Ināianei *Wāhitau o nāianei: Cologne 50931, Tiamana, Cologne Excellence Cluster Research on Cellular Stress Response in Pakeketanga-E pā ana ki ngā Mate (CECAD).
E kiia ana ko te ngoikore o te pūtau o ngā mate mitochondrial he mea kore e taea te whakakore nā te mea he iti te kirihou o te pūtau io, engari kāore i te tino mārama te pānga o te ngoikore o te pūtau mitochondrial ki te mana motuhake o te pūtau o te pākia o te pūtau io i roto i te tinana. I konei, ka whakaurua e mātou te proteome motuhake-pūtau o ngā pūtau io Purkinje me te ngoikore haere o te OXPHOS i puta mai i te whakararuraru o te hononga mitochondrial. I kitea e mātou ko te ngoikore o te pūtau mitochondrial i whakaoho i te huringa nui i roto i te mara o te proteomics, i te mutunga ka arahina ki te whakahohenga raupapa o ngā kaupapa pākia tika i mua i te matenga o te pūtau. I te mea ohorere, i kitea e mātou te whakaoho mārama o te pyruvate carboxylase (PCx) me ētahi atu whākōkī ārai-pakeke e tāpiri ana ki ngā takawaenga o te huringa TCA. Ko te aukati i te PCx i whakanui ake i te ahotea oxidative me te neurodegeneration, e tohu ana he pānga tiaki te atherosclerosis i roto i ngā pūtau io kāore he OXPHOS. Ko te whakahokinga mai o te hononga mitochondrial i roto i ngā pūtau io kua pirau ka tino hurihia ēnei āhuatanga pākia, ā, ka ārai i te matenga o te pūtau. Ka tautuhia e ā mātou kitenga ngā ara kāore i te mōhiotia i mua e whakarato ana i te manawanui ki te ngoikore o te pūtau mitochondrial, ā, e whakaatu ana ka taea te whakakore i te neurodegeneration ahakoa i ngā wāhanga whakamutunga o te mate.
Ko te tūranga matua o te mitochondria ki te pupuri i te pākia o te pūngao neuronal e whakanuia ana e ngā tohu neurological whānui e pā ana ki ngā mate mitochondrial tangata. Ko te nuinga o ēnei mate ka puta mai i ngā whakarerekētanga ira e whakahaere ana i te whakaaturanga ira mitochondrial (1, 2) te whakangaromanga ira rānei e pā ana ki ngā mahi mitochondrial, e pā kino ana ki te pumau o te DNA mitochondrial (mtDNA) (3, 4). Kua whakaatuhia e te mahi i roto i ngā tauira kararehe hei whakautu ki te ngoikore o te mitochondrial i roto i ngā kiko e karapoti ana, ka taea te whakahohe i ngā ara pākia ā-pūmau (5-7), e whakarato ana i ngā mōhiohio nui mō te māramatanga hohonu ki te pathogenesis o ēnei mate uaua. He rerekē rawa atu, ko tō tātou māramatanga ki ngā huringa pākia o ngā momo pūtau motuhake i puta mai i te korenga whānui o te hanga adenosine triphosphate (ATP) mitochondrial roro he mea nui (8), e whakanui ana i te hiahia ki te tautuhi i ngā whāinga rongoā ka taea te whakamahi hei ārai, hei ārai rānei i te mate. Ārai i te neurodegeneration (9). Ko te kore o ngā mōhiohio ko te meka e kiia whānuitia ana he iti rawa te ngāwari pākia o ngā pūtau io ki ngā momo pūtau o ngā kiko e karapoti ana (10). I te mea he mahi nui tā ēnei pūtau ki te whakarite i te tuku o ngā matū matū ki ngā neuron hei whakatairanga i te tuku hononga honohono me te urupare ki ngā āhuatanga whara me ngā mate, ko te kaha ki te urutau i te pākia o te pūtau ki ngā āhuatanga uaua o te kiko roro he tata ki ngā pūtau glial anake (11-14). Hei tāpiri, ko te rerekētanga pūtau o te kiko roro e whakararuraru nui ana i te rangahau i ngā huringa pākia e puta ana i roto i ngā rōpū neuron motuhake. Nō reira, he iti noa te mōhiotia mō ngā hua tuturu o te kore mahi mitochondrial i roto i ngā neuron.
Hei mārama ki ngā hua o te koretake o te mahi mitochondrial, i wehea e mātou ngā neuron Purkinje (PN) i ngā wāhanga rerekē o te neurodegeneration i puta mai i te whakangaromanga o te hononga membrane o waho o te mitochondrial (Mfn2). Ahakoa he hononga ngā whakarerekētanga Mfn2 i roto i te tangata ki tētahi momo neuropathy sensory motor ira e mōhiotia ana ko te momo Charcot-Marie-Tooth 2A (15), ko te whakangaromanga ā-tikanga o Mfn2 i roto i ngā kiore he tikanga induction of oxidation Phosphorylation (OXPHOS) dysfunction e mōhiotia whānuitia ana. Ko ngā momo neuronal momo (16-19) me te momo neurodegenerative hua ka haere tahi me ngā tohu neurological ahunga whakamua, pērā i ngā mate nekehanga (18, 19) te cerebellar ataxia rānei (16). Mā te whakamahi i te huinga o te proteomics quantitative kore-tapanga (LFQ), metabolomics, whakaahua, me ngā tikanga virological, ka whakaatuhia e mātou ko te neurodegeneration ahunga whakamua ka kaha te whakaoho i te pyruvate carboxylase (PCx) me ētahi atu mea e uru ana ki te arteriosclerosis o ngā PN in vivo Te whakaaturanga o ngā whākōkī. Hei whakamana i te hiranga o tēnei kitenga, i whakaitihia e mātou te whakaaturanga o PCx i roto i ngā PN ngoikore-Mfn2, ā, i kitea ko tēnei mahi i whakanui ake i te ahotea waikura me te tere ake o te neurodegeneration, ā, e whakaatu ana ko te azoospermia ka homai te mate pūtau. Te urutau metabolic. Ka taea e te whakaaturanga nui o MFN2 te whakaora katoa i te PN pirau mutunga me te ngoikore nui o OXPHOS, te kai nui o te DNA mitochondrial, me te whatunga mitochondrial pakaru, e whakanui ana i te kaha o tēnei momo neurodegeneration ki te ora anō i te wāhanga matatau o te mate i mua i te matenga o te pūtau.
Hei tiro i ngā mitochondria i roto i ngā Mfn2 knockout PN, i whakamahia e mātou he momo kiore e āhei ai ngā mitochondria Cre-dependent ki te aro ki te pūmua fluorescent kōwhai (YFP) (mtYFP) (20) Cre expression, ā, i tirohia te āhua o te mitochondrial in vivo. I kitea e mātou ko te whakangaromanga o te ira Mfn2 i roto i ngā PN ka arahi ki te wehenga āta haere o te whatunga mitochondrial (Pikitia S1A), ā, i kitea te huringa tuatahi i te 3 wiki o te pakeketanga. He rerekē, ko te pirau nui o te paparanga pūtau PN, e whakaatuhia ana e te ngaronga o te Calbindin immunostaining, kāore i tīmata tae noa ki te 12 wiki o te pakeketanga (Pikitia 1, A me B). Ko te kore taurite o te wā i waenga i ngā huringa tuatahi o te āhua o te mitochondrial me te tīmatanga kitea o te mate neuronal i akiaki i a mātou ki te rangahau i ngā huringa metabolic i puta mai i te ngoikore o te mitochondrial i mua i te matenga o te pūtau. I whakawhanakehia e mātou he rautaki whakarōpū pūtau whakahohe-whakaata (FACS) hei wehe i te PN e whakaatu ana i te YFP (YFP+) (Pikitia 1C), me ngā kiore whakahaere (Mfn2 + / loxP :: mtYFP loxP- stop-loxP: : L7-cre), e kiia nei i muri nei ko CTRL (Pikitia S1B). Mā te arotautanga o te rautaki kuaha i runga i te kaha whanaunga o te tohu YFP ka taea e mātou te pure i te tinana YFP+ (YFPhigh) o ngā PN mai i ngā kore-PN (YFPneg) (Pikitia S1B) ngā kongakonga axon/dendritic fluorescent pea rānei (YFPlow; Pikitia S1D, maui), i whakaūtia e te karuārai confocal (Pikitia S1D, matau). Hei manatoko i te tuakiri o te taupori kua whakarōpūtia, i whakahaerehia e mātou he proteomics LFQ me te tātaritanga wāhanga matua, ā, i kitea he wehewehenga mārama i waenga i ngā pūtau YFPhigh me YFPneg (Pikitia S1C). I whakaatuhia e ngā pūtau YFPhigh te whakarei ake i ngā tohu PN e mōhiotia ana (arā, Calb1, Pcp2, Grid2 me Itpr3) (21, 22), engari kāore he whakarei ake i ngā pūmua e whakaatuhia ana i roto i ngā neuron, i ētahi atu momo pūtau rānei (Pikitia 1D)). I whakaatuhia e te whakataurite i waenga i ngā tauira i roto i ngā pūtau YFPhigh kua whakarōpūtia i kohia i roto i ngā whakamātautau motuhake he tauwehenga hononga > 0.9, e whakaatu ana i te pai o te whakaputa uri i waenga i ngā tārua koiora (Pikitia S1E). Hei whakarāpopototanga, i whakamanahia e ēnei raraunga tā mātou mahere mō te wehenga ohorere me te motuhake o te PN ka taea. Nā te mea ko te pūnaha taraiwa L7-cre i whakamahia ka whakaoho i te whakakotahitanga mosaic i te wiki tuatahi i muri i te whānautanga (23), ka tīmata mātou ki te tango i ngā kiore mai i te CTRL me te (Mfn2 loxP / loxP :: mtYFP loxP-stop-loxP :: L7-cre) Kohikohi neurons. I muri i te otinga o te whakakotahitanga, ka kiia ko Mfn2cKO i te 4 wiki o te pakeke. Hei whakamutunga, i whiriwhiria e mātou te 8 wiki o te pakeketanga i te wā e mau tonu ana te paparanga PN ahakoa te pakaru o te mitochondrial (Pikitia 1B me te Pikitia S1A). I ​​te katoa, i tatauhia e mātou he 3013 ngā pūmua katoa, ā, tata ki te 22% o ēnei i ahu mai i ngā tuhipoka MitoCarta 2.0 i runga i te proteome mitochondrial hei mitochondria (Pikitia 1E) (Pikitia 1E) (24). Ko te tātaritanga whakapuakanga ira rerekē i mahia i te wiki 8 i whakaatu ko te 10.5% anake o ngā pūmua katoa he nui ngā huringa (Pikitia 1F me te Pikitia S1F), ā, 195 ngā pūmua i whakahekehia te taumata, ā, 120 ngā pūmua i piki ake te taumata (Pikitia 1F). He mea tika kia mōhio ko te "tātaritanga ara auaha" o tēnei huinga raraunga e whakaatu ana ko ngā ira rerekē te whakapuakanga he mea nō tētahi huinga iti o ngā ara pākia motuhake (Pikitia 1G). He mea whakamīharo, ahakoa te whakahekenga o ngā ara e pā ana ki te OXPHOS me te tohu konupūmā e whakaū ana i te whakaoho o te ngoikore o te mitochondrial i roto i ngā PN ngoikore-whakakotahi, ko ētahi atu kāwai e uru nui ana ki te pākia o te waikawa amino ka tino piki ake, e hangai ana ki te pākia e puta ana i roto i ngā PN mitochondrial. He rite tonu te ngoikore o te hononga.
(A) Ngā whakaahua huna o ngā wāhanga roro iti o ngā kiore CTRL me Mfn2cKO e whakaatu ana i te ngaronga haere o ngā PN (calbindin, hina); i tāpirihia ngā karihi ki te DAPI. (B) Te ine i te (A) (tātari rerekētanga kotahi-ara, ***P<0.001; n = 4 ki te 6 porowhita mai i ngā kiore e toru). (C) Te rere mahi whakamātautau. (D) Te tohatoha mahere wera o ngā tohu motuhake ki a Purkinje (runga) me ētahi atu momo pūtau (waenganui). (E) Te tūtohi Venn e whakaatu ana i te maha o ngā pūmua mitochondrial i tautuhia i roto i te PN kua whakarōpūtia. (F) Te kauwhata puia o ngā pūmua rerekē te whakaaturanga i roto i ngā neuron Mfn2cKO i te 8 wiki (te uara tapahi hiranga o te 1.3). (G) E whakaatu ana te tātari ara auaha i ngā ara e rima tino nui o te whakahaere-ake (whero) me te whakahaere-iho (kikorangi) i roto i te PN Mfn2cKO kua whakarōpūtia hei 8 wiki. Ka whakaaturia te taumata whakaaturanga toharite o ia pūmua kua kitea. Mahere wera hina-tauine: uara P kua whakatikatikaina. ns, kāore i te hiranga.
I whakaatuhia e ngā raraunga proteomics te hekenga haere o te whakaaturanga pūmua o ngā matatini I, III, me te IV. Kei roto i ngā matatini I, III, me te IV ngā waeine matua kua whakakōwaetia e te mtDNA, ko te matatini II, he mea whakakōwaetia e te karihi anake, kāore i pāngia (Pikitia 2A me te Pikitia S2A). . E ai ki ngā hua proteomics, i whakaatuhia e te immunohistochemistry o ngā wāhanga kiko cerebellar te hekenga haere o te taumata waeine MTCO1 (mitochondrial cytochrome C oxidase waeine 1) o te matatini IV i roto i te PN (Pikitia 2B). I tino heke te waeine Mtatp8 kua whakakōwaetia e te mtDNA (Pikitia S2A), ko te taumata pumau o te waeine ATP synthase kua whakakōwaetia e te karihi i noho tonu kāore i rerekē, e rite ana ki te matatini F1 ATP synthase pumau e mōhiotia ana ina pumau te whakaaturanga mtDNA. He rite tonu te hanganga. Whakamutu (7). I whakaūtia e te aromatawai i te taumata mtDNA i roto i ngā Mfn2cKO PN kua whakarōpūtia mā te tauhohenga mekameka polymerase wā-tūturu (qPCR) te hekenga āta haere o te maha o ngā tārua mtDNA. Ki te whakatauritea ki te rōpū whakahaere, i te 8 wiki o te pakeketanga, tata ki te 20% noa iho o te taumata mtDNA i puritia (Pikitia 2C). E ai ki ēnei hua, i whakamahia te tae microscopy confocal o ngā Mfn2cKO PN hei kite i te DNA, e whakaatu ana i te kai i ngā nucleotides mitochondrial e whakawhirinaki ana ki te wā (Pikitia 2D). I kitea e mātou ko ētahi noa iho o ngā kaitono e uru ana ki te whakahekenga pūmua mitochondrial me te urupare ahotea i piki ake te whakahaere, tae atu ki a Lonp1, Afg3l2 me Clpx, me ngā āhuatanga huihuinga matatini OXPHOS. Kāore i kitea he huringa nui i roto i ngā taumata o ngā pūmua e uru ana ki te apoptosis (Pikitia S2B). Waihoki, i kitea e mātou he iti noa iho ngā huringa o ngā hongere mitochondria me ngā hongere reticulum endoplasmic e uru ana ki te kawe konupūmā (Pikitia S2C). Hei tāpiritanga, kāore i kitea he huringa nui i roto i te aromatawai i ngā pūmua e pā ana ki te autophagy, he mea e rite ana ki te whakaurunga kitea o ngā autophagosome i kitea i roto i te tinana ora e te immunohistochemistry me te electron microscopy (Pikitia S3). Heoi, ko te ngoikore haere o te OXPHOS i roto i ngā PN e haere tahi ana me ngā huringa mitochondrial ultrastructure kitea. Ka kitea ngā tautau mitochondrial i roto i ngā tinana pūtau me ngā rākau dendritic o ngā Mfn2cKO PN e 5 me te 8 wiki te pakeke, ā, kua pāngia te hanganga membrane o roto e ngā huringa nui (Pikitia S4, A me B). E rite ana ki ēnei huringa ultrastructure me te hekenga nui o te mtDNA, i whakaatuhia e te tātaritanga o ngā poro cerebellar cerebral acute me te tetramethylrhodamine methyl ester (TMRM) kua tino heke te pūmanawa membrane mitochondrial i roto i ngā Mfn2cKO PN (Pikitia S4C).
