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: firstname.lastname@example.org
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
| February 26, 2013
| vol. 110
| no. 9
CNS of a Mouse Model of Gaucher Disease Exhibits Accumulation of
Tau Aggregates. Accumulation of α-synuclein and tau inclusions
with resultant dementia are the hallmarks of a number of neu-
rodegenerative diseases, including PD and DLB (22, 25, 26). We
have reported that a mouse model of Gaucher disease har-
boring a single point mutation in the murine Gba1 locus
(Gba1D409V/D409V) exhibits progressive, marked accumulation
of α-synuclein/ubiquitin aggregates in the CNS and a measurable
deficit in hippocampal memory (10). To determine whether mu-
tations in Gba1 with resultant loss of glucocerebrosidase activity
also promote the accumulation of tau in the CNS, brain sections
of 12-mo-old Gba1D409V/D409Vmice were examined immunohis-
tochemically with a specific anti-tau antibody. Marked punctate
staining was noted primarily in the hippocampal regions (Fig. 1A),
although immunoreactivity was also observed in other areas, in-
cluding cerebral cortex and cerebellum. The onset and rate of ac-
cumulation of the tau aggregates in the brains of Gba1D409V/D409V
mice were determined as well. At 2 mo of age, tau immunore-
activity in Gba1D409V/D409Vmice was not different from that in
wild-type controls (Fig. 1 A and B). However, the level of tau
staining in 6-mo-old Gba1D409V/D409Vmice was significantly
higher than in the age-matched controls. Accumulation was pro-
gressive, with 12-mo-old Gba1D409V/D409Vmice displaying higher
amounts of tau aggregates (Fig. 1 A and B).
A common finding in tauopathies and related neurodegener-
ative diseases is an increase in hyperphosphorylated tau com-
prising the neurofibrillary tangles (27, 28). These phosphory-
lated species can be detected with specific antibodies, such as
AT270 (Thr-181), AT8 (Ser-202/Thr-205), and AT180 (Thr-231).
To probe the phosphorylation status of the tau aggregates
in the CNS of Gba1D409V/D409Vmice, Western blot analysis was
performed on hippocampal lysates from 18-mo-old mice. Staining
the blots with the Tau-5 antibody that recognizes all tau species
revealed that the overall levels of the protein were not different
between Gba1D409V/D409Vand wild-type mice (Fig. 1C), nor were
differences in the extent of staining observed between controls
and age-matched Gba1D409V/D409Vmice when the blots were probed
with AT180 or AT270 antibodies (Fig. 1C). However, AT8
staining, which detects phosphorylation on Ser-202 and Thr-205,
was modestly but significantly increased in the lysates of
Gba1D409V/D409Vmice (1.3 ± 0.1 compared with wild-type, n = 6,
P < 0.05; Fig. 1C). This increased phosphorylation on Ser-202
and Thr-205, coupled with the progressive accumulation of the
tau aggregates (in addition to α-synuclein), indicates that the
CNS of Gba1D409V/D409Vmice recapitulates pathological features
in PD and DLB patients.
Administration of Glucocerebrosidase into the Hippocampus Re-
verses the Biochemical and Memory Aberrations of Postsymptomatic
Gba1D409V/D409VMice. To determine whether reconstitution of the
CNS with recombinant glucocerebrosidase can correct the bio-
chemical aberrations and memory deficits of symptomatic
Gba1D409V/D409Vmice, a recombinant self-complementary AAV
vector (serotype 1) encoding human glucocerebrosidase (AAV–
GBA1) was administered bilaterally into the hippocampi of early
and late symptomatic mice (4- and 12-mo-old, respectively). Im-
munohistochemical examination of the CNS of Gba1D409V/D409V
mice that had been administered AAV–GBA1 at 12 mo of age
and were analyzed 6 mo later revealed abundant and widespread
hippocampal expression of glucocerebrosidase (Fig. 2A). Mice
treated with a control virus that did not encode a transgene
(AAV-EV) showed no staining (Fig. 2A Inset). The enzymatic
activity in AAV–GBA1-treated (Fig. 2B, red bar) mice was ∼10-
fold higher than the baseline value (Fig. 2B, black bar) and the
activity in Gba1D409V/D409Vmice administered AAV-EV (Fig. 2B,
blue bar). A similar distribution of the enzyme was noted in the
CNS of Gba1D409V/D409Vmice treated at 4 mo of age and analyzed
6 mo posttreatment. Expression of glucocerebrosidase in the 12-
mo-old mice was associated with normalization of the hyper-
elevated levels of brain GlcSph after 6 mo (Fig. 2C, red bar). In
contrast, Gba1D409V/D409Vmice treated with the control virus
exhibited continued accumulation of the proinflammatory lipid
over the same time interval (Fig. 2C, blue bar). Glucosylceramide
(GlcCer), another glucocerebrosidase substrate, was not affected
by any of the treatments or genotypes (Fig. S1).
