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Hoʻohana ʻia ka waikawa propionic (PPA) e aʻo i ke kuleana o ka hana ʻino mitochondrial i nā maʻi neurodevelopmental e like me ka maʻi autism spectrum. Ua ʻike ʻia ʻo PPA e hoʻopilikia i ka biogenesis mitochondrial, metabolism, a me ka turnover. Eia nō naʻe, pilikia mau nā hopena o PPA ma ka dynamics mitochondrial, fission a me ka fusion ma muli o ke ʻano paʻakikī o kēia mau mīkini. Maanei, hoʻohana mākou i nā ʻenehana kiʻi quantitative complementary e noiʻi pehea e pili ai ka PPA i ka mitochondrial ultrastructure, morphology, a me dynamics i nā cell SH-SY5Y e like me neuron. Ua hoʻoulu ʻo PPA (5 mM) i ka emi nui ʻana o ka ʻāpana mitochondrial (p < 0.01), ke anawaena a me ke anawaena o Feret (p < 0.05), a me ka ʻāpana 2 (p < 0.01). Ua hōʻike ka loiloi Mitochondrial event locator i ka piʻi nui ʻana (p < 0.05) i nā hanana fission a me fusion, no laila e mālama ana i ka pono o ka pūnaewele mitochondrial ma lalo o nā kūlana kaumaha. Eia kekahi, ua hoʻemi nui ʻia ka hōʻike ʻana o ka mRNA o cMYC (p < 0.0001), NRF1 (p < 0.01), TFAM (p < 0.05), STOML2 (p < 0.0001) a me OPA1 (p < 0.05). 01). Hōʻike kēia i ka hana hou ʻana o ke ʻano mitochondrial, biogenesis a me nā dynamics e mālama i ka hana ma lalo o nā kūlana kaumaha. Hāʻawi kā mākou ʻikepili i ka ʻike hou i nā hopena o PPA ma nā dynamics mitochondrial a hōʻike i ka pono o nā ʻenehana kiʻi no ke aʻo ʻana i nā ʻano hoʻoponopono paʻakikī e pili ana i nā pane kaumaha mitochondrial.
He mau mea komo koʻikoʻi nā Mitochondria i nā ʻano hana cellular like ʻole ma mua o kā lākou mau kuleana maʻamau i ka hana ikehu a me ka biosynthesis. ʻO ka metabolism Mitochondrial kahi mea hoʻoponopono koʻikoʻi o ka hōʻailona calcium, metabolic a me redox homeostasis, inflammatory signaling, epigenetic modifications, cell proliferation, differentiation a me ka programmed cell death1. Ma ke ʻano kūikawā, he mea koʻikoʻi ka metabolism mitochondrial no ka ulu ʻana o nā neuronal, ke ola a me ka hana a ua pili nui ʻia i nā ʻano hōʻike like ʻole o ka neuropathology2,3,4.
I loko o nā makahiki he ʻumi i hala iho nei, ua kū mai ke kūlana metabolic ma ke ʻano he mea hoʻoponopono kikowaena o ka neurogenesis, differentiation, maturation a me ka plasticity5,6. I kēia manawa, ua lilo ka morphology a me ka dynamics mitochondrial i mau ʻāpana koʻikoʻi o ka mitosis, kahi hana ikaika e mālama ai i kahi waihona o nā mitochondria olakino i loko o nā cell. Hoʻoponopono ʻia nā dynamics mitochondrial e nā ala paʻakikī e pili ana i ka biogenesis mitochondrial a me bioenergetics a hiki i ka mitochondrial fission, fusion, transport and clearance7,8. ʻO ka hoʻopilikia ʻana o kekahi o kēia mau ʻano hoʻohui e hoʻopilikia i ka mālama ʻana i nā pūnaewele mitochondrial olakino a he hopena hana koʻikoʻi no ka neurodevelopment9,10. ʻOiaʻiʻo, ʻike ʻia ka dysregulation o ka mitochondrial dynamics i nā maʻi psychiatric, neurodegenerative a me neurodevelopmental he nui, me nā maʻi autism spectrum (ASD)11,12.
He maʻi neurodevelopmental heterogeneous ka ASD me kahi hoʻolālā genetic a me epigenetic paʻakikī. ʻAʻole i hoʻopaʻapaʻa ʻia ka hoʻoilina o ASD, akā ʻaʻole maopopo loa ke kumu molecular etiology. ʻO ka hōʻiliʻili ʻana i ka ʻikepili mai nā hiʻohiʻona preclinical, nā haʻawina lapaʻau, a me nā ʻikepili molecular multi-omics e hāʻawi i ka hōʻike e hoʻonui nei i ka hana mitochondrial ma ASD13,14. Ua hana mua mākou i kahi pale methylation DNA genome-ākea i loko o kahi cohort o nā maʻi me ASD a ua ʻike i nā genes methylated ʻokoʻa i hui pū ʻia ma nā ala metabolic mitochondrial15. Ma hope mai ua hōʻike mākou i ka methylation ʻokoʻa o nā mea hoʻoponopono waena o ka biogenesis mitochondrial a me ka dynamics, kahi i pili me ka hoʻonui ʻia o ka helu kope mtDNA a me ka hoʻololi ʻana i ka ʻikepili metabolic urinary ma ASD16. Hāʻawi kā mākou ʻikepili i ka hōʻike e hoʻonui ana i ka mitochondrial dynamics a me ka homeostasis e pāʻani i kahi kuleana koʻikoʻi i ka pathophysiology o ASD. No laila, ʻo ka hoʻomaikaʻi ʻana i ka ʻike mechanistic o ka pilina ma waena o ka mitochondrial dynamics, morphology, a me ka hana he pahuhopu nui ia o ka noiʻi mau ʻana i nā maʻi neurological i hōʻike ʻia e ka hana mitochondrial lua.
Hoʻohana pinepine ʻia nā ʻano hana molekala e aʻo ai i ke kuleana o nā genes kikoʻī i nā pane hoʻoluhi mitochondrial. Eia nō naʻe, hiki ke kaupalena ʻia kēia ʻano e ke ʻano multifaceted a me ka manawa o nā ʻano hana hoʻomalu mitotic. Eia kekahi, ʻo ka hōʻike ʻokoʻa o nā genes mitochondrial he hōʻailona pololei ʻole ia o nā loli hana, ʻoiai ʻo ka helu palena wale nō o nā genes i kālailai pinepine ʻia. No laila, ua hāpai ʻia nā ʻano hana pololei no ke aʻo ʻana i ka hana mitochondrial a me ka bioenergetics17. Pili loa ke ʻano mitochondrial i ka dynamics mitochondrial. He mea koʻikoʻi ke ʻano mitochondrial, ka pilina, a me ke ʻano no ka hana ʻana i ka ikehu a me ke ola mitochondrial a me ke kelepona5,18. Eia kekahi, ʻo nā ʻāpana like ʻole o ka mitosis e kālele ana i nā loli i ke ʻano mitochondrial, hiki ke lawelawe ma ke ʻano he mau hopena pono o ka hana ʻole o ka mitochondrial a hāʻawi i ke kumu no nā haʻawina mechanistic ma hope.
Hiki ke nānā pololei ʻia ke ʻano o ka mitochondrial me ka hoʻohana ʻana i ka microscopy electron transmission (TEM), e ʻae ana i ka noiʻi kikoʻī o ka ultrastructure cellular. Hōʻike pololei ʻo TEM i ke ʻano, ke ʻano a me ke ʻano o ka cristae mitochondrial ma ka hoʻonā ʻana o nā mitochondria pākahi, ma mua o ka hilinaʻi wale ʻana i ka transcription gene, ka hōʻike ʻana o ka protein a i ʻole nā ​​​​​​palapala hana mitochondrial i nā heluna cell17,19,20. Eia kekahi, hoʻomaʻamaʻa ʻo TEM i ke aʻo ʻana i nā pilina ma waena o mitochondria a me nā organelles ʻē aʻe, e like me ka endoplasmic reticulum a me nā autophagosomes, e pāʻani ana i nā kuleana koʻikoʻi i ka hana mitochondrial a me ka homeostasis21,22. No laila, ʻo kēia ka mea e hoʻolilo ai iā TEM i wahi hoʻomaka maikaʻi no ke aʻo ʻana i ka hana ʻino mitochondrial ma mua o ka nānā ʻana i nā ala kikoʻī a i ʻole nā ​​​​​​genes. I ka lilo ʻana o ka hana mitochondrial i mea pili loa i ka neuropathology, aia kahi pono maopopo e hiki ke aʻo pololei a me ka quantitative i ke ʻano mitochondrial a me nā dynamics i nā hiʻohiʻona neuronal in vitro.
