The Journal of Neuroscience, February 11, 2009 • 29(6):1773–1783 • 1773

Cellular/Molecular

Pharmacological Inhibition of mTORC1 Suppresses
Anatomical, Cellular, and Behavioral Abnormalities in
Neural-Specific Pten Knock-Out Mice
Jing Zhou,1 Jacqueline Blundell,2 Shiori Ogawa,3 Chang-Hyuk Kwon,1 Wei Zhang,1 Christopher Sinton,3
Craig M. Powell,2,4 and Luis F. Parada1
1

Department of Developmental Biology and Kent Waldrep Foundation Center for Basic Neuroscience Research on Nerve Growth and Regeneration, andDepartments of 2Neurology, 3Internal Medicine, and 4Psychiatry, University of Texas Southwestern Medical Center, Dallas, Texas 75390

PTEN (phosphatase and tensin homolog deleted on chromosome ten) is a lipid phosphatase that counteracts the function ofphosphatidylinositol-3 kinase (PI3K). Loss of function of PTEN results in constitutive activation of AKT and downstream effectors andcorrelates with many human cancers, as well as various brain disorders, including macrocephaly, seizures, Lhermitte–Duclos disease,and autism. We previously generated a conditional Pten knock-out mouse line with Pten loss in limited postmitotic neurons in the cortex
and hippocampus. Pten-null neurons developed neuronal hypertrophy and loss of neuronal polarity. The mutant mice exhibited macrocephaly and behavioral abnormalities reminiscent of certain features of human autism. Here, we report that rapamycin, a specificinhibitor of mammalian target of rapamycin complex 1 (mTORC1), can prevent and reverse neuronal hypertrophy, resulting in theamelioration of a subset of PTEN-associated abnormal behaviors, providing evidence that the mTORC1 pathway downstream of PTEN is
critical for this complex phenotype.
Key words: PTEN; tuberous sclerosis complex; autism; macrocephaly; neuronal hypertrophy; neuronal polarity

Introduction
PTEN (phosphatase and tensin homolog deleted on chromosome ten) is a major negative regulator of thephosphatidylinositol-3 kinase (PI3K)/AKT pathway. Its primaryfunction is to counteract the kinase function of PI3K and therefore inhibit the activation of AKT. Individuals bearing germ-line
PTEN mutations are prone to many human cancers (Ali et al.,1999), as well as mental disorders, including macrocephaly, seizures, mental retardation, and Lhermitte–Duclos disease (Marshet al., 1999; Zhou et al., 2003). Recently, PTEN mutations havealso been linked to cases of autism, particularly in a subset ofpatients with macrocephaly, suggesting that mutations in thisgene might be one of the genetic risk factors for human autism
spectrum disorders (ASDs) (Goffin et al., 2001; Butler et al., 2005;
Herman et al., 2007a,b).
We reported previously a strain of conditionalNse– cre;PtenloxP/loxP knock-out mice (Pten mutant mice hereafter) with Pten ablation in a subset of postmitotic neurons in thecortex and hippocampus. Pten mutant mice develop macrocephReceived Nov. 28, 2008; accepted Dec. 23, 2008.
This work was supported by the Simons Foundation (L.F.P.) and Autism Speaks (C.M.P.). We thank Dr. Tak Mak for
the PtenloxP mice, Dr. David Kwiatkowski for the Tsc1loxP mice, Dr. Suzanne J. Baker for sharing the Nse– cre mice, Dr.
Renee McKay for helpful advice, and Yanjiao Li, Shawna Kennedy, and Steve McKinnon for technical assistance. We
thank Sharon Matheny for helpful discussions.
Correspondence should be addressed to Luis F. Parada, Department of Developmental Biology and Kent Waldrep
Foundation Center for Basic Neuroscience Research on Nerve Growth and Regeneration, University of Texas Southwestern Medical Center, Dallas, TX 75390. E-mail: luis.parada@utsouthwestern.edu.
DOI:10.1523/JNEUROSCI.5685-08.2009
Copyright © 2009 Society for Neuroscience 0270-6474/09/291773-11$15.00/0

aly and additional behavioral abnormalities reminiscent of human autism, including reduced social activity, increased anxiety,and sporadic seizures (Kwon et al., 2006a; Ogawa et al., 2007). At
the cellular level, we observed two major morphological changes
after in vivo loss of Pten: loss of neuronal polarity and neuronal
hypertrophy.
One important question is which of the downstream pathwaysof PI3K/AKT mediates these cellular and behavioral changes.
Among the various PI3K/AKT downstream pathways, tuberous
sclerosis complex 1/2 (TSC1/2) is an attractive candidate. It is
known that TSC1 and TSC2 function together as a dimer. AKT
directly phosphorylates TSC2 and releases the suppression of
TSC1/TSC2 on mammalian target of rapamycin complex 1(mTORC1) (Inoki et al., 2002; Manning et al., 2002; Potter et al.,2002). We were particularly interested in this AKT downstream
pathway because of two existing facts. First, mutation of either
TSC1 or TSC2 gene causes tuberous sclerosis in humans, in which⬃25–50% of patients show autistic syndrome (Smalley et al.,
1992; Smalley, 1998; Wiznitzer, 2004). Second, it has been shownthat loss of Tsc1 results in neuronal hypertrophy both in vitro andin vivo (Jaworski et al., 2005; Tavazoie et al., 2005; Meikle et al.,2007). In addition, a recent study suggests that the TSC/
mTORC1 pathway is involved in controlling neuronal polarity(Choi et al., 2008).
In the current study, we examined the role of the TSC/
mTORC1 pathway in Pten mutant mice through the use of a
specific mTORC1 inhibitor, rapamycin. Our results indicate amajor contribution of this pathway to the abnormal cellular mor-1774 • J. Neurosci., February 11, 2009 • 29(6):1773–1783

Zhou et al. • Role of mTORC1 in Pten cKO Mutant Phenotype

phology. Pharmacologic attenuation of
the pathway in Pten mutants by rapamycin
has surprising consequences on neuronal
hypertrophy, seizures, and social behavior.
In addition, we show that ablation of Tsc1
using the identical Nse– cre transgenic
mouse line mimics the morphological features observed with the Pten mutant mice.
These data point to a common signaltransduction pathway that is potentially
responsible for the autism-like symptoms
in individuals bearing TSC1/2 or PTEN
mutations and suggest a possible treatment strategy for this subset of autism
patients.

