Iminosugar-based inhibitors of glucosylceramide synthase increase brain glycosphingolipids and survival in a mouse model of Sandhoff disease.
ABSTRACT The neuropathic glycosphingolipidoses are a subgroup of lysosomal storage disorders for which there are no effective therapies. A potential approach is substrate reduction therapy using inhibitors of glucosylceramide synthase (GCS) to decrease the synthesis of glucosylceramide and related glycosphingolipids that accumulate in the lysosomes. Genz-529468, a blood-brain barrier-permeant iminosugar-based GCS inhibitor, was used to evaluate this concept in a mouse model of Sandhoff disease, which accumulates the glycosphingolipid GM2 in the visceral organs and CNS. As expected, oral administration of the drug inhibited hepatic GM2 accumulation. Paradoxically, in the brain, treatment resulted in a slight increase in GM2 levels and a 20-fold increase in glucosylceramide levels. The increase in brain glucosylceramide levels might be due to concurrent inhibition of the non-lysosomal glucosylceramidase, Gba2. Similar results were observed with NB-DNJ, another iminosugar-based GCS inhibitor. Despite these unanticipated increases in glycosphingolipids in the CNS, treatment nevertheless delayed the loss of motor function and coordination and extended the lifespan of the Sandhoff mice. These results suggest that the CNS benefits observed in the Sandhoff mice might not necessarily be due to substrate reduction therapy but rather to off-target effects.
- SourceAvailable from: Katrina L Paumier[Show abstract] [Hide abstract]
ABSTRACT: Aggregate-prone mutant proteins, such as α-synuclein and huntingtin, play a prominent role in the pathogenesis of various neurodegenerative disorders; thus, it has been hypothesized that reducing the aggregate-prone proteins may be a beneficial therapeutic strategy for these neurodegenerative disorders. Here, we identified two previously described glucosylceramide (GlcCer) synthase inhibitors, DL-threo-1-Phenyl-2-palmitoylamino-3-morpholino-1-propanol (PPMP) and Genz-123346(Genz), as enhancers of autophagy flux. We also demonstrate that GlcCer synthase inhibitors exert their effects on autophagy by inhibiting AKT-mTOR signaling. More importantly, siRNA knock down of GlcCer synthase had the similar effect as pharmacological inhibition, confirming the on-target effect. In addition, we discovered that inhibition of GlcCer synthase increased the number and size of lysosomal/late endosomal structures. Although inhibition of GlcCer synthase decreases levels of mutant α-synuclein in neurons, it does so, according to our data, through autophagy-independent mechanisms. Our findings demonstrate a direct link between glycosphingolipid biosynthesis and autophagy in primary neurons, which may represent a novel pathway with potential therapeutic value for the treatment of Parkinson's disease. This article is protected by copyright. All rights reserved.Journal of Neurochemistry 02/2014; · 3.97 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Gaucher disease (GD) and Fabry disease (FD) are two relatively common inherited glycosphingolipidoses caused by deficiencies in the lysosomal glycosidases glucocerebrosidase and alpha-galactosidase A, respectively. For both diseases enzyme supplementation is presently used as therapy. Cells and tissues of GD and FD patients are uniformly deficient in enzyme activity, but the two diseases markedly differ in cell types showing lysosomal accumulation of the glycosphingolipid substrates glucosylceramide and globotriaosylceramide, respectively. The clinical manifestation of Gaucher disease and Fabry disease is consequently entirely different and the response to enzyme therapy is only impressive in the case of GD patients. This review compares both glycosphingolipid storage disorders with respect to similarities and differences. Presented is an update on insights regarding pathophysiological mechanisms as well as recently available biochemical markers and diagnostic tools for both disorders. Special attention is paid to sphingoid bases of the primary storage lipids in both diseases. The value of elevated glucosylsphingosine in Gaucher disease and globotriaosylsphingosine in Fabry disease for diagnosis and monitoring of disease is discussed as well as the possible contribution of the sphingoid bases to (patho)physiology. This article is part of a Special Issue entitled New frontiers in sphingolipid biology.Biochimica et Biophysica Acta 11/2013; · 4.66 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Fabry disease is a lysosomal storage disorder in which neutral glycosphingolipids, predominantly globotriaosylceramide (Gb3), accumulate due to deficient α-galactosidase A (α-Gal A) activity. The α-Gal A-knockout (GLAko) mouse has been used as a model for Fabry disease, but it does not have any symptomatic abnormalities. In this study, we generated a symptomatic mouse model (G3Stg/GLAko) by crossbreeding GLAko mice with transgenic mice expressing human Gb3 synthase. G3Stg/GLAko mice had high Gb3 levels in major organs, and their serum Gb3 level at 5-25 weeks of age was 6-10 times higher than that in GLAko mice of the same age. G3Stg/GLAko mice showed progressive renal impairment, with albuminuria at 3 weeks of age, decreased urine osmolality at 5 weeks, polyuria at 10 weeks, and increased blood urea nitrogen at 15 weeks. The urine volume and urinary albumin concentration were significantly reduced in the G3Stg/GLAko mice when human recombinant α-Gal A was administered intravenously. These data suggest that Gb3 accumulation is a primary pathogenic factor in the symptomatic phenotype of G3Stg/GLAko mice, and that this mouse line is suitable for studying the pathogenesis of Fabry disease and for preclinical studies of candidate therapies.Biochemical Journal 10/2013; · 4.65 Impact Factor
Iminosugar-Based Inhibitors of Glucosylceramide
Synthase Increase Brain Glycosphingolipids and Survival
in a Mouse Model of Sandhoff Disease
Karen M. Ashe1, Dinesh Bangari1, Lingyun Li1, Mario A. Cabrera-Salazar1, Scott D. Bercury1, Jennifer B.
Nietupski1, Christopher G. F. Cooper1, Johannes M. F. G. Aerts2, Edward R. Lee1, Diane P. Copeland1,
Seng H. Cheng1, Ronald K. Scheule1, John Marshall1*
1Genzyme Corporation, Framingham, Massachusetts, United States of America, 2Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands
The neuropathic glycosphingolipidoses are a subgroup of lysosomal storage disorders for which there are no effective
therapies. A potential approach is substrate reduction therapy using inhibitors of glucosylceramide synthase (GCS) to
decrease the synthesis of glucosylceramide and related glycosphingolipids that accumulate in the lysosomes. Genz-529468,
a blood-brain barrier-permeant iminosugar-based GCS inhibitor, was used to evaluate this concept in a mouse model of
Sandhoff disease, which accumulates the glycosphingolipid GM2 in the visceral organs and CNS. As expected, oral
administration of the drug inhibited hepatic GM2 accumulation. Paradoxically, in the brain, treatment resulted in a slight
increase in GM2 levels and a 20-fold increase in glucosylceramide levels. The increase in brain glucosylceramide levels might
be due to concurrent inhibition of the non-lysosomal glucosylceramidase, Gba2. Similar results were observed with NB-DNJ,
another iminosugar-based GCS inhibitor. Despite these unanticipated increases in glycosphingolipids in the CNS, treatment
nevertheless delayed the loss of motor function and coordination and extended the lifespan of the Sandhoff mice. These
results suggest that the CNS benefits observed in the Sandhoff mice might not necessarily be due to substrate reduction
therapy but rather to off-target effects.
