Pharmacological Chaperones and Coenzyme Q10 Treatment Improves Mutant β-Glucocerebrosidase Activity and Mitochondrial Function in Neuronopathic Forms of Gaucher Disease

Abstract

Gaucher disease (GD) is caused by mutations in the GBA1 gene, which encodes lysosomal β-glucocerebrosidase. Homozygosity for the L444P mutation in GBA1 is associated with high risk of neurological manifestations which are not improved by enzyme replacement therapy. Alternatively, pharmacological chaperones (PCs) capable of restoring the correct folding and trafficking of the mutant enzyme represent promising alternative therapies.Here, we report on how the L444P mutation affects mitochondrial function in primary fibroblast derived from GD patients. Mitochondrial dysfunction was associated with reduced mitochondrial membrane potential, increased reactive oxygen species (ROS), mitophagy activation and impaired autophagic flux.Both abnormalities, mitochondrial dysfunction and deficient β-glucocerebrosidase activity, were partially restored by supplementation with coenzyme Q10 (CoQ) or a L-idonojirimycin derivative, N-[N'-(4-adamantan-1-ylcarboxamidobutyl)thiocarbamoyl]-1,6-anhydro-L-idonojirimycin (NAdBT-AIJ), and more markedly by the combination of both treatments. These data suggest that targeting both mitochondria function by CoQ and protein misfolding by PCs can be promising therapies in neurological forms of GD.

Figure 1. Mitochondrial dysfunction in Gaucher fibroblasts.

(a) Mitochondrial enzymatic activities of complex I, II, III and II+III in control and Gaucher fibroblasts was determined as described in Materials and Methods. (b) CoQ levels in control and Gaucher fibroblasts were determined by hexane extraction and HPLC separation as described in Materials and Methods. (c) Mitochondrial membrane potential (ΔΨm) was assessed by flow cytometry using MitoTracker Red. A clear decrease of ΔΨm was observed in Gaucher fibroblasts. (d) ATP levels in control and Gaucher fibroblasts. A significant decrease of ATP levels was observed in Gaucher fibroblasts (e) Representative images of MitoTracker staining in control and Gaucher fibroblasts. Scale bar=15 μm. (f) Magnification of a small area in a Gaucher fibroblast. Red arrow indicates tubular mitochondria with normal polarization. Yellow arrow indicates small mitochondria with a striking depolarization in Gaucher fibroblasts. Scale bar=5 μm. (g) Quantification of depolarized mitochondria in control and Gaucher fibroblasts by fluorescence microscopy determined from 100 randomly selected cells by fluorescence imaging analysis. For control cells, the data are the mean±SD for experiments conducted on 3 different control cell lines. Data represent the mean±SD of 3 separate experiments. *p < 0.05 between control and Gaucher fibroblasts. ^**p < 0.01 between control and Gaucher fibroblasts.

Figure 2. Mitochondrial function in Gaucher fibroblasts is recovered by CoQ and PC NAdBT-AIJ treatments.

Control and Gaucher-B fibroblasts were cultured in the absence or presence of CoQ (25 μM), PC NAdBT-AIJ (25 μM) or CoQ + PC (25 μM + 25 μM) for 96 h. (a) Mitochondrial membrane potential (ΔΨm) in control and Gaucher fibroblasts. ΔΨm was assessed by flow cytometry using MitoTracker Red staining. (b) Adenosine-5’-triphosphate (ATP) levels in control and Gaucher fibroblasts. (c) Cell proliferation of control and Gaucher fibroblasts. For the control cells the data are the means±SD of three different control cell lines. Data represent the mean±SD of 3 separate experiments. *p < 0.01 between control and Gaucher fibroblasts. ^ap < 0.05 between the presence and the absence of CoQ. ^bp < 0.05 between the presence and the absence of PC. ^cp < 0.05 between the presence and the absence of CoQ+PC. ^#p < 0.05 between CoQ+PC and CoQ or PC treatment.

Figure 3. ROS production in Gaucher fibroblasts.

