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Propionic acid (PPA) e sebelisetsoa ho ithuta karolo ea ho se sebetse hantle ha mitochondrial mathateng a nts'etsopele ea methapo a kang autism spectrum disorder. PPA e tsejoa e sitisa biogenesis ea mitochondrial, metabolism, le phetoho. Leha ho le joalo, litlamorao tsa PPA ho matla a mitochondrial, karohano le ho kopana li ntse li le bothata ka lebaka la tlhaho e rarahaneng ea nako ea mekhoa ena. Mona, re sebelisa mekhoa ea ho etsa litšoantšo tse tlatsanang ho batlisisa hore na PPA e ama sebopeho sa mitochondrial ultrastructure, morphology, le matla joang liseleng tsa SH-SY5Y tse kang neuron. PPA (5 mM) e bakile phokotso e kholo sebakeng sa mitochondrial (p < 0.01), bophara ba Feret le sedikadikwe (p < 0.05), le sebaka sa 2 (p < 0.01). Tlhahlobo ea sebaka sa liketsahalo tsa mitochondrial e bontšitse keketseho e kholo (p < 0.05) liketsahalong tsa ho arohana le ho kopana, ka hona e boloka botšepehi ba marang-rang a mitochondrial tlas'a maemo a khatello. Ho phaella moo, tlhahiso ea mRNA ea cMYC (p < 0.0001), NRF1 (p < 0.01), TFAM (p < 0.05), STOML2 (p < 0.0001) le OPA1 (p < 0.05) e fokotsehile haholo. 01). Sena se bontša ho nchafatsoa ha sebopeho sa mitochondrial, biogenesis le matla a ho boloka ts'ebetso tlas'a maemo a khatello. Lintlha tsa rona li fana ka temohisiso e ncha mabapi le litlamorao tsa PPA ho matla a mitochondrial 'me li totobatsa bohlokoa ba mekhoa ea ho etsa litšoantšo bakeng sa ho ithuta mekhoa e rarahaneng ea taolo e amehang likarabelong tsa khatello ea mitochondrial.
Mitochondria ke barupeluoa ba bohlokoa mesebetsing e fapaneng ea sele e fetang mesebetsi ea bona e tloaelehileng tlhahisong ea matla le biosynthesis. Metabolism ea Mitochondrial ke molaoli oa bohlokoa oa calcium signaling, metabolic le redox homeostasis, inflammatory signaling, epigenetic diphetoho, ho ata ha sele, dipharologanyo le programmed cell death1. Haholo-holo, mitochondrial metabolism e bohlokoa bakeng sa nts'etsopele ea neuronal, ho phela le ts'ebetso 'me e ameha haholo lipontšong tse fapaneng tsa neuropathology2,3,4.
Lilemong tse leshome tse fetileng, boemo ba metabolism bo hlahile e le molaoli ea ka sehloohong oa neurogenesis, phapang, khōlo le plasticity5,6. Haufinyane tjena, sebopeho sa mitochondrial le matla a eona li fetohile likarolo tsa bohlokoa haholo tsa mitosis, ts'ebetso e matla e bolokang letamo la mitochondria e phetseng hantle ka har'a lisele. Matla a mitochondrial a laoloa ke litsela tse rarahaneng tse itšetlehileng ka tse ling ho tloha ho biogenesis ea mitochondrial le bioenergetics ho isa ho finsion ea mitochondrial, fusion, transport le clearance7,8. Ho sitisoa ha mekhoa efe kapa efe ea ho kopanya ho sitisa tlhokomelo ea marang-rang a mitochondrial a phetseng hantle 'me ho na le liphello tse tebileng tsa ts'ebetso bakeng sa nts'etsopele ea methapo9,10. Ka 'nete, ho se laolehe ha matla a mitochondrial ho bonoa mafung a mangata a kelello, a methapo ea kutlo le a nts'etsopele ea methapo, ho kenyeletsoa le mathata a autism spectrum (ASD)11,12.
ASD ke bothata ba ntshetsopele ya methapo bo sa tshwaneng bo nang le sebopeho se rarahaneng sa lefutso le epigenetic. Ho futsa ha ASD ha ho na pelaelo, empa sesosa sa motheo sa dimolek'hule se ntse se sa utlwisisehe hantle. Ho bokella tlhahisoleseding ho tswa mehlala ya pele ho kliniki, dithuto tsa kliniki, le di-dataset tsa dimolek'hule tse ngata di fana ka bopaki bo ntseng bo eketseha ba ho se sebetse hantle ha mitochondrial ho ASD13,14. Pele re entse tlhahlobo ya methylation ya DNA ka bophara ba genome sehlopheng sa bakudi ba nang le ASD mme ra hlwaya dijini tse nang le methylated ka tsela e fapaneng tse kopaneng ditseleng tsa metabolism ya mitochondrial15. Ka mora moo re tlalehile methylation e fapaneng ya balaodi ba bohareng ba biogenesis ya mitochondrial le dynamics, e neng e amana le palo e eketsehileng ya kopi ya mtDNA le boemo bo fetotsweng ba metabolism ya moroto ho ASD16. Lintlha tsa rona di fana ka bopaki bo ntseng bo eketseha ba hore dynamics ya mitochondrial le homeostasis di bapala karolo ya bohlokwa pathophysiology ya ASD. Ka hona, ho ntlafatsa kutlwisiso ya mekgwa ya kamano pakeng tsa dynamics ya mitochondrial, morphology, le tshebetso ke sepheo sa bohlokwa sa dipatlisiso tse tswelang pele mabapi le mafu a methapo a kgethollwang ke ho se sebetse hantle ha mitochondrial ya bobedi.
Mekhoa ea limolek'hule e atisa ho sebelisoa ho ithuta karolo ea liphatsa tsa lefutso tse itseng likarabelong tsa khatello ea mitochondrial. Leha ho le joalo, mokhoa ona o ka fokotsoa ke mofuta o nang le mahlakore a mangata le oa nako oa mekhoa ea taolo ea mitotic. Ho feta moo, tlhaloso e fapaneng ea liphatsa tsa lefutso tsa mitochondrial ke sesupo se sa tobang sa liphetoho tsa ts'ebetso, haholo-holo kaha hangata ho hlahlojoa palo e fokolang ea liphatsa tsa lefutso. Ka hona, mekhoa e tobileng haholoanyane ea ho ithuta ts'ebetso ea mitochondrial le bioenergetics e se e sisintsoe17. Sebopeho sa mitochondrial se amana haufi-ufi le matla a mitochondrial. Sebopeho sa mitochondrial, khokahano, le sebopeho li bohlokoa bakeng sa tlhahiso ea matla le ho phela ha mitochondrial le sele5,18. Ho feta moo, likarolo tse fapaneng tsa mitosis li shebana le liphetoho tsa sebopeho sa mitochondrial, tse ka sebetsang e le lintlha tse thusang tsa ho se sebetse hantle ha mitochondrial le ho fana ka motheo oa lithuto tse latelang tsa mechini.
Sebopeho sa mitochondrial se ka shejoa ka ho toba ho sebediswa microscopy ya phetisetso ya electron (TEM), e leng se lumellang ho ithuta ka botlalo sebopeho sa ultrastructure ya sele. TEM e bona ka ho toba sebopeho, sebopeho le sebopeho sa mitochondrial cristae qetong ya mitochondria ka nngwe, ho ena le ho itshetleha feela hodima phetolelo ya gene, tlhahiso ya protheine kapa diparamithara tsa tshebetso ya mitochondrial ho baahi ba sele17,19,20. Ho phaella moo, TEM e thusa ho ithuta dikamano pakeng tsa mitochondria le di-organelle tse ding, tse kang endoplasmic reticulum le autophagosomes, tse bapalang karolo ya bohlokwa mosebetsing wa mitochondrial le homeostasis21,22. Kahoo, sena se etsa hore TEM e be sebaka se setle sa ho qala ho ithuta ho se sebetse hantle ha mitochondrial pele ho tsepamiswa maikutlo ditseleng kapa dijining tse itseng. Ha mosebetsi wa mitochondrial o ntse o ameha haholo ho neuropathology, ho na le tlhoko e hlakileng ya ho kgona ho ithuta sebopeho sa mitochondrial ka ho toba le ka bongata mehlala ya neuronal ya in vitro.
