Vigabatrin Inhibits Seizures and mTOR Pathway
Activation in a Mouse Model of Tuberous Sclerosis
Bo Zhang, Sharon S. McDaniel, Nicholas R. Rensing, Michael Wong*
Department of Neurology and the Hope Center for Neurological Disorders, Washington University School of Medicine, St. Louis, Missouri, United States of America
Epilepsy is a common neurological disorder and cause of significant morbidity and mortality. Although antiseizure
medication is the first-line treatment for epilepsy, currently available medications are ineffective in a significant percentage
of patients and have not clearly been demonstrated to have disease-specific effects for epilepsy. While seizures are usually
intractable to medication in tuberous sclerosis complex (TSC), a common genetic cause of epilepsy, vigabatrin appears to
have unique efficacy for epilepsy in TSC. While vigabatrin increases gamma-aminobutyric acid (GABA) levels, the precise
mechanism of action of vigabatrin in TSC is not known. In this study, we investigated the effects of vigabatrin on epilepsy in
a knock-out mouse model of TSC and tested the novel hypothesis that vigabatrin inhibits the mammalian target of
rapamycin (mTOR) pathway, a key signaling pathway that is dysregulated in TSC. We found that vigabatrin caused a modest
increase in brain GABA levels and inhibited seizures in the mouse model of TSC. Furthermore, vigabatrin partially inhibited
mTOR pathway activity and glial proliferation in the knock-out mice in vivo, as well as reduced mTOR pathway activation in
cultured astrocytes from both knock-out and control mice. This study identifies a potential novel mechanism of action of an
antiseizure medication involving the mTOR pathway, which may account for the unique efficacy of this drug for a genetic
Citation: Zhang B, McDaniel SS, Rensing NR, Wong M (2013) Vigabatrin Inhibits Seizures and mTOR Pathway Activation in a Mouse Model of Tuberous Sclerosis
Complex. PLoS ONE 8(2): e57445. doi:10.1371/journal.pone.0057445
Editor: Gilberto Fisone, Karolinska Inst, Sweden
Received October 1, 2012; Accepted January 24, 2013; Published February 20, 2013
Copyright: ? 2013 Zhang 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: This work was supported by the National Institutes of Health (R01 NS056872 and P20 NS080199 to M.W.; NIH Neuroscience Blueprint Core Grant
NS057105 to Washington University). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: email@example.com
Epilepsy is one of the most common neurological disorders and
is characterized by recurrent seizures, which may result in
significant morbidity and mortality. The first-line treatment for
epilepsy is antiseizure medication . While over twenty such
medications exist and are effective in many cases, available
medications have significant limitations. About one-third of
patients with epilepsy are intractable to all medications [1–3].
Even when effective, current medications act primarily as
symptomatic treatments in suppressing seizures, but do not
actually prevent epilepsy . While antiseizure medications target
a number of mechanisms of action in the brain, most medications
directly inhibit neuronal activity, primarily via modulation of ion
channels or neurotransmitter receptors. Although some medica-
tions are better for particular types of seizures or epilepsy
syndromes, overall all medications are relatively equivalent and
non-specific in their efficacy for different types of epilepsy .