Materials and Methods
Mice and histology. PtenloxP mice (Suzuki et al.,2001) were a gift from Dr. Tak Mak (Universityof Toronto, Toronto, Ontario, Canada), andthe Nse– cre line was generated in the laboratoryof Dr. Suzanne J. Baker (St. Jude Children’s Research Hospital, Memphis, TN) and characterized in our laboratory (Kwon et al., 2006b).
Both lines have been maintained in C57BL/6 Figure 1. Rapamycin effectively blocks mTORC1 signaling in the brain. A, Dose-dependent suppression of P-S6 levels in theinbred background. Pten mutant mice (Nse– cortex and hippocampus after rapamycin injection. Rapamycin was applied to 2-month-old wild-type mice daily for 5 d. B, C,
cre;PtenloxP /loxP) were generated by breeding Rapamycin (Rapa) at 10 mg/kg effectively reduced P-S6 levels in both hippocampus and cortex of Pten mutant mice, as shown by
male PtenloxP /loxP mice and female Nse– cre; Western blotting (B) and IHC (C). ctr, Control; mut, mutant. Control and mutant mice at 10 weeks of age were injected with either
PtenloxP /⫹ mice. Controls used here were litter- vehicle or 10 mg/kg rapamycin daily for 5 d. Scale bar, 100 ␮m.
mates either without cre or in a few cases Nse–sections, we used microwave antigen retrieval for all antibodies. We vicre;PtenloxP /⫹. Tsc1loxP mice were a gift from Dr. David Kwiatkowski
sualized the primary antibodies by treating the sections with biotinylated(Harvard Medical School, Boston, MA) and were crossed with the Nse–
secondary antibody, followed by amplification with peroxidasecre line by a similar strategy. For preparation of brain sections, mice wereconjugated avidin and DAB substrate (Vector Laboratories). DABintracardially perfused with ice-cold PBS, followed by 4% paraformaldestained sections were counterstained with hematoxylin. For vibratome
hyde (PFA). Brains were dissected out immediately after perfusion andsections, we detected the primary antibodies by secondary antibodies
kept in 4% PFA at 4°C for additional use. For paraffin sectioning, halfconjugated with cyanine 3 or cyanine 2 (Jackson ImmunoResearch).
brains were processed, embedded in paraffin, and sectioned sagittally at 5
Measurements. All the measurements were performed using the Meta␮m thickness. For vibratome floating sectioning, brains were embedded
Morph software (Universal Imaging). We measured nuclear diameter ofin 3% agarose and then cut coronally at 50 ␮m thickness. All mousedentate granule neurons as an indication of soma size (Kwon et al., 2003).
protocols were approved by the Institutional Animal Care and Research
To estimate the thickness of the mossy fiber tract, we measured the width
Advisory Committee at the University of Texas Southwestern Medicalof out-coming axonal bundles from the dentate gyrus that were double
Center.
labeled with Synapsin I and Calbindin. To assess dendritic size, we meaRapamycin injection. Rapamycin powder (Sirolimus; LC Laboratories)
sured the length of Calbindin-stained neuronal processes into the mowas dissolved in ethanol and stored at a stock concentration of 25 mg/ml
lecular layer (ML). For cortical neurons, we measured SMI 311-positivein aliquots at ⫺20°C. Working solution was prepared freshly before use
neurons in wild-type controls and compared them with SMI 311-positive
with a final concentration of 1 mg/ml rapamycin in 4% ethanol, 5%
and PTEN-negative neurons in Pten mutants. For each animal, only
Tween 80, and 5% PEG400 (Kwon et al., 2003). Mice were injected
neurons in somatosensory cortex were analyzed. All data were analyzed
intraperitoneally with either rapamycin (10 mg/kg body weight) or vehiwith ANOVA, followed by post hoc Student’s t test.
cle once per day for 5 consecutive days per week (Monday to Friday).
Golgi staining. We performed Golgi staining, image analysis, and denWestern blotting. Hippocampus and cortex were isolated separately
dritic caliber and spine density quantification as described previously
from mouse brain and snap frozen in liquid nitrogen. To prepare the(Luikart et al., 2005).
lysate, frozen tissues were homogenized in lysis buffer containing 20 mM
Timm’s staining. For Timm’s staining, we intracardially perfused theTris-HCl, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100,
mice with ice-cold 0.37% (w/v) sodium sulfide, followed by 4% PFA. We
proteinase inhibitor (Roche), Ser/Thr phosphatase inhibitor, and Tyr
dissected out the brain, postfixed in 4% PFA overnight, and cryoprophosphatase inhibitor (Millipore). Homogenate was centrifuged at
tected in 30% (w/v) sucrose in PBS for 2–3 d. We made 16-␮m-thick
14,000 rpm for 30 min and supernatant was collected. Protein concencoronal cryostat sections and performed a modified Timm’s staining as
tration was determined by using BCA kit (Pierce). For Western blotting,described previously (Danscher et al., 2004).
20 or 50 ␮g of sample was loaded onto SDS-PAGE gel. Antibodies used
Behavioral tests. Mutant mice were studied along with littermate confor Western blotting were against P-AKT–Ser473, AKT, P-S6 (Ser235/
trols in four groups: vehicle-treated controls, vehicle-treated Pten mu236), or S6 (Cell Signaling Technology), or ␤-actin (Sigma). Signal wastant mice, rapamycin-treated controls, and rapamycin-treated Pten mudeveloped by ChemiGlow West reagent (Alpha Innotech) and visualizedtant mice. Behavioral tests were performed as described previously
by Eastman Kodak Image Station 2000R.
(Kwon et al., 2006a). The open-field test was performed for 10 min in a
Immunohistochemistry. Triplicate sections with comparable anatomy
brightly lit, 48 ⫻ 48 cm 2 white plastic arena with a center zone defined aswere chosen from controls and mutants. Antibodies used for immunoa 15 ⫻ 15 cm 2 square. Time spent in the center zone and total travel
histochemistry (IHC) were against PTEN, P-AKT, P-S6 (Cell Signaling
distance were recorded using video-tracking software (Noldus). The soTechnology), Calbindin (Swant), Synapsin I (Millipore Bioscience Recial interaction tests were performed in a neutral cage in the dark undersearch Reagents), or SMI 311 (Covance Research Products). For paraffinZhou et al. • Role of mTORC1 in Pten cKO Mutant Phenotype

