Mprempren *Mprempren address: Cologne 50931, Germany, Cologne Excellence Cluster Nhwehwɛmu a Ɛfa Nkwammoaa mu Adwennwen Ho Mmuae wɔ Nyarewa a Ɛfa Onyin Ho (CECAD).
Wobu mitochondrial nyarewa a ɛma ntini mu yare no sɛe sɛ wontumi nsakra efisɛ ntini ahorow no nipadua mu nneɛma a ɛsakra no sua, nanso wontumi nte nkɛntɛnso a mitochondrial a entumi nyɛ adwuma yiye nya wɔ nkwammoaa a ɛma ntini mu nneɛma yɛ adwuma wɔ nipadua no mu no ahofadi so no ase yiye. Ɛha yi, yɛde nkwammoaa pɔtee proteome a ɛwɔ Purkinje ntini ahorow a OXPHOS a enni hɔ nkakrankakra a mitochondrial fusion dynamics a asɛe de ba no ba. Yehui sɛ mitochondrial a entumi nyɛ adwuma yiye no maa nsakrae kɛse baa proteomics mu, na awiei koraa no ɛmaa nipadua mu nneɛma a ɛsakra no nhyehyɛe ahorow a ɛyɛ pɛpɛɛpɛ no yɛɛ adwuma nnidiso nnidiso ansa na nkwammoaa awuwu. Nea yɛnhwɛ kwan no, yehuu sɛnea ɛda adi pefee sɛ pyruvate carboxylase (PCx) ne enzyme afoforo a ɛko tia onyin a ɛka TCA kyinhyia no ntam nneɛma ho no ba. PCx a wosiw ano no maa oxidative stress ne neurodegeneration yɛɛ kɛse, na ɛkyerɛ sɛ atherosclerosis wɔ ahobammɔ tumi wɔ ntini ahorow a OXPHOS nni mu no mu. Mitochondrial fusion a wɔsan de ba ntini a asɛe koraa mu no dan saa nipadua mu nneɛma a ɛsakra no koraa, na ɛnam so siw nkwammoaa a ewuwu ano. Nea yɛahu no kyerɛ akwan a kan no na wonnim a ɛma wotumi gyina mitochondrial a entumi nyɛ adwuma yiye ano na ɛkyerɛ sɛ wobetumi asan ntini mu yare no wɔ yare no awiei mpo.
Dwuma titiriw a mitochondria di wɔ ntini mu ahoɔden a ɛkɔ so yɛ adwuma no mu no, ntini mu sɛnkyerɛnne a ɛtrɛw a ɛbata nnipa mitochondria nyarewa ho no si so dua. Saa nyarewa yi mu dodow no ara fi awosu mu nkwaadɔm mu nsakrae a ɛhwɛ mitochondrial awosu mu nkwaadɔm a wɔda no adi so (1, 2) anaasɛ awosu mu nkwaadɔm a wɔsɛe no a ɛfa mitochondrial dynamics ho, a ɛka mitochondrial DNA (mtDNA) a egyina pintinn wɔ ɔkwan a ɛnteɛ so (3, 4). Adwuma a wɔyɛ wɔ mmoa nhwɛso mu no ada no adi sɛ wɔ mitochondrial a entumi nyɛ adwuma yiye wɔ ntini a atwa ho ahyia mu ho mmuaema mu no, wobetumi ama nipadua mu nneɛma a ɛsakra no akwan a ɛyɛ katee (5-7) ayɛ adwuma, na ɛma wonya nsɛm a ɛho hia ma ntease a emu dɔ wɔ sɛnea nyarewa a ɛyɛ den yi ba no ho. Nea ɛne eyi bɔ abira koraa no, ntease a yɛwɔ wɔ nsakrae a ɛba wɔ nkwammoaa ahorow pɔtee bi mu a ɛnam amemene mu mitochondrial adenosine triphosphate (ATP) a wɔyɛ no huammɔdi kɛse so de ba no ho no yɛ ade titiriw (8), na esi hia a ehia sɛ yehu ayaresa botae ahorow a wobetumi de asiw yare ano anaasɛ wɔasiw ano no so dua. Siw ntini mu yare ano (9). Nsɛm a wonni ne nokwasɛm a ɛyɛ sɛ wobu ntini mu nkwammoaa sɛ ɛwɔ nipadua mu nkwaadɔm a ɛyɛ mmerɛw koraa sɛ wɔde toto nkwammoaa ahorow a ɛwɔ ntini a atwa ho ahyia no ho a ( 10 ). Esiane sɛ saa nkwammoaa yi di dwuma titiriw wɔ nneɛma a ɛma nipadua no yɛ adwuma a wɔde ma ntini ahorow no mu de hyɛ synaptic transmission ho nkuran na wɔyɛ ho biribi wɔ opira ne yare tebea horow ho nti, ɛkame ayɛ sɛ tumi a wotumi sesa nkwammoaa mu nneɛma a ɛsakra no ma ɛne amemene no mu ntini tebea horow a emu yɛ den no hyia no yɛ glial nkwammoaa nkutoo (11-14). Afei nso, amemene mu ntini ahorow a ɛwɔ nkwammoaa mu a ɛsono emu biara no siw nsakrae a ɛba nipadua mu nneɛma mu a ɛba wɔ ntini nketewa akuw pɔtee bi mu ho adesua no kwan kɛse. Ne saa nti, wonnim nea efi nkwammoaa ne nipadua mu nneɛma a ɛsakra no mu ba ankasa wɔ mitochondrial a entumi nyɛ adwuma yiye wɔ ntini ahorow mu no ho.
Sɛnea ɛbɛyɛ a yɛbɛte nea efi mitochondrial a entumi nyɛ adwuma yiye mu ba wɔ nipadua mu nneɛma mu ase no, yɛyii Purkinje ntini (PNs) a ɛwɔ ntini mu yare a ɛsono emu biara a mitochondrial akyi ntini a ɛka bom (Mfn2) a wɔsɛe no de ba no fii mu. Ɛwom sɛ Mfn2 mu nsakrae a ɛba nnipa mu no ne awosu mu ntini mu nkate mu ntini mu yare bi a wonim no sɛ Charcot-Marie-Tooth type 2A ( 15 ) wɔ abusuabɔ de, nanso Mfn2 a wɔsɛe no wɔ tebea bi mu wɔ mprako mu no yɛ ɔkwan a wogye tom yiye sɛ ɛde oxidation Phosphorylation (OXPHOS) a ɛnyɛ adwuma yiye ba. Ntini mu subtypes ahorow (16-19) ne neurodegenerative phenotype a efi mu ba no, ntini mu sɛnkyerɛnne ahorow a ɛkɔ so nkakrankakra, te sɛ kankyee mu ɔhaw ahorow (18, 19) anaa amemene mu ataxia (16) ka ho. Ɛdenam label-free quantitative (LFQ) proteomics, metabolomics, mfoninitwa, ne virological akwan a wɔaka abom a yɛde di dwuma so no, yɛkyerɛ sɛ ntini mu yare a ɛkɔ so no ma pyruvate carboxylase (PCx) ne nneɛma afoforo a ɛka ho wɔ arteriosclerosis of PNs in vivo The expression of enzymes no denneennen. Sɛnea ɛbɛyɛ na yɛahwɛ sɛnea nea wɔahu yi ho hia no, yɛmaa PCx a wɔda no adi wɔ PN ahorow a enni Mfn2 mu no kɔɔ fam pɔtee, na yehui sɛ saa oprehyɛn yi maa oxidative adwennwen yɛɛ kɛse na ɛmaa neurodegeneration yɛɛ kɛse, na ɛnam so daa no adi sɛ azoospermia ma nkwammoaa wu Metabolic adaptability. MFN2 a wɔda no adi denneennen no betumi agye PN a ɛyɛ mmerɛw a ɛsen biara a OXPHOS a enni mu kɛse, mitochondrial DNA a wodi no kɛse, ne mitochondrial ntam a ɛda adi sɛ abubu no koraa, na ɛno si so dua bio sɛ ​​saa ntini mu yare yi betumi asan anya ahoɔden mpo wɔ yare no fã a ɛkɔ akyiri ansa na nkwammoaa awuwu.
Sɛnea ɛbɛyɛ a yebetumi ayɛ mitochondria a ɛwɔ Mfn2 knockout PNs mu no ho mfonini wɔ yɛn adwenem no, yɛde mmoawa bi a ɛma mitochondria a egyina Cre so no de wɔn ani si yellow fluorescent protein (YFP) (mtYFP) (20) Cre nkyerɛkyerɛmu so na yɛhwɛɛ mitochondria no nsɛso wɔ vivo mu. Yehui sɛ Mfn2 awosu mu abɔde a wɔsɛe no wɔ PN ahorow mu no bɛma mitochondrial ntam nkitahodi no mu apaapae nkakrankakra (Mfonini S1A), na wohuu nsakrae a edi kan koraa wɔ adapɛn 3 a wɔadi mu. Nea ɛne eyi bɔ abira no, PN nkwammoaa no fã a ɛsɛe kɛse, sɛnea Calbindin immunostaining a wɔhwere no di ho adanse no, amfi ase kosii sɛ odii adapɛn 12 (Mfonini 1, A ne B). Bere a enhyia a ɛda nsakrae a edi kan a ɛbaa mitochondrial nsɛso mu ne ntini mu owu a wofi ase a wotumi hu ntam no kanyan yɛn ma yɛhwehwɛɛ nsakrae a ɛba nipadua mu nneɛma a mitochondrial a entumi nyɛ adwuma yiye de ba ansa na nkwammoaa awuwu no mu. Yɛyɛɛ fluorescence-activated cell sorting (FACS)-gyina kwan a wɔfa so yi YFP (YFP +)-da PN adi (Mfonini 1C), ne wɔ control mice (Mfn2 + / loxP :: mtYFP loxP- stop-loxP: : L7-cre), wɔ ha a wɔbɛfrɛ no CTRL (Mfonini S1B). Optimization a gating kwan a egyina relative ahoɔden a YFP sɛnkyerɛnne ma yetumi tew YFP + nipadua (YFPhigh) PNs fi non-PNs (YFPneg) (Mfonini S1B) anaasɛ putative fluorescent axon / dendritic asinasin (YFPlow; Mfonini S1D, benkum), si so dua denam confocal microscope (Mfonini S1D, nifa). Sɛnea ɛbɛyɛ a yɛbɛhwɛ sɛ nnipa dodow a wɔakyekyɛ wɔn mu no yɛ nokware no, yɛyɛɛ LFQ proteomics ne afei principal component analysis, na yehui sɛ mpaapaemu a ɛda adi pefee wɔ YFPhigh ne YFPneg nkwammoaa ntam (Mfonini S1C). YFPhigh nkwammoaa daa net enrichment a wonim PNs agyiraehyɛde (kyerɛ sɛ Calb1, Pcp2, Grid2 ne Itpr3) (21, 22), nanso enrichment biara proteins a wɔtaa da adi wɔ neurons anaa nkwammoaa ahorow afoforo (Mfonini 1D) ). Ntotoho a ɛda nhwɛsode ahorow a ɛwɔ YFPhigh nkwammoaa a wɔakyekyɛ mu a wɔaboaboa ano wɔ sɔhwɛ ahorow a wɔde wɔn ho mu no kyerɛe sɛ abusuabɔ nsusuwii> 0.9, a ɛkyerɛ sɛ wotumi san yɛ bio yiye wɔ abɔde a nkwa wom a wɔayɛ no foforo ntam (Mfonini S1E). Sɛ yɛbɛbɔ no mua a, saa nsɛm yi sii yɛn nhyehyɛe a yɛyɛe sɛ yɛbɛtew PN a ebetumi aba no afi nnipa mu denneennen ne nea ɛyɛ pɔtee ho no so dua. Esiane sɛ L7-cre draiver nhyehyɛe a wɔde dii dwuma no ma mosaic recombination wɔ dapɛn a edi kan wɔ awo akyi ( 23 ) nti, yefii ase kum mprako fii CTRL ne tebea mu (Mfn2 loxP / loxP :: mtYFP loxP-stop-loxP :: L7-cre) Boaboa ntini ahorow ano. Sɛ wɔsan ka bom wie a, wɔfrɛ no Mfn2cKO bere a wadi adapɛn 4 no. Sɛ́ awiei no, yɛpaw adapɛn 8 a yɛadi bere a na PN layer no nni dɛm ɛmfa ho sɛ ɛda adi sɛ mitochondrial no mu apaapae (Mfonini 1B ne Mfonini S1A). Sɛ yɛka ne nyinaa bom a, yɛbuu protein 3013 a ne nyinaa yɛ dodow, a emu bɛyɛ 22% gyina MitoCarta 2.0 nkyerɛkyerɛmu a egyina mitochondrial proteome so sɛ mitochondria (Mfonini 1E) (Mfonini 1E) (24). Nsonsonoeɛ a ɛwɔ gene expression nhwehwɛmu a wɔyɛeɛ wɔ dapɛn 8 mu no kyerɛɛ sɛ protein nyinaa mu 10.5% pɛ na ɛwɔ nsakraeɛ kɛseɛ (Mfonini 1F ne Mfonini S1F), a emu protein 195 yɛ down-regulated na 120 proteins yɛ up-regulated (Mfonini 1F). Ɛfata sɛ yɛhyɛ no nsow sɛ “akwan foforo nhwehwɛmu” a wɔyɛe wɔ saa nsɛm a wɔahyehyɛ yi mu no kyerɛ sɛ awosu mu nkwaadɔm a wɔda no adi wɔ ɔkwan soronko so no yɛ akwan pɔtee bi a wɔabara a ɛma nipadua no yɛ adwuma no dea titiriw (Mfonini 1G). Nea ɛyɛ anigye no, ɛwom sɛ akwan a ɛfa OXPHOS ne calcium sɛnkyerɛnne ho a wɔma ɛkɔ fam no si so dua sɛ mitochondrial dysfunction ba induction wɔ fusion-deficient PNs mu de, nanso akuw afoforo a ɛfa amino acid metabolism ho titiriw no yɛ upregulated kɛse, a ɛne metabolism a ɛkɔ so wɔ mitochondrial metabolis mu no hyia Rewiring is consistent. adwumayɛ a ɛnkɔ yiye.
(A) Ananmusifo confocal mfonini ahorow a ɛkyerɛ CTRL ne Mfn2cKO mprako amemene afã horow a ɛkyerɛ sɛ PNs (calbindin, grey) a wɔhwere nkakrankakra; wɔde DAPI yɛɛ nuklea ahorow no counterstained. (B) (A) dodow (ɔkwan biako so nhwehwɛmu a ɛfa nsonsonoe ho, ***P <0.001; n = 4 kosi 6 kurukuruwa a efi mprako abiɛsa mu). (C) Nsɔhwɛ adwumayɛ nhyehyɛe. (D) Ɔhyew map kyekyɛ a ɛkyerɛ agyiraehyɛde ahorow a ɛfa Purkinje pɔtee (atifi) ne nkwammoaa ahorow afoforo (mfinimfini). (E) Venn mfonini a ɛkyerɛ mitochondrial protein dodow a wɔahu wɔ PN a wɔakyekyɛ mu no. (F) Ogya bepɔw so mfonini a ɛkyerɛ protein ahorow a wɔda no adi wɔ ɔkwan soronko so wɔ Mfn2cKO ntini mu wɔ adapɛn 8 mu (nkyerɛase a wɔatwa no bo a ɛsom yɛ 1.3). (G) Adebɔ kwan nhwehwɛmu no kyerɛ akwan anum a ɛho hia sen biara a ɛkɔ soro (kɔkɔɔ) ne nea ɛkɔ fam (blue) wɔ Mfn2cKO PN a wɔakyekyɛ mu sɛ adapɛn 8 no mu. Wɔakyerɛ protein biara a wɔahu no nkyerɛkyerɛmu dodow a wɔkyekyem pɛpɛɛpɛ. Grayscale ɔhyew map: P bo a wɔayɛ mu nsakrae. ns, ɛnyɛ nea ɛho hia.
