Cerebellar contribution to autism-relevant behaviors in fragile X syndrome models.

Cerebellar dysfunction has been linked to autism spectrum disorders (ASDs). Although cerebellar pathology has been observed in individuals with fragile X syndrome (FXS) and in mouse models of the disorder, a cerebellar functional contribution to ASD-relevant behaviors in FXS has yet to be fully characterized. In this study, we demonstrate a critical cerebellar role for Fmr1 (fragile X messenger ribonucleoprotein 1) in ASD-relevant behaviors. First, we identify reduced social behaviors, sensory hypersensitivity, and cerebellar dysfunction, with loss of cerebellar Fmr1. We then demonstrate that cerebellar-specific expression of Fmr1 is sufficient to impact social, sensory, cerebellar dysfunction, and cerebro-cortical hyperexcitability phenotypes observed in global Fmr1 mutants. Moreover, we demonstrate that targeting the ASD-implicated cerebellar region Crus1 ameliorates behaviors in both cerebellar-specific and global Fmr1 mutants. Together, these results demonstrate a critical role for the cerebellar contribution to FXS-related behaviors, with implications for future therapeutic strategies.

Synaptic BMAL1 phosphorylation controls circadian hippocampal plasticity.

The time of day strongly influences adaptive behaviors like long-term memory, but the correlating synaptic and molecular mechanisms remain unclear. The circadian clock comprises a canonical transcription-translation feedback loop (TTFL) strictly dependent on the BMAL1 transcription factor. We report that BMAL1 rhythmically localizes to hippocampal synapses in a manner dependent on its phosphorylation at Ser42 [pBMAL1(S42)]. pBMAL1(S42) regulates the autophosphorylation of synaptic CaMKIIα and circadian rhythms of CaMKIIα-dependent molecular interactions and LTP but not global rest/activity behavior. Therefore, our results suggest a model in which repurposing of the clock protein BMAL1 to synapses locally gates the circadian timing of plasticity.

Increased glycine contributes to synaptic dysfunction and early mortality in Nprl2 seizure model.

Targeted therapies for epilepsies associated with the mTORC1 signaling negative regulator GATOR1 are lacking. NPRL2 is a subunit of the GATOR1 complex and mutations in GATOR1 subunits, including NPRL2, are associated with epilepsy. To delineate the mechanisms underlying NPRL2-related epilepsies, we created a mouse (Mus musculus) model with neocortical loss of Nprl2. Mutant mice have increased mTORC1 signaling and exhibit spontaneous seizures. They also display abnormal synaptic function characterized by increased evoked and spontaneous EPSC and decreased evoked and spontaneous IPSC frequencies, respectively. Proteomic and metabolomics studies of Nprl2 mutants revealed alterations in known epilepsy-implicated proteins and metabolic pathways, including increases in the neurotransmitter, glycine. Furthermore, glycine actions on the NMDA receptor contribute to the electrophysiological and survival phenotypes of these mice. Taken together, in this neuronal Nprl2 model, we delineate underlying molecular, metabolic, and electrophysiological mechanisms contributing to mTORC1-related epilepsy, providing potential therapeutic targets for epilepsy.

A Critical Period for Development of Cerebellar-Mediated Autism-Relevant Social Behavior.

The cerebellum has been increasingly implicated in autism spectrum disorder (ASD) with many ASD-linked genes impacting both cerebellar function and development. However, the precise timing and critical periods of when abnormal cerebellar neurodevelopment contributes to ASD-relevant behaviors remains poorly understood. In this study, we identify a critical period for the development of ASD-relevant behaviors in a cerebellar male mouse model of tuberous sclerosis complex (TSC), by using the mechanistic target of rapamycin (mTOR) inhibitor, rapamycin, to pharmacologically inhibit dysregulated downstream signaling. We find independent critical periods during which abnormal ASD-relevant behaviors develop for the two core ASD diagnostic criteria, social impairments and behavioral flexibility, and delineate an anatomic, physiological, and behavioral framework. These findings not only further our understanding of the genetic mechanisms underlying the timing of ASD-relevant behaviors but also have the capacity to inform potential therapies to optimize treatment interventions.SIGNIFICANCE STATEMENT No targeted treatments currently exist for autism spectrum disorder (ASD). This complex developmental disorder has established links to genetic and circuit aberrations, yet the precise timing and coordination of these underlying mechanisms that contribute to the spectrum of physiological and behavioral abnormalities remains unclear. Cerebellar pathology is consistently seen in ASD individuals; therefore, we sought to identify the specific windows for cerebellar involvement in the development of ASD-relevant behaviors. Using pharmacologic treatment paradigms, we outline distinct critical periods of developmental vulnerability for ASD-relevant social and inflexible behaviors. From this study, we posit a refined window of time during which ASD symptoms develop that will inform therapeutic timing.

