Jooni *Adresse jooni: Cologne 50931, Almaañ, Wiɗtooji njuɓɓudi ɓurndi moƴƴude e Cologne ko faati e jaabawol stres cellal e ñawuuji jowitiiɗi e duuɓi (CECAD).
Neurodegeneration ñawuuji mitokondriyaal ina hiisee wonde ko huunde nde waawataa wayleede sabu plastisite metabolik neeruuji ina famɗi, kono batte ŋakkeende golle mitokondriyaal e autonomie cellal mbayliigu neeruuji e nder ɓanndu ina famɗi no feewi. Ɗoo, eɗen naatna heen poroteyiin keeriiɗo cellal e nder neronuuji Purkinje jogiiɗi ŋakkeende OXPHOS yahrude yeeso, sabu dinamiik fusiyoŋ mitokondriyaal bonɗo. En tawii wonde ŋakkeende golle mitokondriyeeji ina addana waylo-waylo luggiɗngo e fannu poroteyiin, ko ɗum addani ɗum en waɗde feere haa mbaawa huutoraade porogaraamuuji mbayliigu laaɓtuɗi hade maayde cellal. Ko min cikkaano, min teskiima ƴellitaare laaɓtunde e karboksilaas piruwat (PCx) e enzimuuji goɗɗi kaɓtotooɗi duuɓi, ɓeydooji hakkundeeji cirƴam TCA. Remde PCx ina ɓeyda baasal oksidaatik e ɓuuɓri ƴiiƴam, ko ɗum hollirta wonde aterosklerosis ina jogii batte ndeenka e nder ƴiiƴam ɗam alaa OXPHOS. Restoraade jokkondire mitokondriyaal e nder neronuuji ɓuuɓɗi haa laaɓi ina wayla ɗeen sifaaji mbaylaandi haa laaɓa cer, ɗum noon ina haɗa maayde ɓiɗɓe leɗɗe. Ko min tawi heen koo ina hollita laabi ɗi nganndaaka tawo, baɗooji semmbe e ŋakkeende golle mitokondriyee, ina hollita kadi wonde mboros oo ina waawi wayleede hay e nder daawe cakkitiiɗe ñawu nguu.
Darnde mawnde mitokondriyaaji e nder jogaade mbayliigu semmbe neeruuji ina teeŋtinaa e maale neeruuji yaajɗe jowitiiɗe e ñawuuji mitokondriyaaji aadee. Ko ɓuri heewde e ɗeeɗoo ñawanɗe ina ngadda waylooji taƴe gonɗi e reentaade baylagol taƴe mitokondriyaal (1, 2) walla halkaare taƴe jowitiiɗe e dinamiik mitokondriyaal, tawi noon ina toɗɗii darnde ADN mitokondriyaal (mtDNA) (3, 4). Golle e nder mbaydiiji daabaaji kollitii wonde e jaabawol ŋakkeende mitokondriyee e nder ƴiye taariiɗe, laabi mbayliigu (5-7) ina mbaawi huutoreede, ko ɗum addanta humpitooji teeŋtuɗi ngam faamde no feewi pathogenesis ɗeen ñawanɗe caɗtuɗe. E ko feewti heen, faamde men waylooji mbaylaandi e nder sifaaji cellal keertiiɗi baɗɗi ŋakkeende kuuɓtodinnde peewnugol adenosin trifosfaat (ATP) e nder ngaandi, ina teeŋti (8), teeŋti noon e ŋakkeende anndude paandaale safaara baawɗe huutoreede ngam haɗde walla haɗde ñawu nguu. Haɗde ɓuuɓri ƴiiƴam (9). Ɓooygol humpito ko wonde ƴiye nerwooje ina keewi miijaade wonde ina njogii ɓuuɓri mbayliigu no feewi so yerondiraama e sifaaji ƴiye gonɗe e ƴiye taariiɗe ɗum en (10). E teskaade wonde ɗeen ƴiye ina njogii darnde mawnde e yuɓɓinde dokkal mbayliigaaji e nder neeruuji ngam wallitde jokkondire sinapseeji e jaabaade ngonkaaji ñawu e ñawu, mbaawka jogaade mbaylaandi ƴiye e ngonkaaji caɗtuɗi e nder ƴiye ngaandi ina famɗi ɗo haaɗi e ƴiye glial (11-14). Yanti heen, heterojeneeji cellal gonɗi e nder ƴiye ngaandi ina kaɗa no feewi janngude waylooji mbaylaandi baɗeteeɗi e nder dente neeruuji keertiiɗi. Ko ɗum waɗi, ko seeɗa anndaa e batte cellal e mbaylaandi tigi rigi e ŋakkeende golle mitokondriyaal e nder neronuuji.
Ngam faamde batte mbayliigu mbayliigu mitokondriyaal, min ceerndii neronuuji Purkinje (PNs) e nder daawe ceertuɗe e mbayliigu neeruuji baɗeteeɗi e halkaare fusiyoŋ membrane yaajɗo mitokondriyaal (Mfn2). Hay so tawii noon waylooji Mfn2 e nder aadee ina njokkondiri e sifaa neuropati sensorik mooftirɗo ndonaandi ganndiraaɗo Charcot-Marie-Tooth sifaa 2A (15), halkaare sarɗiyankoore Mfn2 e nder ɓowɗi ko huunde anndaande no feewi e ustude golle fosforilaasiyoŋ oksidaasiyoŋ (OXthoPHOS). Subtipeeji neeruuji ceertuɗi ɗii (16-19) e fenotipe neeruuji gonɗi heen ɗii ina njahda e maale neeruuji jahduɗe yeeso, ko wayi no ŋakkeende dille (18, 19) walla ataksi cerebellar (16). E huutoraade denndaangal laabi proteomik limtilimtinɗi (LFQ) ɗi ngalaa etirɗe, metabolomik, natal, e laabi viroloji, eɗen kollita wonde neurodegeneraasiyoŋ yahruɗo yeeso ina addana karboksilaas piruwat (PCx) e geɗe goɗɗe jowitiiɗe e arterioskleroosis PNs e nder nguurndam (in vivo) Feññinde enzim en. Ngam hoolkisaade nafoore ndeeɗoo yiytunde, min njuurniima teeŋti noon e baylagol PCx e nder PNs ŋakkuɗi Mfn2, min tawi wonde ndeeɗoo feere ina ɓeyda baasal oksidaatik e yaawnaade ustude ƴiiƴam, ɗum noon ina hollita wonde azoospermia ina rokka maayde cellal Metabolic adaptability. Feññinde MFN2 no feewi ina waawi hisnude haa laaɓa cer PN gonɗo e ŋakkeende OXPHOS tiiɗnde, e ñaamde ADN mitokondriyee keewɗo, e jokkondire mitokondriyeeje ɗe nganndu-ɗaa ina taƴaa, ko ɗum ɓeydi teeŋtinde wonde ooɗoo sifaa degeneraasiyoŋ neurodegeneraasiyoŋ ina waawi hay sellude e daawal cellal ɓalliwal yahrude yeeso e hitaande.
Ngam yiytude mitokondriyaaji e nder PNs knockout Mfn2, min kuutoriima mbaydi mooso baawndi rokkude mitokondriyaaji baɗɗi Cre ngam yiylaade poroteyiin ɓuuɓɗo (YFP) (mtYFP) (20) baylagol Cre e ƴeewtaade morfolooji mitokondriyaaji e nder nguurndam. En tawii wonde halkaare taƴre Mfn2 e nder PNs ina addana feccere seeɗa-seeɗa e nder laylaytol mitokondriyaal (Natal S1A), waylo-waylo gadano ngoo tawaama e duuɓi 3. E ko feewti heen, ŋakkeende mawnde e nder laylaytol PN, hono no seedaa e ŋakkeende immunostaining Calbindin nii, fuɗɗoraaki haa jonte 12 (Natal 1, A e B). Soklaaji waktuuji hakkunde mbayliigaaji gadani e nder morfolooji mitokondriyaal e fuɗɗoode maayde neeruuji yiyteteeɗi, addani en wiɗtude mbayliigaaji mbaylaandi ɗi ŋakkeende golle mitokondriyaal addata hade maayde cellal. En mbaɗii feere tuugornde e ƴeewndo ƴiiƴam (FACS) ngam seerndude PN peeñninoowo YFP (YFP+) (Natal 1C), e nder ɓowɗi ƴeewndotooɗi (Mfn2 + / loxP :: mtYFP loxP- stop-loxP: : L7-cre), caggal ɗuum ko CT1BRL (Natal). Ko ɓuri moƴƴude e peeje gating tuugiiɗe e tiiɗnaare gonnde e mbaydi YFP ina addana en laaɓtinde ɓanndu YFP+ (YFPhigh) e PNs e ɓe ngonaa PNs (YFPneg) (Natal S1B) walla e pecce akson/dendritik ɓuuɓɗe teskinɗe (YFPlow; Natal S1D, confirmed byNatal S1D, confirmed microscopecal), nano). Ngam hoolkisaade hol ko woni yimɓe renndinaaɓe ɓee, min mbaɗii proteomics LFQ caggal ɗuum min mbaɗii ƴeewndo komponenteeji mawɗi, min tawi ina woodi seerndi laaɓtundi hakkunde ƴiye YFPhigh e ƴiye YFPneg (Natal S1C). Piɓaaji YFPhigh kollitii alɗude laaɓtunde e maandeeji PNs anndaaɗi (ɗum woni Calb1, Pcp2, Grid2 e Itpr3) (21, 22), kono alaa alɗude poroteyiinuuji keewɗi feeñde e neeruuji walla e sifaaji piɓaaji goɗɗi (Natal 1D) ). Njiylawu hakkunde sampleeji e nder geese YFPhigh ceertuɗe mooftaaɗe e jarribooji keertiiɗi hollirii koyeera korrelaasiyoŋ> 0.9, kolliroowo jibinannde moƴƴere hakkunde rewtinnde biyoloji (Natal S1E). E raɓɓiɗinaade, ɗeeɗoo dokke ina kollita peeje men ngam seerndude PN baawɗo waɗeede oo. Sabu mbaydi difoore L7-cre huutorteende ina addana mosaïque mbayliigu e nder yontere adannde caggal jibinannde (23), min puɗɗiima ustude ɓoccooɗe ummoraade e CTRL e sarɗiiji (Mfn2 loxP / loxP :: mtYFP loxP-stop-loxP :: L7-cre) Mooftude neronuuji. Caggal nde mbayliigu nguu timmi, ngu wiyetee ko Mfn2cKO e nder yontere 4. Ko ɗum woni toɓɓere joofnirde, min cuɓii ko yontere 8 nde laral PN ngal woni e ɓuuɓde hay so tawii ko peccitagol mitokondriyeeji laaɓtuɗi (Natal 1B e Natal S1A). E nder ɗuum, min limtii fotde 3013 poroteyiin, heen fotde 22% tuugii ko e annotaaji MitoCarta 2.0 tuugiiɗi e poroteyiin mitokondriyeeji hono mitokondriyeeji (Natal 1E) (Natal 1E) (24). Anndugol baylagol taƴe ceertuɗe waɗaangol e yontere 8 ndee hollitii wonde ko 10,5% tan e poroteyiinuuji ɗii fof njogii waylooji mawɗi (Natal 1F e Natal S1F), tawi heen 195 poroteyiin ina njiyloo les, 120 ina njiyloo dow (Natal 1F). Ina teskaa wonde « ƴeewndo laabi kesi » e ndeeɗoo fedde doosɗe ina hollita wonde taƴe ceertuɗe peeñɗe ɗee ɓuri jeyaa ko e fedde keeriinde e laabi mbayliigu keertiiɗi (Natal 1G). Ko ɓuri haawnaade heen, hay so tawii dowlaaji jowitiiɗi e OXPHOS e baylagol kalsiyum ina tabitina udditgol ŋakkeende golle mitokondriyaal e nder PNs ŋakkuɗi fusiyoŋ, cate goɗɗe ɓurɗe waawde huutoraade mbayliigu asid amino ina ƴellitoo no feewi, ɗum noon ina yahdi e mbayliigu mitokondriyaal conwiring P. ŋakkeende golle.
(A) Fotooji konfokaal wakiliiji taƴe cerebral CTRL e Mfn2cKO kollitooje ŋakkeende PNs (calbindin, gray); nukkuuji ɗii njuumtaama e DAPI. (B) Limto (A) (anndinde feere wootere e mbayliigu, ***P<0.001; n = 4 haa 6 taarik ummoraade e ɓowɗi tati). (C) Golle jarriborɗe. (D) Peccitagol kartal nguleeki maandeeji keertiiɗi e Purkinje (dow) e sifaaji cellal goɗɗi (hakkunde). (E) Natal Venn hollirngal limoore poroteyiin mitokondriyeeji cifaaɗi e nder PN renndinaaɗo oo. (F) Volcano plot e poroteyiinuuji peeñɗi e nder newon Mfn2cKO e nder yontere 8 (nafoore taƴnde nafoore 1,3). (G) Anndugol laawol tagoore ina hollita laabi joy ɓurɗi teeŋtude dow (boɗeeji) e les (bulo) e nder Mfn2cKO PN renndinaaɗi e yontereeji 8. Hakindo tolno baylagol kala poroteyiin yiytaa ina hollitee. Kartal nguleeki grayscale : teddeendi P toɗɗaandi. ns, alaa ko nafata.
