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I-propionic acid (PPA) isetjenziselwa ukufunda indima yokungasebenzi kuhle kwe-mitochondrial eemrarweni zokukhula kwemizwa ezifana nokuphazamiseka kwe-autism spectrum. I-PPA yaziwa ngokuphazamisa ukwakheka kwe-mitochondrial, ukugaya ukudla, nokutjhuguluka. Kodwana, imiphumela ye-PPA ekuguquguqukeni kwe-mitochondrial, ukuhlukana nokuhlanganiswa kuhlala kuyinkinga ngebanga lobujamo besikhathi obubudisi beendlela lezi. Lapha, sisebenzisa amaqhinga wokuthatha iinthombe ukuhlolisisa bona i-PPA ithinta njani i-mitochondrial ultrastructure, i-morphology, kanye nokuguquguquka kwamaseli we-SH-SY5Y afana ne-neuron. I-PPA (5 mM) yabangela ukwehla okukhulu kwendawo ye-mitochondrial (p < 0.01), ububanzi be-Feret kanye nomjikelezo (p < 0.05), kanye nendawo 2 (p < 0.01). Ukuhlaziywa kwe-mitochondrial event locator kutjengise ukwanda okukhulu (p < 0.05) kwezehlakalo zokuhlukana nokuhlanganiswa, ngaleyo ndlela kugcinwe ubuqotho be-mitochondrial network ngaphasi kweemeko zokugandeleleka. Ukungezelela, ukuvezwa kwe-mRNA ye-cMYC (p < 0.0001), NRF1 (p < 0.01), TFAM (p < 0.05), STOML2 (p < 0.0001) kanye ne-OPA1 (p < 0.05) kwehliswe khulu. 01). Lokhu kutjengisa ukulungiswa kabutjha kwe-mitochondrial morphology, biogenesis kanye nokuguquguquka ukugcina ukusebenza ngaphasi kweemeko zokugandeleleka. Idatha yethu inikela ukuzwisisa okutjha ngemiphumela ye-PPA ekuguquguqukeni kwe-mitochondrial begodu iveza ukusetjenziswa kwamaqhinga wokuthatha iinthombe wokufunda iindlela eziyinkimbinkimbi zokulawula ezibandakanyeka ekuphenduleni kokugandeleleka kwe-mitochondrial.
Ama-mitochondria abahlanganyeli abaqakathekileko emisebenzini ehlukahlukeneko yamaseli ngaphezu kweendima zawo ezijayelekileko ekukhiqizeni amandla kanye nokwakhiwa kwezinto eziphilako. I-mitochondrial metabolism mlawuli oqakathekileko we-calcium, i-metabolic kanye ne-redox homeostasis, i-inflammatory, ukutjhugululwa kwe-epigenetic, ukwanda kwamaseli, ukuhlukana nokufa kwamaseli okuhleliweko1. Khulukhulu, ukugaya ukudla kwe-mitochondrial kuqakathekile ekuthuthukisweni kwe-neuronal, ukuphila nokusebenza begodu kufaka hlangana ukubonakaliswa okuhlukahlukeneko kwe-neuropathology2,3,4.
Eminyakeni elitjhumi edlulileko, ubujamo be-metabolic buvele njengomlawuli omkhulu we-neurogenesis, ukuhlukaniswa, ukuvuthwa kanye nobupulasitiki5,6. Mhlapha nje, ukwakheka kwe-mitochondria nokutjhuguluka kwayo sekuyizinto eziqakatheke khulu ze-mitosis, ikambiso enamandla egcina idamu lama-mitochondria aphilileko ngaphakathi kwamaseli. I-mitochondrial dynamics ilawulwa ziindlela ezihlukahlukeneko ezithembele komunye nomunye ezisuka ku-mitochondrial biogenesis kanye ne-bioenergetics ukuya e-mitochondrial fission, ukuhlangana, ukuthuthwa nokuhlanzwa7,8. Ukuphazamiseka kwanoma ngiyiphi yalezi zindlela zokuhlanganisa kuphazamisa ukugcinwa kwamanethiwekhi we-mitochondrial aphilileko begodu kunemiphumela emikhulu yokusebenza ekuthuthukisweni kwemizwa9,10. Kwamambala, ukungalawuleki kwamandla we-mitochondrial kubonakala emirarweni eminengi yengqondo, ye-neurodegenerative kanye ne-neurodevelopmental, okufaka hlangana ukuphazamiseka kwe-autism spectrum (ASD)11,12.
I-ASD mkhuhlane ohlukileko wokuthuthuka kwemizwa onesakhiwo esiyinkimbinkimbi sezakhi zofuzo kanye ne-epigenetic. Ukutholakala kwe-ASD akuphikiswa, kodwana i-etiology yamamolekhyuli ayikazwisiseki kuhle. Ukubuthelela idatha evela kumamodeli wangaphambi kwezokwelapha, iimfundo zokwelapha, kanye namasethi wedatha yamamolekhyuli we-multi-omics kunikela ubufakazi obukhulako bokungasebenzi kuhle kwe-mitochondrial ku-ASD13,14. Ngaphambilini senze i-genome-wide DNA methylation screen esiqhemeni sezigulani ezine-ASD begodu sabona amajini ahlukeneko ahlanganiswe eendleleni ze-mitochondrial metabolic15. Ngemva kwalokho sabika ukuhlukahluka kwe-methylation yama-regulator aphakathi we-mitochondrial biogenesis kanye nokuguquguquka, okwahlotjaniswa nokwanda kwenani lamakhophi we-mtDNA kanye nokutjhuguluka kwephrofayili yokugaya umchamo ku-ASD16. Idatha yethu inikela ubufakazi obukhulako bokuthi ukuguquguquka kwe-mitochondrial kanye ne-homeostasis kudlala indima eqakathekileko ku-pathophysiology ye-ASD. Ngalokho-ke, ukwenza ngcono ukuzwisisa kwendlela yokusebenza kobudlelwano phakathi kwamandla we-mitochondrial, ukwakheka, nomsebenzi kungumnqopho oqakathekileko wokurhubhulula okuragela phambili ngamalwelwe wezinzwa abonakala ngokungasebenzi kuhle kwe-mitochondrial yesibili.
Amaqhinga wamamolekhyuli avamise ukusetjenziswa ukufunda indima yamajini athileko ekuphenduleni ukugandeleleka kwe-mitochondrial. Kodwana, indlela le ingaba nomkhawulo ngobujamo obunengi nobujamo besikhathi beendlela zokulawula i-mitotic. Ngaphezu kwalokho, ukuvezwa okuhlukileko kwamajini we-mitochondrial kuyinkomba engaqondileko yokutjhuguluka kokusebenza, khulukhulu ngombana inani elilinganiselwe lamajini livamise ukuhlaziywa. Ngalokho-ke, iindlela eziqondileko zokufunda ukusebenza kwe-mitochondrial kanye ne-bioenergetics ziphakanyisiwe17. I-mitochondrial morphology ihlotjaniswa khulu nokutjhuguluka kwe-mitochondrial. Ubujamo be-mitochondrial, ukuxhumana, nesakhiwo kuqakathekile ekukhiqizeni amandla nokusinda kwe-mitochondrial namaseli5,18. Ngaphezu kwalokho, iingcenye ezihlukahlukeneko ze-mitosis zidzimelele ematjhugulukweni we-mitochondrial morphology, okungasebenza njengeziphetho eziwusizo zokungasebenzi kuhle kwe-mitochondrial begodu zinikele isisekelo sezifundo ezilandelako ze-mechanistic.
I-mitochondrial morphology ingabonwa ngqo ngokusebenzisa i-transmission electron microscopy (TEM), evumela isifundo esinabileko se-ultrastructure yamaseli. I-TEM itjengisa ngokunqophileko ukwakheka, ukuma nokwakheka kwe-mitochondrial cristae ekuxazululeni kwe-mitochondria ngayinye, kunokobana ithembele ekutlolweni kwezakhi zofuzo, ukuvezwa kwamaphrotheyini nofana amapharamitha wokusebenza kwe-mitochondrial emaseli17,19,20. Ukungezelela, i-TEM yenza bona kube lula ukufunda ukusebenzisana hlangana nama-mitochondria namanye ama-organelles, afana ne-endoplasmic reticulum kanye nama-autophagosomes, adlala indima eqakathekileko ekusebenzeni kwe-mitochondrial kanye ne-homeostasis21,22. Ngalokho, lokhu kwenza i-TEM ibe yindawo ehle yokuthoma yokufunda ukungasebenzi kuhle kwe-mitochondrial ngaphambi kokutjheja iindlela ezithileko nofana amajini. Njengoba umsebenzi we-mitochondrial uya ngokuya uqakatheka khulu ku-neuropathology, kunesidingo esicacileko sokuthi sikghone ukufunda ngokunqophileko nangobunengi i-mitochondrial morphology kanye nokuguquguquka kwayo kumamodeli we-in vitro neuronal.
