Autism spectrum disorders (ASDs) are highly prevalent neurodevelopmental disorders
1
, but the underlying pathogenesis remains poorly understood. Recent studies have implicated
the cerebellum in these disorders with post-mortem studies in ASD patients demonstrating
cerebellar Purkinje cell (PC) loss
2,3
, while isolated cerebellar injury has been associated with a higher incidence of
ASDs
4
. However, the extent of cerebellar contribution to the pathogenesis of ASDs remains
unclear. Tuberous Sclerosis Complex (TSC) is a genetic disorder with high rates of
comorbid ASDs
5
that results from mutation of either TSC1 or TSC2, whose protein products dimerize
and negatively regulate mTOR signaling. TSC is an intriguing model to investigate
the cerebellar contribution to the underlying pathogenesis of ASDs, as recent studies
in TSC patients demonstrate cerebellar pathology
6
and correlate cerebellar pathology with increased ASD symptomatology
7,8
. TSC patients with ASDs also display hypermetabolism in deep cerebellar structures
on functional imaging when compared to TSC patients without ASDs
9
. However, to date, Tsc1's roles and the sequelae of Tsc1 dysfunction in the cerebellum
have not been investigated. Here we show that both heterozygous and homozygous loss
of Tsc1 in mouse cerebellar PCs results autistic-like behaviors, including abnormal
social interaction, repetitive behavior, and vocalizations, in addition to decreased
PC excitability. Treatment of mutants with the mTOR inhibitor, rapamycin, prevented
the pathological and behavioral deficits. These findings demonstrate novel roles for
Tsc1 in PC function and define, for the first time, a molecular basis for a cerebellar
contribution to cognitive disorders such as autism.
To evaluate Tsc1's role in cerebellar PCs, we generated mice with Tsc1 deleted in
cerebellar PCs (L7Cre;Tsc1flox/+
(het) or L7Cre;Tsc1flox/flox
(mutant))
10
. Cre expression is high in PCs with expression noted by post natal day (P)6
11
. Tsc1+/+
(WT), L7Cre;Tsc1+/+
(L7Cre), Tsc1flox/flox
(Flox), het, and mutant mice did not show reduced survival, and weights were comparable
across genotypes, unlike the severe phenotype of neuronal or glial Tsc mutants (Figure
Supplemental (S)1)
12
.
To insure that TSC1 function was impaired in PCs, we evaluated staining of phospho-S6
(pS6) – a downstream effector of mTOR signaling. We expected TSC1 dysfunction to result
in increased mTOR activity and indeed detected increased pS6 staining in het and mutant
PCs (Figures S2-4). To assess the specificity of Cre-mediated recombination, we crossed
L7Cre
and Rosa26 reporter mice and found only infrequent, scattered recombination in non-cerebellar
areas as previously described (Figure S5)
11
. We also examined pS6 staining in other brain regions but found no differences between
mutants and controls, except in cerebellar PCs (Figure S6).
One of the most consistent pathologic findings in post-mortem studies of ASD patients
is reduced cerebellar PC numbers
2
. In the mutant cerebellum, while basic cellular architecture was maintained in adult
mice, the PC layer was abnormal with increased soma area and reduced PC numbers when
compared to control or het littermates (Figure 1A, S7). To investigate why PCs were
decreased in mutants, we quantified PC numbers throughout development. Decreased cell
numbers were first noted at 2 months of age with further reduction by 4 months of
age, a reduction not seen in hets (Figure 1B). As these findings suggested cell loss,
we investigated markers of apoptosis and found increased TUNEL and cleaved caspase
3 staining in mutant PCs at 7-8 weeks (Figure 1C, S8-9). Recently, neuronal stress
in the cerebellum has been implicated in ASD pathogenesis
13
while studies have demonstrated critical roles for the TSC/mTOR pathway in mediating
neuronal stress responses
14,15
. To investigate whether similar mechanisms were involved in Tsc1 mutant PC death,
we evaluated markers for both ER (GRP78) and oxidative (Heme Oxygenase 1) stress and
found significantly elevated levels of both markers (Figure S9).
