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Propionic acid (PPA) hi autism spectrum disorder ang chi neurodevelopmental disorder-a mitochondrial dysfunction chanvo zir chianna atan hman a ni. PPA hian mitochondrial biogenesis, metabolism leh turnover a tibuai tih hriat a ni. Mahse, PPA hian mitochondrial dynamics, fission leh fusion a nghawng dan chu heng mechanisms te hi complex temporal nature avang hian harsatna a la awm reng a ni. Hetah hian complementary quantitative imaging technique hmangin neuron ang chi SH-SY5Y cell-a PPA hian mitochondrial ultrastructure, morphology, leh dynamics a nghawng dan kan zirchiang a ni. PPA (5 mM) hian mitochondrial area (p < 0.01), Feret diameter leh circumference (p < 0.05), leh area 2 (p < 0.01) a tihhniam nasa hle. Mitochondrial event locator analysis chuan fission leh fusion event-ah nasa takin a pung (p < 0.05) a, chu chuan stress condition hnuaiah mitochondrial network integrity a vawng reng a ni. Hei bakah hian cMYC (p < 0.0001), NRF1 (p < 0.01), TFAM (p < 0.05), STOML2 (p < 0.0001) leh OPA1 (p < 0.05) te mRNA expression pawh nasa takin a tlahniam bawk. 01). Hei hian stress condition hnuaia function vawng reng turin mitochondrial morphology, biogenesis leh dynamics te remodeling a entir a ni. Kan data hian PPA hian mitochondrial dynamics a nghawng dan chungchangah hriatna thar a pe a, mitochondrial stress response-a inrawlh complex regulatory mechanism zir chianna atana imaging technique hman tangkai dan a tarlang bawk.
Mitochondria hi energy siamchhuah leh biosynthesis-a an chanvo pangngai aia cellular function hrang hrangah pawh integral participant an ni. Mitochondrial metabolism hi calcium signaling, metabolic leh redox homeostasis, inflammatory signaling, epigenetic modifications, cell proliferation, differentiation leh programmed cell death te titu pawimawh tak a ni A bik takin mitochondrial metabolism hi neuronal hmasawnna, dam khawchhuahna leh hnathawhna atan a pawimawh hle a, neuropathology lan chhuahna hrang hrangah a inrawlh nasa hle2,3,4.
Kum sawm kalta chhung khan metabolic status chu neurogenesis, differentiation, maturation leh plasticity central regulator angin a lo chhuak a5,6. Tun hnaiah chuan mitochondrial morphology leh dynamics chu mitosis-a thil pawimawh tak a lo ni ta a, hei hi cell chhunga mitochondria hrisel tak takte pool vawng rengtu dynamic process a ni. Mitochondrial dynamics hi mitochondrial biogenesis leh bioenergetics atanga mitochondrial fission, fusion, transport leh clearance thlengin complex interdependent pathways hmanga enkawl a ni7,8. Heng integrative mechanism eng pawh tihbuai hian mitochondrial network hrisel tak tak enkawlna a tichhe a, neurodevelopment-ah pawh functional consequence thuk tak a nei a ni9,10. Dik tak chuan mitochondrial dynamics dysregulation hi psychiatric, neurodegenerative leh neurodevelopmental disorder tam takah hmuh a ni a, autism spectrum disorders (ASD)11,
ASD hi heterogeneous neurodevelopmental disorder a ni a, a genetic leh epigenetic architecture complex tak a ni. ASD hi heritability hi sawisel theih a ni lo a, mahse a hnuaia molecular etiology hi hriat chian a la ni lo. Preclinical model, clinical study, leh multi-omics molecular dataset atanga data khawlkhawm hian ASD-ah mitochondrial dysfunction a awm tih finfiahna a pung zel a ni13,14. Tun hmain ASD vei cohort-ah genome-wide DNA methylation screen kan nei a, mitochondrial metabolic pathways-a clustered differentially methylated genes kan hmuchhuak a ni15. Chumi hnuah mitochondrial biogenesis leh dynamics-a central regulator-te differential methylation kan report a, hei hi mtDNA copy number tihpun leh ASD16-a urinary metabolic profile tihdanglamna nen a inzawm a ni. Kan data hian ASD pathophysiology-ah mitochondrial dynamics leh homeostasis te hian hmun pawimawh tak an chang tih finfiahna a pe nasa hle. Chuvangin, mitochondrial dynamics, morphology leh function inzawmna chungchanga mechanistic understanding tihchangtlun hi secondary mitochondrial dysfunction-a hriat hlawh neurological diseases chungchanga zirchianna kalpui mek tum ber a ni.
Mitochondrial stress response-a gene bikte chanvo zir chianna atan hian molecular technique hman a ni fo thin. Mahse, he approach hi mitotic control mechanisms multifaceted leh temporal nature avang hian a tihkhawtlai thei a ni. Chubakah, mitochondrial genes differential expression hi functional changes indirect indicator a ni a, a bik takin genes tlemte chauh an zirchian tlangpui avangin. Chuvangin, mitochondrial function leh bioenergetics zir chianna atan direct methods zawk an rawn rawt ta a ni17. Mitochondrial morphology hi mitochondrial dynamics nen a inzawm tlat a ni. Mitochondrial pianzia, inzawmna leh structure te hi energy siamchhuahna leh mitochondrial leh cell dam khawchhuahna atan a pawimawh hle5,18. Chubakah, mitosis component hrang hrangte hian mitochondrial morphology inthlak danglamna lam an ngaihtuah a, chu chu mitochondrial dysfunction endpoint tangkai tak a ni thei a, a hnu lama mechanistic study neihna tur bulpui a ni thei bawk.
Mitochondrial morphology hi transmission electron microscopy (TEM) hmangin direct-a enfiah theih a ni a, cellular ultrastructure chipchiar taka zirchian theih a ni. TEM hian cell population-a gene transcription, protein expression emaw mitochondrial functional parameters chauh rinchhan ai chuan, mitochondria pakhat zel resolution-ah mitochondrial cristae morphology, shape leh structure chu direct-in a hmu thei a ni17,19, Chu bakah, TEM hian mitochondria leh organelle dang, endoplasmic reticulum leh autophagosomes te inkara inzawmna zir chianna a ti awlsam a, chungte chuan mitochondrial function leh homeostasis-ah hmun pawimawh tak an chang a ni21, Chutiang chuan hei hian TEM hi kawng bik emaw, gene emaw bik ngaihtuah hmain mitochondrial dysfunction zir chianna atana bul tanna tha tak a siam a ni. Mitochondrial function hi neuropathology nena inzawm a nih chhoh zel avangin in vitro neuronal model-a mitochondrial morphology leh dynamics direct leh quantitative-a zir chian a ngai tih a chiang hle.
