Yonago Acta medica 1998;41:55–63

Tuberous Sclerosis 2 Gene Is Expressed at High Levels in SpecificTypes of Neurons in the Mouse Brain
Makoto Saji, Mitsuhiro Kimura, Hirotoshi Maki, Kouichi Nakayama, Takanori
Nomura, Eiji Nanba* and Kousaku Ohno
Department of Neurobiology, Faculty of Medicine and *Gene Research Center, Tottori
University, Yonago 683-0826, Japan
Tuberous sclerosis (TSC) is an autosomal dominant disorder characterized by mental
retardation, epilepsy and hamartomatous growth in many tissues. The gene (TSC2)
encoding a tumor suppressor protein whose mutations cause TSC, has been demonstrated to be expressed at high levels in the adult and developing brain, raising thequestion of whether or not the TSC2 gene product has unique roles in differentiation
related to cytoskeletal interactions within the central nervous system, in addition to atumor suppressor function. To determine the expression of TSC2 in functionally distinct
neuron types of the mouse brain, we carried out in situ hybridization with digoxigeninlabeled riboprobes for the detection of TSC2 mRNA. High levels of the TSC2 gene
were in neurons of the pyramidal and dentate granular layer in the hippocampus, cerebellar Purkinje cells, neurons of the piriform cortex, motor neurons in the medulla andinterneurons in the striatum, while intermediate levels were in cortical neurons, striatal
neurons, septal neurons, thalamic neurons and neurons in the substantia nigra compacta.
Thus, the high expression of the TSC2 gene has restricted distribution in specific neuronal types which are characterized by well-developed dendrites and rich in usedependent long-term changes in synaptic efficacy. These results suggest that the functionof the TSC2 gene product may be involved on a cellular basis in neuronal plasticity andrelevant to mental retardation observed in TSC patients.
Key words: tuberous sclerosis gene; cytoskeletal interaction; neuronal plasticity; in situ hybridization; mouse brain

Tuberous sclerosis complex (TSC) is an autosomal dominant disorder characterized by mental retardation, epilepsy and development of tumors termed hamartomas such as angiofibromaof the face and on the skin, cardiac rhabdomyoma, ungal fibroma, phakoma of the retina
and angiomyolipoma of the kidney (Gomez,
1988). In the central nervous system, cortical
tubers in the cerebrum and cerebellum, periventricular nodules and giant cell astrocytoma
are characteristic lesions. The frequency of
TSC among the general population is about 1 in

10,000 to 30,000 and it occurs in all racial
groups (Gomez, 1988; Ohno et al, 1982). Linkage studies have shown two loci for TSC: one
on chromosome 9q34.3 (TSC1) and another onchromosome 16p13.3 (TSC2) (Fryer et al.,
1987; Kandts et al., 1992; Povey et al., 1994).
Recently, the TSC2 gene has been isolated andcharacterized (The European Chromosome
Tuberous Sclerosis Consortium, 1993). The
gene for the TSC1 locus on chromosome 9q34
has not yet been identified. Several mutationsin the TSC2 gene of TSC patients have been

Abbreviations: bp, base pair; cDNA, complementary DNA; dCTP, deoxy-CTP; DEPC, diethyl pyrocarbonate; kb, kilobase; mRNA, messenger RNA; LTD, long-term depression; LTP, long-term potentiation;
NBT, nitroblue tetrazolium salt; RT-PCR, reverse transcription-coupled polymerase chain reaction; tRNA,transfer RNA; TSC, tuberous sclerosis complex; TSC1, locus on chromosome 9q34.3 for TSC; TSC2, locus
on chromosome 16q13.3 for TSC

