Panopa *Adilesi yapano: Cologne 50931, Germany, Cologne Excellence Cluster Research on Cellular Stress Response in Aging-related Diseases (CECAD).
Kuwonongeka kwa mitsempha ya mitochondrial kumaonedwa kuti sikungasinthe chifukwa kagayidwe kake ka ma neuron ndi kochepa, koma zotsatira za kusagwira bwino ntchito kwa mitochondrial pa kudziyimira pawokha kwa kagayidwe ka mitsempha ya m'thupi sizikumveka bwino. Pano, tikuyambitsa proteome yeniyeni ya ma neuron a Purkinje yokhala ndi kusowa kwa OXPHOS komwe kumachitika chifukwa cha kusokonezeka kwa kayendedwe ka mitochondrial fusion. Tapeza kuti kusagwira bwino ntchito kwa mitochondrial kunayambitsa kusintha kwakukulu m'munda wa proteomics, zomwe pamapeto pake zidapangitsa kuti mapulogalamu enieni a kagayidwe ka mitsempha ayambe kugwira ntchito maselo asanafe. Mosayembekezereka, tinapeza kuyambitsidwa koonekeratu kwa pyruvate carboxylase (PCx) ndi ma enzyme ena oletsa ukalamba omwe amathandizira pakati pa TCA cycle. Kuletsa PCx kunakulitsa kupsinjika kwa okosijeni ndi kuwonongeka kwa mitsempha, zomwe zikusonyeza kuti atherosclerosis ili ndi mphamvu yoteteza ma neuron omwe alibe OXPHOS. Kubwezeretsedwa kwa kusakanikirana kwa mitochondrial mu ma neuron omwe adawonongeka kotheratu kumasinthiratu machitidwe a kagayidwe ka mitsempha, motero kumaletsa kufa kwa maselo. Zomwe tapeza zikuwonetsa njira zomwe sizinadziwike kale zomwe zimapangitsa kuti kusagwira bwino ntchito kwa mitochondrial kukhale kolimba ndipo zikuwonetsa kuti kuwonongeka kwa mitsempha kumatha kubwezeretsedwa ngakhale kumapeto kwa matendawa.
Udindo waukulu wa mitochondria pakusunga kagayidwe ka mphamvu ya mitsempha umagogomezedwa ndi zizindikiro zazikulu za mitsempha zomwe zimagwirizanitsidwa ndi matenda a mitochondrial a anthu. Matenda ambiriwa amayamba chifukwa cha kusintha kwa majini komwe kumalamulira kufalikira kwa majini a mitochondrial (1, 2) kapena kuwonongeka kwa majini okhudzana ndi mphamvu za mitochondrial, zomwe zimakhudza mwachindunji kukhazikika kwa DNA ya mitochondrial (mtDNA) (3, 4). Ntchito yogwira ntchito m'mafano a nyama yawonetsa kuti poyankha kusagwira bwino ntchito kwa mitochondrial m'minofu yozungulira, njira zosungira kagayidwe kachakudya (5-7) zitha kuyatsidwa, zomwe zimapereka chidziwitso chofunikira kuti timvetsetse bwino za matenda ovuta awa. Mosiyana kwambiri, kumvetsetsa kwathu kusintha kwa kagayidwe kachakudya kwa mitundu inayake ya maselo komwe kumachitika chifukwa cha kulephera kwa kupanga kwa adenosine triphosphate (ATP) muubongo ndikofunikira (8), kugogomezera kufunika kozindikira zolinga zochiritsira zomwe zingagwiritsidwe ntchito popewa kapena kupewa matenda. Kuletsa kuwonongeka kwa mitsempha (9). Kusowa kwa chidziwitso ndikuti maselo amitsempha amaonedwa kuti ali ndi kusinthasintha kochepa kwambiri kwa kagayidwe kachakudya poyerekeza ndi mitundu ya maselo a minofu yozungulira (10). Popeza maselo amenewa amagwira ntchito yofunika kwambiri pogwirizanitsa kuperekedwa kwa ma metabolites ku ma neuron kuti alimbikitse kufalikira kwa synaptic ndikuyankha kuvulala ndi matenda, kuthekera kosintha kagayidwe ka maselo ku zovuta za minofu ya ubongo kumakhala pafupifupi kwa maselo a glial (11-14). Kuphatikiza apo, kusiyana kwa maselo a minofu ya ubongo kumalepheretsa kwambiri kuphunzira kusintha kwa kagayidwe kachakudya komwe kumachitika m'magulu enaake a neuron. Zotsatira zake, sizikudziwika zambiri za zotsatira zenizeni za maselo ndi kagayidwe kachakudya ka mitochondrial mu ma neuron.
Pofuna kumvetsetsa zotsatira za kagayidwe kachakudya ka mitochondrial, tinasiyanitsa ma neuron a Purkinje (PNs) m'magawo osiyanasiyana a kuwonongeka kwa mitsempha komwe kumachitika chifukwa cha kuwonongeka kwa fusion ya membrane yakunja ya mitochondrial (Mfn2). Ngakhale kusintha kwa Mfn2 mwa anthu kumalumikizidwa ndi mtundu wa neuropathy yobadwa nayo ya sensory motor yotchedwa Charcot-Marie-Tooth type 2A (15), kuwonongeka kwa Mfn2 m'makoswe ndi njira yodziwika bwino yoyambitsa oxidation​​​ Phosphorylation (OXPHOS) dysfunction method. Mitundu yosiyanasiyana ya neuronal (16-19) ndi neurodegenerative phenotype yomwe imabwera chifukwa chake imayenderana ndi zizindikiro za neurological progressive, monga mayendedwe osokoneza (18, 19) kapena cerebellar ataxia (16). Pogwiritsa ntchito kuphatikiza kwa proteomics ya quantitative-free (LFQ), metabolomics, imaging, ndi virological methods, tikuwonetsa kuti progressive neurodegeneration imayambitsa kwambiri pyruvate carboxylase (PCx) ndi zinthu zina zomwe zimakhudzana ndi arteriosclerosis ya ma PNs mu vivo The expression of enzymes. Kuti titsimikizire kufunika kwa zomwe tapezazi, tinachepetsa makamaka momwe PCx imagwirira ntchito mu ma PN omwe alibe Mfn2, ndipo tinapeza kuti opaleshoniyi inawonjezera kupsinjika kwa okosijeni komanso kufulumizitsa kuwonongeka kwa mitsempha, motero kutsimikizira kuti azoospermia imapangitsa kuti maselo afe. ​​Kuwonjezeka kwa MFN2 kwambiri kumatha kupulumutsa kwathunthu kuwonongeka kwa PN komwe kumakhala ndi kusowa kwakukulu kwa OXPHOS, kugwiritsa ntchito kwambiri DNA ya mitochondrial, komanso maukonde a mitochondrial omwe akuoneka kuti asweka, zomwe zikugogomezeranso kuti mtundu uwu wa kuwonongeka kwa mitsempha ukhoza kuchira ngakhale pamlingo wapamwamba wa matenda maselo asanafe.
Pofuna kuwona mitochondria mu Mfn2 knockout PNs, tinagwiritsa ntchito mtundu wa mbewa womwe umalola mitochondria yodalira Cre kuyang'ana puloteni yachikasu ya fluorescent (YFP) (mtYFP) (20) Cre expression ndipo tinayang'ana mawonekedwe a mitochondria mu vivo. Tinapeza kuti kuwonongedwa kwa jini la Mfn2 mu PNs kungapangitse kuti ma network a mitochondrial agawanike pang'onopang'ono (Chithunzi S1A), ndipo kusintha koyambirira kunapezeka ali ndi masabata atatu. Mosiyana ndi zimenezi, kuwonongeka kwakukulu kwa gawo la maselo a PN, monga momwe zasonyezedwera ndi kutayika kwa Calbindin immunostaining, sikunayambe mpaka masabata 12 (Chithunzi 1, A ndi B). Kusagwirizana kwa nthawi pakati pa kusintha koyambirira kwa mawonekedwe a mitochondrial ndi kuyamba kooneka kwa imfa ya mitsempha kunatipangitsa kufufuza kusintha kwa kagayidwe kachakudya komwe kumayamba chifukwa cha kusagwira ntchito kwa mitochondrial maselo asanafe. Tinapanga njira yopangira maselo opangidwa ndi fluorescence-activated cell sorting (FACS) kuti tipeze YFP (YFP+)-expressing PN (Chithunzi 1C), ndi mbewa zolamulira (Mfn2 + / loxP :: mtYFP loxP- stop-loxP: : L7-cre), yomwe tsopano ikutchedwa CTRL (Chithunzi S1B). Kukonza njira yopangira gating kutengera mphamvu ya chizindikiro cha YFP kumatithandiza kuyeretsa thupi la YFP+ (YFPhigh) la ma PN kuchokera ku ma non-PN (YFPneg) (Chithunzi S1B) kapena zidutswa za fluorescent axon/dendritic (YFPlow; Chithunzi S1D, kumanzere), zomwe zatsimikiziridwa ndi microscope ya confocal (Chithunzi S1D, kumanja). Pofuna kutsimikizira kudziwika kwa anthu odziwika, tinachita LFQ proteomics kenako kusanthula kwa zigawo zazikulu, ndipo tinapeza kuti pali kusiyana komveka pakati pa maselo a YFPhigh ndi YFPneg (Chithunzi S1C). Maselo a YFPhigh adawonetsa kuchuluka kwa ma PN markers odziwika bwino (monga Calb1, Pcp2, Grid2 ndi Itpr3) (21, 22), koma palibe kuchuluka kwa mapuloteni omwe amafotokozedwa m'ma neuron kapena mitundu ina ya maselo (Chithunzi 1D)). Kuyerekeza pakati pa zitsanzo m'maselo a YFPhigh omwe adasonkhanitsidwa mu zoyeserera zodziyimira pawokha kunawonetsa coefficient yolumikizana> 0.9, kuwonetsa kuberekana bwino pakati pa ma replicates achilengedwe (Chithunzi S1E). Mwachidule, deta iyi idatsimikizira dongosolo lathu la kudzipatula kwa PN koyenera komanso kwapadera. Chifukwa dongosolo loyendetsa la L7-cre lomwe limagwiritsidwa ntchito limayambitsa kubwerezabwereza kwa mosaic sabata yoyamba mutabereka (23), tinayamba kuchotsa mbewa kuchokera ku CTRL ndi conditional (Mfn2 loxP / loxP :: mtYFP loxP-stop-loxP :: L7-cre) Sungani ma neuron. Kubwerezabwereza kukatha, kumatchedwa Mfn2cKO ali ndi masabata anayi. Pomaliza, tinasankha masabata 8 a msinkhu pamene gawo la PN linali lonse ngakhale kuti panali kugawikana kwa mitochondrial (Chithunzi 1B ndi Chithunzi S1A). Ponseponse, tinayesa mapuloteni 3013, omwe pafupifupi 22% adachokera ku MitoCarta 2.0 annotations kutengera proteome ya mitochondrial ngati mitochondria (Chithunzi 1E) (Chithunzi 1E) (24). Kusanthula kwa majini osiyanasiyana komwe kunachitika pa sabata la 8 kunawonetsa kuti 10.5% yokha ya mapuloteni onse anali ndi kusintha kwakukulu (Chithunzi 1F ndi Chithunzi S1F), pomwe mapuloteni 195 adachepetsedwa ndipo mapuloteni 120 adakwezedwa (Chithunzi 1F). Ndikofunikira kudziwa kuti "kusanthula kwatsopano kwa njira" kwa deta iyi kukuwonetsa kuti majini omwe amawonetsedwa mosiyana makamaka ndi a gulu lochepa la njira zinazake za kagayidwe kachakudya (Chithunzi 1G). Chochititsa chidwi n'chakuti, ngakhale kuchepetsa njira zokhudzana ndi OXPHOS ndi calcium signaling kumatsimikizira kuyambitsa kusagwira bwino ntchito kwa mitochondrial mu ma PN omwe alibe fusion, magulu ena omwe makamaka amakhudza kagayidwe ka amino acid amawonjezeka kwambiri, zomwe zikugwirizana ndi kagayidwe kamene kamachitika mu ma PN a mitochondrial.
(A) Zithunzi zoyimira zamkati za magawo a cerebellar a mbewa za CTRL ndi Mfn2cKO zomwe zikuwonetsa kutayika pang'onopang'ono kwa ma PN (calbindin, imvi); ma nuclei adayesedwa ndi DAPI. (B) Kuyeza kwa (A) (kusanthula kwa njira imodzi kwa kusiyana, ***P<0.001; n = mabwalo 4 mpaka 6 kuchokera ku mbewa zitatu). (C) Kuyenda kwa mayeso. (D) Kugawa mapu a kutentha kwa zizindikiro zapadera ku Purkinje (pamwamba) ndi mitundu ina ya maselo (pakati). (E) Chithunzi cha Venn chomwe chikuwonetsa kuchuluka kwa mapuloteni a mitochondrial omwe adapezeka mu PN yodziwika. (F) Chithunzi cha volcano cha mapuloteni owonetsedwa mosiyanasiyana mu ma neuron a Mfn2cKO pa masabata 8 (mtengo wodulidwa wa 1.3). (G) Kusanthula njira yolenga kukuwonetsa njira zisanu zofunika kwambiri zokwezera (zofiira) ndi zotsika (zabuluu) mu Mfn2cKO PN yodziwika ngati masabata 8. Mulingo wapakati wowonetsera wa puloteni iliyonse yodziwika ukuwonetsedwa. Mapu a kutentha a Grayscale: P value yosinthidwa. ns, sikofunikira.