Kulesi sihloko, sihlola ukuguquguquka kwe-mitochondrial kumodeli ye-neuronal yokungasebenzi kuhle kwe-mitochondrial ku-autism spectrum disorder. Ngaphambilini sibike ukuhlukahluka kwe-methylation ye-propionyl-CoA carboxylase beta (PCCB) ku-ASD15, iyunithi ye-mitochondrial propionyl-CoA carboxylase enzyme PCC. Ukungalawuleki kwe-PCC kwaziwa ngokubangela ukubuthelela okunobuthi kwe-propionyl derivatives, okufaka hlangana i-propionic acid (PPA)23,24,25. I-PPA itjengiswe bona iphazamisa ukusebenza kwemithambo yeenzwa begodu itjhugulula ukuziphatha kwe-vivo begodu yimodeli yesilwana ehleliweko yokufunda iindlela zokuthuthukiswa kwemizwa ezithintekako ku-ASD26,27,28. Ukungezelela, i-PPA ibikwe bona iphazamisa amandla we-mitochondrial membrane, i-biogenesis nokuphefumula ku-vitro begodu isetjenziswe khulu ukumodela ukungasebenzi kuhle kwe-mitochondrial kuma-neuron29,30. Kodwana, umthelela wokungasebenzi kuhle kwe-mitochondrial okubangelwa yi-PPA ekubunjweni kwe-mitochondrial kanye nokuguquguquka kwayo kuhlala kungakazwisiseki kuhle.
Irhubhululo lisebenzisa amaqhinga wokuthatha iinthombe ukulinganisa imiphumela ye-PPA ebujameni be-mitochondrial, ukuguquguquka, nokusebenza kwamaseli we-SH-SY5Y. Kokuthoma, sakha indlela ye-TEM ukubona amatjhuguluko we-mitochondrial morphology kanye ne-ultrastructure17,31,32. Ngokuya ngobujamo be-mitochondria33, sisebenzise nokuhlaziywa kwe-mitochondrial event localizer (MEL) ukulinganisa amatjhuguluko ekulinganiseni hlangana nezehlakalo zokuhlukana nokuhlanganiswa, inani le-mitochondria kanye nevolumu ngaphasi kokugandeleleka kwe-PPA. Ekugcineni, sihlole bona i-mitochondrial morphology nokutjhuguluka kwayo kuhlotjaniswa na nokutjhuguluka kokuvezwa kwamajini abandakanyeka ekubunjweni kwezinto eziphilako, ukuhlukana, nokuhlanganiswa. Nakuthathwa ndawonye, ​​idatha yethu itjengisa ubudisi bokucacisa ubudisi beendlela ezilawula amandla we-mitochondrial. Siveza ukusetjenziswa kwe-TEM ekufundeni ubujamo be-mitochondrial njengesiphetho esilinganisekako se-mitosis kumaseli we-SH-SY5Y. Phezu kwalokho, siveza bona idatha ye-TEM inikela ilwazi elinengi khulu lokha nayihlanganiswa namaqhinga wokuthatha iinthombe athatha izehlakalo eziguquguqukako ngokuphendula ekugandelelekeni kwe-metabolic. Ukuhlathulula okungeziweko kweendlela zokulawula amamolekhyuli ezisekela i-mitosis yamaseli we-neuronal kunganikela ukuzwisisa okuqakathekileko ngengcenye ye-mitochondrial yesistimu yezinzwa kanye namalwelwe we-neurodegenerative.
Ukubanga ukugandeleleka kwe-mitochondrial, amaseli we-SH-SY5Y alatjhwa nge-PPA kusetjenziswa i-3 mM kanye ne-5 mM ye-sodium propionate (NaP). Ngaphambi kwe-TEM, amasampula bekafakwa ngaphasi kokulungiselela isampula ye-cryogenic kusetjenziswa ukutjhisa okuphezulu nokuqanda (Umfanekiso 1a). Sakha umtjhini wokuhlaziya iinthombe ze-mitochondrial ezizenzakalelako ukulinganisa amapharamitha abunane we-morphological yabantu be-mitochondrial kizo zoke iindlela ezintathu eziphindaphindiweko. Sithole bona ukwelatjhwa nge-PPA kutjhugulule khulu amapharamitha amane: indawo 2, indawo, umkhawulo, kanye nobubanzi be-Feret (Umfanekiso 1b-e). Indawo 2 yehle khulu ngokwelatjhwa nge-3 mM kanye ne-5 mM ye-PPA (p = 0.0183 kanye ne-p = 0.002, ngokulandelana) (Umfanekiso 1b), ngesikhathi indawo (p = 0.003), umkhawulo (p = 0.0106) kanye nobubanzi be-Feret zoke zehle khulu. Kwaba nokwehla okukhulu (p = 0.0172) esiqhemeni sokwelatjhwa nge-5 mM nayiqathaniswa nesiqhema sokulawula (Umfanekiso 1c–e). Ukwehla okukhulu kwendawo nomjikelezo kutjengise bona amaseli aphathwe nge-5 mM PPA bekanama-mitochondria amancani, ayindilinga, begodu ama-mitochondria la bekangade khulu kunalawo aseseli yokulawula. Lokhu kuhambisana nokwehla okukhulu kobubanzi be-Feret, ipharamitha ezimeleko etjengisa ukwehla kwebanga elikhulu khulu hlangana namaphethelo weenhlayiya. Amatjhuguluko ekwakhiweni kwe-cristae kwabonwa: i-cristae yaba ncani ngaphasi kwethonya lokugandeleleka kwe-PPA (Umfanekiso 1a, iphaneli B). Kodwana, akusizo zoke iinthombe ebezitjengisa kuhle ubujamo be-cristae, njeke ukuhlaziywa okulinganiselweko kwamatjhuguluko la akuzange kwenziwe. Idatha ye-TEM ingatjengisa ubujamo obuthathu obungaba khona: (1) i-PPA ikhulisa ukuhlukana nofana ivimbele ukuhlanganiswa, okwenza bona ama-mitochondria akhona anciphe ngobukhulu; (2) i-biogenesis ethuthukisiweko yenza ama-mitochondria amatjha, amancani nofana (3) yenza zombili iindlela ngasikhathi sinye. Nanyana ubujamo lobu bungakghoni ukuhlukaniswa yi-TEM, amatjhuguluko aqakathekileko we-morphological atjengisa amatjhuguluko we-mitochondrial homeostasis kanye nokuguquguquka kwawo ngaphasi kokugandeleleka kwe-PPA. Ngemva kwalokho sahlola amanye amapharamitha ukuragela phambili nokuhlathulula ama-dynamics la kanye neendlela ezingaba khona ezisekela kiwo.
I-propionic acid (PPA) yenza kabutjha ubujamo be-mitochondrial. (a) Iinthombe ezijameleko ze-elekthronikhi ye-elekthronikhi (TEM) ezitjengisa bona ubukhulu be-mitochondria buyancipha begodu i-mitochondria iba mincani begodu iba yindilinga ngokukhula kokwelatjhwa nge-PPA; 0 mM (engakaphathwa), 3 mM kanye ne-5 mM, ngokulandelana. Imitshoko ebomvu itjengisa ama-mitochondria. (b–e) Amaseli we-SH-SY5Y aphathwe nge-PPA ama-awa ama-24 alungiselelwe i-TEM begodu imiphumela yahlaziywa kusetjenziswa i-Fiji/ImageJ. Amapharamitha amane kwabunane atjengisa umehluko oqakathekileko hlangana namaseli wokulawula (angakaphathwa, 0 mM PPA) kanye namaseli alatjhwako (3 mM kanye ne-5 mM PPA). (b) Isifunda 2, (c) Indawo, (d) Umkhawulo, (e) Ububanzi be-Feret. Ukuhlaziywa kwendlela eyodwa yokuhluka (ukulawula vs. ukwelapha) kanye nokuhlolwa kokumadanisa okunengi kwaka-Dunnett kwasetjenziselwa ukuthola umehluko oqakathekileko (p <0.05). Amaphuzu wedatha ajamele inani eliphakathi le-mitochondrial leseli ngalinye, begodu imigoqo yephutha imele inani eliphakathi ± SEM. Idatha etjengisiweko imele n = 3, okungenani amaseli ama-24 ngokuphindaphinda; kwahlaziywa iinthombe ezima-266; * itjengisa i-p < 0.05, ** itjengisa i-p < 0.01.
