From genetic predisposition to biochemical validation

: investigating the role of LRRK2 signalling network in Parkinson’s Disease

Abstract

Affecting millions worldwide, Parkinson’s disease (PD) remains one of the most common neurodegenerative movement disorders and currently lacks a definitive cure. While the majority PD cases are idiopathic, a subset can now be attributed to rare pathogenic variants in a defined set of genes (e.g. LRRK2, PRKN, SNCA), however these explain only a small fraction of hereditary cases, for other cases with a predicted genetic component, the underlying genetic basis remains unresolved. Pathogenic missense variants in leucine-rich repeat kinase 2 (LRRK2) (e.g., G2019S, R1441G/C and Y1699C) represent a leading cause for monogenetic forms of PD. These variants enhance LRRK2 kinase activity and result in hyperphosphorylation of its endogenous substrates, Rab GTPases, including Rab10. To date, studies have reported >200 LRRK2 variants in PD patients with majority of them classified as variants of uncertain significance (VUS), with functional data lacking. Defining their pathogenic potential is essential, not only in cellular systems but also in patients to guide clinical interpretation and therapeutic targeting.
As such, Chapter 3 of this thesis examined whether LRRK2 kinase activation induced by LRRK2 variants in cellular overexpression systems could also be detected in patient-derived neutrophils and monocytes in patients carrying these variants. This work built on ongoing functional studies in Dr. Esther Sammler’s laboratory where now over 350 LRRK2 variants have been systematically evaluated using a robust cellular overexpression assay in HEK293 cells to identify 64 novel LRRK2 kinase activating variants as measured by enhanced LRRK2-dependent Rab10 phosphorylation (including 23 previously published). This chapter aimed to extend those findings into patient samples. Through collaborations with PD experts across Europe patients carrying pathogenic LRRK2 variants and rare VUS were recruited, and fresh blood was collected for neutrophil and monocyte isolations. Strongly activating LRRK2 variants such as Y1699C and I2020T produced a corresponding increase in Rab10 phosphorylation in both cellular assays and patient-derived samples. In contrast, variants with milder activation in cellular assays including LRRK2 R1325Q and G2019S did not demonstrate a detectable phenotype in patient neutrophil and monocytes. Additionally, integration of genetic, structural and functional data enabled reclassification of rare LRRK2 variants V1447L and F1700L to likely pathogenic and pathogenic, respectively. This framework underscored the value of combining cellular and patient-derived analyses for LRRK2 variant interpretation which in the future will have direct implications for genetic counselling and patient stratification.
Emerging evidence suggests that LRRK2 plays an important role in PD beyond that driven by rare LRRK2 variants. This is most evident in the case of VPS35 D620N, a pathogenic PD variant that has been previously demonstrated to lead to the activation of LRRK2 signalling pathway. To address this hypothesis the next chapter examined LRRK2 kinase activation status in a cohort of 181 PD patients with suspected genetic predisposition (early PD onset and/or family history) as well as healthy controls. PD patients were stratified by their LRRK2 kinase activity by measuring LRRK2-dependent Rab10Thr73 phosphorylation in peripheral blood neutrophils and monocytes via quantitative immunoblot assay. Elevated LRRK2 kinase activity was observed in 26 patients in whom genetic testing did not identify any known pathogenic PD variants. For selected individuals, next generation sequencing data was reviewed to identify rare genetic variants potentially contributing to LRRK2 kinase pathway activation in these patients and a strategy for prioritizing variants for functional validation was outlined. Finally, the stability of phosphorylated Rab10 levels was assessed longitudinally in neutrophils and monocytes of three healthy individuals revealing a degree of technical variability in Rab10 phosphorylation and emphasizing the importance of repeated independent sampling for validation.
Next the aim was to functionally validate prioritized candidate genes to examine whether they underlie LRRK2 kinase activation observed in PD patients harbouring these variants. Firstly, a knock-in A549 cell line was generated with a known PD-causing and LRRK2-activating mutation, VPS35 D620N, to validate CRISPR/Cas9-mediated gene editing as a suitable complimentary tool for functional validation. Next, functional validation of variants in PPM1M, a phosphatase known to dephosphorylate Rab10, identified several phosphatase inactivating variants including PPM1M D440N, for which there was sufficient evidence to associate it with an increased risk of PD. In contrast, variants in SORL1 and WASH complex component, WASHC4, could not be attributed to LRRK2 kinase activation in patients carrying these variants. Finally, an siRNA screen targeting genes with rare variants in four top candidate patients revealed several interesting hits including, NECAP2 and EIF5A2, whose abolished expression led to an increase in LRRK2-dependent Rab10 phosphorylation suggesting their potential as novel regulators of the LRRK2 signalling pathway.
Overall, research outlined in this thesis demonstrated how integrating genetics, functional biomarker assays in patient-derived samples, and cell-based models can enhance our understanding of LRRK2’s role in genetically predisposed PD. This will not only improve patient stratification for clinical trials but also be critical for advancing the understanding of PD pathogenesis and development of novel therapeutic strategies.

Date of Award	2026
Original language	English
Awarding Institution	University of Dundee
Supervisor	Esther Sammler (Supervisor) & Dario Alessi (Supervisor)