The Effect Of AAV9-IGF1 On Threshold Potential Of Motor Neurons In SOD1G93A Mice

SOD1 gene mutations have been identified more than 100,account for~20% of family ALS patients.Clinical phenotype and pathological basis of human SOD1 mouse model is most similar to sporadic and family ALS patients'.Among them,three(SOD1G85R,SOD1G37 R,and SOD1G93A)have been widely applied in the transgenic mouse model of ALS.SOD1 is homodimer enzyme that expresses 153 amino acid polypeptide and each unit combines with a copper and a zinc ion.Copper is responsible for the catalytic activity of enzymes and zinc is responsible to stablilize the protein structure.Main function of SOD1 include the removal of free radicals by catalyzing superoxide anion into oxygen and hydrogen peroxide,through glutathione peroxidase and catalase decomposing into water.SOD1 widely exist,mainly in the cytoplasm,and can also be detected in the mitochondria and other organelles.AAV is a kind of small,no envelope virus and has been the theme in the field of gene therapy research.Mediated gene transduction by AAV is safe and effective and is also a kind of treatment tools as clinical research.Efficacy and safety of the r AAV has been indicated by significant evidence of a large number of animal models.Among them,injecting r AAV-IGF1 into respiratory and hind leg muscle prolongs the survival and delays the cutting of the movement.AAV carrier has the character of reverse transmission,such as the selective target tissue spinal cord neurons,allowing IGF1 of local secretion widely affecting the surrounding cells and is not confined to transfer virus to the motor neuron cells.In the central nervous system,1,2,5,8,9 and recombination are the most studied in AAV serotypes.The effect of serotypes depends on brain distribution,species,and target cell types.The serotype was transduced effectively to neurons,but the transduction of astrocytes,oligodendrocytes,and microglia is restricted,which can be improved by usingcell promoters.Studies confirm that AAV9 can pass the blood brain barrier and effectively target to neurons and astrocytes in the brain and spinal cord.In the mice,AAV9 mediated ways through the medulla pool to target gene delivery,which transfer better and cross the blood brain barrier better in the central nervous system compared with other AAV serotype.To explore electrophysiology change of ALS motor neuron at the stage before symptom and impaction of IGF1 on electrophysiology,this experiment adopts two ways,such as lateral ventricle injection,parenchymal injection,to inject AAV9-IGF1 and AAV9-GFP into SOD1G93 A newborn mice,choosing the method of IGF1 more expression to inject AAV9-IGF1 and AAV9-GFP through observing the expression of motor cortex M2 area,and recording action potential of M2 area in 20 to 40 days SOD1G93 A mice to analyze the impaction of AAV9-IGF1 on action potential of SOD1G93 A mice motor neuron at the stage before symptom.Objective:1 We record action potential of SOD1G93 A transgenic and non-tansgenic mice to compare whether there is difference in threshold potential,frequency,amplitude spike latency and clamping speed of action potential;2 We inject AAV9-IGF1/AAV9-GFP into SOD1G93 A newborn mice(born within 24 hours)by lateral ventricle or parenchymal to observe the expression quantity in motor cortex M2 area;3 We record action potential of 20 to 40 days SOD1G93 A transgenic mice injected AAV9-IGF1/AAV9-GFP by parenchymal to compare whether there is difference in threshold potential,frequency,amplitude,spike latency and clamping speed of action potential,to further explore the impaction of IGF1 at early stage before symptom in ALS.Methods: This experiment applys C57/6 mice and SOD1G93 A transgenic and non-transgenic littermate control mice,and SOD1G93 A transgenic mice are identified by the method of PCR amplification.We adopt two ways,such as lateral ventricle injection,parenchymal injection,to inject AAV9-IGF1 and AAV9-GFP.Then we choose brain slices of 20 to 40 daysC57/6 mice and SOD1G93 A transgenic and non-transgenic,applying the methods of immunohistochemistry and immunofluorescence to observe the expression of AAV9-IGF1 and AAV9-GFP in motor cortex M2 area,choosing the method of IGF1 more expression to inject AAV9-IGF1 and AAV9-GFP into SOD1G93 A transgenic newborn mice.We employ whole cell patch clamp technique to record the action potential of M2 in 20 to 40 days mice brain slices to analyze statistically.Results:1 We employed whole cell patch clamp technique to record the action potential of motor cortex M2 area in 20 to 40 days SOD1G93 A transgenic(h SOD1G93A)and non-transgenic mice brain slices: threshold potential between two groups had some difference,but there was no statistical significance;Frequency in SOD1G93 A transgenic group was higher than that in littermate control group,and statistical significance was indicated with injected currents 150 p A(150p A,P=0.04<0.05),but with 200-500 PA currents stimulating,there was no statistical significance;There was no statistical significance for action potential amplitude between the two groups;There was no statistical significance for spike latency between the two groups;Clamping speed in SOD1G93 A transgenic group was higher than that in littermate control group(except 450 p A),and there was statistical significance with injected currents 250 p A(250p A,P=0.032<0.05).2 We adopted two ways,such as lateral ventricle injection,parenchymal injection,to inject AAV9-IGF1 and AAV9-GFP,and applied methods of immunohistochemistry and immunofluorescence to observe the expression of IGF1 in motor cortex M2 area of 20 to 40 days mice brain slices.In motor cortex M2 area of mice brain slices,compared with lateral ventricle injection,the method of parenchymal injection has higher IGF1 expression,and statistical significance was indicated.3 We empolyed whole cell patch clamp technique to record the action potential of motor cortex M2 area in 20 to 40 days SOD1G93 A transgenic mice injected AAV9-IGF1/AAV9-GFP by the method of parenchymalinjection: compared with AAV9-GFP group,threshold potential in AAV9-IGF1 group was significantly lower,and there was statistical significance;There was no statistical significance for freqency between the two groups;Compared to AAV9-GFP group,action potential amplitude in AAV9-IGF1 group was decreased,but there was no statistical significance.Compared to AAV9-GFP group,spike latency in AAV9-IGF1 group was shortened,and there was statistical significance with injected currents 150 p A and 500 p A(P<0.05);There was no statistical significance for clamping speed between the two groups.Conclusions:1 Compared with littermate control group,SOD1G93 A transgenic mice showed tendency to high excitability at the early stage before symptom.2 Compared with lateral ventricle injection,the method of parenchymal injection had higher IGF1 expression in M2 area of 20 to 40 days mice.3 IGF1 reduced threshold potential in SOD1G93 A transgenic mice,and played the neuroprotective effects.