Cinwaanka hadda jira *Cinwaankan hadda jira: Cologne 50931, Jarmalka, Cologne Excellence Cluster Research on Response Cell Stress in Diseases la xiriira Gabowga (CECAD).
Niyad-jabka cudurada mitochondrial waxaa loo arkaa mid aan dib loo celin karin sababtoo ah dabacsanaanta dheef-shiid kiimikaadka ee neerfayaasha ayaa xaddidan, laakiin saameynta cilladda mitochondrial ee madaxbannaanida unugyada ee dheef-shiid kiimikaadka neerfaha ee jirka si liidata ayaa loo fahmay. Halkan, waxaan ku soo bandhigaynaa borotiinka gaarka ah ee unugyada ee neerfayaasha Purkinje oo leh yaraanta OXPHOS ee horumarsan oo ay keento dhaqdhaqaaqa isku-dhafka mitochondrial ee khalkhalsan. Waxaan ogaanay in cilladda mitochondrial ay kicisay isbeddel qoto dheer oo ku yimid goobta proteomics, taasoo ugu dambeyntii keentay kicinta isku xigxiga ee barnaamijyada dheef-shiid kiimikaadka saxda ah ka hor dhimashada unugyada. Si lama filaan ah, waxaan go'aaminay kicinta cad ee pyruvate carboxylase (PCx) iyo ensaymada kale ee ka hortagga gabowga kuwaas oo kabaya dhexdhexaadinta wareegga TCA. Joojinta PCx oo sii xumeysay walbahaarka oksaydhka iyo neerfaha, taasoo muujinaysa in atherosclerosis uu leeyahay saameyn ilaalin ah oo ku saabsan neerfayaasha aan lahayn OXPHOS. Soo celinta isku-dhafka mitochondrial ee neerfayaasha si kama dambays ah u burburay ayaa si buuxda u beddelaya astaamahan dheef-shiid kiimikaadka, taasoo ka hortagaysa dhimashada unugyada. Natiijooyinkayagu waxay aqoonsadaan waddooyin aan hore loo aqoon oo siiya adkeysiga cilladda mitochondrial waxayna muujinayaan in neerfaha la beddeli karo xitaa marxaladaha dambe ee cudurka.
Doorka dhexe ee mitochondria ee ilaalinta dheef-shiid kiimikaadka tamarta neerfaha waxaa xoojiyay calaamadaha neerfaha ee ballaaran ee la xiriira cudurrada mitochondrial-ka aadanaha. Inta badan cudurradan waxaa sababa isbeddellada hidda-wadaha ee nidaamiya muujinta hidda-wadaha mitochondrial (1, 2) ama burburka hidda-wadaha ee la xiriira dhaqdhaqaaqa mitochondrial, kuwaas oo si dadban u saameeya xasilloonida DNA-da mitochondrial (mtDNA) (3, 4). Shaqada moodooyinka xayawaanka ayaa muujisay in jawaab celinta cilladda mitochondrial ee unugyada ku xeeran, waddooyinka dheef-shiid kiimikaadka ee ilaaliya (5-7) la hawlgelin karo, taas oo bixisa macluumaad muhiim ah si loo fahmo qoto dheer oo ku saabsan cudurkan adag. Si ka duwan, fahamkeenna isbeddellada dheef-shiid kiimikaadka ee noocyada unugyada gaarka ah ee ay sababaan guuldarrada guud ee soo saarista adenosine triphosphate (ATP) ee maskaxda waa aasaasi (8), iyadoo xoogga la saarayo baahida loo qabo in la aqoonsado bartilmaameedyada daaweynta ee loo isticmaali karo in looga hortago ama looga hortago cudurka. Ka hortagga neerfaha (9). La'aanta macluumaadka waa xaqiiqda ah in unugyada neerfaha si weyn loogu tixgeliyo inay leeyihiin dabacsanaan dheef-shiid kiimikaad oo aad u xaddidan marka la barbar dhigo noocyada unugyada ee unugyada ku xeeran (10). Maadaama unugyadani ay door muhiim ah ka ciyaaraan isku-dubaridka sahayda metabolites-ka ee neerfayaasha si kor loogu qaado gudbinta synaptic-ga iyo ka jawaabista xaaladaha dhaawaca iyo cudurrada, awoodda lagu waafajin karo dheef-shiid kiimikaadka unugyada xaaladaha adag ee unugyada maskaxda waxay ku dhowdahay inay ku kooban tahay unugyada glial (11-14). Intaa waxaa dheer, kala duwanaanshaha unugyada ee unugyada maskaxda ee ku jira unugyada maskaxda ayaa inta badan caqabad ku ah daraasadda isbeddellada dheef-shiid kiimikaadka ee ka dhaca kooxo gaar ah oo neerfayaasha ah. Natiijo ahaan, wax yar ayaa laga ogyahay cawaaqibka saxda ah ee unugyada iyo dheef-shiid kiimikaadka ee cilladda mitochondrial ee neerfayaasha.
Si loo fahmo cawaaqib xumada dheef-shiid kiimikaadka ee cilladda mitochondrial, waxaan kala soocnay neerfayaasha Purkinje (PNs) marxalado kala duwan oo neerfaha ah oo ay sababto burburka isku-dhafka xuubka dibadda ee mitochondrial (Mfn2). Inkasta oo isbeddellada Mfn2 ee aadanaha ay la xiriiraan nooc ka mid ah neerfayaasha dareenka dhaqdhaqaaqa ee dhaxalka ah oo loo yaqaan nooca Charcot-Marie-Tooth 2A (15), burburka shuruudaysan ee Mfn2 ee jiirarka waa hab si fiican loo aqoonsan yahay oo lagu kicinayo oksaydheynta Phosphorylation (OXPHOS). Noocyada kala duwan ee neerfayaasha (16-19) iyo nooca neurodegenerative ee ka dhasha waxaa la socda calaamadaha neerfaha ee horumarsan, sida khalkhalka dhaqdhaqaaqa (18, 19) ama cerebellar ataxia (16). Adigoo isticmaalaya isku-darka proteomics-ka aan calaamadda lahayn (LFQ), metabolomics, sawir-qaadista, iyo hababka virological, waxaan muujineynaa in neurodegeneration-ka horumarsan uu si xooggan u kiciyo pyruvate carboxylase (PCx) iyo arrimo kale oo ku lug leh arteriosclerosis ee PNs in vivo Muujinta enzymes-ka. Si loo xaqiijiyo muhiimadda natiijadan, waxaan si gaar ah hoos ugu dhignay muujinta PCx ee PN-yada aan Mfn2 lahayn, waxaana ogaanay in hawlgalkani uu sii xumeeyay cadaadiska oksaydhka iyo dardargelinta neerfaha, taasoo caddaynaysa in azoospermia ay bixiso dhimashada unugyada la qabsiga dheef-shiid kiimikaadka. Muujinta daran ee MFN2 waxay si buuxda u badbaadin kartaa PN-ka burburka terminal-ka oo leh yaraanta OXPHOS ee daran, isticmaalka badan ee DNA-da mitochondrial, iyo shabakadda mitochondrial oo si muuqata u jabtay, taas oo sii xoojinaysa in qaabkan neerfaha uu xitaa ka soo kaban karo marxaladda hore ee cudurka ka hor dhimashada unugyada.
Si aan u sawirno mitochondria-ka ku jira PN-yada Mfn2 ee knockout-ka ah, waxaan isticmaalnay nooc jiir ah oo u oggolaanaya mitochondria-ka ku tiirsan Cre inuu bartilmaameedsado borotiinka jaalaha ah ee fluorescent (YFP) (mtYFP) (20) Muujinta Cre waxaana hubinay qaab-dhismeedka mitochondrial-ka in vivo. Waxaan ogaanay in burburka hidda-wadaha Mfn2 ee PN-yada uu horseedi doono kala-qaybsanaanta tartiib tartiib ah ee shabakadda mitochondrial (Jaantuska S1A), isbeddelka ugu horreeyana waxaa la helay 3 toddobaad jir. Taas bedelkeeda, burburka weyn ee lakabka unugyada PN, sida lagu caddeeyay lumitaanka Calbindin immunostaining, ma uusan bilaaban ilaa 12 toddobaad jir (Jaantuska 1, A iyo B). Is-waafajinta waqtiga ee u dhexeeya isbeddellada ugu horreeya ee qaab-dhismeedka mitochondrial iyo bilawga muuqda ee dhimashada neerfaha ayaa naga dhigtay inaan baarno isbeddellada dheef-shiid kiimikaadka ee ay kiciso cilladda mitochondrial ka hor dhimashada unugyada. Waxaan samaynay istaraatiijiyad ku salaysan kala soocidda unugyada ee firfircoon (FACS) si loo kala saaro YFP (YFP+)-muujinaysa PN (Jaantuska 1C), iyo jiirka xakamaynta (Mfn2 + / loxP :: mtYFP loxP-stop-loxP: : L7-cre), oo halkan loogu yeero CTRL (Jaantuska S1B). Hagaajinta istaraatiijiyadda gate-ka iyadoo lagu salaynayo xoogga qaraabada ah ee calaamadda YFP waxay noo ogolaanaysaa inaan nadiifino jirka YFP+ (YFPhigh) ee PN-yada aan ahayn PN-yada (YFPneg) (Jaantuska S1B) ama jajabyada axon/dendritic ee fluorescent-ka ah (YFPlow; Sawirka S1D, bidix), oo lagu xaqiijiyay mikroskoobka confocal (Jaantuska S1D, midig). Si loo xaqiijiyo aqoonsiga dadka la kala saaray, waxaan sameynay proteomics LFQ ka dibna falanqaynta qaybaha ugu muhiimsan, waxaanan ogaanay inay jirto kala soocid cad oo u dhaxaysa unugyada YFPhigh iyo YFPneg (Jaantuska S1C). Unugyada sare ee YFP waxay muujiyeen kobcinta saafiga ah ee calaamadaha PN-yada la yaqaan (tusaale ahaan Calb1, Pcp2, Grid2 iyo Itpr3) (21, 22), laakiin ma jirin koboc borotiinno ah oo si caadi ah loogu muujiyo neerfayaasha ama noocyada kale ee unugyada (Jaantuska 1D)). Isbarbardhigga u dhexeeya muunado ku jira unugyada sare ee YFP ee la kala soocay ee lagu soo ururiyay tijaabooyin madax-bannaan ayaa muujiyay isku-xidhnaan > 0.9, taasoo muujinaysa dib-u-soo-saar wanaagsan oo u dhexeeya nuqullada bayoolojiga (Jaantuska S1E). Marka la soo koobo, xogtani waxay xaqiijisay qorshahayaga go'doominta degdegga ah iyo tan gaarka ah ee PN-ka suurtagalka ah. Sababtoo ah nidaamka darawalka L7-cre ee la isticmaalay wuxuu kiciyaa dib-u-soo-celinta mosaic usbuuca ugu horreeya ka dib dhalmada (23), waxaan bilownay inaan jiirarka ka jarno CTRL iyo shuruudaysan (Mfn2 loxP / loxP :: mtYFP loxP-stop-loxP :: L7-cre) Ururi neerfayaasha. Ka dib marka dib-u-soo-celinta la dhammeeyo, waxaa loo yaqaan Mfn2cKO 4 toddobaad jir. Barta ugu dambeysa, waxaan doorannay 8 toddobaad oo da'da ah markii lakabka PN uu ahaa mid aan waxba tarayn inkastoo uu si cad u kala qaybsan yahay mitochondrial (Jaantuska 1B iyo Sawirka S1A). Guud ahaan, waxaan tirinnay wadarta 3013 borotiin, kuwaas oo qiyaastii 22% ay ku salaysnaayeen sharraxaadda MitoCarta 2.0 oo ku salaysan borotiinka mitochondrial sida mitochondria (Jaantuska 1E) (Jaantuska 1E) (24). Falanqaynta muujinta hidda-wadaha ee kala duwan ee la sameeyay usbuuca 8 waxay muujisay in 10.5% oo keliya dhammaan borotiinnada ay lahaayeen isbeddello muhiim ah (Jaantuska 1F iyo Sawirka S1F), kuwaas oo 195 borotiinno ah hoos loo dhigay oo 120 borotiinno ah la hagaajiyay (Jaantuska 1F). Waxaa xusid mudan in "falanqaynta waddo cusub" ee xogtan ay muujinayso in hiddo-wadaha kala duwan ee la muujiyay ay inta badan ka tirsan yihiin qaybo xaddidan oo ah waddooyin dheef-shiid kiimikaad oo gaar ah (Jaantuska 1G). Waxaa xiiso leh, inkastoo hoos u dhaca waddooyinka la xiriira OXPHOS iyo calaamadaynta kaalshiyamka ay xaqiijinayso kicinta cillad mitochondrial ah oo ku timaadda PN-yada aan lahayn isku-darka, qaybaha kale ee inta badan ku lug leh dheef-shiid kiimikaadka amino acid-ka ayaa si weyn loo hagaajiyay, taas oo la jaanqaadaysa dheef-shiid kiimikaadka ka dhaca PN-yada mitochondrial. Dib-u-hagaajinta waa mid joogto ah. cillad.
(A) Sawirrada matalaadda ee qaybaha maskaxda ee jiirka CTRL iyo Mfn2cKO oo muujinaya luminta sii socota ee PNs (calbindin, cawl); xudunta waxaa lagu lid ku ahaa DAPI. (B) Tirada (A) (falanqaynta hal-dhinac ee kala duwanaanshaha, ***P<0.001; n = 4 ilaa 6 goobood oo ka yimid saddex jiir). (C) Socodka shaqada ee tijaabada ah. (D) Qaybinta khariidadda kulaylka ee calaamadaha gaarka u ah Purkinje (sare) iyo noocyada kale ee unugyada (dhexe). (E) Jaantuska Venn oo muujinaya tirada borotiinnada mitochondrial ee lagu aqoonsaday PN-ka la kala saaray. (F) Sawirka foolkaano ee borotiinnada si kala duwan loo muujiyey ee neerfaha Mfn2cKO 8 toddobaad (qiimaha goynta muhiimka ah ee 1.3). (G) Falanqaynta waddada hal-abuurka waxay muujinaysaa shanta waddo ee ugu muhiimsan ee kor u qaadista (casaanka) iyo hoos u dhigista (buluugga) ee Mfn2cKO PN oo loo kala saaray 8 toddobaad. Heerka muujinta celceliska ee borotiin kasta oo la ogaaday ayaa la muujiyay. Khariidadda kulaylka ee cabbirka cawlan: qiimaha P ee la hagaajiyay. ns, muhiim maaha.
