Prɛzɛnt *Adrɛs naw: Kɔlɔn 50931, Jamani, Kɔlɔn Ɛksɛlɛns Klɔsta Risach pan Sɛlular Strɛs Rispɔns insay Ɛj-rilayt Disiz (CECAD).
di nyurodijεnεreshכn fכ di maytochכndrial sik dεm dεn kכnsidr am se i nכ go rεvεr biכs di mεtabolik plastisiti fכ nyuron dεm sכmtεm, bכt di ifekt we di maytochכndrial disfכnkshכn de gi pan di sεl כtonomi fכ nyuronal mεtabolism na di bכdi dεn nכ כndastand am fayn. na ya, wi introdכks di sεl-spεsifi k proteom fכ Purkinje nyuron dεm wit prכgrεsiv OXPHOS dεfisiεns we kכz bay disrupted mitochondrial fכshכn dynamics. wi fכnd se di maytochכndrial disfכnkshכn trigεr wan dip chenj na di fil fכ proteomics, we ultimately lid to di sikεnshal aktibכshכn fכ prεsis mεtabolik program bifo sεl dεd. we wi nכ bin εkspεkt, wi bin disayd di klia indukshכn fכ pyruvate carboxylase (PCx) εn כda anti-aging εnzym dεm we de supliment di intamεdiεt dεm fכ di TCA saykl. inhibishכn fכ PCx exacerbate oksidativ strεs εn nyurodijεnεreshכn, we sho se atεrosklεrosis gεt protεktiv ifekt in nyuron dεm we nכ gεt OXPHOS. di ristכreshכn fכ di maytochכndrial fכs insay di tεminal dεjεnεret nyuron dεm de kכmplit wan rivεs dεn mεtabolik kכntribyushכn dεm ya, we de mek di sεl dεm nכ de day. di tin dεm we wi fכnshכn de sho di path dεm we wi nכ bin no bifo we de gi rεsiliεns to mitochondrial dysfunction εn sho se nyurodijεnεreshכn kin rivεs ivin insay di let stej dεm fכ di sik.
di sεntri rol we maytochכndria de ple fכ mεnten nyuronal enεji mεtabolism de εnfaz bay di εksεnshכn nyurolכjik simptom dεm we de asai wit hכman maytochכndrial sik dεm. כl dis sik dεm de kכz fכ jin mכtεshכn we de rεgεl di maytochכndrial jin εksprεshכn (1, 2) כ jin dεstrukshכn we riliyt to maytochכndrial dinamik, we indayktli afekt di stεbiliti fכ maytochכndrial DNA (mtDNA) (3, 4). di wok we dεn du pan animal mכdel dεn sho se in rispכns to di maytochכndrial disfכnkshכn na di tisu dεm we de rawnd, kכnsyuv mεtabolik path dεm (5-7) kin aktibכt, we de gi imכtant infכmeshכn fכ dip כndastandin fכ di patכjεnεsis fכ dεn kכmpleks sik dεm ya. difrεnt frכm dat, di כndastandin we wi כndastand di mεtabolik chenj dεm fכ spεsifi k sεl tכp dεm we di jεnarכl fεil fכ di bren mitochondrial adenosine triphosphate (ATP) prodakshכn na fכndamεnt (8), we de εnfaz di nid fכ no di tεrapi tכgεt dεm we dεn kin yuz fכ prεvεnt כ prεvεnt sik. Prɛvɛnt nyurodijɛnɛreshɔn (9). di infכmeshכn we nכ de na di fכt se dεn kin kכnsidr di nεv sεl dεm fכ gεt vεri limited mεtabolik fleksibiliti kכmpεr wit di sεl tכp dεm fכ di tisu dεm we de rawnd (10). givεn se dεn sεl dεm ya de ple wan sεntri rol fכ kכdכna di saplai fכ mεtabolit to nyuron dεm fכ protεkt sinaptik transmishכn εn rεspכnd to injuri εn sik kכndishכn dεm, di abiliti fכ adap sεl mεtabolism to di chalenj kכndishכn dεm na di bren tisu na כlmost limited to glial sεl dεm (11-14). apat frכm dat, di inhεrent sεlyul hεterojεnεiti fכ di bren tisu de bכku bכku wan de hεndεr di stכdi fכ mεtabolik chenj dεm we de apin insay spεsifi k nyuronal sכbgrכp dεm. as a rizulכt, dεn nכ no bכku bכt di εksakεt sεlyul εn mεtabolik kכnsekvεns dεm we di maytochכndrial dεm we de wok fayn na di nyuron dεm.
fכ כndastand di mεtabolik kכnsekvεns dεm fכ di maytochכndrial disfכnkshכn, wi aylכt Purkinje nyuron dεm (PN) insay difrεn stej dεm fכ nyurodijεnεreshכn we kכz bay di dεstrukshכn fכ di maytochכndrial כta mεmbran fכshכn (Mfn2). pan tap we Mfn2 mכtayshכn insay mכtalman de sho se i gεt wan fכm fכ hεridita mכtalman sεns nyuropati we dεn kכl Charcot-Marie-Tooth tayp 2A (15), di kכndishכnal dεstrukshכn fכ Mfn2 insay mays na wan we dεn no gud gud wan indukshכn fכ כksidεshכn Fכsfכrayleshכn (OXPHOS) disfכnshכn mεtכd. di difrεn nyuronal sכbtayp dεm (16-19) εn di nyurodijεnεraytiv fεnotayp we de kכmכt de kכmכn wit prכgrεsiv nyurolכjik simptom dεm, lεk muvmεnt dizכrd (18, 19) כ sεribεl ataxia (16). bay we wi yuz wan kכmbaynshכn fכ lεbul-fri kwantiti (LFQ) proteomics, mεtabolכmiks, imej, εn vayrכlכjik mεtכd dεm, wi sho se prכgrεsiv nyurodijεnεreshכn strכng wan de indyuz pyruvate carboxylase (PCx) εn כda fכktכ dεm we involv in arteriosklerosis fכ PN dεm in vivo Di εksprεshכn fכ εnzym dεm. fכ vεrifik di rilevans fכ dis fכnd, wi spεshal dכwn-rεgulεt di εksprεshכn fכ PCx in Mfn2-dεficient PN dεm, εn fכnd se dis כpεreshכn aggravated כksidεtiv strεs εn aksεlεrat nyurodijεnεreshכn, so dat pruv se azoospermia de kכnfεr sεl dεd Mεtabolik adaptabiliti. siriכs εksprεshכn fכ MFN2 kin kכmplitli rεsכk di tεrminal dijεnεreshכn PN wit siriכs OXPHOS dεfisiεns, masiv kכnsכmshכn fכ maytochכndrial DNA, εn i tan lεk se i brok maytochכndrial nεtwכk, we de εnfaz fכs se dis fכm fכ nyurodijεnεreshכn kin ivin rεkכv insay di advans stej fכ sik bifo sεl dεd.
In orda fכ visualize di mitochondria in Mfn2 knockout PNs, wi yuz wan maws strayn we alaw Cre-dipεndεnt mitochondria fכ tכk bכt yכlכ fluorescent protin (YFP) (mtYFP) (20) Cre εksprεshכn εn chεk di mitochondrial mכfכlכji in vivo. wi fכnd se di dεstrukshכn fכ di Mfn2 jin insay PN dεm go lid to di sכmtεm divεlכpmεnt fכ di maytochכndrial nεtwכk (Figure S1A), εn di fכs chenj dεn fכnshכn we i ol 3 wik. difrεnt frכm dat, di sכbstanshכl dεjεnεreshכn fכ di PN sεl layεr, as i pruv bay di lכs fכ Calbindin immunostaining, nכ bigin te 12 wik εj (Figure 1, A εn B). di tεm mismatch bitwin di fכs chenj dεm na di maytochכndrial mכfכlכji εn di visible onset fכ nyuronal dεd promp wi fכ invεstigat di mεtabolik chenj dεm we di maytochכndrial disfכnkshכn trigεr bifo sεl dεd. We divεlכp wan fכrosεns-aktivεt sεl sכt (FACS)-bεys stratεji fכ aylכt YFP (YFP +)-εksprεs PN (Figכ 1C), εn in kכntrכl mays (Mfn2 + / loxP :: mtYFP loxP- stכp-loxP: : L7-cre), we dεn kכl afta dis CTRL (Figכ S1B). Di optimayzeshɔn fɔ di gating strateji bays pan di rilitiv intensiti fɔ di YFP signal alaw wi fɔ purify di YFP+ bɔdi (YFPhigh) fɔ PNs frɔm nɔ-PNs (YFPneg) (Figure S1B) ɔ putativ fluorescent axon/dendritic fragments (YFPlow; Figure S1D, lɛft), kɔnfɔm bay kɔnfɔkal maykroskɔp (Figure S1D, rayt). fכ vεrifik di aydentiti fכ di klas populeshכn, wi kכnεkt LFQ proteomics εn afta dat prinsipal kכmכpכnt analisis, εn fכnd se klia separeshכn de bitwin YFPhigh εn YFPneg sεl dεm (Figure S1C). YFPhigh sel dεm sho nεt εnrichmεnt fכ PNs mak dεm we dεn no (dat na Calb1, Pcp2, Grid2 εn Itpr3) (21, 22), bכt nכ enrichmεnt fכ protin dεm we kכmכn εksprεs insay nyuron כ כda sεl tכp dεm (Figure 1D) ). wan kכmpεreshכn bitwin sεmpl dεm na klas YFPhigh sεl dεm we dεn kכlekt in indipεndεnt εkspεriεns sho wan kכrεleshכn kכfishεnt> 0.9, we de sho gud rεprכdaktiviti bitwin bayolojikal rεplikεt dεm (Figure S1E). In sɔmari, dɛn data ya validet wi plan fɔ akyu ɛn spɛsifi k ayzolayshɔn fɔ fisibul PN. biכs di L7-cre drayva sistεm we dεn yuz de indyuz mosayk rεkombinεshכn in di fכs wik afta dεlivεri (23), wi stat fכ kכl mays frכm CTRL εn kכndishכnal (Mfn2 loxP / loxP :: mtYFP loxP-stop-loxP :: L7-cre) Kכlekt nyuron dεm. afta dεn kכmplit rεkombinεshכn, dεn kכl am Mfn2cKO we i rich 4 wik. as di εnd pכynt, wi pik 8 wik εj we di PN layεr bin intakt pan כl we di klia maytochכndrial fragmεntεshכn (Figure 1B εn Figure S1A). In totכl, wi kwantifay wan totכl 3013 protin dεm, we bכt 22% pan dεm bin bays pan MitoCarta 2.0 anoteshכn bays pan di maytochכndrial proteom as maytochכndria (Figure 1E) (Figure 1E) (24). di difrεnt jin εksprεshכn analisis we dεn du na wik 8 sho se na כnli 10.5% pan כl di protin dεm bin gεt sכm chenj dεm (Figure 1F εn Figure S1F), we 195 protin dεm bin dכn-rεgεlεt εn 120 protin dεm bin כp-rεgulεt (Figure 1F). i fayn fכ no se di “innovativ pathεy analisis” fכ dis data sεt sho se di difrεntli εksprεs jin dεm mεnli de pan wan rεstrikt sεt fכ spεsifi k mεtabolik path dεm (Figure 1G). i intrestin fכ no se pan כl we di dכwnrεgulεshכn fכ di path dεm we riliyt to OXPHOS εn kalsiכm signal de kכnfכm di indukshכn fכ maytochכndrial disfכnkshכn insay fכshכn-dεficient PN dεm, כda kεtכgכri dεm we mεnli involv amino asid mεtabolism de sכmtεm כprεgulεt, we de in layn wit di mεtabolism we de apin insay maytochכndrial mεtabolism Riwayring na kכnsistεnt. disfɔkshɔn.
(A) Riprεsεntεtiv kכnfכkal fכtכgraf dεm fכ sεribεl sεkshכn dεm fכ CTRL εn Mfn2cKO mays dεm we de sho prכgrεsiv lכs fכ PN dεm (calbindin, grey); nyuklios dɛn bin kɔntrastayn wit DAPI. (B) Kwantifikeshɔn fɔ (A) (wan-way analisis fɔ varyans, ***P<0.001; n = 4 to 6 sɛrkɛl frɔm tri mays). (C) Ekspirimɛnt wokflɔ. (D) Hεt map distribushכn fכ mak dεm we spεsifi k to Purkinje (tכp) εn כda sεl tכp dεm (midul). (E) Venn dayagram we de sho di nכmba fכ di maytochכndrial protin dεm we dεn aydentify insay di klas PN. (F) Vכlkeno plot fכ difrεntli εksprεs protin dεm na Mfn2cKO nyuron dεm na 8 wik (signifikεns kכt-כf valyu fכ 1.3). (G) di kriaytiv path analisis sho di fayv mכst imכtant כp-rεgulεshכn (rεd) εn dכwn-rεgulεshכn (blu) path dεm na di Mfn2cKO PN we dεn klas as 8 wik. di avrej εksprεshכn lεvεl fכ εvri protin we dεn dεtekt dεn sho. Greyskayl ɔt map: adjɔst P valyu. ns, nɔ impɔtant.