As TSC-mTOR signaling plays important roles in neuronal morphology/function
16,17
, we also investigated whether Tsc1 loss resulted in morphological changes in PCs
at 4 weeks. TSC has known roles in the regulation of cell size
17,18
, and PC soma area was significantly increased in mutant, but not het, mice (Figure
1D, S10). TSC has also been implicated in regulating dendritic spine numbers
19
, and we found increased spine density on het and mutant PC dendrites (Figure 1D,
E). Interestingly, decreased spine density has been reported in hippocampal and cortical
neurons with Tsc loss
17,19,20
, suggesting diverse mechanisms underlying TSC1/2's regulation of dendritic spines.
We also found numerous axonal varicosities and abnormal axonal collaterals in mutants
(Figure S11), consistent with known roles for TSC in regulating axonal morphology
16,21
.
To investigate whether PC Tsc1 mutants might demonstrate abnormal behaviors found
in ASDs, we first evaluated social interaction, using a three chambered assay of social
approach and preference for social novelty. We found social impairment in both het
and mutant animals with no significant differences found between time spent in the
chamber or interacting with the novel mouse versus novel object (Figure 2A). Subsequently,
in a social novelty paradigm, while control animals spent significantly more time
in the chamber and in close interaction with the novel animal, het, and mutant animals
displayed no significant preference for social novelty by either measure (Figure 2B).
We further tested whether mutants would have impaired social interaction in male –
female interactions and observed significant reductions in mutant interaction time
compared with controls (Figure S12).
With the cerebellum's role in motor functions, we investigated whether motor deficits
contributed to social impairment. Mutants’ motor activity was indistinguishable from
littermates until approximately 7-8 weeks of age when mutants displayed initial signs
of ataxia. Ataxia progressed and by four months there were marked changes in gait
parameters (Figure S13). Hets, however, displayed no ataxia (Figure S13) and locomotion
during social testing and open field testing was not significantly different between
genotypes (Figure S14-15), suggesting that motor impairments were not responsible
for observed social deficits.
In rodents, social interaction largely depends on olfactory cues. We observed comparable
time spent investigating three non-social olfactory cues – water, almond extract,
and banana extract (Figure S16), indicating that olfactory function in mutants is
intact. However, consistent with observed social impairment phenotypes, het and mutant
mice demonstrated reduced investigation of social odors compared to controls, suggesting
that impaired discrimination of social olfactory cues contributed to social deficits
in mutants.
ASD patients also display repetitive behaviors and cognitive, behavioral inflexibility.
To model the perseverative thinking and cognitive inflexibility exhibited by patients
with ASDs, we tested animals in a reversal learning paradigm using a water T maze.
Mutant animals demonstrated similar acquisition learning of a submerged, escape platform
location (days 1-3) to control littermates (Figure 2C, S17), using two measures of
learning performance – correct trials and trials needed before 5 consecutive correct
trials. However, when the escape platform location was reversed, mutant animals demonstrated
significantly impaired learning of the new platform location. We also examined repetitive
behavior in a repetitive grooming task and found significantly increased self-grooming
rates in hets and mutants (Figure 2D).
ASD patients also demonstrate deficits in communication. Murine pups use ultrasonic
vocalizations (USV) to communicate with their mothers, and abnormal mother-pup communication
has recently been demonstrated in Tsc2+/- mice
22
. We evaluated USV from P5-12 and, similar to reported ASD mouse models
23
, found increased vocalizations in both hets and mutants (Figure 2E). Consistent with
roles for Tsc1 in regulating these early phenotypes, pS6 levels were elevated by P7
in mutant PCs (Figure S3). Motor deficits are also found in over 50% of patients with
ASDs. To evaluate whether mutants have impaired motor learning, we evaluated mutant
animals prior to ataxia onset on the accelerating rotarod and found significantly
impaired motor learning in mutants (Figure S18).
