Ubu *Aderesi y’ubu: Cologne 50931, Ubudage, Cologne Ubushakashatsi bw’Ishirahamwe ry’Ivyiza ku bijanye n’Inyishu y’Imibabaro y’Imitsi mu ndwara ziterwa n’Ugukura (CECAD).
Ugusenyuka kw’imitsi y’ingwara z’imitochondrie bifatwa nk’ibidashobora gusubira inyuma kubera ko ubuhinga bwo guhindura ibiribwa bw’imitsi y’ubwonko bufise aho bugarukira, ariko ingaruka z’ukudakora neza kw’imitsi y’ubwonko ku kwigenga kw’ingingo z’imitsi y’ubwonko mu mubiri ntizitahurwa neza. Aha, turashira ahabona proteome yihariye y’ingingo z’umubiri zifise ubukene bwa OXPHOS butera imbere buterwa n’uguhungabana kw’inguvu z’ugukorana kw’imitsi y’umubiri. Twasanze ukudakora neza kw’imitochondrie kwatumye haba ihinduka rikomeye mu bijanye n’ubuhinga bwa proteomics, ivyo bikaba vyatumye amaherezo porogarama zitomoye z’uguhindura ibiribwa zikora zikurikirana imbere y’uko utugingo ngengabuzima dupfa. Mu buryo tutavyiteze, twabonye ukuntu bigaragara ko pyruvate carboxylase (PCx) n’izindi nzitizi zirwanya ubusaza zishigikira ibintu biri hagati y’ingendo ya TCA. Inhibition ya PCx yatumye habaho uguhagarika umutima n’ugusenyuka kw’imitsi, ivyo bikaba vyerekana ko indwara y’imitsi ifise ingaruka zo gukingira imitsi idafise OXPHOS. Gusubira gukorana kw’imitsi y’ubwonko mu mitsi y’ubwonko yacitse intege cane, birahindura burundu ivyo bimenyetso vy’imibiri, gutyo bikaba bituma ingingo ngenderwako zidapfa. Ivyo twabonye birerekana inzira zitazwi mbere zituma umuntu ashobora kwihanganira ukudakora neza kw’imitsi y’ubwonko kandi vyerekana ko ugusenyuka kw’imitsi bishobora gusubira inyuma mbere no mu bice vya nyuma vy’iyo ndwara.
Uruhara nyamukuru rw’imitochondrie mu kubungabunga ubushobozi bw’imitsi y’ubwonko rurashimikwako cane n’ibimenyetso vyinshi vy’imitsi y’ubwonko bifitaniye isano n’ingwara z’imitsi y’ubwonko z’abantu. Akenshi muri izo ndwara ziterwa n’uguhinduka kw’imitsi y’umubiri itunganya ivy’imitsi y’umubiri (1, 2) canke ugusenyuka kw’imitsi y’umubiri bifitaniye isano n’inguvu z’imitsi y’umubiri, ivyo bikaba bigira ingaruka zitaziguye ku gushikama kwa ADN y’imitsi y’umubiri (mtDNA) (3, 4). Ibikorwa vyakozwe ku bikoko vyerekanye ko mu kwishura ku kudakora neza kw’imitochondrie mu bihimba bikikije, inzira z’imibiri (5-7) zishobora gukora, ivyo bikaba bitanga amakuru ahambaye kugira ngo umuntu atahure neza ingene izo ndwara zikomeye zitera. Mu buryo butandukanye n’ubwo, ugutahura kwacu ku bijanye n’ihinduka ry’imibiri y’ubwoko bumwebumwe bw’ingingo ngenderwako ziterwa n’ukunanirwa muri rusangi kw’imitsi y’ubwonko yitwa mitochondrie adenosine triphosphate (ATP) ni ngirakamaro (8), bikaba bishimika ku nkenerwa yo kumenya ibintu bishobora gukoreshwa mu gukingira canke gukingira indwara. Gukingira ububabare bw’imitsi (9). Kubura amakuru ni uko ingingo z’imitsi zibonwa cane ko zifise ubushobozi buke cane bwo guhindura ibiribwa ugereranyije n’ubwoko bw’ingingo z’ibihimba bikikije (10). Kubera ko izo ngirabuzimafatizo zifise uruhara runini mu guhuza ivy’ugutanga ibihimba vy’umubiri ku mitsi y’ubwonko kugira ngo ziteze imbere ugutanga ibihimba vy’umubiri no kwishura ku bikomere n’ingwara, ubushobozi bwo guhindura ibihimba vy’umubiri ku bihe bitoroshe vy’imitsi y’ubwonko buragarukira hafi ku ngingo z’umubiri zitwa glial cells (11-14). Vyongeye, ukuntu utugingo ngengabuzima two mu bwonko dutandukanye cane, biratuma ahanini bidashobora kwiga ibijanye n’uguhinduka kw’ibifungurwa bibera mu migwi yihariye y’utugingo ngengabuzima. Ivyo vyatumye ata vyinshi bizwi ku bijanye n’ingaruka nyazo ziterwa n’ukudakora neza kw’imitochondrie mu mitsi y’ubwonko.
Kugira ngo dutahure ingaruka z’imibiri ziterwa n’ukudakora neza kw’imitochondrie, twatandukanije imitsi ya Purkinje (PNs) mu bice bitandukanye vy’ugusenyuka kw’imitsi bitewe n’ugusenyuka kw’imitsi y’inyuma y’imitsi (Mfn2). Naho ihinduka rya Mfn2 mu bantu rifitaniye isano n’ubwoko bw’indwara y’imitsi y’umubiri izwi nka Charcot-Marie-Tooth type 2A (15), ugusambura kwa Mfn2 mu mbeba ni ikintu cemewe neza co gutuma umubiri udakora neza (OXthoPHOS). Ubwoko butandukanye bw’imitsi y’ubwonko (16-19) be n’ivyo bivako biva ku mitsi y’ubwonko, bijana n’ibimenyetso vy’imitsi y’ubwonko bigenda biratera imbere, nk’uguhungabana kw’imitsi (18, 19) canke ataxia cerebellar (16). Mu gukoresha uburyo bwo gupima poroteyine zidafise ikimenyetso (LFQ), uburyo bwo gupima ibiribwa, uburyo bwo gufata amashusho, n’uburyo bwo gupima imigera, twerekana ko ugusenyuka kw’imitsi gutera imbere gutuma habaho pyruvate carboxylase (PCx) n’ibindi bintu bifasha mu gutuma imitsi y’umubiri igira ububabare bw’imitsi y’umubiri. Kugira ngo tugenzure akamaro k’ivyo vyavumbuwe, twagabanyije canecane ukuntu PCx igaragara mu PNs zidafise Mfn2, maze tubona ko iyo nzira yongerera umubabaro w’imitsi n’ugusenyuka kw’imitsi, gutyo bikaba vyerekana ko azoospermia itanga urupfu rw’ingingo z’umubiri. Ivyerekanwa bikomeye vya MFN2 birashobora gukiza burundu PN y’ugusenyuka kw’iherezo n’ubukene bukomeye bwa OXPHOS, ukurya cane ADN y’imitochondrie, n’urusobe rw’imitochondrie bisa n’uko rwavunitse, ivyo bikaba bikomeza gushimika ku vy’uko ubwo buryo bwo gusenyuka kw’imitsi bushobora mbere gukira imbere y’uko indwara y’ingingo ngenderwako itera imbere.
Kugira ngo tubone mu ciyumviro imitochondrie iri muri Mfn2 knockout PNs, twakoresheje ubwoko bw’imbeba butuma mitochondrie zishingiye kuri Cre zishobora gutera umuhondo w’umuhondo (YFP) (mtYFP) (20) Cre expression maze dusuzuma imiterere y’imitochondrie in vivo. Twasanze ugusambura umugera wa Mfn2 mu PNs vyotuma urusobe rw’imitochondrie rugabanywa buhoro buhoro (Ishusho S1A), kandi ihinduka rya mbere ryabonetse ku myaka 3 y’amavuka. Mu buryo bunyuranye, ugusenyuka gukomeye kw’uruzitiro rw’ingingo za PN, nk’uko bigaragazwa n’ugutakaza immunostaining ya Calbindin, ntikwatanguye gushika umuntu afise amayinga 12 (Ishusho 1, A na B). Igihe kidahuye hagati y’amahinduka ya mbere mu mibumbe y’imitochondrie be n’ugutangura kugaragara kw’urupfu rw’imitsi y’ubwonko vyatumye dukora ubushakashatsi ku mahinduka y’imibiri aterwa n’ukudakora neza kw’imitochondrie imbere y’urupfu rw’ingingo ngenderwako. Twarateguye ubuhinga bushingiye ku gutoranya utugingo ngengabuzima (FACS) kugira ngo dutandukanye PN igaragaza YFP (YFP+) (Ishusho 1C), no mu mbeba zigenzura (Mfn2 + / loxP :: mtYFP loxP- stop-loxP: : L7-cre), ubu zizokwitwa CT1BRL (Ishusho). Ivyo bikaba vyatumye dushobora gusukura umubiri wa YFP+ (YFPhigh) w’ibihimba vy’umubiri (YFPhigh) biva ku bitari PN (YFPneg) (Ishusho S1B) canke ibice vy’umubiri vy’umubiri (YFPlow; Ishusho S1D, confopecal) (YFPlow; Ishusho S1D, confopecal). Kugira ngo tugenzure akaranga k’abantu bashizwe mu rwego, twakoze LFQ proteomics hanyuma dukora isesengura ry’ibice nyamukuru, maze tubona ko hariho itandukaniro rigaragara hagati y’ingingo za YFPhigh na YFPneg (Ishusho S1C). Utugingo ngengabuzima twa YFPhigh twarerekanye ugutunganywa kw’ibimenyetso vya PNs bizwi (ni ukuvuga Calb1, Pcp2, Grid2 na Itpr3) (21, 22), ariko nta gutunganywa kw’amaproteine ​​asanzwe agaragara mu mitsi y’ubwonko canke ubundi bwoko bw’utugingo ngengabuzima (Ishusho 1D) ). Igereranyo hagati y’ibigereranyo mu ngirabuzima fatizo za YFPhigh zashizwe mu migwi zakoranijwe mu ngeragezwa zigenga vyerekanye ko hariho isano> 0.9, vyerekana ko hariho ubushobozi bwo kuvyara neza hagati y’ibinyabuzima (Ishusho S1E). Mu ncamake, ayo makuru yemeje umugambi wacu wo kwitandukanya n’abandi bishoboka. Kubera ko uburyo bwo gutwara L7-cre bukoreshwa butera ugusubira gukorana kw’imitsi mu ndwi ya mbere inyuma y’ukuvyara (23), twatanguye kwica imbeba zivuye muri CTRL n’izifise ivyangombwa (Mfn2 loxP / loxP :: mtYFP loxP-stop-loxP :: L7-cre) Gukorakoranya imitsi y’ubwonko. Inyuma y’aho gusubira gukorana, yitwa Mfn2cKO iyo ifise amayinga 4. Nk’iherezo, twahisemwo amayinga 8 y’imyaka igihe uruzitiro rwa PN rwari rudasenyutse naho hariho uguca ibice kw’imitochondrie bigaragara (Ishusho 1B n’Ishusho S1A). Muri rusangi, twagereranyije umubare wose w’amaproteine ​​3013, muri yo nka 22% yari ashingiye ku nsobanuro za MitoCarta 2.0 zishingiye ku poroteyine y’imitochondrie nk’imitochondrie (Ishusho 1E) (Ishusho 1E) (24). Isesengura ry’imiterere y’imitsi itandukanye ryakozwe mu ndwi ya 8 ryerekanye ko 10,5% gusa vy’amaproteine ​​yose vyari bifise amahinduka akomeye (Ishusho 1F n’Igishushanyo S1F), muri ayo maproteine ​​195 yari afise ubushobozi bwo guhindura ibintu, 120 na yo akaba yari afise ubushobozi bwo guhindura ibintu (Ishusho 1F). Birabereye kumenya ko “isesengura ry’inzira nshasha” ry’iyi nkuru ryerekana ko izo nzira zitandukanye zigaragazwa ahanini ziri mu nzira zidasanzwe z’imibiri (Ishusho 1G). Ikintu gitangaje ni uko naho nyene ugugabanya inzira zijanye n’ibimenyetso vya OXPHOS be n’ibimenyetso vya calcium vyemeza ko imitochondrie idakora neza mu PNs zidafise ubushobozi bwo gukorana, ibindi bice bikora canecane mu guhindura ibiribwa vy’acides aminés birahindurwa cane, ivyo bikaba bihuye n’uguhindura ibiribwa mu mitochondrial conwiring PN. ukudakora neza.
(A) Amafoto yerekana ibice vy’ubwonko vy’imbeba CTRL na Mfn2cKO yerekana ko PNs zigenda zitakaza (calbindin, gray); nuclei zari zigizwe n’ibara rya DAPI. (B) Igitigiri ca (A) (isesengura ry’uburyo bumwe ry’ugutandukanya, ***P<0.001; n = 4 gushika ku 6 z’inziga zivuye mu mbeba zitatu). (C) Inzira y’ibikorwa vy’igerageza. (D) Ugukwiragizwa kw’ikarata y’ubushuhe kw’ibimenyetso vyihariye kuri Purkinje (hejuru) n’ubundi bwoko bw’ingingo (hagati). (E) Igishushanyo ca Venn kigaragaza igitigiri c’amaproteine ​​y’imitochondrie yerekanwa muri PN yashizwe mu rwego. (F) Igishushanyo c’ikirunga c’amaproteine ​​agaragara mu buryo butandukanye mu mitsi ya Mfn2cKO ku ndwi 8 (agaciro k’agaciro ka 1.3). (G) Isesengura ry’inzira y’ubuhinga ryerekana inzira zitanu zihambaye cane zo gutera imbere (umutuku) n’ugutera hasi (ubururu) muri Mfn2cKO PN zishizwe mu rwego rw’indwi 8. Urugero rw’imvugo rwa mwayeni rwa poroteyine yose yabonetse ruragaragazwa. Ikarata y’ubushuhe bw’ibara ry’umweru: agaciro ka P kahinduwe. ns, si ngombwa.
Amakuru yerekeye ivy’amaproteine ​​yerekanye ko buhorobuhoro ukuntu poroteyine zigaragara mu bihimba vy’umubiri bita complexes I, III na IV zigenda zigabanuka. Ivyumba I, III na IV vyose vyari birimwo ibice bihambaye vy’ubuhinga bwa mtDNA, mu gihe ivyumba II, vyari bifise ubuhinga bwa nucleaire gusa, mu vy’ukuri ntivyagira ico bikoze (Ishusho 2A n’Ishusho S2A). . Mu buryo buhuye n’ivyavuye mu bikorwa vy’ubuhinga bwa proteomics, ubuhinga bwo gupima umubiri bw’abantu bwagaragaje ko urugero rwa MTCO1 (mitochondrial cytochrome C oxidase subunit 1) rw’igice ca IV muri PN rwagabanutse buhoro buhoro (Ishusho 2B). Igice ca mtDNA-encoded subunit Mtatp8 caragabanutse cane (Ishusho S2A), mu gihe urugero rw’ubuzima buhoraho rw’igice ca ATP synthase gifise kode ya nucleaire rwagumye rudahindutse, ivyo bikaba bihuye n’igice ca ATP synthase subunit F1 kizwi ko gikomeye iyo mtDNA igaragara. Ivyo guhingura birahuye. Guciramwo (7). Igenzura ry’urugero rwa mtDNA mu PNs za Mfn2cKO zatoranijwe hakoreshejwe ubuhinga bwa polymerase chain reaction (qPCR) ryemeje ko umubare w’amakopi ya mtDNA ugabanuka buhoro buhoro. Ugereranije n’umugwi w’abagenzuzi, ku ndwi 8, nk’ibice 20% gusa vy’urugero rwa mtDNA ni vyo vyagumyeho (Ishusho 2C). Mu buryo buhuye n’ivyo bimenyetso, ubuhinga bwo gutera ibara ry’imikorosikopi y’ibihimba vy’umubiri (confocal microscopy) bwa Mfn2cKO PNs bwakoreshejwe kugira ngo hamenyekane ADN, vyerekana igihe c’ugukoresha kw’ibihimba vy’umubiri vy’imitochondrie (Ishusho 2D). Twasanze abantu bamwebamwe gusa bafise uruhara mu gusenyura poroteyine z’imitochondrie no mu gusubiza ivy’imibabaro ari bo bari bafise ivyiyumviro vyiza, harimwo Lonp1, Afg3l2 na Clpx, n’ibintu bikomeye vy’iteraniro rya OXPHOS. Nta mpinduka zikomeye zabonetse mu rugero rw’amaproteine ​​agira uruhara mu gutuma umubiri upfa (Ishusho S2B). Na twebwe nyene, twabonye ko imirongo y’imitochondrie n’iya endoplasmic reticulum ikora mu gutwara calcium ifise gusa amahinduka makeyi (Ishusho S2C). Ikindi, isuzuma ry’amaproteine ​​ajanye n’ugupfa kw’umubiri nta mpinduka zikomeye ryabonye, ​​ivyo bikaba bihuye n’ugutera kw’ibihimba vy’umubiri bigaragara vy’umubiri vyerekanwa mu buzima biciye ku buhinga bwa immunohistochemie n’ubuhinga bwa electron microscopy (Ishusho S3). Ariko rero, ukudakora neza kwa OXPHOS mu PNs kujana n’amahinduka agaragara y’imiterere y’imitsi y’umubiri. Ivyiyumviro vy’imitochondrie birashobora kubonwa mu mibiri y’ingingo n’ibiti vy’imitsi vy’imitsi ya Mfn2cKO PNs ifise imyaka 5 n’umunani, kandi imiterere y’uruzitiro rw’imbere yarahindutse cane (Ishusho S4, A na B). Mu bijanye n’ayo mahinduka y’imiterere y’umubiri be n’ukugabanya cane mtDNA, isesengura ry’ibice vy’ubwonko bukomeye bifise tetramethylrhodamine methyl ester (TMRM) vyerekanye ko ubushobozi bw’uruzitiro rw’imitochondrie mu Mfn2cKO PNs bwagabanutse cane (Ishusho S4C).
