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Aside ya Propionic (PPA) ikoreshwa mu kwiga uruhare rw'imikorere mibi ya mitochondrial mu ndwara z'imitsi nk'indwara ya autism spectrum. PPA izwiho kubangamira imiterere ya mitochondrial, metabolism, no guhinduka kwayo. Ariko, ingaruka za PPA ku mikorere ya mitochondrial, fission na fusion ziracyari ikibazo bitewe n'imiterere y'igihe cy'iyi mikorere. Aha, dukoresha uburyo bwuzuzanya bwo gupima kugira ngo dusuzume uburyo PPA igira ingaruka ku miterere ya mitochondrial ultrastructure, morphology, na dynamics mu turemangingo twa SH-SY5Y dusa na neuron. PPA (5 mM) yateje igabanuka rikomeye ry'agace ka mitochondrial (p < 0.01), umurambararo wa Feret n'umuzenguruko (p < 0.05), n'agace ka 2 (p < 0.01). Isesengura rya Mitochondrial event locator ryagaragaje ubwiyongere bugaragara (p < 0.05) mu bikorwa byo gucikamo ibice no gucikamo ibice, bityo bigakomeza ubusugire bw'urusobe rwa mitochondrial mu bihe by'imihangayiko. Byongeye kandi, imiterere ya mRNA ya cMYC (p < 0.0001), NRF1 (p < 0.01), TFAM (p < 0.05), STOML2 (p < 0.0001) na OPA1 (p < 0.05) yagabanutse cyane. 01). Ibi bigaragaza ivugurura ry'imiterere ya mitochondrial, biogenesis na dynamics kugira ngo imikorere ikomeze mu bihe by'imihangayiko. Amakuru yacu atanga ubumenyi bushya ku ngaruka za PPA ku mihindagurikire ya mitochondrial kandi agaragaza akamaro k'ubuhanga bwo gufata amashusho mu kwiga uburyo bugoye bwo kugenzura bukoreshwa mu gusubiza imihangayiko ya mitochondrial.
Mitochondria ni ingenzi mu mikorere itandukanye y’uturemangingo uretse uruhare rwayo rusanzwe mu gukora ingufu no gukora biosynthesis. Metabolism ya Mitochondria ni ingenzi mu kugenzura ibimenyetso bya kalisiyumu, metabolic na redox homeostasis, inflammatory signaling, epigenetic modifications, growth of cells, differentiation and programmed sexuals death1. By’umwihariko, metabolism ya mitochondria ni ingenzi cyane mu iterambere ry’imitsi, kubaho no gukora neza, kandi igaragara cyane mu bimenyetso bitandukanye bya neuropathology2,3,4.
Mu myaka icumi ishize, imiterere y’imikorere y’umubiri yagaragaye nk’igikoresho cy’ingenzi mu kugenzura imitsi, ihinduka ry’uturemangingo, gukura no guhindagurika kwa pulasitiki5,6. Vuba aha, imiterere n’imikorere ya mitochondrial byabaye ibice by’ingenzi cyane bya mitochondrial, inzira ihinduka ikomeza ikigega cya mitochondrial nzima mu turemangingo. Imiterere ya mitochondrial igenzurwa n’inzira zigoye zishingiyeho kuva kuri mitochondrial biogenesis na bioenergetics kugeza kuri mitochondrial fission, fusion, transportation and clearance7,8. Guhagarika uburyo ubwo aribwo bwose bwo guhuza ibintu bibangamira kubungabunga imiyoboro mitochondrial myiza kandi bigira ingaruka zikomeye ku iterambere ry’ubwonko9,10. Mu by’ukuri, kudakora neza kwa mitochondrial dynamics kugaragara mu ndwara nyinshi zo mu mutwe, ubwonko bwangiritse ndetse n’iz’imitsi, harimo n’indwara za autism spectrum (ASD)11,12.
ASD ni indwara y’imitsi itandukanye ifite imiterere igoye ya genetiki na epigenetic. Inkomoko ya ASD ntabwo ishidikanywaho, ariko impamvu nyamukuru ya molekile ntirasobanuka neza. Gukusanya amakuru aturutse mu byiciro bya mbere y’ubuvuzi, ubushakashatsi bw’ubuvuzi, n’amakuru ya molekile ya multi-omics bitanga ibimenyetso byiyongera by’imikorere mibi ya mitochondrial muri ASD13,14. Mbere twakoze isuzuma rya methylation ya ADN mu itsinda ry’abarwayi ba ASD maze tubona gene za methylation zitandukanye zihuriweho mu nzira za mitochondrial metabolic15. Nyuma twatangaje methylation itandukanye y’abagenzura imiterere ya mitochondrial biogenesis na dynamics, byajyanaga no kwiyongera kwa mtDNA copy no guhinduka kwa metabolic porofil y’inkari muri ASD16. Amakuru yacu atanga ibimenyetso byiyongera byerekana ko imikorere ya mitochondrial na homeostasis bigira uruhare runini mu ndwara za ASD. Kubwibyo, kunoza imyumvire y’imikorere y’imitsi ku isano iri hagati ya mitochondrial dynamics, imiterere yayo, n’imikorere yayo ni intego y’ingenzi y’ubushakashatsi bukomeje ku ndwara z’imitsi zirangwa no kudakora neza kwa mitochondrial.
Ubuhanga bwa molekile bukunze gukoreshwa mu kwiga uruhare rwa za jeni runaka mu gusubiza ibibazo bya mitochondrial. Ariko, ubu buryo bushobora kugabanywa n’imiterere myinshi n’iy’igihe cy’uburyo bwo kugenzura mitochondrial. Byongeye kandi, itandukaniro ry’imiterere ya za jeni za mitochondrial ni ikimenyetso kitaziguye cy’impinduka mu mikorere, cyane cyane ko umubare muto wa za jeni ukunze gusesengurwa. Kubwibyo, hatanzwe uburyo butaziguye bwo kwiga imikorere ya mitochondrial na bioenergies17. Imiterere ya mitochondrial ifitanye isano rya hafi n’imikorere ya mitochondrial. Imiterere ya mitochondrial, isano, n’imiterere ni ingenzi mu gukora ingufu no kubaho kwa mitochondrial n’uturemangingo5,18. Byongeye kandi, ibice bitandukanye bya mitochondrial byibanda ku mpinduka mu miterere ya mitochondrial, bishobora kuba ingenzi ku iherezo ry’imikorere mibi ya mitochondrial kandi bigatanga ishingiro ry’ubushakashatsi bwa tekiniki bukurikira.
Imiterere ya mitochondrial ishobora kubonwa mu buryo butaziguye hakoreshejwe microscopy ya transmission electron (TEM), bigatuma habaho inyigo yimbitse ku miterere ya ultracell structure. TEM yerekana mu buryo butaziguye imiterere, imiterere n'imiterere ya mitochondrial cristae mu gihe mitochondria imwe imwe yabonetse, aho kwishingikiriza gusa ku ihindurwa rya gene, poroteyine cyangwa ibipimo by'imikorere ya mitochondrial mu turemangingo 17,19,20. Byongeye kandi, TEM yorohereza inyigo y'imikoranire hagati ya mitochondria n'izindi organelles, nka endoplasmic reticulum na autophagosomes, bigira uruhare runini mu mikorere ya mitochondrial na homeostasis21,22. Bityo, ibi bituma TEM iba ahantu heza ho gutangira kwiga imikorere mibi ya mitochondrial mbere yo kwibanda ku nzira cyangwa genes runaka. Uko imikorere ya mitochondrial igenda irushaho kuba ingenzi kuri neuropathology, hari ngombwa cyane kuba ushobora kwiga mu buryo butaziguye kandi bungana imiterere ya mitochondrial na dynamics muri models za neuronal za in vitro.
