Acta Neuropathol (2013) 126:207–218
DOI 10.1007/s00401-013-1135-4
ORIGINAL PAPER
mTOR‑dependent abnormalities in autophagy characterize
human malformations of cortical development: evidence from
focal cortical dysplasia and tuberous sclerosis
Shireena A. Yasin · Abu M. Ali · Mathew Tata · Simon R. Picker · Glenn W. Anderson ·
Elizabeth Latimer‑Bowman · Sarah L. Nicholson · William Harkness ·
J. Helen Cross · Simon M. L. Paine · Thomas S. Jacques Received: 14 February 2013 / Revised: 23 May 2013 / Accepted: 24 May 2013 / Published online: 2 June 2013
© Springer-Verlag Berlin Heidelberg 2013
Abstract Focal cortical dysplasia (FCD) is a localized
malformation of cortical development and is the commonest cause of severe childhood epilepsy in surgical practice.
Children with FCD are severely disabled by their epilepsy,
presenting with frequent seizures early in life. The commonest form of FCD in children is characterized by the
presence of an abnormal population of cells, known as balloon cells. Similar pathological changes are seen in the cortical malformations that characterize patients with tuberous
sclerosis complex (TSC). However, the cellular and molecular mechanisms that underlie the malformations of FCD
and TSC are not well understood. We provide evidence for
a defect in autophagy in FCD and TSC. We have found that
balloon cells contain vacuoles that include components of
the autophagy pathway. Specifically, we show that balloon
cells contain prominent lysosomes by electron microscopy, immunohistochemistry for LAMP1 and LAMP2,
Electronic supplementary material The online version of this
article (doi:10.1007/s00401-013-1135-4) contains supplementary
material, which is available to authorized users.
S. A. Yasin · A. M. Ali · M. Tata · S. R. Picker · S. M. L. Paine ·
T. S. Jacques (*) Neural Development Unit, Birth Defects Research Centre,
UCL Institute of Child Health, 30 Guilford Street,
London WC1N 1EH, UK
e-mail: t.jacques@ucl.ac.uk
S. A. Yasin · S. R. Picker · G. W. Anderson · E. Latimer‑Bowman ·
S. L. Nicholson · S. M. L. Paine · T. S. Jacques Department of Histopathology, Great Ormond Street Hospital
for Children NHS Foundation Trust, Great Ormond Street,
London WC1N 3JH, UK
S. R. Picker MRC National Institute for Medical Research, The Ridgeway,
Mill Hill, London NW7 1AA, UK
LysoTracker labelling and enzyme histochemistry for acid
phosphatase. Furthermore, we found that balloon cells contain components of the ATG pathway and that there is cytoplasmic accumulation of the regulator of autophagy, DOR.
Most importantly we found that there is abnormal accumulation of the autophagy cargo protein, p62. We show that
this defect in autophagy can be, in part, reversed in vitro by
inhibition of the mammalian target of rapamycin (mTOR)
suggesting that abnormal activation of mTOR may contribute directly to a defect in autophagy in FCD and TSC.
Keywords Autophagy · Epilepsy · Balloon cells · Focal
cortical dysplasia · Tuberous sclerosis
Introduction
Epilepsy is the commonest severe neurological disease of
children [18, 22]. Some children are severely disabled by
their epilepsy, presenting with frequent seizures early in life.
W. Harkness · J. H. Cross Neurosciences Unit, UCL Institute
of Child Health, 30 Guilford Street,
London WC1N 1EH, UK
W. Harkness Department of Neurosurgery, Great Ormond Street Hospital
for Children NHS Foundation Trust, Great Ormond Street,
London WC1N 3JH, UK
J. H. Cross Department of Neurology, Great Ormond Street Hospital
for Children NHS Foundation Trust, Great Ormond Street,
London WC1N 3JH, UK
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In this group of children, structural abnormalities of the brain
are frequent [5] and focal cortical dysplasia (FCD) is the most
common [7]. FCD is a localized malformation of the cerebral
cortex [7]. Children with FCD develop frequent seizures, for
which treatment with standard anti-epileptic drugs in isolation or combination is usually ineffective, and they often
require surgery. The cause of FCD is unknown. Therefore,
there is a significant clinical need to understand the biology
underlying this disease. Not only is FCD an important clinical problem, but also a disease characterized by fundamental,
but poorly understood, abnormalities of human cortical development including regulation of cell size [7].
