4.8 Article

A versatile fluorescence-quenched substrate for quantitative measurement of glucocerebrosidase activity within live cells

Publisher

NATL ACAD SCIENCES
DOI: 10.1073/pnas.2200553119

Keywords

Parkinson disease; glycoside hydrolase; enzyme kinetics; fluorescence-quenched substrate; flow cytometry

Funding

  1. Natural Sciences and Engineering Research Council (NSERC) of Canada [RGPIN-05426]
  2. Michael J. Fox Foundation (MJFF) for Parkinson Research [16536, 14249]
  3. Canada Research Chairs program
  4. NSERC PGS-D scholarship
  5. Canada Foundation for Innovation grant [38026]
  6. Canadian Glycomics Network [RG-1]

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In this study, a selective fluorescence-quenched substrate, LysoFQ-GBA, was developed to measure the levels of lysosomal GCase activity in living cells. The substrate can be used with fluorescence microscopy or flow cytometry to study the effects of chemical or genetic perturbations on GCase activity. The measurements made using LysoFQ-GBA were validated with different cell types and were shown to accurately report on GCase inhibitor target engagement and the GBA allele status of cells.
Loss of activity of the lysosomal glycosidase beta-glucocerebrosidase (GCase) causes the lysosomal storage disease Gaucher disease (GD) and has emerged as the greatest genetic risk factor for the development of both Parkinson disease (PD) and dementia with Lewy bodies. There is significant interest into how GCase dysfunction contributes to these diseases, however, progress toward a full understanding is complicated by presence of endogenous cellular factors that influence lysosomal GCase activity. Indeed, such factors are thought to contribute to the high degree of variable penetrance of GBA mutations among patients. Robust methods to quantitatively measure GCase activity within lysosomes are therefore needed to advance research in this area, as well as to develop clinical assays to monitor disease progression and assess GCase-directed therapeutics. Here, we report a selective fluorescence-quenched substrate, LysoFQ-GBA, which enables measuring endogenous levels of lysosomal GCase activity within living cells. LysoFQ-GBA is a sensitive tool for studying chemical or genetic perturbations of GCase activity using either fluorescence microscopy or flow cytometry. We validate the quantitative nature of measurements made with LysoFQ-GBA using various cell types and demonstrate that it accurately reports on both target engagement by GCase inhibitors and the GBA allele status of cells. Furthermore, through comparisons of GD, PD, and control patient-derived tissues, we show there is a close correlation in the lysosomal GCase activity within monocytes, neuronal progenitor cells, and neurons. Accordingly, analysis of clinical blood samples using LysoFQ-GBA may provide a surrogate marker of lysosomal GCase activity in neuronal tissue.

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