(A) Isesengura ry’igihe ry’urugero rw’imvugo y’umugwi wa OXPHOS. Niwiyumvire gusa poroteyine zifise P<0.05 ku ndwi 8 (ANOVA y’inzira zibiri). Umurongo w’utudomo: Nta guhindura ugereranije na CTRL. (B) Ibubamfu: Akarorero k’igice c’ubwonko gifise ikimenyetso c’umubiri urwanya MTCO1 (umurongo w’urugero, 20 μm). Aho imibiri y’utugingo ngengabuzima twa Purkinje ifise, hapfutse umuhondo. Iburyo: Gupima ingero za MTCO1 (isesengura ry’uburyo bumwe ry’ugutandukanya; n = 7 gushika ku 20 z’ingingo zasuzumwe zivuye mu mbeba zitatu). (C) qPCR isesengura ry’umubare w’amakopi ya mtDNA mu PN yatoranijwe (isesengura ry’inzira imwe ry’ugutandukana; n = 3 gushika ku 7 imbeba). (D) Ibubamfu: Akarorero k’igice c’ubwonko buto bufise ikimenyetso c’umubiri urwanya ADN (umurongo w’urugero, 20 μm). Aho imibiri y’utugingo ngengabuzima twa Purkinje ifise, hapfutse umuhondo. Iburyo: Gupima igitigiri c’ibikomere vya mtDNA (isesengura ry’uburyo bumwe ry’ugutandukanya; n = 5 gushika ku 9 z’ingingo zivuye mu mbeba zitatu). (E) Akarorero k’igice c’ubwonko gikomeye kigaragaza utugingo ngengabuzima twa mitoYFP + Purkinje (umwampi) mu kwandika utugingo ngengabuzima twose. (F) Gupima igitigiri c’umurongo wa IV. (G) Ivyanditswe vyerekana urushinge rw’amashanyarazi atera ububabare mu ngirabuzimafatizo za CTRL na Mfn2cKO Purkinje. Top trace: Impumuro ya mbere yatumye AP ivuka. Ivyiyumviro vyo hasi: Incuro nyinshi cane za AP. (H) Gupima igitigiri c’ibintu biva mu mubiri (sPSPs). Igishushanyo c’ugufata amajwi giserukira n’igipimo caco co gukura vyerekanywe muri (I). Isesengura ry’uburyo bumwe ry’uguhinduka ryasesenguye n = 5 gushika ku 20 z’ingingo z’imbeba zitatu. Amakuru agaragazwa nk’uko ari hagati±SEM; *P<0,05; **P<0,01; ***P<0,001. (J) Ivyiyumviro vyerekana AP yihuta vyanditswe hakoreshejwe uburyo bwo gufata igipande gitoboye. Igikurikira co hejuru: Incuro nyinshi cane za AP. Igikurikira: zoom ya AP imwe. (K) Gupima incuro AP zisanzwe n’izirengeye hakurikijwe (J). Igerageza rya Mann-Whitney; n = 5 z’ingingo zasuzumwe zivuye mu mbeba zine. Amakuru agaragazwa nk’uko ari hagati±SEM; si ikintu gihambaye.
Ivyonona bigaragara vya OXPHOS vyabonetse mu Mfn2cKO PN y’indwi 8, bikaba vyerekana ko igikorwa c’umubiri c’imitsi y’ubwonko kidasanzwe cane. Ku bw’ivyo, twasuzumye ibiranga umuyagankuba udakora vy’imitsi y’ubwonko idafise OXPHOS ku ndwi 4 gushika ku 5 no ku ndwi 7 gushika ku 8 mu gukora amajwi y’ibihimba vy’umubiri vyose mu bice vy’ubwonko bukomeye (Ishusho 2E). Mu buryo butategerezwa, ubushobozi bw’uruzitiro rwo kuruhuka n’ubushobozi bwo kwinjira bw’imitsi ya Mfn2cKO vyari bisa n’ivyo mu gihe c’igenzura, naho nyene hariho itandukaniro ritoyi hagati y’ingingo (Imbonerahamwe 1). Na vyo nyene, ku ndwi 4 gushika ku 5, nta mpinduka zikomeye zabonetse mu bijanye n’ubucuti buri hagati y’umuriro n’umuriro (IV curve) (Ishusho 2F). Ariko rero, nta n’imwe mu mitsi ya Mfn2cKO y’indwi 7 gushika ku 8 yarokotse iyo nzira ya IV (intambwe y’ugutera imbere cane), ivyo bikaba vyerekana ko hariho ubushobozi bwo gutera imbere cane muri iki gihe c’iherezo. Mu buryo bunyuranye, mu mitsi ya Mfn2cKO, imirongo y’imitsi y’umubiri (depolarizing currents) itera gusubiramwo ubushobozi bwo gukora (AP) irashobora kwihanganirwa neza, ivyo bikaba vyerekana ko uburyo bwo gusohoka kwayo muri rusangi butandukanye cane n’ubw’imitsi y’umubiri igenzura ivy’indwi 8 (Imbonerahamwe 1 n’Igishushanyo 2G). Na vyo nyene, incuro n’uburemere bw’imipfunda y’inyuma y’ugutera imbere (sPSCs) vyari bigereranywa n’ivyo mu mugwi w’abagenzuzi, kandi incuro z’ibintu vyariyongereye kuva ku ndwi 4 gushika ku ndwi 5 gushika ku ndwi 7 gushika ku ndwi 8 n’ukwiyongera nk’ukwo (Ishusho 2, H na I). Igihe c’ugukura kw’imitsi y’umubiri mu PNs (25). Ivyo nyene vyabonetse inyuma y’ugutera ibipande vya PNs vyatobowe. Ivyo bibuza gusubiza inyuma utunenge twa ATP tw’ingingo ngenderwako, nk’uko vyoshobora gushika mu kwandika amajambo y’ingingo ngenderwako yose. Cane cane, ubushobozi bw’uruzitiro rwo kuruhuka n’incuro z’ugutera kw’imitsi ya Mfn2cKO ntivyagira ico bikoze (Ishusho 2, J na K). Mu ncamake, ivyo bimenyetso vyerekana ko PNs zifise ubumuga bugaragara bwa OXPHOS zishobora guhangana n’imirongo y’ugusohoka kw’incuro nyinshi, bikaba vyerekana ko hari uburyo bwo gusubiza inyuma buzifasha kuguma zifise inyishu zisanzwe ziri hafi y’ivyo zisanzwe zikora.
Amakuru agaragazwa nk’uko ari hagati ± SEM (isesengura ry’uburyo bumwe ry’ugutandukanya, ikigeragezo c’ukugereranya kenshi ca Holm-Sidak; *P<0.05). Igitigiri c’ibice kigaragazwa n’ibifunga.
Twarashizeho itohoza nimba urutonde urwo ari rwo rwose mu rutonde rw’amakuru y’ivy’amaproteine ​​(Ishusho 1G) rurimwo inzira zishobora kurwanya ubukene bukomeye bwa OXPHOS, gutyo tugasigura igituma PN ishobora kuguma ifise ubuhinga bw’amashanyarazi hafi y’ubusanzwe (Ishusho 2, E gushika kuri K). . Isesengurwa ry’ivy’amaproteine ​​ryerekanye ko enzymes zija mu guhindura amashami y’ama aside amino (BCAA) zari zifise ubushobozi bwo gutera imbere cane (Ishusho 3A n’Ishusho S5A), kandi igicuruzwa ca nyuma citwa acetyl-CoA (CoA) canke succinyl CoA gishobora kwongereza ama tricarboxylates mu ndwara y’imitsi (arterioscléroïde). Twasanze ibirimwo BCAA transaminase 1 (BCAT1) na BCAT2 vyose vyiyongereye. Batera intambwe ya mbere y’uguhindura BCAA mu gutuma habaho glutamate ivuye muri α-ketoglutarate (26). Ivyiyumviro vyose bigize uruzitiro rw’amashami rwitwa keto acid dehydrogenase (BCKD) biratunganirizwa (urwo ruzitiro ruratuma habaho uguhinduka kw’imitsi y’umubiri y’umubiri w’umubiri w’umubiri w’umubiri) (Ishusho 3A n’Ishusho S5A). Ariko rero, nta mpinduka zigaragara muri BCAA ubwayo zabonetse muri PN yatoranijwe, ivyo bishobora kuba bivuye ku kwiyongera kw’ingingo z’umubiri zifata izo aside amino zihambaye canke gukoresha izindi nzira (glucose canke aside lactic) kugira ngo zishigikire inzira ya TCA (Ishusho S5B). PNs zitagira OXPHOS na zo nyene zerekanye ko zigira ibikorwa vyinshi vyo kubora no guhindura glutamine iyo zifise amayinga 8, ivyo bishobora kugaragazwa n’uguhinduka kw’imitsi y’imitsi y’umubiri yitwa glutaminase (GLS) na glutamine pyruvate transaminase 2 (GPT2) (Ishusho 3, A na C). Ni vyiza kumenya ko uguhinduka kwa GLS kugarukira ku isoform glutaminase C (GLS-GAC) (uguhinduka kwa Mfn2cKO/CTRL ni nk’incuro 4,5, P = 0,05), kandi uguhinduka kwayo kwihariye mu bihimba vya kanseri Bishobora gufasha imitonergyondrial. (27).
(A) Ikarata y’ubushuhe yerekana ihinduka ry’incuro mu rugero rwa poroteyine ku nzira yatanzwe ku ndwi 8. (B) Akarorero k’igice c’ubwonko buto bufise ikimenyetso c’umubiri urwanya PCx (umurongo w’urugero, 20 μm). Umwampi w’umuhondo werekana umubiri w’ingingo ya Purkinje. (C) Isesengura ry’imvugo y’amaproteine ​​y’igihe ryerekanwa ko ari ikintu gihambaye co gutera indwara y’imitsi (t-test myinshi, *FDR <5%; n = 3-5 imbeba). (D) Hejuru: Igishushanyo c’urutonde kigaragaza uburyo butandukanye bwo kwinjira muri carbone ifise ikimenyetso iri mu [1-13C]pyruvate tracer (ni ukuvuga, biciye ku nzira ya PDH canke y’inzira y’amaraso). Inyuma: Igishushanyo c’inanga kirerekana ijana kw’ijana ry’umuyagankuba (M1) ufise ikimenyetso kimwe wahinduwe aside aspartique, aside citrice na aside malique inyuma yo gushirako ikimenyetso ku bice vy’ubwonko bukomeye bikoresheje [1-13C]pyruvate (paired t-test; ** P <0.01). (E) Isesengura ry’amateka y’igihe ry’inzira yerekanwa. Niwiyumvire gusa poroteyine zifise P<0.05 ku ndwi 8. Umurongo w’agacavutu: nta gaciro ko guhindura (isesengura ry’inzira zibiri ry’ugutandukana; * P <0.05; *** P <0.001). Amakuru agaragazwa nk’uko ari hagati ± SEM.
Mu icegeranyo cacu, BCAA catabolism yacitse imwe mu nzira nyamukuru zo gutera imbere. Ivyo bigaragaza cane ko umubare w’impwemu winjira mu nzira ya TCA ushobora guhinduka muri PN idafise OXPHOS. Ivyo bishobora guserukira uburyo buhambaye bwo guhindura imitsi y’ubwonko, bishobora kugira ingaruka zitaziguye ku mibiri y’ubwonko no ku buzima mu gihe co kubungabunga ubumuga bukomeye bwa OXPHOS. Mu buryo buhuye n’ico ciyumviro, twabonye ko umuti nyamukuru urwanya indwara y’imitsi PCx uhindurwa (Mfn2cKO/CTRL uhinduka nk’incuro 1,5; Igishushanyo 3A), ivyo bikaba bituma pyruvate ihinduka oxaloacetate (28), ivyo bikaba vyemerwa ko biri mu brain, 30). Mu buryo buhuye n’ibivuye mu bipimo vya proteomics, confocal microscopy yerekanye ko PCx yariyongereye cane kandi cane mu PNs zidafise OXPHOS, mu gihe PCx reactivity yari igarukiye canecane ku nsoro za Bergmann glial ziri hafi y’ivyo bipimo (Ishusho 3B). Kugira ngo tugerageze ubushobozi bwa PCx, twavuye ibice vy’ubwonko bukomeye n’umuti [1-13C]pyruvate tracer. Igihe pyruvate yahindurwa n’umugera wa pyruvate dehydrogenase (PDH), ikimenyetso cayo c’isotope carazimiye , Ariko kija mu bihimba vy’umubiri vy’inzira ya TCA igihe pyruvate ihindurwa n’imitsi (Ishusho 3D). Mu gushigikira amakuru yacu y’ubuhinga bwa proteomics, twabonye umubare munini w’ibimenyetso biva kuri iyo tracer muri aside aspartique y’ibice vya Mfn2cKO, mu gihe aside citric na aside malic na vyo nyene vyari bifise umurongo uringaniye, naho bitagira akamaro (Ishusho 3D).
Mu mitsi y’umubiri y’imbeba za MitoPark zifise ubumuga bwo mu mutwe buterwa n’imitsi y’umubiri isenya canecane umugera wa mitochondrie (Tfam) (Ishusho S6B), PCx expression na yo nyene yari itunganijwe cane (31), ivyo bikaba vyerekana ko ubumuga bwo mu mutwe butera ubumuga bwo mu mutwe buterwa n’imitsi y’umubiri. OXPHOS mu mubiri. Birabereye kumenya ko vyabonetse ko enzymes zidasanzwe (32-34) zishobora kugaragara mu mitsi y’ubwonko zishobora kuba zifitaniye isano n’indwara y’imitsi y’amaraso zishobora guterwa imbere cane mu PNs zidafise OXPHOS, nk’iyitwa propionyl-CoA carboxylase (PCC-A), Malonyl-CoA mitochylCoA zihindura propionyl-CoA zikaba . enzyme 3 (ME3), uruhara rwayo nyamukuru ni ugukuraho pyruvate mu malate (Ishusho 3, A na C) (33, 35). Ikindi, twabonye ko hariho ukwiyongera gukomeye kw’intungamubiri ya Pdk3, ikora phosphorylates gutyo igatuma PDH idakora (36), mu gihe ata mpinduka zabonetse mu ntungamubiri ya Pdp1 ikora PDH canke mu ntungamubiri ya PDH ubwayo (Ishusho 3A). Mu buryo buhuye, mu PNs za Mern2cKO, uguhinduka kw’igice ca α1 (PDHE1α) c’igice ca pyruvate dehydrogenase E1 c’igice ca PDH kiri muri Ser293 (kizwi ko kibuza igikorwa c’intungamubiri za PDH) kwari kwarongerekanye ( Igishushanyo S6C) (Igishushanyo S6C). Pyruvate nta n’aho ishobora gushika mu mitsi.