Hippocampal memory was evaluated with the novel object
recognition test. Testing of 4-mo-old Gba1D409V/D409Vmice before
treatment confirmed that they exhibited impairments in novel
object recollection (Fig. 2D). Treatment of these mice with AAV–
GBA1 reversed memory deficits when the mice were tested 2 mo
later (at 6 mo of age; Fig. 2E, red bars; n = 10, P < 0.05), whereas
Gba1D409V/D409Vmice treated with the control viral vector showed
no discernible improvement (Fig. 2E, blue bars; n = 9). A similar
result was obtained in a separate cohort of Gba1D409V/D409Vmice
preexisting pathology) and tested 2 mo later (Fig. 2F, red bars, n =
glucocerebrosidase activity in postsymptomatic Gba1D409V/D409V
mice corrected the pathological accumulation of GlcSph and,
importantly, their memory impairments.
Administration of Glucocerebrosidase into the Hippocampus of Symp-
tomatic Gba1D409V/D409VMice Reduces the Levels of Aggregated
Proteins in the Brain. Because Gba1D409V/D409Vmice exhibit re-
duced glucocerebrosidase activity and progressive accumulation
of ubiquitin, α-synuclein, and tau aggregates in the hippocampus,
we sought to test whether augmenting glucocerebrosidase levels
in the brain would decrease the levels of these aberrant pro-
teinaceous materials in symptomatic animals. The hippocampi of
4- and 12-mo-old Gba1D409V/D409Vmice (the latter presented with
Gba1D409V/D409Vmice. (A) Images show immunostaining with an anti-tau
serum (green) and nuclear staining (DAPI; blue) in the hippocampi of 2-, 6-,
and 12-mo-old Gba1D409V/D409Vand age-matched wild-type (WT) mice. (Scale
bar, 500 μm.) (B) Quantification of Tau-5 immunoreactivity in WT and
Gba1D409V/D409Vhippocampi at 2, 6, and 12 mo shows progressive accumu-
lation of aggregates with age (n ≥ 5 per group). (C) Shown are represen-
tative immunoblots of hippocampal lysates from 18-mo-old Gba1D409V/D409V
mice and age-matched controls for AT8, AT180, AT270, Tau-5, and β-tubulin.
Each lane represents an independent mouse brain. Clone AT8 antibody
shows increased tau phosphorylation (S202/T205) in aged Gba1D409V/D409V
mice. No differences between mutant and wild-type mice were observed in
total tau levels (Tau-5) or other phosphorylated species (AT180 or AT270).
The results are represented as the means ± SEM. Bars marked with different
letters are significantly different from each other (P < 0.05).
Progressive accumulation of tau aggregates in the brains of
| www.pnas.org/cgi/doi/10.1073/pnas.1220464110Sardi et al.
