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I-Propionic acid (PPA) isetshenziselwa ukutadisha indima yokungasebenzi kahle kwe-mitochondrial ezinkingeni zokuthuthukiswa kwe-neurodevelopmental njenge-autism spectrum disorder. I-PPA yaziwa ngokuphazamisa i-mitochondrial biogenesis, i-metabolism, kanye nokujika kwayo. Kodwa-ke, imiphumela ye-PPA ku-mitochondrial dynamics, i-fission kanye ne-fusion isalokhu iyinkinga ngenxa yemvelo yesikhashana eyinkimbinkimbi yalezi zindlela. Lapha, sisebenzisa amasu okulinganisa ahambisanayo ukuze sihlole ukuthi i-PPA ithinta kanjani isakhiwo se-mitochondrial ultrastructure, i-morphology, kanye ne-dynamics kumaseli e-SH-SY5Y afana ne-neuron. I-PPA (5 mM) ibangele ukwehla okukhulu endaweni ye-mitochondrial (p < 0.01), ububanzi be-Feret kanye ne-circumference (p < 0.05), kanye nendawo 2 (p < 0.01). Ukuhlaziywa kwe-mitochondrial event locator kubonise ukwanda okukhulu (p < 0.05) emicimbini ye-fission kanye ne-fusion, ngaleyo ndlela kugcinwa ubuqotho benethiwekhi ye-mitochondrial ngaphansi kwezimo zokucindezeleka. Ngaphezu kwalokho, ukuvezwa kwe-mRNA kwe-cMYC (p < 0.0001), i-NRF1 (p < 0.01), i-TFAM (p < 0.05), i-STOML2 (p < 0.0001) kanye ne-OPA1 (p < 0.05) kuncishiswe kakhulu. 01). Lokhu kubonisa ukuvuselelwa kwesimo se-mitochondrial, i-biogenesis kanye ne-dynamics ukuze kulondolozwe ukusebenza ngaphansi kwezimo zokucindezeleka. Idatha yethu inikeza ukuqonda okusha ngemiphumela ye-PPA ku-mitochondrial dynamics futhi iqokomisa ukusetshenziswa kwamasu okuthwebula izithombe okutadisha izindlela eziyinkimbinkimbi zokulawula ezihilelekile ekuphenduleni kokucindezeleka kwe-mitochondrial.
Ama-Mitochondria angabahlanganyeli ababalulekile emisebenzini eyahlukene yamaseli ngaphandle kwezindima zawo ezijwayelekile ekukhiqizweni kwamandla kanye nokwenziwa kwezinto eziphilayo. I-metabolism ye-Mitochondrial iwumlawuli oyinhloko we-calcium signaling, i-metabolic kanye ne-redox homeostasis, i-inflammatory signaling, ukuguqulwa kwe-epigenetic, ukwanda kwamaseli, ukuhlukanisa kanye nokufa kwamaseli okuhleliwe1. Ikakhulukazi, i-mitochondrial metabolism ibalulekile ekuthuthukisweni kwe-neuronal, ukusinda kanye nokusebenza futhi ihileleke kakhulu ekubonakalisweni okuhlukahlukene kwe-neuropathology2,3,4.
Eminyakeni eyishumi edlule, isimo se-metabolic sivele njengomlawuli oyinhloko we-neurogenesis, ukuhlukanisa, ukuvuthwa kanye ne-plasticity5,6. Muva nje, i-mitochondrial morphology kanye ne-dynamics kube yizingxenye ezibaluleke kakhulu ze-mitosis, inqubo eguquguqukayo egcina ichibi le-mitochondria enempilo ngaphakathi kwamaseli. I-mitochondrial dynamics ilawulwa yizindlela eziyinkimbinkimbi ezincikene kusukela ku-mitochondrial biogenesis kanye ne-bioenergetics kuya ekuqhekekeni kwe-mitochondrial, ukuhlanganiswa, ukuthutha kanye nokususwa7,8. Ukuphazamiseka kwanoma yiziphi zalezi zindlela zokuhlanganisa kuphazamisa ukugcinwa kwamanethiwekhi e-mitochondrial anempilo futhi kunemiphumela ejulile yokusebenza kwe-neurodevelopment9,10. Ngempela, ukungasebenzi kahle kwe-mitochondrial dynamics kubonakala ezinkingeni eziningi zengqondo, ze-neurodegenerative kanye neze-neurodevelopmental, kufaka phakathi i-autism spectrum disorders (ASD)11,12.
I-ASD iyisifo esingavamile sokuthuthukiswa kwemizwa esinokwakheka okuyinkimbinkimbi kwezakhi zofuzo kanye ne-epigenetic. Ufuzo lwe-ASD alunakuphikiswa, kodwa imbangela yamangqamuzana ayisisekelo ayisaqondakali kahle. Ukuqoqa idatha evela kumamodeli angaphambi kokwelashwa, izifundo zezokwelapha, kanye namasethi edatha ama-molecule e-multi-omics kunikeza ubufakazi obandayo bokungasebenzi kahle kwe-mitochondrial ku-ASD13,14. Ngaphambilini senze ukuhlolwa kwe-DNA methylation ebanzi ku-cohort yeziguli ezine-ASD futhi sathola izakhi zofuzo ezixutshwe ngendlela ehlukile ezihlanganiswe ngezindlela ze-mitochondrial metabolic15. Ngemuva kwalokho sabika nge-methylation ehlukile yabalawuli abaphakathi be-mitochondrial biogenesis kanye ne-dynamics, eyayihlotshaniswa nenombolo yekhophi ye-mtDNA ekhuphukile kanye nephrofayili ye-metabolic yomchamo eshintshiwe ku-ASD16. Imininingwane yethu inikeza ubufakazi obandayo bokuthi i-mitochondrial dynamics kanye ne-homeostasis kudlala indima ebalulekile ku-pathophysiology ye-ASD. Ngakho-ke, ukuthuthukisa ukuqonda kwe-mechanistic kobudlelwano phakathi kwe-mitochondrial dynamics, i-morphology, kanye nomsebenzi kuyinjongo eyinhloko yocwaningo oluqhubekayo ngezifo ze-neurological ezibonakaliswa ukungasebenzi kahle kwe-mitochondrial yesibili.
Amasu e-molecular avame ukusetshenziswa ukutadisha indima yezakhi zofuzo ezithile ekuphenduleni kokucindezeleka kwe-mitochondrial. Kodwa-ke, le ndlela ingase ikhawulelwe uhlobo olunezinhlangothi eziningi nolwesikhathi lwezindlela zokulawula i-mitotic. Ngaphezu kwalokho, ukubonakaliswa okuhlukile kwezakhi zofuzo ze-mitochondrial kuyinkomba engaqondile yezinguquko zokusebenza, ikakhulukazi njengoba inani elilinganiselwe lezakhi zofuzo livame ukuhlaziywa. Ngakho-ke, izindlela eziqondile kakhulu zokufunda umsebenzi we-mitochondrial kanye ne-bioenergetics ziye zaphakanyiswa17. Ukwakheka kwe-mitochondrial kuhlobene kakhulu nokuguquguquka kwe-mitochondrial. Ukuma kwe-mitochondrial, ukuxhumana, kanye nesakhiwo kubalulekile ekukhiqizweni kwamandla kanye nokusinda kwe-mitochondrial kanye neseli5,18. Ngaphezu kwalokho, izingxenye ezahlukene ze-mitosis zigxila ekushintsheni kokwakheka kwe-mitochondrial, okungase kusebenze njengeziphetho eziwusizo zokungasebenzi kahle kwe-mitochondrial futhi kuhlinzeke ngesisekelo sezifundo ezilandelayo ze-mechanistic.
Ukwakheka kwe-mitochondrial kungabonwa ngqo kusetshenziswa i-transmission electron microscopy (TEM), okuvumela ukutadisha okuningiliziwe kwesakhiwo se-cellular ultrastructure. I-TEM ibona ngqo ukwakheka, ukuma kanye nesakhiwo se-mitochondrial cristae ekuxazululweni kwe-mitochondria ngayinye, kunokuthembela kuphela ekubhalweni kwezakhi zofuzo, ukubonakaliswa kwephrotheni noma amapharamitha okusebenza kwe-mitochondrial kumaseli17,19,20. Ngaphezu kwalokho, i-TEM yenza kube lula ukutadisha ukusebenzisana phakathi kwe-mitochondria nezinye i-organelles, njenge-endoplasmic reticulum kanye ne-autophagosomes, ezidlala indima ebalulekile emsebenzini we-mitochondrial kanye ne-homeostasis21,22. Ngakho-ke, lokhu kwenza i-TEM ibe yindawo enhle yokuqala yokufunda ukungasebenzi kahle kwe-mitochondrial ngaphambi kokugxila ezindleleni ezithile noma izakhi zofuzo. Njengoba umsebenzi we-mitochondrial uba ngowokubaluleka kakhulu kwi-neuropathology, kunesidingo esicacile sokukwazi ukufunda ngokuqondile nangobuningi ukwakheka kwe-mitochondrial kanye ne-dynamics kumamodeli e-neuronal e-in vitro.