Proteomics data sho se di protin εksprεshכn fכ kכmpleks I, III, εn IV de dכn sכmtεm. kכmpleks I, III, εn IV כl bin kכntεn εsεnsial mtDNA-εnkod sεbyunit dεm, we kכmpleks II, we bin כnli nyuklia-kכd, bin bεsכl unafεkt (Figure 2A εn Figure S2A). . kכnsist wit di proteomiks risכlt, imyunohistochεmistri fכ di sεribεl tisu sεkshכn dεm sho se di MTCO1 (mitochondrial cytochrome C oxidase subunit 1) sכbyunit lεvεl fכ kכmpleks IV insay PN sכmtεm dכn dכn (Figure 2B). di mtDNA-εnkod sכbyunit Mtatp8 bin sכmtεm rεdכks (Figure S2A), we di stedi-stεt lεvεl fכ di nyuklia-εnkod ATP sεntaz sεbyunit bin rεmain כnchεnj, we kכnsist wit di stebul ATP sεntaz sכbasembly F1 kכmpleks we dεn no we mtDNA εksprεshכn stebul. Di fɔmɛshɔn de kɔnsistɛns. Intarapt (7). Evalueshכn כf di mtDNA lεvεl in di sכt Mfn2cKO PN dεm bay rial-taym polimεrayz chen riakshכn (qPCR) kכnfכm di sכmtεm dεkrεshכn in mtDNA kכpi nכmba. we yu kכmpεr wit di kכntrol grup, we i ol 8 wik, na כnli lεk 20% pan di mtDNA lεvεl bin rεtεn (Figure 2C). kכnsistεnt wit dεn risכlt ya, di kכnfכkal maykroskכpi stεyning fכ Mfn2cKO PN dεm bin yuz fכ dεtekt DNA, we sho di tεm-dipεndεnt kכnsכmshכn fכ maytochכndrial nyukliotayd dεm (Figure 2D). wi fכnd se כnli sכm kandidet dεm we involv in mitochondrial protin dεgradashכn εn strεs rεspכns bin כp-rεgulεt, inklud Lonp1, Afg3l2 εn Clpx, εn OXPHOS kכmpleks assembly fכktכr dεm. nכ signifyant chenj dεm na di lεvεl dεm fכ di protin dεm we involv insay apoptosis dεn bin dεtekt (Figure S2B). sεm kayn we, wi fכnshכn se di maytochכndria εn εndoplasmik rεtikul chεnal dεm we involv insay kalsiכm transpכt gεt כnli sכm sכm chenj dεm (Figure S2C). apat frכm dat, di evalueshכn fכ כtofagi-rεlatεd protin dεm fכn nכ sכm sכm chenj dεm, we kכnsist wit di visible indukshכn fכ כtofagכsכm dεm we dεn obsεv in vivo bay immunohistochemistry εn εlektrכn maykroskכpi (Figure S3). כltu, di prכgrεsiv OXPHOS disfכnkshכn insay PN dεm de kכmכn wit klia ultrastrכkchכral maytochכndrial chenj dεm. yu kin si maytochכndrial kכlכsta dεm na di sεl bכdi dεm εn di dεndritik tik dεm fכ Mfn2cKO PN dεm we ol 5 εn 8 wik, εn di insay mεmbran strכkchכ dεn dכn כnda dip chenj dεm (Figure S4, A εn B). kכnsistεnt wit dεn ultrastrכkchכral chenj dεm ya εn wan siknifikant dεkrεshכn pan mtDNA, analisis fכ akyu sεribral sεribεl slais wit tεtramethylrhodamine mεtyl εsta (TMRM) sho se di maytochכndrial mεmbran pכtεnshal in Mfn2cKO PN dεm bin sכmtεm dεkrεs (Figure S4C).
(A) Taym kɔs analisis fɔ di ɛksprɛshɔn lɛvɛl fɔ OXPHOS kɔmpleks. כnli tink bכt protin dεm wit P<0.05 na 8 wik (tu-way ANOVA). Dotted line: Nɔ ajɔstmɛnt nɔ kɔmpia to CTRL. (B) Lεft: wan egzampl fכ wan sεribra sεkshכn we dεn lεbul wit anti-MTCO1 antibodi (skel bar, 20 μm). di εria we di Purkinje sεl bכdi dεm de kכba am wit yכlכ. Rayt: Kwantifikεshכn כf MTCO1 lεvεl dεm (wan-way analisis כf vεryans; n = 7 to 20 sεl dεm we dεn analכz frכm tri mays). (C) qPCR analisis fכ mtDNA kכpi nכmba in di sכt PN (wan-way analisis fכ vεryans; n = 3 to 7 mays). (D) Lεft: wan egzampl fכ wan sεribra slais we dεn lεbul wit anti-DNA antibodi (skel bar, 20 μm). di εria we di Purkinje sεl bכdi dεm de kכba am wit yכlכ. Rayt: Kwantifikεshכn כf mtDNA lεshכn dεm (wan-way analisis כf vεryans; n = 5 to 9 sεl dεm frכm tri mays). (E) wan egzampl fכ wan akyu sεribεl sεkshכn we de sho mitoYFP + Purkinje sεl dεm (arכ) insay wan ol sεl patch klem rεkכdin. (F) Kwantifikeshɔn fɔ IV kɔv. (G) Riprεzεntεtiv rεkכdin dεm fכ depolarizing kכrant injεkshכn in CTRL εn Mfn2cKO Purkinje sεl dεm. Top trace: Di fכs puls we trigεr AP. Bottom trace: Maksimal AP frikshɔn. (H) Kwantifikeshɔn fɔ di postsinaptik spɔntan input dɛn (sPSP dɛn). Di ripɔtmɛnt rikodin trays ɛn in zum rεshɔn de sho na (I). Wan-way analisis fɔ varyans analayz n = 5 to 20 sɛl frɔm tri mays. Data dεn εksprεs as min±SEM; *P <0.05, ɛn di ɔda wan dɛn; **P <0.01, ɛn di ɔda wan dɛn; ***P<0.001 na di s. (J) Riprεzεntεtiv trays dεm fכ spontan AP we dεn rεkכd yuz di pεrforεt patch klem mכd. Top trace: Maksimal AP frikshɔn. Bottom trace: zoom fɔ wan AP. (K) Kwantifay di avrej ɛn maksimal AP frikshɔn akɔdin to (J). Man-Whitney tɛst we dɛn bin de du; n = 5 sel dεm bin analכz frכm fכ mays. Data dεn εksprεs as min±SEM; nɔ impɔtant.
dεn dεtekt klia OXPHOS dεmεj insay di 8 wik ol Mfn2cKO PN, we sho se di fysiolojikal fכnshכn fכ nyuron dεm na siriכs abnכmal. כl dis mek wi analכz di pasiv ilektrikal kכntribyushכn dεm fכ OXPHOS-deficient nyuron dεm na 4 to 5 wik εn 7 to 8 wik bay we wi du ol-sεl patch klem rεkכdin insay akyu sεribεl slais dεm (Figure 2E). we dεn nכ bin εkspεkt, di avεj rεst mεmbran pכtεnshal εn input rεsistεns fכ Mfn2cKO nyuron dεm bin sεm lεk di kכntrol, pan כl we sכbtil difrεns bin de bitwin sεl dεm (Tεbul 1). semweso, we i ol 4 to 5 wik, dεn nכ fכn nכ sכm chenj dεm na di kכrant-vכltεj rilayshכn (IV kכv) (Figure 2F). כltu, nכ Mfn2cKO nyuron dεm we ol 7 to 8 wiks nכ bin sev di IV rεjim (haypapolarizeshכn stεp), we sho se klia sεnsitiviti de fכ haypapolarizeshכn pכtεnshal na dis let stej. difrεnt frכm dat, insay Mfn2cKO nyuron dεm, di depolarizing kכrant dεm we de mek rεpεtitiv akshכn pכtεnshal (AP) dischaj dεm de tכlerεt fayn fayn wan, we sho se dεn כvala dischaj patεn nכ sכmtεm difrεnt frכm di wan dεm fכ 8 wik ol kכntrol nyuron dεm (Tεbul 1 εn Fig 2G). Semweso, di frɛkuɛns ɛn amplityudɔ fɔ spɔntan postsinaptik kɔrɛnt (sPSC) bin kɔmparabl wit di wan dɛn we de na di kɔntrol grup, ɛn di frɛkuɛns fɔ ivin inkris frɔm 4 wik to 5 wik to 7 wik to 8 wik wit di sem kayn inkris (Figure 2, H ɛn I). di pεriכd fכ sinaptik mεchurεshכn in PN dεm (25). Similar risalts wεn כbtain afta perforated PNs patch. dis kכnfigureshכn de prεvεnt di posεbul kכmpεnsεshכn fכ sεlyul ATP difεkt dεm, lεk aw i kin apin insay di ol-sεl patch klem rεkכdin. in patikyula, di rεst mεmbran pכtεnshal εn spontan fayrin frεkwεns fכ Mfn2cKO nyuron dεm nכ bin afekt (Figure 2, J εn K). in sכmari, dεn risכlt ya sho se PN dεm we gεt klia OXPHOS disfכnkshכn kin kכp wit hכy-frikyuεns dischaj patεn dεm fayn fayn wan, we sho se kכmpεnsεshכn mεkanism de we de alaw dεm fכ mεnten nia-nכmal εlektrofysiolojikal rispכns dεm.
Data dεn εksprεs as min ± SEM (wan-way analisis כf vεryans, Holm-Sidak in mכltipכl kכmpεreshכn tεst; *P<0.05). Di yunit nɔmba de sho wit brakɛt.
wi set aut fכ invεstigat if eni kεtכgrεf insay di proteomics dataset (Figure 1G) inklud path dεm we kin kכntrכl siriכs OXPHOS dεfisiεns, we de εksplen wetin mek afekt PN kin mεnten nia-nכmal εlektrofysioloji (Figure 2, E to K). . Proteomics analisis sho se di enzym dεm we involv in di katabolism fכ branch chen amino asid dεm (BCAA) bin sכmtεm כp-rεgulεt (Figure 3A εn Figure S5A), εn di fainal prodakt asetil-CoA (CoA) כ succinyl CoA kin supliment di tricarboxylates in arteriosclerosis Acid (TCA) saykl. Wi fכnd se di kכntεnt fכ BCAA transaminase 1 (BCAT1) εn BCAT2 כl tu inkrεs. dεn de katalayz di fכs stεp fכ BCAA katabolism bay we dεn de jεnarεt glutamεyt frכm α-ketoglutarate ( 26 ). כl di sכbyunit dεm we mek di branch chen kεto asid dεhaydrojenεz (BCKD) kכmpleks de upregulated (di kכmpleks de katalayz di sכbsεkvεnt εn irεvεrsibl dεkabכksilayshכn fכ di rizultant BCAA kכbכn skel) (Figure 3A εn Figure S5A). כltu, dεn fכn nכ klia chenj in BCAA insεf insay di sכt PN, we kin bi biכs fכ di inkrεs sεlyul כptek fכ dεn imכtant amino asid dεm ya כ di yus כf כda sכs dεm (glukoz כ laktik asid) fכ supliment di TCA saykl (Figure S5B). PN dεm we nכ gεt OXPHOS sho bak inkrεs glutamin dεkomכshכn εn transaminεshכn aktiviti dεm we dεn ol 8 wik, we kin sho bay di כp-rεgulεshכn fכ di mitochondrial εnzym dεm glutaminase (GLS) εn glutamin pyruvate transaminase 2 (GPT2) (Figure 3, A εn C). i fayn fכ no se di כp-rεgulεshכn fכ GLS de limited to di splais isoform glutaminase C (GLS-GAC) (di chenj fכ Mfn2cKO/CTRL na aprכksimatli 4.5-fol, P = 0.05), εn in spεsifi k כp-rεguleshכn insay kεnsar tisu dεm kin sכpכt maytochכndrial bayoεnεji. (27) we dɛn kɔl.
(A) di hεt map sho di fold chenj in protin lεvεl fכ di spεsifi k rod na 8 wik. (B) εgzampl fכ wan sεribra slais we dεn lεbul wit anti-PCx antibodi (skel bar, 20 μm). Di yɔlɔ aro de sho di Purkinje sɛl bɔdi. (C) Tεm kכs protin εksprεshכn analisis we dεn aydentify as imכtant kandidet fכ atεrosklεrosis (mכltipכl t-tεst, *FDR <5%; n = 3-5 mays). (D) oba: wan skematik dayagram we de sho di difrεn we dεm fכ enta di lεbul kכbכn we de insay di [1-13C]pyruvate tracer (dat na, via PDH כ trans-arterial route). bכtכm: di vaylin chכt sho di pasεnshכn fכ singl-lεbul kכbכn (M1) we kכnvכlt to aspartik asid, sitrik asid εn malik asid afta dεn lεbul akyu sεribεl slais wit [1-13C]pyruvate (paired t-test; ** P <0.01). (E) Kɔmprɛhɛnsif tɛm istri analisis fɔ di rod we dɛn sho. כnli tink bכt protin dεm wit P<0.05 na 8 wik . Dash layn: nɔ ajɔstmɛnt valyu (tu-way analisis fɔ varyans; * P <0.05; *** P <0.001). Data dεn εksprεs as min±SEM.
insay wi analisis, BCAA katabolism dכn bi wan pan di ki כp-rεguleshכn path dεm. dis fכt de sho strכng wan se di vεntileshכn vכlyum we de enta di TCA saykl kin chenj insay PN we nכ gεt OXPHOS. dis kin riprizent wan mεjכr fכm fכ nyuronal mεtabolik riwayring, we kin gεt dayrekt impak pan nyuronal fysioloji εn sכvayv di tεm we dεn de mεnten siriכs OXPHOS disfכnkshכn. kכnsistεnt wit dis haypothεsis, wi fכnd se di mεn anti-atherosclerotic εnzym PCx de כp-rεgεlεt (Mfn2cKO/CTRL chenj aprכksimatli 1.5 tεm; Figure 3A), we de katalayz di kכnvכshכn fכ pyruvate to oxaloacetate (28), we dεn biliv se de insay bren tisu Di εksprεshכn in de rεstrikt to astrocytes (29, 30). Konsistεnt wit di proteomics risכlt, kכnfכkal maykroskכpi sho se PCx εksprεshכn bin spεshal εn sכmtεm inkrεs in OXPHOS-deficient PNs, we PCx riaktiviti bin mεnli rεstrikt to di adjasent Bergmann glial sεl dεm fכ di kכntrכl (Figure 3B). Fכ fכnshכnal tεst di obsכv upregulashכn fכ PCx, wi trit akyu sεribεl slais wit [1-13C]pyruvate tracer. we di pyruvate bin oksidayz bay pyruvate dehydrogenase (PDH), in isotope label disappear , Bɔt i inkɔrpɔret insay di TCA saykl intamɛdiet dɛn we pyruvate de mɛtabolayz bay vaskul riakshɔn (Figure 3D). in sכpכt fכ wi proteomics data, wi obsεv wan big nכmba כf makεr frכm dis traysa insay di aspartik asid fכ Mfn2cKO slais dεm, we sitrik asid εn malik asid tu bin gεt mכdarεt trεnd, pan כl we i nכ sכmtin (Figure 3D).
insay di dopamin nyuron dεm fכ MitoPark mays wit maytochכndrial disfכnkshכn we dεn kכz bay dopamin nyuron dεm we spεshal wan destroy di mitochondrial transkripshכn fכktכ A jin (Tfam) (Figure S6B), PCx εksprεshכn bin כlso sכmtεm כp-rεgulεt (31), we sho se aseton asid atεriosklεrosis Di כkכrεshכn fכ di sik de rεgεl di tεm we di nyuronal disfכnkshכn de OXPHOS na di bɔdi. i fayn fכ no se dεn fכnshכn se yunik εnzym dεm (32-34) we kin εksprεs insay nyuron dεm we kin kכnεkt wit atεriosklεrosis de sכmtεm כp-rεgulεt insay PN dεm we nכ gεt OXPHOS, lεk propionyl-CoA carboxylase (PCC-A), Malonyl-CoA de kכnvכlt propionyl-CoA to succinyl-CoA εn mitochondrial malic 3 (ME3), εnzym . we in men wok na fכ rεkכv di pyruvate frכm malat (Figure 3, A εn C) (33, 35). apat frכm dat, wi fכnd wan sכm inkrεs pan di Pdk3 εnzym, we de fכsfכraylet εn so inaktivat PDH ( 36 ), we nכ chenj dεn bin dεtekt insay di Pdp1 εnzym we de aktibכt PDH כ di PDH εnzym kכmpleks insεf (Figure 3A). kכnsistεntli, in Mern2cKO PN dεm, di fכsfכrayleshכn fכ di α1 sabyunit α (PDHE1α) sabyunit fכ di pyruvate dehydrogenase E1 kכmכpכnt fכ di PDH kכmpleks in Ser293 (we dεn no fכ inhεbit di εnzym aktvכti fכ PDH) bin εnhans ( Figure S6C) (Figure S6C). Pyruvate nɔ gɛt ɛni vaskul akses.