Sehloohong sena, re hlahloba diphetoho tsa mitochondrial mohlaleng wa methapo ya kutlo wa ho se sebetse hantle ha mitochondrial ho autism spectrum disorder. Re kile ra tlaleha differential methylation ya propionyl-CoA carboxylase beta (PCCB) ho ASD15, e leng karoloana ya mitochondrial propionyl-CoA carboxylase enzyme PCC. Ho se sebetse hantle ha PCC ho tsebahala ho baka ho bokellana ha chefo ha di-propionyl derivatives, ho kenyeletswa le propionic acid (PPA)23,24,25. PPA e bontshitswe e sitisa metabolism ya neuronal le ho fetola boitshwaro ka mmele mme ke mohlala o thehilweng wa phoofolo bakeng sa ho ithuta mekgwa ya ntshetsopele ya methapo e amehang ho ASD26,27,28. Ho feta moo, PPA e tlalehilwe e sitisa bokgoni ba lera la mitochondrial, biogenesis le ho hema ka hare ho mmele mme e sebediseditswe haholo ho etsa mohlala wa ho se sebetse hantle ha mitochondrial ho di-neuron29,30. Leha ho le jwalo, tshusumetso ya ho se sebetse hantle ha mitochondrial ho bakwang ke PPA hodima sebopeho sa mitochondrial le diphetoho e ntse e sa utlwisisehe hantle.
Phuputso ena e sebedisa mekgwa e tlatsanang ya ho nka ditshwantsho ho lekanya ditlamorao tsa PPA hodima sebopeho sa mitochondrial, matla, le tshebetso diseleng tsa SH-SY5Y. Taba ya pele, re ntshetsitse mokgwa wa TEM ho bona diphetoho sebopehong sa mitochondrial le sebopeho sa ultrastructure17,31,32. Ka lebaka la tlhaho e fetohang ya mitochondria33, re boetse re sebedisitse tlhahlobo ya mitochondrial event localizer (MEL) ho lekanya diphetoho tekanyong pakeng tsa diketsahalo tsa ho arohana le ho kopana, palo ya mitochondrial le bophahamo tlasa kgatello ya PPA. Qetellong, re hlahlobile hore na sebopeho sa mitochondrial le matla di amana le diphetoho polelong ya dijini tse amehang ho biogenesis, ho arohana le ho kopana. Ha di kopantswe, data ya rona e bontsha phephetso ya ho hlakisa ho rarahana ha mekgwa e laolang matla a mitochondrial. Re totobatsa thuso ya TEM ho ithuteng sebopeho sa mitochondrial e le ntlha e ka lekanngwang ya ho kopana ha mitosis diseleng tsa SH-SY5Y. Ho feta moo, re totobatsa hore data ya TEM e fana ka tlhahisoleseding e ruileng ka ho fetisisa ha e kopanngwa le mekgwa ya ho nka ditshwantsho e boetse e hapa diketsahalo tse fetohang ho arabela kgatello ya metabolism. Tlhaloso e eketsehileng ea mekhoa ea taolo ea limolek'hule e tšehetsang mitosis ea lisele tsa neuronal e ka fana ka temohisiso ea bohlokoa mabapi le karolo ea mitochondrial ea tsamaiso ea methapo le mafu a methapo a senyehang.
Ho baka kgatello ya mitochondrial, disele tsa SH-SY5Y di ile tsa phekolwa ka PPA ho sebediswa 3 mM le 5 mM sodium propionate (NaP). Pele ho TEM, disampole di ile tsa lokiswa ka ho lokisa sampole ya cryogenic ho sebediswa ho hoamisa le ho hoamisa ka kgatello e hodimo (Setshwantsho sa 1a). Re ntsheditse pele phaephe ya tlhahlobo ya setshwantsho sa mitochondrial e iketsang ho lekanya diparamente tse robedi tsa sebopeho sa baahi ba mitochondrial ho pholletsa le dikopi tse tharo tsa baeloji. Re fumane hore kalafo ya PPA e fetotse diparamente tse nne haholo: sebaka sa 2, sebaka, perimeter, le bophara ba Feret (Setshwantsho sa 1b–e). Sebaka sa 2 se fokotsehile haholo ka kalafo ya 3 mM le 5 mM PPA (p = 0.0183 le p = 0.002, ka ho latellana) (Setshwantsho sa 1b), ha sebaka (p = 0.003), perimeter (p = 0.0106) le bophara ba Feret kaofela di fokotsehile haholo. Ho bile le phokotso e kgolo (p = 0.0172) sehlopheng sa kalafo sa 5 mM ha ho bapiswa le sehlopha sa taolo (Setshwantsho sa 1c–e). Phokotso e kholo sebakeng le selikalikoeng e bontšitse hore lisele tse phekoloang ka 5 mM PPA li ne li e-na le mitochondria e nyane, e chitja haholoanyane, le hore mitochondria ena e ne e le telele hanyane ho feta tse liseleng tsa taolo. Sena se boetse se lumellana le phokotso e kholo ea bophara ba Feret, e leng paramethara e ikemetseng e bontšang phokotso ea sebaka se seholohali pakeng tsa mathoko a likaroloana. Liphetoho sebopehong se seholo sa cristae li ile tsa bonoa: cristae e ile ea fokola haholo tlas'a tšusumetso ea khatello ea PPA (Setšoantšo sa 1a, phanele ea B). Leha ho le joalo, ha se litšoantšo tsohle tse bonts'itseng ka ho hlaka sebopeho se seholo sa cristae, kahoo tlhahlobo ea bongata ea liphetoho tsena ha ea ka ea etsoa. ​​Lintlha tsena tsa TEM li ka bonts'a maemo a mararo a ka bang teng: (1) PPA e ntlafatsa karohano kapa e thibela ho kopana, e leng se etsang hore mitochondria e teng e fokotsehe ka boholo; (2) biogenesis e ntlafalitsoeng e hlahisa mitochondria e ncha, e nyane kapa (3) e baka mekhoa ka bobeli ka nako e le 'ngoe. Le hoja maemo ana a ke ke a khetholloa ke TEM, liphetoho tse kholo tsa sebopeho li bontša liphetoho ho homeostasis ea mitochondrial le matla a tlas'a khatello ea PPA. Ka mor'a moo re ile ra hlahloba liparamethara tse ling ho hlalosa haholoanyane matla ana le mekhoa e ka bang teng e li tšehetsang.
Propionic acid (PPA) e nchafatsa sebopeho sa mitochondrial. (a) Litšoantšo tsa microscopy ea elektrone ea phetisetso (TEM) tse bontšang hore boholo ba mitochondrial boa fokotseha 'me mitochondria e ba nyane ebile e chitja haholoanyane ka kalafo e ntseng e eketseha ea PPA; 0 mM (e sa phekoloang), 3 mM le 5 mM, ka ho latellana. Metsu e khubelu e bontša mitochondria. (b–e) Lisele tsa SH-SY5Y tse phekoloang ka PPA lihora tse 24 li ile tsa lokisetsoa TEM 'me liphetho li ile tsa hlahlojoa ho sebelisoa Fiji/ImageJ. Liparamitha tse 'ne ho tse robeli li bontšitse liphapang tse kholo lipakeng tsa lisele tsa taolo (tse sa phekoloang, 0 mM PPA) le lisele tse phekoloang (3 mM le 5 mM PPA). (b) Sebaka sa 2, (c) Sebaka, (d) Perimeter, (e) Bophara ba Feret. Tlhahlobo ea tsela e le 'ngoe ea phapang (taolo khahlanong le kalafo) le teko ea papiso e mengata ea Dunnett li sebelisitsoe ho fumana liphapang tse kholo (p < 0.05). Lintlha tsa data li emela boleng bo tloaelehileng ba mitochondrial bakeng sa sele ka 'ngoe, 'me mekoallo ea liphoso e emela karolelano ea ± SEM. Lintlha tse bontšitsoeng li emela n = 3, bonyane lisele tse 24 ka kopitso; kakaretso ea litšoantšo tse 266 li hlahlobiloe; * e bontša p < 0.05, ** e bontša p < 0.01.
