The Effect Of Melatonin On Improving Core Symptoms In CTNND2-Deficient Autism Model Mice And Its Mechanism

Autism spectrum disorder（ASD）is a group of neurodevelopmental disorders which begin in early childhood and continue throughout whole life,affecting more males than females.The core symptoms of ASD are social deficits（including impaired verbal and non-verbal communication）,narrow interests and restrictive or repetitive behaviors.The prevalence of ASD has increased dramatically throughout the last decades.Research has shown that the prevalence of ASD among children in the United States is about 1.9%,and more than 1%of the world’s population are thought to receive a diagnosis of ASD.Due to poor understanding of the pathogenesis of ASD and the lack of targeted therapy,the increasing societal cost of ASD has become a global public problem.In mammals,the prefrontal cortex（PFC）is a critical part of higher cognitive and social functioning which plays a key role in making decision,regulating emotion,attention control,and working memory.Its dynamic changes are associated with social dysfunction in ASD.The causes of ASD include genetic,immune,and environmental factors.In most cases of ASD,genetic factors play an important role.Researches have shown that many ASD-related gene mutations can cause reduced dendritic branches,abnormal spine density and morphological defects of spines,and affect synaptic plasticity at different levels.Studies suggest that the formation of abnormal neuronal synapses and dendritic spines is involved in the pathogenesis of ASD.Catenin delta 2（CTNND2）is located on chromosome 5p15.2 of human and chromosome 15 B2 of mice.The proteinδ-catenin encoded by CTNND2 gene is one of the members of p-120 catenin（p120ctn）family,which is mainly expressed in neurons of cerebral cortex and hippocampus.The expression level ofδ-catenin began to rise on the thirteenth day of embryo,reached the peak on the seventh day after birth,and then dropped to a certain degree in adulthood,which was mainly distributed in dendrites.δ-catenin plays a key role in maintaining dendrites and dendritic spines of mature cortex.The research team has successfully established a mouse model with CTNND2 gene deletion,and confirmed that it has ASD-like behaviors.Melatonin（MT）is the main hormone secreted by the pineal gland,which has the effects of neuroprotection and regulating circadian rhythm.Research has found that MT can improve the abnormal behaviors of patients with ASD,while the mechanism by which MT improves the core symptoms of ASD has rarely been reported.Part One To observe the ASD-like behaviors and changes of dendritic spines in CTNND2-deficient miceObjective:To verify that CTNND2 gene deletion(CTNND2^-/-)mice have ASD-like behaviors,and observe whether the development of dendritic spines in PFC neurons is impaired.Methods:1.The genotypes of mice were detected with Polymerase Chain Reaction（PCR）.2.Groups:CTNND2^-/-group and wild-type（WT）group.3.Behavior tests:three-chamber sociability test（PN50d）,juvenile play test（PN52d）,open field test（PN54d）.4.Golgi-cox staining（PN56d）:The density and area of dendritic spines in PFC neurons were analyzed.Results:1.Results of behavior tests（1）In three-chamber sociability test,compared with WT mice,interaction time with stranger 1 mouse was reduced and time spent in center chamber was increased in CTNND2^-/-mice at the stage of social interaction test.At the stage of novelty preference test,CTNND2^-/-mice showed reduced interaction time with stranger 2 mouse when compared with WT mice.（2）In juvenile play test,there was a decrease in time of social interaction in CTNND2^-/-mice when compared with WT mice.