The Electrophysiologic Function Of CaBP4 Gene Mutation In Autosomal Dominant Noctumal Frontal Lobe Epilepsy

Epilepsy is one of the most common chronic neurological disease. Many factors can cause neurons abnormal synchronous firing that results in repeated seizures and transient central nervous system dysfunction.World Health Organization (WHO) estimated that there were about seventy million epilepsy patients all around the world. The number of patients with epilepsy is about 9 million in China and the prevalence of epilepsy is now up to 4‰～7‰ according to incomplete statistics, in which children with epilepsy account for two-thirds.Repeated seizures may increase the central nervous system damage,delay their cognitive, mental,development,affect their quality of life, and even can cause disable or life-threatening.However,the pathogenesis of epilepsy still remains unclear.Recent studies show that genetic factor is a major cause of epilepsy,which is particularly significant in the incidence of idiopathic epilepsy.The studies found that more than one thousand kinds of gene mutations can cause seizures and about 150 kinds of epilepsy present monogenic hereditary mode.These Mendelian seizures are mainly ion channel disease,in which encoding the voltage-gated ion channels and ligand-gated ion channel mutation is relatively to clinical phenotype.The autosomal dominant nocturnal frontal epilepsy(ADNFLE) is the first identified single-gene genetic focal epilepsy syndrome.Its characteristic is cluster, frequent and short (a few seconds to several minutes) nocturnal movement attack, including dystonia (or) tonic and excessive exercise.Recent studies show that about 12 percent of ADNFLE families were related to encoding neuronal acetylcholine receptors (nAChRs) subunit gene mutations.So far, there are three nAChRs loci been found associated with ADNFLE, including ENFL1, ENFL3 and ENFL4.The ENFL1 is located on chromosome 20q13, which contains encoding acetylcholine receptor a4 subunit CHRNA4 gene.Up to date,five CHRNA4 gene mutations have been founded, which included four missense mutation (S248F, S252L, T265I and R308H) and one insertion mutation (259InsL).Moreover,CHRNA4 gene mutation exhibits genetic heterogeneity in different pedigrees that is consistent with its autosomal dominant mode of inheritance.The ENFL3 is located on chromosome Iq21,which contains the encoding AChR β2 subunit CHRNB2 gene.There are six identified CHRNB2 mutations which all are missense mutations, gene mutation V287L,V287M,L301V, V308A,I312M and V337G included.Some scholars had found a new heterozygous missense mutation (I279N) of CHRNA2 gene in 2006,which encodes the AChRa2 subunit in ENFL4 located on chromosome 8p21. However, CHRNA4, CHRNA2 and CHRNB2 gene mutations covers only a small part of the ADNFLE families. Researchers also found other ADNFLE possible causative genes,including corticotropin releasing hormone (CRH) gene, encoding the sodium-gated potassium channel KCNT1 gene and DEPDC5 gene respectively.The studies on ADNFLE mutations function found that ADNFLE mutations can enhance receptor function and reduce calcium ions induced increase in acetylcholine receptor response.Extracellular Ca2+ has a dual effect on neuronal nAChRs.At concentrations of 1-8 mM, it potentiates the Ach response,but at concentrations of 10-20 mM, it blocks the human α4β2 ACh response.Presynaptic nAChRs facilitate both excitatory and inhibitory transmitter release in the cortex. Ca2+ modulation may act as a negative feedback mechanism to prevent presynaptic α4β2 nAChRs at central excitatory synapses from overstimulating glutamate release during repetitive synaptic firing.But ADNFLE mutation can increase neuronal excitatory neurotransmitter release by negative feedback regulation of calcium ions that lead to increase neuronal excitability.Some studies found that Ca2+ could block a4b2 nAChRs by binding to a site in the channel pore (channel block) or at some other site. Above findings prompted calcium plays an important role in the pathogenesis ADNFLE.In our previous study,we found a CaBP4 gene novel missense mutation (CaBP4 gene p.G155D mutations) in a ADNFLE family by using whole-genome exon sequencing technology and Sanger sequencing method.And the CaBP4 gene mutation was not found in unaffected family members and 200 controls healthy people.CaBP4 gene with a total length of 828bp is located on chromosome 11q1 3.2, which contains six exons encoding region and its product is calcium binging protein 4(CaBP4) containing 275 amino acids.CaBP4 is a neuronal calcium connexin protein that is similar to the calcium-regulating and it participates in the regulation of L-type voltage-gated calcium channels and neurotransmitters release.CaBP4 mutations had been found that they can not only delay the inactivation of calcium channel result in an increase currents influx,decrease calcium affinity or calcium-binding capacity to reduce the outflow, but also can affect the stability of the protein structure itself,other proteins interacting signaling pathways and neurotransmitter release.But CaBP4 gene mutation has not been reported in epilepsy. Combined with bioinformatics analysis,we hypothesized CaBP4 gene mutation (p.G155D) may cause ADNFLE. Its possible pathogenic mechanisms may be that CaBP4 new missense mutation (p.G155D) changes the normal structure of CaBP4 protein resulting in abnormal regulation of calcium channel.The mutant CaBP4 protein may cause neurons calcium homeostasis imbalance and abnormal neurons synchronized discharge to cause epilepsy seizure.This study aims to detect how CaBP4 gene mutation p.G155D changes hippocampal pyramidal cell membrane potential by the patch-clamp technique and vetify the electrophysiological function of CaBP4 p.G155D gene mutation in the pathogenesis ADNFLE.