He thuziak ah hian autism spectrum disorder-a mitochondrial dysfunction neuronal model-ah mitochondrial dynamics kan enfiah a. Tun hmain mitochondrial propionyl-CoA carboxylase enzyme PCC subunit ASD15-ah propionyl-CoA carboxylase beta (PCCB) differential methylation kan lo report tawh a. PCC dysregulation hian propionyl derivatives te toxic accumulation a thlen tih hriat a ni a, chung zingah chuan propionic acid (PPA)23,24,25 te pawh a tel. PPA hian neuronal metabolism a tibuai a, in vivo-ah nungchang a tidanglam tih hmuhchhuah a ni a, ASD-a inrawlh neurodevelopmental mechanism zir chianna atana animal model din tawh a ni Tin, PPA hian mitochondrial membrane potential, biogenesis leh respiration in vitro a tibuai tih an sawi a, neurons-a mitochondrial dysfunction model atan pawh hman a ni nasa hle bawk29, Mahse, PPA-induced mitochondrial dysfunction hian mitochondrial morphology leh dynamics a nghawng dan hi hriat chian a la ni lo.
He zirchianna hian complementary imaging technique hmangin SH-SY5Y cell-a mitochondrial morphology, dynamics leh function-a PPA-in nghawng a neih dan chu a chhut chhuak a ni. A hmasa berin mitochondrial morphology leh ultrastructure inthlak danglamna hmuh theih nan TEM method kan siam a17,31,32. Mitochondria33 dynamic nature ngaihtuah hian mitochondrial event localizer (MEL) analysis hmangin fission leh fusion events inkara balance, PPA stress hnuaia mitochondrial number leh volume inthlak danglamna chu kan quantified bawk. A tawp berah chuan mitochondrial morphology leh dynamics te hi biogenesis, fission leh fusion-a inrawlh genes expression inthlak danglamna nen a inzawm em tih kan enfiah a. Kan data zawng zawng hi kan han khaikhawm chuan mitochondrial dynamics regulating mechanisms complexity chiang taka hriat chian a harsatzia a tilang chiang hle. SH-SY5Y cell-a mitosis convergent endpoint teh theih anga mitochondrial morphology zir chianna atana TEM hman tangkai dan kan tarlang a ni. Tin, TEM data hian metabolic stress chhanna atana dynamic events pawh capture thei imaging technique nena khaikhin chuan information hautak ber a pe tih kan tarlang bawk. Neuronal cell mitosis thlawptu molecular regulatory mechanisms characterization dang neih belh chuan nervous system leh neurodegenerative diseases-a mitochondrial component chungchangah hriatna pawimawh tak a pe thei ang.
Mitochondrial stress thlen nan SH-SY5Y cell te chu 3 mM leh 5 mM sodium propionate (NaP) hmangin PPA hmangin an enkawl a. TEM hma hian sample te chu high-pressure freezing leh freezing hmangin cryogenic sample preparation an nei a (Fig. 1a). Biological replicate pathum atanga mitochondrial population morphological parameter pariat tehna tur automated mitochondrial image analysis pipeline kan siam a. PPA enkawlna hian parameter pali nasa takin a thlak danglam tih kan hmuchhuak a, chungte chu area 2, area, perimeter, leh Feret diameter te an ni (Fig. 1b–e). Area 2 chu 3 mM leh 5 mM PPA hmanga enkawlna pahnih hmangin nasa takin a tlahniam a (p = 0.0183 leh p = 0.002, a hnuaia mi ang hian) (Fig. 1b), area (p = 0.003), perimeter (p = 0.0106) leh Feret diameter te chu a tlahniam vek thung. 5 mM treatment group-ah hian control group nen khaikhin chuan a tlahniam nasa hle (p = 0.0172) (Fig. 1c–e). Area leh circumference tlahniam nasa tak chuan 5 mM PPA hmanga enkawl cell-te chu mitochondria te zawk, round zawk an nei tih hmuhchhuah a ni a, heng mitochondria te hi control cell-a mitochondria te aiin an sei lo zawk tih hmuhchhuah a ni. Hei hi Feret diameter tlahniam nasa tak nen pawh a inmil a, independent parameter hian particle edge inkar hlat zawng lian ber tlahniam a tilang a ni. Cristae-te ultrastructure-a inthlak danglamna hmuh a ni: PPA stress-in a nghawng avangin cristae-te chu a langsar lo hle (Fig. 1a, panel B). Mahse, thlalak zawng zawng hian cristae ultrastructure chiang takin a lantir vek lo va, chuvangin heng inthlak danglamna te hi quantitative analysis an nei lo. Heng TEM data te hian thil awm thei pathum a tarlang thei a ni: (1) PPA hian fission a tichak emaw, fusion a titawp emaw a, mitochondria awmsa te chu a lian lo hle a (2) enhanced biogenesis hian mitochondria thar, te zawk a siam emaw (3) mechanism pahnih hi a rualin a induce emaw a ni. Heng dinhmunte hi TEM hmanga thliar hran theih ni lo mah se, morphological changes lian tham tak chuan PPA stress hnuaia mitochondrial homeostasis leh dynamics inthlak danglamna a tilang a ni. Chumi hnuah chuan heng dynamics leh a hnuaia awm awm thei mechanism te hi characterize belh zel turin parameter dang kan dap leh a.
Propionic acid (PPA) hian mitochondrial morphology a siam thar leh thin. (a) Representative transmission electron microscopy (TEM) image-a PPA enkawlna tihpun a nih chuan mitochondrial size a tlahniam a, mitochondria chu a te leh a round zawk tih hmuhchhuah a ni 0 mM (enkawl loh), 3 mM leh 5 mM te a ni. Arrow sen hian mitochondria a tarlang a. (b–e) PPA hmanga 24 h enkawl SH-SY5Y cell te chu TEM atan buatsaih a ni a, Fiji/ImageJ hmangin result chu an zirchiang a. Parameter pariat zinga pali chuan control (untreated, 0 mM PPA) leh treated (3 mM leh 5 mM PPA) cells te inkarah danglamna nasa tak a nei a ni. (b) Region 2, (c) Area, (d) Perimeter, (e) Feret diameter. One-way analysis of variance (control vs. treatment) leh Dunnett’s multiple comparison test hmangin danglamna lian tham (p < 0.05) an hmuchhuak a ni. Data point hian cell pakhat zel mitochondrial value average a entir a, error bar hian mean ± SEM a entir bawk. Data tarlan te hian n = 3 a entir a, replicate khatah cell 24 tal a awm a; a vaiin thlalak 266 an zirchiang a; * hian p < 0.05 a tarlang a, ** hian p < 0.01 a tarlang bawk.
