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Ka whakamahia te waikawa propionic (PPA) hei ako i te mahi a te ngoikore o te mitochondrial i roto i ngā mate whanaketanga neuropērā i te mate autism spectrum. E mōhiotia ana ka whakararu te PPA i te koiora mitochondrial, te pākia o te pūtau, me te huringa. Heoi, kei te raru tonu ngā pānga o te PPA ki ngā mahi mitochondrial, te wehenga me te whakakotahitanga nā te mea he uaua te āhua o ēnei tikanga. I konei, ka whakamahia e mātou ngā tikanga whakaahua taunga hei tirotiro i te pānga o te PPA ki te hanganga ultra-hanganga mitochondrial, te āhua, me te whakakotahitanga i roto i ngā pūtau SH-SY5Y rite-neuron. Nā te PPA (5 mM) i heke te rohe mitochondrial (p < 0.01), te diameter me te porowhita Feret (p < 0.05), me te rohe 2 (p < 0.01). I whakaatuhia e te tātaritanga tūponotanga mitochondrial he pikinga nui (p < 0.05) i roto i ngā tūponotanga wehenga me te whakakotahitanga, nā reira ka mau tonu te pono o te whatunga mitochondrial i raro i ngā āhuatanga ahotea. Hei tāpiritanga, i tino heke te whakaaturanga mRNA o cMYC (p < 0.0001), NRF1 (p < 0.01), TFAM (p < 0.05), STOML2 (p < 0.0001) me OPA1 (p < 0.05). 01). E whakaatu ana tēnei i te whakahounga o te āhua o te mitochondrial, te koiora me ngā hihiri hei pupuri i te mahi i raro i ngā āhuatanga ahotea. Ka whakaratohia e ā mātou raraunga he tirohanga hou ki ngā pānga o te PPA ki ngā hihiri mitochondrial, ā, e whakaatu ana i te whai huatanga o ngā tikanga whakaahua mō te ako i ngā tikanga whakahaere matatini e uru ana ki ngā urupare ahotea mitochondrial.
He wāhi nui ngā mitochondria ki ngā mahi pūtau maha atu i ō rātou tūranga noa i roto i te whakaputa pūngao me te biosynthesis. Ko te pākia o te mitochondria he kaiwhakahaere matua o te tohu konupūmā, te homeostasis pākia me te redox, te tohu mumura, ngā whakarerekētanga epigenetic, te tipu pūtau, te rerekētanga me te mate pūtau kua whakaritea1. Ina koa, he mea nui te pākia o te mitochondria mō te whanaketanga o ngā neuron, te oranga me te mahi, ā, he whānuitia te whai pānga ki ngā momo whakaaturanga o te neuropathology2,3,4.
I roto i te tekau tau kua pahure ake nei, kua puta te mana o te pākia o te pūtau hei kaiwhakahaere matua o te neurogenesis, te rerekētanga, te pakeketanga me te kirihou5,6. Nō nā tata nei, kua noho te āhua me te hihiri o te mitochondrial hei wāhanga tino nui o te mitosis, he tukanga hihiri e pupuri ana i te puna o ngā mitochondria hauora i roto i ngā pūtau. Ko ngā hihiri o te mitochondrial e whakahaerehia ana e ngā ara uaua e whakawhirinaki ana tetahi ki tetahi mai i te mitochondrial biogenesis me te bioenergetics ki te mitochondrial fission, fusion, transport and clearance7,8. Ko te whakakorenga o tētahi o ēnei tikanga whakauru ka whakararu i te pupuri i ngā whatunga mitochondrial hauora, ā, he nui ngā hua mahi mō te whanaketanga o te roro9,10. Ina hoki, ka kitea te kore whakahaere o ngā hihiri o te mitochondrial i roto i te maha o ngā mate hinengaro, neurodegenerative me te whanaketanga o te roro, tae atu ki ngā mate autism spectrum (ASD)11,12.
He mate whanaketanga roro rerekē te ASD, he hanganga ira me te epigenetic matatini. Kāore e taea te whakahē i te tukunga iho o te ASD, engari kāore i te tino mārama te take ngota ngota. Ko te kohikohi raraunga mai i ngā tauira haumanu-mua, ngā rangahau haumanu, me ngā huinga raraunga ngota ngota maha-omics e whakarato ana i te taunakitanga piki haere o te ngoikore o te mitochondrial i roto i te ASD13,14. I mua i whakahaerehia e mātou he tirotiro methylation DNA whānui-ira i roto i tētahi rōpū tūroro me te ASD, ā, i tautuhia ngā ira methylated rerekē e huihui ana i ngā ara metabolic mitochondrial15. I muri mai ka ripoatahia e mātou te methylation rerekē o ngā kaiwhakahaere pokapū o te biogenesis me te hihiri mitochondrial, i hono atu ki te pikinga o te maha o ngā tārua mtDNA me te whakarerekētanga o te kōtaha metabolic mimi i roto i te ASD16. Ka whakaratohia e ā mātou raraunga he taunakitanga piki haere ko te hihiri mitochondrial me te homeostasis he mahi nui ki te pathophysiology o te ASD. Nō reira, ko te whakapai ake i te māramatanga tikanga o te whanaungatanga i waenga i te hihiri mitochondrial, te morphology, me te mahi he whāinga matua o te rangahau tonu ki ngā mate neurological e tohuhia ana e te ngoikore o te mitochondrial tuarua.
He maha ngā wā ka whakamahia ngā tikanga ngota ngota hei ako i te mahi a ngā ira motuhake i roto i ngā urupare ahotea mitochondrial. Heoi, tera pea ka whakawhāitihia tēnei huarahi e te āhua maha-āhua me te wā o ngā tikanga whakahaere mitotic. Heoi anō, ko te rerekētanga o te whakaaturanga o ngā ira mitochondrial he tohu ā-korekore o ngā huringa mahi, inā koa he iti noa te maha o ngā ira e tātarihia ana. Nō reira, kua whakaarohia he tikanga tika ake mō te ako i te mahi mitochondrial me te bioenergetics17. He hononga tata te āhua o te mitochondrial ki ngā whanaketanga mitochondrial. He mea nui te āhua, te hononga, me te hanganga o te mitochondrial mō te whakaputa pūngao me te oranga o te mitochondrial me te pūtau5,18. Heoi anō, ko ngā wāhanga rerekē o te mitosis e arotahi ana ki ngā huringa o te āhua o te mitochondrial, ka noho hei pito mutunga whai hua mō te koretake o te mitochondrial me te whakarato i te pūtake mō ngā rangahau tikanga e whai ake nei.
Ka taea te tirotiro tika i te āhua o te mitochondrial mā te whakamahi i te karu hiko tuku (TEM), e āhei ai te ako taipitopito i te hanganga pūtau. Ka whakaatuhia tika e te TEM te āhua, te āhua me te hanganga o ngā cristae mitochondrial i te whakataunga o ngā mitochondria takitahi, kaua e whakawhirinaki noa ki te tuhi ira, te whakaaturanga pūmua, ngā tawhā mahi mitochondrial rānei i roto i ngā taupori pūtau17,19,20. Hei tāpiri, ka āwhina te TEM i te ako i ngā taunekeneke i waenga i te mitochondria me ētahi atu whekau, pērā i te reticulum endoplasmic me ngā autophagosomes, e whai wāhi nui ana ki te mahi mitochondrial me te homeostasis21,22. Nō reira, ka waiho te TEM hei tīmatanga pai mō te ako i te ngoikore o te mitochondrial i mua i te arotahi ki ngā ara, ki ngā ira rānei. I te mea ka nui haere te hiranga o te mahi mitochondrial ki te neuropathology, he mārama te hiahia kia taea te ako tika me te rahinga i te āhua me te hihiri o te mitochondrial i roto i ngā tauira neuronal in vitro.
