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

Aslı Erdoğan Öner; Cira Dansokho; Jari Koistinaho; Sarka Lehtonen; Seyhun Solakoğlu; Michael Thomas Heneka

Makale detayları

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

Yazarlar:

Aslı Erdoğan Öner | Cira Dansokho | Jari Koistinaho | Sarka Lehtonen | Seyhun Solakoğlu | Michael Thomas Heneka

Yayıncı:

Istanbul University

Ana Başlıklar:

Sağlık Hizmetleri ve Sistemleri (Diğer)

Dergi:

İstanbul Tıp Fakültesi Dergisi Vol.87 Issue 4, pp.299-311

Belge Tipi:

Makale

Yayın Tarihi:

10/2024

Belge Dili:

İngilizce

Document ID (Org No.):

2001340561

pISSN:

1305-6441

Anahtar kelimeler:

Özet:

 Objective: Alzheimers 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.

 Objective: Alzheimers 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.

 Objective: Alzheimers 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.

 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, AHde 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 İPKHler 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 İPKHden farklılaştırılan mikroglia hücrelerinin, mikroglianın işlevsel özelliklerini taşıması nedeniyle NLRP3 yolağı ile ilişkili hastalık mekanizmalarının ve tedavi seçeneklerinin araştırılmasında kullanılabileceğini göstermektedir.

Makale detayları

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

Yazarlar:

Aslı Erdoğan Öner | Cira Dansokho | Jari Koistinaho | Sarka Lehtonen | Seyhun Solakoğlu | Michael Thomas Heneka

Yayıncı:

Istanbul University

Ana Başlıklar:

Sağlık Hizmetleri ve Sistemleri (Diğer)

Dergi:

İstanbul Tıp Fakültesi Dergisi Vol.87 Issue 4, pp.299-311

Belge Tipi:

Makale

Yayın Tarihi:

10/2024

Belge Dili:

İngilizce

Document ID (Org No.):

2001340561

pISSN:

1305-6441

Microglia

induced pluripotent stem cells

neuroinflammation

NLRP3 inflammasome

amyloid betaMikroglia

indüklenmiş pluripotent kök hücreler

nöroinflamasyon

NLRP3 inflamazomu

amiloid beta

Sayı

Makale detayları

Yazarlar: Aslı Erdoğan Öner | Cira Dansokho | Jari Koistinaho | Sarka Lehtonen | Seyhun Solakoğlu | Michael Thomas Heneka

Yayıncı: Istanbul University

Ana Başlıklar: Sağlık Hizmetleri ve Sistemleri (Diğer)

Dergi: İstanbul Tıp Fakültesi Dergisi Vol.87 Issue 4, pp.299-311

Belge Tipi: Makale

Yayın Tarihi: 10/2024

Belge Dili: İngilizce

Document ID (Org No.): 2001340561

pISSN: 1305-6441

Sayı

Yazarlar:

Aslı Erdoğan Öner | Cira Dansokho | Jari Koistinaho | Sarka Lehtonen | Seyhun Solakoğlu | Michael Thomas Heneka

Yayıncı:

Istanbul University

Ana Başlıklar:

Sağlık Hizmetleri ve Sistemleri (Diğer)

Dergi:

İstanbul Tıp Fakültesi Dergisi Vol.87 Issue 4, pp.299-311

Belge Tipi:

Makale

Yayın Tarihi:

10/2024

Belge Dili:

İngilizce

Document ID (Org No.):

2001340561

pISSN:

1305-6441