A frequent pathological feature of FCD is the presence
of a population of cells with enlarged ballooned cytoplasm,
known as balloon cells (BC) [7]. A similar cell type is seen in
the cortex of patients with tuberous sclerosis complex (TSC)
[12, 36]. FCD with BCs (FCD type IIb) is one of the commonest forms of FCD in children. Importantly, several studies
have suggested that BCs are related to neural precursors or
stem cells [10, 23, 37, 39, 40]. This suggests that an understanding of BCs will indicate how FCD arises. For example, one possibility is that BCs are abnormal stem cells that
contribute directly to the disease. Alternatively, they may be
derived from abnormal stem cells and will provide an insight
into the primary abnormality affecting those stem cells [39].
Despite its clinical importance, the signalling pathways
that underlie the development of FCD or BCs are unknown.
The best described candidate mechanism in FCD is activation of the serine/threonine kinase, mammalian target of
rapamycin (mTOR) [11]. Several lines of evidence indicate
a role for mTOR in FCD. First, there is increased phosphorylation of downstream targets of mTOR in FCD and TSC
when compared to control brain tissue [3, 24, 31, 32, 39].
Second, in TSC patients, mutations are found in the TSC1
and TSC2 genes that encode for hamartin and tuberin,
inhibitors of mTOR [19]. Furthermore, in some patients
with sporadic FCD, there are allelic variants in TSC1 that
change the cellular localization of hamartin and its interaction with tuberin [4, 25]. Indeed, some studies have found
reduced expression of tuberin or hamartin in FCD [14] or
TSC [14, 32, 33]. Finally recent data raises the possibility
that mTOR may be activated in FCD by congenital infection by human papilloma virus (HPV) [8].
However, the specific contribution of mTOR to the
pathogenesis of FCD is unclear. The published studies have
been descriptive reports of mTOR activity and a functional
role for mTOR activity has not been proven. In addition,
several of the studies have suggested differences in the activation of downstream targets between sporadic FCD and
TSC [3, 31, 35, 39]. There is a suggestion in one report that
mTOR activation is a feature of TSC but not FCD [3].
Given these potential differences between FCD and
TSC, it is possible that a number of mechanisms may act
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Acta Neuropathol (2013) 126:207–218
on a common final cellular process that is responsible for
the pathogenesis of disease. However, the downstream consequences of mTOR activation in FCD and TSC are poorly
understood.
To elucidate these processes, we have focused on
autophagy. Autophagy is a process for destroying organelles and proteins through the lysosomal system [13].
mTOR signalling has an important role in regulating
autophagy [15, 16, 20, 21, 41]. We have found previously
that BCs contain abundant mitochondria, intermediate
filament proteins and vesicles [39]. We hypothesized that
these observations were in keeping with an abnormality of autophagy; for example by reducing mitochondrial
degradation (mitophagy) and the accumulation of vesicle
components of the autophagy pathway. This would be supported by recent studies suggesting a defect in autophagy
in TSC [29, 30]. In this study, we demonstrate that there is
an abnormality in autophagy in BCs and that activation of
mTOR is, at least in part, responsible for this defect.
Materials and methods
Tissue samples
Tissue was obtained from the archives of the department of
histopathology at Great Ormond Street Hospital for Children NHS Foundation Trust from patients who had been
treated at the supra-regional epilepsy service as described
previously [39]. All samples were from surgical cases. All
cases were reviewed by a senior paediatric neuropathologist (TJ) and classified according to the ILAE classification
of FCD [7]. Clinical details of the samples are included
in Supplementary Table 1. Control samples were from the
temporal neocortex of children undergoing temporal lobectomy for hippocampal pathology. Controls were included
if the neocortex was normally formed but were excluded if
there was tumour, encephalitis or cortical dysplasia (including FCD type III).
Immunohistochemistry
Paraffin-embedded sections were cut at 4–7 μm, dewaxed
in xylene (10 min), rehydrated through a graded alcohol
series, blocked in hydrogen peroxide (10 % hydrogen peroxide in PBS) and then rinsed in dH20.
