Adreesị dị ugbu a: Cologne 50931, Germany, Cologne Excellence Cluster Research on Cellular Stress Response in Threat-Related Attacks (CECAD).
A na-ewere neurodegeneration nke ọrịa mitochondrial dị ka ihe a na-apụghị ịgbagha agbagha n'ihi na plasticity metabolic nke neurons pere mpe, mana mmetụta nke mitochondrial dysfunction na nnwere onwe sel nke metabolism neuronal n'ahụ adịghị mma. N'ebe a, anyị na-ewebata proteome nke mkpụrụ ndụ Purkinje neurons nwere ụkọ OXPHOS na-aga n'ihu nke mitochondrial fusion dynamics mebiri emebi. Anyị chọpụtara na mitochondrial dysfunction kpalitere nnukwu mgbanwe n'ọhịa nke proteomics, nke mechara duga na mmalite nke mmemme metabolic ziri ezi tupu ọnwụ mkpụrụ ndụ. N'atụghị anya ya, anyị chọpụtara na mkpali doro anya nke pyruvate carboxylase (PCx) na enzymes ndị ọzọ na-egbochi ịka nká nke na-agbakwunye etiti nke usoro TCA. Mgbochi nke PCx mere ka nrụgide oxidative na neurodegeneration ka njọ, na-egosi na atherosclerosis nwere mmetụta nchebe na neurons na-enweghị OXPHOS. Mweghachi nke mitochondrial fusion na neurons ndị mebiri emebi na-agbanwe àgwà metabolic ndị a kpamkpam, si otú a na-egbochi ọnwụ mkpụrụ ndụ. Nchọpụta anyị na-achọpụta ụzọ ndị a na-amaghị na mbụ nke na-enye iguzogide nsogbu mitochondrial ma na-egosi na enwere ike ịgbanwe neurodegeneration ọbụna n'oge ikpeazụ nke ọrịa ahụ.
Ọrụ dị mkpa nke mitochondria n'ịnọgide na-enwe metabolism ike neuronal bụ ihe mgbaàmà akwara dị iche iche metụtara ọrịa mitochondrial mmadụ na-emesi ike. Ọtụtụ n'ime ọrịa ndị a na-akpata site na mgbanwe mkpụrụ ndụ ihe nketa nke na-achịkwa ngosipụta mkpụrụ ndụ ihe nketa mitochondrial (1, 2) ma ọ bụ mbibi mkpụrụ ndụ ihe nketa metụtara mitochondrial dynamics, nke na-emetụta nkwụsi ike nke mitochondrial DNA (mtDNA) n'ụzọ na-apụtaghị ìhè (3, 4). Ọrụ na ụdị anụmanụ egosila na na nzaghachi maka nsogbu mitochondrial na anụ ahụ gbara ya gburugburu, enwere ike ịgbalite ụzọ metabolic na-agbanwe agbanwe (5-7), nke na-enye ozi dị mkpa maka nghọta miri emi nke pathogenesis nke ọrịa ndị a dị mgbagwoju anya. N'ụzọ dị iche, nghọta anyị banyere mgbanwe metabolic nke ụdị mkpụrụ ndụ kpọmkwem nke ọdịda izugbe nke mmepụta adenosine triphosphate mitochondrial (ATP) nke ụbụrụ kpatara bụ isi (8), na-emesi ike mkpa ọ dị ịchọpụta ebumnuche ọgwụgwọ nke enwere ike iji gbochie ma ọ bụ gbochie ọrịa. Gbochie neurodegeneration (9). Enweghị ozi bụ eziokwu ahụ bụ na a na-ewerekarị mkpụrụ ndụ akwara dị ka ndị nwere obere mgbanwe metabolic ma e jiri ya tụnyere ụdị mkpụrụ ndụ nke anụ ahụ gbara ya gburugburu (10). Ebe ọ bụ na mkpụrụ ndụ ndị a na-arụ ọrụ dị mkpa n'ịhazi ọkọnọ nke metabolites nye neurons iji kwalite nnyefe synaptic ma meghachi omume na ọnọdụ mmerụ ahụ na ọrịa, ikike ime mgbanwe metabolism cell na ọnọdụ siri ike nke anụ ahụ ụbụrụ fọrọ nke nta ka ọ bụrụ naanị mkpụrụ ndụ glial (11-14). Na mgbakwunye, ọdịiche dị n'etiti sel nke anụ ahụ ụbụrụ na-egbochikarị ọmụmụ nke mgbanwe metabolic nke na-eme na obere obere neuronal. N'ihi ya, a maghị ihe gbasara kpọmkwem ihe na-esi na cellular na metabolic pụta nke mitochondrial dysfunction na neurons.
Iji ghọta ihe ndị na-esi na nsogbu mitochondrial apụta, anyị kewapụrụ akwara Purkinje (PNs) n'ọkwa dị iche iche nke neurodegeneration nke mbibi nke mitochondrial outer membrane fusion (Mfn2) kpatara. Ọ bụ ezie na mgbanwe Mfn2 n'ime mmadụ na-ejikọta ya na ụdị neuropathy moto nketa nke a maara dị ka Charcot-Marie-Tooth type 2A (15), mbibi ọnọdụ nke Mfn2 n'ime òké bụ usoro a maara nke ọma maka mmetọ oxidation Phosphorylation (OXPHOS). Ụdị neuronal dị iche iche (16-19) na ụdị neurodegenerative na-esite na ya na-esochi mgbaàmà akwara na-aga n'ihu, dị ka nsogbu mmegharị (18, 19) ma ọ bụ cerebellar ataxia (16). Site na iji ngwakọta nke proteomics, metabolomics, onyonyo, na usoro virological, anyị na-egosi na neurodegeneration na-aga n'ihu na-akpali pyruvate carboxylase (PCx) na ihe ndị ọzọ metụtara arteriosclerosis nke PNs na vivo Ngosipụta nke enzymes. Iji chọpụta mkpa nchọpụta a dị, anyị belatara ngosipụta nke PCx na PN ndị na-enweghị Mfn2, ma chọpụta na ọrụ a mere ka nrụgide oxidative ka njọ ma mee ka neurodegeneration dịkwuo ngwa ngwa, si otú a na-egosi na azoospermia na-enye ọnwụ mkpụrụ ndụ mgbanwe metabolism. Ngosipụta siri ike nke MFN2 nwere ike ịzọpụta PN kpamkpam mmebi ikpeazụ yana oke OXPHOS, oke oriri nke mitochondrial DNA, na netwọk mitochondrial mebiri emebi, nke na-emesi ike na ụdị neurodegeneration a nwere ike ịgbake n'ọkwa dị elu nke ọrịa tupu ọnwụ mkpụrụ ndụ.
Iji hụ mitochondria na PN ndị na-anaghị agagharị na Mfn2, anyị jiri ụdị òké nke na-enye ohere ka mitochondria nke Cre-dependent lekwasị anya na protein fluorescent odo (YFP) (mtYFP) (20) Ngosipụta Cre ma lelee ọdịdị mitochondrial na vivo. Anyị chọpụtara na mbibi nke mkpụrụ ndụ ihe nketa Mfn2 na PN ga-eduga na nkewa nwayọ nke netwọk mitochondrial (Foto S1A), a hụkwara mgbanwe mbụ mgbe izu atọ gachara. N'ụzọ dị iche, nnukwu mmebi nke oyi akwa sel PN, dịka egosiri site na mfu nke Calbindin immunostaining, amaliteghị ruo izu iri na abụọ (Foto 1, A na B). Oge adịghị n'etiti mgbanwe mbụ na ọdịdị mitochondrial na mmalite a na-ahụ anya nke ọnwụ neuronal mere ka anyị nyochaa mgbanwe metabolic nke ọrịa mitochondrial kpatara tupu ọnwụ sel. Anyị mepụtara usoro nhazi mkpụrụ ndụ (FACS) nke dabere na fluorescence-activated cell sorting (YFP+) iji kewapụ YFP (YFP+)-na-egosipụta PN (Foto 1C), na n'ime òké njikwa (Mfn2 + / loxP :: mtYFP loxP-stop-loxP: : L7-cre), nke a na-akpọ CTRL (Foto S1B). Nhazi nke atụmatụ gating dabere na ike dị n'etiti mgbaàmà YFP na-enye anyị ohere ịsacha ahụ YFP+ (YFPhigh) nke PNs site na ndị na-abụghị PN (YFPneg) (Foto S1B) ma ọ bụ iberibe axon/dendritic fluorescent putative (YFPlow; Foto S1D, aka ekpe), nke ejiri igwe microscope confocal kwado (Foto S1D, aka nri). Iji chọpụta njirimara nke ndị mmadụ edepụtara, anyị mere proteomics LFQ wee nyochaa akụkụ bụ isi, wee chọpụta na enwere nkewa doro anya n'etiti mkpụrụ ndụ YFPhigh na YFPneg (Foto S1C). Mkpụrụ ndụ YFP dị elu gosiri mmụba dị ukwuu nke ihe nrịbama PN a ma ama (dịka Calb1, Pcp2, Grid2 na Itpr3) (21, 22), mana ọ dịghị mmụba nke protein ndị a na-egosipụtakarị na neurons ma ọ bụ ụdị mkpụrụ ndụ ndị ọzọ (Foto 1D)). Ntụnyere dị n'etiti ihe nlele na mkpụrụ ndụ YFP dị elu anakọtara na nnwale ndị nọọrọ onwe ha gosiri ọnụọgụ njikọ > 0.9, na-egosi ezigbo mmụba n'etiti ihe ndị na-emepụta ihe dị ndụ (Foto S1E). Na nchịkọta, data ndị a kwadoro atụmatụ anyị maka ikewapụ PN dị oke na nke a kapịrị ọnụ. N'ihi na sistemụ ọkwọ ụgbọala L7-cre ejiri mee ihe na-akpali njikọta mosaic n'izu mbụ mgbe ebusịrị ya (23), anyị malitere igbutu òké site na CTRL na ọnọdụ (Mfn2 loxP / loxP :: mtYFP loxP-stop-loxP :: L7-cre) Chịkọta neurons. Mgbe emechara njikọta, a na-akpọ ya Mfn2cKO mgbe ọ dị izu anọ. Dịka isi mmalite, anyị họọrọ izu asatọ nke afọ mgbe oyi akwa PN dịgidere n'agbanyeghị nkewa mitochondrial doro anya (Foto 1B na Foto S1A). Na mkpokọta, anyị tụlere ngụkọta protein 3013, nke ihe dị ka 22% dabere na nkọwa MitoCarta 2.0 dabere na mitochondrial proteome dị ka mitochondria (Foto 1E) (Foto 1E) (24). Nyocha ngosipụta mkpụrụ ndụ ihe nketa dị iche emere na izu nke 8 gosiri na naanị 10.5% nke protein niile nwere mgbanwe dị ukwuu (Foto 1F na Foto S1F), nke protein 195 dara ada na protein 120 ka achịkwara (Foto 1F). Ọ dị mkpa ịmara na "nyocha ụzọ ọhụrụ" nke data a na-egosi na mkpụrụ ndụ ihe nketa ndị a na-egosipụta dị iche iche bụ isi na usoro mgbochi nke ụzọ metabolic kpọmkwem (Foto 1G). Ọ dị mma ịmara na ọ bụ ezie na mbelata nke ụzọ ndị metụtara OXPHOS na signaling calcium na-egosi na mitochondrial dysfunction na PN ndị na-enweghị fusion, ụdị ndị ọzọ nke gụnyere amino acid metabolism na-amụba nke ukwuu, nke kwekọrọ na metabolism nke na-eme na mitochondrial PNs Rewiring na-agbanwe agbanwe. adịghị arụ ọrụ.
(A) Foto ndị a na-anọchite anya akụkụ ụbụrụ nke òké CTRL na Mfn2cKO na-egosi mfu PN (calbindin, isi awọ); e jiri DAPI kpụọ nuclei ndị ahụ. (B) Ngụkọta nke (A) (nnyocha otu ụzọ nke mgbanwe, ***P<0.001; n = okirikiri 4 ruo 6 site na òké atọ). (C) Usoro ọrụ nnwale. (D) Nkesa maapụ ọkụ nke ihe nrịbama kpọmkwem maka Purkinje (elu) na ụdị sel ndị ọzọ (etiti). (E) eserese Venn na-egosi ọnụọgụ protein mitochondrial achọpụtara na PN nkewa. (F) Atụmatụ ugwu mgbawa nke protein ndị a na-egosipụta dị iche iche na neurons Mfn2cKO na izu 8 (uru mbelata dị mkpa nke 1.3). (G) Nyocha ụzọ okike na-egosi ụzọ ise kachasị mkpa maka iwuli elu (uhie) na iwuli ala (acha anụnụ anụnụ) na Mfn2cKO PN nkewapụtara dị ka izu 8. Egosiri ọkwa ngosipụta nke protein ọ bụla achọpụtara. Maapụ okpomọkụ grayscale: uru P agbanwere. ns, ọ bụghị ihe dị mkpa.
