Construction Of Overexpressed SOD1G41S And G41D Recombinant Adeno-associated Virus Vectors And Their Roles In Amyotrophic Lateral Sclerosis N2a Cell Model

Amyotrophic lateral sclerosis(ALS)is a serious degenerative neurological degenerative disease that selectively inflicts upper and lower motor neurons in the spinal cord,brainstem and cerebral cortex.The study found that 10%ALS was familial ALS(fALS),of which about 20%fALS associated with superoxide dismutase 1(SOD1)gene mutation.There were two mutations SOD1G41S and G41D on the SOD1 gene.The clinical manifestations of patients with these two mutations were significantly different.The disease course of fALS with SOD1G41S mutant was rapid and the average survival time was about 11.6 Months.The patients with SOD1 G41D mutations had a slow progress,and the average survival time was about 17 years.Since Rosen et al in 1993 found that mutations in SOD1 gene related to ALS pathogenesis,the researchers carried out a series of studies about this pathogenesis,but it is not clear.It is thought that oxidative stress,misfolded protein aggregation,mitochondrial dysfunction,glutamate excitotoxicity,axonal transport damage,non-neuronal cell function are related to the pathogenesis of ALS.In addition,clinical studies have found that patients with ALS may appear high excitability of the neuron axons and increased persistent sodium current,the patients showed muscle spasms and muscle bundle tremor.Previous studies also found that neuronal hyperexcitability has been documented in primary spinal motor neuron cultures,in ALS motor neurons derived from induced pluripotential stem cells and in slice preparations from transgenic mice carrying the fALS mutation G93A.The voltage-gated sodium channel(VGSC)is an important component of the neuronal excitatory and action potential generation and conduction.Therefore,the change of the electro-physiological properties of the channel will affect the excitability of the neurons,and lead to the occurrence of the disease.Researchers also found that disturbances in voltage-gated sodium channel currents at early stage of the disease in ALS SOD1G93A model mice.However,there is no study to explore the characteristics of VGSC in SOD1G41S and G41D mutations.Therefore,in order to explore the pathogenesis of SOD1G41S and G41D mutations in ALS further,this study was divided into two parts.The first part,the wild type(WT)SOD1,SOD1G41S and G41D recombinant adeno-associated virus(rAAV)vectors were constructed.The second part,the mouse neuroblastoma N2a cells(N2a)was infected by the rAAV to construct ALS cell model,then observing the impacts of electrophysiological properties of VGSC and the expression of apoptotic molecules after overexpression of the two mutant proteins in vitro.Part? Construction and identification of rAAV vectors of SOD1WT,SOD1 G41S and SOD1G41DObjective:To construct overexpressed SOD1WT,SOD1G41S and SOD1G41D rAAV vectors.Methods:First,the target gene fragments were amplified by PCR,then the pMT121 vector(pAAV-hSyn-bGlobin inton-EGFP-pA)was digested with EcoR I and Hind HI endonucleases,and the purified linearized expression vector was recovered.Furthermore,DH5? competent cells were transformed after homologous recombination in the recovered linearized expression vector and target gene fragments under the action of the seamless reaction solution.Then the transformants were sequenced after identification using colony PCR If the results of sequencing were right,then we started to plasmid extraction.Finally the target plasmids and helper plasmids was co-transfected into HEK293 cells for virus packaging.Then the virus particles were purified,concentrated,and titrated by RT-PCR.Results:PCR results showed that specific RFP,GFP,SOD1WT,5'SOD1G41S/G41D,3'SOD1G41S/G41D bands were found at about 765 bp,792 bp,521 bp,175 bp,367 bp.We recovered 4470 bp linearized vector after using EcoR ? and Hind ?endonucleases to digest the pMT121 vector.The results of colony PCR showed that the positive SOD1 WT rAAV plasmid was 711 bp,and the positive SOD1G41S and G41D were 721 bp.In addition,the result of sequencing were right.The SOD1 WT,SOD1G41S and G41D rAAV particles were obtained after 72 h,and their titers were 2.43×1012 v.g./mL,2.28×1012 v.g./mL,and 2.52×1012 v.g./mL respectively.Lastly,the virus particles were packaged and stored at-80?.Conclusion:The rAAV vectors,which overexpress SOD1WT,SOD1G41S and SOD1G41D,has been successfully constructed.What's more,the virus purification,concentration and titer meet the requirements of subsequent experiments.Part II The role of SOD1WT,SOD1G41S and SOD1G41D rAAV vectors in ALS cell modelObjective:To explore the differences of VGSCs' electrophysiological characteristics in neurons and the possible molecular mechanisms between two mutations G41S and G41D in SOD1 in ALS.Methods:N2a cells were infected with SOD1WT,SOD1G41S and-G4ID rAAV vectors in vitro and divided into three experimental groups:SOD1WT group,SOD1G41S and SOD1G41D group,and N2a cells infected with rAAV without target gene were negative Control group.The VGSC currents of four groups of cells were recorded by whole-cell patch clamp technique,then the curves of channel fast activation and steady-state inactivation were drawn and related parameters were analyzed Moreover,the expression of apoptotic molecular Cleaved caspase-3 was compared after 24,48,72h of virus infection.Results:Compared with Control and SOD1WT cells,the peak currents of infected SOD1G41S and G41D cells were increased significantly(P<0.05),and G41S were significantly higher than G41D(P<0.01).Potentials for half-maximal activation and inactivation of SOD1G41S and G41D were significantly higher than the other two groups(P<0.05),but potentials for half-maximal activation of G41S were higher than G41D(P<0.05),while there were no differences of potentials for half-maximal inactivation between SOD1G41S and G41D groups(P>0.05).And there were no differences of slope factors of activation and inactivation curves in each group(P>0.05).Although there were no differences of the expression of Cleaved caspase-3 after 24,48h of infection in SOD1WT,G41S,G41D groups,but the expression of Cleaved caspase-3 in SOD1G41S,G41D groups higher than that of SOD1WT group after 72h,and SOD1G41S group were higher than G41D group.Conclusion:SOD1G41S and G41D mutations improve the activity of neurons by accelerating the process of VGSC fast activation and inhibiting the process of voltage-gated sodium channel inactivation,and VGSC activity of G41D was significantly higher than that of G41S mutation.The expression of apoptotic molecular Cleaved caspase-3 in G41S were higher than G41D mutation.