Sweswi *Adirese ya sweswi: Cologne 50931, Germany, Cologne Vulavisisi bya Ntlawa wa Vutshila eka Nhlamulo ya Ntshikelelo wa Tisele eka Mavabyi lama fambelanaka na ku Dyuhala (CECAD).
Ku onhaka ka misiha ya mavabyi ya mitochondrial swi tekiwa swi nga hundzuki hikuva metabolic plasticity ya ti neurons yi ringaniseriwile, kambe vuyelo bya mitochondrial dysfunction eka cell autonomy ya neuronal metabolism emirini a byi twisiseki kahle. Laha, hi nghenisa proteome leyi kongomeke ya tisele ya tinyiyoni ta Purkinje leti nga na ku pfumaleka ka OXPHOS loku yaka emahlweni loku vangiwaka hi ku kavanyeteka ka mitochondrial fusion dynamics. Hi kume leswaku ku nga tirhi kahle ka mitochondrial ku pfuxe ku cinca lokukulu eka ntirho wa proteomics, eku heteleleni ku yise eka ku tirhisiwa loku landzelelanaka ka minongonoko leyi kongomeke ya metaboliki emahlweni ka ku fa ka tisele. Swi nga languteriwanga, hi kumile induction leyi nga erivaleni ya pyruvate carboxylase (PCx) na ti peroxidases tin’wana leti fambelanaka na ku engetela ti TCA cycle intermediates. Ku sivela ka PCx swi nyanyise ntshikilelo wa oxidative na neurodegeneration, leswi kombisaka leswaku atherosclerosis yina mbuyelo wo sirhelela eka ti neurons leti pfumalaka OXPHOS. Ku vuyiseriwa ka mitochondrial fusion eka ti neurons leti onhakeke swinene swi tlherisela endzhaku hi ku helela swihlawulekisi leswi swa metabolic, xisweswo swi sivela ku fa ka tisele. Swikumiwa swa hina swi kombisa tindlela leti a ti nga tiviwi khale leti nyikaka ku tiyisela eka ku nga tirhi kahle ka mitochondrial naswona swi kombisa leswaku ku onhaka ka misiha ku nga tlheriseriwa endzhaku hambi ku ri eka swiyimo swa le ndzhaku swa vuvabyi.
Ntirho wa le xikarhi wa mitochondria eku hlayiseni ka metabolism ya matimba ya tinyiyoni wu kandziyisiwa hi swikombiso swo anama swa misiha leswi fambelanaka na mavabyi ya mitochondria ya vanhu. Vunyingi bya mavabyi lawa byi vangiwa hi ku cinca ka tijini loku lawulaka ku paluxiwa ka tijini ta mitochondrial (1, 2) kumbe ku onhiwa ka tijini loku fambelanaka na dynamics ya mitochondrial, leswi khumbaka hi ndlela leyi nga kongomangiki ku tshamiseka ka DNA ya mitochondrial (mtDNA) (3, 4). Ntirho eka timodeli ta swiharhi wu kombisile leswaku ku hlamula eka ku nga tirhi kahle ka mitochondrial eka tinyama leti rhendzeleke, tindlela ta metabolic leti hlayisaka (5-7) ti nga tirhisiwa, xisweswo ti nyika vutivi bya nkoka eka pathogenesis ya mavabyi lawa yo rharhangana. Ku hambana swinene, ku twisisa ka hina ku cinca ka metaboliki ya tinxaka to karhi ta tisele loku vangiwaka hi ku tsandzeka hi ku angarhela ka vuhumelerisi bya mitochondrial adenosine triphosphate (ATP) ya byongo i swa nkoka (8), leswi kandziyisaka xilaveko xo kuma swikongomelo swa vutshunguri leswi nga tirhisiwaka ku sivela kumbe ku sivela mavabyi. Sivela ku onhaka ka misiha (9). Ku pfumaleka ka rungula i mhaka ya leswaku tisele ta misiha ti tekiwa ngopfu tanihi leti nga ni ku cinca-cinca loku nga nyawuriki swinene ka metaboliki loko ti pimanisiwa ni tinxaka ta tisele ta tinyama leti rhendzeleke ( 10 ). Hi ku nyikiwa leswaku tisele leti ti tlanga xiave xa nkoka eku hlanganiseni ka mphakelo wa ti-metabolite eka tinyiyoni ku tlakusa ku hundziseriwa ka ti-synaptic na ku hlamula eka swiyimo swa ku vaviseka na mavabyi, vuswikoti byo cinca-cinca metabolism ya tisele eka swiyimo swo tika swa tinyama ta byongo byi lava ku va byi pimiwile eka tisele ta glial (11-14). Ku engetela kwalaho, ku hambana ka tisele loku nga kona ka tinyama ta byongo ngopfu-ngopfu ku sivela dyondzo ya ku cinca ka metaboliki loku humelelaka eka mintlawa leyitsongo yo karhi ya tinyiyoni. Hikwalaho ka sweswo, a ku tiviwi swo tala hi vuyelo lebyi kongomeke bya tisele ni bya metabolic bya ku nga tirhi kahle ka mitochondrial eka tinyiyoni.
Leswaku hi twisisa vuyelo bya metabolic bya mitochondrial dysfunction, hi hambanise ti neurons ta Purkinje (PNs) eka switeji swo hambana swa neurodegeneration leswi vangiwaka hi ku onhiwa ka mitochondrial outer membrane fusion (Mfn2). Hambi leswi ku cinca ka Mfn2 eka vanhu ku fambelanisiwaka na xivumbeko xa hereditary motor sensory neuropathy lexi tivekaka tani hi Charcot-Marie-Tooth type 2A (15), ku onhiwa ka xiyimo xa Mfn2 eka timbeva i induction leyi tivekaka kahle ya oxidation Phosphorylation (OXPHOS) dysfunction method. Tinxaka letitsongo to hambana ta tinyiyoni (16-19) na phenotype ya neurodegenerative leyi humelelaka swi fambisana na swikombiso swa misiha leswi yaka emahlweni, swo tanihi swiphiqo swa ku fambafamba (18, 19) kumbe cerebellar ataxia (16). Hi ku tirhisa nkatsakanyo wa label-free quantitative (LFQ) proteomics, metabolomics, swifaniso, na tindlela ta virological, hi kombisa leswaku ku onhaka ka misiha loku yaka emahlweni ku hlohlotela swinene pyruvate carboxylase (PCx) na swin’wana leswi katsekaka eka arteriosclerosis ya PNs in vivo Ku paluxiwa ka ti enzymes. Ku tiyisisa ku yelana ka ku kumiwa loku, hi ku kongoma hi lawurile ehansi ku paluxiwa ka PCx eka ti-PN leti pfumalaka Mfn2, naswona hi kumile leswaku vuhandzuri lebyi byi nyanyisa ntshikilelo wa oxidative na ku hatlisisa ku onhaka ka misiha, xisweswo hi kombisa leswaku azoospermia yi nyika ku fa ka tisele Metabolic adaptability. Ku paluxiwa lokukulu ka MFN2 ku nga ponisa hi ku helela PN ya ku onhaka ka makumu leyi nga na ku pfumaleka lokukulu ka OXPHOS, ku dyiwa lokukulu ka DNA ya mitochondrial, na ku vonaka ku tshoveka ka netiweke ya mitochondrial, leswi yaka emahlweni swi kandziyisa leswaku xivumbeko lexi xa ku onhaka ka misiha xi nga ha hola hambi ku ri eka xiteji lexi yaka emahlweni xa vuvabyi emahlweni ka ku fa ka tisele.
Leswaku hi vona hi mahlo ya mianakanyo mitochondria eka Mfn2 knockout PNs, hi tirhise muxaka wa mbeva lowu pfumelelaka mitochondria leyi titshegeke hi Cre ku kongomisa eka yellow fluorescent protein (YFP) (mtYFP) (20) Cre expression na ku kambela mitochondrial morphology in vivo. Hi kumile leswaku ku onhiwa ka jini ya Mfn2 eka ti PNs swita yisa eka ku avana hakatsongotsongo ka netiweke ya mitochondrial (Xifaniso S1A), naswona ku cinca kosungula ku kumiwile eka 3 wa mavhiki ya vukhale. Kuhambana na sweswo, ku onhaka lokukulu ka leyara ya tisele ta PN, tani hileswi swi kombisiweke hi ku lahlekeriwa hi Calbindin immunostaining, a swi sungulanga ku fikela eka 12 wa mavhiki ya vukhale (Xifaniso 1, A na B). Ku nga fambisani ka nkarhi exikarhi ka ku cinca ko sungula eka xivumbeko xa mitochondrial na ku sungula loku vonakaka ka rifu ra tinyiyoni swi hi susumetele ku lavisisa ku cinca ka metabolic loku hlohloteriwaka hi ku nga tirhi kahle ka mitochondrial emahlweni ka rifu ra tisele. Hi tumbuluxile maqhinga lama sekeriweke eka ku hlawula tisele leti tirhisiwaka hi fluorescence (FACS) ku hambanyisa YFP (YFP +)-expressing PN (Xifaniso 1C), na le ka timbeva to lawula (Mfn2 + / loxP :: mtYFP loxP- stop-loxP: : L7-cre), laha ku nga ta vuriwa CTRL (Xifaniso S1B). Ku antswisiwa ka maqhinga ya gating lama sekeriweke eka matimba lama ringanaka ya xikombiso xa YFP swi hi pfumelela ku basisa miri wa YFP + (YFPhigh) wa ti-PN ku suka eka leti nga riki ta PN (YFPneg) (Xifaniso S1B) kumbe swiphemu leswi ehleketiwaka swa fluorescent axon/dendritic (YFPlow; Xifaniso S1D, ximatsi), leswi tiyisisiweke hi microscope ya confocal (Xifaniso S1D, xinene). Leswaku hi tiyisisa vutivi bya vaaki lava hlawuriweke, hi endlile LFQ proteomics ivi endzhaku ka sweswo nxopaxopo wa swiphemu swa nkoka, naswona hi kumile leswaku ku na ku hambana loku nga erivaleni exikarhi ka tisele ta YFPhigh na YFPneg (Xifaniso xa S1C). Tisele ta YFPhigh ti kombisile ku fuwa ka net ya swikombiso swa PNs leswi tivekaka (i.e. Calb1, Pcp2, Grid2 na Itpr3) (21, 22), kambe ku hava ku fuwa ka tiphrotheyini leti talaka ku kombisiwa eka tinyiyoni kumbe tinxaka tin’wana ta tisele (Xifaniso 1D) ). Ku pimanisiwa exikarhi ka tisampulu eka ti cells ta YFPhigh leti hlayiweke leti hlengeletiweke eka swikambelo leswi tiyimeleke swi kombisile correlation coefficient> 0.9, leswi kombisaka ku tlheriseriwa lokunene exikarhi ka ti biological replicates (Xifaniso xa S1E). Hi ku komisa, datha leyi yi tiyisisile pulani ya hina ya ku hambanisiwa ka xihatla na loku kongomeke ka PN leyi kotekaka. Hikuva sisiteme ya muchayeri wa L7-cre leyi tirhisiweke yi hlohlotela ku hlanganisiwa nakambe ka mosaic eka vhiki ro sungula endzhaku ka ku velekiwa (23), hi sungule ku dlaya timbeva ku suka eka CTRL na ku va hi xiyimo (Mfn2 loxP / loxP :: mtYFP loxP-stop-loxP :: L7-cre) Ku hlengeleta tinyiyoni. Endzhaku kaloko ku hlanganisiwa nakambe ku hetisiwile, yi vuriwa Mfn2cKO eka 4 wa mavhiki ya vukhale. Tanihi makumu, hi hlawurile 8 wa mavhiki ya vukhale loko PN layer ya ha ri kona hambi leswi swiphemu swa mitochondrial leswi nga erivaleni (Xifaniso 1B na Xifaniso xa S1A). Hi ku angarhela, hi pimile nhlayo hinkwayo ya 3013 wa tiphrotheyini, laha kwalomu ka 22% a ti sekeriwe eka MitoCarta 2.0 annotations leyi sekeriweke eka mitochondrial proteome tanihi mitochondria (Xifaniso 1E) (Xifaniso 1E) (24). Nxopaxopo wa ku hambana ka ku paluxiwa ka tijini lowu endliweke hi vhiki ra vu 8 wu kombisile leswaku i 10.5% ntsena wa tiphrotheyini hinkwato leti nga na ku cinca lokukulu (Xifaniso 1F na Xifaniso xa S1F), laha 195 wa tiphrotheyini a ti lawuriwa ehansi kasi 120 wa tiphrotheyini a ti lawuriwa ehenhla (Xifaniso 1F). Swi fanerile ku xiya leswaku “nxopaxopo wa tindlela ta vutumbuluxi” wa sete leyi ya datha wu kombisa leswaku tijini leti kombisiweke hi ndlela yo hambana ngopfungopfu ti wela eka sete leyi siveleriweke ya tindlela to karhi ta metaboliki (Xifaniso 1G). Lexi tsakisaka, hambi leswi ku lawuriwa ehansi ka tindlela leti fambelanaka na OXPHOS na ku nyika swikombiso swa calcium swi tiyisisaka ku hlohloteriwa ka ku nga tirhi kahle ka mitochondrial eka ti-PN leti pfumalaka fusion, swiyenge swin’wana leswi ngopfu-ngopfu swi katsaka metabolism ya amino acid swi lawuriwa ngopfu ehenhla, leswi fambisanaka na metabolism leyi humelelaka eka ti-PN ta mitochondrial. Rewiring swi tshamisekile. ku nga tirhi kahle.
(A) Swifaniso swa confocal leswi yimelaka swiphemu swa cerebellar swa CTRL na Mfn2cKO mice leswi kombisaka ku lahlekeriwa loku yaka emahlweni ka PNs (calbindin, grey); ti nuclei ti counterstained hi DAPI. (B) Ku pima nhlayo ya (A) (nxopaxopo wa ndlela yin’we wa ku hambana, ***P<0.001; n = 4 ku ya eka 6 wa swirhendzevutana ku suka eka timbeva tinharhu). (C) Mafambiselo ya ntirho ya ku ringeta. (D) Ku hangalasiwa ka mepe wo hisa wa swimaki leswi kongomeke eka Purkinje (ehenhla) na tinxaka tin’wana ta tisele (exikarhi). (E) Xifaniso xa Venn lexi kombisaka nhlayo ya tiphrotheyini ta mitochondrial leti hlawuriweke eka PN leyi hlawuriweke. (F) Ploto ya vholkheno ya tiphrotheyini leti kombisiweke hi ndlela yo hambana eka tinyiyoni ta Mfn2cKO eka mavhiki ya 8 (nxavo wo tsema wa nkoka wa 1.3). (G) Nxopaxopo wa ndlela ya vutumbuluxi wu kombisa tindlela ta ntlhanu ta nkoka swinene ta up-regulation (red) na down-regulation (blue) eka Mfn2cKO PN leyi hlayiweke tanihi 8 wa mavhiki. Level ya xikarhi ya expression ya protein yin’wana na yin’wana leyi kumiweke ya kombisiwa. Mepe wa ku hisa wa xikalo xa greyscale: nhlayo ya P leyi lulamisiweke. ns, a hi swa nkoka.
