Autophagy analysis in Neuro 2a cell line with CRISPR/Cas9 mediated optineurin deletion

Autofagija je eukariotski stanični proces kojim se uklanja nepotreban i toksičan unutarstanični sadržaj. Optineurin je ubikvitin vezujući adaptorski protein koji u procesu autofagije veže stanični sadržaj te omogućuje njegovu lizosomalnu razgradnju. Nekoliko istraživanja pokazalo je da nedostatak optineurina u stanicama uzrokuje poremećaj autofagije te time omogućuje nakupljanje proteinskih agregata. Budući da su proteinski agregati obilježje neurodegenerativnih bolesti poput amiotrofične lateralne skleroze (ALS), predloženo je da mutacije optineurina u bolesnika s ALS-om doprinose razvoju bolesti zbog poremećaja autofagije. Međutim, točan mehanizam patogeneze ALS-a kod mutacija optineurina nije poznat jer je potonji multifunkcionalni adaptor u više koraka autofagije, ali i drugim procesima poput regulacije prijenosa upalnih signala i prometa staničnih vezikula. Za ispitivanje posljedice nedostatka optineurina na tijek bazalne autofagije i autofagije inducirane različitim stimulansima koristili smo staničnu liniju Neuro 2a, u kojoj je pomoću CRISPR/Cas9 metode napravljena delecija gena za optineurin. Pokazali smo kako optineurin nije nužan za induciranje autofagije gladovanjem ili tretmanom s interferonom-β (IFN-β), ali da u stanicama s delecijom optineurina dolazi do blagog nakupljanja markera autofagije p62 i LC3-II te autofagosoma što sugerira da njegov nedostatak može uzrokovati djelomični blok tijeka autofagije. Također, pokazali smo kako nedostatak optineurina uzrokuje nakupljanje LC3-II u nestimuliranim stanicama, što sugerira na djelomični blok tijeka bazalne autofagije. Pritom smo opazili da u stanicama divljeg soja u bazalnoj autofagiji ne dolazi do razgradnje optineurina, što potencijalno sugerira da optineurin nije selektivni adaptor autofagije te da u autofagiji sudjeluje putem drugih mehanizama. Naši rezultati sugeriraju da optineurin ima ulogu u pokretanju i nesmetanom odvijanju bazalne i inducirane autofagiji te da bi njegov nedostatak mogao biti jedan od uzroka poremećene autofagije i nakupljanja agregiranih proteina u neurodegenerativnim bolestima.Autophagy is a eukaryotic cellular process that removes unnecessary and toxic cellular content. Optineurin is a ubiquitin-binding protein that binds cellular content in the autophagy process and allows its lysosomal degradation. Lack of optineurin causes autophagy related disorders and possible protein aggregation. Since protein aggregates are a hallmark of neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS), it is suggested that optineurin mutations in ALS patients lead to disease due to loss of autophagy. However, the exact mechanism of ALS pathogenesis in patients with optineurin mutations is not known. Optineurin is a multifunctional autophagy adaptor, but also has a role in other processes such as inflammation and vesicle trafficking. To investigate the role of optineurin in basal and induced autophagy, we used Neuro 2a cell line in which optineurin gene was deleted by CRISPR/Cas9 method. We have shown that optineurin is not necessary for inducing autophagy by starvation or IFN-β stimulation, but there was a mild accumulation of autophagy markers p62, LC3-II and autophagosomes in optineurin knockout cells, suggesting that optineurin deficiency can cause partial block of autophagy flow. However, we have shown that lack of optineurin caused the accumulation of LC3-II in non-stimulated cells, suggesting a partial block of basal autophagy flow. In addition, we noticed that there was no optineurin depletion in the wild type cells in basal autophagy, which potentially suggests that optineurin is not a selective autophagy adaptor and that it participates in autophagy through other mechanisms. Our results suggest that optineurin has a role in inducing and proper functioning of basal and induced autophagy and that its deficiency could be one of the causes of disrupted autophagy and the protein aggregation in neurodegenerative diseases