I kēia manawa *He wahi noho o kēia manawa: Cologne 50931, Kelemānia, Cologne Excellence Cluster Research on Cellular Stress Response in Aging-related Diseases (CECAD).
Ua manaʻo ʻia ka neurodegeneration o nā maʻi mitochondrial he mea hiki ʻole ke hoʻihoʻi ʻia no ka mea ua kaupalena ʻia ka plasticity metabolic o nā neurons, akā ʻaʻole maopopo loa ka hopena o ka hana ʻino mitochondrial ma ke kūʻokoʻa o ke kelepona o ka metabolism neuronal i loko o ke kino. Maanei, ke hoʻolauna nei mākou i ka proteome kikoʻī o ke kelepona o nā neurons Purkinje me ka hemahema OXPHOS holomua i hoʻokumu ʻia e ka dynamics fusion mitochondrial i hoʻopilikia ʻia. Ua ʻike mākou ua hoʻoulu ka hana ʻino mitochondrial i kahi loli nui i ke kahua o proteomics, ka mea i alakaʻi hope loa i ka hoʻāla ʻana o nā papahana metabolic pololei ma mua o ka make ʻana o ke kelepona. Me ka manaʻo ʻole, ua hoʻoholo mākou i ka hoʻokomo maopopo ʻana o ka pyruvate carboxylase (PCx) a me nā enzymes anti-aging ʻē aʻe e hoʻohui i nā waena o ka pōʻaiapuni TCA. ʻO ka pale ʻana o PCx i hoʻonui i ke kaumaha oxidative a me ka neurodegeneration, e hōʻike ana he hopena pale ko ka atherosclerosis i nā neurons nele i ka OXPHOS. ʻO ka hoʻihoʻi ʻana o ka fusion mitochondrial i loko o nā neurons degenerated terminally e hoʻohuli loa i kēia mau ʻano metabolic, no laila e pale ana i ka make ʻana o ke kelepona. ʻIke kā mākou mau ʻike i nā ala i ʻike ʻole ʻia ma mua e hāʻawi ana i ka kūpaʻa i ka hana ʻino mitochondrial a hōʻike e hiki ke hoʻohuli ʻia ka neurodegeneration ʻoiai i nā pae hope o ka maʻi.
ʻO ke kuleana koʻikoʻi o ka mitochondria i ka mālama ʻana i ka metabolism ikehu neuronal e hoʻokūpaʻa ʻia e nā hōʻailona neurological nui e pili ana i nā maʻi mitochondrial kanaka. ʻO ka hapa nui o kēia mau maʻi i hoʻokumu ʻia e nā mutations gene e hoʻoponopono i ka hōʻike ʻana o ka gene mitochondrial (1, 2) a i ʻole ka luku ʻana o ka gene e pili ana i ka dynamics mitochondrial, kahi e hoʻopilikia pololei ʻole ai i ke kūpaʻa o ka mitochondrial DNA (mtDNA) (3, 4). Ua hōʻike ʻia ka hana ma nā hiʻohiʻona holoholona i ka pane ʻana i ka hana ʻino o ka mitochondrial i nā ʻiʻo a puni, hiki ke hoʻāla ʻia nā ala metabolic conservative (5-7), kahi e hāʻawi ai i ka ʻike koʻikoʻi no ka hoʻomaopopo hohonu ʻana i ka pathogenesis o kēia mau maʻi paʻakikī. I ka hoʻohālikelike ʻana, ʻo ko mākou ʻike ʻana i nā loli metabolic o nā ʻano cell kikoʻī i hoʻokumu ʻia e ka hāʻule ʻana o ka hana ʻana o ka adenosine triphosphate mitochondrial lolo (ATP) he mea nui (8), e hoʻokūpaʻa ana i ka pono e ʻike i nā pahuhopu therapeutic hiki ke hoʻohana ʻia e pale aku ai a pale aku paha i ka maʻi. Kāohi i ka neurodegeneration (9). ʻO ka nele o ka ʻike ʻo ia ka ʻoiaʻiʻo ua manaʻo nui ʻia nā cell nerve he palena iki loa ka maʻalahi metabolic i hoʻohālikelike ʻia me nā ʻano cell o nā ʻiʻo a puni (10). ʻOiai he kuleana koʻikoʻi kēia mau pūnaewele i ka hoʻonohonoho ʻana i ka hoʻolako ʻana i nā metabolites i nā neurons e hoʻolaha i ka hoʻoili synaptic a pane i nā kūlana hōʻeha a me nā maʻi, ʻo ka hiki ke hoʻololi i ka metabolism cell i nā kūlana paʻakikī o ka ʻiʻo lolo ua aneane kaupalena ʻia i nā cell glial (11-14). Eia kekahi, ʻo ka heterogeneity cellular kūlohelohe o ka ʻiʻo lolo e keakea nui nei i ke aʻo ʻana i nā loli metabolic e hana ʻia ana i nā hui neuronal kikoʻī. ʻO ka hopena, ʻaʻole ʻike nui ʻia e pili ana i nā hopena cellular a me metabolic o ka hana ʻole o ka mitochondrial i loko o nā neurons.
I mea e hoʻomaopopo ai i nā hopena metabolic o ka hana ʻole o ka mitochondrial, ua hoʻokaʻawale mākou i nā neurons Purkinje (PNs) i nā pae like ʻole o ka neurodegeneration i hoʻokumu ʻia e ka luku ʻia ʻana o ka mitochondrial outer membrane fusion (Mfn2). ʻOiai ʻo nā mutations Mfn2 i loko o ke kanaka e pili ana me kahi ʻano o ka hoʻoilina motor sensory neuropathy i ʻike ʻia ʻo Charcot-Marie-Tooth type 2A (15), ʻo ka luku kūlana o Mfn2 i loko o nā ʻiole he ʻano hana hoʻokomo i ʻike maikaʻi ʻia o ka oxidation Phosphorylation (OXPHOS). ʻO nā ʻano neuronal like ʻole (16-19) a me ka phenotype neurodegenerative hopena e hele pū ʻia me nā hōʻailona neurological holomua, e like me nā maʻi neʻe (18, 19) a i ʻole cerebellar ataxia (16). Ma ka hoʻohana ʻana i ka hui pū ʻana o ka label-free quantitative (LFQ) proteomics, metabolomics, imaging, a me nā ʻano virological, hōʻike mākou e hoʻoulu ikaika ka neurodegeneration holomua i ka pyruvate carboxylase (PCx) a me nā mea ʻē aʻe e pili ana i ka arteriosclerosis o PNs in vivo ʻO ka hōʻike ʻana o nā enzymes. No ka hōʻoia ʻana i ka pilina o kēia ʻike, ua hoʻohaʻahaʻa pono mākou i ka hōʻike ʻana o PCx i loko o nā PN nele o Mfn2, a ua ʻike ʻia ua hoʻonui kēia hana i ke kaumaha oxidative a hoʻolalelale i ka neurodegeneration, no laila e hōʻike ana e hāʻawi ana ka azoospermia i ka make ʻana o nā cell i ka Metabolic adaptability. Hiki i ka hōʻike koʻikoʻi o MFN2 ke hoʻopakele piha i ka PN degeneration terminal me ka hemahema OXPHOS koʻikoʻi, ka hoʻopau nui ʻana i ka DNA mitochondrial, a me ka pūnaewele mitochondrial i haki ʻia, kahi e hoʻoikaika hou aku ai e hiki i kēia ʻano o ka neurodegeneration ke hoʻōla i ke kahua holomua o ka maʻi ma mua o ka make ʻana o nā cell.
I mea e ʻike ai i ka mitochondria ma Mfn2 knockout PNs, ua hoʻohana mākou i kahi ʻano ʻiole e hiki ai i ka mitochondria Cre-dependent ke kuhikuhi i ka protein fluorescent melemele (YFP) (mtYFP) (20) Cre expression a nānā i ke ʻano mitochondrial in vivo. Ua ʻike mākou ʻo ka luku ʻia ʻana o ka gene Mfn2 ma PNs e alakaʻi i ka mahele mālie o ka pūnaewele mitochondrial (Kiʻi S1A), a ua ʻike ʻia ka loli mua loa i 3 mau pule o ka makahiki. I ka hoʻohālikelike ʻana, ʻo ka degeneration nui o ka papa cell PN, e like me ka hōʻike ʻana o ka nalowale ʻana o ka immunostaining Calbindin, ʻaʻole i hoʻomaka a hiki i 12 mau pule o ka makahiki (Kiʻi 1, A a me B). ʻO ka manawa like ʻole ma waena o nā loli mua loa i ka morphology mitochondrial a me ka hoʻomaka ʻana o ka make neuronal i hoʻoikaika iā mākou e noiʻi i nā loli metabolic i hoʻoulu ʻia e ka hana ʻino mitochondrial ma mua o ka make ʻana o ke kelepona. Ua hoʻomohala mākou i kahi hoʻolālā hoʻokaʻawale cell fluorescence-activated (FACS) e hoʻokaʻawale iā YFP (YFP+)-expressing PN (Kiʻi 1C), a i loko o nā ʻiole kaohi (Mfn2 + / loxP :: mtYFP loxP- stop-loxP: : L7-cre), ma hope aku nei i kapa ʻia ʻo CTRL (Kiʻi S1B). ʻO ka hoʻonui ʻana o ke hoʻolālā gating e pili ana i ka ikaika pili o ka hōʻailona YFP e hiki ai iā mākou ke hoʻomaʻemaʻe i ke kino YFP+ (YFPhigh) o nā PN mai nā non-PNs (YFPneg) (Kiʻi S1B) a i ʻole nā ​​​​​​ʻāpana fluorescent axon/dendritic putative (YFPlow; Kiʻi S1D, hema), i hōʻoia ʻia e ka microscope confocal (Kiʻi S1D, ʻākau). I mea e hōʻoia ai i ka ʻike o ka heluna kanaka i hoʻokaʻawale ʻia, ua hana mākou i ka LFQ proteomics a laila ka loiloi ʻāpana nui, a ua ʻike ʻia aia kahi kaʻawale maopopo ma waena o nā cell YFPhigh a me YFPneg (Kiʻi S1C). Ua hōʻike nā pūnaewele YFPhigh i kahi hoʻonui ʻupena o nā māka PN i ʻike ʻia (ʻo ia hoʻi ʻo Calb1, Pcp2, Grid2 a me Itpr3) (21, 22), akā ʻaʻohe hoʻonui o nā protein i hōʻike pinepine ʻia i loko o nā neurons a i ʻole nā ​​ʻano cell ʻē aʻe (Kiʻi 1D)). Ua hōʻike kahi hoʻohālikelike ma waena o nā laʻana i nā pūnaewele YFPhigh i hoʻokaʻawale ʻia i hōʻiliʻili ʻia i nā hoʻokolohua kūʻokoʻa i kahi coefficient correlation> 0.9, e hōʻike ana i ka reproducibility maikaʻi ma waena o nā replicates biological (Kiʻi S1E). I ka hōʻuluʻulu manaʻo, ua hōʻoia kēia mau ʻikepili i kā mākou hoʻolālā no ka hoʻokaʻawale koke a kikoʻī o ka PN hiki. No ka mea, ʻo ka ʻōnaehana hoʻokele L7-cre i hoʻohana ʻia e hoʻoulu i ka mosaic recombination i ka pule mua ma hope o ka hānau ʻana (23), ua hoʻomaka mākou e culling i nā ʻiole mai CTRL a me conditional (Mfn2 loxP / loxP :: mtYFP loxP-stop-loxP :: L7-cre) E hōʻiliʻili i nā neurons. Ma hope o ka pau ʻana o ka recombination, ua kapa ʻia ʻo Mfn2cKO i 4 mau pule o ka makahiki. Ma ke ʻano he hopena, ua koho mākou i 8 mau pule o ka makahiki i ka wā i paʻa ai ka papa PN ʻoiai ua ʻike ʻia ka ʻāpana mitochondrial (Kiʻi 1B a me ke Kiʻi S1A). Ma ka huina, ua helu mākou i ka huina o 3013 mau protein, ma kahi o 22% i hoʻokumu ʻia ma nā annotations MitoCarta 2.0 e pili ana i ka proteome mitochondrial ma ke ʻano he mitochondria (Kiʻi 1E) (Kiʻi 1E) (24). Ua hōʻike ka loiloi hōʻike gene ʻokoʻa i hana ʻia ma ka pule 8 he 10.5% wale nō o nā protein āpau i loaʻa nā loli koʻikoʻi (Kiʻi 1F a me ke Kiʻi S1F), kahi i hoʻohaʻahaʻa ʻia ai he 195 mau protein a ua hoʻonui ʻia he 120 mau protein (Kiʻi 1F). He mea kūpono ke hoʻomaopopo ʻana ʻo ka "loiloi ala hou" o kēia ʻikepili e hōʻike ana ʻo nā genes i hōʻike ʻokoʻa ʻia no kahi hui i kaupalena ʻia o nā ala metabolic kikoʻī (Kiʻi 1G). ʻO ka mea hoihoi, ʻoiai ʻo ka hoʻohaʻahaʻa ʻana o nā ala e pili ana i ka OXPHOS a me ka hōʻailona calcium e hōʻoia i ka hoʻoulu ʻana o ka hana ʻole o ka mitochondrial i nā PN fusion-deficient, ʻo nā mahele ʻē aʻe e pili nui ana i ka metabolism amino acid ua hoʻonui nui ʻia, kahi e kūlike me ka metabolism e hana ʻia ana i nā PN mitochondrial. He kūlike ka hana.
(A) Nā kiʻi confocal hōʻike o nā ʻāpana cerebellar o nā ʻiole CTRL a me Mfn2cKO e hōʻike ana i ka nalowale holomua o nā PN (calbindin, hina); ua hoʻoluʻu ʻia nā nuclei me DAPI. (B) Ka helu ʻana o (A) (ka nānā ʻana i ka ʻokoʻa o ke ala hoʻokahi, ***P<0.001; n = 4 a 6 mau pōʻai mai ʻekolu mau ʻiole). (C) Kaʻina hana hoʻokolohua. (D) Ka hoʻolaha ʻana o ka palapala wela o nā māka kikoʻī iā Purkinje (luna) a me nā ʻano cell ʻē aʻe (waena). (E) Kiʻikuhi Venn e hōʻike ana i ka helu o nā protein mitochondrial i ʻike ʻia ma ka PN i hoʻokaʻawale ʻia. (F) Ka palapala lua pele o nā protein i hōʻike ʻokoʻa ʻia ma nā neurons Mfn2cKO ma 8 mau pule (ka waiwai ʻoki koʻikoʻi o 1.3). (G) Hōʻike ka loiloi ala hana i nā ala ʻelima koʻikoʻi loa o ka up-regulation (ʻulaʻula) a me ka down-regulation (uliuli) ma ka Mfn2cKO PN i hoʻokaʻawale ʻia he 8 mau pule. Hōʻike ʻia ka pae hōʻike awelika o kēlā me kēia protein i ʻike ʻia. Palapala wela Grayscale: ka waiwai P i hoʻoponopono ʻia. ns, ʻaʻole koʻikoʻi.