Citation: Ashe KM, Bangari D, Li L, Cabrera-Salazar MA, Bercury SD, et al. (2011) Iminosugar-Based Inhibitors of Glucosylceramide Synthase Increase Brain
Glycosphingolipids and Survival in a Mouse Model of Sandhoff Disease. PLoS ONE 6(6): e21758. doi:10.1371/journal.pone.0021758
Editor: Raphael Schiffmann, Baylor Research Institute, United States of America
Received May 16, 2011; Accepted June 6, 2011; Published June 29, 2011
Copyright: ? 2011 Ashe et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: All support for these studies was provided by Genzyme Corporation, through the employment of Karen M. Ashe, Dinesh Bangari, Lingyun Li, Mario A.
Cabrera-Salazar, Scott D. Bercury, Jennifer B. Nietupski, Christopher G.F. Cooper, Edward R. Lee, Diane P. Copeland, Seng H. Cheng, Ronald K. Scheule and John
Marshall, who contributed to all aspects of the study. The described studies were part of the drug development program at Genzyme Corporation. The internal
publication review committee approved the manuscript for publication.
Competing Interests: The authors have read the journal’s policy and have the following conflicts: Karen Ashe, Dinesh Bangari, Lingyun Li, Mario Cabrera-Salazar,
Scott Bercury, Jennifer Nietupski, Christopher Cooper, Edward Lee, Diane Copeland, Seng Cheng, Ronald Scheule and John Marshall are all employees of Genzyme
Corporation (a subsidiary of Sanofi-aventis Group). Johannes Aerts has received reimbursements of expenses and honoraria for lectures on lysosomal storage
diseases from Genzyme Corporation, Shire HGT and Actelion. Honoraria have been transferred to the Gaucher Stichting, a foundation that supports research in
the field of lysosomal storage disorders. Johannes Aerts is inventor on a patent (US Patent # 6,177,447 B1 issued Jan. 23, 2001) covering therapeutic applications
for Genz-529468 and has received an unrestricted study grant from Genzyme Corporation to investigate modulation of sphingolipid metabolism. This does not
alter the authors’ adherence to all the PLoS ONE policies on sharing data and materials.
* E-mail: firstname.lastname@example.org
Sandhoff disease, or type 2 GM2 gangliosidosis, is caused by
mutations in the HEXB gene and the resultant deficiency in b-
hexosaminidase activity. This deficiency causes aberrant lysosomal
accumulation of the ganglioside GM2, b-N-acetylgalactosamine-
terminal glycolipids and b-N-acetylglucosamine-terminal oligosac-
charides . Sandhoff disease manifests primarily as a neuropathic
disease of infants, though subjects exhibit a range of severities, as
noted for most lysosomal storage disorders. Presently, there are no
approved therapies for Sandhoff disease.
Glycosphingolipidoses that do not present with neuropathic
disease (such as type 1 Gaucher disease and Fabry disease) can be
treated effectively by enzyme-replacement therapy (ERT) by
periodic intravenous infusions of the respective recombinant
enzymes [2,3]. However, ERT does not address the CNS
manifestations of neuropathic glycosphingolipidoses (e.g. types 2
and 3 Gaucher disease, GM1 and GM2 gangliosidoses) since the
lysosomal enzymes are unable to traverse the blood-brain barrier
[4,5]. An alternative therapeutic strategy is to target glucosylcer-
amide synthase (GCS), the enzyme that catalyzes the first step in
the biosynthesis of glycosphingolipids. Inhibition of GCS effects
what is commonly referred to as substrate reduction therapy
(SRT). SRT is designed to abate the synthesis of glucosylceramide,
and by extension the aberrant lysosomal storage of glycosphingo-
lipids. Indeed, preclinical and clinical studies in type 1 Gaucher
disease have shown that such inhibitors significantly improve the
disease manifestations in the viscera [6,7]. Preclinical studies with
a GCS inhibitor have also shown that SRT is potentially
therapeutic in Fabry disease .
Presently, one GCS inhibitor (miglustat) has been approved for
use in mild to moderate type 1 Gaucher patients for whom ERT is
not a therapeutic option and in Niemann-Pick C patients [6,9].
Another, eliglustat tartrate, is in phase 3 clinical trials for type 1
Gaucher patients . Miglustat but not eliglustat tartrate is able to
traverse the blood-brain barrier and thus might be used to treat
PLoS ONE | www.plosone.org1 June 2011 | Volume 6 | Issue 6 | e21758
the neuropathic glycosphingolipidoses. While excessive inhibition
of the glycosphingolipid biosynthetic pathway could be detrimen-
tal to neuronal development and stabilization [10–12], the goal is
to effect a partial reduction such that the rate of synthesis is
matched by the residual capacity of the cells to degrade the
substrates. Accordingly, SRT is arguably best suited for those
indications that retain some measure of residual enzyme activity as
in type 3 Gaucher patients and late-onset Tay-Sachs diseases.
As iminosugar-based GCS inhibitors (N-butyl-deoxynojirimycin
(NB-DNJ), or miglustat) have been shown capable of entering the
brain parenchyma and improving disease outcomes in mouse
models of lysosomal storage disorders [13–15], we have elected to
evaluate structural analogs that are reportedly more potent. One
such molecule is AMP-DMP or Genz-529468 [16,17] as it is
referred to in this report. Testing was performed in a mouse model
of Sandhoff disease  that lacks b-hexosaminidase activity and
accumulates GM2 and GA2 throughout the CNS, liver and
kidney. CNS manifestations are apparent by 3 months of age and
progressive, with death occurring at 4–5 months of age. The CNS
involvement is the likely cause of death, as the peripheral nervous
system shows no significant abnormalities . The potential
utility of SRT in Sandhoff disease was elegantly demonstrated by
the generation of a mouse that was deficient in both b-
hexosaminidase and GM2/GA2 synthase . The resulting
double knockout mouse no longer accumulated glycosphingolipids
and exhibited an increased lifespan.
Previous studies have shown survival benefit when Sandhoff
mice were treated with the GCS inhibitor NB-DNJ or its galactose
analog [13,21,22]. As Genz-529468 is approximately 250-fold
more potent as a GCS inhibitor than NB-DNJ , we sought to
evaluate its therapeutic potential for neuropathic glycosphingoli-
pidoses. Our studies showed that Genz-529468 was comparable to
NB-DNJ at increasing the survival of Sandhoff mice, but could do
so at much lower doses. However, notable treatment-induced
increases in CNS glycosphingolipids, particularly of glucosylcer-
amide, suggest that the mechanism of action of the iminosugar
GCS inhibitors is not necessarily through the anticipated
mechanism of substrate reduction.
Iminosugar-based glucosylceramide synthase inhibitors
reduce GM2 levels in the livers of Sandhoff mice
To test the inhibitory activities of the GCS inhibitors, Genz-
529468 and NB-DNJ, were administered to Sandhoff mice
through their food (100 mg/kg/day and 600 mg/kg/day, respec-
tively) starting at 25 days of age. As a comparator, eliglustat
tartrate (Genz-112638), which is non blood-brain barrier perme-
ant, was included in the study. Analysis of glycosphingolipid levels
in the livers of drug-treated Sandhoff mice showed a 40–60%
reduction in GM2 levels when compared to age-matched
untreated Sandhoff mice at 112 days of age [data not shown
and 21]. Hence, the formulations of Genz-529468 and NB-DNJ
used in these studies were equally active at inhibiting non-CNS
GCS despite using a 6-fold lower dose of Genz-529468.
