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A na-eji Propionic acid (PPA) amụ ọrụ nke nsogbu mitochondrial na nsogbu neurodevelopment dịka autism spectrum disorder. A maara PPA na-emebi mitochondrial biogenesis, metabolism, na turnover. Agbanyeghị, mmetụta nke PPA na mitochondrial dynamics, fission na fusion ka dị nsogbu n'ihi ọdịdị oge dị mgbagwoju anya nke usoro ndị a. N'ebe a, anyị na-eji usoro onyonyo quantitative eme ihe iji nyochaa otu PPA si emetụta mitochondrial ultrastructure, morphology, na dynamics na mkpụrụ ndụ SH-SY5Y dị ka neuron. PPA (5 mM) kpatara nnukwu mbelata na mpaghara mitochondrial (p < 0.01), dayameta na okirikiri Feret (p < 0.05), na mpaghara 2 (p < 0.01). Nyocha ihe omume Mitochondrial gosiri mmụba dị ukwuu (p < 0.05) na ihe omume fission na fusion, si otú a na-ejigide iguzosi ike n'ezi ihe nke netwọk mitochondrial n'okpuru ọnọdụ nrụgide. Tinyere nke ahụ, ngosipụta mRNA nke cMYC (p < 0.0001), NRF1 (p < 0.01), TFAM (p < 0.05), STOML2 (p < 0.0001) na OPA1 (p < 0.05) belatara nke ukwuu. 01). Nke a na-egosi mgbanwe nke mitochondrial morphology, biogenesis na dynamics iji nọgide na-arụ ọrụ n'okpuru ọnọdụ nrụgide. Data anyị na-enye nghọta ọhụrụ banyere mmetụta nke PPA na mitochondrial dynamics ma na-egosipụta uru nke usoro onyonyo maka ịmụ usoro iwu dị mgbagwoju anya metụtara nzaghachi nrụgide mitochondrial.
Mitochondria bụ akụkụ dị mkpa nke ọrụ sel dị iche iche karịa ọrụ ha na-arụ na mmepụta ike na biosynthesis. Mitochondria metabolism bụ isi ihe na-achịkwa signaling calcium, metabolic na redox homeostasis, inflammatory signaling, epigenetic modifications, cell proliferation, difference and programmed cell death1. Karịsịa, mitochondrial metabolism dị oke mkpa maka mmepe neuronal, ndụ na ọrụ ma na-emetụta ya nke ukwuu na ngosipụta dị iche iche nke neuropathology2,3,4.
N'ime afọ iri gara aga, ọnọdụ metabolic apụtala dị ka ihe na-achịkwa neurogenesis, differentiation, maturation na plasticity5,6. N'oge na-adịbeghị anya, mitochondrial morphology na dynamics aghọwo akụkụ dị mkpa karịsịa nke mitosis, usoro ike nke na-edobe ọdọ mmiri nke mitochondria dị mma n'ime sel. A na-achịkwa mitochondrial dynamics site na ụzọ dị mgbagwoju anya dị iche iche site na mitochondrial biogenesis na bioenergetics ruo mitochondrial fission, fusion, transport and clearance7,8. Nkwụsị nke ọ bụla n'ime usoro njikọta ndị a na-emebi mmezi nke netwọk mitochondrial dị mma ma nwee nnukwu nsonaazụ ọrụ maka mmepe neuro9,10. N'ezie, a na-ahụ mmebi nke mitochondrial dynamics n'ọtụtụ nsogbu uche, neurodegenerative na neurodevelopment, gụnyere autism spectrum disorders (ASD)11,12.
ASD bụ nsogbu neurodevelopment dị iche iche nke nwere usoro mkpụrụ ndụ ihe nketa na epigenetic dị mgbagwoju anya. A ka na-agbaghaghị ikike nke ASD, mana isi ihe kpatara ya ka aghọtachaghị nke ọma. Ịchịkọta data sitere na ụdị preclinical, ọmụmụ ihe gbasara ahụike, na datasets molecular multi-omics na-enye ihe akaebe na-abawanye nke nsogbu mitochondrial na ASD13,14. Anyị mere nyocha methylation DNA nke genome-wide n'ime otu ndị ọrịa nwere ASD ma chọpụta mkpụrụ ndụ ihe nketa methylated dị iche iche nke jikọtara n'akụkụ ụzọ metabolic mitochondrial15. Anyị mechara kọọrọ akụkọ methylation dị iche iche nke ndị na-achịkwa etiti nke mitochondrial biogenesis na dynamics, nke jikọtara ya na mmụba nke ọnụọgụ mtDNA na profaịlụ metabolic urinary gbanwere na ASD16. Data anyị na-enye ihe akaebe na-abawanye na mitochondrial dynamics na homeostasis na-arụ ọrụ dị mkpa na pathophysiology nke ASD. Ya mere, imeziwanye nghọta mechanistic nke mmekọrịta dị n'etiti mitochondrial dynamics, morphology, na ọrụ bụ isi ihe mgbaru ọsọ nke nyocha na-aga n'ihu na ọrịa akwara nke a na-ahụ site na dysfunction mitochondrial nke abụọ.
A na-ejikarị usoro molekul eme ihe iji mụọ ọrụ nke mkpụrụ ndụ ihe nketa kpọmkwem na nzaghachi nrụgide mitochondrial. Agbanyeghị, ụzọ a nwere ike ịnwe oke site na ụdị dị iche iche na nke oge nke usoro njikwa mitotic. Ọzọkwa, ngosipụta dị iche iche nke mkpụrụ ndụ ihe nketa mitochondrial bụ ihe ngosi na-apụtaghị ìhè nke mgbanwe ọrụ, ọkachasị ebe ọ bụ na a na-enyochakarị ọnụọgụgụ mkpụrụ ndụ ihe nketa ole na ole. Ya mere, atụpụtala ụzọ ndị ọzọ kpọmkwem maka ịmụ ọrụ mitochondrial na bioenergetics17. Ọdịdị mitochondrial nwere njikọ chiri anya na ọnọdụ mitochondrial. Ọdịdị mitochondrial, njikọ, na nhazi dị oke mkpa maka mmepụta ike na ndụ mitochondrial na mkpụrụ ndụ5,18. Ọzọkwa, akụkụ dị iche iche nke mitosis na-elekwasị anya na mgbanwe na ọnọdụ mitochondrial, nke nwere ike ije ozi dị ka njedebe bara uru nke nsogbu mitochondrial ma nye ntọala maka ọmụmụ ihe mekanistic na-esote.
Enwere ike ịhụ ọdịdị nke mitochondrial ozugbo site na iji transmission electron microscopy (TEM), na-enye ohere ka e mụọ nkọwa zuru ezu banyere ọdịdị nke ultrastructure cellular. TEM na-ahụ ọdịdị, ọdịdị na nhazi nke mitochondrial cristae ozugbo na mkpebi nke mitochondria nke ọ bụla, kama ịdabere naanị na mkpụrụ ndụ ihe nketa, ngosipụta protein ma ọ bụ paramita ọrụ mitochondrial na ọnụọgụgụ mkpụrụ ndụ17,19,20. Na mgbakwunye, TEM na-eme ka ọmụmụ ihe dị n'etiti mitochondria na organelles ndị ọzọ dị mfe, dị ka endoplasmic reticulum na autophagosomes, nke na-arụ ọrụ dị mkpa na ọrụ mitochondrial na homeostasis21,22. Ya mere, nke a na-eme TEM ka ọ bụrụ ezigbo ebe mmalite maka ịmụ banyere nsogbu mitochondrial tupu ilekwasị anya na ụzọ ma ọ bụ mkpụrụ ndụ ihe nketa ụfọdụ. Ka ọrụ mitochondrial na-aghọwanye ihe dị mkpa na neuropathology, enwere mkpa doro anya inwe ike ịmụ kpọmkwem na ọnụọgụgụ na morphology mitochondrial na ụdị neuronal in vitro.