There are very few, if any, examples of specific targeted therapies
for epilepsy with unique effectiveness based on mechanism of
Tuberous sclerosis complex (TSC) is one of the most common
genetic causes of epilepsy [6,7]. The seizures in TSC frequently
present in childhood, can be of multiple types and are often
associated with other neurological problems, such as developmen-
tal delay and autism. Infantile spasms, a particularly devastating
form of seizures in infants, occur in about one-third of TSC
patients. Overall, the majority of patients with TSC and epilepsy
have medically-intractable epilepsy . Interestingly, however,
seizures in TSC are highly-responsive to the drug, vigabatrin
(VGB), with a ,95% efficacy in stopping infantile spasms in TSC
patients [8,9]. Furthermore, resolution of seizures is often
associated with improved developmental progress. Recently it
has been proposed that starting VGB at an early age, at or prior to
the onset of clinical seizures, may improve the long-term outcome
of epilepsy and neurodevelopment in TSC patients [10,11]. Thus,
VGB may represent a rare example of a medication that has
specific efficacy for a particular type or cause of epilepsy. VGB is
known to have antiseizure effects by elevating brain gamma-
aminobutyric acid (GABA) levels via inhibition of its breakdown
by GABA transaminase [12–14]. However, since VGB and other
GABA-modulating drugs are not as effective in other types of
epilepsy, whether this or some other mechanism accounts for
VGB’s unique effectiveness for seizures in TSC is poorly
In addition to epilepsy, developmental delay, and autism, TSC
is characterized by the tendency to form tumors in the brain and
other organs . Recently, significant advances in understanding
the genetics and molecular pathophysiology of TSC have been
made, which largely explain the mechanistic basis of tumorigenesis
in this disease. Two genes, TSC1 and TSC2, cause TSC and
PLOS ONE | www.plosone.org1 February 2013 | Volume 8 | Issue 2 | e57445
encode the proteins, hamartin and tuberin, respectively, which
bind together to form a functional complex that inhibits the
mammalian target of rapamycin (mTOR) pathway. As the mTOR
pathway is normally involved in stimulation of cell growth and
proliferation, mutation of one of the TSC genes leads to
disinhibition of the mTOR pathway, which promotes excessive
cell growth and tumor formation in TSC. The mTOR pathway,
particularly mTOR complex 1 (mTORC1), is involved in a variety
of other functions and has also been implicated in epileptogenesis
in TSC [16,17]. mTORC1 inhibitors are proven therapies for
tumor growth in TSC  and are being investigated as
treatments for epilepsy and other neurological complications of
TSC. Our novel hypothesis is that vigabatrin has its unique
efficacy for seizures in TSC by modulating the mTOR pathway.
In this study, we examined the effect of VGB on epilepsy in a
mouse model of TSC, including possible interactions with the
Materials and Methods
Care and use of animals were conducted according to an animal
protocol approved by the Washington University Animal Studies
Committee (IACUC #A-3381-01, Approval #2010-0235). All
efforts were made to minimize animal discomfort and reduce the
number of animals used.
Animals and drug treatment
Tsc1flox/flox-GFAP-Cre knock-out (Tsc1GFAPCKO) mice with
conditional inactivation of the Tsc1 gene predominantly in glia
were generated as described previously . Tsc1flox/+-GFAP-Cre
and Tsc1flox/floxlittermates have previously been found to have no
abnormal phenotype and were used as control animals in these
Three-week-old Tsc1GFAPCKO mice were treated with vehicle
(saline) or VGB at different doses (50, 100, 200 mg/kg/day, i.p)
for one week for Western blot and GABA concentrations, for four
weeks (200 mg/kg/day) for histology and immunohistochemistry
analysis, and for up to 10 weeks (200 mg/kg/day) for video-EEG
monitoring. Three weeks of age is just prior to the previously-
documented onset of seizures and pathological abnormalities in
Tsc1GFAPCKO mice [19–21]; thus this protocol has the potential
to prevent the onset of neurological abnormalities in these mice, as
previously demonstrated for rapamycin . The dosing for the
histology and video-EEG experiments were selected to maximize
the chance of detecting an effect based on the western blot
experiments, as well as on clinically-relevant dosing. Vehicle-
treated non-KO littermates served as additional controls. Other
vehicle or VGB-treated Tsc1GFAP1CKO mice and control mice
were monitored for body and brain weight measurements (for up
to 4 weeks) or for survival analysis.
Vehicle- and VGB-treated Tsc1GFAPCKO mice underwent
weekly video-EEG monitoring starting at 3 weeks of age, using
established methods for implanting epidural electrodes and
performing continuous video-EEG recordings, as described
previously [20,21]. Briefly, mice were anesthetized with isoflurane
and placed in a stereotaxic frame. Epidural screw electrodes were
surgically implanted and secured using dental cement for long
term EEG recordings. Four electrodes were placed on the skull:
one right and one left central electrodes (1 mm lateral to midline,
2 mm posterior to bregma), one frontal electrode (0.5 mm anterior
and 0.5 mm to the right or left of bregma) and one occipital
electrode (0.5 mm posterior and 0.5 mm to the right or left
lambda). The typical recording montage involved two EEG
channels with the right and left central ‘‘active’’ electrodes being
compared to either the frontal or occipital ‘‘reference’’ electrode.
Video and EEG data were acquired simultaneously with an
XLTEK video-EEG system. Forty-eight-hour epochs of continu-
ous video-EEG data were obtained once a week from each mouse,
for the life of the animal, and were analyzed for seizures.