Data ya proteomics yi kombisile leswaku ku paluxiwa ka ti protein ta ti complexes I, III, na IV ku hungutekile hakatsongotsongo. Complexes I, III, na IV hinkwato a ti ri na ti subunits ta nkoka leti khodiweke hi mtDNA, kasi complex II, leyi a yi ri na khodi ya nyutliya ntsena, hi xisekelo a yi khumbekanga (Xifaniso 2A na Xifaniso xa S2A). . Ku fambisana na mbuyelo wa proteomics, immunohistochemistry ya swiphemu swa tinyama ta cerebellar yi kombisile leswaku MTCO1 (mitochondrial cytochrome C oxidase subunit 1) subunit level ya complex IV eka PN yi hungutekile hakatsongotsongo (Xifaniso 2B). Subunit leyi khodiweke hi mtDNA Mtatp8 yi hungutiwile swinene (Xifaniso S2A), kasi levele ya xiyimo xo tiya ya subunit ya ATP synthase leyi khodiweke hi nyutliya yi tshamile yi nga cinci, leswi fambelanaka na stable ATP synthase subassembly F1 complex leyi tivekaka loko mtDNA expression yi tshamisekile. Xivumbeko xa kona xi nga cinci. Ku kavanyeta (7). Ku kamberiwa ka levele ya mtDNA eka ti Mfn2cKO PNs leti hleriweke hi real-time polymerase chain reaction (qPCR) swi tiyisisile ku hunguteka hakatsongotsongo ka nhlayo ya khopi ya mtDNA. Loko ku pimanisiwa na ntlawa wa vulawuri, eka mavhiki ya 8 hi vukhale, ntsena kwalomu ka 20% wa mpimo wa mtDNA Wu hlayisiwile (Xifaniso 2C). Ku fambisana na mbuyelo lowu, confocal microscopy staining ya Mfn2cKO PNs ku kuma DNA yi kombisa ku tirhisiwa loku titshegeke hi nkarhi ka mitochondrial nucleotides (Xifaniso 2D). Hi kumile leswaku i vahlawuri van’wana ntsena lava katsekaka eka ku onhiwa ka tiphrotheyini ta mitochondrial na nhlamulo ya ntshikilelo lava a va lawuriwa ehenhla, ku katsa na Lonp1, Afg3l2 na Clpx, na OXPHOS complex assembly factors. Ku hava ku cinca lokukulu eka ti levele ta ti protein leti katsekaka eka apoptosis loku kumiweke (Figure S2B). Hilaha ku fanaka, hi kumile leswaku mitochondria na tichannel ta endoplasmic reticulum leti katsekaka eka ku fambisiwa ka calcium ti na ku cinca kutsongo ntsena (Xifaniso xa S2C). Ku engetela kwalaho, ku kamberiwa ka tiphrotheyini leti fambelanaka na autophagy a ku kumanga ku cinca lokukulu, leswi fambelanaka na induction leyi vonakaka ya ti autophagosomes leyi voniweke in vivo hi immunohistochemistry na electron microscopy (Xifaniso S3). Hambiswiritano, ku nga tirhi kahle ka OXPHOS loku yaka emahlweni eka ti-PN ku fambisana na ku cinca loku nga erivaleni ka mitochondrial ya ultrastructural. Swirhabyani swa mitochondrial swinga voniwa eka mimiri ya ti cell na misinya ya dendritic ya Mfn2cKO PNs ya malembe ya 5 na 8 wa mavhiki, naswona xivumbeko xa membrane yale ndzeni xi endle ku cinca lokukulu (Xifaniso xa S4, A na B). Ku fambisana na ku cinca loku ka ultrastructural na ku hunguteka lokukulu ka mtDNA, nxopaxopo wa swilayi swa acute cerebral cerebellar leswi nga na tetramethylrhodamine methyl ester (TMRM) swi kombisile leswaku vuswikoti bya mitochondrial membrane eka Mfn2cKO PNs byi hungutekile swinene (Xifaniso S4C).
(A) Nxopaxopo wa khoso ya nkarhi wa xiyimo xa ku paluxiwa ka OXPHOS complex. Languta ntsena ti protein letingana P<0.05 eka 8 wa mavhiki (two-way ANOVA). Layini ya mathonsi: Ku hava ku lulamisiwa loko ku pimanisiwa na CTRL. (B) Hi ximatsi: Xikombiso xa xiphemu xa cerebellar lexi leteriweke hi anti-MTCO1 antibody (scale bar, 20 μm). Ndzhawu leyi tekiweke hi mimiri ya ti cell ta Purkinje yifunengetiwe hi muhlovo wotshuka. Exineneni: Ku pima mpimo wa swiyimo swa MTCO1 (nxopaxopo wa ndlela yin’we wa ku hambana; n = 7 ku ya eka 20 wa tisele leti kamberiweke ku suka eka timbeva tinharhu). (C) nxopaxopo wa qPCR wa nomboro ya khopi ya mtDNA eka PN leyi hleriweke (nxopaxopo wa ndlela yin’we wa ku hambana; n = 3 ku ya eka 7 wa timbeva). (D) Eximatsini: Xikombiso xa xiphemu xa cerebellar lexi leteriweke hi anti-DNA antibody (scale bar, 20 μm). Ndzhawu leyi tekiweke hi mimiri ya ti cell ta Purkinje yifunengetiwe hi muhlovo wotshuka. Exineneni: Ku pima nhlayo ya swirhumbana swa mtDNA (nxopaxopo wa ndlela yin’we wa ku hambana; n = 5 ku ya eka 9 wa tisele ku suka eka timbeva tinharhu). (E) Xikombiso xa xiphemu xa acute cerebellar lexi kombisaka tisele ta mitoYFP + Purkinje (nseve) eka rhekhodo ya patch clamp ya sele hinkwayo. (F) Ku pima nhlayo ya IV curve. (G) Tirhekhodo leti yimelaka ta ku cheriwa ka sweswi ka depolarizing eka tisele ta CTRL na Mfn2cKO Purkinje. Top trace: I pulse yo sungula leyi nga pfuxa AP. Bottom trace: Nhlayo ya le henhla ya AP. (H) Ku pima nhlayo ya swingheniso swa postsynaptic spontaneous inputs (sPSPs). Trace yo rhekhoda leyi yimelaka na mpimo wa yona wa ku kurisa swi kombisiwile eka (I). Nxopaxopo wa ndlela yin’we wa ku hambana wu xopaxophile n = 5 ku ya eka 20 wa tisele ku suka eka timbeva tinharhu. Data yi kombisiwa tanihi mean±SEM; *P<0.05; **P<0.01 ya xirhendzevutani; ***P<0.001 ya xirhendzevutani. (J) Milandzu leyi yimelaka ya AP leyi tiendlekelaka leyi rhekhodiweke hi ku tirhisa movha wa perforated patch clamp. Top trace: Nhlayo ya le henhla ya AP. Bottom trace: ku kurisa ka AP yin’we. (K) Ku pima nhlayo ya xikarhi na ya le henhla ya AP frequency ku ya hi (J). Xikambelo xa Mann-Whitney; n = 5 wa ti cells ti kamberiwile kusuka eka mune wa timbeva. Data yi kombisiwa tanihi mean±SEM; a hi swa nkoka.
Ku onhaka loku nga erivaleni ka OXPHOS ku kumiwile eka Mfn2cKO PN ya mavhiki ya 8, leswi kombisaka leswaku ntirho wa physiological wa ti neurons a wu tolovelekanga swinene. Hikwalaho, hi kambisisile swihlawulekisi swa gezi leswi nga tirhiki swa tinyiyoni leti pfumalaka OXPHOS eka mavhiki ya 4 ku ya eka 5 na mavhiki ya 7 ku ya eka 8 hi ku endla tirhekhodo ta ti-all-cell patch clamp eka swilayi swa acute cerebellar (Xifaniso 2E). Swi nga languteriwanga, avhareji ya vuswikoti bya membrane yo wisa na ku lwisana na ku nghenisa ka tinyiyoni ta Mfn2cKO a swi fana na swa vulawuri, hambi leswi a ku ri na ku hambana loku nga vonakiki exikarhi ka tisele ( Tafula 1 ). Hilaha ku fanaka, eka 4 kuya eka 5 wa mavhiki ya vukhale, akuri hava ku cinca lokukulu eka vuxaka bya current-voltage (IV curve) loku kumiweke (Xifaniso 2F). Hambiswiritano, ku hava tinyiyoni ta Mfn2cKO ta mavhiki ya 7 ku ya eka 8 leti ponile eka rhejimen ya IV (goza ra hyperpolarization), leswi kombisaka leswaku ku na ku twisisa loku nga erivaleni eka vuswikoti bya hyperpolarization eka xiyimo lexi xa le ndzhaku. Kuhambana na sweswo, eka ti neurons ta Mfn2cKO, ti depolarizing currents leti vangaka ku humesiwa ka repetitive action potential (AP) ti tiyiseleriwa kahle, leswi kombisaka leswaku ti discharge patterns ta tona hinkwato ati hambananga ngopfu na leti ta ti control neurons ta 8 wa mavhiki (Table 1 na Figure 2G). Hilaha ku fanaka, nhlayo na amplitude ya spontaneous postsynaptic currents (sPSCs) a yi ringanisiwa na ya ntlawa wa vulawuri, naswona nhlayo ya swiendlakalo yi tlakuke ku suka eka 4 wa mavhiki ku ya eka 5 wa mavhiki ku ya eka 7 wa mavhiki ku ya eka 8 wa mavhiki hi ku tlakuka loku fanaka (Xifaniso 2, H na I). Nkarhi wa ku vupfa ka synaptic eka PNs (25). Mbuyelo lowu fanaka wu kumiwile endzhaku ka ti perforated PNs patches. Xivumbeko lexi xi sivela ku hakeriwa loku nga kotekaka ka swihoxo swa ATP ya tisele, tanihilaha swi nga humelelaka hakona eka ku rhekhoda ka xikhomela-ndhawu xa patch ya sele hinkwayo. Ngopfu-ngopfu, vuswikoti bya membrane yo wisa na ku duvula hi ku tisungulela ka tinyiyoni ta Mfn2cKO a swi khumbekanga (Xifaniso 2, J na K). Hi ku komisa, mimbuyelo leyi yi kombisa leswaku ti-PN leti nga na ku nga tirhi kahle ka OXPHOS loku nga erivaleni ti nga langutana na swivumbeko swa ku humesiwa ka frequency ya le henhla kahle, leswi kombisaka leswaku ku na endlelo ro hakela leri ti pfumelelaka ku hlayisa tinhlamulo ta electrophysiological leti nga ekusuhi na ntolovelo.
Data yi kombisiwa tanihi xiringaniso ± SEM (nxopaxopo wa ndlela yin’we wa ku hambana, xikambelo xa ku pimanisa ko tala xa Holm-Sidak; *P<0.05). Nomboro ya yuniti yi kombisiwa hi swikhomela-ndhawu.
Hi vekile ku lavisisa loko xiyenge xihi kumbe xihi eka dataset ya proteomics (Xifaniso 1G) xi katsa tindlela leti nga lwisanaka na ku pfumaleka lokukulu ka OXPHOS, hi ndlela yoleyo hi hlamusela leswaku hikokwalaho ka yini PN leyi khumbekaka yi nga hlayisa electrophysiology leyi nga ekusuhi na ntolovelo (Xifaniso 2, E ku ya eka K). . Nxopaxopo wa proteomics wu kombisile leswaku ti enzymes leti katsekaka eka catabolism ya branched chain amino acids (BCAA) ati lawuriwa swinene ehenhla (Xifaniso 3A na Xifaniso xa S5A), naswona xiendliwa xohetelela acetyl-CoA (CoA) kumbe succinyl CoA swinga engetela ti tricarboxylates eka xirhendzevutani xa arteriosclerosis Acid (TCA). Hi kumile leswaku nhlayo ya BCAA transaminase 1 (BCAT1) na BCAT2 hivumbirhi bya yona yi tlakukile. Va catalyzed goza ro sungula ra BCAA catabolism hi ku humesa glutamate ku suka eka α-ketoglutarate ( 26 ). Ti subunits hinkwato leti endlaka branched chain keto acid dehydrogenase (BCKD) complex ti upregulated (complex yi hlohletela decarboxylation leyi landzelaka na leyi nga hundzukiki ya BCAA carbon skeleton leyi humelelaka) (Xifaniso 3A na Xifaniso xa S5A). Hambiswiritano, ku hava ku cinca loku nga erivaleni eka BCAA hi yoxe loku kumiweke eka PN leyi hleriweke, leswi nga ha vaka swi vangiwa hi ku engeteleka ka ku amukeriwa ka tisele ka ti amino acid leti ta nkoka kumbe ku tirhisiwa ka swihlovo swin’wana (glucose kumbe lactic acid) ku engetela xirhendzevutani xa TCA (Xifaniso xa S5B). Ti PNs leti pfumalaka OXPHOS titlhele ti kombisa ku tlakuka ka ku bola ka glutamine na migingiriko ya transamination eka 8 wa mavhiki ya vukhale, leswinga kombisiwa hi up-regulation ya ti mitochondrial enzymes glutaminase (GLS) na glutamine pyruvate transaminase 2 (GPT2) (Xifaniso 3, A na C). Swi fanerile ku xiya leswaku up-regulation ya GLS yi pimiwile eka spliced ​​isoform glutaminase C (GLS-GAC) (ku cinca ka Mfn2cKO/CTRL i kwalomu ka 4.5-fold, P = 0.05), naswona up-regulation ya yona yo karhi eka tinyama ta khensa Swi nga seketela mitochondrial bioenergy. (27).
(A) Mepe wo hisa wu kombisa ku cinca ka fold eka levele ya ti protein eka ndlela leyi boxiweke eka 8 wa mavhiki. (B) Xikombiso xa xiphemu xa cerebellar lexi leteriweke hi anti-PCx antibody (scale bar, 20 μm). Museve wo tshwuka wu kombetela eka miri wa sele ya Purkinje. (C) Nxopaxopo wa ku paluxiwa ka tiphrotheyini wa khoso ya nkarhi lowu hlawuriweke tanihi n’wandla wa nkoka wa atherosclerosis (multiple t-test, *FDR <5%; n = 3-5 mice). (D) Laha henhla: Xifaniso xa xikimi lexi kombisaka tindlela to hambana to nghena eka khaboni leyi leteriweke leyi nga eka [1-13C]pyruvate tracer (i.e., hi ku tirhisa PDH kumbe ndlela ya trans-arterial). Ehansi: Chati ya violin yi kombisa tiphesente ta khaboni leyi leteriweke yin’we (M1) leyi hundzuriweke ku va aspartic acid, citric acid na malic acid endzhaku ko lebula swilayi swa acute cerebellar hi [1-13C]pyruvate (paired t-test; ** P <0.01). (E) Nxopaxopo wa matimu ya nkarhi lowu heleleke wa ndlela leyi kombisiweke. Languta ntsena ti protein letingana P<0.05 eka 8 wa mavhiki . Layini leyi nga na swiphemuphemu: ku hava ntikelo wa ku lulamisa (nxopaxopo wa tindlela timbirhi wa ku hambana; * P <0.05; *** P <0.001). Data yi kombisiwa tanihi mean±SEM.
Eka nxopaxopo wa hina, BCAA catabolism yi hundzuke yin’wana ya tindlela ta nkoka ta up-regulation. Mhaka leyi yi ringanyeta swinene leswaku volume ya ventilation leyi nghenaka eka xirhendzevutani xa TCA yinga cinciwa eka PN leyi pfumalaka OXPHOS. Leswi swi nga yimela xivumbeko lexikulu xa neuronal metabolic rewiring, leswi nga vaka na nkucetelo wo kongoma eka physiology ya ti neuronal na ku hanya hi nkarhi wa ku hlayisiwa ka ku tsandzeka lokukulu ka OXPHOS. Hi ku fambisana na xiringanyeto lexi, hi kumile leswaku enzyme leyikulu ya anti-atherosclerotic PCx yi up-regulated (Mfn2cKO/CTRL yi cinca kwalomu ka 1.5 wa minkarhi; Xifaniso 3A), leswi catalyzes ku hundzuriwa ka pyruvate ku va oxaloacetate (28), leswi ku tshembiwaka leswaku swi le ka tinyama ta byongo The expression in is restricted to astrocytes (29, 30). Ku fambisana na mbuyelo wa proteomics, confocal microscopy yi kombisile leswaku ku paluxiwa ka PCx ku engetelekile hi ku kongoma naswona swinene eka ti-PN leti pfumalaka OXPHOS, kasi ku angula ka PCx ngopfu-ngopfu a ku siveriwa eka tisele ta Bergmann glial leti nga ekusuhi ta vulawuri (Xifaniso 3B). Ku kambela hi ndlela yo tirha upregulation leyi xiyiweke ya PCx, hi tshungule swilayi swa acute cerebellar hi [1-13C]pyruvate tracer. Loko pyruvate yi oxidized hi pyruvate dehydrogenase (PDH), isotope label ya yona yi nyamalarile , Kambe yi nghenisiwa eka ti TCA cycle intermediates loko pyruvate yi metabolized hi vascular reactions (Xifaniso 3D). Ku seketela data ya hina ya proteomics, hi vonile nhlayo leyikulu ya swimaki kusuka eka tracer leyi eka aspartic acid ya swilayi swa Mfn2cKO, kasi citric acid na malic acid na swona swiri na ntshamiseko wo ringanela, hambi leswi swinga riki swa nkoka (Xifaniso 3D).
