Augmenting CNS glucocerebrosidase activity as
a therapeutic strategy for parkinsonism and other
S. Pablo Sardia,1, Jennifer Clarkea, Catherine Viela, Monyrath Chana, Thomas J. Tamsetta, Christopher M. Treleavena,
Jie Bua, Lindsay Sweeta, Marco A. Passinia, James C. Dodgea, W. Haung Yub, Richard L. Sidmanc,1, Seng H. Chenga,
and Lamya S. Shihabuddina
aGenzyme, a Sanofi Company, Framingham, MA 01701;bTaub Institute for Research on Alzheimer’s Disease, Columbia University Medical Center, NY 10032;
andcBeth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215
Contributed by Richard L. Sidman, December 7, 2012 (sent for review September 14, 2012)
Mutations of GBA1, the gene encoding glucocerebrosidase, repre-
sent a common genetic risk factor for developing the synucleino-
pathies Parkinson disease (PD) and dementia with Lewy bodies. PD
patients with or without GBA1 mutations also exhibit lower enzy-
matic levels of glucocerebrosidase in the central nervous system
(CNS), suggesting a possible link between the enzyme and the
development of the disease. Previously, we have shown that early
treatment with glucocerebrosidase can modulate α-synuclein ag-
gregation in a presymptomatic mouse model of Gaucher-related
synucleinopathy (Gba1D409V/D409V) and ameliorate the associated
cognitive deficit. To probe this link further, we have now evalu-
ated the efficacy of augmenting glucocerebrosidase activity in the
CNS of symptomatic Gba1D409V/D409Vmice and in a transgenic
mouse model overexpressing A53T α-synuclein. Adeno-associated
virus-mediated expression of glucocerebrosidase in the CNS of
symptomatic Gba1D409V/D409Vmice completely corrected the aber-
rant accumulation of the toxic lipid glucosylsphingosine and re-
duced the levels of ubiquitin, tau, and proteinase K-resistant
α-synuclein aggregates. Importantly, hippocampal expression of
glucocerebrosidase in Gba1D409V/D409Vmice (starting at 4 or 12
mo of age) also reversed their cognitive impairment when exam-
ined using a novel object recognition test. Correspondingly, over-
expression of glucocerebrosidase in the CNS of A53T α-synuclein
mice reduced the levels of soluble α-synuclein, suggesting that
increasing the glycosidase activity can modulate α-synuclein pro-
cessing and may modulate the progression of α-synucleinopathies.
Hence, increasing glucocerebrosidase activity in the CNS repre-
sents a potential therapeutic strategy for GBA1-related and non-
GBA1–associated synucleinopathies, including PD.
lysosomal storage diseases|mouse models|MAPT|memory defect
pathies such as Parkinson disease (PD) and dementia with Lewy
bodies (DLB) (1–5). The central nervous system (CNS) of Gaucher
patients and carriers who present with parkinsonism and dementia
harbor deposits of α-synuclein–positive Lewy bodies (LBs) and
Lewy neurites (LNs) in hippocampal neurons and their processes
resembling those noted in patients with classical PD and DLB (6,
7). Aspects of these characteristics have also been noted in the CNS
of several mouse models of neuropathic and nonneuropathic
Gaucher disease (8–10). Consequently, a causal relationship has
been suggested between the loss of glucocerebrosidase activity or
the lysosomal accumulation of undegraded metabolites and the
development of PD and DLB. A more direct link between glu-
cocerebrosidase activity and α-synuclein metabolism has been
highlighted by studies of Gaucher cells and mice indicating that
a reduction in glucocerebrosidase activity by pharmacological or
genetic interventions resulted in increased levels of α-synuclein
aggregates (9–12). Moreover, a decrease in glucocerebrosidase
activity has been noted in cerebrospinal fluid (CSF) and brain
utations in the gene for glucocerebrosidase (GBA1) present
the highest genetic risk factor for developing synucleino-
samples from patients with PD and DLB (regardless of whether
they harbor mutations in GBA1), suggesting that a reduction in
glucocerebrosidase activity may contribute to the development of
A role for glucocerebrosidase in the development of synuclei-
nopathies is further supported by clinical observations of patients
with Gaucher-associated parkinsonism. These individuals present
with increased frequencies and severities of nonmotor symptoms
(e.g., cognitive impairment) that substantially erode their quality
of life (16, 17). Individuals harboring mutations in GBA1 also
have a higher incidence of dementia that is correlated with the