Data Proteomics gosiri na ngosipụta protein nke complexes I, III, na IV ji nwayọọ nwayọọ belata. Complexes I, III, na IV niile nwere subunits dị mkpa nke mtDNA-encoded, ebe complex II, nke bụ naanị koodu nuklia, enweghị mmetụta ọ bụla (Foto 2A na Foto S2A). . N'ikwekọ na nsonaazụ proteomics, immunohistochemistry nke ngalaba anụ ahụ cerebellar gosiri na ọkwa subunit MTCO1 (mitochondrial cytochrome C oxidase subunit 1) nke complex IV na PN ji nwayọọ nwayọọ belata (Foto 2B). Subunit mtDNA-encoded Mtatp8 belatara nke ukwuu (Foto S2A), ebe ọkwa ọnọdụ kwụsiri ike nke subunit ATP synthase nke nuklia-encoded ka agbanweghị agbanwe, nke kwekọrọ na complex F1 a maara nke ọma nke ATP synthase subassembly mgbe ngosipụta mtDNA kwụsiri ike. Nhazi ahụ na-agbanwe agbanwe. Nkwụsị (7). Nyocha nke ọkwa mtDNA na Mfn2cKO PNs a haziri ahazi site na mmeghachi omume polymerase chain real-time (qPCR) kwadoro mbelata nwayọ nwayọ na nọmba mtDNA. Ma e jiri ya tụnyere otu njikwa, mgbe ọ dị izu asatọ, naanị ihe dị ka 20% nke ọkwa mtDNA ka ejirigide (Foto 2C). Dịka nsonaazụ ndị a si dị, ejiri confocal microscopy staining nke Mfn2cKO PNs chọpụta DNA, na-egosi oriri nke mitochondrial nucleotides dabere na oge (Foto 2D). Anyị chọpụtara na naanị ụfọdụ ndị sonyere na mitochondrial protein degeneration na nzaghachi nrụgide ka achịkwara, gụnyere Lonp1, Afg3l2 na Clpx, na ihe ndị mejupụtara OXPHOS. Achọpụtaghị mgbanwe dị mkpa na ọkwa protein ndị metụtara apoptosis (Foto S2B). N'otu aka ahụ, anyị chọpụtara na ọwa mitochondria na endoplasmic reticulum metụtara na njem calcium nwere naanị obere mgbanwe (Foto S2C). Tinyere nke ahụ, nyocha nke protein ndị metụtara autophagy ahụghị mgbanwe dị mkpa ọ bụla, nke kwekọrọ na mmụba a na-ahụ anya nke autophagosomes nke a hụrụ na vivo site na immunohistochemistry na electron microscopy (Foto S3). Agbanyeghị, nsogbu OXPHOS na-aga n'ihu na PNs na-esochi mgbanwe mitochondrial doro anya. Enwere ike ịhụ ụyọkọ mitochondrial na ahụ sel na osisi dendritic nke Mfn2cKO PNs dị afọ 5 na 8, nhazi akpụkpọ ahụ dị n'ime agbanweela nnukwu mgbanwe (Foto S4, A na B). N'ikwekọ na mgbanwe ultrastructural ndị a na mbelata dị ukwuu na mtDNA, nyocha nke iberibe cerebellar cerebel na tetramethylrhodamine methyl ester (TMRM) gosiri na ikike mitochondrial membrane na Mfn2cKO PNs belatara nke ukwuu (Foto S4C).
(A) Nyocha oge nke ọkwa ngosipụta nke mgbagwoju anya OXPHOS. Tụlee naanị protein nwere P<0.05 na izu 8 (ANOVA ụzọ abụọ). Ahịrị ntụpọ: Enweghị mgbanwe ma e jiri ya tụnyere CTRL. (B) Aka ekpe: Ihe atụ nke ngalaba cerebellar ejiri antibody anti-MTCO1 mee akara (ogwe nha, 20 μm). Ebe ahụ nke ahụ mkpụrụ ndụ Purkinje bi na ya bụ odo. Aka nri: Nha nke ọkwa MTCO1 (nyocha otu ụzọ nke mgbanwe; n = mkpụrụ ndụ 7 ruo 20 nyochachara site na òké atọ). (C) nyocha qPCR nke nọmba mtDNA depụta na PN a haziri ahazi (nyocha otu ụzọ nke mgbanwe; n = òké 3 ruo 7). (D) Aka ekpe: Ihe atụ nke iberibe cerebellar ejiri antibody anti-DNA mee akara (ogwe nha, 20 μm). Ebe ahụ nke ahụ mkpụrụ ndụ Purkinje bi na ya bụ odo. Aka nri: Nha nke mmerụ ahụ mtDNA (nyocha otu ụzọ nke mgbanwe; n = mkpụrụ ndụ 5 ruo 9 site na òké atọ). (E) Ihe atụ nke ngalaba cerebellar dị nkọ nke na-egosi mkpụrụ ndụ mitoYFP + Purkinje (akụ) na ndekọ mkpọchi mkpọchi mkpụrụ ndụ dum. (F) Ngụkọta nke usoro IV. (G) Ndekọ nnọchite anya nke ntinye ugbu a na-ewepụ polarizing na mkpụrụ ndụ CTRL na Mfn2cKO Purkinje. Ngụkọta elu: Mkpụrụ mbụ nke kpalitere AP. Ngụkọta ala: Oke ugboro AP. (H) Ngụkọta nke ntinye postsynaptic sponset (sPSPs). E gosipụtara usoro ndekọ nnọchite anya na oke mbugharị ya na (I). Nnyocha otu ụzọ nke mgbanwe nyochachara n = 5 ruo 20 mkpụrụ ndụ site na òké atọ. A na-egosipụta data dị ka nkezi ±SEM; *P<0.05; **P<0.01; ***P<0.001. (J) Ngụkọta nnọchite anya nke AP sponset dekọọ site na iji ọnọdụ mkpọchi oghere. Ngụkọta elu: Oke ugboro AP. Ngụkọta ala: mbugharị nke otu AP. (K) Ngụkọta nkezi na oke ugboro AP dịka (J) si dị. Nnwale Mann-Whitney; n = 5 mkpụrụ ndụ nyochachara site na òké anọ. A na-egosi data dị ka nkezi ± SEM; ọ dịghị mkpa.
Achọpụtara mmebi OXPHOS doro anya na Mfn2cKO PN nke dị izu asatọ, nke na-egosi na ọrụ nke akwara ozi adịghị mma nke ukwuu. Ya mere, anyị nyochachara njirimara eletriki na-agafe agafe nke akwara ozi na-enweghị OXPHOS n'izu 4 ruo 5 na izu 7 ruo 8 site na ịme ndekọ mkpọchi mkpọchi mkpụrụ ndụ dum na obere akụkụ ụbụrụ (Foto 2E). N'atụghị anya ya, nkezi ikike izu ike na nguzogide ntinye nke akwara ozi Mfn2cKO yiri njikwa ahụ, ọ bụ ezie na enwere obere ọdịiche dị n'etiti mkpụrụ ndụ (Tebụl 1). N'otu aka ahụ, na izu 4 ruo 5 nke afọ, a hụghị mgbanwe dị mkpa na mmekọrịta voltaji ugbu a (usoro IV) (Foto 2F). Agbanyeghị, ọ dịghị akwara ozi Mfn2cKO dị izu 7 ruo 8 lanarịrị usoro IV (usoro hyperpolarization), na-egosi na enwere mmetụta doro anya na ikike hyperpolarization n'oge ikpeazụ a. N'ụzọ dị iche, na mkpụrụ ndụ akwara Mfn2cKO, a na-anabata mmiri ndị na-eme ka mmiri ghara ịdịghachi ugboro ugboro (AP), nke na-egosi na usoro mmiri ha niile adịghị iche na nke mkpụrụ ndụ akwara ndị dị izu asatọ (Tebụl 1 na Foto 2G). N'otu aka ahụ, ugboro na oke nke mmiri ndị na-eme ka mmiri ghara ịdị iche na nke mkpụrụ ndụ akwara ndị dị izu asatọ (Tebụl 1 na Foto 2G). N'otu aka ahụ, ugboro na oke nke mmiri ndị na-eme ka mmiri ghara ịdị na-apụta ìhè (sPSCs) yiri nke otu njikwa ahụ, ugboro ole ihe na-eme mụbara site na izu anọ ruo izu ise ruo izu asaa ruo izu asatọ na mmụba yiri nke ahụ (Foto 2, H na I). ​​Oge ntozu synaptic na PNs (25). Enwetara nsonaazụ ndị yiri ya mgbe ihe ndị a gbawara agbawa gasịrị. Nhazi a na-egbochi ụgwọ ọrụ nke ntụpọ ATP cellular, dịka ọ nwere ike ime na ndekọ mkpọchi patch cell dum. Karịsịa, ike akpụkpọ ahụ na-ezu ike na ugboro ọkụ nke mkpụrụ ndụ akwara Mfn2cKO emetụtaghị ya (Foto 2, J na K). Na nchịkọta, nsonaazụ ndị a na-egosi na PN nwere nsogbu OXPHOS doro anya nwere ike ịnagide usoro mwepụ ugboro ugboro dị elu nke ọma, na-egosi na enwere usoro nkwụghachi ụgwọ nke na-enye ha ohere ịnọgide na-enwe mmeghachi omume electrophysiological fọrọ nke nta ka ọ bụrụ nkịtị.
A na-egosi data dị ka nkezi ± SEM (nnyocha otu ụzọ nke mgbanwe, ule ntụnyere ọtụtụ Holm-Sidak; *P<0.05). A na-egosi nọmba nkeji site na brackets.
Anyị malitere inyocha ma ụdị ọ bụla dị na dataset proteomics (Foto 1G) nwere ụzọ ndị nwere ike igbochi oke OXPHOS, si otú a na-akọwa ihe kpatara PN metụtara ji nwee ike ịnọgide na-enwe electrophysiology fọrọ nke nta ka ọ bụrụ nkịtị (Foto 2, E ruo K). . Nyocha proteomics gosiri na enzyme ndị dị na catabolism nke branched yinye amino acids (BCAA) ka achịkwa nke ọma (Foto 3A na Foto S5A), na ngwaahịa ikpeazụ acetyl-CoA (CoA) ma ọ bụ succinyl CoA nwere ike ịgbakwunye tricarboxylates na okirikiri arteriosclerosis Acid (TCA). Anyị chọpụtara na ọdịnaya nke BCAA transaminase 1 (BCAT1) na BCAT2 mụbara. Ha na-akwalite nzọụkwụ mbụ nke catabolism BCAA site na imepụta glutamate site na α-ketoglutarate (26). A na-achịkwa obere akụkụ niile mejupụtara keto acid dehydrogenase (BCKD) nke nwere alaka alaka (mgbagwoju anya ahụ na-akpali mwepụta na-esote na nke a na-apụghị ịgbanwe agbanwe nke ọkpụkpụ carbon BCAA nke na-esi na ya apụta) (Foto 3A na Foto S5A). Agbanyeghị, a hụghị mgbanwe doro anya na BCAA n'onwe ya na PN a haziri ahazi, nke nwere ike ịbụ n'ihi mmụba nke nnabata sel nke amino acid ndị a dị mkpa ma ọ bụ ojiji nke isi mmalite ndị ọzọ (glucose ma ọ bụ lactic acid) iji gbakwunye okirikiri TCA (Foto S5B). PN ndị na-enweghị OXPHOS gosikwara mmụba nke mmebi glutamine na ọrụ transamination na izu asatọ nke afọ, nke enwere ike igosipụta site na mmụba nke enzymes mitochondrial glutaminase (GLS) na glutamine pyruvate transaminase 2 (GPT2) (Foto 3, A na C). Ọ dị mkpa ịmara na mmụba nke GLS bụ naanị na isoform glutaminase C (GLS-GAC) nke a na-agbakọta (mgbanwe nke Mfn2cKO/CTRL dị ihe dị ka okpukpu 4.5, P = 0.05), yana mmụba kpọmkwem ya na anụ ahụ kansa nwere ike ịkwado ike mitochondrial bioenergy. (27).
(A) Maapụ okpomọkụ na-egosi mgbanwe mpịachi na ọkwa protein maka ụzọ akọwapụtara na izu 8. (B) Ihe atụ nke ibe cerebellar nke ejiri antibody anti-PCx mee akara (ogwe nha, 20 μm). Akụ odo na-egosi ahụ mkpụrụ ndụ Purkinje. (C) Nyocha ngosipụta protein oge achọpụtara dị ka ihe dị mkpa maka atherosclerosis (ọtụtụ t-test, *FDR <5%; n = òké 3-5). (D) N'elu: Eserese eserese na-egosi ụzọ dị iche iche esi abanye carbon akpọrọ dị na [1-13C]pyruvate tracer (ya bụ, site na PDH ma ọ bụ ụzọ trans-arterial). Ala: Chaatị violin na-egosi pasent nke otu carbon akpọrọ (M1) nke agbanwere na aspartic acid, citric acid na malic acid mgbe ejiri [1-13C]pyruvate (ule t jikọtara ọnụ; ** P <0.01). (E) Nyocha akụkọ ihe mere eme oge zuru oke nke ụzọ egosiri. Tụlee naanị protein nwere P<0.05 na izu 8. Ahịrị e ji akara mee: enweghị uru mgbanwe (nnyocha nke ọdịiche ụzọ abụọ; * P <0.05; *** P <0.001). A na-egosipụta data dị ka nkezi ± SEM.
Na nyocha anyị, BCAA catabolism aghọwo otu n'ime ụzọ ndị dị mkpa iji kwalite usoro. Eziokwu a na-egosi nke ọma na olu ikuku na-abanye na okirikiri TCA nwere ike ịgbanwe na PN na-enweghị OXPHOS. Nke a nwere ike ịnọchite anya ụdị dị mkpa nke mgbanwe metabolic neuronal, nke nwere ike inwe mmetụta kpọmkwem na physiology neuronal na ndụ n'oge mmezi nke nsogbu OXPHOS siri ike. Dịka echiche a si dị, anyị chọpụtara na enzyme anti-atherosclerotic bụ isi PCx na-achịkwa (Mfn2cKO/CTRL na-agbanwe ihe dị ka ugboro 1.5; Foto 3A), nke na-akwalite mgbanwe nke pyruvate ka ọ bụrụ oxaloacetate (28), nke a kwenyere na ọ dị na anụ ahụ ụbụrụ. Okwu in bụ naanị astrocytes (29, 30). Dabere na nsonaazụ proteomics, microscopy confocal gosiri na ngosipụta PCx mụbara nke ọma na PN ndị na-enweghị OXPHOS, ebe mmeghachi omume PCx bụ naanị na mkpụrụ ndụ Bergmann glial dị nso nke njikwa (Foto 3B). Iji nwalee mmụba a hụrụ na PCx, anyị jiri [1-13C] pyruvate tracer gwọọ mpekere cerebellar dị nkọ. Mgbe pyruvate dehydrogenase (PDH) mere ka pyruvate dị oxidized, akara isotope ya kwụsịrị, Mana etinyere ya n'ime etiti okirikiri TCA mgbe mmeghachi omume vascular na-eme ka pyruvate dịghachi ndụ (Foto 3D). Iji kwado data proteomics anyị, anyị hụrụ ọtụtụ ihe nrịbama sitere na tracer a na aspartic acid nke mpekere Mfn2cKO, ebe citric acid na malic acid nwekwara usoro dị mma, ọ bụ ezie na ọ bụghị ihe dị mkpa (Foto 3D).