Xogta Proteomics waxay muujisay in muujinta borotiinka ee isku-dhafka I, III, iyo IV ay si tartiib tartiib ah hoos ugu dhacday. Isku-dhafka I, III, iyo IV dhammaantood waxay ka koobnaayeen subunits muhiim ah oo mtDNA-ku-qoran, halka isku-dhafka II, kaas oo ahaa kaliya nuclear-ku-qoran, asal ahaan aan saameyn ku yeelan (Jaantuska 2A iyo Sawirka S2A). . Iyada oo la raacayo natiijooyinka proteomics, immunohistochemistry ee qaybaha unugyada maskaxda ayaa muujiyay in heerka subunit MTCO1 (mitochondrial cytochrome C oxidase subunit 1) ee isku-dhafka IV ee PN uu si tartiib tartiib ah hoos ugu dhacay (Jaantuska 2B). Subunit Mtatp8 ee mtDNA-ku-qoran ayaa si weyn hoos ugu dhacay (Jaantuska S2A), halka heerka xaaladda deggan ee subunit ATP synthase ee nuclear-ku-qoran uu weli isbeddelin, kaas oo la jaanqaadaya isku-dhafka F1 ee ATP synthase ee deggan marka muujinta mtDNA ay deggan tahay. Samaynta waa mid joogto ah. Jooji (7). Qiimaynta heerka mtDNA ee PN-yada Mfn2cKO ee kala soocay iyadoo la adeegsanayo falgalka silsiladda polymerase-ka waqtiga-dhabta ah (qPCR) waxay xaqiijisay hoos u dhaca tartiib tartiib ah ee lambarka nuqulka mtDNA. Marka la barbardhigo kooxda xakamaynta, da'da 8 toddobaad, kaliya qiyaastii 20% heerka mtDNA ayaa la hayay (Jaantuska 2C). Iyada oo la raacayo natiijooyinkan, rinjiyeynta mikroskoobka confocal ee Mfn2cKO PNs ayaa loo isticmaalay in lagu ogaado DNA-da, taasoo muujinaysa isticmaalka ku-tiirsanaanta waqtiga ee nucleotides-ka mitochondrial (Jaantuska 2D). Waxaan ogaanay in musharixiinta qaarkood oo ku lug leh burburka borotiinka mitochondrial iyo jawaabta walbahaarka la hagaajiyay, oo ay ku jiraan Lonp1, Afg3l2 iyo Clpx, iyo arrimaha isku-dhafka isku-dhafka ah ee OXPHOS. Isbeddello muhiim ah laguma arag heerarka borotiinnada ku lug leh apoptosis (Jaantuska S2B). Sidoo kale, waxaan ogaanay in kanaallada mitochondria iyo endoplasmic reticulum ee ku lug leh gaadiidka kalsiyum ay leeyihiin oo keliya isbeddello yar yar (Jaantuska S2C). Intaa waxaa dheer, qiimeynta borotiinnada la xiriira autophagy ma helin wax isbeddello muhiim ah, taas oo la jaanqaadaysa kicinta muuqata ee autophagosomes-ka lagu arkay in vivo iyadoo la adeegsanayo immunohistochemistry iyo electron mikroskoob (Jaantuska S3). Si kastaba ha ahaatee, cilladda OXPHOS ee sii socota ee PNs waxaa la socda isbeddello muuqda oo mitochondrial ah. Kooxaha Mitochondrial waxaa lagu arki karaa jirka unugyada iyo geedaha dendritic ee Mfn2cKO PNs da'doodu u dhaxayso 5 iyo 8 toddobaad, qaab-dhismeedka xuubka gudahana wuxuu maray isbeddello qoto dheer (Jaantuska S4, A iyo B). Iyada oo la raacayo isbeddelladan ultrastructural iyo hoos u dhac weyn oo ku yimid mtDNA, falanqaynta xaleefyada maskaxda ee ba'an oo leh tetramethylrhodamine methyl ester (TMRM) waxay muujisay in awoodda xuubka mitochondrial ee Mfn2cKO PNs ay si weyn hoos ugu dhacday (Jaantuska S4C).
(A) Falanqaynta koorsada waqtiga ee heerka muujinta isku-dhafka OXPHOS. Kaliya tixgeli borotiinnada leh P<0.05 8 toddobaad (ANOVA laba-geesood ah). Khadadka dhibcaha leh: Ma jiro hagaajin marka la barbar dhigo CTRL. (B) Bidix: Tusaale qayb cerebellar ah oo lagu calaamadeeyay antibody-ka anti-MTCO1 (bar cabir, 20 μm). Aagga ay ku jiraan unugyada Purkinje waxaa daboolay jaalle. Midig: Tirada heerarka MTCO1 (falanqaynta hal-dhinac ee kala duwanaanshaha; n = 7 ilaa 20 unug oo laga falanqeeyay saddex jiir). (C) falanqaynta qPCR ee lambarka nuqulka mtDNA ee PN-ka la kala saaray (falanqaynta hal-dhinac ee kala duwanaanshaha; n = 3 ilaa 7 jiir). (D) Bidix: Tusaale jeex cerebellar ah oo lagu calaamadeeyay antibody-ka anti-DNA (bar cabir, 20 μm). Aagga ay ku jiraan unugyada Purkinje waxaa daboolay jaalle. Midig: Tirada dhaawacyada mtDNA (falanqaynta hal-dhinac ee kala duwanaanshaha; n = 5 ilaa 9 unug oo ka yimid saddex jiir). (E) Tusaale ahaan qayb maskaxda ah oo degdeg ah oo muujinaysa unugyada mitoYFP + Purkinje (fallaadho) oo ku jirta duubista qabsashada qabsashada unugyada oo dhan. (F) Tirada qalooca IV. (G) Diiwaanada matalaadda ee duritaanka hadda ee depolarizing ee unugyada CTRL iyo Mfn2cKO Purkinje. Raad-raaca sare: Garaaca ugu horreeya ee kiciyay AP. Raad-raaca hoose: Soo noqnoqoshada AP ugu badan. (H) Tirada soo-gelinta iswada ee postsynaptic (sPSPs). Raad-raaca duubista ee wakiilka ah iyo saamiga zoom-keeda waxaa lagu muujiyay (I). Falanqaynta hal-dhinac ah ee kala duwanaanshaha la falanqeeyay n = 5 ilaa 20 unug oo ka yimid saddex jiir. Xogta waxaa lagu muujiyay celcelis ahaan ± SEM; *P<0.05; **P<0.01; ***P<0.001. (J) Raad-raacyo matalaad ah oo AP iswada ah oo la duubay iyadoo la adeegsanayo qaabka qabsashada qabsashada ee godan. Raad-raaca sare: Soo noqnoqoshada AP ugu badan. Raad-raaca hoose: zoom-ka hal AP. (K) Tirada celceliska iyo soo noqnoqoshada AP ugu badan sida waafaqsan (J). Tijaabada Mann-Whitney; n = 5 unug ayaa laga falanqeeyay afar jiir. Xogta waxaa loo muujiyaa celcelis ahaan ± SEM; muhiim maaha.
Waxyeellada muuqata ee OXPHOS ayaa lagu ogaaday Mfn2cKO PN oo 8 toddobaad jir ah, taasoo muujinaysa in shaqada jir ahaaneed ee neerfayaasha ay aad u liidato. Sidaa darteed, waxaan falanqaynay astaamaha korontada ee aan firfircoonayn ee neerfayaasha aan lahayn OXPHOS 4 ilaa 5 toddobaad iyo 7 ilaa 8 toddobaad annagoo samaynayna duubista isku-xidhka unugyada oo dhan ee xaleefyada maskaxda ee degdegga ah (Jaantuska 2E). Si aan la filayn, celceliska awoodda xuubka nasashada iyo iska caabbinta gelinta neerfayaasha Mfn2cKO waxay la mid ahaayeen xakamaynta, inkastoo ay jireen kala duwanaansho yar oo u dhexeeya unugyada (Jadwalka 1). Sidoo kale, da'da 4 ilaa 5 toddobaad, wax isbeddello muhiim ah laguma helin xiriirka hadda jira-danab (qallooca IV) (Jaantuska 2F). Si kastaba ha ahaatee, ma jirin neerfayaasha Mfn2cKO 7 ilaa 8 toddobaad jir ah oo ka badbaaday nidaamka IV (tallaabada hyperpolarization), taasoo muujinaysa in ay jirto xasaasiyad cad oo ku aaddan awoodda hyperpolarization marxaladdan dambe. Taas bedelkeeda, neerfaha Mfn2cKO, qulqulka depolarizing-ka ee sababa sii deynta awoodda ficilka soo noqnoqda (AP) si fiican ayaa loo dulqaatay, taasoo muujinaysa in qaababka sii deynta guud aysan si weyn uga duwanayn kuwa neerfaha xakamaynta ee 8-toddobaad jir ah (Jadwalka 1 iyo Jaantuska 2G). Sidoo kale, soo noqnoqoshada iyo baaxadda qulqulka postsynaptic-ka ee iskiis u dhaca (sPSCs) waxay la mid ahaayeen kuwa kooxda xakamaynta, soo noqnoqoshada dhacdooyinkana waxay ka korodhay 4 toddobaad ilaa 5 toddobaad ilaa 7 toddobaad ilaa 8 toddobaad iyadoo koror la mid ah la sameeyay (Jaantuska 2, H iyo I). Muddada bislaanshaha synaptic ee PNs (25). Natiijooyin la mid ah ayaa la helay ka dib markii la sameeyay balastar PNs oo daloolsan. Qaabeyntani waxay ka hortagtaa magdhowga suurtagalka ah ee cilladaha ATP ee unugyada, sida laga yaabo inay ka dhacaan duubista isku-xidhka balastar-ka unugyada oo dhan. Gaar ahaan, awoodda xuubka nasashada iyo soo noqnoqoshada rasaasta iskeed u dhacda ee neerfayaasha Mfn2cKO ma saameyn (Jaantuska 2, J iyo K). Marka la soo koobo, natiijooyinkani waxay muujinayaan in PN-yada leh cilladda OXPHOS ee muuqata ay si fiican ula qabsan karaan qaababka dheecaanka soo noqnoqda ee soo noqnoqda, taasoo muujinaysa in ay jirto hab magdhow ah oo u oggolaanaya inay ilaaliyaan jawaabaha elektrofiziyoolajiyadeed ee ku dhow caadiga.
Xogta waxaa lagu muujiyaa celcelis ahaan ± SEM (falanqaynta hal-dhinac ee kala duwanaanshaha, imtixaanka isbarbardhigga badan ee Holm-Sidak; *P<0.05). Lambarka cutubka waxaa lagu tilmaamay qaws.
Waxaan u diyaarsannay inaan baarno in qayb kasta oo ka mid ah xogta proteomics (Jaantuska 1G) ay ku jiraan waddooyin ka hortagi kara yaraanta OXPHOS ee daran, taasoo sharraxaysa sababta PN-ka saameeyay uu u ilaalin karo electrophysiology-ga caadiga ah ee ku dhow (Jaantuska 2, E ilaa K). . Falanqaynta Proteomics waxay muujisay in enzymes-ka ku lug leh catabolism-ka amino acids silsilad laamood leh (BCAA) si weyn loo habeeyay (Jaantuska 3A iyo Jaantuska S5A), badeecada ugu dambeysa ee acetyl-CoA (CoA) ama succinyl CoA waxay ku dari kartaa tricarboxylates wareegga arteriosclerosis Acid (TCA). Waxaan ogaanay in waxa ku jira BCAA transaminase 1 (BCAT1) iyo BCAT2 labaduba ay kordheen. Waxay kiciyaan tallaabada ugu horreysa ee catabolism-ka BCAA iyagoo soo saaraya glutamate ka yimaada α-ketoglutarate (26). Dhammaan qaybaha hoose ee ka kooban isku-dhafka silsiladda laanta ah ee keto acid dehydrogenase (BCKD) waa la hagaajiyay (isku-dhafka ayaa kiciya decarboxylation-ka xiga iyo kan aan dib loo celin karin ee qalfoofka kaarboon ee BCAA ee ka dhashay) (Jaantuska 3A iyo Jaantuska S5A). Si kastaba ha ahaatee, isbeddello muuqda oo ku yimid BCAA lafteeda laguma helin PN-ka la kala saaray, taasoo laga yaabo inay sabab u tahay kororka qaadashada unugyada ee asiidhyadan muhiimka ah ee amino ama isticmaalka ilo kale (glucose ama lactic acid) si loogu kabo wareegga TCA (Jaantuska S5B). PN-yada ka maqan OXPHOS waxay sidoo kale muujiyeen burburka glutamine iyo hawlaha transamination ee kordhay 8 toddobaad jir, taas oo laga arki karo kor u qaadista enzymes-ka mitochondrial glutaminase (GLS) iyo glutamine pyruvate transaminase 2 (GPT2) (Jaantuska 3, A iyo C). Waxaa xusid mudan in kor u qaadista GLS ay ku kooban tahay glutaminase isoform C (GLS-GAC) oo isku dhafan (isbeddelka Mfn2cKO/CTRL waa qiyaastii 4.5-laab, P = 0.05), iyo kor u qaadista gaarka ah ee unugyada kansarka Waxay taageeri kartaa tamarta biochondroial. (27).
(A) Khariidadda kulaylka waxay muujinaysaa isbeddelka laablaabka heerka borotiinka ee wadada la cayimay 8 toddobaad. (B) Tusaale jeex cerebellar ah oo lagu calaamadeeyay antibody-ka ka hortagga PCx (bar miisaanka, 20 μm). Fallaadha jaalaha ah waxay tilmaamaysaa jirka unugyada Purkinje. (C) Falanqaynta muujinta borotiinka ee waqtiga koorsada oo loo aqoonsaday musharax muhiim u ah atherosclerosis (tijaabo t-badan, *FDR <5%; n = 3-5 jiir). (D) Kor: Jaantus sawireed oo muujinaya siyaabaha kala duwan ee loo galo kaarboonka calaamadaysan ee ku jira raad-raaca pyruvate [1-13C] (tusaale ahaan, iyada oo loo marayo PDH ama wadada halbowlaha). Hoos: Jaantuska violin wuxuu muujinayaa boqolleyda kaarboonka hal-calaamadda leh (M1) oo loo beddelay aspartic acid, citric acid iyo malic acid ka dib markii lagu calaamadeeyay xaleefyada cerebellar ee daran oo leh [1-13C]pyruvate (tijaabo t-lammaane ah; ** P <0.01). (E) Falanqaynta taariikhda waqtiga oo dhammaystiran ee wadada la tilmaamay. Kaliya tixgeli borotiinnada leh P<0.05 8 toddobaad. Xariiqda la jaray: qiimo hagaajin la'aan ah (falanqaynta laba-geesoodka ah ee kala duwanaanshaha; * P <0.05; *** P <0.001). Xogta waxaa loo muujiyaa celcelis ahaan ± SEM.
Falanqaynteenna, catabolism-ka BCAA wuxuu noqday mid ka mid ah waddooyinka ugu muhiimsan ee kor u qaadista. Xaqiiqadani waxay si xooggan u soo jeedinaysaa in mugga hawo-qaadashada ee galaya wareegga TCA laga yaabo in lagu beddelo PN oo aan lahayn OXPHOS. Tani waxay matali kartaa qaab weyn oo dib-u-habaynta dheef-shiid kiimikaadka neerfaha, kaas oo saameyn toos ah ku yeelan kara fiisiyoolajiyadda neerfaha iyo badbaadada inta lagu jiro ilaalinta cilladda daran ee OXPHOS. Iyada oo la raacayo mala-awaalkan, waxaan ogaanay in ensaymka ugu weyn ee ka hortagga atherosclerosis PCx uu kor u kacay (Mfn2cKO/CTRL wuxuu isbeddelaa qiyaastii 1.5 jeer; Jaantuska 3A), kaas oo kiciya beddelka pyruvate ilaa oxaloacetate (28), kaas oo la rumeysan yahay inuu ku jiro unugyada maskaxda. Muujinta gudaha waxay ku kooban tahay astrocytes (29, 30). Iyada oo la raacayo natiijooyinka proteomics, mikroskoobka confocal wuxuu muujiyay in muujinta PCx ay si gaar ah oo si weyn u korodhay PN-yada OXPHOS-ka ee aan lahayn, halka falcelinta PCx ay inta badan ku koobnayd unugyada Bergmann glial ee ku xiga ee xakamaynta (Jaantuska 3B). Si aan si firfircoon u tijaabino kor u kaca la arkay ee PCx, waxaan xaleefyada cerebellar ee ba'an ku daweynay raadraaca pyruvate [1-13C]. Markii pyruvate lagu oksaydhiyay pyruvate dehydrogenase (PDH), calaamadda isotope-keeda way baaba'day , Laakiin waxaa lagu daray dhexdhexaadiyeyaasha wareegga TCA marka pyruvate lagu dheefshiido falcelinta xididdada dhiigga (Jaantuska 3D). Si aan u taageerno xogtayada proteomics, waxaan aragnay tiro badan oo calaamado ah oo ka yimid raadraacan oo ku jira aashitada aspartic ee xaleefyada Mfn2cKO, halka citric acid iyo malic acid ay sidoo kale lahaayeen isbeddel dhexdhexaad ah, inkastoo aysan muhiim ahayn (Jaantuska 3D).
Neef-sidayaasha dopamine ee jiirka MitoPark oo leh cillad mitochondrial ah oo ay sababaan neerfayaasha dopamine si gaar ah u burburiya hidda-wadaha mitochondrial transcription factor A (Tfam) (Jaantuska S6B), muujinta PCx ayaa sidoo kale si weyn loo hagaajiyay (31), taasoo muujinaysa in acetone acid arteriosclerosis Dhacitaanka cudurka waxaa lagu xakameeyaa inta lagu jiro cilladda neerfayaasha OXPHOS ee jirka. Waxaa xusid mudan in la ogaaday in ensaymyada gaarka ah (32-34) ee laga yaabo in lagu muujiyo neerfayaasha lala xiriirin karo arteriosclerosis ay si weyn u kor u kaceen PN-yada ka maqan OXPHOS, sida propionyl-CoA carboxylase (PCC-A), Malonyl-CoA waxay propionyl-CoA u beddeshaa succinyl-CoA iyo mitochondrial malic enzyme 3 (ME3), kuwaas oo doorkooda ugu weyn uu yahay inay ka soo kabtaan pyruvate malate (Jaantuska 3, A iyo C) (33, 35). Intaa waxaa dheer, waxaan helnay koror muhiim ah oo ku yimid enzyme-ka Pdk3, kaas oo fosfooraska kiciya sidaas darteedna joojiya PDH (36), halka aan wax isbeddel ah laga helin enzyme-ka Pdp1 ee dhaqaajiya PDH ama isku-dhafka enzyme-ka PDH laftiisa (Jaantuska 3A). Si joogto ah, Mern2cKO PNs, fosfooraska qaybta α1 ee α (PDHE1α) ee qaybta pyruvate dehydrogenase E1 ee isku-dhafka PDH ee Ser293 (oo loo yaqaan inay joojiso dhaqdhaqaaqa enzyme-ka ee PDH) ayaa la xoojiyay (Jaantuska S6C) (Jaantuska S6C). Pyruvate ma laha marin xididada ah.