N'isiokwu a, anyị na-enyocha usoro mitochondrial n'ime ụdị neuronal nke dysfunction mitochondrial na autism spectrum disorder. Anyị kọrọ na mbụ methylation dị iche iche nke propionyl-CoA carboxylase beta (PCCB) na ASD15, obere nkeji nke enzyme mitochondrial propionyl-CoA carboxylase PCC. A maara na mmebi nke PCC na-akpata mkpokọta nsí nke ihe ndị sitere na propionyl, gụnyere propionic acid (PPA)23,24,25. Egosila na PPA na-akpaghasị metabolism neuronal ma gbanwee omume na vivo ma bụrụ ihe nlereanya anụmanụ edobere maka ịmụ usoro mmepe neurodevelopment metụtara na ASD26,27,28. Na mgbakwunye, akọpụtala na PPA na-emebi ikike membrane mitochondrial, biogenesis na iku ume na vitro ma ejiri ya mee ihe nke ukwuu iji gosipụta nsogbu mitochondrial na neurons29,30. Agbanyeghị, mmetụta nke nsogbu mitochondrial nke PPA kpatara na morphology na dynamics mitochondrial ka aghọtachaghị nke ọma.
Ọmụmụ ihe a na-eji usoro onyonyo ndị ọzọ iji chọpụta mmetụta nke PPA na mitochondrial morphology, dynamics, na ọrụ na mkpụrụ ndụ SH-SY5Y. Nke mbụ, anyị mepụtara usoro TEM iji hụ mgbanwe na mitochondrial morphology na ultrastructure17,31,32. N'iburu n'uche ụdị ike nke mitochondria33, anyị jikwa nyocha mitochondrial event localizer (MEL) iji tụọ mgbanwe na nguzozi dị n'etiti ihe omume fission na fusion, ọnụọgụ mitochondrial na olu n'okpuru nrụgide PPA. N'ikpeazụ, anyị lere anya ma morphology na dynamics mitochondrial jikọtara ya na mgbanwe na ngosipụta nke mkpụrụ ndụ ihe nketa metụtara biogenesis, fission, na fusion. A gbakọtara ọnụ, data anyị na-egosi ihe ịma aka nke ịkọwa mgbagwoju anya nke usoro na-achịkwa ike mitochondrial. Anyị na-egosipụta uru nke TEM n'ịmụ mitochondrial morphology dị ka njedebe convergent nke mitosis na mkpụrụ ndụ SH-SY5Y. Na mgbakwunye, anyị na-eme ka ọ pụta ìhè na data TEM na-enye ozi kachasị mma mgbe ejikọtara ya na usoro onyonyo nke na-ejidekwa ihe omume ike na nzaghachi maka nrụgide metabolic. Nkọwa ọzọ nke usoro nchịkwa molekul nke na-akwado mitosis mkpụrụ ndụ neuronal nwere ike inye nghọta dị mkpa na akụkụ mitochondrial nke sistemụ akwara na ọrịa neurodegenerative.
Iji kpalite nrụgide mitochondrial, ejiri PPA gwọọ mkpụrụ ndụ SH-SY5Y site na iji 3 mM na 5 mM sodium propionate (NaP). Tupu TEM, a na-eji nnukwu nrụgide na-eme ihe nlele eme ihe nlele cryogenic (Foto 1a). Anyị mepụtara ọkpọkọ nyocha onyonyo mitochondrial akpaaka iji tụọ paramita morphological asatọ nke ndị mitochondrial n'ofe ihe ndị dị ndụ atọ. Anyị chọpụtara na ọgwụgwọ PPA gbanwere paramita anọ nke ukwuu: mpaghara 2, mpaghara, gburugburu, na dayameta Feret (Foto 1b–e). Mpaghara 2 belatara nke ukwuu site na ọgwụgwọ PPA 3 mM na 5 mM (p = 0.0183 na p = 0.002, n'otu n'otu) (Foto 1b), ebe mpaghara (p = 0.003), gburugburu (p = 0.0106) na dayameta Feret ebelatala nke ukwuu. E nwere mbelata dị ukwuu (p = 0.0172) na otu ọgwụgwọ 5 mM ma e jiri ya tụnyere otu njikwa (Foto 1c–e). Mbelata dị ukwuu na mpaghara na gburugburu gosiri na mkpụrụ ndụ ejiri 5 mM PPA gwọọ nwere obere mitochondria gbara gburugburu karịa, nakwa na mitochondria ndị a erughị ogologo karịa ndị dị na mkpụrụ ndụ njikwa. Nke a kwekọrọ na mbelata dị ukwuu na dayameta Feret, paramita nọọrọ onwe ya nke na-egosi mbelata na anya kachasị ukwuu n'etiti nsọtụ ihe. A hụrụ mgbanwe na ultrastructure nke cristae: cristae ahụ adịchaghị mma n'okpuru mmetụta nke nrụgide PPA (Fig. 1a, panel B). Agbanyeghị, ọ bụghị onyonyo niile gosipụtara ultrastructure nke cristae nke ọma, yabụ emebeghị nyocha ọnụọgụ nke mgbanwe ndị a. Data TEM ndị a nwere ike igosipụta ọnọdụ atọ nwere ike ime: (1) PPA na-eme ka fission dịkwuo elu ma ọ bụ na-egbochi njikọ, na-eme ka mitochondria dị adị daa na nha; (2) biogenesis emelitere na-emepụta mitochondria ọhụrụ, obere ma ọ bụ (3) na-akpali usoro abụọ ahụ n'otu oge. Ọ bụ ezie na TEM enweghị ike ịmata ọnọdụ ndị a, mgbanwe morphological dị mkpa na-egosi mgbanwe na homeostasis mitochondrial na dynamics n'okpuru nrụgide PPA. Anyị mechara nyochaa paramita ndị ọzọ iji kọwaa dynamics ndị a na usoro nwere ike ịbụ nke dị n'okpuru ha.
Propionic acid (PPA) na-agbanwegharị ọdịdị mitochondrial. (a) Foto elektrọn nnyefe nnọchite anya (TEM) na-egosi na nha mitochondrial na-ebelata ma mitochondria na-adị obere ma gbaa gburugburu ka ọgwụgwọ PPA na-abawanye; 0 mM (anaghị agwọ), 3 mM na 5 mM, n'otu n'otu. Akụ uhie na-egosi mitochondria. (b–e) A kwadebere mkpụrụ ndụ SH-SY5Y ejiri PPA gwọọ ruo awa 24 maka TEM ma nyochaa nsonaazụ ya site na iji Fiji/ImageJ. Anọ n'ime paramita asatọ ahụ gosiri nnukwu ọdịiche dị n'etiti mkpụrụ ndụ njikwa (anaghị agwọ, 0 mM PPA) na mkpụrụ ndụ PPA a gwọrọ (3 mM na 5 mM PPA). (b) Mpaghara 2, (c) Mpaghara, (d) Oke, (e) Dayameta Feret. Ejiri otu ụzọ nyocha nke mgbanwe (njikwa vs. ọgwụgwọ) na ule ntụnyere ọtụtụ Dunnett chọpụta nnukwu ọdịiche (p < 0.05). Isi ihe data na-anọchite anya nkezi uru mitochondrial maka mkpụrụ ndụ ọ bụla, ogwe njehie na-anọchite anya nkezi ± SEM. Data egosiri na-anọchite anya n = 3, opekata mpe mkpụrụ ndụ 24 kwa otu; E nyochachara mkpokọta foto 266; * na-egosi p < 0.05, ** na-egosi p < 0.01.
