Effects And Mechanism Of Overexpression Of MAD2B In Hippocampus On Learning And Memory Of Mice

Objective: Cognitive disorder(CD)can be found in aging or pathological conditions,and its main clinical features are spatial learning disorder and working memory disorder which reduced people's qualities of life.The hippocampus is the center of memory storage,and its structure and function plays important roles in learning and memory consolidation.It has been proved that hippocampus damage is closely related to the occurrence of cognitive disorder.Mitotic arrest deficient-like2(MAD2B)is a kind of mitotic arrest deficient protein which is widely expressed in hippocampus.High glucose can induce MAD2B expression and lead to neuronal damage in primary neurons.Meanwhile,the complex formed by MAD2 B and Single-minded 2(SIM2)can damage synaptic plasticity in Down's syndrome(DS).Although these studies suggested that expression of MAD2 B in neurons may result in neuronal damage,whether overexpression of MAD2 B would affect learning and memory function at animal level remains unclear.Therefore,this study intends to explore the effects and mechanism of overexpression of MAD2 B on learning and memory in mice.Methods: Forty four adult C57BL/6J mice were divided into control group and experimental group randomly,each group contains 22 mice.They received stereotaxic injections of equal amount and titer of the AAV-MAD2 B vector or control vector,AAV-GFP into the hippocampus.Three weeks later,to harvest hippocampal tissue,eight mice in each group were euthanized using cervical dislocation and the left and right hippocampi were extracted and frozen at-80 ?.Using immunofluorescence,Western Blot and qRT-PCR to confirm whether the overexpression of MAD2 B is successful and the injection position is correct.The rest mice were evaluated the effects of MAD2 B overexpression on the motor function,anxiety and depression and learning and memory ability by open field,rotarod test,elevated plus maze,tail suspension test,forced swimming test,Morris water maze testand contextual fear conditioning in turn.We further investigated the molecular mechanisms using RNA sequencing methods.Results:1.The immunofluorescence,Western Blot and qRT-PCR confirmed that the injection position was correct,and the expression of MAD2 B was increased by injection of MAD2 B AAV in the hippocampus.2.There are no difference in the total distance and average speed of the control group and MAD2 B overexpressing group in the open field.There are no difference in the retention time between the two groups in the rotarod test,which indicated that the overexpression of MAD2 B may had no effect on motor ability.3.There are no difference in the retention time between the two groups in the center zone and corner zone in the open field.Meanwhile,there are no difference in the entries of open arm and closed arm,as well as the retention time in the open arm and closed arm in the elevated plus maze.This indicates that MAD2 B overexpression may has no effect on anxiety in mice.4.In the tail suspension test and the forced swimming test,there are no difference in immobility time in MAD2 B overexpression group compared to the control group.his indicates that MAD2 B overexpression may has no effect on depression in mice.5.In the Morris water maze,the latency time to find the platform position throughout the training phase is similar between mice overexpressing MAD2 B and control mice.Moreover,there are no difference in the retention time in the target quadrant between two grops in the post-training probe trail.But in the reversal learning phase,mice overexpressing MAD2B spend more time to find the platform position,and they swim less total distance and spend less time in the target quadrant,as well as less times of crossing the target quadrant in the post-reversal learning probe trial;In contextual fear conditioning,mice overexpressing MAD2 B have shorter freezing time than the control mice.This suggests that MAD2 B overexpression may leads to a decline in learning and memory ability.6.The RNA sequencing results showed that MAD2 B regulates the expression of 134 genes,and Gene Ontology(GO)enrichment analysis and Kyoto Gene and Genomic Encyclopedia(KEGG)analysis shows that these genes are involved in a variety of biological processes.Conclusion: Overexpression of MAD2 B in hippocampus may leads to a decrease in learning and memory ability in mice by regulating the expression of multiple genes and various biological processes,which may plays an important role in regulating cognitive function.