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DOI :10.26650/IUITFD.1451011   IUP :10.26650/IUITFD.1451011    Full Text (PDF)

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

Aslı Erdoğan, Cira Dansokho, Jari Koıstınaho, Sarka Lehtonen, Seyhun Solakoğlu, Michael Thomas Heneka

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.

Keywords: Microglia, induced pluripotent stem cells, neuroinflammation, NLRP3 inflammasome, amyloid beta
DOI :10.26650/IUITFD.1451011   IUP :10.26650/IUITFD.1451011    Full Text (PDF)

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

Aslı Erdoğan, Cira Dansokho, Jari Koıstınaho, Sarka Lehtonen, Seyhun Solakoğlu, Michael Thomas Heneka

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.

Keywords: Mikroglia, indüklenmiş pluripotent kök hücreler, nöroinflamasyon, NLRP3 inflamazomu, amiloid beta

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References

  • Li T, Lu L, Pember E, Li X, Zhang B, Zhu Z. New Insights into neuroinflammation involved in pathogenic mechanism of Alzheimer’s disease and its potential for therapeutic intervention. Cells 2022;11:1925. [CrossRef] google scholar
  • 2022 Alzheimer’s disease facts and figures. Alzheimer’s and Dementia 2022;18(4):700-89. [CrossRef] google scholar
  • McManus RM. The Role of Immunity in Alzheimer’s Disease. Adv Biol 2022;6(5):2101166. [CrossRef] google scholar
  • Crews L, Masliah E. Molecular mechanisms of neurodegeneration in Alzheimer’s disease. Hum Mol Genet 2010;19(R1):R12-20. [CrossRef] google scholar
  • Heneka MT, Carson MJ, Khoury J El, Landreth GE, Brosseron F, Feinstein DL, et al. Neuroinflammation in Alzheimer’s disease. Lancet Neurol 2015;14(4):388-405. [CrossRef] google scholar
  • Heppner FL, Ransohoff RM, Becher B. Immune attack: The role of inflammation in Alzheimer disease. Nat Rev Neurosci 2015;16(6):358-72. [CrossRef] google scholar
  • Li JW, Zong Y, Cao XP, Tan L, Tan L. Microglial priming in Alzheimer’s disease. Ann Transl Med 2018;6(10):176. [CrossRef] google scholar
  • Kettenmann H, Hanisch UK, Noda M, Verkhratsky A. Physiology of microglia. Physiol Rev 2011;91(2):461-553. [CrossRef] google scholar
  • Fan Y, Xie L, Chung CY. Signaling pathways controlling microglia chemotaxis. Mol Cells 2017;40(3):163-8. [CrossRef] google scholar
  • Heneka MT, Kummer MP, Stutz A, Delekate A, Schwartz S, Vieira-Saecker A, et al. NLRP3 is activated in Alzheimer’s disease and contributes to pathology in APP/PS1 mice. Nature 2013;493(7434):674-8. [CrossRef] google scholar
  • Rui W, Xiao H, Fan Y, Ma Z, Xiao M, Li S, et al. Systemic inflammasome activation and pyroptosis associate with the progression of amnestic mild cognitive impairment and Alzheimer’s disease. J Neuroinflammation 2021;18(1):280. [CrossRef] google scholar
  • Dansokho C, Heneka MT. Neuroinflammatory responses in Alzheimer’s disease. J Neural Transm 2018;125(5):771-9. [CrossRef] google scholar
  • Swanson K, Deng M, Ting JPY. The NLRP3 inflammasome: molecular activation and regulation to therapeutics. Nat Rev Immunol 2019;19:477-89. [CrossRef] google scholar
  • Liang T, Zhang Y, Wu S, Chen Q, Wang L. The Role of NLRP3 Inflammasome in Alzheimer’s Disease and Potential Therapeutic Targets. Front Pharmacol 2013;13:845185. [CrossRef] google scholar
  • Daniels MJD, Rivers-Auty J, Schilling T, Spencer NG, Watremez W, Fasolino V, et al. Fenamate NSAIDs inhibit the NLRP3 inflammasome and protect against Alzheimer’s disease in rodent models. Nat Commun 2016;7:12504. [CrossRef] google scholar
  • Yin J, Zhao F, Chojnacki JE, Fulp J, Klein WL, Zhang S, et al. NLRP3 inflammasome inhibitor ameliorates amyloid pathology in a mouse model of Alzheimer’s disease. Mol Neurobiol 2018;55(3):1977-87. [CrossRef] google scholar