Proteomics ho nsɛm kyerɛe sɛ protein a wɔda no adi wɔ complexes I, III, ne IV mu no so tew nkakrankakra. Na complex I, III, ne IV nyinaa kura subunits a ɛho hia a wɔde mtDNA ahyɛ mu, bere a complex II a na nuklea nko ara na ɛwɔ mu no, na ennyaa nkɛntɛnso titiriw (Mfonini 2A ne Mfonini S2A). . Nea ɛne proteomics aba no hyia no, immunohistochemistry a wɔyɛe wɔ amemene mu ntini afã horow ho no kyerɛe sɛ MTCO1 (mitochondrial cytochrome C oxidase subunit 1) subunit dodow a ɛwɔ complex IV mu wɔ PN mu no so tew nkakrankakra (Mfonini 2B). MtDNA-encoded subunit Mtatp8 no so tew kɛse (Mfonini S2A), bere a steady-state level a nuklea-encoded ATP synthase subunit no kɔɔ so yɛɛ nea ɛnsakra, a ɛne ATP synthase subassembly F1 complex a wonim sɛ ɛyɛ pintinn no hyia bere a mtDNA nkyerɛkyerɛmu no gyina hɔ pintinn no. Nneɛma a wɔahyehyɛ no yɛ nea ɛkɔ so daa. Twitwa nsɛm mu (7). Nhwehwɛmu a wɔyɛe wɔ mtDNA dodow a ɛwɔ Mfn2cKO PN ahorow a wɔahyehyɛ no mu denam bere ankasa mu polymerase nkɔnsɔnkɔnsɔn adeyɛ (qPCR) so sii so dua sɛ mtDNA mfonini dodow so tew nkakrankakra. Sɛ wɔde toto kuw a wɔhwɛ so no ho a, wɔ adapɛn 8 mu no, mtDNA dodow no bɛyɛ 20% pɛ na wɔkoraa so (Mfonini 2C). Nea ɛne nea efii mu bae yi hyia no, wɔde confocal microscopy staining a ɛwɔ Mfn2cKO PNs mu no dii dwuma de huu DNA, a ɛkyerɛ bere a wɔde di mitochondrial nucleotides (Mfonini 2D). Yehui sɛ candidates binom nkutoo na wɔde wɔn ho hyɛ mitochondrial protein a wɔsɛe no ne adwennwen mmuae mu no kɔɔ soro, a Lonp1, Afg3l2 ne Clpx, ne OXPHOS complex assembly factors ka ho. Wɔanhu nsakrae titiriw biara wɔ protein dodow a ɛka apoptosis ho no mu (Mfonini S2B). Saa ara nso na yehui sɛ mitochondria ne endoplasmic reticulum akwan a ɛka ho wɔ calcium akwantu mu no wɔ nsakrae nketenkete bi (Mfonini S2C). Bio nso, protein ahorow a ɛfa autophagy ho nhwehwɛmu no anhu nsakrae titiriw biara, a ɛne autophagosomes a wotumi hu a wɔde immunohistochemistry ne electron microscopy hui wɔ vivo mu no hyia (Mfonini S3). Nanso, nsakrae a ɛda adi pefee wɔ ultrastructural mitochondrial mu ka OXPHOS a ɛnyɛ adwuma yiye a ɛkɔ so wɔ PN ahorow mu no ho. Wobetumi ahu mitochondrial akuwakuw wɔ nkwammoaa nipadua ne dendritic nnua a ɛwɔ Mfn2cKO PNs a wɔadi adapɛn 5 ne 8 mu, na nsakrae kɛse aba mu ntini nhyehyɛe no mu (Mfonini S4, A ne B). Nea ɛne saa ultrastructural nsakrae yi ne mtDNA a ɛso tew kɛse hyia no, nhwehwɛmu a wɔyɛe wɔ amemene mu amemene mu ntini a ɛyɛ hu a wɔde tetramethylrhodamine methyl ester (TMRM) yɛe no kyerɛe sɛ mitochondrial membrane tumi a ɛwɔ Mfn2cKO PNs mu no so tew kɛse (Mfonini S4C).
(A) Bere kwan nhwehwɛmu a ɛfa OXPHOS complex no nkyerɛkyerɛmu dodow ho. Susuw protein ahorow a P<0.05 wɔ adapɛn 8 mu nkutoo ho (akwan abien ANOVA). Dotted line: Nsakrae biara nni hɔ sɛ wɔde toto CTRL ho a. (B) Benkum so: Nhwɛso a ɛkyerɛ amemene no fã bi a wɔde anti-MTCO1 antibody (scale bar, 20 μm) ahyɛ so. Beae a Purkinje nkwammoaa nipadua te no yɛ kɔkɔɔ akata so. Nifa so: MTCO1 dodow dodow a wɔkyerɛ (ɔkwan biako so nhwehwɛmu a ɛfa nsonsonoe ho; n = nkwammoaa 7 kosi 20 a wɔyɛɛ mu nhwehwɛmu fii mprako abiɛsa mu). (C) qPCR nhwehwɛmu a ɛfa mtDNA mfonini dodow a ɛwɔ PN a wɔahyehyɛ no mu (ɔkwan biako nhwehwɛmu a ɛfa nsonsonoe ho; n = mprako 3 kosi 7). (D) Benkum so: Nhwɛso a ɛkyerɛ amemene mu slice a wɔde anti-DNA antibody (scale bar, 20 μm) ahyɛ so. Beae a Purkinje nkwammoaa nipadua te no yɛ kɔkɔɔ akata so. Nifa so: mtDNA akuru dodow a wɔkyerɛ (ɔkwan biako so nhwehwɛmu a wɔyɛ wɔ nsonsonoe ho; n = nkwammoaa 5 kosi 9 a efi mprako abiɛsa mu). (E) Nhwɛso a ɛkyerɛ amemene no fã a emu yɛ den a ɛkyerɛ mitoYFP + Purkinje nkwammoaa (agyan) wɔ nkwammoaa mũ no nyinaa patch clamp kyerɛwtohɔ mu. (F) IV curve no dodow a wɔkyerɛ. (G) Ananmusifo kyerɛwtohɔ ahorow a ɛfa depolarizing mprempren injection wɔ CTRL ne Mfn2cKO Purkinje nkwammoaa mu. Top trace: Pulse a edi kan a ɛkanyan AP. Bottom trace: AP mpɛn dodow a ɛsen biara. (H) Dodow a wɔde kyerɛ postsynaptic spontaneous inputs (sPSPs). Wɔakyerɛ ananmusifoɔ recording trace ne ne zoom ratio wɔ (I) mu. Ɔkwan biako so nhwehwɛmu a wɔyɛe wɔ nsakrae ho no yɛɛ nkwammoaa 5 kosi 20 a efi mprako abiɛsa mu mu nhwehwɛmu. Wɔda nsɛm adi sɛ mean±SEM; *P <0.05 na ɛwɔ hɔ; **P <0.01 na ɛwɔ hɔ; ***P <0.001 na ɛwɔ hɔ. (J) Ananmusifoɔ nsɛnkyerɛnne a ɛkyerɛ AP a ɛba ara kwa a wɔde perforated patch clamp mode dii dwuma. Top trace: AP mpɛn dodow a ɛsen biara. Bottom trace: zoom a ɛyɛ AP baako. (K) Kyerɛ AP frequency a ɛwɔ ntam ne nea ɛboro so sɛnea (J) kyerɛ. Mann-Whitney sɔhwɛ; Wɔyɛɛ n = 5 nkwammoaa mu nhwehwɛmu fii mprako anan mu. Wɔda nsɛm adi sɛ mean±SEM; ɛnyɛ nea ɛho hia.
Wohuu OXPHOS sɛe a ɛda adi pefee wɔ Mfn2cKO PN a adi adapɛn 8 no mu, na ɛkyerɛ sɛ ntini ahorow no nipadua mu dwumadi yɛ nea ɛnteɛ koraa. Enti, yɛyɛɛ anyinam ahoɔden su a ɛnyɛ adwuma a OXPHOS-enni ntini ahorow no mu nhwehwɛmu wɔ adapɛn 4 kosi 5 ne adapɛn 7 kosi 8 denam nkwammoaa mũ no nyinaa patch clamp recordings a yɛyɛe wɔ acute cerebellar slices mu (Mfonini 2E). Nea wɔnhwɛ kwan no, sɛ wɔkyekyem pɛpɛɛpɛ a, na Mfn2cKO ntini ahorow no tumi a ɛwɔ ahomegye mu ntini mu tumi ne nea wɔde hyɛ mu no te sɛ nea wɔde di dwuma no, ɛwom sɛ na nsonsonoe a ɛyɛ anifere wɔ nkwammoaa no ntam de (Table 1). Saa ara nso na wɔ adapɛn 4 kosi 5 mu no, wɔanhu nsakrae titiriw biara wɔ mprempren ne ahoɔden ntam abusuabɔ (IV curve) mu (Mfonini 2F). Nanso, Mfn2cKO ntini biara a adi adapɛn 7 kosi 8 annya nkwa wɔ IV nhyehyɛe no mu (hyperpolarization step), a ɛkyerɛ sɛ nkate a ɛda adi pefee wɔ hyperpolarization tumi ho wɔ saa bere a etwa to yi mu. Nea ɛne eyi bɔ abira no, wɔ Mfn2cKO ntini mu no, wɔma depolarizing currents a ɛde repetitive action potential (AP) discharges ba no ho kwan yiye, a ɛkyerɛ sɛ wɔn discharge nhyehyɛe nyinaa nyɛ soronko koraa wɔ control neurons a adi adapɛn 8 no ho (Table 1 ne Mfonini 2G). Saa ara nso na mpɛn dodow ne amplitude a spontaneous postsynaptic currents (sPSCs) no yɛ nea wotumi de toto kuw a wɔhwɛ so no de ho, na mpɛn dodow a nsɛm a esisii no kɔɔ soro fi adapɛn 4 koduu adapɛn 5 koduu adapɛn 7 kosii adapɛn 8 a ɛkɔɔ soro saa ara (Mfonini 2, H ne I). Bere a synaptic nyin wɔ PNs ( 25 ). Wonyaa nea ɛte saa ara wɔ PNs a wɔatoto mu akyi. Saa nhyehyɛe yi siw nkwammoaa mu ATP sintɔ ahorow ho akatua a ebetumi aba no ano, sɛnea ebetumi aba wɔ nkwammoaa mũ no nyinaa patch clamp kyerɛwtohɔ mu no. Titiriw no, ahomegye membrane tumi ne spontaneous tuo mpɛn dodow a Mfn2cKO ntini ahorow no annya nkɛntɛnso (Mfonini 2, J ne K). Sɛ yɛbɛbɔ no mua a, nea afi mu aba yi kyerɛ sɛ PN ahorow a ɛda adi pefee sɛ OXPHOS ntumi nyɛ adwuma yiye no betumi agyina mpɛn dodow a ɛkɔ soro no ho nhyehyɛe ahorow ano yiye, na ɛkyerɛ sɛ ɔkwan bi wɔ hɔ a wɔfa so tua ka a ɛma wotumi kura electrophysiological mmuae a ɛkame ayɛ sɛ ɛyɛ nea ɛfata mu.
Wɔda nsɛm adi sɛ mfinimfini ± SEM (ɔkwan biako so nhwehwɛmu a ɛfa nsonsonoe ho, Holm-Sidak ntotoho pii sɔhwɛ; *P<0.05). Wɔde nkahyemde kyerɛ unit nɔma no.
Yɛde sii yɛn ani so sɛ yɛbɛhwehwɛ sɛ ebia ɔfã biara a ɛwɔ proteomics dataset (Mfonini 1G) no mu no ka akwan a ebetumi ako atia OXPHOS a enni hɔ a emu yɛ den no ho, na ɛnam so akyerɛkyerɛ nea enti a PN a ɛka no betumi akura electrophysiology a ɛkame ayɛ sɛ ɛyɛ nea ɛfata mu (Mfonini 2, E kosi K). . Proteomics nhwehwɛmu kyerɛe sɛ enzymes a ɛka ho wɔ branched chain amino acids (BCAA) catabolism mu no kɔɔ soro kɛse (Mfonini 3A ne Mfonini S5A), na ade a etwa to acetyl-CoA (CoA) anaa succinyl CoA betumi ayɛ tricarboxylates a ɛwɔ arteriosclerosis Acid (TCA) kyinhyia mu no ho. Yehui sɛ BCAA transaminase 1 (BCAT1) ne BCAT2 nyinaa mu nneɛma kɔɔ soro. Wɔn catalyze anammɔn a edi kan a BCAA catabolism denam glutamate a wonya fi α-ketoglutarate ( 26 ). Subunits a ɛka bom branched chain keto acid dehydrogenase (BCKD) complex no nyinaa yɛ upregulated (complex no ma decarboxylation a edi hɔ na wontumi nsakra a ɛba BCAA carbon nnompe a efi mu ba no mu ba) (Mfonini 3A ne Mfonini S5A). Nanso, wɔanhu nsakrae biara a ɛda adi pefee wɔ BCAA ankasa mu wɔ PN a wɔahyehyɛ no mu, a ebia efi nkwammoaa a wɔde amino acid a ɛho hia yi gye kɛse anaasɛ nneɛma afoforo (glucose anaa lactic acid) a wɔde di dwuma de ka TCA kyinhyia no ho (Mfonini S5B). PNs a enni OXPHOS nso daa glutamine a ɛporɔw ne transamination dwumadi ahorow a ɛkɔ soro adi wɔ adapɛn 8 a wɔadi mu, a ebetumi ada adi denam mitochondrial enzymes glutaminase (GLS) ne glutamine pyruvate transaminase 2 (GPT2) a ɛkɔ soro no so (Mfonini 3, A ne C). Ɛfata sɛ yɛhyɛ no nsow sɛ GLS no up-regulation no yɛ spliced ​​isoform glutaminase C (GLS-GAC) nkutoo (Mfn2cKO/CTRL nsakrae no bɛyɛ mpɛn 4.5, P = 0.05), na ne up-regulation pɔtee wɔ kokoram ntini mu Betumi aboa mitochondrial bioenergy. (27) no.
(A) Ɔhyew map no kyerɛ nsakrae a ɛba mpɛn pii wɔ protein dodow mu ma ɔkwan a wɔakyerɛ no wɔ adapɛn 8 mu. (B) Nhwɛsoɔ a ɛfa amemene mu slice a wɔde anti-PCx antibody (scale bar, 20 μm) ahyɛ so. Agyan kɔkɔɔ no kyerɛ Purkinje nkwammoaa nipadua no. (C) Bere kwan protein nkyerɛkyerɛmu nhwehwɛmu a wɔkyerɛe sɛ ɛyɛ ɔkannifo a ɛho hia ma atherosclerosis (multiple t-test, *FDR <5%; n = 3-5 mprako). (D) Atifi hɔ: Mfonini a wɔayɛ a ɛkyerɛ akwan horow a wɔfa so hyɛn carbon a wɔakyerɛw so a ɛwɔ [1-13C]pyruvate tracer no mu (kyerɛ sɛ, ɛnam PDH anaa trans-arterial kwan so). Ase: Violin nhyehyɛe no kyerɛ carbon a wɔakyerɛw so biako (M1) a wɔdan no aspartic acid, citric acid ne malic acid ɔha biara mu nkyem 100 bere a wɔde [1-13C]pyruvate ahyɛ amemene mu ntini a ɛyɛ hu no agyirae akyi (paired t-test; ** P <0.01). (E) Bere abakɔsɛm nhwehwɛmu a edi mũ a ɛfa ɔkwan a wɔakyerɛ no ho. Susuw protein ahorow a P<0.05 wɔ adapɛn 8 mu nkutoo ho . Dashed line: nsakraeɛ boɔ biara nni hɔ (akwan mmienu nhwehwɛmu a ɛfa nsonsonoeɛ ho; * P <0.05; *** P <0.001). Wɔda nsɛm adi sɛ mean±SEM.
Wɔ yɛn nhwehwɛmu mu no, BCAA catabolism abɛyɛ akwan titiriw a wɔfa so kɔ soro no mu biako. Saa nokwasɛm yi kyerɛ denneennen sɛ mframa dodow a ɛhyɛn TCA kyinhyia no mu no betumi asesa wɔ PN a OXPHOS nni mu no mu. Ebia eyi begyina hɔ ama ntini mu nkwaadɔm mu nhama a wɔde hyɛ nipadua no mu titiriw bi, a ebetumi anya ntini ahorow no nipadua ne nkwa a wonya so nkɛntɛnso tẽẽ bere a OXPHOS a ɛnyɛ adwuma yiye a emu yɛ den no kɔ so tra hɔ no. Nea ɛne saa adwene yi hyia no, yehui sɛ titiriw anti-atherosclerotic enzyme PCx yɛ up-regulated (Mfn2cKO / CTRL sesa bɛyɛ mpɛn 1.5; Mfonini 3A), a catalyzes dan pyruvate dan oxaloacetate ( 28 ), a wogye di sɛ ɛwɔ amemene ntini mu The expression in is restricted to astrocytes (29, 30). Nea ɛne proteomics aba no hyia no, confocal microscopy kyerɛe sɛ PCx nkyerɛkyerɛmu no kɔɔ soro pɔtee na ɛkɔɔ soro kɛse wɔ PNs a enni OXPHOS mu, bere a na PCx reactivity no anohyeto titiriw wɔ Bergmann glial nkwammoaa a ɛbɛn a ɛwɔ control no mu (Mfonini 3B). Sɛnea ɛbɛyɛ na yɛasɔ PCx upregulation a wɔahu no ahwɛ wɔ dwumadi mu no, yɛde [1-13C]pyruvate tracer saa amemene mu ntini a ɛyɛ hu no yare. Bere a wɔde pyruvate dehydrogenase (PDH) yɛɛ oxidized no, ne isotope label yerae , Nanso wɔde ka TCA kyinhyia ntamgyinafo no ho bere a pyruvate nam ntini mu nneyɛe so yɛ adwuma no (Mfonini 3D). Wɔ yɛn proteomics data no akyi no, yehuu agyiraehyɛde dodow bi a efi saa tracer yi mu wɔ aspartic acid a ɛwɔ Mfn2cKO slices mu, bere a citric acid ne malic acid nso nyaa su a ɛkɔ fam, ɛwom sɛ na ɛnyɛ nea ɛho hia de (Mfonini 3D).