Electrographic seizures were identified by their characteristic
pattern of discrete periods of rhythmic spike discharges that
evolved in frequency and amplitude lasting at least 10 seconds,
typically ending with repetitive burst discharges and voltage
suppression. On video analysis, the behavioral correlate to these
seizures typically involve head bobbing, rearing with forelimb
clonus, and occasional generalized convulsive activity. Seizure
frequency (number of seizures per 48-hour period, based on
analysis of the entire EEG record) was calculated from each 48-
After one week of VGB or vehicle treatment, western blot
analysis was used to measure the ratio of phospho-S6 (P-S6,
Ser240/244) and total S6 in the neocortex and hippocampus of
Tsc1GFAPCKO mice, as an assessment of mTOR pathway activity.
Western blotting was performed using standard methods as
described previously . Briefly, neocortex and hippocampus
were dissected and homogenized separately. Equal amounts of
total protein extract were separated by gel electrophoresis and
transferred to nitrocellulose membranes. After incubating with
primary antibodies to P-S6 (Ser240/244), S6, or beta-actin
(1:1,000, Cell Signaling Technology, Beverly, MA), the mem-
branes were reacted with a peroxidase-conjugated secondary
antibody (Cell Signaling Technology). Signals were detected by
enzyme chemiluminescence (Pierce, Rockford, IL) and quantita-
tively analyzed with ImageJ software (NIH, Bethesda, MD).
Measurement of brain GABA concentration in
After one week of VGB or vehicle treatment, brain GABA
concentrations were measured using a mouse gamma-aminobu-
tyric acid (GABA) ELISA kit (Novatein Biosciences, Cambridge
MA; sensitivity 2.5–80 ng/ml) according to the manufacturer’s
instructions. One week of VGB treatment was used before
assaying GABA levels, which should start to increase within one
day and reach a steady state after a few days of VGB treatment
based on previous studies (14). Briefly, Tsc1GFAPCKO mice and
non-KO littermates were treated with VGB (200 mg/kg/day) or
vehicle (saline) for one week. Neocortex and hippocampus were
dissected and homogenized separately and a centrifuged superna-
tant of each sample was used to measure the GABA level by using
the GABA ELISA kit. The protein concentration of each
supernatant was determined using a BCA protein assay (Thermo-
scientific, Rockford, IL).
After four weeks of VGB or vehicle treatment, histological
analysis was performed to assess glial proliferation and neuronal
organization by standard methods, as previously described .
Four weeks of VGB treatment was used based on the expected
time course to detect differences in these histological assays from
previous studies [19,21]. In brief, brains were perfusion-fixed with
4% paraformaldehyde and cut into 50 mm sections with a
vibratome. Some sections were stained with 0.5% cresyl violet.
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Other sections were labeled with GFAP antibody (anti–rabbit;
1:500; Sigma) and then rhodamine-conjugated anti–rabbit IgG
(1:500; Sigma, St. Louis, MO). Images were acquired with a
Nanozoomer HT system (Hamamatsu, Bridgewater, NJ). In
images from coronal sections at approximately 2 mm posterior
to bregma and approximately 1 mm from midline, regions of
interest were marked in neocortex by a 200 mm-wide box
spanning from the neocortical surface to the bottom of layer VI
and in hippocampus by a 2006200 mm2box within the striatum
radiatum of CA1 and dentate gyrus. GFAP-immunoreactive cells
were quantified in the regions of interest from two sections per
mouse from a total of four to five mice per group.
Astrocyte culture and mTOR activity in vitro
Astrocytes were obtained from mixed cell cultures of the
forebrains as described previously . The method was slightly
modified to remove microglial cells completely. Briefly, the
forebrains of newborn Tsc1GFAP1CKO mice and non-KO
littermates were dissected and the dissociated brain cells were
seeded in a poly-D-lysine-coated 75 cm2-culture flask (Becton
Figure 1. VGB treatment inhibited seizures and moderately
improved survival in Tsc1GFAPCKO mice. (A) Representative EEG
recordings of Tsc1GFAPCKO mice treated with vehicle or vigabatrin. (B)
Seizures started to develop in vehicle-treated Tsc1GFAPCKO mice (Fig. 1A,
KO + Veh) around 3 weeks and became progressively more frequent with
age. VGB treatment (KO + VGB) almost completely suppressed the
development of seizures in Tsc1GFAPCKO mice (*p,0.05 by one-way
ANOVA, n=13 mice/group). (C) Survival analysis showed that vehicle-
treated Tsc1GFAPCKO mice die prematurely with 50% mortality around 7
weeks of age and 100% mortality by 11 weeks. VGB treatment modestly
improved the survival of Tsc1GFAPCKO mice compared to the vehicle
groups, n=13mice/group),but all VGB-treated micestill died by age of 14
weeks. KO=Tsc1GFAPCKO mice, Veh = vehicle, VGB = vigabatrin.
