Nazish, Iqra; (2020) Investigating Leucine-rich repeat kinase 2 (LRRK2) function: implications for cellular immune responses. Doctoral thesis (Ph.D), UCL (University College London).

Preview

Text Nazish_10118169_thesis_sig_removed.pdf Download (10MB) | Preview

Abstract

Mutations in the Leucine-rich repeat kinase 2 (LRRK2) gene are the most common cause of familial and sporadic Parkinson’s disease. LRRK2 protein is expressed prominently in immune cells, cell types whose contribution to LRRK2-associated Parkinson’s disease is increasingly being recognised. For my PhD, I investigated the effect of inflammatory stimulation using murine RAW264.7 wild-type, T1348N-LRRK2 (artificial mutation which abrogates GTP binding) and LRRK2 knockout macrophage cell lines and wild-type and G2019S-LRRK2 (natural most common PD mutation) primary microglia, as model systems. Immunoblotting and ELISA-based assays were used to investigate a detailed time course of TLR2/TLR4 stimulation with LPS and zymosan by studying LRRK2 phosphorylation at its specific phosphorylation sites, Rab and MAPK proteins and by measuring cytokine release in RAW264.7 macrophages and primary microglia. Four selective LRRK2 kinase inhibitors were also employed to understand LRRK2’s role in immune cell function and finally some bioinformatics work was performed to identify differentially expressed genes using RNA Sequencing. The latter part of my work was conducted at National Institutes of Health, USA. Data presented in this thesis is over three experimental chapters with first two chapters investigating LRRK2 functionality in terms of phosphorylation and cytokine release in RAW264.7 macrophages and the final chapter investigating it in terms of phosphorylation, cytokine release and transcriptomics work in primary microglia. RAW264.7 work confirmed Rab8 and Rab10 as substrates of LRRK2 activity with a differential response of dephosphorylated Rab10 seen with LPS stimulation. Loss of GTP binding due to T1348N and LRRK2 knockout coupled with LRRK2 kinase inhibitors and different inflammatory stimuli exhibited distinct responses in cytokine secretion, particularly with IL-10. Primary microglia work with zymosan and LRRK2 kinase inhibition also revealed varying patterns in cytokine secretion and transcriptomics work identified a wide range of differentially expressed genes with various enriched biological processes. Overall, my data demonstrates for the first time the dynamics observed in LRRK2 and Rab phosphorylation and cytokine release under different experimental conditions in these cellular models. This provides further insight into the function of LRRK2 in immune response and has relevance to understanding cellular dysfunctions when developing LRRK2 based inhibitors for clinical treatment of Parkinson’s disease.

Download activity - last month

Export as JSONXMLCSV

Download activity - last 12 months

Export as CSVJSONXML

Downloads by country - last 12 months

Export as CSVJSONXML

Archive Staff Only

View Item