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Ana amfani da Propionic acid (PPA) don nazarin rawar da rashin aikin mitochondrial ke takawa a cikin cututtukan ci gaban jijiyoyi kamar cutar autism. An san PPA yana kawo cikas ga biogenesis na mitochondrial, metabolism, da juyawa. Duk da haka, tasirin PPA akan mitochondrial dynamics, fission da fusion har yanzu suna da matsala saboda yanayin lokaci mai rikitarwa na waɗannan hanyoyin. A nan, muna amfani da dabarun daukar hoto na adadi mai dacewa don bincika yadda PPA ke shafar tsarin mitochondrial, morphology, da dynamics a cikin ƙwayoyin SH-SY5Y masu kama da jijiyoyi. PPA (5 mM) ya haifar da raguwa mai yawa a yankin mitochondrial (p < 0.01), diamita da kewaye na Feret (p < 0.05), da yanki na 2 (p < 0.01). Binciken gano abubuwan da suka faru na Mitochondrial ya nuna ƙaruwa mai yawa (p < 0.05) a cikin abubuwan da suka faru na fission da fusion, don haka yana kiyaye amincin cibiyar sadarwar mitochondrial a ƙarƙashin yanayin damuwa. Bugu da ƙari, an rage yawan mRNA na cMYC (p < 0.0001), NRF1 (p < 0.01), TFAM (p < 0.05), STOML2 (p < 0.0001) da OPA1 (p < 0.05) sosai. 01). Wannan yana nuna sake fasalin yanayin mitochondrial, biogenesis da dynamics don ci gaba da aiki a ƙarƙashin yanayin damuwa. Bayananmu suna ba da sabon haske game da tasirin PPA akan mitochondrial dynamics kuma suna nuna amfanin dabarun hoto don nazarin hanyoyin kulawa masu rikitarwa da ke tattare da martanin damuwa na mitochondrial.
Mitochondria suna da hannu a cikin ayyuka daban-daban na ƙwayoyin halitta fiye da matsayinsu na yau da kullun a cikin samar da makamashi da kuma biosynthesis. Mitochondria metabolism muhimmin abu ne na daidaita siginar calcium, metabolism da redox homeostasis, siginar kumburi, gyare-gyaren epigenetic, yaduwar ƙwayoyin halitta, bambance-bambance da mutuwar ƙwayoyin halitta da aka tsara1. Musamman ma, mitochondrial metabolism yana da mahimmanci ga ci gaban jijiyoyi, rayuwa da aiki kuma yana da tasiri sosai a cikin bayyanar cututtuka daban-daban na neuropathology2,3,4.
A cikin shekaru goma da suka gabata, yanayin metabolism ya bayyana a matsayin babban mai kula da neurogenesis, bambance-bambance, balaga da kuma plasticity5,6. Kwanan nan, yanayin mitochondrial da dynamics sun zama muhimman abubuwan da ke haifar da mitosis, wani tsari mai ƙarfi wanda ke kula da tarin mitochondria mai lafiya a cikin ƙwayoyin halitta. Ana tsara yanayin mitochondrial ta hanyar hanyoyin da suka dogara da juna waɗanda suka kama daga biogenesis na mitochondrial da bioenergetics zuwa fission na mitochondrial, fusion, sufuri da clearance7,8. Rushewar kowane ɗayan waɗannan hanyoyin haɗin gwiwa yana lalata kula da cibiyoyin sadarwa na mitochondrial masu lafiya kuma yana da mummunan sakamako ga ci gaban neuro9,10. Hakika, ana lura da rashin daidaituwar yanayin mitochondrial a cikin cututtukan tabin hankali da yawa, neurodegenerative da neurodevelopment, gami da cututtukan autism spectrum (ASD)11,12.
ASD cuta ce mai bambancin ci gaban jijiyoyi tare da tsarin kwayoyin halitta da na epigenetic mai rikitarwa. Ba a jayayya game da gadon ASD ba, amma tushen ilimin kwayoyin halitta har yanzu ba a fahimce shi sosai ba. Tarin bayanai daga samfuran preclinical, nazarin asibiti, da kuma bayanan kwayoyin halitta masu yawa suna ba da ƙarin shaida na rashin aikin mitochondrial a cikin ASD13,14. A baya mun yi gwajin methylation na DNA mai faɗi a cikin tarin marasa lafiya da ASD kuma mun gano kwayoyin halittar methylated daban-daban waɗanda aka haɗa tare da hanyoyin metabolism na mitochondrial15. Daga baya mun bayar da rahoton bambancin methylation na masu kula da tsakiya na biogenesis da dynamics na mitochondrial, wanda ke da alaƙa da ƙaruwar lambar kwafin mtDNA da canjin bayanin metabolism na fitsari a cikin ASD16. Bayananmu suna ba da ƙarin shaida cewa dynamics na mitochondrial da homeostasis suna taka muhimmiyar rawa a cikin ilimin cututtukan ASD. Saboda haka, inganta fahimtar hanyoyin sadarwa game da alaƙar da ke tsakanin dynamics na mitochondrial, morphology, da aiki babban burin ci gaba da bincike kan cututtukan jijiyoyi waɗanda ke da alaƙa da rashin aikin mitochondrial na biyu.
Sau da yawa ana amfani da dabarun kwayoyin halitta don nazarin rawar da takamaiman kwayoyin halitta ke takawa a cikin martanin damuwa na mitochondrial. Duk da haka, wannan hanyar na iya iyakance ta hanyar yanayin fuskoki da yawa na hanyoyin sarrafa mitotic. Bugu da ƙari, bambancin bayyanar kwayoyin halittar mitochondrial alama ce ta canje-canjen aiki kai tsaye, musamman tunda galibi ana yin nazari kan adadin kwayoyin halitta kaɗan. Saboda haka, an gabatar da ƙarin hanyoyi kai tsaye don nazarin aikin mitochondrial da bioenergetics17. Tsarin halittar mitochondrial yana da alaƙa da yanayin mitochondrial. Siffar mitochondrial, haɗi, da tsari suna da mahimmanci don samar da makamashi da rayuwar mitochondrial da ƙwayoyin halitta5,18. Bugu da ƙari, sassa daban-daban na mitosis suna mai da hankali kan canje-canje a cikin yanayin halittar mitochondrial, wanda zai iya zama maƙasudin amfani na rashin aikin mitochondrial kuma ya samar da tushe don nazarin injiniya na gaba.
Ana iya lura da yanayin halittar mitochondrial kai tsaye ta amfani da na'urar daukar hoto ta hanyar watsawa (TEM), wanda ke ba da damar yin cikakken bincike kan yanayin halittar tantanin halitta. TEM tana hango yanayin halittar tantanin halitta, siffa da tsarin tantanin halitta a lokacin da aka gano mitochondria na mutum ɗaya, maimakon dogaro kawai da kwafi na kwayar halitta, bayyanar furotin ko sigogin aiki na mitochondrial a cikin yawan tantanin halitta17,19,20. Bugu da ƙari, TEM yana sauƙaƙa nazarin hulɗa tsakanin mitochondria da sauran organelles, kamar endoplasmic reticulum da autophagosomes, waɗanda ke taka muhimmiyar rawa a cikin aikin mitochondrial da homeostasis21,22. Don haka, wannan ya sa TEM ya zama kyakkyawan wurin farawa don nazarin rashin aikin mitochondrial kafin mai da hankali kan takamaiman hanyoyi ko kwayoyin halitta. Yayin da aikin mitochondrial ya zama mai mahimmanci ga cututtukan jijiyoyi, akwai buƙatar a sami damar yin nazarin yanayin mitochondrial da yanayin aiki kai tsaye da adadi a cikin samfuran neuronal na in vitro.