Figure 2. VGB treatment increased brain GABA concentrations
in Tsc1GFAPCKO and control mice. GABA levels were measured
using a commercial mouse GABA ELISA kit. VGB treatment for one week
increased the GABA levels in both neocortex (A) and hippocampus (B)
of Tsc1GFAPCKO mice (KO+VGB), compared with the vehicle-treated
Tsc1GFAPCKO group (KO+Veh). Similar effects of VGB were observed in
control mice in hippocampus only (Cont+VGB versus Cont+Veh). Data
were derived from three separate experiments. *p,0.05, **p,0.01
versus vehicle-treated mice by two-way ANOVA (n=6–7 mice/group).
Cont = control mice, KO = Tsc1GFAPCKO mice, Veh = vehicle, VGB =
Vigabatrin Inhibits Seizures and mTOR in TSC
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Dickinson Labware, Franklin Lakes, NJ). Cells were cultured for
8–10 days until being confluent, then they were vigorously hand-
shaken for 0.5–1 min to remove microglial cells and O-2A lineage
cells that were present on the astrocyte monolayer, followed by
medium exchange and incubation overnight in a CO2incubator.
The purification procedure was repeated three times during the
subsequent 3 days. Finally, the flask was washed with DMEM
several times and the medium were changed to Neurobasal
medium for 6 hours before treatment.
VGB at a concentration of 0.06, 0.3 or 0.6 mM or phenobar-
bital at 0.01, 0.1, and 1 mM was added to the medium and
incubated for 16 hours. VGB dosing was based on previous in
vitro physiology studies on inhibiting GABA transaminase [23,24]
and phenobarbital dosing was based on potentiation of GABA
inhibition in neurons and related pharmacological effects on
astrocyte cultures [25–27]. Samples were collected after trypsin-
ization with 0.25% trypsin-EDTA (Invitrogen, Grand Island, NY),
and then western blotting analysis was done to measure the ratio of
phospho-S6 (P-S6) and total S6 as described above.
All statistical analysis was performed using GraphPad Prism
(GraphPad Software). Quantitative differences between groups
were analyzed by one-way ANOVA with Tukey’s multiple
comparisons post hoc tests when comparing one factor over more
than two groups or by repeated measures two-way ANOVA when
comparing multiple treatment variables (e.g. effect of treatment
and genotype). Comparable non-parametric tests were used when
data did not fit a normal distribution. Chi-Square test was used for
survival analysis. Quantitative data are expressed as mean 6
SEM. Statistical significance was defined as p,0.05.
VGB treatment decreased the development of seizures
and modestly improved survival in Tsc1GFAPCKO mice
Tsc1GFAPCKO mice develop a progressive epilepsy starting
around 4 weeks of life and then die prematurely by 3 months of
age [19–21]. Consistent with previous studies, video-EEG showed
that vehicle-treated Tsc1GFAPCKO mice developed seizures at
around 3–5 weeks of life (1.060.4 seizures/48 hours), which
became progressively more frequent with age (11.363.9 seizures/
48 hours at 8 weeks). In contrast, VGB treatment (200 mg/kg/
day) almost completely suppressed seizures in Tsc1GFAPCKO
mice, as monitored by video-EEG between 3 and 13 weeks of age
(0.060.0 and 0.160.1 seizure/48 hours at 3 and 8 weeks of age,
respectively; Fig 1A,B). Eleven out of thirteen mice were found to
have seizures in vehicle- treated group while only seven out of
thirteen VGB-treated Tsc1GFAPCKO mice had seizures. Survival
analysis confirmed previous studies demonstrating that vehicle-
treated Tsc1GFAPCKO mice die prematurely, with 50% mortality
around 7 weeks of age and 100% mortality by 11 weeks. VGB
treatment caused a modest, but significant, increase in survival of
Tsc1GFAPCKO mice compared to vehicle-treated Tsc1GFAPCKO
mice (Fig 1C). However, all VGB-treated mice still died by 14
weeks of age.
