| Objective:In this study,we constructed a juvenile rat disease model with the core symptoms of Autism spectrum disorder(ASD),and detected synaptic development in prefrontal cortex,microglia polarization phenotype and the expression of trigger receptor expressed on myoid cells 2(TREM2)in the critical period of nervous system development,to explore the effectss of targeted regulation of Trem2 on microglia polarization in the early stage of neurodevelopment,and to preliminarily study its role in synaptic development and the possible molecular mechanisms,so as to provide a theoretical basis for supplementing the pathogenesis of ASD and exploring targeted therapy.Methods:1.In vivo experiments: The neonatal rats exposed to valproic acid(VPA)before delivery were taken as the research subjects to observe the growth and development,behavioral characteristics and synaptic development of the neonatal rats:(1)On the12.5th day of pregnancy Embryonic day(E),the pregnant Wistar female rats were randomly divided into two groups: the model group(VPA): a single intraperitoneal injection of 600mg/kg VPA solution,and the neonatal rats produced were recorded as VPA group;Control group(Con): Rats were intraperitoneally injected with the same dose of normal saline,and the neonatal rats were recorded as Con group.The growth and development of neonatal rats in each group were monitored,including postnatal survival rate,tail deformity rate,body weight gain,eye opening test,negative orientation test and swimming test.Behavioral tests: social behavior,open field test,elevated cross,self-grooming test,bead burying test,and water maze;(2)On postnatal day PN7 and PN28,prefrontal cortex(PFC)was taken to detect the protein expression and relative m RNA content of synapse-related proteins PSD-95,SYN,and Gephyrin by Western blot,real-time polymerase chain reaction(q RT-PCR),and immunohistochemistry,and synaptic ultrastructure was observed by transmission electron microscopy;2.The microglia polarizing phenotype and TREM2 expression were detected in PN7 and PN28:(1)Enzyme-linked immunosorbent assay(ELISA)was used to compare the expression levels of inflammatory factor IL-1β and anti-inflammatory factor IL-4between the two groups.(2)Western blot and q RT-PCR were used to detect the protein expression levels and m RNA relative contents of M1 microglia marker CD86 and M2 microglia marker CD206,and immunofluorescence was used to verify colocalization and expression.(3)The protein levels and m RNA relative contents of TREM2 and DAP12 were determined by 3)Western blot and q RT-PCR,and colocalization and expression were verified by immunofluorescence assay.3.In vitro experiments: Primary neurons and microglia of neonatal rats in Con and ASD groups were isolated and cultured.Transwell mode was adopted for coculture and the microglia were transfected with adenovirus vector to establish TREM2 overexpression and interference model,which was divided into five groups: Con group,ASD group,ASD+Trem2-NC group,ASD+Trem2-OE group and ASD+Trem2-si RNA group:(1)ELISA was used to detect the expression levels of inflammatory factor IL-1β and anti-inflammatory factor IL-4 in each group;(2)Western blot and q RT-PCR were used to compare the expression levels of M1 microglia marker CD86 and M2 microglia marker CD206 in each group,and double-labeled immunofluorescence was used to observe the co-localization of CD86 and CD206.(3)Western blot was used to detect the expression levels of Synapse-related proteins PSD-95,syn and Gephyrin,and the expression changes of p-p38,p38,p-Elk-1 and Elk-1 proteins.Result:1.Prenatally VPA exposure can induce neonatal rats to replicate the core symptoms and behaviors of ASD:(1)The VPA group had high post-natal mortality,high rate of tail deformity,abnormal growth and development,and delayed nervous system development;(2)Behavioral test results showed that compared with Con group,VPA group had social dysfunction(P<0.05)with social novelty defect(P<0.05),significant anxiety,repetitive and rigid behaviors(P < 0.001),and decreased learning,memory and cognitive levels((P<0.001).2.Prenatally VPA exposure resulted in abnormal synaptic development in neonatal rats:(1)Western blot and q RT-PCR showed that the protein expression and m RNA content of PSD-95 in PN28,Con group and VPA group were higher than those in PN7(P<0.05),and the relative content of SYN m RNA in VPA group was higher than that in PN7(P < 0.05).The results of comparison between the two groups showed that PN7 lasted until PN28,and the relative contents of PSD-95,SYN protein expression and m RNA in the VPA group were significantly higher than those in the Con group(P<0.05).The m RNA level of Gephyrin was lower than that in the Con group,and the differences between the groups were statistically significant(P<0.05).