Download Mietzsch U, McKenna J 3rd, Reith RM, Way SW, Gambello MJ. Comparative analysis of Tsc1 and Tsc2 single and double radial glial cell mutants. J Comp Neurol. 2013 Nov. 521(16):3817-31.

R E S EA R C H A R T I C L E
Comparative Analysis of Tsc1 and Tsc2 Single and
Double Radial Glial Cell Mutants
Ulrike Mietzsch,1 James McKenna III,2 R. Michelle Reith,3 Sharon W. Way,4 and Michael J. Gambello2*
1
Department of Pediatrics/Neonatology, Indiana School of Medicine, Indianapolis, Indiana 46202
Department of Human Genetics, Emory University School of Medicine, Atlanta, Georgia 30322
3
Program in Human and Molecular Genetics, University of Texas Health Science Center at Houston, Houston, Texas 77030
4
Department of Neurology, University of Chicago, Chicago, Illinois 60637
2
ABSTRACT
Tuberous sclerosis complex (TSC) is a neurodevelopmental disorder with variable expressivity. Heterozygous
mutations in either of two genes, TSC1 (hamartin) or
TSC2 (tuberin), are responsible for most cases. Hamartin and tuberin form a heterodimer that functions as a
major cellular inhibitor of the mammalian target of
rapamycin complex 1 (mTORC1) kinase. Genotypephenotype studies suggest that TSC2 mutations are
associated with a more severe neurologic phenotype,
although the biologic basis for the difference between
TSC1- and TSC2-based disease is unclear. Here we performed a study to compare and contrast the brain phenotypes of Tsc1 and Tsc2 single and double mutants.
Using Tsc1 and Tsc2 floxed alleles and a radial glial
transgenic Cre driver (FVB-Tg(GFAP-cre)25Mes/J), we
deleted Tsc1 and/or Tsc2 in radial glial progenitor cells.
Single and double mutants had remarkably similar phenotypes: early postnatal mortality, brain overgrowth,
laminar disruption, astrogliosis, a paucity of oligodendroglia, and myelination defects. Double Tsc1/Tsc2
mutants died earlier than single mutants, and single
mutants showed differences in the location of heterotopias and the organization of the hippocampal stratum
pyramidale. The differences were not due to differential
mTORC1 activation or feedback inhibition on Akt. These
data provide further genetic evidence for individual
hamartin and tuberin functions that may explain some
of the genotype–phenotype differences seen in the
human disease. J. Comp. Neurol. 521:3817–3831,
2013.
C 2013 Wiley Periodicals, Inc.
V
INDEXING TERMS: tuberin; hamartin; genotype; phenotype; brain
Tuberous sclerosis complex (TSC) is an autosomal
dominant disorder with an incidence of 1 in 6,000 live
births (Gomez, 1999; Crino et al., 2006). The tumors or
cysts associated with TSC most commonly affect the
brain, kidney, skin, heart, and lung. Greater than 95% of
patients have some degree of anatomical brain pathology that can include cortical or cerebellar tubers, subependymal
nodules,
subependymal
giant
cell
astrocytomas, white matter abnormalities, and other
lesions (DiMario, 2004). These lesions are associated
with seizures, intellectual disability, hydrocephalus,
autism spectrum disorders, as well as other neurocognitive disturbances (DiMario, 2004; Crino et al., 2006).
TSC is caused by mutations in either of two genes:
TSC1 or TSC2 (Consortium, 1993; van Slegtenhorst
et al., 1997). The TSC1 gene encodes the 130-kDa
protein hamartin; the TSC2 gene encodes the larger
200-kDa protein tuberin. The two proteins form a TSC1TSC2 heterodimer (TSC Complex) that regulates the
C 2013 Wiley Periodicals, Inc.
V
insulin-signaling mammalian target of rapamycin complex 1 (mTORC1) pathway, which controls translation,
cell growth, and proliferation (Plank et al., 1998; Sarbassov et al., 2005; Huang et al., 2008). This functional
interaction explains why mutations in either gene result
in a similar, albeit often not identical disease phenotype. The Ras-like protein, Rheb, activates the mTORC1
pathway in the GTP-bound state. The TSC complex negatively regulates the mTORC1 pathway by promoting
the conversion of Rheb-GTP to Rheb-GDP. This
The first two authors contributed equally.
Grant sponsor: National Institutes of Health (NIH); Grant number:
R01NS060804 (to M.J.G.).
*CORRESPONDENCE TO: Michael J. Gambello, MD, PhD, Department of
Human Genetics, Whitehead Biomedical Research Building, 615 Michael
St., Ste. 301, Atlanta, GA 30322. E-mail: [email protected]
Received August 29, 2012; Revised February 27, 2013;
Accepted May 24, 2013.
DOI 10.1002/cne.23380
Published online June 8, 2013 in Wiley
(wileyonlinelibrary.com)
Online
The Journal of Comparative Neurology | Research in Systems Neuroscience 521:3817–3831 (2013)
Library
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U. Mietzsch et al.
conversion is effected by a GTPase activating domain of
the carboxy terminus of Tsc2. The TSC complex acts as
a molecular rheostat, responding to growth factor stimulation, cellular energy status, and oxygen tension (Han
and Sahin, 2011). A two-hit model is believed to be a
major mechanism behind TSC pathogenesis (Henske
et al., 1997; Au et al., 1999): 85% of TSC patients have
an identifiable germline inactivating mutation in one
allele of TSC1 or TSC2 in all cells, and stochastic loss
of the second allele in a somatic cell results in the
complete absence of either hamartin or tuberin, leading
to activated mTORC1. Many TSC lesions show loss of
heterozygosity (LOH) as well as activated mTORC1
(El-Hashemite et al., 2003; Crino, 2004; Crino et al.,
2006).