Ua hōʻike ʻia nā ʻikepili Proteomics ua emi mālie ka hōʻike ʻana o ka protein o nā complexes I, III, a me IV. Loaʻa i nā Complexes I, III, a me IV nā subunits koʻikoʻi i hoʻopili ʻia e ka mtDNA, ʻoiai ʻo ka complex II, kahi i hoʻopili wale ʻia e ka nuclear, ʻaʻole i hoʻopilikia nui ʻia (Kiʻi 2A a me Kiʻi S2A). . E like me nā hopena proteomics, ua hōʻike ka immunohistochemistry o nā ʻāpana ʻiʻo cerebellar ua emi mālie ka pae subunit MTCO1 (mitochondrial cytochrome C oxidase subunit 1) o ka complex IV ma PN (Kiʻi 2B). Ua emi nui ka subunit Mtatp8 i hoʻopili ʻia e ka mtDNA (Kiʻi S2A), ʻoiai ʻaʻole i loli ka pae paʻa o ka subunit ATP synthase i hoʻopili ʻia e ka nuclear, kahi e kūlike me ka complex ATP synthase subassembly F1 i ʻike ʻia i ka wā e paʻa ai ka hōʻike ʻana o ka mtDNA. Kūlike ka hoʻokumu ʻana. Hoʻopau (7). Ua hōʻoia ka loiloi ʻana o ka pae mtDNA i loko o nā Mfn2cKO PN i hoʻonohonoho ʻia e ka reaction chain polymerase manawa maoli (qPCR) i ka emi mālie ʻana o ka helu kope mtDNA. Ke hoʻohālikelike ʻia me ka hui kaohi, i 8 mau pule o ka makahiki, ua mālama ʻia ma kahi o 20% wale nō o ka pae mtDNA (Kiʻi 2C). E kūlike me kēia mau hopena, ua hoʻohana ʻia ka hoʻoluʻu microscopy confocal o Mfn2cKO PNs e ʻike i ka DNA, e hōʻike ana i ka hoʻopau ʻana i ka manawa o nā nucleotides mitochondrial (Kiʻi 2D). Ua ʻike mākou ua hoʻonui ʻia kekahi mau moho i komo i ka hoʻohaʻahaʻa protein mitochondrial a me ka pane stress, me Lonp1, Afg3l2 a me Clpx, a me nā mea hoʻākoakoa paʻakikī OXPHOS. ʻAʻohe loli koʻikoʻi i nā pae o nā protein i komo i ka apoptosis i ʻike ʻia (Kiʻi S2B). Pēlā nō, ua ʻike mākou he mau loli liʻiliʻi wale nō nā kahawai mitochondria a me endoplasmic reticulum i komo i ka lawe ʻana o ka calcium (Kiʻi S2C). Eia kekahi, ʻaʻole i loaʻa i ka loiloi ʻana o nā protein e pili ana i ka autophagy nā loli koʻikoʻi, kahi e kūlike me ka hoʻokomo ʻia ʻana o nā autophagosomes i ʻike ʻia i loko o vivo e ka immunohistochemistry a me ka microscopy electron (Kiʻi S3). Eia nō naʻe, ʻo ka holomua ʻole o OXPHOS i nā PN e hele pū ʻia me nā loli mitochondrial ultrastructural maopopo. Hiki ke ʻike ʻia nā hui mitochondrial i loko o nā kino cell a me nā lāʻau dendritic o Mfn2cKO PNs i piha i 5 a me 8 mau pule, a ua loli nui ke ʻano o ka membrane o loko (Kiʻi S4, A a me B). E kūlike me kēia mau loli ultrastructural a me ka emi nui ʻana o ka mtDNA, ua hōʻike ka nānā ʻana o nā ʻāpana cerebellar cerebral acute me ka tetramethylrhodamine methyl ester (TMRM) ua emi nui ka hiki ke hoʻopili ʻia ka mitochondrial membrane i Mfn2cKO PNs (Kiʻi S4C).
(A) Ka loiloi manawa o ka pae hōʻike o ka paʻakikī OXPHOS. E noʻonoʻo wale i nā protein me P<0.05 ma 8 mau pule (ʻelua ala ANOVA). Laina kiko: ʻAʻohe hoʻoponopono i hoʻohālikelike ʻia me CTRL. (B) Hema: He laʻana o kahi ʻāpana cerebellar i kapa ʻia me ka antibody anti-MTCO1 (ka pā unahi, 20 μm). Uhi ʻia ka wahi i noho ʻia e nā kino cell Purkinje e ka melemele. ʻĀkau: Ka helu ʻana o nā pae MTCO1 (ka loiloi hoʻokahi ala o ka variance; n = 7 a 20 mau cell i kālailai ʻia mai ʻekolu mau ʻiole). (C) Ka loiloi qPCR o ka helu kope mtDNA ma ka PN i hoʻonohonoho ʻia (ka loiloi hoʻokahi ala o ka variance; n = 3 a 7 mau ʻiole). (D) Hema: He laʻana o kahi ʻāpana cerebellar i kapa ʻia me kahi antibody anti-DNA (ka pā unahi, 20 μm). Uhi ʻia ka wahi i noho ʻia e nā kino cell Purkinje e ka melemele. ʻĀkau: Ka helu ʻana o nā lesions mtDNA (ka loiloi hoʻokahi ala o ka variance; n = 5 a 9 mau cell mai ʻekolu mau ʻiole). (E) He laʻana o kahi ʻāpana cerebellar acute e hōʻike ana i nā cell mitoYFP + Purkinje (pua) i loko o kahi hoʻopaʻa leo ʻana o ka patch clamp cell holoʻokoʻa. (F) Ka helu ʻana o ke kiʻikuhi IV. (G) Nā hoʻopaʻa leo ʻana o ka injection depolarizing current ma CTRL a me Mfn2cKO Purkinje cell. Kaha kiʻekiʻe: ʻO ka pulse mua i hoʻoulu ai i ka AP. Kaha lalo: Alapine AP kiʻekiʻe loa. (H) Ka helu ʻana o nā hoʻokomo spontaneous postsynaptic (sPSPs). Hōʻike ʻia ka kaha hoʻopaʻa leo ʻana a me kāna lakio zoom ma (I). Ua kālailai ʻia ka loiloi hoʻokahi ala o ka variance n = 5 a 20 mau cell mai ʻekolu mau ʻiole. Hōʻike ʻia nā ʻikepili ma ke ʻano he mean ± SEM; *P<0.05; **P<0.01; ***P<0.001. (J) Nā kaha kiʻekiʻe o ka AP spontaneous i hoʻopaʻa ʻia me ka hoʻohana ʻana i ke ʻano perforated patch clamp. Kaha kiʻekiʻe: Alapine AP kiʻekiʻe loa. Kaha lalo: zoom o kahi AP hoʻokahi. (K) E helu i ka awelika a me ka alapine AP kiʻekiʻe loa e like me (J). Hoʻāʻo Mann-Whitney; ua kālailai ʻia nā cell n = 5 mai ʻehā mau ʻiole. Ua hōʻike ʻia nā ʻikepili ma ke ʻano he awelika ± SEM; ʻaʻole koʻikoʻi.
Ua ʻike ʻia ka pōʻino OXPHOS maopopo i loko o ka Mfn2cKO PN 8 pule, e hōʻike ana he ʻano ʻē loa ka hana physiological o nā neurons. No laila, ua kālailai mākou i nā ʻano uila passive o nā neurons OXPHOS-deficient i 4 a 5 mau pule a me 7 a 8 mau pule ma ka hana ʻana i nā hoʻopaʻa leo patch clamp cell holoʻokoʻa i nā ʻāpana cerebellar acute (Kiʻi 2E). Me ka manaʻo ʻole ʻia, ua like ka awelika o ka membrane hoʻomaha a me ke kūʻē ʻana o ka hoʻokomo o nā neurons Mfn2cKO me ka mana, ʻoiai he mau ʻokoʻa liʻiliʻi ma waena o nā cell (Papa 1). Pēlā nō, i ka 4 a 5 mau pule o ka makahiki, ʻaʻohe loli koʻikoʻi i ka pilina o kēia manawa-voltage (IV curve) i loaʻa (Kiʻi 2F). Eia naʻe, ʻaʻohe neurons Mfn2cKO 7 a 8 mau pule i ola i ka regimen IV (kaʻina hyperpolarization), e hōʻike ana he ʻike maopopo i ka hiki ke hyperpolarization i kēia pae hope. I ka hoʻohālikelike ʻana, i loko o nā neurons Mfn2cKO, ua ʻae maikaʻi ʻia nā kahe depolarizing e hoʻoulu ai i nā hoʻokuʻu hana hana hou (AP), e hōʻike ana ʻaʻole ʻokoʻa loa kā lākou mau ʻano hoʻokuʻu holoʻokoʻa mai nā neurons hoʻomalu 8-pule (Papa 1 a me ke Kiʻi 2G). Pēlā nō, ua like ke alapine a me ka amplitude o nā kahe postsynaptic spontaneous (sPSCs) me nā mea o ka hui hoʻomalu, a ua piʻi ke alapine o nā hanana mai 4 mau pule a i 5 mau pule a i 7 mau pule a i 8 mau pule me ka piʻi like (Kiʻi 2, H a me I). ʻO ka manawa o ka synaptic maturation ma PNs (25). Ua loaʻa nā hopena like ma hope o nā ʻāpana PNs perforated. Pale kēia hoʻonohonoho i ka uku hiki ke uku ʻia o nā hemahema ATP cellular, e like me ka mea e hiki ke hana ma ka hoʻopaʻa ʻana i ka patch clamp cell holoʻokoʻa. Ma ke ʻano kūikawā, ʻaʻole i hoʻopilikia ʻia ka hiki ke hoʻomaha ʻia o ka membrane a me ke alapine firing spontaneous o nā neurons Mfn2cKO (Kiʻi 2, J a me K). I ka hōʻuluʻulu manaʻo, hōʻike kēia mau hopena e hiki i nā PN me ka hana hewa OXPHOS ke hoʻoponopono maikaʻi i nā ʻano hoʻokuʻu alapine kiʻekiʻe, e hōʻike ana aia kahi ʻano uku e hiki ai iā lākou ke mālama i nā pane electrophysiological kokoke i ka maʻamau.
Ua hōʻike ʻia nā ʻikepili ma ke ʻano he awelika ± SEM (hoʻokahi ala loiloi o ka ʻokoʻa, hoʻāʻo hoʻohālikelike lehulehu a Holm-Sidak; *P<0.05). Hōʻike ʻia ka helu ʻāpana e nā pale.
Ua hoʻomaka mākou e noiʻi inā paha kekahi mahele i loko o ka ʻikepili proteomics (Kiʻi 1G) e komo pū ana me nā ala e hiki ke pale aku i ka hemahema OXPHOS koʻikoʻi, no laila e wehewehe ana i ke kumu e hiki ai i ka PN i hoʻopilikia ʻia ke mālama i ka electrophysiology kokoke i ka maʻamau (Kiʻi 2, E a i K). . Ua hōʻike ka loiloi Proteomics ua hoʻonui nui ʻia nā enzymes i komo i ka catabolism o nā waikawa amino kaulahao lālā (BCAA) (Kiʻi 3A a me Kiʻi S5A), a ʻo ka huahana hope loa acetyl-CoA (CoA) a i ʻole succinyl CoA hiki ke hoʻohui i nā tricarboxylates i loko o ke kaʻina arteriosclerosis Acid (TCA). Ua ʻike mākou ua hoʻonui ʻia ka ʻike o BCAA transaminase 1 (BCAT1) a me BCAT2. Hoʻoulu lākou i ka hana mua o ka catabolism BCAA ma ka hana ʻana i ka glutamate mai α-ketoglutarate (26). Ua hoʻonui ʻia nā subunits āpau e hana ana i ka branched chain keto acid dehydrogenase (BCKD) complex (hoʻoulu ka complex i ka decarboxylation ma hope a hiki ʻole ke hoʻihoʻi ʻia o ka iwi kalapona BCAA i hopena ʻia) (Kiʻi 3A a me Kiʻi S5A). Eia naʻe, ʻaʻohe loli maopopo i loko o BCAA iho i loaʻa i ka PN i hoʻonohonoho ʻia, hiki ke kumu o ka hoʻonui ʻia ʻana o ka lawe ʻana o ke kelepona o kēia mau waikawa amino koʻikoʻi a i ʻole ka hoʻohana ʻana i nā kumu ʻē aʻe (glucose a lactic acid paha) e hoʻohui i ka pōʻaiapuni TCA (Kiʻi S5B). Ua hōʻike pū nā PN nele i ka OXPHOS i ka hoʻonui ʻia ʻana o ka glutamine decomposition a me nā hana transamination i 8 mau pule o ka makahiki, hiki ke hōʻike ʻia e ka hoʻonui ʻia ʻana o nā enzymes mitochondrial glutaminase (GLS) a me glutamine pyruvate transaminase 2 (GPT2) (Kiʻi 3, A a me C). He mea kūpono ke hoʻomaopopo ʻana ua kaupalena ʻia ka piʻi ʻana o GLS i ka spliced ​​​​isoform glutaminase C (GLS-GAC) (ʻo ka loli o Mfn2cKO/CTRL ma kahi o 4.5-fold, P = 0.05), a ʻo kāna piʻi kikoʻī i loko o nā ʻiʻo kanesa Hiki ke kākoʻo i ka bioenergy mitochondrial. (27).
(A) Hōʻike ka palapala wela i ka loli o ka pae protein no ke ala i kuhikuhi ʻia ma 8 mau pule. (B) Laʻana o kahi ʻāpana cerebellar i kapa ʻia me ka antibody anti-PCx (pahu unahi, 20 μm). Kuhikuhi ka pua melemele i ke kino cell Purkinje. (C) Ka loiloi hōʻike protein papa manawa i ʻike ʻia he moho koʻikoʻi no ka atherosclerosis (hoʻāʻo t-maha, *FDR <5%; n = 3-5 mau ʻiole). (D) Ma luna: He kiʻikuhi schematic e hōʻike ana i nā ʻano like ʻole o ke komo ʻana i ke kalapona i kapa ʻia i loko o ka tracer [1-13C]pyruvate (ʻo ia hoʻi, ma o PDH a i ʻole ke ala trans-arterial). Lalo: Hōʻike ka pakuhi violin i ka pakeneka o ke kalapona i kapa ʻia hoʻokahi (M1) i hoʻololi ʻia i ka waikawa aspartic, ka waikawa citric a me ka waikawa malic ma hope o ka lepili ʻana i nā ʻāpana cerebellar acute me [1-13C]pyruvate (paired t-test; ** P <0.01). (E) Ka loiloi mōʻaukala manawa piha o ke ala i hōʻike ʻia. E noʻonoʻo wale i nā protein me P<0.05 ma 8 mau pule. Laina haki: ʻaʻohe waiwai hoʻoponopono (loiloi ʻelua ala o ka ʻokoʻa; * P <0.05; *** P <0.001). Hōʻike ʻia nā ʻikepili ma ke ʻano he awelika ± SEM.
I kā mākou loiloi, ua lilo ka catabolism BCAA i hoʻokahi o nā ala hoʻonui koʻikoʻi. Hōʻike ikaika kēia ʻoiaʻiʻo e loli paha ka nui o ka ea e komo ana i ke kaʻina TCA ma PN nele i ka OXPHOS. Hiki i kēia ke hōʻike i kahi ʻano nui o ka hoʻololi hou ʻana i ka metabolic neuronal, kahi e loaʻa ai ka hopena pololei i ka physiology neuronal a me ke ola ʻana i ka wā o ka mālama ʻana i ka hana koʻikoʻi o OXPHOS. E kūlike me kēia kuhiakau, ua ʻike mākou ua hoʻonui ʻia ka enzyme anti-atherosclerotic nui PCx (hoʻololi ʻo Mfn2cKO/CTRL ma kahi o 1.5 mau manawa; Kiʻi 3A), ka mea e hoʻoulu ai i ka hoʻololi ʻana o ka pyruvate i oxaloacetate (28), ka mea i manaʻo ʻia aia i loko o ka ʻiʻo lolo. Ua kaupalena ʻia ka hōʻike ʻana i loko i nā astrocytes (29, 30). E kūlike me nā hopena proteomics, ua hōʻike ka microscopy confocal ua hoʻonui kūikawā ʻia ka hōʻike ʻana o PCx i nā PN nele i ka OXPHOS, ʻoiai ua kaupalena nui ʻia ka reactivity PCx i nā cell glial Bergmann kokoke o ka mana (Kiʻi 3B). No ka hoʻāʻo ʻana i ka piʻi ʻana o PCx i ʻike ʻia, ua mālama mākou i nā ʻāpana cerebellar acute me ka [1-13C]pyruvate tracer. I ka wā i hoʻopau ʻia ai ka pyruvate e ka pyruvate dehydrogenase (PDH), ua nalowale kona lepili isotope , Akā ua hoʻokomo ʻia i loko o nā waena o ke kaʻina hana TCA i ka wā e metabolized ai ka pyruvate e nā hopena vascular (Kiʻi 3D). I ke kākoʻo ʻana i kā mākou ʻikepili proteomics, ua ʻike mākou i kahi nui o nā māka mai kēia tracer i ka waikawa aspartic o nā ʻāpana Mfn2cKO, ʻoiai ʻo ka waikawa citric a me ka waikawa malic he ʻano kaulike hoʻi, ʻoiai ʻaʻole koʻikoʻi (Kiʻi 3D).
