Fig 4 - uploaded by Matthew Lorincz
Content may be subject to copyright.
Source publication
Gaucher disease type 1 results from the accumulation of glucocerebroside in macrophages of the reticuloendothelial system, as a consequence of a deficiency in glucocerebrosidase (GC) activity. Recent improvements in the methodologies for introducing foreign genes into bone marrow stem cells have prompted several groups to test the efficacy of gene...
Context in source publication
Context 1
... the presence of PFBFDGlu and the inhibitor CBE, both staining. A MoAb recognizing CD14, a marker present on patient and control samples show similar fluorescence distri- the majority of myeloid-lineage cells in peripheral blood, 27,28 butions (Fig 4), suggesting that the remaining fluorescence stained greater than 95% of the large, fluorescein bright is due to hydrolytic activity independent of GC, or to a mononuclear cells (data not shown). These results indicate fluorescent contaminant in the substrate preparation, or both. that CD14 / cells have higher GC activity, and/or higher PBMC were obtained from a total of six unrelated Gaucher endocytic capacity than lymphocytes, both properties that type 1 patients and seven controls, and analyzed by flow have previously been reported for monocytes. 29,30 Addition cytometry in four independent experiments (Fig 5). Gener- of chloroquine 20 minutes before the addition of PFBFDGlu ally, freshly isolated PBMC from two patient samples and inhibited the hydrolytic activity detected in the CD14 / popu- a minimum of two control samples were analyzed in each experiment. By subtracting the MF of the monocytes incu- lation approximately 10-fold (data not shown), indicating AID Blood 0033 / 5h34$$$642 04-01-97 13:33:59 blda WBS: Blood with the MUGlu lysate assay conducted in parallel in the presence of the GC ''activator'' TC, in which both Gaucher patients showed õ10% of the activity of normal control PBMC (data not shown). This inconsistency may be explained by the fact that the level of residual GC activity is highly dependent on the conditions used in the lysate assay. 22,31,32 As the FACS assay is conducted with living cells, the residual activity detected may be more physiologi- cally relevant than that determined in the presence of the various activators and inhibitors used in the lysate ...
Similar publications
Retroviral gene transfer has been used successfully to correct the glucocerebrosidase (GCase) deficiency in primary hematopoietic cells from patients with Gaucher disease. For this model of somatic gene therapy, we developed a high-titer, amphotropic retroviral vector designated NTG in which the human GCase gene was driven by the mutant polyoma vir...
Citations
... One way to assess the activity of GCase in situ in the lysosome in cellular models is with the selective GCase substrate 5-(pentafluorobenzoylamino)fluorescein di-D-glucopyranoside (PFB-FDGlu). The PFB-FDGlu probe enters the cell via pinocytosis and travels to the lysosome, where it is metabolized by GCase to release fluorescein (Lorincz et al., 1997). The fluorescence intensity of fluorescein as measured by flow cytometry has been used to assess GCase activity in peripheral immune cells of patients with Gaucher disease (Rudensky et al., 2003), a lysosomal storage disease caused by homozygous mutations in GBA1 (Hruska et al., 2008;Riboldi and Di Fonzo, 2019), as well as to demonstrate that GCase activity is reduced in monocytes from patients with PD (Atashrazm et al., 2018;Hughes et al., 2021;Wallings et al., 2022). ...
Introduction
Heterozygous mutations in GBA1 , which encodes the lysosomal hydrolase glucocerebrosidase (GCase), are a common risk factor for the neurodegenerative movement disorder Parkinson's disease (PD). Consequently, therapeutic options targeting the GCase enzyme are in development. An important aspect of this development is determining the effect of potential modifying compounds on GCase activity, which can be complicated by the different methods and substrate probes that are commonly employed for this purpose.
Methods
In this study, we employed the GCase substrate probe 5-(pentafluorobenzoylamino)fluorescein di-D-glucopyranoside (PFB-FDGlu) in combination with live cell imaging to measure GCase activity in situ in the lysosome.
Results
The live cell assay was validated using the GCase inhibitor conduritol-B-epoxide and with GBA1 knockout neural cells and was then used to assess GCase activity in iPSC differentiated into neural stem cells and neurons that were obtained from idiopathic PD patients and PD patients with the LRRK2 G2019S and GBA N370S mutations, as well as controls ( n = 4 per group). Heterogeneity in GCase activity was observed across all groups. However, a significant inverse correlation between GCase activity and levels of alpha-synuclein protein was observed.
Discussion
The live cell imaging assay for GCase activity could be useful for further understanding the role of GCase in PD and screening potential modifying compounds in differentiated human cell models.