A hallmark of TSC is extreme inter- and intrafamilial
phenotypic variability (Sancak et al., 2005; Au et al.,
2007). Differences in phenotypes within a family are
likely related to stochastic events, modifier genes, and
possibly environmental influences. Genotype–phenotype
studies suggest that TSC2-based disease is more
severe and associated with a higher burden of brain
pathology and intellectual disability (Jones et al., 1997,
1999; Dabora et al., 2001; Lewis et al., 2004; Au et al.,
2007). We cannot yet account for these genotype–
phenotype data, but different molecular effects from
the loss of hamartin versus tuberin might explain the
differences in disease severity.
Several brain-specific mouse models of TSC have
made it possible to study the roles of hamartin and
tuberin in both normal brain development and in the
neuropathology of TSC (Uhlmann et al., 2002; Meikle
et al., 2007; Way et al., 2009; Carson et al., 2012). In
particular, mouse models provide the unique opportunity to compare and contrast brain-specific deletions of
Tsc1 and/or Tsc2. Analysis of these model organisms
helps determine whether there might be in vivo differences among single or double Tsc1/Tsc2 mutants. For
example, comparison of astrocyte-specific knockout
(KO) mice, Tsc1GFAP1 CKO and Tsc2GFAP1 CKO, revealed
a more severe brain phenotype in the Tsc2 animals
(Zeng et al., 2011). The Tsc2GFAP1 CKO mice had an
earlier onset and higher frequency of seizures and more
severe histologic deficits than Tsc1 animals. Moreover,
increased mTORC1 activation was seen in the Tsc2
brains and may have led to the more severe phenotypes. In this report, we further analyze genotype–phenotype correlations using radial glial-specific mutant
animals. We have reported before on the Tsc2 radial
glial model of TSC, in which Tsc2 was deleted from
radial glial progenitor cells early in development using
an hGFAP-cre line (designated Tsc2 f/-;cre in this article) (Way et al., 2009). These animals recapitulated
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many aspects of the human disease, including megalencephaly, clinical seizures, cortical and hippocampal
migration defects, enlarged cells, and white matter
defects (Gomez, 1999). The Tsc2 f/-;cre animals are different from previously reported astrocyte-specific
mutants (Uhlmann et al., 2002; Zeng et al., 2011). In
the radial glial mutants reported here, both neurons
and glia lack either Tsc1 and/or Tsc2. We generated
Tsc1f/-;cre, Tsc2f-;cre, and Tsc1f/-; Tsc2f-;cre animals.
Our analysis indicates that loss of Tsc1 and/or Tsc2 in
radial glia cause comparable brain pathology, but the
loss of both proteins appears to have an additive effect
on mortality. There were also some notable differences
in hippocampal organization that did not appear to be
due to differences in mTORC1 activation. These results
provide genetic evidence for functional differences
between hamartin and tuberin and may help explain the
genotype–phenotype differences seen in the human
disease.
MATERIALS AND METHODS
All animal experimentation was approved by the University of Texas Health Science Center (UTHSC) Animal
Welfare Committee and carried out in accordance with
the Guide for the Humane Use and Care of Laboratory
Animals. We generated Tsc11/- mice by crossing mice
with a floxed Tsc1 allele (Tsc1f/f 5 Tsc1tmDjk; Jackson
Laboratories Stock number 005680, Bar Harbor, ME)
and CMV-cre mice (C57BL/6J). The F1 generation was
then crossed again with a C57BL/6J mouse to eliminate the CMV-cre. Tsc11/- mice were crossed with an
hGFAP-cre mouse (FVB-Tg(GFAP-cre)25Mes/J, Jackson
Labs) to generate male Tsc11/-;hGFAP-cre drivers that
were crossed with Tsc1f/f females to generate
Tsc1f/-;cre mice. Tsc2f/-;cre mice were generated as
previously described. Approximate genetic backgrounds
are: Tsc1f/-;cre 5 56.9% C57BL/6, 30.2%129X1/SvJ;
12.5% FVB; Tsc2f/-;cre 5 45% C57BL/6; 30% 129X1/
SvJ; 25% FVB; Double mutant 5 56.9% C57BL/6,
30.2%129X1/SvJ; 12.5% FVB. Mice were genotyped as
previously reported (Uhlmann et al., 2002; Way et al.,
2009).
Histological studies
Mice were anesthetized with 2.5% Avertin and then
perfused with ice-cold phosphate-buffered saline (PBS;
pH 5 7.4) and 4% paraformaldehyde (PFA). Brains were
postfixed overnight in 4% PFA at 4 C and stored in 70%
ethanol. Tissue was then dehydrated and embedded in
paraffin. Paraffin blocks were then sectioned at 5 lm
with a Leica Rotary Microtome RM2245 (Richmond, IL).
The Journal of Comparative Neurology | Research in Systems Neuroscience
TSC1/TSC2 single/double radial glial mutants
Slides were rehydrated and stained with hematoxylin
and eosin.