Kulesi sihloko, sihlola i-mitochondrial dynamics kumodeli ye-neuronal yokungasebenzi kahle kwe-mitochondrial ku-autism spectrum disorder. Ngaphambilini sibike nge-differential methylation ye-propionyl-CoA carboxylase beta (PCCB) ku-ASD15, iyunithi encane ye-mitochondrial propionyl-CoA carboxylase enzyme PCC. Ukungasebenzi kahle kwe-PCC kwaziwa ukuthi kubangela ukuqongelela okunobuthi kwe-propionyl derivatives, okuhlanganisa ne-propionic acid (PPA)23,24,25. I-PPA iboniswe ukuthi iphazamisa i-neuronal metabolism futhi ishintsha ukuziphatha emzimbeni futhi iyimodeli yesilwane esisunguliwe yokufunda izindlela zokuthuthukiswa kwe-neurodevelopmental ezihilelekile ku-ASD26,27,28. Ngaphezu kwalokho, i-PPA ibikwe ukuthi iphazamisa amandla e-mitochondrial membrane, i-biogenesis kanye nokuphefumula emzimbeni emzimbeni futhi isetshenziswe kabanzi ukulingisa ukungasebenzi kahle kwe-mitochondrial kuma-neurons29,30. Kodwa-ke, umthelela wokungasebenzi kahle kwe-mitochondrial okubangelwa yi-PPA ku-morphology ye-mitochondrial kanye ne-dynamics usalokhu ungaqondakali kahle.
Lolu cwaningo lusebenzisa amasu okudweba ahambisanayo ukuze kulinganiswe imiphumela ye-PPA ku-morphology ye-mitochondrial, i-dynamics, kanye nomsebenzi kumaseli e-SH-SY5Y. Okokuqala, sakha indlela ye-TEM ukuze sibone izinguquko ku-morphology ye-mitochondrial kanye ne-ultrastructure17,31,32. Njengoba sibheka i-dynamics ye-mitochondria33, siphinde sasebenzisa ukuhlaziywa kwe-mitochondrial event localizer (MEL) ukuze silinganise izinguquko ebhalansi phakathi kwezehlakalo ze-fission kanye ne-fusion, inombolo ye-mitochondrial kanye nevolumu ngaphansi kokucindezeleka kwe-PPA. Okokugcina, sihlole ukuthi i-morphology ye-mitochondrial kanye ne-dynamics kuhlotshaniswa yini nezinguquko ekubonakalisweni kwezakhi zofuzo ezihilelekile ku-biogenesis, i-fission, kanye ne-fusion. Uma sihlanganisa, idatha yethu ibonisa inselele yokucacisa ubunzima bezindlela ezilawula i-mitochondrial dynamics. Siqokomisa ukusetshenziswa kwe-TEM ekutadisheni i-morphology ye-mitochondrial njenge-endpoint elinganisekayo ye-mitosis kumaseli e-SH-SY5Y. Ngaphezu kwalokho, siqokomisa ukuthi idatha ye-TEM inikeza ulwazi olucebile kakhulu uma ihlanganiswa namasu okudweba aphinde abambe izehlakalo eziguquguqukayo ekuphenduleni ukucindezeleka kwe-metabolic. Ukucaciswa okwengeziwe kwezindlela zokulawula ama-molecule ezisekela i-neuronal cell mitosis kunganikeza ukuqonda okubalulekile ngengxenye ye-mitochondrial yesimiso sezinzwa kanye nezifo ze-neurodegenerative.
Ukuze kubangele ukucindezeleka kwe-mitochondrial, amaseli e-SH-SY5Y aphathwa nge-PPA kusetshenziswa i-3 mM kanye ne-5 mM sodium propionate (NaP). Ngaphambi kwe-TEM, amasampula ayelungiswa ngesampula ye-cryogenic kusetshenziswa ukuqandisa okuphezulu kanye nokuqandisa (Isithombe 1a). Sakha ipayipi lokuhlaziya isithombe se-mitochondrial elizenzakalelayo ukuze silinganise amapharamitha ayisishiyagalombili e-morphological emiphakathini ye-mitochondrial kuzo zonke izinhlobo ezintathu zebhayoloji. Sithole ukuthi ukwelashwa kwe-PPA kushintshe kakhulu amapharamitha amane: indawo 2, indawo, umjikelezo, kanye nobubanzi be-Feret (Isithombe 1b–e). Indawo 2 yehle kakhulu kokubili ngokwelashwa kwe-PPA kwe-3 mM kanye ne-5 mM (p = 0.0183 kanye ne-p = 0.002, ngokulandelana) (Isithombe 1b), kuyilapho indawo (p = 0.003), umjikelezo (p = 0.0106) kanye nobubanzi be-Feret konke kwehle kakhulu. Kube nokwehla okukhulu (p = 0.0172) eqenjini lokwelashwa kwe-5 mM uma kuqhathaniswa neqembu lokulawula (Isithombe 1c–e). Ukwehla okukhulu kwendawo kanye nokujikeleza kubonise ukuthi amaseli aphathwe nge-5 mM PPA ayene-mitochondria encane, eyindilinga kakhulu, nokuthi la ma-mitochondria ayengaphakeme kangako kunalawo asemaseli okulawula. Lokhu kuhambisana nokwehla okukhulu kobubanzi be-Feret, ipharamitha ezimele ekhombisa ukwehla kwebanga elikhulu phakathi kwemiphetho yezinhlayiya. Izinguquko kusakhiwo se-cristae zabonwa: i-cristae yaba ncane kakhulu ngaphansi kwethonya lokucindezeleka kwe-PPA (Isithombe 1a, iphaneli B). Kodwa-ke, akuzona zonke izithombe ezibonise ngokucacile isakhiwo se-cristae, ngakho-ke ukuhlaziywa kobuningi kwalezi zinguquko akwenziwanga. Le datha ye-TEM ingabonisa izimo ezintathu ezingaba khona: (1) I-PPA ithuthukisa ukuhlukana noma ivimbela ukuhlangana, okubangela ukuthi i-mitochondria ekhona inciphe ngobukhulu; (2) i-biogenesis ethuthukisiwe idala i-mitochondria entsha, encane noma (3) ibangela zombili izindlela ngesikhathi esisodwa. Nakuba lezi zimo zingenakuhlukaniswa yi-TEM, izinguquko ezibalulekile zesimo somzimba zibonisa izinguquko ku-homeostasis ye-mitochondrial kanye ne-dynamics ngaphansi kokucindezeleka kwe-PPA. Ngemva kwalokho sihlole amapharamitha engeziwe ukuze sichaze kabanzi la ma-dynamics kanye nezindlela ezingaba khona ezingaphansi kwawo.
I-Propionic acid (PPA) ivuselela isimo se-mitochondrial. (a) Izithombe ze-transmission electron microscopy (TEM) ezibonisa ukuthi usayizi we-mitochondrial uyancipha futhi i-mitochondria iba ncane futhi izungeza kakhulu ngokwelashwa kwe-PPA okwandayo; 0 mM (engelashwanga), 3 mM kanye no-5 mM, ngokulandelana. Imicibisholo ebomvu ikhombisa i-mitochondria. (b–e) Amaseli e-SH-SY5Y aphathwe nge-PPA amahora angama-24 alungiselelwe i-TEM futhi imiphumela yahlaziywa kusetshenziswa i-Fiji/ImageJ. Amane kumapharamitha ayisishiyagalombili abonise umehluko omkhulu phakathi kwamaseli okulawula (angelashwanga, 0 mM PPA) kanye namaseli aphathwe (3 mM kanye no-5 mM PPA). (b) Isifunda 2, (c) Indawo, (d) I-Perimeter, (e) Ububanzi be-Feret. Ukuhlaziywa kwendlela eyodwa kokuhlukahluka (ukulawula vs. ukwelashwa) kanye nokuhlolwa kokuqhathanisa okuningi kukaDunnett kwasetshenziswa ukuthola umehluko obalulekile (p < 0.05). Amaphuzu edatha amelela inani elimaphakathi le-mitochondrial leseli ngalinye, kanti amabha amaphutha amelela isilinganiso ± SEM. Idatha ebonisiwe imelela u-n = 3, okungenani amaseli angama-24 ngokuphindaphindwa ngakunye; kuhlaziywe izithombe ezingu-266; * ikhombisa u-p < 0.05, ** ikhombisa u-p < 0.01.