Ho hlalosa haholoanyane kamoo diphetoho tsa mitochondrial di arabelang kateng ho PPA, re silafaditse mitochondria ka tetramethylrhodamine ethyl ester (TMRE) mme ra sebedisa timing-lapse microscopy le tlhahlobo ya MEL ho fumana le ho lekanya mitochondria kamora dihora tse 24 ho 3 le 5 mM PPA. Kalafo ya diketsahalo tsa ho arohana le ho kopana. (Setshwantsho sa 2a). Kamora tlhahlobo ya MEL, mitochondria e ile ya hlahlojwa hape ho lekanya palo ya dibopeho tsa mitochondrial le bophahamo ba tsona bo tloaelehileng. Re bone keketseho e nyane empa e le ea bohlokoa palong ea liketsahalo tsa karohano tse etsahalang ho 3 mM [4.9 ± 0.3 (p < 0.05)] ha ho bapisoa le karohano [5.6 ± 0.3 (p < 0.05) )] le kopano [5.4 ± 0.5 (p < 0.05)] le kopano [5.4 ± 0.5 (p < 0.05)] 0.05)] <0.05)] liketsahalo li eketsehile haholo ho 5 mM ha ho bapisoa le taolo (Setšoantšo sa 3b). Palo ea mitochondria e eketsehile haholo ho 3 ka bobeli [32.6 ± 2.1 (p < 0.05)] le 5 mM [34.1 ± 2.2 (p < 0.05)] (Setšoantšo sa 3c), ha bophahamo bo tloaelehileng ba sebopeho se seng le se seng sa mitochondrial bo ntse bo sa fetohe (Setšoantšo sa 3c). 3d). Ha ho kopanngoa, sena se fana ka maikutlo a hore ho nchafatsa di-dynamics tsa mitochondrial ho sebetsa e le karabelo e buseletsang e bolokang botšepehi ba marangrang a mitochondrial ka katleho. Keketseho ea palo ea liketsahalo tsa ho arohana ha 3 mM PPA e bontša hore keketseho ea palo ea mitochondrial e bakoa ke ho arohana ha mitochondrial, empa kaha karolelano ea bophahamo ba mitochondrial e lula e sa fetohe, biogenesis e ke ke ea qheleloa ka thoko e le karabelo e eketsehileng ea puseletso. Leha ho le joalo, lintlha tsena li lumellana le meaho e menyenyane, e chitja ea mitochondrial e bonoang ke TEM 'me hape li bontša liphetoho tse kholo di-dynamics tsa mitochondrial tse bakoang ke PPA.
Propionic acid (PPA) e susumetsa ho tsosolosoa ha mitochondrial ka matla ho boloka botsitso ba marang-rang. Lisele tsa SH-SY5Y li ile tsa lengoa, tsa phekoloa ka 3 le 5 mM PPA ka lihora tse 24 'me tsa dauoa ka TMRE le Hoechst 33342 tse lateloang ke tlhahlobo ea MEL. (a) Litšoantšo tse emelang nako ea microscopy tse bontšang 'mala le likhakanyo tsa matla a maholo tse nang le binarized ka nako ea 2 (t2) bakeng sa boemo bo bong le bo bong. Libaka tse khethiloeng tse bontšitsoeng setšoantšong se seng le se seng sa binary lia ntlafatsoa 'me li bontšoa ka 3D ka liforeimi tse tharo tse fapaneng tsa nako (t1-t3) ho bontša matla ha nako e ntse e ea; liketsahalo tsa ho kopanya li totobatsoa ka botala; liketsahalo tsa ho arohana li totobatsoa ka botala. Li bontšoa ka bofubelu. (b) Palo e tloaelehileng ea liketsahalo tse matla ka boemo bo bong le bo bong. (c) Palo e tloaelehileng ea mehaho ea mitochondrial ka sele. (d) Bophahamo bo tloaelehileng (µm3) ba sebopeho se seng le se seng sa mitochondrial ka sele. Lintlha tse bontšitsoeng li emela lisele tse n = 15 ka sehlopha sa kalafo. Li-error bar tse bontšitsoeng li emela karolelano ea ± SEM, scale bar = 10 μm, * p < 0.05.
Propionic acid (PPA) e baka kgatello ya transcriptional ya dijini tse amanang le di-mitochondrial dynamics. Disele tsa SH-SY5Y di ile tsa phekolwa ka 3 le 5 mM PPA ka dihora tse 24. Tekanyo ya dijini tse amanang e entswe ka ho sebedisa RT-qPCR mme ya fetolwa ho ba B2M. Dijini tsa Mitochondrial biogenesis (a) cMYC, (b) TFAM, (c) NRF1 le (d) NFE2L2. Dijini tsa Mitochondrial fusion le fission (e) STOML2, (f) OPA1, (g) MFN1, (h) MFN2 le (i) DRP1. Dipharologanyo tse kgolo (p < 0.05) di ile tsa lekoa ho sebediswa ANOVA ya tsela e le nngwe (taolo vs. kalafo) le teko ya papiso e ngata ya Dunnett: * e bontsha p < 0.05, ** e bontsha p < 0.01, mme **** e bontsha p < 0.0001. Dibara di emela polelo e mahareng ± SEM. Lintlha tse bontšitsoeng li emela n = 3 (STOML2, OPA1, TFAM), n = 4 (cMYC, NRF1, NFE2L2), le n = 5 (MFN1, MFN2, DRP1) likopi tsa baeloji.
Lintlha tse tsoang ho tlhahlobo ea TEM le MEL hammoho li bontša hore PPA e fetola sebopeho sa mitochondrial le matla a eona. Leha ho le joalo, mekhoa ena ea ho nka litšoantšo ha e fane ka temohisiso mabapi le mekhoa e ka sehloohong e tsamaisang lits'ebetso tsena. Ka hona, re hlahlobile polelo ea mRNA ea balaoli ba bohlokoa ba robong ba matla a mitochondrial, biogenesis, le mitosis ho arabela kalafo ea PPA. Re lekantse cell myeloma oncogene (cMYC), nuclear respiratory factor (NRF1), mitochondrial transcription factor 1 (TFAM), NFE2-like transcription factor BZIP (NFE2L2), gastrin-like protein 2 (STOML2), optic nerve atrophy 1 (OPA1), Mitofusin 1 (MFN1), Mitofusin 2 (MFN2) le dynamin-related protein 1 (DRP1) kamora lihora tse 24 tsa kalafo ka 3 mM le 5 mM PPA. Re bone 3 mM (p = 0.0053, p = 0.0415 le p < 0.0001, ka ho latellana) le 5 mM (p = 0.0031, p = 0.0233, p < 0.0001) kalafo ea PPA. (Setšoantšo sa 3a–c). Ho fokotseha ha tlhahiso ea mRNA ho ne ho itšetlehile ka tekanyo: polelo ea cMYC, NRF1 le TFAM e fokotsehile ka makhetlo a 5.7, 2.6 le 1.9 ho 3 mM, ka ho latellana, le ka makhetlo a 11.2, 3 le 2.2 ho 5 mM. Ka lehlakoreng le leng, gene ea redox biogenesis ea bohareng NFE2L2 ha ea ka ea fetoloa ka tekanyo efe kapa efe ea PPA, leha mokhoa o tšoanang o itšetlehileng ka tekanyo oa tlhahiso e fokotsehileng o ile oa bonoa (Setšoantšo sa 3d).