  • Zahid A, Li B, Kombe AJK, Jin T, Tao J. Pharmacological inhibitors of the NLRP3 inflammasome. Front Immunol 2019;10:2538. [CrossRef] google scholar
  • Coll RC, Hill JR, Day CJ, Zamoshnikova A, Boucher D, Massey NL, et al. MCC950 directly targets the NLRP3 ATP-hydrolysis motif for inflammasome inhibition. Nat Chem Biol 2019;15(6):556-9. [CrossRef] google scholar
  • Barczuk J, Siwecka N, Lusa W, Rozpçdek-Kaminska W, Kucharska E, Majsterek I. Targeting NLRP3-Mediated Neuroinflammation in Alzheimer’s Disease Treatment. Int Journal of Mol Sci 2022;23(16):8979. [CrossRef] google scholar
  • Galatro TF, Holtman IR, Lerario AM, Vainchtein ID, Brouwer N, Sola PR, et al. Transcriptomic analysis of purified human cortical microglia reveals age-associated changes. Nat Neurosci 2017;20(8):1162-71. [CrossRef] google scholar
  • Washer SJ, Perez-Alcantara M, Chen Y, Steer J, James WS, Trynka G, et al. Single-cell transcriptomics defines an improved, validated monoculture protocol for differentiation of human iPSC to microglia. Sci Rep 2022;12(1):19454. [CrossRef] google scholar
  • Hasselmann J, Blurton-Jones M. Human iPSC-derived microglia: A growing toolset to study the brain’s innate immune cells. Glia 2020;68(4):721-39. [CrossRef] google scholar
  • McQuade A, Coburn M, Tu CH, Hasselmann J, Davtyan H, Blurton-Jones M. Development and validation of a simplified method to generate human microglia from pluripotent stem cells. Mol Neurodegener 2018;13:67. [CrossRef] google scholar
  • Leng F, Edison P. Neuroinflammation and microglial activation in Alzheimer disease: where do we go from here? Nat Rev Neurol 2021;17:157-72. [CrossRef] google scholar
  • Pocock JM, Piers TM. Modelling microglial function with induced pluripotent stem cells: An update. Nat Rev Neurosci 2018;19(8):445-52. [CrossRef] google scholar
  • Friedman BA, Srinivasan K, Ayalon G, Meilandt WJ, Lin H, Huntley MA, et al. Diverse brain myeloid expression profiles reveal distinct microglial activation states and aspects of Alzheimer’s disease not evident in mouse models. Cell Rep 2018;22(3):832-47. [CrossRef] google scholar
  • Kierdorf K, Erny D, Goldmann T, Sander V, Schulz C, Perdiguero EG, et al. Microglia emerge from erythromyeloid precursors via Pu.1-and Irf8-dependent pathways. Nat Neurosci 2013;16(3):273-80. [CrossRef] google scholar
  • Ginhoux F, Greter M, Leboeuf M, Nandi S, See P, Gokhan S, et al. Fate mapping analysis reveals that adult microglia derive from primitive macrophages. Science 2010;330(6005):841-5. [CrossRef] google scholar
  • Haenseler W, Sansom SN, Buchrieser J, Newey SE, Moore CS, Nicholls FJ, et al. A Highly Efficient Human Pluripotent Stem Cell Microglia Model Displays a Neuronal-Co-cultureSpecific Expression Profile and Inflammatory Response. Stem Cell Rep 2017;8(6):1727-42. [CrossRef] google scholar
  • Pandya H, Shen MJ, Ichikawa DM, Sedlock AB, Choi Y, Johnson KR, et al. Differentiation of human and murine induced pluripotent stem cells to microglia-like cells. Nat Neurosci 2017;20(5):753-9. [CrossRef] google scholar
  • Takata K, Kozaki T, Lee CZW, Thion MS, Otsuka M, Lim S, et al. Induced-Pluripotent-Stem-Cell-Derived Primitive Macrophages Provide a Platform for Modeling TissueResident Macrophage Differentiation and Function. Immunity 2017;47(1):183-98. [CrossRef] google scholar
  • Abud EM, Ramirez RN, Martinez ES, Healy LM, Nguyen CHH, Newman SA, et al. iPSC-derived human microglia-like cells to study neurological diseases. Neuron 2017;94(2):278-93. [CrossRef] google scholar
  • Konttinen H, Cabral-da-Silva M e. C, Ohtonen S, Wojciechowski S, Shakirzyanova A, Caligola S, et al. PSEN1AE9, APPswe, and APOE4 Confer Disparate Phenotypes in Human iPSC-Derived Microglia. Stem Cell Rep 2019;13(4):669-83. [CrossRef] google scholar