Ugu dambeyntii, waxaan ogaanay in wadada ugu sarreysa ee biosynthesis-ka serine iyo glycine, wareegga mitochondrial folate (1C) ee la xiriira iyo biosynthesis-ka proline (Jaantuska 1G iyo Sawirka S5C) dhammaantood si weyn ayaa loo habeeyay, sida laga soo xigtay warbixinnada, inta lagu jiro habka kicinta. Unugyada ku hareeraysan waxaa lagu kiciyaa cillad mitochondrial (5-7). Falanqaynta Confocal ee taageerta xogtan proteomics waxay muujisay in PN oo ay ku maqan yihiin OXPHOS, xaleefyada cerebellar ee jiirarka 8-toddobaad jirka ah ayaa lagu sameeyay serine hydroxymethyltransferase 2 (SHMT2), oo ah ensaym muhiim ah oo ka mid ah wareegga folate-ka mitochondrial. Jawaab celin difaac oo muhiim ah (Jaantuska S5D). 13 xaleef oo cerebellar ah oo CU-glucose ah oo lagu daray gulukooska, tijaabooyinka raadinta dheef-shiid kiimikaadka ayaa sii xaqiijiyay kor u qaadista xakamaynta biosynthesis-ka serine iyo proline, taasoo muujinaysa in qulqulka isoforms-ka kaarboonka ee serine iyo proline uu kordhay (Jaantuska S5E). Maadaama falcelinta ay dhiirrigelisay GLS iyo GPT2 ay mas'uul ka yihiin isku-darka glutamate-ka glutamine iyo isu-beddelka u dhexeeya glutamate iyo α-ketoglutarate, kor u qaadistooda waxay muujinaysaa in neerfayaasha aan OXPHOS lahayn ay leeyihiin baahi kordhay oo loo qabo glutamate. Tani waxaa laga yaabaa in loogu talagalay in la ilaaliyo biosynthesis-ka sii kordhaya ee proline (Jaantuska S5C). Marka la barbardhigo isbeddelladan, falanqaynta proteomic ee astrocytes-ka cerebellar ee ka imanaya jiirarka Mfn2cKO ee gaarka u ah PN ayaa muujisay in waddooyinkan (oo ay ku jiraan dhammaan antiperoxidases) aysan si weyn isu beddelin muujinta, sidaas darteed waxay muujineysaa Dib-u-habayntan dheef-shiid kiimikaadka ayaa ah mid xulasho u ah PN-ka burburay (Jaantuska S6, D ilaa G).
Marka la soo koobo, falanqayntani waxay muujisay qaabab aad u kala duwan oo firfircooni ku meel gaar ah oo ku saabsan dariiqooyinka dheef-shiid kiimikaadka gaarka ah ee PN-yada. Inkasta oo shaqada mitochondrial-ka ee aan caadiga ahayn ay horseedi karto atherosclerosis hore iyo dib-u-habeyn 1C ah (Jaantuska 3E iyo Jaantuska S5C), iyo xitaa isbeddellada la saadaalin karo ee muujinta isku-dhafka I iyo IV, isbeddellada ku yimaada isku-darka serine de novo waa kaliya Waxay muuqatay oo keliya marxaladaha dambe. Cilladda OXPHOS (Jaantuska 3E iyo Jaantuska S5C). Natiijooyinkani waxay qeexayaan hab isku xigxig ah oo mitochondrial-ka uu kiciyay walbahaarka (wareegga 1C) iyo cytoplasmic (biosynthesis serine) ay si isku mid ah uga jawaabaan kororka atherosclerosis ee wareegga TCA si dib loogu qaabeeyo dheef-shiid kiimikaadka neerfaha.
PN-yada OXPHOS ee 8-toddobaad jir ah waxay ilaalin karaan dhaqdhaqaaqa kicinta soo noqnoqda badan waxayna mari karaan dib-u-xiriir dheef-shiid kiimikaad oo muhiim ah si loo magdhabo cilladda mitochondrial. Daahfurkani wuxuu soo bandhigayaa suurtagal xiiso leh in xitaa waqtigan xaadirka ah, unugyadani ay sidoo kale heli karaan faragelin daaweyn ah si loo dib u dhigo ama looga hortago neerfaha. Dambe. Waxaan xallinay suurtagalnimadan iyada oo loo marayo laba faragelin oo madax-bannaan. Habka ugu horreeya, waxaan u qaabaynay fayras la xiriira adeno-ku-tiirsan Cre-dependent adeno (AAV) si MFN2 loogu muujiyo PN-yada aan lahayn OXPHOS in vivo (Jaantuska S7A). AAV-ka codeeya MFN2 iyo hidda-wadaha wariyaha fluorescent-ka mCherry (Mfn2-AAV) ayaa lagu xaqiijiyay dhaqamada neerfaha ee aasaasiga ah in vitro, taas oo keentay in MFN2 lagu muujiyo si ku-tiirsan Cre waxayna badbaadisay qaab-dhismeedka mitochondrial, taasoo ka hortagaysa neuromutation ee neerfayaasha Mfn2cKO (Jaantuska S7, B, D iyo E). Marka xigta, waxaan sameynay tijaabooyin in vivo ah si aan si qoto dheer ah ugu gaarsiino Mfn2-AAV oo 8-toddobaad jir ah xuubka maskaxda ee Mfn2cKO iyo xakamaynta jiirka, waxaana falanqeynay jiirarka 12-toddobaad jir ah (Jaantuska 4A). Jiirarka Mfn2cKO ee la daweeyay ayaa dhintay (Jaantuska 1, A iyo B) (16). Gudbinta fayraska ee in vivo waxay keentay muujinta xulashada ee PN ee wareegyada maskaxda qaarkood (Jaantuska S7, G iyo H). Duritaanka AAV ee xakamaynta oo muujinaya mCherry oo keliya (Ctrl-AAV) saameyn muhiim ah kuma yeelan heerka neerfaha ee xayawaanka Mfn2cKO. Taas bedelkeeda, falanqaynta Mfn2cKOs ee lagu sameeyay Mfn2-AAV waxay muujisay saameyn ilaalin oo muhiim ah oo lakabka unugyada PN ah (Jaantuska 4, B iyo C). Gaar ahaan, cufnaanta neerfaha waxay u muuqataa mid aan laga sooci karin xayawaanka xakamaynta (Jaantuska 4, B iyo C, iyo Sawirka S7, H iyo I). Muujinta MFN1 laakiin aan ahayn MFN2 si isku mid ah ayay waxtar ugu leedahay badbaadinta dhimashada neerfaha (Jaantuska 4C iyo Jaantuska S7, C iyo F), taas oo tilmaamaysa in muujinta ectopic MFN1 ay si wax ku ool ah u buuxin karto la'aanta MFN2. Falanqayn dheeraad ah oo lagu sameeyay heerka PN-ka keliya ayaa muujisay in Mfn2-AAV ay si weyn u badbaadisay qaab-dhismeedka sare ee mitochondria, heerarka mtDNA ee caadiga ahaa, waxayna rogtay muujinta sare ee calaamadda ka hortagga angiogenesis-ka PCx (Jaantuska 4, C ilaa E). Kormeerka muuqaalka ee jiirka Mfn2cKO ee la badbaadiyay ee xaalad nasasho ah ayaa muujisay in qaab-dhismeedkooda iyo calaamadaha dhaqdhaqaaqa (dhaqdhaqaaqa S1 ilaa S3) la hagaajiyay. Gunaanadkii, tijaabooyinkani waxay muujinayaan in dib u soo celinta dib u soo celinta MFN2 ee PN-yada oo si daran ugu yar OXPHOS ay ku filan tahay in la beddelo isticmaalka mtDNA oo la kiciyo atherosclerosis, taasoo ka hortagaysa burburka axon iyo dhimashada neerfaha ee jirka.
(A) Qorshe muujinaya jadwalka tijaabada ah ee lagu durayo AAV codeynta MFN2 marka marin-haweedka dheef-shiid kiimikaadka ee la tilmaamay la hawlgeliyo. (B) Sawirrada isku-dhafka ah ee godadka maskaxda ee 12-toddobaad jir ah ee lagu sameeyay 8 toddobaad jiirarka Mfn2cKO oo lagu calaamadeeyay antibody-ga ka hortagga Calbindin. Midig: Cabbirka fiilooyinka axon. Cabbirka zoom-ka axon waa 450 iyo 75 μm. (C) Bidix: Tirada cufnaanta unugyada Purkinje ee wareegga wareejinta AAV (AAV+) (falanqaynta hal-dhinac ee kala duwanaanshaha; n = 3 jiir). Midig: falanqaynta diiradda mtDNA ee PN-ka la beddelay usbuuca 12 (tijaabada t-ga aan la isku-dubaridka ahayn; n = 6 unug oo ka yimid saddex jiir). * P <0.05; ** P <0.01. (D) Micrographs elektaroonik ah oo gudbin wakiil ah oo PNs ah oo ka mid ah qaybaha maskaxda Mfn2cKO oo lagu beddelay vectors-ka fayraska ee la tilmaamay. Maaskarada casaanka ah waxay muujinaysaa aagga ay ku jiraan dendrites, afargeeska dhibcaha jaalaha ahina wuxuu muujinayaa zoom-ka ku yaal dhinaca midig; n wuxuu matalaa xudunta. Barka cabbirka, 1μm. (E) wuxuu muujinayaa tusaale rinjiyeynta PCx ee PN ee la sameeyay 12 toddobaad. Barka cabbirka, 20μm. OE, muujinta xad dhaafka ah; FC, isbeddelka laablaabka.
Ugu dambeyntii, waxaan baarnay muhiimadda ay leedahay badbaadada unugyada ee uu keeno peroxidase ee PN-yada kuwaas oo la kulmay cillad OXPHOS ah. Waxaan soo saarnay mCherry oo codeynaya AAV-shRNA (RNA gaaban oo timaha lagu xiro) oo si gaar ah u bartilmaameedsanaya jiirka PCx mRNA (AAV-shPCx), waxaanan ku durnay fayraska ama xakamaynta la isku qasay (AAV-scr) maskaxda jiirarka Mfn2cKO. Cirbadda waxaa la sameeyay usbuuca afraad ee da'da (Jaantuska 5A) si loo gaaro hoos u dhac PCx ah oo wax ku ool ah inta lagu jiro xilligii muujinta PCx ay korodhay (Jaantuska 3C) lakabka unugyada PN-na uu weli ahaa mid aan waxba tarayn (Jaantuska 1A). Waxaa xusid mudan in hoos u dhaca PCx (Jaantuska S8A) uu horseedo dardargelin weyn oo dhimashada PN ah, taas oo ku xaddidan giraanta cudurka qabta (Jaantuska 5, B iyo C). Si loo fahmo habka saamaynta dheef-shiid kiimikaadka ee ay keento kor u qaadista PCx, waxaan barannay xaaladda redox ee PNs ka dib markii PCx la lumiyay iyo dareemayaasha noolaha indhaha ee AAV-dhexdhexaadka ah Grx1-roGFP2 ayaa isku mar la muujiyay (Jaantuska S8, B ilaa D) si loo qiimeeyo glutathione Isbeddelka qaraabada ah ee awoodda redox-ka peptide (38). Kadib, waxaan sameynay mikroskoob sawir-qaadis oo laba-foton ah oo iftiin leh (FLIM) oo ku jira xaleefyada maskaxda ee daran ee Mfn2cKO 7-toddobaad jir ah ama kuwa xakamaynta si loo ogaado isbeddellada suurtagalka ah ee xaaladda redox-ka cytoplasmic ka dib markii la xaqiijiyay xaaladaha FLIM (Jaantuska S8, E ilaa G). Falanqaynta waxay muujisay koror weyn oo ku yimid xaaladda oksaydhka ee hal PN oo Mfn2cKO ah oo aan lahayn muujinta PCx, taas oo ka duwan neerfayaasha xakamaynta ama Mfn2cKO PNs oo muujinaya shRNA oo keliya oo la burburiyay (Jaantuska 5, D iyo E). Markii muujinta PCx hoos loo dhigay, boqolleyda Mfn2cKO PNs ee muujinaya xaalad aad u oksaydhsan ayaa kordhay in ka badan saddex jeer (Jaantuska 5E), taasoo muujinaysa in kor u qaadista PCx ay ilaalisay awoodda redox ee neerfayaasha qalloocan.
(A) Qorshe muujinaya jadwalka tijaabada ah ee lagu durayo AAV codeynta shPCx marka marinnada dheef-shiid kiimikaadka ee la tilmaamay la hawlgeliyo. (B) Sawirrada confocal ee matalaadda qaybaha maskaxda ee 8-toddobaad jir ah ee jiirarka Mfn2cKO ee la soo saaray oo lagu calaamadeeyay antibody-ka ka hortagga calcineurin 4 toddobaad. Barka cabbirka, 450μm. (C) Tirada cufnaanta unugyada Purkinje ee wareegyada la soo saaray ee AAV (falanqaynta hal-dhinac ee kala duwanaanshaha; n = 3 ilaa 4 jiir). Xogta waxaa lagu muujiyay celcelis ahaan ±SEM; ***P<0.001. (D) Sawirka matalaadda FLIM wuxuu muujinayaa celceliska cimriga ee PN-ga 7-toddobaad jirka ah ee muujinaya glutathione redox sensor Grx1-roGFP2 xaaladaha tijaabada ee la cayimay. Saamiga LUT (miiska raadinta): muddada waqtiga badbaadada (picoseconds). Barka cabbirka, 25μm. (E) Histogram-ku wuxuu muujinayaa qaybinta qiimayaasha cimriga Grx1-roGFP2 laga bilaabo (D) (n=158 ilaa 368 unug oo ku jira laba jiir xaalad kasta). Jaantuska pie-ka ee ka sarreeya histogram kasta: wuxuu muujinayaa tirada unugyada leh qiimayaasha cimriga oo aad u dheer (casaan, oksaydhaysan) ama gaaban (buluug, la dhimay), kuwaas oo ka badan 1 SD oo ah celceliska qiimaha cimriga ee CTRL-AAV-scr. (F) Qaabka la soo jeediyay wuxuu muujinayaa saameynta ilaalinta ee kor u qaadista PCx-ga neerfaha.
Guud ahaan, xogta aan halkan ku bixinno waxay muujinaysaa in dib-u-muujinta MFN2 ay si buuxda u badbaadin karto PN-ka horumarsan iyadoo ay jirto yaraanta OXPHOS ee daran, yaraanta mtDNA ee daran, iyo qaab-dhismeedka ista-la midka ah ee aan caadiga ahayn, taasoo keenta horumar joogto ah xitaa cudurrada horumarsan. Burburka neerfaha wuxuu bixiyaa caddayn dib loo celin karo oo ku saabsan marxaladda ka hor dhimashada unugyada. Heerkan dabacsanaanta dheef-shiid kiimikaadka waxaa sii xoojinaya awoodda neerfayaasha inay kiciyaan atherosclerosis (dib-u-warshadaynta wareegga TCA), kaas oo joojiya muujinta PCx ee PN-yada aan lahayn OXPHOS wuxuuna kordhiyaa dhimashada unugyada, sidaas darteedna wuxuu ciyaaraa door ilaalin ah (Jaantuska 5F).