Ukuragela phambili nokuhlathulula bona ama-mitochondrial dynamics aphendula njani ku-PPA, sifake ama-mitochondria nge-tetramethylrhodamine ethyl ester (TMRE) begodu sasebenzisa i-time-lapse microscopy kanye nokuhlaziywa kwe-MEL ukuthola nokulinganisa ama-mitochondria ngemva kwama-awa ama-24 ku-3 kanye ne-5 mM PPA. Ukwelatjhwa kwezehlakalo zokuhlukana nokuhlanganiswa. (Umfanekiso 2a). Ngemva kokuhlaziywa kwe-MEL, ama-mitochondria ahlaziywa godu ukulinganisa inani lezakhiwo ze-mitochondrial kanye nevolumu yawo ephakathi. Sibone ukwanda okuncani kodwana okuqakathekileko kwenani lezehlakalo zokuhlukana ezenzeka ku-3 mM [4.9 ± 0.3 (p < 0.05)] nayiqathaniswa nokuhlukana [5.6 ± 0.3 (p < 0.05) )] nokuhlanganiswa [5.4 ± 0.5 (p < 0.05)] nokuhlanganiswa [5.4 ± 0.5 (p < 0.05)] 0.05)] <0.05)] izenzakalo zanda khulu ku-5 mM nayimadaniswa nokulawula (Isithombe 3b). Inani lama-mitochondria lakhula khulu ku-3 [32.6 ± 2.1 (p < 0.05)] kanye no-5 mM [34.1 ± 2.2 (p < 0.05)] (Umfanekiso 3c) , ngesikhathi ivolumu ephakathi yesakhiwo ngasinye se-mitochondria ihlala ingatjhuguluki (Isithombe 3c). 3d). Nakuthathwa koke, lokhu kuveza bona ukulungiswa kabutjha kwe-mitochondrial dynamics kusebenza njengependulo yokubuyisela egcina ngempumelelo ubuqotho be-mitochondrial network. Ukwanda kwenani lezehlakalo zokuhlukana ku-3 mM PPA kuveza bona ukwanda kwenani le-mitochondrial kungenxa yokuhlukana kwe-mitochondrial, kodwana nakuqalwa bona ivolumu ye-mitochondrial ihlala ingatjhuguluki, i-biogenesis angeze yakhishwa njengempendulo eyengeziweko. Kodwana, idatha le ihambisana nezakhiwo ezincani, eziyindilinga ze-mitochondrial ezibonwe yi-TEM begodu zitjengisa amatjhuguluko aqakathekileko emandleni we-mitochondrial abangelwa yi-PPA.
I-propionic acid (PPA) yenza bona i-mitochondrial ilungiswe kabutjha ukugcina ubuqotho benethiwekhi. Amaseli we-SH-SY5Y akhuliswa, aphathwa nge-3 kanye ne-5 mM PPA ama-awa ama-24 begodu afakwa ibala nge-TMRE ne-Hoechst 33342 kwalandela ukuhlaziywa kwe-MEL. (a) Iinthombe ezijamele i-time-lapse microscopy eziveza umbala kanye nokuqagela okuphezulu okukhulu ngesikhathi 2 (t2) sesimo ngasinye. Iindawo ezikhethiweko ezitjengiswe emfanekisweni ngamunye we-binary zithuthukiswa begodu zitjengiswa nge-3D ngeenkhathi ezintathu ezihlukileko (t1-t3) ukutjengisa ukutjhuguluka kwesikhathi; izenzakalo zokuhlanganiswa zivezwe ngohlaza satjani; izehlakalo zokuhlukana zivezwe ngohlaza satjani. Itjengiswa ngombala obomvu. (b) Inani eliphakathi lezehlakalo eziguquguqukako ngobujamo ngabunye. (c) Inani eliphakathi lezakhiwo ze-mitochondrial ngeseli ngalinye. (d) Ivolumu ephakathi (μm3) yesakhiwo ngasinye se-mitochondrial ngeseli. Idatha etjengisiweko ijamele amaseli we-n = 15 ngesiqhema ngasinye sokwelatjhwa. Imigoqo yephutha etjengisiweko imele i-mean ± SEM, ibha yesikali = 10 μm, * p < 0.05.
I-propionic acid (PPA) ibangela ukugandeleleka kokutloliswa kwamajini ahlotjaniswa nokusebenza kwe-mitochondrial. Amaseli we-SH-SY5Y alatjhwa nge-3 kanye ne-5 mM PPA ama-awa ama-24. Ukulinganisa izakhi zofuzo kwenziwa kusetjenziswa i-RT-qPCR begodu kwajwayelekile ku-B2M. Amajini we-mitochondrial biogenesis (a) i-cMYC, (b) i-TFAM, (c) i-NRF1 kanye (d) ne-NFE2L2. Amajini we-mitochondrial fusion nokuhlukana (e) STOML2, (f) OPA1, (g) MFN1, (h) MFN2 kanye (i) DRP1. Umehluko oqakathekileko (p < 0.05) wahlolwa kusetjenziswa i-ANOVA yendlela eyodwa (ukulawula vs. ukwelapha) kanye nokuhlolwa kokumadanisa okunengi kuka-Dunnett: * kutjengisa p < 0.05, ** kutjengisa p < 0.01, begodu **** kutjengisa p < 0.0001. Amabha ajamele ukuvezwa okuphakathi ± SEM. Idatha etjengisiweko ijamele i-n = 3 (STOML2, OPA1, TFAM), n = 4 (cMYC, NRF1, NFE2L2), kanye ne-n = 5 (MFN1, MFN2, DRP1) ukuphindaphinda kwezinto eziphilako.
Idatha evela ekuhlaziyweni kwe-TEM ne-MEL ndawonye itjengisa bona i-PPA itjhugulula ukwakheka kwe-mitochondrial namandla. Kodwana, amaqhinga wokuthatha iinthombe awanikeli ukuzwisisa ngeendlela eziqakathekileko eziqhuba iinkambo lezi. Ngalokho-ke sihlole ukuvezwa kwe-mRNA kwabalawuli abalithoba abaqakathekileko be-mitochondrial dynamics, i-biogenesis, kanye ne-mitosis ngokuphendula ekwelapheni 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), ne-Mitofusin (MFN2) iphrotheyini ehlobene ne-dynamin 1 (DRP1) ngemva kwama-awa ama-24 wokwelatjhwa 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. (Umfanekiso 3a-c). Ukwehla kokuvezwa kwe-mRNA bekuthembele emthamo: ukuvezwa kwe-cMYC, NRF1 kanye ne-TFAM kwehle nge-5.7, 2.6 kanye ne-1.9 izikhathi ku-3 mM, ngokulandelana, begodu nge-11.2, 3 kanye ne-2.2 izikhathi ku-5 mM. Ngokuphikisako, isakhi sofuzo se-redox biogenesis esiphakathi se-NFE2L2 azange sitjhugululwe nanyana ngikuphi ukugxila kwe-PPA, nanyana umkhuba ofanako othembele emthamo wokuncipha kokuvezwa kwabonwa (Umfanekiso 3d).
Siphinde sahlola ukuvezwa kwamajini weklasikhi abandakanyeka ekulawuleni ukuhlukana nokuhlanganiswa. I-STOML2 kucatshangwa bona ibandakanyeka ekuhlanganisweni, ekudleni kwe-mitophagy kanye ne-biogenesis, begodu ukuvezwa kwayo kwehliswe khulu (p < 0.0001) nge-3 mM (2.4-fold change) kanye ne-5 mM (2.8-fold change) PPA (Umfanekiso 1). 3d). Ngokufanako, ukuvezwa kwezakhi zofuzo ezihlanganisiweko ze-OPA1 kwehla ku-3 mM (ukutjhuguluka okuphindwe ka-1.6) kanye no-5 mM (ukutjhuguluka okuphindwe ka-1.9) PPA (p = 0.006 no-p = 0.0024, ngokulandelana) (Umfanekiso 3f). Kodwana, azange sithole umehluko oqakathekileko ekuvezweni kwamajini we-fusion MFN1, MFN2 nofana amajini we-fission DRP1 ngaphasi kokugandeleleka kwe-24-h PPA (Umfanekiso 3g–i). Ukungezelela, sithole bona amazinga wamaphrotheyini amane wokuhlanganiswa nokuhlukana (OPA1, MFN1, MFN2 kanye ne-DRP1) azange atjhuguluke ngaphasi kweemeko ezifanako (Umfanekiso 4a–d). Kuqakathekile ukutjheja bona idatha le itjengisa isikhathi esisodwa begodu kungenzeka ingabonisi amatjhuguluko ekuvezweni kwamaphrotheyini nofana amazinga womsebenzi ngesikhathi sesigaba sokuthoma sokugandeleleka kwe-PPA. Kodwana, ukwehla okukhulu kokuvezwa kwe-cMYC, NRF1, TFAM, STOML2, kanye ne-OPA1 kutjengisa ukungalungi okukhulu kokutlolwa kwe-mitochondrial metabolism, biogenesis, kanye nokuguquguquka. Ukungezelela, idatha le iveza ukusetjenziswa kwamaqhinga wokuthatha iinthombe ukufunda ngokunqophileko amatjhuguluko wobujamo bokuphela emsebenzini we-mitochondrial.