greater accumulation of aggregates and pathology) were stereo-
taxically injected bilaterally with 2E11 DNase-resistant particles
(drp) of AAV–GBA1 or –EV. Analysis of brain tissues of
Gba1D409V/D409Vmice at the start of the study (at 4 and 12 mo of
age) and at 6 mo postinjection with the control AAV–EV vector
showed accumulation of ubiquitin, α-synuclein, and tau aggre-
gates over this period (Fig. 3). In contrast, gene delivery of AAV-
GBA1 into the 4-mo-old Gba1D409V/D409Vmice led to reductions
of hippocampal ubiquitin, proteinase K-resistant α-synuclein, and
tau aggregates (Fig. 3). However, the reduction of ubiquitin, but
not the reductions in α-synuclein or tau, reached statistical sig-
nificance.CNSexpressionofglucocerebrosidase intheolder (12-
mo-old) mice produced a similar, but more modest, effect than
of accumulation of tau and α-synuclein but had no effect on
ubiquitin levels, suggesting that the mechanisms for accumu-
lation of these proteins may be different. It is possible that the
higher levels of aggregates present in the older animals require
a longer period or more glucocerebrosidase to be efficiently
reduced. Nevertheless, the data suggest that augmenting glu-
cocerebrosidase activity in the CNS can retard the extent of accu-
mulation of pathologically misfolded protein aggregates in symp-
CNS of Transgenic A53T α-Synuclein Mice Is Associated with Lower
Glucocerebrosidase Activities. Analyses of CSF and brain samples
of patients with PD or DLB have shown that glucocerebrosidase
activity is lower in affected than in unaffected individuals, sug-
gesting a causal role of the lysosomal enzyme in the development
that α-synuclein has the capacity to inhibit lysosomal glucocere-
brosidase activity (12, 29). To determine whether overexpression
of α-synuclein negatively affects the activity of glucocerebrosidase,
brain lysates from transgenic A53T α-synuclein mice (expressing
mutant human α-synuclein bearing the A53T mutation) were
studied (30). Similar to findings in PD patients without mutations
lysosomal glucocerebrosidase activity than did wild-type animals
(Fig. 4A). This effect was dependent on the levels of α-synuclein,
because the CNS of homozygous A53T α-synuclein mice showed
greater reductions in enzymatic activity than their (Het) litter-
mates who expressed lower levels of α-synuclein (Fig. 4A, hatched
bars). This decrease was selectively associated with glucocere-
hexosaminidase and β-galactosidase) were unaffected (Fig. 4A).
These results support the contention that high levels of α-synuclein
can inhibit lysosomal glucocerebrosidase activity, because greater
inhibition was correlated with higher levels of α-synuclein.
AAV-Mediated Expression of Glucocerebrosidase in the CNS of
Transgenic A53T α-Synuclein Mice Lowers α-Synuclein Levels. Ear-
lier, we noted that overexpression of glucocerebrosidase reduced
the accumulation of α-synuclein aggregates in the CNS of symp-
potential of glucocerebrosidase in moderating the accumulation
realized in A53T α-synuclein mice (30). The striatum of 4-mo-old
heterozygous A53T α-synuclein mice was unilaterally injected
with either AAV–GBA1 or a control virus encoding GFP (AAV–
GFP). As expected, glucocerebrosidase activity was significantly
increased (approximately sevenfold) in the ipsilateral striatum of
AAV–GBA1-injected mice compared with the contralateral side
or to AAV–GFP-injected controls (Fig. 4B). Striatal tissue homo-
genates were also subjected to serial fractionation (9) to separate
the cytosolic-soluble, membrane-associated, and cytosolic-insoluble
forms of α-synuclein. Quantitation by ELISA revealed that the
levels of cytosolic soluble α-synuclein were significantly reduced
(86 ± 3% of control, n = 5, P < 0.01) by striatal expression of
glucocerebrosidase (Fig. 4B). The levels of membrane-associated
n = 5, P = 0.07) upon expression of glucocerebrosidase (Fig. 4B).
However, the amount of the insoluble fraction was unchanged by
levels and reverses memory deficits. Four-mo-old and 12-mo-
old Gba1D409V/D409Vmice were given bilateral hippocampal
injections of either AAV–EV or AAV–GBA1. Uninjected
Gba1D409V/D409Vlittermates were euthanized at the time of
surgeries as baselines for biochemical and histological end-
points (n = 8). Age-matched, uninjected wild-type (WT; n = 9)
mice were used as a positive control. In both cohorts, tissues
were collected for biochemical and pathological analysis at
6 mo postinjection. (A) Hippocampal expression of the recombi-
nant enzyme 6 mo after stereotaxic injections. Image shows
glucocerebrosidase immunoreactivity (red) and nuclear (DAPI;
blue) stains in an AAV–GBA1-injected Gba1D409V/D409Vmouse.
