Mechanism And Regulation Of Brain-Derived Endothelial Cdk5 Deficit In Mediating Epilepsy Through CXCL1/CXCR2

Background:Epilepsy is the most common chronic neurological disease with significant morbidity and mortality.In China,epilepsy has become the second neurological disorder after"migraine".A clinical feature of epilepsy is the abnormal firing of neurons from epileptic foci and spreads to other areas of the brain.According to the initial brain region and mechanisms of epilepsy,the treatment of epilepsy in clinic is mainly divided into surgical treatment and drug treatment.Although surgical treament relatively thorough,the difficulty to localize the seizure onset zone（SOZ）and irritative zone（IZ）bring challenge for surgical treatment.At present,the treatment for epilepsy is still mainly focus on pharmacological treatments.Now,the commonly used antiepileptic drugs include phenytoin,phenobarbital,diazepam and valproate.Almost all available drug treatment either act on synaptic mechanisms to restore the balance between excitation and inhibition or on voltage-dependent ion channels to reduce neuronal excitability.However,the available drug treatments target its symptoms rather than the underlying cause.As a result,approximately one-third of the patients affected suffer from therapy-resistant epilepsy.Therefore,elucidating the potential mechanism of epilepsy is helpful to find and develop new drug targets for epilepsy treatment and provide new ideas for better treatment of epilepsy.Cyclin-dependent kinase5（Cdk5）,is a member protein from cell cycle protein family.In contrast to other cell cycle families,the function of Cdk5 is not the regulation of cell cycle.Therefore,Cdk5 plays a critical role in neurons in central nervous system.Upon activation of the upstream p35/p39,Cdk5 is recruited to some neurodegenerative diseases.However,the function of Cdk5 on other cell types has not been reported yet.Previous studies showed that Cdk5 in endothelial cells can be involved in several biological processes,including cell migration,proliferation and sprouting,but whether these biological processes are related to some neurological diseases is not clear.Therefore,we used mice with endothelial cell specific Cdk5 knockout to explore the key function of endothelial Cdk5 in the brain and to investigate whether it has a regulatory effect on epilepsy.It will provide a new candidate target for the development of drugs to treat epilepsy.The research of current study mainly contains the following four parts:1.Endothelial conditional deletion of Cdk5 induces spontaneous seizuresObjective:To investigate whether the endothelial Cdk5 deficit can lead to some neurological diseases.Whether this neurological disease is related to impaired development of conditional knockout mice or to peripheral Cdk5 knockout.Methods and results:Endothelial Cdk5 specific knockout mice(Cdh5-Cre;Cdk5^f/f)were obtained by Cre-loxp system.Next,through 24-hour video monitoring and EEG recording.Cdh5-Cre;Cdk5^f/f mice developed spontaneous epilepsy at 2 months（8.3%）,and the seizure rate reached 80%at 6 months.However,there was no significant epilepsy seizures at 1 month,but the PTZ-induced epilepsy seizures in knockout mice was significantly increased at 1 month compared to control mice.Further,in order to eliminate the influence of puberty and the influence of the peripheral system,we adopt tamoxifen induced knockout and expressed Cre recombinase enzyme using adeno-associated virus in the vascular endothelial cells（AAV-BR1-iCre）in Cdk5^f/f mice at 1 month,the spontaneous epilepsy was also found in these knockout mices through 24-hour video monitoring and EEG recording.Conclusion:The lack of Cdk5 in the endothelial cells,leads to epilepsy seizures in hippocampus in mice more than 2 months.No significant electroencephalographic（EEG）changes were observed in the cortex.For mice with no spontaneous seizures at 1 month,their sensitivity to the convulsant drug pentylenetetrazol（PTZ）was increased.The spontaneous epilepsy was not caused by developmental effects or the Cdk5 knockout in the peripheral nervous system.2.Deletion of endothelial Cdk5 induces neuronal hyperexcitability,progressive astrogliosis and impairs astroglial GLT1 functionObjective:To explore the function of pyramidal neurons and glial cells in the hippocampus,that is,whether Cdk5 knockout in endothelial cells would affect the activity of neurons and the function of glial cells.Methods and results:Firstly,the concentration of glutamate in the hippocampus of knockout mice and control mice was determined by microdialysis.Using microdialysis in freely moving mice,we found a significant increase in extracellular glutamate in the hippocampus in Cdh5-Cre;Cdk5^f/f mice at 1 month and 4 months.Next,we used patch-clamp technique to investigate the excitability of pyramidal neurons in the hippocampus and cortex.Whole-cell recordings in brain slices showed an increased frequency of action potential and excitatory synaptic transmission in hippocampal pyramidal neurons in Cdh5-Cre;Cdk5^f/f mice.There were no significant differences in intrinsic membrane properties of pyramidal neurons and in excitability in medial prefrontal cortex pyramidal neurons between the two groups.Morever,endothelial Cdk5 knockout had no effect on spontaneous and miniature inhibitory postsynaptic current（sIPSC and mIPSC）.This increase in excitability was blocked by the glutamate receptor antagonist.These results suggest that the concentration of glutamate in endothelial Cdk5 knockout mice is increased,acting on excitatory postsynaptic receptors,thus increased excitatory synaptic transmission and action potential frequency,which ultimately leads to hyperexcitability of neurons and the generation of epilepsy.Immunohistochemical technique was used to investigate the morphology of glial cells in the hippocampus and cortex.The result showed a significant change in quantity and morphology