The Role Of Caspr2 In The Mophology And Excitability Of Neurons

ObjectiveEpilepsy is a neurological disease caused by abnormal discharge of neurons in the brain.During the onset,the neural network is in a state of excitatory-inhibitory imbalance.There are many causes of epilepsy,genetic factors for the main cause of primary epilepsy,cerebral hemorrhage,brain tumors,brain trauma,inflammation,infections,etc.are common causes of secondary epilepsy.Many studies have reported that abnormal CNTNAP2 gene could involved in the development of epilepsy;the protein Caspr2 encoded by CNTNAP2 played a role in the regulation of central nervous system excitability.We also found a new mutant CNTNAP2(R777G)in a epilepsy patient.We speculated that Caspr2 may be involved in the nervous system excitatory-inhibitory balance,and when the gene mutation occurs,which resulting in neural network excitatory conduction abnormalities,is likely to lead to epilepsy.Therefore,this study preliminary explored the effect of Caspr2 on the morphological structure and excitability of neurons,providing a theoretical basis for the study of the pathogenesis of epilepsy.MethodsWe constructed plasmids with human wild-type CNTNAP2 gene or mutant human CNTNAP2(R777G)gene,which can emission green fluorescent,then transfected them into cell lines HEK293T,SH-SY5Y and mice primary cultured neurons,respectively.By Western blot and fluorescence quantification PCR,the expression of human wild-type Caspr2 and mutant protein Caspr2(R777G)was detected;the changes of morphological structure of wild-type Caspr2 and mutant protein Caspr2(R777G)in primary cultured neurons were observed by fluorescence microscopy;the amplitude and frequency of spontaneous postsynaptic currents(sEPSC and sIPSC)of culture neurons were recorded by electrophysiological patch-clamp technique,and the release of inducted APs in neurons were also recorded.We compared the excitability of primary cultured neurons overexpresseing wild-type Caspr2 with mutant protein Caspr2(R777G)by electrophysiological patch-clamp technique.ResultsWe successfully constructed pEGFP-N1-CNTNAP2 plasmids and the pEGFP-N1-CNTNAP2(R777G)plasmids,respectively.The transfection efficiency was extremely low,and protein expression was not detected by Western blot.However,the increase of mRNA was detected by quantitative PCR;these plasmids were transfected into mouse primary culture neurons,respectively.It was observed that morphology of neurons overexpressing Caspr2 protein may slightly change under the fluorescence microscope,while neurons expressing Caspr2(R777G)protein changes their structure:dendritic reduction,abnormal dendritic spine morphology.The electrophysiological whole-cell patch clamp records also showed that the amplitude and frequency of sEPSC and sIPSC in primary cultured neurons overexpressing Caspr2 protein were higher than those in the other two groups,so did the number and increasing tendency of APs as.The excitability of the primary cultured neurons expressing the mutant protein Caspr2(R777G)was not different from that of the control group,but the overall trend decline.ConclusionCaspr2 plays an important role in the excitatory-inhibitory balance.Our experimental results show that Caspr2 is involved in maintaining the structure and morphology of neurons and has the effect of promoting neuronal excitation.The mutant protein Caspr2(R777G)can change the morphology and structure of neurons:dendrites reduction,abnormal dendritic spines.Though the excitability of mutant neurons did not decrease,the trend was lower than that of the control group.We speculate that when the structure and function of Caspr2 are changed,it is possible to affect the E/I balance mechanism of the nervous system by increasing the abnormal excitability of the neuron or by participating in the formation of the morphologyl and structure of the neuron,directly or indirectly,then epilepsy may occur.