.1; 0% B ku 19.1 kuya ku 28 imizuzu (55, 56). I-QE-HF mass spectrometer isebenta ngendlela ye-ionization lenhle ngeluhlu lwe-m / z (silinganiso se-mass / charge) se-50 kuya ku-750. Lomtfombo we-ionization ye-electrospray leshisako (ESI) usebenta ngemandla ekufafata langu-3.5 kV, lizinga lekushisa le-capillary lelingu-250°C, kuhamba kwemoya we-sheath lokungu-60 AU (emayunithi langakhetsi), kanye nemoya lositako longu-20 AU. 250°C. I lens ye S isethwe ku 60 AU.
Kuhlatiya kwe-anion chromatography-MS kwe-13C lebhalwe nge-esidi lephilako. Lokusele kwe metabolite (55μl) kwahlaziywa kusetjentiswa luhlelo lwe Dionex ion chromatography (ICS 5000+, Isayensi Yekushisa) lehlanganiswe ne QE-HF mass spectrometer (Isayensi Yekushisa). Ngamafuphi, 5μl ye-metabolite lekhishiwe yajovwa kukholomu ye-Dionex IonPac AS11-HC lefakwe i-HPLC (2 mm × 250 mm, bukhulu betinhlayiya 4μm, i-Thermo Fisher Scientific) ngendlela yekuphusha-ngekhatsi kweluphu leyingcenye ngesilinganiso sekugcwalisa se-1. 4μm, Umdobi Loshisako Wesayensi). Lokushisa kwekholomu kugcinwa ku 30°C, bese i-autosampler isethwa ku 6°C. Sebentisa ikhatriji ye potassium hydroxide leniketwa ngemanti lakhiwe nge deionized kwentela kukhicita umkhawulo we potassium hydroxide ngekusebentisa umshini we eluent. Kuhlukaniswa kwema metabolites ngesilinganiso sekugeleta se 380μl/min, kusetjentiswa le gradient lelandzelako: 0 kuya ku 3 imizuzu, 10 mM KOH; 3 kuya ku-12 imizuzu, 10 kuya ku-50 mM KOH; 12 kuya ku-19 imizuzu, 50 kuya ku-100 mM KOH; 19 kuya ku-21 imizuzu , 100 mM KOH; 21 kuya ku-21.5 imizuzu, 100 kuya ku-10 mM KOH. Le kholomu yaphindze yalinganiswa ngaphansi kwe-10 mM KOH imizuzu lengu-8.5.
Letintfo letilahliwe tihlanganiswa ne 150μl/min isopropanol yekwengeta umfudlana ngemuva kwekholomu bese icondziswa ku spectrometer yebukhulu lesetulu lesebenta ngendlela ye ionization lembi. MS ugadza uhla mass kusuka m / z 50 kuya 750 ngesisombululo 60,000. I-AGC isethwe ku-1 × 106, futsi sikhatsi lesikhulu se-ion sigcinwa ku-100 ms. Umtfombo we-ESI loshisako bewusebenta ngemandla ekufafata langu-3.5 kV. Letinye tilungiselelo temtfombo we-ion nguleti letilandzelako: lizinga lekushisa le-capillary 275°C; kugeleta kwegesi yesikhumba, 60 AU; kugeleta kwegesi lokusitako, 20 AU ku 300°C, kanye nekubeka lens ye S ku 60 AU.
Kuhlatiya imininingwane ye-13C lebhalwe nge-metabolites. Sebentisa luhlelo lwe-TraceFinder (inguqulo 4.2, I-Thermo Fisher Yesayensi) yekuhlatiya idatha yesilinganiso se-isotope. Bunikati be-compound ngayinye bacinisekiswa yi-compound yekubhekisa letsembekile futsi bahlatiya ngekutimela. Kute kwentiwe kuhlatiya kwekucebisa kwe-isotope, indzawo ye-ion chromatogram lekhishiwe (XIC) ye-isotope ngayinye ye-13C (Mn) yakhishwa ku [M + H] +, lapho khona n yinombolo yekhabhoni yekhompawundi lehlosiwe, lesetjentiselwa kuhlatiya ema-amino acid noma [ MH] + isetjentiselwa kuhlatiya ema-anions. Kunemba kwe-mass kwe-XIC kungaphansi kwetincenye letisihlanu ngesigidzi, futsi kunemba kwe-RT kuyimizuzu lengu-0.05. Lokuhlatiya kwekucebisa kwentiwa ngekubala silinganiso se-isotope ngayinye letfolakele kuya esibalweni sawo onkhe ema-isotopes e-compound lehambisanako. Leti ratios tiniketwa njengemanani emaphesenti e-isotope ngayinye, futsi imiphumela ivetwa njengekucebisa emaphesenti e-molar (MPE), njengoba kuchaziwe ngaphambilini (42).
