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.
Highlights
The autophagy–lysosome system plays a key role in degrading the misfolded proteins that form the abnormal protein accumulations that occur in the common late onset neurodegenerative diseases
Glucosylceramidase activity is decreased in fibroblasts from patients with Gaucher disease and heterozygous GBA mutation carriers with and without Parkinson’s disease
Western blotting revealed a significant reduction in glucosylceramidase protein levels in all Gaucher disease, Parkinson’s disease with GBA mutation, E326K/E326K and non-manifesting carrier cell lines (Fig. 1A and B)
Summary
The autophagy–lysosome system plays a key role in degrading the misfolded proteins that form the abnormal protein accumulations that occur in the common late onset neurodegenerative diseases. In concert with the proteasome, the autophagy–lysosome system degrades tau, the proteins that form neurofibrillary tangles in Alzheimer’s disease (Lee et al, 2013). Markers of dysfunctional autophagy have been described in motor neuron disease spinal cord (Otomo et al, 2012), and the autophagy–lysosome system plays a role in degrading superoxide dismutase 1 and TBP43 ( known as TARDBP) (Otomo et al, 2012). Accumulation of p62 and LC3-II, markers of dysfunction of the autophagy–lysosome system, has been described in post-mortem Parkinson’s disease brain (Alvarez-Erviti et al, 2010; Dehay et al, 2013). Mutations in the glucocerebrosidase gene (GBA), which encodes the lysosomal hydrolase glucosylceramidase deficient in Gaucher disease, have been identified as a risk factor for the development of Parkinson’s disease (Sidransky et al, 2009; Sidransky and Lopez, 2012), dementia with Lewy bodies (Nalls et al, 2013) and a subtype of Alzheimer’s disease (Tsuang et al, 2012)
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