Enkosi ngokutyelela iNature.com. Inguqulelo yesikhangeli osisebenzisayo inenkxaso encinci yeCSS. Ukuze ufumane iziphumo ezilungileyo, sicebisa ukuba usebenzise inguqulelo entsha yesikhangeli sakho (okanye uvale iMowudi yoKuhambelana kwi-Internet Explorer). Okwangoku, ukuqinisekisa inkxaso eqhubekayo, sibonisa indawo ngaphandle kwesitayile okanye iJavaScript.
I-Propionic acid (PPA) isetyenziselwa ukufunda indima yokungasebenzi kakuhle kwe-mitochondrial kwizifo zophuhliso lwe-neurodevelopmental ezifana ne-autism spectrum disorder. I-PPA yaziwa ngokuphazamisa i-mitochondrial biogenesis, i-metabolism, kunye ne-turnover. Nangona kunjalo, iziphumo ze-PPA kwi-mitochondrial dynamics, i-fission kunye ne-fusion zihlala ziyingxaki ngenxa yendalo enzima yexesha lale ndlela. Apha, sisebenzisa iindlela zokulinganisa ezipheleleyo ukuze sihlolisise indlela i-PPA echaphazela ngayo i-mitochondrial ultrastructure, i-morphology, kunye ne-dynamics kwiiseli ze-SH-SY5Y ezifana ne-neuron. I-PPA (5 mM) ibangele ukwehla okukhulu kwindawo ye-mitochondrial (p < 0.01), ububanzi be-Feret kunye ne-circumference (p < 0.05), kunye nendawo yesi-2 (p < 0.01). Uhlalutyo lwe-Mitochondrial event locator lubonise ukwanda okukhulu (p < 0.05) kwiziganeko ze-fission kunye ne-fusion, ngaloo ndlela kugcinwa ingqibelelo yenethiwekhi ye-mitochondrial phantsi kweemeko zoxinzelelo. Ukongeza, ukubonakaliswa kwe-mRNA kwe-cMYC (p < 0.0001), i-NRF1 (p < 0.01), i-TFAM (p < 0.05), i-STOML2 (p < 0.0001) kunye ne-OPA1 (p < 0.05) kuncitshiswe kakhulu. 01). Oku kubonisa ukuhlaziywa kwe-mitochondrial morphology, i-biogenesis kunye ne-dynamics ukugcina umsebenzi phantsi kweemeko zoxinzelelo. Idatha yethu inika ulwazi olutsha malunga nemiphumo ye-PPA kwi-mitochondrial dynamics kwaye igxininisa ukusetyenziswa kweendlela zokwenza imifanekiso ekufundeni iindlela ezintsonkothileyo zokulawula ezibandakanyekayo kwiimpendulo zoxinzelelo lwe-mitochondrial.
I-Mitochondria ngabathathi-nxaxheba ababalulekileyo kwimisebenzi eyahlukeneyo yeselula ngaphaya kwendima yayo eqhelekileyo kwimveliso yamandla kunye ne-biosynthesis. I-Mitochondrial metabolism ngumlawuli ophambili we-calcium signaling, i-metabolic kunye ne-redox homeostasis, i-inflammatory signaling, i-epigenetic modifications, i-cell proliferation, i-differentiation kunye ne-programmed cell death1. Ngokukodwa, i-mitochondrial metabolism ibalulekile kuphuhliso lwe-neuronal, ukusinda kunye nokusebenza kwaye ibandakanyeka kakhulu kwiimpawu ezahlukeneyo ze-neuropathology2,3,4.
Kwiminyaka elishumi edlulileyo, imeko ye-metabolic iye yavela njengomlawuli ophambili we-neurogenesis, umahluko, ukuvuthwa kunye ne-plasticity5,6. Kutshanje, i-mitochondrial morphology kunye ne-dynamics ziye zaba zizinto ezibaluleke kakhulu ze-mitosis, inkqubo eguqukayo egcina ichibi le-mitochondria enempilo ngaphakathi kweeseli. I-mitochondrial dynamics ilawulwa ziindlela ezintsonkothileyo ezixhomekeke phakathi kwazo ukusuka kwi-mitochondrial biogenesis kunye ne-bioenergetics ukuya kwi-mitochondrial fission, fusion, transport and clearance7,8. Ukuphazamiseka kwayo nayiphi na kwezi ndlela zokudibanisa kuphazamisa ukugcinwa kwenethiwekhi ye-mitochondrial enempilo kwaye kunemiphumo ebalulekileyo yokusebenza kwi-neurodevelopment9,10. Enyanisweni, ukuphazamiseka kwe-mitochondrial dynamics kubonwa kwiingxaki ezininzi zengqondo, ze-neurodegenerative kunye ne-neurodevelopmental, kubandakanya ne-autism spectrum disorders (ASD)11,12.
I-ASD sisifo se-neurodevelopmental esahlukileyo esine-architecture enzima ye-genetic kunye ne-epigenetic. Ufuzo lwe-ASD alunakuphikiswa, kodwa i-etiology ye-molecular esisiseko ayikaqondwa kakuhle. Ukuqokelela idatha evela kwiimodeli zangaphambi kweklinikhi, izifundo zeklinikhi, kunye neeseti zedatha ze-molecular ze-multi-omics zibonelela ngobungqina obandayo bokungasebenzi kakuhle kwe-mitochondrial kwi-ASD13,14. Ngaphambili senze uvavanyo lwe-genome-wide DNA methylation kwiqela lezigulane ezine-ASD kwaye sachonga ii-genes ze-methylated ezahlukeneyo eziqokelelwe kwiindlela ze-metabolism ze-mitochondrial15. Emva koko saxela i-methylation eyahlukileyo yabalawuli abaphambili be-mitochondrial biogenesis kunye ne-dynamics, eyayinxulunyaniswa nenani lekopi ye-mtDNA eyongeziweyo kunye notshintsho kwiprofayili ye-metabolic yomchamo kwi-ASD16. Idatha yethu inikezela ngobungqina obandayo bokuba i-mitochondrial dynamics kunye ne-homeostasis zidlala indima ephambili kwi-pathophysiology ye-ASD. Ke ngoko, ukuphucula ukuqonda kobuchwephesha kobudlelwane phakathi kwe-mitochondrial dynamics, i-morphology, kunye nomsebenzi yinjongo ephambili yophando oluqhubekayo kwizifo ze-neurological ezibonakaliswa kukungasebenzi kakuhle kwe-mitochondrial yesibini.
Iindlela zemolekyuli zihlala zisetyenziselwa ukufunda indima yezakhi zofuzo ezithile kwiimpendulo zoxinzelelo lwe-mitochondrial. Nangona kunjalo, le ndlela inokuthintelwa bubume obunemilo emininzi nobexesha leendlela zokulawula i-mitotic. Ngaphezu koko, ukubonakaliswa okwahlukileyo kwezakhi zofuzo ze-mitochondrial luphawu olungangqalanga lotshintsho olusebenzayo, ngakumbi kuba kuphela inani elincinci lezakhi zofuzo elihlala lihlalutywa. Ke ngoko, iindlela ezithe ngqo zokufunda umsebenzi we-mitochondrial kunye ne-bioenergetics ziye zacetyiswa17. Imo ye-mitochondrial inxulumene kakhulu ne-mitochondrial dynamics. Imo ye-mitochondrial, uqhagamshelwano, kunye nesakhiwo zibalulekile kwimveliso yamandla kunye nokusinda kwe-mitochondrial kunye neseli5,18. Ngaphezu koko, izinto ezahlukeneyo ze-mitosis zigxila kutshintsho kwimo ye-mitochondrial, ezinokusebenza njengeendawo eziluncedo zokungasebenzi kakuhle kwe-mitochondrial kwaye zibonelele ngesiseko sezifundo ezilandelayo ze-mechanistic.
Imo ye-mitochondrial inokubonwa ngokuthe ngqo kusetyenziswa i-transmission electron microscopy (TEM), nto leyo evumela uphononongo oluneenkcukacha lwe-cellular ultrastructure. I-TEM ibona ngokuthe ngqo imo, imo kunye nesakhiwo se-mitochondrial cristae kwisisombululo se-mitochondria nganye, endaweni yokuxhomekeka kuphela kwi-gene transcription, i-protein expression okanye ii-mitochondrial functional parameters kwi-cell populations17,19,20. Ukongeza, i-TEM iququzelela uphononongo lokusebenzisana phakathi kwe-mitochondria kunye nezinye ii-organelles, ezifana ne-endoplasmic reticulum kunye ne-autophagosomes, ezidlala indima ebalulekileyo kumsebenzi we-mitochondrial kunye ne-homeostasis21,22. Ke ngoko, oku kwenza i-TEM ibe yindawo elungileyo yokuqala yokufunda ukungasebenzi kakuhle kwe-mitochondrial ngaphambi kokugxila kwiindlela ezithile okanye ii-genes. Njengoko umsebenzi we-mitochondrial usiba luncedo ngakumbi kwi-neuropathology, kukho imfuneko ecacileyo yokukwazi ukufunda ngokuthe ngqo nangobuninzi imo ye-mitochondrial kunye ne-dynamics kwiimodeli ze-neuronal ze-in vitro.