(A) Tātaritanga wā o te taumata whakaaturanga o te matatini OXPHOS. Whakaarohia noa ngā pūmua me te P<0.05 i te 8 wiki (ANOVA rua-ara). Rārangi iraira: Kāore he whakatikatika i whakaritea ki te CTRL. (B) Mauī: He tauira o te wāhanga roro iti kua tohua ki te paturopi anti-MTCO1 (pae tauine, 20 μm). Ko te rohe e nohoia ana e ngā tinana pūtau Purkinje kua hipokina e te kōwhai. Matau: Te ine i ngā taumata MTCO1 (tātaritanga rerekētanga kotahi-ara; n = 7 ki te 20 pūtau i tātarihia mai i ngā kiore e toru). (C) Tātaritanga qPCR o te tau tārua mtDNA i roto i te PN kua whakarōpūtia (tātaritanga rerekētanga kotahi-ara; n = 3 ki te 7 kiore). (D) Mauī: He tauira o te poro roro iti kua tohua ki te paturopi anti-DNA (pae tauine, 20 μm). Ko te rohe e nohoia ana e ngā tinana pūtau Purkinje kua hipokina e te kōwhai. Matau: Te ine i ngā whara mtDNA (tātaritanga rerekētanga kotahi-ara; n = 5 ki te 9 pūtau mai i ngā kiore e toru). (E) He tauira o te wāhanga roro nui e whakaatu ana i ngā pūtau mitoYFP + Purkinje (pere) i roto i te tuhi rawhi papaki pūtau-katoa. (F) Te ine i te pihi IV. (G) Ngā tuhi tohu o te werohanga iahiko depolarizing i roto i ngā pūtau Purkinje CTRL me Mfn2cKO. Aromatawai o runga: Ko te pao tuatahi i whakaoho i te AP. Aromatawai o raro: Auautanga AP mōrahi. (H) Te ine i ngā whakaurunga tūpono postsynaptic (sPSP). Ko te aromatawai tuhi tohu me tōna ōwehenga whakanui e whakaaturia ana i roto i te (I). Te tātaritanga kotahi-ara o te rerekētanga i tātarihia n = 5 ki te 20 pūtau mai i ngā kiore e toru. Ka whakaatuhia ngā raraunga hei toharite ± SEM; *P<0.05; **P<0.01; ***P<0.001. (J) Ngā tohu tohu o te AP tūpono i tuhia mā te whakamahi i te aratau rawhi papaki poka. Aromatawai o runga: Auautanga AP mōrahi. Aromatawai o raro: whakanui o te AP kotahi. (K) Te ine i te auau AP toharite me te mōrahi e ai ki (J). Whakamātautau Mann-Whitney; n = 5 ngā pūtau i tātarihia mai i ngā kiore e whā. Ka whakaatuhia ngā raraunga hei toharite ± SEM; kāore i te hiranga.
I kitea te kino OXPHOS mārama i roto i te Mfn2cKO PN 8-wiki te pakeke, e tohu ana he tino rerekē te mahi ā-tinana o ngā neurons. Nō reira, i tātarihia e mātou ngā āhuatanga hiko korekore o ngā neurons OXPHOS-deficient i te 4 ki te 5 wiki me te 7 ki te 8 wiki mā te mahi i ngā rekoata papaki pūtau-katoa i roto i ngā poro cerebellar acute (Pikitia 2E). I te mea ohorere, he rite te pūmanawa membrane okiokinga toharite me te ātete whakauru o ngā neurons Mfn2cKO ki te mana whakahaere, ahakoa he rerekētanga ngawari i waenga i ngā pūtau (Ripanga 1). Waihoki, i te 4 ki te 5 wiki o te pakeke, kāore i kitea he huringa nui i roto i te whanaungatanga au-ngaohiko (piko IV) (Pikitia 2F). Heoi, kāore he neurons Mfn2cKO 7 ki te 8 wiki te pakeke i ora i te regimen IV (taahiraa hyperpolarization), e tohu ana he mārama te aro ki te pūmanawa hyperpolarization i tēnei wāhanga mutunga. He rerekē, i roto i ngā neurons Mfn2cKO, he pai te manawanui ki ngā iahiko whakaheke-polarizing e puta ai ngā tukunga pūmanawa mahi auau (AP), e tohu ana kāore ō rātou tauira tukunga whānui i te tino rerekē mai i ērā o ngā neurons whakahaere 8-wiki te pakeke (Ripanga 1 me te Pikitia 2G). Waihoki, ko te auau me te kaha o ngā iahiko postsynaptic tūpono (sPSC) he rite ki ērā o te rōpū whakahaere, ā, i piki ake te auau o ngā huihuinga mai i te 4 wiki ki te 5 wiki ki te 7 wiki ki te 8 wiki me te pikinga rite (Pikitia 2, H me te I). Te wā o te pakeketanga synaptic i roto i ngā PN (25). I whiwhihia ngā hua rite i muri i ngā papaki PN kua pokaia. Ka ārai tēnei whirihoranga i te utu pea o ngā hapa ATP pūtau, pērā i te mea ka tupu pea i roto i te tuhi papaki pūtau-katoa. Ina koa, kāore i pāngia te pūmanawa membrane okiokinga me te auau pupuhi tūpono o ngā neurons Mfn2cKO (Pikitia 2, J me te K). Hei whakarāpopototanga, e whakaatu ana ēnei hua ka taea e ngā PN me te ngoikore o te OXPHOS te aro atu ki ngā tauira tukunga auau-tiketike, e tohu ana he tikanga utu e taea ai e rātou te pupuri i ngā urupare hiko-tinana tata ki te noa.
Ka whakaatuhia ngā raraunga hei toharite ± SEM (tātari rerekētanga kotahi-ara, whakamātautau whakatairite maha a Holm-Sidak; *P<0.05). Ka tohua te tau waeine mā roto i ngā pūrua.
I tīmata mātou ki te tirotiro mēnā kei roto i tētahi kāwai i roto i te huinga raraunga proteomics (Pikitia 1G) ngā ara ka taea te ārai i te ngoikore nui o te OXPHOS, ā, mā reira ka taea e te PN e pāngia ana te pupuri i te electrophysiology tata ki te noa (Pikitia 2, E ki K). . I whakaatuhia e te tātaritanga proteomics ko ngā whākōkī e whai wāhi ana ki te catabolism o ngā waikawa amino mekameka peka (BCAA) i tino piki ake (Pikitia 3A me te Pikitia S5A), ā, ko te hua whakamutunga ko te acetyl-CoA (CoA) te succinyl CoA rānei ka taea te tāpiri i ngā tricarboxylates i roto i te huringa arteriosclerosis Acid (TCA). I ​​kitea e mātou kua piki ake te ihirangi o te BCAA transaminase 1 (BCAT1) me te BCAT2. Ka whakakorikorihia e rāua te taahiraa tuatahi o te catabolism BCAA mā te whakaputa glutamate mai i te α-ketoglutarate (26). Ka piki ake ngā wae katoa e hanga ana i te matatini keto acid dehydrogenase (BCKD) mekameka peka (ka whakaoho te matatini i te decarboxylation o muri mai me te kore e taea te huri o te anga waro BCAA) (Pikitia 3A me te Pikitia S5A). Heoi, kāore i kitea he huringa mārama i roto i te BCAA i roto i te PN kua whakarōpūtia, tera pea nā te pikinga ake o te tango pūtau o ēnei waikawa amino nui, te whakamahinga rānei o ētahi atu puna (huka, waikawa lactic rānei) hei tāpiri i te huringa TCA (Pikitia S5B). Ko ngā PN kāore i te OXPHOS i whakaatu hoki i te pikinga ake o ngā mahi wetewete glutamine me te transamination i te 8 wiki o te pakeke, ka taea te whakaata i roto i te pikinga ake o ngā whākōkī mitochondrial glutaminase (GLS) me te glutamine pyruvate transaminase 2 (GPT2) (Pikitia 3, A me C). He mea tika kia mōhiotia ko te pikinga ake o te GLS e herea ana ki te spliced ​​​​isoform glutaminase C (GLS-GAC) (ko te huringa o Mfn2cKO/CTRL he tata ki te 4.5-ngā wā, P = 0.05), ā, ko tōna pikinga ake motuhake i roto i ngā kiko matepukupuku ka taea te tautoko i te bioenergy mitochondrial. (27).
(A) E whakaatu ana te mahere wera i te panonitanga o te taumata pūmua mō te ara kua tohua i te 8 wiki. (B) Tauira o te poro cerebellar kua tohua ki te paturopi anti-PCx (pae tauine, 20 μm). E tohu ana te pere kōwhai ki te tinana pūtau Purkinje. (C) Tātaritanga whakaaturanga pūmua i te wā i tohua hei kaitono nui mō te atherosclerosis (whakamātautau-t maha, *FDR <5%; n = 3-5 kiore). (D) I runga ake: He hoahoa kauwhata e whakaatu ana i ngā huarahi rerekē o te whakauru i te waro kua tohua kei roto i te tohu [1-13C]pyruvate (arā, mā te PDH, mā te ara trans-arterial rānei). I raro: E whakaatu ana te tūtohi vaiorine i te ōrau o te waro kotahi-tohu (M1) i hurihia ki te waikawa aspartic, te waikawa citric me te waikawa malic i muri i te tohu i ngā poro cerebellar whakapeka ki te [1-13C]pyruvate (whakamātautau-t takirua; ** P <0.01). (E) Tātaritanga hītori wā whānui o te ara kua tohua. Whakaarohia noa ngā pūmua me te P<0.05 i te 8 wiki​​. Rārangi pūmau: kāore he uara whakatikatika (tātaritanga rerekētanga-rua; * P <0.05; *** P <0.001). Ka whakaatuhia ngā raraunga hei toharite±SEM.
I roto i tā mātou tātaritanga, kua riro te whakapōrearea o te BCAA hei tētahi o ngā ara matua mō te pikinga ake o te pūnaha io. E whakaatu ana tēnei meka he rerekē pea te nui o te hau e uru ana ki te huringa TCA i roto i te PN kāore he OXPHOS. Tērā pea he momo nui tēnei o te whakahounga o te pūnaha io, ka pā tika pea ki te āhua o te pūnaha io me te oranga tonutanga i te wā e mau tonu ana te ngoikore o te OXPHOS. E rite ana ki tēnei whakapae, i kitea e mātou ko te whākōkī matua anti-atherosclerotic PCx kua piki ake (ka huri te Mfn2cKO/CTRL tata ki te 1.5 ngā wā; Pikitia 3A), e whakaoho ana i te hurihanga o te pyruvate ki te oxaloacetate (28), e whakaponohia ana kei roto i te kiko roro. Ko te whakaaturanga i roto he mea here ki ngā astrocytes (29, 30). E rite ana ki ngā hua o te proteomics, i whakaatuhia e te karuārai confocal he tino piki ake te whakaaturanga PCx i roto i ngā PN kāore he OXPHOS, ko te tauhohenga PCx he mea here ki ngā pūtau glial Bergmann tata o te mana whakahaere (Pikitia 3B). Hei whakamātautau i te pikinga o te PCx i kitea, i rongoatia e mātou ngā poro cerebellar acute ki te [1-13C]pyruvate tracer. I te wā i whakahāngaihia ai te pyruvate e te pyruvate dehydrogenase (PDH), ka ngaro tōna tapanga isotope , Engari ka whakauruhia ki roto i ngā takawaenga huringa TCA ina whakahāngaihia te pyruvate e ngā tauhohenga toto (Pikitia 3D). Hei tautoko i ā mātou raraunga proteomics, i kitea e mātou he maha ngā tohu mai i tēnei tracer i roto i te waikawa aspartic o ngā poro Mfn2cKO, i te mea he āhua taurite te waikawa citric me te waikawa malic, ahakoa kāore i te tino nui (Pikitia 3D).
I roto i ngā neuron dopamine o ngā kiore MitoPark me te ngoikore o te mitochondrial i puta mai i ngā neuron dopamine e whakangaro ana i te ira mitochondrial transcription factor A (Tfam) (Pikitia S6B), i tino piki ake te whakapuakanga PCx (31), e tohu ana ko te putanga mai o te mate ka whakahaeretia i te wā e ngoikore ana te neuronal OXPHOS i roto i te tinana. He mea tika kia mōhio kua kitea ko ngā whākōkī ahurei (32-34) ka whakaatuhia pea i roto i ngā neuron e hono ana ki te arteriosclerosis ka tino piki ake i roto i ngā PN kāore i roto i te OXPHOS, pērā i te propionyl-CoA carboxylase (PCC-A), ka huri te Malonyl-CoA i te propionyl-CoA ki te succinyl-CoA me te mitochondrial malic enzyme 3 (ME3), ko tana mahi matua ko te whakaora i te pyruvate mai i te malate (Pikitia 3, A me C) (33, 35). Hei tāpiritanga, i kitea e mātou he pikinga nui o te whākōkī Pdk3, e whakapūtā ana, ā, e whakakore ana i te PDH (36), engari kāore i kitea he huringa i roto i te whākōkī Pdp1 e whakahohe ana i te PDH, i te matatini whākōkī PDH rānei (Pikitia 3A). I ​​roto i ngā PN Mern2cKO, i whakanuia te whakapūtātanga o te waeine α1 α (PDHE1α) o te wāhanga pyruvate dehydrogenase E1 o te matatini PDH i roto i te Ser293 (e mōhiotia ana hei aukati i te mahi whākōkī o te PDH) (Pikitia S6C) (Pikitia S6C). Kāore he urunga toto o te Pyruvate.