Re boetse ra hlahloba tlhahiso ea liphatsa tsa lefutso tsa khale tse amehang taolong ea karohano le ho kopana. Ho nahanoa hore STOML2 e ameha ho kopaneng, mitophagy le biogenesis, 'me tlhahiso ea eona e fokotsehile haholo (p < 0.0001) ka 3 mM (phetoho ea makhetlo a 2.4) le 5 mM (phetoho ea makhetlo a 2.8) PPA (Setšoantšo sa 1). 3d). Ka mokhoa o ts'oanang, tlhahiso ea liphatsa tsa lefutso tsa fusion ea OPA1 e fokotsehile ho 3 mM (phetoho ea makhetlo a 1.6) le 5 mM (phetoho ea makhetlo a 1.9) PPA (p = 0.006 le p = 0.0024, ka ho latellana) (Setšoantšo sa 3f). Leha ho le joalo, ha rea ​​​​ka ra fumana liphapang tse kholo tlhahisong ea liphatsa tsa lefutso tsa fusion tsa MFN1, MFN2 kapa fission gene DRP1 tlas'a khatello ea PPA ea lihora tse 24 (Setšoantšo sa 3g–i). Ho phaella moo, re fumane hore maemo a diprotheine tse nne tsa fusion le fission (OPA1, MFN1, MFN2 le DRP1) ha a fetoha tlasa maemo a tshwanang (Setshwantsho sa 4a–d). Ho bohlokwa ho hlokomela hore dintlha tsena di bontsha ntlha e le nngwe ka nako mme di kanna tsa se bontshe diphetoho tlhahisong ya protheine kapa maemo a tshebetso nakong ya mehato ya pele ya kgatello ya PPA. Leha ho le jwalo, phokotso e kgolo tlhahisong ya cMYC, NRF1, TFAM, STOML2, le OPA1 e bontsha ho se sebetse hantle ha transcriptional metabolism ya mitochondrial, biogenesis, le dynamics. Ho phaella moo, dintlha tsena di totobatsa tshebediso ya mekgwa ya ho etsa ditshwantsho ho ithuta ka ho toba diphetoho tsa boemo ba qetello mosebetsing wa mitochondrial.
Maemo a protheine ya motsoako le fission factor ha a a ka a fetoha kamora kalafo ya propionic acid (PPA). Disele tsa SH-SY5Y di ile tsa phekolwa ka 3 le 5 mM PPA ka dihora tse 24. Maemo a protheine a ile a lekanngwa ka tlhahlobo ya Western blot, mme maemo a tlhahiso a ile a tloaeleha ho ba protheine yohle. Ponahalo e tloaelehileng ya protheine le dibloto tse emelang tsa Western blots tsa target le total protein di a bontshwa. a – OPA1, b – MFN1, c – MFN2, d – DRP1. Dibara di emela mean ± SEM, mme data e bontshitsweng e emela n = dikopi tse 3 tsa baeloji. Dipapiso tse ngata (p < 0.05) di entswe ka ho sebedisa tlhahlobo ya tsela e le nngwe ya phapang le teko ya Dunnett. Gel ya pele le blot di bontshitswe ho Setšoantšo sa S1.
Ho se sebetse hantle ha methapo ya madi ho amana le mafu a mangata a tsamaiso ya mmele ho tloha mafung a metabolism, pelo le mesifa ho isa mafung a methapo ya kutlo1,10. Mafu a mangata a methapo ya kutlo le a methapo ya kutlo a amana le ho se sebetse hantle ha methapo ya kutlo, e leng se totobatsang bohlokwa ba di-organelle tsena nakong yohle ya bophelo ba boko. Mafu ana a kenyeletsa lefu la Parkinson, lefu la Alzheimer le ASD3,4,18. Leha ho le jwalo, phihlello ya dinama tsa boko ho ithuta mafu ana e thata, haholoholo maemong a mekeniki, e leng se etsang hore ditsamaiso tsa mohlala wa sele e be mokhoa o mong o hlokahalang. Thutong ena, re sebedisa sistimi ya mohlala wa sele e sebedisang disele tsa SH-SY5Y tse phekolwang ke PPA ho hlalosa hape ho se sebetse hantle ha methapo ya kutlo ho bonweng mafung a methapo ya kutlo, haholoholo mathata a autism spectrum. Ho sebedisa mohlala ona wa PPA ho ithuta di-dynamics tsa mitochondrial di-neuron ho ka fana ka temohisiso mabapi le sesosa sa ASD.
Re hlahlobile monyetla oa ho sebelisa TEM ho bona liphetoho mofuteng oa mitochondrial. Ho bohlokoa ho hlokomela hore TEM e tlameha ho sebelisoa ka nepo ho eketsa katleho ea eona. Ho lokisoa ha li-cryo-specimens ho lumella poloko e betere ea meaho ea neuronal ka ho lokisa likarolo tsa sele ka nako e le 'ngoe le ho fokotsa ho thehoa ha lintho tsa khale34. Tumellanong le sena, re hlokometse hore lisele tsa SH-SY5Y tse kang neuron li ne li e-na le li-organelle tse ka tlas'a sele tse sa senyehang le mitochondria e telele (Setšoantšo sa 1a). Sena se totobatsa thuso ea mekhoa ea ho lokisa cryogenic bakeng sa ho ithuta mofuteng oa mitochondrial mehlala ea lisele tsa neuronal. Le hoja litekanyo tsa bongata li le bohlokoa bakeng sa tlhahlobo e nepahetseng ea data ea TEM, ho ntse ho se na tumellano mabapi le hore na ke liparamente life tse itseng tse lokelang ho lekanyetsoa ho netefatsa liphetoho tsa mofuteng oa mitochondrial. Ho latela palo e kholo ea lithuto tse hlahlobileng mofuteng oa mitochondrial17,31,32 ka bongata, re hlahisitse phaephe ea tlhahlobo ea setšoantšo sa mitochondrial e iketsang e lekanyang liparamente tse robeli tsa mofuteng, e leng: sebaka, sebaka2, karolelano ea ponahalo, perimeter, circularity, degree , Feret bophara. le circularness.
Har'a tsona, PPA e fokolitse sebaka sa 2 haholo, sebaka, potoloho, le bophara ba Feret (Setšoantšo sa 1b–e). Sena se bontšitse hore mitochondria e ile ea e-ba nyane 'me ea ba chitja haholoanyane, e leng se lumellanang le lithuto tse fetileng tse bontšang phokotseho ea sebaka sa mitochondrial kamora lihora tse 72 tsa khatello ea mitochondrial e bakoang ke PPA30. Litšobotsi tsena tsa sebopeho li ka bontša ho arohana ha mitochondrial, ts'ebetso e hlokahalang ea ho hula likarolo tse senyehileng marang-rang a mitochondrial ho khothaletsa ho senyeha ha tsona ka mitophagy35,36,37. Ka lehlakoreng le leng, phokotseho ea boholo ba mitochondrial e tloaelehileng e ka amahanngoa le keketseho ea biogenesis, e fellang ka ho thehoa ha mitochondria e nyane e sa tsoa hlaha. Ho eketseha ha ho arohana kapa biogenesis ho emela karabelo ea puseletso ho boloka mitosis khahlanong le khatello ea mitochondrial. Leha ho le joalo, ho fokotseha ha kholo ea mitochondrial, ho senyeha ha motsoako, kapa maemo a mang ho ke ke ha qheleloa ka thoko.
Leha litšoantšo tse nang le qeto e phahameng tse entsoeng ke TEM li lumella ho fumana litšobotsi tsa sebopeho boemong ba mitochondria ka 'ngoe, mokhoa ona o hlahisa linepe tse peli ka nako e le 'ngoe. Ho ithuta likarabo tse matla khatellong ea metabolism, re silafalitse mitochondria ka TMRE 'me ra sebelisa microscopy ea nako e telele ka tlhahlobo ea MEL, e lumellang pono e phahameng ea 3D ea liphetoho marangrang a mitochondrial ha nako e ntse e ea33,38. Re bone liphetoho tse poteletseng empa tse bohlokoa ho matla a mitochondrial tlas'a khatello ea PPA (Setšoantšo sa 2). Ho 3 mM, palo ea liketsahalo tsa ho arohana e eketsehile haholo, ha liketsahalo tsa ho kopana li ntse li tšoana le tse taolong. Keketseho ea palo ea liketsahalo tsa ho arohana le ho kopana e bonoe ho 5 mM PPA, empa liphetoho tsena li ne li batla li lekana, li fana ka maikutlo a hore kinetics ea ho arohana le ho kopana e fihlella tekano maemong a holimo (Setšoantšo sa 2b). Bophahamo ba mitochondrial bo tloaelehileng bo ile ba lula bo sa fetohe ho 3 le 5 mM PPA, e leng se bontšang hore botšepehi ba marangrang a mitochondrial bo bolokiloe (Setšoantšo sa 2d). Sena se bonts'a bokhoni ba marangrang a matla a mitochondrial ho arabela khatellong e bobebe ea metabolism ho boloka homeostasis ka katleho ntle le ho baka karohano ea marang-rang. Ho 3 mM PPA, keketseho ea karohano e lekane ho khothaletsa phetoho ho ea ho tekano e ncha, empa ho hlokahala phetoho e tebileng ea kinetic ho arabela khatellong e bakoang ke maemo a holimo a PPA.