Daraasaddan, waxaan ku bixinay caddayn muujinaysa in jawaabta PNs ee cilladda OXPHOS ay tahay in si tartiib tartiib ah loogu daro wareegga TCA atherosclerosis iyada oo loo marayo marin-hawleedka kala duwan ee firfircoonida oo ay kiciso barnaamijyada dheef-shiid kiimikaadka. Waxaan xaqiijinay falanqaynta proteomic iyadoo la adeegsanayo habab badan oo is-waafaqsan waxaanan shaaca ka qaadnay in marka ay caqabad ku noqoto cillad mitochondrial daran, neerfayaashu ay leeyihiin qaab aan hore loo aqoon oo dabacsanaan dheef-shiid kiimikaad ah. Si la yaab leh, habka dib-u-warshadaynta oo dhan daruuri maaha inuu calaamadeeyo xaaladda dheef-shiid kiimikaadka ee ugu dambeysa ee la socota neerfaha si tartiib tartiib ah oo aan laga noqon karin, laakiin xogteennu waxay soo jeedinaysaa inay noqon karto neerfayaasha dayactirka xitaa marxaladda ka hor dhimashada unugyada Habka magdhowga shaqada. Natiijadani waxay muujinaysaa in neerfayaashu ay leeyihiin heer aad u badan oo ah dabacsanaanta dheef-shiid kiimikaadka jirka. Xaqiiqadani waxay caddaynaysaa in dib-u-soo-saarka dambe ee MFN2 uu beddeli karo muujinta calaamadaha dheef-shiid kiimikaadka muhiimka ah wuxuuna ka hortagi karaa burburka PN. Taas beddelkeeda, waxay joojisaa atherosclerosis waxayna dardar gelisaa neerfayaasha. transsexual.
Mid ka mid ah natiijooyinka ugu xiisaha badan ee cilmi-baaristeena ayaa ah in PN-yada aan lahayn OXPHOS ay wax ka beddeli karaan dheef-shiid kiimikaadka wareegga TCA iyagoo kor u qaadaya enzymes-ka si gaar ah u kiciya arteriosclerosis. Dib-u-habaynta dheef-shiid kiimikaadka waa astaamo caadi ah oo unugyada kansarka ah, kuwaas oo qaarkood ku tiirsan glutamine si ay u kabaan dhexdhexaadiyeyaasha wareegga TCA si ay u soo saaraan kuwa u dhigma kuwa yareeya, kuwaas oo wada silsiladda neefsashada oo ilaaliya soo saarista lipid iyo preselectors biosynthesis nucleotide (39, 40). Daraasad dhowaan la sameeyay ayaa muujisay in unugyada durugsan ee la kulma cillad la'aanta OXPHOS, dib-u-xidhka dheef-shiid kiimikaadka glutamine/glutamate sidoo kale waa astaamo caan ah (5, 41), halkaas oo jihada gelitaanka glutamine ee wareegga TCA ay ku xiran tahay Sababtoo ah darnaanta dhaawaca OXPHOS (41). Si kastaba ha ahaatee, waxaa jira caddayn cad oo ku saabsan isku mid ahaanshaha dabacsanaanta dheef-shiid kiimikaadka neerfaha ee jirka iyo muhiimaddeeda suurtagalka ah ee xaaladda cudurka. Daraasad dhowaan lagu sameeyay vitro, neerfayaasha cortical-ka aasaasiga ah ayaa la muujiyay inay abaabulaan barkadaha glutamate ee gudbinta neerfaha, taasoo kor u qaadaysa dheef-shiid kiimikaadka oksaydhka iyo atherosclerosis xaaladaha walaaca dheef-shiid kiimikaadka (42). Waxaa xusid mudan in iyadoo la raacayo joojinta daawada ee enzyme-ka wareegga TCA succinate dehydrogenase, pyruvate carboxylation waxaa la rumeysan yahay inay ilaaliso isku-darka oxaloacetate ee neerfayaasha granule-ka ee la beeray (34). Si kastaba ha ahaatee, muhiimadda jir ahaaneed ee hababkani u leeyihiin unugyada maskaxda (halkaas oo atherosclerosis-ka la rumeysan yahay inuu inta badan ku kooban yahay astrocytes) wali waxay leedahay muhiimad jireed oo muhiim ah (43). Xaaladdan, xogteennu waxay muujinaysaa in PN-yada ay waxyeelleeyeen OXPHOS ee jirka loo beddeli karo burburka BCAA iyo carboxylation pyruvate, kuwaas oo ah labada ilo ee ugu muhiimsan ee kaabista dhexdhexaadinta barkadda TCA. Inkasta oo la soo jeediyay ka-qaybgalka catabolism-ka BCAA ee dheef-shiid kiimikaadka tamarta neerfaha, marka lagu daro doorka glutamate iyo GABA ee gudbinta neerfaha (44), weli ma jiraan wax caddayn ah oo ku saabsan farsamooyinkan gudaha jirka. Sidaa darteed, way fududahay in la qiyaaso in PN-yada aan shaqaynayn ay si toos ah u magdhabi karaan isticmaalka dhexdhexaadinta TCA ee ay horseeddo habka isku-darka iyadoo la kordhinayo atherosclerosis. Gaar ahaan, kor u qaadista PCx waxaa laga yaabaa in loo baahdo si loo ilaaliyo baahida sii kordheysa ee aspartic acid, taas oo lagu soo jeediyay unugyada sii kordhaya ee qaba cillad mitochondrial (45). Si kastaba ha ahaatee, falanqayntayada dheef-shiid kiimikaadka ma aysan muujin wax isbeddello muhiim ah oo ku yimid heerka xaaladda deggan ee aspartic acid ee Mfn2cKO PNs (Jaantuska S6A), kaas oo u muuqda inuu ka tarjumayo isticmaalka dheef-shiid kiimikaadka kala duwan ee aspartic acid ee u dhexeeya unugyada sii kordhaya iyo neerfayaasha ka dambeeya mitotic. Inkasta oo habka saxda ah ee kor u qaadista PCx ee neerfayaasha aan shaqaynayn ee ku jira vivo uu weli yahay mid la garanayo, waxaan muujinay in jawaabtan degdega ah ay door muhiim ah ka ciyaarto ilaalinta xaaladda redox ee neerfayaasha, taas oo lagu muujiyay tijaabooyinka FLIM ee xaleefyada maskaxda. Gaar ahaan, ka hortagga PNs inay kor u qaadaan PCx waxay keeni kartaa xaalad oksaydh badan waxayna dedejin kartaa dhimashada unugyada. Hawlgalka burburka BCAA iyo karboksylation-ka pyruvate maaha siyaabo lagu garto unugyada durugsan ee cillad mitochondrial (7). Sidaa darteed, waxay u muuqdaan inay yihiin astaamo mudnaan leh oo neerfayaasha aan lahayn OXPHOS, xitaa haddii aysan ahayn astaamaha kaliya, taas oo muhiim u ah neerfayaasha. .
Cudurka maskaxda ku dhaca waa nooc ka mid ah cudurrada neerfaha ee kala duwan kaas oo badanaa u muuqda ataxia oo inta badan waxyeeleeya PN-yada (46). Dadkan neerfaha ku jira ayaa si gaar ah ugu nugul cilladda mitochondrial sababtoo ah kala-goysyadooda xulashada ee jiirarka ayaa ku filan inay soo saaraan calaamado badan oo dhaqdhaqaaqa ah oo lagu garto ataxia spinocerebellar aadanaha (16, 47, 48). Sida laga soo xigtay warbixinnada, qaabka jiirka ee transgenic-ka ah oo leh hiddo-wadaha isbeddelay ayaa lala xiriiriyaa ataxia spinocerebellar aadanaha wuxuuna leeyahay cillad mitochondrial (49, 50), isagoo xoogga saaraya muhiimadda ay leedahay barashada cawaaqibka ka dhasha yaraanta OXPHOS ee PNPH. Sidaa darteed, waxay si gaar ah ugu habboon tahay in si wax ku ool ah loo go'doomiyo oo loo barto dadkan neerfaha ee gaarka ah. Si kastaba ha ahaatee, maadaama PN-yadu ay aad ugu nugul yihiin cadaadiska oo ay xisaabiyaan qayb hoose oo ka mid ah dadka unugyada maskaxda oo dhan, daraasado badan oo ku salaysan omics, kala-soocidda iyaga oo ah unugyada oo dhan weli waa dhinac adag. In kasta oo ay ku dhowdahay wax aan macquul ahayn in la gaaro la'aanta wasakhowga noocyada kale ee unugyada (gaar ahaan unugyada dadka waaweyn), waxaan isku darnay tallaabo kala-goyn wax ku ool ah oo FACS ah si aan u helno tiro ku filan oo neerfayaasha nool ee falanqaynta proteomics-ka ee hoose, waxaanan leenahay dabool borotiin aad u sarreeya (qiyaastii 3000 oo borotiin ah) marka la barbar dhigo xogta hadda jirta ee cerebellum-ka oo dhan (51). Annagoo ilaalinayna jiritaanka unugyada oo dhan, habka aan halkan ku bixinno wuxuu noo oggolaanayaa inaan hubinno isbeddellada ku yimaada waddooyinka dheef-shiid kiimikaadka ee mitochondria, laakiin sidoo kale inaan hubinno isbeddellada ku yimaada dhiggeeda cytoplasmic, kaas oo dhammaystiraya isticmaalka calaamadaha xuubka mitochondrial si loo kobciyo nooca unugyada Habka cusub ee tirada mitochondria ee unugyada adag (52, 53). Habka aan qeexnay kuma koobna oo keliya daraasadda unugyada Purkinje, laakiin si fudud ayaa loogu dabaqi karaa nooc kasta oo unug ah si wax looga qabto isbeddellada dheef-shiid kiimikaadka ee maskaxda buka, oo ay ku jiraan moodooyinka kale ee cilladda mitochondrial.
Ugu dambeyntii, waxaan aqoonsannay daaqad daaweyn ah inta lagu jiro habkan dib-u-habaynta dheef-shiid kiimikaadka kaas oo si buuxda u beddeli kara calaamadaha muhiimka ah ee walbahaarka unugyada isla markaana ka hortagi kara burburka neerfaha. Sidaa darteed, fahamka saameynta shaqada ee dib-u-hagaajinta halkan lagu sharraxay waxay bixin kartaa aragtiyo aasaasi ah oo ku saabsan daaweynta suurtagalka ah ee lagu ilaalinayo jiritaanka neerfaha inta lagu jiro cilladda mitochondrial. Cilmi-baaris mustaqbalka ah oo loogu talagalay in lagu kala saaro isbeddellada ku yimaada dheef-shiid kiimikaadka tamarta ee noocyada kale ee unugyada maskaxda ayaa loo baahan yahay si si buuxda loogu muujiyo ku-dhaqanka mabda'an ee cudurada kale ee neerfaha.
Jiirarka MitoPark ayaa hore loogu sharraxay (31). Jiirarka C57BL/6N oo leh hiddo-sideyaal Mfn2 oo ku yaal dhinaca loxP ayaa hore loo sharraxay (18) waxaana lagu dhex mariyay jiirarka L7-Cre (23). Dhalmada heterozygous ee laba-jibbaaran ayaa markaa laga gudbay jiirarka homozygous Mfn2loxP/Mfn2loxP si loo soo saaro hidde-sideyaal gaar ah oo Purkinje ah oo loogu talagalay Mfn2 (Mfn2loxP/Mfn2loxP; L7-cre). Qayb ka mid ah isku-xidhka, allele Gt (ROSA26) SorStop-mito-YFP (stop-mtYFP) ayaa lagu soo bandhigay isgoysyo dheeraad ah (20). Dhammaan hababka xayawaanka waxaa lagu sameeyay iyadoo la raacayo tilmaamaha Yurub, qaranka iyo hay'adaha waxaana ansixiyay LandesamtfürNatur ee Umwelt iyo Verbraucherschutz, North Rhine-Westphalia, Jarmalka. Shaqada xayawaanku waxay sidoo kale raacdaa tilmaamaha Ururka Yurub ee Sayniska Xoolaha Shaybaarka.
Ka dib marka la suuxiyo kala-baxa afka ilmo-galeenka ee haweeneyda uurka leh, embriyaha jiirka ayaa la go'doomiyaa (E13). Qolofka waxaa lagu kala gooyay Xalka Cusbada Dheelitiran ee Hanks (HBSS) oo lagu daray 10 mM Hepes waxaana lagu shubay Dulbecco's Modified Eagle's Medium oo ay ku jiraan papain (20 U/ml) iyo cysteine ​​​​(1μg/ml). Ku rid unugyada DMEM) oo kala goo iyadoo la isticmaalayo dheefshiidka enzymatic. Ml) heerkul ah 37°C muddo 20 daqiiqo ah, ka dibna si farsamo ah ayaa loogu shiilay DMEM oo lagu daray 10% serum lo'da uurjiifka ah. Unugyada waxaa lagu beeray dhalooyinka daboolka ah ee lagu dahaadhay polylysine cufnaanta 2×106 saxan kasta oo 6 cm ah ama cufnaanta 0.5×105 unug/cm2 si loo sawiro falanqaynta. 4 saacadood ka dib, daawada waxaa lagu beddelay daawo aan lahayn serum Neurobasal oo ka kooban 1% B27 dheeri ah iyo 0.5 mM GlutaMax. Dabadeed neerfayaasha waxaa lagu hayay heerkul ah 37°C iyo 5% CO2 intii tijaabada lagu jiray, waxaana la quudin jiray hal mar usbuucii. Si loo abuuro isku-darka in vitro, 3μl (saxan dhaqameed 24-ceel ah) ama 0.5μl (saxan 24-ceel ah) oo ka mid ah vector-ka fayraska AAV9 ee soo socda ayaa loo isticmaalay in lagu daaweeyo neerfayaasha maalintii labaad in vitro: AAV9.CMV.PI.eGFP. WPRE.bGH (Addgene, lambarka buugga 105530-AAV9) iyo AAV9.CMV.HI.eGFP-Cre.WPRE.SV40 (Addgene, lambarka buugga 105545-AAV9).
DNA-da is-waafajinta ee Mouse Mfn1 iyo Mfn2 (oo laga helay plasmid Addgene #23212 iyo #23213, siday u kala horreeyaan) waxaa lagu calaamadeeyay taxanaha V5 (GKPIPNPLLGLDST) ee C-terminus, waxaana lagu dhex daray mCherry qaab-dhismeedka iyada oo loo marayo taxanaha T2A. Grx1-roGFP2 waa hadiyad ka timid Heidelberg TP Dick DFKZ (Deutsches Krebsforschungszentrum). Iyadoo lagu beddelayo cajaladda tdTomato iyadoo la adeegsanayo hababka cloning-ka caadiga ah, cajaladda waxaa lagu hoos duubay laf-dhabarta pAAV-CAG-FLEX-tdTomato (lambarka tixraaca Addgene 28306) si loo soo saaro vectors-ka pAAV-CAG-FLEX-mCherry-T2A-MFN2-V5, pAAV-CAG-FLEX-mCherry-T2A-MFN1-V5 iyo pAAV-CAG-FLEX-Grx-roGFP2. Istaraatiijiyad la mid ah ayaa loo isticmaalay in lagu soo saaro vector-ka xakamaynta pAAV-CAG-FLEX-mCherry. Si loo soo saaro dhismaha AAV-shPCx, vector-ka plasmid AAV (VectorBuilder, pAAV [shRNA] -CMV-mCherry-U6-mPcx- [shRNA#1]) ayaa loo baahan yahay, kaas oo ka kooban taxanaha DNA-da ee ku jira jiirka bartilmaameedka shRNA PCx (5′CTTTCGCTCTAAGGTGCTAAACTCGAGTTTAGCACCTTAGAGCGAAAG 3′) Iyada oo la raacayo xakamaynta hormoodka U6, mCherry waxaa loo isticmaalaa iyada oo la raacayo xakamaynta hormoodka CMV. Soo saarista vector-yada AAV ee kaaliyaha ah waxaa la sameeyay iyadoo la raacayo tilmaamaha soo saaraha (Cell Biolabs). Marka la soo koobo, isticmaal plasmid wareejin ah oo sita mCherry-T2A-MFN2-V5 (pAAV-CAG-FLEX-mCherry-T2A-MFN2-V5), mCherry-T2A-MFN1-V5 (pAAV-CAG-FLEX-mCherry) si ku meel gaar ah loogu beddelayo unugyada 293AAV-T2A-MFN1-V5), mCherry (pAAV-CAG-FLEX-mCherry) ama Grx-roGFP2 (pAAV-CAG-FLEX-Grx-roGFP2) oo lagu beddelayo hiddo-wadaha, iyo sidoo kale lagu beddelayo borotiinka capsid ee AAV1 iyo borotiinka dheeraadka ah. Baakaynta plasmid-ka plasmid-ka, iyadoo la adeegsanayo habka kalsiyum fosfate. Fayraska cayriin ee ka sarreeya waxaa lagu helay wareegyada barafaysan ee lagu dhalaaliyo qubeyska barafka/ethanol qalalan iyo unugyada la lysed ee saline-ka phosphate buffered (PBS). Vektor-ka AAV waxaa lagu nadiifiyay ultracentrifugation iodixanol gradient oo aan kala joogsi lahayn (24 saacadood 32,000 rpm iyo 4°C) waxaana lagu ururiyay iyadoo la isticmaalayo shaandhada centrifugal-ka Amicon ultra-15. Titer-ka genome-ka ee AAV1-CAG-FLEX-mCherry-T2A-MFN2-V5 [2.9×1013 nuqulka genome-ka (GC)/ml], AAV1-CAG-FLEX-mCherry (6.1×1012 GC/ml), AAV1-CAG-FLEX waxaa loo sameeyay sidii hore loogu sharaxay (54), iyadoo lagu cabbiray PCR-ka waqtiga-dhabta ah (qPCR) -MFN1-V5 (1.9×1013 GC/ml) iyo AAV1-CAG-FLEX-Grx-roGFP2 (8.9×1012 GC/ml).
