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The influence that these modifiers have on lysosomal GCase activity is typically characterized by using standard assays that use simple fluorogenic glucosides to measure its activity in tissue homogenates where lysosomes have been broken open. The regulation of lysosomal activity of GCase is accordingly not fully understood ( 16– 19). These proteins, and likely several others, act as modifiers that influence lysosomal GCase activity and in doing so influence the severity and penetrance of GD and PD ( 15, 16). Most notably, these factors include lysosomal integral membrane protein 2 (LIMP-2), which aids in trafficking GCase from the endoplasmic reticulum (ER) to lysosomes, and Saposin C, which modulates its lysosomal activity ( 12– 14). Studying GCase activity within lysosomes is complicated by cellular factors that regulate the activity of this enzyme. Nevertheless, the precise molecular mechanism by which loss of GCase activity within lysosomes contributes to these diseases remains a topic of intense interest ( 10, 11). A mechanistic role for loss of GCase activity contributing to PD pathogenesis is supported by observations that either chemical inhibition or loss of function mutations in GCase accelerates PD phenotypes ( 8, 9). Underscoring the importance of GCase function within brain, its dysfunction has also emerged as the most prevalent genetic risk factor for Parkinson disease (PD), with 5–10% of PD patients carrying a mutation in GBA ( 6, 7). Notably, GD patients show considerable phenotypic variability, particularly in the presence and severity of neuropathic symptoms, which are only weakly correlated to patient genotype ( 3– 5). The accumulation of these substrates is thought to drive development of the lysosomal storage disease known as Gaucher disease (GD) ( 2). Biallelic loss of function mutations in the gene GBA, which encodes GCase, lead to the accumulation of GlcCer and its downstream metabolite glucosyl sphingosine (GlcSph) within lysosomes of various tissues. The principle substrate of the lysosomal glycoside hydrolase β-glucocerebrosidase (GCase, CAZY family GH30) is the sphingolipid glucosylceramide (GlcCer) ( 1). Accordingly, analysis of clinical blood samples using LysoFQ-GBA may provide a surrogate marker of lysosomal GCase activity in neuronal tissue. 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. 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. LysoFQ-GBA is a sensitive tool for studying chemical or genetic perturbations of GCase activity using either fluorescence microscopy or flow cytometry. Here, we report a selective fluorescence-quenched substrate, LysoFQ-GBA, which enables measuring endogenous levels of lysosomal GCase activity within living cells. 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. Indeed, such factors are thought to contribute to the high degree of variable penetrance of GBA mutations among patients.

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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. 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.