Changes In Hippo Signaling Pathway Core Proteins At Different Time Points Of Postnatal Brain Development In VPA Autistic Mice

Objective: To investigate the role of Hippo signaling pathway core proteins in the postnatal brain development of Valproic acid(VPA)autistic mice and the mechanism of action.Methods: Adult C57BL/6 mice were caged 2:1male to female for 24 hours,observed for pubic bolus,and recorded as E(Embryonic,E)for 1 day after seeing pubic bolus,and females were housed separately in separate cages.The pregnant female rats were randomly divided into two groups,one group was injected intraperitoneally with 0.6 mg/g of VPA(prepared by 0.9% NaCl solution)on day E12.5 as the experimental group,and one group was injected intraperitoneally with an equal amount of 0.9% NaCl solution on day E12.5 as the control group.The offspring produced by female rats in the experimental group were VPA autistic mice;the offspring produced by female rats in the control group were recorded as normal control group.Postnatal(P)3 days,6 days and 9 days were considered as the early postnatal period and 8 weeks as the maturation period.Materials were taken at these four time points.Four mice in each group were extracted from prefrontal proteins,and the expression of MST1,LATS1,P-YAP-S127,and YAP,the core proteins of Hippo signaling pathway,were detected by protein immunoblotting in the prefrontal lobe of mice;three mice were perfused to take frozen sections of brain,and the expression of microglia in the prefrontal secondary motor cortex(M2)was detected by immunofluorescence,Three mice were perfused with frozen sections,and the distribution of YAP in microglia,neuronal cells and neuronal cells in the dentate gyrus(DG)of the hippocampus was localized and analyzed by immunofluorescence.Results: The results of protein immunoblotting showed that the expression of core proteins LATS1,MST1 and P-YAP-S127 of Hippo signaling pathway in VPA autism mice decreased with the increase of mouse age;there was no significant difference in the expression of YAP in the early postnatal period and significantly decreased in the mature period;compared with the control group,the expression of core proteins LATS1,MST1 and P-YAP-S127 of Hippo signaling pathway in VPA autism mice increased in the early postnatal period.MST1 and P-YAP-S127 expressions were increased in the early postnatal period compared with the control group,while the expressions of LATS1,MST1 and P-YAP-S127 in the Hippo signaling pathway core proteins were not significantly different in the mature stage;there was no significant difference in the expression of YAP protein in VPA autistic mice.Compared with the control group of immunofluorescence results: the number of microglia in VPA autistic mice increased and the percentage of YAP in the nucleus increased at P3 d and P6 d,and no significant difference at P9 d and P8w;the number of neuronal cells in the prefrontal M2 area decreased and the percentage of YAP in the nucleus increased;the number of newborn neurons in the DG area increased at P6 d and decreased at P9d;concomitant with P3 d,the percentage of YAP in the nucleus of newborn neurons decreased,while the percentage of YAP in the nucleus of newborn neurons increased at P6 d and P9 d,and there was no significant difference at P8 w.Conclusions:(1)Hippo signaling pathway inhibits microglia repopulation in VPA autistic mice in the early postnatal period;(2)Hippo signaling pathway adjusts the number of neurons by regulating the number of neonatal neurons in VPA autistic mice.