Title Utjecaj nedostatka optineurina u autofagiji induciranoj s IFN-β
Title (english) Effect of optineurin deficiency on IFN-β-induced autophagy
Author Rozalija Šajn
Mentor Ivana Munitić (mentor)
Committee member Jelena Ban (predsjednik povjerenstva)
Committee member Antonija Jurak Begonja (član povjerenstva)
Granter University of Rijeka (Faculty of Biotechnology and Drug Development) Rijeka
Defense date and country 2020-09-29, Croatia
Scientific / art field, discipline and subdiscipline BIOTECHNICAL SCIENCES Biotechnology Molecular Biotechnology
Abstract Učinci različitih neurodegenerativnih bolesti na živčane stanice međusobno se bitno razlikuju, međutim nakupljanje unutarstaničnih, toksičnih agregata krivo smotanih proteina i posljedično gubitak funkcije stanica, zajednička su obilježja svih neurodegenerativnih bolesti. Osnovni razlog prisutnosti agregata nije poznat u velikoj većini slučajeva, ali dio mutacija u genima povezanim s neurodegenerativnim bolestima veže se uz autofagiju. Autofagija je evolucijski očuvani proces kojim se putem lizosoma razgrađuje citoplazmatski sadržaj poput proteinskih agregata, patogena i oštećenih organela. Pritom adaptori autofagije prepoznaju označeni sadržaj za razgradnju i dostavljaju ga do vezikula s dvostrukom membranom koje se zovu autofagosomi. Autofagosomi se spajaju s lizosomima što dovodi do stvaranja autolizosoma gdje se sadržaj razgrađuje. U adaptore autofagije ubrajaju se i dva proteina čije su mutacije pronađene u bolesnika s neurodegenerativnom bolešću amiotrofičnom lateralnom sklerozom (ALS), optineurin (OPTN) i sekvestosom 1 (SQSTM1 ili p62). Za OPTN je poznato da osim kao adaptor autofagije, sudjeluje u još dva koraka autofagije: inicijaciji autofagije i sazrijevanju autofagosoma. Stoga smo u ovom radu htjeli ispitati utjecaj OPTN-a na autofagiju u staničnoj liniji neuroblastoma N2a, koristeći N2a stanice u kojima je OPTN uklonjen CRISPR-Cas9 metodom. Analizirali smo bazalnu autofagiju i autofagiju induciranu s interferonom (IFN)-β, citokinom za koji je nedavno pokazano da regulira autofagiju u neuronima. Koristili smo standardne testove za praćenje autofagije poput mjerenja količine markera autofagosoma LC3-II (engl. microtubule associated protein light chain 3), adaptora p62, i OPTN-a Western blot metodom, te brojanje LC3 pozitivnih vezikula imunofluorescencijom ili fluorescentno označenim LC3 konstruktom. Iz naših rezultata zaključujemo da u OPTN-deficijentnim stanicama postoji djelomični blok autofagije u bazalnim uvjetima kojeg vidimo kao povećani broj i veličinu autofagosoma te povećano nakupljanje LC3-II. IFN-β je u N2a stanicama inducirao autofagiju, ali nismo uočili nakupljanje OPTN i p62 što može značiti da nisu adaptori autofagije potaknute s IFN-β te da djeluju na autofagiju u nekom drugom koraku. Nadalje, moguće je i da su razgrađeni prije naše analize te da pokuse treba modificirati. Zbog djelomičnog bloka u autofagiji pri nedostatku OPTN-a, moguće je da iz tog razloga dolazi do nakupljanja proteinskih agregata u ALS-u. U budućim pokusima važno je stoga dokazati da opaženi poremećaj autofagije dovodi do agregacije proteina.
Abstract (english) The effects of different neurodegenerative diseases on neurons substantially differ, but the accumulation of intracellular toxic aggregates of misfolded proteins and the consequent loss of cell function are common features of all neurodegenerative diseases. The underlying reason for the presence of aggregates is not known in the vast majority of cases, but some genes mutated in neurodegenerative diseases are linked to autophagy. Autophagy is an evolutionarily conserved process by which noxious cytoplasmic contents such as protein aggregates, pathogens, and damaged organelles are broken down in lysosomes. Autophagy adaptors recognize the cargo labeled for degradation and deliver it to double-membrane vesicles called autophagosomes. Autophagosomes fuse with lysosomes leading to the formation of autolysosomes where the cargo is degraded. Autophagy adapters also include two proteins whose mutations have been found in patients with the neurodegenerative disease amyotrophic lateral sclerosis (ALS), optineurin (OPTN) and sequestosome 1 (SQSTM1 or p62). In addition to its adaptor role, OPTN has also been described to participate in two other autophagy steps: autophagy initiation and autophagosome maturation. Therefore, in this paper, we examined the effect of OPTN on autophagy in the N2a neuroblastoma cell line, using N2a cells in which OPTN was removed by the CRISPR-Cas9 approach. We analyzed basal autophagy and autophagy induced by interferon (IFN)-, a cytokine recently shown to regulate autophagy in neurons. We used standard tests to monitor autophagy such as measuring the amount of LC3-II (microtubule associated protein light chain 3), p62 adaptor, and OPTN by Western blotting, and counting LC3 positive vesicles by immunofluorescence or via a fluorescently labeled LC3 construct. We observed a partial block of basal autophagy in the OPTN-deficient cells manifested as an increased number and size of autophagosomes and increased accumulation of LC3-II. IFN-β induced autophagy in N2a cells, but we did not observe the accumulation of OPTN and p62 which may mean that they do not serve as autophagy adaptors upon IFN-β-treatment or that they act on autophagy in some other step(s). Furthermore, it is possible that they were degraded before our analysis and that our experimental approach needs to be modified. Due to the partial block in autophagy in the absence of OPTN, it is possible that this leads to an accumulation of protein aggregates in ALS. In future experiments, it is therefore important to test if this observed autophagy dysregulation in OPTN-deficient cells leads to protein aggregation.
Keywords
optineurin
autofagija
IFN-β
Keywords (english)
optineurin
autophagy
IFN-β
Language croatian
URN:NBN urn:nbn:hr:193:229829
Study programme Title: Biotechnology in medicine Study programme type: university Study level: graduate Academic / professional title: magistar/magistra biotehnologije u medicini (magistar/magistra biotehnologije u medicini)
Type of resource Text
File origin Born digital
Access conditions Embargoed access Embargo expiration date: 2030-09-29
Terms of use
Repository Repository of the University of Rijeka, Faculty of Biotechnology and Drug Development
Created on 2020-09-28 08:52:46