Hei whakamutunga, i kitea e mātou ko te ara super o te biosynthesis serine me te glycine, te huringa mitochondrial folate (1C) e pā ana me te biosynthesis proline (Pikitia 1G me te Pikitia S5C) kua tino piki ake, e ai ki ngā pūrongo, i te wā o te tukanga whakahohenga. Ka whakahohehia ngā kiko e karapoti ana me te korenga o te mahi mitochondrial (5-7). Ko te tātari confocal e tautoko ana i ēnei raraunga proteomics i whakaatu i roto i te PN me te ngaro o te OXPHOS, i tukuna ngā poro cerebellar o ngā kiore 8-wiki te serine hydroxymethyltransferase 2 (SHMT2), he whākōkī matua o te huringa folate mitochondrial. He urupare ārai mate nui (Pikitia S5D). I roto i te 13 poro cerebellar acute i whakatōkia ki te CU-glucose, i whakaūhia e ngā whakamātautau aroturuki metabolic te pikinga o te biosynthesis serine me te proline, e tohu ana i te pikinga o te rere o ngā isoforms waro ki roto i te serine me te proline (Pikitia S5E). I te mea ko ngā tauhohenga i whakatairangahia e GLS me GPT2 te kawenga mō te hanganga o te glutamate mai i te glutamine me te whakawhiti i waenga i te glutamate me te α-ketoglutarate, ko tā rātou pikinga ake e tohu ana he nui ake te hiahia o ngā neuron OXPHOS-deficient mō te glutamate. Tērā pea ko te whāinga o tēnei ko te pupuri i te pikinga o te biosynthesis o te proline (Pikitia S5C). He rerekē ki ēnei huringa, i whakaatuhia e te tātaritanga proteomic o ngā astrocytes cerebellar mai i ngā kiore Mfn2cKO motuhake ki te PN kāore i tino rerekē te whakaaturanga o ēnei ara (tae atu ki ngā antiperoxidases katoa), e whakaatu ana he mea whiriwhiri tēnei huringa metabolic ki te PN kua heke (Pikitia S6, D ki G).
Hei whakarāpopototanga, i whakaatuhia e ēnei tātaritanga he rerekētanga nui o ngā tauira whakahohenga wā o ngā ara pākia motuhake i roto i ngā PN. Ahakoa ka taea e te mahi mitochondrial neuronal rerekē te arahi ki te atherosclerosis wawe me te hanga hou 1C (Pikitia 3E me te Pikitia S5C), tae atu ki ngā huringa matapae i roto i te whakaaturanga o ngā matatini I me IV, ko ngā huringa i roto i te hanganga serine de novo he mea noa iho i ngā wāhanga whakamutunga. I kitea noa i ngā wāhanga whakamutunga. Ko te ngoikore o te OXPHOS (Pikitia 3E me te Pikitia S5C). Ka tautuhia e ēnei kitenga he tukanga raupapa e whakautu tahi ana te mitochondrial (huringa 1C) me te cytoplasmic (biosynthesis serine) i puta mai i te ahotea me te pikinga o te atherosclerosis i roto i te huringa TCA ki te hanga anō i te metabolism neuronal.
Ka taea e ngā PN ngoikore-OXPHOS e 8 wiki te pakeke te pupuri i te mahi whakaoho auau teitei, ā, ka honoa anō te pūnaha matū hei utu mō te ngoikore o te mitochondrial. Mā tēnei kitenga ka ara ake he tūponotanga whakamere ahakoa i tēnei wā tonu, ka whiwhi pea ēnei pūtau i te wawaotanga rongoā hei whakaroa, hei ārai rānei i te neurodegeneration. I muri mai. I whakatauhia e mātou tēnei tūponotanga mā roto i ngā wawaotanga motuhake e rua. I te tikanga tuatahi, i hangaia e mātou he vector huaketo adeno-associated (AAV) e whakawhirinaki ana ki a Cre kia taea ai te whakaatu motuhake i te MFN2 i roto i ngā PN ngoikore-OXPHOS i roto i te tinana ora (Pikitia S7A). Ko te AAV e whakamārama ana i te MFN2 me te ira kairīpoata fluorescent mCherry (Mfn2-AAV) i manatokohia i roto i ngā ahurea neuron tuatahi i roto i te vitro, i meinga ai te whakaaturanga o te MFN2 i roto i te tikanga Cre-dependent, ā, i whakaora i te āhua mitochondrial, nā reira i ārai ai i te neuromutation i roto i ngā neuron Mfn2cKO (Pikitia S7, B, D me E). Muri iho, i whakahaerehia e mātou ngā whakamātautau in vivo hei tuku i te Mfn2-AAV 8-wiki te pakeke ki te cortex cerebellar o te Mfn2cKO me ngā kiore whakahaere, ā, i tātarihia ngā kiore 12-wiki te pakeke (Pikitia 4A). I ​​mate ngā kiore Mfn2cKO i rongoatia (Pikitia 1, A me B) (16). Nā te whakawhiti huaketo in vivo i puta ai te whakaaturanga whiriwhiri o te PN i roto i ētahi porowhita cerebellar (Pikitia S7, G me H). Kāore he pānga nui o te werohanga o te AAV whakahaere e whakaatu ana i te mCherry anake (Ctrl-AAV) ki te taumata o te neurodegeneration i roto i ngā kararehe Mfn2cKO. He rerekē, ko te tātaritanga o ngā Mfn2cKO i whakawhitihia ki te Mfn2-AAV i whakaatu i te pānga tiaki nui o te paparanga pūtau PN (Pikitia 4, B me C). Ina koa, ko te kiato neuron te āhua nei kāore e taea te wehewehe mai i ngā kararehe whakahaere (Pikitia 4, B me C, me te Pikitia S7, H me I). He rite tonu te whai huatanga o te whakaaturanga o te MFN1, engari kāore i te MFN2, ki te whakaora i te mate o ngā neuron (Pikitia 4C me te Pikitia S7, C me te F), e tohu ana ka taea e te whakaaturanga o te ectopic MFN1 te tāpiri i te korenga o te MFN2. I whakaatuhia e te tātaritanga anō i te taumata PN kotahi, i whakaora nui a Mfn2-AAV i te hanganga ultra o te mitochondria, i whakahou i ngā taumata mtDNA, ā, i whakakore i te whakaaturanga teitei o te tohu anti-angiogenesis PCx ​​(Pikitia 4, C ki E). I whakaatuhia e te tirotiro tirohanga o ngā kiore Mfn2cKO i whakaorangia i roto i te āhua okiokinga kua pai ake te tū me ngā tohu nekehanga (nekehanga S1 ki S3). Hei whakamutunga, e whakaatu ana ēnei whakamātautau he rawaka te whakaurunga whakaroa o te MFN2 ki roto i ngā PN he nui te ngoikore o te OXPHOS hei whakakore i te kai mtDNA me te whakaoho i te atherosclerosis, ā, ka ārai i te pirau axon me te mate neuron i roto i te tinana.
(A) He kaupapa e whakaatu ana i te kaupapa whakamātautau mō te werohanga i te AAV e whakamārama ana i te MFN2 ina whakahohehia te ara pākia kua tohua. (B) Ngā whakaahua huna o ngā poro cerebellar 12-wiki te pakeke i whakawhitihia i te 8 wiki i roto i ngā kiore Mfn2cKO me te tapanga ki te paturopi anti-Calbindin. Matau: Te tauine o ngā muka axon. Ko te tauine o te whakanui axon he 450 me te 75 μm. (C) Mauī: Te ine i te kiato pūtau Purkinje i roto i te koropiko whakawhiti AAV (AAV+) (tātaritanga rerekētanga kotahi-ara; n = 3 kiore). Matau: tātaritanga arotahi mtDNA i roto i te PN i whakawhitihia i te wiki 12 (whakamātautau-t kore takirua; n = 6 pūtau mai i ngā kiore e toru). * P <0.05; ** P <0.01. (D) Ngā whakaahua hiko tuku tohu o ngā PN o ngā wāhanga cerebellar Mfn2cKO i whakawhitihia me ngā kawe huaketo kua tohua. Ko te kopare māwhero e whakaatu ana i te rohe e nohoia ana e ngā dendrite, ā, ko te tapawhā ira kōwhai e whakaatu ana i te whakanui i whakaratohia i te taha matau; ko n te tohu o te karihi. Ko te pae tauine, 1μm. (E) e whakaatu ana i tētahi tauira o te tae PCx i roto i te PN i whakawhitia i te 12 wiki. Pae tauine, 20μm. OE, te whakanui rawa; FC, te huringa takai.
Hei whakamutunga, i tirotirohia e mātou te hiranga o te oranga tonutanga o ngā pūtau i puta mai i te peroxidase i roto i ngā PN kua pāngia e te ngoikore o te OXPHOS. I hangaia e mātou he mCherry e whakamārama ana i te AAV-shRNA (RNA makawe poto) e aro ana ki te mRNA PCx kiore (AAV-shPCx), ā, i werohia te huaketo, tōna mana whakahaere rānei (AAV-scr) ki roto i te roro iti o ngā kiore Mfn2cKO. I mahia te werohanga i te wiki tuawhā o te pakeketanga (Pikitia 5A) hei whakatutuki i te whakahekenga PCx whai hua i te wā i piki ake ai te whakaaturanga PCx (Pikitia 3C) me te paparanga pūtau PN kei ​​te ora tonu (Pikitia 1A). He mea tika kia mōhio ko te whakahekenga i te PCx (Pikitia S8A) ka arahi ki te tere tere o te mate PN, e herea ana ki te mowhiti pangia (Pikitia 5, B me C). Hei mārama ki te tikanga o ngā pānga pākia i puta mai i te pikinga o te PCx, i akohia e mātou te āhua redox o ngā PN i muri i te whakaaturanga o te PCx knockdown me te AAV-mediated optical biosensor Grx1-roGFP2 i te wā kotahi (Pikitia S8, B ki D) hei aromatawai i te glutathione. Ko te huringa whanaunga o te pūmanawa redox peptide (38). Kātahi ka whakahaerehia e mātou he rua-photon fluorescence lifetime imaging microscopy (FLIM) i roto i ngā poro roro koi o te Mfn2cKO 7-wiki te pakeke, ngā hoa whakahaere rānei hei kite i ngā huringa pea o te āhua redox cytoplasmic i muri i te manatoko i ngā āhuatanga FLIM (Pikitia S8, E ki G). I whakaatuhia e te tātaritanga he pikinga nui o te āhua waikura o tētahi PN Mfn2cKO kotahi kāore he whakaaturanga PCx, he rerekē tēnei i ngā neuron whakahaere, i ngā PN Mfn2cKO rānei e whakaatu ana i te shRNA kua whakaranua anake (Pikitia 5, D me E). I te whakahekenga o te whakapuakanga PCx, ka piki ake te ōrau o ngā Mfn2cKO PN e whakaatu ana i te āhua tino waikura mā te neke atu i te toru ngā wā (Pikitia 5E), e tohu ana ko te whakanuinga o te PCx i pupuri i te kaha redox o ngā neuron kua pakaru.
(A) He kaupapa e whakaatu ana i te kaupapa whakamātautau mō te werohanga i te AAV e whakawaehere ana i te shPCx ina whakahohehia te ara pākia kua tohua. (B) Ngā whakaahua huna o ngā wāhanga cerebellar 8-wiki te pakeke i roto i ngā kiore Mfn2cKO i whakawhitihia, i tapaina hoki ki te paturopi anti-calcineurin i te 4 wiki. Pae tauine, 450μm. (C) Te ine i te kiato pūtau Purkinje i roto i ngā koropiko AAV-transduced (tātaritanga rerekētanga kotahi-ara; n = 3 ki te 4 kiore). Ka whakaatuhia ngā raraunga hei toharite±SEM; ***P<0.001. (D) E whakaatu ana te pikitia FLIM e whakaatu ana i te roanga ora toharite o te PN 7-wiki te pakeke e whakaatu ana i te pūoko glutathione redox Grx1-roGFP2 i raro i ngā tikanga whakamātautau kua tohua. Ōwehenga LUT (ripanga tirotiro): te wā ora (i roto i ngā pikohekona). Pae tauine, 25μm. (E) E whakaatu ana te kauwhata i te tohatoha o ngā uara oranga o Grx1-roGFP2 mai i (D) (n=158 ki te 368 pūtau i roto i ngā kiore e rua i raro i ia āhuatanga). Ko te tūtohi porowhita i runga ake i ia kauwhata: e whakaatu ana i te maha o ngā pūtau he roa ake (whero, waikura) he poto ake rānei (kikorangi, whakaitihia), e neke atu ana i te 1 SD o te uara oranga toharite i roto i te CTRL-AAV-scr. (F) E whakaatu ana te tauira kua whakaarohia i te pānga tiaki o te pikinga ake o te PCx io.
I te katoa, e whakaatu ana ngā raraunga e tukuna ana e mātou i konei ka taea e te whakaputanga anō o MFN2 te whakaora katoa i te PN matatau me te ngoikore nui o te OXPHOS, te whakahekenga nui o te mtDNA, me te āhua rerekē rawa o te ista, ā, ka whakarato i te ahunga whakamua tonu ahakoa i roto i ngā mate matatau. Ka whakaratohia e te neurodegeneration he taunakitanga whakahurihuri o te wāhanga i mua i te matenga o te pūtau. Ka whakanuia ake tēnei taumata o te ngāwari o te pākia e te kaha o ngā neuron ki te whakaoho i te atherosclerosis (he hononga anō o te huringa TCA), e aukati ana i te whakaaturanga PCx i roto i ngā PN kāore he OXPHOS, ā, ka whakanui ake i te matenga o te pūtau, ā, ka whai wāhi tiaki (Pikitia 5F).
I roto i tēnei rangahau, i whakaratohia e mātou he taunakitanga ko te urupare a ngā PN ki te ngoikore o te OXPHOS ko te āta hono atu ki te atherosclerosis huringa TCA mā te ara whakahohenga rerekētanga e whakahohehia ana e ngā kaupapa pākia. I whakaūhia e mātou te tātari proteomic me ngā tikanga tautoko maha, ā, i whakakitea mai i te wā e whakawerohia ana e te ngoikore nui o te mitochondrial, he momo ngāwari pākia ngā neurons kāore i te mōhiotia i mua. Ki tā mātou miharo, ko te tukanga whakahou katoa kāore e tohu i te āhua pākia mutunga e haere tahi ana me te neurodegeneration āta haere me te kore e taea te hoki, engari e kī ana ā mātou raraunga tera pea he neuron tiaki ahakoa i te wā i mua i te matenga o te pūtau Tikanga utu mahi. E tohu ana tēnei kitenga he nui te kirihou pākia o ngā neurons i roto i te tinana. E whakaatu ana tēnei meka ka taea e te whakaurunga anō o MFN2 i muri mai te huri i te whakaaturanga o ngā tohu pākia matua me te aukati i te pirau PN. Engari, ka aukati i te atherosclerosis me te whakateretere i ngā io. transsexual.