Amazinga wamaphrotheyini we-fusion no-fission factor azange atjhuguluke ngemva kokwelatjhwa nge-propionic acid (PPA). Amaseli we-SH-SY5Y alatjhwa nge-3 kanye ne-5 mM PPA ama-awa ama-24. Amazinga wamaphrotheyini alinganiswa ngokuhlaziywa kwe-Western blot, begodu amazinga wokuvezwa ajwayelekileko abe maphrotheyini woke. Ukuvezwa kwamaphrotheyini okuphakathi nendawo kanye nama-Western blots ajameleko wamaphrotheyini ahlosiweko kanye namaphrotheyini woke kutjengiswa. a – OPA1, b – MFN1, c – MFN2, d – DRP1. Amabha ajamele i-mean ± SEM, begodu idatha etjengisiweko ijamele n = 3 ukuphindaphinda kwezinto eziphilako. Ukumadanisa okunengi (p <0.05) kwenziwa kusetjenziswa ukuhlaziywa kwendlela eyodwa yokuhlukahluka nokuhlolwa kwaka-Dunnett. Ijeli yasekuthomeni ne-blot zitjengiswe kuMfanekiso S1.
Ukungasebenzi kuhle kwe-mitochondrial kuhlotjaniswa namalwelwe amanengi ukusuka emzimbeni we-metabolic, weenhliziyo kanye nemisipha ukuya ekuguleni kwemizwa1,10. Amalwelwe amanengi wokuwohloka kwemizwa nokuwohloka kwemizwa ahlotjaniswa nokungasebenzi kuhle kwe-mitochondrial, okuveza ukuqakatheka kwama-organelles la esikhathini soke sokuphila kobuchopho. Amalwelwe la afaka hlangana isifo se-Parkinson, isifo se-Alzheimer kanye ne-ASD3,4,18. Kodwana, ukufikelela emathitjhu wengqondo ukufunda amalwelwe la kunzima, khulukhulu ezingeni lendlela, okwenza bona amahlelo wemodeli yamaseli abe ngenye indlela etlhogekako. Kulesi sifundo, sisebenzisa ihlelo lamaseli sisebenzisa amaseli we-SH-SY5Y aphathwe nge-PPA ukubuyekeza ukungasebenzi kuhle kwe-mitochondrial okubonwe emalwelweni we-neuronal, khulukhulu ukuphazamiseka kwe-autism spectrum. Ukusebenzisa imodeli ye-PPA ukufunda ukuguquguquka kwe-mitochondrial kuma-neurons kunganikela ukuzwisisa nge-etiology ye-ASD.
Sihlolisise ithuba lokusebenzisa i-TEM ukubona amatjhuguluko we-mitochondrial morphology. Kuqakathekile ukutjheja bona i-TEM kufanele isetjenziswe kuhle ukukhulisa ukusebenza kwayo. Ukulungiselela ama-cryo-specimen kuvumela ukulondolozwa okungcono kwezakhiwo ze-neuronal ngokulungisa ngasikhathi sinye iingcenye zamaseli nokunciphisa ukwakheka kwezinto ezibunjiweko34. Ngokuvumelana nalokhu, sabona bona amaseli we-SH-SY5Y afana ne-neuron anama-organelles angaphansi kwamaseli kanye nama-mitochondria amade (Umfanekiso 1a). Lokhu kuveza ukusetjenziswa kwamaqhinga wokulungiselela we-cryogenic wokufunda ukwakheka kwe-mitochondrial kumamodeli wamaseli we-neuronal. Nanyana ukulinganisa okulinganiselweko kuqakathekile ekuhlaziyweni okuqondileko kwedatha ye-TEM, akukabi nokuvumelana ngokuthi ngimiphi imikhawulo ekufuze ilinganiswe ukuqinisekisa amatjhuguluko we-mitochondrial. Ngokuya ngenani elikhulu leemfundo ezihlolisise ubujamo be-mitochondrial17,31,32, sakha iphayiphi yokuhlaziywa kwemifanekiso ye-mitochondrial ezenzakalelayo elinganisa amapharamitha abunane we-morphological, okungukuthi: indawo, indawo2, isilinganiso se-aspect, umngcele, ukujikeleza, izinga, ububanzi be-Feret. kanye nobuyindilinga.
Hlangana nazo, i-PPA yehlise khulu indawo 2, indawo, umkhawulo, kanye nobubanzi be-Feret (Umfanekiso 1b-e). Lokhu kutjengise bona i-mitochondria yaba yincani begodu yayindilinga, okuhambisana neemfundo zangaphambilini ezitjengisa ukwehla kwendawo ye-mitochondria ngemva kwama-awa ama-72 wokugandeleleka kwe-mitochondria okubangelwa yi-PPA30. Iimfanelo zesakhiwo zingatjengisa ukuhlukana kwe-mitochondrial, ikambiso etlhogekako yokuhlukanisa iingcenye ezilimeleko kunethiwekhi ye-mitochondrial ukukhuthaza ukuwohloka kwazo nge-mitophagy35,36,37. Ngakelinye ihlangothi, ukwehla kobukhulu be-mitochondria kungahlotjaniswa nokwanda kwe-biogenesis, okurholela ekwakhiweni kwe-mitochondria encani. Ukwanda kokuhlukana nofana ukwakheka kwezinto eziphilako kujamele ipendulo yokubuyisela ukugcina i-mitosis ngokujamelana nokugandeleleka kwe-mitochondrial. Kodwana, ukwehla kokukhula kwe-mitochondrial, ukuhlangana okukhubazekileko, nofana ezinye iimeko angeze zakhishwa ngaphandle.
Nanyana iinthombe ezine-resolution ephezulu ezenziwe yi-TEM zivumela ukunqunywa kweempawu ze-morphological ezingeni le-mitochondria ngayinye, indlela le yenza iinthombe ezimbili ngesikhathi esisodwa. Ukufunda iimpendulo eziguquguqukako zokugandeleleka kwe-metabolic, sifake i-mitochondria nge-TMRE begodu sasebenzisa i-time-lapse microscopy ngokuhlaziywa kwe-MEL, okuvumela ukubonwa kwe-3D okuphezulu kwamatjhuguluko enethiwekhi ye-mitochondrial ngokukhamba kwesikhathi33,38. Sibone amatjhuguluko angabonakaliko kodwana aqakathekileko emandleni we-mitochondrial ngaphasi kokugandeleleka kwe-PPA (Umfanekiso 2). Ku-3 mM, inani lezehlakalo zokuhlukana lakhula khulu, ngesikhathi izehlakalo zokuhlanganiswa zihlala zifana nezokulawula. Ukwanda kwenani lezehlakalo zokuhlukana nokuhlanganiswa kwabonwa ku-5 mM PPA, kodwana amatjhuguluko la bekalingana, okutjengisa bona ukuhlukana nokuhlangana kufinyelela ukulingana emazingeni aphezulu (Umfanekiso 2b). Ivolumu ephakathi ye-mitochondrial yahlala ingatjhuguluki ku-3 kanye ne-5 mM PPA, okutjengisa bona ukuthembeka kwenethiwekhi ye-mitochondrial kwalondolozwa (Umfanekiso 2d). Lokhu kutjengisa ikghono lamanethiwekhi we-mitochondrial aguquguqukako wokuphendula ekugandelelekeni okuncani kwe-metabolic ukugcina kuhle i-homeostasis ngaphandle kokubangela ukuhlukana kwenethiwekhi. Ku-3 mM PPA, ukwanda kokuhlukana kwanele ukukhuthaza ukutjhugulukela ekulinganiseni okutjha, kodwana ukulungiswa okujulileko kwe-kinetic kuyatlhogeka ukuphendula ukugandeleleka okubangelwa ziinqhema eziphezulu ze-PPA.
Inani lama-mitochondria lakhula kiwo womabili amazinga wokugandeleleka kwe-PPA, kodwana ivolumu ye-mitochondria ephakathi azange itjhuguluke khulu (Umfanekiso 2c). Lokhu kungabangelwa kukwanda kwe-biogenesis nofana ukwanda kokuhlukana; nanyana kunjalo, lokha nakungabi nokwehla okukhulu kwevolumu ye-mitochondrial, kungenzeka bona i-biosynthesis ikhuphuke. Kodwana, idatha ekuMfanekisweni 2 isekela ubukhona beendlela ezimbili zokubuyisela: ukwanda kwenani lezehlakalo zokuhlukana, okuhambisana nokulawulwa kokuhlukana kwe-mitochondrial, kanye nokwanda kwenani lezehlakalo, okuhambisana ne-biogenesis ye-mitochondrial. Ekugcineni, ukubhadela okunamandla kokugandeleleka okuncani kungaba neenkambo ezithatha isikhathi sinye ezibandakanya ukuhlukana, ukuhlanganiswa, ukwakheka kwezinto eziphilako, kanye ne-mitophagy. Nanyana abatloli bangaphambilini batjengise bona i-PPA ikhuphula i-mitosis30,39 kanye ne-mitophagy29, sinikela ubufakazi bokubuyiselwa kwe-mitochondrial fission kanye nokuhlangana kwamandla ngokuphendula i-PPA. Idatha le iqinisekisa amatjhuguluko wesakhiwo esibonwe yi-TEM begodu inikela ngokuzwisisa okungeziweko ngeendlela ezihlobene nokungasebenzi kuhle kwe-mitochondrial okubangelwa yi-PPA.