A cikin wannan labarin, mun bincika yanayin mitochondrial a cikin samfurin neuronal na rashin aikin mitochondrial a cikin matsalar autism. A baya mun bayar da rahoton bambancin methylation na propionyl-CoA carboxylase beta (PCCB) a cikin ASD15, wani yanki na mitochondrial propionyl-CoA carboxylase enzyme PCC. An san cewa rashin aiki na PCC yana haifar da tarin guba na abubuwan da aka samo daga propionyl, gami da propionic acid (PPA)23,24,25. An nuna cewa PPA yana wargaza metabolism na neuronal da canza hali a cikin vivo kuma samfurin dabba ne da aka kafa don nazarin hanyoyin ci gaban neuro da ke da hannu a cikin ASD26,27,28. Bugu da ƙari, an ruwaito cewa PPA yana wargaza yuwuwar membrane na mitochondrial, biogenesis da numfashi a cikin vitro kuma an yi amfani da shi sosai don yin kwaikwayon rashin aikin mitochondrial a cikin neurons29,30. Duk da haka, tasirin rashin aikin mitochondrial da PPA ke haifarwa akan yanayin mitochondrial da dynamics har yanzu ba a fahimce shi sosai ba.
Wannan binciken yana amfani da dabarun daukar hoto masu dacewa don tantance tasirin PPA akan yanayin mitochondrial, kuzari, da aiki a cikin ƙwayoyin SH-SY5Y. Da farko, mun ƙirƙiri hanyar TEM don hango canje-canje a cikin yanayin mitochondrial da ultrastructure17,31,32. Ganin yanayin motsi na mitochondria33, mun kuma yi amfani da nazarin localizer na taron mitochondrial (MEL) don auna canje-canje a cikin daidaito tsakanin abubuwan da suka faru na fission da fusion, adadin mitochondrial da girma a ƙarƙashin damuwa na PPA. A ƙarshe, mun bincika ko yanayin mitochondrial da kuzari suna da alaƙa da canje-canje a cikin bayyanar kwayoyin halitta da ke da hannu a cikin biogenesis, fission, da fusion. Idan aka haɗa su, bayananmu suna nuna ƙalubalen bayyana sarkakiyar hanyoyin da ke daidaita yanayin mitochondrial. Mun haskaka amfanin TEM wajen nazarin yanayin mitochondrial a matsayin ma'aunin haɗuwa na mitosis a cikin ƙwayoyin SH-SY5Y. Bugu da ƙari, mun haskaka cewa bayanan TEM suna ba da mafi kyawun bayanai lokacin da aka haɗa su da dabarun daukar hoto waɗanda kuma ke kama abubuwan da suka faru masu ƙarfi don mayar da martani ga damuwa na rayuwa. Ƙarin bayanin hanyoyin sarrafa kwayoyin halitta waɗanda ke tallafawa mitosis na ƙwayoyin jijiyoyi na iya ba da muhimmiyar fahimta game da ɓangaren mitochondrial na tsarin juyayi da cututtukan neurodegenerative.
Don haifar da damuwa a cikin mitochondria, an yi wa ƙwayoyin SH-SY5Y magani da PPA ta amfani da sodium propionate 3 mM da 5 mM (NaP). Kafin TEM, an yi wa samfuran shirye-shiryen samfurin cryogenic ta amfani da daskarewa da daskarewa mai ƙarfi (Hoto na 1a). Mun ƙirƙiri bututun nazarin hoton mitochondrial ta atomatik don auna sigogi takwas na yanayin halittar yawan mitochondrial a cikin kwafi uku na halittu. Mun gano cewa maganin PPA ya canza sigogi huɗu sosai: yanki na 2, yanki, kewaye, da diamita na Feret (Hoto na 1b–e). Yankin na 2 ya ragu sosai tare da maganin PPA 3 mM da 5 mM (p = 0.0183 da p = 0.002, bi da bi) (Hoto na 1b), yayin da yanki (p = 0.003), kewaye (p = 0.0106) da diamita na Feret duk sun ragu sosai. An sami raguwa mai yawa (p = 0.0172) a cikin rukunin jiyya na 5 mM idan aka kwatanta da rukunin kulawa (Hoto na 1c–e). Ragewar da aka samu a yanki da kewaye ya nuna cewa ƙwayoyin da aka yi wa magani da 5 mM PPA suna da ƙananan mitochondria masu zagaye, kuma waɗannan mitochondria ba su da tsayi fiye da waɗanda ke cikin ƙwayoyin sarrafawa. Wannan kuma ya yi daidai da raguwa mai mahimmanci a cikin diamita na Feret, siga mai zaman kanta da ke nuna raguwar mafi girman nisa tsakanin gefuna na barbashi. An lura da canje-canje a cikin tsarin ultrastructure na cristae: cristae ya zama ƙasa da bayyana a ƙarƙashin tasirin damuwa na PPA (Hoto na 1a, panel B). Duk da haka, ba duk hotuna ba ne suka nuna tsarin ultrastructure na cristae a sarari, don haka ba a gudanar da nazarin adadi na waɗannan canje-canje ba. Waɗannan bayanan TEM na iya nuna yanayi uku masu yiwuwa: (1) PPA yana haɓaka fission ko hana haɗuwa, yana sa mitochondria da ke akwai ya ragu a girma; (2) haɓaka biogenesis yana ƙirƙirar sabbin, ƙananan mitochondria ko (3) yana haifar da duka hanyoyin biyu a lokaci guda. Kodayake ba za a iya bambance waɗannan yanayi ta hanyar TEM ba, manyan canje-canje na morphological suna nuna canje-canje a cikin homeostasis na mitochondria da dynamics a ƙarƙashin damuwa na PPA. Daga baya mun bincika ƙarin sigogi don ƙara fayyace waɗannan dynamics da hanyoyin da za su iya haifar da su.
Propionic acid (PPA) yana sake fasalin yanayin mitochondrial. (a) Hotunan da aka nuna ta hanyar amfani da na'urar daukar hoto ta lantarki (TEM) suna nuna cewa girman mitochondrial yana raguwa kuma mitochondria ya zama ƙarami da zagaye tare da ƙara yawan maganin PPA; 0 mM (ba a yi wa magani ba), 3 mM da 5 mM, bi da bi. Kibiyoyi ja suna nuna mitochondria. (b–e) An shirya ƙwayoyin SH-SY5Y da aka yi wa magani da PPA na tsawon awanni 24 don TEM kuma an yi nazarin sakamakon ta amfani da Fiji/ImageJ. Huɗu daga cikin sigogi takwas sun nuna manyan bambance-bambance tsakanin ƙwayoyin sarrafawa (ba a yi wa magani ba, 0 mM PPA) da ƙwayoyin PPA da aka yi wa magani (3 mM da 5 mM PPA). (b) Yanki na 2, (c) Yanki, (d) Gefen, (e) Diamita na Feret. An yi amfani da nazarin bambanci na hanya ɗaya (ikon sarrafawa da magani) da gwajin kwatantawa da yawa na Dunnett don tantance manyan bambance-bambance (p < 0.05). Ma'aunin bayanai suna wakiltar matsakaicin ƙimar mitochondrial ga kowane ƙwayar halitta, kuma sandunan kuskure suna wakiltar matsakaicin ± SEM. Bayanan da aka nuna suna wakiltar n = 3, aƙalla ƙwayoyin halitta 24 a kowace kwafi; An yi nazarin jimillar hotuna 266; * yana nuna p < 0.05, ** yana nuna p < 0.01.
