Autism-like behaviors and cerebellar dysfunction in the Bmal1 mutant mouse ameliorated by mTORC1 inhibition

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. total RNA-seq and footprint experiments from the cerebellar tissue of Bmal1 knockout and wildtype mice, 3 biological replicates form each condition for RNA-seq; 2 biological replicates from each condition for footprint experiments.尽管昼夜节律紊乱与睡眠障碍常与孤独症谱系障碍（Autism Spectrum Disorders, ASD）相关联，但时钟基因功能失调是否会诱导动物产生类孤独症表型，目前仍未明确。核心时钟基因Bmal1已被证实与人类社交能力密切相关，且其错义突变已在ASD患者中被鉴定得到。本研究发现，全身性Bmal1敲除会导致小鼠出现显著的社交障碍、过度刻板与重复行为，以及运动学习能力缺陷，上述表型均与ASD的核心行为缺陷高度相似。进一步研究显示，Bmal1敲除（knockout, KO）小鼠的小脑浦肯野细胞（cerebellar Purkinje cells, PCs）存在细胞密度异常与树突棘形态不成熟的特征。电生理记录结果表明，Bmal1 KO小鼠的浦肯野细胞兴奋性与抑制性突触传递均增强，且放电频率降低。在Bmal1 KO小鼠的小脑组织中，我们鉴定出ASD与共济失调相关基因（Ntng2、Mfrp、Nr4a2、Thbs1、Atxn1及Atxn3）的差异表达，以及翻译调控通路的失调，其中包括过度激活的雷帕霉素靶蛋白复合物1（mammalian target of rapamycin complex 1, mTORC1）信号通路。有趣的是，抗糖尿病药物二甲双胍可逆转mTORC1的过度激活状态，并缓解Bmal1 KO小鼠的主要行为缺陷与浦肯野细胞异常。值得注意的是，仅在小脑浦肯野细胞中条件性敲除Bmal1，即可重现全身性Bmal1 KO小鼠中观察到的类孤独症行为与细胞变化。综上，本研究揭示了此前未被报道的Bmal1功能失调在小脑功能异常与类孤独症行为中的作用。我们的研究结果为昼夜节律时钟基因表达失调参与ASD发病机制提供了实验证据。本数据集包含来自Bmal1敲除与野生型小鼠小脑组织的总RNA测序（total RNA-seq）及足迹实验数据：RNA-seq每组设置3个生物学重复，足迹实验每组设置2个生物学重复。