Genz-529468 and NB-DNJ significantly increase
glucosylceramide levels in the brains of Sandhoff mice
To evaluate whether the inhibitors (Genz-529468 and NB-DNJ)
were also active in the CNS of Sandhoff mice, their brains were
weighed and analyzed for changes in glycosphingolipid levels.
Brain weight as a ratio to body weight was not significantly
different between wild-type and Sandhoff mice at 112 days of age,
and was unaffected by treatment with Genz-529468 or NB-DNJ
(data not shown). Glycosphingolipid analysis was focused on the
proximal target of the inhibitors, namely glucosylceramide (GL1),
together with the two main storage products found in Sandhoff
disease, the gangliosides GM2 and GA2. Untreated Sandhoff and
wild-type mice harbor similar levels of GL1, but the former have
.100-fold higher levels of GM2 and GA2 (data not shown).
Contrary to our expectations, Sandhoff mice treated with Genz-
529468 beginning at 25 days of age showed a rapid increase in
brain GL1 levels, with levels rising to .10-fold those of untreated
mice after 2–3 days of treatment (Figure 1A). These GL1 levels
continued to increase further, rising to .20-fold those of untreated
mice at all subsequent time points assayed (56, 84 and 112 days of
age). Significant increases in GA2 and GM2 were also observed
(Figure 1A), though these increases were more modest (120–
150% those of untreated Sandhoff mice). Sandhoff mice treated
with NB-DNJ resulted in a similar temporal profile of brain
glycolipids, with GL1 levels increasing to .10-fold higher than
those of untreated Sandhoff control mice (Figure 1B), However,
in contrast to the effects of Genz-529468, NB-DNJ effected a
modest but significant reduction in GM2 (,90% of untreated
mice) at 84 and 112 days of age, and had no effect on GA2 levels.
Treating Sandhoff mice with the non-CNS permeant GCS
inhibitor Genz-112638 (eliglustat tartrate) had no impact on brain
In a separate study, Sandhoff mice were treated with Genz-
529468 as in Figure 1A, and brain tissue at day 112 was analyzed
for the levels of a panel of glycosphingolipids and other lipids.
Figure 1C shows that in addition to the .20-fold increase in GL1
levels in the treated mice (as in Figure 1A), the relative levels of
other glycosphingolipids in the pathway (GL2, GM3, GM2, GA2
and GM1) were also increased to 120–150% of untreated mice.
Genz-529468 treatment had no effect on the relative brain levels
of ceramide (Cer), sphingomyelin (SPM), phosphatidylcholine (PC)
and galactosylceramide (Gal), but sphingosine-1-phosphate (S1P)
levels were reduced to about 75% those of untreated Sandhoff
mice (Figure 1D). Hence, enteric administration of the
iminosugar-based GCS inhibitors Genz-529468 and NB-DNJ
acted to increase rather than decrease glycosphingolipid levels in
the brains of Sandhoff mice. In particular, GL1, the proximal
glycosphingolipid in the GCS pathway, was increased many fold
by both inhibitors. This effect on brain GL1 levels did not appear
to be restricted to the Sandhoff mouse, as similar increases in brain
GL1 levels were found in other mouse strains following treatment
with Genz-529468 or NB-DNJ (data not shown).
Treatment with iminosugar-based GCS inhibitors delays
the development of pathology in Sandhoff mouse brain
To evaluate the effects of the iminosugar-based GCS inhibitors
on the brain pathology of Sandhoff mice, time-dependent changes
in CD68 immunopositive cells, glial fibrillary acidic protein
(GFAP) expression, and a-synuclein immunopositive cells were
determined. CD68 is a marker for cells of the monocyte lineage
(macrophages, dendritic cells, microglia), granulocytes and acti-
vated T cells. In wild-type mice, CD68+cells were absent or rare
within the neuroparenchyma, though a few scattered CD68+cells
were occasionally observed in leptomeninges. In contrast, Sandh-
off mice exhibited significant numbers of CD68+cells in the brain
stem, cerebellum, hippocampus and thalamus. A few scattered
CD68+cells were also observed in the cerebral cortex and
leptomeninges. Figure 2A shows the time-dependent changes in
CD68+staining in the brain stem of untreated wild-type and
Sandhoff mice and Sandhoff mice treated with Genz-529468 or
NB-DNJ. Untreated Sandhoff mice exhibited a higher number of
CD68+cells than age-matched wild-type mice at all ages. This
Small Molecule Therapy of Sandhoff Disease
PLoS ONE | www.plosone.org2 June 2011 | Volume 6 | Issue 6 | e21758
increase was exacerbated as the Sandhoff mice aged, with a
dramatic increase in CD68+cells noted between 84 and 112 days
of age. Treatment with either Genz-529468 or NB-DNJ appeared
to reduce the number of CD68+cells at each time point. This
reduction was also apparent in other regions of the brain
(Figure 2B). Quantitative analysis of the cerebellum, hippocam-
pus and thalamus of Sandhoff mice treated with Genz-529468 or
NB-DNJ at day 112 showed a similar decrease in the number of
CD68+cells noted in the brain stem. These observations are
consistent with previous studies with GCS inhibitors [21,24] or
bone marrow transplantation .
Glial fibrillary acidic protein (GFAP) is an astrocyte marker that
increases during astrocytic activation, including in response to
neurodegeneration. Elevations in GFAP staining in the brains of
Sandhoff mice have been reported previously [24,26]. In
untreated Sandhoff mice, the most prominent GFAP immunola-
beling was observed in the brain stem, thalamus, cerebellum,
hippocampus and cerebral cortex. As with the CD68 marker,
there was a temporal increase in GFAP immunolabeling within
these brain regions, especially in the brain stem and thalamus.
Immunohistochemical analysis of the brain stem and thalamus of
Sandhoff mice treated with either Genz-529468 or NB-DNJ (at
day 112) showed a reduction in GFAP staining when compared
with age-matched wild type mice (Figure 3A). Quantitation of the
number of GFAP-positive cells confirmed these apparent histo-
logical reductions in drug-treated animals (Figure 3B).
Accumulation of a-synuclein in the CNS is a common feature of
many neurodegenerative diseases including Sandhoff disease .
Staining of cortical sections from 112 day-old wild type mice
showed no detectable a-synuclein staining (Figure 4A; panel i).