Le pellet ye neuron lefriziwe yahlanganiswa ku 80% methanol (v/v) lebandza njenge ayisi, yavutsiwe, futsi yafutfunyelwa ku -20°C imizuzu lengemashumi lamatsatfu. Vortex lesampula futsi bese uyavusa ku +4°C imizuzu lengemashumi lamatsatfu. Lesampula yahlanganiswa ku 21,000 g imizuzu lesihlanu ku 4°C, bese le supernatant lephumako yabutfwa yaphindze yomiswa kusetjentiswa iSpeedVac concentrator ku 25°C kwentela kuhlatiya lokulandzelako. Njengobe kuchaziwe ngenhla, kuhlatiya kwe-LC-MS kwentiwa kuma-amino acid emangcamuzane lahlukaniswako. Kusebentisa iTraceFinder (inguqulo 4.2, Thermo Fisher Yesayensi), kuhlatiya idatha kwentiwa kusetjentiswa bukhulu be-monoisotopic be-compound ngayinye. Kujwayelekile kwemininingwane ye-metabolite kwentiwa kusetjentiswa iphakheji yesoftware ye-preprocessCore (57).
Kulungiselela tincetu. Leligundvwane lasheshe labulawa buhlungu nge-carbon dioxide futsi lancunywa inhloko, ingcondvo yasuswa ngekushesha esikhalweni, futsi ummese lodvudvutako logcwele ice (HM-650 V, Thermo Fisher Scientific, Walldorf, Jalimane) wasetjentiswa kulisika waba tincenye letingu-300 kuya ku-375 μm CO2) I-Ca2 + ACSF lephansi (125.0 mM NaCl, 2.5 mM KCl, 1.25 mM sodium phosphate buffer, 25.0 mM NaHCO3, 25.0 mM d-glucose, 1.0 mM CaCl2 kanye ne-6.0 mM Mg301 kuya ku-pH302. mOsm). Dlulisela tincetu tebuchopho letitfolakele egumbini lelicuketse i-Ca2 + ACSF lephakeme (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 pHC2l) kanye ne-310 kuya ku-320 mOsm). Gcina letincetu imizuzu lengu 20 kuya ku 30 kute tibuyiselwe ngaphambi kwekubhala.
lokucophako. Sigaba sesibonakhulu lesifakwe igumbi lekucopha lelicinile kanye ne lens yekucwilisa emantini le 20x (Scientifica) yasetjentiswa kukho konkhe lokucophako. Emaseli ePurkinje latsatfwako akhonjwa nge (i) bukhulu bemtimba, (ii) indzawo ye-anatomical ye-cerebellum, kanye (iii) nekuveta lifutsi lembiko le-mtYFP lekhanyako. I-pipette yesichibi lenesihloko sekumelana kwema-megohms lasihlanu kuya kulalishumi nakunye idvonswa yi-capillary yengilazi ye-borosilicate (GB150-10, 0.86 mm×1.5 mm×100 mm, Imikhicito Yesayensi, eHofheim, eJalimane) kanye nephayiphi levundlile Tintfo Tekusebenta (P-1000). Konkhe lokucoshiwe kwentiwa yi-ELC-03XS npi i-electronic clamp amplifier (i-GmbH ye-elekthronikhi ye-npi, i-Tam, eJalimane), lebeyilawulwa yi-software Signal (inguqulo 6.0, i-Elekthronikhi yaseCambridge, eCambridge, e-UK). Lokuhlolwa kwabhalwa ngesilinganiso sesampula se-12.5 kHz. Lesibonakaliso sihlungwa ngetihlungi letimbili te-Bessel letidlulako letifishane letinemvamisa yekusika lengu-1.3 na-10 kHz ngekulandzelana. I capacitance ulwelwesi kanye pipette kunxephezelwa isekhethe sinxephetelo kusetjentiswa amplifier. Konkhe lokuhlolwa kwentiwa ngaphansi kwekulawula kwekhamera ye-Orca-Flash 4.0 (eHamamatsu, eJalimane), lebeyilawulwa yi-software ye-Hokawo (inguqulo 2.8, eHamamatsu, eJalimane).