... We were inspired by previous work on fluorogenic substrates that enable detecting enzyme activity directly within cells and tissues. [23][24][25][26][27][28][29] We recognized, however, that most of these substrates lead to products that rapidly diffuse out of the cell. This undesirable diffusion leads to the loss of both spatial information regarding the enzymatic activity and the accuracy of activity measurements. ...
Fluorogenic substrates are emerging tools that enable studying enzymatic processes within their native cellular environments. However, fluorogenic substrates that function within live cells are generally incompatible with cellular fixation, preventing their tandem application with fundamental cell biology methods such as immunocytochemistry. Here we report a simple approach to enable the chemical fixation of a dark‐to‐light substrate, LysoFix‐GBA, which enables quantification of glucocerebrosidase (GCase) activity in both live and fixed cells. LysoFix‐GBA enables measuring responses to both chemical and genetic perturbations to lysosomal GCase activity. Further, LysoFix‐GBA permits simple multiplexed co‐localization studies of GCase activity with subcellular protein markers. This tool will aid studying the role of GCase activity in Parkinson's Disease, creating new therapeutic approaches targeting the GCase pathway. This approach also lays the foundation for an approach to create fixable substrates for other lysosomal enzymes.
... The first factor is that because it is functionalized with two glucose residues, it produces two distinct enzymatic products that have different photophysical properties, leading to complex kinetics (27,28). The second factor is that the fluorescence intensity of fluorescein is highly pH-dependent due its ionization within a physiological range (pK a = 6.4), such that variations in lysosomal acidity impact the accuracy of measurements (29). Finally, and perhaps most problematic, is that PFB-FDGlu is not selective for GCase and in cell lines expressing other β-glucosidases, including neurons, complicated subtractive assays are needed (25,30). ...
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.
... 4-methylumbelliferone (4-MU) [17,18], and C12glucocerebroside, which is hydrolyzed to C12 ceramide and quantified by mass spectrometry [19,20]. However, the cell permeable quenched substrate PFB-FDGlu, which is hydrolyzed by GCase to release fluorescein, has an advantage over other substrates in that it can be used to directly assess GCase activity in situ in living single cells [21,22]. Using the PFB-FDGlu substrate in combination with flow cytometry and surface marker antibodies to delineate PBMC types, we have previously shown that GCase activity is specifically reduced in monocytes from PD patients [15]. ...
Background:
Reduced activity of lysosomal glucocerebrosidase is found in brain tissue from Parkinson's disease patients. Glucocerebrosidase is also highly expressed in peripheral blood monocytes where its activity is decreased in Parkinson's disease patients, even in the absence of GBA mutation.
Objective:
To measure glucocerebrosidase activity in cryopreserved peripheral blood monocytes from 30 Parkinson's disease patients and 30 matched controls and identify any clinical correlation with disease severity.
Methods:
Flow cytometry was used to measure lysosomal glucocerebrosidase activity in total, classical, intermediate, and non-classical monocytes. All participants underwent neurological examination and motor severity was assessed by the Movement Disorders Society Unified Parkinson's Disease Rating Scale.
Results:
Glucocerebrosidase activity was significantly reduced in the total and classical monocyte populations from the Parkinson's disease patients compared to controls. GCase activity in classical monocytes was inversely correlated to motor symptom severity.
Conclusion:
Significant differences in monocyte glucocerebrosidase activity can be detected in Parkinson's disease patients using cryopreserved mononuclear cells and monocyte GCase activity correlated with motor features of disease. Being able to use cryopreserved cells will facilitate the larger multi-site trials needed to validate monocyte GCase activity as a Parkinson's disease biomarker.
... We have previously found that these neurons faithfully recapitulate PD disease phenotypes 19,26 . Lysosomal GCase activity in live cells was measured using the fluorescent quenched substrate PFB-FDGlu that enables real-time analysis of lysosome-specific GCase activity 27 , unlike traditional approaches which measure activity in lysed cells. Using this approach, we examined the effects of LRRK2 G2019S mutations on GCase activity in mutant versus control DA neurons and observed a significant reduction in GCase activity in two independent LRRK2 G2019S iPSC neuronal lines relative to two distinct healthy controls (Fig. 1a). ...
Mutations in LRRK2 and GBA1 are common genetic risk factors for Parkinson's disease (PD) and major efforts are underway to develop new therapeutics that target LRRK2 or glucocerebrosidase (GCase). Here we describe a mechanistic and therapeutic convergence of LRRK2 and GCase in neurons derived from patients with PD. We find that GCase activity was reduced in dopaminergic (DA) neurons derived from PD patients with LRRK2 mutations. Inhibition of LRRK2 kinase activity results in increased GCase activity in DA neurons with either LRRK2 or GBA1 mutations. This increase is sufficient to partially rescue accumulation of oxidized dopamine and alpha-synuclein in PD patient neurons. We have identified the LRRK2 substrate Rab10 as a key mediator of LRRK2 regulation of GCase activity. Together, these results suggest an important role of mutant LRRK2 as a negative regulator of lysosomal GCase activity.
