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Propionic acid (PPA) cu autism spectrum zawtnak tibantuk thluak tthanchonak zawtnak ah mitochondrial riantuan ningcang lo riantuannak hlathlainak caah hman a si. PPA nih hin mitochondrial biogenesis, metabolism le turnover a hrawh tiah theih a si. Sihmanhsehlaw, PPA nih mitochondrial thlennak, tthencheunak le fonhnak cung i a chuahpimi thil hna cu hi thil kalning hna i caan karlak sining a harmi ruangah buainak a chuahter peng. Hika ah, PPA nih neuron-bantuk SH-SY5Y cells ah mitochondrial ultrastructure, morphology le dynamics zeitindah a thlen timi hlathlainak caah aa pehtlaimi zatlang hmanthlaknak thiamnak kan hman. PPA (5 mM) nih mitochondrial area (p < 0.01), Feret diameter le circumference (p < 0.05), le area 2 (p < 0.01) ah a zorter ngaingai. Mitochondrial thil cangmi kawlnak hlathlainak nih fission le fusion thil cangmi ah a langmi karhternak (p < 0.05) a langhter, cucaah lungretheihnak dirhmun tangah mitochondrial network a fekmi a kilven. Cun, cMYC (p < 0.0001), NRF1 (p < 0.01), TFAM (p < 0.05), STOML2 (p < 0.0001) le OPA1 (p < 0.05) mRNA langhternak cu a zor ngaingai. 01). Mah nih hin lungretheihnak dirhmun tangah riantuannak kilven khawhnak ding caah mitochondrial morphology, biogenesis le dynamics remhthannak a langhter. Kan data nih PPA nih mitochondrial dynamics cung ah a chuahpimi thil kongah hmuhnak thar a kan pek i mitochondrial lungretheihnak lehrulhnak ah aa telmi a harmi uknak lamthluan hna hlathlainak caah hmanthlaknak thiamnak hna hmannak kha a langhter.
Mitochondria cu thazaang chuahnak le biosynthesis ah an riantuannak lengah cell riantuannak phunphun ah aa telmi an si. Mitochondrial metabolism cu calcium signaling, metabolic le redox homeostasis, inflammatory signaling, epigenetic remhnak, cell karhternak, thleidannak le program tuahmi cell thihnak1 ah a biapi bikmi uktu a si. A bik in, mitochondrial metabolism cu neuronal tthanchonak, nunnak le riantuannak caah a biapi tukmi a si i neuropathology langhternak phunphun ah aa tel ngaingai2,3,4.
A luancia kum hra chung ah, metabolic dirhmun cu neurogenesis, thleidannak, tthanchonak le thlen khawhnak ah a biapi bikmi uknak ah a hung chuak5,6. Nai ah, mitochondrial morphology le dynamics cu mitosis ah a biapi tukmi thilri ah an cang, cu cu cells chung i a ngandammi mitochondria pawl a kilvengtu a tthawngmi thil a si. Mitochondrial dynamics cu mitochondrial biogenesis le bioenergetics in mitochondrial fission, fusion, transport le clearance tiang a harmi pakhat le pakhat aa hngatmi lam hna nih an uk hna7,8. Hi fonhmi thilri hna lakah pakhatkhat hrawhnak nih a ngandammi mitochondrial network zohkhenhnak a hrawh i thluak tthanchonak caah riantuannak ah a thukmi thil a chuahter9,10. A ngaingai ti ahcun, mitochondrial dynamics thlennak cu lungthin lei, thluak rawhnak le thluak tthanchonak lei zawtnak tampi ah hmuh khawh a si, autism spectrum zawtnak (ASD)11,12 zong aa tel.
ASD cu aa khat lomi thluak tthanchonak zawtnak a si i a harmi thlennawnnak le thlennak sining a ngeimi a si. ASD hrinsor khawhnak cu al khawh a si lo, asinain a tanglei molecular etiology cu ttha tein theihthiam a si rih lo. Preclinical models, clinical studies, le multi-omics molecular datasets in data laknak nih ASD13,14 ah mitochondrial riantuan ningcang lo tehte a karhter. A hlan ah ASD zawtnak a ngeimi mizaw phu pakhat ah genome-wide DNA methylation screen kan tuah i mitochondrial metabolic lamthluan ah aa fonhmi aa dangmi methylated genes15 kan hmuh. Cu hnu ah mitochondrial biogenesis le dynamics i a biapi bikmi uknak hna i aa dangmi methylation kha kan thanh, cucu ASD16 ah mtDNA copy zat karhternak le zunthlum thlennak profile he aa pehtlaimi a si. Kan data nih mitochondrial dynamics le homeostasis nih ASD zawtnak thlennak ah a biapi tukmi hmunhma an lak ti a langhter chin lengmang. Cucaah, mitochondrial riantuan ning, a muisam le riantuannak karlak pehtlaihnak kong theihthiamnak tthanchoter cu a pahnihnak mitochondrial riantuan ningcang lo in a langmi thluak lei zawtnak kong hlathlainak tuah cuahmahmi ah a biapi bikmi tinhmi a si.
Mitochondrial lungretheihnak lehrulhnak ah a hleiin genes hna an riantuannak hlathlainak caah molecular thiamnak hna kha hman a si tawn. Sihmanhsehlaw, hi lamthluan hi mitotic uknak mechanism hna i a phunphun le caan tawite chung lawng a ummi nih a kham men lai. Cun, mitochondrial genes i aa dangmi langhternak cu riantuannak thlennak a langhtertu a si, a hlei in genes tlawmte lawng an hlathlai tawn caah a si. Cucaah, mitochondrial riantuannak le bioenergetics hlathlainak caah a dik deuhmi lam pawl kha an chuahpi cang17. Mitochondrial morphology cu mitochondrial dynamics he aa pehtlai ngaingai. Mitochondrial muisam, pehtlaihnak le dirhmun cu thazaang chuah khawhnak le mitochondrial le cell nunnak caah a biapi tukmi an si5,18. Cun, mitosis a phunphun nih mitochondrial morphology thlennak ah an i hngat, cucu mitochondrial riantuan ningcang lo caah a ttha mi donghnak ah a cang kho i a hnu i mechanistic hlathlainak caah hrampi a si kho.
Mitochondrial morphology cu transmission electron microscopy (TEM) hmangin direct in hmuh khawh a si, cu nih cun cellular ultrastructure kong ttha tein hlathlainak a tuah khawh. TEM nih hin mitochondrial cristae hna i an muisam, an muisam le an dirhmun kha mitochondria pakhat cio i an i tthennak ah direct in a hmuh khawh, cell population chung i gene ṭialnak, protein langhternak asiloah mitochondrial rianṭuannak parameters lawng i bochan loin17,19,20. Cun, TEM nih mitochondria le a dang organelles, endoplasmic reticulum le autophagosomes tibantuk karlak pehtlaihnak kong hlathlainak a bawmh, cu hna nih cun mitochondrial riantuannak le homeostasis ah a biapi tukmi rian an ttuan21,22. Cucaah, hi nih hin TEM cu a hleiin lam asiloah genes ah lungthin pe hlan ah mitochondrial riantuan ningcang lo kong hlathlainak caah hramthawknak tha ah a ser. Mitochondrial riantuannak cu thluak lei zawtnak he aa pehtlaimi a si chin lengmang tikah, in vitro neuronal model ah mitochondrial morphology le dynamics kha direct le quantitative in hlathlai khawh a herh ti cu a fiang ko.