Dowlaaji poroteyiin kollitii wonde baylagol poroteyiin e nder komplekseeji I, III, e IV ina ustoo seeɗa seeɗa. Kompleks I, III, e IV fof ina njogii ɓuuɓri gonndi e mtDNA, tawi noon kompleks II, gonɗo e ɓuuɓri nukliyeer tan, ko ɓuri heewde e mum alaa ko battini heen (Natal 2A e Natal S2A). . E tuugnaade e njeñtudi proteomik, immunohistokimikaaji taƴe ƴiye cerebellar hollitii wonde tolno MTCO1 (mitokondriyaal sitokrom C oksidaas subunit 1) subunit kompleks IV e nder PN ina ustoo seeɗa seeɗa (Natal 2B). Subunit Mtatp8 mo mtDNA-koddi oo ustiima no feewi (Natal S2A), tawi noon tolno steady-state mo subunit ATP sintase mo nukliyeer oo waylaaki, ɗum noon ina yahdi e mbaydi F1 anndaandi e ATP sintase gonndi e mbaydi gonndi e ɓuuɓri so tawii baylagol mtDNA ina dartii. Sosngo ngoo ina yahdi. Taƴu (7). Ƴeewndo tolno mtDNA e nder PNs Mfn2cKO sortaaɗi ɗii e mbaydi mbayliigu (qPCR) hollitii ustaare seeɗa-seeɗa e limoore koppiiji mtDNA. So en tuugniima e fedde toppitiinde ndee, e nder yontere 8, ko hedde 20% tan e tolno mtDNA Was heddii (Natal 2C). E tuugnaade e ɗeen geɗe, ƴeewndo mikroskoop konfokaal PNs Mfn2cKO huutoraa ngam yiytude ADN, kolliroowo ñaamde nukleotideeji mitokondriyeeji fawaade e sahaa (Natal 2D). En tawii ko won e kanndidaaji tan jeyaaɗi e ustude poroteyiin mitokondriyee e jaabawol stres, ina heen Lonp1, Afg3l2 e Clpx, e geɗe mooɓondiral kuuɓtodinɗe OXPHOS. Alaa fof waylo waylo mawngo yiytaa e toɓɓe poroteyiinuuji baɗɗi e apoptosis (Natal S2B). Ko noon kadi, en njiyii wonde laabi retikulum mitokondriyaaji e endoplasmaaji baɗɗi e yah-ngartaa kalsiyum ina njogii tan mbayliigaaji tokoosi (Natal S2C). Yanti heen, ƴeewndo poroteyiinuuji jowitiiɗi e otofaaji ɗii, alaa ko wayli heen, ɗum noon ina yahdi e ƴeewndo yiyngo otofagosomeeji yiytaaɗi e nder nguurndam (in vivo) e immunohistochemistry e mikroskopi elektron (Natal S3). Kono tan, ŋakkeende OXPHOS yahrude yeeso e nder PN ina yahdi e mbayliigaaji mitokondriyeeji ultrastructural laaɓtuɗi. Kosam mitokondriyaal ina waawi yiyeede e ɓalli cellal e leɗɗe dendritik Mfn2cKO PNs duuɓi 5 e 8, kadi mbaydi ɓalndu ndernderiiru waɗii waylooji luggiɗɗi (Natal S4, A e B). E tuugnaade e ɗeen mbayliigaaji ultrastructural e ustaare mawnde e mtDNA, ƴeewndo ƴiye cerebral cerebral acute e tetrametilrodamin metil ester (TMRM) hollitii wonde mbaawka membrane mitokondriyee e nder PNs Mfn2cKO ina usta no feewi (Natal S4C).
(A) Ƴeewndo sahaa e tolno baylagol kompleks OXPHOS. Ƴeewee tan poroteyiinuuji jogiiɗi P<0.05 e nder yontere 8 (ANOVA laabi ɗiɗi). Laral toɓɓere: Alaa njuɓɓudi so yerondiraama e CTRL. (B) Nano : Yeru taƴre cerebral nde limtaa e antibiotik MTCO1 (barre njuuteendi, 20 μm). Nokku mo ɓalli cellal Purkinje njogii oo ina suddii oole. Ñaamo : Ƴeewndo tolnooji MTCO1 (anndugol feere wootere ; n = 7 haa 20 cellal ƴeewndaaɗe e ɓowɗi tati). (C) ƴeewndo qPCR limoore koppi mtDNA e nder PN sortaa (anndugol feere wootere e mbaydi; n = 3 haa 7 ɓowɗi). (D) Nano : Yeru ƴiiƴam cereeli gonɗam e ƴiiƴam gonɗam e ADN (barre échelle, 20 μm). Nokku mo ɓalli cellal Purkinje njogii oo ina suddii oole. Ñaamo : Ƴeewndogol mborosaaji mtDNA (anndinde mbayliigu laawol gootol; n = 5 haa 9 ƴiiƴam ummoraade e ɓowɗi tati). (E) Yeru taƴre cerebellar akute hollirnde ƴiye mitoYFP + Purkinje (arrow) e nder binndol ƴiye patch clamp. (F) Ƴeewndo IV kurjuru. (G) Winnditooji wakilaaji gonɗi e nder ƴiiƴam CTRL e Mfn2cKO Purkinje. Top trace: Pulse gadano mo AP ummini. Tolno les: Ɓuri heewde e AP. (H) Ƴeewndogol naatgol gonngol e hoore mum (sPSPs). Trace winnditorde wakiliijo e ratio zoom mum kolliraama e (I). Anndugol feere wootere e mbaydi anndinde n = 5 haa 20 cellal ummoraade e ɓowɗi tati. Dataaji ɗii kolliraama ko no mbaydi ± SEM ; *P<0,05; **P<0,01 ko; ***P<0,001. (J) Batte AP gonɗe e hoore mum mbinndaaɗe e kuutoragol mbaadi jokkere enɗam perforated. Top trace: Ko ɓuri heewde e AP. Toɓɓere les: zoom AP gooto. (K) Limto AP hakkundeejo e ɓurɗo mawnude fawaade e (J). Teskuyaaji Mann-Witney; n = 5 cellal ƴeewtaama e nder ɓowɗi nay. Dataaji ɗii kolliraama ko no mbaydi ± SEM ; wonaa huunde himmunde.
Bonannde OXPHOS laaɓtunde yiytaa ko e Mfn2cKO PN mo duuɓi 8, ko ɗum hollirta wonde golle fijirde neeruuji ina ŋakki no feewi. Ko ɗum waɗi, min ƴeewtindorii ko sifaaji kuuɓtodinɗi (passifs) gonɗi e neeruuji ɗi ngalaa OXPHOS e nder yontere 4 haa 5 e yontere 7 haa 8, e waɗde binnditagol patch clamp (patch clamp) e nder ƴiye cerebellar acute (Natal 2E). Ko sikkanooka, cakkital mbaawka membrane fooftere e reentaade naatgol neeruuji Mfn2cKO ina nanndi e ƴeewndo, hay so tawii noon ina waɗi ceertugol seeɗa hakkunde ƴiye (Tablo 1). Ko noon kadi, e yontere 4 haa 5, alaa fof waylo waylo mawngo tawaa e jokkondire hakkunde doole e doole (curve IV) (Natal 2F). Kono tan, alaa neron Mfn2cKO mo duuɓi 7 haa 8 wuuri e mbaydi IV (taƴre hiperpolarisaasiyoŋ), ko ɗum hollirta wonde ina woodi sensitiviteeji laaɓtuɗi e mbaawka hiperpolarisaasiyoŋ e ooɗoo sahaa cakkitiiɗo. E ko feewti heen, e nder neronuuji Mfn2cKO, doole depolarizing (depolarizing currents) baɗɗe ɓuuɓol potngol huutoreede (AP) ina muña no feewi, ko ɗum hollirta wonde ɓuuɓol mum en fof seerndaani no feewi e ɓuuɓol neronuuji toppitiiɗi jonte 8 (Tablo 1 e Natal 2G). Ko noon kadi, keewal e amplitude doole guurɗe (sPSCs) ina nanndi e ɗe fedde toppitiinde ndee, tee keewal kewuuji ɗii ɓeydiima gila e yontere 4 haa yontere 5 haa yontere 7 haa yontere 8 e ɓeydagol nanndungol (Natal 2, H e I). Mudda ɓuuɓgol sinapseeji e nder PNs (25). Ko nanndi e ɗuum, njeñtudi keɓaama caggal paɗe PNs perforated. Ndeeɗoo feere haɗata ko yoɓde ŋakkeende ATP cellal, hono no waawi waɗde e binndol patch clamp cellal fof. Haa teeŋti noon, mbaawka membrane fooftere e keewal firo gonndi e neeruuji Mfn2cKO ɗii, mettinaani (Natal 2, J e K). E tonngol, ɗeeɗoo geɗe kollitii wonde PN-ji jogiiɗi ŋakkeende OXPHOS laaɓtunde ina mbaawi daɗde e mbaydiiji ɓuuɓɗi toowɗi no moƴƴi, ko ɗum hollirta wonde ina woodi feere mbayliigu baawnde rokkude ɗum en jaabawuuli elektorofisiyoloji ɓadtiiɗi no feewi.
Dowlaaji ɗii kolliraama ko no mbaydi ± SEM (anndinde mbayliigu laawol gootol, jarribo ƴeewndo keewngo Holm-Sidak; *P<0.05). Limre yuɓɓo ngoo hollirtee ko e jokkorgal.
En puɗɗiima wiɗtude so tawii kala cate e nder doggol dokke proteomics (Natal 1G) ina waɗi laabi baawɗi haɓaade ŋakkeende OXPHOS tiiɗnde, tawa noon ina paamnina ko waɗi PN toɗɗaaɗo oo ina waawi jogaade elektorofisiyoloji ɓadiiɗo no woorunoo (Natal 2, E haa K). . Annduɓe proteomik kollitii wonde enzimuuji baɗɗi e katabolism asideeji amino cakkitiiɗi (BCAA) ina keewi ƴellitaade (Natal 3A e Natal S5A), tee geɗel cakkitiingel ngel asetil-KoA (CoA) walla suksinil CoA ina waawi ɓeydude trikarboksilaat e nder arteriosklerosis). En tawii wonde ko woni e nder BCAA 1 (BCAT1) e BCAT2 fof ina ɓeydoo. Ɓe kataliis daawal gadanal katabolism BCAA e feewnude glutamat ummoraade e α-ketoglutaraat (26). Denndaangal ɓuuɓri gonndi e ɓuuɓri keto asid dehidrogenase (BCKD) ina njuɓɓinee (koolondiral ngal ina kataliisa dekarboksilaasiyoŋ caggal mum mo waylaaki e ƴiye karbon BCAA keɓtinaaɗe ɗee) (Natal 3A e Natal S5A). Kono tan, alaa waylooji laaɓtuɗi tawaa e BCAA e hoore mum e nder PN sortuɗo oo, ina waawi tawa ko sabu ɓeydagol ƴellitaare cellal ɗeeɗoo asideeji aminooji baɗɗi faayiida walla huutoraade geɗe goɗɗe (glukos walla asid laktik) ngam ɓeydude cirƴam TCA (Natal S5B). PNs ɗi ngalaa OXPHOS kollitii kadi ɓeydagol golle ɓuuɓgol e mbayliigu glutamin e nder yontere 8, ɗum ina waawi feeñde e ƴellitgol enzim mitokondriyeeji glutaminase (GLS) e glutamin piruwat mbayliigu 2 (GPT2) (Natal 3, A e C). Ina teskaa wonde ƴellitaare GLS ina fawii e isoforme glutaminase C (GLS-GAC) gonɗo e ɓuuɓri (mbayliigu Mfn2cKO/CTRL ina sowoo laabi 4,5, P = 0,05), e ƴellitaare mum keeriinde e nder ƴiye kanseer Waawaa wallitde mitonerjiyondriyaal. (27).
(A) Kartal nguleeki hollirta ko waylo-waylo follere to bannge poroteyiin wonande laawol toɗɗaangol ngol e nder yontere 8. (B) Yeru ƴiiƴam cereeli gonɗam e ƴiiƴam PCx (barre échelle, 20 μm). Ɓoggi daneeji ɗii ina kollita ɓanndu cellal Purkinje. (C) Anndugol baylagol poroteyiin e nder sahaaji, anndaaɗi ko kanndidaa teeŋtuɗo wonande ateroskleroosis (t-test keewɗo, *FDR <5%; n = 3-5 ɓowɗi). (D) Dow : Natal skematik hollitoore laabi ceertuɗi naatgol karbon markeeji gonɗi e nder [1-13C]pyruvate tracer (ɗum woni, rewrude e PDH walla laawol trans-arterial). Les : Natal violon hollirta ko teemedere karbon (M1) mo alaa ko woni e mum so wonaa asid aspartik, asid sitiriik e asid malik caggal nde limtaa e ƴiye cerebellar akute e [1-13C]pyruvate (t-test paired; ** P <0.01). (E) Ƴeewndo daartol sahaa kuuɓtodinngo laawol hollirngol ngol. Ƴeewee tan poroteyiinuuji jogiiɗi P<0.05 e nder yontere 8 . Laral gonngal e ŋorol : alaa nafoore njuɓɓudi (anndinde mbayliigu laabi ɗiɗi; * P <0,05; *** P <0,001). Dataaji ɗii kolliraama ko no mbaydi±SEM.
E nder wiɗto amen, katabolism BCAA wontii gooto e laabi ɓurɗi teeŋtude ngam ƴellitde. Ndeeɗoo hujja ina hollita no feewi wonde ɓuuɓol weeyo naatngol e cirƴam TCA ina waawi waylaade e PN mo alaa OXPHOS. Ɗum ina waawi wonde sifaa mawɗo mbayliigu mbayliigu neeruuji, baawɗo jogaade batte to bannge fijirde neeruuji e nguurndam e nder toppitagol ŋakkeende OXPHOS tiiɗnde. E tuugnaade e ngool miijo, en njiyii wonde enzim PCx mawɗo kaɓtotooɗo aterosklerosis ina ƴellitoo (Mfn2cKO/CTRL ina wayloo fotde laabi 1,5; Natal 3A), ko ɗum addanta waylude piruwat e oksaloasetaat (28), ko ɗum sikkaa ko e nder brain, ko tissucy a2). 30). E tuugnaade e njeñtudi proteomik, mikroskopi konfokaal hollitii wonde baylagol PCx ina ɓeydoo teeŋti e no feewi e nder PNs ŋakkuɗi OXPHOS, tawi noon baylagol PCx ɓuri heewde ko e ƴiye Bergmann glial ɓadiiɗe e ƴeewndo (Natal 3B). Ngam ƴeewde no feewi ƴellitaare PCx yiytaande ndee, min njuurnitii ƴiye cerebellar ɓuuɓɗe e [1-13C]pyruvate tracer. Nde piruwat oksidaa e piruwat dehidrogenase (PDH), mbaydi isotop mum majjii , Kono ina naata e nder hakkundeeji cirƴam TCA nde piruwat wayletee e mbaydiiji ƴiiƴam (Natal 3D). E ballondiral e dokke men proteomik, en teskiima maandeeji keewɗi ummoraade e ooɗoo tracer e nder asid aspartik e nder ƴiye Mfn2cKO, tawi asid sitiriik e asid malik ina njogii kadi mbaydi teskinndi, hay so tawii noon alaa ko nafata (Natal 3D).
E nder neronuuji dopamin ɗi ɓowɗi MitoPark jogiiɗi ŋakkeende golle mitokondriyaal saabiinde neronuuji dopamin teeŋti noon e halkude taƴre A (Tfam) (Natal S6B), baylagol PCx kadi ina ƴellitoo no feewi (31), ko ɗum hollirta wonde asid asetoon ina ustoo e nder mboros oo OXPHOS e nder ɓanndu. Ina teskaa wonde ina teskaa wonde enzim keeriiɗo (32-34) baawɗo feeñde e neronuuji baawɗi jokkondirde e arterioskleroosis ina ɓeydoo ƴellitaade no feewi e nder PNs ɗi ngalaa OXPHOS, ko wayi no karboksilaas propionil-CoA (PCC-A), Malonil-CoA mitochilCoA ina wayla propionil-CoA to propionyl-CoA enzim 3 (ME3), mo darnde mum mawnde woni heɓtude piruwat e malaat (Natal 3, A e C) (33, 35). Yanti heen, en njiyii ɓeydagol mawngol e nder enzim Pdk3, fosforilaatoowo, ɗum noon ina ustoo PDH (36), tawi noon alaa ko waylii e enzim Pdp1 ina ustoo PDH walla e kompleks enzim PDH e hoore mum (Natal 3A). E nder ko jokki, e nder PNs Mern2cKO, fosforilaasiyoŋ α1 α (PDHE1α) gonɗo e nder feccere piruwat dehidrogenase E1 e nder kompleks PDH e nder Ser293 (anndiraaɗo haɗde golle enzim PDH) ɓeydaama ( Natal S6C) (Natal S6C). Piruwat alaa ɗo haaɗi e ƴiiƴam.
