GBA1 and Parkinson’s Disease: Development of a Comprehensive Assay Framework and its Application in Clinical Samples

Abstract

Parkinson’s disease (PD) is a neurodegenerative movement disorder that is becoming increasingly prevalent in ageing populations worldwide, yet there is no cure. The underlying disease mechanisms are still not fully understood, however, are thought to involve complex interactions between genetic susceptibility, environmental risk factors, and ageing. Among several pathogenic pathways proposed, lysosomal dysfunction has emerged as a central disease mechanism, closely linked to genetic variants in the PD-associated genes LRRK2 and GBA1. GBA1 encodes a lysosomal enzyme glucocerebrosidase (GCase), which is critical for maintaining lysosomal function and lipid homeostasis. Pathogenic loss-of-function GBA1 variants result in reduced GCase enzymatic activity leading to lysosomal dysfunction implicated in PD. As a result, GCase has emerged as a promising target for disease-modifying therapies as well as a useful readout for studying the underlying disease mechanisms. However, inconsistencies in GCase activity measurements, likely due to methodological differences, have led to conflicting results across many research studies, highlighting the need for more standardised analytical approaches.

To provide a comprehensive overview of the advantages and limitations of currently available methods for enzymatic GCase activity measurements, I compared plate reader, live cell imaging, and flow cytometry-based approaches, using various fluorescent probes and sample types. Each method was assessed for specificity, sensitivity, data reproducibility, and potential utility in a clinical setting. A flow cytometry assay was further adapted for the use in human PBMCs, with particular emphasis on refining multiple assay parameters and assessing the impact of cryopreservation on sample quality, ensuring reliable analysis of clinical sample collections.

A comprehensive evaluation of GCase functional profiles was performed in cryopreserved PBMCs derived from PD patients with heterozygous GBA1 variants, including T408M, E365K, and L483P as well as healthy controls. The optimised flow cytometry GCase activity assay revealed a significant reduction in lysosomal GCase activity in severe L483P variant carrier group, which was not observed by the plate reader assay measuring total cellular GCase activity, highlighting the importance of methodological choice. GBA1 protein abundance and maturation status were also evaluated to provide additional context for the observed mutation-specific changes in GCase activity. The results were further validated in HEK293-FT landing pad cell lines, which supported the clinical findings, however, also highlighted potential limitations of the flow cytometry based GBA1 protein quantification technique.

GBA1 and LRRK2 are both associated with lysosomal impairment in PD, which suggests a potential functional interplay. To explore this further, GCase activity was measured in mouse embryonic fibroblasts (MEFs) with LRRK2 kinase activating mutations, including LRRK2 G2019S, R1441C, and VPS35 D620N, by both plate reader and flow cytometry assays. The findings revealed an inverse relationship between LRRK2 kinase activity and GCase function, however, the observed effect was assay dependent. The effects of LRRK2 kinase activity on GCase function were also assessed in cryopreserved PBMCs derived from PD patients with heterozygous LRRK2 kinase activating variants, including LRRK2 G2019S, Y1699C, and VPS35 D620N, however, no clear association was observed, likely due to small sample size, interindividual variability, and heterozygous carrier status.

Together, this project presents a systematic overview of several GCase activity measurement methods currently used in the field, providing new insights into how methodological differences may influence experimental outcomes. GCase functional profiling in clinical samples demonstrated the suitability of the optimised flow cytometry assay for the analysis of human biosamples and advanced the understanding of the effects of GBA1 variants on lysosomal function in PD.

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