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

Abstract

Loss of activity of the lysosomal glycosidase β-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.

LRRK2 Kinase Activity Regulates Parkinson’s Disease-Relevant Lipids at the Lysosome

“…We employed a recently-described activity-based probe to measure lysosomal GCase activity (LysoFQ-GBA) that has superior signal to noise and retention in lysosomes compared to other probes tested to address this question (44). We confirmed that LysoFQ-GBA specifically detected GCase signal as no detectable signal was observed in GBA1 KO A549 cells (Fig.…”

“…A clear understanding of the relationship between LRRK2 and lysosomal GCase activity has been hindered by limitations in existing methods to measure GCase activity with respect to sensitivity, selectivity, and spatial resolution. We employed a recently-described activity-based probe to measure lysosomal GCase activity (LysoFQ-GBA) that has superior signal to noise and retention in lysosomes compared to other probes tested to address this question (44). We confirmed that LysoFQ-GBA specifically detected GCase signal as no detectable signal was observed in GBA1 KO A549 cells (Fig.…”

“…Our data demonstrate that LRRK2 exerts its effects on glycosphingolipid levels by modulating GCase activity within lysosomes, and we propose that reduced GCase activity may be a primary driver of LRRK2-dependent lysosomal dysfunction. Using a recently-described activity-based probe to measure lysosomal GCase activity, we observed an approximately 50% decrease in lysosomal GCase activity in LRRK2 variant cell models, including iMicroglia(44). We established that this reduction in GCase activity correlated with accumulation of GCase substrates and BMP in lysosomes and with defects in lysosomal proteolysis in cellular models expressing disease-associated LRRK2 variants.…”

“…LysoFQ-GBA was recently described and obtained from the laboratory of David Vocadlo (44). Briefly, cells were seeded into 96-well PDL-coated plates.…”

Targeting neuronal lysosomal dysfunction caused by β-glucocerebrosidase deficiency with an enzyme-based brain shuttle construct

“…We next exploited a GCase-specific fluorescence-quenched substrate LysoFQ-GBA 29 to measure increase in lysosomal GCase activity in neurons. Cell imaging revealed that treating Gba -deficient immortalised murine cortical neurons for 2 h with mGCase or mGCase-mBS could restore lysosomal GCase activity to WT levels.…”