Endothelial Cdk5 Deficit Involved In The Pathological Process Of Epilepsy

Epilepsy is one of major neuropsychiatric diseases with feature of sudden brain neuron abnormal discharge.Even under formal antiepileptic drug treatment,there are still about 25%of patients with drug refractory epilepsy.Further understanding of the underlying molecular mechanisms of epilepsy is likely to provide important impetus for the development of new pharmacological treatment strategies.Accumulating evidences indicated that cerebrovascular factors play an important role in epilepsy.However,the cerebrovascular endothelium etiology of epilepsy and cerebrovascular endothelium therapy method or medicine to cure epilepsy is still enigmatic.Cdk5(Cyclin-dependent kinase 5)is one of the members of the serine/threonine kinase family,which is mainly distributed in the nervous system.Cdk5 in neurons is mainly involved in the migration and differentiation of neurons,axonal growth,synaptogenesis and so on.In addition to neurons,Cdk5 is also expressed in vascular endothelial cells,tumor cells,epithelial cells,endocrine tissue,etc.Cell experiments show that Cdk5 is mainly involved in endothelial cell migration,proliferation,inflammation activation and aging,but rare in vivo experiments are avilable.In this study,the occurrence of spontaneous epilepsy was observed in endothelial Cdk5 deficit mice,and the potential mechanisms were elucidated.The main research contents of this subject are as follows:1.Generation and characterization of Cdk5 conditional knockout mice.Objective:To construct endothelium knockout Cdk5 mice by Cre/loxP system.The various behavior test and EEG recording were used to validity the seizure in Cdh5Cre;Cdk5f/f mice.Methods and results:Cdh5Cre;Cdk5f/n mice were harvested by crossbreeding Cdh5Cre mice with Cdk5f/f mice.Then Cdh5Cre;Cdk5f/f mice were obtained by hybridization between Cdh5Cre;Cdk5f/n mice.The genotype was determined by PCR and agarose gel electrophoresis.Together with the control mice.4 week and 12 week old Cdh5Cre;Cdk5f/fmice were subjected to various behavioral experiments,including open field,Y maze,novel object recognize test,elevated O maze,tail suspension,forced swimming and so on.Once accidental seizure was observed,4 week,8 week,16 week,24 week mice were videoed,the seizure occurrence rate and frequency of daily seizures were calculated.EEG monitoring was performanced to comfirm the phenotype as epilepsy.PET test was carried to determine the location of epilepsy lesions.The moving distance and speed in the open field increased significantly in Cdh5Cre;Cdk5f/f mice,in comapared to control mice.There is no difference in the fast-mobilty time in the tail suspension test between the two groups,indicating Cdh5Cre;Cdk5f/f mice did not take manic disorder.No changes in activity count in 24 h locomotion test excluded the existence of ADHD in Cdh5Cre;Cdk5f/f mice.The probability of seizures and the frequency of episodes occur every day increased with age in Cdh5Cre;Cdk5f/f mice.Typical epileptic discharged spikes appeared in the EEG of the Cdh5Cre;Cdkf/f mice,identifying the behavior as epilepsy.PET results showed that epilepsic focal is mainly in the hippocampus of Cdh5Cre;Cdk5f/f mice,consistent with decreased hippocampus-related learning and memory levels.Conclusion:Endothelium Cdk5-knockout mice were successfully constructed.Epilepsy is the main behavioral phenotype of Cdh5Cre;Cdk5f/f micte2.Study on neuronal excitability and astrogliosis in Cdh5Cre;Cdk5f/f mice.Objective:To study the changes of neuronal passive membrane characteristics and synaptic transmission characteristics and astrocytes in the vascular endothelial CdkS deficit mice by electrophysiological and immunohistochemoistry method.Methods and results:Detect the passive membrane properties,EPSC,IPSC of hippocampal CA1 neurons in Cdh5Cre;Cdk5f/f and control mice.GFAP,S100?expression were quantified by immunofluorescence staining in 4 week,8 week,16 week knockout and control animals.Compared with the control group,the action potential frequency of hippocampal CA1 region in Cdh5Cre;Cdk5f/f mice increased significantly,and the frequency of sEPSC and mEPSC increased but the amplitude did not change in Cdh5Cre;Cdk5f/f mice.The frequency and amplitude of both sIPSC and mIPSC did not change.Endothelial-specific inactivation of Cdk5 results in progressive astrogliosis in vivo,mainly in the lacunar molecular layer of hippocampus.Conclusion:Endothelial Cdk5 deficit results in increased neuronal excitability,which is due to increased excitatory synaptic transmission but not inhibitory synaptic transmission.The increased excitatory synaptic transmission is not related to increased presynaptic membrane neurotransmitter release.Endothelial Cdk5 deficit mice show progressive astrogliosis and activation,mainly in the lacunar molecular layer of hippocampus.We suggested that pathologic events are involved in mediating seizures.3.Molecular mechanism of endothelium Cdk5 deficit in initiating pathological process of epilepsy.Objective:In order to reveal the mechanism underlying endothelial conditional knockout Cdk5 leads to epileptogenesis,BBB permeability and astrocytes function on glutamate,potassium ion reuptake,etc were detected.At the same time,the gene expression level of Cdk5 deficit primary endothelial cells was studied by transcriptomics method.Methods and results:BBB permeability was assayed by Evans blue injection and fluorescence imaging.The Kir4.1 current,GLAST+GLT1 current and GLT1 current in astrocytes were electrophysiological detected in hippocampus stratum radiatum in p18-23d knockout and control mice.Fluorescence imaging of Evans blue revealed BBB permeability increased in knockout animal,mainly in the luminal molecular layer of hippocampus.The electrophysiology of astrocytes showed that the Kir4.1 current of Cdh5Cre;Cdk5f/f mice was lower than that of the control group.The GLT1 current decreased obviously in endothelial Cdk5 deficit mice,compared to control group.GLT1 activator ceftriaxone can rescue the increased action potential frequency and reduced GLT1 current in Cdh5Cre;Cdk5f/f mice.Transcriptome results showed that nectin related adhesion juntion was alert and the leukocyte-to-endothelial cell infiltration increased in the endothelial cell of Cdh5Cre;Cdk5f/f mice.Conclusion:BBB permeability was increased in hippocampal lumens molecular layer of Cdh5Cre;Cdk5f/f mice.Dysfunction of Kir4.1 and GLT1 of astrocytes,blocked potassium and weaken glutamate buffer capacity thereof,lead to increased neuronal excitation and epileptogenesis.The change of BBB permeability may be related to the decrease of endothelial adhesion junction and more leukocyte infiltration.In summary,we uncovered a role for endothelial Cdk5 in regulating several of the neurochemical functions of the brain and initiate pathological process of epilepsy.Endothelial Cdk5 deficit results in increased neuronal excitability,at least in part,associate with increased BBB permeability and reactive astrogliosis.Dysfunction of astrocytes Kir4.1 and GLT1 contribute to increased neuronal excitation and epileptogenesis.The present study provides an important impetus for the development of new pharmacological treatment strategies for epilepsy.