Ambroxol improves lysosomal biochemistry in glucocerebrosidase mutation-linked Parkinson disease cells

Abstract

Gaucher disease is caused by mutations in the glucocerebrosidase gene, which encodes the lysosomal hydrolase glucosylceramidase. Patients with Gaucher disease and heterozygous glucocerebrosidase mutation carriers are at increased risk of developing Parkinson's disease. Indeed, glucocerebrosidase mutations are the most frequent risk factor for Parkinson's disease in the general population. Therefore there is an urgent need to understand the mechanisms by which glucocerebrosidase mutations predispose to neurodegeneration to facilitate development of novel treatments. To study this we generated fibroblast lines from skin biopsies of five patients with Gaucher disease and six heterozygous glucocerebrosidase mutation carriers with and without Parkinson's disease. Glucosylceramidase protein and enzyme activity levels were assayed. Oxidative stress was assayed by single cell imaging of dihydroethidium. Glucosylceramidase enzyme activity was significantly reduced in fibroblasts from patients with Gaucher disease (median 5% of controls, P = 0.0001) and heterozygous mutation carriers with (median 59% of controls, P = 0.001) and without (56% of controls, P = 0.001) Parkinson's disease compared with controls. Glucosylceramidase protein levels, assessed by western blot, were significantly reduced in fibroblasts from Gaucher disease (median glucosylceramidase levels 42% of control, P < 0.001) and heterozygous mutation carriers with (median 59% of control, P < 0.001) and without (median 68% of control, P < 0.001) Parkinson's disease. Single cell imaging of dihydroethidium demonstrated increased production of cytosolic reactive oxygen species in fibroblasts from patients with Gaucher disease (dihydroethidium oxidation rate increased by a median of 62% compared to controls, P < 0.001) and heterozygous mutation carriers with (dihydroethidium oxidation rate increased by a median of 68% compared with controls, P < 0.001) and without (dihydroethidium oxidation rate increased by a median of 70% compared with controls, P < 0.001) Parkinson's disease. We hypothesized that treatment with the molecular chaperone ambroxol hydrochloride would improve these biochemical abnormalities. Treatment with ambroxol hydrochloride increased glucosylceramidase activity in fibroblasts from healthy controls, Gaucher disease and heterozygous glucocerebrosidase mutation carriers with and without Parkinson's disease. This was associated with a significant reduction in dihydroethidium oxidation rate of ∼50% (P < 0.05) in fibroblasts from controls, Gaucher disease and heterozygous mutation carriers with and without Parkinson's disease. In conclusion, glucocerebrosidase mutations are associated with reductions in glucosylceramidase activity and evidence of oxidative stress. Ambroxol treatment significantly increases glucosylceramidase activity and reduces markers of oxidative stress in cells bearing glucocerebrosidase mutations. We propose that ambroxol hydrochloride should be further investigated as a potential treatment for Parkinson's disease.

Keywords: Gaucher disease; Parkinson’s disease; ambroxol; glucocerebrosidase; lysosome.

Figure 1

Glucosylceramidase protein, activity and transcript levels. (A) Representative western blots of glucosylceramidase protein. Note reduction in glucosylceramidase protein levels in Gaucher disease (GD) fibroblasts with a lesser degree of reduction in Parkinson’s disease with glucocerebrosidase mutations (PD-GBA), non-manifesting carrier (NMC) and the E326K/E326K fibroblasts. (B) Bar chart summarizing glucosylceramidase protein levels as assessed by western blot normalized to control. Bars represent median value and 95% confidence interval for all cell lines in each group; each line was measured on three separate blots. There are significant reductions in Gaucher disease (Mann-Whitney U-test P < 0.001), Parkinson’s disease with glucocerebrosidase mutations (P < 0.001), non-manifesting carrier (P = 0.004) and E326K/E326K lines (P = 0.003). (C) Bar chart summarizing glucosylceramidase activity levels (nmol/h/mg). Enzyme activity was significantly reduced in fibroblasts from patients with Gaucher disease (t-test P = 0.001), patients with Parkinson’s disease with glucocerebrosidase mutations (P = 0.004), non-manifesting carriers (P = 0.009) and E326k/E326K lines (P = 0.001) compared with controls. Each bar represents all cell lines in each group. Results are mean ± 1 SD of three separate experiments done for each line. (D) GBA transcript levels in disease lines compared to controls (n = 3), there were reduced transcript levels in GD01, GD04, GD05, and PD03. *P < 0.05.

Figure 2

Evidence of endoplasmic reticulum retention of glucosylceramidase in Gaucher disease. (A) Top: immunofluorescence of control cells showing vesicular staining of glucosylceramidase (green) at the periphery of the cell and perinuclear, reticular staining of calnexin (red, endoplasmic reticulum marker). Bottom: co-localization of glucosylceramidase and calnexin represented by yellow perinuclear staining with minimal green vesicular staining pattern in GD02 and GD03 cell lines. (B) Representative western blots of cell lysate treated with endoglycosidase-H for 12 h. Note appearance of low molecular weight band in Gaucher disease samples (arrow), which represents endoplasmic reticulum retained glucosylceramidase protein. ‘+’ lanes were treated with endoglycosidase-H. (C) Bar chart summarizing percentage of endoglycosidase-H sensitive glucosylceramidase in Gaucher disease (GD) (t-test P = 0.0001), Parkinson’s disease with glucocerebrosidase mutations (PD-GBA) (P = 0.01), non-manifesting carriers (NMC) and E326K/E326K (P = 0.001) compared with controls. Results are mean of three experiments. (D) Top: representative western blot of BiP expression. Bottom: representative western blot of calnexin expression. There was significant elevation of endoplasmic reticulum stress markers BiP and calnexin expression in all Gaucher disease, Parkinson’s disease with GBA mutation and non-manifesting carrier lines.