fכ finish, wi fכnd se di supεr path fכ serin εn glycine bayosεntesis, di rilayt mitochondrial folate (1C) saykl εn prolin bayosεntesis (Figure 1G εn Figure S5C) כl dεn sכmtεm כp-rεgεlεt, akɔdin to ripot dεm, di tεm we di aktibכshכn prכsεs de. di tisu dεm we de rawnd de aktibכt wit maytochכndrial disfכnkshכn (5-7). kכnfכkal analisis we sכpכt dεn proteomics data ya sho se in PN wit OXPHOS we nכ de, sεribεl slais dεm fכ 8 wik ol mays dεm bin sכbjεkt to serin hydroxymethyltransferase 2 (SHMT2), we na wan kכl εnzym fכ di mitochondrial folate saykl. Sigifikan imyun rεspכns (Figכ S5D). insay 13 CU-glukoz-inkubayt akyu sεribεl slais, mεtabolik traysin εkspεriεns dεm fכs kכnfכm di כp-rεguleshכn fכ serin εn prolin bayosεntesis, we sho se di fכlכ fכ kabכn isoform dεm insay sεrin εn prolin inkrεs (Figure S5E). sכm di riakshכn dεm we GLS εn GPT2 de promuot na dεn rispansabl fכ di sεntez fכ glutamεyt frכm glutamin εn di transaminεshכn bitwin glutamεyt εn α-ketoglutarate, dεn upregulashכn de sho se OXPHOS-dεficient nyuron dεm gεt inkrεs dimand fכ glutamεyt , Dis kin bi fכ mεnten di inkrεs bayosεntesis fכ prolin (Figure S5C). difrεnt frכm dεn chenj dεm ya, wan proteomik analisis fכ sεribεl astrosayt dεm frכm PN-spεsifi k Mfn2cKO mays dεm sho se dεn path dεm ya (inklud כl di antipεrכksidayz dεm) nכ chenj sכmtεm insay εksprεshכn, so dat de sho Dis mεtabolik ridayrεkshכn na sεlektiv to dεgrad PN (Fig. S6, D to G).
in sכmari, dεn analisis ya rivεl signifyant difrεnt patεn dεm fכ tεmporal aktibכshכn fכ spεsifi k mεtabolik path dεm na PN dεm. pan tap we abnכmal nyuronal maytochכndrial fכnshכn kin mek di atεrosklεrosis εli 1C rimכdelin (Figure 3E εn Figure S5C), εn ivin prεdiktibכl chenj dεm na di εksprεshכn fכ I εn IV kכmpleks dεm, di chenj dεm na di serin de novo sεntesis na כnli I bin כnli bi klia insay di let stej dεm. OXPHOS disfכnkshכn (Figכ 3E εn Fig S5C). dis fכnd dεm de difayn wan sikεnshal prכsεs we di strεs-indyus maytochכndrial (1C saykl) εn saytoplasmik (serine bayosεntesis) rεspכns sinagεstik wit di inkrεs pan atεrosklεrosis insay di TCA saykl fכ rishεp nyuronal mεtabolism.
8 wik ol OXPHOS-dεficient PN dεm kin mεnten hεy-frikyuεns εksayteshכn aktiviti εn כndago sכm mεtabolik rεkכnekshכn fכ kכmpεns fכ maytochכndrial disfכnkshכn. dis diskכvri de rayz wan intrestin posisibul se ivin na dis mכment, dεn sεl dεm ya kin כlso Risiv tεrapi intavεnshכn fכ delay כ prεvεnt nyurodijεnεreshכn. Let. Wi bin sɔlv dis pɔsibul tru tu indipɛndɛnt intavɛnshɔn dɛn. In di fכs mεtכd, wi disayn wan Cre-dipεndεnt adeno-asכsiet vayrus (AAV) vεktכr so dat MFN2 kin sεlektivli εksprεs in OXPHOS-deficient PNs in vivo (Figure S7A). di AAV we de kכd MFN2 εn di fluorescent rεpכta jin mCherry (Mfn2-AAV) bin vεrifik in praymar nyuron kכltכr in vitro, we mek MFN2 εksprεs in wan Cre-dipεndεnt we εn rεsכk di maytochכndrial mכfכlכji, we de prεvεnt nyuromyushכn in Mfn2cKO nyuron dεm ( Figure S7, B, D εn E). Neks, wi kכnεkt in vivo εkspεriεns fכ stεriכtaktikli delivεr 8 wik ol Mfn2-AAV to di sεribεl kכtεks fכ Mfn2cKO εn kכntrכl mays, εn analכz 12 wik ol mays (Figure 4A). Di Mfn2cKO mays dɛn we dɛn trit day (Figure 1, A ɛn B) (16). vayral transdyushכn in vivo rεsult in sεlektiv εksprεshכn fכ PN in sכm sεribεl sεkshכn dεm (Figure S7, G εn H). di injεkshכn כf di kכntrol AAV we εksprεs כnli mCherry (Ctrl-AAV) nכ bin gεt nכ signifyant ifekt pan di digri כf nyurodijεnεreshכn in Mfn2cKO animal dεm. In kontrεst, di analisis כf Mfn2cKOs transdyus wit Mfn2-AAV sho wan signifyant prכtektiv ifekt כf di PN sεl layεr (Figure 4, B εn C). in patikyula, i lεk se di nyuron dεnsiti kכlכsכl nכ de difrεnt frכm di kכntrol animal dεm (Figure 4, B εn C, εn Figure S7, H εn I). di εksprεshכn fכ MFN1 bכt nכto MFN2 ikwal ifektiv fכ sev nyuronal dεd (Figure 4C εn Figure S7, C εn F), we sho se di εksprεshכn fכ εktopik MFN1 kin ifektivli supliment di lכk fכ MFN2. fכda analisis na di singl PN lεvεl sho se Mfn2-AAV big wan rεsכk di ultrastrכkchכ fכ maytochכndria, nכmal mtDNA lεvεl, εn rivεs di hεy εksprεshכn fכ di anti-angiojεnεsis mak PCx (Figכ 4, C to E ). Visual inspekshɔn fɔ di Mfn2cKO mays dɛn we dɛn rɛskɔ insay wan rɛst stet sho se dɛn postɔ ɛn mɔta simptom dɛn (muvmɛnt S1 to S3) bin impɔtant. fכ kכnklud, dεn εkspεriεns dεm ya sho se dilayed riintrodכksכshכn fכ MFN2 insay PN dεm we siriכs dεficient in OXPHOS na infεkt fכ rivεs mtDNA kכnsכmshכn εn indyuz atεrosklεrosis, we de prεvεnt aksכn dεjεnεreshכn εn nyuronal dεd in vivo.
(A) Wan skim we de sho di ekspirimental schedule fכ injεkt AAV we de kכd MFN2 we di indikεt mεtabolik path de aktibכt. (B) Riprεsεntεtiv kכnfכkal imej dεm fכ 12 wik ol sεribεl slais dεm we transdyus na 8 wik in Mfn2cKO mays dεm εn lεbul wit anti-Calbindin antibodi. Rayt: Skel we di aksɔn fayv dɛn de skel. di skel fכ di aksכn zum na 450 εn 75 μm. (C) Lεft: Kwantifikεshכn כf Purkinje sεl dεnsiti in di AAV transdyushכn lכp (AAV+) (wan-way analisis כf vεryans; n = 3 mays). Rayt: mtDNA fכkus analisis in transdyus PN na wik 12 (unpaired t-tεst; n = 6 sεl frכm tri mays). * P <0.05, ɛn di ɔda wan dɛn; ** P <0.01. Na di s. (D) Riprεsεntεtiv transmishכn εlektrכn maykrograf dεm fכ PN dεm fכ Mfn2cKO sεribεl sεkshכn dεm we transdyus wit di indikεt vayral vεktכr dεm. Di pink mask de sho di eria we di dendrite dɛn de, ɛn di yɔlɔ dɔt skwea de sho di zum we dɛn gi na di rayt say; n de sho di nyuklios. Skel bar, 1μm. (E) sho wan egzampl fכ PCx stεyning in PN we dεn transdyuz na 12 wik. Skel bar, 20μm. OE, ɔva ɛksprɛshɔn; FC, fold chenj.
fכ finish, wi invεstigat di imכpכtants fכ pεrכksidayz-indyus sεl sכvayv in PN dεm we dεn εkspiriεns OXPHOS disfכnkshכn. Wi jεnarεt mCherry we de kכd AAV-shRNA (sכt hairpin RNA) we spεshal tכgεt maus PCx mRNA (AAV-shPCx), εn injεkt di vayrus כ in skrambl kכntrכl (AAV-scr) insay di sεribεl כf Mfn2cKO mays. di injεkshכn bin du insay di fכs wik we i ol (Figure 5A) fכ achy ifektiv PCx knockdown di tεm we PCx εksprεshכn inkrεs (Figure 3C) εn di PN sel layεr bin stil intakt (Figure 1A). i fayn fכ no se we yu kכnכk PCx dכn (Figure S8A) i de mek PN day sכmtεm aksεlεrayshכn, we de limited to di infεkt ring (Figure 5, B εn C). In orda fכ כndastand di mεkanism כf di mεtabolik ifekt dεm we PCx כp-rεguleshכn indyuz, wi stכdi di rεdכks stetכs כf PNs afta PCx kכnכk dכwn εn AAV-mεdiεt כptik bayosεns Grx1-roGFP2 bin simultaneously εksprεs (Figure S8, B to D) fכ evaluate glutathione Di rilitiv chenj כf pεptida rεdכks pכtεnshal (38). Afta dat, wi du tu-fכtכn fכrosεns layftaym imej maykroskכpi (FLIM) insay akyu bren slais dεm fכ 7 wik ol Mfn2cKO כ kכntrol littermates fכ dεtekt pכtεnshal chenj dεm na saytoplasmik rεdכks stetכs afta wi vεrifik FLIM kכndishכn dεm (Figure S8, E to G). di analisis sho wan signifyant inkris in di oksidashכn stet fכ wan singl Mfn2cKO PN dεm we nכ gεt PCx εksprεshכn, we difrεnt frכm kכntrol nyuron כ Mfn2cKO PN dεm we de εksprεs כnli skrambl shRNA (Figure 5, D εn E). we PCx εksprεshכn bin dכn-rεgulεt, di pasεnshכn fכ Mfn2cKO PN dεm we sho wan hεli כksidayz stet inkrεs bay mכr dan tri tεm (Figure 5E), we sho se PCx כp-rεgulεshכn mεnten di rεdכks kapasiti fכ dijεnεret nyuron dεm.
(A) wan skim we de sho di εkspiriεnshכnal schedule fכ injεkt AAV we de kכd shPCx we di indikεt mεtabolik path de aktibכt. (B) Riprεsεntεtiv kכnfכkal fכtכgraf dεm fכ 8 wik ol sεribεl sεkshכn dεm in Mfn2cKO mays dεm we dεn transdyuz εn lεbul wit anti-kalsinyurin antibodi na 4 wik. Skel bar, 450μm. (C) Kwantifikeshכn כf Purkinje sεl dεnsiti in AAV-transdyus lכp (wan-way analisis כf vεryans; n = 3 to 4 mays). Data dεn εksprεs as min±SEM; ***P<0.001 na di s. (D) Riprεsεntεtiv FLIM pikchכ sho di avεrej layf span fכ 7 wik ol PN we de εksprεs glutathione rεdכks sεns Grx1-roGFP2 כnda di spεsifi k εkspεriεns kכndishכn dεm. LUT (luk-ap tebul) rεshכ: sכvayv tεm intaval (insay pikosεkכnd). Skel bar, 25μm. (E) Di histogram sho di distribushכn כf Grx1-roGFP2 layftaym valyu frכm (D) (n=158 to 368 sεl dεm in tu mays כnda εvri kכndishכn). di pie chart oba εvri histogram: sho di nכmba fכ di sεl dεm we gεt signifyant lכng (rεd, כksidayz) כ sכt (blu, rεdכks) layfspan valyu, we pas 1 SD fכ di avεrej layfspan valyu insay CTRL-AAV-scr. (F) di prכpos mכdel sho di prכtektiv ifekt fכ upregulashכn fכ nyuronal PCx.
כl in כl, di data we wi gi ya sho se di ri-εksprεshכn fכ MFN2 kin komplitli rεsכk advans PN wit siriכs OXPHOS dεfisiεns, siriכs mtDNA dεplishכn, εn ekstrim abnכmal ista-lεk mכfכlכji, we de gi kכntinyu prכgrεs ivin insay advans sik dεm. Nyurodijεnεreshכn de gi rivεrsibl pruf fכ di stej bifo sεl dεm day. dis digri fכ mεtabolik fleksibiliti de fכs εnfaz bay di abiliti fכ nyuron dεm fכ indyuz atεrosklεrosis (a riwayring fכ di TCA saykl), we de inhεbit PCx εksprεshכn insay PN dεm we nכ gεt OXPHOS εn de εnhans sεl dεd, we de ple wan prכtektiv rol (Figure 5F).
insay dis stכdi, wi gi pruf se di rispכns fכ PN dεm to OXPHOS disfכnkshכn na fכ kכnvεrj sכmtεm to TCA saykl atεrosklεrosis tru di difrεnt aktibכshכn path we mεtabolik program dεm de aktibכt. wi kכnfכm di proteomik analisis wit plεnti komplimentari mεtכd dεm εn rivεl se we dεn chalenj bay siriכs maytochכndrial disfכnkshכn, nyuron dεm gεt wan fכm we dεn nכ bin no bifo fכ mεtabolik εlastik. fכ wi sכprayz, di כl riwayring prכsεs nכ nכ de mak di tεminal mεtabolik stet we de akkompani nyurodijεnεreshכn sכmtεm εn i nכ de rεvεrsibl, bכt wi data sho se i kin kכnstityut wan mεntenans nyuron ivin insay di stej bifo sεl dεd Fכnshכnal kכmpεnsεshכn mεkanism. dis fכnshכn sho se nyuron dεm gεt bכku digri fכ mεtabolik plastisiti na di bכdi. dis fכs pruv se di leta riintrodכks fכ MFN2 kin rivεs di εksprεshכn fכ di ki mεtabolik mak dεm εn fכ mek PN dεjεnεreshכn. Difrɛn frɔm dat, i de mek di at nɔ de rɔtin ɛn i de mek di nerv dɛn kwik kwik wan. transseksuɛl.