Mitochondrial dynamics-in PPA a chhan dan chiang zawka hriat theih nan mitochondria chu tetramethylrhodamine ethyl ester (TMRE) hmangin kan stain a, time-lapse microscopy leh MEL analysis hmangin darkar 24 hnuah 3 leh 5 mM PPA-ah mitochondria chu localize leh quantify turin kan hmang a ni. Fission leh fusion thil thleng enkawl dan. (Fig. 2a) a ni. MEL analysis hnuah mitochondria te chu mitochondrial structure awm zat leh an average volume tehna atan an zirchiang leh a. Fission [5.6 ± 0.3 (p < 0.05) )] leh fusion [5.4 ± 0.5 (p < 0.05)] leh fusion [5.4 ± 0.5 (p < 0.05)] leh fusion [5.4 ± 0.5 (p <.05)] nena khaikhin chuan 3 mM-a fission event thleng zat chu tlem mahse nasa takin a pung tih kan hmu a 0.05)] 0.05)] <0.05)] events chu control nen khaikhin chuan 5 mM-ah nasa takin a pung a (Fig. 3b). Mitochondria zat chu 3 [32.6 ± 2.1 (p < 0.05)] leh 5 mM [34.1 ± 2.2 (p < 0.05)]-ah te nasa takin a pung a (Fig. 3c) , mitochondria structure tinte average volume erawh a danglam lo thung (Fig. 3c). 3d). Heng zawng zawng hi han khaikhawm ila, mitochondrial dynamics remodeling hian compensatory response angin a thawk a, chu chuan mitochondrial network integrity chu hlawhtling takin a vawng reng tih a tilang a ni. 3 mM PPA-a fission events tam zel hian mitochondrial number tihpun chhan hi mitochondrial fission vang a ni tih a tilang a, mahse mitochondrial volume average chu a bul berah chuan a danglam lo tih ngaihtuah chuan biogenesis hi additional compensatory response anga sawi theih a ni lo. Mahse, heng data te hi TEM hmanga mitochondrial structure tenau zawk, round tak tak hmuhchhuah te nen a inmil a, PPA induced mitochondrial dynamics ah pawh danglamna nasa tak a awm tih a tilang bawk.
Propionic acid (PPA) hian network integrity vawng reng turin dynamic mitochondrial remodeling a tichhuak a. SH-SY5Y cells te chu culture a ni a, 3 leh 5 mM PPA hmangin darkar 24 chhung enkawl a ni a, TMRE leh Hoechst 33342 hmangin stain a ni a, chumi hnuah MEL analysis neih a ni. (a) Condition tin atan hun 2 (t2)-a color leh binarized maximum intensity projection tarlanna representative time-lapse microscopy images. Binary image tina region thlan bik tarlan te chu tihchangtlun a ni a, hun kal zelah dynamics entir nan time frame hrang hrang pathum (t1-t3)-ah 3D-in tarlan a ni a fusion thilthlengte chu green-in a langsar a; fission events te chu green in a highlight a ni. Red in a lang. (b) Condition khata dynamic event awm zat zatve. (c) Cell khata mitochondrial structure awm zat zatve. (d) Cell khata mitochondrial structure tinte volume (μm3) zatve. Data tarlan te hi treatment group khatah n = 15 cells aiawh a ni. Error bar tarlan te hian mean ± SEM, scale bar = 10 μm, * p < 0.05 a entir a ni.
Propionic acid (PPA) hian mitochondrial dynamics nena inzawm genes te transcriptional suppression a thlen thin. SH-SY5Y cells te chu 3 leh 5 mM PPA hmangin 24 h chhung an enkawl a. Relative gene quantification chu RT-qPCR hmangin an ti a, B2M-ah normalized an ni. Mitochondrial biogenesis genes (a) cMYC, (b) TFAM, (c) NRF1 leh (d) NFE2L2 te hi a ni. Mitochondrial fusion leh fission genes (e) STOML2, (f) OPA1, (g) MFN1, (h) MFN2 leh (i) DRP1 te hi a ni. One-way ANOVA (control vs. treatment) leh Dunnett’s multiple comparison test hmangin danglamna lian tham (p < 0.05) an test a: * hian p < 0.05 a tarlang a, ** hian p < 0.01 a tarlang a, **** hian p < 0.0001 a tarlang bawk. Bars hian mean expression ± SEM a entir a ni. Data tarlan te hian biological replicate n = 3 (STOML2, OPA1, TFAM), n = 4 (cMYC, NRF1, NFE2L2), leh n = 5 (MFN1, MFN2, DRP1) te a entir a ni.
TEM leh MEL analysis atanga data lakkhawm chuan PPA hian mitochondrial morphology leh dynamics a tidanglam tih a tarlang. Mahse, heng imaging technique te hian heng process te khalhtu underlying mechanisms te hi a pe lo. Chuvangin PPA enkawlna chhanna atana mitochondrial dynamics, biogenesis leh mitosis key regulator 9 mRNA expression kan enfiah a. Cell myeloma oncogene (cMYC), nuclear respiratory factor (NRF1), mitochondrial transcription factor 1 (TFAM), NFE2 ang chi transcription factor BZIP (NFE2L2), gastrin ang chi protein 2 (STOML2), optic nerve atrophy 1 (OPA1 ), Mitofusin 1 (MFN1), Mitofusin 2 (MFN2) leh dynamin-related protein 1 (DRP1) chu darkar 24 chhung 3 mM leh 5 mM PPA hmanga enkawl hnuah a ni. 3 mM (p = 0.0053, p = 0.0415 leh p < 0.0001, a hnuaia mi ang hian) leh 5 mM (p = 0.0031, p = 0.0233, p < 0.0001) PPA hmanga enkawlna kan hmu a. (Fig. 3a–c) ah hian a lang. mRNA expression tlahniam hi dose-ah a innghat a: cMYC, NRF1 leh TFAM expression chu 3 mM-ah vawi 5.7, 2.6 leh 1.9-in a tlahniam a, 5 mM-ah vawi 11.2, 3 leh 2.2-in a tlahniam bawk. Chumi danglamna chu, central redox biogenesis gene NFE2L2 chu PPA concentration eng pawhah a danglam lo a, mahse dose-dependent trend ang chiah, expression tlahniam chu hmuh a ni (Fig. 3d).