(Scale bar, 400 μm.) Inset depicts glucocerebrosidase and nu-
clear staining in an AAV–EV-injected mouse. (B and C) Hippo-
campal administration of AAV–GBA1 into Gba1D409V/D409Vmice
increased glucocerebrosidase activity (B, red; n = 11, P < 0.05)
and promoted clearance of GlcSph to WT levels (C, red; n = 11,
P < 0.05), whereas AAV–EV-treated Gba1D409V/D409Vmice
showed no change in glucocerebrosidase activity (B, blue; n =
12, P > 0.05) and continued to accumulate GluSph compared
with baseline levels (C, black; n = 8, P < 0.05). (D) Presurgical
evaluation of 4-mo-old wild-type (WT) and Gba1D409V/D409Vmice revealed no object preference when exposed to two identical objects. The results from trial
1 (training) are shown as white (WT) and purple (Gba1D409V/D409Vmice) filled bars. After a 24-h retention period, mice were presented with a novel object. In
trial 2 (testing, hatched bars), WT mice investigated the novel object significantly more frequently (n = 9, P < 0.05). In contrast, Gba1D409V/D409Vmice (n = 11;
purple hatched bar) showed no preference for the novel object, indicating a cognitive impairment. (E) At 2 mo postinjection, mice were subjected to the novel
object recognition (NOR) test. AAV–GBA1-treated Gba1D409V/D409Vmice (n = 10; blue hatched bar), but not AAV–EV-treated animals (n = 9; red hatched bar),
exhibited a reversal of their memory deficit when presented with the novel object during the testing trial. (F) A separate cohort of 12-mo-old Gba1D409V/D409V
mice were injected with AAV–EV (n = 12) or AAV–GBA1 (n = 12). Similar to the 4-mo-old cohort, reversal of the memory dysfunction was observed when these
animals were tested at 2 mo postinjection (14 mo of age). The results are represented as means ± SEM. (D–F) The horizontal line demarcates 50% target
investigations, which represents no preference for either object (*, significantly different from 50%, P < 0.05). (B and C) Bars with different letters are sig-
nificantly different from each other (P < 0.05).
CNS administration of AAV–GBA1 reduces GlcSph
Sardi et al.PNAS
| February 26, 2013
| vol. 110
| no. 9
The efficacy of glucocerebrosidase in reducing α-synuclein
levels in the spinal cord of A53T α-synuclein mice was also de-
termined. Newborn A53T α-synuclein mice were injected with
AAV–GBA1 or –GFP into both cerebral lateral ventricles and
the upper lumbar spinal cord for a total dose of 3E11 drp per
pup (31). As expected, robust expression of glucocerebrosidase
(approximately threefold higher than controls) in the spinal cord
was achieved following administration of AAV–GBA1 but not
the control vector (Fig. 4C). Human α-synuclein mRNA levels
were similar in control and treated mouse brains (control, 100 ±
5%; AAV-GBA1, 95 ± 5%). Analogous to the striatal injections,
administration of AAV–GBA1 lowered α-synuclein levels in the
soluble fraction to 67 ± 7% of control (P < 0.01; Fig. 4C).
However, despite these reductions in α-synuclein, we did not
observe a significant survival benefit [the median survival of
AAV–GFP-treated mice was 290 d (n = 13) vs. 313 for AAV–
GBA1-treated mice (n = 18)]. Nevertheless, these results in-
dicate that augmenting the activity of glucocerebrosidase can
lower α-synuclein levels in the CNS of A53T α-synuclein mice.
for PD and DLB, findings from several independent studies have
supported a role for glucocerebrosidase in the pathogenesis of
these devastating diseases (4). A decrease in glucocerebrosidase
activity and presence of mutant enzyme can purportedly induce
increases in CNS α-synuclein/ubiquitin aggregates (8–12). Analy-
ses of mouse Gaucher models harboring mutations in Gba1 sug-
gest that a decrease in enzymatic activity promotes neuronal
protein misprocessing and cognitive deficits, two characteristics of
PD and DLB (8–10, 32). However, the extent to which Gba1 de-
ficiency contributes to the pathogenesis of these ailments has not
been established. This study provides further support for a role of
glucocerebrosidase in α-synuclein processing, confirms a potential
levels, and validates glucocerebrosidase augmentation in the CNS
as a therapeutic approach for diseases associated with α-synuclein
misprocessing, such as PD and DLB.