E joofnirde, en njiyii wonde laawol mawngol biyosintesis serine e glisin, cirƴam folate (1C) mitokondriyee jokkondirɗam e biyosintesis prolin (Natal 1G e Natal S5C) fof ina ƴellitoo no feewi, e fawaade e ciimtol, e nder golle kuutoragol. Ɓowɗi taariiɗi ɗii ina kuutoree e ŋakkeende golle mitokondriyaal (5-7). Anndugol konfokaal ballitoowo ɗeeɗoo dokke proteomik hollitii wonde e nder PN mo OXPHOS ŋakki, ƴiye cerebellaaji ɓowɗi duuɓi 8 ina mbaɗee e serine hidroksimetiltransferase 2 (SHMT2), woni enzim kuuɓtodinɗo e cirƴam folate mitokondriyee. Jaabawol immuno maantinngol (Natal S5D). E nder 13 ƴiye cerebellar acute gonɗe e CU-glucose, jarribooji ƴeewndorɗe mbaylaandi ɓeydiima tabitinde ƴellitaare biyosintesis serin e prolin, ko ɗum hollirta wonde ɓuuɓri isoformeeji karbon e nder serin e prolin ina ɓeydoo (Natal S5E). Nde tawnoo ko GLS e GPT2 njiylotoo koo ina addana sintesis glutamat ummoraade e glutamin e transamination hakkunde glutamat e α-ketoglutarat, upregulation mum en ina hollita wonde neronuuji ɗi ngalaa OXPHOS ina njogii ɓeydagol ɗaɓɓaande glutamat , Ɗum ina waawi wonde fawaade e jokkude ɓeydagol biyosintesis Staureline (Fig5). E ko feewti e ɗeen mbayliigaaji, wiɗto proteomik astrositteeji cerebellar ummoriiɗi e ɓowɗi Mfn2cKO keertiiɗi PN hollitii wonde ɗeen laabi (ina heen denndaangal antiperoksidaaseeji) mbaylaaki no feewi e baylagol, ɗum noon ina hollita Ngolɗoo mbayliigu metabolik ina suɓoo PN mo ŋakkaani (Fig. S6, G6).
E raɓɓiɗinaade, ɗeeɗoo wiɗtooji kollitii sifaaji ceertuɗi no feewi e ƴellitgol sahaa e sahaa fof laabi mbayliigu keertiiɗi e nder PNs. Hay so tawii golle mitokondriyaal neuronal ɗe ngonaa no haanirta nii ina mbaawi addande ateroskleroosis gadano e mbayliigu 1C (Natal 3E e Natal S5C), e hay waylooji teskaaɗi e baylagol komplekseeji I e IV, waylooji e sintesis serine de novo ko tan Ko ɗum tan feeñi e sta gees cakkitiiɗo. OXPHOS ina ŋakki golle (Natal 3E e Natal S5C). Ɗee geɗe ina cifa peeje jokkondire ɗe mitokondriyeeji (cirko 1C) e sitoplasma (biosintesis serine) baɗɗi stres njaabotoo e nder nanondiral e ɓeydagol aterosklerosis e nder cirko TCA ngam waylude mbaadi mbayliigu neeruuji.
PNs ŋakkuɗi OXPHOS duuɓi 8 ina mbaawi jogaade golle ɓuuɓɗe toowɗe, kadi ina mbaawi jogaade jokkondire keewɗe e nder ɓalndu ngam ustude ŋakkeende golle mitokondriyaal. Ndee yiytunde ina addana en miijaade wonde hay e oo sahaa, ɗeen ceŋorɗe ina mbaawi kadi Heɓde ballal safaara ngam leeltinde walla haɗde mboros oo. Maynama. Min ndartinii ndee feere e nder golle ɗiɗi ceertuɗe. E nder feere adannde ndee, min mbaɗii mbaydi mboros (AAV) jokkondirɗo e adeno (adeno) fawaade e Cre, mbele MFN2 ina waawa feeñde e PNs ŋakkuɗi OXPHOS e nder nguurndam (Natal S7A). AAV koddiroowo MFN2 e taƴre kabaaru mCherry (Mfn2-AAV) hoolkisaama e nder pine neeruuji adanɗi e nder geese, ko ɗum addani MFN2 feeñde e mbaadi fawaande e Cre, kadi hisna morfolooji mitokondriyeeji, ɗum noon haɗde neeruuji Mfn, D Figure S7). Ko adii ɗuum, min mbaɗii jarribooji in vivo ngam stereotaktik yettinde Mfn2-AAV mo jonte 8 e nder ɓalndu Mfn2cKO e ɓowɗi toppitiiɗi, min ƴeewtindii ɓowɗi jonte 12 (Natal 4A). Miijooji Mfn2cKO safrooɓe ɓee maayii (Natal 1, A e B) (16). Ko waylude mboros e nder nguurndam (in vivo) addani PN feeñde e won e cirkooji cerebellar (Natal S7, G e H). Injection AAV ƴeewndotooɗo holliroowo tan mCherry (Ctrl-AAV) alaa ko battini e tolno ɓuuɓri ƴiiƴam e nder daabaaji Mfn2cKO. E ko feewti heen, wiɗto Mfn2cKOs mbaylaaɗi e Mfn2-AAV hollitii batte ndeenka mawɗe e laral cellal PN (Natal 4, B e C). Haa teeŋti noon, denndaangal neeruuji ɗii ina nanndi e ko famɗi fof ko seerndaani e daabaaji toppitiiɗi ɗii (Natal 4, B e C, e Natal S7, H e I). Feññinde MFN1 kono wonaa MFN2 ina foti kadi wallitde e hisnude maayde neeruuji (Natal 4C e Natal S7, C e F), ko ɗum hollirta wonde feeñninde MFN1 ektopik ina waawi ɓeydude no feewi ŋakkeende MFN2. Ko adii fof, wiɗtooji goɗɗi e tolno PN gooto kollitii wonde Mfn2-AAV ina hisna no feewi ultrastructure mitokondriyaaji, ina normalisee tolnooji mtDNA, ina wayla baylagol toowngol maande PCx (Natal 4, C haa E ). Ƴeewndo yiyngo ɓowɗi Mfn2cKO kisnaaɗi ɗii e nder ngonka fooftere hollitii wonde darnde mum en e maale mooftugol mum en (dille S1 haa S3) ina moƴƴi. E joofnirde, ɗeeɗoo jarribooje kollitii wonde leeltude naatgol MFN2 e nder PNs ŋakkuɗi no feewi e OXPHOS ina heddinoo ngam waylude ñaamde mtDNA e saabaade ateroskleroosis, ɗum noon haɗde ŋakkeende akson e maayde neeruuji e nder nguurndam.
(A) Feere hollitoore doggol jarriborgol ngam innirde AAV koɗki MFN2 so laawol mbayliigu holliraaɗo ngol huutoraama. (B) Nate konfokaal wakiliije ƴiye cerebellar duuɓi 12, mbaylaaɗe e yontere 8 e nder ɓowɗi Mfn2cKO, ɗe limtaaɗe e antibiotik anti-Calbindin. Ñaamo: Ƴeewndo fiɓnde akson. Njaajeendi zoom akson ko 450 e 75 μm. (C) Nano : Ƴeewndo njuuteendi ƴiiƴam Purkinje e nder loowdi mbayliigu AAV (AAV+) (anndinde mbayliigu laawol gootol; n = 3 ɓowɗi). Ñaamo : ƴeewndo focus mtDNA e nder PN mbayliigu e yontere 12 (t-test mo alaa ko jokkondiri; n = 6 cellal ummoraade e ɓowɗi tati). * P <0,05; ** P <0,01. (D) Mikrogaraamuuji elektoronik gonɗi e jolngo PNs taƴe cerebellar Mfn2cKO jolnaaɗe e jolngooji viral kollitaaɗi ɗii. Mask roosa oo ina hollita nokku mo dendriteeji ɗii njogii, e kaaƴel toɓɓere geese ina hollita zoom dokkaaɗo to bannge ñaamo oo ; n ina hollira ɓuuɓri. Barme njuuteendi, 1μm. (E) hollitii yeru PCx mbayliigu e nder PN mbayliigu e nder yontere 12. Barme njuuteendi, 20μm. OE, ko ɓuri heewde e haala; FC, waylo folde.
E joofnirde, min njuurniima nafoore nguurndam celule peroksidaas e nder PNs keɓtuɗe ŋakkeende OXPHOS. En peewnii mCherry koddiroowo AAV-shRNA (RNA ɓuuɓɗo) mo teeŋti noon e mRNA PCx (AAV-shPCx) peƴooji, en mbaɗii mboros oo walla ƴeewndo mum ɓuuɓngo (AAV-scr) e nder ɓalndu peƴooji Mfn2cKO. Injection oo waɗiraa ko e yontere nayaɓere duuɓi (Natal 5A) ngam heɓde PCx knockdown moƴƴo e nder sahaa mo PCx ɓeydotoo (Natal 3C) tawa laral cellal PN ina heddii haa jooni (Natal 1A). Ina teskaa wonde ustude PCx (Natal S8A) ina addana maayde PN yaawde no feewi, tawi noon ko e nder cirƴam ɗam (Natal 5, B e C). Ngam faamde no batte mbayliigu PCx ƴellitortoo, min njanngii ngonka redox PNs caggal nde PCx yani e redox biosensor optik mo AAV-mediated Grx1-roGFP2 hollitaama e sahaa gooto (Natal S8, B haa D) ngam ƴeewde glutathione potential of3). Ndeen, min mbaɗii mikroskoop ƴeewndo nguurndam (FLIM) e nder ƴiye ngaandiiji ɓuuɓɗi Mfn2cKO mo jonte 7 walla littermates ƴeewndo ngam yiytude waylooji baawɗi wonde e ngonka redox sitoplasma caggal nde min hoolkisii ngonka FLIM (Natal S8, E haa G). Njiylawu oo hollitii ɓeydagol mawngol e ngonka oksidaasiyoŋ PN gooto Mfn2cKO mo alaa baylagol PCx, ko ɗum seerti e neronuuji toppitiiɗi walla PN Mfn2cKO bayyinoowo shRNA ɓuuɓɗo tan (Natal 5, D e E). Nde baylagol PCx ngol ustaa, keewal PNs Mfn2cKO kollitooji ngonka oksidaa toowka ɓeydiima ko ɓuri laabi tati (Natal 5E), ko ɗum hollirta wonde PCx dow-dow ina jokki mbaawka redox neronuuji ɓuuɓɗi.
(A) Feere hollitoore doggol jarriborgol ngam innde AAV koɗki shPCx nde laawol mbayliigu hollirteengol ngol huutoraa. (B) Fotooji konfokaal wakiliiji taƴe cerebellar duuɓi 8 e nder ɓowɗi Mfn2cKO mbaylaaɗi e limtaaɗi e antibiotik anti-kalsineurin e nder yontere 4. Barme njuuteendi, 450μm. (C) Ƴeewndogol njuuteendi ƴiiƴam Purkinje e nder loopeeji baylaaɗi AAV (anndinde mbayliigu laawol gootol; n = 3 haa 4 ɓowɗi). Dataaji ɗii kolliraama ko no mbaydi ± SEM ; ***P<0,001. (D) Natal FLIM wakiliijo hollirta ko tolno nguurndam PN mo yontere 7 holliroowo sensor redox glutathione Grx1-roGFP2 e nder sarɗiiji jarribo toɗɗaaɗi ɗii. LUT (tablo ƴeewndo) njuuteendi : hakkunde waktuuji nguurndam (e pikosekon). Barme njuuteendi, 25μm. (E) Histogram ina hollita peccitagol teddeendi nguurndam Grx1-roGFP2 gila e (D) (n=158 haa 368 ƴiiƴam e nder ɓowɗi ɗiɗi e nder kala ngonka). Natal piye dow kala histogram : hollirta ko limoore celule jogiiɗe teddeendi nguurndam ɓurndi juutde (boɗeeri, oksidaa) walla ɓurndi juutde (bulo, ustaare) no feewi, ɓurndi 1 SD e teddeendi nguurndam hakkundeeri e nder CTRL-AAV-scr. (F) Model gonaaɗo oo ina hollita batte ndeenka upregulation PCx neuronal.
Ko ɗum fof e wayde noon, dokke ɗe min ndokki ɗoo ɗee ina kollita wonde feeñninde MFN2 ina waawi hisnude haa timma PN jahruɗo yeeso e ŋakkeende OXPHOS tiiɗnde, ŋakkeende mtDNA tiiɗnde, e morfolooji nannduɗi e ista ɗi ngalaa no feewi, ɗum noon ina addana ɗum yahrude yeeso jokkondirnde hay e nder ñawuuji jahruɗi yeeso. Neurodegeneration ina rokka seedeeji mbayliigu e daawal hade maayde cellal. Ndeeɗoo tolno ɓuuɓri mbayliigu ina ɓeydoo teeŋtineede e mbaawka neronuuji ngam saabaade aterosklerosis (rewiring cirƴam TCA), ko ɗum haɗata baylagol PCx e nder PNs ɗi ngalaa OXPHOS, ina ɓeyda maayde ƴiiƴam, ɗum noon ina jogii darnde ndeenka (Natal 5F).
E nder ndeeɗoo wiɗto, min ndokkii seedeeji wonde jaabawol PNs e ŋakkeende OXPHOS ko jokkondirde seeɗa-seeɗa e aterosklerosis cirƴam TCA rewrude e laawol gonngol e mbaydi gonndi e porogaraamuuji mbayliigu. En tabitinii ƴeewndo proteomik ngoo e laabi keewɗi timmuɗi, en peeñninii wonde so tawii ko ŋakkeende mitokondriyaal tiiɗnde, neeruuji ina njogii sifaa ɓuuɓɗo mo anndaaka tawo. Haawnaaki tawa golle rewiring ɗee fof, alaa e sago maande ngonka mbaylaandi (metabolisme) timmungal, tawa ina yahdi e neurodegeneraasiyoŋ seeɗa-seeɗa tawa ina waawi waylude, kono doosɗe men ina kollita wonde ina waawi wonde neuron toppitiiɗo hay e nder daawal hade maayde cellal Mekanisme compensation fonctionnel. Ndeeɗoo yiytunde ina hollita wonde neeruuji ina njogii tolno keewɗo e nder ɓanndu (plastisite métabolique). Ngolɗoo laawol ina hollita wonde naatgol MFN2 caggal ɗuum ina waawi waylude baylagol maandeeji mbayliigu teeŋtuɗi, haɗde PN ustaade. Ko wonaa ɗum koo, ina haɗa ateroskleroosis, ina yaawi neruuji. transsexual.