Kucushwa kweseli lonkhe lokujwayelekile kanye nekuhlatiya. Ngekushesha ngaphambi kwekubhala, gcwalisa iphayiphi ngesisombululo sangekhatsi lesicuketse letintfo letilandzelako: 4.0 mM KCl, 2.0 mM NaCl, 0.2 mM EGTA, 135.0 mM potassium gluconate, 10.0 mM Hepes, 4.0 mM Guphano. (Na) kanye ne 10.0 mM ye creatinine phosphate kwalungiswa kwaba yi pH 7.25, futsi umfutfo we osmotic bewungu 290 mOsm (i-sucrose). Ngekushesha ngemuva kwekusebentisa emandla e 0 pA kutsi adzabule ulwelwesi, emandla elulwelwesi lwekuphumula alinganiswa. Lokumelana lokufakwako kulinganiswa ngekusebentisa imisinga le-hyperpolarized ye--40, -30, -20, kanye ne--10 pA. Kala bukhulu bekuphendvula kwevolthi bese usebentisa umtsetfo wa-Ohm kubala kumelana kwekufaka. Umsebenti lotentekelako wabhalwa ku-clamp yevoltheji imizuzu le-5, futsi i-sPSC yakhonjwa futsi yalinganiswa ku-Igor Pro (inguqulo 32 7.01, WaveMetrics, Lake Oswego, Oregon, USA) kusetjentiswa umbhalo wekubonwa lokuzenzakalelayo. I-IV curve kanye ne-steady-state yamanje kulinganiswa ngekutsi kucindzetelwe ibhethri ngemandla lehlukene (kucala kusuka ku--110 mV) bese kukhuliswa i-voltage ngetinyatselo te-5 mV. Kukhicitwa kwe-AP kwahlolwa ngekusebentisa umfutfo we-depolarizing. Bopha iseli ku -70 mV ngesikhatsi ufaka i-pulse yamanje le-depolarizing. Lungisa usayizi wesinyatselo seyunithi ngayinye yekubhala ngekwehlukana (10 kuya ku-60 pA). Bala imvamisa lephakeme ye-AP ngekubala ngesandla ema-pulse spikes labangela imvamisa lephakeme ye-AP. Umkhawulo we-AP uhlaziywa ngekusebentisa lokususelwa kwesibili kwe-pulse ye-depolarization lecala kuvusa i-AP yinye noma ngetulu.
Kucushwa kwesichibi lesibhobokile kanye nekuhlatiya. Yenta kubhalwa kwema-patch labhobokile usebentisa imigomo lejwayelekile. Sebentisa i-pipette lete i-ATP- kanye ne-GTP lete letintfo letilandzelako: 128 mM gluconate K, 10 mM KCl, 10 mM Hepes, 0.1 mM EGTA kanye na 2 mM MgCl2, bese ulungisa i-pH 7.2 (kusebentisa i-KOH). I-ATP ne-GTP iyashiywa kulesisombululo se-intracellular kuvikela kungena lokungalawuleki kwe-membrane yemaseli. Le pipette yesichibi igcwaliswa ngesisombululo sangekhatsi lesine amphotericin (cishe 200 kuya ku 250 μg/ml; G4888, Sigma-Aldrich) kutfola irekhodi lesichibi lesishaywe ngesibhakela. I-Amphotericin yancibilikiswa ku-dimethyl sulfoxide (kugcila kwekugcina: 0.1 kuya ku-0.3%; i-DMSO; i-D8418, i-Sigma-Aldrich). Lokucuketfwe kwe-DMSO lesetjentisiwe bekute umphumela lomkhulu kuma-neurons lafundvwako. Ngesikhatsi senchubo yekushaya ngesibhakela, kumela kwesiteshi (Ra) kwachubeka kugadvwa, futsi lokuhlolwa kwacala ngemuva kwekutsi amplitude ye Ra ne AP izinzile (imizuzu lengu 20-40). Umsebenti lotentakalelako ukalwa ku voltage kanye/noma clamp yamanje imizuzu lemibili kuya kulemihlanu. Kuhlatiya imininingwane kwentiwa kusetjentiswa i-Igor Pro (inguqulo 7.05.2, WaveMetrics, eMelika), i-Excel (inguqulo 2010, Inkampani yeMicrosoft, eRedmond, eMelika) kanye ne-GraphPad Prism (inguqulo 8.1.2, I-GraphPad Software Inc. eMelika). Kute kutfolakale ema-AP latentekelako, kusetjentiswa ipulagi ye-IgorPro ye-NeuroMatic v3.0c. Hlola ngekuzenzakalela ema-AP usebentisa umkhawulo loniketiwe, lolungiswa ngamunye ngelirekhodi ngalinye. Kusebentisa sikhatsi se-spike, tfola imvamisa ye-spike ngemvamisa lephakeme ye-spike lesheshako kanye nemvamisa ye-spike lesemkhatsini.
