Objective: The objective of this project is to investigate the effect of Leucine Rich-Repeat Kinase 2 (LRRK2) dysfunction in the signalling mechanisms in macrophages and the pathological processes in Parkinson’s disease (PD).

Background: Mutations in the LRRK2 gene constitute the highest genetic risk factor for early-onset PD. LRRK2 is expressed strongly in macrophages and microglia indicating a link between LRRK2 and inflammation playing a critical role in PD neuropathology. However, there is still ambiguity in how LRRK2 dysfunction causes PD. The two LRRK2 kinase and GTPase enzymatic domains are highly druggable targets for potential PD therapies. LRRK2 plays a wide range of cellular functions via different molecular mechanisms within the immune system which needs to be fully deciphered to understand LRRK2-related PD pathogenesis.

Method: We investigated LRRK2 phosphorylation dynamics in RAW264.7 macrophage cell lines (wild-type, T1348N-LRRK2 (artificial mutation that ablates GTP binding) and KO-LRRK2) following stimulation with lipopolysaccharide (LPS) and zymosan and pharmacologically inhibiting with four specific LRRK2 kinase inhibitors, GNE-7915, GNE-9605, MLi-2 and PF-06447475. LRRK2 phosphorylation was monitored at specific phosphorylation sites (Ser935, Ser955 and Ser910) using standard immunoblot procedures and secreted cytokines (Tumour Necrosis Factor-α (TNF-α), interleukin-6 (IL-6) and interleukin-10 (IL-10)) were measured using Enzyme-linked Immunosorbent Assay (ELISA). ANOVA and T-test with Graph-PAD prism was used to analyse the statistically significant differences.

Results: In wild-type cells, we observed a significant effect of both LPS (100mg/ml) and zymosan (200µg/ml) in phosphorylating LRRK2 at Ser935 and Ser955 in both dose (50 – 500ng/ml for LPS and 50 – 500µg/ml for zymosan) and time (2h – 24h) dependent manners with the four LRRK2 kinase inhibitors reversing the effect of LPS. Furthermore, there is an aberrant basal secretion of TNFα, IL-6 and IL-10 in the modified cells.

Conclusion: LPS and zymosan significantly upregulate LRRK2 phosphorylation in wild-type cells with LPS comparatively showing a more sustained upregulation over time. All four specific LRRK2 kinase inhibitors downregulated phosphorylation at Ser935 and Rab8 which are readouts of LRRK2 kinase activity. Our data of aberrant cytokine basal secretion in modified cells indicate the involvement of LRRK2 and inhibitors in the immune processes of macrophages.

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MDS Abstracts - https://www.mdsabstracts.org/abstract/investigating-the-cellular-role-of-lrrk2-in-the-immune-system/