Antigen retrieval was performed by pressure-cooking
sections in EDTA-Citrate buffer pH 6.2 for the following
antigens: DOR, p62, ATG5, LC3/ATG8, ATG12 and Beclin-1/ATG6. Sections were then washed in PBS (2 × 3 min)
and once in 0.1 % Tween/PBS (3 min). Primary antibodies [p62 (1:1000, BD Biosciences), DOR (1:400 a gift
from Prof. Zorzano, Barcelona [2]) ATG5 (1:125, Abgent),
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LC3/ATG8 (1:125, Abgent), ATG12 (1:100, Abgent) and
Beclin-1 (1:100, Abgent)] were applied to sections, which
were then incubated at room temperature for 60 min in a
humid slide chamber. Sections were rinsed with dH2O,
washed in PBS (2 × 3 min) and once in 0.1 % Tween/PBS
(3 min). After washing with PBS, biotinylated secondary antibodies were subsequently applied to sections and
incubated for 30 min at room temperature in a humid slide
chamber. Sections were rinsed with dH2O, washed in PBS
(2 × 3 min) and once in 0.1 % Tween/PBS (3 min). Detection was performed with Vectorstain ABC kit (Vector) and
0.5 % 3,3′ diaminobenzidine tetrahydrochloride (DAB) for
5 min. Sections were then rinsed with dH2O. Sections were
counterstained with Mayer’s Haematoxylin for 1 min then
washed in warm tap water for 1 min. Sections were dehydrated through a graded alcohol series (1 min in 70 % alcohol, 2 min in absolute alcohol and 2 min in xylene) before
being mounted with DPX (BDH). The ATG antibodies
were also stained on an intelliPATH FLX staining system
(A. Menarini Diagnostics), which produced similar results.
Additional immunohistochemistry was performed on a
DAKO Bond-Max automated stainer: LAMP1 (Abcam)
1:200 HIER 10 ER2 Bond-Max protocol F, LAMP2 (Abcam)
1:100 HIER 20 ER2 Bond-Max protocol F, Ubiquitin (Dako)
1:1200 HIER 20 ER1 Bond-Max protocol F and p62 (BD
Biosciences) 1:1000 HIER 20 ER1 Bond-Max protocol F.
The numbers of cases stained are indicated in the “Results”
section. For each antibody, similar staining was seen in all
stained cases of a given diagnosis unless specified in the text.
For immunofluorescence, sections were dewaxed and
washed in 0.1 % Tween/PBS (2 × 3 min). Sections were
then blocked using 10 % heat-inactivated sheep serum/PBS
for at least 10 min and rinsed with dH2O. Sections were
again washed in 0.1 % Tween/PBS (2 × 3 min) before
being incubated with primary antibody (anti-DOR 1:400 in
0.1 % BSA/PBS) at 4 °C overnight in a sealed, humid slide
chamber. Sections were rinsed with dH2O, washed in 0.1 %
Tween/PBS (2 × 3 min) and then incubated with Alexa
Fluor 488 secondary antibody (1:240, Molecular Probes)
in 1 % BSA/PBS in the dark for 90 min at room temperature. Slides were washed in PBS (3 × 3 min) and mounted
in Vectashield aqueous mounting medium containing
4′, 6-diamidino-2-phenylindole/DAPI (Vector).
10 μm cryostat sections were allowed to thaw and 0.5 ml
of incubation medium was applied to each slide. The slides
were incubated at 37 °C for 2 h. The sections were washed,
counterstained with Carazzi’s haematoxylin and dehydrated
through a graded alcohol series before being mounted with
DPX.
Acid phosphatase staining
Thawed BCs were allowed to settle overnight, the following day they were plated onto 5 μg/ml PDL coated 1 N
HCL treated 13 mm glass coverslips and allowed to adhere
for 2.5 h. The cells were then treated with 100 or 500 nM
rapamycin diluted in dimethyl sulphoxide (DMSO) for
either 6 or 24 h prior to being fixed and stained. Control
BCs were treated with vehicle alone (DMSO) diluted in
media for the same lengths of time prior to fixation in 4 %
PFA. Rapamycin was diluted in sterile DMSO to a final
To make the incubation solution, 5 mg Naphthol AS-BI
phosphate was dissolved in 2–3 drops of dimethylformaldehyde, 2.5 ml of 0.1 M Acetate buffer, 6 ml of distilled
water and 0.4 ml of hexazotized pararosaniline (HPR). The
HPR solution was prepared by mixing equal volumes of
40 mg of basic fuchsin dissolved in 1 ml 2 M HCl with
40 mg of sodium nitrate dissolved in 1 ml distilled water.