Ina jeyaa e ko ɓuri teeŋtude e ko min keɓi e wiɗto amen, wonde PNs ɗi ngalaa OXPHOS ina mbaawi waylude mbaydi mbaylaandi (métabolisme) e nder cirƴam TCA, tawa ina kuutoroo enzimuuji baawɗi ƴellitde ƴiiƴam (arteriosclerosis) teeŋti noon. Reentaade mbayliigu ko huunde woowaande e nder geese kanseer, won heen tuugii ko e glutamin ngam ɓeydude hakkundeeji cirƴam TCA ngam feewnude ko nanndi e ustooje, ko ɗum addanta jokkere enɗam foofaandu e jogaade peewnugol prekursoruuji biyosintesis lipid e nukleotiid (39 , 40). Wiɗto sakkitiingo hollitii wonde e nder ƴiye periferik keɓtinaaɗe e ŋakkeende OXPHOS, jokkondire hakkunde mbayliigu glutamine/glutamate kadi ko huunde maantiniinde (5, 41), ɗo feere naatgol glutamine e nder cirƴam TCA fawii ko e Sabu tiiɗnaare ñawu OXPHOS (41). ). Kono tan, ŋakkeende hujjaaji laaɓtuɗi ina ngoodi ko fayti e kala nanndugol e mbaydi mbaylaandi (plasticité métabolique neuronale) e nder ɓanndu e no ɗum waawi jogaade batte e nder ngonka ñawu nguu. E nder wiɗto sakkitiingo e nder geese (in vitro), kolliraama wonde neronuuji kortikol adanɗi ina mbaawi mooftude poolguuji glutamate ngam ƴellitde neeruuji, ɗum noon ina ɓeyda mbayliigu oksidaaji e ateroskleroosis e nder ngonkaaji teskinɗi mbayliigu (42). Ina teskaa wonde e les njiimaandi farmakoloji enzim suksinaat dehidrogenaas cirƴam TCA, ina sikkaa karboksilaasiyoŋ piruwat ina jogoo sintesis oksaloasetat e nder neronuuji ƴiiƴam cerebellar pinal (34). Kono tan, jokkondire fisiyoloji ɗeen peeje e ƴiye ngaandi (ɗo aterosklerosis ɓuri siforaade ko e astrositteeji) ina njogii haa hannde nafoore fisiyoloji teeŋtunde (43). E nder ɗuum, dokke men ina kollita wonde PNs bonɗi e OXPHOS e nder ɓanndu ina mbaawi wayleede e ustude BCAA e karboksilaasiyoŋ piruwat, ko ɗiin ngoni ƴoogirɗe ɗiɗi mawɗe ɓeydugol hakkundeeje poolgu TCA. Hay so tawii darnde katabolism BCAA e nder mbayliigu semmbe neeruuji ina miijanoo, ko jiidaa e darnde glutamate e GABA ngam mbayliigu neeruuji (44), haa jooni alaa ko holliti ɗeen peeje e nder nguurndam. Ko ɗum waɗi, ina weeɓi miijaade wonde PN-ji ɗi ngonaa gollotooɗi ina mbaawi ustude otooji mum en e ñaamde hakkundeeji TCA ɗi laawol assimilaasiyoŋ ngol dognata e ɓeydagol aterosklerosis. Haa teeŋti noon, upregulation PCx ina waawi ɗaɓɓude ngam tabitinde ɓeydagol ɗaɓɓaande asid aspartik, ko ɗum miijotoo e nder ɓeydagol celule jogiiɗe ŋakkeende mitokondriyee (45). Kono tan, wiɗto men metabolomik hollirtaa waylooji keewɗi e tolno steady-state asid aspartik e nder Mfn2cKO PNs (Natal S6A), ko ɗum sikkaa ina hollita kuutoragol metabolik ceertuɗo asid aspartik hakkunde ƴiye ɓeydotooɗe e neronuuji caggal mitosis . Hay so tawii noon feere laaɓtunde PCx upregulation e nder neronuuji ɗi ngonaa gollotooɗi e nder nguurndam ina heddii e sifaade, min kollitii wonde ngool jaabawol ɓooyngol ina jogii darnde mawnde e jogaade ngonka redox neronuuji, ko ɗum hollitaama e jarribooji FLIM e dow ƴiye cerebellar. Haa teeŋti noon, haɗde PNs ƴellitde PCx ina waawi addande ɗum ngonka ɓurka oksidaade, kadi ina yaawi maayde cellal. Ko ustude BCAA e karboksilaasiyoŋ piruwat wonaa laabi ngam sifaade ƴiye periferik ŋakkere golle mitokondriyee (7). Ko ɗum waɗi, ina wayi no ko ɗe sifaa ɓurɗo teeŋtude e neeruuji ɗi ngalaa OXPHOS, hay so tawii noon wonaa sifaa tan, ko huunde himmunde e neurodegeneraasiyoŋ. .
Ñawu cerebellar ko ñawu nguu ina heewi feeñde e ataxia, ina heewi bonnude PNs (46). Ndeeɗoo fedde neeruuji ina teskaa e ŋakkeende golle mitokondriyaal sabu ŋakkeende cuɓagol mum en e nder ɓowɗi ina heewi jibinde maale mooftugol keewɗe, baɗɗe ataksi spinocerebellar neɗɗo (16, 47, 48). E wiyde ciimtol, mbaydi mooso transgenic jogiindi taƴre muusnde ina jokkondiri e ataxia spinocerebellar neɗɗo, ina jogii kadi ŋakkeende mitokondriyee (49, 50), teeŋtini nafoore janngude batte ŋakkeende OXPHOS e nder PNPH. Ko ɗum waɗi, ina teskaa wonde ina moƴƴi seerndude e janngude ndeeɗoo fedde neeruuji keertiiɗi. Kono, nde tawnoo PN ina heewi ɓuuɓde e ɓuuɓde, ina waɗi kadi ko famɗi fof e nder denndaangal celule cerebellar, wonande wiɗtooji keewɗi baɗɗi omics, seerndude ɗum en e celule guurɗe ko huunde nde haa jooni ina saɗti. Hay so tawii ina gasa tawa ina gasa tawa ina waawi heɓde ŋakkeende timmunde e nder sifaaji cellal goɗɗi (haa teeŋti e ƴiye mawɗe), min kawrii e ɗaɗol diisnondiral moƴƴal e FACS ngam heɓde limoore neeruuji baawɗi wuurde ngam yuurnitaade proteomiik dowri, e jogaade ɓuuɓri protein toowndi no feewi (fotde 3000 the cerebel the datastingle) (51). E reende nguurndam ƴiye guurɗe, feere nde min ndokki ɗoo ndee ina addana en ƴeewtaade waylooji e nder laabi mbayliigu e nder mitokondriyaaji, kono kadi ƴeewtaade waylooji e nder mbaydiiji mum sitoplasmaaji, ko ɗum timminirta huutoraade tagɗe membrane mitokondriyaal ngam alɗinde sifaa cellal forx tissumber michond (52 e 53). Laawol ngol min cifotoo ngol, jokkondirtaa tan e wiɗto ƴiye Purkinje, kono ina waawi huutoreede e kala sifaa ƴiye ngam safrude waylooji mbaylaandi e nder ngaandiiji ñawɗi, haa arti noon e mbaydiiji goɗɗi gonɗi e ŋakkeende golle mitokondriyeeji.
E joofnirde, en njiytii windo safaara e nder ndeeɗoo feere mbayliigu mbaylaandi, baawɗo waylude haa laaɓa cer maale teeŋtuɗe gonɗe e ŋakkere cellal, haɗde ŋakkere neeruuji. Ko ɗum waɗi, faamde batte golle rewiring sifotooɗe ɗoo ɗee ina waawi addande en humpitooji keewɗi ko fayti e safaaraaji baawɗi wonde ngam hisnude nguurndam neeruuji e nder sahaa ŋakkeende golle mitokondriyaal. Wiɗtooji garooji baɗɗi faayiida ngam ƴeewde waylooji mbayliigu semmbe e nder sifaaji cellal ngaandi goɗɗi ina njiɗi feeñninde no feewi kuutoragol ndeeɗoo feere e ñawuuji goɗɗi gonɗi e nder ɓalndu.
Miijooji MitoPark ina cifaa ko adii (31). Peƴooji C57BL/6N jogiiɗi taƴe Mfn2 gonɗe e loxP ina cifaa ko adii (18) e peƴooji L7-Cre (23). Ndeen ɓiɗɓe ɗiɗo heterozigoteeji keɓtinaaɗi ɗii, njiytiraa ko ɓowɗi Mfn2loxP/Mfn2loxP ngam heɓde mbaydiiji taƴe Purkinje keertiiɗe wonande Mfn2 (Mfn2loxP/Mfn2loxP; L7-cre). E nder feccere jokkere enɗam, allele Gt (ROSA26) SorStop-mito-YFP (stop-mtYFP) naatnaa e jokkondire ɓeydooje (20). Denndaangal laabi daabaaji mbaɗaama e fawaade e kuule Orop, ngenndiije e juɓɓule, ɗe LandesamtfürNatur mo Umwelt e Verbraucherschutz, to Rhein-Westfalie worgo, to Almaañ njaɓi ɗum en. Golle daabaaji kadi ina njokki e yamiroore Fedde Oropnaare Fedde Ganndal Daabaaji Laboratoori.
Caggal nde ƴiiƴam debbo reedu oo ɓuuɓnaa, ɓiɗɗo mooso oo seertinee (E13). Korte ɗee ƴettaa ko e ɓuuɓri Hanks (HBSS) ɓeydaandi e Hepes 10 mM, rewnaa e ɓuuɓri Dulbecco mo papain (20 U/ml) e sisteyin (1 μg/ml). Ƴeewee ƴiiƴam ɗam e nder DMEM) e seerndude ɗum e ɓuuɓri enzimatik. Ml) e 37°C fotde hojomaaji 20, caggal ɗuum ɓuuɓna ɗum e DMEM ɓeydaaɗo e 10% e ƴiiƴam nagge ɓiɗɗo. Piɓaaji ɗii njibinaama e dow ɓuuɓri gilaas ɓuuɓndi polilisiin e ɓuuɓri 2×106 e kala diƴƴe pinal 6 cm walla e ɓuuɓri 0,5×105 piɓaaji/cm2 ngam ƴeewndaade natal. Caggal waktuuji 4, ɓuuɓri ndii lomtinaama e ɓuuɓri Neurobasal ndi alaa serum, ndi 1% e ɓeydagol B27 e 0,5 mM GlutaMax. Ndeen neeruuji ɗii njooɗiima e 37°C e 5% CO2 e nder jarribo ngoo fof, ina ñamlee laawol gootol e nder yontere. Ngam addande mbayliigu e nder geese, 3μl (diƴƴe pinal 24-ɓundu) walla 0,5μl (ɓundu 24-ɓundu) e mboros AAV9 gonɗo les oo huutoraa ngam safrude neeruuji ñalnde ɗiɗmere e nder geese : AAV9.CMV.PI.eGFP. WPRE.bGH (Addgene, limoore catal 105530-AAV9) e AAV9.
ADN timmuɗo Mfn1 e Mfn2 mooso (keɓaaɗo e pilasmid Addgene #23212 e #23213, no mbaydiiji ɗii fof) ina maantiniree mbaydi V5 (GKPIPNPLLGLDST) to C-terminus, ina jokkondiri e mCherry e nder kaɓirgal rewrude e mbaydi T2A. Grx1-roGFP2 ko dokkal ummoraade e TP Dick DFKZ (Centre wiɗtooji Almaañ). E lomtinande kaset tdTomato e kuutoragol laabi tafngo gaadoraaɗi, kaset oo taƴaa e nder ɓalndu pAAV-CAG-FLEX-tdTomato (limre tuugorgal Adgene 28306) ngam feewnude pAAV-CAG-FLEX-mCherry-T2A-MFNGAV-V5, pAAV-CAG-FLEX-tdTomato. Ɓoornugol pAAV-CAG-FLEX-Grx-roGFP2. Ko nanndi e ɗuum, feere huutoraa ngam feewnude mbaydi pAAV-CAG-FLEX-mCherry. Ngam feewnude konngol AAV-shPCx, ina haani jokkorgal AAV (VectorBuilder, pAAV [shRNA] -CMV-mCherry-U6-mPcx- [shRNA#1]), ngal ina waɗi jokkorgal ADN koɗki shRNA gonɗo e PCx mooso (5′CTAGCTAGGTGTGCTACTACTA 3′) E les njiimaandi ƴellitoowo U6, mCherry ina huutoree e les njiimaandi ƴellitoowo CMV. Pewnugol vektooruuji AAV ballitooji ɗii waɗiraa ko no peewnoowo oo yamiri nii (Cell Biolabs). E raɓɓiɗinaade, huutoroo pilaasmid mbayliigu jogiiɗo mCherry-T2A-MFN2-V5 (pAAV-CAG-FLEX-mCherry-MFN2-V5), mCherry-T2A-MFN1-V5 (pAAV-CAG-FLEX-mCherry) e nder sahaa gooto, mbayliigu cellal 29MFC-MHEAV2-2 (pAAV-CAG-FLEX-mCherry) walla Grx-roGFP2 (pAAV-CAG-FLEX-Grx-roGFP2) taƴre koɗki, kam e koɗki poroteyiin kapsid AAV1 e poroteyiin kuutortooɗo Pilasmid pilasmid paaspoor, kuutortooɗo feere fosfaat kalsiyum. Virus ɓuuɓɗo oo heɓaama e cirkooji ɓuuɓɗi-ɓuuɓɗi e nder lootorde glaas/etanol yoornde e ƴiye limtaaɗe e nder saline tampon fosfaat (PBS). Vektor AAV oo laaɓtinaama e ɓuuɓri gonndi e ɓuuɓri gonndi e ɓuuɓri (waktuuji 24 e 32 000 rpm e 4°C) e ɓuuɓri gonndi e ɓuuɓri Amicon ultra-15. Titer genome AAV1-CAG-FLEX-mCherry-T2A-MFN2-V5 [2,9×1013 koppi genome (GC)/ml], AAV1-CAG-FLEX-mCherry (6,1×1012 GC/ml), AAV1-CAG-FLEX-mCherry (6,1×1012 GC/ml), AAV1-CAG-FLEX limtilimtinɗo (PCR) ina fotnoo ƴeewteede no fotiri nii (qPCR) -MFN1-V5 (1,9 × 1013 GC/ml) e AAV1-CAG-FLEX-Grx-roGFP2 (8,9 × 1012 GC/ml).