VGB treatment did not affect the brain and body weight
of Tsc1GFAPCKO mice
Tsc1GFAPCKO mice developed dramatic, diffuse megalencephaly
(brain weight=50169 mg at 7 weeks of age) compared with non-
KO control mice (41663 mg; p,0.05). VGB treatment at dose of
200 mg/kg/day for four weeks did not prevent the megalence-
phaly in Tsc1GFAPCKO mice (48964; p,0.05 compared with
non-KO control mice). VGB treatment also had no significant
effect on body weight of Tsc1GFAPCKO mice. At 7 weeks of age,
there was a trend towards a decrease in body weight in vehicle-
treated Tsc1GFAPCKO mice (16.260.7 g) compared with non-KO
control mice (19.160.8 g) and an increase with VGB treatment in
Tsc1GFAPCKO mice (18.561.20 g), but these were not statistically
significant. Furthermore, there was no obvious effect of VGB on
behavior or activity.
VGB treatment increased brain GABA concentration in
Tsc1GFAPCKO and control mice
Brain gamma-aminobutyric acid (GABA) concentrations in
neocortex and hippocampus were measured using a mouse GABA
ELISA kit. GABA levels in neocortex (Fig. 2A) and hippocampus
(Fig. 2B)were similarin
Tsc1GFAPCKO mice. VGB treatment for one week increased
GABA levels in neocortex and hippocampus of Tsc1GFAPCKO
mice and hippocampus of control mice.
Figure 3. VGB treatment decreased the number of GFAP
positive cells in hippocampus of Tsc1GFAPCKO mice. (A) Vehicle-
treated Tsc1GFAPCKO mice (KO + Veh) displayed a diffuse increase in
GFAP-positive cells in neocortex and hippocampus compared with the
vehicle-treated control mice (Cont + Veh). VGB treatment partially
prevented this increase in GFAP-positive cells in Tsc1GFAPCKO mice (KO
+ VGB). (B) Quantitative analysis demonstrated a 2–2.5 fold increase in
GFAP-positive cell in vehicle-treated Tsc1GFAPCKO group (KO + Veh)
compared with vehicle-treated control group (Cont + Veh) in neocortex
(CT), dentate gyrus (DG) and CA1 of hippocampus. #p,0.05, ##
p,0.01, ### p,0.001 versus vehicle-treated control mice by two-way
ANOVA (n=4–5 mice/group). VGB treatment decreased GFAP-positive
cells in Tsc1GFAPCKO mice (KO+VGB). **p,0.01 versus vehicle-treated
Tsc1GFAPCKO mice by two-way ANOVA (n=4–5 mice/group). Scale
bars=500 mm. Cont = control mice, KO = Tsc1GFAPCKO mice, Veh =
vehicle, VGB = vigabatrin, CT = neocortex, DG = dentate gyrus, CA1
= CA1 pyramidal cell layer of hippocampus.
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VGB treatment decreased the number of GFAP positive
cells in hippocampus of Tsc1GFAPCKO mice
Tsc1GFAPCKO mice exhibit a progressive, diffuse increase in
GFAP-positive astrocytes related to excessive glial proliferation
Tsc1GFAPCKO mice showed a large increase in GFAP-immuno-
reactive cells compared with non-KO control mice (Fig. 3A, B).
VGB treatment caused a significant decrease in the number of
GFAP positive cells in hippocampus of Tsc1GFAPCKO mice.
VGB treatment did not prevent neuronal disorganization
in Tsc1GFAPCKO mice
Tsc1GFAPCKO mice exhibit disorganization and dispersion of
the pyramidal cell layer of hippocampus [19,21]. Consistent with
previous studies, Cresyl violet staining demonstrated that vehicle-
treated Tsc1GFAPCKO mice had widely dispersed pyramidal cell
layers (Fig. 4, arrows in the middle panels) in all regions of
hippocampus (CA1–CA4) compared with control mice. VGB
treatment had no apparent effect on this neuronal disorganization
in Tsc1GFAPCKO mice (Fig. 4, arrows in right panels).