J. Neurosci., February 11, 2009 • 29(6):1773–1783 • 1775

Figure 2. Rapamycin can prevent or reverse macrocephaly in Pten mutant mice. A, Rapamycin injection strategy. Rapamycin was applied to two cohorts of mice at different ages: a young group(5– 6 weeks old) and an adult group (10 –12 weeks old). Mutants and controls injected with either vehicle or 10 mg/kg rapamycin were analyzed after 4 or 6 weeks of injections. B, Rapamycin
prevents or reverses the brain enlargement of Pten mutant mice. The effect of rapamycin on brain and body weight was measured after 4 weeks for the young group or after 6 weeks for the adultgroup (n ⫽ 15 mice per group for body weight measurement, n ⫽ 8 mice per group for brain/body ratio). Data are mean ⫾ SEM and were analyzed by paired t test. *p ⬍ 0.05 and **p ⬍ 0.01
compared with control mice treated with vehicle or between groups as indicated. C, Representative Nissl staining shows that rapamycin (Rapa) treatment suppressed the enlargement of the cortex(scale bar, 200 ␮m) and hippocampus (scale bar, 500 ␮m) when applied to young Pten mutant mice for 4 weeks. Arrows show thickness of cortical layers II–VI.
red light by pairing a test mouse with a male juvenile mouse. During a 2
min time window, the time that the testing mouse actively approachedand sniffed the social target was recorded. After this initial interaction,
the test was repeated after 3 d by reintroducing the same social target. The
above behavioral tests were performed blindly to genotype andtreatment.
Electroencephalogram/electromyogram recording. For electroencephalogram/electromyogram (EEG/EMG) analysis, Pten mutant (n ⫽ 12) andwild-type (n ⫽ 3) mice were anesthetized and surgically implanted forlong-term EEG/EMG monitoring as described previously (Chemelli etal., 1999). The design for the EEG/EMG implant allowed precise insertion of electrodes, targeting the frontal and occipital cortices at a consistent depth, just touching the dura, while minimizing surgical trauma.
Mice were housed individually under a 12 h light/dark cycle at 25°C
[lights on, circadian time (CT) 0000], with food and water being replenished as necessary at CT 1200 each day, but they were not otherwise
disturbed. Mice were habituated to the recording conditions for 2 weeks
before baseline EEG/EMG signals were recorded over a period of 3 d,
beginning at lights off (CT 1200). In the following week, the mice weredivided into two groups: a rapamycin treatment group (n ⫽ 6 mutants,n ⫽ 3 controls) and a vehicle control group (n ⫽ 6 mutants). Each week,
the animals were intraperitoneally injected with either rapamycin or vehicle once daily between 10:00 and 11:00 A.M. for 5 successive days. The
EEG/EMG was recorded from the second day of the injection continuingfor 3 d, and this procedure was repeated for 4 weeks to evaluate thechronic effect of the drug. No adverse effects from the injection wereobserved in either group, and the body weights remained stable throughout the study. Subsequently, the EEG/EMG record was visually screened
for seizure epochs. Seizures were characterized as a spike-wave pattern on
the EEG, typically accompanied by atonic periods or sustained rhythmic
contractions on the EMG. Each seizure lasting for 2 s or more was noted.
The total number of seizures during the 12 h dark and light periods wasanalyzed, as well as the time of occurrence and the duration of each
seizure. One-way ANOVA with Bonferroni’s multiple comparison as apost hoc test was applied to analyze differences within groups.

Results
Rapamycin effectively blocks mTORC1 signaling in the CNS
A dose–response experiment was performed to determine an effective rapamycin concentration that would inhibit mTORC1
activity in mouse cortex and hippocampus. Rapamycin (Sirolimus) (Heitman et al., 1991) was administered by intraperitoneal
injection for 5 consecutive days at various concentrations. Mice
were killed on the last day of injection for analysis. As readout for
mTORC1 activity, we assayed levels of phospho-Ser235/236 –S6(P-S6 hereafter) and determined that 10 mg/kg rapamycin effectively reduced mTORC1 signaling in both hippocampus and cortex (Fig. 1 A). In addition, 10 mg/kg rapamycin was also sufficient
to attenuate mTORC1 signaling in Pten mutant brains, in which
mTORC1 was highly activated (Fig. 1 B, C). We therefore adopted
this rapamycin dosage in all subsequent studies.
Rapamycin can prevent or reverse macrocephaly in Pten
mutant mice
In our mouse model, deletion of Pten is mainly restricted to subsets of postmitotic neurons in cortex and hippocampus (Kwon etal., 2006b). By using a Rosa26 –stop–lacZ reporter mouse line
(Soriano, 1999), we found that cre activity in the brain mainly
developed postnatally (supplemental Fig. 1, available at www.
jneurosci.org as supplemental material). Only minimal cre activity could be detected at early postnatal days but increased dramatically during the second and third postnatal weeks, reaching
maximal activity at 4 weeks.
In our previous study, Pten mutant mice were carefully monitored and shown to progressively develop macrocephaly that is
detectable as early as 4 –5 weeks of age, reaching statistical significance at 2 months of age (Kwon et al., 2006a). This timing correlates with the appearance of abnormal behavior, which appears