Immunofluorescence was performed by blocking in
10% serum from an animal in which secondary antibody
was raised and 0.05% Triton X-100 in 13 PBS for 1
hour. Primary antibody was allowed to incubate overnight
in a humidified chamber at 4 C. Sections were washed
in 13 PBS followed by secondary antibody incubation
for 1 hour at room temperature. Nuclei were stained by
incubating the slides for 10 minutes in 1:1,000 Hoechst
in 13 PBS. Tissue images were analyzed by direct visualization with an Olympus BX51 or IX81 microscope. Photos were captured with a Q-Imaging RETIGA-2000RV
digital camera. Digital images were processed using
Adobe Photoshop CS4 (San Jose, CA).
Antibody characterization
Antibodies used in this study are commercially available. Table 1 provides antibody information, including
immunogens, host species, commercial sources, catalog
numbers, and dilutions for single antigen immunohistochemistry (IHC). Hoechst 33258 (Invitrogen, La Jolla,
CA) was used for nuclear staining and coverslipping of
postnatal tissue.
All primary antibodies used here have been previously used and validated in peer-reviewed publications.
The antibodies for CC1 (Calbiochem, La Jolla, CA;
#OP80), Cux-1 (SantaCruz, Santa Cruz, CA; #sc13024), FoxP2 (Abcam, Cambridge, MA; #ab16046),
GFAP (Sigma, St. Louis, MO; #G 3893), and MBP
(Chemicon, Temecula, CA; #AB980) are all found in the
JCN Antibody Database and were utilized in a comparable application.
The rabbit polyclonal AKT1 antibody (SantaCruz, #sc1618-R) shows a distinct band at 62 kDa on western
blots from mouse brain lysates. This corresponds to a
band visualized on western blots run on NIH/3T3,
MCF7, and KNRK whole cell lysates (manufacturer provided). It has also been previously characterized using
western blot (DiNardo et al., 2009; Li et al., 2011). The
rabbit monoclonal alpha-tubulin antibody (Cell Signaling,
Beverly, MA; #2125) shows a clear band at 52 kDa on
western blots from mouse brain lysates. This corresponds to a band visualized on western blots run on
C6, COS-7, and HeLa cell extracts (manufacturer provided). Flow cytometric analysis of HeLa cells using this
antibody also shows a specific binding profile when
compared to nonspecific negative control (manufacturer
provided). The rabbit polyclonal hamartin antibody (Cell
Signaling, #4906) shows a distinct band at 150 kDa on
western blots from control and Tsc2f/-;cre brain lysates,
and shows diminished signal in the Tsc1f/-;cre and
Tsc1f/-; Tsc2 f-;cre animals. This band corresponds to
western blots run on NIH/3T3 and PC12 cell extracts
(manufacturer provided). Flow cytometric analysis of
NIH/3T3 cells using this antibody has shown a specific
binding profile when compared to a nonspecific negative control (manufacturer provided). The mouse monoclonal Olig2 antibody (Millipore, Billerica, MA;
#MABN50) allowed visualization of a distinct 37 kDa
band on western blot from mouse brain lysate (manufacturer provided). Also, this antibody has been shown
to detect Olig2 in coronal brain tissue as reported by
an independent laboratory (Cai et al., 2007).
The rabbit monoclonal phospho-AKT antibody (Cell Signaling, #4060) shows a clear band at 60 kDa on western
blots from mouse brain lysates. This band corresponds
to western blots run on PC-3 cell extracts, and this band
is abolished when cells are treated with the PI3K antagonist wortmannin (manufacturer provided). Immunohistochemical analysis using this antibody shows positive
signal in LNCaP cells, but it is greatly diminished in
LY294002-treated cells (manufacturer provided).
The rabbit polyclonal phospho-S6 (Ser235/236) antibody (Cell Signaling, #2211) shows a clear band at 32
kDa in mouse brain lysates. The Tsc1f/-;cre, Tsc2f/-;cre,
and Tsc1f/-; Tsc2f/-;cre brain lysates all show highly
upregulated levels of phospho-S6(Ser235/236) compared to control. This band corresponds to western blots
run on 293 cells which were treated with 20% fetal
bovine serum (FBS) over time and show an increase in
protein synthesis (manufacturer provided). Immunofluorescent analysis of HeLa cells untreated, 20% serum
treated, and rapamycin pretreated shows signal only in
the 20% serum treated condition (manufacturer provided). The rabbit polyclonal phospho-S6 (Ser240/244)
antibody (Cell Signaling, #2215) shows a distinct band
at 32 kDa in mouse brain lysates. The Tsc1f/-;cre,
Tsc2f/-;cre, and Tsc1f/-; Tsc2f/-;cre brain lysates all show
highly upregulated levels of phospho-S6(Ser240/244)
compared to control. This band corresponds to western
blots run on 293 cells which were treated with 20% FBS
over time and show an increase in protein (manufacturer
provided). Other laboratories have also demonstrated the
specificity of this antibody via western blot (Fonseca
et al., 2011; Goto et al., 2011). The rabbit monoclonal
S6 antibody (Cell Signaling, #2217) shows a distinct
band at 32 kDa in mouse brain lysates, which is consistent for all genotypes. A corresponding band on western
blot has been observed in HeLa, NIH/3T3, PC12, and
COS cell extracts (manufacturer provided). Other laboratories have also demonstrated the specificity of this antibody via western blot (Possemato et al., 2011; Yang
et al., 2011). The rabbit polyclonal tuberin antibody (Cell
Signaling, #3612) shows a distinct band at 200 kDa in
mouse brain lysates, and it was diminished in Tsc1f/-;cre,
The Journal of Comparative Neurology | Research in Systems Neuroscience
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2
1
Immunogen
synthetic peptide mapping at C-terminus of human AKT1 from aa
430–480
synthetic peptide (KLH-coupled) corresponding to sequence
CQPDGQMPSDKTIGGGDDS of human alpha-tubulin
recombinant mouse protein consisting of aa 1–226 of APC (CC-1)
synthetic peptide, aa 1111–1332 of CDP from mouse
synthetic peptide conjugated to KLH derived from within aa 703–
715 to C-terminus of human FOXP2
Purified full length GFAP from pig spinal cord
synthetic peptide (KLH-coupled) corresponding to sequence
CPRGGFDSPFYRDSLPGSQRK of human hamartin
Full length human myelin basic protein from brain
recombinant protein corresponding to N terminus from aa 10–110
of human Olig2
synthetic phospo-peptide(KLH-coupled) corresponding to sequence
CHFPQFsYSAS of human Akt
synthetic phospho-peptide(KLH-coupled) corresponding to sequence
CRRLssLRASTSKSES of human ribosomal protein S6
synthetic phospo-peptide(KLH-coupled) corresponding to sequence
CRRLSSLRAsTSKsES of human S6 ribosomal protein
synthetic peptide (KLH-coupled) corresponding to sequence
CLQHKRRRIALKKQRTKKNKEE of human S6 ribosomal protein
synthetic peptide (KLH-coupled) corresponding to sequence
CQRKRLISSVEDFTEFV of human tuberin
Antibody was used for western blotting.
