Okwangoku *Idilesi yangoku: Cologne 50931, eJamani, iCologne Excellence Cluster Research on Cellular Stress Response in Aging-related Diseases (CECAD).
Ukuwohloka kwemithambo-luvo kwezifo ze-mitochondrial kuthathwa njengokungalungisekiyo kuba i-metabolic plasticity yee-neurons incinci, kodwa isiphumo sokungalungelelani kwe-mitochondrial kwi-cell autonomy ye-neuronal metabolism emzimbeni asiqondwa kakuhle. Apha, sazisa i-proteome yeseli ethile yee-neurons zePurkinje ene-progressive OXPHOS deficiency ebangelwa kukuphazamiseka kwe-mitochondrial fusion dynamics. Sifumanise ukuba ukungasebenzi kakuhle kwe-mitochondrial kubangele utshintsho olukhulu kwicandelo le-proteomics, ekugqibeleni okukhokelele ekusebenzeni ngokulandelelana kweenkqubo ze-metabolic ezichanekileyo ngaphambi kokufa kweseli. Ngokungalindelekanga, sifumanise ukungeniswa okucacileyo kwe-pyruvate carboxylase (PCx) kunye nezinye ii-enzymes zokulwa nokuguga ezongeza ii-intermediates zomjikelo we-TCA. Ukuthintelwa kwe-PCx kubangele uxinzelelo lwe-oxidative kunye ne-neurodegeneration, okubonisa ukuba i-atherosclerosis inefuthe lokukhusela kwii-neurons ezingenayo i-OXPHOS. Ukubuyiselwa kwe-mitochondrial fusion kwii-neurons eziwohloka ngokupheleleyo kuguqula ngokupheleleyo ezi mpawu ze-metabolic, ngaloo ndlela kuthintele ukufa kweseli. Iziphumo zethu zichonga iindlela ezazingaziwa ngaphambili ezinika ukuqina kokungasebenzi kakuhle kwe-mitochondrial kwaye zibonisa ukuba i-neurodegeneration inokuguqulwa kwanakwizigaba zokugqibela zesifo.
Indima ephambili ye-mitochondria ekugcineni imetabolism yamandla e-neuronal igxininiswa ziimpawu ezininzi ze-neurological ezinxulumene nezifo ze-mitochondrial zabantu. Uninzi lwezi zifo lubangelwa kukuguquka kwezakhi zofuzo okulawula ukubonakaliswa kwezakhi zofuzo ze-mitochondrial (1, 2) okanye ukutshatyalaliswa kwezakhi zofuzo ezinxulumene ne-mitochondrial dynamics, ezichaphazela ngokungathanga ngqo ukuzinza kwe-mitochondrial DNA (mtDNA) (3, 4). Umsebenzi kwiimodeli zezilwanyana ubonise ukuba ukuphendula ukungasebenzi kakuhle kwe-mitochondrial kwizicubu ezijikelezileyo, iindlela ze-metabolism ezigcinayo (5-7) zingasetyenziswa, nto leyo ebonelela ngolwazi olubalulekileyo lokuqonda ngokunzulu i-pathogenesis yezi zifo zintsonkothileyo. Ngokwahlukileyo koko, ukuqonda kwethu utshintsho lwe-metabolism kwiintlobo ezithile zeeseli olubangelwa kukusilela ngokubanzi kwemveliso ye-brain mitochondrial adenosine triphosphate (ATP) kubalulekile (8), kugxininisa imfuneko yokuchonga iinjongo zonyango ezinokusetyenziselwa ukuthintela okanye ukuthintela izifo. Ukuthintela ukuwohloka kwe-neuro (9). Ukunqongophala kolwazi kukuba iiseli zemithambo-luvo zithathwa ngokubanzi njengezinokuguquguquka okuncinci kakhulu kwe-metabolism xa kuthelekiswa neentlobo zeeseli zezicubu ezijikelezileyo (10). Njengoko ezi seli zidlala indima ephambili ekuququzeleleni ukuhanjiswa kwee-metabolites kwii-neurons ukukhuthaza ukudluliselwa kwe-synaptic kunye nokuphendula kwiimeko zokwenzakala kunye nezifo, amandla okulungelelanisa imetabolism yeseli kwiimeko ezinzima zezicubu zobuchopho phantse aphelele kwiiseli ze-glial (11-14). Ukongeza, ukungafani kweeseli zezicubu zobuchopho kuthintela kakhulu ukufundwa kotshintsho lwemetabolism olwenzeka kumaqela athile e-neuronal. Ngenxa yoko, kuncinci okwaziwayo malunga nemiphumo echanekileyo yeseli kunye nemetabolism yokungasebenzi kakuhle kwe-mitochondrial kwii-neurons.
Ukuze siqonde iziphumo ze-metabolic zokungasebenzi kakuhle kwe-mitochondrial, sazihlukanisa ii-neuron zePurkinje (ii-PN) kwizigaba ezahlukeneyo zokuwohloka kwe-neuron okubangelwa kukutshatyalaliswa kwe-mitochondrial outer membrane fusion (i-Mfn2). Nangona utshintsho lwe-Mfn2 ebantwini lunxulunyaniswa nohlobo lwe-hereditary motor sensory neuropathy eyaziwa ngokuba yi-Charcot-Marie-Tooth type 2A (15), ukutshatyalaliswa okunemiqathango kwe-Mfn2 kwiimpuku yindlela eyaziwayo yokungenisa i-oxidation​​ Phosphorylation (OXPHOS) dysfunction method. Ii-neuronal subtypes ezahlukeneyo (16-19) kunye ne-neurodegenerative phenotype ephumayo zihamba kunye neempawu ze-neurological eziqhubekayo, ezifana nokuphazamiseka kokuhamba (18, 19) okanye i-cerebellar ataxia (16). Ngokusebenzisa indibaniselwano ye-proteomics ye-quantitative-free label (LFQ), i-metabolomics, i-imaging, kunye neendlela ze-virological, sibonisa ukuba i-progressive neurodegeneration ibangela ngamandla i-pyruvate carboxylase (PCx) kunye nezinye izinto ezibandakanyekayo kwi-arteriosclerosis ye-PNs in vivo The expression yee-enzymes. Ukuqinisekisa ukubaluleka kolu phando, sinciphise ngokukodwa ukubonakaliswa kwe-PCx kwi-Mfn2-deficient PNs, saza safumanisa ukuba olu tyando lonyusa uxinzelelo lwe-oxidative kwaye lwakhawulezisa ukubola kwe-neuron, nto leyo ebonisa ukuba i-azoospermia ibangela ukufa kweseli Ukuzivumelanisa neMetabolic. Ukubonakaliswa okuqatha kwe-MFN2 kunokuhlangula ngokupheleleyo ukubola kwe-terminal PN ngokunqongophala okukhulu kwe-OXPHOS, ukusetyenziswa kakhulu kwe-DNA ye-mitochondrial, kunye nenethiwekhi ye-mitochondrial ebonakala ngathi yaphukile, nto leyo egxininisa ngakumbi ukuba olu hlobo lokubola kwe-neuron lunokuphinda lubuyele kwimeko yesiqhelo yesifo ngaphambi kokuba iseli ife.
Ukuze sibone i-mitochondria kwi-Mfn2 knockout PNs, sisebenzise uhlobo lwegundane oluvumela i-Cre-dependent mitochondria ukuba ijolise kwi-yellow fluorescent protein (YFP) (mtYFP) (20) Cre expression kwaye sajonga i-mitochondrial morphology in vivo. Sifumanise ukuba ukutshatyalaliswa kwe-Mfn2 gene kwi-PNs kuya kukhokelela ekwahlulweni kancinci kwenethiwekhi ye-mitochondrial (Umfanekiso S1A), kwaye utshintsho lokuqala lufunyenwe kwiiveki ezi-3 ubudala. Ngokwahlukileyo koko, ukuwohloka okukhulu komaleko weseli ye-PN, njengoko kuboniswe kukulahleka kwe-Calbindin immunostaining, akuzange kuqale de kube ziiveki ezili-12 ubudala (Umfanekiso 1, A kunye no-B). Ukungafani kwexesha phakathi kotshintsho lokuqala kwi-mitochondrial morphology kunye nokuqala okubonakalayo kokufa kwe-neuronal kusishukumisele ukuba siphande utshintsho lwe-metabolic olubangelwa kukungasebenzi kakuhle kwe-mitochondrial ngaphambi kokufa kweseli. Siphuhlise icebo elisekelwe kwi-fluorescence-activated cell sorting (FACS) ukuze sahlukanise i-YFP (YFP+)-expressing PN (Umfanekiso 1C), kunye neempuku ezilawulayo (Mfn2 + / loxP :: mtYFP loxP- stop-loxP: : L7-cre), emva koko ebizwa ngokuba yi-CTRL (Umfanekiso S1B). Ukulungiswa kwecebo lokuvala ngokusekelwe kubunzulu besignali ye-YFP kusivumela ukuba sicoce umzimba we-YFP+ (YFPhigh) wee-PN kwii-non-PNs (YFPneg) (Umfanekiso S1B) okanye iziqwenga ze-fluorescent axon/dendritic (YFPlow; Umfanekiso S1D, ekhohlo), eziqinisekiswe yi-confocal microscope (Umfanekiso S1D, ekunene). Ukuze siqinisekise ubuwena boluntu oluhleliweyo, senze i-LFQ proteomics kwaye emva koko sahlalutya i-principal component, saza safumanisa ukuba kukho ukwahlukana okucacileyo phakathi kwee-YFPhigh kunye nee-YFPneg cells (Umfanekiso S1C). Iiseli ze-YFPhigh zibonise ukutyeba okune-net kweempawu ze-PN ezaziwayo (oko kukuthi iCalb1, iPcp2, iGrid2 kunye ne-Itpr3) (21, 22), kodwa akukho kutyeba kweeproteni ezibonakaliswa rhoqo kwii-neurons okanye kwezinye iintlobo zeeseli (Umfanekiso 1D)). Uthelekiso phakathi kweesampuli kwiiseli ze-YFPhigh eziqokelelwe kwiimvavanyo ezizimeleyo lubonise i-coefficient yokuhambelana> 0.9, ebonisa ukuphindaphinda okuhle phakathi kwee-replicates zebhayoloji (Umfanekiso S1E). Ngamafutshane, ezi datha ziqinisekisile isicwangciso sethu sokwahlulwa okukhawulezileyo nokuthe ngqo kwe-PN enokwenzeka. Ngenxa yokuba inkqubo yomqhubi we-L7-cre esetyenzisiweyo ibangela ukutyeba kwe-mosaic kwiveki yokuqala emva kokuzalwa (23), saqala ukubulala iimpuku kwi-CTRL kunye ne-conditional (Mfn2 loxP / loxP :: mtYFP loxP-stop-loxP :: L7-cre) Qokelela ii-neurons. Emva kokuba ukutyeba kugqityiwe, kubizwa ngokuba yi-Mfn2cKO kwiiveki ezi-4 ubudala. Njengesiphelo, sikhethe iiveki ezisi-8 ubudala xa umaleko we-PN wawungaphelelanga nangona kukho ukuqhekeka kwe-mitochondrial okubonakalayo (Umfanekiso 1B kunye noMfanekiso S1A). Lilonke, silinganise iiproteni ezingama-3013, apho malunga nama-22% asekelwe kwi-MitoCarta 2.0 annotations esekelwe kwi-mitochondrial proteome njenge-mitochondria (Umfanekiso 1E) (Umfanekiso 1E) (24). Uhlalutyo lokubonakaliswa kwezakhi zofuzo ezahlukeneyo olwenziwe kwiveki yesi-8 lubonise ukuba yi-10.5% kuphela yazo zonke iiproteni ezazinotshintsho olubalulekileyo (Umfanekiso 1F kunye noMfanekiso S1F), apho iiproteni ezili-195 zaziphantsi kwaye iiproteni ezili-120 zaziphezulu (Umfanekiso 1F). Kubalulekile ukuqaphela ukuba "uhlalutyo lwendlela entsha" lwale seti yedatha lubonisa ukuba ii-genes ezichazwe ngokwahlukileyo ikakhulu zezeseti ethintelweyo yeendlela ezithile ze-metabolic (Umfanekiso 1G). Okunomdla kukuba, nangona ukwehla kweendlela ezinxulumene ne-OXPHOS kunye ne-calcium signaling kuqinisekisa ukungeniswa kokungasebenzi kakuhle kwe-mitochondrial kwi-PN ezingenayo i-fusion, ezinye iindidi ezibandakanya kakhulu i-amino acid metabolism zinyuswa kakhulu, nto leyo ehambelana ne-metabolism eyenzeka kwi-mitochondrial PNs.
(A) Iifoto ezimeleyo ze-confocal ze-cerebellar sections ze-CTRL kunye ne-Mfn2cKO iimpuku ezibonisa ukulahleka okuqhubekayo kwe-PNs (calbindin, grey); ii-nuclei zichasene ne-DAPI. (B) Ukulinganiswa kwe-(A) (uhlalutyo lwendlela enye yokwahluka, ***P<0.001; n = izangqa ezi-4 ukuya kwezi-6 ezivela kwiimpuku ezintathu). (C) Umsebenzi wovavanyo. (D) Ukusasazwa kwemephu yobushushu beemaki ezithile kwiPurkinje (phezulu) nakwezinye iintlobo zeeseli (phakathi). (E) Umzobo weVenn obonisa inani leeproteni ze-mitochondrial ezichongiweyo kwi-PN ekhethiweyo. (F) Iploti yevolcano yeeproteni ezichazwe ngokwahlukileyo kwi-Mfn2cKO neurons kwiiveki ezi-8 (ixabiso lokunqunyulwa kokubaluleka le-1.3). (G) Uhlalutyo lwendlela yokudala lubonisa iindlela ezintlanu ezibalulekileyo zokunyusa ukulawulwa (obomvu) kunye nokwehla ukulawulwa (oluhlaza okwesibhakabhaka) kwi-Mfn2cKO PN echazwe njengeeveki ezi-8. Inqanaba eliqhelekileyo lokubonakaliswa kweproteni nganye efunyenweyo liyaboniswa. Imephu yobushushu yeGreyscale: ixabiso le-P elilungisiweyo. ns, alibalulekanga.
