Title Karakterizacija uloge optineurina u regulaciji upalnih signala u mikroglijalnim i neuronalnim staničnim linijama
Title (english) Characterizing the role of optineurin in inflammatory signaling in microglial and neuronal cell lines
Author Matea Rob
Mentor Ivana Munitić (mentor)
Committee member Nicholas Bradshaw (predsjednik povjerenstva)
Committee member Jelena Ban (član povjerenstva)
Committee member Ivana Munitić (član povjerenstva)
Granter University of Rijeka (Faculty of Biotechnology and Drug Development) Rijeka
Defense date and country 2018-09-26, Croatia
Scientific / art field, discipline and subdiscipline BIOTECHNICAL SCIENCES Biotechnology
Abstract Mutations in the optineurin gene have recently been linked to amyotrophic lateral sclerosis (ALS), a progressive motor neuron degenerative disease. Optineurin has been implicated in the regulation of many cellular processes such as inflammation, autophagy, Golgi maintenance, and vesicular trafficking. However, the exact physiological function of optineurin remains unknown. Since motor neuron degeneration in ALS has been shown to be driven by microglia, the resident immune cells of the brain, we focused here on the role of optineurin in the regulation of inflammatory signaling. Optineurin deficiency has been proposed to drive neuronal degeneration by overactivation of pro-inflammatory NF-κB signaling and by the insufficient activation of protective TBK1 signaling. However, discrepancies in findings exist between cell lines and animal models and the reason behind it is unclear. In order to contribute to understanding the role of optineurin in these pathways, we established novel microglial (BV2) and neuronal (Neuro2A) optineurin knockout (Optn KO) cell lines by employing the CRISPR/Cas9 technique. We tested the early markers of NF-κB activation, IκB degradation and p65 phosphorylation upon LPS or TNF-α treatment and found no difference in Optn KO compared to WT in neither BV2 or Neuro2A cell lines. We also tested for TBK1 activation in BV2 after LPS treatment and found that TBK1 and IRF3 phosphorylation was substantially impaired in the absence of optineurin. Furthermore, we set up a flow-cytometry based phagocytic assay to test the phagocytic properties of Optn KO BV2 cell line. We found phagocytosis was mildly impaired in optineurin deficient conditions. TNF-α secretion was found to be severely impaired upon LPS treatment in optineurin KO BV2 as well. Our results suggest that optineurin deficiency potentially might drive the progression of ALS in more ways than previously described. In conclusion, in this study, we confirmed previous findings of the role of optineurin in TBK1 activation and opened a new
possibility that insufficient phagocytic clearance by microglia may contribute to motor neuron damage.
Abstract (croatian) Mutacije optineurina nedavno su pronađene u bolesnika s amiotrofičnom lateralnom sklerozom (ALS), fatalnom bolesti motornih neurona. Za optineurin se smatra da sudjeluje u regulaciji nekoliko staničnih procesa kao što su upala, autofagija, promet vezikula te održavanje strukture Golgijeva tjelešca. Međutim, točna fiziološka uloga optineurina još uvijek je nepoznata. Pokazano je da su mikroglija stanice, koje su glavne imunološke stanice u mozgu, važne u procesu degeneracije motornih neurona u ALS-u te smo se zbog toga u ovom radu usredotočili na ulogu optineurina u upalnoj signalizaciji. Zapaženo je da deficijencija optineurina prekomjerno aktivira upalni NF-κB put te istovremeno nepotpuno aktivira potencijalno protektivnu TBK1 signalizaciju. Međutim, rezultati dobiveni na staničnim linijama ne slažu se s onima dobivenima na primarnim stanicama te je nejasno zašto. Kako bismo pridonijeli razjašnjavanju uloge optineurina u ovim signalnim putevima, uspostavili smo nove, optineurin deficijentne (Optn KO) mikroglijalne (BV2) i neuronalne (Neuro2A) stanične linije pomoću CRISPR/Cas9 tehnologije. Testirali smo razine ranih markera nakon NF-κB aktivacije, poput degradacije IκB te fosforilacije p65 nakon tretmana TNF-om ili LPS-om, no nismo zamijetili razliku niti između neuronalnih niti mikroglijalnih Optn KO i WT linija. Također, testirali smo TBK1 aktivaciju u BV2 staničnoj liniji te smo zamijetili pad u TBK1 i IRF3 fosforilaciji u Optn KO u usporedbi s WT stanicama. Nadalje, uspostavili smo esej fagocitoze baziran na protočnoj citometriji kako bismo testirali fagocitičke sposobnosti BV2 KO stanične linije. Primijetili smo da je fagocitoza u uvjetima bez optineurina bila blago smanjena. Sekrecija citokina TNF-α nakon tretmana LPS-om također je bila smanjena u Optn KO BV2 stanicama. Naši rezultati sugeriraju da deficijencija optineurina potencijalno posreduje progresiju ALS-a na više načina nego što je do sada opisano. Zaključno, ovo istraživanje potvrđuje ulogu optineurina kao pozitivnog regulatora TBK1 aktivacije te otvara novu mogućnost da
nedostatna mikroglijalna fagocitoza staničnog otpada pridonosi oštećenju motornih neurona.
Keywords
optineurin
neurodegeneration
inflammation
NF-κB
TBK1
phagocytosis
Keywords (croatian)
optineurin
neurodegeneracija
upala
NF-κB
TBK1
fagocitoza
Language english
URN:NBN urn:nbn:hr:193:162533
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 Closed access
Terms of use
Created on 2018-10-31 08:51:27