Ubwa nyuma, twabonye ko inzira nziza cane y’uguhingura serine na glycine, uruziga rw’imitochondrie folate (1C) n’uguhingura proline (Ishusho 1G n’Ishusho S5C) vyose bihinduka cane, nk’uko bivugwa na raporo, mu gihe c’ugukora. Ivyo bihimba bikikije birakorana n’ukudakora neza kw’imitochondrie (5-7). Isesengurwa ry’ibintu bimwe bimwe rishigikira ayo makuru y’ivy’amaproteine ​​ryerekanye ko muri PN ata OXPHOS iriho, ibice vy’ubwonko vy’imbeba z’indwi 8 vyakoreshejwe serine hydroxymethyltransferase 2 (SHMT2), enzyme nyamukuru y’ingendo y’imitochondrie. Inyishu ihambaye y’ubudahangarwa bw’umubiri (Ishusho S5D). Mu bice 13 vy’ubwonko bukomeye bwari bwatewe n’isukari CU, igerageza ryo gukurikirana ivy’imibiri ryaremeje kandi ko ivy’uguhingura ivyuma vy’umubiri vy’umubiri vy’umubiri vy’umubiri vy’umubiri vy’umubiri vy’umubiri vy’umubiri vy’umubiri vy’umubiri vy’umubiri vy’umubiri vy’umubiri vy’umubiri vy’umubiri vy’umubiri vyiyongera (Ishusho S5E). Kubera ko inyishu ziterwa imbere na GLS na GPT2 ari zo zituma habaho ugukora glutamate iva muri glutamine be n’uguhinduranya hagati ya glutamate na α-ketoglutarate, uguhindura kwavyo kwerekana ko imitsi y’ubwonko idafise OXPHOS ifise ivyifuzo vyinshi vy’ivyo bita glutamate , Ivyo bishobora kuba bigamije kubungabunga ivyimburwa vy’umubiri vyiyongereye (Ishusho5). Mu buryo butandukanye n’ayo mahinduka, isesengura ry’amaproteine ​​y’ibihimba vy’umubiri vy’ubwonko bivuye mu mbeba za Mfn2cKO zidasanzwe za PN ryerekanye ko izo nzira (harimwo n’ivyo bita antiperoxidases zose) zitahindutse cane mu guserura, gutyo bikaba vyerekana ko iyo nzira y’uguhindura ibiribwa ari iyo guhitamwo PN yasenyutse gushika kuri G (Ishusho S6, G).
Mu ncamake, izo nseguro zarerekanye uburyo butandukanye cane bwo gukora kw’igihe c’inzira zimwe zimwe z’imibiri mu PNs. Naho igikorwa kidasanzwe c’imitsi y’ubwonko gishobora gutuma umuntu agira indwara y’imitsi y’ubwonko n’uguhindura 1C (Ishusho 3E n’Ishusho S5C), eka mbere n’amahinduka ashobora gutegekanirwa mu bijanye n’uguserura imitsi ya I na IV, amahinduka mu gukora serine de novo synthesis ni gusa Yabonetse mu myaka ya nyuma. OXPHOS idakora neza (Ishusho 3E n’Ishusho S5C). Ivyo bimenyetso bisobanura urutonde rw’imitsi y’umubiri (1C cycle) n’imitsi y’umubiri (serine biosynthesis) bitera umubabaro bifatanya n’ukwiyongera kw’indwara y’imitsi mu nzinguzingu ya TCA kugira ngo bihindure imiterere y’imitsi.
PNs zifise ubukene bwa OXPHOS zifise indwi 8 zishobora kuguma zikora igikorwa co gukangura incuro nyinshi kandi zigasubira gukorana cane kugira ngo zisubize inyuma ukudakora neza kw’imitochondrie. Ivyo bivumbuwe biratuma habaho ubushobozi bushimishije bw’uko no muri iki gihe, izo ngingo zishobora no kuronka ubuvuzi kugira ngo zicereze canke zikingire ugusenyuka kw’imitsi. Biratevye. Ivyo twabitorera umuti biciye mu nzira zibiri zigenga. Mu buryo bwa mbere, twateguye umugera w’indwara y’ibihimba vy’irondoka (AAV) ushingiye kuri Cre kugira ngo MFN2 ishobore guserurwa mu buryo butoranijwe mu PNs zidafise OXPHOS in vivo (Ishusho S7A). AAV ikoresha MFN2 n’umugera w’umumenyeshamakuru w’umuco mCherry (Mfn2-AAV) vyasuzumwe mu mico kama y’imitsi y’ubwonko y’intango in vitro, ivyo vyatumye MFN2 igaragara mu buryo bushingiye kuri Cre kandi irokora imiterere y’imitsi y’ubwonko, gutyo bikaba bibuza guhinduka kw’imitsi y’ubwonko ( MfnKO, B Igishushanyo S7). Inyuma y’aho, twakoze igerageza ry’ubuzima kugira ngo dushikirize mu buryo butari bwo Mfn2-AAV y’indwi 8 ku rwego rw’ubwonko rwa Mfn2cKO n’imbeba zigenzura, maze dusuzuma imbeba z’indwi 12 (Ishusho 4A). Imbeba za Mfn2cKO zavuwe zapfuye (Ishusho 1, A na B) (16). Uguhindura umugera mu buzima bwatumye PN igaragara mu buryo bubereye mu mizingi imwe imwe y’ubwonko (Ishusho S7, G na H). Injection y’ubugenzuzi AAV igaragaza gusa mCherry (Ctrl-AAV) nta ngaruka zikomeye zagize ku rugero rw’ugusenyuka kw’imitsi mu bikoko vya Mfn2cKO. Mu buryo bunyuranye, isesengura rya Mfn2cKOs ryahinduwe na Mfn2-AAV ryerekanye ingaruka zikomeye zo gukingira uruzitiro rw’ingingo za PN (Ishusho 4, B na C). Cane cane, uburemere bw’imitsi y’ubwonko busa n’ubudashobora gutandukanywa n’ibikoko bigenzurwa (Ishusho 4, B na C, n’Ishusho S7, H na I). Invugo ya MFN1 ariko atari MFN2 irakora neza mu gukiza urupfu rw’imitsi (Ishusho 4C n’Ishusho S7, C na F), ivyo bikaba vyerekana ko invugo ya ectopic MFN1 ishobora kwongereza neza ukubura kwa MFN2. Ibindi bipimo ku rugero rwa PN rumwe vyerekanye ko Mfn2-AAV yarokoye cane cane imiterere y’imbere y’imitochondrie, ihindura urugero rwa mtDNA, kandi ihindura ivyerekanwa vyinshi vy’ikimenyetso co kurwanya angiogenesis PCx (Ishusho 4, C gushika kuri E ). Gusuzuma n’amaso imbeba za Mfn2cKO zarokowe ziri mu gihe c’akaruhuko vyerekanye ko ukuntu zihagaze n’ibimenyetso vy’imitsi (ukwinyiganza S1 gushika S3) vyari vyiza. Mu gusozera, izo ngeragezwa zirerekana ko gutevya gusubira kwinjiza MFN2 mu PNs zidafise cane OXPHOS bihagije kugira ngo bihindure ukuntu mtDNA ikoreshwa no gutuma habaho indwara y’imitsi, gutyo bikabuza gusenyuka kw’imitsi y’umubiri n’urupfu rw’imitsi y’umubiri in vivo.
(A) Igishushanyo kigaragaza urutonde rw’igerageza rwo gutera AAV ikoresha MFN2 igihe inzira y’imibiri yerekanwa ikora. (B) Amashusho aserukira ivyiyumviro vy’ibice vy’ubwonko vy’indwi 12 vyahinduwe ku ndwi 8 mu mbeba za Mfn2cKO kandi vyashizweko ikimenyetso c’umubiri urwanya Calbindin. Iburyo: Gupima intsinga z’imitsi y’umubiri. Urugero rw’ivyo bihimba vy’umubiri ni 450 na 75 μm. (C) Ibubamfu: Gupima uburemere bw’ingingo za Purkinje mu nzira y’uguhindura AAV (AAV+) (isesengura ry’uburyo bumwe ry’uguhinduka; n = 3 imbeba). Iburyo: isesengura ry’ivyiyumviro vya mtDNA mu PN yahinduwe ku ndwi ya 12 (ikigeragezo ca t kitagira ababiri; n = 6 utugingo ngengabuzima tuvuye mu mbeba zitatu). * P <0,05; ** P <0,01. (D) Ivyerekanwa vy’ugutanga amakuru y’imigera y’ibice vy’ubwonko bwa Mfn2cKO vyahinduwe n’ibimenyetso vy’imigera vyerekanwa. Ico gipfukisho c’ibara ry’agahama kirerekana ahantu hari ama dendrite, kandi ikibanza c’umuhondo gifise utudomo kigaragaza ukuntu umuntu ashobora gukura ibintu iburyo; n igereranya umubiri. Umurongo w’urugero, 1μm. (E) yerekana akarorero k’ugutera ibara rya PCx muri PN yahinduwe ku ndwi 12. Igiti c’urugero, 20μm. OE, guserura ivyiyumviro birenze urugero; FC, guhindura.
Ubwa nyuma, twarondeye akamaro k’ukurokoka kw’ingingo ngenderwako ziterwa na peroxidase mu PNs zashikiwe n’ubumuga bwa OXPHOS. Twarakoze mCherry ikoresha AAV-shRNA (ARN y’umusatsi mugufi) yibanda canecane ku mRNA y’imbeba PCx (AAV-shPCx), maze dutera uwo mugera canke uburyo bwo kuwugenzura (AAV-scr) mu bwonko bw’imbeba za Mfn2cKO. Iryo rushinge ryakozwe mu ndwi ya kane y’ubukure (Ishusho 5A) kugira ngo PCx ishobore gutera neza mu kiringo PCx yariko iraseruka (Ishusho 3C) kandi igice c’ingingo za PN cari kikiriho (Ishusho 1A). Birabereye kumenya ko gutera hasi PCx (Ishusho S8A) bituma urupfu rwa PN rwihuta cane, rukaba ruri ku mpeta yanduye gusa (Ishusho 5, B na C). Kugira ngo dutahure uburyo ingaruka z’imibiri ziterwa n’uguhindura PCx zikora, twarize ivyerekeye redox y’ibihimba vy’umubiri inyuma y’aho PCx iterewe hasi, kandi n’ivyo bihimba vy’umubiri bikoreshwa na AAV Grx1-roGFP2 vyaseruwe icarimwe (Ishusho S8, B gushika kuri D) kugira ngo dusuzume glutathione potential of3). Hanyuma, twakoze ubuhinga bwo gupima ubuzima bwose bw’amafoto abiri (FLIM) mu bice vy’ubwonko vy’abantu b’indwi 7 Mfn2cKO canke abagenzuzi b’ibikoko kugira ngo tumenye amahinduka yoshobora kubaho mu bijanye n’imimerere y’ubuzima bw’umubiri inyuma yo kugenzura imimerere ya FLIM (Ishusho S8, E gushika kuri G). Iryo sesangura ryerekanye ko hariho ukwiyongera gukomeye kw’imimerere y’uguhinduka kw’umubiri kw’umubiri umwe wa Mfn2cKO PNs udafise PCx, ivyo bikaba bitandukanye n’imitsi y’umubiri canke Mfn2cKO PNs ziserura shRNA gusa (Ishusho 5, D na E). Igihe PCx yariko iragabanywa, ijana kw’ijana ry’ibihimba vya Mfn2cKO PNs vyerekana ko bifise ubuzima bwiza cane vyariyongereye incuro zirenga zitatu (Ishusho 5E), bikaba vyerekana ko PCx up-regulation yagumye ifise ubushobozi bwo guhindura ibara ry’umubiri bw’imitsi y’ubwonko yasenyutse.
(A) Igishushanyo kigaragaza urutonde rw’igerageza rwo gutera AAV ikoresha shPCx igihe inzira y’imibiri yerekanwa ikora. (B) Amafoto yerekana ibice vy’ubwonko vy’indwi 8 mu mbeba za Mfn2cKO zahinduwe kandi zishirwako ikimenyetso c’umubiri urwanya calcineurin ku ndwi 4. Igiti c’urugero, 450μm. (C) Gupima uburemere bw’ingingo za Purkinje mu nzitiro zahinduwe na AAV (isesengura ry’uburyo bumwe ry’ugutandukana; n = 3 gushika ku 4 z’imbeba). Amakuru agaragazwa nk’uko ari hagati±SEM; ***P<0,001. (D) Ishusho ya FLIM iserukira yerekana igihe c’ubuzima c’indwi 7 PN yerekana glutathione redox sensor Grx1-roGFP2 mu bihe vy’igerageza vyavuzwe. LUT (imeza yo kuraba) igipimo: igihe co kurokoka (mu masegonda). Igiti c’urugero, 25μm. (E) Histogram yerekana ukuntu Grx1-roGFP2 igabanywa n’agaciro k’ubuzima kuva kuri (D) (n=158 gushika ku 368 utugingo ngengabuzima mu mbeba zibiri mu gihe c’imimerere imwe imwe). Igishushanyo c’ibipande kiri hejuru y’igishushanyo cose: kigaragaza igitigiri c’ingingo zifise agaciro k’ubuzima burebure cane (umutuku, bufise umwuka) canke bugufi (ubururu, bugabanutse), burengeye 1 SD y’agaciro k’ubuzima busanzwe muri CTRL-AAV-scr. (F) Ico kigereranyo cashikirijwe kirerekana ingaruka zo gukingira PCx y’imitsi.
Muri vyose, amakuru dutanga hano yerekana ko gusubira kugaragaza MFN2 bishobora gukiza burundu PN iteye imbere ifise ubukene bukomeye bwa OXPHOS, ukubura mtDNA gukomeye, n’imiterere idasanzwe cane imeze nk’iya ista, gutyo bikaba bitanga iterambere ridahera mbere no mu ndwara ziteye imbere. Ugusenyuka kw’imitsi y’ubwonko gutanga ikimenyamenya gishobora gusubirwamwo c’inzira y’imbere y’urupfu rw’ingingo ngenderwako. Uwo murongo w’uguhinduranya ibiribwa urashimikwako cane n’ubushobozi bw’imitsi y’ubwonko bwo gutuma habaho indwara y’imitsi (ugusubira guhindura umurongo wa TCA), ivyo bikaba bibuza PCx guseruka muri PNs zitagira OXPHOS kandi bigatuma utugingo ngengabuzima dupfa, gutyo bikagira uruhara rwo gukingira (Ishusho 5F).
Muri iki cigwa, twatanze ikimenyamenya c’uko inyishu ya PNs ku bijanye n’ukudakora neza kwa OXPHOS ari ugutera imbere buhoro buhoro ku ndwara y’imitsi y’amaraso y’ingendo ya TCA biciye mu nzira y’ugukora itandukanye ikoreshwa na porogarama z’imibiri. Twemeje ko isesengura ry’amaproteine ​​n’uburyo bwinshi bwo kwuzuzanya kandi twahishuye ko iyo umuntu ahanganye n’ukudakora neza kw’imitochondrie, imitsi y’ubwonko ifise uburyo butazwi mbere bwo guhindura ibiribwa. Turatangazwa n’uko iyo nzira yose yo gusubiramwo amawaya idashobora gutuma umuntu agira ikimenyetso c’iherezo ry’imibiri ijana n’ugusenyuka kw’imitsi buhorobuhoro kandi ataco ihinduye, ariko amakuru yacu yerekana ko yoshobora kuba ari umutsi w’umubiri ushobora gukingira mbere no mu gihe c’imbere y’urupfu rw’ingingo Uburyo bwo gusubiza inyuma. Ivyo vyavumbuwe vyerekana ko utugingo ngengabuzima dufise urugero runini rw’ubushobozi bwo guhindura ibiribwa mu mubiri. Ivyo vyerekana ko gusubira gukoresha MFN2 mu nyuma bishobora guhindura ukuntu ibimenyetso nyamukuru vy’imibiri bigaragara no gukingira PN gusenyuka. Ahubwo riho, irabuza indwara y’imitsi y’amaraso kandi igatuma imitsi yihuta. abahindura igitsina.
