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Propionic acid (PPA) inoshandiswa kudzidza basa rekusashanda zvakanaka kwemitochondrial muzvirwere zve neurodevelopmental zvakadai se autism spectrum disorder. PPA inozivikanwa nekukanganisa mitochondrial biogenesis, metabolism, uye turnover. Zvisinei, migumisiro yePPA pa mitochondrial dynamics, fission uye fusion inoramba iri dambudziko nekuda kwehunhu hwakaoma hwenguva hwema mechanisms aya. Pano, tinoshandisa matekiniki e complementary quantitative imaging kuti tiongorore kuti PPA inokanganisa sei mitochondrial ultrastructure, morphology, uye dynamics mumasero e SH-SY5Y akafanana ne neuron. PPA (5 mM) yakakonzera kudzikira kukuru kwenzvimbo ye mitochondrial (p < 0.01), dhayamita yeFeret uye circumference (p < 0.05), uye nzvimbo 2 (p < 0.01). Kuongororwa kweMitochondrial event locator kwakaratidza kuwedzera kukuru (p < 0.05) muzviitiko zve fission uye fusion, nokudaro ichichengetedza kusimba kwe network ye mitochondrial pasi pemamiriro ekumanikidzwa. Pamusoro pezvo, kuratidzwa kwe mRNA kwe cMYC (p < 0.0001), NRF1 (p < 0.01), TFAM (p < 0.05), STOML2 (p < 0.0001) uye OPA1 (p < 0.05) kwakaderedzwa zvakanyanya. 01). Izvi zvinoratidza kugadziriswa kwechimiro che mitochondrial, biogenesis uye dynamics kuti irambe ichishanda mumamiriro ekumanikidzwa. Data redu rinopa nzwisiso itsva pamusoro pemhedzisiro yePPA pa mitochondrial dynamics uye rinoratidza kushanda kwehunyanzvi hwekufungidzira pakudzidza nzira dzakaoma dzekutonga dzinobatanidzwa mumhinduro dzekushushikana kwe mitochondrial.
Mitochondria inhengo dzakakosha mumabasa akasiyana-siyana emasero kunze kwebasa rawo rakajairika mukugadzira simba uye biosynthesis. Mitochondria metabolism ndiyo inonyanya kukosha mukutonga kwecalcium signaling, metabolic uye redox homeostasis, inflammatory signaling, epigenetic modifications, masero ekuwedzera, differentiation uye programmed cell death1. Kunyanya, mitochondrial metabolism yakakosha pakukura kwetsinga, kurarama uye kushanda uye inonyanya kutaurwa muzviratidzo zvakasiyana-siyana zve neuropathology2,3,4.
Mumakore gumi apfuura, mamiriro emetabolism akabuda senzira huru yekudzora neurogenesis, differentiation, maturation uye plasticity5,6. Munguva pfupi yapfuura, mitochondrial morphology uye dynamics zvave zvikamu zvakakosha zve mitosis, nzira inochinja inochengetedza dziva re mitochondria ine hutano mukati memasero. Mitochondrial dynamics inodzorwa nenzira dzakaoma dzinoenderana kubva pa mitochondrial biogenesis uye bioenergetics kusvika pa mitochondrial fission, fusion, transport and clearance7,8. Kukanganiswa kwechero yeiyi integrative mechanisms kunokanganisa kuchengetedza hutano hwe mitochondrial networks uye kune mhedzisiro yakakura yekushanda kwe neurodevelopment9,10. Zvechokwadi, kusashanda zvakanaka kwe mitochondrial dynamics kunoonekwa muzvirwere zvakawanda zvepfungwa, neurodegenerative uye neurodevelopmental, kusanganisira autism spectrum disorders (ASD)11,12.
ASD chirwere chepfungwa chakasiyana-siyana chine maitiro akaomarara emajini uye epigenetic. Kugara nhaka kweASD hakuna mubvunzo, asi chikonzero chemorekuru chiripo hachina kunzwisiswa zvakanaka. Kuunganidza data kubva kumamodheru epamberi pekiriniki, zvidzidzo zvekiriniki, uye multi-omics molecular datasets zvinopa humbowo huri kuwedzera hwekusashanda zvakanaka kwemitochondrial muASD13,14. Takamboita genome-wide DNA methylation screen muboka revarwere vane ASD uye takaona majini ane methylated akasiyana-siyana akaunganidzwa munzira dzemitochondrial metabolic15. Takazotaura nezve differential methylation yepakati pemitochondrial biogenesis uye dynamics, iyo yakabatana nekuwedzera kwenhamba yekopi yemtDNA uye kuchinja kwehuwandu hwemetabolism yeurinary muASD16. Data redu rinopa humbowo huri kuwedzera hwekuti mitochondrial dynamics uye homeostasis zvinoita basa guru mu pathophysiology yeASD. Nokudaro, kuvandudza kunzwisisa kwemechanistic kwehukama huripo pakati pemitochondrial dynamics, morphology, uye basa chinangwa chikuru chekutsvagisa kuri kuenderera mberi muzvirwere zvetsinga zvinoratidzwa nekusashanda zvakanaka kwemitochondrial secondary.
Maitiro emamorekuru anowanzo shandiswa kudzidza basa remajini chaiwo mumhinduro dzekushushikana kwemitochondrial. Zvisinei, nzira iyi inogona kuganhurirwa nehunhu hwakawanda uye hwenguva hwemitotic control mechanisms. Uyezve, kusiyana kwemajini emitochondrial chiratidzo chisina kunanga chekuchinja kwebasa, kunyanya sezvo huwandu hushoma hwemajini hunowanzo ongororwa. Nokudaro, nzira dzakananga dzekudzidza basa remitochondrial uye bioenergetics dzakakurudzirwa17. Maumbirwo emitochondrial ane hukama hwakanyanya nekuchinjana kwemitochondrial. Chimiro chemitochondrial, kubatana, uye chimiro zvakakosha pakugadzirwa kwesimba uye kupona kwemitochondrial nemasero5,18. Uyezve, zvikamu zvakasiyana-siyana zvemitosis zvinotarisa pakuchinja muumbirwo wemitochondrial, izvo zvinogona kushanda semagumo anobatsira ekusashanda zvakanaka kwemitochondrial uye zvinopa hwaro hwezvidzidzo zvinotevera zvemakanika.
Maumbirwo eMitochondrial anogona kuonekwa zvakananga uchishandisa transmission electron microscopy (TEM), zvichibvumira kudzidza kwakadzama kwe cellular ultrastructure. TEM inoona zvakananga maumbirwo, chimiro uye chimiro che mitochondrial cristae pakugadziriswa kwe mitochondria yega yega, pane kuvimba chete ne gene transcription, protein expression kana mitochondrial functional parameters mu cell populations17,19,20. Pamusoro pezvo, TEM inobatsira kudzidza kudyidzana pakati pe mitochondria nedzimwe organelles, dzakadai se endoplasmic reticulum uye autophagosomes, dzinoita basa guru mu mitochondrial function uye homeostasis21,22. Saka, izvi zvinoita kuti TEM ive nzvimbo yakanaka yekutanga kudzidza kusashanda zvakanaka kwe mitochondrial usati watarisa panzira kana majini chaiwo. Sezvo basa re mitochondrial richiva rakakosha kune neuropathology, pane chikonzero chakajeka chekukwanisa kudzidza zvakananga uye nehuwandu hwe mitochondrial morphology uye dynamics mu in vitro neuronal models.
