Zikomo pochezera Nature.com. Mtundu wa msakatuli womwe mukugwiritsa ntchito uli ndi chithandizo chochepa cha CSS. Kuti mupeze zotsatira zabwino, tikukulimbikitsani kuti mugwiritse ntchito mtundu watsopano wa msakatuli wanu (kapena letsani Compatibility Mode mu Internet Explorer). Pakadali pano, kuti tiwonetsetse kuti chithandizo chikupitilira, tikuonetsa tsamba lino popanda kukongoletsa kapena JavaScript.
Propionic acid (PPA) imagwiritsidwa ntchito pophunzira udindo wa kusokonekera kwa mitochondrial mu matenda a neurodevelopmental monga autism spectrum disorder. PPA imadziwika kuti imasokoneza biogenesis ya mitochondrial, kagayidwe kachakudya, ndi kusintha kwa maselo. Komabe, zotsatira za PPA pa mitochondrial dynamics, fission ndi fusion zimakhalabe zovuta chifukwa cha zovuta za nthawi ya njirazi. Pano, tikugwiritsa ntchito njira zowerengera zowerengera kuti tifufuze momwe PPA imakhudzira mitochondrial ultrastructure, morphology, ndi dynamics m'maselo a SH-SY5Y ofanana ndi neuron. PPA (5 mM) idapangitsa kuchepa kwakukulu kwa malo a mitochondrial (p < 0.01), m'mimba mwake ndi m'zigawo (p < 0.05), ndi dera 2 (p < 0.01). Kusanthula kwa malo a mitochondrial event locator kunawonetsa kuwonjezeka kwakukulu (p < 0.05) mu zochitika za fission ndi fusion, motero kusunga umphumphu wa netiweki ya mitochondrial pansi pa zovuta. Kuphatikiza apo, kuchuluka kwa mRNA kwa cMYC (p < 0.0001), NRF1 (p < 0.01), TFAM (p < 0.05), STOML2 (p < 0.0001) ndi OPA1 (p < 0.05) kunachepetsedwa kwambiri. 01). Izi zikuwonetsa kusintha kwa mawonekedwe a mitochondrial, biogenesis ndi dynamics kuti zisunge ntchito pansi pa mikhalidwe yopsinjika. Deta yathu imapereka chidziwitso chatsopano pa zotsatira za PPA pa mitochondrial dynamics ndipo ikuwonetsa kufunika kwa njira zojambulira zithunzi pophunzira njira zovuta zowongolera zomwe zimakhudzidwa ndi mayankho a mitochondrial stress.
Mitochondria ndi gawo lofunika kwambiri pa ntchito zosiyanasiyana za maselo kuposa ntchito zawo zachizolowezi popanga mphamvu ndi biosynthesis. Kagayidwe ka mitochondrial ndi gawo lofunika kwambiri pa calcium signaling, metabolic ndi redox homeostasis, inflammatory signaling, epigenetic modifications, cell expansion, differentiation and programmed cell death1. Makamaka, kagayidwe ka mitochondrial ndi kofunikira kwambiri pakukula kwa mitsempha, kupulumuka ndi kugwira ntchito ndipo kumakhudzidwa kwambiri ndi zizindikiro zosiyanasiyana za neuropathology2,3,4.
M'zaka khumi zapitazi, kagayidwe kachakudya kakhala ngati chowongolera chachikulu cha neurogenesis, kusiyanitsa, kukhwima ndi plasticity5,6. Posachedwapa, mawonekedwe a mitochondrial ndi mphamvu zake zakhala zigawo zofunika kwambiri za mitosis, njira yosinthasintha yomwe imasunga dziwe la mitochondria yathanzi mkati mwa maselo. Mphamvu za mitochondrial zimayendetsedwa ndi njira zovuta zodalirana kuyambira mitochondrial biogenesis ndi bioenergetics mpaka mitochondrial fission, fusion, transport and clearance7,8. Kusokonezeka kwa njira iliyonse yolumikizirana iyi kumasokoneza kusungidwa kwa maukonde a mitochondrial athanzi ndipo kumakhala ndi zotsatirapo zazikulu pakukula kwa mitsempha9,10. Zoonadi, kusokonekera kwa mphamvu za mitochondrial kumawonedwa m'matenda ambiri amisala, neurodegenerative ndi neurodevelopmental, kuphatikizapo autism spectrum disorders (ASD)11,12.
ASD ndi matenda osiyanasiyana a neurodevelopmental omwe ali ndi kapangidwe kovuta ka majini ndi epigenetic. Kubadwa kwa ASD sikunatsutsidwe, koma chifukwa cha mamolekyu omwe ali pansi pake sichikumveka bwino. Kusonkhanitsa deta kuchokera ku zitsanzo zoyambilira, maphunziro azachipatala, ndi ma data a mamolekyu a multi-omics kumapereka umboni wowonjezereka wa kusagwira bwino ntchito kwa mitochondrial mu ASD13,14. Kale tidachita kafukufuku wa DNA methylation wa genome-wide mu gulu la odwala omwe ali ndi ASD ndipo tidapeza majini osiyanasiyana a methylated omwe amasonkhana motsatira njira za metabolic za mitochondrial15. Pambuyo pake tidanena za methylation yosiyana ya olamulira apakati a mitochondrial biogenesis ndi dynamics, zomwe zimagwirizanitsidwa ndi kuchuluka kwa mtDNA copy number ndi kusintha kwa mbiri ya metabolic ya mkodzo mu ASD16. Deta yathu imapereka umboni wowonjezereka wakuti mitochondrial dynamics ndi homeostasis zimagwira ntchito yayikulu mu pathophysiology ya ASD. Chifukwa chake, kukonza kumvetsetsa kwaukadaulo kwa ubale pakati pa mitochondrial dynamics, morphology, ndi ntchito ndi cholinga chachikulu cha kafukufuku wopitilira pa matenda amitsempha omwe amadziwika ndi kusagwira bwino ntchito kwa mitochondrial secondary.
Njira zama molekyulu nthawi zambiri zimagwiritsidwa ntchito pophunzira ntchito ya majini enaake pa mayankho a kupsinjika kwa mitochondrial. Komabe, njira iyi ikhoza kuchepetsedwa ndi mawonekedwe ambiri komanso anthawi ya njira zowongolera mitotic. Kuphatikiza apo, kufotokozera kosiyana kwa majini a mitochondrial ndi chizindikiro chosalunjika cha kusintha kwa magwiridwe antchito, makamaka popeza majini ochepa okha ndi omwe amawunikidwa nthawi zambiri. Chifukwa chake, njira zolunjika kwambiri zophunzirira ntchito ya mitochondrial ndi bioenergetics zaperekedwa17. Kapangidwe ka mitochondrial kamagwirizana kwambiri ndi mphamvu za mitochondrial. Kapangidwe ka mitochondrial, kulumikizana, ndi kapangidwe kake ndizofunikira kwambiri pakupanga mphamvu ndi kupulumuka kwa mitochondrial ndi maselo5,18. Kuphatikiza apo, zigawo zosiyanasiyana za mitosis zimayang'ana kwambiri kusintha kwa mawonekedwe a mitochondrial, komwe kungakhale ngati mapeto othandiza a kusagwira bwino ntchito kwa mitochondrial ndikupereka maziko a maphunziro otsatira a makina.
Kapangidwe ka mitochondrial kangathe kuwonedwa mwachindunji pogwiritsa ntchito ma microscopy a ma electron otumizira (TEM), zomwe zimathandiza kuphunzira mwatsatanetsatane za kapangidwe ka maselo. TEM imayang'ana mwachindunji mawonekedwe, mawonekedwe ndi kapangidwe ka mitochondrial cristae pakutha kwa mitochondria ya munthu aliyense, m'malo mongodalira kokha kulembedwa kwa majini, kufotokozera mapuloteni kapena magawo ogwirira ntchito a mitochondrial m'magulu a maselo17,19,20. Kuphatikiza apo, TEM imathandizira kuphunzira kuyanjana pakati pa mitochondria ndi ma organelle ena, monga endoplasmic reticulum ndi autophagosomes, omwe amagwira ntchito yofunika kwambiri mu ntchito ya mitochondrial ndi homeostasis21,22. Chifukwa chake, izi zimapangitsa TEM kukhala poyambira pabwino pophunzira kusokonekera kwa mitochondrial musanayang'ane njira kapena majini enaake. Pamene ntchito ya mitochondrial ikukhala yofunikira kwambiri ku neuropathology, pali kufunikira kodziwikiratu kuti athe kuphunzira mwachindunji komanso mochuluka mawonekedwe a mitochondrial ndi mphamvu mu zitsanzo za neuronal za in vitro.