In contrast, the cortex of age-matched Sandhoff mice exhibited
clear indications of positive a-synuclein staining (Figure 4A;
panel ii). Similar results were seen in the hippocampus and brain
stem of Sandhoff mice, with more infrequent positive staining in
Figure 1. Iminosugar-based GCS inhibitors increase brain glycosphingolipid levels. Beginningat 25 daysofage,Sandhoffmiceweretreated
Brain levels of GL1, GM2 and GA2 are shown relative to amounts in untreated age-matched controls. A larger range of brain (C) glycosphingolipids and (D)
lipids, are shown following treatment with Genz-529468 at 112 days of age. Cer=ceramide, SPM=sphingomyelin, PC=phosphatidylcholine,
Gal=galactosylceramide, S1P=sphingosine-1-phosphate. Statistics were performed between Sandhoff mice that were untreated and treated with drug,
and were determined using the Graphpad Prism software t test; *=p,0.05, **=p,0.01, ***=p,0.001. Error bars indicate SEM.
Small Molecule Therapy of Sandhoff Disease
PLoS ONE | www.plosone.org3 June 2011 | Volume 6 | Issue 6 | e21758
Small Molecule Therapy of Sandhoff Disease
PLoS ONE | www.plosone.org4 June 2011 | Volume 6 | Issue 6 | e21758
the striatum and thalamus (data not shown). Intense immuno-
staining for a-synuclein was observed in the cytoplasm of neurons
in these areas. Immunoreactivity was also observed in a few
cortical neurons containing cytoplasmic vacuoles (suggesting
substrate accumulation). Sandhoff mice treated with Genz-
529468 or NB-DNJ showed a reduced number of cells staining
positive for a-synuclein and the intensity of staining was also lower
(Figure 4A; panels iii and iv). Quantitation of the number of a-
synuclein-positive cells in the cortex of 112 day-old animals
showed that treatment had reduced the number by ,50%
(Figure 4B). Together, these data suggest that the iminosugar-
based inhibitors of GCS were able to reduce the extent of
inflammation and neurodegeneration in multiple brain regions of
Genz-529468 and NB-DNJ significantly delay the rate of
loss of motor-coordination and locomotion in Sandhoff
Mouse locomotor ability was measured using an activity
chamber apparatus (open-field) in which the mice were allowed
to explore freely for 30 min. Figure 5A shows that at the 112 day
time point, both NB-DNJ and Genz-529468 treated mice
exhibited significantly greater ambulatory activity (as measured
by the distance traversed and the number of rearing events) than
the untreated Sandhoff controls. There was no significant
difference between the groups treated with NB-DNJ and Genz-
529468. These results are consistent with previous reports of
Sandhoff mice treated with iminosugar-based GCS inhibitors
Mice were also evaluated using the elevated plus maze
(habituation) and rotarod (motor coordination) assays. Motor
coordination deficits have previously been demonstrated in the
Sandhoff mouse model [18,20,26,28]. Testing was initiated prior
to the onset of overt disease symptoms and continued until either
the mice were unable to perform the test, or there were fewer than
8 mice/group surviving. No measurable deficit was detected in the
Sandhoff mice using the elevated plus maze test. However,
untreated Sandhoff mice showed a loss of motor coordination
starting at 100 days of age when tested with the rotarod assay
(Figure 5B). Treatment with Genz-529468 significantly delayed
the onset (by approximately 2 weeks) and rate of loss in motor
coordination. Sandhoff mice treated with NB-DNJ showed a
similar if not better benefit than those treated with Genz-529468
(Figure 5B). Age-matched wild-type mice showed no loss in
motor coordination over the same period.
Genz-529468 and NB-DNJ are equally effective at
increasing the longevity of Sandhoff mice
Sandhoff mice were administered the maximal effective dose of
either Genz-529468 or NB-DNJ in their diet; for Genz-529468 this
was ,100 mg/kg/day (determined empirically) and for NB-DNJ
,600 mg/kg/day . A dose of 1200 mg/kg/day of NB-DNJ
caused diarrhea and resulted in no survival benefit, although this and
higher doses have previously been reported as beneficial [13,21].
Body weights and food intake were the same between Sandhoff and
wild-type mice (through day 100), and were unaffected by treatment.
In agreement with previous studies [22,28,29], untreated Sandhoff
mice becamemoribund (criterion forsacrifice)at amedian age of135
days (Figure 6). Treatment with either iminosugar-based GCS
inhibitor significantly (p,0.0001) increased their lifespan; with Genz-
529468, the median lifespan was 181 days and with NB-DNJ it was
191 days, which represented increases of 34% and 41%, respectively.
There was no significant difference in survival between mice treated
with NB-DNJ and Genz-529468. This enhancement of survival is in
concordance with published reports using NB-DNJ [13,21,22,29].
Delivery of these inhibitors across the blood-brain barrier was key to
their efficacy, since Genz-112638 (eliglustat tartrate), which is non
blood-brain barrier-permeant [7,8] showed no survival benefit
(median lifespan of 132 days).
Substrate reduction therapy has shown promise in preclinical
studies as a therapeutic approach for several lysosomal storage
diseases, including Gaucher [6,7], Fabry , Pompe , and the
gangliosidoses [13,14]. However, as a tolerated dose of SRT is
unlikely to completely block the synthesis of the respective storage
products, this strategy is expected to be most applicable to diseases
where there is some residual enzyme activity or in which an
alternative mechanism or pathway exists for substrate removal. In
this regard, neuropathic glycosphingolipidoses that are likely to
benefit from this therapeutic paradigm are type 3 Gaucher disease,
and late-onset Sandhoff and Tay-Sachs diseases [31,32]. However,
clinical trials with miglustat (NB-DNJ) have demonstrated limited
success [33–36]. A suggestion was that the drug might not have
sufficient potency at the tolerated doses. Consequently, we elected
to evaluate the therapeutic potential of Genz-529468, another
iminosugar-based inhibitor of GCS, whose IC50is 250-fold greater
than that of miglustat .
Sandhoff mice treated with either Genz-529468 or NB-DNJ (at
their maximal effective doses) showed similar improvements in a
number of parameters assayed, perhaps reflecting a shared
mechanism of action for these two structurally related molecules.
These improvements included a delay in the loss of motor function
and coordination, reduced neuroinflammation and histopatholo-
gy, as well as increased survival. Several of these observations are
consistent with those reported previously for NB-DNJ [13,21].
However, it would appear that the use of Genz-529468, a more
potent GCS inhibitor than NB-DNJ, provided no additional
benefit in the parameters measured, suggesting that a maximal
therapeutic effect might have been attained with this class of GCS
Profiling the glycosphingolipids in the livers of Sandhoff mice
treated with the GCS inhibitors revealed the expected lowering of
the levels of the glycosphingolipids GL1, GM2 and GA2.
Paradoxically, analysis of brain lipids from treated Sandhoff mice
showed a dramatic increase in the GL1 levels as well as significant
increases in other glycosphingolipids. This finding of higher lipid
levels (GM2) in the brains of drug-treated Sandhoff mice had been
reported previously  and also for another iminosugar-based
GCS inhibitor, NB-DGJ , though these were both end-stage
measurements. The basis for the observed increase in brain GL1
levels by these iminosugar-based GCS inhibitors was likely due to
its reported secondary inhibitory activity of the non-lysosomal
enzyme b-glucosidase 2 (Gba2) [16,37,38]. Gba2 is a plasma
Figure 2. Iminosugar-based GCS inhibitors decrease the number of brain CD68 positive cells. (A) CD68 immunolabeling of brain stem
from 28, 56, 84 and 112 day old Sandhoff mice, untreated or treated with either Genz-529468 or NB-DNJ. Dark brown cells are positive for CD68; scale
bar=50 mm. (B) Quantification of CD68+cell counts in the brain stem, cerebellum, hippocampus and thalamus of 112 day old drug-treated Sandhoff
mice (n=4–5 mice per group). Cell counts are presented relative to those in untreated Sandhoff mice. Statistics are between untreated and treated
Sandhoff mice, and were determined using the Graphpad Prism software t test; *=p,0.05. Error bars indicate SEM.