Kule nqaku, sihlola i-mitochondrial dynamics kwimodeli ye-neuronal yokungasebenzi kakuhle kwe-mitochondrial kwi-autism spectrum disorder. Ngaphambili sibike nge-differential methylation ye-propionyl-CoA carboxylase beta (PCCB) kwi-ASD15, iyunithi encinci ye-mitochondrial propionyl-CoA carboxylase enzyme PCC. Ukungasebenzi kakuhle kwe-PCC kuyaziwa ukuba kubangela ukuqokelelwa kwetyhefu kwe-propionyl derivatives, kubandakanya i-propionic acid (PPA)23,24,25. I-PPA ibonakalisiwe ukuba iphazamisa i-neuronal metabolism kwaye itshintsha indlela yokuziphatha kwi-vivo kwaye yimodeli yezilwanyana esekwe ekufundeni iindlela zophuhliso lwe-neurodevelopmental ezibandakanyeka kwi-ASD26,27,28. Ukongeza, i-PPA ibikwe ukuba iphazamisa amandla e-mitochondrial membrane, i-biogenesis kunye nokuphefumla kwi-vitro kwaye isetyenzisiwe kakhulu ukubonisa ukungasebenzi kakuhle kwe-mitochondrial kwi-neurons29,30. Nangona kunjalo, impembelelo yokungasebenzi kakuhle kwe-mitochondrial okubangelwa yi-PPA kwi-morphology ye-mitochondrial kunye ne-dynamics ayikaqondwa kakuhle.
Olu phononongo lusebenzisa iindlela zokulinganisa ezongezelelweyo ukuze kulinganiswe imiphumo ye-PPA kwimo ye-mitochondrial, ii-dynamics, kunye nomsebenzi kwiiseli ze-SH-SY5Y. Okokuqala, siphuhlise indlela ye-TEM ukuze sibone utshintsho kwimo ye-mitochondrial kunye ne-ultrastructure17,31,32. Ngenxa yendalo ye-dynamic ye-mitochondria33, sikwasebenzise uhlalutyo lwe-mitochondrial event localizer (MEL) ukuze kulinganiswe utshintsho kwibhalansi phakathi kweziganeko ze-fission kunye ne-fusion, inani le-mitochondrial kunye nomthamo phantsi koxinzelelo lwe-PPA. Okokugqibela, sihlolisise ukuba imo ye-mitochondrial kunye ne-dynamics zidibene natshintsho ekubonakalisweni kwezakhi zofuzo ezibandakanyeka kwi-biogenesis, i-fission, kunye ne-fusion. Xa zidibene, idatha yethu ibonisa umngeni wokucacisa ubunzima beendlela ezilawula ii-mitochondrial dynamics. Sigxininisa ukusetyenziswa kwe-TEM ekufundeni imo ye-mitochondrial njengesiphelo esilinganiselweyo se-mitosis kwiiseli ze-SH-SY5Y. Ukongeza, sigxininisa ukuba idatha ye-TEM ibonelela ngolwazi olutyebileyo xa idityaniswe neendlela zokujonga ezibamba neziganeko ze-dynamic ekuphenduleni uxinzelelo lwe-metabolic. Ukucaciswa ngakumbi kweendlela zokulawula iimolekyuli ezixhasa i-neuronal cell mitosis kunokubonelela ngengcaciso ebalulekileyo malunga nenxalenye ye-mitochondrial yenkqubo yemithambo-luvo kunye nezifo ze-neurodegenerative.
Ukuze kubangele uxinzelelo lwe-mitochondrial, iiseli ze-SH-SY5Y zanyangwa nge-PPA kusetyenziswa i-3 mM kunye ne-5 mM sodium propionate (NaP). Ngaphambi kwe-TEM, iisampuli zazilungiswa ngesampuli ye-cryogenic kusetyenziswa ukuqandisa nokufaka ubushushu obuphezulu (Umzobo 1a). Siphuhlise umbhobho wohlalutyo lomfanekiso we-mitochondrial ozenzekelayo ukulinganisa iiparameter ezisibhozo ze-morphological zabemi be-mitochondrial kuzo zonke iikopi ezintathu zebhayoloji. Sifumanise ukuba unyango lwe-PPA lutshintshe kakhulu iiparameter ezine: indawo yesi-2, indawo, umjikelezo, kunye nobubanzi be-Feret (Umzobo 1b–e). Indawo yesi-2 yehle kakhulu ngonyango lwe-3 mM kunye ne-5 mM PPA (p = 0.0183 kunye ne-p = 0.002, ngokulandelelana) (Umzobo 1b), ngelixa indawo (p = 0.003), umjikelezo (p = 0.0106) kunye nobubanzi be-Feret zonke zehle kakhulu. Bekukho ukwehla okukhulu (p = 0.0172) kwiqela lonyango lwe-5 mM xa kuthelekiswa neqela lolawulo (Umzobo 1c–e). Ukuncipha okukhulu kwendawo kunye nokujikeleza kubonise ukuba iiseli eziphathwe nge-5 mM PPA zazine-mitochondria encinci, ejikelezileyo, kwaye ezi mitochondria zazinde kancinci kunezo zikwiiseli zolawulo. Oku kuhambelana nokuncipha okukhulu kububanzi beFeret, ipharamitha ezimeleyo ebonisa ukwehla komgama omkhulu phakathi kwemiphetho yamasuntswana. Utshintsho kwisakhiwo se-cristae esingaphezulu kwesangqa lubonwe: i-cristae yaba yinto engacacanga kangako phantsi kwempembelelo yoxinzelelo lwe-PPA (Umzobo 1a, iphaneli B). Nangona kunjalo, ayizizo zonke iifoto ezibonakalise ngokucacileyo isakhiwo se-cristae esingaphezulu kwesangqa, ngoko ke uhlalutyo lobungakanani lwale nguqu aluzange lwenziwe. Le datha ye-TEM inokubonisa iimeko ezintathu ezinokwenzeka: (1) I-PPA inyusa ukuqhekeka okanye ithintele ukudibana, ibangela ukuba i-mitochondria ekhoyo inciphe ngobukhulu; (2) i-biogenesis ephuculweyo idala i-mitochondria entsha, encinci okanye (3) ibangela zombini iindlela ngaxeshanye. Nangona ezi meko zingenakwahlulwa yi-TEM, utshintsho olukhulu lwe-morphological lubonisa utshintsho kwi-homeostasis ye-mitochondrial kunye ne-dynamics phantsi koxinzelelo lwe-PPA. Emva koko sihlolisise iiparameter ezongezelelweyo ukuze sichaze ngakumbi ezi dynamics kunye neendlela ezinokubakho eziphantsi kwazo.
I-Propionic acid (PPA) ilungisa imo ye-mitochondrial. (a) Imifanekiso emele i-transmission electron microscopy (TEM) ebonisa ukuba ubungakanani be-mitochondrial buyancipha kwaye i-mitochondria iba ncinci kwaye ijikeleze ngakumbi ngonyango lwe-PPA olukhulayo; 0 mM (enganyangwanga), 3 mM kunye ne-5 mM, ngokulandelanayo. Iintolo ezibomvu zibonisa i-mitochondria. (b–e) Iiseli ze-SH-SY5Y ezinyangwe nge-PPA iiyure ezingama-24 zalungiselelwa i-TEM kwaye iziphumo zahlalutywa kusetyenziswa iFiji/ImageJ. Iiparameter ezine kwezisibhozo zibonise umahluko obalulekileyo phakathi kweeseli zolawulo (ezinganyangwanga, 0 mM PPA) kunye neeseli ezinyangiweyo (3 mM kunye ne-5 mM PPA). (b) Ummandla 2, (c) Indawo, (d) I-Perimeter, (e) Ububanzi be-Feret. Uhlalutyo lwendlela enye yokwahluka (ulawulo vs. unyango) kunye novavanyo lukaDunnett lokuthelekisa okuninzi zisetyenzisiwe ukufumanisa umahluko obalulekileyo (p < 0.05). Amanqaku edatha amele ixabiso eliphakathi le-mitochondrial kwiseli nganye, kwaye iibha zempazamo zimele i-mean ± SEM. Idatha ebonisiweyo imele i-n = 3, ubuncinane iiseli ezingama-24 ngokuphindaphindwayo; imifanekiso engama-266 iyonke ihlalutywe; * ibonisa u-p < 0.05, ** ibonisa u-p < 0.01.