Kimwe mu bintu biteye umunezero cane twabonye mu bushakashatsi bwacu ni uko PNs zidafise OXPHOS zishobora guhindura imigenderanire y’imitsi ya TCA biciye mu gutuma enzymes zitera canecane indwara y’imitsi. Gusubira gutunganya ivy’umubiri ni ikintu gisanzwe mu ngingo za kanseri, zimwe muri zo zikaba zishingiye kuri glutamine kugira ngo zishiremwo ibintu bifatanya n’ibindi bigabanya, ivyo bikaba bituma uruhererekane rw’uguhema rugenda neza kandi bikaba bigumya ivyimburwa vy’ibihimba vy’umubiri bita lipid na nucleotide biosynthesis (39 , 40). Icigwa giherutse gukorwa cerekanye ko mu bihimba vy’inyuma bishikirwa n’ubumuga bwa OXPHOS, ugusubira gufatanya kw’imibiri ya glutamine/glutamate na vyo nyene ni ikintu gihambaye (5, 41), aho uburyo glutamine yinjira mu nzinguzingu ya TCA buvana n’Ukubera uburemere bw’ibikomere vya OXPHOS (41). ). Ariko rero, nta bimenyamenya bitomoye biriho ku bijanye n’ugusa kwose kw’ubuhinga bwo guhindura ibiribwa mu mubiri be n’akamaro kavyo mu bijanye n’ingwara. Mu cigwa cakozwe vuba cane, imitsi y’imbere y’umubiri yerekanwe ko ikoranya ibidengeri vya glutamate kugira ngo igere ku nzira y’imitsi, gutyo igatuma habaho uguhinduka kw’imitsi n’ugukomera kw’imitsi mu bihe vy’imibabaro (42). Ni vyiza kumenya ko mu gihe c’uguhagarika kw’imiti kw’intungamubiri y’ingendo ya TCA yitwa succinate dehydrogenase, pyruvate carboxylation yizerwa ko igumya ugukorana kwa oxaloacetate mu mitsi y’ubwonko (34). Ariko rero, ukuntu izo nzira zifitaniye isano n’imitsi y’ubwonko (aho indwara y’imitsi y’ubwonko yizera ko ahanini igarukira ku mitsi y’inyenyeri) iracari n’agaciro kanini mu mitsi y’ubwonko (43). Muri ivyo, amakuru yacu yerekana ko PNs zononekaye na OXPHOS mu mubiri zishobora guhindurwa zigasenyuka BCAA n’ugusenyuka kwa pyruvate carboxylation, ivyo bikaba ari vyo bibiri nyamukuru bifasha mu kwongerako ivyuma vyo hagati mu mazi ya TCA. Naho uruhara rw’ihinduka ry’inguvu z’imitsi y’ubwonko rwavuzwe, uretse uruhara rwa glutamate na GABA mu guhindura inguvu z’ubwonko (44), nta kimenyamenya kihari c’uko ubwo buryo bukora in vivo. Ku bw’ivyo, biroroshe guca irya n’ino ko PNs zidakora neza zishobora kwishura ubwo nyene ku bijanye n’ugufungura kw’ibintu vyo hagati vya TCA biterwa n’ingendo y’ugufatanya n’ibindi bihimba vy’umubiri mu kwongerera indwara y’imitsi. Cane cane, upregulation ya PCx ishobora gukenerwa kugira ngo umuntu agumane ivyifuzo vyinshi vy’aside aspartique, ivyo bikaba bivugwa mu kwiyongera kw’ingingo zifise ubumuga bw’imitochondrie (45). Ariko rero, isesengura ryacu ry’ivy’imibiri ntiryerekanye amahinduka akomeye mu rugero rw’ubuzima buhoraho bw’aside ya aspartic mu Mfn2cKO PNs (Ishusho S6A), ivyo bikaba vyerekana ko aside ya aspartic ikoreshwa hagati y’ingingo zigwira n’imitsi y’inyuma y’imitotic. Naho uburyo nyabwo bwo guhindura PCx mu mitsi idakora neza in vivo buguma bumenyekana, twarerekanye ko iyo nyishu itarashika ifise uruhara runini mu kubungabunga ubuzima bw’imitsi, ivyo bikaba vyerekanwa mu ngeragezwa za FLIM ku bice vy’ubwonko. Cane cane, kubuza PNs guhindura PCx bishobora gutuma umuntu agira ubuzima bwiza cane kandi bigatuma utugingo ngengabuzima twihuta gupfa. Ivyo bikaba vyatumye BCAA igira ubushobozi bwo gusenyuka be n’uko pyruvate ikora carboxylation si uburyo bwo kumenya ko imitsi y’inyuma y’umubiri idakora neza (7). Ku bw’ivyo, bisa n’uko ari ikintu gihambaye cane mu mitsi y’ubwonko idafise OXPHOS, naho yoba atari co kintu conyene, kikaba ari ikintu gihambaye ku bijanye n’ugusenyuka kw’imitsi. .
Indwara y’ubwonko ni ubwoko butandukanye bw’ingwara y’ubwonko ikunda kwigaragaza nk’ataxia kandi akenshi yononekaza PNs (46). Ivyo bice vy’imitsi y’ubwonko birashobora cane gushikirwa n’ukudakora neza kw’imitsi y’ubwonko kubera ko ugusenyuka kwavyo mu mbeba guhagije kugira ngo bivyare ibimenyetso vyinshi vy’imitsi y’ubwonko biranga ataxia spinocerebellar y’umuntu (16, 47, 48). Dukurikije amakuru, ikigereranyo c’imbeba gihinduwe n’umubiri gifise umugera uhinduka gifitaniye isano n’ubumuga bwo mu mutwe bw’umuntu kandi gifise ubumuga bwo mu mutwe (49, 50), ivyo bikaba bishimika ku kamaro ko kwiga ingaruka z’ukubura OXPHOS muri PNPH. Ku bw’ivyo, birabereye canecane gutandukanya neza no kwiga iyo nkuru idasanzwe y’imitsi y’ubwonko. Ariko rero, kubera ko PNs zikunda cane gushikirwa n’imikazo kandi zikaba zigize igice gitoyi c’ingingo zose z’ubwonko, ku nyigisho nyinshi zishingiye ku buhinga bwa omics, kuzitandukanya nk’ingingo zose biracari ikintu kigoye. Naho ari hafi bidashoboka ko umuntu ashobora kutagira ubuhumane bw’ibindi bihimba vy’umubiri (na canecane utugingo ngengabuzima tw’abantu bakuze), twahuje intambwe nziza yo gutandukanya n’ibindi bihimba vy’umubiri kugira ngo turonke umubare uhagije w’imitsi y’ubwonko ishobora gukoreshwa mu gusesangura ivy’amaproteine, kandi dufise proteine ​​nyinshi cane zigereranywa n’ivy’amaproteine ​​(nk’ibice 3000 vy’amaproteine) (51). Mu kuzigama ubuzima bw’ingingo ngenderwako zose, uburyo dutanga ngaha ntibutuma tudashobora gusa kugenzura ihinduka ry’inzira z’imibiri mu mitochondrie, ariko kandi tugasuzuma n’ihinduka ry’ingingo ngenderwako z’ingingo ngenderwako, ivyo bikaba bifasha gukoresha ibimenyetso vy’ingingo ngenderwako kugira ngo dutunganye ubwoko bw’ingingo z’umubiri The new method (52, 53). Uburyo tudondora ntibujanye n’ukwiga gusa ingingo za Purkinje, ariko burashobora gukoreshwa mu buryo bworoshe ku bwoko bwose bw’ingingo kugira ngo bufashe mu gutorera umuti ihinduka ry’imibiri mu bwonko burwaye, harimwo n’ibindi bigereranyo vy’ukudakora neza kw’imitochondrie.
Ubwa nyuma, twabonye idirisha ryo kuvura muri iyo nzira yo gusubira gutunganya ivy’umubiri rishobora guhindura burundu ibimenyetso nyamukuru vy’uguhagarika umutima kw’ingingo ngenderwako no gukingira ugusenyuka kw’ingingo ngenderwako. Ku bw’ivyo, gutahura ingaruka z’ibikorwa vy’ugusubira gukoresha amawaya bivugwa ngaha birashobora gutanga ubumenyi bw’ishimikiro ku bijanye n’ubuvuzi bushoboka bwo kubungabunga ubuzima bw’imitsi y’ubwonko mu gihe imitsi y’ubwonko idakora neza. Ubushakashatsi bwo muri kazoza bugamije gucapura ihinduka ry’uguhindura inguvu mu bindi bihimba vy’ubwonko burakenewe kugira ngo umuntu amenye neza ukuntu iyo ngingo ngenderwako ishobora gukoreshwa ku zindi ndwara zifata ubwonko.
Imbeba za MitoPark zaravuzwe kera (31). Imbeba C57BL/6N zifise loxP iruhande y’imitsi ya Mfn2 zari zaradondowe mbere (18) kandi zijabukana n’imbeba L7-Cre (23). Ivyo bihimba bibiri vy’umubiri bitandukanye vyaciye bijabukwa n’imbeba zifise umubiri umwe Mfn2loxP/Mfn2loxP kugira ngo bivemwo ivy’uruvyaro rwa Purkinje vy’umubiri wa Mfn2 (Mfn2loxP/Mfn2loxP; L7-cre). Mu gice c’uguhuza ibitsina, umugera wa Gt (ROSA26) SorStop-mito-YFP (stop-mtYFP) warashizwemwo biciye mu kujabukana kw’inyongera (20). Ivyo vyose vyakozwe ku bikoko vyakozwe hakurikijwe amabwirizwa y’Uburaya, y’igihugu n’ay’inzego kandi vyemejwe na LandesamtfürNatur yo muri Umwelt na Verbraucherschutz, muri Rhénanie du Nord-Westphalie, mu Budagi. Ibikorwa vy’ibikoko na vyo nyene bikurikiza ubuyobozi bw’Ishirahamwe ry’Uburaya ry’Ivy’Ibikoko vyo mu Laboratwari.
Amaze gutera umuti w’ububabare ku nzira y’umugore atwite, urusoro rw’imbeba ruratandukanwa (E13). Ico gice c’umubiri caciwe mu munyu w’umunyu wa Hanks (HBSS) wongereweko Hepes 10 mM hanyuma gica ku munyu wa Dulbecco wahinduwe w’inkukuma urimwo papaine (20 U/ml) na cysteine ​​(1μg/ml). Ivyo bihimba bishire mu DMEM) hanyuma bikabitandukanya biciye mu gufata ibiribwa bikoresheje enzyme. Ml) ku bushuhe bwa 37°C mu minota 20, hanyuma bagaseya mu buryo bw’imashini mu DMEM yongeweko 10% y’amaraso y’inka y’uruhinja. Utugingo ngengabuzima twatewe ku bipande vy’ibirahuri vyuzuye polylysine ku buremere bwa 2×106 ku gitereko c’ivyatsi c’amasentimetero 6 canke ku buremere bwa 0,5×105 utugingo ngengabuzima/cm2 kugira ngo hakorwe isesengura ry’amashusho. Inyuma y’amasaha 4, iyo miti yasubirijwe n’imiti idafise serum ya Neurobasal irimwo 1% y’inyongera ya B27 na 0,5 mM ya GlutaMax. Ivyo bihimba vy’umubiri vyaciye bigumwa ku bushuhe bwa 37°C na 5% CO2 mu kiringo cose c’igerageza, hanyuma bikagaburirwa rimwe mu ndwi. Kugira ngo haboneke ugusubira gukorana mu bipimo vy’ubushakashatsi, 3μl (isahani y’imico kama y’amariba 24) canke 0.5μl (isahani y’amariba 24) y’umugera wa AAV9 ukurikira vyakoreshejwe mu kuvura imitsi y’ubwonko ku musi ugira kabiri mu bipimo vy’ubushakashatsi: AAV9.CMV.PI.eGFP. 105530-AAV9) na AAV9.CMV.HI.eGFP-Cre.WPRE.SV40.
ADN y’imbeba Mfn1 na Mfn2 (ivuye muri plasmide ya Addgene #23212 na #23213, uko bikurikirana) zishirwako ikimenyetso c’urutonde rwa V5 (GKPIPNPLLGLDST) ku mpera ya C, kandi zifatanywa na mCherry mu gicapo biciye ku rutonde rwa T2A. Grx1-roGFP2 ni ingabirano yatanzwe na TP Dick DFKZ wo mu gisagara ca Heidelberg. Mu gusubirira kaseti ya tdTomato hakoreshejwe uburyo busanzwe bwo guhingura, iyo kaseti yarahinduwe mu mugongo wa pAAV-CAG-FLEX-tdTomato (inomero y’ishingiro ya Addgene 28306) kugira ngo haboneke pAAV-CAG-FLEX-mCherry-T2A-MFNGAV-V5, pAAV-CAG-FLEX-mCherry-T2A-MFNGAV2-V5, Ivyo bimenyetso vy’ubuhinga bwa none. Ingamba nk’izo nyene zakoreshejwe kugira ngo haboneke umurongo w’ubugenzuzi pAAV-CAG-FLEX-mCherry. Kugira ngo umuntu ashobore gukora iyo nyubakwa ya AAV-shPCx, birakenewe ko habaho umugera wa AAV (VectorBuilder, pAAV [shRNA] -CMV-mCherry-U6-mPcx- [shRNA#1]), ukaba urimwo urutonde rwa ADN rushiramwo shRNA igenewe PCx y’imbeba (5′CTAGCTAGGTTCGTAGCTAACTA 3′) Mu kugenzura kw’umurongozi wa U6, mCherry ikoreshwa munsi y’uburongozi bw’umurongozi wa CMV. Ivyo gukora ama vecteurs y’inyongera ya AAV vyakozwe hakurikijwe amabwirizwa y’uwabikoze (Cell Biolabs). Muri make, ukoreshe plasmide yo kwimurira itwara mCherry-T2A-MFN2-V5 (pAAV-CAG-FLEX-mCherry-MFN2-V5), mCherry-T2A-MFN1-V5 (pAAV-CAG-FLEX-mCherry) mu buryo bw’igihe gito-Uguhindura utugingo ngengabuzima tw’umugera wa 2900. (pAAV-CAG-FLEX-mCherry) canke Grx-roGFP2 (pAAV-CAG-FLEX-Grx-roGFP2) bikora amakode y’umugera, hamwe n’ugukora amakode y’ivyo bita AAV1 n’ivyo bita proteine ​​y’ibikoresho. Ivyo bihimba vy’umubiri vy’umugera wa crude supernatant vyaronswe biciye mu nzira y’ugukonja no gukonja mu bwogero bw’urubura/ethanol n’ingingo z’umubiri zigasenyuka mu mazi y’umunyu (PBS). Ico gikoresho ca AAV carasukuwe hakoreshejwe ubuhinga bwo gukoresha iodixanol (amasaha 24 ku 32.000 rpm na 4°C) hanyuma gica gikoreshwa hakoreshejwe akayunguruzo ka Amicon ultra-15. Igitigiri c’ibihimba vy’umubiri vy’umubiri wa AAV1-CAG-FLEX-mCherry-T2A-MFN2-V5 [2,9×1013 kopi y’umubiri (GC)/ml], AAV1-CAG-FLEX-mCherry (6,1×1012 GC/ml), AAV1-CAG-FLEX-mCherry (6,1×1012 GC/ml), AAV1-CAG-FLEX-mCherry (6,1×1012 GC/ml), AAV1-CAG-FLEX-mCherry yagereranywa nk’uko vyari vyagenze imbere (qPCR) -MFN1-V5 (1,9×1013 GC/ml) na AAV1-CAG-FLEX-Grx-roGFP2 (8,9×1012 GC/ml).