Muri iyi nkuru, turasuzuma imikorere ya mitochondrial mu buryo bw'imitsi y'ubwonko bw'imikorere mibi ya mitochondrial mu ndwara ya autism spectrum disorder. Twavuze mbere ko methylation ya propionyl-CoA carboxylase beta (PCCB) inyuranye muri ASD15, igice gito cya enzyme ya mitochondrial propionyl-CoA carboxylase PCC. Kugabanuka kwa PCC bizwiho gutera uburozi bw'ibikomoka kuri propionyl, harimo na aside propionic (PPA)23,24,25. PPA byagaragaye ko ibangamira imikorere y'imitsi y'ubwonko kandi igahindura imyitwarire mu mubiri kandi ni icyitegererezo cy'inyamaswa cyo kwiga uburyo bwo guteza imbere ubwonko bugira uruhare muri ASD26,27,28. Byongeye kandi, PPA byavuzwe ko ibangamira ubushobozi bwa mitochondrial membrane, biogenesis na respiracy in vitro kandi yakoreshejwe cyane mu kwerekana imikorere mibi ya mitochondrial mu mitsi y'ubwonko29,30. Ariko, ingaruka z'imikorere mibi ya mitochondrial iterwa na PPA ku miterere na dynamics za mitochondrial ntizisobanutse neza.
Ubu bushakashatsi bukoresha uburyo bwuzuzanya bwo gushushanya kugira ngo bupime ingaruka za PPA ku miterere ya mitochondrial, dynamics, n'imikorere mu turemangingo twa SH-SY5Y. Ubwa mbere, twakoze uburyo bwa TEM bwo kureba impinduka mu miterere ya mitochondrial na ultrastructure17,31,32. Bitewe n'imiterere ya mitochondrial33, twakoresheje kandi isesengura rya mitochondrial event localizer (MEL) kugira ngo dupime impinduka mu kuringaniza hagati y'ibikorwa byo gucikamo ibice no guhurirana, umubare wa mitochondrial n'ingano munsi ya stress ya PPA. Amaherezo, twasuzumye niba imiterere ya mitochondrial na dynamics bifitanye isano n'impinduka mu mikorere ya za gene zigize uruhare mu biogenesis, gucikamo ibice, no guhurirana. Dushyize hamwe, amakuru yacu agaragaza imbogamizi yo gusobanura uburyo bugoye bwo kugenzura dynamics ya mitochondrial. Turagaragaza akamaro ka TEM mu kwiga morphology ya mitochondrial nk'iherezo rishobora gupimwa rya mitosis mu turemangingo twa SH-SY5Y. Byongeye kandi, turagaragaza ko amakuru ya TEM atanga amakuru menshi cyane iyo ahujwe n'ubuhanga bwo gufotora bunafata ibintu bihinduka mu gusubiza stress ya metabolism. Kugaragaza uburyo bwo kugenzura molekile bushyigikira mitose y'uturemangingo tw'imitsi bishobora gutanga ubumenyi bw'ingenzi ku gice cya mitochondrial cy'imitsi n'indwara zo kwangirika kw'imitsi.
Kugira ngo habeho stress ya mitochondrial, uturemangingo twa SH-SY5Y twavuwe na PPA hakoreshejwe 3 mM na 5 mM sodium propionate (NaP). Mbere ya TEM, ingero zakoreweho isuzuma ry’icyitegererezo hakoreshejwe gukonjesha no gukonjesha (Ishusho ya 1a). Twakoze umuyoboro wo gusesengura amashusho ya mitochondrial wikora kugira ngo upime ibipimo umunani by’imiterere y’uturemangingo twa mitochondrial mu duce dutatu tw’ibinyabuzima. Twasanze uburyo bwo kuvura PPA bwahinduye cyane ibipimo bine: agace ka 2, agace, umuzenguruko, n’umurambararo wa Feret (Ishusho ya 1b–e). Agace ka 2 karagabanutse cyane hamwe n’uburyo bwo kuvura 3 mM na 5 mM PPA (p = 0.0183 na p = 0.002, uko bikurikirana) (Ishusho ya 1b), mu gihe agace (p = 0.003), umuzenguruko (p = 0.0106) n’umurambararo wa Feret byose byagabanutse cyane. Habayeho kugabanuka gukomeye (p = 0.0172) mu itsinda rya 5 mM rivurwa ugereranije n’itsinda rigenzura (Ishusho ya 1c–e). Kugabanuka gukomeye k'ubuso n'umuzenguruko byagaragaje ko uturemangingo twavuwe na PPA ya 5 mM twari dufite mitoyalike ntoya kandi izengurutse, kandi ko izi mitoyalike zari ndende cyane ugereranyije n'izo mu turemangingo ducunga. Ibi kandi bihuye no kugabanuka gukomeye k'umurambararo wa Feret, ikimenyetso cyigenga kigaragaza kugabanuka kw'intera nini hagati y'inkombe z'uduce. Impinduka mu miterere ya cristae zagaragaye: cristae ntiyagaragaye cyane bitewe n'ingaruka za stress ya PPA (Ishusho ya 1a, agace ka B). Ariko, si amashusho yose yagaragaje neza imiterere ya cristae, bityo isesengura ry'ibi bihinduka ntiryakozwe. Aya makuru ya TEM ashobora kugaragaza ibintu bitatu bishoboka: (1) PPA yongera ubwiyongere bw'icyuho cyangwa ikabuza ihuriro, bigatuma mitoyalike isanzwe igabanuka mu bunini; (2) biogenesis yongerewe irema mitoyalike nshya, ntoya cyangwa (3) itera uburyo bwombi icyarimwe. Nubwo ibi bintu bidashobora gutandukanywa na TEM, impinduka zikomeye mu miterere zigaragaza impinduka mu mitoyalike homeostasis na dynamics munsi ya stress ya PPA. Nyuma yaho twasuzumye ibindi bipimo kugira ngo turusheho kugaragaza izi dynamics n'uburyo bushoboka buzishingiyeho.
Aside ya Propionic (PPA) ivugurura imiterere ya mitochondrial. (a) Amashusho ya mikroskopiya ya elegitoroniki (TEM) agaragaza ko ingano ya mitochondrial igabanuka kandi mitochondrial irushaho kuba ntoya kandi ikagira uruziga rwinshi hamwe n’uburyo bwo kongera kuvurwa na PPA; 0 mM (itavuwe), 3 mM na 5 mM, uko bikurikirana. Utwambi dutukura tugaragaza mitochondrial. (b–e) Uturemangingo twa SH-SY5Y twavuwe na PPA mu gihe cy’amasaha 24 twateguwe kuri TEM kandi ibyavuye muri TEM byasesenguwe hakoreshejwe Fiji/ImageJ. Ibipimo bine mu munani byagaragaje itandukaniro rikomeye hagati y’uturemangingo two kugenzura (tutavuwe, 0 mM PPA) n’uturemangingo two kuvurwa (3 mM na 5 mM PPA). (b) Akarere ka 2, (c) Akarere, (d) Perimeter, (e) Feret diameter. Isesengura ry’uburyo bumwe bwo guhindagurika (gusuzuma ugereranije n’ubuvuzi) n’isuzuma rya Dunnett ryo kugereranya byinshi byakoreshejwe kugira ngo hamenyekane itandukaniro rikomeye (p < 0.05). Ingingo z’amakuru zigaragaza agaciro mpuzandengo ka mitochondrial kuri buri turemangingo, naho uduce tw’amakosa tugaragaza impuzandengo ya ± SEM. Amakuru yagaragajwe ahagarariye n = 3, nibura uturemangingo 24 kuri buri dusubizo; amashusho 266 yose yasesenguwe; * igaragaza p < 0.05, ** igaragaza p < 0.01.
