Effects Of Hippocampal Neurogenesis Promotion On Social Deficits In Autism Mice Model And Its Underlying Mechanisms

Background:Autism is a group of severe neurodevelopmental disorders,the prevalence of which is about 1:59 and is increasing year by year.The typical clinical manifestations of autism are speech difficulties,social interaction disorders,repetitive behaviors,and narrow and intense interest abnormalities.The severity of symptoms varies from mild to disabling depending on the level of individual development.People with autism often have other psychiatric symptoms such as attention deficit disorder,anxiety,tics,sleep disorders and epilepsy.At present,symptoms are improved mainly through behavior correction,and there is no effective drug for prevention and treatment.The etiology of autism is complex,and thousands of related pathogenic genes have been confirmed,including MECP2,SHANK3,MEF2 C,ELF1,etc.The abnormality of a single target gene cannot be one-to-one corresponding to the clinical phenotype,making it difficult to become a breakthrough point in the treatment of autism.Clinical data show that autism often causes structural abnormalities in several important brain areas of the central nervous system,including cortex,hippocampus and cerebellum,which are also important pathological basis for clinical manifestations of autism.Improving developmental abnormalities in these structures is an important strategy for correcting autismlike behavior.Hippocampus,an important brain region with neurogenesis potential and high plasticity,affects learning,memory and emotion regulation.In humans and mammals,the subgranular zone(SGZ)in the hippocampus continues to produce neurons both postnatal and adult,with a small number of granular cells.These new neurons are highly plastic and can be integrated into neural circuits to affect higher brain functions.Promoting hippocampal neurogenesis can enhance learning and memory,as well as improve anxiety and depression-like behavior.It has been confirmed in human and autistic mice that hippocampal neurogenetic abnormalities are highly correlated with the occurrence of ASD,and the promotion of hippocampal neurogenesis can improve ASD to some extent.Therefore,strategies to promote hippocampal neurogenesis may be a new way to modify autism-like behavior.Estrogen receptor ? is the dominant estrogen receptor distributed in the brain.Significant down-regulation of ER? and aromatase(estrogen-synthesis rate-limiting enzyme)was detected in the cerebral cortex of autistic patients,suggesting that ER is down-regulated as a high-risk factor for autism.Endogenous ER? activation promotes neurogenesis,enhances hippocampal plasticity,and improves cognitive function in hippocampal dentate gyrus.Invitro studies have shown that ER? promotes proliferation and differentiation of neural stem cells.It has been confirmed that ER is highly elpressed in the hippocampal dentate gyrus during early hippocampal development,and whether ER? activation in the hippocampal dentate gyrus can improve autistic behavior by promoting neurogenesis remains to be further clarified.Stem cell transplantation is widely used in regenerative medicine.Exogenous stem cells can not only serve as an important supplementary pathway of cell origin,but also promote the proliferation and differentiation of endogenous neural stem cells through autocrine or paracrine action,which has a broad application prospect in the treatment of central nervous system diseases.Human amniotic epithelial cells(hAEC)isolated from placenta are unique and ideal cell sources with multipotent differentiation and low risk of graft rejection and tumorigenesis.HAEC transplantation can repair brain injury and neurodegenerative diseases,but whether hAEC transplantation can improve autistic behavior by promoting neurogenesis in the hippocampal dentate gyrus,and the regulatory mechanism of promoting neurogenesis in the hippocampal dentate gyrus is still unclear.To elucidate the effect of ER agonist treatment and hAEC transplantation on the improvement of autism-like behavior is expected to elucidate the mechanism of autism treatment from the hippocampal neurogenetic pathway.Method:1.C57 and BTBR mice were first pretreated with ER agonist Ly3201 by intraperitoneal injection 2 days after birth.The expression of ER was detected in the cortex and hippocampus of mice at P14 to clarify the activation of ER by Ly3201.The expression of BrdU and DCX in hippocampal dentate gyrus was analyzed to clarify the regulation of Ly3201 on hippocampal neurogenesis.At the age of 2 months,three boxes of social interaction,grooming,opening,and elevated behaviors