Fission leh fusion regulation-a inrawlh classical genes expression pawh kan enfiah bawk. STOML2 hi fusion, mitophagy leh biogenesis-ah a inrawlh nia ngaih a ni a, a expression chu 3 mM (2.4-fold change) leh 5 mM (2.8-fold change) PPA hmangin nasa takin a tlahniam (p < 0.0001) (Fig. 1). 3d). Chutiang bawkin OPA1 fusion gene expression pawh 3 mM (1.6-fold change) leh 5 mM (1.9-fold change) PPA (p = 0.006 leh p = 0.0024, a hnuaia mi ang hian)-ah a tlahniam bawk (Fig. 3f). Mahse, 24-h PPA stress hnuaia fusion genes MFN1, MFN2 emaw fission gene DRP1 emaw expression-ah danglamna lian tham kan hmu lo (Fig. 3g–i). Chu bakah, fusion leh fission protein pali (OPA1, MFN1, MFN2 leh DRP1) level chu dinhmun inangah a danglam lo tih kan hmu bawk (Fig. 4a–d). Heng data te hian hun bi khat chauh a lantir a, PPA stress hmasa berah protein expression emaw activity level inthlak danglamna a lantir lo thei tih hriat a pawimawh hle. Mahse, cMYC, NRF1, TFAM, STOML2, leh OPA1 expression tlahniam nasa tak chuan mitochondrial metabolism, biogenesis, leh dynamics-a transcriptional dysregulation nasa tak a awm tih a tilang. Chu bakah, heng data te hian mitochondrial function-a end-state changes direct-a zir chianna atana imaging technique hman tangkai dan a tarlang bawk.
Propionic acid (PPA) hmanga enkawl hnuah fusion leh fission factor protein level a danglam lo. SH-SY5Y cells te chu 3 leh 5 mM PPA hmangin 24 h chhung an enkawl a. Western blot analysis hmangin protein level chu quantified a ni a, expression level chu total protein-ah normalized a ni. Protein expression average leh target leh total protein representative Western blot te tarlan a ni. a – OPA1, b – MFN1, c – MFN2, d – DRP1 te an ni. Bars hian mean ± SEM a entir a, data tarlan te hi n = 3 biological replicate aiawhtu a ni. One-way analysis of variance leh Dunnett’s test hmangin multiple comparisons (p < 0.05) an nei a. Original gel leh blot chu Figure S1-ah hian kan hmu a.
Mitochondrial dysfunction hi multisystem natna, metabolic, cardiovascular leh muscular natna atanga thluak lam natna thlengin a inzawm a ni1,10. Neurodegenerative leh neurodegenerative natna tam tak hi mitochondrial dysfunction nen a inzawm a, hei hian thluak dam chhung zawnga heng organelles te hi an pawimawhzia a tilang chiang hle. Heng natnate hi Parkinson natna, Alzheimer natna leh ASD3,4,18 te an ni. Mahse, heng natnate zir chianna tur thluak tissue hmuh hi a harsa a, a bik takin mechanistic level-ah chuan cellular model systems chu a tul zawk a ni. He zirchiannaah hian PPA hmanga enkawl SH-SY5Y cells hmanga cellular model system hmangin neuronal diseases, a bik takin autism spectrum disorders-a mitochondrial dysfunction hmuhchhuah te chu recapitulate turin kan hmang a ni. He PPA model hmanga neurons-a mitochondrial dynamics zir chianna hian ASD etiology hriat theihna a pe thei ang.
Mitochondrial morphology inthlak danglamna thlir nan TEM hman theih dan tur kan zirchiang a. TEM hi a hlawhtlinna sang ber neih theih nan dik taka hman a ngai tih hriat a pawimawh. Cryo-specimens buatsaih hian cellular components te chu a ruala fixing leh artifacts siam chhuahna tihtlem hmangin neuronal structures te chu a humhalh tha zawk thei a ni Chumi nena inmil takin neuron ang chi SH-SY5Y cells te hian intact subcellular organelles leh elongated mitochondria an nei tih kan hmu a (Fig. 1a). Hei hian neuronal cell model-a mitochondrial morphology zir chianna atana cryogenic preparation technique hman tangkaizia a tilang chiang hle. TEM data objective analysis atan quantitative measurement hi a pawimawh hle nachungin, mitochondrial morphological changes nemnghet turin eng parameter bik nge teh tur tih chungchangah hian inremna a la awm lo. Mitochondrial morphology17,31,32 quantitative-a zirchianna tam tak atanga chhut chuan automated mitochondrial image analysis pipeline kan siam a, chu chuan morphological parameter pariat a teh a, chungte chu: area, area2, aspect ratio, perimeter, circularity, degree , Feret diameter te an ni. leh roundness te pawh a awm bawk.
Chung zingah chuan PPA hian area 2, area, perimeter, leh Feret diameter te nasa takin a tihtlem a ni (Fig. 1b–e). Hei hian mitochondria chu a te zawkin a round zawk tih a tarlang a, hei hi zirchianna hmasa lama PPA30-induced mitochondrial stress darkar 72 hnuah mitochondrial area a tlahniam tih hmuhchhuah tawh nen a inmil hle. Heng morphological features te hian mitochondrial fission a tilang thei a, hei hi mitophagy hmanga an tihchhiat theihna tura mitochondrial network atanga component chhia te sequester nana thil tul tak a ni35,36, A lehlamah chuan mitochondrial size average tlahniam hi biogenesis tihpun nen a inzawm thei a, chu chuan nascent mitochondria te tak te a siam chhuak thei a ni. Fission emaw biogenesis tihpun hian mitochondrial stress laka mitosis vawng reng turin compensatory response a entir a ni. Mahse, mitochondrial growth tlahniam, fusion tihchhiat emaw, thil dang emaw chu a huam lo thei lo.
TEM hmanga high-resolution image siam hian mitochondria pakhat zel level-ah morphological characteristics hriat theihna a siam thei a, mahse he method hian hun khatah two-dimensional snapshot a siam chhuak thei a ni. Metabolic stress-a dynamic response zir chian nan mitochondria chu TMRE hmangin kan stain a, MEL analysis hmangin time-lapse microscopy kan hmang a, hei hian hun kal zelah mitochondrial network-a inthlak danglamna high-throughput 3D visualization a siam thei a ni PPA stress hnuaiah mitochondrial dynamics-ah inthlak danglamna fiah lo mahse langsar tak kan hmu a (Fig. 2). 3 mM-ah chuan fission events a pung nasa hle a, fusion events erawh control-a awm ang thovin a awm thung. Fission leh fusion event pahnih a punna chu 5 mM PPA-ah hmuh a ni a, mahse heng inthlak danglamna te hi a proportional vel a ni a, hei hian fission leh fusion kinetics hian concentration sang zawkah equilibrium a thleng tih a tilang (Fig. 2b). 3 leh 5 mM PPA-ah te hian mitochondrial volume average chu a danglam lo va, hei hian mitochondrial network integrity chu a humhim tih a tilang (Fig. 2d). Hei hian dynamic mitochondrial network-te’n network fragmentation awm lovin homeostasis tha taka an vawn theih nan mild metabolic stress an chhan theihna a lantir a ni. 3 mM PPA-ah chuan fission tihpun hi equilibrium thara inthlakna tipung turin a tawk a, mahse PPA concentration sang zawk avanga stress awm chhanna atan kinetic remodeling thuk zawk a ngai a ni.