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found (The European Chromosome Tuberous
Sclerosis Consortium, 1993; Kuma et al., 1995a,
1995b).
Loss of heterozygosity on chromosome
16p13.3 has been observed in the hamartomas
from TSC2 patients, suggesting the role of tumor suppressor function of the TSC2 gene product (Green et al., 1994). The Eker rat, one typeof animal model for dominantly inherited cancer, which bears a gene mutation in the TSC2
gene and develops tumors in kidneys (renal carcinoma) and other tissues has also been found to
exhibit a loss of heterozygosity in tumors (Hinoet al., 1994; Kobayashi et al., 1995; Kubo et al.,1995).
The TSC2 gene encodes a 1784-amino acidprotein, tuberin, which shares a region of homology with the GTPase-activating protein for
rap1 (rap1-GAP) (Wienecke et al., 1995). It has
been predicted that this region of homology
interacts with rap1 (p21-ras-related protein)which functions to regulate cytoskeletal interactions both in mammalian platelets and in thebudding pathway of yeast cells (McCabe et al.,
1992; White et al., 1992). The expression of
rap1-GAP especially suppresses the random
budding due to the expression of rap1, suggesting that rap1-GAP may be required for nonrandom selective budding in yeast cells(McCabe et al., 1992). Recently, it has been demonstrated by Northern blot analysis and in situ
hybridization for TSC2 mRNA that the TSC2
gene is expressed at high levels in specific regions of the adult and developing brain (Geist
and Gutmann, 1995). Together with the putative function of rap1-GAP as a regulatory factor
in budding, finding on the high expression of
the TSC2 gene in the mammalian brain raises a
new question of whether or not the TSC2 geneproduct tuberin has unique roles in the development and cellular basis of synaptic plasticity of
the central nervous system which are bothclosely related to cytoskeletal interactions, inaddition to the tumor suppressor function.
In this study, using the in situ hybridization
technique, we have described the neuronal pattern of the TSC2 gene expression in the mousebrain in order to examine which type of neurons
express the most abundant TSC2 gene and havediscussed the possible function of the TSC2 inthe central nervous system.

Materials and Methods

Production and subcloning of the TSC2
cDNA fragment
Total cellular RNA from a human normal skinfibroblast cell line was purified by the acidguanidium thiocyanate-phenolchloroform extraction method followed by the elimination of
genomic DNA using RNase-free DNase I. The
TSC2 cDNA fragment (810 bp of human TSC2
cDNA) was generated from the total RNA by
the reverse transcription-coupled polymerase
chain reaction (RT-PCR) method using forward
primer (F: 5'-AGAAGGCTTCTCCAG-AACT3') and reverse primer (R: 5'-GGAGGTGGA
CCT-CGAGTGCCTT-3'). The RT-PCR primers were designed to amplify a region of 810
bases corresponding to bases 2280–3090 of thehuman TSC2 sequence (The European Chromosome Tuberus Sclerosis Consortium, 1993). The
TSC2 cDNA fragment thus produced was subcloned into pGEM-5Zf(+) plasmid (Promega).

Northern blot analysis
Tissues: RNA was extracted from brain
regions and other peripheral tissues of normal
C57 BL/KsJ mice (10–12 weeks old) using thecesium chloride ultra centrifugation method.
For each sample, 45–135 µg of total RNA wasdenatured by heating at 65˚C for 10 min with
50% formamide, separated electrophoreticallyon a 1% denaturing formaldehyde agarose gel
and transferred to nitrocellulose (Hybond-N+,
Amersham, Bucks, United Kingdom) by Northern transfer.
Probes: For all mouse tissues, the TSC2
cDNA probes used for Northern blot analysis
were the inserts from the cloned human TSC2
cDNA fragment. The TSC2 cDNA probe and γ
actin cDNA probe were labeled with [α32P]CTP using the random primer reaction.