I loko o nā neurons dopamine o nā ʻiole MitoPark me ka hana ʻole o ka mitochondrial i hoʻokumu ʻia e nā neurons dopamine e hoʻopau pono ana i ka mitochondrial transcription factor A gene (Tfam) (Kiʻi S6B), ua hoʻonui nui ʻia ka hōʻike ʻana o PCx (31), e hōʻike ana i ka acetone acid arteriosclerosis. Hoʻoponopono ʻia ka hanana o ka maʻi i ka wā o ka hana ʻole o ka neuronal OXPHOS i loko o ke kino. He mea kūpono ke hoʻomaopopo ʻana ua ʻike ʻia ua hoʻonui nui ʻia nā enzymes kūikawā (32-34) i hiki ke hōʻike ʻia i loko o nā neurons i pili paha me ka arteriosclerosis i nā PNs nele i ka OXPHOS, e like me ka propionyl-CoA carboxylase (PCC-A), Malonyl-CoA hoʻololi i ka propionyl-CoA i succinyl-CoA a me ka mitochondrial malic enzyme 3 (ME3), nona ke kuleana nui e hoʻihoʻi i ka pyruvate mai ka malate (Kiʻi 3, A a me C) (33, 35). Eia kekahi, ua ʻike mākou i ka piʻi nui ʻana o ka enzyme Pdk3, ka mea e phosphorylates a no laila e hoʻopau i ka PDH (36), ʻoiai ʻaʻohe loli i ʻike ʻia i loko o ka enzyme Pdp1 e hoʻāla ana iā PDH a i ʻole ka complex enzyme PDH ponoʻī (Kiʻi 3A). Ma ke ʻano mau, ma Mern2cKO PNs, ua hoʻonui ʻia ka phosphorylation o ka subunit α1 α (PDHE1α) subunit o ka pyruvate dehydrogenase E1 component o ka PDH complex ma Ser293 (i ʻike ʻia e kāohi i ka hana enzyme o PDH) (Kiʻi S6C) (Kiʻi S6C). ʻAʻohe komo vascular o Pyruvate.
ʻO ka mea hope loa, ua ʻike mākou ua hoʻonui nui ʻia ke ala nui o ka biosynthesis serine a me glycine, ka pōʻaiapuni folate mitochondrial (1C) pili a me ka biosynthesis proline (Kiʻi 1G a me ke Kiʻi S5C), e like me nā hōʻike, i ka wā o ke kaʻina hana hoʻāla. Hoʻāla ʻia nā ʻiʻo a puni me ka hana ʻole o ka mitochondrial (5-7). Ua hōʻike ka loiloi confocal e kākoʻo ana i kēia ʻikepili proteomics i loko o PN me ka nalowale ʻana o OXPHOS, ua hoʻokomo ʻia nā ʻāpana cerebellar o nā ʻiole 8 pule i ka serine hydroxymethyltransferase 2 (SHMT2), kahi enzyme koʻikoʻi o ka pōʻaiapuni folate mitochondrial. Pane pale koʻikoʻi (Kiʻi S5D). I loko o 13 mau ʻāpana cerebellar acute i hoʻokomo ʻia e CU-glucose, ua hōʻoia hou nā hoʻokolohua metabolic tracing i ka hoʻonui ʻia ʻana o ka biosynthesis serine a me proline, e hōʻike ana ua hoʻonui ʻia ke kahe ʻana o nā isoforms carbon i loko o ka serine a me ka proline (Kiʻi S5E). ʻOiai ʻo nā hopena i hoʻolaha ʻia e GLS a me GPT2 ke kuleana no ka synthesis o ka glutamate mai ka glutamine a me ka transamination ma waena o ka glutamate a me ka α-ketoglutarate, hōʻike ko lākou upregulation he nui ka koi o nā neurons OXPHOS-deficient no ka glutamate, hiki ke kuhikuhi ʻia kēia i ka mālama ʻana i ka hoʻonui ʻia o ka biosynthesis o ka proline (Kiʻi S5C). I ka hoʻohālikelike ʻana i kēia mau loli, ua hōʻike kahi loiloi proteomic o nā astrocytes cerebellar mai nā ʻiole Mfn2cKO kikoʻī PN ʻaʻole i loli nui kēia mau ala (me nā antiperoxidases āpau) i ka hōʻike ʻana, no laila e hōʻike ana he koho kēia metabolic redirection i ka PN degradable (Kiʻi S6, D a i G).
I ka hōʻuluʻulu manaʻo, ua hōʻike kēia mau loiloi i nā ʻano hana like ʻole o ka hoʻoulu ʻana i ka manawa o nā ala metabolic kikoʻī ma PNs. ʻOiai hiki i ka hana mitochondrial neuronal maʻamau ke alakaʻi i ka atherosclerosis mua a me ka hoʻoponopono hou ʻana o 1C (Kiʻi 3E a me Kiʻi S5C), a hiki i nā loli i wānana ʻia i ka hōʻike ʻana o nā paʻakikī I a me IV, ʻo nā loli i ka synthesis serine de novo wale nō Ua ʻike wale ʻia i nā pae hope. ʻO ka hana ʻino OXPHOS (Kiʻi 3E a me Kiʻi S5C). Ho'ākāka kēia mau ʻike i kahi kaʻina hana maʻamau kahi e pane ai ka mitochondrial i hoʻokomo ʻia e ka stress (cycle 1C) a me ka cytoplasmic (serine biosynthesis) me ka hoʻonui ʻana o ka atherosclerosis i ka pōʻaiapuni TCA e hoʻoponopono hou i ka metabolism neuronal.
Hiki i nā PN nele OXPHOS 8-pule ke mālama i ka hana hoʻonāukiuki alapine kiʻekiʻe a hana i ka hoʻopili hou ʻana i ka metabolic e uku no ka hana ʻino mitochondrial. Hāpai kēia ʻike i kahi hiki ke hoihoi a hiki i kēia manawa, hiki i kēia mau cell ke loaʻa i ka hana therapeutic e hoʻopaneʻe a pale paha i ka neurodegeneration. Lohi. Ua hoʻoholo mākou i kēia hiki ma o ʻelua mau hana kūʻokoʻa. Ma ke ʻano mua, ua hoʻolālā mākou i kahi vector virus adeno-associated (AAV) Cre-dependent i hiki ke hōʻike koho ʻia ʻo MFN2 i nā PN nele OXPHOS i vivo (Kiʻi S7A). Ua hōʻoia ʻia ka AAV encoding MFN2 a me ka fluorescent reporter gene mCherry (Mfn2-AAV) i nā moʻomeheu neuron mua i vitro, ka mea i hoʻoulu ai i ka hōʻike ʻana o MFN2 ma ke ʻano Cre-dependent a hoʻopakele i ke ʻano mitochondrial, no laila e pale ana i ka neuromutation i nā neurons Mfn2cKO (Kiʻi S7, B, D a me E). ʻO ka mea aʻe, ua hana mākou i nā hoʻokolohua in vivo e hoʻouna i ka Mfn2-AAV 8 pule i ka cortex cerebellar o Mfn2cKO a me nā ʻiole kaohi, a ua kālailai i nā ʻiole 12 pule (Kiʻi 4A). Ua make nā ʻiole Mfn2cKO i mālama ʻia (Kiʻi 1, A a me B) (16). ʻO ka hoʻoili ʻana o ka viral in vivo i hopena i ka hōʻike koho ʻana o PN i kekahi mau pōʻai cerebellar (Kiʻi S7, G a me H). ʻO ka injection o ka AAV kaohi e hōʻike wale ana iā mCherry (Ctrl-AAV) ʻaʻohe hopena koʻikoʻi i ke kiʻekiʻe o ka neurodegeneration i nā holoholona Mfn2cKO. I ka hoʻohālikelike ʻana, ua hōʻike ka nānā ʻana o Mfn2cKOs i hoʻoili ʻia me Mfn2-AAV i kahi hopena pale koʻikoʻi o ka papa cell PN (Kiʻi 4, B a me C). Ma ke ʻano kūikawā, ʻike ʻia ka nui o ka neuron mai nā holoholona kaohi (Kiʻi 4, B a me C, a me Kiʻi S7, H a me I). ʻO ka hōʻike ʻana o MFN1 akā ʻaʻole ʻo MFN2 he like ka hopena i ka hoʻopakele ʻana i ka make neuronal (Kiʻi 4C a me ke Kiʻi S7, C a me F), e hōʻike ana e hiki i ka hōʻike ʻana o ka ectopic MFN1 ke hoʻopiha pono i ka nele o MFN2. Ua hōʻike ʻia ka loiloi hou ʻana ma ka pae PN hoʻokahi ua hoʻopakele nui ʻo Mfn2-AAV i ka ultrastructure o mitochondria, hoʻoponopono i nā pae mtDNA, a hoʻohuli i ka hōʻike kiʻekiʻe o ka māka anti-angiogenesis PCx ​​(Kiʻi 4, C a i E). Ua hōʻike ka nānā ʻana o nā ʻiole Mfn2cKO i hoʻopakele ʻia i kahi kūlana hoʻomaha ua hoʻomaikaʻi ʻia ko lākou kūlana a me nā hōʻailona motor (neʻe S1 a i S3). I ka hopena, hōʻike kēia mau hoʻokolohua ua lawa ka hoʻokomo hou ʻana o MFN2 i loko o nā PN i nele loa i ka OXPHOS e hoʻohuli i ka hoʻohana ʻana o ka mtDNA a hoʻoulu i ka atherosclerosis, no laila e pale ana i ka degeneration axon a me ka make neuronal i vivo.
(A) He hoʻolālā e hōʻike ana i ka papa hana hoʻokolohua no ka hoʻokomo ʻana i ka AAV encoding MFN2 i ka wā e hoʻāla ʻia ai ke ala metabolic i hōʻike ʻia. (B) Nā kiʻi confocal hōʻike o nā ʻāpana cerebellar 12-pule i hoʻololi ʻia i 8 mau pule i nā ʻiole Mfn2cKO a ua lepili ʻia me ka antibody anti-Calbindin. ʻĀkau: Scaling o nā fibers axon. ʻO ke ana o ka zoom axon he 450 a me 75 μm. (C) Hema: Quantification o ka nui o nā cell Purkinje ma ka AAV transduction loop (AAV+) (hoʻokahi ala loiloi o ka variance; n = 3 ʻiole). ʻĀkau: mtDNA focus analysis ma ka PN i hoʻololi ʻia i ka pule 12 (unpaired t-test; n = 6 mau cell mai ʻekolu ʻiole). * P <0.05; ** P <0.01. (D) Nā micrographs electron transmission hōʻike o nā PN o nā ʻāpana cerebellar Mfn2cKO i hoʻololi ʻia me nā vectors viral i hōʻike ʻia. Hōʻike ka pale maka poni i ka wahi i noho ʻia e nā dendrites, a hōʻike ka huinaha kiko melemele i ka zoom i hāʻawi ʻia ma ka ʻākau; hōʻike ʻo n i ka nucleus. ʻO ka pā unahi, 1μm. (E) hōʻike i kahi laʻana o ka pena ʻana o PCx ma PN i hoʻololi ʻia i 12 mau pule. ʻO ka pā unahi, 20μm. OE, overexpression; FC, hoʻololi pelu.
ʻO ka mea hope loa, ua noiʻi mākou i ke koʻikoʻi o ke ola ʻana o nā cell i hoʻokomo ʻia e ka peroxidase i nā PN i loaʻa i ka hana hewa OXPHOS. Ua hana mākou i ka mCherry encoding AAV-shRNA (short hairpin RNA) e kuhikuhi pono ana i ka mouse PCx mRNA (AAV-shPCx), a hoʻokomo i ka maʻi a i ʻole kona scrambled control (AAV-scr) i loko o ka cerebellum o nā ʻiole Mfn2cKO. Ua hana ʻia ka injection i ka pule ʻehā o ka makahiki (Kiʻi 5A) e hoʻokō i ka PCx knockdown maikaʻi i ka wā i hoʻonui ʻia ai ka hōʻike ʻana o PCx (Kiʻi 3C) a ua paʻa mau ka papa cell PN (Kiʻi 1A). He mea kūpono ke hoʻomaopopo ʻana ʻo ka hoʻokuʻu ʻana iā PCx (Kiʻi S8A) e alakaʻi i ka wikiwiki nui o ka make PN, i kaupalena ʻia i ke apo i loaʻa i ka maʻi (Kiʻi 5, B a me C). I mea e hoʻomaopopo ai i ke ʻano o nā hopena metabolic i hoʻokumu ʻia e ka PCx up-regulation, ua aʻo mākou i ke kūlana redox o PNs ma hope o ka PCx knockdown a me ka AAV-mediated optical biosensor Grx1-roGFP2 i hōʻike like ʻia (Kiʻi S8, B a D) e loiloi i ka glutathione ʻO ka loli pili o ka peptide redox potential (38). A laila, ua hana mākou i ka ʻelua-photon fluorescence lifetime imaging microscopy (FLIM) i nā ʻāpana lolo koʻikoʻi o Mfn2cKO 7-pule a i ʻole nā ​​​​​​littermates kaohi e ʻike i nā loli hiki ke loaʻa i ke kūlana redox cytoplasmic ma hope o ka hōʻoia ʻana i nā kūlana FLIM (Kiʻi S8, E a G). Ua hōʻike ka loiloi i kahi piʻi nui o ke kūlana oxidation o hoʻokahi Mfn2cKO PNs nele i ka hōʻike PCx, ʻokoʻa ia mai nā neurons kaohi a i ʻole Mfn2cKO PNs e hōʻike wale ana i ka shRNA scrambled (Kiʻi 5, D a me E). I ka wā i hoʻohaʻahaʻa ʻia ai ka hōʻike ʻana o PCx, ua piʻi ka pakeneka o nā Mfn2cKO PN e hōʻike ana i kahi kūlana oxidized kiʻekiʻe ma mua o ʻekolu manawa (Kiʻi 5E), e hōʻike ana ua mālama ka PCx up-regulation i ka mana redox o nā neurons degenerated.
(A) He hoʻolālā e hōʻike ana i ka papa hana hoʻokolohua no ka hoʻokomo ʻana i ka AAV encoding shPCx i ka wā e hoʻāla ʻia ai ke ala metabolic i hōʻike ʻia. (B) Nā kiʻi confocal hōʻike o nā ʻāpana cerebellar 8-pule i nā ʻiole Mfn2cKO i hoʻololi ʻia a lepili ʻia me ka antibody anti-calcineurin i 4 mau pule. Kaula unahi, 450μm. (C) Ka helu ʻana o ka nui o nā cell Purkinje i nā loops AAV-transduced (hoʻokahi ala loiloi o ka variance; n = 3 a 4 mau ʻiole). Hōʻike ʻia nā ʻikepili ma ke ʻano he mean ± SEM; ***P<0.001. (D) Hōʻike ke kiʻi FLIM hōʻike i ka awelika o ke ola o ka PN 7-pule e hōʻike ana i ka glutathione redox sensor Grx1-roGFP2 ma lalo o nā kūlana hoʻokolohua i kuhikuhi ʻia. LUT (papa nānā): ka manawa ola (ma picoseconds). Kaula unahi, 25μm. (E) Hōʻike ka histogram i ka hoʻolaha ʻana o nā waiwai ola Grx1-roGFP2 mai (D) (n=158 a 368 mau pūnaewele i ʻelua mau ʻiole ma lalo o kēlā me kēia kūlana). ʻO ka pakuhi pai ma luna o kēlā me kēia histogram: hōʻike i ka helu o nā pūnaewele me nā waiwai ola lōʻihi loa (ʻulaʻula, oxidized) a i ʻole pōkole (uliuli, hoʻemi ʻia), ʻoi aku ma mua o 1 SD o ka waiwai ola awelika ma CTRL-AAV-scr. (F) Hōʻike ke kumu hoʻohālike i manaʻo ʻia i ka hopena pale o ka hoʻonui ʻana o ka neuronal PCx.
Ma keʻano holoʻokoʻa, hōʻike ka ʻikepili a mākou e hāʻawi nei ma ʻaneʻi e hiki i ka hoʻopuka hou ʻana o MFN2 ke hoʻopakele piha i ka PN holomua me ka hemahema OXPHOS koʻikoʻi, ka hoʻopau nui ʻana o ka mtDNA, a me ke ʻano like ʻole o ka ista, no laila e hāʻawi ana i ka holomua mau ʻana i nā maʻi holomua. Hāʻawi ka Neurodegeneration i nā hōʻike hoʻohuli o ke kahua ma mua o ka make ʻana o ke kelepona. Hoʻoikaika hou ʻia kēia kekelē o ka maʻalahi metabolic e ka hiki o nā neurons ke hoʻoulu i ka atherosclerosis (kahi hoʻopili hou ʻana o ke kaʻina TCA), kahi e kāohi ai i ka hōʻike ʻana o PCx i nā PN nele i ka OXPHOS a hoʻonui i ka make ʻana o ke kelepona, no laila e pāʻani ana i kahi kuleana pale (Kiʻi 5F).