Wɔ dopamine ntini a ɛwɔ MitoPark mprako a mitochondrial a entumi nyɛ adwuma yiye a dopamine ntini a ɛsɛe mitochondrial transcription factor A gene (Tfam) (Mfonini S6B) no pɔtee na ɛde ba no mu no, na PCx nkyerɛkyerɛmu nso kɔɔ soro kɛse (31), a ɛkyerɛ sɛ acetone acid arteriosclerosis Wɔhwɛ yare no a ɛba no so bere a ntini no ntumi nyɛ adwuma yiye no OXPHOS a ɛwɔ nipadua no mu. Ɛfata sɛ yɛhyɛ no nsow sɛ wɔahu sɛ enzymes soronko (32-34) a ebia ɛda adi wɔ ntini ahorow a ebia ɛne ntini mu yare wɔ abusuabɔ mu no kɔ soro kɛse wɔ PN ahorow a enni OXPHOS mu, te sɛ propionyl-CoA carboxylase (PCC-A), Malonyl-CoA dan propionyl-CoA ma ɛbɛyɛ succinyl-CoA ne mitochondrial malic enzyme 3 (ME3), . a ne dwumadie titire ne sɛ ɔbɛsan anya pyruvate afiri malate mu (Mfonini 3, A ne C) (33, 35). Afei nso, yehuu nkɔanim kɛse wɔ Pdk3 enzyme a ɛma phosphorylate na ɛnam so ma PDH yɛ adwuma no mu ( 36 ), bere a wɔanhu nsakrae biara wɔ Pdp1 enzyme a ɛma PDH anaa PDH enzyme complex no ankasa yɛ adwuma no mu ( Mfonini 3A ). Daa, wɔ Mern2cKO PNs mu no, phosphorylation a ɛwɔ α1 subunit α (PDHE1α) subunit a ɛwɔ pyruvate dehydrogenase E1 fã a ɛwɔ PDH complex a ɛwɔ Ser293 (a wonim sɛ esiw enzyme dwumadi ano wɔ PDH) no yɛɛ kɛse ( Mfonini S6C) (Mfonini S6C). Pyruvate nni ntini biara a ɛkɔ mu.
Awiei koraa no, yehui sɛ super kwan a serine ne glycine biosynthesis, mitochondrial folate (1C) kyinhyia a ɛfa ho ne proline biosynthesis (Mfonini 1G ne Mfonini S5C) nyinaa yɛ kɛse up-regulated, sɛnea amanneɛbɔ ahorow kyerɛ no, wɔ activation nhyehyɛe no mu. Ntini a atwa ho ahyia no yɛ adwuma denam mitochondrial a entumi nyɛ adwuma yiye so ( 5-7 ). Confocal nhwehwɛmu a ɛfoa proteomics data yi so kyerɛe sɛ wɔ PN a OXPHOS nni mu no, wɔde mprako a wɔadi adapɛn 8 no amemene mu ntini a wɔakyekyɛm no hyɛɛ serine hydroxymethyltransferase 2 (SHMT2), a ɛyɛ mitochondrial folate kyinhyia no mu enzyme titiriw bi mu. Nkwammoaa a ɛko tia nyarewa a ɛho hia (Mfonini S5D). Wɔ 13 CU-glucose-incubated acute cerebellar slices, metabolic tracing nhwehwɛmu san sii so dua sɛ serine ne proline biosynthesis up-regulation, a ɛkyerɛ sɛ carbon isoforms a ɛkɔ serine ne proline mu no kɔɔ soro (Mfonini S5E). Esiane sɛ nneyɛe a GLS ne GPT2 hyɛ ho nkuran no na ɛma glutamate a efi glutamine mu ba ne transamination a ɛda glutamate ne α-ketoglutarate ntam nti, wɔn upregulation kyerɛ sɛ OXPHOS-deficient neurons wɔ glutamate ahwehwɛde a ɛkɔ soro , Ebia eyi atirimpɔw ne sɛ ɛbɛma proline biosynthesis a ɛkɔ soro no akɔ so (Mfonini S5C). Nea ɛne nsakrae yi bɔ abira no, proteomic nhwehwɛmu a wɔyɛe wɔ cerebellar astrocytes a efi PN-specific Mfn2cKO mprako mu no kyerɛe sɛ saa akwan yi (a antiperoxidases nyinaa ka ho) ansakra kɛse wɔ nkyerɛkyerɛmu mu, ma enti ɛdaa adi sɛ Saa metabolic redirection yi paw PN a asɛe (Fig. S6, D to G).
Sɛ yɛbɛbɔ no mua a, saa nhwehwɛmu ahorow yi daa nhwɛso ahorow a ɛsono kɛse adi wɔ bere mu dwumadie a ɛfa nipadua mu nkwaadɔm akwan pɔtee bi a ɛwɔ PNs mu no ho. Ɛwom sɛ ntini mu mitochondrial dwumadi a ɛnteɛ betumi ama ntini mu ntini a ɛyɛ den ntɛm ne 1C a wɔsan yɛ no foforo (Mfonini 3E ne Mfonini S5C), ne nsakrae a wotumi hyɛ ho nkɔm mpo wɔ I ne IV complexes a wɔda no adi mu de, nanso nsakrae a ɛba serine de novo synthesis mu no yɛ nea ɛdaa adi wɔ akyiri yi nkutoo. OXPHOS a ɛnyɛ adwuma yiye (Mfonini 3E ne Mfonini S5C). Saa nsɛm yi kyerɛkyerɛ adeyɛ a ɛtoatoa so a mitochondrial (1C kyinhyia) ne cytoplasmic (serine biosynthesis) a adwennwen de ba no yɛ wɔn ade wɔ biakoyɛ mu ne ntini mu yare a ɛkɔ soro wɔ TCA kyinhyia no mu de san hyehyɛ ntini mu nneɛma a ɛsakra no.
PN ahorow a OXPHOS nnim a wɔadi adapɛn 8 no betumi akura mpɛn dodow a ɛkanyan dwumadi a ɛkɔ soro mu na wɔasan akɔ nipadua no mu nsakrae kɛse mu de atua mitochondrial a entumi nyɛ adwuma yiye no ho ka. Saa ade a wɔahu yi ma asɛm bi a ɛyɛ anigye sɔre sɛ saa bere yi mpo no, ebia saa nkwammoaa yi nso Benya ayaresa mu dwumadie de akyɛ anaasɛ wɔasiw ntini mu yareɛ ano. Aka akyi. Yɛnam nneɛma abien a yɛde wɔn ho hyɛɛ mu a yɛde wɔn ho hyɛɛ mu so siesiee saa asɛm a ebetumi aba yi. Wɔ ɔkwan a edi kan no mu no, yɛyɛɛ Cre-dependent adeno-associated virus (AAV) vector sɛnea ɛbɛyɛ a wobetumi apaw MFN2 ada no adi wɔ OXPHOS-a enni PNs mu wɔ vivo (Mfonini S7A). Wɔdii AAV a ɛkyerɛw MFN2 ne fluorescent reporter gene mCherry (Mfn2-AAV) no mu adanseɛ wɔ primary neuron cultures mu wɔ vitro, a ɛmaa MFN2 daa adi wɔ Cre-dependent kwan so na ɛgyee mitochondrial morphology, na ɛnam so siw neuromutation wɔ Mfn2cKO neurons mu ( Mfonini S7, B, D ne E). Afei, yɛyɛɛ in vivo nhwehwɛmu ahorow de stereotactically de Mfn2-AAV a adi adapɛn 8 kɔ Mfn2cKO ne mprako a wɔhwɛ so no amemene no mu, na yɛyɛɛ mprako a wɔadi adapɛn 12 mu nhwehwɛmu (Mfonini 4A). Mfn2cKO mprako a wɔsaa wɔn yare no wuwui (Mfonini 1, A ne B) (16). Viral transduction in vivo maa PN a wɔpaw no daa no adi wɔ amemene mu nkuruwankuruwa bi mu (Mfonini S7, G ne H). AAV a wɔde dii dwuma a ɛda mCherry (Ctrl-AAV) nkutoo adi no a wɔde guu mu no annya nkɛntɛnso titiriw biara wɔ sɛnea ntini mu yare no te wɔ Mfn2cKO mmoa mu no so. Nea ɛne eyi bɔ abira no, nhwehwɛmu a wɔyɛe wɔ Mfn2cKOs a wɔde Mfn2-AAV ayɛ no ho no kyerɛe sɛ PN nkwammoaa a ɛwɔ nipadua no mu no nya ahobammɔ kɛse (Mfonini 4, B ne C). Titiriw no, ɛte sɛ nea ɛkame ayɛ sɛ wontumi nhu nsonsonoe a ɛda ntini a ɛwɔ nipadua no mu no mu wɔ mmoa a wɔde di dwuma no ho (Mfonini 4, B ne C, ne Mfonini S7, H ne I). MFN1 a wɔda no adi nanso ɛnyɛ MFN2 no tu mpɔn saa ara wɔ ntini mu owu a wogye mu (Mfonini 4C ne Mfonini S7, C ne F), a ɛkyerɛ sɛ MFN1 a wɔda no adi wɔ awotwaa no akyi no betumi de ahyɛ MFN2 a enni hɔ no mu yiye. Nhwehwɛmu foforo a wɔyɛe wɔ PN gyinabea biako no kyerɛe sɛ Mfn2-AAV gyee mitochondria no ultrastructure kɛse, maa mtDNA dodow yɛɛ nea ɛfata, na ɛdanee anti-angiogenesis agyiraehyɛde PCx a wɔda no adi kɛse no (Mfonini 4, C kosi E ). Mfn2cKO mprako a wogyee wɔn no a wɔde aniwa hwɛɛ wɔn wɔ ahomegye tebea mu no kyerɛe sɛ wɔn gyinabea ne wɔn nipadua mu sɛnkyerɛnne ahorow (kankyee S1 kosi S3) no tu mpɔn. Sɛ yɛde rewie a, saa nhwehwɛmu ahorow yi kyerɛ sɛ MFN2 a wɔsan de ba PN ahorow a OXPHOS nni mu kɛse mu a wɔkyɛ no dɔɔso sɛ ɛbɛdan mtDNA a wɔde di dwuma no na ama ntini mu yare ayɛ kɛse, na ɛnam so asiw axon a ɛsɛe ne ntini mu owu ano wɔ vivo.
(A) Nhyehyɛe a ɛkyerɛ sɔhwɛ nhyehyɛe a wɔde bɛhyɛ AAV a ɛkyerɛw MFN2 mu bere a wɔde metabolic kwan a wɔakyerɛ no ayɛ adwuma no. (B) Ananmusifo confocal mfonini ahorow a adi adapɛn 12 cerebellar slices transduced wɔ adapɛn 8 wɔ Mfn2cKO mprako mu na wɔde anti-Calbindin antibody ahyɛ no agyirae. Nifa so: Axon nhama a ɛyɛ mmerɛw. Axon zoom no nsenia yɛ 450 ne 75 μm. (C) Benkum: Purkinje nkwammoaa dodow a wɔkyerɛ wɔ AAV transduction loop (AAV +) mu (ɔkwan biako so nhwehwɛmu a ɛfa nsonsonoe ho; n = mprako 3). Nifa so: mtDNA adwene nhwehwɛmu wɔ transduced PN mu wɔ dapɛn 12 (unpaired t-test; n = 6 nkwammoaa fi mprako abiɛsa). * P <0.05 na ɛwɔ hɔ; ** P <0.01 na ɛwɔ hɔ. (D) Ananmusifoɔ transmission electron micrographs a ɛkyerɛ PNs a ɛwɔ Mfn2cKO cerebellar afã horow a wɔde mmoawa a wɔde mmoawa a wɔakyerɛ no transduced no. Akataso a ɛyɛ pink no kyerɛ beae a dendrite ahorow no, na ahinanan a ɛyɛ kɔkɔɔ a nsensanee wɔ so no kyerɛ sɛnea wɔde ma wɔ nifa so no; n gyina hɔ ma nuklea no. Nsεmfua a εwכ hכ, 1μm. (E) kyerɛ PCx staining ho nhwɛso wɔ PN a wɔdannan no wɔ adapɛn 12 mu. Scale bar, 20μm. OE, nsɛm a wɔda no adi boro so; FC, mpopaho nsakrae.
Awiei koraa no, yɛhwehwɛɛ hia a ɛho hia sɛ nkwammoaa a peroxidase ma ɛtra ase wɔ PN ahorow a wɔanya OXPHOS a entumi nyɛ adwuma yiye no mu. Yɛyɛɛ mCherry a ɛkyerɛw AAV-shRNA (nwi tiawa RNA) a ɛde n’ani si mmoawa PCx mRNA (AAV-shPCx) so titiriw, na yɛde mmoawa no anaa nea wɔabɔ no afrafra (AAV-scr) guu Mfn2cKO mprako amemene mu. Wɔyɛɛ injection no wɔ dapɛn a ɛto so anan a wɔadi mu (Mfonini 5A) sɛnea ɛbɛyɛ a wobenya PCx knockdown a etu mpɔn wɔ bere a PCx nkyerɛkyerɛmu kɔɔ soro (Mfonini 3C) na na PN nkwammoaa no da so ara yɛ nea enni dɛm (Mfonini 1A). Ɛfata sɛ yɛhyɛ no nsow sɛ PCx a wɔtow gu fam (Mfonini S8A) no ma PN wu no kɔ ntɛmntɛm kɛse, a ɛyɛ ring a ɛwɔ ɔyare no nkutoo (Mfonini 5, B ne C). Sɛnea ɛbɛyɛ a yɛbɛte ɔkwan a metabolic nsunsuanso a PCx up-regulation de ba no ase no, yesuaa redox tebea a PNs wɔ akyi PCx knockdown ne AAV-ntamgyinafo optical biosensor Grx1-roGFP2 daa no adi bere koro mu (Mfonini S8, B to D) de hwɛɛ glutathione The relative change of peptide redox potential (38). Afei, yɛyɛɛ abien-photon fluorescence nkwa nna mfonini microscopy (FLIM) wɔ acute amemene slices 7-adapɛn Mfn2cKO anaasɛ control littermates de huu nsakrae a ebetumi aba wɔ cytoplasmic redox tebea mu bere a yɛadi FLIM tebea horow ho adanse akyi (Mfonini S8, E kosi G). Nhwehwɛmu no kyerɛɛ nkɔanim kɛse wɔ oxidation tebea a ɛwɔ Mfn2cKO PNs biako a enni PCx nkyerɛkyerɛmu mu, a ɛyɛ soronko wɔ control neurons anaa Mfn2cKO PNs a ɛda scrambled shRNA nkutoo adi (Mfonini 5, D ne E). Bere a PCx nkyerɛkyerɛmu no kɔɔ fam-regulated, Mfn2cKO PNs ɔha biara mu nkyem a ɛkyerɛ tebea a oxidized kɛse no kɔɔ soro bɛboro mprɛnsa (Mfonini 5E), a ɛkyerɛ sɛ PCx up-regulation kura redox tumi a ɛwɔ ntini a asɛe no mu.
(A) Nhyehyɛe a ɛkyerɛ sɔhwɛ nhyehyɛe a wɔde bɛhyɛ AAV a ɛkyerɛw shPCx mu bere a wɔde metabolic kwan a wɔakyerɛ no ayɛ adwuma no. (B) Ananmusifo confocal mfonini ahorow a adi adapɛn 8 amemene no afã horow wɔ Mfn2cKO mprako a wɔde anti-calcineurin antibody transduced na wɔahyɛ no agyirae wɔ adapɛn 4 mu. Scale bar, 450μm. (C) Purkinje nkwammoaa dodow a wɔkyerɛ wɔ AAV-transduced loops mu (ɔkwan biako so nhwehwɛmu a ɛfa nsonsonoe ho; n = 3 kosi 4 mprako). Wɔda nsɛm adi sɛ mean±SEM; ***P <0.001 na ɛwɔ hɔ. (D) Ɔnanmusifo FLIM mfonini kyerɛ nkwa nna a PN a wadi adapɛn 7 a ɛda glutathione redox sensor Grx1-roGFP2 adi wɔ sɔhwɛ tebea horow a wɔakyerɛ no mu. LUT (hwɛ pon) ratio: nkwagye bere ntam (wɔ picoseconds mu). Scale bar, 25μm, na ɛyɛ 25μm. (E) Histogram no kyerɛ Grx1-roGFP2 nkwa nna mu gyinapɛn ahorow a wɔkyekyɛ fi (D) (n=158 kosi 368 nkwammoaa wɔ mprako abien mu wɔ tebea biara mu). Pie chart a ɛwɔ histogram biara atifi: kyerɛ nkwammoaa dodow a wɔn nkwa nna tenten (kɔkɔɔ, oxidized) anaa tiawa (blue, wɔatew so) kɛse, a ɛboro 1 SD wɔ nkwa nna bo a wɔkyekyem pɛpɛɛpɛ wɔ CTRL-AAV-scr mu. (F) Nhwɛso a wɔahyɛ ho nyansa no kyerɛ ahobammɔ nkɛntɛnso a ɛwɔ upregulation of neuronal PCx so.