Don ƙarin bayani game da yadda tasirin mitochondrial ke amsawa ga PPA, mun yi wa mitochondria fenti da tetramethylrhodamine ethyl ester (TMRE) kuma mun yi amfani da na'urar hangen nesa ta lokaci-lokaci da kuma nazarin MEL don gano da kuma auna mitochondria bayan awanni 24 a 3 da 5 mM PPA. Maganin fashewar abubuwa da haɗuwa. (Hoto na 2a). Bayan nazarin MEL, an ƙara yin nazarin mitochondria don ƙididdige adadin tsarin mitochondrial da matsakaicin girman su. Mun lura da ƙaramin ƙaruwa mai mahimmanci a cikin adadin fashewar abubuwa da ke faruwa a 3 mM [4.9 ± 0.3 (p < 0.05)] idan aka kwatanta da fashewar abubuwa [5.6 ± 0.3 (p < 0.05) )] da haɗuwa [5.4 ± 0.5 (p < 0.05)] da haɗuwa [5.4 ± 0.5 (p < 0.05)] 0.05)] abubuwan da suka faru <0.05)] sun ƙaru sosai a 5 mM idan aka kwatanta da sarrafawa (Hoto na 3b). Adadin mitochondria ya ƙaru sosai a duka 3 [32.6 ± 2.1 (p < 0.05)] da 5 mM [34.1 ± 2.2 (p < 0.05)] (Hoto na 3c), yayin da matsakaicin girman kowane tsarin mitochondrial ya kasance ba a canza shi ba (Hoto na 3c). 3d). Idan aka haɗa, wannan yana nuna cewa sake fasalin yanayin mitochondrial yana aiki azaman martanin diyya wanda ya sami nasarar kiyaye amincin hanyar sadarwar mitochondrial. Ƙara yawan abubuwan da suka faru na fission a 3 mM PPA yana nuna cewa ƙaruwar adadin mitochondrial wani ɓangare ne saboda fission na mitochondrial, amma idan aka yi la'akari da cewa matsakaicin girman mitochondrial ya kasance ba a canza shi ba, ba za a iya kawar da biogenesis a matsayin ƙarin martanin diyya ba. Duk da haka, waɗannan bayanan sun yi daidai da ƙananan tsarin mitochondrial zagaye da TEM ta lura kuma suna nuna manyan canje-canje a cikin yanayin mitochondrial wanda PPA ya haifar.
Propionic acid (PPA) yana haifar da sake fasalin mitochondrial mai ƙarfi don kiyaye amincin hanyar sadarwa. An haɓaka ƙwayoyin SH-SY5Y, an yi musu magani da 3 da 5 mM PPA na tsawon awanni 24 kuma an yi musu fenti da TMRE da Hoechst 33342 sannan aka yi musu nazarin MEL. (a) Hotunan microscopy masu wakiltar lokaci-lokaci waɗanda ke nuna launi da hasashen ƙarfin binary a lokaci na 2 (t2) ga kowane yanayi. An inganta yankunan da aka zaɓa a cikin kowane hoto na binary kuma an nuna su a cikin 3D a cikin firam ɗin lokaci uku daban-daban (t1-t3) don nuna yanayin aiki akan lokaci; abubuwan da suka haɗu an haskaka su da kore; abubuwan da suka faru na fission an haskaka su da kore. An nuna su da ja. (b) Matsakaicin adadin abubuwan da suka faru na ƙarfi a kowane yanayi. (c) Matsakaicin adadin tsarin mitochondrial a kowace tantanin halitta. (d) Matsakaicin girma (µm3) na kowane tsarin mitochondrial a kowace tantanin halitta. Bayanan da aka nuna suna wakiltar n = ƙwayoyin halitta 15 a kowace ƙungiyar magani. Sandunan kuskure da aka nuna suna wakiltar matsakaicin ± SEM, sandar sikelin = 10 μm, * p < 0.05.
Propionic acid (PPA) yana haifar da danne kwayoyin halitta da ke da alaƙa da yanayin mitochondrial. An yi wa ƙwayoyin SH-SY5Y magani da 3 da 5 mM PPA na tsawon awanni 24. An yi amfani da RT-qPCR wajen ƙididdige kwayoyin halitta kuma an daidaita su zuwa B2M. Kwayoyin halittar halittar mitochondrial (a) cMYC, (b) TFAM, (c) NRF1 da (d) NFE2L2. Kwayoyin halittar haɗakar mitochondrial da fission (e) STOML2, (f) OPA1, (g) MFN1, (h) MFN2 da (i) DRP1. An gwada bambance-bambance masu mahimmanci (p < 0.05) ta amfani da ANOVA ta hanya ɗaya (ikon sarrafawa vs. magani) da gwajin kwatantawa da yawa na Dunnett: * yana nuna p < 0.05, ** yana nuna p < 0.01, kuma **** yana nuna p < 0.0001. Sanduna suna wakiltar matsakaicin magana ± SEM. Bayanan da aka nuna suna wakiltar n = 3 (STOML2, OPA1, TFAM), n = 4 (cMYC, NRF1, NFE2L2), da n = 5 (MFN1, MFN2, DRP1) kwafi na halitta.
Bayanai daga nazarin TEM da MEL tare sun nuna cewa PPA yana canza yanayin mitochondrial da yanayin aiki. Duk da haka, waɗannan dabarun daukar hoto ba su ba da haske game da hanyoyin da ke haifar da waɗannan hanyoyin ba. Saboda haka, mun bincika bayyanar mRNA na manyan masu kula da yanayin aiki na mitochondrial guda tara, biogenesis, da mitosis a martanin maganin PPA. Mun ƙididdige myeloma oncogene ta cell (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) da dynamin-related protein 1 (DRP1) bayan awanni 24 na magani tare da 3 mM da 5 mM PPA. Mun lura da 3 mM (p = 0.0053, p = 0.0415 da p < 0.0001, bi da bi) da kuma 5 mM (p = 0.0031, p = 0.0233, p < 0.0001) maganin PPA. (Hoto na 3a–c). Raguwar bayyanar mRNA ta dogara ne akan kashi: bayyanar cMYC, NRF1 da TFAM ta ragu da sau 5.7, 2.6 da 1.9 a 3 mM, bi da bi, da kuma sau 11.2, 3 da 2.2 a 5 mM. Sabanin haka, kwayar halittar redox biogenesis ta tsakiya NFE2L2 ba ta canza ba a kowane yawan PPA, kodayake an lura da irin wannan yanayin raguwar bayyanar da ya dogara da kashi (Hoto na 3d).