Munkhaniyi, tikuyang'ana momwe mitochondrial imagwirira ntchito mu njira ya neuronal ya mitochondrial dysfunction mu autism spectrum disorder. Kale tinanena za differential methylation ya propionyl-CoA carboxylase beta (PCCB) mu ASD15, gawo laling'ono la mitochondrial propionyl-CoA carboxylase enzyme PCC. Kusokonekera kwa PCC kumadziwika kuti kumayambitsa kusonkhanitsa poizoni kwa propionyl derivatives, kuphatikizapo propionic acid (PPA)23,24,25. PPA yawonetsedwa kuti imasokoneza kagayidwe ka mitsempha ya m'mitsempha ndikusintha khalidwe la m'thupi ndipo ndi chitsanzo chodziwika bwino cha nyama chophunzirira njira zopititsira patsogolo mitsempha zomwe zimakhudzidwa ndi ASD26,27,28. Kuphatikiza apo, PPA yanenedwa kuti imasokoneza mphamvu ya mitochondrial membrane, biogenesis ndi kupuma mu vitro ndipo yagwiritsidwa ntchito kwambiri poyesa kusagwira ntchito kwa mitochondrial mu ma neuron29,30. Komabe, zotsatira za kusagwira ntchito kwa mitochondrial komwe kumabwera chifukwa cha PPA pa mawonekedwe a mitochondrial ndi mphamvu zake sizikumveka bwino.
Kafukufukuyu akugwiritsa ntchito njira zojambulira zithunzi kuti apeze zotsatira za PPA pa mawonekedwe a mitochondrial, mphamvu, ndi ntchito m'maselo a SH-SY5Y. Choyamba, tinapanga njira ya TEM kuti tiwone kusintha kwa mawonekedwe a mitochondrial ndi kapangidwe kake ka ultrastructure17,31,32. Popeza mitochondria33 ndi yosinthasintha, tinagwiritsanso ntchito kusanthula kwa mitochondrial event localizer (MEL) kuti tipeze kusintha pakati pa zochitika za fission ndi fusion, chiwerengero cha mitochondrial ndi voliyumu pansi pa PPA stress. Pomaliza, tinafufuza ngati mawonekedwe a mitochondrial ndi mphamvu zake zikugwirizana ndi kusintha kwa ma gene omwe amakhudzidwa ndi biogenesis, fission, ndi fusion. Ponseponse, deta yathu ikuwonetsa vuto lofotokozera zovuta za njira zomwe zimalamulira mphamvu za mitochondrial. Tikuwonetsa kufunika kwa TEM pophunzira mawonekedwe a mitochondrial ngati gawo loyezeka la mitosis m'maselo a SH-SY5Y. Kuphatikiza apo, tikugogomezera kuti deta ya TEM imapereka chidziwitso chochuluka kwambiri ikaphatikizidwa ndi njira zojambulira zithunzi zomwe zimagwiranso ntchito poyankha kupsinjika kwa kagayidwe kachakudya. Kufotokozera kwina za njira zowongolera mamolekyu zomwe zimathandizira mitosis ya maselo a mitsempha kungapereke chidziwitso chofunikira pa gawo la mitochondrial la dongosolo lamanjenje ndi matenda amitsempha.
Pofuna kuyambitsa kupsinjika kwa mitochondrial, maselo a SH-SY5Y adachiritsidwa ndi PPA pogwiritsa ntchito 3 mM ndi 5 mM sodium propionate (NaP). Tisanayambe TEM, zitsanzo zinkakonzedwa pogwiritsa ntchito cryogenic sample precipitation pogwiritsa ntchito high-pressure ndi kuzizira (Chithunzi 1a). Tinapanga automated mitochondrial image analysis pipeline kuti tiyese magawo asanu ndi atatu a mitochondrial populations pa ma biological replicates atatu. Tinapeza kuti PPA treatment inasintha kwambiri magawo anayi: dera 2, dera, perimeter, ndi Feret diameter (Chithunzi 1b–e). Dera 2 linachepa kwambiri ndi 3 mM ndi 5 mM PPA treatment (p = 0.0183 ndi p = 0.002, motsatana) (Chithunzi 1b), pomwe dera (p = 0.003), perimeter (p = 0.0106) ndi Feret diameter zonse zachepa kwambiri. Panali kuchepa kwakukulu (p = 0.0172) mu gulu la 5 mM treatment group poyerekeza ndi gulu lolamulira (Chithunzi 1c–e). Kuchepa kwakukulu kwa malo ndi kuzungulira kunasonyeza kuti maselo omwe adalandira chithandizo cha 5 mM PPA anali ndi mitochondria yocheperako komanso yozungulira, ndipo mitochondria iyi inali yochepa kwambiri kuposa yomwe ili m'maselo olamulira. Izi zikugwirizananso ndi kuchepa kwakukulu kwa m'mimba mwake wa Feret, chizindikiro chodziyimira pawokha chomwe chikuwonetsa kuchepa kwa mtunda waukulu pakati pa tinthu tating'onoting'ono. Kusintha kwa kapangidwe ka cristae kunawonedwa: cristae sinali yodziwika bwino chifukwa cha kupsinjika kwa PPA (Chithunzi 1a, gulu B). Komabe, si zithunzi zonse zomwe zikuwonetsa bwino kapangidwe ka cristae, kotero kusanthula kochuluka kwa kusinthaku sikunachitike. Deta ya TEM iyi ikhoza kuwonetsa zochitika zitatu zomwe zingatheke: (1) PPA imakulitsa kugawanika kapena kuletsa kusakanikirana, zomwe zimapangitsa kuti mitochondria yomwe ilipo ichepe kukula; (2) biogenesis yowonjezereka imapanga mitochondria yatsopano, yaying'ono kapena (3) imayambitsa njira zonse ziwiri nthawi imodzi. Ngakhale kuti mikhalidwe iyi singathe kusiyanitsa ndi TEM, kusintha kwakukulu kwa mawonekedwe kumasonyeza kusintha kwa homeostasis ya mitochondrial ndi mphamvu pansi pa kupsinjika kwa PPA. Pambuyo pake tinafufuza magawo ena kuti tidziwitse mphamvu izi ndi njira zomwe zingayambitse.
Propionic acid (PPA) imakonzanso mawonekedwe a mitochondrial. (a) Zithunzi zoyimira transmission electron microscopy (TEM) zomwe zikuwonetsa kuti kukula kwa mitochondrial kumachepa ndipo mitochondria imakhala yocheperako komanso yozungulira kwambiri ndi chithandizo cha PPA chowonjezeka; 0 mM (yosachiritsidwa), 3 mM ndi 5 mM, motsatana. Mivi yofiira imasonyeza mitochondria. (b–e) Maselo a SH-SY5Y omwe adathandizidwa ndi PPA kwa maola 24 adakonzedwa kuti agwiritsidwe ntchito pa TEM ndipo zotsatira zake zidasanthulidwa pogwiritsa ntchito Fiji/ImageJ. Zinayi mwa magawo asanu ndi atatu adawonetsa kusiyana kwakukulu pakati pa maselo olamulira (osachiritsidwa, 0 mM PPA) ndi maselo ochiritsidwa (3 mM ndi 5 mM PPA). (b) Chigawo 2, (c) Chigawo, (d) Mzere, (e) M'mimba mwake wa Feret. Kusanthula kwa njira imodzi kwa kusiyana (kulamulira vs. chithandizo) ndi mayeso oyerekeza angapo a Dunnett adagwiritsidwa ntchito kuti adziwe kusiyana kwakukulu (p < 0.05). Mfundo za data zikuyimira mtengo wapakati wa mitochondrial pa selo lililonse, ndipo mipiringidzo yolakwika ikuyimira mean ± SEM. Deta yomwe yawonetsedwa ikuyimira n = 3, osachepera maselo 24 pa kubwerezabwereza; Zithunzi zonse 266 zinasanthulidwa; * ikusonyeza p < 0.05, ** ikusonyeza p < 0.01.
