A Preliminary Study On The Improvement Of Sleep-wake Disorder In Ctnnd2 Knockout Mice By Melatonin And Its Mechanism

ASD is a general term for a wide range of neurodevelopmental disorders.Its clinical manifestations mainly consist of two core symptoms:early social interaction and communication disorders and repetitive stereotypical behaviors.In recent years,the incidence of ASD has been rising,and the prognosis is poor with serious impact on the quality of the patient and their family life.In addition to the core symptoms of ASD,it accompanied by many comorbidities such as abnormal movement disorders,sleep-wake disturbances,gastrointestinal disorders,epilepsy,mental retardation and hyperesthesia.These comorbidities may exacerbate the abnormal behavior of patients with ASD.According to many studies,many ASD patients experienced sleep problems,and the insomnia（the inability to fall asleep or maintain a stable sleep state）and sleep-wake disorders caused by circadian rhythm abnormalities are the most common.Sleep-wake disturbances in normal children not only caused irritability,anxiety and other emotional problems during the daytime,but also adversely affect the development of the brain,leading to damaged learning and memory function.The adverse effects caused by sleep problems in ASD patients are more serious than those in normal people.Besides the impact on emotional and cognitive development,sleep problems could exacerbate the core symptoms of ASD patients.However,for ASD patients,sleep problems had got inadequately recognized,no enough attention and had no method for treating.The consistency of sleep in animal models also provides an opportunity for us to study sleep problems in ASD patients.At present,there have been many studies on the sleep problems of different mice models with ASD and they were accompanied by different degrees of sleep problems.We screened an ASD-relative gene CTNND2,maps in the short arm of human chromosome 5 and in mice chromosome 15.The CTNND2gene has been reported to be highly associated with autism,therefore we constructed the Ctnnd2 KO mice by CRISPR/Cas9 technology and our previously experimental results had confirmed that Ctnnd2 KO mice showing core symptoms of ASD as well as the damaged ability of learning and memory.However,whether CTNND2 is involved in the regulation of sleep has not been reported.Whether Ctnnd2 KO induces sleep-wake disorder in mice,and whether the application of sleep regulator melatonin（MT）could improve sleep-wake disorder is also lacking of experimental evidence.E/I imbalance is not only one of the etiological hypotheses for the occurrence of the core symptoms in autism,but also one of the pathological mechanisms of sleep-wake disorders.GABAergic interneurons are crucial for the induction and maintenance of sleep.Therefore,in our study,an autism model of Ctnnd2 KO mice was established,and EEG-EMG records were used to determine whether there were sleep-wake disorders in Ctnnd2KO model mice,and how the effect of MT on sleep in Ctnnd2 KO mice.On this basis,this paper also conducted a preliminary assessment of GABAergic transmitter system,in order to screen out the brain regions with the largest changes in GABAergic system as the targeted research brain regions.Finally,we analyzed whether MT could regulate the synaptic transmission efficiency of GABAergic neurons by combining with MT-R on GABAergic neurons in the targeted brain region and identified the molecular mechanism of the effect that MT improving sleep-wake disorders in Ctnnd2 KO mice.Part One MT improved sleep-wake disturbances in Ctnnd2 KO miceObjective:Whether the application of MT on Ctnnd2 KO mice improve sleep-wake disorders of Ctnnd2 KO mice.Methods:1.Genotyping of mice was confirmed by PCR.2.EEG-EMG were recorded to evaluate sleep-wake status of mice.3.Detection indicators:（1）The percentage of time spent in Wake,NREM and REM sleep of mice.（2）The number of episodes in Wake,NREM and REM sleep of mice.（3）The mean duration of episodes in Wake,NREM and REM sleep of mice.（4）The analysis of EEG nomalized spectral activities in Wake,NREM and REM sleep of mice.Results:1.Compared with WT mice,Ctnnd2 KO mice exhibited sleep-wake disorders:（1）The percentage of time spent in Wake was increased（P<0.05）and the percentage of time spent in NREM（P<0.05）and REM（P<0.001）sleep were decreased in KO mice.（2）KO mice entered REM sleep less frequently（P<0.01）.（3）The mean duration of Wake was increased（P<0.05）and the mean duration of REM sleep（P<0.05）was decreased in KO mice.（4）The EEG normalized spectral activities in Wake（P<0.01）and REM sleep（P<0.05）of KO mice were changed.2.Compared with Ctnnd2 KO mice,MT improved the sleep-wake disorders of Ctnnd2 KO mice.（1）The percentage of time spent in REM sleep was increased（P<0.05）in KO mice treated with MT.（2）KO mice treated with MT entered REM sleep more frequently（P<0.05）.（3）The mean duration of Wake was decreased and the mean duration of REM sleep was increased in KO mice treated with MT（P<0.05）.（4）The EEG normalized spectral activities in KO mice treated with MT had no changes.Conclusion:Ctnnd2 KO mice exhibited sleep-wake disorders including prolonged Wake time,decreased NREM and REM sleep time and impaired sleep quality.However,after the administration of MT,the sleep-wake disorders of Ctnnd2 KO mice were alleviated including the increased sleep time,numbers of episodes and mean duration of episodes in REM,the decreased mean duration of episodes in Wake.However,there was no significant effect on their sleep quality.Part Two To verify whether MT enhanced synaptic transmission efficiency of GABAergic to improve sleep-wake disorders of Ctnnd2 KO mice by binding with MT-R and activating PI3K/Akt pathwayObjective:To investigate the possible molecular mechanism of MT in improving sleep-wake disorders of Ctnnd2 KO miceMethod:1.Western blot was used to detect the expression of GAD67 and GAD65in the whole brain of WT and Ctnnd2 KO mice,and the best brain regions were selected.2.The PFC of Ctnnd2 KO mice was micro-injected with different drugs using brain stereotaxic,and the brain tissue was sacrificed 2 h later.3.Western blot was used to detect the expression of GAD67,p-Syn,ELKS and PI3K/Akt signaling pathway in PFC of mice.4.Immunofluorescence was used to co-label MT₁R,MT₂R and GAD67separately in PFC of WT and Ctnnd2 KO mice.5.Immunofluorescence was used to co-label p-Syn,ELKS and GAD67separately in the PFC of mice to determine the changes in the synaptic transmission efficiency of GABAergic neurons.Result:1.Compared with WT mice,the expression of GAD67 in PFC of Ctnnd2KO mice was significantly decreased（P<0.001）.2.After local injection of MT into PFC of Ctnnd2 KO mice,MT enhanced the synaptic transmission efficiency of neurons by binding with MT-R through activation of PI3K/Akt signaling.3.After local injection of MT into PFC of Ctnnd2 KO mice,MT activated PI3K/Akt signaling pathway to enhance the synaptic transmission efficiency of GABA neurons by binding with MT-R.Conclusion:By combining with MT-R,MT activated PI3K/Akt signaling pathway to enhance the synaptic transmission efficiency of GABAergic neurons,thus may improve sleep-wake disorders in Ctnnd2 KO mice.