Ivyuma vy’ubwonko vya mbere vyaciwe mu mazi akonje nk’urubura 1x PBS, bishirwa mu bipande, hanyuma bihindurwa bimwe mu 0,5% Triton X-100 / 0,5% sodium deoxycholate/PBS lysis buffer irimwo phosphatase na protease inhibiteur (Roche). Ivyo bipimo vy’amaproteine ​​vyakozwe hakoreshejwe ubuhinga bwo gupima aside bicinchonique (Thermo Fisher Scientific). Ivyo bihimba vy’umubiri vyaciye bitandukanywa hakoreshejwe ubuhinga bwa SDS-polyacrylamide, hanyuma bica bishirwa ku gipfukisho c’umuyagankuba (GE Healthcare). Gufunga ahantu hatahariye no gutera n’umubiri w’intango (raba imbonerahamwe S1 ku bijanye n’ibindi) mu mata 5% muri TBST (Tris-buffered saline na Tween), intambwe zo kwoza n’umubiri wa kabiri mu TBST Incubate. Gutera n’umubiri w’intango ijoro ryose ku +4°C. Uhejeje kwoza, ushireko umuti w’indwara ya kabiri amasaha 2 mu bushuhe bwo mu cumba. Hanyuma, mu gutera iyo nkoko nyene n’umubiri urwanya β-actin, ivyo bintu nyene vyaremejwe. Gutahura mu guhindura mu guhindura umuco w’imiti no kwongera umuco w’imiti (GE Healthcare).
Ivyo bihimba vy’umubiri vyari vyaratewe mbere ku bipfukisho vy’ibirahuri vyashizweko 4% vya paraformaldehyde (PFA)/PBS ku gihe categekanijwe ku bushuhe bwo mu cumba iminota 10. Ivyo bipfukisho bibanza guterwa n’amazi y’inka 0,1% Triton X-100/PBS mu minota 5 mu bushuhe bwo mu cumba, hanyuma bishirwa mu mazi yo gukingira [3% y’amaraso y’inka (BSA)/PBS]. Ku musi wa kabiri, ivyo bipande vy’ibipfukisho vyarasukuwe n’umuti wo gukingira umubiri, hanyuma bishirwa mu mfuruka y’umubiri n’umuti wo gukingira umubiri ukwiye (fluorophore-conjugated secondary antibody) mu kiringo c’amasaha 2 mu bushuhe bwo mu cumba; Ubwa nyuma, ivyerekanwa vyarasukuwe neza muri PBS n’umuti witwa 4′,6-diamidino-2 -Phenylindole (DAPI) hanyuma ugashirwa ku gicapo c’imikorosikope n’umuti witwa Aqua-Poly/Mount.
Imbeba (iz’igitsina-gabo n’iz’igitsina-gore) zari zatewe ububabare bw’umubiri biciye mu guterwa umuti witwa ketamine (130 mg/kg) n’umuti witwa xylazine (10 mg/kg) hanyuma ziterwa munsi y’urukoba n’umuti w’ububabare witwa carprofen (5 mg/kg) , Hanyuma zishirwa mu gikoresho co gutera ububabare (Kopd a pamf) equi. Gushira ahabona igihanga maze ukoreshe igikoresho co gutera amenyo kugira ngo ushiremwo igice c’umubiri w’ubwonko gihuye n’igufa rya mis (kuva kuri lambda: umurizo 1,8, uruhande 1, gihuye n’ibice IV na V). Koresha urushinge rw’urushinge rugoramye kugira ngo ukore witonze akabombo gatoyi mu gihanga kugira ngo ntuhungabanye imitsi iri musi. Hanyuma iyo nzira y’ibirahuri ikwezwe buhoro buhoro yinjizwa buhoro buhoro mu mwobo mutoyi (kuva kuri -1,3 gushika kuri -1 ku ruhande rw’imbere rw’igihimba c’umubiri gikomeye), hanyuma 200 gushika kuri 300 nl AAV igaterwa mu gikoresho co gutera umuti (Narishige) hakoreshejwe inshinge z’amaboko (Narishige tisure) ku kiringo c’umuvuduko mutoyi gushika kuri -1. idirisha ry’iminota. Inyuma yo gutera umuti, ushire iyo capillaire mu yindi minota 10 kugira ngo uwo mugera ukwiragire burundu. Inyuma y’aho izo nzira z’amaraso zikuwemwo, urukoba ruradogwa neza kugira ngo umururumba w’ibikomere ugabanuke, maze igikoko kibe gikira. Ivyo bikoko vyavuwe n’imiti igabanya ububabare (caspofen) imisi myinshi inyuma y’aho bibazwe, muri ico kiringo bikaba vyari bikurikiranywe neza uko umubiri wavyo umeze, hanyuma bica bipfa ku gihe cavuzwe. Ivyo vyose vyakozwe hakurikijwe amabwirizwa y’Uburayi, y’igihugu n’ay’inzego kandi vyemejwe na LandesamtfürNatur yo muri Umwelt na Verbraucherschutz, mu Buraruko bwa Rhénanie-Westphalie, mu Budagi.
Ivyo bikoko vyatewe ububabare n’imiti yitwa ketamine (100 mg/kg) na xylazine (10 mg/kg), umutima ubanza guterwa n’imiti 0,1 M PBS, hanyuma ushirwamwo 4% PFA muri PBS. Ivyo bihimba vyaciwemwo ibice bishirwa mu 4% PFA/PBS ijoro ryose ku 4°C. Imbugita izunguruka (Leica Microsystems GmbH, Vienne, Otirishiya) yarakoreshejwe mu gutegura ibice vy’umubiri (uburebure bwa 50 μm) bivuye mu bwonko bugumye buri muri PBS. Keretse iyo bivuzwe ukundi, gutera amabara ku bice bireremba ata co bimaze vyakozwe nk’uko vyavuzwe haruguru (13) mu bushuhe bwo mu cumba no mu gutera imbere. Muri make, ubwa mbere, izo nkoko zaronswe zaciwemwo 0,5% Triton X-100/PBS mu minota 15 ku bushuhe bwo mu cumba; ku bihimba bimwebimwe (Pcx na Shmt2), ukoresheje mu buffer ya tris-EDTA ku 80°C (PH 9) gushushanya ibice mu minota 25 aho gukoresha iyo ntambwe. Inyuma y’aho, ivyo bice vyatewe n’umubiri urwanya indwara (raba urutonde S1) mu gikoresho co gukingira (3% BSA/PBS) ku 4°C ijoro ryose bivugururwa. Bukeye, ivyo bice vyarasukuwe n’umuti wo gukingira indwara, hanyuma biterwa n’umuti wo gukingira indwara ukwiye, ukoresheje fluorophore, mu kiringo c’amasaha 2 mu bushuhe bwo mu cumba; amaherezo, ivyo bice vyarasukuwe neza muri PBS, bica bishirwako ibara ry’umubiri ryitwa DAPI, hanyuma bishirwako ibara ry’umubiri ryitwa AquaPolymount Ku gicapo c’imikorosikope.
Igikoresho co gupima umuco w’ibara ry’agahama (TCS SP8-X canke TCS Digital Light Sheet, Leica Microsystems) gifise umuco w’umuco w’umweru n’umuco w’umuco w’umuco w’amadiode 405 ni co cakoreshejwe kugira ngo umuntu abone ishusho y’ico kigereranyo. Mu gutera umunezero fluorophore no kwegeranya ikimenyetso hakoreshejwe Hybrid Detector (HyDs), porogarama ya LAS-X yarakoreshejwe mu kwegeranya amashusho ateretse ahuye n’ugutora ivyerekanwa vya Nyquist mu buryo bukurikirana: ku bipande bitagira igitigiri, ni ibimenyetso bifise ubushobozi bwinshi (nk’akarorero, mu ngingo z’umubiri n’ivy’imitsi) mt Uburyo bwa BrightR). Gating kuva kuri 0.3 gushika kuri 6 ns irakoreshwa kugira ngo hagabanuke inyuma.
Igihe nyaco c’amashusho y’ingingo zatoranijwe. Inyuma yo gutoranya mu gikoresho ca Neurobasal-A kirimwo 1% y’inyongera ya B27 na 0,5 mM ya GlutaMax, utugingo ngengabuzima twaciye duterwa ku bipande vy’ibirahuri vyuzuye poly-l-lysine (μ-Slide8 Iriba, Ibidi, inomero y’urutonde 80826) , Hanyuma tukabigumya ku 1% ho°C na 300. kugira ngo babeho. Ivyo bishushanyo vy’igihe nyaco vyakozwe ku gikoresho citwa Leica SP8 gikoreshwa n’umuyagankuba gifise umuyagankuba w’umweru, HyD, 63×[1.4 numerical aperture (NA)] c’amavuta y’ibarabara n’ibarabara ry’ugushushanya.
Imbeba yaciye iterwa umuti w’ububabare bwihuse n’umuyagankuba maze iracirwa umutwe, ubwonko bwayo burakurwa mu gihanga, hanyuma buratemwa igice c’umubiri c’uburebure bwa 200μm (ku bijanye n’igerageza ry’ugushirako ikimenyetso ca Fi3C) canke 275μm (ku bijanye n’igerageza ry’amafotoni abiri) c’igice c’umubiri c’umubiri cuzuye ibikoresho bikurikira (The VHr50). Scientific, Walldorf, mu Budagi) yuzuye ibi bikurikira: 125 mM y’urubura, yuzuye carbone (95% O2 na 5% CO2) Ca2 nke + amazi y’ubwonko akozwe n’abantu (ACSF) NaCl, 2,5 mM KCl, 1,25 mM Na3O m2, pho5. glucose, 0,5 mM CaCl2 na 3,5 mM MgCl2 (umuvuduko w’amazi uva kuri 310 gushika kuri 330 mmol). Ivyo bice vy’ubwonko vyaronswe, bishire mu cumba c’imbere y’ugutera ivyatsi birimwo Ca2 + ACSF nyinshi (NaCl 125,0 mM, KCl 2,5 mM, umuti wo gukingira amazi 1,25 mM, NaHCO3 25,0 mM, Ca2.glucose 25,0 mM, Ca2.glucose 100 mM). MgCl2) Iringaniye) pH 7,4 na 310 gushika kuri 320 mmol).
Mu gihe c’ugufata amashusho, ivyo bipande vyari vyimuriwe mu cumba cagenewe gufata amashusho, kandi igerageza ryakozwe mu gihe c’uguterwa ACSF ubudasiba ku bushuhe buhoraho bwa 32° gushika kuri 33°C. Igikoresho co gupima amafoto menshi (TCS SP8 MP-OPO, Leica Microsystems) gifise urushinge rwo gupima amashusho rw’ibintu vyinshi (NA 0,95, amazi), urushinge rw’ibara ry’agahama rwitwa Ti: Sapphire (Chameleon Vision II, Coherent) rwakoreshejwe mu gufata amashusho y’ibice. Igikoresho ca FLIM (Igiharuro c'Igiharuro300, Igiharuro c'Igiharuro).
FLIM ya Grx1-roGFP2. Impinduka ziri mu bijanye n’imiterere y’imitsi y’umubiri (cytoplasmic redox state) zagereranywa n’ivyuma bibiri vy’amafotoni FLIM mu bice vy’ubwonko vy’umubiri, aho igikoresho citwa Grx1-roGFP2 biosensor cari kigamije PNs. Mu rwego rwa PN, ahantu ho kuronka hatorwa hafi 50 gushika kuri 80 μm munsi y’uburebure bw’igice kugira ngo haboneke ko hariho PN ishobora kubaho (ni ukuvuga, ukubura imiterere y’amabuye canke amahinduka y’imiterere y’imitsi y’ubwonko ku ruhande rw’ibihimba vy’umubiri) be n’ivyo gucungera roGFP2 sensor n’ivyo gucungera AAVx-RNA canke sh mCeresi). Kwegeranya amashusho y’ikirundo kimwe afise 2x digital zoom [uburebure bw’umuvuduko w’ugukangura: 890 nm; 512 nm 512 n’ibipimo]. Ivyerekeye: HyD yo mu mutima, umugwi w’iyunguruzo w’isothiocyanate (FITC)] n’ugupima ishusho mu minota 2 canke 3 birakoreshwa kugira ngo hamenyekane ko amafotoni ahagije yegeranywa (amafotoni 1000 yose hamwe) kugira ngo ashobore guhuza umurongo. Ivyo bikaba vyatumye habaho ugukurikirana agaciro k’ubuzima bwa roGFP2 igihe umuntu yongerako 10 mM H2O2 ku ACSF (kugira ngo umuntu ashobore gutera imbere cane, ivyo bikaba bituma umuntu agira ubuzima burengeye ubw’ibindi bihimba vy’umubiri), hanyuma hakongerako mregreethizol of2. kugabanya, bikaba bituma ubuzima bugabanuka) (Ishusho S8, D gushika kuri G). Koresha porogarama ya FLIMfit 5.1.1 kugira ngo usesengure ibisubizo vyaronswe, ushire umurongo umwe w’ugusenyuka kw’ishusho yose ku IRF yagereranywa (igikorwa c’inyishu y’igikoresho), kandi χ2 ni nk’1. ikoreshwa mu gupima igitigiri.
Isesengura ry’ubushobozi bw’imitochondrie. Inyuma y’aho igice c’umubiri gikomeye giteye TMRM 100 nM yongewe ku ACSF yashizwemwo iminota 30, amahinduka y’ubushobozi bw’imitochondrie ya PNs yapimwa n’igikoresho citwa microscope c’amafotoni abiri. Ivyo bishushanyo vya TMRM vyakozwe mu gutera umunezero igikoresho co gupima ku 920 nm no gukoresha HyD yo mu mutima (isothiocyanate de tetraméthylrhodamine: 585/40 nm) kugira ngo hakusanyirizwe hamwe ibimenyetso; mu gukoresha uburebure bw’umuvuduko w’ugukangura bumwe ariko hakoreshejwe HyD y’imbere itandukanye (FITC :525/50) kugira ngo mtYFP ifate ishusho. Koresha igikoresho co guharura ishusho ca ImageJ kugira ngo usuzume ubushobozi bw’imitochondrie ku rugero rw’ingingo imwe. Muri make, ihanganywa ry’ivyuma: ikimenyetso = min (mtYFP, TMRM) rikoreshwa mu kumenya akarere k’imitochondrie kerekana ikimenyetso ca TMRM mu rurimi rwa Purkinje Somali mu gishushanyo c’umurongo umwe w’umurongo uhuye. Hanyuma, uburebure bw’ibipimo biri mu gipfukisho kivuyemwo buragereranywa, hanyuma bugasanzwe ku gishushanyo c’uruzitiro rumwe rw’umurongo wa mtYFP kugira ngo haboneke igice c’imitochondrie kigaragaza ubushobozi bw’imitochondrie.
Iryo shusho ryaciwemwo ibintu bitari vyo hakoreshejwe porogarama yitwa Huygens Pro (Igishushanyo c’Igitabu c’Ivyuma). Ku mashusho y’amabuye y’agaciro, uguteranya amabuye y’agaciro rimwe bikorwa hakoreshejwe ubuhinga bwo gudodera bwikora butangwa na porogarama ya LAS-X. Inyuma yo gupima ishusho, koresha ImageJ na Adobe Photoshop kugira ngo urushirize gukora ishusho no gutunganya mu buryo bumwe umuco n’ubudasa. Koresha igishushanyo ca Adobe kugira ngo utegure ibishushanyo.
mtDNA isesengura ry’ibintu. Igitigiri c’ibikomere vya mtDNA caragereranywa ku bice vy’ubwonko bwari bwanditsweko imiti irwanya ADN hakoreshejwe igikoresho citwa confocal microscope. Buri kibanza c’intumbero cararemewe umubiri w’ingingo n’umubiri w’ingingo imwe imwe, kandi ico kibanza cari giharuwe hakoreshejwe ubuhinga bwa Multi Measure plug-in (ImageJ software). Kura ahantu h’umubiri w’ingingo kugira ngo ubone ahantu h’umubiri w’ingingo. Ubwa nyuma, igikoresho citwa Analyse Particles plug-in (ImageJ software) carakoreshejwe kugira ngo gipime ubwaco uturongo twa ADN tw’umubiri twerekana mtDNA ku ishusho y’imbere, maze ivyo vyabonetse vyarasanzwe ku rugero rwa PN rw’imbeba za CTRL. Ivyo bivamwo bigaragazwa nk’umubare w’ibintu bita nucleosides biri ku ngingo ngenderwako imwe imwe.