Ma kēia ʻatikala, ke nānā nei mākou i nā dynamics mitochondrial i loko o kahi kumu hoʻohālike neuronal o ka hana ʻole o ka mitochondrial i ka maʻi autism spectrum. Ua hōʻike mua mākou i ka methylation differential o ka propionyl-CoA carboxylase beta (PCCB) ma ASD15, kahi subunit o ka mitochondrial propionyl-CoA carboxylase enzyme PCC. Ua ʻike ʻia ka dysregulation o PCC e hoʻoulu ai i ka hōʻiliʻili ʻawahia o nā propionyl derivatives, me ka propionic acid (PPA)23,24,25. Ua hōʻike ʻia ʻo PPA e hoʻopilikia i ka metabolism neuronal a hoʻololi i ke ʻano in vivo a he kumu hoʻohālike holoholona i hoʻokumu ʻia no ke aʻo ʻana i nā ʻano neurodevelopmental i komo i ka ASD26,27,28. Eia kekahi, ua hōʻike ʻia ʻo PPA e hoʻopilikia i ka hiki ke hoʻopili ʻia ka mitochondrial membrane, biogenesis a me ka hanu ʻana i loko o vitro a ua hoʻohana nui ʻia e hoʻohālike i ka hana ʻole o ka mitochondrial i nā neurons29,30. Eia naʻe, ʻaʻole maopopo ka hopena o ka hana ʻole o ka mitochondrial i hoʻokomo ʻia e PPA ma ke ʻano mitochondrial a me nā dynamics.
Hoʻohana kēia haʻawina i nā ʻano hana kiʻi hoʻohui e helu i nā hopena o ka PPA ma ke ʻano mitochondrial, dynamics, a me ka hana ma nā cell SH-SY5Y. ʻO ka mea mua, ua hoʻomohala mākou i kahi ʻano TEM e ʻike i nā loli i ke ʻano mitochondrial a me ka ultrastructure17,31,32. Ma muli o ke ʻano ikaika o mitochondria33, ua hoʻohana pū mākou i ka loiloi mitochondrial event localizer (MEL) e helu i nā loli i ke kaulike ma waena o nā hanana fission a me fusion, ka helu mitochondrial a me ka nui ma lalo o ke kaumaha PPA. ʻO ka hope loa, ua nānā mākou inā pili ka morphology a me ka dynamics mitochondrial me nā loli i ka hōʻike ʻana o nā genes i komo i ka biogenesis, fission, a me ka fusion. I ka hui pū ʻia, hōʻike kā mākou ʻikepili i ka paʻakikī o ka wehewehe ʻana i ka paʻakikī o nā ʻano hana e hoʻoponopono ana i ka dynamics mitochondrial. Hōʻike mākou i ka pono o TEM i ke aʻo ʻana i ka morphology mitochondrial ma ke ʻano he hopena convergent convergent o mitosis ma nā cell SH-SY5Y. Eia kekahi, hōʻike mākou e hāʻawi ka ʻikepili TEM i ka ʻike waiwai loa i ka wā e hui pū ʻia me nā ʻano hana kiʻi e hopu pū ana i nā hanana dynamic i ka pane ʻana i ke kaumaha metabolic. ʻO ka wehewehe hou ʻana o nā ʻano hana hoʻoponopono molecular e kākoʻo ana i ka neuronal cell mitosis e hāʻawi i ka ʻike koʻikoʻi i ka ʻāpana mitochondrial o ka ʻōnaehana nerve a me nā maʻi neurodegenerative.
No ka hoʻoulu ʻana i ke kaumaha mitochondrial, ua mālama ʻia nā pūnaewele SH-SY5Y me PPA me ka hoʻohana ʻana i ka 3 mM a me 5 mM sodium propionate (NaP). Ma mua o ka TEM, ua hoʻokomo ʻia nā laʻana i ka hoʻomākaukau laʻana cryogenic me ka hoʻohana ʻana i ka hoʻopaʻa ʻana a me ka hoʻopaʻa ʻana i ke kaomi kiʻekiʻe (Kiʻi 1a). Ua hoʻomohala mākou i kahi pipeline loiloi kiʻi mitochondrial automated e ana i ʻewalu mau palena morphological o nā heluna mitochondrial ma waena o ʻekolu mau replicates biological. Ua ʻike mākou ua hoʻololi nui ka mālama ʻana o PPA i ʻehā mau palena: kahi 2, kahi, perimeter, a me ke anawaena Feret (Kiʻi 1b–e). Ua emi nui ka wahi 2 me ka mālama ʻana o 3 mM a me 5 mM PPA (p = 0.0183 a me p = 0.002, kēlā me kēia) (Kiʻi 1b), ʻoiai ua emi nui ka wahi (p = 0.003), perimeter (p = 0.0106) a me ke anawaena Feret. He emi nui (p = 0.0172) ma ka hui mālama 5 mM i hoʻohālikelike ʻia me ka hui kaohi (Kiʻi 1c–e). Ua hōʻike ʻia nā hōʻemi koʻikoʻi ma ka ʻāpana a me ke anapuni he mitochondria liʻiliʻi a ʻoi aku ka poepoe o nā cell i mālama ʻia me 5 mM PPA, a ʻoi aku ka liʻiliʻi o ka elongated o kēia mau mitochondria ma mua o nā cell kaohi. He kūlike nō hoʻi kēia me ka emi nui ʻana o ke anawaena Feret, kahi palena kūʻokoʻa e hōʻike ana i ka emi ʻana o ka mamao nui loa ma waena o nā lihi o ka ʻāpana. Ua ʻike ʻia nā loli i ka ultrastructure o ka cristae: ua emi iki ka ʻike ʻia o ka cristae ma lalo o ka mana o ke kaumaha PPA (Fig. 1a, panel B). Eia naʻe, ʻaʻole i hōʻike maopopo nā kiʻi āpau i ka ultrastructure o ka cristae, no laila ʻaʻole i hana ʻia kahi loiloi quantitative o kēia mau loli. Hiki i kēia ʻikepili TEM ke hōʻike i ʻekolu mau hiʻohiʻona hiki: (1) hoʻonui ka PPA i ka fission a pale paha i ka fusion, e hoʻemi ana i ka nui o ka mitochondria e kū nei; (2) hoʻonui ʻia ka biogenesis e hana i nā mitochondria hou a liʻiliʻi a i ʻole (3) hoʻoulu i nā ʻano hana ʻelua i ka manawa like. ʻOiai ʻaʻole hiki ke hoʻokaʻawale ʻia kēia mau kūlana e TEM, hōʻike nā loli morphological koʻikoʻi i nā loli i ka homeostasis mitochondrial a me nā dynamics ma lalo o ke kaumaha PPA. Ma hope mai ua ʻimi mākou i nā palena hou aʻe e wehewehe hou i kēia mau dynamics a me nā ʻano hana e pili ana iā lākou.
Hoʻoponopono hou ka waikawa propionic (PPA) i ke ʻano mitochondrial. (a) Nā kiʻi microscopy electron transmission (TEM) e hōʻike ana e emi ana ka nui o ka mitochondrial a lilo ka mitochondria i mea liʻiliʻi a ʻoi aku ka poepoe me ka hoʻonui ʻana i ka mālama ʻana o ka PPA; 0 mM (ʻaʻole i mālama ʻia), 3 mM a me 5 mM, kēlā me kēia. Hōʻike nā pua ʻulaʻula i ka mitochondria. (b–e) Ua hoʻomākaukau ʻia nā pūnaewele SH-SY5Y i mālama ʻia me ka PPA no 24 h no TEM a ua kālailai ʻia nā hopena me ka hoʻohana ʻana iā Fiji/ImageJ. ʻEhā o nā ʻāpana ʻewalu i hōʻike i nā ʻokoʻa koʻikoʻi ma waena o nā pūnaewele kaohi (ʻaʻole i mālama ʻia, 0 mM PPA) a me nā pūnaewele i mālama ʻia (3 mM a me 5 mM PPA). (b) Māhele 2, (c) ʻĀpana, (d) Perimeter, (e) Anawaena Feret. Ua hoʻohana ʻia ka loiloi hoʻokahi ala o ka variance (mana vs. mālama ʻana) a me ka hoʻāʻo hoʻohālikelike lehulehu a Dunnett e hoʻoholo i nā ʻokoʻa koʻikoʻi (p < 0.05). Hōʻike nā helu ʻikepili i ka waiwai mitochondrial awelika no kēlā me kēia cell pākahi, a hōʻike nā kaha hewa i ka awelika ± SEM. Hōʻike nā ʻikepili i hōʻike ʻia iā n = 3, ma ka liʻiliʻi he 24 mau cell no kēlā me kēia replicate; ua kālailai ʻia he 266 mau kiʻi; * hōʻike iā p < 0.05, ** hōʻike iā p < 0.01.