Neerfayaasha aasaasiga ah waxaa lagu xoqay PBS baraf-qabow 1x ah, oo la jarjaray, ka dibna lagu daray isku-dhafan 0.5% Triton X-100 / 0.5% sodium deoxycholate/PBS lysis buffer oo ka kooban phosphatase iyo protease inhibitor (Roche). Qiyaasidda borotiinka waxaa lagu sameeyay iyadoo la adeegsanayo baaritaanka bicinchoninic acid (Thermo Fisher Scientific). Borotiinnada waxaa markaa lagu kala saaray electrophoresis jel SDS-polyacrylamide, ka dibna lagu tirtiray xuubka polyvinylidene fluoride (GE Healthcare). Xidh meelaha aan gaarka ahayn oo ku dhex geli antibody-ga aasaasiga ah (fiiri Jadwalka S1 wixii faahfaahin ah) caanaha 5% ee TBST (Tris-buffered saline with Tween), tallaabooyinka dhaqidda iyo antibody-ga labaad ee TBST Incubate. Ku dhex geli antibody-ga aasaasiga ah habeenkii +4°C. Ka dib markaad dhaqdo, mari antibody-ga labaad 2 saacadood heerkulka qolka. Ka dib, adigoo isla blot-ka ku dhejinaya antibody-ga anti-β-actin, isla rarka ayaa la xaqiijiyay. Ogaanshaha iyadoo loo beddelayo kiimikada kiimikada iyo kor u qaadista kiimikada kiimikada (GE Healthcare).
Neerfayaasha hore loogu beeray muraayadaha daboolka galaaska waxaa lagu hagaajiyay 4% paraformaldehyde (PFA)/PBS waqtiga loo cayimay heerkulka qolka muddo 10 daqiiqo ah. Daloolada daboolka waxaa marka hore lagu shubaa 0.1% Triton X-100/PBS muddo 5 daqiiqo ah heerkulka qolka, ka dibna waxaa lagu shubaa baffer xannibaya [3% albumin serum lo'da ah (BSA)/PBS]. Maalintii labaad, daloolada daboolka waxaa lagu dhaqay baffer xannibaya waxaana lagu shubay antibody-ga labaad ee fluorophore-conjugated ee ku habboon muddo 2 saacadood ah heerkulka qolka; ugu dambeyntii, muunado si fiican ayaa loogu dhaqay PBS iyadoo la isticmaalayo 4′,6-diamidino-2 -Phenylindole (DAPI) oo lagu dhejiyay wasakh ka dibna lagu dhejiyay mikroskoobka iyadoo la adeegsanayo Aqua-Poly/Mount.
Jiirarka (lab iyo dheddig) waxaa lagu suuxiyay duritaan gudaha caloosha ah oo ketamine ah (130 mg/kg) iyo xylazine (10 mg/kg) ah, waxaana lagu siiyay subcutaneous analgesic carprofen (5 mg/kg), Waxaana lagu dhejiyay qalab stereotactic ah (Kopf) oo lagu rakibay suuf diirran. Soo bandhig madaxa oo isticmaal dalool ilko si aad u khafiifiso qaybta maskaxda ee u dhiganta lafta mis (laga bilaabo lambda: dabada 1.8, dhinaca 1, oo u dhiganta lobules IV iyo V). Isticmaal irbad irbad qaloocan si aad si taxaddar leh u abuurto god yar oo ku yaal madaxa si aad uga fogaato carqaladaynta xididdada dhiigga ee hoose. Kadib xididka galaaska ee khafiifka ah ayaa si tartiib tartiib ah loogu gelinayaa godka yar (laga bilaabo -1.3 ilaa -1 dhinaca ventral ee dura mater), 200 ilaa 300 nl AAV ayaa lagu duraa micro-injector (Narishige) iyadoo la isticmaalayo irbado gacanta lagu qaato (Narishige) dhowr jeer cadaadis hooseeya muddo 10 ilaa 20 daqiiqo ah. Ka dib marka la geliyo xididka, dhig xididka muddo 10 daqiiqo ah si fayrasku si buuxda ugu faafo. Ka dib marka xididdada dhiigga laga saaro, maqaarka si taxaddar leh ayaa loo tolaa si loo yareeyo bararka nabarka loona oggolaado in xayawaanku uu soo kabsado. Xayawaanka waxaa lagu daweeyay dawooyin xanuun baabi'iye ah (caspofen) dhowr maalmood ka dib qalliinka, muddadaas oo xaaladdooda jireed si taxaddar leh loola socday ka dibna la dilay waqtigii la cayimay. Dhammaan hababka waxaa loo sameeyay si waafaqsan tilmaamaha Yurub, qaranka iyo kuwa hay'adaha waxaana ansixiyay LandesamtfürNatur oo ka socda Umwelt iyo Verbraucherschutz, Waqooyiga Rhine-Westphalia, Jarmalka.
Xayawaanka waxaa lagu suuxiyay ketamine (100 mg/kg) iyo xylazine (10 mg/kg), wadnahana waxaa lagu shubay 0.1 M PBS marka hore, ka dibna 4% PFA oo PBS ah. Unugyada ayaa la kala qaaday oo lagu hagaajiyay 4% PFA/PBS habeenkii heerkulkiisu ahaa 4°C. Mindi gariiraysa (Leica Microsystems GmbH, Vienna, Austria) ayaa loo isticmaalay in lagu diyaariyo qaybaha sagittal (50 μm dhumucdiisuna) maskaxda go'an ee PBS. Haddii aan si kale loo cayimin, rinjiyeynta qaybaha xorta ah ee sabeynaya ayaa la sameeyay sida kor lagu sharaxay (13) heerkulka qolka oo la walaaqo. Marka la soo koobo, marka hore, xaleefyada la helay waxaa lagu shubay 0.5% Triton X-100/PBS muddo 15 daqiiqo ah heerkulka qolka; qaar ka mid ah epitopes (Pcx iyo Shmt2), iyadoo lagu shubay tris-EDTA buffer 80°C (PH9) waxay kululeeyaan xaleefyada 25 daqiiqo halkii ay ka ahaan lahayd tallaabadan. Marka xigta, qaybaha waxaa lagu shubay antibody-ga aasaasiga ah (fiiri Jadwalka S1) oo ku jira kaydka xannibaya (3% BSA/PBS) 4°C habeenkii iyadoo la walaaqayo. Maalintii xigtay, qaybaha waxaa lagu dhaqay kaydka xannibaya waxaana lagu shubay antibody-ga labaad ee fluorophore-conjugated ee ku habboon muddo 2 saacadood ah heerkulka qolka; ugu dambeyntii, qaybaha waxaa si fiican loogu dhaqay PBS, oo lagu rinjiyeeyay DAPI, ka dibna waxaa lagu hagaajiyay sawir mikroskoob ah oo AquaPolymount ah.
Microscope sawir-qaadis ah oo laysar ah oo confocal ah (TCS SP8-X ama TCS Digital Light Sheet, Leica Microsystems) oo lagu qalabeeyay laysar iftiin cad iyo laysar ultraviolet diode 405 ah ayaa loo isticmaalay in lagu sawiro muunadda. Iyadoo la dhiirrigelinayo fluorophore-ka iyo ururinta calaamadda iyadoo la adeegsanayo Hybrid Detector (HyDs), barnaamijka LAS-X waxaa loo isticmaalay in lagu ururiyo sawirro la isku dhejiyay oo u dhigma muunadda Nyquist qaab isku xigxiga: loogu talagalay guddiyada aan tirada lahayn, waa calaamado aad u firfircoon (tusaale ahaan, unugyada somatic iyo dendrites) mtYFP) Isticmaal HyD si aad u ogaato tirada PN-yada ku jira qaabka BrightR). Albaabka laga bilaabo 0.3 ilaa 6 ns ayaa la adeegsadaa si loo yareeyo asalka.
Sawir-qaadista waqtiga-dhabta ah ee unugyada la kala saaray. Ka dib markii lagu kala saaray Neurobasal-A oo ka kooban 1% B27 dheeri ah iyo 0.5 mM GlutaMax, unugyada ayaa isla markiiba lagu beeray sawirro galaas ah oo poly-l-lysine ah (μ-Slide8 Well, Ibidi, lambarka buugga 80826), Kadibna ku hay 37°C iyo 5% CO2 muddo 1 saac ah si ay unugyadu u degaan. Sawir-qaadista waqtiga-dhabta ah waxaa lagu sameeyay mikroskoobka sawir-qaadista leysarka ee Leica SP8 oo ku qalabaysan leysarka cad, HyD, muraayadda saliidda ee 63×[1.4 aperture tirsi ah (NA)] iyo marxaladda kululaynta.
Jiirka si dhakhso ah ayaa loo suuxiyay kaarboon laba ogsaydh oo madaxa laga jaray, maskaxdana si dhakhso ah ayaa looga saaray madaxa, waxaana loo jaray 200μm oo dhumuc ah (tijaabada calaamadaynta 13C) ama 275μm oo dhumuc ah (laba tijaabo oo photon ah) oo lagu buuxiyay agabkan soo socda Jalaatada (HM-650 V, Thermo Fisher Scientific, Walldorf, Jarmalka) waxaa ka buuxa walxahan soo socda: 125 mM baraf-qabow, kaarboon-buuxsan (95% O2 iyo 5% CO2) Ca2 hooseeya + dareeraha maskaxda ee macmalka ah (ACSF) NaCl, 2.5 mM KCl, 1.25 mM sodium phosphate buffer, 25 mM NaHCO3, 25 mM glucose, 0.5 mM CaCl2 iyo 3.5 mM MgCl2 (cadaadiska osmotic ee 310 ilaa 330 mmol). Qaybaha maskaxda ee la helay u wareeji qol kahor-ku-shubista oo ka kooban Ca2 + ACSF sare (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 iyo 2.0 mM MgCl2) Dhexdhexaad) pH 7.4 iyo 310 ilaa 320 mmol).
Inta lagu guda jiray habka sawir-qaadista, xaleefyada waxaa loo raray qol sawir-qaadis oo gaar ah, tijaabadana waxaa lagu sameeyay iyadoo la adeegsanayo cadar joogto ah oo ACSF ah heerkul joogto ah oo ah 32° ilaa 33°C. Microscope sawir-qaadis ah oo laser ah oo badan (TCS SP8 MP-OPO, Leica Microsystems) oo lagu qalabeeyay muraayad Leica 25x ah (NA 0.95, biyo), Ti: Sapphire laser (Chameleon Vision II, Coherent) ayaa loo isticmaalay sawir-qaadista jajabka. Module FLIM (PicoHarp300, PicoQuant).
FLIM ee Grx1-roGFP2. Isbeddellada ku yimid xaaladda redox-ka cytoplasmic ee PNs waxaa lagu cabbiray FLIM laba-photon ah oo ku jira qaybaha maskaxda ee sagittal, halkaas oo dareemayaasha bayoolojiga ee Grx1-roGFP2 ay bartilmaameedsadeen PNs. Lakabka PN, goobta helitaanka waxaa lagu xushay qiyaastii 50 ilaa 80 μm oo ka hooseysa dusha sare ee jeexka si loo hubiyo inuu jiro PN oo la heli karo (taasi waa, la'aanta qaab-dhismeedka looxa ama isbeddellada qaab-dhismeedka neerfaha ee ku teedsan dendrites) iyo dareemayaasha roGFP2 ee labanlaab ah iyo AAV oo ku qeexaya shRNA PCx ama taxanaha xakamaynta (mid kasta oo mCherry ah oo wada muujinaya). Ururi sawirro hal-tuubo ah oo leh 2x zoom dijitaal ah [mowjadda kicinta: 890 nm; 512 nm 512 pixels]. Ogaanshaha: HyD gudaha, kooxda shaandhada fluorescein isothiocyanate (FITC)] iyo celceliska sawirka 2 ilaa 3 daqiiqo gudahood ayaa loo isticmaalaa si loo hubiyo in photons ku filan la ururiyo (1000 photons guud ahaan) si loogu habeeyo qalooca. Xasaasiyadda baaritaanka Grx1-roGFP2 iyo xaqiijinta xaaladaha FLIM waxaa lagu sameeyay iyadoo la kormeerayo qiimaha cimriga ee roGFP2 marka lagu daro 10 mM H2O2 oo dibadda ah ACSF perfusion (si loo kordhiyo oksaydhka, taasoo keentay cimriga oo kordha), ka dibna lagu daro 2 mM dithiothreitol (wuxuu yareeyaa heerka dhimista, taasoo keenta hoos u dhac cimriga) (Jaantuska S8, D ilaa G). Adeegso barnaamijka FLIMfit 5.1.1 si aad u falanqeyso natiijooyinka la helay, ku habboon qalooca qudhunka ee hal-dheer ee sawirka oo dhan IRF-ga la cabbiray (shaqada jawaabta qalabka), χ2-na waa qiyaastii 1. Si loo xisaabiyo cimriga hal PN ah, maaskarada ku wareegsan jirka neerfaha ayaa gacanta lagu sawiray, celceliska cimriga maaskaro kastana waxaa loo isticmaalay cabbir.
Falanqaynta kartida Mitochondrial. Ka dib markii qaybta degdegga ah lagu shubay 100 nM TMRM si toos ah loogu daray ACSF-ga la shubay muddo 30 daqiiqo ah, isbeddellada suurtagalka ah ee mitochondrial ee PN-yada waxaa lagu cabbiray mikroskoob laba-foton ah. Sawirka TMRM waxaa lagu sameeyay iyadoo la dhiirrigelinayo baaritaanka 920 nm iyo iyadoo la adeegsanayo HyD gudaha ah (tetramethylrhodamine isothiocyanate: 585/40 nm) si loo ururiyo calaamadaha; iyadoo la adeegsanayo hirarka kicinta isku midka ah laakiin la adeegsanayo HyD gudaha ah oo kala duwan (FITC :525/50) si loo sawiro mtYFP. Isticmaal qalabka ImageJ's Image Calculator si aad u qiimeyso awoodda mitochondrial heerka hal unug. Marka la soo koobo, isle'egta fiilada: calaamad = min (mtYFP, TMRM) waxaa loo isticmaalaa in lagu aqoonsado gobolka mitochondrial ee muujinaya calaamadda TMRM ee Purkinje Somali sawirka hal-istaag ee kanaalka u dhigma. Dabadeed aagga pixel-ka ee maaskarada ka dhalatay ayaa la cabbiraa, ka dibna waxaa lagu caadiyeeyaa sawirka hal-istaag ee u dhigma ee kanaalka mtYFP si loo helo jajabka mitochondrial oo muujinaya awoodda mitochondrial.
Sawirka waxaa lagu furfuray barnaamijka Huygens Pro (Scientific Volume Imaging). Sawirrada la sawiray ee taayirada, isku-darka hal taayir waxaa lagu sameeyaa iyadoo la adeegsanayo algorithm-ka tolida otomaatiga ah ee ay bixiso barnaamijka LAS-X. Ka dib marka la saxo sawirka, isticmaal ImageJ iyo Adobe Photoshop si aad u sii farsamayso sawirka oo aad si isku mid ah u hagaajiso dhalaalka iyo isbarbardhigga. Isticmaal Adobe Illustrator si aad u diyaariso sawirka.