Mun kuma bincika bayyanar kwayoyin halittar gargajiya da ke da hannu a cikin daidaita fission da fusion. Ana tsammanin STOML2 yana da hannu a cikin fusion, mitophagy da biogenesis, kuma bayyanarsa ta ragu sosai (p < 0.0001) ta hanyar 3 mM (canjin ninki 2.4) da 5 mM (canjin ninki 2.8) PPA (Hoto na 1). 3d). Hakazalika, bayyanar halittar fusion OPA1 ta ragu a 3 mM (canjin ninki 1.6) da 5 mM (canjin ninki 1.9) PPA (p = 0.006 da p = 0.0024, bi da bi) (Hoto na 3f). Duk da haka, ba mu sami bambance-bambance masu mahimmanci a cikin bayyanar kwayoyin halittar fusion MFN1, MFN2 ko fission DRP1 a ƙarƙashin damuwa na PPA na awanni 24 ba (Hoto na 3g–i). Bugu da ƙari, mun gano cewa matakan furotin guda huɗu na haɗuwa da fission (OPA1, MFN1, MFN2 da DRP1) ba su canza ba a ƙarƙashin yanayi ɗaya (Hoto na 4a–d). Yana da mahimmanci a lura cewa waɗannan bayanan suna nuna lokaci ɗaya a cikin lokaci kuma ƙila ba za su nuna canje-canje a cikin bayyanar furotin ko matakan aiki ba a lokacin farkon damuwa na PPA. Duk da haka, raguwa mai mahimmanci a cikin bayyanar cMYC, NRF1, TFAM, STOML2, da OPA1 suna nuna babban raguwar rubutun bayanai na metabolism na mitochondrial, biogenesis, da dynamics. Bugu da ƙari, waɗannan bayanan suna nuna amfanin dabarun hoto don nazarin canje-canje kai tsaye a cikin aikin mitochondrial.
Matakan furotin da suka haɗu da juna da kuma fission factor ba su canza ba bayan maganin propionic acid (PPA). An yi wa ƙwayoyin SH-SY5Y magani da 3 da 5 mM PPA na tsawon awanni 24. An auna matakan furotin ta hanyar nazarin Western blot, kuma an daidaita matakan bayyanar zuwa jimlar furotin. An nuna matsakaicin bayyanar furotin da kuma wakilcin Western blots na manufa da jimlar furotin. a – OPA1, b – MFN1, c – MFN2, d – DRP1. Sanduna suna wakiltar matsakaicin ± SEM, kuma bayanan da aka nuna suna wakiltar n = kwafi 3 na halittu. An yi kwatancen da yawa (p < 0.05) ta amfani da nazarin bambanci na hanya ɗaya da gwajin Dunnett. An nuna asalin gel da blot a cikin Hoto na S1.
Rashin aikin mitochondrial yana da alaƙa da cututtukan tsarin da yawa, tun daga cututtukan metabolism, cututtukan zuciya da jijiyoyin jini zuwa cututtukan jijiyoyi1,10. Yawancin cututtukan neurodegenerative da neurodegenerative suna da alaƙa da rashin aikin mitochondrial, wanda ke nuna mahimmancin waɗannan ƙwayoyin halitta a tsawon rayuwar kwakwalwa. Waɗannan cututtukan sun haɗa da cutar Parkinson, cutar Alzheimer da ASD3,4,18. Duk da haka, samun damar shiga kyallen kwakwalwa don nazarin waɗannan cututtuka yana da wahala, musamman a matakin injiniya, yana mai da tsarin ƙirar salula madadin da ake buƙata. A cikin wannan binciken, muna amfani da tsarin ƙirar salula ta amfani da ƙwayoyin SH-SY5Y da aka yi wa magani da PPA don sake dawo da rashin aikin mitochondrial da aka lura a cikin cututtukan jijiyoyi, musamman cututtukan autism. Amfani da wannan samfurin PPA don nazarin yanayin mitochondrial a cikin jijiyoyi na iya samar da haske game da asalin ASD.
Mun bincika yiwuwar amfani da TEM don ganin canje-canje a cikin yanayin mitochondrial. Yana da mahimmanci a lura cewa dole ne a yi amfani da TEM daidai don haɓaka ingancinsa. Shirya samfuran cryo yana ba da damar kiyaye tsarin jijiyoyi ta hanyar daidaita abubuwan da ke cikin tantanin halitta a lokaci guda da rage samuwar kayan tarihi34. Dangane da wannan, mun lura cewa ƙwayoyin SH-SY5Y masu kama da jijiyoyi suna da ƙwayoyin halitta marasa lalacewa da kuma dogon mitochondria (Hoto na 1a). Wannan yana nuna amfanin dabarun shirye-shiryen cryogenic don nazarin yanayin mitochondrial a cikin samfuran ƙwayoyin jijiyoyi. Kodayake ma'aunin adadi yana da mahimmanci don nazarin bayanai na TEM, har yanzu babu wata yarjejeniya kan takamaiman sigogi da ya kamata a auna don tabbatar da canje-canjen yanayin mitochondrial. Dangane da adadi mai yawa na bincike waɗanda suka bincika yanayin mitochondrial a adadi17,31,32, mun haɓaka bututun nazarin hoto na mitochondrial mai sarrafa kansa wanda ke auna sigogi takwas na yanayin halitta, wato: yanki, yanki2, rabon al'amari, kewaye, zagaye, digiri, diamita na Feret. da zagaye.
Daga cikinsu, PPA ta rage yanki na 2, yanki, kewaye, da diamita na Feret sosai (Hoto na 1b–e). Wannan ya nuna cewa mitochondria ta zama ƙarami kuma ta yi zagaye, wanda ya yi daidai da binciken da aka yi a baya wanda ya nuna raguwar yankin mitochondrial bayan awanni 72 na damuwa ta mitochondrial da PPA30 ta haifar. Waɗannan fasalulluka na iya nuna fission na mitochondrial, wani tsari da ake buƙata don nemo abubuwan da suka lalace daga hanyar sadarwar mitochondrial don haɓaka lalacewarsu ta hanyar mitophagy35,36,37. A gefe guda kuma, raguwar matsakaicin girman mitochondrial na iya dangantawa da ƙaruwar biogenesis, wanda ke haifar da samuwar ƙaramin mitochondria na asali. Ƙara fission ko biogenesis yana wakiltar martanin diyya don kiyaye mitosis akan damuwa ta mitochondrial. Duk da haka, raguwar girma na mitochondrial, rashin daidaituwar haɗuwa, ko wasu yanayi ba za a iya cire su ba.
Duk da cewa hotunan da aka ƙirƙira masu ƙuduri masu girma da TEM ke bayarwa suna ba da damar tantance halayen siffofi a matakin mitochondria na mutum ɗaya, wannan hanyar tana samar da hotuna masu girma biyu a lokaci guda. Don nazarin martanin da ke da ƙarfi ga damuwa ta rayuwa, mun shafa wa mitochondria fenti da TMRE kuma mun yi amfani da na'urar hangen nesa ta lokaci tare da nazarin MEL, wanda ke ba da damar hangen nesa na 3D mai girma na canje-canje a cikin hanyar sadarwar mitochondrial akan lokaci33,38. Mun lura da ƙananan canje-canje masu mahimmanci a cikin yanayin mitochondrial ƙarƙashin damuwa na PPA (Hoto na 2). A 3 mM, adadin abubuwan da suka faru na fission sun ƙaru sosai, yayin da abubuwan da suka faru na fission suka kasance iri ɗaya kamar yadda suke a cikin sarrafawa. An lura da ƙaruwa a cikin adadin abubuwan da suka faru na fission da fusion a 5 mM PPA, amma waɗannan canje-canjen sun kasance daidai gwargwado, suna nuna cewa fission da fusion kinetics sun kai daidaito a mafi yawan taro (Hoto na 2b). Matsakaicin girman mitochondrial bai canza ba a duka 3 da 5 mM PPA, yana nuna cewa an kiyaye amincin hanyar sadarwar mitochondrial (Hoto na 2d). Wannan yana nuna ikon cibiyoyin sadarwa na mitochondrial masu ƙarfi don mayar da martani ga ɗan damuwa na metabolism don kiyaye homeostasis yadda ya kamata ba tare da haifar da rarrabuwar hanyar sadarwa ba. A 3 mM PPA, ƙaruwar fission ya isa don haɓaka sauyawa zuwa sabon daidaito, amma ana buƙatar ƙarin sake fasalin motsi mai zurfi don mayar da martani ga damuwa da yawan PPA ya haifar.