PN kuhlukaniswa. Ngekuvumelana ne-protocol leyashicilelwa ngaphambilini, i-PNs yahlanjululwa kusuka ku-cerebellum yegundvwane esigabeni lesitsite (58). Ngamafuphi, i-cerebellum yahlukaniswa futsi yagaywa ngendlela yekuhlukana lebandza njengeqhwa [ngaphandle kwe-HBSS Ca2 + ne-Mg2 +, lengetiwe nge-20 mM glucose, i-penicillin (50 U / ml) kanye ne-streptomycin (0.05 mg / ml)], bese igaywa lendzawo nge-mgSS1-1. / ml), i-papain (16 U / ml) kanye ne-deoxyribonuklease I (i-DNase I; 0.1 mg/ml)] Lapha imizuzu lengu-30 ku-30°C. Kwekucala geza ticubu endzaweni ye HBSS lecuketse i-mucus yemacandza (10 mg/ml), i-BSA (10 mg/ml) kanye ne-DNase (0.1 mg/ml) ngesikhatsi sekushisa kweligumbi kuvikela kugaya kwe-enzymatic, bese kusemkhatsini we-HBSS lecuketse 20 mM glucose (20 mM glucose), kugaya ngebumnene ku-HBlinm5. (0.05 mg/ml) kanye ne-DNase (0.1 mg/ml) kukhipha emaseli langawodvwa. Lokumiswa kwemaseli lokuphumelako kwahlungwa ngesihlungi semaseli lesingu 70μm, bese lamaseli ahlanganiswa ngekutsi ahlanganiswe (1110 rpm, imizuzu lesihlanu, 4°C) bese aphindze amiswa ngendlela yekuhlunga [HBSS, lengetiwe nge 20 mM glucose, 20% fetal streemyin / peptomylin ) Serum, (0.05 mg/mli)]; hlola kusebenta kwemaseli nge propidium iodide bese ulungisa buningi bemaseli ku 1×106 kuya ku 2×106 emaseli/ml. Ngaphambi kwekugeleta kwe-cytometry, lokumiswa kwakuhlungwa nge-50 μm cell strainer.
I-cytometer yekugeleta. Kuhlunga emaseli kwentiwa ku 4°C kusetjentiswa umshini we FACSAria III (BD Tisayensi Tekuphila) kanye nesoftware ye FACSDiva (BD Tisayensi Tilwane, inguqulo 8.0.1). Lokumiswa kwemaseli kwahlukaniswa kusetjentiswa umlomo we 100 μm ngaphansi kwemfutfo we 20 psi ngesilinganiso se ~ 2800 imicimbi / sec. Njengoba indlela yekuvula emasango yendabuko (bukhulu bemaseli, kubandlululwa kwe-bimodal, kanye netimphawu tekusakateka) ayikhoni kucinisekisa kuhlukaniswa lokufanele kwe-PN kuletinye tinhlobo temaseli, lisu lekuvula lisango libekwa ngekusebentisa kucatsanisa lokucondzile kwemandla e-YFP kanye ne-autofluorescence ku-mitoYFP + kanye nekulawula i-MitoYFP −ce. YFP ujabule ngekutsi kukhanye isampula nge 488 nm laser umugca, futsi luphawu lutfolakala kusetjentiswa 530/30 nm band pass filter. Emagundvwaneni e-mitoYFP +, emandla lahlobene e-Rosa26-mitoYFP gene yembiko aphindze asetjentiswe kwehlukanisa umtimba we-neuronal kanye netincetu te-axon. 7-AAD ujabulile nge 561 nm laser lephuti futsi kutfolakala nge 675/20 nm bandpass luhlu kukhipha emaseli lafile. Kute kwehlukaniswe ema-astrocytes ngesikhatsi sinye, kumiswa kwemaseli kwagcotjwa nge-ACSA-2-APC, kwase kutsi lesampula yakhishwa ngemugca we-laser we-640 nm, futsi kwasetjentiswa luhlu lwe-bandpass lwe-660/20 nm kutfola lesibonakaliso.