Neronuuji gadani ɗii njuppaa e PBS 1x ɓuuɓɗo, njuppaa, caggal ɗuum njuppaa e 0,5% Triton X-100 / 0,5% deoksikolaat sodium/tampon lisiyoŋ PBS ina waɗi fosfataas e ɓuuɓri protease (Roche). Ko limtugol poroteyiin ngol waɗiraa ko e ƴeewndo asid bisinkonik (Thermo Fisher Scientific). Ndeen poroteyiin oo seerndaama e elektoroforesis gel SDS-poliakrilamid, caggal ɗuum ɓuuɓnaa e dow ɓuuɓri poliviniliden fluoriid (GE Healthcare). Uddude nokkuuji ɗi ngonaa keertiiɗi e ɓuuɓnude e antibiotik gadano oo (ƴeew alluwal S1 ngam humpitaade) e nder 5% kosam e nder TBST (saline Tris-buffered e Tween), laabi loowdi e antibiotik ɗiɗaɓo e nder TBST Incubate. Inkubaasiyoŋ e antibiotik gadano jamma e +4°C. Caggal nde lootaa, waɗ heen antibiotik ɗiɗaɓo oo fotde waktuuji 2 e nguleeki suudu. Caggal ɗuum, e ɓuuɓnude oon nokku gooto e antibiotik anti-β-actin, ko noon kadi loowdi ndii tabitinaa. Yiytude ɗum e waylude ɗum e kemikal e ɓeydude kemikal (GE Healthcare).
Neronuuji ɗi aawnanoo ko adii e dow ɓuuɓri gilaasi ɗii, ndartinaama e 4% paraformaldehiid (PFA)/PBS e sahaa toɗɗaaɗo oo e nguleeki suudu fotde hojomaaji 10. Ko adii fof, ɓuuɓri ndii ina ɓuuɓnee e 0,1% Triton X-100/PBS fotde hojomaaji 5 e nguleeki suudu, caggal ɗuum e nder tampon ɓuuɓɗo [3% albumin serum bovine (BSA)/PBS]. Ñalnde ɗiɗmere ndee, ɓuuɓri ndii loowaa e tampon ɓuuɓɗo, ɓuuɓnaa e antibiotik ɗiɗaɓo mo fluorophore-conjugué potɗo, fotde waktuuji 2 e nguleeki suudu ; e joofnirde, ƴeewndorɗe ɗee loowaa no feewi e PBS e 4′,6-diyamidino-2 -Fenilindol (DAPI) ina ɓuuɓna, caggal ɗuum ina fiyree e dow laylaytol mikroskoop e Aqua-Poly/Mount.
Miijooji (gorko e debbo) ina njuurnitee e nder reedu, ina njuutee e ɓuuɓri ketamin (130 mg/kg) e xilazin (10 mg/kg) e nder reedu, ina njuutee e ɓuuɓri karprofen (5 mg/kg) , ina mbaɗee e nder kuutorgal stereotaktik (Kopd a pamf) war equi. Feññinde ɓalndu nduu, huutoroo ƴulɓe dental ngam ɓuuɓnude feccere e ɓalndu nduu ko nanndi e ƴiyal mis (gila e lambda : wutte 1,8, lateral 1, ko nanndi e lobule IV e V). Huutoroo ƴulɓe siriŋ ɓuuɓɗe ngam waɗde e hakkille ɓuuɓol tokosol e nder ɓalndu nduu ngam waasde bonnude ƴiiƴam les ɗam. Ndeen kapilaar gilaas ɓuuɓɗo mo ɓuuɓnaaka oo, ina waɗee seeɗa seeɗa e nder ɓuuɓri mikroo (tuggi -1,3 haa -1 to bannge ventral dura mater), 200 haa 300 nl AAV ina waɗee e nder mikroo-injektor (Narishige) e ɓuuɓri junngo (Narishige tisure) e dumunna ɓuuɓɗo sever10 winndannde hojomaaji. Caggal nde ɓuuɓri ndii waɗi, waɗ heen kapilaar oo fotde hojomaaji 10 goɗɗi ngam newnude mboros oo saabaade ɗum haa laaɓa. Caggal nde kapilaaruuji ɗii njaltinaa, ɓalndu nduu suuɗee no feewi ngam ustude ɓuuɓri gaañiiɓe ɓee, addana daaba oo sellude. Daabaaji ɗii njaltinaama e leɗɗe cafrorteeɗe (caspofen) balɗe keewɗe caggal nde ɗi ngoppitaa, e nder ɗuum, ngonka ɓalli mum en ina ƴeewtee no feewi, caggal ɗuum ɗi mbaraa e sahaa gonaaɗo oo. Laabi ɗii fof mbaɗaama e fawaade e kuule Orop, ngenndiije e juɓɓule, tawi ko LandesamtfürNatur mo Umwelt e Verbraucherschutz, to Rhein-Westfalie worgo, Almaañ njaɓi ɗum en.
Daabaaji ɗii njuurnitee ko e ketamin (100 mg/kg) e xylazine (10 mg/kg), ɓernde ndee ina ɓuuɓnee e 0,1 M PBS ko adii, caggal ɗuum e 4% PFA e nder PBS. Ɗaɗi ɗii njuppaa, njuppaa e 4% PFA/PBS jamma e 4°C. Laɓi dimmbotooɗi (Leica Microsystems GmbH, Wiyen, Otiris) kuutoraa ngam feewnude taƴe sagittal (50 μm tekkeendi) ummoraade e ngaandi tabitndi e nder PBS. So wonaa tawa ko noon hollirtee, mbayliigu taƴe ɓuuɓɗe ɗee mbaɗaama no siforaa dow nii (13) e nguleeki suudu e ɓuuɓri. E raɓɓiɗinaade, ko adii fof, ƴiye keɓtinaaɗe ɗee njuppaama e 0,5% Triton X-100/PBS fotde hojomaaji 15 e nguleeki suudu ; won e epitopeeji (Pcx e Shmt2), e nder tampon tris-EDTA to 80°C (PH 9) ɓuuɓna ƴiye ɗee fotde hojomaaji 25 e nokku ndeeɗoo ɗatal. Ko adii ɗuum, taƴe ɗee njuppaa e antibiotik gadano (ƴeew alluwal S1) e nder tampon ɓuuɓɗo (3% BSA/PBS) e nguleeki 4°C jamma e ñalawma tawa ina ɓuuɓna. Ñalnde heen, taƴe ɗee loowaa e tampon ɓuuɓɗo, ɓuuɓnaa e antibiotik ɗiɗaɓo mo fluorophore-conjugué potɗo, fotde waktuuji 2 e nguleeki suudu ; e joofnirde, taƴe ɗee loowaa no feewi e PBS, njuppaa e DAPI, caggal ɗuum njuppaa e AquaPolymount E dow laylaytol mikroskoop.
Mikroskoop konfokaal ƴeewndotooɗo laser (TCS SP8-X walla TCS Lefol annoore dijital, Leica Microsystems) jogiiɗo laser annoore daneejo e laser 405 diyoode ultraviolet huutoraa ngam ƴeewde sample oo. E ɓuuɓnude fluorofoor oo e mooftude maande ndee e Hybrid Detector (HyDs), lowre LAS-X huutoraa ngam mooftude natal gonngal e mbaydi njuɓɓudi Nyquist e mbaadi jokkondire : wonande panneeji ɗi ngonaa limtooji, ko maandeeji diƴƴe no feewi (yeru, e nder ƴiye somatik e dendriteeji) dect e UFPY to mt Mode BrightR). Gating gila 0,3 haa 6 ns ina huutoree ngam ustude caggal.
Natal e sahaa goonga e cellal sortaangal. Caggal nde cirƴaa e nder lowre Neurobasal-A ina waɗi 1% ɓeydaare B27 e 0,5 mM GlutaMax, ƴiiƴam ɗam ƴettaa ɗoon e ɗoon e dow ɓulli gilaasi ɓuuɓɗi (μ-Slide8 Weli, Ibidi, limoore catal 80826) , E caggal ɗuum ɓulli ɗii njuppaa e a1% CO2 ngam jooɗaade. Natal e sahaa goonga waɗiraa ko e mikroskoop konfokaal ƴeewndotooɗo laser Leica SP8, baɗɗo laser daneejo, HyD, 63×[1,4 aperture numérique (NA)] lens objektif nebam e dingiral nguleeki.
Moƴƴere ndee yaawi anestetik e gaas karbonik, taƴaa hoore mum, ngaandi ndii yaawi ittude e ɓalndu nduu, taƴaa e 200μm (ngam jarribo 13C martaba) walla 275μm (ngam jarribooji foton ɗiɗi) taƴre sagittal heewnde e ɗeeɗoo geɗe crece The VH.5 Scientific, Walldorf, Almaañ) ina heewi ɗeeɗoo geɗe : 125 mM ɓuuɓɗo no geec nii, ɓuuɓɗo karbon (95% O2 e 5% CO2) Ca2 seeɗa + ndiyam ɓalndu artificiel (ACSF) NaCl, 2,5 mM KCl, 1,25 mM tampon Na3O m2, 1,25 mM tampon Na3O m2, 1,25 mM tampon Na3O m2, 1,25 mM tampon Na3O, 1,25 mM tampon sodium suukara, CaCl2 0,5 mM e MgCl2 3,5 mM (toɓɓere osmotik 310 haa 330 mmol). Waɗde, ƴettude ƴiye ngaandi keɓtinaaɗe ɗee e nder suudu hade mum ɓuuɓde, tawa ina waɗi Ca2 + ACSF ɓurɗo toowde (125,0 mM NaCl, 2,5 mM KCl, 1,25 mM tampon fosfaat sodium, 25,0 mM NaHCO3, 25,0 mM d-glucose Ca2.0M, 100 mM d-glucose MgCl2) Hakkunde) pH 7,4 e 310 haa 320 mmol).
E nder golle ƴeewndo, ƴiye ɗee njaltinaa e suudu ƴeewndo keeriiɗo, jarribo ngoo waɗaama e les ɓuuɓri ACSF jokkondirndi e nguleeki 32° haa 33°C. Mikroskoop ƴeewndo laser keewɗo foton (TCS SP8 MP-OPO, Leica Microsystems) jogiiɗo ƴulɓe 25x (NA 0,95, ndiyam), laser Ti: Sapphire (Cameleon Vision II, Coherent) huutoraa ngam ƴeewde ƴiye. Module FLIM (PikoHarp300, PikoKuan).
FLIM mo Grx1-roGFP2. Waylooji ngonka redox sitoplasma PNs ɗii, peeñninaama e FLIM foton ɗiɗi e nder ƴiye ngaandi sagittal, ɗo biosensor Grx1-roGFP2 ƴetti PNs. E nder laral PN, fannu keɓgol ngol suɓaama fotde 50 haa 80 μm les ɓuuɓri slice ngam tabitinde wonde PN ina waawi wuurde (ɗum woni ŋakkeende mbaydi beɗi walla waylooji morfolooji neeruuji e dow dendriteeji ɗii) e sensor roGFP2 moƳƳo ɗiɗi ( sensor roGFP2 co-presseach co-contrôleur PCx ​​or shcoding PCx mCeri). Moftu natal gonngal e mbaydi gootiri e zoom dijital 2x [njuuteendi ɓoggi ɓuuɓɗi: 890 nm; 512 nm ko 512 piksel]. Yiytude : HyD ndernderiijo, fedde filtirde isotiyosiyanat fluoresein (FITC)] e natal hakkundeewal e nder hojomaaji 2 haa 3 ina kuutoree ngam tabitinde wonde fotooje keewɗe ina mooftee (1000 fotooje e nder kala) ngam fiɓnde kurjuruuji. Sensitibilite sonndu Grx1-roGFP2 e hoolkiso sarɗiiji FLIM mbaɗaama e ƴeewtaade nafoore nguurndam roGFP2 nde ɓeydata H2O2 eksoojen 10 mM e perfusion ACSF (ngam ɓeydude oksidaasiyoŋ, ko ɗum addanta ɓeydagol nguurndam), caggal ɗuum ɓeydaa mrethizol of2 ustaare, ɗum addanta ustaare nguurndam) (Natal S8, D haa G). Huutoro lowre FLIMfit 5.1.1 ngam yuurnitaade njeñtudi keɓtinaandi, fittaandu mbaydi mbaylaandi esponensiyel gootol e natal ngal fof e IRF fotnoo (golle jaabawol kuutorgal), e χ2 ko hedde 1. Ngam hiisaade nguurndam maskn gooto, mask wonnoo ko junngo e average life e nder nerve huutortee ko ngam limtude.
Ƴeewndo mbaawka mitokondriyaal. Caggal nde taƴre ɓuuɓnde ndee ɓuuɓnaa e TMRM 100 nM ɓeydaama to bannge ACSF perfuse fotde hojomaaji 30, mbayliigaaji potɗi mitokondriyaal PNs ɗii pooɗanoo ko e mikroskoop foton ɗiɗi. Natal TMRM ngal waɗiraa ko e ɓuuɓnude sonndu nduu e 920 nm e huutoraade HyD nder mum (isotiyosiyanat tetrametilrodamin : 585/40 nm) ngam mooftude maandeeji ; e huutoraade njuuteendi ɓoggi ɓuuɓɗi gooti kono huutoraade HyD nder mum ceertuɗo (FITC :525/50) ngam naatde e mtYFP. Huutoro plug-in Image Calculator ngam ƴeewtaade mbaawka mitokondriyaal to tolno cellal gootal. E raɓɓiɗinaade, pottital plug-in : maande = min (mtYFP, TMRM) huutortee ko ngam anndude nokku mitokondriyaal holliroowo maande TMRM e nder Purkinje Somali e nder natal konfokaal gonngal e mbaydi gonndi e mum. Ndeen nokku pixel e nder mask keɓaaɗo oo ina limtee, caggal ɗuum ina normalisee e dow natal gonngal e mbaydi mtYFP ngam heɓde feccere mitokondriyee hollitoore mbaawka mitokondriyee.
Natal ngal ƴettaa ko e lowre Huygens Pro (Natal njuuteendi ganndal). Wonande nataluuji skanndeeji kaɓirɗe, montage kaɓirɗe goote ina mbaɗee e kuutoragol algoritme stitching automatique mo lowre LAS-X rokkata. Caggal kalibraasiyoŋ natal ngal, huutoro ImageJ e Adobe Photoshop ngam ɓeydude ƴellitde natal ngal e yuɓɓinde no fotiri jalbugol e mbaydi. Huutoro Adobe Illustrator ngam feewnude nate.
mtDNA ƴeewndo focus. Limre mborosaaji mtDNA limtaama e taƴe cerebellar ɗe limtaaɗe e antibiotikuuji ADN e mikroskoop konfokaal. Kala nokku njiylawu oo sosaa ko e ɓanndu cellal e ƴiiƴam kala, nokku oo hiisaama e kuutoragol plug-in Multi Measure (software ImageJ). Ittu nokku nukkuur e nokku ɓanndu cellal ngam heɓde nokku sitoplasma. E joofnirde, plug-in Analyse Particles (software ImageJ) huutoraama ngam limtude otomatikk toɓɓe ADN sitoplasma kollitooje mtDNA e dow natal gonngal e ŋorol ngol, tawa njeñtudi keɓtinaandi ndii ina normalisee e tolno PN mo ɓowɗi CTRL. Njiylawu oo hollirtee ko no limoore nukleosiiduuji e nder cellal kala.