Idatha yeProteomics ibonise ukuba ukubonakaliswa kweproteni yee-complexes I, III, kunye ne-IV kuye kwehla kancinci kancinci. IiComplexes I, III, kunye ne-IV zonke beziqulathe ii-subunits ezibalulekileyo ze-mtDNA, ngelixa i-complex II, eyayine-nuclear-coded kuphela, yayingachaphazeleki (Umfanekiso 2A kunye noMfanekiso S2A). . Ngokuhambelana neziphumo zeproteomics, i-immunohistochemistry yamacandelo ezicubu ze-cerebellar ibonise ukuba inqanaba le-MTCO1 (mitochondrial cytochrome C oxidase subunit 1) subunit ye-complex IV kwi-PN liye lehla kancinci (Umfanekiso 2B). I-subunit ye-mtDNA-encoded Mtatp8 yehlisiwe kakhulu (Umfanekiso S2A), ngelixa inqanaba le-steady-state le-subunit ye-ATP synthase enee-nuclear-encoded ATP synthase lihlala lingatshintshanga, elihambelana ne-complex eyaziwayo ye-ATP synthase subassembly F1 xa ukubonakaliswa kwe-mtDNA kuzinzile. Ukwakheka kuyahambelana. Interrupt (7). Uvavanyo lwenqanaba le-mtDNA kwii-PN ze-Mfn2cKO ezihleliweyo nge-real-time polymerase chain reaction (qPCR) luqinisekisile ukwehla kancinci kwenani lekopi ye-mtDNA. Xa kuthelekiswa neqela lolawulo, kwiiveki ezi-8 ubudala, malunga ne-20% kuphela yenqanaba le-mtDNA eligcinwe (Umfanekiso 2C). Ngokuhambelana nezi ziphumo, i-confocal microscopy staining ye-Mfn2cKO PNs yasetyenziswa ukufumanisa i-DNA, ebonisa ukusetyenziswa kwexesha kwee-nucleotides ze-mitochondrial (Umfanekiso 2D). Sifumanise ukuba kuphela abanye abaviwa ababandakanyeka kwi-mitochondrial protein degradation kunye nempendulo yoxinzelelo abaphuculweyo, kubandakanya i-Lonp1, i-Afg3l2 kunye ne-Clpx, kunye ne-OXPHOS complex assembly factors. Akukho tshintsho lubalulekileyo kumanqanaba eeproteni ezibandakanyeka kwi-apoptosis ezifunyenweyo (Umfanekiso S2B). Ngokufanayo, sifumanise ukuba i-mitochondria kunye ne-endoplasmic reticulum channels ezibandakanyeka ekuthuthweni kwe-calcium zinenguqu ezincinci kuphela (Umfanekiso S2C). Ukongeza, uvavanyo lweeproteni ezinxulumene ne-autophagy alufumananga tshintsho lubalulekileyo, oluhambelana nokungeniswa okubonakalayo kwe-autophagosomes ebonwe kwi-vivo yi-immunohistochemistry kunye ne-electron microscopy (Umfanekiso S3). Nangona kunjalo, ukungasebenzi kakuhle kwe-OXPHOS okuqhubekayo kwi-PNs kuhamba notshintsho olucacileyo lwe-ultrastructural mitochondrial. Amaqela e-Mitochondrial anokubonwa kwimizimba yeseli kunye nemithi ye-dendritic ye-Mfn2cKO PNs eneminyaka eyi-5 neyesi-8, kwaye isakhiwo se-inner membrane siye satshintsha kakhulu (Umfanekiso S4, A kunye no-B). Ngokuhambelana nolu tshintsho lwe-ultrastructural kunye nokwehla okukhulu kwe-mtDNA, uhlalutyo lwe-acute cerebellar slices ene-tetramethylrhodamine methyl ester (TMRM) lubonise ukuba amandla e-mitochondrial membrane kwi-Mfn2cKO PNs ayehla kakhulu (Umfanekiso S4C).
(A) Uhlalutyo lwexesha lenqanaba lokubonakaliswa kwe-OXPHOS complex. Cinga kuphela ngeeproteni ezine-P<0.05 kwiiveki ezi-8 (i-ANOVA eneendlela ezimbini). Umgca onamachaphaza: Akukho hlengahlengiso xa kuthelekiswa ne-CTRL. (B) Ekhohlo: Umzekelo wecandelo le-cerebellar elibhalwe nge-anti-MTCO1 antibody (ibha yesikali, 20 μm). Indawo ehlala iiseli zePurkinje igqunywe ngomthubi. Ekunene: Ukulinganiswa kwamanqanaba e-MTCO1 (uhlalutyo lwendlela enye yokwahluka; n = iiseli ezi-7 ukuya kwezingama-20 ezihlalutyiweyo kwiigundane ezintathu). (C) Uhlalutyo lwe-qPCR lwenombolo yekopi ye-mtDNA kwi-PN ehleliweyo (uhlalutyo lwendlela enye yokwahluka; n = iimpuku ezi-3 ukuya kwezi-7). (D) Ekhohlo: Umzekelo wesilayi se-cerebellar esibhalwe nge-anti-DNA antibody (ibha yesikali, 20 μm). Indawo ehlala iiseli zePurkinje igqunywe ngomthubi. Ekunene: Ukulinganiswa kwezilonda ze-mtDNA (uhlalutyo lwendlela enye yokwahluka; n = iiseli ezi-5 ukuya kwezi-9 ezivela kwiigundane ezintathu). (E) Umzekelo wecandelo le-cerebellar elibukhali elibonisa iiseli ze-mitoYFP + Purkinje (utolo) kwirekhodi ye-clamp ye-patch yeseli yonke. (F) Ubungakanani be-IV curve. (G) Iirekhodi ezimele i-depolarizing current injection kwiiseli ze-CTRL kunye ne-Mfn2cKO Purkinje. Ubuninzi be-trace: I-pulse yokuqala eyabangela i-AP. Ubuninzi be-trace ye-AP. (H) Ubungakanani be-postsynaptic spontaneous inputs (sPSPs). Ubuninzi be-trace yokurekhoda kunye nomlinganiselo wayo we-zoom ziboniswe kwi-(I). Uhlalutyo lwendlela enye lokungafani oluhlalutyiweyo n = iiseli ezi-5 ukuya kwezingama-20 ezivela kwiimpuku ezintathu. Idatha ichazwa njenge-mean±SEM; *P<0.05; **P<0.01; ***P<0.001. (J) Iirekhodi ezimele i-AP spontaneous ezirekhodwe kusetyenziswa imo ye-clamp ye-patch eneembobo. Ubuninzi be-AP frequency. Ubuninzi be-AP frequency. Ubunzima be-AP: ukusondeza kwe-AP enye. (K) Ubungakanani be-avareji kunye ne-avareji ye-AP ngokutsho kwe-(J). Uvavanyo lweMann-Whitney; n = iiseli ezi-5 zihlalutywe kwiimpuku ezine. Idatha ichazwa njenge-mean±SEM; ayibalulekanga.
Umonakalo ocacileyo we-OXPHOS ufunyenwe kwi-Mfn2cKO PN eneeveki ezisi-8 ubudala, nto leyo ebonisa ukuba umsebenzi we-physiological wee-neurons awuqhelekanga kakhulu. Ke ngoko, sihlalutye iimpawu zombane ezingasebenziyo zee-neurons ezinqongopheleyo ze-OXPHOS kwiiveki ezi-4 ukuya kwezi-5 kunye neeveki ezi-7 ukuya kwezi-8 ngokwenza iirekhodi ze-clamp ye-patch yeseli yonke kwiislayi ze-cerebellar ezibukhali (Umfanekiso 2E). Ngokungalindelekanga, i-avareji ye-resting membrane potential kunye ne-input resistance yee-neurons ze-Mfn2cKO zazifana nolawulo, nangona kwakukho umahluko omncinci phakathi kweeseli (Itheyibhile 1). Ngokufanayo, kwiiveki ezi-4 ukuya kwezi-5 ubudala, akukho tshintsho lubalulekileyo kubudlelwane be-current-voltage (i-IV curve) olufunyenweyo (Umfanekiso 2F). Nangona kunjalo, akukho neurons ze-Mfn2cKO ezineeveki ezi-7 ukuya kwezi-8 ubudala ezisindileyo kwi-IV regimen (inyathelo le-hyperpolarization), nto leyo ebonisa ukuba kukho ukuvakalelwa okucacileyo kwi-hyperpolarization potential kweli nqanaba lokugqibela. Ngokwahlukileyo koko, kwii-neurons ze-Mfn2cKO, ii-currents ezibangela ukukhutshwa kwe-repetitive action potential (AP) ziyanyamezela, nto leyo ebonisa ukuba iipatheni zazo zokukhupha azihlukanga kakhulu kwezo zee-neurons zolawulo zeeveki ezi-8 ubudala (Itheyibhile 1 kunye noMfanekiso 2G). Ngokufanayo, i-frequency kunye ne-amplitude ye-spontaneous postsynaptic currents (sPSCs) yayifana neyeqela lolawulo, kwaye i-frequency yeziganeko yanda ukusuka kwiiveki ezi-4 ukuya kwiiveki ezi-5 ukuya kwiiveki ezi-7 ukuya kwiiveki ezi-8 kunye nokwanda okufanayo (Umfanekiso 2, H kunye no-I). Ixesha lokuvuthwa kwe-synaptic kwi-PNs (25). Iziphumo ezifanayo zifunyenwe emva kwee-patches ze-PN ezigqobhoziweyo. Olu qwalaselo luthintela imbuyekezo enokwenzeka yeempazamo ze-ATP zeseli, njengoko kunokwenzeka ekurekhodweni kwe-clamp ye-patch yeseli yonke. Ngokukodwa, i-resting membrane potential kunye ne-spontaneous firing frequency ye-Mfn2cKO neurons azichaphazelekanga (Umfanekiso 2, J kunye no-K). Ngamafutshane, ezi ziphumo zibonisa ukuba ii-PNs ezinengxaki ecacileyo ye-OXPHOS zinokuhlangabezana kakuhle neepateni zokukhupha eziphuma rhoqo, nto leyo ebonisa ukuba kukho indlela yokubuyisela evumela ukuba zigcine iimpendulo ze-electrophysiological eziphantse zibe zesiqhelo.
Idatha ichazwa njenge-mean ± SEM (uhlalutyo lwendlela enye yokwahluka, uvavanyo lokuthelekisa okuninzi lukaHolm-Sidak; *P<0.05). Inombolo yeyunithi iboniswa zizibiyeli.
Sizimisele ukuphanda ukuba ngaba naluphi na udidi kwi-dataset ye-proteomics (Umfanekiso 1G) luquka iindlela ezinokulwa nokungabikho okukhulu kwe-OXPHOS, ngaloo ndlela sichaza isizathu sokuba i-PN echaphazelekayo inokugcina i-electrophysiology ephantse ibe yeqhelekileyo (Umfanekiso 2, E ukuya kwi-K). . Uhlalutyo lwe-Proteomics lubonise ukuba ii-enzymes ezibandakanyeka kwi-catabolism ye-branched chain amino acids (BCAA) ziphuculwe kakhulu (Umfanekiso 3A kunye nomfanekiso S5A), kwaye imveliso yokugqibela i-acetyl-CoA (CoA) okanye i-succinyl CoA inokongeza ii-tricarboxylates kumjikelo we-arteriosclerosis Acid (TCA). Sifumanise ukuba umxholo we-BCAA transaminase 1 (BCAT1) kunye ne-BCAT2 zombini zanda. Zivuselela inyathelo lokuqala le-BCAA catabolism ngokuvelisa i-glutamate kwi-α-ketoglutarate (26). Zonke ii-subunits ezenza i-branched chain keto acid dehydrogenase (BCKD) complex ziyanda (le complex ivuselela i-decarboxylation elandelayo nengaguqukiyo ye-BCAA carbon skeleton ephumayo) (Umfanekiso 3A kunye noMfanekiso S5A). Nangona kunjalo, akukho tshintsho lucacileyo kwi-BCAA ngokwayo olufunyenweyo kwi-PN ehleliweyo, enokubangelwa kukwanda kokuthathwa kwezi amino acids ezibalulekileyo kwiseli okanye ukusetyenziswa kwezinye imithombo (i-glucose okanye i-lactic acid) ukongeza umjikelo we-TCA (Umfanekiso S5B). Ii-PN ezingenayo i-OXPHOS nazo zibonise ukwanda kwe-glutamine decomposition kunye nemisebenzi ye-transamination kwiiveki ezi-8 ubudala, nto leyo enokubonakaliswa kukunyuka kwe-enzymes ye-mitochondrial glutaminase (GLS) kunye ne-glutamine pyruvate transaminase 2 (GPT2) (Umfanekiso 3, A kunye no-C). Kubalulekile ukuqaphela ukuba ukuphuculwa kwe-GLS kunqunyelwe kwi-isoform glutaminase C (GLS-GAC) edibeneyo (utshintsho lwe-Mfn2cKO/CTRL lumalunga ne-4.5-fold, P = 0.05), kwaye ukuphuculwa kwayo okuthe ngqo kwizicubu zomhlaza Kungaxhasa i-mitochondrial bioenergy. (27).
(A) Imephu yobushushu ibonisa utshintsho olugobileyo kwinqanaba leproteni kwindlela echaziweyo kwiiveki ezi-8. (B) Umzekelo wesilayi se-cerebellar esibhalwe nge-anti-PCx antibody (isikali sebha, i-20 μm). Utolo olutyheli lukhomba umzimba weseli yePurkinje. (C) Uhlalutyo lokubonakaliswa kweprotheni yexesha oluchongiweyo njengomviwa obalulekileyo we-atherosclerosis (uvavanyo lwe-t oluninzi, *i-FDR <5%; n = iimpuku ezi-3-5). (D) Ngaphezulu: Umzobo weskimu obonisa iindlela ezahlukeneyo zokungena kwikhabhoni ebhalwe ilebhile equlethwe kwi-[1-13C]pyruvate tracer (oko kukuthi, nge-PDH okanye indlela ye-trans-arterial). Ezantsi: Itshathi ye-violin ibonisa ipesenti yekhabhoni enelebhile enye (M1) eguqulwe yaba yi-aspartic acid, i-citric acid kunye ne-malic acid emva kokubhala iilebhile ze-cerebellar ezibukhali nge-[1-13C]pyruvate (uvavanyo lwe-t oludibeneyo; ** P <0.01). (E) Uhlalutyo olupheleleyo lwembali yexesha lendlela echaziweyo. Cinga kuphela ngeeproteni ezine-P <0.05 kwiiveki ezi-8. Umgca oqhekekileyo: akukho xabiso lohlengahlengiso (uhlalutyo lweendlela ezimbini zokwahluka; * P <0.05; *** P <0.001). Idatha ivezwa njenge-mean±SEM.
Kuhlalutyo lwethu, i-BCAA catabolism ibe yenye yeendlela eziphambili zokunyusa ukulawulwa. Le nyaniso ibonisa ngamandla ukuba umthamo womoya ongena kumjikelo we-TCA unokutshintshwa kwi-PN engenayo i-OXPHOS. Oku kunokumela uhlobo oluphambili lwe-neuronal metabolic rewiring, olunokuba nefuthe ngqo kwi-neuronal physiology kunye nokusinda ngexesha lokugcinwa kokungasebenzi kakuhle kwe-OXPHOS. Ngokuhambelana nale ngcamango, sifumanise ukuba i-enzyme ephambili yokulwa ne-atherosclerotic PCx ilawulwa ngokuphezulu (i-Mfn2cKO/CTRL itshintsha malunga namaxesha ali-1.5; Umfanekiso 3A), ekhuthaza ukuguqulwa kwe-pyruvate kwi-oxaloacetate (28), ekholelwa ukuba ikwizicubu zobuchopho. Intetho ethi in ikhawulelwe kwi-astrocytes (29, 30). Ngokuhambelana neziphumo ze-proteomics, i-confocal microscopy ibonise ukuba ukubonakaliswa kwe-PCx kwandiswe ngokukodwa kwaye kakhulu kwi-PNs ezingenayo i-OXPHOS, ngelixa i-PCx reactivity yayikhawulelwe kakhulu kwiiseli ze-glial zeBergmann ezikufutshane zolawulo (Umfanekiso 3B). Ukuvavanya ngokusebenzayo ukunyuka okubonweyo kwe-PCx, sinyange izilayi ze-cerebellar ezibukhali nge-[1-13C]pyruvate tracer. Xa i-pyruvate yafakwa i-oxidized yi-pyruvate dehydrogenase (PDH), ilebhile yayo ye-isotope yanyamalala, Kodwa ifakwe kwi-intermediates yomjikelo we-TCA xa i-pyruvate iguqulwa yi-vascular reactions (Umfanekiso 3D). Ukuxhasa idatha yethu ye-proteomics, sibone inani elikhulu leempawu ezivela kule tracer kwi-aspartic acid yezilayi ze-Mfn2cKO, ngelixa i-citric acid kunye ne-malic acid nazo zazine-trend ephakathi, nangona zingabonakali kangako (Umfanekiso 3D).
