Ubururu bwa metilene na autofaji: AMPK, mTOR, n'amagerageza ku turemangingo tw'imyakura

Tekereza ko ukora igerageza ku turemangingo, ukongeramo ubururu bwa metilene, maze ukabona ko umurongo wa poroteyine yitwa LC3-II urushaho kwijima ku gupima kwa blot. Biroroshye guhita uvuga ko icyo kinyabutabire "cyongera autofaji" maze ukikomereza. Uwo mwanzuro uba ufashwe hakiri kare. Umurongo wa LC3-II wijimye kurushaho ushobora kuvuga ko akaremangingo gakora udusaho twinshi two gukuraho imyanda, cyangwa ko utwo dusaho twirundanya kubera ko intambwe yo kuyisenya yahagaze. Ibyo bisobanuro byombi byerekeza ku myanzuro inyuranye, kandi inyandiko z'ubushakashatsi ku bururu bwa metilene mu turemangingo tw'imyakura zirimo ingero z'ibimenyetso bya buri bwoko. Iyi ngingo isobanura ibyapimwe mu by'ukuri, igipimo ku kindi.

Macroautofaji, ari bwo bwoko bwizwe hano, ni uburyo akaremangingo kakoresha mu gusenya no kongera gukoresha ibice byinshi icyarimwe. Agasaho kagizwe n'agahu kitwa fagofore gakura gakikije poroteyine n'uduce dukora imirimo yihariye mu karemangingo byashaje, kagafunga kakaba autofagozome, kagahuza na lizozome, maze ibikarimo bigasenywa bikarekurwa kugira ngo byongere gukoreshwa. Uturemangingo tw'imyakura twishingikiriza kuri ubu buryo kuko tutigabanya, bityo tukaba tudashobora kugabanya ubwinshi bw'ibice byangiritse binyuze mu kwigabanya. Iyo ubwo buryo bwo gusenya no kongera gukoresha ibice buhagaze, imyanda irirundanya. Iyo bukora, akaremangingo kari mu bihe bigoye kongera kubona ibikoresho by'ibanze byo kwiyubaka.

Ibyagaragajwe n'igerageza rimwe ku turemangingo twa hipokampe

Ubushakashatsi bw'ingenzi ku gutangiza autofaji bwakoresheje uturemangingo twa HT22 two muri hipokampe y'imbeba, twashyizwe mu bihe bigoye twimwa serumu mu gihe cy'amasaha 24. Ibyo bikuraho ibitera gukura n'ubutumwa bufasha uturemangingo kubaho, bigatangiza apopitozi, ari rwo rupfu rw'akaremangingo ruteguwe, na autofaji. Abashakashatsi batangaje ko ubururu bwa metilene bwagabanyije impfu z'uturemangingo hakurikijwe ingano yabwo yakoreshejwe, kandi ko ubwo burinzi bwajyanaga n'ibimenyetso bya macroautofaji.

Ibimenyetso byabonetse byari bisobanutse. Irangi rya LC3B ryari ritatanye ryarongeye kwikusanyiriza mu tudomo dukikije intima y'akaremangingo, guhera hafi ya 20 nM z'ubururu bwa metilene; impinduka zikomeye kurusha izindi muri LC3-I na LC3-II zabonetse hafi ya 1 μM. LC3B-I na LC3B-II byombi byariyongereye hakurikijwe ingano yakoreshejwe n'igihe cyari gishize. Icy'ingenzi ni uko itsinda ritagarukiye ku gupima LC3 hifashishijwe blot yerekana uko ibintu byari bimeze mu kanya kamwe. Bongeyemo 10 μM za chloroquine, ibuza lizozome gukuraho ibirimo, maze basanga LC3-II yiyongereye na p62 iguma ku rugero rwayo mu turemangingo twari twahawe ubururu bwa metilene. Ibyo bishyigikira ko gusenya no gusimbuza ibice byari bishingiye kuri lizozome, aho kuba ukwirundanya gusa. Banagaragaje ko chloroquine yagabanyije igice cy'ingaruka zo kubuza apopitozi, bikaba bihuza ubwo burinzi n'inzira ya autofaji na lizozome muri ubu buryo bw'igerageza.