Deta ya ma proteomics inasonyeza kuti kuchuluka kwa mapuloteni a ma complexes I, III, ndi IV kunachepa pang'onopang'ono. Ma Complexes I, III, ndi IV onse anali ndi ma subunits ofunikira a mtDNA, pomwe complex II, yomwe inali ndi ma nuclear-coded okha, sinakhudzidwe kwenikweni (Chithunzi 2A ndi Chithunzi S2A). . Mogwirizana ndi zotsatira za ma proteomics, immunohistochemistry ya magawo a minofu ya cerebellar inasonyeza kuti kuchuluka kwa MTCO1 (mitochondrial cytochrome C oxidase subunit 1) subunit ya complex IV mu PN kunachepa pang'onopang'ono (Chithunzi 2B). Subunit ya mtDNA-encoded Mtatp8 inachepa kwambiri (Chithunzi S2A), pomwe kuchuluka kwa steady-state kwa nuclear-encoded ATP synthase subunit sikunasinthe, zomwe zikugwirizana ndi stable ATP synthase subassembly F1 complex yodziwika bwino pamene mtDNA expression ili yokhazikika. Kapangidwe kake ndi kokhazikika. Interrupt (7). Kuwunika kwa mulingo wa mtDNA mu ma PN a Mfn2cKO osankhidwa ndi real-time polymerase chain reaction (qPCR) kunatsimikizira kuchepa pang'onopang'ono kwa chiwerengero cha makope a mtDNA. Poyerekeza ndi gulu lolamulira, ali ndi masabata 8, pafupifupi 20% yokha ya mulingo wa mtDNA idasungidwa (Chithunzi 2C). Mogwirizana ndi zotsatirazi, kupendekera kwa confocal microscopy kwa ma PN a Mfn2cKO kunagwiritsidwa ntchito kuzindikira DNA, kusonyeza kugwiritsidwa ntchito kwa mitochondrial nucleotides kumadalira nthawi (Chithunzi 2D). Tapeza kuti anthu ena okha omwe adakhudzidwa ndi kuwonongeka kwa mapuloteni a mitochondrial ndi kuyankha kwa kupsinjika ndi omwe adakwezedwa, kuphatikiza Lonp1, Afg3l2 ndi Clpx, ndi OXPHOS complex assembly factors. Palibe kusintha kwakukulu pamlingo wa mapuloteni omwe amakhudzidwa ndi apoptosis komwe kudapezeka (Chithunzi S2B). Mofananamo, tapeza kuti mitochondria ndi endoplasmic reticulum channels zomwe zimakhudzidwa ndi calcium transport zili ndi kusintha pang'ono chabe (Chithunzi S2C). Kuphatikiza apo, kuwunika kwa mapuloteni okhudzana ndi autophagy sikunapeze kusintha kwakukulu, komwe kumagwirizana ndi kulowetsedwa kowoneka kwa autophagosomes komwe kumawonedwa mu vivo ndi immunohistochemistry ndi electron microscopy (Chithunzi S3). Komabe, kusagwira ntchito bwino kwa OXPHOS mu PNs kumayenderana ndi kusintha koonekeratu kwa mitochondrial. Magulu a mitochondrial amatha kuwoneka m'maselo ndi mitengo ya dendritic ya Mfn2cKO PNs azaka za masabata 5 ndi 8, ndipo kapangidwe ka mkati mwa nembanemba kasintha kwambiri (Chithunzi S4, A ndi B). Mogwirizana ndi kusintha kwa ultrastructural kumeneku komanso kuchepa kwakukulu kwa mtDNA, kusanthula kwa magawo a acute cerebellar cerebral cerebral ndi tetramethylrhodamine methyl ester (TMRM) kunawonetsa kuti kuthekera kwa nembanemba ya mitochondrial mu Mfn2cKO PNs kunachepa kwambiri (Chithunzi S4C).
(A) Kusanthula nthawi ya kuchuluka kwa mafotokozedwe a OXPHOS complex. Ganizirani mapuloteni okha ndi P<0.05 pa masabata 8 (ANOVA ya njira ziwiri). Mzere wolembedwa: Palibe kusintha poyerekeza ndi CTRL. (B) Kumanzere: Chitsanzo cha gawo la cerebellar lolembedwa ndi anti-MTCO1 antibody (scale bar, 20 μm). Malo omwe ali ndi matupi a maselo a Purkinje ali ndi chikasu. Kumanja: Kuyeza kuchuluka kwa MTCO1 (kusanthula kusiyana kwa njira imodzi; n = maselo 7 mpaka 20 omwe amafufuzidwa kuchokera ku mbewa zitatu). (C) Kusanthula kwa qPCR kwa nambala ya kopi ya mtDNA mu PN yosankhidwa (kusanthula kusiyana kwa njira imodzi; n = mbewa 3 mpaka 7). (D) Kumanzere: Chitsanzo cha chidutswa cha cerebellar cholembedwa ndi anti-DNA antibody (scale bar, 20 μm). Malo omwe ali ndi matupi a maselo a Purkinje ali ndi chikasu. Kumanja: Kuyeza kuchuluka kwa zilonda za mtDNA (kusanthula kusiyana kwa njira imodzi; n = maselo 5 mpaka 9 ochokera ku mbewa zitatu). (E) Chitsanzo cha gawo la cerebellar lowonetsa maselo a mitoYFP + Purkinje (muvi) mu kujambula kwa clamp ya patch ya selo lonse. (F) Kuyeza kwa IV curve. (G) Zolemba zoyimira jakisoni wa depolarizing current mu maselo a CTRL ndi Mfn2cKO Purkinje. Zotsatira Zapamwamba: Kugunda koyamba komwe kunayambitsa AP. Zotsatira Zapansi: Mafupipafupi a AP ambiri. (H) Kuyeza kwa zolowetsa za postsynaptic (sPSPs). Zotsatira zoyimira zolembera ndi chiŵerengero chake cha zoom zikuwonetsedwa mu (I). Kusanthula kwa njira imodzi kwa kusiyana komwe kunawunikidwa n = maselo 5 mpaka 20 kuchokera ku mbewa zitatu. Deta imafotokozedwa ngati mean±SEM; *P<0.05; **P<0.01; ***P<0.001. (J) Zotsatira zoyimira za AP yodzidzimutsa yojambulidwa pogwiritsa ntchito njira ya clamp ya patch yobowoka. Zotsatira Zapamwamba: Mafupipafupi a AP ambiri. Zotsatira Zapansi: zoom ya AP imodzi. (K) Kuyeza mafupipafupi apakati ndi apamwamba a AP malinga ndi (J). Mayeso a Mann-Whitney; n = maselo 5 adawunikidwa kuchokera ku mbewa zinayi. Deta imafotokozedwa ngati yapakati ± SEM; sikofunikira.
Kuwonongeka koonekeratu kwa OXPHOS kunapezeka mu Mfn2cKO PN ya masabata 8, zomwe zikusonyeza kuti ntchito ya ma neuron ndi yosazolowereka kwambiri. Chifukwa chake, tinasanthula makhalidwe amagetsi osagwira ntchito a ma neuron osowa OXPHOS pa masabata 4 mpaka 5 ndi masabata 7 mpaka 8 pochita zojambula za patch clamp ya cell yonse mu magawo a cerebellar (Chithunzi 2E). Mosayembekezereka, mphamvu yapakati yopumula ya membrane ndi kukana kwa ma neuron a Mfn2cKO zinali zofanana ndi zowongolera, ngakhale panali kusiyana pang'ono pakati pa maselo (Table 1). Mofananamo, pa masabata 4 mpaka 5 azaka, palibe kusintha kwakukulu mu ubale wamagetsi (IV curve) komwe kunapezeka (Chithunzi 2F). Komabe, palibe ma neuron a Mfn2cKO a masabata 7 mpaka 8 omwe adapulumuka IV regimen (gawo la hyperpolarization), zomwe zikusonyeza kuti pali kukhudzidwa komveka bwino kwa hyperpolarization potential kumapeto kwa gawoli. Mosiyana ndi zimenezi, mu ma neuron a Mfn2cKO, ma currents a depolarizing omwe amachititsa kuti ma AP atuluke (repetitive action potential) amaloledwa bwino, zomwe zikusonyeza kuti ma currents awo onse sasiyana kwambiri ndi ma neuron olamulira a masabata 8 (Table 1 ndi Chithunzi 2G). Mofananamo, ma frequency ndi amplitude a ma currents a postsynaptic currents (sPSCs) anali ofanana ndi a gulu lolamulira, ndipo ma frequency a zochitika adakwera kuchokera pa masabata 4 mpaka masabata 5 mpaka masabata 7 mpaka masabata 8 ndi kuwonjezeka kofanana (Chithunzi 2, H ndi I). Nthawi ya ma synaptic maturation mu PNs (25). Zotsatira zofananazo zidapezeka pambuyo pa ma PNs obowoka. Kapangidwe kameneka kamaletsa kubweza zolakwika za ATP zama cell, monga momwe zingachitikire pakujambula clamp ya cell yonse. Makamaka, mphamvu ya resting membrane ndi ma frequency a automatic firing a ma neuron a Mfn2cKO sizinakhudzidwe (Chithunzi 2, J ndi K). Mwachidule, zotsatirazi zikusonyeza kuti ma PN omwe ali ndi vuto lodziwika bwino la OXPHOS amatha kuthana bwino ndi njira zotulutsira madzi pafupipafupi, zomwe zikusonyeza kuti pali njira yolipirira yomwe imawalola kuti asunge mayankho amagetsi pafupifupi abwinobwino.
Deta imafotokozedwa ngati mean ± SEM (kusanthula kwa njira imodzi kwa kusiyana, mayeso oyerekeza angapo a Holm-Sidak; *P<0.05). Nambala ya unit ikuwonetsedwa ndi mabulaketi.
Tinayamba kufufuza ngati gulu lililonse mu deta ya proteomics (Chithunzi 1G) likuphatikizapo njira zomwe zingagonjetse kusowa kwakukulu kwa OXPHOS, potero kufotokoza chifukwa chake PN yomwe yakhudzidwa imatha kusunga electrophysiology yapafupifupi yachibadwa (Chithunzi 2, E mpaka K). . Kusanthula kwa proteomics kunawonetsa kuti ma enzyme omwe amagwira ntchito mu catabolism ya amino acids (BCAA) anali ochulukirapo kwambiri (Chithunzi 3A ndi Chithunzi S5A), ndipo chinthu chomaliza cha acetyl-CoA (CoA) kapena succinyl CoA chingathandize ma tricarboxylates mu arteriosclerosis Acid (TCA) cycle. Tapeza kuti kuchuluka kwa BCAA transaminase 1 (BCAT1) ndi BCAT2 zonse zinawonjezeka. Zimathandizira gawo loyamba la catabolism ya BCAA popanga glutamate kuchokera ku α-ketoglutarate (26). Ma subuniti onse omwe amapanga nthambi ya keto acid dehydrogenase (BCKD) complex amakwezedwa (complex iyi imayambitsa decarboxylation yotsatira komanso yosasinthika ya mafupa a kaboni a BCAA omwe amachokera) (Chithunzi 3A ndi Chithunzi S5A). Komabe, palibe kusintha koonekeratu mu BCAA yokha komwe kunapezeka mu PN yosankhidwa, yomwe ikhoza kukhala chifukwa cha kuchuluka kwa ma amino acid ofunikira awa m'maselo kapena kugwiritsa ntchito magwero ena (glucose kapena lactic acid) kuti awonjezere kayendedwe ka TCA (Chithunzi S5B). Ma PN omwe alibe OXPHOS adawonetsanso kuchuluka kwa glutamine decomposition ndi transamination activity ali ndi masabata 8, zomwe zitha kuwonetsedwa ndi kukwera kwa ma enzymes a mitochondrial glutaminase (GLS) ndi glutamine pyruvate transaminase 2 (GPT2) (Chithunzi 3, A ndi C). Ndikofunikira kudziwa kuti kukwera kwa GLS kumangokhala pa spliced ​​​​isoform glutaminase C (GLS-GAC) (kusintha kwa Mfn2cKO/CTRL kuli pafupifupi 4.5-fold, P = 0.05), ndipo kukwera kwake kwapadera m'maselo a khansa Kungathandize bioenergy ya mitochondrial. (27).
(A) Mapu a kutentha akuwonetsa kusintha kwa kuchuluka kwa mapuloteni pa njira yomwe yatchulidwa pa masabata 8. (B) Chitsanzo cha chidutswa cha cerebellar cholembedwa ndi anti-PCx antibody (scale bar, 20 μm). Muvi wachikasu umaloza ku thupi la selo la Purkinje. (C) Kusanthula kwa nthawi yogwiritsira ntchito mapuloteni komwe kwadziwika ngati kofunikira pa atherosclerosis (mayeso angapo a t, *FDR <5%; n = mbewa 3-5). (D) Pamwambapa: Chithunzi chosonyeza njira zosiyanasiyana zolowera kaboni wolembedwa womwe uli mu [1-13C]pyruvate tracer (mwachitsanzo, kudzera mu PDH kapena trans-arterial route). Pansi: Tchati cha violin chikuwonetsa kuchuluka kwa kaboni wolembedwa (M1) wosinthidwa kukhala aspartic acid, citric acid ndi malic acid mutalemba magawo a cerebellar acute ndi [1-13C]pyruvate (mayeso ophatikizidwa a t; ** P <0.01). (E) Kusanthula kwathunthu kwa mbiri ya nthawi ya njira yomwe yatchulidwa. Ganizirani mapuloteni okha omwe ali ndi P <0.05 pa masabata 8. Mzere wodulidwa: palibe kusintha (kusanthula kwa kusiyana kwa mbali ziwiri; * P <0.05; *** P <0.001). Deta imafotokozedwa ngati apakati±SEM.
Mu kusanthula kwathu, BCAA catabolism yakhala imodzi mwa njira zofunika kwambiri zokwezera malamulo. Izi zikusonyeza mwamphamvu kuti kuchuluka kwa mpweya wolowa mu TCA kungasinthidwe mu PN yopanda OXPHOS. Izi zitha kuyimira mtundu waukulu wa neuronal metabolic rewiring, zomwe zitha kukhala ndi zotsatira zachindunji pa physiology ya neuronal ndi kupulumuka panthawi yosunga kusokonekera kwakukulu kwa OXPHOS. Mogwirizana ndi lingaliro ili, tapeza kuti enzyme yayikulu yotsutsana ndi atherosclerotic PCx imakwezedwa (Mfn2cKO/CTRL imasintha pafupifupi nthawi 1.5; Chithunzi 3A), chomwe chimalimbikitsa kusintha kwa pyruvate kukhala oxaloacetate (28), yomwe imakhulupirira kuti ili mu minofu ya ubongo. Kufotokozera kwa in kumalekeredwa ku astrocytes (29, 30). Mogwirizana ndi zotsatira za proteomics, confocal microscopy idawonetsa kuti kufotokozera kwa PCx kudakulitsidwa makamaka komanso mokulira mu ma PN osowa a OXPHOS, pomwe reactivity ya PCx idalekeredwa makamaka ku maselo apafupi a Bergmann glial a control (Chithunzi 3B). Kuti tiyese bwino momwe PCx imakulirakulira, tinachiza magawo a cerebellar acute ndi [1-13C]pyruvate tracer. Pamene pyruvate idasungunuka ndi pyruvate dehydrogenase (PDH), chizindikiro chake cha isotope chinatha, Koma chimaphatikizidwa mu TCA cycle intermediates pamene pyruvate imasinthidwa ndi mitsempha yamagazi (Chithunzi 3D). Pothandizira deta yathu ya proteomics, tinawona zizindikiro zambiri kuchokera ku tracer iyi mu aspartic acid ya Mfn2cKO slices, pomwe citric acid ndi malic acid nazonso zinali ndi chizolowezi chocheperako, ngakhale sizinali zofunikira (Chithunzi 3D).