I roto i tēnei tuhinga, ka tirohia e mātou ngā mahi mitochondrial i roto i tētahi tauira neuronal o te ngoikore o te mitochondrial i roto i te mate autism spectrum. I mua ake nei, i kōrerohia e mātou te rerekētanga o te methylation o te propionyl-CoA carboxylase beta (PCCB) i roto i te ASD15, he wāhanga iti o te whākōkī propionyl-CoA carboxylase PCC. E mōhiotia ana ko te ngoikore o te PCC ka puta he kohikohinga paitini o ngā propionyl derivatives, tae atu ki te waikawa propionic (PPA)23,24,25. Kua whakaaturia te PPA ki te whakararuraru i te pākia o te neuronal me te whakarerekē i te whanonga i roto i te tinana, ā, he tauira kararehe kua whakapumautia mō te ako i ngā tikanga whanaketanga neuro e uru ana ki te ASD26,27,28. Hei tāpiri, kua pūrongohia te PPA ki te whakararuraru i te pūmanawa membrane mitochondrial, te biogenesis me te manawa i roto i te vitro, ā, kua whakamahia whānuitia hei tauira i te ngoikore o te mitochondrial i roto i ngā neuron29,30. Heoi, kāore anō kia tino mārama te pānga o te ngoikore o te mitochondrial i puta mai i te PPA ki te āhua me te hihiri o te mitochondrial.
Ka whakamahia e tēnei rangahau ngā tikanga whakaahua tautoko hei ine i ngā pānga o te PPA ki te āhua o te mitochondrial, te hihiri, me te mahi i roto i ngā pūtau SH-SY5Y. Tuatahi, i whakawhanakehia e mātou he tikanga TEM hei whakaatu i ngā huringa o te āhua o te mitochondrial me te hanganga ultra17,31,32. I runga i te āhua hihiri o te mitochondria33, i whakamahia hoki e mātou te tātaritanga mitochondrial event localizer (MEL) hei ine i ngā huringa i roto i te taurite i waenga i ngā huihuinga fission me te fusion, te maha me te rōrahi o te mitochondrial i raro i te ahotea PPA. Hei whakamutunga, i tirotirohia e mātou mēnā he hononga te āhua me te hihiri o te mitochondrial ki ngā huringa o te whakaaturanga o ngā ira e whai wāhi ana ki te biogenesis, fission, me te fusion. Ina huihuia, ka whakaatuhia e ā mātou raraunga te wero o te whakamārama i te uaua o ngā tikanga e whakahaere ana i ngā hihiri mitochondrial. Ka whakanuia e mātou te whai huatanga o te TEM ki te ako i te āhua o te mitochondrial hei pito mutunga ine o te mitosis i roto i ngā pūtau SH-SY5Y. Hei tāpiri, ka whakanuia e mātou ko ngā raraunga TEM e whakarato ana i ngā mōhiohio tino nui ina honoa ki ngā tikanga whakaahua e hopu ana hoki i ngā huihuinga hihiri hei whakautu ki te ahotea metabolic. Ko te whakaahuatanga atu o ngā tikanga whakahaere ngota ngota e tautoko ana i te mitosis pūtau neuronal ka whakarato i te māramatanga nui ki te wāhanga mitochondrial o te pūnaha io me ngā mate neurodegenerative.
Hei whakaoho i te ahotea mitochondrial, i tukinotia ngā pūtau SH-SY5Y ki te PPA mā te whakamahi i te 3 mM me te 5 mM konutai propionate (NaP). I mua i te TEM, i tukuna ngā tauira ki te whakarite tauira cryogenic mā te whakamahi i te tio me te tio pēhanga teitei (Pikitia 1a). I whakawhanakehia e mātou he paipa tātari whakaahua mitochondrial aunoa hei ine i ngā tawhā āhuahanga e waru o ngā taupori mitochondrial puta noa i ngā tārua koiora e toru. I kitea e mātou he nui te whakarerekētanga o ngā tawhā e whā i te maimoatanga PPA: te rohe 2, te rohe, te taha, me te diameter Feret (Pikitia 1b–e). I tino heke te rohe 2 me te maimoatanga PPA 3 mM me te 5 mM (p = 0.0183 me te p = 0.002, ia) (Pikitia 1b), i te mea kua tino heke te rohe (p = 0.003), te taha (p = 0.0106) me te diameter Feret. He whakahekenga nui (p = 0.0172) i roto i te rōpū maimoatanga 5 mM ki te whakataurite ki te rōpū whakahaere (Pikitia 1c–e). I whakaatuhia e ngā whakahekenga nui o te horahanga me te porowhita he iti ake, he porotaka ake ngā mitochondria o ngā pūtau i rongoatia ki te 5 mM PPA, ā, he iti ake te roa o ēnei mitochondria i ērā o ngā pūtau whakahaere. He rite anō hoki tēnei ki te whakahekenga nui o te diameter Feret, he tawhā motuhake e tohu ana i te whakahekenga o te tawhiti nui rawa atu i waenga i ngā taha matūriki. I kitea ngā huringa o te ultrastructure o te cristae: i iti ake te kitea o te cristae i raro i te awe o te ahotea PPA (Pikitia 1a, röpü B). Heoi, kāore i te whakaata mārama ngā whakaahua katoa i te ultrastructure o te cristae, nō reira kāore i mahia he tātaritanga tauanga o ēnei huringa. Ka whakaata pea ēnei raraunga TEM i ngā āhuatanga e toru: (1) ka whakarei ake te PPA i te wehenga, ka aukati rānei i te hanumi, ka whakaiti i te rahi o ngā mitochondria o nāianei; (2) ka hangaia e te biogenesis whakarei ake he mitochondria hou, he iti ake rānei (3) ka whakaoho i ngā tikanga e rua i te wā kotahi. Ahakoa kāore e taea te wehewehe i ēnei āhuatanga e te TEM, ko ngā huringa morphological nui e tohu ana i ngā huringa o te homeostasis mitochondrial me ngā hihiri i raro i te ahotea PPA. I muri mai ka tūhuratia e mātou ētahi atu tawhā hei whakaahua ake i ēnei hihiri me ngā tikanga pea kei raro i a rātou.
Ka hangaia anōtia te āhua o te mitochondria e te waikawa propionic (PPA). (a) Ngā whakaahua o te karu hiko tuku (TEM) e whakaatu ana ka heke te rahi o te mitochondria, ka iti haere, ka porotaka ake ngā mitochondria me te piki haere o te maimoatanga PPA; 0 mM (kāore i rongoatia), 3 mM me te 5 mM, ia. Ko ngā pere whero e tohu ana i ngā mitochondria. (b–e) Ko ngā pūtau SH-SY5Y i rongoatia ki te PPA mō te 24 hāora i whakaritea mō te TEM, ā, i tātarihia ngā hua mā te whakamahi i a Fiji/ImageJ. E whā o ngā tawhā e waru i whakaatu i ngā rerekētanga nui i waenga i ngā pūtau mana (kāore i rongoatia, 0 mM PPA) me ngā pūtau i rongoatia (3 mM me te 5 mM PPA). (b) Rohe 2, (c) Rohe, (d) Tahataha, (e) Diameter Feret. I whakamahia te tātaritanga kotahi-ara o te rerekētanga (mana vs. maimoatanga) me te whakamātautau whakatairite maha a Dunnett hei whakatau i ngā rerekētanga nui (p < 0.05). Ko ngā tohu raraunga e tohu ana i te uara mitochondrial toharite mō ia pūtau takitahi, ā, ko ngā pae hapa e tohu ana i te toharite ± SEM. Ko ngā raraunga e whakaaturia ana e tohu ana i te n = 3, neke atu i te 24 ngā pūtau mō ia tāruarua; e 266 ngā whakaahua katoa i tātarihia; * e tohu ana ko te p < 0.05, ** e tohu ana ko te p < 0.01.