Ngombana ukuhlaziywa kwe-TEM nofana kwe-MEL akuzange kunikele ubufakazi obuqondileko beendlela zokulawula izakhi zofuzo ezisekela amatjhuguluko we-morphological abonwako, sihlole ukuvezwa kwe-RNA yamajini abandakanyeka ekusebenzeni kwe-mitochondrial, i-biogenesis, kanye nokuguquguquka. I-cMYC proto-oncogene yi-transcription factor ebandakanyeka ekulawuleni i-mitochondria, i-glycolysis, i-amino acid kanye ne-fatty acid metabolism40. Ukungezelela, i-cMYC yaziwa ngokuthi ilawula ukuvezwa kwamajini we-mitochondrial angaba ma-600 abandakanyeka ekutlolweni kwe-mitochondrial, ukutjhugululwa, nokuhlanganiswa okuhlukahlukeneko, kufaka hlangana i-NRF1 ne-TFAM41. I-NRF1 ne-TFAM ziinlawuli ezimbili eziqakathekileko ze-mitosis, ezisebenza phasi kwe-PGC-1α ukuvuselela ukuphindaphindwa kwe-mtDNA. Indlela le yenziwa yi-cAMP ne-AMPK begodu iyazwela ekusetjenzisweni kwamandla nokugandeleleka kwe-metabolic. Sibuye sahlolisisa i-NFE2L2, umlawuli we-redox we-mitochondrial biogenesis, ukuthola bona imiphumela ye-PPA ingalawulwa kukugandeleleka kwe-oxidative.
Nanyana ukuvezwa kwe-NFE2L2 kungatjhuguluki, sithole ukwehla okungaguqukiko okuthembele emthamo ekuvezweni kwe-cMYC, NRF1 kanye ne-TFAM ngemva kwama-awa ama-24 wokwelatjhwa nge-3 mM ne-5 mM PPA (Umfanekiso 3a–c). Ukunciphisa ukuvezwa kwe-cMYC kuke kwabikwa ngaphambilini njengempendulo yokugandeleleka kwe-mitochondrial42, begodu ngokuphambene nalokho, ukunciphisa ukuvezwa kwe-cMYC kungabangela ukungasebenzi kuhle kwe-mitochondrial ngokuvuselela umzimba we-mitochondrial, ukuxhumana kwenethiwekhi, nokuhlukaniswa kwe-membrane43. Ngokuthakazelisako, i-cMYC nayo ibandakanyeka ekulawuleni ukuhlukaniswa kwe-mitochondrial nokuhlanganiswa42,43 begodu yaziwa ngokukhuphula i-DRP1 phosphorylation kanye nokuhlala kwe-mitochondrial ngesikhathi sokuhlukaniswa kwamaseli44, kanye nokulamula ukulungiswa kabutjha kwe-mitochondrial kumaseli we-neuronal45. Kwamambala, i-cMYC-deficient fibroblasts itjengisa ukwehlisa ubukhulu be-mitochondrial, okuhambisana namatjhuguluko abangelwa kugandeleleka kwe-PPA43. Idatha le itjengisa ubudlelwano obuthakazelisako kodwana obungakacaci hlangana ne-cMYC kanye nokuguquguquka kwe-mitochondrial, okunikela umnqopho othakazelisako weemfundo zesikhathi esizako zokuvuselelwa okubangelwa kugandeleleka kwe-PPA.
Ukwehliswa kwe-NRF1 ne-TFAM kuhambisana nendima ye-cMYC njengesisebenzi esiqakathekileko sokutlola. Idatha le iyahambisana neemfundo zangaphambilini emaseli womdlavuza wekoloni wabantu ezitjengisa bona i-PPA yehlisa ukuvezwa kwe-NRF1 mRNA emahoreni ama-22, ebekuhlotjaniswa nokuphelelwa yi-ATP begodu kwandisa i-ROS46. Abatloli laba baphinde babika bona ukuvezwa kwe-TFAM kwanda emahoreni ayi-8.5 kodwana kwabuyela emazingeni wokuthoma emahoreni ama-22. Ngokuphikisako, u-Kim et al. (2019) watjengisa bona ukuvezwa kwe-TFAM mRNA kwehle khulu ngemva kwama-awa ama-4 wokugandeleleka kwe-PPA kumaseli we-SH-SY5Y; kodwana, ngemva kwama-awa ama-72, ukuvezwa kwamaphrotheyini we-TFAM kwakhuphuka khulu begodu inani lekhophi ye-mtDNA lakhuphuka khulu. Ngalokho, ukwehla kwenani lamajini we-mitochondrial biogenesis esiwabonileko ngemva kwama-awa ama-24 akutjho bona ukwanda kwenani lama-mitochondria kuhlotjaniswa nokusebenza kwe-biogenesis ngesikhathi sangaphambilini. Iimfundo zangaphambilini zitjengise bona i-PPA ikhuphula khulu i-PGC-1α mRNA namaphrotheyini kumaseli we-SH-SY5Y emahoreni ama-4 nemizuzu ema-30, ngesikhathi i-propionic acid ikhuphula i-mitochondrial biogenesis kuma-hepatocytes wethole nge-PGC-1α ema-awa ali-12 nama-39 wemizuzu. Ngokukarisako, i-PGC-1α akusiwo umlawuli wokutlola oqondileko we-NRF1 ne-TFAM kwaphela, kodwana itjengiswe nokuthi ilawula umsebenzi we-MFN2 ne-DRP1 ngokulawula ukuhlukana nokuhlanganiswa47. Nakuthathwa ndawonye, ​​lokhu kuveza ukuhlangana okuseduze kweendlela ezilawula iimpendulo ze-mitochondrial ezibangelwa yi-PPA. Ngaphezu kwalokho, idatha yethu itjengisa ukungalungi okukhulu kokulawulwa kokutloliswa kwe-biogenesis kanye nokugaya ukudla ngaphasi kokugandeleleka kwe-PPA.
Amajini we-STOML2, OPA1, MFN1, MFN2 kanye ne-DRP1 aphakathi kwabalawuli abaphakathi bokuhlukana kwe-mitochondrial, ukuhlanganiswa kanye nokuguquguquka37,48,49. Kunamanye amajini amanengi abandakanyeka ekuguquguqukeni kwe-mitochondrial, nanyana kunjalo, i-STOML2, i-OPA1 kanye ne-MFN2 ngaphambilini sele itholakele bona i-methylated ngokuhlukileko emaqenjini we-ASD,16 begodu iimfundo ezinengi ezizijameleko zibike amatjhuguluko kilezi zinto zokutlola ngokuphendula ukugandeleleka kwe-mitochondrial50,51. 52. Ukuvezwa kwe-OPA1 ne-STOML2 kwehliswe khulu nge-3 mM ne-5 mM ye-PPA (Umfanekiso 3e, f). I-OPA1 ngenye yeenlawuli ezijayelekileko zokuhlanganiswa kwe-mitochondrial ngokusebenzisana ngqo ne-MFN1 ne-2 begodu idlala indima ekuvuseleleni ama-cristae kanye ne-mitochondrial morphology53. Indima eqondileko ye-STOML2 ekuguquguqukeni kwe-mitochondrial ayikacaci, kodwana ubufakazi butjengisa bona idlala indima ekuhlanganisweni kwe-mitochondrial, i-biogenesis, kanye ne-mitophagy.
I-STOML2 ibandakanyeka ekugcineni ukuhlanganiswa kokuphefumula kwe-mitochondrial nokwakheka kwama-respiratory chain complexes54,55 begodu itjengiswe bona itjhugulula khulu ubujamo bokugaya ukudla kwamaseli womdlavuza56. Iimfundo zitjengise bona i-STOML2 ikhuthaza amandla we-mitochondrial membrane kanye ne-biogenesis ngokusebenzisana ne-BAN kanye ne-cardiolipin 55, 57, 58. Ngaphezu kwalokho, iimfundo ezizijameleko zitjengise bona ukusebenzisana hlangana ne-STOML2 ne-PINK1 kulawula i-mitophagy59,60. Ngokuphawulekako, i-STOML2 ibikwe bona isebenzisana ngqo ne-MFN2 begodu idlala indima eqakathekileko ekuzinziseni ama-isoform we-OPA1 amade ngokuvimbela i-protease enesibopho sokuwohloka kwe-OPA153,61,62. Ukwehla kokuvezwa kwe-STOML2 okubonwe ekuphenduleni kwe-PPA kungenza amaphrotheyini we-fusion la abe sengozini yokuwohloka ngeendlela ezithembele ku-ubiquitin- kanye ne-proteasome48. Nanyana indima eqondileko ye-STOML2 ne-OPA1 ekuphenduleni okuguquguqukako kwe-PPA ingacaci, ukwehla kokuvezwa kwamajini we-fusion (Umfanekiso 3) kungaphazamisa ukulingana hlangana nokuhlukana nokuhlanganiswa begodu kurholele ekwehleni kobukhulu be-mitochondrial (Umfanekiso 3). 1).
