Multiomics Profiling Of The Changes Of Choroid Plexus Epithelium And Cerebrospinal Fluid During Aging

Background:The blood cerebrospinal fluid barrier(BCSFB)is a barrier system that maintains brain homeostasis,located in the Choroid plexus(CP)of the ventricles of the brain.The choroid plexus is the primary secretory tissue to produce cerebrospinal fluid(CSF).It consists of a monolayer of cuboidal CP epithelial cells(CPECs),which are interconnected through tight connections to establish the BCSFB and prevent the entry of potentially harmful substances and macromolecules.CPEC are the CP cells responsible for CSF secretion,which can selectively transport solutes into the brain.CPEC also secrete their own synthesized proteins,and neuropeptides into CSF.CPEC and CSF in BCSFB are associated with ageing or ageing-related diseases,especially neurodegenerative diseases.Objective: BCSFB may lose the barrier function and decrease the function of secreting cerebrospinal fluid during aging,leading to the homeostasis disequilibrium of the central nervous system and even cognitive dysfunction.CPEC are one of the less-studied cell types in the brain despite their critical role in maintaining the homeostasis of central nervous system.Therefore,we conducted this study to focus on the multiomic changes of CPEC during aging,construct a global multiomic map of CPEC,elucidate the function and potential molecular mechanisms of CPEC by combining the age-related changes of CSF,and study the mechanism of BCSFB aging.Methods:1.Choroid plexus from mice of different genders and ages were collected,and choroid plexus epithelial cells were sorted.Then,RNA sequencing,whole genome methylation sequencing(WGBS)and mi RNA sequencing were conducted.2.Differentially expressed genes,differentially methylation loci,differentially expressed mi RNAs and integrative analysis were systematically analyzed by bioinformatics methods.3.Cerebrospinal fluid from mice of different genders and ages were collected,and high-throughput targeted small molecule compounds liquid chromatography mass spectrometry were carried out.After screening age-related differential compounds,CPEC and CSF were jointly analyzed to elucidate CPEC functions and underlying molecular mechanism.Results:1.RNA-Seq results: The expression pattern of transcriptome was different between female and male during aging.This study identified a total of 432 age-related differentially expressed genes(DEGs)in females and 1,410 age-related DEGs in males.The activation of the response to virus and interferon pathway,imbalance of the energy metabolism pathway,and dysfunction of substance secretion were the main manifestations of aged mice CPEC.The secreted proteins Lgals3 bp and Spp1 were highly expressed in CPEC of elderly mice,and Spp1 could migrate macrophages.TF analysis found that Stat1、Nrf1、Stat3 and Esr1 may regulate different types of age-related DEGs.2.WGBS results: There was a significant difference in methylation pattern between males and females,with 1,359,358 age-related differential methylation loci(DML)in females and 1,330,967 age-related DML in males.There was a non-linear relationship between these DML and age,with some loci already undergoing changes in middle age,while others only experiencing significant changes in old age.And these DML were mainly distributed in the distal intergenic region,indirectly regulating gene transcription.The combination analysis of two omics screened 3 genes:Sult1c1,Sult1c2 and Lgals3 bp that might be negatively related to DNA methylation and regulate CPEC aging.3.mi RNA-Seq results: A total of 15 mi RNAs highly correlated with CPEC aging were identified.Through joint analysis with transcriptome,173 mi RNA/m RNA interaction pairs were identified that might regulate CPEC aging.The most significant pairs were mmu let-7b-5p/CCnd2,mmu mi R-351-5p/Stat1,mmu mi R-342-3p/Ifit3 and mmu-mi R-181a-5p/Spp1.These four pairs were probably involved in regulating immunity,inflammation,and cell cycle.4.CSF mass spectrometry results: A total of 52 age-related differential compounds were found in females,and 68 in males.Age-related differential compounds mainly consisted of amino acids and organic acids,and microbial metabolites TMAO were enriched in aged CSF.Combined with transcriptome analysis,it was found that the changes in organic acids in CSF may be related to the differential expression of transporters for organic acid on CPEC.The expression of taurine transporter,Slc6a6,was upregulated in compensation during aging,maintaining stable taurine content in CSF.And the expression of uric acid transporter,Abcg2,was upregulated during aging,increasing the uric acid content in CSF.Conclusion:We have provided transcriptomic information,WGBS information,mi RNA information,and small molecule composition of the choroid plexus epithelium and CSF from mice at different ages.Analysis identified age-related differential expressed secreted genes: Lgals3 bp and Spp1.Lgals3 bp may be jointly regulated by TF and DNA methylation,while Spp1 may be jointly regulated by TF and mi RNA.And experiments showed that Spp1 can recruit macrophages,which is related to the inflammatory status of choroid plexus and the integrity of barrier function.In addition,analysis identified TFs related to aging: Stat1,Nrf1,Stat3 and Esr1,and Stat1 may be regulated by mi RNA.Analysis identified differential methylation genes related to choroid plexus epithelial aging:Sult1c1 and Sult1c2.Analysis identified differential mi RNA related to aging: mmu let-7b-5p,mmu mi R-351-5p,mmu mi R-342-3p,and mmu-mi R-181a-5p.And transporter genes related to changes in CSF composition,Slc6a6 and Abcg2,were identified.Therefore,these datasets provide rich resources for further research on the mechanism of choroid plexus epithelial and BCSFB aging.