The Role Of The Parkinson's Disease-Related Protein LRRK2 In Autophagy And Calcium Signalling In Cellular Models

Azeggagh, Sonia (2021). The Role Of The Parkinson's Disease-Related Protein LRRK2 In Autophagy And Calcium Signalling In Cellular Models. PhD thesis The Open University.



Parkinson’s disease (PD) is the second most common neurodegenerative disease worldwide, affecting 3% of individuals of >75 years of age. The disease is relentless and incurable, and the need to understand its causes and develop new treatments is overwhelming. Mutations in the LRRK2 gene are the most common cause of familial PD, and strongly influence the risk of sporadic PD. This study investigates the impact of LRRK2 enzymatic activities and LRRK2 pathogenic and protective variants on autophagy and calcium signalling to gain insight into the early impairments involved in PD pathogenesis. To this end, the initial approach was to develop isogenically matched cell lines using the CRISPR/Cas9 gene-editing technique, which was unsuccessful. Alternative strategies to study LRRK2 were therefore pursued. The LRRK2 kinase and GTPase activities were modulated with pharmacological inhibitors, and the effects of LRRK2 mutations were investigated by transfection of wild-type and mutant LRRK2 constructs. Autophagy assays combined the quantification of GFP-LC3 punctae and endogenous WIPI2 punctae numbers using fluorescence and electron microscopy, whilst intracellular calcium signals were measured using ratiometric Fura-2 imaging following treatment with ATP, CPA or ionomycin. Overall, this project revealed a cell-type specific action of LRRK2 in autophagy, where LRRK2 appears to be pro-autophagic and involved in a kinase and GTPase activity-dependent manner in the early stages of this process in HEK293 GFP-LC3 cells, whilst data from RAW264.7 macrophages do not support a role for Lrrk2 in autophagy. Calcium-related findings indicated that Lrrk2 is involved in the regulation of IP3-R mediated calcium signals in WT RAW264.7 macrophages and suggest that this role for Lrrk2 may be as a scaffolding protein. The data also indicated that the function of LRRK2 in autophagy induction in HEK293 GFP-LC3 cells is unlikely to require intracellular calcium, and LRRK2 was not found to participate in the regulation of lysosomal calcium storage in either cell line. Additional findings revealed that the calcium indicators Fura-2 and Cal-590, as well as BAPTA and the BAPTA analogues di-bromo-BAPTA and di-fluoro-BAPTA, displayed significant off-target effects on autophagy, and indicated that BAPTA is an unsuitable tool to explore the involvement of calcium signals in autophagy. Taken together, this study yields new and important data about the role of LRRK2 kinase and GTPase activities in the early stages of autophagy, and sheds light on the relevance of LRRK2 to the regulation of intracellular calcium signalling.

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