Adadin mitochondria ya ƙaru a duka yawan damuwa na PPA, amma matsakaicin girman mitochondria bai canza sosai ba (Hoto na 2c). Wannan na iya faruwa ne saboda ƙaruwar biogenesis ko ƙaruwar rarrabuwa; duk da haka, idan babu raguwa mai yawa a matsakaicin girman mitochondrial, yana da yuwuwar biosynthesis ya ƙaru. Duk da haka, bayanan da ke cikin Hoto na 2 suna goyon bayan wanzuwar hanyoyin diyya guda biyu: ƙaruwa a cikin adadin abubuwan da suka faru na fission, daidai da haɓaka fission na mitochondrial, da ƙaruwa a cikin adadin abubuwan da suka faru, daidai da biogenesis na mitochondrial. A ƙarshe, diyya mai ƙarfi don ɗan damuwa mai sauƙi na iya ƙunsar matakai a lokaci guda waɗanda suka haɗa da fission, fusion, biogenesis, da mitophagy. Kodayake marubutan da suka gabata sun nuna cewa PPA yana haɓaka mitosis30,39 da mitophagy29, muna ba da shaida don sake fasalin fission da fusion dynamics na mitochondrial don mayar da martani ga PPA. Waɗannan bayanan sun tabbatar da canje-canjen yanayin da TEM ta lura kuma suna ba da ƙarin haske game da hanyoyin da ke da alaƙa da rashin aikin mitochondrial da PPA ke haifarwa.
Domin binciken TEM ko MEL bai bayar da shaida kai tsaye game da hanyoyin daidaita kwayoyin halitta da ke ƙarƙashin canje-canjen siffar halittar da aka lura ba, mun bincika bayyanar RNA na kwayoyin halitta da ke da hannu a cikin metabolism na mitochondrial, biogenesis, da dynamics. CMYC proto-oncogene wani abu ne na rubutu wanda ke da hannu a cikin daidaita mitochondria, glycolysis, amino acid da fatty acid metabolism40. Bugu da ƙari, an san cMYC yana daidaita bayyanar kusan kwayoyin halitta 600 na mitochondrial da ke da hannu a cikin rubutun mitochondrial, fassara, da haɗuwa mai rikitarwa, gami da NRF1 da TFAM41. NRF1 da TFAM sune masu daidaita tsakiya guda biyu na mitosis, suna aiki a ƙasan PGC-1α don kunna kwafin mtDNA. Wannan hanyar tana aiki ta hanyar siginar cAMP da AMPK kuma tana da hankali ga kashe kuzari da damuwa na rayuwa. Mun kuma bincika NFE2L2, mai daidaita redox na biogenesis na mitochondrial, don tantance ko tasirin PPA na iya kasancewa ta hanyar damuwa ta oxidative.
Duk da cewa bayyanar NFE2L2 ba ta canza ba, mun sami raguwar da ta dogara da kashi-kashi a cikin bayyanar cMYC, NRF1 da TFAM bayan awanni 24 na magani tare da 3 mM da 5 mM PPA (Hoto na 3a–c). An bayar da rahoton raguwar bayyanar cMYC a baya a matsayin martani ga damuwa ta mitochondrial42, kuma akasin haka, raguwar bayyanar cMYC na iya haifar da rashin aiki na mitochondrial ta hanyar sake fasalin metabolism na mitochondrial, haɗin hanyar sadarwa, da kuma rarraba membrane43. Abin sha'awa, cMYC kuma yana da hannu a cikin daidaita fission da fusion42,43 kuma an san yana ƙara DRP1 phosphorylation da wurin mitochondrial yayin rarraba tantanin halitta44, da kuma daidaita gyaran yanayin mitochondrial a cikin ƙwayoyin jijiyoyi45. Hakika, fibroblasts marasa ƙarancin cMYC suna nuna raguwar girman mitochondrial, daidai da canje-canjen da damuwar PPA43 ta haifar. Waɗannan bayanan suna nuna alaƙa mai ban sha'awa amma har yanzu ba a fayyace ba tsakanin cMYC da yanayin mitochondrial, suna ba da manufa mai ban sha'awa don nazarin nan gaba na sake fasalin da damuwa ta PPA ta haifar.
Ragewar NRF1 da TFAM ya yi daidai da rawar da cMYC ke takawa a matsayin muhimmin mai kunna bayanai. Waɗannan bayanai sun kuma yi daidai da binciken da aka yi a baya a cikin ƙwayoyin cutar kansar hanji na ɗan adam wanda ke nuna cewa PPA ta rage bayyanar NRF1 mRNA a cikin awanni 22, wanda ke da alaƙa da raguwar ATP da ƙaruwar ROS46. Waɗannan marubutan sun kuma ba da rahoton cewa bayyanar TFAM ta ƙaru da awanni 8.5 amma ta koma matakan farko a awanni 22. Sabanin haka, Kim et al. (2019) sun nuna cewa bayyanar TFAM mRNA ta ragu sosai bayan awanni 4 na damuwa na PPA a cikin ƙwayoyin SH-SY5Y; duk da haka, bayan awanni 72, bayyanar furotin TFAM ta ƙaru sosai kuma lambar kwafin mtDNA ta ƙaru sosai. Don haka, raguwar adadin kwayoyin halittar biogenesis na mitochondrial da muka lura bayan awanni 24 ba ya ware yiwuwar karuwar adadin mitochondria yana da alaƙa da kunna biogenesis a lokutan baya. Nazarin da aka yi a baya ya nuna cewa PPA yana ƙara yawan PGC-1α mRNA da furotin a cikin ƙwayoyin SH-SY5Y a cikin awanni 4 da mintuna 30, yayin da propionic acid ke haɓaka mitochondrial biogenesis a cikin ƙwayoyin hanta ta hanyar PGC-1α a cikin awanni 12 da mintuna 39. Abin sha'awa, PGC-1α ba wai kawai mai tsara rubutun NRF1 da TFAM kai tsaye ba ne, amma kuma an nuna yana daidaita ayyukan MFN2 da DRP1 ta hanyar daidaita fission da fusion47. Idan aka haɗa su, wannan yana nuna kusancin hanyoyin da ke daidaita martanin ramawa na mitochondrial da PPA ke haifarwa. Bugu da ƙari, bayananmu suna nuna raguwar tsarin rubutun biogenesis da metabolism a ƙarƙashin damuwa na PPA.
Kwayoyin halittar STOML2, OPA1, MFN1, MFN2 da DRP1 suna daga cikin manyan masu kula da fission na mitochondrial, fusion da dynamics37,48,49. Akwai wasu kwayoyin halitta da yawa da ke da hannu a cikin mitochondrial dynamics, duk da haka, an gano cewa STOML2, OPA1 da MFN2 suna da bambanci a cikin methylation a cikin ƙungiyoyin ASD,16 kuma wasu bincike masu zaman kansu sun ba da rahoton canje-canje a cikin waɗannan abubuwan rubutu don mayar da martani ga damuwa na mitochondrial50,51. 52. An rage yawan bayyanar OPA1 da STOML2 sosai ta hanyar maganin PPA na 3 mM da 5 mM (Hoto na 3e, f). OPA1 yana ɗaya daga cikin masu kula da haɗakar mitochondrial ta hanyar hulɗa kai tsaye tare da MFN1 da 2 kuma yana taka rawa a cikin sake fasalin cristae da siffar mitochondrial53. Matsayin STOML2 a cikin mitochondrial dynamics har yanzu ba a fayyace shi ba, amma shaida ta nuna cewa yana taka rawa a cikin mitochondrial fusion, biogenesis, da mitophagy.