Isesengura ry’imvugo y’amaproteine. Koresha ImageJ's Image Calculator plug-in kugira ngo usuzume ukuntu poroteyine zigaragara muri PN ku rugero rw'ingingo imwe. Muri make, mw’ishusho y’umurongo umwe w’umurongo uhuye, biciye ku nsiguro: ikimenyetso = min (mtYFP, anticorps), akarere k’imitochondrie kerekana immunoreactivity ku anticorps kanaka muri Purkina karamenyekana. Hanyuma, uburebure bw’ibipimo biri mu gipfukisho kivuyemwo buragereranywa, hanyuma bugasanzwe ku gishushanyo c’uruzitiro rumwe rw’umurongo wa mtYFP kugira ngo haboneke igice c’imitochondrie c’iyi poroteyine yerekanwa.
Isesengura ry’uburemere bw’ingingo za Purkinje. Igikoresho co guharura utugingo ngengabuzima ca ImageJ carakoreshejwe mu gusuzuma uburemere bwa Purkinje mu kugabanya igitigiri c’utugingo ngengabuzima twa Purkinje twaharuwe n’uburebure bw’uruzitiro rw’ubwonko bufise utugingo ngengabuzima twaharuwe.
Gutegura no gutoragura ivyerekanwa. Ubwonko buvuye mu mugwi w’abagenzuzi n’imbeba za Mfn2cKO bwashizwe mu 2% PFA/2,5% glutaraldehyde mu 0,1 M y’amazi y’ivyatsi (PB), hanyuma ibice vy’ivyatsi vy’ivyatsi bitegurwa hakoreshejwe ivyuma vy’ivyatsi vy’ivyatsi (Leica Mikrosysteme GmbH, Vienne, Otirishiya) (Uburebure bw’amazi y’ivyatsi 50 gushika kuri The60). tetraoxide na 1,5% ya ferrocyanide ya potasiyumu mu bushuhe bwo mu cumba mu kiringo c’isaha 1. Ivyo bice vyayozwe incuro zitatu n’amazi ayunguruwe, hanyuma bica biterwa n’amazi y’ivyatsi 70% arimwo uranyl acetate 1% mu minota 20. Ivyo bice vyaciye bikurwamwo amazi mu nzoga zifise ubushobozi bwo gutera, hanyuma bishirwa mu nzira y’amazi y’i Durcupan ACM (Araldite casting resin M) hagati y’ibipande vy’ibirahuri vy’ibara ry’agahama vy’ibara ry’agahama, hanyuma ku bushuhe bwa 60°C bishirwa mu furi. Uburebure bw’ubwonko bwatowe maze ibice vy’uburebure bwa 50 nm bicapwa kuri Leica Ultracut (Leica Mikrosysteme GmbH, Vienne, Otirishiya) hanyuma bitoragurwa ku gicapo c’umuringa c’uburebure bwa 2×1 mm gipfutse igipande c’ivyuma vy’ibara ry’agahama. Ivyo bice vyashizweko ibara ry’umunyu w’amazi y’ibice 4% uranyl acetate mu mazi mu minota 10, biyozwa n’amazi incuro nyinshi, hanyuma biyozwa n’amazi yitwa Reynolds lead citrate mu mazi mu minota 10, hanyuma biyozwa n’amazi incuro nyinshi. Ivyo bishushanyo vyafashwe hakoreshejwe igikoresho co gupima amashusho citwa Philips CM100 (Thermo Fisher Scientific, Waltham, MA, Leta Zunze Ubumwe za Amerika) hakoreshejwe igikoresho co gupima amashusho citwa TemCam-F416 (TVIPS GmbH, Gauting, Leta Zunze Ubumwe za Amerika). Ubudage).
Ku mbeba zandujwe na AAV, ubwonko bwaratandukanijwe maze bugacagurwa mu gice c’umubiri gifise uburebure bwa milimetero 1, hanyuma ubwonko buto burasuzumwa hakoreshejwe igikoresho citwa fluorescence microscope kugira ngo hamenyekane impeta yanduye AAV (ni ukuvuga mCherry expressing). Igerageza gusa aho urushinge rwa AAV rutuma habaho ubushobozi bwo guhindura cane igice c’ingingo za Purkinje (ni ukuvuga hafi igice cose) mu bizingiti bibiri bikurikirana vy’ubwonko ni vyo bikoreshwa. Ivyo bihimba vy’umubiri vyahinduwe na AAV vyaciwemwo ibice bitobito kugira ngo bishobore gukoreshwa ijoro ryose (4% PFA na 2,5% glutaraldehyde mu 0,1 M cocoate buffer) hanyuma bikomeza gutunganirizwa. Ku bijanye no gushiramwo EPON, igihimba gikomeye carayozwe n’amazi y’ikawo 0,1 M (Applichem), hanyuma giterwa n’amazi y’ikawo 0,1 M (Applichem) Amasaha 4, hanyuma kigasukurwa amasaha 2. Subiramwo incuro 3 n’umuti wo gukingira 0,1 M wa cocamide. Hanyuma, urutonde rw’ivyo bihimba vy’umubiri biduga rwakoreshejwe kugira ngo umuti wose w’ivyo bihimba vy’umubiri utererwe ku bushuhe bwa 4°C mu minota 15 kugira ngo umubiri ushiremwo amazi. Ivyo bihimba vy’umubiri vyaciye bijanwa mu mfuruka y’umubiri, hanyuma bishirwa mu mfuruka y’umubiri (EPON) (Sigma-Aldrich) ku bushuhe bwa 4°C. Ico gipande gishire muri EPON nshasha mu bushuhe bwo mu cumba amasaha 2, hanyuma ugishire ku bushuhe bwa 62°C amasaha 72. Koresha igikoresho co gupima amabuye y’agaciro (Leica Microsystems, UC6) n’icumu ry’amadayimoni (Diatome, Biel, Ubusuwisi) kugira ngo uce ibice vy’amabuye y’agaciro bifise uburebure bwa 70 nm, hanyuma ushireko ibara ry’umunyu w’amabuye y’agaciro 1,5% mu minota 15 ku bushuhe bwa 37°C, hanyuma ushireko ibara ry’umunyu w’amabuye y’agaciro y’amabuye y’agaciro iminota 4. Ivyo bishushanyo vyafashwe hakoreshejwe igikoresho citwa JEM-2100 Plus gifise ubuhinga bwa Camera OneView 4K 16-bit (Gatan) hamwe n’ubuhinga bwa DigitalMicrograph (Gatan). Kugira ngo hasuzumwe, amafoto y’ivyuma vy’ubuhinga bwa none yararonswe akoresheje ubuhinga bwo gukuraho 5000× canke 10.000×.
Isesengura ry’imiterere y’imitochondrie. Ku bijanye n’isesengura ryose, imirongo y’imitochondrie imwe imwe yashizwe ahabona n’amaboko mu mashusho y’ivyuma hakoreshejwe porogarama ya ImageJ. Ivyiyumviro bitandukanye vy’imiterere y’umubiri birasuzumwa. Uburemere bw’imitochondrie bugaragazwa nk’ijanisha ry’ijanisha riboneka mu kugabanya uburebure bwose bw’imitochondrie y’ingingo imwe imwe n’uburebure bwa sitoplasma (uburebure bw’ingingo = uburebure bw’ingingo-uburebure bw’ingingo) × 100. Uburebure bw’imitokondriya buharurwa hakoreshejwe ubuhinga [4π∙2(uburebure)]./perimeter. Igishushanyo c’imitochondrie citwa ista carasuzumwe maze kigabanwa mu mice ibiri (“tubular” na “blister”) hakurikijwe imiterere yavyo nyamukuru.
Igitigiri c’ibinyabuzima bita autofagosome/lysosome n’isesengura ry’uburemere bwavyo. Koresha porogarama ya ImageJ kugira ngo ushire ahabona n’amaboko imirongo y’ibihimba vy’umubiri vyose biri mw’ishusho y’ivyuma. Uburebure bw’umubiri bugaragazwa nk’ijanisha ry’ijanisha riharuwe mu kugabanya uburebure bwose bw’imiterere y’umubiri w’ingingo imwe imwe n’uburebure bw’umubiri (uburebure bw’umubiri=uburebure bw’umubiri-uburebure bw’umubiri)×100. Ubwinshi bw’ibihimba vy’umubiri bifata umubiri (autophagosomes/lysosomes) buharurwa mu kugabanya umubare wose n’umubare w’imiterere y’ibihimba vy’umubiri bifata umubiri (autophagosomes/lysosomes) ku nsoro (mu bijanye n’uburebure bw’umubiri) (uburebure bw’umubiri = uburebure bw’umubiri-uburebure bw’umubiri).
Gushirako amazina y’ugucapura n’ugutegura ivyerekanwa. Ku ngeragezwa zisaba gushirwako ikimenyetso c’isukari, ushire ibice vy’ubwonko bikomeye mu cumba c’imbere y’ugutera, kirimwo carbone yuzuye (95% O2 na 5% CO2), Ca2 + ACSF nyinshi (125,0 mM NaCl, 2,5 mM KCl, 1,25 mM sodium phosphate Na3O, 1,25 mM sodium phosphate. 25,0 mM d-isukari, 1,0 mM CaCl 2 na 2,0 mM MgCl 2, zihinduwe ku pH 7,4 na 310 gushika kuri 320 mOsm), aho isukari ari 13 C 6- Gusubirira isukari (Eurisotop, inomero y’urutonde69-1). Ku ngeragezwa zisaba gushirwako ikimenyetso c’ivyuma bita pyruvate, ushire ibice vy’ubwonko bikomeye mu mazi menshi cane Ca2 + ACSF (125,0 mM NaCl, 2,5 mM KCl, 1,25 mM buffer de phosphate de sodium, 25,0 mM NaHCO3, 25,0 mM d-glucose d2C, 1 MgCl2, uyihindure pH 7,4 na 310 gushika kuri 320mOsm), hanyuma wongereko 1-[1-13C]pyruvate (Eurisotop, inomero y’urutonde CLM-1082). Ivyo bice ubishire mu minota 90 ku bushuhe bwa 37°C. Igerageza ryarangiye, ivyo bice vyarasukuwe ningoga n’umunyu w’amazi (pH 7,4) urimwo 75 mM y’umunyu w’amazi, hanyuma bica bihurizwa hamwe mu 40:40:20 (v:v:v) acetonitrile (ACN): methanol: amazi. Ivyo bice bimaze guterwa ku barafu iminota 30, ivyo bipimo vyaciye bishirwa mu gitereko c’amazi ku 21.000 g mu minota 10 ku 4°C, hanyuma ivyo bihimba vy’umubiri vyari bigaragara vyumwa mu gikoresho co gutera amazi citwa SpeedVac. Ivyo bihimba vy’umubiri vyumye vyabitswe ku -80°C gushika bisuzumwe.
Icegeranyo c’amazi c’ibara ry’umubiri-uburemere bw’ibara ry’umubiri c’acides aminés 13 zifise ikimenyetso ca C. Ku bijanye n’isesengura ry’amazi (LC-MS), igice c’umubiri c’ivyo bihimba vy’umubiri carasubiwemwo mu mazi 75μl y’agaciro ka LC-MS (Honeywell). Inyuma yo gucapura ku 21.000 g mu minota 5 ku 4°C, 20 μl y’ivyo bihimba vy’umubiri vyari vyarasobanutse vyakoreshejwe mu gusuzuma uruja n’uruza rw’acides aminés, mu gihe ibindi bihimba vy’umubiri vyakoreshejwe mu gupima anions (raba munsi). Isesengurwa ry’acides aminés ryakozwe hakoreshejwe uburyo bwavuzwe mbere bwo gukuraho benzoyl chloride (55, 56). Mu ntambwe ya mbere, 10μl y’umunyu w’amazi 100 mM (Sigma-Aldrich) yongewe ku 20μl y’umunyu w’ibinyabuzima, hanyuma 10μl y’amazi y’ibinyabuzima 2% (Sigma-Aldrich) yongewe ku ACN y’urugero rwa LC. Ico gipimo carazungurutswe mu gihe gitoyi hanyuma gica gihindurwa n’amazi ku 21.000 g mu minota 5 ku 20°C. Ivyo bihimba vy’umubiri vyari vyarasukuwe, bishire mu gitereko c’amazi c’imiliyoni 2 gifise ikintu co kwinjizamwo ikirahuri c’imirongo (200 μl volume). Ivyo bipimo vyasuzumwe hakoreshejwe ubuhinga bwa Acquity iClass bukora neza cane (Waters) bufatanye n’ubuhinga bwa Q-Exactive (QE)-HF (Ultra High Field Orbitrap) bufise ubushobozi bwo gupima uburemere bw’ibintu (Thermo Fisher Scientific). Kugira ngo hakorwe isesengura, 2μl y’ivyo bihimba vyavuye mu bihimba vy’umubiri vyashizwe mu nkingi ya silica T3 (Waters) y’uburemere bwinshi y’iminota 100×1,0 irimwo ibice vy’iminota 1,8. Igitigiri c’amazi atemba ni 100μl/min, kandi uburyo bwo gukingira bugizwe n’ububiko A (10 mM ya formate d’amonium na 0,15% y’aside formique mu mazi) n’ububiko B (ACN). Igishushanyo c’ibara ry’umubiri ni iki: 0%B ku minota 0; 0%B. 0 gushika kuri 15% B ku minota 0 gushika kuri 0,1; 15 gushika kuri 17% B ku minota 0,1 gushika kuri 0,5; B ku 17 gushika ku 55% ku minota 0,5 gushika ku 14; B ku 55 gushika ku 70% ku minota 14 gushika ku 14,5; ku 14,5 gushika ku 70 gushika ku 100% B ku minota 18; 100% B ku minota 18 gushika ku 19; 100 gushika kuri 0% B ku minota 19 gushika kuri 19,1; 0% B ku minota 19,1 gushika ku minota 28 (55, 56). Igikoresho co gupima uburemere bw’ibintu (QE-HF) gikora mu buryo bw’iyonization nziza gifise urutonde rw’uburemere rwa m/z (igipimo c’uburemere/uburemere) kuva kuri 50 gushika kuri 750. Igisubizo gikoreshwa ni 60.000, kandi intumbero y’iyon y’ubugenzuzi bw’inyungu (AGC) ironswa ni 3×106, kandi igihe c’iyon gisumba ibindi vyose ni millico1. Isoko y’iyonization y’umuyagankuba (ESI) ishushe ikora ku nkomezi z’umuyagankuba zingana na 3,5 kV, ubushuhe bw’amazi bugera kuri 250°C, umuyaga wo mu gipfukisho ugera kuri 60 AU (ibice bitari vyo), n’umuyaga w’inyongera ugera kuri 20 AU. 250°C. Igikoresho ca S gishirwa kuri 60 AU.
Ivyigwa vy’ibara ry’umubiri-MS vy’aside z’ibinyabuzima zifise ikimenyetso ca 13C. Ivyo bihimba vy’umubiri vyasigaye (55μl) vyasuzumwe hakoreshejwe ubuhinga bwo gupima uburemere bw’ibintu (ICS 5000+, Thermo Fisher Scientific) bufatanye n’igikoresho co gupima uburemere bw’ibintu (QE-HF). Muri make, 5μl y’ibiva mu bihimba vy’umubiri vyashizwe mu nkingi ya Dionex IonPac AS11-HC ifise HPLC (2 mm×250 mm, ubunini bw’ibice 4μm, Thermo Fisher Scientific) mu buryo bwo gusunika mu gice c’uruzitiro n’igipimo co kwuzuza 1. 4μm, Ivy’ubuhinga bw’abarovyi b’ubushuhe). Ubushuhe bw’inkingi buguma kuri 30°C, kandi igikoresho co gufata ivyerekanwa kigashirwa kuri 6°C. Koresha igikoresho c’amazi y’ipotasiyumu gifise amazi atagira iyoni kugira ngo ushireho umurongo w’amazi y’ipotasiyumu biciye ku gikoresho co gutanga amazi y’ipotasiyumu. Gutandukanya ibintu bihinduka mu mubiri ku rugero rw’uruja n’uruza rwa 380μl/min, hakoreshejwe urugero rukurikira: iminota 0 gushika kuri 3, 10 mM KOH; Iminota 3 gushika ku 12, 10 gushika ku 50 mM KOH; iminota 12 gushika ku 19, 50 gushika ku 100 mM KOH; 19 gushika ku minota 21 , 100 mM KOH; 21 gushika ku minota 21,5, 100 gushika ku 10 mM KOH. Iyi nkingi yongeye gutunganirizwa munsi ya 10 mM KOH mu minota 8,5.
