Liver X Receptor ? Regulates Early Development Of Hippocampus And Autistic-like Behaviors In The Mice

Background:Autism spectrum disorder?ASD?is a group of severe developmental disorders,including autism,asperger's syndrome,childhood disintegrating disorder,and unclassified pervasive developmental disorders.The clinical characteristics of ASD include impaired social interaction and communication,as well as restricted interests and repetitive behaviors.The causes of the ASD are very complex,including both environmental and genetic factors.About one thousand genes that are known to contribute to autism,but how these genes interact with environmental factors remains unclear.Recently,several lines of evidence have indicated that the deficits of neurogenesis may be associated with ASD.Throughout life,the active neurogenesis is mainly found in the subventricular zone?SVZ?and subgranular zone?SGZ?of mammalian brain.Abnormalities found in these regions may affect brain functions and result in neuropsychiatric disorders.The hippocampus is closely related to higher cognitive functions of the brain,such as learning and memory,and plays an important role in the regulation of emotions.Abnormal hippocampal neurogenesis is closely related to cognitive dysfunction caused by alzheimer's disease and may be involved in the occurrence of anxiety-like and depression-like behaviors.Pathological changes in autism occur in multiple brain regions of the central nervous system,and hippocampus is one of the major brain regions.One clinical study showed that autistic patients had a significant anatomical abnormality in the area dentata?AD;DG+CA4?.Another clinical study demonstrated that autistic teenagers?aged 12 to 18 years?had larger amygdala and hippocampus compared to the healthy controls.Additionally,one autopsy study found that multiregional abnormalities in neuronal migration,maturation and neurogenesis?including dentate gyrus,DG?in the ASD brain.Recently,many autistic mouse models have shown that there are significant changes in the neurogenesis of the hippocampus.Notably,new neurons continuously generated in the subgranular area of the hippocampal gyrus and can be integrated into the functional loop,thus providing an important pathway for functional repair and reconstruction.Promoting hippocampal neurogenesis has been shown to enhance cognitive functions such as learning and memory,and to improve anxiety-like and depression-like behaviors.Elucidation of the pathways and mechanisms of abnormal neurogenesis is helpful to understand the pathogenesis of autism.Due to its strong plasticity,the hippocampal DG may be new target for for the intervention and treatment of ASD.Liver X receptors?LXRs?belong to superfamily of nuclear receptors which are ligand-activated transcription factors,including LXR?and LXR?.Mounting of data have demonstrated that LXRs can modulate the lipid homeostasis and metabolism,and inflammatory responses.It has been demonstrated that LXR?expression is mainly expressed in lipid tissues,while LXR?is mainly expressed in the young and adult rodent brain.LXR?plays an important role in the formation of lamellar structures such as cerebral cortex,cerebellar cortex and spinal cord gray matter.Loss of LXR?could result in thinning of cerebral cortex and obstacles in migration,differentiation and maturation of neurons;developmental deficit in cerebellar purkinje cell and the migration of granule neurons;abnormalities in development and maturation of gray matter interneurons in spinal cord.LXR?affects the development of lamellar structure through regulating radial glial cells?Radial glia cells,RGCs?.Our another study showed that LXR?was involved in white matter development and central nervous system myelin formation by improcving RGC differentiation into oligodendrocytes,and the absence of LXR?resulted in abnormal motor function in mice.Hippocampal DG has a typical lamellar structure.During the formation of DG,RGC mediates the generation and migration of neural precursor cells and neurons in the early stage,and is also responsible for the formation of granular cell layer and subgranular zone in DG in the later stage.The neural progenitor cells retained in the subgranular area of postnatal and adult hippocampal DG come from the early RGC differentiation,which provides the hippocampus lifelong neurogenesis potential.While LXR?has an important role in the development and differentiation of RGC,it can be speculated that LXR?is involved in the lamellar development of the hippocampal DG,and the mechanisms have not been reported.In the present study,we first found that LXR?deletion can lead to early hippocampal development disorders in mice and affect the generation,differentiation and maturation of RGC.Autistic behavior changes were observed in adult LXR?-knockout mice.Activation of LXR receptors with LXR agonist TO901317 significantly alleviated social behavior deficits and reduced repetitive and stereotyped behaviors in autistic mouse models,and to some extent repaired hippocampal neurodevelopmental deficits.Methods and results:1.Mechanism of LXR?regulating the early development of hippocampal DG in mice?1?The BLBP was used to labele RGCs in the hippocampus of E15.5 and E18.5 mice,and it was found that the BLBP-labeled RGCs in E15.5 wild type?WT?mice were enriched in the fimbriodentate junction?FDJ?,and the LXR?deletion significantly reduced the number of RGCs in FDJ.In the hippocampal dentate gyrus of E18.5 WT mice,BLBP positive cells were mainly located in the hippocampal fissure,and a rich migration flow of BLBP positive cells across the hilus was observed,while the LXR?deletion caused a decrease in the number of BLBP positive cells in the hippocampal fissure of mice,and decreased the expression of the migration flow of BLBP positive cells across the hilus.Ki67 immunofluorescence staining showed that LXR?deletion caused a decrease in proliferating cells in the dentate gyrus.BrdU labeling showed that LXR deletion caused the block of the migration of neuronal precursor cells from VZ to DG.GFAP immunofluorescence