Autism spectrum disorders (ASDs) are highly prevalent neurodevelopmental disorders1, 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) loss2,3, while isolated cerebellar injury has been associated with a higher incidence of ASDs4. 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 ASDs5 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 pathology6 and correlate cerebellar pathology with increased ASD symptomatology7,8. TSC patients with ASDs also display hypermetabolism in deep cerebellar structures on functional imaging when compared to TSC patients without ASDs9. 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)611. 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 numbers2. 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 pathogenesis13 while studies have demonstrated critical roles for the TSC/mTOR pathway in mediating neuronal stress responses14,15. 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/function16,17, we also investigated whether Tsc1 loss resulted in morphological changes in PCs at 4 weeks. TSC has known roles in the regulation of cell size17,18, 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 numbers19, 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 loss17,19,20, 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 morphology16,21.
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+/- mice22. We evaluated USV from P5-12 and, similar to reported ASD mouse models23, 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 properties17,20, 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 neurons17, 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 patients9,25.
To evaluate whether the abnormal phenotypes seen in PC Tsc1 mice were modifiable as demonstrated in other models of increased mTOR signaling12,19,26, 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 interaction27 while cerebellar abnormalities are associated with ASDs as well as cognitive and behavioral disturbances28. 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 networks29. 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 dysmetria30. 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.