Hei whakaahua anō i te urupare a ngā mahi mitochondrial ki te PPA, i pania e mātou ngā mitochondria ki te tetramethylrhodamine ethyl ester (TMRE) ā, i whakamahia te karu tirotiro wā-roa me te tātari MEL hei kimi me te ine i ngā mitochondria i muri i ngā hāora 24 i te 3 me te 5 mM PPA. Te maimoatanga o ngā huihuinga fission me te fusion. (Pikitia 2a). I muri i te tātari MEL, i tātarihia anō ngā mitochondria hei ine i te maha o ngā hanganga mitochondrial me tō rātou rahinga toharite. I kite mātou i tētahi pikinga iti engari nui o te maha o ngā huihuinga fission e puta ana i te 3 mM [4.9 ± 0.3 (p < 0.05)] ki te whakataurite ki te fission [5.6 ± 0.3 (p < 0.05) )] ā, ko ngā huihuinga fusion [5.4 ± 0.5 (p < 0.05)] me te fusion [5.4 ± 0.5 (p < 0.05)] 0.05)] <0.05)] i tino piki ake i te 5 mM ki te whakataurite ki te mana whakahaere (Pikitia 3b). I tino piki ake te maha o ngā mitochondria i te 3 [32.6 ± 2.1 (p < 0.05)] me te 5 mM [34.1 ± 2.2 (p < 0.05)] (Pikitia 3c), i te mea kāore i rerekē te rahinga toharite o ia hanganga mitochondrial (Pikitia 3c). 3d). Ina huihuia, e kī ana tēnei ko te whakahounga o ngā mahi mitochondrial he urupare utu e pupuri angitu ana i te pono o te whatunga mitochondrial. Ko te pikinga o te maha o ngā huihuinga wehenga i te 3 mM PPA e kī ana ko te pikinga o te maha o ngā mitochondrial he wāhanga nā te wehenga mitochondrial, engari i te mea kei te noho tonu te rahinga mitochondrial toharite i te nuinga o te wā, kāore e taea te whakakore i te biogenesis hei urupare utu tāpiri. Heoi, he rite ēnei raraunga ki ngā hanganga mitochondrial iti ake, porowhita i kitea e te TEM, ā, e whakaatu ana hoki i ngā huringa nui o ngā mahi mitochondrial i puta mai i te PPA.
Ka whakaoho te waikawa propionic (PPA) i te whakahounga hihiko o te mitochondrial hei pupuri i te pono o te whatunga. I whakatipuria ngā pūtau SH-SY5Y, i tukinotia ki te 3 me te 5 mM PPA mō ngā haora 24, ā, i pania ki te TMRE me te Hoechst 33342, ā, i muri mai ka tātarihia te MEL. (a) Ngā whakaahua karuārai wā-wā e whakaatu ana i te tae me ngā matapae kaha mōrahi kua whakarōpūtia i te wā 2 (t2) mō ia āhuatanga. Ko ngā rohe kua tohua i roto i ia whakaahua rua ka whakarei ake, ā, ka whakaaturia i roto i te 3D i ngā anga wā rerekē e toru (t1-t3) hei whakaatu i ngā hihiko i roto i te wā; ka whakamāramahia ngā huihuinga hanumi ki te kakariki; ka whakamāramahia ngā huihuinga wehenga ki te kakariki. Ka whakaaturia ki te whero. (b) Te maha toharite o ngā huihuinga hihiko mō ia āhuatanga. (c) Te maha toharite o ngā hanganga mitochondrial mō ia pūtau. (d) Te rōrahi toharite (µm3) o ia hanganga mitochondrial mō ia pūtau. Ko ngā raraunga e whakaaturia ana he tohu mō te n = 15 pūtau mō ia rōpū maimoatanga. Ko ngā pae hapa e whakaaturia ana e tohu ana i te toharite ± SEM, pae tauine = 10 μm, * p < 0.05.
Ko te waikawa propionic (PPA) ka pehia te whakawhitiwhiti kōrero o ngā ira e pā ana ki ngā whanaketanga mitochondrial. I rongoatia ngā pūtau SH-SY5Y ki te 3 me te 5 mM PPA mō te 24 hāora. I mahia te ine ira whanaunga mā te whakamahi i te RT-qPCR, ā, i whakatauritea ki te B2M. Ngā ira mitochondrial biogenesis (a) cMYC, (b) TFAM, (c) NRF1 me (d) NFE2L2. Ngā ira mitochondrial fusion me te fission (e) STOML2, (f) OPA1, (g) MFN1, (h) MFN2 me (i) DRP1. I whakamatautauria ngā rerekētanga nui (p < 0.05) mā te whakamahi i te ANOVA kotahi-ara (mana whakahaere vs. maimoatanga) me te whakamātautau whakataurite maha a Dunnett: * e tohu ana i te p < 0.05, ** e tohu ana i te p < 0.01, ā, **** e tohu ana i te p < 0.0001. Ko ngā pae e tohu ana i te whakapuakitanga toharite ± SEM. Ko ngā raraunga e whakaaturia ana e tohu ana i ngā tāruarua koiora n = 3 (STOML2, OPA1, TFAM), n = 4 (cMYC, NRF1, NFE2L2), me n = 5 (MFN1, MFN2, DRP1).
E whakaatu ana ngā raraunga mai i ngā tātaritanga TEM me MEL e whakarerekē ana te PPA i te āhua me te hihiri o te mitochondrial. Heoi, kāore ēnei tikanga whakaahua e whakarato i te māramatanga ki ngā tikanga matua e akiaki ana i ēnei tukanga. Nō reira, i tirotirohia e mātou te whakaaturanga mRNA o ngā kaiwhakahaere matua e iwa o ngā hihiri mitochondrial, te biogenesis, me te mitosis hei whakautu ki te maimoatanga PPA. I inehia e mātou te oncogene myeloma pūtau (cMYC), te tauwehe manawa nuclear (NRF1), te tauwehe whakawhiti mitochondrial 1 (TFAM), te tauwehe whakawhiti NFE2-rite BZIP (NFE2L2), te pūmua rite gastrin 2 (STOML2), te atrophy optic nerve 1 (OPA1), Mitofusin 1 (MFN1), Mitofusin 2 (MFN2) me te pūmua e pā ana ki te dynamin 1 (DRP1) i muri i te 24 hāora o te maimoatanga me te 3 mM me te 5 mM PPA. I kitea e mātou he 3 mM (p = 0.0053, p = 0.0415 me te p < 0.0001, ia) me te 5 mM (p = 0.0031, p = 0.0233, p < 0.0001) maimoatanga PPA. (Pikitia 3a–c). Ko te hekenga o te whakaaturanga mRNA i whakawhirinaki ki te horopeta: ko te whakaaturanga o cMYC, NRF1 me TFAM i heke mā te 5.7, 2.6 me te 1.9 ngā wā i te 3 mM, ia, me te 11.2, 3 me te 2.2 ngā wā i te 5 mM. He rerekē, ko te ira redox biogenesis pokapū NFE2L2 kāore i whakarerekētia i tetahi kukū o te PPA, ahakoa i kitea he ia rite o te whakahekenga whakaaturanga e whakawhirinaki ana ki te horopeta (Pikitia 3d).
