The Role And Mechanism Of Rictor In Spatial Learning And Memory Impairment In Ctnnd2 KO Mice

Autism,also known as Autism,is a serious developmental disorder of the Nervous System(NS).The incidence of autism is increasing year by year around the world,and males are more than females.The clinical features are memory impairment,social difficulties and repetitive and rigid behaviors.However,because of its unknown causes,there is no effective treatment except early behavioral intervention.Current research suggests that autism spectrum disorder is mainly related to genetic factors,present the polygenic inheritance mode,the interaction between environmental factors and lead to brain developmental disorders,although ASDs are the result of superposition of multiple candidate gene variants.Based on the research of the idiopathic autism spectrum disorder,researchers found a number of single gene mutations,such as CTNND2,UBE3A,NLGN3,NLGN4X,SHANK3,Syn GAP and DLGAP2,these all belong to synapses related genes,participate in the carrying out of the normal functions of the synapses.The CTNND2 gene encodes?-catenin protein and contains 23 exons with a span of at least 640kb,located in the 5p15.2 region of the human chromosome,which is a susceptibility site for autism.Delta-catenin is one of the serial p120 protein family,almost only expressed in nervous system.The interaction of?-catenin with N-cadherin and proteins containing PDZ domains is a key factor regulating synaptic and dendritic spines development and maturation.It is localized in dendrites and dendritic spines,especially in the cerebral cortex,hippocampus and cerebellum,and its loss leads to abnormal synaptic function.The hippocampus is an important target region of?-catenin in the central nervous system,and its synaptic plasticity is the basis of learning,memory and cognition,while ASDs patients are associated with learning and cognitive impairment and other clinical manifestations.Synaptic plasticity is considered to be one of the core mechanisms of learning,memory and cognition.Synaptic plasticity includes changes in synaptic morphology and synaptic transmission efficiency.Nerve microfilaments are the thinnest filamentous structures in neurons,and their main components are actin and myosin.As one of the main components of cytoskeleton,actin mainly maintains cell morphology and plasticity.Actin mainly participates in the movement of synaptic vesicles,the growth of neuronal projections and the formation of membrane specialized structures through the interaction with cell membrane.In developing neurons,actin is mainly distributed in the anterior part of the growth cone and plays a key role in the formation,growth and branching of neurites and synaptogenesis.In mature neurons,actin is the dominant cytoskeletal protein in synapses,occurring at both presynaptic and postsynaptic endings.Most excitatory synaptic transmission in the brain is mediated by actin,and the abnormal expression of actin is significantly associated with the disorder of synaptic plasticity and cognitive impairment.Rapamycin-insensitive companion of m TOR(Rictor),a core component of mammalian target rapamycin protein complex 2(m TORC2),plays a major role in actin skeleton remodeling.Rictor binds to the cell membrane and regulates actin dynamics by specific phosphorylation of Akt at Serine473.Downstream target molecules of m TORC2 include AGC kinases such as Akt,PKC,and SGK1,which play an important role in cytoskeleton formation by regulating signal transduction junction proteins paxillin,GTPase Rho A,Rac1,and cell division control proteins Cdc42,as well as PKC?.Rictor is necessary for dendritic arbor development,and axon growth requires the involvement of p I3K-Rictor-RAC GTPase pathway,which is involved in the Rictor-Actin cytoskeleton.The role of Rictor,the core component of m TORC2,in learning and cognition has been reported in literature,but the role and mechanism of Rictor and other components of m TORC2 in learning and memory cognition of Ctnnd2 KO mice are almost blank.Part One Establishment and behavioral characteristics of Ctnnd2 KO mouse modelObjective:To observe the neurorelated behavioral phenotype of Ctnnd2 KO mice.Methods:1.Polymerase Chain Reaction(PCR)was used to identify the genotypes of the experimental mice.2.Groupings:Ctnnd2 KO group(Ctnnd2^-/-homozygous male C57 mice)and WT group(wild-type male C57 mice).3.Behavioral changes of mice in each group were detected:(1)The Three Chamber Sociability Test was used to detect the social interaction and novelty preference behaviors of two groups of mice.(2)The open-field test was used to detect the spontaneous exploration ability and anxiety of the two groups of mice.(3)The self-grooming test was used to detect the repetitive behaviors of two groups of mice.(4)Morris water maze test was used to test the learning and memory ability of two groups of mice.Results:1.The Three Chamber Sociability Test:The social behavior(p<0.05)and novelty preference(p<0.05)in KO group was significantly decreased.2.The open-field test:The spontaneous exploration behavior of mice in KO group decreased(p<0.01),while the anxiety behavior increased(p<0.01),and the mice in both groups had no obvious tension(p>0.05).3.The self-grooming test:Repetitive and stereotyped behaviors of mice in KO group were significantly increased(p<0.001).4.Morris water maze test:KO group mice had obvious learning memory disabilities(p<0.05),and there was no difference in motor ability between the two groups(p>0.05).Conclusion:Ctnnd2 KO mice showed autism-like behavior and spatial learning and memory impairment.Part Two Expression changes of Rictor and its behavioral function of Ctnnd2 KO miceObjective:Observed the expression of Rictor in postnatal mice and the neurobehavioral changes of Ctnnd2 KO mice after inhibiting Rictor.Methods:1.Groupings:(1)Developmological experiment:WT male C57 mice at different time points after birth were divided into WT group,and Ctnnd2 KO male C57mice at different time points after birth were divided into KO group.