Kuti tifotokoze bwino momwe mitochondria imayankhira ku PPA, tinapaka mitochondria ndi tetramethylrhodamine ethyl ester (TMRE) ndipo tinagwiritsa ntchito time-lapse microscopy ndi MEL analysis kuti tipeze malo ndi kuyeza mitochondria pambuyo pa maola 24 pa 3 ndi 5 mM PPA. Chithandizo cha zochitika za fission ndi fusion. (Chithunzi 2a). Pambuyo pa kusanthula kwa MEL, mitochondria inafufuzidwanso kuti iwerengere kuchuluka kwa kapangidwe ka mitochondria ndi kuchuluka kwake kwapakati. Tinaona kuwonjezeka pang'ono koma kwakukulu kwa chiwerengero cha zochitika za kugawanika kwa maselo zomwe zikuchitika pa 3 mM [4.9 ± 0.3 (p < 0.05)] poyerekeza ndi kugawanika kwa maselo [5.6 ± 0.3 (p < 0.05) )] ndi kusakanikirana [5.4 ± 0.5 (p < 0.05)] ndi kusakanikirana [5.4 ± 0.5 (p < 0.05)] 0.05)] <0.05]] zochitika zinawonjezeka kwambiri pa 5 mM poyerekeza ndi kulamulira (Chithunzi 3b). Chiwerengero cha mitochondria chinawonjezeka kwambiri pa onse 3 [32.6 ± 2.1 (p < 0.05)] ndi 5 mM [34.1 ± 2.2 (p < 0.05)] (Chithunzi 3c), pomwe kuchuluka kwapakati kwa kapangidwe ka mitochondrial sikunasinthe (Chithunzi 3c). 3d). Poganizira zonsezi, izi zikusonyeza kuti kusintha kwa mphamvu za mitochondrial kumagwira ntchito ngati yankho lobwezera lomwe limasunga bwino umphumphu wa netiweki ya mitochondrial. Kuwonjezeka kwa kuchuluka kwa zochitika za kugawanika kwa mitochondrial pa 3 mM PPA kukuwonetsa kuti kuwonjezeka kwa chiwerengero cha mitochondrial kumachitika chifukwa cha kugawanika kwa mitochondrial, koma popeza kuti kuchuluka kwa mitochondrial sikunasinthe kwenikweni, biogenesis singathe kuchotsedwa ngati yankho lowonjezera lobwezera. Komabe, deta iyi ikugwirizana ndi kapangidwe kakang'ono, kozungulira ka mitochondrial komwe kawonedwa ndi TEM ndipo ikuwonetsanso kusintha kwakukulu kwa mphamvu za mitochondrial komwe kumachitika chifukwa cha PPA.
Propionic acid (PPA) imayambitsa kusintha kwa mitochondrial kuti isunge umphumphu wa netiweki. Maselo a SH-SY5Y adakulitsidwa, kupatsidwa chithandizo ndi 3 ndi 5 mM PPA kwa maola 24 ndipo adapakidwa utoto ndi TMRE ndi Hoechst 33342 kutsatiridwa ndi kusanthula kwa MEL. (a) Zithunzi zoyimira nthawi ya microscopy zomwe zikuwonetsa mtundu ndi ma binarized maximum intensity projections pa nthawi 2 (t2) pa vuto lililonse. Madera osankhidwa omwe akuwonetsedwa pachithunzi chilichonse cha binary amakulitsidwa ndikuwonetsedwa mu 3D pa nthawi zitatu zosiyana (t1-t3) kuti awonetse mphamvu pakapita nthawi; zochitika zosakanikirana zimawonetsedwa mu zobiriwira; zochitika za fission zimawonetsedwa mu zobiriwira. Zimawonetsedwa mu zofiira. (b) Chiwerengero chapakati cha zochitika zamphamvu pa vuto lililonse. (c) Chiwerengero chapakati cha kapangidwe ka mitochondrial pa selo lililonse. (d) Chiwerengero chapakati (µm3) cha kapangidwe ka mitochondrial pa selo lililonse. Deta yomwe yawonetsedwa ikuyimira n = maselo 15 pa gulu lililonse la chithandizo. Mipiringidzo yolakwika yomwe yawonetsedwa ikuyimira mean ± SEM, mipiringidzo ya sikelo = 10 μm, * p < 0.05.
Propionic acid (PPA) imayambitsa kutsekeka kwa majini ogwirizana ndi mitochondrial dynamics. Maselo a SH-SY5Y adathandizidwa ndi 3 ndi 5 mM PPA kwa maola 24. Kuyeza kwa majini ogwirizana kunachitika pogwiritsa ntchito RT-qPCR ndipo kunasinthidwa kukhala B2M. Majini a Mitochondrial biogenesis (a) cMYC, (b) TFAM, (c) NRF1 ndi (d) NFE2L2. Majini a Mitochondrial fusion and fission (e) STOML2, (f) OPA1, (g) MFN1, (h) MFN2 ndi (i) DRP1. Kusiyana kwakukulu (p < 0.05) kunayesedwa pogwiritsa ntchito njira imodzi ya ANOVA (control vs. treatment) ndi mayeso a Dunnett oyerekeza angapo: * akuwonetsa p < 0.05, ** akuwonetsa p < 0.01, ndi **** akuwonetsa p < 0.0001. Mipiringidzo ikuyimira mean expression ± SEM. Deta yomwe yawonetsedwa ikuyimira n = 3 (STOML2, OPA1, TFAM), n = 4 (cMYC, NRF1, NFE2L2), ndi n = 5 (MFN1, MFN2, DRP1) ma replicates achilengedwe.
Deta yochokera ku kusanthula kwa TEM ndi MEL ikusonyeza kuti PPA imasintha mawonekedwe a mitochondrial ndi mphamvu zake. Komabe, njira zojambulira izi sizipereka chidziwitso cha njira zomwe zimayambitsa njirazi. Chifukwa chake tidayang'ana momwe mRNA imagwirira ntchito kwa olamulira asanu ndi anayi ofunikira a mitochondrial dynamics, biogenesis, ndi mitosis poyankha chithandizo cha PPA. Tinayesa kuchuluka kwa maselo a myeloma oncogene (cMYC), nuclear respiratory factor (NRF1), mitochondrial transcription factor 1 (TFAM), NFE2-like transcription factor BZIP (NFE2L2), gastrin-like protein 2 (STOML2), optic nerve atrophy 1 (OPA1), Mitofusin 1 (MFN1), Mitofusin 2 (MFN2) ndi dynamin-related protein 1 (DRP1) titatha maola 24 a chithandizo ndi 3 mM ndi 5 mM PPA. Tinaona 3 mM (p = 0.0053, p = 0.0415 ndi p < 0.0001, motsatana) ndi 5 mM (p = 0.0031, p = 0.0233, p < 0.0001) chithandizo cha PPA. (Chithunzi 3a–c). Kuchepa kwa kufotokozedwa kwa mRNA kumadalira mlingo: kufotokozedwa kwa cMYC, NRF1 ndi TFAM kunachepa ndi nthawi 5.7, 2.6 ndi 1.9 pa 3 mM, motsatana, komanso nthawi 11.2, 3 ndi 2.2 pa 5 mM. Mosiyana ndi zimenezi, jini yapakati ya redox biogenesis NFE2L2 sinasinthidwe pa kuchuluka kulikonse kwa PPA, ngakhale kuti kusintha kofananako kumadalira mlingo wa kufotokozedwa kunawonedwa (Chithunzi 3d).
Tinayang'ananso momwe majini akale amagwirira ntchito polamulira kugawanika ndi kusakanikirana. STOML2 ikuganiziridwa kuti imagwira ntchito mu kusakanikirana, mitophagy ndi biogenesis, ndipo mawonekedwe ake adachepetsedwa kwambiri (p < 0.0001) ndi 3 mM (kusintha kwa 2.4-fold) ndi 5 mM (kusintha kwa 2.8-fold) PPA (Chithunzi 1). 3d). Mofananamo, mawonekedwe a majini a OPA1 fusion adachepetsedwa pa 3 mM (kusintha kwa 1.6-fold) ndi 5 mM (kusintha kwa 1.9-fold) PPA (p = 0.006 ndi p = 0.0024, motsatana) (Chithunzi 3f). Komabe, sitinapeze kusiyana kwakukulu pakuwonekera kwa majini a fusion MFN1, MFN2 kapena fission gene DRP1 pansi pa 24-hour PPA stress (Chithunzi 3g–i). Kuphatikiza apo, tapeza kuti kuchuluka kwa mapuloteni anayi a fusion ndi fission (OPA1, MFN1, MFN2 ndi DRP1) sikunasinthe pansi pa mikhalidwe yomweyi (Chithunzi 4a–d). Ndikofunikira kudziwa kuti deta iyi ikuwonetsa nthawi imodzi ndipo singawonetse kusintha kwa protein expression kapena kuchuluka kwa zochita panthawi yoyambirira ya PPA stress. Komabe, kuchepa kwakukulu kwa cMYC, NRF1, TFAM, STOML2, ndi OPA1 kukuwonetsa kusokonekera kwakukulu kwa transcriptional metabolism ya mitochondrial, biogenesis, ndi dynamics. Kuphatikiza apo, deta iyi ikuwonetsa kugwiritsa ntchito njira zojambulira kuti ziphunzire mwachindunji kusintha kwa ma end-state mu ntchito ya mitochondrial.