Ukuze sichaze kabanzi ukuthi i-mitochondrial dynamics isabela kanjani ku-PPA, sifake i-mitochondria i-tetramethylrhodamine ethyl ester (TMRE) futhi sasebenzisa i-time-lapse microscopy kanye nokuhlaziywa kwe-MEL ukuze sithole futhi silinganise i-mitochondria ngemva kwamahora angu-24 ku-3 kanye no-5 mM PPA. Ukwelashwa kwezehlakalo zokuhlukana kanye nokuhlangana. (Isithombe 2a). Ngemva kokuhlaziywa kwe-MEL, i-mitochondria yahlaziywa kabanzi ukuze kulinganiswe inani lezakhiwo ze-mitochondrial kanye nevolumu yazo ephakathi. Sibone ukwanda okuncane kodwa okuphawulekayo kwenani lezehlakalo zokuqhekeka ezenzeka ku-3 mM [4.9 ± 0.3 (p < 0.05)] uma kuqhathaniswa nokuqhekeka [5.6 ± 0.3 (p < 0.05) )] kanye nokuhlanganiswa [5.4 ± 0.5 (p < 0.05)] kanye nokuhlanganiswa [5.4 ± 0.5 (p < 0.05)] 0.05)] <0.05)] izehlakalo zanda kakhulu ku-5 mM uma kuqhathaniswa nokulawula (Isithombe 3b). Inani le-mitochondria landa kakhulu kuzo zombili izigaba 3 [32.6 ± 2.1 (p < 0.05)] kanye no-5 mM [34.1 ± 2.2 (p < 0.05)] (Isithombe 3c), kuyilapho ivolumu evamile yesakhiwo ngasinye se-mitochondrial ihlale ingashintshi (Isithombe 3c). 3d). Uma sibheka konke lokhu, lokhu kusikisela ukuthi ukuvuselelwa kwe-mitochondrial dynamics kusebenza njengempendulo yokubuyisela egcina ngempumelelo ubuqotho benethiwekhi ye-mitochondrial. Ukwanda kwenani lezehlakalo zokuhlukana ku-3 mM PPA kusikisela ukuthi ukwanda kwenani le-mitochondrial kungenxa yokuhlukana kwe-mitochondrial, kodwa uma sibheka ukuthi ivolumu ye-mitochondrial emaphakathi ihlala ingashintshi, i-biogenesis ayinakushiywa ngaphandle njengempendulo eyengeziwe yokubuyisela. Kodwa-ke, le datha ihambisana nezakhiwo ezincane, eziyindilinga ze-mitochondrial ezibonwe yi-TEM futhi ikhombisa nezinguquko ezibalulekile ku-mitochondrial dynamics ezibangelwa yi-PPA.
I-Propionic acid (PPA) ibangela ukuvuselelwa kabusha kwe-mitochondrial okunamandla ukuze kulondolozwe ubuqotho benethiwekhi. Amaseli e-SH-SY5Y akhuliswa, aphathwa nge-3 kanye ne-5 mM PPA amahora angama-24 futhi agcotshwa nge-TMRE kanye ne-Hoechst 33342 kulandelwa ukuhlaziywa kwe-MEL. (a) Izithombe ze-microscopy ezimele isikhathi ezibonisa umbala kanye nokubikezelwa kokuqina okuphezulu okuhlanganisiwe ngesikhathi sesi-2 (t2) sesimo ngasinye. Izindawo ezikhethiwe ezikhonjiswe esithombeni ngasinye se-binary ziyathuthukiswa futhi zikhonjiswa ku-3D ngezikhathi ezintathu ezahlukene (t1-t3) ukuze kuboniswe ukuguquguquka ngokuhamba kwesikhathi; imicimbi yokuhlangana igqanyiswa ngombala oluhlaza okotshani; imicimbi yokuhlukanisa igqanyiswa ngombala oluhlaza okotshani. Ikhonjiswa ngombala obomvu. (b) Inani elimaphakathi lemicimbi eguquguqukayo ngesimo ngasinye. (c) Inani elimaphakathi lezakhiwo ze-mitochondrial ngeseli ngalinye. (d) Ivolumu emaphakathi (µm3) yesakhiwo ngasinye se-mitochondrial ngeseli ngalinye. Idatha ekhonjisiwe imele amaseli angu-n = 15 ngeqembu ngalinye lokwelashwa. Amabha amaphutha abonisiwe amele isilinganiso ± SEM, ibha yesikali = 10 μm, * p < 0.05.
I-Propionic acid (PPA) ibangela ukucindezelwa kokubhalwa kwezakhi zofuzo ezihlotshaniswa ne-mitochondrial dynamics. Amaseli e-SH-SY5Y aphathwe nge-3 kanye ne-5 mM PPA amahora angama-24. Ukulinganiswa kwezakhi zofuzo okuhlobene kwenziwe kusetshenziswa i-RT-qPCR futhi kwenziwa kwaba yi-B2M evamile. Izakhi zofuzo ze-Mitochondrial biogenesis (a) cMYC, (b) TFAM, (c) NRF1 kanye (d) NFE2L2. Izakhi zofuzo ze-Mitochondrial fusion kanye ne-fission (e) STOML2, (f) OPA1, (g) MFN1, (h) MFN2 kanye (i) DRP1. Umehluko omkhulu (p < 0.05) uhlolwe kusetshenziswa i-ANOVA yendlela eyodwa (ukulawula vs. ukwelashwa) kanye nokuhlolwa kokuqhathanisa okuningi kukaDunnett: * kubonisa u-p < 0.05, ** kubonisa u-p < 0.01, kanye no-*** kubonisa u-p < 0.0001. Amabha amelela ukubonakaliswa okuphakathi ± SEM. Idatha ebonisiwe imele i-n = 3 (STOML2, OPA1, TFAM), n = 4 (cMYC, NRF1, NFE2L2), kanye ne-n = 5 (MFN1, MFN2, DRP1) amakhophi ebhayoloji.
Idatha evela ekuhlaziyweni kwe-TEM kanye ne-MEL ndawonye ikhombisa ukuthi i-PPA ishintsha isimo se-mitochondrial kanye ne-dynamics. Kodwa-ke, lawa masu okuthwebula izithombe awanikezi ukuqonda ngezindlela eziyisisekelo eziqhuba lezi zinqubo. Ngakho-ke sihlole ukubonakaliswa kwe-mRNA kwabalawuli abayisishiyagalolunye ababalulekile be-mitochondrial dynamics, i-biogenesis, kanye ne-mitosis ekuphenduleni ukwelashwa kwe-PPA. Silinganise i-cell myeloma oncogene (cMYC), i-nuclear respiratory factor (NRF1), i-mitochondrial transcription factor 1 (TFAM), i-NFE2-like transcription factor BZIP (NFE2L2), i-gastrin-like protein 2 (STOML2), i-optic nerve atrophy 1 (OPA1), i-Mitofusin 1 (MFN1), i-Mitofusin 2 (MFN2) kanye ne-dynamin-related protein 1 (DRP1) ngemuva kwamahora angama-24 okwelashwa nge-3 mM kanye ne-5 mM PPA. Sibone i-3 mM (p = 0.0053, p = 0.0415 kanye ne-p < 0.0001, ngokulandelana) kanye ne-5 mM (p = 0.0031, p = 0.0233, p < 0.0001) ukwelashwa kwe-PPA. (Isithombe 3a–c). Ukwehla kokubonakaliswa kwe-mRNA kwakuncike kumthamo: ukubonakaliswa kwe-cMYC, i-NRF1 kanye ne-TFAM kwehle ngezikhathi ezingu-5.7, 2.6 kanye no-1.9 ku-3 mM, ngokulandelana, kanye nangezikhathi ezingu-11.2, 3 kanye no-2.2 ku-5 mM. Ngokuphambene nalokho, i-central redox biogenesis gene NFE2L2 ayizange ishintshwe kunoma yikuphi ukuhlushwa kwe-PPA, yize kwabonakala umkhuba ofanayo oncike kumthamo wokubonakaliswa okunciphile (Isithombe 3d).