Iji kọwaa etu mitochondrial dynamics si emeghachi omume na PPA, anyị sere mitochondria na tetramethylrhodamine ethyl ester (TMRE) ma jiri microscopy nke oge na nyocha MEL iji chọpụta ma tụọ mitochondria mgbe awa 24 gachara na 3 na 5 mM PPA. Ọgwụgwọ nke ihe omume fission na fusion. (Fig. 2a). Mgbe nyocha MEL gasịrị, a nyochakwuru mitochondria iji tụọ ọnụọgụ nke ihe owuwu mitochondrial na olu nkezi ha. Anyị hụrụ obere mmụba mana dị mkpa na ọnụọgụ nke ihe omume fission na-eme na 3 mM [4.9 ± 0.3 (p < 0.05)] ma e jiri ya tụnyere fission [5.6 ± 0.3 (p < 0.05) )] na fusion [5.4 ± 0.5 (p < 0.05)] na fusion [5.4 ± 0.5 (p < 0.05)] 0.05)] ihe omume <0.05)] mụbara nke ukwuu na 5 mM ma e jiri ya tụnyere njikwa (Fig. 3b). Ọnụọgụgụ mitochondria mụbara nke ukwuu na 3 [32.6 ± 2.1 (p < 0.05)] na 5 mM [34.1 ± 2.2 (p < 0.05)] (Fig. 3c), ebe olu nkezi nke usoro mitochondrial ọ bụla agbanwebeghị (Fig. 3c). 3d). Nke a na-egosi na imeghari usoro mitochondrial na-eje ozi dị ka nzaghachi nkwụghachi ụgwọ nke na-ejigide iguzosi ike n'ezi ihe nke netwọk mitochondrial nke ọma. Mmụba nke ọnụọgụ ihe omume fission na 3 mM PPA na-egosi na mmụba nke ọnụọgụ mitochondrial bụ akụkụ ụfọdụ n'ihi fission mitochondrial, mana ebe ọ bụ na olu mitochondrial nkezi ka na-agbanweghi agbanwe, enweghị ike iwepụ biogenesis dị ka nzaghachi nkwụghachi ọzọ. Agbanyeghị, data ndị a kwekọrọ na obere usoro mitochondrial gburugburu nke TEM hụrụ ma gosipụtakwa mgbanwe dị ukwuu na usoro mitochondrial nke PPA kpaliri.
Propionic acid (PPA) na-akpali mgbanwe mitochondrial dị ike iji nọgide na-enwe iguzosi ike n'ezi ihe nke netwọk. E kụnyere mkpụrụ ndụ SH-SY5Y, gwọọ ya na 3 na 5 mM PPA ruo awa 24 ma tee ya agba na TMRE na Hoechst 33342, wee jiri nyocha MEL mee ya. (a) Foto mikroskopu oge na-anọchi anya nke na-egosi agba na amụma ike kachasị elu nke abụọ na oge 2 (t2) maka ọnọdụ ọ bụla. A na-eme ka mpaghara ahọpụtara egosiri na onyonyo abụọ ọ bụla ka mma ma gosipụta ya na 3D na oge atọ dị iche iche (t1-t3) iji gosipụta ike ka oge na-aga; a na-eme ka ihe omume njikọ pụta ìhè na akwụkwọ ndụ akwụkwọ ndụ; a na-eme ka ihe omume fission pụta ìhè na akwụkwọ ndụ akwụkwọ ndụ. A na-egosi ya na uhie. (b) Ọnụọgụ nkezi nke ihe omume ike kwa ọnọdụ. (c) Ọnụọgụ nkezi nke ihe owuwu mitochondrial kwa sel. (d) Olu nkezi (µm3) nke ihe owuwu mitochondrial ọ bụla kwa sel. Data egosiri bụ ihe nnọchiteanya nke n = mkpụrụ ndụ 15 kwa otu ọgwụgwọ. Ogwe njehie egosiri na-anọchite anya nkezi ± SEM, ogwe nha = 10 μm, * p < 0.05.
Propionic acid (PPA) na-akpata mwepu mkpụrụ ndụ ihe nketa nke metụtara mitochondrial dynamics. E jiri 3 na 5 mM PPA gwọọ mkpụrụ ndụ SH-SY5Y ruo awa 24. Ejiri RT-qPCR mee nhazi mkpụrụ ndụ ihe nketa dịtụ iche ma mee ka ọ bụrụ B2M. Mkpụrụ ndụ ihe nketa Mitochondrial (a) cMYC, (b) TFAM, (c) NRF1 na (d) NFE2L2. Mkpụrụ ndụ ihe nketa Mitochondrial fusion na fission (e) STOML2, (f) OPA1, (g) MFN1, (h) MFN2 na (i) DRP1. E jiri otu ụzọ ANOVA (njikwa vs. ọgwụgwọ) nwalee ọdịiche dị mkpa (p < 0.05) na ule ntụnyere ọtụtụ Dunnett: * na-egosi p < 0.05, ** na-egosi p < 0.01, na **** na-egosi p < 0.0001. Ogwe na-anọchite anya ngosipụta nkezi ± SEM. Data egosiri na-egosi n = 3 (STOML2, OPA1, TFAM), n = 4 (cMYC, NRF1, NFE2L2), na n = 5 (MFN1, MFN2, DRP1) ihe ndị metụtara ndụ.
Data sitere na nyocha TEM na MEL ọnụ na-egosi na PPA na-agbanwe ọdịdị na ike mitochondrial. Agbanyeghị, usoro onyonyo ndị a anaghị enye nghọta n'ime usoro ndị na-akwalite usoro ndị a. Ya mere, anyị lere anya na mRNA ngosipụta nke ihe nchịkwa itoolu dị mkpa nke mitochondrial dynamics, biogenesis, na mitosis dịka nzaghachi nye ọgwụgwọ PPA. Anyị tụlere myeloma oncogene cell (cMYC), nuclear respiratory factor (NRF1), mitochondrial transcription factor 1 (TFAM), NFE2-like transcription factor BZIP (NFE2L2), gastrin-like protein 2 (STOML2), optic nerve atrophy 1 (OPA1), Mitofusin 1 (MFN1), Mitofusin 2 (MFN2) na dynamin-related protein 1 (DRP1) mgbe awa 24 nke ọgwụgwọ na 3 mM na 5 mM PPA gasịrị. Anyị hụrụ 3 mM (p = 0.0053, p = 0.0415 na p < 0.0001, n'otu n'otu) na 5 mM (p = 0.0031, p = 0.0233, p < 0.0001) ọgwụgwọ PPA. (Foto 3a–c). Mbelata na ngosipụta mRNA dabere na dose: ngosipụta nke cMYC, NRF1 na TFAM belatara site na ugboro 5.7, 2.6 na 1.9 na 3 mM, n'otu n'otu, na site na ugboro 11.2, 3 na 2.2 na 5 mM. N'ụzọ dị iche, a gbanweeghị mkpụrụ ndụ ihe nketa redox biogenesis etiti NFE2L2 na mkpokọta ọ bụla nke PPA, ọ bụ ezie na a hụrụ usoro yiri nke ahụ nke mbelata ngosipụta (Foto 3d).