Ma protein a fusion ndi fission factor sanasinthe pambuyo pa chithandizo cha propionic acid (PPA). Maselo a SH-SY5Y adachiritsidwa ndi 3 ndi 5 mM PPA kwa maola 24. Ma protein milingo adayesedwa pogwiritsa ntchito Western blot analysis, ndipo ma protein milingo adasinthidwa kukhala total protein. Avereji ya protein expression ndi ma Western blots oyimira target ndi total protein akuwonetsedwa. a – OPA1, b – MFN1, c – MFN2, d – DRP1. Mipiringidzo ikuyimira mean ± SEM, ndipo deta yomwe yawonetsedwa ikuyimira n = 3 biological replicates. Kuyerekeza kambiri (p < 0.05) kunachitika pogwiritsa ntchito kusanthula kwa njira imodzi kwa kusiyana ndi mayeso a Dunnett. Gel yoyambirira ndi blot zikuwonetsedwa mu Chithunzi S1.
Kusagwira bwino ntchito kwa mitochondrial kumakhudzana ndi matenda osiyanasiyana kuyambira matenda a kagayidwe kachakudya, matenda a mtima ndi minofu mpaka matenda a mitsempha1,10. Matenda ambiri a neurodegenerative ndi neurodegenerative amakhudzana ndi kusagwira bwino ntchito kwa mitochondrial, zomwe zikuwonetsa kufunika kwa ma organelles awa nthawi yonse ya moyo wa ubongo. Matendawa akuphatikizapo matenda a Parkinson, matenda a Alzheimer's ndi ASD3,4,18. Komabe, kupeza minofu ya ubongo kuti muphunzire matendawa n'kovuta, makamaka pamlingo wa makina, zomwe zimapangitsa kuti machitidwe a maselo akhale njira ina yofunikira. Mu kafukufukuyu, timagwiritsa ntchito njira ya maselo yogwiritsira ntchito maselo a SH-SY5Y omwe amathandizidwa ndi PPA kuti tifotokozenso za kusagwira bwino ntchito kwa mitochondrial komwe kumawonedwa m'matenda a neuronal, makamaka matenda a autism spectrum. Kugwiritsa ntchito njira ya PPA iyi pophunzira mphamvu za mitochondrial mu ma neuron kungapereke chidziwitso pa zomwe zimayambitsa ASD.
Tinafufuza kuthekera kogwiritsa ntchito TEM kuti tiwone kusintha kwa mawonekedwe a mitochondrial. Ndikofunikira kudziwa kuti TEM iyenera kugwiritsidwa ntchito moyenera kuti igwire bwino ntchito. Kukonzekera ma cryo-specimens kumathandiza kuti mapangidwe a neuronal asungidwe bwino mwa kukonza nthawi imodzi zigawo za maselo ndikuchepetsa mapangidwe a zinthu zakale34. Mogwirizana ndi izi, tawona kuti maselo a SH-SY5Y ofanana ndi neuron anali ndi ma organelles osasinthika a subcellular ndi mitochondria yayitali (Chithunzi 1a). Izi zikuwonetsa kugwiritsa ntchito njira zokonzekera cryogenic pophunzira mawonekedwe a mitochondrial mu mitundu ya maselo a neuronal. Ngakhale kuti kuyeza kuchuluka ndikofunikira kwambiri pakusanthula deta ya TEM, palibe mgwirizano pa magawo enieni omwe ayenera kuyezedwa kuti atsimikizire kusintha kwa mawonekedwe a mitochondrial. Kutengera ndi kafukufuku wambiri yemwe adafufuza mawonekedwe a mitochondrial17,31,32, tidapanga njira yodziwira zithunzi za mitochondrial yokha yomwe imayesa magawo asanu ndi atatu a mawonekedwe, omwe ndi: dera, dera2, chiŵerengero cha mawonekedwe, perimeter, circularity, digiri , Feret diameter. ndi roundness.
Pakati pawo, PPA inachepetsa kwambiri dera 2, dera, perimeter, ndi Feret diameter (Chithunzi 1b–e). Izi zinasonyeza kuti mitochondria inakhala yocheperako komanso yozungulira, zomwe zikugwirizana ndi kafukufuku wakale wosonyeza kuchepa kwa dera la mitochondria pambuyo pa maola 72 a kupsinjika kwa mitochondria komwe kunayambitsidwa ndi PPA30. Zizindikiro za mawonekedwe awa zitha kusonyeza kugawanika kwa mitochondria, njira yofunikira yochotsera zigawo zowonongeka kuchokera ku netiweki ya mitochondria kuti zithandizire kuwonongeka kwawo kudzera mu mitophagy35,36,37. Kumbali ina, kuchepa kwa kukula kwa mitochondria kwapakati kungagwirizane ndi kuwonjezeka kwa biogenesis, zomwe zimapangitsa kuti mitochondria yaying'ono ipangidwe. Kuwonjezeka kwa kugawanika kapena biogenesis kumayimira yankho lobwezera kuti tisunge mitosis motsutsana ndi kupsinjika kwa mitochondria. Komabe, kuchepa kwa kukula kwa mitochondria, kusokonezeka kwa fusion, kapena zinthu zina sizingachotsedwe.
Ngakhale zithunzi zapamwamba zopangidwa ndi TEM zimalola kudziwa mawonekedwe a morphological pamlingo wa mitochondria ya munthu aliyense, njira iyi imapanga zithunzi zamitundu iwiri panthawi imodzi. Kuti tiphunzire mayankho amphamvu ku metabolic stress, tinapaka mitochondria ndi TMRE ndipo tinagwiritsa ntchito time-lapse microscopy ndi MEL analysis, zomwe zimathandiza kuwona kusintha kwa 3D mu netiweki ya mitochondrial pakapita nthawi33,38. Tinawona kusintha kochepa koma kofunikira mu mitochondrial dynamics pansi pa PPA stress (Chithunzi 2). Pa 3 mM, chiwerengero cha zochitika za fission chinawonjezeka kwambiri, pomwe zochitika za fusion zinakhalabe chimodzimodzi ndi zomwe zinali mu control. Kuwonjezeka kwa chiwerengero cha zochitika za fission ndi fusion kunawonedwa pa 5 mM PPA, koma kusinthaku kunali kofanana, zomwe zikusonyeza kuti fission ndi fusion kinetics zimafika pamlingo wapamwamba (Chithunzi 2b). Volume yapakati ya mitochondrial sinasinthe pa 3 ndi 5 mM PPA, zomwe zikusonyeza kuti umphumphu wa netiweki ya mitochondrial unasungidwa (Chithunzi 2d). Izi zikuwonetsa kuthekera kwa ma network amphamvu a mitochondrial kuyankha kupsinjika pang'ono kwa kagayidwe kachakudya kuti asunge bwino homeostasis popanda kuyambitsa kugawikana kwa ma network. Pa 3 mM PPA, kuwonjezeka kwa kugawikana ndikokwanira kulimbikitsa kusintha kupita ku mgwirizano watsopano, koma kukonzanso kwakukulu kwa kinetic kumafunika poyankha kupsinjika komwe kumachitika chifukwa cha kuchuluka kwa PPA.