Siphinde sahlola ukuvezwa kwezakhi zofuzo zakudala ezihilelekile ekulawulweni kokuhlukana kanye nokuhlangana. I-STOML2 kucatshangwa ukuthi ihilelekile ekuhlanganisweni, ekuhlanganisweni kwezakhi zofuzo kanye nasekwakheni kwezakhi zofuzo, futhi ukuvezwa kwayo kwehliswe kakhulu (p < 0.0001) ngo-3 mM (ushintsho oluphindwe kabili) kanye no-5 mM (ushintsho oluphindwe kabili) i-PPA (Isithombe 1). 3d). Ngokufanayo, ukubonakaliswa kwezakhi zofuzo ze-OPA1 fusion kwehliswe ku-3 mM (ushintsho oluphindwe kabili) kanye no-5 mM (ushintsho oluphindwe kabili) i-PPA (p = 0.006 kanye no-p = 0.0024, ngokulandelana) (Isithombe 3f). Kodwa-ke, asitholanga umehluko omkhulu ekubonakalisweni kwezakhi zofuzo ze-fusion i-MFN1, i-MFN2 noma i-fission gene DRP1 ngaphansi kokucindezeleka kwe-PPA kwamahora angama-24 (Isithombe 3g–i). Ngaphezu kwalokho, sithole ukuthi amazinga amaprotheni amane e-fusion kanye ne-fission (OPA1, MFN1, MFN2 kanye ne-DRP1) awazange ashintshe ngaphansi kwezimo ezifanayo (Isithombe 4a–d). Kubalulekile ukuqaphela ukuthi le datha ibonisa iphuzu elilodwa ngesikhathi futhi ingase ingabonakali izinguquko ekubonakalisweni kwamaprotheni noma amazinga omsebenzi ngesikhathi sokuqala kokucindezeleka kwe-PPA. Kodwa-ke, ukwehla okukhulu ekubonakalisweni kwe-cMYC, NRF1, TFAM, STOML2, kanye ne-OPA1 kubonisa ukuphazamiseka okukhulu kokubhalwa kwe-metabolism ye-mitochondrial, i-biogenesis, kanye ne-dynamics. Ngaphezu kwalokho, le datha igcizelela ukusetshenziswa kwamasu okuthwebula izithombe ukuze kufundwe ngqo izinguquko zesimo sokuphela komsebenzi we-mitochondrial.
Amazinga e-fusion kanye ne-fission factor protein awazange ashintshe ngemva kokwelashwa kwe-propionic acid (PPA). Amaseli e-SH-SY5Y aphathwe nge-3 kanye ne-5 mM PPA amahora angama-24. Amazinga e-protein alinganiswa ngokuhlaziywa kwe-Western blot, futhi amazinga okuvezwa alungiswa abe yi-total protein. Ukuvezwa kwe-protein okumaphakathi kanye nama-Western blots amele i-target kanye ne-total protein kuyaboniswa. a – OPA1, b – MFN1, c – MFN2, d – DRP1. Amabha amele i-mean ± SEM, kanti idatha ebonisiwe imele i-n = 3 biological replicates. Ukuqhathanisa okuningi (p < 0.05) kwenziwa kusetshenziswa ukuhlaziywa kwe-one-way of variance kanye nokuhlolwa kukaDunnett. Ijeli yokuqala kanye ne-blot kuboniswe ku-Figure S1.
Ukungasebenzi kahle kwe-mitochondrial kuhlotshaniswa nezifo eziningi zesistimu kusukela ezifweni ze-metabolic, zenhliziyo nezemisipha kuya ezifweni ze-neurological1,10. Izifo eziningi ze-neurodegenerative kanye neze-neurodegenerative zihlotshaniswa nokungasebenzi kahle kwe-mitochondrial, okugqamisa ukubaluleka kwalezi zitho zomzimba kuyo yonke impilo yobuchopho. Lezi zifo zifaka phakathi isifo sikaParkinson, isifo sika-Alzheimer kanye ne-ASD3,4,18. Kodwa-ke, ukufinyelela ezicutshini zobuchopho ukuze kufundwe lezi zifo kunzima, ikakhulukazi ezingeni lobuchwepheshe, okwenza izinhlelo zemodeli yeselula zibe yindlela edingekayo. Kulesi sifundo, sisebenzisa uhlelo lwemodeli yeselula olusebenzisa amaseli e-SH-SY5Y aphathwe yi-PPA ukuze siphinde sichaze ukungasebenzi kahle kwe-mitochondrial okubonwa ezifweni ze-neuronal, ikakhulukazi izifo ze-autism spectrum. Ukusebenzisa lolu hlobo lwemodeli ye-PPA ukuze kufundwe ukuguquguquka kwe-mitochondrial kuma-neurons kunganikeza ukuqonda ngemvelaphi ye-ASD.
Sihlole ukuthi kungenzeka yini ukusebenzisa i-TEM ukubuka izinguquko ekubumbeni kwe-mitochondrial. Kubalulekile ukuqaphela ukuthi i-TEM kumele isetshenziswe kahle ukuze ikhuphule ukusebenza kwayo. Ukulungiswa kwama-cryo-specimens kuvumela ukulondolozwa okungcono kwezakhiwo ze-neuronal ngokulungisa izingxenye zamaseli ngasikhathi sinye nokunciphisa ukwakheka kwezinto zobuciko34. Ngokuhambisana nalokhu, sibonile ukuthi amaseli e-SH-SY5Y afana ne-neuron ayenama-organelle angaphansi kweseli aphelele kanye ne-mitochondria ende (Isithombe 1a). Lokhu kugqamisa ukusetshenziswa kwamasu okulungiselela i-cryogenic okutadisha ukubumbeka kwe-mitochondrial kumamodeli weseli le-neuronal. Nakuba izilinganiso zobuningi zibalulekile ekuhlaziyweni okuqondile kwedatha ye-TEM, akukabikho ukuvumelana ngokuthi yiziphi izilinganiso ezithile okufanele zilinganiswe ukuqinisekisa izinguquko ze-mitochondrial morphological. Ngokusekelwe enanini elikhulu lezifundo ezihlole ukubumbeka kwe-mitochondrial morphology17,31,32 ngobuningi, sakha ipayipi lokuhlaziya isithombe se-mitochondrial elizenzakalelayo elilinganisa izilinganiso eziyisishiyagalombili ze-morphological, okungukuthi: indawo, indawo2, isilinganiso sesici, umjikelezo, ukujikeleza, izinga, ububanzi be-Feret. kanye nokuzungeza.
Phakathi kwazo, i-PPA inciphise kakhulu indawo 2, indawo, umjikelezo, kanye nobubanzi be-Feret (Isithombe 1b–e). Lokhu kubonise ukuthi i-mitochondria yaba ncane futhi yaba yindilinga, okuhambisana nezifundo zangaphambilini ezibonisa ukwehla kwendawo ye-mitochondrial ngemuva kwamahora angu-72 okucindezeleka kwe-mitochondrial okubangelwa yi-PPA30. Lezi zici ze-morphological zingase zibonise ukuhlukana kwe-mitochondrial, inqubo edingekayo yokususa izingxenye ezonakele kunethiwekhi ye-mitochondrial ukukhuthaza ukuwohloka kwazo nge-mitophagy35,36,37. Ngakolunye uhlangothi, ukwehla kobukhulu be-mitochondrial obujwayelekile kungahlotshaniswa nokwanda kwe-biogenesis, okuholela ekwakhekeni kwe-mitochondria encane esafufusa. Ukwanda kokuhlukana noma i-biogenesis kumelela impendulo yokubuyisela ukugcina i-mitosis ngokumelene nokucindezeleka kwe-mitochondrial. Kodwa-ke, ukukhula okunciphile kwe-mitochondrial, ukuhlanganiswa okuphazamisekile, noma ezinye izimo akunakukhishwa ngaphandle.
Nakuba izithombe ezinesinqumo esiphezulu ezidalwe yi-TEM zivumela ukunqunywa kwezici zesimo somzimba ezingeni le-mitochondria ngayinye, le ndlela ikhiqiza izithombe ezinezilinganiso ezimbili ngesikhathi esisodwa. Ukuze sifunde izimpendulo eziguquguqukayo ekucindezelekeni kwe-metabolic, sidwebe i-mitochondria nge-TMRE futhi sasebenzisa i-time-lapse microscopy enokuhlaziywa kwe-MEL, okuvumela ukubonakala kwe-3D okuphezulu kwezinguquko kunethiwekhi ye-mitochondrial ngokuhamba kwesikhathi33,38. Sibone izinguquko ezicashile kodwa ezibalulekile ku-mitochondrial dynamics ngaphansi kokucindezeleka kwe-PPA (Isithombe 2). Ku-3 mM, inani lemicimbi yokuqhekeka landa kakhulu, kuyilapho imicimbi yokuqhekeka yahlala ifana naleyo ekulawulweni. Ukwanda kwenani lemicimbi yokuqhekeka kanye neyokuhlanganiswa kwabonwa ku-5 mM PPA, kodwa lezi zinguquko zazicishe zilingane, okuphakamisa ukuthi i-fission kanye ne-fusion kinetics zifinyelela ukulingana ekugxilweni okuphezulu (Isithombe 2b). Ivolumu ye-mitochondrial emaphakathi ayizange ishintshe kokubili ku-3 kanye no-5 mM PPA, okubonisa ukuthi ubuqotho benethiwekhi ye-mitochondrial bugcinwe (Isithombe 2d). Lokhu kubonisa ikhono lamanethiwekhi e-mitochondrial ashukumisayo lokuphendula ekucindezelekeni okuncane kwe-metabolic ukuze kugcinwe ngempumelelo i-homeostasis ngaphandle kokubangela ukuhlukana kwenethiwekhi. Ku-3 mM PPA, ukwanda kokuhlukana kwanele ukukhuthaza ukushintshela ekulinganeni okusha, kodwa kudingeka ukulungiswa kabusha kwe-kinetic okujulile ekuphenduleni ukucindezeleka okubangelwa ukugcwala okuphezulu kwe-PPA.