PPA stress concentration pahnihah hian mitochondria zat a pung a, mahse mitochondrial volume average erawh a danglam vak lo (Fig. 2c). Hei hi biogenesis tihpun vang emaw, inthen darh nasa vang emaw pawh a ni thei; mahse, mean mitochondrial volume tlahniam nasa tak a awm loh chuan biosynthesis a pung zawk a ni. Mahse, Figure 2-a data hian compensatory mechanism pahnih a awm tih a nemnghet a, chungte chu fission events pung, mitochondrial fission upregulation nena inmil, leh events pung, mitochondrial biogenesis nena inmil. A tawpah chuan, mild stress-a dynamic compensation chu fission, fusion, biogenesis, leh mitophagy te inzawmkhawmna a ruala thil tih a ni thei. Tun hmaa ziaktute chuan PPA hian mitosis30,39 leh mitophagy29 a tichak tih lo sawi tawh mah se, PPA chhanna atana mitochondrial fission leh fusion dynamics remodeling theihna tur evidence kan pe a ni. Heng data te hian TEM-in a hmuh chhuah morphological changes te chu a nemnghet a, PPA-induced mitochondrial dysfunction nena inzawm mechanisms te pawh a hriat belh zel bawk.
TEM emaw MEL emaw analysis pawhin morphological changes hmuhchhuah hnuaia gene regulatory mechanisms direct evidence a pek loh avangin mitochondrial metabolism, biogenesis, leh dynamics-a inrawlh genes RNA expression kan enfiah a. cMYC proto-oncogene hi mitochondria, glycolysis, amino acid leh fatty acid metabolism regulation-a thawktu transcription factor a ni40. Hei bakah hian cMYC hian mitochondrial transcription, translation, leh complex assembly-a inrawlh mitochondrial gene 600 vel expression a tidanglam tih hriat a ni a, chung zingah chuan NRF1 leh TFAM41 te pawh an tel. NRF1 leh TFAM te hi mitosis central regulator pahnih an ni a, PGC-1α hnuai lamah hna thawkin mtDNA replication an tichak a ni. He pathway hi cAMP leh AMPK signaling hmanga activate a ni a, energy expenditure leh metabolic stress ah a sensitive hle. Mitochondrial biogenesis redox regulator NFE2L2 pawh kan enfiah a, PPA-in nghawng a neih chu oxidative stress-in a mediated thei em tih kan enfiah bawk.
NFE2L2 expression chu danglam lo mah se, 3 mM leh 5 mM PPA hmanga 24 h enkawl hnuah cMYC, NRF1 leh TFAM expression-ah dose-dependent decrease consistent kan hmu a (Fig. 3a–c). cMYC expression downregulation hi mitochondrial stress42 chhanna a nih thu an lo sawi tawh a, a lehlamah chuan cMYC expression downregulation hian mitochondrial metabolism, network connectivity, leh membrane polarization te remodeling hmangin mitochondrial dysfunction a thlen thei a ni Ngaihven awm tak chu cMYC hian mitochondrial fission leh fusion regulation-ah pawh a inrawlh a42,43 a, cell division laiin DRP1 phosphorylation leh mitochondrial localization a tipung tih hriat a ni44 a, chubakah neuronal stem cells-a mitochondrial morphological remodeling mediate a ni bawk Dik tak chuan cMYC tlachham fibroblast te hian mitochondrial size tlahniam an lantir a, hei hi PPA43 stress avanga inthlak danglamna nen a inmil hle. Heng data te hian cMYC leh mitochondrial dynamics inzawmna ngaihnawm tak, mahse la chiang lo tak a lantir a, nakin lawka PPA stress-induced remodeling zirchianna atan target ngaihnawm tak a pe a ni.
NRF1 leh TFAM tihtlem hi cMYC hian transcriptional activator pawimawh tak a nihna nen a inmil hle. Heng data te hi mihring colon cancer cell-a zirchianna hmasa te nen pawh a inmil hle a, PPA hian darkar 22 chhungin NRF1 mRNA expression a tihhniam a, hei hi ATP tlakchhamna leh ROS46 tihpunna nen a inzawm a ni. Heng ziaktute hian TFAM expression hi darkar 8.5-ah a pung a, mahse darkar 22-ah chuan baseline level-ah a kir leh tih an sawi bawk. Chumi danglamna chu Kim et al. (2019) te chuan SH-SY5Y cell-a PPA stress 4 h hnuah TFAM mRNA expression chu nasa takin a tlahniam tih an hmuchhuak a; mahse, darkar 72 hnuah TFAM protein expression chu nasa takin a pung a, mtDNA copy number pawh nasa takin a pung bawk. Chutiang chuan darkar 24 hnua kan hmuh mitochondrial biogenesis genes tlahniam hian mitochondria tamna hi hun hmasa lama biogenesis activation nen a inzawm tih a tichhe lo. Tun hmaa zirchianna hrang hrangah PPA hian darkar 4 leh minute 30 chhungin SH-SY5Y cell-a PGC-1α mRNA leh protein chu nasa takin a upregulate tih hmuhchhuah a ni a, propionic acid hian calf hepatocytes-a mitochondrial biogenesis chu PGC-1α kaltlangin darkar 12 leh minute 39 chhungin a tichak thung. Ngaihven awm tak chu PGC-1α hi NRF1 leh TFAM direct transcriptional regulator chauh ni lovin, fission leh fusion regulate-in MFN2 leh DRP1 activity a regulate tih hmuhchhuah a ni bawk A pum puia kan han khaikhawm chuan, hei hian PPA-in a thlen mitochondrial compensatory responses regulating mechanisms close coupling a tarlang a ni. Chubakah, kan data hian PPA stress hnuaia biogenesis leh metabolism transcriptional regulation dysregulation nasa tak a lantir bawk.
