Study On The Effects And Mechanism Of Insular Cortex Scn1a Gene On Dravet Syndrome

Dravet syndrome(DS)is a rare and serious encephalopathy(DEE)occurring in infancy and childhood.Febrile seizures(FS)or heat-induced epilepsy and general nervous system development disorder were the two most characteristic clinical manifestations of DS.SCN1 A gene is highly specific to DS,and its mutation is the main cause of DS.As it belongs to hereditary epileptic encephalopathy and is rare clinically,there is no fully effective treatment plan at present,and the prognosis is extremely poor,bringing heavy economic burden to the family and society.Insular cortex is closely related to epilepsy.The concept of insular epilepsy has been widely accepted by the academic community and has become a hot topic in the field of epilepsy research.Insula is an important structural and functional unit of epilepsy as the origin of epileptosis(independent epileptosis)and the transmission path of epilepsy(focal complex).Insula is also involved in cognitive control and is associated with cognitive functions such as learning and memory.Insula is considered to be the "bottleneck" of cognitive control.Previous studies of our team found that repeated subthreshold electrical stimulation of the insula cortex could induce epilepsy in rats,and the stimulation of the insula cortex was easier to ignite than the traditional amygdala nuclear ignition epilepsy model.We have also previously found that the insula is involved in learning,memory,and memory consolidation.In recent years,there has been much attention on the relationship between DS and different brain regions.Studies have shown that the clinical phenotypes of DS(e.g.,heatsensitive epilepsy or spontaneous epilepsy,cognitive dysfunction)are specifically related to certain brain regions(e.g.,hippocampus and forebrain base).However,the role of insula in DS has not been studied and described.Based on the above theories,we hypothesized that the insular cortex may play an important role in DS,that is,the loss of Na V1.1 channel function caused by insular Scn1 a knockdown is involved in the occurrence of DS epilepsy and associated cognitive changes.In view of this,in this study,the Scn1 a knock down DS mouse model was constructed using the adenovirus vector system of small interfering RNA(si RNA),and the epileptic behavior and the electrical activity characteristics of cerebral cortex of the model were observed through the heat-induction system and electrophysiology.Smart 3.0 behavioral system was used to detect and evaluate the memory,cognition,anxiety and other behaviors of model mice.Finally,we deliberately selected insular cortex from classical DS model mice with systematic Scn1 a Knock out(KO)for proteomic analysis and protein verification by parallel response monitoring(PRM).To further accurately explore the possible mechanism of insular Scn1 a gene in DS,provide new therapeutic clues and targets for DS,and provide theoretical guidance for clinical diagnosis and treatment.Part ⅠConstruction of a mouse model of insular cortex Scn1 a knockdown Dravet syndromeObjective The Scn1 a knockdown DS mouse model of insular cortex was constructed by the adenovirus vector system of small interfering RNA(si RNA).Methods The Scn1 a interfering adenovirus vector(AD-Scn1a-Si RNA)and the idle adenovirus(AD-Scn1a-Δ si RNA)were respectively injected into the bilateral insular cortex of P28-day-old C57/BL6 J mice by stereoscopic injection technique.To construct the Dravet syndrome model of insula Scn1 a knockdown mice.After 7 days,the expression of SCN1 A protein was detected by immunofluorescence staining and Western-blot.The mice were heated by heat-induced heating system,and the epileptic seizures in each group were observed and recorded.Eeg was used to record the eeg activity of the mice.Results Immunofluorescence results suggested that adenovirus injection into insular cortex could effectively reduce the expression of Scn1 a protein,and the fluorescence density of mice in Ad-Scn1a-Si RNA group was significantly lower than that in Ad-Scn1a-Δsi RNA group(P <0.0001).Western Blot results showed that ad-Scn1a-si RNA adenovirus significantly inhibited the