Ibyabonetse ku butumwa bwo mu karemangingo ni byo bikunze gusubirwamo nabi kurusha ibindi. Ubutumwa bwa AMPK, bwapimwe harebwa AMPK ifite itsinda rya fosifate n'ikintu ikoreraho mu ntambwe zikurikiraho, ACC na yo ifite itsinda rya fosifate, bwariyongereye ku masaha 12 na 24 hakurikijwe ingano yakoreshejwe. Compound C ibuza AMPK gukora, yashyizwe ku rugero rwa 20 μM mu masaha 4 ya nyuma, yagabanyije gushyirwa kwa fosifate kuri ACC n'ikangurwa rya LC3. Nyamara, nta gabanyuka ryashoboye kugaragara muri mTOR ifite itsinda rya fosifate. Bityo, mu turemangingo twa hipokampe twari mu bihe bigoye, icyabonetse ni itangizwa rifitanye isano na AMPK, binyuze mu nzira isa n'itishingikiriza kuri mTOR. Ibisa n'ibyo byanagaragaye mu mbeba nzima, aho gukoresha ubururu bwa metilene byatumye ibimenyetso bya macroautofaji byiyongera mu gice cy'inyuma cy'ubwonko no muri hipokampe.

Gutangiza ntibivuze ko uruhererekane rwose rwarangiye

Aha ni ho gutandukanya izi nyigisho bifite akamaro kanini. Gutangiza bivuze ko uburyo bukora mu ntambwe za mbere butuma hakorwa autofagozome nyinshi. Urujya n'uruza rwuzuye, cyangwa flux, bivuze ko ibigiye gusenywa binyura mu nzira yose: gufatwa no gushyirwa ukwabyo, gukura, guhuza na lizozome, gusenywa, no kongera gukoreshwa. Buri kwiyongera kwa LC3-II kuguma ari ikimenyetso cyo gutangiza kugeza igihe ikizamini cy'uruhererekane rwose cyemeje ko n'izindi ntambwe zakozwe.

Ikizamini gisanzwe cy'uruhererekane rwose gipima LC3-II igihe ibikorwa bya lizozome byabujijwe by'igihe gito, urugero hakoreshejwe bafilomycin A1 cyangwa chloroquine, n'igihe bitabujijwe. Niba ibyakoreshejwe byihutisha koko ikorwa rya autofagozome, kubuza isenywa ryazo byagombye gutuma LC3-II yiyongera kurushaho. Ikimenyetso cya mCherry-GFP-LC3 gihuza ibice bibiri gitanga ubundi buryo bwo kubireba: utwo dusaho turabagirana mu ibara ry'umuhondo mbere yo guhuza na lizozome, tukarabagirana mu mutuku gusa nyuma y'uko aside ihagaritse ikimenyetso cya GFP. Bityo, utudomo twinshi tw'umutuku gusa dushyigikira ko ibyo bikoresho byakomeje bigera muri autolizozome zirimo aside. Kugabanuka kwa poroteyine ya p62 gushobora gushimangira ko ibigiye gusenywa bikurwaho bigasimbuzwa, ariko hakazirikanwa ko kwandukura amakuru ya p62 na byo bihinduka bitewe n'ibihe bigoye, bityo bikaba bigomba kugenzurwa ku rwego rwa mRNA.

Urutonde rukurikira ruhuza buri gipimo cyakoreshejwe muri ubu bushakashatsi n'ibyo gipima, ndetse n'ibyo kidashobora kwemeza cyonyine. Abasomyi bashobora kongera kurukoresha ku kirego icyo ari cyo cyose kizavugwa kuri macroautofaji, harimo ibisobanuro ku kwiyiriza ubusa no kuramba byifashisha ibi bimenyetso.

Zirikana umutego wo gukoresha amagambo iyi mbonerahamwe ifasha kwirinda. ACC ifite itsinda rya fosifate ni uburyo bwayo bufite ibikorwa by'enzime byabujijwe. Kwiyongera kwayo kugaragaza ibiva mu bikorwa bya AMPK, si ikangurwa rya ACC.