Chiwerengero cha mitochondria chinawonjezeka pa kuchuluka kwa PPA stress, koma avareji ya mitochondrial volume sichinasinthe kwambiri (Chithunzi 2c). Izi zitha kukhala chifukwa cha kuwonjezeka kwa biogenesis kapena kugawa kwakukulu; komabe, popanda kuchepa kwakukulu kwa avareji ya mitochondrial volume, ndizotheka kuti biosynthesis imawonjezeka. Komabe, deta yomwe ili mu Chithunzi 2 ikuthandizira kukhalapo kwa njira ziwiri zolipirira: kuwonjezeka kwa chiwerengero cha zochitika za fission, mogwirizana ndi kukwezedwa kwa mitochondrial fission, ndi kuwonjezeka kwa chiwerengero cha zochitika, mogwirizana ndi mitochondrial biogenesis. Pomaliza, dynamic compensation for stress slow can be containing particular processes involving fission, fusion, biogenesis, and mitophagy. Ngakhale olemba akale awonetsa kuti PPA imakulitsa mitosis30,39 ndi mitophagy29, timapereka umboni wokonzanso mitochondrial fission and fusion dynamics poyankha PPA. Deta iyi imatsimikizira kusintha kwa mawonekedwe komwe TEM imawona ndipo imapereka chidziwitso chowonjezereka pa njira zomwe zimagwirizanitsidwa ndi PPA-induced mitochondrial dysfunction.
Popeza kusanthula kwa TEM kapena MEL sikunapereke umboni wolunjika wa njira zowongolera majini zomwe zimayambitsa kusintha kwa mawonekedwe a thupi komwe kwawonedwa, tinayang'ana momwe RNA imawonetsera majini omwe amakhudzidwa ndi kagayidwe ka mitochondrial, biogenesis, ndi dynamics zimagwirira ntchito. CMYC proto-oncogene ndi transcription factor yomwe imakhudzidwa ndi kagayidwe ka mitochondrial, glycolysis, amino acid ndi fatty acid metabolism40. Kuphatikiza apo, cMYC imadziwika kuti imawongolera momwe majini pafupifupi 600 a mitochondrial amagwirira ntchito mu kusindikiza, kumasulira, ndi kusonkhana kovuta kwa mitochondrial, kuphatikiza NRF1 ndi TFAM41. NRF1 ndi TFAM ndi olamulira awiri apakati a mitosis, omwe amagwira ntchito pansi pa PGC-1α kuti ayambitse kubwerezabwereza kwa mtDNA. Njirayi imayatsidwa ndi cAMP ndi AMPK signaling ndipo imakhudzidwa ndi kugwiritsa ntchito mphamvu komanso kupsinjika kwa kagayidwe kachakudya. Tinayang'ananso NFE2L2, redox regulator ya mitochondrial biogenesis, kuti tidziwe ngati zotsatira za PPA zitha kuyambitsidwa ndi kupsinjika kwa okosijeni.
Ngakhale kuti kafotokozedwe ka NFE2L2 sikanasinthe, tinapeza kuchepa kosalekeza kwa kafotokozedwe ka cMYC, NRF1 ndi TFAM pambuyo pa maola 24 a chithandizo ndi 3 mM ndi 5 mM PPA (Chithunzi 3a–c). Kuchepa kwa kafotokozedwe ka cMYC kwanenedwa kale ngati yankho la kupsinjika kwa mitochondrial42, ndipo mosiyana, kuchepetsa kafotokozedwe ka cMYC kungayambitse kusagwira ntchito kwa mitochondrial mwa kusintha kagayidwe ka mitochondrial, kulumikizana kwa netiweki, ndi polarization ya membrane43. Chochititsa chidwi n'chakuti, cMYC imagwiranso ntchito pakulamulira kugawanika kwa mitochondrial ndi fusion42,43 ndipo imadziwika kuti imawonjezera phosphorylation ya DRP1 ndi malo a mitochondrial panthawi yogawa maselo44, komanso imayambitsa kukonzanso kwa mitochondrial morphological mu maselo oyambira a neuronal45. Zoonadi, ma fibroblast osowa cMYC amawonetsa kukula kochepa kwa mitochondrial, mogwirizana ndi kusintha komwe kumachitika chifukwa cha kupsinjika kwa PPA43. Deta iyi ikuwonetsa ubale wosangalatsa koma wosamveka bwino pakati pa cMYC ndi mitochondrial dynamics, zomwe zimapereka cholinga chosangalatsa cha maphunziro amtsogolo a kukonzanso koyambitsidwa ndi kupsinjika kwa PPA.
Kuchepa kwa NRF1 ndi TFAM kukugwirizana ndi ntchito ya cMYC ngati choyambitsa chofunikira cha transcriptional. Deta iyi ikugwirizananso ndi kafukufuku wakale m'maselo a khansa ya m'matumbo a anthu omwe akuwonetsa kuti PPA inachepetsa kufotokozedwa kwa NRF1 mRNA pa maola 22, zomwe zinkakhudzana ndi kuchepa kwa ATP ndikuwonjezera ROS46. Olemba awa adanenanso kuti kufotokozedwa kwa TFAM kunawonjezeka pa maola 8.5 koma kunabwerera ku milingo yoyambira pa maola 22. Mosiyana ndi zimenezi, Kim et al. (2019) adawonetsa kuti kufotokozedwa kwa TFAM mRNA kunachepa kwambiri patatha maola 4 a kupsinjika kwa PPA m'maselo a SH-SY5Y; komabe, patatha maola 72, kufotokozedwa kwa mapuloteni a TFAM kunawonjezeka kwambiri ndipo chiwerengero cha makope a mtDNA chinawonjezeka kwambiri. Chifukwa chake, kuchepa kwa chiwerengero cha majini a mitochondrial biogenesis omwe tidawona patatha maola 24 sikuchotsa kuthekera kwakuti kuwonjezeka kwa chiwerengero cha mitochondria kumalumikizidwa ndi kuyatsidwa kwa biogenesis nthawi yoyambirira. Kafukufuku wakale wasonyeza kuti PPA imakweza kwambiri PGC-1α mRNA ndi mapuloteni m'maselo a SH-SY5Y pa maola 4 ndi mphindi 30, pomwe propionic acid imawonjezera biogenesis ya mitochondrial mu hepatocytes ya calf kudzera mu PGC-1α pa maola 12 ndi mphindi 39. Chochititsa chidwi n'chakuti, PGC-1α sikuti ndi yowongolera mwachindunji transcriptional ya NRF1 ndi TFAM, komanso yawonetsedwa kuti imayang'anira ntchito ya MFN2 ndi DRP1 mwa kuwongolera fission ndi fusion47. Ponseponse, izi zikuwonetsa kuyanjana kwapafupi kwa njira zomwe zimawongolera mayankho a mitochondrial compensatory omwe amachitika chifukwa cha PPA. Kuphatikiza apo, deta yathu ikuwonetsa kusokonekera kwakukulu kwa transcriptional regulation ya biogenesis ndi metabolism pansi pa PPA stress.
Majini a STOML2, OPA1, MFN1, MFN2 ndi DRP1 ndi ena mwa olamulira apakati a mitochondrial fission, fusion ndi dynamics37,48,49. Pali majini ena ambiri omwe amagwira ntchito mu mitochondrial dynamics, komabe, STOML2, OPA1 ndi MFN2 adapezeka kale kuti ali ndi methylated mosiyanasiyana m'magulu a ASD,16 ndipo maphunziro angapo odziyimira pawokha anena za kusintha kwa zinthu izi poyankha kupsinjika kwa mitochondrial50,51. 52. Kuwonetsedwa kwa OPA1 ndi STOML2 konse kunachepetsedwa kwambiri ndi chithandizo cha 3 mM ndi 5 mM PPA (Chithunzi 3e, f). OPA1 ndi imodzi mwa olamulira akale a mitochondrial fusion kudzera mu kulumikizana mwachindunji ndi MFN1 ndi 2 ndipo imagwira ntchito pakukonzanso cristae ndi mawonekedwe a mitochondrial53. Udindo weniweni wa STOML2 mu mitochondrial dynamics sunadziwikebe bwino, koma umboni ukusonyeza kuti imagwira ntchito mu mitochondrial fusion, biogenesis, ndi mitophagy.