Muchinyorwa chino, tinoongorora mitochondrial dynamics mu neuronal model ye mitochondrial dysfunction mu autism spectrum disorder. Takambotaura nezve differential methylation ye propionyl-CoA carboxylase beta (PCCB) mu ASD15, chikamu chidiki che mitochondrial propionyl-CoA carboxylase enzyme PCC. Kusashanda zvakanaka kwePCC kunozivikanwa nekukonzera kuunganidzwa kwechepfu kwepropionyl derivatives, kusanganisira propionic acid (PPA)23,24,25. PPA yakaratidzwa kuti inokanganisa metabolic ye neuronal uye inochinja maitiro mumuviri uye imhando yemhuka yakasimbiswa yekudzidza neurodevelopmental mechanisms inobatanidzwa mu ASD26,27,28. Pamusoro pezvo, PPA yakaonekwa kuti inokanganisa mitochondrial membrane potential, biogenesis uye respiration in vitro uye yakashandiswa zvakanyanya kuratidza kusashanda zvakanaka kwe mitochondrial mu neurons29,30. Zvisinei, kukanganisa kusashanda zvakanaka kwe mitochondrial kunokonzerwa nePPA pa mitochondrial morphology uye dynamics hakusati kwanzwisiswa zvakanaka.
Chidzidzo ichi chinoshandisa matekiniki ekuwedzera mifananidzo kuti chiverengere mhedzisiro yePPA pachimiro chemitochondrial, dynamics, uye mashandiro mumasero eSH-SY5Y. Kutanga, takagadzira nzira yeTEM yekuona shanduko muchimiro chemitochondrial uye ultrastructure17,31,32. Tichifunga nezvehunhu hwekuchinja kwemitochondria33, takashandisawo analysis yemitochondrial event localizer (MEL) kuti tiverengere shanduko mukuenzanisa pakati pezviitiko zve fission ne fusion, nhamba yemitochondrial uye vhoriyamu pasi pekumanikidzwa kwePPA. Pakupedzisira, takaongorora kana chimiro chemitochondrial uye dynamics zvakabatana nekuchinja mukutaura kwemajini anobatanidzwa mu biogenesis, fission, uye fusion. Takatora pamwe chete, data redu rinoratidza dambudziko rekujekesa kuoma kwemaitiro anodzora dynamics yemitochondrial. Tinosimbisa kushanda kweTEM mukudzidza chimiro chemitochondrial semhedzisiro inoyerwa ye mitosis mumasero eSH-SY5Y. Pamusoro pezvo, tinoratidza kuti data reTEM rinopa ruzivo rwakapfuma kana rikabatanidzwa nematekiniki ekufungidzira anobatawo zviitiko zvinoshanduka mukupindura kushushikana kwemetabolism. Kutsanangurwa kwakawanda kwemaitiro ekudzora mamorekuru anotsigira mitosis yemasero etsinga kunogona kupa ruzivo rwakakosha nezvechikamu chemitochondrial chehurongwa hwetsinga uye zvirwere zvetsinga.
Kuti zvikonzere kushushikana kwemitochondrial, masero eSH-SY5Y akarapwa nePPA achishandisa 3 mM uye 5 mM sodium propionate (NaP). TEM isati yasvika, sampuli dzakagadzirwa ne cryogenic sample preparation dzichishandisa high-pressure freezing uye freezing (Mufananidzo 1a). Takagadzira automated mitochondrial image analysis pipeline kuti tiongorore ma parameter masere e morphological e mitochondrial populations mu biological replicates matatu. Takaona kuti PPA treatment yakachinja zvakanyanya ma parameter mana: area 2, area, perimeter, uye Feret diameter (Mufananidzo 1b–e). Area 2 yakaderera zvakanyanya ne 3 mM ne 5 mM PPA treatment (p = 0.0183 uye p = 0.002, zvichiteerana) (Mufananidzo 1b), nepo area (p = 0.003), perimeter (p = 0.0106) uye Feret diameter zvese zvakaderera zvakanyanya. Pakava nekudzikira kukuru (p = 0.0172) mu 5 mM treatment group zvichienzaniswa ne control group (Mufananidzo 1c–e). Kudzikira kukuru kwenzvimbo nekutenderera kwakaratidza kuti masero akarapwa ne5 mM PPA aive ne mitochondria diki, yakatenderera, uye kuti mitochondria idzi dzakanga dzisina kureba zvakanyanya pane dziri mumasero ekudzora. Izvi zvinoenderanawo nekuderera kukuru kwedhayamita yeFeret, parameter yakazvimiririra inoratidza kudzikira kwedaro guru pakati pemipendero yezvikamu. Shanduko muchimiro che cristae chakaonekwa: cristae yakava shoma nekuda kwekumanikidzwa kwePPA (Mufananidzo 1a, panel B). Zvisinei, haasi mifananidzo yese yakaratidza zvakajeka chimiro che cristae, saka ongororo yehuwandu hweshanduko idzi haina kuitwa. Aya data eTEM anogona kuratidza mamiriro matatu anogona kuitika: (1) PPA inowedzera fission kana kudzivirira fusion, zvichiita kuti mitochondria iripo iderere muhukuru; (2) biogenesis yakawedzerwa inogadzira mitochondria itsva, diki kana (3) inokonzera mashandiro ese ari maviri panguva imwe chete. Kunyangwe mamiriro aya asingakwanise kusiyaniswa neTEM, shanduko huru dzechimiro dzinoratidza shanduko mu mitochondrial homeostasis uye dynamics pasi pePPA stress. Takazoongorora mamwe ma parameter kuti tiwedzere kutsanangura mashandiro aya uye nzira dzinogona kuitika dziri pasi pawo.
Propionic acid (PPA) inogadzirisa chimiro chemitochondrial. (a) Mifananidzo yekutumira electron microscopy (TEM) inoratidza kuti saizi yemitochondrial inoderera uye mitochondria inova diki uye yakatenderera zvakanyanya nekurapwa kwePPA kuri kuwedzera; 0 mM (isina kurapwa), 3 mM uye 5 mM, zvichiteerana. Miseve mitsvuku inoratidza mitochondria. (b–e) Masero eSH-SY5Y akarapwa nePPA kwemaawa makumi maviri nemana akagadzirirwa TEM uye mhedzisiro yacho yakaongororwa uchishandisa Fiji/ImageJ. Mana emaparamita mana paasere akaratidza mutsauko mukuru pakati pemasero ekudzora (asina kurapwa, 0 mM PPA) uye akarapwa (3 mM uye 5 mM PPA). (b) Dunhu 2, (c) Nzvimbo, (d) Perimeter, (e) Feret dhayamita. Kuongororwa kwega kwega kwekusiyana (kutonga vs. kurapwa) uye bvunzo yekuenzanisa yakawanda yaDunnett yakashandiswa kuona mutsauko wakakosha (p < 0.05). Mapoinzi edata anomiririra avhareji yeukoshi hwemitochondrial yesero rega rega, uye mitsetse yekukanganisa inomiririra avhareji ± SEM. Data rakaratidzwa rinomiririra n = 3, masero angangoita makumi maviri nemana pakudzokorora; mifananidzo inosvika 266 yakaongororwa; * inoratidza p < 0.05, ** inoratidza p < 0.01.