Kugira ngo turusheho kugaragaza uburyo mitochondria ikora neza kuri PPA, twakoresheje tetramethylrhodamine ethyl ester (TMRE) muri mitochondria, dukoresha mikroskopi ya time-lapse na MEL analysis kugira ngo tumenye aho mitochondria iherereye kandi tuyipime nyuma y'amasaha 24 kuri 3 na 5 mM PPA. Uburyo bwo kuvura icikamo ibice no kuvanga. (Ishusho ya 2a). Nyuma yo gusesengura MEL, mitochondria yongeye gusesengura kugira ngo tumenye umubare w'imiterere ya mitochondria n'ingano yayo mpuzandengo. Twabonye ukwiyongera guto ariko gukomeye mu mubare w'ibintu byacitsemo ibice biba kuri 3 mM [4.9 ± 0.3 (p < 0.05)] ugereranije n'ibicikamo ibice [5.6 ± 0.3 (p < 0.05) )] no guhuza [5.4 ± 0.5 (p < 0.05)] no guhuza [5.4 ± 0.5 (p < 0.05)] 0.05)] <0.05)] ibintu byariyongereye cyane kuri 5 mM ugereranije n'uburyo bwo kugenzura (Ishusho ya 3b). Umubare wa mitochondria wiyongereye cyane kuri 3 [32.6 ± 2.1 (p < 0.05)] na 5 mM [34.1 ± 2.2 (p < 0.05)] (Ishusho ya 3c), mu gihe ingano mpuzandengo ya buri mitochondria itahindutse (Ishusho ya 3c). 3d). Ibi byose hamwe bigaragaza ko kuvugurura imikorere ya mitochondrial bitanga igisubizo cy’ingurane gikomeza neza ubusugire bw’imiyoboro ya mitochondrial. Ukwiyongera k’umubare w’ibikorwa byo gucika kuri 3 mM PPA bigaragaza ko ukwiyongera k’umubare wa mitochondrial biterwa no gucika kwa mitochondrial, ariko bitewe nuko ingano ya mitochondrial isanzwe idahinduka, biogenesis ntishobora gukurwaho nk’igisubizo cy’inyongera cy’ingurane. Ariko, aya makuru ahuye n’imiterere mito ya mitochondrial izengurutse yagaragaye na TEM kandi agaragaza impinduka zikomeye mu mihindagurikire ya mitochondrial iterwa na PPA.
Aside ya Propionic (PPA) ituma habaho kuvugurura mitochondrial kugira ngo ikomeze ubusugire bw'urusobe rw'amakuru. Uturemangingo twa SH-SY5Y twatewe, duvurwa na PPA ya 3 na 5 mM mu gihe cy'amasaha 24 kandi twasizwe irangi rya TMRE na Hoechst 33342 hagakurikiraho isesengura rya MEL. (a) Amashusho ya mikorosikope yerekana igihe agaragaza ibara n'ibipimo by'ubukana ntarengwa ku gihe cya 2 (t2) kuri buri kibazo. Uduce twatoranijwe twagaragajwe muri buri shusho y'ibice bibiri turanozwa kandi tugaragazwa muri 3D ku bihe bitatu bitandukanye (t1-t3) kugira ngo tugaragaze imiterere uko igihe kigenda gihita; ibikorwa byo guhuza bigaragazwa mu cyatsi kibisi; ibikorwa byo gucikamo ibice bigaragazwa mu cyatsi kibisi. Bigaragazwa mu ibara ritukura. (b) Umubare mpuzandengo w'ibikorwa bihinduka kuri buri kibazo. (c) Umubare mpuzandengo w'imiterere ya mitochondrial kuri buri ngirabuzimafatizo. (d) Ingano mpuzandengo (µm3) ya buri ngirabuzimafatizo kuri buri ngirabuzimafatizo. Amakuru yagaragajwe ahagarariye n = uturemangingo 15 kuri buri tsinda ry'ubuvuzi. Uduce tw'amakosa twagaragajwe tugaragaza ikigereranyo cya ± SEM, utwenda = 10 μm, * p < 0.05.
Aside ya Propionic (PPA) ituma habaho gufunga kwa transcriptional kwa genes zijyanye na mitochondrial dynamics. Uturemangingo twa SH-SY5Y twavuwe na 3 na 5 mM PPA mu gihe cy'amasaha 24. Gupima genes bifitanye isano byakozwe hakoreshejwe RT-qPCR kandi bihinduka B2M. Genes za Mitochondrial biogenesis (a) cMYC, (b) TFAM, (c) NRF1 na (d) NFE2L2. Genes za Mitochondrial fusion and fission (e) STOML2, (f) OPA1, (g) MFN1, (h) MFN2 na (i) DRP1. Itandukaniro rikomeye (p < 0.05) ryageragejwe hakoreshejwe one-way ANOVA (control vs. treatment) na multiple comparison test ya Dunnett: * igaragaza p < 0.05, ** igaragaza p < 0.01, na **** igaragaza p < 0.0001. Uduce tugaragaza mean expression ± SEM. Amakuru yagaragajwe agaragaza n = 3 (STOML2, OPA1, TFAM), n = 4 (cMYC, NRF1, NFE2L2), na n = 5 (MFN1, MFN2, DRP1) imiterere y’ibinyabuzima.
Amakuru ava mu isesengura rya TEM na MEL hamwe agaragaza ko PPA ihindura imiterere n'imikorere ya mitochondrial. Ariko, ubu buryo bwo gufata amashusho ntibutanga ibisobanuro ku buryo bw'ibanze buyobora iyi mikorere. Bityo twasuzumye uburyo mRNA igaragaramo ibintu icyenda by'ingenzi bigenzura imikorere ya mitochondrial, biogenesis, na mitosis mu gusubiza PPA. Twapimye cell 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) na dynamin-related protein 1 (DRP1) nyuma y'amasaha 24 yo kuvurwa hakoreshejwe 3 mM na 5 mM PPA. Twabonye 3 mM (p = 0.0053, p = 0.0415 na p < 0.0001, uko bikurikirana) na 5 mM (p = 0.0031, p = 0.0233, p < 0.0001) PPA treatment. (Ishusho ya 3a–c). Kugabanuka kw'igaragara rya mRNA kwaterwaga n'umuti: igaragara rya cMYC, NRF1 na TFAM ryagabanutseho inshuro 5.7, 2.6 na 1.9 kuri 3 mM, uko bikurikirana, naho ku nshuro 11.2, 3 na 2.2 kuri 5 mM. Mu buryo bunyuranye, gene ya redox biogenesis NFE2L2 ntabwo yahindutse ku gipimo icyo ari cyo cyose cya PPA, nubwo hagaragaye icyerekezo nk'icyo cyo kugabanuka kw'igaragara (Ishusho ya 3d).
Twasuzumye kandi uburyo gene za kera zigaragara mu kugenzura ubwiyongere n'ubwiyongere. STOML2 ifatwa nk'ifite uruhare mu guhuza, mitophagy na biogenesis, kandi uburyo igaragaramo bwagabanutse cyane (p < 0.0001) kuri 3 mM (impinduka ku nshuro 2.4) na 5 mM (impinduka ku nshuro 2.8) PPA (Ishusho ya 1). 3d). Mu buryo nk'ubwo, ubwiyongere bwa gene za OPA1 bwagabanutse kuri 3 mM (impinduka ku nshuro 1.6) na 5 mM (impinduka ku nshuro 1.9) PPA (p = 0.006 na p = 0.0024, uko bikurikirana) (Ishusho ya 3f). Ariko, nta tandukaniro rinini twabonye mu buryo gene za fusion MFN1, MFN2 cyangwa fission gene DRP1 zigaragara munsi y'umuvuduko wa PPA w'amasaha 24 (Ishusho ya 3g–i). Byongeye kandi, twasanze urwego rwa poroteyine enye zo guhuza no gucikamo ibice (OPA1, MFN1, MFN2 na DRP1) rutahindutse mu bihe bimwe (Ishusho ya 4a–d). Ni ngombwa kumenya ko aya makuru agaragaza igihe kimwe kandi ashobora kutagaragaza impinduka mu mikorere ya poroteyine cyangwa urwego rw'ibikorwa mu ntangiriro z'umuvuduko wa PPA. Ariko, kugabanuka gukomeye mu mikorere ya cMYC, NRF1, TFAM, STOML2, na OPA1 bigaragaza ko uburyo bwo guhindura imikorere ya mitochondrial, biogenesis, na dynamics ari bubi cyane. Byongeye kandi, aya makuru agaragaza akamaro k'ubuhanga bwo gufata amashusho mu kwiga neza impinduka za nyuma mu mikorere ya mitochondrial.