Ne nyinaa mu no, nsɛm a yɛde ma wɔ ha no kyerɛ sɛ MFN2 a wɔsan da no adi no betumi agye PN a ɛkɔ anim a OXPHOS a enni mu kɛse, mtDNA a ɛso tew kɛse, ne ne nsɛso a ɛte sɛ ista a ɛnyɛ ne kwan so koraa no koraa, na ɛnam so ama nkɔso a ɛkɔ so wɔ nyarewa a akɔ anim mpo mu. Ntini a ɛma nipadua no sɛe no de adanse a wotumi dannan ma wɔ ɔkwan a ɛkɔ so ansa na nkwammoaa awuwu no ho. Saa nipadua mu nkwaadɔm a ɛyɛ mmerɛw yi si so dua bio denam tumi a ntini ahorow no tumi ma ntini mu yare (TCA kyinhyia no mu nhama a wɔde hyɛ mu), a esiw PCx a wɔda no adi wɔ PN ahorow a enni OXPHOS mu no ano na ɛma nkwammoaa wuwu yɛ kɛse, na ɛnam so di dwuma a ɛbɔ ho ban (Mfonini 5F).
Wɔ saa nhwehwɛmu yi mu no, yɛde adanse mae sɛ mmuae a PN ahorow de ma wɔ OXPHOS a entumi nyɛ adwuma yiye ho ne sɛ ɛde nkakrankakra bɛkɔ TCA kyinhyia atherosclerosis mu denam differential activation pathway a metabolic nhyehyɛe ahorow ma ɛyɛ adwuma no so. Yɛde akwan pii a ɛka bom sii proteomic nhwehwɛmu no so dua na yɛdaa no adi sɛ sɛ mitochondrial a ɛnyɛ adwuma yiye a emu yɛ den twa ho mpoa a, ntini ahorow no wɔ ɔkwan a kan no na wonnim a ɛfa nipadua mu nneɛma a ɛsakra ho. Ɛyɛ yɛn nwonwa sɛ, ɛnyɛ nea ɛkyerɛ sɛ nhama a wɔde hyɛ nipadua mu no nyinaa hyɛ nipadua mu nneɛma a ɛsakra no tebea a etwa to a ɛka ntini mu yare ho nkakrankakra na wontumi nsakra no agyirae, nanso yɛn nsɛm a yɛanya no kyerɛ sɛ ebia ɛbɛyɛ ntini a ɛhwɛ so yiye wɔ bere a edi nkwammoaa wu anim mpo mu Functional compensation mechanism. Saa ade a wɔahu yi kyerɛ sɛ ntini ahorow no wɔ nipadua no mu nneɛma a ɛsakra no kɛse. Saa nokwasɛm yi di adanse sɛ MFN2 a wɔsan de ba akyiri yi no betumi adan nipadua mu nneɛma atitiriw a ɛma nipadua no yɛ adwuma no da adi na asiw PN a ɛbɛsɛe no ano. Nea ɛne no bɔ abira no, esiw ntini mu yare ano na ɛma ntini ahorow no yɛ ntɛmntɛm. transsexual.
Ade biako a ɛyɛ anigye sen biara a yɛahu wɔ yɛn nhwehwɛmu no mu ne sɛ PN ahorow a enni OXPHOS no betumi asesa TCA kyinhyia no mu nneɛma a ɛsakra denam enzyme ahorow a ɛkanyan ntini mu yare pɔtee a ɛma ɛkɔ soro no so. Metabolic rearrangement yɛ ade a ɛtaa ba kokoram nkwammoaa mu, a ebinom de wɔn ho to glutamine so de ka TCA cycle intermediates ho ma ɛyɛ reducing equivalents, a ɛma ɔhome nkɔnsɔnkɔnsɔn no kɔ so yɛ lipid ne nucleotide biosynthesis precursors (39 , 40). Nnansa yi nhwehwɛmu bi kyerɛe sɛ wɔ ntini a ɛwɔ akyi a ɛrehyia OXPHOS a ɛnyɛ adwuma yiye mu no, glutamine/glutamate metabolism a ɛsan bata ho no nso yɛ ade titiriw ( 5 , 41 ), baabi a akwankyerɛ a glutamine kɔ TCA kyinhyia no mu no gyina Esiane sɛ OXPHOS opira no mu yɛ den nti ( 41 ). ). Nanso, adanse a emu da hɔ nni hɔ a ɛkyerɛ sɛ ntini mu nkwaadɔm a ɛma nipadua no yɛ adwuma no yɛ pɛpɛɛpɛ wɔ nipadua no mu ne sɛnea ebetumi aba sɛ ɛfa ho wɔ yare no ho. Wɔ nnansa yi nhwehwɛmu bi a wɔyɛe wɔ vitro mu no, wɔkyerɛe sɛ cortical neurons titiriw no boaboa glutamate pools ano ma neurotransmission, na ɛnam so ma oxidative metabolism ne atherosclerosis nya nkɔso wɔ metabolic stress tebea horow mu ( 42 ). Ɛfata sɛ yɛhyɛ no nsow sɛ wɔ nnuru a wɔde siw TCA kyinhyia enzyme succinate dehydrogenase ano ase no, wogye di sɛ pyruvate carboxylation kɔ so yɛ oxaloacetate a wɔyɛ wɔ amemene mu ntini a wɔayɛ no amammerɛ mu ( 34 ). Nanso, nipadua mu mfaso a saa akwan yi wɔ wɔ amemene mu ntini ho (baabi a wogye di sɛ ntini mu yare no yɛ astrocytes nkutoo) da so ara wɔ nipadua mu ntease a ɛho hia ( 43 ). Wɔ saa tebea yi mu no, yɛn data kyerɛ sɛ wobetumi adan PN ahorow a OXPHOS asɛe no wɔ nipadua no mu no akɔ BCAA a wɔsɛe no ne pyruvate carboxylation so, a ɛyɛ nneɛma atitiriw abien a ɛde TCA pool intermediates ka ho. Ɛwom sɛ wɔahyɛ mmoa a wosusuw sɛ BCAA catabolism de ma wɔ ntini mu ahoɔden a wɔde yɛ adwuma mu ho nyansa de, nanso wɔ dwuma a glutamate ne GABA di wɔ ntini mu nkwaadɔm mu ( 44 ) akyi no, adanse biara nni hɔ a ɛkyerɛ sɛ saa akwan yi yɛ adwuma wɔ nipadua mu. Enti, ɛnyɛ den sɛ yebesusuw sɛ PN ahorow a entumi nyɛ adwuma yiye no betumi atua TCA ntamgyinafo a assimilation nhyehyɛe no na ɛkanyan no a wɔde di dwuma no ho ka denam ntini a ɛma mogya tu a ɛkɔ soro no so. Titiriw no, ebia ɛho behia sɛ wɔma PCx kɔ soro na ama wɔakɔ so ahwehwɛ aspartic acid a ɛkɔ soro, a wɔhyɛ nyansa wɔ nkwammoaa a ɛredɔɔso a mitochondrial ntumi nyɛ adwuma yiye mu ( 45 ). Nanso, yɛn metabolomics nhwehwɛmu no ankyerɛ nsakrae titiriw biara wɔ aspartic acid dodow a ɛyɛ gyinabea a ɛwɔ Mfn2cKO PNs mu (Mfonini S6A), a wosusuw sɛ ɛkyerɛ sɛnea wɔde aspartic acid di dwuma wɔ nipadua mu nneɛma mu wɔ nkwammoaa a ɛredɔɔso ne ntini a ɛwɔ mitotic akyi no ntam Ɛwom sɛ ɛda so ara yɛ nea wontumi nhu ɔkwan pɔtee a PCx kɔ soro wɔ ntini a entumi nyɛ adwuma mu wɔ vivo mu de, nanso yɛkyerɛe sɛ saa mmuae a ɛba ntɛm yi di dwuma titiriw wɔ ntini ahorow no redox tebea a wɔda no adi mu, a wɔdaa no adi wɔ FLIM nhwehwɛmu ahorow a wɔyɛe wɔ amemene no mu slices so no mu. Titiriw no, sɛ wosiw PN ahorow ano sɛ wɔbɛma PCx akɔ soro a, ebetumi ama wɔanya tebea a ɛyɛ oxidized kɛse na ama nkwammoaa awuwu ntɛmntɛm. BCAA a wɔsɛe no a wɔma ɛyɛ adwuma ne pyruvate carboxylation nyɛ akwan a wɔfa so kyerɛ ntini a ɛwɔ akyi a ɛwɔ mitochondrial a entumi nyɛ adwuma yiye no su ( 7 ). Enti, ɛte sɛ nea wɔyɛ ade titiriw wɔ ntini a OXPHOS nni mu no mu, sɛ ɛnyɛ ɛno nkutoo mpo a, a ɛho hia ma ntini a ɛyɛ mmerɛw. .
Cerebellar yare yɛ ntini mu yare a ɛsono emu biara a ɛtaa da adi sɛ ataxia na ɛtaa sɛe PNs ( 46 ). Saa ntini ahorow yi yɛ mmerɛw titiriw sɛ mitochondrial ntumi nyɛ adwuma yiye efisɛ wɔn a wɔpaw sɛe wɔ mprako mu no dɔɔso sɛ ɛbɛsan awo ntini mu sɛnkyerɛnne ahorow pii a ɛkyerɛ nnipa spinocerebellar ataxia ( 16 , 47 , 48 ). Sɛnea amanneɛbɔ kyerɛ no, mmoawa a wɔadan wɔn awosu mu nkwaadɔm a wɔde awosu mu nkwaadɔm a wɔayɛ no foforo awosu mu nkwaadɔm ayɛ no ne nnipa spinocerebellar ataxia wɔ abusuabɔ na ɛwɔ mitochondrial dysfunction ( 49 , 50 ), na esi hia a ɛho hia sɛ wosua nea efi OXPHOS a enni hɔ wɔ PNPH mu ba no ho ade so dua. Enti, ɛfata titiriw sɛ wɔayi ntini ahorow a ɛyɛ soronko yi afi nnipa mu yiye na wɔasua ho ade. Nanso, esiane sɛ PN ahorow no te nka kɛse wɔ nhyɛso ho na ɛyɛ amemene mu nkwammoaa dodow no nyinaa fã ketewaa bi nti, wɔ nhwehwɛmu pii a egyina omics so mu no, wɔpaw wɔn mu sɛ nkwammoaa mũ no nyinaa da so ara yɛ ade a ɛyɛ den. Ɛwom sɛ ɛkame ayɛ sɛ ɛrentumi nyɛ yiye sɛ yebenya efĩ a enni hɔ koraa wɔ nkwammoaa ahorow afoforo (titiriw mpanyimfo ntini) mu de, nanso yɛde dissociation anammɔn a etu mpɔn ne FACS boom de nyaa ntini ahorow a ɛtumi tra ase dodow a ɛdɔɔso ma downstream proteomics analysis, na yɛwɔ protein coverage a ɛkorɔn yiye (bɛyɛ proteins 3000) sɛ wɔde toto data set a ɛwɔ hɔ dedaw a ɛfa cerebellum no nyinaa ho (51). Ɛdenam nkwammoaa mũ no nyinaa a yɛbɛkora so no so no, ɛnyɛ sɛ ɔkwan a yɛde ma wɔ ha no ma yɛn kwan ma yɛhwɛ nsakrae a ɛba wɔ nipadua mu nneɛma a ɛsakra no mu wɔ mitochondria no mu nko, na mmom yɛhwɛ nsakrae a ɛba ne mfɛfo cytoplasmic mu nso, a ɛboa mitochondrial membrane tags a wɔde di dwuma de ma nkwammoaa su yɛ kɛse Ɔkwan foforo a wɔfa so yɛ mitochondria dodow wɔ ntini a ɛyɛ den mu (52, 53). Ɔkwan a yɛka ho asɛm no nyɛ Purkinje nkwammoaa ho adesua nko na ɛfa ho, na mmom ɛnyɛ den sɛ wɔde bedi dwuma wɔ nkwammoaa biara mu de adi nsakrae a ɛba nipadua mu nneɛma mu wɔ amemene a ɛyare mu ho dwuma, a mitochondrial dwumadi a entumi nyɛ adwuma yiye ho nhwɛso afoforo ka ho.
Awiei koraa no, yɛahu ayaresa mfɛnsere bi wɔ saa nipadua mu nneɛma a wɔsan hyehyɛ mu yi mu a ebetumi adan nkwammoaa mu nhyɛso ho sɛnkyerɛnne atitiriw no koraa na asiw ntini ahorow a ɛsɛe no ano. Enti, sɛ yɛte dwumadi mu nkyerɛkyerɛmu a ɛwɔ nhama a wɔde hyɛ mu a wɔaka ho asɛm wɔ ha no ase a, ebetumi ama yɛanya nhumu atitiriw wɔ ayaresa ahorow a ebetumi aba a wɔde bɛkɔ so akura ntini ahorow no mu den bere a mitochondrial no ntumi nyɛ adwuma yiye no ho. Nhwehwɛmu a wɔbɛyɛ daakye a wɔde asi wɔn ani so sɛ wɔbɛpaapae nsakrae a ɛba ahoɔden a wɔde di dwuma wɔ amemene mu nkwammoaa ahorow afoforo mu no mu na ama wɔada nnyinasosɛm yi a wɔde bedi dwuma wɔ ntini mu nyarewa afoforo mu adi koraa.
Wɔadi kan aka MitoPark mprako ho asɛm ( 31 ). Wɔadi kan aka C57BL/6N mprako a wɔwɔ loxP flanking Mfn2 awosu mu nkwaadɔm ho asɛm ( 18 ) na wɔde L7-Cre mprako abɔ mu ( 23 ). Afei wɔde asefo a wɔyɛ heterozygous abien a efii mu bae no ne homozygous Mfn2loxP/Mfn2loxP mprako twaa mu de yɛɛ Purkinje-specific gene knockouts maa Mfn2 (Mfn2loxP/Mfn2loxP; L7-cre). Wɔ aware mu kuw ketewaa bi mu no, wɔde Gt (ROSA26) SorStop-mito-YFP allele (stop-mtYFP) no bae denam ntwamu foforo so ( 20 ). Wɔyɛɛ mmoa ho nhyehyɛe nyinaa sɛnea Europa, ɔman ne ahyehyɛde ahorow akwankyerɛ kyerɛ na LandesamtfürNatur a ofi Umwelt ne Verbraucherschutz, North Rhine-Westphalia, Germany, penee so. Mmoa adwuma nso di Europa Mmoa Nyansahu Fekuw a Ɛhwɛ Nhwehwɛmubea Fekuw So no akwankyerɛ akyi.
Bere a wɔama ɔbea a onyinsɛn no awotwaa a atutu no awie no, woyi mmoawa a wɔfrɛ wɔn mouse embryo no fi mu (E13). Wɔtwitwaa cortex no mu wɔ Hanks’ Balanced Salt Solution (HBSS) a wɔde Hepes 10 mM ahyɛ mu ma mu na wɔde maa Dulbecco’s Modified Eagle’s Medium a papain (20 U/ml) ne cysteine ​​(1μg/ml) wom. Fa ntini no to DMEM mu) na fa enzymatic digestion tetew mu. Ml) wɔ 37°C simma 20, na afei wɔde mfiri ayɛ no wɔ DMEM a wɔde awotwaa mu nantwi mogya 10% ahyɛ mu. Wɔde nkwammoaa guu ahwehwɛ a wɔde kata so a wɔde polylysine akata so wɔ 2×106 wɔ 6 cm amammerɛ dish biara mu anaasɛ 0.5×105 nkwammoaa/cm2 mu de yɛɛ mfonini nhwehwɛmu. Nnɔnhwerew 4 akyi no, wɔde Neurobasal serum-free medium a 1% B27 supplement ne 0.5 mM GlutaMax wom sii aduru no ananmu. Afei wɔmaa ntini ahorow no yɛɛ 37°C ne 5% CO2 wɔ sɔhwɛ no nyinaa mu, na wɔmaa no aduan pɛnkoro dapɛn biara. Sɛnea ɛbɛyɛ a wɔbɛkanyan recombination wɔ vitro no, wɔde 3μl (24-well culture dish) anaa 0.5μl (24-well plate) a ɛyɛ AAV9 virus vector a edidi so yi dii dwuma de saa ntini ahorow no yare da a ɛto so abien wɔ vitro: AAV9.CMV.PI.eGFP. WPRE.bGH (Addgene, katalog nɔma 105530-AAV9) ne AAV9.CMV.HI.eGFP-Cre.WPRE.SV40 (Addgene, katalog nɔma 105545-AAV9).