Mu ma neuron a dopamine a mbewa za MitoPark omwe ali ndi vuto la mitochondrial lomwe limayambitsidwa ndi ma neuron a dopamine omwe amawononga makamaka jini la transcription factor A (Tfam) (Chithunzi S6B), kafotokozedwe ka PCx kanakwezedwanso kwambiri (31), zomwe zikusonyeza kuti acetone acid arteriosclerosis Kupezeka kwa matendawa kumayendetsedwa panthawi ya kusagwira ntchito kwa neuronal OXPHOS m'thupi. Ndikofunikira kudziwa kuti zapezeka kuti ma enzyme apadera (32-34) omwe angawonetsedwe mu ma neuron omwe angagwirizane ndi arteriosclerosis amawongoleredwa kwambiri mu ma PN omwe alibe OXPHOS, monga propionyl-CoA carboxylase (PCC-A), Malonyl-CoA amasintha propionyl-CoA kukhala succinyl-CoA ndi mitochondrial malic enzyme 3 (ME3), omwe ntchito yawo yayikulu ndikubwezeretsa pyruvate kuchokera ku malate (Chithunzi 3, A ndi C) (33, 35). Kuphatikiza apo, tapeza kuwonjezeka kwakukulu kwa enzyme ya Pdk3, yomwe imapanga phosphoryl ndikuletsa PDH (36), pomwe palibe kusintha komwe kunapezeka mu enzyme ya Pdp1 yomwe imayambitsa PDH kapena PDH enzyme complex yokha (Chithunzi 3A). Nthawi zonse, mu Mern2cKO PNs, phosphorylation ya α1 subunit α (PDHE1α) subunit ya pyruvate dehydrogenase E1 gawo la PDH complex mu Ser293 (yomwe imadziwika kuti imaletsa ntchito ya enzyme ya PDH) idakulitsidwa (Chithunzi S6C) (Chithunzi S6C). Pyruvate ilibe mwayi wolowera m'mitsempha.
Pomaliza, tinapeza kuti njira yapamwamba ya serine ndi glycine biosynthesis, kayendedwe kogwirizana ka mitochondrial folate (1C) ndi proline biosynthesis (Chithunzi 1G ndi Chithunzi S5C) zonse zawonjezeka kwambiri, malinga ndi malipoti, panthawi yoyambitsa. Minofu yozungulira imayatsidwa ndi vuto la mitochondrial (5-7). Kusanthula kwa Confocal komwe kumathandizira deta ya proteomics iyi kunawonetsa kuti mu PN pomwe OXPHOS ikusowa, magawo a cerebellar a mbewa za masabata 8 adayikidwa serine hydroxymethyltransferase 2 (SHMT2), enzyme yofunika kwambiri ya kayendedwe ka mitochondrial folate. Kuyankha kwakukulu kwa chitetezo chamthupi (Chithunzi S5D). Mu magawo 13 a CU-glucose-incubated acute cerebellar, kuyesa kwa metabolic tracing kunatsimikiziranso kukwera kwa serine ndi proline biosynthesis, kusonyeza kuti kuyenda kwa carbon isoforms kukhala serine ndi proline kunawonjezeka (Chithunzi S5E). Popeza kuti zochita zomwe GLS ndi GPT2 zimathandizira kupanga glutamate kuchokera ku glutamine ndi transamination pakati pa glutamate ndi α-ketoglutarate, kukwera kwawo kukuwonetsa kuti ma neuron omwe alibe OXPHOS ali ndi kufunikira kwakukulu kwa glutamate, Izi zitha kukhala cholinga chosunga kuchuluka kwa biosynthesis ya proline (Chithunzi S5C). Mosiyana ndi kusinthaku, kusanthula kwa proteomic kwa ma astrocyte a cerebellar ochokera ku mbewa za PN-specific Mfn2cKO kunawonetsa kuti njira izi (kuphatikiza ma antiperoxidases onse) sizinasinthe kwambiri pakuonekera, motero zikuwonetsa kuti kusinthaku kwa kagayidwe kachakudya kumasankha PN yowonongeka (Chithunzi S6, D mpaka G).
Mwachidule, kusanthula kumeneku kwawonetsa mitundu yosiyanasiyana ya kuyambitsa kwakanthawi kwa njira zinazake za kagayidwe kachakudya mu ma PN. Ngakhale kuti ntchito yosazolowereka ya mitochondrial ya mitsempha ya mitsempha ingayambitse atherosclerosis yoyambirira ndi kukonzanso kwa 1C (Chithunzi 3E ndi Chithunzi S5C), komanso kusintha komwe kungadziwike bwino pakuwonetsa ma complexes a I ndi IV, kusintha kwa kapangidwe ka serine de novo kokha kunayamba kuonekera kumapeto kwa nthawi. Kusagwira ntchito kwa OXPHOS (Chithunzi 3E ndi Chithunzi S5C). Zomwe zapezekazi zimafotokoza njira yotsatizana momwe mitochondrial yomwe imayambitsa kupsinjika (1C cycle) ndi cytoplasmic (serine biosynthesis) zimayankhira mogwirizana ndi kuwonjezeka kwa atherosclerosis mu TCA cycle kuti zisinthe kagayidwe ka mitsempha.
Ma PN a OXPHOS a masabata 8 amatha kusunga ntchito yolimbikitsa kwambiri komanso kulumikizidwanso kwambiri kuti athetse vuto la mitochondrial. Kupeza kumeneku kukuwonetsa mwayi wosangalatsa wakuti ngakhale pakadali pano, maselowa amathanso kulandira chithandizo chochiritsira kuti achedwetse kapena kupewa kuwonongeka kwa mitsempha. Mochedwa. Tinathetsa mwayiwu kudzera mu njira ziwiri zodziyimira pawokha. Mu njira yoyamba, tinapanga vector ya Cre-dependent adeno-associated virus (AAV) kuti MFN2 iwonetsedwe mosankha mu ma PN a OXPHOS omwe alibe mu vivo (Chithunzi S7A). AAV encoding MFN2 ndi fluorescent reporter gene mCherry (Mfn2-AAV) zinatsimikiziridwa mu primary neuron cultures in vitro, zomwe zinapangitsa kuti MFN2 iwonetsedwe modalira Cre ndikupulumutsa mawonekedwe a mitochondrial, motero kupewa neuromutation mu ma neuron a Mfn2cKO (Chithunzi S7, B, D ndi E). Kenako, tinachita zoyeserera za mu vivo kuti tipereke Mfn2-AAV ya masabata 8 ku cerebellar cortex ya Mfn2cKO ndi mbewa zowongolera, ndipo tinasanthula mbewa za masabata 12 (Chithunzi 4A). Mbewa za Mfn2cKO zomwe zinathandizidwa zinafa (Chithunzi 1, A ndi B) (16). Kutumiza kachilombo mu vivo kunapangitsa kuti PN iwonetsedwe bwino m'mabwalo ena a cerebellar (Chithunzi S7, G ndi H). Kulowetsa AAV yowongolera yomwe imatulutsa mCherry (Ctrl-AAV) yokha sikunakhudze kwambiri kuchuluka kwa kuwonongeka kwa mitsempha mwa nyama za Mfn2cKO. Mosiyana ndi zimenezi, kusanthula kwa Mfn2cKOs komwe kunaperekedwa ndi Mfn2-AAV kunawonetsa chitetezo chachikulu cha gawo la maselo a PN (Chithunzi 4, B ndi C). Makamaka, kuchuluka kwa ma neuron kumawoneka kuti sikusiyana ndi nyama zowongolera (Chithunzi 4, B ndi C, ndi Chithunzi S7, H ndi I). Kuwonetsedwa kwa MFN1 koma osati MFN2 kumathandizanso populumutsa imfa ya mitsempha (Chithunzi 4C ndi Chithunzi S7, C ndi F), zomwe zikusonyeza kuti kufotokozedwa kwa ectopic MFN1 kungathandize bwino kusowa kwa MFN2. Kusanthula kwina pa mlingo umodzi wa PN kunawonetsa kuti Mfn2-AAV inapulumutsa kwambiri kapangidwe ka mitochondria, inasintha kuchuluka kwa mtDNA, ndikuchepetsa kuchuluka kwa chizindikiro cha anti-angiogenesis PCx ​​(Chithunzi 4, C mpaka E). Kuyang'ana kowoneka bwino kwa mbewa za Mfn2cKO zomwe zapulumutsidwa zili mu mkhalidwe wopumula kunawonetsa kuti kaimidwe kawo ndi zizindikiro zawo zoyenda (kuyenda kwa S1 kupita ku S3) zinakula. Pomaliza, zoyeserera izi zikuwonetsa kuti kubwezeretsedwanso kwa MFN2 mu ma PN omwe alibe OXPHOS mokwanira ndikokwanira kusintha kugwiritsidwa ntchito kwa mtDNA ndikuyambitsa atherosclerosis, motero kupewa kuwonongeka kwa axon ndi kufa kwa mitsempha m'thupi.
(A) Ndondomeko yosonyeza ndondomeko yoyesera yojambulira AAV encoding MFN2 pamene njira yodziwikiratu ya kagayidwe kachakudya yayatsidwa. (B) Zithunzi zoyimira zamkati za magawo a cerebellar a masabata 12 obadwa omwe adasinthidwa pa masabata 8 mu mbewa za Mfn2cKO ndikulembedwa ndi anti-Calbindin antibody. Kumanja: Kukula kwa ulusi wa axon. Kukula kwa axon zoom ndi 450 ndi 75 μm. (C) Kumanzere: Kuyeza kuchuluka kwa maselo a Purkinje mu AAV transduction loop (AAV+) (kusanthula kwa njira imodzi kwa kusiyana; n = mbewa 3). Kumanja: kusanthula kwa mtDNA focus mu transduced PN pa sabata 12 (unpaired t-test; n = maselo 6 kuchokera ku mbewa zitatu). * P <0.05; ** P <0.01. (D) Ma micrographs oyimira ma transmission electron a ma PN a Mfn2cKO cerebellar sections transduced ndi ma virus vectors omwe awonetsedwa. Chigoba cha pinki chikuwonetsa malo omwe ali ndi ma dendrite, ndipo sikweya yachikasu yokhala ndi madontho ikuwonetsa zoom yomwe yaperekedwa kumanja; n ikuyimira nucleus. Scale bar, 1μm. (E) ikuwonetsa chitsanzo cha utoto wa PCx mu PN woperekedwa pa masabata 12. Scale bar, 20μm. OE, expression yochulukirapo; FC, kusintha kwa ma fold.
Pomaliza, tinafufuza kufunika kwa kupulumuka kwa maselo opangidwa ndi peroxidase m'ma PN omwe adakumana ndi vuto la OXPHOS. Tinapanga mCherry encoding AAV-shRNA (fupi hairpin RNA) yomwe imayang'ana makamaka PCx mRNA ya mbewa (AAV-shPCx), ndipo tinalowetsa kachilomboka kapena scrambled control (AAV-scr) mu cerebellum ya mbewa za Mfn2cKO. Jakisoniyo adachitika mlungu wachinayi wa kubadwa (Chithunzi 5A) kuti PCx igwetsedwe bwino panthawi yomwe PCx imawonjezeka (Chithunzi 3C) ndipo gawo la maselo a PN linali lisanathe (Chithunzi 1A). Ndikofunikira kudziwa kuti kugwetsa PCx (Chithunzi S8A) kumabweretsa kufa kwa PN mwachangu, komwe kumangokhala mphete yodwala (Chithunzi 5, B ndi C). Kuti timvetse momwe kagayidwe kachakudya kamakhudzira mphamvu ya PCx, tinaphunzira momwe ma PN amagwirira ntchito redox pambuyo pa PCx ​​knockdown ndi AAV-mediated optical biosensor Grx1-roGFP2 zinafotokozedwera nthawi imodzi (Chithunzi S8, B mpaka D) kuti tiwone kusintha kwa peptide redox potential (38). Kenako, tinachita ma microscopy awiri a fluorescence lifetime imaging microscopy (FLIM) mu magawo a ubongo a Mfn2cKO wa masabata 7 kapena kuwongolera ma littermates kuti tizindikire kusintha komwe kungachitike mu cytoplasmic redox status titatsimikizira momwe zinthu zilili mu FLIM (Chithunzi S8, E mpaka G). Kusanthulaku kunawonetsa kuwonjezeka kwakukulu kwa mkhalidwe wa okosijeni wa Mfn2cKO PN imodzi yopanda PCx expression, yomwe ndi yosiyana ndi ma neurons olamulira kapena ma Mfn2cKO PN omwe amatulutsa shRNA yosweka yokha (Chithunzi 5, D ndi E). Pamene kuchuluka kwa ma PCx kunachepetsedwa, kuchuluka kwa ma Mfn2cKO PN omwe amasonyeza kuti ali ndi okosijeni wambiri kunawonjezeka ndi kupitirira katatu (Chithunzi 5E), zomwe zikusonyeza kuti kuwonjezeka kwa PCx kunasunga mphamvu ya redox ya ma neuron omwe adawonongeka.
(A) Ndondomeko yosonyeza nthawi yoyesera yojambulira AAV encoding shPCx pamene njira yodziwikiratu ya kagayidwe kachakudya yayatsidwa. (B) Zithunzi zoyimira zamkati za magawo a cerebellar a masabata 8 mu mbewa za Mfn2cKO zosinthidwa ndikulembedwa ndi anti-calcineurin antibody pa masabata 4. Scale bar, 450μm. (C) Kuyeza kuchuluka kwa maselo a Purkinje mu ma loops osinthidwa a AAV (kusanthula kwa njira imodzi kwa kusiyana; n = mbewa 3 mpaka 4). Deta imafotokozedwa ngati mean±SEM; ***P<0.001. (D) Chithunzi choyimira FLIM chikuwonetsa nthawi yapakati ya PN ya masabata 7 yowonetsa glutathione redox sensor Grx1-roGFP2 pansi pa mikhalidwe yoyesera yomwe yatchulidwa. Chiŵerengero cha LUT (look-up table): nthawi yopulumuka (mu picoseconds). Scale bar, 25μm. (E) Histogram ikuwonetsa kufalikira kwa ma values ​​a moyo wa Grx1-roGFP2 kuchokera ku (D) (n=158 mpaka 368 maselo m'makoswe awiri pansi pa vuto lililonse). Tchati cha pie pamwamba pa histogram iliyonse: chikuwonetsa chiwerengero cha maselo omwe ali ndi ma values ​​a moyo wautali kwambiri (ofiira, okodzetsedwa) kapena afupiafupi (buluu, ochepetsedwa), omwe amaposa 1 SD ya avareji ya moyo wa moyo mu CTRL-AAV-scr. (F) Chitsanzo chomwe chikuperekedwa chikuwonetsa mphamvu yoteteza ya kukwera kwa neuronal PCx.
Zonse pamodzi, deta yomwe timapereka pano ikuwonetsa kuti kubwezeretsedwanso kwa MFN2 kungapulumutse kwathunthu PN yapamwamba yokhala ndi kusowa kwakukulu kwa OXPHOS, kuchepa kwakukulu kwa mtDNA, komanso mawonekedwe osazolowereka kwambiri ofanana ndi ista, motero kumapereka kupita patsogolo kosalekeza ngakhale m'matenda opita patsogolo. Kuwonongeka kwa mitsempha kumapereka umboni wosinthika wa gawo lisanafike imfa ya maselo. Kusinthasintha kumeneku kwa kagayidwe kachakudya kumagogomezedwanso ndi kuthekera kwa ma neuron kuyambitsa atherosclerosis (kubwezeretsanso kwa TCA cycle), komwe kumaletsa kuwonetsedwa kwa PCx mu ma PN omwe alibe OXPHOS ndikuwonjezera kufa kwa maselo, motero kumakhala ndi gawo loteteza (Chithunzi 5F).