(a) Mitochondrial ROS levels in control and Gaucher fibroblasts. Results are expressed as the ratio of MitoSOX signal to 10-N-nonyl acridine orange signal. (b) H₂O₂ levels in control and Gaucher fibroblasts by CMH₂-DCFDA staining coupled with flow cytometry analysis. (c) Mitochondrial ROS levels in control and Gaucher-B fibroblasts cultured, in the absence or presence of CoQ (25 μM), PC NAdBT-AIJ (25 μM) or the combination of both CoQ + PC (25 μM + 25 μM) for 96 h. (d) H₂O₂ levels in control and Gaucher-B fibroblasts cultured, in the absence or presence of CoQ (25 μM), PC NAdBT-AIJ (25 μM) and the combination of both CoQ + PC (25 μM + 25 μM) for 96 h. The mean ± SD of 3 independent experiments are showed. *p < 0.01 between control and Gaucher fibroblasts. ^ap < 0.05 between the presence and the absence of CoQ. ^bp < 0.05 between the presence and the absence of PC. ^cp < 0.05 between the presence and the absence of CoQ + PC. ^#p < 0.05 between CoQ + PC and CoQ or PC treatment.

Figure 4. Increased expression of autophagic markers in Gaucher fibroblasts.

(a) Quantification of acidic vacuoles in control and Gaucher fibroblasts by LysoTracker staining and flow cytometry analysis. (b) Representative LysoTracker staining images in control and Gaucher fibroblasts. Scale bar= 15 μm. (c) Effect of CoQ, PC and CoQ + PC on the amount of acidic vesicules in Gaucher-B fibroblasts. Control and Gaucher fibroblasts were cultured in the presence or absence of CoQ (25 μM), PC NAdBT-AIJ (25 μM) or CoQ + PC (25 μM + 25 μM) for 96 h. Acidic vacuoles were quantified by LysoTracker staining and flow cytometry analysis. For control cells, the data are the mean ± SD for experiments conducted on 2 different control cell lines. Data represent the mean ± SD of 3 separate experiments. *p < 0.01 between control and Gaucher fibroblasts. ^ap < 0.05 between the presence and the absence of CoQ. ^bp < 0.05 between the presence and the absence of PC. ^cp < 0.05 between the presence and the absence of CoQ + PC. ^#p < 0.05 between CoQ + PC and CoQ or PC treatment. (d) The expression levels of LC3-I (upper band) and LC3-II (lower band), ATG12, BECLIN1 and cathepsin D were determined in the control and Gaucher-B fibroblast cultures by Western blotting. The ATG12 band represents the Atg12-Atg5 conjugated form. Fibroblast cultures were grown in normal culture medium or in medium supplemented with CoQ (25 μM), PC (25 μM) or the combination of both CoQ + PC (25 μM + 25 μM) for 96 h. Actin was used as a loading control. The densitometric analysis of Western blottings is showed in Supplementary Figure S6.

Figure 5. Autophagosome and mitochondria markers colocalization in Gaucher fibroblasts.

(a) Image analysis of LC3 and cytochrome c immunostaining in control and Gaucher fibroblasts. Control and Gaucher-B fibroblasts were cultured in the presence or absence of CoQ (25 μM), PC NAdBT-AIJ (25 μM) or CoQ + PC (25 μM + 25 μM) for 96 h. Cells were fixed and immunostained with anti-LC3 (autophagosome marker) and cytochrome c (mitochondrial marker) and examined by fluorescence microscopy. Scale bars=15 (upper and lower pannel) or 5 μm (middle pannel). (b) Magnification of a small area in a Gaucher fibroblast. Yellow arrow shows autophagosomes with LC3 and cytochrome c colocalization. Red arrow shows tubular mitochondria without colocalization with LC3. Colocalization of both markers was assessed using the DeltaVision software and calculating the Pearson´s coefficient of correlation. Scale bar = 5 μm. (c) Quantification of LC3/cytochrome c puntacta in control and Gaucher fibroblasts incubated with or without CoQ, PC and CoQ+PC (n = 100 cells).*p < 0.01 between control and Gaucher fibroblasts. ^ap < 0.05 between the presence and the absence of CoQ. ^bp < 0.05 between the presence and the absence of PC. ^cp < 0.05 between the presence and the absence of CoQ + PC. ^#p < 0.05 between CoQ + PC and CoQ or PC treatment.

Figure 6. Mitophagy and impaired autophagic flux in Gaucher fibroblasts.

