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Investigating the expression and function of Complement Receptor 1 (CR1) on iPS derived microglia

Shaw, Bethany Jane Marshall 2022. Investigating the expression and function of Complement Receptor 1 (CR1) on iPS derived microglia. PhD Thesis, Cardiff University.
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Objectives: GWAS has identified the gene encoding Complement receptor 1 (CR1/CD35) as a high risk locus for the development of Alzheimer’s disease (AD)1–3. CR1 is a receptor and regulator of the complement system, involved in immune complex handling and as co-factor for Factor I (FI) mediated breakdown of C3b4,5. Studies of expression of CR1 within the CNS have been contradictory, some reporting no expression1,5–7. We aimed to investigate the expression of CR1 on CNS cells using induced pluripotent stem cells (iPSC) differentiated to glia. To confirm expression and test function, CR1 KO iPS clones were generated and differentiated. Methods: iPSC were differentiated to microglia and astrocytes using adaptions of published protocols.Expression of CR1 transcript was tested with RT-qPCR and protein using immunofluorescence and western blotting. CR1 was knocked out using CRISPR Cas9. CR1 KO and CR1 WT iPS lines were differentiated to microglia for testing in functional assays, including phagocytosis of pH-Rodo e-coli bioparticles and pro-inflammatory transcript response following acute LPS stimulation. Results: CR1 was expressed on iPS microglia but not on iPS astrocytes. CR1 KO clones were generated and differentiated to microglia. Acute LPS stimulation of CR1 KO and CR1 WT iPS microglia revealed significantly less TNFα and IL‐6 transcript in KO compared to WT cells. In phagocytosis assays, CR1 KO microglia showed a significant reduction in uptake of e-coli bioparticles compared to CR1 WT when the particles were serum opsonised but not in the absence of opsonisation or when the serum source was depleted of FI. Conclusions: We demonstrate that CR1 is expressed on iPS-derived microglia confirmed by CRISPR KO. Functional assays showed an impact of CR1 expression on key iPS microglia functions that are relevant to AD pathology. Together the data provide a starting point to understanding how CR1 variants impact AD pathogenesis.

Item Type: Thesis (PhD)
Date Type: Completion
Status: Unpublished
Schools: Medicine
Date of First Compliant Deposit: 13 July 2022
Last Modified: 12 Jul 2023 01:30

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