Anndugol baylagol poroteyiin. Huutoro jokkorgal ImageJ ngam ƴeewtaade baylagol poroteyiin e nder PN to tolno cellal gootal. E raɓɓiɗinaade, e natal konfokaal laral gootal gonngal e laawol ngol, rewrude e pottital : signal = min (mtYFP, antibiotik), nokku mitokondriyaal holliroowo immunoreaktivite e won e antibiotik e nder Purkina ina anndaa. Ndeen nokku pixel e nder mask keɓaaɗo oo ina limtee, caggal ɗuum ina normalisee e dow natal gonngal e mbaydi mtYFP ngam heɓde feccere mitokondriyee e poroteyiin holliraaɗo oo.
Ƴeewndo denndaangal cellal Purkinje. Plug-in Cell Counter mo ImageJ huutoraa ko ngam ƴeewde densité Purkinje e feccude limoore ƴiye Purkinje limtaaɗe e njuuteendi cirƴam cereeli ɗi ƴiye limtaaɗe ɗee njogii.
Pewnugol e mooftugol sample. Ngaandiiji ummoriiɗi e fedde toppitiinde ndee e ɓowɗi Mfn2cKO ɗii, ndartinaama e 2% PFA/2,5% glutaraldehiid e nder 0,1 M tampon fosfaat (PB), caggal ɗuum taƴe koronaawiris peewniraa ko e kuutoragol ciliateeji (Leica Mikrosysteme GmbH, Wiyen, Otiris) (Tekkeendi 50 haa tampon The60 μm1 oPBn). tetraoksiid e 1,5% ferrosiyaniid potaasiyom e nguleeki suudu fotde waktu gooto. Taƴe ɗee loowaama laabi tati e ndiyam ɓuuɓɗam, caggal ɗuum njuppaa e 70% etanol ina waɗi 1% uranil asetaat fotde hojomaaji 20. Ndeen taƴe ɗee njuumtaama e nder alkol ɓuuɓɗo, njuppaa e nder Durcupan ACM (resin casting Araldite M) e resin epoksi (Ganndal Mikroskopi Elektron, limoore catal 14040) hakkunde ɓuuɓri gilaasi ɓuuɓndi silikon, e nder 60°C Polymerize e nder hours48. Nokku gonɗo e ɓalndu nduu suɓaama, taƴe 50 nm ɗe ɓuuɓɗe taƴaa e Leica Ultracut (Leica Mikrosysteme GmbH, Wiyen, Otiris) ƴettaa e dow ŋoral njamndi 2×1 mm, ɓuuɓngal e filmo polistireen. Taƴe ɗee njuumtaama e ɓuuɓri 4% uranil asetaat e nder H2O fotde hojomaaji 10, loowaa e H2O laabi keewɗi, caggal ɗuum e sitraat plumbum Reynolds e nder H2O fotde hojomaaji 10, caggal ɗuum loowaa e H2O laabi keewɗi. Mikrogaraafiiji ɗii ƴettaa ko e mikroskoop elektoronik gonɗo e jolngo biyeteeɗo Philips CM100 (Thermo Fisher Scientific, Waltham, MA, Amerik) kuutortooɗo kamera dijital TemCam-F416 (TVIPS GmbH, Gauting, Amerik). Almaañ).
Wonande ɓowɗi gonɗi e AAV, ngaandi ndii seerndaama, taƴaa e taƴre sagittal 1 mm, cerebellum oo ƴeewtee ko e mikroskoop ɓuuɓɗo ngam anndude cirƴam gonɗam e AAV (ɗum woni mCherry expressing). Ko jarribooji ɗi injection AAV addanta ƴellitaare toownde no feewi e laral cellal Purkinje (ɗum woni ko famɗi fof laral ngal fof) e nder ko famɗi fof cirƴe cerebellar ɗiɗi jokkondirɗe tan kuutortee. Loop mo AAV wayli oo, ƴettaa ko e mikroo-diisneteeɗo ngam ƴeewndaade ɗum jamma caggal nde o fiyi (4% PFA e 2,5% glutaraldehyde e nder 0,1 M tampon kakawo) e ɓeydaade ƴeewteede. Ngam udditde EPON, ƴiiƴam tabitɗam ɗam loowaama e 0,1 M tampon koko (Applichem), ɓuuɓnaa e 2% OsO4 (os, Sarwisaaji ganndal; Caco) e nder 0,1 M tampon koko (Applichem) Waktuuji 4, caggal ɗuum loowaa waktuuji 2. Fuɗɗito laabi 3 e 0,1 M tampon kokamiid. Caggal ɗuum, seerndoy etanol ngol ina huutoree ngam ustude kala ɓuuɓri etanol e nguleeki 4°C fotde hojomaaji 15 ngam ustude ndiyam e ɓalndu nduu. Ɓoornugol ngol waɗtaa e oksidaa propilen, ɓuuɓnaa jamma e nder EPON (Sigma-Aldrich) e nguleeki 4°C. Waɗde ƴiiƴam ɗam e nder EPON keso e nder nguleeki suudu fotde waktuuji 2, caggal ɗuum ɓuuɓna ɗum e nguleeki 62°C fotde waktuuji 72. Huutoro ultramikrotom (Leica Microsystems, UC6) e laɓi diamant (Diatome, Biel, Siwis) ngam taƴde taƴe ɓutte 70 nm, e ɓuuɓnude e 1,5% asetaat uranil fotde hojomaaji 15 e 37°C, e ɓuuɓnude e ɓuuɓri sitraat plumbum minitaaji 4. Mikrogaraafiiji elektoronik ɗii ƴettaa ko e kuutoragol mikroskoop elektoronik gonɗo e jolngo JEM-2100 Plus (JEOL) jogiiɗo kamera OneView 4K 16-bit (Gatan) e lowre DigitalMicrograph (Gatan). Ngam hiisaade, mikroo-ektoronik keɓaama e 5000× walla 10 000× zoom dijital.
Njiylawu morfolooji mitokondriyaaji. Ngam denndaangal wiɗtooji ɗii, konnguɗi mitokondriyaaji gooto gooto ɗii, kollitaama e junngo e natal dijital kuutortoongal lowre ImageJ. Parametruuji morfolooji ceertuɗi ina ƴeewtee. Denndaangal mitokondriyeeji ina kollitee e teemedere keɓaaɗo e feccude keewal mitokondriyeeji kala e nokkuuji sitoplasma (nokku sitoplasma = nokku cellal-nokku ƴiiƴam) × 100. Rogere mitokondriyaal ina hiisee e foormula [4π∙2(area)]./perimeter. Morfolooji ista mitokondriyaaji ƴeewtaama, feccitaa e cate ɗiɗi (“tubular” e “blister”) fawaade e mbaadiiji mum en mawɗi.
Limre otofagosom/lisosom e ƴeewndo denndaangal. Huutoro lowre ImageJ ngam siifde e junngo maa mbaydiiji kala otofagosom/lisosom e nder natal dijital. Nokku otofagosom/lisosom hollirtee ko e teemedere hiisaande e feccude nokku gonɗo e mbaydi otofagosom/lisosom e kala ƴiiƴam e nokku sitoplasma (nokku sitoplasma=nokku ƴiiƴam-nukleus)×100. Denndaangal otofagosomuuji/lisosoomuuji ina hiisee e feccude limoore ndee fof e limoore mbaydiiji otofagosomeeji/lisosoomuuji e nder cellal kala (to bannge nokku sitoplasma) (nokku sitoplasma = nokku cellal-nokku nukliyeer).
Label ngam taƴde akut e peewnugol sample. Wonande jarribooji baɗɗi limde glukos, nawde ƴiye ngaandi ɓuuɓɗe ɗee e nder suudu ɓuuɓndu, tawa ina waɗi karbon keewɗo (95% O2 e 5% CO2), Ca2 + ACSF toowɗo (125,0 mM NaCl, 2,5 mM KCl, 1,25 mM fosfaat sodium m2O, 1,25 mM fosfaat sodium m2O, 1,25 mM fosfaat sodium m2O. 25,0 mM d-glukoos, 1,0 mM CaCl 2 e 2,0 mM MgCl 2, njuɓɓinaaɗi e pH 7,4 e 310 haa 320 mOsm), e nder ɗuum glukos ko 13 C 6- Lomto glukos (Eurisotop, limoore catal63-1). Ngam jarribooji baɗɗi limde piruwat, ƴettude ƴiye ngaandi ɓuuɓɗe ɗee e Ca2 + ACSF ɓurɗo toowde (125,0 mM NaCl, 2,5 mM KCl, 1,25 mM tampon fosfaat sodium, 25,0 mM NaHCO3, 25,0 mM d-glucose Ad2C e 100 mM MgCl2, njuumtu e pH 7,4 e 310 haa 320 mOsm), ɓeyda heen 1 mM 1-[1-13C]piruwaat (Eurisotop, limoore catal CLM-1082). Ɗaɗi ɗii njuumta fotde hojomaaji 90 e nguleeki 37°C. E joofnirde jarribo ngoo, taƴe ɗee loowetee ko e yaawre e ɓuuɓri ndiyam (pH 7,4) tawa ina waɗi 75 mM karbonaat amoniyum, caggal ɗuum ɗe njuumta e 40:40:20 (v:v:v) asetonitril (ACN) : metaan : ndiyam. Caggal nde taƴe ɗee njuppaa e dow ice fotde hojomaaji 30, ƴeewndorɗe ɗee njuppaa e 21 000 g fotde hojomaaji 10 e 4°C, tawa ko ɓuuɓɗe laaɓtuɗe ɗee njuppaa e nder ɓuuɓri SpeedVac. Pellet metabolit yoorɗo mo heɓaa oo, mooftaa ko e -80°C haa nde ƴeewndaa.
Ƴeewndo spektorometri masse kromatogaraafi ndiyam e asideeji amino 13 markeeji C. Ngam ƴeewde ko wiyetee « LC-MS » (LC-MS), pellet metabolit oo rewnaama e 75μl ndiyam ɗam LC-MS (Honeywell). Caggal nde 21 000 g ɓuuɓnaa e 21 000 g fotde hojomaaji 5 e 4°C, 20 μl e ɓuuɓri laaɓtundi ndii huutoraa ngam ƴeewde ɓuuɓri asid amino, heddii ko ɓuuɓri ndii huutoraa ɗoon e ɗoon ngam ƴeewde aniyon (ƴeew les). Anndugol asid amino waɗiraa ko e kuutoragol porotokol ƴettugol kuloriid benzoil sifotoo ko adii (55, 56). E daawal gadanal ngal, 10μl karbonaat sodium 100 mM (Sigma-Aldrich) ɓeydaama e 20μl ɓuuɓri metabolit, caggal ɗuum 10μl 2% kuloriid benzoil (Sigma-Aldrich) ɓeydaama e ACN tolno LC. Sammba oo ɓuuɓnaama seeɗa, caggal ɗuum ɓuuɓnaama e 21 000 g fotde hojomaaji 5 e nguleeki 20°C. Waɗde, ɓuuɓnude ɓuuɓri laaɓtundi ndii e nder ɓuuɓri autosampler 2 ml tawa ina waɗi ɓuuɓri gilaas konik (200 μl njuuteendi). Sammbaaji ɗii ƴeewtaama e nder njuɓɓudi LC (Ndiyam) jokkondirndi e ƴulɓe toowɗe (Thermo Fisher Scientific) . Ngam ƴeewndaade, 2μl e sample derivatized oo, waɗaama e nder 100×1,0 mm toowɗo semmbe silis T3 (Waters) ina waɗi 1,8μm ɓuuɓri. Ƴeewirde ndee ko 100μl/min, tawa kadi ko mbaydi tampon A (10 mM amoniyum e 0,15% asid formik e nder ndiyam) e tampon B (ACN). Gradient oo ko nii siforii : 0%B e hojomaaji 0 ; 0%B. 0 haa 15% B e hojomaaji 0 haa 0,1 ; 15 haa 17% B e hojomaaji 0,1 haa 0,5 ; B e 17 haa 55% e hojomaaji 0,5 haa 14 ; B e 55 haa 70% e hojomaaji 14 haa 14,5 ; e 14,5 haa 70 haa 100% B e hojomaaji 18 ; 100% B e nder hojomaaji 18 haa 19 ; 100 haa 0% B e hojomaaji 19 haa 19,1 ; 0% B e hojomaaji 19,1 haa 28 (55, 56). Spektromeeter masse QE-HF ina gollira e mbaadi iyonization moƴƴo e njuuteendi masse m/z (ratio masse/charge) 50 haa 750. Resolution kuutorteeɗo oo ko 60 000, e target iyon keɓaaɗo (AGC) keɓaaɗo ko 3×106, e waktu iyon ɓurɗo mawnude ko milliconds1. Fuɗɗoode iyon elektoro-spray (ESI) ngulnde ina gollira e doole ɓuuɓɗe 3,5 kV, nguleeki kapilaar 250°C, ɓuuɓol henndu ɓuuɓngol 60 AU (unités arbitraires), e ɓuuɓol henndu ballitoowo 20 AU. 250°C. Lens S oo ina waɗi 60 AU.
Aniyoŋ kromatogaraafi-MS ƴeewndo asideeji organik 13C markeeji. Heddii ko ɓuuɓri mbaylaandi (55μl) ƴeewtaama e kuutoragol mbaydi mbaylaandi iyon Dionex (ICS 5000+, ganndal Thermo Fisher) jokkondirndi e mbaydi mbaylaandi QE-HF (Thermo Fisher Scientific). E raɓɓiɗinaade, 5μl ƴiiƴam mbaylaandi (metabolit) ɓuuɓnaama e nder ŋoral Dionex IonPac AS11-HC jogingal HPLC (2 mm×250 mm, njaajeendi ŋoral 4μm, Thermo Fisher Scientific) e mbaadi loowdi feccere push-in e ŋoral hebbingol 1. 4μm, Ganndal Liɗɗi Termo). Nguleeki koloñaal ina jogaa e 30°C, otosampler ina jogaa e 6°C. Huutoroo karte hidroksiid potaasiyom dokkaaɗe e ndiyam deyon ngam feewnude mbaydi hidroksiid potaasiyom rewrude e feewnude eluent. Seerndude mbayliigaaji e njaaweeki 380μl/min, tawa ina huutoroo mbaydi mbaylaandi : 0 haa 3 minit, 10 mM KOH ; Hojomaaji 3 haa 12, KOH 10 haa 50 mM ; Hojomaaji 12 haa 19, 50 haa 100 mM KOH ; Hojomaaji 19 haa 21 , 100 mM KOH ; 21 haa 21,5 hojom, 100 haa 10 mM KOH. Kolonel oo artiraa e les 10 mM KOH fotde hojomaaji 8,5.