expression of Scn1 a protein(P <0.0001).With the increase of core temperature in heat-induced mice,the seizure rate increased to 56.6%,while the non-seizure rate decreased to 43.4%.The average temperature of epileptic seizures above grade 4 in mice was 41.5±0.2℃.The eeg monitoring results showed that there was no significant difference between Ad-Scn1a-δ si RNA mice in the heating stage and the adaptation stage,while AdScn1a-Δsi RNA mice in the adaptation stage were basically similar to Ad-Scn1a-Δsi RNA mice in the adaptation stage,but in the heating stage,a large number of seizures with high-spikspike wave were observed.A small number of single high-sharp waves were also seen in the interphase of grand mal seizures.Conclusion1.The mouse model of insular cortex Scn1 a knockdown Dravet syndrome was successfully constructed.2.Local insular cortex Scn1 a deletion may lead to heat-induced epilepsy or febrile convulsion.Part Ⅱ Effects of insular cortex Scn1 a knockdown on cognitive behavior in miceObjective To explore the effects of insular cortex Scn1 a knockdown on cognition,anxiety and social interaction in mice.Methods After the construction of insular cortex Scn1 a knockdown mouse model(Ad-Scn1a-si RNA group)and control group(Ad-Scn1a-Δsi RNA group),The two groups of model mice were subjected to cognitive,anxiety,autism and other behavioral tests,including open field test,elevated cross maze test,new object recognition test,three-box interaction test,water maze test and shuttle box test.Nissl staining and Neu N/GFAP immunofluorescence staining were used to analyze the damage of hippocampal neurons and glial cells,so as to determine whether the possible cognitive dysfunction was related to secondary hippocampal injury caused by epilepsy or the primary loss of Scn1 a in insular cortex.Results1.In MWM(Morris water maze,MWM)navigation experiment,it was found that the escape latency of mice in Ad-Scn1a-Δsi RNA group was significantly longer than that in AdScn1a-Δsi RNA group(P <0.001).Mice in the Ad-Scn1a-si RNA group had to swim longer distances to reach the hidden platform than those in the Ad-Scn1a-Δsi RNA group(P<0.05).In MWM space exploration experiment,the time of crossing the target quadrant and the time spent in the target quadrant within 60 seconds in Ad-Scn1a-Δsi RNA group was significantly lower than that in Ad-Scn1a-Δsi RNA group(P<0.05).In the new object recognition(NOR)test,there was no significant difference in the time of exposure to new objects between the two groups(P>0.05).In the shuttle-box(Shuttle-Box,SB)tests,the active escape times of mice in Ad-Scn1a-Δ si RNA group were significantly lower than those in Ad-Scn1a-Δsi RNA group(P < 0.001).2.We evaluated whether insular Scn1 a knockdown resulted in anxiety-like behavior changes in mice using an open field test and an elevated cross maze test.In the open field test,there were no significant differences between the two groups in total travel distance,stay time(s)in the central region and shuttle times(P> 0.05).In contrast to the open field test,the time and times of stay in the open arm of mice in the Ad-Scn1a-Δsi RNA group were significantly lower than those in the Ad-Scn1a-Δsi RNA group in the elevated maze test.3.In the three-box interaction test,it was found that the two groups of mice were more "willing" to contact with Stranger 1 than the empty cage placed at the opposite corner,and there was a significant difference in the contact time of the two groups of mice to Stranger 1 compared with the empty cage(P< 0.05).However,there was no significant difference in the contact time between ad-SCN1A-δ si RNA group and AD-SCN1A-si RNA group after placing Stranger2(P > 0.05).4.Nissal staining showed that there was no significant loss of neurons in hippocampal CA1,CA3 and DG regions,and there was no significant difference between AdScn1a-Si RNA group and Ad-Scn1a-Δsi RNA group(P > 0.05).5.Compared with ad-Scn1a-Δsi RNA group,the expression of Neun in hippocampus(CA1,CA3 and DG)of Ad-Scn1a-si RNA group was not decreased,that is,the number of neurons was not significantly decreased(P>0.05).GFAP fluorescence did not increase the expression of GFAP in Ad-Scn1a-si RNA group,that is,the