Study On Pathogenesis And Precise Drug Intervention Of Epilepsy Induced By Loss-of-function Mutation Of NMDA Receptor Subunit GluN2A/GRIN2A

Background:N-methyl-D-aspartate(NMDA)receptor(NR)is the main receptorof the excitatory neurotransmitter-glutamate in the brain.As a ligand-gated ion channel,it is mainly involved in mediating the generation and transmission of excitatory postsynaptic current(EPSC)in the synapse,and also participates in the presynaptic GABA release of γ-aminobutyric acid(GABA)outside the synapse,thus affecting the generation of inhibitory postsynaptic current(IPSC).The imbalance between excitatory synaptic signals and inhibitory synaptic signals,which constitute the neuronal network,is an important cause of epilepsy.Therefore,NR should be a key factor affecting the excitatory/inhibitory balance of neural networks.Through some clinical cases and cellular receptor function analysis,it has been confirmed that both gain-of-function(Go F)gene mutation and loss-of-function(Lo F)gene mutation can lead to epilepsy.As one of the subunits of NR,78% of patients with GluN2 A /GRIN2 A subunit mutation show epilepsy,which is the research focus of NR gene mutation-related epilepsy.At present,the pathogenesis of epilepsy caused by Lo F type NR gene mutation is not clear.It is of great significance to research through in vivo/vitro electrophysiological and behavioral studies at the level of transgenic animals,and further clarify the pathogenic characteristics and mechanism of this type of gene mutation.In addition,some allosteric modulators can reverse the functional abnormalities of NR caused by some gene variation.Through cellular screening and animal intervention,positive allosteric modulators(PAMs)that are more effective for Lo F type NR may be screened out,thus providing important theoretical and practical support for clinical precision treatment.Methods:In this study,the typical Lo F mutation GluN2A/GRIN2A-V685 G confirmed by clinical reports and cellular receptor function analysis was used as the study site.First of all,take advantage of Xenopus laevis oocytes to evaluate NR function at a cellular level,and chose pregnenolone sulfate(PS),24(S)hydroxy cholesterol(24(S)-HC),tobramycin,arachidonic acid,D-serine,spermine and glutamic acid,glycine as alternative medicine sensitive drug screening on a cellular level according to the literature.In the end,chose 1 or 2 of the most sensitive drugs as intervention.Then GluN2A/GRIN2A-V685 G transgenic mice were prepared by embryonic microinjection.The mice were divided into wild type group(WT),loss-of-function mutation group(Lo F),wild treatment group(WT+drug),mutation treatment group(Lo F +drug).In terms of behavior,spontaneous epileptic seizures were observed in each group,and epileptic grade was scored.If there was no spontaneous epileptic phenotype,hyperfebrile epileptic model was established in each group to evaluate the threshold of epileptic seizures(onset latency).Then the cognitive status of mice in each group was evaluated by novel object recognition experiment(NOR)and novel object location recognition experiment(NOL).Tail suspension test,sugar water preference test and forced swimming test were used to evaluate the depressive behavior of mice in each group.After that,cortical electroencephalogram abnormal discharge levels were measured in vivo.Then,the brain of each group was taken,and the EPSC mediated by the postsynaptic membrane of pyramidal neurons in hippocampal CA1 region and m IPSC mediated by GABA interneurons were detected by patch clamp technique,and then the action potential of whole pyramidal neurons was detected.Real-time PCR and Western blot were used to detect the expression of GluN2 A subunit.Results:In this study,NR function evaluation was carried out on Xenopus laevis oocytes at the cellular level.The results showed that the NR current on the surface of frog eggs transfected with GRIN2A-V685 G mutant c DNA decreased significantly compared with the control group,and the function almost lost.The results showed that PS and 24(S)-HC could partially reverse the NR function of the mutant,with 24(S)-HC being the most sensitive.According to relevant literature,efavirenz(EFV),which can increase the level of 24(S)-HC in the brain and has been clinically used in anti-HIV therapy,was finally selected as the intervention drug.Then,the animals were divided into WT group,Lo F group,WT+ EFV group,and Lo F + EFV group.The behavioral evaluation showed that no spontaneous seizures occurred in each group.After the establishment of the hyperfebrile convulsion model,the latent period of epileptic seizures in the Lo F group was shorter,while the latent period of epileptic seizures in the Lo F +EFV group was significantly longer than that in the Lo F group.There were no significant differences in the intensity and duration of seizures in each group.In the evaluation of cognitive function,each group in the novel object recognition experiment(NOR)have no obvious difference,in the novel object location identification experiment(NOL),Lo F group had no significant difference with WT group,but the observed trends show the identification index of Lo F group is slightly lower than WT group.The NOL identification index of Lo F+EFV group is more obviously than Lo F group,there are significant difference.Therefore,Lo F mice may be spatial memory loss and EFV can reverse the trend.In the evaluation of depressive behavior,no significant difference was found among all groups,and the immobile time of Lo F group and Lo F +EFV group was shorter in the tail suspension immobile test.In vivo electrophysiological tests showed that there was a significant increase in the frequency of seizure-like events(SLEs)in the LOF group,but less in the LOF +EFV group,indicating that EFV could inhibit the abnormal discharge in the brain of mutant mice.Then using patch clamp tests synaptic NR(EPSC)current level,it shows current amplitude and the maximum current area of Lo F group were significantly lower,Lo F+EFV group is obviously higher than the Lo F group,the differences were significant.m IPSC level detection shows that peak amplitude of the current(peak amplititude)of all groups of found no obvious difference,but in the current distribution of frequency(event frequency)tests,the Lo F group significantly decreased compared with WT group,while Lo F+EFV group without falling.Subsequently,whole-cell action potential detection showed that compared with WT group,action potential threshold of pyramidal cells in hippocampal CA1 region of mice in Lo F group decreased,action potential amplitude increased,action potential input resistance increased,and the number of action potential increased significantly under the same current stimulation.The above indexes of action potential were reversed in the Lo F+EFV group compared with the Lo F group,but the difference was not significant.Real-time PCR and Western blot analysis showed no significant difference in GluN2 A expression among all group.Conclusions:(1)GRIN2A-V685 G mutation can cause dysfunction of the Glun2 A subunit ligand binding domain(ABD),resulting in loss of glutamate potency,thus leading to dysfunction of NR.(2)The NR-mediated EPSC of pyramidal neurons in hippocampal CA1 region of GluN2A/GRIN2A-V685 G transgenic mice was decreased.However,the decrease of m IPSC mediated by GABA-mediated interneurons with the involvement of extra-synaptic NR was more significant.Therefore,the excitability/ inhibition balance of the neuronal network may be disbalanced,leading to increased excitability of pyramidal neurons.(3)GluN2 A /GRIN2A-V685 G transgenic mice are more prone to epileptic seizures and abnormal potential of field potential in the hippocampal CA1 region and cortical electroencephalogram increased in these mice.(4)The anti-HIV drug efavirenz(EFV)can partially restore the function of NR lost by GluN2A/GRIN2A-V685 G mutation by increasing the level of 24(S)hydroxyl-cholesterol in the brain,reducing the abnormal disactivation of hippocampal and cortex,alleviating the seizure of transgenic mice.