Anomalous expression of chloride transporters in the sclerosed hippocampus of mesial temporal lobe epilepsy patients

The Na＋-K＋-CI- cotransporter 1 and K＋-CI- cotransporter 2 regulate the levels of intracellular chloride in hippocampal cells. Impaired chloride transport by these proteins is thought to be involved in the pathophysiological mechanisms of mesial temporal lobe epilepsy. Imbalance in the relative expression of these two proteins can lead to a collapse of CI- homeostasis, resulting in a loss of gamma-aminobutyric acid-ergic inhibition and even epileptiform discharges. In this study, we investigated the expression of Na＋-K＋-CI- cotransporter 1 and K＋-CI- cotransporter 2 in the sclerosed hippocampus of patients with mesial temporal lobe epilepsy, using western blot analysis and immunohistochemistry. Compared with the histologically normal hippocampus, the sclerosed hippocampus showed increased Na＋-K＋-Cl- cotransporter 1 expression and decreased K＋-CI- cotransporter 2 expression, especially in CA2 and the dentate gyrus. The change was more prominent for the Na＋-K＋-CI- cotransporter 1 than for the K＋-CI- cotransporter 2. These experimental findings indicate that the balance between intracellular and extracellular chloride may be disturbed in hippocampal sclerosis, contributing to the hyperexcitability underlying epileptic seizures. Changes in Na＋-K＋-CI-cotransporter 1 expression seems to be the main contributor. Our study may shed new light on possible therapies for patients with mesial temporal lobe epilepsy with hippocampal sclerosis.

Effects of febrile seizures in mesial temporal lobe epilepsy with hippocampal sclerosis on gene expression using bioinformatical analysis

Background:To investigate the effect of long-term febrile convulsions on gene expression in mesial temporal lobe epilepsy with hippocampal sclerosis(MTLE-HS)and explore the molecular mechanism of MTLE-HS.Methods:Microarray data of MTLE-HS were obtained from the Gene Expression Omnibus database.Differentially expressed genes(DEGs)between MTLE-HS with and without febrile seizure history were screened by the GEO2R software.Pathway enrichment and gene ontology of the DEGs were analyzed using the DAVID online database and FunRich software.Protein–protein interaction(PPI)networks among DEGs were constructed using the STRING database and analyzed by Cytoscape.Results:A total of 515 DEGs were identified in MTLE-HS samples with a febrile seizure history compared to MTLEHS samples without febrile seizure,including 25 down-regulated and 490 up-regulated genes.These DEGs were expressed mostly in plasma membrane and synaptic vesicles.The major molecular functions of those genes were voltage-gated ion channel activity,extracellular ligand-gated ion channel activity and calcium ion binding.The DEGs were mainly involved in biological pathways of cell communication signal transduction and transport.Five genes(SNAP25,SLC32A1,SYN1,GRIN1,and GRIA1)were significantly expressed in the MTLE-HS with prolonged febrile seizures.Conclusion:The pathogenesis of MTLE-HS involves multiple genes,and prolonged febrile seizures could cause differential expression of genes.Thus,investigations of those genes may provide a new perspective into the mechanism of MTLE-HS.

Isolation and identification of a novel STR(D17S2204)in the vicinity of NF1 locus

Using a synthetic oligonucleotide （dA-dC）15 as a probe to screen a probe pool made by microdissected human chromosome band 17q11-q12, a DNA fragment containing （CA）16 was isolated, which is a novel short tandem repeat （STR） determined by searching in the GenBank and GDB. It has 8 allelic types with a PIC value of 0.61. The novel STR is conformed with Mendelian segregation according to the linkage analysis of a three-generation neurofibromatosis type Ⅰ （NF1） pedigree. The STR was localized at chromosome band 17q11-q12 in the vicinity of NF1 gene by FISH analysis. The accession number of the STR in GenBank and GDB is G32112 and D17S2204 respectively.

SiRNA-mediated ankyrin-G silence modulates the expression of voltage-gated Na channels in murine hippocampal HT22 cells

Background:Voltage-gated sodium channels are the targets of many commonly used antiepileptic drugs.NaV1.6,encoded by Scn8a,increased in chronic mesial temporal epilepsy animal models and co-localized with Ankyrin-G,encoded by Ank3.We hypothesized that inhibition of Ank3 transcription by siRNA decrease the expression of NaV1.6.Results:We characterized expression of the target genes in hippocampal neuron HT22 cells by Real time-PCR.The melt peak in the resolution curve of Scn1a,Scn8a and Ank3 were all unique.Ank3 transcription was interfered and the relative Ank3 mRNA levels of the three interfered groups compared to GAPDH were 0.89±0.13,0.52±0.07 and 0.26±0.05 while that of the negative control group was 1.01±0.08(P<0.05).When Ank3 transcription was inhibited by siRNA,the relative mRNA levels of Scn8a decreased in the three groups(0.91±0.09,0.33±0.06 and 0.25±0.05),compared to the negative control group(1.10±0.09).Tested by Western blotting,protein levels of ankyrinG and Nav1.6 decreased after ank3-siRNA.Ankyrin-G in negative control group,group1,group2 and group1+2 were 0.813±0.051,0.744±0.041,0.477±0.055 and 0.351±0.190 respectively(P<0.01)while Nav1.6 were 0.934±0.036,0.867±0.078,0.498±0.070 and 0.586±0.180(P<0.01).The quantity analysis of immunofluorescence showed significant decrease of ankyrin-G and Nav1.6(Student’s test,P=0.046 and 0.016 respectively).Conclusion:We therefore concluded that in HT22 cells the expression of Nav1.6 was down-regulated by Ank3 RNA interference.

PRRT2 gene and protein in human:characteristics,evolution and function

Background:This study was designed to characterize human PRRT2 gene and protein,in order to provide theoretical reference for research on regulation of PRRT2 expression and its involvement in the pathogenesis of paroxysmal kinesigenic dyskinesia and other related diseases.Method:Biological softwares Protparam,Protscale,MHMM,SignalP 5.0,NetPhos 3.1,Swiss-Model,Promoter 2.0,AliBaba2.1 and EMBOSS were used to analyze the sequence characteristics,transcription factors of human PRRT2 and their binding sites in the promoter region of the gene,as well as the physicochemical properties,signal peptides,hydrophobicity property,transmembrane regions,protein structure,interacting proteins and functions of PRRT2 protein.Results:(1)Evolutionary analysis of PRRT2 protein showed that the human PRRT2 had closest genetic distance from Pongo abelii.(2)The human PRRT2 protein was an unstable hydrophilic protein located on the plasma membrane.(3)The forms of random coil(67.65%)and alpha helix(23.24%)constituted the main secondary structure elements of PRRT2 protein.There were also multiple potential phosphorylation sites in the protein.(4)The results of ontology analysis showed that the cellular component of PRRT2 protein was located in the plasma membrane;the molecular function of PRRT2 included syntaxin-1 binding and SH3 domain binding;the PRRT2 protein is involved in biological processes of negative regulation of soluble NSF attachment protein receptor(SNAR E)complex assembly and calcium-dependent activation of synaptic vesicle fusion.(5)String database analysis revealed 10 proteins with close interactions with the human PRRT2 protein.(6)There were at least two promoter regions in the PRRT2 gene within 2000 bp upstream the 5'flank,a 304-bp CpG island in the promoter region and four GC boxes in the 5'regulatory region of PRRT2 gene and we found 13 transcription factors that could bind the promoter region of the PRRT2 gene.Conclusion:These results provide important information for further studies on the role of PRRT2 gene and identify their functions.