Ivyo bihimba vy’umubiri bivanwa n’umugezi w’inyongera w’isopropanol 150μl/min inyuma y’inkingi hanyuma bikajanwa ku gikoresho co gupima uburemere bw’ibintu gikora mu buryo bw’iyonization mbi. MS iriko irakurikirana uburemere bw'ibintu kuva kuri m/z 50 gushika kuri 750 n'ubushobozi bwa 60.000. AGC ishirwa kuri 1×106, kandi igihe kinini c’iyon kiguma kuri 100 ms. Iryo soko rya ESI ry’ubushuhe ryakoreshwa ku nguvu z’umuriro w’amazi 3,5 kV. Ibindi bipimo vy’isoko y’iyon ni ibi bikurikira: ubushuhe bw’imitsi 275°C; umuvuduko w’umuyagankuba, 60 AU; n’umuyagankuba w’inyongera, 20 AU ku 300°C, n’ugushinga urushinge rwa S ku 60 AU.
Isesengura ry’amakuru y’ibinyabuzima bifise ikimenyetso ca 13C. Koresha porogarama ya TraceFinder (uburyo bwa 4.2, ubuhinga bwa Thermo Fisher) kugira ngo usesengure amakuru y’igipimo c’isotope. Akaranga k’ikirungo kimwekimwe cose karagenzuwe n’ikirungo c’ishingiro co kwizigirwa maze kigasuzumwa ku giti cabo. Kugira ngo hakorwe isesengura ry’ubutunzi bw’isotope, uburebure bw’iyon chromatogramme (XIC) yakuwemwo y’isotope imwe imwe ya 13C (Mn) bwakuwe muri [M + H] +, aho n ari umubare wa carbone w’ikirungo c’intumbero, ukoreshwa mu gusesangura aside amino canke [ MH] + ukoreshwa mu gusesangura anions. Ugushikama kw’uburemere bwa XIC kuri munsi y’ibice bitanu kuri miliyoni, kandi ukuri kwa RT ni iminota 0,05. Isesengura ry’ubutunzi rikorwa mu kubara igipimo c’isotope imwe imwe ibonetse n’umubare w’isotope zose z’ikirungo gihuye. Ivyo bipimo bitangwa nk’agaciro k’ijanisha ry’isotope imwe imwe, kandi ivyo bivamwo bigaragazwa nk’ubutunzi bw’ijanisha ry’ibara ry’umubiri (MPE), nk’uko vyavuzwe imbere y’aho (42).
Ivyo bihimba vy’umubiri vyari vyakonjeshejwe vyashizwe mu mazi akonje nk’urubura 80% methanol (v/v), bica bihindurwa, hanyuma bishirwa ku -20°C iminota 30. Usubire gutera umuvuduko w’ivyo bihimba hanyuma ubivungure ku +4°C iminota 30. Ico gipimo carashizwe mu gitereko c’amazi ku 21.000 g mu minota 5 ku 4°C, hanyuma ivyuma vyavuyemwo birakorakoranywa maze vyumwa hakoreshejwe igikoresho citwa SpeedVac concentrator ku 25°C kugira ngo bishobore gusuzumwa. Nk’uko vyavuzwe haruguru, isesengura rya LC-MS ryakozwe ku ma aside amino y’ingingo zatoranijwe. Hakoreshejwe TraceFinder (uburyo bwa 4.2, Thermo Fisher Scientific), isesengura ry’amakuru ryakozwe hakoreshejwe uburemere bw’ibintu bimwe bimwe vy’ikirungo kimwekimwe cose. Ivyo bikaba vyakozwe hakoreshejwe porogarama ya preprocessCore (57).
Gutegura ibice. Imbeba yaciye iterwa ububabare bwihuse n’umuyagankuba maze iracirwa umutwe, ubwonko bwayo burakurwa mu gihanga vyihuse, hanyuma hakoreshwa imbugita izunguruka yuzuye urubura (HM-650 V, Thermo Fisher Scientific, Walldorf, Ubudage) kugira ngo bayice mu bice 300 gushika kuri 375 μ% vy’umuyagankuba O5d. CO2) Ca2 + ACSF nto (NaCl 125,0 mM, KCl 2,5 mM, umuti wo gukingira amazi 1,25 mM, NaHCO3 25,0 mM, d-glucose 25,0 mM, CaCl2 1,0 mM na Adjust 6,0 mM ku pH372. mOsm). Ivyo bice vy’ubwonko vyaronswe, bishire mu cumba kirimwo Ca2 + ACSF nyinshi (NaCl 125,0 mM, KCl 2,5 mM, umuti wo gukingira amazi 1,25 mM, NaHCO3 25,0 mM, d-glucose 25,0 mM, pH42 mM Ca3l5C 4,0 mM. na 310 gushika kuri 320 mOsm). Bika ivyo bipande mu minota 20 canke 30 kugira ngo bishobore gusubirwamwo imbere y’uko ubifata amajwi.
gufata amajwi. Igikoresho co gufata amajwi gifise icumba co kwandikako kidahinduka be n’urushinge rwo gufata amajwi rwo mu mazi incuro 20 (Scientifica) rwakoreshejwe mu kwandika vyose. Utugingo ngengabuzima twa Purkinje twamenyekanye biciye ku (i) ubunini bw’umubiri, (ii) aho ubwonko buto buteye, be n’ukuntu (iii) ukuntu umugera w’umumenyeshamakuru witwa mtYFP ugaragara. Igikoresho co gupima gifise ubushobozi bwo guhangana n’isonga ry’ama megohms 5 gushika ku 11 gikurwa n’igikoresho c’ibirahuri c’ibara ry’agahama (GB150-10, 0,86 mm×1,5 mm×100 mm, Ibikoresho vya Siyansi, Hofheim, Ubudage) n’igikoresho c’ipipe gihagaze (P-1000). Ivyo vyose vyafashwe vyakozwe n’igikoresho co gutera amajwi citwa ELC-03XS (npi electronic GmbH, Tam, mu Budagi), kikaba cari kigenzurwa na porogarama yitwa Signal (verisiyo 6.0, Cambridge Electronic, Cambridge, Ubwongereza). Iryo gerageza ryanditswe ku rugero rw’ugutora 12,5 kHz. Ico kimenyetso gicungururwa n’ibiyunguruzo bibiri vya Bessel bica mu gihe gito bifise incuro z’uguca 1,3 na 10 kHz. Ubushobozi bw’uruzitiro n’urw’ipipe burasubirwamwo n’umurongo w’ugusubizwa hakoreshejwe igikoresho co gukomeza ijwi. Ivyo bigeragezo vyose vyakozwe bigenzurwa n’icamera yitwa Orca-Flash 4.0 (Hamamatsu, Gerden, Ubudage), iyo kamera ikaba yari igenzurwa na porogarama ya Hokawo (verisiyo 2.8, Hamamatsu, Gerden, Ubudage).
Gutunganya n’ugusesangura utugingo ngengabuzima twose. Imbere yo kwandika, wuzuze iyo pipete umuti wo mu mutima urimwo ibi bikurikira: KCl 4,0 mM, NaCl 2,0 mM, EGTA 0,2 mM, gluconate ya potassium 135,0 mM, Hepes 10,0 mM, Guphano.TPGsine 4,0 mM (4,0 mM ATP.TPG5g) (Na) na 10,0 mM y’ivyo bita creatinine phosphate vyahinduwe bishika ku pH 7,25, kandi umuvuduko w’amazi wari 290 mOsm (sucrose). Ubwo nyene bamaze gukoresha inguvu 0 pA kugira ngo bacire uruzitiro, ubushobozi bw’uruzitiro rwo kuruhuka burapimwa. Igitero c’injiza gipimwa n’ugukoresha amashanyarazi arengeje urugero -40, -30, -20, na -10 pA. Pima ubunini bw’inyishu y’umuriro maze ukoreshe itegeko rya Ohm kugira ngo ubare uburemere bw’injiza. Ibikorwa vy’ubwisanzure vyanditswe mu gikoresho co gufata umuriro mu minota 5, sPSC na yo iramenyekana kandi ipimwa muri Igor Pro (verisiyo 32 7.01, WaveMetrics, Ikiyaga Oswego, Oregon, Leta Zunze Ubumwe za Amerika) hakoreshejwe inyandiko y’ugutahura ivy’umuriro. Igicapo ca IV n’igicapo c’umuriro gihagaze bipimwa mu gufata bateri ku bushobozi butandukanye (guhera kuri -110 mV) no kwongera umuriro mu ntambwe za 5 mV. Ivyo gukora AP vyageragejwe hakoreshejwe umuriro w’ugukuraho umuriro. Gufata igice c’umubiri ku -70 mV mu gihe ukoresha umuyagankuba w’ugukuraho umuriro. Guhindura ubunini bw’intambwe y’igikoresho cose co kwandika ukwaco (10 gushika kuri 60 pA). Harura incuro nyinshi cane za AP mu guharura n’amaboko ibipimo vy’impumuro bitera incuro nyinshi cane za AP. Igitigiri ca AP gisesangurwa hakoreshejwe igice ca kabiri c’impumuro y’ugukuraho ivyuma (depolarization pulse) ibanza gutera AP imwe canke nyinshi.
Gutunganya n’ugusesangura ibipande vy’ibitobo. Gukora amajwi y’ibipande vy’imyobo ukoresheje amasezerano asanzwe. Koresha ipipe idafise ATP na GTP idafise ibi bikurikira: 128 mM ya gluconate K, 10 mM ya KCl, 10 mM ya Hepes, 0,1 mM ya EGTA na 2 mM ya MgCl2, hanyuma uyihindure pH 7,2 (ukoresheje KOH). ATP na GTP birakurwa mu muti wo mu turemangingo kugira ngo ntihagire ubushobozi bwo guca mu turemangingo butagenzurwa. Igikoresho co gutera ivyatsi cuzuye umuti wo mu mutima urimwo amfoterisine (nk’ibice 200 gushika kuri 250μg/ml; G4888, Sigma-Aldrich) kugira ngo umuntu aronke igitabu c’ivyatsi gipfutse. Amfoterisini yashizwe mu mazi y’ivyatsi (ivy’iherezo: 0,1 gushika kuri 0,3%; DMSO; D8418, Sigma-Aldrich). Igitigiri ca DMSO cakoreshejwe nta ngaruka zikomeye cagize ku mitsi y’ubwonko yigishijwe. Mu gihe c’ugutera ibipfunsi, ubushobozi bwo guhangana n’imiyoboro (Ra) bwaguma bukurikiranirwa hafi, kandi igerageza ryatanguye inyuma y’aho uburemere bwa Ra na AP buhagaze (iminota 20-40). Ibikorwa vy’ubwisanzure bipimwa mu gipimo c’umuriro n’/canke c’amashanyarazi mu minota 2 gushika ku 5. Isesengurwa ry’amakuru ryakozwe hakoreshejwe ubuhinga bwa Igor Pro (uburyo bwa 7.05.2, WaveMetrics, muri Leta Zunze Ubumwe za Amerika), Excel (uburyo bwa 2010, ishirahamwe rya Microsoft, muri Leta Zunze Ubumwe za Amerika) na GraphPad Prism (uburyo bwa 8.1.2, ubuhinga bwa GraphPad Software Inc., La Jolla, CA). Leta Zunze Ubumwe za Amerika). Kugira ngo umuntu amenye APs zidasanzwe, hakoreshwa ubuhinga bwa IgorPro bwa NeuroMatic v3.0c. Ukwitaho kumenya APs ukoresheje urugero rwatanzwe, ruhindurwa ku giti cabo ku nyandiko yose. Ukoresheje ikiringo c’isonga, menya incuro y’isonga n’incuro y’isonga y’aho nyene n’incuro y’isonga y’igihe gito.
PN kwitandukanya. Mu guhindura umurongo wasohowe mbere, PNs zasukuwe zivuye mu bwonko bw’imbeba ku ntambwe yihariye (58). Muri make, ubwonko buto bwaciwe maze bugasembwa mu mazi akonje nk’urubura [atagira HBSS Ca2+ na Mg2+, hashizwemwo 20 mM y’isukari, penisilini (50 U/ml) na streptomycine (0,05 mg/ ml) ( 0,05 mg/ml) ( mgSSHB-cysteine ​​[ / ml), papayini (16 U / ml) na deoksiribonuklease I (DNase I; 0,1 mg/ml)] Uvure iminota 30 ku bushuhe bwa 30°C. Ubanze woze utugingo ngengabuzima mu gikoresho ca HBSS kirimwo umunyu w’amagi (10 mg/ml), BSA (10 mg/ml) na DNase (0,1 mg/ml) mu bushuhe bwo mu cumba kugira ngo ntihagire intungamubiri zifatwa n’indwara, hanyuma woze mu gikoresho ca HBSS kirimwo 20 mM y’isukari, usye mu buryo bwiyoroheje muri HBmyilm5 (0,05 mg/ml) na DNase (0,1 mg/ml) zirekura utugingo ngengabuzima tumwetumwe. Ivyo bihimba vy’umubiri vyavuyemwo vyacunzwe biciye mu gikoresho co gucungera utugingo ngengabuzima c’uburebure bwa 70μm, hanyuma utugingo ngengabuzima twaciwe mu bihimba vy’umubiri bikoresheje ubuhinga bwo gusenyura (1110 rpm, iminota 5, 4°C) hanyuma dusubira gusubirwamwo mu gikoresho co gutoranya [HBSS, yuzuye 20 mM glucose, 20% fetal streptomylin/fetal U5il ) Serum, (0,05 militiro)]; gusuzuma ubushobozi bw’ingingo z’umubiri hakoreshejwe propidium iodide no guhindura uburemere bw’ingingo z’umubiri ku 1×106 gushika ku 2×106 z’ingingo/ml. Imbere y’uko hakorwa flow cytometry, iyo nzira yacunzwe biciye mu gikoresho co gucungera utugingo ngengabuzima c’uburebure bwa 50 μm.
Igipimo c’uruzi. Gutoranya utugingo ngengabuzima vyakozwe ku bushuhe bwa 4°C hakoreshejwe imashini ya FACSAria III (BD Biosciences) na porogarama ya FACSDiva (BD Biosciences, verisiyo 8.0.1). Ivyo bihimba vy’umubiri vyatoranijwe hakoreshejwe umupfunda w’uburebure bwa 100 μm munsi y’umuvuduko wa 20 psi ku rugero rwa ~2800 events/sec. Kubera ko ibipimo vya kera vy’ugufungura (ubunini bw’ingingo, ivangura ry’imirongo ibiri, n’ibiranga ugushwiragira) bidashobora gutuma PN itandukana neza n’ibindi bihimba vy’umubiri, ingamba zo gufungura zishingiye ku kugereranya ata guca ku ruhande uburemere bwa YFP n’uguca ibibatsi kw’umubiri mu mitoYFP+ ​​no mu mitoYFP − igenzura. YFP iravyurirwa umutima mu gutera umuco ku citegererezo n’umurongo wa laser w’uburebure bwa 488 nm, kandi ikimenyetso kigamenyekana hakoreshejwe akayunguruzo k’umurongo w’uburebure bwa 530/30 nm. Mu mbeba zitwa mitoYFP+, inkomezi z’umugera wa Rosa26-mitoYFP na zo nyene zirakoreshwa mu gutandukanya umubiri w’imitsi n’ibice vy’imitsi. 7-AAD iravyurirwa umutima n’umuco w’umuhondo w’uburebure bwa 561 nm kandi igafatwa n’akayunguruzo k’uburebure bwa 675/20 nm kugira ngo ikureho utugingo ngengabuzima twapfuye. Kugira ngo astrocytes zitandukanywe igihe kimwe, iyo cell suspension yarashizweko ibara rya ACSA-2-APC, hanyuma iyo nkuru irashirwako umuco n’umurongo wa laser w’uburebure bwa 640 nm, hanyuma hakoreshwa akayunguruzo k’uburebure bwa 660/20 nm kugira ngo hamenyekane ico kimenyetso.