VGB treatment decreased activation of the mTOR
pathway in vivo
Consistent with previous studies , western blot analysis
showed that the brains of vehicle-treated Tsc1GFAPCKO mice
have increased mTOR pathway activation compared to control
mice, as reflected by an increase in the P-S6 (Ser240/244) to S6
ratio. VGB treatment at doses of 100 and 200 mg/kg/day
decreased activation of the mTOR pathway in both neocortex
(Fig. 5A) and hippocampus (Fig. 5B) by a maximum of about 40%
in Tsc1GFAPCKO mice. Similar effects of VGB were observed in
non-KO control mice. VGB treatment at doses of 100 and
200 mg/kg/day decreased the activation of the mTOR pathway
in both neocortex (Fig. 5C) and hippocampus (Fig. 5D) in control
VGB treatment decreased activation of the mTOR
pathway in cultured astrocytes
Seizures themselves may directly cause acute activation of the
mTOR pathway [28,29]. In order to eliminate the possibility that
VGB secondarily inhibited mTOR activity in vivo via suppression
of seizure activity, the effect of VGB on mTOR activation was also
tested in additional experiments in primary cultured astrocytes.
VGB treatment decreased the activation of mTOR pathway in
both Tsc1GFAPCKO and control astrocytes, as reflected by the P-
S6 (Ser 240/244) to S6 ratio (Fig. 6A). By comparison, the
GABAergic modulator, phenobarbital had no effect on P-S6
expression (Fig. 6B).
VGB is particularly effective for seizures in TSC patients, but
the mechanism of this unique relationship between VGB and TSC
is poorly understood. In this study, we show that VGB strongly
inhibits seizures in a mouse model of TSC. Consistent with its
known mechanisms of action, VGB causes the expected increase
in brain GABA levels in the KO mice. Furthermore, as a novel
and unexpected finding, VGB inhibits mTOR pathway activity,
which could represent an additional mechanism of action that may
contribute to the distinctive efficacy of VGB in TSC.
VGB appears to have unique therapeutic properties in TSC for
a couple of reasons. First, medical intractability is especially
common in epilepsy in TSC, occurring at a much higher rate
(,65%) in epilepsy in TSC than in epilepsy overall (,30%) [2,7].
However, in contrast to most other antiseizure medications, VGB
is particularly effective for seizures in TSC, especially infantile
spasms, a typically devastating type of childhood seizure. VGB has
been reported to eliminate spasms in about 95% of cases, which is
much higher than all other treatments for spasms due to TSC or
any other cause [8,9]. In fact, this special relationship between
VGB and TSC is one of the few documented examples of a
medication having individualized efficacy for a specific epilepsy
syndrome or cause of epilepsy. Furthermore, early treatment with
VGB has been reported to improve the long-term outcome of
Figure 4. VGB treatment did not prevent neuronal disorganization in Tsc1GFAPCKO mice. The left panels show Cresyl violet staining of a
brain section at low (upper) and high (lower) magnification of vehicle-treated control mice (Cont + Veh). Vehicle-treated Tsc1GFAPCKO group (KO +
Veh) exhibited widely dispersed pyramidal cell layers (arrows in the middle panels) in all regions of hippocampus (CA1–CA4) compared with control
mice. As shown in the right panels, VGB treated Tsc1GFAPCKO mice (KO + VGB) had a similar pattern as vehicle-treated Tsc1GFAPCKO group, with no
apparent effect on this neuronal disorganization (arrows in right panel). Scale bar=500 mm. Cont = control mice, KO = Tsc1GFAPCKO mice, Veh =
vehicle, VGB = vigabatrin.
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neurological development and epilepsy in TSC patients [10,11],
although additional controlled clinical trials are needed to confirm
these findings. In the present study in a mouse model of TSC,
VGB was very effective in inhibiting epilepsy, causing almost
complete suppression of seizures. The efficacy of VGB was
comparatively better than other standard antiseizure medications
in Tsc1GFAPCKO mice, such as phenytoin and phenobarbital,
which reduced seizure frequency by 55–68% . Thus, this
mouse model represents an appropriate system to investigate the
mechanisms of action of VGB in TSC.
The standard, accepted mechanism of action of VGB is
inhibition of the catabolism of GABA, the major inhibitory
neurotransmitter in the brain [12,13]. Irreversible inhibition of
GABA transaminase by VGB leads to elevated brain GABA levels
. The resulting potentiation of inhibitory GABA systems in the
brain is a rational explanation for the antiseizure effects of VGB.
However, the effects of VGB on GABA physiology is not
straightforward, as VGB causes a paradoxical reduction in evoked
fast inhibitory postsynaptic potentials of neurons but instead
potentiates tonic GABAergic inhibition . Furthermore,
enhancement of GABA signaling by increased availability of
GABA may not explain the unique effectiveness of VGB for
epilepsy in TSC, as other GABA-potentiating drugs, such as
barbiturates and benzodiazepines, do not show comparable
efficacy in TSC. In Tsc1GFAPCKO mice, a previous study found
no abnormalities in GABAergic synaptic transmission  and the
present study demonstrates that baseline GABA levels in neocortex
and hippocampus of the KO mice are similar to control mice.