Ukuze sichaze ngakumbi indlela i-mitochondrial dynamics ephendula ngayo kwi-PPA, sifake i-mitochondria i-tetramethylrhodamine ethyl ester (TMRE) kwaye sasebenzisa i-time-lapse microscopy kunye nohlalutyo lwe-MEL ukuze sifumane indawo kwaye silinganise i-mitochondria emva kweeyure ezingama-24 kwi-3 kunye ne-5 mM PPA. Unyango lweziganeko zokuqhekeka kunye nokudibana. (Umzobo 2a). Emva kohlalutyo lwe-MEL, i-mitochondria yahlalutywa ngakumbi ukuze kulinganiswe inani lezakhiwo ze-mitochondrial kunye nomthamo wazo ophakathi. Sibone ukwanda okuncinci kodwa okubalulekileyo kwinani leziganeko zokuqhekeka ezenzeka kwi-3 mM [4.9 ± 0.3 (p < 0.05)] xa kuthelekiswa nokuqhekeka [5.6 ± 0.3 (p < 0.05) )] kunye nokuhlanganiswa [5.4 ± 0.5 (p < 0.05)] kunye nokuhlanganiswa [5.4 ± 0.5 (p < 0.05)] 0.05)] <0.05)] iziganeko zonyuswe kakhulu kwi-5 mM xa kuthelekiswa nolawulo (Umzobo 3b). Inani le-mitochondria landa kakhulu kuzo zombini i-3 [32.6 ± 2.1 (p < 0.05)] kunye ne-5 mM [34.1 ± 2.2 (p < 0.05)] (Umzobo 3c), ngelixa umthamo ophakathi wesakhiwo ngasinye se-mitochondrial ungatshintshanga (Umzobo 3c). 3d). Xa zizonke, oku kuthetha ukuba ukuhlaziywa kwe-mitochondrial dynamics kusebenza njengempendulo yokubuyisela egcina ngempumelelo ukuthembeka kwenethiwekhi ye-mitochondrial. Ukwanda kwenani leziganeko zokuqhekeka kwi-3 mM PPA kubonisa ukuba ukwanda kwenani le-mitochondrial kungenxa yokuqhekeka kwe-mitochondrial, kodwa ngenxa yokuba umthamo ophakathi we-mitochondrial uhlala ungatshintshi, i-biogenesis ayinakucinywa njengempendulo eyongezelelweyo yokubuyisela. Nangona kunjalo, ezi datha ziyahambelana nezakhiwo ezincinci, ezijikelezileyo ze-mitochondrial ezibonwe yi-TEM kwaye zibonisa utshintsho olukhulu kwi-mitochondrial dynamics ezibangelwa yi-PPA.
I-Propionic acid (PPA) ikhuthaza ukuvuselelwa kwe-mitochondrial enamandla ukugcina ukuthembeka kwenethiwekhi. Iiseli ze-SH-SY5Y zakhuliswa, zanyangwa nge-3 kunye ne-5 mM PPA kangangeeyure ezingama-24 zaza zafakwa i-TMRE kunye ne-Hoechst 33342 kulandele uhlalutyo lwe-MEL. (a) Imifanekiso ye-microscopy emele ixesha elidlulayo ebonisa umbala kunye ne-binarized maximum intensity projections ngexesha lesi-2 (t2) kwimeko nganye. Iindawo ezikhethiweyo eziboniswe kumfanekiso ngamnye we-binary ziyaphuculwa kwaye ziboniswe kwi-3D ngamaxesha amathathu ahlukeneyo (t1-t3) ukubonisa i-dynamics ngokuhamba kwexesha; iziganeko ze-fusion zigqanyisiwe ngombala oluhlaza; iziganeko ze-fission zigqanyisiwe ngombala oluhlaza. Ziboniswe ngombala obomvu. (b) Inani eliphakathi leziganeko ze-dynamic kwimeko nganye. (c) Inani eliphakathi lezakhiwo ze-mitochondrial ngeseli nganye. (d) Umthamo ophakathi (µm3) wesakhiwo ngasinye se-mitochondrial ngeseli nganye. Idatha ebonisiweyo imele i-n = iiseli ezili-15 kwiqela ngalinye lonyango. Iibha zempazamo ezibonisiweyo zimele i-mean ± SEM, ibha yesikali = 10 μm, * p < 0.05.
I-Propionic acid (PPA) ibangela ukucinezelwa kwe-transcriptional kwezakhi zofuzo ezinxulumene ne-mitochondrial dynamics. Iiseli ze-SH-SY5Y ziphathwe nge-3 kunye ne-5 mM PPA kangangeeyure ezingama-24. Ukulinganiswa kwezakhi zofuzo okunxulumeneyo kwenziwe kusetyenziswa i-RT-qPCR kwaye kwenziwa ngokwesiqhelo kwi-B2M. Izakhi zofuzo ze-Mitochondrial biogenesis (a) cMYC, (b) TFAM, (c) NRF1 kunye (d) NFE2L2. Izakhi zofuzo ze-Mitochondrial fusion kunye ne-fission (e) STOML2, (f) OPA1, (g) MFN1, (h) MFN2 kunye (i) DRP1. Umahluko obalulekileyo (p < 0.05) uvavanywe kusetyenziswa i-ANOVA yendlela enye (ulawulo vs. unyango) kunye novavanyo lokuthelekisa oluninzi lukaDunnett: * ibonisa p < 0.05, ** ibonisa p < 0.01, kunye **** ibonisa p < 0.0001. Iibha zimele i-mean expression ± SEM. Idatha ebonisiweyo imele i-n = 3 (STOML2, OPA1, TFAM), n = 4 (cMYC, NRF1, NFE2L2), kunye ne-n = 5 (MFN1, MFN2, DRP1) iikopi zebhayoloji.
Idatha evela kuhlalutyo lwe-TEM kunye ne-MEL kunye ibonisa ukuba i-PPA itshintsha imo ye-mitochondrial kunye ne-dynamics. Nangona kunjalo, ezi ndlela zokujonga imifanekiso aziboneleli ngengqiqo kwiindlela ezisisiseko eziqhuba ezi nkqubo. Ngoko ke sihlolisise ukubonakaliswa kwe-mRNA kwabalawuli abasithoba abaphambili be-mitochondrial dynamics, i-biogenesis, kunye ne-mitosis ukuphendula unyango lwe-PPA. Silinganise i-cell myeloma oncogene (cMYC), i-nuclear respiratory factor (NRF1), i-mitochondrial transcription factor 1 (TFAM), i-NFE2-like transcription factor BZIP (NFE2L2), i-gastrin-like protein 2 (STOML2), i-optic nerve atrophy 1 (OPA1), i-Mitofusin 1 (MFN1), i-Mitofusin 2 (MFN2) kunye ne-dynamin-related protein 1 (DRP1) emva kweeyure ezingama-24 zonyango nge-3 mM kunye ne-5 mM PPA. Sibone i-3 mM (p = 0.0053, p = 0.0415 kunye ne-p < 0.0001, ngokulandelanayo) kunye ne-5 mM (p = 0.0031, p = 0.0233, p < 0.0001) unyango lwe-PPA. (Umzobo 3a–c). Ukwehla kokubonakaliswa kwe-mRNA bekuxhomekeke kwidosi: ukubonakaliswa kwe-cMYC, i-NRF1 kunye ne-TFAM kwehle ngamaxesha angama-5.7, 2.6 kunye ne-1.9 kwi-3 mM, ngokulandelanayo, kwaye ngamaxesha angama-11.2, 3 kunye ne-2.2 kwi-5 mM. Ngokwahlukileyo koko, i-central redox biogenesis gene NFE2L2 ayizange itshintshe nakweyiphi na i-concentration ye-PPA, nangona kukho umkhwa ofanayo oxhomekeke kwidosi wokubonakaliswa okunciphileyo (Umzobo 3d).
Sikwahlolisise ukubonakaliswa kweejini zakudala ezibandakanyekayo ekulawuleni ukuqhekeka kunye nokuhlanganiswa. I-STOML2 kucingelwa ukuba ibandakanyeka ekuhlanganisweni, kwi-mitophagy nakwi-biogenesis, kwaye ukubonakaliswa kwayo kuncitshiswe kakhulu (p < 0.0001) nge-3 mM (utshintsho oluphindwe kabini) kunye ne-5 mM (utshintsho oluphindwe kabini) i-PPA (Umzobo 1). 3d). Ngokufanayo, ukubonakaliswa kwejini ye-OPA1 fusion kuncitshisiwe kwi-3 mM (utshintsho oluphindwe kabini) kunye ne-5 mM (utshintsho oluphindwe kabini) i-PPA (p = 0.006 kunye ne-p = 0.0024, ngokulandelelana) (Umzobo 3f). Nangona kunjalo, asifumananga mahluko abalulekileyo ekubonakalisweni kweejini ze-fusion i-MFN1, i-MFN2 okanye i-fission gene DRP1 phantsi koxinzelelo lwe-PPA lweeyure ezingama-24 (Umzobo 3g–i). Ukongeza, sifumanise ukuba amanqanaba eeproteni ezine ze-fusion kunye ne-fission (OPA1, MFN1, MFN2 kunye ne-DRP1) awatshintshanga phantsi kweemeko ezifanayo (Umzobo 4a–d). Kubalulekile ukuqaphela ukuba le datha ibonisa inqaku elinye ngexesha kwaye isenokungabonisi utshintsho kwimbonakalo yeproteni okanye amanqanaba omsebenzi ngexesha lamanqanaba okuqala oxinzelelo lwe-PPA. Nangona kunjalo, ukuncipha okubalulekileyo kwimbonakalo ye-cMYC, NRF1, TFAM, STOML2, kunye ne-OPA1 kubonisa ukuphazamiseka okukhulu kwe-transcriptional ye-metabolism ye-mitochondrial, i-biogenesis, kunye ne-dynamics. Ukongeza, le datha igxininisa ukusetyenziswa kweendlela zokujonga ukufunda ngokuthe ngqo utshintsho lwe-end-state kumsebenzi we-mitochondrial.