Eka tinyiyoni ta dopamine ta timbeva ta MitoPark leti nga na ku tsandzeka ku tirha ka mitochondrial loku vangiwaka hi tinyiyoni ta dopamine leti herisaka hi ku kongoma jini ya mitochondrial transcription factor A (Tfam) (Xifaniso S6B), ku paluxiwa ka PCx na kona ku lawuriwe swinene ehenhla (31), leswi kombisaka leswaku acetone acid arteriosclerosis Ku humelela ka vuvabyi ku lawuriwa hi nkarhi wa ku nga tirhi kahle ka tinyiyoni OXPHOS emirini. Swi fanerile ku xiya leswaku ku kumiwile leswaku ti enzymes to hlawuleka (32-34) leti nga kombisiwaka eka ti neurons leti nga fambelanisiwaka na arteriosclerosis ti lawuriwa swinene eka ti PN leti pfumalaka OXPHOS, ku fana na propionyl-CoA carboxylase (PCC-A), Malonyl-CoA yi hundzula propionyl-CoA ku va succinyl-CoA na mitochondrial malic enzyme 3 (ME3), . loyi ntirho wa yena lowukulu ku nga ku vuyisa pyruvate eka malate (Xifaniso 3, A na C) (33, 35). Ku engetela kwalaho, hi kumile ku tlakuka lokukulu eka enzyme ya Pdk3, leyi phosphorylates xisweswo yi endla leswaku PDH yi nga tirhi ( 36 ), kasi ku hava ku cinca loku kumiweke eka enzyme ya Pdp1 leyi endlaka leswaku PDH yi tirha kumbe PDH enzyme complex hi yoxe (Xifaniso 3A). Hi ku landzelelana, eka Mern2cKO PNs, phosphorylation ya α1 subunit α (PDHE1α) subunit ya pyruvate dehydrogenase E1 xiphemu xa PDH complex eka Ser293 (leyi tivekaka ku sivela ntirho wa enzyme ya PDH) yi antswisiwile ( Xifaniso S6C) (Xifaniso S6C). Pyruvate ayi na mfikelelo wa misiha.
Eku heteleleni, hi kumile leswaku ndlela ya super ya serine na glycine biosynthesis, xirhendzevutani xa mitochondrial folate (1C) lexi fambelanaka na proline biosynthesis (Xifaniso 1G na Xifaniso xa S5C) hinkwaswo swi lawuriwa swinene ehenhla, hi ku ya hi swiviko, hi nkarhi wa endlelo ra activation. Tinyama leti rhendzeleke ti tirhisiwa hi ku nga tirhi kahle ka mitochondrial ( 5-7 ). Nxopaxopo wa confocal lowu seketelaka datha leyi ya proteomics wu kombisile leswaku eka PN leyi nga na OXPHOS leyi kayivelaka, swilayi swa cerebellar swa timbeva ta mavhiki ya 8 swi endliwile serine hydroxymethyltransferase 2 (SHMT2), enzyme ya nkoka ya xirhendzevutani xa mitochondrial folate. Nhlamulo ya nkoka ya masocha ya miri (Xifaniso xa S5D). Eka 13 wa CU-glucose-incubated acute cerebellar slices, swikambelo swa metabolic tracing switlhele switiyisisa up-regulation ya serine na proline biosynthesis, leswi kombisaka leswaku ku khuluka ka ti carbon isoforms eka serine na proline swi engetelekile (Figure S5E). Tanihi leswi ti reactions leti hlohloteriwaka hi GLS na GPT2 ti nga na vutihlamuleri eka ku endliwa ka glutamate kusuka eka glutamine na transamination exikarhi ka glutamate na α-ketoglutarate, upregulation ya tona yi kombisa leswaku ti neurons leti pfumalaka OXPHOS tina xilaveko lexi engetelekeke xa glutamate , Leswi swinga kongomisiwa eka ku hlayisa biosynthesis leyi engetelekeke ya proline (Xifaniso S5C). Kuhambana na ku cinca loku, nxopaxopo wa proteomic wa ti cerebellar astrocytes kusuka eka PN-specific Mfn2cKO mice wu kombisile leswaku tindlela leti (kukatsa na ti antiperoxidases hinkwato) ati cincanga ngopfu eka ku paluxiwa, xisweswo swikombisa This metabolic redirection is selective to degraded PN (Fig. S6, D to G).
Hi ku komisa, minxopaxopo leyi yi paluxe swivumbeko swo hambana swinene swa ku tirhisiwa ka nkarhi wa tindlela to karhi ta metaboliki eka ti-PN. Hambi leswi ntirho lowu nga tolovelekangiki wa mitochondrial ya tinyiyoni wu nga vangaka atherosclerosis yo sungula na 1C remodeling (Xifaniso 3E na Xifaniso xa S5C), hambi ku ri ku cinca loku nga languteriwaka eka ku paluxiwa ka I na IV complexes, ku cinca eka serine de novo synthesis i ntsena Swi ve erivaleni ntsena eka switeji swa le ndzhaku. Ku tsandzeka ku tirha ka OXPHOS (Xifaniso 3E na Xifaniso xa S5C). Swikumiwa leswi swi hlamusela endlelo ro landzelelana leri eka rona mitochondrial (1C cycle) na cytoplasmic (serine biosynthesis) leyi hlohloteriwaka hi ntshikilelo yi hlamulaka hi ku tirhisana na ku tlakuka ka atherosclerosis eka xirhendzevutani xa TCA ku vumba hi vuntshwa metabolism ya tinyiyoni.
Ti PNs ta mavhiki ta 8 leti pfumalaka OXPHOS tinga hlayisa ntirho wa high-frequency excitation naswona ti hundza eka ku hlanganisiwa nakambe ka metabolic lokukulu ku hakela ku nga tirhi kahle ka mitochondrial. Ku tshuburiwa loku ku tlakusa ku koteka loku tsakisaka ka leswaku hambi ku ri eka nkarhi lowu, tisele leti ti nga ha tlhela ti Amukela ku nghenelela ka vutshunguri ku hlwela kumbe ku sivela ku onhaka ka misiha. Hlwela. Hi lulamise ku koteka loku hi ku tirhisa ku nghenelela kambirhi loku tiyimeleke. Eka ndlela yo sungula, hi endlile vector leyi titshegeke hi Cre-dependent adeno-associated virus (AAV) leswaku MFN2 yi ta kombisiwa hi ku hlawula eka ti-PN leti pfumalaka OXPHOS eka vivo (Xifaniso S7A). AAV leyi khodaka MFN2 na fluorescent reporter gene mCherry (Mfn2-AAV) swi tiyisisiwile eka mindhavuko ya ti neuron to sungula in vitro, leswi endleke leswaku MFN2 yi kombisiwa hi ndlela leyi titshegeke hi Cre naswona yi ponise mitochondrial morphology, hi ndlela yoleyo yi sivela neuromutation eka ti neurons ta Mfn2cKO ( Xifaniso S7, B, D na E). Endzhaku ka sweswo, hi endlile swikambelo swa in vivo ku yisa hi stereotactically Mfn2-AAV ya mavhiki ya 8 eka cerebellar cortex ya Mfn2cKO na timbeva ta vulawuri, naswona hi xopaxophile timbeva ta mavhiki ya 12 (Xifaniso 4A). Timbeva ta Mfn2cKO leti tshunguriweke ti file (Xifaniso 1, A na B) (16). Ku hundziseriwa ka xitsongwatsongwana eka vivo swi endle leswaku ku va na ku paluxiwa loku hlawulekeke ka PN eka swirhendzevutana swin’wana swa cerebellar (Xifaniso xa S7, G na H). Ku cheriwa ka AAV ya vulawuri leyi kombisaka mCherry ntsena (Ctrl-AAV) akuri na mbuyelo lowukulu eka mpimo wa neurodegeneration eka swifuwo swa Mfn2cKO. Kuhambana na sweswo, nxopaxopo wa Mfn2cKOs lowu hundziseriweke hi Mfn2-AAV wu kombisile mbuyelo lowukulu wo sirhelela wa leyara ya tisele ta PN (Xifaniso 4, B na C). Ngopfu-ngopfu, ku tsindziyela ka tinyiyoni ku vonaka ku nga hambanisiwi ni swiharhi leswi lawuriwaka (Xifaniso 4, B na C, na Xifaniso xa S7, H na I). Ku paluxiwa ka MFN1 kambe ku nga ri MFN2 swi tirha hi ku ringana eku poniseni ka rifu ra tinyiyoni (Xifaniso 4C na Xifaniso xa S7, C na F), leswi kombisaka leswaku ku paluxiwa ka MFN1 ya le handle ka mbeleko swi nga engetela hi ndlela leyinene ku pfumaleka ka MFN2. Nxopaxopo wun’wana eka xiyimo xin’we xa PN wu kombisile leswaku Mfn2-AAV ngopfungopfu yi ponise ultrastructure ya mitochondria, yi normalise ti levele ta mtDNA, naswona yi tlherisele endzhaku ku paluxiwa ka le henhla ka anti-angiogenesis marker PCx ​​(Xifaniso 4, C ku ya eka E ). Ku kamberiwa hi mahlo ka ti Mfn2cKO mice leti ponisiweke eka xiyimo xo wisa swikombe leswaku ku yima ka tona na swikombiso swa motor (ku famba ka S1 kuya eka S3) swi antswisiwile. Hiku gimeta, swikambelo leswi swikombisa leswaku ku hlwela ku nghenisiwa nakambe ka MFN2 eka ti PNs leti pfumalaka swinene eka OXPHOS swiringanerile ku tlherisela endzhaku ku tirhisiwa ka mtDNA naku hlohletela atherosclerosis, hindlela yaleyo ku sivela ku onhaka ka axon na ku fa ka ti neuron in vivo.
(A) Xikimi lexi kombisaka xiyimiso xa ku ringeta xo chela AAV leyi khodaka MFN2 loko ndlela ya metaboliki leyi kombisiweke yi tirhisiwa. (B) Swifaniso swa confocal leswi yimelaka swiphemu swa cerebellar swa mavhiki ya 12 leswi hundziseriweke hi timbeva ta Mfn2cKO eka mavhiki ya 8 naswona swi lebuliwile hi anti-Calbindin antibody. Exineneni: Ku ringanisiwa ka tifibre ta axon. Xikalo xa axon zoom i 450 na 75 μm. (C) Left: Ku pima nhlayo ya ku tsindziyela ka tisele ta Purkinje eka AAV transduction loop (AAV+) (nxopaxopo wa ndlela yin’we wa ku hambana; n = 3 wa timbeva). Exineneni: nxopaxopo wa mtDNA focus eka transduced PN eka vhiki ra 12 (unpaired t-test; n = 6 wa tisele ku suka eka timbeva tinharhu). * P <0.05; ** P <0.01. (D) Ti micrographs ta tielektroni ta ku hundzisela leti yimelaka ta ti-PN ta swiyenge swa cerebellar swa Mfn2cKO leswi hundziseriweke hi ti vectors ta xitsongwatsongwana leti kombisiweke. Xisirhelelo xa xitshopana xi kombisa ndhawu leyi tekiweke hi ti- dendrite, naswona xikwere xa mathonsi yo tshwuka xi kombisa ku kurisa loku nyikeriweke etlhelo ra xinene; n yi yimela nyutliyasi. Bar ya xikalo, 1μm. (E) yi kombisa xikombiso xa PCx staining eka PN leyi hundziseriweke eka 12 wa mavhiki. Bar ya xikalo, 20μm. OE, ku paluxiwa ku tlula mpimo; FC, ku cinca ka swiphemu.
Eku heteleleni, hi lavisisile nkoka wa ku hanya ka tisele leti hlohloteriweke hi peroxidase eka ti PN leti nga hlangana na ku tsandzeka ku tirha ka OXPHOS. Hi endlile mCherry leyi khodaka AAV-shRNA (short hairpin RNA) leyi kongomisaka hi ku kongoma eka mRNA ya PCx ya mbeva (AAV-shPCx), naswona hi chele xitsongwatsongwana kumbe vulawuri bya xona lebyi hlanganisiweke (AAV-scr) eka cerebellum ya timbeva ta Mfn2cKO. Ku cheriwa ku endliwile eka vhiki ra vumune ra malembe (Xifaniso 5A) ku fikelela PCx knockdown leyi tirhaka hi nkarhi lowu ku paluxiwa ka PCx ku engetelekeke (Xifaniso 3C) naswona leyara ya tisele ta PN a ya ha ri kona (Xifaniso 1A). Swi fanerile ku xiya leswaku ku wisela ehansi PCx (Xifaniso xa S8A) swi yisa eka ku hatlisa lokukulu ka rifu ra PN, leri nga rona ntsena ring leyi siveleriweke ya ku tluleriwa (Xifaniso 5, B na C). Leswaku hi twisisa endlelo ra switandzhaku swa metabolic leswi hlohloteriweke hi PCx up-regulation, hi dyondze xiyimo xa redox xa PNs endzhaku ka PCx knockdown na AAV-mediated optical biosensor Grx1-roGFP2 swi kombisiwile hi nkarhi wun’we (Xifaniso S8, B ku ya eka D) ku kambela glutathione Ku cinca loku yelanaka ka vuswikoti bya peptide redox (38). Kutani, hi endlile two-photon fluorescence lifetime imaging microscopy (FLIM) eka swilayi swa byongo swa acute swa 7-week-old Mfn2cKO kumbe control littermates ku kuma ku cinca loku nga vaka kona eka xiyimo xa cytoplasmic redox endzhaku ko tiyisisa swiyimo swa FLIM (Xifaniso S8, E ku ya eka G). Nxopaxopo wu kombisile ku tlakuka lokukulu eka xiyimo xa oxidation xa Mfn2cKO PNs yin’we leyi pfumalaka PCx expression, leswi hambaneke na ti control neurons kumbe Mfn2cKO PNs leti kombisaka ntsena scrambled shRNA (Xifaniso 5, D na E). Loko PCx expression yi lawuriwa ehansi, tiphesente ta Mfn2cKO PNs leti kombisaka xiyimo xa oxidized swinene ti tlakukile hi kutlula kanharhu (Figure 5E), leswi kombisaka leswaku PCx up-regulation yi hlayise vuswikoti bya redox bya ti neurons leti onhakeke.
(A) Xikimi lexi kombisaka xiyimiso xa ku ringeta xo chela AAV leyi khodaka shPCx loko ndlela ya metaboliki leyi kombisiweke yi tirhisiwa. (B) Swifaniso swa confocal leswi yimelaka swiphemu swa cerebellar swa mavhiki ya 8 eka timbeva ta Mfn2cKO leti hundzuriweke no lebuliwa hi anti-calcineurin antibody eka 4 wa mavhiki. Bar ya xikalo, 450μm. (C) Ku pima nhlayo ya ku tsindziyela ka tisele ta Purkinje eka swirhendzevutana leswi hundziseriweke hi AAV (nxopaxopo wa ndlela yin’we wa ku hambana; n = 3 ku ya eka 4 wa timbeva). Data yi kombisiwa tanihi mean±SEM; ***P<0.001 ya xirhendzevutani. (D) Xifaniso xa FLIM lexi yimelaka xi kombisa nkarhi wa vutomi bya xikarhi bya PN ya mavhiki ya 7 leyi kombisaka glutathione redox sensor Grx1-roGFP2 ehansi ka swiyimo leswi boxiweke swa ku ringeta. LUT (tafula ro languta ehenhla) ratio: nkarhi wo hanya (hi ti picoseconds). Bar ya xikalo, 25μm. (E) Histogram yi kombisa ku hangalasiwa ka mimpimo ya vutomi bya Grx1-roGFP2 ku suka eka (D) (n=158 ku ya eka 368 wa tisele eka timbeva timbirhi ehansi ka xiyimo xin’wana na xin’wana). Chati ya phayi ehenhla ka histogram yin’wana na yin’wana: yi kombisa nhlayo ya tisele leti nga na mimpimo ya vutomi byo leha swinene (yo tshwuka, leyi oxidized) kumbe yo koma (ya wasi, leyi hungutiweke), leyi tlulaka 1 SD ya nhlayo ya vutomi bya xikarhi eka CTRL-AAV-scr. (F) Modele lowu ringanyetiweke wu kombisa mbuyelo wo sirhelela wa upregulation ya neuronal PCx.