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Zhou et al. • Role of mTORC1 in Pten cKO Mutant Phenotype

at 5– 6 weeks of age and is fully penetrant
by 2–3 months of age. We chose to apply
rapamycin to two cohorts of mutant andcontrol mice: a presymptomatic younggroup (5– 6 weeks old) and a symptomatic
adult group (10 –12 weeks old). Thus, inthe former group, rapamycin activity
would be present at the earliest manifestation of abnormality and would be tested
for prevention of symptoms. In the lattergroup, rapamycin delivery commenced
when symptoms were fully penetrant totest the potential for reversal. For eachgroup, mutants and age-matched controls
were injected with either vehicle or 10
mg/kg rapamycin. Mice were killed after
either 4 or 6 weeks of drug treatment foranalysis as illustrated in Figure 2 A.
Overall, the mice appeared healthyduring rapamycin administration, although slight growth retardation was observed after long-term treatment. In theyoung group, rapamycin-injected mice
showed a 5–9% reduction in body weightcompared with vehicle-injected controls.
This effect became less obvious in the adultgroup, which shared a similar trend but
did not reach statistical significance. To Figure 3. Rapamycin suppresses soma hypertrophy of dentate granule cells in young Pten mutant mice. A, PTEN immunoaccount for the overall effect of rapamycin staining on dentate granule cells. In Pten mutant mice, Pten-negative cells [mutant Pten (⫺), blue cells] are distinguishable fromon growth, brain/body weight ratio was Pten-positive cells [mutant Pten (⫹), brown cells]. Scale bars: top, 200 ␮m; bottom, 50 ␮m. B, Quantification reveals a significant suppression of soma hypertrophy of Pten-negative neurons after rapamycin treatment. n ⫽ 4 animals per group. Data areused to assess relative brain growth (Fig. mean ⫾ SEM and were analyzed by ANOVA, followed by post hoc t test. **p ⬍ 0.01 compared to control mice treated with vehicle
2 B). In the young group, the vehicle- or between groups as indicated.
treated mutant mice already showed significantly increased brain/body weight rawhereas the soma hypertrophy does not develop in mutant micetio compared with control mice by the fourth week of injection.
undergoing rapamycin treatment (Fig. 3 A, B).
In contrast, the mutants receiving rapamycin showed normal
To examine the effect of the drug on neuronal processes, webrain/body weight ratio. In the adult group, the same effect wasagain took advantage of the well established circuitry in the hipobserved, although the macrocephaly (17% increase in brain
pocampus. The granule cells of the dentate gyrus spread theirweight) was more dramatic than in the young group (7% increase
dendrites out toward the molecular layer, whereas the axons formin brain weight) (Fig. 2 B). As described previously, the enlarged
the mossy fiber tract that projects through the polymorphic layer
brain size is mainly the consequence of Pten null cellto synapse onto the CA3 region. The mossy fiber tract was visuautonomous overgrowth in the two brain regions in which cre
alized by IHC using antibodies to Synapsin I, a presynaptic
activity is confined (cortex and hippocampus). Consistent withmarker, and to Calbindin, which is expressed in both soma andthe brain weight data, the enlargement of cortex and hippocamprocesses. The increased thickness of the mossy fiber tract seen inpus in mutants is controlled by rapamycin, as shown by Nissl
vehicle-treated mutants was dramatically suppressed by rapamystaining of the treated young group of mice (Fig. 2C).
cin administration (Fig. 4 A, B). This result was confirmed by
Rapamycin suppresses hippocampal granule cell hypertrophy
Using various techniques, we have assessed the changes in cellmorphology caused by Pten loss and demonstrated that brain
enlargement is not only attributable to enlarged soma size but
also neuronal process hypertrophy, including increased dendritic
size and thickened axon bundles (Kwon et al., 2006a). We therefore first examined the effect of rapamycin on neuronal hypertrophy in the presymptomatic group of mice.
On tissue sections, the cellular boundary of granule cells in thedentate gyrus is not clearly identifiable because the nuclei of granule neurons are prominent and surrounded by a thin layer ofcytosol. We therefore measured nuclear diameter to reflect soma
size (Kwon et al., 2003, 2006a). PTEN immunostaining illustrates
that Pten-negative cells (blue cells, hematoxylin counterstained)
are obviously larger than Pten-positive cells (brown cells),

Timm’s staining, which specifically highlights the mossy fiber
tract (Fig. 4C). Dendritic growth was measured by the thicknessof the ML, defined here as the length of Calbindin-stained neuronal processes into the ML. The data indicate that dendritic
overgrowth was also reduced by rapamycin (Fig. 4 A, D).
We further observed that, at the end of treatment, four of eightmutants treated with vehicle exhibited ectopic Synapsin I reactivity in the inner ML (Fig. 4 A, arrowheads), reflecting loss of polarity in granule neurons. This is a common feature of older
mutant mice (Kwon et al., 2006a). Interestingly, none of the six
rapamycin-treated mutants showed ectopic Synapsin I. Similar
results were obtained with Timm’s staining, with two of five mutants treated with vehicle showing ectopic staining in the innerML, whereas none of the four mutants treated with rapamycin
showed this ectopic staining. Together, these data indicate that,when administered to presymptomatic mice, rapamycin can sup-

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Figure 4. Rapamycin inhibits dendritic and axonal hypertrophy of dentate granule cells in young Pten mutant mice. A, Immunostaining on 50-␮m-floating sections for Synapsin I (red) andCalbindin (green) shows reduced dendritic and axonal hypertrophy in Pten mutants after rapamycin treatment. Ectopic Synapsin I staining (arrowhead) in the inner molecular layer was observed insome vehicle-treated Pten mutants. Scale bar, 500 ␮m. B, Quantification reveals that increased thickness of mossy fiber tract in Pten mutant mice was significantly reduced after rapamycin
treatment. The thickness of mossy fiber tracts is defined by the width of the axon bundle coming out of the dentate gyrus in Synapsin I and Calbindin double-stained sections. n ⫽ 8 mice forvehicle-treated groups; n ⫽ 6 mice for rapamycin-treated groups. C, Timm’s staining to visualize mossy fiber tract. n ⫽ 4 or 5 mice were examined per group. Scale bar, 500 ␮m. D, Increased
thickness of the molecular layer in Pten mutant mice was significantly reduced after rapamycin treatment. Data are mean ⫾ SEM and were analyzed by ANOVA, followed by post hoc t test. **p ⬍0.01 and ***p ⬍ 0.001 compared with control mice treated with vehicle or between groups as indicated.