Anyị lerekwa anya n'otú mkpụrụ ndụ ihe nketa oge gboo si arụ ọrụ n'ịhazi nkewa na njikọta. A na-eche na STOML2 na-etinye aka na njikọta, mitophagy na biogenesis, a na-ebelatakwa ngosipụta ya nke ukwuu (p < 0.0001) site na 3 mM (mgbanwe okpukpu 2.4) na 5 mM (mgbanwe okpukpu 2.8) PPA (Foto 1). 3d). N'otu aka ahụ, e belatara ngosipụta mkpụrụ ndụ ihe nketa njikọta OPA1 na 3 mM (mgbanwe okpukpu 1.6) na 5 mM (mgbanwe okpukpu 1.9) PPA (p = 0.006 na p = 0.0024, n'otu n'otu) (Foto 3f). Agbanyeghị, anyị ahụtaghị nnukwu ọdịiche na ngosipụta nke mkpụrụ ndụ ihe nketa njikọta MFN1, MFN2 ma ọ bụ mkpụrụ ndụ ihe nketa njikọta DRP1 n'okpuru nrụgide PPA awa 24 (Foto 3g–i). Na mgbakwunye, anyị chọpụtara na ọkwa nke protein njikọ anọ na fission (OPA1, MFN1, MFN2 na DRP1) agbanweghị n'otu ọnọdụ ahụ (Foto 4a–d). Ọ dị mkpa ịmara na data ndị a na-egosipụta otu oge na ọ nwere ike ọ gaghị egosipụta mgbanwe na ngosipụta protein ma ọ bụ ọkwa ọrụ n'oge mmalite nke nrụgide PPA. Agbanyeghị, mbelata dị ukwuu na ngosipụta nke cMYC, NRF1, TFAM, STOML2, na OPA1 na-egosi nnukwu mmebi ederede nke mitochondrial metabolism, biogenesis, na dynamics. Na mgbakwunye, data ndị a na-egosipụta uru nke usoro onyonyo iji mụọ kpọmkwem mgbanwe ọnọdụ njedebe na ọrụ mitochondrial.
Ọkwa protein nke ngwakọta na fission factor agbanweghị mgbe ọgwụgwọ propionic acid (PPA) gasịrị. E jiri 3 na 5 mM PPA gwọọ mkpụrụ ndụ SH-SY5Y ruo awa 24. E jiri nyocha Western blot tụọ ọkwa protein, a na-emekwa ka ọkwa ngosipụta dị ka protein zuru oke. E gosipụtara ngosipụta protein nkezi na Western blots nke protein a na-elekwasị anya na ya. a – OPA1, b – MFN1, c – MFN2, d – DRP1. Ogwe ndị ahụ na-anọchite anya nkezi ± SEM, data egosiri na-anọchitekwa anya n = 3 bio replicates. E mere ọtụtụ ntụnyere (p < 0.05) site na iji nyocha otu ụzọ nke mgbanwe na ule Dunnett. E gosiri gel na blot mbụ na Foto S1.
Nsogbu nke mitochondria na-ejikọta ya na ọrịa dị iche iche, site na ọrịa metabolic, obi na akwara ruo na ọrịa akwara ozi1,10. Ọtụtụ ọrịa neurodegenerative na neurodegenerative na-ejikọta ya na nsogbu mitochondrial, na-egosipụta mkpa nke akụkụ ahụ ndị a n'oge ndụ ụbụrụ. Ọrịa ndị a gụnyere ọrịa Parkinson, ọrịa Alzheimer na ASD3,4,18. Agbanyeghị, ịnweta anụ ahụ ụbụrụ iji mụọ ọrịa ndị a siri ike, karịsịa na ọkwa mechanistic, na-eme ka sistemụ nlereanya cellular bụrụ ihe ọzọ dị mkpa. N'ọmụmụ ihe a, anyị na-eji sistemụ nlereanya cellular nke ejiri mkpụrụ ndụ SH-SY5Y gwọọ PPA iji weghachite nsogbu mitochondrial a hụrụ na ọrịa neuronal, ọkachasị nsogbu autism spectrum. Iji ụdị PPA a mụọ usoro mitochondrial na neurons nwere ike inye nghọta na isi ihe kpatara ASD.
Anyị lere anya n'ihe gbasara iji TEM lee mgbanwe dị na mitochondrial morphology. Ọ dị mkpa ịmara na a ga-eji TEM nke ọma iji mee ka ọ dịkwuo irè. Nkwadebe nke cryo-specimens na-enye ohere maka nchekwa ka mma nke usoro neuronal site n'ịhazi ihe ndị dị na sel n'otu oge ma belata mmepụta nke ihe ochie34. N'ikwekọ na nke a, anyị hụrụ na mkpụrụ ndụ SH-SY5Y dị ka neuron nwere organelles dị n'okpuru sel na mitochondria ogologo (Fig. 1a). Nke a na-egosi uru nke usoro nkwadebe cryogenic maka ịmụ morphology mitochondrial na ụdị sel neuronal. Ọ bụ ezie na nha ọnụọgụ dị oke mkpa maka nyocha ebumnuche nke data TEM, enweghị nkwekọrịta na paramita kpọmkwem a ga-atụ iji kwado mgbanwe morphological mitochondrial. Dabere na ọtụtụ ọmụmụ ndị nyochaworo morphology mitochondrial n'ụzọ ọnụọgụ17,31,32, anyị mepụtara ọkpọkọ nyocha onyonyo mitochondrial akpaaka nke na-atụ paramita morphological asatọ, ya bụ: mpaghara, mpaghara2, nha akụkụ, gburugburu, okirikiri, ogo, dayameta Feret. na gburugburu.
N'ime ha, PPA belatara nke ukwuu mpaghara 2, mpaghara, gburugburu, na dayameta Feret (Foto 1b–e). Nke a gosiri na mitochondria ghọrọ obere ma gbaa gburugburu, nke kwekọrọ na ọmụmụ ihe ndị gara aga na-egosi mbelata na mpaghara mitochondrial mgbe awa 72 nke nrụgide mitochondrial nke PPA30 kpatara. Atụmatụ ọdịdị ndị a nwere ike igosi mitochondrial fission, usoro dị mkpa iji wepụ ihe ndị mebiri emebi site na netwọk mitochondrial iji kwalite mmebi ha site na mitophagy35,36,37. N'aka nke ọzọ, mbelata na nkezi nha mitochondrial nwere ike ijikọ ya na mmụba biogenesis, nke na-ebute ịmepụta obere mitochondria ọhụrụ. Mmụba fission ma ọ bụ biogenesis na-anọchite anya nzaghachi nkwụghachi iji nọgide na-enwe mitosis megide nrụgide mitochondrial. Agbanyeghị, enweghị ike iwepụ uto mitochondrial belatara, njikọ na-adịghị mma, ma ọ bụ ọnọdụ ndị ọzọ.
Ọ bụ ezie na onyonyo dị elu nke TEM mepụtara na-enye ohere ịchọpụta njirimara morphological na ọkwa mitochondria nkeonwe, usoro a na-emepụta foto abụọ n'otu oge. Iji mụọ mmeghachi omume na-agbanwe agbanwe maka nrụgide metabolic, anyị sere mitochondria na TMRE ma jiri microscopy oge na nyocha MEL, nke na-enye ohere ka a hụ mgbanwe dị elu na netwọk mitochondrial 3D nke mgbanwe na netwọk mitochondrial ka oge na-aga33,38. Anyị hụrụ mgbanwe dị obere mana dị mkpa na mitochondrial dynamics n'okpuru nrụgide PPA (Fig. 2). Na 3 mM, ọnụọgụ nke ihe omume fission mụbara nke ukwuu, ebe ihe omume fission nọgidere otu ihe ahụ dị na njikwa ahụ. A hụrụ mmụba na ọnụọgụ nke ihe omume fission na fusion na 5 mM PPA, mana mgbanwe ndị a dị ihe dị ka nha nhata, na-atụ aro na kinetics fission na fusion na-eru nha nhata na oke dị elu (Fig. 2b). Olu mitochondrial nkezi agbanwebeghị na ma 3 na 5 mM PPA, na-egosi na echekwara iguzosi ike n'ezi ihe nke netwọk mitochondrial (Fig. 2d). Nke a na-egosipụta ikike nke netwọk mitochondrial na-agbanwe agbanwe iji meghachi omume na nrụgide metabolic dị nro iji nọgide na-enwe homeostasis nke ọma n'enweghị nsogbu nkewa netwọk. Na 3 mM PPA, mmụba nke fission zuru ezu iji kwalite mgbanwe gaa na nhazi ọhụrụ, mana ọ dị mkpa ka e mee mgbanwe kinetic miri emi karịa iji zaghachi nrụgide nke oke PPA kpatara.