STOML2 imagwira ntchito yosunga kulumikizana kwa kupuma kwa mitochondrial ndi kupanga ma complexes a unyolo wopumira54,55 ndipo yawonetsedwa kuti ikusintha kwambiri mawonekedwe a kagayidwe kachakudya ka maselo a khansa56. Kafukufuku wasonyeza kuti STOML2 imalimbikitsa kuthekera kwa membrane ya mitochondrial ndi biogenesis kudzera mu kuyanjana ndi BAN ndi cardiolipin 55, 57, 58. Kuphatikiza apo, kafukufuku wodziyimira pawokha wasonyeza kuti kuyanjana pakati pa STOML2 ndi PINK1 kumawongolera mitophagy59,60. Chodziwika bwino n'chakuti, STOML2 yanenedwa kuti imagwirizana mwachindunji ndi kukhazikika kwa MFN2 ndipo imagwiranso ntchito yofunika kwambiri pakukhazikitsa ma isoform atali a OPA1 poletsa protease yomwe imayambitsa kuwonongeka kwa OPA153,61,62. Kuchepa kwa mawonekedwe a STOML2 omwe amawonedwa mu PPA reactions kungapangitse mapuloteni osakanikirana awa kukhala osavuta kuwonongeka kudzera mu ubiquitin- ndi proteasome-dependent pathways48. Ngakhale kuti udindo weniweni wa STOML2 ndi OPA1 pa yankho lamphamvu ku PPA sudziwika bwino, kuchepa kwa kufotokozera kwa majini osakanikirana awa (Chithunzi 3) kungasokoneze mgwirizano pakati pa kugawanika ndi kusakanikirana ndi kutsogolera kuchepa kwa kukula kwa mitochondrial (Chithunzi 3). 1).
Kumbali inayi, kagwiritsidwe ntchito ka mapuloteni a OPA1 kanasinthabe patatha maola 24, pomwe kuchuluka kwa mRNA ndi mapuloteni a MFN1, MFN2 kapena DRP1 sikunasinthe kwambiri pambuyo pa chithandizo cha PPA (Chithunzi 3g-i, Chithunzi 4). Izi zitha kusonyeza kuti palibe kusintha komwe kwachitika pakulamulira zinthu izi zomwe zimakhudzidwa ndi kusakanikirana kwa mitochondrial ndi kugawanika kwa mitochondrial. Komabe, ndikofunikira kudziwa kuti majini onse anayiwa amawongoleredwanso ndi kusintha kwa posttranscriptional (PTMs) komwe kumawongolera ntchito ya mapuloteni. OPA1 ili ndi mitundu isanu ndi itatu ya splice yomwe imadulidwa mu mitochondria kuti ipange ma isoform awiri osiyana 63. Kugwirizana pakati pa ma isoform ataliatali ndi afupi pamapeto pake kumatsimikiza udindo wa OPA1 pakusakanikirana kwa mitochondrial ndi kusamalira network ya mitochondrial64. Ntchito ya DRP1 imawongoleredwa ndi calcium/calmodulin-dependent protein kinase II (CaMKII) phosphorylation, pomwe kuwonongeka kwa DRP1 kumawongoleredwa ndi ubiquitination ndi SUMOylation65. Pomaliza, DRP1 ndi MFN1/2 zonse ndi ma GTPases, kotero ntchito yake ingakhudzidwe ndi kuchuluka kwa kupanga kwa GTP mu mitochondria 66. Chifukwa chake, ngakhale kuti ma protein awa amakhalabe osasintha, izi sizingasonyeze ntchito ya mapuloteni osasinthika kapena malo67,68. Zoonadi, ma repertoire a mapuloteni a PTM omwe alipo nthawi zambiri amakhala ngati mzere woyamba wa chitetezo womwe umayang'anira mayankho a kupsinjika kwadzidzidzi. Pakakhala kupsinjika pang'ono kwa kagayidwe kachakudya mu chitsanzo chathu, ndizotheka kuti PTM imalimbikitsa ntchito yowonjezera ya mapuloteni osakanikirana ndi odulidwa kuti abwezeretse mokwanira umphumphu wa mitochondrial popanda kufunikira kuyambitsanso majini awa pamlingo wa mRNA kapena mapuloteni.
Ponseponse, deta yomwe ili pamwambapa ikuwonetsa kusinthasintha kovuta komanso kodalira nthawi kwa mawonekedwe a mitochondrial komanso zovuta zowunikira njirazi. Kuti tiphunzire momwe majini amagwirira ntchito, choyamba ndikofunikira kuzindikira majini enieni omwe ali munjirayo. Komabe, deta yathu ikuwonetsa kuti majini omwe ali munjira yomweyo sayankha mofanana ndi kupsinjika komweko. Ndipotu, kafukufuku wakale wasonyeza kuti majini osiyanasiyana omwe ali munjira yomweyo akhoza kuwonetsa ma profiles osiyanasiyana a nthawi 30,46. Kuphatikiza apo, pali njira zovuta zolembera pambuyo pa kulembedwa zomwe zimasokoneza ubale pakati pa kulembedwa ndi ntchito ya majini. Maphunziro a proteomic angapereke chidziwitso cha momwe ma PTM ndi ntchito ya mapuloteni zimakhudzira, koma amabweretsanso zovuta kuphatikiza njira zotsika, kuchuluka kwa ma signal-to-noise ratios, komanso kusakhazikika bwino.
Pachifukwa ichi, kuphunzira za kapangidwe ka mitochondrial pogwiritsa ntchito TEM ndi MEL kuli ndi kuthekera kwakukulu koyankha mafunso ofunikira okhudza ubale pakati pa mphamvu za mitochondrial ndi ntchito yake komanso momwe izi zimakhudzira matenda. Chofunika kwambiri, TEM imapereka njira yolunjika yoyezera kapangidwe ka mitochondrial ngati gawo logwirizana la kusagwira bwino ntchito kwa mitochondrial ndi mphamvu zake51. MEL imaperekanso njira yolunjika yowonera zochitika za fission ndi fusion m'malo mwa maselo amitundu itatu, zomwe zimathandiza kuwerengera kusintha kwa mitochondrial ngakhale popanda kusintha kwa majini33. Apa tikuwonetsa kugwiritsa ntchito njira zojambulira mitochondrial m'matenda achiwiri a mitochondrial. Matendawa nthawi zambiri amadziwika ndi kupsinjika pang'ono kwa kagayidwe kachakudya komwe kumadziwika ndi kusintha pang'ono kwa maukonde a mitochondrial m'malo mwa kuwonongeka kwakukulu kwa mitochondrial. Komabe, kubweza kwa mitochondrial komwe kumafunika kuti mitochondrial ikhalebe ndi kupsinjika kosatha kumakhala ndi zotsatirapo zazikulu pantchito. Pankhani ya neuroscience, kumvetsetsa bwino njira izi zolipirira kungapereke chidziwitso chofunikira chokhudza pleiotropic neuropathology yokhudzana ndi kusagwira bwino ntchito kwa mitochondrial.
Pomaliza, deta yathu ikuwonetsa kufunika kwa njira zojambulira zithunzi kuti timvetsetse zotsatira za kuyanjana kovuta pakati pa kufotokozera majini, kusintha kwa mapuloteni, ndi ntchito ya mapuloteni yomwe imalamulira mphamvu za mitochondrial za neuronal. Tinagwiritsa ntchito PPA kuti tiyerekezere kusagwira ntchito kwa mitochondrial mu chitsanzo cha maselo a neuronal kuti tidziwe bwino gawo la mitochondrial la ASD. Maselo a SH-SY5Y omwe amathandizidwa ndi PPA adawonetsa kusintha kwa mawonekedwe a mitochondrial: mitochondria inakhala yaying'ono komanso yozungulira, ndipo ma cristae sanadziwike bwino akawonedwa ndi TEM. Kusanthula kwa MEL kukuwonetsa kuti kusinthaku kumachitika nthawi imodzi ndi kuwonjezeka kwa zochitika za fission ndi fusion kuti asunge netiweki ya mitochondrial poyankha kupsinjika pang'ono kwa kagayidwe kachakudya. Kuphatikiza apo, PPA imasokoneza kwambiri malamulo olembera kagayidwe ka mitochondrial ndi homeostasis. Tinazindikira cMYC, NRF1, TFAM, STOML2, ndi OPA1 ngati olamulira ofunikira a mitochondrial omwe asokonezedwa ndi kupsinjika kwa PPA ndipo atha kukhala ndi gawo lothandizira kusintha komwe kumabwera chifukwa cha PPA mu mawonekedwe a mitochondrial ndi ntchito. Maphunziro amtsogolo akufunika kuti afotokoze bwino kusintha kwa nthawi komwe kumabwera chifukwa cha PPA mu kufotokozera majini ndi ntchito ya mapuloteni, malo, ndi kusintha pambuyo pa kumasulira. Deta yathu ikuwonetsa kusinthasintha ndi kudalirana kwa njira zowongolera zomwe zimathandizira kuyankha kwa kupsinjika kwa mitochondrial ndikuwonetsa kugwiritsa ntchito kwa TEM ndi njira zina zojambulira zithunzi pamaphunziro owunikira kwambiri.