N'ime akwara dopamine nke oke MitoPark nwere nsogbu mitochondrial nke dopamine neurons kpatara kpọmkwem mbibi mitochondrial transcription factor A gene (Tfam) (Foto S6B), a na-achịkwa ngosipụta PCx nke ọma (31), na-egosi na acetone acid arteriosclerosis A na-achịkwa ihe na-eme na ọrịa ahụ n'oge nsogbu nke neuronal OXPHOS n'ahụ. Ọ dị mkpa ịmara na achọpụtala na enzymes pụrụ iche (32-34) nke enwere ike igosipụta na neurons nke nwere ike ijikọ na arteriosclerosis na-achịkwa nke ukwuu na PN ndị na-enweghị OXPHOS, dị ka propionyl-CoA carboxylase (PCC-A), Malonyl-CoA na-agbanwe propionyl-CoA ka ọ bụrụ succinyl-CoA na mitochondrial malic enzyme 3 (ME3), nke ọrụ ya bụ isi bụ iweghachite pyruvate site na malate (Foto 3, A na C) (33, 35). Tinyere nke a, anyị hụrụ mmụba dị ukwuu na enzyme Pdk3, nke na-eme ka PDH ghara ịrụ ọrụ (36), ebe achọpụtaghị mgbanwe ọ bụla na enzyme Pdp1 nke na-eme ka PDH ma ọ bụ enzyme PDH dị mgbagwoju anya rụọ ọrụ (Foto 3A). N'otu aka ahụ, na Mern2cKO PNs, phosphorylation nke α1 subunit α (PDHE1α) nke ihe mejupụtara pyruvate dehydrogenase E1 nke PDH dị mgbagwoju anya na Ser293 (a maara na ọ na-egbochi ọrụ enzyme nke PDH) ka mma (Foto S6C) (Foto S6C). Pyruvate enweghị ohere ịbanye n'ime akwara.
N'ikpeazụ, anyị chọpụtara na ụzọ kachasị elu nke serine na glycine biosynthesis, usoro mitochondrial folate (1C) metụtara ya na proline biosynthesis (Foto 1G na Foto S5C) niile ka achịkwa nke ọma, dịka akụkọ si kwuo, n'oge usoro mmalite. A na-eme ka anụ ahụ gbara ya gburugburu rụọ ọrụ site na nsogbu mitochondrial (5-7). Nyocha Confocal nke na-akwado data proteomics ndị a gosiri na na PN na OXPHOS na-efu, iberibe cerebellar nke òké dị izu asatọ ka e tinyere serine hydroxymethyltransferase 2 (SHMT2), enzyme dị mkpa nke usoro mitochondrial folate. Mmeghachi omume mgbochi dị mkpa (Foto S5D). Na mpekere cerebellar 13 nke CU-glucose-incubated, nnwale ndị metụtara metabolic kwadoro mmụba nke serine na proline biosynthesis, na-egosi na mmụba nke isoforms carbon n'ime serine na proline mụbara (Foto S5E). Ebe ọ bụ na mmeghachi omume nke GLS na GPT2 kwalitere bụ maka mmepụta glutamate site na glutamine na mgbanwe n'etiti glutamate na α-ketoglutarate, mmụba ha na-egosi na akwara ndị na-enweghị OXPHOS nwere mmụba nke ọchịchọ maka glutamate. Nke a nwere ike ịbụ iji nọgide na-enwe mmụba nke biosynthesis nke proline (Foto S5C). N'adịghị ka mgbanwe ndị a, nyocha proteomic nke cerebellar astrocytes sitere na òké Mfn2cKO nke PN-specific gosiri na ụzọ ndị a (gụnyere antiperoxidases niile) agbanweghị nke ukwuu na ngosipụta, si otú a na-egosi na ntụgharị metabolic a na-ahọrọ PN mebiri emebi (Foto S6, D ruo G).
Na nchịkọta, nyocha ndị a gosiri ụdị dị iche iche nke mkpali oge nke ụzọ metabolic kpọmkwem na PNs. Ọ bụ ezie na ọrụ mitochondrial neuronal na-adịghị mma nwere ike ibute atherosclerosis mbụ na mgbanwe 1C (Foto 3E na Foto S5C), yana ọbụna mgbanwe a na-atụ anya ya na ngosipụta nke mgbagwoju anya I na IV, mgbanwe na njikọ serine de novo bụ naanị Ọ pụtara ìhè naanị na oge ikpeazụ. Nsogbu OXPHOS (Foto 3E na Foto S5C). Nchọpụta ndị a na-akọwa usoro usoro nke mitochondrial nke nrụgide kpatara (1C cycle) na cytoplasmic (serine biosynthesis) na-azaghachi n'ụzọ kwekọrọ na mmụba nke atherosclerosis na okirikiri TCA iji gbanwee metabolism neuronal.
PN ndị dị izu asatọ nke OXPHOS enweghị ike ịnọgide na-enwe ọrụ mkpali ugboro ugboro ma na-enweta njikọ dị ukwuu nke metabolic iji kwụọ ụgwọ maka nsogbu mitochondrial. Nchọpụta a na-eweta ohere na-adọrọ mmasị na ọbụna n'oge a, mkpụrụ ndụ ndị a nwekwara ike ịnweta ọgwụgwọ iji gbuo oge ma ọ bụ gbochie neurodegeneration. N'oge na-adịghị anya. Anyị doziri ohere a site na ntinye aka abụọ nọọrọ onwe ha. Na usoro mbụ, anyị chepụtara vektọ nje adeno-associated (AAV) nke Cre-dependent ka e wee nwee ike igosipụta MFN2 na PN ndị na-enweghị OXPHOS na vivo (Foto S7A). E gosipụtara AAV na-agbanwe MFN2 na mkpụrụ ndụ ihe nketa fluorescent reporter mCherry (Mfn2-AAV) na omenala neuron bụ isi na vitro, nke mere ka a gosipụta MFN2 n'ụzọ Cre-dependent ma zọpụta mitochondrial morphology, si otú a gbochie neuromutation na Mfn2cKO neurons (Foto S7, B, D na E). Mgbe nke ahụ gasịrị, anyị mere nnwale in vivo iji nye Mfn2-AAV dị izu asatọ n'ime cerebellar cortex nke Mfn2cKO ma chịkwaa òké, ma nyochaa òké dị izu iri na abụọ (Foto 4A). Òké Mfn2cKO a gwọrọ ọrịa nwụrụ (Foto 1, A na B) (16). Mbugharị nje na vivo mere ka PN pụta ìhè n'ụfọdụ okirikiri cerebellar (Foto S7, G na H). Ịgba AAV njikwa nke na-egosipụta naanị mCherry (Ctrl-AAV) enweghị mmetụta dị ukwuu na ọkwa neurodegeneration na anụmanụ Mfn2cKO. N'ụzọ dị iche, nyocha nke Mfn2cKOs ejiri Mfn2-AAV mee gosipụtara mmetụta dị mkpa nke oyi akwa mkpụrụ ndụ PN (Foto 4, B na C). Karịsịa, njupụta neuron yiri ka ọ fọrọ nke nta ka ọ ghara ịdị iche na anụmanụ ndị a na-achịkwa (Foto 4, B na C, na Foto S7, H na I). Ngosipụta nke MFN1 mana ọ bụghị MFN2 dị irè n'ịzọpụta ọnwụ akwara ozi (Foto 4C na Foto S7, C na F), nke na-egosi na ngosipụta nke ectopic MFN1 nwere ike imeju enweghị MFN2 nke ọma. Nyocha ọzọ na ọkwa PN otu gosiri na Mfn2-AAV zọpụtara nnukwu nhazi nke mitochondria, mee ka ọkwa mtDNA dị mma, ma gbanwee ngosipụta dị elu nke akara anti-angiogenesis PCx ​​(Foto 4, C ruo E). Nnyocha anya nke òké Mfn2cKO a zọpụtara n'ọnọdụ izu ike gosiri na ọnọdụ ha na mgbaàmà moto ha ka mma (mmegharị S1 ruo S3). Na mmechi, nnwale ndị a na-egosi na mwepụghachi MFN2 n'ime PNs nke na-adịghị oke njọ na OXPHOS zuru oke iji weghachite oriri mtDNA ma kpalite atherosclerosis, si otú a gbochie mmebi axon na ọnwụ neuronal na vivo.
(A) Atụmatụ na-egosi usoro nnwale maka ịgba AAV koodu MFN2 mgbe a na-arụ ọrụ ụzọ metabolic egosiputara. (B) Foto confocal nke ndị na-anọchite anya iberibe cerebellar dị izu iri na abụọ nke a gbanwere na izu asatọ na òké Mfn2cKO ma jiri antibody anti-Calbindin dee ya. Aka nri: Ịhazi eriri axon. Ọnụọgụ nke axon zoom bụ 450 na 75 μm. (C) Aka ekpe: Ọnụọgụ nke njupụta mkpụrụ ndụ Purkinje na loop transduction AAV (AAV+) (nnyocha otu ụzọ nke mgbanwe; n = òké 3). Aka nri: nyocha mtDNA lekwasịrị anya na PN a gbanwere na izu nke 12 (ule t-enweghị njikọ; n = mkpụrụ ndụ 6 sitere na òké atọ). * P <0.05; ** P <0.01. (D) Micrographs elektrọn nnyefe nnọchite anya nke PNs nke ngalaba cerebellar Mfn2cKO nke ejiri vektọ nje egosiputara mee ihe. Ihe mkpuchi ihu pink na-egosi ebe dendrites bi, square odo nwere ntụpọ na-egosi zoom enyere n'aka nri; n na-anọchite anya nucleus. Ogwe nha, 1μm. (E) na-egosi ihe atụ nke ntụpọ PCx na PN nke a gbanwere na izu iri na abụọ. Ogwe nha, 20μm. OE, oke ngosipụta; FC, mgbanwe mpịachi.
N'ikpeazụ, anyị nyochachara mkpa ọ dị ndụ mkpụrụ ndụ peroxidase kpatara na PN ndị nwere nsogbu OXPHOS. Anyị mepụtara mCherry nke na-akpọ AAV-shRNA (mkpụmkpụ ntutu isi mkpụmkpụ) nke na-elekwasị anya kpọmkwem na òké PCx mRNA (AAV-shPCx), ma gbanye nje ahụ ma ọ bụ njikwa ya (AAV-scr) n'ime cerebellum nke òké Mfn2cKO. A gbara ọgwụ ahụ n'izu nke anọ nke afọ (Foto 5A) iji nweta mbelata PCx dị irè n'oge ahụ mgbe ngosipụta PCx mụbara (Foto 3C) na oyi akwa mkpụrụ ndụ PN ka dịgidere (Foto 1A). Ọ dị mkpa ịmara na ịkụda PCx (Foto S8A) na-eduga na mmụba dị ukwuu nke ọnwụ PN, nke bụ naanị na mgbanaka ahụ butere ọrịa (Foto 5, B na C). Iji ghọta usoro nke mmetụta metabolic nke PCx na-akwalite, anyị mụrụ ọnọdụ redox nke PNs mgbe PCx knockdown gasịrị ma gosipụta ihe mmetụta biosensor optical nke AAV na-akwado Grx1-roGFP2 n'otu oge (Foto S8, B ruo D) iji nyochaa glutathione. Mgbanwe dị n'etiti ikike peptide redox (38). Mgbe ahụ, anyị mere microscopy onyonyo ndụ abụọ nke photon fluorescence (FLIM) na iberibe ụbụrụ dị nkọ nke Mfn2cKO dị izu 7 ma ọ bụ ndị na-achịkwa littermates iji chọpụta mgbanwe nwere ike ime na ọnọdụ redox cytoplasmic mgbe enyochachara ọnọdụ FLIM (Foto S8, E ruo G). Nyocha ahụ gosiri mmụba dị ukwuu na ọnọdụ oxidation nke otu PN Mfn2cKO na-enweghị ngosipụta PCx, nke dị iche na neurons njikwa ma ọ bụ Mfn2cKO PNs na-egosipụta naanị shRNA a gbajiri agbaji (Foto 5, D na E). Mgbe e belatara ngosipụta PCx, pasentị nke Mfn2cKO PNs nke na-egosi ọnọdụ oxidized nke ukwuu mụbara site na ihe karịrị ugboro atọ (Foto 5E), nke na-egosi na nhazi PCx nọgidere na-enwe ikike redox nke neurons mebiri emebi.
(A) Atụmatụ na-egosi usoro nnwale maka ịgba ọgwụ AAV na-agbanwe shPCx mgbe a na-arụ ọrụ ụzọ metabolic egosiputara. (B) Foto confocal nke ndị na-anọchite anya akụkụ cerebellar dị izu 8 na òké Mfn2cKO e tinyere ma tinye antibody anti-calcineurin na izu 4. Ogwe nha, 450μm. (C) Ngụkọta nke njupụta mkpụrụ ndụ Purkinje na loops AAV gbanwere (nyocha otu ụzọ nke mgbanwe; n = òké 3 ruo 4). A na-egosipụta data dị ka nkezi ±SEM; ***P<0.001. (D) Foto FLIM nke nnọchi anya na-egosi nkezi ndụ nke PN dị izu 7 nke na-egosipụta glutathione redox sensọ Grx1-roGFP2 n'okpuru ọnọdụ nnwale akọwapụtara. Oke LUT (tebụl nlele-elu): oge ​​ndụ (na picoseconds). Ogwe nha, 25μm. (E) Histogram ahụ na-egosi nkesa nke uru ndụ Grx1-roGFP2 site na (D) (n=158 ruo mkpụrụ ndụ 368 n'ime òké abụọ n'okpuru ọnọdụ ọ bụla). Chaatị achịcha dị n'elu histogram ọ bụla: na-egosi ọnụọgụ nke mkpụrụ ndụ nwere uru ndụ dị ogologo (uhie, oxidized) ma ọ bụ mkpụmkpụ (acha anụnụ anụnụ, ebelatara) nke gafere 1 SD nke uru ndụ nkezi na CTRL-AAV-scr. (F) Ụdị atụpụtara na-egosi mmetụta nchebe nke mmụba nke PCx neuronal.
