The Role Of Glial Cells And Their Associated Proteins In Epilepsy

Epilepsy is a chronic,recurrent transient brain dysfunction syndrome characterized by abnormal epileptic neuronal discharges that cause recurrent epileptic seizures.Epilepsy is a neurological disorder with a high prevalence in different countries around the world.Approximately 30% of people with epilepsy are unable to stop their seizures after regular treatment with antiepileptic drugs,and have ≥4 seizures per month,which is called intractable epilepsy(IE)or drug-resistant epilepsy.The majority of these are temporal lobe epilepsy.In recent years,proteomics-based approaches have opened up greater possibilities for discovering new drug targets in intractable epilepsy.One such technique,Data independent acquisition(DIA)quantitative proteomics,is a targeted proteomics technique that allows for the efficient determination of protein molecules with very low abundance in complex samples by fragmentation and secondary mass spectrometry of all ions in a selected massto-charge ratio(m/z)range,thus improving the confidence of quantitative analysis.with greater quantitative accuracy and reproducibility.A growing number of studies are now finding that aberrant neuroglia function may cause dysfunctional excitation and inhibition in the central nervous system and alterations in the neuronal cell microenvironment,which may trigger epilepsy.Neuroglia play an important role in epilepsy susceptibility and the development of epilepsy,and are potential drug targets for the treatment of epilepsy and other central nervous system disorders.In this study,brain tissue specimens obtained surgically from patients with temporal lobe epilepsy were subjected to DIA proteomics analysis,and numerous proteins with significantly different expression were identified by comparing the core and border regions of the medial temporal lobe epileptic foci in epileptic patients and by comparing the medial temporal lobe resected tissue in epileptic patients with the medial temporal lobe tissues from the nonepileptic patients.Considering the important role of glial cells in the pathogenesis of epilepsy,the 412 differentially expressed proteins obtained from epileptic and non-epileptic patients were compared with the protein data related to human astrocytes,microglia and oligodendrocytes in Uniprot.The role of these three proteins and associated glial cells in epilepsy was explored in this study.Objective: Exploring the role of glial cells and their associated proteins in the pathogenesis of epilepsy.Methods:1.Tissues from the core and the border regions of the medial temporal lobe epileptic foci in epileptic patients and the medial temporal lobe region in non-epileptic patients were subjected to DIA proteomics analysis to screen for differentially expressed proteins between the groups and analyses them bioinformatically.2.The above differentially expressed proteins were compared with the protein data of three human glial cells in the Uniprot database to screen for differentially expressed proteins related to glial cells.3.Qualitative and quantitative studies of the differentially expressed proteins screened in 2 in human brain tissue sections using immunohistochemistry and immunofluorescence techniques.4.Silencing of the SORT1 gene using sh RNA AAV9 virus to explore its effect in susceptibility to epilepsy in a mouse model of epilepsy.Results:1.More than 5,000 proteins were identified by DIA proteomics analysis in each of the three groups of tissue from the core and border regions of epileptic foci in the medial temporal lobe of patients with epilepsy,and in the medial temporal lobe of non-epileptic patients;protein clustering analysis showed that these proteins were clearly classified between the three groups and were more consistently expressed within the groups.2.The differentially expressed proteins between the three groups described above,as well as differentially expressed proteins between epileptic and nonepileptic patients,were screened for differential expression based on differential expression fold(1.2-fold or 2-fold)and a statistical threshold of P < 0.05.3.The differentially expressed proteins from epileptic and non-epileptic patients were compared to the Uniprot Protein Data Bank data on human astrocyte,microglia and oligodendrocyte-associated proteins,and a protein associated with each of these three glial cell types was identified:metallothionein-3(MT-3),a protein associated with astrocytes;sortilin 1(SORT1),a protein associated with microglia;and FYN,a protein associated with oligodendrocytes.Sortilin1(SORT1),and Src family tyrosine kinase(FYN),a protein associated with oligodendrocytes.Compared to non-epileptic patients,MT-3 and FYN protein expression was upregulated and SORT1 was downregulated in epileptic patients4.In paraffin sections of human brain tissue,astrocytes and microglia were found to be abnormally proliferating in epileptic patients,especially in the core of the lesion,with a lower degree of lesioning in the border region.In contrast,oligodendrocytes showed weaker positive expression in epilepsy,particularly in the core of epileptic foci,and more often showed myelin deficiency.5.Quantitative expression of the three glial cell-associated proteins on brain tissue sections was more consistent with the data from proteomic analysis.6.SORT1 gene silencing in the hippocampal brain region of mice resulted in decreased seizure extent and frequency and reduced epilepsy susceptibility.Conclusions:1.Glial cells and their associated molecules may be involved in the pathological process of epilepsy onset and development.2.Three glial cell-associated protein molecules,MT-3,SORT1 and FYN,may be one of the molecular mechanisms of glial cell involvement in epileptic pathology.3.SORT1 gene silencing reduces epilepsy susceptibility in mice.