The changes in PC morphology, combined with previous reports that Tsc1 loss can alter
synaptic properties
17,20
, suggested that synaptic inputs to PCs might also be affected. PCs receive a single,
strong climbing fiber (CF) input and many weak granule cell-parallel fiber (PF) inputs
(Figure 3A). However, we found no difference in the amplitude of single fiber CF inputs
between mutant and littermate controls (Figure 3B) at P28. In control animals, when
synapses are stimulated twice in rapid succession, CF synapses depress, whereas PF
synapses facilitate, consistent with the high and low release probabilities of these
synapses, respectively (Figure 3B, left). The same characteristic plasticity was observed
in mutants (Figure 3B, right). We also stimulated PFs, which produce both a direct
excitatory short-latency PF EPSC and a disynaptic IPSC that arises from PF activation
of molecular layer interneurons (Figure 3C, left). There was a trend towards a reduction
in the ratio of the amplitudes of the EPSCs and IPSCs recorded in PCs, but it was
not statistically significant (Figure 3C, right). Although it is difficult to exclude
a subtle effect on synaptic properties, these results suggest that in spite of morphological
differences, synaptic function in mutants appears normal.
Previous studies of Tsc1 have also focused on neurons that are quiescent in the absence
of excitatory input, whereas PCs fire spontaneous action potentials even in the absence
of synaptic inputs. Because PC firing rate is thought to be critical for encoding
cerebellar output in deep cerebellar nuclei (DCN)
24
, we examined the intrinsic excitability of PCs using extracellular recordings, and
found a significantly lower, graded spontaneous spiking rate in hets and mutants (Figure
3D, left). Moreover, also in graded fashion, current injection evoked fewer action
potentials in het and mutant PCs (Figure 3E). A plot of firing frequency versus injected
current shows that het and mutant PCs were significantly less excitable than controls
(Figure 3E, right). Injection of small hyperpolarizing currents resulted in smaller
voltage changes in mutant and het PCs suggesting a decrease in the effective input
resistance (Figure S19A), which has been described previously for hippocampal neurons
17
, likely contributed to the reduced excitability of PCs in mutant and het animals.
By 6 weeks of age there was an even more profound reduction in excitability in mutant
mice (Figure S19B). Hence, despite receiving seemingly normal functioning synaptic
inputs, the output of the cerebellar cortex of het and mutant animals appears to be
strongly reduced, both tonically and in response to incoming excitatory drive. Our
findings implicate reduced PC excitability as a potential mechanism underlying the
abnormal behaviors in PC Tsc1 mice, consistent with clinical observations of impaired
cerebellar function in ASD patients
9,25
.
To evaluate whether the abnormal phenotypes seen in PC Tsc1 mice were modifiable as
demonstrated in other models of increased mTOR signaling
12,19,26
, we treated animals with the mTOR inhibitor, rapamycin, starting at P7. Whereas vehicle
treatment resulted in identical phenotypes to untreated cohorts, rapamycin treatment
prevented the development of pathologic deficits in mutant animals, with mutant soma
size and PC numbers indistinguishable from controls (Figure 4A, S20).
We subsequently evaluated whether the abnormal behaviors could also be rescued with
rapamycin treatment. In vehicle treated mice, behavioral phenotypes were identical
to untreated cohorts (Figure 4B-C, S21-23); however, rapamycin treatment ameliorated
the motor phenotypes seen in mutant animals in gait testing and the rotarod (Figure
S21, S24). Rapamycin treatment also prevented deficits in the water T Maze with no
significant differences seen between rapamycin treated mutants and controls in both
acquisition and reversal learning (Figure 4B, S22). In addition, following rapamycin
treatment, mutants displayed comparable social behaviors to controls in both social
approach and social novelty assays (Figure 4C, S23). Thus, rapamycin prevented both
pathologic and behavioral phenotypes in Tsc1 PC mutants, supporting the possibility
of a therapeutic role for mTOR inhibition.