No ka wehewehe hou ʻana i ke ʻano o ka pane ʻana o ka mitochondrial dynamics i ka PPA, ua hoʻoluʻu mākou i ka mitochondria me ka tetramethylrhodamine ethyl ester (TMRE) a hoʻohana i ka microscopy time-lapse a me ka MEL analysis e localize a helu i ka mitochondria ma hope o 24 mau hola ma 3 a me 5 mM PPA. Ka mālama ʻana i nā hanana fission a me fusion. (Kiʻi 2a). Ma hope o ka nānā ʻana o MEL, ua kālailai hou ʻia nā mitochondria e helu i ka helu o nā ʻano mitochondrial a me ko lākou nui awelika. Ua ʻike mākou i kahi piʻi liʻiliʻi akā koʻikoʻi i ka helu o nā hanana fission e hana ʻia ana ma 3 mM [4.9 ± 0.3 (p < 0.05)] i hoʻohālikelike ʻia me ka fission [5.6 ± 0.3 (p < 0.05) )] a me ka fusion [5.4 ± 0.5 (p < 0.05)] a me ka fusion [5.4 ± 0.5 (p < 0.05)] 0.05)] <0.05)] ua hoʻonui nui ʻia nā hanana ma 5 mM i hoʻohālikelike ʻia me ka mana (Kiʻi 3b). Ua piʻi nui ka nui o nā mitochondria ma 3 [32.6 ± 2.1 (p < 0.05)] a me 5 mM [34.1 ± 2.2 (p < 0.05)] (Kiʻi 3c), ʻoiai ʻaʻole i loli ka nui o kēlā me kēia ʻano mitochondrial (Kiʻi 3c). 3d). I ka hui pū ʻia ʻana, hōʻike kēia e lawelawe ana ka hana hou ʻana o nā dynamics mitochondrial ma ke ʻano he pane compensatory e mālama pono ana i ka pono o ka pūnaewele mitochondrial. ʻO ka hoʻonui ʻana o ka nui o nā hanana fission ma 3 mM PPA e hōʻike ana ʻo ka hoʻonui ʻana o ka helu mitochondrial ma muli o ka fission mitochondrial, akā no ka mea ʻo ka nui o ka mitochondrial awelika e noho mau ana i ka loli ʻole, ʻaʻole hiki ke kāpae ʻia ka biogenesis ma ke ʻano he pane compensatory hou. Eia nō naʻe, kūlike kēia mau ʻikepili me nā ʻano mitochondrial liʻiliʻi a puni i ʻike ʻia e TEM a hōʻike pū i nā loli koʻikoʻi i nā dynamics mitochondrial i hoʻokumu ʻia e PPA.
Hoʻoulu ka waikawa Propionic (PPA) i ka hoʻoponopono hou ʻana o ka mitochondrial ikaika e mālama i ka pono o ka pūnaewele. Ua hoʻoulu ʻia nā pūnaewele SH-SY5Y, mālama ʻia me 3 a me 5 mM PPA no 24 mau hola a ua hoʻoluʻu ʻia me TMRE a me Hoechst 33342 a ukali ʻia e ka loiloi MEL. (a) Nā kiʻi microscopy manawa-lapse e hōʻike ana i ke kala a me nā hoʻolālā ikaika binarized i ka manawa 2 (t2) no kēlā me kēia kūlana. Hoʻonui ʻia nā wahi i koho ʻia i hōʻike ʻia i kēlā me kēia kiʻi binary a hōʻike ʻia ma 3D ma ʻekolu mau manawa like ʻole (t1-t3) e hōʻike i ka dynamics i ka hala ʻana o ka manawa; hōʻike ʻia nā hanana fusion i ka ʻōmaʻomaʻo; hōʻike ʻia nā hanana fission i ka ʻōmaʻomaʻo. Hōʻike ʻia i ka ʻulaʻula. (b) Ka awelika o nā hanana ikaika no kēlā me kēia kūlana. (c) Ka awelika o nā ʻano mitochondrial no kēlā me kēia cell. (d) Ka nui awelika (µm3) o kēlā me kēia ʻano mitochondrial no kēlā me kēia cell. ʻO nā ʻikepili i hōʻike ʻia he ʻelele o n = 15 mau cell no kēlā me kēia hui lapaʻau. Hōʻike nā kaha hewa i hōʻike ʻia i ka awelika ± SEM, kaha unahi = 10 μm, * p < 0.05.
ʻO ka waikawa propionic (PPA) ke kumu o ka hoʻopau ʻana i ka transcriptional o nā genes e pili ana i ka dynamics mitochondrial. Ua mālama ʻia nā cell SH-SY5Y me 3 a me 5 mM PPA no 24 h. Ua hana ʻia ka helu ʻana o ka gene pili me ka hoʻohana ʻana iā RT-qPCR a ua hoʻonohonoho ʻia i B2M. Nā genes biogenesis mitochondrial (a) cMYC, (b) TFAM, (c) NRF1 a me (d) NFE2L2. Nā genes fusion a me ka fission mitochondrial (e) STOML2, (f) OPA1, (g) MFN1, (h) MFN2 a me (i) DRP1. Ua hoʻāʻo ʻia nā ʻokoʻa koʻikoʻi (p < 0.05) me ka hoʻohana ʻana i hoʻokahi ala ANOVA (mana vs. mālama) a me ka hoʻāʻo hoʻohālikelike lehulehu a Dunnett: * hōʻike p < 0.05, ** hōʻike p < 0.01, a **** hōʻike p < 0.0001. Hōʻike nā bars i ka awelika hōʻike ± SEM. Hōʻike nā ʻikepili i hōʻike ʻia iā n = 3 (STOML2, OPA1, TFAM), n = 4 (cMYC, NRF1, NFE2L2), a me n = 5 (MFN1, MFN2, DRP1) i nā kope olaola.
Hōʻike nā ʻikepili mai nā loiloi TEM a me MEL e hoʻololi ana ka PPA i ke ʻano a me ka dynamics mitochondrial. Eia naʻe, ʻaʻole hāʻawi kēia mau ʻano hana kiʻi i ka ʻike i nā ʻano hana kumu e hoʻokele nei i kēia mau kaʻina hana. No laila ua nānā mākou i ka hōʻike ʻana o ka mRNA o ʻeiwa mau mea hoʻoponopono koʻikoʻi o ka mitochondrial dynamics, biogenesis, a me ka mitosis i ka pane ʻana i ka mālama ʻana iā PPA. Ua helu mākou i ka cell myeloma oncogene (cMYC), nuclear respiratory factor (NRF1), mitochondrial transcription factor 1 (TFAM), NFE2-like transcription factor BZIP (NFE2L2), gastrin-like protein 2 (STOML2), optic nerve atrophy 1 (OPA1), Mitofusin 1 (MFN1), Mitofusin 2 (MFN2) a me ka dynamin-related protein 1 (DRP1) ma hope o 24 mau hola o ka mālama ʻana me 3 mM a me 5 mM PPA. Ua nānā mākou i ka 3 mM (p = 0.0053, p = 0.0415 a me p < 0.0001, kēlā me kēia) a me 5 mM (p = 0.0031, p = 0.0233, p < 0.0001) lapaʻau PPA. (Kiʻi 3a–c). ʻO ka emi ʻana o ka hōʻike ʻana o ka mRNA ua hilinaʻi ʻia e ka lāʻau lapaʻau: ua emi ka hōʻike ʻana o cMYC, NRF1 a me TFAM ma 5.7, 2.6 a me 1.9 mau manawa ma 3 mM, kēlā me kēia, a ma 11.2, 3 a me 2.2 mau manawa ma 5 mM. I ka hoʻohālikelike ʻana, ʻaʻole i loli ka gene biogenesis redox waena ʻo NFE2L2 ma kekahi ʻano o ka PPA, ʻoiai ua ʻike ʻia kahi ʻano like o ka hōʻike ʻana e hilinaʻi ana i ka lāʻau lapaʻau (Kiʻi 3d).
Ua nānā pū mākou i ka hōʻike ʻana o nā genes kuʻuna i komo i ka hoʻoponopono ʻana o ka fission a me ka fusion. Manaʻo ʻia ʻo STOML2 e komo i ka fusion, mitophagy a me ka biogenesis, a ua hoʻemi nui ʻia kona hōʻike ʻana (p < 0.0001) e 3 mM (2.4-fold loli) a me 5 mM (2.8-fold loli) PPA (Kiʻi 1). 3d). Pēlā nō, ua hoʻemi ʻia ka hōʻike ʻana o ka gene fusion OPA1 ma 3 mM (1.6-fold loli) a me 5 mM (1.9-fold loli) PPA (p = 0.006 a me p = 0.0024, kēlā me kēia) (Kiʻi 3f). Eia naʻe, ʻaʻole mākou i ʻike i nā ʻokoʻa koʻikoʻi i ka hōʻike ʻana o nā genes fusion MFN1, MFN2 a i ʻole fission gene DRP1 ma lalo o ke kaumaha PPA 24-h (Kiʻi 3g–i). Eia kekahi, ua ʻike mākou ʻaʻole i loli nā pae o nā protein fusion a me fission ʻehā (OPA1, MFN1, MFN2 a me DRP1) ma lalo o nā kūlana like (Kiʻi 4a-d). He mea nui e hoʻomaopopo he hōʻike kēia mau ʻikepili i hoʻokahi kiko i ka manawa a ʻaʻole paha e hōʻike i nā loli i ka hōʻike ʻana o ka protein a i ʻole nā ​​​​pae hana i nā wā mua o ke kaumaha PPA. Eia nō naʻe, ʻo ka emi nui ʻana o ka hōʻike ʻana o cMYC, NRF1, TFAM, STOML2, a me OPA1 e hōʻike ana i ka dysregulation transcriptional koʻikoʻi o ka metabolism mitochondrial, biogenesis, a me dynamics. Eia kekahi, hōʻike kēia mau ʻikepili i ka pono o nā ʻenehana kiʻi e aʻo pololei i nā loli hope-state i ka hana mitochondrial.
