A Systems Biology Framework For Dissecting Mechanisms Of Epileptogenesis And Identifying Antiepileptogenic Drugs

Epilepsy is a common chronic nervous system disease that affects about 50 million people worldwide.Although a series of disease-causing genes for genetic epilepsy have been identified,the molecular mechanisms underlying the occurrence and development of acquired epilepsy remain unclear.In addition,about one-third of epilepsy patients do not respond to current anti-epileptic drugs.Therefore,there is an urgent need to reveal the molecular regulatory mechanisms of the epileptogenic process and to identify new anti-epileptogenic drugs.In this study,a systems biology framework that integrates gene expression signatures,gene co-expression network,cellular regulatory network and protein-protein interaction network was constructed to reveal the network functional dynamics and regulatory mechanisms of epileptogenesis and to screen potential anti-epileptogenic drugs from traditional Chinese medicine Ginkgo biloba.The main research results are as follows:Network functional dynamics during epileptogenesis.Using time-specific hippocampal transcriptome profiles from several different animal epilepsy models,an integrated meta-analysis was performed for each of the three epileptogenesis stages(acute,latent and chronic stage)to obtain stage-specific gene expression signatures.To clarify the functional organization and connection of genes in the context of epileptogenesis,a genome-wide gene co-expression network(containing 8,384 genes)was constructed and 13 functional modules were identified.Subsequently,a module association score(MAS)was defined to quantify the degree of association between the module and each stage,and the dynamic changes of the gene network during epileptogenesis were depicted.It was found that the inflammatory and immune response processes(module M1)in the brain were positively associated with all three epileptogenesis stages,while synaptic transmission related modules(M6 and M7)showed negative association.The dysregulations of these modules constituted the key functional changes in the epileptogenic process.It is worth noting that a new module M8,which is involved in intracellular signal transduction related functions,was found to be gradually upregulated during epileptogenesis.Identification of gene regulators that drive epileptogenesis.Using a pre-defined set of regulators(including synaptic proteins,signal proteins,transcription factors,etc.)and a dataset covering the entire epileptogenesis stages,a gene regulatory network,which comprises 1,493 regulators and 5,695 target genes,and 41,364 interactions between them,was constructed via the ARACNe algorithm.Based on the gene regulatory network and the stage-specific gene expression signatures of epileptogenesis,the relative protein activity of the regulator in each stage was inferred using the VIPER model.214,198 and 156 key regulators with significant protein activity changes were obtained for acute,latent and chronic periods,respectively.43%(113/265)of the key regulators were common to all three stages.The changing pattern of the activity of key regulators during epileptogenesis is in line with the MAS changes of the modules,confirming the reasonability and reliability of the results.Subsequently,using RNA sequencing data from epilepsy patients with different seizure frequencies and patients with/without hippocampus sclerosis,72 key regulators were found to be associated with seizure frequency and/or hippocampus sclerosis.Importantly,four new regulators involving in nervous system development,i.e.FAM107 A,LAMB2,LTBP1 and TGIF1,which have not been related to epilepsy previously,were found to show significant upregulated activity in both conditions.Efficacy screening of Ginkgo biloba(GBL)chemical ingredients based on key regulators of epileptogenesis.225 chemical components in GBL were collected from the TCMSP database and related literature,and 31 potential active compounds were screened out through systematic ADME evaluation.These compounds include terpene trilactones(TTLs),flavonoids and carboxylic acids,etc.,in which TTLs have good oral activity and blood-brain barrier permeability.The 86 direct binding protein targets of active compounds were obtained by retrieving experimental protein-binding data and using SEA algorithm.Then,based on the key regulators and functional modules of epileptogenesis,the regulatory effects of GBL active compounds on key modules were quantified by calculating the network proximity in human interactome.The results showed that TTLs were relatively close to module M1,while the flavonoids were closer to modules M3 and M8.Further analysis showed that Ginkgolide B regulates the key regulator ARR3 in module M1 by targeting PTAFR,and thus modulates the entire M1 regulator network.Finally,a glutamate-induced excitotoxicity model was constructed to preliminarily verify the effects of Ginkgolide B in protecting nerve cells from apoptosis through regulating M1.In summary,based on a systems biology framework,this study revealed the gene network dynamic changes during epilepsy development,identified key gene regulators driving epileptogenesis,and further screened potential anti-epileptogenic molecules in Ginkgo biloba.These findings present a global landscape for the molecular evolution process of acquired epilepsy,and provide potential protein targets and drug molecules for future anti-epileptic therapy development.