Ma kēia haʻawina, ua hāʻawi mākou i nā hōʻike e pili ana i ka pane ʻana o nā PN i ka hana ʻole o OXPHOS, ʻo ia ka hui mālie ʻana i ka atherosclerosis pōʻaiapuni TCA ma o ke ala hoʻāla ʻokoʻa i hoʻāla ʻia e nā papahana metabolic. Ua hōʻoia mākou i ka loiloi proteomic me nā ʻano hana hoʻopihapiha he nui a ua hōʻike ʻia i ka wā e hoʻokūkū ʻia e ka hana ʻino mitochondrial koʻikoʻi, loaʻa i nā neurons kahi ʻano elasticity metabolic i ʻike ʻole ʻia ma mua. I ko mākou kahaha, ʻaʻole pono ke kaʻina hana rewiring holoʻokoʻa e hōʻailona i ka mokuʻāina metabolic hope e hele pū me ka neurodegeneration mālie a me ka hiki ʻole ke hoʻihoʻi ʻia, akā hōʻike kā mākou ʻikepili he neuron mālama paha ia ma ke kahua ma mua o ka make ʻana o ke kelepona. ʻO ka ʻano uku hana. Hōʻike kēia ʻike he nui ke kiʻekiʻe o ka plasticity metabolic o nā neurons i loko o ke kino. Hōʻike kēia ʻoiaʻiʻo e hiki i ka hoʻolauna hou ʻana o MFN2 ke hoʻohuli i ka hōʻike ʻana o nā māka metabolic koʻikoʻi a pale i ka degeneration PN. Ma ke ʻano ʻē, pale ia i ka atherosclerosis a hoʻolalelale i nā aʻalolo. transsexual.
ʻO kekahi o nā mea hoihoi loa i kā mākou noiʻi ʻana, ʻo ia ka hiki i nā PN nele i ka OXPHOS ke hoʻololi i ka metabolism cycle TCA ma o ka hoʻonui ʻana i nā enzymes e hoʻoulu pono i ka arteriosclerosis. ʻO ka hoʻonohonoho hou ʻana o ka metabolic kahi hiʻohiʻona maʻamau o nā cell cancer, ʻo kekahi o ia mau mea e hilinaʻi nei i ka glutamine e hoʻohui i nā intermediates cycle TCA e hana i nā mea like e hōʻemi ana, e hoʻokele ana i ke kaulahao hanu a mālama i ka hana ʻana o nā lipid a me nā nucleotide biosynthesis precursors (39, 40). Ua hōʻike ʻia kahi noiʻi hou i loko o nā ʻiʻo peripheral e ʻike nei i ka hana ʻole o OXPHOS, ʻo ka hoʻopili hou ʻana o ka metabolism glutamine/glutamate he hiʻohiʻona koʻikoʻi nō hoʻi (5, 41), kahi e hilinaʻi ai ke kuhikuhi o ke komo ʻana o ka glutamine i loko o ka cycle TCA Ma muli o ke koʻikoʻi o ka hōʻeha OXPHOS (41). ). Eia nō naʻe, ʻaʻohe hōʻike maopopo e pili ana i kekahi ʻano like o ka neuronal metabolic plasticity i loko o ke kino a me kona pilina hiki i ka pōʻaiapili maʻi. I loko o kahi noiʻi in vitro hou, ua hōʻike ʻia nā neurons cortical mua e hoʻoneʻe i nā loko glutamate no ka neurotransmission, no laila e hoʻolaha ana i ka metabolism oxidative a me ka atherosclerosis ma lalo o nā kūlana stress metabolic (42). He mea kūpono ke hoʻomaopopo ʻana ma lalo o ka pharmacological inhibition o ka TCA cycle enzyme succinate dehydrogenase, ua manaʻo ʻia ka pyruvate carboxylation e mālama i ka synthesis o oxaloacetate i loko o nā neurons granule cerebellar i hoʻoulu ʻia (34). Eia nō naʻe, ʻo ka pilina physiological o kēia mau mīkini i ka ʻiʻo lolo (kahi i manaʻo ʻia ai ka atherosclerosis e kaupalena nui ʻia i nā astrocytes) he mea nui nō ia i ka physiological (43). I kēia hihia, hōʻike kā mākou ʻikepili e hiki ke hoʻololi ʻia nā PN i hōʻino ʻia e OXPHOS i loko o ke kino i ka BCAA degradation a me ka pyruvate carboxylation, ʻo ia nā kumu nui ʻelua o ka hoʻohui ʻana o nā intermediates pool TCA. ʻOiai ua hāpai ʻia ka hāʻawi putative o BCAA catabolism i ka metabolism ikehu neuronal, me ke kuleana o ka glutamate a me GABA no ka neurotransmission (44), ʻaʻohe hōʻike no kēia mau mīkini i vivo. No laila, he mea maʻalahi ke kuhi e hiki i nā PN dysfunctional ke uku koke no ka hoʻopau ʻana i nā intermediates TCA i hoʻokele ʻia e ke kaʻina hana assimilation ma o ka hoʻonui ʻana i ka atherosclerosis. ʻO ka mea nui, pono paha ka hoʻonui ʻana o PCx e mālama i ka hoʻonui ʻia o ke koi no ka waikawa aspartic, kahi i manaʻo ʻia i nā cell proliferating me ka mitochondrial dysfunction (45). Eia nō naʻe, ʻaʻole i hōʻike kā mākou loiloi metabolomics i nā loli koʻikoʻi i ka pae paʻa o ka waikawa aspartic ma Mfn2cKO PNs (Kiʻi S6A), kahi e hōʻike ana i ka hoʻohana metabolic like ʻole o ka waikawa aspartic ma waena o nā cell proliferating a me nā neurons post-mitotic. ʻOiai ʻo ke ʻano kikoʻī o ka PCx upregulation i nā neurons dysfunctional in vivo e mau ana ke ʻano, ua hōʻike mākou he kuleana koʻikoʻi kēia pane mua i ka mālama ʻana i ke kūlana redox o nā neurons, i hōʻike ʻia ma nā hoʻokolohua FLIM ma nā ʻāpana cerebellar. ʻO ka mea nui, ʻo ka pale ʻana i nā PN mai ka hoʻonui ʻana i ka PCx hiki ke alakaʻi i kahi kūlana oxidized a hoʻolalelale i ka make ʻana o nā cell. ʻO ka hoʻoulu ʻana o ka BCAA degradation a me ka carboxylation o pyruvate ʻaʻole ia he mau ala e wehewehe ai i nā ʻiʻo peripheral o ka mitochondrial dysfunction (7). No laila, me he mea lā he hiʻohiʻona koʻikoʻi lākou o nā neurons OXPHOS-deficient, ʻoiai ʻaʻole ʻo ia wale nō ka hiʻohiʻona, he mea nui ia no ka neurodegeneration. .
ʻO ka maʻi Cerebellar kahi ʻano heterogeneous o ka maʻi neurodegenerative e hōʻike pinepine ʻia me ka ataxia a hōʻino pinepine i nā PN (46). He palupalu loa kēia heluna neuron i ka hana mitochondrial no ka mea ua lawa ko lākou degeneration koho i nā ʻiole e hana hou i nā hōʻailona motor he nui e hōʻike ana i ka ataxia spinocerebellar kanaka (16, 47, 48). Wahi a nā hōʻike, ua pili kahi kumu hoʻohālike ʻiole transgenic me kahi gene mutant me ka ataxia spinocerebellar kanaka a he hana mitochondrial (49, 50), e hōʻike ana i ke koʻikoʻi o ke aʻo ʻana i nā hopena o ka hemahema OXPHOS ma PNPH. No laila, kūpono loa ia e hoʻokaʻawale a aʻo pono i kēia heluna neuron kū hoʻokahi. Eia nō naʻe, no ka mea he naʻau nui nā PN i ke kaomi a helu i kahi hapa haʻahaʻa o ka heluna cell cerebellar holoʻokoʻa, no nā haʻawina he nui e pili ana i ka omics, ʻo ka hoʻokaʻawale koho ʻana iā lākou ma ke ʻano he mau cell holoʻokoʻa he ʻano paʻakikī nō ia. ʻOiai ua aneane hiki ʻole ke hoʻokō i ka nele loa o ka haumia o nā ʻano cell ʻē aʻe (ʻoi aku hoʻi nā ʻiʻo makua), ua hoʻohui mākou i kahi ʻanuʻu dissociation kūpono me FACS e loaʻa ai kahi helu lawa o nā neurons ola no ka nānā ʻana o ka proteomics ma lalo, a he kiʻekiʻe loa ka uhi protein (ma kahi o 3000 mau protein) i hoʻohālikelike ʻia me ka ʻikepili o kēia manawa o ka cerebellum holoʻokoʻa (51). Ma ka mālama ʻana i ke ola o nā cell holoʻokoʻa, ʻo ke ʻano a mākou e hāʻawi nei ma ʻaneʻi e ʻae iā mākou ʻaʻole wale e nānā i nā loli i nā ala metabolic i loko o ka mitochondria, akā e nānā pū i nā loli i kona mau hoa cytoplasmic, kahi e hoʻopiha ai i ka hoʻohana ʻana i nā lepili membrane mitochondrial e hoʻonui i ke ʻano cell ʻO ke ʻano hou no ka helu o mitochondria i nā ʻiʻo paʻakikī (52, 53). ʻO ke ʻano a mākou e wehewehe nei ʻaʻole pili wale i ke aʻo ʻana i nā cell Purkinje, akā hiki ke hoʻopili maʻalahi ʻia i kekahi ʻano cell e hoʻoponopono i nā loli metabolic i nā lolo maʻi, me nā hiʻohiʻona ʻē aʻe o ka hana mitochondrial.
ʻO ka mea hope loa, ua ʻike mākou i kahi puka makani therapeutic i loko o kēia kaʻina hana hoʻonohonoho metabolic hiki ke hoʻohuli loa i nā hōʻailona koʻikoʻi o ke kaumaha cellular a pale i ka degeneration neuronal. No laila, ʻo ka hoʻomaopopo ʻana i nā hopena hana o ka rewiring i wehewehe ʻia ma ʻaneʻi e hāʻawi i nā ʻike kumu i nā lāʻau lapaʻau hiki ke mālama i ka ola neuronal i ka wā o ka hana ʻole o ka mitochondrial. Pono ka noiʻi e hiki mai ana e kuhikuhi ana i ka wehewehe ʻana i nā loli i ka metabolism ikehu i nā ʻano cell lolo ʻē aʻe e hōʻike piha i ka hoʻohana ʻana o kēia kumumanaʻo i nā maʻi neurological ʻē aʻe.
Ua wehewehe mua ʻia nā ʻiole MitoPark (31). Ua wehewehe mua ʻia nā ʻiole C57BL/6N me nā genes Mfn2 e pili ana i ka loxP (18) a ua hoʻohui ʻia me nā ʻiole L7-Cre (23). A laila ua hoʻohui ʻia nā hua heterozygous pālua i loaʻa me nā ʻiole homozygous Mfn2loxP/Mfn2loxP e hana i nā knockouts gene Purkinje-specific no Mfn2 (Mfn2loxP/Mfn2loxP; L7-cre). Ma kahi hui o ka male ʻana, ua hoʻolauna ʻia ka allele SorStop-mito-YFP Gt (ROSA26) (stop-mtYFP) ma o nā hoʻohui hou ʻana (20). Ua hana ʻia nā kaʻina hana holoholona āpau e like me nā alakaʻi ʻEulopa, aupuni a me nā ʻoihana a ua ʻae ʻia e LandesamtfürNatur o Umwelt a me Verbraucherschutz, North Rhine-Westphalia, Kelemānia. Ke hahai nei hoʻi ka hana holoholona i ke alakaʻi a ka European Federation of Laboratory Animal Sciences Associations.
Ma hope o ka hoʻomaʻemaʻe ʻana i ka dislocation cervical o ka wahine hāpai, ua hoʻokaʻawale ʻia ka embryo ʻiole (E13). Ua ʻoki ʻia ka cortex i loko o ka Hanks' Balanced Salt Solution (HBSS) i hoʻohui ʻia me 10 mM Hepes a ua hoʻouna ʻia ma ka Dulbecco's Modified Eagle's Medium e loaʻa ana ka papain (20 U/ml) a me ka cysteine ​​​​(1μg/ml). E hoʻomoʻa i ka ʻiʻo i loko o DMEM) a hoʻokaʻawale iā ia ma o ka ʻai enzymatic. Ml) ma 37°C no 20 mau minuke, a laila wili ʻia me ka mīkini i loko o DMEM i hoʻohui ʻia me 10% fetal bovine serum. Ua lūlū ʻia nā cell ma nā uhi aniani i uhi ʻia me ka polylysine ma ka density o 2 × 106 no kēlā me kēia kīʻaha moʻomeheu 6 cm a i ʻole ma ka density o 0.5 × 105 cells/cm2 no ka nānā ʻana i ke kiʻi. Ma hope o 4 mau hola, ua pani ʻia ka medium me ka Neurobasal serum-free medium e loaʻa ana ka 1% B27 supplement a me 0.5 mM GlutaMax. A laila ua mālama ʻia nā neurons ma 37°C a me 5% CO2 i loko o ka hoʻokolohua, a ua hānai ʻia i hoʻokahi manawa i ka pule. I mea e hoʻoulu ai i ka recombination in vitro, ua hoʻohana ʻia ʻo 3μl (24-well culture dish) a i ʻole 0.5μl (24-well plate) o ka vector virus AAV9 e mālama ai i nā neurons i ka lua o ka lā in vitro: AAV9.CMV.PI.eGFP. WPRE.bGH (Addgene, helu papa inoa 105530-AAV9) a me AAV9.CMV.HI.eGFP-Cre.WPRE.SV40 (Addgene, helu papa inoa 105545-AAV9).
Ua hōʻailona ʻia ka DNA hoʻopihapiha ʻo Mouse Mfn1 a me Mfn2 (i loaʻa mai ka plasmid Addgene #23212 a me #23213, kēlā me kēia) me ka moʻo V5 (GKPIPNPLLGLDST) ma ka C-terminus, a ua hoʻohui ʻia me mCherry i loko o ke kiʻi ma o ka moʻo T2A. ʻO Grx1-roGFP2 kahi makana mai Heidelberg TP Dick DFKZ (Deutsches Krebsforschungszentrum). Ma ka hoʻololi ʻana i ka cassette tdTomato me ka hoʻohana ʻana i nā ʻano cloning maʻamau, ua subcloned ʻia ka cassette i loko o ka iwi kuamoʻo pAAV-CAG-FLEX-tdTomato (helu kuhikuhi Addgene 28306) e hana i nā vectors pAAV-CAG-FLEX-mCherry-T2A-MFN2-V5, pAAV-CAG-FLEX-mCherry-T2A-MFN1-V5 a me pAAV-CAG-FLEX-Grx-roGFP2. Ua hoʻohana ʻia kekahi ʻano hana like e hana i ka vector control pAAV-CAG-FLEX-mCherry. I mea e hana ai i ke kūkulu AAV-shPCx, pono kahi vector plasmid AAV (VectorBuilder, pAAV [shRNA] -CMV-mCherry-U6-mPcx- [shRNA#1]), nona ka moʻo DNA e hoʻopili ana i ka shRNA targeting mouse PCx (5′CTTTCGCTCTAAGGTGCTAAACTCGAGTTTAGCACCTTAGAGCGAAAG 3′) Ma lalo o ka mana o ka mea hoʻolaha U6, hoʻohana ʻia ʻo mCherry ma lalo o ka mana o ka mea hoʻolaha CMV. Ua hoʻokō ʻia ka hana ʻana o nā vectors AAV kōkua e like me nā kuhikuhi a ka mea hana (Cell Biolabs). I ka pōkole, e hoʻohana i kahi plasmid hoʻoili e lawe ana iā mCherry-T2A-MFN2-V5 (pAAV-CAG-FLEX-mCherry-T2A-MFN2-V5), mCherry-T2A-MFN1-V5 (pAAV-CAG-FLEX-mCherry) transiently Transfection o 293AAV cells-T2A-MFN1-V5), mCherry (pAAV-CAG-FLEX-mCherry) a i ʻole Grx-roGFP2 (pAAV-CAG-FLEX-Grx-roGFP2) coding gene, a me ke coding AAV1 capsid protein a me ka accessory protein Packaging plasmid plasmid, me ka hoʻohana ʻana i ke ʻano calcium phosphate. Ua loaʻa ka supernatant virus maka ma o nā pōʻaiapuni freeze-thaw i loko o kahi ʻauʻau hau maloʻo/ethanol a me nā cell lysed i loko o ka phosphate buffered saline (PBS). Ua hoʻomaʻemaʻe ʻia ka vector AAV e ka iodixanol gradient ultracentrifugation discontinuous (24 mau hola ma 32,000 rpm a me 4°C) a ua hoʻohuihui ʻia me ka hoʻohana ʻana i kahi kānana centrifugal Amicon ultra-15. ʻO ka titer genome o AAV1-CAG-FLEX-mCherry-T2A-MFN2-V5 [2.9 × 1013 kope genome (GC) / ml], AAV1-CAG-FLEX-mCherry (6.1 × 1012 GC / ml), ʻo AAV1-CAG-FLEX e like me ka mea i wehewehe mua ʻia (54), i ana ʻia e ka real-time quantitative PCR (qPCR) -MFN1-V5 (1.9 × 1013 GC / ml) a me AAV1-CAG-FLEX-Grx-roGFP2 (8.9 × 1012 GC / ml).