Electron microscopy
All samples were fixed in 2.5 % glutaraldehyde in 0.1 M
cacodylate buffer followed by secondary fixation in 1.0 %
osmium tetroxide. Tissues were dehydrated in graded ethanol, transferred to propylene oxide and then infiltrated and
embedded in Agar 100 epoxy resin. Polymerization was at
60 °C for 48 h. 90 nm ultrathin sections were then cut using
a Diatome diamond knife on a Leica Ultracut UCT ultramicrotome. Sections were picked up on copper grids and
stained with alcoholic uranyl acetate and Reynold’s lead
citrate. The samples were examined in a JEOL transmission electron microscope.
Cell culture immunocytochemistry
BCs were cultured as previously described [39]. Dissociated cells from the TS tuber were plated onto 5 μg/ml PDL
coated 1 N HCL treated 13 mm glass coverslips 1 day after
dissociation and allowed to adhere for 2.5 h before fixation in 4 % PFA. Frozen cells from the FCDIIb case were
allowed to settle in culture media overnight at 37 °C following thawing and were then plated onto PDL-coated coverslips and fixed as above. Some BCs could still be seen
in culture 21 days after dissociation of the FCDIIb tissue.
These cells were plated onto coverslips, allowed to adhere
and fixed as above.
Fixed cells were stained as described in [39] with antibodies to Beclin-1/ATG6 (1:50, Abgent), LC3/ATG8
(1:400, Abgent), ATG12 (1:50, Abgent), DOR (1:500 a gift
from Prof. Zorzano, Barcelona), LAMP1 (1:200, Abcam),
LAMP2 (1:100, Abcam), p62 (1:50, BD Biosciences),
phospho-S6 (Ser235/236 1:100, Cell Signalling) and phospho-4EBP1 (Thr37/46 1:100, Cell Signalling).
Cell treatments
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concentration of 100 μM. For final concentrations this
stock was then diluted to 1 μl/ml media for a final concentration of 100 nM and 5 μl/ml media for a final concentration of 500 nM. Control conditions were 5 μl DMSO only/
ml media (0.5 % DMSO).
Acta Neuropathol (2013) 126:207–218
Results
Lysosomes accumulate in balloon cells
The lysosomes of cultured BCs (n = 3 cultures) were
labelled by incubating them in LysoTracker dye (1 μM
in media, Molecular Probes) for 5–10 min at 37 °C. Cells
were washed with media and re-suspended in fresh media
and the fluorescence observed under a time-lapse microscope using Volocity software (Improvision, UK).
In order to explore the role of autophagy in FCD, we tested
the hypothesis that BCs accumulate cellular structures
normally consumed during autophagy. If autophagy were
defective in BCs, one would anticipate an accumulation of
these components. First, we explored the lysosomal component of BCs. Histochemical staining for the lysosomal
enzyme, acid phosphatase, showed strong positive reactivity within BCs (n = 6 cases, FCDIIb) (Fig. 1a). There was
weaker staining of some neurons and glia in FCDIIb. Staining of normally formed cortex from temporal lobectomy
samples (n = 5 cases) showed relatively weak staining of
neurons and glia (Fig. 1d).
This lysosomal accumulation was confirmed by immunohistochemistry for the two major lysosomal glycoproteins, LAMP1 and LAMP2. BCs in FCDIIb and TSC
showed prominent and consistent expression of LAMP1 and
LAMP2 (FCDIIb n = 3 cases, TSC n = 4 cases) (Fig. 1b, c).
Some BCs showed paranuclear or central staining for
LAMP. In contrast, there was notably less expression of
LAMP1 in normally formed cortex and white matter (n = 5
cases) than in cortical dysplasia (Fig. 1e). LAMP2 showed
variable labelling of glia and neurons in normally formed
cortex (Fig. 1f) and in cortical dysplasia (Fig. 1c).