... Molecular probe 69 is also commonly used in ELISA for detecting βgalactosidase positive cells using fluorescence microscopy 167 and flow cytometry. 146,147 Furthermore, this reagent has been successfully utilized to detect the recombinant herpesvirus infected cells 148 for identifying the unique patterns of βgalactosidase expression in live transgenic zebrafish embryos 149 and also for studying the β-galactosidase expression in bacteria. 150,151 This similar ideology was exploited for developing several molecular probes (70−72) for β-galactosidase for commercial exploitation. ...
All cellular processes are the results of synchronized actions of several intracellular biochemical pathways. Recent emphasis is to visualize such pathways using appropriate small molecular reagents, dye-labeled proteins, and genetically encoded fluorescent biosensors that produce a luminescence ON response either on selective binding or on reacting with an analyte that is produced through a specific biochemical/enzymatic transformation. Studying such enzymatic processes by probing the fluorescence response as the read-out signal is expected to provide important insights into crucial biochemical transformations induced by an enzyme in its native form. Many of such studies are extended for monitoring enzymatic transformations under in vitro or in vivo condition. A few of the recent reports reveal that such molecular probes are even capable of quantifying abnormal levels of enzymes in real-time and is linked to the key area of clinical diagnostics and chemical biology. A synchronized analysis of all such reports helps in developing a rationale for designing purpose-built molecular probes or chemodosimeters as well as newer reagents for studying crucial enzymatic process or quantification of the respective enzyme. In this review, an attempt will be there to highlight several recent bioimaging reagents and studies that have provided insights into crucial biochemical or enzymatic transformations.
... Flow cytometry measurement of GCase activity. GCase enzymatic activity was measured using a flow cytometry based assay, similar to assays used previously to assess GCase activity, including in samples from patients with Gaucher's disease 29,[31][32][33][34] incubation, 1 mL of ice-cold flow buffer (1 X PBS, 1 mM EDTA, 25 mM HEPES, 1% heat inactivated FBS, pH 7.0) was added and the cells pelleted by centrifugation at 350 × g for 5 min. Cells were then resuspended in flow buffer and stained with anti-CD14 monoclonal antibody conjugated to PEcy7, anti-CD3 monoclonal antibody conjugated to V450, and anti-CD19 monoclonal antibody conjugated to APC (BD Biosciences) for 30 min at 4 °C. ...
Abstract Missense mutations in glucocerebrosidase (GBA1) that impair the activity of the encoded lysosomal lipid metabolism enzyme (GCase) are linked to an increased risk of Parkinson’s disease. However, reduced GCase activity is also found in brain tissue from Parkinson’s disease patients without GBA1 mutations, implicating GCase dysfunction in the more common idiopathic form of Parkinson’s disease. GCase is very highly expressed in monocytes, and thus we measured GCase activity in blood samples from recently diagnosed Parkinson’s disease patients. Flow cytometry and immunoblotting assays were used to measure levels of GCase activity and protein in monocytes and lymphocytes from patients with Parkinson’s disease (n = 48) and matched controls (n = 44). Gene sequencing was performed to screen participants for GBA1 missense mutations. In the Parkinson’s disease patients, GCase activity was significantly reduced in monocytes, but not lymphocytes, compared to controls, even when GBA1 mutation carriers were excluded. Monocyte GCase activity correlated with plasma ceramide levels in the Parkinson’s disease patients. Our results add to evidence for GCase dysfunction in idiopathic Parkinson’s disease and warrant further work to determine if monocyte GCase activity associates with Parkinson’s disease progression.
... and -Galactosidase-After incubation with  2 m samples for 24 h, cells were washed and incubated with either 58 M 5-(pentafluorobenzoylamino)fluorescein di--D-glucopyranoside (Invitrogen) or 33 M 5-dodecanoylaminofluorescein di--D-galactopyranoside (C 12 FDG, Invitrogen) for 1 h at 37°C. -glucocerebrosidase cleaves the non-fluorescent substrate 5-(pentafluorobenzoylamino)fluorescein di--D-glucopyranoside to yield the green-fluorescent 5-(pentafluorobenzoylamino)fluorescein dye, whereas -galactosidase cleaves the nonfluorescent substrate C 12 FDG to yield fluorescent fluorescein that can be detected by flow cytometry (37,38). As a control, cells were preincubated for 1 h with 1 M conduritol B epoxide (Sigma-Aldrich) and 1 mM phenylethyl -D-thiogalactopyranoside (Invitrogen) to inhibit the activities of -glucocerebrosidase and -galactosidase, respectively (39,40). ...