I tirotirohia hoki e mātou te whakaaturanga o ngā ira tawhito e whai wāhi ana ki te whakahaere i te wehenga me te whakakotahitanga. E whakaarohia ana ko STOML2 te mea e whai wāhi ana ki te whakakotahitanga, te mitophagy me te biogenesis, ā, i tino heke tōna whakaaturanga (p < 0.0001) mā te 3 mM (huringa 2.4-ngā-wā) me te 5 mM (huringa 2.8-ngā-wā) PPA (Pikitia 1). 3d). Waihoki, i heke te whakaaturanga o te ira whakakotahitanga OPA1 i te 3 mM (huringa 1.6-ngā-wā) me te 5 mM (huringa 1.9-ngā-wā) PPA (p = 0.006 me te p = 0.0024, ia) (Pikitia 3f). Heoi, kāore mātou i kite i ngā rerekētanga nui i roto i te whakaaturanga o ngā ira whakakotahitanga MFN1, MFN2, ira wehenga rānei DRP1 i raro i te ahotea PPA 24-h (Pikitia 3g–i). Hei tāpiritanga, i kitea e mātou kāore i rerekē ngā taumata o ngā pūmua hanumi me te wehenga e whā (OPA1, MFN1, MFN2 me te DRP1) i raro i ngā āhuatanga ōrite (Pikitia 4a–d). He mea nui kia mōhio ko ēnei raraunga e whakaata ana i tētahi wā kotahi, ā, kāore pea e whakaata i ngā huringa o te whakaaturanga pūmua, ngā taumata mahi rānei i ngā wāhanga tōmua o te ahotea PPA. Heoi, ko ngā whakahekenga nui o te whakaaturanga o cMYC, NRF1, TFAM, STOML2, me te OPA1 e tohu ana i te koretake o te whakawhitiwhiti kōrero o te pākia o te mitochondrial, te biogenesis, me ngā hihiri. Hei tāpiritanga, e whakaatu ana ēnei raraunga i te whai huatanga o ngā tikanga whakaahua hei ako tika i ngā huringa mutunga-āhua i roto i te mahi mitochondrial.
Kāore i rerekē ngā taumata pūmua whakakotahi me te wehenga i muri i te maimoatanga waikawa propionic (PPA). I ​​rongoatia ngā pūtau SH-SY5Y ki te 3 me te 5 mM PPA mō te 24 hāora. I inehia ngā taumata pūmua mā te tātaritanga Western blot, ā, i whakatauritehia ngā taumata whakapuakanga ki te pūmua katoa. Kei te whakaatuhia te whakapuakanga pūmua toharite me ngā Western blot e tohu ana i te pūmua ūnga me te pūmua katoa. a – OPA1, b – MFN1, c – MFN2, d – DRP1. Ko ngā pae e tohu ana i te toharite ± SEM, ā, ko ngā raraunga e whakaatuhia ana he tohu mō ngā tāruarua koiora n = 3. I mahia ngā whakatairite maha (p < 0.05) mā te whakamahi i te tātaritanga rerekētanga kotahi-ara me te whakamātautau a Dunnett. Kei te whakaatuhia te tiēre me te blot taketake i te Pikitia S1.
Ko te ngoikore o te mitochondrial e pā ana ki ngā mate maha-pūnaha mai i ngā mate pākia o te pūnaha, te mate ngakau me te mate uaua ki ngā mate pūnaha io1,10. He maha ngā mate neurodegenerative me ngā mate neurodegenerative e pā ana ki te ngoikore o te mitochondrial, e whakaatu ana i te hiranga o ēnei whekau puta noa i te roanga o te oranga o te roro. Kei roto i ēnei mate ko te mate a Parkinson, te mate a Alzheimer me te ASD3,4,18. Heoi, he uaua te urunga atu ki te kiko roro hei ako i ēnei mate, inā koa i te taumata miihini, e tika ana kia waiho ngā pūnaha tauira pūtau hei huarahi kē atu. I roto i tēnei rangahau, ka whakamahia e mātou he pūnaha tauira pūtau e whakamahi ana i ngā pūtau SH-SY5Y kua rongoatia ki te PPA hei whakahoki anō i te ngoikore o te mitochondrial i kitea i roto i ngā mate pūtau io, inā koa ngā mate autism spectrum. Mā te whakamahi i tēnei tauira PPA hei ako i ngā mahi mitochondrial i roto i ngā neuron ka taea te whakarato māramatanga ki te pūtake o te ASD.
I tūhuratia e mātou te āheinga o te whakamahi i te TEM hei tiro i ngā panonitanga o te āhua o te mitochondrial. He mea nui kia mōhiotia me whakamahi tika te TEM kia pai ake ai tōna whai huatanga. Mā te whakarite i ngā tauira cryo-specimen ka taea te tiaki pai ake i ngā hanganga neuronal mā te whakatika i ngā wāhanga pūtau me te whakaiti i te hanganga o ngā taonga34. E rite ana ki tēnei, i kitea e mātou he whekau iti-pūtau tonu me ngā mitochondria roa o ngā pūtau SH-SY5Y rite-neuron (Pikitia 1a). E whakaatu ana tēnei i te whai huatanga o ngā tikanga whakarite cryogenic mō te ako i te āhua o te mitochondrial i roto i ngā tauira pūtau neuronal. Ahakoa he mea nui ngā inenga tau mō te tātari whāinga o ngā raraunga TEM, kāore anō kia whakaaetia he aha ngā tawhā motuhake me ine hei whakaū i ngā panonitanga āhua o te mitochondrial. I runga i te maha o ngā rangahau kua tirotiro i te āhua o te mitochondrial17,31,32, i whakawhanakehia e mātou he paipa tātari whakaahua mitochondrial aunoa e ine ana i ngā tawhā tau e waru, arā: te horahanga, te horahanga2, te ōwehenga āhua, te taha, te porowhita, te nekehanga , te diameter Feret. me te porowhita.
I roto i ēnei, i tino whakaitihia e te PPA te rohe 2, te horahanga, te taha, me te diameter o te Feret (Pikitia 1b–e). I whakaatuhia e tēnei kua iti ake, kua porotaka ake ngā mitochondria, he rite ki ngā rangahau o mua e whakaatu ana i te hekenga o te rohe mitochondrial i muri i te 72 hāora o te ahotea mitochondrial i puta mai i te PPA30. Tērā pea ka tohu ēnei āhuatanga āhua i te wehenga mitochondrial, he tukanga e tika ana hei tango i ngā wāhanga kua pakaru mai i te whatunga mitochondrial hei whakatairanga i tō rātou pirau mā te mitophagy35,36,37. I tētahi atu taha, ko te hekenga o te rahi mitochondrial toharite ka hono pea ki te pikinga o te biogenesis, ka hua ake ai te hanganga o ngā mitochondria iti e tipu ake ana. Ko te pikinga o te wehenga, te biogenesis rānei, he urupare utu hei pupuri i te mitosis ki te ahotea mitochondrial. Heoi, kāore e taea te whakakore i te hekenga o te tipu mitochondrial, te ngoikore o te hanumi, me ētahi atu āhuatanga rānei.
Ahakoa ka taea e ngā whakaahua taumira teitei i hangaia e te TEM te whakatau i ngā āhuatanga āhua i te taumata o ngā mitochondria takitahi, ka puta mai i tēnei tikanga ngā whakaahua rua-ahu i te wā kotahi. Hei ako i ngā urupare hihiri ki te ahotea pākia, i pania e mātou te mitochondria ki te TMRE, ā, i whakamahia te karuārai wā-roa me te tātari MEL, e taea ai te whakaatu 3D tere-rahi i ngā huringa i roto i te whatunga mitochondrial i roto i te wā33,38. I kite mātou i ngā huringa ngawari engari he nui i roto i ngā hihiri mitochondrial i raro i te ahotea PPA (Pikitia 2). I te 3 mM, i piki ake te maha o ngā huihuinga wehenga, i te mea i noho tonu ngā huihuinga hanumi ki te mana whakahaere. I kitea he pikinga o te maha o ngā huihuinga wehenga me te hanumi i te 5 mM PPA, engari he rite tonu ēnei huringa, e kī ana ka eke te kinetics wehenga me te hanumi ki te taurite i ngā kukū teitei ake (Pikitia 2b). I noho tonu te rōrahi mitochondrial toharite i te 3 me te 5 mM PPA, e tohu ana i te tiakitanga o te whatunga mitochondrial (Pikitia 2d). E whakaatu ana tēnei i te kaha o ngā whatunga mitochondrial hihiri ki te urupare ki te ahotea pākia ngawari ki te pupuri i te homeostasis me te kore e pakaru te whatunga. I te 3 mM PPA, he rawaka te pikinga o te wehenga hei whakatairanga i te whakawhiti ki tētahi taurite hou, engari me nui ake te whakahounga nekenekehanga hei whakautu ki te ahotea i puta mai i ngā kukū teitei ake o te PPA.
