Research On The Mechanism Of HMGB1/RAGE Mediated Neurogenesis In The Development Of Cortical Dysplasia Epilepsy

Objective:Focal cortical dysplasias（FCDs）are a group of heterogeneous developmental disorders that are caused by germline or somatic mutations and characterized by abnormal cortical lamination,cell morphology（e.g.,cell enlargement）,and cell polarity.FCDs are highly associated with drug-resistant epilepsy and the most common cause of neocortical epilepsy in children.Previous studies have shown that abnormal cells in epileptogenic lesions of FCDs are mainly derived from the abnormal migration and differentiation of subventricular zone-derived neural progenitor cells（SVZ-NPCs）.However,the mechanisms involved in the regulation of abnormal SVZ-NPCs migration still need to be explored.High mobility group box 1（HMGB1）is a nuclear protein with multiple functions.It is well known that under normal conditions,HMGB1 is mainly located in the nucleus as a nonacetylated and thiol form but released from dead and dying cells after tissue injury and can be further converted to disulfide HMGB1.In addition,after cell activation or injury,translocation of HMGB1 from the nucleus to the cytoplasm may occur,followed by inflammasome activation and pyroptosis.In addition,growing evidence suggests that HMGB1 can regulate cell proliferation,migration and differentiation.As one of the receptors of HMGB1,receptor for advanced glycation end products（RAGE）can promote the migration of many kinds of cells,including neural stem cells.Evidence suggests that the expression of RAGE is restricted in the undifferentiated neural stem/progenitor cells of the mouse adult SVZ neurogenic region and adult SVZ-derived neurospheres,and the HMGB1-RAGE pathway can promote the proliferation and differentiation of SVZ-NPCs.Furthermore,the m RNA and protein levels of HMGB1 and RAGE were significantly upregulated in FCDs cortical epileptic foci.Therefore,we proposed that the HMGB1-RAGE pathway is involved in the pathophysiological process of abnormal SVZ-NPCs migration and hyperexcitability of cortical lesion in FCDs.In this study,we established a freezing lesion-cortical dysplasia（FL-CD）model to observe the expression of HMGB1 and RAGE in the cortical lesion of FL-CD animal models,its potential role in regulating the migration of SVZ-NPCs,and its influence on cortical epileptiform discharge in mice.Methods:1.We established a FL-CD model on Nestin-GFP^Tg/+mice,and observed the expression of SVZ-NPCs and their migration by suing immunofluorescence staining technology.2.We analyzed the protein expression of HMGB1 and RAGE in FL-CD model by Western Blot and immunofluorescence staining.3.The HMGB1/RAGE pathway antagonist FPS-ZM1 was injected intraperitoneally to block the combination of HMGB1 and RAGE in Nestin-GFP^Tg/+mice.And Nestin-GFP ^Tg/+/RAGE^-/-hybrid mice which are crossbred from Nestin-GFP^Tg/+mice and RAGE^-/-mice,were also used.Observe the changes of SVZ-NPCs migration in the FL-CD model under different conditions.4.The FL-CD modeled Nestin-GFP^Tg/+/RAGE^-/-mice and Nestin-GFP^Tg/+mice were used for cortical electrodes implantation and in vivo video electroencephalogram recording to observe the mouse cortical excitability features.Results:1.The result of immunofluorescence staining showed that in the FL-CD model,SVZ-NPCs would form a“migration flow”and migrate to the cortical freezing lesion.And the phenomenon of migration was most obvious at P5,and decreased with time（P15,P30）.2.Western Blot results showed that the overall expression of HMGB1 protein in the FL-CD group and the sham group decreased with time,and the expression of RAGE protein increased with time,but there was no significant difference between the FL-CD group and sham group.The results of immunofluorescence staining showed that HMGB and RAGE were highly expressed in the center of the cortical freezing lesion,and the distribution of HMGB1in the cells around cortical freezing lesion changed.The HMGB1 expressed in the cytoplasm was significantly increased in the FL-CD group,and this phenomenon was most obvious at P5,and decreased over time（P15,P30）.At the same time,it was observed that a large number of nestin positive cells on the“migration flow”expressed HMGB1 and RAGE.3.After the establishment of FL-CD model,compared with Nestin-GFP^Tg/+mice,thenumber of nestin positive cells in the“migration flow”of Nestin-GFP^Tg/+/RAGE^-/-mice was significant reduced at P5,but it started to be up-regulated at P15 and was significantly higher than that of the Nestin-GFP^Tg/+group until P30.However,the trend of the number of nestin positive cells in Nestin-GFP^Tg/+mice after FPS-ZM1 intervention was similar to that of Nestin-GFP^Tg/+/RAGE^-/-group,that is lower than Nestin-GFP^Tg/+mice at P5 and higher at P15,but there were no statistical significances.According to the analysis of the number of cells with colocalization of DCX and Nestin,the numbers in Nestin-GFP^Tg/+/FPS-ZM1 group and the Nestin-GFP^Tg/+/RAGE^-/-group were both significantly lower than the Nestin-GFP^Tg/+group.And this difference changes at P15 and P30,that is,both groups are slightly higher than the Nestin-GFP^Tg/+group,but the difference is not statistically significant.4.The Nestin-GFP^Tg/+/RAGE^-/-mice in the FL-CD model showed a significant decrease in the level of epileptiform discharges,and the duration of epileptiform discharge was also significantly lower than that of the Nestin-GFP^Tg/+mice.Video electroencephalogram（v EEG）results and time-frequency analysis showed that the degree of epileptiform discharge in Nestin-GFP^Tg/+/RAGE^-/-mice was significant reduced.And the coastline bursting index of Nestin-GFP^Tg/+/RAGE^-/-mice was significantly lower than that of Nestin-GFP^Tg/+mice in two time periods of 0-10 min and 40-50 min after pentetrazol（PTZ）injection,and there was no difference before PTZ injection and other time periods within one hour after PTZ injection.Conclusions:1.SVZ-NPCs participate in the formation of microgyrus in FL-CD model mice.2.After FL,the distribution pattern of HMGB1 and RAGE in the peripheral cortex of the microgyrus were changed.3.Antagonizing the HMGB1-RAGE pathway by FPS-ZM1 or absence of Rage could affect the abnormal migration of SVZ-NPCs.4.HMGB1/RAGE signaling pathway could affect the degree of epileptic cortical discharge induced by PTZ in FL-CD model mice.In conclusion,HMGB1/RAGE mediated the abnormal migration of SVZ-NPCs in FL-CD model mice at the early stage of development,which may be involved in the generation of epileptiform discharge.Therefore,intervention in the HMGB1-RAGE signaling pathway may be a preventive strategy to prevent the occurrence of FCDS refractory epilepsy.