(2)Rictor virus interference experiment:Male C57 Ctnnd2 KO mice undergoing Sham operation at postnatal 21 day were divided into Sham group,Vector group after stereotropic injection of virus,and Sh Rictor group after stereotropic injection of Rictor inhibitory virus.(3)Behavioral science and molecular biology experiment:WT+Vector group,KO+Vector group,WT+Sh Rictor group,KO+Sh Rictor group.2.Behavioral and molecular biology experiments of Ctnnd2 KO mice after Rictor interference:Fluorescence microscopy determined the site for stereoscopic injection of adeno-associated virus.WB was used to detect the efficacy of sh Rictor.The changes of autism-like behavior and learning and memory were detected after Rictor inhibition.Results:1.The expression of Rictor in the hippocampus of postnatal mice decreased gradually with development and tended to be stable at 21 days.The expression of Rictor in Ctnnd2 KO group was lower than that in WT group(p<0.05).2.The green fluorescence carried by the virus was observed under fluorescence microscope in the hippocampus of mice,indicating that the injection location was correct and the virus was successfully transfected into the hippocampus of mice.WB method detected that the expression of Rictor in Sh Rictor group was significantly lower than that in Sham group(p<0.05),while the expression of Rictor in Vector group had no significant change compared with that in Sham group(p>0.05).These results indicated that Rictor could successfully transfect the virus and achieve the inhibitory effect,and mice could be used for subsequent experiments.3.The results of the Three Chamber Sociability Test showed that after Rictor inhibition,there were no statistically significant differences in social and novelty preference experiments in WT and KO group compared with WT+Vector and KO+Vector groups,respectively(p>0.05).These results suggest that Rictor inhibition has no effect on social preference in mice.4.The open-field test results showed that after Rictor inhibition in WT and KO mice,there was no statistically significant difference in all observation indexes compared with WT+Vector and KO+Vector groups,respectively(p>0.05).These results suggest that Rictor inhibition has no effect on anxiety behavior,exploration behavior and nervous emotion in mice.5.The self-grooming test showed that after Rictor inhibition,there was no statistically significant difference in the repetitive and rigid behavior of WT and KO mice compared with WT+Vector and KO+Vector groups,respectively(p>0.05).These results suggest that Rictor inhibition does not affect the stereotyped repetitive behavior in mice.6.Morris water maze test showed that after Rictor inhibition,the spatial learning and memory abilities of WT and KO mice were reduced compared with WT+Vector group and KO+Vector group,respectively(p<0.05).There was no significant difference in swimming speed and distance,and no difference in motor ability in each group(p>0.05).The results indicated that the memory ability of KO group was significantly delayed,and the memory ability was further decreased after inhibiting Rictor.Conclusion:Rictor gradually decreases with the development of mice after birth and plays a regulatory role in the development and maturation of hippocampus.Rictor is a key regulator of spatial cognition in Ctnnd2 KO mice,but inhibition of Rictor does not affect ASD-like behavior.Part Three Regulatory role of Rictor in the remodeling of hippocampal actin cytoskeleton in Ctnnd2 KO miceObjective:To investigate whether Rictor affects spatial cognition by regulating the dynamic changes of actin cytoskeleton in Ctnnd2 KO mice.Methods:1.Groupings:WT+Vector group,KO+Vector group,WT+Sh Rictor group,KO+Sh Rictor group.2.WB was used to detect the expression changes of Rictor and p-AKT in hippocampal region.3.The expression of profilin-1 and Cofilin,the regulatory proteins of actin cytoskeleton polymerization were detected by WB.4.WB was used to detect the ratio of F-actin/G-actin in mice hippocampus.5.Golgi silver staining was used to detect the density of dendritic spines in hippocampal CA1 region.Results:1.In Ctnnd2 KO mice,the expression of Rictor(p<0.01)and p-AKT(p<0.05)was further decreased.2.Inhibition of Rictor in the hippocampus of Ctnnd2 KO mice further reduced the expression of Profilin-1(p<0.05),the regulatory protein of actin cytoskeleton polymerization,and there was no significant difference in the expression of Cofilin(p>0.05).3.In Ctnnd2 KO mice,the ratio of F-actin/G-actin in the hippocampus decreased further after inhibiting Rictor(p<0.05).4.In Ctnnd2 KO mice,the density of dendritic spines in hippocampal CA1region decreased further after inhibiting Rictor(p<0.05).Conclusion:Rictor is a key regulator of the dynamic changes of the actin cytoskeleton in the hippocampal neurons of Ctnnd2 KO mice,affecting its structural synaptic plasticity.Part Four Role of Rictor in regulating synaptic density and related proteins in Ctnnd2 KO miceObjective:To investigate whether Rictor affects spatial cognition by regulating the expression of synaptic density,postsynaptic membrane thickness and synaptic related proteins in Ctnnd2 KO mice.Methods:1.Groupings:WT+Vector group,KO+Vector group,WT+Sh Rictor group,KO+Sh Rictor group.2.The changes of synaptic density and postsynaptic membrane thickness in hippocampal CA1 region were detected by transmission electron microscopy.3.WB was used to detect the changes in the expression of synapse-related proteins in the hippocampus of mice.Results:1.In Ctnnd2 KO mice,the synaptic density in hippocampal CA1 region was further decreased after inhibiting Rictor(p<0.05).2.In Ctnnd2 KO mice,the postsynaptic membrane thickness of hippocampal CA1 region was further decreased after inhibiting Rictor(p<0.05).3.After inhibiting Rictor in the hippocampus of Ctnnd2 KO mice,the expressions of synapse-related proteins Gl UR1(p<0.01)and ELKs(p<0.01)were further decreased,and there was no significant difference in the expression of Synapsin-1(p>0.05).Conclusion:Rictor is a key regulator of synaptic density and postsynaptic membrane changes in the hippocampus of Ctnnd2 KO mice,which is involved in the regulation of spatial cognition.