Hinkwaswo hi ku angarhela, datha leyi hi yi nyikaka laha yi kombisa leswaku ku paluxiwa nakambe ka MFN2 ku nga ponisa hi ku helela PN ya le henhla leyi nga na ku pfumaleka lokukulu ka OXPHOS, ku hela lokukulu ka mtDNA, na xivumbeko lexi nga tolovelekangiki swinene xo fana na ista, hi ndlela yoleyo ku nyika nhluvuko lowu yaka emahlweni hambi eka mavabyi lama yaka emahlweni. Ku onhaka ka misiha ku nyikela vumbhoni lebyi nga hundzukiki bya xiteji xa le mahlweni ka ku fa ka tisele. Digiri leyi ya ku cinca-cinca ka metabolic yi tlhela yi kandziyisiwa hi vuswikoti bya ti neurons byo hlohlotela atherosclerosis (a rewiring ya xirhendzevutani xa TCA), leswi sivelaka ku paluxiwa ka PCx eka ti PNs leti pfumalaka OXPHOS naswona swi ndlandlamuxa ku fa ka tisele, hi ndlela yaleyo swi tlanga xiave xo sirhelela (Xifaniso 5F).
Eka dyondzo leyi, hi nyikile vumbhoni bya leswaku nhlamulo ya ti-PN eka ku tsandzeka ku tirha ka OXPHOS i ku hlangana hakatsongo-tsongo eka TCA cycle atherosclerosis hi ku tirhisa ndlela ya differential activation leyi tirhisiwaka hi minongonoko ya metabolic. Hi tiyisisile nxopaxopo wa proteomic hi tindlela to tala to tatisela naswona hi paluxe leswaku loko hi tlhontlhiwa hi ku tsandzeka lokukulu ka mitochondrial, tinyiyoni ti na xivumbeko lexi nga tiviwiki khale xa metabolic elasticity. Lexi hi hlamariseke, endlelo hinkwaro ra ku hlanganisa tintambhu a ri funghi xiyimo xa metabolic xa makumu lexi fambisanaka na ku onhaka ka misiha hakatsongo-tsongo naswona hi ndlela leyi nga hundzukiki, kambe datha ya hina yi ringanyeta leswaku yi nga ha vumba nyuroni yo hlayisa hambi ku ri eka xiteji xa le mahlweni ka ku fa ka tisele Functional compensation mechanism. Ku kumiwa loku ku kombisa leswaku ti neurons tina mpimo lowukulu wa metabolic plasticity emirini. Mhaka leyi yi tiyisisa leswaku ku nghenisiwa nakambe ka MFN2 endzhaku swi nga tlherisela endzhaku ku paluxiwa ka swikombiso swa nkoka swa metabolic na ku sivela ku onhaka ka PN. Ku hambana ni sweswo, yi sivela ku tsana ka misiha naswona yi hatlisisa misiha. transsexual.
Xin’wana xa swikumiwa leswi tsakisaka swinene eka ndzavisiso wa hina hileswaku ti PN leti pfumalaka OXPHOS ti nga cinca metabolism ya xirhendzevutani xa TCA hi ku up-regulating enzymes leti hi ku kongoma ti hlohlotelaka arteriosclerosis. Ku lulamisiwa nakambe ka metabolic i xivumbeko lexi tolovelekeke xa tisele ta khensa, tin’wana ta tona ti titshege hi glutamine ku engetela switirhisiwa swa le xikarhi swa xirhendzevutani xa TCA ku humesa leswi ringanaka leswi hungutaka, leswi fambisaka nxaxamelo wo hefemula na ku hlayisa vuhumelerisi bya ti-lipid na ti-nucleotide biosynthesis precursors (39 , 40). Dyondzo ya sweswinyana yi kombisile leswaku eka tinyama ta le matlhelo leti hlanganaka na ku tsandzeka ku tirha ka OXPHOS, ku hlanganisiwa nakambe ka metabolism ya glutamine/glutamate na swona i xivumbeko lexi xiyekaka ( 5 , 41 ), laha nkongomiso wa ku nghena ka glutamine eka xirhendzevutani xa TCA wu titshegeke hi Hikwalaho ka ku tika ka ku vaviseka ka OXPHOS ( 41 ). ). Hambiswiritano, ku pfumaleka vumbhoni lebyi nga erivaleni eka ku fana kwihi na kwihi ka neuronal metabolic plasticity emirini na ku yelana ka yona loku nga kotekaka eka xiyimo xa vuvabyi. Eka dyondzo ya sweswinyana ya in vitro, tinyiyoni ta cortical to sungula ti kombisiwile ku hlengeleta swidziva swa glutamate eka neurotransmission, hi ndlela yoleyo ti tlakusa oxidative metabolism na atherosclerosis ehansi ka swiyimo swa ntshikilelo wa metabolic ( 42 ). Swi fanerile ku xiya leswaku ehansi ka ku sivela ka mirhi ka enzyme ya xirhendzevutani xa TCA succinate dehydrogenase, pyruvate carboxylation yi tshembiwa ku hlayisa ku hlanganisiwa ka oxaloacetate eka tinyiyoni ta cerebellar granule leti kurisiweke ( 34 ). Hambiswiritano, ku yelana ka physiological ka tindlela leti eka tinyama ta byongo (laha atherosclerosis yi tshembiwaka leswaku ngopfu-ngopfu yi pfaleriwile eka astrocytes) ya ha ri na nkoka wa nkoka wa physiological ( 43 ). Eka xiyimo lexi, data ya hina yi kombisa leswaku ti PNs leti onhiweke hi OXPHOS emirini tinga cinciwa kuya eka ku onhaka ka BCAA na pyruvate carboxylation, leswinga swihlovo swimbirhi leswikulu swa ku engeteriwa ka ti TCA pool intermediates. Hambi leswi ku hoxa xandla loku ehleketiwaka ka BCAA catabolism eka neuronal energy metabolism ku ringanyetiweke, ku engetela eka ntirho wa glutamate na GABA eka neurotransmission ( 44 ), ka ha ri hava vumbhoni bya tindlela leti eka vivo. Hikwalaho, swa olova ku ringanyeta leswaku ti-PN leti nga tirhiki kahle ti nga ha hakela hi ku tisungulela ku dyiwa ka switirhisiwa swa le xikarhi swa TCA leswi fambiwaka hi endlelo ra assimilation hi ku engetela atherosclerosis. Ngopfu-ngopfu, upregulation ya PCx yi nga ha laveka ku hlayisa xilaveko lexi engetelekeke xa aspartic acid, leswi kombisiweke eka tisele leti andzaka leti nga na mitochondrial dysfunction ( 45 ). Hambiswiritano, nxopaxopo wa hina wa metabolomics a wu paluxa ku cinca lokukulu eka mpimo wa xiyimo xo tiya xa aspartic acid eka Mfn2cKO PNs (Xifaniso S6A), leswi ku ehleketiwaka leswaku swi kombisa ku tirhisiwa ko hambana ka metabolic ka aspartic acid exikarhi ka tisele leti andzaka na tinyiyoni ta le ndzhaku ka mitotic . Hambi leswi endlelo leri kongomeke ra PCx upregulation eka tinyiyoni leti nga tirhiki kahle eka vivo ri nga si tivekaka, hi kombisile leswaku nhlamulo leyi ya ka ha ri emahlweni yi tlanga xiave xa nkoka eku hlayiseni ka xiyimo xa redox xa tinyiyoni, leswi kombisiweke eka swikambelo swa FLIM eka swilayi swa cerebellar. Ngopfu ngopfu, ku sivela ti PNs kuva ti up-regulating PCx swinga endla leswaku kuva na xiyimo xa oxidized swinene xikan’we naku hatlisisa ku fa ka ti cell. Ku tirhisiwa ka ku onhaka ka BCAA na carboxylation ya pyruvate a hi tindlela to hlamusela tinyama ta le matlhelo ta mitochondrial dysfunction ( 7 ). Hikwalaho, swi vonaka swi ri nchumu lowu rhangisaka emahlweni eka tinyiyoni leti pfumalaka OXPHOS, hambiloko ku nga ri wona ntsena xivumbeko, lexi nga xa nkoka eka ku onhaka ka misiha. .
Vuvabyi bya cerebellar i muxaka wa heterogeneous wa vuvabyi bya neurodegenerative lebyi hi ntolovelo byi vonakaka tani hi ataxia naswona byi tala ku onha ti PNs ( 46 ). Nhlayo leyi ya ti neuron yi le khombyeni ngopfu eka ku nga tirhi kahle ka mitochondrial hikuva ku hohloka ka tona loku hlawulaka eka timbeva ku ringanerile ku humesa swikombiso swo tala swa motor leswi hlawulaka ataxia ya spinocerebellar ya munhu ( 16 , 47 , 48 ). Hi ku ya hi swiviko, modele wa mbeva ya transgenic leyi nga na jini ya mutant yi fambelanisiwa na human spinocerebellar ataxia naswona yi na mitochondrial dysfunction ( 49 , 50 ), leswi kandziyisaka nkoka wo dyondza vuyelo bya ku pfumaleka ka OXPHOS eka PNPH. Hikwalaho, swi fanerile ngopfu ku hambanyisa ni ku dyondza hi ndlela leyi humelelaka nhlayo leyi yo hlawuleka ya tinyiyoni. Hambiswiritano, loko hi tekela enhlokweni leswaku ti PNs ti na vuxiyaxiya swinene eka ntshikelelo naswona ti hlamusela xiphemu xale hansi xa nhlayo hinkwayo ya ti cell ta cerebellar, eka tidyondzo totala leti simekiweke eka omics, ku hambanisiwa hiku hlawula ka tona tani hi ti cells hinkwato swahari xiphemu xa ntlhontlho. Hambi leswi swi nga ha kotekiki ku fikelela ku pfumaleka loku heleleke ka ku thyakisa ka tinxaka tin’wana ta tisele (ngopfu-ngopfu tinyama letikulu), hi hlanganisile goza leri tirhaka ra ku hambanisiwa na FACS ku kuma nhlayo leyi eneleke ya tinyiyoni leti hanyaka ta nxopaxopo wa proteomics ya le hansi, naswona hi va na ku funengetiwa ka tiphrotheyini ta le henhla swinene (kwalomu ka 3000 wa tiphrotheyini) loko ku pimanisiwa na sete ya datha leyi nga kona ya cerebellum hinkwayo ( 51 ). Hi ku hlayisa ku hanya ka tisele hinkwato, ndlela leyi hi yi nyikaka laha a yi hi pfumeleli ntsena ku kambela ku cinca eka tindlela ta metabolic eka mitochondria, kambe na ku kambela ku cinca eka ti-cytoplasmic counterparts ta yona, leswi hetisisaka ku tirhisiwa ka mitochondrial membrane label enrichment specific to cell types Ndlela leyintshwa ya nhlayo ya mitochondria eka tinyama leti rharhanganeke (52, 53). Ndlela leyi hi yi hlamuselaka a yi fambelani ntsena na dyondzo ya tisele ta Purkinje, kambe yi nga tirhisiwa hi ku olova eka muxaka wihi na wihi wa sele ku lulamisa ku cinca ka metaboliki eka byongo lebyi vabyaka, ku katsa na timodeli tin’wana ta ku tsandzeka ku tirha ka mitochondrial.
Eku heteleleni, hi kumile fasitere ra vutshunguri hi nkarhi wa endlelo leri ra ku lulamisiwa nakambe ka metaboliki leri nga tlheriselaka endzhaku hi ku helela swikombiso swa nkoka swa ntshikilelo wa tisele na ku sivela ku hohloka ka tinyiyoni. Hikwalaho, ku twisisa switandzhaku swa ntirho swa ku hlanganisiwa ka tintambhu leswi hlamuseriweke laha swi nga nyika vutivi bya xisekelo eka vutshunguri lebyi nga kotekaka byo hlayisa ku hanya ka tinyiyoni hi nkarhi wa ku nga tirhi kahle ka mitochondrial. Vulavisisi bya nkarhi lowu taka lebyi kongomisiweke eka ku tsema ku cinca eka metabolism ya eneji eka tinxaka tin’wana ta tisele ta byongo bya laveka ku paluxa hi ku helela ku tirhisiwa ka nsinya lowu wa nawu eka mavabyi man’wana ya misiha.
MitoPark mice yi hlamuseriwile khale ( 31 ). Timbeva ta C57BL/6N leti nga na tijini ta loxP flanking Mfn2 ti hlamuseriwile khale ( 18 ) naswona ti hlanganisiwa na timbeva ta L7-Cre ( 23 ). Kutani vatukulu va double heterozygous lava humeleleke va hlanganisiwile na ti homozygous Mfn2loxP/Mfn2loxP mice ku tumbuluxa Purkinje-specific gene knockouts ya Mfn2 (Mfn2loxP/Mfn2loxP; L7-cre). Eka ntlawa lowutsongo wa ku hlangana, Gt (ROSA26) SorStop-mito-YFP allele (stop-mtYFP) yi nghenisiwile hi ku tirhisa swihambano swo engetela ( 20 ). Maendlelo hinkwawo ya swiharhi ya endliwile kuya hi swiletelo swa Europe, tiko na nhlangano naswona ya pasisiwile hi LandesamtfürNatur ya Umwelt na Verbraucherschutz, North Rhine-Westphalia, Germany. Ntirho wa swiharhi wu tlhela wu landzela nkongomiso wa European Federation of Laboratory Animal Sciences Associations.
Endzhaku ko titivata ku tshoveka ka khwiri ra wansati la tikeke, embryo ya mbeva ya hambanisiwa (E13). Cortex yi tsemiwile eka Hanks’ Balanced Salt Solution (HBSS) leyi engeteriweke hi 10 mM Hepes naswona yi hundziseriwe eka Dulbecco’s Modified Eagle’s Medium leyingana papain (20 U/ml) na cysteine ​​(1μg/ml). Hlanganisa tissue eka DMEM) naswona u yi hambanyisa hi enzymatic digestion. Ml) eka 37°C kuringana 20 wa timinete, kutani yi gayeriwa hi muchini eka DMEM leyi engeteriweke hi 10% fetal bovine serum. Ti cells ti byariwile eka ti glass coverslips leti pfanganisiweke hi polylysine eka density ya 2×106 eka 6 cm culture dish kumbe eka density ya 0.5×105 cells/cm2 kuva yi endliwa nxopaxopo wa swifaniso. Endzhaku ka 4 wa tiawara, xitirhisiwa lexi xi cinciwile hi Neurobasal serum-free medium leyi nga na 1% B27 supplement na 0.5 mM GlutaMax. Kutani ti neurons ti hlayisiwile eka 37°C na 5% CO2 eka nkarhi hinkwawo wa xikambelo, naswona ti phameriwile kan’we hi vhiki. Leswaku ku hlohleteriwa ku hlanganisiwa nakambe eka vitro, 3μl (24-well culture dish) kumbe 0.5μl (24-well plate) ya vector ya xitsongwatsongwana xa AAV9 leyi landzelaka yi tirhisiwile ku tshungula ti neurons hi siku ra vumbirhi eka vitro: AAV9.CMV.PI.eGFP. WPRE.bGH (Addgene, nomboro ya khathalogi 105530-AAV9) na AAV9.CMV.HI.eGFP-Cre.WPRE.SV40 (Addgene, nomboro ya khathalogi 105545-AAV9).