I piki ake te maha o ngā mitochondria i ngā kukū ahotea PPA e rua, engari kāore i tino rerekē te rahinga mitochondrial toharite (Pikitia 2c). Tērā pea nā te pikinga o te biogenesis, te pikinga rānei o te wehenga; heoi, i te korenga o te hekenga nui o te rahinga mitochondrial toharite, he nui ake te tūponotanga ka piki ake te biosynthesis. Heoi, e tautoko ana ngā raraunga i te Pikitia 2 i te noho o ngā tikanga utu e rua: he pikinga o te maha o ngā huihuinga fission, e rite ana ki te pikinga o te fission mitochondrial, me te pikinga o te maha o ngā huihuinga, e rite ana ki te biogenesis mitochondrial. Hei whakamutunga, ko te utu hihiri mō te ahotea ngawari ka uru pea ki ngā tukanga tukutahi e uru ana ki te fission, te fusion, te biogenesis, me te mitophagy. Ahakoa kua whakaatuhia e ngā kaituhi o mua ka whakarei ake te PPA i te mitosis30,39 me te mitophagy29, ka whakaratohia e mātou he taunakitanga mō te whakahou i te fission mitochondrial me ngā hihiri fusion hei whakautu ki te PPA. Ka whakaūhia e ēnei raraunga ngā huringa morphological i kitea e te TEM, ā, ka whakarato i ētahi atu māramatanga ki ngā tikanga e pā ana ki te ngoikore o te mitochondrial i puta mai i te PPA.
Nā te mea kāore i whakaratohia e te tātaritanga TEM me te MEL he taunakitanga tika mō ngā tikanga whakahaere ira e whai pānga ana ki ngā huringa āhua i kitea, i tirotirohia e mātou te whakaaturanga RNA o ngā ira e whai wāhi ana ki te pākia o te mitochondria, te biogenesis, me ngā mahi hihiri. Ko te proto-oncogene cMYC he take whakawhiti e whai wāhi ana ki te whakahaere i te mitochondria, te glycolysis, te waikawa amino me te pākia o te waikawa ngako40. Hei tāpiri, e mōhiotia ana ko te cMYC te whakahaere i te whakaaturanga o te tata ki te 600 ira mitochondrial e whai wāhi ana ki te whakawhiti mitochondrial, te whakamāoritanga, me te huihuinga matatini, tae atu ki te NRF1 me te TFAM41. Ko te NRF1 me te TFAM e rua ngā kaiwhakahaere matua o te mitosis, e mahi ana i raro i te PGC-1α hei whakahohe i te tāruarua mtDNA. Ka whakahohehia tēnei ara e te tohu cAMP me te AMPK, ā, he tairongo ki te whakapaunga pūngao me te ahotea pākia. I tirotirohia hoki e mātou te NFE2L2, he kaiwhakahaere redox o te biogenesis mitochondrial, hei whakatau mena ka taea te whakahaere i ngā pānga o te PPA e te ahotea oxidative.
Ahakoa i noho tonu te whakaaturanga o te NFE2L2 i roto i te rerekētanga, i kitea e mātou he hekenga pumau i roto i te whakaaturanga o te cMYC, NRF1 me te TFAM i muri i te 24 haora o te maimoatanga me te 3 mM me te 5 mM PPA (Pikitia 3a–c). Kua pūrongohia i mua te hekenga o te whakaaturanga cMYC hei urupare ki te ahotea mitochondrial42, ā, i tetahi atu taha, ko te hekenga o te whakaaturanga cMYC ka taea te whakararu i te mahi mitochondrial mā te whakahou i te metabolism mitochondrial, te hononga whatunga, me te polarization membrane43. He mea whakamīharo, kei roto hoki te cMYC i te whakahaere i te wehenga mitochondrial me te whakakotahitanga42,43 ā, e mōhiotia ana ka whakanui ake i te phosphorylation DRP1 me te localization mitochondrial i te wā o te wehenga pūtau44, me te whakahaere hoki i te whakahounga morphological mitochondrial i roto i ngā pūtau take neuronal45. Ina hoki, ka whakaatuhia e ngā fibroblasts cMYC-deficient te rahi mitochondrial kua whakaitihia, e rite ana ki ngā huringa i puta mai i te ahotea PPA43. E whakaatu ana ēnei raraunga i tētahi whanaungatanga whakamere engari kāore anō kia mārama i waenga i te cMYC me ngā mahi mitochondrial, e whakarato ana i tētahi whāinga whakamere mō ngā rangahau a meake nei mō te whakahounga i puta mai i te ahotea PPA.
Ko te whakahekenga o te NRF1 me te TFAM he rite tonu ki te mahi a te cMYC hei kaiwhakaoho whakawhiti kōrero nui. He rite anō hoki ēnei raraunga ki ngā rangahau o mua i roto i ngā pūtau matepukupuku koroni tangata e whakaatu ana i whakaitihia e te PPA te whakaaturanga mRNA NRF1 i te 22 hāora, i honoa ki te whakahekenga ATP me te pikinga o te ROS46. I kōrero anō hoki ēnei kaituhi i piki ake te whakaaturanga TFAM i te 8.5 hāora engari i hoki ki ngā taumata taketake i te 22 hāora. He rerekē, i whakaatu a Kim et al. (2019) i tino heke te whakaaturanga mRNA TFAM i muri i te 4 hāora o te ahotea PPA i roto i ngā pūtau SH-SY5Y; heoi, i muri i te 72 hāora, i tino piki ake te whakaaturanga pūmua TFAM, ā, i tino piki ake te maha o ngā tārua mtDNA. Nō reira, ko te whakahekenga o te maha o ngā ira biogenesis mitochondrial i kitea e mātou i muri i te 24 hāora kāore e whakakore i te tūponotanga ko te pikinga o te maha o ngā mitochondria e hono ana ki te whakahohenga o te biogenesis i ngā wā o mua. Kua whakaatuhia e ngā rangahau o mua he nui ake te pikinga ake o te PPA i te PGC-1α mRNA me te pūmua i roto i ngā pūtau SH-SY5Y i te 4 hāora 30 meneti, ko te waikawa propionic ia e whakarei ake ana i te biogenesis mitochondrial i roto i ngā hepatocytes kuao kau mā te PGC-1α i te 12 hāora 39 meneti. He mea whakamīharo, ehara i te mea ko te PGC-1α he kaiwhakahaere whakawhiti tika o NRF1 me TFAM anake, engari kua whakaaturia hoki he whakahaere i te mahi a MFN2 me DRP1 mā te whakahaere i te wehenga me te whakakotahitanga47. Ina huihuia, e whakaatu ana tēnei i te hononga tata o ngā tikanga e whakahaere ana i ngā urupare utu mitochondrial i puta mai i te PPA. Heoi anō, e whakaatu ana ā mātou raraunga i te korenga o te whakahaere whakawhiti o te biogenesis me te metabolism i raro i te ahotea PPA.
Ko ngā ira STOML2, OPA1, MFN1, MFN2 me DRP1 kei roto i ngā kaiwhakahaere matua o te wehenga mitochondrial, te whakakotahitanga me te hihiri37,48,49. He maha atu ngā ira e whai wāhi ana ki ngā hihiri mitochondrial, heoi, kua kitea i mua ko STOML2, OPA1 me MFN2 he rerekē te methylation i roto i ngā rōpū ASD,16 ā, he maha ngā rangahau motuhake kua pūrongo i ngā huringa o ēnei take whakawhiti hei whakautu ki te ahotea mitochondrial50,51. 52. Ko te whakapuakanga o OPA1 me STOML2 i tino whakaitihia e te maimoatanga PPA 3 mM me te 5 mM (Pikitia 3e, f). Ko OPA1 tētahi o ngā kaiwhakahaere matarohia o te whakakotahitanga mitochondrial mā te taunekeneke tika ki a MFN1 me te 2, ā, he wāhi tōna ki te whakahou i te cristae me te morphology mitochondrial53. Kāore anō kia mārama te tūranga tika o STOML2 i roto i ngā hihiri mitochondrial, engari e kī ana ngā taunakitanga he wāhi tōna ki te whakakotahitanga mitochondrial, te biogenesis, me te mitophagy.