Falanqaynta diiradda mtDNA. Tirada dhaawacyada mtDNA waxaa lagu cabiray qaybaha maskaxda ee lagu calaamadeeyay unugyada difaaca jirka ee ka soo horjeeda DNA iyadoo la adeegsanayo mikroskoobka isku dhafan. Meel kasta oo bartilmaameed ah waxaa loo sameeyay jirka unugyada iyo xudunta unug kasta, aagga u dhigmana waxaa lagu xisaabiyay iyadoo la adeegsanayo qalabka Multi Measure-ka (software-ka ImageJ). Ka jar aagga nukliyeerka aagga jirka unugyada si loo helo aagga cytoplasmic. Ugu dambeyntii, qalabka Falanqaynta Qaybaha (software-ka ImageJ) ayaa loo isticmaalay in si toos ah loogu cabbiro dhibcaha DNA-da cytoplasmic ee tilmaamaya mtDNA sawirka bilowga ah, natiijooyinka la helayna waxaa loo beddelay celceliska PN ee jiirka CTRL. Natiijooyinka waxaa lagu muujiyay tirada celceliska nucleosides unug kasta.
Falanqaynta muujinta borotiinka. Adeegso qalabka Xisaabiyaha Sawirka ee ImageJ si aad u qiimeyso muujinta borotiinka ee PN heerka hal unug. Marka la soo koobo, sawirka hal lakabka ah ee kanaalka u dhigma, iyada oo loo marayo isle'egta: calaamad = min (mtYFP, antibody), gobolka mitochondrial ee muujiya firfircoonida difaaca jirka ee antibody gaar ah ee Purkina ayaa la aqoonsaday. Kadib aagga pixel ee maaskarada ka dhalatay ayaa la cabbiraa, ka dibna caadi ayaa loo dhigaa sawirka hal-istaag ee u dhigma ee kanaalka mtYFP si loo helo jajabka mitochondrial ee borotiinka la soo bandhigay.
Falanqaynta cufnaanta unugyada Purkinje. Ku-darka Counter Cell ee ImageJ waxaa loo isticmaalay in lagu qiimeeyo cufnaanta Purkinje iyadoo loo qaybinayo tirada unugyada Purkinje ee lagu tiriyo dhererka giraanta cerebellar ee ay ku jiraan unugyada la tiriyey.
Diyaarinta iyo ururinta muunadda. Maskaxda kooxda xakamaynta iyo jiirka Mfn2cKO waxaa lagu hagaajiyay 2% PFA/2.5% glutaraldehyde oo ku jirta 0.1 M phosphate buffer (PB), ka dibna qaybaha coronal waxaa lagu diyaariyey iyadoo la isticmaalayo ciliates (Leica Mikrosysteme GmbH, Vienna, Austria) (Dhumucdiisuna waa 50 ilaa 60 μm). Ka dibna ku rid PB buffer 1% os tetraoxide iyo 1.5% potassium ferrocyanide heerkulka qolka 1 saac. Qaybaha waxaa saddex jeer lagu dhaqay biyo la sifeeyay, ka dibna waxaa lagu rinjiyeeyay 70% ethanol oo ka kooban 1% uranyl acetate muddo 20 daqiiqo ah. Qaybaha waxaa lagu qalajiyay aalkolo la qiimeeyay waxaana lagu dhex geliyay Durcupan ACM (Araldite casting resin M) epoxy resin (Electron Microscopy Sciences, lambarka buugga 14040) inta u dhaxaysa muraayadaha galaaska ee silicon lagu dahaadhay, ugu dambayntiina 60°C Polymerize foornada muddo 48 saacadood ah. Aagga xuubka maskaxda ayaa la doortay, qaybaha 50 nm ee aadka u khafiifsan ayaa laga jaray Leica Ultracut (Leica Mikrosysteme GmbH, Vienna, Austria) waxaana lagu soo qaaday shabag naxaas ah oo 2×1 mm ah oo lagu dahaadhay filim polystyrene ah. Qaybaha waxaa lagu rinjiyeeyay xal 4% uranyl acetate ah oo ku jira H2O muddo 10 daqiiqo ah, dhowr jeerna waxaa lagu dhaqay H2O, ka dibna waxaa lagu dhaqay Reynolds lead citrate oo ku jira H2O muddo 10 daqiiqo ah, ka dibna waxaa lagu dhaqay H2O dhowr jeer. Micrographs waxaa lagu qaaday mikroskoob elektaroonig ah oo gudbin ah Philips CM100 (Thermo Fisher Scientific, Waltham, MA, USA) iyadoo la isticmaalayo kamarad dijitaal ah oo TVIPS (Tietz Video and Image Processing System) oo ah TemCam-F416 (TVIPS GmbH, Gauting, USA). Jarmalka).
Jiirarka qaba AAV, maskaxda ayaa la kala saaray oo loo jarjaray qayb sagittal ah oo 1 mm qaro weyn leh, cerebellum-kana waxaa lagu baaray mikroskoob fluorescence si loo aqoonsado giraanta uu ku dhacay AAV (taas oo ah, muujinta mCherry). Kaliya tijaabooyin ay duritaanka AAV ku keento hufnaan aad u sarreysa oo ah lakabka unugyada Purkinje (tusaale ahaan ku dhawaad ​​​​lakabka oo dhan) ugu yaraan laba faraanti oo cerebellar ah oo isku xiga ayaa la isticmaalaa. Wareegga AAV-ga lagu beddelay ayaa la yareeyay si loogu sameeyo habeenimo kadib hagaajinta (4% PFA iyo 2.5% glutaraldehyde oo ku jira 0.1 M cocoate buffer) ka dibna si dheeraad ah ayaa loo farsameeyay. Ku darista EPON, unugyada go'an waxaa lagu dhaqay 0.1 M sodium cocoate buffer (Applichem), waxaana lagu daray 2% OsO4 (os, Adeegyada Sayniska; Caco) oo ku jira 0.1 M sodium cocoate buffer (Applichem) 4 saacadood, ka dibna dhaq 2 saacadood. Ku celi 3 jeer oo leh 0.1 M cocamide buffer. Dabadeed, taxanaha kor u kaca ee ethanol ayaa loo isticmaalay in lagu kululeeyo xal kasta oo ethanol ah heerkul ah 4°C muddo 15 daqiiqo ah si loo nuugo unugyada. Unugyada waxaa loo wareejiyay oxide propylene waxaana lagu kululeeyay habeenkii EPON (Sigma-Aldrich) heerkul ah 4°C. Dhig unugyada EPON cusub heerkulka qolka muddo 2 saacadood ah, ka dibna ku dheji 62°C muddo 72 saacadood ah. Isticmaal ultramicrotome (Leica Microsystems, UC6) iyo mindi dheeman ah (Diatome, Biel, Switzerland) si aad u jarto qaybo 70 nm ah oo aad u khafiif ah, oo ku wasakhee 1.5% uranyl acetate muddo 15 daqiiqo ah heerkul ah 37°C, oo ku wasakhee xalka citrate-ka rasaasta 4 daqiiqo. Micrographs-ka elektaroonigga ah waxaa lagu qaaday iyadoo la isticmaalayo mikroskoob elektaroonig ah oo gudbin ah oo JEM-2100 Plus ah (JEOL) oo lagu qalabeeyay Camera OneView 4K 16-bit (Gatan) iyo software DigitalMicrograph (Gatan). Falanqaynta, mikroograafka elektaroonigga ah waxaa lagu helay 5000× ama 10,000× zoom dijitaal ah.
Falanqaynta qaab-dhismeedka mitochondria. Dhammaan falanqaynta, qaababka mitochondria-ga shaqsiga ah waxaa gacanta lagu qeexay sawirro dhijitaal ah iyadoo la adeegsanayo barnaamijka ImageJ. Xuduudo kala duwan oo qaab-dhismeed ayaa la falanqeeyay. Cufnaanta mitochondrial waxaa lagu muujiyaa boqolkiiba iyadoo loo qaybinayo wadarta guud ee aagga mitochondrial ee unug kasta aagga cytoplasm (aagga cytoplasm = aagga unugyada-unugyada xudunta) × 100. Wareegga mitochondria waxaa lagu xisaabiyaa qaacidada [4π∙(aagga/wareegga 2)]. Qaab-dhismeedka ista ee mitochondria waxaa la falanqeeyay oo loo qaybiyay laba qaybood ("tubular" iyo "blister") iyadoo loo eegayo qaababkooda ugu muhiimsan.
Falanqaynta tirada iyo cufnaanta ee Autophagosome/lysosome. Adeegso barnaamijka ImageJ si aad gacanta ugu sharaxdo qaababka autophagosome/lysosome kasta oo ku jira sawirka dijitaalka ah. Aagga Autophagosome/lysosome waxaa lagu muujiyaa boqolkiiba iyadoo la xisaabinayo iyadoo loo qaybinayo wadarta guud ee aagga qaab-dhismeedka autophagosome/lysosome ee unug kasta iyadoo loo eegayo aagga cytoplasm (aagga cytoplasm=aagga unugyada-aagga nucleus) × 100. Cufnaanta autophagosomes/lysosomes waxaa lagu xisaabiyaa iyadoo tirada guud loo qaybinayo tirada qaab-dhismeedka autophagosome/lysosome unug kasta (marka loo eego aagga cytoplasmic) (aagga cytoplasmic = aagga unugyada-aagga nukliyeerka).
Calaamadaynta qaybaha degdegga ah iyo diyaarinta muunadda. Tijaabooyinka u baahan calaamadaynta gulukooska, u wareeji xaleefyada maskaxda ee degdegga ah qol kahor-ku-shubista, kaas oo ka kooban kaarboon buuxa (95% O2 iyo 5% CO2), Ca2 + ACSF sare (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 iyo 2.0 mM MgCl2, oo loo habeeyay pH 7.4 iyo 310 ilaa 320 mOsm), kaas oo gulukoosku yahay 13 C 6- Beddelka Gulukooska (Eurisotop, lambarka buugga CLM-1396). Tijaabooyinka u baahan calaamadaynta pyruvate, u wareeji xaleefyada maskaxda ee degdegga ah Ca2 + ACSF sare (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 oo ku dar 2.0mM MgCl2, ku hagaaji pH 7.4 iyo 310 ilaa 320mOsm), oo ku dar 1mM 1-[1-13C]pyruvate (Eurisotop, lambarka buugga CLM-1082). Ku rid qaybaha 90 daqiiqo heerkul ah 37°C. Dhammaadka tijaabada, qaybaha si dhakhso ah ayaa loogu dhaqay xal biyo ah (pH 7.4) oo ka kooban 75 mM ammonium carbonate, ka dibna lagu daray 40:40:20 (v:v:v) acetonitrile (ACN): methanol: biyo. Ka dib markii qaybaha lagu kululeeyay barafka muddo 30 daqiiqo ah, muunado ayaa lagu kululeeyay heerkul ah 21,000 g muddo 10 daqiiqo ah heerkul ah 4°C, ka dibna dareeraha cad ayaa lagu qalajiyay isku-darka SpeedVac. Kiniinka metabolite-ka ee la qalajiyey ee ka dhashay waxaa lagu kaydiyay -80°C ilaa la falanqeeyo.
Falanqaynta dareeraha ah ee chromatography-mass spectrometry ee 13 amino acids oo calaamadeysan C. Falanqaynta dareeraha ah ee chromatography-mass spectrometry (LC-MS), pellet-ka metabolite-ka ayaa dib loogu celiyey 75μl oo biyo ah heerka LC-MS (Honeywell). Ka dib markii la centrifugation lagu sameeyay 21,000 g muddo 5 daqiiqo ah heerkul ah 4°C, 20 μl oo ka mid ah supernatant-ka la caddeeyey ayaa loo isticmaalay falanqaynta qulqulka amino acid, halka inta soo hartayna isla markiiba loo isticmaalay falanqaynta anion (hoos ka eeg). Falanqaynta amino acid waxaa lagu sameeyay iyadoo la adeegsanayo hab-raaca derivatization benzoyl chloride ee hore loo sharraxay (55, 56). Tallaabada ugu horreysa, 10μl oo ka mid ah 100 mM sodium carbonate (Sigma-Aldrich) ayaa lagu daray 20μl oo ka mid ah soo saarista metabolite-ka, ka dibna 10μl oo ka mid ah 2% benzoyl chloride (Sigma-Aldrich) ayaa lagu daray heerka LC ACN. Muunadda waxaa si kooban loo rogay ka dibna lagu dhex shubay 21,000 g muddo 5 daqiiqo ah heerkul ah 20°C. Ku wareeji dusha sare ee la nadiifiyey dhalo otomaatig ah oo 2 ml ah oo leh galaas koontarool leh (mugga 200 μl). Muunadaha waxaa lagu falanqeeyay iyadoo la adeegsanayo nidaamka LC ee waxqabadka sare leh ee Acquity iClass (Waters) oo ku xiran Q-Exactive (QE)-HF (Ultra High Field Orbitrap) oo cabbir cabbir sare leh (Thermo Fisher Scientific). Falanqaynta, 2μl oo ka mid ah muunadda la soo saaray ayaa lagu duray tiir 100 × 1.0 mm oo xoog badan oo silica T3 ah (Waters) oo ka kooban walxo 1.8μm ah. Heerka socodka waa 100μl/daqiiqo, nidaamka kaydka wuxuu ka kooban yahay baffer A (10 mM ammonium formate iyo 0.15% formic acid oo biyo ku jira) iyo baffer B (ACN). Jaangooyadu waa sida soo socota: 0%B 0 daqiiqo; 0%B. 0 ilaa 15% B marka ay tahay 0 ilaa 0.1 daqiiqo; 15 ilaa 17% B marka ay tahay 0.1 ilaa 0.5 daqiiqo; B marka ay tahay 17 ilaa 55% marka ay tahay 0.5 ilaa 14 daqiiqo; B marka ay tahay 55 ilaa 70% marka ay tahay 14 ilaa 14.5 daqiiqo; marka ay tahay 14.5 ilaa 70 ilaa 100% B marka ay tahay 18 daqiiqo; 100% B marka ay tahay 18 ilaa 19 daqiiqo; 100 ilaa 0% B marka ay tahay 19 ilaa 19.1 daqiiqo; 0% B marka ay tahay 19.1 ilaa 28 daqiiqo (55, 56). Qalabka cabbira cufka QE-HF wuxuu ku shaqeeyaa qaabka ionization togan oo leh tiro cuf ah oo ah m/z (saamiga cufka/kharashka) oo ah 50 ilaa 750. Xalka la adeegsaday waa 60,000, bartilmaameedka ion-ka xakamaynta (AGC) ee la helayna waa 3×106, waqtiga ugu badan ee ion-kuna waa 100 milise ilbiriqsi. Isha ionization-ka korantada ee kululaysa (ESI) waxay ku shaqeysaa danab buufin ah oo ah 3.5 kV, heerkul kali ah oo ah 250°C, hawada qolofta ah oo ah 60 AU (cutubyo aan kala sooc lahayn), iyo hawada kaalmaysan oo ah 20 AU. 250°C. Muraayadda S waxaa loo dejiyay 60 AU.
Falanqaynta koromatografiga Anion-MS ee asiidhyada dabiiciga ah ee 13C lagu calaamadeeyay. Soo-baxa metabolite-ka ee soo haray (55μl) waxaa lagu falanqeeyay iyadoo la adeegsanayo nidaamka koromatografiga ion Dionex (ICS 5000+, Thermo Fisher Scientific) oo ku xiran cabbiraha cufka QE-HF (Thermo Fisher Scientific). Marka la soo koobo, 5μl oo ah soosaarka metabolite-ka ayaa lagu duray tiir Dionex IonPac AS11-HC ah oo ku qalabaysan HPLC (2 mm × 250 mm, cabbirka walxaha 4μm, Thermo Fisher Scientific) oo ku jira qaabka wareegga qayb ahaan riixista oo leh saamiga buuxinta ee 1.) Tiirka ilaalada Dionex IonPac AG11-HC (2 mm x 50 mm, 4μm, Thermo Fisher Scientific). Heerkulka tiirka waxaa lagu hayaa 30°C, sawir-qaadaha otomaatiga ahna waxaa loo dejiyay 6°C. Isticmaal kartoon potassium hydroxide ah oo lagu siiyay biyo la gooyay si aad u soo saarto jajab potassium hydroxide ah iyada oo loo marayo matoorka eluent. Kala soocidda metabolites-ka heerka socodka oo ah 380μl/daqiiqo, iyadoo la adeegsanayo heerka soo socda: 0 ilaa 3 daqiiqo, 10 mM KOH; 3 ilaa 12 daqiiqo, 10 ilaa 50 mM KOH; 12 ilaa 19 daqiiqo, 50 ilaa 100 mM KOH; 19 ilaa 21 daqiiqo, 100 mM KOH; 21 ilaa 21.5 daqiiqo, 100 ilaa 10 mM KOH. Tiirka waxaa dib loogu miisaamay 10 mM KOH muddo 8.5 daqiiqo ah.