Hi capar ah, autism spectrum zawtnak ah mitochondrial riantuan ningcang lo neuronal model ah mitochondrial dynamics kan zohfel. A hlan ah ASD15, mitochondrial propionyl-CoA carboxylase enzyme PCC subunit ah propionyl-CoA carboxylase beta (PCCB) aa dangmi methylation kong kan rak langhter cang. PCC a ttha lonak nih propionyl derivatives, propionic acid (PPA)23,24,25 telh in, a ttha lomi khonnak a chuahter ti a si. PPA nih hin neuronal metabolism a hrawh i in vivo ah ziaza a thlen ti a si i ASD26,27,28 ah aa telmi thluak tthanchonak lamthluan hlathlainak caah dirhmi saram model a si. Cun, PPA nih mitochondrial membrane thilti khawhnak, biogenesis le thawchuahnak kha in vitro ah a hrawh tiah report an tuah i neurons ah mitochondrial riantuan ningcang lo zohchunh awk ah tampi hman a si29,30. Sihmanhsehlaw, PPA-induced mitochondrial riantuan ningcang lo nih mitochondrial morphology le dynamics cungah a chuahpimi thil cu ttha tein theihthiam a si rih lo.
Hi hlathlainak nih hin SH-SY5Y cells chung i mitochondrial morphology, dynamics le riantuannak cung ah PPA nih a chuahpimi thil kha tuaktan awkah aa pehtlaimi hmanthlaknak thiamnak a hman. Pakhatnak ah, mitochondrial morphology le ultrastructure thlennak hmuh khawhnak ding caah TEM phunglam kan ser17,31,32. Mitochondria33 i aa thleng lengmangmi sining zoh in, PPA hnahnawhnak tangah fission le fusion thil cangmi, mitochondrial zat le volume karlak i tlukruannak thlennak kha tuaktan awkah mitochondrial event localizer (MEL) hlathlainak zong kan hman. A donghnak ah, mitochondrial morphology le dynamics cu biogenesis, fission le fusion ah aa telmi genes langhternak thlennak he aa pehtlai maw ti kha kan zoh. A dihlak in lak ahcun, kan data nih mitochondrial dynamics a ukmi thilri hna i a harnak fianternak ah a harnak kha a langhter. SH-SY5Y cells ah mitosis i tah khawhmi aa fonhmi donghnak pakhat in mitochondrial morphology hlathlainak ah TEM hmannak kha kan langhter. Cun, TEM data nih hin metabolic stress lehrulhnak ah aa thleng lengmangmi thil cangmi zong a tlaihmi hmanthlaknak thiamnak he fonh tikah a rum bikmi thawngpang a pek ti kha kan langhter. Neuronal cell mitosis a bawmmi molecule uknak lamthluan hna kha a dang tein fianternak nih thluak riantuan ning le thluak rawhnak zawtnak hna i mitochondrial tthencheunak kong ah a biapi mi hmuhnak a pek khawh.
Mitochondrial stress chuahter awkah, SH-SY5Y cells cu 3 mM le 5 mM sodium propionate (NaP) hmangin PPA in an thlop hna. TEM hlan ah, sample pawl cu high-pressure lumternak le lumternak hmangin cryogenic sample timhtuahnak ah an chiah hna (Fig. 1a). Mitochondrial mibu hna i an muisam sining pariat kha biological replicate pathum ah tah khawhnak ding caah automated mitochondrial hmanthlak hlathlainak pipeline kan ser. PPA thlopnak nih parameter pali a thlen ngaingai ti kan hmuh: area 2, area, perimeter, le Feret diameter (Fig. 1b–e). Area 2 cu 3 mM le 5 mM PPA thlopnak pahnih in a zor ngaingai (p = 0.0183 le p = 0.002, ciocio) (Fig. 1b), area (p = 0.003), a pawngkam (p = 0.0106) le Feret diameter vialte cu a zor ngaingai. mM thlopnak phu ah control phu he tahchunh tikah a langmi zornak (p = 0.0172) a um (Fig. 1c–e). A kauhnak le a velchum ah a langmi zornak nih 5 mM PPA in thlopmi cells pawl cu a hme deuhmi, a tlong deuhmi mitochondria an ngei ti a langhter, cun mah mitochondria pawl cu control cells nakin a sau deuh lo. Mah cu Feret diameter a zornak he zong aa tlak, mah cu a dang tein a ummi parameter nih particle tthennak karlak a ngan bikmi hlatnak a zornak a langhter. Cristae i a sining thlennak cu hmuh a si: PPA hnahnawhnak nih a ukmi cristae cu a lang lo deuh (Fig. 1a, panel B). Sihmanhsehlaw, hmanthlak vialte nih cristae hna i an sining kha fiang tein an langhter lo caah, hi thlennak hna hi zatlang hlathlainak tuah a si lo. Hi TEM data nih hin thil sining pathum a langhter khawh: (1) PPA nih tthennak a thanchoter asiloah fonhnak a kham, cu nih cun a um cangmi mitochondria kha a hmeter; (2) a tthanchomi biogenesis nih a thar, a hme deuhmi mitochondria a ser asiloah (3) a pahnih in caankhatte ah a chuahter. Hi thil sining hna hi TEM in thleidan khawh an si lo nain, a biapi mi sining thlennak nih PPA hnahnawhnak tangah mitochondrial homeostasis le dynamics thlennak a langhter. Cu hnu ah hi thil sining hna le an hnulei ah a ummi thil sining hna kha langhter chin awkah a dang parameters pawl kha kan kawl.
Propionic acid (PPA) nih mitochondrial sining a remh. (a) PPA thlopnak a karh tikah mitochondrial ngan a zor i mitochondria cu a hme deuh i a tlong deuh ti a langhtermi aiawhtu thlennak electron microscopy (TEM) hmanthlak hna; 0 mM (thlop lomi), 3 mM le 5 mM, ciocio. A senmi thal nih mitochondria a langhter. (b–e) SH-SY5Y cells cu PPA in 24 h chung thlopmi hna cu TEM caah timhtuahnak an ngei i a phichuak cu Fiji/ImageJ hmangin an hlathlai. Parameter pariat chungin pali nih control (thlop lomi, 0 mM PPA) le thlopmi (3 mM le 5 mM PPA) cells karlak ah a biapi mi thleidannak an langhter. (b) Ramkulh 2, (c) Kawng, (d) A velchum, (e) Feret a kauhnak. A biapi mi thleidannak (p < 0.05) hngalh khawhnak ding caah aa dannak (uknak le thlopnak) le Dunnett's tahchunhnak hneksaknak phunphun kha lam khatlei in hlathlainak an tuah. Data points nih cell pakhat cio caah a tlangpi mitochondrial man a langhter, cun palhnak bar nih a tlangpi ± SEM a langhter. Langhtermi data nih n = 3 a langhter, a tlawmbik ah 24 cells pakhat ah; a dihlak ah hmanthlak 266 an hlathlai; * nih p < 0.05 a langhter, ** nih p < 0.01 a langhter.