press the development of neuronal process hypertrophy and theloss of neuronal polarity caused by Pten ablation.
Rapamycin inhibits cortical neuron hypertrophy
Another region affected in the Pten mutant mice is the cortex, in
which cre expression is restricted mainly to layers III–V with
⬃30 – 60% neurons bearing cre activity. We examined pyramidalcells in somatosensory cortex of the presymptomatic group of
mice. Neuronal morphology was visualized by IHC with antibodies against SMI 311 and PTEN. SMI 311 is a pan-neuronal neurofilament antibody that detects a subset of pyramidal cells in
cortical layers III and V (Ulfig et al., 1998). In mutant mice, mostPten-null pyramidal cells, if not all, had obviously larger somata
and were often accompanied by brighter staining of SMI 311 (Fig.
5A, arrowhead). Similar phenomena have been reported for neurons that lack Tsc1, suggesting neuronal hypertrophy with increased expression of neurofilament (Meikle et al., 2007). Compared with controls, the mutant samples showed overall
increased SMI 311 staining, whereas with rapamycin treatment,
the SMI 311 signal was reduced, as were soma size and thicknessof the dendritic caliber (Fig. 5B–D, arrow). Therefore, rapamycin
impedes development of abnormalities caused by Pten loss incortical neurons as well.
Rapamycin reverses dentate gyrus enlargement
In later stages, we observed more dramatic neuronal abnormalities in the Pten mutants. By 4 months of age, mutants showedabnormal foliation of the hippocampal dentate granule layer andcompression of the CA1 region (Kwon et al., 2006a). To evaluate
whether rapamycin could reverse already established morphological abnormalities, we treated a second cohort of mice begin-ning at 10 –12 weeks of age (adult group) for 4 – 6 weeks. Our
results indicate that rapamycin was able to significantly reverse
neuronal hypertrophy in older mice, resulting in restoration ofmuch of the hippocampal structure (Fig. 6 A). However, somecompression of the CA1 region still remained. In contrast to theyounger cohort study, ectopic Synapsin I staining in the ML persisted in the drug-treated cohort even after 6 weeks of rapamycin
treatment (Fig. 6 A, arrowheads). These data indicate the sustained loss of polarity of granular cells. Nonetheless, the extent of
the Synapsin I staining in the ML was reduced and mainly restricted to the inner ML. The enlarged cell size of Pten-null granule cells was also progressively reversed by rapamycin treatment(Fig. 6 B) (supplemental Fig. 2, available at www.jneurosci.org as
supplemental material). In addition, the granule cell layer partially regained its tight organization, with less extracellular spacethat was probably previously occupied by the enlarged dendritic
processes. To further visualize individual cell morphology, we
performed Golgi staining. Consistent with our previous data
(Kwon et al., 2006a), granule cells in mutant mice are globallyenlarged. After rapamycin treatment, enlarged dendritic size wasreversed, accompanied by reduced dendritic caliber thickness
and spine density (Fig. 6C,D). However, in both vehicle- andrapamycin-treated samples, granule cells with multiple processes
into polymorphic layers were occasionally observed, suggesting
loss of polarity (Fig. 6C, arrowhead). This, together with the Synapsin I staining result, suggests that rapamycin, when applied toolder mice, cannot reverse loss of polarity. It is possible that, inlater stages, once the neurons lose polarity it is not fully reversible
in vivo. However, we cannot exclude the possibility that other
AKT downstream effectors are also involved.

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Zhou et al. • Role of mTORC1 in Pten cKO Mutant Phenotype

Beneficial effects of rapamycin
on behavior
We previously demonstrated that Pten
mutant mice have increased anxiety andimpaired social behavior in a series of experimental paradigms (Kwon et al.,2006a). We therefore tested whether rapamycin treatment in a young cohort could
prevent development of these abnormal
behaviors. Because, at the cellular level, theeffects of rapamycin were gradual, we
chose to examine behaviors during the 4th
week of drug administration.
Restricted by the narrow time window,
we performed specific behavioral tests to
examine the effects of rapamycin treatment mainly on anxiety and social interestin mice. The open-field test measures anxiety in mice. Consistent with our previous
findings, vehicle-treated mutant micespent most of the time in the corner oralong the edge of the open field and significantly less time in the center comparedwith controls, indicating increased anxiety
in the mutants (Fig. 7A). Interestingly, after treatment with rapamycin, this difference was no longer apparent: a clear trendof increased time spent in the center wasobserved in mutant mice treated with
rapamycin versus mutants treated with vehicle. As a control for possible locomotor
defects, we measured the distances traveled by the four cohorts of mice. We foundthat rapamycin-treated mice traveled
comparable distances in the test as vehicle- Figure 5. Rapamycin inhibits development of cortical neuron hypertrophy in young Pten mutant mice. A, B, Floating sections
treated controls, suggesting normal loco- were immunostained with PTEN (green) and SMI 311 (red). A, Immunostaining on Pten mutants. Arrowheads highlight Pten-nullmotor activity (supplemental Fig. 3, avail- hypertrophic neurons with bright SMI 311 staining. B, Representative double-staining pictures show inhibition of dendritic
able at www.jneurosci.org as supplemental hypertrophy by rapamycin (Rapa) treatment on Pten-null neurons. Arrows highlight the primary dendrites of SMI 311-positive
material). Therefore, rapamycin treat- neurons. Scale bar, 20 ␮m. C, D, Quantification shows that rapamycin inhibits neuronal hypertrophy of cortical neurons byment reduced anxiety of Pten mutant decreasing soma hypertrophy and dendritic hypertrophy. n ⫽ 4 brains analyzed for each group. For wild-type controls, SMI 311
and PTEN double-positive cells were analyzed; for Pten mutants, SMI 311-positive and PTEN-negative neurons were examined.
mice.
Data
are mean ⫾ SEM and were analyzed by ANOVA, followed by post hoc t test. *p ⬍ 0.05, **p ⬍ 0.01, and ***p ⬍ 0.001
Mice were also examined in a test forreciprocal social interaction. As before, compared with control mice treated with vehicle or between groups as indicated.
vehicle-treated mutants showed a signifizure occurrence. As shown in Figure 7C, EEG/EMG monitoring
cant decrease in social interaction compared with controls. Rapawas
performed over a period of 5 weeks for each mouse. EEG/
mycin treatment produced a clear increase in social interaction in
EMG signals were recorded each week on 3 consecutive days
the mutants. This effect of rapamycin on Pten mutant mice oc(Tuesday to Thursday). For each mouse, baseline EEG/EMG wascurred despite its opposite effect on control mice (Fig. 7B). This
recorded at the first week. Then mutant mice were divided into
trend was also seen when the mice were reexposed to the sametwo groups that were treated with either rapamycin or vehiclesocial target 3 d later. Thus, rapamycin appears to specificallystarting at the beginning of the second week. As anticipated, mostprevent development of anxiety and social behavior abnormalimutant mice (n ⫽ 11) developed spontaneous seizures during theties in Pten mutants when administered early.
period of study, with the exception of one mouse in the drugWe attempted to perform these same tests on the older grouptreated group that only displayed occasional epileptiform activityof treated mice; however, the older mutant mice were more vul(e.g., spikes and spike waves) but not full seizures. During the
nerable to handling, and increased mortality during the period of
seizures, repetitive spike-wave patterns were noted, sometimes
injection was observed, making the tests difficult to perform andaccompanied by rhythmic slow activity. Continuous spike-wave
analyze. As we suspected, chronic daily injection of rapamycin
bursting was also seen. In the baseline EEG/EMG recording, alaltered behavior directly as a consequence of stress placed on thethough the severity of the seizures varied between individual anmice. This effect was apparently amplified in the mutant mice
imals, there was no significant difference in either frequency orthat already exhibited a dramatic increase in anxiety.
duration of the seizures between vehicle- and rapamycin-treatedPten mutant mice also develop an age-related increase ofgroups. However, compared with baseline, the mean duration of
spontaneous seizures (Ogawa et al., 2007). We therefore investithe seizures decreased significantly in the drug-treated group in
gated whether rapamycin exposure would have an effect on sei-Zhou et al. • Role of mTORC1 in Pten cKO Mutant Phenotype