Fig. 1 Balloon cells accumulate lysosomes. Acid phosphatase staining of balloon cells in FCDIIb (a) compared with normally formed
cortex (d). LAMP1 staining of balloon cells in FCDIIb (b) com-
pared with normally formed cortex (e). LAMP2 staining of balloon
cells in FCDIIb (c) compared with normally formed cortex (f). Scale
bar = 50 μm
Confocal microscopy
Confocal images of immunostained BCs were acquired
with a Zeiss LSM710 microscope (Zen2009, Zeiss) using
oil immersion (63×, N.A. 1.4) and water immersion (25×,
N.A 0.8). Z-projections of confocal stacks were created in ImageJ. Images of single BCs in immunopanels
show a Z-projection of all channels around the nucleus/
nuclei of cells. For the quantitative analysis fluorescence
was measured per cell using an entire tile scan (10 × 10
grids at 20×) over 3 coverslips from 2 temporally separate
experiments.
LysoTracker labelling of cultured BC
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Fig. 2 The prominent lysosomal component of balloon
cells is maintained in culture.
Confocal projection of balloon
cells at low power (a, c) and at
high power (b, d) stained with
antibodies against LAMP1
(a, b) and LAMP2 (c, d). Scale
bars in a, c = 25 μm, scale
bars in b, d = 10 μm
BCs isolated in vitro also showed extensive immunoreactivity for LAMP1 and LAMP2. LAMP1 and LAMP2
were strongly expressed in a punctate pattern, which in
some cases was found within the centre of the cells (n = 3,
1 FCDIIb after 1DIV, 1 FCDIIb 21DIV and 1TSC after 1
DIV) (Fig. 2). Furthermore, live cell staining with the lysosomal stain, LysoTracker showed frequent mobile punctate
structures with a central accumulation (n = 3) (Supplementary Video 1).
These data suggest that there is a significant accumulation of lysosomes in BCs. To confirm this, we undertook electron microscopy on BCs in vivo (n = 3 FCDIIb,
n = 2 TSC). BCs in vivo could be identified by their
enlarged cytoplasm, eccentric nuclei with prominent
nucleoli and lack of features of neuronal or astrocytic
differentiation. Similar to our previous in vitro findings,
BCs in vivo contained prominent mitochondria, intermediate filaments and vesicles, many of which were lysosomes (Fig. 3). Taken together, these data support the
hypothesis that BCs showed a significant accumulation
of lysosomes.
Components of the autophagy cascade accumulate
in balloon cells
Next, we tested the hypothesis that components of the
mechanisms that mediate autophagy also accumulate
in BCs. A protein cascade that directs the formation of
autophagosomes initiates autophagy. We examined the
expression of four components of this pathway, ATG5,
LC3/ATG8, ATG12 and Beclin-1/ATG6 in FCD with (IIb,
n = 5) and without BCs (IIa, n = 4), TSC (n = 5) and normally formed cortex (n = 5). These components were chosen, not only because they are critical parts of the machinery
of autophagy, but also because previous studies had indicated there was low or absent immunoreactivity in normal
cortex and their accumulation had been taken to indicate an
abnormality of autophagy [26].
All four proteins showed prominent accumulation in a
proportion of BCs from FCDIIb or TSC and this was readily distinguished from the comparatively low levels of
expression seen in normally formed cortex (Fig. 4) or in
FCD lacking BCs (FCDIIa). In these control cases, there
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Fig. 3 Ultrastructural examination by electron microscopy
shows that the balloon cells
contain abundant mitochondria, intermediate filament and
vesicles that included prominent dense cored lysosomes
(arrows). The images show
electron micrographs taken at
low power (showing the entire
balloon cell) (a, b) and at high
power (showing the organelles)
(c, d). Cells from both TSC
(a, c) and FCDIIb (b, d)
are shown. Scale bars in a,
b = 2 μm and in c, d = 500 nm
Fig. 4 Balloon cells accumulate components of the autophagy pathway. a–d FCDIIb, e–h TS and i–l control tissue (normally formed
cortex from temporal lobectomies). Balloon cells in FCDIIb and TS
tissue sections express the autophagy markers ATG5 (a, e), BECN-
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1/ATG6 (b, f), LC3/ATG8 (c, g) and ATG12 (d, h). Normally formed
cortex and white matter showed low levels or negative expression of
these proteins (i–l). Scale bar = 100 μm
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was some expression of ATG proteins (mostly of ATG5 and
ATG8) that was predominantly in neurons. This was quite
distinct from the strong reactivity of BCs. Immunoreactivity for Beclin-1/ATG6, ATG12 and LC3/ATG8 was confirmed within cultured BCs (Fig. 5). Notably, the strongest
reactivity was demonstrated for ATG12. The staining of
the ATG proteins was punctate and predominantly distributed throughout the cytoplasm, raising the possibility of
autophagosome formation.