Our study demonstrates critical, novel roles for the TSC-mTOR pathway in cerebellar
PCs. We find that mice with homozygous loss of Tsc1 in PCs (mutant) demonstrated social
impairment, restrictive behavior, and abnormal vocalizations – representative of the
three core deficits in ASDs. Mutants also displayed pathologic features found in ASD
post-mortem studies with reduced PC numbers and evidence of increased neuronal stress.
While PC loss has been reported in postmortem studies of ASD patients, several lines
of evidence suggest that PC death cannot fully explain the abnormal behaviors seen
in PC Tsc1 mice. Prior to PC death, mutants displayed abnormal vocalizations and motor
learning impairments. In addition, mice with heterozygous loss of Tsc1 displayed no
evidence of PC loss yet displayed autistic-like behaviors.
In this study, we also demonstrate that loss of Tsc1 from cerebellar PCs is sufficient
to result in abnormal autistic-like behaviors. These findings implicate the cerebellum
in the neural circuitry mediating core features of autism. The cerebellum has been
previously suggested to play roles in social interaction
27
while cerebellar abnormalities are associated with ASDs as well as cognitive and behavioral
disturbances
28
. How the cerebellum modulates the abnormal behaviors of autism remains a topic of
intense investigation. Autism has been proposed to be a disorder of abnormally distributed
networks
29
. The cerebellum, via the DCN, is connected to these networks and cortical areas implicated
in ASDs. Akin to its role in motor coordination, the cerebellum has been proposed
to modulate these cognitive networks, with dysfunction resulting in abnormally regulated
behaviors comparable to cognitive, behavioral dysmetria
30
. Our data displayed markedly impaired PC excitability in both hets and mutants. As
PC firing rates are critical determinants of DCN output, by affecting DCN activity,
PC dysfunction could be postulated to alter these downstream neuronal networks, thereby
contributing to abnormal autistic-like behaviors. Therefore, PC Tsc1 mutants should
provide a valuable experimental system to investigate the effects of PC dysfunction
on these neuronal networks and other mechanisms contributing to the pathogenesis of
ASDs.
METHODS
Mice
L7
cre; Tsc1
flox/flox (mutant)animals were generated by crossing L7/Pcp2-Cre (L7Cre
) transgenic mice
11
with floxed Tsc1 mice (Tsc1flox/flox
)
10
to yield L7Cre;Tsc1 flox/+
progeny. These progeny were then crossed with one another or with Tsc1
flox/flox animals to yield litters consisting of control (Tsc1+/+
(WT), Tsc1flox/+
, L7Cre;Tsc+/+
(L7Cre), or Tsc1flox/flox
(Flox)) mice, heterozygous (L7Cre;Tsc1flox/+
(het)) mice, or mutant (L7Cre;Tsc1flox/flox
(mutant)) mice. Only male animals were used for behavioral experiments except for
ultrasonic vocalizations where both male and female pups were utilized in the analysis.
Mice were of mixed genetic backgrounds (C57Bl/6J, 129 SvJae, BALB/cJ). During analysis,
germline deletion was discovered to occur in the mouse colony at a frequency of ~5%.
Genotyping for het mice would exclude inclusion of these animals in this cohort, but
it is possible that a small percentage of L7Cre;Tsc1flox/-
mice were included in the mutant cohort. As such, we repeated behavioral analysis
in a cohort of mutant (L7Cre;Tsc1flox/flox
[LFF]) mice and littermate controls and found no significant differences with the
previous mutant (LFF*) cohort (Figure S25-26), and thus cohorts were combined for
analysis. As behavioral data revealed comparable behavioral phenotypes between all
untreated control (Tsc1+/+
, Tsc1flox/flox, L7Cre;Tsc+/+
) genotypes, these genotypes were pooled for behavioral studies involving rapamycin
treatment. All experimental protocols were approved by the Animal Research at Children's
Hospital Committee.
