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Optineurin is a multifunctional ubiquitin (Ub)-binding protein that was proposed to regulate numerous cellular processes including inflammatory signalling. Around 30 mutations in optineurin, which are thought to act by loss-of-function, have been found in patients with amyotrophic lateral sclerosis (ALS), a neurodegenerative disease of motor neurons. Because neuroinflammation is one of the earliest and the most consistent neuropathological hallmarks of ALS, we aimed to investigate if the loss of optineurin function leads to heightened neuroinflammation. To assess this, we analysed primary brain cells and tissues from optineurin insufficiency mouse model with C-terminal truncation of Ub-binding region (Optn470T) upon stimulation with inflammatory stimuli and during aging. We showed that optineurin played a key role in activating the Tank binding kinase 1 (TBK1) signalling pathway upon Toll-like receptor (TLR) stimulation and thus upregulated interferon-beta (IFN-β) production in primary neonatal microglia. The repercussions of decreased IFN-β production were seen in diminished activation of the Signal transducer and activator of transcription 1 (STAT1) and disbalance in the expression of several pro- and anti-inflammatory factors including interferon regulatory factor 7 (IRF7), nitric oxide synthetase 2 (NOS2), interleukin 10 (IL-10) and C-XC motif chemokine 1 (CXCL1). Cytokine and chemokine expression was successfully restored upon addition of recombinant IFN-β, suggesting that optineurin-supported IFN-β secretion upon TLR activation is necessary for optimal inflammatory response. Similar phenotype of diminished TBK1 signalling activation has also been found in Optn470T primary neurons. Despite differences in microglial inflammatory response in vitro, similar level of microgliosis was found in wild-type (WT) and Optn470T mice upon short-term systemic LPS application. In contrast, cytokine array analysis of the whole brain lysates from Optn470T mice upon short-term systemic LPS application showed increased production of granulocyte and monocyte chemoattractants. However, Optn470T mice showed diminished infiltration of the peripheral myeloid cells into the brain, suggesting that the role of optineurin in regulation of neuroinflammation in vivo is more complex than expected from the microglial activation patterns in vitro. The analysis of aged Optn470T mice did not show signs of microgliosis and neurodegeneration in comparison to WT control. For these reasons we hypothesize that additional and/or chronic stimuli are needed for manifestation of neurodegenerative disease and/or neuroinflammation in the mouse optineurin insufficiency model.Optineurin je multifunkcionalni ubikvitin (Ub)-vezujući protein za koji je predloženo da regulira niz staničnih procesa, uključujući prijenos signala tijekom upale. U pacijenata s amiotrofičnom lateralnom sklerozom (ALS), neurodegenerativnom bolešću motornih neurona, pronađeno je više od 30 mutacija u optineurinu. Smatra se da te mutacije uzrokuju bolest gubitkom funkcije optineurina. Jedno od najranijih i najkonzistentnijih neuropatoloških obilježja ALS-a je neuroinflamacija. Kako bismo ispitali uzrokuje li gubitak optineurina pojačanu neuroinflamaciju, koristili smo primarne stanice i tkiva izolirane iz mozgova miševa s insuficijencijom optineurina uslijed C-terminalne trunkacije Ub-vezujuće regije (Optn470T) nakon poticanja upale s različitim stimulansima te tijekom starenja. Pokazali smo da optineurin ima ključnu ulogu u aktivaciji signalnog puta TANK-vezujuće kinaze 1 (TBK1) nakon stimulacije receptora sličnih Toll-u (TLR) te da potiče proizvodnju interferona-beta (IFN- β) u primarnim mikroglija stanicama. Smanjena proizvodnja IFN-β rezultirala je smanjenom aktivacijom STAT1 (engl. signal transducer and activator of transcription 1) i poremećajem u ekspresiji nekoliko pro- i anti-inflamatornih gena uključujući interferon regulirajući factor 7 (IRF7), sintetazu dušikovog oksida 2 (NOS2), kemokinski ligand s C-X-C motivom 1 (CXCL1) i interleukin 10 (IL-10). Dodatkom IFN-β, ekspresija citokina i kemokina je uspješno vraćena na razinu WT (engl. wild-type) stanica, ukazujući da je sekrecija IFN-β regulirana od strane optineurina nakon aktivacije TLR-a nužna za optimalni upalni odgovor. Sukladno tome, primarni Optn470T neuroni su pokazali smanjenu aktivaciju TBK1 signalnog puta. Unatoč pronađenim razlikama u primarnim stanicama, akutna sistemska primjena LPS-a rezultirala je jednakim morfološkim znakovima aktivacije mikroglije in vivo. Međutim, analiza citokina u ukupnom lizatu mozga tako tretiranih Optn470T miševa u usporedbi s WT miševima pokazala je povećanu proizvodnju kemokina koji privlače granulocite i monocite na mjesto upale. Usprkos tome, Optn470T miševi su pokazali smanjenu infiltraciju perifernih mijeloidnih stanica u mozak nakon tretmana s LPS-om, što sugerira da je uloga optineurina u regulaciji neuroinflamacije in vivo znatno složenija od njegove uloge u aktivaciji mikroglije in vitro. Nadalje, ostarjeli Optn470T miševi nisu pokazali znakove mikroglioze i neurodegeneracije u odnosu na WT kontrole. Iz tih razloga pretpostavljamo da su u mišjem modelu insuficijencije optineurina za manifestaciju neurodegeneracije i/ili neuroinflamacije potrebni dodatni ili kronični stimulansi