Kwi-dopamine neurons yeempuku zeMitoPark ezine-mitochondrial dysfunction ezibangelwa yi-dopamine neurons ezitshabalalisa ngokukodwa i-mitochondrial transcription factor A gene (Tfam) (Umfanekiso S6B), ukubonakaliswa kwe-PCx nako kwanyuswa kakhulu (31), okubonisa ukuba i-acetone acid arteriosclerosis Ukwenzeka kwesi sifo kulawulwa ngexesha lokungasebenzi kakuhle kwe-neuronal OXPHOS emzimbeni. Kubalulekile ukuqaphela ukuba kufunyenwe ukuba ii-enzymes ezikhethekileyo (32-34) ezinokubonakaliswa kwii-neurons ezinokunxulunyaniswa ne-arteriosclerosis zinyuswa kakhulu kwi-PNs ezingenayo kwi-OXPHOS, njenge-propionyl-CoA carboxylase (PCC-A), iMalonyl-CoA iguqula i-propionyl-CoA ibe yi-succinyl-CoA kunye ne-mitochondrial malic enzyme 3 (ME3), enendima ephambili ekubuyiseleni i-pyruvate kwi-malate (Umfanekiso 3, A kunye no-C) (33, 35). Ukongeza, sifumene ukwanda okukhulu kwi-enzyme ye-Pdk3, ephosphates kwaye ngaloo ndlela ingasebenzi i-PDH (36), ngelixa kungekho tshintsho lufunyenweyo kwi-enzyme ye-Pdp1 esebenza kwi-PDH okanye kwi-enzyme ye-PDH ngokwayo (Umfanekiso 3A). Ngokuqhubekayo, kwi-Mern2cKO PNs, i-phosphorylation ye-α1 subunit α (PDHE1α) subunit ye-pyruvate dehydrogenase E1 component ye-PDH complex kwi-Ser293 (eyaziwa ngokuthintela umsebenzi we-enzyme ye-PDH) yandiswa (Umfanekiso S6C) (Umfanekiso S6C). I-Pyruvate ayinayo i-vascular access.
Ekugqibeleni, sifumanise ukuba indlela ephezulu ye-serine kunye ne-glycine biosynthesis, umjikelo we-mitochondrial folate (1C) ohambelanayo kunye ne-proline biosynthesis (Umfanekiso 1G kunye nomfanekiso S5C) zonke ziphuculwe kakhulu, ngokweengxelo, ngexesha lenkqubo yokusebenza. Izicubu ezijikelezileyo ziyasebenza ngenxa yokungasebenzi kakuhle kwe-mitochondrial (5-7). Uhlalutyo lwe-Confocal oluxhasa le datha ye-proteomics lubonise ukuba kwi-PN ene-OXPHOS engekhoyo, izilayi ze-cerebellar zeempuku ezineeveki ezi-8 ubudala zifakwe kwi-serine hydroxymethyltransferase 2 (SHMT2), i-enzyme ephambili yomjikelo we-mitochondrial folate. Impendulo ebalulekileyo yomzimba (Umfanekiso S5D). Kwizilayi ze-cerebellar ezi-13 ze-CU-glucose-incubated acute cerebellar, iimvavanyo zokulandelela i-metabolic ziqinisekisile ngakumbi ukunyuka kwe-serine kunye ne-proline biosynthesis, okubonisa ukuba ukuhamba kwee-isoforms ze-carbon kwi-serine kunye ne-proline kwanda (Umfanekiso S5E). Ekubeni ii-reactions ezikhuthazwa yi-GLS kunye ne-GPT2 zinoxanduva lokwenziwa kwe-glutamate kwi-glutamine kunye nokudluliselwa kwe-glutamate phakathi kwe-glutamate kunye ne-α-ketoglutarate, ukunyuka kwazo kubonisa ukuba ii-neurons ezingenayo i-OXPHOS zinesidingo esikhulu se-glutamate, Oku kunokuthi kujoliswe ekugcineni ukwanda kwe-biosynthesis ye-proline (Umfanekiso S5C). Ngokungafaniyo nolu tshintsho, uhlalutyo lwe-proteomic lwee-astrocytes ze-cerebellar ezivela kwiimpuku ze-Mfn2cKO ezithile ze-PN lubonise ukuba ezi ndlela (kuquka zonke ii-antiperoxidases) azitshintshanga kakhulu ekubonakalisweni, ngaloo ndlela zibonisa ukuba olu tshintsho lwe-metabolic lukhetha i-PN ewohlokileyo (Umfanekiso S6, D ukuya ku-G).
Ngamafutshane, olu hlalutyo lutyhile iipatheni ezahlukeneyo kakhulu zokusebenza kwexesha leendlela ezithile ze-metabolic kwi-PNs. Nangona umsebenzi ongaqhelekanga we-neuronal mitochondrial unokukhokelela kwi-atherosclerosis yokuqala kunye nokulungiswa kwe-1C (Umfanekiso 3E kunye noMfanekiso S5C), kunye notshintsho oluqikelelweyo ekubonakalisweni kwee-complexes ze-I kunye ne-IV, utshintsho kwi-serine de novo synthesis lubonakala kuphela kwizigaba zokugqibela. Ukungasebenzi kakuhle kwe-OXPHOS (Umfanekiso 3E kunye noMfanekiso S5C). Ezi ziphumo zichaza inkqubo elandelelanayo apho i-mitochondrial ebangelwa luxinzelelo (umjikelo we-1C) kunye ne-cytoplasmic (serine biosynthesis) zisabela ngokubambisana kunye nokwanda kwe-atherosclerosis kumjikelo we-TCA ukuze kuphinde kuguqulwe i-metabolism ye-neuronal.
Ii-PN ze-OXPHOS ezineeveki ezi-8 ubudala zinokugcina umsebenzi wokuvusa rhoqo kwaye zenze uxhulumaniso olukhulu lwe-metabolic ukuze kulungiswe ukungasebenzi kakuhle kwe-mitochondrial. Oku kufunyaniswa kuphakamisa ithuba elinomdla lokuba nangona ngoku, ezi seli zinokufumana ukungenelela konyango ukulibazisa okanye ukuthintela ukuwohloka kwemithambo-luvo. Kade. Sisombulule eli thuba ngeendlela ezimbini ezizimeleyo. Kwindlela yokuqala, siyile i-vector ye-adeno-associated virus (AAV) exhomekeke kwi-Cre ukuze i-MFN2 ikwazi ukubonakaliswa ngokukhetha kwii-PN ze-OXPHOS ezinqongopheleyo kwi-vivo (Umfanekiso S7A). I-AAV encoding MFN2 kunye ne-fluorescent reporter gene mCherry (Mfn2-AAV) ziqinisekiswe kwiinkcubeko ze-neuron eziphambili kwi-vitro, nto leyo eyabangela ukuba i-MFN2 ibonakaliswe ngendlela exhomekeke kwi-Cre kwaye yasindisa i-morphology ye-mitochondrial, ngaloo ndlela ithintela i-neuromutation kwi-neurons ze-Mfn2cKO (Umfanekiso S7, B, D kunye no-E). Okulandelayo, senze uvavanyo lwe-in vivo ukuze sihambise i-Mfn2-AAV eneeveki ezisi-8 ubudala kwi-cerebellar cortex ye-Mfn2cKO kunye neempuku zokulawula, saza sahlalutya iimpuku ezineeveki ezili-12 ubudala (Umfanekiso 4A). Iimpuku ze-Mfn2cKO ezinyangiweyo zafa (Umfanekiso 1, A kunye no-B) (16). Ukudluliselwa kwe-viral kwi-vivo kubangele ukubonakaliswa kwe-PN okhethiweyo kwezinye izangqa ze-cerebellar (Umfanekiso S7, G kunye no-H). Ukufakwa kwe-AAV yokulawula eveza kuphela i-mCherry (Ctrl-AAV) akubanga nampembelelo ibalulekileyo kwinqanaba lokuwohloka kwe-neurogeneity kwizilwanyana ze-Mfn2cKO. Ngokwahlukileyo koko, uhlalutyo lwe-Mfn2cKOs oludluliselwe kwi-Mfn2-AAV lubonise impembelelo ebalulekileyo yokukhusela yomaleko weseli ye-PN (Umfanekiso 4, B kunye no-C). Ngokukodwa, ubuninzi be-neuron bubonakala bungahlukani nezilwanyana zokulawula (Umfanekiso 4, B kunye no-C, kunye nomfanekiso S7, H kunye no-I). Ukubonakaliswa kwe-MFN1 kodwa kungekhona i-MFN2 kusebenza ngokulinganayo ekusindiseni ukufa kwe-neuronal (Umfanekiso 4C kunye noMfanekiso S7, C kunye no-F), nto leyo ebonisa ukuba ukubonakaliswa kwe-ectopic MFN1 kunokongeza ngempumelelo ukungabikho kwe-MFN2. Uhlalutyo olongezelelweyo kwinqanaba le-PN enye lubonise ukuba i-Mfn2-AAV isindise kakhulu isakhiwo se-mitochondria, yalungisa amanqanaba e-mtDNA, kwaye yaguqula ukubonakaliswa okuphezulu kwe-anti-angiogenesis marker PCx ​​​​(Umfanekiso 4, C ukuya ku-E). Ukuhlolwa okubonakalayo kweempuku ze-Mfn2cKO ezisindisiweyo kwimeko yokuphumla kubonise ukuba ukuma kwazo kunye neempawu zokunyakaza (intshukumo ye-S1 ukuya kwi-S3) ziphuculwe. Ukuqukumbela, ezi mvavanyo zibonisa ukuba ukubuyiselwa kwakhona kwe-MFN2 kwi-PNs okusilela kakhulu kwi-OXPHOS kwanele ukuguqula ukusetyenziswa kwe-mtDNA kunye nokudala i-atherosclerosis, ngaloo ndlela kuthintele ukuwohloka kwe-axon kunye nokufa kwe-neuronal emzimbeni.
(A) Iskimu esibonisa ishedyuli yovavanyo yokufaka i-AAV encoding MFN2 xa indlela ebonisiweyo ye-metabolic ivuliwe. (B) Imifanekiso emele i-confocal yee-cerebellar slices zeeveki ezili-12 ubudala eziguqulwe kwiiveki ezisi-8 kwiimpuku ze-Mfn2cKO kwaye zibhalwe nge-anti-Calbindin antibody. Ekunene: Ukulinganiswa kwee-axon fibers. Isikali se-axon zoom yi-450 kunye ne-75 μm. (C) Ekhohlo: Ukulinganiswa koxinano lweeseli zePurkinje kwi-AAV transduction loop (AAV+) (uhlalutyo lwendlela enye yokwahluka; n = 3 iimpuku). Ekunene: Uhlalutyo lokugxila kwe-mtDNA kwi-PN eguqulweyo kwiveki ye-12 (uvavanyo lwe-t olungalinganiyo; n = iiseli ezi-6 ezivela kwiimpuku ezintathu). * P <0.05; ** P <0.01. (D) Ii-micrographs ze-electron ezimele ii-PNs ze-Mfn2cKO cerebellar sections eziguqulwe ngee-vectors ze-viral ezibonisiweyo. Imaski epinki ibonisa indawo ehlala ii-dendrites, kwaye isikwere esityheli esinamachaphaza sibonisa i-zoom enikiweyo ngasekunene; u-n umele i-nucleus. Ibha yesikali, 1μm. (E) ibonisa umzekelo wombala we-PCx kwi-PN eguqulwe kwiiveki ezili-12. Ibha yesikali, 20μm. OE, ukubonakaliswa okugqithisileyo; i-FC, utshintsho olugobileyo.
Okokugqibela, siphande ngokubaluleka kokusinda kweeseli ezibangelwa yi-peroxidase kwii-PN eziye zahlangabezana nokungasebenzi kakuhle kwe-OXPHOS. Sivelise i-mCherry encoding AAV-shRNA (i-short hairpin RNA) ejolise ngokukodwa kwi-PCx mRNA yegundane (AAV-shPCx), saza safaka intsholongwane okanye ulawulo lwayo oluqhekekileyo (AAV-scr) kwi-cerebellum yeempuku ze-Mfn2cKO. Inaliti yenziwe kwiveki yesine ubudala (Umfanekiso 5A) ukuze kufezekiswe i-PCx knockdown esebenzayo ngexesha apho ukubonakaliswa kwe-PCx kwanda (Umfanekiso 3C) kwaye umaleko weseli ye-PN wawusaphelele (Umfanekiso 1A). Kubalulekile ukuqaphela ukuba ukuwisa i-PCx (Umfanekiso S8A) kukhokelela ekukhawulezisweni okukhulu kokufa kwe-PN, okukhawulelwe kwiringi eyosulelekileyo (Umfanekiso 5, B kunye no-C). Ukuze siqonde indlela esebenza ngayo imiphumo ye-metabolic ebangelwa kukunyuka kwe-PCx, sifunde imeko ye-redox yee-PN emva kokuba i-PCx ​​knockdown kunye ne-AAV-mediated optical biosensor Grx1-roGFP2 zibonakaliswe ngaxeshanye (Umfanekiso S8, B ukuya ku-D) ukuvavanya utshintsho oluhambelanayo lwe-peptide redox potential (38). Emva koko, senze i-two-photon fluorescence lifetime imaging microscopy (FLIM) kwiislices zobuchopho ezibukhali ze-Mfn2cKO yeeveki ezi-7 ubudala okanye ii-control littermates ukuze sibone utshintsho olunokwenzeka kwi-cytoplasmic redox status emva kokuqinisekisa iimeko ze-FLIM (Umfanekiso S8, E ukuya ku-G). Uhlalutyo lubonise ukwanda okukhulu kwimeko ye-oxidation ye-Mfn2cKO PN enye engenazo ii-PCx expression, eyahlukileyo kwii-control neurons okanye ii-Mfn2cKO PNs eziveza kuphela i-shRNA eqhekekileyo (Umfanekiso 5, D kunye no-E). Xa ukubonakaliswa kwe-PCx kwehliswa, ipesenti yee-Mfn2cKO PNs ezibonisa imeko ye-oxidized ephezulu yanda ngaphezu kwezihlandlo ezithathu (Umfanekiso 5E), nto leyo ebonisa ukuba ukulawulwa kwe-PCx okuphezulu kwagcina amandla e-redox ee-neurons eziwohlokileyo.
(A) Iskimu esibonisa ishedyuli yovavanyo yokufaka i-AAV encoding shPCx xa indlela ye-metabolic ebonisiweyo ivuliwe. (B) Iifoto ezimeleyo ze-confocal ze-cerebellar sections zeeveki ezi-8 ubudala kwiimpuku ze-Mfn2cKO eziguqulweyo zaza zalebheliswa nge-anti-calcineurin antibody kwiiveki ezi-4. Ibha yesikali, 450μm. (C) Ubungakanani boxinano lweeseli zePurkinje kwi-AAV-transduced loops (uhlalutyo lwendlela enye yokwahluka; n = iimpuku ezi-3 ukuya kwezi-4). Idatha ichazwa njenge-mean±SEM; ***P<0.001. (D) Umfanekiso we-FLIM omeleyo ubonisa ubude bobomi be-PN yeeveki ezi-7 ubudala eveza i-glutathione redox sensor Grx1-roGFP2 phantsi kweemeko zovavanyo ezichaziweyo. Umlinganiselo we-LUT (itheyibhile yokukhangela): ixesha lokusinda (kwii-picoseconds). Ibha yesikali, 25μm. (E) I-histogram ibonisa ukusasazwa kwamaxabiso obomi be-Grx1-roGFP2 ukusuka kwi-(D) (n=158 ukuya kwiiseli ezingama-368 kwiimpuku ezimbini phantsi kwemeko nganye). Itshati yephayi engaphezulu kwe-histogram nganye: ibonisa inani leeseli ezinexabiso elide kakhulu (elibomvu, elixidiziweyo) okanye elifutshane (eliluhlaza okwesibhakabhaka, elincitshisiweyo), elidlula i-1 SD yexabiso eliqhelekileyo lobomi kwi-CTRL-AAV-scr. (F) Imodeli ecetywayo ibonisa isiphumo sokukhusela sokwandiswa kwe-neuronal PCx.
Lilonke, idatha esiyinika apha ibonisa ukuba ukubonakaliswa kwakhona kwe-MFN2 kunokuhlangula ngokupheleleyo i-PN ephucukileyo ene-OXPHOS enqongopheleyo, ukuncipha okukhulu kwe-mtDNA, kunye ne-morphology engaqhelekanga efana ne-ista, ngaloo ndlela ibonelela ngenkqubela phambili eqhubekayo nakwizifo eziphambili. Ukonakala kwe-neurogene kubonelela ngobungqina obuguqukayo besigaba ngaphambi kokufa kweseli. Eli nqanaba lokuguquguquka kwe-metabolic ligxininiswa ngakumbi kubuchule bee-neurons bokubangela i-atherosclerosis (ukufakelwa kwakhona kwentambo yomjikelo we-TCA), okuthintela ukubonakaliswa kwe-PCx kwi-PN ezingenayo i-OXPHOS kwaye kwandisa ukufa kweseli, ngaloo ndlela kudlala indima yokukhusela (Umfanekiso 5F).