Lamangcamuzane labutfwe abekwa ema pellets ngekutsi ahlanganiswe (1110 rpm, imizuzu lesihlanu, 4°C) bese agcinwa ku -80°C kuze kube ngulapho asetjentiswa. Emagundvwane eMfn2cKO kanye nemidlwane yawo yekudla ihlukaniswe ngelilanga linye kwentela kunciphisa kugucugucuka kwenchubo. Kwetfulwa kwemininingwane ye-FACS kanye nekuhlatiya kwentiwa kusetjentiswa isoftware ye-FlowJo (FlowJo LLC, e-Ashland, e-Oregon, eMelika).
Njengobe kushiwo ngenhla (59), i-PCR yesikhatsi sangempela isetjentiselwa kuhlukanisa i-DNA kusuka kuma-neurons lahlukaniswako kute kube nekulinganisa kwe-mtDNA lokulandzelako. Le linearity kanye nekuzwela kwemkhawulo kwahlolwa ekucaleni ngekugijima qPCR etinombolweni letehlukene temangcamuzane. Ngalokufishane, hlanganisa ema PN lamakhulu lamatsatfu ku buffer ye lysis lefaka 50 mM tris-HCl (pH 8.5), 1 mM EDTA, 0.5% Tween 20 kanye ne proteinase K (200 ng/ml) bese uyafukamela ku 55°C imizuzu lelishumi nakubili. Lamangcamuzane aphindze afutfunyelwa ku 95°C imizuzu lelishumi kwentela kucinisekisa kungasebenti lokuphelele kwe proteinase K. Kusebentisa luphenyo lwe TaqMan (Thermo Fisher) lolucondzile ku mt-Nd1, i mtDNA yalinganiswa nge PCR lesemkhatsini wesilinganiso ku 7900HT Real-Time PCR Sluhlelo (Ther). Isayensi, inombolo yekhathalogu Mm04225274_s1), mt-Nd6 (Isayensi Yemdobi Loshisako, inombolo yekhathalogu AIVI3E8) kanye ne 18S (Isayensi Yemdobi Loshisako, inombolo yekhathalogu Hs99999901_s1) emajini.
Kulungiselela isampula ye-proteome. Ngekushisa lesisombululo ku-95 ° C imizuzu lelishumi bese sikhipha umsindvo, ku-buffer ye-lysis [6 M guanidine chloride, 10 mM tris (2-carboxyethyl) phosphine hydrochloride, 10 mM chloroacetamide kanye ne-100 mM tris- Lyse pellets ku-neuron lefriziwe]. Ku Bioruptor (Diagenode) imizuzu lengu 10 (imizuzwane lengu 30 i-pulse / imizuzwane lengu 30 sikhatsi sekuma). Lesampula yahlanjululwa nga 1:10 ku 20 mM tris-HCl (pH 8.0), yahlanganiswa ne 300 ng ye trypsin igolide (Promega), yase iyafutfunyelwa busuku bonkhe ku 37°C kwentela kufinyelela kugaya lokuphelele. Ngelilanga lesibili, lesampula yafakwa ku 20,000 g imizuzu lengu 20. Lendzawo lesetulu yahlanjululwa nge 0.1% we formic acid, bese lesisombululo sasuswa luswayi nge StageTips leyentiwe. Lesampula yomiswa ngetintfo tekusebenta te SpeedVac (umgcini we Eppendorf kanye na 5305) ku 45°C, bese le peptide yamiswa ku 0.1% we formic acid. Onkhe emasampula alungiselelwe ngaso sonkhe sikhatsi ngumuntfu munye. Kute kuhlaziywe emasampula e-astrocyte, ema-peptide lakhiwe ngeluswayi langu-4 μg abekwa ngethegi yebuningi be-tandem (TMT10plex, inombolo yekhathalogu 90110, i-Thermo Fisher yesayensi) ngesilinganiso se-peptide kuya ku-TMT reagent se-1:20. Kulebula kwe TMT, 0.8 mg we TMT reagent yaphindze yamiswa ku 70 μl we ACN lengenamanti, bese le peptide leyomile yaphindze yahlanganiswa yaba ngu 9 μl we 0.1 M TEAB (triethylammonium bicarbonate), lapho khona 7 μl we TMT reagent ku ACN yengetwa khona. Lokucuketfwe kwaba ngu-43.75%. Ngemuva kwemizuzu lengu 60 yekufukamela, lokuphendvula kwacinywa nge 2 μl ye 5% hydroxylamine. Ema peptides labhalwe emagama ahlanganiswa, omiswa, aphindze ahlanganiswa ku 200μl