STOML2 yana da hannu wajen kula da haɗin gwiwar numfashi na mitochondrial da kuma samar da hadaddun sarkar numfashi54,55 kuma an nuna cewa yana canza halayen metabolism na ƙwayoyin cutar kansa56. Bincike ya nuna cewa STOML2 yana haɓaka yuwuwar membrane na mitochondrial da biogenesis ta hanyar hulɗa da BAN da cardiolipin 55, 57, 58. Bugu da ƙari, bincike mai zaman kansa ya nuna cewa hulɗar da ke tsakanin STOML2 da PINK1 yana daidaita mitophagy59,60. Abin lura, an ruwaito cewa STOML2 yana hulɗa kai tsaye da kuma daidaita MFN2 kuma yana taka muhimmiyar rawa wajen daidaita dogayen isoforms na OPA1 ta hanyar hana protease da ke da alhakin lalata OPA153,61,62. Rage bayyanar STOML2 da aka lura a cikin halayen PPA na iya sa waɗannan furotin na haɗuwa su fi sauƙin lalacewa ta hanyar hanyoyin ubiquitin da proteasome48. Ko da yake ainihin rawar da STOML2 da OPA1 ke takawa a cikin martanin da ake samu ga PPA ba a fayyace ta ba, raguwar bayyanar waɗannan kwayoyin halittar haɗuwa (Hoto na 3) na iya kawo cikas ga daidaito tsakanin fission da haɗuwa da kuma haifar da raguwar girman mitochondrial (Hoto na 3). 1).
A gefe guda kuma, bayyanar furotin na OPA1 bai canza ba bayan awanni 24, yayin da matakan mRNA da furotin na MFN1, MFN2 ko DRP1 ba su canza sosai ba bayan maganin PPA (Hoto na 3g-i, Hoto na 4). Wannan na iya nuna cewa babu canje-canje a cikin tsarin waɗannan abubuwan da ke da alaƙa da haɗakar mitochondrial da fission. Duk da haka, ya kamata a lura cewa kowanne daga cikin waɗannan kwayoyin halitta guda huɗu kuma ana sarrafa shi ta hanyar gyare-gyaren bayan rubutu (PTMs) waɗanda ke sarrafa ayyukan furotin. OPA1 yana da wasu bambance-bambancen splice guda takwas waɗanda aka raba su ta hanyar proteolytic a cikin mitochondria don samar da isoforms guda biyu daban-daban 63. Daidaito tsakanin isoforms masu tsayi da gajeru a ƙarshe yana ƙayyade rawar da OPA1 ke takawa a cikin haɗakar mitochondrial da kuma kula da hanyar sadarwar mitochondrial64. Ayyukan DRP1 ana tsara su ta hanyar phosphorylation na furotin kinase II (CaMKII) mai dogara da calcium/calmodulin, yayin da lalata DRP1 ana tsara shi ta hanyar ubiquitination da SUMOylation65. A ƙarshe, duka DRP1 da MFN1/2 GTPases ne, don haka aikin na iya shafar yawan samar da GTP a cikin mitochondria 66. Saboda haka, kodayake bayyanar waɗannan sunadarai ya kasance iri ɗaya, wannan bazai nuna aikin furotin mara canzawa ko wurin zama ba67,68. Hakika, jerin sunadaran PTM da ke akwai galibi suna aiki azaman layin farko na kariya wanda ke da alhakin magance martanin damuwa mai tsanani. A gaban matsakaicin damuwa na rayuwa a cikin samfurinmu, yana yiwuwa PTM yana haɓaka aikin haɗuwa da furotin na fission don dawo da daidaiton mitochondrial ba tare da buƙatar ƙarin kunnawa na waɗannan kwayoyin halitta a matakin mRNA ko furotin ba.
Idan aka haɗa su wuri ɗaya, bayanan da ke sama suna nuna tsari mai sarkakiya da dogaro da lokaci na tsarin mitochondrial da ƙalubalen fayyace waɗannan hanyoyin. Don nazarin bayyanar kwayoyin halitta, da farko ya zama dole a gano takamaiman kwayoyin halitta da ake nufi a cikin hanyar. Duk da haka, bayananmu sun nuna cewa kwayoyin halitta a cikin hanya ɗaya ba sa amsawa ta hanya ɗaya ga damuwa iri ɗaya. A zahiri, binciken da aka yi a baya ya nuna cewa kwayoyin halitta daban-daban a cikin hanya ɗaya na iya nuna bayanan amsawa na ɗan lokaci daban-daban30,46. Bugu da ƙari, akwai hanyoyin da suka yi rikitarwa bayan rubuta bayanai waɗanda ke wargaza alaƙar da ke tsakanin kwafi da aikin kwayoyin halitta. Nazarin Proteomic na iya ba da haske game da tasirin PTMs da aikin furotin, amma kuma suna haifar da ƙalubale gami da hanyoyin samar da ƙarancin aiki, babban rabon sigina zuwa hayaniya, da rashin mafita.
A cikin wannan mahallin, nazarin yanayin mitochondrial ta amfani da TEM da MEL yana da babban damar magance tambayoyi masu mahimmanci game da alaƙar da ke tsakanin yanayin mitochondrial da aiki da kuma yadda wannan ke shafar cuta. Mafi mahimmanci, TEM tana ba da hanya kai tsaye don auna yanayin mitochondrial a matsayin maƙasudin haɗuwa na rashin aikin mitochondrial da dynamics51. MEL kuma tana ba da hanya kai tsaye don hango abubuwan da suka faru na fission da haɗaɗɗen yanayi a cikin yanayin ƙwayoyin halitta mai girma uku, yana ba da damar ƙididdige gyaran mitochondrial mai ƙarfi koda kuwa babu canje-canje a cikin bayyanar kwayoyin halitta33. A nan mun haskaka amfanin dabarun hoton mitochondrial a cikin cututtukan mitochondrial na biyu. Waɗannan cututtukan galibi ana siffanta su da damuwa mai sauƙi na rayuwa wanda ke haifar da sake fasalin hanyoyin sadarwa na mitochondrial maimakon lalacewar mitochondrial mai tsanani. Duk da haka, diyya ta mitochondrial da ake buƙata don kula da mitosis a ƙarƙashin damuwa na yau da kullun yana da sakamako mai zurfi na aiki. A cikin mahallin neuroscience, fahimtar waɗannan hanyoyin diyya na iya samar da mahimman bayanai game da ilimin cututtukan jijiyoyi na pleiotropic da ke da alaƙa da rashin aikin mitochondrial.
