Use this URL to cite or link to this record in EThOS: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.789134
Title: Generation and characterisation of an in vitro TREM2-deficient microglia model
Author: Lim, Yau Mun
ISNI:       0000 0004 8499 9914
Awarding Body: King's College London
Current Institution: King's College London (University of London)
Date of Award: 2018
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Abstract:
Rare variants of TREM2 have been associated with increased risk of developing several neurodegenerative diseases. In particular, the heterozygous p.R47H variant (rs75932628) has been associated with approximately three times increased risk to develop Alzheimer's disease (AD). Other homozygous TREM2 variants have been associated with developing Nasu-Hakola disease and frontotemporal-like dementia. This PhD project aimed to develop an in vitro TREM2 model endogeneously expressing the disease-associated variants with intact downstream signalling pathways. This cell model would be suitable in screening assays including for use to identify disease-modifying therapeutic compounds. CRISPR/Cas9 genome editing was used to knock in the disease-associated variants into BV2 immortalised mouse microglia cell line. Various steps in the CRISPR/Cas9 workflow were optimised for use with BV2 cells. Despite four iterative improvements in experimental design, knock-in of the variants were unsuccessful. This has been attributed to very low efficiency of homogeneous editing in individual cells with chromosomal instability and hyperploidy that frequently occur in immortalised cell lines. Nevertheless, four clonal lines with homogeneous editing resulting in indels in Trem2 were generated and predicted to result in loss of functional TREM2 expression. Post-mortem hippocampal brain tissue from AD cases with and without a disease-associated TREM2 variant were immunostained for functional microglial activation markers: CD68, HLA-DP, -DQ, -DR and Iba-1 to investigate the effects of these variants on microglia. AD cases with disease-associated TREM2 variants, particularly p.R47H, have decreased CD68 expression and fewer HLA-DP, -DQ, -DR-expressing cells compared to those without variants in hippocampal CA4 despite no differences in the density of Iba-1-expressing cells. DNA indels generated in the Trem2-edited cell lines were stable and the correct clonal lines were selected and expanded from cryopreservation in 96-well culture plates. Protein-coding Trem2 transcripts were disrupted in these cells and functional TREM2 protein was not expressed as predicted from changes in Trem2 DNA sequence. Functional characterisation of these cell lines using a TREM2-activating antibody revealed potential co-regulation of shared immunoreceptor tyrosine-based activation motif (ITAM)-mediated signalling pathways downstream of TREM2 and FcgR. Furthermore, TREM2 deficiency did not affect protein levels of CD68 and Iba-1 after stimulation with the TREM2-activating antibody, unlike in AD cases with a disease-associated TREM2 variant. Low or tonic ligand-binding avidity from monoclonal antibody stimulation or amino acid variants of these receptors that decrease ligand-binding may result in inhibitory ITAM signals that can suppress innate immune responses. This suggests a disease-causing mechanism conveyed by TREM2 variants that impair ligand binding, such as the p.R47H, p.R62H and p.D87N variants.
Supervisor: Hodges, Angela Kaye ; Killick, Richard Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.789134  DOI: Not available
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