Adaptive Prediction for Social Contexts: The Cerebellar Contribution to Typical and Atypical Social Behaviors.

Social interactions involve processes ranging from face recognition to understanding others' intentions. To guide appropriate behavior in a given context, social interactions rely on accurately predicting the outcomes of one's actions and the thoughts of others. Because social interactions are inherently dynamic, these predictions must be continuously adapted. The neural correlates of social processing have largely focused on emotion, mentalizing, and reward networks, without integration of systems involved in prediction. The cerebellum forms predictive models to calibrate movements and adapt them to changing situations, and cerebellar predictive modeling is thought to extend to nonmotor behaviors. Primary cerebellar dysfunction can produce social deficits, and atypical cerebellar structure and function are reported in autism, which is characterized by social communication challenges and atypical predictive processing. We examine the evidence that cerebellar-mediated predictions and adaptation play important roles in social processes and argue that disruptions in these processes contribute to autism.

Cerebellar Dysfunction in Autism Spectrum Disorders: Deriving Mechanistic Insights from an Internal Model Framework.

Autism spectrum disorders (ASD) are highly prevalent neurodevelopmental disorders; however, the neurobiological mechanisms underlying disordered behavior in ASD remain poorly understood. Notably, individuals with ASD have demonstrated difficulties generating implicitly derived behavioral predictions and adaptations. Although many brain regions are involved in these processes, the cerebellum contributes an outsized role to these behavioral functions. Consistent with this prominent role, cerebellar dysfunction has been increasingly implicated in ASD. In this review, we will utilize the foundational, theoretical contributions of the late neuroscientist Masao Ito to establish an internal model framework for the cerebellar contribution to ASD-relevant behavioral predictions and adaptations. Additionally, we will also explore and then apply his key experimental contributions towards an improved, mechanistic understanding of the contribution of cerebellar dysfunction to ASD.

Regulation of autism-relevant behaviors by cerebellar-prefrontal cortical circuits.

Cerebellar dysfunction has been demonstrated in autism spectrum disorders (ASDs); however, the circuits underlying cerebellar contributions to ASD-relevant behaviors remain unknown. In this study, we demonstrated functional connectivity between the cerebellum and the medial prefrontal cortex (mPFC) in mice; showed that the mPFC mediates cerebellum-regulated social and repetitive/inflexible behaviors; and showed disruptions in connectivity between these regions in multiple mouse models of ASD-linked genes and in individuals with ASD. We delineated a circuit from cerebellar cortical areas Right crus 1 (Rcrus1) and posterior vermis through the cerebellar nuclei and ventromedial thalamus and culminating in the mPFC. Modulation of this circuit induced social deficits and repetitive behaviors, whereas activation of Purkinje cells (PCs) in Rcrus1 and posterior vermis improved social preference impairments and repetitive/inflexible behaviors, respectively, in male PC-Tsc1 mutant mice. These data raise the possibility that these circuits might provide neuromodulatory targets for the treatment of ASD.

Therapeutic Targeting of mTORC2 in mTORopathies.

Dysregulated mTOR contributes to neurodevelopmental dysfunction. A new study (Chen et al., 2019) demonstrates that suppression of mTORC2, not mTORC1, ameliorates survival, seizures, and abnormal behaviors in a Pten mutant model, highlighting mTORC2 as a potential therapeutic target in mTORopathies.

Sensitive Periods for Cerebellar-Mediated Autistic-like Behaviors.