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Expression of tuberin mRNA in tissues

Hybridization conditions: The blots wereprehybridized with a buffer containing 50%
formamide, 5 × SSC, 5 × Denhardt, 50 mM
NaH2PO4, 0.5% SDS, 0.25 mg/mL denatured
salmon sperm at 42˚C for 2 h and then hybridized at 42˚C overnight in the same buffer containing labeled cDNA probes (4 × 108 cpm/mL).
Blots were first washed 3 times at room temperature in 2 × SSC for 15 min and then, for higher
stringency, 3 times at 62–65˚C in 0.2 × SSC, 1.0%
SDS for 30–60 min. Finally, blots were rinsed
with 2 × SSC at room temperature for 10 min.
The blots were apposed to a Bio-rad molecularimaging plate for 4 days to visualize the intensity and localization of the probe. To normalize
the quality of the RNA, a 32P-labeled actin cDNA
probe was used for each blot at 2 × 108 cpm/mL
with the same hybridization conditions.

Fig. 1. Expression of TSC2 mRNA in mouse
tissues. A poly(A) +RNA Northern blot obtained
from various mouse tissues was probed with the
TSC2 cDNA probe. The blot was reprobed with anactin cDNA probe as a control for quantity of RNA.
Each lane indicates a blot from: 1) the liver, 2) thebrain, 3) the kidney, 4) the spleen and 5) the heart,respectively. There is wide spread TSC2 mRNA
expression in many tissues with high levels of
expression (4.8–3.4% of actin) in the liver, brain andspleen and lower levels (1.2% of actin) in kidney,when corrected for amount of RNA by expression ofactin mRNA.

In situ hybridization
Tissues: Normal C57BL/KsJ mice (12–15
weeks old) were deeply anesthetized with sodium pentobarbital and perfused transcardiallywith ice-cold 20 mL heparinized saline followed by ice-cold 50 mL phosphate buffered 4%paraformaldehyde. The brains were removed,
postfixed in the fixative for 1 h, saturated in
30% sucrose prepared by diethyl pyrocarbonate(DEPC) treated water overnight in the cold
(4˚C). The brains were cut into the serial coronal sections (40 µm thickness) on a freezing
microtome and free-floated in 0.1 M phosphatebuffer prepared by DEPC water.

substrates (RNA labeling Kit, Boehringer
Mannheim, Germany).
Hybridization conditions: The sections
were processed by a free-floating method previously described (Saji et al., 1995). Briefly,
the free-floating sections were prehybridized at
55˚C for 3 h in 50% formamide, 10% dextran, 2
× Denhart’s solution, 2 × SSC (1 × SSC is 0.15
M sodium chloride + 0.015 M sodium citrate),
0.5 mg/mL denatured salmon sperm DNA, 1
mg/mL yeast tRNA and 0.25% SDS. Hybridization with digoxigenin-labeled riboprobes
was performed by adding the probes (10 ng/mL) to the prehybridization buffer containing
the sections (50–100 µL/section) and incubating at 55˚C overnight. The sections were washed with the incubation in 50% formamide, 2 ×
SSC at 45˚C for 20 min and in 2 × SSC at 37˚C
for 10 min, followed by RNase A treatment (20mg/mL in 2 × SSC) at 37˚C for 30 min and byrinsing with a dilutional series of SSC solution
at room temperature (2 × SSC twice, 1 × SSC
twice, 0.5 × SSC for 10 min each). Then thesections were processed for immunohistochemistry using anti-digoxigenin alkaline phosphatase

