Genotype-phenotypic Characteristics And Underlying Mechanisms Of Developmental And Epileptic Encephalopathy Associated With KCNA2

Objective:Kv1.2 channels,encoded by KCNA2,play critical roles in regulating neuronal excitability and neurotransmission.KCNA2mutations are associated with a spectrum of neurodevelopmental disorders,including epilepsy,mental retardation,ataxia and behavioral disorders.According to the changes in biophysical properties of muted Kv1.2 channels,KCNA2 mutations could be divided into three groups,which are loss of function（LOF）,gain of function（GOF）,loss-and gain of function（LOF and GOF）.The substantial phenotypes of KCNA2-related epileptic encephalopathy caused by different mutations overlap,but they differ in some aspects.For example,LOF mutations lead to a relatively mild phenotype,while GOF mutations lead to more severe phenotypes.As the association between genotype and phenotype of KCNA2-related epileptic encephalopathy is not fully clarified due to the lack of cases,the diagnosis and treatment of this disease are facing a great dilemma.In addition,epilepsy is a chronic brain disease caused by the abnormal firing of neurons in the brain,which is contributed by excitatory/inhibitory imbalance.Both the actions of excitatory and inhibitory neurons play essential roles in the homeostasis of neural networks.Kv1.2 channels are highly expressed in these neurons,however,the effects of deficiency of Kv1.2 on excitatory or inhibitory neurons have not been elucidated.Methods:（1）clinical data were collected from patients carrying KCNA2 variants,who visited Xiangya hospital and other hospitals in China from 2015 to 2022.（2）KCNA2 wild-type and mutant plasmids were constructed and transfected into Chinese hamster ovary cells（CHO）.Immunofluorescence and Western-blot experiments were used to study the effects of KCNA2 mutation on Kv1.2 protein expression.Patch-clamp experiments were used to study the biophysical properties of Kv1.2mutated channels in homozygous and heterozygous conditions.（3）The protein expression in neurons was detected by immunofluorescence experiments,and the neuronal function was examined by electrophysiological experiments.（4）The KCNA2 floxp mice were constructed and hybridized with Nestin-Cre mice,Camk IIα-Cre mice,PV-Cre mice and SST-Cre mice in order to obtain conditional knockout mice.（5）Kv1.2 knockout was identified by RT-PCR and Western-blot.（6）Phenotypes,body weight,spontaneous epilepsy and survival time of mice in each group were monitored.Video electroencephalogram（EEG）was used to detect epileptic seizures and epileptic discharge in mice.The sensitivity of KCNA2 knockout mice to febrile convulsions was investigated by high-temperature induction.（7）HE staining was used to evaluate whether the structures in the brain and cerebellum were changed in each group.The astrocytes of wild-type,Nestin-KCNA2^-/-and Camk2α-KCNA2^-/-mice were studied by immunofluorescence experiments.（8）Patch-clamp experiments were used to study the excitability of pyramidal neurons in the hippocampal CA1 region and 2/3layer of the cortex of Nestin-KCNA2^-/-and Camk2α-KCNA2^-/-mice.Results:（1）In this study,we reported nine different KCNA2variants in nine different individuals（R294H,H310R,S392F,V399E,A403V,F413Y,N414K,R354Q,F333L）.（2）The protein expression was reduced in CHO cells and neurons expressing KCNA2 mutants,and the function of Kv1.2 channels was impaired.（3）KCNA2 variants with different biophysical properties exert different effects on neuronal excitability.（4）In conditioned knockout（c KO）mice,Nestin-KCNA2^-/-and Camk2α-KCNA2^-/-mice showed spontaneous seizures,early death and epileptic charges observed by synchronous video EEG,while no spontaneous seizures or epileptic charges were detected in PV-KCNA2^-/-and SST-KCNA2^-/-mice.（5）Nestin-KCNA2^+/-and Camk2α-KCNA2^+/-mice showed seizures triggered by high temperature,with an average latency of 225 s and 539 s,respectively.（6）HE staining results showed that the brain structures of Nestin-KCNA2^-/-,Nestin-KCNA2^+/-,Camk2α-KCNA2^-/-,PV-KCNA2^-/-and SST-KCNA2^-/-mice were normal.（7）Immunofluorescence results showed that the GFAP expression was increased in the hippocampus of Nestin-KCNA2^-/-and Camk2α-KCNA2^-/-mice.（8）Electrophysiological results showed the increased excitability of hippocampal CA1 pyramidal neurons and pyramidal neurons in 2/3 layer of the cortex in Nestin-KCNA2^-/-mice.In Camk2α-KCNA2^-/-mice,the excitability of pyramidal neurons in the hippocampal CA1 region was not impaired,but that of pyramidal neurons in the 2/3 layer of the cortex was increased.Conclusions:（1）The present study reported novel likely pathogenic/pathogenic mutations of KCNA2 and expanded the phenotypic spectrum.The reduced protein expression and altered biophysical properties contributed to the impairment of neuronal excitability,thus leading to epilepsy.（2）The KCNA2 conditional knockout mice were constructed successfully,and we confirmed that Kv1.2 deficiency was an important cause of KCNA2-related epilepsy by inducing neuronal hyperexcitability.（3）The deficiency of Kv1.2 in forebrain pyramidal neurons plays pivotal roles in the pathogenesis of epilepsy,whereas the deficiency of Kv1.2 in PV or SST neurons is not sufficient to cause epilepsy.