Antibody was used for IHC staining.
Tuberin1
S61
ps6(240/244)1
ps6 (235/236)1
pAKT (Ser473)1
MBP2
Olig22
GFAP2
Hamartin1
CC12
Cux-1(CDP)2
FOXP22
alpha-tubulin1
AKT1
1
Antigen
TABLE 1.
List of Antibodies
Cell Signaling, Rabbit IgG, Polyclonal, #3612
Cell Signaling, Rabbit IgG, Monoclonal, #2217
Cell Signaling, Rabbit, Polyclonal, #2215
Cell Signaling, Rabbit IgG, Polyclonal, #2211
Cell Signaling, Rabbit IgG, Monoclonal, #4060
Chemicon, Rabbit IgG, Polyclonal, #AB980
Millipore, Mouse IgG2aK, Monoclonal, #MABN50, lot# ng1852660
Sigma, Mouse IgG1, Monoclonal, #G 3893
Cell Signaling, Rabbit IgG, Polyclonal, #4906
Calbiochem, Mouse IgG2b, Monoclonal, #OP80 Lot#D00082502
Santa Cruz Biotechnology, Rabbit, Polyclonal, #sc-13024
Abcam, Rabbit,Polyclonal, #ab16046
Cell Signaling, Rabbit IgG, Monoclonal, #2125
Santa Cruz Biotechnology, Rabbit, Polyclonal, #sc-1618-R
Source, species., mono/poly-clonal, cat. no.
1:1,000
1:2,000
1:1,000
1:1,000
1:1,000
1:200
1:200
1:400
1:1,000
1:50
1:50
1:400
1:2,000
1:1,000
Dilution
U. Mietzsch et al.
TSC1/TSC2 single/double radial glial mutants
Tsc2f/-;cre, and Tsc1f/-; Tsc2f/-;cre brain lysates. A corresponding band on western blot has been observed in
NIH/3T3 and MCF-7 cell extracts (manufacturer provided). Other laboratories have also demonstrated the
specificity of this antibody via western blot (Wu et al.,
2010; Park et al., 2011).
Quantitative analysis
Two or three serial sections from each mouse were
used for analysis unless otherwise noted. For postnatal cell
counts, images were captured at 103 magnification, and
equal-sized areas spanning the complete thickness of the
cortex were cropped for counting. Marker-labeled cells with
visible nuclei were manually counted using Adobe Photoshop CS4 (San Jose, CA). One-way analysis of variance
(ANOVA) with an alpha level of 0.05 was used to analyze
weight, cortical thickness, neuronal size, Cux-1 and FoxP2
cell counts, and western blot densitometry. Tukey and
Scheffe post-hoc analyses were performed when ANOVA
indicated a significant difference between groups. A logrank test was performed on the survival data. A Fisher
exact test was conducted for the pyramidal layer analysis.
Data were analyzed using IBM SPSS Statistics 20 (Armonk,
NY) and Microsoft Excel 2010 (Seattle, WA).
Protein analysis
Whole cell lysates were made from brains that were
quick-frozen in liquid nitrogen, then homogenized with a
dounce homogenizer using 10 volumes of cerebrospinal
fluid (CSF) buffer with protease inhibitor cocktail and
phosphatase inhibitor cocktail (Sigma). Protein lysates
were centrifuged at 4 C, sonicated, and stored at 280 C
until use. Protein concentrations were determined with a
BCA reagent kit (Pierce, ThermoFisher Scientific, Rockford,
IL). Equal amounts of protein were separated on a denaturing 4–12% gradient gel (Invitrogen) and transferred to a
PVDF membrane, pore size 0.45 lm (Immobilon Transfer
Membranes, Millipore). The membrane was incubated
with the desired antibody using a stripping procedure
after each experiment, if necessary. Secondary antibodies
were horseradish peroxidase-conjugated. Visualization was
developed via the SuperSignal West Pico ECL kit (Thermo
Scientific, Rockford, IL) and KonicaMinolta Tabletop Processor SRX-101A (Konica Minolta, Wayne, NJ).