Mitochondrial dynamics nih PPA zeitindah a lehrulh timi hngalh khawhnak ding caah, mitochondria cu tetramethylrhodamine ethyl ester (TMRE) in kan rong i 3 le 5 mM PPA ah suimilam 24 hnu ah mitochondria hmunhma le zat tuaktannak caah time-lapse microscopy le MEL hlathlainak kan hman. Fission le fusion thil cangmi thlopnak. (Fig. 2a). MEL hlathlainak hnu ah, mitochondria cu mitochondrial sining zat le an tlawmtam zat tuaktan awkah an hlathlai tthan hna. Fission [5.6 ± 0.3 (p < 0.05)]] le fusion [5.4 ± 0.5 (p < 0.05)] le fusion [5.4 ± 0.5 (p < 0.05)] le fusion [5.4 ± 0.5 (p < 0.05)] he tahchunh tikah a tlawm nain a biapi tukmi karhnak kan hmuh. 0.05)] <0.05)] thil cangmi cu 5 mM ah control he tahchunh tikah a karh ngaingai (Fig. 3b). Mitochondria zat cu 3 [32.6 ± 2.1 (p < 0.05)] le 5 mM [34.1 ± 2.2 (p < 0.05)] pahnih ah a karh ngaingai (Fig. 3c) , cu lio ah mitochondrial sining pakhat cio i a tlangpi volume cu aa thleng lo (Fig. 3c). 3d). A dihlak in lak ahcun, hi nih hin mitochondrial dynamics remhthannak nih hin mitochondrial network a tthatnak kha hlawhtling tein a kilvenmi tlukruannak a si tiah a langhter. mM PPA ah tthencheunak thil cangmi a karhmi nih mitochondrial tthencheunak a karhmi cu mitochondrial tthencheunak ruangah a si tiah a langhter, asinain a tlangpi in mitochondrial volume cu aa thleng lo caah, biogenesis cu a dang tlukruannak lehrulhnak ah hrial khawh a si lo. Sihmanhsehlaw, hi data hna hi TEM nih a hmuhmi a hme deuhmi, a tlongmi mitochondrial sining he aa tlak i PPA nih a chuahtermi mitochondrial thlennak ah a biapi mi thlennak zong a langhter.
Propionic acid (PPA) nih network tthatnak kilven awkah aa thleng lengmangmi mitochondrial remhnak a chuahter. SH-SY5Y cells cu an cin hna, 3 le 5 mM PPA in suimilam 24 chung an zohkhenh hna i TMRE le Hoechst 33342 in an thuh hna hnu ah MEL hlathlainak an tuah hna. (a) Caan-lapse microscopy hmanthlak nih rong le binarized a sangbik tthawnnak langhternak kha caan 2 (t2) ah thil sining pakhat cio caah a langhter. Binary hmanthlak pakhat cio ah langhtermi thimcia mi hmun pawl cu caan chung i aa thlengmi langhternak caah caan karlak aa dangmi pathum (t1-t3) ah 3D in an ṭhanchoter hna i an langhter hna; fusion thil cangmi hna cu a sen in langhter an si; fission thil cangmi hna cu a sen in an langhter. A sen in langhter. (b) Thil sining pakhat ah aa thleng lengmangmi thil cangmi zat tlawmpal. (c) Cell pakhat ah mitochondrial sining zat tlawmpal. (d) Cell pakhat ah mitochondrial sining pakhat cio i a tlangpi in a ngan le a hme (μm3). Langhtermi data cu thlopnak phu pakhat ah n = 15 cells aiawhtu an si. Langhtermi palhnak tthen hna nih a tlangpi ± SEM, tahfung tthen = 10 μm, * p < 0.05 an langhter.
Propionic acid (PPA) nih mitochondrial dynamics he aa pehtlaimi genes hna kha ttialnak khamnak a chuahter. SH-SY5Y cells cu 3 le 5 mM PPA in 24 h chung an thlop hna. RT-qPCR hmangin aa pehtlaimi gene zat tuaktannak tuah a si i B2M ah thlen a si. Mitochondrial biogenesis genes (a) cMYC, (b) TFAM, (c) NRF1 le (d) NFE2L2. Mitochondrial fusion le fission genes (e) STOML2, (f) OPA1, (g) MFN1, (h) MFN2 le (i) DRP1. A biapi mi thleidannak (p < 0.05) cu one-way ANOVA (control vs. treatment) le Dunnett's multiple comparison test hmangin hneksaknak an tuah: * nih p < 0.05 a langhter, ** nih p < 0.01 a langhter, cun **** nih p < 0.0001 a langhter. Bars nih a tlangpi in langhternak ± SEM a langhter. Langhtermi data nih n = 3 (STOML2, OPA1, TFAM), n = 4 (cMYC, NRF1, NFE2L2), le n = 5 (MFN1, MFN2, DRP1) biological replicates a aiawh.
TEM le MEL hlathlainak fonh in data nih PPA nih mitochondrial sining le cawlcanghnak a thlen ti a langhter. Sihmanhsehlaw, hi hmanthlaknak thiamnak hna nih hin hi thil kalning a hruaitu a tanglei thilri hna kong ah theihhngalhnak a pe lo. Cucaah PPA thlopnak ah mitochondrial dynamics, biogenesis le mitosis a biapi bikmi uktu pakua hna i mRNA langhternak kha kan zoh. Cell myeloma oncogene (cMYC), nuclear thawchuahnak 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), le Mitofusin (2) le Mitofusin 2 kha kan tuak dynamin he aa pehtlaimi protein 1 (DRP1) cu 3 mM le 5 mM PPA in suimilam 24 chung thlop hnu ah. mM (p = 0.0053, p = 0.0415 le p < 0.0001, ciocio) le 5 mM (p = 0.0031, p = 0.0233, p < 0.0001) PPA thlopnak kan hmuh. (Fig. 3a–c). mRNA langhternak a zornak cu a zat cungah aa hngat: cMYC, NRF1 le TFAM langhternak cu 3 mM ah 5.7, 2.6 le 1.9 in a zor, cun 5 mM ah 11.2, 3 le 2.2 in a zor. Cu he aa dang in, central redox biogenesis gene NFE2L2 cu PPA tlawmtam ah aa thleng lo, asinain aa lo ngaimi dose-dependent trend a zormi langhternak hmuh a si (Fig. 3d).