were tested to further clarify the effect of Ly3201 preconditioning on the improvement of ASD-like behaviors in the early postnatal period.2.BTBR mice were injected with human amniotic epithelial stem cells(hAEC)in the lateral ventricle 60 days after birth(P60),and the control group was injected with volumetric preservation solution.One month after injection,three boxes of behavioral tests,such as social contact,grooming,opening,and elevated,were performed to determine the therapeutic effect of hAEC on autistic behavior in BTBR mice.The expression of BrdU and DCX in hippocampal dentate gyrus was analyzed to clarify the regulation of hAEC on hippocampal neurogenesis in BTBR mice.The double-labeled SOX2 and GFAP cells identified the nerve precursor cells of the gyrus dentate,and combined with the detection of Nestin,DCX,SOX2,BDNF and TrkB mRNA levels,the regulation of hAEC on the nerve precursor cells of the hippocampal gyrus was determined.The immunomodulatory effect of hAEC on hippocampus was determined by fluorescence quantitative PCR assay on IL6,IL1,IL10,NF-kB and TNF mRNA levels of microglia labeled with IBa1.Results:· Therapeutic effects of estrogen receptor agonists on autism model mice through hippocampal neurogenesis promotion(1)At P14,the expression level of ER in the hippocampus and cortex of BTBR mice was significantly lower than that of C57 mice.Ly3201 significantly increased ER expression in hippocampus and cortex of BTBR and C57 mice,suggesting Ly3201 pretreatment at P2 significantly activates endogenous ER.(2)In the three-chamber social test,BTBR mice showed typical social dysfunction,and ly3201 pretreatment significantly improved the social dysfunction of BTBR mice in the neonatal period.In the experiments of grooming and bead embedding,the time of grooming and bead embedding of BTBR mice was longer than that of C57 mice,but the repetitive behavior of BTBR mice was not improved after ly3201 treatment.Ly3201 pretreatment had no effect on the motor function and anxious behavior of BTBR mice,suggesting that ly3201 specifically improved the social behavior of BTBR mice.(3)Immunohistochemical staining of BrdU and DCX was used to reflect the neurogenesis of hippocampal dentate gyrus.The immunoreactive cells of BrdU and DCX in the hippocampal dentate gyrus of BTBR mice were significantly less than those in the C57 group,and ly3201 in the neonatal stage promoted the neurogenesis of the hippocampal dentate gyrus of BTBR mice.2.Therapeutic effect of human amniotic epithelial cells on autism model mice through hippocampal neurogenesis promotion(1)hAEC injection into the lateral ventricle of BTBR mice significantly improved the social dysfunction of BTBR mice after 1 month.There was no significant effect on repetitive behavior,motor function and anxious behavior of BTBR mice,suggesting that hAEC injection specifically improved the social deficits of BTBR mice.(2)Compared with C57 mice,neurogenesis in hippocampal dentate gyrus was inhibited,and the number of neural precursor cells was significantly reduced.hAEC injection significantly increased the immunoreactive cells of BrdU and DCX in the hippocampal dentate gyrus of BTBR mice,and the neural precursor cells labeled with both SOX2 and GFAP,suggesting that hAEC injection significantly increased the neurogenesis in the hippocampal dentate gyrus of BTBR mice.Fluorescence quantitative PCR revealed that hAEC injection significantly increased the expression of stem cell markers in the hippocampus of BTBR mice,while the levels of BDNF and TrkB were significantly increased,suggesting that hAEC promoted the neurogenesis of the hippocampal dentate gyrus by upregulating the level of BDNF in the hippocampus.(3)Immunlogical regulation can also affect the neurogenesis of the hippocampal dentate gyrus.Iba1 was used to label microglia,and the number of microglia in the hippocampal dentate gyrus of BTBR mice was significantly higher than that of C57 group.The hAEC treatment group had no significant effect on the Iba1 immunoreactive cells in the hippocampus of BTBR mice.In addition,the hAEC treatment group had no significant effect on the mRNA levels of IL1,IL6,IL10,TNF and NF-kB in the hippocampus of BTBR mice,suggesting that hAEC had no significant correlation with the regulation of hippocampal dentate neurogenesis and immune regulation in BTBR mice.Conclusions:The results suggest that ly3201 pretreatment and adult hAEC transplantation can specifically improve the social deficits of BTBR mice,and this effect is closely related to the promotion of hippocampal neurogenesis in BTBR mice,which provides a new way for the improvement of social behaviors.