(a) Western blot analysis of mitochondrial (complex I, 30 kDa subunit; complex II, 30 kDa subunit; complex III, core 1 subunit; complex IV, COX II subunit; and porin), Golgi (Golgi marker), endoplasmic reticulum (PDI), and peroxisome (catalase) proteins in control and Gaucher-B fibroblasts treated with CoQ (25 μM), PC NAdBT-AIJ (25 μM) or CoQ + PC (25 μM+25 μM) for 96 h. Alpha-tubulin was used as loading control. (b) Autophagy flux. Determination of LC3-II, porin and cytochrome c levels in the presence and absence of bafilomycin A1 in control and Gaucher-B fibroblasts. Control and Gaucher fibroblasts were incubated with bafilomycin A1 (100 nM for 12 h). Total cellular extracts were analyzed by immunoblotting with antibodies against LC3, porin and cytochrome c. α-Tubulin was used as a loading control. (c) Densitometry of Western blotting was performed using the ImageJ software. Data represent the mean ± SD of three separate experiments. *p < 0.01 between control and Gaucher fibroblasts. ^ap < 0.05 between the presence and the absence of bafilomycin A1. (d) Apoptosis is increased in GCase-deficient Atg5-/- cells. Apoptosis is increased in GCase-deficient Atg5-/- cells. GCase deficiency in wild-type and Atg5-/- MEFs was induced by treatment with CBE 2,5 mM for 48 hours. Apoptosis was assessed in both autophagy proficient (wild-type), and autophagy-deficient cells (Atg5-/-) treated with CBE as described in Material and Methods. CBE-treated MEFs were also supplemented with, CoQ+PC NAdBT-AIJ for 96 hours to verify the specificity of apoptosis induction by GCase deficiency. Results are expressed as mean ± SD of three independent experiments *p < 0.01 between Atg5-/- and wild-type MEFs. ^ap < 0.01 between the presence and the absence of CBE . ^bp < 0.01 between Atg5-/- in the presence or absence of CoQ+PC.

Figure 7. Treatment of Gaucher fibroblasts with CoQ and PC NAdBT-AIJ increases GCase activity.

(a) GCase activities in control and Gaucher-B fibroblasts cultured in the absence or presence of CoQ (25 μM), PC NAdBT-AIJ (25 μM) and CoQ + PC (25 μM + 25 μM) for 96 h. GCase activities increase after CoQ, PC and markedly after CoQ+PC supplementation. Data, expressed as Gcase activity (% of control), represent the mean ± SD of 3 separate experiments. (b) GCase expression levels determined in control and Gaucher-B fibroblasts by Western blotting. Control and Gaucher fibroblast cultures were grown in normal culture medium or in medium supplemented with CoQ, PC and CoQ + PC for 96 h. Fibroblast protein extracts (50 μg) were separated on a 12.5% SDS-polyacrylamide gel and immunostained with an antibody against GCase. α-Tubulin was used as a loading control. (c) Densitometric analysis of Western blottings. Data represent the mean ± SD of 3 separate experiments. *p < 0.01 between control and Gaucher fibroblasts. ^ap < 0.05 between the presence and the absence of CoQ. ^bp < 0.05 between the presence and the absence of PC. ^cp < 0.05 between the presence and the absence of CoQ + PC. ^#p < 0.05 between CoQ + PC and CoQ or PC treatment.

Figure 8. Treatment with CoQ and PC NAdBT-AIJ changes the intracellular localization of GCase in Gaucher fibroblasts.

Effects of CoQ and PC NAdBT-AIJ on the traffic of GCase from the ER to lysosomes in Gaucher-B fibroblasts. Gaucher fibroblasts were treated with CoQ + PC (25 μM + 25 μM. (a) ER marker, PDI, or GCase are visualized as red or green, respectively. In the merged images, yellow denotes colocalization in the ER. Scale bar=15 μm. (b) Lysosomal marker, LAMP-1, or GCase are visualized as red or green, respectively. In the merged images, yellow denotes colocalization in lysosomes. Colocalization of both markers was assessed using the DeltaVision software and calculating the Pearson´s coefficient of correlation. Scale bar = 15 μm. (c) Representative Western blots of cell lysates treated with Endo-H. Cell lysates from control and Gaucher fibroblasts treated with CoQ, PC and CoQ + PC were subjected to Endo-H digestion and Western blot analysis with anti GCase antibody. Note the appearance, after Endo-H treatment, of a low molecular weight band in Gaucher disease samples (grey arrow), which represents endoplasmic reticulum retained GCase protein (Endo-H-S) and a high molecular weight band (Endo-H-R) which represents mature GCase (black arrow). GAPDH was used as a loading control. (d) Densitometric analysis of Western blotting. Quantification of Endo H assay using ImageJ analysis software. The gray portion of each bar represents Endo H-S bands and the black portion of the bar represents Endo H-R bands.