Mzere wa maselo a SH-SY5Y (ECACC, 94030304-1VL) unagulidwa kuchokera ku Sigma-Aldrich. Maselo a SH-SY5Y anakulitsidwa mu Dulbecco's modified Eagle's medium/F-12 nutrient mixture (DMEM/F-12) ndi L-glutamine (SC09411, ScienCell) m'mabotolo a 25 cm2 owonjezeredwa ndi 20% fetal bovine serum (FBS) (10493106, ThermoFisher Scientific) ndi 1% penicillin-streptomycin (P4333-20ML, Sigma-Aldrich) pa 37 °C, 5% CO2. Maselo anakulitsidwa pang'ono kufika pa 80% confluence pogwiritsa ntchito 0.05% trypsin-EDTA (15400054, ThermoFisher Scientific), centrifuged pa 300 g ndipo anaikidwa pa kuchuluka kwa maselo pafupifupi 7 × 105/ml. Mayeso onse anachitika pa maselo osagawanika a SH-SY5Y pakati pa njira 19-22. PPA imaperekedwa ngati NaP. Sungunulani ufa wa NaP (CAS No. 137-40-6, formula ya mankhwala C3H5NaO2, P5436-100G, Sigma-Aldrich) m'madzi ofunda a MilliQ mpaka 1 M ndikusunga pa 4 °C. Patsiku la chithandizo, sakanizani yankho ili ndi 1 M PPA mpaka 3 mM ndi 5 mM PPA mu seramu yopanda seramu (DMEM/F-12 ndi L-glutamine). Kuchuluka kwa chithandizo pa mayeso onse sikunali PPA (0 mM, control), 3 mM, ndi 5 mM PPA. Mayeso anachitika m'magawo osachepera atatu achilengedwe.
Maselo a SH-SY5Y adabzalidwa m'mabotolo a 25 cm5 pamlingo wa 5.5 × 105 maselo/ml ndipo adakula kwa maola 24. Chithandizo cha PPA chidawonjezedwa mu botolo musanafike maola 24 oyambira kubzala. Sonkhanitsani ma pellets a maselo motsatira njira zabwinobwino za nyama zoyamwitsa (zomwe zafotokozedwa pamwambapa). Bwezeretsani pellet ya maselo mu 100 µl 2.5% glutaraldehyde, 1 × PBS ndikusunga pa 4 °C mpaka mutakonza. Maselo a SH-SY5Y adayikidwa pang'ono kuti achotse ma cell ndikuchotsa 2.5% glutaraldehyde, 1 × PBS yankho. Bwezeretsani sediment mu 4% agarose gel yokonzedwa m'madzi osungunuka (chiŵerengero cha agarose ndi sediment voliyumu ndi 1:1). Zidutswa za agarose zidayikidwa pa grids pa mbale zathyathyathya ndikupakidwa ndi 1-hexadecene musanazimitse kwambiri. Zitsanzo zidazimitsidwa mu 100% acetone youma pa -90°C kwa maola 24. Kenako kutentha kunakwezedwa kufika pa -80°C ndipo yankho la 1% osmium tetroxide ndi 0.1% glutaraldehyde linawonjezedwa. Zitsanzo zinasungidwa pa -80°C kwa maola 24. Pambuyo pake, kutentha kunawonjezeka pang'onopang'ono kufika pa kutentha kwa chipinda kwa masiku angapo: kuyambira -80 °C mpaka -50 °C kwa maola 24, mpaka -30 °C kwa maola 24, mpaka -10 °C kwa maola 24 ndipo potsiriza kufika pa kutentha kwa chipinda.
Pambuyo pokonzekera cryogenic, zitsanzozo zinalowetsedwa ndi utomoni ndipo zigawo zoonda kwambiri (~100 nm) zinapangidwa pogwiritsa ntchito Leica Reichert UltracutS ultramicrotome (Leica Microsystems). Zigawozo zinapakidwa utoto wa 2% uranyl acetate ndi lead citrate. Zitsanzozo zinawonedwa pogwiritsa ntchito FEI Tecnai 20 transmission electron microscope (ThermoFisher (yomwe kale inali FEI), Eindhoven, The Netherlands) yomwe imagwira ntchito pa 200 kV (Lab6 transmitter) ndi kamera ya Gatan CCD (Gatan, UK) yokhala ndi fyuluta ya mphamvu ya Tridiem.
Mu chithunzi chilichonse chaukadaulo, zithunzi zosachepera 24 za selo limodzi zinapezeka, zomwe zinapanga zithunzi 266. Zithunzi zonse zinasanthulidwa pogwiritsa ntchito macro ya Region of Interest (ROI) ndi macro ya Mitochondria. Macro ya mitochondrial imachokera ku njira zofalitsidwa17,31,32 ndipo imalola kukonzedwa kwa zithunzi za TEM ku Fiji/ImageJ69 motsatira njira zodziyimira pawokha. Mwachidule: chithunzicho chimasinthidwa ndikusinthidwa pogwiritsa ntchito kuchotsera kumbuyo kwa mpira wozungulira (ma pixel 60 radius) ndi fyuluta ya FFT bandpass (pogwiritsa ntchito malire a pixel 60 ndi 8 apamwamba ndi otsika motsatana) ndikuchepetsa mzere wowongoka ndi kulolerana kwa 5%. Chithunzi chokonzedwacho chimayikidwa chokha pogwiritsa ntchito algorithm yayikulu ya entropy ndipo chigoba cha binary chimapangidwa. Zigawo zazithunzi zomwe zimagwirizanitsidwa ndi ma ROI osankhidwa ndi manja muzithunzi za TEM zosaphika zinachotsedwa, zomwe zimasiyanitsa mitochondria ndikuchotsa nembanemba ya plasma ndi madera ena osiyana kwambiri. Pa ROI iliyonse yochotsedwa, tinthu ta binary toposa ma pixel 600 tinasanthulidwa, ndipo dera la tinthu, perimeter, major and minor axes, Feret diameter, roundness, and circularity zinayesedwa pogwiritsa ntchito ntchito zoyezera za Fiji/ImageJ. Kutsatira Merrill, Flippo, ndi Strack (2017), dera lachiwiri, particle aspect ratio (major to minor axis ratio), ndi shape factor (FF) zinawerengedwa kuchokera ku deta iyi, komwe FF = perimeter 2/4pi x area. Tanthauzo la parametric formula lingapezeke mu Merrill, Flippo, ndi Strack (2017). Ma macros omwe atchulidwa akupezeka pa GitHub (onani Data Availability Statement). Pa avareji, pafupifupi tinthu ta 5,600 tinasanthulidwa pa PPA iliyonse, kuti tinthu ta 17,000 tinthu tonse (deta siinawonetsedwe).
Maselo a SH-SH5Y anaikidwa m'mbale zosungiramo zinthu za zipinda 8 (ThermoFisher, #155411) kuti alole kuti azimatirira usiku wonse kenako n’kuikidwa ndi TMRE 1:1000 (ThermoFisher, #T669) ndi Hoechst 33342 1:200 (Sigma-Aldrich, H6024). Kupaka utoto kwa zithunzi kunapezeka pogwiritsa ntchito ma laser a 405 nm ndi 561 nm pa malo okwana mphindi 10, ndipo zithunzi zosaphika zinapezeka ngati z-stacks zokhala ndi ma micrograph 10 azithunzi okhala ndi az step ya 0.2 μm pakati pa mafelemu azithunzi pa nthawi 12 zotsatira. Zithunzi zinasonkhanitsidwa pogwiritsa ntchito nsanja ya Carl Zeiss LSM780 ELYRA PS.1 super-resolution (Carl Zeiss, Oberkochen, Germany) pogwiritsa ntchito lenzi ya LCI Plan Apochromate 100x/1.4 Oil DIC M27. Zithunzi zinasanthulidwa mu ImageJ pogwiritsa ntchito njira yomwe yafotokozedwa kale komanso pulogalamu yowonjezera ya ImageJ kuti iyese zochitika zosakanikirana ndi zogawanika, kuchuluka kwa kapangidwe ka mitochondrial, ndi kuchuluka kwa mitochondrial pa selo lililonse33. Ma macro a MEL akupezeka pa GitHub (onani Chidziwitso cha Kupezeka kwa Deta).