staining was used to mark the transient neurogenic region?SPZ?enriched by RGCs in the hippocampal dentate gyrus of P2 mice.The loss of LXR?caused a reduction of SPZ area and the number of RGC.Using Tbr2 to lable intermediate progenitor cells in the hippocampal DG,it was found that LXR?deletion caused a decrease in the number of Tbr2 positive cells in P2 and P7 DG,and a large number of Tbr2positive cells were blocked in the granule layer at P7.Prox1 was used to labele granule neurons in DG,and LXR?deletion significantly reduced the number of prox1-labeled granular neurons.?2?WB technology was used to screen out the signaling pathways.Smad4?Reelin?P27^kip1?pERK1/2and ERK1/2 did not change in the knockout mice.Notch1 immunofluorescence staining showed higher expression of Notch1 in dentate gyrus of wild mice,while lower expression in LXR?KO mice.The expressions of Jagged1,NICD,BLBP and Hes1 were detected by WB and they were all decreased in the KO mice.WT mice were intraperitoneally injected with DAPT and TO901317,and results showed that the neurogenesis promotion effect of TO901317 was inhibited by DAPT.?3?Mice received BrdU injection intraperitoneally at P5,P6 and then brain tissue samples were collected at P14.BrdU,GFAP and Prox1 immunofluorescence staining was used to assess the differentiation of neural precursors.Data showed LXR?knockout could promote the differentiation of neural stem cells to glial cell,and inhibit the differentiation to granule neurons.2.LXR?deficiency leads to autistic behavior in mice?1?In the second phase of the three-chamber social test,WT mice spent more time exploring novel mice,while LXR?KO mice spent about eaqual amount of time exploring novel mice and new objects.In the third phase of the social test,WT mice spent more time exploring unfamiliar mouse,while LXR?KO mice spent about eaqual amount of time exploring familiar mouse and unfamiliar mouse.?2?Morris water maze test showed that there was no difference in the time of finding the platform between LXR?KO mice and WT mice at the training stage?day 1-5?,and no difference in the number of times crossing the platform between the two groups at the detection stage?day 6?.However,in the reversal Morris water maze,LXR KO mice spent significant more time in finding the plateform than WT mice.Grooming experiments showed that LXR?knockout significantly increased the grooming time of mice.?3?Novel object recognition and location recognition experiments showed that LXR?KO mice showed no significant difference in recognition index compared to the wild group.Tail suspension and forced swimming tests showed no difference in the immobility time between LXR?KO mice and WT mice.Olfactory tests showed no difference in the time to find food between LXR?KO mice and WT mice.3.Effect of LXR?deletion on hippocampal gene expression in mice?1?RNA samples were submitted to perform quality analysis and it meet the detection standards.?2?The sequencing results showed 717 differentially expressed genes in the hippocampus of mice induced by LXR?knockout.By comparing these differentially expressed genes with the database of genes related to autism,19 genes were found.Real-time PCR technology was applied to verify 8 genes of them,and 6 genes had good consistency.?3?GO analysis of differentially expressed genes showed that LXR?knockout resulted in up-regulation of many immune-related genes in the hippocampus of mice,and down-regulation of many genes related to lipid metabolism.?4?Pathway analysis of differentially expressed genes showed that LXR?knockout could lead to up-regulation of pathways related to cell adhesion molecules and down-regulation of signal pathways related to steroid synthesis.4.Liver X receptor agonists could improve autism-like behaviors of mice?1?Intraperitoneal injection of TO in mice had no effect on the expression of LXR?in the hippocampus,while the expression of LXR?,ABCA1 and ABCG1 increased significantly.?2?In three-chamber social test,WT mice spent more time exploring novel mice,while BTBR mice spent the same amount of time exploring novel mice and novel objects.After TO injection,the social interaction of WT mice was not affected,while the time of BTBR mice to explore novel mice increased significantly.In grooming experiment,the grooming time of mice in the BTBR group was significantly increased compared with that in the WT group,while it was significantly decreased after TO treatment.?3?In the social interaction experiment,mice in the VPA exposure model group spent the same time exploring novel objects and unfamiliar mice,and TO injection could improve the social ability of VPA exposed mice.The social novelty preference experiment showed that there was no difference in the time of exploring familiar mice and strange mice in the VPA exposure model group,while the VPA exposed mice spent more time exploring strange mice after TO treatment.Compared with the control group,the grooming time of VPA exposed mice was significantly increased,and TO could reduce the grooming time of VPA exposed mice.?4?BrdU and DCX staining showed that the number of BrdU and DCX positive cells in the hippocampus of BTBR mice was significantly reduced compared with that of WT mice,and the number of positive cells could be significantly increased by TO treatment.?5?BrdU and DCX staining showed that the number of BrdU and DCX positive cells in the hippocampus of VPA-exposed mice was significantly lower than that of the control group,and the number of positive cells could be significantly increased by TO treatment.Conclusions:The above experimental results showed that:LXR?could regulate early hippocampal development through Notch1,and the absence of LXR?could result in abnormal hippocampal development thus lead to deficits in social behaviors and repetitive,stereotyped behavior in mice,as well as the alteration of autism-related genes expression in mouse hippocampus.Liver X receptor agonist TO901317 promoted hippocampal development in both BTBR and VPA-exposed mce,improved social ability and reduced repetitive stereotypical behaviors in these mice.