STOML2, OPA1, MFN1, MFN2 leh DRP1 gene te hi mitochondrial fission, fusion leh dynamics central regulator zinga mi an ni37,48,49. Mitochondrial dynamics-a inrawlh gene dang tam tak a awm a, mahse, a hmain STOML2, OPA1 leh MFN2 te hi ASD cohort-ah hian differentially methylated an nih thu hmuhchhuah a ni tawh a,16 independent study engemaw zatah chuan mitochondrial stress chhanna atan heng transcription factors-ah hian inthlak danglamna a awm tih an sawi bawk50, 52. OPA1 leh STOML2 pahnih expression chu 3 mM leh 5 mM PPA treatment hmangin nasa takin a tlahniam a (Fig. 3e, f). OPA1 hi MFN1 leh 2 nena direct interaction hmanga mitochondrial fusion classical regulator zinga mi a ni a, cristae remodeling leh mitochondrial morphology-ah pawh a thawhhlawk hle Mitochondrial dynamics-a STOML2 chanvo dik tak hi hriat chian a la ni lo a, mahse evidence chuan mitochondrial fusion, biogenesis leh mitophagy-ah te chanvo a nei tih a tarlang.
STOML2 hian mitochondrial respiratory coupling leh respiratory chain complex siamnaah a inrawlh a54,55 a, cancer cells metabolic characteristics nasa takin a tidanglam tih hmuhchhuah a ni Zirna hrang hrangah STOML2 hian BAN leh cardiolipin nena inzawmna hmangin mitochondrial membrane potential leh biogenesis a tichak tih hmuhchhuah a ni Hriat tur pawimawh tak chu STOML2 hian MFN2 nen direct-in a inzawm a, a ti nghet tih an sawi a, OPA1 isoform sei tak takte stabilize-naah pawh OPA1 tihchhiatna atana mawhphurtu protease a titawp a, a ti nghet thei tih an sawi bawk53,61, PPA reaction-a STOML2 expression tlahniam hmuhchhuah hian heng fusion protein te hi ubiquitin- leh proteasome-dependent pathways hmanga tihchhiat awlsam zawkah a siam thei a ni PPA laka dynamic response-a STOML2 leh OPA1 te chanvo dik tak chu chiang lo mah se, heng fusion genes te expression tlahniam hian (Figure 3) fission leh fusion inkara balance a tibuai thei a, mitochondrial size tlahniam a thlen thei bawk (Figure 3). 1).
A lehlamah chuan OPA1 protein expression chu 24 h hnuah pawh a danglam lo va, PPA treatment hnuah MFN1, MFN2 emaw DRP1 mRNA leh protein level erawh a danglam vak lo thung (Fig. 3g-i, Fig. 4). Hei hian mitochondrial fusion leh fission-a inrawlh heng thilte regulation-ah hian danglamna a awm lo tih a tilang thei. Mahse, hriat tur pawimawh tak chu heng gene pali zinga pakhat zel hi protein activity control tu posttranscriptional modifications (PTMs) hmanga control a ni bawk. OPA1 hian splice variant dang pariat a nei a, chungte chu mitochondria-ah proteolytically cleaved in isoform hrang hrang pahnih siam a ni 63 . Isoform sei leh tawi inkara balance hian a tawpah chuan mitochondrial fusion leh mitochondrial network enkawlna kawnga OPA1 chanvo a tichiang a ni64. DRP1 activity hi calcium/calmodulin-dependent protein kinase II (CaMKII) phosphorylation hmanga tihregulate a ni a, DRP1 degradation hi ubiquitination leh SUMOylation hmanga control a ni thung A tawp berah chuan DRP1 leh MFN1/2 te hi GTPase an nih avangin mitochondria-a GTP siam chhuah rate hian activity a nghawng thei a ni 66 . Chuvangin, heng proteins te expression hi awm reng mahse, hei hian protein activity danglam lo emaw localization emaw a lantir lo thei67,68. Dik tak chuan, PTM protein repertoire awmsa te hi acute stress response mediating-a mawhphurtu line of defense hmasa ber atan an thawk fo thin. Kan model-a metabolic stress hniam tak a awm chuan PTM hian fusion leh fission proteins activity tihpun a tichak a, chu chuan mitochondrial integrity a siam that tawk a, mRNA emaw protein level-ah heng genes te hi activation dang a ngai lo.
A chunga data zawng zawng hi khaikhawm ila, mitochondrial morphology regulation complex leh time-dependent regulation leh heng mechanisms te chiang zawka hriat theihna tura harsatna awmte a tarlang a ni. Gene expression zir chian nan chuan pathway-a target gene bikte hriatchhuah hmasak a ngai a ni. Mahse, kan data atanga a lan dan chuan kawng khata awm gene te hian stress inang chiah chiah hi an chhang let ve lo. Dik tak chuan, zirchianna hmasaah chuan pathway khata gene hrang hrangte hian temporal response profile hrang hrang an lantir thei tih hmuhchhuah a ni30,46. Chu bakah, transcription leh gene function inzawmna tibuaitu post-transcriptional mechanism complex tak tak a awm bawk. Proteomic study hian PTMs leh protein function-in nghawng a neih dan tur hriatna a pe thei a, mahse harsatna a thlen bawk a, chung zingah chuan low-throughput method, signal-to-noise ratio sang, leh resolution tha lo te pawh a tel.
Hemi kawngah hian TEM leh MEL hmanga mitochondrial morphology zir chianna hian mitochondrial dynamics leh function inzawmna leh hei hian natna a nghawng dan chungchanga zawhna bulpui tak takte chhanna kawngah theihna nasa tak a nei a ni. A pawimawh ber chu TEM hian mitochondrial dysfunction leh dynamics convergent endpoint angin mitochondrial morphology tehna atana direct method a pe a ni MEL hian three-dimensional cellular environment-a fission leh fusion events hmuh theihna tur direct method a pe bawk a, gene expression-a inthlak danglamna awm lo pawhin dynamic mitochondrial remodeling quantification a phalsak bawk Hetah hian secondary mitochondrial natnaah mitochondrial imaging technique hman tangkai dan kan tarlang a ni. Heng natnate hi a tlangpuiin chronic mild metabolic stress a ni a, chu chu acute mitochondrial damage aiin mitochondrial networks subtle remodeling a ni. Mahse, chronic stress hnuaia mitosis vawng reng tura mitochondrial compensation mamawh hian functional consequence thuk tak a nei a ni. Neuroscience lam hawi chuan heng compensatory mechanisms te hi hriatthiamna tha zawk neih chuan mitochondrial dysfunction nena inzawm pleiotropic neuropathology chungchangah thu pawimawh tak a pe thei a ni.