hippocampal astrocytes were not damaged(P > 0.05).Conclusion1.Insular Scn1 a knockdown in mice can cause two comorbidities of DS,namely cognitive dysfunction and anxiety-like phenotype,but does not cause social interaction disorders.2.The cognitive impairment and anxiety-like manifestations in DS Scn1 a knockdown insula mice were not associated with secondary hippocampus injury,but with the loss of primary insula Nav1.1 sodium channel.Part ⅢMechanism of insular cortex Scn1 a gene in Dravet syndromeObjective To explore the possible mechanisms of insula in Dravet syndrome in Scn1 a knockout mice.Methods A Scn1 a gene knockout Dravet syndrome(Scn1a+/-KO)mouse model was constructed.The experiment was divided into wild group(WT)and KO group.1.Breeding and identification of mice: KO and WT mice were first bred and identified.2.The 5-week-old WT and KO mice were identified for heat induction,and the epileptic behavior and Electroencephalogram(EEG)activity of each mouse were recorded.3.Three KO and three WT mice were randomly selected from the two groups of mice,and the insular cortex tissues were dissected,and the differentially expressed proteins between the two groups were analyzed by tandem mass spectrometry(TMT).Functional enrichment(GO)and pathway enrichment(KEGG)of genes were analyzed by bioinformatics tools to screen out relevant pathways and capture relevant important differential proteins,and then parallel response monitoring(PRM)was used to verify the differential expression of proteins.Results1.According to the first exon sequence of Scn1 a Dravet syndrome mouse gene,a common primer,WT reverse primer and mutant reverse primer were designed to amplify THE DNA fragment by polymerase chain reaction(PCR).Because the resistance box of the first exon of the mouse Scn1 a gene was replaced by neomycin,the expected PCR products of WT mice were 357 BP,while those of heterozygous mice were 200 BP and 357 BP.2.The incidence of epilepsy in Scn1a+/-KO mice was up to 80% under heat-induced conditions,while no seizures of any grade or form were observed in WT mice.The mean temperature of epileptic seizure induced by heat in KO mice was 40.0±0.4℃.EEG showed that KO mice showed a large amount of high-spikthorn wave discharge,while WT mice showed no epileptic wave during heat induction.3.A total of 260 differentially expressed proteins were identified in the insular cortex of mice,of which 140 proteins were up-regulated(53.8%)and 120 proteins were down-regulated(46.2%).Thirty of the 260 differential proteins(except Scn1 a gene)belonged to epilepsy and seizure-related genes and developmental genes.The top 5 most significant and most relevant molecular functions of GO biological enrichment are protein transport regulation,peptide transport regulation,heart development,muscle cell differentiation and cell response to heat regulation.In terms of biological processes,the top 5 most significant and most relevant activities are lyase activity,carboxypeptidase activity,carboxylyase activity GDP-dissociative inhibitory protein binding and cysteine peptidase activity.KEGG analysis showed that the most significant pathways were Ras signaling pathway,GABAergic synapse,Glutamatergic synapse,Wnt signaling pathway and retrograde enannabinoid signaling pathway.PRM verified that the expression changes of epilepsy and developmental related proteins in Ntrk2,Epha4,Asns,Cacna2d2,Dnajc6,Cadps2,Ank2 and Atp1a1 were consistent with TMT results.Conclusion1.The insula organization proteomic analysis showed that the Ras signaling pathways,GABAergic synapses,Wnt signaling pathways,retrograde endocannabinoids signaling pathways closely related to these four pathways and epilepsy,and factors related to these signaling pathways or protein may play a role in the occurrence of DS epilepsy,also for the treatment of DS may provide valuable potential targets.2.Glutamatergic synaptic signaling pathway may play a limited or even insignificant role in the occurrence of DS.3.SCN1 A gene mutations lead to other related epilepsy and developmental proteins including Ntrk2,Epha4,Asns,Cacna2d2,Dnajc6,Cadps2,Ank2 and Atp1a1,which provide further research directions for the subsequent treatment of DS.