... To determine whether the activity of other lysosomal hydrolases was affected by fragmented  2 m fibrils, the cellular activities of the glycosidases -glucocerebrosidase and -galactosidase were assayed by flow cytometry using the substrates 5-(pentafluorobenzoylamino)fluorescein di--D-glucopyranoside and C 12 FDG, respectively (37,38). The -glucocerebrosidase inhibitor conduritol B epoxide, but not fragmented  2 m fibrils, caused a significant reduction in the hydrolysis of 5-(pentafluorobenzoylamino)fluorescein di--D-glucopyranoside by SH-SY5Y cells (Fig. 10A). ...
Fragmentation of amyloid fibrils produces fibrils that are reduced in length but have an otherwise unchanged molecular architecture.
The resultant nanoscale fibril particles inhibit the cellular reduction of the tetrazolium dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT), a substrate commonly used to measure cell viability, to a greater extent than unfragmented fibrils. Here we
show that the internalization of β2-microglobulin (β2m) amyloid fibrils is dependent on fibril length, with fragmented fibrils being more efficiently internalized by cells. Correspondingly,
inhibiting the internalization of fragmented β2m fibrils rescued cellular MTT reduction. Incubation of cells with fragmented β2m fibrils did not, however, cause cell death. Instead, fragmented β2m fibrils accumulate in lysosomes, alter the trafficking of lysosomal membrane proteins, and inhibit the degradation of a
model protein substrate by lysosomes. These findings suggest that nanoscale fibrils formed early during amyloid assembly reactions
or by the fragmentation of longer fibrils could play a role in amyloid disease by disrupting protein degradation by lysosomes
and trafficking in the endolysosomal pathway.
... GBA activity was measured in monocytes, lymphocytes, granulocytes, CD34 + cells and MSCs with PFB-FDGlu (59pentafluorobenzoylaminofluorescein-di-b-D-glucoside) as substrate. Fluorescence was measured on the FL-1 emission channel by flow cytometry as previously described [16,17,18]. Results were expressed as an index defined as the ratio between median fluorescence intensity (MFI) of cells incubated with PFB-FDGlu alone and median fluorescence intensity of cells incubated with CBE and PFB-FDGlu. ...
Gaucher disease (GD) is an autosomal recessive disorder characterized by lysosomal glucocerebrosidase (GBA) deficiency leading to hematological and skeletal manifestations. Mechanisms underlying these symptoms have not yet been elucidated. In vivo, bone marrow (BM) mesenchymal stem cells (MSCs) have important role in the regulation of bone mass and in the support of hematopoiesis, thus representing potential candidate that could contribute to the disease. GBA deficiency may also directly impair hematopoietic stem/progenitors cells (HSPCs) intrinsic function and induce hematological defect. In order to evaluate the role of BM stem cells in GD pathophysiology, we prospectively analyzed BM-MSCs and HSPCs properties in a series of 10 patients with type 1 GD. GBA activity was decreased in all tested cell subtypes. GD-MSCs had an impaired growth potential, morphological and cell cycle abnormalities, decreased capacities to differentiate into osteoblasts. Moreover, GD-MSCs secreted soluble factors that stimulated osteoclasts resorbing activities. In vitro and in vivo primitive and mature hematopoiesis were similar between patients and controls. However, GD-MSCs had a lower hematopoietic supportive capacity than those from healthy donors. These data suggest that BM microenvironment is altered in GD and that MSCs are key components of the manifestations observed in GD.
... The reaction was stopped with addition of 3 mL of ice-cold PBS and PFB-F fluorescence was measured by FACS. 20 When necessary, cells were pre-incubated for 1 hour at 37°C with 500M conduritol B epoxyde (CBE), a GluCerase inhibitor. ...
Gaucher disease (GD) is a lysosomal storage disorder caused by glucocerebrosidase deficiency. It is notably characterized by splenomegaly, complex skeletal involvement, ischemic events of the spleen and bones, and the accumulation of Gaucher cells in several organs. We hypothesized that red blood cells (RBC) might be involved in some features of GD and studied the adhesive and hemorheological properties of RBC from GD patients. Hemorheological analyses revealed enhanced blood viscosity, increased aggregation and disaggregation threshold of GD RBC as compared to control (CTR) RBC. GD RBC also exhibited more frequent morphological abnormalities and lower deformability. Under physiological flow conditions, GD RBC adhered more strongly to human microvascular endothelial cells and to laminin than CTR. We showed that Lu/BCAM, the unique erythroid laminin receptor, is overexpressed and highly phosphorylated in GD RBC, and may play a major role in the adhesion process. The demonstration that GD RBC have abnormal rheological and adhesion properties suggests that they may trigger ischemic events in GD, and possibly phagocytosis by macrophages, leading to the appearance of pathogenic Gaucher cells.