Kolu phononongo, sinike ubungqina bokuba impendulo ye-PNs kwi-OXPHOS dysfunction kukudibana kancinci kancinci kwi-TCA cycle atherosclerosis ngendlela ye-differential activation pathway evuselelwa ziinkqubo ze-metabolic. Siqinisekisile uhlalutyo lwe-proteomic ngeendlela ezininzi ezincedisayo kwaye satyhila ukuba xa ijongene nokungasebenzi kakuhle kwe-mitochondrial, ii-neurons zinendlela engaziwayo ngaphambili ye-metabolic elasticity. Okumangalisayo kukuba, yonke inkqubo yokubuyisela i-wiring ayibonisi imeko ye-terminal metabolic ehamba ne-neurodegeneration kancinci kancinci nangokungenakuguquguquka, kodwa idatha yethu ibonisa ukuba inokuba yi-neuron yokugcina nakwinqanaba langaphambi kokufa kweseli. Indlela yokubuyisela ukusebenza. Oku kufunyanisiweyo kubonisa ukuba ii-neurons zinomlinganiselo omkhulu we-metabolic plasticity emzimbeni. Le nyaniso ingqina ukuba ukubuyiselwa kwakhona kwe-MFN2 kamva kunokuguqula ukubonakaliswa kweempawu eziphambili ze-metabolic kwaye kuthintele ukuwohloka kwe-PN. Ngokuchaseneyo, ithintela i-atherosclerosis kwaye ikhawulezise imithambo-luvo.
Enye yezona zinto zibalulekileyo ezifunyenweyo kuphando lwethu kukuba ii-PN ezingenayo i-OXPHOS zinokutshintsha imetabolism yomjikelo we-TCA ngokunyusa ii-enzymes ezikhuthaza ngokukodwa i-arteriosclerosis. Ukulungiswa kwakhona kwe-metabolic luphawu oluqhelekileyo lweeseli zomhlaza, ezinye zazo ezixhomekeke kwi-glutamine ukongeza ii-intermediates zomjikelo we-TCA ukuvelisa izinto ezinciphisayo, eziqhuba ikhonkco lokuphefumla kwaye zigcine imveliso ye-lipid kunye ne-nucleotide biosynthesis precursors (39, 40). Uphononongo lwakutshanje lubonise ukuba kwizicubu ezingaphandle ezihlangabezana nokungasebenzi kakuhle kwe-OXPHOS, ukudibana kwakhona kwe-glutamine/glutamate metabolism nako luphawu oluphambili (5, 41), apho indlela yokungena kwe-glutamine kumjikelo we-TCA ixhomekeke Ngenxa yobunzima bokulimala kwe-OXPHOS (41). Nangona kunjalo, akukho bungqina bucacileyo kuyo nayiphi na into efana ne-neuronal metabolic plasticity emzimbeni kunye nokubaluleka kwayo kwimeko yesifo. Kwisifundo sakutshanje se-in vitro, ii-cortical neurons eziphambili ziboniswe ukuba zihambisa amachibi e-glutamate ukuze adlulisele i-neurotransmission, ngaloo ndlela zikhuthaza imetabolism ye-oxidative kunye ne-atherosclerosis phantsi kweemeko zoxinzelelo lwe-metabolic (42). Kubalulekile ukuqaphela ukuba phantsi kokuthintelwa kwe-enzyme ye-TCA cycle succinate dehydrogenase, i-pyruvate carboxylation kukholelwa ukuba igcina ukwenziwa kwe-oxaloacetate kwi-cerebellar granule neurons ezikhuliswe kakuhle (34). Nangona kunjalo, ukubaluleka kwe-physiological kwezi ndlela kwizicubu zobuchopho (apho i-atherosclerosis kukholelwa ukuba ixhomekeke kakhulu kwi-astrocytes) kusenokubaluleka okubalulekileyo kwi-physiological (43). Kule meko, idatha yethu ibonisa ukuba ii-PN ezonakele yi-OXPHOS emzimbeni zinokutshintshelwa kwi-BCAA degradation kunye ne-pyruvate carboxylation, eziyimithombo emibini ephambili yokongeza ii-TCA pool intermediates. Nangona igalelo elibonakalayo le-BCAA catabolism kwi-metabolism yamandla e-neuronal liye lacetyiswa, ukongeza kwindima ye-glutamate kunye ne-GABA yokudluliselwa kwe-neurotransmission (44), akukho bungqina bezi ndlela kwi-vivo. Ke ngoko, kulula ukuqikelela ukuba ii-PN ezingasebenzi kakuhle zinokubuyisela ngokuzenzekelayo ukusetyenziswa kwee-TCA intermediates eziqhutywa yinkqubo yokuhlanganisa ngokunyusa i-atherosclerosis. Ngokukodwa, ukwandiswa kwe-PCx kunokufuneka ukuze kugcinwe imfuno eyongeziweyo ye-aspartic acid, ecetyiswa kwiiseli ezikhulayo ezine-mitochondrial dysfunction (45). Nangona kunjalo, uhlalutyo lwethu lwe-metabolomics aluzange lubonise naluphi na utshintsho olubalulekileyo kwinqanaba elizinzileyo le-aspartic acid kwi-Mfn2cKO PNs (Umfanekiso S6A), onokuthi ubonise ukusetyenziswa okwahlukileyo kwe-metabolic ye-aspartic acid phakathi kweeseli ezikhulayo kunye nee-neuron ze-post-mitotic. Nangona indlela echanekileyo yokunyuka kwe-PCx kwi-neurons ezingasebenzi kakuhle kwi-vivo isaza kuchazwa, sibonise ukuba le mpendulo yangaphambi kwexesha idlala indima ebalulekileyo ekugcineni imeko ye-redox yee-neurons, eyaboniswa kuvavanyo lwe-FLIM kwizilayi ze-cerebellar. Ngokukodwa, ukuthintela ii-PN kwi-PCx elawulayo kunokukhokelela kwimeko ye-oxidized ngakumbi kwaye kukhawulezise ukufa kweeseli. Ukusebenza kokonakala kwe-BCAA kunye ne-carboxylation ye-pyruvate azizo iindlela zokubonakalisa izicubu ezingaphandle kokungasebenzi kakuhle kwe-mitochondrial (7). Ngoko ke, zibonakala ziluphawu oluphambili lwee-neurons ezingenayo i-OXPHOS, nokuba ayilulo lodwa uphawu, olubalulekileyo ekuwohlokeni kwemithambo-luvo.
Isifo seCerebellar sisifo esahlukileyo se-neurodegenerative esidla ngokubonakala njenge-ataxia kwaye sidla ngokonakalisa ii-PN (46). Olu luntu lwe-neuron lusengozini enkulu yokungasebenzi kakuhle kwe-mitochondrial kuba ukonakala kwazo okukhethayo kwiimpuku kwanele ukuvelisa uninzi lweempawu zomzimba ezibonisa i-human spinocerebellar ataxia (16, 47, 48). Ngokweengxelo, imodeli yegundane eguqulweyo ene-gene eguquliweyo inxulunyaniswa ne-human spinocerebellar ataxia kwaye inokungasebenzi kakuhle kwe-mitochondrial (49, 50), nto leyo egxininisa ukubaluleka kokufunda iziphumo zokunqongophala kwe-OXPHOS kwi-PNPH. Ke ngoko, kufanelekile ngokukodwa ukwahlula nokufunda olu luntu lukhethekileyo lwe-neuron. Nangona kunjalo, ngenxa yokuba ii-PN zinovelwano kakhulu kuxinzelelo kwaye zibangela inani eliphantsi lalo lonke uluntu lwe-cerebellar cell, kwizifundo ezininzi ezisekelwe kwi-omics, ukwahlulahlula kwazo njengeeseli ezipheleleyo kusengumbandela onzima. Nangona kungenakwenzeka ukufikelela ekunqongophaleni ngokupheleleyo kwezinye iintlobo zeeseli (ingakumbi izicubu zabantu abadala), sidibanise inyathelo elisebenzayo lokuhlukana ne-FACS ukuze sifumane inani elaneleyo lee-neurons ezisebenzayo zohlalutyo lwe-proteomics olusezantsi, kwaye sibe neprotheyini ephezulu kakhulu (malunga neeprotheyini ezingama-3000) xa kuthelekiswa nedatha ekhoyo ye-cerebellum yonke (51). Ngokugcina ubomi beeseli ezipheleleyo, indlela esiyinikezelayo apha ivumela ukuba singajongi nje kuphela utshintsho kwiindlela ze-metabolic kwi-mitochondria, kodwa sikwajonga utshintsho kwi-cytoplasmic counterparts zayo, ezizalisa ukusetyenziswa kweethegi ze-mitochondrial membrane ukutyebisa uhlobo lweseli Indlela entsha yenani le-mitochondria kwizicubu ezintsonkothileyo (52, 53). Indlela esiyichazayo ayinxulumananga kuphela nophando lweeseli zePurkinje, kodwa ingasetyenziswa ngokulula kulo naluphi na uhlobo lweseli ukujongana notshintsho lwe-metabolic kwiingqondo ezigulayo, kubandakanya nezinye iimodeli zokungasebenzi kakuhle kwe-mitochondrial.
Okokugqibela, sifumene ithuba lokunyanga ngexesha lenkqubo yokulungiswa kwakhona kwe-metabolic enokuguqula ngokupheleleyo iimpawu eziphambili zoxinzelelo lweseli kwaye ithintele ukuwohloka kwe-neuronal. Ke ngoko, ukuqonda iimpembelelo zomsebenzi wokuphinda udibane okuchazwe apha kunokubonelela ngengqiqo esisiseko malunga nonyango olunokwenzeka lokugcina impilo ye-neuronal ngexesha lokungasebenzi kakuhle kwe-mitochondrial. Uphando lwexesha elizayo olujolise ekuhlalutyeni utshintsho kwi-metabolism yamandla kwezinye iintlobo zeeseli zobuchopho luyafuneka ukuze kutyhileke ngokupheleleyo ukusebenza kwalo mgaqo kwezinye izifo ze-neurological.
Iimpuku zeMitoPark zichazwe ngaphambili (31). Iimpuku zeC57BL/6N ezine-loxP flanking Mfn2 genes zichazwe ngaphambili (18) zaza zadityaniswa neempuku zeL7-Cre (23). Inzala ephindwe kabini ene-heterozygous emva koko yadityaniswa neempuku zeMfn2loxP/Mfn2loxP ezine-homozygous ukuze kuveliswe i-Purkinje-specific gene knockouts yeMfn2 (Mfn2loxP/Mfn2loxP; L7-cre). Kwiqela elincinci lokudibana, i-Gt (ROSA26) SorStop-mito-YFP allele (stop-mtYFP) yaziswa ngokudityaniswa okongezelelweyo (20). Zonke iinkqubo zezilwanyana zenziwa ngokuhambelana nezikhokelo zaseYurophu, zesizwe nezeziko kwaye zavunywa yiLandesamtfürNatur yase-Umwelt naseVerbraucherschutz, eNorth Rhine-Westphalia, eJamani. Umsebenzi wezilwanyana ulandela ulwalathiso lwe-European Federation of Laboratory Animal Sciences Associations.
Emva kokuthomalalisa ukudumba komlomo wesibeleko komfazi okhulelweyo, i-embryo yempuku iyahlukaniswa (E13). I-cortex ihlinzwe kwi-Hanks' Balanced Salt Solution (HBSS) eyongezwe yi-10 mM Hepes yaza yadluliselwa kwi-Dulbecco's Modified Eagle's Medium equlethe i-papain (20 U/ml) kunye ne-cysteine ​​​​(1μg/ml). Faka izicubu kwi-DMEM) kwaye uyihlukanise ngokugaya i-enzyme. Ml) kwi-37°C imizuzu engama-20, uze ucolwe ngoomatshini kwi-DMEM eyongezwe yi-10% ye-fetal bovine serum. Iiseli zityalwe kwiiglasi ezigqunywe yi-polylysine kubuninzi be-2×106 kwisitya sokukhulisa esingu-6 cm okanye kubuninzi be-0.5×105 cells/cm2 ukuze kuhlalutywe imifanekiso. Emva kweeyure ezi-4, i-medium yatshintshwa yi-Neurobasal serum-free medium equlethe i-1% B27 supplement kunye ne-0.5 mM GlutaMax. Emva koko ii-neurons zagcinwa kwi-37°C kunye ne-5% CO2 kulo lonke uvavanyo, zaze zondliwa kanye ngeveki. Ukuze kukhuthazwe ukuphinda kuhlanganiswe kwi-vitro, i-3μl (isitya sokukhulisa ii-24-well) okanye i-0.5μl (ipleyiti ye-24-well) ye-vector ye-AAV9 elandelayo yasetyenziswa ukunyanga ii-neurons ngosuku lwesibini kwi-vitro: AAV9.CMV.PI.eGFP. WPRE.bGH (Addgene, inombolo yekhathalogu 105530-AAV9) kunye ne-AAV9.CMV.HI.eGFP-Cre.WPRE.SV40 (Addgene, inombolo yekhathalogu 105545-AAV9).
I-DNA eyongezelelekileyo ye-Mfn1 kunye ne-Mfn2 (efunyenwe kwi-Addgene plasmid #23212 kunye ne-#23213, ngokulandelelana) ziphawulwe ngolandelelwano lwe-V5 (GKPIPNPLLGLDST) kwi-C-terminus, kwaye zidityaniswe ne-mCherry kwisakhelo ngokusebenzisa ulandelelwano lwe-T2A. I-Grx1-roGFP2 sisipho esivela ku-Heidelberg TP Dick DFKZ (Deutsches Krebsforschungszentrum). Ngokutshintsha ikhasethi ye-tdTomato kusetyenziswa iindlela ze-cloning eziqhelekileyo, ikhasethi yafakwa kwi-pAAV-CAG-FLEX-tdTomato backbone (inombolo yesalathiso ye-Addgene 28306) ukuvelisa i-pAAV-CAG-FLEX-mCherry-T2A-MFN2-V5, i-pAAV-CAG-FLEX-mCherry-T2A-MFN1-V5 kunye ne-pAAV-CAG-FLEX-Grx-roGFP2 vectors. Icebo elifanayo lisetyenzisiwe ukuvelisa i-control vector pAAV-CAG-FLEX-mCherry. Ukuze kuveliswe i-AAV-shPCx construct, kufuneka i-plasmid AAV vector (iVectorBuilder, i-pAAV [shRNA] -CMV-mCherry-U6-mPcx- [shRNA#1]), equlathe ulandelelwano lwe-DNA olufaka i-shRNA targeting mouse PCx (5′CTTTCGCTCTAAGGTGCTAAACTCGAGTTTAGCACCCTTAGAGCGAAAG 3′) Phantsi kolawulo lwe-U6 promoter, i-mCherry isetyenziswa phantsi kolawulo lwe-CMV promoter. Ukuveliswa kwee-AAV vectors ezincedisayo kwenziwe ngokwemiyalelo yomenzi (iiCell Biolabs). Ngamafutshane, sebenzisa i-plasmid yokudlulisa ephethe i-mCherry-T2A-MFN2-V5 (pAAV-CAG-FLEX-mCherry-T2A-MFN2-V5), mCherry-T2A-MFN1-V5 (pAAV-CAG-FLEX-mCherry) okwethutyana Ukudluliselwa kweeseli ezingama-293AAV-T2A-MFN1-V5), mCherry (pAAV-CAG-FLEX-mCherry) okanye i-Grx-roGFP2 (pAAV-CAG-FLEX-Grx-roGFP2) i-code gene, kunye nokubhala i-AAV1 capsid protein kunye ne-accessory protein Ukupakisha i-plasmid plasmid, kusetyenziswa indlela ye-calcium phosphate. I-crude virus supernatant ifunyenwe nge-freeze-thaw cycles kwi-dry ice/ethanol bath kunye nee-lysed cells kwi-phosphate buffered saline (PBS). I-AAV vector icocwe nge-discontinuous iodixanol gradient ultracentrifugation (iiyure ezingama-24 kwi-32,000 rpm kunye ne-4°C) yaze yaxutywa kusetyenziswa isihluzi se-Amicon ultra-15 centrifugal. I-Genome titer ye-AAV1-CAG-FLEX-mCherry-T2A-MFN2-V5 [2.9×1013 genome copy (GC)/ml], AAV1-CAG-FLEX-mCherry (6.1×1012 GC/ml), AAV1-CAG-FLEX yayinjengoko kuchaziwe ngaphambili (54), ilinganiswa yi-real-time quantitative PCR (qPCR) -MFN1-V5 (1.9×1013 GC/ml) kunye ne-AAV1-CAG-FLEX-Grx-roGFP2 (8.9×1012 GC/ml).