Figure 3

Oxidative stress assays in Gaucher and Parkinson’s disease fibroblasts. (A) Graph summarizing increases in rates of dihydroethidium oxidation rates for Gaucher disease (GD) (Mann-Whitney U-test P < 0.001), Parkinson’s disease with glucocerebrosidase mutations (PD-GBA) (P < 0.001), non-manifesting carriers (NMC) (P < 0.001) and E326K/E326K (P < 0.001) compared with control cells. (B) Graph demonstrating significant reduction in dihydroethidium oxidation rates for controls (n = 2), Gaucher disease (n = 3) and Parkinson’s disease with glucocerebrosidase mutations fibroblasts (n = 2) treated with ambroxol compared to untreated cells. Results expressed as median rate of dihydroethidium oxidation ± 95% confidence interval.

Figure 4

Analysis of lysosomal markers and autophagy. (A) Bar chart summarizing results for LAMP1 western blot, LysoID assay and beta-galactosidase activity. There is no significant difference in any of these variables, suggesting lysosomal mass is not altered. For each variable results are expressed as a percentage of results from control cells (n = 3). (B) Representative western blots demonstrating increased cathepsin D in Gaucher disease and Parkinson’s disease with GBA mutations compared to controls (top panel) and increased LAMP1 expression after ambroxol treatment (lanes marked ‘+’, bottom panel). (C) Bar chart summarizing increase in total beta-hexosaminidase activity in Gaucher disease (GD), Parkinson’s disease with GBA mutations (PD-GBA) and non-manifesting carriers (NMC) compared with controls. *P < 0.05. (D) Represenative western blots demonstrating LC3-II levels (top) and p62 (bottom). There was no significant difference in either marker in Gaucher disease or Parkinson’s disease with GBA mutations compared to control.

Figure 5

Ambroxol treatment significantly increases glucosylceramidase protein levels. Representative western blots showing increased glucosylceramidase protein levels and decreased cathepsin D levels with ambroxol treatment. ‘+’ represents lanes treated with ambroxol. Ambroxol treatment resulted in a significant elevation of glucosylceramidase protein levels in control [median increase 30% of untreated cells (IQR 15–40%), Mann-Whitney U-test P = 0.0085], Gaucher disease (GD) [median increase 100% of untreated (IQR 87–200% increase), P = 0.004], Parkinson’s disease with GBA mutations (PD-GBA) [median increase 50% of untreated (IQR 40–130%), P = 0.04] and non-manifesting carrier (NMC) fibroblasts [median increase 35% of untreated cells (IQR 25–40% increase), P = 0.008]. Results are derived from all cell lines in each group and represent median value and IQR.

Figure 6

Ambroxol treatment significantly increases glucosylceramidase enzyme activity. Bar chart demonstrating significant increase in glucosylceramidase activity (nmol/mg/h) with ambroxol treatment in controls (t-test P = 0.001), Gaucher disease (GD) (P = 0.0001) and Parkinson’s disease with glucocerebrosidase mutations (PD-GBA) (P = 0.0001). ‘+’ = with ambroxol treatment. Results are derived from all cell lines in each group and represent the mean of three experiments ± 1 standard deviation.

Figure 7

Ambroxol treatment significantly increases glucosylceramidase transcript levels. Bar chart demonstrating significant increase in glucosylceramidase (GCase) messenger RNA levels after ambroxol treatment in controls (Mann-Whitney U-test, P = 0.001), Gaucher disease (GD) (P = 0.0015) and Parkinson’s disease with glucocerebrosidase mutations (PD-GBA) (P = 0.001). Results are median of three experiments ± 95% confidence interval.

Figure 8

Ambroxol significantly increases TFEB transcript levels. Bar chart summarizing increase (fold increase) in TFEB transcript levels in cells treated with ambroxol, data are from two cell lines in each group. There was a significant increase in TFEB transcript levels for control (Mann-Whitney U-test P = 0.02), Gaucher disease (GD) (P = 0.01) and Parkinson’s disease with glucocerebrosidase mutations (PD-GBA) (P = 0.03) after treatment with ambroxol.

Figure 9

Effect of ambroxol on alpha-synuclein overexpressing neuroblastoma cell lines. (A) Representative western blots demonstrating disappearance of endoglycosidase-H sensitive band (arrow) in ambroxol treated Gaucher disease cell lines. ‘+’ = treated with ambroxol and the arrow indicates the endoglycosidase-H sensitive fraction. (B) Bar chart summarizing significant increase in glucosylceramidase and LAMP1 and significant decrease in alpha-synuclein (SNCA) after ambroxol treatment (bars marked ‘+’). (C) Representative western blots demonstrating increased LAMP1 and glucosylceramidase (GCase) after ambroxol treatment and decreased HA-tagged alpha-synuclein. ‘ambroxol’ = cells treated with ambroxol.