  • Douvaras P, Sun B, Wang M, Kruglikov I, Lallos G, Zimmer M, et al. Directed Differentiation of Human Pluripotent Stem Cells to Microglia. Stem Cell Rep 2017;8(6):1516-24. [CrossRef] google scholar
  • Foudah D, Monfrini M, Donzelli E, Niada S, Brini AT, Orciani M, et al. Expression of neural markers by undifferentiated mesenchymal-like stem cells from different sources. J Immunol Res 2014;2014:987678. [CrossRef] google scholar
  • Bianchi F, Malboubi M, Li Y, George JH, Jerusalem A, Szele F, et al. Rapid and efficient differentiation of functional motor neurons from human iPSC for neural injury modelling. Stem Cell Res 2018;32:126-34. [CrossRef] google scholar
  • Böttcher C, Schlickeiser S, Sneeboer MAM, Kunkel D, Knop A, Paza E, et al. Human microglia regional heterogeneity and phenotypes determined by multiplexed single-cell mass cytometry. Nat Neurosci 2019;22(1):78-90. [CrossRef] google scholar
  • Melief J, Sneeboer MAM, Litjens M, Ormel PR, Palmen SJMC, Huitinga I, et al. Characterizing primary human microglia: A comparative study with myeloid subsets and culture models. Glia 2016;64(11):1857-68. [CrossRef] google scholar
  • Garcia-Reitboeck P, Phillips A, Piers TM, Villegas-Llerena C, Butler M, Mallach A, et al. Human Induced Pluripotent Stem Cell-Derived Microglia-Like Cells Harboring TREM2 Missense Mutations Show Specific Deficits in Phagocytosis. Cell Rep 2018;24(9):2300-11. [CrossRef] google scholar
  • Satoh J, Kino Y, Asahina N, Takitani M, Miyoshi J, Ishida T, et al. TMEM119 marks a subset of microglia in the human brain. Neuropathology 2016;36(1):39-49. [CrossRef] google scholar
  • Bohnert S, Seiffert A, Trella S, Bohnert M, Distel L, Ondruschka B, et al. TMEM119 as a specific marker of microglia reaction in traumatic brain injury in postmortem examination. Int J Legal Med 2020;134(6):2167-76. [CrossRef] google scholar
  • Murai N, Mitalipova M, Jaenisch, R. Functional analysis of CX3CR1 in human induced pluripotent stem (iPS) cell-derived microglia-like cells. Eur J Neurosci 2020;52(7):3667-78. [CrossRef] google scholar
  • Morillas AG, Besson VC, Lerouet D. Microglia and neuroinflammation: What place for p2ry12? Int J Mol Sci 2021;22(4):1-16. [CrossRef] google scholar
  • Luciûnaité A, McManus RM, Jankunec M, Racz I, Dansokho C, Dalgédiené I, et al. Soluble Aß oligomers and protofibrils induce NLRP3 inflammasome activation in microglia. J Neurochem 2020;155(6):650-61. [CrossRef] google scholar
  • Dempsey C, Rubio Araiz A, Bryson KJ, Finucane O, Larkin C, Mills EL, et al. Inhibiting the NLRP3 inflammasome with MCC950 promotes non-phlogistic clearance of amyloid—P and cognitive function in APP/PS1 mice. Brain Behav Immun 2017;61:306-16. [CrossRef] google scholar
  • Mouton-Liger F, Rosazza T, Sepulveda-Diaz J, Ieang A, Hassoun SM, Claire E, et al. Parkin deficiency modulates NLRP3 inflammasome activation by attenuating an A20-dependent negative feedback loop. Glia 2018;66(8):1736-51. [CrossRef] google scholar
  • Clénet ML, Keaney J, Gillet G, Valadas JS, Langlois J, Cardenas A, et al. Divergent functional outcomes of NLRP3 blockade downstream of multi-inflammasome activation: therapeutic implications for ALS. Front Immunol 2023;14:1190219. [CrossRef] google scholar
  • Baik SH, Kang S, Son SM, Mook-Jung I. Microglia contributes to plaque growth by cell death due to uptake of amyloid P in the brain of Alzheimer’s disease mouse model. Glia 2016;64(12):2274-90. [CrossRef] google scholar
  • Huang Y, Happonen KE, Burrola PG, O’Connor C, Hah N, Huang L, et al. Microglia use TAM receptors to detect and engulf amyloid P plaques. Nat Immunol 2021;22(5):586-94. [CrossRef] google scholar
  • Xu M, Zhang L, Liu G, Jiang N, Zhou W, Zhang Y. Pathological Changes in Alzheimer’s Disease Analyzed Using Induced Pluripotent Stem Cell-Derived Human Microglia-Like Cells. J Alzheimer’s Dis 2019;67(1):357-68. [CrossRef] google scholar