Small Molecule Therapy of Sandhoff Disease
PLoS ONE | www.plosone.org5 June 2011 | Volume 6 | Issue 6 | e21758
Small Molecule Therapy of Sandhoff Disease
PLoS ONE | www.plosone.org6 June 2011 | Volume 6 | Issue 6 | e21758
membrane-associated enzyme involved in GL1 homeostasis and is
expressed maximally in testis and brain tissue . Consistent with
this suggestion is the observation that Gba2 knockout mice develop
elevated levels of GL1 in the brain, though with no apparent
detrimental effects on health .
GL1 accumulation has also been previously reported in the
testis and brain tissue of wild-type mice treated with this class of
GCS inhibitors . This increase in GL1 levels probably led to
the observed increased levels of the additional complex glyco-
sphingolipids, presumably through greater synthesis. Previous
studies using NB-DNJ in the Sandhoff mouse had not reported
altered brain GL1 levels [13,21,22,40], possibly because some
assay methods do not easily differentiate galactosylceramide from
glucosylceramide, and galactosylceramide is generally present in a
10–20 fold excess over GL1 in the mouse CNS. These data suggest
that the survival benefit elicited by the iminosugar-based GCS
inhibitors might not be primarily due to substrate reduction in the
CNS. It is possible that the increase in survival reflected a delay in
the onset or severity of disease manifestations in the visceral
organs. Indeed, bone marrow transplantation of Sandhoff mice
 has been shown to reduce storage pathology in the visceral
organs but not the brain but nevertheless conferred a 3 month
extension in longevity . However, as the non-CNS permeant
GCS inhibitor (Genz-112638) did not provide the same improve-
ments noted with the CNS-permeant inhibitors (Genz-529468 and
NB-DNJ), this could not be the sole explanation.
The documented pathophysiology of neuropathic diseases such
as Sandhoff  and the complex roles of gangliosides in the CNS
 provide some potential mechanisms of action through which
the iminosugar-based GCS inhibitors might have worked to effect
the observed positive outcomes. For example, it is possible that
their activities altered the extent of neurodegeneration, inflamma-
tion, autophagy and intracellular calcium regulation. Changing
the lipid profiles in the brain to contain higher levels of GM1 and
GL1 and lower levels of sphingosine-1-phosphate could have
contributed to moderating disease severity. GM1 has been shown
to enhance the functional recovery of damaged neurons , and
GL1 reportedly can stimulate neuronal growth and development
. The noted Genz-529468-mediated reduction in sphingosine-
1-phosphate levels could also have translated to a reduction in
astroglial proliferation in the Sandhoff mice as suggested
previously . As inflammation is a major pathophysiologic
feature of Sandhoff disease [24,45] and a contributor to
neurodegeneration or apoptosis , these inhibitors could also
be acting to limit the inflammatory response. Anti-inflammatory
drugs have been reported to provide a survival benefit in the
Sandhoff mouse [26,29]. Similarly, survival benefit following
bone-marrow transplantation in Sandhoff mice has been postu-
lated as being through an anti-inflammatory mechanism [22,28].
Genz-529468 exhibits systemic anti-inflammatory properties
[47,48], which raises the possibility that this might be part of the
basis for the improved survival seen in the treated Sandhoff mice.
Brains of animals treated with Genz-529468 showed less
astrogliosis and microglial activation, which in turn might have
reduced the degree of neuronal damage. Treatment also caused
significant reductions in both the intensity and number of a-
synuclein positive aggregates in the brain. In murine models of
Parkinson’s disease, aggregates of a-synuclein have been shown to
activate microglia and amplify neurodegenerative processes
In summary, these studies clearly demonstrated and confirmed
the ability of iminosugar-based GCS inhibitors to delay the onset
of disease and increase the longevity of a mouse model of Sandhoff
disease. However, contrary to prior suggestions [13,21,22] it
would appear that these benefits are unrelated to substrate
reduction therapy, since treatment led to elevated levels of
glycosphingolipids in the brain. Potential alternate mechanisms
to explain the observed benefits of this class of drugs might be
through their ability to (i) lessen the extent of a-synuclein
aggregation, (ii) act as an anti-inflammatory agent or (iii) inhibit
the non-lysosomal b-glucosidase resulting in altered levels of
neuronal glycosphingolipids. Further studies are necessary to
elucidate fully the basis for the neurologic benefits of this class of
GCS inhibitors in Sandhoff mice.
Materials and Methods
Ethics Statement: Procedures involving mice were reviewed and
approved by Genzyme Corporation’s Institutional Animal Care
and Use Committee (Protocol 07-1115-2-BC) following guidelines
established by the Association for Assessment of Accreditation of
Laboratory Animal Care. The review board specifically approved
all the studies (identification numbers 09-3706, 09-3784, 09-4157,
09-4231) reported in this manuscript. Sandhoff mice  were
purchased from Jackson Labs (Bar Harbor, ME) and contract bred
at Charles River Labs (Bedford, MA). This mouse model develops
neurodegenerative disease and exhibits physical difficulties in
feeding, drinking and grooming at about 100 days of age. To
minimize the potential for suffering, mice were assessed daily from
day 80 and euthanized when they were unable to right themselves
from a supine position within 30 sec.
Beginning at 3–4 weeks of age, animals were given drug as a
component of the pellet food diet. Drugs were formulated at
0.05% (Genz-529468) or 0.3% (NB-DNJ) w/w in a standard
mouse chow (LabDiet 5053, TestDiet, Richmond, IN) and
provided ad libitum. This formulation was calculated to provide
100 mg/kg of Genz-529468 or 600 mg/kg of NB-DNJ per day for
a 25 g mouse eating 5 g of food per day. These doses of the GCS
inhibitors were considered maximal based on pilot tolerability and
Mice were evaluated for motor coordination and locomotion
using accelerating rotarod and open-field assays, respectively.
Tests were run weekly at the same time of day on each occasion.
The rotarod assay consisted of placing the animals on a 30 mm
diameter spindle at a height of 30 cm. The Smartrod program
(AccuScan Instruments, Columbus, OH) controlled the accelera-
tion from 0–15 rpm over 60 s. The time to fall (latency) was
automatically recorded by a light beam sensor underneath the
spindle. Each animal was subjected to 4 trials at each time point
Figure 3. Iminosugar-based GCS inhibitors decrease brain GFAP positive cells. (A) GFAP immunolabeling of brain stem (left panels) or
thalamus (right panels) of 112 day old wild-type or Sandhoff mice (untreated or treated with either Genz-529468 or NB-DNJ). Dark brown cells are
positive for GFAP; scale bar=50 mm. (B) Quantitative analysis of GFAP staining in brain stem and thalamus of drug-treated Sandhoff mice (n=4–5
mice per group). GFAP stained area is presented relative to that of untreated Sandhoff mice. Statistics are between untreated and treated Sandhoff
mice, and were determined using the Graphpad Prism software t test; *=p,0.05, ***=p,0.001. Error bars indicate SEM.