Balloon cells show cytoplasmic expression of the diabetes
and obesity‑related gene (DOR)
We examined the localization of the product of the diabetes and obesity-related gene (DOR) in BCs. DOR
is a nuclear cofactor that is involved in the initiation of
autophagy. Stimulation of autophagy is thought to cause
DOR to shuttle from the nucleus to the cytoplasm where
it binds to autophagosomes and facilitates the initiation
of autophagy [27]. We hypothesized that the localization
of DOR may be interpreted as an indication of the state
of autophagy in BCs. Staining of FCDIIb cases (n = 4)
with an antibody against DOR showed prominent cytoplasmic expression and variable nuclear expression in
dysplastic cells that included BCs and dysmorphic neurons (Fig. 6c). The principal normal cell type expressing
DOR were small glial cells, which in contrast, predominantly showed nuclear staining (Fig. 6a, b). Morphologically normal neurons showed a more variable, often weak,
pattern of staining. Confocal microscopy confirmed that
the predominant pattern of DOR expression in BCs in
vivo and in vitro was as cytoplasmic puncta (Fig. 6d, e).
These observations suggest that there is an accumulation
of autophagosomes in BCs.
Balloon cells accumulate p62
To test the hypothesis that autophagy is inhibited in BCs,
we examined the expression of the adaptor protein, p62.
p62 marks proteins for degradation via autophagy and
is itself destroyed in the process. The turnover of p62
is rapid in normal cells (e.g. pulse chase experiments in
culture suggest a half life of 6 h with complete loss in
24 h [6]) and expression is not detectable in the normal
brain by immunohistochemistry on paraffin-embedded
tissue. Furthermore, p62 levels rise following inhibition
of autophagy [17]. Therefore, we hypothesized that BCs
would show an accumulation of p62 compared to normally formed brain.
Immunohistochemistry for p62 revealed strong staining of BCs in FCDIIb (n = 5) and TSC (n = 5), indicating
an accumulation of p62. Staining was present in the cytoplasm and the nucleus of cells and occasional surrounding
processes (Fig. 6g, h). Weaker staining was seen in occasional dysmorphic neurons but this was not a consistent
finding. The dysplastic cortex of FCDIIa showed very little
p62 expression, with most cases either entirely negative or
with weak neuronal staining (n = 4). Normally formed cortex was negative for p62 (n = 4) (Fig. 6f) (the association
between p62 reactivity in BC-cases vs. normally formed
cortex is significant, p = 0.001 Fisher’s exact test). These
data show that p62 accumulation occurs in BCs supporting
the view that autophagy is defective in these cells. Staining of balloon cells in culture showed prominent punctate
staining for p62 in the cytoplasm and nucleus. To exclude
the alternative possibility that p62 accumulation reflects a
defect in the ubiquitin–proteasome system, we undertook
immunohistochemistry for ubiquitin. While occasional
cells showed weak staining similar to the controls, there
was no reproducible specific staining of BCs (n = 5) (data
not shown).
mTOR activity is required for defective autophagy
in balloon cells
A major regulator of autophagy is the mTOR pathway
and a number of reports have suggested that mTOR activation characterizes BCs in FCD [16, 20, 21, 35, 39, 41].
We wished to determine whether this activation of mTOR
in BCs contributes to the defect in autophagy or if other
mechanisms are responsible.
First, to test the hypothesis that mTOR is activated in
BCs from FCDIIb, we measured immunoreactivity for
phosphorylation of S6, a downstream target of mTOR,
in BCs after treatment with the mTOR inhibitor, rapamycin. Six hours after treatment with rapamycin, there was
no detectable staining for phosphorylated S6 in the cultured BCs confirming that phosphorylation of S6 in BCs
is driven by mTOR (p < 0.001 ANOVA with Dunnet’s
post hoc testing) (Fig. 7, Supplementary Figure 1 and
Figure 8).