Kuti tiwedzere kutsanangura kuti mitochondrial dynamics inopindura sei kuPPA, takaisa mitochondria ne tetramethylrhodamine ethyl ester (TMRE) uye takashandisa time-lapse microscopy uye MEL analysis kuti tiwane nzvimbo uye tione mitochondria mushure memaawa makumi maviri nemana pa3 ne5 mM PPA. Kurapa kwezviitiko zve fission ne fusion. (Mufananidzo 2a). Mushure mekuongorora MEL, mitochondria yakaongororwa zvakare kuti ione huwandu hwezvimiro zvemitochondrial uye huwandu hwazvo hweavhareji. Takaona kuwedzera kudiki asi kwakakosha muhuwandu hwezviitiko zvekupatsanurwa kwemasero zvichiitika pa3 mM [4.9 ± 0.3 (p < 0.05)] zvichienzaniswa nekupatsanurwa [5.6 ± 0.3 (p < 0.05) )] uye kusangana [5.4 ± 0.5 (p < 0.05)] uye kusangana [5.4 ± 0.5 (p < 0.05)] 0.05)] <0.05)] zviitiko zvakawedzera zvakanyanya pa5 mM zvichienzaniswa nekudzora (Mufananidzo 3b). Huwandu hwemitochondria hwakawedzera zvakanyanya pa3 [32.6 ± 2.1 (p < 0.05)] uye 5 mM [34.1 ± 2.2 (p < 0.05)] (Mufananidzo 3c), nepo huwandu hweavhareji hwechimiro chega chega chemitochondrial hwakaramba husina kuchinja (Mufananidzo 3c). 3d). Zvichitorwa pamwe chete, izvi zvinoratidza kuti kugadziriswa kwesimba remitochondrial kunoshanda semhinduro yekutsiva iyo inobudirira kuchengetedza kusimba kwenetwork yemitochondrial. Kuwedzera kwehuwandu hwezviitiko zvekupatsanurwa pa3 mM PPA kunoratidza kuti kuwedzera kwenhamba yemitochondrial kunokonzerwa nekupatsanurwa kwemitochondrial, asi tichifunga kuti avhareji yehuwandu hwemitochondrial inoramba isina kuchinja, biogenesis haigone kubviswa semhinduro yekuwedzera yekutsiva. Zvisinei, data iri rinoenderana nezvikamu zvidiki, zvakatenderera zvemitochondrial zvakaonekwa neTEM uye zvinoratidzawo shanduko huru musimba remitochondrial rinokonzerwa nePPA.
Propionic acid (PPA) inokonzera dynamic mitochondrial remodeling kuchengetedza network integrity. Masero eSH-SY5Y akarimwa, akarapwa ne3 ne5 mM PPA kwemaawa makumi maviri nemana uye akaiswa mavara eTMRE neHoechst 33342 zvichiteverwa neMEL analysis. (a) Mifananidzo ye time-lapse microscopy inoratidza ruvara uye binarized maximum intensity projections panguva 2 (t2) yemamiriro ese ezvinhu. Nzvimbo dzakasarudzwa dzakaratidzwa mumufananidzo wega wega webinary dzakasimbiswa uye dzinoratidzwa mu3D panguva nhatu dzakasiyana dzenguva (t1-t3) kuratidza maitiro ekufamba kwenguva; zviitiko zve fusion zvinoratidzwa mugirini; zviitiko zve fission zvinoratidzwa mugirini. Zvinoratidzwa mutsvuku. (b) Avhareji yenhamba yezviitiko zve dynamic pamamiriro ese ezvinhu. (c) Avhareji yenhamba ye mitochondrial structures pasero. (d) Avhareji yevhoriyamu (µm3) yechimiro chega chega che mitochondrial pasero. Data rinoratidzwa rinomiririra n = 15 masero paboka rekurapa. Error bars dzakaratidzwa dzinomiririra avhareji ± SEM, scale bar = 10 μm, * p < 0.05.
Propionic acid (PPA) inokonzera kudzvinyirirwa kwemajini ane chekuita nekuchinjana kwemasero emuviri (mitochondrial dynamics). Masero eSH-SY5Y akarapwa ne3 ne5 mM PPA kwemaawa makumi maviri nemana. Kuyerwa kwemajini ehukama kwakaitwa uchishandisa RT-qPCR uye kwakajairwa kuita B2M. Majini eMitochondrial biogenesis (a) cMYC, (b) TFAM, (c) NRF1 uye (d) NFE2L2. Majini eMitochondrial fusion and fission (e) STOML2, (f) OPA1, (g) MFN1, (h) MFN2 uye (i) DRP1. Misiyano yakakosha (p < 0.05) yakaedzwa uchishandisa one-way ANOVA (control vs. treatment) uye bvunzo yekuenzanisa yaDunnett yakawanda: * inoratidza p < 0.05, ** inoratidza p < 0.01, uye **** inoratidza p < 0.0001. Mipira inomiririra mean expression ± SEM. Data rakaratidzwa rinomiririra n = 3 (STOML2, OPA1, TFAM), n = 4 (cMYC, NRF1, NFE2L2), uye n = 5 (MFN1, MFN2, DRP1) makopi ezvebhayoloji.
Data kubva kuongororo dzeTEM neMEL pamwe chete rinoratidza kuti PPA inochinja chimiro chemitochondrial uye dynamics. Zvisinei, matekiniki aya ekufungidzira haape ruzivo nezvenzira dziri pasi dzinotungamira maitiro aya. Saka takaongorora mRNA expression yezvikamu zvipfumbamwe zvakakosha zvemitochondrial dynamics, biogenesis, uye mitosis mukupindura kurapwa kwePPA. Takayera 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) uye dynamin-related protein 1 (DRP1) mushure memaawa makumi maviri nemana ekurapwa ne3 mM uye 5 mM PPA. Takaona 3 mM (p = 0.0053, p = 0.0415 uye p < 0.0001, zvichiteerana) uye 5 mM (p = 0.0031, p = 0.0233, p < 0.0001) kurapwa kwePPA. (Mufananidzo 3a–c). Kuderera kwekutaura kwemRNA kwaienderana nedosi: kutaura kwe cMYC, NRF1 uye TFAM kwakadzikira nenguva 5.7, 2.6 uye 1.9 pa3 mM, zvichiteerana, uye nenguva 11.2, 3 uye 2.2 pa5 mM. Kusiyana neizvi, jini repakati redox biogenesis NFE2L2 harina kuchinjwa pane chero huwandu hwePPA, kunyangwe maitiro akafanana ekuderedza kutaura akaonekwa (Mufananidzo 3d).
Takaongororawo kuratidzwa kwemajini ekare anobatanidzwa mukudzora kupatsanurwa nekusanganiswa. STOML2 inofungidzirwa kuti ine chekuita nekusanganiswa, mitophagy uye biogenesis, uye kuratidzwa kwayo kwakaderedzwa zvakanyanya (p < 0.0001) ne3 mM (2.4-fold change) uye 5 mM (2.8-fold change) PPA (Mufananidzo 1). 3d). Saizvozvowo, OPA1 fusion gene expression yakaderedzwa pa3 mM (1.6-fold change) uye 5 mM (1.9-fold change) PPA (p = 0.006 uye p = 0.0024, zvichiteerana) (Mufananidzo 3f). Zvisinei, hatina kuwana mutsauko wakakura mukuratidzwa kwemajini efusion MFN1, MFN2 kana fission gene DRP1 pasi pe24-h PPA stress (Mufananidzo 3g–i). Pamusoro pezvo, takaona kuti mazinga emapuroteni mana ekubatanidza uye ekuputika (OPA1, MFN1, MFN2 uye DRP1) haana kuchinja pasi pemamiriro akafanana (Mufananidzo 4a–d). Zvakakosha kucherechedza kuti data iri rinoratidza nguva imwe chete uye rinogona kusaratidza shanduko mukutaura kweprotein kana mazinga ekuita panguva yekutanga kwekushushikana kwePPA. Zvisinei, kudzikira kwakakosha mukutaura kwe cMYC, NRF1, TFAM, STOML2, uye OPA1 kunoratidza kusagadzikana kwakakosha kwekushandurwa kwemetabolism yemitochondrial, biogenesis, uye dynamics. Pamusoro pezvo, data iri rinoratidza kushandiswa kwehunyanzvi hwekufungidzira kudzidza zvakananga shanduko dzemagumo mukushanda kwemitochondrial.