wan pan di mכst fascinating fכnd dεm na wi risεch na dat PN dεm we nכ gεt OXPHOS kin mכdify di TCA saykl mεtabolism bay we dεn de כp-rεgεl εnzym dεm we spεshal wan de stimulat arteriosclerosis. mεtabolik riarenjmεnt na kכmכn fכm fכ kεnsar sεl dεm, sכm pan dεm de dip pan glutamin fכ supliment TCA saykl intamεdiεt fכ prodyuz ridyus ikwal, we de drεb di rεspiretכri chen εn mεnten di prodyushכn fכ lipid εn nyukliotayd bayosεntesis prεkursכr dεm (39 , 40). wan stכdi we dεn jכs du sho se insay pεrifεral tisu dεm we de εkspiriεns OXPHOS disfכnkshכn, di rεkכnekshכn fכ glutamin/glutamate mεtabolism na כlso wan prכminεnt fכm (5, 41), usay di dεyshכn fכ glutamin εntri insay di TCA saykl dipכnt pan Biכs fכ di siriכs we OXPHOS injuri de (41). ). כltu, i nכ de fכ klia pruf fכ eni similariti fכ nyuronal mεtabolik plastisiti na di bכdi εn in posεbul rilevans insay di sik kכntεks. in wan rεsεnt in vitro stכdi, dεn sho se praymar kכrtikכl nyuron dεm de mobiliz glutamεyt pul fכ nyurotransmishכn, we de protεkt כksidεtiv mεtabolism εn atεrosklεrosis כnda mεtabolik strεs kכndishכn dεm ( 42 ). i fayn fכ no se כnda di fכmakכlכjik inhibishכn fכ di TCA saykl εnzym succinate dehydrogenase, dεn biliv se pyruvate carboxylation de mεnten di sεntesis fכ oxaloacetate in kכlt sεribεl granul nyuron dεm (34). כltu, di fysiolojikal rilevans fכ dεn mεkanism dεm ya to di bren tisu (we dεn biliv se atεrosklεrosis de mεnli kכnfyus to astrosayt dεm) stil gεt imכtant fysiolojikal siginifikεns (43). insay dis kes, wi data sho se PN dεm we OXPHOS dεm dεm na di bכdi kin swich to BCAA dεgradashכn εn pyruvate kכbכksilayshכn, we na di tu men sכs dεm fכ supliment TCA pul intamεdiεt dεm. pan tap we dεn bin prכpos di putativ kכntribyushכn fכ BCAA katabolism to nyuronal εnεji mεtabolism, in ad pan di rol we glutamεyt εn GABA de ple fכ nyurotransmishכn ( 44 ), nכ pruf stil de fכ dεn mεkanism dεm ya in vivo. fכ dat, i izi fכ spεkul se disfכnkshכnal PN dεm kin כtomatכk kכmpεns fכ di kכnsכmshכn fכ TCA intamεdiεt dεm we di asimilεshכn prכsεs de drεb bay we dεn de inkrεs atεrosklεrosis. in patikyula, upregulashכn fכ PCx kin nid fכ mεnten inkrεs dimand fכ aspartik asid, we dεn sכgεst in proliferating sεl dεm wit maytochכndrial disfכnkshכn ( 45 ). כltu, wi mεtabolכmiks analisis nכ rivεl eni signifyant chenj in di stedi-stet lεvεl fכ aspartik asid in Mfn2cKO PN dεm (Figure S6A), we presumably rεflεkt di difrεnt mεtabolik yutilizeshכn fכ aspartik asid bitwin proliferating sεl dεm εn post-mitotic nyuron dεm . pan ɔl we di εksakεt mεkanism fכ PCx upregulation in dysfunctional nyuron dεm in vivo stil de fכ kכntribyut, wi dכn sho se dis prεmature rεspכns de ple imכtant rol fכ mεnten di rεdכks stet fכ nyuron dεm, we dεn dכn sho insay FLIM εkspεriεns pan sεribεl slais dεm. in patikyula, fכ mek di PN dεm nכ de כp-rεgεl PCx kin mek i gεt mכr כksidayz stet εn aksεlεrat sεl dεd. di aktibכshכn fכ BCAA dεgradashכn εn di kכbכksilayshכn fכ pyruvate nכto we fכ kכl di pεrifεral tisu dεm fכ di maytochכndrial disfכnkshכn (7). fכ dat, i lεk se dεn na di prayoritכt fכm fכ di nyuron dεm we nכ gεt OXPHOS, ivin if nכto di כnli fכm, we imכtant fכ nyurodijεnεreshכn. .
sεribεl sik na hεtirojεnik tכp fכ nyurodijεnεraytiv sik we kin sho as ataxia εn bכku tεm i kin damej PN dεm ( 46 ). dis nyuron populeshכn de patikyuכl vulnerable fכ mitochondrial dysfunction biכs dεn sεlektiv dεjεnεreshכn in mays na infεkt fכ rεprכdyuz plεnti pan di mכtal simptom dεm we de kכl hכman spinocerebellar ataxia ( 16 , 47 , 48 ). akɔdin to ripɔt, wan transjɛnik maws mɔdel wit wan mutant jin gɛt fɔ du wit mɔtalman spinosɛribɛl ataxia ɛn i gɛt maytochondrial disfɔkshɔn ( 49 , 50 ), we de ɛmpɛsh di impɔtant tin fɔ stɔdi di kɔnsɛkshɔn dɛn we OXPHOS nɔ gɛt na PNPH. כl dis mek i fayn fכ aylכt εn stכdi dis yכnik nyuron populeshכn fayn fayn wan. כltu, givεn se PN dεm rili sεnsitiv to prεshכn εn akכnt fכ lכw prכpכshכn fכ di כl sεribεl sεl populeshכn, fכ plεnti omiks-bεys stכdi dεm, sεlektiv separeshכn fכ dεm as ol sεl dεm stil na wan chalεnj aspek. pan tap we i kכlכsכl impכsibul fכ achy absolyut lכk fכ kכntaminεshכn כda sεl tכp dεm (especially adכlt tisu dεm), wi kכmbayn wan ifektiv dissociation stεp wit FACS fכ gεt wan sufayf nכmba fכ viable nyuron fכ dכwnstrim proteomics analisis, εn gεt Kwik hεy protin kכvεrej (bכt 3000 protin dεm) kכmpεr wit di egzistin data sεt fכ di ol sεribεl (51). bay we wi de kip di viabiliti fכ di ol sεl dεm, di mεtכd we wi de gi ya de alaw wi nכ nכ de כnli fכ chεk di chenj dεm na di mεtabolik path dεm na di maytochכndria, bכt fכ chεk di chenj dεm na in saytoplasmik kכwntεrpat dεm, we de kompliment di yus fכ di maytochכndrial mεmbran tεg dεm fכ εnrich sεl tכp Di nyu mεtכd fכ di nכmba fכ di maytochכndria dεm na di kכmpleks tisu dεm (52, 53). di mεtכd we wi de dεskrεb nכto nכmכ de riliyt to di stכdi fכ di Purkinje sεl dεm, bכt i kin izi fכ aplay to eni tכp sεl fכ adrεs mεtabolik chenj dεm na di sik bren, inklud כda mכdel dεm fכ di maytochכndrial disfכnkshכn.
fכ finish, wi dכn aydentify wan tεrapi winda di tεm we dis mεtabolik riarenjmεnt prכsεs de kכmplit wan rivεs di kכl sayn dεm fכ sεlyul strεs εn fכ mek nyuronal dεjεnεreshכn nכ de. fכ dat, fכ כndastand di fכnshכnal implεkshכn dεm fכ di riwayring we dεn dεskrεb ya kin gi fכndamεntכl insayt dεm fכ posεbul tritmεnt fכ mεnten nyuronal viabiliti di tεm we di maytochכndrial disfכnkshכn de. fכs risεch we dεn aim fכ dεsεkt chenj dεm na di εnεji mεtabolism insay כda bren sεl tכp dεm nid fכ fulכp fכ sho di aplikεbiliti fכ dis prinsipul to כda nyurolכjik sik dεm.
MitoPark mays dεn bin dεskrεb bifo ( 31 ). C57BL/6N mays wit loxP flanking Mfn2 jin dεm dεn bin dεskrεb bifo ( 18 ) εn kכros wit L7-Cre mays ( 23 ). di rizultant dכbl hεterozaygכs projεni bin kכros wit homozaygכs Mfn2loxP/Mfn2loxP mays fכ jεnarεt Purkinje-spεsifi k jin kכnכt fכ Mfn2 (Mfn2loxP/Mfn2loxP; L7-cre). In wan sabsεt fכ mating, di Gt (ROSA26) SorStop-mito-YFP alεl (stכp-mtYFP) bin introdכks tru adishכnal krכsin ( 20 ). Dɛn bin du ɔl di animal prosidyushɔn dɛn akɔdin to di Yuropian, nashɔnal ɛn institiushɔnal gaydlayn dɛn ɛn LandesamtfürNatur we kɔmɔt na Umwelt ɛn Verbraucherschutz, Nɔt Rayn-Wɛstfalia, Jamani bin gri fɔ dɛn. Animal wok de fala bak di gayd we di European Federation of Laboratory Animal Sciences Associations de gi.
afta dεn anεsthεz di bεlε uman in sεvikal dislכkeshכn, dεn de aylכt di maws εmbriyo (E13). di kכtεks bin dεsεkt insay Hanks in Balanced Salt Solution (HBSS) we dεn supliment wit 10 mM Hepes εn pas pan Dulbecco in Modified Eagle's Medium we kכntεn papain (20 U/ml) εn sistayn (1μg/ml). inkubayt di tisu insay DMEM) εn dissosiayt am bay εnzym dεyjεshכn. Ml) na 37°C fכ 20 minit, εn afta dat dεn mεkanikal mil insay DMEM we dεn supliment wit 10% fetal bovin sεrum. Sεl dεm bin sid pan glas kכvaslip dεm we dεn kכt wit polilaysin pan wan dεnsiti we na 2×106 pan 6 cm kכlt dish כ pan wan dεnsiti we na 0.5×105 sεl/cm2 fכ imej analisis. Afta 4 awa, dεn riples di mεdiכm wit Nyurobasal sεrum-fri mεdiכm we kכntεn 1% B27 supliment εn 0.5 mM GlutaMax. afta dat dεn bin mεnten di nyuron dεm na 37°C εn 5% CO2 tru di εkspεriεns, εn dεn bin fεd dεm wan tεm insay di wik. fכ mek dεn indyuz rεkombinεshכn in vitro, dεn yuz 3μl (24-wεl kכltכr dish) כ 0.5μl (24-wεl plet) fכ di fכlכw AAV9 vayrus vεktכr fכ trit nyuron dεm pan di sεkכn de in vitro: AAV9.CMV.PI.eGFP. WPRE.bGH (Addgene, katalog nɔmba 105530-AAV9) ɛn AAV9.CMV.HI.eGFP-Cre.WPRE.SV40 (Addgene, katalog nɔmba 105545-AAV9).
di maws Mfn1 εn Mfn2 komplimentari DNA (we dεn gεt frכm Addgene plasmid #23212 εn #23213, rispεktivli) dεn mak wit di V5 sikεns (GKPIPNPLLGLDST) na di C-tεrminus, εn dεn fכs wit mCherry in fכm tru di T2A sikεns. Grx1-roGFP2 na gift frɔm Haydelberg T. P. Dik DFKZ (Doyches Krebsforschungszentrum). Bay we dɛn riples di tdTomato kasɛt we dɛn yuz kɔvɛnshɔnal klonin we dɛn de yuz, dɛn bin sabklɔn di kasɛt insay di pAAV-CAG-FLEX-tdTomato bakbon (Addgene rɛfrɛns nɔmba 28306) fɔ mek pAAV-CAG-FLEX-mCherry-T2A-MFN2-V5, pAAV-CAG- FLEX-mCherry-T2A-MFN1-V5 ɛn pAAV-CAG-FLEX-Grx-roGFP2 vektɔr dɛn. A similar strategy wεn yuz fכ jεnarεt di kכntrכl vεktכr pAAV-CAG-FLEX-mCherry. fכ mek dεn jεnarεt di AAV-shPCx kכnstrכkt, wan plasmid AAV vεktכr (VectorBuilder, pAAV [shRNA] -CMV-mCherry-U6-mPcx- [shRNA#1]) nid fכ, we kכntεn di DNA sikεns we de kכd di shRNA we de tכgεt maws PCx (5′CTTTCGCTCTAAGGTGCTAAACTCGAGTTTAGCACCTTAGAGCGAAAG 3′) כnda di kכntrכl fכ di U6 prכmotεr, dεn de yuz mCherry כnda di kכntrכl fכ di CMV prכmotεr. Di prodakshɔn fɔ ɔgzilari AAV vektɔ dɛn bin kɔmɔt akɔdin to di manifakta in instrɔkshɔn (Cell Biolabs). In shot, yuz wan transfכm plasmid we de kכri mCherry-T2A-MFN2-V5 (pAAV-CAG-FLEX-mCherry-T2A-MFN2-V5), mCherry-T2A-MFN1-V5 (pAAV-CAG-FLEX-mCherry) transiεntli Transfεkshכn כf 293AAV sεl dεm-T2A-MFN1-V5), mCherry (pAAV-CAG-FLEX-mCherry) כ Grx-roGFP2 (pAAV-CAG-FLEX-Grx-roGFP2) we de kכd jin, εn bak we de kכd AAV1 kapsid protin εn aksesכri protin Packaging plasmid plasmid, we de yuz kalsiכm fכsfεt mεtכd. Di krud vayrɔs supamantant bin gɛt bay friz-thaw saykl dɛn insay wan dray ays/ɛtanol bat ɛn layz sɛl dɛn insay fɔsfɛt bafa salin (PBS). Di AAV vektɔ bin purify bay diskɔntinyu ayodiksanol gradient ɔltrasɛntrifugayshɔn (24 awa na 32,000 rpm ɛn 4°C) ɛn kɔnsɛntret yuz wan Amicon ɔltra-15 sɛntrifugal filta. Jεnom taytεr כf AAV1-CAG-FLEX-mCherry-T2A-MFN2-V5 [2.9×1013 jεnom kכpi (GC)/ml], AAV1-CAG-FLEX-mCherry (6.1×1012 GC/ml), AAV1-CAG- FLEX bin lεk aw dεn bin dεskrεb am bifo (54), we dεn mεzj bay rial-taym kwantiti PCR (qPCR) -MFN1-V5 (1.9×1013 GC/ml) ɛn AAV1-CAG-FLEX-Grx-roGFP2 (8.9×1012 GC/ml).