Behavioral Analysis
Social Interaction
Animals were tested for social interaction in the three chambered apparatus (Dold
Labs) as previously described
31
. Time in chambers and number of crossings between chambers was recorded in an automated
manner (National Instruments). Time spent interacting with the novel animal and object
was recorded by the examiner with stopwatch. Animals were tested between 7-9 weeks
of age. For male-female interaction, tested males were placed into an open field with
control females and evaluated for male-initiated interaction over a five minute period.
All behavioral assays (including social interaction) were performed by examiner blinded
to genotype.
Gait Analysis
Animals were placed into the end of an apparatus 5 cm in width (preventing animals
from turning around), 56 cm in length with paper placed along the entire course of
the apparatus. Apparatus walls consisted of opaque material and were 15 cm in height
preventing animals from looking or escaping beyond walls. Paws were painted with red
(Forepaws) and black (Hindpaws) ink. Length of stride was measured from the longest
stride for each trial. Stride width was also measured between hind paws at time of
the longest stride. Measurements were taken from 14-16 week old animals.
Open Field
Open field testing was performed as described for 15 minute period
32
. Movement and time spent in center quadrants were recorded by video camera and analyzed
by Noldus (Virginia) analysis software. Measurements were taken from animals aged
7-10 weeks.
Olfaction
Olfaction was tested as previously described
33
. Animals were presented sequentially with odors on cotton tipped applicators: first
non-social, then social odors. Odors were presented in 3 consecutive trials per odorant
stimulus (2 minutes/trial) in the following order: water, almond extract, banana extract,
social odor 1, and lastly social odor 2. Social odors were swipes from cages containing
unfamiliar, gender (male), and age matched animals. Measurements were taken from animals
aged 8-12 weeks.
Grooming
After habituation, animals were observed for 10 minutes, and time spent grooming was
recorded as described
34
. Measurements were taken from animals aged 8-12 weeks.
Water T Maze
Reversal learning was tested using the water T maze as described
35
. On Days 1-3, mice were given 15 trials and tasked to locate a submerged platform
placed in one of the maze arms. After 15 trials on Day 3, the platform was changed
to the other T arm. Mice were then tested for 15 additional trials (Reversal (R)Day1).
Then for 2 subsequent days (Reversal (R)Day 2-3), mice were given 15 trials/day. Number
of correct trials and number of trials required to achieve 5 consecutive correct trials
were recorded. Measurements were taken from animals aged 8-12 weeks.
Ultrasonic Vocalizations
Ultrasonic Vocalizations were tested as described on postnatal days 5-12
23
. Pups were removed individually from their mother and placed inside a soundproof
container where 3 detectors were used to monitor vocalizations for 5 minutes. Calls
were recorded using Ultravox recording software (Noldus)
36
. Maternal genotype in all experiments was L7Cre;Tsc1flox/+
.
Accelerating Rotarod
Animals were tested using the Accelerating Rotarod as described over 5 consecutive
days
37
. Animals were tested prior to overt ataxia between 5-7 weeks of age.
Slices
Acute sagittal slices (250-300 μm thick) were prepared from the cerebellar vermis
of 4 and 6 week old mutant and control littermates. Slices were cut in an ice cold
artificial cerebrospinal fluid (ACSF) solution consisting of (mM): 125 NaCl, 26 NaHCO3,
1.25 NaH2PO4, 2.5 KCl, 1 MgCl2, 2 CaCl2, and 25 glucose (pH 7.3, osmolarity 310) equilibrated
with 95% O2 and 5% CO2. Slices were initially incubated at 34° C for 25 minutes, and
then at room temperature prior to recording in the same ACSF.
Recordings
Visually guided (infrared DIC videomicroscopy and water-immersion 40x objective) whole-cell
recordings were obtained with patch pipettes (2-4 MΩ) pulled from borosilicate capillary
glass (World Precision Instruments) with a Sutter P-97 horizontal puller. Electrophysiological
recordings were performed at 31-33° C.