Na mkpokọta, data anyị nyere ebe a na-egosi na ngosipụtaghachi nke MFN2 nwere ike ịzọpụta PN dị elu kpamkpam na oke OXPHOS dị oke njọ, oke mbelata mtDNA, na ọdịdị ista dị oke njọ, si otú a na-enye ọganihu na-aga n'ihu ọbụlagodi na ọrịa ndị dị elu. Mmebi akwara na-enye ihe akaebe a na-agbanwe agbanwe nke ọkwa tupu ọnwụ mkpụrụ ndụ. A na-emesikwu ike ọkwa mgbanwe metabolic a site n'ike nke neurons iji kpalite atherosclerosis (mmegharị nke usoro TCA), nke na-egbochi ngosipụta PCx na PNs na-enweghị OXPHOS ma na-eme ka ọnwụ mkpụrụ ndụ dịkwuo mma, si otú a na-arụ ọrụ nchebe (Foto 5F).
N'ọmụmụ ihe a, anyị nyere ihe akaebe na mmeghachi omume nke PNs na nsogbu OXPHOS bụ iji nwayọọ nwayọọ na-agbakọta na TCA cycle atherosclerosis site na ụzọ ọrụ dị iche iche nke mmemme metabolic na-arụ ọrụ. Anyị kwadoro nyocha proteomic site na ọtụtụ ụzọ ndị ọzọ ma kpughee na mgbe nsogbu mitochondrial siri ike na-ama aka, neurons nwere ụdị mgbanwe metabolic a na-amaghị na mbụ. Ihe ijuanya nye anyị bụ na usoro rewiring dum apụtaghị na ọnọdụ metabolic ikpeazụ nke na-eso neurodegeneration nwayọ nwayọ na enweghị mgbagha, mana data anyị na-egosi na ọ nwere ike ịbụ neuron mmezi ọbụlagodi na ọkwa tupu ọnwụ sel Usoro nkwụghachi ọrụ. Nchọpụta a na-egosi na neurons nwere oke nke plasticity metabolic n'ahụ. Eziokwu a na-egosi na MFN2 emesịa nwere ike ịgbanwe ngosipụta nke ihe nrịbama metabolic dị mkpa ma gbochie mmebi PN. N'ụzọ megidere nke ahụ, ọ na-egbochi atherosclerosis ma na-eme ka akwara dịkwuo ngwa ngwa. transsexual.
Otu n'ime ihe ndị kacha adọrọ mmasị na nnyocha anyị bụ na PNs enweghị OXPHOS nwere ike ịgbanwe metabolism okirikiri TCA site na ịbawanye enzymes nke na-akpali arteriosclerosis kpọmkwem. Nhazi metabolic bụ ihe a na-ahụkarị na mkpụrụ ndụ kansa, ụfọdụ n'ime ha na-adabere na glutamine iji gbakwunye etiti okirikiri TCA iji mepụta ihe nhata na-ebelata, nke na-akwali agbụ iku ume ma na-eme ka mmepụta nke lipid na nucleotide biosynthesis precursors dịgide (39, 40). Nnyocha e mere n'oge na-adịbeghị anya gosiri na n'ime anụ ahụ dị n'akụkụ na-enwe nsogbu OXPHOS, njikọta nke metabolism glutamine/glutamate bụkwa ihe a ma ama (5, 41), ebe ntụziaka nke ntinye glutamine n'ime okirikiri TCA dabere na N'ihi oke mmerụ ahụ OXPHOS (41). Agbanyeghị, enweghị ihe akaebe doro anya na myirịta ọ bụla nke plasticity metabolic neuronal n'ahụ na mkpa ya nwere ike ịdị na ọnọdụ ọrịa ahụ. Na ọmụmụ in vitro na-adịbeghị anya, egosiri na neurons cortical bụ isi na-akwalite ọdọ mmiri glutamate maka neurotransmission, si otú a na-akwalite metabolism oxidative na atherosclerosis n'okpuru ọnọdụ nrụgide metabolic (42). Ọ dị mkpa ịmara na n'okpuru mgbochi ọgwụ nke enzyme okirikiri TCA succinate dehydrogenase, a kwenyere na pyruvate carboxylation na-eme ka njikọ nke oxaloacetate dịgide na mkpụrụ ndụ cerebellar granule a zụlitere (34). Agbanyeghị, mkpa nke usoro ndị a na anụ ahụ ụbụrụ (ebe a kwenyere na atherosclerosis bụ naanị na astrocytes) ka nwere mkpa dị mkpa nke physiological (43). N'okwu a, data anyị na-egosi na enwere ike ịgbanwe PNs nke OXPHOS mebiri n'ahụ gaa na mbibi BCAA na pyruvate carboxylation, nke bụ isi mmalite abụọ nke mgbakwunye nke TCA pool intermediates. Ọ bụ ezie na atụpụtala ntinye aka nke BCAA catabolism na metabolism ike neuronal, na mgbakwunye na ọrụ nke glutamate na GABA maka neurotransmission (44), ọ ka enweghị ihe akaebe maka usoro ndị a na vivo. Ya mere, ọ dị mfe iche na PN ndị na-adịghị arụ ọrụ nwere ike ịkwụghachi akpaghị aka maka oriri nke TCA intermediates nke usoro assimilation na-akwalite site na ịbawanye atherosclerosis. Karịsịa, a pụrụ ịchọ mmụba nke PCx iji nọgide na-enwe mmụba nke ọchịchọ maka aspartic acid, nke a na-atụ aro na mkpụrụ ndụ ndị na-amụba nke nwere nsogbu mitochondrial (45). Agbanyeghị, nyocha metabolomics anyị ekpugheghị mgbanwe dị mkpa na ọkwa aspartic acid na-adịgide adịgide na Mfn2cKO PNs (Foto S6A), nke nwere ike igosipụta ojiji dị iche iche nke aspartic acid n'etiti mkpụrụ ndụ na-amụba na mkpụrụ ndụ post-mitotic. Ọ bụ ezie na usoro kpọmkwem nke mmụba PCx na mkpụrụ ndụ na-adịghị arụ ọrụ na vivo ka a ka ga-amata, anyị gosipụtara na nzaghachi a na-aga n'ihu na-arụ ọrụ dị mkpa n'ịnọgide na-enwe ọnọdụ redox nke mkpụrụ ndụ, nke egosiri na nnwale FLIM na mpekere cerebellar. Karịsịa, igbochi PNs ịbawanye PCx nwere ike ibute ọnọdụ oxidized karịa ma mee ka ọnwụ mkpụrụ ndụ dịkwuo ngwa ngwa. Mmelite nke mbibi BCAA na carboxylation nke pyruvate abụghị ụzọ isi mara anụ ahụ dị n'akụkụ nke nsogbu mitochondrial (7). Ya mere, ha yiri ka ha bụ ihe kacha mkpa nke neurons enweghị OXPHOS, ọbụlagodi na ọ bụghị naanị njirimara, nke dị mkpa maka neurodegeneration. .
Ọrịa cerebellar bụ ụdị ọrịa neurodegenerative dị iche iche nke na-apụtakarị dị ka ataxia ma na-emebi PNs (46). Ọnụ ọgụgụ neuron a na-adịkarị mfe ịnweta nsogbu mitochondrial n'ihi na mmebi nhọrọ ha na òké zuru oke iji mepụta ọtụtụ ihe mgbaàmà moto nke na-akọwapụta spinocerebellar ataxia mmadụ (16, 47, 48). Dịka akụkọ si kwuo, ụdị òké transgenic nwere mkpụrụ ndụ ihe nketa mutant jikọtara ya na spinocerebellar ataxia mmadụ ma nwee nsogbu mitochondrial (49, 50), na-emesi ike mkpa ọ dị ịmụ ihe ga-esi na enweghị OXPHOS pụta na PNPH. Ya mere, ọ dị mma karịsịa ịkewapụ ma mụọ ọnụọgụ neuron pụrụ iche a nke ọma. Agbanyeghị, ebe ọ bụ na PNs na-enwe mmetụta dị ukwuu na nrụgide ma na-aza maka obere akụkụ nke ọnụọgụ sel cerebellar dum, maka ọtụtụ ọmụmụ dabere na omics, nkewa ha dị ka mkpụrụ ndụ dum ka bụ akụkụ siri ike. Ọ bụ ezie na ọ fọrọ nke nta ka ọ ghara ikwe omume imezu enweghị mmetọ zuru oke nke ụdị mkpụrụ ndụ ndị ọzọ (karịsịa anụ ahụ ndị toro eto), anyị jikọtara nzọụkwụ nkewa dị irè na FACS iji nweta ọnụọgụgụ zuru oke nke neurons dị irè maka nyocha proteomics dị ala, ma nwee oke mkpuchi protein dị elu (ihe dị ka protein 3000) ma e jiri ya tụnyere data dị ugbu a nke cerebellum dum (51). Site n'ichekwa ndụ nke mkpụrụ ndụ dum, usoro anyị na-enye ebe a na-enye anyị ohere ọ bụghị naanị ịlele mgbanwe na ụzọ metabolic na mitochondria, kamakwa ịlele mgbanwe na ndị ogbo cytoplasmic ya, nke na-emeju ojiji nke mitochondrial membrane tag iji mee ka ụdị mkpụrụ ndụ baa ọgaranya Usoro ọhụrụ maka ọnụọgụ mitochondria na anụ ahụ dị mgbagwoju anya (52, 53). Usoro anyị na-akọwa abụghị naanị metụtara ọmụmụ nke mkpụrụ ndụ Purkinje, mana enwere ike itinye ya n'ọrụ ngwa ngwa na ụdị mkpụrụ ndụ ọ bụla iji dozie mgbanwe metabolic na ụbụrụ ọrịa, gụnyere ụdị ndị ọzọ nke dysfunction mitochondrial.
N'ikpeazụ, anyị achọpụtala usoro ọgwụgwọ n'oge usoro nhazi metabolic a nke nwere ike ịgbanwe ihe ịrịba ama dị mkpa nke nrụgide mkpụrụ ndụ ma gbochie mmebi akwara. Ya mere, ịghọta ihe ọrụ nke rewiring akọwara ebe a nwere ike inye nghọta bụ isi banyere ọgwụgwọ enwere ike iji nọgide na-enwe ike ịrụ ọrụ neuronal n'oge nsogbu mitochondrial. Nnyocha n'ọdịnihu nke ebumnuche ya bụ ịchọpụta mgbanwe na metabolism ike na ụdị mkpụrụ ndụ ụbụrụ ndị ọzọ dị mkpa iji kpughee nke ọma ojiji nke ụkpụrụ a na ọrịa akwara ndị ọzọ.
A kọwaala òké MitoPark na mbụ (31). A kọwaala òké C57BL/6N nwere mkpụrụ ndụ ihe nketa Mfn2 dị n'akụkụ loxP (18) ma jiri òké L7-Cre gafere ya (23). E jiri òké homozygous Mfn2loxP/Mfn2loxP kewaa ụmụ heterozygous abụọ si na ya pụta iji mepụta mkpụrụ ndụ ihe nketa Purkinje kpọmkwem maka Mfn2 (Mfn2loxP/Mfn2loxP; L7-cre). Na obere njikọ, e webatara allele Gt (ROSA26) SorStop-mito-YFP (stop-mtYFP) site na ngafe ndị ọzọ (20). Emere usoro anụmanụ niile dịka ntuziaka Europe, mba na ụlọ ọrụ si dị ma LandesamtfürNatur nke Umwelt na Verbraucherschutz, North Rhine-Westphalia, Germany kwadoro ya. Ọrụ anụmanụ na-agbasokwa nduzi nke European Federation of Laboratory Animal Sciences Associations.
Mgbe emechara ka nwanyị dị ime nwee nkụchi n'ọkpụkpụ olu, a na-ewepụ nwa ebu n'afọ òké (E13). A gbawara akụkụ ahụ n'ime Hanks' Balanced Salt Solution (HBSS) nke e tinyere 10 mM Hepes ma nye ya Dulbecco's Modified Eagle's Medium nke nwere papain (20 U/ml) na cysteine ​​​​(1μg/ml). Tinye anụ ahụ na DMEM) ma kewaa ya site na mgbaze enzymatic. Ml) na 37°C maka nkeji iri abụọ, wee jiri igwe gwerie ya na DMEM nke e tinyere 10% serum ehi nwa ebu n'afọ. A gbanyere mkpụrụ ndụ ahụ na mkpuchi iko nke e ji polylysine kpuchie na njupụta nke 2 × 106 kwa efere ọdịbendị 6 cm ma ọ bụ na njupụta nke 0.5 × 105 sel/cm2 maka nyocha onyonyo. Mgbe awa anọ gachara, ejiri usoro Neurobasal serum-free medium nke nwere 1% B27 mgbakwunye na 0.5 mM GlutaMax dochie ihe ahụ. E wee nọgide na-enwe akwara ndị ahụ na 37°C na 5% CO2 n'oge nnwale ahụ, ma nye ha nri otu ugboro n'izu. Iji mee ka a jikọta ha na vitro, e jiri 3μl (nri omenala olulu 24) ma ọ bụ 0.5μl (nri olulu 24) nke nje AAV9 a gwọọ akwara ndị ahụ n'ụbọchị nke abụọ na vitro: AAV9.CMV.PI.eGFP. WPRE.bGH (Adgene, nọmba katalọgụ 105530-AAV9) na AAV9.CMV.HI.eGFP-Cre.WPRE.SV40 (Adgene, nọmba katalọgụ 105545-AAV9).