RESULTS
Comparison of health, growth, and survival
in single and double mutants
All mutants were born in the expected Mendelian
ratios, indicating that deletion of Tsc1 and/or Tsc2 in
radial glial cells was not embryonic lethal. By postnatal
day (P)15 all mutants were significantly smaller and
weighed less than control animals (F(3,30) 5 4.93, P 5
0.007) (Fig. 1A–E). There was no significant difference
in weight among the mutants as determined by posthoc tests (Tukey HSD and Scheffe). All mutants were
sick, with splayed feet, tremors, domed heads, and
occasional seizure-like activity. All mutants died prematurely compared to controls (P < 0.001, log-rank test),
either close to or shortly after weaning. Dead animals
were often found in extensor position, suggesting they
died of seizures. Kaplan-Meier survival curves were similar for the Tsc1f/-;cre and the Tsc2f/-;cre animals (Fig.
1F). A small cohort of the Tsc1f/-;cre lived until P45,
whereas a small cohort of the Tsc2f/-;cre lived until
P39; this difference was not statistically significant (P
5 0.61, log-rank test). All double mutants,
Tsc1f/-;Tsc2f/-;cre, died at or before P23, significantly
younger (P < 0.001, log-rank test) than single mutants.
These data show there is little difference in morbidity
or mortality between the Tsc1 and Tsc2 single radial
glial knockouts, but loss of both genes seemed to have
an additive effect that resulted in earlier mortality.
Similar effects on brain size and cortical
disorganization among single and double
mutants
All mutant brains had significantly increased cortical
thickness compared to control brains (F(3,19) 5 7.130,
P < 0.002), demonstrating well-established roles of the
TSC genes in regulating organ size (Fig. 2) (Potter
et al., 2001). Although the area of the cortical neurons
in the mutants did not show a statistically significant
increase from controls (P 5 0.08), median neuron area
tended to be greater in mutants than controls (Fig. 3).
Since TSC is often considered a neuronal migration
defect, we assessed cortical lamination using layerspecific transcription factors. We previously showed
that the Tsc2f/-;cre mouse had lamination abnormalities
(Way et al., 2009), and we expected similar defects in
Tsc1 and double mutants. Neurons in layer VI of the
cortex represent early-born neurons. To assess their
appropriate position and number, we performed IHC for
the layer VI-specific transcription factor FoxP2. We
found that the number of FoxP2-positive cells was significantly lower in all mutants versus control mice
(F(3,6) 5 48.52, P < 0.001), but there was no statistically significant difference among the three mutant
genotypes (Fig. 4E–H,L). The location of FoxP2-labeled
cells was no different from control, suggesting that loss
of Tsc1/2 does not affect migration into layer VI. Many
late-born neurons migrate to the upper layers of the
cortex. To assess the number and position of these
late-born neurons in the cortex, we performed IHC for
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U. Mietzsch et al.
Figure 1. Growth curves and longevity of Tsc1 and Tsc2 single and double mutants. A–D: All mutant mice were smaller than control (A)
(Tsc11/1;Tsc21/f) animals at P21. The physical appearance of the Tsc1f/-;cre (B), Tsc2f/-;cre, (C) and Tsc1f/-;Tsc2f/-;cre (D) was identical
at weaning. E: Growth curves of control, Tsc1f/-;cre, Tsc2f/-;cre,and Tsc1f/-;Tsc2f/-;cre. All mutant mice had similar growth curves, indicating poor weight gain compared to control (black) animals. F: The Kaplan–Meier survival curves are shown for all genotypes. None of the
control mice died prematurely. The Tsc1f/-;cre and the Tsc2f/-;cre mutant mice showed similar survival; 50% of all animals in these two
groups died at or before P23, 75% died at or before P27 in the Tsc1f/-;cre and P26 for the Tsc2f/-;cre mutant cohort. While a small number of Tsc1f/-;cre mutant mice survived until P41, this was not statistically significant (P 5 0.61). The most striking differences were found
between the two single mutant mice and the Tsc1f/-;Tsc2f/-;cre mutant mice. Of all double mutants, 50%, died at or before P20, 75% died
at or prior to P21, and only 5% survived until P23. These differences were all statistically significant (P < 0.001).
Cux1, a transcription factor that labels neurons positioned in the cortical layers II–IV. We found that, in contrast to the decreased number of FoxP2 neurons in
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layer VI of mutant brains, the number of Cux1-positive
cells was similar among all four genotypes (F(3,9) 5
1.29, P 5 0.337) (Fig. 4A–D,I); however, we found a
The Journal of Comparative Neurology | Research in Systems Neuroscience
TSC1/TSC2 single/double radial glial mutants
Figure 2. Whole-brain anatomy and cortical histology of P21 animals. A–D: Hematoxylin and eosin staining of coronal sections of cerebral
cortex of control (A), Tsc1f/-;cre (B), Tsc2f/-;cre (C) and Tsc1f/-;Tsc2f/-;cre (D) mice. (F) All mutant cerebral cortex sections were thicker
than control scale bar = 100 microns. Cortical neurons of all mutants tended to be larger than control (black arrows, insets) inset scale
bar 50 lm. E: All mutant brains were larger than control, and occasionally the double mutant was larger than either single mutant. Scale
bar 5 2 mm in E. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
population of Cux1-labeled cells in the lower layers of
the cortex in all mutant mice (Fig. 4J). The ratio of
ectopic Cux1-positive/Total Cux1-positive cells was similar among the three mutant genotypes, and statistically
greater than control (F(3,9) 5 4.40, P < 0.04) (Fig. 4K).