Utugingo ngengabuzima twakoranijwe twaciwe mu bipande bikoresheje ubuhinga bwo gucapura (1110 rpm, iminota 5, 4°C) hanyuma tubika ku bushuhe bwa -80°C gushika dukoreshejwe. Imbeba za Mfn2cKO n’imbeba zazo zishirwa mu migwi ku musi umwe kugira ngo zigabanye uguhinduka kw’uburyo bwo gukora. Ivyerekanwa n’isesengura ry’amakuru ya FACS vyakozwe hakoreshejwe porogarama ya FlowJo (FlowJo LLC, Ashland, Oregon, Leta Zunze Ubumwe za Amerika).
Nk’uko twabibonye haruguru (59), PCR y’igihe nyaco ikoreshwa mu gutandukanya ADN n’imitsi y’ubwonko yatoranijwe kugira ngo hakurikizwe gupima mtDNA. Ivyo bimenyetso vy’umurongo n’ivy’ugutahura ivy’imbere vyageragejwe mu ntango hakoreshejwe qPCR ku mibare itandukanye y’ingingo. Muri make, ushire hamwe 300 PN mu gitereko c’amazi gifise tris-HCl 50 mM (pH 8,5), EDTA 1 mM, Tween 20 0,5% na proteinase K (200 ng/ml) hanyuma ubishire ku bushuhe bwa 55°C iminota 120. Utugingo ngengabuzima twaciye duterwa ku bushuhe bwa 95°C mu minota 10 kugira ngo proteinase K ntikore burundu. Ivy’ubuhinga, inomero y’urutonde Mm04225274_s1), mt-Nd6 (Ivya Siyansi vy’Umurovyi, inomero y’urutonde AIVI3E8) na 18S (Ivya Siyansi vy’Umurovyi, inomero y’urutonde Hs99999901_s1) vy’imitsi.
Gutegura ivyerekanwa vya proteome. Mu gushushanya umuti ku 95°C mu minota 10 no gukoresha amajwi, mu gikoresho co gukingira [6 M kloride ya guanidine, 10 mM ya tris (2-carboxyethyl) hydrochloride ya phosphine, 10 mM ya kloroacetamide na 100 mM ya tris-HC Lyse mu nzira y’imitsi y’umubiri]. Ku Bioruptor (Diagenode) mu minota 10 (amasegonda 30 y’umutima / amasegonda 30 y’igihe co guhagarara). Ico gipimo caciwemwo 1:10 muri tris-HCl 20 mM (pH 8.0), kivangwa n’inzahabu ya trypsine 300 ng (Promega), hanyuma gica giterwa ijoro ryose ku 37°C kugira ngo kigere ku gufungura neza. Ku musi ugira kabiri, iyo nkuru yarashizwe mu gitereko c’amazi (centrifuge) ku g 20.000 mu minota 20. Ivyo bihimba vy’umubiri vyari hejuru y’ivyo bihimba vyaciwemwo aside formique 0,1%, hanyuma ivyo bihimba vy’umubiri bica bikurwako umunyu hakoreshejwe StageTips yikoreye. Ico gipimo cayumye mu gikoresho ca SpeedVac (igikoresho ca Eppendorf co gushiramwo 5305) ku bushuhe bwa 45°C, hanyuma iyo peptide ihagarikwa mu 0,1% y’aside formique. Ivyitegererezo vyose vyateguwe icarimwe n’umuntu umwe. Kugira ngo hasuzumwe ivyerekanwa vy’inyenyeri, 4 μg y’ibinyabuzima bitagira umunyu vyashizweko ikimenyetso c’uburemere bw’ibinyabuzima (TMT10plex, inomero y’urutonde 90110, Thermo Fisher Scientific) n’igipimo c’ibinyabuzima bifise umunyu n’ibinyabuzima 1:20. Ku bijanye no gushirako ikimenyetso ca TMT, 0,8 mg y’igikoresho ca TMT carasubiwemwo mu 70 μl ya ACN idafise amazi, hanyuma iyo peptide yumutse isubizwamwo 9 μl ya TEAB 0,1 M (triethylammonium bicarbonate), aho 7 μl y’igikoresho ca TMT kiri muri ACN yongeweko. Ivyo vyiyumviro vyari 43,75%. Inyuma y’iminota 60 y’ugutera, iyo nzira yazimye hakoreshejwe 2 μl y’amazi y’umunyu 5%. Ivyo bihimba vy’umubiri vyari vyerekanwa vyakwegeranijwe, vyumwa, bisubira gushirwa mu 200μl y’aside formique (FA) 0,1%, bigabanywamwo bibiri, hanyuma bikurwako umunyu hakoreshejwe ubuhinga bwa StageTips bwikoreye. Hakoreshejwe igikoresho co gupima amazi gifise ubushobozi bwinshi cane citwa UltiMate 3000 (igikoresho co gupima amazi gifise ubushobozi bwinshi cane citwa UltiMate 3000), kimwe muri ivyo bice bibiri caraciwemwo ibice ku nkingi ya Acquity y’uburebure bwa milimetero 1 x 150 yuzuye ibice vya C18 vy’uburebure bwa 130Å1.7μm (NoWaters, catalog150). Ivy’ubuhinga bw’umurovyi w’umuriro). Gutandukanya peptides ku rugero rw’uruja n’uruza rwa 30μl/min, gutandukanya kuva kuri 1% gushika kuri 50% y’umunyu B mu minota 85 n’intambwe ku yindi y’iminota 96, kuva kuri 50% gushika ku 95% y’umunyu B mu minota 3, hanyuma iminota 8 ku 95 % y’umunyu B; Igikoresho co gukingira A ni 5% ACN na 10 mM y’ibikarbonate y’amoniyumu (ABC), igikoresho co gukingira B ni 80% ACN na 10 mM ABC. Ukorakoranye imice buri minota 3 hanyuma uyishire mu mirwi ibiri (1 + 17, 2 + 18, n’ibindi) hanyuma uyiyume mu gikoresho citwa centrifuge c’ubusa.
Isesengura rya LC-MS/MS. Ku bijanye n’ugupima uburemere bw’ibintu, izo peptide (umubare r119.aq) zatandukanijwe ku nkingi y’isesengura y’uburebure bwa cm 25, 75 μm y’uburebure bw’imbere (ibara rishasha ry’intumbero, inomero y’igice PF7508250) ifise uburebure bwa 1,9 μm Ma ReproSil-Pur C. EASY-nLC 1200 (Ivy’ubuhinga bw’abarovyi b’umuriro, mu Budagi). Iyo nkingi yagumye iri ku bushuhe bwa 50°C. Ivyo gukingira A na B ni 0,1% vy’aside formique mu mazi na 0,1% vy’aside formique mu 80% vya ACN, uko bikurikirana. Peptides zatandukanijwe kuva kuri 6% gushika kuri 31% y’umunyu B mu minota 65, kuva kuri 31% gushika ku 50% y’umunyu B mu minota 5 n’urugero rwa 200 nl/min. Ivyo bihimba vy’umubiri vyasuzumwe ku gikoresho citwa Orbitrap Fusion (Thermo Fisher Scientific). Ipima ry’imbere y’ivyo bita peptide m/z rikorwa n’ubushobozi bwo gupima 120.000 mu rugero rwa 350 na 1500 m/z. Ukoresheje 27% y’inguvu z’ugutombora zisanzwe, ikintu gikomeye kuruta ibindi vyose gifise ikibanza c’umuriro kuva kuri 2 gushika kuri 6 kiratorwa ku nguvu nyinshi C trap dissociation (HCD) cleavage. Igihe c’ingendo gishirwa kuri 1 s. Agaciro ka m/z k’igice ca peptide kagereranywa mu mutego w’iyon hakoreshejwe intumbero ntoyi cane ya AGC ya 5×104 n’igihe kinini co gutera urushinge ca 86 ms. Inyuma y’uguca ibice, umurongo w’imbere yashizwe ku rutonde rw’abakurwako b’inguvu mu kiringo c’amasegonda 45. Ivyo bihimba vy’umubiri vyerekanwa na TMT vyatanzwe ku nkingi ya Acclaim PepMap y’uburebure bwa cm 50, 75 μm (Thermo Fisher Scientific, inomero y’urutonde 164942), kandi imirongo y’ukwimuka yasuzumwe ku gipimo c’uburemere bw’imirongo y’imirongo y’imirongo y’imirongo y’imirongo y’imirongo y’imirongo y’imirongo y’imirongo y’imirongo y’imirongo y’imirongo y’imirongo (FAIMS) ibikoresho (Thermo Fisher Scientific) bikora ku nkomezi zibiri z’ugusubiza inyuma −50 na −70 V. MS3 yatowe ishingiye ku ntumbero y’uguhuza ikoreshwa mu gupima ikimenyetso c’iyon ca raporo ya TMT. Ivyo gutandukanya peptide vyakozwe kuri EASY-nLC 1200, hakoreshejwe uburyo bwo gukuraho 90% bw’umurongo, n’uburyo bwo gukingira 6% gushika kuri 31%; ububiko A bwari 0,1% FA, ububiko B bwari 0,1% FA na 80% ACN. Inkingi y’isesengura ikoreshwa ku bushuhe bwa 50°C. Koresha FreeStyle (uburyo 1.6, Thermo Fisher Scientific) kugira ngo ugabanye dosiye y’intango hakurikijwe umuriro w’indishi wa FAIMS.
Imenyekanisha ry’amaproteine ​​n’ingero. Hakoreshejwe ubuhinga bwo gushakisha bwa Andromeda, amakuru y’intango yasuzumwe hakoreshejwe ubuhinga bwa MaxQuant 1.5.2.8 (https://maxquant.org/). Uretse urutonde rwa Cre recombinase na YFP rwaronswe muri Aequorea victoria, imirongo y’ibice vya peptide yararondewe urutonde rw’ivyagezwe n’urutonde rw’isoforme y’imbeba (Proteome ID UP000000589, yavanwe kuri UniProt77 muri Rusama2020). Ivyo bihinduka bihinduka; ivy’uguhindura cysteine ​​carbamoyl vyashizweho nk’ibintu bihinduka bihoraho. Ivyo bipimo vy’ugufata ibiribwa bishirwa ku “vyihariye” no ku “trypsine/P”. Igitigiri gitoyi c’amapeptide n’amapeptide y’inzobe akoreshwa mu kumenya poroteyine ni 1; umubare mutoyi w'amapeptide yihariye ni 0. Mu gihe c'uguhuza ikarita y'amapeptide, igipimo co kumenya poroteyine cari 0.01. Ihitamwo ry’“Peptide ya kabiri” rirakora. Koresha “uguhuza hagati y’imigenderanire” kugira ngo ushire ibimenyetso vyiza hagati y’amadosiye atandukanye y’umwimerere. Koresha LFQ igiharuro gitoyi c’igiharuro 1 ku bijanye n’ugupima ata n’ikimenyetso (LFQ) (60). Inkomezi za LFQ zicungururwa nibura ku mibare ibiri ibereye mu mugwi umwe w’ubwoko bw’umubiri ku gihe cose, kandi zigakurwa mu gukwiragira gusanzwe n’ubwaguke bwa. 0.3 hanyuma ukamanuka 1.8. Koresha urubuga rw’ubuhinga bwa none rwa Perseus (https://maxquant.net/perseus/) na R (https://r-projet.org/) kugira ngo usesengure ibivuye mu bibazo vya LFQ. Igerageza ry’inzira zibiri ry’uburinganire ry’iporogarama ya limma ryakoreshejwe mu gusesangura invugo zitandukanye (61). Isesengura ry’amakuru y’ubushakashatsi rikorwa hakoreshejwe ggplot, FactoMineR, factoextra, GGally na pheatmap. Amakuru y'ubuhinga bwa poroteyine ashingiye kuri TMT yasuzumwe hakoreshejwe verisiyo ya MaxQuant 1.6.10.43. Rondera amakuru y’ivy’amaproteine ​​atagiramwo ivyatsi avuye mu rutonde rw’amakuru y’ivy’amaproteine ​​y’abantu rwa UniProt, rwavanwe muri Nzero 2018. Iryo sesangura ririmwo ikintu co gukosora ubusuku bw’isotope catanzwe n’uwagikoze. Koresha limma muri R ku bijanye n’isesengura ry’imvugo itandukanye. Amakuru y'intango, ibisubizo vy'ubushakashatsi, n'ibikorwa vy'isesengura ry'amakuru n'ibisubizo vyose bibikwa mu muryango wa ProteomeXchange biciye mu bubiko bw'abafatanyabikorwa ba PRIDE n'ikimenyetso c'amakuru PXD019690.
Ivyiyumviro bikora biratuma isesengura rigira akamaro. Igikoresho co gusesangura inzira y’ubuhinga (QIAGEN) carakoreshejwe kugira ngo hamenyekane ubutunzi bw’amajambo y’insobanuro y’ibikorwa vy’amakuru yashizweho ku ndwi 8 (Ishusho 1). Muri make, urutonde rw’ingero z’amaproteine ​​rwaronswe mu gusesangura amakuru ya LC-MS/MS (tandem mass spectrometry) rukoreshwa n’ibi bikurikira: Mus musculus itoranywa nk’ubwoko n’inyuma, kandi urutonde rwerekana agaciro ka P kahinduwe na Benjamini kugira ngo hatunganywe 0.05 canke hasi kabonwa ko ari akahambaye. Kuri iyi graphe, ivyiciro bitanu vy’imbere vy’ibirenze urugero muri buri kigwi bishingiye ku gaciro ka P kahinduwe biragaragazwa. Hakoreshejwe ikigeragezo ca t-test kinini, hakoreshejwe porogarama y’ugutera imbere kw’umurongo w’inzira zibiri ya Benjamini, Krieger, na Yekutieli (Q = 5%), isesengura ry’imvugo y’amaproteine ​​mu gihe c’igihe rikorwa ku bantu bahambaye bamenyekanye mu rwego rumwe rumwe, kandi umurongo umwumwe wose usesengurwa ukwawo. Nta nkenerwa yo kwemera SD idahinduka.
Kugira ngo tugereranye ivyavuye muri iri koraniro n’ibiharuro vyasohowe maze dushireho igishushanyo ca Venn kiri ku gishushanyo ca 1, twahuje urutonde rw’amaproteine ​​y’ingero n’ibisobanuro vya MitoCarta 2.0 (24). Koresha igikoresho co kuri interineti Gushushanya igishushanyo ca Venn (http://amakuru y’ibinyabuzima.psb.ugent.be/ibikoresho vyo ku rubuga/Venn/) kugira ngo ubone igishushanyo.
Kugira ngo uronke amakuru arambuye yerekeye uburyo bw’imibare bukoreshwa mu gusesangura ivy’amaproteine, urashobora kuraba igice gihuye n’ico c’Ibikoresho n’Uburyo. Ku bindi bigeragezo vyose, amakuru arambuye araboneka mu nkuru y’imigani ihuye. Keretse iyo bigaragajwe ukundi, amakuru yose agaragazwa nk’uko ari hagati ± SEM, kandi isesengura ryose ry’imibare ryakozwe hakoreshejwe porogarama ya GraphPad Prism 8.1.2.
Ku bijanye n’ibikoresho vy’inyongera vy’iyi ngingo, raba kuri http://advances.sciencemag.org/cgi/ibirimwo/vyuzuye/6/35/eaba8271/DC1.
Iyi ni ingingo ishobora gukoreshwa n’abantu bose igakwiragizwa hakurikijwe amategeko y’uruhusha rwo gukoresha, gukwiragiza no gusubiramwo mu buryo ubwo ari bwo bwose, igihe cose ikoreshwa rya nyuma ritari ryo gushaka inyungu z’ubudandaji kandi ikintu nyamukuru ari uko igikorwa c’intango ari co. Urutonde.
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Ivyo vyanditswe na E. Motori, I. Atanassov, SMV Kochan, K. Folz-Donahue, V. Sakthivelu, P. Giavalisco, N. Toni, J. Puyal, N.-G. Larson
Isesengurwa ry’imitsi y’ubwonko ridakora neza ryerekanye ko porogarama z’imitsi zikora kugira ngo zirwanye ukuntu imitsi idakora neza.
Ivyo vyanditswe na E. Motori, I. Atanassov, SMV Kochan, K. Folz-Donahue, V. Sakthivelu, P. Giavalisco, N. Toni, J. Puyal, N.-G. Larson
Isesengurwa ry’imitsi y’ubwonko ridakora neza ryerekanye ko porogarama z’imitsi zikora kugira ngo zirwanye ukuntu imitsi idakora neza.
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