A na-eji usoro V5 (GKPIPNPLLGLDST) akara òké na Mfn1 na Mfn2 (nke e nwetara site na Addgene plasmid #23212 na #23213, n'otu n'otu) na C-terminus, a na-ejikọtakwa ya na mCherry n'ime etiti site na usoro T2A. Grx1-roGFP2 bụ onyinye sitere na Heidelberg TP Dick DFKZ (Deutsches Krebsforschungszentrum). Site na iji usoro cloning nkịtị dochie kaseti tdTomato, e tinyere kaseti ahụ n'ime azụ pAAV-CAG-FLEX-tdTomato (nọmba ntụaka Addgene 28306) iji mepụta vektọ pAAV-CAG-FLEX-mCherry-T2A-MFN2-V5, pAAV-CAG-FLEX-mCherry-T2A-MFN1-V5 na pAAV-CAG-FLEX-Grx-roGFP2. E jiri usoro yiri nke ahụ mepụta vektọ njikwa pAAV-CAG-FLEX-mCherry. Iji mepụta nhazi AAV-shPCx, achọrọ vektọ plasmid AAV (VectorBuilder, pAAV [shRNA] -CMV-mCherry-U6-mPcx- [shRNA#1]), nke nwere usoro DNA nke na-akọwapụta òké pRNA na-elekwasị anya (5′CTTTCGCTCTAAGGTGCTAAACTCGAGTTTAGCACCTTAGAGCGAAAG 3′) N'okpuru njikwa nke onye na-akwalite U6, a na-eji mCherry n'okpuru njikwa nke onye na-akwalite CMV. Emere mmepụta nke vektọ AAV enyemaka dịka ntuziaka nke onye nrụpụta (Cell Biolabs) si dị. Na nkenke, jiri plasmid mbufe nke nwere mCherry-T2A-MFN2-V5 (pAAV-CAG-FLEX-mCherry-T2A-MFN2-V5), mCherry-T2A-MFN1-V5 (pAAV-CAG-FLEX-mCherry) nke nwere obere oge. Mgbanwe mkpụrụ ndụ 293AAV-T2A-MFN1-V5, mCherry (pAAV-CAG-FLEX-mCherry) ma ọ bụ Grx-roGFP2 (pAAV-CAG-FLEX-Grx-roGFP2), yana idetuo protein capsid na protein mgbakwunye. Nkwakọ ngwaahịa plasmid plasmid, site na iji usoro calcium phosphate. E nwetara ihe na-esi na nje virus a na-akpọ crude-thaw site na usoro friza-thaw na bath ice/ethanol kpọrọ nkụ na mkpụrụ ndụ lysed na phosphate buffered saline (PBS). E jiri ihe nzacha ultracentrifugation nke iodixanol na-adịghị agbanwe agbanwe (awa 24 na 32,000 rpm na 4°C) sachaa vektọ AAV ma tinye ya na nzacha centrifugal Amicon ultra-15. Titer genome nke 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 dịka akọwara na mbụ (54), a tụrụ ya site na real-time quantitative PCR (qPCR) -MFN1-V5 (1.9×1013 GC/ml) na AAV1-CAG-FLEX-Grx-roGFP2 (8.9×1012 GC/ml).
A kpụchapụrụ akwara ndị bụ isi na PBS oyi 1x, kpọchie ha, wee mee ka ha dị ka otu n'ime ihe nchekwa 0.5% Triton X-100 / 0.5% sodium deoxycholate/PBS lysis nke nwere phosphatase na protease inhibitor (Roche). E jiri nyocha bicinchoninic acid (Thermo Fisher Scientific) mee nhazi protein. E jiri electrophoresis gel SDS-polyacrylamide kewaa protein ndị ahụ, wee hichaa ha na polyvinylidene fluoride membrane (GE Healthcare). Gbochie ebe ndị na-abụghị kpọmkwem ma tinye antibody mbụ (lee Tebụl S1 maka nkọwa) na mmiri ara ehi 5% na TBST (Tris-buffered saline with Tween), usoro ịsa ahụ na antibody nke abụọ na TBST Incubate. Tinye antibody mbụ n'abalị na +4°C. Mgbe ịsachara ahụ, tinye antibody nke abụọ maka awa 2 na okpomọkụ ụlọ. Mgbe nke ahụ gasịrị, site na itinye otu blot ahụ na antibody anti-β-actin, a kwadoro otu ibu ahụ. Nchọpụta site n'ịgbanwe na chemiluminescence na ime ka chemiluminescence dịkwuo mma (GE Healthcare).
E tinyere 4% paraformaldehyde (PFA)/PBS n'ime akwara ndị e tinyere na iko mkpuchi n'oge a kapịrị ọnụ na okpomọkụ ụlọ maka nkeji iri. A na-ebu ụzọ tinye 0.1% Triton X-100/PBS n'ime mkpuchi ahụ maka nkeji ise na okpomọkụ ụlọ, wee tinye 3% bovine serum albumin (BSA)/PBS). N'ụbọchị nke abụọ, a sachara mkpuchi ahụ na ihe mgbochi ma tinye ya na ihe mgbochi nke abụọ fluorophore-conjugated kwesịrị ekwesị maka awa abụọ na okpomọkụ ụlọ; n'ikpeazụ, a sachara ihe nlele ahụ nke ọma na PBS na 4′,6-diamidino-2 -Phenylindole (DAPI) a na-egbochi ya ma tinyezie ya na microscope slide na Aqua-Poly/Mount.
A gbara òké (nwoke na nwanyị) ọgwụ site n'ịgba ọgwụ ketamine (130 mg/kg) na xylazine (10 mg/kg) n'ime afọ ime, ma nye ha ọgwụ analgesic carprofen (5 mg/kg) n'okpuru ahụ. E tinyekwa ha n'ime ngwa stereotactic (Kopf) nke nwere ihe mkpuchi ọkụ. Kpuchie okpokoro isi ahụ ma jiri ihe eji eme ezé mee ka akụkụ nke cerebellar cortex nke kwekọrọ na ọkpụkpụ mis (site na lambda: ọdụ 1.8, n'akụkụ 1, nke kwekọrọ na lobules IV na V). Jiri agịga sirinji gbagọrọ agbagọ mepụta obere oghere n'isi isi iji zere imebi vasculature dị n'okpuru. Mgbe ahụ, a na-etinye obere capillary iko a dọtara n'ime obere oghere (site na -1.3 ruo -1 n'akụkụ ventral nke dura mater), a na-etinyekwa 200 ruo 300 nl AAV n'ime obere injector (Narishige) na sirinji aka (Narishige) ọtụtụ ugboro na obere nrụgide n'ime oge nkeji iri ruo iri abụọ. Mgbe a mịnyechara ya, tinye mkpuchi ahụ ruo nkeji iri ọzọ ka nje ahụ wee gbasaa kpamkpam. Mgbe a mịpụrụ mkpuchi ahụ, a na-akpacha akpụkpọ ahụ nke ọma iji belata mbufụt ọnya ahụ ma kwe ka anụmanụ ahụ gbakee. E jiri ọgwụ mgbu (caspofen) gwọọ anụmanụ ndị ahụ ruo ọtụtụ ụbọchị mgbe a wachara ha ahụ, n'oge ahụ a na-enyocha ọnọdụ ahụ ha nke ọma wee gbue ha n'oge a kapịrị ọnụ. Emere usoro niile dịka ntuziaka Europe, mba na ụlọ ọrụ si dị, LandesamtfürNatur nke Umwelt na Verbraucherschutz, North Rhine-Westphalia, Germany kwadoro ya.
E jiri ketamine (100 mg/kg) na xylazine (10 mg/kg) mee ka anụmanụ ndị ahụ nwee nkụchi, e wee jiri 0.1 M PBS tee obi ha mmanụ, wee jiri 4% PFA na PBS tee ha mmanụ. E jiri mma na-ama jijiji (Leica Microsystems GmbH, Vienna, Austria) kwadebe akụkụ sagittal (50 μm dị arọ) site na ụbụrụ edobere na PBS. Ọ gwụla ma akọwapụtara ya, e mere ntụpọ nke akụkụ ndị na-ese n'elu dịka akọwara n'elu (13) na okpomọkụ ụlọ ma na-akpali ha. Na nkenke, nke mbụ, e tinyere 0.5% Triton X-100/PBS n'ime iberibe ndị e nwetara ruo nkeji iri na ise na okpomọkụ ụlọ; maka ụfọdụ epitopes (Pcx na Shmt2), site na tris-EDTA buffer na 80°C (PH9) kpoo iberibe ndị ahụ ọkụ maka nkeji iri abụọ na ise kama nzọụkwụ a. Mgbe nke ahụ gasịrị, e tinyere ọgwụ mgbochi mbụ (lee Tebụl S1) n'ime ihe nchekwa mgbochi (3% BSA/PBS) na 4°C n'abalị na-akpali ya. N'echi ya, e ji ihe nchekwa mgbochi saa akụkụ ndị ahụ ma jiri ọgwụ mgbochi nke abụọ fluorophore-conjugated kwesịrị ekwesị tinye ha n'ime mmiri ruo awa abụọ n'ọnọdụ okpomọkụ ụlọ; n'ikpeazụ, e ji PBS sachaa akụkụ ndị ahụ nke ọma, tinye DAPI n'ime ha, wee jiri ihe ngosi AquaPolymount On microscope dozie ha.
E jiri igwe onyonyo laser confocal scanning (TCS SP8-X ma ọ bụ TCS Digital Light Sheet, Leica Microsystems) nke nwere laser ọkụ ọcha na laser ultraviolet diode 405 iji see ihe nlele ahụ. Site n'ịkpalite fluorophore ahụ ma nakọta mgbaàmà ahụ na Hybrid Detector (HyDs), ejiri ngwanrọ LAS-X chịkọta onyonyo ndị kwekọrọ na Nyquist sample na usoro usoro: maka panel ndị na-abụghị ọnụọgụgụ, ọ bụ akara ngosi dị elu (dịka ọmụmaatụ, na mkpụrụ ndụ somatic na dendrites) mtYFP) Jiri HyD chọpụta ọnụọgụ PN na ọnọdụ BrightR). A na-etinye ọnụ ụzọ site na 0.3 ruo 6 ns iji belata ndabere.
Foto nke mkpụrụ ndụ ndị a haziri n'oge. Mgbe e mechara nhazi ha na Neurobasal-A medium nke nwere 1% B27 mgbakwunye na 0.5 mM GlutaMax, a kụrụ mkpụrụ ndụ ozugbo na ihe ngosi iko poly-l-lysine (μ-Slide8 Well, Ibidi, nọmba katalọgụ 80826), wee debe ya na 37°C na 5% CO2 ruo otu awa iji kwe ka mkpụrụ ndụ ndị ahụ daa. E mere onyonyo oge ahụ na igwe nyocha laser Leica SP8 confocal nke nwere laser ọcha, HyD, 63×[1.4 numerical aperture (NA)] na ogbo ọkụ.
E jiri carbon dioxide mee ka òké ahụ nwee nkụchi obi ngwa ngwa ma bepụ isi ya, e wepụrụ ụbụrụ ya ngwa ngwa n'okpo isi ya, wee bepụ ya n'ime ọkpụrụkpụ 200μm (maka nnwale labeling 13C) ma ọ bụ ọkpụrụkpụ 275μm (maka nnwale photon abụọ) nke e ji ihe ndị a mee. Aịs krim (HM-650 V, Thermo Fisher Scientific, Walldorf, Germany) jupụtara na ihe ndị a: 125 mM ice-cold, carbon-saturated (95% O2 na 5% CO2) obere Ca2 + cerebrospinal fluid (ACSF) NaCl, 2.5 mM KCl, 1.25 mM sodium phosphate buffer, 25 mM NaHCO3, 25 mM glucose, 0.5 mM CaCl2 na 3.5 mM MgCl2 (nrụgide osmotic nke 310 ruo 330 mmol). Bufee iberibe ụbụrụ e nwetara na ime ụlọ tupu incubation nke nwere Ca2 + ACSF dị elu (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 2.0 mM MgCl2) Medium) pH 7.4 na 310 ruo 320 mmol).
N'oge usoro onyonyo ahụ, e bufere iberibe ndị ahụ n'ime ụlọ onyonyo raara onwe ya nye, e mekwara nnwale ahụ n'okpuru mgbasa ACSF na-aga n'ihu na okpomọkụ na-adịgide adịgide nke 32° ruo 33°C. Ejiri igwe onyonyo nyocha laser multiphoton (TCS SP8 MP-OPO, Leica Microsystems) nke nwere lensi Leica 25x (NA 0.95, mmiri), Ti: Sapphire laser (Chameleon Vision II, Coherent) mee ihe maka onyonyo slice. Modulu FLIM (PicoHarp300, PicoQuant).
FLIM nke Grx1-roGFP2. A tụrụ mgbanwe ndị dị na ọnọdụ redox cytoplasmic nke PNs site na iji FLIM abụọ na iberibe ụbụrụ sagittal, ebe ihe mmetụta Grx1-roGFP2 lekwasịrị anya na PNs. Na oyi akwa PN, a na-ahọrọ ubi nnweta ihe dị ka 50 ruo 80 μm n'okpuru elu iberibe iji hụ na enwere PN dị irè (ya bụ, enweghị nhazi beaded ma ọ bụ mgbanwe morphological neuronal n'akụkụ dendrites) na ihe mmetụta roGFP2 abụọ dị mma na AAV na-agbanwe shRNA PCx ma ọ bụ usoro njikwa ya (mCherry ọ bụla na-egosipụta otu). Chịkọta onyonyo otu-stack nwere mbugharị dijitalụ 2x [ogologo oge mkpali: 890 nm; 512 nm 512 pikselụ]. Nchọpụta: HyD dị n'ime, otu nzacha fluorescein isothiocyanate (FITC)] na nha onyonyo n'ime nkeji 2 ruo 3 ka ejiri iji hụ na anakọtara photons zuru oke (fotons 1000 na mkpokọta) maka nhazi usoro. E mere mmetụta nke nyocha Grx1-roGFP2 na nkwenye nke ọnọdụ FLIM site na ileba anya na uru ndụ nke roGFP2 mgbe etinyere 10 mM H2O2 nke mpụga na ACSF perfusion (iji mee ka oxidation dịkwuo elu, na-ebute ndụ ka mma), wee tinye 2 mM dithiothreitol (na-ebelata ogo mbelata, na-ebute mbelata na ndụ) (Foto S8, D ruo G). Jiri ngwanrọ FLIMfit 5.1.1 nyochaa nsonaazụ enwetara, tinye otu usoro ire ere exponential nke onyonyo dum na IRF a tụrụ (ọrụ nzaghachi ngwa), na χ2 bụ ihe dị ka 1. Iji gbakọọ ndụ nke otu PN, e sere ihe mkpuchi ahụ gbara akwara gburugburu aka, e jirikwa aka see ndụ nkezi na ihe mkpuchi ọ bụla maka nha.
Nnyocha ike Mitochondrial. Mgbe etinyere akụkụ acute ahụ na 100 nM TMRM ozugbo na ACSF perfused ruo nkeji iri atọ, e ji igwe microscope abụọ-photon tụọ mgbanwe ike mitochondrial nke PNs. E mere onyonyo TMRM site n'ịkụnye ihe nyocha ahụ na 920 nm ma jiri HyD dị n'ime (tetramethylrhodamine isothiocyanate: 585/40 nm) iji chịkọta akara; site na iji otu ogologo oge mkpali mana iji HyD dị n'ime dị iche (FITC :525/50) iji gosipụta mtYFP. Jiri ihe mgbakwunye Image Calculator Image Plug-in nke ImageJ iji nyochaa ike mitochondrial na ọkwa otu sel. Na nkenke, a na-eji nha nhata plug-in: signal = min (mtYFP, TMRM) iji chọpụta mpaghara mitochondrial nke na-egosi akara TMRM na Purkinje Somali na onyonyo confocal otu-stack nke ọwa kwekọrọ. Mgbe ahụ, a na-agbakọ mpaghara pikselụ dị na ihe mkpuchi ahụ, wee mee ka ọ dị mma na onyonyo otu-stack kwekọrọ na ọwa mtYFP iji nweta akụkụ mitochondrial nke na-egosi ike mitochondrial.