Mapuroteni eFusion neFission Factor haana kuchinja mushure mekurapwa nepropionic acid (PPA). Masero eSH-SY5Y akarapwa ne3 ne5 mM PPA kwemaawa makumi maviri nemana. Mapuroteni akayerwa neWestern blot analysis, uye ma expression levels akajairwa kuita total protein. Avhareji yeprotein expression uye inomiririra Western blots yetarget ne total protein zvinoratidzwa. a – OPA1, b – MFN1, c – MFN2, d – DRP1. Mabhawa anomiririra mean ± SEM, uye data rinoratidzwa rinomiririra n = 3 biological replicates. Kuenzanisa kwakawanda (p < 0.05) kwakaitwa uchishandisa one-way analysis ye variance uye bvunzo yaDunnett. Gel yekutanga ne blot zvinoratidzwa muMufananidzo S1.
Kusashanda zvakanaka kwemasero eropa (Mitochondrial dysfunction) kunobatanidzwa nezvirwere zvakawanda kubva kuzvirwere zve metabolic, cardiovascular, uye tsandanyama kusvika kuzvirwere zve neurological1,10. Zvirwere zvakawanda zve neurodegenerative uye neurodegenerative zvine chekuita nekusashanda zvakanaka kwemasero eropa (mitochondrial dysfunction), zvichiratidza kukosha kwemasero aya muhupenyu hwese hweuropi. Zvirwere izvi zvinosanganisira chirwere cheParkinson, chirwere cheAlzheimer, uye ASD3,4,18. Zvisinei, kuwana masero europi kuti adzidze nezvezvirwere izvi kwakaoma, kunyanya padanho re mechanistic, zvichiita kuti ma cellular model systems ave imwe nzira inodiwa. Muchidzidzo ichi, tinoshandisa cellular model system tichishandisa PPA-treated SH-SY5Y cells kuti titsanangurezve kusashanda zvakanaka kwemasero eropa (mitochondrial dysfunction) kunoonekwa muzvirwere zve neuronal, kunyanya autism spectrum disorders. Kushandisa iyi PPA model kudzidza mitochondrial dynamics mu neuron kunogona kupa ruzivo nezve etiology yeASD.
Takaongorora mukana wekushandisa TEM kuti tione shanduko mu mitochondrial morphology. Zvakakosha kuziva kuti TEM inofanira kushandiswa nemazvo kuti iwedzere kushanda kwayo. Kugadzirira cryo-specimens kunobvumira kuchengetedzwa kuri nani kwezvimiro zve neuron nekugadzirisa zvikamu zvemasero panguva imwe chete uye kuderedza kuumbwa kwezvinhu zvakagadzirwa34. Zvichienderana neizvi, takaona kuti masero eSH-SY5Y akafanana ne neuron aive ne subcellular organelles dzisina kusimba uye mitochondria yakareba (Mufananidzo 1a). Izvi zvinoratidza kushanda kwe cryogenic preparation techniques pakudzidza mitochondrial morphology muma neuronal cell models. Kunyangwe zviyero zvehuwandu zvakakosha pakuongorora data reTEM, hapasati pave nekubvumirana pamusoro pekuti ndeapi ma parameter chaiwo anofanira kuyerwa kusimbisa shanduko dze mitochondrial morphological. Zvichibva pahuwandu hwakawanda hwezvidzidzo zvakaongorora mitochondrial morphology17,31,32, takagadzira automated mitochondrial image analysis pipeline inoyera ma parameter masere e morphological, anoti: area, area2, aspect ratio, perimeter, circularity, degree , Feret diameter. uye roundness.
Pakati pazvo, PPA yakaderedza zvakanyanya nzvimbo 2, nzvimbo, perimeter, uye dhayamita yeFeret (Mufananidzo 1b–e). Izvi zvakaratidza kuti mitochondria yakava diki uye yakatenderera, izvo zvinoenderana nezvidzidzo zvekare zvinoratidza kudzikira kwenzvimbo yemitochondria mushure memaawa makumi manomwe nemaviri ekumanikidzwa kwemitochondrial kwakakonzerwa nePPA30. Maonero aya echimiro anogona kuratidza kupatsanurwa kwemitochondrial, maitiro anodiwa ekubvisa zvikamu zvakakuvara kubva kune network yemitochondrial kukurudzira kuora kwazvo kuburikidza ne mitophagy35,36,37. Kune rumwe rutivi, kudzikira kweavhareji yehukuru hwemitochondrial kunogona kusanganiswa nekuwedzera kwe biogenesis, izvo zvinoguma nekuumbwa kwe mitochondria diki iri kubuda. Kuwedzera kwe fission kana biogenesis kunomiririra mhinduro yekuchengetedza mitosis kurwisa mitochondrial stress. Zvisinei, kudzikira kwekukura kwemitochondrial, kukanganisa fusion, kana zvimwe zvirwere hazvigone kubviswa.
Kunyangwe mifananidzo yepamusoro yakagadzirwa neTEM ichibvumira kusarudzwa kwehunhu hwechimiro padanho remitochondria yega yega, nzira iyi inogadzira mifananidzo miviri-dimensional panguva imwe chete. Kuti tidzidze mhinduro dzinochinja kune metabolic stress, takaisa mitochondria neTMRE uye takashandisa time-lapse microscopy neMEL analysis, iyo inobvumira high-throughput 3D visualization yekuchinja mu mitochondrial network nekufamba kwenguva33,38. Takaona shanduko dzakadzika asi dzakakosha mu mitochondrial dynamics pasi pePPA stress (Mufananidzo 2). Pa3 mM, huwandu hwezviitiko zvefission hwakawedzera zvakanyanya, nepo zviitiko zvefusion zvakaramba zvakafanana nezviri mukutonga. Kuwedzera kwehuwandu hwezviitiko zvefission nefusion kwakaonekwa pa5 mM PPA, asi shanduko idzi dzaive dzakafanana, zvichiratidza kuti fission nefusion kinetics zvinosvika pakuenzana pahuwandu hwakakwira (Mufananidzo 2b). Avhareji yehuwandu hwemitochondrial yakaramba isina kuchinja pa3 ne5 mM PPA, zvichiratidza kuti kusimba kwenetwork yemitochondrial kwakachengetedzwa (Mufananidzo 2d). Izvi zvinoratidza kugona kwema network e mitochondrial anochinja-chinja kupindura kushushikana kwe metabolic kudiki kuti arambe ari homeostasis pasina kukonzera kupatsanuka kwe network. Pa 3 mM PPA, kuwedzera kwe fission kwakakwana kukurudzira shanduko kuenda kune imwe equilibrium, asi kinetic remodeling yakadzama inodiwa mukupindura kushushikana kunokonzerwa nehuwandu hwakawanda hwePPA.