Mu kafukufukuyu, tapereka umboni wakuti momwe ma PN amayankhira ku vuto la OXPHOS limagwirira ntchito pang'onopang'ono kupita ku TCA cycle atherosclerosis kudzera mu njira yosiyana yogwirira ntchito yomwe imayendetsedwa ndi mapulogalamu a metabolic. Tatsimikizira kusanthula kwa proteomic ndi njira zambiri zowonjezera ndipo tawonetsa kuti pamene ma neuron akuvutika ndi vuto lalikulu la mitochondrial, ma neuron amakhala ndi mawonekedwe osadziwika kale a metabolic elasticity. Chodabwitsa n'chakuti, njira yonse yolumikizira mawaya sikutanthauza kuti metabolic state yomwe imabwera ndi neurodegeneration pang'onopang'ono komanso mosasinthika, koma deta yathu ikuwonetsa kuti ikhoza kukhala neuron yosamalira ngakhale siteji isanafike imfa ya maselo. Njira yogwirira ntchito yolipirira ntchito. Kupeza kumeneku kukuwonetsa kuti ma neuron ali ndi kuchuluka kwakukulu kwa metabolic plasticity m'thupi. Izi zikutsimikizira kuti kubwezeretsedwanso kwa MFN2 pambuyo pake kumatha kusintha mawonekedwe a zizindikiro zazikulu za metabolic ndikuletsa kuwonongeka kwa PN. M'malo mwake, kumaletsa atherosclerosis ndikufulumizitsa mitsempha.
Chimodzi mwa zinthu zosangalatsa kwambiri zomwe tapeza mu kafukufuku wathu ndichakuti ma PN omwe alibe OXPHOS amatha kusintha kagayidwe ka TCA cycle mwa kuwonjezera ma enzyme omwe amalimbikitsa arteriosclerosis. Kukonzanso kagayidwe kachakudya ndi chinthu chofala kwambiri m'maselo a khansa, ena mwa iwo amadalira glutamine kuti awonjezere ma TCA cycle intermediates kuti apange ma reducing equivalents, omwe amayendetsa unyolo wopumira ndikusunga kupanga kwa lipid ndi nucleotide biosynthesis precursors (39, 40). Kafukufuku waposachedwa wasonyeza kuti m'maselo am'mbali omwe ali ndi vuto la OXPHOS, kulumikizidwanso kwa glutamine/glutamate metabolism ndi chinthu chofunikira kwambiri (5, 41), komwe njira yolowera glutamine mu TCA cycle imadalira Chifukwa cha kuopsa kwa kuvulala kwa OXPHOS (41). Komabe, palibe umboni womveka bwino wokhudza kufanana kulikonse kwa neuronal metabolic plasticity m'thupi komanso kufunika kwake pa matenda. Mu kafukufuku waposachedwa wa in vitro, ma primary cortical neurons adawonetsedwa kuti amalimbikitsa ma glutamate pools kuti azitha kufalikira kwa mitsempha, motero amalimbikitsa kagayidwe ka okosijeni ndi atherosclerosis pansi pa zovuta za metabolic (42). Ndikofunikira kudziwa kuti pansi pa kuletsa kwa mankhwala kwa enzyme ya TCA cycle succinate dehydrogenase, pyruvate carboxylation imakhulupirira kuti imasunga kapangidwe ka oxaloacetate mu ma neuron a cerebellar granule (34). Komabe, kufunika kwa thupi kwa njira izi ku minofu ya ubongo (komwe atherosclerosis imakhulupirira kuti imangokhala ndi astrocytes) kudakali ndi kufunika kofunikira kwa thupi (43). Pankhaniyi, deta yathu ikuwonetsa kuti ma PN omwe awonongeka ndi OXPHOS m'thupi amatha kusinthidwa kukhala BCAA degradation ndi pyruvate carboxylation, omwe ndi magwero awiri akuluakulu othandizira ma TCA pool intermediates. Ngakhale kuti BCAA catabolism ikuthandizira pa kagayidwe ka mphamvu ya neuronal yaperekedwa, kuwonjezera pa ntchito ya glutamate ndi GABA pa neurotransmission (44), palibe umboni wa njirazi mu vivo. Chifukwa chake, n'zosavuta kuganiza kuti ma PN osagwira ntchito amatha kulipira okha kugwiritsa ntchito ma TCA intermediates omwe amayendetsedwa ndi njira yolumikizirana powonjezera atherosclerosis. Makamaka, kukwezedwa kwa PCx kungafunike kuti pakhale kufunikira kwakukulu kwa aspartic acid, komwe kumaganiziridwa m'maselo omwe akuchulukirachulukira omwe ali ndi vuto la mitochondrial (45). Komabe, kusanthula kwathu kwa metabolomics sikunawonetse kusintha kulikonse kwakukulu mu mulingo wokhazikika wa aspartic acid mu Mfn2cKO PNs (Chithunzi S6A), chomwe mwina chikuwonetsa kagwiritsidwe ntchito kosiyanasiyana ka aspartic acid pakati pa maselo omwe akuchulukirachulukira ndi ma neuron a post-mitotic. Ngakhale kuti njira yeniyeni ya kukwezedwa kwa PCx mu ma neuron osagwira ntchito bwino mu vivo ikadali yodziwikiratu, tawonetsa kuti kuyankha koyambirira kumeneku kumachita gawo lofunikira pakusunga mkhalidwe wa redox wa ma neuron, zomwe zidawonetsedwa mu zoyeserera za FLIM pazidutswa za cerebellar. Makamaka, kuletsa ma PN kuti asakweze PCx kungayambitse mkhalidwe wowonjezereka ndikufulumizitsa kufa kwa maselo. Kuyambitsa kuwonongeka kwa BCAA ndi carboxylation ya pyruvate si njira zodziwira minofu ya m'mphepete mwa mitochondrial dysfunction (7). Chifukwa chake, zikuwoneka kuti ndi chinthu chofunikira kwambiri pa ma neuron omwe alibe OXPHOS, ngakhale si chinthu chokhacho, chomwe ndi chofunikira pakukula kwa mitsempha.
Matenda a Cerebellar ndi matenda osiyanasiyana a neurodegenerative omwe nthawi zambiri amaonekera ngati ataxia ndipo nthawi zambiri amawononga ma PN (46). Gulu la ma neuron awa limakhala pachiwopsezo chachikulu cha kusagwira bwino ntchito kwa mitochondrial chifukwa kusagwira bwino ntchito kwawo mwa mbewa ndikokwanira kubereka zizindikiro zambiri za kayendedwe ka thupi zomwe zimasonyeza ataxia ya spinocerebellar ya anthu (16, 47, 48). Malinga ndi malipoti, chitsanzo cha mbewa chopangidwa ndi transgenic chokhala ndi jini yosinthika chimagwirizanitsidwa ndi ataxia ya spinocerebellar ya anthu ndipo chimakhala ndi vuto la mitochondrial (49, 50), zomwe zikugogomezera kufunika kophunzira zotsatira za kusowa kwa OXPHOS mu PNPH. Chifukwa chake, ndikofunikira kwambiri kudzipatula ndikuwerenga bwino gulu lapadera la ma neuron. Komabe, popeza ma PN ndi osavuta kwambiri kukakamizidwa ndipo amawerengera gawo lochepa la gulu lonse la ma cell a cerebellar, pamaphunziro ambiri ozikidwa pa omics, kulekanitsa kwawo ngati maselo athunthu kudakali vuto. Ngakhale kuti n'zosatheka kupeza kuipitsidwa kwathunthu kwa mitundu ina ya maselo (makamaka minofu ya akuluakulu), tinaphatikiza gawo lothandiza lolekanitsa ndi FACS kuti tipeze ma neuron okwanira kuti tipeze kusanthula kwa proteomics, ndipo tili ndi mapuloteni ambiri (pafupifupi mapuloteni 3000) poyerekeza ndi deta yomwe ilipo ya cerebellum yonse (51). Mwa kusunga moyo wa maselo onse, njira yomwe timapereka pano imatithandiza osati kungoyang'ana kusintha kwa njira zamagetsi mu mitochondria, komanso kuyang'ana kusintha kwa maselo ake a cytoplasmic, zomwe zimathandizira kugwiritsa ntchito ma tag a mitochondrial membrane kuti tiwonjezere mtundu wa maselo Njira yatsopano ya kuchuluka kwa mitochondria m'maselo ovuta (52, 53). Njira yomwe tikufotokoza sikuti imangogwirizana ndi kuphunzira maselo a Purkinje, komanso ingagwiritsidwe ntchito mosavuta ku mtundu uliwonse wa selo kuti ithetse kusintha kwa kagayidwe kachakudya muubongo wodwala, kuphatikizapo mitundu ina ya kusagwira bwino ntchito kwa mitochondrial.
Pomaliza, tapeza njira yochiritsira panthawiyi yokonzanso kagayidwe kachakudya komwe kungasinthe kwathunthu zizindikiro zazikulu za kupsinjika kwa maselo ndikuletsa kuwonongeka kwa mitsempha. Chifukwa chake, kumvetsetsa momwe mawaya olumikizirana omwe afotokozedwa pano angakhalire ndi mphamvu yogwiritsira ntchito mawaya ena kungapereke chidziwitso chofunikira pamankhwala omwe angathandize kuti mitsempha ikhale ndi moyo wabwino panthawi ya kusagwira ntchito kwa mitochondrial. Kafukufuku wamtsogolo wofuna kusanthula kusintha kwa kagayidwe ka mphamvu m'maselo ena a ubongo akufunika kuti awulule mokwanira momwe mfundoyi ingagwiritsidwe ntchito pa matenda ena amitsempha.
Mbewa za MitoPark zafotokozedwa kale (31). Mbewa za C57BL/6N zokhala ndi majini a Mfn2 okhala ndi loxP m'mbali mwake zafotokozedwa kale (18) ndipo zaphatikizidwa ndi mbewa za L7-Cre (23). Kenako mbewa ziwiri zomwe zinatulukazo zinaphatikizidwa ndi mbewa za Mfn2loxP/Mfn2loxP zomwe zimakhala ndi homozygous kuti zipange majini a Purkinje enieni a Mfn2 (Mfn2loxP/Mfn2loxP; L7-cre). Mu gawo la kukwatirana, Gt (ROSA26) SorStop-mito-YFP allele (stop-mtYFP) inayambitsidwa kudzera mu njira zina zosakanikirana (20). Njira zonse za nyama zinachitidwa motsatira malangizo aku Europe, dziko komanso mabungwe ndipo zavomerezedwa ndi LandesamtfürNatur wa Umwelt ndi Verbraucherschutz, North Rhine-Westphalia, Germany. Ntchito za nyama zimatsatiranso malangizo a European Federation of Laboratory Animal Sciences Associations.
Pambuyo pochepetsa ululu wa m'mimba mwa mayi wapakati, mbewa imachotsedwa (E13). Kotekisiyo inadulidwa mu Hanks' Balanced Salt Solution (HBSS) yowonjezeredwa ndi 10 mM Hepes ndipo inaperekedwa kwa Dulbecco's Modified Eagle's Medium yokhala ndi papain (20 U/ml) ndi cysteine ​​​​(1μg/ml). Ikani minofu mu DMEM ndikuilekanitsa ndi enzyme digestion. Ml) pa 37°C kwa mphindi 20, kenako n’kuphwanyidwa ndi DMEM yowonjezeredwa ndi 10% fetal bovine serum. Maselo anabzalidwa pa magalasi ophimba ndi polylysine pamlingo wa 2×106 pa mbale yokulirapo ya 6 cm kapena pamlingo wa 0.5×105 cells/cm2 kuti awonetse zithunzi. Patatha maola 4, chotchingacho chinasinthidwa ndi chotchinga chopanda seramu cha Neurobasal chokhala ndi 1% B27 supplement ndi 0.5 mM GlutaMax. Kenako ma neuron anasungidwa pa 37°C ndi 5% CO2 panthawi yonse yoyesera, ndipo anadyetsedwa kamodzi pa sabata. Pofuna kuyambitsa kuyanjananso mu vitro, 3μl (mbale yolerera ya zitsime 24) kapena 0.5μl (mbale ya zitsime 24) ya vector yotsatira ya kachilombo ka AAV9 idagwiritsidwa ntchito pochiza ma neuron patsiku lachiwiri mu vitro: AAV9.CMV.PI.eGFP. WPRE.bGH (Addgene, nambala ya catalog 105530-AAV9) ndi AAV9.CMV.HI.eGFP-Cre.WPRE.SV40 (Addgene, nambala ya catalog 105545-AAV9).
DNA yowonjezera ya Mfn1 ndi Mfn2 (yomwe idapezedwa kuchokera ku Addgene plasmid #23212 ndi #23213, motsatana) imalembedwa ndi V5 sequence (GKPIPNPLLGLDST) pa C-terminus, ndipo imaphatikizidwa ndi mCherry mu chimango kudzera mu T2A sequence. Grx1-roGFP2 ndi mphatso yochokera kwa Heidelberg TP Dick DFKZ (Deutsches Krebsforschungszentrum). Posintha kaseti ya tdTomato pogwiritsa ntchito njira zachikhalidwe zokopera, kasetiyo idalowetsedwa mu pAAV-CAG-FLEX-tdTomato backbone (Addgene reference number 28306) kuti ipange pAAV-CAG-FLEX-mCherry-T2A-MFN2-V5, pAAV-CAG-FLEX-mCherry-T2A-MFN1-V5 ndi pAAV-CAG-FLEX-Grx-roGFP2 vectors. Njira yofananayo idagwiritsidwa ntchito popanga vekitala yowongolera pAAV-CAG-FLEX-mCherry. Kuti apange kapangidwe ka AAV-shPCx, vekitala ya plasmid AAV (VectorBuilder, pAAV [shRNA] -CMV-mCherry-U6-mPcx- [shRNA#1]) imafunika, yomwe ili ndi DNA sequence yomwe imalemba mbewa ya shRNA yolunjika PCx (5′CTTTCGCTCTAAGGTGCTAAACTCGAGTTTAGCACCCTTAGAGCGAAAG 3′) Motsogozedwa ndi wolimbikitsa wa U6, mCherry imagwiritsidwa ntchito motsogozedwa ndi wolimbikitsa wa CMV. Kupanga mavekitala othandizira a AAV kunachitika motsatira malangizo a wopanga (Cell Biolabs). Mwachidule, gwiritsani ntchito plasmid yonyamula mCherry-T2A-MFN2-V5 (pAAV-CAG-FLEX-mCherry-T2A-MFN2-V5), mCherry-T2A-MFN1-V5 (pAAV-CAG-FLEX-mCherry) Kusamutsa maselo 293AAV-T2A-MFN1-V5), mCherry (pAAV-CAG-FLEX-mCherry) kapena jini yolemba Grx-roGFP2 (pAAV-CAG-FLEX-Grx-roGFP2), komanso kulemba mapuloteni a capsid a AAV1 ndi mapuloteni othandizira. Kuyika plasmid plasmid, pogwiritsa ntchito njira ya calcium phosphate. Supernatant ya kachilombo koyipa idapezeka mwa kuzizira-kusungunuka mu bafa youma ya ayezi/ethanol ndi maselo osungunuka mu phosphate buffered saline (PBS). Vekitala ya AAV inayeretsedwa pogwiritsa ntchito discontinuous iodixanol gradient ultracentrifugation (maola 24 pa 32,000 rpm ndi 4°C) ndipo inagwiritsidwa ntchito pogwiritsa ntchito fyuluta ya Amicon ultra-15 centrifugal. Genome titer ya 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 inali monga momwe tafotokozera kale (54), yoyesedwa ndi real-time quantitative PCR (qPCR) -MFN1-V5 (1.9×1013 GC/ml) ndi AAV1-CAG-FLEX-Grx-roGFP2 (8.9×1012 GC/ml).