Ngakelinye ihlangothi, ukuvezwa kwamaphrotheyini we-OPA1 kwahlala kungatjhuguluki ngemva kwama-awa ama-24, ngesikhathi amazinga we-mRNA namaphrotheyini we-MFN1, MFN2 nofana i-DRP1 azange atjhuguluke khulu ngemva kokwelatjhwa nge-PPA (Umfanekiso 3g-i, Umfanekiso 4). Lokhu kungatjengisa bona akukho amatjhuguluko ekulawulweni kwalezi zinto ezibandakanyeka ekuhlanganisweni kwe-mitochondrial nokuhlukana. Kodwana, kufanele kuqashelwe bona ngalinye lamajini la amane nalo lilawulwa ziintjhuguluko ze-posttranscriptional (PTMs) ezilawula ukusebenza kwamaphrotheyini. I-OPA1 inezinye iinhlobo ezisibhozo ezihlukaniswa nge-proteolytically ku-mitochondria ukukhiqiza ama-isoform amabili ahlukileko 63 . Ukulinganisela hlangana nama-isoform amade namafitjhani ekugcineni kunquma indima ye-OPA1 ekuhlanganisweni kwe-mitochondrial nokugcinwa kwenethiwekhi ye-mitochondrial64. Umsebenzi we-DRP1 ulawulwa yi-calcium/calmodulin-dependent protein kinase II (CaMKII) phosphorylation, ngesikhathi ukuwohloka kwe-DRP1 kulawulwa yi-ubiquitination kanye ne-SUMOylation65. Ekugcineni, zombili i-DRP1 ne-MFN1/2 zii-GTPases, ngalokho umsebenzi ungathonywa lizinga lokukhiqizwa kwe-GTP ku-mitochondria 66 . Ngalokho-ke, nanyana ukuvezwa kwamaphrotheyini la kuhlala kunjalo, lokhu kungenzeka kungatjengisi umsebenzi wamaphrotheyini ongatjhugulukiko nofana ukubekwa endaweni67,68. Kwamambala, amaphrotheyini we-PTM akhona kanengi asebenza njengendlela yokuthoma yokuzivikela enesibopho sokulawula iimpendulo zokugandeleleka okubukhali. Nakube nokugandeleleka okulinganiselweko kwe-metabolic emfanekisweni wethu, kungenzeka bona i-PTM ikhuthaza ukusebenza okukhulu kwamaphrotheyini we-fusion no-fission ukubuyisela ngokwaneleko ubuqotho be-mitochondrial ngaphandle kokutlhoga ukusebenza okungezelelweko kwamajini la ezingeni le-mRNA nofana lamaphrotheyini.
Nakuthathwa ndawonye, ​​idatha engenhla iveza ukulawulwa okuyinkimbinkimbi nokuthembele esikhathini kwe-mitochondrial morphology kanye neentjhijilo zokucacisa iindlela lezi. Ukufunda ukuvezwa kwezakhi zofuzo, kutlhogeka kokuthoma ukukhomba amajini athileko ahlosiweko endleleni. Kodwana, idatha yethu itjengisa bona amajini asendleleni efanako awaphenduli ngendlela efanako ekugandelelekeni okufanako. Kuhlekuhle, iimfundo zangaphambilini zitjengise bona amajini ahlukileko endleleni efanako angatjengisa amaphrofayili ahlukileko wokuphendula kwesikhatjhana30,46. Ukungezelela, kuneendlela eziyinkimbinkimbi zokutlolwa ngemva kokutlolwa eziphazamisa ubudlelwano hlangana nokutlolwa nokusebenza kwezakhi zofuzo. Iimfundo ze-proteomic zinganikela ukuzwisisa ngomthelela wama-PTM nomsebenzi wamaphrotheyini, kodwana ziveza neentjhijilo ezifaka hlangana iindlela eziphasi, amazinga aphezulu wesignali-kuya-nomsindo, kanye nokulungiswa okumbi.
Ngalokhu, ukufunda ubujamo be-mitochondrial ngokusebenzisa i-TEM ne-MEL kunekghono elikhulu lokuphendula imibuzo eqakathekileko malungana nobudlelwano hlangana nokusebenza kwe-mitochondrial nokuthi lokhu kunomthelela njani emalwelweni. Okuqakatheke khulu, i-TEM inikela ngendlela eqondileko yokulinganisa ukubunjwa kwe-mitochondrial njengesiphetho esihlanganako sokungasebenzi kuhle kwe-mitochondrial51. I-MEL godu inikela ngendlela eqondileko yokubona ngelihlo lengqondo izehlakalo zokuhlukana nokuhlanganiswa endaweni yamaseli enemikhakha emithathu, okuvumela ukulinganisa ukuvuselelwa kwama-mitochondrial ngitjho nanyana kungekho amatjhuguluko ekuvezweni kwezakhi zofuzo33. Lapha siveza ukusetjenziswa kwamaqhinga wokuthatha iinthombe ze-mitochondrial emalwelweni wesibili we-mitochondrial. Amalwelwe la avamise ukubonakala ngokugandeleleka okuncani okungapheliko okuvezwa kuvuselelwa okufihlakeleko kwamanethiwekhi we-mitochondrial kunokulimala okubukhali kwe-mitochondrial. Kodwana, ukubuyiselwa kwe-mitochondrial okutlhogekako ukugcina i-mitosis ngaphasi kokugandeleleka okungapheliko kunemiphumela emikhulu yokusebenza. Ngokwesayensi yezinzwa, ukuzwisisa ngcono iindlela zokubuyisela kunganikela ilwazi eliqakathekileko nge-pleiotropic neuropathology ehlotjaniswa nokungasebenzi kuhle kwe-mitochondrial.
Ekugcineni, idatha yethu iveza ukusetjenziswa kwamaqhinga wokuthatha iinthombe ukuzwisisa imiphumela yokusebenza yokusebenzisana okuhlukahlukeneko hlangana nokuvezwa kwezakhi zofuzo, ukutjhugululwa kwamaphrotheyini, nomsebenzi wamaphrotheyini olawula amandla we-neuronal mitochondrial. Sisebenzise i-PPA ukumodela ukungasebenzi kuhle kwe-mitochondrial kumodeli yamaseli we-neuronal ukuthola ilwazi ngengcenye ye-mitochondrial ye-ASD. Amaseli we-SH-SY5Y aphathwe nge-PPA atjengisa amatjhuguluko ebujameni be-mitochondrial: ama-mitochondria aba mancani begodu ayindilinga, begodu ama-cristae bekangahlathululi kuhle lokha nakabonwa yi-TEM. Ukuhlaziywa kwe-MEL kutjengisa bona amatjhuguluko la enzeka ngesikhathi sinye nokwanda kwezehlakalo zokuhlukana nokuhlanganiswa ukugcina inethiwekhi ye-mitochondrial ngokuphendula ukugandeleleka okuncani kwe-metabolic. Ngaphezu kwalokho, i-PPA iphazamisa khulu ukulawulwa kokutlolwa kwe-mitochondrial metabolism kanye ne-homeostasis. Sithole i-cMYC, i-NRF1, i-TFAM, i-STOML2, kanye ne-OPA1 njengeenlawuli eziqakathekileko ze-mitochondrial eziphazanyiswa kugandeleleka kwe-PPA begodu zingadlala indima ekulamuleni amatjhuguluko abangelwa yi-PPA ekubunjweni kwe-mitochondrial nomsebenzi. Iimfundo zesikhathi esizako ziyatlhogeka ukuhlathulula ngcono amatjhuguluko wesikhatjhana abangelwa yi-PPA ekuvezweni kwezakhi zofuzo nomsebenzi wamaphrotheyini, ukubekwa endaweni, nokutjhuguluka ngemva kokuhumusha. Idatha yethu iveza ubudisi nokuthembelana kweendlela zokulawula ezilawula ukuphendula kokugandeleleka kwe-mitochondrial begodu itjengisa ukusetjenziswa kwe-TEM namanye amaqhinga wokuthatha iinthombe eenhlolweni eziqondiswe khulu.