we 0.1% formic acid (FA), ahlukaniswe aba mabili, bese asuswa luswayi asebentisa ema StageTips lakhiwe ngekwawo. Kusebentisa i-UltiMate 3000 lesetulu kakhulu lesetulu kakhulu (i-UltiMate 3000 lesetulu kakhulu), lenye yaletihhafu letimbili yahlukaniswa kukholomu ye-Aquity le-1mm x 150mm legcwele tinhlayiya te-C18 letingu-130Å1.7μm (NoWaters, catalog150). I-Thermo Fisher Yesayensi). Hlukanisa ema peptides ngesilinganiso sekugeleta se 30μl/min, hlukanisa kusuka ku 1% kuya ku 50% buffer B imizuzu lengu 85 ngekuhamba ngetinyatselo kwemizuzu lengu 96, kusuka ku 50% kuya ku 95% buffer B imizuzu lemitsatfu, bese kuba yimizuzu lengu 8 ku 95 % Buffer B; Sivikeli A ngu 5% ACN kanye na 10 mM we ammonium bicarbonate (ABC), bese sivikelo B ngu 80% ACN kanye na 10 mM ABC. Hlanganisa tincenye njalo ngemuva kwemizuzu lemitsatfu bese uyatihlanganisa tibe ngemacembu lamabili (1 + 17, 2 + 18, njll) bese uyawomisa ku vacuum centrifuge.
Kuhlatiya kwe-LC-MS / MS. Kute kuhlolwe bukhulu, ema-peptides (inombolo r119.aq) ahlukaniswe kukholomu yekuhlatiya ye-PicoFrit lenebubanti bangekhatsi lobungama-25 cm, 75 μm (lensi lensha lehlosiwe, inombolo yencenye PF7508250) lefakwe 1.9 μm Ma KULULA-nLC 1200 (Isayensi Yemdobi Wekushisa, eJalimane). Lekholomu beyigcinwa ku 50°C. Ema buffers A na B ngu 0.1% we formic esidi emantini kanye na 0.1% we formic esidi ku 80% we ACN, ngekulandzelana. Ema peptides ahlukaniswe kusuka ku 6% kuya ku 31% buffer B imizuzu lengu 65 bese kusuka ku 31% kuya ku 50% buffer B imizuzu lesihlanu ngekutsi kube ne gradient lengu 200 nl/min. Ema-peptides lahlanjululwe ahlaziywa ku-Orbitrap Fusion mass spectrometer (i-Thermo Fisher yesayensi). Peptide precursor m / z kukala kwentiwa ngesisombululo 120,000 ebangeni 350 kuya 1500 m / z. Kusebentisa 27% emandla ekushayisana lajwayelekile, umlandzeli locinile lonesimo sekushaja se 2 kuya ku 6 ukhetselwa emandla laphakeme C trap dissociation (HCD) cleavage. Sikhatsi semjikeleto sisethwe ku 1 s. Inani le m/z lesicucu se peptide lalinganiswa esicupheni se ion kusetjentiswa lisu lelincane kakhulu le AGC le 5 × 104 kanye nesikhatsi lesikhulu sekujova se 86 ms. Ngemuva kwekuhlukaniswa, le-precursor yabekwa eluhlwini lwekungafakwa lolushintjashintjako lwe-45 s. Ema-peptides labhalwe nge-TMT ahlukaniswe kukholomu ye-50 cm, 75 μm ye-Acclaim PepMap (i-Thermo Fisher Scientific, inombolo yekhathalogu 164942), futsi imibala yekufuduka yahlaziywa ku-Orbitrap Lumos Tribrid mass spectrometer (i-Thermo Fisher Scientific-equimetric lesetulu) (FAIMS) imishini (Thermo Fisher Scientific) isebenta emavoltheji lamabili ekunxephetela −50 kanye −70 V. MS3 lekhetsiwe ngekusebentisa umlandzeli wekuvumelanisa isetjentiselwa kukala kwesiginali ye-ion yembiko we-TMT. Lokuhlukaniswa kwe peptide kwentiwa ku EASY-nLC 1200, kusetjentiswa i-90% ye-elution ye-gradient lesemgceni, ngekuhlanganiswa kwe-buffer lokungu-6% kuya ku-31%; sivikelo A bekungu 0.1% FA, bese sivikelo B bekungu 0.1% FA kanye na 80% ACN. Lekholomu yekuhlatiya isebenta ku 50°C. Sebentisa i-FreeStyle (inguqulo 1.6, I-Thermo Fisher Yesayensi) kuhlukanisa lifayela lekucala ngekuya kwevolthi yesinxephetelo ye-FAIMS.