J. Neurosci., February 11, 2009 • 29(6):1773–1783 • 1779

Figure 6. Rapamycin reverses dentate gyrus enlargement but not ectopic axonal projections when applied to adult Pten mutant mice. A, Immunostaining shows that rapamycin (Rapa)
substantially reverses dentate gyrus hypertrophy, but axonal ectopia (arrowheads) still persist after 6 weeks of treatment in adult Pten mutant mice. Scale bar, 500 ␮m. B, PTEN immunostaining ofdentate granule cells demonstrates reversal of soma hypertrophy of Pten-negative cells and restored granular layer organization after rapamycin treatment. Scale bar, 50 ␮m. C, Representative
pictures of Golgi staining show reversal of neuronal hypertrophy in the dentate gyrus of Pten mutant mice after 4 weeks of rapamycin treatment. PML, Polymorphic layer; GL, granular layer. Scalebar, 50 ␮m. D, E, Quantification shows that increases in spine density and dendritic caliber in Pten mutant mice were decreased after rapamycin treatment. n ⫽ 3 brains were analyzed for eachgroup. Five to ten primary dendritic branches were counted for each brain. Data are mean ⫾ SEM and were analyzed by ANOVA, followed by post hoc t test. *p ⬍ 0.05 and **p ⬍ 0.01 comparedwith control mice treated with vehicle or between groups as indicated.

week 3 and 4 (Fig. 6 D) (F(4,25) ⫽ 3.172; p ⬍ 0.05). This decrease
was not observed in the vehicle-treated group (Fig. 7D). In addition, the tendency toward reduced seizure occurrence approached significance in the drug-treated group, despite large
individual variance (Fig. 7D). It is also worth mentioning that
two mutant mice from the vehicle group developed severe statusepilepticus as the study progressed, resulting in premature death
during the third week of treatment. Wild-type control mice (n ⫽3) treated with rapamycin did not develop seizures or display
adverse effects. Thus, pharmacologic blockade of mTORC1 was
sufficient to quantitatively reverse seizure behavior in Pten mutant mice.
Feedback effects on AKT
It has been suggested that chronic rapamycin exposure can havenegative effects on AKT activity (Sarbassov et al., 2005, 2006).
Studies show that mTORC2 can effectively phosphorylate AKT atserine 473, which is important for AKT activity. Although only
mTORC1 is rapamycin sensitive, long-term rapamycin treatment can cause the depletion of mTOR from mTORC2, thereby
negatively affecting AKT activity. However, this feedback effect iscontext dependent, because in vitro not every cell line exposed to

rapamycin results in downregulation of P-AKT–S473 levels (Sarbassov et al., 2006). To examine potential chronic effects of rapamycin on AKT activity in our system, we collected cortical andhippocampal tissues at different postinjection time points forWestern blotting analysis of P-AKT–S473 levels. The data indicate that AKT–S473 phosphorylation is reduced by chronic rapamycin exposure in both treated controls and mutants (Fig. 8 A).
This trend is clearly seen after the first week of drug exposure andpersists in the following injection weeks. However, the overall
P-AKT–S473 level in drug-treated mutant mice remained higher
than that in vehicle-treated controls, as also supported by PAKT–S473 immunostaining of the dentate gyrus (Fig. 8 B). A
similar effect was observed in the cortex (data not shown). Therefore, we are unable to exclude the possibility that rapamycin, inpart, exerts its effect on cellular morphology and behavior
through this feedback action, thus potentially implicating additional effectors downstream of AKT.
TSC1-mediated neuronal hypertrophy in vivo
Previous in vitro studies have reported that loss of TSC1/2 oroveractivation of mTOR signaling causes neuronal hypertrophy
and increased dendritic arborization (Jaworski et al., 2005; Ku-