Palo ea mitochondria e eketsehile maemong a mabeli a khatello ea PPA, empa bophahamo ba mitochondrial bo tloaelehileng ha boa fetoha haholo (Setšoantšo sa 2c). Sena se ka bakoa ke keketseho ea biogenesis kapa karohano e eketsehileng; leha ho le joalo, ha ho se na phokotso e kholo ea bophahamo ba mitochondrial bo tloaelehileng, ho ka etsahala hore biosynthesis e eketsehe. Leha ho le joalo, lintlha tse ho Setšoantšo sa 2 li tšehetsa boteng ba mekhoa e 'meli ea puseletso: keketseho palong ea liketsahalo tsa ho arohana, tse tsamaellanang le ho eketseha ha ho arohana ha mitochondrial, le keketseho palong ea liketsahalo, tse tsamaellanang le biogenesis ea mitochondrial. Qetellong, puseletso e matla bakeng sa khatello e bobebe e ka kenyelletsa lits'ebetso tse ts'oanang tse kenyeletsang ho arohana, ho kopana, biogenesis le mitophagy. Le hoja bangoli ba pejana ba bontšitse hore PPA e ntlafatsa mitosis30,39 le mitophagy29, re fana ka bopaki ba ho nchafatsa ho arohana ha mitochondrial le fusion dynamics ho arabela PPA. Lintlha tsena li tiisa liphetoho tsa sebopeho tse bonoang ke TEM 'me li fana ka temohisiso e eketsehileng mabapi le mekhoa e amanang le ho se sebetse hantle ha mitochondrial ho bakoang ke PPA.
Hobane tlhahlobo ea TEM kapa MEL ha ea ka ea fana ka bopaki bo tobileng ba mekhoa ea taolo ea liphatsa tsa lefutso e thehiloeng liphetohong tse bonoang tsa sebopeho, re hlahlobile polelo ea RNA ea liphatsa tsa lefutso tse amehang metabolism ea mitochondrial, biogenesis, le dynamics. Proto-oncogene ea cMYC ke ntho ea transcription e amehang taolong ea mitochondria, glycolysis, amino acid le metabolism ea mafura a acid40. Ho phaella moo, cMYC e tsejoa e laola polelo ea liphatsa tsa mitochondrial tse ka bang 600 tse amehang ho transcription ea mitochondrial, phetolelo le kopano e rarahaneng, ho kenyeletsoa NRF1 le TFAM41. NRF1 le TFAM ke balaoli ba babeli ba bohareng ba mitosis, ba sebetsang tlase ho PGC-1α ho kenya tšebetsong replication ea mtDNA. Tsela ena e kenngoa tšebetsong ke cAMP le AMPK signaling 'me e na le kutloelo-bohloko tšebelisong ea matla le khatello ea metabolism. Re boetse re hlahlobile NFE2L2, molaoli oa redox oa biogenesis ea mitochondrial, ho fumana hore na liphello tsa PPA li ka 'na tsa tsamaisoa ke khatello ea oxidative.
Leha polelo ea NFE2L2 e sa ka ea fetoha, re fumane phokotso e tsitsitseng e itšetlehileng ka tekanyo tlhahisong ea cMYC, NRF1 le TFAM kamora lihora tse 24 tsa kalafo ka 3 mM le 5 mM PPA (Setšoantšo sa 3a–c). Ho fokotseha ha polelo ea cMYC ho tlalehiloe pele e le karabelo khatellong ea mitochondrial42, 'me ka lehlakoreng le leng, ho fokotseha ha polelo ea cMYC ho ka baka ho se sebetse hantle ha mitochondrial ka ho fetola metabolism ea mitochondrial, khokahano ea marang-rang, le polarization ea membrane43. Hoa thahasellisa hore cMYC e boetse e ameha taolong ea ho arohana ha mitochondrial le fusion42,43 'me e tsejoa e eketsa phosphorylation ea DRP1 le sebaka sa mitochondrial nakong ea karohano ea sele44, hammoho le ho lokisa sebopeho sa mitochondrial liseleng tsa methapo ea methapo45. Ka 'nete, li-fibroblast tse haelloang ke cMYC li bontša boholo bo fokotsehileng ba mitochondrial, bo lumellanang le liphetoho tse bakoang ke khatello ea PPA43. Lintlha tsena li bontša kamano e khahlisang empa e ntse e sa hlaka pakeng tsa cMYC le mitochondrial dynamics, e fanang ka sepheo se khahlisang bakeng sa lithuto tsa nakong e tlang tsa ntlafatso e bakoang ke khatello ea PPA.
Phokotso ea NRF1 le TFAM e tsamaellana le karolo ea cMYC e le sesebelisoa sa bohlokoa sa ho qapa mongolo. Lintlha tsena li boetse li tsamaellana le lithuto tse fetileng liseleng tsa mofetše oa mala a motho tse bontšang hore PPA e fokolitse tlhahiso ea NRF1 mRNA ka lihora tse 22, e leng se neng se amahanngoa le ho fokotseha ha ATP le ho eketseha ha ROS46. Bangoli bana ba boetse ba tlalehile hore tlhahiso ea TFAM e eketsehile ka lihora tse 8.5 empa e khutletse maemong a motheo ka lihora tse 22. Ka lehlakoreng le leng, Kim et al. (2019) ba bontšitse hore tlhahiso ea TFAM mRNA e fokotsehile haholo kamora lihora tse 4 tsa khatello ea PPA liseleng tsa SH-SY5Y; leha ho le joalo, kamora lihora tse 72, tlhahiso ea protheine ea TFAM e eketsehile haholo 'me palo ea kopi ea mtDNA e eketsehile haholo. Kahoo, phokotso ea palo ea liphatsa tsa lefutso tsa mitochondrial biogenesis tseo re li boneng kamora lihora tse 24 ha e qhelele ka thoko monyetla oa hore keketseho ea palo ea mitochondria e amahanngoa le ts'ebetso ea biogenesis linakong tsa pele. Lithuto tse fetileng li bontšitse hore PPA e eketsa PGC-1α mRNA le protheine haholo liseleng tsa SH-SY5Y ka lihora tse 4 le metsotso e 30, ha propionic acid e eketsa biogenesis ea mitochondrial ho li-hepatocytes tsa namane ka PGC-1α ka lihora tse 12 le metsotso e 39. Hoa thahasellisa hore PGC-1α ha se feela molaoli oa transcriptional o tobileng oa NRF1 le TFAM, empa hape ho bontšitsoe hore e laola ts'ebetso ea MFN2 le DRP1 ka ho laola karohano le fusion47. Ha e kopanngoa, sena se totobatsa ho kopana ho haufi ha mekhoa e laolang likarabo tsa puseletso ea mitochondrial tse bakoang ke PPA. Ho feta moo, lintlha tsa rona li bonts'a ho se sebetse hantle ha taolo ea transcriptional ea biogenesis le metabolism tlas'a khatello ea PPA.
Lijini tsa STOML2, OPA1, MFN1, MFN2 le DRP1 ke tse ling tsa balaoli ba mantlha ba ho arohana ha mitochondrial, ho kopana le ho fetoha ha dijini37,48,49. Ho na le dijini tse ding tse ngata tse amehang ho feto-fetoha ha dijini tsa mitochondrial, leha ho le jwalo, STOML2, OPA1 le MFN2 di kile tsa fumanwa di na le methylated ka tsela e fapaneng dihlopheng tsa ASD,16 mme dithuto tse mmalwa tse ikemetseng di tlalehile diphetoho dinthong tsena tsa phetolelo ho arabela kgatellong ya mitochondrial50,51. 52. Ponahalo ya OPA1 le STOML2 ka bobedi e fokoditswe haholo ke kalafo ya 3 mM le 5 mM PPA (Setshwantsho sa 3e, f). OPA1 ke e nngwe ya balaoli ba kgale ba ho kopana ha dijini tsa mitochondrial ka ho sebelisana ka kotloloho le MFN1 le 2 mme e bapala karolo ho ntlafatseng cristae le sebopeho sa dijini tsa mitochondrial53. Karolo e nepahetseng ya STOML2 ho diphetoho tsa dijini tsa mitochondrial e ntse e sa hlaka, empa bopaki bo bontsha hore e bapala karolo ho kopanyeng ha dijini tsa mitochondrial, biogenesis, le mitophagy.