These results suggest that neuronal migration is similarly
affected in single and double mutants. Deletion of Tsc1
and/or Tsc2 from radial glial cells during embryonic
development does not appear to affect the number
of neurons originating from the SVZ, but mTORC1 activation does regulate their appropriate positioning in the
cortex. In summary, deletion of Tsc1 and/or Tsc2 from
radial glial cells affects the number of early-born,
layer VI neurons, and the position of later-born, layer
II–IV neurons.
and several subtle
types. Overall, the
disorganized in all
mice (Fig. 5A–P).
differences among the three genostratum pyramidale was markedly
mutants compared to the control
The Tsc1f/-;cre mice had ectopic
Differences in hippocampal development
among single and double mutants
In hGFAP-Cre mice, Cre expression begins at embryonic day (E)12.5 in the radial glial cells of the developing hippocampus (Malatesta et al., 2003). In all
mutants, this timing of Cre expression and subsequent
Tsc1 and/or Tsc2 deletion is concurrent with hippocampal development. Histologic analysis of the hippocampus at P21 showed many pathologic similarities
Figure 3. Cell size of NeuN-positive cells in cortical Layer II. Boxplot showing the distribution of cell areas (lm2) of NeuN-positive
cells in cortical layer II for control and mutant mice (n 5 3).
Lines represent the median value.
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U. Mietzsch et al.
Figure 4. Cortical layer formation. A–D: Upper-layer neurons were stained with Cux1 in Control, Tsc1f/-;cre, Tsc2f/-;cre, Tsc1f/-;Tsc2f/-;cre
left to right. (I) The number of Cux1-positive cells was comparable among all genotypes. Cux1-positive cells in the control mice were found
as expected in layers II–IV of cerebral cortex. In the Tsc1f/-;cre, Tsc2f/-;cre, and Tsc1f/-;Tsc2f/-;cre mutant mice, we also found most of
the Cux1-labeled cells in layers II–IV, but a significantly higher number of upper-layer neurons were found in the lower layers. J: The number of these ectopic upper-layer neurons was comparable in the Tsc1f/-;cre and Tsc2f/-;cre mutant mice, but significantly higher in the
Tsc1f/-;Tsc2f/-;cre mice. K: The ratio of ectopic to total Cux1-labeled cells. E–H: Layer VI neurons were labeled with FoxP2 in Control,
Tsc1ff/-;cre, Tsc2ff/-;cre, Tsc1ff/-;Tsc2f/-;cre left to right. L: We found that the number of FoxP2-labeled cells was significantly decreased in
Tsc1f/-;cre, Tsc2f/-;cre, and Tsc1f/-;Tsc2f/-;cre mice compared to the control mice. Scale bars 5 100 lm in A–D,E–H;. [Color figure can be
viewed in the online issue, which is available at wileyonlinelibrary.com.]
neurons scattered more uniformly throughout the stratum oriens (Fig. 5B,F) compared to the other mutants.
In contrast, the pyramidal layer of the Tsc2f/-;cre and
the double mutant while disorganized, was better
formed, and the ectopic cells seemed to form their own
layer (Fig. 5C,D,G,H). This double-layer phenotype was
observed significantly more in the Tsc2f/-;cre and double KO than the Tsc1f/-;cre (P < 0.045, FET) The results
are summarized in Table 2.
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One common feature of TSC is the subependymal
nodule (DiMario, 2004; Crino et al., 2006). We previously reported ring, or nodular heterotopia in the stratum lacunosum moleculare of the Tsc2f/-;cre mice, and
observed this phenotype again in Tsc2f/-;cre in our
present study (Fig. 5C,K). We also observed ring heterotopia in the double mutant (Fig. 5D,L), but never in the
Tsc1f/-;cre animals (Fig. 5B,J; Table 2). However, we did
detect ectopic clusters of neurons along the ventricles
The Journal of Comparative Neurology | Research in Systems Neuroscience
TSC1/TSC2 single/double radial glial mutants
Figure 5. Hippocampal histology. A–P: H&E staining of the hippocampus of the control (A, E, I, M), Tsc1f/-;cre (B, F, J, N), Tsc2f/-;cre (C,
G, K, O), and Tsc1f/-;Tsc2f/-;cre (D, H, L, P) mice. A disorganized stratum pyramidale (sp) is noted in all three mutant genotypes. B-F: The
stratum pyramidale in the Tsc1f/-;cre mutant mice is diffusely disrupted (B, F), whereas in the Tsc2f/-;cre and double mutant mice, it
appeared to have formed a second layer. (C, D, G, H) Ring heterotopia were often found in the stratum lacunosum moleculare (slm) of Tsc2f/-;cre
and double mutants, but never in Tsc1f/-;cre animals. (C, D, K, L) In the Tsc1f/-;cre and double mutant mice, we found heterotopias located around
the ventricles, resembling subependymal nodules seen in the human patients (B, D, N, P). Low-magnification scale bar 5 200 mm; highmagnification scale bar 5 50 lm. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
of the Tsc1f/-;cre and double mutant mice (Fig. 5N,P).
These results point to individual functions for hamartin,
tuberin, and the hamartin-tuberin complex in the development of the hippocampus.
No differences in effects on glial cells or
myelination among the three genotypes
Loss of Tsc1 or Tsc2 in astrocytes caused an astrogliosis and marked overexpression of cortical GFAP
(Uhlmann et al., 2002). We found a similar astrogliosis
in all three genotypes (Fig. 6A–D). We next examined
mature oligodendroglia using the markers olig2 and
CC1 and found a marked decrease in the number of
mature oligodendroglia in all mutant genotypes (Fig.