E jiri ngwanrọ Huygens Pro (Scientific Volume Imaging) wepụ onyonyo ahụ. Maka foto ndị a na-enyocha nke taịlị, a na-eji algọridim ịkwa akwa akpaka nke ngwanrọ LAS-X nyere mee ntinye nke otu taịlị. Mgbe emechara nhazi onyonyo, jiri ImageJ na Adobe Photoshop hazie onyonyo ahụ nke ọma ma gbanwee nchapụta na ọdịiche ahụ n'otu aka ahụ. Jiri Adobe Illustrator mee nkwadebe eserese.
Nyocha mtDNA lekwasịrị anya. A tụrụ ọnụọgụ mmerụ ahụ mtDNA na ngalaba cerebellar nke ejiri ọgwụ mgbochi megide DNA kpọọ site na iji microscope confocal. E kere mpaghara ebumnuche ọ bụla maka ahụ sel na nucleus nke sel ọ bụla, a gbakọọkwa mpaghara nke ọ bụla site na iji Multi Measure plug-in (ngwanrọ ImageJ). Wepụ mpaghara nuklia site na mpaghara sel ahụ iji nweta mpaghara cytoplasmic. N'ikpeazụ, ejiri Analyse Particles plugins (ngwanrọ ImageJ) tụọ isi ihe DNA cytoplasmic na-egosi mtDNA na onyonyo njedebe, a na-emekwa ka nsonaazụ enwetara bụrụ nkezi PN nke òké CTRL. A na-egosipụta nsonaazụ ahụ dị ka ọnụọgụ nkezi nke nucleosides kwa sel.
Nyocha ngosipụta protein. Jiri ihe mgbakwunye Image Calculator nke ImageJ iji nyochaa ngosipụta protein na PN na ọkwa otu sel. Na nkenke, na onyonyo confocal nke otu oyi akwa nke ọwa kwekọrọ, site na nha nhata: signal = min (mtYFP, antibody), a na-achọpụta mpaghara mitochondrial nke na-egosi immunoreactivity na antibody ụfọdụ na Purkina. Mgbe ahụ, a na-agbakọ mpaghara pixel dị na ihe mkpuchi ahụ, wee mee ka ọ dị mma na onyonyo otu stack kwekọrọ na ọwa mtYFP iji nweta akụkụ mitochondrial nke protein egosiri.
Nnyocha njupụta mkpụrụ ndụ Purkinje. E jiri ihe mgbakwunye Cell Counter nke ImageJ nyochaa njupụta Purkinje site n'ikewa ọnụọgụ nke mkpụrụ ndụ Purkinje a gụrụ site na ogologo mgbanaka cerebellar nke mkpụrụ ndụ a gụrụ bi.
Nkwadebe na nchịkọta ihe nlele. E tinyere ụbụrụ sitere na otu njikwa na òké Mfn2cKO na 2% PFA/2.5% glutaraldehyde na 0.1 M phosphate buffer (PB), wee kwadebe akụkụ coronal site na iji ciliates (Leica Mikrosysteme GmbH, Vienna, Austria) (Ọkpụrụkpụ 50 ruo 60 μm) wee tinye na PB buffer na 1% os tetraoxide na 1.5% potassium ferrocyanide na okpomọkụ ụlọ maka otu awa. E jiri mmiri a gwakọtara agwakọta saa akụkụ ndị ahụ ugboro atọ, wee tee ha na 70% ethanol nwere 1% uranyl acetate maka nkeji iri abụọ. E mesịa, e tinyere akụkụ ndị ahụ na mmanya a kpara akpa ma tinye ha na Durcupan ACM (Araldite casting resin M) epoxy resin (Electron Microscopy Sciences, nọmba katalọgụ 14040) n'etiti slide iko a kpuchiri silicon, wee mechaa na 60°C Polymerize na oven maka awa iri anọ na asatọ. A họọrọ mpaghara cerebellar cortex wee bepụ akụkụ 50 nm ultrathin na Leica Ultracut (Leica Mikrosysteme GmbH, Vienna, Austria) ma họpụta ha na grid ọla kọpa 2 × 1 mm nke e ji ihe nkiri polystyrene kpuchie. E ji mmiri uranyl acetate 4% tee akụkụ ndị ahụ na H2O ruo nkeji iri, saa ha ọtụtụ ugboro, wee jiri Reynolds lead citrate na H2O ruo nkeji iri, wee jiri H2O saa ha ọtụtụ ugboro. E jiri igwe microscope elektrọn transmission Philips CM100 (Thermo Fisher Scientific, Waltham, MA, USA) were obere micrographs site na iji igwefoto dijitalụ TVIPS (Tietz Video and Image Processing System) TemCam-F416 (TVIPS GmbH, Gauting, USA). Germany.
Maka òké ndị nwere AAV, e kewara ụbụrụ ha ma bee ha n'ime akụkụ sagittal dị 1 mm n'obosara, e wee jiri igwe nyocha fluorescence lee cerebellum ahụ iji chọpụta mgbanaka nwere AAV (ya bụ, mCherry na-egosipụta). Naanị nnwale ndị a na-eji ntụtụ AAV eme ka mkpụrụ ndụ Purkinje dị elu (ya bụ, ihe fọrọ nke nta ka ọ bụrụ oyi akwa ahụ dum) n'ime opekempe mgbanaka cerebellar abụọ na-esochi onwe ha ka a na-eji. E jiri obere akụkụ AAV-transduced loop mee ihe maka n'abalị mgbe a rụchara ya (4% PFA na 2.5% glutaraldehyde na 0.1 M cocoate buffer) ma hazie ya ọzọ. Maka itinye EPON, e ji 0.1 M sodium cocoate buffer (Applichem) saa anụ ahụ e doziri edozi, ma tinye 2% OsO4 (os, Science Services; Caco) na 0.1 M sodium cocoate buffer (Applichem) awa 4, wee saa ya awa 2. Megharịa ya ugboro atọ na 0.1 M cocamide buffer. Mgbe nke ahụ gasịrị, e jiri usoro ethanol na-arị elu mee ka mmiri ethanol ọ bụla dị na 4°C ruo nkeji iri na ise iji mee ka mmiri ghara ịdị n'ime anụ ahụ. E zigara anụ ahụ na propylene oxide ma tinye ya n'ime EPON (Sigma-Aldrich) n'abalị na 4°C. Debe anụ ahụ na EPON ọhụrụ na okpomọkụ ụlọ ruo awa abụọ, wee tinye ya na 62°C ruo awa 72. Jiri ultramicrotome (Leica Microsystems, UC6) na mma diamond (Diatome, Biel, Switzerland) bee akụkụ 70 nm ultrathin, ma tee ya na 1.5% uranyl acetate maka nkeji iri na ise na 37°C, ma tee ya na mmiri lead citrate maka nkeji anọ. E jiri igwe microscope electron transmission JEM-2100 Plus (JEOL) nke nwere Camera OneView 4K 16-bit (Gatan) na DigitalMicrograph software (Gatan) were micrograph electron ndị ahụ. Maka nyocha, e ji 5000 × ma ọ bụ 10,000 × zoom dijitalụ zụta micrograph elektrọn.
Nnyocha morphological nke mitochondria. Maka nyocha niile, ejiri aka depụta nhazi nke mitochondria nke ọ bụla na onyonyo dijitalụ site na iji ngwanrọ ImageJ. A na-enyocha paramita morphological dị iche iche. A na-egosipụta njupụta mitochondrial dị ka pasentị enwetara site na ikewa mpaghara mitochondrial zuru oke nke sel ọ bụla site na mpaghara cytoplasm (mpaghara cytoplasm = mpaghara sel-mpaghara nucleus cell) × 100. A na-agbakọ gburugburu mitochondria na usoro [4π∙(mpaghara/akụkụ 2)]. A nyochachara ma kewaa morphology nke mitochondria n'ime ụdị abụọ ("tubular" na "blister") dịka ọdịdị ha si dị.
Nyocha ọnụọgụgụ na njupụta nke Autophagosome/lysosome. Jiri ngwanrọ ImageJ mee ka aka gị depụta nhazi nke autophagosome/lysosome ọ bụla na onyonyo dijitalụ. A na-egosipụta mpaghara Autophagosome/lysosome dị ka pasentị agbakọrọ site na ikewa mpaghara nhazi autophagosome/lysosome nke sel ọ bụla site na mpaghara cytoplasm (mpaghara cytoplasm=mpaghara sel-mpaghara nucleus) ×100. A na-agbakọ njupụta nke autophagosomes/lysosomes site na ikewa ọnụọgụgụ mkpokọta site na ọnụọgụgụ nke nhazi autophagosome/lysosome kwa sel (n'ihe gbasara mpaghara cytoplasmic) (mpaghara cytoplasmic = mpaghara sel-mpaghara nuclear).
Ndekọ maka nhazi nkewa dị oke njọ na nkwadebe ihe nlele. Maka nnwale ndị chọrọ akara glucose, bufee iberibe ụbụrụ dị oke njọ gaa na ụlọ tupu itinye ihe, nke nwere carbon juru (95% O2 na 5% CO2), Ca2 + ACSF dị elu (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 2.0 mM MgCl2, agbanwere na pH 7.4 na 310 ruo 320 mOsm), ebe glucose bụ 13 C 6- mgbanwe glucose (Eurisotop, nọmba katalọgụ CLM-1396). Maka nnwale ndị chọrọ akara pyruvate, bufee iberibe ụbụrụ dị nkọ gaa na Ca2 + ACSF dị elu (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 ma tinye 2.0mM MgCl2, gbanwee ya na pH 7.4 na 310 ruo 320mOsm), ma tinye 1mM 1-[1-13C]pyruvate (Eurisotop, nọmba katalọgụ CLM-1082). Kpoo akụkụ ndị ahụ maka nkeji 90 na 37°C. Na ngwụcha nnwale ahụ, ejiri mmiri mmiri (pH 7.4) nke nwere 75 mM ammonium carbonate saa akụkụ ndị ahụ ngwa ngwa, wee mee ka ha dị ka otu na 40:40:20 (v:v:v) acetonitrile (ACN): methanol: mmiri. Mgbe e tinyere akụkụ ndị ahụ n'ime ice ruo nkeji iri atọ, a na-etinye ihe nlele ahụ na centrifuge na 21,000 g maka nkeji iri na okpomọkụ 4°C, a na-akpọkwa ihe na-enwu gbaa n'ime ihe nchekwa SpeedVac. E debere pellet metabolite kpọrọ nkụ nke sitere na ya na -80°C ruo mgbe a ga-enyocha ya.
Nnyocha spectrometry nke mmiri mmiri chromatography-mass nke amino acid 13 ejiri C-labeled mee. Maka nyocha chromatography-mass spectrometry (LC-MS), a kpọghachiri pellet metabolite na 75μl nke mmiri LC-MS (Honeywell). Mgbe centrifugation gasịrị na 21,000 g maka nkeji ise na 4°C, ejiri 20 μl nke supernatant a kọwara maka nyocha amino acid flux, ebe ejiri ihe ndị ọzọ fọdụrụ mee nyocha anion ozugbo (lee n'okpuru). Ejiri usoro derivatization benzoyl chloride akọwapụtara na mbụ mee nyocha amino acid (55, 56). Na nzọụkwụ mbụ, etinyere 10μl nke 100 mM sodium carbonate (Sigma-Aldrich) na 20μl nke metabolite extract, wee tinye 10μl nke 2% benzoyl chloride (Sigma-Aldrich) na ọkwa LC ACN. A gbanyere ihe nlele ahụ obere oge wee tinye ya na centrifuge na 21,000 g maka nkeji ise na 20°C. Bufee ihe mkpuchi ahụ a kpochapụrụ na karama autosampler 2 ml nwere ihe ntinye iko conical (olu 200 μl). Ejiri sistemụ Acquity iClass ultra-high performance LC (Waters) jikọtara na Q-Exactive (QE)-HF (Ultra High Field Orbitrap) high-resolution precision mass spectrometer (Thermo Fisher Scientific) nyochaa ihe nlele ndị a. Maka nyocha, a gbanyere 2μl nke ihe nlele ahụ n'ime kọlụm silica T3 dị elu nke 100 × 1.0 mm (Waters) nke nwere ihe ndị dị na 1.8μm. Ọnụego mmiri ahụ bụ 100μl/min, sistemụ buffer ahụ nwekwara buffer A (10 mM ammonium formate na 0.15% formic acid na mmiri) na buffer B (ACN). Gradient bụ dịka ndị a: 0%B na nkeji 0; 0%B. 0 ruo 15% B na nkeji 0 ruo 0.1; 15 ruo 17% B na nkeji 0.1 ruo 0.5; B na nkeji 17 ruo 55% na nkeji 0.5 ruo 14; B na nkeji 55 ruo 70% na nkeji 14 ruo 14.5; na nkeji 14.5 ruo 70 ruo 100% B na nkeji 18; 100% B na nkeji 18 ruo 19; 100 ruo 0% B na nkeji 19 ruo 19.1; 0% B na nkeji 19.1 ruo 28 (55, 56). Igwe nlele QE-HF na-arụ ọrụ na ọnọdụ ionization dị mma yana oke oke m/z (oke oke/oke chajị) nke 50 ruo 750. Mkpebi etinyere bụ 60,000, ebe ebumnuche njikwa uru (AGC) enwetara bụ 3×106, oge ion kachasị bụ 100 milliseconds. Isi iyi ionization electrospray (ESI) na-ekpo ọkụ na-arụ ọrụ na voltaji ịgba nke 3.5 kV, okpomọkụ capillary nke 250°C, ikuku sheath nke 60 AU (nkeji na-enweghị usoro), na ikuku enyemaka nke 20 AU. 250°C. A na-edozi anya S ka ọ bụrụ 60 AU.