Fission le fusion uknak ah aa telmi classical genes langhternak zong kan zohfel. STOML2 cu fonhnak, mitophagy le biogenesis ah aa tel tiah ruah a si, cun a langhternak cu 3 mM (2.4-fold thlennak) le 5 mM (2.8-fold thlennak) PPA (Fig. 1) in a zor ngaingai (p < 0.0001). 3d). Cu bantuk cun, OPA1 fusion gene langhternak cu 3 mM (1.6-fold thlennak) le 5 mM (1.9-fold thlennak) PPA (p = 0.006 le p = 0.0024, ciocio) ah a zor (Fig. 3f). Sihmanhsehlaw, 24-h PPA stress tangah fusion genes MFN1, MFN2 asiloah fission gene DRP1 langhternak ah a biapi mi thleidannak kan hmu lo (Fig. 3g–i). Cun, fonhmi le tthencheunak protein pali (OPA1, MFN1, MFN2 le DRP1) hna cu thil sining aa khatmi tangah an i thleng lo ti zong kan hmuh (Fig. 4a–d). Hi data nih hin caan pakhat lawng a langhter i PPA lungretheihnak a hramthawk lio ah protein langhternak asiloah cawlcanghnak tlukruannak thlennak a langhter lo ti kha hngalh a biapi. Sihmanhsehlaw, cMYC, NRF1, TFAM, STOML2, le OPA1 langhternak ah a langmi zornak nih mitochondrial metabolism, biogenesis le dynamics ah a langmi thlennak thlennak a langhter. Cun, hi data nih hin mitochondrial riantuannak ah a donghnak dirhmun thlennak kha direct in hlathlainak tuah khawhnak ding caah hmanthlaknak thiamnak hmannak a langhter.
Propionic acid (PPA) thlopnak hnu ah fusion le fission factor protein dat cu aa thleng lo. SH-SY5Y cells cu 3 le 5 mM PPA in 24 h chung an thlop hna. Protein dat cu Western blot hlathlainak in tah a si, cun langhternak dat cu protein dihlak ah aa tlak. Target le protein dihlak i a tlangpi in protein langhternak le aiawhtu Western blots pawl kha langhter an si. a – OPA1, b – MFN1, c – MFN2, d – DRP1. Bars nih a tlangpi ± SEM a aiawh, cun langhtermi data cu n = 3 biological replicates aiawhtu a si. Tahchunhnak tampi (p < 0.05) cu lam khatlei thleidannak le Dunnett's hneksaknak hmangin tuah a si. A hramthawk gel le blot cu hmanthlak S1 ah hmuh khawh a si.
Mitochondrial riantuan ningcang lo cu thilri thlennak, lungthin le thahri zawtnak in thluak lei zawtnak tiang a phunphun in a chuakmi zawtnak he aa pehtlai1,10. Thluak rawhnak le thluak rawhnak zawtnak tampi cu mitochondrial riantuan ningcang lo he aa pehtlaimi an si, cu nih cun thluak nunchung vialte hi organelles hna an biapitnak a langhter. Cu zawtnak hna cu Parkinson zawtnak, Alzheimer zawtnak le ASD3,4,18 hna an si. Sihmanhsehlaw, hi zawtnak hna hlathlainak caah thluak tissue luh khawhnak cu a har, a hlei in mechanistic level ah, cellular model systems kha a herhmi lam dang ah a ser. Hi hlathlainak ah, PPA-thlopmi SH-SY5Y cells hmangin cellular model system kan hman i, neuronal zawtnak, a bik in autism spectrum zawtnak ah hmuhmi mitochondrial riantuan ningcang lo kha kan tuaktan ṭhan. Hi PPA model hmangin neurons chung i mitochondrial dynamics hlathlainak nih ASD a chuahtertu kong ah theihhngalhnak a pek khawh.
Mitochondrial morphology thlennak zoh khawhnak ding caah TEM hman khawhnak lam kan kawl. TEM cu a tthatnak tam bik hmuh khawhnak dingah a dikthliar in hman a hau ti kha hngalh a biapi. Cryo-specimens timhtuahnak nih hin cellular thilri pawl kha caankhatte ah remhnak le thilri sernak a zorternak thawngin neuronal sining pawl kha ttha deuh in kilven khawhnak a tuah34. Cu he aa tlak in, neuron-like SH-SY5Y cells nih a rawk lomi subcellular organelles le a saumi mitochondria an ngei ti kha kan hmuh (Fig. 1a). Mah nih hin neuronal cell model ah mitochondrial morphology hlathlainak caah cryogenic timhtuahnak thiamnak hmannak a langhter. TEM data zohfelnak caah zatlang tahnak cu a biapi tuk ko nain, mitochondrial morphological thlennak fehternak caah zeibantuk parameters dah tah ding a si timi kongah lungtlinnak a um rih lo. Mitochondrial morphology kha zat in a zohfelmi hlathlainak tampi cungah hram bunh in17,31,32, kannih nih mitochondrial hmanthlak hlathlainak pipeline pakhat kan ser i mah nih cun morphological parameters pariat a tah, cu hna cu: area, area2, aspect ratio, perimeter, circularity, degree , Feret diameter. le a tlongmi.
Cu hna lakah, PPA nih hmun 2, hmun, pawngkam, le Feret diameter kha a zorter ngaingai (Fig. 1b–e). Mah nih hin mitochondria cu a hme deuh i a tlong deuh ti a langhter, cucu PPA30-induced mitochondrial stress suimilam 72 hnu ah mitochondrial area a zornak a langhtermi hlan hlathlainak he aa tlak. Hi sining hna nih hin mitochondrial tthencheunak a langhter khawh, mitochondrial network chungin a rawkmi thilri pawl kha mitophagy hmangin an rawhnak thanchoternak caah a herhmi thil kalning a si35,36,37. Khatlei kam in, a tlangpi in mitochondrial ngan a zornak cu biogenesis a karhmi he aa pehtlaimi a si kho, cu nih cun mitochondria hmete a chuahter. A karhmi tthencheunak asiloah biogenesis nih mitochondrial hnahnawhnak dohnak ah mitosis kilvennak caah tlukruannak lehrulhnak a aiawh. Sihmanhsehlaw, mitochondrial tthanchonak a zormi, fonhnak a rawkmi, asiloah a dang thil sining hna cu hrial khawh an si lo.
TEM nih a sermi a ttha ngaimi hmanthlak nih mitochondria pakhat cio tlukruannak ah a sining hngalh khawhnak a pek ko nain, hi lam nih hin caan khat te ah a tthen hnih in hmanthlak a chuahter. Metabolic stress ah aa thleng lengmangmi lehrulhnak hlathlainak tuah awkah, mitochondria kha TMRE in kan thuh i MEL hlathlainak he time-lapse microscopy kan hman, mah nih caan saupi chung mitochondrial network thlennak kha a sangmi 3D hmuh khawhnak a kan pek33,38. PPA hnahnawhnak tangah mitochondrial thlennak ah a fiang lomi asinain a biapi tukmi thlennak kan hmuh (Fig. 2). mM ah, tthencheunak thil cangmi hna cu a karh ngaingai, cu lio ah fonhnak thil cangmi hna cu control bantuk tein an um. Fission le fusion thil cangmi pahnih an karh ning cu 5 mM PPA ah hmuh a si, asinain hi thlennak hna hi aa tluk tein an si, cu nih cun fission le fusion kinetics nih a sang deuhmi concentration ah tlukruannak an phan ti a langhter (Fig. 2b). A tlangpi in mitochondrial volume cu 3 le 5 mM PPA pahnih ah aa thleng lo, cu nih cun mitochondrial network a fekmi a kilven ti a langhter (Fig. 2d). Mah nih hin aa thleng lengmangmi mitochondrial network hna nih network tthencheunak chuahter loin homeostasis ttha tein kilven khawhnak ding caah a nemmi metabolic stress lehrulhnak an tuah khawhnak kha a langhter. mM PPA ah, tthennak a karhmi cu tlukruannak thar ah thlennak a chuahter khawh, asinain PPA a tam deuhmi nih a chuahtermi lungretheihnak lehrulhnak ah a thuk deuhmi kinetic remhthannak a herh.