Although the precise pathologies of PD and LBD remain un-
clear, the findings of progressive accumulation of α-synuclein and
other proteins in LBs have implicated protein misfolding as a po-
tential causative mechanism (25, 33). This proteinopathy is repli-
cated in the Gba1D409V/D409Vmouse model of Gaucher disease,
which shows a progressive accumulation of tau in addition to the
described increases in α-synuclein and ubiquitin aggregates (10).
Tau and α-synuclein likely play pivotal roles in disparate sets of
neurodegenerative diseases (25). Mutations in their genes, MAPT
and SNCA, that lead to formation of tau and α-synuclein, re-
spectively, result in Alzheimer’s disease, PD, DLB, and fronto-
temporal dementia (21, 25, 34). Although the mechanisms by
which theseproteinsaggregate appeartobedifferent, α-synuclein,
for example, is spontaneously self-polymerizing (35), whereas tau
requires the presence of an inducing agent (36, 37). However, the
disease mechanisms need further clarification, because α-synuclein
fibrils may be able to promote the polymerization of tau (24, 38),
so the observed tau aggregation in the CNS of Gba1D409V/D409V
mice may be occurring secondary to α-synuclein fibrillation.
may present various degrees of cognitive impairment, including
lower cognitive scores than their non-GBA1-mutation–bearing
counterparts, suggesting that altered GBA1 increases susceptibility
to development of cognitive deficits (20). The Gba1D409V/D409V
Gaucher mouse model recapitulates many of the aberrant bio-
chemical characteristics noted in brains from PD and DLB
patients and also features measurable deficits in memory. We
dase in symptomatic Gba1D409V/D409V
mouse hippocampi slows accumulation
of aggregated α-synuclein and tau. Two
cohorts of Gba1D409V/D409Vmice were
injected with either AAV–EV or –GBA1
bilaterally into the hippocampus at 4
or 12 mo of age. Age-matched, unin-
jected WT mice were left untreated as
positive controls. Gba1D409V/D409Vlitter-
mates were harvested at the time of
the injections as a baseline group.
Injected animals were killed 6 mo af-
ter surgery. Graphs represent hippo-
proteinase K-resistant α-synuclein (B),
and tau immunoreactivity (C) for the
mo of age. Glucocerebrosidase aug-
mentation in the CNS of symptomatic
Gba1D409V/D409Vmice reduced the levels
ofaggregated proteins, although this
treatment was less effective in older
(A, green), proteinase K-resistant α-syn-
uclein (B, red) and tau (C, green) immu-
noreactivity in the hippocampi of 18-
mo-old Gba1D409V/D409Vmice treated
with AAV–EV or –GBA1. DAPI nuclear
staining is shown in blue. (Scale bar,
100 μm.) The results are represented
as means ± SEM, with n ≥ 8 per group.
Bars with different letters are sig-
nificantly different from each other
(P < 0.05).
Expression of glucocerebrosi-
| www.pnas.org/cgi/doi/10.1073/pnas.1220464110Sardi et al.
have shown that these CNS manifestations can be ameliorated in
presymptomatic animals by supplementation with exogenous
enzyme (10). Because very few patients carrying GBA1 muta-
tions will develop cognitive impairment, it was pertinent to test
whether the same salutary effects can also be realized in animals
with overt disease. We showed that AAV-mediated expres-
sion of glucocerebrosidase in both early and late symptomatic
Gba1D409V/D409Vmice was also effective in reversing cognitive
impairment. This recovery in cognition was associated with
complete clearance of GlcSph and measurable reductions in
the accumulation of the pathological aggregates. Augmenting
glucocerebrosidase activity in the CNS of Gba1D409V/D409Vmice
may possibly reduce the levels of “toxic” metabolites and thereby
improve lysosomal function, a requirement for correct synaptic
activity (39, 40) and proper functioning of pathways that degrade
aggregated proteins (41, 42). Importantly, these results strongly
suggest that augmenting CNS glucocerebrosidase activity may
impede progression of (and perhaps even reverse) some aspects of
Gaucher-related parkinsonism and associated synucleinopathies.