A tawpah chuan kan data hian neuronal mitochondrial dynamics control tu gene expression, protein modification leh protein activity inkara complex interactions te functional consequences hriatthiamna atana imaging technique hman tangkaizia a tarlang a ni. ASD-a mitochondrial component hriatthiamna kan neih theih nan neuronal cell model-a mitochondrial dysfunction model nan PPA kan hmang a. PPA hmanga enkawl SH-SY5Y cell-te chuan mitochondrial morphology-ah inthlak danglamna an nei a: mitochondria chu a te leh a zungbun a lo ni ta a, TEM hmanga enfiah chuan cristae chu a chiang lo hle bawk. MEL analysis atanga a lan dan chuan heng inthlak danglamna te hi mild metabolic stress chhanna atana mitochondrial network vawng reng turin fission leh fusion events pung zel nen a inzawm tlat a ni. Chubakah, PPA hian mitochondrial metabolism leh homeostasis transcriptional regulation nasa takin a tibuai bawk. CMYC, NRF1, TFAM, STOML2, leh OPA1 te hi PPA stress avanga tihbuai mitochondrial regulator pawimawh tak an nih thu kan hmuchhuak a, PPA-in mitochondrial morphology leh function a inthlak danglamna mediating-ah pawh chanvo an nei thei a ni. PPA-induced temporal changes in gene expression leh protein activity, localization, leh post-translational modifications te chu a characterize tha zawk theih nan nakin lawka zirchianna neih a ngai a ni. Kan data hian mitochondrial stress response mediating regulatory mechanisms complexity leh interdependence a tarlang a, targeted mechanistic study tam zawk neihna atana TEM leh imaging technique dangte hman tangkai dan a tilang bawk.
SH-SY5Y cell line (ECACC, 94030304-1VL) hi Sigma-Aldrich atanga lei a ni. SH-SY5Y cells chu Dulbecco’s modified Eagle’s medium/F-12 nutrient mixture (DMEM/F-12) leh L-glutamine (SC09411, ScienCell)-ah 25 cm2 flask-ah 20% fetal bovine serum (FBS) (10493106, ThermoFisher Scientific) leh 1% penicillin-streptomycin (P4333-20ML, Sigma-Aldrich) chu 37 °C-ah dahin, 5% CO2 a ni. Cell te chu 0.05% trypsin-EDTA (15400054, ThermoFisher Scientific) hmangin 80% confluence thlengin subculture an ni a, 300 g-ah centrifuged an ni a, density 7 × 105 cells/ml velin plate an ni. Experiment zawng zawng hi undifferentiated SH-SY5Y cells-ah passage 19–22 inkarah an ti vek a ni. PPA hi NaP angin pek a ni. NaP powder (CAS No. 137-40-6, chemical formula C3H5NaO2, P5436-100G, Sigma-Aldrich) chu MilliQ tui lum takah 1 M concentration-ah hmin la, 4 °C-ah dah rawh. Treatment ni chuan he solution hi 1 M PPA to 3 mM leh 5 mM PPA hmangin serum-free medium (DMEM/F-12 with L-glutamine) ah dilute tur a ni. Experiment zawng zawnga enkawlna concentration chu PPA (0 mM, control), 3 mM, leh 5 mM PPA a ni. Experiment hi biological replicate pathum tal hmangin an ti a.
SH-SY5Y cells chu 25 cm5 flask-ah 5.5 × 105 cells/ml rate-in seeded niin darkar 24 chhung an tipung a. PPA treatment chu 24 h incubation hmain flask-ah dah a ni. Mammalian tissue subculture protocol pangngai (a chunga kan sawi tawh ang khan) zawm chungin cell pellets te chu khawlkhawm rawh. Cell pellet chu 100 μl 2.5% glutaraldehyde, 1× PBS-ah suspend leh la, processing hma chu 4 °C-ah dah rawh. SH-SY5Y cell te chu rei vak lo centrifuged hmangin cell te chu pellet a ni a, 2.5% glutaraldehyde, 1× PBS solution chu lakchhuah a ni. Distilled water-a siam 4% agarose gel-ah (agarose leh sediment volume ratio chu 1:1 a ni)-ah sediment chu resuspend leh rawh. Agarose piece te chu flat plate-a grid-ah dahin high-pressure freezing hmain 1-hexadecene hmanga coated a ni. Sample te chu 100% dry acetone ah -90°C ah darkar 24 chhung frozen a ni. Chumi hnuah -80°C-ah tihsan a ni a, 1% osmium tetroxide leh 0.1% glutaraldehyde solution chu dah a ni. Sample te chu -80°C ah darkar 24 chhung dah a ni. Hemi hnu hian ni engemaw zat chhung chu room temperature-ah zawi zawiin tihsan a ni a: – 80 °C atanga – 50 °C thleng darkar 24 chhung, – 30 °C thleng darkar 24 chhung, – 10 °C thleng darkar 24 chhung leh a tawpah chuan room temperature-ah tihsan a ni. lum leh vawt tehna.
Cryogenic preparation hnuah sample te chu resin hmanga impregnate a ni a, Leica Reichert UltracutS ultramicrotome (Leica Microsystems) hmangin ultrathin section (∼100 nm) siam a ni. Section hrang hrangte chu 2% uranyl acetate leh lead citrate hmangin an stain a. Sample te chu FEI Tecnai 20 transmission electron microscope (ThermoFisher (FEI hlui), Eindhoven, The Netherlands) 200 kV-a thawk (Lab6 transmitter) leh Gatan CCD camera (Gatan, UK) Tridiem energy filter hmanga thuam hmangin an enfiah a ni.
Technical replicate tinah hian single cell image 24 tal lak a ni a, a vaiin image 266 a ni. Thlalak zawng zawng hi Region of Interest (ROI) macro leh Mitochondria macro hmangin an zirchiang vek a ni. Mitochondrial macro hi published methods17,31,32 hmanga siam a ni a, Fiji/ImageJ69-a TEM images te semi-automated batch processing a phalsak a ni. A tawi zawngin: image chu rolling ball background subtraction (60 pixel radius) leh FFT bandpass filter (60 leh 8 pixel upper leh lower bounds hmangin) leh vertical line suppression hmangin invert leh inverted a ni a, orientation tolerance 5% a ni. Processed image chu maximum entropy algorithm hmangin automatic in threshold a ni a, binary mask a siam a ni. Raw TEM image-a manually selected ROIs nena inzawm image region te chu lakchhuah a ni a, mitochondria characterize a ni a, plasma membrane leh high-contrast region dangte chu paih chhuah a ni. Extracted ROI tin atan binary particles pixels 600 aia lian te chu an zirchiang a, Fiji/ImageJ-a built-in measurement functions hmangin particle area, perimeter, major leh minor axes, Feret diameter, roundness, leh circularity te chu teh a ni. Merrill, Flippo, leh Strack (2017) te hnung zuiin heng data atang hian area 2, particle aspect ratio (major to minor axis ratio), leh shape factor (FF) te chu chhut a ni a, FF = perimeter 2/4pi x area a ni. Parametric formula awmzia hi Merrill, Flippo, leh Strack (2017) te hian kan hmu thei a ni. Macro tarlan te hi GitHub ah a awm a (Data Availability Statement en rawh). A vaiin PPA treatment khatah particle 5,600 vel an zirchiang a, a vaiin particle 17,000 vel an zirchiang a ni (data tarlan loh).