7A–L), consistent with a crucial role for the Tsc1-Tsc2
complex and mTORC1 regulation in the control of oligodendrocyte maturation (Tyler et al., 2009).
Abnormalities in myelination are also seen in the
brains of patients with TSC (Ridler et al., 2001; Makki
et al., 2007). Tsc2 deletion from radial glial cells or
postmitotic neurons causes hypomyelination (Meikle
et al., 2007; Way et al., 2009) Here we found a comparable decrease in cortical myelination in all three
mutant genotypes compared to controls. Myelin
remains present within the corpus callosum, but does
not progress into the cortex (Fig. 8A–D). These results
suggest minimal differences in myelination among the
three genotypes.
No differences in mTORC1 activation
or feedback inhibition of Akt among
the genotypes
We examined protein expression in whole brain
lysates from the different genotypes to assess the level
of mTORC1 activation and feedback inhibition of Akt.
Expression of tuberin was decreased in brain lysates of
Tsc2 f/-;cre, Tsc1f/-;Tsc2f/-;cre, and Tsc1f/-;cre compared to control. The decrease in tuberin in Tsc1f/-;cre
TABLE 2.
Penetrance of Hippocampal Phenotypes
Phenotype
Heterotopia in SLM
Scattered Pym. Layer
Double Pym. Layer
Tsc1
;cre
f/-
Tsc2
;cre
Tsc1 f/-;
Tsc2 f/-;cre
0/7
8/9
1/9
15/15
2/6
4/6
7/7
0/7
7/7
f/-
The Journal of Comparative Neurology | Research in Systems Neuroscience
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U. Mietzsch et al.
Figure 6. GFAP immunostaining. A–D: IHC for GFAP on cortex proximal to the midline was performed on control, Tsc1f/-;cre, Tsc2f/-;cre,
and Tsc1f/-; Tsc2f/-;cre mice (left to right). Scale bar 5 50 lm. [Color figure can be viewed in the online issue, which is available at
wileyonlinelibrary.com.]
lysates is consistent with the well-established role of
Tsc1 in stabilizing Tsc2. Hamartin expression was
decreased in Tsc1f/-;cre and Tsc1f/-;Tsc2f/-;cre animals,
but unaffected in Tsc2f/-;cre. Tuberin does not appear
to be necessary for hamartin stability.
A major cellular consequence of the loss of Tsc1 or
Tsc2 is activation of the mTORC1 kinase. Activation of
mTORC1 has been demonstrated in human TSC samples based on increased levels of the downstream substrate phosphorylated ribosomal protein S6 (Chan
et al., 2004; Crino, 2004). Activated ribosomal S6 can
be phosphorylated at several sites, including Ser 235/
236 and Ser 240/244. Using phosphospecifc antibodies, we found a significant upregulation of pS6-Ser235/
236 (F(3,8) 5 4.94, P < 0.032) and pS6-Ser240/244
(F(3,8) 5 9.38, P < 0.005) in brain lysates of all
mutant genotypes (Fig. 9). These data indicate that differential mTORC1 activation is not the principal mechanism for the histologic differences seen among the
genotypes. The activation of mTORC1 is accompanied
by considerable feedback inhibition on the PI3K pathway and mTORC2, another form of mTOR (Huang and
Manning, 2009). Both PI3 kinase and mTORC2 serve to
activate Akt by phosphorylation of Ser473. The TSC
complex activates mTORC2 (Huang et al., 2008); consequently, loss of the TSC complex causes a decrease in
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Akt Ser-473 by decreasing mTORC2 activity, as well as
by feedback inhibition of PI3 kinase by S6 kinase (Dibble et al., 2009). We examined how the loss of Tsc1
and/or Tsc2 affected levels of Akt-Ser473 and found a
similar decrease in activated Akt among the three
genotypes.
DISCUSSION
TSC, a disease associated with serious neuropathology, is caused by mutations in either TSC1 or TSC2 in
about 85% of cases, underscoring the biochemical association between hamartin (Tsc1) and tuberin (Tsc2) to
form the TSC complex. Based on this association, the
loss of either binding partner should produce a very
similar, if not identical, phenotype; however, genotype–
phenotype studies reveal that TSC2-based disease is
linked to more severe neurologic signs and symptoms
(Jones et al., 1997; Dabora et al., 2001; Au et al.,
2007). The reasons for this remain obscure due to variations in mutation type, cell-specific stochastic events
leading to LOH in some lesions, environmental effects,
allele-specific expression differences, genetic modifiers,
and other potential mechanisms. To explore the genetic
basis of genotype–phenotype differences, we created
Tsc1, Tsc2, and Tsc1;Tsc2 brain-specific KO models
The Journal of Comparative Neurology | Research in Systems Neuroscience
TSC1/TSC2 single/double radial glial mutants
Figure 7. Oligodendrocyte maturation. A–C: Medial corpus callosum (CC) in control mice shows abundant Olig2 (green) and CC1
(magenta) immunostaining at P21. D–L: Note that all three mutant genotypes, Tsc1f/-;cre (D-F), Tsc2ff/-;cre (G-I), Tsc1f/-; Tsc2f/-;cre (J-L),
display a decreased number of cells staining for both markers in medial CC. The localization of the respective markers in the Olig21/
CC11 cells do appear normal in the mutant genotypes (insets). Scale bars 5 50 lm; 20 lm in insets.