PPA stress concentration pahnih ah mitochondria zat cu a karh, asinain a tlangpi in mitochondrial volume cu aa thleng tuk lo (Fig. 2c). Mah cu biogenesis a karhmi asiloah tthencheunak a karhmi ruangah a si kho; sihmanhsehlaw, a tlangpi in mitochondrial volume ah a tlawm deuhmi a um lo ahcun, biosynthesis a karh deuh lai ti a si. Sihmanhsehlaw, hmanthlak 2 chung i data nih hin tlukruannak lam pahnih a um ti kha a dirpi: mitochondrial tthennak upregulation he aa tlakmi fission thil cangmi a karh, cun mitochondrial biogenesis he aa tlakmi thil cangmi a karh. A donghnak ah, lungretheihnak hmete caah aa thleng lengmangmi tlukruannak cu tthencheunak, fonhnak, biogenesis le mitophagy aa telmi caankhatte ah a cangmi thil sining hna in aa tel kho. A hlan catialtu hna nih PPA nih mitosis30,39 le mitophagy29 a ṭhanchoter tiah an langhter ko nain, PPA lehrulhnak ah mitochondrial tthennak le fonhnak thlennak remhnak caah tehte kan pek. Hi data nih hin TEM nih a hmuhmi sining thlennak kha a fehter i PPA-induced mitochondrial riantuan ningcang lo he aa pehtlaimi thil kalning kong ah theihhngalhnak a pek.
TEM le MEL hlathlainak nih hmuhmi sining thlennak tangah a ummi gene uknak phunglam hna kha direct in tehte an pek lo caah, mitochondrial metabolism, biogenesis le dynamics ah aa telmi genes hna i RNA langhternak kha kan zoh. cMYC proto-oncogene cu mitochondria, glycolysis, amino acid le fatty acid metabolism40 uknak ah aa telmi ttialnak thilri a si. Cun, cMYC cu NRF1 le TFAM41 telh in mitochondrial ṭialnak, lehnak le a harmi fonhnak ah aa telmi mitochondrial genes 600 hrawng langhternak kha a uk khawh tiah theih a si. NRF1 le TFAM cu mitosis uknak ah a biapi bikmi pahnih an si, PGC-1α hnulei ah mtDNA thlennak a chuahtertu ah an cawlcang. Hi lam hi cAMP le AMPK signaling nih a cawlcangter i thazaang hmannak le metabolic stress ah a tuar khawh. Cun, NFE2L2, mitochondrial biogenesis redox regulator zong kan zoh, PPA nih a chuahpimi thil cu oxidative stress nih a hruaimi a si kho maw ti hngalh khawhnak ding caah.
NFE2L2 langhternak cu aa thleng lo nain, 3 mM le 5 mM PPA in 24 h thlop hnu ah cMYC, NRF1 le TFAM langhternak ah aa khatmi dose-dependent zornak kan hmuh (Fig. 3a–c). cMYC langhternak tlawmternak cu a hlan ah mitochondrial stress42 lehrulhnak ah report a rak si cang, cun a dang tein, cMYC langhternak tlawmternak nih mitochondrial metabolism, network pehtlaihnak le membrane polarization remhnak in mitochondrial riantuan ningcang lo a chuahter khawh43. A hngalh a nuammi cu, cMYC cu mitochondrial tthennak le fonhnak uknak ah aa tel ve42,43 i cell tthennak chungah DRP1 phosphorylation le mitochondrial hmunhma a karhter ti a si44, cun neuronal stem cells45 ah mitochondrial morphological remhnak zong a tuah. A ngaingai ti ahcun, cMYC-tlawmmi fibroblasts nih mitochondrial ngan a zorter, PPA43 lungretheihnak nih a chuahtermi thlennak he aa tlak. Hi data nih hin cMYC le mitochondrial dynamics karlak ah a hngalh a nuammi asinain a fiang rih lomi pehtlaihnak a langhter, PPA lungretheihnak nih a chuahtermi remhthannak kong hmailei hlathlainak caah a nuammi tinhmi a pek.
NRF1 le TFAM zorternak cu cMYC nih a biapi mi ttialmi riantuannak a ttuanmi he aa tlak. Hi data hna hi minung pawpi cancer cells ah PPA nih suimilam 22 ah NRF1 mRNA langhternak a zorter ti a langhtermi hlathlainak hna he zong aa tlak, cucu ATP zorternak le ROS46 karhternak he aa pehtlaimi a si. Hi caṭialtu hna nih TFAM langhternak cu suimilam 8.5 ah a karh nain suimilam 22 ah a hramthawk dirhmun ah a kir ṭhan tiah an langhter. Cu he aa dang in, Kim le a hawile. (2019) nih SH-SY5Y cells ah PPA stress 4 h hnu ah TFAM mRNA langhternak cu a zor ngaingai ti a langhter; sihmanhsehlaw, suimilam 72 hnu ah, TFAM protein langhternak cu a karh ngaingai i mtDNA copy zat cu a karh ngaingai. Cucaah, suimilam 24 hnu ah kan hmuhmi mitochondrial biogenesis genes a zornak nih mitochondria a karhmi cu a hlan deuh caan ah biogenesis riantuannak he aa pehtlaimi a si khomi kha a hrial lo. Hlan lio hlathlainak nih a langhter ning ahcun PPA nih SH-SY5Y cells ah PGC-1α mRNA le protein kha suimilam 4 le minutes 30 ah a tthawnter ngaingai, cu lio ah propionic acid nih suimilam 12 le minutes 39 ah PGC-1α hmangin calf hepatocytes ah mitochondrial biogenesis a tthanchoter. A hngalh a nuammi cu, PGC-1α cu NRF1 le TFAM i direct in ṭialmi uknak lawng si loin, MFN2 le DRP1 cawlcanghnak kha fission le fusion uknak in a uk khawh ti zong hmuh a si47. A dihlak in lak ahcun, hi nih hin PPA nih a chuahtermi mitochondrial tlukruannak lehrulhnak a ukmi thilri hna i naihniam tein pehtlaihnak a langhter. Cun, kan data nih PPA lungretheihnak tangah biogenesis le metabolism i ttialmi uknak ah a biapi tukmi thlennak a langhter.
STOML2, OPA1, MFN1, MFN2 le DRP1 genes hna cu mitochondrial tthennak, fonhnak le thlennak ah a biapi bikmi uknak ah an i tel37,48,49. Mitochondrial dynamics ah aa telmi gene dang tampi an um, sihmanhsehlaw, STOML2, OPA1 le MFN2 cu ASD cohorts ah aa dang tein methylated an si ti kha hmuh a si cang,16 cun aa hngatmi hlathlainak zeimawzat nih mitochondrial stress lehrulhnak ah hi transcription factors ah thlennak an langhter50,51. 52. OPA1 le STOML2 pahnih langhternak cu 3 mM le 5 mM PPA thlopnak nih a zorter ngaingai (Fig. 3e, f). OPA1 cu MFN1 le 2 he direct in pehtlaihnak thawngin mitochondrial fonhnak a uktu hlun pakhat a si i cristae remhnak le mitochondrial morphology ah rian a ttuan53. STOML2 nih mitochondrial dynamics ah a ttuanvo taktak cu a fiang rih lo, asinain tehte nih mitochondrial fonhnak, biogenesis le mitophagy ah rian a ttuan ti a langhter.
