Fig 1.
Glucosylceramide (GC) metabolism in Gaucher diseases.
Ceramide, glucosylceramide shift between the Golgi apparatus and lysosomes. Ceramides, generated in the ER, are transported to the Golgi apparatus, where UDP-glucosylceramide synthase (UGCG synthase) converts Cer to GC on the cytosolic side of the Golgi [19]. After, GC transport back into the ER. To transport ceramide to lysosome, GS, GCase, and saposin C (reaction facilitator) are embedded within intralysosomal membrane where cleavage of lipid tail will occur [20]. Eliglustat inhibits UGCG synthase. Ambroxol increases GCase enzyme activity.
Table 1.
Demographics, clinical and molecular characteristics of subjects with GD.
Fig 2.
Assessing GCase activity in primary fibroblasts treated with AMB and EGT.
A. Fibroblasts derived from healthy controls and GD3 patients with the genotypes L444P/L444P and L444P/L444P/A456P were cultured for 5 days in the presence of increasing concentrations of AMB or EGT. Relative GCase enzyme activity was estimated as a percentage towards untreated control. Each bar represents the average +/- STDEV. * p<0.05 compared with an untreated group. B. Fibroblasts derived from GD2 with different GBA mutations, as indicated, were treated for 5 days in the presence of AMB and EGT. Relative GCase enzyme activity was estimated as a percentage towards untreated control. Each bar represents the average +/- STDEV. * p<0.05 compared with an untreated group. C. Comparing GCase enzyme activity estimated as nmol/μg/hr in healthy control fibroblasts and GD3 fibroblasts.
Fig 3.
AMB and EGT induce lysosomal trafficking and LAMP-1 level in primary fibroblasts.
A-B. Fluorescence microscopy images of control fibroblast (A) and GD2 fibroblast (B). The cells were treated with 10 μM AMB and 10 μM EGT for five days. Each set of three side-by-side images shows anti-GBA (red), anti-LAMP1 (green color) antibodies, and merged images. The yellow color indicates colocalization of GBA and LAMP1 in the lysosome. (C) The interactive 3D color inspector plots displayed a three-dimensional graph of pixel distribution of images of GD2 fibroblasts. Nucleus (blue), GBA (red) and LAMP1 (green) colocalization. D. Top row represents the western blot of LAMP1 in fibroblasts derived from GD3 patients. Actin is used as the loading control. P1, P2, P4, and P5 (n = 4). * p<0.05 compared with an untreated group. E. The top: western blot of LAMP1 in GD2 fibroblasts derived from patient 8 with L444P/L444P;D409H;A456P genotype. The bottom: quantification of the relative level of LAMP1/actin from P8. Each bar represents the average +/- STDEV from three independent experiments. * p<0.05 compared with an untreated group.
Fig 4.
EGT and AMB improve autophagy and lysosomal dynamics.
A. The lysosome (red) and autophagy (green) fluorescent staining in live control and GD3 (P5) fibroblasts with L444P/L444P GBA mutation after AMB and EGT treatments for five days. B. Lysosome and autophagy colocalization analysis in GD2 fibroblasts after treatment with AMB and EGT for five days. C. Quantification of fluorescence intensity of lysosomes. The signal intensity in untreated cells was set at 100%. The graph indicates the relative intensity value of fluorescence signal related to LysoTracker in control fibroblasts, GD3 fibroblasts with L444P/L444P, and GD2 fibroblasts. Values are expressed as average ± STDEV. D, E, and F. Following AMB and EGT treatments, representative western blots showing LC3-I/LC3-II protein expression level in GD3 fibroblast derived from patients: P1 (D), P5 (E), and GD2 patient P8 (F). G. Quantification of the relative level of LC3-II to LC3-I. Each bar represents the average +/- SEM. * p<0.05 compared with an untreated group. Student T-test, 2 tail, 2 type.
Fig 5.
Assessment of cell viability and metabolic status in fibroblasts.
A. Control (WT) fibroblasts were treated with 1, 10 or 100 μM of AMB and 0.1, 1 or 10 μM of EGT for 5 days and were submitted to the CCK-8 cell viability assay and ATP content. The obtain cell counting assay, CCK-8, and ATP results were normalized to the untreated cells. Additionally, the ratio ATP/CCK-8 (cell viability) was estimated. Values are expressed as average ±SEM, n = 3. * p<0.05 compared with an untreated group. B. Fibroblasts derived from three GD2 patients, P6 with L444P/L444P;RecΔ55;RecNCiI, P7 with L444P/L444P;R495P/R495P;A456P GBA variations, P9 with L444P/D409H, and one GD3 patient with L444P/L444P mutation, (P5) were treated with 1, 10, 100 μM of AMB and 0.1, 1,10 μM of EGT for 5 days. The CCK-8 cell viability assay and ATP content were analyzed. The obtained cell counting assay, CCK-8, and ATP results were normalized in relationship to the untreated cells. ATP/CCK-8 (cell viability) ratio was estimated. Values are expressed as average ±SEM, n = 4. * p<0.05 compared with an untreated group.
Fig 6.
MitoTracker deep red staining in AMB and EGT treated fibroblasts.
(A) The mitochondrial visualization in live control (WT) fibroblasts with AMB and EGT treatment for five days. Scale bar represents 400 μm. (B) Quantification of fluorescent intensities of mitochondria. The signal intensity in untreated cells was set at 100%. The graph indicates the relative intensity value of the fluorescence signal related to MitoTracker Red in control fibroblasts. Values are expressed as average ±STDEV. (C) The mitochondrial visualization in GD fibroblasts with AMB and EGT treatment for five days. Scale bar represents 100 μm. (D) Quantification of fluorescent intensities of mitochondria in GD cells. The signal intensity in untreated cells was set at 100%. Values are expressed as average ±STDEV. * p<0.05 compared with an untreated group. (E) The mitochondrial visualization in live GD3 fibroblasts. Representative images were assessed in regards to the degree of mitochondrial branching.
Fig 7.
The skeleton algorithm identifies mitochondrial density in sample images from cells.
(A) Post-processed images of living mitochondria stained with MitoTracker. Control fibroblasts were treated with AMB and EGT for five days. Two-tone color (blue turn to pink) represents the intensity of density. (B) Results of the corresponding mitochondrial assessment using skeleton 2D/3D analysis.
Fig 8.
The effects of EGT and AMB on mitochondrial membrane potential in GD.
Fibroblasts (A) and PBMC (B) were treated with increasing concentrations of AMB or EGT for five days, then were stained with Mito-ID Membrane Potential reagent and visualized by fluorescence microscopy. Green represents mitochondria with low membrane potential. Highly polarized mitochondria exhibit red color.