IPSCs were recorded at the EPSC reversal potential, and PF EPSCs were recorded at
the IPSC reversal potential. To measure climbing fiber synaptic inputs, 500 nM NBQX
was used to reduce the size of synaptic currents, and picrotoxin (20 μM) was used
to block GABAergic inhibition. For experiments recorded at the EPSC reversal potential
and for CF EPSCs, the internal pipette solution contained (in mM): 140 Cs-methanesulfonate,
15 HEPES, 0.5 EGTA, 2 TEA-Cl, 2 MgATP, 0.3 NaGTP, 10 phosphocreatine-tris2, 2 QX 314-Cl.
pH was adjusted to 7.2 with CsOH. Membrane potentials were not corrected for the liquid
junction potential. The EPSC and IPSC reversal potentials were determined in each
experiment by adjusting the membrane potential until no EPSC or IPSC was evident,
and was approximately +15 mV for the EPSC reversal, and -65 mV for the IPSC reversal.
For current-clamp recordings, the internal solution contained (in mM): 150 K-gluconate,
3 KCl, 10 HEPES, 0.5 EGTA, 3 MgATP, 0.5 GTP, 5 phosphocreatine-tris2, and 5 phosphocreatine-Na2.
pH was adjusted to 7.2 with NaOH. Current-clamp and extracellular recordings were
performed in NBQX (5 μM), R-CPP (2.5 μM), and picrotoxin (20 μM) to block AMPA receptors,
NMDA receptors, and GABAA receptors respectively. All drugs were purchased from Sigma-Aldrich
or Tocris. Electrophysiolgical data were acquired as described previously
38
.
Rapamycin Treatment
Rapamycin was dissolved in 0.25% polyethylene glycol and 0.25% tween prior to usage.
Vehicle or rapamycin was administered intraperitoneally every Monday, Wednesday, and
Friday with rapamycin dosed at 6 mg/kg per injection starting at P7. As behavioral
data revealed comparable behavioral phenotypes between all untreated control (Tsc1+/+,
Tsc1flox/flox, L7Cre;Tsc+/+
) genotypes, these genotypes were pooled for behavioral studies involving rapamycin
treatment.
Immunohistochemistry
Mice were perfused and post-fixed with 4% paraformaldehyde. Sections were prepared
by cryostat sectioning and were stained with the following antibodies: PhosphoS6 (Cell
Signaling), calbindin (Sigma, Cell Signaling), GRP78 (Stressgen), Heme Oxygenase-1
(Stressgen), Cleaved Caspase 3 (Cell Signaling). TUNEL staining was performed per
manufacturer recommendations (Millipore).
Microscopy
Intracellular labeling of Purkinje cells was accomplished using recording pipettes
filled with 0.05% biocytin (Tocris). Neurons in deeper portions of the Purkinje cell
layer were targeted and filled for 3 min, and then the pipette was slowly withdrawn
so that the cell membrane could reseal. Slices (250 μm thick) were then fixed in 4%
paraformaldehyde in 0.1 M phosphate buffer for 24 hours, rinsed thoroughly in PBS,
and incubated for 90 min in a PBS solution containing 0.5% Triton-X, 10% normal goat
serum and streptavidin Alexa Fluor 488 conjugate (1:500, Life Technologies). Slices
were then rinsed in PBS, mounted on Superfrost Plus slides (VWR International), air-dried,
and cover-slipped in Vectashield mounting media (Vector Labs). Immunohistochemical
studies were captured using Zeiss Confocal LSM710. Images were processed and morphology
quantified using ImageJ software with studies performed by examiner blinded to genotypes.
Statistics
Data are reported as mean ± SEM, and statistical analysis was carried out with GraphPad
Prism software using one- and two-way ANOVA with Bonferroni's multiple comparison
tests for post hoc analysis unless otherwise specified.
Supplementary Material
1