STOML2 cu mitochondrial thawchuahnak pehtlaihnak le thawchuahnak hri complex sernak ah aa tel54,55 i cancer cells hna i an thlennak sining kha fakpi in a thlen ti a si56. Hlathlainak nih a langhter ning ahcun STOML2 nih BAN le cardiolipin 55, 57, 58 he pehtlaihnak thawngin mitochondrial membrane thilti khawhnak le biogenesis a thanchoter. Cun, mah tein hlathlainak nih STOML2 le PINK1 karlak pehtlaihnak nih mitophagy59,60 a uk ti a langhter. Hngalh awk ngai a simi cu, STOML2 cu MFN2 he direct in pehtlaihnak a tuah i a dirhmun a fehter tiah report an tuah i OPA1 rawhnak caah tuanvo a ngeimi protease kha khamnak in OPA1 isoform sau dirhmun ah a biapi tukmi rian a ttuan53,61,62. PPA lehrulhnak ah hmuhmi STOML2 langhternak a zornak nih hin hi fonhmi protein pawl hi ubiquitin- le proteasome-a hngatchanmi lam hna in rawhnak ah a fawi deuhmi a siter khawh48. PPA cung i aa thleng lengmangmi lehrulhnak ah STOML2 le OPA1 riantuannak taktak cu a fiang lo nain, hi fusion genes (Figure 3) langhternak a zormi nih fission le fusion karlak i tlukruannak kha a hrawh khawh i mitochondrial ngan a zorter khawh (Figure 3). 1).
Khatlei kam in, OPA1 protein langhternak cu 24 h hnu zongah aa thleng lo, cu lio ah MFN1, MFN2 asiloah DRP1 mRNA le protein dat cu PPA thlopnak hnu ah a dang tein aa thleng lo (Fig. 3g-i, Fig. 4). Mah nih hin mitochondrial fonhnak le tthennak ah aa telmi hi thil hna uknak ah thlennak a um lo ti a langhter men lai. Sihmanhsehlaw, hi gene pali hna hi pakhat cio cu protein cawlcanghnak a ukmi posttranscriptional remhnak (PTMs) nih a uk hna ti zong hi hngalh awk tlak a si. OPA1 nih a dang splice phun 8 a ngei i cu hna cu mitochondria ah proteolytically in an i tthen i aa dangmi isoform pahnih an chuahter 63 . Isoform sau le tawi karlak i tlukruannak nih a donghnak ah mitochondrial fonhnak le mitochondrial network zohkhenhnak ah OPA1 riantuannak kha a khiah64. DRP1 cawlcanghnak cu calcium/calmodulin-dependent protein kinase II (CaMKII) phosphorylation nih a uk, DRP1 rawhnak cu ubiquitination le SUMOylation65 nih a uk. A donghnak ah, DRP1 le MFN1/2 pahnih hi GTPases an si, cucaah cawlcanghnak cu mitochondria ah GTP chuahternak rate nih a thlen khawh 66 . Cucaah, hi protein pawl langhternak cu aa thleng lo nain, mah nih hin aa thleng lomi protein cawlcanghnak asiloah hmunhma a langhter lo men lai67,68. A ngaingai ti ahcun, a um cangmi PTM protein repertoires pawl cu a fakmi lungretheihnak lehrulhnak hna caah rian a ttuanmi ralvennak hmasa bik ah an ṭuan tawn. Kan model ah a tlawm deuhmi metabolic stress a um tikah, PTM nih mRNA asiloah protein level ah hi genes pawl riantuannak chap a hau loin mitochondrial tthatnak tling tein remh khawhnak ding caah fusion le fission proteins cawlcanghnak a karhter lai ti a si.
A dihlak in lak ahcun, a cunglei data nih hin mitochondrial morphology a harmi le caan cung ah aa hngatmi uknak le hi thil kalning hna fianternak ah a harmi thil hna kha a langhter. Gene langhternak hlathlainak tuah awkah, lamthluan chung i a bik in target genes pawl kha hngalh hmasa a herh. Sihmanhsehlaw, kan data nih a langhter mi cu lam pakhat chung i a ummi genes pawl nih lungretheihnak pakhat kha aa khatmi lam in an lehrulh lo. A ngaingai ti ahcun, a hlan i hlathlainak nih lam pakhat chung i aa dangmi genes nih caan tawite chung lehnak profile aa dangmi an langhter khawh ti kha a langhter30,46. Cun, cafang ttialnak le gene riantuannak karlak pehtlaihnak a hrawktu a harmi cafang ttial hnu thil sining hna an um. Proteomic hlathlainak nih PTMs le protein riantuannak nih a chuahpimi thil sining kong ah theihhngalhnak a pek khawh, asinain a tlawmmi chuah khawhnak lam, a sangmi signal-to-noise ratio, le a ttha lomi thlennak hna telh in harnak zong an chuahpi.
Hi kong he pehtlai in, TEM le MEL hmangin mitochondrial morphology hlathlainak nih mitochondrial dynamics le riantuannak karlak pehtlaihnak kong le zeitindah zawtnak a thlen timi kong he pehtlai in a hrampi biahalnak hna kha leh khawhnak thazaang tampi a ngei. A biapi bikmi cu, TEM nih mitochondrial riantuan ningcang lo le thlennak aa fonhmi donghnak ah mitochondrial morphology tahnak caah direct lam a pek51. MEL nih hin tthenthumnak cell pawngkam ah tthencheunak le fonhnak thil cangmi hmuh khawhnak lam zong a pek, cu nih cun gene langhternak ah thlennak a um lo hmanh ah aa thleng lengmangmi mitochondrial remhnak zat tuak khawhnak a tuah33. Hika ah hin a pahnihnak mitochondrial zawtnak ah mitochondrial hmanthlaknak thiamnak hmannak kha kan langhter. Hi zawtnak hna hi a tlangpi in a fakmi mitochondrial rawhnak nakin mitochondrial network hna i a fiang lomi remhnak in a langmi a nemmi metabolic stress in a lang. Sihmanhsehlaw, caan saupi lungretheihnak tangah mitosis kilven awkah a herhmi mitochondrial tlukruannak nih riantuannak ah a thukmi thil a chuahter. Neuroscience kongkau ah, hi tlukruannak lamthluan hna hngalhthiamnak nih hin mitochondrial riantuan ningcang lo he aa pehtlaimi pleiotropic neuropathology kong ah a biapi mi thawngpang a kan pek khawh.