ʻAʻole i loli nā pae protein fusion a me ka fission factor ma hope o ka mālama ʻana i ka propionic acid (PPA). Ua mālama ʻia nā cell SH-SY5Y me 3 a me 5 mM PPA no 24 hola. Ua helu ʻia nā pae protein e ka loiloi Western blot, a ua hoʻohālikelike ʻia nā pae hōʻike i ka protein holoʻokoʻa. Hōʻike ʻia ka awelika o ka hōʻike protein a me nā Western blots hōʻike o ka target a me ka protein holoʻokoʻa. a - OPA1, b - MFN1, c - MFN2, d - DRP1. Hōʻike nā pā i ka awelika ± SEM, a ʻo ka ʻikepili i hōʻike ʻia he hōʻike ia o n = 3 mau replicates biological. Ua hana ʻia nā hoʻohālikelike he nui (p < 0.05) me ka hoʻohana ʻana i ka loiloi hoʻokahi ala o ka variance a me ka hoʻāʻo ʻana o Dunnett. Hōʻike ʻia ka gel a me ka blot kumu ma ke Kiʻi S1.
Pili ka hana ʻino o ka mitochondrial me nā maʻi multisystem mai nā maʻi metabolic, cardiovascular a me nā muscular a hiki i nā maʻi neurological1,10. Nui nā maʻi neurodegenerative a me nā neurodegenerative e pili ana me ka hana ʻino mitochondrial, e hōʻike ana i ke koʻikoʻi o kēia mau organelles i ke ola o ka lolo. ʻO kēia mau maʻi e komo pū ana me ka maʻi Parkinson, ka maʻi Alzheimer a me ASD3,4,18. Eia nō naʻe, he paʻakikī ke komo ʻana i ka ʻiʻo lolo e aʻo ai i kēia mau maʻi, ʻoi aku hoʻi ma ka pae mechanistic, e hana ana i nā ʻōnaehana hoʻohālike cellular i mea koho ʻē aʻe. Ma kēia haʻawina, hoʻohana mākou i kahi ʻōnaehana hoʻohālike cellular e hoʻohana ana i nā cell SH-SY5Y i mālama ʻia e PPA e hana hou i ka hana ʻino mitochondrial i ʻike ʻia i nā maʻi neuronal, ʻoi aku hoʻi nā maʻi autism spectrum. ʻO ka hoʻohana ʻana i kēia kumu hoʻohālike PPA e aʻo ai i nā dynamics mitochondrial i loko o nā neurons e hāʻawi i ka ʻike i ka etiology o ASD.
Ua noiʻi mākou i ka hiki ke hoʻohana i ka TEM e nānā i nā loli i ke ʻano mitochondrial. He mea nui e hoʻomaopopo he pono e hoʻohana pono ʻia ka TEM e hoʻonui i kona pono. ʻO ka hoʻomākaukau ʻana o nā cryo-specimens e ʻae ai i ka mālama maikaʻi ʻana i nā ʻano neuronal ma o ka hoʻopaʻa ʻana i nā ʻāpana cellular a me ka hōʻemi ʻana i ke kūkulu ʻia ʻana o nā artifacts34. E kūlike me kēia, ua ʻike mākou he mau organelles subcellular i hoʻopaʻa ʻia nā cell SH-SY5Y e like me neuron a me ka mitochondria elongated (Fig. 1a). Hōʻike kēia i ka pono o nā ʻano hana hoʻomākaukau cryogenic no ke aʻo ʻana i ke ʻano mitochondrial i nā hiʻohiʻona cell neuronal. ʻOiai he mea koʻikoʻi nā ana quantitative no ka nānā pono ʻana i ka ʻikepili TEM, ʻaʻohe manaʻo like e pili ana i nā ʻano kikoʻī e pono e ana ʻia e hōʻoia i nā loli morphological mitochondrial. Ma muli o ka nui o nā haʻawina i nānā pono i ka morphology mitochondrial17,31,32, ua hoʻomohala mākou i kahi pipeline loiloi kiʻi mitochondrial automated e ana i ʻewalu mau ʻano morphological, ʻo ia hoʻi: wahi, wahi2, lakio hiʻohiʻona, perimeter, circularity, degree , Feret diameter. a me ka poepoe.
I waena o lākou, ua hoʻemi nui ka PPA i ka ʻāpana 2, ka ʻāpana, ka perimeter, a me ke anawaena Feret (Kiʻi 1b-e). Ua hōʻike kēia ua lilo ka mitochondria i mea liʻiliʻi a ʻoi aku ka poepoe, kahi e kūlike me nā haʻawina mua e hōʻike ana i ka emi ʻana o ka ʻāpana mitochondrial ma hope o 72 mau hola o ke kaumaha mitochondrial i hoʻokumu ʻia e PPA30. Hiki i kēia mau hiʻohiʻona morphological ke hōʻike i ka fission mitochondrial, kahi hana e pono ai e hoʻokaʻawale i nā ʻāpana i hōʻino ʻia mai ka pūnaewele mitochondrial e hoʻolaha i ko lākou hōʻino ʻana ma o mitophagy35,36,37. Ma ka ʻaoʻao ʻē aʻe, ʻo ka emi ʻana o ka nui mitochondrial awelika e pili ana paha me ka hoʻonui ʻia o ka biogenesis, kahi e hopena ai i ka hoʻokumu ʻia ʻana o nā mitochondria nascent liʻiliʻi. ʻO ka hoʻonui ʻia ʻana o ka fission a i ʻole biogenesis e hōʻike ana i kahi pane compensatory e mālama i ka mitosis e kūʻē i ke kaumaha mitochondrial. Eia nō naʻe, ʻaʻole hiki ke hoʻokaʻawale ʻia ka emi ʻana o ka ulu ʻana o ka mitochondrial, ka fusion i hoʻopilikia ʻia, a i ʻole nā ​​​​​​kūlana ʻē aʻe.
ʻOiai ʻo nā kiʻi hoʻonā kiʻekiʻe i hana ʻia e TEM e ʻae i ka hoʻoholo ʻana i nā ʻano morphological ma ka pae o kēlā me kēia mitochondria, hana kēia ʻano hana i nā kiʻi ʻelua-dimensional i hoʻokahi manawa. No ke aʻo ʻana i nā pane dynamic i ke kaumaha metabolic, ua hoʻoluʻu mākou i ka mitochondria me TMRE a hoʻohana i ka microscopy time-lapse me ka nānā ʻana o MEL, kahi e ʻae ai i ka nānā ʻana o 3D kiʻekiʻe o nā loli i ka pūnaewele mitochondrial i ka hala ʻana o ka manawa33,38. Ua ʻike mākou i nā loli maalea akā koʻikoʻi i ka dynamics mitochondrial ma lalo o ke kaumaha PPA (Kiʻi 2). Ma 3 mM, ua piʻi nui ka nui o nā hanana fission, ʻoiai ua like nā hanana fusion me ka mana. Ua ʻike ʻia ka piʻi ʻana o ka helu o nā hanana fission a me fusion ma 5 mM PPA, akā ua like kēia mau loli me ka like, e hōʻike ana ua hiki i ka fission a me ka fusion kinetics ke kaulike ma nā ʻano kiʻekiʻe (Kiʻi 2b). Ua noho mau ka nui mitochondrial awelika ma 3 a me 5 mM PPA, e hōʻike ana ua mālama ʻia ka pono o ka pūnaewele mitochondrial (Kiʻi 2d). Hōʻike kēia i ka hiki o nā pūnaewele mitochondrial dynamic ke pane i ke kaumaha metabolic akahai e mālama pono i ka homeostasis me ka ʻole o ka hoʻoulu ʻana i ka ʻāpana pūnaewele. Ma 3 mM PPA, ua lawa ka hoʻonui ʻia o ka fission e hoʻolaha i ka hoʻololi ʻana i kahi kaulike hou, akā koi ʻia ka hoʻoponopono kinetic hohonu hou aʻe i ka pane ʻana i ke kaumaha i hoʻokumu ʻia e nā kiʻekiʻe kiʻekiʻe o ka PPA.