Ua ʻoki ʻia nā neurons mua i loko o ka 1x PBS anuanu, pelleted, a laila homogenized i loko o 0.5% Triton X-100 / 0.5% sodium deoxycholate/PBS lysis buffer e loaʻa ana ka phosphatase a me ka protease inhibitor (Roche). Ua hana ʻia ka helu ʻana o ka protein ma ka hoʻohana ʻana i ka bicinchoninic acid assay (Thermo Fisher Scientific). A laila ua hoʻokaʻawale ʻia nā protein e ka SDS-polyacrylamide gel electrophoresis, a laila holoi ʻia ma luna o kahi membrane polyvinylidene fluoride (GE Healthcare). E ālai i nā wahi kikoʻī ʻole a hoʻoulu me ka antibody mua (e ʻike i ka Papa S1 no nā kikoʻī) i loko o ka waiū 5% i loko o TBST (Tris-buffered saline me Tween), nā ʻanuʻu holoi a me ka antibody lua i loko o TBST Incubate. E hoʻoulu me ka antibody mua i ka pō ma +4°C. Ma hope o ka holoi ʻana, e hoʻopili i ka antibody lua no 2 mau hola ma ka mahana o ka lumi. Ma hope iho, ma ka hoʻoulu ʻana i ka blot like me kahi antibody anti-β-actin, ua hōʻoia ʻia ka ukana like. ʻIke ʻia ma ka hoʻololi ʻana i ka chemiluminescence a me ka hoʻonui ʻana i ka chemiluminescence (GE Healthcare).
Ua hoʻopaʻa ʻia nā neurons i lūlū mua ʻia ma nā uhi aniani me 4% paraformaldehyde (PFA)/PBS ma kahi manawa i kuhikuhi ʻia ma ka mahana o ka lumi no 10 mau minuke. Hoʻokomo mua ʻia nā uhi me 0.1% Triton X-100/PBS no 5 mau minuke ma ka mahana o ka lumi, a laila i loko o ka buffer blocking [3% bovine serum albumin (BSA)/PBS]. I ka lua o ka lā, ua holoi ʻia nā uhi me ka buffer blocking a hoʻomoʻa ʻia me ka antibody lua fluorophore-conjugated kūpono no 2 mau hola ma ka mahana o ka lumi; ʻo ka hope loa, ua holoi maikaʻi ʻia nā laʻana ma PBS me 4′,6-diamidino-2-Phenylindole (DAPI) i counterstained ʻia a laila hoʻopaʻa ʻia ma ka slide microscope me Aqua-Poly/Mount.
Ua hoʻomaʻamaʻa ʻia nā ʻiole (kāne a me ka wahine) e ka injection intraperitoneal o ketamine (130 mg/kg) a me xylazine (10 mg/kg) a lawelawe ʻia ma lalo o ka ʻili me ka carprofen analgesic (5 mg/kg), a waiho ʻia i loko o kahi mea hana stereotactic (Kopf) i lako me kahi pad mahana. E hōʻike i ke poʻo a hoʻohana i ka wili niho e hoʻomāmā i ka ʻāpana o ka cortex cerebellar e pili ana i ka iwi mis (mai lambda: huelo 1.8, ʻaoʻao 1, e pili ana i nā lobules IV a me V). E hoʻohana i kahi nila syringe curved e hana pono i kahi lua liʻiliʻi i loko o ke poʻo i ʻole e hoʻopilikia i ka vasculature ma lalo. A laila hoʻokomo mālie ʻia ke aniani capillary i huki ʻia i loko o ka micro-hole (mai -1.3 a i -1 ma ka ʻaoʻao ventral o ka dura mater), a ua hoʻokomo ʻia he 200 a 300 nl AAV i loko o ka micro-injector (Narishige) me nā syringes manual (Narishige) i nā manawa he nui ma ke kaomi haʻahaʻa ma luna o kahi manawa 10 a 20 mau minuke puka makani. Ma hope o ke kāwili ʻana, e kau i ke capillary no 10 mau minuke hou aʻe e ʻae i ka maʻi e pālahalaha loa. Ma hope o ka wehe ʻia ʻana o nā capillaries, ua humuhumu pono ʻia ka ʻili e hōʻemi i ka mumū o ka ʻeha a hiki i ka holoholona ke hoʻōla. Ua mālama ʻia nā holoholona me nā analgesics (caspofen) no kekahi mau lā ma hope o ke ʻoki ʻana, i loko o ia manawa ua nānā pono ʻia ko lākou kūlana kino a laila ua pepehi ʻia lākou i ka manawa i ʻōlelo ʻia. Ua hoʻokō ʻia nā kaʻina hana āpau e like me nā alakaʻi ʻEulopa, aupuni a me nā ʻoihana a ua ʻae ʻia e LandesamtfürNatur o Umwelt a me Verbraucherschutz, North Rhine-Westphalia, Kelemānia.
Ua hoʻomaʻemaʻe ʻia nā holoholona me ketamine (100 mg/kg) a me xylazine (10 mg/kg), a ua hoʻopiha mua ʻia ka puʻuwai me 0.1 M PBS, a laila me 4% PFA ma PBS. Ua ʻoki ʻia ka ʻiʻo a hoʻopaʻa ʻia i loko o 4% PFA/PBS i ka pō ma 4°C. Ua hoʻohana ʻia kahi pahi haʻalulu (Leica Microsystems GmbH, Vienna, Austria) e hoʻomākaukau i nā ʻāpana sagittal (50 μm ka mānoanoa) mai ka lolo paʻa ma PBS. Inā ʻaʻole i kuhikuhi ʻia, ua hana ʻia ke kala ʻana o nā ʻāpana lana manuahi e like me ka mea i wehewehe ʻia ma luna (13) ma ka mahana o ka lumi a me ka hoʻoulu ʻana. I ka pōkole, ʻo ka mea mua, ua permeabilized nā ʻāpana i loaʻa me 0.5% Triton X-100/PBS no 15 mau minuke ma ka mahana o ka lumi; no kekahi mau epitopes (Pcx a me Shmt2), ma o ka tris-EDTA buffer ma 80°C (PH 9) e hoʻomehana i nā ʻāpana no 25 mau minuke ma kahi o kēia ʻanuʻu. ʻO ka mea aʻe, ua hoʻomoʻa ʻia nā ʻāpana me ka antibody mua (e nānā i ka Papa S1) i loko o ka buffer blocking (3% BSA/PBS) ma 4°C i ka pō me ka hoʻoulu ʻana. I kekahi lā aʻe, ua holoi ʻia nā ʻāpana me ka buffer blocking a ua hoʻomoʻa ʻia me ka antibody lua fluorophore-conjugated kūpono no 2 mau hola ma ka mahana o ka lumi; ʻo ka mea hope loa, ua holoi maikaʻi ʻia nā ʻāpana i loko o PBS, i hoʻoluʻu ʻia me DAPI, a laila hoʻopaʻa ʻia me AquaPolymount Ma kahi slide microscope.
Ua hoʻohana ʻia kahi microscope confocal scanning laser (TCS SP8-X a i ʻole TCS Digital Light Sheet, Leica Microsystems) i lako me kahi laser kukui keʻokeʻo a me kahi laser ultraviolet 405 diode e kiʻi i ka laʻana. Ma ka hoʻonāukiuki ʻana i ka fluorophore a me ka hōʻiliʻili ʻana i ka hōʻailona me Hybrid Detector (HyDs), ua hoʻohana ʻia ka polokalamu LAS-X e hōʻiliʻili i nā kiʻi i hoʻopaʻa ʻia e kūlike ana me ka Nyquist sampling ma ke ʻano sequential: no nā panela non-quantitative, he mau hōʻailona dynamic loa ia (no ka laʻana, ma nā cell somatic a me nā dendrites) mtYFP) E hoʻohana iā HyD e ʻike i ka helu o nā PN ma ke ʻano BrightR). Hoʻopili ʻia ka Gating mai 0.3 a 6 ns e hōʻemi i ke kua.
Kiʻi manawa maoli o nā pūnaewele i hoʻonohonoho ʻia. Ma hope o ka hoʻokaʻawale ʻana i loko o ka medium Neurobasal-A e loaʻa ana ka 1% B27 supplement a me 0.5 mM GlutaMax, ua lūlū koke ʻia nā pūnaewele ma luna o nā paheʻe aniani i uhi ʻia me ka poly-l-lysine (μ-Slide8 Well, Ibidi, helu papa inoa 80826), A laila e mālama iā ia ma 37°C a me 5% CO2 no 1 hola e ʻae i nā pūnaewele e noho. Ua hana ʻia ke kiʻi manawa maoli ma kahi microscope confocal scanning laser Leica SP8 i lako me kahi laser keʻokeʻo, HyD, 63×[1.4 helu aperture (NA)] lens objective aila a me kahi kahua hoʻomehana.
Ua hoʻomaʻū koke ʻia ka ʻiole me ke kalapona ʻokikene a ua ʻoki ʻia ke poʻo, ua wehe koke ʻia ka lolo mai ka iwi poʻo, a ua ʻoki ʻia i loko o 200μm ka mānoanoa (no ka hoʻokolohua lepili 13C) a i ʻole 275μm ka mānoanoa (no ʻelua mau hoʻokolohua photon) ʻāpana sagittal i hoʻopiha ʻia me nā mea aʻe. Ua hoʻopiha ʻia ka hau kalima (HM-650 V, Thermo Fisher Scientific, Walldorf, Kelemānia) me kēia mau mea: 125 mM hau anuanu, kalapona-saturated (95% O2 a me 5% CO2) haʻahaʻa Ca2 + wai cerebrospinal artificial (ACSF) NaCl, 2.5 mM KCl, 1.25 mM sodium phosphate buffer, 25 mM NaHCO3, 25 mM glucose, 0.5 mM CaCl2 a me 3.5 mM MgCl2 (kaomi osmotic o 310 a 330 mmol). E hoʻoili i nā ʻāpana lolo i loaʻa i kahi keʻena hoʻomoʻa mua e loaʻa ana iā Ca2 + ACSF kiʻekiʻe aʻe (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 a me 2.0 mM MgCl2) Medium) pH 7.4 a me 310 a 320 mmol).
I ke kaʻina hana kiʻi, ua hoʻoneʻe ʻia nā ʻāpana i loko o kahi lumi kiʻi i hoʻolaʻa ʻia, a ua hana ʻia ka hoʻokolohua ma lalo o ka perfusion ACSF hoʻomau ma kahi mahana mau o 32° a 33°C. Ua hoʻohana ʻia kahi microscope scanning laser multiphoton (TCS SP8 MP-OPO, Leica Microsystems) i lako me kahi lens objective Leica 25x (NA 0.95, wai), Ti: Sapphire laser (Chameleon Vision II, Coherent) no ke kiʻi ʻana i nā ʻāpana. Module FLIM (PicoHarp300, PicoQuant).
ʻO FLIM o Grx1-roGFP2. Ua ana ʻia nā loli i ke kūlana redox cytoplasmic o nā PN e ʻelua-photon FLIM i nā ʻāpana lolo sagittal, kahi i kuhikuhi ai ka biosensor Grx1-roGFP2 i nā PN. Ma ka papa PN, ua koho ʻia ke kahua loaʻa ma kahi o 50 a 80 μm ma lalo o ka ʻili ʻāpana e hōʻoia i ka loaʻa ʻana o kahi PN ola (ʻo ia hoʻi, ka nele o ke ʻano beaded a i ʻole nā ​​​​loli morphological neuronal ma nā dendrites) a me ka sensor roGFP2 pālua maikaʻi a me AAV e hoʻopili ana iā shRNA PCx a i ʻole kāna kaʻina hoʻomalu (ʻo kēlā me kēia mCherry e hōʻike pū ana). E hōʻiliʻili i nā kiʻi hoʻokahi-stack me 2x digital zoom [kaha nalu hoʻonāukiuki: 890 nm; 512 nm 512 pixels]. ʻIke: hoʻohana ʻia ka HyD kūloko, ka hui kānana fluorescein isothiocyanate (FITC)] a me ka awelika kiʻi i loko o 2 a 3 mau minuke e hōʻoia i ka hōʻiliʻili ʻia ʻana o nā photons lawa (1000 photons i ka huina) no ke kūpono ʻana o ke kuʻe. Ua hana ʻia ka ʻike o ka probe Grx1-roGFP2 a me ka hōʻoia ʻana o nā kūlana FLIM ma ka nānā ʻana i ka waiwai ola o roGFP2 i ka wā e hoʻohui ai i ka 10 mM H2O2 exogenous i ka perfusion ACSF (e hoʻonui i ka oxidation, e hopena ana i ka hoʻonui ʻia ʻana o ke ola), a laila e hoʻohui i ka 2 mM dithiothreitol (e hoʻemi i ke kiʻekiʻe o ka hōʻemi ʻana, e hopena ana i ka emi ʻana o ke ola) (Kiʻi S8, D a i G). E hoʻohana i ka polokalamu FLIMfit 5.1.1 e kālailai i nā hopena i loaʻa, e hoʻokomo i ka piʻo decay exponential hoʻokahi o ke kiʻi holoʻokoʻa i ka IRF i ana ʻia (hana pane mea hana), a ʻo χ2 ma kahi o 1. No ka helu ʻana i ke ola o kahi PN hoʻokahi, ua kaha lima ʻia ka mask a puni ke kino nerve, a ua hoʻohana ʻia ke ola awelika i kēlā me kēia mask no ka helu ʻana.
Ka loiloi hiki ke mitochondrial. Ma hope o ka hoʻoulu ʻia ʻana o ka ʻāpana ʻoi me 100 nM TMRM i hoʻohui pololei ʻia i ka ACSF perfused no 30 mau minuke, ua ana ʻia nā loli hiki ke mitochondrial o PNs e kahi microscope ʻelua-photon. Ua hana ʻia ke kiʻi ʻana o TMRM ma ka hoʻonāukiuki ʻana i ka probe ma 920 nm a me ka hoʻohana ʻana i ka HyD kūloko (tetramethylrhodamine isothiocyanate: 585/40 nm) e hōʻiliʻili i nā hōʻailona; ma ka hoʻohana ʻana i ka nalu hoʻonāukiuki like akā me ka hoʻohana ʻana i kahi HyD kūloko ʻokoʻa (FITC: 525/50) e kiʻi iā mtYFP. E hoʻohana i ka plug-in Image Calculator o ImageJ e loiloi i ka hiki ke mitochondrial ma ka pae cell hoʻokahi. I ka pōkole, hoʻohana ʻia ka hoʻohālikelike plug-in: signal = min (mtYFP, TMRM) e ʻike i ka ʻāpana mitochondrial e hōʻike ana i ka hōʻailona TMRM ma Purkinje Somali ma ke kiʻi confocal hoʻokahi-stack o ke kahawai pili. A laila ua helu ʻia ka ʻāpana pixel i loko o ka mask hopena, a laila ua hoʻonohonoho ʻia ma ke kiʻi paepae hoʻokahi-stack o ke kahawai mtYFP e kiʻi i ka ʻāpana mitochondrial e hōʻike ana i ka hiki mitochondrial.