Igipimo cya poroteyine ya Fusion na Fission Factor ntabwo cyahindutse nyuma yo kuvurwa na aside propionic (PPA). Uturemangingo twa SH-SY5Y twavuwe na PPA ya 3 na 5 mM mu gihe cy'amasaha 24. Igipimo cya poroteyine cyapimwe hakoreshejwe isesengura rya Western blot, kandi igipimo cy'igaragazwa cyashyizwe kuri poroteyine yose. Impuzandengo ya poroteyine n'ibipimo by'impande zombi bya target na poroteyine yose biragaragazwa. a – OPA1, b – MFN1, c – MFN2, d – DRP1. Uduce tugaragaza impuzandengo ± SEM, kandi amakuru yagaragajwe agaragaza n = 3 biological replicates. Igereranya ryinshi (p < 0.05) ryakozwe hakoreshejwe isesengura ry'uburyo bumwe bw'impinduka hamwe n'ikizamini cya Dunnett. Gel y'umwimerere na blot bigaragara ku Ishusho S1.
Indwara za Mitochondria zifitanye isano n'indwara nyinshi kuva ku ndwara za metabolisme, iz'umutima n'imitsi kugeza ku ndwara z'imitsi1,10. Indwara nyinshi za neurodegenerative na neurodegenerative zifitanye isano n'ihindagurika rya mitochondrial, bigaragaza akamaro k'izi organelles mu gihe cyose cy'ubwonko. Izi ndwara zirimo indwara ya Parkinson, indwara ya Alzheimer na ASD3,4,18. Ariko, kubona ingirabuzima fatizo z'ubwonko kugira ngo zige izi ndwara biragoye, cyane cyane ku rwego rwa mechanistic, bigatuma sisitemu y'ingirabuzima fatizo iba ubundi buryo bukenewe. Muri ubu bushakashatsi, dukoresha sisitemu y'ingirabuzima fatizo ikoresha uturemangingo twa SH-SY5Y twavuwe na PPA kugira ngo twongere ibisobanuro ku ihindagurika rya mitochondrial rigaragara mu ndwara z'imitsi, cyane cyane indwara za autism spectrum. Gukoresha iyi moderi ya PPA kugira ngo twige imikorere ya mitochondrial mu mitsi bishobora gutanga ibisobanuro ku mpamvu ya ASD.
Twasesenguye uburyo bwo gukoresha TEM kugira ngo turebe impinduka mu miterere ya mitochondrial. Ni ngombwa kumenya ko TEM igomba gukoreshwa neza kugira ngo yongere imikorere yayo. Gutegura cryo-specimens bituma imiterere ya neuronal ibungabungwa neza binyuze mu gusana ibice by'ingirabuzimafatizo icyarimwe no kugabanya ikorwa ry'ibintu34. Dukurikije ibi, twabonye ko uturemangingo twa SH-SY5Y tumeze nk'uturemangingo tw'ingirabuzimafatizo twari dufite organelles zitari zisanzwe na mitochondrial ndende (Ishusho ya 1a). Ibi bigaragaza akamaro k'ubuhanga bwo gutegura cryogenic mu kwiga imiterere ya mitochondrial mu miterere y'uturemangingo tw'ingirabuzimafatizo. Nubwo ibipimo by'ingano ari ingenzi mu gusesengura amakuru ya TEM, nta cyumvikanyweho ku bipimo byihariye bigomba gupimwa kugira ngo byemeze impinduka mu miterere ya mitochondrial. Dushingiye ku bushakashatsi bwinshi bwakozwe mu mibare bwasuzumye imiterere ya mitochondrial17,31,32, twakoze umuyoboro wo gusesengura ishusho ya mitochondrial wikora upima ibipimo umunani by'imiterere, ari byo: agace, agace2, igipimo cy'imiterere, uruziga, dogere , umurambararo wa Feret. n'ubuzengurutse.
Muri byo, PPA yagabanyije cyane ubuso bwa 2, ubuso, uruziga, na Feret diameter (Ishusho ya 1b–e). Ibi byagaragaje ko mitochondria yabaye nto kandi ikagira uruziga rwinshi, ibi bikaba bihuye n’ubushakashatsi bwabanje bwerekanaga ko ubuso bwa mitochondria bwagabanutse nyuma y’amasaha 72 y’umuvuduko wa mitochondria watewe na PPA30. Ibi bintu bishobora kugaragaza ko mitochondria yacitse, inzira ikenewe yo gukura ibice byangiritse mu mitochondria kugira ngo birusheho kwangirika binyuze muri mitophagy35,36,37. Ku rundi ruhande, kugabanuka k’ingano mpuzandengo ya mitochondria bishobora gufitanye isano no kwiyongera kwa biogenesis, bigatuma mitochondria ntoya ikiri kuvuka. Ukwiyongera kwa fission cyangwa biogenesis bigaragaza igisubizo cyo gukomeza mitosis mu kurwanya imitochondria. Ariko, kugabanuka k’umuvuduko wa mitochondria, ihuriro ritameze neza, cyangwa izindi ndwara ntibishobora gukurwaho.
Nubwo amashusho afite ubushobozi bwo hejuru yakozwe na TEM yemerera kumenya imiterere y’imiterere y’umubiri ku rwego rwa mitochondria imwe, ubu buryo butanga amashusho y’ibice bibiri mu gihe kimwe. Kugira ngo twige ibisubizo bihindagurika ku muvuduko wa metabolike, twakoresheje mitochondria TMRE kandi dukoresha mikorosikope igenda ihinduka hifashishijwe MEL analysis, ituma habaho kureba impinduka mu buryo bwa 3D mu mitochondria mu gihe runaka33,38. Twabonye impinduka zigaragara ariko zikomeye mu mihindagurikire ya mitochondria munsi ya PPA stress (Ishusho ya 2). Kuri 3 mM, umubare w’ibikorwa byo gucikamo ibice wiyongereye cyane, mu gihe ibikorwa byo gucikamo ibice byakomeje kuba bimwe no mu kugenzura. Ubwiyongere bw’umubare w’ibikorwa byo gucikamo ibice no gucikamo ibice byagaragaye kuri 5 mM PPA, ariko izi mpinduka zari hafi ku rugero, bigaragaza ko fission na fusion kinetics bigera kuri equilibrium ku gipimo cyo hejuru (Ishusho ya 2b). Impuzandengo y’ingano ya mitochondria ntiyahindutse kuri 3 na 5 mM PPA, bigaragaza ko ubusugire bw’imitochondria bwari bukomeje (Ishusho ya 2d). Ibi bigaragaza ubushobozi bw'imiyoboro ya mitochondrial ikora mu gusubiza ku muvuduko muto wa metabolike kugira ngo ikomeze kugira homeostasis idatera gucikamo ibice. Kuri 3 mM PPA, kwiyongera kwa fission birahagije kugira ngo habeho impinduka mu buryo bushya, ariko hakenewe kuvugurura kinetic mu buryo bwimbitse mu gusubiza umuvuduko uterwa n'ubwinshi bwa PPA.