STOML2 e ameha ho bolokeng ho kopana ha ho hema ha mitochondrial le ho thehwa ha di-complex tsa ketane ya ho hema54,55 mme ho bontshitswe hore e fetola ka botebo dibopeho tsa metabolism tsa disele tsa mofetshe56. Diphuputso di bontshitse hore STOML2 e kgothaletsa bokgoni ba membrane ya mitochondrial le biogenesis ka ho sebelisana le BAN le cardiolipin 55, 57, 58. Ho feta moo, diphuputso tse ikemetseng di bontshitse hore tshebedisano pakeng tsa STOML2 le PINK1 e laola mitophagy59,60. Haholo-holo, STOML2 e tlalehilwe e sebelisana ka ho toba le ho tsitsisa MFN2 mme hape e bapala karolo ya bohlokwa ho tsitsiseng di-isoform tse telele tsa OPA1 ka ho thibela protease e ikarabellang bakeng sa ho senyeha ha OPA153,61,62. Phokotso ya tlhahiso ya STOML2 e bonwang dikarabelong tsa PPA e ka etsa hore diprotheine tsena tsa fusion di be kotsing ya ho senyeha ka ditsela tse itshetlehileng ka ubiquitin le proteasome48. Leha karolo e nepahetseng ea STOML2 le OPA1 karabelong e matla ho PPA e sa hlaka, tlhahiso e fokotsehileng ea liphatsa tsena tsa lefutso tsa ho kopana (Setšoantšo sa 3) e ka sitisa botsitso lipakeng tsa ho arohana le ho kopana 'me ea lebisa ho fokotseha ha boholo ba mitochondrial (Setšoantšo sa 3). 1).
Ka lehlakoreng le leng, tlhahiso ea protheine ea OPA1 e ile ea lula e sa fetohe kamora lihora tse 24, ha maemo a mRNA le protheine a MFN1, MFN2 kapa DRP1 a sa ka a fetoha haholo kamora kalafo ea PPA (Setšoantšo sa 3g-i, Setšoantšo sa 4). Sena se ka 'na sa bontša hore ha ho na liphetoho taolong ea lintlha tsena tse amehang ho kopaneng ha mitochondrial le ho arohana ha eona. Leha ho le joalo, hoa bohlokoa ho hlokomela hore e 'ngoe le e 'ngoe ea liphatsa tsena tse 'ne e boetse e laoloa ke liphetoho tsa posttranscriptional (PTMs) tse laolang ts'ebetso ea protheine. OPA1 e na le mefuta e robeli ea splice e meng e arotsoeng ka proteolytically mitochondria ho hlahisa li-isoform tse peli tse fapaneng 63. Tekanyo pakeng tsa li-isoform tse telele le tse khutšoane qetellong e khetholla karolo ea OPA1 ho kopaneng ha mitochondrial le tlhokomelong ea marang-rang a mitochondrial64. Ts'ebetso ea DRP1 e laoloa ke phosphorylation ea calcium/calmodulin-dependent protein kinase II (CaMKII), ha ho senyeha ha DRP1 ho laoloa ke ubiquitination le SUMOylation65. Qetellong, DRP1 le MFN1/2 ka bobeli ke li-GTPase, kahoo ts'ebetso e ka susumetsoa ke sekhahla sa tlhahiso ea GTP ho mitochondria 66. Ka hona, leha polelo ea liprotheine tsena e lula e tsitsitse, sena se kanna sa se bontše ts'ebetso e sa fetoheng ea protheine kapa sebaka sa eona67,68. Ka 'nete, li-repertoire tsa protheine tsa PTM tse teng hangata li sebetsa e le mola oa pele oa tšireletso o ikarabellang bakeng sa ho rarolla likarabo tsa khatello e matla. Ha ho na le khatello e itekanetseng ea metabolism mohlaleng oa rona, ho ka etsahala hore PTM e khothaletse ts'ebetso e eketsehileng ea liprotheine tsa ho kopanya le ho arohana ho khutlisetsa botšepehi ba mitochondrial ka ho lekaneng ntle le ho hloka ts'ebetso e eketsehileng ea liphatsa tsena tsa lefutso boemong ba mRNA kapa protheine.
Ha ho kopanngoa, lintlha tse ka holimo li totobatsa taolo e rarahaneng le e itšetlehileng ka nako ea sebopeho sa mitochondrial le liphephetso tsa ho hlakisa mekhoa ena. Ho ithuta polelo ea liphatsa tsa lefutso, ho hlokahala pele ho khetholla liphatsa tsa lefutso tse itseng tse reriloeng tseleng. Leha ho le joalo, lintlha tsa rona li bontša hore liphatsa tsa lefutso tse tseleng e tšoanang ha li arabele ka tsela e tšoanang khatellong e tšoanang. Ha e le hantle, lithuto tse fetileng li bontšitse hore liphatsa tsa lefutso tse fapaneng tseleng e tšoanang li ka bontša liprofaele tse fapaneng tsa karabelo ea nako30,46. Ho phaella moo, ho na le mekhoa e rarahaneng ea kamora ho ngolisoa e sitisang kamano pakeng tsa ho ngoloa le mosebetsi oa liphatsa tsa lefutso. Lithuto tsa proteomic li ka fana ka temohisiso mabapi le tšusumetso ea li-PTM le mosebetsi oa protheine, empa li boetse li hlahisa liphephetso ho kenyeletsoa mekhoa ea tlhahiso e tlase, likarolelano tse phahameng tsa lets'oao-lerata, le tharollo e fokolang.
Moelelong ona, ho ithuta sebopeho sa mitochondrial ho sebedisa TEM le MEL ho na le bokgoni bo boholo ba ho araba dipotso tsa motheo mabapi le kamano pakeng tsa diphetoho tsa mitochondrial le tshebetso le kamoo sena se amang lefu kateng. Habohlokoa le ho feta, TEM e fana ka mokgwa o tobileng wa ho lekanya sebopeho sa mitochondrial e le ntlha e kopaneng ya ho se sebetse hantle ha mitochondrial le diphetoho51. MEL e boetse e fana ka mokgwa o tobileng wa ho bona ka mahlo a kelello diketsahalo tsa karohano le ho kopana tikolohong ya sele ya mahlakore a mararo, e leng se dumellang ho lekanya ho ntjhafatswa ha mitochondrial ho matla leha ho se na diphetoho polelong ya lefutso33. Mona re totobatsa thuso ya mekgwa ya ho nka ditshwantsho tsa mitochondrial mafung a bobedi a mitochondrial. Mafu ana hangata a tshwantshwa ka kgatello e fokolang ya metabolism e tshwantshwang ka ho ntjhafatswa ho poteletseng ha marangrang a mitochondrial ho ena le tshenyo e matla ya mitochondrial. Leha ho le jwalo, puseletso ya mitochondrial e hlokahalang ho boloka mitosis tlasa kgatello e sa foleng e na le ditlamorao tse tebileng tsa tshebetso. Moelelong wa neuroscience, kutlwisiso e betere ya mekgwa ena ya puseletso e ka fana ka tlhahisoleseding ya bohlokwa mabapi le neuropathology ya pleiotropic e amanang le ho se sebetse hantle ha mitochondrial.