Inani le-mitochondria landa kokubili amazinga okucindezeleka kwe-PPA, kodwa ivolumu ye-mitochondrial ejwayelekile ayizange ishintshe kakhulu (Isithombe 2c). Lokhu kungase kube ngenxa yokwanda kwe-biogenesis noma ukwanda kokuhlukana; nokho, uma kungekho ukwehla okukhulu kwevolumu ye-mitochondrial ephakathi, kungenzeka kakhulu ukuthi i-biosynthesis iyanda. Kodwa-ke, idatha eku-Figure 2 isekela ukuba khona kwezindlela ezimbili zokubuyisela: ukwanda kwenani lezehlakalo zokuhlukana, okuhambisana nokwenyuka kokuhlukana kwe-mitochondrial, kanye nokwanda kwenani lezehlakalo, okuhambisana ne-mitochondrial biogenesis. Ekugcineni, isinxephezelo esinamandla sokucindezeleka okuncane singase sibe nezinqubo ezihambisanayo ezibandakanya ukuhlukana, ukuhlanganiswa, i-biogenesis, kanye ne-mitophagy. Nakuba abalobi bangaphambilini bebonise ukuthi i-PPA ithuthukisa i-mitosis30,39 kanye ne-mitophagy29, sinikeza ubufakazi bokuguqulwa kwe-mitochondrial fission kanye ne-fusion dynamics ekuphenduleni i-PPA. Le datha iqinisekisa izinguquko zesimo ezibonwe yi-TEM futhi inikeza ukuqonda okwengeziwe ngezindlela ezihlotshaniswa nokungasebenzi kahle kwe-mitochondrial okubangelwa yi-PPA.
Ngenxa yokuthi ukuhlaziywa kwe-TEM noma kwe-MEL akuhlinzekanga ubufakazi obuqondile bezindlela zokulawula izakhi zofuzo ezisekela izinguquko zesimo somzimba ezibonwe, sihlole ukubonakaliswa kwe-RNA kwezakhi zofuzo ezihilelekile ekusetshenzisweni kwe-mitochondrial, i-biogenesis, kanye ne-dynamics. I-cMYC proto-oncogene iyisici sokubhala esihilelekile ekulawulweni kwe-mitochondria, i-glycolysis, i-amino acid kanye ne-fatty acid metabolism40. Ngaphezu kwalokho, i-cMYC yaziwa ngokulawula ukubonakaliswa kwezakhi zofuzo ze-mitochondrial ezingaba ngu-600 ezihilelekile ekubhalweni kwe-mitochondrial, ekuhumusheni, nasekuhlanganisweni okuyinkimbinkimbi, okuhlanganisa i-NRF1 kanye ne-TFAM41. I-NRF1 kanye ne-TFAM zingabalawuli ababili abaphakathi be-mitosis, abasebenza ngaphansi kwe-PGC-1α ukuze kusebenze ukuphindaphindwa kwe-mtDNA. Le ndlela ivuselelwa yi-cAMP kanye ne-AMPK signaling futhi ibucayi ekusetshenzisweni kwamandla kanye nokucindezeleka kwe-metabolic. Siphinde sahlola i-NFE2L2, umlawuli we-redox we-mitochondrial biogenesis, ukuze sinqume ukuthi imiphumela ye-PPA ingase iqondiswe ukucindezeleka kwe-oxidative.
Nakuba ukubonakaliswa kwe-NFE2L2 kungashintshile, sithole ukwehla okuqhubekayo okuncike kumthamo ekubonakalisweni kwe-cMYC, i-NRF1 kanye ne-TFAM ngemuva kwamahora angama-24 okwelashwa nge-3 mM kanye ne-5 mM PPA (Isithombe 3a–c). Ukwehla kokubonakaliswa kwe-cMYC kuye kwabikwa ngaphambilini njengempendulo ekucindezelekeni kwe-mitochondrial42, futhi ngokuphambene nalokho, ukwehla kokubonakaliswa kwe-cMYC kungabangela ukungasebenzi kahle kwe-mitochondrial ngokuguqula i-metabolism ye-mitochondrial, ukuxhumana kwenethiwekhi, kanye ne-membrane polarization43. Ngokuthakazelisayo, i-cMYC nayo ihilelekile ekulawulweni kokuqothuka kwe-mitochondrial kanye ne-fusion42,43 futhi yaziwa ngokwandisa i-DRP1 phosphorylation kanye nendawo ye-mitochondrial ngesikhathi sokuhlukaniswa kweseli44, kanye nokulungisa kabusha i-mitochondrial morphological kumaseli e-neuronal stem45. Ngempela, ama-fibroblast angenayo i-cMYC abonisa usayizi we-mitochondrial onciphile, okuhambisana nezinguquko ezibangelwa ukucindezeleka kwe-PPA43. Le datha ibonisa ubudlelwano obuthakazelisayo kodwa obungakacaci phakathi kwe-cMYC kanye ne-mitochondrial dynamics, okunikeza inhloso ethakazelisayo yezifundo zesikhathi esizayo zokuguqulwa kokuvuselelwa okubangelwa ukucindezeleka kwe-PPA.
Ukwehla kwe-NRF1 kanye ne-TFAM kuhambisana nendima ye-cMYC njenge-activator ebalulekile yokubhala. Le datha ihambisana nezifundo zangaphambilini kumaseli omdlavuza wamathumbu abantu ezibonisa ukuthi i-PPA yehlise ukubonakaliswa kwe-NRF1 mRNA emahoreni angama-22, okwakuhlotshaniswa nokuncipha kwe-ATP kanye nokwanda kwe-ROS46. Laba babhali babike nokuthi ukubonakaliswa kwe-TFAM kwanda emahoreni angama-8.5 kodwa kwabuyela emazingeni okuqala emahoreni angama-22. Ngokuphambene nalokho, uKim et al. (2019) babonise ukuthi ukubonakaliswa kwe-TFAM mRNA kwehle kakhulu ngemva kwamahora ama-4 okucindezeleka kwe-PPA kumaseli e-SH-SY5Y; nokho, ngemva kwamahora angama-72, ukubonakaliswa kwephrotheni ye-TFAM kwanda kakhulu futhi inombolo yekhophi ye-mtDNA yanda kakhulu. Ngakho-ke, ukwehla kwenani lezakhi zofuzo ze-mitochondrial biogenesis esizibonile ngemva kwamahora angama-24 akukhiphi ngaphandle ithuba lokuthi ukwanda kwenani le-mitochondria kuhlotshaniswa nokusebenza kwe-biogenesis ezikhathini zangaphambilini. Izifundo zangaphambilini zikhombisile ukuthi i-PPA ikhulisa kakhulu i-PGC-1α mRNA kanye neprotheyini kumaseli e-SH-SY5Y emahoreni ama-4 nemizuzu engama-30, kuyilapho i-propionic acid ithuthukisa i-mitochondrial biogenesis kuma-hepatocytes ethole nge-PGC-1α emahoreni ayi-12 nemizuzu engama-39. Ngokuthakazelisayo, i-PGC-1α ayiyona nje kuphela isilawuli esiqondile se-transcriptional se-NRF1 kanye ne-TFAM, kodwa futhi iboniswe ukuthi ilawula umsebenzi we-MFN2 kanye ne-DRP1 ngokulawula ukuhlukana kanye nokuhlangana47. Uma kuhlanganiswa, lokhu kugqamisa ukuhlangana okuseduze kwezindlela ezilawula izimpendulo ze-mitochondrial compensatory ezibangelwa yi-PPA. Ngaphezu kwalokho, idatha yethu ibonisa ukungasebenzi kahle kokulawulwa kwe-transcriptional kwe-biogenesis kanye ne-metabolism ngaphansi kokucindezeleka kwe-PPA.