Dheelitirka la soo saaray waxaa lagu daraa durdur dheeri ah oo isopropanol ah oo 150μl/daqiiqo ah ka dib tiirka ka dibna waxaa loo jiheeyaa spectrometer cufnaan sare leh oo ku shaqeeya qaabka ionization taban. MS waxay la socotaa baaxadda cufnaanta laga bilaabo m/z 50 ilaa 750 iyadoo leh xallinta 60,000. AGC waxaa loo dejiyay 1×106, waqtiga ugu badan ee ion-kana waxaa lagu hayaa 100 ms. Isha ESI ee kulul waxaa lagu shaqeeyay danab buufin ah oo ah 3.5 kV. Dejinta kale ee isha ion-ka waa sida soo socota: heerkulka capillary 275°C; socodka gaaska ee qolofta, 60 AU; socodka gaaska ee caawiya, 20 AU at 300°C, iyo dejinta muraayadda S ilaa 60 AU.
Falanqaynta xogta ee metabolites-ka calaamadaysan ee 13C. Isticmaal barnaamijka TraceFinder (nooca 4.2, Thermo Fisher Scientific) si aad u hesho falanqaynta xogta ee saamiga isotope-ka. Aqoonsiga isku-darka kasta waxaa lagu xaqiijiyay isku-darka tixraaca lagu kalsoonaan karo oo si madax-bannaan loo falanqeeyay. Si loo sameeyo falanqaynta kobcinta isotope-ka, aagga chromatogram-ka ion-ka ee la soo saaray (XIC) ee isotope kasta oo 13C ah (Mn) ayaa laga soo saaray [M + H] +, halkaas oo n uu yahay tirada kaarboonka ee isku-darka bartilmaameedka ah, oo loo isticmaalay in lagu falanqeeyo amino acids ama [MH] + ayaa loo isticmaalaa in lagu falanqeeyo anions-ka. Saxnaanta cufka ee XIC waa wax ka yar shan qaybood halkii milyan, saxnaanta RT-na waa 0.05 daqiiqo. Falanqaynta kobcinta waxaa lagu sameeyaa iyadoo la xisaabinayo saamiga isotope kasta oo la ogaaday iyo wadarta dhammaan isotope-yada isku-darka u dhigma. Saamiyadan waxaa loo bixiyaa qiimayaal boqolkiiba ah isotope kasta, natiijooyinkana waxaa lagu muujiyaa kobcinta boqolkiiba molar (MPE), sida hore loogu sharraxay (42).
Kiniinka neerfaha ee la qaboojiyey ayaa lagu daray 80% methanol baraf ah (v/v), lagu shubay, ka dibna lagu shubay -20°C muddo 30 daqiiqo ah. Mar kale ku shub muunadda oo ku walaaq +4°C muddo 30 daqiiqo ah. Muunadda waxaa lagu kululeeyay 21,000 g muddo 5 daqiiqo ah heerkul ah 4°C, ka dibna waxa ka soo baxay ayaa la soo ururiyay oo la qalajiyey iyadoo la isticmaalayo isku-darka SpeedVac oo ah 25°C si loo falanqeeyo. Sida kor lagu sharaxay, falanqaynta LC-MS waxaa lagu sameeyay amino acids-ka unugyada la kala saaray. Iyadoo la adeegsanayo TraceFinder (nooca 4.2, Thermo Fisher Scientific), falanqaynta xogta waxaa lagu sameeyay iyadoo la isticmaalayo cufka monoisotopic ee isku-darka kasta. Caadiyeynta tirada xogta metabolite-ka waxaa lagu sameeyay iyadoo la isticmaalayo xirmada software-ka preprocessCore (57).
Diyaarinta jarjarka. Jiirka si dhakhso ah ayaa loo suuxiyay kaarboon laba ogsaydh oo madaxa laga jaray, maskaxdana si dhakhso ah ayaa looga saaray madaxa, mindidii gariirka barafka ku jirtayna (HM-650 V, Thermo Fisher Scientific, Walldorf, Jarmalka) ayaa loo isticmaalay in lagu jaro qaybo sagittal ah oo 300 ilaa 375 μm ah oo gaas kaarboon qabow ah (95% O2 iyo 5% CO2) Ca2 hooseeya + 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 iyo 6.0 mM MgCl2 La qabso pH 7.4 iyo 310 ilaa 330 mOsm). Jeexjeexyada maskaxda ee la helay u wareeji qol ay ku jiraan Ca2 + ACSF sare (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 iyo mM 3.5 MgCl2) pH 7.4 iyo 310 ilaa 320 mOsm). Ku kaydi xaleefyada 20 ilaa 30 daqiiqo si loo soo celiyo ka hor inta aan la duubin.
duubista. Marxal mikroskoob ah oo lagu qalabeeyay qol duubis oo go'an iyo muraayad ujeedo biyo-ku-dhex-gelis ah oo 20x ah (Scientifica) ayaa loo isticmaalay dhammaan duubista. Unugyada Purkinje ee la soo bandhigay waxaa lagu aqoonsaday (i) cabbirka jirka, (ii) goobta anatomical ee cerebellum, iyo (iii) muujinta hidda-wadaha mtYFP ee fluorescent reporter. Pipette-ka balastar-ka oo leh iska caabin caaradda ah oo ah 5 ilaa 11 megohms waxaa soo saaraya xidid galaas borosilicate ah (GB150-10, 0.86 mm×1.5 mm×100 mm, Science Products, Hofheim, Jarmalka) iyo Qalabka Pipette-ka ee toosan (P-1000, Sutter), Novato, CA). Dhammaan duubista waxaa sameeyay ELC-03XS npi patch clamp amplifier (npi electronic GmbH, Tam, Jarmalka), kaas oo ay xakamaysay barnaamijka Signal (nooca 6.0, Cambridge Electronic, Cambridge, UK). Tijaabada waxaa lagu duubay heer muunad ah oo ah 12.5 kHz. Calaamadda waxaa lagu shaandheeyaa laba shaandho oo Bessel ah oo gaaban oo leh mawjado goyn ah oo ah 1.3 iyo 10 kHz siday u kala horreeyaan. Awoodda xuubka iyo tuubada waxaa lagu magdhabaa wareegga magdhowga iyadoo la adeegsanayo cod-weyneeyaha. Dhammaan tijaabooyinka waxaa lagu sameeyay iyadoo la adeegsanayo kamarad Orca-Flash 4.0 ah (Hamamatsu, Gerden, Jarmalka), oo ay maamulaysay barnaamijka Hokawo (nooca 2.8, Hamamatsu, Gerden, Jarmalka).
Qaabeynta iyo falanqaynta unugyada oo dhan ee caadiga ah. Isla markiiba ka hor inta aan la duubin, ku buuxi tuubada xalka gudaha ee ka kooban walxaha soo socda: 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) iyo 10.0 mM creatinine phosphate ayaa loo hagaajiyay pH 7.25, cadaadiska osmotic-gana wuxuu ahaa 290 mOsm (sucrose). Isla markii la mariyo xoog 0 pA ah si loo jebiyo xuubka, awoodda xuubka nasashada ayaa la cabbiray. Iska caabbinta gelinta waxaa lagu cabbiraa iyadoo la adeegsanayo durdurro hyperpolarized ah oo ah -40, -30, -20, iyo -10 pA. Cabbir baaxadda jawaabta danabka oo isticmaal sharciga Ohm si aad u xisaabiso iska caabbinta gelinta. Dhaqdhaqaaqa iskiis ah ayaa lagu duubay qabsashada danab muddo 5 daqiiqo ah, sPSC-na waxaa lagu aqoonsaday oo lagu cabiray Igor Pro (nooca 32 7.01, WaveMetrics, Lake Oswego, Oregon, USA) iyadoo la adeegsanayo qoraal aqoonsi oo nus-otomaatig ah. Qallooca IV-ga iyo hadda taagan waxaa lagu cabbiraa iyadoo batteriga lagu xirayo awoodo kala duwan (laga bilaabo -110 mV) iyo kordhinta danabka 5 mV tallaabooyin. Soo saarista AP waxaa lagu tijaabiyay iyadoo la adeegsanayo hadda depolarizing. Ku dheji unugga -70 mV inta lagu jiro garaaca hadda depolarizing. Hagaaji cabbirka tallaabada ee cutub kasta oo duubis ah si gaar ah (10 ilaa 60 pA). Xisaabi inta jeer ee ugu badan AP adigoo gacanta ku tirinaya garaaca garaaca ee sababa inta jeer ee ugu badan AP. Heerka AP waxaa lagu falanqeeyaa iyadoo la isticmaalayo derivative labaad ee garaaca depolarization ee marka hore kiciya hal ama in ka badan APs.
Qaabeynta iyo falanqaynta balastar-ka daloolsan. Samee duubista balastar-ka daloolsan adoo isticmaalaya hab-raacyada caadiga ah. Isticmaal balaastar-ka aan lahayn ATP iyo GTP oo aan ku jirin maaddooyinka soo socda: 128 mM gluconate K, 10 mM KCl, 10 mM Hepes, 0.1 mM EGTA iyo 2 mM MgCl2, oo la qabso pH 7.2 (iyadoo la adeegsanayo KOH). ATP iyo GTP waa laga saaray xalka gudaha si looga hortago marin-u-helka aan la xakamayn karin ee xuubka unugyada. Balaastar-ka balastar-ka waxaa ka buuxa xal gudaha ah oo ay ku jirto amphotericin (qiyaastii 200 ilaa 250μg/ml; G4888, Sigma-Aldrich) si loo helo diiwaan balastar ah oo feeran. Amphotericin waxaa lagu milmay dimethyl sulfoxide (fiirsashada kama dambaysta ah: 0.1 ilaa 0.3%; DMSO; D8418, Sigma-Aldrich). Fiirsashada DMSO ee la isticmaalay saameyn muhiim ah kuma yeelan neerfayaasha la bartay. Inta lagu jiro habka feerka, iska caabbinta kanaalka (Ra) si joogto ah ayaa loola socday, tijaabadana waxaa la bilaabay ka dib markii baaxadda Ra iyo AP ay xasilloonayd (20-40 daqiiqo). Dhaqdhaqaaqa iskiis ah waxaa lagu cabbiraa danab iyo/ama qabsasho hadda ah 2 ilaa 5 daqiiqo. Falanqaynta xogta waxaa lagu sameeyay iyadoo la adeegsanayo Igor Pro (nooca 7.05.2, WaveMetrics, USA), Excel (nooca 2010, Microsoft Corporation, Redmond, USA) iyo GraphPad Prism (nooca 8.1.2, GraphPad Software Inc., La Jolla, CA). Mareykanka). Si loo aqoonsado AP-yada iskiis ah, waxaa la isticmaalaa qalabka IgorPro ee NeuroMatic v3.0c. Si toos ah u aqoonso AP-yada adoo isticmaalaya heer la bixiyay, kaas oo si gaar ah loogu habeeyey diiwaan kasta. Adigoo isticmaalaya muddada kor u kaca, go'aami inta jeer ee kor u kaca leh inta jeer ee kor u kaca ugu badan iyo celceliska soo noqnoqoshada kor u kaca.
Go'doominta PN. Iyadoo la waafajinayo hab-maamuuska hore loo daabacay, PN-yada waxaa laga nadiifiyay cerebellum-ka jiirka marxalad cayiman (58). Marka la soo koobo, cerebellum-ka waa la kala gooyay oo lagu jarjaray dhexdhexaad baraf-qabow ah [iyada oo aan lahayn HBSS Ca2+ iyo Mg2+, lagu daray 20 mM glucose, penicillin (50 U/ml) iyo streptomycin (0.05 mg/ml)], ka dibna lagu dheefshiido daawada papain [HBSS, oo lagu daray 1-cysteine·HCl (1 mg / ml), papain (16 U/ml) iyo deoxyribonuclease I (DNase I; 0.1 mg/ml)] Ku dawee 30 daqiiqo heerkul ah 30°C. Marka hore unugyada ku dhaq HBSS oo ay ku jiraan xabka ukunta (10 mg/ml), BSA (10 mg/ml) iyo DNase (0.1 mg/ml) heerkulka qolka si looga hortago dheefshiidka enzymatic, ka dibna HBSS oo ay ku jiraan 20 mM glucose si tartiib ah ugu shiidi HBSS, penicillin (50 U/ml), streptomycin (0.05 mg/ml) iyo DNase (0.1 mg/ml) unugyo hal ah. Joojinta unugyada ka dhalatay waxaa lagu sifeeyay shaandho unug oo 70μm ah, ka dibna unugyada waxaa lagu xoqay centrifugation (1110 rpm, 5 daqiiqo, 4°C) ka dibna dib loogu celiyey kala soocidda [HBSS, oo lagu kabay 20 mM glucose, 20% fetal bovine) Serum, penicillin (50 U/ml) iyo streptomycin (0.05 mg/ml)]; qiimee jiritaanka unugyada iyadoo la adeegsanayo propidium iodide oo cufnaanta unugyada u hagaaji 1×106 ilaa 2×106 unugyo/ml. Kahor inta aan la isticmaalin cytometry-ga socodka, joojinta waxaa lagu sifeeyay shaandho unug oo 50 μm ah.
Cytometer-ka socodka. Kala soocidda unugyada waxaa lagu sameeyay 4°C iyadoo la adeegsanayo mashiinka FACSAria III (BD Biosciences) iyo software-ka FACSDiva (BD Biosciences, nooca 8.0.1). Joojinta unugyada waxaa lagu kala saaray iyadoo la isticmaalayo tuubo 100 μm ah iyadoo cadaadiskeedu yahay 20 psi oo ah heer dhan ~2800 dhacdo/ilbiriqsi. Maadaama shuruudaha kala soocidda dhaqameed (cabbirka unugyada, kala soocidda bimodal, iyo astaamaha kala firdhinta) aysan hubin karin go'doominta saxda ah ee PN noocyada kale ee unugyada, istaraatiijiyadda kala soocidda waxaa lagu dejiyaa iyadoo lagu saleynayo isbarbardhigga tooska ah ee xoogga YFP iyo iftiinka otomaatiga ah ee mitoYFP+ ​​​​iyo mice-ka xakamaynta mitoYFP − Mice. YFP waxay ku faraxsan tahay iyadoo muunadda ku iftiiminaysa xariiq laysar ah oo 488 nm ah, calaamaddana waxaa lagu ogaadaa iyadoo la adeegsanayo shaandhada band 530/30 nm. Jiirarka mitoYFP+, xoogga qaraabada ah ee hidda-wadaha wariyaha Rosa26-mitoYFP ayaa sidoo kale loo isticmaalaa in lagu kala saaro qaybaha neerfaha iyo axon-ka. 7-AAD waxay ku faraxsan tahay laysarka jaalaha ah ee 561 nm waxaana lagu ogaaday shaandhada bandpass-ka 675/20 nm si looga saaro unugyada dhintay. Si loo kala saaro astrocytes-ka isla waqtigaas, joojinta unugyada waxaa lagu rinjiyeeyay ACSA-2-APC, ka dibna muunadda waxaa lagu iftiimiyay xariiq laysar ah oo 640 nm ah, waxaana loo isticmaalay shaandhada bandpass-ka 660/20 nm si loo ogaado calaamadda.
Unugyada la ururiyay waxaa lagu xoqay centrifugation (1110 rpm, 5 daqiiqo, 4°C) waxaana lagu keydiyay -80°C ilaa la isticmaalo. Jiirarka Mfn2cKO iyo eeyaha yaryar ee ay dhaleen waxaa lagu kala saaraa isla maalintaas si loo yareeyo kala duwanaanshaha hab-raaca. Soo bandhigida xogta FACS iyo falanqaynta waxaa lagu sameeyay iyadoo la adeegsanayo software-ka FlowJo (FlowJo LLC, Ashland, Oregon, USA).
