Mol Psychiatry︱The role of biological clock gene Bmal1 in mouse models of autism and cerebellar ataxia

Written by ︱ Liu Dong, editor in charge ︱ Wang Sizhen Biological circadian rhythms (circadian rhythms) is a common phenomenon that exists in a variety of organisms
.

The circadian rhythm is generated by the circadian clock and plays an important role in regulating biological gene expression, neural activity and individual behavior
.

At the cellular level, the circadian clock is driven by a transcription-translation feedback loop composed of a set of clock genes
.

In mammals, BMAL1 forms a dimer with CLOCK or NPAS2 to regulate rhythmic transcription.
Bmal1 gene is the only circadian clock gene that can cause complete loss of biological rhythm after gene knockout [1]
.

Not only that, BMAL1 can also act as a translation factor to regulate protein synthesis [2]
.

 Autism spectrum disorders (ASDs) are disorders related to brain development that affect the way a person perceives others and socializes, causing social and communication problems
.

The disorder also includes limited and repetitive behavioral patterns
.

Previous studies have found that loss of function mutation occurs in multiple biological clock genes in autistic people [3-5]
.

In addition, hormonal circadian rhythm disturbances and sleep disturbances are common manifestations of autism
.

Recent studies have found that BMAL1 is a gene that is significantly correlated with human social behavior, and significant Bmal1 mutations have also been found in autistic patients [5,6]
.

All this evidence suggests that Bmal1 may be involved in the development of autism, but there is no experimental evidence to prove this
.

 On March 17, 2022, the team of Prof.
Ruifeng Cao from the University of Minnesota School of Medicine and Prof.
Christos G.
Gkogkas from the University of Edinburgh, UK published a paper entitled "Autistic-like behavior and cerebellar dysfunction in Bmal1 mutant mice ameliorated by mTORC1 inhibition in Molecular Psychiatry.
"The article first revealed the mechanism of Bmal1 gene in autism and cerebellar ataxia, and these studies support that circadian clock gene expression disorder may play a significant role in autism
.

In this article, the authors firstly used the mouse social behavior detection method and found that after Bmal1 knockout (KO), the sociability of mice was significantly reduced (Fig.
1a, b), and the olfactory adaptation experiment also proved that Bmal1 KO Mice showed a lack of interest in the scent of unfamiliar mice (Fig.
1c)
.

At the same time, Bmal1 KO mice also exhibited excessive grooming (Fig.
1d) and stereotyped activity trajectories (Fig.
1e)
.

These results all support that Bmal1 KO mice exhibit significant autism-related behavioral disorders
.

Figure 1 Bmal1 knockout mice exhibit autistic behavioral characteristics (Source: D Liu et al.
, Mol Psychiatry, 2022) Since children with autism often have motor dysfunction at the same time, the authors also detected Bmal1 KO mice Movement function
.

During training with the Rotarod device, Bmal1 KO mice exhibited markedly reduced motor learning and motor coordination dysfunction, suggesting the presence of cerebellar ataxia (Fig.
2a)
.

Histological studies showed that the cerebellar Purkinje cells of Bmal1 KO mice were denser and longer than normal mice (Fig.
2b), and the dendritic spines of Purkinje cells also showed dysplastic morphology and higher density (Fig.
2c)
.

These results suggest that Bmal1 knockout significantly affects both the morphology and function of Purkinje cells in the mouse cerebellum
.

Figure 2 Bmal1 knockout mice showed motor learning impairment and morphological changes in cerebellar Purkinje neurons (Source: D Liu et al.
, Mol Psychiatry, 2022) Further neuroelectrophysiological studies showed that Bmal1 KO mice had The amplitudes of excitatory postsynaptic potentials (EPSCs) recorded in Kenye cells were approximately 2-fold higher than normal (Fig.
3c, d)
.

At the same time, the inhibitory postsynaptic potential (IPSC) amplitude was also about 2-fold higher than normal (Fig.
3e), but the excitatory/inhibitory potential ratio (E:I ratio) did not change significantly (Fig.
3f)
.

In addition, the spontaneous firing frequencies of Purkinje cells were significantly reduced after Bmal1 KO (Fig.
3g, h)
.

These results demonstrate that the altered electrophysiological properties of Purkinje cells in Bmal1 KO mice may be the mechanism responsible for cerebellar dysfunction after Bmal1 knockout
.

Figure 3 Electrophysiological changes of Purkinje neurons in Bmal1 knockout mice (Source: D Liu et al.
, Mol Psychiatry, 2022) Since Bmal1 can regulate protein synthesis, the authors carried out Bmal1 knockout mice and wild-type small Ribo-seq (ribosome imprinting sequencing technology) of mouse cerebellum tissue found 304 differentially translated genes (DTGs), of which 12 genes were autism risk genes (SFARI genes)
.