Imvugo ngo "kubuza macroautofaji gukorwa byakuyeho uburinzi bw'uturemangingo tw'imyakura" na yo ikwiye gusobanurwa neza. Mu buryo bw'igerageza bwa HT22, bivuze ko kubangamira inzira ya autofaji na lizozome byakuyeho inyungu ubururu bwa metilene bwatangaga mu gukomeza kubaho igihe uturemangingo twari twimwe serumu. Ishyigikira ko autofaji yari ikenewe cyangwa yagiraga uruhare muri ako gakoresho k'igerageza. Ntiyemeza ko autofaji ari bwo buryo bwonyine icyo kinyabutabire kirindamo uturemangingo, ntigaragaza ko chloroquine ikora gusa binyuze muri autofaji, kandi kugira ngo ikirego cy'isano y'impamvu n'ingaruka gikomere, byakenera guhindura imikorere ya jeni za ATG5, ATG7 cyangwa ULK1.

Inzira ebyiri zigana ku buryo bumwe bwo gukora

Igishushanyo cyoroheje kirafasha. Kubura ingufu byongera ibikorwa bya AMPK, AMPK igakangura uruhurirane rwa ULK1, maze autofaji igatangira. Ibitera gukura n'intungamubiri byongera ibikorwa bya mTORC1, mTORC1 ikabuza ULK1 gukora, maze autofaji ntitangire. Mu ntambwe zikurikira ULK1, uruhurirane rwa Beclin-1 na VPS34 rutangiza ikorwa rya fagofore, LC3-I ikongerwaho lipide ikaba LC3-II, autofagozome igafunga, igahuza na lizozome, maze ibikarimo bigasenywa, p62 na yo igasenywa igasimbuzwa.

Igishushanyo gishyira buri cyabonetse ku bururu bwa metilene kuri iyi nzira: ibyabonetse muri hipokampe biri iruhande rw'ishami rya AMPK, naho ibyabonetse mu turemangingo tubyara utundi biri iruhande rw'ishami rya mTOR.

Ubu buryo bwo kubisobanura bukemura igisa n'ukutumvikana n'ubushakashatsi bwa kabiri bwa Xie. Mu turemangingo tubyara uturemangingo tw'imyakura tw'imbeba nini twari turi kwiyongera, irindi gerageza ryasanze 5 μM z'ubururu bwa metilene zarabujije kwiyongera kandi zigatuma tujya mu mimerere yo kudakomeza kwigabanya. Uturemangingo twagaragazaga Ki67 twaragabanutse ku munsi wa 1 n'uwa 3, mu gihe inzira yo guhinduka uturemangingo tw'imyakura yari yaratangiye itahungabanye. Aho, uburyo bw'imikorere bwanyuze mu kugabanya ubutumwa bwa mTOR: mTOR yose hamwe na mTOR ifite itsinda rya fosifate byaragabanutse, hamwe no kugabanuka kw'ikorwa rya p70S6K na 4EBP1, ndetse n'igabanywa ry'ikorwa rya sikiline E1, B1, D1 na D2. Imiterere y'ingenzi yagaragaye yari iyo kuba uturemangingo tubyara utundi tutakomeje kwigabanya; abanditsi bayihuje no gutinda gusaza, aho kuba ugukiza uturemangingo twa hipokampe twari mu bihe bigoye.