Ma neuron oyambira adachotsedwa mu 1x PBS yozizira kwambiri, kenako adayikidwa mu pellet, kenako adasinthidwa kukhala homogenized mu 0.5% Triton X-100 / 0.5% sodium deoxycholate/PBS lysis buffer yokhala ndi phosphatase ndi protease inhibitor (Roche). Kuyeza kwa mapuloteni kunachitika pogwiritsa ntchito bicinchoninic acid assay (Thermo Fisher Scientific). Kenako mapuloteniwo adalekanitsidwa ndi SDS-polyacrylamide gel electrophoresis, kenako adachotsedwa pa polyvinylidene fluoride membrane (GE Healthcare). Tsekani malo osakhala enieni ndikuyikamo ndi antibody yoyamba (onani Table S1 kuti mudziwe zambiri) mu mkaka wa 5% mu TBST (Tris-buffered saline with Tween), masitepe otsukira ndi antibody yachiwiri mu TBST Incubate. Ikanimo ndi antibody yoyamba usiku wonse pa +4°C. Mukatsuka, ikani antibody yachiwiri kwa maola awiri kutentha kwa chipinda. Pambuyo pake, poyikamo blot yomweyo ndi antibody ya anti-β-actin, katundu womwewo unatsimikiziridwa. Kuzindikira mwa kusintha kukhala chemiluminescence ndikuwonjezera chemiluminescence (GE Healthcare).
Ma neuron omwe adabzalidwa kale pa glaze coverlip adakhazikika ndi 4% paraformaldehyde (PFA)/PBS panthawi yomwe yatchulidwa kutentha kwa chipinda kwa mphindi 10. Cover lip poyamba imalowetsedwa ndi 0.1% Triton X-100/PBS kwa mphindi 5 kutentha kwa chipinda, kenako mu blocking buffer [3% bovine serum albumin (BSA)/PBS]. Pa tsiku lachiwiri, cover lip adatsukidwa ndi blocking buffer ndikuyikidwa ndi fluorophore-conjugated secondary antibody yoyenera kwa maola awiri kutentha kwa chipinda; pamapeto pake, zitsanzozo zidatsukidwa bwino mu PBS ndi 4′,6-diamidino-2-Phenylindole (DAPI) yophimbidwa kenako yokhazikika pa microscope slide ndi Aqua-Poly/Mount.
Mbewa (zachimuna ndi zazikazi) zinapatsidwa mankhwala oletsa ululu pobayira ketamine (130 mg/kg) ndi xylazine (10 mg/kg) mkati mwa peritoneal ndipo zinaperekedwa pansi pa khungu ndi carprofen analgesic (5 mg/kg), ndipo zinayikidwa mu chipangizo choyezera ululu (Kopf) chokhala ndi chofunda chofunda. Tsegulani chigazacho ndikugwiritsa ntchito chobowolera mano kuti muchepetse gawo la cerebellar cortex lofanana ndi fupa la mis (kuchokera ku lambda: mchira 1.8, mbali 1, yofanana ndi lobules IV ndi V). Gwiritsani ntchito singano yokhota ya syringe kuti mupange kabowo kakang'ono m'chigaza kuti mupewe kusokoneza mitsempha yamagazi yomwe ili pansipa. Kenako capillary yopyapyala yokokedwa imalowetsedwa pang'onopang'ono mu kabowo kakang'ono (kuchokera -1.3 mpaka -1 kumbali ya ventral ya dura mater), ndipo 200 mpaka 300 nl AAV imalowetsedwa mu micro-injector (Narishige) ndi ma syringe amanja (Narishige) kangapo pa mphamvu yochepa kwa nthawi ya mphindi 10 mpaka 20. Pambuyo poika mankhwala, ikani capillary kwa mphindi zina 10 kuti kachilomboka kafalikire kwathunthu. Pambuyo pochotsa mitsempha yamagazi, khungu limakulungidwa mosamala kuti lichepetse kutupa kwa bala ndikulola nyamayo kuchira. Nyamazo zinapatsidwa mankhwala ochepetsa ululu (caspofen) kwa masiku angapo pambuyo pa opaleshoni, ndipo panthawiyi thanzi lawo linayang'aniridwa mosamala kenako n’kuphedwa panthawi yomwe yatchulidwa. Njira zonse zinachitidwa motsatira malangizo a ku Ulaya, dziko ndi mabungwe ndipo zinavomerezedwa ndi LandesamtfürNatur a Umwelt ndi Verbraucherschutz, North Rhine-Westphalia, Germany.
Nyamazo zinapatsidwa mankhwala oletsa ululu ndi ketamine (100 mg/kg) ndi xylazine (10 mg/kg), ndipo mtima unathiridwa ndi 0.1 M PBS poyamba, kenako 4% PFA mu PBS. Minofuyo inadulidwa ndikukhazikika mu 4% PFA/PBS usiku wonse pa 4°C. Mpeni wogwedezeka (Leica Microsystems GmbH, Vienna, Austria) unagwiritsidwa ntchito kukonzekera magawo a sagittal (50 μm makulidwe) kuchokera ku ubongo wokhazikika mu PBS. Pokhapokha ngati tanena mwanjira ina, kuyika utoto wa magawo oyandama momasuka kunachitika monga tafotokozera pamwambapa (13) kutentha kwa chipinda ndikusakaniza. Mwachidule, choyamba, zidutswa zomwe zapezeka zinalowetsedwa ndi 0.5% Triton X-100/PBS kwa mphindi 15 kutentha kwa chipinda; kwa ena a epitopes (Pcx ndi Shmt2), ndi mu tris-EDTA buffer pa 80°C (PH 9) tenthetsani zidutswazo kwa mphindi 25 m'malo mwa sitepe iyi. Kenako, magawowo anaikidwa mu chivundikiro cha antibody yoyamba (onani Table S1) mu chivundikiro chotseka (3% BSA/PBS) pa 4°C usiku wonse ndi kusakaniza. Tsiku lotsatira, magawowo anatsukidwa ndi chivundikiro chotseka ndipo anaikidwa mu chivundikiro cha antibody yachiwiri cholumikizidwa ndi fluorophore kwa maola awiri kutentha kwa chipinda; pamapeto pake, magawowo anatsukidwa bwino mu PBS, anapakidwa ndi DAPI, kenako anakonzedwa ndi AquaPolymount On the microscope slide.
Makina oonera zithunzi a laser (TCS SP8-X kapena TCS Digital Light Sheet, Leica Microsystems) okhala ndi laser yoyera ndi laser ya 405 diode ultraviolet anagwiritsidwa ntchito kujambula chitsanzocho. Mwa kusangalatsa fluorophore ndikusonkhanitsa chizindikirocho ndi Hybrid Detector (HyDs), pulogalamu ya LAS-X idagwiritsidwa ntchito kusonkhanitsa zithunzi zodzaza zomwe zikugwirizana ndi Nyquist sampling mu sequentially mode: pa mapanelo osachuluka, ndi zizindikiro zamphamvu kwambiri (mwachitsanzo, mu maselo a somatic ndi dendrites) mtYFP) Gwiritsani ntchito HyD kuti mudziwe kuchuluka kwa ma PN mu BrightR mode). Gating kuyambira 0.3 mpaka 6 ns imagwiritsidwa ntchito kuti muchepetse maziko.
Kujambula maselo osankhidwa nthawi yeniyeni. Pambuyo posankha Neurobasal-A medium yokhala ndi 1% B27 supplement ndi 0.5 mM GlutaMax, maselowo adabzalidwa nthawi yomweyo pa magalasi okhala ndi poly-l-lysine (μ-Slide8 Well, Ibidi, catalog number 80826), Kenako sungani pa 37°C ndi 5% CO2 kwa ola limodzi kuti maselowo akhazikike. Kujambula kwa nthawi yeniyeni kunachitika pa maikulosikopu ya Leica SP8 laser scanning confocal yokhala ndi laser yoyera, HyD, 63×[1.4 numeral aperture (NA)] oil objective lens ndi gawo lotenthetsera.
Mbewayo inapatsidwa mankhwala oletsa ululu mwachangu ndi carbon dioxide ndipo inadulidwa mutu, ubongo unachotsedwa mwachangu kuchokera ku chigaza, ndipo unadulidwa kukhala makulidwe a 200μm (pa kuyesa kwa 13C) kapena makulidwe a 275μm (pa kuyesa kawiri kwa photon) gawo la sagittal lodzazidwa ndi zinthu zotsatirazi. Ayisikilimu (HM-650 V, Thermo Fisher Scientific, Walldorf, Germany) ili ndi zinthu zotsatirazi: 125 mM yozizira kwambiri, yodzaza ndi carbon (95% O2 ndi 5% CO2) Ca2 yotsika + madzi opangidwa ndi cerebrospinal fluid (ACSF) NaCl, 2.5 mM KCl, 1.25 mM sodium phosphate buffer, 25 mM NaHCO3, 25 mM glucose, 0.5 mM CaCl2 ndi 3.5 mM MgCl2 (osmotic pressure ya 310 mpaka 330 mmol). Tumizani zidutswa za ubongo zomwe mwapeza ku chipinda chosungiramo zinthu zomwe zili ndi Ca2 + ACSF yapamwamba (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 ndi 2.0 mM MgCl2) Medium) pH 7.4 ndi 310 mpaka 320 mmol).
Pa nthawi yojambula zithunzi, zidutswazo zinasamutsidwira ku chipinda chodzipangira zithunzi, ndipo kuyeseraku kunachitika pansi pa ACSF perfusion yosalekeza pa kutentha kosalekeza kwa 32° mpaka 33°C. Microscope yojambulira ya laser ya multiphoton (TCS SP8 MP-OPO, Leica Microsystems) yokhala ndi lenzi ya Leica 25x objective (NA 0.95, madzi), Ti: Sapphire laser (Chameleon Vision II, Coherent) idagwiritsidwa ntchito pojambula zithunzi za zidutswa. FLIM module (PicoHarp300, PicoQuant).
FLIM ya Grx1-roGFP2. Kusintha kwa momwe ma PN amagwirira ntchito m'thupi kunayezedwa ndi ma photon awiri a FLIM m'magawo a ubongo wa sagittal, pomwe Grx1-roGFP2 biosensor inkayang'ana ma PN. Mu gawo la PN, gawo lopezera limasankhidwa pafupifupi 50 mpaka 80 μm pansi pa gawolo kuti zitsimikizire kuti pali PN yogwira ntchito (ndiko kuti, kusowa kwa kapangidwe kokhala ndi mikanda kapena kusintha kwa ma neuronal morphological m'mbali mwa ma dendrites) ndi sensa ya roGFP2 yokhala ndi double positive ndi AAV encoding shRNA PCx kapena njira yake yowongolera (mCherry iliyonse yolumikizana). Sonkhanitsani zithunzi za single-stack ndi 2x digital zoom [excitation wavelength: 890 nm; 512 nm 512 pixels]. Kuzindikira: HyD yamkati, gulu losefera la fluorescein isothiocyanate (FITC)] ndi avareji ya chithunzi mkati mwa mphindi 2 mpaka 3 zimagwiritsidwa ntchito kuti zitsimikizire kuti ma photon okwanira asonkhanitsidwa (ma photon 1000 onse) kuti agwirizane ndi ma curve. Kuzindikira kwa probe ya Grx1-roGFP2 ndi kutsimikizira kwa mikhalidwe ya FLIM kunachitika poyang'anira kuchuluka kwa moyo wa roGFP2 powonjezera 10 mM H2O2 yakunja ku ACSF yotulutsa mpweya (kuti iwonjezere okosijeni, zomwe zimapangitsa kuti moyo ukhale wochuluka), kenako ndikuwonjezera 2 mM dithiothreitol (kumachepetsa kuchuluka kwa kuchepa, zomwe zimapangitsa kuti moyo ukhale wochepa) (Chithunzi S8, D mpaka G). Gwiritsani ntchito pulogalamu ya FLIMfit 5.1.1 kuti mufufuze zotsatira zomwe zapezedwa, kuyika mzere umodzi wa exponential decay wa chithunzi chonse ku IRF yoyezedwa (ntchito yoyankhira chida), ndipo χ2 ndi pafupifupi 1. Kuti muwerengere nthawi ya moyo wa PN imodzi, chigoba chozungulira thupi la mitsempha chinajambulidwa pamanja, ndipo nthawi ya moyo wapakati mu chigoba chilichonse idagwiritsidwa ntchito poyesa.
Kusanthula kwa mphamvu ya mitochondrial. Pambuyo poti gawo la acute laikidwa mu incubation ndi 100 nM TMRM yowonjezeredwa mwachindunji ku ACSF yophikidwa kwa mphindi 30, kusintha kwa mphamvu ya mitochondrial ya ma PN kunayesedwa ndi maikulosikopu awiri a photon. Kujambula kwa TMRM kunachitika poyambitsa probe pa 920 nm ndikugwiritsa ntchito HyD yamkati (tetramethylrhodamine isothiocyanate: 585/40 nm) kuti itenge zizindikiro; pogwiritsa ntchito kutalika komweko kwa excitation koma pogwiritsa ntchito HyD yamkati yosiyana (FITC :525/50) kuti iwonetse mtYFP. Gwiritsani ntchito pulogalamu ya ImageJ's Image Calculator kuti muyese mphamvu ya mitochondrial pamlingo wa selo limodzi. Mwachidule, equation ya plug-in: signal = min (mtYFP, TMRM) imagwiritsidwa ntchito kuzindikira dera la mitochondrial lomwe likuwonetsa chizindikiro cha TMRM mu Purkinje Somali mu chithunzi cha confocal cha single-stack cha njira yofananira. Kenako dera la pixel mu chigoba chomwe chikubweracho limayesedwa, kenako limasinthidwa kukhala labwinobwino pachithunzi chofanana cha threshold single-stack cha mtYFP channel kuti mupeze gawo la mitochondrial lomwe likuwonetsa kuthekera kwa mitochondrial.
Chithunzicho chinachotsedwa ndi pulogalamu ya Huygens Pro (Scientific Volume Imaging). Pazithunzi za matailosi zojambulidwa, mawonekedwe a matailosi amodzi amapangidwa pogwiritsa ntchito njira yosokera yokha yoperekedwa ndi pulogalamu ya LAS-X. Pambuyo pokonza chithunzi, gwiritsani ntchito ImageJ ndi Adobe Photoshop kuti mupitirize kukonza chithunzicho ndikusintha kuwala ndi kusiyana kwake mofanana. Gwiritsani ntchito Adobe Illustrator pokonzekera zithunzi.
