Roles Of CSDE1 In Neurogenesis And Autistic Behaviors

Background:Autism spectrum disorder(ASD)represents a group of neurodevelopmental disorders with substantial genetic and clinical heterogeneity.The core behavioral symptoms are characterized by impairments in social communication and interaction,restricted interests,and repetitive behaviors.In addition to the core symptoms,there are many co-occurring conditions.Family and twin studies show that ASD is highly heritable(ranging from 60% to 90%).Our previous genome-wide association study suggested CSDE1 as a potential ASD risk gene.However,the genetic and molecular mechanisms of CSDE1 in ASD risk are still unclear.Up to now,over 300 high-confidence risk genes of ASD have been identified.Most of them are functional as gene expression regulators.Magnetic resonance imaging,post-mortem studies,and other studies have revelated that dysregulation of fetal cortical development is one of the important pathologies of ASD.This study aims to explore the effects of CSDE1 on early neurogenesis and autistic-like behavior and the underlying molecular mechanisms.Methods:1.Based on Autism Clinical and Genetic Resources in China(ACGC)and international cohort of patients in the ASD/ID network,identified CSDE1 variants by targeted sequencing based on single-molecule molecular inversion probes technology.Collect more patients with CSDE1 variants and detailed clinical information through the Gene Matcher.We curated the clinical information of the ASD patients to investigate the detailed genotype-phenotype relationships.2.To explore the roles of Csde1 in cortical neurogenesis,we constructed Csde1 conditional knock out mice(Nestin-Cre).During the perinatal period,the number of neurons in deep layer(Tbr1,Ctip2)and upper layer(Brn2,Satb2)of the mouse cortex were detected by immunofluorescence staining.At the neurogenesis peak period embryonic16.5,we detected the numbers of radial glial cell(RGC)and intermediate progenitor cells(IPC)by immunofluorescence staining and examined the proliferation by Brd U incorporation experiment.3.RNA-seq was used to detect the differentially expressed genes in embryonic cortex resulted from Csde1 knock out.GO and KEGG were performed to investigate the enrichment of differentially expressed genes.RIP-q PCR was used to verify the potential targets of Csde1.4.A series of behavioral paradigms,such as Three-chamber Social test,Open Field test,Elevated Plus Maze test,Y-maze test and Morris Water Maze test,were used to examine whether Csde1 conditional knockout mice exhibited autism-like behaviors.Result:1.Three patients with likely gene-disruptive(LGD)variants of CSDE1 were identified in the Autism Clinical and Genetic resources in China(ACGC).Five patients with LGD variants of CSDE1 were identified in a larger international ASD/ID cohort.Ten patients with LGD variants of CSDE1 were collected through Gene Matcher.These patients displayed relatively consistent phenotypes,such as ASD,mild to severe intellectual disability,and language and motor delay.These data supported the association of both inherited and de novo LGD variants in CSDE1 with ASD and related NDDs.2.Immunofluorescence staining of specific cortex layers showed that the number of neurons in both deeper layers and upper layers were reduced in Csde1 conditional knockout mice.Immunofluorescence staining of progenitor cells revealed a significant reduction in the number of radial glial cells and intermediate progenitor cells.Brd U incorporation experiments showed that the proliferation of progenitors decreased.These data suggested that the proliferation deficits of progenitors may contribute to the decreased number of cortical neurons in Csde1 conditional knockout mice.3.RNA-seq of embryonic cerebral cortex showed that compared with wild-type mice,Csde1 conditional knockout mice have over 1800 differentially expressed genes,including 865 down-regulated genes and941 up-regulated genes.The enrichment analysis of 865 down-regulated genes showed that they are mainly enriched in cell cycle-related pathways,and the gene with the largest fold change was Cdk6.RIP-q PCR revealed that Csde1 directly binds to the m RNA of Cdk6.This data suggested that Csde1 may be involved in early neurogenesis by regulating the cell cyclerelated gene Cdk6.4.The lack of Csde1 led to embryonic lethality at perinatal period.Thus,we performed behavioral analysis using Csde1 haploinsufficiency mice.Three-chamber test,grooming test,open field test,elevated plus maze test,Y maze,and morris water maze test suggested that Csde1 heterozygous mice have no obvious autistic-like phenotypes.Considering Csde1 is a key component and regulator of stress granules,we proposed that CSDE1 variants might interact with stress factors to increase ASD risk.We intragastrically inject pregnant mice with sodium arsenite to mimic oxidative stress effect.We found that Csde1 haploinsufficiency mice showed mild social impairment and anxiety-like behavior.These results suggested that Csde1 variants cause autistic-like behaviors through interacting with environmental factors,such as oxidative stress.Conclusion:This study suggested that disruption of CSDE1 increases ASD risk.Csde1 participates in early neurogenesis likely through regulating the cell cycle related gene Cdk6.Csde1 haploinsufficiency in mice might lead to autistic-like behaviors through gene-environment interaction.The underlying molecular mechanisms need further study.