Ọnụọgụgụ mitochondria mụbara na mkpokọta nrụgide PPA abụọ ahụ, mana nkezi olu mitochondrial agbanweghị nke ukwuu (Foto 2c). Nke a nwere ike ịbụ n'ihi mmụba nke biogenesis ma ọ bụ mmụba nkewa; agbanyeghị, na enweghị mbelata dị ukwuu na nkezi olu mitochondrial, o yikarịrị ka biosynthesis na-abawanye. Agbanyeghị, data dị na Foto 2 na-akwado ịdị adị nke usoro nkwụghachi ụgwọ abụọ: mmụba na ọnụọgụ nke ihe omume fission, kwekọrọ na mmụba nke mitochondrial fission, na mmụba na ọnụọgụ nke ihe omume, kwekọrọ na mitochondrial biogenesis. N'ikpeazụ, ụgwọ ọrụ dị ike maka nrụgide dị nro nwere ike ịgụnye usoro n'otu oge gụnyere fission, fusion, biogenesis, na mitophagy. Ọ bụ ezie na ndị edemede gara aga egosila na PPA na-eme ka mitosis30,39 na mitophagy29 dịkwuo mma, anyị na-enye ihe akaebe maka imegharị mitochondrial fission na fusion dynamics na nzaghachi na PPA. Data ndị a na-akwado mgbanwe morphological nke TEM hụrụ ma na-enye nghọta ọzọ na usoro metụtara ọrụ mitochondrial nke PPA kpatara.
Ebe ọ bụ na nyocha TEM ma ọ bụ MEL enyeghị ihe akaebe kpọmkwem nke usoro nhazi mkpụrụ ndụ ihe nketa nke na-adabere na mgbanwe morphological a hụrụ, anyị nyochara ngosipụta RNA nke mkpụrụ ndụ ihe nketa metụtara na mitochondrial metabolism, biogenesis, na dynamics. CMYC proto-oncogene bụ ihe ntụgharị okwu nke metụtara nhazi nke mitochondria, glycolysis, amino acid na fatty acid metabolism40. Na mgbakwunye, a maara cMYC ka ọ na-achịkwa ngosipụta nke ihe fọrọ nke nta ka ọ bụrụ mkpụrụ ndụ ihe nketa mitochondrial 600 metụtara na mitochondrial transcription, translation, na mgbakọ mgbagwoju anya, gụnyere NRF1 na TFAM41. NRF1 na TFAM bụ ndị na-achịkwa etiti abụọ nke mitosis, na-arụ ọrụ n'okpuru PGC-1α iji mee ka mmịpụta mtDNA rụọ ọrụ. A na-eme ka ụzọ a rụọ ọrụ site na cAMP na AMPK signaling ma na-enwe mmetụta maka mmefu ike na nrụgide metabolic. Anyị nyochakwara NFE2L2, onye na-achịkwa redox nke mitochondrial biogenesis, iji chọpụta ma mmetụta nke PPA nwere ike ịbụ nke nrụgide oxidative kpatara.
Ọ bụ ezie na ngosipụta NFE2L2 agbanwebeghị, anyị hụrụ mbelata dabere na dose mgbe niile na ngosipụta nke cMYC, NRF1 na TFAM mgbe awa 24 nke ọgwụgwọ na 3 mM na 5 mM PPA (Foto 3a–c). Akọpụtala mbelata nke ngosipụta cMYC dị ka nzaghachi maka nrụgide mitochondrial42, n'aka nke ọzọ, mbelata nke ngosipụta cMYC nwere ike ibute nsogbu mitochondrial site na imeghari metabolism mitochondrial, njikọ netwọk, na polarization membrane43. N'ụzọ na-adọrọ mmasị, cMYC na-etinyekwa aka na nhazi nke mitochondrial fission na fusion42,43 ma a maara na ọ na-eme ka DRP1 phosphorylation na mpaghara mitochondrial dịkwuo elu n'oge nkewa sel44, yana imezigharị mitochondrial morphological na mkpụrụ ndụ akwara neuronal45. N'ezie, fibroblasts ndị na-enweghị cMYC na-egosipụta obere nha mitochondrial, kwekọrọ na mgbanwe nke nrụgide PPA43 kpatara. Data ndị a na-egosi mmekọrịta na-adọrọ mmasị mana nke edoghị anya n'etiti cMYC na mitochondrial dynamics, na-enye ihe mgbaru ọsọ na-adọrọ mmasị maka ọmụmụ ihe n'ọdịnihu nke nhazi nrụgide PPA kpatara.
Mbelata nke NRF1 na TFAM kwekọrọ na ọrụ nke cMYC dị ka ihe na-eme ka mkpụrụ ndụ kansa eriri afọ mmadụ rụọ ọrụ. Data ndị a kwekọkwara na ọmụmụ ihe gara aga na mkpụrụ ndụ kansa eriri afọ mmadụ na-egosi na PPA belatara ngosipụta NRF1 mRNA na awa 22, nke jikọtara ya na mbelata ATP na mmụba ROS46. Ndị edemede a kọkwara na ngosipụta TFAM mụbara na awa 8.5 mana ọ laghachiri na ọkwa mmalite na awa 22. N'ụzọ dị iche, Kim et al. (2019) gosiri na ngosipụta TFAM mRNA belatara nke ukwuu mgbe awa 4 nke nrụgide PPA gasịrị na mkpụrụ ndụ SH-SY5Y; agbanyeghị, mgbe awa 72 gasịrị, ngosipụta protein TFAM mụbara nke ukwuu ma ọnụọgụ mtDNA mụbara nke ukwuu. Ya mere, mbelata na ọnụọgụ mkpụrụ ndụ ihe nketa biogenesis mitochondrial nke anyị hụrụ mgbe awa 24 gachara anaghị ewepụ ohere na mmụba nke ọnụọgụ mitochondria jikọtara ya na mkpali nke biogenesis na oge mbụ. Nnyocha ndị gara aga egosila na PPA na-eme ka PGC-1α mRNA na protein dịkwuo elu nke ukwuu na mkpụrụ ndụ SH-SY5Y n'ime awa 4 na nkeji 30, ebe propionic acid na-eme ka mitochondrial biogenesis dịkwuo mma na calf hepatocytes site na PGC-1α na awa 12 na nkeji 39. N'ụzọ na-akpali mmasị, PGC-1α abụghị naanị onye na-achịkwa NRF1 na TFAM kpọmkwem, kamakwa egosila na ọ na-achịkwa ọrụ nke MFN2 na DRP1 site na ịchịkwa fission na fusion47. Nke a na-egosipụta njikọ chiri anya nke usoro na-achịkwa nzaghachi nkwụghachi mitochondrial nke PPA kpatara. Ọzọkwa, data anyị na-egosipụta nnukwu mmebi nke iwu ntụgharị nke biogenesis na metabolism n'okpuru nrụgide PPA.
Mkpụrụ ndụ ihe nketa STOML2, OPA1, MFN1, MFN2 na DRP1 so n'ime ndị na-achịkwa mitochondrial fission, fusion na dynamics37,48,49. E nwere ọtụtụ mkpụrụ ndụ ihe nketa ndị ọzọ metụtara mitochondrial dynamics, agbanyeghị, achọpụtala na mbụ na STOML2, OPA1 na MFN2 nwere methylation dị iche iche na ASD cohorts,16 na ọtụtụ ọmụmụ onwe ha akọpụtala mgbanwe na ihe ndị a na-edegharị na nzaghachi maka nrụgide mitochondrial50,51. 52. Ọgwụgwọ PPA 3 mM na 5 mM belatara ngosipụta nke ma OPA1 na STOML2 nke ukwuu (Fig. 3e, f). OPA1 bụ otu n'ime ndị na-achịkwa mitochondrial fusion site na mmekọrịta kpọmkwem na MFN1 na 2 ma na-arụ ọrụ na mgbanwe cristae na mitochondrial morphology53. Ọrụ kpọmkwem nke STOML2 na mitochondrial dynamics ka edoghị anya, mana ihe akaebe na-egosi na ọ na-arụ ọrụ na mitochondrial fusion, biogenesis, na mitophagy.