A donghnak ah, kan data nih hin gene langhternak, protein remhnak le neuronal mitochondrial dynamics a ukmi protein cawlcanghnak karlak ah a harmi pehtlaihnak hna i riantuannak a phichuak hngalhthiamnak caah hmanthlaknak thiamnak hmannak a langhter. ASD mitochondrial component kong theihthiamnak hmuh khawhnak ding caah neuronal cell model ah mitochondrial riantuan ningcang lo zohchunh awk ah PPA kan hman. PPA in thlopmi SH-SY5Y cells nih mitochondrial morphology ah thlennak an langhter: mitochondria cu a hme i a tlong, cun TEM in zoh tikah cristae cu a fiang lo. MEL hlathlainak nih a langhter mi cu hi thlennak hna hi a nemmi metabolic stress lehrulhnak ah mitochondrial network kilvennak caah fission le fusion thil cangmi a karh he aa pehtlaimi a si. Cun, PPA nih mitochondrial metabolism le homeostasis i ttialmi phunglam kha tampi in a hrawh. cMYC, NRF1, TFAM, STOML2, le OPA1 cu PPA lungretheihnak nih a hrawhmi a biapi mi mitochondrial uknak ah kan hmuh hna i PPA nih a chuahtermi mitochondrial sining le riantuannak ah thlennak a chuahtertu ah rian a ttuan kho men. Hmailei hlathlainak hna cu PPA-induced caan karlak thlennak hna kha gene langhternak le protein cawlcanghnak, hmunhma, le lehnak hnu thlennak hna ah ttha deuh in langhter a herh. Kan data nih mitochondrial stress lehrulhnak a tawlreltu uknak lamthluan hna i a harmi le pakhat le pakhat i hngatchannak kha a langhter i TEM le a dang hmanthlaknak thiamnak hna kha a biapi deuhmi mechanistic hlathlainak caah hman khawhnak kha a langhter.
SH-SY5Y cell line (ECACC, 94030304-1VL) cu Sigma-Aldrich in cawkmi a si. SH-SY5Y cells cu Dulbecco's modified Eagle's medium/F-12 nutrient mixture (DMEM/F-12) le L-glutamine (SC09411, ScienCell) ah 25 cm2 flasks ah 20% fetal bovine serum (FBS) (193013, 1936) he an cawhmi ah an cin hna 1% penicillin-streptomycin (P4333-20ML, Sigma-Aldrich) 37 °C ah, 5% CO2. Cell pawl cu 0.05% trypsin-EDTA (15400054, ThermoFisher Scientific) hmangin 80% fonh tiang an cawm hna, 300 g ah an cawh hna i 7 × 105 cells/ml hrawng ah an cawh hna. Hneksaknak vialte cu 19-22 karlak ah aa thleidang lomi SH-SY5Y cells cungah tuah a si. PPA cu NaP in pek a si. NaP powder (CAS No. 137-40-6, chemical formula C3H5NaO2, P5436-100G, Sigma-Aldrich) kha MilliQ ti lum ah 1 M tiang cawh law 4 °C ah chiah. Thlopnak ni ah, hi cawhmi cu 1 M PPA in 3 mM le 5 mM PPA in serum-free medium (DMEM/F-12 with L-glutamine) ah cawh. Hneksaknak vialte caah thlopnak tlawmpal cu PPA (0 mM, control), 3 mM, le 5 mM PPA a um lo. Hneksaknak cu a tlawmbik biological tlukruannak pathum ah tuah a si.
SH-SY5Y cells cu 25 cm5 flasks ah 5.5 × 105 cells/ml in an cin hna i suimilam 24 chung an cin hna. PPA thlopnak cu 24 h chiah hlan ah flask chungah an cawh. A tlangpi in saram tissue subculture phunglam (a cunglei ah langhtermi) zul in cell pellets kha lak. Cell pellet cu 100 μl 2.5% glutaraldehyde, 1× PBS ah chiah tthan law 4 °C ah tuah hlan tiang chiah. SH-SY5Y cells cu cells pawl kha pellet tuah awk le 2.5% glutaraldehyde, 1× PBS cawhnuk chuah awkah caan tawite chung centrifuge an tuah hna. Ti thianghlim ah timhmi 4% agarose gel ah vawlei kha hnim ṭhan (agarose le vawlei zat zat cu 1:1 a si). Agarose tthen pawl cu a tlawmmi changreu cung i hri cung ah chiah an si i 1-hexadecene in an thuh hna hnu ah a fakmi lumter an si. Sample pawl cu 100% a ro cangmi acetone ah -90°C ah suimilam 24 chung an hnim hna. Cu hnu ah lumnak cu -80°C tiang an kaiter i 1% osmium tetroxide le 0.1% glutaraldehyde cawhmi cawhmi kha an cawh. Samples cu -80°C ah suimilam 24 chung chiah an si. Cu hnu ah, ni zeimawzat chung khaan lumnak tiang zaangfah tein an karhter: suimilam 24 chung – 80 °C in – 50 °C tiang, suimilam 24 chung – 30 °C tiang, suimilam 24 chung – 10 °C tiang le a donghnak ah khaan lumnak tiang. a kih a lum.
Cryogenic timhtuahnak hnu ah, sample pawl cu resin in an cawh hna i Leica Reichert UltracutS ultramicrotome (Leica Microsystems) hmangin a tlawm tukmi tthen (∼100 nm) an tuah. Cheukhat cu 2% uranyl acetate le lead citrate in an thuh hna. FEI Tecnai 20 transmission electron microscope (ThermoFisher (a hlan ah FEI), Eindhoven, The Netherlands) 200 kV (Lab6 transmitter) le Tridiem thazaang filter he aa pehtlaimi Gatan CCD camera (Gatan, UK) hmangin zohchunhmi hna cu zoh an si.
Technical replicate pakhat cio ah, a tlawmbik ah cell pakhat hmanthlak 24 an hmuh, a dihlak ah hmanthlak 266 an si. Hmanthlak vialte cu Region of Interest (ROI) macro le Mitochondria macro hmangin hlathlainak tuah an si. Mitochondrial macro cu chuahmi lamthluan17,31,32 cungah hram aa bunh i Fiji/ImageJ69 ah TEM hmanthlak pawl kha amah tein a tlawmpal in a tthencheu in thlennak a tuah khawh. A tawinak in chim ahcun: hmanthlak cu a hnulei ah a tlongmi ball hnulei tlawmternak (60 pixel radius) le FFT bandpass filter (60 le 8 pixel cunglei le a tanglei ramri hmangin) le 5% tluk tuar khawhnak he a dirmi line khamnak hmangin a hnulei le a hnulei ah a kir. A ttha bikmi hmanthlak cu a sang bik entropy algorithm hmangin amah tein threshold tuah a si i binary mask a chuak. Raw TEM hmanthlak chung i kut in thimmi ROIs he aa pehtlaimi hmanthlak hmun pawl cu an chuah hna, mitochondria sining langhternak le plasma membrane le a dang aa dannak sangmi hmun pawl telh loin. ROI chuahmi pakhat cio caah, 600 pixels nak ngan deuhmi binary particles pawl kha an hlathlai hna, cun particle area, perimeter, a ngan le a hme axes, Feret diameter, a tlongmi, le a tlongmi kha Fiji/ImageJ i a chunglei tahnak riantuannak hmangin tah an si. Merrill, Flippo, le Strack (2017) hnu zul in, area 2, particle aspect ratio (major in minor axis ratio), le shape factor (FF) hna cu hi data chungin tuak an si, cuka ahcun FF = perimeter 2/4pi x area. Parametric formula fianternak cu Merrill, Flippo, le Strack (2017) ah hmuh khawh a si. A langhtermi macros pawl cu GitHub ah hmuh khawh an si (Data Hmuh khawhnak Cathanh zoh). A tlangpi in, PPA thlopnak pakhat ah 5,600 hrawng cu an hlathlai hna, a dihlak ah 17,000 hrawng an si (data langhter lo).
