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Title: Investigating the role of Leucine Rich Repeat Kinase 2 (LRRK2) in human induced pluripotent stem cell derived macrophages
Author: Lee, Heyne (Cecilia)
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 2017
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Mutations in Leucine Rich Repeat Kinase 2 (LRRK2) are the most prevalent cause of familial PD. Genome Wide Association Studies (GWAS) have linked its variants with increased risk of developing sporadic PD, inflammatory bowel disease, and leprosy, diseases commonly associated with inflammation and immune dysfunction. LRRK2 is predominantly expressed in a subset of immune cells, notably macrophages and microglia and has been implicated in innate immunity. Yet, most studies report conflicting results, and data are based on mouse models or in immortalised cell lines which do not faithfully recapitulate all aspects of tissue macrophages. In this thesis, macrophages and microglia differentiated from human induced pluripotent stem cells (hiPSCs) have been used as a genetically tractable tool that expresses LRRK2 at physiological levels from its endogenous promoter in its normal human chromosomal location. Using this physiologically relevant cell model, the aim of this thesis was to investigate the expression, subcellular localisation and role of LRRK2 protein in macrophages and microglia. This thesis maps the cleavage region of an N-terminal proteolytically cleaved form of LRRK2 in macrophages and shows that LRRK2 can form heterodimers of full-length and cleaved forms. My results demonstrate that LRRK2 is recruited to late phagosomes and that treatment of LRRK2 kinase inhibitors leads to the formation of LRRK2 super-coated phagosomes. However, I demonstrate that LRRK2 is not required for the acidification of phagosomes, and its role at the phagolysosome remains unresolved. Lastly, using hiPSC-microglia in co-culture with iPSC-cortical neurons, I demonstrate that in human cells, LRRK2 is robustly expressed in microglia and is undetectable in cortical neurons. In this co-culture model, under the conditions tested, inflammatory cytokine release was broadly unaffected by LRRK2 mutation or KO, although two different TNFα assay approaches gave conflicting results. This thesis provides novel insights into the role of LRRK2 in macrophages and microglia, and shows that hiPSC-macrophages and microglia can serve as a valuable platform to examine LRRK2 biology.
Supervisor: Cowley, Sally A. ; James, William S. Sponsor: Wellcome Trust ; Oxford Martin School ; EU IMI StemBANCC
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available