[Methods](1) We construct mutant CaBP4 gene recombinant plasmid (pEGFP-N1-CaBP4mu) and wild-type CaBP4 gene recombinant plasmid (pEGFP-N1-CaBP4wt). Then we amplificate glycerol bacteria and extract plasmid.The digested plasmid products are identified by agar gel electrophoresis experiment.Furthermore,we validate the gene sequence of construct mutant and wild-type CaBP4 gene recombinant plasmid by sequencing respectively.(2) After taking postnatal brain from 0 to 24 hours SD neonatal rat hippocampus,we scattere the SD neonatal rat hippocampus into individual cells by trypsin digestion and percussion.The primary hippocampal neurons culture is applied for serum-free medium in accordance with 700,000/well density. After 6 to 7 days cultured, we detecte and analyze the purity of primary cultured hippocampal neurons by immunofluorescence.(3) By using X-tremeGENE, the constructed pEGFP-N1-CaBP4mu and pEGFP-N1-CaBP4wt plasmids were transfected into primary cultured hippocampal neurons,which are divided into experimental group (hippocampal neurons transfected with mutant CaBP4 gene recombinant plasmid) and the control group (hippocampal neurons transfected with wild-type CaBP4 gene recombinant plasmid). We detect GFP expression after 72 hours transfection by inverted fluorescence microscope and analyze transfection efficiency.(4) The transfected hippocampal neurons in the experimental group and the control group which expresses green fluorescent were recorded the currents and action potential of L-type calcium channels by whole-cell patch clamp recording techniques.[Results](1) Primary cultured hippocampal neurons mostly adhered to six-well plates and extended protrusion in the first day when its cell oval or pyramidal shape body increased, when cultured for 3d, hippocampal neurons were almost pyramidal cells with a plurality of projections and surrounded by a halo.Its neurite significantly elongated and form a sparse network between adjacent cells.When cultured for 5d, the cell body is further increased and the neurite further elongated to form a dense network between neurons, when cultured for 7d,neuronal cells have grown more mature and its halo was clear.Some hippocampal neurons were concentrated distribution trend.The purity of hippocampal neurons was 83.69% ± 3.55% by immunofluorescence.(2) The mutant CaBP4 recombinant plasmid (pEGFP-N1- CaBP4mu) and wild-type CaBP4 recombinant plasmid (pEGFP-N1-CaBP4wt) were digested by Xhol and HindⅢ restriction endonuclease and the digested products were subjected to agarose gel electrophoresis experimental.The result showed that there are two bands which respectively presents about 4.72kb and 840 bp band.We further validated CaBP4 gene recombinant plasmid sequences by using gene sequencing and the resule is totally conformed designed to the design sequence.(3) The mutant and wild-type CaBP4 recombinant plasmids were transfected into primary cultured hippocampal neurons respectively that were divided into experimental group (transfected with mutant CaBP4 gene recombinant plasmid) and control group (transfected with wild-type CaBP4 gene recombinant plasmid). We detected the expression of green fluorescent of the hippocampus in the experimental group and the control group after transfection for 72 hours with the application of an inverted fluorescence microscope.The transfection efficiency of the experimental group was 11.88% ± 2.44% and that of the control group was 12.09% ± 2.09%. Moreover,compared to the control group,more hippocampus neurons in the experimental group died.(4) We detect hippocampal neurons L-type calcium currents by whole-cell patch clamp recording techniques.Gived the same stimulation, L-type calcium current in the experimental group is more increased than that of control group. According to the IV curve,when the voltage is about-40mV,the hippocampal neurons L-type calcium channels open and when the voltage is about 10 mV, the channel current reaches to the peak.And L-type calcium current peak of hippocampal neurons in the experimental group and control group was-13.15 ± 4.35pA (n= 5) and-7.67 ± 5.11 pA (n= 6) respectively. Moreover, the Ⅳ curve of the experimental group changes to the hyperpolarized direction.(5) We found that potential firing frequency of hippocampal neurons in the experimental group increased significantly.[Conclusions](1) This is the first study on the electrophysiological function of CaBP4 p.G155D gene mutations in autosomal dominant nocturnal frontal lobe epilepsy by whole-cell patch clamp recording technique.(2) The result of whole-cell patch clamp recording is that L-type calcium current in the experimental group is more increased than that of control group.Moreover, the potential firing frequency of hippocampal neurons in the experimental group increased significantly.So we speculate that CaBP4 mutations p. G155D can alter CaBP4 protein structure or function to change the regulation of hippocampal neurons L- type calcium channels which causes an increase in the calcium influx flow from extracellular and further triggers synaptic terminal neurotransmitter release. Thus it increases the excitability of hippocampal neurons to cause seizures.(3) There are more transfected hippocampus neurons died in the experimental group than that of control group. We speculate that CaBP4 gene mutations p.G155D may cause intracellular calcium overload by increasing extracellular calcium influx, thus it may produce neurons neuron toxicity to increase neuronal the injury and the promote neurons death.