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APA

Erdoğan, A., Dansokho, C., Koıstınaho, J., Lehtonen, S., Solakoğlu, S., & Heneka, M.T. (2024). INDUCED PLURIPOTENT STEM CELL-DERIVED MICROGLIA TO STUDY NLRP3 INFLAMMASOME ACTIVATION IN ALZHEIMER’S DISEASE. Journal of Istanbul Faculty of Medicine, 87(4), 299-311. https://doi.org/10.26650/IUITFD.1451011


AMA

Erdoğan A, Dansokho C, Koıstınaho J, Lehtonen S, Solakoğlu S, Heneka M T. INDUCED PLURIPOTENT STEM CELL-DERIVED MICROGLIA TO STUDY NLRP3 INFLAMMASOME ACTIVATION IN ALZHEIMER’S DISEASE. Journal of Istanbul Faculty of Medicine. 2024;87(4):299-311. https://doi.org/10.26650/IUITFD.1451011


ABNT

Erdoğan, A.; Dansokho, C.; Koıstınaho, J.; Lehtonen, S.; Solakoğlu, S.; Heneka, M.T. INDUCED PLURIPOTENT STEM CELL-DERIVED MICROGLIA TO STUDY NLRP3 INFLAMMASOME ACTIVATION IN ALZHEIMER’S DISEASE. Journal of Istanbul Faculty of Medicine, [Publisher Location], v. 87, n. 4, p. 299-311, 2024.