Ii-neurons eziphambili zakhutshelwa kwi-1x PBS ebandayo yomkhenkce, zaza zaxutywa, zaza zafakwa kwi-homogenized kwi-0.5% Triton X-100 / 0.5% sodium deoxycholate/PBS lysis buffer equlethe i-phosphatase kunye ne-protease inhibitor (Roche). Ukulinganiswa kweproteni kwenziwe ngokusebenzisa uvavanyo lwe-bicinchoninic acid (Thermo Fisher Scientific). Ezi protein zahlulwa yi-SDS-polyacrylamide gel electrophoresis, zaza zacinywa kwi-polyvinylidene fluoride membrane (GE Healthcare). Vimba iindawo ezingezizo ezithile kwaye uzifake kwi-primary antibody (jonga iTheyibhile S1 ngeenkcukacha) kubisi lwe-5% kwi-TBST (Tris-buffered saline ene-Tween), amanyathelo okuhlamba kunye ne-secondary antibody kwi-TBST Incubate. Zifake kwi-primary antibody ubusuku bonke kwi-+4°C. Emva kokuhlamba, faka i-secondary antibody iiyure ezi-2 kubushushu begumbi. Emva koko, ngokufaka i-blot efanayo nge-antibody ye-anti-β-actin, umthwalo ofanayo waqinisekiswa. Ukufunyanwa ngokuguqula i-chemiluminescence kunye nokuphucula i-chemiluminescence (GE Healthcare).
Ii-neurons ezazityalwe ngaphambili kwi-glass coverlips zilungisiwe nge-4% ye-paraformaldehyde (PFA)/PBS ngexesha elichaziweyo kubushushu begumbi imizuzu eli-10. I-coverlips ziqala zifakwe i-0.1% yeTriton X-100/PBS imizuzu emi-5 kubushushu begumbi, emva koko zifakwe kwi-blocking buffer [3% ye-bovine serum albumin (BSA)/PBS]. Ngosuku lwesibini, i-coverlips zahlanjwa nge-blocking buffer zaza zafakwa kwi-incubator efanelekileyo ye-fluorophore-conjugated secondary antibody kangangeeyure ezi-2 kubushushu begumbi; ekugqibeleni, iisampulu zahlanjwa kakuhle kwi-PBS nge-4′,6-diamidino-2-Phenylindole (DAPI) echatshazelweyo yaze yabekwa kwi-microscope slide nge-Aqua-Poly/Mount.
Iimpuku (eziziinkunzi neziziimazi) zathotywa i-anesthesia ngokujova i-ketamine (130 mg/kg) kunye ne-xylazine (10 mg/kg) ngaphakathi kwesisu kwaye zanikwa ngaphantsi kwesisu nge-carprofen analgesic (5 mg/kg), kwaye zafakwa kwisixhobo se-stereotactic (Kopf) esine-pad efudumeleyo. Vula ukhakhayi kwaye usebenzise i-dental drill ukuze unciphise inxalenye ye-cerebellar cortex ehambelana nethambo le-mis (ukusuka kwi-lambda: umsila 1.8, ecaleni 1, ehambelana ne-lobules IV kunye ne-V). Sebenzisa inaliti yesirinji egobileyo ukwenza ngononophelo umngxuma omncinci kwi-skull ukuze uphephe ukuphazamisa imithambo yegazi engezantsi. Emva koko i-capillary yeglasi ebhityileyo etsaliweyo ifakwa kancinci kwi-micro-hole (ukusuka kwi--1.3 ukuya kwi--1 kwicala le-ventral le-dura mater), kwaye i-200 ukuya kwi-300 nl AAV ifakwa kwi-micro-injector (Narishige) ngeesirinji ezenziwe ngesandla (Narishige) izihlandlo ezininzi ngoxinzelelo oluphantsi kangangexesha elithile imizuzu eli-10 ukuya kwengama-20 iwindow. Emva kokufakwa kwe-infusion, beka i-capillary eminye imizuzu eli-10 ukuze intsholongwane isasazeke ngokupheleleyo. Emva kokuba i-capillaries isusiwe, ulusu luyathungwa ngononophelo ukunciphisa ukudumba kwamanxeba nokuvumela isilwanyana ukuba siphile. Izilwanyana zanyangwa ngamayeza okunciphisa iintlungu (caspofen) kangangeentsuku ezimbalwa emva kotyando, apho imeko yazo yomzimba yayijongwa ngononophelo kwaye emva koko zabulawa ngexesha elichaziweyo. Zonke iinkqubo zenziwa ngokuhambelana nezikhokelo zaseYurophu, zesizwe nezeziko kwaye zavunywa yiLandesamtfürNatur yase-Umwelt naseVerbraucherschutz, eNorth Rhine-Westphalia, eJamani.
Izilwanyana zafakwa i-anesthesia nge-ketamine (100 mg/kg) kunye ne-xylazine (10 mg/kg), kwaye intliziyo yafakwa i-0.1 M PBS kuqala, yaza emva koko yafakwa i-4% PFA kwi-PBS. Izicubu zanqunyulwa zaza zafakwa kwi-4% PFA/PBS ubusuku bonke kwi-4°C. Imela engcangcazelayo (Leica Microsystems GmbH, eVienna, eAustria) yasetyenziswa ukulungiselela amacandelo e-sagittal (50 μm ubukhulu) ukusuka kwingqondo ezinzileyo kwi-PBS. Ngaphandle kokuba kuchazwe ngenye indlela, ukudaywa kwamacandelo ahamba ngokukhululekileyo kwenziwa njengoko kuchaziwe apha ngasentla (13) kubushushu begumbi kunye nokuxukuxa. Ngamafutshane, okokuqala, izilayi ezifunyenweyo zafakwa i-0.5% Triton X-100/PBS imizuzu eli-15 kubushushu begumbi; kwezinye ii-epitopes (Pcx kunye neShmt2), nge-tris-EDTA buffer kwi-80°C (PH 9) fudumeza izilayi imizuzu engama-25 endaweni yale nyathelo. Okulandelayo, la macandelo afakwe i-antibody yokuqala (jonga iTheyibhile S1) kwi-blocking buffer (3% BSA/PBS) kwi-4°C ubusuku bonke ngokuxukuxa. Ngosuku olulandelayo, la macandelo ahlanjwa nge-blocking buffer kwaye afakwa i-incubator efanelekileyo ye-fluorophore-conjugated secondary antibody kangangeeyure ezi-2 kubushushu begumbi; ekugqibeleni, la macandelo ahlanjwa kakuhle kwi-PBS, afakwe i-counter-stain nge-DAPI, aze alungiswe nge-AquaPolymount kwi-microscope slide.
I-laser scanning confocal microscope (TCS SP8-X okanye TCS Digital Light Sheet, Leica Microsystems) exhotyiswe nge-white light laser kunye ne-405 diode ultraviolet laser yasetyenziswa ukuzoba isampuli. Ngokushukumisa i-fluorophore kunye nokuqokelela isignali nge-Hybrid Detector (HyDs), isoftware ye-LAS-X yasetyenziswa ukuqokelela imifanekiso eqingqiweyo ehambelana nesampulu yeNyquist kwimo yokulandelelana: kwiiphaneli ezingezizo ezobuninzi, ziisignali ezinamandla kakhulu (umzekelo, kwiiseli ze-somatic kunye ne-dendrites) mtYFP) Sebenzisa i-HyD ukubona inani lee-PN kwimo yeBrightR). Ukulinganisa ukusuka kwi-0.3 ukuya kwi-6 ns kusetyenziswa ukunciphisa imvelaphi.
Umzobo wexesha langempela weeseli ezihleliweyo. Emva kokuhlelwa kwi-Neurobasal-A medium equlethe i-1% B27 supplement kunye ne-0.5 mM GlutaMax, iiseli zatyalwa ngoko nangoko kwiislayidi zeglasi ezine-poly-l-lysine (μ-Slide8 Well, Ibidi, inombolo yekhathalogu 80826), Emva koko zigcinwe kwi-37°C kunye ne-5% CO2 kangangeyure e-1 ukuvumela iiseli ukuba zihlale. Umzobo wexesha langempela wenziwe kwi-microscope ye-Leica SP8 laser scanning confocal exhotyiswe nge-laser emhlophe, i-HyD, ilensi yeoyile ye-63×[1.4 numerical aperture (NA)] kunye nesigaba sokufudumeza.
Impuku yakhawuleziswa yaza yanqunyulwa intloko, ingqondo yasuswa ngokukhawuleza ekhayeni, yaza yanqunyulwa yaba bubukhulu obuyi-200μm (kwi-13C labeling experiment) okanye ubukhulu obuyi-275μm (kwi-photon experiments ezimbini) icandelo le-sagittal elizaliswe zezi zinto zilandelayo. I-ayisikhrim (HM-650 V, Thermo Fisher Scientific, Walldorf, eJamani) izaliswe zezi zinto zilandelayo: 125 mM ice-cold, carbon-saturated (95% O2 kunye ne-5% CO2) iphantsi 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 kunye ne-3.5 mM MgCl2 (uxinzelelo lwe-osmotic lwe-310 ukuya kwi-330 mmol). Dlulisa izilayi zobuchopho ezifunyenweyo kwigumbi lokufunxa ngaphambi kokufunxa eliqulethe i-Ca2 + ACSF ephezulu (125.0 mM NaCl, 2.5 mM KCl, 1.25 mM sodium phosphate buffer, 25.0 mM NaHCO3, 25.0 mM d-glucose, 1.0 mM CaCl2 kunye ne-2.0 mM MgCl2) Medium) pH 7.4 kunye ne-310 ukuya kwi-320 mmol).
Ngexesha lenkqubo yokufota, izilayi zafuduselwa kwigumbi elikhethekileyo lokufota, kwaye uvavanyo lwenziwa phantsi kokuphefumla okuqhubekayo kwe-ACSF kubushushu obungaguqukiyo obuyi-32° ukuya kwi-33°C. I-microscope yokuskena nge-laser ene-multiphoton (TCS SP8 MP-OPO, Leica Microsystems) exhotyiswe ngelensi yeLeica 25x objective (NA 0.95, water), i-Ti: Sapphire laser (Chameleon Vision II, Coherent) yasetyenziswa kwimifanekiso yezilayi. Imodyuli yeFLIM (PicoHarp300, PicoQuant).
I-FLIM ye-Grx1-roGFP2. Utshintsho kwimeko ye-cytoplasmic redox ye-PNs lulinganiswe yi-FLIM ye-photon ezimbini kwiislices zobuchopho be-sagittal, apho i-Grx1-roGFP2 biosensor ijolise kwi-PNs. Kumaleko we-PN, intsimi yokufumana ikhethwa malunga ne-50 ukuya kwi-80 μm ngaphantsi komphezulu wesilayi ukuqinisekisa ukuba kukho i-PN esebenzayo (oko kukuthi, ukungabikho kwesakhiwo esineentsimbi okanye utshintsho lwe-neuronal morphological ecaleni kwe-dendrites) kunye ne-double positive roGFP2 sensor kunye ne-AAV encoding shRNA PCx okanye ulandelelwano lwayo lolawulo (i-mCherry nganye eveza ngokubambisana). Qokelela imifanekiso ye-single-stack ene-2x digital zoom [ububanzi bokuchukumisa: 890 nm; 512 nm 512 pixels]. Ukufumanisa: i-HyD yangaphakathi, iqela lesihluzi se-fluorescein isothiocyanate (FITC)] kunye ne-avarage yomfanekiso ngaphakathi kwemizuzu emi-2 ukuya kwemi-3 isetyenziselwa ukuqinisekisa ukuba ii-photon ezaneleyo ziqokelelwe (ii-photon ezili-1000 zizonke) zokufakela i-curve. Uvakalelo lweprobe yeGrx1-roGFP2 kunye nokuqinisekiswa kweemeko zeFLIM kwenziwe ngokujonga ixabiso lobomi be-roGFP2 xa kongezwa i-10 mM H2O2 yangaphandle kwi-ACSF yokuphefumla (ukwandisa i-oxidation, okubangela ukwanda kobomi), uze wongeze i-2 mM dithiothreitol (kunciphisa inqanaba lokunciphisa, okubangela ukwehla kobomi) (Umfanekiso S8, D ukuya ku-G). Sebenzisa isoftware yeFLIMfit 5.1.1 ukuhlalutya iziphumo ezifunyenweyo, ulungelelanise i-single exponential decay curve yomfanekiso wonke kwi-IRF elinganisiweyo (umsebenzi wokuphendula kwesixhobo), kwaye i-χ2 imalunga ne-1. Ukubala ubomi be-PN enye, imaski ejikeleze umzimba wemithambo-luvo yatsalwa ngesandla, kwaye ubomi obuqhelekileyo kwimaski nganye yasetyenziselwa ukulinganisa.
Uhlalutyo lwe-Mitochondrial potential. Emva kokuba i-acute section ifakwe i-100 nM TMRM yongezwe ngqo kwi-ACSF ecociweyo kangangemizuzu engama-30, utshintsho lwe-mitochondrial potential lwe-PNs lwalinganiswa nge-microscope ye-two-photon. I-TMRM imaging yenziwe ngokushukumisa i-probe kwi-920 nm kunye nokusebenzisa i-HyD yangaphakathi (tetramethylrhodamine isothiocyanate: 585/40 nm) ukuqokelela imiqondiso; ngokusebenzisa ubude be-excitation obufanayo kodwa kusetyenziswa i-HyD yangaphakathi eyahlukileyo (FITC :525/50) kumfanekiso we-mtYFP. Sebenzisa i-plug-in ye-ImageJ's Image Calculator ukuvavanya amandla e-mitochondrial kwinqanaba leseli enye. Ngamafutshane, i-plug-in equation: signal = min (mtYFP, TMRM) isetyenziselwa ukuchonga ummandla we-mitochondrial obonisa uphawu lwe-TMRM kwiPurkinje Somali kumfanekiso we-single-stack confocal wetshaneli ehambelanayo. Emva koko indawo ye-pixel kwimaski ephumayo iyalinganiswa, ize imiselwe kumfanekiso ohambelanayo we-threshold single-stack wetshaneli ye-mtYFP ukuze kufunyanwe i-mitochondrial fraction ebonisa amandla e-mitochondrial.
Umfanekiso ususwe kwi-software yeHuygens Pro (Scientific Volume Imaging). Kwimifanekiso eskeniweyo yeethayile, i-montage yethayile enye yenziwa kusetyenziswa i-algorithm yokuthunga ngokuzenzekelayo ebonelelwe yisoftware ye-LAS-X. Emva kokulinganisa umfanekiso, sebenzisa i-ImageJ kunye ne-Adobe Photoshop ukuze uqhubeke nokucubungula umfanekiso kwaye ulungise ukukhanya kunye nomahluko ngokulinganayo. Sebenzisa i-Adobe Illustrator ukulungiselela imizobo.