（3）In open field test,compared with WT mice,there were decreases in numbers of cross grid,cross center grid,vertical and climbing of CTNND2^-/-mice.CTNND2^-/-mice showed increased time of self-grooming.2.Results of Golgi-cox staining:Total spine density and density of mushroom spine and stubby spine,as well as the area of mushroom spine and stubby spine in CTNND2^-/-mice were less than those of WT mice,there was no difference in density and area of filopodia spine between WT and CTNND2^-/-mice.Conclusion:The CTNND2^-/-mice have ASD-like behaviors and impaired dendritic spines of PFC neurons,which can be used as mice model with ASD.Part two The improvement of melatonin on core symptoms of ASD in CTNND2-deficient miceObjective:To investigate whether MT can improve the core symptoms of ASD in CTNND2^-/-mice,and select the dose with most obvious effect for subsequent experiments.Methods:1.The genotypes of mice were detected with Polymerase Chain Reaction（PCR）.2.Groups:CTNND2^-/-group,wild-type（WT）group,CTNND2^-/-+5mg/kg MT group,CTNND2^-/-+10mg/kg MT group and CTNND2^-/-+50mg/kg MT group.3.MT intervention:MT was administered by gavage at 0.01ml/g body weight from PN22d to PN49d.4.Behavior tests:three-chamber sociability test（PN50d）,juvenile play test（PN52d）,open field test（PN54d）.Results:1.Results of behavior tests（1）In three-chamber sociability test,at the stage of social interaction test,compared with CTNND2^-/-mice,interaction time with stranger 1mouse was increased in groups of three doses of MT,and time spent in center chamber was decreased in 5mg/kg MT and 50mg/kg MT groups.At the stage of novelty preference test,interaction time with stranger 2 mouse was increased in 10mg/kg MT group when compared with CTNND2^-/-mice,5mg/kg MT group showed reduced time spent in center chamber.（2）In juvenile play test,there was an increase in time of social interaction of 5mg/kg MT and 10mg/kg MT groups when compared with CTNND2^-/-mice.（3）In open field test,compared with CTNND2^-/-mice,there were increases in numbers of cross grid and cross center grid in 10mg/kg MT group,there was also an increase in number of cross grid in 50mg/kg MT group.Groups of three doses of MT showed decreased time of self-grooming when compared with CTNND2^-/-mice.Conclusion:Three doses of MT could improve the ASD-like behaviors of CTNND2^-/-mice in different degrees.With the best effect of 10mg/kg MT,this group was selected for the subsequent experiments.Part three The mechanism of melatonin improving core symptoms of ASD in CTNND2-deficient miceObjective:To observe whether MT could promote synthesis of synapse-associated proteins and improve development of dendritic spine by upregulating the protein expression of PI3K/Akt/m TOR signaling pathway in PFC of CTNND2^-/-mice,and to preliminarily explore the possible mechanism of MT to improve core symptoms of ASD.Methods:1.Groups:CTNND2^-/-group,wild-type（WT）group,CTNND2^-/-+10mg/kg MT group,CTNND2^-/-+DMSO group,CTNND2^-/-+MT group and CTNND2^-/-+Wortmannin+MT group.2.Stereotactic injection of cerebra（PN56d）:injected with 5%DMSO,MT and Wortmannin into PFC respectively.3.Western blot was performed to detect the expression levels of p-PI3K,PI3K,p-Akt,Akt,p-m TOR and m TOR proteins of PFC（PN56d）.4.Western blot was performed to detect the expression levels of p-Syn,Syn,ELKS and PSD95 proteins of PFC（PN56d）.5.Golgi-cox staining（PN56d）:The density and area of dendritic spines in PFC neurons were analyzed.Results:1.The ratio of p-PI3K/PI3K,p-Akt（Ser473）/Akt,p-Akt（Thr308）/Akt and p-m TOR/m TOR in PFC of CTNND2^-/-mice were less than those of WT mice.The ratio of p-PI3K/PI3K,p-Akt（Ser473）/Akt,p-Akt（Thr308）/Akt and p-m TOR/m TOR were increased in PFC of 10mg/kg MT group when compared with CTNND2^-/-mice.2.The ratio of p-Syn/Syn,as well as the expression levels of ELKS and PSD95 in PFC of CTNND2^-/-mice were lower than those of WT mice.The ratio of p-Syn/Syn,as well as the expression levels of ELKS and PSD95 were increased in PFC of 10mg/kg MT group when compared with CTNND2^-/-mice.3.Results of Golgi-cox staining:Total spine density and density of mushroom spine and stubby spine,as well as the area of mushroom spine and stubby spine were increased in 10mg/kg MT group when compared with CTNND2^-/-mice,there was no difference in density and area of filopodia spine between 10mg/kg MT group and CTNND2^-/-mice.4.After injected with MT and Wortmannin into PFC of CTNND2^-/-mice,the activation of PI3K/Akt/m TOR signaling pathway by MT can be inhibited by Wortmannin,and the promoting effect of MT on synapse-associated protein synthesis can also be inhibited by Wortmannin.Conclusion:MT could promote synthesis of synapse-associated proteins and improve development of dendritic spine by upregulating the expression of PI3K/Akt/m TOR signaling pathway in PFC of CTNND2^-/-mice.