Qetellong, data ea rona e totobatsa bohlokoa ba mekhoa ea ho nka litšoantšo bakeng sa ho utloisisa liphello tsa ts'ebetso ea likamano tse rarahaneng lipakeng tsa tlhahiso ea liphatsa tsa lefutso, liphetoho tsa protheine, le ts'ebetso ea protheine e laolang matla a mitochondrial ea neuronal. Re sebelisitse PPA ho etsa mohlala oa ho se sebetse hantle ha mitochondrial mohlaleng oa sele ea neuronal ho fumana temohisiso mabapi le karolo ea mitochondrial ea ASD. Lisele tsa SH-SY5Y tse phekoloang ka PPA li bontšitse liphetoho mofuteng oa mitochondrial: mitochondria e ile ea e-ba nyane ebile e chitja, 'me cristae li ne li sa hlalosoa hantle ha li bonoa ke TEM. Tlhahlobo ea MEL e bontša hore liphetoho tsena li etsahala ka nako e le 'ngoe le keketseho ea liketsahalo tsa ho arohana le ho kopana ho boloka marang-rang a mitochondrial ho arabela khatello e bobebe ea metabolism. Ho feta moo, PPA e sitisa haholo taolo ea transcriptional ea metabolism ea mitochondrial le homeostasis. Re hlwaile cMYC, NRF1, TFAM, STOML2, le OPA1 e le balaoli ba bohlokoa ba mitochondrial ba sitisitsoeng ke khatello ea PPA 'me ba ka bapala karolo ho buelleng liphetoho tse bakoang ke PPA mofuteng oa mitochondrial le ts'ebetso. Lithuto tsa nakong e tlang lia hlokahala ho hlalosa hamolemo liphetoho tsa nako tse bakoang ke PPA tlhahisong ea liphatsa tsa lefutso le ts'ebetso ea protheine, sebaka, le liphetoho tsa kamora phetolelo. Lintlha tsa rona li totobatsa ho rarahana le ho itšetleha ha mekhoa ea taolo e sebetsanang le karabelo ea khatello ea mitochondrial 'me li bontša thuso ea TEM le mekhoa e meng ea ho nka litšoantšo bakeng sa lithuto tse shebaneng haholo le mechini.
Mohala oa sele oa SH-SY5Y (ECACC, 94030304-1VL) o rekiloe ho Sigma-Aldrich. Lisele tsa SH-SY5Y li holisitsoe ka har'a motsoako oa limatlafatsi oa Dulbecco o fetotsoeng oa Eagle's medium/F-12 (DMEM/F-12) le L-glutamine (SC09411, ScienCell) ka har'a li-flask tsa 25 cm2 tse tlatsitsoeng ka 20% fetal bovine serum (FBS) (10493106, ThermoFisher Scientific) le 1% penicillin-streptomycin (P4333-20ML, Sigma-Aldrich) mochesong oa 37 °C, 5% CO2. Lisele li ile tsa lengoa ka tlase ho 80% ho sebelisoa 0.05% trypsin-EDTA (15400054, ThermoFisher Scientific), tsa kenngoa ka centrifuge ho 300 g 'me tsa penteloa ka bongata ba lisele tse ka bang 7 × 105/ml. Liteko tsohle li entsoe liseleng tse sa arohaneng tsa SH-SY5Y lipakeng tsa liphaseje tsa 19-22. PPA e fanoa e le NaP. Qhibilihisa phofo ea NaP (CAS No. 137-40-6, foromo ea lik'hemik'hale C3H5NaO2, P5436-100G, Sigma-Aldrich) metsing a futhumetseng a MilliQ ho fihlela ho 1 M 'me u boloke mochesong oa 4 °C. Letsatsing la kalafo, hlapolla tharollo ena ka 1 M PPA ho isa ho 3 mM le 5 mM PPA ka har'a serum-free medium (DMEM/F-12 ka L-glutamine). Maqhubu a kalafo bakeng sa liteko tsohle e ne e se PPA (0 mM, taolo), 3 mM, le 5 mM PPA. Liteko li entsoe bonyane ka likopi tse tharo tsa baeloji.
Lisele tsa SH-SY5Y li ile tsa jaloa ka har'a liflask tsa 25 cm5 ka sekhahla sa lisele tse 5.5 × 105/ml 'me tsa holisoa lihora tse 24. Kalafo ea PPA e ile ea eketsoa ka har'a flask pele ho lihora tse 24 tsa ho ikatisa. Bokella li-pellet tsa sele ho latela mekhoa e tloaelehileng ea ho lema lisele tsa mammalian (e hlalositsoeng kaholimo). Hlakola pellet ea sele hape ka 100 µl 2.5% glutaraldehyde, 1 × PBS 'me u e boloke mochesong oa 4 °C ho fihlela e sebetsoa. ​​Lisele tsa SH-SY5Y li ile tsa kenngoa ka setering ka nakoana ho hlakola lisele le ho tlosa 2.5% glutaraldehyde, 1 × tharollo ea PBS. Hlakola sediment hape ka gel ea agarose ea 4% e lokisitsoeng ka metsi a hloekisitsoeng (karolelano ea agarose le bophahamo ba sediment ke 1:1). Likotoana tsa agarose li ile tsa beoa holim'a li-grid holim'a lipoleiti tse bataletseng 'me tsa koaheloa ka 1-hexadecene pele ho hoamisoa ka khatello e phahameng. Lisampole li ile tsa hoamisoa ka 100% acetone e omileng ho -90°C ka lihora tse 24. Mocheso o ile oa phahamisoa ho -80°C 'me tharollo ea 1% osmium tetroxide le 0.1% glutaraldehyde ea eketsoa. ​​Lisampole li ile tsa bolokoa ho -80°C ka lihora tse 24. Kamora sena, mocheso o ile oa eketseha butle-butle ho ea mochesong oa kamore ka matsatsi a 'maloa: ho tloha ho -80 °C ho ea ho -50 °C ka lihora tse 24, ho ea ho -30 °C ka lihora tse 24, ho ea ho -10 °C ka lihora tse 24 'me qetellong mochesong oa kamore.
Kamora ho lokisetsoa cryogenic, disampole di ile tsa tlatswa ka resin mme dikarolo tse tshesane haholo (~100 nm) tsa etswa ho sebediswa Leica Reichert UltracutS ultramicrotome (Leica Microsystems). Dikarolo di ile tsa tshwaetswa ka 2% uranyl acetate le lead citrate. Disampole di ile tsa bonwa ho sebediswa microscope ya FEI Tecnai 20 transmission electron (ThermoFisher (eo pele e neng e le FEI), Eindhoven, The Netherlands) e sebetsang ho 200 kV (Lab6 transmitter) le khamera ya Gatan CCD (Gatan, UK) e nang le sefe ya matla a Tridiem.
Ho kopi e 'ngoe le e 'ngoe ea tekheniki, bonyane litšoantšo tse 24 tsa sele e le 'ngoe li fumanoe, bakeng sa kakaretso ea litšoantšo tse 266. Litšoantšo tsohle li hlahlobiloe ho sebelisoa macro ea Region of Interest (ROI) le macro ea Mitochondria. Macro ea mitochondrial e thehiloe mekhoeng e phatlalalitsoeng17,31,32 'me e lumella ts'ebetso ea sehlopha sa litšoantšo tsa TEM ka mokhoa o ikemetseng Fiji/ImageJ69. Ka bokhutšoanyane: setšoantšo se khelositsoe le ho khelosoa ho sebelisoa ho tlosa bokamorao ba bolo e phutholohang (radius ea 60 pixel) le sefe sa FFT bandpass (ho sebelisoa meeli e holimo le e tlase ea pixel ea 60 le 8 ka ho latellana) le ho hatelloa ha mola o otlolohileng ka mamello ea tataiso ea 5%. Setšoantšo se sebetsitsoeng se thibeloa ka bohona ho sebelisoa algorithm e phahameng ea entropy 'me maske ea binary ea hlahisoa. Libaka tsa litšoantšo tse amanang le li-ROI tse khethiloeng ka letsoho litšoantšong tsa TEM tse tala li ile tsa ntšoa, tsa khetholla mitochondria le ho qhelela ka thoko lera la plasma le libaka tse ling tse fapaneng haholo. Bakeng sa ROI e 'ngoe le e 'ngoe e ntšitsoeng, likaroloana tsa binary tse kholo ho feta li-pixel tse 600 li ile tsa hlahlojoa, 'me sebaka sa likaroloana, perimeter, li-axes tse kholo le tse nyane, bophara ba Feret, chitja, le circularity li ile tsa lekanngoa ho sebelisoa mesebetsi ea tekanyo e hahiloeng ea Fiji/ImageJ. Ho latela Merrill, Flippo, le Strack (2017), sebaka sa 2, karolelano ea likaroloana (karoloana e kholo ho ea ho e nyane), le shape factor (FF) li ile tsa baloa ho tsoa ho data ena, moo FF = perimeter 2/4pi x area. Tlhaloso ea foromo ea parametric e ka fumanoa ho Merrill, Flippo, le Strack (2017). Li-macro tse boletsoeng li fumaneha ho GitHub (sheba Polelo ea ho Fumaneha ha Data). Ka karolelano, likaroloana tse ka bang 5,600 li ile tsa sekasekoa ka kalafo ea PPA, bakeng sa kakaretso ea likaroloana tse ka bang 17,000 (data ha e bontšoe).