Amanqanaba eprotheyini ye-Fusion kunye ne-fission factor awatshintshanga emva konyango lwe-propionic acid (PPA). Iiseli ze-SH-SY5Y zanyangwa nge-3 kunye ne-5 mM PPA kangangeeyure ezingama-24. Amanqanaba eprotheyini alinganiswe ngohlalutyo lwe-Western blot, kwaye amanqanaba okubonakaliswa ahlengahlengiswa kwi-total protein. I-avareji ye-protein expression kunye ne-Western blots emele i-target kunye ne-total protein ziyaboniswa. a – OPA1, b – MFN1, c – MFN2, d – DRP1. Iibha zimele i-mean ± SEM, kwaye idatha ebonisiweyo imele i-n = 3 biological replicates. Uthelekiso oluninzi (p < 0.05) lwenziwe kusetyenziswa uhlalutyo lwe-one-way of variance kunye novavanyo lukaDunnett. Ijeli yokuqala kunye ne-blot ziboniswe kuMfanekiso S1.
Ukungasebenzi kakuhle kwe-Mitochondrial kunxulunyaniswa nezifo ezininzi eziqala kwizifo ze-metabolic, zentliziyo kunye nezemisipha ukuya kwizifo ze-neurological1,10. Izifo ezininzi ze-neurodegenerative kunye neze-neurodegenerative zinxulunyaniswa nokungasebenzi kakuhle kwe-mitochondrial, nto leyo egxininisa ukubaluleka kwezi organelles kulo lonke ixesha lobomi bengqondo. Ezi zifo ziquka isifo sikaParkinson, isifo sika-Alzheimer kunye ne-ASD3,4,18. Nangona kunjalo, ukufikelela kwizicubu zobuchopho ukuze kufundwe ezi zifo kunzima, ingakumbi kwinqanaba le-mechanistic, okwenza iinkqubo zemodeli yeselula zibe yenye indlela efunekayo. Kolu phononongo, sisebenzisa inkqubo yemodeli yeselula esebenzisa iiseli ze-SH-SY5Y eziphathwe yi-PPA ukuze sichaze kwakhona ukungasebenzi kakuhle kwe-mitochondrial okubonwa kwizifo ze-neuronal, ngakumbi iingxaki ze-autism spectrum. Ukusebenzisa le modeli ye-PPA ukufunda i-mitochondrial dynamics kwi-neurons kunokubonelela ngengqiqo kwi-etiology ye-ASD.
Sihlolisise ithuba lokusebenzisa i-TEM ukujonga utshintsho kwimo ye-mitochondrial. Kubalulekile ukuqaphela ukuba i-TEM kufuneka isetyenziswe ngokuchanekileyo ukuze isebenze kakuhle. Ukulungiswa kwee-cryo-specimens kuvumela ukugcinwa okungcono kwezakhiwo ze-neuronal ngokulungisa ngaxeshanye izinto zeseli kunye nokunciphisa ukwakheka kwezinto ezisetyenzisiweyo34. Ngokuhambelana noku, siqaphele ukuba iiseli ze-SH-SY5Y ezifana ne-neuron zazine-organelles ezingaphantsi kweseli ezingaphelelanga kunye ne-mitochondria ende (Umzobo 1a). Oku kugxininisa ukusetyenziswa kweendlela zokulungiselela i-cryogenic zokufunda imo ye-mitochondrial kwiimodeli zeseli ze-neuronal. Nangona imilinganiselo yobungakanani ibalulekile kuhlalutyo oluchanekileyo lwedatha ye-TEM, akukabikho mvumelwano malunga nokuba zeziphi iiparameter ezithile ekufuneka zilinganiswe ukuqinisekisa utshintsho lwemo ye-mitochondrial. Ngokusekelwe kwinani elikhulu lezifundo ezihlolisise imo ye-mitochondrial ngobuninzi17,31,32, senze umbhobho wohlalutyo lomfanekiso we-mitochondrial ozenzekelayo olinganisa iiparameter ezisibhozo zemo, ezizezi: indawo, indawo2, umlinganiselo wento, umjikelezo, ukujikeleza, idigri, ububanzi be-Feret. kunye nokujikeleza.
Phakathi kwazo, i-PPA inciphise kakhulu indawo yesi-2, indawo, umjikelezo, kunye nobubanzi beFeret (Umzobo 1b–e). Oku kubonise ukuba i-mitochondria yaba ncinci kwaye yajikeleza ngakumbi, nto leyo ehambelana nezifundo zangaphambili ezibonisa ukwehla kwendawo ye-mitochondrial emva kweeyure ezingama-72 zoxinzelelo lwe-mitochondrial olubangelwe yi-PPA30. Ezi mpawu ze-morphological zinokubonisa ukuqhekeka kwe-mitochondrial, inkqubo efunekayo yokususa izinto ezonakeleyo kwinethiwekhi ye-mitochondrial ukukhuthaza ukuwohloka kwazo nge-mitophagy35,36,37. Kwelinye icala, ukwehla kobukhulu be-mitochondrial obuqhelekileyo kunokunxulunyaniswa nokwanda kwe-biogenesis, okubangela ukwakheka kwe-mitochondria encinci esandula ukuzalwa. Ukwanda kokuqhekeka okanye i-biogenesis kubonisa impendulo yokubuyisela ukugcina i-mitosis ngokuchasene noxinzelelo lwe-mitochondrial. Nangona kunjalo, ukukhula okunciphileyo kwe-mitochondrial, ukudibana okungasebenzi kakuhle, okanye ezinye iimeko azinakukhutshelwa ngaphandle.
Nangona imifanekiso enesisombululo esiphezulu eyenziwe yi-TEM ivumela ukumisela iimpawu zesimo somzimba kwinqanaba le-mitochondria nganye, le ndlela ivelisa ii-snapshots ezimbini-dimensional ngexesha elinye. Ukuze sifunde iimpendulo eziguquguqukayo kuxinzelelo lwe-metabolic, sifake i-mitochondria i-TMRE kwaye sasebenzisa i-time-lapse microscopy enohlalutyo lwe-MEL, oluvumela ukubonakala kwe-3D ephezulu yotshintsho kwinethiwekhi ye-mitochondrial ngokuhamba kwexesha33,38. Sibone utshintsho oluncinci kodwa olubalulekileyo kwi-mitochondrial dynamics phantsi koxinzelelo lwe-PPA (Umzobo 2). Kwi-3 mM, inani leziganeko ze-fission landa kakhulu, ngelixa iziganeko ze-fusion zahlala zifana nezolawulo. Ukwanda kwenani leziganeko ze-fission kunye ne-fusion kwabonwa kwi-5 mM PPA, kodwa olu tshintsho lwalumalunga nomlinganiselo, nto leyo ebonisa ukuba i-fission kunye ne-fusion kinetics zifikelela kumlinganiselo ophezulu (Umzobo 2b). Umthamo ophakathi we-mitochondrial awuzange utshintshe kuzo zombini i-3 kunye ne-5 mM PPA, nto leyo ebonisa ukuba ukuthembeka kwenethiwekhi ye-mitochondrial kugcinwe (Umzobo 2d). Oku kubonisa amandla eenethiwekhi ze-mitochondrial eziguqukayo ukuphendula kuxinzelelo oluncinci lwe-metabolic ukugcina i-homeostasis ngokufanelekileyo ngaphandle kokubangela ukuqhekeka kwenethiwekhi. Kwi-3 mM PPA, ukwanda kokuqhekeka kwanele ukukhuthaza utshintsho oluya kumlinganiselo omtsha, kodwa kufuneka ukulungiswa kwakhona kwe-kinetic okunzulu ukuphendula uxinzelelo olubangelwa kukugxila okuphezulu kwe-PPA.