Ongoing investigations continue to provide greater insights into
the relationship between glucocerebrosidase and α-synuclein (4,
32). It is evident that a decrease in glucocerebrosidase activity or
the presence of mutant glucocerebrosidase can promote the ab-
errant accumulation of α-synuclein (32). Reportedly, α-synuclein
the lysosomes or inhibit its activity, thereby exacerbating the dis-
ease state (12, 29). A role for glucocerebrosidase in the disease
process is also supported by findings of decreased glucocere-
brosidase activity in the brains and CSF of sporadic PD patients,
irrespective of whether they harbor GBA1 mutations (15). To
mice that overexpress A53T α-synuclein in the CNS (30). Mea-
surements of brain lysates from A53T α-synuclein mice showed
that mice with higher levels of α-synuclein were correlated with
lower amounts of glucocerebrosidase activity. Importantly, in-
creasing glucocerebrosidase activity in the brains of A53T
α-synuclein mice reduced α-synuclein levels. These resultssuggest
that augmenting glucocerebrosidase activity in the CNS of A53T
α-synuclein mice, through its “synuclease” activity, may interrupt
the deleterious feedback of α-synuclein on glucocerebrosidase ac-
tivity and thereby restore the cell’s capacity to degrade α-synuclein.
Hence, augmenting glucocerebrosidase activity in the CNS via
administration of the recombinant enzyme, gene transfer vectors
encoding the lysosomal enzyme, or small-molecule activators of
thehydrolasemayreducetheextent ofaccumulation ofmisfolded
with or without GBA1 mutations.
In summary, the efficacy of increasing glucocerebrosidase in
modulating the extent of accumulation of aggregates in the CNS
was demonstrated in two murine models of synucleinopathy. In
a symptomatic mouse model of Gaucher-related parkinsonism and
proteinopathy, augmenting glucocerebrosidase activity in the CNS
corrected the aberrant storage of lipids, reversed cognitive dys-
function, and reduced the levels of aggregated α-synuclein and tau.
Increasing glucocerebrosidase levels in the CNS was also effective
in decreasing α-synuclein levels in the A53T α-synuclein mouse
model, indicating that the positive feedback between α-synuclein
increase and loss of glucocerebrosidase activity (10, 12) can be
lysosomes. Together, these results provide further support for the
and related synucleinopathies.
Materials and Methods
Animals. The Institutional Animal Care and Use Committee at Genzyme,
a Sanofi Company, approved all procedures. The Gba1D409V/D409Vmouse
model of Gaucher disease harbors a point mutation at residue 409 in the
murine Gba1 gene (43). Transgenic A53T α-synuclein mice express human
mouse brain decreases α-synuclein levels. A53T α-synuclein
transgenic mice exhibit decreased brain glucocerebrosidase
activity. (A) The activity of various lysosomal enzymes was
determined in cortical homogenates from homozygous (n = 9)
and heterozygous (n = 8) α-synuclein transgenics and wild-type
littermates (n = 9). Glucocerebrosidase activity was inversely
correlated with α-synuclein levels, with homozygous mice
showing a greater reduction of hydrolase activity. The enzy-
matic activities of two other lysosomal hydrolases, hexosa-
minidase and β-galactosidase, remained unchanged by the
expression of A53T α-synuclein. (B) Four-mo-old A53T α-synu-
clein mice were each injected with either AAV–GFP (n = 6) or
AAV–GBA1 (n = 5) unilaterally into the right striatum. The left
striatum was used as an uninjected control for each animal to
reduce the variability in α-synuclein levels between subjects.
Four mo later, mice were euthanized, and both striata were
collected. Robust glucocerebrosidase activity was observed in
the AAV–GBA1-injected striatum (sevenfold over the uninjected
contralateral side). Expression of glucocerebrosidase promoted
decreased α-synuclein levels in the cytosolic fraction (Tris-solu-
ble, non-membrane–associated; P < 0.05). (C) Newborn (P0)
A53T α-synuclein mice were injected with either AAV–GFP or
–GBA1 into the lumbar spinal cord. As expected, robust gluco-
cerebrosidase activity was noted in AAV–GBA1-injected mice
(threefold over controls). As in the striatum, expression of glu-
cocerebrosidase reduced α-synuclein levels in the cytosolic frac-
tion (Tris-soluble, nonmembrane associated; n = 7 per group,
P < 0.05). Data are represented as means ± SEM. *P < 0.05.
Glucocerebrosidase augmentation in A53T α-synuclein
Sardi et al.PNAS
| 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.
ACKNOWLEDGMENTS. We thank Tatyana Taksir, Kuma Misra, Denise Wood-
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.