Kei roto a STOML2 i te pupuri i te hononga manawa mitochondrial me te hanganga o ngā matatini mekameka manawa54,55 ā, kua whakaaturia he tino whakarerekē i ngā āhuatanga pākia o ngā pūtau matepukupuku56. Kua whakaatuhia e ngā rangahau ka whakatairanga a STOML2 i te pūmanawa membrane mitochondrial me te biogenesis mā te taunekeneke ki te BAN me te cardiolipin 55, 57, 58. Hei tāpiri, kua whakaatuhia e ngā rangahau motuhake ko te taunekeneke i waenga i a STOML2 me PINK1 e whakahaere ana i te mitophagy59,60. He mea nui, kua pūrongohia a STOML2 ki te taunekeneke tika me te whakaū i te MFN2, ā, he mahi nui anō hoki tana ki te whakaū i ngā isoforms OPA1 roa mā te aukati i te protease e kawenga ana mō te whakahekenga OPA153,61,62. Ko te whakahekenga o te whakaaturanga STOML2 i kitea i roto i ngā tauhohenga PPA ka nui ake te ngawari o ēnei pūmua hanumi ki te whakahekenga mā roto i ngā ara e whakawhirinaki ana ki te ubiquitin me te proteasome48. Ahakoa kāore i te mārama te tūranga tuturu o STOML2 me OPA1 i roto i te urupare hihiri ki te PPA, ko te whakahekenga o te whakaaturanga o ēnei ira whakakotahitanga (Pikitia 3) ka whakararuraru pea i te taurite i waenga i te wehenga me te whakakotahitanga, ā, ka heke te rahi o te mitochondrial (Pikitia 3). 1).
I tētahi atu taha, kāore i rerekē te whakaaturanga pūmua OPA1 i muri i te 24 hāora, engari kāore i tino rerekē ngā taumata mRNA me te pūmua o MFN1, MFN2, DRP1 rānei i muri i te maimoatanga PPA (Pikitia 3g-i, Pikitia 4). Tērā pea he tohu tēnei kāore he huringa i roto i te whakahaere i ēnei āhuatanga e uru ana ki te whakakotahitanga me te wehenga mitochondrial. Heoi, he mea tika kia mōhiotia ko ia o ēnei ira e whā ka whakahaeretia hoki e ngā whakarerekētanga muri-whakawhitinga (PTM) e whakahaere ana i te mahi pūmua. E waru ngā momo hononga rerekē a OPA1 ka tapahia kia rite ki te proteolytic i roto i te mitochondria hei whakaputa i ngā isoforms motuhake e rua 63 . Ko te taurite i waenga i ngā isoforms roa me te poto ka whakatau i te tūranga o OPA1 i roto i te whakakotahitanga mitochondrial me te pupuri i te whatunga mitochondrial64. Ko te mahi DRP1 ka whakahaeretia e te phosphorylation protein kinase II (CaMKII) e whakawhirinaki ana ki te calcium/calmodulin, ko te whakahekenga DRP1 ka whakahaeretia e te ubiquitination me te SUMOylation65. Hei whakamutunga, he GTPase ngā DRP1 me MFN1/2, nō reira ka pāngia pea te mahi e te tere o te hanga GTP i roto i te mitochondria 66. Nō reira, ahakoa kei te pumau tonu te whakaaturanga o ēnei pūmua, kāore pea tēnei e whakaata i te mahi pūmua kāore i rerekē, i te wāhi rānei67,68. Ina hoki, ko ngā pūmua PTM o nāianei he maha ngā wā ka noho hei rārangi parepare tuatahi e kawenga ana mō te takawaenga i ngā urupare ahotea ohorere. I te aroaro o te ahotea pākia ngawari i roto i tā mātou tauira, he tūponotanga ka whakatairangahia e te PTM te pikinga o te mahi o ngā pūmua hanumi me te wehenga kia tino whakahokia mai te pono o te mitochondrial me te kore e hiahiatia he whakahohenga tāpiri o ēnei ira i te taumata mRNA, i te taumata pūmua rānei.
Ina huihuia, e whakaatu ana ngā raraunga o runga ake nei i te whakahaere uaua me te wā-e pā ana ki te āhua o te mitochondrial me ngā wero o te whakamārama i ēnei tikanga. Hei ako i te whakaaturanga ira, me tautuhi tuatahi i ngā ira ūnga motuhake i roto i te ara. Heoi, e whakaatu ana ā mātou raraunga kāore ngā ira i roto i te ara kotahi e urupare i te ara kotahi ki te ahotea kotahi. Inaa, kua whakaatuhia e ngā rangahau o mua he rerekē ngā kōtaha urupare wā o ngā ira rerekē i roto i te ara kotahi30,46. Hei tāpiri, he maha ngā tikanga whai muri i te whakawhiti e whakararuraru ana i te whanaungatanga i waenga i te whakawhiti me te mahi ira. Ka taea e ngā rangahau proteomic te whakarato māramatanga ki te pānga o ngā PTM me te mahi pūmua, engari he wero anō hoki kei roto ko ngā tikanga iti-te-whakaputa, ngā ōwehenga tohu-ki-haruru teitei, me te whakataunga ngoikore.
I roto i tēnei horopaki, he nui te kaha o te ako i te āhua o te mitochondrial mā te whakamahi i te TEM me te MEL ki te whakautu i ngā pātai matua mō te whanaungatanga i waenga i ngā mahi me te mahi a te mitochondrial, me pēhea hoki tēnei e awe ai i te mate. Ko te mea nui, ka whakaratohia e te TEM he tikanga tika mō te ine i te āhua o te mitochondrial hei pito mutunga o te ngoikore me te hihiri o te mitochondrial51. Ka whakaratohia hoki e te MEL he tikanga tika mō te whakaatu i ngā huihuinga wehenga me te whakakotahitanga i roto i te taiao pūtau toru-ahu, e āhei ai te ine i te whakahounga mitochondrial hihiri ahakoa te kore o ngā huringa i roto i te whakaaturanga ira33. I konei ka whakanuia e mātou te whai huatanga o ngā tikanga whakaahua mitochondrial i roto i ngā mate mitochondrial tuarua. Ko ēnei mate ka tohuhia e te ahotea metabolic ngawari mau tonu e tohuhia ana e te whakahounga ngawari o ngā whatunga mitochondrial kaua ki te kino mitochondrial tere. Heoi, ko te utu mitochondrial e hiahiatia ana hei pupuri i te mitosis i raro i te ahotea mau tonu he hua mahi nui. I roto i te horopaki o te neuroscience, ko te mārama ake ki ēnei tikanga utu ka whakarato i ngā mōhiohio nui mō te neuropathology pleiotropic e pā ana ki te ngoikore o te mitochondrial.