STOML2 na-etinye aka n'ịnọgide na-enwe njikọ iku ume mitochondrial na ịmepụta mgbagwoju anya nke usoro iku ume54,55 ma egosila na ọ na-agbanwe àgwà metabolic nke mkpụrụ ndụ kansa56. Ọmụmụ ihe egosila na STOML2 na-akwalite ikike nke membrane mitochondrial na biogenesis site na mmekọrịta ya na BAN na cardiolipin 55, 57, 58. Na mgbakwunye, ọmụmụ ihe onwe onye egosila na mmekọrịta dị n'etiti STOML2 na PINK1 na-achịkwa mitophagy59,60. N'ụzọ dị mkpa, akọpụtala na STOML2 na-emekọrịta ihe ozugbo na ime ka MFN2 kwụsie ike ma na-arụkwa ọrụ dị mkpa n'ịkwado isoforms OPA1 ogologo site na igbochi protease nke na-akpata mbibi OPA153,61,62. Mbelata na ngosipụta STOML2 a hụrụ na mmeghachi omume PPA nwere ike ime ka protein njikọ ndị a dịkwuo mfe ịdaba site na ụzọ ubiquitin na proteasome-dependent48. Ọ bụ ezie na ọrụ kpọmkwem nke STOML2 na OPA1 na nzaghachi ike na PPA edoghị anya, mbelata ngosipụta nke mkpụrụ ndụ ihe nketa njikọta ndị a (Foto 3) nwere ike imebi nguzozi dị n'etiti fission na fusion ma mee ka nha mitochondrial belata (Foto 3). 1).
N'aka nke ọzọ, ngosipụta protein OPA1 agbanweghị agbanwe mgbe awa 24 gachara, ebe ọkwa mRNA na protein nke MFN1, MFN2 ma ọ bụ DRP1 agbanweghị nke ukwuu mgbe ọgwụgwọ PPA gasịrị (Foto 3g-i, Fig. 4). Nke a nwere ike igosi na enweghị mgbanwe ọ bụla na nhazi nke ihe ndị a metụtara njikọ na fission mitochondrial. Agbanyeghị, ọ dị mkpa ịmara na a na-achịkwa mkpụrụ ndụ ihe nketa anọ a site na mgbanwe posttranscriptional (PTMs) nke na-achịkwa ọrụ protein. OPA1 nwere ụdị splice asatọ ọzọ nke kewara na mitochondria iji mepụta isoforms abụọ dị iche 63. Nguzozi dị n'etiti isoforms ogologo na mkpụmkpụ na-ekpebi ọrụ nke OPA1 na njikọ mitochondrial na mmezi nke netwọk mitochondrial64. A na-achịkwa ọrụ DRP1 site na calcium/calmodulin-dependent protein kinase II (CaMKII) phosphorylation, ebe ubiquitination na SUMOylation65 na-achịkwa mmebi DRP1. N'ikpeazụ, ma DRP1 na MFN1/2 bụ GTPases, yabụ ọrụ nwere ike imetụta ọnụego mmepụta GTP na mitochondria 66. Ya mere, ọ bụ ezie na ngosipụta nke protein ndị a ka na-adịgide adịgide, nke a nwere ike ọ gaghị egosipụta ọrụ protein na-agbanweghi agbanwe ma ọ bụ mpaghara67,68. N'ezie, nchịkọta protein PTM dị adị na-abụkarị ụzọ mbụ nke nchebe nke na-ahụ maka nzaghachi nrụgide dị oke njọ. N'inwe nrụgide metabolic dị ala na ụdị anyị, o yikarịrị ka PTM na-akwalite mmụba nke ọrụ nke fusion na protein fission iji weghachite iguzosi ike n'ezi ihe mitochondrial nke ọma na-achọghị mmegharị ọzọ nke mkpụrụ ndụ ihe nketa ndị a na ọkwa mRNA ma ọ bụ protein.
Ijikọta data dị n'elu, data ndị a na-egosipụta usoro mgbagwoju anya na nke dabere na oge nke mitochondrial morphology na ihe ịma aka nke ịkọwa usoro ndị a. Iji mụọ ngosipụta mkpụrụ ndụ ihe nketa, ọ dị mkpa mbụ ịchọpụta mkpụrụ ndụ ihe nketa kpọmkwem n'ụzọ ahụ. Agbanyeghị, data anyị na-egosi na mkpụrụ ndụ ihe nketa dị n'otu ụzọ ahụ anaghị azaghachi otu nrụgide ahụ. N'ezie, ọmụmụ ihe ndị gara aga egosila na mkpụrụ ndụ ihe nketa dị iche iche n'otu ụzọ ahụ nwere ike igosipụta profaịlụ nzaghachi oge dị iche iche30,46. Na mgbakwunye, enwere usoro mgbagwoju anya mgbe edechara ihe nke na-emebi mmekọrịta dị n'etiti ntụgharị na ọrụ mkpụrụ ndụ ihe nketa. Ọmụmụ ihe Proteomic nwere ike inye nghọta banyere mmetụta nke PTMs na ọrụ protein, mana ha na-ewetakwa ihe ịma aka gụnyere ụzọ mmepụta dị ala, oke mgbaàmà-na-mkpọtụ dị elu, na mkpebi na-adịghị mma.
N'ọnọdụ a, ịmụ gbasara ọdịdị mitochondrial site na iji TEM na MEL nwere nnukwu ikike ịza ajụjụ ndị bụ isi gbasara mmekọrịta dị n'etiti ọnọdụ mitochondrial na ọrụ na otu nke a si emetụta ọrịa. Nke kachasị mkpa, TEM na-enye ụzọ kpọmkwem maka ịtụ ọdịdị mitochondrial dị ka njedebe njikọta nke nsogbu mitochondrial na ọnọdụ dynamics51. MEL na-enyekwa ụzọ kpọmkwem maka ịhụ ihe omume fission na fusion na gburugburu sel atọ, na-enye ohere ịhazi mgbanwe mitochondrial na-agbanwe agbanwe ọbụlagodi na enweghị mgbanwe na ngosipụta mkpụrụ ndụ ihe nketa33. N'ebe a, anyị na-akọwapụta uru nke usoro onyonyo mitochondrial na ọrịa mitochondrial nke abụọ. A na-ahụkarị ọrịa ndị a site na nrụgide metabolic dị nro nke a na-akọwa site na mgbanwe dị nro nke netwọk mitochondrial kama mmebi mitochondrial dị njọ. Agbanyeghị, nkwụghachi mitochondrial achọrọ iji nọgide na-enwe mitosis n'okpuru nrụgide na-adịghị ala ala nwere nnukwu nsonaazụ ọrụ. N'ihe gbasara neuroscience, nghọta ka mma nke usoro nkwụghachi ụgwọ ndị a nwere ike inye ozi dị mkpa gbasara neuropathology pleiotropic metụtara dysfunction mitochondrial.