Uhlalutyo lokugxila kwe-mtDNA. Inani lezilonda ze-mtDNA lilinganiswe kwiindawo ze-cerebellar ezibhalwe ngee-antibodies ezichasene ne-DNA nge-confocal microscope. Indawo nganye ekujoliswe kuyo yenzelwe umzimba weseli kunye ne-nucleus yeseli nganye, kwaye indawo efanelekileyo yabalwa kusetyenziswa i-Multi Measure plug-in (isoftware ye-ImageJ). Susa indawo yenyukliya kwindawo yomzimba weseli ukuze ufumane indawo ye-cytoplasmic. Okokugqibela, i-Analyze Particles plug-in (isoftware ye-ImageJ) yasetyenziswa ukulinganisa ngokuzenzekelayo amanqaku e-cytoplasmic DNA abonisa i-mtDNA kumfanekiso ongaphantsi, kwaye iziphumo ezifunyenweyo zahlengahlengiswa zibe ngumyinge we-PN weempuku ze-CTRL. Iziphumo zibonakaliswa njengenani eliqhelekileyo lee-nucleosides ngeseli nganye.
Uhlalutyo lokubonakaliswa kweproteni. Sebenzisa i-plug-in ye-ImageJ's Image Calculator ukuvavanya ukubonakaliswa kweproteni kwi-PN kwinqanaba leseli enye. Ngamafutshane, kumfanekiso we-confocal we-single-layer yetshaneli ehambelanayo, ngokusebenzisa i-equation: signal = min (mtYFP, antibody), ummandla we-mitochondrial obonisa ukusabela komzimba kwi-antibody ethile ePurkina uyachongwa. Emva koko indawo ye-pixel kwimaski ephumayo iyalinganiswa, ize imiselwe kumfanekiso we-single-stack ohambelanayo wetshaneli ye-mtYFP ukuze kufunyanwe i-mitochondrial fraction yeproteni ebonisiweyo.
Uhlalutyo loxinano lweeseli zePurkinje. I-plug-in yeCell Counter yeImageJ isetyenziselwe ukuvavanya uxinano lwePurkinje ngokwahlula inani leeseli zePurkinje ezibalwe ngobude beringi yecerebellar ehlala iiseli ezibaliweyo.
Ukulungiswa kunye nokuqokelelwa kweesampuli. Ubuchopho obuvela kwiqela lolawulo kunye neempuku zeMfn2cKO zilungisiwe kwi-2% PFA/2.5% glutaraldehyde kwi-0.1 M phosphate buffer (PB), kwaye emva koko kwalungiswa iindawo ze-coronal kusetyenziswa ii-ciliates (Leica Mikrosysteme GmbH, eVienna, eAustria) (Ubukhulu obuyi-50 ukuya kwi-60 μm). Emva koko lungisa kwi-PB buffer kwi-1% os tetraoxide kunye ne-1.5% potassium ferrocyanide kubushushu begumbi kangangeyure e-1. Ezi ndawo zahlanjwa kathathu ngamanzi acocekileyo, zaza zafuthwa nge-70% ethanol equlethe i-1% uranyl acetate imizuzu engama-20. Ezi ndawo zaza zasuswa emanzini kwi-graded alcohol zaza zafakwa kwi-Durcupan ACM (Araldite casting resin M) epoxy resin (Electron Microscopy Sciences, inombolo yekhathalogu 14040) phakathi kwee-slides zeglasi ezine-silicon-coated, kwaye ekugqibeleni kwi-60°C Polymerize kwi-oven iiyure ezingama-48. Indawo ye-cerebellar cortex yakhethwa kwaye iindawo ezincinci kakhulu ezingama-50 nm zasikwa kwiLeica Ultracut (Leica Mikrosysteme GmbH, eVienna, eAustria) zaze zakhethwa kwigridi ye-copper slit ye-2×1 mm egqunywe ngefilimu ye-polystyrene. Ezi ndawo zafakwa idayi ngesisombululo se-4% uranyl acetate kwi-H2O kangangemizuzu eli-10, zahlanjwa nge-H2O izihlandlo ezininzi, emva koko zahlanjwa nge-lead citrate kaReynolds kwi-H2O kangangemizuzu eli-10, zaza zahlanjwa nge-H2O izihlandlo ezininzi. Ii-Micrographs zathathwa nge-transmission electron microscope uPhilips CM100 (Thermo Fisher Scientific, Waltham, MA, USA) kusetyenziswa ikhamera yedijithali ye-TVIPS (Tietz Video and Image Processing System) iTemCam-F416 (TVIPS GmbH, Gauting, USA). Germany).
Kwiimpuku ezine-AAV, ubuchopho bahlulwe baza banqunyulwa baba yinxalenye yesagittal enobukhulu obuyi-1 mm, kwaye i-cerebellum yahlolwa kusetyenziswa i-microscope ye-fluorescence ukuchonga iringi ene-AAV (oko kukuthi, i-mCherry expressing). Kuphela kuvavanyo apho i-AAV injection iphumela ekusebenzeni kakuhle kakhulu kwe-transduction yomaleko weseli yePurkinje (oko kukuthi phantse umaleko wonke) ubuncinane kwiiringi ezimbini ezilandelelanayo ze-cerebellar ezisetyenziswayo. I-AAV-transduced loop yanqunyulwa kwi-microscope ukuze ilungiswe ubusuku bonke (4% PFA kunye ne-2.5% glutaraldehyde kwi-0.1 M cocoate buffer) kwaye yaqhubekeka isetyenzwa. Kwi-EPON embedding, i-fixed tissue yahlanjwa nge-0.1 M sodium cocoate buffer (Applichem), yaza yafakwa kwi-2% OsO4 (os, Science Services; Caco) kwi-0.1 M sodium cocoate buffer (Applichem) Iiyure ezi-4, emva koko ihlanjwe iiyure ezi-2. Phinda amaxesha ama-3 nge-0.1 M cocamide buffer. Emva koko, uthotho lwe-ethanol olunyukayo lwasetyenziswa ukufunxa isisombululo ngasinye se-ethanol kwi-4°C imizuzu eli-15 ukuze kunyibilikiswe izicubu. Izicubu zadluliselwa kwi-propylene oxide zaza zafakwa ubusuku bonke kwi-EPON (Sigma-Aldrich) kwi-4°C. Beka izicubu kwi-EPON entsha kubushushu begumbi iiyure ezi-2, uze uzifake kwi-62°C iiyure ezingama-72. Sebenzisa i-ultramicrotome (Leica Microsystems, UC6) kunye nemela yedayimani (Diatome, Biel, Switzerland) ukusika amacandelo ama-70 nm acetate acetate, uze ufake i-1.5% uranyl acetate imizuzu eli-15 kwi-37°C, uze ufake i-lead citrate solution imizuzu emi-4. Ii-electron micrographs zathathwa kusetyenziswa i-JEM-2100 Plus transmission electron microscope (JEOL) exhotyiswe ngeCamera OneView 4K 16-bit (Gatan) kunye ne-DigitalMicrograph software (Gatan). Uhlalutyo, ii-micrographs ze-electron zifunyenwe nge-5000× okanye i-10,000× digital zoom.
Uhlalutyo lwe-morphological ye-mitochondria. Kuzo zonke ii-analysis, ii-contours ze-mitochondria nganye zichazwe ngesandla kwimifanekiso yedijithali kusetyenziswa isoftware ye-ImageJ. Iiparameter ezahlukeneyo ze-morphological ziyahlalutywa. Uxinano lwe-mitochondrial lubonakaliswa njengepesenti efunyenwe ngokwahlula indawo iyonke ye-mitochondrial yeseli nganye ngendawo ye-cytoplasm (indawo ye-cytoplasm = indawo yeseli-nucleus yeseli) × 100. Ukujikeleza kwe-mitochondria kubalwa ngefomula [4π∙ (indawo/i-perimeter 2)]. I-ista morphology ye-mitochondria ihlalutywe yaza yahlulwahlulwa yaba ngamacandelo amabini ("tubular" kunye "ne-blister") ngokweemilo zazo eziphambili.
Uhlalutyo lwenani le-Autophagosome/lysosome kunye noxinano. Sebenzisa isoftware ye-ImageJ ukuchaza ngesandla imilo ye-autophagosome/lysosome nganye kumfanekiso wedijithali. Indawo ye-Autophagosome/lysosome ichazwa njengepesenti ebalwe ngokwahlula indawo iyonke yesakhiwo se-autophagosome/lysosome yeseli nganye ngendawo ye-cytoplasm (indawo ye-cytoplasm=indawo yeseli-nucleus)×100. Uxinano lwee-autophagosomes/lysosomes lubalwa ngokwahlula inani lilonke ngenani lezakhiwo ze-autophagosome/lysosome ngeseli nganye (ngokwendawo ye-cytoplasmic) (indawo ye-cytoplasmic = indawo yeseli-indawo yenyukliya).
Ukubhala iilebheli zokwahlulahlula ngokukhawuleza kunye nokulungiselela isampuli. Kwizilingo ezifuna ukulebhelishwa kweglucose, thumela izilayi zobuchopho ezibukhali kwigumbi langaphambi kokufunxa, eliqulethe ikhabhoni eyomileyo (95% O2 kunye ne-5% CO2), iCa2 + ACSF ephezulu (125.0 mM NaCl, 2.5 mM KCl, 1.25 mM sodium phosphate buffer, 25.0 mM NaHCO3, 25.0 mM d-glucose, 1.0 mM CaCl2 kunye ne-2.0 mM MgCl2, ehlengahlengisiweyo kwi-pH 7.4 kunye ne-310 ukuya kwi-320 mOsm), apho iglucose yi-13 C 6- Ukutshintshwa kweglucose (i-Eurisotop, inombolo yekhathalogu CLM-1396). Kwizilingo ezifuna ukulebhelwa kwe-pyruvate, dlulisela izilayi zobuchopho ezibukhali kwi-Ca2 + ACSF ephezulu (125.0 mM NaCl, 2.5 mM KCl, 1.25 mM sodium phosphate buffer, 25.0 mM NaHCO3, 25.0 mM d-glucose, 1.0 mM CaCl2 kunye ne-Add 2.0mM MgCl2, lungisa kwi-pH 7.4 kunye ne-310 ukuya kwi-320mOsm), kwaye wongeze i-1mM 1-[1-13C]pyruvate (i-Eurisotop, inombolo yekhathalogu CLM-1082). Faka ezi nxalenye imizuzu engama-90 kwi-37°C. Ekupheleni kovavanyo, la macandelo ahlanjwe ngokukhawuleza ngesisombululo samanzi (pH 7.4) esine-75 mM ammonium carbonate, aze ahlanjululwe kwi-40:40:20 (v:v:v) acetonitrile (ACN): methanol: amanzi. Emva kokuba la macandelo efakwe kwiqhwa imizuzu engama-30, iisampulu zafakwa kwi-centrifuge kwi-21,000 g imizuzu eli-10 kwi-4°C, kwaye i-clear supernatant yomiswa kwi-SpeedVac concentrator. I-metabolite pellet eyomileyo ephumeyo igcinwe kwi--80°C de kube luhlalutyo.
Uhlalutyo lwe-chromatography-mass spectrometry lwe-13 C-labeled amino acids. Kuhlalutyo lwe-liquid chromatography-mass spectrometry (LC-MS), i-metabolite pellet yaphinda yaxhonywa kwi-75μl yamanzi e-LC-MS grade (Honeywell). Emva kwe-centrifugation kwi-21,000 g imizuzu emi-5 kwi-4°C, i-20 μl ye-cleared supernatant yasetyenziswa kuhlalutyo lwe-amino acid flux, ngelixa yonke i-extract yasetyenziswa ngoko nangoko kuhlalutyo lwe-anion (jonga ngezantsi). Uhlalutyo lwe-amino acid lwenziwe kusetyenziswa i-benzoyl chloride derivatization protocol echazwe ngaphambili (55, 56). Kwinyathelo lokuqala, i-10μl ye-100 mM sodium carbonate (Sigma-Aldrich) yongezwa kwi-20μl ye-metabolite extract, kwaye emva koko i-10μl ye-2% benzoyl chloride (Sigma-Aldrich) yongezwa kwi-LC grade ACN. Isampuli ixutywe okwethutyana yaze yafakwa kwi-centrifuge kwi-21,000 g imizuzu emi-5 kwi-20°C. Dlulisa i-supernatant ecocekileyo kwi-2 ml autosampler vial ene-conical glass insert (200 μl volume). Iisampulu zihlalutywe kusetyenziswa inkqubo ye-Acquity iClass ultra-high performance LC (Waters) eqhagamshelwe kwi-Q-Exactive (QE)-HF (Ultra High Field Orbitrap) high-resolution precision mass spectrometer (Thermo Fisher Scientific). Uhlalutyo, i-2μl yesampulu ekhutshweyo ifakwe kwikholamu ye-silica T3 (Waters) eyi-100×1.0 mm high-strength (Waters) equlethe ii-particles ze-1.8μm. Isantya sokuhamba yi-100μl/min, kwaye inkqubo ye-buffer ine-buffer A (10 mM ammonium formate kunye ne-0.15% formic acid emanzini) kunye ne-buffer B (ACN). I-gradient yile ilandelayo: 0%B kwimizuzu eyi-0; 0%B. 0 ukuya kwi-15% B kwimizuzu eyi-0 ukuya kwi-0.1; 15 ukuya kwi-17% B kwimizuzu eyi-0.1 ukuya kwi-0.5; B kwi-17 ukuya kwi-55% kwimizuzu eyi-0.5 ukuya kwi-14; B kwi-55 ukuya kwi-70% kwimizuzu eyi-14 ukuya kwi-14.5; kwi-14.5 ukuya kwi-70 ukuya kwi-100% B kwimizuzu eyi-18; 100% B kwimizuzu eyi-18 ukuya kwi-19; 100 ukuya kwi-0% B kwimizuzu eyi-19 ukuya kwi-19.1; 0% B kwimizuzu eyi-19.1 ukuya kwi-28 (55, 56). I-QE-HF mass spectrometer isebenza kwimo ye-ionization elungileyo enoluhlu lobunzima be-m/z (umlinganiselo wobunzima/wetshaja) oluyi-50 ukuya kwi-750. Isisombululo esisetyenzisiweyo yi-60,000, kwaye ithagethi ye-gain control (AGC) ion efunyenweyo yi-3×106, kwaye ixesha eliphezulu le-ion liyi-100 milliseconds. Umthombo we-heated electrospray ionization (ESI) usebenza kwi-spray voltage ye-3.5 kV, ubushushu be-capillary obuyi-250°C, i-sheath airflow engama-60 AU (iiyunithi ezingacwangciswanga), kunye ne-airflow encedisayo engama-20 AU. 250°C. Ilensi ye-S isetelwe kwi-60 AU.
Uhlalutyo lwe-anion chromatography-MS lwe-13C organic acids ebhalwe. I-metabolite precipitate eseleyo (55μl) ihlalutywe kusetyenziswa inkqubo ye-Dionex ion chromatography (ICS 5000+, Thermo Fisher Scientific) eqhagamshelwe kwi-QE-HF mass spectrometer (Thermo Fisher Scientific). Ngamafutshane, i-5μl ye-metabolite extract ifakwe kwikholamu ye-Dionex IonPac AS11-HC exhotyiswe nge-HPLC (2 mm×250 mm, ubukhulu be-particle 4μm, Thermo Fisher Scientific) kwimo ye-push-in partial loop ene-filling ratio ye-1. ) Ikholamu yomlindi ye-Dionex IonPac AG11-HC (2 mm x 50 mm, 4μm, Thermo Fisher Scientific). Ubushushu bekholamu bugcinwa kwi-30°C, kwaye i-autosampler isethwe kwi-6°C. Sebenzisa i-potassium hydroxide cartridge enikwe amanzi acocekileyo ukuvelisa i-potassium hydroxide gradient nge-eluent generator. Ukwahlulwa kwee-metabolites ngesantya sokuhamba se-380μl/min, kusetyenziswa le gradient ilandelayo: imizuzu eyi-0 ukuya kweyi-3, i-10 mM KOH; imizuzu emi-3 ukuya kweyi-12, i-10 ukuya kweyi-50 mM KOH; imizuzu eli-12 ukuya kweyi-19, i-50 ukuya kweyi-100 mM KOH; imizuzu eli-19 ukuya kweyi-21, i-100 mM KOH; imizuzu engama-21 ukuya kweyi-21.5, i-100 ukuya kweyi-10 mM KOH. Ikholamu yaphinda yalinganiswa phantsi kwe-10 mM KOH imizuzu eyi-8.5.