Huwandu hwemitochondria hwakawedzera pahuwandu hwePPA stress concentrations, asi avhareji yehuwandu hwemitochondrial haina kuchinja zvakanyanya (Mufananidzo 2c). Izvi zvinogona kunge zvichikonzerwa nekuwedzera kwebiogenesis kana kuwedzera kwekupatsanurwa; zvisinei, kana pasina kudzikira kukuru kwehuwandu hwemitochondrial, zvingangoita kuti biosynthesis iwedzere. Zvisinei, data riri muMufananidzo 2 rinotsigira kuvapo kwenzira mbiri dzekugadzirisa: kuwedzera kwehuwandu hwezviitiko zvefission, zvinoenderana nekuwedzera kwemitochondrial fission, uye kuwedzera kwehuwandu hwezviitiko, zvinoenderana nemitochondrial biogenesis. Pakupedzisira, dynamic compensation yekushushikana kudiki inogona kunge iri maitiro panguva imwe chete anosanganisira fission, fusion, biogenesis, uye mitophagy. Kunyangwe vanyori vekare vakaratidza kuti PPA inowedzera mitosis30,39 uye mitophagy29, tinopa humbowo hwekugadzirisa mitochondrial fission uye fusion dynamics mukupindura kuPPA. Aya data anosimbisa shanduko dze morphological dzakaonekwa neTEM uye dzinopa rumwe ruzivo nezve nzira dzine chekuita ne PPA-induced mitochondrial dysfunction.
Nekuti TEM kana MEL ongororo haina kupa humbowo hwakananga hwemajini anotonga maitiro ari pasi pekuchinja kwechimiro kwakacherechedzwa, takaongorora kuratidzwa kweRNA kwemajini anobatanidzwa mu metabolism yemitochondrial, biogenesis, uye dynamics. Iyo cMYC proto-oncogene chinhu chinoshandura majini chinobatanidzwa mukutonga kwemitochondria, glycolysis, amino acid uye fatty acid metabolism40. Pamusoro pezvo, cMYC inozivikanwa nekudzora kuratidzwa kwemajini anoda kusvika mazana matanhatu emitochondrial anobatanidzwa mukunyora, kushandura, uye kuunganidza kwakaoma kwemitochondrial, kusanganisira NRF1 neTFAM41. NRF1 neTFAM ndiwo maviri mapakati ekutonga mitosis, anoshanda pasi pePGC-1α kuti aite mtDNA replication. Iyi nzira inogadziriswa ne cAMP ne AMPK signaling uye inonzwa kushandiswa kwesimba uye metabolic stress. Takaongororawo NFE2L2, redox regulator ye mitochondrial biogenesis, kuti tione kana mhedzisiro yePPA ingangove yakakonzerwa nekushushikana kwe oxidative.
Kunyangwe kutaura kweNFE2L2 kwakaramba kusina kuchinja, takaona kudzikira kwakasimba kwekutaura kwe cMYC, NRF1 neTFAM mushure memaawa makumi maviri nemana ekurapwa ne3 mM ne5 mM PPA (Mufananidzo 3a–c). Kudzikira kwekutaura kwe cMYC kwakambotaurwa semhinduro yekushushikana kwe mitochondrial42, uye zvakasiyana, kudzikira kwekutaura kwe cMYC kunogona kukonzera kusashanda zvakanaka kwe mitochondrial nekugadzirisa metabolism ye mitochondrial, network connectivity, uye membrane polarization43. Zvinofadza kuti cMYC inobatanidzwawo mukutonga kwe mitochondrial fission ne fusion42,43 uye inozivikanwa nekuwedzera DRP1 phosphorylation uye mitochondrial localization panguva yekupatsanurwa kwemasero44, pamwe nekubatanidza mitochondrial morphological remodeling mu neuronal stem cells45. Zvechokwadi, cMYC-deficient fibroblasts inoratidza kuderera kwehukuru hwe mitochondrial, zvinoenderana nekuchinja kunokonzerwa nePPA43 stress. Aya data anoratidza hukama hunonakidza asi husati hwajeka pakati pe cMYC ne mitochondrial dynamics, zvichipa chinangwa chinonakidza chezvidzidzo zvemangwana zvePPA stress-induced remodeling.
Kuderedzwa kweNRF1 neTFAM kunoenderana nebasa re cMYC semushandi anobatsira kunyora zvinyorwa. Data iri rinoenderanawo nezvidzidzo zvekare mumasero ekenza yemudumbu remunhu zvichiratidza kuti PPA yakaderedza kuratidzwa kweNRF1 mRNA pamaawa makumi maviri nemaviri, izvo zvakabatana nekudzikira kweATP uye kuwedzera kweROS46. Vanyori ava vakataurawo kuti kuratidzwa kweTFAM kwakawedzera pamaawa masere nehafu asi kwakadzokera padanho rekutanga pamaawa makumi maviri nemaviri. Kusiyana neizvi, Kim et al. (2019) vakaratidza kuti kuratidzwa kweTFAM mRNA kwakadzikira zvakanyanya mushure memaawa mana ekumanikidzwa kwePPA mumasero eSH-SY5Y; zvisinei, mushure memaawa makumi manomwe nemaviri, kuratidzwa kweTFAM protein kwakawedzera zvakanyanya uye nhamba yekopi yemtDNA yakawedzera zvakanyanya. Saka, kudzikira kwehuwandu hwemajini emitochondrial biogenesis atakaona mushure memaawa makumi maviri nemana hakubvisi mukana wekuti kuwedzera kwehuwandu hwemitochondria kwakabatana nekuitwa kwebiogenesis panguva dzekare. Zvidzidzo zvekare zvakaratidza kuti PPA inowedzera zvakanyanya PGC-1α mRNA uye mapuroteni mumasero eSH-SY5Y mumaawa mana nemaminitsi makumi matatu, nepo propionic acid ichivandudza mitochondrial biogenesis mu calf hepatocytes kuburikidza nePGC-1α mumaawa gumi nemaviri nemaminitsi makumi matatu nemapfumbamwe. Zvinofadza kuti, PGC-1α haisi chete murairidzi we transcriptional weNRF1 neTFAM, asi yakaratidzwawo kuti inotonga mashandiro eMFN2 neDRP1 nekutonga fission ne fusion47. Kana zvabatanidzwa, izvi zvinoratidza kubatana kwakasimba kwemaitiro anodzora mhinduro dzemitochondrial compensatory dzinokonzereswa nePPA. Uyezve, data redu rinoratidza kusagadzikana kukuru kwe transcriptional regulation ye biogenesis uye metabolism pasi pekumanikidzwa kwePPA.
Majini eSTOML2, OPA1, MFN1, MFN2 uye DRP1 ari pakati pevanodzora mitochondrial fission, fusion uye dynamics37,48,49. Kune mamwe majini akawanda anobatanidzwa mumitochondrial dynamics, zvisinei, STOML2, OPA1 uye MFN2 zvakamboonekwa kuti zvine methylation yakasiyana muASD cohorts,16 uye zvidzidzo zvakati wandei zvakashuma shanduko mune izvi transcription factors mukupindura kushushikana kwemitochondrial50,51. 52. Kuonekwa kweOPA1 neSTOML2 kwakaderedzwa zvakanyanya nekurapwa kwe3 mM ne5 mM PPA (Mufananidzo 3e, f). OPA1 ndeimwe yevanodzora mitochondrial fusion kuburikidza nekudyidzana zvakananga neMFN1 ne2 uye inoita basa mukugadzirisa cristae uye mitochondrial morphology53. Basa chairo reSTOML2 mumitochondrial dynamics harisati rajeka, asi humbowo hunoratidza kuti inoita basa mumitochondrial fusion, biogenesis, uye mitophagy.
