Uloga optineurina u fagocitozi fibrilarnog amiloida b u makrofagima i dendritičkim stanicama proizvedenim iz koštane srži

Abstract

Optineurin is a multifunctional ubiquitin-binding adaptor protein involved in regulation of cellular processes, for instance vesicle trafficking, autophagy and inflammatory signalling. The mutations of the OPTN gene that encodes for optineurin were linked to several neurodegenerative diseases, the most important being amyotrophic lateral sclerosis (ALS), which leads to destruction of motor neurons. Research has shown that phagocytosis, a process of internalizing and degrading particles larger than 0.5 µm in diameter, is disrupted in various neurodegenerative diseases, including ALS and Alzheimer’s disease (AD). Previous research in our lab showed no effect of optineurin during phagocytosis of dead neurons and pHrodo™ Green E. coli BioParticles™. The main goal of this thesis was to investigate the role optineurin has in phagocytosis of fibrillar amyloid beta (A-beta) . To investigate this, we performed a flow cytometry analysis of the phagocytosis assay of fluorescent A-beta in wild type (WT) and optineurin truncation Optn470T-carrying bone marrow-derived macrophages (BMDMs) and dendritic cells (BMDCs). Optn470T mice mimic the C-terminal truncations found in a small percentage of ALS patients. Our results showed no difference between BMDM genotypes in the number of phagocytic cells, but there was a decrease of ~20% in the phagocytic capacity of Optn470T cells compared to WT BMDMs. There was also a very significant difference between WT and Optn470T BMDC in both the number of phagocytosing cells and their phagocytic ability. Optn470T BMDCs had a ~15-20% lower number of phagocytosing cells than WT BMDCs. Their phagocytic ability was ~30-40% lower than that of WT cells. The differences between the genotypes did not significantly increase if the cells were pretreated with inflammatory stimuli. In conclusion, this study demonstrates that optineurin has a role in phagocytosis of fibrillar fA-beta in BMDCs, opening a possibility that this is the mechanism by which optineurin contributes to disease pathogenesis.Optineurin je multifunkcionalni adapterski protein koji veže ubikvitin. Uključen je u regulaciju staničnih procesa kao što je upalna signalizacija, promet vezikulama i autofagija. Mutacije u OPTN genu koji kodira za optineurin povezane su s nekoliko neurodegenerativnih bolesti, od kojih je najvažnija amiotrofična lateralna skleroza (ALS), koja dovodi do odumiranja motornih neurona. Istraživanja su pokazala da je fagocitoza, proces internalizacije i razgradnje čestica većih od 0,5 µm u promjeru, poremećena u raznim neurodegenerativnim bolestima uključujući ALS i Alzheimerovu bolest (AD). Prethodna istraživanja u našem laboratoriju nisu pokazala nikakav učinak optineurina tijekom fagocitoze mrtvih neurona i pHrodo™ Green E. coli BioParticles™. Cilj ovog diplomskog rada bio je istražiti ulogu optineurina u fagocitozi fibrilarnog amiloida beta (A-beta). U tu smo svrhu proveli analizu fagocitoze fluorescentnog A-beta na stanicama divljeg tipa (WT) i stanicama s trunkacijom optineurina (Optn470T) na temelju protočne citometrije. Korišteni su makrofagi (BMDM) i dendritičke stanicame (BMDC) porijeklom iz koštane srži. Optn470T miševi oponašaju skraćenja C-terminala pronađena u maloj podskupini pacijenata s ALS-om. Naši rezultati nisu pokazali razliku između genotipova BMDM u broju fagocitnih stanica, ali došlo je do smanjenja od ~20% u fagocitnom kapacitetu Optn470T stanica u usporedbi s WT BMDM-ovima. Nadalje, postojala je značajna razlika između WT i Optn470T BMDC-a u broju fagocitoznih stanica i njihovoj fagocitnoj sposobnosti. Optn470T BMDC-i imali su ~15-20% niži broj stanica koje fagocitiraju od WT BMDC. Njihova fagocitna sposobnost bila je ~30-40% niža nego kod WT stanica. Razlike između genotipova nisu se značajno povećale ako su stanice prethodno tretirane upalnim podražajima. Zaključno, ova studija pokazuje da optineurin ima ulogu u fagocitozi fibrilarnog fA-beta u BMDC, otvarajući mogućnost da je to mehanizam kojim optineurin doprinosi patogenezi bolesti