Sida kor ku xusan (59), PCR-ka waqtiga-dhabta ah waxaa loo isticmaalaa in lagu kala saaro DNA-da neerfayaasha la kala soocay si loo helo qiyaasta mtDNA ee xigta. Xasaasiyadda toosan iyo dareenka heerka waxaa markii hore lagu tijaabiyay iyadoo la adeegsanayo qPCR tirooyin kala duwan oo unugyo ah. Marka la soo koobo, ku ururi 300 PN oo ku jira lysis buffer oo ka kooban 50 mM tris-HCl (pH 8.5), 1 mM EDTA, 0.5% Tween 20 iyo proteinase K (200 ng/ml) oo ku kululee 55°C 120 daqiiqo. Unugyada waxaa lagu sii kululeeyay 95°C muddo 10 daqiiqo ah si loo hubiyo in si buuxda loo joojiyo firfircoonida proteinase K. Iyadoo la adeegsanayo baaritaanka TaqMan (Thermo Fisher) oo gaar u ah mt-Nd1, mtDNA waxaa lagu cabiray PCR-ka semi-quantitative ee nidaamka PCR-ga waqtiga-dhabta ah ee 7900HT (Thermo Fisher Scientific). Sayniska, lambarka buugga Mm04225274_s1), mt-Nd6 (Thermo Fisher Scientific, lambarka buugga AIVI3E8) iyo 18S (Thermo Fisher Scientific, lambarka buugga Hs99999901_s1) hiddo-sideyaasha.
Diyaarinta muunadda borotiinka. Iyadoo xalka lagu kululeynayo 95°C muddo 10 daqiiqo ah oo lagu shubayo, ku jira lysis buffer [6 M guanidine chloride, 10 mM tris(2-carboxyethyl) phosphine hydrochloride, 10 mM chloroacetamide iyo 100 mM tris-Lyse pellets neuron qaboojiyey oo ku jira HCl]. Bioruptor (Diagenode) muddo 10 daqiiqo ah (30 ilbiriqsi garaaca wadnaha / 30 ilbiriqsi hakad muddo ah). Muunadda waxaa lagu qasi jiray 1:10 20 mM tris-HCl (pH 8.0), waxaana lagu qasay 300 ng trypsin gold (Promega), waxaana lagu kululayn jiray habeenkii 37°C si loo gaaro dheefshiid dhammaystiran. Maalintii labaad, muunadda waxaa lagu kululayn jiray 20,000 g muddo 20 daqiiqo ah. Subagga waxaa lagu qasi jiray 0.1% formic acid, xalkana waxaa lagu nadiifin jiray StageTips oo iskiis u sameeyay. Shaybaarka waxaa lagu qalajiyey qalab SpeedVac ah (Eppendorf concentrator plus 5305) heerkul ah 45°C, ka dibna peptide-ka waxaa lagu laalay 0.1% formic acid. Dhammaan muunadaha waxaa isku mar diyaariyey isla qofkaas. Si loo falanqeeyo muunadaha astrocyte-ka, 4 μg oo ah peptides desalted ayaa lagu calaamadeeyay calaamad cuf ah oo tandem ah (TMT10plex, lambarka buugga 90110, Thermo Fisher Scientific) oo leh saamiga peptide ilaa TMT reagent oo ah 1:20. Calaamadaynta TMT, 0.8 mg oo ah TMT reagent ayaa dib loogu celiyey 70 μl oo ah ACN aan biyo lahayn, peptide-ka la qalajiyeyna waxaa dib loogu celiyey 9 μl oo ah 0.1 M TEAB (triethylammonium bicarbonate), kaas oo lagu daray 7 μl oo ah TMT reagent oo ku jira ACN. Xoogga wuxuu ahaa 43.75%. Ka dib 60 daqiiqo oo la shiiday, falcelinta waxaa lagu damiyey 2 μl oo ah 5% hydroxylamine. Peptides-ka calaamadaysan ayaa la soo ururiyay, la qalajiyey, dib loogu celiyey 200μl oo ah 0.1% aashitada formic (FA), oo loo qaybiyey laba, ka dibna milix lagu shubay iyadoo la isticmaalayo StageTips-ka iskiis u sameeyay. Iyada oo la adeegsanayo UltiMate 3000 ultra high performance liquid chromatograph (UltiMate 3000 ultra high performance liquid chromatograph), mid ka mid ah labada qaybood ayaa lagu kala qaybiyey tiir chromatographic Acquity 1mm x 150mm ah oo lagu buuxiyey walxo 130Å1.7μm C18 ah (Biyaha, buugga Lambarka SKU: 186006935). Thermo Fisher Scientific). Kala sooc peptides heerka socodka 30μl/daqiiqo, kala sooc 1% ilaa 50% buffer B muddo 85 daqiiqo ah oo leh jaantus tallaabo-waji ah oo ah 96 daqiiqo, laga bilaabo 50% ilaa 95% buffer B muddo 3 daqiiqo ah, ka dibna 8 daqiiqo 95% Buffer B; Kaydka A waa 5% ACN iyo 10 mM ammonium bicarbonate (ABC), kaydka B-na waa 80% ACN iyo 10 mM ABC. Soo ururi jajabyada saddexdii daqiiqoba mar oo isku dar laba kooxood (1 + 17, 2 + 18, iwm.) oo ku qalaji centrifuge faakiyuum ah.
Falanqaynta LC-MS/MS. Si loo sameeyo cabbiraadda cufka, peptide-yada (lambarka r119.aq) waxaa lagu kala saaray tiir falanqayn ah oo dhexroorkiisu yahay 25 cm, 75 μm oo PicoFrit ah (muraayadaha cusub ee ujeedka leh, lambarka qaybta PF7508250) oo lagu qalabeeyay 1.9 μm ReproSil-Pur 120 C18-AQ (Dr. Maisch, mat), Isticmaal EASY-nLC 1200 (Thermo Fisher Scientific, Jarmalka). Tiirka waxaa lagu hayay 50°C. Kaydka A iyo B waa 0.1% aashitada formic ee biyaha iyo 0.1% aashitada formic ee 80% ACN, siday u kala horreeyaan. Peptides-ka waxaa laga soocay 6% ilaa 31% baytariga B muddo 65 daqiiqo ah iyo 31% ilaa 50% baytariga B muddo 5 daqiiqo ah iyadoo leh jaantus 200 nl/daqiiqo ah. Peptides-ka la soo saaray waxaa lagu falanqeeyay spectrometer-ka Orbitrap Fusion mass spectrometer (Thermo Fisher Scientific). Cabbirka m/z ee horudhaca ah ee peptide waxaa lagu sameeyaa xallinta 120,000 oo u dhaxaysa 350 ilaa 1500 m/z. Iyada oo la adeegsanayo 27% tamar isku dhac caadi ah, horudhaca ugu xooggan ee leh xaalad dallac ah oo ah 2 ilaa 6 ayaa loo xushay kala-goynta dabinnada C ee tamarta sare (HCD). Waqtiga wareegga waxaa loo dejiyay 1 ilbiriqsi. Qiimaha m/z ee jajabka peptide waxaa lagu cabbiray dabinka ion iyadoo la adeegsanayo bartilmaameedka ugu yar ee AGC ee 5 × 104 iyo waqtiga ugu badan ee duritaanka oo ah 86 ms. Ka dib kala-goynta, horudhaca waxaa la dhigay liiska ka-saarista firfircoon muddo 45 ilbiriqsi ah. Peptides-ka calaamadaysan ee TMT ayaa lagu kala saaray tiir 50 cm, 75 μm ah oo Acclaim PepMap ah (Thermo Fisher Scientific, lambarka buugga 164942), waxaana lagu falanqeeyay spectra-ka socdaalka ee Orbitrap Lumos Tribrid mass spectrometer (Thermo Fisher Scientific) oo ku qalabaysan qalabka ions-ka mawjadaha aan sinnayn ee goobta sare (FAIMS) (Thermo Fisher Scientific) oo ku shaqeeya laba danab magdhow ah oo ah −50 iyo −70 V. MS3 oo lagu xushay iyadoo lagu saleynayo horudhaca isku-dubaridka ayaa loo isticmaalaa cabbirka calaamadda ion warbixinta TMT. Kala-soocidda peptide-ka waxaa lagu sameeyay EASY-nLC 1200, iyadoo la adeegsanayo 90% kor u qaadis toosan oo ah gradient, iyadoo xoogga buffer-ka uu yahay 6% ilaa 31%; buffer A wuxuu ahaa 0.1% FA, buffer B-na wuxuu ahaa 0.1% FA iyo 80% ACN. Tiirka falanqaynta waxaa lagu shaqeeyaa 50°C. Isticmaal FreeStyle (nooca 1.6, Thermo Fisher Scientific) si aad u kala qaybiso faylka asalka ah iyadoo loo eegayo danabka magdhowga FAIMS.
Aqoonsiga Borotiinka iyo tirada. Iyadoo la adeegsanayo mashiinka raadinta ee isku dhafan ee Andromeda, xogta asalka ah waxaa lagu falanqeeyay iyadoo la adeegsanayo nooca MaxQuant 1.5.2.8 (https://maxquant.org/). Marka lagu daro taxanaha Cre recombinase iyo YFP ee laga helay Aequorea victoria, spectra jajabka peptide ayaa laga raadiyay taxanaha canonical iyo taxanaha isoform ee proteome-ka tixraaca jiirka (Proteome ID UP000000589, oo laga soo dejiyay UniProt bishii Maajo 2017). Oxidation-ka Methionine iyo borotiinka N-terminal acetylation waxaa loo dejiyay isbeddello isbeddelaya; cysteine ​​​​carbamoyl methylation waxaa loo dejiyay isbeddello go'an. Xuduudaha dheefshiidka waxaa loo dejiyay "gaar ahaan" iyo "trypsin/P". Tirada ugu yar ee peptides iyo peptides-ka radar ee loo isticmaalo aqoonsiga borotiinka waa 1; tirada ugu yar ee peptides-ka gaarka ah waa 0. Xaaladaha isku-dhafka khariidadda peptide, heerka aqoonsiga borotiinka wuxuu ahaa 0.01. Ikhtiyaarka "Peptide Labaad" waa la awoodsiiyay. Isticmaal ikhtiyaarka "isku dheelitirka u dhexeeya orodka" si aad ugu wareejiso aqoonsiyada guuleysta faylasha asalka ah ee kala duwan. Isticmaal tirada saamiga ugu yar ee LFQ 1 ee qiyaasta aan calaamadda lahayn (LFQ) (60). Xoogga LFQ waxaa lagu sifeeyaa ugu yaraan laba qiime oo ansax ah ugu yaraan hal koox oo ah nooca genotype wakhti kasta, waxaana laga soo saaraa qaybin caadi ah oo leh ballac ah. 0.3 oo hoos ugu dhaqaaq 1.8. Isticmaal madal xisaabinta Perseus (https://maxquant.net/perseus/) iyo R (https://r-project.org/) si aad u falanqeyso natiijooyinka LFQ. Tijaabo t oo dhexdhexaad ah oo laba-geesood ah oo laga sameeyay xirmada software-ka limma ayaa loo isticmaalay falanqaynta muujinta kala duwan (61). Falanqaynta xogta sahaminta waxaa lagu sameeyaa iyadoo la adeegsanayo ggplot, FactoMineR, factoextra, GGally iyo pheatmap. Xogta proteomics-ka ku salaysan TMT waxaa lagu falanqeeyay iyadoo la adeegsanayo nooca MaxQuant 1.6.10.43. Ka raadi xogta proteomics cayriin ee xogta aadanaha ee UniProt, kaas oo la soo dejiyay Sebtembar 2018. Falanqaynta waxaa ku jira qodobka sixitaanka daahirnimada isotope ee uu bixiyay soo-saaraha. Isticmaal limma ee R si aad u hesho falanqaynta muujinta kala duwan. Xogta asalka ah, natiijooyinka raadinta xogta, iyo socodka shaqada falanqaynta xogta iyo natiijooyinka dhammaantood waxaa lagu kaydiyaa isbahaysiga ProteomeXchange iyada oo loo marayo kaydka lammaanaha PRIDE oo leh aqoonsiga dejinta xogta PXD019690.
Qoraallada shaqaynta ayaa kobciya falanqaynta. Qalabka Falanqaynta Waddada Ingenuity (QIAGEN) ayaa loo isticmaalay in lagu go'aamiyo hodannimada shuruudaha sharraxaadda shaqaynta ee xogta la dejiyay 8 toddobaad (Jaantuska 1). Marka la soo koobo, liiska borotiinka tirada leh ee laga helay falanqaynta xogta LC-MS/MS (tandem mass spectrometry) waxaa loo isticmaalaa shuruudaha shaandhaynta soo socda: Musculus-ka Mus waxaa loo xushay noocyada iyo asalka, qaybtana waxay muujinaysaa qiimaha P ee uu Benjamini u hagaajiyay kobcinta 0.05 ama ka hooseeya waxaa loo arkaa mid muhiim ah. Garaafkan, shanta qaybood ee dheeraadka ah ee ugu sarreeya koox kasta oo ku salaysan qiimaha P ee la hagaajiyay ayaa la muujiyay. Iyada oo la adeegsanayo tijaabo t- badan, iyadoo la adeegsanayo barnaamijka kor u qaadista toosan ee laba-marxal ah ee Benjamini, Krieger, iyo Yekutieli (Q = 5%), falanqaynta muujinta borotiinka waqtiga-koorsada waxaa lagu sameeyaa musharixiinta muhiimka ah ee lagu aqoonsaday qayb kasta, saf kastana si gaar ah ayaa loo falanqeeyaa. Looma baahna in la qaato SD joogto ah.
Si aan natiijooyinka daraasaddan ula barbar dhigno xogta la daabacay oo aan u soo saarno jaantus Venn ah Jaantuska 1aad, waxaan isku darnay liiska borotiinka tirada leh iyo qoraallada MitoCarta 2.0 (24). Adeegso qalabka khadka tooska ah ee Sawir Jaantuska Venn (http://bioinformatics.psb.ugent.be/webtools/Venn/) si aad u soo saarto jaantuska.
Wixii macluumaad faahfaahsan oo ku saabsan hababka tirakoobka ee loo isticmaalo falanqaynta proteomics, fadlan tixraac qaybta u dhiganta ee Agabka iyo Hababka. Dhammaan tijaabooyinka kale, macluumaad faahfaahsan waxaa laga heli karaa halyeeyga u dhigma. Haddii aan si kale loo cayimin, dhammaan xogta waxaa lagu muujiyay celcelis ahaan ± SEM, dhammaan falanqaynta tirakoobkana waxaa lagu sameeyay iyadoo la adeegsanayo barnaamijka GraphPad Prism 8.1.2.
Wixii agab dheeraad ah ee maqaalkan, fadlan eeg http://advances.sciencemag.org/cgi/content/full/6/35/eaba8271/DC1
Kani waa maqaal furan oo loo qaybiyay shuruudaha Ruqsadda Hal-abuurka ee Aan Ganacsiga ahayn, kaas oo u oggolaanaya isticmaalka, qaybinta iyo taranka wax kasta oo warbaahin ah, ilaa inta isticmaalka kama dambaysta ah uusan ahayn mid loogu talagalay faa'iido ganacsi iyo ujeeddada laga leeyahay in shaqadii asalka ahayd ay sax tahay. Tixraac.
Fiiro gaar ah: Waxaan kaa codsaneynaa oo keliya inaad bixiso cinwaankaaga emaylka si qofka aad bogga kula taliso uu u ogaado inaad rabto inuu arko emaylka iyo in uusan ahayn spam. Ma qaban doonno wax cinwaanada emaylka ah.
Su'aashan waxaa loo isticmaalaa in lagu tijaabiyo inaad tahay booqde iyo in laga hortago gudbinta spam-ka ee otomaatiga ah.
Waxaa qoray E. Motori, I. Atanassov, SMV Kochan, K. Folz-Donahue, V. Sakthivelu, P. Giavalisco, N. Toni, J. Puyal, N.-G. Larson
Falanqaynta proteomics ee neerfayaasha aan shaqaynayn waxay muujisay in barnaamijyada dheef-shiid kiimikaadka la hawlgeliyo si looga hortago neerfayaasha.
Waxaa qoray E. Motori, I. Atanassov, SMV Kochan, K. Folz-Donahue, V. Sakthivelu, P. Giavalisco, N. Toni, J. Puyal, N.-G. Larson
Falanqaynta proteomics ee neerfayaasha aan shaqaynayn waxay muujisay in barnaamijyada dheef-shiid kiimikaadka la hawlgeliyo si looga hortago neerfayaasha.
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