Kusanthula kwa mtDNA focus. Chiwerengero cha zotupa za mtDNA chinayesedwa pa magawo a cerebellar olembedwa ndi ma antibodies motsutsana ndi DNA pogwiritsa ntchito microscope ya confocal. Malo aliwonse omwe adapangidwira adapangidwira thupi la selo ndi nucleus ya selo lililonse, ndipo malo omwewo adawerengedwa pogwiritsa ntchito Multi Measure plug-in (ImageJ software). Chotsani dera la nyukiliya kuchokera kudera la thupi la selo kuti mupeze dera la cytoplasmic. Pomaliza, pulogalamu ya Analyse Particles plug-in (ImageJ software) idagwiritsidwa ntchito poyesa zokha mfundo za cytoplasmic DNA zomwe zikuwonetsa mtDNA pachithunzi cholowera, ndipo zotsatira zomwe zapezeka zidasinthidwa kukhala avareji ya PN ya mbewa za CTRL. Zotsatira zake zimafotokozedwa ngati chiwerengero chapakati cha ma nucleoside pa selo iliyonse.
Kusanthula momwe mapuloteni amaonekera. Gwiritsani ntchito pulogalamu ya ImageJ's Image Calculator kuti muyese momwe mapuloteni amaonekera mu PN pa mulingo wa selo limodzi. Mwachidule, mu chithunzi cha single-layer confocal cha njira yofananira, kudzera mu equation: signal = min (mtYFP, antibody), dera la mitochondrial lomwe limasonyeza kuti chitetezo cha mthupi chimagwirira ntchito ku antibody inayake mu Purkina limazindikirika. Kenako dera la pixel mu chigoba chomwe chimachokera limawerengedwa, kenako limasinthidwa kukhala labwinobwino pachithunzi chofanana cha single-stack cha njira ya mtYFP kuti mupeze gawo la mitochondrial la mapuloteni omwe akuwonetsedwa.
Kusanthula kuchuluka kwa maselo a Purkinje. Pulogalamu ya Cell Counter ya ImageJ idagwiritsidwa ntchito poyesa kuchuluka kwa maselo a Purkinje pogawa chiwerengero cha maselo a Purkinje owerengedwa ndi kutalika kwa mphete ya cerebellar yomwe ili ndi maselo owerengedwa.
Kukonzekera ndi kusonkhanitsa zitsanzo. Ubongo wochokera ku gulu lolamulira ndi mbewa za Mfn2cKO unakhazikika mu 2% PFA/2.5% glutaraldehyde mu 0.1 M phosphate buffer (PB), kenako magawo a coronal anakonzedwa pogwiritsa ntchito ciliates (Leica Mikrosysteme GmbH, Vienna, Austria) (Kukhuthala 50 mpaka 60 μm). Kenako sungani mu PB buffer mu 1% os tetraoxide ndi 1.5% potassium ferrocyanide kutentha kwa chipinda kwa ola limodzi. Magawowo anatsukidwa katatu ndi madzi osungunuka, kenako anapaka utoto wa 70% ethanol wokhala ndi 1% uranyl acetate kwa mphindi 20. Magawowo kenako anasungunuka mu mowa wokonzedwa bwino ndikuyikidwa mu Durcupan ACM (Araldite casting resin M) epoxy resin (Electron Microscopy Sciences, catalog number 14040) pakati pa magalasi ophimbidwa ndi silicon, ndipo potsiriza pa 60°C Polymerize mu uvuni kwa maola 48. Malo a cerebellar cortex adasankhidwa ndipo magawo opyapyala a 50 nm adadulidwa pa Leica Ultracut (Leica Mikrosysteme GmbH, Vienna, Austria) ndikusankhidwa pa gridi ya mkuwa ya 2 × 1 mm yokutidwa ndi polystyrene film. Magawowo adapakidwa ndi yankho la 4% uranyl acetate mu H2O kwa mphindi 10, kutsukidwa ndi H2O kangapo, kenako ndi Reynolds lead citrate mu H2O kwa mphindi 10, kenako kutsukidwa ndi H2O kangapo. Ma Micrograph adatengedwa ndi microscope ya transmission electron Philips CM100 (Thermo Fisher Scientific, Waltham, MA, USA) pogwiritsa ntchito TVIPS (Tietz Video and Image Processing System) TemCam-F416 digital camera (TVIPS GmbH, Gauting, USA). Germany).
Pa mbewa zomwe zili ndi AAV, ubongo unalekanitsidwa ndikudulidwa m'gawo la sagittal lokhala ndi makulidwe a 1 mm, ndipo cerebellum inawunikidwa pogwiritsa ntchito maikulosikopu ya fluorescence kuti izindikire mphete yodwala AAV (ndiko kuti, mCherry expressing). Kuyesera kokha komwe jakisoni wa AAV kumapangitsa kuti gawo la maselo a Purkinje likhale logwira ntchito bwino kwambiri (monga pafupifupi gawo lonse) m'magawo awiri otsatizana a cerebellar ndi komwe kumagwiritsidwa ntchito. Chizunguliro chopangidwa ndi AAV chinadulidwa pang'onopang'ono kuti chikhale chokhazikika usiku wonse (4% PFA ndi 2.5% glutaraldehyde mu 0.1 M cocoate buffer) ndikukonzedwanso. Pakuyika kwa EPON, minofu yokhazikika idatsukidwa ndi 0.1 M sodium cocoate buffer (Applichem), ndikuyikidwa ndi 2% OsO4 (os, Science Services; Caco) mu 0.1 M sodium cocoate buffer (Applichem) Maola 4, kenako sambani kwa maola awiri. Bwerezani katatu ndi 0.1 M cocamide buffer. Pambuyo pake, ethanol yomwe inakwera inagwiritsidwa ntchito kuyika madzi mu ethanol iliyonse pa 4°C kwa mphindi 15 kuti isungunule madzi mu minofu. Minofuyo inasamutsidwira ku propylene oxide ndipo inayikidwa mu EPON (Sigma-Aldrich) usiku wonse pa 4°C. Ikani minofuyo mu EPON yatsopano kutentha kwa chipinda kwa maola awiri, kenako ikani mu 62°C kwa maola 72. Gwiritsani ntchito ultramicrotome (Leica Microsystems, UC6) ndi mpeni wa diamondi (Diatome, Biel, Switzerland) kudula magawo a 70 nm ultrathin, ndikuyika utoto ndi 1.5% uranyl acetate kwa mphindi 15 pa 37°C, ndikuyika utoto ndi lead citrate solution kwa mphindi 4. Ma electron micrographs adatengedwa pogwiritsa ntchito JEM-2100 Plus transmission electron microscope (JEOL) yokhala ndi Camera OneView 4K 16-bit (Gatan) ndi DigitalMicrograph software (Gatan). Pofufuza, ma micrograph a ma electron adapezeka pogwiritsa ntchito zoom ya digito ya 5000× kapena 10,000×.
Kusanthula kwa mawonekedwe a mitochondria. Pa kusanthula konse, mawonekedwe a mitochondria iliyonse adafotokozedwa pamanja m'zithunzi za digito pogwiritsa ntchito pulogalamu ya ImageJ. Ma parameter osiyanasiyana a mawonekedwe amawunikidwa. Kuchuluka kwa mitochondria kumawonetsedwa ngati peresenti yomwe imapezeka pogawa dera lonse la mitochondria la selo lililonse ndi dera la cytoplasm (dera la cytoplasm = dera la selo-dera la selo) × 100. Kuzungulira kwa mitochondria kumawerengedwa ndi fomula [4π∙ (dera/perimeter 2)]. Mawonekedwe a ista a mitochondria adawunikidwa ndikugawidwa m'magulu awiri ("tubular" ndi "blister") malinga ndi mawonekedwe awo akuluakulu.
Kusanthula kwa chiwerengero cha Autophagosome/lysosome ndi kuchuluka kwa maselo. Gwiritsani ntchito pulogalamu ya ImageJ kuti mufotokozere pamanja mawonekedwe a autophagosome/lysosome iliyonse pachithunzi cha digito. Malo a Autophagosome/lysosome amafotokozedwa ngati peresenti yowerengedwa pogawa dera lonse la kapangidwe ka autophagosome/lysosome la selo lililonse ndi malo a cytoplasm (malo a cytoplasm=malo a selo-nucleus)×100. Kuchuluka kwa autophagosomes/lysosomes kumawerengedwa pogawa chiwerengero chonse ndi chiwerengero cha kapangidwe ka autophagosome/lysosome pa selo lililonse (malinga ndi malo a cytoplasmic) (malo a cytoplasmic = malo a selo-nucleus).
Zolemba zogaŵira mwachangu ndi kukonzekera zitsanzo. Pa zoyeserera zomwe zimafuna kulemba shuga m'magazi, tumizani zidutswa za ubongo wa acute ku chipinda chosungiramo shuga, chomwe chili ndi kaboni wokhuta (95% O2 ndi 5% CO2), Ca2 + ACSF yapamwamba (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 ndi 2.0 mM MgCl2, yosinthidwa kukhala pH 7.4 ndi 310 mpaka 320 mOsm), momwe shuga ndi 13 C 6- M'malo mwa shuga (Eurisotop, nambala ya katalogi CLM-1396). Pa mayeso omwe amafunikira kulemba zilembo za pyruvate, sinthani magawo a ubongo wa acute ku Ca2 + ACSF yapamwamba (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 ndi Add 2.0mM MgCl2, sinthani ku pH 7.4 ndi 310 mpaka 320mOsm), ndikuwonjezera 1mM 1-[1-13C]pyruvate (Eurisotop, nambala ya catalog CLM-1082). Ikani magawowo kwa mphindi 90 pa 37°C. Pamapeto pa kuyesaku, magawowo anatsukidwa mwachangu ndi yankho lamadzi (pH 7.4) lomwe lili ndi 75 mM ammonium carbonate, kenako anasinthidwa kukhala homogenized mu 40:40:20 (v:v:v) acetonitrile (ACN): methanol: madzi. Magawowo atayikidwa pa ayezi kwa mphindi 30, zitsanzozo zinayikidwa centrifuge pa 21,000 g kwa mphindi 10 pa 4°C, ndipo supernatant yoyera inaumitsidwa mu SpeedVac concentrator. Pellet youma ya metabolite yomwe inatuluka inasungidwa pa -80°C mpaka itafufuzidwa.
Kusanthula kwa madzi a chromatography-mass spectrometry a ma amino acid 13 olembedwa C. Pa kusanthula kwa madzi a chromatography-mass spectrometry (LC-MS), metabolite pellet inabwezeretsedwanso mu 75μl ya madzi a LC-MS grade (Honeywell). Pambuyo pa centrifugation pa 21,000 g kwa mphindi 5 pa 4°C, 20 μl ya supernatant yofotokozedwa idagwiritsidwa ntchito posanthula amino acid flux, pomwe chotsalacho chinagwiritsidwa ntchito nthawi yomweyo posanthula anion (onani pansipa). Kusanthula kwa amino acid kunachitika pogwiritsa ntchito njira yofotokozera kale ya benzoyl chloride derivatization (55, 56). Mu gawo loyamba, 10μl ya 100 mM sodium carbonate (Sigma-Aldrich) idawonjezedwa ku 20μl ya metabolite extract, kenako 10μl ya 2% benzoyl chloride (Sigma-Aldrich) idawonjezedwa ku LC grade ACN. Chitsanzocho chinasunthidwa kwa kanthawi kenako chinayikidwa mu centrifuge pa 21,000 g kwa mphindi 5 pa 20°C. Samutsani supernatant yochotsedwayo ku vial ya 2 ml autosampler yokhala ndi conical glass insert (200 μl voliyumu). Zitsanzozo zinawunikidwa pogwiritsa ntchito Acquity iClass ultra-high performance LC system (Waters) yolumikizidwa ku Q-Exactive (QE)-HF (Ultra High Field Orbitrap) high-resolution precision mass spectrometer (Thermo Fisher Scientific). Kuti ziwunikidwa, 2μl ya chitsanzo chochotsedwacho inalowetsedwa mu 100×1.0 mm high-strength silica T3 column (Waters) yokhala ndi tinthu ta 1.8μm. Kuthamanga kwa madzi ndi 100μl/min, ndipo dongosolo la buffer limapangidwa ndi buffer A (10 mM ammonium formate ndi 0.15% formic acid m'madzi) ndi buffer B (ACN). Gradient ndi iyi: 0%B pa mphindi 0; 0%B. 0 mpaka 15% B pa mphindi 0 mpaka 0.1; 15 mpaka 17% B pa mphindi 0.1 mpaka 0.5; B pa 17 mpaka 55% pa mphindi 0.5 mpaka 14; B pa 55 mpaka 70% pa mphindi 14 mpaka 14.5; pa 14.5 mpaka 70 mpaka 100% B pa mphindi 18; 100% B pa mphindi 18 mpaka 19; 100 mpaka 0% B pa mphindi 19 mpaka 19.1; 0% B pa mphindi 19.1 mpaka 28 (55, 56). QE-HF mass spectrometer imagwira ntchito mu positive ionization mode yokhala ndi mass range ya m/z (mass/charge ratio) ya 50 mpaka 750. Resolution yogwiritsidwa ntchito ndi 60,000, ndipo gain control (AGC) ion target yomwe yapezeka ndi 3×106, ndipo nthawi yayitali ya ion ndi 100 milliseconds. Gwero la heated electrospray ionization (ESI) limagwira ntchito pa spray voltage ya 3.5 kV, capillary temperature ya 250°C, sheath airflow ya 60 AU (arbitrary units), ndi assistant airflow ya 20 AU. 250°C. S lens imayikidwa pa 60 AU.
Kusanthula kwa Anion chromatography-MS kwa ma organic acids olembedwa ndi 13C. Ma metabolite precipitate otsala (55μl) adawunikidwa pogwiritsa ntchito Dionex ion chromatography system (ICS 5000+, Thermo Fisher Scientific) yolumikizidwa ndi QE-HF mass spectrometer (Thermo Fisher Scientific). Mwachidule, 5μl ya metabolite extract idalowetsedwa mu Dionex IonPac AS11-HC column yokhala ndi HPLC (2 mm×250 mm, particle size 4μm, Thermo Fisher Scientific) mu push-in partial loop mode yokhala ndi chiŵerengero chodzaza cha 1. ) Dionex IonPac AG11-HC guard column (2 mm x 50 mm, 4μm, Thermo Fisher Scientific). Kutentha kwa column kumasungidwa pa 30°C, ndipo autosampler imayikidwa pa 6°C. Gwiritsani ntchito cartridge ya potassium hydroxide yoperekedwa ndi madzi oyeretsedwa kuti mupange potassium hydroxide gradient kudzera mu eluent generator. Kulekanitsa ma metabolites pa liwiro la 380μl/min, pogwiritsa ntchito njira yotsatirayi: mphindi 0 mpaka 3, 10 mM KOH; mphindi 3 mpaka 12, 10 mpaka 50 mM KOH; mphindi 12 mpaka 19, 50 mpaka 100 mM KOH; mphindi 19 mpaka 21, 100 mM KOH; mphindi 21 mpaka 21.5, 100 mpaka 10 mM KOH. Mzatiwo unakonzedwanso pansi pa 10 mM KOH kwa mphindi 8.5.