A ƙarshe, bayananmu sun nuna amfanin dabarun daukar hoto don fahimtar sakamakon aiki na hulɗar da ke tsakanin bayyanar kwayoyin halitta, gyare-gyaren furotin, da ayyukan furotin da ke sarrafa yanayin mitochondrial na jijiyoyi. Mun yi amfani da PPA don yin kwaikwayon rashin aikin mitochondrial a cikin samfurin ƙwayoyin jijiyoyi don samun fahimta game da ɓangaren mitochondrial na ASD. Kwayoyin SH-SY5Y da aka yi wa magani da PPA sun nuna canje-canje a cikin yanayin mitochondrial: mitochondria ya zama ƙarami da zagaye, kuma cristae ba a bayyana shi da kyau ba lokacin da TEM ta lura da shi. Binciken MEL ya nuna cewa waɗannan canje-canje suna faruwa tare da ƙaruwa a cikin abubuwan da suka faru na fission da haɗuwa don kula da hanyar sadarwar mitochondrial don mayar da martani ga ɗan damuwa na metabolism. Bugu da ƙari, PPA yana da matukar kawo cikas ga tsarin rubutu na metabolism na mitochondrial da homeostasis. Mun gano cMYC, NRF1, TFAM, STOML2, da OPA1 a matsayin manyan masu kula da mitochondrial waɗanda damuwar PPA ta lalata kuma suna iya taka rawa wajen daidaita canje-canjen da PPA ta haifar a cikin yanayin mitochondrial da aiki. Ana buƙatar nazarin gaba don siffanta canje-canje na ɗan lokaci da PPA ta haifar a cikin bayyanar kwayoyin halitta da aikin furotin, wurin zama, da gyare-gyare bayan fassarar. Bayananmu sun nuna sarkakiya da dogaro da hanyoyin da ke daidaita martanin damuwa na mitochondrial kuma suna nuna amfanin TEM da sauran dabarun daukar hoto don ƙarin nazarin injiniya.
An sayi layin ƙwayoyin SH-SY5Y (ECACC, 94030304-1VL) daga Sigma-Aldrich. An shuka ƙwayoyin SH-SY5Y a cikin cakuda abinci mai gina jiki na Dulbecco's modified Eagle's medium/F-12 (DMEM/F-12) da L-glutamine (SC09411, ScienCell) a cikin kwalba 25 cm2 waɗanda aka ƙara musu 20% serum na shanu na tayi (FBS) (10493106, ThermoFisher Scientific) da 1% penicillin-streptomycin (P4333-20ML, Sigma-Aldrich) a zafin jiki na 37 °C, 5% CO2. An haɓaka ƙwayoyin zuwa kashi 80% na haɗuwa ta amfani da 0.05% trypsin-EDTA (15400054, ThermoFisher Scientific), an sanya su a centrifuge a 300 g kuma an rufe su da yawa na kimanin ƙwayoyin 7 × 105/ml. An yi dukkan gwaje-gwajen akan ƙwayoyin SH-SY5Y marasa bambanci tsakanin sassan 19-22. Ana ba da PPA a matsayin NaP. Narke foda na NaP (CAS No. 137-40-6, dabarar sinadarai C3H5NaO2, P5436-100G, Sigma-Aldrich) a cikin ruwan MilliQ mai ɗumi zuwa yawan M1 kuma a adana a 4 °C. A ranar maganin, a narkar da wannan maganin da 1 M PPA zuwa 3 mM da 5 mM PPA a cikin matsakaici mara serum (DMEM/F-12 tare da L-glutamine). Yawan magani ga duk gwaje-gwajen ba su da PPA (0 mM, control), 3 mM, da 5 mM PPA. An gudanar da gwaje-gwajen a cikin aƙalla kwafi uku na halitta.
An shuka ƙwayoyin SH-SY5Y a cikin kwalba mai girman cm5 25 a cikin adadin ƙwayoyin 5.5 × 105/ml kuma an girma su na tsawon awanni 24. An ƙara maganin PPA a cikin kwalbar kafin awanni 24 na haɗuwa. Tattara ƙwayoyin tantanin halitta bisa ga ka'idojin al'ada na ƙwayoyin dabbobi masu shayarwa (wanda aka bayyana a sama). Sake dasa ƙwayar tantanin halitta a cikin 100 µl 2.5% glutaraldehyde, 1 × PBS kuma a adana a 4 °C har sai an sarrafa su. An ɗan daɗe ana amfani da ƙwayoyin SH-SY5Y don su lalata ƙwayoyin kuma a cire 2.5% glutaraldehyde, maganin PBS 1 ×. Sake dasa laka a cikin gel na agarose 4% wanda aka shirya a cikin ruwan da aka tace (rabo daga agarose zuwa girman laka shine 1:1). An sanya guntun Agarose a kan grid akan faranti masu faɗi kuma an shafa su da 1-hexadecene kafin daskarewa mai ƙarfi. An daskare samfuran a cikin busasshen acetone 100% a -90°C na tsawon awanni 24. Daga nan aka ɗaga zafin zuwa -80°C sannan aka ƙara ruwan maganin osmium tetroxide 1% da glutaraldehyde 0.1%. An adana samfuran a -80°C na tsawon awanni 24. Bayan haka, an ƙara zafin a hankali zuwa zafin ɗaki a cikin kwanaki da yawa: daga - 80°C zuwa - 50°C na tsawon awanni 24, zuwa - 30°C na tsawon awanni 24, zuwa - 10°C na tsawon awanni 24 sannan a ƙarshe zuwa zafin ɗaki.
Bayan shirye-shiryen cryogenic, an saka samfuran da resin kuma an yi sassan da suka yi laushi (kimanin 100 nm) ta amfani da Leica Reichert UltracutS ultramicrotome (Leica Microsystems). An yi wa sassan fenti da kashi 2% na uranyl acetate da citrate na gubar. An lura da samfuran ta amfani da na'urar hangen nesa ta FEI Tecnai 20 (ThermoFisher (wanda a da FEI ne), Eindhoven, Netherlands) wacce ke aiki a 200 kV (mai watsawa na Lab6) da kyamarar Gatan CCD (Gatan, UK) wacce aka sanya matatar makamashi ta Tridiem.
A cikin kowace kwafin fasaha, an samo aƙalla hotunan tantanin halitta guda 24, jimillar hotuna 266. An yi nazarin dukkan hotunan ta amfani da macro na Yankin Sha'awa (ROI) da macro na Mitochondria. Macro na mitochondrial ya dogara ne akan hanyoyin da aka buga17,31,32 kuma yana ba da damar sarrafa hotunan TEM na rabin-atomatik a Fiji/ImageJ69. A taƙaice: hoton yana juyawa kuma yana juyawa ta amfani da ragewar bango na ƙwallon birgima (radius na pixel 60) da matattarar bandpass FFT (ta amfani da iyakokin sama da ƙasa na pixel 60 da 8 bi da bi) da kuma danne layin tsaye tare da juriyar daidaitawa na 5%. Hoton da aka sarrafa ana iyakance shi ta atomatik ta amfani da matsakaicin algorithm na entropy kuma ana samar da abin rufe fuska na binary. An cire yankunan hoto da ke da alaƙa da ROIs da aka zaɓa da hannu a cikin hotunan TEM marasa inganci, suna siffanta mitochondria kuma ba tare da membrane na plasma da sauran yankuna masu babban bambanci ba. Ga kowace ROI da aka cire, an yi nazarin barbashi biyu da suka fi girman pixels 600, kuma an auna yankin barbashi, kewaye, manyan da ƙananan gatari, diamita na Feret, zagaye, da zagaye ta amfani da ayyukan aunawa da aka gina a ciki na Fiji/ImageJ. Bayan Merrill, Flippo, da Strack (2017), yanki na 2, rabon ɓangaren barbashi (babban rabon axis zuwa ƙaramin rabo), da kuma siffa (FF) an ƙididdige su daga waɗannan bayanan, inda FF = kewaye 2/4pi x yanki. Ana iya samun ma'anar dabarar parametric a cikin Merrill, Flippo, da Strack (2017). Macros da aka ambata suna samuwa akan GitHub (duba Bayanin Samun Bayanai). A matsakaici, an yi nazarin kusan barbashi 5,600 a kowace maganin PPA, don jimlar kusan barbashi 17,000 (bayanan da ba a nuna ba).