Metaboliituuji ɓuuɓɗi ɗii kawra e 150μl/min e ɓuuɓri ɓeydundi isopropanol caggal koloñaal, caggal ɗuum ndartinaa e spektorooter masse toowɗo golloowo e mbaadi iyonization negatif. MS ina ƴeewa tolno masse m/z 50 haa 750 e resolution 60 000. AGC ina waɗee 1×106, waktu iyon ɓurɗo mawnude ina jogoo 100 ms. Fuɗɗoode ESI ɓuuɓnde ndee golloraa ko e doole ɓuuɓɗe 3,5 kV. Settings keddiiɗi ɗii e ɓuuɓri iyon ko nii : nguleeki kapilaar 275°C ; ɓuuɓol gaas ɓuuɓngol, 60 AU; ɓuuɓol gaas ballitoowo, 20 AU e 300°C, e lenyol S gonngol e 60 AU.
Ƴeewndo doosɗe 13C limtaaɗe e metaboliteeji. Huutoro lowre TraceFinder (mbaydi 4.2, ganndal Thermo Fisher) ngam yuurnitaade dokke njuuteendi isotopeeji. Anndinde kala kompoññe hoolkisaama e kompoññe tuugorgal koolniingal, ƴeewtaama e hoore mum. Ngam waɗde wiɗto alɗugol isotopeeji, nokku gonɗo e kromatogram iyon (XIC) ƴettaaɗo e kala isotop 13C (Mn) ƴettaa ko e [M + H] +, ɗo n woni limoore karbon gonɗo e mbaydi njiylawu, huutorteeɗo ngam ƴeewndaade asideeji amino walla [ MH] + huutorteeɗo ngam ƴeewndaade aniyon. Njuuteeki masse XIC ina famɗi e pecce joy e nder miliyoŋ, njuuteeki RT ko 0,05 minit. Anndugol alɗugol ngol waɗata ko e hiisaade tolno kala isotope yiytaa e denndaangal isotopeeji gonɗi e mbaydi gonndi e mum. Ɗeeɗoo limlebbi ina ndokkee no teddeendi teemedere wonande kala isotope, tawa njeñtudi ndii ina hollitee no teddeendi teemedere e nder teemedere (MPE), hono no siforaa adan nii (42).
Pellet neuron ɓuuɓɗo oo homogenisee e nder metaan 80% (v/v) ɓuuɓɗo no ice nii, ɓuuɓnaa, ɓuuɓnaa e -20°C fotde hojomaaji 30. Vortex sample oo kadi, ɓuuɓna ɗum e +4°C fotde hojomaaji 30. Sammba oo senndiraama e 21 000 g fotde hojomaaji 5 e 4°C, caggal ɗuum, ko heddii koo mooftaa, yoora e kuutoragol ƴulɓe SpeedVac e 25°C ngam ƴeewndaade caggal ɗuum. No siforaa dow nii, ƴeewndo LC-MS waɗiraa ko e asideeji amino ɗi ƴiye sortaaɗe ɗee. Huutoraade TraceFinder (mbaydi 4.2, Thermo Fisher Scientific), ƴeewndo doosɗe waɗiraa ko e huutoraade masse monoisotopeeji kala kompoññe. Normalisaasiyoŋ limtilimtinɗo e dokke metabolit waɗiraa ko e kuutoragol lowre preprocessCore (57).
Peewnugol slice. Moƴƴere ndee yaawi ɓuuɓneede e gaas karbonik, taƴaa hoore mum, ngaandi ndii yaawi ittude e ɓalndu nduu, laɓi diƴƴe keewɗi ƴiiƴam (HM-650 V, Thermo Fisher Scientific, Walldorf, Almaañ) huutoraa ngam taƴde ɗum e 300 haa 375 μm gaas karbon O5d. CO2) Ca2 + ACSF mo famɗaani (NaCl 125,0 mM, KCl 2,5 mM, tampon fosfaat sodium 1,25 mM, NaHCO3 25,0 mM, d-glukoos 25,0 mM, CaCl2 1,0 mM e Adjust 6,0 mM to pH370 mOsm). Waɗde, ɓuuɓnude ƴiye ngaandi keɓtinaaɗe ɗee e nder suudu tawa ina waɗi Ca2 + ACSF ɓurɗo toowde (125,0 mM NaCl, 2,5 mM KCl, 1,25 mM tampon fosfaat sodium, 25,0 mM NaHCO3.25,0 mM d-glucose, 4,0 mM pH42l5C e 310 haa 320 mOsm). Siftor limre ndee fotde hojomaaji 20 haa 30 mbele ina waawa artireede hade mum winndeede.
winnditaade. Dingiral mikroskoop jogingal suudu winnditorde tabitndu e 20x lens objectif immersion d’eau (Scientifica) huutoraa ngam winnditaade denndaangal. Piɓaaji Purkinje teskinɗi ɗii nganndiraa ko (i) mawneeki ɓanndu, (ii) nokku ɗo ƴiiƴam ɗam woni ɗoo, e (iii) baylagol taƴre mtYFP ɓuuɓnde. Pipet patch mo njuuteendi mum tolnii e 5 haa 11 megohm, ina yaltinee e kapilaar gilaas borosilikat (GB150-10, 0,86 mm×1,5 mm×100 mm, geɗe ganndal, Hofheim, Almaañ) e pipet gonɗo e dow ŋoral (P-1000). Denndaangal binndanɗe ɗee mbaɗiraa ko amplifier npi npi (npi elektoronik GmbH, Tam, Almaañ), mo lowre Signal (mbaydi 6.0, elektoronik Cambridge, Cambridge, Angalteer) jogori huutoraade. Njiylawu oo winndaa ko e tolno 12,5 kHz. Signal oo ina filtinee e filtoraaji ɗiɗi Bessel juutɗi, tawi njuuteendi mum en ko 1,3 e 10 kHz. Capanɗe membrane e pipet ina njuutee e cirƴe njuumri kuutortooɗe amplifier. Jarribooji ɗii fof mbaɗaama e les njiimaandi kamera biyeteeɗo Orca-Flash 4.0 (Hamamatsu, Gerden, Almaañ), tawi ko lowre Hokawo (mbaydi 2.8, Hamamatsu, Gerden, Almaañ) ɗownoo ɗum.
Koolkisagol e ƴeewndo cellal fof ko woowaa. Ɗoon e ɗoon hade winnditaade, hebbinee pipet oo e ɓuuɓri ndernderi ndi ina waɗi ɗeeɗoo geɗe : 4,0 mM KCl, 2,0 mM NaCl, 0,2 mM EGTA, 135,0 mM glukonaat potaasiyom, 10,0 mM Hepes, 4,0 mM Guphano.TPG5s (4,0 mM ATP.TPM) (Na) e fosfaat kreatinin 10,0 mM njuɓɓinaama haa pH 7,25, tawi noon ɓuuɓol osmotik ngol ko 290 mOsm (sukrose). Ɗoon e ɗoon caggal nde doole 0 pA mbaɗa ngam ruttaade e ɓuuɓri ndii, doole ɓuuɓri fooftere ndee fotnoo. Reentaade naatgol ngol ina fotndee e huutoraade doole hiperpolariseeji -40, -30, -20, e -10 pA. Fotde mawneeki jaabawol doolnugol ngol e huutoraade sariya Ohm ngam hiisaade doolnugol naatgol ngol. Golle gonɗe e hoore mum mbinndaama e nder ƴulɓe 5, sPSC anndaama, fotndaama e Igor Pro (mbaydi 32 7.01, WaveMetrics, Lake Oswego, Oregon, Amerik) e kuutoragol binndol anndinde semi-otomatik. Kuraana IV e jolngo steady-state ina pooɗee e ɓuuɓnude batiri oo e mbaadiiji ceertuɗi (fuɗɗoraade -110 mV) e ɓeydude jolngo ngoo e daawal 5 mV. Pewnugol AP ƴeewtaama e huutoraade doole depolarizing. Ɗaɓɓu cellal ngal e -70 mV tawa aɗa waɗa pulse depolarizing. Reento njaajeendi taƴre kala njuɓɓudi winnditorde ceertundi (10 haa 60 pA). Hiisaade AP ɓurɗo mawnude e limtude e junngo ɓuuɓol pulseeji baɗooji AP ɓurɗo toowde. Ɗatal AP ina hiisee e huutoraade ƴellitaare ɗiɗmere pulse depolarisaasiyoŋ nde adii ustude AP gooto walla ko ɓuri ɗum.
Konfiguraasiyoŋ e ƴeewndo patch perforated. Waɗde winnditaade patch perforated huutoraade porotokolle kuuɓtodinɗe. Huutoro pipet mo alaa ATP e GTP, mo alaa ko woni e mum so wonaa ɗeeɗoo geɗe : 128 mM glukonaat K, 10 mM KCl, 10 mM Hepes, 0,1 mM EGTA e 2 mM MgCl2, njuɓɓina ɗum haa pH 7,2 (kuutoragol KOH). ATP e GTP ina njaltinee e ɓuuɓri nder ɓalndu ngam haɗde ɓuuɓri ɓalndu ndi rewaani laawol. Pipet paɗe oo ina heewi ɓuuɓri ndernderi ndi amfoterisin ina waɗi (ko ina wona 200 haa 250μg/ml; G4888, Sigma-Aldrich) ngam heɓde winndannde paɗe punndi. Amfoterisin oo ɓuuɓnaama e dimetil sulfoksiid (ɓurɗo heewde : 0,1 haa 0,3% ; DMSO ; D8418, Sigma-Aldrich). Ko heewi e DMSO kuutorteeɗo oo alaa ko battini e neronuuji janngeteeɗi ɗii. E nder golle punndi, reentaare kanndaa (Ra) ina jokki e ƴeewteede, jarribo ngoo fuɗɗotoo ko caggal nde amplitude Ra e AP dartii (20-40 minit). Golle gonɗe e hoore mum ina pooɗee e nder laylaytol (voltage) e/walla laylaytol (current) fotde hojomaaji 2 haa 5. Njiylawu doosɗe ɗee waɗiraa ko e kuutoragol Igor Pro (mbaydi 7.05.2, WaveMetrics, Amerik), Excel (mbaydi 2010, Sosiyetee Microsoft, Redmond, Amerik) e GraphPad Prism (mbaydi 8.1.2, mbaydi GraphPad Software Inc. Dowlaaji Dentuɗi Amerik). Ngam anndude AP-ji gonɗi e hoore mum, kuutorgal IgorPro ngal NeuroMatic v3.0c huutortee. Anndinde APs otomatik kuutortooɗo limre dottaande, nde njuɓɓintee gooto gooto e kala winndannde. Huutoraade hakkundeere ɓuuɓri, anndu ɓuuɓri ɓuuɓri e ɓuuɓri ɓuuɓri ɓurndi heewde e sahaa gooto e ɓuuɓri hakkundeeri.
PN seerndude. E huutoraade porotokol bayyinaango ko adii, PNs laaɓtinaama e cerebellum mooso e daawal toɗɗangal (58). E raɓɓiɗinaade, ƴiiƴam ɗam ƴettaa, ɓuuɓnaa e nder weeyo ɓuuɓngo [tawa HBSS Ca2+ e Mg2+ alaa heen, ɓeydaa heen 20 mM glukos, penisilin (50 U/ml) e streptomisin (0,05 mg./ ml)], caggal ɗuum ɓuuɓnaa e nder weeyo ngo mgSS1-sisteinin, ɓeydee heen mgSS1 / ml), papain (16 U / ml) e deoksiribonuklease I (DNase I; 0,1 mg/ml)] Ƴeewee fotde hojomaaji 30 e nguleeki 30°C. Ko adii fof lootde ƴiye ɗee e nder ɓuuɓri HBSS ina waɗi ɓuuɓri eggudu (10 mg/ml), BSA (10 mg/ml) e DNase (0,1 mg/ml) e nguleeki suudu ngam haɗde ɓuuɓri enzim, caggal ɗuum e nder ɓuuɓri HBSS ina waɗi 20 mM glukos (10 mg/ml) , ɓuuɓna ɗum no feewi e nder HBmylm5 (0,05 mg/ml) e DNase (0,1 mg/ml) ina njaltina ƴiye goote. Ko heɓaa heen koo, ƴiiƴam ɗam filtiraa ko e 70μm, caggal ɗuum ƴiiƴam ɗam ɓuuɓnaa e ɓuuɓri (1110 rpm, hojomaaji 5, 4°C) ɗam ɓuuɓnaa kadi e nder ɓuuɓri ɓuuɓri [HBSS, ɓeydaama e 20 mM glukos, 20% stremylin bovine fetal) Serum50 (0,05 mg/ml)]; ƴeewtaade nguurndam ƴiiƴam e yodiid propidium e ƴeewtaade njuuteendi ƴiiƴam haa 1×106 haa 2×106 ƴiiƴam/ml. Ko adii nde flow cytometry waɗata, ɓuuɓri ndii ina filtiree e nder ɓuuɓri 50 μm.
Sitomeeter ɓuuɓɗo. Soklaaji cellal mbaɗaama e 4°C e kuutoragol masiŋ FACSAria III (BD Biosciences) e lowre FACSDiva (BD Biosciences, mbaydi 8.0.1). Suspension cellal oo renndinaama e kuutoragol nofru 100 μm e les tolno 20 psi e tolno ~2800 kewu/sec. Nde tawnoo laabi gating gaadanteeji (mawneeki cellal, diisnondiral bimodal, e sifaaji cirƴagol) mbaawaa tabitinde seerndude PN no moƴƴi e sifaaji cellal goɗɗi, feere gating ndee tuugii ko e ƴeewndo toowngo tiiɗndam YFP e otofluoresans e nder mitoYFP+ ​​e MitoYFP − control mitoYFP. YFP ina weltina e ɓuuɓnude sample oo e laana laser 488 nm, tawa kadi maande ndee ina yiytee e kuutoragol filtirde band pass 530/30 nm. E nder ɓowɗi mitoYFP+, semmbe gonɗo e taƴre Rosa26-mitoYFP ina huutoree kadi ngam seerndude ɓanndu neeruuji e pecce akson. 7-AAD ina ɓuuɓna e laser oole 561 nm, ina yiytee e filtirde bandpass 675/20 nm ngam ittude ƴiye maayɗe. Ngam seerndude astrocytes e sahaa gooto, ɓuuɓri cellal ndii ɓuuɓnaama e ACSA-2-APC, caggal ɗuum, sample oo ƴettaa e laylaytol laser 640 nm, filtirde bandpass 660/20 nm huutoraa ngam yiytude maande ndee.