Ua piʻi ka nui o nā mitochondria ma nā ʻano koʻikoʻi PPA ʻelua, akā ʻaʻole i loli nui ka nui o ka mitochondrial awelika (Kiʻi 2c). Hiki paha kēia ma muli o ka hoʻonui ʻia ʻana o ka biogenesis a i ʻole ka hoʻonui ʻia ʻana o ka mahele; akā naʻe, i ka loaʻa ʻole o ka emi nui ʻana o ka nui o ka mitochondrial awelika, ʻoi aku ka nui o ka piʻi ʻana o ka biosynthesis. Eia nō naʻe, kākoʻo ka ʻikepili ma ke Kiʻi 2 i ke ola ʻana o ʻelua mau ʻano hana compensatory: ka hoʻonui ʻana i ka nui o nā hanana fission, e kūlike me ka upregulation o ka mitochondrial fission, a me ka hoʻonui ʻana i ka nui o nā hanana, e kūlike me ka mitochondrial biogenesis. I ka hopena, hiki i ka uku hoʻololi no ke kaumaha iki ke komo pū me nā kaʻina hana like e pili ana i ka fission, fusion, biogenesis, a me ka mitophagy. ʻOiai ua hōʻike nā mea kākau mua e hoʻonui ka PPA i ka mitosis30,39 a me ka mitophagy29, hāʻawi mākou i nā hōʻike no ka hana hou ʻana o ka mitochondrial fission a me ka fusion dynamics i ka pane ʻana i ka PPA. Hōʻoia kēia mau ʻikepili i nā loli morphological i ʻike ʻia e TEM a hāʻawi i ka ʻike hou aku i nā ʻano hana e pili ana me ka hana ʻole o ka mitochondrial i hoʻokomo ʻia e PPA.
No ka mea, ʻaʻole i hāʻawi ka loiloi TEM a me MEL i nā hōʻike pololei o nā ʻano hoʻoponopono gene ma lalo o nā loli morphological i ʻike ʻia, ua nānā mākou i ka hōʻike ʻana o ka RNA o nā genes i komo i ka metabolism mitochondrial, biogenesis, a me dynamics. ʻO ka cMYC proto-oncogene kahi mea hoʻololi i komo i ka hoʻoponopono ʻana o mitochondria, glycolysis, amino acid a me ka metabolism fatty acid40. Eia kekahi, ua ʻike ʻia ʻo cMYC e hoʻoponopono i ka hōʻike ʻana o kahi kokoke i 600 mau genes mitochondrial i komo i ka mitochondrial transcription, translation, a me ka hui paʻakikī, me NRF1 a me TFAM41. ʻO NRF1 a me TFAM ʻelua mau mea hoʻoponopono kikowaena o ka mitosis, e hana ana ma hope o PGC-1α e hoʻāla i ka replication mtDNA. Hoʻāla ʻia kēia ala e ka cAMP a me AMPK signaling a he naʻau ia i ka hoʻolilo ʻana i ka ikehu a me ke kaumaha metabolic. Ua nānā pū mākou iā NFE2L2, kahi mea hoʻoponopono redox o ka mitochondrial biogenesis, e hoʻoholo ai inā paha e hoʻopili ʻia nā hopena o PPA e ka oxidative stress.
ʻOiai ua noho mau ka hoʻololi ʻana o ka hōʻike ʻana o NFE2L2, ua ʻike mākou i ka emi mau ʻana o ka hōʻike ʻana o cMYC, NRF1 a me TFAM ma hope o 24 mau hola o ka mālama ʻana me 3 mM a me 5 mM PPA (Kiʻi 3a-c). Ua hōʻike mua ʻia ka emi ʻana o ka hōʻike ʻana o cMYC ma ke ʻano he pane i ka pilikia mitochondrial42, a ma ke ʻano ʻē aʻe, hiki i ka emi ʻana o ka hōʻike ʻana o cMYC ke hana i ka hana ʻino mitochondrial ma o ka hoʻoponopono hou ʻana i ka metabolism mitochondrial, ka pilina pūnaewele, a me ka polarization membrane43. ʻO ka mea hoihoi, pili pū ʻo cMYC i ka hoʻoponopono ʻana o ka fission mitochondrial a me ka fusion42,43 a ua ʻike ʻia e hoʻonui i ka phosphorylation DRP1 a me ka localization mitochondrial i ka wā o ka mahele cell44, a me ka hoʻoponopono ʻana i ka hoʻoponopono morphological mitochondrial i loko o nā cell kumu neuronal45. ʻOiaʻiʻo, hōʻike nā fibroblasts cMYC-deficient i ka nui mitochondrial i hoʻemi ʻia, e kūlike me nā loli i hoʻokumu ʻia e ke kaumaha PPA43. Hōʻike kēia mau ʻikepili i kahi pilina hoihoi akā ʻaʻole maopopo ma waena o cMYC a me nā dynamics mitochondrial, e hāʻawi ana i kahi pahuhopu hoihoi no nā haʻawina e hiki mai ana o ka hoʻoponopono hou ʻana i hoʻokumu ʻia e ka PPA.
ʻO ka hōʻemi ʻana o NRF1 a me TFAM e kūlike me ke kuleana o cMYC ma ke ʻano he mea hoʻoulu transcriptional koʻikoʻi. Kūlike pū kēia mau ʻikepili me nā haʻawina mua i nā cell cancer colon kanaka e hōʻike ana ua hōʻemi ka PPA i ka hōʻike ʻana o ka NRF1 mRNA i 22 mau hola, kahi i pili me ka hoʻopau ʻana o ATP a me ka hoʻonui ʻana o ROS46. Ua hōʻike pū kēia mau mea kākau ua piʻi ka hōʻike ʻana o TFAM i 8.5 mau hola akā ua hoʻi i nā pae kumu i 22 mau hola. I ka hoʻohālikelike ʻana, ua hōʻike ʻo Kim et al. (2019) ua emi nui ka hōʻike ʻana o TFAM mRNA ma hope o 4 h o ke kaumaha PPA i nā cell SH-SY5Y; eia naʻe, ma hope o 72 mau hola, ua hoʻonui nui ʻia ka hōʻike ʻana o ka protein TFAM a ua hoʻonui nui ʻia ka helu kope mtDNA. No laila, ʻo ka emi ʻana o ka helu o nā genes biogenesis mitochondrial a mākou i ʻike ai ma hope o 24 mau hola ʻaʻole ia e kāpae i ka hiki ke pili ka hoʻonui ʻana o ka helu o mitochondria me ka hoʻoulu ʻana o ka biogenesis i nā manawa mua. Ua hōʻike ʻia nā haʻawina mua e hoʻonui nui ana ka PPA i ka PGC-1α mRNA a me ka protein i loko o nā cell SH-SY5Y i 4 mau hola 30 mau minuke, ʻoiai ʻo ka propionic acid e hoʻonui i ka biogenesis mitochondrial i loko o nā hepatocytes bipi ma o PGC-1α i 12 mau hola 39 mau minuke. ʻO ka mea hoihoi, ʻaʻole wale ʻo PGC-1α he mea hoʻoponopono transcriptional pololei o NRF1 a me TFAM, akā ua hōʻike ʻia hoʻi e hoʻoponopono i ka hana o MFN2 a me DRP1 ma o ka hoʻoponopono ʻana i ka fission a me ka fusion47. I ka hui pū ʻia ʻana, hōʻike kēia i ka pili kokoke o nā ʻano hana e hoʻoponopono ana i nā pane compensatory mitochondrial i hoʻokumu ʻia e PPA. Eia kekahi, hōʻike kā mākou ʻikepili i ka dysregulation koʻikoʻi o ka hoʻoponopono transcriptional o ka biogenesis a me ka metabolism ma lalo o ke kaumaha PPA.
ʻO nā genes STOML2, OPA1, MFN1, MFN2 a me DRP1 kekahi o nā mea hoʻoponopono waena o ka mitochondrial fission, fusion a me dynamics37,48,49. Nui nā genes ʻē aʻe i komo i ka mitochondrial dynamics, akā naʻe, ua ʻike mua ʻia ʻo STOML2, OPA1 a me MFN2 he ʻokoʻa ka methylation i loko o nā cohorts ASD,16 a ua hōʻike kekahi mau haʻawina kūʻokoʻa i nā loli i kēia mau mea transcription i ka pane ʻana i ka mitochondrial stress50,51. 52. Ua hoʻemi nui ʻia ka hōʻike ʻana o OPA1 a me STOML2 e ka 3 mM a me 5 mM PPA treatment (Fig. 3e, f). ʻO OPA1 kekahi o nā mea hoʻoponopono maʻamau o ka mitochondrial fusion ma o ka launa pū ʻana me MFN1 a me 2 a he kuleana ko ia i ka hoʻoponopono hou ʻana o cristae a me ka mitochondrial morphology53. ʻAʻole maopopo ka kuleana pololei o STOML2 i ka mitochondrial dynamics, akā hōʻike nā hōʻike he kuleana ko ia i ka mitochondrial fusion, biogenesis, a me mitophagy.
