The Study Of HCMV IE2 Effect On Mood And Cognition In UL122 Transgenic Mice

Infection with human cytomegalovirus(HCMV)increases the risk of a range of neurological abnormalities,including mood disorders and cognitive decline.FGF2 is a member of the fibroblast growth factor family involved in early brain development and repair of the adult brain.There is growing evidence that FGF2 protein plays a key role in the development of mood and cognitive disorders by regulating neurogenesis and synaptic plasticity.Mature neuronal marker(NeuN)and astrocyte marker(GFAP)involving in cognitive and emotional regulation have important effects on neurodevelopment and hippocampal synaptic plasticity.IE2 is a 579 amino acid protein encoded by gene UL122;it is the most important protein in HCMV incubation period and replication.The mechanism of IE2's role in HCMV-induced mental illness has not been elucidated,although there is considerable evidence that HCMV infection is closely related to mood and cognitive impairment.HCMV has strict host species specificity,which is the main obstacle to the study of the pathogenicity of HCMV in vivo.Current studies are mainly conducted at the cellular and molecular levels.Objective:In order to overcome the limitation of species specificity of HCMV,we constructed a transgenic mouse model of UL122.Using this transgenic mouse model that can continuously and stably express IE2 protein,a series of animal behavior experiments were conducted on the whole level to investigate whether the continuous expression of IE2 can cause emotional and cognitive impairment.To study whether the continuous expression of IE2 affects the expression of FGF2,NeuN and GFAP and the morphological development of hippocampal neurons in mice at the tissue and cell levels.And to examine whether synaptic plasticity,which is closely related to mood and memory,is impaired.Methods:1.Immunohistochemistry and PCR were used to identify the transgenic mouse model of UL122 expressing IE2 protein.UL122 transgenic positive mice were selected as the experimental group and C57BL/6 wild type mice as the control group.Each group is equal to 16.2.The open mine(OF),elevated cross maze(EPM),and tail suspension(TST)experiments were used to measure anxiety and depression-related mood disorders.3.Morris water maze(MWM)and new object recognition(NOR)explored the cognitive behavior of mice.4.Illumina Hiseq platform was used for microarray analysis to find differentially expressed genes by comparing the expression profiles of the two groups of mice.5.HE staining was used to observe the effect of IE2 on hippocampal neuron morphology.6.The expressions of IE2,NeuN,GFAP and FGF2 mRNA levels in hippocampus were detected by real-time fluorescence quantitative PCR.7.Western Blot was applied to study the protein levels of FGF2,GFAP,NeuN,PSD-95 and SYP in the hippocampus of mice.8.IHC method was used to detect the expression and distribution of FGF2,GFAP and NeuN in the hippocampus of mice.Results:1.PCR and IHC results showed that the model of UL122 transgenic mice was successful.2.Emotional behavior: open field test results: the experimental group in the central area of the time significantly lower than the control group,and the experimental group have a longer-term a period of stagnation in the peripheral time,the differences are significant(P<0.001);Results of the elevated plus-maze test: mice of IE group spent very short period of time in the open area and spent significantly more time in the closed area(P<0.001).Results of tail suspension test: Compared with the NC group,the UL122 group have longer immobility period(P<0.001).3.In terms of cognition behavior,Morris water maze: compared with the control group,the number of times of platform crossing was less in the experimental group(P<0.001),but there was no significant difference in average speed and escape latency between the experimental group and the control group(P>0.05).New object recognition experiment: compared with the control group,transgenic mice showed no difference in overall exploration,indicating that there was no spontaneous bias(P>0.05).The recognition index was sharply down-regulated in the positive group(P<0.001).4.Illumina Hiseq platform microarray data analysis showed that,compared with the control group,the neurogene-related genes Fgf2,Foxo3,Cdkl3,Apoe,Cpeb3,Etv1,Ilk and Mef2 c in the hippocampus region of the IE group were diminished at the mRNA level(P<0.001).5.HE staining consequence reveal that the hippocampal neuron structure of the experimental group was derangement and sparse,some neurons were damaged,and the intercellular space was larger than that of the control group.6.The results of fluorescence quantitative PCR showed that the mRNA levels of NeuN,GFAP and FGF2 in the hippocampal tissues of the experimental group were negatively correlated with IE2,and their mRNA levels decreased with the increase of IE2 mRNA levels(P<0.001).7.Western Blot results showed that the experimental group expression levels of FGF2,GFAP,NeuN,PSD-95 and SYP were dropped significantly(P<0.001).8.Immunohistochemical results showed that the IE positive group expression levels of IE2,FGF2,GFAP and NeuN proteins were markedly decreased(P<0.001).Conclusions:1.In the animal behavior experiment of UL122 transgenic mice,they showed depression,anxiety and cognitive impairment,thus indicating that there is a close relationship between HCMV IE2 and emotional cognitive function.2.The decreased expressions of FGF2,NeuN and GFAP in UL122 transgenic mice affect the function of synaptic plasticity,and synaptic plasticity is closely related to the development of the nervous system,thus suggesting that HCMV IE2 may cause depression,anxiety and cognitive impairment by inhibiting synaptic plasticity.In this study,the establishment of the UL122 genetically modified mice model overcame species specificity,and it was concluded that HCMV induced emotional and cognitive abnormalities may be realized by IE2 proteins indirectly affecting the function of synaptic plasticity by down-regulating the expression of FGF2,NeuN and GFAP.This study will provide new ideas for further elucidating the molecular mechanism of HCMV IE2 causing emotional and cognitive abnormalities,and it will also lay a theoretical foundation for the drug development of antidepressants and cognitive degenerative diseases.