Dissertation

Compound Screening in a TREM2-Deficient Microglial Cell Model

Rutter, Hannah

2020

Overview

The identification of rare heterozygous TREM2 gene variants that increase the susceptibility of developing sporadic Alzheimer’s disease has reinvigorated research into the contribution of neuroinflammation to Alzheimer’s disease pathology and identified the TREM2 signalling pathway as a novel therapeutic target. The aim of this thesis was to identify TREM2-dependent phenotypes that could be used to identify tool compounds capable of restoring the normal function of TREM2 in the absence of functional TREM2 protein. Any identified tool compounds may serve to further elucidate the function of TREM2 in healthy and diseased states, and identify novel downstream therapeutic targets for the treatment of AD. To achieve this aim compounds with the greatest potential of directly or indirectly targeting the TREM2 signalling pathway were selected, primarily, using SPIEDw to match correlated patterns of gene expression within the connectivity map database. Then, the structure and function of our CRISPR TREM2 knockout BV2 microglial cell lines were characterised to identify TREM2-dependent phenotypes that could be utilised for the development of compound screening assays. Finally, the selected compounds were screened in the developed phenotypic assays to identify tool compounds capable of restoring the normal function of TREM2 in the absence of functional TREM2 protein. Characterisation of the CRISPR TREM2 knockout cells showed that the TREM2-deficient cells possessed significantly shorter and fewer microglial processes, and significantly up-regulated uptake of the Toll-like receptor 2 agonist zymosan, relative to TREM2 wild-type cells. Though it has been well characterised that TREM2 regulates TLR-mediated pro-inflammatory cytokine production, the effect of TREM2 on TLR-mediated uptake has not been investigated previously. The identified uptake phenotype was then utilised as the outcome measure for the primary compound screening assay. During the screen, four hit compounds capable of inhibiting the uptake of zymosan in TREM2 knockout cells, whilst having no effect on TREM2 wild-type, were identified. Then, in a secondary compound screen aimed at promoting the formation of microglial processes in the TREM2 knockout cells, two of the hit compounds from the primary screen also significantly increased the formation of microglial processes in TREM2 knockout. Based upon what is known about the pharmacological activity of these two compounds, it was speculated that they were likely mediating their functions through inhibiting the phosphorylation of ERK. Further investigations may determine that these compounds are suitable tools to further investigate the function of TREM2 in other model systems.

• Publisher: ProQuest Dissertations & Theses