1780 • J. Neurosci., February 11, 2009 • 29(6):1773–1783

Zhou et al. • Role of mTORC1 in Pten cKO Mutant Phenotype

mar et al., 2005; Tavazoie et al., 2005). In
vivo, embryonic loss of Tsc1 in neurons
driven by Synapsin I– cre causes severe seizures in mice, although only one of two
independent studies observed obvious
cortical tubers similar to those found in
human TSC patients (Meikle et al., 2007;
Wang et al., 2007). To further confirm therole of the mTOR pathway in cell morphology in our system, we generated Tsc1
conditional knock-out mice (Tsc1 mutant
mice hereafter) using our postnatal Nse–cre line. Although the progeny of this crosshad reduced viability (data not shown), wewere able to examine three Tsc1 mutants
that survived to 10 weeks of age. Interestingly, although the Tsc1 mutants examined were ⬃20% (20.1 ⫾ 9.2%) smaller
than their littermate controls, they showed
a 36% (36.5 ⫾ 14.8%) increase in brain/body weight ratio. Closer examination
demonstrated giant neurons in cortex(supplemental Fig. 4 A, arrowhead, available at www.jneurosci.org as supplemental
material), which resembled the cortical tubers observed in human TSC patients. In
addition, the dentate gyrus in Tsc1 mutants was obviously enlarged (supplemental Fig. 4 A, available at www.jneurosci.org
as supplemental material). Additional
analysis indicated significantly increased Figure 7. Rapamycin treatment reduces anxiety, improves social activity, and controls seizures. A, In the open-field test,
thickness of mossy fiber tract ( p ⬍ 0.01) rapamycin (Rapa)-treated Pten mutant mice showed no significant difference from rapamycin-treated controls, whereas vehicletreated Pten mutants showed statistically significant decrease in center time compared with vehicle-treated control mice. B,
and molecular layers ( p ⬍ 0.05), suggestRapamycin treatment increases social interaction time of Pten mutants and has the opposite effect on control mouse social
ing axonal and dendritic hypertrophy (Fig. interaction. n ⫽ 9 for vehicle-treated groups, n ⫽ 12 for rapamycin-treated control, and n ⫽ 10 for rapamycin-treated Pten
8C). Also, P-S6 levels were increased in the mutants. *p ⬍ 0.05 compared with control mice treated with vehicle or between groups as indicated. C, Strategy of EEG/EMG
cortex and hippocampus of Tsc1 mutant monitoring. D, Rapamycin injection progressively reduces seizure duration and frequency of Pten mutant mice. n ⫽ 6 mice per
mice, reflecting robust activation of group. *p ⬍ 0.05 compared between vehicle- and rapamycin-treated mutants. Data are mean ⫾ SEM and were analyzed by
mTOR signaling (supplemental Fig. 4 B, ANOVA, followed by post hoc t test.
available at www.jneurosci.org as supplemental material). All of these changes werebrain development could be one of the key factors in the pathosimilar to those observed in Pten mutant mice. These indepengenesis of autism. Thus, one hypothesis would be that pathways
dent genetic data further support the key role of the PI3K/AKT/
that control growth could play an important role in the etiology
mTOR pathway in mediating the anatomical defects seen in ourof autism. One such pathway is the PI3K/AKT pathway, whichmutant mice.
regulates cell growth and division. Mutations in PTEN, the negative regulator of this pathway, have been found to cause macroDiscussion
cephaly in both human and mouse. In addition, increasing clinThe development and use of murine models for the purpose ofical reports link germ-line PTEN mutations to autism patients
replicating and better understanding human disease has gainedwith macrocephaly (Goffin et al., 2001; Butler et al., 2005; Herconsiderable ascendance in recent years. Here, we take advantage
man et al., 2007a,b). Consistent with these clinical reports, weof murine models to attempt to better understand the etiology ofdemonstrated previously that mice with Pten loss in postmitotic
human macrocephaly and autism when associated with the PI3K/
neurons in the cortex and hippocampus develop macrocephaly
AKT signaling pathway.
and additional behavioral phenotypes that are reminiscent of huASDs represent a broad range of developmental brain disorman autism (Kwon et al., 2006a). This information, coupled withders characterized by impairment of communication and social
the link of autism to tuberous sclerosis, indicates that dysregulainteractions and frequently accompanied by restricted and repettion of the PI3K/AKT/mTORC1 pathway might be responsible
itive behaviors. One of the frequent physical traits associated withfor the pathogenesis of a subset of autism patients.
autism is abnormal brain size. Both macrocephaly and microIn our system, the abnormal behaviors observed in Pten mucephaly have been reported in autism patients. We are particutant mice are accompanied by an overgrowth of the cortex andlarly interested in the 10 –20% of autism patients with associatedhippocampus and changes in neuronal morphology, including
macrocephaly (Piven et al., 1995; Davidovitch et al., 1996; Lainneuronal hypertrophy and loss of neuronal polarity. Blocking thehart et al., 1997; Stevenson et al., 1997; Fidler et al., 2000; Deutsch
TSC/mTORC1 downstream pathway in Pten mutants dramatiand Joseph, 2003; Lainhart, 2003). A recent review fromcally reversed the soma, dendritic, and axonal hypertrophy in
Courchesne et al. (2007) suggests that overgrowth during early