Despite a prevalence exceeding 1%, mechanisms underlying autism spectrum disorders (ASDs) are poorly understood, and targeted therapies and guiding parameters are urgently needed. We recently demonstrated that cerebellar dysfunction is sufficient to generate autistic-like behaviors in a mouse model of tuberous sclerosis complex (TSC). Here, using the mechanistic target of rapamycin (mTOR)-specific inhibitor rapamycin, we define distinct sensitive periods for treatment of autistic-like behaviors with sensitive periods extending into adulthood for social behaviors. We identify cellular and electrophysiological parameters that may contribute to behavioral rescue, with rescue of Purkinje cell survival and excitability corresponding to social behavioral rescue. In addition, using anatomic and diffusion-based MRI, we identify structural changes in cerebellar domains implicated in ASD that correlate with sensitive periods of specific autism-like behaviors. These findings thus not only define treatment parameters into adulthood, but also support a mechanistic basis for the targeted rescue of autism-related behaviors.

Author Correction: Altered cerebellar connectivity in autism and cerebellar-mediated rescue of autism-related behaviors in mice.

In the version of this article initially published, the Simons Foundation was missing from the list of sources of support to P.T.T. in the Acknowledgments. The error has been corrected in the HTML and PDF versions of the article.

Altered cerebellar connectivity in autism and cerebellar-mediated rescue of autism-related behaviors in mice.

Cerebellar abnormalities, particularly in Right Crus I (RCrusI), are consistently reported in autism spectrum disorders (ASD). Although RCrusI is functionally connected with ASD-implicated circuits, the contribution of RCrusI dysfunction to ASD remains unclear. Here neuromodulation of RCrusI in neurotypical humans resulted in altered functional connectivity with the inferior parietal lobule, and children with ASD showed atypical functional connectivity in this circuit. Atypical RCrusI-inferior parietal lobule structural connectivity was also evident in the Purkinje neuron (PN) TscI ASD mouse model. Additionally, chemogenetically mediated inhibition of RCrusI PN activity in mice was sufficient to generate ASD-related social, repetitive, and restricted behaviors, while stimulation of RCrusI PNs rescued social impairment in the PN TscI ASD mouse model. Together, these studies reveal important roles for RCrusI in ASD-related behaviors. Further, the rescue of social behaviors in an ASD mouse model suggests that investigation of the therapeutic potential of cerebellar neuromodulation in ASD may be warranted.

Aberrant Proteostasis of BMAL1 Underlies Circadian Abnormalities in a Paradigmatic mTOR-opathy.

Tuberous sclerosis complex (TSC) is a neurodevelopmental disorder characterized by mutations in either the TSC1 or TSC2 genes, whose products form a critical inhibitor of the mechanistic target of rapamycin (mTOR). Loss of TSC1/2 gene function renders an mTOR-overactivated state. Clinically, TSC manifests with epilepsy, intellectual disability, autism, and sleep dysfunction. Here, we report that mouse models of TSC have abnormal circadian rhythms. We show that mTOR regulates the proteostasis of the core clock protein BMAL1, affecting its translation, degradation, and subcellular localization. This results in elevated levels of BMAL1 and a dysfunctional clock that displays abnormal timekeeping under constant conditions and exaggerated responses to phase resetting. Genetically lowering the dose of BMAL1 rescues circadian behavioral phenotypes in TSC mouse models. These findings indicate that BMAL1 deregulation is a feature of the mTOR-activated state and suggest a molecular mechanism for mitigating circadian phenotypes in a neurodevelopmental disorder.

Autism and cerebellar dysfunction: Evidence from animal models.

Autism is a prevalent neurodevelopmental disorder whose origins are not well understood. Cerebellar involvement has been implicated in the pathogenesis of autism spectrum disorders with increasing evidence from both clinical studies and animal models supporting an important role for cerebellar dysfunction in autism spectrum disorders. This article discusses the various cerebellar contributions to autism spectrum disorders. Both clinical and preclinical studies are discussed and future research directions highlighted.

Cerebellar associative sensory learning defects in five mouse autism models.

