Uloga autofagije u neurotoksičnom delovanju vanćelijskog alfa sinukleina na SH-SY5Y ćelije humanog neuroblastoma

Abstract

Uvod. U osnovi patogenetskog mehanizma Parkinsonove bolesti leži povećano nakupljanje proteina α-sinukleina (ASYN) u dopaminergičkim neuronima substantiae nigrae, što, između ostalog, dovodi i do smrti ćelija. Iako je ranije smatran isključivo unutarćelijskim proteinom, novijim istraživanjima je pokazano da se ASYN nakuplja i u vanćelijskom prostoru. Do nakupljanja ASYN može da dođe usled oštećenja sistema za razgradnju proteina, od kojih je najznačajniji mehanizam autofagija. Autofagija je regulisana proizvodima ATG gena (engl. autophagy-related genes). Jedan od njih, ATG7, ima bitnu ulogu u formiranju i sazrevanju autofagozoma. Cilj. Cilj ovog rada je bio da se ispita uloga autofagije u neurotoksičnosti vanćelijskog ASYN na SH-SY5Y ćelije humanog neuroblastoma, diferentovanih u neuronski fenotip. Materijali i metode. Svi eksperimenti su rađeni na SH-SY5Y ćelijskoj liniji humanog neuroblastoma. Ćelije su diferentovane 'all-trans' retinoičnom kiselinom i tretirane medijumom koji sadrži vanćelijski ASYN. Imunoblot metodom je potvrđeno prisustvo vanćelijskog ASYN i praćena promena eskpresije markera autofagije, proteina LC3-II i beklin-1. Metodom transfekcije sa malom interferirajućom RNK inhibirana je ekspresija ATG7 proteina. Vijabilitet i broj ćelija određeni su kristal violet testom. Rezultati. Vanćelijski ASYN dovodi do značajnog smanjenja vijabiliteta SH-SY5Y ćelija, što je praćeno povećanjem nivoa markera autofagije, proteina LC3-II i beklin-1. Analizom ćelijskog vijabiliteta primećen je značajan pad u broju živih ćelija kod kojih je utišan gen za ATG7, a koje su gajene u prisustvu vanćelijskog ASYN. Zaključak. Vanćelijski ASYN dovodi do smanjenog preživljavanja SH-SY5Y ćelija diferentovanih u neuronski fenotip, što je praćeno indukcijom autofagije. Inhibicija autofagije preko utišavanja ATG7 gena dovela je do povećane osetljivosti ćelija gajenih u prisustvu vanćelijskog ASYN, što ukazuje na moguću protektivnu ulogu autofagije u neurotoksičnosti, izazvanoj nagomilavanjem ASYN u vanćelijskom prostoru.

Introduction: The accumulation of alpha-synuclein (ASYN) in susceptible neurons is considered to be a major contributing factor in pathogenesis of Parkinson’s disease. Although ASYN was considered as an intracellular protein, recent data suggest that it can be detected extracellularly. Autophagy plays an important role in ASYN degradation; therefore, impairment of autophagy could be an important contributor to ASYN accumulation. ATG (autophagy-related genes) proteins function at several physiologically continuous steps in autophagy, and ATG7 is considered as essential in autophagosome formation and maturation. Aim: The aim of this study was to investigate the role of autophagy in neurotoxicity, caused by extracellular ASYN. Material and methods: All experiments were conducted in all-trans retinoic acid-differentiated human neuroblastoma SH-SY5Y cells, that were exposed to extracellular ASYN. The presence of extracellular ASYN and the expression of autophagy markers, beclin-1 and LC3-II, were monitored using immunoblotting. Transfection, with small interfering RNA (siRNA), was used to knock down ATG7 gene. Cell viability was assessed using crystal violet dye exclusion assay. Results: Extracellular ASYN caused significant loss of viability in differentiated SH-SY5Y cells, accompanied by the increase in expression of beclin-1 and in conversion of LC3-I to autophagosome-associated LC3-II. The RNA interference-mediated knock-down of ATG7 increased the sensitivity of SH-SY5Y cells to the extracellular ASYN-induced toxicity. Conclusion: Extracellular ASYN caused loss of viability in differentiated SH-SY5Y cells accompanied by autophagy induction. Having in mind that inhibition of autophagy, through ATG7 knock-down increased cell death, we can conclude that autophagy could have a protective role in the harmful effect of extracellular ASYN.