Umubare wa mitochondria wiyongereye ku gipimo cya PPA, ariko ingano ya mitochondria isanzwe ntabwo yahindutse cyane (Ishusho ya 2c). Ibi bishobora guterwa no kwiyongera kwa biogenesis cyangwa ukwiyongera kw'igabana; icyakora, mu gihe hatabayeho kugabanuka gukomeye k'ingano ya mitochondria, birashoboka cyane ko biosynthesis yiyongera. Ariko, amakuru ari ku Ishusho ya 2 ashyigikira ko hariho uburyo bubiri bwo kwishyura: kwiyongera k'umubare w'ibikorwa byo gucikamo ibice, bijyanye no kwiyongera kwa mitochondria, no kwiyongera k'umubare w'ibintu, bijyanye na biogenesis ya mitochondria. Amaherezo, indishyi ihindagurika ku muvuduko woroheje ishobora kuba igizwe n'ibikorwa icyarimwe birimo gucikamo ibice, guhuza, biogenesis, na mitophagy. Nubwo abanditsi ba mbere bagaragaje ko PPA yongera mitochondria30,39 na mitophagy29, dutanga ibimenyetso byo kuvugurura mitochondria fission na fusion dynamics mu gusubiza PPA. Aya makuru yemeza impinduka za morphological zagaragaye na TEM kandi atanga ibisobanuro birambuye ku buryo bujyanye n'imikorere mibi ya mitochondria iterwa na PPA.
Kubera ko isesengura rya TEM cyangwa MEL nta bimenyetso byatanze byerekana uburyo bwo kugenzura imiterere y’uturemangingo bushingiye ku mpinduka zagaragaye mu miterere y’uturemangingo, twasuzumye uburyo RNA igaragara mu mikorere ya mitochondrial, biogenesis, na dynamics. CMYC proto-oncogene ni transcription factor ikora mu kugenzura mitochondrial, glycolysis, aside amine na metabolism ya aside fatty40. Byongeye kandi, cMYC izwiho kugenga uburyo gene hafi 600 za mitochondrial zikora mu guhindura, guhindura, no guteranya ibintu mu buryo bugoye, harimo NRF1 na TFAM41. NRF1 na TFAM ni inkingi ebyiri z’ingenzi zigenzura mitosis, zikora nyuma ya PGC-1α kugira ngo zikore replication ya mtDNA. Iyi nzira ikorwa na cAMP na AMPK signaling kandi irashishikajwe n’ikoreshwa ry’ingufu n’ihungabana rya metabolism. Twasuzumye kandi NFE2L2, redox regulator ya mitochondrial biogenesis, kugira ngo tumenye niba ingaruka za PPA zishobora guterwa n’ihungabana rya oxidative.
Nubwo imiterere ya NFE2L2 itahindutse, twabonye ko imiterere ya cMYC, NRF1 na TFAM igabanuka bitewe n’umuti nyuma y’amasaha 24 yo kuvurwa hakoreshejwe 3 mM na 5 mM PPA (Ishusho ya 3a–c). Kugabanuka kw’imiterere ya cMYC byavuzwe mbere nk'igisubizo ku stress ya mitochondrial42, kandi ku rundi ruhande, kugabanuka kw’imiterere ya cMYC bishobora gutera imikorere mibi ya mitochondrial binyuze mu kuvugurura imikorere ya mitochondrial, guhuza urusobe rw’amakuru, no gutandukanya membrane43. Igishimishije ni uko cMYC inagira uruhare mu kugenga gucika no guhuza mitochondrial42,43 kandi izwiho kongera phosphorylation ya DRP1 n’aho mitochondrial iherereye mu gihe cyo kugabana uturemangingo44, ndetse no guhuza imiterere ya mitochondrial mu turemangingo tw’imitsi y’ubwonko45. Mu by’ukuri, fibroblast zidafite cMYC zigaragaza ingano ya mitochondrial yagabanutse, ijyanye n’impinduka ziterwa na stress ya PPA43. Aya makuru agaragaza isano ishimishije ariko itarasobanuka neza hagati ya cMYC na mitochondrial dynamics, itanga intego ishimishije y’ubushakashatsi bw’ejo hazaza ku kuvugurura stress ya PPA.
Kugabanuka kwa NRF1 na TFAM bihuye n'uruhare rwa cMYC nk'ikintu cy'ingenzi gitera imbaraga mu kwandika. Aya makuru kandi ahuye n'ubushakashatsi bwakozwe mbere ku turemangingo twa kanseri y'amara y'umuntu bwerekana ko PPA yagabanyije igaragara rya NRF1 mRNA mu masaha 22, ibyo bikaba byarajyanye no kugabanuka kwa ATP no kwiyongera kwa ROS46. Aba banditsi banavuze ko igaragara rya TFAM ryiyongereye mu masaha 8.5 ariko rigaruka ku rugero rw'ibanze mu masaha 22. Mu buryo bunyuranye, Kim et al. (2019) bagaragaje ko igaragara rya TFAM mRNA ryagabanutse cyane nyuma y'amasaha 4 y'umuvuduko wa PPA mu turemangingo twa SH-SY5Y; icyakora, nyuma y'amasaha 72, igaragara rya poroteyine ya TFAM ryiyongereye cyane kandi umubare wa kopi ya mtDNA wongewe cyane. Bityo, kugabanuka k'umubare wa genes za mitochondrial biogenesis twabonye nyuma y'amasaha 24 ntibikuraho ko kwiyongera k'umubare wa mitochondrial bifitanye isano no gukora kwa biogenesis mu bihe byabanje. Ubushakashatsi bwakozwe mbere bwagaragaje ko PPA yongera cyane PGC-1α mRNA na poroteyine mu turemangingo twa SH-SY5Y mu masaha 4 n'iminota 30, mu gihe aside propionic yongera biogenesis ya mitochondrial mu turemangingo tw'amaraso tw'inyana binyuze muri PGC-1α mu masaha 12 n'iminota 39. Igishimishije ni uko PGC-1α atari umugenzuzi w'inyandiko ya NRF1 na TFAM gusa, ahubwo byagaragaye ko igenzura imikorere ya MFN2 na DRP1 binyuze mu kugenzura icikamo ry'imitsi n'ihuza ryayo47. Ibi byose hamwe bigaragaza isano ya hafi y'uburyo bugenga ibisubizo bya mitochondrial byishyurwa na PPA. Byongeye kandi, amakuru yacu agaragaza ihindagurika rikomeye ry'amabwiriza ya transcription ya biogenesis na metabolism mu gihe cy'ihungabana rya PPA.
Jène za STOML2, OPA1, MFN1, MFN2 na DRP1 ziri mu zigenzura ry’ihinduka rya mitochondrial, fusion na dynamics37,48,49. Hariho izindi jeni nyinshi zigira uruhare mu mihindagurikire ya mitochondrial, icyakora, STOML2, OPA1 na MFN2 byagaragaye ko bifite methylation itandukanye mu matsinda ya ASD,16 kandi ubushakashatsi bwinshi bwigenga bwagaragaje impinduka muri izi nyuguti z’inyandiko mu gusubiza stress ya mitochondrial50,51. 52. Igaragara rya OPA1 na STOML2 ryagabanutse cyane ku buvuzi bwa PPA bwa 3 mM na 5 mM (Ishusho ya 3e, f). OPA1 ni imwe mu zigenzura imikorere ya mitochondrial binyuze mu mikoranire itaziguye na MFN1 na 2 kandi igira uruhare mu kuvugurura cristae no mu miterere ya mitochondrial53. Uruhare nyarwo rwa STOML2 mu mihindagurikire ya mitochondrial ntirurasobanuka neza, ariko ibimenyetso bigaragaza ko igira uruhare mu mihindagurikire ya mitochondrial, biogenesis, na mitophagy.