Sensory integration difficulties have been reported in autism, but their underlying brain-circuit mechanisms are underexplored. Using five autism-related mouse models, Shank3+/ΔC, Mecp2(R308/Y), Cntnap2-/-, L7-Tsc1 (L7/Pcp2(Cre)::Tsc1(flox/+)), and patDp(15q11-13)/+, we report specific perturbations in delay eyeblink conditioning, a form of associative sensory learning requiring cerebellar plasticity. By distinguishing perturbations in the probability and characteristics of learned responses, we found that probability was reduced in Cntnap2-/-, patDp(15q11-13)/+, and L7/Pcp2(Cre)::Tsc1(flox/+), which are associated with Purkinje-cell/deep-nuclear gene expression, along with Shank3+/ΔC. Amplitudes were smaller in L7/Pcp2(Cre)::Tsc1(flox/+) as well as Shank3+/ΔC and Mecp2(R308/Y), which are associated with granule cell pathway expression. Shank3+/ΔC and Mecp2(R308/Y) also showed aberrant response timing and reduced Purkinje-cell dendritic spine density. Overall, our observations are potentially accounted for by defects in instructed learning in the olivocerebellar loop and response representation in the granule cell pathway. Our findings indicate that defects in associative temporal binding of sensory events are widespread in autism mouse models.

Both maternal and pup genotype influence ultrasonic vocalizations and early developmental milestones in tsc2 (+/-) mice.

Tuberous sclerosis complex (TSC) is an autosomal dominant disorder characterized by tumor growth and neuropsychological symptoms such as autistic behavior, developmental delay, and epilepsy. While research has shed light on the biochemical and genetic etiology of TSC, the pathogenesis of the neurologic and behavioral manifestations remains poorly understood. TSC patients have a greatly increased risk of developmental delay and autism spectrum disorder, rendering the relationship between the two sets of symptoms an extremely pertinent issue for clinicians. We have expanded on previous observations of aberrant vocalizations in Tsc2 (+/-) mice by testing vocalization output and developmental milestones systematically during the early postnatal period. In this study, we have demonstrated that Tsc2 haploinsufficiency in either dams or their pups results in a pattern of developmental delay in sensorimotor milestones and ultrasonic vocalizations.

Neuronal Tsc1/2 complex controls autophagy through AMPK-dependent regulation of ULK1.

Tuberous sclerosis complex (TSC) is a disorder arising from mutation in the TSC1 or TSC2 gene, characterized by the development of hamartomas in various organs and neurological manifestations including epilepsy, intellectual disability and autism. TSC1/2 protein complex negatively regulates the mammalian target of rapamycin complex 1 (mTORC1) a master regulator of protein synthesis, cell growth and autophagy. Autophagy is a cellular quality-control process that sequesters cytosolic material in double membrane vesicles called autophagosomes and degrades it in autolysosomes. Previous studies in dividing cells have shown that mTORC1 blocks autophagy through inhibition of Unc-51-like-kinase1/2 (ULK1/2). Despite the fact that autophagy plays critical roles in neuronal homeostasis, little is known on the regulation of autophagy in neurons. Here we show that unlike in non-neuronal cells, Tsc2-deficient neurons have increased autolysosome accumulation and autophagic flux despite mTORC1-dependent inhibition of ULK1. Our data demonstrate that loss of Tsc2 results in autophagic activity via AMPK-dependent activation of ULK1. Thus, in Tsc2-knockdown neurons AMPK activation is the dominant regulator of autophagy. Notably, increased AMPK activity and autophagy activation are also found in the brains of Tsc1-conditional mouse models and in cortical tubers resected from TSC patients. Together, our findings indicate that neuronal Tsc1/2 complex activity is required for the coordinated regulation of autophagy by AMPK. By uncovering the autophagy dysfunction associated with Tsc2 loss in neurons, our work sheds light on a previously uncharacterized cellular mechanism that contributes to altered neuronal homeostasis in TSC disease.

A magnetic resonance imaging study of cerebellar volume in tuberous sclerosis complex.

The cerebellum plays an important role in motor learning and cognition, and structural cerebellar abnormalities have been associated with cognitive impairment. In tuberous sclerosis complex, neurologic outcome is highly variable, and no consistent imaging or pathologic determinant of cognition has been firmly established. The cerebellum calls for specific attention because mouse models of tuberous sclerosis complex have demonstrated a loss of cerebellar Purkinje cells, and cases of human histologic data have demonstrated a similar loss in patients. We hypothesized that there might be a common cerebellar finding in tuberous sclerosis complex that could be measured as morphometric changes with magnetic resonance imaging. Using a robust, automated image analysis procedure, we studied 36 patients with tuberous sclerosis complex and age-matched control subjects and observed significant volume loss among patients in the cerebellar cortices and vermis. Furthermore, this effect was strongest in a subgroup of 19 patients with a known, pathogenic mutation of the tuberous sclerosis 2 gene and impacted all cerebellar structures. We conclude that patients with tuberous sclerosis complex exhibit volume loss in the cerebellum, and this loss is larger and more widespread in patients with a tuberous sclerosis 2 mutation.