Mouse Mfn1 na Mfn2 complementary DNA (leyi kumiweke eka Addgene plasmid #23212 na #23213, hi ku landzelelana) yi funghiwile hi ntlhandlamano wa V5 (GKPIPNPLLGLDST) eka C-terminus, naswona yi hlanganisiwa na mCherry eka furemu hi ku tirhisa ntlhandlamano wa T2A. Grx1-roGFP2 i nyiko leyi humaka eka Heidelberg T. P. Dick DFKZ (Deutsches Krebsforschungszentrum). Hiku cinca khasete ya tdTomato hiku tirhisa tindlela ta ntolovelo ta cloning, khasete yi subcloned eka pAAV-CAG-FLEX-tdTomato backbone (Addgene reference number 28306) ku tumbuluxa pAAV-CAG-FLEX-mCherry-T2A-MFN2-V5, pAAV-CAG- FLEX-mCherry-T2A-MFN1-V5 na switsongwatsongwana swa pAAV-CAG-FLEX-Grx-roGFP2. Qhinga leri fanaka ri tirhisiwile ku tumbuluxa vector ya vulawuri pAAV-CAG-FLEX-mCherry. Leswaku ku endliwa xivumbeko xa AAV-shPCx, vector ya plasmid AAV (VectorBuilder, pAAV [shRNA] -CMV-mCherry-U6-mPcx- [shRNA#1]) ya laveka, leyi nga na ntlhandlamano wa DNA lowu khodaka shRNA leyi kongomisaka eka PCx ya mbeva (5′CTTTCGCTCTCTAAGGTGCTAACCTCGAGTTTAGCACCTTAGAGCGAAAG 3′) Ehansi ka vulawuri bya U6 promoter, mCherry yi tirhisiwa ehansi ka vulawuri bya CMV promoter. Ku endliwa ka ti auxiliary AAV vectors swiendliwile kuya hi swiletelo swa muendli (Cell Biolabs). Hi ku komisa, tirhisa plasmid yo hundzisela leyi rhwalelaka mCherry-T2A-MFN2-V5 (pAAV-CAG-FLEX-mCherry-T2A-MFN2-V5), mCherry-T2A-MFN1-V5 (pAAV-CAG-FLEX-mCherry) swa xinkarhana Ku hundziseriwa ka tisele ta 293AAV-T2A-MFN1-V5), mCherry (pAAV-CAG-FLEX-mCherry) kumbe Grx-roGFP2 (pAAV-CAG-FLEX-Grx-roGFP2) leyi khodaka jini, xikan’we na ku khoda AAV1 capsid protein na phrotheyini yo pfuneta Ku paka plasmid plasmid, hi ku tirhisa ndlela ya calcium phosphate. Crude virus supernatant yikumeke hiku tirhisa freeze-thaw cycles eka dry ice/ethanol bath na ti cells leti lysed eka phosphate buffered saline (PBS). Vector ya AAV yi basisiwile hi discontinuous iodixanol gradient ultracentrifugation (24 wa tiawara eka 32,000 rpm na 4°C) naswona yi hlanganisiwile hiku tirhisa Amicon ultra-15 centrifugal filter. Titer ya genome ya AAV1-CAG-FLEX-mCherry-T2A-MFN2-V5 [2.9×1013 khopi ya genome (GC)/ml], AAV1-CAG-FLEX-mCherry (6.1×1012 GC/ml), AAV1-CAG- FLEX a yi ri tanihilaha swi hlamuseriweke hakona khale (54), yi pimiwe hi nkarhi wa xiviri wa nhlayo ya PCR (qPCR) -MFN1-V5 (1,9×1013 GC/ml) na AAV1-CAG-FLEX-Grx-roGFP2 (8,9×1012 GC/ml) na swona.
Ti neurons tosungula ti khwaxiwile eka ice-cold 1x PBS, ti pelleted, kutani ti homogenized eka 0.5% Triton X-100 / 0.5% sodium deoxycholate/PBS lysis buffer leyingana phosphatase na protease inhibitor (Roche). Ku pima nhlayo ya ti protein swiendliwile hiku tirhisa bicinchoninic acid assay (Thermo Fisher Scientific). Kutani ti protein ti hambanisiwile hi SDS-polyacrylamide gel electrophoresis, kutani ti blotted eka polyvinylidene fluoride membrane (GE Healthcare). Sivela tindzhawu letinga kongomangiki naswona u pfalela na antibody yosungula (vona Tafula ra S1 kuva u kuma vuxokoxoko) eka masi ya 5% eka TBST (Tris-buffered saline with Tween), magoza yo hlamba na antibody ya vumbirhi eka TBST Incubate. Hlayisa na primary antibody vusiku hinkwabyo eka +4°C. Endzhaku ko hlamba, chela secondary antibody kuringana 2 wa tiawara eka mahiselo ya kamara. Endzhaku ka sweswo, hiku pfaleriwa ka blot leyi fanaka na anti-β-actin antibody, ku layicha loku fanaka ku tiyisisiwile. Ku kumiwa hi ku hundzula eka chemiluminescence na ku ndlandlamuxa chemiluminescence (GE Healthcare).
Ti neurons letinga byariwa khale eka ti glass coverslips ti lulamisiwile hi 4% paraformaldehyde (PFA)/PBS eka nkarhi lowu vekiweke eka mahiselo ya kamara kuringana 10 wa timinete. Ti coverslips tisungula ku nghenisiwa hi 0.1% Triton X-100/PBS kuringana 5 wa timinete eka mahiselo ya kamara, kutani eka blocking buffer [3% bovine serum albumin (BSA)/PBS]. Hi siku ravumbirhi, ti coverslips ti hlantswiwile hi blocking buffer naswona ti pfaleriwile na fluorophore-conjugated secondary antibody leyi faneleke kuringana 2 wa tiawara eka mahiselo ya kamara; ekuheteleleni, tisampulu ti hlantswiwile kahle eka PBS hi 4′,6-diamidino-2 -Phenylindole (DAPI) yi counterstained kutani yi vekiwa eka microscope slide hi Aqua-Poly/Mount.
Timbeva (ta xinuna na ta xisati) ti anesthetized hi intraperitoneal injection ya ketamine (130 mg/kg) na xylazine (10 mg/kg) naswona ti nyikiwile ehansi ka nhlonge na carprofen analgesic (5 mg/kg) , Naswona ti vekiwa eka xitirhisiwa xa stereotactic (Kopf) lexi hlomisiweke hi pad yo kufumela. Pfuxeta xikandza kutani u tirhisa dental drill ku olovisa xiphemu xa cerebellar cortex lexi fambelanaka na mis bone (kusuka eka lambda: tail 1.8, lateral 1, leyi fambelanaka na lobules IV na V). Tirhisa naliti ya syringe leyi govekeke ku endla hi vukheta khumbi lerintsongo eka khwiri ku papalata ku kavanyeta misiha yale hansi. Kutani capillary ya nghilazi leyi kokiwaka yo olova yi nghenisiwa hiku nonoka eka micro-hole (kusuka eka -1.3 kuya eka -1 eka tlhelo ra ventral ra dura mater), naswona 200 kuya eka 300 nl ya AAV yi cheriwa eka micro-injector (Narishige) hi ti manual syringes (Narishige) minkarhi yotala eka ntshikelelo wale hansi eka nkarhi wa 10 kuya eka 20 wa timinete ta fasitere. Endzhaku ka ku cheriwa, veka capillary kuringana 10 wa timinete tin’wana leswaku xitsongwatsongwana xi hangalaka hiku hetiseka. Endzhaku kaloko ti capillaries ti hoxiwile, nhlonge yi rhungiwile hi vukheta ku hunguta ku pfimba ka swilondza naswona swipfumelela leswaku xifuwo xi hola. Swifuwo swi tshunguriwile hi switlhavi (caspofen) kuringana masiku yo hlaya endzhaku ka vuhandzuri, hi nkarhi walowo xiyimo xa swona xa miri xi langutisiwile hi vukheta kutani swi dlayiwa hi nkarhi lowu boxiweke. Maendlelo hinkwawo ya endliwile hi ku landza swiletelo swa le Yuropa, swa tiko na swa nhlangano naswona ya amukeriwile hi LandesamtfürNatur ya le Umwelt na Verbraucherschutz, North Rhine-Westphalia, Germany.
Swifuwo swi anesthetized hi ketamine (100 mg/kg) na xylazine (10 mg/kg), naswona mbilu yi cheriwile 0.1 M PBS kusungula, kutani yi cheriwa 4% PFA eka PBS. Tissue ti tsemiwile kutani ti vekiwa eka 4% PFA/PBS vusiku hinkwabyo eka 4°C. Banga leri rhurhumelaka (Leica Microsystems GmbH, Vienna, Austria) ritirhisiwile ku lulamisa swiphemu swa sagittal (50 μm hiku enta) kusuka eka byongo lebyi nga cinciki eka PBS. Handle ka loko swi boxiwile hi ndlela yin’wana, ku pendiwa ka swiphemu leswi papamalaka hi ku ntshunxeka swi endliwile tanihilaha swi hlamuseriweke hakona laha henhla (13) eka mahiselo ya kamara na ku hlanganisiwa. Hiku komisa, xosungula, swilayi leswi kumiweke swi permeabilized hi 0.5% Triton X-100/PBS kuringana 15 wa timinete eka mahiselo ya kamara; eka ti epitopes tin’wana (Pcx na Shmt2), hi eka tris-EDTA buffer eka 80°C (PH 9) hisa swilayi kuringana 25 wa timinete ematshan’wini ya goza leri. Endzhaku ka sweswo, swiphemu swi pfaleriwile na primary antibody (vona Tafula ra S1) eka blocking buffer (3% BSA/PBS) eka 4°C vusiku hinkwabyo hiku hlanganisiwa. Hi siku leri landzelaka, swiphemu swi hlantswiwile hi blocking buffer naswona swi pfaleriwile na fluorophore-conjugated secondary antibody leyi faneleke kuringana 2 wa tiawara eka mahiselo ya kamara; ekuheteleleni, swiphemu swi hlantswiwile kahle eka PBS, swi counter-stained hi DAPI, kutani swi lulamisiwa hi AquaPolymount Eka microscope slide.
Laser scanning confocal microscope (TCS SP8-X kumbe TCS Digital Light Sheet, Leica Microsystems) leyi hlomisiweke hi laser yo voninga yo basa na 405 diode ultraviolet laser yitirhisiwile ku teka xifaniso xa xikombiso. Hiku tsakisa fluorophore naku hlengeleta xigingi hi Hybrid Detector (HyDs), software ya LAS-X yitirhisiwile ku hlengeleta swifaniso leswi stacked leswi fambelanaka na Nyquist sampling eka sequential mode: eka ti non-quantitative panels, i swikombiso leswi cinca cinca swinene (xikombiso, eka ti somatic cells na dendrites) mtYFP) Tirhisa HyD ku kuma nhlayo ya PNs eka BrightR mode). Ku tirhisiwa gating ya 0.3 kuya eka 6 ns ku hunguta background.
Ku teka swifaniso hi nkarhi wa xiviri swa tisele leti hleriweke. Endzhaku ko hlela eka Neurobasal-A medium leyingana 1% B27 supplement na 0.5 mM GlutaMax, ti cells ti hatle ti byariwa eka ti poly-l-lysine-coated glass slides (μ-Slide8 Well, Ibidi, catalog number 80826) , Kutani ti hlayisiwa eka 37°C na 5% CO2 kuringana 1 wa awara kuva ti pfumelela ti cells kuva ti tshamiseka. Ku teka swifaniso swa nkarhi wa xiviri swi endliwile eka microscope ya Leica SP8 laser scanning confocal leyi hlomisiweke hi laser yo basa, HyD, 63×[1.4 numerical aperture (NA)] oil objective lens na xiteji xo kufumeta.
Mbeva yi hatle yi anesthetized hi carbon dioxide kutani yi tsemiwa nhloko, byongo byi hatlisile byi susiwa eka khwiri, naswona byi tsemiwa byi va 200μm hiku enta (eka 13C labeling experiment) kumbe 275μm hiku enta (eka ti photon experiments timbirhi) sagittal section leyi tatiweke hi switirhisiwa leswi landzelaka The ice cream (HM-650 V, Thermo Fisher Scientific, Walldorf, Germany) . yi tatiwa hi swilo leswi landzelaka: 125 mM yo titimela ya ayisi, leyi taleke hi khaboni (95% O2 na 5% CO2) yale hansi ya Ca2 + artificial cerebrospinal fluid (ACSF) NaCl, 2.5 mM KCl, 1.25 mM sodium phosphate buffer, 25 mM NaHCO3, 25 mM glucose, 0.5 mM CaCl2 na 3.5 mM MgCl2 (ntshikelelo wa osmotic wa 310 kuya eka 330 mmol). Tlhuvulela swilayi swa byongo leswi kumiweke eka kamara ra pre-incubation leri nga na Ca2 + ACSF yale henhla (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 na 2.0 mM MgCl2) Xikarhi) pH 7.4 na 310 kuya eka 320 mmol).
Hi nkarhi wa endlelo ro teka swifaniso, swilayi swi rhurhiseriwe eka kamara leri tinyiketeleke ro teka swifaniso, naswona ku ringeta ku endliwile ehansi ka ACSF perfusion leyi yaka emahlweni eka mahiselo lawa ya nga cinciki ya 32° kuya eka 33°C. Microscope ya multiphoton laser scanning (TCS SP8 MP-OPO, Leica Microsystems) leyi hlomisiweke hi Leica 25x objective lens (NA 0.95, mati), Ti: Sapphire laser (Chameleon Vision II, Coherent) yitirhisiwile eka ku teka swifaniso swa swilayi. Modyuli ya FLIM (PicoHarp300, PicoQuant).
FLIM ya Grx1-roGFP2. Ku cinca eka xiyimo xa cytoplasmic redox xa ti PNs swi pimiwe hi two-photon FLIM eka swilayi swa byongo bya sagittal, laha Grx1-roGFP2 biosensor yi kongomisiweke eka ti PNs. Eka PN layer, nsimu ya acquisition yi hlawuriwa kwalomu ka 50 kuya eka 80 μm ehansi ka slice surface kutiyisisa leswaku kuna PN leyi hanyaka (leswivulaka leswaku, ku pfumaleka ka xivumbeko xa beaded kumbe ku cinca ka neuronal morphological etlhelo ka ti dendrites) na double positive roGFP2 sensor na AAV encoding shRNA PCx kumbe ntlhandlamano wa yona wa vulawuri (each co-expressing mCherry). Hlengeleta swifaniso swa xirhendzevutani xin’we hi 2x digital zoom [excitation wavelength: 890 nm; 512 nm 512 wa tipikseli]. Ku kumiwa: HyD yale ndzeni, ntlawa wa sefo ya fluorescein isothiocyanate (FITC)] na ku averiwa ka swifaniso eka timinete ta 2 kuya eka 3 switirhisiwa ku tiyisisa leswaku ti photons leti ringaneleke ti hlengeletiwile (1000 photons hiku angarhela) kuva ti ringanela curve. Ku twisisa ka Grx1-roGFP2 probe xikan’we naku tiyisisiwa ka swiyimo swa FLIM swiendliwile hiku langutisisa nkoka wa vutomi bya roGFP2 loko ku engeteriwa exogenous 10 mM H2O2 eka perfusion ACSF (ku kurisa oxidation, leswi endlaka leswaku kuva na ku engeteleriwa ka vutomi), kutani ku engeteriwa 2 mM dithiothreitol (ku hunguta mpimo wa kuhunguteka, leswi endlaka leswaku kuva na kuhunguteka eka vutomi) (Xifaniso xa S8, D ku ya eka G). Tirhisa software ya FLIMfit 5.1.1 ku xopaxopa mbuyelo lowu kumiweke, ku ringanisa curve yin’we ya exponential decay ya xifaniso hinkwaxo eka IRF leyi pimiweke (ntirho wa nhlamulo ya xitirhisiwa), naswona χ2 i kwalomu ka 1. Ku hlayela vutomi bya PN yin’we, xisirhelelo lexi rhendzeleke miri wa misiha xi dirowiwile hi voko, naswona vutomi bya xikarhi eka xisirhelelo xin’wana na xin’wana byi tirhisiwile ku pima nhlayo.
Nxopaxopo wa vuswikoti bya mitochondrial. Endzhaku kaloko xiphemu xa acute xi pfaleriwile na 100 nM TMRM leyi engeteriweke hiku kongoma eka ACSF leyi cheriweke kuringana 30 wa timinete, ku cinca ka mitochondrial potential ya PNs swipimiwile hi microscope ya ti photon timbirhi. TMRM imaging yi endliwile hiku tsakisa probe eka 920 nm naku tirhisa HyD yale ndzeni (tetramethylrhodamine isothiocyanate: 585/40 nm) ku hlengeleta swikombiso; hi ku tirhisa wavelength ya excitation leyi fanaka kambe hi ku tirhisa HyD ya le ndzeni yo hambana (FITC :525/50) ku endla xifaniso xa mtYFP. Tirhisa plug-in ya ImageJ’s Image Calculator ku kambela vuswikoti bya mitochondrial eka xiyimo xa sele yin’we. Hi ku komisa, xiringaniso xa plug-in: signal = min (mtYFP, TMRM) xi tirhisiwa ku kuma ndhawu ya mitochondrial leyi kombisaka xikombiso xa TMRM hi Purkinje Somali eka xifaniso xa single-stack confocal xa xiteji lexi fambelanaka. Kutani ndzhawu ya pixel eka mask leyi humelelaka yi pimiwa, kutani yi normaliseriwa eka xifaniso xa threshold lexi fambelanaka xa single-stack xa mtYFP channel ku kuma xiphemu xa mitochondrial lexi kombisaka vuswikoti bya mitochondrial.