Small Molecule Therapy of Sandhoff Disease
PLoS ONE | www.plosone.org7June 2011 | Volume 6 | Issue 6 | e21758
Small Molecule Therapy of Sandhoff Disease
PLoS ONE | www.plosone.org8 June 2011 | Volume 6 | Issue 6 | e21758
(the first result on each assay day was discarded), with an ,30 min
rest period between each trial. The open-field assay (Med
Associates, Georgia, VT) was used to measure locomotor activity.
Mice were placed individually into a 30 cm/side square high-
walled arena. Movement was automatically detected using a series
of sensor light beams to measure horizontal and vertical
movement. Trials were performed for 30 min in a noise-controlled
room. Data were analyzed using Activity Monitor software (Med
Associates, St. Albans, VT) to evaluate total ambulatory
movement and rearing.
Tissue processing and immunohistochemistry
Mice at 28, 56, 84 and 112 days of age were transcardially
perfused with 0.9% sodium chloride solution and tissues fixed in
fresh 4% paraformaldehyde for 2 days at 4uC. Tissues were
embedded in paraffin and 5 mm sections were cut. CD68 staining
was performed using the Bond Polymer Refine Detection System
(BPRDS; Leica Microsystems, Bannockburn, IL). Brain sections
were incubated with Proteinase K (DAKO, Carpinteria, CA) for
5 min for antigen retrieval prior to staining. Sections were then
incubated with either an anti-CD68 antibody (clone FA-11;
Serotec, Raleigh, NC) or an isotype-matched non-specific
antibody (rat IgG2a; Serotec, Raleigh, NC). Secondary detection
was with a rabbit anti-rat antibody (Vector Labs, Burlingame,
CA). Glial Fibrillary Acidic Protein (GFAP) staining was
performed using the BPRDS and an anti-GFAP antibody (DAKO,
Carpinteria, CA). An isotype-matched, non-specific antibody
(rabbit IgG, Jackson Immunoresearch, West Grove, PA) was used
as the negative control. For a-synuclein staining, antigen retrieval
was achieved by treatment with 70% formic acid (Sigma, St.
Louis, MO) for 10 min prior to boiling in Antigen Unmasking
Solution (Vector Labs, Burlingame, CA) for 30 min. Endogenous
peroxidase activity was quenched by immersion in 0.3% hydrogen
peroxide (Sigma) in methanol for 30 min and a-synuclein was
detected by incubating with rabbit anti-a-synuclein antibody
(Sigma) overnight at 4uC. Visualization was achieved using goat
Figure 4. Iminosugar-based GCS inhibitors decrease brain a-synuclein aggregates. (A) Immunolabeling of a-synuclein in the cortex (Cx)
adjacent to the corpus callosum (CC) of 112 day old (i) wild-type or (ii) untreated Sandhoff mice or (iii) Sandhoff mice treated with Genz-529468 or (iv)
Sandhoff mice treated with NB-DNJ. Dark cells (arrows) are positive for a-synuclein; scale bar=50 mm. Inset shows a further 36magnification of a-
synuclein positive cells. (B) Quantitation of a-synuclein positive cells in the cortex of untreated or drug-treated 112 day old Sandhoff mice (n=4–5
mice per group). Statistics are between untreated and treated Sandhoff mice, and were determined using the Graphpad Prism software t test;
***=p,0.001. Error bars indicate SEM.
Figure 5. Iminosugar-based GCS inhibitors improve Sandhoff
mouse function. (A) Mice were evaluated in an open-field assay at
112 days of age. Total distance traversed (ambulatory distance) and the
number of times the mice raised onto their hind legs (rearing events)
over 30 min are shown. (n=15/group. Statistics are between untreated
and treated Sandhoff mice, and were determined using the Graphpad
Prism software t test; **=p,0.01, ***=p,0.001. Error bars indicate
SEM). (B) Mice were evaluated for motor coordination using the rotarod
assay. The amount of time (in secs) the mice remained on the rotarod is
reported as the latency. Latency is shown for wild-type mice, Genz-
529468- and NB-DNJ-treated Sandhoff mice and untreated Sandhoff
mice. (n=15/group). Statistics compared untreated Sandhoff to Genz-
529468-treated Sandhoff mice, and were determined using the
Graphpad Prism software t test; * = p,0.05, ** = p,0.01,
***=p,0.001. Error bars indicate SEM.
Figure 6. Iminosugar-based GCS inhibitors increase Sandhoff
mouse survival. Mice were monitored daily from 80 days of age and
euthanized when they became moribund and were unable to right
themselves from a supine position within 30 sec. Untreated mice
displayed a median survival of 135 days; Sandhoff mice treated with
Genz-5294468 or NB-DNJ had median survivals of 181 days and 191
days, respectively. Both iminosugar-based GCS inhibitors significantly
(p,0.0001) increased survival relative to that of untreated Sandhoff
Small Molecule Therapy of Sandhoff Disease
PLoS ONE | www.plosone.org9 June 2011 | Volume 6 | Issue 6 | e21758
anti-rabbit-HRP followed by diaminobenzidine (DAB) and
counterstained with hematoxylin.
For quantitation, three non-overlapping fields of view for each
brain region were examined at 4006magnification (for counting
CD68 positive cells) or 2006 (for counting a-synuclein positive
cells) with an n of at least 3 per group. GFAP immunopositive
areas were quantitated at 4006 in the brain stem and thalamus
using the MetaMorph image analysis software (Molecular Devices,
Inc., Sunnyvale, CA). The images were thresholded for brown
areas corresponding to GFAP immunostaining. GFAP immuno-
positive area in each section was determined using three
representative, non-overlapping images.
Quantitative analysis of sphingolipids was performed by liquid
chromatography and tandem mass spectrometry (LC/MS/MS)
. Briefly, tissue was homogenized in 10 times its volume of
water (w/v) and 10 ml of homogenate was extracted with 1 ml of
an organic solvent mixture (acetonitrile, methanol, acetic acid, 50/
50/1:v/v/v) for 10 min under strong vortex. For sphingomyelin,
phosphatidylcholine, GL2, GM3, GM2, GA2 and GM1 analyses,
an Acquity HILIC column (2.16100 mm, Waters Corp., Milford,
MA) was used to separate the glycosphingolipids and phospholip-
ids that were then analyzed by triple quadrupole tandem mass
spectrometry (API 4000, Applied Biosystems/MDS SCIEX,
Carlsbad, CA) using MRM mode. An Atlantis HILIC column
(Waters Corp., Milford, MA) was used to separate GL1 and
galactosylceramide prior to detection by tandem mass spectrom-
etry (API 4000). For ceramide and sphingosine-1-phosphate
analyses, a reverse phase column (Acquity C8 2.16100 mm,
Waters Corp., Milford, MA) was used to separate different
isoforms of ceramide before analysis by tandem mass spectrometry
(API 5000 detector). Sphingolipid standards were obtained from
Matreya, LLC (Pleasant Gap, PA).