Probes: For in situ hybridization, digoxigenin-labeled antisense and sense riboprobes to
TSC2 mRNA were synthetized as follows: thedigoxigenin-labeled TSC2 antisense riboprobes
were synthesized by the transcription of a human TSC2 cDNA fragment (810 bp) (amplified
by RT-PCR and inserted into the pGEM-5Zf(+)
plasmid as described above) with SP6 polymerase after linearization of the template DNA
by restriction enzyme SphI, while the digoxigenin-labeled TSC2 sense riboprobes weresynthesized with T7 polymerase after linearization of the template DNA by restriction enzyme EcoRI, using digoxigenin-labeled UTP as
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dentate molecular layer in the hippocampus (r,o in Figs. 2C to E, and m in Fig. 2E), there werea few TSC2 gene expressing cells.
In the coronal sections hybridized with antisense TSC2 probes, large interneurons in the
striatum (arrows in Fig. 3B), large neurons of
the Purkinje cell layer in the cerebellum (p inFig. 3E), neurons of the piriform cortex (Fig.
3D) and large neurons of the facial nucleus in
the medulla (f in Fig. 3F) exhibited expressionof TSC mRNA at high levels with the intensely
purple-colored cytoplasm. On the other hand,most neurons in the cerebral cortex (Fig. 3A),
septum (not shown), thalamus (not shown),
striatum (Fig. 3B), substantia nigra compacta (cin Fig. 3C), and reticular formation in the medulla (Fig. 3F) expressed moderate or slight levels
of TSC2 mRNA, having their cytoplasm weakly stained with purple-colored TSC2 signals(Fig. 3). As seen in Fig. 3E, the cerebellar cortex had a typical laminar distribution of TSC2mRNA signals which consisted of a moderately
signal expressing granular layer (g in Fig. 3E), ahighly expressing Purkinje cell layer (p in Fig.
3E) and a molecular layer (m in Fig. 3E) withthe slightest expression of TSC2 signals. Besides the cerebellar molecular layer and hippocampal striatal areas, other reticular areas likethe substantia nigra reticulata (r in Fig. 2E) exhibited only the slightest expression of TSC2mRNA.
The degree of TSC2 gene expression in various types of neurons is summarized in Table 1.

conjugate (1:500, Boehringer Mannheim). To
visualize the hybrid molecules, the sections
were incubated in a reaction solution with Xphosphate and nitroblue tetrazolium salt (NBT)
for 24 h and mounted on subbed slides.

Results

Expression of TSC2 gene in the various
mouse tissues analyzed by Northern blot
Northern blot analysis of poly (A)+RNA fromseveral mouse tissues (liver, brain, kidney,spleen and heart) revealed that the TSC2mRNA was widely expressed in many tissues
including the brain. In all tissues examined,
there was one transcript evident as a 5.5 kb
RNA signal (Fig. 1). When corrected for amountof RNA by expression of actin mRNA as acontrol, high levels of TSC2 mRNA wereexpressed in the liver (4.8% of actin), brain(3.4% of actin), spleen (4.2% of actin), withmoderate levels in the heart (2.9% of actin) andlower levels in the kidney (1.2% of actin).

Neuron type specific expression of the
TSC2 gene in the brain demonstrated byin situ hybridization
Figure 2 shows coronal sections of the hippocampus hybridized for TSC2 mRNA with antisense TSC2 riboprobes or sense TSC2 riboprobes. As seen in Figs. 2A and F, the section
hybridized with sense TSC2 riboprobes indicated the red colored background and neurons
which were weakly stained by non-specificbinding of labeled riboprobes. In addition to
the red colored background staining by nonspecific binding of labeled probes shown in the
hippocampal section hybridized with sense
TSC2 probes (Fig. 2A), the hippocampal section hybridized with antisense TSC2 probes
demonstrated neurons with a purple coloredcytoplasm in the CA1-CA4 pyramidal layers,
dentate granular layer and hilus (Figs. 2B to E)
which expressed TSC2 mRNA at a high level.
In the dendritic area or area rich in fiber tracts
like the striatum radiatum, striatum oriens andDiscussion
An essential step in unraveling the functions of
the TSC2 gene product tuberin in the mature
mammalian brain is to determine which
neuronal types show the most abundant
expression of the TSC2 gene and to establish
the common morphological features of the high
TSC2 expressing neurons. The present study
demonstrates (i) that TSC2 signals are
expressed in the pyramidal neurons of the
hippocampal CA1-CA4, granule cells of thedentate gyrus, neurons of the piriform cortex,
large interneurons of the striatum, Purkinje
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