SH-SH5Y cells cu 8-chamber culture dishes (ThermoFisher, #155411) ah an chiah hna i zankhat chung an i hngat khawh nakhnga an chiah hna i cu hnu ah TMRE 1:1000 (ThermoFisher, #T669) le Hoechst 33342 1:200 (Sigma-Aldrich, #15411) he an cawh hna. rong suai. Hmanthlak pawl cu 405 nm le 561 nm laser hmangin 10 min pawngkam ah an lak hna, cun raw hmanthlak pawl cu a hnu caan 12 ah hmanthlak frame karlak ah 0.2 μm az step he hmanthlak hmete 10 aa telmi z-stacks in an lak hna. Hmanthlak pawl cu LCI Plan Apochromate 100x/1.4 Oil DIC M27 lens hmangin Carl Zeiss LSM780 ELYRA PS.1 super-resolution platform (Carl Zeiss, Oberkochen, Germany) hmangin an lak hna. Hmanthlak pawl cu a hlan ah langhtermi pipeline le ImageJ plugin hmangin ImageJ ah fusion le fission thil cangmi, mitochondrial sining zat zat, le cell pakhat ah mitochondrial zat zat tahnak caah an hlathlai hna33. MEL macros cu GitHub ah hmuh khawh a si (Data Hmuh khawhnak Cathanh zoh).
SH-SY5Y cells cu thlopbulnak tuah hlan suimilam 24 chung 0.3 × 106 cells/mL density in six-well plates ah an cin hna. RNA cu Quick-RNATM Miniprep protocol (ZR R1055, Zymo Research) hmangin tlawmpal remhnak he chuah a si: chuah hlan ah tikhor pakhat cio ah RNA lysis buffer 300 μl chap law DNase/RNase elution 30 μl he a donghnak ah sample pakhat cio kha lyse tuah. -man loin ti. Sample vialte cu NanoDrop ND-1000 UV-Vis Spectrophotometer hmangin an zat le an tthatnak zohfel an si. Cell lysates chungin protein dihlak cu 200 μl RIPA lysis buffer hmangin hmuh a si, cun protein tlawmtam cu Bradford protein assay70 hmangin tah a si.
cDNA sernak cu TetroTM cDNA sernak thilri (BIO-65043, Meridian Bioscience) hmangin sertu nih a chimmi ningin remhnak cheukhat he tuah a si. cDNA cu 0.7 in 1 μg dihlak RNA hmangin 20-μl thlennak ah ser a si. Primers cu a hlan ah chuahmi cauk 42, 71, 72, 73, 74, 75, 76, 77, 78 (Table S1) chungin thim an si i aa pehtlaimi probe pawl cu Integrated DNA Technologies in PrimerQuest thilri hmangin an suai hna. Lungthawhnak a ngeimi gene vialte cu nuclear B2M gene ah an i thlen. STOML2, NRF1, NFE2L2, TFAM, cMYC le OPA1 gene langhternak cu RT-qPCR in tah a si. A tlangpi cawhmi ah LUNA Taq polymerase (M3003L, New England Biolabs), 10 μM hmailei le hnulei primers, cDNA, le PCR-grade ti aa tel i a donghnak volume 10 μL kha thlennak pakhat cio ah a chuak. Tthennak le tthennak genes (DRP1, MFN1/2) langhternak cu TaqMan multiplex hneksaknak hmangin tah a si. Luna Universal Probe qPCR Master Mix (M3004S, New England Biolabs) cu a sertu nih a chimmi ningin remhnak hmete he hman a si. Multiplex RT-qPCR master mix ah 1X LUNA Taq polymerase, 10 μM hmailei le hnulei primers, 10 μM probe, cDNA, le PCR-grade ti aa tel, cu nih cun a donghnak ah 20 μL a chuahter. RT-qPCR cu Rotor-Gene Q 6-plex (QIAGEN RG—serial number: R0618110) hmangin tuah a si. Cycle mawngh ningcang cu Table S1 ah langhter a si. cDNA sample vialte cu a let thum in an karhter hna i a let hra tlawmternak hmangin a tlarimi tthencheunak pakhat an ser. Data chuah khawhnak ding caah cycle threshold standard deviation (Ct) >0.5 a ngeimi triplicate sample chung i a lenglei pawl cu hlathlainak chungin an chuah hna30,72. Aa pehtlaimi gene langhternak cu 2-ΔΔCt79 phunglam hmangin tuak a si.
Protein sample (60 μg) cu Laemmli loading buffer he 2:1 ratio in an cawh i 12% rong ngei lomi protein gel (Bio-Rad #1610184) ah an hman. Protein pawl cu Trans-Blot Turbo system (#170-4155, Bio-Rad) hmangin PVDF (polyvinylidene fluoride) membrane (#170-84156, Bio-Rad) ah an thlen hna. Membrane cu an phih i aa tlakmi a hrampi antibodies (OPA1, MFN1, MFN2, le DRP1) (1:1000 in cawhmi) he suimilam 48 chung an chiah, cun a pahnihnak antibodies (1:10,000) he suimilam 1 chung an chiah. Membranes cu Clarity Western ECL Substrate (#170-5061, Bio-Rad) hmangin hmanthlak an thlak hna i Bio-Rad ChemiDoc MP system hmangin an khumh hna. ImageLab version 6.1 cu Western blot hlathlainak caah hman a si. A hramthawk gel le blot cu hmanthlak S1 ah hmuh khawh a si. Antibody konglam cu Table S2 ah pek a si.
Data set pawl cu a tlawmbik aa hngatmi sample pathum i a tlangpi le a tlangpi palhnak (SEM) in langhter an si. Data set pawl cu Gaussian phawtzamhnak le aa tlukmi tlukruannak thleidannak le hlathlainak pehzulh hlan ah Shapiro-Wilks hneksaknak (a dang tein langhter a si lo ahcun) hmangin a tlarimi sinak caah hneksaknak an tuah. Fisher's MEL LSD (p < 0.05), one-way ANOVA (treatment vs. control mean), le Dunnett's multiple comparison test hmangin a sullam hngalh khawhnak (p < 0.05) hmangin data set hlathlainak tuah chap ah. A biapi mi p man hna cu graph ah *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 tiah langhter an si. Statistical hlathlainak le graph vialte cu GraphPad Prism 9.4.0 hmangin tuah le chuah an si.
TEM hmanthlak hlathlainak caah Fiji/ImageJ macros cu GitHub ah mipi hmuh khawh a si: https://github.com/caaja/TEMMitoMacro. Mitochondrial Event Locator (MEL) macro cu GitHub ah mipi nih hmuh khawh a si: https://github.com/rensutheart/MEL-Fiji-Plugin.
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