Maselo a SH-SY5Y adakulitsidwa m'magawo asanu ndi limodzi okhala ndi kachulukidwe ka 0.3 × 106 maselo/mL kwa maola 24 chithandizo chisanachitike. RNA idachotsedwa pogwiritsa ntchito Quick-RNA™ Miniprep protocol (ZR R1055, Zymo Research) ndi zosintha pang'ono: onjezerani 300 μl ya RNA lysis buffer pachitsime chilichonse musanachotsedwe ndikuyika chitsanzo chilichonse ngati gawo lomaliza ndi 30 μl ya DNase/RNase elution. -madzi opanda DNase/RNase. Zitsanzo zonse zidayang'aniridwa kuchuluka ndi mtundu pogwiritsa ntchito NanoDrop ND-1000 UV-Vis Spectrophotometer. Mapuloteni onse ochokera ku maselo adapezeka pogwiritsa ntchito 200 μl RIPA lysis buffer, ndipo kuchuluka kwa mapuloteni kudayesedwa pogwiritsa ntchito Bradford protein assay70.
Kupanga kwa cDNA kunachitika pogwiritsa ntchito Tetro™ cDNA Synthesis Kit (BIO-65043, Meridian Bioscience) malinga ndi malangizo a wopanga ndi zosintha zina. cDNA idapangidwa mu 20-μl reactions pogwiritsa ntchito 0.7 mpaka 1 μg ya RNA yonse. Ma primers adasankhidwa kuchokera m'mapepala omwe adasindikizidwa kale 42, 71, 72, 73, 74, 75, 76, 77, 78 (Table S1) ndipo ma probes otsatira adapangidwa pogwiritsa ntchito chida cha PrimerQuest kuchokera ku Integrated DNA Technologies. Majini onse ofunikira adasinthidwa kukhala majini a nuclear B2M. Kufotokozera kwa majini a STOML2, NRF1, NFE2L2, TFAM, cMYC ndi OPA1 kudayesedwa ndi RT-qPCR. Kusakaniza kwakukulu kunaphatikizapo LUNA Taq polymerase (M3003L, New England Biolabs), ma primer 10 μM forward ndi reverse, cDNA, ndi madzi a PCR-grade kuti apereke voliyumu yomaliza ya 10 μL pa reaction iliyonse. Kuwonetsa majini ogawa ndi fission (DRP1, MFN1/2) kunayesedwa pogwiritsa ntchito mayeso a TaqMan multiplex. Luna Universal Probe qPCR Master Mix (M3004S, New England Biolabs) idagwiritsidwa ntchito motsatira malangizo a wopanga ndi zosintha zazing'ono. Kusakaniza kwakukulu kwa RT-qPCR kumaphatikizapo 1X LUNA Taq polymerase, ma primer 10 μM forward ndi reverse, probe 10 μM, cDNA, ndi madzi a PCR-grade, zomwe zinapangitsa kuti voliyumu yomaliza ya 20 μL pa reaction iliyonse. RT-qPCR idachitika pogwiritsa ntchito Rotor-Gene Q 6-plex (QIAGEN RG—serial number: R0618110). Mikhalidwe ya njinga ikuwonetsedwa mu Table S1. Zitsanzo zonse za cDNA zinakulitsidwa mu katatu ndipo curve yokhazikika idapangidwa pogwiritsa ntchito mndandanda wa ma dilutions khumi. Ma outliers mu zitsanzo zitatu zokhala ndi cycle threshold standard deviation (Ct) >0.5 adachotsedwa mu kusanthula kuti atsimikizire kubwerezabwereza kwa deta30,72. Kufotokozera kwa majini ogwirizana kunawerengedwa pogwiritsa ntchito njira ya 2-ΔΔCt79.
Zitsanzo za mapuloteni (60 μg) zinasakanizidwa ndi Laemmli loading buffer pa chiŵerengero cha 2:1 ndipo zinagwiritsidwa ntchito pa 12% colorless protein gel (Bio-Rad #1610184). Mapuloteni anasamutsidwira ku PVDF (polyvinylidene fluoride) membrane (#170-84156, Bio-Rad) pogwiritsa ntchito Trans-Blot Turbo system (#170-4155, Bio-Rad). Nembanembayo inatsekedwa ndikuyikidwa ndi ma antibodies oyambira oyenera (OPA1, MFN1, MFN2, ndi DRP1) (yochepetsedwa 1:1000) kwa maola 48, kutsatiridwa ndi incubation ndi ma antibodies achiwiri (1:10,000) kwa ola limodzi. Kenako ma membrane anajambulidwa pogwiritsa ntchito Clarity Western ECL Substrate (#170-5061, Bio-Rad) ndikujambulidwa pogwiritsa ntchito Bio-Rad ChemiDoc MP system. ImageLab version 6.1 idagwiritsidwa ntchito pofufuza Western blot. Gel yoyambirira ndi blot zawonetsedwa pa Chithunzi S1. Zambiri za ma antibodies zaperekedwa mu Table S2.
Ma data akuwonetsedwa ngati mean ndi standard error ya mean (SEM) ya zitsanzo zitatu zodziyimira pawokha. Ma data ayesedwa kuti awone ngati ali bwino pogwiritsa ntchito mayeso a Shapiro-Wilks (pokhapokha atanenedwa mwanjira ina) asanaganize za kugawa kwa Gaussian ndi kupotoka kofanana ndi kusanthula. Kuphatikiza pa kusanthula deta pogwiritsa ntchito Fisher's MEL LSD (p < 0.05), one-way ANOVA (mankhwala vs. control mean), ndi mayeso oyerekeza angapo a Dunnett kuti adziwe kufunika (p < 0.05). P values ​​​​zofunikira zikuwonetsedwa mu graph monga *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Kusanthula konse kwa ziwerengero ndi ma graph kunachitika ndi kupangidwa pogwiritsa ntchito GraphPad Prism 9.4.0.
Ma macro a Fiji/ImageJ owunikira zithunzi za TEM akupezeka pagulu pa GitHub: https://github.com/caaja/TEMMitoMacro. Macro a Mitochondrial Event Locator (MEL) amapezeka pagulu pa GitHub: https://github.com/rensutheart/MEL-Fiji-Plugin.
Meiliana A., Devi NM ndi Vijaya A. Mitochondria: akatswiri owongolera kagayidwe kachakudya, homeostasis, kupsinjika, ukalamba ndi epigenetics. Indonesian. Sayansi ya Zamankhwala. J. 13, 221–241 (2021).
Ben-Shachar, D. Kusokonekera kwa mitochondrial kwa mbali zambiri mu schizophrenia, complex I ngati cholinga cha matenda. Schizophrenia. gwero. 187, 3–10 (2017).
Bose, A. ndi Beal, MF Kulephera kwa Mitochondrial mu matenda a Parkinson. J. Neurochemistry. 139, 216–231 (2016).
Sharma VK, Singh TG ndi Mehta V. Mitochondria yopsinjika: zolinga zolowerera matenda a Alzheimer's. Mitochondria 59, 48–57 (2021).
Belenguer P., Duarte JMN, Shook PF ndi Ferreira GK Mitochondria ndi ubongo: bioenergetics ndi zina zambiri. Neurotoxins. resource. 36, 219–238 (2019).
Rangaraju, V. et al. Pleiotropic mitochondria: momwe mitochondria imakhudzira chitukuko cha mitsempha ndi matenda. J. Neuroscience. 39, 8200–8208 (2019).
Cardaño-Ramos, C. ndi Morais, VA Mitochondrial biogenesis mu ma neuron: momwe ndi komwe. dziko lonse lapansi. J. Mohr. sayansi. 22, 13059 (2021).
Yu, R., Lendahl, U., Nister, M. ndi Zhao, J. Kulamulira kwa mphamvu za mitochondrial za nyamakazi: mwayi ndi zovuta. kutsogolo. endocrine. (Lausanne) 11, 374 (2020).
Khacho, M. ndi Slack, RS Mitochondrial dynamics mu regulation of neurogenesis: kuyambira pakukula kwa ubongo kupita ku wamkulu. development. dynamic. 247, 47–53 (2018).