Ma metabolites opangidwa ndi eluted amaphatikizidwa ndi mtsinje wowonjezera wa isopropanol wa 150μl/min pambuyo pa column kenako n’kuwongoleredwa ku high-resolution mass spectrometer yomwe imagwira ntchito mu negative ionization mode. MS ikuyang'anira kuchuluka kwa misa kuyambira m/z 50 mpaka 750 ndi resolution ya 60,000. AGC imayikidwa pa 1×106, ndipo nthawi yayitali ya ion imasungidwa pa 100 ms. Gwero la ESI lotenthedwa linkagwiritsidwa ntchito pa mphamvu ya spray ya 3.5 kV. Makonzedwe ena a gwero la ion ndi awa: kutentha kwa capillary 275°C; kuyenda kwa mpweya wa sheath, 60 AU; kuyenda kwa mpweya wothandiza, 20 AU pa 300°C, ndi S lens yokhazikika pa 60 AU.
Kusanthula deta ya metabolites yolembedwa ndi 13C. Gwiritsani ntchito pulogalamu ya TraceFinder (mtundu 4.2, Thermo Fisher Scientific) kuti muwunikenso deta ya chiŵerengero cha isotope. Kudziwika kwa chinthu chilichonse kunatsimikiziridwa ndi gulu lodalirika lofotokozera ndipo kunasanthulidwa paokha. Pofuna kuchita kusanthula kwa isotope enrichment, dera la ion chromatogram (XIC) yochotsedwa ya isotope iliyonse ya 13C (Mn) inachotsedwa kuchokera ku [M + H]+, komwe n ndi nambala ya kaboni ya gulu lofunidwa, lomwe limagwiritsidwa ntchito kusanthula amino acid kapena [MH] + limagwiritsidwa ntchito kusanthula anions. Kulondola kwa XIC ndi kochepera magawo asanu pa miliyoni, ndipo kulondola kwa RT ndi mphindi 0.05. Kusanthula kwa enrichment kumachitika powerengera chiŵerengero cha isotope iliyonse yomwe yapezeka ndi kuchuluka kwa isotope zonse za gulu lofanana. Ma ratios awa amaperekedwa ngati ma peresenti a isotope iliyonse, ndipo zotsatira zake zimafotokozedwa ngati molar percentage enrichment (MPE), monga tafotokozera kale (42).
Pellet ya neuron yozizirayo inasinthidwa kukhala homogenized mu 80% methanol (v/v) yozizira kwambiri, kuyikidwa mu vortex, ndikuyikidwa mu incubation pa -20°C kwa mphindi 30. Vortex chitsanzo kachiwiri ndikusakaniza pa +4°C kwa mphindi 30. Chitsanzocho chinayikidwa mu centrifuge pa 21,000 g kwa mphindi 5 pa 4°C, kenako supernatant yomwe inatuluka inasonkhanitsidwa ndikuumitsidwa pogwiritsa ntchito SpeedVac concentrator pa 25°C kuti iwunikenso pambuyo pake. Monga tafotokozera pamwambapa, kusanthula kwa LC-MS kunachitika pa amino acid a maselo osankhidwa. Pogwiritsa ntchito TraceFinder (version 4.2, Thermo Fisher Scientific), kusanthula deta kunachitika pogwiritsa ntchito monoisotopic mass ya compound iliyonse. Quantile normalization ya metabolite data idachitika pogwiritsa ntchito preprocessCore software package (57).
Kukonzekera magawo. Mbewa inapatsidwa mankhwala oletsa ululu mwachangu ndi carbon dioxide ndikudulidwa mutu, ubongo unachotsedwa mwachangu pachigaza, ndipo mpeni wogwedezeka wodzazidwa ndi ayezi (HM-650 V, Thermo Fisher Scientific, Walldorf, Germany) unagwiritsidwa ntchito kuudula m'magawo a sagittal a 300 mpaka 375 μm. Kuzizira kwa mpweya wa carbon (95% O2 ndi 5% CO2) Kutsika kwa Ca2 + ACSF (125.0 mM NaCl, 2.5 mM KCl, 1.25 mM sodium phosphate buffer, 25.0 mM NaHCO3, 25.0 mM d-glucose, 1.0 mM CaCl2 ndi 6.0 mM MgCl2 Sinthani kukhala pH 7.4 ndi 310 mpaka 330 mOsm). Tumizani zidutswa za ubongo zomwe mwapeza ku chipinda chokhala ndi Ca2 + ACSF yapamwamba (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 ndi mM 3.5 MgCl2) pH 7.4 ndi 310 mpaka 320 mOsm). Sungani zidutswazo kwa mphindi 20 mpaka 30 kuti zibwezeretsedwe musanazilembe.
kujambula. Gawo la maikulosikopu lokhala ndi chipinda chojambulira chokhazikika ndi lenzi yowunikira ya 20x immersion water (Scientifica) linagwiritsidwa ntchito pojambula zonse. Maselo a Purkinje omwe amaganiziridwa kuti ndi ofunikira adazindikirika ndi (i) kukula kwa thupi, (ii) malo a cerebellum, ndi (iii) mawonekedwe a fluorescent mtYFP reporter gene. Patch pipette yokhala ndi nsonga yolimba ya 5 mpaka 11 megohms imatulutsidwa ndi borosilicate glass capillary (GB150-10, 0.86 mm×1.5 mm×100 mm, Science Products, Hofheim, Germany) ndi horizontal pipette Instruments (P-1000, Sutter), Novato, CA). Zojambula zonse zidachitidwa ndi ELC-03XS npi patch clamp amplifier (npi electronic GmbH, Tam, Germany), yomwe idayang'aniridwa ndi pulogalamu ya Signal (version 6.0, Cambridge Electronic, Cambridge, UK). Kuyeseraku kudalembedwa pamlingo wa 12.5 kHz. Chizindikirocho chimasefedwa ndi zosefera ziwiri za Bessel zodutsa pang'ono zokhala ndi ma frequency odulira a 1.3 ndi 10 kHz motsatana. Mphamvu ya nembanemba ndi pipette imalipidwa ndi dera lolipirira pogwiritsa ntchito amplifier. Kuyesera konse kunachitika motsogozedwa ndi kamera ya Orca-Flash 4.0 (Hamamatsu, Gerden, Germany), yomwe inkayang'aniridwa ndi pulogalamu ya Hokawo (mtundu 2.8, Hamamatsu, Gerden, Germany).
Kapangidwe ka maselo onse ndi kusanthula. Nthawi yomweyo musanalembe, dzazani pipette ndi yankho lamkati lomwe lili ndi zinthu zotsatirazi: 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) ndi 10.0 mM creatinine phosphate zinasinthidwa kukhala pH 7.25, ndipo kuthamanga kwa osmotic kunali 290 mOsm (sucrose). Nthawi yomweyo mutagwiritsa ntchito mphamvu ya 0 pA kuti muswe nembanemba, mphamvu ya nembanemba yopumula inayesedwa. Kukana kwa input kumayesedwa pogwiritsa ntchito ma currents a hyperpolarized a -40, -30, -20, ndi -10 pA. Yesani kukula kwa mphamvu ya voltage ndikugwiritsa ntchito lamulo la Ohm kuti muwerengere kukana kwa input. Ntchito yodzidzimutsa inalembedwa mu cholumikizira chamagetsi kwa mphindi 5, ndipo sPSC inazindikirika ndikuyesedwa mu Igor Pro (mtundu 32 7.01, WaveMetrics, Lake Oswego, Oregon, USA) pogwiritsa ntchito semi-automatic recognition script. Mzere wa IV ndi mphamvu yokhazikika zimayesedwa poyika batri pa potentials zosiyanasiyana (kuyambira -110 mV) ndikuwonjezera mphamvu mu masitepe 5 mV. Kupanga kwa AP kunayesedwa pogwiritsa ntchito mphamvu yochotsera mphamvu. Ikani selo pa -70 mV pamene mukugwiritsa ntchito mphamvu yochotsera mphamvu. Sinthani kukula kwa sitepe ya gawo lililonse lojambulira padera (10 mpaka 60 pA). Werengerani kuchuluka kwa AP mwa kuwerengera pamanja ma pulse spikes omwe amayambitsa ma AP frequency apamwamba kwambiri. Mzere wa AP umasanthulidwa pogwiritsa ntchito derivative yachiwiri ya depolarization pulse yomwe imayambitsa AP imodzi kapena zingapo.
Kapangidwe ndi kusanthula kwa chigamba chobowoka. Chitani kujambula kwa chigamba chobowoka pogwiritsa ntchito njira zokhazikika. Gwiritsani ntchito pipette yopanda ATP ndi GTP yomwe ilibe zosakaniza izi: 128 mM gluconate K, 10 mM KCl, 10 mM Hepes, 0.1 mM EGTA ndi 2 mM MgCl2, ndikusinthira ku pH 7.2 (pogwiritsa ntchito KOH). ATP ndi GTP zimachotsedwa mu yankho la mkati mwa maselo kuti zisalowerere bwino mu nembanemba ya selo. Pipette yobowoka imadzazidwa ndi yankho lamkati lomwe lili ndi amphotericin (pafupifupi 200 mpaka 250μg/ml; G4888, Sigma-Aldrich) kuti mupeze mbiri ya chigamba chobowoka. Amphotericin inasungunuka mu dimethyl sulfoxide (kuchuluka komaliza: 0.1 mpaka 0.3%; DMSO; D8418, Sigma-Aldrich). Kuchuluka kwa DMSO komwe kunagwiritsidwa ntchito sikunakhudze kwambiri ma neuron omwe adaphunziridwa. Panthawi yobowola, kukana kwa njira (Ra) kunayang'aniridwa mosalekeza, ndipo kuyesera kunayamba pambuyo poti kuchuluka kwa Ra ndi AP kwakhazikika (mphindi 20-40). Ntchito yodzidzimutsa imayesedwa mu voltage ndi/kapena current clamp kwa mphindi 2 mpaka 5. Kusanthula deta kunachitika pogwiritsa ntchito Igor Pro (mtundu 7.05.2, WaveMetrics, USA), Excel (mtundu 2010, Microsoft Corporation, Redmond, USA) ndi GraphPad Prism (mtundu 8.1.2, GraphPad Software Inc., La Jolla, CA). United States). Kuti mudziwe ma AP odzidzimutsa, pulagi ya IgorPro's NeuroMatic v3.0c imagwiritsidwa ntchito. Dziwani ma AP okha pogwiritsa ntchito malire operekedwa, omwe amasinthidwa payekhapayekha pa cholembedwa chilichonse. Pogwiritsa ntchito nthawi yodzidzimutsa, dziwani kuchuluka kwa spike ndi kuchuluka kwa spike komwe kumakhalapo nthawi yomweyo komanso kuchuluka kwa spike komwe kumakhalapo nthawi yomweyo.
Kupatula kwa PN. Mwa kusintha njira yomwe idasindikizidwa kale, ma PN adayeretsedwa kuchokera ku cerebellum ya mbewa pa siteji inayake (58). Mwachidule, cerebellum idadulidwa ndikudulidwa mu malo ozizira osakanikirana [popanda HBSS Ca2+ ndi Mg2+, yowonjezera ndi shuga wa 20 mM, penicillin (50 U/ml) ndi streptomycin (0.05 mg/ml)], kenako ndikugaya malo mu papain [HBSS, yowonjezera ndi 1-cysteine·HCl (1 mg/ml), papain (16 U / ml) ndi deoxyribonuclease I (DNase I; 0.1 mg/ml)] Chitani kwa mphindi 30 pa 30°C. Choyamba sambitsani minofu mu HBSS medium yokhala ndi dzira (10 mg/ml), BSA (10 mg/ml) ndi DNase (0.1 mg/ml) kutentha kwa chipinda kuti mupewe kugaya kwa enzymatic, kenako mu HBSS medium yokhala ndi shuga wa 20 mM. Gwirani pang'onopang'ono mu HBSS, penicillin (50 U/ml), streptomycin (0.05 mg/ml) ndi DNase (0.1 mg/ml) kutulutsa maselo amodzi. Kuyimitsidwa kwa maselo komwe kunachokera kunasefedwa kudzera mu 70μm cell strainer, kenako maselowo anathiridwa ndi centrifugation (1110 rpm, mphindi 5, 4°C) ndikuyimitsidwanso mu sorting medium [HBSS, yowonjezera ndi 20 mM glucose, 20% fetal bovine ) Serum, penicillin (50 U/ml) ndi streptomycin (0.05 mg/ml)]; fufuzani momwe maselo amagwirira ntchito ndi propidium iodide ndikusintha kuchuluka kwa maselo kukhala 1 × 106 mpaka 2 × 106 cells/ml. Asanayambe flow cytometry, suspension imasefedwa kudzera mu 50 μm cell strainer.
Kusanthula kwa ma cell kunachitika pa 4°C pogwiritsa ntchito makina a FACSAria III (BD Biosciences) ndi pulogalamu ya FACSDiva (BD Biosciences, mtundu 8.0.1). Kuyimitsidwa kwa ma cell kunasankhidwa pogwiritsa ntchito nozzle ya 100 μm pansi pa kupanikizika kwa 20 psi pamlingo wa ~2800 events/sec. Popeza njira zachikhalidwe zowunikira ma cell (kukula kwa ma cell, kusankhana kwa ma bimodal, ndi makhalidwe obalalika) sizingatsimikizire kuti PN yachotsedwa bwino kuchokera ku mitundu ina ya ma cell, njira yowunikira ma cell imakhazikitsidwa potengera kufananiza kwa YFP intensity ndi autofluorescence mu mitoYFP+ ​​​​ndi control mitoYFP − Mbewa. YFP imakondwera powunikira chitsanzocho ndi mzere wa laser wa 488 nm, ndipo chizindikirocho chimazindikirika pogwiritsa ntchito fyuluta ya 530/30 nm band pass. Mu mitoYFP+ ​​​​mbewa, mphamvu ya Rosa26-mitoYFP reporter gene imagwiritsidwanso ntchito kusiyanitsa ziwalo za neuronal body ndi axon. 7-AAD imayatsidwa ndi laser yachikasu ya 561 nm ndipo imapezedwa ndi fyuluta ya bandpass ya 675/20 nm kuti ichotse maselo akufa. Pofuna kulekanitsa ma astrocyte nthawi yomweyo, kuimitsidwa kwa selo kunapakidwa utoto wa ACSA-2-APC, kenako chitsanzocho chinayatsidwa ndi mzere wa laser wa 640 nm, ndipo fyuluta ya bandpass ya 660/20 nm inagwiritsidwa ntchito kuzindikira chizindikirocho.
Maselo osonkhanitsidwawo anathiridwa ndi centrifugation (1110 rpm, mphindi 5, 4°C) ndipo anasungidwa pa -80°C mpaka atagwiritsidwa ntchito. Mbewa za Mfn2cKO ndi ana awo aakazi zimayikidwa m'magulu tsiku lomwelo kuti zichepetse kusinthasintha kwa njira. Kuwonetsa ndi kusanthula deta ya FACS kunachitika pogwiritsa ntchito pulogalamu ya FlowJo (FlowJo LLC, Ashland, Oregon, USA).