Zhou et al. • Role of mTORC1 in Pten cKO Mutant Phenotype

J. Neurosci., February 11, 2009 • 29(6):1773–1783 • 1781

about the precise nature of direct versus
indirect defects. Kwon et al. (2003) further
used a rapamycin analog, CCI-779, to treat
the mGFAP– cre;PtenloxP/loxP mutant miceand reported partial inhibition of cell size
growth in soma. This study further qualitatively described appearance of seizures(by investigator observation) in mutant
mice that was reduced after drug
treatment.
The present study is consistent withprevious reports, but it has several important distinctions. First, in contrast to broadand early deletion of the tumor suppressors, we restricted Pten and Tsc1 ablation
to a limited number of postnatal, postmitotic hippocampal and cortical neurons.
Second, our study on neuronal morphology is not only restricted to soma hypertrophy but also extends to dendritic andaxonal hypertrophy and neuronal polarity. Third, we observed similar anatomical
Figure 8. Long-term rapamycin treatment has a feedback effect on AKT activity and loss of Tsc1 causes a similar neuronalcellular hypertrophy in Nse– cre;
hypertrophy phenotype as Pten loss. A, Rapamycin (Rapa) treatment has a negative feedback effect on P-AKT–S473 level as and loxP/loxP
Tsc1
mice, providing additional geshown by Western blotting on hippocampus. n ⫽ 3 mice per group. Data are mean ⫾ SEM and analyzed by t test. *p ⬍ 0.05
compared with control mice treated with vehicle. B, P-AKT–S473 immunostaining of the dentate gyrus of adult mice treated with netic support for the role of this pathway
either vehicle or rapamycin for 4 weeks. Scale bar, 200 ␮m. C, Representative immunostaining pictures show increased thickness in the phenotype. Finally, we found theof mossy fiber tract (arrowheads) and molecular layer (arrows) in Tsc1 mutant mice. Scale bar, 500 ␮m. n ⫽ 3 pairs of mice were cellular effects of rapamycin could relate to
analyzed.
quantifiable behavioral outcomes including reduced anxiety, increased social interboth young and old groups. However, the loss of polarity was not
est, and control of seizure duration and frequency measured byalways inhibited by rapamycin treatment. When applied toobjective EEG measurements. Therefore, our mouse models perasymptomatic Pten mutants, rapamycin suppressed the appearmit us to examine the mutant neurons and to identify the anaance of Synapsin I staining in the molecular layer, a sign of loss of
tomical regions responsible for the defects. With that, we are able
polarity of granule cells, whereas in older mice, ectopic Synapsin
to assert that postmitotic neurons can undergo dramatic morI staining was reduced but not eliminated. The overall inhibitory
phological changes during deregulation of the PI3K/AKT patheffect of rapamycin on ectopic axonal projection is consistent
way and relate these changes to abnormal behaviors.
with a recent study on the role of the TSC/mTORC1 pathway in
Although our Tsc1 mutant mice failed to thrive, our conclucontrolling neuronal polarity (Choi et al., 2008). It is not likely
sion regarding the role of Tsc1 in the observed neuronal phenosolely attributable to the feedback effect, because the basal level oftypes is supported by an increasing number of reports from otherpAKT–S473 in drug-treated Pten mutants is still higher than thatgroups that suggest that deletion of Tsc1 in nervous system causes
in vehicle-treated controls. There are a couple of possible explaneuronal hypertrophy and seizures (Uhlmann et al., 2002; Meikle
nations for why rapamycin treatment did not fully reverse theet al., 2007; Wang et al., 2007). As our manuscript was in prepaectopic projection phenotype in older mice. We suspect that esration, studies from other laboratories reported that applying
tablished polarity loss may become fixed and not subject to rerapamycin could control seizures in human GFAP– cre;Tsc1loxP/loxP
mice (Zeng et al., 2008) and prolong the survival of Synapse I– cre;versibility in vivo. Alternatively, other PI3K/AKT downstream
Tsc1loxP/loxP mutant mice (Meikle et al., 2008), further supporting
pathways may also be at play. Thus, ectopic axonal projection
our findings here. Twenty to twenty-five percent of autism patients
may result from activation of multiple AKT downstream pathexperience seizures, which is life threatening (Canitano, 2007). In
ways, including the TSC/mTORC1 pathway. The fact that we did
our study, we used EEG recording, the most objective measurement
not observe ectopic Synapsin I staining in our Tsc1 mutants
method, to monitor seizure activity. We were surprised by the degree
would be consistent with this latter explanation. However, weto which rapamycin administration could reverse both the seizure
should mention that the appearance of ectopic Synapsin I stainfrequency and duration. The anti-seizure effect of the drug was obing was mainly observed in older Pten mutant mice. Unfortuserved as early as the first week of treatment and remained throughnately, the failure of Tsc1 mutant mice to thrive, leading to deathout the 4 week drug administration period. In contrast, Pten mutantsof the mice before adulthood, precluded this analysis. We believe
treated with vehicle demonstrated higher seizure frequency and duthat death results from kidney failure, although this has yet to beration, with two of six mice perishing during the injection period.
rigorously ascertained (data not shown).
These data reveal that rapamycin is a potentially useful seizure treatPrevious studies have conditionally ablated Pten in the brain
ment modality for autism patients, particularly for those bearing
using a murine GFAP– cre transgenic line (mGFAP– cre) (Backgerm-line Pten or Tsc1/2 mutations. More importantly, a recent
man et al., 2001; Kwon et al., 2001). The mutant mice developed
study suggested that Tsc2⫹/⫺ mice show deficits in learning anddramatic brain abnormalities especially in the cerebellum andmemory that could be reversed by rapamycin treatment (Ehninger
hippocampus, accompanied by macrocephaly, severe seizures,
et al., 2008). Similarly, Tsc1⫹/⫺ mice display cognitive deficits, inand increased morbidity. The broad and early expression of thecluding impaired social behaviors (Goorden et al., 2007). These data
cre transgene used for the studies, however, limited interpretation

1782 • J. Neurosci., February 11, 2009 • 29(6):1773–1783

indicate the possible involvement of the TSC/mTORC1 pathway inautism-related behaviors in human. In our social tests, rapamycin
improved the social interaction behavior of Pten mutant mice despite an opposite effect on wild-type controls, which not only is
consistent with these previous reports but also suggests the importance of this pathway in maintaining normal social activity.
We should mention that rapamycin can also modify the activity of ryanodine receptors (RyRs), a family of calcium-releasechannels. It is known that FKBP12 binds to members of the RyR
family and that binding of rapamycin to FKBP12 releases it from
RyRs, resulting in increased channel-opening probability (Chelu
et al., 2004). Although to date there is no evidence suggesting that
RyRs regulate neuronal morphology, they may influence synaptic
activity under certain circumstances (Shimizu et al., 2008).
Therefore, we cannot exclude the possibility that rapamycin
functions through RyRs as well.
In conclusion, we performed the present study in the context
of seeking possible mechanisms underlying PI3K pathwayassociated human autism. This approach may help to shed lighton the anatomical and cellular underpinnings of related brain
disorders and, furthermore, provides evidence for a potential
therapeutic window in certain cases of ASD.

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