The authors would like to thank the sterling work performed by the
department of Comparative Medicine at Genzyme, especially Leah Curtin,
Terry Hartnett, Erik Zarazinski and Christina Norton. We also wish to
acknowledge the expertise of Kerry McEachern from the Applied
Discovery Research group and Jonathan Fidler, Lisa Stanek, Jennifer
Matthews and Lamya Shihabuddin from the Neuroscience group, Eva
Budman from the Analytical Chemistry department, John Lydon and the
other members of the Histology and Pathology departments.
Conceived and designed the experiments: KMA DB DPC SHC RKS JM.
Performed the experiments: KMA DB LL MAC-S SDB JBN JM. Analyzed
the data: KMA DB LL JMFGA SHC RKS JM. Contributed reagents/
materials/analysis tools: DB CGFC JMFGA ERL JM. Wrote the paper:
SHC RKS JM.
1. Gravel RA, Kaback MM, Proia RL, Sandhoff K, Suzuki K, et al. (2001) The
GM2 Gangliosidoses. In Scriver C, Beaudet A, Sly W, Valle D, eds. The
Metabolic and Molecular Basis of Inherited Disease (8thed.), McGraw-Hill, New
York. pp 3827–3876.
2. Weinreb NJ, Charrow J, Andersson HC (2002) Effectiveness of enzyme
replacement therapy in 1028 patients with type 1 Gaucher disease after 2–5
years of treatment: a report from the Gaucher Registry. Am J Med 113:
3. Wilcox WR, Banikazemi M, Guffon N, Waldek S, Lee P, et al. (2004) Long-term
safety and efficacy of enzyme replacement therapy for Fabry disease. Am J Hum
Genet 75: 65–74.
4. Davies EH, Erikson A, Collin-Histed T, Mengel E, Tylki-Szymanska A, et al.
(2007) Outcome of type III Gaucher disease on enzyme replacement therapy:
review of 55 cases. J Inherit Metab Dis 30(6): 935–942.
5. Zimran A, Elstein D (2007) No justification for very high-dose enzyme therapy
for patients with type III Gaucher disease. J Inherit Metab Dis 30(6): 843–844.
6. Lachmann RH, Platt FM (2001) Substrate reduction therapy for glycosphingo-
lipid storage disorders. Expert Opinions Investig Drugs 10: 455–466.
7. Lukina E, Watman N, Arreguin EA, Banikazemi M, Dragosky M, et al. (2010) A
Phase 2 study of eliglustat tartrate (Genz-112638), an oral substrate reduction
therapy for Gaucher disease type 1. Blood 116(6): 893–899.
8. Marshall J, Ashe KM, Bangari D, McEachern K, Chuang W-L, et al. (2010)
Substrate reduction augments the efficacy of enzyme therapy in a mouse model
of Fabry disease. PLoS ONE 5(11): e15033.
9. Wraith JE, Vecchio D, Jacklin E, Abel L, Chadha-Boreham H, et al. (2010)
Miglustat in adult and juvenile patients with Niemann-Pick disease type C: long-
term data from a clinical trial. Molec Gen Metab 99(4): 351–357.
10. Yamashita T, Wu YP, Sandhoff R, Werth N, Mizukami H, et al. (2005)
Interruption of ganglioside synthesis produces central nervous system degener-
ation and altered axon-glial interactions. Proc Natl Acad Sci USA 102:
11. Jennemann R, Sandhoff R, Wang S, Kiss E, Gretz N, et al. (2005) Cell-specific
deletion of glucosylceramide synthase in brain leads to severe neural defects after
birth. Proc Natl Acad Sci USA 102(35): 12459–12464.
12. Yamashita T, Allende ML, Kalkofen DN, Werth N, Sandhoff K, et al. (2005)
Conditional LoxP-flanked glucosylceramide synthase allele controlling glyco-
sphingolipid synthesis. Genesis (New York, NY : 2000) 43(4): 175–180.
13. Jeyakumar M, Butters TD, Cortina-Borja M, Hunnam V, Proia RL, et al. (1999)
Delayed symptom onset and increased life expectancy in Sandhoff disease mice
treated withN-butyldeoxynojirimycin. Proc Natl Acad Sci USA 96(11):6388–6393.
14. Platt FM, Jeyakumar M, Andersson U, Heare T, Dwek RA, et al. (2003)
Substrate reduction therapy in mouse models of the glycosphingolipidoses.
Philosophical transactions of the Royal Society of London Series B, Biological
sciences, 358(1433): 947–954.
15. Kasperzyk JL, d’Azzo A, Platt FM, Alroy J, Seyfried TN (2005) Substrate
reduction reduces gangliosides in postnatal cerebrum-brainstem and cerebellum
in GM1 gangliosidosis mice. J Lipid Res 46(4): 744–751.
16. Overkleeft HS, Renkema GH, Neele J, Vianello P, Hung IO, et al. (1998)
Generation of specific deoxynojirimycin-type inhibitors of the non-lysosomal
glucosylceramidase. J Biol Chem 273(41): 26522–26527.
17. Aerts JM, Ottenhoff R, Powlson AS, Grefhorst A, van Eijk M, et al. (2007)
Pharmacological inhibition of glucosylceramide synthase enhances insulin
sensitivity. Diabetes 56(5): 1341–1349.
18. Sango K, Yamanaka S, Hoffmann A, Okuda Y, Grinberg A, et al. (1995) Mouse
models of Tay-Sachs and Sandhoff diseases differ in neurologic phenotype and
ganglioside metabolism. Nature Gen 11(2): 170–176.
19. McNally MA, Baek RC, Avila RL, Seyfried TN, Strichartz GR, et al. (2007)
Peripheral nervous system manifestations in a Sandhoff disease mouse model:
nerve conduction, myelin structure, lipid analysis. J Neg Results Biomed 6: 8.
20. Liu Y, Wada R, Kawai H, Sango K, Deng C, et al. (1999) A genetic model of
substrate deprivation therapy for a glycosphingolipid storage disorder. J Clin
Invest 103(4): 497–505.
21. Andersson U, Smith D, Jeyakumar M, Butters TD, Cortina-Borja M, et al.
(2004) Improved outcome of N-butyldeoxygalactonojirimycin-mediated sub-
strate reduction therapy in a mouse model of Sandhoff disease. Neurobiol Dis
22. Jeyakumar M, Norflus F, Tifft CJ, Cortina-Borja M, Butters TD, et al. (2001)
Enhanced survival in Sandhoff disease mice receiving a combination of substrate
deprivation therapy and bone marrow transplantation. Blood 97(1): 327–329.
23. Wennekes T, Meijer AJ, Groen AK, Boot RG, Groener JE, et al. (2010) Dual-
action lipophilic iminosugar improves glycemic control in obese rodents by
reduction of visceral glycosphingolipids and buffering of carbohydrate
assimilation. J Med Chem 53(2): 689–698.
24. Jeyakumar M, Thomas R, Elliot-Smith E, Smith DA, van der Spoel AC, et al.
(2003) Central nervous system inflammation is a hallmark of pathogenesis in
mouse models of GM1 and GM2 gangliosidosis. Brain 126(Pt 4): 974–987.