In order to determine if the defect in autophagy in BCs
is dependent on mTOR, we stained BCs for p62 with or
without treatment with rapamycin. In the absence of
rapamycin, p62 was expressed in strong punctate structures scattered throughout the cell (Fig. 6i, j). The intensity of staining, density of puncta and distribution of p62
varied considerably between cells. p62 aggregates were
seen throughout the cytoplasm but in addition, in shortterm cultures we often found large aggregates of p62 in
the nucleus. Rapamycin treatment lead to a significant
decrease in p62 immunoreactivity within 24 h indicating
that the defect in autophagy is at least in part dependent on mTOR activity (p < 0.001 ANOVA with Dunnet’s
post hoc testing) (Fig. 7, Supplementary Figure 1 and
Fig. 8).
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Fig. 5 Cultured balloon cells
expressed markers of the
autophagy cascade. Confocal images a–i = FCDIIb and
j–r = TS. Cells expressed
BECN-1/ATG6 (a–c and j–l),
LC3/ATG8 (d–f and m–o) and
ATG12 (g–i and p–r). These
markers were expressed in a
punctate pattern at high density
in the cytoplasm of cells
from FCDIIb and TS. Scale
bar = 10 μm
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Acta Neuropathol (2013) 126:207–218
Fig. 6 Balloon cells show cytoplasmic accumulation of DOR (a–e)
and nuclear and cytoplasmic accumulation of p62 (f–j). Immunohistochemistry for DOR in tissue sections shows frequent strong nuclear
staining in small glial cells (arrows), variable staining in neurons
(arrow heads) (a and b adjacent non-dysplastic regions of an FCDIIb
case) and strong cytoplasmic staining in balloon cells (c). Confocal
microscopy of tissue sections shows that this staining has a punctate
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cytoplasmic pattern (d). Similar punctate cytoplasmic staining was
seen in balloon cells in culture (e). p62 was strongly expressed in the
cytoplasm and nucleus of balloon cells from FCDIIb (g) and TSC
(h), but was not expressed in control cortex (f). There are aggregates of p62, in the nucleus and cytoplasm of cultured balloon cells.
(i FCDIIb) and (j TSC). Scale bars a–c and f–h = 50 μm; d, e, i,
j = 10 μm
Fig. 7 Balloon cells show
down-regulation of pS6 and
p62 in response to rapamycin.
Immunofluorescence for pS6
(a, c) and p62 (b, d) in control
media (upper row) and rapamycin (500 nM) (lower row). The
images are counterstained with
DAPI. See Fig. 8 for quantification and supplementary figure 1
for a high power image. Scale
bar = 100 μm
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Fig. 8 Balloon cells show down-regulation of pS6 and p62 in
response to rapamycin. Fluorescence intensity of pS6 (a) and p62
(b), in balloon cells grown in control conditions (DMSO, blue
bars) and in 2 concentrations of rapamycin (100 nM green bars and
500 nM rapamycin brown bars) at 6 and 24 h post-treatment. pS6 is
almost completely switched off in both concentrations of rapamycin at both time points (p < 0.001 ANOVA with Dunnett’s post hoc
testing). p62 shows significant down-regulation by 24 h at both concentrations (p < 0.001 ANOVA with Dunnett’s post hoc testing). The
images shown are the average fluorescence signal per cell and have
been normalized to the control sample
Discussion
We have provided evidence that FCD with BCs shows an
abnormality in autophagy. We have also confirmed for the
first time functionally that mTOR is over-activated in BCs
and that the abnormality in autophagy is at least in part
dependent on this mTOR activity. This raises the possibility that FCD arises due to a defect in autophagy during
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development that is at least in part driven by dysregulation
of the mTOR pathway.
Several lines of evidence support the view that
autophagy is dysregulated in FCD. The most direct evidence is that the protein, p62 accumulates in BCs. p62 is
normally turned over rapidly by autophagy and inhibition
of autophagy (e.g. by knocking out components of the
autophagy pathway) leads to p62 accumulation [17]. p62
is a ubiquitin-binding protein and a defect in the ubiquitin–
proteasome system may also lead to accumulation of p62.
However, we do not favour this explanation in view of the
lack of accumulation of ubiquitinated proteins in FCD. We
cannot exclude entirely the possibility that p62 has additional roles acting through other mechanisms in FCD or
TSC or that there are more subtle effects on ubiquitinated
proteins that we have not been able to detect.
