Melatonin ameliorates sleep-wake disturbances and autism-like behaviors in the Ctnnd2 knock out mouse model of autism spectrum disorders

Abstract

Autism spectrum disorder (ASD) is a prevalent neurodevelopmental disorder characterized by atypical patterns of social interaction and communication, as well as restrictive and repetitive behaviors. In addition, patients with ASD often presents with sleep disturbances. Delta (δ) catenin protein 2 (CTNND2) encodes δ-catenin protein, a neuron-specific catenin implicated in many complex neuropsychiatric diseases. Our previous study demonstrated that the deletion of Ctnnd2 in mice led to autism-like behaviors. However, to our knowledge, no study has investigated the effects of Ctnnd2 deletion on sleep in mice. In this study, we investigated whether the knockout (KO) of exon 2 of the Ctnnd2 gene could induce sleep-wake disorders in mice and identified the effects of oral melatonin (MT) supplementation on Ctnnd2 KO mice. Our results demonstrated that the Ctnnd2 KO mice exhibited ASD-like behaviors and sleep-wake disorders that were partially attenuated by MT supplementation. Overall, our current study is the first to identify that knockdown of Ctnnd2 gene could induce sleep-wake disorders in mice and suggests that treatment of sleep-wake disturbances by MT may benefit to autism-like behaviors causing by Ctnnd2 gene deletion.

Keywords: Ctnnd2; autism spectrum disorder; melatonin; mice; sleep-wake disorders.

FIGURE 1

Ctnnd2 KO mice display ASD‐like behaviors. (A) Time line of experiments. (B, C) Schematic view and quantification of the first 10 min (B) and second 10 min (C) in the three‐chamber sociability assay. (D) Schematic view and quantification of the direct social interaction test. (E) Schematic view of the open field test. (F) KO mice exhibit more grooming behaviors than WT mice. (G) Quantification of the numbers of cross grids, cross center grids, vertical and climbing movements in the open field test. All groups: n = 8. Data are presented as the mean ± standard error. *p < 0.05, **p < 0.01, ****p < 0.0001 versus WT group (two‐tailed unpaired t‐test). ASD, autism spectrum disorder; EEG, electroencephalogram; EMG, electromyography; KO, knock out; PND, postnatal day; WT, wild type.

FIGURE 2

Ctnnd2 KO mice exhibit prolonged wakefulness and reduced NREM and REM sleep. (A) EEG power spectrums and hypnograms in mice during 24 h period; red, wakefulness; blue, NREM sleep; green, REM sleep. (B) Percentage of time spent in each state during the 24 h, daytime, and nighttime periods (two‐tailed unpaired t‐test). (C) Hourly percentage of time spent in wakefulness, NREM and REM sleep across the 24‐h sleep–wake status (Two‐way ANOVA test followed by multiple's comparison). (D, E) The number (D) and mean duration (E) of bouts in each state during the 24 h (left), daytime (center) and nighttime (right) periods (two‐tailed unpaired t‐test). All groups: n = 8. Data are presented as the mean ± standard error. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 versus WT group. EEG, electroencephalogram; KO, knock out; NREM, non‐REM; REM, rapid eye movement; WT, wild type.

FIGURE 3

Ctnnd2 KO mice exhibit abnormal normalized EEG power spectrum. (A, D, G) Schematic view of EEG and EMG in wakefulness (A), NREM (D) and REM (G) sleep. (B, E, H) Normalized EEG power density of wakefulness (B), NREM (E) and REM (H) sleep during the light phase (Two‐way ANOVA test followed by multiple's comparison). (C, F, I) Quantitative analysis of the power of wakefulness (C), NREM (F) and REM (I) sleep during the light phase in different frequency bands (two‐tailed unpaired t‐test). All groups: n = 6–9. Data are presented as the mean ± standard error. *p < 0.05, **p < 0.01 versus WT group. EEG, electroencephalogram; EMG, electromyography; KO, knock out; NREM, non‐REM; REM, rapid eye movement; WT, wild type.

FIGURE 4

The effect of MT on sleep–wake disorders in Ctnnd2 KO mice. (A) Percentage of time spent in REM sleep during the 24 h (left), daytime and nighttime (right) periods (one‐way ANOVA test followed by multiple's comparison). (B) From left to right: hourly percentage of time spent in REM sleep across the 24‐h sleep–wake between WT mice and KO mice and between KO mice and KO mice treated with three dosages of MT separately (two‐way ANOVA test followed by multiple's comparison). (C) The number of bouts in REM sleep during the 24 h (left), daytime and nighttime (right) periods. (D) The mean bouts duration of wakefulness during the 24 h (left), daytime and nighttime (right) periods. (E) The mean bouts duration of REM sleep during the (one‐way ANOVA test followed by multiple's comparison). All groups: n = 8. Data are presented as the mean ± standard error. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 versus WT group; ^# p < 0.05, ^## p < 0.01, ^### p < 0.001, ^#### p < 0.0001 versus KO group. KO, knock out; MT, melatonin; REM, rapid eye movement; WT, wild type.

FIGURE 5

The effect of MT on ASD‐like behaviors in Ctnnd2 KO mice. (A) Time line of experiments. (B, C) Schematic view and quantification of the first 10 min (B) and second 10 min (C) in the three‐chamber sociability assay. (D) Schematic view and quantification of the direct social interaction test. (E) KO mice exhibit more grooming behaviors than WT mice in the open field test. (F) Schematic view and quantification of the numbers of cross grids, cross center grids, vertical and climbing movements in the open field test. All groups: n = 8–10. Data are presented as the mean ± standard error. *p < 0.05, **p < 0.01, ****p < 0.0001 versus WT group; ^# p < 0.05, ^## p < 0.01, ^#### p < 0.0001 versus KO group (one‐way ANOVA test followed by multiple's comparison). ASD, autism spectrum disorder; EEG, electroencephalogram; EMG, electromyography; KO, knock out; MT, melatonin; PND, postnatal day; WT, wild type.