STOML2 igira uruhare mu kubungabunga uburyo bwo guhumeka bwa mitochondrial no gushyiraho uburyo bwo guhumeka54,55 kandi byagaragaye ko ihindura cyane imiterere y’uturemangingo twa kanseri56. Ubushakashatsi bwagaragaje ko STOML2 iteza imbere ubushobozi bwa mitochondrial membrane na biogenesis binyuze mu gukorana na BAN na cardiolipin 55, 57, 58. Byongeye kandi, ubushakashatsi bwigenga bwagaragaje ko imikoranire hagati ya STOML2 na PINK1 igenga mitophagy59,60. By’umwihariko, byatangajwe ko STOML2 ikorana neza na MFN2 kandi igatuza kandi ikagira uruhare runini mu gutuma isoforms ndende za OPA1 zihagarara binyuze mu gukumira protease itera kwangirika kwa OPA153,61,62. Kugabanuka kwa STOML2 kugaragara mu bikorwa bya PPA bishobora gutuma izi poroteyine zo mu bwoko bwa fusion zishobora kwangirika binyuze mu nzira zishingiye kuri ubiquitin na proteasome48. Nubwo uruhare nyarwo rwa STOML2 na OPA1 mu gusubiza ibintu kuri PPA rutarasobanuka neza, kugabanuka k’uburyo izi gene zihurirana (Ishusho ya 3) bishobora kubangamira uburinganire hagati yo gucikamo ibice no guhuzwa kw’uturemangingo ndetse bigatera kugabanuka k’ingano ya mitochondrial (Ishusho ya 3). 1).
Ku rundi ruhande, imiterere ya poroteyine ya OPA1 ntiyahindutse nyuma y'amasaha 24, mu gihe urugero rwa mRNA na poroteyine bya MFN1, MFN2 cyangwa DRP1 bitahindutse cyane nyuma yo kuvurwa na PPA (Ishusho ya 3g-i, Ishusho ya 4). Ibi bishobora kwerekana ko nta mpinduka zabayeho mu miyoborere y'ibi bintu bigira uruhare mu guhuza no gucika kwa mitochondrial. Ariko, ni ngombwa kumenya ko buri gene muri izi enye igenzurwa na modifications za posttranscriptional (PTMs) zigenzura imikorere ya poroteyine. OPA1 ifite ubwoko umunani bw'inyongera bugabanijwe muri mitochondria kugira ngo butange isoforms ebyiri zitandukanye 63. Uburinganire hagati ya isoforms ndende n'igufi bugena uruhare rwa OPA1 mu guhuza mitochondrial no kubungabunga network ya mitochondrial64. Umurimo wa DRP1 ugenzurwa na phosphorylation ya poroteyine kinase II (CaMKII) ishingiye kuri calcium/calmodulin, mu gihe ukwangirika kwa DRP1 kugenzurwa na ubiquitination na SUMOylation65. Amaherezo, DRP1 na MFN1/2 byombi ni GTPases, bityo ibikorwa bishobora guterwa n'umuvuduko w'umusaruro wa GTP muri mitochondria 66. Kubwibyo, nubwo iyi poroteyine ikomeza kubaho, ibi bishobora kutaba bigaragaza imikorere ya poroteyine idahinduka cyangwa aho iherereye67,68. Mu by'ukuri, poroteyine za PTM zisanzweho akenshi zikora nk'umurongo wa mbere w'ubwirinzi bushinzwe gusubiza ibibazo bikomeye. Mu gihe hari stress iri hagati ya metabolisme muri model yacu, birashoboka ko PTM itera imbaraga imikorere ya poroteyine zo guhuza no gucikamo ibice kugira ngo zigarure bihagije ubusugire bwa mitochondria hatabayeho kongera gukora kw'izi gene ku rwego rwa mRNA cyangwa poroteyine.
Iyo mibare yose hamwe igaragaza uburyo ibintu bihinduka kandi bigashingira ku gihe ku miterere ya mitochondrial ndetse n'imbogamizi zo gusobanura ubu buryo. Kugira ngo twige uburyo gene igaragara, ni ngombwa kubanza kumenya gene zihariye mu nzira. Ariko, amakuru yacu agaragaza ko gene ziri mu nzira imwe zidasubiza kimwe ku muvuduko umwe. Mu by'ukuri, ubushakashatsi bwabanje bwagaragaje ko gene zitandukanye mu nzira imwe zishobora kugaragaza uburyo butandukanye bwo gusubiza ibintu mu gihe30,46. Byongeye kandi, hari uburyo bugoye bwo guhindura ibintu nyuma yo kwandika bubangamira isano iri hagati yo kwandika no gukora gene. Ubushakashatsi bwa poroteyine bushobora gutanga ibisobanuro ku ngaruka za PTM na poroteyine, ariko nanone butera imbogamizi zirimo uburyo bwo gutanga amakuru make, ikigereranyo kinini cy'urusaku hagati y'ibimenyetso, no gukemura nabi.
Muri uru rwego, kwiga imiterere ya mitochondrial hakoreshejwe TEM na MEL bifite ubushobozi bukomeye bwo gusubiza ibibazo by'ibanze ku isano iri hagati y'imikorere ya mitochondrial n'imikorere yayo n'uburyo ibi bigira ingaruka ku ndwara. Icy'ingenzi kurushaho, TEM itanga uburyo butaziguye bwo gupima imiterere ya mitochondrial nk'iherezo ry'imikorere mibi ya mitochondrial n'imikorere51. MEL kandi itanga uburyo butaziguye bwo kwiyumvisha ibintu byacitsemo ibice no guhuzwa mu ngirabuzimafatizo mu buryo butatu, bigatuma hagaragazwa uburyo mitochondrial ihinduka nubwo nta mpinduka mu mikorere ya gene33 zibaho. Aha turagaragaza akamaro k'ubuhanga bwo gufata amashusho ya mitochondrial mu ndwara za mitochondrial. Izi ndwara ubusanzwe zirangwa n'umuvuduko udakira wa metabolike urangwa no kuvugurura urusobe rw'imitochondrial aho kwangirika gukabije kwa mitochondrial. Ariko, indishyi ya mitochondrial isabwa kugira ngo mitochondrial ikomeze kubaho mu gihe cy'imihangayiko idakira igira ingaruka zikomeye ku mikorere yayo. Mu rwego rw'ubuhanga mu by'imitsi, gusobanukirwa neza izi nzira zo kwishyura bishobora gutanga amakuru y'ingenzi ku bijyanye n'indwara ya pleiotropic neuropathology ijyanye n'imikorere mibi ya mitochondrial.
Amaherezo, amakuru yacu agaragaza akamaro k'ubuhanga bwo gufata amashusho mu gusobanukirwa ingaruka z'imikorere y'imikoranire ikomeye hagati y'ishyirwa mu bikorwa rya gene, guhindura poroteyine, n'imikorere ya poroteyine igenzura imikorere ya mitochondrial y'imitsi y'ubwonko. Twakoresheje PPA kugira ngo dukore isuzuma ry'imikorere mibi ya mitochondrial mu buryo bw'uturemangingo tw'imitsi y'ubwonko kugira ngo tumenye neza igice cya mitochondrial cya ASD. Uturemangingo twa SH-SY5Y twavuwe na PPA twagaragaje impinduka mu miterere ya mitochondrial: mitochondria yabaye nto kandi izengurutse, kandi cristae ntiyasobanuwe neza iyo yabonwaga na TEM. Isesengura rya MEL ryerekana ko izi mpinduka ziba rimwe n'ubwiyongere bw'ibikorwa byo gucika no guhuza kugira ngo imiyoboro ya mitochondrial ikomeze kugirana ingaruka ku muvuduko woroheje wa metabolic. Byongeye kandi, PPA ibangamira cyane uburyo bwo kwandika imikorere ya mitochondrial na homeostasis. Twasanze cMYC, NRF1, TFAM, STOML2, na OPA1 ari ingenzi mu kugenzura mitochondrial byahungabanyijwe n'umuvuduko wa PPA kandi bishobora kugira uruhare mu guhuza impinduka ziterwa na PPA mu miterere ya mitochondrial n'imikorere yayo. Ubushakashatsi bw'ejo hazaza burakenewe kugira ngo hamenyekane neza impinduka ziterwa na PPA mu mikorere ya gene n'imikorere ya poroteyine, aho iherereye, n'impinduka nyuma yo guhindura. Amakuru yacu agaragaza uburyo uburyo bwo kugenzura butuma habaho igisubizo cy’imihangayiko ya mitochondrial burushaho kuba bwiza, kandi agaragaza akamaro ka TEM n’ubundi buryo bwo gufata amashusho mu bushakashatsi bw’ikoranabuhanga bwibanda ku bintu byihariye.
