| BackgroundEpilepsy is a severe chronic neurological disease that affects more than 70million people worldwidely.It is caused by highly abnormal synchronous discharge of neurons in the brain.Its main clinical features include recurrent seizures,sensory and conscious disturbances.At present,the clinically proven effective treatments consist of drug therapy,surgery,physical support and diet intervention,among which drug treatment represent the most common one.However,the current clinically used drugs are only effective to about 70%of epilepsy patients.Thus,it remains a critical scientific question in the epilepsy field as to explore the underlying pathological mechanisms of epileptogenesis and identify new and more effective treatment targets.Astrocyte,a type of cell that is most abundant in the brain,plays important roles in not only supporting,comparting and suppling nutrition to neurons,but also in regulation of synaptogenesis,maturation and synaptic transmission.Numerous studies indicate that dysfunction of astrocytes may participate in epileptogenesis.However,the underlying mechanisms are not clear.It has been known that proteoglycan-agrin and its receptor-Lrp4（low density lipoprotein receptor related protein 4）function as critical regulators of Neuromuscular junction formation,differention and maintainence.In the brain,agrin was reported to mainly expressed in the neuron,heterogenous deletion of agrin led to resistance to neurotoxicity and increased threshold to seizure.On the other hand,previous study had indicated that Lrp4 was enriched in the astrocytes in the hippocampus.Deletion of Lrp4 caused decreased firing frequency of hippocampal excitatory neurons and increased threshold to induce seizure.These observations suggest agrin/Lrp4 signaling may play a role in epileptogensis through regulating neuronal activity.However,the direct evidence is lacking.Aim:This project aims to explore whether agrin/Lrp4 signaling in astrocytes plays an important role in epileptogenesis.Results will shed light on the pathological mechanisms of epileptogenesis and provide new therapeutic targets.Methods:An seizure model was generated by injecting kainic acid（KA）stereotactically into amygdala in the brain of C57 wildtype mice.We used western blot（WB）and quantatitive real-time PCR（RT-PCR）to monitor the expression of agrin and Lrp4 at differnent time points after seizure induction.We generated brain-specific Lrp4knockout mice(GFAP-Cre;Lrp4 f/f)as well as specific knockdown of Lrp4 in hippocampal astrocytes by virus to monitor their seizure threshold and following spontaneous recurrent seizures（SRS）.We also injected agrin protein to lateral ventricles to examine its effect on SRS.Result:Seizure activity increased levels of agrin and Lrp4 in the hippocampus,which were recovered gradually after they reached peak at 12 hours.Compared with control,GFAP-Cre;Lrp4 f/f mice exhibited enhanced threshold to seizure and reduced SRS frequency,with no change in stage and duration.In addition,the level of mossy fiber sprouting in hippocampal dentate gyrus（DG）was decreased.Deletion of Lrp4specifically in excitatory neurons(NEX-Cre;Lrp4 f/f)caused no dramatic changes in above parameters.However,knockdown of Lrp4 in hippocampal astrocytes by virus significantly reduced SRS frequency.Last,injection of agrin into lateral ventricle increased SRS frequency.Conclusion:These observations indicated that expression levels of agrin and Lrp4 is positively correlated to seizure activity,revealed the unrecognized function of agrin/Lrp4 signaling in astrocytes in regulating epileptogenesis.Our results provide new targets for clinical epilepsy therapies. |
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