Hei whakamutunga, e whakaatu ana ā mātou raraunga i te whai huatanga o ngā tikanga whakaahua hei mārama ki ngā hua mahi o ngā taunekeneke matatini i waenga i te whakaaturanga ira, ngā whakarerekētanga pūmua, me te mahi pūmua e whakahaere ana i ngā whanaketanga mitochondrial neuronal. I whakamahia e mātou te PPA hei tauira i te ngoikore o te mitochondrial i roto i te tauira pūtau neuronal hei whiwhi māramatanga ki te wāhanga mitochondrial o te ASD. Ko ngā pūtau SH-SY5Y i rongoatia ki te PPA i whakaatu i ngā huringa o te āhua mitochondrial: ka iti, ka porotaka te mitochondria, ā, kāore i tino tautuhia ngā cristae i te kitenga e te TEM. E whakaatu ana te tātaritanga MEL ka puta ēnei huringa i te wā kotahi me te pikinga o ngā huihuinga wehenga me te whakakotahitanga hei pupuri i te whatunga mitochondrial hei whakautu ki te ahotea pākia ngawari. Heoi anō, ka tino whakararuraru te PPA i te whakahaeretanga whakawhiti o te pākia me te homeostasis mitochondrial. I tautuhia e mātou ko cMYC, NRF1, TFAM, STOML2, me OPA1 hei kaiwhakahaere mitochondrial matua i whakararuraruhia e te ahotea PPA, ā, tera pea ka whai wāhi ki te takawaenga i ngā huringa i puta mai i te PPA i roto i te āhua me te mahi mitochondrial. Me whai rangahau ā muri ake nei hei whakaahua pai ake i ngā huringa wā i puta mai i te PPA i roto i te whakaaturanga ira me te mahi pūmua, te rohe, me ngā whakarerekētanga i muri i te whakamaoritanga. E whakaatu ana ā mātou raraunga i te uauatanga me te whakawhirinakitanga o ngā tikanga whakahaere e takawaenga ana i te urupare ahotea mitochondrial, ā, e whakaatu ana i te whai huatanga o te TEM me ētahi atu tikanga whakaahua mō ngā rangahau tikanga kua arotahi ake.
I hokona te raina pūtau SH-SY5Y (ECACC, 94030304-1VL) mai i Sigma-Aldrich. I whakatipuria ngā pūtau SH-SY5Y i roto i te ranunga matūkai reo/F-12 a Dulbecco (DMEM/F-12) me te L-glutamine (SC09411, ScienCell) i roto i ngā ipu 25 cm2 i tāpirihia ki te 20% fetal bovine serum (FBS) (10493106, ThermoFisher Scientific) me te 1% penicillin-streptomycin (P4333-20ML, Sigma-Aldrich) i te 37 °C, 5% CO2. I whakatōkia ngā pūtau ki raro ki te 80% te huihuinga mā te whakamahi i te 0.05% trypsin-EDTA (15400054, ThermoFisher Scientific), i poipoia ki te 300 g, ā, i pania ki te matotoru tata ki te 7 × 105 pūtau/ml. I whakahaerehia ngā whakamātautau katoa ki ngā pūtau SH-SY5Y kāore anō kia rerekē i waenganui i ngā ara 19–22. Ka tukuna te PPA hei NaP. Whakarewahia te paura NaP (CAS Nama 137-40-6, tātai matū C3H5NaO2, P5436-100G, Sigma-Aldrich) ki roto i te wai MilliQ mahana ki te kukū o te 1 M ka rongoa ki te 4 °C. I te rā o te maimoatanga, whakaranua tēnei otinga ki te 1 M PPA ki te 3 mM me te 5 mM PPA i roto i te reo kore-toto (DMEM/F-12 me te L-glutamine). Ko ngā kukū maimoatanga mō ngā whakamātautau katoa kāore he PPA (0 mM, mana whakahaere), 3 mM, me te 5 mM PPA. I whakahaerehia ngā whakamātautau i roto i te iti rawa e toru ngā tāruarua koiora.
I whakatōngia ngā pūtau SH-SY5Y ki roto i ngā ipu 25 cm5 i te tere o te 5.5 × 105 pūtau/ml, ā, i whakatipuria mō ngā hāora 24. I tāpirihia te maimoatanga PPA ki te ipu i mua i te 24 hāora o te whakatō. Kohia ngā pirepire pūtau mā te whai i ngā tikanga whakatipu kiko mammal noa (kua whakaahuahia i runga ake nei). Whakatārewatia anō te pirepire pūtau ki roto i te 100 µl 2.5% glutaraldehyde, 1× PBS, ā, rongoatia ki te 4 °C tae noa ki te tukatuka. I whakatōngia ngā pūtau SH-SY5Y mō te wā poto hei whakatō i ngā pūtau, hei tango i te 2.5% glutaraldehyde, 1× otinga PBS. Whakatārewatia anō te paru ki roto i te 4% agarose gel i whakaritea ki roto i te wai whakapūkara (ko te ōwehenga o te agarose ki te rōrahi paru he 1:1). I whakatakotoria ngā wahi agarose ki runga i ngā mata i runga i ngā pereti papatahi, ā, i pania ki te 1-hexadecene i mua i te whakatio i te pēhanga teitei. I whakatiohia ngā tauira ki roto i te 100% acetone maroke i te -90°C mō ngā hāora 24. Kātahi ka whakapiki ake i te pāmahana ki te -80°C, ā, ka tāpirihia he otinga o te 1% osmium tetroxide me te 0.1% glutaraldehyde. I rongoatia ngā tauira ki te -80°C mō ngā hāora 24. Whai muri i tēnei, ka piki haere te pāmahana ki te pāmahana rūma mō ētahi rā: mai i te –80 °C ki te –50 °C mō ngā hāora 24, ki te –30 °C mō ngā hāora 24, ki te –10 °C mō ngā hāora 24, ā, i te mutunga ki te pāmahana rūma.
I muri i te whakaritenga cryogenic, i whakakīia ngā tauira ki te kapia, ā, i hangaia ngā wāhanga tino angiangi (∼100 nm) mā te whakamahi i te Leica Reichert UltracutS ultramicrotome (Leica Microsystems). I pania ngā wāhanga ki te 2% uranyl acetate me te lead citrate. I tirohia ngā tauira mā te whakamahi i te karu hiko tuku FEI Tecnai 20 (ThermoFisher (i mua ko FEI), Eindhoven, Netherlands) e mahi ana i te 200 kV (kamupene tuku Lab6) me te kāmera Gatan CCD (Gatan, UK) kua rite ki te tātari pūngao Tridiem.
I roto i ia tāruarua hangarau, neke atu i te 24 ngā whakaahua pūtau kotahi i kohia, kia 266 katoa ngā whakaahua. I tātarihia ngā whakaahua katoa mā te whakamahi i te Rohe o te Hiahia (ROI) me te Mitochondria macro. Ko te macro mitochondrial e ahu mai ana i ngā tikanga kua whakaputaina17,31,32, ā, ka taea te tukatuka puranga aunoa-haurua o ngā whakaahua TEM i Fiji/ImageJ69. I roto i te poto: ka hurihia te whakaahua mā te whakamahi i te tango papamuri pōro hurihuri (60 te radius pika) me te tātari bandpass FFT (mā te whakamahi i ngā rohe o runga me raro o te pika 60 me te 8) me te pehi raina poutū me te manawanui aronga o te 5%. Ka whakawhāitihia aunoatia te whakaahua tukatuka mā te whakamahi i te pūnaha entropy mōrahi, ā, ka hangaia he kopare rua. I tangohia ngā rohe whakaahua e pā ana ki ngā ROI kua tīpakohia ā-ringa i roto i ngā whakaahua TEM mata, e whakaahua ana i ngā mitochondria me te tango i te kiriuhi plasma me ētahi atu rohe rerekētanga teitei. Mō ia ROI i tangohia, i tātarihia ngā matūriki rua neke atu i te 600 pika, ā, i inehia te horahanga matūriki, te taha, ngā tuaka matua me ngā tuaka iti, te whānui o te Feret, te porowhita, me te porowhita mā te whakamahi i ngā mahi ine kua hangaia ki roto o Fiji/ImageJ. Whai muri i a Merrill, Flippo, me Strack (2017), i tatauhia te horahanga 2, te ōwehenga āhua matūriki (ōwehenga tuaka matua ki te tuaka iti), me te tauwehenga āhua (FF) mai i ēnei raraunga, ko FF = taha 2/4pi x horahanga. Ka kitea te whakamāramatanga o te tātai tawhā i roto i a Merrill, Flippo, me Strack (2017). Kei te wātea ngā makaro kua whakahuatia i runga i a GitHub (tirohia te Tauākī Wātea Raraunga). I te toharite, tata ki te 5,600 ngā matūriki i tātarihia mō ia maimoatanga PPA, mō te tapeke o te tata ki te 17,000 ngā matūriki (kāore i whakaaturia ngā raraunga).