25. Wada R, Tifft CJ, Proia RL (2000) Microglial activation precedes acute
neurodegeneration in Sandhoff disease and is suppressed by bone marrow
transplantation. PNAS USA 97(20): 10954–10959.
26. Wu YP, Proia RL (2004) Deletion of macrophage-inflammatory protein 1 alpha
retards neurodegeneration in Sandhoff disease mice. Proc Natl Acad Sci USA
27. Suzuki K, Iseki E, Katsuse O, Yamaguchi A, Katsuyama K, et al. (2003)
Neuronal accumulation of a- and b-synucleins in the brain of a GM2
gangliosidosis mouse model. Clin Neurosci Neuropath 14(4): 551–554.
28. Norflus F, Tifft CJ, McDonald MP, Goldstein G, Crawley JN, et al. (1998) Bone
marrow transplantation prolongs life span and ameliorates neurologic
manifestations in Sandhoff disease mice. J Clin Invest 101(9): 1881–1888.
Small Molecule Therapy of Sandhoff Disease
PLoS ONE | www.plosone.org10June 2011 | Volume 6 | Issue 6 | e21758
29. Jeyakumar M, Smith DA, Williams IM, Borja MC, Neville DC, et al. (2004)
NSAIDs increase survival in the Sandhoff disease mouse: synergy with N-
butyldeoxynojirimycin. Ann Neurol 56(5): 642–649.
30. Ashe KM, Taylor KM, Chu Q, Meyers E, Ellis A, et al. (2010) Inhibition of
glycogen biosynthesis via mTORC1 suppression as an adjunct therapy for
Pompe disease. Molec Gen Metab 100: 309–315.
31. Lachmann R (2009) Substrate-reduction therapy with miglustat for glycosphin-
golipid storage disorders affecting the brain. Expert Rev Endocrin Metab 4(3):
32. Schiffmann R (2010) Therapeutic approaches for neuronopathic lysosomal
storage disorders. J Inherit Metab Dis 33(4): 373–379.
33. Schiffmann R, Fitzgibbon E, Harris C, Devile C, Davies E, et al. (2008)
Randomized, controlled trial of miglustat in Gaucher’s disease type 3. Annals
Neurol 64(5): 514–222.
34. Shapiro BE, Pastores GM, Gianutsos J, Luzy C, Kolodny EH (2009) Miglustat
in late-onset Tay-Sachs disease: a 12-month, randomized, controlled clinical
study with 24 months of extended treatment. Genetics in Medicine 11(6):
35. Maegawa GH, Banwell BL, Blaser S, Sorge G, Toplak M, et al. (2009) Substrate
reduction therapy in juvenile GM2 gangliosidosis. Molec Gen Metab 98(1–2):
36. Masciullo M, Santoro M, Modoni A, Ricci E, Guitton J, et al. (2010) Substrate
reduction therapy with miglustat in chronic GM2 gangliosidosis type Sandhoff:
results of a 3-year follow-up. J Inherit Metab Dis 33: DOI 10.1007/s10545-010-
37. Yildiz Y, Matern H, Thompson B, Allegood JC, Warren RL, et al. (2006)
Mutation of beta-glucosidase 2 causes glycolipid storage disease and impaired
male fertility. J Clin Invest 116(11): 2985–2994.
38. Boot RG, Verhoek M, Donker-Koopman W, Strijland A, van Marle J, et al.
(2007) Identification of the non-lysosomal glucosylceramidase as b-glucosidase 2.
J Biol Chem 282(2): 1305–1312.
39. Walden CM, Sandhoff R, Chuang CC, Yildiz Y, Butters TD, et al. (2007)
Accumulation of glucosylceramide in murine testis, caused by inhibition of beta-
glucosidase 2: implications for spermatogenesis. J Biol Chem 282(45):
40. Pelled D, Lloyd-Evans E, Riebeling C, Jeyakumar M, Platt FM, et al. (2003)
Inhibition of calcium uptake via the sarco/endoplasmic reticulum Ca2+-ATPase
in a mouse model of Sandhoff disease and prevention by treatment with N-
butyldeoxynojirimycin. J Biol Chem 278(32): 29496–29501.
41. Bellettato C, Scarpa M (2010) Pathophysiology of neuropathic lysosomal storage
disorders. J Inherit Metab Dis 33(4): 347–362.
42. Geisler FH, Dorsey FC, Coleman WP (1991) Recovery of motor function after
spinal-cord injury–a randomized, placebo-controlled trial with GM-1 ganglio-
side. New Engl J Med 324(26): 1829–1838.
43. Bodennec J, Pelled D, Riebeling C, Trajkovic S, Futerman AH (2002)
Phosphatidylcholine synthesis is elevated in neuronal models of Gaucher disease
due to direct activation of CTP:phosphocholine cytidylyltransferase by
glucosylceramide. FASEB 16(13): 1814–1816.
44. Wu YP, Mizugishi K, Bektas M, Sandhoff R, Proia RL (2008) Sphingosine
kinase 1/S1P receptor signaling axis controls glial proliferation in mice with
Sandhoff disease. Hum Molec Gen 17(15): 2257–2264.
45. Myerowitz R, Lawson D, Mizukami H, Mi Y, Tifft CJ, et al. (2002) Molecular
pathophysiology in Tay-Sachs and Sandhoff diseases as revealed by gene
expression profiling. Hum Molec Gen 11(11): 1343–1350.
46. Huang JQ, Trasler JM, Igdoura S, Michaud J, Hanal N, et al. (1997) Apoptotic
cell death in mouse models of GM2 gangliosidosis and observations on human
Tay-Sachs and Sandhoff diseases. Hum Molec Gen 6(11): 1879–1885.
47. Shen C, Bullens D, Kasran A, Maerten P, Boon L, et al. (2004) Inhibition of
glycolopid bisynthesis by N-(5-adamantane-1-yl-methoxy-pentyl)-deoxynojirimy-
cin protects against the inflammatory response in hapten-induced colitis. Int
Immunopharma 4: 939–951.
48. Van Eijk M, Aten J, Bijl N, Ottenhoff R, van Roomen CPAA, et al. (2009)
Reducing glycosphingolipid content in adipose tissue of obese mice restores
insulin sensitivity, adipogenesis and reduces inflammation. PLoS ONE 4(3):
49. Zhang W, Wang T, Pei Z, Miller DS, Wu X, et al. (2005) Aggregated a-
synuclein activates microglia: a process leading to disease progression in
Parkinson’s disease. FASEB J 19: 533–542.
50. Lee E-J, Woo M-S, Moon P-G, Baek M-C, Choi I-Y, et al. (2010) a-Synuclein
activates microglia by inducing the expressions of matrix metalloproteinases and
the subsequent activation of protease-activated receptor-1. J Immunol 185:
51. Merrill AH, Sullards MC, Allegood JC, Kelly S, Wang E (2005) Sphingolipi-
domics: high-throughput, structure-specific, and quantitative analysis of
sphingolipids by liquid chromatography tandem mass spectrometry. Methods
(San Diego, Calif) 36(2): 207–224.
Small Molecule Therapy of Sandhoff Disease
PLoS ONE | www.plosone.org11 June 2011 | Volume 6 | Issue 6 | e21758