Ɓowɗi mooftaaɗi ɗii pelletee ko e ɓuuɓri (1110 rpm, hojomaaji 5, 4°C) ɗi ndewa e -80°C haa ɗi kuutoree. Miijooji Mfn2cKO e ɓiɗɓe mum en ina njuutee e ñalawma gooto ngam ustude waylo-waylo weeyo. Kollitgol e ƴeewndo dokke FACS mbaɗaama e kuutoragol lowre FlowJo (FlowJo LLC, Ashland, Oregon, Amerik).
No mbiyru-ɗen dow nii (59), PCR e sahaa goonga ina huutoree ngam seerndude ADN e neeruuji ceertuɗi ngam ƴeewde limto mtDNA caggal ɗuum. Linearite e sensitivite threshold ƴeewtaama e fuɗɗoode ko e dogginde qPCR e dow limooje cellal ceertuɗe. E raɓɓiɗinaade, mooftu 300 PN e nder tampon lisiyoŋ tawa ina waɗi 50 mM tris-HCl (pH 8,5), EDTA 1 mM, Tween 20 0,5% e poroteyiin K (200 ng/ml) ɓuuɓna ɗum e nguleeki 55°C fotde hojomaaji 120. Piɓaaji ɗii ɓeydaama kadi ɓuuɓde e nguleeki 95°C fotde hojomaaji 10 ngam tabitinde ŋakkeende golle timmunde e nder poroteyiin K. E kuutoragol sonndu TaqMan (Thermo Fisher) keeriiɗo e mt-Nd1, mtDNA fotnoo ko ƴeewteede e PCR semi-kantitatiif e nder njuɓɓudi PCR 7900HT Real-Time Ficientimosher (Ther). Ganndal, limoore catal Mm04225274_s1), mt-Nd6 (Ganndal Termo Fisher, limoore catal AIVI3E8) e 18S (Ganndal Termo Fisher, limoore catal Hs99999901_s1) taƴe.
Pewnugol sample proteome. E ɓuuɓnude ɓuuɓri ndii e 95°C fotde hojomaaji 10 e sonikaade, e nder tampon lisiyoŋ [6 M kuloriid guanidin, 10 mM kuloriid tris(2-karboksitil) fosfin hidrokloriid, 10 mM kuloroasetamid e 100 mM tris- Lyse pellet e nder neron ɓuuɓɗo]. E dow Bioruptor (Diagenode) fotde hojomaaji 10 (30 leƴƴannde pulse / 30 leƴƴannde dumunna fooftere). Sammba oo ɓuuɓnaama 1:10 e nder tris-HCl 20 mM (pH 8,0), jillondiraa e 300 ng kaŋŋe tripsin (Promega), ɓuuɓnaa jamma e 37°C ngam heɓde ɓuuɓri timmundi. Ñalnde ɗiɗmere ndee, ƴiiƴam ɗam ƴettaa e 20 000 g fotde hojomaaji 20. Ko heddii koo, ɓuuɓnaama e asid formik 0,1%, ɓuuɓri ndii ɓuuɓnaama e StageTips mo hoore mum waɗi. Sammba oo yoornaa e kuutorgal SpeedVac (konsentrator Eppendorf ɓeydi heen 5305) e nguleeki 45°C, caggal ɗuum peptiid oo ɓuuɓnaa e asid formik 0,1%. Sammbaaji ɗii fof ko neɗɗo gooto peewni ɗum en e sahaa gooto. Ngam yuurnitaade seedanteeje astrosit, 4 μg peptideeji ɓuuɓɗi, mbinndaa ko e tagaaɗo masse tandem (TMT10plex, limoore catal 90110, Thermo Fisher Scientific) tawa ko 1:20 hakkunde peptideeji e TMT. Ngam limtude TMT, 0,8 mg e mbaydi TMT ina rewnee e 70 μl ACN mo alaa ndiyam, peptiid yoorɗo oo rewnaa e 9 μl TEAB 0,1 M (bikarbonaat trietilammoniyum), tawi heen 7 μl e nder ACN ina ɓeydee heen. Hakindo ngo ko 43,75 %. Caggal 60 minit njuumri, njuumri ndii ɓuuɓnaa e 2 μl hidroksilamin 5%. Peptideeji limtaaɗi ɗii mooftaa, njoornaa, njuppaa e 200μl asid formik (FA) 0,1%, peccaa e ɗiɗi, caggal ɗuum njuppaa e kuutoragol StageTips mo hoore mum waɗi. E kuutoragol ƴoogirde ndiyam toownde (UltiMate 3000), gooto e feccereeji ɗiɗi ɗii feccitaama e dow ƴoogirde Acquity 1mm x 150mm, keewnde 130Å1,7μm C18 (NoWaters, catalog150). Termo Fisher ganndal). Seerndude peptideeji e njaaweeki 30μl/min, seerndude e 1% haa 50% e tampon B fotde hojomaaji 85 tawa ina waɗi njuuteendi 96 minit, e 50% haa 95% e tampon B fotde hojomaaji 3, caggal ɗuum hojomaaji 8 e nder 95 % e tampon B ; Tampon A ko 5% ACN e 10 mM bikarbonaat amoniyum (ABC), tampon B ko 80% ACN e 10 mM ABC. Moftu pecce kala hojomaaji 3, kawra e pecce ɗiɗi (1 + 17, 2 + 18 ekn) e yoorde ɗe e nder lowre ɓuuɓnde.
Ƴeewndo LC-MS/MS. Ngam ƴeewde ƴulɓe, peptideeji ɗii (limre r119.aq) ceerndaama e dow 25 cm, 75 μm njaajeendi mum ndernderi, (lens objektif keso, limre feccere PF7508250) jogiinde 1,9 μm ReproSil-Pur C EASY-nLC 1200 (Ganndal Liɗɗi Termo, Almaañ). Kolonel oo ina joginoo 50°C. Tampon A e B ko 0,1% asid formik e nder ndiyam e 0,1% asid formik e nder 80% ACN, kam en fof. Peptideeji ɗii ceerndaama e 6% haa 31% e tampon B fotde hojomaaji 65 e gila e 31% haa 50% e tampon B fotde hojomaaji 5 e njuuteendi 200 nl/min. Peptideeji ɓuuɓɗi ɗii ƴeewtaama e ƴulɓe ƴulɓe Orbitrap Fusion (Thermo Fisher Scientific). Fotde m/z gonɗo e peptide ina waɗee e njuuteendi 120 000 e nder 350 haa 1500 m/z. Huutoraade 27% semmbe colli normalisée, ko ɓuri semmbe e ngonka charge 2 haa 6 ina suɓee ngam semmbe toowɗo C trap dissociation (HCD) cleavage. Waktuuji cirƴam ɗii ko 1 s. Nafoore m/z feccere peptiid ndee fotnoo ko e nder laylaytol iyon huutortoo ko AGC ɓurɗo famɗude 5×104 e waktu ɓurɗo mawnude e 86 ms. Caggal peccitagol ngol, prekursor oo waɗtaa e doggol limtooji dinamiik fotde 45 s. Peptideeji gonɗi e TMT ɗii ceerndaama e dow 50 cm, 75 μm e dow ŋoral Acclaim PepMap (Thermo Fisher Scientific, limoore catal 164942), e dow ŋoral eggugol ngol ƴeewtaama e dow ŋoral masse Orbitrap Lumos Tribrid (Thermo Fisher Scientific equimmetric toowɗo) Kaɓirɗe (FAIMS) (Thermo Fisher Scientific) ina ngolloo e jolɗe ɗiɗi limtooje −50 e −70 V. MS3 cuɓaaɗo tuugnaade e jolngo sinkronisaasiyoŋ huutortee ko ngam fooɗtude maande iyon ciimtol TMT. Seerndude peptiid oo waɗiraa ko e EASY-nLC 1200, e kuutoragol 90% e ɓuuɓri lineer, tawa ɓuuɓri tampon ina waɗi 6% haa 31% ; tampon A ko 0,1% FA, tampon B ko 0,1% FA e 80% ACN. Kolonel analitik oo ina golloree e 50°C. Huutoro FreeStyle (mbaydi 1.6, ganndal Thermo Fisher) ngam feccude fiilngo asliijo fawaade e jolngo FAIMS.
Anndinde e limtude poroteyiin. Huutoraade njiylawu Andromeda kuutorteeɗo, dokke asliije ɗee ƴeewtaama e kuutoragol mbaydi MaxQuant 1.5.2.8 (https://maxquant.org/). Ko jiidaa e jokkondire Cre rekombinaas e YFP keɓtinaaɗe e Aequorea victoria, spekteeruuji pecce peptideeji njiylawu ngam ƴeewde jokkondire kanyon e jokkondire isoformeeji poroteyiin tuugnorgal mooso (ID poroteyiin UP000000589, ƴettaa e UniProt e lewru mee 2020. Oksidaasiyoŋ metionin e asetilaasiyoŋ N-terminal poroteyiin mbaɗtaa ko waylooji baylotooɗi ; metilaasiyoŋ sisteen karbamoil waɗaama e waylooji tabitɗi. Parametruuji diƴƴe ɗee ko “keertinaango” e “tripsin/P”. Limre ɓurnde famɗude e peptideeji e peptideeji rasooji kuutorteeɗi ngam anndude poroteyiin ko 1 ; limoore peptideeji keertiiɗi ɓurɗi famɗude ko 0. E nder sarɗiiji jokkondire karte peptideeji, tolno anndinde poroteyiin ko 0,01. Cuɓal “Peptide ɗiɗaɓo” ina softina. Huutoro cuɓal “jokkondiral hakkunde doge” ngam waylude anndinde jaajɗe hakkunde piille asliije ceertuɗe. Huutoro LFQ limre 1 ɓurnde famɗude ngam limde ko aldaa e etiket (LFQ) (60). Tiiɗnaare LFQ ina filtiree ko famɗi fof nafooje ɗiɗi laaɓtuɗe e nder ko famɗi fof fedde genotype wootere e kala sahaa, ina ekstrapolee e peccitagol no woorunoo e njaajeendi... 0.3 e yahde les 1.8. Huutoroo lowre limto Perseus (https) e R (https/project.org/) ngam yuurnitaade njeñtudi LFQ. Test t moderate laabi ɗiɗi ummoraade e lowre limma huutoraa ngam yuurnitaade baylagol feere (61). Ƴeewndo doosɗe wiɗto waɗaama e kuutoragol ggplot, FactoMineR, faktoekstra, GGally e pheatmap. Dowlaaji poroteyiin tuugiiɗi e TMT ɗii ƴeewtaama e nder mbaydi MaxQuant 1.6.10.43. Yiylo dokke proteomics raw ummoraade e lowre proteomics aadee UniProt, nde ƴettaa e lewru suwee 2018. Anndugol ngol ina waɗi faktoriyaal peewnugol laaɓal isotope mo peewnoowo oo rokki. Huutoro limma e nder R ngam ƴeewtaade baylagol feere. Dowlaaji asliiji, njeñtudi njiylawu domen, e golle e njeñtudi ƴeewndo domen fof ina ndesndaa e nder fedde ProteomeXchange rewrude e defterdu gollodiiɓe PRIDE e anndinde domen PXD019690.
Annotaaji gollorɗi ina alɗina ƴeewndo ngoo. Kuutorgal ƴeewndo laawol ganndal (QIAGEN) huutoraama ngam anndude ngalu sarɗiiji annotaasiyoŋ gollorɗi e doggol doggi baɗaaɗi e yontere 8 (Natal 1). E raɓɓiɗinaade, doggol poroteyiinuuji keewɗi keɓaaɗi e ƴeewndo doosɗe LC-MS/MS (tandem mass spectrometry) huutortee ko e ɗeeɗoo sifaaji filtoraade : Mus musculus ina suɓee ngam wonde sifaa e mbaydi, tawa cate ɗee ina kollita teddeendi P ndi Benjamini yuɓɓini ngam alɗude 0,05 walla les ina hiisee ina maantini. Ngalɗoo natal, cate joy ɓurɗe toowde e nder kala njuɓɓudi tuugiindi e tolno P toɗɗaaɗo oo kolliraama. Huutoraade t-test keewɗo, huutoraade porogaraam boost linear mo Benjamini, Krieger, e Yekutieli (Q = 5%), ƴeewndo baylagol poroteyiin e nder sahaa ina waɗa e dow kanndidaaji teeŋtuɗi cifaaɗi e kala cate, kala rowre ina ƴeewtee heen seeɗa. Alaa ko haani ƴettude SD jokkondirɗo.
Ngam renndinde njeñtudi ndeeɗoo wiɗto e binndanɗe bayyinaaɗe e feewnude natal Venn e Natal 1, min kawrii e doggol poroteyiinuuji keewɗi e annotaaji MitoCarta 2.0 (24). Huutoro kuutorgal internet Siftorde Natal Venn (http://bioinformatik.psb.ugent.be/webtools/Venn/) ngam feewnude natal ngal.
Ngam heɓde humpitooji laaɓtuɗi e laabi limtooji kuutorteeɗi ngam ƴeewndaade proteomik, ƴeewto-ɗee toɓɓere jokkondirnde e Kaɓirɗe e Laabi. Ngam jarribooji goɗɗi ɗii fof, humpitooji laaɓtuɗi ina tawee e lefol ngol. So wonaa tawa ko noon hollirtee, denndaangal dokke ɗee kolliraama ko no ± SEM nii, denndaangal wiɗtooji limtooji ɗii mbaɗaama e nder lowre GraphPad Prism 8.1.2.
Ngam heɓde geɗe ɓeydooje e ndee winndannde, ƴeew http://advances.sciencemag.org/cgi/content/full/6/35/eaba8271/DC1
Ko ɗum winndannde udditiinde renndinde e nder sarɗiiji Creative Commons Attribution-Non-Commercial License, baawɗo huutoraade, renndinde e ƴellitde e kala fannu, so tawii kuutoragol timmungol ngol wonaa ngam heɓde nafoore njulaagu, tawa kadi ko miijo ngo woni wonde golle asliije ɗee ko goonga. Tendinowo.
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Ko E. Motori, I. Atanassov, SMV Kochan, K. Folz-Donahue, V. Saktivelu, P. Giavalisko, N. Toni, J. Puyal, N.-G. Larson
Anndugol proteomics e neeruuji ɗi ngonaa gollotooɗi hollitii wonde porogaraamuuji mbaylaandi ina kuutoree ngam haɓaade mborosaaji.
Ko E. Motori, I. Atanassov, SMV Kochan, K. Folz-Donahue, V. Saktivelu, P. Giavalisko, N. Toni, J. Puyal, N.-G. Larson
Anndugol proteomics e neeruuji ɗi ngonaa gollotooɗi hollitii wonde porogaraamuuji mbaylaandi ina kuutoree ngam haɓaade mborosaaji.
©2020 Fedde Ameriknaare ngam ɓamtaare ganndal. hakkeeji fof ina njogii. AAAS ko gollodiiɗo e HINARI, AGORA, OARE, KOOR, CLOCKSS, CrossRef e COUNTER. Ganndal ina yahra yeeso ISSN 2375-2548.