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

Öz

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.

Anahtar Kelimeler

• Microglia
• induced pluripotent stem cells
• neuroinflammation
• NLRP3 inflammasome
• amyloid beta

İNDÜKLENMİŞ PLURİPOTENT KÖK HÜCRE KAYNAKLI MİKROGLİA HÜCRELERİNİN ALZHEİMER HASTALIĞINDA NLRP3 İNFLAMAZOM AKTİVASYONU ARAŞTIRMALARINDA KULLANIMI

Öz

Amaç: Alzheimer hastalığı (AH) geri dönüşümsüz ve ilerleyici bir nörodejeneratif hastalıktır. Amiloid beta (Aβ) ve tau birikimlerinin yanı sıra, inflamasyon da AH patogenezinde rol oynamaktadır. Mikroglia hücrelerinde NLR ailesi pirin domain içeren 3 (NLRP3) inflamazomunun aktivasyonunun AH ile ilişkili olduğu düşünülmektedir. Hastalığın hayvan modelleri insandaki patolojiyi tam olarak yansıtamadığından, çalışmamızda insan kaynaklı indüklenmiş pluripotent kök hücreler (İPKH) mikrogliaya farklılaştırılarak, AH’de NLRP3 yolağı ilişkili mekanizmaların araştırılmasındaki kullanım potansiyeli incelenmiştir. Gereç ve Yöntem: AH, izogenik ve kontrol genotipteki iPKH hücre hatları mikrogliaya farklılaştırılmış ve farklılaşmanın çeşitli aşamalarındaki hücreler akış sitometrisi, gerçek zamanlı kantitatif polimeraz zincir reaksiyonu (RT-qPCR), immünositokimya ve Western blot yöntemleriyle karakterize edilmiştir. NLRP3 yolağı ile ilişkili proteinlerin ekspresyonu Western blot ile incelenmiştir. Fonksiyonel analizler için, NLRP3 inflamazom aktivatörleri (lipopolisakkarid (LPS), Aβ) ve inhibitörü (sitokin salgılanmasını inhibe edici ilaç 3, CRID3) varlığında sitokin salımı ELISA ile ölçülmüştür. pHrodo partiküllerinin ve amiloid betanın fagositozu ise akış sitometrisi, floresan mikroskobu ve canlı hücre görüntüleme sistemi ile değerlendirilmiştir. Bulgular: Çalışmamızda farklı genotiplerdeki İPKH’ler başarılı biçimde mikrogliaya farklılaştırılmıştır. Elde edilen mikroglia hücrelerinin çeşitli mikroglia ve NLRP3 inflamazomu ilişkili belirteçleri eksprese ettiği ve pHrodo ile amiloid betayı fagosite edebildiği gösterilmiştir. Mikroglia hücrelerinin LPS ve amiloid beta ile uyarılması IL-1beta ve IL-18 salımına neden olurken, CRID3 uygulanması bu etkiyi tersine çevirmiştir. Sonuç: Çalışmamız İPKH’den farklılaştırılan mikroglia hücrelerinin, mikroglianın işlevsel özelliklerini taşıması nedeniyle NLRP3 yolağı ile ilişkili hastalık mekanizmalarının ve tedavi seçeneklerinin araştırılmasında kullanılabileceğini göstermektedir.

Anahtar Kelimeler

• Mikroglia
• indüklenmiş pluripotent kök hücreler
• nöroinflamasyon
• NLRP3 inflamazomu
• amiloid beta

Kaynakça

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