Izakhi zofuzo ze-STOML2, OPA1, MFN1, MFN2 kanye ne-DRP1 ziphakathi kwabalawuli abaphakathi bokuhlukana kwe-mitochondrial, ukuhlangana kanye ne-dynamics37,48,49. Kunezinye izakhi zofuzo eziningi ezihilelekile ku-mitochondrial dynamics, nokho, i-STOML2, OPA1 kanye ne-MFN2 zitholakale ngaphambilini zine-methylated ehlukile kuma-cohorts e-ASD,16 futhi izifundo eziningana ezizimele zibike izinguquko kulezi zici zokubhala ukuze ziphendule ekucindezelekeni kwe-mitochondrial50,51.52. Ukuvezwa kokubili kwe-OPA1 kanye ne-STOML2 kwehliswe kakhulu ngokwelashwa kwe-3 mM kanye ne-5 mM PPA (Isithombe 3e, f). I-OPA1 ingenye yabalawuli be-classical bokuhlanganiswa kwe-mitochondrial ngokusebenzisana okuqondile ne-MFN1 kanye no-2 futhi idlala indima ekulungisweni kwe-cristae kanye ne-mitochondrial morphology53. Indima eqondile ye-STOML2 ku-mitochondrial dynamics ayikacaci, kodwa ubufakazi busikisela ukuthi idlala indima ekuhlanganisweni kwe-mitochondrial, i-biogenesis, kanye ne-mitophagy.
I-STOML2 ihilelekile ekugcineni ukuhlangana kokuphefumula kwe-mitochondrial kanye nokwakhiwa kwama-complex e-respiratory chain54,55 futhi iboniswe ukuthi ishintsha kakhulu izici ze-metabolic zamaseli omdlavuza56. Izifundo zibonise ukuthi i-STOML2 ikhuthaza amandla e-mitochondrial membrane kanye ne-biogenesis ngokusebenzisana ne-BAN kanye ne-cardiolipin 55, 57, 58. Ngaphezu kwalokho, izifundo ezizimele zibonise ukuthi ukusebenzisana phakathi kwe-STOML2 ne-PINK1 kulawula i-mitophagy59,60. Okuphawulekayo ukuthi i-STOML2 ibikwe ukuthi isebenzisana ngqo futhi izinzise i-MFN2 futhi idlala indima ebalulekile ekuzinziseni ama-isoform amade e-OPA1 ngokuvimbela i-protease ebangela ukubola kwe-OPA153,61,62. Ukuncipha kokubonakaliswa kwe-STOML2 okubonwe ekuphenduleni kwe-PPA kungenza lawa maprotheni e-fusion abe sengozini enkulu yokubola ngezindlela ezincike ku-ubiquitin kanye ne-proteasome48. Nakuba indima eqondile ye-STOML2 kanye ne-OPA1 ekuphenduleni okuguquguqukayo ku-PPA ingacacile, ukwehla kokubonakaliswa kwalezi zakhi zofuzo zokuhlanganiswa (Isithombe 3) kungaphazamisa ibhalansi phakathi kokuhlukana nokuhlanganiswa futhi kuholele ekunciphiseni usayizi we-mitochondrial (Isithombe 3). 1).
Ngakolunye uhlangothi, ukubonakaliswa kwephrotheni ye-OPA1 kwahlala kungashintshile ngemva kwamahora angama-24, kuyilapho amazinga e-mRNA kanye namaprotheni e-MFN1, MFN2 noma i-DRP1 engashintshanga kakhulu ngemva kokwelashwa kwe-PPA (Isithombe 3g-i, Isithombe 4). Lokhu kungabonisa ukuthi azikho izinguquko ekulawulweni kwalezi zici ezihilelekile ekuhlanganisweni nasekuqhekekeni kwe-mitochondrial. Kodwa-ke, kubalulekile ukuqaphela ukuthi ngayinye yalezi zakhi zofuzo ezine ilawulwa futhi ukuguqulwa kwe-posttranscriptional (PTMs) okulawula umsebenzi wephrotheni. I-OPA1 inezinhlobo eziyisishiyagalombili ze-splice ezihlukile eziqhekeka nge-proteolytically ku-mitochondria ukuze kukhiqizwe ama-isoform amabili ahlukene 63. Ibhalansi phakathi kwama-isoform amade namafushane ekugcineni inquma indima ye-OPA1 ekuhlanganisweni kwe-mitochondrial kanye nokugcinwa kwenethiwekhi ye-mitochondrial64. Umsebenzi we-DRP1 ulawulwa yi-calcium/calmodulin-dependent protein kinase II (CaMKII) phosphorylation, kuyilapho ukuwohloka kwe-DRP1 kulawulwa yi-ubiquitination kanye ne-SUMOylation65. Okokugcina, kokubili i-DRP1 kanye ne-MFN1/2 ziyi-GTPases, ngakho-ke umsebenzi ungathonywa izinga lokukhiqizwa kwe-GTP ku-mitochondria 66. Ngakho-ke, nakuba ukubonakaliswa kwalawa maprotheni kuhlala kungaguquki, lokhu kungase kungabonakali umsebenzi weprotheyini ongashintshi noma indawo67,68. Ngempela, ama-repertoire amaprotheni e-PTM akhona avame ukusebenza njengomugqa wokuqala wokuzivikela obhekene nokuxazulula izimpendulo zokucindezeleka okukhulu. Uma kukhona ukucindezeleka okulinganiselwe kwe-metabolic kumodeli yethu, kungenzeka ukuthi i-PTM ikhuthaza umsebenzi owandisiwe wamaprotheni e-fusion kanye ne-fission ukuze abuyisele ngokwanele ubuqotho be-mitochondrial ngaphandle kokudinga ukusebenza okwengeziwe kwalawa ma-gene ezingeni le-mRNA noma leprotheyini.
Uma kuhlanganiswa, idatha engenhla iqokomisa ukulawulwa okuyinkimbinkimbi nokuncike esikhathini kwesimo se-mitochondrial kanye nezinselele zokucacisa lezi zindlela. Ukuze kufundwe ukubonakaliswa kwezakhi zofuzo, kubalulekile kuqala ukuhlonza izakhi zofuzo ezithile eziqondiwe endleleni. Kodwa-ke, idatha yethu ikhombisa ukuthi izakhi zofuzo ezisendleleni efanayo aziphenduli ngendlela efanayo ekucindezelekeni okufanayo. Eqinisweni, izifundo zangaphambilini zibonise ukuthi izakhi zofuzo ezahlukene endleleni efanayo zingase zibonise amaphrofayili ahlukene okuphendula kwesikhashana30,46. Ngaphezu kwalokho, kunezindlela eziyinkimbinkimbi zangemva kokubhalwa kwezakhi zofuzo eziphazamisa ubudlelwano phakathi kokubhalwa kwezakhi zofuzo kanye nomsebenzi wezakhi zofuzo. Izifundo ze-Proteomic zinganikeza ukuqonda ngomthelela wama-PTM kanye nomsebenzi weprotheyini, kodwa futhi ziletha izinselele kufaka phakathi izindlela zokukhipha okuphansi, izilinganiso eziphezulu zesignali-kuya-kumsindo, kanye nokuxazululwa okubi.
Kulo mongo, ukufunda ngesimo se-mitochondrial kusetshenziswa i-TEM ne-MEL kunamandla amakhulu okuphendula imibuzo eyisisekelo mayelana nobudlelwano phakathi kwe-mitochondrial dynamics kanye nomsebenzi nokuthi lokhu kuthonya kanjani isifo. Okubaluleke kakhulu, i-TEM inikeza indlela eqondile yokulinganisa isimo se-mitochondrial njengendawo ehlanganayo yokungasebenzi kahle kwe-mitochondrial kanye ne-dynamics51. I-MEL iphinde inikeze indlela eqondile yokubona ngeso lengqondo izehlakalo zokuhlukana kanye nokuhlangana endaweni yamaseli enezinhlangothi ezintathu, okuvumela ukulinganiswa kokuguqulwa kabusha kwe-mitochondrial okuguquguqukayo ngisho noma kungekho zinguquko ekubonakalisweni kwezakhi zofuzo33. Lapha siqokomisa ukusetshenziswa kwamasu okuthwebula izithombe ze-mitochondrial ezifweni ze-mitochondrial zesibili. Lezi zifo zivame ukubonakala ngokucindezeleka okungapheli kwe-metabolic okubonakala ngokuguqulwa okucashile kwamanethiwekhi e-mitochondrial kunomonakalo omkhulu we-mitochondrial. Kodwa-ke, isinxephezelo se-mitochondrial esidingekayo ukugcina i-mitosis ngaphansi kokucindezeleka okungapheli kunemiphumela ejulile yokusebenza. Kumongo we-neuroscience, ukuqonda okungcono kwalezi zindlela zokubuyisela kunganikeza ulwazi olubalulekile mayelana ne-pleiotropic neuropathology ehlobene nokungasebenzi kahle kwe-mitochondrial.