Monga tafotokozera pamwambapa (59), PCR yeniyeni imagwiritsidwa ntchito kupatula DNA kuchokera ku ma neuron osankhidwa kuti iwerengedwe pambuyo pake. Kuzindikira kwa mzere ndi kutha kwa threshold kunayesedwa poyamba poyendetsa qPCR pa maselo osiyanasiyana. Mwachidule, sonkhanitsani 300 PN mu lysis buffer yokhala ndi 50 mM tris-HCl (pH 8.5), 1 mM EDTA, 0.5% Tween 20 ndi proteinase K (200 ng/ml) ndikuyika mu incubator pa 55°C kwa mphindi 120. Maselowo adayikidwanso mu incubator pa 95°C kwa mphindi 10 kuti atsimikizire kuti proteinase K yatha. Pogwiritsa ntchito probe ya TaqMan (Thermo Fisher) yapadera kwa mt-Nd1, mtDNA idayesedwa ndi semi-quantitative PCR mu 7900HT Real-Time PCR system (Thermo Fisher Scientific). Sayansi, nambala ya katalogi Mm04225274_s1), mt-Nd6 (Thermo Fisher Scientific, nambala ya katalogi AIVI3E8) ndi 18S (Thermo Fisher Scientific, nambala ya katalogi Hs99999901_s1) majini.
Kukonzekera chitsanzo cha Proteome. Mwa kutentha yankho pa 95°C kwa mphindi 10 ndikupatsa sonicating, mu lysis buffer [6 M guanidine chloride, 10 mM tris(2-carboxyethyl) phosphine hydrochloride, 10 mM chloroacetamide ndi 100 mM tris-Lyse frozen neuron pellets mu HCl]. Pa Bioruptor (Diagenode) kwa mphindi 10 (masekondi 30 pulse / masekondi 30 pause period). Chitsanzocho chinachepetsedwa 1:10 mu 20 mM tris-HCl (pH 8.0), chosakanizidwa ndi 300 ng trypsin gold (Promega), ndipo chinayikidwa usiku wonse pa 37°C kuti chikwaniritse kugaya bwino. Pa tsiku lachiwiri, chitsanzocho chinayikidwa centrifuge pa 20,000 g kwa mphindi 20. Supernatant inachepetsedwa ndi 0.1% formic acid, ndipo yankholo linachotsedwa mchere ndi StageTips yopangidwa yokha. Chitsanzocho chinaumitsidwa mu chipangizo cha SpeedVac (Eppendorf concentrator plus 5305) pa 45°C, kenako peptide inaimitsidwa mu 0.1% formic acid. Zitsanzo zonse zinakonzedwa nthawi imodzi ndi munthu yemweyo. Pofuna kusanthula zitsanzo za astrocyte, ma peptide 4 μg ochotsedwa mchere adalembedwa ndi tandem mass tag (TMT10plex, catalog number 90110, Thermo Fisher Scientific) ndi chiŵerengero cha peptide ku TMT reagent cha 1:20. Pa kulemba TMT, 0.8 mg ya reagent ya TMT idabwezeretsedwanso mu 70 μl ya ACN yopanda madzi, ndipo peptide youmayo idapangidwanso kukhala 9 μl ya 0.1 M TEAB (triethylammonium bicarbonate), pomwe 7 μl ya reagent ya TMT mu ACN idawonjezedwa. Kuchuluka kwake kunali 43.75%. Pambuyo pa mphindi 60 zoyamwitsa, yankholo linazimitsidwa ndi 2 μl ya 5% hydroxylamine. Ma peptide olembedwawo anasonkhanitsidwa, kuumitsidwa, kubwezeretsedwa mu 200μl ya 0.1% formic acid (FA), kugawidwa m'magulu awiri, kenako n’kuchotsedwa mchere pogwiritsa ntchito StageTips yopangidwa yokha. Pogwiritsa ntchito UltiMate 3000 ultra high performance liquid chromatograph (UltiMate 3000 ultra high performance liquid chromatograph), imodzi mwa magawo awiriwa inagawidwa pa 1mm x 150mm Acquity chromatographic column yodzazidwa ndi tinthu ta 130Å1.7μm C18 (Waters, catalog No. SKU: 186006935). Thermo Fisher Scientific). Siyanitsani ma peptide pamlingo woyenda wa 30μl/min, patulani 1% mpaka 50% buffer B kwa mphindi 85 ndi gradient yoyenda pang'onopang'ono ya mphindi 96, kuyambira 50% mpaka 95% buffer B kwa mphindi 3, kenako mphindi 8 za 95% Buffer B; Buffer A ndi 5% ACN ndi 10 mM ammonium bicarbonate (ABC), ndipo buffer B ndi 80% ACN ndi 10 mM ABC. Sonkhanitsani magawo mphindi zitatu zilizonse ndikusakaniza m'magulu awiri (1 + 17, 2 + 18, etc.) ndikuwumitsa mu vacuum centrifuge.
Kusanthula kwa LC-MS/MS. Pa mass spectrometry, ma peptide (nambala r119.aq) adalekanitsidwa pa 25 cm, 75 μm inner diameter PicoFrit analytical column (lens yatsopano, gawo nambala PF7508250) yokhala ndi 1.9 μm ReproSil-Pur 120 C18-AQ medium (Dr. Maisch, mat), Use EASY-nLC 1200 (Thermo Fisher Scientific, Germany). Column idasungidwa pa 50°C. Ma Buffers A ndi B ndi 0.1% formic acid m'madzi ndi 0.1% formic acid mu 80% ACN, motsatana. Ma Peptides adalekanitsidwa kuchokera ku 6% mpaka 31% buffer B kwa mphindi 65 ndi kuchokera ku 31% mpaka 50% buffer B kwa mphindi 5 ndi gradient ya 200 nl/min. Ma peptides osadziwika adasanthulidwa pa Orbitrap Fusion mass spectrometer (Thermo Fisher Scientific). Kuyeza kwa peptide precursor m/z kumachitika ndi resolution ya 120,000 pakati pa 350 mpaka 1500 m/z. Pogwiritsa ntchito mphamvu yokhazikika ya 27%, precursor yamphamvu kwambiri yokhala ndi charge state ya 2 mpaka 6 imasankhidwa kuti pakhale cleavage ya C trap dissociation (HCD) yamphamvu kwambiri. Nthawi yozungulira imayikidwa pa 1 s. Mtengo wa m/z wa peptide fragment unayesedwa mu ion trap pogwiritsa ntchito AGC target yaying'ono kwambiri ya 5×104 ndi nthawi yayikulu yobayira ya 86 ms. Pambuyo pa kugawikana, precursor inayikidwa pamndandanda wochotsa mphamvu kwa masekondi 45. Ma peptides okhala ndi zilembo za TMT adalekanitsidwa pa 50 cm, 75 μm Acclaim PepMap column (Thermo Fisher Scientific, catalog number 164942), ndipo ma spectra osamukira adawunikidwa pa Orbitrap Lumos Tribrid mass spectrometer (Thermo Fisher Scientific) yokhala ndi zida za high-field asymmetric waveform ions (FAIMS) (Thermo Fisher Scientific) zomwe zimagwira ntchito pa ma voltage awiri a −50 ndi −70 V. MS3 yosankhidwa kutengera synchronization precursor yomwe imagwiritsidwa ntchito poyesa TMT report ion signal. Kulekanitsidwa kwa peptide kunachitika pa EASY-nLC 1200, pogwiritsa ntchito 90% linear gradient elution, yokhala ndi buffer concentration ya 6% mpaka 31%; buffer A inali 0.1% FA, ndipo buffer B inali 0.1% FA ndi 80% ACN. Column yowunikira imagwira ntchito pa 50°C. Gwiritsani ntchito FreeStyle (mtundu 1.6, Thermo Fisher Scientific) kuti mugawanitse fayilo yoyambirira malinga ndi mphamvu ya FAIMS.
Kuzindikira ndi kuwerengera mapuloteni. Pogwiritsa ntchito injini yosakira ya Andromeda yolumikizidwa, deta yoyambirira idasanthulidwa pogwiritsa ntchito MaxQuant version 1.5.2.8 (https://maxquant.org/). Kuphatikiza pa ma Cre recombinase ndi ma YFP sequences omwe adapezeka kuchokera ku Aequorea victoria, ma peptide fragment spectra adafufuzidwa kuti apeze ma canonical sequence ndi isoform sequence ya mbewa yofotokozera proteome (Proteome ID UP000000589, idatsitsidwa kuchokera ku UniProt mu Meyi 2017). Methionine oxidation ndi protein N-terminal acetylation zidakhazikitsidwa ngati zosintha zosinthika; cysteine ​​​​carbamoyl methylation idakhazikitsidwa ngati zosintha zokhazikika. Ma parameter a digestion amakhazikitsidwa ku "specificity" ndi "trypsin/P". Chiwerengero chochepa cha ma peptide ndi ma razor peptides omwe amagwiritsidwa ntchito pozindikira mapuloteni ndi 1; chiwerengero chochepa cha ma peptides apadera ndi 0. Pansi pa mikhalidwe yofananiza mapu a peptide, kuchuluka kwa kuzindikira mapuloteni kunali 0.01. Njira ya "Second Peptide" imayatsidwa. Gwiritsani ntchito njira ya "match between run" kuti musamutse zizindikiritso zopambana pakati pa mafayilo osiyanasiyana oyambira. Gwiritsani ntchito LFQ minimum ratio count 1 ya quantification yopanda zilembo (LFQ) (60). Mphamvu ya LFQ imasefedwa kuti ipeze ma values ​​awiri ovomerezeka mu gulu limodzi la genotype nthawi iliyonse, ndipo imachotsedwa kuchokera ku distribution yachizolowezi yokhala ndi m'lifupi wa. 0.3 ndikusunthira pansi 1.8. Gwiritsani ntchito nsanja ya Perseus computing (https://maxquant.net/perseus/) ndi R (https://r-project.org/) kuti mufufuze zotsatira za LFQ. Mayeso a two-way moderate t ochokera ku limma software package adagwiritsidwa ntchito posanthula ma differential expression (61). Kusanthula deta yofufuza kumachitika pogwiritsa ntchito ggplot, FactoMineR, factoextra, GGally ndi pheatmap. Deta ya proteomics yochokera ku TMT idasanthulidwa pogwiritsa ntchito MaxQuant version 1.6.10.43. Fufuzani deta ya proteomics yosaphika kuchokera ku database ya proteomics ya anthu ya UniProt, yomwe idatsitsidwa mu Seputembala 2018. Kusanthulaku kumaphatikizapo chinthu chowongolera kuyera kwa isotope chomwe chinaperekedwa ndi wopanga. Gwiritsani ntchito limma mu R kuti muwunikenso kusiyana kwa mawu. Deta yoyambirira, zotsatira zakusaka kwa database, ndi njira yowunikira deta ndi zotsatira zake zonse zimasungidwa mu mgwirizano wa ProteomeXchange kudzera mu PRIDE partner repository yokhala ndi chidziwitso cha seti ya data PXD019690.
Mafotokozedwe ogwira ntchito amawonjezera kusanthula. Chida cha Ingenuity Pathway Analysis (QIAGEN) chinagwiritsidwa ntchito kudziwa kuchuluka kwa mawu ofotokozera ntchito a deta yomwe idakhazikitsidwa pa masabata 8 (Chithunzi 1). Mwachidule, mndandanda wa mapuloteni ochulukirapo womwe udapezeka kuchokera ku kusanthula kwa deta ya LC-MS/MS (tandem mass spectrometry) umagwiritsidwa ntchito ndi njira zotsatirazi zofufuzira: Mus musculus imasankhidwa ngati mtundu ndi maziko, ndipo gululi likuwonetsa kuti P value yosinthidwa ndi Benjamini kuti iwonjezere 0.05 kapena kutsika imawonedwa kuti ndi yofunika kwambiri. Pa graph iyi, magulu asanu ochulukirapo apamwamba mu gulu lililonse kutengera P value yosinthidwa akuwonetsedwa. Pogwiritsa ntchito mayeso angapo a t, pogwiritsa ntchito pulogalamu ya Benjamini, Krieger, ndi Yekutieli (Q = 5%), kusanthula kwa nthawi yowonetsa mapuloteni kumachitika pa anthu ofunikira omwe apezeka mu gulu lililonse, ndipo mzere uliwonse umasanthulidwa padera. Palibe chifukwa chogwiritsa ntchito SD yokhazikika.
Pofuna kuyerekeza zotsatira za kafukufukuyu ndi ma database ofalitsidwa ndikupanga chithunzi cha Venn chomwe chili mu Chithunzi 1, tinaphatikiza mndandanda wa mapuloteni ochulukirapo ndi zolemba za MitoCarta 2.0 (24). Gwiritsani ntchito chida cha pa intaneti cha Draw Venn Diagram (http://bioinformatics.psb.ugent.be/webtools/Venn/) kuti mupange chithunzicho.
Kuti mudziwe zambiri zokhudza njira zowerengera zomwe zimagwiritsidwa ntchito posanthula ma proteomics, chonde onani gawo lofanana la Zipangizo ndi Njira. Pa zoyeserera zina zonse, zambiri zatsatanetsatane zitha kupezeka mu nthano yofananira. Pokhapokha ngati zanenedwa mwanjira ina, deta yonse imafotokozedwa ngati mean ± SEM, ndipo kusanthula konse kwa ziwerengero kunachitika pogwiritsa ntchito pulogalamu ya GraphPad Prism 8.1.2.
Kuti mudziwe zambiri zowonjezera pa nkhaniyi, chonde onani http://advances.sciencemag.org/cgi/content/full/6/35/eaba8271/DC1
Iyi ndi nkhani yotseguka yogawidwa motsatira malamulo a Creative Commons Attribution-Non-Commercial License, yomwe imalola kugwiritsa ntchito, kufalitsa ndi kubwerezanso mu njira iliyonse, bola ngati kugwiritsidwa ntchito komaliza sikuli kopindulitsa pamalonda ndipo mfundo ndi yakuti ntchito yoyambirira ndi yolondola.
Dziwani: Tikukupemphani kuti mupereke imelo yanu kuti munthu amene mwamulimbikitsa patsamba lino adziwe kuti mukufuna kuti aone imeloyo ndipo si sipamu. Sitidzalemba ma adilesi aliwonse a imelo.
Funso ili limagwiritsidwa ntchito poyesa ngati ndinu mlendo ndikuletsa kutumiza sipamu yokha.
Wolemba E. Motori, I. Atanassov, SMV Kochan, K. Folz-Donahue, V. Sakthivelu, P. Giavalisco, N. Toni, J. Puyal, N.-G. Larson
Kusanthula kwa ma protein a ma neuron osagwira ntchito bwino kwawonetsa kuti mapulogalamu a kagayidwe kachakudya amayambitsidwa kuti athane ndi kuwonongeka kwa mitsempha.
Wolemba E. Motori, I. Atanassov, SMV Kochan, K. Folz-Donahue, V. Sakthivelu, P. Giavalisco, N. Toni, J. Puyal, N.-G. Larson
Kusanthula kwa ma protein a ma neuron osagwira ntchito bwino kwawonetsa kuti mapulogalamu a kagayidwe kachakudya amayambitsidwa kuti athane ndi kuwonongeka kwa mitsempha.
©2020 American Association for the Advancement of Science. maumwini onse ndi otetezedwa. AAAS ndi mnzake wa HINARI, AGORA, OARE, CHORUS, CLOCKSS, CrossRef ndi COUNTER. ScienceAdvances ISSN 2375-2548.