SH-SH5Y cells chu 8-chamber culture dish (ThermoFisher, #155411)-ah zan khat chhung adhesion theih nan dah a ni a, chutah chuan TMRE 1:1000 (ThermoFisher, #T669) leh Hoechst 33342 1:200 (Sigma-Aldrich, H6024) hmangin incubate a ni. dyeing tih a ni. 405 nm leh 561 nm laser hmangin 10 min environment-ah images lak a ni a, raw images chu z-stack angin lak a ni a, chutah chuan image micrograph 10 a awm a, az step chu image frame inkarah 0.2 μm a ni a, a hnu lama time point 12-ah Hmanlai thlalak te hi Carl Zeiss LSM780 ELYRA PS.1 super-resolution platform (Carl Zeiss, Oberkochen, Germany) hmangin LCI Plan Apochromate 100x/1.4 Oil DIC M27 lens hmangin lakkhawm a ni. ImageJ-ah hian a hmaa kan sawi tawh pipeline leh ImageJ plugin hmangin fusion leh fission events, mitochondrial structures average number leh cell khata mitochondrial volume average te tehna atan imageJ-ah an zirchiang a ni MEL macros hi GitHub ah a awm a (Data Availability Statement en rawh).
SH-SY5Y cells chu well paruk awmna plate-ah density 0.3 × 106 cells/mL-ah darkar 24 chhung enkawl hmain an enkawl a. RNA chu Quick-RNATM Miniprep protocol (ZR R1055, Zymo Research) hmangin siam danglam tlem a ni a: lakchhuah hmain well tinah RNA lysis buffer 300 μl dah la, sample tin chu final step atan DNase/RNase elution 30 μl hmangin lyse a ni. -free tui. Sample zawng zawng hi NanoDrop ND-1000 UV-Vis Spectrophotometer hmangin quantity leh quality enfiah vek a ni. Cell lysates atanga protein zawng zawng chu 200 μl RIPA lysis buffer hmangin lakchhuah a ni a, Bradford protein assay hmangin protein concentration chu quantified a ni70.
cDNA siamna chu siamtute thupek angin TetroTM cDNA Synthesis Kit (BIO-65043, Meridian Bioscience) hmangin siamthatna engemaw zat nen an ti a. cDNA chu 20-μl reaction-ah RNA zawng zawng 0.7 atanga 1 μg hmangin siam a ni. Primer hi a hmaa paper tihchhuah tawh 42, 71, 72, 73, 74, 75, 76, 77, 78 (Table S1) atanga thlan niin, a kaihhnawih probes te chu Integrated DNA Technologies atanga PrimerQuest tool hmangin design a ni. Gene ngaihven zawng zawng chu nuclear B2M gene-ah normalized vek a ni. STOML2, NRF1, NFE2L2, TFAM, cMYC leh OPA1 te gene expression chu RT-qPCR hmangin an teh a. Master mix-ah hian LUNA Taq polymerase (M3003L, New England Biolabs), forward leh reverse primers 10 μM, cDNA, leh PCR-grade water te a tel a, reaction tin atan final volume 10 μL a pe chhuak a ni. Division leh fission genes (DRP1, MFN1/2) expression chu TaqMan multiplex assay hmangin an teh a. Luna Universal Probe qPCR Master Mix (M3004S, New England Biolabs) chu siamtu thupek angin hman a ni a, siamthatna tenau te a awm bawk. Multiplex RT-qPCR master mix-ah hian 1X LUNA Taq polymerase, 10 μM forward leh reverse primers, 10 μM probe, cDNA, leh PCR-grade water te a awm a, hei hian reaction tin atan final volume 20 μL a siam chhuak a ni. RT-qPCR hi Rotor-Gene Q 6-plex (QIAGEN RG—serial number: R0618110) hmangin an ti a. Cycling condition chu Table S1-ah hian tarlan a ni. cDNA sample zawng zawng chu triplicate-in amplified vek a ni a, standard curve chu a let sawma dilution series hmangin siam a ni. Data reproducibility enfiah nan cycle threshold standard deviation (Ct) >0.5 nei sample triplicate-a outlier te chu analysis atang hian lakchhuah a ni30,72. Relative gene expression chu 2-ΔΔCt79 method hmangin chhut a ni.
Protein sample (60 μg) chu Laemmli loading buffer nen 2:1 ratio-in an pawlh a, 12% colorless protein gel (Bio-Rad #1610184)-ah an kalpui a ni. Protein te chu Trans-Blot Turbo system (#170-4155, Bio-Rad) hmangin PVDF (polyvinylidene fluoride) membrane (#170-84156, Bio-Rad) ah an dah a. Membrane chu block a ni a, primary antibodies dik tak (OPA1, MFN1, MFN2, leh DRP1) (diluted 1:1000) nen darkar 48 chhung incubate a ni a, chumi hnuah secondary antibodies (1:10,000) nen darkar 1 chhung incubate a ni. Chumi hnuah Clarity Western ECL Substrate (#170-5061, Bio-Rad) hmangin membrane te chu imaged a ni a, Bio-Rad ChemiDoc MP system hmangin record a ni. Western blot analysis atan hian ImageLab version 6.1 hman a ni. Original gel leh blot chu Figure S1-ah hian kan hmu a. Antibody chungchang hi Table S2-ah hian tarlan a ni.
Data set te hi independent sample pathum tal mean leh standard error of the mean (SEM) anga tarlan a ni. Data set te chu Gaussian distribution leh standard deviation inang anga ngaih a, analysis te kalpui hmain Shapiro-Wilks test hmangin (a dang sawi loh chuan) normality test a ni. Fisher-a MEL LSD (p < 0.05), one-way ANOVA (treatment vs. control mean), leh Dunnett-a multiple comparison test hmanga data set thlirletna bakah hian significance hriat theihna (p < 0.05). Graph-ah hian p value pawimawh tak takte chu *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 angin tarlan a ni. Statistical analysis leh graph zawng zawng hi GraphPad Prism 9.4.0 hmanga tih leh siam a ni.
TEM image analysis atana Fiji/ImageJ macros te hi GitHub: https://github.com/caaja/TEMMitoMacro ah mipui hriat theih a ni. Mitochondrial Event Locator (MEL) macro hi GitHub-ah mipui hriat theih a ni a: https://github.com/rensutheart/MEL-Fiji-Plugin.
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