STOML2 inobatsira mukuchengetedza mitochondrial respiratory coupling uye kuumbwa kwe respiratory chain complexes54,55 uye yakaratidzwa kuti inochinja zvakanyanya maitiro e metabolic emasero ekenza56. Zvidzidzo zvakaratidza kuti STOML2 inokurudzira mitochondrial membrane potential uye biogenesis kuburikidza nekudyidzana neBAN ne cardiolipin 55, 57, 58. Pamusoro pezvo, zvidzidzo zvakazvimiririra zvakaratidza kuti kudyidzana pakati peSTOML2 nePINK1 kunodzora mitophagy59,60. Zvinonyanya kukosha kuti STOML2 yakataurwa kuti inosangana zvakananga ne MFN2 uye inosimbisa uye inoitawo basa rakakosha mukusimbisa ma OPA1 isoforms marefu nekudzivisa protease inokonzera OPA1 degradation53,61,62. Kuderedzwa kwekutaura kweSTOML2 kunoonekwa muPPA reactions kunogona kuita kuti ma fusion proteins aya ave nyore kukuvadzwa kuburikidza ne ubiquitin- uye proteasome-dependent pathways48. Kunyangwe basa chairo reSTOML2 neOPA1 mukupindura kwePPA risingajekeswi, kudzikira kwekuratidzwa kwemajini aya ekubatanidza (Mufananidzo 3) kunogona kukanganisa kuenzana pakati pekuputika nekusanganiswa uye kutungamira kukudzikira kwehukuru hwemitochondrial (Mufananidzo 3). 1).
Kune rumwe rutivi, kuratidzwa kweprotein yeOPA1 kwakaramba kusina kuchinja mushure memaawa makumi maviri nemana, nepo mRNA uye mazinga eprotein eMFN1, MFN2 kana DRP1 asina kuchinja zvakanyanya mushure mekurapwa kwePPA (Mufananidzo 3g-i, Mufananidzo 4). Izvi zvinogona kuratidza kuti hapana shanduko mukudzora kwezvinhu izvi zvinobatanidzwa mukusanganiswa kwemitochondrial uye fission. Zvisinei, zvakakosha kuziva kuti rimwe nerimwe remajini mana aya rinodzorwawo ne posttranscriptional modifications (PTMs) dzinodzora mashandiro eprotein. OPA1 ine ma alternative splice variants masere akachekwa proteolytically mu mitochondria kuti agadzire isoforms mbiri dzakasiyana 63. Kuenzana pakati peisoforms refu nepfupi kunozopedzisira kwasarudza basa reOPA1 mukubatanidzwa kwemitochondrial uye kuchengetedza mitochondrial network64. Basa reDRP1 rinodzorwa ne calcium/calmodulin-dependent protein kinase II (CaMKII) phosphorylation, nepo DRP1 degradation ichidzorwa ne ubiquitination uye SUMOylation65. Pakupedzisira, DRP1 neMFN1/2 maGTPases, saka mashandiro anogona kukanganiswa nehuwandu hwekugadzirwa kweGTP mu mitochondria 66. Nokudaro, kunyange zvazvo kuratidzwa kwemapuroteni aya kuchiramba kuripo, izvi zvingasaratidze mashandiro epuroteni asina kuchinja kana localization67,68. Zvechokwadi, mapuroteni ePTM aripo anowanzo shanda senzira yekutanga yekudzivirira ine basa rekugadzirisa mhinduro dzekushushikana kwakanyanya. Kana paine kushushikana kwemetabolism kuri pakati nepakati mumuenzaniso wedu, zvingangoita kuti PTM inokurudzira kuwedzera kwebasa remapuroteni ekubatanidza uye fission kuti adzorere zvakakwana mitochondrial integrity pasina kuda kuwedzerwa kushanda kwemajini aya padanho remRNA kana protein.
Kana zvabatanidzwa, data riri pamusoro apa rinoratidza kurongeka kwakaoma uye kunoenderana nenguva kwechimiro chemitochondrial uye matambudziko ekutsanangura mashandiro aya. Kuti tidzidze kutaurwa kwemajini, zvinotanga zvakakosha kuziva majini chaiwo ari munzira. Zvisinei, data redu rinoratidza kuti majini ari munzira imwe chete haapindure nenzira imwechete kune kushushikana kumwe chete. Kutaura zvazviri, zvidzidzo zvekare zvakaratidza kuti majini akasiyana ari munzira imwe chete anogona kuratidza maitiro akasiyana emhinduro yenguva30,46. Pamusoro pezvo, kune maitiro akaomarara ekunyora mushure mekunyora anovhiringidza hukama huripo pakati pekunyora uye mashandiro emajini. Zvidzidzo zveProteomic zvinogona kupa nzwisiso pamusoro pemhedzisiro yePTMs uye mashandiro emapuroteni, asi zvinounzawo matambudziko anosanganisira nzira dzisina simba, huwandu hwesignali-ku-ruzha, uye kusagadziriswa kwakashata.
Munyaya iyi, kudzidza chimiro chemitochondrial uchishandisa TEM neMEL kune mukana mukuru wekugadzirisa mibvunzo mikuru nezvehukama huripo pakati pesimba remitochondrial uye mashandiro ayo uye kuti izvi zvinokanganisa sei chirwere. Chinonyanya kukosha, TEM inopa nzira yakananga yekuyera chimiro chemitochondrial semhedzisiro inopindirana yekusashanda zvakanaka kwemitochondrial uye dynamics51. MEL inopawo nzira yakananga yekuona zviitiko zve fission uye fusion munzvimbo yemasero ine mativi matatu, zvichibvumira kuyera dynamic mitochondrial remodeling kunyangwe pasina shanduko mukutaura kwemajini33. Pano tinoratidza kushandiswa kwehunyanzvi hwemitochondrial imaging muzvirwere zvechipiri zvemitochondrial. Zvirwere izvi zvinowanzoonekwa nekushushikana kusingaperi kwemetabolism kunoratidzwa nekuchinja zvishoma kwenetwork yemitochondrial pane kukuvara kwakanyanya kwemitochondrial. Zvisinei, muripo wemitochondrial unodiwa kuchengetedza mitosis pasi pekushushikana kusingaperi kune mhedzisiro yakakura yekushanda. Mumamiriro e neuroscience, kunzwisisa zviri nani kwemaitiro aya ekubhadhara kunogona kupa ruzivo rwakakosha nezve pleiotropic neuropathology inobatanidzwa nekusashanda zvakanaka kwemitochondrial.