While Tsc1GFAPCKO mice did show a better response to VGB in
GABA levels in neocortex than control mice, this was a modest
difference, and the effects of VGB on GABA levels in hippocam-
pus were similar in control and in Tsc1GFAPCKO mice. Thus,
other mechanisms of action independent of GABA may need to be
Figure 5. VGB decreased activation of the mTOR pathway in vivo. (A, B) Western blotting shows P-S6 (Ser240/244), total S6, and beta-actin
expression in neocortex (A) and hippocampus (B) of control mice and Tsc1GFAPCKO mice treated with vehicle or VGB at daily doses of 50, 100 and
200 mg/kg for 1 week. Quantitative summary demonstrates that vehicle-treated Tsc1GFAPCKO mice have significantly increased P-S6 levels compared
with control mice (# p,0.05, ### p,0.001 versus control mice by two-way ANOVA, n=5–9 mice/group), and VGB inhibited the activation of S6 in
Tsc1GFAPCKO mice in a dose-dependent fashion (* p,0.05 versus vehicle-treated Tsc1GFAPCKO mice by two-way ANOVA, n=5–9/group). The ratio of
P-S6/total S6 was normalized to the vehicle-treated Tsc1GFAPCKO group. (C, D) Western blotting shows P-S6 (Ser240/244), total S6 and beta-actin
expression in non-KO control mice administered vehicle or VGB at 50, 100 and 200 mg/kg/day for 1 week starting at age of three weeks. Quantitative
summary demonstrates that VGB inhibited the activation of S6 in control mice in a dose-dependent fashion. The ratio of P-S6/total S6 was normalized
to the vehicle-treated control group. *p,0.05, **p,0.01, *** p,0.001 versus vehicle-treated non-KO control mice by one-way ANOVA (n=5–9 mice/
group). Cont = control mice, KO = Tsc1GFAPCKO mice, Veh = vehicle.
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considered in order to account for the unique efficacy of VGB for
seizures in TSC.
Recent evidence suggests that abnormal mTOR pathway
activity is critical for epileptogenesis in TSC and that mTORC1
inhibitors, such as rapamycin, may be particularly effective
therapies for epilepsy, not just as an anticonvulsant to treat
seizures, but also as antiepileptogenic therapy to prevent epilepsy
in TSC [16,17]. mTOR is a protein kinase, which normally
regulates a number of important functions, such as cell growth,
proliferation, metabolism, and protein synthesis, many of which
could affect epileptogenesis under pathological conditions. Thus,
the possibility that VGB could exert at least some of its effects on
epilepsy in TSC via interaction with the mTOR pathway is an
intriguing, but previously untested, hypothesis. In the present
study, we have provided evidence that VGB can inhibit the
mTOR pathway. First, mTORC1 pathway activity, as reflected by
downstream S6 phosphorylation, was decreased by VGB admin-
istered to Tsc1GFAPCKO mice in vivo. It is important to note that
other signaling pathways, such as ribosomal s6 kinases (RSK) and
(ERK) kinases, may also modulate S6 phosphorylation indepen-
dent of mTOR. However, this mTORC1-independent phosphor-
ylation appears to only involve the Ser235/236 phosphorylation
site of S6, not the Ser240/244 site . Thus, our finding that
VGB decreases Ser240/244 phosphorylation supports the involve-
ment of the mTORC1 pathway. As seizures themselves can cause
mTOR activation [28,29], one simplistic explanation for the
apparent mTOR pathway inhibition by VGB in the TSC mouse
model is that VGB’s inhibition of seizures secondarily decreased
mTOR activity. However, this possibility is basically excluded by
the similar findings of mTOR pathway inhibition by VGB in
normal control mice in vivo, as well as in cultured astrocytes from
both control and Tsc1GFAPCKO mice. By comparison, the GABA
potentiating drug, phenobarbital, inhibits seizures, but has no
effect on mTOR activity . Furthermore, the inhibition of
mTOR pathway activity by VGB in cultures in vitro also eliminates
other confounding factors in the brain in vivo, suggesting that VGB
directly inhibits the mTOR pathway.