Ua wehe ʻia ke kiʻi me ka polokalamu Huygens Pro (Scientific Volume Imaging). No nā kiʻi i scan ʻia o nā tile, hana ʻia ka montage o hoʻokahi tile me ka hoʻohana ʻana i ka algorithm humuhumu aunoa i hāʻawi ʻia e ka polokalamu LAS-X. Ma hope o ka hoʻoponopono ʻana i ke kiʻi, e hoʻohana iā ImageJ a me Adobe Photoshop e hana hou aku i ke kiʻi a hoʻoponopono like i ka ʻōlinolino a me ke ʻano ʻokoʻa. E hoʻohana iā Adobe Illustrator no ka hoʻomākaukau kiʻi.
ka loiloi kiko mtDNA. Ua helu ʻia ka helu o nā ʻeha mtDNA ma nā ʻāpana cerebellar i hōʻailona ʻia me nā antibodies e kūʻē i ka DNA e ka microscope confocal. Ua hana ʻia kēlā me kēia wahi i manaʻo ʻia no ke kino o ke kelepona a me ka nucleus o kēlā me kēia kelepona, a ua helu ʻia ka wahi pili me ka hoʻohana ʻana i ka Multi Measure plug-in (polokalamu ImageJ). E unuhi i ka wahi nucleus mai ka wahi o ke kino o ke kelepona e loaʻa ai ka wahi cytoplasmic. ʻO ka hope loa, ua hoʻohana ʻia ka Analyze Particles plug-in (polokalamu ImageJ) e helu ponoʻī i nā kiko DNA cytoplasmic e hōʻike ana i ka mtDNA ma ke kiʻi paepae, a ua hoʻohālikelike ʻia nā hopena i loaʻa i ka awelika PN o nā ʻiole CTRL. Hōʻike ʻia nā hopena ma ke ʻano he helu awelika o nā nucleosides no kēlā me kēia kelepona.
Ka loiloi ʻana i ka hōʻike ʻana o ka protein. E hoʻohana i ka plug-in Image Calculator a ImageJ e loiloi i ka hōʻike ʻana o ka protein ma PN ma ka pae cell hoʻokahi. I ka pōkole, ma ke kiʻi confocal papa hoʻokahi o ke kahawai pili, ma o ka hoʻohālikelike: hōʻailona = min (mtYFP, antibody), ʻike ʻia ka ʻāpana mitochondrial e hōʻike ana i ka immunoreactivity i kahi antibody ma Purkina. A laila ua helu ʻia ka ʻāpana pixel i loko o ka mask hopena, a laila hoʻonohonoho ʻia ma ke kiʻi paepae hoʻokahi o ke kahawai mtYFP e loaʻa ai ka hapa mitochondrial o ka protein i hōʻike ʻia.
Ka loiloi ʻana o ka nui o nā pūnaewele Purkinje. Ua hoʻohana ʻia ka plug-in Cell Counter o ImageJ e loiloi i ka nui o Purkinje ma ka puʻunaue ʻana i ka helu o nā pūnaewele Purkinje i helu ʻia e ka lōʻihi o ke apo cerebellar i noho ʻia e nā pūnaewele i helu ʻia.
Hoʻomākaukau a me ka hōʻiliʻili ʻana i nā laʻana. Ua hoʻopaʻa ʻia nā lolo mai ka hui kaohi a me nā ʻiole Mfn2cKO i loko o 2% PFA/2.5% glutaraldehyde i loko o 0.1 M phosphate buffer (PB), a laila ua hoʻomākaukau ʻia nā ʻāpana coronal me ka hoʻohana ʻana i nā ciliates (Leica Mikrosysteme GmbH, Vienna, Austria) (Mānoanoa 50 a 60 μm) A laila hoʻopaʻa i loko o ka PB buffer i loko o 1% os tetraoxide a me 1.5% potassium ferrocyanide ma ka mahana o ka lumi no 1 hola. Ua holoi ʻia nā ʻāpana i ʻekolu manawa me ka wai i hoʻoheheʻe ʻia, a laila ua hoʻoluʻu ʻia me 70% ethanol e loaʻa ana he 1% uranyl acetate no 20 mau minuke. A laila ua hoʻomaloʻo ʻia nā ʻāpana i loko o ka waiʻona i hoʻokaʻawale ʻia a hoʻokomo ʻia i loko o Durcupan ACM (Araldite casting resin M) epoxy resin (Electron Microscopy Sciences, helu papa inoa 14040) ma waena o nā paheʻe aniani i uhi ʻia me ka silicon, a ma ka hope loa ma 60°C Polymerize i loko o ka umu no 48 mau hola. Ua koho ʻia ka ʻāpana cortex cerebellar a ua ʻoki ʻia nā ʻāpana ultrathin 50 nm ma Leica Ultracut (Leica Mikrosysteme GmbH, Vienna, Austria) a ua ʻoki ʻia ma kahi grid slit keleawe 2 × 1 mm i uhi ʻia me ka polystyrene film. Ua hoʻoluʻu ʻia nā ʻāpana me kahi hopena o 4% uranyl acetate ma H2O no 10 mau minuke, holoi ʻia me H2O i nā manawa he nui, a laila me Reynolds lead citrate ma H2O no 10 mau minuke, a laila holoi ʻia me H2O i nā manawa he nui. Ua lawe ʻia nā micrographs me kahi microscope electron transmission Philips CM100 (Thermo Fisher Scientific, Waltham, MA, USA) me ka hoʻohana ʻana i kahi kāmela kikohoʻe TVIPS (Tietz Video and Image Processing System) TemCam-F416 (TVIPS GmbH, Gauting, USA). Kelemānia).
No nā ʻiole i loaʻa i ka AAV, ua hoʻokaʻawale ʻia ka lolo a ʻokiʻoki ʻia i loko o kahi ʻāpana sagittal 1 mm ka mānoanoa, a ua nānā ʻia ka cerebellum me ka hoʻohana ʻana i kahi microscope fluorescence e ʻike ai i ke apo i loaʻa i ka AAV (ʻo ia hoʻi, ka hōʻike ʻana o mCherry). ʻO nā hoʻokolohua wale nō i loaʻa ai ka hopena o ka injection AAV i kahi transduction efficiency kiʻekiʻe loa o ka papa cell Purkinje (ʻo ia hoʻi, kokoke i ka papa holoʻokoʻa) ma ka liʻiliʻi he ʻelua mau apo cerebellar i hoʻohui ʻia. Ua microdissected ʻia ka loop AAV-transduced no ka hoʻopaʻa ʻana i ka pō (4% PFA a me 2.5% glutaraldehyde i loko o 0.1 M cocoate buffer) a hana hou ʻia. No ka hoʻokomo ʻana o EPON, ua holoi ʻia ka ʻiʻo paʻa me 0.1 M sodium cocoate buffer (Applichem), a ua hoʻomoʻa ʻia me 2% OsO4 (os, Science Services; Caco) i loko o 0.1 M sodium cocoate buffer (Applichem) 4 hola, a laila holoi no 2 hola. E hana hou i 3 mau manawa me 0.1 M cocamide buffer. Ma hope iho, ua hoʻohana ʻia ke ʻano piʻi o ka ethanol e hoʻomoʻa i kēlā me kēia hopena ethanol ma 4°C no 15 mau minuke e hoʻomaloʻo i ke kino. Ua hoʻoili ʻia ke kino i ka propylene oxide a hoʻomoʻa ʻia i ka pō ma EPON (Sigma-Aldrich) ma 4°C. E kau i ke kino i loko o ka EPON hou ma ka mahana o ka lumi no 2 mau hola, a laila e hoʻokomo iā ia ma 62°C no 72 mau hola. E hoʻohana i kahi ultramicrotome (Leica Microsystems, UC6) a me kahi pahi daimana (Diatome, Biel, Switzerland) e ʻoki i nā ʻāpana lahilahi 70 nm, a hoʻoluʻu me 1.5% uranyl acetate no 15 mau minuke ma 37°C, a hoʻoluʻu me ka hopena citrate kēpau 4 mau minuke. Ua lawe ʻia nā micrographs electron me ka hoʻohana ʻana i kahi microscope electron transmission JEM-2100 Plus (JEOL) i lako me Camera OneView 4K 16-bit (Gatan) a me ka polokalamu DigitalMicrograph (Gatan). No ka nānā ʻana, ua kiʻi ʻia nā micrographs electron me ka zoom kikohoʻe 5000× a i ʻole 10,000×.
ʻO ka loiloi morphological o ka mitochondria. No nā loiloi āpau, ua hoʻākāka lima ʻia nā contours o kēlā me kēia mitochondria i nā kiʻi kikohoʻe me ka hoʻohana ʻana i ka polokalamu ImageJ. Hoʻopili ʻia nā ʻano morphological like ʻole. Hōʻike ʻia ka nui o ka mitochondrial ma ke ʻano he pakeneka i loaʻa ma ka puʻunaue ʻana i ka ʻāpana mitochondrial holoʻokoʻa o kēlā me kēia cell e ka ʻāpana cytoplasm (ʻāpana cytoplasm = ʻāpana cell-ʻāpana nucleus cell) × 100. Ua helu ʻia ka poepoe o ka mitochondria me ke ʻano [4π∙(ʻāpana/perimeter 2)]. Ua hoʻopili ʻia ke ʻano ista o ka mitochondria a ua māhele ʻia i ʻelua mau māhele ("tubular" a me "blister") e like me ko lākou mau ʻano nui.
Ka helu autophagosome/lysosome a me ka loiloi density. E hoʻohana i ka polokalamu ImageJ e hōʻike lima i nā contours o kēlā me kēia autophagosome/lysosome i ke kiʻi kikohoʻe. Hōʻike ʻia ka ʻāpana autophagosome/lysosome ma ke ʻano he pakeneka i helu ʻia ma ka puʻunaue ʻana i ka huina o ka ʻāpana autophagosome/lysosome o kēlā me kēia cell e ka ʻāpana cytoplasm (ʻāpana cytoplasm=ʻāpana cell-nucleus area) × 100. Ua helu ʻia ka nui o nā autophagosomes/lysosome ma ka puʻunaue ʻana i ka huina o ka helu e ka helu o nā ʻano autophagosome/lysosome i kēlā me kēia cell (ma ke ʻano o ka ʻāpana cytoplasmic) (ʻāpana cytoplasmic = ʻāpana cell-nuclear area).
Ke kāpili ʻana no ka ʻoki ʻoki a me ka hoʻomākaukau ʻana i ka laʻana. No nā hoʻokolohua e pono ai ke kāpili ʻana i ka glucose, e hoʻoili i nā ʻāpana lolo ʻoki i kahi keʻena hoʻomoʻa mua, kahi i loaʻa ai ke kalapona saturated (95% O2 a me 5% CO2), Ca2 + ACSF kiʻekiʻe (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 a me 2.0 mM MgCl2, i hoʻoponopono ʻia i ka pH 7.4 a me 310 a 320 mOsm), kahi glucose he 13C 6- Glucose substitution (Eurisotop, helu papa inoa CLM-1396). No nā hoʻokolohua e pono ai ka lepili pyruvate, e hoʻoili i nā ʻāpana lolo ʻoi i ka Ca2 + ACSF kiʻekiʻe (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 a hoʻohui i 2.0mM MgCl2, hoʻoponopono i ka pH 7.4 a me 310 a i 320mOsm), a hoʻohui i ka 1mM 1-[1-13C]pyruvate (Eurisotop, helu papa inoa CLM-1082). E hoʻomoʻa i nā ʻāpana no 90 mau minuke ma 37°C. I ka hopena o ka hoʻokolohua, ua holoi koke ʻia nā ʻāpana me kahi hopena wai (pH 7.4) nona ka 75 mM ammonium carbonate, a laila homogenized i 40:40:20 (v:v:v) acetonitrile (ACN): methanol: wai. Ma hope o ka hoʻomoʻa ʻia ʻana o nā ʻāpana ma luna o ka hau no 30 mau minuke, ua centrifuged ʻia nā laʻana ma 21,000 g no 10 mau minuke ma 4°C, a ua hoʻomaloʻo ʻia ka supernatant maopopo i loko o kahi SpeedVac concentrator. Ua mālama ʻia ka pellet metabolite maloʻo i loaʻa ma -80°C a hiki i ka nānā ʻana.
ʻO ka loiloi ʻana o ka chromatography wai-spectrometry mass o 13 mau waikawa amino i kapa ʻia ʻo C. No ka loiloi ʻana o ka chromatography wai-spectrometry mass (LC-MS), ua hoʻoulu hou ʻia ka pellet metabolite i loko o 75μl o ka wai LC-MS (Honeywell). Ma hope o ka centrifugation ma 21,000 g no 5 mau minuke ma 4°C, ua hoʻohana ʻia he 20 μl o ka supernatant i hoʻomālamalama ʻia no ka loiloi flux waikawa amino, ʻoiai ua hoʻohana koke ʻia ke koena o ka extract no ka loiloi anion (e nānā ma lalo). Ua hana ʻia ka loiloi waikawa Amino me ka hoʻohana ʻana i ke kaʻina hana derivatization benzoyl chloride i wehewehe mua ʻia (55, 56). I ka hana mua, ua hoʻohui ʻia he 10μl o 100 mM sodium carbonate (Sigma-Aldrich) i 20μl o ka metabolite extract, a laila ua hoʻohui ʻia he 10μl o 2% benzoyl chloride (Sigma-Aldrich) i ka LC grade ACN. Ua vortexed pōkole ʻia ka hāpana a laila centrifuged ma 21,000 g no 5 mau minuke ma 20°C. E hoʻoili i ka supernatant i hoʻomaʻemaʻe ʻia i kahi vial autosampler 2 ml me kahi hoʻokomo aniani conical (200 μl volume). Ua kālailai ʻia nā hāpana me ka hoʻohana ʻana i ka ʻōnaehana Acquity iClass ultra-high performance LC (Waters) i hoʻopili ʻia i ka Q-Exactive (QE)-HF (Ultra High Field Orbitrap) high-resolution precision mass spectrometer (Thermo Fisher Scientific). No ka nānā ʻana, ua hoʻokomo ʻia ka 2μl o ka hāpana i hoʻokaʻawale ʻia i loko o kahi kolamu silica T3 ikaika kiʻekiʻe 100 × 1.0 mm (Waters) e loaʻa ana nā ʻāpana 1.8μm. ʻO ka nui o ke kahe ʻana he 100μl/min, a ʻo ka ʻōnaehana buffer he buffer A (10 mM ammonium formate a me 0.15% formic acid i loko o ka wai) a me buffer B (ACN). Penei ke ʻano o ka gradient: 0%B ma 0 mau minuke; 0%B. 0 a i 15% B ma 0 a i 0.1 mau minuke; 15 a i 17% B ma 0.1 a i 0.5 mau minuke; B ma 17 a i 55% ma 0.5 a i 14 mau minuke; B ma 55 a i 70% ma 14 a i 14.5 mau minuke; ma 14.5 a i 70 a i 100% B ma 18 mau minuke; 100% B ma 18 a i 19 mau minuke; 100 a i 0% B ma 19 a i 19.1 mau minuke; 0% B ma 19.1 a i 28 mau minuke (55, 56). Hana ka spectrometer mass QE-HF ma ke ʻano ionization maikaʻi me kahi pae mass o m/z (lakio mass/charge) o 50 a 750. ʻO ka hoʻonā i hoʻopili ʻia he 60,000, a ʻo ka pahuhopu ion gain control (AGC) i loaʻa he 3 × 106, a ʻo ka manawa ion kiʻekiʻe loa he 100 milliseconds. Hana ka puna electrospray ionization (ESI) i hoʻomehana ʻia ma kahi volta spray o 3.5 kV, kahi mahana capillary o 250 ° C, kahi kahe ea sheath o 60 AU (nā ʻāpana arbitrary), a me kahi kahe ea kōkua o 20 AU. 250 ° C. Ua hoʻonohonoho ʻia ka lens S i 60 AU.
ʻO ka loiloi anion chromatography-MS o nā waikawa organik i kapa ʻia ʻo 13C. Ua kālailai ʻia ke koena metabolite precipitate (55μl) me ka hoʻohana ʻana i kahi ʻōnaehana Dionex ion chromatography (ICS 5000+, Thermo Fisher Scientific) i hoʻopili ʻia i kahi spectrometer mass QE-HF (Thermo Fisher Scientific). I ka pōkole, ua hoʻokomo ʻia he 5μl o ka metabolite extract i loko o kahi kolamu Dionex IonPac AS11-HC i lako me HPLC (2 mm × 250 mm, ka nui o ka ʻāpana 4μm, Thermo Fisher Scientific) ma ke ʻano loop hapa push-in me ka lakio hoʻopiha o 1. ) Kolum kiaʻi Dionex IonPac AG11-HC (2 mm x 50 mm, 4μm, Thermo Fisher Scientific). Mālama ʻia ka mahana o ke kolamu ma 30°C, a ua hoʻonohonoho ʻia ka autosampler i 6°C. E hoʻohana i kahi cartridge potassium hydroxide i hāʻawi ʻia me ka wai deionized e hana i kahi gradient potassium hydroxide ma o ka mea hana eluent. Ka hoʻokaʻawale ʻana o nā metabolites ma kahi kahe o 380μl/min, e hoʻopili ana i ka gradient penei: 0 a 3 mau minuke, 10 mM KOH; 3 a 12 mau minuke, 10 a 50 mM KOH; 12 a 19 mau minuke, 50 a 100 mM KOH; 19 a 21 mau minuke, 100 mM KOH; 21 a 21.5 mau minuke, 100 a 10 mM KOH. Ua hoʻoponopono hou ʻia ke kolamu ma lalo o 10 mM KOH no 8.5 mau minuke.