Ko tētahi o ngā kitenga tino whakamīharo i roto i tā mātou rangahau ko te kaha o ngā PN kāore i te OXPHOS ki te whakarerekē i te pākia o te huringa TCA mā te whakanui ake i ngā whākōkī e whakaoho ana i te arteriosclerosis. Ko te whakarerekētanga pākia he āhuatanga noa o ngā pūtau matepukupuku, ko ētahi o ēnei e whakawhirinaki ana ki te glutamine hei tāpiri i ngā takawaenga huringa TCA hei whakaputa i ngā mea rite, e akiaki ana i te mekameka manawa me te pupuri i te hanga o ngā matū whakaputanga lipid me te nucleotide (39, 40). I whakaatuhia e tētahi rangahau tata nei i roto i ngā kiko ā-taha e pāngia ana e te ngoikore o te OXPHOS, ko te hononga anō o te pākia o te glutamine/glutamate he āhuatanga nui hoki (5, 41), kei reira te ahunga o te urunga o te glutamine ki te huringa TCA e whakawhirinaki ana ki te kaha o te whara OXPHOS (41). Heoi, kāore he taunakitanga mārama mō tētahi ōrite o te kirihou pākia o ngā neuron i roto i te tinana me tōna hiranga pea i roto i te horopaki mate. I roto i tētahi rangahau in vitro tata nei, i whakaaturia ngā neuron cortical tuatahi ki te whakaoho i ngā puna glutamate mō te neurotransmission, nā reira ka whakatairanga i te pākia o te oxidative me te atherosclerosis i raro i ngā āhuatanga ahotea pākia (42). He mea tika kia mōhiotia i raro i te aukati rongoā o te whākōkī huringa TCA succinate dehydrogenase, e whakaponohia ana ka pupuri te pyruvate carboxylation i te hanganga o te oxaloacetate i roto i ngā neurons granule cerebellar kua whakatipuria (34). Heoi, he mea nui tonu te hiranga ā-tinana o ēnei tikanga ki te kiko roro (kei reira e whakaponohia ana ko te atherosclerosis te nuinga o te wā e herea ana ki ngā astrocytes) (43). I tēnei take, e whakaatu ana ā mātou raraunga ka taea te huri i ngā PN i pakaruhia e OXPHOS i roto i te tinana ki te whakahekenga BCAA me te pyruvate carboxylation, koinei ngā puna matua e rua o te tāpiritanga o ngā takawaenga puna TCA. Ahakoa kua whakaarohia te takoha whakapae o te catabolism BCAA ki te metabolism pūngao neuronal, hei tāpiri atu ki te mahi a te glutamate me te GABA mō te neurotransmission (44), kāore anō kia kitea he taunakitanga mō ēnei tikanga in vivo. Nō reira, he ngāwari ki te whakapae ka taea e ngā PN kore mahi te utu aunoa mō te kai o ngā takawaenga TCA e peia ana e te tukanga whakauru mā te whakanui ake i te atherosclerosis. Ina koa, me whakanui ake te PCx hei pupuri i te hiahia nui mō te waikawa aspartic, e whakaarohia ana i roto i ngā pūtau e tipu haere ana me te ngoikore o te mitochondrial (45). Heoi, kāore i kitea e tā mātou tātaritanga metabolomics he huringa nui i roto i te taumata pumau o te waikawa aspartic i roto i ngā Mfn2cKO PN (Pikitia S6A), e whakaatu ana pea i te whakamahinga rerekē o te waikawa aspartic i waenga i ngā pūtau e tipu haere ana me ngā neuron post-mitotic. Ahakoa me tautuhi tonu te tikanga tika o te whakanui ake i te PCx i roto i ngā neuron kore mahi i roto i te vivo, i whakaatuhia e mātou he mahi nui tēnei urupare wawe ki te pupuri i te āhua redox o ngā neuron, i whakaaturia i roto i ngā whakamātautau FLIM i runga i ngā poro cerebellar. Ina koa, ko te aukati i ngā PN mai i te whakanui ake i te PCx ka arahi ki te āhua oxidized me te whakatere i te mate pūtau. Ko te whakahohenga o te whakahekenga BCAA me te carboxylation o te pyruvate ehara i te huarahi ki te tautuhi i ngā kiko peripheral o te ngoikore o te mitochondrial (7). Nō reira, te āhua nei he āhuatanga matua rātou o ngā neuron OXPHOS-deficient, ahakoa ehara i te mea ko te āhuatanga anake, he mea nui mō te neurodegeneration. .
Ko te mate roro he momo mate neurodegenerative rerekē e puta pinepine ana hei ataxia, ā, he maha ngā wā ka pakaru ngā PN (46). He tino ngoikore tēnei taupori neuron ki te ngoikore o te mitochondrial nā te mea he nui te pirau whiriwhiri i roto i ngā kiore hei whakaputa i te maha o ngā tohu motuka e tohu ana i te ataxia spinocerebellar tangata (16, 47, 48). E ai ki ngā pūrongo, he hononga kei waenganui i tētahi tauira kiore transgenic me tētahi ira whakarerekētanga ki te ataxia spinocerebellar tangata, ā, he ngoikore mitochondrial tōna (49, 50), e whakanui ana i te hiranga o te ako i ngā hua o te korenga OXPHOS i roto i te PNPH. Nō reira, he tino pai ki te wehe me te ako i tēnei taupori neuron ahurei. Heoi, i te mea he tino tairongo ngā PN ki te pēhanga, ā, he iti noa te ōwehenga o te taupori pūtau roro katoa, mō te maha o ngā rangahau e hangai ana ki te omics, he āhuatanga wero tonu te wehenga whiriwhiri o rātou hei pūtau katoa. Ahakoa he tata kore e taea te whakatutuki i te korenga o te poke o ētahi atu momo pūtau (ina koa ko ngā kiko pakeke), i whakakotahi mātou i tētahi taahiraa wehewehe whai hua me te FACS kia whiwhi ai i te maha o ngā neuron ora mō te tātari proteomics o raro, ā, he tino teitei te kapinga pūmua (tata ki te 3000 pūmua) ki te whakataurite ki te huinga raraunga o nāianei o te cerebellum katoa (51). Mā te tiaki i te oranga o ngā pūtau katoa, mā te tikanga e whakaratohia ana e mātou i konei ka taea e mātou te tirotiro i ngā huringa o ngā ara pākia i roto i te mitochondria, engari me te tirotiro hoki i ngā huringa o ōna hoa cytoplasmic, e tautoko ana i te whakamahinga o ngā tohu membrane mitochondrial hei whakarei ake i te momo pūtau. Ko te tikanga hou mō te maha o ngā mitochondria i roto i ngā kiko uaua (52, 53). Ko te tikanga e whakaahuahia ana e mātou ehara i te mea e pā ana ki te ako i ngā pūtau Purkinje anake, engari ka taea te whakamahi ngāwari ki tētahi momo pūtau hei aro atu ki ngā huringa pākia i roto i ngā roro mate, tae atu ki ētahi atu tauira o te ngoikore o te mitochondria.
Hei whakamutunga, kua kitea e mātou he matapihi rongoā i roto i tēnei tukanga whakarerekētanga pākia e taea ai te whakakore katoa i ngā tohu matua o te ahotea pūtau me te ārai i te pirau o ngā neuron. Nō reira, mā te mārama ki ngā pānga mahi o te hononga anō kua whakaahuatia i konei ka taea te whakarato i ngā māramatanga taketake ki ngā maimoatanga pea hei pupuri i te oranga o ngā neuron i te wā o te ngoikore o te mitochondrial. Me rangahau ā muri ake nei e aro ana ki te wetewete i ngā huringa o te pākia o te pūngao i roto i ētahi atu momo pūtau roro kia tino kitea ai te whai huatanga o tēnei mātāpono ki ētahi atu mate neurological.
Kua whakaahuatia ngā kiore MitoPark i mua (31). Kua whakaahuatia ngā kiore C57BL/6N me ngā ira Mfn2 e taha ana i te loxP (18) ā, kua whakawhitia ki ngā kiore L7-Cre (23). Ko ngā uri takirua heterozygous i puta mai i whakawhitia ki ngā kiore homozygous Mfn2loxP/Mfn2loxP hei whakaputa i ngā ira Purkinje-motuhake mō Mfn2 (Mfn2loxP/Mfn2loxP; L7-cre). I roto i tētahi wāhanga o te marena, i whakaurua te allele SorStop-mito-YFP Gt (ROSA26) (stop-mtYFP) mā roto i ngā whakawhiti tāpiri (20). I mahia ngā tikanga kararehe katoa i runga anō i ngā aratohu a Ūropi, ā-motu me ngā aratohu whakahaere, ā, i whakaaetia e LandesamtfürNatur o Umwelt me ​​Verbraucherschutz, North Rhine-Westphalia, Tiamana. Ka whai hoki te mahi kararehe i ngā aratohu a te European Federation of Laboratory Animal Sciences Associations.
I muri i te whakamoe i te piko o te kaki o te wahine hapu, ka wehea te kukune kiore (E13). I tapahia te kiri o te kiore ki roto i te Hanks' Balanced Salt Solution (HBSS) i tāpirihia ki te 10 mM Hepes, ā, i tukuna ki te Dulbecco's Modified Eagle's Medium kei roto ko te papain (20 U/ml) me te cysteine ​​​​(1μg/ml). Whakatōkia te kiko ki roto i te DMEM) ka wehea mā te nakunaku whākōkī. Ml) i te 37°C mō te 20 meneti, kātahi ka mirahia mā te miihini ki roto i te DMEM i tāpirihia ki te 10% serum kau kukune. I whakatōkia ngā pūtau ki runga i ngā uhi karāhe kua pania ki te polylysine i te kiato o te 2×106 mō ia rihi ahurea 6 cm, i te kiato rānei o te 0.5×105 pūtau/cm2 mō te tātari whakaahua. I muri i te 4 hāora, i whakakapia te medium ki te medium kore-serum Neurobasal kei roto ko te 1% tāpiringa B27 me te 0.5 mM GlutaMax. Kātahi ka puritia ngā neuron ki te 37°C me te 5% CO2 puta noa i te whakamātautau, ā, ka whāngaihia kotahi i te wiki. Hei whakaoho i te whakakotahitanga i roto i te vitro, i whakamahia te 3μl (riihi ahurea 24-pū) 0.5μl rānei (pereti 24-pū) o te kawe huaketo AAV9 e whai ake nei hei rongoā i ngā neuron i te rā tuarua i roto i te vitro: AAV9.CMV.PI.eGFP. WPRE.bGH (Addgene, nama rarangi 105530-AAV9) me te AAV9.CMV.HI.eGFP-Cre.WPRE.SV40 (Addgene, nama rarangi 105545-AAV9).
Ko te DNA tautoko a te kiore Mfn1 me te Mfn2 (i tangohia mai i te plasmid Addgene #23212 me te #23213) kua tohua ki te raupapatanga V5 (GKPIPNPLLGLDST) i te pito-C, ā, kua honoa ki te mCherry i roto i te anga mā te raupapatanga T2A. Ko Grx1-roGFP2 he koha mai i Heidelberg TP Dick DFKZ (Deutsches Krebsforschungszentrum). Mā te whakakapi i te rīpene tdTomato mā te whakamahi i ngā tikanga tāruarua tikanga, i tāruaruatia te rīpene ki roto i te tuara pAAV-CAG-FLEX-tdTomato (nama tohutoro Addgene 28306) hei whakaputa i ngā wetereo pAAV-CAG-FLEX-mCherry-T2A-MFN2-V5, pAAV-CAG-FLEX-mCherry-T2A-MFN1-V5 me te pAAV-CAG-FLEX-Grx-roGFP2. I whakamahia tētahi rautaki ōrite hei whakaputa i te ira whakahaere pAAV-CAG-FLEX-mCherry. Hei whakaputa i te hanganga AAV-shPCx, me whai i tētahi ira plasmid AAV (VectorBuilder, pAAV [shRNA] -CMV-mCherry-U6-mPcx- [shRNA#1]), kei roto nei te raupapatanga DNA e whakamārama ana i te kiore PCx e aro ana ki te shRNA (5′CTTTCGCTCTAAGGTGCTAAACTCGAGTTTAGCACCTTAGAGCGAAAG 3′). I raro i te mana o te kaiwhakatairanga U6, ka whakamahia a mCherry i raro i te mana o te kaiwhakatairanga CMV. I mahia te whakaputa i ngā ira āwhina AAV e ai ki ngā tohutohu a te kaihanga (Cell Biolabs). Hei whakarāpopototanga, whakamahia he plasmid whakawhiti e mau ana i te mCherry-T2A-MFN2-V5 (pAAV-CAG-FLEX-mCherry-T2A-MFN2-V5), mCherry-T2A-MFN1-V5 (pAAV-CAG-FLEX-mCherry) Whakawhitiwhitinga wā poto o ngā pūtau 293AAV-T2A-MFN1-V5), mCherry (pAAV-CAG-FLEX-mCherry) Grx-roGFP2 (pAAV-CAG-FLEX-Grx-roGFP2 rānei) ira whakakōwae, me te whakakōwae hoki i te pūmua capsid AAV1 me te pūmua tāpiri. Te tākai plasmid plasmid, mā te whakamahi i te tikanga konupūmā phosphate. I whiwhihia te waikeri mata o te huaketo mā te huringa tio-rewa i roto i te kaukau hukapapa/ethanol maroke, ā, i wehea ngā pūtau ki roto i te tote phosphate buffered (PBS). I purea te AAV vector mā te iodixanol gradient ultracentrifugation motumotu (24 hāora i te 32,000 rpm me te 4°C) ā, i whakatōtōhia mā te whakamahi i te tātari Amicon ultra-15 centrifugal. Ko te taitara ira o te AAV1-CAG-FLEX-mCherry-T2A-MFN2-V5 [2.9×1013 tārua ira (GC)/ml], AAV1-CAG-FLEX-mCherry (6.1×1012 GC/ml), ko te AAV1-CAG-FLEX i rite ki te whakaahuatanga o mua (54), i inehia mā te PCR tauanga wā-tūturu (qPCR) -MFN1-V5 (1.9×1013 GC/ml) me te AAV1-CAG-FLEX-Grx-roGFP2 (8.9×1012 GC/ml).