The Journal of Comparative Neurology | Research in Systems Neuroscience
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U. Mietzsch et al.
Figure 8. MBP immunostaining. A–D: IHC for MBP in cortex and corpus callosum was performed on control (A), Tsc1f/-;cre (B), Tsc2f/-;cre
(C), and Tsc1f/-;Tsc2f/-;cre (D) mice. Scale bar 5 50 lm. [Color figure can be viewed in the online issue, which is available at
wileyonlinelibrary.com.]
Figure 9. Immunoblot analysis of whole-brain extracts from control, Tsc1f/-;cre, Tsc2f/-;cre, and Tsc1f/-;Tsc2f/-;cre double mutant mice, left
to right. Phospho-S6 (Ser235/236) and Phospho-S6 (Ser240/244) were quantitated after adjusting for total S6 levels, and densitometry
readings were analyzed using ImageJ v. 1.46 (W. Rasband, National Institutes of Health, Bethesda, MD).
3828
The Journal of Comparative Neurology | Research in Systems Neuroscience
TSC1/TSC2 single/double radial glial mutants
using the same hGFAP-cre driver to delete either or
both genes in radial glial cells. This strategy allowed us
to compare phenotypes while keeping other parameters
relatively constant such as mutation type (deletion), cell
type, mode of the second hit (radial glial cell and deletion), environment, and genetic background. We found
that the brain phenotypes of all genotypes were
remarkably similar, albeit with some subtle differences.
In spite of the similarities mentioned, there were
notable phenotypic differences among the genotypes
that suggest independent functions of hamartin and
tuberin. Although the median survival of the mutants
was similar, all the double Tsc1/Tsc2 mutants died
much sooner than either single mutant. Also noteworthy
are the ring heterotopia in the stratum lacunosum
moleculare (SLM) seen only in the Tsc2 and the double
mutants; the Tsc1 mutants had nodule-like lesions
along the ventricles, but no distinctive ring heterotopias. There were also organizational differences in the
pyramidal layer. Tuberin might have an important functional role in neuronal migration, independent of its
association with hamartin. Tsc2 is known to interact
with many other proteins (Rosner et al., 2008). One
explanation for the differences seen here is that modifier proteins that specifically interact with tuberin might
be responsible for the histological differences seen in
the hippocampus. Another consideration that might
contribute to subtle phenotypic differences is the timing
of Cre recombination between the Tsc1flox and the
Tsc2flox alleles. We have not investigated this possibility. Lastly, while we made every effort to keep genetic
backgrounds similar, there were slight differences. Performing such genetic experiments on an isogenic background would be ideal, although daunting given the
variety of strains used in transgenic technology.
An earlier study compared the neurologic phenotypes
of Tsc1 and Tsc2 loss in astrocytes (Zeng et al., 2011),
and while there were many similarities between the
Tsc1 and Tsc2 mice, the Tsc2 model exhibited more
severe epilepsy and histologic abnormalities. The more
severe phenotype was attributed to differential mTORC1
activation between the genotypes, and rapamycin rescued the phenotypes in both models. In our study using
radial glial cells, although the SLM heterotopia in the
Tsc2 f/-;cre could be considered “more severe,” there
was no significant difference in the sick appearance or
the median survival of the single mutants. Furthermore,
we found no difference in mTORC1 activation among
any of the mutant genotypes as measured by pS6
235/236 and pS6 240/244. There are several possible
explanations for these differences. In the models presented here, genes are deleted from all progeny of
radial glia, both neurons and glia of the cortex and
hippocampus. The severity of these models might preclude detecting a minor effect on longevity, although
we did see earlier mortality in the double KO. The
decreased severity of our Tsc1 mutant could be due to
residual GAP activity of tuberin, although we were not
able to detect this (Zeng et al., 2011). In astrocytes,
tuberin may be more stable in the absence of hamartin
than in neurons. This would explain our inability to
detect a difference in mTORC1 activation.
Using the TSC pathogenesis model of LOH, we found
few differences among the Tsc1, Tsc2, and Tsc1/2 double KO animals. Perhaps differences in genomic stability
at the human TSC2 locus predisposes to LOH in some
CNS lesions, such as subependymal nodules. Deep
sequencing of tubers from human patients suggests
LOH may not be the primary pathogenic event in the
genesis of tubers (Qin et al., 2012). In that case, differences in the interaction of hamartin and tuberin with
alternate binding partners could be an important determinant in phenotypic differences in tuber burden. Further analysis of the independent cellular functions of
hamartin and tuberin, as well as proteomic analysis
among the different genotypes, might reveal important
pathogenic pathways that are responsible for the genotype–phenotype correlations seen in human patients.
Knowledge of these subtle differences might lead to
adjunct therapies that could synergize with mTORC1
inhibition.
ACKNOWLEDGMENTS
The authors thank Cheryl Strauss for professional editorial services.
CONFLICT OF INTEREST
The authors state that there is no conflict of interest.
ROLE OF AUTHORS
All authors had full access to all the data in the
study and take responsibility for the integrity of the
data and the accuracy of the data analysis. Study concept and design: UM and MJG. Acquisition of data: UM,
JM, RMR, SW. Analysis and interpretation of data: UM,
JM, RMR, SW, MJG. Drafting of the article: UM, JM,
MJG. Critical revision of the article for important intellectual content: MJG. Statistical analysis: JM. Obtained
funding: MJG. Administrative, technical, and material
support: JM. Study supervision: MJG.
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