Inani le-mitochondria landa kuzo zombini iindawo ezixineneyo ze-PPA, kodwa umyinge we-mitochondrial volume awutshintshanga kakhulu (Umzobo 2c). Oku kusenokuba kungenxa yokwanda kwe-biogenesis okanye ukwanda kolwahlulo; nangona kunjalo, xa kungekho kwehla okukhulu kumyinge we-mitochondrial volume, kunokwenzeka ukuba i-biosynthesis iyanda. Nangona kunjalo, idatha ekuMfanekiso 2 ixhasa ukubakho kweendlela ezimbini zokuhlawula: ukwanda kwenani leziganeko zokuqhekeka, ezihambelana nokunyuka kokuqhekeka kwe-mitochondrial, kunye nokwanda kwenani leziganeko, ezihambelana ne-mitochondrial biogenesis. Ekugqibeleni, imbuyekezo enamandla yoxinzelelo oluncinci inokuba neenkqubo ngaxeshanye ezibandakanya ukuqhekeka, ukuhlanganiswa, i-biogenesis, kunye ne-mitophagy. Nangona ababhali bangaphambili bebonise ukuba i-PPA iphucula i-mitosis30,39 kunye ne-mitophagy29, sinika ubungqina bokulungiswa kwe-mitochondrial fission kunye ne-fusion dynamics ukuphendula kwi-PPA. Ezi datha ziqinisekisa utshintsho lwe-morphological olubonwe yi-TEM kwaye zibonelela ngolwazi oluthe kratya malunga neendlela ezinxulumene nokungasebenzi kakuhle kwe-mitochondrial okubangelwa yi-PPA.
Ngenxa yokuba uhlalutyo lwe-TEM kunye ne-MEL aluzange lubonelele ubungqina obuthe ngqo beendlela zokulawula iijini eziphantsi kotshintsho olubonakalayo lwe-morphological, sihlolisise ukubonakaliswa kwe-RNA kweejini ezibandakanyeka kwi-metabolism ye-mitochondrial, i-biogenesis, kunye ne-dynamics. I-cMYC proto-oncogene yinto ebangela ukubhalwa kwe-mitochondria, i-glycolysis, i-amino acid kunye ne-fatty acid metabolism40. Ukongeza, i-cMYC yaziwa ngokulawula ukubonakaliswa kweejini ze-mitochondrial eziphantse zibe yi-600 ezibandakanyeka kwi-mitochondrial transcription, translation, kunye ne-complex assembly, kubandakanya i-NRF1 kunye ne-TFAM41. I-NRF1 kunye ne-TFAM ngabalawuli ababini abaphambili be-mitosis, abasebenza phantsi kwe-PGC-1α ukuze kusebenze ukuphindaphinda kwe-mtDNA. Le ndlela isebenza nge-cAMP kunye ne-AMPK signaling kwaye inovelwano kwinkcitho yamandla kunye noxinzelelo lwe-metabolic. Sikwahlolisise i-NFE2L2, umlawuli we-redox we-mitochondrial biogenesis, ukumisela ukuba iziphumo ze-PPA zinokulawulwa luxinzelelo lwe-oxidative.
Nangona ukubonakaliswa kwe-NFE2L2 kungatshintshanga, sifumene ukwehla okuxhomekeke kwidosi ekubonakalisweni kwe-cMYC, i-NRF1 kunye ne-TFAM emva kweeyure ezingama-24 zonyango nge-3 mM kunye ne-5 mM PPA (Umzobo 3a–c). Ukwehla kokubonakaliswa kwe-cMYC kuye kwaxelwa ngaphambili njengempendulo kuxinzelelo lwe-mitochondrial42, kwaye ngokuchaseneyo, ukwehla kokubonakaliswa kwe-cMYC kunokubangela ukungasebenzi kakuhle kwe-mitochondrial ngokuguqula imetabolism ye-mitochondrial, uqhagamshelo lwenethiwekhi, kunye ne-membrane polarization43. Okunomdla kukuba, i-cMYC ikwabandakanyeka ekulawulweni kwe-mitochondrial fission kunye ne-fusion42,43 kwaye yaziwa ngokunyusa i-DRP1 phosphorylation kunye nendawo ye-mitochondrial ngexesha lokwahlulwa kweseli44, kunye nokulungisa i-mitochondrial morphological kwiiseli ze-neuronal stem45. Enyanisweni, ii-fibroblasts ezingenayo i-cMYC zibonisa ubungakanani obunciphileyo be-mitochondrial, obuhambelana notshintsho olubangelwa luxinzelelo lwe-PPA43. Ezi datha zibonisa ubudlelwane obunomdla kodwa obungacacanga phakathi kwe-cMYC kunye ne-mitochondrial dynamics, ebonelela ngenjongo enomdla kwizifundo ezizayo zokulungiswa koxinzelelo lwe-PPA.
Ukuncipha kwe-NRF1 kunye ne-TFAM kuhambelana nendima ye-cMYC njenge-activator ebalulekileyo yokubhala. Le datha ikwahambelana nezifundo zangaphambili kwiiseli zomhlaza wamathumbu omntu ezibonisa ukuba i-PPA inciphise ukubonakaliswa kwe-NRF1 mRNA kwiiyure ezingama-22, nto leyo eyayinxulunyaniswa nokuncipha kwe-ATP kunye nokwanda kwe-ROS46. Aba babhali baxele nokuba ukubonakaliswa kwe-TFAM kwanda kwiiyure eziyi-8.5 kodwa kwabuyela kumanqanaba okuqala kwiiyure ezingama-22. Ngokwahlukileyo koko, uKim et al. (2019) babonise ukuba ukubonakaliswa kwe-TFAM mRNA kwehle kakhulu emva kweeyure ezi-4 zoxinzelelo lwe-PPA kwiiseli ze-SH-SY5Y; nangona kunjalo, emva kweeyure ezingama-72, ukubonakaliswa kweproteni ye-TFAM kwanda kakhulu kwaye inani lekopi ye-mtDNA landa kakhulu. Ke ngoko, ukwehla kwenani le-mitochondrial biogenesis genes esizibonileyo emva kweeyure ezingama-24 akukhuphi ngaphandle ithuba lokuba ukwanda kwenani le-mitochondria kunxulunyaniswa nokusebenza kwe-biogenesis kwiindawo zangaphambili. Izifundo zangaphambili zibonise ukuba i-PPA inyusa kakhulu i-PGC-1α mRNA kunye neproteni kwiiseli ze-SH-SY5Y kwiiyure ezi-4 nemizuzu engama-30, ngelixa i-propionic acid inyusa i-mitochondrial biogenesis kwi-calf hepatocytes nge-PGC-1α kwiiyure ezili-12 nemizuzu engama-39. Okunomdla kukuba, i-PGC-1α ayisiyo kuphela i-transcriptional regulator ye-NRF1 kunye ne-TFAM, kodwa ikwabonakalisiwe ukuba ilawula umsebenzi we-MFN2 kunye ne-DRP1 ngokulawula ukuqhekeka kunye nokudibana47. Xa zizonke, oku kugxininisa ukudibana okusondeleyo kweendlela ezilawula iimpendulo ze-mitochondrial compensatory ezibangelwa yi-PPA. Ngaphezu koko, idatha yethu ibonisa ukuphazamiseka okukhulu kokulawulwa kwe-transcriptional ye-biogenesis kunye ne-metabolism phantsi koxinzelelo lwe-PPA.
Iijini ze-STOML2, OPA1, MFN1, MFN2 kunye ne-DRP1 ziphakathi kwabalawuli abaphambili be-mitochondrial fission, fusion kunye ne-dynamics37,48,49. Kukho ezinye iijini ezininzi ezibandakanyeka kwi-mitochondrial dynamics, nangona kunjalo, i-STOML2, OPA1 kunye ne-MFN2 zifunyenwe ngaphambili ukuba zine-methylated ngokwahlukileyo kwii-cohorts ze-ASD,16 kwaye izifundo ezininzi ezizimeleyo zibike utshintsho kwezi zinto zokubhala ukuphendula uxinzelelo lwe-mitochondrial50,51.52. Ukubonakaliswa kwe-OPA1 kunye ne-STOML2 kuncitshiswe kakhulu ngonyango lwe-3 mM kunye ne-5 mM PPA (Umzobo 3e, f). I-OPA1 yenye yabalawuli beklasikhi be-mitochondrial fusion ngokusebenzisana ngqo ne-MFN1 kunye ne-2 kwaye idlala indima ekulungisweni kwe-cristae kunye ne-mitochondrial morphology53. Indima echanekileyo ye-STOML2 kwi-mitochondrial dynamics ayikacaci, kodwa ubungqina bubonisa ukuba idlala indima kwi-mitochondrial fusion, i-biogenesis, kunye ne-mitophagy.