Umugqa wamaseli we-SH-SY5Y (ECACC, 94030304-1VL) wathengwa ku-Sigma-Aldrich. Amaseli we-SH-SY5Y akhuliswa ku-Dulbecco's modified Eagle's medium/F-12 nutrient mixture (DMEM/F-12) kanye ne-L-glutamine (SC09411, ScienCell) ngaphakathi kwamaflaski angama-25 cm2 angezelelwe nge-20% ye-fetal bovine serum (FBS) (1936) kanye ne-ScienCell 1% i-penicillin-streptomycin (P4333-20ML, Sigma-Aldrich) ku-37 °C, 5% CO2. Amaseli akhuliswa ngaphasi kwe-80% ye-confluence kusetjenziswa i-0.05% trypsin-EDTA (15400054, ThermoFisher Scientific), ahlanganiswa nge-300 g begodu afakwe nge-density engaba ngu-7 × 105 amaseli/ml. Zoke iimhloliso zenziwa kumaseli we-SH-SY5Y angakahlukaniswa hlangana namavesi 19 ukuya ku-22. I-PPA iphathwa njenge-NaP. Ncibilikisa ipuyere ye-NaP (i-CAS No. 137-40-6, ifomula yamakhemikhali C3H5NaO2, P5436-100G, i-Sigma-Aldrich) emanzini afuthumeleko we-MilliQ ukuya ekuhlanganisweni kwe-1 M bese uyigcina ku-4 °C. Ngelanga lokwelatjhwa, nciphisa isisombululo lesi nge-1 M PPA ukuya ku-3 mM kanye ne-5 mM PPA ngaphakathi kwe-serum-free medium (DMEM/F-12 nge-L-glutamine). Ukugxila kokwelatjhwa kwazo zoke iimhloliso bekungekho PPA (0 mM, ukulawula), 3 mM, kanye ne-5 mM PPA. Iimhloliso zenziwa okungenani ekuphindaphindweni okuthathu kwezinto eziphilako.
Amaseli we-SH-SY5Y atjalwa ngaphakathi kwamaflaski ama-25 cm5 ngezinga elingu-5.5 × 105 amaseli/ml begodu akhuliswa ama-awa ama-24. Ukwelatjhwa kwe-PPA kwafakwa eflaskini ngaphambi kwama-awa ama-24 wokufukamela. Buthelela ama-pellets wamaseli ngokulandela amaphrothokholi ajayelekileko wokukhula kwamathitjhu weenlwana ezimunyisako (echazwe ngehla). Faka kabutjha i-cell pellet ku-100 μl 2.5% glutaraldehyde, 1× PBS bese uyigcina ku-4 °C bekube kulapho isetjenziswa. Amaseli we-SH-SY5Y ahlanganiswa kafitjhani ukukhipha amaseli begodu asuse i-2.5% ye-glutaraldehyde, isisombululo se-1× PBS. Faka kabutjha inkunkuma ku-4% ye-agarose gel elungiselelwe emanzini ahlanjululweko (isilinganiso se-agarose nomthamo we-sediment ngu-1:1). Iingcezu ze-agarose zafakwa emagridini emapuletini aphasi begodu zagcotshwa nge-1-hexadecene ngaphambi kokuqandisa okuphezulu. Amasampula afriziwe ku-100% ye-asetoni eyomileko ku--90°C ama-awa ama-24. Izinga lokutjhisa laphakanyiswa laya ku--80°C begodu kwafakwa isisombululo se-1% ye-osmium tetroxide kanye ne-0.1% ye-glutaraldehyde. Amasampula agcinwe ku--80°C ama-awa ama-24. Ngemva kwalokhu, izinga lokutjhisa lakhuphuka kancanikancani laya emazingeni womtjhiso wegumbi emalangeni ambalwa: ukusuka ku-80 °C ukuya ku-50 °C ama-awa ama-24, ukuya ku-30 ​​°C ama-awa ama-24, ukuya ku-10 °C ama-awa ama-24 begodu ekugcineni ukuya emazingeni womtjhiso wegumbi. izinga lokutjhisa.
Ngemva kokulungiselela kwe-cryogenic, amasampula afakwe nge-resin begodu iingaba ezincani khulu (∼100 nm) zenziwa kusetjenziswa i-Leica Reichert UltracutS ultramicrotome (Leica Microsystems). Iingcenye zafakwa ibala nge-2% ye-uranyl acetate kanye ne-lead citrate. Amasampula atjhejwa kusetjenziswa i-FEI Tecnai 20 transmission electron microscope (i-ThermoFisher (gade ibiyi-FEI), e-Eindhoven, i-The Netherlands) esebenza ku-200 kV (i-Lab6 transmitter) kanye nekhamera ye-Gatan CCD (i-Gatan, UK) ene-Tridiem energy filter.
Ekuphindaphindweni ngalinye kwezobuchwepheshe, okungenani iinthombe ezima-24 zeseli elilodwa zatholwa, okwenza iinthombe ezima-266. Zoke iinthombe zahlaziywa kusetjenziswa i-Region of Interest (ROI) macro kanye ne-Mitochondria macro. I-mitochondrial macro isekelwe eendleleni ezigadangisiweko17,31,32 begodu ivumela ukucubungula okuzenzakalelayo kweenthombe ze-TEM ku-Fiji/ImageJ69. Ngamafuphi: isithombe sitjhugululwa begodu sitjhugululwa kusetjenziswa ukususwa kwesizinda sebhola elijikajikako (irediyasi yamaphikseli ama-60) kanye nesefo ye-FFT bandpass (ngokusebenzisa imikhawulo ephezulu nephansi yamaphikseli ama-60 nama-8 ngokulandelana) kanye nokugandeleleka kwelayini eqondileko ngokubekezelela ukuqondiswa kwe-5%. Isithombe esisetjenzisiweko sibekelwe umkhawulo ngokuzenzakalelako kusetjenziswa i-algorithm ye-entropy ephezulu begodu imaski ye-binary iyakhiwa. Iindawo zesithombe ezihlobene nama-ROI akhethwe ngesandla eenthombeni ze-TEM ezihlaza zakhishwa, ziveza i-mitochondria begodu zingafaki i-plasma membrane nezinye iindawo ezine-contrast ephezulu. Ku-ROI ngayinye ekhutjhweko, iinhlayiya ezimbili ezikulu ukudlula amaphikseli ama-600 zahlaziywa, begodu indawo yeenhlayiya, umkhawulo, ama-axes amakhulu namancani, ububanzi be-Feret, ubuyindilinga, nokujikeleza kwalinganiswa kusetjenziswa imisebenzi yokulinganisa eyakhelwe ngaphakathi ye-Fiji/ImageJ. Ukulandela u-Merrill, u-Flippo, no-Strack (2017), indawo 2, isilinganiso se-particle (i-major to minor axis ratio), kanye ne-shape factor (FF) zabalwa ukusuka kuledatha, lapho i-FF = umkhawulo 2/4pi x indawo. Ihlathululo yefomula yepharamitha ingatholakala ku-Merrill, Flippo, no-Strack (2017). Ama-macros akhulunywe ngawo ayatholakala ku-GitHub (qala isitatimende sokutholakala kwedatha). Ngokwesilinganiso, pheze iinhlayiya ezima-5,600 zahlaziywa ngokwelashwa kwe-PPA, ukuthola inani elingaba ziinhlayiya eziyi-17,000 (idatha ayitjengiswanga).
Amaseli we-SH-SH5Y afakwa eendaweni ezine-8-chamber culture dishes (ThermoFisher, #155411) ukuvumela ukunamathela ubusuku boke bese afukanywa nge-TMRE 1:1000 (ThermoFisher, #T669) kanye ne-Hoechst 33342 1:200 (Sigma-Aldrich, #155411). ukudaya. Iinthombe zatholwa kusetjenziswa ama-laser we-405 nm kanye ne-561 nm phezu kwebhoduluko lemizuzu eli-10, begodu iinthombe ezingakalungiswa zatholwa njenge-z-stacks eneenthombe ezili-10 zemifanekiso ene-az step ye-0.2 μm hlangana namafreyimu wesithombe emaphuzwini wesikhathi ali-12 alandelako. Iinthombe zabuthelelwa kusetjenziswa i-Carl Zeiss LSM780 ELYRA PS.1 super-resolution platform (Carl Zeiss, Oberkochen, Germany) kusetjenziswa i-LCI Plan Apochromate 100x/1.4 Oil DIC M27 lens. Iinthombe zahlaziywa ku-ImageJ kusetjenziswa iphayiphi echazwe ngaphambilini kanye ne-plugin ye-ImageJ ukulinganisa izehlakalo zokuhlanganiswa nokuhlukana, inani eliphakathi lezakhiwo ze-mitochondrial, kanye nevolumu ye-mitochondrial ephakathi ngeseli33. I-MEL macros iyatholakala ku-GitHub (qala isitatimende sokutholakala kwedatha).