Ke komo nei ʻo STOML2 i ka mālama ʻana i ka hui ʻana o ka hanu mitochondrial a me ka hoʻokumu ʻana o nā complexes chain hanu54,55 a ua hōʻike ʻia e hoʻololi nui i nā ʻano metabolic o nā cell cancer56. Ua hōʻike ʻia nā haʻawina e hoʻolaha ana ʻo STOML2 i ka hiki ke hoʻopili ʻia ka mitochondrial membrane a me ka biogenesis ma o ka launa pū ʻana me BAN a me cardiolipin 55, 57, 58. Eia kekahi, ua hōʻike ʻia nā haʻawina kūʻokoʻa e hoʻoponopono ka launa pū ʻana ma waena o STOML2 a me PINK1 i ka mitophagy59,60. ʻIke ʻia, ua hōʻike ʻia ʻo STOML2 e launa pū pololei me a hoʻopaʻa iā MFN2 a he kuleana koʻikoʻi hoʻi i ka hoʻopaʻa ʻana i nā isoforms OPA1 lōʻihi ma ka pale ʻana i ka protease kuleana no ka hoʻohaʻahaʻa ʻana o OPA153,61,62. ʻO ka emi ʻana o ka hōʻike ʻana o STOML2 i ʻike ʻia i nā hopena PPA e hoʻolilo i kēia mau protein fusion i mea maʻalahi i ka hoʻohaʻahaʻa ʻia ma o nā ala ubiquitin- a me proteasome-dependent48. ʻOiai ʻaʻole maopopo ka hana pololei o STOML2 a me OPA1 i ka pane ikaika i ka PPA, ʻo ka emi ʻana o ka hōʻike ʻana o kēia mau genes fusion (Kiʻi 3) e hoʻopilikia paha i ke kaulike ma waena o ka fission a me ka fusion a alakaʻi i ka emi ʻana o ka nui mitochondrial (Kiʻi 3). 1).
Ma ka ʻaoʻao ʻē aʻe, ua noho mau ka hoʻololi ʻana o ka protein OPA1 ma hope o 24 mau hola, ʻoiai ʻaʻole i loli nui nā pae mRNA a me ka protein o MFN1, MFN2 a i ʻole DRP1 ma hope o ka mālama ʻana iā PPA (Kiʻi 3g-i, Kiʻi 4). Hōʻike paha kēia ʻaʻohe loli i ka hoʻoponopono ʻana o kēia mau mea i pili i ka mitochondrial fusion a me ka fission. Eia nō naʻe, he mea kūpono ke hoʻomaopopo ʻana ua hoʻoponopono ʻia kēlā me kēia o kēia mau genes ʻehā e nā hoʻololi posttranscriptional (PTMs) e kāohi ana i ka hana protein. Loaʻa iā OPA1 ʻewalu mau ʻano splice ʻē aʻe i ʻoki ʻia me ka proteolytically i loko o mitochondria e hana i ʻelua isoforms ʻokoʻa 63. ʻO ke kaulike ma waena o nā isoforms lōʻihi a me ka pōkole e hoʻoholo hope loa i ke kuleana o OPA1 i ka mitochondrial fusion a me ka mālama ʻana i ka pūnaewele mitochondrial64. Hoʻoponopono ʻia ka hana DRP1 e ka phosphorylation calcium/calmodulin-dependent protein kinase II (CaMKII), ʻoiai ʻo ka hoʻohaʻahaʻa ʻana o DRP1 e hoʻoponopono ʻia e ka ubiquitination a me SUMOylation65. ʻO ka mea hope loa, ʻo DRP1 a me MFN1/2 he GTPases, no laila hiki ke hoʻopili ʻia ka hana e ka wikiwiki o ka hana ʻana o GTP i loko o mitochondria 66. No laila, ʻoiai ke mau nei ka hōʻike ʻana o kēia mau protein, ʻaʻole paha kēia e hōʻike i ka hana protein i hoʻololi ʻole ʻia a i ʻole ka localization67,68. ʻOiaʻiʻo, ʻo nā repertoire protein PTM e kū nei e lawelawe pinepine ma ke ʻano he laina mua o ka pale e kuleana no ka hoʻoponopono ʻana i nā pane koʻikoʻi koʻikoʻi. I ke alo o ke kaumaha metabolic waena i kā mākou kumu hoʻohālike, he mea hiki paha iā PTM ke hoʻolaha i ka hoʻonui ʻia o ka hana o ka fusion a me nā protein fission e hoʻihoʻi pono i ka pono mitochondrial me ka ʻole o ka koi ʻana i ka hoʻoulu hou ʻana o kēia mau genes ma ka mRNA a i ʻole ka pae protein.
I ka hui pū ʻia ʻana, hōʻike ka ʻikepili ma luna i ka hoʻoponopono paʻakikī a me ka manawa e pili ana i ke ʻano o ka mitochondrial a me nā pilikia o ka wehewehe ʻana i kēia mau ʻano hana. No ke aʻo ʻana i ka hōʻike ʻana o ka gene, pono mua e ʻike i nā genes i manaʻo ʻia ma ke ala. Eia nō naʻe, hōʻike kā mākou ʻikepili ʻaʻole pane like nā genes ma ke ala like i ke kaumaha like. ʻO ka ʻoiaʻiʻo, ua hōʻike nā haʻawina mua e hōʻike paha nā genes like ʻole ma ke ala like i nā ʻano pane manawa like ʻole30,46. Eia kekahi, aia nā ʻano hana post-transcriptional paʻakikī e hoʻopilikia i ka pilina ma waena o ka transcription a me ka hana gene. Hiki i nā haʻawina Proteomic ke hāʻawi i ka ʻike i ka hopena o nā PTM a me ka hana protein, akā ke hōʻike nei hoʻi lākou i nā pilikia e pili ana i nā ʻano hana haʻahaʻa, nā lakio signal-to-noise kiʻekiʻe, a me ka hoʻonā maikaʻi ʻole.
Ma kēia ʻano, ʻo ke aʻo ʻana i ke ʻano mitochondrial me ka hoʻohana ʻana iā TEM a me MEL he mana nui ia e hoʻoponopono i nā nīnau kumu e pili ana i ka pilina ma waena o ka mitochondrial dynamics a me ka hana a pehea e hoʻohuli ai kēia i ka maʻi. ʻO ka mea nui loa, hāʻawi ʻo TEM i kahi ʻano pololei no ke ana ʻana i ke ʻano mitochondrial ma ke ʻano he hopena hui o ka hana ʻole o ka mitochondrial a me ka dynamics51. Hāʻawi pū ʻo MEL i kahi ʻano pololei no ka nānā ʻana i nā hanana fission a me fusion i loko o kahi ʻano cellular ʻekolu-dimensional, e ʻae ana i ka helu ʻana o ka hoʻoponopono hou ʻana o ka mitochondrial dynamic ʻoiai me ka ʻole o nā loli i ka hōʻike ʻana o ka gene33. Maanei mākou e hōʻike nei i ka pono o nā ʻenehana kiʻi mitochondrial i nā maʻi mitochondrial lua. ʻO ka maʻamau, ʻike ʻia kēia mau maʻi e ke kaumaha metabolic akahai mau i hōʻike ʻia e ka hoʻoponopono hou ʻana o nā pūnaewele mitochondrial ma mua o ka hōʻino mitochondrial acute. Eia nō naʻe, ʻo ka uku mitochondrial e pono ai e mālama i ka mitosis ma lalo o ke kaumaha mau he hopena hana koʻikoʻi. Ma ke ʻano o ka neuroscience, ʻo ka hoʻomaopopo maikaʻi ʻana i kēia mau ʻano hana compensatory e hāʻawi i ka ʻike koʻikoʻi e pili ana i ka neuropathology pleiotropic e pili ana me ka hana ʻole o ka mitochondrial.
