Background

Glucocerebrosidase (GCase), encoded by the GBA1 gene, is a lysosomal hydrolase that converts glucosylceramide to glucose and ceramide. Loss of function homozygous, and compound heterozygous missense mutations in GBA1 are associated with the accumulation of glucosylceramide and the lysosomal storage disorder Gaucher’s disease (Sidransky, 2012). Whereas heterozygous mutations in GBA1 are associated with increased risk of developing the neurodegenerative movement disorder Parkinson’s disease (PD) (Sidransky and Lopez, 2012). While GBA1 mutations occur in approximately 5-15% of PD cases (Neumann et al., 2009; Sidransky et al., 2009; Lesage et al., 2011), reduced GCase activity is also observed in PD patients without GBA1 mutations (Murphy et al., 2014; Alcalay et al., 2015; Atashrazm et al., 2018), and there is increasing evidence supporting the notion that restoring GCase function is a valid therapeutic option for PD (Schapira 2015; Sardi et al., 2018). Subsequently, a number of small molecule chaperones have been described that can restore GCase function (Khanna et al., 2010 and 2012; Patnaik et al., 2012; McNeill et al., 2014).

The assessment of GCase activity in PD patients can help inform which patients may benefit from such therapies, by identifying those with reduced enzyme activity. Assessment of GCase activity could also be used to gauge the efficacy of proposed therapies to restore GCase function. The efficacy of chaperone-based therapies for restoring GCase function have predominantly been assessed by measuring changes in GCase protein by immunoblotting, or by indirectly measuring GCase activity using mass spectrometry-based assays. Although these approaches have proven useful and do have advantages (Wolf et al., 2018), they are also limited in their ability to distinguish between blood cell types, assay GCase activity in intact living cells, and are potentially less clinically amenable than other techniques. Another current approach to measuring GCase activity involves the use of the non-natural substrate 4-MUG (4-Methylumbelliferyl β-D-galactopyranoside), which when cleaved by GCase produces the fluorescent product 4-methylumbelliferon (4-MU). However, 4-MUG is also a substrate for non-lysosomal GCase (encoded by GBA2) (Boot et al., 2007) requiring the purification of lysosomes or performing the assay at distinguishing pH to add specificity.

The current protocol presented here utilises a selective lysosomal GCase substrate 5-(Pentafluorobenzoylamino) Fluorescein Di-beta-D-Glucopyranoside (PFB-FDGlu), which is metabolised by GCase to yield fluorescein. PFB-FDGlu is cell permeable and can be used with a flow cytometer to measure GCase activity in living cells on a single-cell basis. This protocol has been adapted from earlier studies describing and validating the use of PFB-FDGlu for assessing GCase activity, primarily in the context of Gaucher’s disease (Lorincz et al., 1997; van Es et al., 1997; Rudensky et al., 2003). The ability to combine PFB-FDGlu with other phenotypic indicators, such as a viability stain and antibodies to label cell surface markers is advantageous, as it enables identification of distinct target cell populations in which GCase activity may be modulated.