A number of additional observations also support the
view that autophagy is abnormal in these cells. First, we
have shown that lysosomes, an essential precursor organelle
for autophagy, are a prominent cytoplasmic component of
BCs. This would be in keeping with an accumulation of the
precursor organelle following a block in autophagy. Similarly, there is a prominent punctate expression of autophagy
cascade proteins in the cytoplasm of BCs. This expression
was mirrored by the cytoplasmic expression of DOR. Several studies have shown that DOR shuttles between the
cytoplasm and the nucleus and that one of its cytoplasmic
functions is to regulate autophagosome formation [27, 28].
It is not possible to compare the expression of these components directly with a ‘normal comparison’ cell as we do
not know the cell of origin for BCs but the accumulation
of these components, compared to normally formed cortex, supports the hypothesis that autophagy is inhibited in
FCD. However, the punctate cytoplasmic staining suggests
that autophagy has been initiated as autophagosomes are
accumulating.
Our data supports the hypothesis that activation of mTOR
is a key pathway in the pathogenesis of FCD. Several previous publications have implicated over-activation of mTOR
in FCD. Previously, this has been supported by the histological and biological similarity of FCD with BCs to the
cortical pathology seen in TSC, which is due to mutations
in the genes encoding for the inhibitors of mTOR, hamartin (TSC1) and tuberin (TSC2) [36]. Furthermore, polymorphisms in the TSC1 gene have been described in FCD [4].
Finally, there is phosphorylation of targets of mTOR in BCs
in FCD [3, 24, 31, 34, 39]. However, the pattern of target
phosphorylation differs in some studies between FCD and
TSC [3, 31, 34, 39]. For example activation of pS6 kinase
and pS6, which are not entirely specific to mTOR, are common to both diseases but activation of the more specific target of mTOR, 4E-BP1 is either variable or absent in FCD
depending on the study [3, 39]. This has led some authors
Acta Neuropathol (2013) 126:207–218
to suggest that TSC but not FCD is characterized by mTOR
activation [3]. Therefore, our cell culture data is important as
we have found that phosphorylation of S6 is rapidly ablated
by the addition of the specific mTOR inhibitor, rapamycin.
This strongly supports the view that S6 is phosphorylated
in FCD by activation of mTOR and supports the view that
mTOR activation characterizes FCD. Interestingly, as with
the in vivo studies, we have not found that phosphorylation of 4E-BP1 is a reliable marker of BCs (in vitro) (data
not shown). The reason for the difference between S6 and
4E-BP1 is not clear but data in other cell types suggest that
phosphorylation by mTOR of the pS6 kinase/pS6 system
can be unlinked to phosphorylation of 4E-BP1 [9].
As several mechanisms, including mTOR, regulate
autophagy, it was important to determine if the abnormalities in autophagy in FCD are dependent on the activation of
mTOR seen in FCD or if other pathways are responsible.
We found that a significant reduction in p62 accumulation
was seen following mTOR inhibition. This result indicates
that reversing the activation of mTOR, at least in part, can
reverse the defect in autophagy. This raises the possibility that over-activation of mTOR in the developing cortex leads to defective autophagy and may be an important
mechanism in the pathogenesis of FCD. We cannot exclude
the possibility that other pathways contribute to the inhibition of autophagy in BCs, as we were unable to eliminate
p62 accumulation entirely in these experiments.
Taken together, these data raise two possible mechanisms
linking autophagy and the pathogenesis of FCD. In the first,
over-activation of mTOR during cortical development leads
to a defect in autophagy that directly impairs normal development. The role of autophagy in normal cortical development is unclear but several studies have suggested that
autophagy regulates normal progenitor cell function [1, 38].
The second possibility is that the defect in autophagy and
mTOR is responsible for specific aspects of the phenotype,
for example, abnormalities in this pathway would easily
explain the abnormalities of cell size that characterize several forms of FCD and that are not restricted to BCs.
Acknowledgments TSJ was in receipt of funding from the Great
Ormond Street Hospital Children’s Charity and holds a HEFCE Clinical Senior Lecturer Award. This report is independent research supported by the National Institute for Health Research Great Ormond
Street Hospital Biomedical Research Centre. The views expressed in
this publication are those of the author(s) and not necessarily those of
the NHS, the National Institute for Health Research or the Department
of Health. We are grateful to Prof. Zorzano, Institute of Research in
Biomedicine, Barcelona for the DOR antibody.
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