presence of neocortical accumulation of aggregates of α-synuclein
(18, 19). Indeed, mutations in GBA1 are now recognized as an
independent risk factor for development of cognitive impairment
in PD patients (20). Another gene associated with an increased
risk for dementia in PD is MAPT (21), the gene encoding the
microtubule-associated protein tau, which helps maintain cyto-
skeletal organization and integrity. Tau-associated and α-synu-
clein–associated pathology frequently occurs in patients with PD
and LBD (22–24) although the relative roles of these proteins
are not well defined. Tau is more explicitly involved in Alzheimer’s
We previously described a Gaucher-related synucleinopathy in
mice with progressive CNS accumulation of proteinase K-resistant
α-synuclein/ubiquitin aggregates reminiscent of LNs (10). These
mice also have high CNS levels of the neurotoxin glucosyl-
sphingosine (GlcSph) and a hippocampal memory deficit. We
showed these biochemical and behavioral aberrations to be
ameliorated by CNS administration into presymptomatic animals
of a recombinant adeno-associated viral (AAV) vector encoding
human glucocerebrosidase. The present study further character-
izes the pathological features in this Gaucher-associated synu-
cleinopathy model, adding increased protein tau and demon-
strating cognitive improvement and moderation of CNS pathology
when glucocerebrosidase was administered at a clinically relevant
postsymptomatic stage. Finally, to further probe the glucocere-
brosidase/α-synuclein relationship, the effect of the lysosomal
hydrolase on α-synuclein levels in the A53T α-synuclein mouse was
Author contributions: S.P.S., S.H.C., and L.S.S. designed research; S.P.S., J.C., C.V., M.C., T.J.T.,
C.M.T., J.B., L.S., M.A.P., J.C.D., and W.H.Y. performed research; S.P.S., J.C., C.V., M.C., W.H.Y.,
R.L.S., and L.S.S. analyzed data; and S.P.S., R.L.S., S.H.C., and L.S.S. wrote the paper.
The authors declare no conflict of interest.
Freely available online through the PNAS open access option.
See Commentary on page 3214.
1To whom correspondence may be addressed. E-mail: email@example.com
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
| February 26, 2013
| vol. 110
| no. 9
A53T α-synuclein (line M83) under the transcriptional control of the murine
PrP promoter (30).
AAV Vectors and Injections. A detailed description of the AAV vectors and
the injection procedures used in these studies is available in SI Materials
Western Blotting. Western blotting procedures are described in SI Materials
Measurements of Glucocerebrosidase Activity and Glycosphingolipid Levels.
Brain and hippocampal glucocerebrosidase activities were determined as
described with 4-methylumbelliferyl (4-MU)-β-D-glucoside as the artificial
substrate (10). Hexosaminidase and β-galactosidase activities were de-
termined with 4-MU-N-acetyl-β-D-glucosaminide and 4-MU-β-D-galactopyr-
anoside, respectively. Tissue GlcCer and GlcSph levels were measured by
mass spectrometry as described (10).
Immunohistochemistry. Immunohistochemistry procedures are described in SI
Materials and Methods.
Mice BehavioralTests.A detailed descriptionof thebehavioraltests is available
in SI Materials and Methods.
Fractionation and Quantification of α-Synuclein. Striatum and spinal cord from
A53T–α-synuclein mice were homogenized as described (9) to obtain three
fractions: cytosolic (Tris-soluble), membrane-associated (Triton X-100–solu-
ble), and insoluble (SDS-soluble). The concentration of human α-synuclein in
the different fractions was quantified by sandwich ELISA (Invitrogen). Pro-
tein concentration was determined by the microBCA assay (Pierce).
Statistical Analysis. Statistical analyses were performed by Student’s t test or
ANOVA followed by Newman–Keuls’ post hoc test. Preference for novelty
was defined as investigating the novel object >50% of the time by a one-
sample t test. All statistical analyses were performed with GraphPad Prism
v4.0 (GraphPad Software). P < 0.05 was considered significant.
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cock, Shelley Nass, Brenda Burnham, Maryellen O’Neill, Wendy Yang, Mario
Cabrera-Salazar, Katie Kinnecom, Matthew DeRiso, Michael Phipps, Leah Cur-
tin, JoAnne Fagan, and David Lee-Parritz for technical assistance and support.
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| www.pnas.org/cgi/doi/10.1073/pnas.1220464110Sardi et al.