N'ikpeazụ, data anyị na-egosi uru nke usoro onyonyo maka ịghọta ihe na-esi na mmekọrịta dị mgbagwoju anya dị n'etiti ngosipụta mkpụrụ ndụ ihe nketa, mgbanwe protein, na ọrụ protein nke na-achịkwa usoro mitochondrial neuronal pụta. Anyị jiri PPA mee ihe nlereanya mitochondrial dysfunction na ụdị mkpụrụ ndụ neuronal iji nweta nghọta na akụkụ mitochondrial nke ASD. Mkpụrụ ndụ SH-SY5Y ejiri PPA gwọọ gosiri mgbanwe na morphology mitochondrial: mitochondria ghọrọ obere ma gbaa gburugburu, a kọwaghịkwa cristae nke ọma mgbe TEM hụrụ ya. Nyocha MEL na-egosi na mgbanwe ndị a na-eme n'otu oge na mmụba na ihe omume fission na fusion iji nọgide na-enwe netwọk mitochondrial na nzaghachi maka nrụgide metabolic dị nro. Ọzọkwa, PPA na-akpaghasị iwu ntụgharị nke mitochondrial metabolism na homeostasis nke ukwuu. Anyị chọpụtara cMYC, NRF1, TFAM, STOML2, na OPA1 dị ka ndị na-achịkwa mitochondrial bụ isi nke nrụgide PPA mebiri ma nwee ike ịrụ ọrụ n'ịgbaso mgbanwe PPA kpatara na morphology na ọrụ mitochondrial. Ọmụmụ ihe ndị ga-eme n'ọdịnihu dị mkpa iji kọwaa mgbanwe oge nke PPA kpatara na ngosipụta mkpụrụ ndụ ihe nketa na ọrụ protein, mpaghara, na mgbanwe mgbe ntụgharị. Data anyị na-egosipụta mgbagwoju anya na njikọ aka nke usoro iwu na-eme ka nzaghachi nrụgide mitochondrial dị irè ma gosipụta uru nke TEM na usoro onyonyo ndị ọzọ maka ọmụmụ ihe mekaniki lekwasịrị anya.
A zụtara mkpụrụ ndụ SH-SY5Y (ECACC, 94030304-1VL) site na Sigma-Aldrich. A zụlitere mkpụrụ ndụ SH-SY5Y na ngwakọta nri etiti/F-12 nke Dulbecco gbanwere (DMEM/F-12) na L-glutamine (SC09411, ScienCell) na karama 25 cm2 nke e tinyere 20% fetal bovine serum (FBS) (10493106, ThermoFisher Scientific) na 1% penicillin-streptomycin (P4333-20ML, Sigma-Aldrich) na 37 °C, 5% CO2. E tinyere mkpụrụ ndụ ndị a n'ime 80% confluence site na iji 0.05% trypsin-EDTA (15400054, ThermoFisher Scientific), centrifuged na 300 g ma kpuchie ya na njupụta nke ihe dị ka 7 × 105 mkpụrụ ndụ/ml. Emere nnwale niile na mkpụrụ ndụ SH-SY5Y na-enweghị ọdịiche dị n'etiti ngafe 19-22. A na-enye PPA dị ka NaP. Na-agbaze ntụ NaP (CAS Nke 137-40-6, usoro kemịkalụ C3H5NaO2, P5436-100G, Sigma-Aldrich) na mmiri MilliQ ọkụ ruo njupụta nke 1 M ma chekwaa na 4 °C. N'ụbọchị ọgwụgwọ ahụ, gwakọta ngwọta a na 1 M PPA ruo 3 mM na 5 mM PPA na ihe na-enweghị serum (DMEM/F-12 na L-glutamine). Ọnụọgụ ọgwụgwọ maka nnwale niile abụghị PPA (0 mM, njikwa), 3 mM, na 5 mM PPA. Emere nnwale na opekata mpe ihe atọ dị ndụ.
A kụrụ mkpụrụ ndụ SH-SY5Y n'ime karama 25 cm5 na ọnụego nke mkpụrụ ndụ 5.5 × 105/ml ma too ha ruo awa 24. Agbakwunyere ọgwụgwọ PPA na karama ahụ tupu awa 24 nke ịbanye. Chịkọta mkpụrụ ndụ dịka usoro omenala anụ anụmanụ si dị (akọwapụtara n'elu). Tinyeghachi mkpụrụ ndụ ahụ na 100 µl 2.5% glutaraldehyde, 1 × PBS ma chekwaa ya na 4 °C ruo mgbe a ga-ahazi ya. E tinyere mkpụrụ ndụ SH-SY5Y obere oge iji mee ka mkpụrụ ndụ ahụ dị nro ma wepụ 2.5% glutaraldehyde, 1 × PBS ngwọta. Tinyeghachi mkpụrụ ndụ ahụ na gel agarose 4% nke a kwadebere na mmiri a gwakọtara agwakọta (oke nke agarose na olu sediment bụ 1:1). E tinyere iberibe Agarose na grid na efere dị larịị ma tee ya 1-hexadecene tupu a kpụọ ya n'elu nrụgide dị elu. E tinyere ihe nlele n'ime acetone akọrọ 100% na -90°C maka awa 24. E wee bulie okpomọkụ ahụ ruo -80°C ma tinye ihe ngwọta nke 1% osmium tetroxide na 0.1% glutaraldehyde. E debere ihe nlele na -80°C ruo awa 24. Mgbe nke a gasịrị, a na-amụba okpomọkụ ahụ nwayọ nwayọ ruo okpomọkụ ụlọ ruo ọtụtụ ụbọchị: site na - 80°C ruo - 50°C ruo awa 24, ruo - 30°C maka awa 24, ruo - 10°C maka awa 24 na n'ikpeazụ ruo okpomọkụ ụlọ.
Mgbe nkwadebe cryogenic gasịrị, e tinyere resin n'ime ihe nlele ndị ahụ ma jiri Leica Reichert UltracutS ultramicrotome (Leica Microsystems) mee akụkụ ultrathin (ihe dị ka nm 100). E jiri 2% uranyl acetate na lead citrate mee akụkụ ndị ahụ. E ji igwe microscope elektrọn nnyefe FEI Tecnai 20 (ThermoFisher (nke bụbu FEI), Eindhoven, Netherlands) na-arụ ọrụ na 200 kV (onye na-ebufe Lab6) na igwefoto Gatan CCD (Gatan, UK) nke nwere nzacha ike Tridiem hụ ihe nlele ndị ahụ.
N'ime ihe nlereanya teknụzụ ọ bụla, e nwetara opekata mpe onyonyo otu sel 24, nke ruru mkpokọta onyonyo 266. E jiri macro Mpaghara Mmasị (ROI) na macro Mitochondria nyochaa onyonyo niile. Mitochondria macro dabere na usoro ebipụtara17,31,32 ma na-enye ohere nhazi ogbe ọkara akpaaka nke onyonyo TEM na Fiji/ImageJ69. Na nkenke: a na-atụgharị onyonyo ahụ ma tụgharịa ya site na iji mwepu ndabere bọọlụ rolling (radius pixel 60) na nzacha bandpass FFT (na-eji oke elu na ala 60 na 8 pixel n'otu n'otu) na mwepu ahịrị kwụ ọtọ yana nnabata ntụzịaka nke 5%. A na-eji algọridim entropy kachasị elu eme ihe onyonyo a na-ahazi na akpaghị aka ma mepụta ihe mkpuchi binary. E wepụtara mpaghara onyonyo ejikọtara ya na ROI ahọpụtara aka na onyonyo TEM raw, na-akọwa mitochondria ma wepụ akpụkpọ ahụ plasma na mpaghara ndị ọzọ dị oke iche. Maka ROI ọ bụla ewepụtara, a tụlere ihe dị ka pikselụ abụọ karịrị 600, a tụkwara mpaghara ihe dị ka mpekere, gburugburu, axes ukwu na obere, dayameta Feret, gburugburu, na okirikiri site na iji ọrụ nha arụnyere n'ime nke Fiji/ImageJ. Site na Merrill, Flippo, na Strack (2017), mpaghara 2, oke akụkụ ihe dị ka mpekere (oke ruo obere axes), na ihe na-egosi ọdịdị (FF) site na data ndị a, ebe FF = gburugburu 2/4pi x mpaghara. Enwere ike ịchọta nkọwa nke usoro parametric na Merrill, Flippo, na Strack (2017). Macros ndị a kpọtụrụ aha dị na GitHub (lee Nkwupụta Nnweta Data). Na nkezi, a tụlere ihe dị ka mpekere 5,600 kwa ọgwụgwọ PPA, maka ngụkọta ihe dị ka mpekere 17,000 (egosighi data).
