Mechanism Of DISC1 Regulating The Newborn Neurons Migration In Mouse Hippocampus After Status Epilepticus

Part one:The effects of DISC1 on neuronal migration in hippocampus after epilepsyObjectives:1.To investigate the regulation of DISC1 protein on neurogenesis in the dentate gyrus(DG)of mouse hippocampus after status epilepticus(SE);2.To elucidate the relationship between DISC1 and Reelin,ApoER2,EphB2,Integrin β1,Integrin α5 after SE。Methods:Adult male C57BL/6 mice(4-6 weeks,18-22g)were used to kindle seizures by intraperitoneal injection of pilocarpine(260-275mg/kg).Those mice who suffered grade 4 seizures were included according to modified Racine classification.The control group was intraperitoneally injected by the same volume of sterilized saline.Hippocampi were collected 1,2,4 and 8 weeks after SE and the following methods were performed:(1)Immunofluorescence was adopted to detect co-expression of DISC1 and several proteins that were specific during different stages of neurogenesis,such as DCX,Prox1,GFAP,NeuN,BLBP and Nestin;to observe the co-expression of DISC1 and Reelin,EphB2,Integrin β1,and Integrin α5;(2)Western Blot was used to test the expression of DISC1,Reelin,ApoER2,EphB2,Integrin β1 and Integrin α5 in whole hippocampi 1,2,3 and 4 weeks after SE;(3)Realtime PCR was used to test the mRNA expression of DISC1,Reelin,ApoER2,EphB2,Integrin β1and Integrin α5 in whole hippocampi 1,2,3 and 4 weeks after SE.Results:1.The kindled rate of epilepsy was 89%,and the mortality rate was 45%.The peak of death was within 1 week after pilocarpine injection.2.GFAP+cells embraced DISC1+cells in the Hilus after SE;DISC1 co-expressed with BLBP,Nestin,Prox1 and NeuN.3.Western Blot and Realtime PCR showed that the protein and mRNA expression of DISC1 was down-regulated,similar to that of Reelin,ApoER2,and EphB2,while opposite to that of Integrin β1 and Integrin α5.4.Immunofluorescence indicated that DISC1 co-expressed with Reelin,EphB2,Integrin β1 and Integrin α5 in Hilus after SE.Conclusion:1.DISC1 may have a regulatory role in hippocampal neurogenesis after epilepsy.2.The effect of DISC1 on the neuronal migration in the hippocampus after seizures is highly correlated to Reelin-ApoER2/EphB2 and Reelin-Integrin a5/β1 signaling pathways.Part two:The regulation mechanism of DISC1 on neuron migration in hippocampus after epilepsyObjective:To explore the role of DISC1 on the modulation of newborn neurons’migration through Reelin-ApoER2/EphB2 and Reelin-Integrin β1/Integrin α5 in DG after SE.Methods:Seizures were kindled the same as previous.Mice were randomly divided into over-expression group,negative control group and negative control+green fluorescent group 1 week after SE.HBAAV2/9-Ta1-m-Disc1-3xflag-Null,HBAAV2/9-Ta1-Null and HBAAV2/9-Tal-ZsGreen were stereotactically transfected into hippocampi bilaterally(2ul/site).Hippocampi were collected 3 and 6 weeks after viral injection and the following methods were performed:(1)Western Blot was used to detect the expression of DISC1,Reelin,ApoER2,EphB2,Integrin β1 and Integrin α5 3 and 6 weeks after AVV injection;(2)Realtime PCR was used to detect the mRNA expression of DISC1,Reelin,ApoER2,EphB2,and Integrin β1 and Integrin α5 at 3 and 6 weeks after AVV injection;(3)Immunofluorescence was used to observe the co-expression of DISC1 and Reelin,EphB2,Integrin β1 and Integrin α5.Results:1.The successful rate of AAV injection was 99%,the mortality rate after AAV injection was 14%.2.Western Blot and Realtime PCR showed that the protein and mRNA expression of DISC1 were effectively up-regulated in the hippocampus after AVV injection.3.The protein and mRNA expressions of Reelin,ApoER2 and EphB2 were significantly increased after the up-regulation of DISC1 expression,while the expressions of Integrin β1 and Integrin α5 were obviously decreased.4.Immunofluorescence indicated that the number of DISC1+/EphB2+cells was increased significantly in the Hilus after DISC1 over-expression.Conclusions:1.DISC1 regulates newborn neurons migration after epilepsy through Reelin-ApoER2/EphB2 signaling pathway.2.DISC1 may play a regulatory role in neurogenesis in the hippocampus after epilepsy through Reelin-integrin α5/β1 signaling pathway.