ʻO ka mea hope loa, hōʻike kā mākou ʻikepili i ka pono o nā ʻano hana kiʻi no ka hoʻomaopopo ʻana i nā hopena hana o nā pilina paʻakikī ma waena o ka hōʻike ʻana o ka gene, nā hoʻololi protein, a me ka hana protein e kāohi ana i ka neuronal mitochondrial dynamics. Ua hoʻohana mākou iā PPA e hoʻohālike i ka hana ʻino mitochondrial i loko o kahi kumu hoʻohālike cell neuronal e loaʻa ai ka ʻike i ka ʻāpana mitochondrial o ASD. Ua hōʻike nā cell SH-SY5Y i mālama ʻia me PPA i nā loli i ke ʻano mitochondrial: ua liʻiliʻi a poepoe ka mitochondria, a ua wehewehe maikaʻi ʻole ʻia ka cristae i ka wā i nānā ʻia e TEM. Hōʻike ka loiloi MEL e hana pū ana kēia mau loli me ka hoʻonui ʻana i nā hanana fission a me fusion e mālama i ka pūnaewele mitochondrial i ka pane ʻana i ke kaumaha metabolic akahai. Eia kekahi, hoʻopilikia nui ka PPA i ka hoʻoponopono transcriptional o ka metabolism mitochondrial a me ka homeostasis. Ua ʻike mākou iā cMYC, NRF1, TFAM, STOML2, a me OPA1 ma ke ʻano he mau mea hoʻoponopono mitochondrial koʻikoʻi i hoʻopilikia ʻia e ke kaumaha PPA a hiki ke pāʻani i kahi kuleana i ka hoʻoponopono ʻana i nā loli i hoʻokomo ʻia e PPA i ke ʻano a me ka hana mitochondrial. Pono nā haʻawina e hiki mai ana e wehewehe maikaʻi i nā loli manawa i hoʻokomo ʻia e PPA i ka hōʻike ʻana o ka gene a me ka hana protein, localization, a me nā hoʻololi ma hope o ka unuhi ʻana. Hōʻike kā mākou ʻikepili i ka paʻakikī a me ka hilinaʻi like ʻana o nā ʻōnaehana hoʻoponopono e hoʻopili ana i ka pane koʻikoʻi mitochondrial a hōʻike i ka pono o TEM a me nā ʻano hana kiʻi ʻē aʻe no nā haʻawina mechanistic i hoʻolālā hou ʻia.
Ua kūʻai ʻia ka laina cell SH-SY5Y (ECACC, 94030304-1VL) mai Sigma-Aldrich. Ua ulu ʻia nā cell SH-SY5Y i loko o ka hui meaʻai medium/F-12 a Dulbecco i hoʻololi ʻia (DMEM/F-12) a me L-glutamine (SC09411, ScienCell) i loko o nā ʻōmole 25 cm2 i hoʻohui ʻia me 20% fetal bovine serum (FBS) (10493106, ThermoFisher Scientific) a me 1% penicillin-streptomycin (P4333-20ML, Sigma-Aldrich) ma 37 °C, 5% CO2. Ua hoʻoulu ʻia nā cell i 80% hui ʻana me ka hoʻohana ʻana i ka 0.05% trypsin-EDTA (15400054, ThermoFisher Scientific), centrifuged ma 300 g a ua hoʻopili ʻia ma kahi density o 7 × 105 cells/ml. Ua hana ʻia nā hoʻokolohua āpau ma nā pūnaewele SH-SY5Y i hoʻokaʻawale ʻole ʻia ma waena o nā alahele 19-22. Hoʻokele ʻia ka PPA ma ke ʻano he NaP. E hoʻoheheʻe i ka pauka NaP (CAS No. 137-40-6, ke ʻano kemika C3H5NaO2, P5436-100G, Sigma-Aldrich) i loko o ka wai MilliQ mehana i kahi nui o 1 M a mālama ma 4 °C. I ka lā o ka mālama ʻana, e hoʻoheheʻe i kēia hopena me 1 M PPA i 3 mM a me 5 mM PPA i loko o ka serum-free medium (DMEM/F-12 me L-glutamine). ʻAʻohe PPA nā nui o ka mālama ʻana no nā hoʻokolohua āpau (0 mM, kaohi), 3 mM, a me 5 mM PPA. Ua hana ʻia nā hoʻokolohua ma ka liʻiliʻi he ʻekolu mau replicates biological.
Ua lūlū ʻia nā pūnaewele SH-SY5Y i loko o nā ʻōmole 25 cm5 ma ka wikiwiki o 5.5 × 105 mau pūnaewele/ml a ua ulu ʻia no 24 mau hola. Ua hoʻohui ʻia ka lāʻau PPA i ka ʻōmole ma mua o 24 mau hola o ka hoʻoulu ʻana. E hōʻiliʻili i nā pellets cell ma hope o nā kaʻina hana subculture maʻamau o nā mammalian tissue (i wehewehe ʻia ma luna). E hoʻokuʻu hou i ka pellet cell i loko o 100 µl 2.5% glutaraldehyde, 1 × PBS a mālama ma 4 °C a hiki i ka hana ʻana. Ua centrifuged pōkole ʻia nā pūnaewele SH-SY5Y e pellet i nā pūnaewele a wehe i ka 2.5% glutaraldehyde, 1 × PBS solution. E hoʻokuʻu hou i ka sediment i loko o kahi gel agarose 4% i hoʻomākaukau ʻia i loko o ka wai i hoʻoheheʻe ʻia (ʻo ka lakio o ka agarose i ka nui o ka sediment he 1:1). Ua kau ʻia nā ʻāpana Agarose ma nā grids ma nā papa pālahalaha a ua uhi ʻia me 1-hexadecene ma mua o ka hoʻopaʻa ʻana i ke kaomi kiʻekiʻe. Ua hoʻopaʻa ʻia nā laʻana i loko o 100% acetone maloʻo ma -90 °C no 24 mau hola. A laila ua hoʻokiʻekiʻe ʻia ka mahana i -80°C a ua hoʻohui ʻia kahi hopena o 1% osmium tetroxide a me 0.1% glutaraldehyde. Ua mālama ʻia nā laʻana ma -80°C no 24 mau hola. Ma hope o kēia, ua hoʻonui mālie ʻia ka mahana i ka mahana o ka lumi i kekahi mau lā: mai – 80 °C a i – 50 °C no 24 mau hola, a i – 30 °C no 24 mau hola, a i – 10 °C no 24 mau hola a ma ka hope loa i ka mahana o ka lumi.
Ma hope o ka hoʻomākaukau ʻana i ka cryogenic, ua hoʻopiha ʻia nā laʻana me ka resin a ua hana ʻia nā ʻāpana ultrathin (∼100 nm) me ka hoʻohana ʻana i ka Leica Reichert UltracutS ultramicrotome (Leica Microsystems). Ua hoʻoluʻu ʻia nā ʻāpana me 2% uranyl acetate a me ke lead citrate. Ua nānā ʻia nā laʻana me ka hoʻohana ʻana i kahi microscope electron transmission FEI Tecnai 20 (ThermoFisher (FEI ma mua), Eindhoven, Netherlands) e hana ana ma 200 kV (Lab6 transmitter) a me kahi kāmela Gatan CCD (Gatan, UK) i lako me kahi kānana ikehu Tridiem.
Ma kēlā me kēia hana hou ʻenehana, ua loaʻa ma ka liʻiliʻi he 24 mau kiʻi kelepona hoʻokahi, no ka huina o 266 mau kiʻi. Ua kālailai ʻia nā kiʻi a pau me ka hoʻohana ʻana i ka macro Region of Interest (ROI) a me ka macro Mitochondria. Hoʻokumu ʻia ka macro mitochondrial ma nā ʻano i paʻi ʻia17,31,32 a ʻae i ka hana ʻana o ka hapa-automated o nā kiʻi TEM ma Fiji/ImageJ69. I ka pōkole: ua hoʻohuli ʻia ke kiʻi a hoʻohuli ʻia me ka hoʻohana ʻana i ka unuhi ʻana o ka pōpō ʻōwili (60 pixel radius) a me kahi kānana bandpass FFT (me ka hoʻohana ʻana i nā palena kiʻekiʻe a me lalo o 60 a me 8 pixel) a me ka hoʻopau ʻana i ka laina kū pololei me ka hoʻomanawanui o orientation o 5%. Hoʻopaʻa ʻia ke kiʻi i hana ʻia me ka hoʻohana ʻana i kahi algorithm entropy kiʻekiʻe a hana ʻia kahi mask binary. Ua unuhi ʻia nā ʻāpana kiʻi e pili ana me nā ROI i koho lima ʻia i nā kiʻi TEM maka, e hōʻike ana i ka mitochondria a me ka hoʻokaʻawale ʻana i ka membrane plasma a me nā ʻāpana ʻokoʻa kiʻekiʻe ʻē aʻe. No kēlā me kēia ROI i unuhi ʻia, ua kālailai ʻia nā ʻāpana binary i ʻoi aku ma mua o 600 mau pikela, a ua ana ʻia ka ʻāpana ʻāpana, ka perimeter, nā axes nui a me nā liʻiliʻi, ke anawaena Feret, ka poepoe, a me ke kaʻapuni me ka hoʻohana ʻana i nā hana ana i kūkulu ʻia o Fiji/ImageJ. Ma hope o Merrill, Flippo, a me Strack (2017), ua helu ʻia ka ʻāpana 2, ka lakio ʻaoʻao ʻāpana (ka lakio axis nui i ka liʻiliʻi), a me ke kumu hoʻohālike (FF) mai kēia mau ʻikepili, kahi FF = perimeter 2/4pi x wahi. Hiki ke loaʻa ka wehewehe ʻana o ke ʻano parametric ma Merrill, Flippo, a me Strack (2017). Loaʻa nā macros i ʻōlelo ʻia ma GitHub (e nānā i ka ʻŌlelo Loaʻa ʻIkepili). Ma ka awelika, ua kālailai ʻia ma kahi o 5,600 mau ʻāpana no kēlā me kēia mālama PPA, no ka huina o kahi o 17,000 mau ʻāpana (ʻaʻole i hōʻike ʻia ka ʻikepili).