Hoʻohui ʻia nā metabolites i hoʻoheheʻe ʻia me kahi kahawai hoʻohui isopropanol 150μl/min ma hope o ke kolamu a laila kuhikuhi ʻia i kahi spectrometer mass hoʻonā kiʻekiʻe e hana ana ma ke ʻano ionization maikaʻi ʻole. Ke nānā nei ʻo MS i ka pae mass mai m/z 50 a 750 me ka hoʻonā o 60,000. Hoʻonohonoho ʻia ka AGC i 1 × 106, a mālama ʻia ka manawa ion kiʻekiʻe loa ma 100 ms. Ua hana ʻia ke kumu ESI wela ma kahi volta pīpī o 3.5 kV. ʻO nā hoʻonohonoho ʻē aʻe o ke kumu ion penei: ka mahana capillary 275 ° C; ke kahe kinoea sheath, 60 AU; ke kahe kinoea kōkua, 20 AU ma 300 ° C, a me ka hoʻonohonoho lens S i 60 AU.
ʻIkepili loiloi o nā metabolites i kapa ʻia he 13C. E hoʻohana i ka polokalamu TraceFinder (mana 4.2, Thermo Fisher Scientific) no ka loiloi ʻikepili o ka lakio isotope. Ua hōʻoia ʻia ka ʻike o kēlā me kēia hui e kahi hui kuhikuhi hilinaʻi a ua kālailai kūʻokoʻa ʻia. I mea e hana ai i ka loiloi hoʻonui isotope, ua unuhi ʻia ka ʻāpana o ka chromatogram ion i unuhi ʻia (XIC) o kēlā me kēia isotope 13C (Mn) mai [M + H] +, kahi ʻo n ka helu kalapona o ka hui i manaʻo ʻia, i hoʻohana ʻia e kālailai i nā waikawa amino a i ʻole [MH] + i hoʻohana ʻia e kālailai i nā anions. ʻOi aku ka pololei o ka nuipa o XIC ma mua o ʻelima mau ʻāpana no ka miliona, a ʻo ka pololei o RT he 0.05 mau minuke. Hana ʻia ka loiloi hoʻonui ma ka helu ʻana i ka lakio o kēlā me kēia isotope i ʻike ʻia i ka huina o nā isotopes āpau o ka hui like. Hāʻawi ʻia kēia mau lakio ma ke ʻano he mau waiwai pakeneka no kēlā me kēia isotope, a ua hōʻike ʻia nā hopena ma ke ʻano he molar percent enrichment (MPE), e like me ka mea i wehewehe mua ʻia (42).
Ua hoʻohuihui ʻia ka pellet neuron hau i loko o ka 80% methanol (v/v) anuanu, vortexed, a hoʻomoʻa ʻia ma -20°C no 30 mau minuke. E vortex hou i ka laʻana a hoʻoulu ma +4°C no 30 mau minuke. Ua centrifuged ʻia ka laʻana ma 21,000 g no 5 mau minuke ma 4°C, a laila ua hōʻiliʻili ʻia ka supernatant hopena a hoʻomaloʻo ʻia me ka hoʻohana ʻana i kahi SpeedVac concentrator ma 25°C no ka nānā ʻana ma hope. E like me ka mea i wehewehe ʻia ma luna, ua hana ʻia ka nānā ʻana o LC-MS ma nā waikawa amino o nā cell i hoʻokaʻawale ʻia. Me ka hoʻohana ʻana iā TraceFinder (mana 4.2, Thermo Fisher Scientific), ua hana ʻia ka nānā ʻana i ka ʻikepili me ka hoʻohana ʻana i ka nui monoisotopic o kēlā me kēia hui. Ua hana ʻia ka normalization quantile o ka ʻikepili metabolite me ka hoʻohana ʻana i ka pūʻolo polokalamu preprocessCore (57).
Hoʻomākaukau ʻoki. Ua hoʻomaʻemaʻe koke ʻia ka ʻiole me ke kalapona ʻokikene a ua ʻoki ʻia ke poʻo, ua wehe koke ʻia ka lolo mai ka iwi poʻo, a ua hoʻohana ʻia ka pahi haʻalulu i piha i ka hau (HM-650 V, Thermo Fisher Scientific, Walldorf, Kelemānia) e ʻoki iā ia i loko o 300 a 375 μm mau ʻāpana sagittal. ʻO ke kinoea kalapona anuanu (95% O2 a me 5% CO2) Haʻahaʻa Ca2 + ACSF (125.0 mM NaCl, 2.5 mM KCl, 1.25 mM sodium phosphate buffer, 25.0 mM NaHCO3, 25.0 mM d-glucose, 1.0 mM CaCl2 a me 6.0 mM MgCl2 E hoʻoponopono i ka pH 7.4 a me 310 a 330 mOsm). E hoʻoili i nā ʻāpana lolo i loaʻa i loko o kahi keʻena e loaʻa ana ka Ca2 + ACSF kiʻekiʻe aʻe (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 a me mM 3.5 MgCl2) pH 7.4 a me 310 a 320 mOsm). E mālama i nā ʻāpana no 20 a 30 mau minuke i hiki ai ke hoʻihoʻi ʻia ma mua o ka hoʻopaʻa ʻana.
hoʻopaʻa leo. Ua hoʻohana ʻia kahi kahua microscope i lako me kahi keʻena hoʻopaʻa leo paʻa a me kahi aniani hoʻokomo wai 20x (Scientifica) no nā hoʻopaʻa leo āpau. Ua ʻike ʻia nā cell Purkinje putative e (i) ka nui o ke kino, (ii) kahi anatomical o ka cerebellum, a me (iii) ka hōʻike ʻana o ka fluorescent mtYFP reporter gene. ʻO ka patch pipette me kahi kū'ē tip o 5 a 11 megohms i huki ʻia e kahi capillary aniani borosilicate (GB150-10, 0.86 mm × 1.5 mm × 100 mm, Science Products, Hofheim, Kelemānia) a me kahi pipette horizontal Instruments (P-1000, Sutter), Novato, CA). Ua hana ʻia nā hoʻopaʻa leo āpau e ka ELC-03XS npi patch clamp amplifier (npi electronic GmbH, Tam, Kelemānia), i kāohi ʻia e ka polokalamu Signal (mana 6.0, Cambridge Electronic, Cambridge, UK). Ua hoʻopaʻa ʻia ka hoʻokolohua ma kahi helu laʻana o 12.5 kHz. Kānana ʻia ka hōʻailona me ʻelua mau kānana Bessel pōkole me nā alapine ʻoki o 1.3 a me 10 kHz. Hoʻoponopono ʻia ka capacitance o ka membrane a me ka pipette e ke kaapuni hoʻoponopono me ka hoʻohana ʻana i ka amplifier. Ua hana ʻia nā hoʻokolohua āpau ma lalo o ka mana o kahi kāmela Orca-Flash 4.0 (Hamamatsu, Gerden, Kelemānia), i kāohi ʻia e ka polokalamu Hokawo (mana 2.8, Hamamatsu, Gerden, Kelemānia).
ʻO ka hoʻonohonoho ʻana a me ka nānā ʻana o ke kelepona holoʻokoʻa maʻamau. Ma mua koke o ke hoʻopaʻa ʻana, e hoʻopiha i ka pipette me ka hopena kūloko e loaʻa ana i kēia mau mea: 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) a me 10.0 mM creatinine phosphate i hoʻoponopono ʻia i ka pH 7.25, a ʻo ke kaomi osmotic he 290 mOsm (sucrose). Ma hope koke iho o ka hoʻopili ʻana i ka ikaika o 0 pA e haki ai ka membrane, ua ana ʻia ka hiki ke hoʻomaha o ka membrane. Ana ʻia ke kū'ē komo ma ka hoʻopili ʻana i nā kahe hyperpolarized o -40, -30, -20, a me -10 pA. E ana i ka nui o ka pane voltage a hoʻohana i ke kānāwai o Ohm e helu i ke kū'ē komo. Ua hoʻopaʻa ʻia ka hana kūʻokoʻa i loko o kahi ʻūmi uila no 5 mau minuke, a ua ʻike ʻia a ana ʻia ka sPSC ma Igor Pro (mana 32 7.01, WaveMetrics, Lake Oswego, Oregon, USA) me ka hoʻohana ʻana i kahi palapala ʻike semi-automatic. Ana ʻia ke kiʻikuhi IV a me ke au paʻa ma ke ʻūmi ʻana i ka pila ma nā potentials like ʻole (e hoʻomaka ana mai -110 mV) a me ka hoʻonui ʻana i ka uila i 5 mV mau ʻanuʻu. Ua hoʻāʻo ʻia ka hana ʻana o AP ma ka hoʻopili ʻana i kahi au depolarizing. E hoʻopaʻa i ke kelepona ma -70 mV i ka wā e hoʻopili ana i kahi pulse au depolarizing. E hoʻoponopono i ka nui o ke ʻanuʻu o kēlā me kēia ʻāpana hoʻopaʻa leo ma ke kaʻawale (10 a 60 pA). E helu i ka alapine AP kiʻekiʻe loa ma ka helu lima ʻana i nā spike pulse e hoʻoulu ai i ke alapine AP kiʻekiʻe loa. Hoʻopili ʻia ka paepae AP ma ka hoʻohana ʻana i ka lua o ka derivative o ka pulse depolarization e hoʻoulu mua i hoʻokahi a ʻoi aku paha AP.
Hoʻonohonoho ʻana a me ka nānā ʻana i ka ʻāpana perforated. E hana i ka hoʻopaʻa ʻana i ka ʻāpana perforated me ka hoʻohana ʻana i nā protocols maʻamau. E hoʻohana i kahi pipette ATP- a me GTP-free ʻaʻohe ona mea hoʻohui: 128 mM gluconate K, 10 mM KCl, 10 mM Hepes, 0.1 mM EGTA a me 2 mM MgCl2, a hoʻoponopono i ka pH 7.2 (me ka hoʻohana ʻana iā KOH). Ua kāpae ʻia ʻo ATP a me GTP mai ka hopena intracellular e pale aku i ka permeability i kāohi ʻole ʻia o ka membrane cell. Ua hoʻopiha ʻia ka pipette patch me kahi hopena kūloko i loaʻa ka amphotericin (ma kahi o 200 a 250μg/ml; G4888, Sigma-Aldrich) e loaʻa ai kahi moʻolelo patch punched. Ua hoʻoheheʻe ʻia ʻo Amphotericin i loko o dimethyl sulfoxide (ka nui hope loa: 0.1 a 0.3%; DMSO; D8418, Sigma-Aldrich). ʻAʻohe hopena koʻikoʻi o ka nui o DMSO i hoʻohana ʻia ma nā neurons i aʻo ʻia. I ka wā o ke kaʻina hana punching, ua nānā mau ʻia ke kū'ē ʻana o ke kahawai (Ra), a ua hoʻomaka ʻia ka hoʻokolohua ma hope o ka paʻa ʻana o ka amplitude o Ra a me AP (20-40 mau minuke). Ana ʻia ka hana kūʻokoʻa i loko o kahi clamp voltage a/a i ʻole ke au no 2 a 5 mau minuke. Ua hana ʻia ka loiloi ʻikepili me ka hoʻohana ʻana iā Igor Pro (mana 7.05.2, WaveMetrics, USA), Excel (mana 2010, Microsoft Corporation, Redmond, USA) a me GraphPad Prism (mana 8.1.2, GraphPad Software Inc., La Jolla, CA). ʻAmelika Hui Pū ʻIa). I mea e ʻike ai i nā AP kūʻokoʻa, hoʻohana ʻia ka plug-in NeuroMatic v3.0c a IgorPro. E ʻike pono i nā AP me ka hoʻohana ʻana i kahi paepae i hāʻawi ʻia, kahi i hoʻoponopono ʻia no kēlā me kēia moʻolelo. Me ka hoʻohana ʻana i ka manawa spike, e hoʻoholo i ka alapine spike me ka alapine spike koke loa a me ka awelika o ka alapine spike.
Hoʻokaʻawale ʻana o PN. Ma ka hoʻololi ʻana i ke kaʻina hana i paʻi mua ʻia, ua hoʻomaʻemaʻe ʻia nā PN mai ka cerebellum ʻiole ma kahi pae i kuhikuhi ʻia (58). I ka pōkole, ua ʻoki ʻia ka cerebellum a wili ʻia i loko o ka medium dissociation anu hau [me ka ʻole o HBSS Ca2+ a me Mg2+, i hoʻohui ʻia me 20 mM glucose, penicillin (50 U/ml) a me streptomycin (0.05 mg/ml)], a laila e hoʻoheheʻe i ka medium i loko o ka papain [HBSS, i hoʻohui ʻia me 1-cysteine·HCl (1 mg/ml), papain (16 U/ml) a me deoxyribonuclease I (DNase I; 0.1 mg/ml)] E mālama no 30 mau minuke ma 30°C. E holoi mua i nā ʻiʻo i loko o ka medium HBSS e loaʻa ana ka mucus hua manu (10 mg/ml), BSA (10 mg/ml) a me DNase (0.1 mg/ml) ma ka mahana o ka lumi e pale ai i ka ʻeli ʻana o ka enzymatic, a laila i loko o ka medium HBSS e loaʻa ana ka 20 mM glucose. E wili mālie i loko o ka HBSS, penicillin (50 U/ml), streptomycin (0.05 mg/ml) a me DNase (0.1 mg/ml) e hoʻokuʻu i nā cell hoʻokahi. Ua kānana ʻia ka hopena o ka hoʻokuʻu ʻana o nā cell ma o kahi kānana cell 70μm, a laila ua pelleted ʻia nā cell ma o ka centrifugation (1110 rpm, 5 mau minuke, 4°C) a hoʻokuʻu hou ʻia i loko o ka medium hoʻokaʻawale [HBSS, i hoʻohui ʻia me 20 mM glucose, 20% fetal bovine) Serum, penicillin (50 U/ml) a me streptomycin (0.05 mg/ml)]; e loiloi i ke ola o nā cell me ka propidium iodide a hoʻoponopono i ka nui o nā cell i 1 × 106 a i 2 × 106 cells/ml. Ma mua o ke kahe ʻana o ka cytometry, ua kānana ʻia ka hoʻokuʻu ʻana ma o kahi kānana cell 50 μm.
ʻO ka cytometer kahe. Ua hana ʻia ka hoʻokaʻawale ʻana o nā cell ma 4°C me ka hoʻohana ʻana i ka mīkini FACSAria III (BD Biosciences) a me ka polokalamu FACSDiva (BD Biosciences, mana 8.0.1). Ua hoʻokaʻawale ʻia ka hoʻokuʻu ʻana o nā cell me ka hoʻohana ʻana i kahi nozzle 100 μm ma lalo o ke kaomi o 20 psi ma ka wikiwiki o ~2800 mau hanana/sec. ʻOiai ʻaʻole hiki i nā pae hoʻokaʻawale kuʻuna (ka nui o nā cell, ka hoʻokaʻawale bimodal, a me nā ʻano hoʻopuehu) ke hōʻoia i ka hoʻokaʻawale pololei ʻana o PN mai nā ʻano cell ʻē aʻe, ua hoʻonohonoho ʻia ke ʻano hoʻokaʻawale ma muli o ka hoʻohālikelike pololei ʻana o ka ikaika YFP a me ka autofluorescence ma mitoYFP+ ​​​​a me ka mitoYFP − Mice mana. Hoʻonāukiuki ʻia ʻo YFP ma ka hoʻomālamalama ʻana i ka laʻana me kahi laina laser 488 nm, a ua ʻike ʻia ka hōʻailona me ka hoʻohana ʻana i kahi kānana band pass 530/30 nm. I loko o nā mitoYFP+ ​​​​mice, hoʻohana pū ʻia ka ikaika pili o ka gene reporter Rosa26-mitoYFP e hoʻokaʻawale i nā ʻāpana kino neuronal a me nā axon. Hoʻonāukiuki ʻia ʻo 7-AAD me kahi laser melemele 561 nm a ʻike ʻia me kahi kānana bandpass 675/20 nm e kāpae i nā cell make. I mea e hoʻokaʻawale ai i nā astrocytes i ka manawa like, ua hoʻoluʻu ʻia ka hoʻokuʻu ʻana o ke cell me ACSA-2-APC, a laila ua hoʻomālamalama ʻia ka laʻana me kahi laina laser 640 nm, a ua hoʻohana ʻia kahi kānana bandpass 660/20 nm e ʻike i ka hōʻailona.