Kukhonjwa kwemaprotein kanye nekulinganisa. Kusetjentiswa injini yekuphenya lehlanganisiwe ye-Andromeda, idatha yekucala yahlaziywa kusetjentiswa inguqulo ye-MaxQuant 1.5.2.8 (https/maxquant.org/). Ngetulu kwekulandzelana kwe Cre recombinase kanye ne YFP lokutfolakala ku Aequorea victoria, ema spectra etincenye te peptide aseshwa ngekulandzelana kwe canonical kanye nekulandzelana kwe isoform ye proteome yekubhekisa yeligundvwane (I-Proteome ID UP000000589, lelandvwe ku UniProt ngenyanga yeNkhwenkhweti20. I-oxidation ye-methionine kanye ne-acetylation yemaprotheyini N-terminal kwabekwa njengekuguculwa lokugucukako; i-cysteine ​​carbamoyl methylation yabekwa njengekuguculwa lokucinile. Emapharamitha ekugaya asethwe ku “kucacile” kanye “ne-trypsin/P”. Inani lelincane lema peptides kanye nema peptides esikele lasetjentiselwa kukhomba emaprotein ngu 1; inombolo lencane yema peptides lahlukile ngu 0. Ngaphansi kwetimo tekuhambisana kwemephu yema peptide, lizinga lekukhonjwa kwemaprotheyini belingu 0.01. Inketho letsi “Peptide Yesibili” inikwe emandla. Sebentisa inketho “yekuhambisana emkhatsini wekugijima” kudlulisela kukhonjwa lokuphumelelako emkhatsini wemafayela lehlukene ekucala. Sebentisa LFQ silinganiso lesincane sekubala 1 sekulinganisa ngaphandle kwemalebuli (LFQ) (60). Le LFQ intensity ihlungwa lokungenani emanani lamabili lasemtsetfweni lokungenani ecenjini linye le genotype ngesikhatsi ngasinye, futsi i extrapolated kusuka ekusabalalisweni lokujwayelekile ngebubanti be. 0.3 bese uya phansi 1.8. Sebentisa iplatifomu yekucomputer ye-Perseus () kanye ne-R () kuhlatiya imiphumela ye-LFQ. I-t test lelinganiselwe yetindlela letimbili kusuka kuphakheji yesoftware ye-limma yasetjentiswa kuhlatiya kuveta umehluko (61). Kuhlatiya imininingwane yekuhlola kwentiwa kusetjentiswa i-ggplot, i-FactoMineR, i-factoextra, i-GGally kanye ne-pheatmap. Idatha ye-TMT lesekelwe ku-proteomics yahlaziywa kusetjentiswa inguqulo ye-MaxQuant 1.6.10.43. Sesha idatha ye-proteomics leluhlata kusuka ku-database ye-proteomics yebantfu ye-UniProt, leyalandvwa ngenyanga yeNyoni nga 2018. Lokuhlatiya kufaka ekhatsi sici sekulungisa bucwebe be-isotope lesiniketwe ngumkhiciti. Sebentisa limma ku R kuhlatiya inkulumo umehluko. Idatha yekucala, imiphumela yekuphenya kwedathabheyisi, kanye nekuhamba kwemsebenti wekuhlatiya idatha kanye nemiphumela konkhe kugcinwa ku-ProteomeXchange alliance ngekusebentisa indzawo yekugcina umlingani we-PRIDE ngesikhombi sedatha se-PXD019690.
