The Effect And Potential Mechanism Of Muatnt KIF1A Involved In Epilepsy

BackgroundEpilepsy is one of the common diseases of the nervous system,affecting over than 50 million people worldwide,and genetic factors are one of the main causes of epilepsy.According to the latest definition of the International League Against Epilepsy(ILAE),genetic epilepsy is defined as the direct results of a known or presumed genetic defect where seizures are the core symptom of the disorder,these mutant genes causes dysfunction of neuronal development or neuronal migration,alteration of ion channel property,abnormal synaptic transmission(excitatory and inhibitory synapse).KIF1 A,encoding by KIF1 A gene,is responsible for the transport of membranous organelles and synaptic vesicles protein and plays a significant role in brain development and synaptic plasticity.Mutation in KIF1 A have been associated with hereditary spastic paraplegia,hereditary sensory and autonomic neuropathy type 2,PEHO syndrome,Rett syndrome,Autism spectrum disorders and intellectual disability.Only a handful of reports have related KIF1 A gene mutation to epileptic seizure.Thus,we hypothesis that mutations in this gene may involved in epilepsythrough causing brain development disorder or imbalance between excitatory and inhibitory synaptic transmission.ObjectiveTo determine whether the KIF1 A gene mutation will affect the susceptibility to epilepsy and the possible mechanism.MethodsWe conducted a gene sequence in a family with six patients with generalized epilepsy over three generations and identified a rare heterozygous mutation(c.1190C>A,p.Ala397Asp)in KIF1 A.Bioinformatics analysis found that this gene mutation is harmful.First,the zebrafish model was constructed using Tol2 expression vector,at 5 days postfertilization(d.p.f.),locomotion tracking plot and local field potential recording were used to verify the epilepsy phenotype of the KIF1 A gene mutation(c.1190 C> A,p.A397D)in the family.Then the primary neuron culture technique,whole-cell patch clamp technique,immunofluorescence technique were used to explore its possible mechanism.Human wild-type KIF1A(397Ala)plasmid and the human mutant KIF1A(397Asp)plasmid were transfected into primary cultured neurons,then resting membrane potential,rheobase,action potential,miniature excitatory post-synaptic currents(mEPSCs)and miniature inhibitory post-synaptic currents(mIPSCs)of these neurons were measured by whole cell patch clamp technique.Finally,immunofluorescence technique was introduced for theassessment of the density of excitatory synapse and neuronal morphology in neurons that transfected with human wild-type KIF1A(397Ala)plasmid and human mutant KIF1A(397Asp)plasmid.Results1.No obvious morphological abnormalities were found in zebrafish larvae.In zebrafish larvae with overexpressed mutant kif1 a,40.6% of zebrafish movements can be defined increased significantly,and 39.1% of zebrafish movements can be characterized as baseline movements;In zebrafish larvae with overexpressed WT kif1 a,11.5% of zebrafish displayed a significant increase in movement,and 67.2% of the zebrafish had baseline movement(40.6%VS11.5%,P<0.05,39.1% VS67.2%,P<0.05).2.In local field potential recording,we observed spontaneous epileptiform activity(Polyspiking discharges)in 42.2% of mutant larvae and 9.8% of WT larvae(42.2%VS9.8%,P<0.05).3.There were no difference in resting membrane potential,rheobase,and frequency of action potential in neurons that transfected with human WT KIF1A(397Ala)plasmid and human mutant KIF1A(397Asp)plasmid.4.Compared with the primary neurons transfected with human WT KIF1A(397Ala)plasmid,the frequency of mEPSCs were increased significantly in neurons that transfected with human mutant KIF1A(397Asp)plasmid.There were no difference in frequency and amplitude of mIPSCs between two groups.5.Compared with neurons transfected with human WT KIF1A(397Ala)plasmid,the density of dendritic spines,vGLUT,vGLUT-positive and PSD-95 clusters were increased significantly in neurons that transfected with human mutant KIF1A(397Asp)plasmid.6.There were no difference in neurons complexity,and number of primary neurites and secondary neurites between two groups.ConclusionThe mutant KIF1A(c.1190 C> A,p.A397D)increased the susceptibility to epilepsy by increasing the density of mature excitatory synapses.