I waruhia ngā neuron tuatahi ki roto i te 1x PBS makariri, i whakapūpūhia, kātahi ka whakakoitia ki roto i te 0.5% Triton X-100 / 0.5% sodium deoxycholate/PBS lysis buffer kei roto ko te phosphatase me te protease inhibitor (Roche). I mahia te ine pūmua mā te whakamahi i te whakamātautau waikawa bicinchoninic (Thermo Fisher Scientific). Kātahi ka wehea ngā pūmua mā te SDS-polyacrylamide gel electrophoresis, kātahi ka pania ki runga i te membrane polyvinylidene fluoride (GE Healthcare). Āraia ngā wāhi kore-motuhake ka whakatōkia ki te paturopi tuatahi (tirohia te Ripanga S1 mō ētahi atu kōrero) ki roto i te miraka 5% i roto i te TBST (Tris-buffered saline me Tween), ngā mahi horoi me te paturopi tuarua i roto i te TBST Incubate. Whakatōkia ki te paturopi tuatahi mō te pō i te +4°C. Whai muri i te horoinga, tāpirihia te paturopi tuarua mō ngā hāora e 2 i te pāmahana rūma. Muri iho, mā te whakatō i te paninga kotahi ki te paturopi anti-β-actin, ka whakaūhia te utaina kotahi. Te kitenga mā te huri ki te chemiluminescence me te whakarei ake i te chemiluminescence (GE Healthcare).
Ko ngā neuron i whakatōngia i mua ki runga i ngā uhi karāhe i whakapūmauhia ki te 4% paraformaldehyde (PFA)/PBS i te wā kua tohua i te pāmahana rūma mō te 10 meneti. Ka whakapūmauhia ngā uhi ki te 0.1% Triton X-100/PBS mō te 5 meneti i te pāmahana rūma, kātahi ki roto i te aukati ārai [3% bovine serum albumin (BSA)/PBS]. I te rua o ngā rā, i horoia ngā uhi ki te aukati ārai, ā, i whakatōkia ki te paturopi tuarua kua honoa ki te fluorophore e tika ana mō te 2 hāora i te pāmahana rūma; hei whakamutunga, i horoia pai ngā tauira ki roto i te PBS me te 4′,6-diamidino-2-Phenylindole (DAPI) kua tāpirihia he tae anō, kātahi ka whakapūmauhia ki runga i te kiriata karuārai me te Aqua-Poly/Mount.
I whakamoea ngā kiore (tāne me te wahine) mā te werohanga ketamine (130 mg/kg) me te xylazine (10 mg/kg) ki roto i te puku, ā, i tukuna ki raro i te kiri me te carprofen analgesic (5 mg/kg). I whakanohoia ki roto i tētahi taputapu stereotactic (Kopf) he papa mahana tōna. Whakaaturia te angaanga, ā, whakamahia he wiri niho hei whakaangawari i te wāhanga o te cortex cerebellar e rite ana ki te wheua mis (mai i te lambda: hiku 1.8, taha 1, e rite ana ki ngā lobules IV me V). Whakamahia he ngira werohanga piko hei hanga āta i tētahi kōhao iti ki roto i te angaanga kia kore ai e whakararuraru i te rere o te toto i raro. Kātahi ka whakaurua mārire te capillary karāhe angiangi ki roto i te kōhao moroiti (mai i te -1.3 ki te -1 i te taha ventral o te dura mater), ā, ka werohangahia he 200 ki te 300 nl AAV ki roto i te micro-injector (Narishige) me ngā werohanga ā-ringa (Narishige) i ētahi wā i te pēhanga iti i roto i tētahi wā 10 ki te 20 meneti te roa. I muri i te werohanga, waiho te pūwero mō ētahi atu 10 meneti kia horapa katoa ai te huaketo. I muri i te tangohanga o ngā pūwero, ka tuituihia te kiri kia āta whakaitihia te mumura o te patunga, kia ora ai te kararehe. I rongoatia ngā kararehe ki ngā rongoā whakamamae (caspofen) mō ētahi rā i muri i te pokanga, ā, i taua wā i āta tirotirohia ai tō rātou āhua tinana, kātahi ka whakamatea i te wā kua whakaritea. I mahia ngā tikanga katoa i runga anō i ngā aratohu a Ūropi, ā-motu me ngā whare whakahaere, ā, i whakaaetia e LandesamtfürNatur o Umwelt me ​​Verbraucherschutz, North Rhine-Westphalia, Tiamana.
I whakamoea ngā kararehe ki te ketamine (100 mg/kg) me te xylazine (10 mg/kg), ā, i whakakīia te ngākau ki te 0.1 M PBS i te tuatahi, kātahi ki te 4% PFA i roto i te PBS. I wehea te kiko, ā, i whakapūmautia ki roto i te 4% PFA/PBS i te pō i te 4°C. I whakamahia he maripi wiri (Leica Microsystems GmbH, Vienna, Austria) hei whakarite i ngā wāhanga sagittal (50 μm te matotoru) mai i te roro kua whakapūmautia i roto i te PBS. Ki te kore e tohua tētahi atu mea, i mahia te tae o ngā wāhanga e mānu ana i runga ake nei (13) i te pāmahana rūma me te whakaoho. I te poto, tuatahi, i whakapūmautia ngā poro i whiwhi ki te 0.5% Triton X-100/PBS mō te 15 meneti i te pāmahana rūma; mō ētahi epitopes (Pcx me Shmt2), mā te whakapūmau i te tris-EDTA i te 80°C (PH 9) whakamahanahia ngā poro mō te 25 meneti hei utu mō tēnei taahiraa. Muri iho, i whakatōngia ngā wāhanga ki te paturopi tuatahi (tirohia te Ripanga S1) i roto i te arai ārai (3% BSA/PBS) i te 4°C i te pō me te whakaoho. I te rā i muri mai, i horoia ngā wāhanga ki te arai ārai, ā, i whakatōngia ki te paturopi tuarua kua honoa ki te fluorophore e tika ana mō ngā hāora e 2 i te pāmahana rūma; hei whakamutunga, i horoia pai ngā wāhanga ki te PBS, i pania ki te DAPI, kātahi ka whakapirihia ki te AquaPolymount i runga i te kiriata karuārai.
I whakamahia he karuārai matawai taiaho (TCS SP8-X, TCS Digital Light Sheet rānei, Leica Microsystems) me te taiaho mārama mā me te taiaho ultraviolet 405 diode hei whakaahua i te tauira. Mā te whakaoho i te fluorophore me te kohikohi i te tohu ki te Hybrid Detector (HyDs), i whakamahia te pūmanawa LAS-X hei kohikohi i ngā whakaahua whakarārangi e rite ana ki te tauira Nyquist i te aratau raupapa: mō ngā panera kore-tau, he tohu tino hihiri (hei tauira, i roto i ngā pūtau somatic me ngā dendrites) mtYFP) Whakamahia te HyD hei kite i te maha o ngā PN i te aratau BrightR). Ka whakamahia te kuaha mai i te 0.3 ki te 6 ns hei whakaiti i te papamuri.
Whakaahua wā-tūturu o ngā pūtau kua whakarōpūtia. Whai muri i te whakarōpūtanga ki roto i te reo Neurobasal-A kei roto ko te 1% tāpiringa B27 me te 0.5 mM GlutaMax, ka whakatōngia tonutia ngā pūtau ki runga i ngā kiriata karāhe kua pania ki te poly-l-lysine (μ-Slide8 Well, Ibidi, nama rarangi 80826), Kātahi ka pupuri i te 37°C me te 5% CO2 mō te 1 hāora kia tau ai ngā pūtau. I mahia te whakaahua wā-tūturu ki runga i te karuārai matawai taiaho Leica SP8 kua rite ki te taiaho mā, HyD, te arotahi arotahi hinu 63×[1.4 tauārai (NA)] me te atamira whakamahana.
I tere te whakamoe i te kiore ki te hauhā waro, ā, i tapahia te upoko, i tere te tango i te roro i te angaanga, ā, i tapahia kia 200μm te matotoru (mō te whakamātautau tapanga 13C) kia 275μm te matotoru rānei (mō ngā whakamātautau photon e rua) te wāhanga sagittal kua whakakiia ki ngā rauemi e whai ake nei. Ko te aihikirīmi (HM-650 V, Thermo Fisher Scientific, Walldorf, Tiamana) kua whakakiia ki ēnei matū: 125 mM hukapapa-makariri, waro-kū (95% O2 me te 5% CO2) iti Ca2 + wai cerebrospinal horihori (ACSF) NaCl, 2.5 mM KCl, 1.25 mM konutai phosphate buffer, 25 mM NaHCO3, 25 mM hukahuka, 0.5 mM CaCl2 me te 3.5 mM MgCl2 (pēhanga osmotic o 310 ki te 330 mmol). Whakawhitia ngā poro roro kua riro mai ki tētahi rūma i mua i te whakatōnga kei roto ko te Ca2 + ACSF teitei ake (125.0 mM NaCl, 2.5 mM KCl, 1.25 mM konutai phosphate buffer, 25.0 mM NaHCO3, 25.0 mM d-glucose, 1.0 mM CaCl2 me te 2.0 mM MgCl2) (Waenga) pH 7.4 me te 310 ki te 320 mmol).
I te wā o te tukanga whakaahua, i nekehia ngā poro ki roto i tētahi rūma whakaahua motuhake, ā, i whakahaerehia te whakamātautau i raro i te rere toto ACSF tonu i te pāmahana pumau o te 32° ki te 33°C. I whakamahia he karu tirotiro taiaho maha-whakaahua (TCS SP8 MP-OPO, Leica Microsystems) me te arotahi whāinga Leica 25x (NA 0.95, wai), taiaho Ti: Sapphire (Chameleon Vision II, Coherent) mō te whakaahua poro. He waeine FLIM (PicoHarp300, PicoQuant).
FLIM o Grx1-roGFP2. I inehia ngā panonitanga o te āhua redox cytoplasmic o ngā PN mā te FLIM photon-rua i roto i ngā poro roro sagittal, i reira ka arohia e te pūoko koiora Grx1-roGFP2 ngā PN. I roto i te paparanga PN, ka tīpakohia te mara hopu i te 50 ki te 80 μm i raro i te mata poro hei whakarite kei reira he PN ora (arā, te korenga o te hanganga piripiri, ngā panonitanga āhua neuronal rānei i te taha o ngā dendrite) me te pūoko roGFP2 pai rua me te AAV e whakawaehere ana i te shRNA PCx, i tana raupapa whakahaere rānei (ia mCherry e whakaatu tahi ana). Kohia ngā whakaahua kotahi-pūranga me te whakanui mamati 2x [roaroaroa whakaoho: 890 nm; 512 nm 512 pika]. Kitenga: HyD ā-roto, rōpū tātari fluorescein isothiocyanate (FITC)] me te toharite whakaahua i roto i te 2 ki te 3 meneti ka whakamahia hei whakarite kia nui ngā photon kua kohia (1000 photon katoa) mō te taunga pihi. I mahia te aro o te pūwero Grx1-roGFP2 me te manatoko i ngā āhuatanga FLIM mā te aroturuki i te uara roa o te ora o te roGFP2 i te tāpiri i te 10 mM H2O2 o waho ki te ACSF toto (hei whakanui ake i te waikura, ka hua ake he roa ake te ora), kātahi ka tāpirihia he 2 mM dithiothreitol (ka whakaiti i te nui o te whakahekenga, ka hua ake he hekenga o te roa o te ora) (Pikitia S8, D ki G). Whakamahia te pūmanawa FLIMfit 5.1.1 hei tātari i ngā hua i whiwhihia, whakauruhia te pihi pirau taupū kotahi o te ahua katoa ki te IRF i inehia (mahi urupare taputapu), ā, ko te χ2 he tata ki te 1. Hei tatau i te roa o te ora o te PN kotahi, i tuhia ā-ringa te kopare huri noa i te tinana io, ā, i whakamahia te roanga o te ora toharite i roto i ia kopare hei ine.
Te tātari pūmanawa mitochondrial. I muri i te whakatōngia o te wāhanga koi ki te 100 nM TMRM i tāpirihia tika ki te ACSF kua whakakīia mō te 30 meneti, ka inehia ngā huringa pūmanawa mitochondrial o ngā PN mā te whakamahi i te karuārai-rua-photon. I mahia te whakaahua TMRM mā te whakaoho i te probe i te 920 nm me te whakamahi i te HyD ā-roto (tetramethylrhodamine isothiocyanate: 585/40 nm) hei kohi tohu; mā te whakamahi i te roangaru whakaoho kotahi engari he rerekē te HyD ā-roto (FITC: 525/50) hei whakaahua mtYFP. Whakamahia te mono-mai a te Image Calculator a ImageJ hei aromatawai i te pūmanawa mitochondrial i te taumata pūtau kotahi. I roto i te poto, ka whakamahia te whārite mono-mai: tohu = min (mtYFP, TMRM) hei tautuhi i te rohe mitochondrial e whakaatu ana i te tohu TMRM i roto i te Purkinje Somali i roto i te whakaahua confocal kotahi-pūranga o te hongere e rite ana. Kātahi ka inehia te horahanga pika i roto i te kopare hua, kātahi ka whakatauritea ki te paepae e rite ana o te ahua puranga-kotahi o te hongere mtYFP hei tiki i te wāhanga mitochondrial e whakaatu ana i te pūmanawa mitochondrial.
I wetewetehia te ahua mā te pūmanawa Huygens Pro (Scientific Volume Imaging). Mō ngā pikitia kua matawaihia o ngā taera, ka hangaia te whakakotahitanga o tētahi taera kotahi mā te whakamahi i te raupapa tuitui aunoa i whakaratohia e te pūmanawa LAS-X. Whai muri i te whakatikatika ahua, whakamahia a ImageJ me Adobe Photoshop hei tukatuka anō i te ahua me te whakarite ōrite i te kanapa me te rerekētanga. Whakamahia a Adobe Illustrator mō te whakarite whakairoiro.
Tātaritanga arotahi mtDNA. I tauahia te maha o ngā whara mtDNA i runga i ngā wāhanga roro iti kua tohua ki ngā paturopi ki te DNA mā te karuārai confocal. I hangaia ia wāhi ūnga mō te tinana pūtau me te karihi o ia pūtau, ā, i tatauhia te wāhi e tika ana mā te whakamahi i te mono-ipurangi Multi Measure (pūmanawa ImageJ). Tangohia te wāhi karihi mai i te wāhi tinana pūtau hei whiwhi i te wāhi cytoplasmic. Hei whakamutunga, i whakamahia te mono-ipurangi Analyze Particles (pūmanawa ImageJ) hei tauanga aunoa i ngā pūwāhi DNA cytoplasmic e tohu ana i te mtDNA i runga i te ahua paepae, ā, i whakatauritea ngā hua i whiwhi ki te toharite PN o ngā kiore CTRL. Ka whakaatuhia ngā hua hei te maha toharite o ngā nucleosides mō ia pūtau.