Wɔde V5 ntoatoaso (GKPIPNPLLGLDST) ahyɛ mmoawa Mfn1 ne Mfn2 a ɛka bom DNA (a wonya fii Addgene plasmid #23212 ne #23213, sɛnea ɛte biara) agyirae wɔ C-awiei no, na wɔde mCherry afrafra wɔ frame mu denam T2A ntoatoaso no so. Grx1-roGFP2 yɛ akyɛde a efi Heidelberg T. P. Dick DFKZ (Deutsches Krebsforschungszentrum) hɔ. Ɛnam sɛ wɔde akwan a wɔtaa fa so yɛ cloning sesaa tdTomato cassette no so no, wɔde cassette no subcloned kɔɔ pAAV-CAG-FLEX-tdTomato akyi dompe (Addgene reference number 28306) mu de yɛɛ pAAV-CAG-FLEX-mCherry-T2A-MFN2-V5, pAAV-CAG- FLEX-mCherry-T2A-MFN1-V5 ne pAAV-CAG-FLEX-Grx-roGFP2 mmoawa a wɔde nyarewa ba. Wɔde ɔkwan a ɛte saa ara dii dwuma de yɛɛ control vector pAAV-CAG-FLEX-mCherry. Sɛnea ɛbɛyɛ a wobetumi ayɛ AAV-shPCx construct no, plasmid AAV vector (VectorBuilder, pAAV [shRNA] -CMV-mCherry-U6-mPcx- [shRNA#1]) a ɛho hia, a DNA ntoatoaso a ɛkyerɛw shRNA a ɛde n’ani si mmoawa PCx so (5′CTTTCGCTCTAAGGTGCTAAACTCGAGTTTAGCACCTTAGAGCGAAAG) wom 3′) Wɔ U6 promoter no tumi ase no, wɔde mCherry di dwuma wɔ CMV promoter no tumi ase. Wɔyɛɛ AAV mmoawa a wɔboa no sɛnea nea ɔyɛe no akwankyerɛ kyerɛ (Cell Biolabs). Ne tiawa mu no, fa transfer plasmid a ɛkura mCherry-T2A-MFN2-V5 (pAAV-CAG-FLEX-mCherry-T2A-MFN2-V5), mCherry-T2A-MFN1-V5 (pAAV-CAG-FLEX-mCherry) bere tiaa mu Transfection of 293AAV cells-T2A-MFN1-V5), mCherry di dwuma (pAAV-CAG-FLEX-mCherry) anaa Grx-roGFP2 (pAAV-CAG-FLEX-Grx-roGFP2) a ɛkyerɛw awosu mu abɔde, ne afei nso a ɛkyerɛw AAV1 capsid protein ne protein a ɛka ho Packaging plasmid plasmid, a wɔde calcium phosphate kwan di dwuma. Wɔnam freeze-thaw cycles a wɔde yɛ dry ice/ethanol bath ne lysed cells wɔ phosphate buffered saline (PBS) mu so nyaa virus supernatant a ɛnyɛ den no. Wɔnam iodixanol gradient ultracentrifugation a ɛnkɔ so ( nnɔnhwerew 24 wɔ 32,000 rpm ne 4°C) so tew AAV vector no ho na wɔde Amicon ultra-15 centrifugal filter yɛɛ no ​​den. AAV1-CAG-FLEX-mCherry-T2A-MFN2-V5 [2.9×1013 genome copy (GC)/ml], AAV1-CAG-FLEX-mCherry (6.1×1012 GC/ml), AAV1-CAG- FLEX no genome titer yɛ sɛnea wɔadi kan aka ho asɛm (54), a wɔde bere ankasa dodow PCR (qPCR) susuw -MFN1-V5 (1.9 × 1013 GC / ml) ne AAV1-CAG-FLEX-Grx-roGFP2 (8.9 × 1012 GC / milili).
Wɔpopaa ntini atitiriw no wɔ 1x PBS a ɛyɛ nsukyenee a ɛyɛ nwini mu, yɛɛ no ​​pelleted, na afei wɔde yɛɛ biako wɔ 0.5% Triton X-100 / 0.5% sodium deoxycholate/PBS lysis buffer a phosphatase ne protease inhibitor (Roche) wom mu. Wɔde bicinchoninic acid nhwehwɛmu (Thermo Fisher Scientific) na ɛyɛɛ protein dodow. Afei wɔde SDS-polyacrylamide gel electrophoresis yii protein ahorow no mu, na afei wɔde popaa polyvinylidene fluoride membrane (GE Healthcare) so. Siw mmeae a ɛnyɛ pɔtee no ano na fa antibody titiriw (hwɛ Table S1 ma nsɛm no mu nsɛm) to nufusu 5% mu wɔ TBST (Tris-buffered saline with Tween), ahohoro anammɔn ne antibody a ɛto so abien wɔ TBST Incubate mu. Fa antibody titiriw no to mu anadwo wɔ +4°C. Sɛ wohohoro wie a, fa antibody a ɛto so abien no gu so nnɔnhwerew 2 wɔ dan mu hyew mu. Akyiri yi, ɛdenam blot koro no ara a wɔde anti-β-actin antibody guu mu so no, wosii adesoa koro no ara so dua. Detection denam dan a wɔdan kɔ chemiluminescence ne enhancing chemiluminescence (GE Healthcare) so.
Wɔde 4% paraformaldehyde (PFA)/PBS hyehyɛɛ ntini a wɔadi kan ayɛ wɔ ahwehwɛ a wɔde kata so no wɔ bere a wɔakyerɛ no mu wɔ dan mu hyew mu simma 10. Wodi kan de Triton X-100/PBS 0.1% hyɛ nkataso no mu simma 5 wɔ dan mu hyew mu, na afei wɔde blocking buffer [3% bovine serum albumin (BSA)/PBS] gu mu. Da a ɛto so abien no, wɔde blocking buffer hohoroo nkataso no na wɔde fluorophore-conjugated secondary antibody a ɛfata no too mu nnɔnhwerew 2 wɔ dan mu hyew mu; awiei koraa no, wɔhohoroo nhwɛsode ahorow no yiye wɔ PBS mu a wɔde 4′,6-diamidino-2 -Phenylindole (DAPI) yɛ counterstained na afei wɔde Aqua-Poly/Mount si microscope slide no so.
Wɔde ketamine (130 mg/kg) ne xylazine (10 mg/kg) a wɔde guu mprako (mmarima ne mmea) aduru a ɛma obi dwensɔ na wɔde carprofen aduru a ɛma ɛyaw ano brɛ ase (5 mg/kg) , Na wɔde guu stereotactic adwinnade (Kopf) a wɔde aduru a ɛyɛ hyew ahyɛ mu mu. Da ti nhwi no adi na fa aduru a wɔde twitwiw sẽ di dwuma de yɛ amemene no fã a ɛne mis dompe no hyia (efi lambda: dua 1.8, akyi 1, a ɛne lobules IV ne V hyia) no yɛ teateaa. Fa syringe needle a ɛyɛ kurukuruwa di dwuma de ahwɛyiye yɛ tokuru ketewaa bi wɔ ti nhwi no mu na amma woanhaw ntini a ɛwɔ ase no. Afei wɔde ahwehwɛ a wɔatwe no teateaa no hyɛ micro-hole no mu nkakrankakra (efi -1.3 kosi -1 wɔ dura mater no ventral fã), na wɔde AAV 200 kosi 300 nl gu micro-injector (Narishige) no mu denam nsa syringe (Narishige) so mpɛn pii wɔ nhyɛso a ɛba fam mu wɔ bere tenten a ɛyɛ simma 10 kosi 20 mfɛnsere mu. Sɛ wode mogya no ahyɛ wo mu wie a, fa ntini no gu simma 10 bio na ama mmoawa no atrɛw koraa. Sɛ wɔtwe ntini nketenkete no wie a, wɔde ahwɛyiye pam honam ani sɛnea ɛbɛyɛ a kuru no renhyew na aboa no ho atɔ no. Wɔde nnuru a wɔde kum ɛyaw (caspofen) saa mmoa no yare nna pii wɔ oprehyɛn no akyi, na saa bere no mu no, wɔhwɛɛ wɔn nipadua tebea yiye na afei wokum wɔn wɔ bere a wɔakyerɛ no mu. Wɔyɛɛ akwan no nyinaa sɛnea Europa, ɔman ne ahyehyɛde ahorow akwankyerɛ kyerɛ na LandesamtfürNatur a ofi Umwelt ne Verbraucherschutz, North Rhine-Westphalia, Germany na ɛpene so.
Wɔde ketamine (100 mg/kg) ne xylazine (10 mg/kg) guu mmoa no mu, na wɔde 0.1 M PBS dii kan guu koma no mu, na afei wɔde 4% PFA a ɛwɔ PBS mu guu koma no mu. Wɔtwitwaa ntini no mu na wɔde sii hɔ wɔ 4% PFA/PBS mu anadwo biako wɔ 4°C. Wɔde sekan a ɛwosow (Leica Microsystems GmbH, Vienna, Austria) siesiee sagittal afã horow (a ne kɛse yɛ 50 μm) fii amemene a ɛyɛ pintinn wɔ PBS mu no mu. Gye sɛ wɔakyerɛ sɛ ɛnte saa, na wɔde staining yɛɛ afã horow a ɛsensɛn hɔ a ɛnyɛ den sɛnea yɛaka ho asɛm wɔ atifi hɔ (13) wɔ dan mu hyew mu na wɔkanyan no. Ne tiawa mu no, nea edi kan no, wɔde 0.5% Triton X-100/PBS maa nkuruwankuruwa a wonyae no yɛɛ permeabilized simma 15 wɔ dan mu hyew mu; ma epitopes binom (Pcx ne Shmt2), denam wɔ tris-EDTA buffer a ɛwɔ 80°C (PH 9) mu no, fa slices no hyew simma 25 sen sɛ wobɛfa anammɔn yi. Afei, wɔde afã horow no too antibody titiriw (hwɛ Table S1) mu wɔ blocking buffer (3% BSA/PBS) mu wɔ 4°C anadwo biako a wɔkanyan no. Ade kyee no, wɔde blocking buffer hohoroo afã horow no na wɔde fluorophore-conjugated secondary antibody a ɛfata no guu mu nnɔnhwerew 2 wɔ dan mu hyew mu; awiei koraa no, wɔhohoroo afã horow no yiye wɔ PBS mu, de DAPI yɛɛ counter-stained, na afei wɔde AquaPolymount On a microscope slide siesiee.
Wɔde laser scanning confocal microscope (TCS SP8-X anaa TCS Digital Light Sheet, Leica Microsystems) a wɔde hann fitaa laser ne 405 diode ultraviolet laser ahyɛ mu yɛɛ nhwɛsode no ho mfonini. Ɛdenam fluorophore no a wɔkanyan no na wɔde Hybrid Detector (HyDs) boaboaa sɛnkyerɛnne no ano so no, wɔde LAS-X software dii dwuma de boaboaa mfonini ahorow a wɔaboaboa ano a ɛne Nyquist nhwɛsode hyia wɔ nnidiso nnidiso kwan so: wɔ panel ahorow a ɛnyɛ dodow mu no, ɛyɛ nsɛnkyerɛnne a ɛyɛ nnam kɛse (sɛ nhwɛso no, wɔ somatic nkwammoaa ne dendrites mu) mtYFP) Fa HyD di dwuma de hu PN dodow wɔ BrightR mode mu). Wɔde gating a efi 0.3 kosi 6 ns di dwuma de tew akyi.
Nkwammoaa a wɔahyehyɛ no yiye ho mfonini a wɔyɛ wɔ bere ankasa mu. Bere a wɔhyehyɛe wɔ Neurobasal-A medium a 1% B27 supplement ne 0.5 mM GlutaMax wom mu akyi no, wɔde nkwammoaa no guu poly-l-lysine-coated glass slides (μ-Slide8 Well, Ibidi, catalog number 80826) so ntɛm ara , Na afei wɔde siee 37°C ne 5% CO2 dɔnhwerew 1 na ama nkwammoaa no atumi atena hɔ. Wɔyɛɛ bere ankasa mu mfonini wɔ Leica SP8 laser scanning confocal microscope a wɔde laser fitaa, HyD, 63×[1.4 numerical aperture (NA)] ngo botae ahwehwɛ ne ɔhyew stage ahyɛ mu ma so.
Wɔde carbon dioxide yɛɛ mmoawa no aduru ntɛmntɛm na wotwitwaa ne ti, woyii amemene no fii ne ti nhwi no mu ntɛmntɛm, na wotwitwaa no yɛɛ no ​​200μm (ma 13C labeling sɔhwɛ) anaa 275μm (ma photon sɔhwɛ abien) sagittal fã a wɔde nneɛma a edidi so yi ahyɛ mu ma The ice cream (HM-650 V, Thermo Fisher Scientific, Walldorf, Germany) nneɛma a edidi so yi ahyɛ mu ma: 125 mM nsukyenee a ɛyɛ nwini, carbon a ɛyɛ hyew (95% O2 ne 5% CO2) a ɛba fam Ca2 + amemene mu nsu a wɔde ayɛ (ACSF) NaCl, 2.5 mM KCl, 1.25 mM sodium phosphate buffer, 25 mM NaHCO3, 25 mM glucose, 0.5 mM CaCl2 ne 3.5 mM MgCl2 (osmotic nhyɛso a ɛyɛ 310 kosi 330 mmol). Fa amemene no mu nsensanee a woanya no kɔ dan a wɔadi kan ahyɛ mu a Ca2 + ACSF a ɛkorɔn (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 ne 2.0 mM MgCl2) wom mu. Medium) pH 7.4 ne 310 kosi 320 mmol).
Wɔ mfoninitwa nhyehyɛe no mu no, wɔde slices no kɔɔ mfoninitwa dan a wɔahyira so mu, na wɔyɛɛ sɔhwɛ no wɔ ACSF perfusion a ɛkɔ so wɔ ɔhyew a ɛkɔ so daa a ɛyɛ 32° kosi 33°C ase. Wɔde multiphoton laser scanning microscope (TCS SP8 MP-OPO, Leica Microsystems) a wɔde Leica 25x botaeɛ ahwehwɛ (NA 0.95, nsuo) ahyɛ mu, Ti: Sapphire laser (Chameleon Vision II, Coherent) dii dwuma de yɛɛ slice mfonini. FLIM module (PicoHarp300, PicoQuant) na ɛyɛ adwuma wɔ ɔkwan a ɛyɛ nwonwa so.
FLIM a ɛfa Grx1-roGFP2 ho. Wɔde twen abien FLIM susuw nsakrae a ɛba PNs cytoplasmic redox tebea mu wɔ sagittal amemene slices mu, faako a Grx1-roGFP2 biosensor no de n’ani sii PNs so. Wɔ PN layer no mu no, wɔpaw acquisition field no bɛyɛ 50 kosi 80 μm wɔ slice no ani de hwɛ hu sɛ PN a ɛyɛ adwuma (kyerɛ sɛ, beaded nhyehyɛe anaa neuronal morphological nsakrae a enni dendrites no ho) ne double positive roGFP2 sensor ne AAV encoding shRNA PCx anaa ne control sequence (biara co-expressing mCherry) wɔ hɔ. Boaboa mfonini ahorow a wɔaboaboa ano biako ano denam 2x dijitaal zoom [excitation wavelength: 890 nm; 512 nm 512 piksel]. Detection: wɔde HyD a ɛwɔ mu, fluorescein isothiocyanate (FITC) filter group] ne mfonini a wɔde simma 2 kosi 3 na ɛyɛ averaging di dwuma de hwɛ hu sɛ wɔaboaboa photons a ɛdɔɔso ano (foton 1000 ne nyinaa) ama curve fitting. Wɔyɛɛ Grx1-roGFP2 nhwehwɛmu no nkate ne FLIM tebea horow no mu nokwaredi denam roGFP2 nkwa nna bo a wɔhwɛɛ so bere a wɔde exogenous 10 mM H2O2 ka perfusion ACSF no ho (de ma oxidation yɛ kɛse, na ɛma nkwa nna kɔ soro), na afei wɔde 2 mM dithiothreitol kaa ho (ɛtew dodow a ɛso tew so, na ɛma ɛso tew wɔ nkwa nna mu) (Mfonini S8, D kosi G). Fa FLIMfit 5.1.1 softwea no hwehwɛ nea wonyae no mu, fa mfonini no nyinaa mu exponential decay curve biako no hyia IRF a wɔasusuw no (adwinnade mmuae dwumadi), na χ2 bɛyɛ 1. Sɛ yɛbɛbu PN biako nkwa nna a, wɔde nsa twee akatasoɔ a ɛtwa ntini nipadua no ho hyia no, na wɔde nkwa nna a ɛwɔ akatasoɔ biara mu no dii dwuma de kyerɛɛ dodoɔ.