Nyocha chromatography-MS nke asịd organic nke 13C akpọrọ aha ya. E jiri sistemụ chromatography Dionex ion (ICS 5000+, Thermo Fisher Scientific) nke ejikọtara na QE-HF mass spectrometer (Thermo Fisher Scientific) nyochaa ihe fọdụrụ na metabolite precipitate (55μl) na kọlụm Dionex IonPac AS11-HC nke nwere HPLC (2 mm × 250 mm, nha ihe 4μm, Thermo Fisher Scientific) na ọnọdụ mkpọchi akụkụ nke push-in yana oke njupụta nke 1. ) Kọlụm nche Dionex IonPac AG11-HC (2 mm x 50 mm, 4μm, Thermo Fisher Scientific). A na-edobe okpomọkụ kọlụm ahụ na 30°C, a na-edobekwa autosampler ahụ na 6°C. Jiri katrij potassium hydroxide nke enyere na mmiri deionized iji mepụta gradient potassium hydroxide site na jenerato eluent. Nkewa nke metabolites na ọsọ mmiri nke 380μl/min, na-etinye gradient ndị a: nkeji 0 ruo 3, 10 mM KOH; nkeji 3 ruo 12, nkeji 10 ruo 50 mM KOH; nkeji 12 ruo 19, 50 ruo 100 mM KOH; nkeji 19 ruo 21, 100 mM KOH; nkeji 21 ruo 21.5, nkeji 100 ruo 10 mM KOH. E megharịrị kọlụm ahụ n'okpuru 10 mM KOH maka nkeji 8.5.
A na-ejikọta metabolites ndị a gbapụrụ agbapụ na iyi mgbakwunye isopropanol 150μl/min mgbe kọlụm ahụ gasịrị, wee gaa na spectrometer mass dị elu nke na-arụ ọrụ na ọnọdụ ionization na-adịghị mma. MS na-enyocha oke oke site na m/z 50 ruo 750 yana mkpebi nke 60,000. A na-edozi AGC ka ọ bụrụ 1 × 106, a na-edebekwa oge ion kachasị na 100 ms. A na-arụ ọrụ isi iyi ESI a na-ekpo ọkụ na voltaji ịgba nke 3.5 kV. Ntọala ndị ọzọ nke isi iyi ion bụ ndị a: okpomọkụ capillary 275°C; mmiri gas sheath, 60 AU; mmiri gas enyemaka, 20 AU na 300°C, na ntọala oghere S na 60 AU.
Nyocha data nke metabolites akara 13C. Jiri ngwanrọ TraceFinder (ụdị 4.2, Thermo Fisher Scientific) maka nyocha data nke oke isotope. Ejiri ihe ntụaka a pụrụ ịdabere na ya kwado njirimara nke ihe mejupụtara ọ bụla ma nyochaa ya n'adabereghị onwe ya. Iji mee nyocha utotope, e si na [M + H] + wepụta mpaghara nke ion chromatogram ewepụtara (XIC) nke isotope 13C ọ bụla (Mn), ebe n bụ ọnụọgụ carbon nke ihe mejupụtara ebumnuche, nke e ji nyochaa amino acid ma ọ bụ [MH] + nyochaa anions. Izi ezi nke XIC erughị akụkụ ise kwa nde, izi ezi nke RT bụkwa nkeji 0.05. A na-eme nyocha uto site na ịgbakọ oke nke isotope ọ bụla achọpụtara na mkpokọta isotope niile nke ihe mejupụtara kwekọrọ. A na-enye oke ndị a dị ka uru pasent maka isotope ọ bụla, a na-egosipụtakwa nsonaazụ ya dị ka mmụba pasent molar (MPE), dịka akọwara na mbụ (42).
E tinyere mkpụrụ neuron oyi kpọnwụrụ akpọnwụ n'ime otu ihe na methanol 80% oyi (v/v), gbanye ya, ma tinye ya na -20°C maka nkeji iri atọ. Tinyegharịa ihe nlele ahụ ọzọ ma gwakọta ya na +4°C maka nkeji iri atọ. E tinyere centrifuge na 21,000 g maka nkeji ise na 4°C, wee jiri SpeedVac concentrator chịkọta ihe na-esi na ya pụta ma kpọọ nkụ site na iji ihe nchekwa SpeedVac na 25°C maka nyocha ọzọ. Dịka akọwara n'elu, emere nyocha LC-MS na amino acid nke mkpụrụ ndụ ndị a haziri ahazi. Site na iji TraceFinder (ụdị 4.2, Thermo Fisher Scientific), emere nyocha data site na iji monoisotopic mass nke ihe ọ bụla mejupụtara. Ejiri ngwugwu ngwanrọ preprocessCore (57) mee nhazi data metabolite.
Nkwadebe nke a gbubiri agbubi. E jiri carbon dioxide mee ka òké ahụ nwee ọgwụ mgbu ngwa ngwa ma bepụ isi ya, e wepụrụ ụbụrụ ya ngwa ngwa n'okpo isi ya, e jiri mma na-ama jijiji nke jupụtara na ice (HM-650 V, Thermo Fisher Scientific, Walldorf, Germany) bee ya n'ime akụkụ sagittal 300 ruo 375 μm nke gas carbon oyi (95% O2 na 5% CO2) Ca2 + ACSF dị ala (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 6.0 mM MgCl2 Gbanwee na pH 7.4 na 310 ruo 330 mOsm). Bufee iberibe ụbụrụ e nwetara n'ime ụlọ nwere Ca2 + ACSF dị elu (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 na mM 3.5 MgCl2) pH 7.4 na 310 ruo 320 mOsm). Debe iberibe ahụ maka nkeji 20 ruo 30 ka e wee weghachite ha tupu e dekọọ ha.
Ndekọ. E jiri igwe microscope nke nwere ụlọ ndekọ edobere na oghere anya mmiri nke 20x (Scientifica) maka ndekọ niile. A chọpụtara mkpụrụ ndụ Purkinje ndị a site na (i) nha ahụ, (ii) ebe anatomical nke cerebellum dị, na (iii) ngosipụta nke mkpụrụ ndụ ihe nketa mtYFP fluorescent reporter. A na-eji okpu iko borosilicate (GB150-10, 0.86 mm×1.5 mm×100 mm, Science Products, Hofheim, Germany) na ngwa pipette kwụ ọtọ (P-1000, Sutter), Novato, CA dọpụta patch pipette nwere nguzogide isi nke 5 ruo 11 megohms. E mere ndekọ niile site na ELC-03XS npi patch clamp amplifier (npi electronic GmbH, Tam, Germany), nke ngwanrọ Signal (ụdị 6.0, Cambridge Electronic, Cambridge, UK) na-achịkwa. E dekọrọ nnwale ahụ na ọnụego nlele nke 12.5 kHz. A na-eji ihe nzacha Bessel abụọ dị mkpụmkpụ nke na-agafe agafe, nke nwere ugboro 1.3 na 10 kHz n'otu n'otu. A na-eji ihe na-eme ka ike nke akpụkpọ ahụ na pipette kwụghachi ike ya. E mere nnwale niile n'okpuru njikwa nke igwefoto Orca-Flash 4.0 (Hamamatsu, Gerden, Germany), nke ngwanrọ Hokawo (ụdị 2.8, Hamamatsu, Gerden, Germany) na-achịkwa.
Nhazi na nyocha nke mkpụrụ ndụ dum. Ozugbo e dekọrọ ya, jupụta paịpụ ahụ na mmiri dị n'ime nke nwere ihe ndị a: 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) na 10.0 mM creatinine phosphate ka agbanwere na pH 7.25, nrụgide osmotic bụ 290 mOsm (sucrose). Ozugbo e tinyere ike nke 0 pA iji gbawaa akpụkpọ ahụ, a tụrụ ikike akpụkpọ ahụ fọdụrụ. A na-atụ nguzogide ntinye site na itinye oke ikuku nke -40, -30, -20, na -10 pA. Tụọ oke nzaghachi voltaji ma jiri iwu Ohm gbakọọ nguzogide ntinye. E dekọrọ ọrụ mberede na ihe mkpuchi voltaji ruo nkeji ise, achọpụtara ma tụọ sPSC na Igor Pro (mbipụta 32 7.01, WaveMetrics, Lake Oswego, Oregon, USA) site na iji edemede njirimara ọkara-akpaka. A na-atụ usoro IV na ugbu a kwụsiri ike site na ijide batrị na ikike dị iche iche (malite na -110 mV) ma na-amụba voltaji na usoro 5 mV. A nwalere mmepụta nke AP site na itinye ugbu a depolarizing. Kpuchie sel ahụ na -70 mV mgbe ị na-etinye pulse ugbu a depolarizing. Gbanwee nha nzọụkwụ nke nkeji ndekọ ọ bụla iche iche (10 ruo 60 pA). Gbakọọ ugboro AP kachasị site na iji aka gụọ pulse spikes nke na-akpata ugboro AP kachasị elu. A na-enyocha oke AP site na iji ihe nke abụọ nke depolarization pulse nke na-akpali otu ma ọ bụ karịa APs.
Nhazi na nyocha nke patch gbara okirikiri. Mee ndekọ patch gbara okirikiri site na iji usoro ọkọlọtọ. Jiri pipette ATP na GTP na-enweghị nke na-enweghị ihe ndị a: 128 mM gluconate K, 10 mM KCl, 10 mM Hepes, 0.1 mM EGTA na 2 mM MgCl2, ma gbanwee ya na pH 7.2 (site na iji KOH). A na-ewepụ ATP na GTP na ngwọta intracellular iji gbochie ikike nke membrane cell. A na-ejupụta patch pipette ahụ na ngwọta dị n'ime nke nwere amphotericin (ihe dị ka 200 ruo 250μg/ml; G4888, Sigma-Aldrich) iji nweta ndekọ patch gbajiri agbaji. A gbazere Amphotericin na dimethyl sulfoxide (njupụta ikpeazụ: 0.1 ruo 0.3%; DMSO; D8418, Sigma-Aldrich). Ntinye nke DMSO ejiri mee enweghị mmetụta dị ukwuu na neurons ndị a mụrụ. N'oge usoro ịkụ ọkpọ, a na-enyocha nguzogide ọwa (Ra) mgbe niile, a malitekwara nnwale ahụ mgbe oke Ra na AP kwụsiri ike (nkeji 20-40). A na-atụ ọrụ mberede na voltaji na/ma ọ bụ mkpọchi ugbu a maka nkeji 2 ruo 5. E jiri Igor Pro (nsụgharị 7.05.2, WaveMetrics, USA), Excel (nsụgharị 2010, Microsoft Corporation, Redmond, USA) na GraphPad Prism (nsụgharị 8.1.2, GraphPad Software Inc., La Jolla, CA) mee nyocha data. Iji chọpụta AP ndị na-apụtaghị ìhè, a na-eji nkwụnye NeuroMatic v3.0c nke IgorPro. Chọpụta AP na akpaghị aka site na iji oke enyere, nke a na-agbanwe iche maka ndekọ ọ bụla. Site na iji oge mgbawa, chọpụta ugboro mgbawa na ugboro mmụba kachasị elu na ugboro mmụba nkezi.
Nnọpụiche PN. Site na imeghari usoro e bipụtara na mbụ, a sachara PNs site na cerebellum òké n'oge akọwapụtara (58). Na nkenke, a gbubiri cerebellum ma gwerie ya na ihe na-eme ka oyi na-atụ [na-enweghị HBSS Ca2+ na Mg2+, tinye 20 mM glucose, penicillin (50 U/ml) na streptomycin (0.05 mg/ml)], wee gbarie ihe na papain [HBSS, nke ejiri 1-cysteine·HCl (1 mg/ml), papain (16 U/ml) na deoxyribonuclease I (DNase I; 0.1 mg/ml)] gwọọ ya maka nkeji iri atọ na 30°C. Buru ụzọ saa anụ ahụ n'ime ihe mkpuchi HBSS nke nwere imi àkwá (10 mg/ml), BSA (10 mg/ml) na DNase (0.1 mg/ml) n'ọnọdụ okpomọkụ ụlọ iji gbochie mgbaze enzymatic, wee jiri ihe mkpuchi HBSS nke nwere glucose 20 mM gwerie ya nwayọ nwayọ na HBSS, penicillin (50 U/ml), streptomycin (0.05 mg/ml) na DNase (0.1 mg/ml) wepụta otu mkpụrụ ndụ. A na-enyocha ihe mkpuchi mkpụrụ ndụ nke sitere na ya site na ihe nzacha mkpụrụ ndụ 70μm, wee jiri centrifugation (1110 rpm, nkeji 5, 4°C) kpụọ mkpụrụ ndụ ahụ ma tinyeghachi ha na nhazi [HBSS, nke ejiri 20 mM glucose, 20% fetal bovine ) Serum, penicillin (50 U/ml) na streptomycin (0.05 mg/ml)]; nyochaa ndụ mkpụrụ ndụ site na iji propidium iodide ma gbanwee njupụta mkpụrụ ndụ ahụ ka ọ bụrụ 1 × 106 ruo 2 × 106 mkpụrụ ndụ/ml. Tupu usoro cytometry flow, a na-enyocha nkwụsịtụ ahụ site na ihe nzacha mkpụrụ ndụ 50 μm.
Sistemụ mmiri. E mere nhazi mkpụrụ ndụ na 4°C site na iji igwe FACSAria III (BD Biosciences) na ngwanrọ FACSDiva (BD Biosciences, ụdị 8.0.1). E ji ihe dị ka 100 μm chịkọta nkwụsịtụ mkpụrụ ndụ ahụ n'okpuru nrụgide nke 20 psi na ọnụego nke ihe omume ~2800/sekọnd. Ebe ọ bụ na njirisi nhazi ọdịnala (nha mkpụrụ ndụ, ịkpa ókè bimodal, na njirimara ịgbasa) enweghị ike ijide n'aka na enwere ikewapụ PN na ụdị mkpụrụ ndụ ndị ọzọ, a na-ahazi atụmatụ nhazi ahụ dabere na ntụnyere kpọmkwem nke ike YFP na autofluorescence na mitoYFP+ ​​​​na njikwa mitoYFP − Mice. YFP na-enwe obi ụtọ site na iji ahịrị laser 488 nm kpughee ihe nlele ahụ, a na-achọpụtakwa mgbaàmà ahụ site na iji nzacha ngafe band 530/30 nm. Na mitoYFP+ ​​​​òké, a na-ejikwa ike dị n'etiti mkpụrụ ndụ ihe nketa Rosa26-mitoYFP iji mata akụkụ ahụ neuronal na iberibe axon. A na-eji laser odo 561 nm 7-AAD eme ihe ma jiri nzacha bandpass 675/20 nm chọpụta ya iji wepụ mkpụrụ ndụ nwụrụ anwụ. Iji kewaa astrocytes n'otu oge ahụ, a na-eji ACSA-2-APC tee ihe mgbochi sel ahụ, wee jiri eriri laser 640 nm kpughee ihe nlele ahụ, ma jiri nzacha bandpass 660/20 nm chọpụta ihe mgbaàmà ahụ.