Tātaritanga o te whakaaturanga pūmua. Whakamahia te monomai Image Calculator a ImageJ hei aromatawai i te whakaaturanga pūmua i roto i te PN i te taumata pūtau kotahi. I roto i te poto, i roto i te ahua confocal paparanga kotahi o te hongere e rite ana, mā te whārite: tohu = min (mtYFP, paturopi), ka tautuhia te rohe mitochondrial e whakaatu ana i te urupare ārai mate ki tētahi paturopi i roto i a Purkina. Kātahi ka inehia te horahanga pika i roto i te kopare hua, kātahi ka whakatauritea ki runga i te paepae e rite ana o te ahua puranga kotahi o te hongere mtYFP hei tiki i te wāhanga mitochondrial o te pūmua e whakaaturia ana.
Te tātaritanga kiato pūtau Purkinje. I whakamahia te monomai Kaitatau Pūtau o ImageJ hei aromatawai i te kiato Purkinje mā te wehewehe i te maha o ngā pūtau Purkinje i tatauhia mā te roa o te mowhiti roro iti e nohoia ana e ngā pūtau i tatauhia.
Te whakarite tauira me te kohikohinga. I whakapūmauhia ngā roro o te rōpū whakahaere me ngā kiore Mfn2cKO ki roto i te 2% PFA/2.5% glutaraldehyde i roto i te 0.1 M phosphate buffer (PB), kātahi ka whakapūmauhia ngā wāhanga korona mā te whakamahi i ngā ciliates (Leica Mikrosysteme GmbH, Vienna, Austria) (Matotoru 50 ki te 60 μm). Kātahi ka whakapūmauhia ki roto i te PB buffer ki roto i te 1% os tetraoxide me te 1.5% potassium ferrocyanide i te pāmahana rūma mō te 1 hāora. E toru ngā wā i horoia ai ngā wāhanga ki te wai whakapūmau, kātahi ka pania ki te 70% ethanol kei roto ko te 1% uranyl acetate mō te 20 meneti. Kātahi ka whakamaroketia ngā wāhanga ki roto i te waipiro kua whakarōpūtia, ā, ka tākaihia ki roto i te Durcupan ACM (Araldite casting resin M) epoxy resin (Electron Microscopy Sciences, nama rārangi 14040) i waenganui i ngā kiriata karāhe kua pania ki te silicon, ā, i te mutunga i te 60°C Polymerize ki roto i te oumu mō te 48 hāora. I tīpakohia te rohe o te cortex cerebellar, ā, i tapahia ngā wāhanga tino angiangi 50 nm ki runga i te Leica Ultracut (Leica Mikrosysteme GmbH, Vienna, Austria) ā, i tangohia ki runga i te mata kohao parahi 2×1 mm kua pania ki te kiriata polystyrene. I pania ngā wāhanga ki te otinga o te 4% uranyl acetate i roto i te H2O mō ngā meneti 10, i horoia ki te H2O i ngā wā maha, kātahi ki te Reynolds lead citrate i roto i te H2O mō ngā meneti 10, kātahi ka horoia ki te H2O i ngā wā maha. I tangohia ngā maikoropōro ki te whakamahi i te karu hiko tuku Philips CM100 (Thermo Fisher Scientific, Waltham, MA, USA) mā te whakamahi i te kāmera matihiko TVIPS (Tietz Video and Image Processing System) TemCam-F416 (TVIPS GmbH, Gauting, USA). Tiamana).
Mō ngā kiore i pangia e te AAV, i wehea te roro, ā, i tapatapahia kia rite ki te wāhanga sagittal 1 mm te matotoru, ā, i tirohia te roro iti mā te whakamahi i te karuārai fluorescence hei tautuhi i te mowhiti i pangia e te AAV (arā, te mCherry expressing). Ko ngā whakamātautau anake i whakamahia ai te werohanga AAV i te pai o te whakawhiti o te paparanga pūtau Purkinje (arā, tata ki te paparanga katoa) i roto i te iti rawa e rua ngā mowhiti roro iti karapīpiti. I tapahia te koropiko i whakawhitihia e te AAV mō te pō i muri i te whakapiringa (4% PFA me te 2.5% glutaraldehyde i roto i te 0.1 M cocoate buffer) ā, i tukatukahia anō. Mō te whakauru EPON, i horoia te kiko kua whakapirihia ki te 0.1 M sodium cocoate buffer (Applichem), ā, i whakatōkia ki te 2% OsO4 (os, Science Services; Caco) i roto i te 0.1 M sodium cocoate buffer (Applichem) mō te 4 hāora, kātahi ka horoia mō te 2 hāora. Whakahokia kia 3 ngā wā me te 0.1 M cocamide buffer. I muri mai, i whakamahia te raupapa piki o te etanoru hei whaowhia i ia otinga etanoru i te 4°C mō te 15 meneti hei whakamaroke i te kiko. I whakawhitia te kiko ki te propylene oxide, ā, i whaowhia mō te pō i roto i te EPON (Sigma-Aldrich) i te 4°C. Whakanohoia te kiko ki roto i te EPON hou i te pāmahana rūma mō te 2 hāora, kātahi ka tāpui i te 62°C mō te 72 hāora. Whakamahia he ultramicrotome (Leica Microsystems, UC6) me te maripi taimana (Diatome, Biel, Switzerland) hei tapahi i ngā wāhanga tino angiangi 70 nm, ā, pania ki te 1.5% uranyl acetate mō te 15 meneti i te 37°C, ā, pania ki te otinga lead citrate mō te 4 meneti. I tangohia ngā maikorohiko hiko mā te whakamahi i te karu hiko tuku JEM-2100 Plus (JEOL) kua rite ki te Camera OneView 4K 16-bit (Gatan) me te pūmanawa DigitalMicrograph (Gatan). Mō te tātari, i tangohia mai ngā maikorohiko hiko mā te whakanui mamati 5000×, 10,000× rānei.
Te tātari āhua o ngā mitochondria. Mō ngā tātaritanga katoa, i tuhia ā-ringa ngā āhua o ia mitochondria ki ngā whakaahua matihiko mā te whakamahi i te pūmanawa ImageJ. Ka tātarihia ngā tawhā āhua rerekē. Ka whakaatuhia te kiato mitochondrial hei ōrau mā te wehewehe i te horahanga mitochondrial katoa o ia pūtau ki te horahanga cytoplasm (horahanga cytoplasm = horahanga pūtau-horahanga karihi pūtau) × 100. Ka tatauhia te porowhita o ngā mitochondria mā te tātai [4π∙(horahanga/tawhiti 2)]. I tātarihia te āhua ista o ngā mitochondria, ā, i wehea kia rua ngā wāhanga ("pūpū" me te "pupuhi") e ai ki ō rātou āhua matua.
Te tātaritanga o te maha me te mātotoru o te autophagosome/lysosome. Whakamahia te pūmanawa ImageJ hei whakaahua ā-ringa i ngā āhua o ia autophagosome/lysosome i roto i te whakaahua matihiko. Ka whakaatuhia te horahanga o te autophagosome/lysosome hei ōrau mā te wehewehe i te horahanga hanganga autophagosome/lysosome katoa o ia pūtau ki te horahanga cytoplasm (horahanga cytoplasm=horahanga pūtau-horahanga karihi)×100. Ka tatauhia te mātotoru o ngā autophagosome/lysosome mā te wehewehe i te maha katoa ki te maha o ngā hanganga autophagosome/lysosome mō ia pūtau (e ai ki te horahanga cytoplasmic) (horahanga cytoplasmic = horahanga pūtau-horahanga karihi).
Te tapanga mō te tapahi whakapeka me te whakarite tauira. Mō ngā whakamātautau e hiahia ana kia tapangahia te hukahuka, whakawhitihia ngā poro roro whakapeka ki tētahi rūma i mua i te whakatōnga, kei roto ko te waro kukū (95% O2 me te 5% CO2), te Ca2 + ACSF teitei (125.0 mM NaCl, 2.5 mM KCl, 1.25 mM konutai phosphate buffer, 25.0 mM NaHCO3, 25.0 mM d-huka, 1.0 mM CaCl2 me te 2.0 mM MgCl2, kua whakatikatikaina ki te pH 7.4 me te 310 ki te 320 mOsm), kei roto ko te hukahuka he 13C6- te whakakapinga hukahuka (Eurisotop, nama rarangi CLM-1396). Mō ngā whakamātautau e hiahia ana kia tāpirihia he tapanga pyruvate, whakawhitihia ngā poro roro koi ki te Ca2 + ACSF teitei ake (125.0 mM NaCl, 2.5 mM KCl, 1.25 mM konutai phosphate buffer, 25.0 mM NaHCO3, 25.0 mM d-glucose, 1.0 mM CaCl2 me te Tāpiri 2.0mM MgCl2, whakatikatika ki te pH 7.4 me te 310 ki te 320mOsm), ka tāpirihia te 1mM 1-[1-13C]pyruvate (Eurisotop, nama rarangi CLM-1082). Whakatōngia ngā wāhanga mō te 90 meneti i te 37°C. I te mutunga o te whakamātautau, i horoia wawetia ngā wāhanga ki te wairewa wai (pH 7.4) kei roto ko te 75 mM ammonium carbonate, kātahi ka whakaranua ki te 40:40:20 (v:v:v) acetonitrile (ACN): methanol: wai. I muri i te whaowhia o ngā wāhanga ki runga i te hukapapa mō te 30 meneti, ka whakatōngia ngā tauira ki te 21,000 karamu mō te 10 meneti i te 4°C, ā, ka whakamaroketia te wai mārama i roto i te pūwero SpeedVac. Ko te pirepire matū maroke i puta mai i rongoatia ki te -80°C tae noa ki te tātaritanga.
Tātaritanga chromatography wai-spectrometry papatipu o ngā waikawa amino 13 kua tohua ki te C. Mō te tātaritanga chromatography wai-spectrometry papatipu (LC-MS), i whakatārewatia anō te pirepire metabolite ki roto i te 75μl o te wai karaehe LC-MS (Honeywell). I muri i te centrifugation i te 21,000 g mō te 5 meneti i te 4°C, i whakamahia te 20 μl o te supernatant kua whakamāramahia mō te tātaritanga rerenga waikawa amino, ko te toenga o te tangohanga i whakamahia tonutia mō te tātaritanga anion (tirohia i raro). I whakahaerehia te tātaritanga waikawa amino mā te whakamahi i te kawa tangohanga benzoyl chloride kua whakaahuatia i mua (55, 56). I te taahiraa tuatahi, i tāpirihia te 10μl o te 100 mM konutai kāponāte (Sigma-Aldrich) ki te 20μl o te tangohanga metabolite, kātahi ka tāpirihia te 10μl o te 2% benzoyl chloride (Sigma-Aldrich) ki te ACN karaehe LC. I whakakorikoria te tauira mō te wā poto, kātahi ka whakatōkia ki te 21,000 karamu mō te 5 meneti i te 20°C. Whakawhitihia te wai i horoia ki roto i tētahi ipu tauira aunoa 2 ml me tētahi whakaurunga karāhe kōniko (rahi 200 μl). I tātarihia ngā tauira mā te whakamahi i te pūnaha LC mahi tino teitei Acquity iClass (Waters) e hono ana ki te ine papatipu tino tika Q-Exactive (QE)-HF (Ultra High Field Orbitrap) (Thermo Fisher Scientific). Mō te tātari, i werohia te 2μl o te tauira i tangohia ki roto i tētahi pou T3 silica kaha-teitei 100×1.0 mm (Waters) kei roto ngā matūriki 1.8μm. Ko te tere rere he 100μl/min, ā, ko te pūnaha parepare he mea hanga mai i te parepare A (10 mM ammonium formate me te 0.15% waikawa formic i roto i te wai) me te parepare B (ACN). Ko te rōnaki e whai ake nei: 0%B i te 0 meneti; 0%B. 0 ki te 15% B i te 0 ki te 0.1 meneti; 15 ki te 17% B i te 0.1 ki te 0.5 meneti; B i te 17 ki te 55% i te 0.5 ki te 14 meneti; B i te 55 ki te 70% i te 14 ki te 14.5 meneti; i te 14.5 ki te 70 ki te 100% B i te 18 meneti; 100% B i te 18 ki te 19 meneti; 100 ki te 0% B i te 19 ki te 19.1 meneti; 0% B i te 19.1 ki te 28 meneti (55, 56). Ka mahi te ine papatipu QE-HF i roto i te aratau ionization pai me te whānuitanga papatipu o te m/z (ōwehenga papatipu/utu) o te 50 ki te 750. Ko te taumira i whakamahia ko te 60,000, ā, ko te whāinga ion mana whakanui (AGC) i whiwhi ko te 3×106, ā, ko te wā ion mōrahi ko te 100 mirihekona. Ka mahi te pūtake ionization rehu hiko wera (ESI) i te ngaohiko rehu o te 3.5 kV, te pāmahana capillary o te 250°C, te rere o te hau i roto i te uhi o te 60 AU (ngā waeine ā-wātea), me te rere o te hau āwhina o te 20 AU. 250°C. Kua whakatakotoria te arotahi S ki te 60 AU.
Tātaritanga chromatography anion-MS o ngā waikawa waro kua tohua ki te 13C. I tātarihia te toenga o te matū matū (55μl) mā te whakamahi i te pūnaha chromatography ion Dionex (ICS 5000+, Thermo Fisher Scientific) e hono ana ki te ine papatipu QE-HF (Thermo Fisher Scientific). I roto i te poto, i werohia te 5μl o te tango matū matū ki roto i te pou Dionex IonPac AS11-HC kua rite ki te HPLC (2 mm × 250 mm, te rahi matūriki 4μm, Thermo Fisher Scientific) i roto i te aratau porowhita wāhanga pana-roto me te ōwehenga whakakī o te 1. ) Pou tiaki Dionex IonPac AG11-HC (2 mm x 50 mm, 4μm, Thermo Fisher Scientific). Ka puritia te pāmahana pou ki te 30°C, ā, kua whakatakotoria te autosampler ki te 6°C. Whakamahia he kātereti pāhare pākawa kua whakaratohia me te wai deionized hei whakaputa i te gradient pāhare pākawa mā te kaihanga eluent. Te wehenga o ngā matū matū i te tere rere o te 380μl/min, me te whakamahi i te pikinga e whai ake nei: 0 ki te 3 meneti, 10 mM KOH; 3 ki te 12 meneti, 10 ki te 50 mM KOH; 12 ki te 19 meneti, 50 ki te 100 mM KOH; 19 ki te 21 meneti, 100 mM KOH; 21 ki te 21.5 meneti, 100 ki te 10 mM KOH. I whakatauritea anō te pou i raro i te 10 mM KOH mō te 8.5 meneti.