Mitochondrial tumi nhwehwɛmu. Bere a wɔde TMRM 100 nM a wɔde ahyɛ ACSF a wɔde ahyɛ mu no mu tẽẽ no ahyɛ ɔfã a emu yɛ den no mu simma 30 akyi no, wɔde photon abien microscope susuw nsakrae a ɛba wɔ mitochondrial tumi mu wɔ PN ahorow mu no. Wɔyɛɛ TMRM mfoniniyɛ denam nhwehwɛmu a wɔkanyan no wɔ 920 nm na wɔde HyD a ɛwɔ mu (tetramethylrhodamine isothiocyanate: 585/40 nm) dii dwuma de boaboaa nsɛnkyerɛnne ano; denam excitation wavelength koro no ara a wɔde bedi dwuma nanso wɔde HyD soronko a ɛwɔ mu (FITC :525/50) bedi dwuma de ayɛ mtYFP mfonini no so. Fa ImageJ no Image Calculator plug-in no di dwuma de susuw mitochondrial tumi a ɛwɔ nkwammoaa biako gyinabea no ho. Ne tiawa mu no, wɔde plug-in equation: signal = min (mtYFP, TMRM) di dwuma de kyerɛ mitochondrial mpɔtam a ɛkyerɛ TMRM sɛnkyerɛnne wɔ Purkinje Somali mu wɔ single-stack confocal mfonini a ɛwɔ channel a ɛne no hyia no mu. Afei wɔkyerɛ pixel beae a ɛwɔ akataso a efi mu ba no mu dodow, na afei wɔma ɛyɛ nea ɛfata wɔ mtYFP kwan no threshold single-stack mfonini a ɛne no hyia no so na ama wɔanya mitochondrial fã a ɛkyerɛ mitochondrial tumi no.
Wɔde Huygens Pro (Scientific Volume Imaging) softwea yii mfonini no mu. Wɔ tiles mfonini ahorow a wɔayɛ no scan no ho no, wɔde automatic stitching algorithm a LAS-X software de ama no na ɛyɛ tile biako montage. Sɛ woyɛ mfonini no calibration wie a, fa ImageJ ne Adobe Photoshop di dwuma kɔ so yɛ mfonini no ho adwuma na yɛ nsakrae wɔ hann ne nsonsonoe no mu pɛpɛɛpɛ. Fa Adobe Illustrator di dwuma ma mfonini siesie.
mtDNA adwene a wɔde si biribi so nhwehwɛmu. Wɔde confocal microscope buu mtDNA akuru dodow wɔ amemene no afã horow a wɔde nnuru a ekum DNA a wɔakyerɛw so no so. Wɔyɛɛ beae biara a wɔde wɔn ani asi so maa nkwammoaa nipadua ne nkwammoaa biara nucleus, na wɔde Multi Measure plug-in (ImageJ software) buu beae a ɛwɔ hɔ no ho akontaa. Twe nuklea beae no fi nkwammoaa nipadua no fã no mu na woanya cytoplasmic beae no. Awiei koraa no, wɔde Analyze Particles plug-in (ImageJ software) no dii dwuma de kyerɛɛ cytoplasmic DNA nsɛntitiriw a ɛkyerɛ mtDNA wɔ threshold mfonini no so no dodow, na wɔde nea efii mu bae no yɛɛ nea ɛfata maa CTRL mprako PN average. Wɔda nea efi mu ba no adi sɛ sɛ wɔkyekyem pɛpɛɛpɛ a, nucleoside dodow a ɛwɔ nkwammoaa biara mu.
Protein a wɔda no adi ho nhwehwɛmu. Fa ImageJ no Image Calculator plug-in no hwɛ protein a wɔda no adi wɔ PN mu wɔ nkwammoaa biako gyinabea. Ne tiawa mu no, wɔ confocal mfonini a ɛwɔ ɔfa biako a ɛwɔ ɔkwan a ɛne no hyia no so no, ɛnam nsɛso no so: sɛnkyerɛnne = min (mtYFP, antibody) no, wɔkyerɛ mitochondrial mpɔtam a ɛkyerɛ immunoreactivity ma antibody pɔtee bi wɔ Purkina. Afei wɔkyerɛ pixel beae a ɛwɔ akataso a efi mu ba no mu dodow, na afei wɔma ɛyɛ nea ɛfata wɔ mtYFP kwan no threshold single-stack mfonini a ɛne no hyia no so na ama wɔanya protein a wɔada no adi no mitochondrial fã.
Purkinje nkwammoaa mu density nhwehwɛmu. Wɔde Cell Counter plug-in a ɛwɔ ImageJ mu no dii dwuma de hwɛɛ Purkinje density denam Purkinje nkwammoaa dodow a wɔkan no a wɔkyekyɛɛ mu denam amemene no mu mprɛte a nkwammoaa a wɔakan no agye no tenten so.
Nhwɛsode ahosiesie ne nea wɔboaboa ano. Wɔde amemene a efi kuw a wɔhwɛ so ne Mfn2cKO mprako mu no sii hɔ wɔ 2% PFA/2.5% glutaraldehyde mu wɔ 0.1 M phosphate buffer (PB) mu, na afei wɔde ciliates (Leica Mikrosysteme GmbH, Vienna, Austria) (Ne kɛse yɛ 50 kosi 60 μm) siesiee coronal afã horow no Afei siesiee wɔ PB buffer mu wɔ 1% os tetraoxide ne 1.5% potassium ferrocyanide wɔ dan mu hyew mu dɔnhwerew 1. Wɔde nsu a wɔahoro hohoroo afã horow no mprɛnsa, na afei wɔde 70% ethanol a 1% uranyl acetate wom guu so simma 20. Afei wɔde nsuo fii afã horow no mu wɔ nsa a wɔahyɛ da ayɛ mu na wɔde guu Durcupan ACM (Araldite casting resin M) epoxy resin (Electron Microscopy Sciences, catalog number 14040) mu wɔ ahwehwɛ a wɔde silicon akata so ntam, na awiei koraa no wɔde 60°C Polymerize wɔ fononoo mu nnɔnhwerew 48. Wɔpaw amemene no fã a ɛwɔ amemene no mu no na wotwitwaa afã horow a ɛyɛ teateaa kɛse a ne kɛse yɛ 50 nm wɔ Leica Ultracut (Leica Mikrosysteme GmbH, Vienna, Austria) so na wɔpaw no wɔ 2×1 mm kɔbere slit grid a wɔde polystyrene film akata so so. Wɔde 4% uranyl acetate aduru a ɛwɔ H2O mu yɛɛ afã horow no fĩ simma 10, wɔde H2O hohoroo ho mpɛn pii, afei wɔde Reynolds lead citrate a ɛwɔ H2O mu simma 10, na afei wɔde H2O hohoroo ho mpɛn pii. Wɔde TVIPS (Tietz Video and Image Processing System) TemCam-F416 dijitaal mfoninitwa afiri (TVIPS GmbH, Gauting, U.S.A.) na ɛyɛɛ mfonini nketenkete. Germany).
Wɔ mprako a wɔanya AAV no fam no, wɔtetew amemene no mu na wɔtwitwaa mu yɛɛ no ​​sagittal fã a ne kɛse yɛ mm 1, na wɔde fluorescence microscope hwehwɛɛ amemene no mu de huu ring a AAV ayɛ wɔn (kyerɛ sɛ, mCherry a ɛda adi). Nsɔhwɛ ahorow a AAV a wɔde twitwiw nipadua no ma Purkinje nkwammoaa no fã (kyerɛ sɛ ɛkame ayɛ sɛ ɔfa no nyinaa) a ɛkɔ soro yiye wɔ anyɛ yiye koraa no amemene mu nkaa abien a ɛtoatoa so mu nkutoo na wɔde di dwuma. Wɔyɛɛ AAV-transduced loop no microdissected maa anadwo post-fixation (4% PFA ne 2.5% glutaraldehyde wɔ 0.1 M cocoate buffer) na wɔsan yɛɛ ho adwuma. Sɛ wɔde EPON hyɛ mu a, wɔde 0.1 M sodium cocoate buffer (Applichem) hohoroo ntini a ɛyɛ pintinn no, na wɔde 2% OsO4 (os, Science Services; Caco) too mu wɔ 0.1 M sodium cocoate buffer (Applichem) mu Nnɔnhwerew 4, afei wɔhohoroo ho nnɔnhwerew 2. Fa 0.1 M cocamide buffer san yɛ no mpɛn 3. Akyiri yi, wɔde ethanol a ɛforo kɔ soro no dii dwuma de ethanol aduru biara too 4°C so simma 15 de maa nsu fii ntini no mu. Wɔde ntini no guu propylene oxide mu na wɔde too EPON (Sigma-Aldrich) mu anadwo biako wɔ 4°C. Fa ntini no gu EPON foforo mu wɔ dan mu hyew mu nnɔnhwerew 2, na afei fa hyɛ mu wɔ 62°C nnɔnhwerew 72. Fa ultramicrotome (Leica Microsystems, UC6) ne diamond sekan (Diatome, Biel, Switzerland) twa afã horow a ɛyɛ 70 nm ultrathin, na fa 1.5% uranyl acetate gu so simma 15 wɔ 37°C, na fa lead citrate solution gu so simma 4. Wɔde JEM-2100 Plus transmission electron microscope (JEOL) a wɔde Camera OneView 4K 16-bit (Gatan) ne DigitalMicrograph software (Gatan) ahyɛ mu ma na ɛyɛɛ ɛlɛtrɔnik mfonini nketenkete no. Sɛnea ɛbɛyɛ na wɔayɛ nhwehwɛmu no, wɔde 5000× anaa 10,000× dijitaal zoom nyaa ɛlɛtrɔnik mfonini nketenkete.
Mitochondria ahorow no nsɛso nhwehwɛmu. Wɔ nhwehwɛmu nyinaa mu no, wɔde nsa kyerɛkyerɛɛ mitochondria ankorankoro no nsusuwii mu wɔ dijitaal mfonini ahorow mu denam ImageJ softwea so. Wɔyɛ nsɛsoɔ ahodoɔ mu nhwehwɛmu. Wɔda mitochondrial density adi sɛ ɔha biara mu nkyem a wonya denam nkwammoaa biara mitochondrial beae nyinaa a wɔkyekyɛ mu denam cytoplasm beae (cytoplasm area = cell area-cell nucleus area) × 100. Wɔde formula [4π∙(beae/perimeter 2)] na ebu mitochondria no kurukuruwa. Wɔyɛɛ mitochondria ista nsɛso mu nhwehwɛmu na wɔkyekyɛɛ mu akuw abien (“tubular” ne “blister”) sɛnea wɔn nsusuwii atitiriw te.
Autophagosome/lysosome dodow ne density nhwehwɛmu. Fa ImageJ software di dwuma de nsa kyerɛ autophagosome/lysosome biara contours wɔ digital mfonini no mu. Wɔda autophagosome/lysosome beae adi sɛ ɔha biara mu nkyem a wɔabu ho akontaa denam autophagosome/lysosome nhyehyɛe beae a ɛwɔ nkwammoaa biara mu nyinaa a wɔkyekyɛ mu denam cytoplasm beae (cytoplasm area=cell area-nucleus area)×100 so. Wɔnam dodow no nyinaa a wɔkyekyɛ mu de autophagosome/lysosome nhyehyɛe dodow a ɛwɔ nkwammoaa biara mu (wɔ cytoplasmic area) (cytoplasmic area = cell area-nuclear area) so na ebu autophagosomes/lysosomes dodow.
Labeling ma acute sectioning ne nhwɛsode ahosiesie. Sɛ wopɛ sɔhwɛ ahorow a ɛhwehwɛ sɛ wɔde glucose ahyɛ so a, fa amemene no mu nkuruwankuruwa a ɛyɛ hu no kɔ dan a wɔadi kan ahyɛ mu, a carbon a wɔahyɛ no ma (95% O2 ne 5% CO2), Ca2 + ACSF a ɛdɔɔso (125.0 mM NaCl, 2.5 mM KCl, 1.25 mM sodium phosphate buffer, 25.0 mM NaHCO 3, 25.0 mM) wom d-glucose, 1.0 mM CaCl 2 ne 2.0 mM MgCl 2, a wɔayɛ nsakraeɛ wɔ pH 7.4 ne 310 kɔsi 320 mOsm), a glucose yɛ 13 C 6- Glucose a wɔde si ananmu (Eurisotop, katalog nɔma CLM-1396). Sɛ wopɛ sɔhwɛ ahorow a ɛhwehwɛ sɛ wɔde pyruvate ahyɛ so a, fa amemene no mu nkuruwankuruwa a ɛyɛ hu no kɔ Ca2 + ACSF a ɛkorɔn (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 na Fa 2.0mM ka ho MgCl2, yɛ nsakraeɛ kɔ pH 7.4 ne 310 kɔsi 320mOsm), na fa 1mM 1-[1-13C]pyruvate (Eurisotop, katalog nɔma CLM-1082) ka ho. Fa afã horow no to 37°C so simma 90. Wɔ sɔhwɛ no awiei no, wɔde nsu a wɔde afra (pH 7.4) a ammonium carbonate 75 mM wom hohoroo afã horow no ntɛmntɛm, na afei wɔde yɛɛ biako wɔ 40:40:20 (v:v:v) acetonitrile (ACN): methanol: nsu mu. Bere a wɔde afã horow no too nsukyenee so simma 30 akyi no, wɔde nhwɛsode ahorow no guu centrifuged wɔ 21,000 g mu simma 10 wɔ 4°C, na wɔde nsu a ɛwɔ soro a emu da hɔ no wee wɔ SpeedVac concentrator mu. Wɔde metabolite pellet a ayow a efii mu bae no siee -80°C kosii sɛ wɔyɛɛ mu nhwehwɛmu.
Nsu chromatography-mass spectrometry nhwehwɛmu a wɔyɛe wɔ amino acid 13 a wɔakyerɛw so C ho. Sɛ wopɛ nsuo chromatography-mass spectrometry (LC-MS) nhwehwɛmu a, wɔsan de metabolite pellet no guu 75μl LC-MS grade nsuo (Honeywell) mu. Bere a wɔde centrifugation a ɛyɛ 21,000 g simma 5 wɔ 4°C akyi no, wɔde supernatant a wɔama emu ada hɔ no 20 μl dii dwuma de yɛɛ amino acid flux nhwehwɛmu, bere a wɔde nea aka no dii dwuma ntɛm ara de yɛɛ anion nhwehwɛmu (hwɛ ase hɔ). Wɔde benzoyl chloride derivatization protocol a wɔadi kan aka ho asɛm no na ɛyɛɛ amino acid nhwehwɛmu ( 55 , 56 ). Wɔ anammɔn a edi kan no mu no, wɔde 10μl 100 mM sodium carbonate (Sigma-Aldrich) guu 20μl metabolite extract mu, na afei wɔde 10μl 2% benzoyl chloride (Sigma-Aldrich) guu LC grade ACN no mu. Wɔde vortex yɛɛ nhwɛsode no bere tiaa bi na afei wɔde centrifuged wɔ 21,000 g mu simma 5 wɔ 20°C. Fa nsuo a ɛwɔ soro a wɔayiyi no kɔ 2 ml autosampler vial a conical glass insert (200 μl volume) wom mu. Wɔde Acquity iClass ultra-high performance LC system (Waters) a ɛbata Q-Exactive (QE)-HF (Ultra High Field Orbitrap) high-resolution precision mass spectrometer (Thermo Fisher Scientific) ho na ɛyɛɛ nhwɛsoɔ no mu nhwehwɛmu. Sɛ wɔbɛyɛ nhwehwɛmu a, wɔde 2μl guu derivatized nhwɛsode no mu 100×1.0 mm ahoɔden kɛse silica T3 adum (Nsu) a 1.8μm asinasin wom. Nsuo a ɛsen no yɛ 100μl/min, na buffer nhyehyɛeɛ no yɛ buffer A (10 mM ammonium formate ne 0.15% formic acid wɔ nsuo mu) ne buffer B (ACN). Gradient no te sɛ nea edidi so yi: 0%B wɔ simma 0 mu; 0%B. 0 kosi 15% B wɔ simma 0 kosi 0.1 mu; 15 kosi 17% B wɔ simma 0.1 kosi 0.5 mu; B wɔ 17 kosi 55% wɔ simma 0.5 kosi 14; B wɔ 55 kosi 70% wɔ simma 14 kosi 14.5; wɔ 14.5 kosi 70 kosi 100% B wɔ simma 18 mu; 100% B wɔ simma 18 kosi 19 mu; 100 kosi 0% B wɔ simma 19 kosi 19.1 mu; 0% B wɔ simma 19.1 kosi 28 (55, 56). QE-HF mass spectrometer no yɛ adwuma wɔ ionization mode pa mu a ne mass range yɛ m/z (mass/charge ratio) yɛ 50 kosi 750. Resolution a wɔde di dwuma no yɛ 60,000, na gain control (AGC) ion botae a wonya no yɛ 3×106, na ion bere a ɛsen biara yɛ 100 milliseconds. Heated electrospray ionization (ESI) fibea no yɛ adwuma wɔ spray voltage a ɛyɛ 3.5 kV, capillary temperature a ɛyɛ 250°C, sheath airflow a ɛyɛ 60 AU (arbitrary units), ne auxiliary airflow a ɛyɛ 20 AU. 250°C na ɛwɔ hɔ. Wɔde S lens no asi hɔ sɛ 60 AU.