Ekugcineni, idatha yethu iqokomisa ukusetshenziswa kwamasu okuthwebula izithombe ukuqonda imiphumela yokusebenza kokuxhumana okuyinkimbinkimbi phakathi kokubonakaliswa kwezakhi zofuzo, ukuguqulwa kwamaprotheni, kanye nomsebenzi wamaprotheni olawula amandla e-neuronal mitochondrial. Sisebenzise i-PPA ukulingisa ukungasebenzi kahle kwe-mitochondrial kumodeli weseli le-neuronal ukuze sithole ukuqonda ngengxenye ye-mitochondrial ye-ASD. Amaseli e-SH-SY5Y aphathwe nge-PPA abonise izinguquko ekubunjweni kwe-mitochondrial: i-mitochondria yaba ncane futhi iyindilinga, kanti ama-cristae awachazwanga kahle lapho ebonwa yi-TEM. Ukuhlaziywa kwe-MEL kukhombisa ukuthi lezi zinguquko zenzeka ngesikhathi esifanayo nokwanda kwezehlakalo ze-fission kanye ne-fusion ukuze kugcinwe inethiwekhi ye-mitochondrial ekuphenduleni ukucindezeleka okuncane kwe-metabolic. Ngaphezu kwalokho, i-PPA iphazamisa kakhulu ukulawulwa kokubhalwa kwe-metabolism ye-mitochondrial kanye ne-homeostasis. Sithole i-cMYC, i-NRF1, i-TFAM, i-STOML2, kanye ne-OPA1 njengezilawuli ezibalulekile ze-mitochondrial eziphazanyiswa ukucindezeleka kwe-PPA futhi zingadlala indima ekuxazululeni izinguquko ezibangelwa yi-PPA ekubunjweni kwezakhi zofuzo kanye nomsebenzi wamaprotheni. Izifundo zesikhathi esizayo ziyadingeka ukuze kuchazwe kangcono izinguquko zesikhathi ezibangelwa yi-PPA ekubunjweni kwezakhi zofuzo kanye nomsebenzi wamaprotheni, indawo, kanye nokuguqulwa kwangemva kokuhumusha. Idatha yethu iqokomisa ubunzima kanye nokuncika kwezindlela zokulawula ezilawula impendulo yokucindezeleka kwe-mitochondrial futhi ikhombisa ukusetshenziswa kwe-TEM kanye nezinye izindlela zokuthwebula izithombe ezifundweni zemishini eziqondiswe kakhulu.
Umugqa weseli we-SH-SY5Y (ECACC, 94030304-1VL) uthengwe ku-Sigma-Aldrich. Amaseli e-SH-SY5Y akhuliswe engxubeni yezakhamzimba ye-Eagle's medium/F-12 eguquliwe kaDulbecco (DMEM/F-12) kanye ne-L-glutamine (SC09411, ScienCell) kumaflaski angu-25 cm2 afakwe i-20% ye-fetal bovine serum (FBS) (10493106, ThermoFisher Scientific) kanye ne-1% ye-penicillin-streptomycin (P4333-20ML, Sigma-Aldrich) ku-37 °C, 5% CO2. Amaseli akhuliswe ngaphansi kokukhula kwafinyelela ku-80% confluence kusetshenziswa i-0.05% trypsin-EDTA (15400054, ThermoFisher Scientific), agxishwe ku-300 g futhi afakwa ku-density cishe yamaseli angu-7 × 105/ml. Zonke izivivinyo zenziwe kumaseli e-SH-SY5Y angahlukaniswanga phakathi kwezindlela 19–22. I-PPA inikezwa njenge-NaP. Ncibilikisa i-NaP powder (CAS No. 137-40-6, ifomula yamakhemikhali C3H5NaO2, P5436-100G, Sigma-Aldrich) emanzini afudumele e-MilliQ kuze kube yilapho kugxilwe ku-1 M bese ugcina ku-4 °C. Ngosuku lokwelashwa, ncibilikisa lesi sixazululo nge-1 M PPA kuya ku-3 mM kanye ne-5 mM PPA endaweni engena-serum medium (DMEM/F-12 ene-L-glutamine). Izinga lokwelashwa lazo zonke izivivinyo lalingekho i-PPA (0 mM, control), 3 mM, kanye ne-5 mM PPA. Izivivinyo zenziwe okungenani eziphindaphindweni ezintathu zebhayoloji.
Amaseli e-SH-SY5Y ahlwanyelwe kuma-flask angu-25 cm5 ngesilinganiso samaseli angu-5.5 × 105/ml futhi akhuliswa amahora angu-24. Ukwelashwa kwe-PPA kwenezelwa ebhodleleni ngaphambi kwamahora angu-24 okufukamela. Qoqa ama-pellets amaseli ngokulandela izinqubo ezijwayelekile zokukhulisa izicubu zezilwane ezincelisayo (ezichazwe ngenhla). Phinda umise i-pellet yamaseli ku-100 µl 2.5% glutaraldehyde, 1 × PBS bese ugcina ku-4 °C kuze kube yilapho kucutshungulwa. Amaseli e-SH-SY5Y afakwe isikhashana ukuze afake amaseli futhi asuse i-2.5% glutaraldehyde, 1 × PBS solution. Phinda umise i-sediment ku-4% agarose gel elungiselelwe emanzini acwecwekile (isilinganiso se-agarose kuya ku-sediment volume singu-1:1). Izingcezu ze-Agarose zabekwa kuma-grid ezingcwecweni ezisicaba futhi zambozwa nge-1-hexadecene ngaphambi kokubanda ngomfutho ophezulu. Amasampula aqandisiwe ku-100% e-acetone eyomile ku--90°C amahora angu-24. Izinga lokushisa labe selikhushulwa laya ku-80°C kwase kungezwa isixazululo se-1% osmium tetroxide kanye ne-0.1% glutaraldehyde. Amasampula agcinwa ku-80°C amahora angama-24. Ngemva kwalokhu, izinga lokushisa lakhushulwa kancane kancane laya ekushiseni kwegumbi izinsuku eziningana: kusukela ku-80 °C kuya ku-50 °C amahora angama-24, kuya ku-30 ​​°C amahora angama-24, kuya ku-10 °C amahora angama-24 futhi ekugcineni laya ekushiseni kwegumbi.
Ngemva kokulungiswa kwe-cryogenic, amasampula afakwa i-resin futhi izingxenye ezincane kakhulu (~100 nm) zenziwa kusetshenziswa i-Leica Reichert UltracutS ultramicrotome (Leica Microsystems). Izingxenye zafakwa i-uranyl acetate engu-2% kanye ne-lead citrate. Amasampula abonwa kusetshenziswa i-FEI Tecnai 20 transmission electron microscope (ThermoFisher (eyayikade iyi-FEI), Eindhoven, eNetherlands) esebenza ku-200 kV (Lab6 transmitter) kanye nekhamera ye-Gatan CCD (Gatan, UK) efakwe isihlungi samandla se-Tridiem.
Kukopishi ngayinye yobuchwepheshe, okungenani izithombe zeseli elilodwa ezingama-24 zatholakala, okwaba yizithombe ezingama-266 sezizonke. Zonke izithombe zahlaziywa kusetshenziswa i-macro yeSifunda Sentshisekelo (ROI) kanye ne-macro ye-Mitochondria. I-macro ye-mitochondrial isekelwe ezindleleni ezishicilelwe17,31,32 futhi ivumela ukucutshungulwa kwe-batch okuzenzakalelayo kwezithombe ze-TEM eFiji/ImageJ69. Kafushane: isithombe siguqulwe futhi siguqulwe kusetshenziswa ukususwa kwengemuva lebhola eligoqayo (irediyasi yamaphikseli angama-60) kanye nesihlungi se-FFT bandpass (sisebenzisa imingcele ephezulu nephansi yamaphikseli angama-60 kanye nama-8 ngokulandelana) kanye nokucindezelwa komugqa oqondile ngokubekezelela ukuqondiswa okungu-5%. Isithombe esicutshunguliwe sinqunyelwa ngokuzenzakalelayo kusetshenziswa i-algorithm ephezulu ye-entropy futhi kukhiqizwa imaski enama-binary. Izifunda zesithombe ezihlotshaniswa nama-ROI akhethwe ngesandla ezithombeni ze-TEM ezingavuthiwe zakhishwa, zachaza i-mitochondria futhi zakhipha i-plasma membrane nezinye izifunda ezihlukile kakhulu. Ku-ROI ngayinye ekhishwe, kwahlaziywa izinhlayiya ezimbili ezinkulu kunamaphikseli angu-600, futhi indawo yezinhlayiya, umjikelezo, ama-axes amakhulu namancane, ububanzi be-Feret, ukujikeleza, kanye nokujikeleza kwalinganiswa kusetshenziswa imisebenzi yokulinganisa eyakhelwe ngaphakathi ye-Fiji/ImageJ. Kulandela i-Merrill, Flippo, kanye ne-Strack (2017), indawo 2, isilinganiso se-particle aspect (isilinganiso esikhulu kuya kwe-axis encane), kanye ne-shape factor (FF) kubalwe kusukela kule datha, lapho i-FF = indawo ye-perimeter 2/4pi x. Incazelo yefomula ye-parametric ingatholakala ku-Merrill, Flippo, kanye ne-Strack (2017). Ama-macro okukhulunywe ngawo ayatholakala ku-GitHub (bheka Isitatimende Sokutholakala Kwedatha). Ngokwesilinganiso, cishe izinhlayiya ezingu-5,600 zahlaziywa ngokwelashwa kwe-PPA ngakunye, kwaba cishe izinhlayiya ezingu-17,000 (idatha ayiboniswanga).