I whakanohoia ngā pūtau SH-SH5Y ki roto i ngā rihi ahurea 8-ruma (ThermoFisher, #155411) kia piri ai te kiri i te pō, kātahi ka whakatōkia ki te TMRE 1:1000 (ThermoFisher, #T669) me te Hoechst 33342 1:200 (Sigma-Aldrich, H6024). I tangohia ngā whakaahua mā te whakamahi i ngā taiaho 405 nm me te 561 nm mō te 10 meneti, ā, i tangohia ngā whakaahua mata hei puranga-z kei roto ngā whakaahua iti 10 me te taahiraa az o te 0.2 μm i waenga i ngā anga whakaahua i ngā wā 12 i muri mai. I kohia ngā whakaahua mā te whakamahi i te tūāpapa taumira-rahi Carl Zeiss LSM780 ELYRA PS.1 (Carl Zeiss, Oberkochen, Tiamana) mā te whakamahi i te arotahi LCI Plan Apochromate 100x/1.4 Oil DIC M27. I tātarihia ngā whakaahua i roto i te ImageJ mā te whakamahi i tētahi pipeline kua whakamāramahia i mua me te monomai ImageJ hei ine i ngā huihuinga hanumi me te wehenga, te toharite o ngā hanganga mitochondrial, me te rahinga mitochondrial toharite mō ia pūtau33. Kei te wātea ngā macros MEL i runga i te GitHub (tirohia te Tauākī Wātea Raraunga).
I whakatipuria ngā pūtau SH-SY5Y i roto i ngā pereti ono-pū i te kiato o te 0.3 × 106 pūtau/mL mō ngā hāora 24 i mua i te maimoatanga. I tangohia te RNA mā te whakamahi i te kawa Quick-RNA™ Miniprep (ZR R1055, Zymo Research) me ngā whakarerekētanga iti: tāpirihia he 300 μl o te RNA lysis buffer ki ia pū i mua i te tangohanga, ā, i wetewetehia ia tauira hei taahiraa whakamutunga me te 30 μl o te DNase/RNase elution. -wai kore utu. I tirohia ngā tauira katoa mō te rahinga me te kounga mā te whakamahi i te NanoDrop ND-1000 UV-Vis Spectrophotometer. I whiwhihia te pūmua katoa mai i ngā lysates pūtau mā te whakamahi i te 200 μl RIPA lysis buffer, ā, i inehia te kukū pūmua mā te whakamahi i te Bradford protein assay70.
I whakahaerehia te hanga cDNA mā te whakamahi i te Kete Hanga cDNA Tetro™ (BIO-65043, Meridian Bioscience) e ai ki ngā tohutohu a te kaihanga me ētahi whakarerekētanga. I hangaia te cDNA i roto i ngā tauhohenga 20-μl mā te whakamahi i te 0.7 ki te 1 μg o te RNA katoa. I tīpakohia ngā primer mai i ngā pepa kua whakaputaina i mua 42, 71, 72, 73, 74, 75, 76, 77, 78 (Ripanga S1) ā, i hangaia ngā probe e haere tahi ana mā te whakamahi i te taputapu PrimerQuest mai i Integrated DNA Technologies. I whakatauritea ngā ira katoa e hiahiatia ana ki te ira karihi B2M. I inehia te whakaaturanga ira o STOML2, NRF1, NFE2L2, TFAM, cMYC me OPA1 mā te RT-qPCR. I roto i te ranunga matua ko te LUNA Taq polymerase (M3003L, New England Biolabs), he 10 μM ngā parai whakamua me ngā parai whakamuri, te cDNA, me te wai taumata-PCR hei whakaputa i te rōrahi whakamutunga o te 10 μL mō ia tauhohenga. I inehia te whakaaturanga o ngā ira wehenga me te wehenga (DRP1, MFN1/2) mā te whakamahi i ngā whakamātautau maha TaqMan. I whakamahia te Luna Universal Probe qPCR Master Mix (M3004S, New England Biolabs) e ai ki ngā tohutohu a te kaihanga me ngā whakarerekētanga iti. Kei roto i te ranunga matua RT-qPCR maha te 1X LUNA Taq polymerase, he 10 μM ngā parai whakamua me ngā parai whakamuri, he 10 μM ngā parai, te cDNA, me te wai taumata-PCR, ā, i hua ake ai te rōrahi whakamutunga o te 20 μL mō ia tauhohenga. I mahia te RT-qPCR mā te whakamahi i te Rotor-Gene Q 6-plex (QIAGEN RG—tau rangatū: R0618110). Kei te whakaatuhia ngā āhuatanga huringa i roto i te Ripanga S1. I whakanuia ngā tauira cDNA katoa i roto i ngā tāruarua e toru, ā, i hangaia he pihi paerewa mā te whakamahi i tētahi raupapa o ngā whakarea tekau-whakarea. I tangohia ngā mea rerekē i roto i ngā tauira tāruarua me te paepae huringa paerewa rerekētanga (Ct) >0.5 mai i te tātari hei whakarite i te āheinga ki te whakaputa anō i ngā raraunga30,72. I tatauhia te whakaaturanga ira whanaunga mā te whakamahi i te tikanga 2-ΔΔCt79.
I konatunatua ngā tauira pūmua (60 μg) ki te Laemmli loading buffer i te ōwehenga 2:1, ā, i whakahaerehia ki runga i te 12% te tae kore-tae o te pūmua gel (Bio-Rad #1610184). I whakawhitia ngā pūmua ki te kiriuhi PVDF (polyvinylidene fluoride) (#170-84156, Bio-Rad) mā te whakamahi i te pūnaha Trans-Blot Turbo (#170-4155, Bio-Rad). I āraia te kiriuhi, ā, i whakatōkia ki ngā paturopi tuatahi e tika ana (OPA1, MFN1, MFN2, me te DRP1) (i whakananua ki te 1:1000) mō ngā hāora 48, ā, i muri mai ka whakatōkia ki ngā paturopi tuarua (1:10,000) mō te hāora 1. Kātahi ka whakaahuahia ngā kiriuhi mā te whakamahi i te Clarity Western ECL Substrate (#170-5061, Bio-Rad) ā, i tuhia mā te whakamahi i te pūnaha Bio-Rad ChemiDoc MP. I whakamahia te ImageLab putanga 6.1 mō te tātaritanga Western blot. Kei te Whakaahua S1 te tiēre taketake me te ira. Kei te Ripanga S2 ngā mōhiohio mō te paturopi.
Ka whakaaturia ngā huinga raraunga hei toharite me te hapa paerewa o te toharite (SEM) o te iti rawa e toru ngā tauira motuhake. I whakamātauria ngā huinga raraunga mō te ōritetanga mā te whakamahi i te whakamātautau Shapiro-Wilks (ki te kore e kīia tētahi atu mea) i mua i te whakaaro he tohatoha Gaussian me ngā paerewa rerekētanga ōrite me te haere tonu me ngā tātaritanga. Haunga te tātari i te huinga raraunga mā te whakamahi i te MEL LSD a Fisher (p < 0.05), te ANOVA kotahi-ara (te toharite maimoatanga vs. mana whakahaere), me te whakamātautau whakataurite maha a Dunnett hei whakatau i te hiranga (p < 0.05). Ka whakaaturia ngā uara p hiranga i roto i te kauwhata hei *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. I mahia, ā, i whakaputahia ngā tātaritanga tatauranga me ngā kauwhata katoa mā te whakamahi i te GraphPad Prism 9.4.0.
Kei te wātea ngā makaro Fiji/ImageJ mō te tātari whakaahua TEM ki te marea i runga i te GitHub: https://github.com/caaja/TEMMitoMacro. Kei te wātea hoki te makaro Mitochondrial Event Locator (MEL) ki te marea i runga i te GitHub: https://github.com/rensutheart/MEL-Fiji-Plugin.
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