I-STOML2 ibandakanyeka ekugcineni ukudibana kwe-mitochondrial respiratory coupling kunye nokwakhiwa kwee-respiratory chain complexes54,55 kwaye ibonakalisiwe ukuba itshintsha kakhulu iimpawu ze-metabolic zeeseli zomhlaza56. Izifundo zibonise ukuba i-STOML2 ikhuthaza amandla e-mitochondrial membrane kunye ne-biogenesis ngokusebenzisana ne-BAN kunye ne-cardiolipin 55, 57, 58. Ukongeza, izifundo ezizimeleyo zibonise ukuba ukusebenzisana phakathi kwe-STOML2 kunye ne-PINK1 kulawula i-mitophagy59,60. Okuphawulekayo kukuba, i-STOML2 ibikwe ukuba isebenzisana ngokuthe ngqo kwaye izinzise i-MFN2 kwaye idlala indima ebalulekileyo ekuzinziseni ii-isoforms ezinde ze-OPA1 ngokuthintela i-protease enoxanduva lokubola kwe-OPA153,61,62. Ukunciphisa ukubonakaliswa kwe-STOML2 okubonwa kwiimpendulo ze-PPA kunokwenza ezi proteins ze-fusion zibe sesichengeni sokubola ngeendlela ezixhomekeke kwi-ubiquitin kunye ne-proteasome48. Nangona indima echanekileyo ye-STOML2 kunye ne-OPA1 kwimpendulo eguqukayo kwi-PPA ingacacanga, ukwehla kokubonakaliswa kwezi genes ze-fusion (Umfanekiso 3) kunokuphazamisa ibhalansi phakathi kokuqhekeka kunye ne-fusion kwaye kukhokelele ekunciphiseni ubungakanani be-mitochondrial (Umfanekiso 3). 1).
Kwelinye icala, ukubonakaliswa kweprotheyini ye-OPA1 akutshintshanga emva kweeyure ezingama-24, ngelixa amanqanaba e-mRNA kunye neeprotheyini ze-MFN1, MFN2 okanye i-DRP1 engatshintshanga kakhulu emva konyango lwe-PPA (Umzobo 3g-i, Umzobo 4). Oku kunokubonisa ukuba akukho tshintsho kulawulo lwezi zinto zibandakanyekayo ekuhlanganiseni nasekuqhekekeni kwe-mitochondrial. Nangona kunjalo, kubalulekile ukuqaphela ukuba nganye kwezi jini zine ilawulwa ziinguqu ze-posttranscriptional (PTMs) ezilawula umsebenzi weprotheyini. I-OPA1 inee-splice variants ezisibhozo ezikhethiweyo kwi-mitochondria ukuvelisa ii-isoforms ezimbini ezahlukeneyo 63. Ibhalansi phakathi kwee-isoforms ezinde nezimfutshane ekugqibeleni imisela indima ye-OPA1 ekuhlanganiseni kwe-mitochondrial kunye nokugcinwa kwenethiwekhi ye-mitochondrial64. Umsebenzi we-DRP1 ulawulwa yi-calcium/calmodulin-dependent protein kinase II (CaMKII) phosphorylation, ngelixa ukubola kwe-DRP1 kulawulwa yi-ubiquitination kunye ne-SUMOylation65. Okokugqibela, zombini i-DRP1 kunye ne-MFN1/2 zii-GTPases, ngoko ke umsebenzi unokuchatshazelwa lizinga lemveliso ye-GTP kwi-mitochondria 66. Ke ngoko, nangona ukubonakaliswa kwezi proteni kuhlala kungaguquki, oku kusenokungabonisi umsebenzi weproteni ongaguqukiyo okanye indawo67,68. Enyanisweni, ii-repertoires zeproteni ze-PTM ezikhoyo zihlala zisebenza njengomgca wokuqala wokhuselo onoxanduva lokulawula iimpendulo zoxinzelelo olukhawulezileyo. Ukuba kukho uxinzelelo oluphakathi lwe-metabolic kwimodeli yethu, kusenokwenzeka ukuba i-PTM ikhuthaza umsebenzi owandisiweyo weeproteni ze-fusion kunye ne-fission ukubuyisela ngokwaneleyo ukuthembeka kwe-mitochondrial ngaphandle kokufuna ukusebenza okongezelelweyo kwezi gene kwinqanaba le-mRNA okanye leproteni.
Xa zizonke, idatha engentla igxininisa ukulawulwa okuntsonkothileyo nokuxhomekeke kwixesha kwe-mitochondrial morphology kunye nemingeni yokucacisa ezi ndlela. Ukuze kufundwe ukubonakaliswa kwezakhi zofuzo, kufuneka kuqala kuchongwe izakhi zofuzo ezithile ezijoliswe kuzo kwindlela. Nangona kunjalo, idatha yethu ibonisa ukuba izakhi zofuzo ezikwindlela efanayo aziphenduli ngendlela efanayo kuxinzelelo olufanayo. Enyanisweni, izifundo zangaphambili zibonise ukuba izakhi zofuzo ezahlukeneyo kwindlela efanayo zinokubonisa iiprofayili ezahlukeneyo zempendulo yexesha30,46. Ukongeza, kukho iindlela ezinzima emva kokubhalwa kwezakhi zofuzo eziphazamisa ubudlelwane phakathi kokubhalwa kwezakhi zofuzo kunye nomsebenzi wezakhi zofuzo. Izifundo zeProteomic zinokubonelela ngengqiqo ngempembelelo ye-PTM kunye nomsebenzi weproteni, kodwa zikwabangela imingeni equka iindlela eziphantsi zokukhupha, umlinganiselo ophezulu wesignali-kwingxolo, kunye nesisombululo esibi.
Kule meko, ukufunda imo ye-mitochondrial usebenzisa i-TEM kunye ne-MEL kunamandla amakhulu okuphendula imibuzo esisiseko malunga nolwalamano phakathi kwe-mitochondrial dynamics kunye nomsebenzi kunye nendlela oku kuchaphazela ngayo isifo. Okubaluleke kakhulu, i-TEM ibonelela ngendlela ethe ngqo yokulinganisa imo ye-mitochondrial njengesiphelo esidibeneyo sokungasebenzi kakuhle kwe-mitochondrial kunye ne-dynamics51. I-MEL ikwabonelela ngendlela ethe ngqo yokubona iziganeko zokuqhekeka kunye nokudibana kwindawo yeseli enamacala amathathu, okuvumela ukubalwa kokulungiswa kwakhona kwe-mitochondrial dynamic nokuba akukho tshintsho kwi-gene expression33. Apha sigxininisa ukusetyenziswa kweendlela ze-mitochondrial imaging kwizifo ze-mitochondrial yesibini. Ezi zifo zihlala zibonakaliswa luxinzelelo olungapheliyo lwe-metabolic olubonakaliswa kukulungiswa okuncinci kwenethiwekhi ye-mitochondrial endaweni yomonakalo omkhulu we-mitochondrial. Nangona kunjalo, imbuyekezo ye-mitochondrial efunekayo ukugcina i-mitosis phantsi koxinzelelo olungapheliyo inemiphumo emibi yokusebenza. Kwimeko ye-neuroscience, ukuqonda okungcono kwezi ndlela zokuhlawula kunokubonelela ngolwazi olubalulekileyo malunga ne-pleiotropic neuropathology enxulumene nokungasebenzi kakuhle kwe-mitochondrial.
Ekugqibeleni, idatha yethu igxininisa ukusetyenziswa kweendlela zokuthatha imifanekiso ukuqonda iziphumo zokusebenza konxibelelwano oluntsonkothileyo phakathi kokubonakaliswa kwezakhi zofuzo, ukuguqulwa kweproteni, kunye nomsebenzi weproteni olawula i-neuronal mitochondrial dynamics. Sisebenzise i-PPA ukwenza umzekelo wokungasebenzi kakuhle kwe-mitochondrial kwimodeli yeseli ye-neuronal ukuze sifumane ulwazi malunga nenxalenye ye-mitochondrial ye-ASD. Iiseli ze-SH-SY5Y ezinyangwe nge-PPA zibonise utshintsho kwimo ye-mitochondrial: i-mitochondria yaba ncinci kwaye ijikelezile, kwaye ii-cristae azichazwanga kakuhle xa zijongwa yi-TEM. Uhlalutyo lwe-MEL lubonisa ukuba olu tshintsho lwenzeka ngaxeshanye nokwanda kweziganeko ze-fission kunye ne-fusion ukugcina inethiwekhi ye-mitochondrial isabela kuxinzelelo oluncinci lwe-metabolic. Ngaphezu koko, i-PPA iphazamisa kakhulu ukulawulwa kwe-transcriptional ye-mitochondrial metabolism kunye ne-homeostasis. Sichonge i-cMYC, i-NRF1, i-TFAM, i-STOML2, kunye ne-OPA1 njengabalawuli abaphambili be-mitochondrial abaphazanyiswa luxinzelelo lwe-PPA kwaye banokudlala indima ekulawuleni utshintsho olubangelwa yi-PPA kwimo ye-mitochondrial kunye nomsebenzi. Izifundo zexesha elizayo ziyafuneka ukuze kuchazwe ngcono utshintsho lwexesha olubangelwa yi-PPA kwimo yezakhi zofuzo kunye nomsebenzi weprotheni, indawo, kunye notshintsho lwasemva koguqulelo. Idatha yethu igxininisa ubunzima kunye nokuxhomekeka kweendlela zokulawula ezilawula impendulo yoxinzelelo lwe-mitochondrial kwaye ibonisa ukusetyenziswa kwe-TEM kunye nezinye iindlela zokwenza imifanekiso kwizifundo zoomatshini ezijolise ngakumbi.