Praymari nyuron dεm bin skrap כf insay ays-kol 1x PBS, pεlεt, εn afta dat homojεnayz insay 0.5% Triton X-100 / 0.5% sכdyכm dεoxycholate/PBS laysis bכfa we kכntεn fכsfataz εn proteas inhibito (Roche). Protein kwantifiyeshɔn bin de bay we dɛn yuz di bicinchoninic acid assay (Thermo Fisher Scientific). afta dat, dεn separet di protin dεm bay SDS-polyacrylamide jel εlektrofכresis, εn afta dat dεn blot dεm pan wan polyvinylidene fluoride membran (GE Healthcare). Blɔk sayt dɛn we nɔ spɛshal ɛn inkubayt wit di praymari antibodi (luk Tebul S1 fɔ di ditel) insay 5% milk insay TBST (Tris-buffered saline wit Tween), was stɛp ɛn sɛkɔndari antibodi insay TBST Inkubayt. Inkubayt wit praymari antibodi ovanayt na +4°C. afta yu was, put di sεkכnd antibodi fכ 2 awa na rum tεmprachכ. Afta dat, bay we dɛn inkubayt di sem blɔt wit wan anti-β-aktin antibodi, dɛn kɔnfyus di sem lod. Ditekshכn bay we dεn kכnvכlt to kεmiluminesεns εn εnhans kεmiluminesεns (GE Healthcare).
di nyuron dεm we dεn bin sid bifo pan glas kכvaslip dεm bin fiks wit 4% paraformaldehyde (PFA)/PBS na di spεsifi k tεm pכynt na rum tεmprachכ fכ 10 minit. di kכvaslip dεm fכs dεn kin pεrmiet wit 0.1% Triton X-100/PBS fכ 5 minit na rum tεmprachכ, εn afta dat insay blכk bכfa [3% bovin sεrum albumin (BSA)/PBS]. Na di sɛkɔn de, dɛn was di kɔvaslip dɛn wit blɔk bafa ɛn inkubayt wit di aprɔpriet fluorophore-conjugated sɛkɔndari antibodi fɔ 2 awa na rum tɛmpracha; fכ finish, dεn was di sεmpl dεm gud gud wan insay PBS wit 4′,6-diamidino-2 -Phenylindole (DAPI) dεn kכntrεstεn εn afta dat dεn fiks dεm pan di maykroskכp slayd wit Aqua-Poly/Mount.
Mice (man ɛn uman) bin anestez bay intraperitoneal injɛkshɔn fɔ ketamin (130 mg/kg) ɛn xylazine (10 mg/kg) ɛn administret dɛn sabkyutan wit carprofen analgesic (5 mg/kg) , Ɛn put dɛn insay wan stiriɔtaktik inschrumɛnt (Kopf) we gɛt wam pad. εkspכz di sכkul εn yuz dεntal drכl fכ tכn di pat pan di sεribra kכtεks we kכrεkt to di mis bon (frכm lambda: tel 1.8, lateral 1, we kכrεspכnd to di lכbul dεm IV εn V). Yuz wan kɔba sirinj nidul fɔ tek tɛm mek wan smɔl ol na di skel fɔ mek i nɔ ambɔg di vaskul we de dɔŋ. afta dat dεn de put di tin we dεn drכ glas kapilari sכmtεm insay di maykro-ol (frכm -1.3 to -1 na di ventral sayd fכ di dura mater), εn dεn injεkt 200 to 300 nl AAV insay di maykro-injεktכr (Narishige) wit manual sirinj dεm (Narishige) sεvεra tεm pan lכw prεshכn ova wan tεm 10 to 20 minit winda. afta di infushכn, put di kapilari fכ כda 10 minit fכ alaw di vayrus fכ spre kכmplit wan. afta dεn dכn pul di kapilari dεm, dεn kin tek tεm sכt di skin fכ mek di wund dεm nכ inflameshn εn alaw di animal fכ rεkכva. Dɛn bin trit di animal dɛn wit analjɛsik (caspofen) fɔ sɔm dez afta di ɔpreshɔn, ɛn insay da tɛm de dɛn bin tek tɛm wach dɛn bɔdi kɔndishɔn ɛn afta dat dɛn bin kil dɛn na di tɛm we dɛn bin dɔn tɔk bɔt. Dɛn bin du ɔl di prosidur dɛn akɔdin to di Yuropian, nashɔnal ɛn institiushɔnal gaydlayn dɛn ɛn na LandesamtfürNatur we de na Umwelt ɛn Verbraucherschutz, Nɔt Rayn-Wɛstfalia, Jamani bin gri fɔ dɛn.
Dɛn bin anestez di animal dɛn wit ketamin (100 mg/kg) ɛn xylazine (10 mg/kg), ɛn dɛn bin perfyuz di at wit 0.1 M PBS fɔs, ɛn afta dat wit 4% PFA insay PBS. Di tisu bin dissect ɛn fiks insay 4% PFA/PBS ɔvnayt na 4°C. wan vaybret naif (Leica Microsystems GmbH, Vienna, Austria) bin yuz fכ pripia sajital sekshכn dεm (50 μm tik) frכm di fiks bren insay PBS. If dɛn nɔ tɔk ɔda we, dɛn bin de du stɛyn pan fri-flɔt sɛkshɔn dɛn lɛk aw dɛn bin dɔn tɔk bɔt am ɔp (13) na rum tɛmpracha ɛn stir. In shot, fכs, di slais dεm we dεn gεt bin permeabilized wit 0.5% Triton X-100/PBS fכ 15 minit na rum tεmprachכ; fכ sכm εpitכp dεm (Pcx εn Shmt2), bay in tris-EDTA bכfa na 80°C (PH 9) hεt di slais dεm fכ 25 minit insted fכ dis stεp. Neks, di sekshכn dεm bin inkכba wit praymar antibodi (luk Tεbul S1) insay blכk bכfa (3% BSA/PBS) na 4°C כvnayt wit stir. di nεks de, dεn was di sεkshכn dεm wit blכk bכfa εn inkכba wit di aprכpriet fluorophore-conjugated sεkכnd antibodi fכ 2 awa na rum tεmprachכ; fכ finish, dεn was di sεkshכn dεm gud gud wan insay PBS, kכntrכl stεyn wit DAPI, εn afta dat dεn fiks dεm wit AquaPolymount On wan maykroskכp slayd.
Dɛn yuz wan lɛsa skan kɔnfɔkal maykroskɔp (TCS SP8-X ɔ TCS Dijital Layt Shit, Leica Microsystems) we gɛt wayt layt lɛsa ɛn 405 dayɔd ɔltravayolɛt lɛsa fɔ imej di sampul. Bay we dɛn ɛksayt di fluorophore ɛn gɛda di signal wit Hybrid Detector (HyDs), dɛn yuz LAS-X softwe fɔ gɛda stak imej dɛn we kɔnfɔm to Nyquist sampling insay sikwinshal mɔd: fɔ nɔ-kwantiti panɛl dɛn, na ayli dinamik signal dɛn (fɔ ɛgzampul, insay somatik sɛl ɛn dendrites) mtYFP) Yuz HyD fɔ detekt di nɔmba fɔ PN dɛn na BrightR mod). Gating frɔm 0.3 to 6 ns de aplay fɔ ridyus bakgrɔn.
Rial-taym imej fɔ di sɛl dɛn we dɛn dɔn sɔt. Afta we dɛn sɔt insay Nyurobasal-A mɛdiɔm we gɛt 1% B27 sɔpɔt ɛn 0.5 mM GlutaMax, dɛn sid di sɛl dɛn wantɛm wantɛm pan poly-l-laysin-kɔt glas slayd (μ-Slide8 Well, Ibidi, katalog nɔmba 80826) , Ɛn afta dat dɛn kip am na 37°C ɛn 5% CO2 fɔ 1 awa fɔ alaw di sɛl dɛn fɔ sɛtul. Rial-taym imej bin de pan wan Leica SP8 laser skan kɔnfɔkal maykroskɔp we gɛt wan wayt lɛsa, HyD, 63×[1.4 numɛrik aperchɔ (NA)] ɔyl ɔbjɛktiv lens ɛn wan ɔt stej.
Dɛn bin anestez di maws kwik kwik wan wit kabɔn dayɔgzayd ɛn kɔt in ed, dɛn pul di bren kwik kwik wan frɔm di skel, ɛn kɔt am insay 200μm tik (fɔ 13C lɛbl ɛkspiriɛns) ɔ 275μm tik (fɔ tu fɔtɔn ɛkspiriɛns) sajital sɛkshɔn we ful-ɔp wit di tin dɛn we de dɔŋ ya Di ayskrim (HM-650 V, Thermo Fisher Scientific, Walldorf, Germany) de fulכp wit dεn tin ya: 125 mM ays-kol, kabכn-sεtyuret (95% O2 εn 5% CO2) lכw Ca2 + atifishal sεribrospεnal fכluid (ACSF) NaCl, 2.5 mM KCl, 1.25 mM sכdyכm fכsfεt bכfa, 25 mM NaHCO3, 25 mM glukכs, 0.5 mM CaCl2 εn 3.5 mM MgCl2 (osmotik prεshכn 310 to 330 mmol). Transfεr di bren slais dεm we yu gεt to wan prε-inkכbeshכn chεmba we gεt hכy Ca2 + ACSF (125.0 mM NaCl, 2.5 mM KCl, 1.25 mM sכdiכm fכsfεt bכfa, 25.0 mM NaHCO3, 25.0 mM d-glukoz, 1.0 mM CaCl2 εn 2.0 mM MgCl2) Medium) pH 7.4 ɛn 310 to 320 mmol).
Durin di imej prכsεs, dεn muv di slais dεm insay wan dεdiket imej rum, εn di εkspεriεns bin du כnda kכntinyu ACSF pεrfyushכn pan wan kכnstant tεmprachכ we de 32° to 33°C. Wan maltifɔtɔn lɛsa skan maykroskɔp (TCS SP8 MP-OPO, Leica Microsystems) we gɛt Leica 25x ɔbjɛktiv lens (NA 0.95, wata), Ti: Safaya lɛsa (Chameleon Vision II, Coherent) bin yuz fɔ slais imej. FLIM modul (PikoHarp300, PikoKwant).
FLIM di Grx1-roGFP2. di chenj dεm na di saytoplasmik rεdכks stet fכ PN dεm bin mεzj bay tu-fכtכn FLIM insay sajital bren slais, usay di Grx1-roGFP2 bayosεns tכgεt PN dεm. insay di PN layεr, dεn sεlekt di akwyushכn fil bכt 50 to 80 μm dכn di slais sεf fכ mek sכh se wan viable PN de (dat na, di lכk fכ biad strכkchכ כ nyuronal mכfכlכjik chenj dεm along di dεndrit dεm) εn di dכbl positifu roGFP2 sεns εn AAV we de kכd shRNA PCx כ in kכntrכl sikεns (εvri wan we de kכ-εksprεs mCherry). Kɔlkt singl-stak imej dɛn wit 2x dijital zum [ɛksayteshɔn wɛvlɛnj: 890 nm; 512 nm 512 piksɛl dɛn]. Ditekshכn: intanεt HyD, fluorescein isothiocyanate (FITC) filta grup] εn imej avεrej insay 2 to 3 minit dεn yuz fכ mek sכh se dεn kכlekt inof fכtכn dεm (1000 fכtכn in tכtal) fכ kכv fitin. di sεnsitiviti כf di Grx1-roGFP2 prob εn di vεrifikεshכn כf FLIM kכndishכn dεm bin de du bay we dεn mכnitor di layfspan valyu fכ roGFP2 we dεn ad εksojen 10 mM H2O2 to di pεrfyushכn ACSF (fכ maksimayz כksidεshכn, we rεsult in inkrεs layfspan), εn afta dat ad 2 mM dithiothreitol (minimiz di digri כf rεdukshכn, rεsult in a dεkrεs in layfspan) (Figure S8, D to G). Yuz FLIMfit 5.1.1 softwe fɔ analayz di rizɔlt dɛn we dɛn gɛt, fit di singl ɛkspɔnɛnshal dɛkayshɔn kɔv fɔ di ɔl imej to di IRF we dɛn mɛzhɔ (instrumɛnt rispɔns fɛnshɔn), ɛn χ2 na lɛk 1. Fɔ kɔl di layf tɛm fɔ wan PN, dɛn drɔ di mask rawnd di nɛv bɔdi wit an, ɛn dɛn yuz di avɛrej layf tɛm insay ɛni mask fɔ kwantifay.
Mitochondrial pכtεnshal analisis. Afta di akyu sekshכn bin inkכba wit 100 nM TMRM we dεn ad dairekt to di pεrfyuz ACSF fכ 30 minit, dεn mεzj di maytochכndrial pכtεnshal chenj dεm fכ PN dεm bay tu-fכtכn maykroskכp. TMRM imej bin de du bay we dɛn ɛksayz di prob na 920 nm ɛn yuz intanɛnt HyD (tetramethylrhodamine isothiocyanate: 585/40 nm) fɔ gɛda signal dɛn; bay we yu yuz di sem εksayz wevlεngth bכt yuz difrεnt intanεt HyD (FITC :525/50) fכ imej mtYFP. yuz ImageJ in Imej Kכlkyulet pכlכg-in fכ evaluate di maytochכndrial pכtεnshal na di singl sεl lεvεl. in sכt, di pכl-in ikwyulet: signal = min (mtYFP, TMRM) dεn de yuz fכ aydentify di maytochכndrial rijyכn we de sho di TMRM signal insay Purkinje Somali insay di singl-stak kכnfכkal imej fכ di kכrεspכndεnt chεnal. afta dat dεn kכntify di piksεl εria na di rizultant mask, εn dεn nכmal pan di kכrεspכndεnt thrεshold singl-stεk imej fכ di mtYFP chεnal fכ gεt di maytochכndrial frakshכn we de sho di maytochכndrial pכtεnshal.
Dɛn bin dekɔnvɔlɔt di pikchɔ wit Huygens Pro (Scientific Volume Imaging) softwe. Fɔ di pikchɔ dɛn we dɛn dɔn skan fɔ tayl dɛn, dɛn kin mek di montaj fɔ wan tayl bay we dɛn yuz di ɔtomatik stich algɔritm we LAS-X softwe dɔn gi. Afta yu dɔn kalibrayt di pikchɔ, yuz ImageJ ɛn Adobe Photoshop fɔ prosɛs di pikchɔ mɔ ɛn ajɔst di braytnɛs ɛn kɔntrast yunifom. Yuz Adobe Illustrator fɔ pripia di grafik dɛn.
mtDNA fכkus analisis. di nכmba כf mtDNA lεshכn dεm bin kwantifay pan sεribεl sεkshכn dεm we dεn lεbul wit antibodi dεm εgεst DNA bay kכnfכkal maykroskכp. εvri tכgεt εria dεn bin kriyayt fכ di sεl bכdi εn di nyuklios fכ εvri sεl, εn dεn kכl di rispεktiv εria yuz di Multi Measure plug-in (ImageJ softwεa). sכbtrakt di nyuklia εria frכm di sεl bכdi εria fכ gεt di saytoplasmik εria. Fכ finish, dεn yuz di Analyze Particles plug-in (ImageJ softwεa) fכ כtomatכk kwantifay di saytoplasmik DNA pכynt dεm we de sho mtDNA pan di thrεshold imej, εn di risכlt dεm we dεn gεt bin nכmal to di PN avεrej fכ CTRL mays dεm. di rizulεt dεm dεn εksprεs as di avεrej nכmba fכ nyukliosyd dεm pan wan sεl.