E tinyere mkpụrụ ndụ SH-SH5Y n'ime efere ọdịbendị nke nwere ọnụ ụlọ asatọ (ThermoFisher, #155411) iji kwe ka ha rapara n'otu abalị wee tinye ha na TMRE 1:1000 (ThermoFisher, #T669) na Hoechst 33342 1:200 (Sigma-Aldrich, H6024). agba agba. E ji laser 405 nm na 561 nm nweta onyonyo n'ime nkeji iri, e wee nweta onyonyo ndị a na-anaghị ahụ anya dị ka z-stacks nwere micrograph onyonyo 10 nwere usoro az nke 0.2 μm n'etiti etiti onyonyo na oge 12 sochirinụ. E jiri ikpo okwu Carl Zeiss LSM780 ELYRA PS.1 super-resolution (Carl Zeiss, Oberkochen, Germany) chịkọta onyonyo ndị a. E jiri usoro e ji arụ ọrụ na ImageJ na mgbakwunye ImageJ nyochaa onyonyo ndị a iji tụọ ihe omume njikọ na mgbawa, ọnụọgụgụ nke nhazi mitochondrial, na nkezi olu mitochondrial kwa sel33. Macros MEL dị na GitHub (lee Nkwupụta Nnweta Data).
A zụlitere mkpụrụ ndụ SH-SY5Y n'ime efere olulu isii na njupụta nke mkpụrụ ndụ 0.3 × 106/mL ruo awa 24 tupu a gwọọ ha. E jiri usoro Quick-RNA™ Miniprep (ZR R1055, Zymo Research) wepụta RNA ya na obere mgbanwe: tinye 300 μl nke RNA lysis buffer na olulu mmiri ọ bụla tupu ewepụ ya ma tee ihe nlele ọ bụla dịka nzọụkwụ ikpeazụ site na iji 30 μl nke mmiri DNase/RNase. -free. E jiri NanoDrop ND-1000 UV-Vis Spectrophotometer lelee ihe nlele niile maka ọnụọgụ na ịdị mma ha. E jiri 200 μl RIPA lysis buffer nweta protein zuru oke sitere na mkpụrụ ndụ lysates, e wee tụọ mkpokọta protein site na iji nnwale protein Bradford70.
E ji Tetro™ cDNA Synthesis Kit (BIO-65043, Meridian Bioscience) mee njikọ cDNA dịka ntuziaka onye nrụpụta si dị, ma gbanwee ụfọdụ mgbanwe. E ji 0.7 ruo 1 μg nke RNA zuru oke mepụta cDNA na mmeghachi omume 20-μl. E si na akwụkwọ ebipụtara na mbụ 42, 71, 72, 73, 74, 75, 76, 77, 78 (Tebụl S1) họrọ primers, e jikwa ngwa PrimerQuest sitere na Integrated DNA Technologies mepụta nyocha ndị so ya. E mere ka mkpụrụ ndụ ihe nketa niile dị mkpa ka ọ bụrụ mkpụrụ ndụ ihe nketa nuklia B2M. E jiri RT-qPCR tụọ ngosipụta mkpụrụ ndụ ihe nketa nke STOML2, NRF1, NFE2L2, TFAM, cMYC na OPA1. Ngwakọta ukwu ahụ gụnyere LUNA Taq polymerase (M3003L, New England Biolabs), 10 μM praịmarị n'ihu na nke azụ, cDNA, na mmiri PCR iji nye olu ikpeazụ nke 10 μL maka mmeghachi omume ọ bụla. Ejiri nnwale TaqMan multiplex tụọ ngosipụta nke mkpụrụ ndụ ihe nketa nkewa na nkewa (DRP1, MFN1/2). Ejiri ntuziaka onye nrụpụta Luna Universal Probe qPCR Master Mix (M3004S, New England Biolabs) mee ihe dịka ntuziaka onye nrụpụta si dị yana obere mgbanwe. Ngwakọta ukwu RT-qPCR multiplex gụnyere 1X LUNA Taq polymerase, 10 μM praịmarị n'ihu na nke azụ, 10 μM probe, cDNA, na mmiri PCR, nke butere olu ikpeazụ nke 20 μL maka mmeghachi omume ọ bụla. Ejiri Rotor-Gene Q 6-plex (QIAGEN RG—nọmba usoro: R0618110) mee RT-qPCR. E gosiri ọnọdụ ịgba ígwè na Tebụl S1. E mere ka ihe nlele cDNA niile dịkwuo elu n'ụzọ atọ, e wee jiri usoro nke ịgbakọta okpukpu iri mepụta usoro ọkọlọtọ. E wepụrụ ihe ndị dị n'ime ihe nlele atọ nwere mgbanwe ọkọlọtọ okirikiri (Ct) >0.5 na nyocha ahụ iji hụ na enwere ike imegharị data30,72. E jiri usoro 2-ΔΔCt79 gbakọọ ngosipụta mkpụrụ ndụ ihe nketa dị n'otu.
E tinyere ihe nlele protein (60 μg) na ihe nchekwa ibu Laemmli na nha 2:1 ma jiri jel protein 12% na-enweghị agba (Bio-Rad #1610184) mee ihe. E zigara protein na membrane PVDF (polyvinylidene fluoride) (#170-84156, Bio-Rad) site na iji sistemụ Trans-Blot Turbo (#170-4155, Bio-Rad). E mechiri akpụkpọ ahụ ma tinye ọgwụ mgbochi mbụ kwesịrị ekwesị (OPA1, MFN1, MFN2, na DRP1) (agbazere 1:1000) ruo awa 48, wee tinye ya na ọgwụ mgbochi nke abụọ (1:10,000) ruo otu awa. E jiri Clarity Western ECL Substrate (#170-5061, Bio-Rad) see foto akpụkpọ ahụ ma dekọọ ya site na iji sistemụ Bio-Rad ChemiDoc MP. E jiri ụdị ImageLab 6.1 mee ihe maka nyocha Western blot. E gosiri gel na blot mbụ ahụ na Foto S1. E nyere ozi gbasara ihe ndị na-egbochi mmadụ na Tebụl S2.
A na-egosi nhazi data dị ka mmejọ nkezi na ọkọlọtọ nke nkezi (SEM) nke ihe atụ atọ nọọrọ onwe ha ma ọ dịkarịa ala. E jiri ule Shapiro-Wilks nwalee nhazi data maka ịdị n'otu tupu e were nkesa Gaussian na nha nhata ọkọlọtọ ma gaa n'ihu na nyocha ahụ. Na mgbakwunye na inyocha nhazi data site na iji Fisher's MEL LSD (p < 0.05), otu ụzọ ANOVA (ọgwụgwọ vs. njikwa nkezi), na ule ntụnyere ọtụtụ Dunnett iji chọpụta mkpa (p < 0.05). Egosiri ụkpụrụ p dị mkpa na eserese ahụ dị ka *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. E mere ma mepụta nyocha na eserese ọnụ ọgụgụ niile site na iji GraphPad Prism 9.4.0.
Enwere ike ịchọta makros Fiji/ImageJ maka nyocha onyonyo TEM n'ihu ọha na GitHub: https://github.com/caaja/TEMMitoMacro. A na-enweta makros Mitochondrial Event Locator (MEL) n'ihu ọha na GitHub: https://github.com/rensutheart/MEL-Fiji-Plugin.
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