Ua hoʻokomo ʻia nā pūnaewele SH-SH5Y i loko o nā kīʻaha moʻomeheu 8-lumi (ThermoFisher, #155411) e ʻae i ka hoʻopili ʻana i ka pō a laila hoʻomoʻa ʻia me TMRE 1:1000 (ThermoFisher, #T669) a me Hoechst 33342 1:200 (Sigma-Aldrich, H6024). dyeing. Ua loaʻa nā kiʻi me ka hoʻohana ʻana i nā lasers 405 nm a me 561 nm ma luna o kahi kaiapuni 10 min, a ua loaʻa nā kiʻi maka ma ke ʻano he z-stacks e loaʻa ana he 10 mau micrographs kiʻi me ka az step o 0.2 μm ma waena o nā mōlina kiʻi ma 12 mau manawa ma hope. Ua hōʻiliʻili ʻia nā kiʻi me ka hoʻohana ʻana i kahi kahua hoʻonā super Carl Zeiss LSM780 ELYRA PS.1 (Carl Zeiss, Oberkochen, Kelemānia) me ka hoʻohana ʻana i kahi aniani LCI Plan Apochromate 100x/1.4 Oil DIC M27. Ua kālailai ʻia nā kiʻi ma ImageJ me ka hoʻohana ʻana i kahi pipeline i wehewehe mua ʻia a me ka plugin ImageJ e ana i nā hanana fusion a me fission, ka helu awelika o nā ʻano mitochondrial, a me ka nui mitochondrial awelika no kēlā me kēia cell33. Loaʻa nā macros MEL ma GitHub (e nānā i ka ʻŌlelo Loaʻa ʻIkepili).
Ua ulu ʻia nā pūnaewele SH-SY5Y i loko o nā pā ʻeono-pūpū ma ka nui o 0.3 × 106 mau pūnaewele/mL no 24 mau hola ma mua o ka mālama ʻana. Ua unuhi ʻia ka RNA me ka hoʻohana ʻana i ka protocol Quick-RNA™ Miniprep (ZR R1055, Zymo Research) me nā hoʻololi iki: e hoʻohui i 300 μl o ka RNA lysis buffer i kēlā me kēia pūpū ma mua o ka wehe ʻana a lyse i kēlā me kēia laʻana ma ke ʻano he hana hope loa me 30 μl o ka DNase/RNase elution. -wai ʻole. Ua nānā ʻia nā laʻana a pau no ka nui a me ka maikaʻi me ka hoʻohana ʻana i ka NanoDrop ND-1000 UV-Vis Spectrophotometer. Ua loaʻa ka protein holoʻokoʻa mai nā lysates cell me ka hoʻohana ʻana i ka 200 μl RIPA lysis buffer, a ua helu ʻia ka nui o ka protein me ka hoʻohana ʻana i ka Bradford protein assay70.
Ua hana ʻia ka synthesis cDNA me ka hoʻohana ʻana i ka Tetro™ cDNA Synthesis Kit (BIO-65043, Meridian Bioscience) e like me nā kuhikuhi a ka mea hana me kekahi mau hoʻololi. Ua synthesized ʻia ka cDNA ma nā hopena 20-μl me ka hoʻohana ʻana i ka 0.7 a i ka 1 μg o ka RNA holoʻokoʻa. Ua koho ʻia nā Primers mai nā pepa i paʻi mua ʻia 42, 71, 72, 73, 74, 75, 76, 77, 78 (Papa S1) a ua hoʻolālā ʻia nā probes e hele pū ana me ka hoʻohana ʻana i ka mea hana PrimerQuest mai Integrated DNA Technologies. Ua hoʻonohonoho ʻia nā genes āpau o ka hoihoi i ka gene B2M nuklea. Ua ana ʻia ka hōʻike ʻana o ka gene o STOML2, NRF1, NFE2L2, TFAM, cMYC a me OPA1 e RT-qPCR. ʻO ka hui haku i hoʻokomo pū ʻia me LUNA Taq polymerase (M3003L, New England Biolabs), 10 μM mau primers mua a me hope, cDNA, a me ka wai PCR-grade e loaʻa ai ka nui hope loa o 10 μL no kēlā me kēia hopena. Ua ana ʻia ka hōʻike ʻana o nā genes mahele a me ka fission (DRP1, MFN1/2) me ka hoʻohana ʻana i nā assays TaqMan multiplex. Ua hoʻohana ʻia ʻo Luna Universal Probe qPCR Master Mix (M3004S, New England Biolabs) e like me nā kuhikuhi a ka mea hana me nā hoʻololi liʻiliʻi. ʻO ka hui haku multiplex RT-qPCR e komo pū ana me 1X LUNA Taq polymerase, 10 μM mau primers mua a me hope, 10 μM probe, cDNA, a me ka wai PCR-grade, e hopena ana i ka nui hope loa o 20 μL no kēlā me kēia hopena. Ua hana ʻia ʻo RT-qPCR me ka hoʻohana ʻana i ka Rotor-Gene Q 6-plex (QIAGEN RG—serial number: R0618110). Hōʻike ʻia nā kūlana paikikala ma ka Papa S1. Ua hoʻonui ʻia nā laʻana cDNA āpau i ka triplicate a ua hana ʻia kahi piʻo maʻamau me ka hoʻohana ʻana i kahi moʻo o nā dilutions he ʻumi manawa. Ua wehe ʻia nā outliers i nā laʻana triplicate me ka paepae cycle standard deviation (Ct) >0.5 mai ka nānā ʻana e hōʻoia i ka hana hou ʻana o ka ʻikepili30,72. Ua helu ʻia ka hōʻike ʻana o ka gene pili me ka hoʻohana ʻana i ke ʻano 2-ΔΔCt79.
Ua hui pū ʻia nā hāpana protein (60 μg) me ka Laemmli loading buffer ma ka lakio 2:1 a holo ma luna o kahi gel protein kala ʻole 12% (Bio-Rad #1610184). Ua hoʻoili ʻia nā protein i kahi membrane PVDF (polyvinylidene fluoride) (#170-84156, Bio-Rad) me ka hoʻohana ʻana i ka ʻōnaehana Trans-Blot Turbo (#170-4155, Bio-Rad). Ua ālai ʻia ka membrane a ua hoʻoulu ʻia me nā antibodies mua kūpono (OPA1, MFN1, MFN2, a me DRP1) (diluted 1:1000) no 48 mau hola, a ukali ʻia e ka hoʻoulu ʻana me nā antibodies lua (1:10,000) no 1 hola. A laila ua kiʻi ʻia nā membrane me ka hoʻohana ʻana iā Clarity Western ECL Substrate (#170-5061, Bio-Rad) a hoʻopaʻa ʻia me ka hoʻohana ʻana i kahi ʻōnaehana Bio-Rad ChemiDoc MP. Ua hoʻohana ʻia ʻo ImageLab mana 6.1 no ka nānā ʻana o Western blot. Ua hōʻike ʻia ka gel mua a me ka blot ma ke Kiʻi S1. Hāʻawi ʻia ka ʻike antibody ma ka Papa S2.
Hōʻike ʻia nā ʻikepili ma ke ʻano he awelika a me ka hewa maʻamau o ka awelika (SEM) o ka liʻiliʻi he ʻekolu mau laʻana kūʻokoʻa. Ua hoʻāʻo ʻia nā ʻikepili no ka maʻamau me ka hoʻohana ʻana i ka hoʻāʻo Shapiro-Wilks (ke ʻole i ʻōlelo ʻia) ma mua o ka manaʻo ʻana i kahi hoʻolaha Gaussian a me nā ʻokoʻa maʻamau like a me ka hoʻomau ʻana me nā loiloi. Ma waho aʻe o ka loiloi ʻana i ka ʻikepili me ka hoʻohana ʻana iā Fisher's MEL LSD (p < 0.05), hoʻokahi ala ANOVA (lāʻau lapaʻau vs. kaohi awelika), a me ka hoʻāʻo hoʻohālikelike lehulehu a Dunnett e hoʻoholo ai i ke koʻikoʻi (p < 0.05). Hōʻike ʻia nā waiwai p koʻikoʻi ma ka pakuhi ma ke ʻano he *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Ua hana ʻia a hana ʻia nā loiloi helu a me nā pakuhi āpau me ka hoʻohana ʻana iā GraphPad Prism 9.4.0.
Loaʻa i ka lehulehu nā macros Fiji/ImageJ no ka loiloi kiʻi TEM ma GitHub: https://github.com/caaja/TEMMitoMacro. Loaʻa i ka lehulehu ka macro Mitochondrial Event Locator (MEL) ma GitHub: https://github.com/rensutheart/MEL-Fiji-Plugin.
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