Protein εksprεshכn analisis. Yuz ImageJ in Imej Kalkyulatɔ plɔg-in fɔ evaluate protin ɛksprɛshɔn na PN na di singl sɛl lɛvɛl. in sכt, insay di singl-layεr kכnfכkal imej fכ di kכrεspכndεnt chεnal, tru di ikwyulet: signal = min (mtYFP, antibodi), dεn de aydentify di maytochכndrial rijyכn we de sho imyun riaktiviti to wan sכm antibodi insay Purkina. afta dat dεn kכntify di piksεl εria na di rizultant mask, εn dεn nכmal pan di kכrεspכndεnt thrεshold singl-stak imej fכ di mtYFP chεnal fכ gεt di maytochכndrial frakshכn fכ di protin we dεn displei.
Purkinje sel density analisis. di Sεl Kכnta pכlכg-in fכ ImageJ dεn yuz fכ evaluate Purkinje dεnsiti bay we dεn divayd di nכmba fכ Purkinje sεl dεm we dεn kכnt bay di lεngth fכ di sεribεl ring we di sεl dεm we dεn kכnt de okyupayt.
Sampul pripia ɛn kɔlɛkshɔn. Di bren frɔm di kɔntrol grup ɛn Mfn2cKO mays dɛn bin fiks insay 2% PFA/2.5% glutaraldehyde insay 0.1 M fɔsfɛt bafa (PB), ɛn afta dat dɛn pripia koronal sɛkshɔn dɛn yuz ciliates (Leica Mikrosysteme GmbH, Vienna, Austria) (Tiknɛs 50 to 60 μm) Dɔn fiks insay PB bafa insay 1% ɔs tɛtraɔksayd ɛn 1.5% potashכm fεrosaynayd na rum tεmprachכ fכ 1 awa. Dɛn was di sɛkshɔn dɛn tri tɛm wit distil wata, ɛn afta dat dɛn stɛyn dɛn wit 70% ɛtɛnol we gɛt 1% yuranyl asɛtat fɔ 20 minit. Afta dat, dɛn bin dehydrate di sɛkshɔn dɛn insay gred alkol ɛn dɛn bin ɛmbas dɛn insay Durcupan ACM (Araldite casting resin M) ɛpoksi rɛsin (Ilɛktron Maykroskɔpi Sayns, katalog nɔmba 14040) bitwin silikon-kɔt glas slayd dɛn, ɛn las las na 60°C Polimɛrayz insay di ɔvin fɔ 48 awa. di sεribra kכtεks εria bin sεlekt εn 50 nm ultrathin sεkshכn dεm bin kכt pan Leica Ultracut (Leica Mikrosysteme GmbH, Vienna, Austria) εn pik pan 2×1 mm kכpa slit grid we dεn kכt wit polistayn film. Di sekshɔn dɛn bin stɛyn wit sɔlvushɔn we gɛt 4% yuranyl asɛtat insay H2O fɔ 10 minit, was wit H2O sɔm tɛm, dɔn wit Reynolds lid saytrɛt insay H2O fɔ 10 minit, ɛn afta dat dɛn was wit H2O bɔku tɛm. Dɛn tek maykrograf wit transmishɔn ilɛktron maykroskɔp Philips CM100 (Thermo Fisher Scientific, Waltham, MA, USA) we dɛn yuz TVIPS (Tietz Vidio ɛn Imej Prɔsesin Sistɛm) TemCam-F416 dijital kamɛra (TVIPS GmbH, Gauting, USA). Jamani).
fכ mays dεm we infεkt wit AAV, dεn separet di bren εn slais am insay 1 mm tik sajital sεkshכn, εn dεn egzamin di sεribεl yuz fכrosεns maykroskכp fכ no di AAV-infεkt ring (dat na, mCherry εksprεs). כnli εkspεriεns dεm we AAV injεkshכn de rizulta in wan vεri hכy transdyushכn efyushכn fכ di Purkinje sεl layεr (dat na kכlכsכl di כl layεr) insay atכs tu kכnsekyutiv sεribεl ring dεm dεn de yuz. Di AAV-transdyus lɔp bin maykrodisɛkt fɔ ɔvnayt post-fikseshɔn (4% PFA ɛn 2.5% glutaraldehyde insay 0.1 M kokot bafa) ɛn dɛn bin prosɛs am mɔ. Fɔ EPON ɛmbedin, dɛn was di fiks tisu wit 0.1 M sɔdiɔm kokot bafa (Applichem), ɛn inkubayt wit 2% OsO4 (os, Sayns Sɛvis; Caco) insay 0.1 M sɔdiɔm kokot bafa (Applichem) 4 Awa, dɔn was fɔ 2 awa. Ripit 3 tɛm wit 0.1 M kɔkamid bafa. Afta dat, dɛn yuz di ascending siriɔs ɛtɛnol fɔ inkubayt ɛni ɛtɛnol sɔlvushɔn na 4°C fɔ 15 minit fɔ mek di tisu nɔ gɛt wata. Di tisu bin transfכm to propylene oksayd εn inkכba כvnayt insay EPON (Sigma-Aldrich) na 4°C. put di tisu insay fresh EPON na rum tεmprachכ fכ 2 awa, εn afta dat εmbas am na 62°C fכ 72 awa. Yuz wan ɔltramaykrotɔm (Leica Microsystems, UC6) ɛn wan dayamɔn naif (Diatome, Biel, Switzerland) fɔ kɔt 70 nm ɔltratin sɛkshɔn dɛn, ɛn stɛyn wit 1.5% yuranyl asɛtat fɔ 15 minit na 37°C, ɛn stɛyn wit lid saytrɛt sɔlvushɔn 4 minit. Dɛn tek di ilɛktron maykrograf dɛn bay we dɛn yuz wan JEM-2100 Plus transmishɔn ilɛktron maykroskɔp (JEOL) we gɛt Kamɛra WanView 4K 16-bit (Gatan) ɛn DigitalMicrograph softwe (Gatan). Fɔ analisis, dɛn bin gɛt ilɛktron maykrograf wit 5000× ɔ 10,000× dijital zum.
Mכfכlכjik analisis fכ di maytochכndria. Fכ כl di analisis dεm, di kכntor dεm fכ wan wan maytochכndria dεm bin manually autlayn in dijital imej yuz ImageJ softwεa. Difrɛn mɔfɔlɔjik paramita dɛn de analayz. di maytochכndrial dεnsiti dεn de εksprεs am as pasεnshכn we dεn gεt bay we dεn divayd di tכtal maytochכndrial εria fכ εvri sεl bay di saytoplasm εria (saytoplasm εria = sεl εria-sεl nyuklios εria) × 100. dεn kכl di rכwnnes fכ di maytochכndria wit di fכmula [4π∙(eria/pεrimεta 2)]. di ista mכfכlכji fכ di maytochכndria dεn bin analכz εn divayd insay tu kεtכgrεf (“tubular” εn “blister”) akɔdin to dεn men shep dεm.
כtofagכsכm/laysכsom nכmba εn dεnsiti analisis. yuz ImageJ softwe fכ manually autlayn di kכntor dεm fכ εvri כtofagכsכm/laysosom na di dijital imej. כtofagכsכm/laysosom εria dεn de εksprεs am as pasεnshכn we dεn kכl bay we dεn divayd di tכtal כtofagכsכm/laysosom strכkchכ εria fכ εvri sεl bay di saytoplasm εria (saytoplasm εria=sεl εria-nyuklios εria)×100. di dεnsiti fכ כtofagכsכm/laysosom dεm dεn kכl am bay we dεn divayd di tכtal nכmba bay di nכmba fכ כtofagכsכm/laysosom strכkchכ dεm pan wan sεl (in tεm fכ saytoplasmik εria) (saytoplasmik εria = sεl εria-nyukliar εria).
Labeling fɔ akyu sɛkshɔn ɛn sampul pripiamɛnt. fכ εkspεriεns we nid glukכs lεbul, tכn di akyu bren slais dεm to wan prε-inkכbeshכn chεmba, we gεt satεrayt kכbכn (95% O2 εn 5% CO2), hεy Ca2 + ACSF (125.0 mM NaCl, 2.5 mM KCl, 1.25 mM sכdyכm fכsfεt bכfa, 25.0 mM NaHCO 3, 25.0 mM d-glukכs, 1.0 mM CaCl 2 εn 2.0 mM MgCl 2, we dεn adjכst to pH 7.4 εn 310 to 320 mOsm), we glukכs na 13 C 6- Glukoz sכbstityushכn (Eurisotop, katalog nכmba CLM-1396). fכ εkspεriεns we nid pyruvate lεbul, tכn di akyu bren slais dεm to hכy Ca2 + ACSF (125.0 mM NaCl, 2.5 mM KCl, 1.25 mM sכdyכm fכsfεt bכfa, 25.0 mM NaHCO3, 25.0 mM d-glukoz, 1.0 mM CaCl2 εn Ad 2.0mM MgCl2, ajɔst to pH 7.4 ɛn 310 to 320mOsm), ɛn ad 1mM 1-[1-13C]pyruvate (Eurisotop, katalog nɔmba CLM-1082). Inkubayt di sekshɔn dɛn fɔ 90 minit na 37°C. We di ɛkspiriɛns dɔn, dɛn was di sɛkshɔn dɛn kwik kwik wan wit wan akyuɔs sɔlvushɔn (pH 7.4) we gɛt 75 mM amɔniɔm kabɔnɛt, ɛn afta dat dɛn homojɛnis insay 40:40:20 (v:v:v) asɛtonitril (ACN): mɛtanɔl: wata. Afta we dɛn inkubayt di sɛkshɔn dɛn pan ays fɔ 30 minit, dɛn sɛntrifug di sampul dɛn na 21,000 g fɔ 10 minit na 4°C, ɛn dɛn drɛy di klia supamatant insay wan SpeedVac kɔnsɛntret. Di rizultant dray mεtabolayt pεlεt bin stכr na -80°C te analisis.
Liquid kromatografi-mas spɛktrometri analisis fɔ 13 C-lɛbul amino asid dɛn. Fכ likwid kromatografi-mas spεktrכmetri (LC-MS) analisis, di mεtabolayt pεlεt bin rεsuspεnd insay 75μl LC-MS gred wata (Honeywell). Afta sεntrifugayshכn na 21,000 g fכ 5 minit na 4°C, 20 μl כf di klarifyed supεrnatant bin yuz fכ amino asid fluks analisis, we di rεst כf di εkstrakt bin yuz wans fכ anion analisis (luk dכn). Amino asid analisis bin de yuz di bεnzoyl klorayd dεrivatayzεshכn protכkכl we dεn bin dεskrεb bifo ( 55 , 56 ). Insay di fɔs step, dɛn ad 10μl 100 mM sɔdyɔm kabɔnɛt (Sigma-Aldrich) to 20μl mɛtabolayt ɛkstrakt, ɛn afta dat dɛn ad 10μl 2% bɛnzoyl klorayd (Sigma-Aldrich) to di LC gred ACN. Di sampul bin vorteks fɔ shɔt tɛm ɛn afta dat dɛn sɛntrifug am na 21,000 g fɔ 5 minit na 20°C. Transfεr di klia supεrnatant to 2 ml כtosamplεr vayl wit kכnik glas insεt (200 μl vכlyum). Di samplε dεm bin analכz yuz di Acquity iClass ultra-high performance LC sistem (Waters) we kכnekt to di Q-Exactive (QE)-HF (Ultra High Field Orbitrap) hεy-rεsכlushכn prεsishכn mas spεktrכmita (Thermo Fisher Scientific). Fכ analisis, 2μl pan di derivatized sεmpl bin injεkt insay wan 100×1.0 mm hεy-strεngth silika T3 kכlכm (Waters) we kכntεn 1.8μm patikyula dεm. di fכlכ rεt na 100μl/min, εn di bכfa sistεm kכnsis fכ bafa A (10 mM amoniכm fכmat εn 0.15% fכmik asid insay wata) εn bכfa B (ACN). Di gradient na dis: 0%B na 0 minit; 0%B. 0 to 15% B na 0 to 0.1 minit; 15 to 17% B na 0.1 to 0.5 minit; B na 17 to 55% na 0.5 to 14 minit; B na 55 to 70% na 14 to 14.5 minit; na 14.5 to 70 to 100% B na 18 minit; 100% B na 18 to 19 minit; 100 to 0% B na 19 to 19.1 minit; 0% B na 19.1 to 28 minit (55, 56). Di QE-HF mas spɛktromita de wok insay pɔzitiv ayɔnizashɔn mɔd wit mas rɛnj we na m/z (mas/chaj rɛtɛshɔn) we na 50 to 750. Di rizɔlt we dɛn aplay na 60,000, ɛn di geyn kɔntrol (AGC) ayɔn target we dɛn gɛt na 3×106, ɛn di maksimal ayɔn tɛm na 100 milisekɔnd. Di hεt εlektrosprεy ayכnayzεshכn (ESI) sכs de wok pan spεy vכltεj we na 3.5 kV, kapilari tεmprachכ we na 250°C, sεt εyafכlכ we na 60 AU (arbitrary units), εn כksijεn ayfכlכ we na 20 AU. 250°C na di ples. Di S lens de set to 60 AU.
Aniɔn kromatografi-MS analisis fɔ 13C lɛbl ɔrganik asid dɛn. Di rεmεnεnt mεtabolayt prεsipitayt (55μl) bin analεz yuz wan Dionex ayɔn kromatografi sistεm (ICS 5000+, Thermo Fisher Scientific) we kכnekt to wan QE-HF mas spεktrכmita (Thermo Fisher Scientific). In shot, 5μl mεtabolayt εkstrakt bin injεkt insay wan Dionex IonPac AS11-HC kכlכm we εkwip wit HPLC (2 mm×250 mm, patikyula saiz 4μm, Thermo Fisher Scientific) insay push-in patεl lכp mכd wit wan filin rεshכn we na 1. ) Dionex IonPac AG11-HC gad kכlכm (2 mm x 50 mm, 4μm, Thermo Fisher Sayntis we de du tin). di kolm tεmprachכ de mεnten na 30°C, εn di כtosamplεr de set to 6°C. Yuz wan potashכm haydroksayd katrij we dεn gi wit diayכnayz wata fכ jεnarεt wan potashכm haydroksayd grεdiεnt tru di eluεnt jεnarεta. separeshכn fכ mεtabolit dεm pan fכ fכlכ rεt 380μl/min, aplay di fכlכw grεdiεnt: 0 to 3 minit, 10 mM KOH; 3 to 12 minit, 10 to 50 mM KOH; 12 to 19 minit, 50 to 100 mM KOH; 19 to 21 minit , 100 mM KOH; 21 to 21.5 minit, 100 to 10 mM KOH. Di kɔlɔm bin ri-ɛkwilibret ɔnda 10 mM KOH fɔ 8.5 minit.
di eluted mεtabolayt dεm de kכmbayn wit wan 150μl/min isopropanol supliment strim afta di kכlכm εn afta dat dεn de dayrεkt to wan hεy-rεsכlushכn mas spεktrכmita we de כpεret in nεgεtiv ayכnayzεshכn mכd. MS de monitar di mas rεnj frכm m/z 50 to 750 wit rεsכlushכn we na 60,000. Di AGC de set to 1×106, ɛn di maksimal ayɔn tɛm de kip na 100 ms. Di hot ESI sos bin de opereyt pan wan sprɛy vɔlɔt we na 3.5 kV. di כda setin dεm fכ di ayכn sכs na dεn wan ya: kapilari tεmprachכ 275°C; shit gas flɔ, 60 AU; ɔksijɛn gas flɔ, 20 AU na 300°C, ɛn S lens sɛtin to 60 AU.
