Title In vitro characterization of TNF-mediated programmed death in cells with optineurin deficiency Title (croatian) In vitro karakterizacija TNF-om posredovane programirane stanične smrti u stanicama s deficijencijom optineurina Author Karla Dubaić Mentor Ivana Munitić (mentor) Committee member Rozi Andretić Waldowski (predsjednik povjerenstva) Committee member Christian Andrew Reynolds (član povjerenstva) Granter University of Rijeka Defense date and country 2020-09-16, Croatia Scientific / art field, BIOTECHNICAL SCIENCES Abstract In recent years neuroinflammation was found to be a major contributor to disease progression in neurodegenerative disorders such as amyotrophic laterals sclerosis (ALS). Inflammatory cytokines like tumor necrosis factor (TNF) can push cells into programmed cell death, such as apoptosis or necroptosis, which are mediated by receptor-interacting protein kinase 1 (RIPK1). It is not entirely clear what pushes cells to different forms of cell death. A recent study proposed that RIPK1 turnover is mediated by an adaptor protein optineurin, which is included in various cellular signaling and trafficking processes. Loss of optineurin was reported to be associated with enhanced motor neuron death and axonal degeneration, both of which are part of ALS pathology. However, this was not consistently reported in other studies, so the exact mechanism of action of optineurin in neuroprotection is still debated. Here, we focused on characterizing TNF-mediated programmed cell death in various optineurin-deficient cell lines. To address the role of optineurin in apoptosis and necroptosis, we first generated a novel mouse fibroblast (L929) optineurin knockout (Optn KO) cell line using CRISPR/Cas9 technique. L929 cells have been reported to be permissive to necroptotic cell death, so they were used to set up the assays. We induced necroptosis and apoptosis in wild-type (WT) and Optn KO L929 cells to determine the effect Optn KO on cell survival. Furthermore, we tested if cell types of the central nervous system (CNS), such as microglial (BV2) cells and neuronal (Neuro2A) cells, are dying by necroptosis. Our study confirmed that L929 cells are highly susceptible to necroptotic death. However, the survival rate of WT and Optn KO L929 cells was similar, which differs from previously published results. In BV2 and Neuro2A cells we detected a significant decrease in cell survival upon stimulating apoptosis and necroptosis, which was not rescued by caspase or RIPK1 inhibition. Notably, we did not detect any difference between WT and Optn KO BV2 cells, suggesting that optineurin deficiency does not sensitize microglia to TNF-mediated apoptosis and/or necroptosis. In addition, our data suggest that another TNF-mediated mechanism may push cells to death, which is independent of caspases or RIPK1.
Abstract (croatian) Tijekom posljednjih godina je utvrđeno da neuroinflamacija snažno utječe na progresiju neurodegenerativnih bolesti poput amiotrofične lateralne skleroze (ALS). Upalni citokini poput faktora tumorske nekroze (TNF) mogu usmjeriti stanice u različite oblike stanične smrti uključujući apoptozu i nekroptozu, koje su posredovane protein-kinazom RIPK1 (prema engl. receptor-interacting protein kinase 1). Nije još u potpunosti razjašnjeno što tjera stanice u različite oblike smrti. Nedavna istraživanja predlažu da adaptorski protein optineurin, koji je uključen u niz signalnih i transportnih procesa u stanicama, regulira obrtaj RIPK1. Nedavno je objavljeno da je gubitak optineurina povezan s pojačanom smrti motorničkih neurona i aksonalnom degeneracijom, procesima koji su dio patologije ALS-a. Međutim, ti rezultati su opovrgnuti u nekoliko drugih studija i točan mehanizam optineurina u neuroprotekciji je još nejasan. U ovom radu, naglasak je stavljen na karakterizaciju TNF-om posredovane stanične smrti u različitim staničnim linijama u kojima je deletiran optineurin. Kako bismo analizirali ulogu optineurina u apoptozi i nekroptozi, prvo smo stvorili novu mišju fibroblastnu (L929) optineurin deficijentnu (Optn KO, prema engl. knockout) staničnu liniju pomoću CRISPR/Cas9 tehnologije. L929 stanice su sklone nekroptozi te su iz toga razloga korištene kao pozitivna kontrola. Potaknuli smo nekroptozu i apoptozu u stanicama divljeg tipa (WT) i Optn KO stanicama te promatrali preživljenje stanica. Osim toga, zanimalo nas je da li stanice bitne za središnji živčani sustav umiru nekroptozom, poput mikroglijalne (BV2) i WT neuronalne (Neuro2A) stanične linije. Potvrdili smo da su L929 stanice sklone nekroptotskoj smrti. Međutim, postotak preživljenja WT i Optn KO stanica bio je sličan, suprotno prije objavljenim rezultatima. U BV2 i Neuro2A stanicama smo primijetili značajno smanjeno preživljenje stanica nakon stimulacije apoptoze i nekroptoze, koje se nije poboljšalo nakon inhibicije kaspaza ili RIPK1. Značajno je da nismo otkrili razliku između WT i Optn KO BV2 stanica, što upućuje da deficijencija optineurina ne senzibilizira mikrogliju prema TNF-om posredovanoj apoptozi i/ili nekroptozi. Štoviše, naši rezultati otvaraju mogućnost da postoji još jedan TNF-om posredovan mehanizam koji tjera stanice u smrt, neovisan o kaspazama ili RIPK1.
Keywords
neuroinflammation
programmed cell death
optineurin
neurodegeneration
Keywords (croatian)
neuroinflamacija
programirana stanična smrt
optineurin
neurodegeneracija
Language english URN:NBN urn:nbn:hr:193:761888 Study programme Title: Biotechnology in medicine Type of resource Text File origin Born digital Access conditions Open access Terms of use Created on 2020-09-15 13:07:35