Xifaniso lexi xi susiwile hi software ya Huygens Pro (Scientific Volume Imaging). Eka swifaniso leswi skeniweke swa tithayile, montage ya tile yin’we yi endliwa hiku tirhisa algorithm ya automatic stitching leyi nyikiwaka hi software ya LAS-X. Endzhaku ka ku kalibhara xifaniso, tirhisa ImageJ na Adobe Photoshop ku ya emahlweni u tirhisa xifaniso na ku lulamisa hi ndlela leyi fanaka ku vonakala na ku hambana. Tirhisa Adobe Illustrator ku lunghiselela swifaniso.
nxopaxopo wa mtDNA lowu kongomisiweke. Nhlayo ya swirhumbana swa mtDNA yi pimiwile eka swiphemu swa cerebellar leswi leteriweke hi ti antibodies leti lwisanaka na DNA hi confocal microscope. Ndzhawu yin’wana na yin’wana leyi kongomisiweke yi endleriwe miri wa sele na nyutliyasi ya sele yin’wana na yin’wana, naswona ndzhawu leyi faneleke yi hlayiwile hiku tirhisa Multi Measure plug-in (ImageJ software). Susa ndzhawu ya nyutliya eka ndzhawu ya miri wa sele kuva u kuma ndzhawu ya cytoplasmic. Eku heteleleni, Analyze Particles plug-in (ImageJ software) yi tirhisiwile ku tisungulela ku pima nhlayo ya tinhla ta cytoplasmic DNA leti kombisaka mtDNA eka xifaniso xa threshold, naswona mbuyelo lowu kumiweke wu normalised eka PN average ya CTRL mice. Vuyelo byi kombisiwa tani hi nhlayo ya xikarhi ya ti nucleosides hi sele.
Nxopaxopo wa ku paluxiwa ka tiphrotheyini. Tirhisa plug-in ya ImageJ’s Image Calculator ku kambela ku paluxiwa ka tiphrotheyini eka PN eka xiyimo xa sele yin’we. Hiku komisa, eka xifaniso xa confocal xa single-layer xa channel leyi fambelanaka, hiku tirhisa equation: signal = min (mtYFP, antibody), ndzhawu ya mitochondrial leyi kombisaka immunoreactivity eka antibody yokarhi eka Purkina ya tiveka. Kutani ndzhawu ya pixel eka mask leyi humelelaka yi pimiwa, kutani yi normaliseriwa eka xifaniso xa threshold lexi fambelanaka xa single-stack xa mtYFP channel ku kuma xiphemu xa mitochondrial xa protein leyi kombisiweke.
Nxopaxopo wa ku tsindziyela ka tisele ta Purkinje. Plug-in ya Cell Counter ya ImageJ yi tirhisiwile ku kambela ku tsindziyela ka Purkinje hi ku avanyisa nhlayo ya tisele ta Purkinje leti hlayiweke hi ku leha ka ring ya cerebellar leyi tekiweke hi tisele leti hlayiweke.
Ku lulamisiwa ka xikombiso na ku hlengeletiwa. Byongo kusuka eka ntlawa wa vulawuri na Mfn2cKO mice byi lulamisiwile eka 2% PFA/2.5% glutaraldehyde eka 0.1 M phosphate buffer (PB), kutani swiphemu swa coronal swi lulamisiwile hiku tirhisa ciliates (Leica Mikrosysteme GmbH, Vienna, Austria) (Thickness 50 kuya eka 60 μm) Kutani swi lulamisiwile eka PB buffer eka 1% os tetraoxide na 1.5% potassium ferrocyanide eka mahiselo ya kamara kuringana 1 wa awara. Swiphemu swi hlantswiwile kanharhu hi mati ya distilled, kutani swi cheriwa 70% ethanol leyingana 1% uranyl acetate kuringana 20 wa timinete. Kutani swiphemu swi susiwile mati eka byala lebyi ringanisiweke kutani swi nghenisiwa eka Durcupan ACM (Araldite casting resin M) epoxy resin (Electron Microscopy Sciences, catalog number 14040) exikarhi ka ti silicon-coated glass slides, naswona ekuheteleleni eka 60°C Polymerize eka oven kuringana 48 wa tiawara. Ndzhawu ya cerebellar cortex yi hlawuriwile naswona swiphemu swa 50 nm ultrathin swi tsemiwile eka Leica Ultracut (Leica Mikrosysteme GmbH, Vienna, Austria) naswona swi tshoveriwile eka 2×1 mm copper slit grid leyi pfanganisiweke hi polystyrene film. Swiphemu swi cheriwe hi solution ya 4% uranyl acetate eka H2O kuringana 10 wa timinete, swi hlantswiwa hi H2O minkarhi yo hlaya, kutani swi hlantswiwa hi Reynolds lead citrate eka H2O kuringana 10 wa timinete, kutani swi hlantswiwa hi H2O minkarhi yo hlaya. Ti micrographs ti tekiwile hi microscope ya electron yo hundzisela Philips CM100 (Thermo Fisher Scientific, Waltham, MA, USA) hiku tirhisa TVIPS (Tietz Video and Image Processing System) TemCam-F416 digital camera (TVIPS GmbH, Gauting, USA). Jarimani).
Eka timbeva leti tluleriweke hi AAV, byongo byi hambanisiwile kutani byi tsemiwa eka xiphemu xa sagittal xo enta xa 1 mm, naswona cerebellum yi kamberiwile hiku tirhisa microscope ya fluorescence ku kuma ring leyi tluleriweke hi AAV (hileswaku, mCherry expressing). Ku tirhisiwa ntsena swikambelo leswi eka swona ku cheriwa ka AAV ku endlaka leswaku ku va na vuswikoti bya le henhla swinene bya transduction bya leyara ya tisele ta Purkinje (i.e. kwalomu ka leyara hinkwayo) eka kwalomu ka swirhendzevutana swimbirhi leswi landzelelanaka swa cerebellar leswi tirhisiwaka. Loop leyi hundzuriweke hi AAV yi microdissected kuva yiva vusiku hinkwabyo endzhaku ka fixation (4% PFA na 2.5% glutaraldehyde eka 0.1 M cocoate buffer) naswona yitlhele yi lulamisiwa. Eka EPON embedding, tissue leyi nga cinciki yi hlantswiwile hi 0.1 M sodium cocoate buffer (Applichem), naswona yi pfaleriwile na 2% OsO4 (os, Science Services; Caco) eka 0.1 M sodium cocoate buffer (Applichem) 4 Hours, kutani yi hlantswiwa kuringana 2 wa tiawara. Phindha minkarhi ya 3 hi 0.1 M cocamide buffer. Endzhaku ka sweswo, nxaxamelo lowu tlhandlukaka wa ethanol wutirhisiwile ku pfalela solution yin’wana na yin’wana ya ethanol eka 4°C kuringana 15 wa timinete kuva ku susa mati eka tissue. Ti tissue yi hundziseriwe eka propylene oxide kutani yi pfaleriwa vusiku hinkwabyo eka EPON (Sigma-Aldrich) eka 4°C. Veka tissue eka EPON leyintshwa eka mahiselo ya kamara kuringana 2 wa tiawara, kutani u yi nghenisa eka 62°C kuringana 72 wa tiawara. Tirhisa ultramicrotome (Leica Microsystems, UC6) na banga ra dayimani (Diatome, Biel, Switzerland) ku tsema swiphemu swa 70 nm ultrathin, naswona u peta hi 1.5% uranyl acetate kuringana 15 wa timinete eka 37°C, naswona u peta hi lead citrate solution 4 wa timinete. Ti micrographs ta electron ti tekiwile hiku tirhisa JEM-2100 Plus transmission electron microscope (JEOL) leyi hlomisiweke hi Camera OneView 4K 16-bit (Gatan) na software ya DigitalMicrograph (Gatan). Ku kamberiwa, ti electron micrographs ti kumiwile hi 5000× kumbe 10,000× digital zoom.
Nxopaxopo wa xivumbeko xa mitochondria. Eka minxopaxopo hinkwayo, ti contours ta mitochondria yin’wana na yin’wana ti andlariwile hi voko eka swifaniso swa xidijitali hiku tirhisa software ya ImageJ. Ku xopaxopiwa tipharamitha to hambana ta xivumbeko. Ku tsindziyela ka mitochondria ku kombisiwa tanihi phesente leyi kumiweke hi ku avanyisa ndhawu hinkwayo ya mitochondrial ya sele yin’wana na yin’wana hi ndhawu ya cytoplasm (ndhawu ya cytoplasm = ndhawu ya sele-ndhawu ya nyutliyasi ya sele) × 100. Ku rhendzeleka ka mitochondria ku hlayeriwa hi fomula ya [4π∙(ndhawu/perimeter 2)]. Xivumbeko xa ista xa mitochondria xi kamberiwile kutani xi avanyisiwa hi swiyenge swimbirhi (“tubular” na “blister”) kuya hi swivumbeko swa swona leswikulu.
Nhlayo ya autophagosome/lysosome na nxopaxopo wa density. Tirhisa software ya ImageJ ku andlala hi voko ti contours ta autophagosome/lysosome yin’wana na yin’wana eka xifaniso xa xidijitali. Ndzhawu ya autophagosome/lysosome yi kombisiwa tani hi tiphesente leti hlayiweke hiku avanyisa ndzhawu hinkwayo ya xivumbeko xa autophagosome/lysosome xa sele yin’wana na yin’wana hi ndzhawu ya cytoplasm (ndhawu ya cytoplasm=ndhawu ya sele-ndhawu ya nyutliyasi)×100. Ku tsindziyela ka ti autophagosomes/lysosomes swi hlayeriwa hiku avanyisa nhlayo hinkwayo hi nhlayo ya swivumbeko swa autophagosome/lysosome hi sele (hi ku ya hi ndzhawu ya cytoplasmic) (cytoplasmic area = cell area-nuclear area).
Ku lebula ka acute sectioning xikan’we naku lulamisela xikombiso. Eka swikambelo leswi lavaka ku lebula ka glucose, hundzisela swilayi swa byongo bya acute eka kamara ra pre-incubation, leri nga na saturated carbon (95% O2 na 5% CO2), Ca2 + ACSF yale henhla (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 na 2.0 mM MgCl 2, leyi lulamisiweke kuya eka pH 7.4 na 310 kuya eka 320 mOsm), laha glucose yinga 13 C 6- Ku cinciwa ka glucose (Eurisotop, nomboro ya khathalogi CLM-1396). Eka swikambelo leswi lavaka ku lebula ka pyruvate, hundzisela swilayi swa byongo bya acute eka Ca2 + ACSF yale henhla (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 naswona Chela 2.0mM MgCl2, lulamisa kuya eka pH 7.4 na 310 kuya eka 320mOsm), naswona chela 1mM 1-[1-13C]pyruvate (Eurisotop, nomboro ya khathalogi CLM-1082). Hlayisa swiphemu kuringana 90 wa timinete eka 37°C. Eku heleni ka xikambelo, swiphemu swi hatlisile swi hlantswiwile hi aqueous solution (pH 7.4) leyinga na 75 mM ammonium carbonate, kutani swi homogenized eka 40:40:20 (v:v:v) acetonitrile (ACN): methanol: mati. Endzhaku kaloko swiphemu swi pfaleriwile eka ayisi kuringana 30 wa timinete, tisampulu ti centrifuged eka 21,000 g kuringana 10 wa timinete eka 4°C, naswona supernatant leyinga basa yi omisiwa eka SpeedVac concentrator. Pellet ya metabolite leyi omisiweke leyi humelelaka yi hlayisiwile eka -80°C kufikela loko yi kamberiwa.
Nxopaxopo wa liquid chromatography-mass spectrometry wa 13 wa ti amino acids leti leteriweke hi C. Eka nxopaxopo wa liquid chromatography-mass spectrometry (LC-MS), pellet ya metabolite yitlhele yi suspended eka 75μl ya mati ya LC-MS grade (Honeywell). Endzhaku ka centrifugation eka 21,000 g kuringana 5 wa timinete eka 4°C, 20 μl ya supernatant leyi hlawuriweke yitirhisiwile eka nxopaxopo wa amino acid flux, kasi leswin’wana hinkwaswo leswinga humesiwa switirhisiwile hiku hatlisa eka nxopaxopo wa anion (vona laha hansi). Nxopaxopo wa amino acid wu endliwile hi ku tirhisa protocol ya benzoyl chloride derivatization leyi hlamuseriweke khale ( 55 , 56 ). Eka goza rosungula, 10μl ya 100 mM sodium carbonate (Sigma-Aldrich) yi cheriwile eka 20μl ya metabolite extract, kutani 10μl ya 2% benzoyl chloride (Sigma-Aldrich) yi cheriwile eka LC grade ACN. Xikombiso xi vortexed swa xinkarhana kutani xi centrifuged eka 21,000 g kuringana 5 wa timinete eka 20°C. Tlhuvulela supernatant leyi basisiweke eka 2 ml ya autosampler vial leyingana conical glass insert (200 μl volume). Tisampulu ti kamberiwile hiku tirhisa Acquity iClass ultra-high performance LC system (Waters) leyi hlanganisiweke na Q-Exactive (QE)-HF (Ultra High Field Orbitrap) high-resolution precision mass spectrometer (Thermo Fisher Scientific). Eka nxopaxopo, 2μl ya xikombiso lexi derivatized yi cheriwile eka 100×1.0 mm high-strength silica T3 column (Mati) leyingana 1.8μm particles. Nhlayo ya ku khuluka i 100μl/min, naswona sisiteme ya buffer yina buffer A (10 mM ammonium formate na 0.15% formic acid ematini) na buffer B (ACN). Gradient yi le ka xiyimo lexi landzelaka: 0%B eka 0 wa timinete; 0%B. 0 kuya eka 15% B eka 0 kuya eka 0.1 wa timinete; 15 kuya eka 17% B eka 0.1 kuya eka 0.5 wa timinete; B eka 17 kuya eka 55% eka 0.5 kuya eka 14 wa timinete; B eka 55 kuya eka 70% eka 14 kuya eka 14.5 wa timinete; eka 14.5 kuya eka 70 kuya eka 100% B eka 18 wa timinete; 100% B eka 18 kuya eka 19 wa timinete; 100 kuya eka 0% B eka 19 kuya eka 19.1 wa timinete; 0% B eka 19.1 kuya eka 28 wa timinete (55, 56). QE-HF mass spectrometer yi tirha eka positive ionization mode na mass range ya m/z (mass/charge ratio) ya 50 kuya eka 750. Resolution leyi tirhisiweke i 60,000, naswona gain control (AGC) ion target leyi kumiweke i 3×106, naswona nkarhi wa ion lowukulu i 100 milliseconds. Xihlovo xa heated electrospray ionization (ESI) xi tirha eka voltage yo fafazela ya 3.5 kV, mahiselo ya capillary ya 250°C, sheath airflow ya 60 AU (arbitrary units), na moya wo pfuneta wa 20 AU. 250°C. Lens ya S yi vekiwile eka 60 AU.