Ka honoa ngā matū matū kua whakawāteatia ki tētahi awa tāpiri isopropanol 150μl/min i muri i te pou, kātahi ka tukuna ki tētahi ine papatipu taumira teitei e mahi ana i te aratau ionization kino. Kei te aroturuki a MS i te awhe papatipu mai i te m/z 50 ki te 750 me te taumira o te 60,000. Kua whakatakotoria te AGC ki te 1×106, ā, ka puritia te wā ion mōrahi ki te 100 ms. I whakahaerehia te pūtake ESI wera i te ngaohiko rehu o te 3.5 kV. Ko ētahi atu tautuhinga o te pūtake ion koia ēnei: ​​te pāmahana capillary 275°C; te rere hau o te uhi, 60 AU; te rere hau āwhina, 20 AU i te 300°C, me te tautuhinga arotahi S ki te 60 AU.
Te tātari raraunga o ngā matū matū kua tohua ki te 13C. Whakamahia te pūmanawa TraceFinder (putanga 4.2, Thermo Fisher Scientific) mō te tātari raraunga o te ōwehenga isotope. I manatokohia te tuakiri o ia pūhui e tētahi pūhui tohutoro pono, ā, i tātarihia motuhaketia. Hei whakahaere i te tātari whakarei isotope, i tangohia te horahanga o te chromatogram katote (XIC) i tangohia o ia isotope 13C (Mn) mai i [M + H] +, ko n te tau waro o te pūhui ūnga, e whakamahia ana hei tātari i ngā waikawa amino, [MH] + rānei hei tātari i ngā anion. Ko te tika o te papatipu o XIC he iti iho i te rima wāhanga ia miriona, ā, ko te tika o te RT he 0.05 meneti. Ka mahia te tātari whakarei mā te tatau i te ōwehenga o ia isotope kua kitea ki te tapeke o ngā isotope katoa o te pūhui e rite ana. Ka hoatu ēnei ōwehenga hei uara ōrau mō ia isotope, ā, ka whakaatuhia ngā hua hei whakarei ōrau molar (MPE), e ai ki te whakaahuatanga o mua (42).
I whakaranua te pirepire neuron tio ki roto i te methanol 80% makariri (v/v), i whakakorikoria, ā, i whaowhia ki te -20°C mō te 30 meneti. Whakakorikoria anō te tauira, ka whakaohokia ki te +4°C mō te 30 meneti. I whakakorikoria te tauira ki te 21,000 karamu mō te 5 meneti i te 4°C, kātahi ka kohia te wai i puta mai, ā, ka whakamarokehia mā te whakamahi i te SpeedVac concentrator i te 25°C mō te tātaritanga i muri mai. E ai ki te whakaahuatanga i runga ake nei, i mahia te tātaritanga LC-MS ki ngā waikawa amino o ngā pūtau kua whakarōpūtia. Mā te whakamahi i te TraceFinder (putanga 4.2, Thermo Fisher Scientific), i mahia te tātaritanga raraunga mā te whakamahi i te papatipu monoisotopic o ia pūhui. I mahia te whakataurite ine o ngā raraunga metabolite mā te whakamahi i te kete pūmanawa preprocessCore (57).
Te whakarite i te tapatapahi. I whakamoea wawetia te kiore ki te hauhā waro, ā, i tapahia te upoko, i tangohia wawetia te roro i te angaanga, ā, i whakamahia te maripi wiri kī tonu i te huka (HM-650 V, Thermo Fisher Scientific, Walldorf, Tiamana) hei tapahi kia 300 ki te 375 μm ngā wāhanga porowhita. Te whakapūngao waro matao (95% O2 me te 5% CO2) Iti Ca2 + ACSF (125.0 mM NaCl, 2.5 mM KCl, 1.25 mM konutai phosphate buffer, 25.0 mM NaHCO3, 25.0 mM d-glucose, 1.0 mM CaCl2 me te 6.0 mM MgCl2. Whakaritea ki te pH 7.4 me te 310 ki te 330 mOsm). Whakawhitia ngā poro roro kua riro mai ki tētahi rūma kei roto ko te Ca2 + ACSF teitei ake (125.0 mM NaCl, 2.5 mM KCl, 1.25 mM konutai phosphate buffer, 25.0 mM NaHCO3, 25.0 mM d-glucose, 4.0 mM CaCl2 me te mM 3.5 MgCl2) pH 7.4 me te 310 ki te 320 mOsm). Penapenahia ngā poro mō te 20 ki te 30 meneti kia taea ai te whakahoki mai i mua i te tuhi.
te tuhi. I whakamahia he atamira karuārai kua rite ki tētahi rūma tuhi pūmau me tētahi karāhe arotahi rumaki wai 20x (Scientifica) mō ngā tuhi katoa. I tautuhia ngā pūtau Purkinje e whakaarohia ana mā te (i) rahi o te tinana, (ii) te taunga hanganga o te roro iti, me te (iii) te whakaaturanga o te ira pūrongo mtYFP kanapa. Ko te pipette papaki me te ātete pito o te 5 ki te 11 megohms ka unuhia e tētahi capillary karāhe borosilicate (GB150-10, 0.86 mm×1.5 mm×100 mm, Science Products, Hofheim, Tiamana) me tētahi pipette whakapae Instruments (P-1000, Sutter), Novato, CA). I ​​mahia ngā tuhi katoa e te whakakaha papaki npi ELC-03XS (npi electronic GmbH, Tam, Tiamana), i whakahaeretia e te pūmanawa Signal (putanga 6.0, Cambridge Electronic, Cambridge, UK). I tuhia te whakamātautau i te tere tauira o te 12.5 kHz. Ka tātarihia te tohu mā te whakamahi i ngā tātari poto-poto e rua a Bessel me ngā auau tapahi o te 1.3 me te 10 kHz. Ka whakaea te kaha o te membrane me te pipette e te ara iahiko whakaea mā te whakamahi i te whakakaha. I whakahaerehia ngā whakamātautau katoa i raro i te mana o te kāmera Orca-Flash 4.0 (Hamamatsu, Gerden, Tiamana), i whakahaeretia e te pūmanawa Hokawo (putanga 2.8, Hamamatsu, Gerden, Tiamana).
Te whirihoranga me te tātaritanga pūtau-katoa auau. I mua tonu i te tuhi, whakakīia te pipette ki te otinga o roto kei roto ko ēnei matū e whai ake nei: 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) me te 10.0 mM creatinine phosphate i whakatikatikaina ki te pH 7.25, ā, ko te pēhanga osmotic he 290 mOsm (sucrose). I muri tonu i te whakamahi i te kaha o te 0 pA hei pakaru i te membrane, ka inehia te pūmanawa membrane okiokinga. Ka inehia te ātete whakauru mā te whakamahi i ngā iahiko hyperpolarized o -40, -30, -20, me te -10 pA. Inehia te rahi o te urupare ngaohiko ka whakamahia te ture a Ohm hei tatau i te ātete whakauru. I tuhia te ngohe tūpono i roto i te rawhi ngaohiko mō te 5 meneti, ā, i tautuhia te sPSC me te ine i roto i te Igor Pro (putanga 32 7.01, WaveMetrics, Lake Oswego, Oregon, USA) mā te whakamahi i tētahi tuhinga mōhiotanga haurua-aunoa. Ka inehia te pihi IV me te iahiko pumau mā te rawhi i te pākahiko i ngā pūmanawa rerekē (tīmata mai i te -110 mV) me te whakanui ake i te ngaohiko i roto i ngā taahiraa 5 mV. I whakamatautauria te whakaputanga o te AP mā te whakamahi i te iahiko whakaheke-polarizing. Rawhihia te pūtau ki te -70 mV i te wā e whakamahi ana i te pā o te iahiko whakaheke-polarizing. Whakatikatikahia te rahi taahiraa o ia wae tuhi motuhake (10 ki te 60 pA). Tātaihia te auau AP mōrahi mā te tatau ā-ringa i ngā pīka pā e hua ai te auau AP teitei rawa. Ka tātarihia te paepae AP mā te whakamahi i te tuarua o ngā pānga o te pā o te whakaheke-polarization e whakaoho tuatahi ana i tētahi, neke atu rānei o ngā AP.
Whirihoranga me te tātari i te papaki pokaia. Whakahaerehia te tuhi papaki pokaia mā te whakamahi i ngā tikanga paerewa. Whakamahia he pipette kore-ATP me te GTP kāore i roto i ēnei kai e whai ake nei: 128 mM gluconate K, 10 mM KCl, 10 mM Hepes, 0.1 mM EGTA me te 2 mM MgCl2, ka whakatikatika ki te pH 7.2 (mā te whakamahi i te KOH). Ka tangohia te ATP me te GTP mai i te otinga ā-roto hei ārai i te urunga kore mana o te kiriuhi pūtau. Ka whakakīia te pipette papaki ki te otinga ā-roto kei roto te amphotericin (tata ki te 200 ki te 250μg/ml; G4888, Sigma-Aldrich) hei whiwhi i te tuhi papaki pokaia. I whakarewaina te amphotericin ki roto i te dimethyl sulfoxide (te kukū whakamutunga: 0.1 ki te 0.3%; DMSO; D8418, Sigma-Aldrich). Kāore he pānga nui o te kukū o te DMSO i whakamahia ki ngā neuron i rangahauhia. I te wā o te mahi wero, i aroturukitia tonutia te ātete o te awa (Ra), ā, i tīmata te whakamātautau i muri i te pumau o te kaha o Ra me te AP (20-40 meneti). Ka inehia te mahi tūpono i roto i te rawhi ngaohiko me/ranei te iahiko mō te 2 ki te 5 meneti. I whakahaerehia te tātari raraunga mā te whakamahi i a Igor Pro (putanga 7.05.2, WaveMetrics, USA), Excel (putanga 2010, Microsoft Corporation, Redmond, USA) me GraphPad Prism (putanga 8.1.2, GraphPad Software Inc., La Jolla, CA). Te Hononga o Amerika). Hei tautuhi i ngā AP tūpono, ka whakamahia te monomai NeuroMatic v3.0c a IgorPro. Tāutuhia aunoa ngā AP mā te whakamahi i tētahi paepae kua hoatu, ka whakarerekētia takitahi mō ia rekoata. Mā te whakamahi i te wā tūpono, whakatauhia te auau tūpono me te auau tūpono mōrahi me te auau tūpono toharite.
Te wehenga PN. Mā te urutau ki te kawa i whakaputaina i mua, i purea ngā PN mai i te roro iti o te kiore i tētahi wāhanga kua tohua (58). I roto i te poto, i wehea te roro iti, ā, i tapatapahia ki roto i te reo wehewehe makariri [kāore he HBSS Ca2+ me te Mg2+, i tāpirihia ki te 20 mM glucose, penicillin (50 U/ml) me te streptomycin (0.05 mg/ml)], kātahi ka nakunaku te reo ki roto i te papain [HBSS, i tāpirihia ki te 1-cysteine·HCl (1 mg/ml), papain (16 U/ml) me te deoxyribonuclease I (DNase I; 0.1 mg/ml)] Whakaorahia mō te 30 meneti i te 30°C. Horoia tuatahitia ngā kiko ki roto i te reo HBSS kei roto te hūpē hua manu (10 mg/ml), te BSA (10 mg/ml) me te DNase (0.1 mg/ml) i te pāmahana rūma hei ārai i te nakunaku whākōkī, kātahi ki roto i te reo HBSS kei roto te 20 mM huka. Huri mārire ki roto i te HBSS, ka tukuna ngā pūtau takitahi e te penicillin (50 U/ml), te streptomycin (0.05 mg/ml) me te DNase (0.1 mg/ml). I tātarihia te hua o te whakatārewatanga pūtau mā roto i te tātari pūtau 70μm, kātahi ka whakapūpūtia ngā pūtau mā te centrifugation (1110 rpm, 5 meneti, 4°C) ā, ka whakatārewatia anō ki roto i te reo whakarōpū [HBSS, tāpirihia ki te 20 mM huka, 20% kukune kau) serum, penicillin (50 U/ml) me te streptomycin (0.05 mg/ml)]; arotakehia te oranga o te pūtau ki te propidium iodide me te whakatikatika i te kiato o te pūtau ki te 1×106 ki te 2×106 pūtau/ml. I mua i te tātaritanga rere, i tātarihia te whakatārewatanga mā roto i tētahi tātari pūtau 50 μm.
Ine rere. I whakahaerehia te whakarōpūtanga pūtau i te 4°C mā te whakamahi i te mīhini FACSAria III (BD Biosciences) me te pūmanawa FACSDiva (BD Biosciences, putanga 8.0.1). I whakarōpūtia te whakatārewatanga pūtau mā te whakamahi i te pūwero 100 μm i raro i te pēhanga o te 20 psi i te tere o te ~2800 ngā huihuinga/hēkona. Nā te mea kāore e taea e ngā paearu whakawātea tuku iho (te rahi o te pūtau, te wehewehe bimodal, me ngā āhuatanga marara) te whakarite i te wehenga tika o te PN mai i ētahi atu momo pūtau, ka whakatakotoria te rautaki whakawātea i runga i te whakataurite tika o te kaha YFP me te autofluorescence i roto i te mitoYFP+ ​​​​me te mitoYFP − Mice whakahaere. Ka whakaohokia te YFP mā te whakamārama i te tauira ki te raina laser 488 nm, ā, ka kitea te tohu mā te whakamahi i te tātari paahi roopu 530/30 nm. I roto i ngā kiore mitoYFP+, ka whakamahia hoki te kaha whanaunga o te ira pūrongo Rosa26-mitoYFP hei wehewehe i ngā kongakonga tinana neuronal me te axon. Ka whakaohokia te 7-AAD ki te whakamahi i te taiaho kōwhai 561 nm, ā, ka kitea mā te tātari whakawhiti roopu 675/20 nm hei ārai i ngā pūtau mate. Hei wehewehe i ngā pūtau whetu i te wā kotahi, ka pania te whakatārewatanga pūtau ki te ACSA-2-APC, kātahi ka whakamātaohia te tauira ki te aho taiaho 640 nm, ā, ka whakamahia he tātari whakawhiti roopu 660/20 nm hei kite i te tohu.
I whakapūpūhia ngā pūtau i kohia mā te centrifugation (1110 rpm, 5 meneti, 4°C) ā, i rongoatia ki te -80°C tae noa ki te whakamahinga. Ka whakarōpūtia ngā kiore Mfn2cKO me ā rātou uri i te rā kotahi hei whakaiti i te rerekētanga o ngā tikanga. I whakahaerehia te whakaaturanga me te tātari raraunga FACS mā te whakamahi i te pūmanawa FlowJo (FlowJo LLC, Ashland, Oregon, USA).
