Effects Of YAP Gene Deletion In Pericytes Of Adult Mice On Cognition And Fear Memory

Objective:This paper’s purpose is clearing YAP gene expression in central nervous system of adult mice,By constructing a mouse model that conditional knockout YAP gene in pericytes during adulthood,and revealing the role and mechanism of learning cognition ability and fear memory on account of YAP gene in pericytes of adult mice.This study provides a new target and direction for the prevention and treatment of neuropsychiatric diseases caused by cognitive impairment and emotional disorders.Methods:1.We detected the expression of YAP gene in pericytes（PDGFRβ⁺cells）,endothelial cells（CD31⁺cells）,OPCs（PDGFRα⁺cells）and neurons（NeuN⁺cells）of adult mice by immunofluorescence staining.2.We constructed the conditional YAP gene knockout model of adult mice pericytes by Cre-lox P gene recombination technology.Then we used Western Blot technology to detect the protein levels of YAP gene in YAP1^ng2-CKO experimental group and its counterpart control YAP1^fl/flgroup.In addition,we further detected the expression of YAP gene on the mice pericytes of two groups by immunofluorescence staining.3.We selected 4-month-old mice as the research objects in YAP1^ng2-CKO group and YAP1^fl/flgroup,and excluded the effects of YAP gene deletion in adult pericytes on motor function and anxiety behavior of mice by detecting the average speed and distance,time spent in the central region and times of crossing the central region in the open field.We also further analyzed the effect of YAP gene deletion in adult pericytes on learning and memory of mice was analyzed by detecting the exploration time of mice to new and old objects;in additon,we analyzed the effect of YAP gene deletion in adult pericytes on the spatial memory of mice by detecting the exploration time of mice to new and old objects.4.We excluded the effect of YAP gene deletion in adult pericytes on sensory function of mice by detecting the time of pain behavior on metal plate at 52℃in YAP1^ng2-CKO mice and YAP1^fl/flmice.We also further analyzed the effect of YAP gene deletion in adult pericytes on fear memory of mice by detecting the time ratio of freezing behavior at 1 day and 28 days after fear experience.5.We selected 4-month-old mice to make hippocampal tissue sections in YAP1^ng2-CKO group and YAP1^fl/flgroup,and detected the effect of YAP gene deletion in adult pericytes on the proliferation of oligodendrocyte precursor cells（OPCs）in ventral and dorsal hippocampal by BrdU and PDGFRαimmunofluorescence staining.Results:1.The results of immunofluorescence staining showed that YAP positive signal was mainly detected PDGFRβ⁺and CD31⁺cells in the brain of adult mice;however,it cannot be detected in PDGFα⁺and NeuN⁺cells.These results indicated that YAP was expressed in pericytes and endothelial cells in the brain of adult mice,but not in OPCs and neurons.2.To test the specific knockout efficiency of YAP gene in adult pericytes.Western Blot results showed that YAP protein levels in cortex and hippocampus of mice in the YAP1^ng2-CKO mice were significantly lower than those in the YAP1^fl/flmice（P<0.01）.Immunofluorescence staining results showed that the expression of YAP positive signal in PDGFRβ⁺cells of mice’s brain was significantly reduced in the YAP1^ng2-CKO mice,which suggested that the model with YAP gene knockout in pericytes of adult mice had been constructed.3.To investigate the effect of YAP gene deletion in adult mice pericytes on learning and cognition ability.The results of the new object recognition test showed that the time of exploring new object in YAP1^ng2-CKO mice was significantly reduced compared with that in YAP1^fl/flmice,the new object recognition index of the YAP1^fl/flmice was 0.66±0.13,while the new object recognition index of the YAP1^ng2-CKO mice was 0.48±0.08.It was suggesting that the YAP1^ng2-CKO mice had learning and cognition ability impairments.Besides,the results of the new position recognition test showed that the time of exploring new position object in YAP1^ng2-CKO mice was much lower than that in YAP1^fl/flmice,and the new position object recognition index of the YAP1^fl/flmice was 0.64±0.08,while the new position object recognition index of the YAP1^ng2-CKO mice was 0.43±0.09,that is to say,the YAP1^ng2-CKO mice had obvious spatial learning and memory impairments.4.To investigate the effect of YAP gene deletion in adult mice pericytes on fear memory.The fear conditioning test results showed that the time ratio of freezing behavior in the YAP1^ng2-CKO mice was obviously lower than that in the YAP1^fl/flmice at 1 day after fear experience（P<0.01）.At 28 days after the fear experience,the time ratio of freezing behavior in the YAP1^ng2-CKO mice was distinctly lower than that in the YAP1^fl/flmice（P<0.01）.In addition,the time ratio of freezing behavior in YAP1^ng2-CKO mice after 28 days was significantly lower than that after 1 day（P<0.05）.These results indicate that YAP1^ng2-CKO mice have significant fear memory impairments.5.To investigate the effect of YAP gene deletion in adult pericytes on the proliferation of OPCs in the ventral and dorsal hippocampus of mice.Immunofluorescence staining results showed that:BrdU⁺/PDGFRα⁺double positive cells numbers in dorsal and ventral hippocampus in the YAP1^ng2-CKO mice were clearly less than those in the YAP1^fl/flmice（P<0.01）,which suggested that OPCs proliferative capacity in dorsal and ventral hippocampus had reduced in the YAP1^ng2-CKO group.These results indicated that YAP gene in pericytesis an essential transcriptional coactivator for the regulation of OPCs proliferation.Conclusion:YAP was expressed in pericytes and endothelial cells in the brains of adult mice,but not in OPCs and neurons.After adult mice pericytes were specifically knocked out YAP gene,their learning cognition ability and fear memory extraction impaired,and the proliferation of OPCs in the ventral and dorsal hippocampus also damaged.Therefore,we concluded that YAP gene in pericytes is an essential transcriptional coactivator that regulates the proliferation of OPCs and thus affects the learning,cognition and fear memory of mice.