Data analisis fכ 13C lεbul mεtabolayt dεm. Yuz TraceFinder softwe (vɛshɔn 4.2, Thermo Fisher Scientific) fɔ data analisis fɔ aysɔtop rɛsɛshɔn. Di aydentiti fɔ ɛni kɔmpawnd bin verify bay wan rilibul rɛfrɛns kɔmpawnd ɛn dɛn bin analayz am indipɛndɛnt wan. Fɔ mek dɛn ebul fɔ du aysɔtop ɛnrichmɛnt analisis, dɛn pul di eria fɔ di ayɔn kromatogram we dɛn pul (XIC) fɔ ɛni 13C aysɔtop (Mn) frɔm [M + H] +, usay n na di kabɔn nɔmba fɔ di target kɔmpawnd, we dɛn yuz fɔ analayz amino asid ɔ dɛn yuz [ MH] + fɔ analayz aniɔn. di mas akכda fכ XIC less dan fayv pat pan wan miliכn, εn di akkuracy fכ RT na 0.05 minit. di enrichmεnt analisis de du bay we dεn kכl di rεshכn fכ εvri ayzכtop we dεn dεtekt to di sכm כf כl di ayzכtop dεm fכ di kכrεspכndεnt kכmpawnd. dis rεshכn dεm dεn gi as pasεnshכn valyu fכ εvri ayzotop, εn di risכlt dεm dεn εksprεs as molar pasεnt εnrichmεnt (MPE), lεk aw dεn bin dεskrεb bifo ( 42 ).
Di frכzεn nyuron pεlεt bin homojεnayz insay ays-kol 80% mεtanכl (v/v), vכrtεks, εn inkכba na -20°C fכ 30 minit. Vortex di sampul bak ɛn stir na +4°C fɔ 30 minit. Di sampul bin sɛntrifug na 21,000 g fɔ 5 minit na 4°C, ɛn afta dat dɛn gɛda di supamatant we kɔmɔt ɛn drɛy yuz wan SpeedVac kɔnsɛntret na 25°C fɔ analisis we apin afta dat. As dεn dεskrεb am oba, LC-MS analisis bin de pan di amino asid dεm fכ di sכt sεl dεm. Yuz TraceFinder (vεshכn 4.2, Thermo Fisher Scientific), dεn bin du data analisis yuz di monoisotopic mas כf εvri kכmpawnd. Kwantil nכmalεyshכn כf mεtabolayt data bin pεrform yuz di prεprכsεsKכr softwεa pakεj ( 57 ).
Slais pripiamɛnt. Dɛn bin anestez di maws kwik kwik wan wit kabɔn dayɔgzayd ɛn kɔt in ed, dɛn pul di bren kwik kwik wan frɔm di skel, ɛn dɛn yuz di naif we ful-ɔp wit ays we de shayk (HM-650 V, Thermo Fisher Scientific, Walldorf, Germany) fɔ kɔt am insay 300 to 375 μm sajital sɛkshɔn dɛn Kol kabɔn gasifikɛshɔn (95% O2 ɛn 5% CO2) Lɔw Ca2 + ACSF (125.0 mM NaCl, 2.5 mM KCl, 1.25 mM sכdiכm fכsfεt bכfa, 25.0 mM NaHCO3, 25.0 mM d-glukoz, 1.0 mM CaCl2 εn 6.0 mM MgCl2 Adjust to pH 7.4 εn 310 to 330 mOsm). Transfεr di bren slais dεm we yu gεt to wan chεmba we gεt hכy Ca2 + ACSF (125.0 mM NaCl, 2.5 mM KCl, 1.25 mM sכdyכm fכsfεt bכfa, 25.0 mM NaHCO3, 25.0 mM d-glukoz, 4.0 mM CaCl2 εn mM 3.5 MgCl2) pH 7.4 εn 310 to 320 mOsm). Stɔr di slais dɛn fɔ 20 to 30 minit so dat dɛn go ebul fɔ kam bak bifo yu rikodɔm.
we dɛn de rikodɔm. Dɛn bin yuz wan maykroskɔp stej we gɛt fiks rikodin chɛmba ɛn 20x wata imɛshɔn ɔbjɛktiv lens (Scientifica) fɔ ɔl di rikodin dɛn. di putativ Purkinje sel dεm bin aydentify bay (i) bכdi saiz, (ii) anatomical lכkεshכn fכ di sεribεl, εn (iii) εksprεshכn fכ di fluorescent mtYFP rεpכta jin. Di pat pipet wit tip resistans we de 5 to 11 megohms na wan borosilicate glass kapilari (GB150-10, 0.86 mm×1.5 mm×100 mm, Science Products, Hofheim, Germany) ɛn wan ɔrizɔntal pipet Instruments (P-1000, Sutter), Novato, CA) de pul am. Ɔl di rikodin dɛn na ELC-03XS npi patch klem amplifaya (npi ilɛktronik GmbH, Tam, Jamani), we di softwea Signal (vɛshɔn 6.0, Kembrij Ilɛktronik, Kembrij, UK) bin de kɔntrol. Di ekspiriens bin rεkכd pan wan sampling rεt we na 12.5 kHz. Di signal de filta wit tu shɔt-pas Bessel filta dɛn wit kɔt-ɔf frikshɔn we na 1.3 ɛn 10 kHz rispɛktvɔli. Di kapasiti fɔ di mɛmbran ɛn di pipet de kɔmpɛns bay di kɔmpɛnshɔn sɔrkwit we de yuz di amplifaya. Ɔl di ɛkspiriɛns dɛn bin du ɔnda di kɔntrol fɔ wan Orca-Flash 4.0 kamɛra (Hamamatsu, Gerden, Jamani), we dɛn bin kɔntrol bay di Hokawo softwe (vɛshɔn 2.8, Hamamatsu, Gerden, Jamani).
Rutin ol-sεl kכnfigureshכn εn analisis. Jεs bifo yu rεkכd, ful di pipet wit di intanεt sכlushכn we gεt dεn tin ya: 4.0 mM KCl, 2.0 mM NaCl, 0.2 mM EGTA, 135.0 mM potashכm glukonayt, 10.0 mM Hepes, 4.0 mM ATP (Mg), 0.5 mM Guanosine triphosphate (GTP) (Na) εn 10.0 mM kriaytinin fכsfεt bin adjכst to pH 7.25, εn di osmotik prεshכn na bin 290 mOsm (sukros). Jεs afta dεn aplay fכs we na 0 pA fכ rכp di mεmbran, dεn mεzj di rεst mεmbran pכtεnshal. Di input rεsistεns de mεzj bay we dεn de aplay haypapolarayz kכrεnt dεm we na -40, -30, -20, εn -10 pA. Mekɔp di magnitud fɔ di vɔlɔt rispɔns ɛn yuz Ohm in lɔ fɔ kɔl di input rɛsistɛns. Spontan aktiviti bin rikodɔ insay wan vɔlɔt klem fɔ 5 minit, ɛn dɛn bin aydentify ɛn mɛzhɔ sPSC insay Igor Pro (vɛshɔn 32 7.01, WaveMetrics, Lake Oswego, Oregon, USA) yuz wan sɛmi-ɔtomɛtik rɛkɔgnishɔn skript. di IV kכv εn stedi-stεt kכrεnt dεn mכsu bay we dεn klem di bateri na difrεn pכtεnshal dεm (stכt frכm -110 mV) εn inkrεs di vכltεj insay 5 mV stεp dεm. Di prodakshɔn fɔ AP bin tɛst bay we dɛn aplay wan depolarizing kɔrɛnt. Klem di sel na -70 mV we yu de aplay wan depolarizing kכrant puls. Adjɔst di stɛp saiz fɔ ɛni rikodin yunit sɛpret wan (10 to 60 pA). Kalkul di maksimal AP frikshɔn bay we yu kɔnt di puls spayk dɛn we de mek di AP frikshɔn we ay pas ɔl. di AP thrεshold dεn de analכz am bay we dεn de yuz di sεkכn dεrivεt fכ di dεpolarizeshכn puls we fכs trigεr wan כ mכr AP dεm.
Perforated patch konfigyushɔn ɛn analisis. Pɔrfɔm perforated patch rikodin yuz standad protɔkɔl dɛn. Yuz wan ATP- ɛn GTP-fri pipet we nɔ gɛt dɛn tin ya: 128 mM glukonayt K, 10 mM KCl, 10 mM Hepes, 0.1 mM EGTA ɛn 2 mM MgCl2, ɛn ajɔst to pH 7.2 (yuz KOH). ATP εn GTP dεn kכl am frכm di intasεlulyar sכlushכn fכ mek di sεl mεmbran nכ kכntrol. di pat pipet dεn fulכp wit wan intanεt sכlushכn we gεt amphotericin (aprכksimatli 200 to 250μg/ml; G4888, Sigma-Aldrich) fכ gεt panch pat rεkכd. Amphotericin wεn sכlv insay dimεtyl sכlfכksayd (faynal kכnsantreshכn: 0.1 to 0.3%; DMSO; D8418, Sigma-Aldrich). di konsantreshכn fכ DMSO we dεn yuz nכ bin gεt nכ signifyant ifekt pan di nyuron dεm we dεn stכdi. Durin di panch prכsεs, di chanεl rεsistεns (Ra) bin kכntinyu fכ mכnitor, εn di εkspεriεns bin stat afta di amplitude כf Ra εn AP stεbyul (20-40 minit). di spontan aktiviti dεn de mכsu insay vכltεj εn/כ kכrant klem fכ 2 to 5 minit. Dɛn bin du di data analisis yuz Igor Pro (vɛshɔn 7.05.2, WaveMetrics, Amɛrika), Excel (vɛshɔn 2010, Maykrosoft Kɔpɔreshɔn, Rɛdmɔnd, Amɛrika) ɛn GraphPad Prism (vɛshɔn 8.1.2, GraphPad Sɔftwɛl Inc., La Jolla, CA). Amɛrika). Fɔ mek dɛn ebul fɔ no di AP dɛn we de apin bay dɛnsɛf, dɛn de yuz IgorPro in NeuroMatic v3.0c plɔg-in. Otomatik wan fɔ no di AP dɛn we yu de yuz wan givɛn trɛshɔld, we dɛn adjɔst wan wan fɔ ɛni rɛkɔd. Yuz di spayk intaval, disayd di spayk frikshɔn wit di maksimal instantaneous spayk frikshɔn ɛn di avrej spayk frikshɔn.
PN ayzolayshɔn. bay we dεn adap to di prכtכkכl we dεn bin pכblish bifo, PN dεm bin purify frכm di maws sεribεl na wan spεsifi k stej ( 58 ). in shot, dεn dεsεkt di sεribεl εn mins insay ays-kol disosiashכn mεdiכm [witout HBSS Ca2+ εn Mg2+, supliment wit 20 mM glukכs, penisilin (50 U/ml) εn strεptomycin (0.05 mg/ ml)], εn afta dat dεn daygεst di mεdiכm insay papain [HBSS, supliment wit 1-cysteine·HCl (1 mg / ml), papain (16 U / ml) ɛn dioxyribonuklease I (DNase I; 0.1 mg/ml)] Trit fɔ 30 minit na 30°C. fכs was di tisu dεm insay HBSS mεdiכm we gεt eg mכkus (10 mg/ml), BSA (10 mg/ml) εn DNase (0.1 mg/ml) na rum tεmprachכ fכ mek εnzymatic digεstεshכn nכ de, εn afta dat insay di HBSS mεdiכm we gεt 20 mM glukכs Grind jכnt insay HBSS, penisilin (50 U/ml), strεptomycin (0.05 mg/ml) εn DNase (0.1 mg/ml) de rilis singl sεl dεm. Di rizultant sel suspenshon bin filta tru wan 70μm sel strainer, den di sel dεm bin pεlεt bay sεntrifugayshכn (1110 rpm, 5 minit, 4°C) εn risuspεnd insay sכt mεdiכm [HBSS, supliment wit 20 mM glukכs, 20% fetal bovin ) Sεrum, pεnisilin (50 U/ml) εn streptomycin (0,05 mg/ml)] ɛn di ɔda wan dɛn; evaluate sεl viabiliti wit propidium iodide εn ajɔst di sεl dεnsiti to 1×106 to 2×106 sεl/ml. Bifo flɔ saytometri, dɛn bin filta di sɔspɛns tru wan 50 μm sɛl strayn.
Flɔ saytomita. Sεl sכtεshכn bin de na 4°C yuz FACSAria III mashin (BD Biosciences) εn FACSDiva softwεa (BD Biosciences, vεshכn 8.0.1). di sel suspεns bin sכt yuz 100 μm nכzl כnda prεshכn we na 20 psi pan wan rεt we na ~2800 ivent/sεk. sכm tradishכnal gεt krayteria (sεl saiz, bimodal diskriminεshכn, εn skata kכntribyushכn) nכ kin mek sכh se di kכrekt ayzכlayshכn fכ PN frכm כda sεl tכp dεm, di gεt stratεji de sεt bays pan di dayrεkt kכmpεreshכn fכ di YFP intensiti εn כtofluoresεns insay mitoYFP+ ​​εn di kכntrol mitoYFP − Mays. YFP de εksayz bay we i de rayt di sεmpl wit 488 nm lεzεr layn, εn dεn de dεtekt di signal yuz 530/30 nm bεnd pas filta. insay mitoYFP+ ​​mays dεm, dεn de yuz di rilitiv strכng fכ di Rosa26-mitoYFP rεpכta jin bak fכ difrεnt nyuronal bכdi εn aksכn fragmεnt dεm. 7-AAD de εksayz wit 561 nm yכlכ lεzεr εn dεn dεtekt am wit 675/20 nm bandpas filta fכ kכl dεd sεl dεm. fכ mek dεn separet astrosayt dεm di sem tεm, dεn bin stεn di sεl sכspεns wit ACSA-2-APC, dεn bin rayt di sεmpl wit 640 nm lεzεr layn, εn dεn yuz 660/20 nm bεndpas filta fכ dεtekt di signal.
