EMX2 Gene Mutation And The Role And Mechanism Of KIF17 In Epilepsy

PART 1: THE POTENTIAL MECHANISM OF EMX2 MUTATION(C.478G>T)AFFECTING SUSCEPTIBILITY TO EPILEPSYBackgroundEpilepsy is a common and frequently occurring neurological disease that seriously endangers human health.Genetic factors are among the causes of epilepsy.We previously applied gene-capture sequencing technology and identified a missense mutation in EMX2(c.478G>T)in a family with generalized epilepsy.This mutation led to an amino acid change from Ala to Ser at position 160(p.Ala160Ser)that may have affected the function of the protein.EMX2 plays a very important role in the development of the nervous system;therefore,mutations in this gene may lead to nervous system developmental disorders or induce abnormal neural network activity that could contribute to the development of epilepsy.ObjectiveTo explore the effect of the EMX2 mutation(c.478G>T)on epileptic susceptibility and the underlying mechanism of this effect.MethodsIn this study,we mainly used molecular biology techniques,primary neuronal cultures,in utero electroporation,a CRISPR/Cas9-based conditional gene knock-in mouse model,stereotaxic injections of Cre recombinase adeno-associated virus,a pentylenetetrazole(PTZ)kindling model and Chromatin Immunoprecipitation-Sequencing(Ch IP-Seq).The EMX2(160Ala)plasmid and the EMX2(160Ser)plasmid were constructed using gene cloning.The plasmids were transfected into HEK293 cells,and the subcellular localization patterns of wild-type and mutant EMX2 were detected by immunofluorescence.Cycloheximide tests were combined with immunoblotting to detect the degradation of wild-type and mutant EMX2.Primary neurons were transfected with plasmids to observe the growth of primary and secondary neurites.Mouse brains were electroporated in utero with EMX2(160Ala)or EMX2(160Ser)plasmids for analysis of the migration of cortical neurons.Conditional gene knock-in mice(Emx2-A161S)were constructed using CRISPR/Cas9 technology.Based on the principles of the Cre/lox P recombinase system,we established Emx2-161S(CKI/WT)and Emx2-161A(CKI/WT)mouse models by injecting a Cre recombinase-associated virus or control adeno-associated virus into the hippocampus of conditional knock-in mice.The mice were intraperitoneally injected with a subthreshold dose of PTZ to establish a chronic kindling model,and seizure scores were then assessed across 15 injections.Finally,we used Ch IP-Seq to analyze the downstream targetgenes of EMX2 in HEK293 cells transfected with EMX2(160Ala)or EMX2(160Ser)plasmids.ResultsThere was no difference in the subcellular localization of the wild-type and mutant EMX2 proteins,and the cycloheximide experiment indicated that there was no significant difference in protein stability between wild-type and mutant EMX2.The evaluated EMX2 mutation affected the growth and development of neuronal primary and secondary neurites and led to migration disorders in cortical neurons.In the PTZ kindling model,epileptogenesis occurred more quickly,and the survival rate was lower in Emx2-161S(CKI/WT)mice than in Emx2-161A(CKI/WT)mice.Ch IP-Seq suggested that this mutation prevents EMX2 from precisely regulating a range of target genes.ConclusionsThe missense mutation c.478G>T may affect the ability of EMX2 to transcriptionally regulate downstream target genes,leading to neuronal migration and developmental disorders that increase epileptic susceptibility.PART 2: THE POTENTIAL MECHANISM OF KIF17 IN MODULATING EPILEPTIC SEIZUREBackgroundEpilepsy is a chronic brain dysfunction syndrome with a variety of causes.Recurrent epileptic seizures can cause serious harm to patients' physical and mental health.Abnormal synchronized neuronal discharge caused by an imbalance between excitation and inhibition of the central nervous system is considered an important mechanism of epilepsy.Further research into the molecular mechanisms that influence this imbalance is necessary to explore new antiepileptic targets.Previous studies have confirmed that KIF17 plays an important role in the transport and regulation of the NR2 B subunit.Therefore,we speculate that KIF17 may modulate epileptic seizures.ObjectiveTo investigate the role and possible mechanism of KIF17 in modulating epileptic seizures.MethodsIn this study,we mainly used molecular biology,recombinant lentiviral vector construction and stereotactic injection,behavioral analysisof the kainic acid(KA)-induced temporal lobe epilepsy(TLE)model,and whole-cell patch-clamp recording.Cortical tissue harvested from the temporal lobes of patients with drug-refractory TLE or brain trauma was collected for detection of KIF17 expression by Western blotting.KIF17 expression was also detected by Western blotting in the hippocampus of the KA-induced TLE mouse model and control mice.The effect of KIF17 on the behavior of the KA-induced epilepsy model was observed by constructing lentiviruses to locally overexpress or knock down KIF17 in the mouse hippocampus.Whole-cell patch-clamp recordings were performed on hippocampal CA1 pyramidal neurons in an Mg2+-free-induced brain slice model of epileptiform activity.The action potentials(APs),miniature excitatory postsynaptic currents(m EPSCs),and miniature inhibitory postsynaptic currents(m IPSCs)were recorded to determine the effect of KIF17 overexpression or knockdown on electrophysiology.ResultsWestern blotting analysis suggested that KIF17 expression levels were increased in brain tissues from pharmacoresistant TLE patients compared with those from trauma patients and in the hippocampus derived from KA-induced epileptic mice with spontaneous recurrent seizures(SRSs)compared with those from control mice.In the KA-induced TLE mouse model,behavioral observation showed that KIF17 overexpressionshortened the latency of seizure onset and increased the number of SRSs,while KIF17 knockdown had the opposite effects.Whole-cell patch-clamp recordings confirmed that AP frequency and m EPSC frequency and amplitude were increased or decreased with KIF17 overexpression or knockdown,respectively.However,neither the frequency nor the amplitude of m IPSCs was affected by upregulation or downregulation of KIF17.ConclusionsKIF17 may modulate epileptic seizures by affecting excitatory synaptic transmission.