Chicago: Author-Date Style

Erdoğan, Aslı, and Cira Dansokho and Jari Koıstınaho and Sarka Lehtonen and Seyhun Solakoğlu and Michael Thomas Heneka. 2024. “INDUCED PLURIPOTENT STEM CELL-DERIVED MICROGLIA TO STUDY NLRP3 INFLAMMASOME ACTIVATION IN ALZHEIMER’S DISEASE.” Journal of Istanbul Faculty of Medicine 87, no. 4: 299-311. https://doi.org/10.26650/IUITFD.1451011


Chicago: Humanities Style

Erdoğan, Aslı, and Cira Dansokho and Jari Koıstınaho and Sarka Lehtonen and Seyhun Solakoğlu and Michael Thomas Heneka. “INDUCED PLURIPOTENT STEM CELL-DERIVED MICROGLIA TO STUDY NLRP3 INFLAMMASOME ACTIVATION IN ALZHEIMER’S DISEASE.” Journal of Istanbul Faculty of Medicine 87, no. 4 (Nov. 2024): 299-311. https://doi.org/10.26650/IUITFD.1451011


Harvard: Australian Style

Erdoğan, A & Dansokho, C & Koıstınaho, J & Lehtonen, S & Solakoğlu, S & Heneka, MT 2024, 'INDUCED PLURIPOTENT STEM CELL-DERIVED MICROGLIA TO STUDY NLRP3 INFLAMMASOME ACTIVATION IN ALZHEIMER’S DISEASE', Journal of Istanbul Faculty of Medicine, vol. 87, no. 4, pp. 299-311, viewed 14 Nov. 2024, https://doi.org/10.26650/IUITFD.1451011


Harvard: Author-Date Style

Erdoğan, A. and Dansokho, C. and Koıstınaho, J. and Lehtonen, S. and Solakoğlu, S. and Heneka, M.T. (2024) ‘INDUCED PLURIPOTENT STEM CELL-DERIVED MICROGLIA TO STUDY NLRP3 INFLAMMASOME ACTIVATION IN ALZHEIMER’S DISEASE’, Journal of Istanbul Faculty of Medicine, 87(4), pp. 299-311. https://doi.org/10.26650/IUITFD.1451011 (14 Nov. 2024).


MLA

Erdoğan, Aslı, and Cira Dansokho and Jari Koıstınaho and Sarka Lehtonen and Seyhun Solakoğlu and Michael Thomas Heneka. “INDUCED PLURIPOTENT STEM CELL-DERIVED MICROGLIA TO STUDY NLRP3 INFLAMMASOME ACTIVATION IN ALZHEIMER’S DISEASE.” Journal of Istanbul Faculty of Medicine, vol. 87, no. 4, 2024, pp. 299-311. [Database Container], https://doi.org/10.26650/IUITFD.1451011


Vancouver

Erdoğan A, Dansokho C, Koıstınaho J, Lehtonen S, Solakoğlu S, Heneka MT. INDUCED PLURIPOTENT STEM CELL-DERIVED MICROGLIA TO STUDY NLRP3 INFLAMMASOME ACTIVATION IN ALZHEIMER’S DISEASE. Journal of Istanbul Faculty of Medicine [Internet]. 14 Nov. 2024 [cited 14 Nov. 2024];87(4):299-311. Available from: https://doi.org/10.26650/IUITFD.1451011 doi: 10.26650/IUITFD.1451011


ISNAD

Erdoğan, Aslı - Dansokho, Cira - Koıstınaho, Jari - Lehtonen, Sarka - Solakoğlu, Seyhun - Heneka, MichaelThomas. “INDUCED PLURIPOTENT STEM CELL-DERIVED MICROGLIA TO STUDY NLRP3 INFLAMMASOME ACTIVATION IN ALZHEIMER’S DISEASE”. Journal of Istanbul Faculty of Medicine 87/4 (Nov. 2024): 299-311. https://doi.org/10.26650/IUITFD.1451011



TIMELINE


Submitted26.03.2024
Accepted24.09.2024
Published Online07.10.2024

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