Study On The Mechanism Of PIK3R4 Participating In Autophagy Formation In SOD1G93A Transgenic Mice

Objective:Amyotrophic lateral sclerosis(ALS)is one of the neurodegenerative diseases characterized by upper and/or lower motor neuron injury.Its prognosis is poor and the average survival period is 3-5 years.There are many popular pathogenic theories,the more popular one is abnormal accumulation of protein.In previous studies,we found that the expression of serine/threonine PI3 kinase regulatory subunit 4(PIK3R4,Vps15)protein decreased in ALS mice.Therefore,we study the molecular structure,function of the protein and its interaction with other important molecules.Methods:The experiment was divided into cell experiment group and animal experiment group.The PC12 cells were divided into siRNA-PIK3R4 transfection group,NC negative control group and normal PC12 cell group.The expression of PIK3R4 mRNA was detected by QPCR before and after transfection.The expression of PIK3R4 protein in each group was detected by Western Blot.CCK-8 was used to observe the effect of cell proliferation of each group.Animal experiment group: 18 Tg(SOD1 * G93A)1Gur mice and 18 WT mice were divided into three groups according to their neurological function and age,six in each group,which were the preonset group(60-70 days),the onset group(90-100days)and the progression group(120-130 days).According to cervical and lumbar enlargement,the spinal cord of mice was divided into three segments: cervical,thoracic and lumbar.We will take lumbar cord as a representative to observe the expression and distribution of PIK3R4 and related proteins of autophagy by Immunofluorescence and Western Blot.Results:Cell experiments showed that:1.Western Blot showed that the down-regulated expression of PIK3R4 protein(siRNA-PIK3R4 group:0.35±0.21;PC12 group:1.51±0.51;p<0.05)making p62protein(siRNA-PIK3R4 group: 0.51±0.11;p<0.01),LC3 protein(siRNA-PIK3R4group: 0.52±0.14;PC12 group:1.33±0.37;p<0.05)and SOD1 protein were down regulated.2.There was no significant difference in cell viability among the three groups.Animal experiments showed that:1.The results of animal's Western Blot showed that in the development of SOD1G93 A mice,the protein of PIK3R4 gradually decreased in the process of disease and was generally lower than wild-type mice.And there was a difference between SOD1G93 A and wild-type mice in preonset period(p<0.05),while p62 protein showed a gradual upward trend without significant statistical difference,and LC3 protein had no significant change.2.Animal Immunofluorescence showed that the number of PIK3R4 protein positive cells decreased in the course of SOD1G93 A mice.The number of PIK3R4 protein positive cells in every stage of SOD1G93 A mice was different from that in the control group(p< 0.05).Most of the co-localization of PIK3R4 protein and NeuN coincided with NeuN protein,and decreased with NeuN reduction to some extent,especially in the anterior horn of spinal cord.Secondly,the expression of PIK3R4 protein was found in the anterior horn of the cervical,thoracic and lumbar cord.The overall trend was that there was no significant difference in the number of PIK3R4 positive cells in all stages of wild type mice,while in SOD1G93 A mice,it decreased with the progress of disease.Among them,there were statistical differences in the number of PIK3R4 positive cells in the anterior horn of the thoracic and lumbar segments between the preonset group and the control group,and there were statistical differences in the number of PIK3R4 positive cells in the anterior horn of the cervical and lumbar segments between the progression group and the control group(p < 0.05).Conclusions:In our study,we observed the changes of PIK3R4 protein in PC12 cells when it was down regulated,which was different from the results of SOD1G93 A ALS mice.The silencing of PIK3R4 gene in cells can cause the down-regulation of autophagy,but the autophagy in the spinal cord of SOD1G93 A mice has no obvious change.In addition,most of the PIK3R4 protein overlaps with NeuN protein in the co-locationof neuron marker NeuN in spinal cord anterior horn cells.Observation of sample shows that it decreases with the decrease of NeuN to a certain extent,indicating that PIK3R4 protein may participate in the autophagy process in ALS disease,so further research can be carried out to clarify its specific mechanism.