Utunyangingo twa SH-SY5Y (ECACC, 94030304-1VL) twaguzwe muri Sigma-Aldrich. Utunyangingo twa SH-SY5Y twakuriye mu mvange y’intungamubiri ya Dulbecco's modified Eagle's medium/F-12 (DMEM/F-12) na L-glutamine (SC09411, ScienCell) mu dupfunyika twa cm2 25 twongewemo 20% by’amaraso y’inka yo mu nda (FBS) (10493106, ThermoFisher Scientific) na 1% ya penicillin-streptomycin (P4333-20ML, Sigma-Aldrich) kuri 37 °C, 5% CO2. Utunyangingo twashyizwe mu byiciro bya 80% hakoreshejwe 0.05% trypsin-EDTA (15400054, ThermoFisher Scientific), dushyirwa kuri centrifuge kuri 300 g hanyuma dushyirwa ku bucucike bwa selile zigera kuri 7 × 105/ml. Igerageza ryose ryakozwe ku tunyangingo twa SH-SY5Y tutarahinduka hagati y’inzira 19-22. PPA itangwa nka NaP. Ishongesha ifu ya NaP (CAS No. 137-40-6, formula ya chimique C3H5NaO2, P5436-100G, Sigma-Aldrich) mu mazi ashyushye ya MilliQ kugeza ku gipimo cya 1 M hanyuma ubibike kuri 4 °C. Ku munsi wo kuvurwa, shyiramo iyi mvange na 1 M PPA kuri 3 mM na 5 mM PPA mu buryo budafite serum (DMEM/F-12 hamwe na L-glutamine). Ingano y’ubuvuzi ku igerageza ryose ntabwo yari PPA (0 mM, igenzura), 3 mM, na 5 mM PPA. Igerageza ryakozwe nibura mu buryo butatu bw’ibinyabuzima.
Uturemangingo twa SH-SY5Y twashyizwe mu dupfunyika twa cm5 25 ku gipimo cya selile 5.5 × 105 / ml hanyuma dukura amasaha 24. Uburyo bwa PPA bwongewe mu icupa mbere y'amasaha 24 yo gukaraba. Kusanya utubuto tw'uturemangingo hakurikijwe uburyo busanzwe bwo korora inyama z'inyamabere (byasobanuwe haruguru). Ongera ushyiremo utubuto tw'uturemangingo muri µl 2.5% ya glutaraldehyde, 1 × PBS hanyuma ubibike kuri 4 °C kugeza igihe bitunganijwe. Uturemangingo twa SH-SY5Y twashyizwemo igihe gito kugira ngo dushyiremo utubuto hanyuma dukuremo 2.5% ya glutaraldehyde, 1 × PBS. Ongera ushyiremo utubuto muri gel ya agarose ya 4% yateguwe mu mazi yaciwe (igipimo cya agarose n'ingano y'utubuto ni 1:1). Uduce twa agarose twashyizwe ku dupfunyika ku masahani arambuye hanyuma dusigwa na 1-hexadecene mbere yo gukonjesha cyane. Ingero zashyizwe muri acetone yumye ya 100% kuri -90 °C mu gihe cy'amasaha 24. Ubushyuhe bwazamuwe bugera kuri -80°C, hashyirwaho umuti wa 1% osmium tetroxide na 0.1% glutaraldehyde. Ingero zabitswe kuri -80°C mu gihe cy'amasaha 24. Nyuma y'ibi, ubushyuhe bwagiye bwongerwa buhoro buhoro bugera ku bushyuhe bw'icyumba mu minsi myinshi: kuva kuri -80°C kugeza kuri -50°C mu gihe cy'amasaha 24, kugeza kuri -30°C mu gihe cy'amasaha 24, kugeza kuri -10°C mu gihe cy'amasaha 24, amaherezo bugera ku bushyuhe bw'icyumba.
Nyuma yo gutegura imashini zikora ubushyuhe, ingero zashyizwemo resin maze ibice bito cyane (~100 nm) bikorwa hakoreshejwe Leica Reichert UltracutS ultramicrotome (Leica Microsystems). Ibice byasizwe irangi rya 2% rya uranyl acetate na citrate y'icyitegererezo. Ingero zabonetse hakoreshejwe mikorosikopi ya FEI Tecnai 20 transmission electron (ThermoFisher (yahoze ari FEI), Eindhoven, mu Buholandi) ikora kuri 200 kV (Lab6 transmitter) na kamera ya Gatan CCD (Gatan, mu Bwongereza) ifite akayunguruzo k'ingufu ka Tridiem.
Muri buri kopi ya tekiniki, habonetse nibura amashusho 24 y’ingirabuzimafatizo imwe, yose hamwe akaba 266. Amashusho yose yasesenguwe hakoreshejwe macro y’Akarere k’Ibyiza (ROI) na macro ya Mitochondria. Macro ya mitochondria ishingiye ku buryo bwasohotse17,31,32 kandi yemerera gutunganya amashusho ya TEM muri Fiji/ImageJ69 mu buryo bwa semi-automated. Muri make: ishusho ihindurirwa inyuma kandi ihindurirwa inyuma hakoreshejwe gukuraho inyuma kw’umupira (radius ya pixel 60) na filter ya FFT bandpass (ikoresha imipaka yo hejuru n’iyo hasi ya pixel 60 na 8) hamwe no gufunga umurongo uhagaze hamwe n’uburyo bwo kwihanganira icyerekezo cya 5%. Ishusho yatunganyijwe yihuta hakoreshejwe algorithme ntarengwa ya entropy hanyuma hagakorwa mask ya binary. Uduce tw’amashusho dufitanye isano na ROI zatoranijwe n’intoki mu mashusho ya TEM yakozwe, hagaragazwa mitochondria kandi hakuwemo membrane ya plasma n’utundi duce dufite itandukaniro rikomeye. Kuri buri ROI yakuwemo, uduce duto twa binary turenze pikseli 600 twarasesenguwe, maze agace k'uduce, uruziga, imigozi minini n'imito, umurambararo wa Feret, uruziga, n'uruziga byarapimwe hakoreshejwe uburyo bwo gupima bwa Fiji/ImageJ. Nyuma ya Merrill, Flippo, na Strack (2017), agace ka 2, igipimo cy'uduce duto (igipimo cy'umurongo munini kugeza ku muto), na factor y'imiterere (FF) byarabazwe uhereye kuri aya makuru, aho FF = uruziga 2/4pi x agace. Ibisobanuro bya formula ya parametric biboneka muri Merrill, Flippo, na Strack (2017). Macros zavuzwe ziraboneka kuri GitHub (reba Itangazo ry'Uko Amakuru Aboneka). Ku mpuzandengo, uduce duto 5.600 twasesenguwe kuri buri vuriro rya PPA, ku bice bigera ku 17.000 (amakuru ntabwo yagaragajwe).