Ii-metabolites eziluted zidityaniswe nomsinga we-isopropanol we-150μl/min emva kwekholamu uze uqondiswe kwi-high-resolution mass spectrometer esebenza kwi-negative ionization mode. I-MS ijonga uluhlu lobunzima ukusuka kwi-m/z 50 ukuya kwi-750 enesisombululo se-60,000. I-AGC imiselwe kwi-1×106, kwaye ixesha eliphezulu le-ion ligcinwa kwi-100 ms. Umthombo we-ESI oshushu usebenze kwi-spray voltage ye-3.5 kV. Ezinye izicwangciso zomthombo we-ion zezi zilandelayo: ubushushu be-capillary 275°C; ukuhamba kwegesi yesheath, 60 AU; ukuhamba kwegesi encedisayo, 20 AU kwi-300°C, kunye nokuseta ilensi ye-S ukuya kwi-60 AU.
Uhlalutyo lwedatha lwee-metabolites ezineelebheli ze-13C. Sebenzisa isoftware yeTraceFinder (uhlobo 4.2, iThermo Fisher Scientific) kuhlalutyo lwedatha ye-isotope ratio. Ubume bekhompawundi nganye buqinisekiswe yikhompawundi ethembekileyo kwaye buhlalutywe ngokuzimeleyo. Ukuze kwenziwe uhlalutyo lwe-isotope enrichment, indawo ye-ion chromatogram ekhutshiweyo (XIC) ye-isotope nganye ye-13C (Mn) ikhutshwe kwi-[M + H]+, apho i-n yinombolo yekhabhoni yekhompawundi ekujoliswe kuyo, esetyenziselwa ukuhlalutya ii-amino acids okanye i-[MH] + isetyenziselwa ukuhlalutya ii-anions. Ukuchaneka kobunzima be-XIC kungaphantsi kweenxalenye ezintlanu kwisigidi, kwaye ukuchaneka kwe-RT yimizuzu eyi-0.05. Uhlalutyo lwe-enrichment lwenziwa ngokubala umlinganiselo we-isotope nganye efunyenweyo kwinani lazo zonke ii-isotopes zekhompawundi ehambelanayo. Ezi ratios zinikwe njengexabiso lepesenti kwi-isotope nganye, kwaye iziphumo zibonakaliswa njenge-molar percentage enrichment (MPE), njengoko kuchaziwe ngaphambili (42).
I-pellet ye-neuron eqandisiweyo yenziwe yafana kwi-80% methanol (v/v) ebandayo njengomkhenkce, yaza yafakwa kwi-vortex, yaza yafakwa kwi--20°C imizuzu engama-30. Phinda ujikeleze isampuli uze uyixube kwi-+4°C imizuzu engama-30. Isampuli ifakwe kwi-centrifuge kwi-21,000 g imizuzu emi-5 kwi-4°C, emva koko i-supernatant ephumayo yaqokelelwa yaza yomiswa kusetyenziswa i-SpeedVac concentrator kwi-25°C ukuze kuhlalutywe okulandelayo. Njengoko kuchaziwe apha ngasentla, uhlalutyo lwe-LC-MS lwenziwe kwii-amino acids zeeseli ezihleliweyo. Kusetyenziswa i-TraceFinder (uhlobo 4.2, i-Thermo Fisher Scientific), uhlalutyo lwedatha lwenziwe kusetyenziswa ubunzima be-monoisotopic bekhompawundi nganye. Ukulungiswa kwe-quantile yedatha ye-metabolite kwenziwe kusetyenziswa iphakheji yesoftware ye-preprocessCore (57).
Ukulungiswa kwesilayi. Impuku yakhawuleziswa yaza yanqunyulwa intloko, ingqondo yasuswa ngokukhawuleza ekhayeni, kwaye imela engcangcazelayo egcwele umkhenkce (HM-650 V, Thermo Fisher Scientific, Walldorf, Germany) yasetyenziswa ukuyinqumla ibe ngama-300 ukuya kuma-375 μm sagittal sections I-Cold carbon gasification (95% O2 kunye ne-5% CO2) I-Ca2 ephantsi + 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 kunye ne-6.0 mM MgCl2 Lungisa kwi-pH 7.4 kunye ne-310 ukuya kwi-330 mOsm). Dlulisa izilayi zobuchopho ezifunyenweyo kwigumbi eliqulethe i-Ca2 + ACSF ephezulu (125.0 mM NaCl, 2.5 mM KCl, 1.25 mM sodium phosphate buffer, 25.0 mM NaHCO3, 25.0 mM d-glucose, 4.0 mM CaCl2 kunye ne-mM 3.5 MgCl2) pH 7.4 kunye ne-310 ukuya kwi-320 mOsm). Gcina izilayi imizuzu engama-20 ukuya kwengama-30 ukuze zibuyiselwe ngaphambi kokuba zirekhodwe.
ukurekhoda. Isigaba semakroskopu esixhotyiswe ngegumbi lokurekhoda elizinzileyo kunye nelensi ye-20x yokuntywiliselwa emanzini (iScientifica) yasetyenziswa kuzo zonke iirekhodi. Iiseli zePurkinje ezicingelwayo zichongiwe (i) ngobukhulu bomzimba, (ii) indawo ye-anatomical ye-cerebellum, kunye (iii) ukubonakaliswa kwe-fluorescent mtYFP reporter gene. I-patch pipette enokumelana nencam ye-5 ukuya kwi-11 megohms itsalwa yi-borosilicate glass capillary (GB150-10, 0.86 mm×1.5 mm×100 mm, Science Products, Hofheim, Germany) kunye ne-horizontal pipette Instruments (P-1000, Sutter), Novato, CA). Zonke iirekhodi zenziwe yi-ELC-03XS npi patch clamp amplifier (npi electronic GmbH, Tam, Germany), eyayilawulwa yisoftware iSignal (inguqulelo 6.0, Cambridge Electronic, Cambridge, UK). Olu vavanyo lurekhodwe ngesantya sesampulu se-12.5 kHz. Isignali ihluzwa ngee-short-pass Bessel filters ezimbini ezine-cutoff frequency ze-1.3 kunye ne-10 kHz ngokulandelanayo. I-capacitance ye-membrane kunye ne-pipette ihlawulwa yi-compension circuit esebenzisa i-amplifier. Zonke iimvavanyo zenziwe phantsi kolawulo lwekhamera ye-Orca-Flash 4.0 (eHamamatsu, eGerden, eJamani), eyayilawulwa yisoftware yeHokawo (uhlobo 2.8, eHamamatsu, eGerden, eJamani).
Yenza uhlengahlengiso lweseli epheleleyo rhoqo kunye nohlalutyo. Ngaphambi kokuba urekhode, gcwalisa i-pipette ngesisombululo sangaphakathi esiqulethe ezi zinto zilandelayo: 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) kunye ne-10.0 mM creatinine phosphate zilungiswe kwi-pH 7.25, kwaye uxinzelelo lwe-osmotic yayiyi-290 mOsm (sucrose). Emva kokusebenzisa amandla e-0 pA ukuqhekeza i-membrane, amandla e-membrane aphumlayo alinganiswa. Ukumelana nokungena kulinganiswa ngokusebenzisa i-hyperpolarized currents ye--40, -30, -20, kunye ne--10 pA. Linganisa ubukhulu bempendulo ye-voltage kwaye usebenzise umthetho we-Ohm ukubala ukumelana nokungena. Umsebenzi ozenzekelayo urekhodwe kwi-voltage clamp imizuzu emi-5, kwaye i-sPSC ichongiwe yaza yalinganiswa kwi-Igor Pro (uhlobo 32 7.01, iWaveMetrics, iLake Oswego, iOregon, e-USA) kusetyenziswa iskripthi sokuqaphela esizenzekelayo. I-IV curve kunye ne-steady-state current zilinganiswa ngokucinezela ibhetri kwii-potentials ezahlukeneyo (ukuqala kwi--110 mV) kunye nokunyusa i-voltage ngamanyathelo ama-5 mV. Ukuveliswa kwe-AP kuhlolwe ngokusebenzisa i-depolarizing current. Cinezela iseli kwi--70 mV ngelixa usebenzisa i-depolarizing current pulse. Lungisa ubungakanani besinyathelo seyunithi nganye yokurekhoda ngokwahlukeneyo (10 ukuya kwi-60 pA). Bala i-AP frequency ephezulu ngokubala ngesandla ii-pulse spikes ezibangela i-AP frequency ephezulu. Umda we-AP uhlalutywa ngokusebenzisa i-derivative yesibini ye-depolarization pulse eqala ngokubangela i-AP enye okanye ezingaphezulu.
Uqwalaselo kunye nohlalutyo lwepatch eneembobo. Yenza ukurekhoda kwepatch eneembobo usebenzisa iiprotokholi eziqhelekileyo. Sebenzisa i-pipette engena-ATP kunye ne-GTP engenazo ezi zithako zilandelayo: i-128 mM gluconate K, i-10 mM KCl, i-10 mM Hepes, i-0.1 mM EGTA kunye ne-2 mM MgCl2, kwaye ulungelelanise kwi-pH 7.2 (usebenzisa i-KOH). I-ATP kunye ne-GTP zisusiwe kwisisombululo sangaphakathi kweseli ukuthintela ukungena okungalawulekiyo kwe-membrane yeseli. I-patch pipette izaliswe yisisombululo sangaphakathi esine-amphotericin (malunga ne-200 ukuya kwi-250μg/ml; G4888, Sigma-Aldrich) ukuze ufumane irekhodi yepatch ebhoboziweyo. I-Amphotericin yanyibilikiswa kwi-dimethyl sulfoxide (uxinzelelo lokugqibela: 0.1 ukuya kwi-0.3%; DMSO; D8418, Sigma-Aldrich). Uxinzelelo lwe-DMSO olusetyenzisiweyo aluzange lube nefuthe libalulekileyo kwii-neurons ezifundwe. Ngexesha lenkqubo yokubhoboza, ukumelana kwetshaneli (iRa) kwajongwa rhoqo, kwaye uvavanyo lwaqalwa emva kokuba i-amplitude yeRa kunye ne-AP izinzisiwe (imizuzu engama-20-40). Umsebenzi ozenzekelayo ulinganiswa kwi-voltage kunye/okanye i-current clamp imizuzu emi-2 ukuya kwemi-5. Uhlalutyo lwedatha lwenziwe kusetyenziswa i-Igor Pro (inguqulelo 7.05.2, iWaveMetrics, i-USA), i-Excel (inguqulelo 2010, iMicrosoft Corporation, iRedmond, i-USA) kunye neGraphPad Prism (inguqulelo 8.1.2, iGraphPad Software Inc., iLa Jolla, CA). eMelika). Ukuze kuchongwe ii-AP ezizenzekelayo, kusetyenziswa i-plug-in ye-IgorPro's NeuroMatic v3.0c. Zichonge ngokuzenzekelayo ii-AP kusetyenziswa umda othile, ohlengahlengiswa ngokwahlukeneyo kwirekhodi nganye. Usebenzisa i-spike interval, misela i-spike frequency enomlinganiselo ophezulu we-spike frequency kunye ne-avareji ye-spike frequency.
Ukuhlukaniswa kwe-PN. Ngokuziqhelanisa neprotokholi epapashwe ngaphambili, ii-PN zacocwa kwi-cerebellum yegundane kwinqanaba elithile (58). Ngamafutshane, i-cerebellum yanqunyulwa yaza yanqunyulwa kwindawo yokwahlulahlula ebandayo [ngaphandle kwe-HBSS Ca2+ kunye ne-Mg2+, yongezwa nge-20 mM glucose, i-penicillin (50 U/ml) kunye ne-streptomycin (0.05 mg/ml)], yaze yagaywa indawo kwi-papain [HBSS, yongezwa nge-1-cysteine·HCl (1 mg/ml), i-papain (16 U / ml) kunye ne-deoxyribonuclease I (DNase I; 0.1 mg/ml)] Nyanga imizuzu engama-30 kwi-30°C. Kuqala hlamba izicubu kwindawo ye-HBSS equlethe i-mucus yeqanda (10 mg/ml), i-BSA (10 mg/ml) kunye ne-DNase (0.1 mg/ml) kubushushu begumbi ukuthintela ukugaywa kwe-enzyme, uze emva koko kwindawo ye-HBSS equlethe i-glucose engama-20 mM. Yigaya kancinci kwi-HBSS, i-penicillin (50 U/ml), i-streptomycin (0.05 mg/ml) kunye ne-DNase (0.1 mg/ml) ikhuphe iiseli ezilodwa. Ukumiswa kweseli okubangelwe yiyo kwahluzwa nge-70μm cell strainer, emva koko iiseli zahluzwa nge-centrifugation (1110 rpm, imizuzu emi-5, 4°C) zaza zaphinda zaxutywa kwindawo yokwahlula [HBSS, yongezwa nge-20 mM glucose, i-20% yenkomo ye-fetal) i-Serum, i-penicillin (50 U/ml) kunye ne-streptomycin (0.05 mg/ml)]; vavanya ukuphila kweseli nge-propidium iodide kwaye ulungise uxinano lweseli ukuya kwi-1×106 ukuya kwi-2×106 cells/ml. Ngaphambi kokuba i-flow cytometry iqale, i-suspension yayihluzwa nge-50 μm cell strainer.
I-Flow cytometer. Ukuhlelwa kweeseli kwenziwe kwi-4°C kusetyenziswa umatshini we-FACSAria III (BD Biosciences) kunye nesoftware ye-FACSDiva (BD Biosciences, inguqulelo 8.0.1). Ukumiswa kweeseli kwahlelwa kusetyenziswa i-nozzle ye-100 μm phantsi koxinzelelo lwe-20 psi ngesantya se-~2800 events/sec. Ekubeni iikhrayitheriya zendabuko zokugada (ubungakanani beeseli, ucalucalulo lwe-bimodal, kunye neempawu zokusasazeka) azinakuqinisekisa ukwahlulwa ngokuchanekileyo kwe-PN kwezinye iintlobo zeeseli, icebo lokugada limiselwe ngokuthelekisa ngokuthe ngqo i-YFP intensity kunye ne-autofluorescence kwi-mitoYFP+ ​​​​kunye ne-control mitoYFP − Mice. I-YFP iyavuya ngokukhanyisela isampuli ngomgca we-laser we-488 nm, kwaye isignali ifunyanwa kusetyenziswa isihluzi se-band pass se-530/30 nm. Kwiimpuku ze-mitoYFP+, amandla ahambelanayo e-Rosa26-mitoYFP reporter gene asetyenziswa ukwahlula iziqwenga zomzimba we-neuronal kunye ne-axon. I-7-AAD ivuselelwe yi-laser etyheli ye-561 nm kwaye ifunyenwe ngesihluzo se-bandpass ye-675/20 nm ukuze kukhutshwe iiseli ezifileyo. Ukuze kwahlulwe ii-astrocytes ngaxeshanye, i-cell suspension yadaywa yi-ACSA-2-APC, emva koko isampuli yatshiswa ngomgca we-laser ye-640 nm, kwaye kwasetyenziswa isihluzo se-bandpass ye-660/20 nm ukufumana isignali.
Iiseli eziqokelelweyo zifakwe i-pellet nge-centrifugation (1110 rpm, imizuzu emi-5, 4°C) zaza zagcinwa kwi--80°C de zisetyenziswe. Iimpuku zeMfn2cKO kunye neenjana zazo zihlelwa ngosuku olunye ukunciphisa ukuguquguquka kwenkqubo. Ukuboniswa kwedatha ye-FACS kunye nohlalutyo lwenziwe kusetyenziswa isoftware yeFlowJo (FlowJo LLC, Ashland, Oregon, USA).
