INDUCED PLURIPOTENT STEM CELL-DERIVED MICROGLIA TO STUDY NLRP3 INFLAMMASOME ACTIVATION IN ALZHEIMER’S DISEASE

Objective: Alzheimer’s disease (AD) is an irreversible and progressive neurodegenerative disease. Besides amyloid beta (Aβ) and tau accumulations, inflammation also contributes to AD pathogenesis. NLR family pyrin domain containing 3 (NLRP3) inflammasome activation in microglia is thought to be associated with AD. Since animal models of AD do not accurately reflect human pathology, in our study, human induced pluripotent stem cells (iPSCs) were differentiated into microglia, and their potential to be used in NLRP3 pathway-related mechanisms in AD was investigated.Material and Method: iPSC cell lines of AD, isogenic, or control genotypes were differentiated into microglia and cells from different stages of the differentiation were characterized by flow cytometry, real time quantintative polymerase chain reaction (RTqPCR), immunocytochemistry, and Western blot. The expression of proteins associated with the NLRP3 pathway was investigated by Western blot. For functional analysis, cytokine release was assessed by Enzyme-linked immunosorbent assay (ELISA) upon NLRP3 inflammasome activators (lipopolysaccharide (LPS), Aβ) and inhibitor (cytokine release inhibitory drug 3, CRID3) treatments. The phagocytosis of pHrodo particles and Aβ were evaluated by flow cytometry, fluorescence microscopy, and live cell imaging system.Result: In our study, we could differentiate microglia from iPSCs derived from different genotypes. These microglia cells expressed various microglia and NLRP3 inflammasome-related markers and were able to phagocytose pHrodo particles and Aβ. The stimulation of the microglia cells with LPS and Aβ caused IL- 1beta and IL-18 release, while CRID3 reversed this effect.Conclusion: Our results show that iPSC-derived microglia generated in this study recapitulate microglia functional characteristics and can therefore be used to study NLRP3 pathway-associated disease mechanisms and treatment options.