Uturemangingo twa SH-SH5Y twashyizwe mu byumba 8 byo gukurura (ThermoFisher, #155411) kugira ngo dufatanye mu ijoro ryose hanyuma dushyirwamo TMRE 1:1000 (ThermoFisher, #T669) na Hoechst 33342 1:200 (Sigma-Aldrich, H6024). Gusiga irangi. Amashusho yabonetse hakoreshejwe laser ya 405 nm na 561 nm mu gihe cy'iminota 10, kandi amashusho yabonetse nk'udukingirizo tw'amashusho 10 dufite mikorografi 10 z'amashusho hamwe n'intambwe ya az ya 0.2 μm hagati y'amashusho ku gihe 12 cyakurikiyeho. Amashusho yakusanyijwe hakoreshejwe urubuga rwa Carl Zeiss LSM780 ELYRA PS.1 super-resolution (Carl Zeiss, Oberkochen, Ubudage) hakoreshejwe lenzi ya LCI Plan Apochromate 100x/1.4 Oil DIC M27. Amashusho yasesenguwe muri ImageJ hakoreshejwe umuyoboro wavuzwe mbere hamwe na plugin ya ImageJ kugira ngo hapimwe ibikorwa byo guhuza no gucikamo ibice, impuzandengo y'umubare w'imiterere ya mitochondrial, n'impuzandengo y'ingano ya mitochondrial kuri buri selile33. Macros za MEL ziraboneka kuri GitHub (reba Raporo y'uko Amakuru Aboneka).
Uturemangingo twa SH-SY5Y twakuriwe mu masahani atandatu y’ibiziba ku bucucike bwa 0.3 × 106 selile/mL mu gihe cy’amasaha 24 mbere yo kuvurwa. RNA yakuwemo hakoreshejwe protocole ya Quick-RNA™ Miniprep (ZR R1055, Zymo Research) hamwe n’impinduka nto: ongeramo 300 μl ya RNA lysis buffer kuri buri riba mbere yo gukuraho hanyuma ushyiremo lyse buri rugero nk’intambwe ya nyuma ukoresheje 30 μl ya DNase/RNase elution. -amazi adakoresha. Ingero zose zagenzuwe ingano n’ubwiza hakoreshejwe NanoDrop ND-1000 UV-Vis Spectrophotometer. Poroteyine yose yavuye muri lysates z’uturemangingo yabonetse hakoreshejwe 200 μl ya RIPA lysis buffer, kandi ingano ya poroteyine yapimwe hakoreshejwe isuzuma rya poroteyine rya Bradford70.
Guhuza cDNA byakozwe hakoreshejwe Tetro™ cDNA Synthesis Kit (BIO-65043, Meridian Bioscience) hakurikijwe amabwiriza y'uruganda, hamwe n'impinduka zimwe na zimwe. cDNA yakozwe mu buryo bwa 20-μl hakoreshejwe 0.7 kugeza kuri 1 μg ya RNA yose. Primers zatoranyijwe mu nyandiko zasohotse mbere 42, 71, 72, 73, 74, 75, 76, 77, 78 (Imbonerahamwe S1) kandi imashini ziherekeje zakozwe hakoreshejwe igikoresho cya PrimerQuest cya Integrated DNA Technologies. Genes zose zishishikaje zahinduwe ku buryo busanzwe kuri gene ya nuclear B2M. Ishusho ya gene ya STOML2, NRF1, NFE2L2, TFAM, cMYC na OPA1 yapimwe hakoreshejwe RT-qPCR. Uruvange rw’ibanze rwarimo LUNA Taq polymerase (M3003L, New England Biolabs), primers 10 μM imbere na nyuma, cDNA, n’amazi yo mu rwego rwa PCR kugira ngo haboneke ingano ya nyuma ya 10 μL kuri buri reaction. Ishusho ya genes zo kugabana no gucika (DRP1, MFN1/2) yapimwe hakoreshejwe TaqMan multiplex assays. Luna Universal Probe qPCR Master Mix (M3004S, New England Biolabs) yakoreshejwe hakurikijwe amabwiriza y’uwakoze hamwe n’impinduka nto. Uruvange rw’ibanze rwa multiplex RT-qPCR rurimo 1X LUNA Taq polymerase, primers 10 μM imbere na nyuma, probe 10 μM, cDNA, n’amazi yo mu rwego rwa PCR, bituma ingano ya nyuma ya 20 μL kuri buri reaction. RT-qPCR yakozwe hakoreshejwe Rotor-Gene Q 6-plex (QIAGEN RG—inomero y’uruhererekane: R0618110). Imiterere y'igare igaragara muri Imbonerahamwe S1. Ingero zose za cDNA zongerewe mu byiciro bitatu kandi hakorwa umurongo usanzwe hakoreshejwe urukurikirane rw'inshuro icumi. Ingero za outliers mu byiciro bitatu bifite uruziga rwo kugabanya (Ct) >0.5 zakuwe mu isesengura kugira ngo harebwe ko amakuru ashobora kongera kuboneka30,72. Imvugo ya gene ijyanye n'uko ibintu bimeze yabazwe hakoreshejwe uburyo bwa 2-ΔΔCt79.
Ingero za poroteyine (60 μg) zavanzwe na Laemmli loading buffer ku gipimo cya 2: 1 kandi zikoreshwa kuri 12% bya poroteyine idafite ibara (Bio-Rad #1610184). Poroteyine zoherejwe kuri PVDF (polyvinylidene fluoride) membrane (#170-84156, Bio-Rad) hakoreshejwe sisitemu ya Trans-Blot Turbo (#170-4155, Bio-Rad). membrane yarafunzwe kandi ishyirwamo antibodies z'ibanze zikwiye (OPA1, MFN1, MFN2, na DRP1) (yavanzwemo 1:1000) mu gihe cy'amasaha 48, hakurikiraho incubation hamwe na antibodies za kabiri (1:10.000) mu gihe cy'isaha 1. Hanyuma membrane zafashwe amashusho hakoreshejwe Clarity Western ECL Substrate (#170-5061, Bio-Rad) hanyuma zifatwa hakoreshejwe sisitemu ya Bio-Rad ChemiDoc MP. ImageLab verisiyo ya 6.1 yakoreshejwe mu isesengura rya Western blot. Jeli y'umwimerere n'agahu kayo bigaragazwa ku Ishusho ya S1. Amakuru y'uturemangingo tw'umubiri atangwa mu mbonerahamwe ya S2.
Amakuru agaragazwa nk'ikosa ry'ikigereranyo n'iry'ikigereranyo (SEM) by'ingero eshatu byigenga nibura. Amakuru yapimwe kugira ngo harebwe ko ari ibintu bisanzwe hakoreshejwe ikizamini cya Shapiro-Wilks (keretse byavuzwe ukundi) mbere yo gufata ikwirakwizwa rya Gaussian n'itandukaniro ringana no gukomeza isesengura. Uretse gusesengura amakuru hakoreshejwe Fisher's MEL LSD (p < 0.05), ANOVA y'inzira imwe (ikigereranyo cyo kuvura ugereranyije n'icyitegererezo cyo kugenzura), n'ikizamini cya Dunnett cyo kugereranya inshuro nyinshi kugira ngo hamenyekane akamaro (p < 0.05). Agaciro k'ingenzi ka p kagaragara muri graph nka *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Isesengura ryose ry'ibarurishamibare n'imbonerahamwe byakozwe kandi byakozwe hakoreshejwe GraphPad Prism 9.4.0.
Macro ya Fiji/ImageJ yo gusesengura amashusho ya TEM aboneka ku mugaragaro kuri GitHub: https://github.com/caaja/TEMMitoMacro. Macro ya Mitochondrial Event Locator (MEL) iboneka ku mugaragaro kuri GitHub: https://github.com/rensutheart/MEL-Fiji-Plugin.
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