E jiri centrifugation (1110 rpm, nkeji 5, 4°C) kpụọ mkpụrụ ndụ ndị a chịkọtara ma chekwaa ha na -80°C ruo mgbe e jiri ha. A na-ekewa òké Mfn2cKO na ụmụ nkịta ha n'otu ụbọchị iji belata mgbanwe usoro. E jiri ngwanrọ FlowJo (FlowJo LLC, Ashland, Oregon, USA) mee ngosipụta na nyocha data FACS.
Dịka ekwuru n'elu (59), a na-eji PCR oge-oge kewapụ DNA site na akwara ndị a haziri ahazi maka nha mtDNA na-esote. E buru ụzọ nwalee linearity na portal sensitivity site na ịgba qPCR na ọnụọgụ sel dị iche iche. Na nkenke, chịkọta 300 PN na lysis buffer nke nwere 50 mM tris-HCl (pH 8.5), 1 mM EDTA, 0.5% Tween 20 na proteinase K (200 ng/ml) wee tinye ya na 55°C nkeji 120. E tinyere mkpụrụ ndụ ndị ahụ na 95°C ruo nkeji 10 iji hụ na proteinase K anaghị arụ ọrụ nke ọma. Site na iji TaqMan probe (Thermo Fisher) nke kpọmkwem na mt-Nd1, a tụrụ mtDNA site na semi-quantitative PCR na sistemụ 7900HT Real-Time PCR (Thermo Fisher Scientific). Sayensị, nọmba katalọgụ Mm04225274_s1), mt-Nd6 (Thermo Fisher Scientific, nọmba katalọgụ AIVI3E8) na 18S (Thermo Fisher Scientific, nọmba katalọgụ Hs99999901_s1) mkpụrụ ndụ ihe nketa.
Nkwadebe ihe nlele proteome. Site n'ikpo ọkụ na mmiri ọkụ na 95°C maka nkeji iri ma na-eme ka ọ dị ọkụ, na ihe nchekwa lysis [6 M guanidine chloride, 10 mM tris(2-carboxyethyl) phosphine hydrochloride, 10 mM chloroacetamide na 100 mM tris-Lyse frozen neuron pellets na HCl]. Na Bioruptor (Diagenode) maka nkeji iri (sekọnd 30 pulse / sekọnd 30 kwụsịtụ oge). A gwakọtara ihe nlele ahụ 1:10 na 20 mM tris-HCl (pH 8.0), gwakọta ya na 300 ng trypsin gold (Promega), ma tinye ya n'ime ya n'abalị na 37°C iji nweta mgbaze zuru oke. N'ụbọchị nke abụọ, a kpụchara ihe nlele ahụ na 20,000 g maka nkeji iri abụọ. A gwakọtara ihe dị n'elu ya na 0.1% formic acid, a kpụkwara ya na StageTips nke e ji aka mee. A kpọrọ ihe nlele ahụ n'ime ngwa SpeedVac (Eppendorf concentrator plus 5305) na 45°C, wee kwụchie peptide ahụ na 0.1% formic acid. Otu onye ahụ kwadebere ihe nlele niile n'otu oge. Iji nyochaa ihe nlele astrocyte, e jiri akara tandem mass tag (TMT10plex, nọmba katalọgụ 90110, Thermo Fisher Scientific) akara 4 μg desalted peptides yana oke reagent peptide na TMT nke 1:20. Maka akara TMT, e weghachitere 0.8 mg nke TMT reagent na 70 μl nke anhydrous ACN, e mekwara peptide kpọrọ nkụ ka ọ bụrụ 9 μl nke 0.1 M TEAB (triethylammonium bicarbonate), nke etinyere 7 μl nke TMT reagent na ACN. Ntinye ahụ bụ 43.75%. Mgbe nkeji 60 nke incubation gasịrị, e jiri 2 μl nke 5% hydroxylamine mechie mmeghachi omume ahụ. A chịkọtara peptides ndị a kpọrọ aha, kpọọ nkụ, ma tinyeghachi ha ọzọ na 200μl nke 0.1% formic acid (FA), kewaa ha abụọ, wee jiri StageTips nke aka ha mee wepụ nnu. Site na iji UltiMate 3000 ultra high performance liquid chromatograph (UltiMate 3000 ultra high performance liquid chromatograph), otu n'ime ọkara abụọ ahụ kewara na kọlụm chromatographic Acquity 1mm x 150mm nke jupụtara na 130Å1.7μm C18 particles (Mmiri, katalọgụ No. SKU: 186006935). Thermo Fisher Scientific). Kewapụ peptides na ọnụego mmiri nke 30μl/min, kewapụ site na 1% ruo 50% buffer B maka nkeji 85 na gradient nzọụkwụ nke nkeji 96, site na 50% ruo 95% buffer B maka nkeji 3, wee kewaa nkeji 8 maka 95% Buffer B; Buffer A bụ 5% ACN na 10 mM ammonium bicarbonate (ABC), ebe buffer B bụ 80% ACN na 10 mM ABC. Chịkọta akụkụ dị iche iche kwa nkeji atọ wee jikọta ha n'ime otu abụọ (1 + 17, 2 + 18, wdg) ma kpoo ha na centrifuge vacuum.
Nyocha LC-MS/MS. Maka nyocha oke, e kewara peptides (nọmba r119.aq) na kọlụm nyocha PicoFrit nke dị sentimita 25, 75 μm (lensi anya ọhụrụ, nọmba akụkụ PF7508250) nke nwere 1.9 μm ReproSil-Pur 120 C18-AQ (Dr. Maisch, mat), Jiri EASY-nLC 1200 (Thermo Fisher Scientific, Germany). E dobere kọlụm ahụ na 50°C. Buffers A na B bụ 0.1% formic acid na mmiri na 0.1% formic acid na 80% ACN, n'otu n'otu. E kewapụrụ Peptides site na 6% ruo 31% buffer B maka nkeji 65 na site na 31% ruo 50% buffer B maka nkeji 5 yana gradient nke 200 nl/min. E nyochachara peptides ndị a na-ebuli elu na Orbitrap Fusion mass spectrometer (Thermo Fisher Scientific). A na-eme nha peptide precursor m/z site na mkpebi nke 120,000 n'etiti 350 ruo 1500 m/z. Site na iji ike nkwekọ 27% nke a na-ahazi, a na-ahọrọ precursor kachasị ike nwere ọnọdụ chajị nke 2 ruo 6 maka ike dị elu nke C trap dissociation (HCD). A na-ahazi oge okirikiri ahụ na 1 s. A tụrụ uru m/z nke iberibe peptide ahụ na ọnyà ion site na iji obere ebumnuche AGC nke 5 × 104 na oge ntụtụ kachasị nke 86 ms. Mgbe e kewara ya, e tinyere precursor na ndepụta mwepu ike maka 45 s. E kewapụrụ peptides TMT akara na kọlụm Acclaim PepMap nke dị sentimita 50, 75 μm (Thermo Fisher Scientific, nọmba katalọgụ 164942), a tụlekwara spectra mbugharị na Orbitrap Lumos Tribrid mass spectrometer (Thermo Fisher Scientific) nke nwere ngwa ions waveform dị elu (FAIMS) (Thermo Fisher Scientific) na-arụ ọrụ na voltaji nkwụghachi ụgwọ abụọ nke −50 na −70 V. A na-eji MS3 ahọpụtara dabere na precursor synchronization maka nha akara ion akụkọ TMT. Emere nkewa peptide na EASY-nLC 1200, site na iji elution gradient 90% linear, yana njupụta buffer nke 6% ruo 31%; buffer A bụ 0.1% FA, buffer B bụkwa 0.1% FA na 80% ACN. A na-arụ ọrụ kọlụm nyocha na 50°C. Jiri FreeStyle (ụdị 1.6, Thermo Fisher Scientific) kewaa faịlụ mbụ dịka voltaji ụgwọ FAIMS si dị.
Nchọpụta na nhazi protein. Site na iji igwe nchọta Andromeda agbakọtara, e jiri MaxQuant version 1.5.2.8 (https://maxquant.org/) nyochaa data mbụ ahụ. Na mgbakwunye na usoro Cre recombinase na YFP enwetara site na Aequorea victoria, a chọtara peptide fragment spectra maka usoro canonical na usoro isoform nke òké reference proteome (Proteome ID UP000000589, ebudatara na UniProt na Mee 2017). E debere methionine oxidation na protein N-terminal acetylation dị ka mgbanwe mgbanwe; e debere cysteine ​​​​carbamoyl methylation dị ka mgbanwe edobere. E debere paramita nri na "specificity" na "trypsin/P". Ọnụọgụ kacha nta nke peptides na razor peptides eji maka njirimara protein bụ 1; ọnụọgụ kacha nta nke peptides pụrụ iche bụ 0. N'okpuru ọnọdụ nke maapụ peptide dakọtara, ọnụego njirimara protein bụ 0.01. Emere nhọrọ "Second Peptide" ka ọ rụọ ọrụ. Jiri nhọrọ "nhazi n'etiti ọsọ" iji bufee njirimara na-aga nke ọma n'etiti faịlụ mbụ dị iche iche. Jiri ọnụọgụgụ LFQ kacha nta nke 1 maka nhazi enweghị akara (LFQ) (60). A na-enyocha ike LFQ maka ma ọ dịkarịa ala uru abụọ dị irè na opekata mpe otu genotype n'oge ọ bụla, a na-ewepụtakwa ya site na nkesa nkịtị nwere obosara nke. 0.3 wee gbadaa 1.8. Jiri ikpo okwu kọmputa Perseus (https://maxquant.net/perseus/) na R (https://r-project.org/) iji nyochaa nsonaazụ LFQ. Ejiri ule t nke dị n'etiti ụzọ abụọ site na ngwugwu ngwanrọ limma mee nyocha ngosipụta dị iche iche (61). A na-eji ggplot, FactoMineR, factoextra, GGally na pheatmap mee nyocha data nyocha. Ejiri MaxQuant version 1.6.10.43 nyochaa data proteomics dabere na TMT. Chọọ data proteomics sitere na nchekwa data proteomics nke UniProt, nke ebudatara na Septemba 2018. Nyocha ahụ gụnyere ihe mmezi ọcha nke isotope nke onye nrụpụta nyere. Jiri limma na R maka nyocha ngosipụta dị iche. A na-echekwa data mbụ, nsonaazụ ọchụchọ nchekwa data, na usoro ọrụ nyocha data na nsonaazụ niile na njikọ ProteomeXchange site na ebe nchekwa mmekọrịta PRIDE yana njirimara set data PXD019690.
Nkọwapụta ọrụ na-eme ka nyocha ahụ ka mma. Ejiri ngwa nyocha ụzọ Ingenuity (QIAGEN) chọpụta akụnụba nke okwu nkọwa ọrụ nke data edobere na izu 8 (Foto 1). Na nkenke, a na-eji ndepụta protein ọnụọgụgụ enwetara site na nyocha data LC-MS/MS (tandem mass spectrometry) yana njirisi nzacha ndị a: A na-ahọrọ musculus dị ka ụdị na ndabere, na otu ahụ na-egosi uru P nke Benjamini gbanwere maka mmụba 0.05 ma ọ bụ ala ka a na-ewere dị ka ihe dị mkpa. Maka eserese a, a na-egosi ụdị ise kachasị elu na otu ọ bụla dabere na uru P agbanwere. Site na iji ọtụtụ t-test, site na iji mmemme nkwalite ahịrị abụọ nke Benjamini, Krieger, na Yekutieli (Q = 5%), a na-eme nyocha ngosipụta protein oge-oge na ndị dị mkpa achọpụtara na otu ọ bụla, a na-enyocha ahịrị ọ bụla iche. Ọ dịghị mkpa ịnakwere SD na-agbanwe agbanwe.
Iji tụnyere nsonaazụ nke ọmụmụ ihe a na nchekwa data ebipụtara ma mepụta eserese Venn na Foto 1, anyị jikọtara ndepụta protein ọnụọgụgụ na nkọwa MitoCarta 2.0 (24). Jiri ngwaọrụ dị n'ịntanetị Draw Venn Diagram (http://bioinformatics.psb.ugent.be/webtools/Venn/) mepụta eserese ahụ.
Maka ozi zuru ezu gbasara usoro ọnụọgụgụ eji eme nyocha proteomics, biko lee ngalaba kwekọrọ na nke Ihe na Usoro. Maka nnwale ndị ọzọ niile, enwere ike ịchọta ozi zuru ezu na akụkọ ifo kwekọrọ. Ọ gwụla ma akọwapụtara ya ọzọ, a na-egosipụta data niile dị ka nkezi ± SEM, e mekwara nyocha ọnụọgụgụ niile site na iji ngwanrọ GraphPad Prism 8.1.2.
Maka ihe ndị ọzọ gbasara akụkọ a, biko lee http://advances.sciencemag.org/cgi/content/full/6/35/eaba8271/DC1
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Site E. Motori, I. Atanassov, SMV Kochan, K. Folz-Donahue, V. Sakthivelu, P. Giavalisco, N. Toni, J. Puyal, N.-G. Larson
Nnyocha proteomics nke neurons na-adịghị arụ ọrụ gosiri na a na-eme ka mmemme metabolic rụọ ọrụ iji lụso neurodegeneration ọgụ.
Site E. Motori, I. Atanassov, SMV Kochan, K. Folz-Donahue, V. Sakthivelu, P. Giavalisco, N. Toni, J. Puyal, N.-G. Larson
Nnyocha proteomics nke neurons na-adịghị arụ ọrụ gosiri na a na-eme ka mmemme metabolic rụọ ọrụ iji lụso neurodegeneration ọgụ.
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