Ua hoʻoheheʻe ʻia nā ʻāpana i hōʻiliʻili ʻia ma o ka centrifugation (1110 rpm, 5 mau minuke, 4°C) a mālama ʻia ma -80°C a hiki i ka hoʻohana ʻana. Hoʻokaʻawale ʻia nā ʻiole Mfn2cKO a me kā lākou mau ʻīlio litter i ka lā hoʻokahi e hōʻemi i ka loli o ke kaʻina hana. Ua hana ʻia ka hōʻike ʻikepili FACS a me ka nānā ʻana me ka hoʻohana ʻana i ka polokalamu FlowJo (FlowJo LLC, Ashland, Oregon, USA).
E like me ka mea i ʻōlelo ʻia ma luna (59), hoʻohana ʻia ka PCR manawa maoli e hoʻokaʻawale i ka DNA mai nā neurons i hoʻonohonoho ʻia no ka helu ʻana o ka mtDNA ma hope. Ua hoʻāʻo mua ʻia ka linearity a me ka threshold sensitivity ma ka holo ʻana i ka qPCR ma nā helu like ʻole o nā cell. I ka pōkole, e hōʻiliʻili i ka 300 PN i loko o kahi lysis buffer i haku ʻia me 50 mM tris-HCl (pH 8.5), 1 mM EDTA, 0.5% Tween 20 a me ka proteinase K (200 ng/ml) a hoʻoulu ʻia ma 55°C 120 mau minuke. Ua hoʻoulu hou ʻia nā cell ma 95°C no 10 mau minuke e hōʻoia i ka inactivation piha o ka proteinase K. Ma ka hoʻohana ʻana i kahi probe TaqMan (Thermo Fisher) kikoʻī iā mt-Nd1, ua ana ʻia ka mtDNA e ka semi-quantitative PCR ma ka ʻōnaehana 7900HT Real-Time PCR (Thermo Fisher Scientific). ʻEpekema, helu papa inoa Mm04225274_s1), mt-Nd6 (Thermo Fisher Scientific, helu papa inoa AIVI3E8) a me 18S (Thermo Fisher Scientific, helu papa inoa Hs99999901_s1) mau genes.
Hoʻomākaukau ʻana i ka laʻana Proteome. Ma ka hoʻomehana ʻana i ka hopena ma 95°C no 10 mau minuke a me ka sonicating, i loko o ka lysis buffer [6 M guanidine chloride, 10 mM tris(2-carboxyethyl) phosphine hydrochloride, 10 mM chloroacetamide a me 100 mM tris-Lyse i hoʻopaʻa ʻia nā pellets neuron ma HCl]. Ma Bioruptor (Diagenode) no 10 mau minuke (30 kekona pulse / 30 kekona hoʻomaha). Ua hoʻoheheʻe ʻia ka laʻana 1:10 i 20 mM tris-HCl (pH 8.0), i hui pū ʻia me 300 ng trypsin gold (Promega), a ua hoʻomoʻa ʻia i ka pō ma 37°C e hoʻokō i ka ʻai piha. I ka lua o ka lā, ua centrifuged ʻia ka laʻana ma 20,000 g no 20 mau minuke. Ua hoʻoheheʻe ʻia ka supernatant me 0.1% formic acid, a ua desalt ʻia ka hopena me StageTips i hana ʻia e ia iho. Ua hoʻomaloʻo ʻia ka hāpana i loko o kahi mea hana SpeedVac (Eppendorf concentrator me 5305) ma 45°C, a laila ua hoʻokuʻu ʻia ka peptide i loko o ka 0.1% formic acid. Ua hoʻomākaukau ʻia nā hāpana a pau i ka manawa like e ke kanaka hoʻokahi. I mea e kālailai ai i nā hāpana astrocyte, ua lepili ʻia nā peptides desalted 4 μg me kahi tandem mass tag (TMT10plex, helu papa inoa 90110, Thermo Fisher Scientific) me ka ratio peptide i TMT reagent o 1:20. No ka lepili TMT, ua hoʻokuʻu hou ʻia ka 0.8 mg o ka reagent TMT i loko o 70 μl o ka ACN anhydrous, a ua hoʻohui hou ʻia ka peptide maloʻo i loko o 9 μl o 0.1 M TEAB (triethylammonium bicarbonate), kahi i hoʻohui ʻia ai ka 7 μl o ka reagent TMT i loko o ACN. ʻO ka nui o ka hui ʻana he 43.75%. Ma hope o 60 mau minuke o ka incubation, ua hoʻopau ʻia ka hopena me 2 μl o 5% hydroxylamine. Ua hōʻiliʻili ʻia nā peptides i hōʻailona ʻia, hoʻomaloʻo ʻia, hoʻoheheʻe hou ʻia i loko o 200μl o 0.1% formic acid (FA), i māhele ʻia i ʻelua, a laila hoʻohemo ʻia ka paʻakai me ka hoʻohana ʻana iā StageTips i hana ponoʻī ʻia. Me ka hoʻohana ʻana i ka UltiMate 3000 ultra high performance liquid chromatograph (UltiMate 3000 ultra high performance liquid chromatograph), ua hoʻokaʻawale ʻia kekahi o nā hapalua ʻelua ma kahi kolamu chromatographic Acquity 1mm x 150mm i hoʻopiha ʻia me nā ʻāpana 130Å1.7μm C18 (Waters, catalog No. SKU: 186006935). Thermo Fisher Scientific). E hoʻokaʻawale i nā peptides ma kahi kahe o 30μl/min, e hoʻokaʻawale mai 1% a 50% buffer B no 85 mau minuke me kahi gradient stepwise o 96 mau minuke, mai 50% a 95% buffer B no 3 mau minuke, a laila 8 mau minuke no 95% Buffer B; ʻO ka Buffer A he 5% ACN a me 10 mM ammonium bicarbonate (ABC), a ʻo ka buffer B he 80% ACN a me 10 mM ABC. E hōʻiliʻili i nā ʻāpana i kēlā me kēia 3 mau minuke a hoʻohui iā lākou i ʻelua mau hui (1 + 17, 2 + 18, a pēlā aku) a hoʻomaloʻo iā lākou i loko o kahi centrifuge vacuum.
ʻO ka loiloi LC-MS/MS. No ka spectrometry mass, ua hoʻokaʻawale ʻia nā peptides (helu r119.aq) ma kahi kolamu analytical PicoFrit 25 cm, 75 μm ke anawaena o loko (lens objective hou, helu ʻāpana PF7508250) i lako me 1.9 μm ReproSil-Pur 120 C18-AQ medium (Dr. Maisch, mat), E hoʻohana iā EASY-nLC 1200 (Thermo Fisher Scientific, Kelemānia). Ua mālama ʻia ke kolamu ma 50°C. ʻO nā Buffers A a me B he 0.1% formic acid i loko o ka wai a me 0.1% formic acid i loko o 80% ACN, kēlā me kēia. Ua hoʻokaʻawale ʻia nā peptides mai 6% a i 31% buffer B no 65 mau minuke a mai 31% a i 50% buffer B no 5 mau minuke me ka gradient o 200 nl/min. Ua kālailai ʻia nā peptides i eluted ma kahi spectrometer mass Orbitrap Fusion (Thermo Fisher Scientific). Hana ʻia ke ana ʻana o ka peptide precursor m/z me ka hoʻonā o 120,000 ma ka laulā o 350 a 1500 m/z. Me ka hoʻohana ʻana i ka ikehu collision normalized 27%, ua koho ʻia ka precursor ikaika loa me kahi kūlana hoʻopiʻi o 2 a 6 no ka ʻoki ʻana o ka dissociation trap C (HCD) ikehu kiʻekiʻe. Ua hoʻonohonoho ʻia ka manawa pōʻaiapuni i 1 s. Ua ana ʻia ka waiwai m/z o ka ʻāpana peptide i loko o ka ion trap me ka hoʻohana ʻana i ka pahuhopu AGC liʻiliʻi loa o 5 × 104 a me ka manawa injection kiʻekiʻe loa o 86 ms. Ma hope o ka fragmentation, ua kau ʻia ka precursor ma ka papa inoa hoʻokoe dynamic no 45 s. Ua hoʻokaʻawale ʻia nā peptides i hōʻailona ʻia me ka TMT ma kahi kolamu Acclaim PepMap 50 cm, 75 μm (Thermo Fisher Scientific, helu papa inoa 164942), a ua kālailai ʻia nā spectra migration ma kahi spectrometer mass Orbitrap Lumos Tribrid (Thermo Fisher Scientific) i lako me nā lako ion waveform asymmetric kiʻekiʻe (FAIMS) (Thermo Fisher Scientific) e hana ana ma ʻelua mau voltages compensation o −50 a me −70 V. ʻO MS3 i koho ʻia ma muli o ka precursor synchronization e hoʻohana ʻia no ke ana ʻana o ka hōʻailona ion hōʻike TMT. Ua hana ʻia ka hoʻokaʻawale peptide ma EASY-nLC 1200, me ka hoʻohana ʻana i kahi elution gradient linear 90%, me kahi buffer concentration o 6% a 31%; ʻo buffer A he 0.1% FA, a ʻo buffer B he 0.1% FA a me 80% ACN. Hana ʻia ke kolamu analytical ma 50°C. E hoʻohana iā FreeStyle (mana 1.6, Thermo Fisher Scientific) e hoʻokaʻawale i ka faila kumu e like me ka voltages compensation FAIMS.
ʻIke ʻana i ka protein a me ka helu ʻana. Me ka hoʻohana ʻana i ka ʻenekini huli Andromeda i hoʻohui ʻia, ua kālailai ʻia ka ʻikepili kumu me ka hoʻohana ʻana i ka mana MaxQuant 1.5.2.8 (https://maxquant.org/). Ma waho aʻe o nā moʻo Cre recombinase a me YFP i loaʻa mai Aequorea victoria, ua ʻimi ʻia nā spectra ʻāpana peptide no ka moʻo canonical a me ka moʻo isoform o ka proteome kuhikuhi ʻiole (Proteome ID UP000000589, i hoʻoiho ʻia mai UniProt i Mei 2017). Ua hoʻonohonoho ʻia ka Methionine oxidation a me ka protein N-terminal acetylation ma ke ʻano he mau hoʻololi loli; ua hoʻonohonoho ʻia ka cysteine ​​​​carbamoyl methylation ma ke ʻano he mau hoʻololi paʻa. Ua hoʻonohonoho ʻia nā palena digestion i "specificity" a me "trypsin/P". ʻO ka helu liʻiliʻi o nā peptides a me nā peptides razor i hoʻohana ʻia no ka ʻike ʻana i ka protein he 1; ʻo ka helu liʻiliʻi o nā peptides kū hoʻokahi he 0. Ma lalo o nā kūlana o ka hoʻohālikelike ʻana o ka palapala ʻāina peptide, ʻo ka helu ʻike protein he 0.01. Ua hoʻāla ʻia ke koho "Second Peptide". E hoʻohana i ke koho "match between runs" e hoʻoili i nā ʻike kūleʻa ma waena o nā faila kumu like ʻole. E hoʻohana i ka helu lakio liʻiliʻi loa LFQ 1 no ka helu ʻana me ka ʻole o ka lepili (LFQ) (60). Kānana ʻia ka ikaika LFQ no ka liʻiliʻi o ʻelua mau waiwai kūpono ma ka liʻiliʻi o hoʻokahi hui genotype i kēlā me kēia manawa, a ua extrapolated mai kahi hoʻolaha maʻamau me ka laulā o . 0.3 a neʻe i lalo 1.8. E hoʻohana i ka paepae helu Perseus (https://maxquant.net/perseus/) a me R (https://r-project.org/) e kālailai i nā hopena LFQ. Ua hoʻohana ʻia kahi hoʻāʻo t moderate ʻelua ala mai ka pūʻolo polokalamu limma no ka nānā ʻana i ka hōʻike ʻokoʻa (61). Hana ʻia ka nānā ʻikepili ʻimi me ka hoʻohana ʻana i ka ggplot, FactoMineR, factoextra, GGally a me pheatmap. Ua kālailai ʻia ka ʻikepili proteomics e pili ana iā TMT me ka hoʻohana ʻana i ka mana MaxQuant 1.6.10.43. E ʻimi i ka ʻikepili proteomics maka mai ka waihona ʻikepili proteomics kanaka a UniProt, i hoʻoiho ʻia i Kepakemapa 2018. Hoʻokomo pū ka loiloi i ka isotope pureness correction factor i hāʻawi ʻia e ka mea hana. E hoʻohana i ka limma ma R no ka loiloi ʻokoʻa. Mālama ʻia ka ʻikepili kumu, nā hopena hulina waihona ʻikepili, a me ke kaʻina hana loiloi ʻikepili a me nā hopena i loko o ka hui ProteomeXchange ma o ka waihona hoa PRIDE me ka mea hōʻike hoʻonohonoho ʻikepili PXD019690.
Hoʻonui nā hōʻike hana i ka loiloi. Ua hoʻohana ʻia ka mea hana Ingenuity Pathway Analysis (QIAGEN) e hoʻoholo i ka waiwai o nā huaʻōlelo hōʻike hana o ka ʻikepili i hoʻonohonoho ʻia ma 8 mau pule (Kiʻi 1). I ka pōkole, hoʻohana ʻia ka papa inoa protein quantitative i loaʻa mai ka loiloi ʻikepili LC-MS/MS (tandem mass spectrometry) me nā pae kānana aʻe: Ua koho ʻia ʻo Mus musculus ma ke ʻano he ʻano a me ke kua, a hōʻike ka mahele i ka waiwai P i hoʻoponopono ʻia e Benjamini no ka hoʻonui ʻana 0.05 a i ʻole ka haʻahaʻa i manaʻo ʻia he koʻikoʻi. No kēia kiʻi, ua hōʻike ʻia nā mahele keu ʻelima kiʻekiʻe ma kēlā me kēia hui e pili ana i ka waiwai P i hoʻoponopono ʻia. Ma ka hoʻohana ʻana i ka hoʻāʻo t he nui, me ka hoʻohana ʻana i ka papahana hoʻonui linear ʻelua-paepae o Benjamini, Krieger, a me Yekutieli (Q = 5%), hana ʻia ka loiloi hōʻike protein manawa-papa ma nā moho koʻikoʻi i ʻike ʻia ma kēlā me kēia mahele, a ua loiloi ʻia kēlā me kēia lālani ma ke kaʻawale. ʻAʻohe pono e lawe i kahi SD kūlike.
I mea e hoʻohālikelike ai i nā hopena o kēia noiʻi me nā waihona ʻikepili i paʻi ʻia a hana i kahi kiʻikuhi Venn ma ke Kiʻi 1, ua hoʻohui mākou i ka papa inoa protein quantitative me nā annotations MitoCarta 2.0 (24). E hoʻohana i ka mea hana pūnaewele Draw Venn Diagram (http://bioinformatics.psb.ugent.be/webtools/Venn/) e hana i ke kiʻikuhi.
No ka ʻike kikoʻī e pili ana i nā kaʻina hana helu i hoʻohana ʻia no ka loiloi proteomics, e ʻoluʻolu e nānā i ka ʻāpana pili o nā Mea Hana a me nā Hana. No nā hoʻokolohua ʻē aʻe a pau, hiki ke loaʻa ka ʻike kikoʻī ma ka moʻolelo pili. Inā ʻaʻole i kuhikuhi ʻia, ua hōʻike ʻia nā ʻikepili a pau ma ke ʻano he mean ± SEM, a ua hana ʻia nā loiloi helu a pau me ka hoʻohana ʻana i ka polokalamu GraphPad Prism 8.1.2.
No nā mea kōkua no kēia ʻatikala, e ʻoluʻolu e nānā i http://advances.sciencemag.org/cgi/content/full/6/35/eaba8271/DC1
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©2020 ʻAmelika Hui Pū ʻIa no ka holomua o ka ʻepekema. Ua mālama ʻia nā pono āpau. ʻO AAAS he hoa pili o HINARI, AGORA, OARE, CHORUS, CLOCKSS, CrossRef a me COUNTER. ScienceAdvances ISSN 2375-2548.