Njengoko kukhankanyiwe apha ngasentla (59), i-PCR yexesha langempela isetyenziselwa ukwahlula i-DNA kwii-neurons ezihleliweyo ukuze kulinganiswe i-mtDNA elandelayo. Ulungelelwaniso kunye novakalelo lwe-threshold kwaqala kwavavanywa ngokusebenzisa i-qPCR kwiinombolo ezahlukeneyo zeeseli. Ngamafutshane, qokelela i-300 PN kwi-lysis buffer equlathe i-50 mM tris-HCl (pH 8.5), i-1 mM EDTA, i-0.5% Tween 20 kunye ne-proteinase K (200 ng/ml) kwaye uyifake kwi-55°C imizuzu eli-120. Iiseli zifakwe kwi-95°C ngaphezulu kangangemizuzu eli-10 ukuqinisekisa ukungasebenzi ngokupheleleyo kwe-proteinase K. Kusetyenziswa i-TaqMan probe (Thermo Fisher) ekhethekileyo kwi-mt-Nd1, i-mtDNA ilinganiswe yi-semi-quantitative PCR kwinkqubo ye-7900HT Real-Time PCR (Thermo Fisher Scientific). Isayensi, inombolo yekhathalogu Mm04225274_s1), mt-Nd6 (Thermo Fisher Scientific, inombolo yekhathalogu AIVI3E8) kunye ne-18S (Thermo Fisher Scientific, inombolo yekhathalogu Hs99999901_s1) iijini.
Ukulungiswa kwesampuli yeProteome. Ngokufudumeza isisombululo kwi-95°C imizuzu eli-10 kunye nokunika i-sonicating, kwi-lysis buffer [6 M guanidine chloride, 10 mM tris(2-carboxyethyl) phosphine hydrochloride, 10 mM chloroacetamide kunye ne-100 mM tris-Lyse frozen neuron pellets kwi-HCl]. Kwi-Bioruptor (Diagenode) imizuzu eli-10 (imizuzwana engama-30 ye-pulse / imizuzwana engama-30 yekhefu). Isampuli yaxutywa nge-1:10 kwi-20 mM tris-HCl (pH 8.0), ixutywe ne-300 ng trypsin gold (Promega), yaza yafakwa ubusuku bonke kwi-37°C ukuze kufezekiswe ukugaywa okupheleleyo. Ngosuku lwesibini, isampuli yaxutywa kwi-centrifuge kwi-20,000 g imizuzu engama-20. I-supernatant yaxutywa nge-0.1% formic acid, kwaye isisombululo saxutywa ngeStageTips ezenziwe ngokuzenzekelayo. Isampuli yomiswe kwisixhobo seSpeedVac (Eppendorf concentrator plus 5305) kwi-45°C, yaze ke ipeptide yaxhonywa kwi-0.1% formic acid. Zonke iisampuli zalungiswa ngaxeshanye ngumntu omnye. Ukuze kuhlalutywe iisampuli ze-astrocyte, ii-4 μg zepeptides ezisusiwe ityuwa zaphawulwa nge-tandem mass tag (TMT10plex, inombolo yekhathalogu 90110, Thermo Fisher Scientific) kunye nomlinganiselo we-peptide ukuya kwi-TMT reagent we-1:20. Kwileyibhile ye-TMT, i-0.8 mg ye-reagent ye-TMT yaphinda yaxhonywa kwi-70 μl ye-ACN engenamanzi, kwaye i-peptide eyomileyo yaphinda yahlanganiswa kwi-9 μl ye-0.1 M TEAB (triethylammonium bicarbonate), apho kongezwe i-7 μl ye-reagent ye-TMT kwi-ACN. Uxinzelelo lwaluyi-43.75%. Emva kwemizuzu engama-60 yokufunxa, impendulo yacinywa nge-2 μl ye-5% ye-hydroxylamine. Iipeptides ezibhalwe amagama zaqokelelwa, zomiswa, zaphinda zaxhonywa kwi-200μl ye-0.1% formic acid (FA), zahlulwahlulwa zibe zimbini, zaza zasuswa ityuwa kusetyenziswa iStageTips ezenziwe ngokuzenzekelayo. Kusetyenziswa i-UltiMate 3000 ultra high performance liquid chromatograph (UltiMate 3000 ultra high performance liquid chromatograph), enye yezi halves zimbini yahlulwahlulwa kwikholamu ye-1mm x 150mm Acquity chromatographic ezaliswe zii-particles ze-130Å1.7μm C18 (Waters, catalog No. SKU: 186006935). Thermo Fisher Scientific). Yahlula iipeptides ngesantya sokuhamba se-30μl/min, yahlula ukusuka kwi-1% ukuya kwi-50% ye-buffer B imizuzu engama-85 kunye ne-stepwise gradient yemizuzu engama-96, ukusuka kwi-50% ukuya kwi-95% ye-buffer B imizuzu emi-3, emva koko imizuzu esi-8 ye-95% ye-Buffer B; I-Buffer A yi-5% ye-ACN kunye ne-10 mM ammonium bicarbonate (ABC), kwaye i-buffer B yi-80% ye-ACN kunye ne-10 mM ABC. Qokelela amaqhezu rhoqo emva kwemizuzu emi-3 uze uwadibanise abe ngamaqela amabini (1 + 17, 2 + 18, njl.njl.) uze uwazomise kwi-vacuum centrifuge.
Uhlalutyo lwe-LC-MS/MS. Kwi-mass spectrometry, iipeptides (inombolo r119.aq) zahlulwe kwikholamu yohlalutyo yePicoFrit engama-25 cm, 75 μm ububanzi bangaphakathi (ilensi entsha yenjongo, inombolo yenxalenye PF7508250) exhotyiswe nge-1.9 μm ReproSil-Pur 120 C18-AQ medium (uGqr. Maisch, mat), Sebenzisa i-EASY-nLC 1200 (iThermo Fisher Scientific, eJamani). Ikholamu igcinwe kwi-50°C. IiBuffers A kunye ne-B ziyi-0.1% formic acid emanzini kunye ne-0.1% formic acid kwi-80% ACN, ngokulandelelana. IiPeptides zahlulwe ukusuka kwi-6% ukuya kwi-31% buffer B imizuzu engama-65 kunye ukusuka kwi-31% ukuya kwi-50% buffer B imizuzu emi-5 nge-gradient ye-200 nl/min. Iipeptides eziluted zahlalutywa kwi-Orbitrap Fusion mass spectrometer (iThermo Fisher Scientific). Umlinganiselo we-Peptide precursor m/z wenziwa ngesisombululo se-120,000 kuluhlu oluphakathi kwe-350 ukuya kwi-1500 m/z. Kusetyenziswa amandla e-27% aqhelekileyo okungqubana, i-precursor enamandla kakhulu ene-charge state ye-2 ukuya kwi-6 ikhethwa kwi-high energy C trap dissociation (HCD) cleavage. Ixesha lomjikelo limiselwe kwi-1 s. Ixabiso le-m/z le-peptide fragment lilinganiswe kwi-ion trap kusetyenziswa eyona target incinci ye-AGC ye-5×104 kunye nexesha eliphezulu lokufaka i-injection ye-86 ms. Emva kokuqhekeka, i-precursor ibekwe kuluhlu lwe-dynamic exclusion kangangemizuzwana engama-45. Iipeptides ezineelebheli ze-TMT zahlulwe kwikholamu ye-Acclaim PepMap engama-50 cm, 75 μm (Thermo Fisher Scientific, inombolo yekhathalogu 164942), kwaye ii-spectra zokufuduka zahlalutywa kwi-Orbitrap Lumos Tribrid mass spectrometer (Thermo Fisher Scientific) exhotyiswe ngezixhobo ze-high-field asymmetric waveform ions (FAIMS) (Thermo Fisher Scientific) ezisebenza kwii-voltage ezimbini ze-−50 kunye ne-−70 V. I-MS3 ekhethiweyo ngokusekelwe kwi-synchronous precursor esetyenziselwa ukulinganisa isignali ye-ion yengxelo ye-TMT. Ukwahlulwa kwe-peptide kwenziwe kwi-EASY-nLC 1200, kusetyenziswa i-90% linear gradient elution, enoxinzelelo lwe-buffer oluyi-6% ukuya kwi-31%; i-buffer A yayiyi-0.1% FA, kwaye i-buffer B yayiyi-0.1% FA kunye ne-80% ACN. Ikholamu yohlalutyo isebenza kwi-50°C. Sebenzisa iFreeStyle (inguqulelo 1.6, iThermo Fisher Scientific) ukuze wahlule ifayile yokuqala ngokwe-voltage yembuyekezo ye-FAIMS.
Ukuchonga iiproteni kunye nokulinganisa. Kusetyenziswa injini yokukhangela ye-Andromeda edibeneyo, idatha yokuqala yahlalutywa kusetyenziswa inguqulelo yeMaxQuant 1.5.2.8 (https://maxquant.org/). Ukongeza kwi-Cre recombinase kunye ne-YFP sequences ezifunyenwe kwi-Aequorea victoria, ii-peptide fragment spectra zakhangelwa ulandelelwano lwe-canonical kunye nolandelelwano lwe-isoform lwe-mouse reference proteome (Proteome ID UP000000589, ekhutshelwe kwi-UniProt ngoMeyi 2017). I-Methionine oxidation kunye ne-protein N-terminal acetylation zamiselwa njengotshintsho oluguquguqukayo; i-cysteine ​​​​carbamoyl methylation yamiselwa njengotshintsho oluzinzileyo. Iiparameter zokugaya zimiselwe kwi-"specificity" kunye ne-"trypsin/P". Inani elincinci lee-peptides kunye ne-razor peptides ezisetyenziselwa ukuchonga iiproteni yi-1; inani elincinci lee-peptides ezizodwa yi-0. Phantsi kweemeko zokudibanisa imephu ye-peptide, izinga lokuchonga iiproteni yayiyi-0.01. Ukhetho lwe-"Second Peptide" luyasebenza. Sebenzisa ukhetho "lokudibanisa phakathi kokubaleka" ukuze udlulisele ukuchonga okuphumelelayo phakathi kweefayile zokuqala ezahlukeneyo. Sebenzisa i-LFQ minimum ratio count 1 kwi-label-free quantification (LFQ) (60). Ubunzulu be-LFQ buhluzwa ubuncinane amaxabiso amabini asebenzayo ubuncinane kwiqela elinye le-genotype kwindawo nganye yexesha, kwaye buthathwa kwi-normal distribution enobubanzi be-. 0.3 kwaye uhlehlise i-1.8. Sebenzisa iqonga le-Perseus computing (https://maxquant.net/perseus/) kunye ne-R (https://r-project.org/) ukuhlalutya iziphumo ze-LFQ. Uvavanyo lwe-t oluphakathi oluvela kwiphakheji yesoftware ye-limma lusetyenziselwe uhlalutyo lokubonisa umahluko (61). Uhlalutyo lwedatha yokuhlola lwenziwa kusetyenziswa i-ggplot, i-FactoMineR, i-factoextra, i-GGally kunye ne-pheatmap. Idatha ye-proteomics esekwe kwi-TMT ihlalutywe kusetyenziswa i-MaxQuant version 1.6.10.43. Khangela idatha yeproteomics eluhlaza kwisiseko sedatha se-proteomics somntu se-UniProt, esakhutshelwa ngoSeptemba 2018. Olu hlalutyo luquka i-isotope purity correction factor ebonelelwe ngumvelisi. Sebenzisa i-limma kwi-R kuhlalutyo lokubonisa umahluko. Idatha yokuqala, iziphumo zokukhangela kwisiseko sedatha, kunye nomsebenzi wohlalutyo lwedatha kunye neziphumo zonke zigcinwe kwi-ProteomeXchange alliance ngokusebenzisa indawo yokugcina amaqabane ye-PRIDE ene-data set identifier PXD019690.
Iinkcazo ezisebenzayo ziyayiphucula indlela ezihlalutywa ngayo. Isixhobo se-Ingenuity Pathway Analysis (QIAGEN) sisetyenzisiwe ukumisela ubutyebi bemigqaliselo yenkcazo yokusebenza yedatha ebekwe kwiiveki ezi-8 (Umfanekiso 1). Ngamafutshane, uluhlu lweprotheyini yobuninzi olufunyenwe kuhlalutyo lwedatha ye-LC-MS/MS (tandem mass spectrometry) lusetyenziswa kunye neendlela ezilandelayo zokucoca: I-Mus musculus ikhethwe njengohlobo kunye nemvelaphi, kwaye udidi lubonisa ixabiso le-P elilungisiweyo nguBenjamini ukuze kuphuculwe i-0.05 okanye ngaphantsi lithathwa njengelibalulekileyo. Kule grafu, iindidi ezintlanu eziphezulu ezingaphezulu kwiqela ngalinye ngokusekelwe kwixabiso le-P elilungisiweyo ziyaboniswa. Kusetyenziswa uvavanyo lwe-t oluninzi, kusetyenziswa inkqubo yokunyusa umgca we-Benjamini, Krieger, kunye neYekutieli (Q = 5%), uhlalutyo lokubonakaliswa kweprotheyini yexesha lwenziwa kubaviwa ababalulekileyo abachongiweyo kudidi ngalunye, kwaye umqolo ngamnye uhlalutywa ngokwahlukeneyo. Akukho mfuneko yokwamkela i-SD ehambelanayo.
Ukuze sithelekise iziphumo zolu phononongo kunye needathabheyisi ezipapashiweyo kwaye senze umzobo weVenn kuMfanekiso 1, sidibanise uluhlu lweeprotheyini zobuninzi kunye neenkcazo zeMitoCarta 2.0 (24). Sebenzisa isixhobo esikwi-intanethi iDraw Venn Diagram (http://bioinformatics.psb.ugent.be/webtools/Venn/) ukuvelisa umzobo.
Ukuze ufumane ulwazi oluneenkcukacha ngeenkqubo zezibalo ezisetyenziselwa uhlalutyo lweproteomics, nceda ujonge kwicandelo elifanelekileyo leZixhobo kunye neendlela. Kuzo zonke ezinye iimvavanyo, ulwazi oluneenkcukacha lunokufumaneka kwintsomi ehambelanayo. Ngaphandle kokuba kuchazwe ngenye indlela, yonke idatha ichazwa njenge-mean ± SEM, kwaye lonke uhlalutyo lwezibalo lwenziwe kusetyenziswa isoftware yeGraphPad Prism 8.1.2.
Ukuze ufumane ezinye izinto ezongezelelweyo kweli nqaku, nceda ujonge ku-http://advances.sciencemag.org/cgi/content/full/6/35/eaba8271/DC1
Eli linqaku elivulelekileyo elisasazwa phantsi kwemigaqo yeLayisensi yeCreative Commons Attribution-Non-Commercial, evumela ukusetyenziswa, ukusasazwa kunye nokuveliswa kwakhona kuyo nayiphi na indlela, lo gama nje ukusetyenziswa kokugqibela kungekuko ukuzuza kwezorhwebo kwaye ingqikelelo kukuba umsebenzi wokuqala uchanekile. Isalathiso.
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Ngu-E. Motori, I. Atanassov, SMV Kochan, K. Folz-Donahue, V. Sakthivelu, P. Giavalisco, N. Toni, J. Puyal, N.-G. Larson
Uhlalutyo lwe-Proteomics lwee-neurons ezingasebenzi kakuhle lubonise ukuba iinkqubo ze-metabolic ziyasebenza ukulwa nokuwohloka kwe-neurological.
Ngu-E. Motori, I. Atanassov, SMV Kochan, K. Folz-Donahue, V. Sakthivelu, P. Giavalisco, N. Toni, J. Puyal, N.-G. Larson
Uhlalutyo lwe-Proteomics lwee-neurons ezingasebenzi kakuhle lubonise ukuba iinkqubo ze-metabolic ziyasebenza ukulwa nokuwohloka kwe-neurological.
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