Pakupedzisira, data redu rinoratidza kushanda kwematekiniki ekufungidzira pakunzwisisa mhedzisiro yekushanda kwekubatana kwakaoma pakati pekugadzirwa kwemajini, kugadziriswa kwemapuroteni, uye mashandiro emapuroteni anodzora mashandiro ema neuronal mitochondrial. Takashandisa PPA kuratidza kusagadzikana kwema mitochondrial mumodhi yemasero e neuronal kuti tinzwisise chikamu che mitochondrial cheASD. Masero eSH-SY5Y akarapwa nePPA akaratidza shanduko muchimiro che mitochondrial: mitochondria yakava diki uye yakatenderera, uye ma cristae haana kutsanangurwa zvakanaka paakaonekwa neTEM. Kuongorora kweMEL kunoratidza kuti shanduko idzi dzinoitika panguva imwe chete nekuwedzera kwezviitiko zve fission uye fusion kuchengetedza mitochondrial network mukupindura kushushikana kwe metabolic kushoma. Uyezve, PPA inokanganisa zvakanyanya kurongeka kwe transcriptional ye metabolism ye mitochondrial uye homeostasis. Takaona cMYC, NRF1, TFAM, STOML2, uye OPA1 sevatongi vakakosha ve mitochondrial vakakanganiswa nekushushikana kwePPA uye vanogona kuita basa mukuyananisa shanduko dzinokonzerwa nePPA muchimiro che mitochondrial uye mashandiro. Zvidzidzo zveramangwana zvinodiwa kuti zvitsanangure zviri nani shanduko dzenguva dzinokonzerwa nePPA mukugadzirwa kwemajini uye mashandiro emapuroteni, localization, uye post-translational modifications. Ruzivo rwedu runoratidza kuomarara uye kudyidzana kwenzira dzinodzora dzinoita kuti pave nekushushikana kwemitochondrial uye runoratidza kushanda kweTEM nedzimwe nzira dzekufungidzira pakuongorora kwakanangana neunyanzvi hwemichina.
Mutsetse wesero weSH-SY5Y (ECACC, 94030304-1VL) wakatengwa kubva kuSigma-Aldrich. Masero eSH-SY5Y akakurira mumusanganiswa weDulbecco's modified Eagle's medium/F-12 nutrient (DMEM/F-12) uye L-glutamine (SC09411, ScienCell) mumaflasks e25 cm2 akawedzerwa ne20% fetal bovine serum (FBS) (10493106, ThermoFisher Scientific) uye 1% penicillin-streptomycin (P4333-20ML, Sigma-Aldrich) pa37 °C, 5% CO2. Masero akasimwa kusvika pa80% confluence achishandisa 0.05% trypsin-EDTA (15400054, ThermoFisher Scientific), akaiswa centrifuge pa300 g uye akaiswa pahuwandu hwe7 × 105 cells/ml. Kuedza kwese kwakaitwa pamasero eSH-SY5Y asina kupatsanurwa pakati penzira 19–22. PPA inopihwa seNaP. Nyungudutsa upfu hweNaP (CAS Nhamba 137-40-6, fomura yemakemikari C3H5NaO2, P5436-100G, Sigma-Aldrich) mumvura inodziya yeMilliQ kusvika pakuwanda kwe1 M woichengeta pa4 °C. Pazuva rekurapwa, sanganisa mushonga uyu ne1 M PPA kusvika 3 mM uye 5 mM PPA mune serum-free medium (DMEM/F-12 neL-glutamine). Kuwanda kwekurapwa kwekuyedza kwese kwaive kusina PPA (0 mM, control), 3 mM, uye 5 mM PPA. Kuedza kwakaitwa mune kanenge katatu biological replicates.
Masero eSH-SY5Y akadyarwa mumaflasks e25 cm5 nechiyero che5.5 × 105 masero/ml uye akakura kwemaawa makumi maviri nemana. Kurapwa kwePPA kwakawedzerwa muflask maawa makumi maviri nemana asati aiswa mudumbu. Unganidza mapellets emasero uchitevera nzira dzakajairika dzekurima tissue subculture (dzakatsanangurwa pamusoro). Isa zvakare pellet yemasero mu100 µl 2.5% glutaraldehyde, 1 × PBS uye chengeta pa4 °C kusvika zvagadziriswa. Masero eSH-SY5Y akatenderedzwa kwenguva pfupi kuti abvise masero uye abvise 2.5% glutaraldehyde, 1 × PBS solution. Isa zvakare sediment mu4% agarose gel yakagadzirwa mumvura yakasanganiswa (chiyero cheagarose kune sediment volume i1:1). Zvidimbu zveAgarose zvakaiswa pamagrids pamaplate akatsetseka uye zvakaputirwa ne1-hexadecene zvisati zvanyatsoomeswa nechando. Samples dzakaomeswa mu100% dry acetone pa -90°C kwemaawa makumi maviri nemana. Tembiricha yakazowedzerwa kusvika -80°C uye mvura ye1% osmium tetroxide ne0.1% glutaraldehyde yakawedzerwa. Mienzaniso yakachengetwa pa -80°C kwemaawa makumi maviri nemana. Mushure meizvi, tembiricha yakawedzerwa zvishoma nezvishoma kusvika patembiricha yemukamuri kwemazuva akati wandei: kubva pa -80 °C kusvika -50 °C kwemaawa makumi maviri nemana, kusvika -30 °C kwemaawa makumi maviri nemana, kusvika -10 °C kwemaawa makumi maviri nemana uye pakupedzisira kusvika patembiricha yemukamuri.
Mushure mekugadzirira cryogenic, sampuro dzakaiswa resin uye zvikamu zvakatetepa zvakanyanya (~100 nm) zvakagadzirwa uchishandisa Leica Reichert UltracutS ultramicrotome (Leica Microsystems). Zvikamu zvakaiswa 2% uranyl acetate uye lead citrate. Sampuro dzakaonekwa uchishandisa FEI Tecnai 20 transmission electron microscope (ThermoFisher (yaimbonzi FEI), Eindhoven, The Netherlands) inoshanda pa200 kV (Lab6 transmitter) uye kamera yeGatan CCD (Gatan, UK) ine Tridiem energy filter.
Mumifananidzo yega yega yehunyanzvi, mifananidzo inosvika makumi maviri nemana yemasero mamwechete yakawanikwa, zvichiita kuti pave nemifananidzo inosvika mazana maviri nemakumi matanhatu nematanhatu. Mifananidzo yese yakaongororwa uchishandisa macro yeRegion of Interest (ROI) uye macro yeMitochondria. Macro yemitochondrial yakavakirwa panzira dzakaburitswa17,31,32 uye inobvumira semi-automated batch processing yemifananidzo yeTEM muFiji/ImageJ69. Muchidimbu: mufananidzo unopindurwa uye unopindurwa uchishandisa rolling ball background subtraction (60 pixel radius) uye FFT bandpass filter (ichishandisa 60 ne8 pixel upper and lower bounds zvichiteerana) uye vertical line depression ine orientation tolerance ye5%. Mufananidzo unogadziriswa unopindirwa otomatiki uchishandisa maximum entropy algorithm uye binary mask inogadzirwa. Mifananidzo yenzvimbo dzakabatana neROIs dzakasarudzwa nemaoko mumifananidzo yeTEM isina kugadzirwa yakabviswa, zvichiratidza mitochondria uye kusabvisa plasma membrane nedzimwe nzvimbo dzine musiyano mukuru. Pa ROI yega yega yakatorwa, zvimedu zve binary zvakakura kupfuura mapikiseli mazana matanhatu zvakaongororwa, uye nzvimbo yezvikamu, perimeter, major ne minor axes, Feret diameter, roundness, uye circularity zvakayerwa uchishandisa mabasa ekuyera akavakirwa mukati eFiji/ImageJ. Zvichitevera Merrill, Flippo, naStrack (2017), nzvimbo 2, particle aspect ratio (major to minor axis ratio), uye shape factor (FF) zvakaverengerwa kubva padata iri, uko FF = perimeter 2/4pi x area. Tsananguro ye parametric formula inogona kuwanikwa muMerrill, Flippo, naStrack (2017). Ma macros ataurwa anowanikwa paGitHub (ona Data Availability Statement). Paavhareji, zvimedu zvinosvika zviuru zvishanu nemazana matanhatu zvakaongororwa pakurapwa kwePPA, kusvika pahuwandu hwezvimedu zvinosvika zviuru gumi nezvinomwe (data harina kuratidzwa).