In addition, signaling pathway analysis found that many DTGs were located in the eIF2 signaling and eIF4E-p70S6K signaling pathways (Fig.
4a, b)
.

Confirmed by immunohistochemistry and western blotting experiments
.

The activity of mTORC1/S6K1 and eIF2 signaling pathways was significantly enhanced after knockdown of Bmal1 (Fig.
4c–e)
.

Figure 4.
Translational characteristics of the cerebellum of Bmal1 KO mice and changes in related pathways (Source: D Liu et al.
, Mol Psychiatry, 2022) Since the mTORC1 signaling pathway is significantly enhanced in the cerebellum of Bmal1 KO mice, studies have shown that a diabetes treatment The first-line drug, metformin, inhibits mTORC1 activity
.

Based on these data, the authors applied metformin to experimentally treat Bmal1 KO mice
.

The authors were pleasantly surprised to find that metformin significantly inhibited mTORC1 pathway activity in the cerebellum and significantly improved social impairment and repetitive behaviors in Bmal1 KO mice (Fig.
5a–f)
.

Electrophysiological results also showed that metformin could increase the spontaneous firing frequency of Purkinje cells in Bmal1 KO mice to normal levels (Fig.
5g)
.

Figure 5 Metformin can improve autism-related behaviors and inhibit the mTORC1 pathway in Bmal1 KO mice (Source: D Liu et al.
, Mol Psychiatry, 2022) Finally, in order to verify whether the expression of Bmal1 in cerebellar Purkinje cells is autonomic Playing a key role in the autism phenotype, the authors established Bmal1 Purkinje cell conditional knockout mice (Bmal1flx/flx:L7-Cre)
.

Compared with control Bmal1flx/flx mice, Bmal1flx/flx:L7-Cre mice also exhibited social novelty deficits, significantly increased repetitive behaviors, and decreased motor coordination during Rotarod training experiments (Fig.
6a).
-d)
.

Electrophysiologically, compared with Bmal1flx/flx Purkinje cells, Bmal1flx/flx:L7-Cre Purkinje cells had significantly higher EPSC and IPSC amplitudes and significantly lower spontaneous firing frequencies (Fig.
6f–h)
.

Meanwhile, the mTORC1 pathway activity in Purkinje cells was significantly enhanced after Bmal1 conditional knockout (Fig.
6i)
.

The dendritic spine morphology of Purkinje cells also showed dysplasia (Fig.
6j)
.

These results suggest that the conditional knockout of Bmal1 Purkinje cells is sufficient to make mice exhibit autism-related performance similar to Bmal1 knockout mice
.

Figure 6 Bmal1flx/flx:L7-Cre conditional knockout mice exhibit autism-related behaviors and changes (Source: D Liu et al.
, Mol Psychiatry, 2022) Conclusions and discussions, inspiration and prospects Conditional knockout of Bmal1 either systemically or only in cerebellar Purkinje cells in a mouse model resulted in autism-related symptoms in mice, including loss of social activity and increased repetitive behaviors
.

Previous studies have found that Bmal1 knockout also affects cognitive and memory functions [7, 8], and leads to astrogliosis [9], which is consistent with some clinically observed manifestations of ASD [10, 11]
.

At the same time, motor function and the role of the cerebellum in the occurrence of autism spectrum disorder (ASD) have also been widely concerned and studied, especially the role of Purkinje cells
.

In Bmal1 knockout mice, the authors observed the morphological and functional changes of Purkinje cells, which further provided evidence for the role of Purkinje cells in the occurrence of ASD
.

Although the effects of Bmal1 systemic knockout and Purkinje cell conditional knockout on social behavior were slightly different, they both significantly affected the function of Purkinje cells and the activity of mTORC1 signaling pathway
.

Not only that, by applying metformin [12], which was previously used to improve social interaction in mice with Fragile X syndrome (Fragile X syndrome), it also played a significant therapeutic role in the Bmal1 knockout social disorder model of autism
.

 This study found that metformin works by reducing the abnormal increase in mTORC1, because metformin is a commonly used drug in clinical practice, and these results suggest that metformin can be used in the treatment of some autistic patients
.

 Overall, this study is the first to demonstrate the role of the key biological clock gene Bmal1 in a mouse model of autism, suggesting that abnormal expression of biological clock genes may play a significant role in the development of autism
.

Link to the original text: https:// The first author of the article Liu Dong (first from the left), the corresponding author Ruifeng Cao (the second from the left) (photo courtesy of Ruifeng Cao's laboratory) Postdoctoral, doctoral, or master students interested in mammalian biological clocks or signal transduction mechanisms in neurological diseases are welcome to join the lab
.

Contact: rcao@umn.
edu
.

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2-3 Tencent Online) References (up and down) 1.
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Plate making︱Sizhen Wang End of this paper