of astrocytes in the hippocampus of 1-month-old Cdh5-Cre;Cdk5^f/f/f mice.However,the morphology of astrocytes in the cortex and microglia in the hippocampus showed no obvious changes in Cdh5-Cre;Cdk5^f/f compared with Cdk5^f/f mice.Based on the morphological changes of astrocytes in the hippocampus,the function of astrocytes in the hippocampus was then recorded by the electrophysiological technique for 1-month-old Cdh5-Cre;Cdk5^f/f and Cdk5^f/f mice.The results indicated that there was a significantly decrease in the GLT1-mediated current but not the GLAST-mediated current.Further,GLT1 was activation byβ-lactam antibiotic ceftriaxone or overexpressed in the hippocampus by virus injection.The two treatment both can significantly reversed the increase in firing frequency of pyramidal neurons and the decrease in GLT1-mediated current through electrophysiological recordings and also delayed the onset of seizures and decreased seizure duration induced by PTZ in Cdh5-Cre;Cdk5^f/f mice compared to Cdk5^f/f/f mice.These results suggest that the increased glutamate concentration in the synaptic mediated by impaired GLT1 is responsible for neuronal hyperexcitability and epilepsy in the hippocampus.Conclusion:Deletion of Cdk5 in endothelial cells increases the concentration of glutamate and the excitability of pyramidal neurons in the hippocampus.This is mainly due to the dysfunction of glutamate transporter GLT1,which ultimately leads to the increase of glutamate concentration and neuronal hyperexcitability.GLT1 was activation byβ-lactam antibiotic ceftriaxone or overexpressed in the hippocampus can effectively reduce the excitability of pyramidal neurons and epileptic phenotype.3.Endothelial Cdk5 deficit in mediating the development of epilepsy through CXCL1 signalingObjective:To investigate the mechanism of epilepsy induced by astrogliosis in endothelial Cdk5 deficit mice.Methods and results:First,We analyzed BBB permeability by Evans blue combined with immunostaining in 1-month-old and 4-months-old Cdh5-Cre;Cdk5^f/f and Cdk5^f/f/f mice.The results showed that BBB leakage evidenced by positive immunostaining for astrogliosis,was present in 4-months-old,but not 1-month-old Cdh5-Cre;Cdk5^f/f mice.Next,using different size of molecular of endogenous and exogenous tracer effusion combined with transmission electron microscopy and western blot experiments further confirmed that BBB damage is unlikely to be responsible for the decreased threshold for PTZ-induced seizures seen in 1-month-old mice since it was not observed at that age.Next,we cultured primary endothelial cells from the cerebral microvessels of 1-month-old Cdh5-Cre;Cdk5^f/f and Cdk5^f/f mice.The results showed that most of these genes related to endothelial activation were upregulated in the Cdh5-Cre;Cdk5^f/f mice when compared with the Cdk5^f/f mice.Consistent with this activation pattern,the level of CXCL1 was found to be significantly increased in Cdh5-Cre;Cdk5^f/f mice.No changes were found in the CXCL16 level.Furthermore,real-time quantitative PCR（qRT-PCR）and enzyme-linked immunosorbent assay（ELISA）confirmed the increase of CXCL1 in transcription level and protein level.In situ hybridization further showed that CXCL1mRNAs existed in endothelial cells by using RNAscope,suggesting its presence in endothelial cells.Next,at the cellular level,the cultured primary astrocytes(from Cdk5^f/f mice about 3weeks)were treated with the recombinant protein（20 ng）of CXCL1 for 6 hours.Immunohistochemical technique and patch-clamp recording indicated that application of recombinant CXCL1 protein directly to cultured hippocampal astrocytes,increased the number of cells expressing the astrocytic markers GFAP and S100β,decreased the GLT1-mediated current.Further,at the animal level,CXCL1 neutralizing antibody was administered via the guide cannula using a micro syringe pump or AAV-BR1-shCXCL1was injected in the hippocampus in Cdh5-Cre;Cdk5^f/f and Cdk5^f/f mice for 1 month.The above treatment was followed by behavioral analysis of PTZ-induced epilepsy and in vitro electrophysiological recordings of brain slices.The results showed that either the virus and CXCL1 neutralizing antibody both can significantly reduced the PTZ-induced epilepsy in endothelial Cdk5 knockout mice at 1 month,and also significantly reduced the proliferation and activation of astrocytes,the dysfunction of GLT1-mediated glutamate transporters and neuronal hyperexcitability.Conclusion:Endothelial Cdk5 knockout resulted in increased CXCL1 expression but did not affect BBB permeability in 1-month-old knockout mice.The increase of CXCL1expression by endothelial cells not only led to the proliferation and activation of astrocyte,but also led to the decrease of current of GLT1-mediated glutamate uptake,which was related to neuronal excitability and the generation of epilepsy.4.Endothelial CXCL1 regulates astrogliosis and astrocytic glutamate uptake via CXCR2 contributes to the development of spontaneous epilepsyObjective:To investigate the function of CXCR2 on neurons and astrocytes.Whether the effect of CXCL1 expressed by endothelial cells on neurons and astrocytes is mediated by binding to its receptor,CXCR2.Methods and results:Cre-dependent shCXCR2 and GFAP-Cre/CaMKII-Cre AAV was injected into hippocampus of Cdh5-Cre;Cdk5^f/f and Cdk5^f/f mice at 1 month,and behavioral tests,immunohistochemical tests and electrophysiological tests were examined 3 weeks later.The results showed that silencing CXCR2 on astrocytes significantly improved PTZ-induced epilepsy,astrocytic activation,GLT1-mediated transporter dysfunction,and decreased excitability of pyramidal neurons in the hippocampus.However,CXCR2 knockdown in pyramidal neurons had no effect on significant changes in the above phenotypes.Conclusion:Regulation of CXCR2 on astrocytes significantly reduced neuronal excitability in endothelial Cdk5 knockout mice and also improved PTZ-induced epilepsy.