(2)It was observed under the transmission electron microscope that the synaptic structure in the VPA group was vague and crowded,and the postsynaptic membrane was not clear at all stages.On PN28,the width of postsynaptic dense substance in the VPA group was not significantly increased as compared with that in PN7,and the width of synaptic cleft was significantly lower than that in the Con group(P<0.05).(3)Immunohistochemistry showed that in PN28,the PSD-95 protein of VPA group was significantly higher than that of Con group(P < 0.01),and the content of Gephyrin was significantly lower than that of Con group(P<0.05).3.Detection of microglia polarization and the expression levels of TREM2 and DAP12 in Con and ASD groups:(1)ELISA showed that in PN28,the expression of IL-1β in ASD group was significantly higher than that in Con group(P < 0.05).(2)Western Blot and q RT-PCR results showed that on PN28,compared with that onPN7,the expression of CD86 protein was down-regulated in Con group(P<0.05),and the relative content of CD206 m RNA was increased(P<0.001).Compared with that on PN7,the expression of CD86 protein and m RNA in ASD group were significantly increased(P < 0.01),and the expression of CD206 protein was significantly decreased(P<0.05).Compared with Con group,the protein expression and m RNA level of CD86 in ASD group were significantly increased(P < 0.01),while the protein expression and m RNA relative content of CD206 were significantly decreased(P<0.01).The differences between the groups were statistically significant in PN28.(2)Immunofluorescence showed that both CD86 and CD206 were colocated in microglia.On PN28,the expression level of CD86 in ASD group was higher than that in Con group(P< 0.05),and the expression level of CD206 was lower than that in Con group(P<0.01).(3)The results of TREM2 assay showed that the expression levels of Trem2 and DAP12 in the Con and ASD groups were increased significantly from PN7 to PN28.Compared with Con group,the protein expressions of TREM2 and DAP12 in ASD group were low at each stage(P<0.05),and the m RNA level was significantly lower than that in Con group(P<0.01).(4)Double-labeling immunofluorescence showed that TREM2 and DAP12 were colocated and highly expressed in microglia.On PN28,the expressions of TREM2 and DAP12 in ASD group were significantly lower than those in Con group(P<0.05).4.Cell experiment:(1)Microglia cells were transfected by adenovirus cell transfection technology to construct TREM2 overexpression and interference model;(2)ELISA showed that compared with the ASD group,the pro-inflammatory factor IL-1β was significantly decreased(P < 0.001)and the anti-inflammatory factor IL-4expression was increased(P<0.05)in the ASD+Trem2+OE group.(3)Western Blot showed that the expression level of CD86 protein in the ASD+Trem2+OE group was significantly lower than that in the ASD group(P<0.05),and the expression level of CD86 protein in the ASD+Trem2+si RNA group was significantly higher than that in the ASD group(P< 0.05).The expression level of CD206 was the highest in the ASD+Trem2+OE group,and the lowest in the ASD+Trem2+si RNA group,and the differences between groups were statistically significant(P < 0.05).(4)Immunofluorescence results showed that both CD86 and CD206 were co-located with Iba-1 and highly expressed in microglia.CD86 was expressed highest in the ASD+Trem2+si RNA group,while CD206 was expressed highest in the ASD+Trem2+OE group,with a statistically significant difference between groups(P< 0.05).(5)The results of synaptophysin-related detection showed that compared with Con group,the expression of Gephyrin protein in ASD group was significantly reduced(P<0.01).The expression of Gephyrin protein in ASD+Trem2+OE group was significantly higher than that in ASD group(P<0.05).The expression level of Gephyrin protein in ASD+Trem2+si RNA group was the lowest in all groups(P<0.05).In the ASD+Trem2+si RNA group,the expression level of PSD-95 was higher than that in Con group(P<0.01),and the expression level of SYN was significantly up-regulated as compared with those in Con groups(P<0.001).(6)Compared with Con group,the phosphorylation of p38(p-p38)was significantly increased in ASD group,and the differences were statistically significant(P < 0.01);the phosphorylation of Elk-1(p-Elk-1)was significantly increased(P<0.05);the p-p38 and p-p38 were significantly down-regulated in ASD+Trem2+OE group as compared with that in ASD group,and the differences between groups were statistically significant(P<0.05)..Conclusion:1.Prenatally VPA exposure cause synaptic dysplasia and autistic-like behavior in the neonatal rats;2.Compared with the control group,the expression of TREM2 in the PFC of neonate rats which exposured to prenatal VPA and was signifficantly downregulated and the microglia tended to activate M1;3.Overexpression of TREM2 can promote microglia to incline to M2 polarization,promote phagocytosis,and improve the development of synaptophysin;4.TREM2 regulates microglia by inhibiting p38/Elk-1 pathway. |
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