Amaseli e-SH-SH5Y abekwe ezitsheni zokukhulisa ezinamakamelo ayi-8 (ThermoFisher, #155411) ukuze avumele ukunamathela ubusuku bonke bese efakwa nge-TMRE 1:1000 (ThermoFisher, #T669) kanye ne-Hoechst 33342 1:200 (Sigma-Aldrich, H6024). Ukudaya izithombe. Izithombe zatholakala kusetshenziswa ama-laser angu-405 nm kanye nama-561 nm endaweni ethatha imizuzu eyi-10, kanti izithombe ezingavuthiwe zatholakala njengezitaki ze-z eziqukethe ama-micrograph ezithombe ayi-10 anesinyathelo se-az esingu-0.2 μm phakathi kwamafreyimu ezithombe ngezikhathi eziyi-12 ezalandela. Izithombe zaqoqwa kusetshenziswa ipulatifomu ye-Carl Zeiss LSM780 ELYRA PS.1 super-resolution (Carl Zeiss, Oberkochen, Germany) kusetshenziswa ilensi ye-LCI Plan Apochromate 100x/1.4 Oil DIC M27. Izithombe zihlaziywe ku-ImageJ kusetshenziswa ipayipi elichazwe ngaphambilini kanye ne-plugin ye-ImageJ ukukala imicimbi yokuhlangana kanye nokuhlukana, inani elimaphakathi lezakhiwo ze-mitochondrial, kanye nevolumu yama-mitochondrial emaphakathi ngeseli ngalinye33. Ama-macro e-MEL ayatholakala ku-GitHub (bheka Isitatimende Sokutholakala Kwedatha).
Amaseli e-SH-SY5Y akhuliswe emapuletini anezimbobo eziyisithupha ngobuningi bamaseli angu-0.3 × 106/mL amahora angama-24 ngaphambi kokwelashwa. I-RNA ikhishwe kusetshenziswa iphrothokholi ye-Quick-RNA™ Miniprep (ZR R1055, Zymo Research) ngokuguqulwa okuncane: engeza i-300 μl ye-RNA lysis buffer emthonjeni ngamunye ngaphambi kokususwa bese ulungisa isampula ngayinye njengesinyathelo sokugcina ngamanzi angenawo ama-30 μl e-DNase/RNase. Wonke amasampula ahlolwe ubuningi nekhwalithi kusetshenziswa i-NanoDrop ND-1000 UV-Vis Spectrophotometer. I-protein ephelele evela kuma-lysates eseli itholakale kusetshenziswa i-200 μl ye-RIPA lysis buffer, futhi ukuhlushwa kwamaprotheni kwalinganiswa kusetshenziswa i-Bradford protein assay70.
Ukwenziwa kwe-cDNA kwenziwa kusetshenziswa i-Tetro™ cDNA Synthesis Kit (BIO-65043, Meridian Bioscience) ngokwemiyalelo yomenzi kanye nokuguqulwa okuthile. I-cDNA yenziwe nge-reactions engu-20-μl kusetshenziswa i-0.7 kuya ku-1 μg ye-RNA iyonke. Ama-primer akhethwe emaphepheni ashicilelwe ngaphambilini angu-42, 71, 72, 73, 74, 75, 76, 77, 78 (Ithebula S1) kanti ama-probe ahambisana nawo aklanywa kusetshenziswa ithuluzi le-PrimerQuest elivela ku-Integrated DNA Technologies. Zonke izakhi zofuzo ezithakaselwayo zalungiswa zaba yizakhi zofuzo ze-nuclear B2M. Ukuvezwa kwezakhi zofuzo ze-STOML2, NRF1, NFE2L2, TFAM, cMYC kanye ne-OPA1 kwalinganiswa yi-RT-qPCR. Ingxube enkulu yayihlanganisa i-LUNA Taq polymerase (M3003L, New England Biolabs), ama-primer angu-10 μM phambili nangemuva, i-cDNA, kanye namanzi ebanga le-PCR ukuze kutholakale ivolumu yokugcina engu-10 μL yokusabela ngakunye. Ukuvezwa kwezakhi zofuzo zokuhlukanisa kanye nokuhlukanisa (DRP1, MFN1/2) kwalinganiswa kusetshenziswa izivivinyo ze-TaqMan multiplex. I-Luna Universal Probe qPCR Master Mix (M3004S, New England Biolabs) yasetshenziswa ngokwemiyalelo yomenzi enezinguquko ezincane. Ingxube enkulu ye-multiplex RT-qPCR ihlanganisa i-1X LUNA Taq polymerase, ama-primer angu-10 μM phambili nangemuva, i-probe engu-10 μM, i-cDNA, kanye namanzi ebanga le-PCR, okwaholela kuvolumu yokugcina engu-20 μL yokusabela ngakunye. I-RT-qPCR yenziwe kusetshenziswa i-Rotor-Gene Q 6-plex (QIAGEN RG—inombolo ye-serial: R0618110). Izimo zokujikeleza ziboniswe kuThebula S1. Wonke amasampula e-cDNA akhuliswe kathathu futhi kwakhiqizwa ijika elijwayelekile kusetshenziswa uchungechunge lwezinciphiso eziphindwe kayishumi. Ama-Outliers kumasampula kathathu anokuhluka okujwayelekile komjikelezo (Ct) >0.5 asusiwe ekuhlaziyweni ukuqinisekisa ukuphindaphindwa kwedatha30,72. Ukuvezwa kwezakhi zofuzo okuhlobene kubalwe kusetshenziswa indlela ye-2-ΔΔCt79.
Amasampula ephrotheni (60 μg) axutshwe ne-Laemmli loading buffer ngesilinganiso esingu-2:1 futhi asebenza ngejeli yephrotheni engenambala engu-12% (i-Bio-Rad #1610184). Amaprotheni adluliselwa kulwelwesi lwe-PVDF (i-polyvinylidene fluoride) (#170-84156, i-Bio-Rad) kusetshenziswa uhlelo lwe-Trans-Blot Turbo (#170-4155, i-Bio-Rad). Ulwelwesi lwavinjwa futhi lwafakwa kuma-antibodies ayisisekelo afanele (i-OPA1, i-MFN1, i-MFN2, kanye ne-DRP1) (axutshwe ngo-1:1000) amahora angu-48, kwalandela ukufuthwa ngama-antibodies esibili (1:10,000) ihora eli-1. Ama-membrane abe esethathwa izithombe kusetshenziswa i-Clarity Western ECL Substrate (#170-5061, i-Bio-Rad) futhi aqoshwa kusetshenziswa uhlelo lwe-Bio-Rad ChemiDoc MP. Inguqulo ye-ImageLab 6.1 yasetshenziselwa ukuhlaziywa kwe-Western blot. Ijeli yokuqala kanye ne-blot kuboniswe kuMfanekiso S1. Ulwazi lwe-antibody lunikezwe kuThebula S2.
Amasethi edatha ethulwa njengephutha eliphakathi nendawo nelijwayelekile lesilinganiso (SEM) okungenani amasampula amathathu azimele. Amasethi edatha ahlolwe ukuthi ajwayelekile yini kusetshenziswa isivivinyo se-Shapiro-Wilks (ngaphandle kokuthi kuchazwe ngenye indlela) ngaphambi kokuthatha ukusatshalaliswa kwe-Gaussian kanye nokuphambuka okujwayelekile okulinganayo nokuqhubeka nokuhlaziya. Ngaphezu kokuhlaziya isethi yedatha kusetshenziswa i-Fisher's MEL LSD (p < 0.05), i-ANOVA yendlela eyodwa (ukwelashwa vs. isilinganiso sokulawula), kanye nokuhlolwa kokuqhathanisa okuningi kukaDunnett ukuthola ukubaluleka (p < 0.05). Amanani abalulekile e-p aboniswa kugrafu njengo-*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Zonke izibalo kanye namagrafu zenziwe futhi zakhiqizwa kusetshenziswa i-GraphPad Prism 9.4.0.
Ama-macro e-Fiji/ImageJ okuhlaziywa kwesithombe se-TEM atholakala esidlangalaleni ku-GitHub: https://github.com/caaja/TEMMitoMacro. I-macro ye-Mitochondrial Event Locator (MEL) itholakala esidlangalaleni ku-GitHub: https://github.com/rensutheart/MEL-Fiji-Plugin.
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