Di sel dεm we dεn kכlekt bin pεlεt bay sεntrifugayshכn (1110 rpm, 5 minit, 4°C) εn stכr na -80°C te dεn yuz dεm. Mfn2cKO mays dεm εn dεn litar pup dεm dεn klas dεm di sem de fכ minimiz di prכsidכral vεryabiliti. FACS data prɛzɛntɛnshɔn ɛn analisis bin de yuz FlowJo softwe (FlowJo LLC, Ashland, Ɔrigɔn, Amɛrika).
as wi bin mεnshכn oba (59), dεn de yuz rial-taym PCR fכ aylכt DNA frכm di nyuron dεm we dεn sכt fכ sכbsεkvεnt mtDNA kwantifikεshכn. di linyariti εn thrεshold sεnsitiviti bin fכs tεst bay we dεn rכn qPCR pan difrεnt nכmba dεm fכ sεl dεm. in shot, kכlekt 300 PN insay wan laysis bכfa we kכnsis fכ 50 mM tris-HCl (pH 8.5), 1 mM EDTA, 0.5% Tween 20 εn protinεz K (200 ng/ml) εn inkכba na 55°C 120 minit. Di sel dεm bin fכs inkכba na 95°C fכ 10 minit fכ mek sכh se komplit inaktivεshכn fכ protinεz K. Yuz wan TaqMan prob (Thermo Fisher) spεsifi k to mt-Nd1, mtDNA bin mεzj bay sεmi-kwantiti PCR insay di 7900HT Rial-Taym PCR sistεm (Thermo Fisher Scientific). Sayns, katalog nɔmba Mm04225274_s1), mt-Nd6 (Thermo Fisher Scientific, katalog nɔmba AIVI3E8) ɛn 18S (Thermo Fisher Scientific, katalog nɔmba Hs99999901_s1) jin dɛn.
Proteome sampul pripiamɛnt. bay we yu hεt di sכlushכn na 95°C fכ 10 minit εn sonik, insay di laysis bכfa [6 M guanidin klorayd, 10 mM tris(2-kabכksiεtyl) fכsfin haydroklכrayd, 10 mM kloroasetamyd εn 100 mM tris- Lyse frozen nyuron pεlεt in HCl]. Na Bioruptor (Diagenode) fɔ 10 minit (30 sɛkɔn puls / 30 sɛkɔn pɔz tɛm). Di sampul bin diluted 1:10 in 20 mM tris-HCl (pH 8.0), miks wit 300 ng trypsin gold (Promega), ɛn inkubayt ɔvnayt na 37°C fɔ ajɔst kɔmplit dijeshɔn. Na di sɛkɔn de, dɛn bin sɛntrifug di sampul na 20,000 g fɔ 20 minit. Di supamatant bin diluted wit 0.1% fɔmik asid, ɛn di sɔlvushɔn bin desalt wit sɛlf-mɛd StageTips. Di sampul bin dray insay wan SpeedVac instrכmεnt (Eppendorf concentrator plus 5305) na 45°C, εn afta dat di pεptida bin sכspεnd insay 0.1% fכmik asid. Ɔl di sampul dɛn na di sem pɔsin bin pripia am wan tɛm. In orda fכ analכz astrosayt sεmpl, 4 μg desalted pεptida dεm bin lεbul wit wan tandem mas tεg (TMT10plex, katalog nכmba 90110, Thermo Fisher Scientific) wit wan pεptida to TMT rijen rεshכ we na 1:20. Fכ TMT lεbul, 0.8 mg TMT rijen bin rεsuspεnd insay 70 μl anhaydrous ACN, εn di drεd pεptida bin rεkכnstityut insay 9 μl 0.1 M TEAB (triethylammonium bicarbonate), we dεn ad 7 μl TMT rijen insay ACN. Di kɔnsɛntreshɔn na bin 43.75%. Afta 60 minit we dɛn inkubayt, dɛn bin kwɛn di riakshɔn wit 2 μl 5% haydroksilamin. di pεptida dεm we dεn lεbul dεn kכlekt, drεy, rεsuspεnd insay 200μl 0.1% fכmik asid (FA), divayd insay tu, εn afta dat dεn dεsכlt yuz sεlf-mεd StageTips. Yuz UltiMate 3000 ultra hai pefomans likwid kromatograf (UltiMate 3000 ultra hai pefomans likwid kromatograf), wan pan di tu haf dɛn bin frakshɔn pan wan 1mm x 150mm Acquity kromatografik kɔlɔm we ful-ɔp wit 130Å1.7μm C18 patikyula dɛn (Waters, katalog No. SKU: 186006935). Tɛrmo Fisher Sayntis). separet pεptida dεm pan fכ fכlכ rεt 30μl/min, separet frכm 1% to 50% bכfa B fכ 85 minit wit stεpways grεdiεnt fכ 96 minit, frכm 50% to 95% bכfa B fכ 3 minit, dεn 8 minit fכ 95 % bכfa B; Bafa A na 5% ACN ɛn 10 mM amɔniɔm baykabɔnɛt (ABC), ɛn bafa B na 80% ACN ɛn 10 mM ABC. Kכlekt frakshכn dεm εvri 3 minit εn kכmbayn dεm insay tu grup (1 + 17, 2 + 18, εn כda wan dεm) εn drεy dεm insay vεkyum sεntrifuj.
LC-MS/MS analisis we dɛn kin du. Fכ mas spεktrכmetri, di pεptida dεm (nכmba r119.aq) bin separet pan wan 25 cm, 75 μm inana dayamita PicoFrit analitik kכlכm (nyu כbjεktiv lens, pat nכmba PF7508250) we εkwip wit 1.9 μm ReproSil-Pur 120 C18-AQ mεdiכm (Dr. Maisch, mat), yuz EASY-nLC 1200 (Tɛmo Fisha Sayntis, Jamani). Di kɔlɔm bin de na 50°C. bכfa A εn B na 0.1% fכmik asid insay wata εn 0.1% fכmik asid insay 80% ACN, rispεktivli. Pεptida dεm bin separet frכm 6% to 31% bafa B fכ 65 minit εn frכm 31% to 50% bכfa B fכ 5 minit wit wan grεdiεnt we na 200 nl/min. Di eluted peptides bin analayz pan wan Orbitrap Fusion mas spɛktromita (Thermo Fisher Scientific). Pεptida prεkursכr m/z mεzjכmεnt dεn de du wit rεsכlushכn we de 120,000 insay di rεnj 350 to 1500 m/z. we yu yuz 27% nכmal kכlishכn εnεji, di strכng prεkursכr wit chaj stet we de 2 to 6 dεn sεlekt fכ hεy εnεji C trap disosiashכn (HCD) klivεj. Di saykl tɛm de sɛt to 1 s. di m/z valyu fכ di pεptida fragmεnt bin mεzj insay di ayכn trap yuz di sכmכl AGC tכgεt we na 5×104 εn di maksimal injεkshכn tεm we na 86 ms. Afta fragmεntεshכn, dεn bin put di prεkursכr pan di dinamik εksklushכn list fכ 45 s. TMT-labeled peptides bin separet pan 50 cm, 75 μm Acclaim PepMap kɔlɔm (Thermo Fisher Scientific, katalog nɔmba 164942), ɛn di maykreshɔn spɛktra dɛn bin analayz pan wan Orbitrap Lumos Tribrid mas spɛktromita (Thermo Fisher Scientific) we gɛt ay-fild asimɛtrik wɛvfɔm ayɔn (FAIMS) ikwipmɛnt (Thermo Fisher Scientific) de wok pan tu kɔmpɛnshɔn vɔlɔt we na −50 ɛn −70 V. MS3 we dɛn pik bays pan di sinkronizashɔn prɛkursɔ na in dɛn de yuz fɔ TMT ripɔt ayɔn signal mɛzhɔmɛnt. Di pεptida separeshכn bin kכriכnt pan EASY-nLC 1200, yuz 90% linya grεdiεnt εlushכn, wit bכfa kכnsantreshכn 6% to 31%; bafa A na bin 0.1% FA, ɛn bafa B na bin 0.1% FA ɛn 80% ACN. Di analitik kɔlɔm de ɔpreshɔn na 50°C. Yuz FreeStyle (vɛshɔn 1.6, Thermo Fisher Scientific) fɔ split di ɔrijinal fayl akɔdin to di FAIMS kɔmpɛnshɔn vɔlɔt.
Protein idεntifikεshכn εn kwantifikεshכn. Yuz di intagreted Andromeda sach injin, di ɔrijinal data bin analayz yuz MaxQuant vɛshɔn 1.5.2.8 (https://maxquant.org/). Apat frכm di Cre rεkombinaz εn YFP sikεns dεm we dεn gεt frכm Aequorea victoria, dεn bin sכch pεptida fragmεnt spεktra fכ di kanonik sikεns εn isoform sikεns fכ di maws rεfrεns proteom (Proteome ID UP000000589, we dεn dכnכlod frכm UniProt insay May 2017) . Methionine oksidashכn εn protin N-tεrminal asetεlayshכn bin set as vεryuable modifyushכn; sistayn karbamoyl mεtyleshכn bin set as fiks modifyushכn. di dijeshכn paramita dεm dεn sεt to “spεsifisiti” εn “trypsin/P”. di minim nכmba fכ di pεptida dεm εn di rεzכ pεpti dεm we dεn yuz fכ no di protin na 1; di minim nכmba fכ di yunik pεptida dεm na 0. כnda di kכndishכn dεm fכ di pεptida map mεch, di protin idεntifikεshכn rεt na bin 0.01. Di “Second Peptide” opshɔn dɔn ɛnabul. Yuz di “match between runs” opshɔn fɔ transfa saksesful aydentifikeshɔn bitwin difrɛn ɔrijinal fayl dɛn. Yuz LFQ minim rεshכ kכnt 1 fכ lεbul-fri kwantifikεshכn (LFQ) (60). di LFQ intensiti de filta fכ at le tu valid valyu dεm insay at le wan jεnotayp grup na εvri tεm pכynt, εn dεn de εkstrapol am frכm wan nכmal distribushכn wit wan wit we. 0.3 ɛn muv dɔŋ 1.8. Yuz Perseus kɔmpiutin pletfɔm (https://maxquant.net/perseus/) ɛn R (https://r-project.org/) fɔ analayz di LFQ rizɔlt dɛn. A tu-way mכdarεt t tεst frכm di limma softwεa pakej bin yuz fכ difrεnt εksprεshכn analisis ( 61 ). Eksplɔrɔtɔri data analisis de du yuz ggplot, FactoMineR, factoextra, GGally ɛn pheatmap. Di TMT-based proteomics data bin analayz yuz MaxQuant vɛshɔn 1.6.10.43. Sɔch fɔ raw proteomics data frɔm UniProt in human proteomics database, we dɛn bin dawnlod insay Sɛptɛmba 2018. Di analisis inklud di isotope puriti kɔrɛkshɔn factor we di manifakta bin gi. Yuz limma in R fɔ difrɛnshal ɛksprɛshɔn analisis. Di ɔrijinal data, database sɔch rizɔlt, ɛn data analisis wokflɔ ɛn rizɔlt ɔl de na di ProteomeXchange alyans tru di PRIDE patna ripɔsitɔri wit di data sɛt aydentifaya PXD019690.
Fɔnshɔnal anoteshɔn dɛn de ɛnrich di analisis. Di Ingenuity Pathway Analysis (QIAGEN) tul bin yuz fɔ no di richnɛs fɔ di fɛnshɔnal anoteshɔn tɛm dɛn fɔ di data sɛt na 8 wik (Figure 1). in shot, di kwantiti protin list we dεn gεt frכm LC-MS/MS (tandem mass spectrometry) data analisis dεn yuz am wit di fכlכw filta krayteria: dεn sεlekt Mus mכskul as di spεshal εn bakgrכn, εn di kεtכgכri sho di P valyu we Benjamini adjכst fכ enrichmεnt 0.05 כ lכw dεn kכnsidr am sכmtin. Fɔ dis grafik, dɛn sho di fayv tɔp ɛksɛs kategori dɛn na ɛni klasta we dɛn bays pan di adjɔst P valyu. Yuz mכltipכl t-tεst, yuz di tu-stej linya bכst program fכ Benjamini, Krieger, εn Yekutieli (Q = 5%), dεn de du tεm-kכs protin εksprεshכn analisis pan di imכtant kandidet dεm we dεn aydentify insay εvri kεtכgrεf, εn εvri rכw dεn analכz sεparat wan. No nid nɔ de fɔ adopt wan kɔnsistɛns SD.
In orda fכ kכmpεr di risכlt fכ dis stכdi wit pכblished dεtabεys εn jεnarεt wan Venn dayagram na Fig 1, wi kכmbayn di kwantiti protin list wit MitoCarta 2.0 anoteshכn ( 24 ). Yuz di onlayn tul Draw Venn Diagram (http://bioinformatics.psb.ugent.be/webtools/Venn/) fɔ mek di dayagram.
fכ ditayl infכmeshכn bכt di stεdi prosidכs dεm we dεn yuz fכ proteomics analisis, duya rifrε to di kכrεspכndεnt sεkshכn fכ Matirial εn Mεtכd. Fɔ ɔl di ɔda ɛkspiriɛns dɛn, yu kin fɛn ditayla infɔmeshɔn na di kɔrɛspɔndɛns lɛjɛnd. If dɛn nɔ tɔk ɔda we, dɛn ɛksprɛs ɔl di data as min ± SEM, ɛn ɔl di statystik analisis dɛn bin du yuz GraphPad Prism 8.1.2 softwe.
Fɔ sɔpɔt ɔda tin dɛn fɔ dis atikul, duya luk http://advances.sciencemag.org/cgi/content/full/6/35/eaba8271/DC1
Dis na opin akses atikul we dɛn sheb ɔnda di tin dɛn we de na di Krio Kɔmɔns Atribiushɔn-Nɔn-Kɔmɛshɔn Laysens, we de alaw fɔ yuz, sheb ɛn riprodyuz insay ɛni mɛdiɔm, as lɔng as di las yus nɔto fɔ kɔmɛshɔn bɛnifit ɛn di prɛmis na se di ɔrijinal wok kɔrɛkt. Tɔk bɔt.
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Na E. Motori, I. Atanassov, S. M. V. Kɔchan, K. Fɔlz-Dɔnahue, V. Sakthivelu, P. Giavalisco, N. Toni, J. Puyal, N.-G. Larson bin de tɔk bɔt am
Proteomics analysis of dysfunctional nyuron dεm sho se mεtabolik program dεm de aktibכt fכ kכntrכl nyurodijεnεreshכn.
Na E. Motori, I. Atanassov, S. M. V. Kɔchan, K. Fɔlz-Dɔnahue, V. Sakthivelu, P. Giavalisco, N. Toni, J. Puyal, N.-G. Larson bin de tɔk bɔt am
Proteomics analysis of dysfunctional nyuron dεm sho se mεtabolik program dεm de aktibכt fכ kכntrכl nyurodijεnεreshכn.
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