Title Impact of optineurin on the basal and IFN-β induced autophagy of Neuro2a cell line Title (croatian) Utjecaj optineurina na bazalnu i IFN-β induciranu autofagiju u Neuro2a staničnim linijama Author Klara Omrčen Mentor Ivana Munitić (mentor) Committee member Anđelka Radojčić-Badovinac (predsjednik povjerenstva) Committee member Nicholas James Bradshaw (član povjerenstva) Committee member Ivana Munitić (član povjerenstva) Granter University of Rijeka Defense date and country 2024-09-24, Croatia Scientific / art field, BIOTECHNICAL SCIENCES Abstract Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by progressive denervation of voluntary muscles. So far, more than 30 genes have been associated with ALS, which participate in many molecular pathways, including autophagy, neuroinflammation and DNA repair. Autophagy is a dynamic process that eliminates damaged organelles, misfolded proteins and pathogens, and ensures nutrients for cell survival. Since autophagy disposes of protein aggregates, its dysfunction is linked to neurodegenerative diseases. Optineurin is an ALS-linked protein involved in cell signalling and autophagy. For this reason, we hypothesized that optineurin deficiency could disturb autophagy flux. To investigate this, we monitored the levels of several autophagy-associated proteins in wild-type (WT) and optineurin-deficient (OPTN-KO) Neuro2a cell lines under basal conditions and upon IFN-β treatment. We observed that the lack of optineurin resulted in increased inhibitory ULK1 phosphorylation and diminished degradation of phosphorylated TBK1 (pTBK1) via autophagy. However, the lack of optineurin did not impact p62, LC3-II and GABARAP levels in the basal state, suggesting that it does not disturb autophagy flux. IFN-β treatment increased TBK1 phosphorylation, but overall, it did not induce autophagy in Neuro2a cells. Next, we inhibited TBK1 activation by BX795 to determine if optineurin phosphorylation is primarily mediated by pTBK1 in neuronal cells and/or if pTBK1 inhibition will have a greater effect on autophagy than optineurin deficiency. We observed diminished phosphorylation of optineurin, demonstrating that optineurin is a TBK1 target in Neuro2a cells. We also observed decreased ULK1 phosphorylation, which has previously not been described. The latter could be caused by either the BX795 off-target effect or the currently unknown TBK1 effect on ULK1 modification. In conclusion, neither optineurin deficiency nor IFN-β affected autophagy flux in Neuro2a cells. However, this study highlights the possibility that optineurin mayregulate ULK1 phosphorylation and pTBK1 turnover under basal conditions, but further research is needed to confirm this.
Abstract (croatian) Amiotrofična lateralna skleroza (ALS) je neurodegenerativna bolest karakterizirana progresivnom denervacijom voljnih mišića. Do sada je više od 30 gena povezano s ALS-om, koji sudjeluju u mnogim molekularnim putevima, uključujući autofagiju, neuroinflamaciju i popravak DNA. Autofagija je dinamičan proces koji eliminira oštećene organele, nepravilno savijene proteine i patogene, te osigurava hranjive tvari za preživljavanje stanica. Budući da autofagija uklanja proteinske agregate, njezina disfunkcija je povezana s neurodegenerativnim bolestima. Optineurin je protein povezan s ALS-om koji sudjeluje u signalnim putevima i autofagiji. Iz tog razloga, pretpostavljamo da bi nedostatak optineurina mogao poremetiti protok autofagije. Kako bismo to istražili, promatrali smo razine nekoliko proteina povezanih s autofagijom u divljem tipu (engl. wild-type (WT)) i optineurindeficijentnim (OPTN-KO) Neuro2a staničnim linijama pri bazalnim uvjetima i nakon IFN-β tretmana. Uočili smo da nedostatak optineurina dovodi do povećane inhibitorne fosforilacije ULK1 i smanjene razgradnje fosforiliranog TBK1 (pTBK1) putem autofagije. Međutim, nedostatak optineurina nije utjecao na razine p62, LC3-II i GABARAP u bazalnom stanju, sugerirajući da ne remeti tok autofagije. IFN-β tretman je povećao fosforilaciju TBK1, ali općenito nije potaknuo autofagiju u Neuro2a stanicama. Zatim smo inhibirali aktivaciju TBK1 pomoću BX795 kako bismo utvrdili je li fosforilacija optineurina primarno posredovana pTBK1 u neuronskim stanicama te hoće li inhibicija pTBK1 imati veći utjecaj na autofagiju u odnosu na nedostatak optineurina. Uočili smo smanjenu fosforilaciju optineurina, pokazujući da je optineurin meta TBK1 u Neuro2a stanicama. Primijetili smo i sniženu fosforilaciju ULK1, što dosada nije bilo opisano. Potonje bi moglo biti uzrokovano nespecifičnim učinkom BX795 ili dosad nepoznatim utjecajem TBK1 na modifikaciju ULK1. Zaključno, ni nedostatak optineurina ni IFN-β nisu utjecali na tok autofagije. Međutim, ovaj rad ističe mogućnost da optineurin može regulirati fosforilaciju ULK1 i promet pTBK1 pri bazalnim uvjetima, ali potrebna su daljnja istraživanja kako bismo to mogli potvrditi.
Keywords
amyotrophic lateral sclerosis
optineurin
autophagy
IFN-β
Keywords (croatian)
amiotrofična lateralna skleroza
optineurin
autofagija
IFN-β
Language english URN:NBN urn:nbn:hr:193:449275 Study programme Title: Medicinal chemistry Type of resource Text File origin Born digital Access conditions Embargoed access Terms of use Created on 2024-09-23 21:16:52