E ai ki te kōrero i runga ake nei (59), ka whakamahia te PCR wā-tūturu hei wehe i te DNA mai i ngā neuron kua whakarōpūtia mō te ine ine mtDNA i muri mai. I te tuatahi i whakamatautauria te raina me te aro o te paepae mā te whakahaere i te qPCR i runga i ngā tau rerekē o ngā pūtau. I roto i te poto, kohia te 300 PN ki roto i te lysis buffer kei roto ko te 50 mM tris-HCl (pH 8.5), 1 mM EDTA, 0.5% Tween 20 me te proteinase K (200 ng/ml) ka whaowhia ki te 55°C mō te 120 meneti. I whaowhia anō ngā pūtau ki te 95°C mō te 10 meneti hei whakarite kia kore rawa e whakahohea te proteinase K. Mā te whakamahi i tētahi whakamātautau TaqMan (Thermo Fisher) motuhake ki te mt-Nd1, i inehia te mtDNA mā te PCR haurua-tau i roto i te pūnaha PCR Wā-Tūturu 7900HT (Thermo Fisher Scientific). Pūtaiao, nama rarangi ingoa Mm04225274_s1), mt-Nd6 (Thermo Fisher Scientific, nama rarangi ingoa AIVI3E8) me ngā ira 18S (Thermo Fisher Scientific, nama rarangi ingoa Hs99999901_s1).
Te whakarite tauira proteome. Mā te whakamahana i te otinga i te 95°C mō te 10 meneti me te sonicating, i roto i te lysis buffer [6 M guanidine chloride, 10 mM tris(2-carboxyethyl) phosphine hydrochloride, 10 mM chloroacetamide me te 100 mM tris-Lyse tio neuron pellets i roto i te HCl]. I runga i te Bioruptor (Diagenode) mō te 10 meneti (30 hēkona pulse / 30 hēkona wā okioki). I whakaranua te tauira 1:10 ki roto i te 20 mM tris-HCl (pH 8.0), i konatunatua ki te 300 ng trypsin koura (Promega), ā, i whaowhia mō te pō i te 37°C kia oti te nakunaku. I te rā tuarua, i centrifugehia te tauira ki te 20,000 g mō te 20 meneti. I whakaranua te supernatant ki te 0.1% waikawa formic, ā, i tangohia te tote o te otinga ki te StageTips i hangaia e ia anō. I whakamaroketia te tauira i roto i te taputapu SpeedVac (Eppendorf concentrator me te 5305) i te 45°C, kātahi ka whakatārewatia te peptide ki roto i te 0.1% waikawa formic. I whakaritea ngā tauira katoa i te wā kotahi e te tangata kotahi. Hei tātari i ngā tauira astrocyte, i tapaina ngā peptide kua tangohia te tote e 4 μg ki te tohu papatipu tandem (TMT10plex, nama rarangi 90110, Thermo Fisher Scientific) me te ōwehenga peptide ki te matū TMT o te 1:20. Mō te tapanga TMT, i whakatārewatia anō te 0.8 mg o te matū TMT ki roto i te 70 μl o te ACN anhydrous, ā, i hangaia anō te peptide maroke ki roto i te 9 μl o te 0.1 M TEAB (triethylammonium bicarbonate), ā, i tāpirihia ki reira te 7 μl o te matū TMT i roto i te ACN. Ko te kukū he 43.75%. I muri i te 60 meneti o te whakatipu, i tineia te tauhohenga ki te 2 μl o te 5% hydroxylamine. I kohia ngā peptide kua tohua, i whakamaroketia, i whakatārewatia anō ki roto i te 200μl o te 0.1% waikawa formic (FA), i wehea kia rua, kātahi ka tangohia te tote mā te whakamahi i a StageTips i hangaia e rātou anō. Mā te whakamahi i te UltiMate 3000 ultra high performance liquid chromatography (UltiMate 3000 ultra high performance liquid chromatography), i wehea tētahi o ngā haurua e rua ki runga i te pou chromatography Acquity 1mm x 150mm kua whakakiia ki ngā matūriki C18 130Å1.7μm (Waters, nama putumōhio SKU: 186006935). Thermo Fisher Scientific). Wehea ngā peptide i te tere rere o te 30μl/min, wehea mai i te 1% ki te 50% buffer B mō te 85 meneti me te pikinga takahanga o te 96 meneti, mai i te 50% ki te 95% buffer B mō te 3 meneti, kātahi ka 8 meneti mō te 95% Buffer B; Ko te pūwero A he 5% ACN me te 10 mM ammonium bicarbonate (ABC), ā, ko te pūwero B he 80% ACN me te 10 mM ABC. Kohia ngā wāhanga i ia 3 meneti ka whakakotahi kia rua ngā rōpū (1 + 17, 2 + 18, me ētahi atu) ka whakamaroke i roto i te mīhini whakapūwero korehau.
Tātari LC-MS/MS. Mō te ine i te papatipu, i wehea ngā peptide (nama r119.aq) i runga i te pou tātari PicoFrit 25 cm, 75 μm te whānui o roto (he karāhe arotahi hou, nama wāhanga PF7508250) kua rite ki te reo ReproSil-Pur 120 C18-AQ 1.9 μm (Dr. Maisch, mat), Whakamahia te EASY-nLC 1200 (Thermo Fisher Scientific, Tiamana). I puritia te pou i te 50°C. Ko ngā pūwero A me B he 0.1% waikawa formic i roto i te wai me te 0.1% waikawa formic i roto i te 80% ACN, ia. I wehea ngā peptide mai i te 6% ki te 31% pūwero B mō te 65 meneti me te 31% pūwero B mō te 5 meneti me te pikinga o te 200 nl/min. I tātarihia ngā peptide i tangohia i runga i te ine ine papatipu Orbitrap Fusion (Thermo Fisher Scientific). Ka mahia te ine i te m/z o te pūmua peptide me te taumira o te 120,000 i te awhe o te 350 ki te 1500 m/z. Mā te whakamahi i te 27% o te kaha tukinga kua whakatauritea, ka tīpakohia te pūmua kaha rawa atu me te āhua utu o te 2 ki te 6 mō te wehenga wehenga mahanga C (HCD) kaha teitei. Kua whakatakotoria te wā huringa ki te 1 s. I inehia te uara m/z o te kongakonga peptide i roto i te mahanga katote mā te whakamahi i te ūnga AGC iti rawa o te 5 × 104 me te wā werohanga mōrahi o te 86 ms. I muri i te wehewehenga, ka whakanohoia te pūmua ki te rārangi aukati hihiri mō te 45 s. I wehea ngā peptide kua tohua ki te TMT i runga i te pou Acclaim PepMap 50 cm, 75 μm (Thermo Fisher Scientific, nama rarangi 164942), ā, i tātarihia ngā hihi hekenga i runga i te ine papatipu Orbitrap Lumos Tribrid (Thermo Fisher Scientific) kua rite ki ngā taputapu ion ngaru kore-taurite teitei (FAIMS) (Thermo Fisher Scientific) e mahi ana i ngā ngaohiko taurite e rua o te −50 me te −70 V. Ko te MS3 i tīpakohia i runga i te mua tukutahi ka whakamahia mō te ine tohu ion pūrongo TMT. I mahia te wehenga peptide i runga i te EASY-nLC 1200, mā te whakamahi i te elution gradient raina 90%, me te kukū parepare o te 6% ki te 31%; ko te parepare A he 0.1% FA, ā, ko te parepare B he 0.1% FA me te 80% ACN. Ka whakahaerehia te pou tātari i te 50°C. Whakamahia a FreeStyle (putanga 1.6, Thermo Fisher Scientific) hei wehe i te kōnae taketake i runga i te ngaohiko taurite FAIMS.
Te tautuhi pūmua me te ine ine. Mā te whakamahi i te miihini rapu Andromeda whakauru, i tātarihia ngā raraunga taketake mā te whakamahi i te MaxQuant putanga 1.5.2.8 (https://maxquant.org/). Haunga ngā raupapatanga Cre recombinase me YFP i whiwhi mai i Aequorea victoria, i rapua ngā momo peptide mokowā mō te raupapatanga matua me te raupapatanga isoform o te proteome tohutoro kiore (Proteome ID UP000000589, i tangohia mai i UniProt i te marama o Mei 2017). I whakatakotoria te waikura methionine me te acetylation N-terminal pūmua hei whakarerekētanga taurangi; i whakatakotoria te methylation cysteine ​​​​carbamoyl hei whakarerekētanga pumau. Kua whakatakotoria ngā tawhā nakunaku ki te "motuhaketanga" me te "trypsin/P". Ko te iti rawa o ngā peptides me ngā peptides razor i whakamahia mō te tautuhi pūmua ko te 1; ko te iti rawa o ngā peptides ahurei ko te 0. I raro i ngā tikanga o te taurite mahere peptide, ko te tere tautuhi pūmua ko te 0.01. Kua whakahohea te kōwhiringa "Peptide Tuarua". Whakamahia te kōwhiringa "taurite i waenga i ngā oma" hei whakawhiti i ngā tautuhi angitu i waenga i ngā kōnae taketake rerekē. Whakamahia te tatau ōwehenga iti rawa LFQ 1 mō te ine kore-tapanga (LFQ) (60). Ka tātarihia te kaha LFQ mō te iti rawa e rua ngā uara tika i roto i te iti rawa kotahi te rōpū ira i ia wā, ā, ka tangohia mai i te tohatoha noa me te whānui o te . 0.3 ka neke ki raro 1.8. Whakamahia te tūāpapa rorohiko Perseus (https://maxquant.net/perseus/) me te R (https://r-project.org/) hei tātari i ngā hua LFQ. I whakamahia he whakamātautau t-waenga rua-ara mai i te kete pūmanawa limma mō te tātari whakapuakanga rerekētanga (61). Ka mahia te tātari raraunga tūhura mā te whakamahi i te ggplot, FactoMineR, factoextra, GGally me te pheatmap. I tātarihia ngā raraunga proteomics e hangai ana ki te TMT mā te whakamahi i te putanga MaxQuant 1.6.10.43. Rapua ngā raraunga proteomics mata mai i te pātengi raraunga proteomics tangata a UniProt, i tangohia i te marama o Hepetema 2018. Kei roto i te tātaritanga te tauwehenga whakatikatika parakore isotope i whakaratohia e te kaihanga. Whakamahia te limma i roto i te R mō te tātaritanga whakapuakanga rerekētanga. Ko ngā raraunga taketake, ngā hua rapu pātengi raraunga, me te reremahi tātari raraunga me ngā hua katoa e rongoa ana i roto i te hononga ProteomeXchange mā te putunga hoa PRIDE me te tohu huinga raraunga PXD019690.
Ka whakarei ake ngā tuhipoka mahi i te tātari. I whakamahia te taputapu Ingenuity Pathway Analysis (QIAGEN) hei whakatau i te momonatanga o ngā kupu tuhipoka mahi o te huinga raraunga i te 8 wiki (Pikitia 1). I roto i te poto, ka whakamahia te rārangi pūmua tauanga i whiwhi mai i te tātari raraunga LC-MS/MS (tandem mass spectrometry) me ngā paearu tātari e whai ake nei: Ka tīpakohia a Mus musculus hei momo me te papamuri, ā, ka whakaatuhia e te kāwai ko te uara P i whakatikatikaina e Benjamini mō te whakarei ake 0.05, iti iho rānei, e kiia ana he mea nui. Mō tēnei kauwhata, ka whakaaturia ngā kāwai taikaha e rima o runga i ia rōpū i runga i te uara P i whakatikatikaina. Mā te whakamahi i te whakamātautau-t maha, mā te whakamahi i te kaupapa whakanui raina-rua-wāhanga a Benjamini, Krieger, me Yekutieli (Q = 5%), ka mahia te tātari whakaaturanga pūmua wā-wā ki ngā kaitono nui kua tautuhia i roto i ia kāwai, ā, ka tātarihia motuhaketia ia rarangi. Kāore he take ki te tango i tētahi SD ōrite.
Hei whakataurite i ngā hua o tēnei rangahau ki ngā pātengi raraunga kua whakaputaina, me te whakaputa i tētahi tūtohi Venn i te Pikitia 1, i whakakotahi mātou i te rārangi pūmua tauanga me ngā tuhipoka MitoCarta 2.0 (24). Whakamahia te taputapu ipurangi Tuhia te tūtohi Venn (http://bioinformatics.psb.ugent.be/webtools/Venn/) hei whakaputa i te tūtohi.
Mō ngā kōrero taipitopito mō ngā tikanga tatauranga e whakamahia ana mō te tātari proteomics, tirohia te wāhanga e pā ana ki ngā Rauemi me ngā Tikanga. Mō ngā whakamātautau katoa, kei roto i te kōrero e pā ana ngā kōrero taipitopito. Ki te kore e tohua tētahi atu mea, ka whakaatuhia ngā raraunga katoa hei toharite ± SEM, ā, i mahia ngā tātari tatauranga katoa mā te whakamahi i te pūmanawa GraphPad Prism 8.1.2.
Mō ngā rauemi tāpiri mō tēnei tuhinga, tirohia http://advances.sciencemag.org/cgi/content/full/6/35/eaba8271/DC1
He tuhinga urunga-tuwhera tēnei e tohatohahia ana i raro i ngā tikanga o te Raihana Creative Commons Attribution-Non-Commercial, e āhei ai te whakamahinga, te tohatoha me te tārua i roto i tētahi momo pāpāho, mena kāore te whakamahinga whakamutunga mō te whai hua arumoni, ā, ko te whakaaro matua he tika te mahi taketake. Tohutoro.
Kia mōhio: Ko tā mātou anake he tuku mai i tō wāhitau īmēra kia mōhio ai te tangata e taunaki ana koe ki te whārangi e hiahia ana koe kia kite rātou i te īmēra, ā, ehara i te pāme. Kāore mātou e hopu i tētahi wāhitau īmēra.
Ka whakamahia tēnei pātai hei whakamātautau mēnā he manuhiri koe, hei ārai hoki i te tuku pāme aunoa.
Na E. Motori, I. Atanassov, SMV Kochan, K. Folz-Donahue, V. Sakthivelu, P. Giavalisco, N. Toni, J. Puyal, N.-G. Larson
I whakaatuhia e te tātaritanga proteomics o ngā neuron kore mahi pai, ka whakahohehia ngā kaupapa pākia hei ārai i te mate neurodegeneration.
Na E. Motori, I. Atanassov, SMV Kochan, K. Folz-Donahue, V. Sakthivelu, P. Giavalisco, N. Toni, J. Puyal, N.-G. Larson
I whakaatuhia e te tātaritanga proteomics o ngā neuron kore mahi pai, ka whakahohehia ngā kaupapa pākia hei ārai i te mate neurodegeneration.
©2020 American Association for the Advancement of Science. mana katoa. Ko AAAS te hoa o HINARI, AGORA, OARE, CHORUS, CLOCKSS, CrossRef me COUNTER. ScienceAdvances ISSN 2375-2548.