Autistic-like behavior and cerebellar dysfunction in Bmal1 mutant mice ameliorated by mTORC1 inhibition

Abstract

Although circadian and sleep disorders are frequently associated with autism spectrum disorders (ASD), it remains elusive whether clock gene disruption can lead to autistic-like phenotypes in animals. The essential clock gene Bmal1 has been associated with human sociability and its missense mutations are identified in ASD. Here we report that global Bmal1 deletion led to significant social impairments, excessive stereotyped and repetitive behaviors, as well as motor learning disabilities in mice, all of which resemble core behavioral deficits in ASD. Furthermore, aberrant cell density and immature morphology of dendritic spines were identified in the cerebellar Purkinje cells (PCs) of Bmal1 knockout (KO) mice. Electrophysiological recordings uncovered enhanced excitatory and inhibitory synaptic transmission and reduced firing rates in the PCs of Bmal1 KO mice. Differential expression of ASD- and ataxia-associated genes ( Ntng2 , Mfrp , Nr4a2 , Thbs1 , Atxn1 , and Atxn3 ) and dysregulated pathways of translational control, including hyperactivated mammalian target of rapamycin complex 1 (mTORC1) signaling, were identified in the cerebellum of Bmal1 KO mice. Interestingly, the antidiabetic drug metformin reversed mTORC1 hyperactivation and alleviated major behavioral and PC deficits in Bmal1 KO mice. Importantly, conditional Bmal1 deletion only in cerebellar PCs was sufficient to recapitulate autistic-like behavioral and cellular changes akin to those identified in Bmal1 KO mice. Together, these results unveil a previously unidentified role for Bmal1 disruption in cerebellar dysfunction and autistic-like behaviors. Our findings provide experimental evidence supporting a putative role for dysregulation of circadian clock gene expression in the pathogenesis of ASD.

Haploinsufficiency of a Circadian Clock Gene Bmal1 (Arntl or Mop3) Causes Brain-Wide mTOR Hyperactivation and Autism-like Behavioral Phenotypes in Mice

“…By Western blotting, we found that the level of Bmal1 was decreased by ~50% in the cerebellum ( p = 0.0019) and ~75% in the forebrain ( p < 0.0001) of the Bmal1 +/− mice as compared to the levels in the WT mice ( Figure 1 A), indicating a haploinsufficiency of Bmal1 . As we found mTOR hyperactivation in the brain of Bmal1 KO mice in our previous study [ 20 ], we further assessed whether the mTORC1 activities were changed in the Bmal1 +/− brain by measuring the levels of phosphorylated S6 ribosomal proteins (p-S6), a sensitive marker of mTORC1. Interestingly, we found that the p-S6 levels were increased by ~50% in the cerebellum (p = 0.0495) and the forebrain ( p = 0.0396) of Bmal1 +/− mice as compared to the levels in the WT mice ( Figure 1 A), indicating pervasive mTORC1 hyperactivation in the brains of Bmal1 +/− mice.…”

“…In the previous study, we tested Bmal1 KO mice for similar behavioral phenotypes [ 20 ]. In the present study, we demonstrated that the haploinsufficiency of Bmal1 can also cause a variety of autism-like phenotypes in mice, akin to those identified in Bmal1 KO mice.…”

“…Metformin treatment can rescue some behavioral deficits in Bmal1 KO mice. Interestingly, the hyperactivation of mTOR but no other pathways are reversed by metformin treatment, suggesting that aberrant mTOR activities may underlie the behavioral changes in Bmal1 KO mice [ 20 ]. In Bmal1 +/− mice, increased p-S6 levels are also found in the forebrain and the cerebellum, suggesting that mTOR hyperactivation may underlie the behavioral changes in Bmal1 +/− mice.…”

“…Although the role of the cerebellum in motor coordination is well-known, how the cerebellum modulates cognition and social behaviors is still an intense topic of discussion [ 39 ]. The electrophysiological data from our previous study demonstrated aberrant synaptic transmission and reduced PC firing rates in Bmal1 KO mice [ 20 ]. Using a conditional Bmal1 deletion in cerebellar PCs, most of the behavioral and molecular phenotypes seen in Bmal1 KO mice can be recapitulated, suggesting a significant role for Bmal1 expression in the cerebellum in generating autism-like phenotypes in Bmal1 KO mice [ 20 ].…”

“…In addition, its missense mutation was identified in ASD [ 6 ], suggesting its disruption could play a role in ASD. In a recent study, we found that global Bmal1 deletion leads to significant impairments in social interactions and recognition and excessive stereotyped and repetitive behaviors, as well as disabilities in motor learning and coordination, all of which are reminiscent of the core behavioral deficits in ASD [ 20 ]. These results suggest that BMAL1 disruption could be involved in the development of ASD.…”