Izi nyandiko zombi ntizivuguruzanya. AMPK na mTOR bihuza ubutumwa hakurikijwe imiterere y'ibiri kuba, ntabwo ari utubuto two gufungura no gufunga tudahinduka. Akaremangingo gasa n'akatw'imyakura kari mu bihe bigoye gashobora gukoresha AMPK nta kubuzwa kwa mTOR gupimika kugaragaye, mu gihe akaremangingo kabyara utundi gatewe kwigabanya n'ibinyabutabire gashobora gusubiza icyo kinyabutabire kimwe kagabanya uburyo bwo gukura bushingiye kuri mTOR. Ubwoko bw'akaremangingo, imiterere y'ibihe bigoye, ubwinshi bw'ikinyabutabire n'igihe cyo gupima ni byo bigena ishami rirushaho gukora. Ni na yo mpamvu ingaruka z'ubururu bwa metilene ku ngufu za mitokondiriya no ku kwamburwa no kwakira electron zigomba gushyirwa muri iyi shusho imwe: nk'uko inyandiko isesengura uburyo bwo kurinda uturemangingo tw'imyakura ibisobanura, icyo kinyabutabire gikora nk'undi mwikorezi wa electron, kigakoresha ubutumwa bwo kurwanya okisidasiyo no kubuza apopitozi, kikabuza MAO na NOS gukora, kandi kigahuza no kuvugurura mitokondiriya binyuze mu gukora izindi nshya no muri autofaji. Autofaji ni ishami rimwe ry'ingaruka nyinshi icyo kinyabutabire kigira, si yo nkuru yose.

Ibyo amagerageza atagaragaza

Kwima uturemangingo serumu bigana ibihe bigoye byo kubura ibidufasha kubaho n'ibyo gukoresha mu mikorere y'ibinyabutabire mu gakoresho ka laboratwari. Ntibigana kwiyiriza ubusa kw'ikinyabuzima cyose: gilikoze, aside amine n'imyunyu biguma mu ruvange rw'ibanze uturemangingo turererwamo, kandi nta myitwarire yo kurya ibaho. Ubwinshi bw'ikinyabutabire bukoreshwa mu korora uturemangingo, kuva kuri 20 nM kugeza kuri 5 μM, ntibushobora guhindurwamo ingano zihabwa abantu hakoreshejwe imibare gusa, kuko kwinjira mu mubiri, gufata kuri poroteyine, imimerere yo kwamburwa no kwakira electron, gutunganywa n'umubiri, kwirundanya mu nyama z'umubiri n'igihe cyo guhura n'ikinyabutabire byose bitandukanye.

Nta gerageza ku bantu ryateguwe rigamije kugaragaza uruhererekane rwose rwa autofaji rwatangijwe n'ubururu bwa metilene mu barwayi. Ibimenyetso ku buryo bw'imikorere biracyaturuka ku bushakashatsi ku turemangingo, ku bice byakuwe mu binyabuzima no ku nyamaswa, kandi inyandiko zisesengura ubushakashatsi ziracyavuga ko ibimenyetso byo kubikoresha ku bantu bikiri iby'ibanze. Bityo, ubu bushakashatsi ntibugaragaza ko ubururu bwa metilene busubiramo ibiba mu kwiyiriza ubusa, bwongera igihe abantu bamara babaho binyuze muri autofaji, cyangwa butuma uruhererekane rwose rwa autofaji rurangira mu bwonko bw'umuntu. Ibivugwa ku kwigana kwiyiriza ubusa no kuramba bikeneye ibimenyetso byabyo byo mu magerageza ku bantu, kandi amagerageza y'ubururu bwa metilene ku bantu agamije ibindi ntabwo ahinduka amagerageza ya autofaji kubera gusa ko bifitanye isano. Ubushakashatsi ku buryo bwo kwigana ibihe bigoye byo kubura intungamubiri no kuramba bukwiye gusomwa hitabwa kuri iyo mbibi.

Ku nyandiko y'ubushakashatsi itaha uzasoma, kurikiza uru rutonde uko rukurikirana. Banza ubaze ishami ryahindutse: AMPK, mTOR, cyangwa nta na rimwe. Hanyuma ubaze niba ibyabonetse kuri LC3 byajyanaga n'ikizamini cy'uruhererekane rwose cyangwa niba byari byonyine. Nyuma yaho, genzura niba kubuza iyo nzira gukorwa byarakuyeho inyungu yari yabonetse, n'uburyo byakoreshejwemo. Ubushakashatsi busubiza ibyo bibazo bitatu byose buba bufite ishingiro rikomeye ryo kuvuga ku buryo bw'imikorere. Ubusubiza icya mbere gusa buba bwaragaragaje ikimenyetso cyemerera inzira gutangira, naho izindi ntambwe zigakomeza kuba zitarasobanuwe.