Amaseli we-SH-SY5Y akhuliswa emapuletini aneenthombe ezisithandathu ngobukhulu obungu-0.3 × 106 amaseli/mL ama-awa ama-24 ngaphambi kokwelatjhwa. I-RNA yakhishwa kusetjenziswa iphrothokholi ye-Quick-RNATM Miniprep (ZR R1055, Zymo Research) ngokutjhugululwa okuncani: faka i-300 μl ye-RNA lysis buffer emthonjeni ngamunye ngaphambi kokususwa bese ukhipha isampula ngayinye njengegadango lokugcina nge-30 μl ye-DNase/RNase elution. -amanzi wamahhala. Woke amasampula ahlolwa ubunengi nekhwalithi kusetjenziswa i-NanoDrop ND-1000 UV-Vis Spectrophotometer. Iphrotheyini epheleleko evela kuma-lysates wamaseli itholwe kusetjenziswa i-200 μl ye-RIPA lysis buffer, begodu ukugxila kwamaphrotheyini kwalinganiswa kusetjenziswa i-Bradford protein assay70.
Ukwenziwa kwe-cDNA kwenziwa kusetjenziswa i-TetroTM cDNA Synthesis Kit (BIO-65043, Meridian Bioscience) ngokuya kwemilayo yomkhiqizi ngokutjhugulula okuthileko. I-cDNA yakhiwe ngama-20-μl wokusabela kusetjenziswa i-0.7 ukuya ku-1 μg ye-RNA epheleleko. Ama-primers akhethwe emaphepheni atjhatjalaliswe ngaphambilini 42, 71, 72, 73, 74, 75, 76, 77, 78 (Ithebula S1) begodu ama-probe akhambisana nawo aklanyelwe kusetjenziswa ithulusi le-PrimerQuest elivela ku-Integrated DNA Technologies. Woke amajini athakazelisako ajwayelekileko abe yijini ye-B2M yenyukliya. Ukuvezwa kwezakhi zofuzo ze-STOML2, NRF1, NFE2L2, TFAM, cMYC kanye ne-OPA1 kwalinganiswa nge-RT-qPCR. Umxube omkhulu ufaka hlangana i-LUNA Taq polymerase (M3003L, New England Biolabs), i-10 μM yama-primers aya phambili nabuyela emuva, i-cDNA, kanye namanzi we-PCR-grade ukukhiqiza ivolumu yokugcina ye-10 μL yokuphendula ngayinye. Ukuvezwa kwamajini wokuhlukana nokuhlukana (DRP1, MFN1/2) kwalinganiswa kusetjenziswa i-TaqMan multiplex assays. I-Luna Universal Probe qPCR Master Mix (M3004S, New England Biolabs) yasetjenziswa ngokuya kwemilayo yomkhiqizi ngokutjhugulula okuncani. I-multiplex RT-qPCR master mix ifaka hlangana i-1X LUNA Taq polymerase, i-10 μM yama-primers aya phambili nabuyela emuva, i-10 μM ye-probe, i-cDNA, kanye namanzi we-PCR-grade, okurholela ekutheni kube nomthamo wokugcina wama-20 μL wokuphendula ngamunye. I-RT-qPCR yenziwa kusetjenziswa i-Rotor-Gene Q 6-plex (QIAGEN RG—inombolo yomlandelande: R0618110). Ubujamo bokukhamba ngebhayisikili butjengiswe kuThebuli S1. Woke amasampula we-cDNA akhuliswa kathathu begodu ijika elijwayelekileko lakhiwa kusetjenziswa uchungechunge lokuhlanjululwa okuphindwe kalitjhumi. Ama-outliers kumasampula aphindwe kathathu ane-cycle threshold standard deviation (Ct) >0.5 asuswe ekuhlaziyweni ukuqinisekisa ukukhiqizwa kwedatha30,72. Ukuvezwa kwezakhi zofuzo okuhlobeneko kwabalwa kusetjenziswa indlela ye-2-ΔΔCt79.
Amasampula wamaphrotheyini (60 μg) ahlanganiswa ne-Laemmli loading buffer nge-2:1 ratio begodu asebenza nge-12% yamaprotheyini angenambala (Bio-Rad #1610184). Amaphrotheyini adluliselwa ku-PVDF (polyvinylidene fluoride) membrane (#170-84156, Bio-Rad) kusetjenziswa i-Trans-Blot Turbo system (#170-4155, Bio-Rad). I-membrane yavalwa begodu yafukanywa ngama-antibodies afaneleko (OPA1, MFN1, MFN2, kanye ne-DRP1) (ehlanjululwe ngo-1:1000) ama-awa ama-48, kwalandela ukufukamela ngama-antibodies wesibili (1:10,000) i-awa elilodwa. Ama-membrane abese athwetshulwa kusetjenziswa i-Clarity Western ECL Substrate (#170-5061, Bio-Rad) begodu arekhodwa kusetjenziswa i-Bio-Rad ChemiDoc MP system. I-ImageLab version 6.1 yasetjenziselwa ukuhlaziywa kwe-Western blot. Ijeli yasekuthomeni ne-blot zitjengiswe kuMfanekiso S1. Ilwazi lama-antibody linikelwe kuThebuli S2.
Amasethi wedatha avezwa njengesilinganiso kanye nephutha elijwayelekileko lesilinganiso (SEM) okungenani samasampula amathathu azijameleko. Amasethi wedatha ahlolwa ukujayeleka kusetjenziswa i-Shapiro-Wilks test (ngaphandle kobana kutjhiwo ngenye indlela) ngaphambi kokuthatha ukusabalalisa kwe-Gaussian nokuphambuka okulinganako begodu kuragele phambili nokuhlaziywa. Ngaphezu kokuhlaziya isethi yedatha kusetjenziswa i-Fisher's MEL LSD (p < 0.05), i-ANOVA yendlela eyodwa (isilinganiso sokwelapha vs. ukulawula), kanye nokuhlolwa kokumadanisa okunengi kwe-Dunnett ukuthola ukuqakatheka (p < 0.05). Amanani we-p aqakathekileko atjengiswa egrafwini njenge-*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Zoke iinhlaziyo zezibalo namagrafu zenziwa begodu zenziwa kusetjenziswa i-GraphPad Prism 9.4.0.
Ama-macro we-Fiji/ImageJ wokuhlaziywa kwemifanekiso ye-TEM atholakala tjhatjhalazi ku-GitHub: https://github.com/caaja/TEMMitoMacro. I-Mitochondrial Event Locator (MEL) itholakala emphakathini ku-GitHub: https://github.com/rensutheart/MEL-Fiji-Plugin.
U-Meiliana A., u-Devi NM no-Vijaya A. I-Mitochondria: abalawuli abakhulu be-metabolism, i-homeostasis, ukugandeleleka, ukuguga kanye ne-epigenetics. Isi-Indonesia. Isayensi yezokwelapha. J. 13, 221–241 (2021).
U-Ben-Shachar, D. Ukungasebenzi kuhle kwe-mitochondrial okuhlukahlukeneko ku-schizophrenia, i-complex I njengethagethi engenzeka. I-Schizophrenia. umthombo. 187, 3–10 (2017).
U-Bose, A. no-Beal, MF Ukungasebenzi kuhle kwe-Mitochondrial ebulwelweni be-Parkinson. J. I-Neurochemistry. 139, 216–231 (2016).
U-Sharma VK, u-Singh TG no-Mehta V. I-mitochondria egandelelekileko: amathagethi wokuhlasela esifo se-Alzheimer. I-Mitochondria 59, 48–57 (2021).
U-Belenguer P., u-Duarte JMN, u-Shook PF no-Ferreira GK I-Mitochondria nobuchopho: i-bioenergetics nokunye. Ama-Neurotoxins. umthombo. 36, 219–238 (2019).
Rangaraju, V. et al. I-Pleiotropic mitochondria: umthelela we-mitochondria ekuthuthukisweni kwe-neuron kanye namalwelwe. J. Isayensi yeenzwa. 39, 8200–8208 (2019).
U-Cardano-Ramos, C. no-Morais, VA I-Mitochondrial biogenesis kuma-neuron: njani begodu kuphi. ubujamo beentjhabatjhaba. J. Mohr. isayensi. 22, 13059 (2021).
U-Yu, R., Lendahl, U., Nister, M. no-Zhao, J. Ukulawulwa kwama-mitochondrial weenlwana ezimunyisako: amathuba neentjhijilo. phambili. i-endocrine. (Lausanne) 11, 374 (2020).
U-Khacho, M. no-Slack, RS Ukuguquguquka kwe-mitochondrial ekulawuleni i-neurogenesis: ukusuka ebuchosheni obukhulako ukuya ebuchosheni bomuntu omdala. ituthuko. ezinamandla. 247, 47–53 (2018).