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BRAIN A JOURNAL OF NEUROLOGY
Ambroxol improves lysosomal biochemistry in glucocerebrosidase mutation-linked Parkinson disease cells Alisdair McNeill,1 Joana Magalhaes,1 Chengguo Shen,2 Kai-Yin Chau,1 Derralyn Hughes,3
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Write My Essay For MeAtul Mehta,3 Tom Foltynie,4 J. Mark Cooper,1 Andrey Y. Abramov,5 Matthew Gegg1 and Anthony H.V. Schapira1
1 Department of Clinical Neurosciences, Institute of Neurology, University College London, UK
2 Bioinformatics Unit, Source Bioscience, Nottingham, UK
3 Lysosomal storage disorders unit, Royal Free Hospital, London, UK
4 Sobell Department of Motor Neuroscience and Movement Disorders, Institute of Neurology, University College London, UK
5 Department of Molecular Neuroscience, Institute of Neurology, University College London, UK
Correspondence to: Professor A.H.V. Schapira,
Chairman and Head of Department,
Department of Clinical Neurosciences,
UCL Institute of Neurology,
Upper level 3,
UCL Medical School,
Royal Free Hospital London NW3 2PF UK
E-mail: a.schapira@ucl.ac.uk
Gaucher disease is caused by mutations in the glucocerebrosidase gene, which encodes the lysosomal hydrolase glucosylcer-
amidase. Patients with Gaucher disease and heterozygous glucocerebrosidase mutation carriers are at increased risk of develop-
ing 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 glucosylcer-
amidase levels 42% of control, P50.001) and heterozygous mutation carriers with (median 59% of control, P5 0.001) and without (median 68% of control, P50.001) 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, P5 0.001) and heterozygous mutation carriers with (dihydroethidium oxidation rate increased by a median of 68% compared with controls, P5 0.001) and without (dihydroethidium oxidation rate increased by a median of 70% compared with controls, P5 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
doi:10.1093/brain/awu020 Brain 2014: 137; 1481–1495 | 1481
Received September 27, 2013. Revised November 26, 2013. Accepted December 15, 2013. Advance Access publication February 25, 2014 � The Author (2014). Published by Oxford University Press on behalf of the Guarantors of Brain. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0/), which permits unrestricted reuse,
distribution, and reproduction in any medium, provided the original work is properly cited.
in dihydroethidium oxidation rate of �50% (P50.05) 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: Parkinson’s disease; ambroxol; lysosome; Gaucher disease; glucocerebrosidase
Abbreviation: CLEAR = coordinated lysosomal expression and regulation
Introduction 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 (now
known as TARDBP) (Otomo et al., 2012). There is also strong
evidence for a role of this system in the aetiopathogenesis of
Parkinson’s disease (Goker-Alpan, et al., 2010; Ebrahimi-Fakhari
et al., 2012; Houlden 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). In addition
cell biology studies have demonstrated that inhibition of
the autophagy–lysosome system, and in particular chaperone-
mediated autophagy, is associated with elevation of alpha-synu-
clein protein levels (Alvarez-Erviti et al., 2010). Recently,
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).
Three broad clinical subtypes of Gaucher disease are recognized
(Balwani et al., 2010). In type I (non-neuronopathic) Gaucher disease,
patients develop combinations of blood dyscrasia, hepato-splenomegaly
and bone disease (Balwani et al., 2010). Patients with types II (acute
neuronopathic) and III (chronic neuronopathic) Gaucher disease present
with a predominantly neurodegenerative syndrome in childhood or
early adulthood (Tajima et al., 2009). Several hundred GBA mutations
have been reported, but the most common are the N370S and L444P
missense mutations (Hruska et al., 2008). There is no precise geno-
type–phenotype correlation but patients with the N370S allele generally
develop non-neuropathic disease (Balwani et al., 2010). The clinical
manifestations of Gaucher disease are associated with lysosomal accu-
mulation of the glucosylceramidase substrates glucosylceramide and
glucosylsphingosine (Balwani et al., 2010). Patients with Gaucher
disease have an 8–12% chance of developing Parkinson’s disease by
age 80 (Rosenbloom et al., 2011) whereas the risk in heterozygous
GBA mutations carriers is 10–15% by age 80 (McNeill et al., 2012).
The mechanisms by which GBA mutations predispose to neuro-
degeneration remain unclear. However, post-mortem studies have
demonstrated severe loss of glucosylceramidase enzyme activity in
brain tissue from patients with Gaucher disease and in the
substantia nigra of patients with Parkinson’s disease (Mazzulli
et al., 2011; Gegg et al., 2012) and patients with Lewy body
dementia (Kurzawa-Akanbi et al., 2012) with and without GBA
mutations. Current evidence thus supports a central role for neur-
onal loss of glucosylceramidase activity in the pathogenesis of
neurodegeneration associated with GBA mutations. The majority
of GBA mutations do not affect the catalytic site of glucosylcer-
amidase, and so other mechanisms must account for loss of
enzyme activity in Parkinson’s disease and Gaucher disease
(Hruska et al., 2008). There is evidence that GBA missense
mutations cause endoplasmic reticulum retention and proteasomal
degradation of the mutant protein (Ron et al., 2005; Sawkar
et al., 2006). Given that there is reduction of glucosylceramidase
activity in brain tissue from patients with Parkinson’s disease and
those with Lewy body dementia, with and without GBA muta-
tions, there is clearly an urgent need to understand how reduced
glucosylceramidase activity causes neurodegeneration to facilitate
development of new treatments.