Lisele tsa SH-SH5Y li ile tsa beoa ka har'a lijana tsa temo tsa likamore tse 8 (ThermoFisher, #155411) ho lumella ho khomarela bosiu bohle ebe li kenngoa ka TMRE 1:1000 (ThermoFisher, #T669) le Hoechst 33342 1:200 (Sigma-Aldrich, H6024). Ho daea. Litšoantšo li ile tsa fumanoa ho sebelisoa li-laser tsa 405 nm le 561 nm tikolohong ea metsotso e 10, 'me litšoantšo tse tala li ile tsa fumanoa e le li-z-stacks tse nang le li-micrograph tsa litšoantšo tse 10 tse nang le mohato oa az oa 0.2 μm lipakeng tsa liforeimi tsa litšoantšo ka linako tse 12 tse latelang. Litšoantšo li ile tsa bokelloa ho sebelisoa sethala sa Carl Zeiss LSM780 ELYRA PS.1 super-resolution (Carl Zeiss, Oberkochen, Jeremane) ho sebelisoa lense ea LCI Plan Apochromate 100x/1.4 Oil DIC M27. Litšoantšo li hlahlobiloe ho ImageJ ho sebelisoa phaephe e hlalositsoeng pele le plugin ea ImageJ ho lekanya liketsahalo tsa ho kopana le ho arohana, palo e tloaelehileng ea meaho ea mitochondrial, le bophahamo ba mitochondrial bo tloaelehileng ka sele33. Li-macro tsa MEL lia fumaneha ho GitHub (sheba Polelo ea ho Fumaneha ha Lintlha).
Lisele tsa SH-SY5Y li holisitsoe ka lipoleiti tse nang le liliba tse tšeletseng ka bongata ba lisele tse 0.3 × 106/mL lihora tse 24 pele ho kalafo. RNA e ntšoa ho sebelisoa protocol ea Quick-RNA™ Miniprep (ZR R1055, Zymo Research) ka liphetoho tse nyane: eketsa 300 μl ea RNA lysis buffer selibeng ka seng pele u tlosa 'me u lyse sampole ka' ngoe e le mohato oa ho qetela ka 30 μl ea DNase/RNase elution. -metsi a se nang DNase/RNase. Lisampole tsohle li hlahlobiloe bongata le boleng ho sebelisoa NanoDrop ND-1000 UV-Vis Spectrophotometer. Kakaretso ea protheine e tsoang ho lysates ea sele e fumanoe ho sebelisoa 200 μl RIPA lysis buffer, 'me mahloriso a protheine a ile a lekanngoa ho sebelisoa Bradford protein assay70.
Ho kopanngoa ha cDNA ho entsoe ho sebelisoa Tetro™ cDNA Synthesis Kit (BIO-65043, Meridian Bioscience) ho latela litaelo tsa moetsi ka liphetoho tse ling. cDNA e entsoe ka li-reaction tsa 20-μl ho sebelisoa 0.7 ho isa ho 1 μg ea kakaretso ea RNA. Li-primer li khethiloe ho tsoa lipampiring tse hatisitsoeng pele 42, 71, 72, 73, 74, 75, 76, 77, 78 (Tafole ea S1) 'me li-probe tse tsamaeang le tsona li entsoe ho sebelisoa sesebelisoa sa PrimerQuest ho tsoa ho Integrated DNA Technologies. Lijini tsohle tse khahlisang li ile tsa fetoloa hore e be jini ea nuclear B2M. Pontšo ea jini ea STOML2, NRF1, NFE2L2, TFAM, cMYC le OPA1 e lekantsoe ka RT-qPCR. Motswako o moholo o ne o kenyelletsa LUNA Taq polymerase (M3003L, New England Biolabs), di-primer tsa pele le tsa morao tsa 10 μM, cDNA, le metsi a sehlopha sa PCR ho hlahisa bophahamo ba ho qetela ba 10 μL bakeng sa karabelo ka 'ngoe. Tlhaloso ea liphatsa tsa lefutso tsa karohano le karohano (DRP1, MFN1/2) e lekantsoe ho sebelisoa liteko tsa multiplex tsa TaqMan. Luna Universal Probe qPCR Master Mix (M3004S, New England Biolabs) e sebelisitsoe ho latela litaelo tsa moetsi ka liphetoho tse nyane. Motswako o moholo oa multiplex oa RT-qPCR o kenyelletsa 1X LUNA Taq polymerase, di-primer tsa pele le tsa morao tsa 10 μM, probe ea 10 μM, cDNA, le metsi a sehlopha sa PCR, e leng se ileng sa fella ka bophahamo ba ho qetela ba 20 μL bakeng sa karabelo ka 'ngoe. RT-qPCR e entsoe ho sebelisoa Rotor-Gene Q 6-plex (QIAGEN RG—nomoro ea serial: R0618110). Maemo a ho palama baesekele a bontshitswe ho Tafole ya S1. Disampole tsohle tsa cDNA di ile tsa eketswa ka hararo mme moedi o tlwaelehileng o ile wa hlahiswa ho sebediswa letoto la dilutions tse leshome. Disampole tse kantle ho hararo tse nang le ho fapoha ho tloaelehileng ha moedi wa potoloho (Ct) >0.5 di ile tsa tloswa tlhahlobong ho netefatsa hore data e ka ikatisa hape30,72. Polelo ya lefutso e amanang e ile ya balwa ho sebediswa mokgwa wa 2-ΔΔCt79.
Disampole tsa protheine (60 μg) di kopantswe le Laemmli loading buffer ka karolelano ya 2:1 mme tsa sebediswa hodima gel ya protheine e se nang mmala ya 12% (Bio-Rad #1610184). Diprotheine di ile tsa fetisetswa ho membrane ya PVDF (polyvinylidene fluoride) (#170-84156, Bio-Rad) ho sebediswa sistimi ya Trans-Blot Turbo (#170-4155, Bio-Rad). Lera le ile la thijwa mme la kenngwa ka di-antibodies tse loketseng tsa mantlha (OPA1, MFN1, MFN2, le DRP1) (e hlapolotsweng ka 1:1000) ka dihora tse 48, mme la latelwa ke ho kenngwa ka di-antibodies tsa bobedi (1:10,000) ka hora e le 1. Di-membrane di ile tsa nkuwa setshwantsho ho sebediswa Clarity Western ECL Substrate (#170-5061, Bio-Rad) mme tsa rekotwa ho sebediswa sistimi ya Bio-Rad ChemiDoc MP. Mofuta wa ImageLab 6.1 o sebediseditswe tlhahlobo ya Western blot. Jel le blot tsa pele di bontshitswe ho Setšoantšo sa S1. Tlhahisoleseding ya antibody e fanoe ho Tafole ya S2.
Disete tsa data di hlahiswa e le phoso ya karolelano le e tloaelehileng ya karolelano (SEM) ya bonyane disampole tse tharo tse ikemetseng. Disete tsa data di ile tsa lekoa bakeng sa ho ba maemong a tlwaelehileng ho sebediswa teko ya Shapiro-Wilks (ntle le haeba ho boletswe ka tsela e nngwe) pele ho nkuwa kabo ya Gaussian le diphetoho tse lekanang tsa maemo mme tsa tswela pele ka ditshekatsheko. Ntle le ho sekaseka sete ya data ho sebediswa Fisher's MEL LSD (p < 0.05), ANOVA ya tsela e le nngwe (kalafo vs. karolelano ya taolo), le teko ya papiso e ngata ya Dunnett ho fumana bohlokwa (p < 0.05). Boleng ba bohlokwa ba p bo bontshwa kerafong e le *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Ditshekatsheko tsohle tsa dipalopalo le dikerafo di entswe mme tsa hlahiswa ho sebediswa GraphPad Prism 9.4.0.
Li-macro tsa Fiji/ImageJ bakeng sa tlhahlobo ea litšoantšo tsa TEM li fumaneha phatlalatsa ho GitHub: https://github.com/caaja/TEMMitoMacro. Macro ea Mitochondrial Event Locator (MEL) e fumaneha phatlalatsa ho GitHub: https://github.com/rensutheart/MEL-Fiji-Plugin.
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