Masero eSH-SH5Y akaiswa mumidziyo yekurima ine makamuri masere (ThermoFisher, #155411) kuti abvumire kunamira husiku hwese uye ozoiswa mu TMRE 1:1000 (ThermoFisher, #T669) uye Hoechst 33342 1:200 (Sigma-Aldrich, H6024). Kudhaya. Mifananidzo yakawanikwa uchishandisa 405 nm uye 561 nm lasers munzvimbo yemaminitsi gumi, uye mifananidzo isina kubikwa yakawanikwa sez-stacks ine 10 image micrographs ine az step ye 0.2 μm pakati pemafuremu emifananidzo panguva gumi nembiri dzakatevera. Mifananidzo yakaunganidzwa uchishandisa Carl Zeiss LSM780 ELYRA PS.1 super-resolution platform (Carl Zeiss, Oberkochen, Germany) uchishandisa LCI Plan Apochromate 100x/1.4 Oil DIC M27 lens. Mifananidzo yakaongororwa muImageJ uchishandisa pipeline yakambotsanangurwa uye ImageJ plugin kuyera zviitiko zvekubatana uye fission, avhareji yehuwandu hwemaumbirwo emitochondrial, uye avhareji yehuwandu hwemitochondrial pasero 33. MaMEL macros anowanikwa paGitHub (ona Data Availability Statement).
Masero eSH-SY5Y akakurira mumaplates matanhatu ane density ye 0.3 × 106 cells/mL kwemaawa makumi maviri nemana kurapwa kusati kwaitwa. RNA yakabviswa uchishandisa Quick-RNA™ Miniprep protocol (ZR R1055, Zymo Research) nekuchinja kudiki: wedzera 300 μl yeRNA lysis buffer pachitubu chimwe nechimwe usati wabvisa uye lyse sample yega yega sedanho rekupedzisira ne 30 μl yeDNase/RNase elution. -isina mvura. Sampuli dzese dzakaongororwa huwandu uye mhando uchishandisa NanoDrop ND-1000 UV-Vis Spectrophotometer. Mapuroteni ese kubva kumasero lysates akawanikwa uchishandisa 200 μl RIPA lysis buffer, uye huwandu hweprotein hwakayerwa uchishandisa Bradford protein assay70.
Kugadzirwa kwe cDNA kwakaitwa uchishandisa Tetro™ cDNA Synthesis Kit (BIO-65043, Meridian Bioscience) zvichienderana nemirairo yemugadziri pamwe nekugadziriswa kwakati. cDNA yakagadzirwa mu 20-μl reactions uchishandisa 0.7 kusvika 1 μg ye total RNA. Ma primer akasarudzwa kubva mumapepa akamboburitswa 42, 71, 72, 73, 74, 75, 76, 77, 78 (Table S1) uye ma probes anoenderana nawo akagadzirwa achishandisa PrimerQuest tool kubva kuIntegrated DNA Technologies. Ma gene ese aifarira akajairwa kuita nuclear B2M gene. Kuonekwa kwema gene eSTOML2, NRF1, NFE2L2, TFAM, cMYC neOPA1 kwakayerwa neRT-qPCR. Musanganiswa mukuru waisanganisira LUNA Taq polymerase (M3003L, New England Biolabs), 10 μM forward and reverse primers, cDNA, uye PCR-grade water kuti pave nehuwandu hwekupedzisira hwe10 μL pakuita kwega kwega. Kuratidzwa kwemajini ekuparadzanisa uye fission (DRP1, MFN1/2) kwakayerwa uchishandisa TaqMan multiplex assays. Luna Universal Probe qPCR Master Mix (M3004S, New England Biolabs) yakashandiswa zvichienderana nemirairo yemugadziri nekuchinja kudiki. Musanganiswa mukuru weRT-qPCR unosanganisira 1X LUNA Taq polymerase, 10 μM forward and reverse primers, 10 μM probe, cDNA, uye PCR-grade water, zvichikonzera huwandu hwekupedzisira hwe20 μL pakuita kwega kwega. RT-qPCR yakaitwa uchishandisa Rotor-Gene Q 6-plex (QIAGEN RG—serial number: R0618110). Mamiriro ekufamba kwemhepo anoratidzwa muTafura S1. Samples dzese dze cDNA dzakawedzerwa muzvikamu zvitatu uye curve yakajairwa yakagadzirwa uchishandisa nzira dzakasiyana-siyana dzekuwedzera huwandu hwezvikamu gumi. Zvikamu zvakasarudzika muzvikamu zvitatu zvine cycle threshold standard deviation (Ct) >0.5 zvakabviswa kubva muongororo kuitira kuve nechokwadi chekuti data rinodzokororwazve30,72. Kutsanangurwa kwemajini ehukama kwakaverengerwa uchishandisa nzira ye2-ΔΔCt79.
Mapuroteni (60 μg) akasanganiswa neLaemmli loading buffer pachiyero che2:1 uye akashandiswa pa12% protein gel isina ruvara (Bio-Rad #1610184). Mapuroteni akaendeswa kuPVDF (polyvinylidene fluoride) membrane (#170-84156, Bio-Rad) achishandisa Trans-Blot Turbo system (#170-4155, Bio-Rad). Kamudzira kakavharirwa ndokuiswa muincubation nema primary antibodies akakodzera (OPA1, MFN1, MFN2, uye DRP1) (yakasanganiswa 1:1000) kwemaawa makumi mana nemasere, zvichiteverwa ne incubation nema secondary antibodies (1:10,000) kweawa imwe chete. Ma Membranes akazotorwa mifananidzo achishandisa Clarity Western ECL Substrate (#170-5061, Bio-Rad) uye akarekodhwa achishandisa Bio-Rad ChemiDoc MP system. ImageLab version 6.1 yakashandiswa pakuongorora Western blot. Gel yekutanga ne blot zvinoratidzwa muMufananidzo S1. Ruzivo rwemasoja ekudzivirira chirwere rwuri muTafura S2.
Seti dzedata dzinoratidzwa sechikanganiso chepakati nepakati chepakati (SEM) chezviyero zvitatu zvakazvimiririra. Seti dzedata dzakaedzwa kuti dzionekwe kuti dzakajairwa sei uchishandisa bvunzo yeShapiro-Wilks (kunze kwekunge zvataurwa neimwe nzira) vasati vatora kugoverwa kweGaussian uye kutsauka kwakaenzana uye kuenderera mberi nekuongorora. Pamusoro pekuongorora seti yedata uchishandisa Fisher's MEL LSD (p < 0.05), one-way ANOVA (kurapa vs. control mean), uye bvunzo yekuenzanisa yakawanda yaDunnett kuti vaone kukosha (p < 0.05). Kukosha kwep kunoratidzwa mugirafu se *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Kuongororwa kwese kwenhamba nemagrafu kwakaitwa uye kwakagadzirwa uchishandisa GraphPad Prism 9.4.0.
Ma macro eFiji/ImageJ ekuongorora mifananidzo yeTEM anowanikwa pachena paGitHub: https://github.com/caaja/TEMMitoMacro. Macro eMitochondrial Event Locator (MEL) anowanikwa pachena paGitHub: https://github.com/rensutheart/MEL-Fiji-Plugin.
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