Nxopaxopo wa anion chromatography-MS wa ti organic acids leti leteriweke hi 13C. Metabolite precipitate leyi saleke (55μl) yi kamberiwile hiku tirhisa Dionex ion chromatography system (ICS 5000+, Thermo Fisher Scientific) leyi hlanganisiweke na QE-HF mass spectrometer (Thermo Fisher Scientific). Hiku komisa, 5μl ya metabolite extract yi cheriwile eka Dionex IonPac AS11-HC column leyi hlomisiweke hi HPLC (2 mm×250 mm, particle size 4μm, Thermo Fisher Scientific) eka push-in partial loop mode leyingana filling ratio ya 1. )on. Kholomo ya varindzi ya Dionex IonPac AG11-HC (2 mm x 50 mm, 4μm, Thermo Fisher Scientific). Mahiselo ya kholomo ya hlayisiwa eka 30°C, naswona autosampler yi vekiwa eka 6°C. Tirhisa potassium hydroxide cartridge leyi nyikiwaka mati lawa yanga deionized kuva u humesa potassium hydroxide gradient hiku tirhisa eluent generator. Ku hambanisiwa ka ti metabolites eka mpimo wa ku khuluka wa 380μl/min, ku cheriwa gradient leyi landzelaka: 0 kuya eka 3 wa timinete, 10 mM KOH; 3 kuya eka 12 wa timinete, 10 kuya eka 50 mM KOH; 12 kuya eka 19 wa timinete, 50 kuya eka 100 mM KOH; 19 ku ya eka 21 wa timinete , 100 mM KOH; 21 kuya eka 21.5 wa timinete, 100 kuya eka 10 mM KOH. Kholomo yitlhele yi ringanisiwa ehansi ka 10 mM KOH kuringana 8.5 wa timinete.
Ti metabolites leti eluted ti hlanganisiwa na 150μl/min isopropanol supplement stream endzhaku ka kholomo kutani ti kongomisiwa eka high-resolution mass spectrometer leyi tirhaka eka negative ionization mode. MS yi veka tihlo eka mass range kusuka eka m/z 50 kuya eka 750 hi resolution ya 60,000. AGC yi vekiwile eka 1×106, naswona nkarhi wa ion lowukulu wu hlayisiwa eka 100 ms. Xihlovo xa ESI lexi hisiweke xi tirhisiwile hi voltage yo fafazela ya 3.5 kV. Swiyimiso swin’wana swa xihlovo xa ion hi leswi landzelaka: mahiselo ya capillary 275°C; ku khuluka ka gasi ya xifunengeto, 60 AU; ku khuluka ka gasi yo pfuneta, 20 AU eka 300°C, na ku vekiwa ka S lens eka 60 AU.
Nxopaxopo wa datha ya ti metabolites leti leteriweke hi 13C. Tirhisa software ya TraceFinder (version 4.2, Thermo Fisher Scientific) eka nxopaxopo wa data ya isotope ratio. Ku tiviwa ka nkatsakanyo wun’wana na wun’wana ku tiyisisiwile hi nkatsakanyo wa ndzavisiso lowu tshembekaka naswona wu kamberiwile hi ku tiyimela. Leswaku ku endliwa nxopaxopo wa ku fuwisa isotope, ndhawu ya chromatogram ya ion leyi humesiweke (XIC) ya isotope yin’wana na yin’wana ya 13C (Mn) yi humesiwile eka [M + H] +, laha n ku nga nomboro ya khaboni ya nkatsakanyo lowu kongomisiweke, lowu tirhisiwaka ku xopaxopa ti-amino acid kumbe [ MH] + yi tirhisiwa ku xopaxopa ti-anion. Ku lulama ka vukulu bya XIC i ehansi ka swiphemu swa ntlhanu hi miliyoni, naswona ku pakanisa ka RT i 0.05 wa timinete. Nxopaxopo wa ku fuwisa wu endliwa hi ku hlayela mpimo wa isotope yin’wana na yin’wana leyi kumiweke eka nhlayo ya ti isotope hinkwato ta nkatsakanyo lowu fambelanaka. Ti ratios leti ti nyikiwa tani hi tiphesente ta mimpimo eka isotope yin’wana na yin’wana, naswona mbuyelo wu kombisiwa tani hi molar percent enrichment (MPE), tani hileswi swi hlamuseriweke khale (42).
Frozen neuron pellet yi homogenized eka ice-cold 80% methanol (v/v), yi vortexed, naswona yi pfaleriwile eka -20°C kuringana 30 wa timinete. Vortex xikombiso nakambe kutani u hlanganisa eka +4°C kuringana 30 wa timinete. Xikombiso xi centrifuged eka 21,000 g kuringana 5 wa timinete eka 4°C, kutani supernatant leyi humelelaka yi hlengeletiwile kutani yi omisiwa hiku tirhisa SpeedVac concentrator eka 25°C kuva yi endliwa nxopaxopo lowu landzelaka. Tani hilaha swi hlamuseriweke hakona laha henhla, nxopaxopo wa LC-MS wu endliwile eka ti amino acids ta ti cells leti hleriweke. Hiku tirhisa TraceFinder (version 4.2, Thermo Fisher Scientific), nxopaxopo wa data wu endliwile hiku tirhisa monoisotopic mass ya compound yin’wana na yin’wana. Quantile normalization ya metabolite data yi endliwile hi ku tirhisa preprocessCore software package ( 57 ).
Ku lulamisiwa ka swilayi. Mbeva yi hatlisile yi anesthetized hi carbon dioxide kutani yi tsemiwa nhloko, byongo byi hatlisile byi susiwa eka khwiri, naswona banga leri rhurhumelaka leri taleke hi ayisi (HM-650 V, Thermo Fisher Scientific, Walldorf, Germany) ri tirhisiwile ku yi tsemiwa yiva 300 kuya eka 375 μm ya swiphemu swa sagittal Cold carbon gasification (95% O2 na 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 na 6.0 mM MgCl2 Lulamisa eka pH 7.4 na 310 kuya eka 330 mOsm). Tlhuvulela swilayi swa byongo leswi kumiweke eka kamara leri nga na Ca2 + ACSF yale henhla (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 na mM 3.5 MgCl2) pH 7.4 na 310 ku ya eka 320 mOsm). Hlayisa swilayi kuringana 20 kuya eka 30 wa timinete leswaku swita vuyiseriwa loko swinga se rhekhoda.
ku rhekhoda. Xiteji xa microscope lexi hlomisiweke hi kamara ro rhekhoda leri nga cinciki na 20x water immersion objective lens (Scientifica) switirhisiwile eka ku rhekhoda hinkwako. Ti cells ta Purkinje leti ehleketiwaka ti tiviwile hi (i) vukulu bya miri, (ii) ndzhawu ya anatomical ya cerebellum, na (iii) ku paluxiwa ka fluorescent mtYFP reporter gene. Pipette ya patch leyingana tip resistance ya 5 kuya eka 11 megohms yi hoxiwa hi borosilicate glass capillary (GB150-10, 0.86 mm×1.5 mm×100 mm, Science Products, Hofheim, Germany) na pipette leyi nga horizontal Instruments (P-1000, Sutter), Novato, CA). Ku rhekhodiwa hinkwako ku endliwile hi ELC-03XS npi patch clamp amplifier (npi electronic GmbH, Tam, Germany), leyi a yi lawuriwa hi software ya Signal (version 6.0, Cambridge Electronic, Cambridge, UK). Xikambelo lexi xi rhekhodiwile hi mpimo wa sampling wa 12.5 kHz. Xigingi xi sefiwile hi ti short-pass Bessel filters timbirhi leti nga na ti cutoff frequencies ta 1.3 na 10 kHz hi ku landzelelana. Capacitance ya membrane na pipette yi tlheriseriwa hi compensation circuit hiku tirhisa amplifier. Swikambelo hinkwaswo swi endliwile ehansi ka vulawuri bya khamera ya Orca-Flash 4.0 (Hamamatsu, Gerden, Germany), leyi a yi lawuriwa hi software ya Hokawo (version 2.8, Hamamatsu, Gerden, Germany).
Ku lulamisiwa ka sele hinkwayo ya ntolovelo na nxopaxopo. Hi ku hatlisa loko u nga si rhekhoda, tata pipette hi xitofu xa le ndzeni lexi nga na swilo leswi landzelaka: 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) na 10.0 mM creatinine phosphate yi lulamisiwile kuya eka pH 7.25, naswona ntshikelelo wa osmotic awuri 290 mOsm (sucrose). Hiku hatlisa endzhaku ko tirhisa matimba ya 0 pA kuva ku tshoveka membrane, vuswikoti bya membrane yo wisa byi pimiwe. Ku tiya ka input ku pimiwa hiku tirhisa ti hyperpolarized currents ta -40, -30, -20, na -10 pA. Pima vukulu bya nhlamulo ya voltage kutani u tirhisa nawu wa Ohm ku hlayela ku lwisana ka input. Ntirho lowu tiendlekelaka wu rhekhodiwile eka voltage clamp kuringana 5 wa timinete, naswona sPSC yi tiviwile no pimiwa eka Igor Pro (version 32 7.01, WaveMetrics, Lake Oswego, Oregon, USA) hiku tirhisa semi-automatic recognition script. IV curve na steady-state current swi pimiwa hiku clamping battery eka ti potentials tohambana hambana (kusukela eka -110 mV) naku engetela voltage hi magoza ya 5 mV. Ku endliwa ka AP swikamberiwile hiku tirhisa depolarizing current. Clamp cell eka -70 mV loko u ri karhi u tirhisa depolarizing current pulse. Lulamisa mpimo wa goza ra yuniti yin’wana na yin’wana yo rhekhoda hi ku hambana (10 ku ya eka 60 pA). Hlela frequency ya AP yale henhla hiku hlayela hi voko ti pulse spikes leti vangaka frequency yale henhla ya AP. Threshold ya AP yi xopaxopiwa hi ku tirhisa derivative ya vumbirhi ya depolarization pulse leyi rhanga yi hlohlotela AP yin’we kumbe ku tlula.
Ku lulamisiwa ka patch leyi pfulekeke na nxopaxopo. Endla ku rhekhoda ka patch leyi pfulekeke hi ku tirhisa tiphrothokholi ta ntolovelo. Tirhisa pipette leyi nga riki na ATP na GTP leyi nga riki na switirhisiwa leswi landzelaka: 128 mM gluconate K, 10 mM KCl, 10 mM Hepes, 0.1 mM EGTA na 2 mM MgCl2, naswona u lulamisa eka pH 7.2 (hi ku tirhisa KOH). ATP na GTP swi tshikiwa eka intracellular solution ku sivela ku nghenelela loku nga lawuriwiki ka cell membrane. Pipette ya patch yi tatiwa hi solution yale ndzeni leyingana amphotericin (kwalomu ka 200 kuya eka 250 μg/ml; G4888, Sigma-Aldrich) kuva ku kumiwa rhekhodo ya patch leyi pfulekeke. Amphotericin yi pfanganisiwile na dimethyl sulfoxide (nhlayo yohetelela: 0.1 kuya eka 0.3%; DMSO; D8418, Sigma-Aldrich). Nhlayo ya DMSO leyi tirhisiweke a yi nga ri na vuyelo lebyikulu eka tinyiyoni leti dyondziweke. Hi nkarhi wa punching process, channel resistance (Ra) ayi tshama yi langutiwa, naswona experiment yisunguriwile endzhaku kaloko amplitude ya Ra na AP yi tshamisekile (20-40 minutes). Ntirho lowu tiendlekelaka wu pimiwa eka voltage na/kumbe current clamp kuringana 2 kuya eka 5 wa timinete. Nxopaxopo wa datha wu endliwile hi ku tirhisa Igor Pro (vuhundzuluxi bya 7.05.2, WaveMetrics, USA), Excel (vuhundzuluxi bya 2010, Microsoft Corporation, Redmond, USA) na GraphPad Prism (vuhundzuluxi bya 8.1.2, GraphPad Software Inc., La Jolla, CA). United States). Leswaku ku kumiwa ti-AP leti ti tiendlekelaka, ku tirhisiwa plug-in ya IgorPro ya NeuroMatic v3.0c. Hlawula hi ku tisungulela ti-AP hi ku tirhisa threshold leyi nyikiweke, leyi lulamisiwaka hi yoxe eka rhekhodo yin’wana na yin’wana. Hiku tirhisa nkarhi wa spike, kumisisa frequency ya spike na frequency ya spike ya xihatla ya xiyimo xale henhla na frequency ya spike ya xikarhi.
PN ku tihambanisa. Hi ku pfumelelana na protocol leyi kandziyisiweke khale, PNs yi basisiwile kusuka eka cerebellum ya mbeva eka xiteji lexi vekiweke ( 58 ). Hiku komisa, cerebellum yi tsemiwile no tsemiwa eka ice-cold dissociation medium [handle ka HBSS Ca2+ na Mg2+, yi engeteriwile hi 20 mM glucose, penicillin (50 U/ml) na streptomycin (0.05 mg/ ml)], kutani yi gayela medium eka papain [HBSS, leyi engeteriweke hi 1-cysteine·HCl (1 mg / ml), papain (16 U / ml) na deoxyribonuclease I (DNase I; 0.1 mg/ml)] Titshungula kuringana 30 wa timinete eka 30°C. Xosungula hlamba ti tissues eka HBSS medium leyingana egg mucus (10 mg/ml), BSA (10 mg/ml) na DNase (0.1 mg/ml) eka mahiselo ya kamara ku sivela ku gayiwa ka enzymatic, kutani eka HBSS medium leyingana 20 mM glucose Hiku sila hiku olova eka HBSS, penicillin (50 U/ml), streptomycin (0.05 mg/ml) na DNase (0.1 mg/ml) yi humesa ti cells tin’we. Ku yimisiwa ka ti cells loku humelelaka ku sefiwile hi 70μm cell strainer, kutani ti cells ti pelleted hi centrifugation (1110 rpm, 5 minutes, 4°C) kutani ti tlhela ti suspended eka sorting medium [HBSS, leyi engeteriweke hi 20 mM glucose, 20% fetal bovine ) Serum, penicillin (50 U/ml) na streptomycin (0,05 mg/ml)]; kambela ku hanya ka ti cell hi propidium iodide naku lulamisa nhlayo ya ti cells kuya eka 1×106 kuya eka 2×106 cells/ml. Loko kungase endliwa flow cytometry, suspension yi sefiwile hiku tirhisa 50 μm cell strainer.
Cytometer ya ku khuluka. Ku hlawuriwa ka ti cell swi endliwile eka 4°C hiku tirhisa muchini wa FACSAria III (BD Biosciences) na software ya FACSDiva (BD Biosciences, version 8.0.1). Ku yimisiwa ka ti cell swi hlawuriwile hiku tirhisa 100 μm nozzle ehansi ka ntshikelelo wa 20 psi hi rivilo ra ~2800 events/sec. Tanihi leswi swipimelo swa ndhavuko swa geting (vukulu bya tisele, xihlawuhlawu xa bimodal, na swihlawulekisi swa ku hangalaka) swi nga tiyisisi ku hambanisiwa lokunene ka PN eka tinxaka tin’wana ta tisele, maqhinga ya geting ya vekiwile hi ku ya hi ku pimanisiwa ko kongoma ka matimba ya YFP na autofluorescence eka mitoYFP+ ​​na mitoYFP − Mice ya vulawuri. YFP yi tsakisiwa hiku irradiating xikombiso hi 488 nm laser line, naswona signal yi voniwa hiku tirhisa 530/30 nm band pass filter. Eka ti mice ta mitoYFP+, matimba yo ringana ya Rosa26-mitoYFP reporter gene ya tlhela ya tirhisiwa ku hambanyisa miri wa ti neuronal na swiphemu swa axon. 7-AAD yi tsakisiwa hi 561 nm yellow laser naswona yi kumiwile hi 675/20 nm bandpass filter ku susa ti cells leti feke. Leswaku ku hambanisiwa ti astrocytes hi nkarhi wun’we, cell suspension yi pfaleriwile hi ACSA-2-APC, kutani xikombiso xi cheriwile hi 640 nm laser line, naswona 660/20 nm bandpass filter yitirhisiwile ku kuma xikombiso.
Ti cells leti hlengeletiweke ti pelleted hi centrifugation (1110 rpm, 5 minutes, 4°C) naswona ti hlayisiwile eka -80°C kufikela loko titirhisiwa. Mfn2cKO mice na ti litter pups ta tona ti hlayiwa hi siku rin’we ku hunguta ku cinca cinca ka maendlelo. Ku nyikiwa ka datha ya FACS na nxopaxopo swi endliwile hi ku tirhisa software ya FlowJo (FlowJo LLC, Ashland, Oregon, USA).