An sanya ƙwayoyin SH-SH5Y a cikin abincin gargajiya mai ɗakuna 8 (ThermoFisher, #155411) don ba da damar mannewa cikin dare ɗaya sannan a saka su da TMRE 1:1000 (ThermoFisher, #T669) da Hoechst 33342 1:200 (Sigma-Aldrich, H6024). Rini. An samo hotunan ta amfani da lasers 405 nm da 561 nm a cikin yanayi na mintuna 10, kuma an samo hotunan da ba a so a matsayin tarin z-stacks waɗanda ke ɗauke da ƙananan hotuna 10 tare da matakin az na 0.2 μm tsakanin firam ɗin hoto a wurare 12 na gaba. An tattara hotunan ta amfani da dandamalin Carl Zeiss LSM780 ELYRA PS.1 mai ƙuduri mai girma (Carl Zeiss, Oberkochen, Jamus) ta amfani da ruwan tabarau na LCI Plan Apochromate 100x/1.4 Oil DIC M27. An yi nazarin hotuna a cikin ImageJ ta amfani da bututun da aka bayyana a baya da kuma plugin ɗin ImageJ don auna abubuwan da suka faru na haɗuwa da fission, matsakaicin adadin tsarin mitochondrial, da matsakaicin girman mitochondrial a kowace tantanin halitta33. Ana samun macros na MEL akan GitHub (duba Bayanin Samun Bayanai).
An shuka ƙwayoyin SH-SY5Y a cikin faranti masu ramuka shida a yawan ƙwayoyin 0.3 × 106/mL na tsawon awanni 24 kafin a yi magani. An cire RNA ta amfani da tsarin Quick-RNA™ Miniprep (ZR R1055, Zymo Research) tare da ɗan gyare-gyare kaɗan: ƙara 300 μl na RNA lysis buffer a kowace rijiya kafin a cire sannan a wanke kowane samfurin a matsayin mataki na ƙarshe tare da 30 μl na DNase/RNase exlution. -free ruwa. An duba duk samfuran don adadi da inganci ta amfani da NanoDrop ND-1000 UV-Vis Spectrophotometer. An samo jimlar furotin daga ƙwayoyin lysates ta amfani da 200 μl RIPA lysis buffer, kuma an auna yawan furotin ta amfani da gwajin furotin na Bradford70.
An yi amfani da Tetro™ cDNA Synthesis Kit (BIO-65043, Meridian Bioscience) bisa ga umarnin masana'anta tare da wasu gyare-gyare. An haɗa cDNA a cikin halayen 20-μl ta amfani da 0.7 zuwa 1 μg na jimlar RNA. An zaɓi primers daga takardu da aka buga a baya 42, 71, 72, 73, 74, 75, 76, 77, 78 (Table S1) kuma an tsara gwaje-gwajen da ke tare da su ta amfani da kayan aikin PrimerQuest daga Integrated DNA Technologies. An daidaita dukkan kwayoyin halittar da ke da sha'awa zuwa kwayar halittar B2M ta nukiliya. An auna bayyanar kwayar halittar STOML2, NRF1, NFE2L2, TFAM, cMYC da OPA1 ta hanyar RT-qPCR. Babban haɗin ya haɗa da LUNA Taq polymerase (M3003L, New England Biolabs), 10 μM na gaba da na baya, cDNA, da ruwan PCR don samar da ƙarar ƙarshe ta 10 μL ga kowane amsawa. An auna bayyanar kwayoyin halittar rarrabuwa da fission (DRP1, MFN1/2) ta amfani da gwaje-gwajen TaqMan multiplex. An yi amfani da Luna Universal Probe qPCR Master Mix (M3004S, New England Biolabs) bisa ga umarnin masana'anta tare da ƙananan gyare-gyare. Multiplex RT-qPCR master cakuda ya haɗa da 1X LUNA Taq polymerase, 10 μM na gaba da na baya primers, 10 μM probe, cDNA, da ruwa na PCR, wanda ya haifar da ƙarar ƙarshe ta 20 μL ga kowane amsawa. An yi RT-qPCR ta amfani da Rotor-Gene Q 6-plex (QIAGEN RG—lambar serial: R0618110). An nuna yanayin keke a cikin Tebur S1. An ƙara girman dukkan samfuran cDNA a cikin sau uku kuma an samar da madaidaicin lanƙwasa ta amfani da jerin rarrabuwar ninki goma. An cire waɗanda ba su dace ba a cikin samfuran sau uku tare da karkacewar ma'aunin zagaye (Ct) >0.5 daga binciken don tabbatar da sake haifuwar bayanai30,72. An ƙididdige bayyanar kwayar halitta ta hanyar amfani da hanyar 2-ΔΔCt79.
An haɗa samfuran furotin (60 μg) da ma'aunin Laemmli a rabo na 2:1 kuma an gudanar da su akan gel ɗin furotin mara launi na 12% (Bio-Rad #1610184). An canja sunadaran zuwa membrane na PVDF (polyvinylidene fluoride) (#170-84156, Bio-Rad) ta amfani da tsarin Trans-Blot Turbo (#170-4155, Bio-Rad). An toshe membrane ɗin kuma an haɗa shi da manyan ƙwayoyin rigakafi (OPA1, MFN1, MFN2, da DRP1) (wanda aka narkar da 1:1000) na tsawon awanni 48, sannan aka haɗa shi da ƙwayoyin rigakafi na biyu (1:10,000) na tsawon awa 1. Daga nan aka ɗauki hotunan membranes ta amfani da Clarity Western ECL Substrate (#170-5061, Bio-Rad) kuma aka yi rikodin su ta amfani da tsarin Bio-Rad ChemiDoc MP. An yi amfani da sigar ImageLab 6.1 don nazarin Western blot. An nuna gel ɗin asali da blot a cikin Hoto na S1. Bayanin antibody an bayar da shi a cikin Tebur S2.
An gabatar da saitin bayanai a matsayin matsakaicin kuskure da daidaitaccen ma'auni na matsakaicin (SEM) na akalla samfuran guda uku masu zaman kansu. An gwada saitin bayanai don daidaito ta amfani da gwajin Shapiro-Wilks (sai dai idan an faɗi akasin haka) kafin a ɗauki rarrabawar Gaussian da daidaitattun karkacewa daidai kuma a ci gaba da nazarin. Baya ga nazarin saitin bayanai ta amfani da Fisher's MEL LSD (p < 0.05), ANOVA ta hanya ɗaya (ma'aunin magani da iko), da gwajin kwatantawa da yawa na Dunnett don tantance mahimmanci (p < 0.05). An nuna mahimman ƙimar p a cikin jadawalin kamar *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. An yi duk nazarin ƙididdiga da zane-zane kuma an samar da su ta amfani da GraphPad Prism 9.4.0.
Ana samun macros na Fiji/ImageJ don nazarin hotuna na TEM a bainar jama'a a GitHub: https://github.com/caaja/TEMMitoMacro. Ana samun macro na Mitochondrial Event Locator (MEL) a bainar jama'a a GitHub: https://github.com/rensutheart/MEL-Fiji-Plugin.
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