Anion chromatography-MS nhwehwɛmu a wɔyɛe wɔ 13C a wɔakyerɛw so wɔ organic acids ho. Wɔde Dionex ion chromatography system (ICS 5000+, Thermo Fisher Scientific) a wɔde abata QE-HF mass spectrometer (Thermo Fisher Scientific) ho yɛɛ metabolite precipitate a aka (55μl) no mu nhwehwɛmu. Ne tiawa mu no, wɔde metabolite a wɔayiyi 5μl guu Dionex IonPac AS11-HC adum a wɔde HPLC (2 mm×250 mm, abɔdeɛ nketenkete kɛseɛ 4μm, Thermo Fisher Scientific) ahyɛ mu wɔ push-in partial loop mode a ne hyɛ mu nsusuiɛ a ɛyɛ 1. ) Dionex IonPac AG11-HC guard column (2 mm x 50 mm, 4μm, Thermo Fisher Nyansahu mu Nsɛm). Wɔma adum no hyew yɛ 30°C, na wɔde autosampler no si 6°C. Fa potassium hydroxide afiri a wɔde nsuo a wɔayi ion afiri mu ama di dwuma na ama potassium hydroxide a ɛkɔ soro denam eluent generator no so. Nkwammoaa mu nneɛma a wɔpaapae mu wɔ nsuo a ɛsen 380μl/simma mu, de gradient a ɛdidi soɔ yi di dwuma: simma 0 kɔsi 3, 10 mM KOH; Simma 3 kosi 12, 10 kosi 50 mM KOH; Simma 12 kosi 19, 50 kosi 100 mM KOH; Simma 19 kosi 21 , 100 mM KOH; Simma 21 kosi 21.5, 100 kosi 10 mM KOH. Wɔsan de adum no yɛɛ pɛ wɔ 10 mM KOH ase simma 8.5.
Wɔde metabolites a wɔahoro no ka 150μl/min isopropanol supplement stream ho wɔ adum no akyi na afei wɔde kyerɛ mass spectrometer a ɛwɔ nsusuwii a ɛkorɔn a ɛyɛ adwuma wɔ ionization mode a enye mu. MS rehwɛ sɛnea mass range fi m/z 50 kosi 750 a resolution yɛ 60,000. Wɔde AGC no asi 1×106, na wɔde ion bere a ɛsen biara no sie 100 ms. Wɔde ESI fibea a ɛyɛ hyew no yɛɛ adwuma wɔ spray voltage a ɛyɛ 3.5 kV. Nsiesiei afoforo a wɔde yɛ ion fibea no te sɛ nea edidi so yi: capillary temperature 275°C; mframa a ɛsen fa sheath mu, 60 AU; gas a ɛboa a ɛsen, 20 AU wɔ 300°C, ne S lens a wɔde si 60 AU.
Data nhwehwɛmu a wɔyɛe wɔ 13C labeled metabolites ho. Fa TraceFinder software (version 4.2, Thermo Fisher Scientific) di dwuma ma data nhwehwɛmu a ɛfa isotope ratio ho. Wɔde aduru a wɔde gyina hɔ ma a wotumi de ho to so dii aduru biara ho adanse na wɔyɛɛ mu nhwehwɛmu wɔ ahofadi mu. Sɛnea ɛbɛyɛ a wɔbɛyɛ isotope enrichment nhwehwɛmu no, woyii 13C isotope (Mn) biara mu ion chromatogram (XIC) a wɔayiyi no fã no fii [M + H] + mu, faako a n yɛ carbon dodow a ɛwɔ aduru a wɔde asi wɔn ani so no mu, a wɔde yɛ amino acid mu nhwehwɛmu anaasɛ [ MH] + na wɔde yɛ anion mu nhwehwɛmu. XIC no mass pɛpɛɛpɛyɛ nnu ɔpepem biara mu nkyem anum, na RT no pɛpɛɛpɛyɛ yɛ simma 0.05. Wɔnam isotope biara a wɔahu no ne isotope a ɛwɔ aduru a ɛne no hyia no nyinaa a wɔaka abom no so na ɛyɛ enrichment nhwehwɛmu no. Wɔde saa nsusuiɛ yi ama sɛ ɔha mu nkyekyɛmu boɔ ma isotope biara, na wɔda nea ɛfiri mu ba no adi sɛ molar ɔha mu nkyekyɛmu enrichment (MPE), sɛdeɛ yɛadi kan aka ho asɛm ( 42 ).
Wɔde neuron pellet a wɔahyɛ no nwini no yɛɛ biako wɔ nsukyenee a ɛyɛ nwini 80% methanol (v/v) mu, vortexed, na wɔde too -20°C simma 30. Vortex nhwɛsode no bio na woakanyan no wɔ +4°C simma 30. Wɔde nhwɛsode no centrifuged wɔ 21,000 g simma 5 wɔ 4°C, na afei wɔboaboaa nsu a ɛwɔ soro a efii mu bae no ano na wɔde SpeedVac concentrator a ɛwɔ 25°C yɛɛ nhwehwɛmu a edi hɔ no wee. Sɛnea yɛaka ho asɛm wɔ atifi hɔ no, wɔyɛɛ LC-MS nhwehwɛmu wɔ amino acid ahorow a ɛwɔ nkwammoaa a wɔahyehyɛ no mu no so. Wɔde TraceFinder (version 4.2, Thermo Fisher Scientific) dii dwuma de yɛɛ data nhwehwɛmu denam monoisotopic mass a ɛwɔ aduru biara mu no so. Wɔde preprocessCore software package ( 57 ) na ɛyɛɛ metabolite data no quantile normalization.
Slice ahosiesie. Wɔde carbon dioxide maa mmoawa no aduru ntɛmntɛm na wotwitwaa ne ti, woyii amemene no fii ne ti nhwi no mu ntɛm ara, na wɔde sekan a ɛwosow a nsukyenee ahyɛ mu ma (HM-650 V, Thermo Fisher Scientific, Walldorf, Germany) twitwaa no yɛɛ no ​​300 kosi 375 μm sagittal afã horow Cold carbon gasification (95% O2 ne 5% CO2) Low 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 ne 6.0 mM MgCl2 Yɛ nsakraeɛ kɔ pH 7.4 ne 310 kɔsi 330 mOsm). Fa amemene no mu nsensanee a woanya no kɔ dan a Ca2 + ACSF a ɛkorɔn (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 ne mM 3.5 MgCl2) pH 7.4 ne 310 kosi 320 mOsm). Fa slices no sie simma 20 kosi 30 sɛnea ɛbɛyɛ a wobetumi asan de aba ansa na woakyere agu hama so.
a wɔkyere gu kasɛt so. Wɔde microscope stage a wɔde afiri a wɔde kyere nsɛm gu kasɛt so a ɛyɛ pintinn ne 20x nsuo mu nsuo mu botaeɛ ahwehwɛ (Scientifica) dii dwuma maa nsɛm a wɔkyere gu kasɛt so nyinaa. Wɔnam (i) nipadua kɛse, (ii) beae a amemene no wɔ wɔ nipadua mu, ne (iii) mtYFP amanneɛbɔfo awosu mu abɔde a ɛma hann no a wɔda no adi so na ɛkyerɛɛ Purkinje nkwammoaa a wosusuw sɛ ɛwɔ hɔ no. Wɔnam borosilicate glass capillary (GB150-10, 0.86 mm×1.5 mm×100 mm, Science Products, Hofheim, Germany) ne pipette a ɛda fam Instruments (P-1000, Sutter), Novato, CA so na ɛtwe patch pipette a ne tip resistance yɛ megohms 5 kosi 11 no fi mu. Wɔde ELC-03XS npi patch clamp amplifier (npi electronic GmbH, Tam, Germany) na ɛyɛɛ nsɛm a wɔakyere agu hama so nyinaa, a na softwea Signal (version 6.0, Cambridge Electronic, Cambridge, UK) na ɛhwɛ so. Wɔkyerɛw sɔhwɛ no wɔ sampling rate a ɛyɛ 12.5 kHz. Wɔde Bessel filter abien a ɛtwam tiawa a cutoff frequency yɛ 1.3 ne 10 kHz na ɛsesa sɛnkyerɛnne no. Capacitance a membrane ne pipette no yɛ compensated denam compensation circuit a wɔde amplifier di dwuma no so. Wɔyɛɛ nhwehwɛmu no nyinaa wɔ Orca-Flash 4.0 mfoninitwa afiri (Hamamatsu, Gerden, Germany) a Hokawo softwea (version 2.8, Hamamatsu, Gerden, Germany) na ɛhwɛ so no ase.
Nkwammoaa mũ no nyinaa nhyehyɛe ne nhwehwɛmu a wɔyɛ no daa. Ansa na wobɛkyere agu hama so ntɛm ara no, fa nsu a ɛwɔ mu a nneɛma a edidi so yi wom no hyɛ pipette no ma: 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) ne Wɔsesaa creatinine phosphate 10.0 mM koduu pH 7.25, na na osmotic nhyɛso no yɛ 290 mOsm (sucrose). Bere a wɔde tumi a ɛyɛ 0 pA dii dwuma de bubuu ntini no akyi pɛɛ no, wɔsusuw tumi a ɛwɔ ntini a ɛregye n’ahome no mu. Wɔnam hyperpolarized currents a ɛyɛ -40, -30, -20, ne -10 pA a wɔde di dwuma so na ɛsusu input resistance. Susuw voltage mmuae no kɛse na fa Ohm mmara no bu input resistance no ho akontaa. Wɔkyerɛw dwumadi a ɛba ara kwa wɔ voltage clamp mu simma 5, na wɔde semi-automatic recognition script na ɛkyerɛɛ sPSC na wɔsusuu wɔ Igor Pro (version 32 7.01, WaveMetrics, Lake Oswego, Oregon, USA) mu. Wɔsusu IV curve ne steady-state current no denam clamping battery no wɔ potentials ahodoɔ (a ɛfiri -110 mV) na wɔma voltage no kɔ soro wɔ 5 mV anammɔn mu. Wɔsɔɔ AP a wɔyɛ no hwɛe denam depolarizing current a wɔde dii dwuma so. Clamp cell no wɔ -70 mV bere a wode depolarizing current pulse di dwuma. Sesa anammɔn kɛse a ɛwɔ afiri biara a wɔde kyere nsɛm gu kasɛt so no mu wɔ ɔkwan soronko so (10 kosi 60 pA). Bu AP frequency a ɛsen biara ho akontaa denam nsa a wode bɛkan pulse spikes a ɛde AP frequency a ɛkorɔn sen biara ba no so. Wɔnam depolarization pulse a edi kan kanyan AP biako anaa nea ɛboro saa no derivative a ɛto so abien a wɔde di dwuma so na ɛyɛ AP threshold no mu nhwehwɛmu.
Perforated patch nhyehyɛe ne nhwehwɛmu. Yɛ perforated patch recording denam standard protocols so. Fa pipette a ATP ne GTP nnim a nneɛma a edidi so yi nni mu di dwuma: 128 mM gluconate K, 10 mM KCl, 10 mM Hepes, 0.1 mM EGTA ne 2 mM MgCl2, na yɛ nsakrae kɔ pH 7.2 (fa KOH di dwuma). Woyi ATP ne GTP fi nkwammoaa mu aduru no mu na amma nkwammoaa no mu ntini no ntumi nkɔ mu a wontumi nni so. Wɔde aduru a ɛwɔ mu a amphotericin wom (bɛyɛ 200 kosi 250μg/ml; G4888, Sigma-Aldrich) hyɛ patch pipette no ma na ama wɔanya patch record a wɔabɔ no akuturuku. Wɔde amphotericin guu dimethyl sulfoxide mu (ne dodow a etwa to: 0.1 kosi 0.3%; DMSO; D8418, Sigma-Aldrich). DMSO dodow a wɔde dii dwuma no annya nkɛntɛnso titiriw biara wɔ ntini ahorow a wɔyɛɛ ho nhwehwɛmu no so. Wɔ punching nhyehyɛe no mu no, wɔkɔɔ so hwɛɛ channel resistance (Ra) no, na wofii sɔhwɛ no ase bere a Ra ne AP amplitude no ayɛ pintinn (simma 20-40). Wɔsusu dwumadie a ɛba ara kwa wɔ voltage ne/anaasɛ current clamp mu simma 2 kɔsi 5. Wɔde Igor Pro (nkyekyɛmu 7.05.2, WaveMetrics, USA), Excel (nsɛmfua 2010, Microsoft Corporation, Redmond, USA) ne GraphPad Prism (nkyekyɛmu 8.1.2, GraphPad Software Inc., La Jolla, CA) na ɛyɛɛ nsɛm nhwehwɛmu. United States). Sɛnea ɛbɛyɛ a wobehu AP ahorow a ɛba ara kwa no, wɔde IgorPro NeuroMatic v3.0c plug-in di dwuma. Fa threshold a wɔde ama no kyerɛ AP ahorow no ankasa, a wɔayɛ nsakrae wɔ mu mmiako mmiako ma kyerɛwtohɔ biara. Fa spike interval no di dwuma, kyerɛ spike frequency a ɛwɔ spike frequency a ɛsen biara a ɛba ntɛm ara ne spike frequency a ɛkyɛn so.
PN a wɔtew wɔn ho fi afoforo ho. Ɛdenam nsakrae a wɔyɛe wɔ protocol a wɔadi kan atintim no ho so no, wɔtew PN ahorow ho fii mmoawa a wɔfrɛ wɔn mouse cerebellum no mu wɔ bere pɔtee bi mu ( 58 ). Ne tiawa mu no, wotwitwaa amemene no mu na wɔtwitwaa mu wɔ nsukyenee a ɛyɛ nwini a wɔde yiyi mu [a HBSS Ca2+ ne Mg2+ nni mu, a wɔde 20 mM glucose, penicillin (50 U/ml) ne streptomycin (0.05 mg/ ml) kaa ho], na afei wɔyam aduru no wɔ papain [HBSS, a wɔde 1-cysteine·HCl aka ho (1 mg / ml), papain (16 U / ml) ne deoxyribonuclease I (DNase I; 0.1 mg/ml)] Fa simma 30 sa yare wɔ 30°C. Di kan hohoro ntini ahorow no wɔ HBSS aduru a ɛmo mu nsu (10 mg/ml), BSA (10 mg/ml) ne DNase (0.1 mg/ml) wom mu wɔ dan mu hyew mu na amma enzymatic digestion, na afei wɔ HBSS aduru a 20 mM glucose wom mu brɛoo yam wɔ HBSS, penicillin (50 U/ml), streptomycin (0.05 mg/ml) ne mu DNase (0.1 mg/ml) ma nkwammoaa biako fi adi. Wɔde 70μm cell strainer yii nkwammoaa a wɔde ahyɛ mu no mu, afei wɔde centrifugation (1110 rpm, simma 5, 4°C) yɛɛ nkwammoaa no pelleted na wɔsan de guu mu wɔ sorting medium [HBSS, a wɔde 20 mM glucose, 20% fetal bovine ) Serum, penicillin (50 U/ml) ne streptomycin kaa ho (0.05 mg/ml)]; fa propidium iodide hwɛ sɛnea nkwammoaa no tumi tra ase na wɔyɛ nsakrae wɔ nkwammoaa dodow mu ma ɛnyɛ 1×106 kosi 2×106 nkwammoaa/ml. Ansa na wɔbɛyɛ flow cytometry no, na wɔde 50 μm cell strainer yiyi suspension no mu.
Nsuo a ɛsen cytometer. Wɔde FACSAria III afiri (BD Biosciences) ne FACSDiva software (BD Biosciences, version 8.0.1) na ɛyɛɛ nkwammoaa mu nhwehwɛmu wɔ 4°C. Wɔde 100 μm nozzle na ɛhyehyɛɛ nkwammoaa a wɔde ahyɛ mu no wɔ nhyɛso a ɛyɛ 20 psi ase wɔ ~2800 events/sec. Esiane sɛ atetesɛm gating gyinapɛn (nkwaboaa kɛse, bimodal nyiyim, ne apete su) ntumi nhwɛ mma wɔbɛma PN afi nkwammoaa ahorow afoforo ho yiye nti, wɔde gating nhyehyɛe no gyina YFP ahoɔden ne autofluorescence a wɔde toto ho tẽẽ wɔ mitoYFP + ne control mitoYFP − Mice mu no so. YFP ani gye denam 488 nm laser line a wɔde hyerɛn nhwɛsode no so, na wɔde 530/30 nm band pass filter na ɛkyerɛ sɛnkyerɛnne no. Wɔ mitoYFP + mprako mu no, wɔde ahoɔden a ɛwɔ Rosa26-mitoYFP amanneɛbɔfo awosu mu abɔde mu nkwaadɔm no nso di dwuma de kyerɛ nsonsonoe a ɛda ntini nipadua ne axon asinasin ntam. 7-AAD no de 561 nm yellow laser ani gye na wɔde 675/20 nm bandpass filter hu de yi nkwammoaa a awuwu fi mu. Sɛnea ɛbɛyɛ a wɔbɛtetew astrocytes mu bere koro no ara mu no, wɔde ACSA-2-APC yɛɛ nkwammoaa a wɔde ahyɛ mu no ho fĩ, afei wɔde 640 nm laser line hyerɛn nhwɛsode no so, na wɔde 660/20 nm bandpass filter dii dwuma de huu sɛnkyerɛnne no.