Umgca weseli we-SH-SY5Y (ECACC, 94030304-1VL) uthengwe kwiSigma-Aldrich. Iiseli ze-SH-SY5Y zikhuliswe kumxube wezondlo we-Eagle's medium/F-12 oguquliweyo (DMEM/F-12) kunye ne-L-glutamine (SC09411, ScienCell) kwiiflaski ezingama-25 cm2 ezongezwe yi-20% ye-fetal bovine serum (FBS) (10493106, ThermoFisher Scientific) kunye ne-1% ye-penicillin-streptomycin (P4333-20ML, Sigma-Aldrich) kwi-37 °C, 5% CO2. Iiseli zahluzwa ukuya kwi-80% confluence kusetyenziswa i-0.05% trypsin-EDTA (15400054, ThermoFisher Scientific), zaxutywa kwi-centrifuge kwi-300 g zaza zagqunywa kuxinano olumalunga ne-7 × 105 cells/ml. Zonke iimvavanyo zenziwe kwiiseli ze-SH-SY5Y ezingachazwanga phakathi kweendlela ze-19–22. I-PPA inikwa njenge-NaP. Nyibilikisa umgubo we-NaP (i-CAS No. 137-40-6, ifomula yekhemikhali i-C3H5NaO2, i-P5436-100G, i-Sigma-Aldrich) emanzini afudumeleyo e-MilliQ ukuya kuxinzelelo lwe-1 M kwaye ugcine kwi-4 °C. Ngomhla wonyango, nyibilikisa esi sisombululo nge-1 M PPA ukuya kwi-3 mM kunye ne-5 mM PPA kwindawo engena-serum medium (i-DMEM/F-12 ene-L-glutamine). Amanqanaba onyango kuzo zonke iimvavanyo ayengekho i-PPA (0 mM, ulawulo), i-3 mM, kunye ne-5 mM PPA. Iivavanyo zenziwe ubuncinane kwiikopi ezintathu zebhayoloji.
Iiseli ze-SH-SY5Y zityalwe kwiiflaski ezingama-25 cm5 ngesantya se-5.5 × 105 cells/ml kwaye zakhuliswa iiyure ezingama-24. Unyango lwe-PPA longezwe kwiflaski ngaphambi kweeyure ezingama-24 zokufunxa. Qokelela iipellets zeseli ngokulandela iinkqubo eziqhelekileyo ze-subculture yezicubu zezilwanyana ezincancisayo (ezichazwe apha ngasentla). Phinda uxhome ipellet yeseli kwi-100 µl ye-2.5% glutaraldehyde, 1 × PBS kwaye uyigcine kwi-4 °C ide icutshungulwe. Iiseli ze-SH-SY5Y zifakwe i-centrifuge okwethutyana ukuze zikhuphe iiseli kwaye zisuse i-2.5% glutaraldehyde, 1 × PBS isisombululo. Phinda uxhome i-sediment kwi-4% agarose gel elungiselelwe emanzini acocekileyo (umlinganiselo we-agarose kwi-sediment volume yi-1:1). Iziqwenga ze-Agarose zibekwe kwiigridi kwiiplate ezithe tyaba kwaye zigqunywe nge-1-hexadecene ngaphambi kokubanda ngoxinzelelo oluphezulu. Iisampulu ziqandisiwe kwi-100% eyomileyo ye-acetone kwi--90°C iiyure ezingama-24. Emva koko ubushushu banyuswa baya kwi -80°C kwaye kongezwa isisombululo se-1% osmium tetroxide kunye ne-0.1% glutaraldehyde. Iisampuli zagcinwa kwi -80°C iiyure ezingama-24. Emva koko, ubushushu bandiswa kancinci kancinci ukuya kubushushu begumbi kwiintsuku ezimbalwa: ukusuka kwi -80 °C ukuya kwi -50 °C iiyure ezingama-24, ukuya kwi -30 °C iiyure ezingama-24, ukuya kwi -10 °C iiyure ezingama-24 kwaye ekugqibeleni ukuya kubushushu begumbi.
Emva kokulungiswa kwe-cryogenic, iisampulu zafakwa i-resin kwaye iinxalenye ezincinci kakhulu (~100 nm) zenziwa kusetyenziswa i-Leica Reichert UltracutS ultramicrotome (Leica Microsystems). Iinxalenye zafakwa i-uranyl acetate eyi-2% kunye ne-lead citrate. Iisampulu zabonwa kusetyenziswa i-FEI Tecnai 20 transmission electron microscope (ThermoFisher (eyayisakuba yi-FEI), e-Eindhoven, eNetherlands) esebenza kwi-200 kV (Lab6 transmitter) kunye nekhamera ye-Gatan CCD (eGatan, e-UK) exhotyiswe ngesihluzi samandla seTridiem.
Kwikopi nganye yobugcisa, ubuncinci imifanekiso engama-24 yeseli enye ifunyenwe, nto leyo eyenza imifanekiso engama-266 iyonke. Yonke imifanekiso ihlalutywe kusetyenziswa i-Region of Interest (ROI) macro kunye ne-Mitochondria macro. I-mitochondrial macro isekelwe kwiindlela ezipapashwe17,31,32 kwaye ivumela ukucutshungulwa kwe-semi-automated batch yemifanekiso ye-TEM eFiji/ImageJ69. Ngamafutshane: umfanekiso uguqulwe kwaye uguqulwe kusetyenziswa ukutsalwa kwebhola eqengqelekayo (i-60 pixel radius) kunye nesihluzi se-FFT bandpass (sisebenzisa imida ephezulu nesezantsi ye-60 kunye ne-8 pixel ngokulandelanayo) kunye nokucinezelwa komgca othe nkqo ngokunyamezelana kokujonga kwe-5%. Umfanekiso ocutshungulweyo unqunyanyiswa ngokuzenzekelayo kusetyenziswa i-algorithm ephezulu ye-entropy kwaye kwenziwa i-binary mask. Imimandla yemifanekiso enxulumene ne-ROIs ezikhethwe ngesandla kwimifanekiso ye-TEM eluhlaza ikhutshiwe, ichaza i-mitochondria kwaye ikhuphe i-plasma membrane kunye neminye imimandla engafaniyo. Kwi-ROI nganye ekhutshiweyo, amasuntswana e-binary amakhulu kunee-pixels ezingama-600 ahlalutywe, kwaye indawo yamasuntswana, i-perimeter, ii-axes ezinkulu nezincinci, ububanzi be-Feret, ukujikeleza, kunye nokujikeleza kwalinganiswa kusetyenziswa imisebenzi yokulinganisa eyakhelwe ngaphakathi yeFiji/ImageJ. Emva kweMerrill, Flippo, kunye neStrack (2017), indawo yesi-2, umlinganiselo we-particle aspect (umlinganiselo omkhulu ukuya kwi-minor axis ratio), kunye ne-shape factor (FF) zibalwe kwezi datha, apho i-FF = i-perimeter 2/4pi x area. Inkcazo yefomula ye-parametric inokufumaneka kwiMerrill, Flippo, kunye neStrack (2017). Ii-macros ezikhankanyiweyo ziyafumaneka kwiGitHub (jonga iNgxelo yokuFumaneka kweDatha). Ngokomyinge, malunga namasuntswana angama-5,600 ahlalutywe ngonyango ngalunye lwe-PPA, malunga namasuntswana angama-17,000 (idatha ayiboniswanga).
Iiseli ze-SH-SH5Y zibekwe kwizitya zokukhulisa iiseli ezinamagumbi asi-8 (ThermoFisher, #155411) ukuze zivumele ukunamathela ubusuku bonke, emva koko zifakwe i-TMRE 1:1000 (ThermoFisher, #T669) kunye ne-Hoechst 33342 1:200 (Sigma-Aldrich, H6024). Ukudaya imifanekiso. Imifanekiso ifunyenwe kusetyenziswa ii-laser ze-405 nm kunye ne-561 nm kwindawo engqongileyo yemizuzu eli-10, kwaye imifanekiso eluhlaza ifunyenwe njenge-z-stacks equlethe ii-micrographs zemifanekiso ezili-10 ezine-az step ye-0.2 μm phakathi kweefreyimu zemifanekiso kwiindawo ezili-12 ezilandelayo. Imifanekiso iqokelelwe kusetyenziswa iqonga le-Carl Zeiss LSM780 ELYRA PS.1 super-resolution (Carl Zeiss, Oberkochen, Germany) kusetyenziswa ilensi ye-LCI Plan Apochromate 100x/1.4 Oil DIC M27. Imifanekiso ihlalutywe kwi-ImageJ kusetyenziswa umbhobho ochazwe ngaphambili kunye ne-plugin ye-ImageJ ukulinganisa iziganeko zokudibana kunye nokuqhekeka, inani eliphakathi lezakhiwo ze-mitochondrial, kunye nomthamo ophakathi we-mitochondrial ngeseli33. Ii-macro ze-MEL ziyafumaneka kwi-GitHub (jonga iNgxelo yokuFumaneka kweDatha).