Tichasiselo letisebentako ticebisa kuhlatiya. Lithuluzi lekuhlatiya indlela yebuhlakani (QIAGEN) lasetjentiswa kutfola bunjinga bemigomo yekuchaza lesebentako yedatha lebekwe emavikini langu-8 (Umfanekiso 1). Ngamafuphi, luhlu lwemaprotein lalinganiselwe latfolakala ku LC-MS/MS (tandem mass spectrometry) kuhlatiya idatha lusetjentiswa ngalendlela yekuhlunga lelandzelako: Mus musculus ukhetwa njengeluhlobo kanye nemlandvo, futsi lesigaba sikhombisa linani le P lelilungiswe ngu Benjamini kwentela kucebisa 0.05 noma ngaphansi kutsatfwa njengalebalulekile. Kuleligrafu, tigaba letisihlanu letihamba embili letengetiwe esigabeni ngasinye ngekuya ngenani le-P lelilungisiwe tiyakhonjiswa. Kusebentisa t-test leminyenti, kusetjentiswa luhlelo lwekukhutsata lwemugca lwetigaba letimbili lwaBenjamini, Krieger, naYekutieli (Q = 5%), kuhlatiya kwekuveta emaprotheni esikhatsi kwentiwa kulabo lababalulekile labakhonjwe esigabeni ngasinye, futsi umugca ngamunye uhlatiya ngekwehlukana. Asikho sidzingo sekwemukela i-SD lehambisanako.
Kute sicatsanise imiphumela yalolucwaningo nema-database lashicilelwe futsi sikhicite umdvwebo we-Venn kuMfanekiso 1, sahlanganisa luhlu lwemaprotheni lalinganiselwe nge-MitoCarta 2.0 tichasiselo (24). Sebentisa lithulusi leliku-inthanethi Dvweba Umdvwebo we-Venn (http://informatics.psb.ugent.be/webtools/Venn/) kute ukhicite lomdvwebo.
Kute utfole lwati lolubanti ngetinchubo tetibalo letisetjentiselwa kuhlatiya i-proteomics, sicela ubheke sigaba lesihambisanako seTintfo neTindlela. Kuto tonkhe letinye tilingo, lwati lolubanti lungatholakala ku-legend lehambisanako. Ngaphandle nangabe kucaciswe ngalenye indlela, yonkhe idatha ivetwa njengekusho ± SEM, futsi konkhe kuhlatiya kwetibalo kwentiwa kusetjentiswa isoftware ye-GraphPad Prism 8.1.2.
Kute utfole tintfo letengetiwe talesihloko, sicela ubuke http://advances.sciencemag.org/cgi.
Lena yindzatjana levulekile lesakatwa ngaphasi kwemigomo ye-Creative Commons Attribution-Non-Commercial License, levumela kusetjentiswa, kusabalaliswa kanye nekukhicita kabusha nganoma nguyiphi indlela, uma nje kusetjentiswa kwekugcina akusiko kwenzuzo yekutsengisa futsi umcondvo kutsi umsebenti wekucala ulungile. Kudlilisela.
Caphela: Sicela kuphela kutsi unikete likheli lakho le-imeyili kute umuntfu lomncoma kulelikhasi ati kutsi ufuna ayibone le-imeyili nekutsi akusiyo i-spam. Angeke sitsatse noma ngumaphi emakheli e-imeyili.
Lombuto usetjentiselwa kuhlola kutsi usivakashi yini kanye nekuvikela kuhanjiswa kwe-spam lokuzenzakalelayo.
Ngu-E. Motori, I. Atanassov, SMV Kochan, K. Folz-Donahue, V. Larson
Kuhlatiya kwe-proteomics kwetinzwa letingasebenti kahle kwaveta kutsi tinhlelo tekugaya kudla tiyasebenta kute tilwe nekubola kwetinzwa.
Ngu-E. Motori, I. Atanassov, SMV Kochan, K. Folz-Donahue, V. Larson
Kuhlatiya kwe-proteomics kwetinzwa letingasebenti kahle kwaveta kutsi tinhlelo tekugaya kudla tiyasebenta kute tilwe nekubola kwetinzwa.
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