The prototypic mTORC1 inhibitor, rapamycin, has potent
effects in preventing epilepsy, prolonging survival, and blocking
associated pathological and molecular changes that promote
epileptogenesis in Tsc1GFAPCKO mice, as well as in other mouse
models of TSC [21,32–34]. In the present study, in addition to
seizures, VGB also had some inhibitory effects on glial prolifer-
ation, at least in hippocampus. As VGB inhibited P-S6 in both
hippocampus and cortex, a similar effect of VGB on astrocyte
number likely also occurred in neocortex, but the sample size may
have been underpowered to reach statistical significance. Such
effects of VGB on glial proliferation can likely be attributed to
mTOR pathway inhibition. However, the effects of VGB were not
as strong or extensive as rapamycin, as VGB only had modest
effects on survival and glial proliferation and no effect on neuronal
organization. Since VGB was effective in inhibiting seizures, this
suggests that the pathological abnormalities in these mice are not
secondary to ongoing seizures. As rapamycin is a very potent
mTORC1 inhibitor , the difference in the effectiveness of
rapamycin and VGB may be due to the milder suppression of
mTOR activity by VGB in vivo, which maximized at approx-
imately 60% of the levels of vehicle-treated Tsc1GFAPCKO mice
(Fig. 5A, B). Given these differences between VGB and rapamycin,
it is possible that VGB does not directly inhibit the mTOR kinase
itself like rapamycin, but may also involve other components of the
mTOR pathway. Future studies are needed to determine the
specific molecular mechanisms by which VGB regulates the
Other limitations of this study include issues intrinsic to the
mouse model. While there are now a variety of animal models of
TSC, involving inactivation of Tsc1 or Tsc2 at different
developmental time points and in different subsets of brain cells,
there is no perfect model that recapitulates all neurodevelopmental
features of TSC. Tsc1GFAPCKO mice involve primarily inactiva-
tion of Tsc1 in glial cells, although a subset of neurons is also
affected. The mechanism of action of VGB in TSC may depend
on the cell type(s) affected, but this issue is not addressed with this
one model of TSC. In addition, in patients with TSC, VGB is
most effective against infantile spasms. Neither Tsc1GFAPCKO
mice nor any other animal model of TSC have been documented
Figure 6. VGB decreased activation of the mTOR pathway in
vitro. A) Western blotting shows P-S6 (Ser 240/244), total S6, and beta-
actin expression in primary cultured astrocytes derived from
Tsc1GFAPCKO and non-KO control mice. Vehicle or VGB at a dose of
0.06, 0.3 and 0.6 mM was added to the culture medium for 16 hours.
Overall, Tsc1GFAPCKO astrocytes showed increased P-S6 expression
compared with astrocytes from control mice. VGB blocked the
activation of P-S6 in a dose-dependent fashion in both control and
KO astrocytes. The ratio of P-S6/total S6 was normalized to the vehicle-
treated control group (Cont) or vehicle-treated Tsc1GFAPCKO group (KO).
Quantitative summary demonstrates that VGB treatment at doses of 0.3
and 0.6 mM significantly inhibits the activation of P-S6 in astrocytes of
both Tsc1GFAPCKO and control mice. B) In contrast to VGB, phenobar-
bital had no effect on P-S6 expression in control astrocytes at doses that
are effective in potentiating GABA-mediated inhibition . *p,0.05,
**p,0.01 versus Veh by one-way ANOVA (n=8 mice/group). Cont =
control mice, KO = Tsc1GFAPCKO mice, Veh = vehicle.
Vigabatrin Inhibits Seizures and mTOR in TSC
PLOS ONE | www.plosone.org7 February 2013 | Volume 8 | Issue 2 | e57445
to have spasm-like seizures. Interestingly, however, rapamycin has Download full-text
been shown to selectively suppress spasms in a non-TSC rat model
of infantile spasms . Finally, the present study has not
determined the relative contribution of GABA potentiation and
mTOR pathway inhibition in decreasing seizures. Future studies
need to define in more detail the specific cell types, seizure types,
and specific mechanisms involved in VGB’s effect in TSC. Despite
these current limitations, the present study is significant in
identifying a potential novel mechanism of action of an antiseizure
medication involving the mTOR pathway. This interaction of
VGB with the mTOR pathway may account for the unique
efficacy of this drug for a common genetic epilepsy.
Conceived and designed the experiments: BZ MW. Performed the
experiments: BZ SM NR. Analyzed the data: BZ MW. Wrote the paper:
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PLOS ONE | www.plosone.org8 February 2013 | Volume 8 | Issue 2 | e57445