Prenatal rapamycin results in early and late behavioral abnormalities in wildtype C57BL/6 mice.

Mammalian target of rapamycin (mTOR) signaling has been shown to be deregulated in a number of genetic, neurodevelopmental disorders including Tuberous Sclerosis Complex, Neurofibromatosis, Fragile X, and Rett syndromes. As a result, mTOR inhibitors, such as rapamycin and its analogs, offer potential therapeutic avenues for these disorders. Some of these disorders-such as Tuberous Sclerosis Complex-can be diagnosed prenatally. Thus, prenatal administration of these inhibitors could potentially prevent the development of the devastating symptoms associated with these disorders. To assess the possible detrimental effects of prenatal rapamycin treatment, we evaluated both early and late behavioral effects of a single rapamycin treatment at embryonic day 16.5 in wildtype C57Bl/6 mice. This treatment adversely impacted early developmental milestones as well as motor function in adult animals. Rapamycin also resulted in anxiety-like behaviors during both early development and adulthood but did not affect adult social behaviors. Together, these results indicate that a single, prenatal rapamycin treatment not only adversely affects early postnatal development but also results in long lasting negative effects, persisting into adulthood. These findings are of importance in considering prenatal administration of rapamycin and related drugs in the treatment of patients with neurogenetic, neurodevelopmental disorders.

Autistic-like behaviour and cerebellar dysfunction in Purkinje cell Tsc1 mutant mice.

Autism spectrum disorders (ASDs) are highly prevalent neurodevelopmental disorders, but the underlying pathogenesis remains poorly understood. Recent studies have implicated the cerebellum in these disorders, with post-mortem studies in ASD patients showing cerebellar Purkinje cell (PC) loss, and isolated cerebellar injury has been associated with a higher incidence of ASDs. 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 that result from mutation of either TSC1 or TSC2, whose protein products dimerize and negatively regulate mammalian target of rapamycin (mTOR) signalling. 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 and correlate cerebellar pathology with increased ASD symptomatology. Functional imaging also shows that TSC patients with ASDs display hypermetabolism in deep cerebellar structures, compared to TSC patients without ASDs. However, the roles of Tsc1 and the sequelae of Tsc1 dysfunction in the cerebellum have not been investigated so far. Here we show that both heterozygous and homozygous loss of Tsc1 in mouse cerebellar PCs results in autistic-like behaviours, including abnormal social interaction, repetitive behaviour and vocalizations, in addition to decreased PC excitability. Treatment of mutant mice with the mTOR inhibitor, rapamycin, prevented the pathological and behavioural deficits. These findings demonstrate new roles for Tsc1 in PC function and define a molecular basis for a cerebellar contribution to cognitive disorders such as autism.

Graded loss of tuberin in an allelic series of brain models of TSC correlates with survival, and biochemical, histological and behavioral features.

Tuberous sclerosis complex (TSC) is a neurodevelopmental disorder with prominent brain manifestations due to mutations in either TSC1 or TSC2. Here, we describe novel mouse brain models of TSC generated using conditional hypomorphic and null alleles of Tsc2 combined with the neuron-specific synapsin I cre (SynIcre) allele. This allelic series of homozygous conditional hypomorphic alleles (Tsc2(c-del3/c-del3)SynICre(+)) and heterozygote null/conditional hypomorphic alleles (Tsc2(k/c-del3)SynICre(+)) achieves a graded reduction in expression of Tsc2 in neurons in vivo. The mice demonstrate a progressive neurologic phenotype including hunchback, hind limb clasp, reduced survival and brain and cortical neuron enlargement that correlates with a graded reduction in expression of Tsc2 in the two sets of mice. Both models also showed behavioral abnormalities in anxiety, social interaction and learning assays, which correlated with Tsc2 protein levels as well. The observations demonstrate that there are graded biochemical, cellular and clinical/behavioral effects that are proportional to the extent of reduction in Tsc2 expression in neurons. Further, they suggest that some patients with milder manifestations of TSC may be due to persistent low-level expression of functional protein from their mutant allele. In addition, they point to the potential clinical benefit of strategies to raise TSC2 protein expression from the wild-type allele by even modest amounts.