Overexpression Of SOD1G41S And SOD1G41D In The MPFC Of Mouse Alter Cognitive Behavior

Amyotrophic lateral sclerosis(ALS)has always been considered as a simple motor neuron disease.Superoxide dismutase 1(SOD1)is also a common mutated gene that causes ALS.we found that some patients with SOD1 gene mutation,such as G41S and G41D were diagnosed with Mild cognitive impairment.To explain the problem,we choosed SOD1G41S and SOD1G41D mutations,carried by building anthropogenic SOD1 WT,SOD1G41S and SOD1G41D recombinant adeno-associated virus,stereotaxic injected into mice bilateral medial prefrontal cortex,to observe the effects of cognitive behavior in mice.The first part:Construction of recombinant adeno-related viruses SOD1 WT,SOD1G41S and SOD1G41DObjective To construct recombinant adeno-associated virus vectors carrying SOD1 WT,SOD1G41S and SOD1G41D and determine their titer、purity.Methods First,the cDNA of SOD1 WT,G41S,G41D,EGFP and m Cherry was synthesized artificially.The plasmid PT-1727 was transformed into a linear vector by Spe I and Eco R I.Homologous recombined of the target gene and the linearized vector,recombinant plasmid transfected stbl3 cells,choosed positive cloning sequence,sequenced and correct after extraction baculovirus shuttle carrier bacmid,baculovirus shuttle carrier bacmid transfected sf9 insect cell packed,tpurificated and concentrated the virus for preparing the final viral vector,Virus titer and purity were determined by q-PCR and SDS-PAGE,and stored at-80℃ for future use.Results The purity and titer of the synthesized,purified and concentrated virus were determined.The titers of SOD1 WT,SOD1G41S and SOD1G41D r AAV were 2.63E+12 vg/L,2.25E+12 vg/L and 2.23E+12 vg/L,respectively.After SDS-PAGE electrophoresis and silver staining,the virus samples showed there were no obvious heteroproteins except the three obvious protein bands of VP1,VP2 and VP3.Conclution Recombinant adeno-related vectors of SOD1 WT,SOD1G41S and SOD1G41D were successfully constructed,and the virus purity and titer met the requirements of subsequent trails.The second part:the effects of SOD1G41S and SOD1G41D overexpression in the medial prefrontal cortex of mice on cognitive behaviorObjective to investigate the effects of SOD1G41S and SOD1G41D on cognitive behavior of mice and related neuropathological changes.Methods The experimental group was divided into SOD1 WT group、SOD1G41S group and SOD1G41D group,and a blank control group was set as the control group(n=16).the 60 n L corresponding virus was injected into the bilateral medial prefrontal cortex.one month later,the mice were subjected to open field test,Y maze spontaneous alternations experiment,three-box social experiment,and trace fear condition test were used to observe the behavioral changes of transgenic mice,and observed the changes in the morphology and number of neurons and glial cell in the brain region of virus injected through Nissl staining and immunofluorescence.Results In the open field test,the moved distance of SOD1 WT group was obviously higher than that of SOD1G41D group(p=0.34).In the Y maze spontaneous alternations experiment,the arm number,maximum alternations and actual alternations of the SOD1 WT group and the SOD1G41S group were significantly higher than that of the SOD1G41D group(P<0.05),indicating that SOD1 WT and SOD1G41S had positive effects on the spontaneous activities of experimental animals(P>0.05).In the three box social experiment,SOD1G41D group showed no special preference for the strange mice and had social defects(p=0.199).In the trace fear conditioning test,the SOD1G41S group showed significantly better memory for context-related fear memory and sound-related fear memory than the other groups(p<0.05).In Nissl staining,The number of neurons in the SOD1G41S group showed significant cell body shrinkage and decreased,while the number of cells in the SOD1G41D group significantly decreased and the staining became lighter.In immunofluorescence staining,the number of microglia in the SOD1G41S group was significantly higher than that in the other groups.Conclusion SOD1G41S and SOD1G41D may cause cognitive and behavioral changes in mice,accompanied by neuropathological changes.