Hilaha swi boxiweke hakona laha henhla (59), PCR ya nkarhi wa xiviri yi tirhisiwa ku hambanyisa DNA eka tinyiyoni leti hleriweke ku endlela ku pima nhlayo ya mtDNA leyi landzelaka. Linearity na threshold sensitivity ekusunguleni swikamberiwile hiku tirhisa qPCR eka tinomboro tohambana hambana ta ti cells. Hiku komisa, hlengeleta 300 PN eka lysis buffer leyingana 50 mM tris-HCl (pH 8.5), 1 mM EDTA, 0.5% Tween 20 na proteinase K (200 ng/ml) kutani u pfaleriwa eka 55°C 120 wa timinete. Ti cells titlhele ti pfaleriwa eka 95°C kuringana 10 wa timinete kutiyisisa ku nga tirhi hiku hetiseka ka proteinase K. Hiku tirhisa TaqMan probe (Thermo Fisher) leyi kongomeke eka mt-Nd1, mtDNA yi pimiwe hi semi-quantitative PCR eka 7900HT Real-Time PCR system (Thermo Fisher Scientific). Sayense, nomboro ya khathalogi Mm04225274_s1), mt-Nd6 (Thermo Fisher Scientific, nomboro ya khathalogi AIVI3E8) na 18S (Thermo Fisher Scientific, nomboro ya khathalogi Hs99999901_s1) tijini.
Ku lulamisiwa ka xikombiso xa proteome. Hiku hisa solution eka 95°C kuringana 10 wa timinete naku sonicating, eka lysis buffer [6 M guanidine chloride, 10 mM tris(2-carboxyethyl) phosphine hydrochloride, 10 mM chloroacetamide na 100 mM tris- Lyse frozen neuron pellets eka HCl]. Eka Bioruptor (Diagenode) kuringana 10 wa timinete (30 wa tisekoni pulse / 30 wa tisekoni pause period). Xikombiso xi cheriwile 1:10 eka 20 mM tris-HCl (pH 8.0), xi hlanganisiwa na 300 ng trypsin gold (Promega), naswona xi pfaleriwile vusiku hinkwabyo eka 37°C kuva ku fikeleriwa ku gayiwa loku heleleke. Hi siku ravumbirhi, xikombiso xi centrifuged eka 20,000 g kuringana 20 wa timinete. Supernatant yi cheriwile hi 0.1% formic acid, naswona solution yi susiwile munyu hi StageTips leyi tiendleke yona. Xikombiso xi omisiwa eka xitirhisiwa xa SpeedVac (Eppendorf concentrator plus 5305) eka 45°C, kutani peptide yi yimisiwile eka 0.1% formic acid. Tisampulu hinkwato ti lulamisiwile hi nkarhi wun’we hi munhu un’we. Ku kota ku xopaxopa swikombiso swa astrocyte, 4 μg ya ti peptides letinga susiwa munyu ti lebuliwile hi tandem mass tag (TMT10plex, nomboro ya catalog 90110, Thermo Fisher Scientific) na peptide kuya eka TMT reagent ratio ya 1:20. Eka ku lebula ka TMT, 0.8 mg ya TMT reagent yitlhele yi suspended eka 70 μl ya anhydrous ACN, naswona peptide leyi omisiweke yi vumbiwile nakambe kuva 9 μl ya 0.1 M TEAB (triethylammonium bicarbonate), laha 7 μl ya TMT reagent eka ACN yinga cheriwa. Nhlayo ya yona a yi ri 43.75%. Endzhaku ka 60 wa timinete ta ku pfaleriwa, reaction yi timiwile hi 2 μl ya 5% hydroxylamine. Ti peptides letinga lebuliwa ti hlengeletiwile, ti omisiwa, titlhela ti suspended eka 200μl ya 0.1% formic acid (FA), ti avanyisiwile hi timbirhi, kutani ti susiwa munyu hiku tirhisa ti StageTips leti ti endleke toxe. Hiku tirhisa UltiMate 3000 ultra high performance liquid chromatograph (UltiMate 3000 ultra high performance liquid chromatograph), yin’wana ya ti hafu timbirhi yi hambanisiwile eka kholomo ya 1mm x 150mm Acquity chromatographic leyi tatiweke hi 130Å1.7μm C18 particles (Waters, catalog No. SKU: 186006935). Thermo Fisher Vutivi bya Sayense). Hluvula ti peptides hi flow rate ya 30μl/min, hambanyisa kusuka eka 1% kuya eka 50% buffer B kuringana 85 wa timinete hi stepwise gradient ya 96 wa timinete, kusuka eka 50% kuya eka 95% buffer B kuringana 3 wa timinete, kutani 8 wa timinete eka 95 % Buffer B; Buffer A i 5% ACN na 10 mM ammonium bicarbonate (ABC), naswona buffer B i 80% ACN na 10 mM ABC. Hlengeleta swiphemuphemu endzhaku ka timinete tin’wana na tin’wana ta 3 kutani u swihlanganisa swiva mintlawa yimbirhi (1 + 17, 2 + 18, na swin’wana) kutani u swi omisa eka vacuum centrifuge.
Nxopaxopo wa LC-MS/MS. Eka mass spectrometry, ti peptides (nomboro r119.aq) ti hambanisiwile eka 25 cm, 75 μm inner diameter PicoFrit analytical column (lens leyintshwa ya xikongomelo, nomboro ya xiphemu PF7508250) leyi hlomisiweke hi 1.9 μm ReproSil-Pur 120 C18-AQ medium (Dr. Maisch, mat), tirhisa EASY-nLC 1200 (Thermo Fisher ya Sayense, eJarimani). Kholomo yi hlayisiwile eka 50°C. Ti buffers A na B i 0.1% wa formic acid ematini na 0.1% wa formic acid eka 80% ACN, hiku landzelelana. Ti peptides ti hambanisiwile hi stepwise gradient ya 200 nl/min kusuka eka 6% kuya eka 31% buffer B kuringana 65 wa timinete, na kusuka eka 31% kuya eka 50% buffer B kuringana 5 wa timinete. Ti peptides letinga eluted ti kamberiwile eka Orbitrap Fusion mass spectrometer (Thermo Fisher Scientific). Ku pimiwa ka peptide precursor m/z ku endliwa hi xiboho xa 120,000 eka nhlayo ya 350 kuya eka 1500 m/z. Hiku tirhisa 27% normalized collision energy, precursor yo tiya swinene leyingana charge state ya 2 kuya eka 6 yi hlawuriwa eka high energy C trap dissociation (HCD) cleavage. Nkarhi wa xirhendzevutani wu vekiwile eka 1 s. Nhlayo ya m/z ya xiphemu xa peptide yi pimiwe eka ion trap hiku tirhisa xikongomelo lexintsongo xa AGC xa 5×104 na nkarhi lowukulu wa injection wa 86 ms. Endzhaku ka ku hambanisiwa, precursor yi vekiwile eka nxaxamelo wa dynamic exclusion kuringana 45 s. Ti peptides leti leteriweke hi TMT ti hambanisiwile eka kholomo ya 50 cm, 75 μm ya Acclaim PepMap (Thermo Fisher Scientific, nomboro ya catalog 164942), naswona ti migration spectra ti kamberiwile eka Orbitrap Lumos Tribrid mass spectrometer (Thermo Fisher Scientific) leyi hlomisiweke hi switirhisiwa swa high-field asymmetric waveform ions (FAIMS). (Thermo Fisher Scientific) yi tirha eka ti voltage timbirhi ta compensation ta −50 na −70 V. MS3 leyi hlawuriweke hi ku ya hi synchronization precursor yi tirhisiwa eka ku pima ka xigingi xa ion xa xiviko xa TMT. Ku hambanisiwa ka peptide swiendliwile eka EASY-nLC 1200, hiku tirhisa 90% linear gradient elution, na buffer concentration ya 6% kuya eka 31%; buffer A a yi ri 0.1% FA, naswona buffer B a yi ri 0.1% FA na 80% ACN. Kholomo ya analytical yi tirhisiwa eka 50°C. Tirhisa FreeStyle (version 1.6, Thermo Fisher Scientific) ku avanyisa fayili yosungula kuya hi voltage ya compensation ya FAIMS.
Ku tivisiwa ka tiphrotheyini na ku pima nhlayo. Hi ku tirhisa njhini yo lavisisa ya Andromeda leyi hlanganisiweke, datha yo sungula yi kamberiwile hi ku tirhisa MaxQuant version 1.5.2.8 (https://maxquant.org/). Ku engetela eka ntlhandlamano wa Cre recombinase na YFP lowu kumiweke kusuka eka Aequorea victoria, ti peptide fragment spectra ti lavisisiwile eka ntlhandlamano wa canonical na ntlhandlamano wa isoform wa proteome ya reference ya mbeva (Proteome ID UP000000589, leyi dawunilodiweke kusuka eka UniProt hi May 2017) . Methionine oxidation na protein N-terminal acetylation swi vekiwile tani hi ku cinca loku cinca-cincaka; cysteine ​​carbamoyl methylation yi vekiwile tanihi ku cinca loku nga cinciki. Ti parameters ta ku gayiwa ka swakudya ti vekiwa eka “specificity” na “trypsin/P”. Nhlayo ya le hansi ya ti peptides na ti razor peptides leti tirhisiwaka ku tiveka ka ti protein i 1; nhlayo ya le hansi ya ti peptides to hlawuleka i 0. Ehansi ka swiyimo swa ku fambelanisiwa ka mepe wa ti peptide, mpimo wa ku tiviwa ka tiphrotheyini a wu ri 0.01. Nhlawulo wa “Second Peptide” wu tirhisiwile. Tirhisa nhlawulo wa “match between runs” ku hundzisela swihlawulekisi leswi humelelaka exikarhi ka tifayela to hambana ta masungulo. Tirhisa LFQ minimum ratio count 1 eka ku pima nhlayo leyi nga riki na malebvu (LFQ) (60). Ntshikelelo wa LFQ wu sefiwile eka kwalomu ka mimpimo yimbirhi leyi faneleke eka kwalomu ka ntlawa wun’we wa genotype eka nkarhi wun’wana na wun’wana, naswona wu humesiwa kusuka eka ku hangalasiwa ka ntolovelo na ku anama ka. 0.3 naswona u ya ehansi 1.8. Tirhisa pulatifomo ya khomphyuta ya Perseus (https://maxquant.net/perseus/) na R (https://r-project.org/) ku xopaxopa mimbuyelo ya LFQ. Xikambelo xa t yo ringanela ya tindlela timbirhi ku suka eka phakeji ya software ya limma xi tirhisiwile eka nxopaxopo wa ku paluxiwa ka ku hambana ( 61 ). Nxopaxopo wa datha ya vukambisisi wu endliwa hi ku tirhisa ggplot, FactoMineR, factoextra, GGally na pheatmap. Data ya proteomics leyi simekiweke eka TMT yi kamberiwile hi ku tirhisa MaxQuant version 1.6.10.43. Ku lavisisa datha ya proteomics yo ka yi nga swekiwanga ku suka eka database ya proteomics ya vanhu ya UniProt, leyi dawunilodiweke hi Ndzati 2018. Nxopaxopo wu katsa xivangelo xo lulamisa ku basa ka isotope lexi nyikiweke hi muendli. Tirhisa limma eka R eka nxopaxopo wa ku paluxiwa ka ku hambana. Data yo sungula, mimbuyelo ya ku lavisisa ya database, na mafambelo ya ntirho ya nxopaxopo wa datha na mimbuyelo hinkwaswo swi hlayisiwile eka ntwanano wa ProteomeXchange hi ku tirhisa vuhlayiselo bya mutirhisani wa PRIDE na xihlawulekisi xa sete ya datha PXD019690.
Tinhlamuselo ta ntirho ti fuwisa nxopaxopo. Xitirhisiwa xa Ingenuity Pathway Analysis (QIAGEN) xi tirhisiwile ku kumisisa ku fuwa ka swipimelo swa nhlamuselo ya ntirho ya sete ya datha eka 8 wa mavhiki (Xifaniso 1). Hiku komisa, nxaxamelo wa ti protein ta nhlayo lowu kumiweke kusuka eka nxopaxopo wa data wa LC-MS/MS (tandem mass spectrometry) wutirhisiwa na swipimelo swa sefo leswi landzelaka: Mus musculus yi hlawuriwa tani hi muxaka na ndzhaku, naswona xiyenge xikombisa nhlayo ya P leyi lulamisiweke hi Benjamini eka ku fuwa ka 0.05 kumbe ehansi yi tekiwa tani hi ya nkoka. Eka girafu leyi, swiyenge swa ntlhanu swa le henhla leswi tlulaka mpimo eka xitluletsongo xin’wana na xin’wana leswi simekiweke eka ntikelo wa P lowu lulamisiweke swa kombisiwile. Hi ku tirhisa multiple t-test, hi ku tirhisa nongonoko wa switeji swimbirhi wa linear boost wa Benjamini, Krieger, na Yekutieli (Q = 5%), nxopaxopo wa time-course protein expression wu endliwa eka vahlawuriwa va nkoka lava hlawuriweke eka xiyenge xin’wana na xin’wana, naswona layini yin’wana na yin’wana yi xopaxopiwa hi ku hambana. A ku na xilaveko xo amukela SD leyi nga cincekiki.
Leswaku hi pimanisa mbuyelo wa ndzavisiso lowu na tidathabeyisi leti kandziyisiweke na ku tumbuluxa xifaniso xa Venn eka Xifaniso 1, hi hlanganisile nxaxamelo wa tiphrotheyini ta nhlayo na tinhlamuselo ta MitoCarta 2.0 ( 24 ). Tirhisa xitirhisiwa xa inthanete xa Draw Venn Diagram (http://bioinformatics.psb.ugent.be/webtools/Venn/) ku tumbuluxa xifaniso.
Ku kuma vuxokoxoko bya vuxokoxoko bya maendlelo ya tinhlayo lama tirhisiwaka eka nxopaxopo wa proteomics, hi kombela u languta eka xiyenge lexi fambelanaka xa Switirhisiwa na Maendlelo. Eka swikambelo swin’wana hinkwaswo, rungula leri nga ni vuxokoxoko ri nga kumeka eka ntsheketo lowu fambisanaka na wona. Handle ka loko swi boxiwile hi ndlela yin’wana, datha hinkwayo yi kombisiwile tanihi xiringaniso ± SEM, naswona minxopaxopo hinkwayo ya tinhlayo yi endliwile hi ku tirhisa sofwere ya GraphPad Prism 8.1.2.
Ku kuma switirhisiwa swo engetela swa xihloko lexi, hi kombela u vona http://advances.sciencemag.org/cgi/content/full/6/35/eaba8271/DC1
Leyi i atikili ya mfikelelo lowu pfulekeke leyi hangalasiweke ehansi ka swipimelo swa Layisense ya Creative Commons Attribution-Non-Commercial, leyi pfumelelaka ku tirhisiwa, ku hangalasiwa na ku humesiwa nakambe eka xitirhisiwa xihi kumbe xihi, ntsena loko ku tirhisiwa ko hetelela ku nga ri ku vuyeriwa ka mabindzu naswona xisekelo xa leswaku ntirho wo sungula wu lulamile. Rheferense.
Xiya: Hi kombela ntsena ku nyika adirese ya wena ya email leswaku munhu loyi u n’wi bumabumelaka eka pheji a swi tiva leswaku u lava leswaku a vona email naswona a hi spam. A hi nge khomi tiadirese tihi na tihi ta imeyili.
Xivutiso lexi xi tirhisiwa ku kambela loko u ri muendzi na ku sivela ku rhumeriwa ka swipamu hi ku tisungulela.
Hi E. Motori, I. Atanassov, S. M. V. Kochan, K. Folz-Donahue, V. Sakthivelu, P. Giavalisco, N. Toni, J. Puyal, N.-G. Larson
Nxopaxopo wa tiproteomics wa tinyiyoni leti nga tirhiki kahle wu paluxe leswaku minongonoko ya metaboliki ya tirhisiwa ku lwisana ni ku onhaka ka misiha.
Hi E. Motori, I. Atanassov, S. M. V. Kochan, K. Folz-Donahue, V. Sakthivelu, P. Giavalisco, N. Toni, J. Puyal, N.-G. Larson
Nxopaxopo wa tiproteomics wa tinyiyoni leti nga tirhiki kahle wu paluxe leswaku minongonoko ya metaboliki ya tirhisiwa ku lwisana ni ku onhaka ka misiha.
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