Iiseli ze-SH-SY5Y zikhuliswe kwiiplates ezineemithombo ezintandathu kubuninzi beeseli eziyi-0.3 × 106/mL kangangeeyure ezingama-24 ngaphambi konyango. I-RNA ikhutshiwe kusetyenziswa i-Quick-RNA™ Miniprep protocol (ZR R1055, Zymo Research) kunye notshintsho oluncinci: yongeza i-300 μl ye-RNA lysis buffer kwiqula ngalinye ngaphambi kokususwa kwaye ucofe isampuli nganye njengenyathelo lokugqibela ngamanzi angena-30 μl ye-DNase/RNase elution. Zonke iisampuli zihlolwe ubungakanani kunye nomgangatho kusetyenziswa i-NanoDrop ND-1000 UV-Vis Spectrophotometer. I-protein iyonke evela kwi-cell lysates ifunyenwe kusetyenziswa i-200 μl ye-RIPA lysis buffer, kwaye uxinzelelo lweproteni lwalinganiswa kusetyenziswa i-Bradford protein assay70.
Ukwenziwa kwe-cDNA kwenziwe kusetyenziswa iTetro™ cDNA Synthesis Kit (BIO-65043, Meridian Bioscience) ngokwemiyalelo yomenzi kunye notshintsho oluthile. I-cDNA yenziwe kwiimpendulo ze-20-μl kusetyenziswa i-0.7 ukuya kwi-1 μg ye-RNA iyonke. Ii-primers zikhethwe kumaphepha apapashwe ngaphambili 42, 71, 72, 73, 74, 75, 76, 77, 78 (Itheyibhile S1) kwaye ii-probes ezihamba nazo ziyilwe kusetyenziswa isixhobo sePrimerQuest esivela kwi-Integrated DNA Technologies. Zonke ii-genes ezinomdla zahlengahlengiswa kwi-nuclear B2M gene. Ukubonakaliswa kwe-gene ye-STOML2, NRF1, NFE2L2, TFAM, cMYC kunye ne-OPA1 kwalinganiswa yi-RT-qPCR. Umxube oyintloko wawuquka i-LUNA Taq polymerase (M3003L, New England Biolabs), ii-primers ze-10 μM phambili nangasemva, i-cDNA, kunye namanzi e-PCR-grade ukuze kuvele umthamo wokugqibela we-10 μL kwimpendulo nganye. Ukubonakaliswa kwe-division and fission genes (DRP1, MFN1/2) kulinganiswe kusetyenziswa iimvavanyo ze-TaqMan multiplex. I-Luna Universal Probe qPCR Master Mix (M3004S, New England Biolabs) isetyenzisiwe ngokwemiyalelo yomenzi kunye notshintsho oluncinci. Umxube oyintloko we-multiplex RT-qPCR uquka i-1X LUNA Taq polymerase, ii-primers ze-10 μM phambili nangasemva, i-10 μM probe, i-cDNA, kunye namanzi e-PCR-grade, nto leyo ebangele umthamo wokugqibela we-20 μL kwimpendulo nganye. I-RT-qPCR yenziwe kusetyenziswa i-Rotor-Gene Q 6-plex (QIAGEN RG—inombolo ye-serial: R0618110). Iimeko zokujikeleza ziboniswe kwiTheyibhile S1. Zonke iisampulu ze-cDNA zandisiwe kwi-triplicate kwaye kwenziwa i-standard curve kusetyenziswa uthotho lwe-tenfold dilutions. Ii-Outliers kwiisampulu ze-triplicate ezine-cycle threshold standard deviation (Ct) >0.5 zisusiwe kuhlalutyo ukuqinisekisa ukuphinda-phinda kwedatha30,72. I-relative gene expression ibalwe kusetyenziswa indlela ye-2-ΔΔCt79.
Iisampulu zeproteni (60 μg) zixutywe ne-Laemmli loading buffer kwi-2:1 ratio kwaye zisebenza kwi-12% yejeli yeproteni engenambala (Bio-Rad #1610184). Iiproteni zidluliselwe kwi-PVDF (polyvinylidene fluoride) membrane (#170-84156, Bio-Rad) kusetyenziswa inkqubo yeTrans-Blot Turbo (#170-4155, Bio-Rad). I-membrane ivalwe kwaye ifakwa kwi-incubated kunye nee-antibodies eziphambili ezifanelekileyo (OPA1, MFN1, MFN2, kunye ne-DRP1) (ixutywe nge-1:1000) iiyure ezingama-48, ilandelwa yi-incubation ene-antibodies yesibini (1:10,000) iyure e-1. Ii-Membranes emva koko zathathwa umfanekiso kusetyenziswa i-Clarity Western ECL Substrate (#170-5061, Bio-Rad) kwaye zarekhodwa kusetyenziswa inkqubo ye-Bio-Rad ChemiDoc MP. Inguqulelo ye-ImageLab 6.1 yasetyenziselwa uhlalutyo lwe-Western blot. Ijeli yokuqala kunye neblot ziboniswe kuMfanekiso S1. Ulwazi lwe-antibody lubonelelwe kwiTheyibhile S2.
Iiseti zedatha ziboniswa njengempazamo ephakathi kunye nesemgangathweni ye-mean (SEM) ubuncinane yeesampulu ezintathu ezizimeleyo. Iiseti zedatha zivavanywe ukufaneleka kusetyenziswa uvavanyo lweShapiro-Wilks (ngaphandle kokuba kuchazwe ngenye indlela) ngaphambi kokuba kuthathwe usasazo lweGaussian kunye nokuphambuka okulinganayo kunye nokuqhubeka nohlalutyo. Ukongeza ekuhlalutyeni iseti yedatha kusetyenziswa iFisher's MEL LSD (p < 0.05), i-ANOVA yendlela enye (unyango vs. i-control mean), kunye novavanyo lokuthelekisa oluninzi lukaDunnett ukufumanisa ukubaluleka (p < 0.05). Amaxabiso abalulekileyo e-p aboniswe kwigrafu njenge *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Zonke ii-statistical analysis kunye neegraphs zenziwe kwaye zenziwa kusetyenziswa iGraphPad Prism 9.4.0.
Iimacro zeFiji/ImageJ zohlalutyo lwemifanekiso yeTEM ziyafumaneka esidlangalaleni kwiGitHub: https://github.com/caaja/TEMMitoMacro. I-macro yeMitochondrial Event Locator (MEL) iyafumaneka esidlangalaleni kwiGitHub: https://github.com/rensutheart/MEL-Fiji-Plugin.
UMeiliana A., uDevi NM kunye noVijaya A. Mitochondria: abalawuli abaphambili bemetabolism, i-homeostasis, uxinzelelo, ukwaluphala kunye ne-epigenetics. Isi-Indonesia. Isayensi yezebhayoloji. J. 13, 221–241 (2021).
UBen-Shachar, D. Ukungasebenzi kakuhle kwe-mitochondrial kwi-schizophrenia, i-complex I njengesixhobo esinokwenzeka se-pathological. I-Schizophrenia. umthombo. 187, 3–10 (2017).
UBose, A. kunye noBeal, MF Ukungasebenzi kakuhle kweMitochondrial kwisifo sikaParkinson. J. Neurochemistry. 139, 216–231 (2016).
USharma VK, uSingh TG kunye noMehta V. I-mitochondria exinezelekileyo: iinjongo zokuhlaselwa sisifo se-Alzheimer. I-Mitochondria 59, 48–57 (2021).
UBelenguer P., uDuarte JMN, uShook PF kunye noFerreira GK I-Mitochondria kunye nobuchopho: i-bioenergetics kunye nokunye. Ii-Neurotoxins. umthombo. 36, 219–238 (2019).
URangaraju, V. et al. I-Pleiotropic mitochondria: impembelelo ye-mitochondria kuphuhliso lwe-neuronal kunye nesifo. J. Neuroscience. 39, 8200–8208 (2019).
UCardaño-Ramos, C. kunye noMorais, VA I-Mitochondrial biogenesis kwii-neurons: njani kwaye phi. ubuzwe. J. Mohr. isayensi. 22, 13059 (2021).
Yu, R., Lendahl, U., Nister, M. kunye noZhao, J. Ulawulo lwe-mammalian mitochondrial dynamics: amathuba kunye nemingeni. phambili. i-endocrine. (Lausanne) 11, 374 (2020).
UKhacho, M. kunye noSlack, RS Iintshukumo zeMitochondrial ekulawuleni i-neurogeneis: ukusuka kwingqondo ephuhlayo ukuya kweyabantu abadala. uphuhliso. i-dynamic. 247, 47–53 (2018).