Wɔde centrifugation (1110 rpm, simma 5, 4°C) yɛɛ nkwammoaa a wɔaboaboa ano no pelleted na wɔde siee -80°C kosii sɛ wɔde bedi dwuma. Wɔkyekyɛ Mfn2cKO mprako ne wɔn nwura mma mu da koro na ama nsakrae a ɛba wɔ ɔkwan a wɔfa so yɛ no mu no ayɛ ketewaa bi. Wɔde FlowJo software (FlowJo LLC, Ashland, Oregon, USA) na ɛyɛɛ FACS data nkyerɛkyerɛmu ne nhwehwɛmu.
Sɛnea yɛaka wɔ atifi hɔ (59) no, wɔde bere ankasa mu PCR di dwuma de yi DNA fi ntini a wɔahyehyɛ no mu ma mtDNA dodow a edi hɔ. Mfiase no wɔsɔɔ linearity ne threshold sensitivity hwɛe denam qPCR a wɔde dii dwuma wɔ nkwammoaa dodow ahorow so no so. Ne tiawa mu no, boaboa PN 300 ano wɔ lysis buffer a 50 mM tris-HCl (pH 8.5), 1 mM EDTA, 0.5% Tween 20 ne proteinase K (200 ng/ml) wom mu na fa to 55°C simma 120. Wɔsan de nkwammoaa no too 95°C simma 10 de hwɛ hui sɛ proteinase K no ntumi nyɛ adwuma koraa. Nyansahu, katalog nɔma Mm04225274_s1), mt-Nd6 (Thermo Fisher Scientific, katalog nɔma AIVI3E8) ne 18S (Thermo Fisher Scientific, katalog nɔma Hs99999901_s1) awosu mu nkwaadɔm.
Proteome nhwɛsode ahosiesie. Ɛdenam ano aduru no a wɔma ɛyɛ hyew wɔ 95°C simma 10 ne sonicating so no, wɔ lysis buffer [6 M guanidine chloride, 10 mM tris (2-carboxyethyl) phosphine hydrochloride, 10 mM chloroacetamide ne 100 mM tris- Lyse frozen neuron pellets wɔ HCl mu]. Wɔ Bioruptor (Diagenode) so simma 10 (sekan 30 pulse / sikani 30 pause bere). Wɔde 1:10 guu nhwɛsode no mu wɔ 20 mM tris-HCl (pH 8.0) mu, de 300 ng trypsin gold (Promega) fraa mu, na wɔde too mu anadwo wɔ 37°C mu sɛnea ɛbɛyɛ a ɛbɛyɛ nea ɛyɛ mmerɛw koraa. Da a ɛto so abien no, wɔde nhwɛsode no guu centrifuge mu wɔ 20,000 g mu simma 20. Wɔde formic acid 0.1% guu nsu a ɛwɔ soro no mu, na wɔde StageTips a wɔn ankasa ayɛ no yii nkyene fii aduru no mu. Wɔde nhwɛsode no wee wɔ SpeedVac adwinnade (Eppendorf concentrator plus 5305) mu wɔ 45°C, na afei wɔde peptide no sɛn 0.1% formic acid mu. Onipa koro na ɔyɛɛ nhwɛsode ahorow no nyinaa bere koro mu. Sɛnea ɛbɛyɛ a wɔbɛhwehwɛ astrocyte nhwɛsode ahorow mu no, wɔde tandem mass tag (TMT10plex, catalog number 90110, Thermo Fisher Scientific) a peptide ne TMT reagent ratio yɛ 1:20 kyerɛw peptide a wɔayi nkyene afi mu 4 μg. Wɔ TMT nkyerɛwde ho no, wɔsan de TMT reagent 0.8 mg guu ACN a nsu nnim 70 μl mu, na wɔsan hyehyɛɛ peptide a ayow no yɛɛ no ​​9 μl 0.1 M TEAB (triethylammonium bicarbonate), a wɔde TMT reagent 7 μl a ɛwɔ ACN mu guu mu. Nneɛma a ɛwɔ mu no yɛ 43.75%. Bere a wɔde simma 60 ahyɛ mu akyi no, wɔde 2 μl 5% hydroxylamine dum adeyɛ no. Wɔboaboaa peptide a wɔakyerɛw so no ano, wee, san de guu 200μl a ɛyɛ 0.1% formic acid (FA) mu, kyekyɛɛ mu abien, na afei wɔde StageTips a wɔn ankasa ayɛ na yii nkyene fii mu. Wɔde UltiMate 3000 ultra high performance liquid chromatograph (UltiMate 3000 ultra high performance liquid chromatograph) dii dwuma no, wɔkyekyɛɛ fã abien no mu biako mu wɔ 1mm x 150mm Acquity chromatographic column a 130Å1.7μm C18 asinasin ahyɛ mu ma (Waters, catalog No. SKU: 186006935). Thermo Fisher Nyansahu mu Ɔbenfo). Tetew peptides mu wɔ nsuo a ɛsen 30μl/min, tew firi 1% kɔsi 50% buffer B simma 85 a anammɔn anammɔn gradient yɛ simma 96, firi 50% kɔsi 95% buffer B simma 3, afei simma 8 ma 95 % Buffer B; Buffer A yɛ 5% ACN ne 10 mM ammonium bicarbonate (ABC), na buffer B yɛ 80% ACN ne 10 mM ABC. Boaboa afã horow no ano simma 3 biara na fa bom yɛ akuw abien (1 + 17, 2 + 18, ne nea ɛkeka ho) na fa vacuum centrifuge ma ɛyow.
LC-MS/MS nhwehwɛmu. Wɔ mass spectrometry mu no, wɔtetew peptides (nɔma r119.aq) no mu wɔ 25 cm, 75 μm mu ntwemu PicoFrit analytical column (botae foforo lens, ɔfã nɔma PF7508250) a wɔde 1.9 μm ReproSil-Pur 120 C18-AQ medium (Dr. Maisch, mat), de EASY-nLC dii dwuma 1200 (Thermo Fisher Nyansahu Nhoma, Germany). Wɔmaa adum no yɛɛ 50°C. Buffers A ne B yɛ 0.1% formic acid wɔ nsuo mu ne 0.1% formic acid wɔ 80% ACN mu, sɛdeɛ ɛteɛ biara. Wɔyii peptides firii 6% kɔsi 31% buffer B simma 65 ne 31% kɔsi 50% buffer B simma 5 a wɔde gradient a ɛyɛ 200 nl/min. Wɔde Orbitrap Fusion mass spectrometer (Thermo Fisher Scientific) yɛɛ peptide ahorow a wɔayiyi no mu nhwehwɛmu. Wɔyɛ peptide precursor m/z susuw a ɛwɔ nsusuwii a ɛyɛ 120,000 wɔ 350 kosi 1500 m/z mu. Sɛ wɔde 27% normalized collision energy di dwuma a, wɔpaw precursor a ɛyɛ den sen biara a ɛwɔ charge tebea 2 kosi 6 ma ahoɔden a ɛkorɔn C trap dissociation (HCD) cleavage. Wɔde cycle bere no asi hɔ sɛ 1 s. Wɔde AGC botaeɛ ketewa a ɛyɛ 5×104 ne berɛ a ɛkyɛn so a wɔde bɛhyɛ mu a ɛyɛ 86 ms na ɛsusuu m/z boɔ a ɛwɔ peptide asinasin no mu wɔ ion afiri no mu. Wɔ mpaapaemu akyi no, wɔde nea edi kan no too dynamic exclusion list no so 45 s. Wɔtetew peptide ahorow a wɔahyɛ no agyirae wɔ 50 cm, 75 μm Acclaim PepMap adum (Thermo Fisher Scientific, katalog nɔma 164942) so, na wɔyɛɛ atutra spektro no mu nhwehwɛmu wɔ Orbitrap Lumos Tribrid mass spectrometer (Thermo Fisher Scientific) a wɔde high-field asymmetric waveform ions (FAIMS) mfiri ahyɛ mu so (Thermo Fisher Scientific) yɛ adwuma wɔ compensation voltages abien a ɛyɛ −50 ne −70 V. MS3 a wɔapaw a egyina synchronization precursor so no na wɔde di dwuma ma TMT amanneɛbɔ ion sɛnkyerɛnne susuw. Wɔyɛɛ peptide mpaapaemu no wɔ EASY-nLC 1200 so, de 90% linear gradient elution dii dwuma, a buffer dodow yɛ 6% kosi 31%; buffer A yɛ 0.1% FA, na buffer B yɛ 0.1% FA ne 80% ACN. Wɔde analytical column no yɛ adwuma wɔ 50°C. Fa FreeStyle (version 1.6, Thermo Fisher Scientific) kyekyɛ fael a edi kan no mu sɛnea FAIMS akatua ahoɔden te.
Protein a wɔde hu ne dodow a wɔde kyerɛ. Wɔde Andromeda nhwehwɛmu afiri a wɔaka abom no dii dwuma de yɛɛ mfitiaseɛ data no mu nhwehwɛmu denam MaxQuant version 1.5.2.8 (https://maxquant.org/) so. Wɔ Cre recombinase ne YFP ntoatoasoɔ a wɔnya firii Aequorea victoria mu akyi no, wɔhwehwɛɛ peptide asinasin spectra hwehwɛɛ canonical ntoatoasoɔ ne isoform ntoatoasoɔ a ɛwɔ mouse reference proteome (Proteome ID UP000000589, a wɔtwe fii UniProt wɔ May 2017) Wɔde methionine oxidation ne protein N-terminal acetylation sii hɔ sɛ nsakrae a ɛsakra; wɔde cysteine ​​carbamoyl methylation sii hɔ sɛ nsakrae a wɔahyɛ da ayɛ. Wɔde digestion parameters no asi hɔ sɛ “specificity” ne “trypsin/P”. Peptide ne razor peptide dodow a ɛba fam koraa a wɔde di dwuma de hu protein ne 1; peptide soronko dodow a ɛba fam koraa ne 0. Wɔ tebea horow a peptide map hyia mu no, na protein a wohu no dodow yɛ 0.01. “Second Peptide” a wobɛpaw no ayɛ adwuma. Fa “match between runs” option no di dwuma fa de nkyerɛkyerɛmu a edi mu kɔ mfitiase fael ahorow ntam. Fa LFQ ratio a ɛsua koraa 1 di dwuma ma label-free quantification (LFQ) (60). Wɔde LFQ ahoɔden no yiyi mu ma anyɛ yiye koraa no gyinapɛn abien a ɛfata wɔ anyɛ yiye koraa no genotype kuw biako mu wɔ bere biara mu, na wɔde extrapolated fi normal distribution a ne trɛw yɛ. 0.3 na ɛkɔ fam 1.8. Fa Perseus kɔmputa so nhyehyɛe (https://maxquant.net/perseus/) ne R (https://r-project.org/) di dwuma de hwehwɛ LFQ aba no mu. Wɔde akwan abien a ɛyɛ mmerɛw t sɔhwɛ a efi limma software package no dii dwuma maa nsonsonoe nkyerɛkyerɛmu nhwehwɛmu ( 61 ). Wɔde ggplot, FactoMineR, factoextra, GGally ne pheatmap na ɛyɛ nhwehwɛmu data nhwehwɛmu. Wɔde MaxQuant version 1.6.10.43 na ɛyɛɛ proteomics data a egyina TMT so no mu nhwehwɛmu. Hwehwɛ raw proteomics data fi UniProt nnipa proteomics database, a wɔtwee wɔ September 2018. Nhwehwɛmu no ka isotope ahotew nteɛso factor a nea ɔyɛe no de mae no ho. Fa limma di dwuma wɔ R mu ma nsonsonoe nkyerɛkyerɛmu nhwehwɛmu. Wɔde mfitiaseɛ data, database hwehwɛ aba, ne data nhwehwɛmu adwumayɛ ne nea ɛfiri mu ba nyinaa sie wɔ ProteomeXchange apam no mu denam PRIDE ahokafoɔ akoraeɛ a ɛwɔ data set identifier PXD019690 so.
Dwumadi mu nkyerɛkyerɛmu ma nhwehwɛmu no yɛ kɛse. Wɔde Ingenuity Pathway Analysis (QIAGEN) adwinnade no dii dwuma de kyerɛɛ sɛnea nsɛm a wɔde kyerɛw nsɛm a ɛyɛ adwuma wɔ data a wɔahyehyɛ no mu wɔ adapɛn 8 mu no dɔɔso (Mfonini 1). Ne tiawa mu no, wɔde protein dodow a wɔahyehyɛ a wonya fii LC-MS/MS (tandem mass spectrometry) data nhwehwɛmu mu no di dwuma denam filter criteria a edidi so yi so: Wɔpaw Mus musculus sɛ ne su ne akyi, na ɔfã no kyerɛ P bo a Benjamini ayɛ nsakrae wɔ enrichment 0.05 anaa nea ɛba fam a wobu no sɛ ɛho hia. Wɔ saa graph yi mu no, wɔakyerɛ akuo anum a ɛboro soɔ a ɛwɔ akuakuo biara mu a egyina P boɔ a wɔayɛ nsakraeɛ so. Wɔde multiple t-test di dwuma, de Benjamini, Krieger, ne Yekutieli (Q = 5%) linear boost nhyehyɛe a ɛwɔ afã abien (Q = 5%) di dwuma no, wɔyɛ bere-kɔ so protein nkyerɛkyerɛmu nhwehwɛmu wɔ wɔn a wɔho hia a wɔakyerɛ wɔn wɔ ɔfã biara mu no so, na wɔyɛ row biara mu nhwehwɛmu wɔ ɔkwan soronko so. Ɛho nhia sɛ wogye SD a ɛkɔ so daa tom.
Sɛnea ɛbɛyɛ a yɛde nea efii nhwehwɛmu yi mu bae bɛtoto databea ahorow a wɔatintim ho na yɛayɛ Venn mfonini wɔ Mfonini 1 mu no, yɛde protein dodow a wɔahyehyɛ no boom ne MitoCarta 2.0 nkyerɛkyerɛmu ahorow ( 24 ). Fa intanɛt so adwinnade Draw Venn Diagram (http://bioinformatics.psb.ugent.be/webtools/Venn/) yɛ mfonini no.
Sɛ wopɛ akontaabu akwan a wɔfa so yɛ proteomics nhwehwɛmu ho nsɛm a ɛkɔ akyiri a, yɛsrɛ wo hwɛ Ɔfã a ɛne no hyia a ɛne Nneɛma ne Akwan a wɔfa so yɛ no. Wɔ sɔhwɛ afoforo nyinaa mu no, wobetumi ahu nsɛm a ɛkɔ akyiri wɔ anansesɛm a ɛne no hyia no mu. Gye sɛ wɔakyerɛ sɛ ɛnte saa, wɔda nsɛm a wɔde ama no nyinaa adi sɛ mfimfini ± SEM, na wɔde GraphPad Prism 8.1.2 softwea na ɛyɛɛ akontabuo nhwehwɛmu nyinaa.
Sɛ wopɛ nneɛma foforo a wode bɛyɛ saa asɛm yi a, yɛsrɛ wo hwɛ http://advances.sciencemag.org/cgi/content/full/6/35/eaba8271/DC1
Eyi yɛ asɛm a wɔabue ano a wɔkyekyɛ wɔ Creative Commons Attribution-Non-Commercial License no nsɛm ase, a ɛma kwan ma wɔde di dwuma, kyekyɛ na wɔsan yɛ wɔ ɔkwan biara so, bere tenten a nea etwa to a wɔde bedi dwuma no nyɛ aguadi mu mfaso na adwene no ne sɛ mfitiase adwuma no teɛ. Mmoa nwoma.
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E. Motori, I. Atanassov, S. M. V. Kochan, K. Folz-Donahue, V. Sakthivelu, P. Giavalisco, N. Toni, J. Puyal, N.-G., na wɔkyerɛwee. Larson na ɔkyerɛwee
Proteomics nhwehwɛmu a wɔyɛe wɔ ntini a entumi nyɛ adwuma yiye ho no daa no adi sɛ wɔma nipadua mu nneɛma a ɛsakra no nhyehyɛe ahorow yɛ adwuma de siw ntini a ɛyɛ mmerɛw ano.
E. Motori, I. Atanassov, S. M. V. Kochan, K. Folz-Donahue, V. Sakthivelu, P. Giavalisco, N. Toni, J. Puyal, N.-G., na wɔkyerɛwee. Larson na ɔkyerɛwee
Proteomics nhwehwɛmu a wɔyɛe wɔ ntini a entumi nyɛ adwuma yiye ho no daa no adi sɛ wɔma nipadua mu nneɛma a ɛsakra no nhyehyɛe ahorow yɛ adwuma de siw ntini a ɛyɛ mmerɛw ano.
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