Using Quantitative Proteomics Study The Impact Of Oxidative Stress On N2a-swe Cells

As a fatal neurodegenerative disease, Alzheimer’s disease(Alzheimer’s disease, AD) is characterized by two pathological protein deposits, the senile plaques composed mainly of amyloid-β(Aβ) peptide and the neurofibrillary tangles(NFT) which are bundles of paired helical filaments of protein tau.Relevant literatures suggest that overexpression of amyloid-β in AD patients’ brain have multiple effects to nerve cells, including calcium metabolism disorders, inflammation, ion channel abnormalities and oxidative stress. However, some people thought that Aβ is only the downstream events in AD pathological process. Aβ is a biomarker rather than a reason in AD pathological process. Therefore, Research on the relationship between Aβ and oxidative stress contributes to a more indepth understanding of the pathogenesis of Alzheimer’s disease.N2a-swe cells are stable cell lines formed by which transferred human APP695 gene into N2 a cells, Mainly overexpression of Aβ, phosphorylated tau protein, β-secretase enzyme, Apo E and other AD related proteins. In this study, we explored different concentrations of H2O2 effect on N2a-swe cells activity, the generation of ROS and Aβ. Found that 1m M H2O2 treat N2a-swe cells with 3 hours which can produce significant oxidative damage to the N2a-swe cells, but a low concentration of μM H2O2 had no significant effect on N2a-swe cells, and low concentrations of H2O2 did not increase the expression of Aβ in N2a-swe cells, but the expression of Aβ had a decreasing trend. however the result had not statistically significant. In this study, we also tried to transfect the whitetip shark methionine reductase A plasmid into N2a-swe cells, target protein expression was not found in N2a-swe cells. Then we tried to constructe stably lentiviral transfected N2a-swe cell lines to express methionine sulfoxide reductase A, Related biological experiments showed that the stably transfected N2a-swe cell lines were successfully constructed. MS(mass spectrometry) technology could not identified the protein, so we could not carry on the related research.To clarify whether Aβ is a direct factor in the pathogenesis of Alzheimer’s disease, we studied N2 a cells and N2a-swe cells by i TRAQ technology, Significant change proteins including Argininosuccinate synthase, Glutathione Stransferase A4, Protein disulfide-isomerase A6 and glucose-6-phosphate dehydrogenase, farnesy l pyrophosphate synthase, Cellular retinoic acid-binding protein 1, Serum albumin, Neural cell adhesion molecule 1, S100A6 protein, retinoic acid binding protein 1 serum albumin, neural cell adhesion molecule 1, Complexin-1, Glucose-6-phosphate 1 dehydrogenase, Calcium-binding mitochondrial carrier protein Aralar1, NADH-cytochrome b5 reductase 1, NADH dehydrogenase 1 beta subcomplex subunit 11, Calcium uniporter regulatory subunit MCUb and so on. The functions of these proteins including oxidation stress, neuronal function and mitochondrial energy metabolism.Secondly, we used i TRAQ technology to identify the change proteins in proteomics level. Resveratrol(RSV), a natural polyphenolic compound exists abundantly in grape skins and wines, has been found to protect against oxidative stress-induced cell injury in many tissues. we found that Peroxisomal targeting signal 1 receptor, Docose Hexaenoie Acid, Presenilins-associated rhomboid-like protein, Microtubuleactin cross linking factor 1were upregulate after resveratrol induced N2a-swe cells, the up-regulation of these proteins suggested that resveratrol could alleviate AD pathological process, at the same time, resveratrol reduced Phosphoglycerate kinase and Triosephosphate isomerase’s expression, the two enzymes are related to glycolytic enzymes, In this case, They would hinder the glycolytic pathway, the low efficiency of glycolysis and ATP depletion are the characteristics of AD pathological process, therefore, The results showed that lower levels of oxidation in vivo does not have a positive effect in delaying AD pathological process, while maintaining the organism oxidation-reduction balance is the key role.Finally, we studied that H2O2 treated N2a-swe cells by quantitative proteomics techniques, H2O2 caused the number of proteins change was much more than the total number of proteins change caused by resveratrol, indicating that oxidants has greater impact on N2a-swe cells, the histone(Histone H2 and Histone H4), Nucleoside diphosphate kinase, Thioredox 1, Peptidyl-prolyl cis-trans isomerase A, Parathymosin, Prothymosin α were significantly increaseing. These downregulated proteins are involved in nerve cell functions, antioxidant functions and protein folding functions, These results showed that H2O2 caused damage to N2a-swe cells by affecting these proteins’ expression.The difference proteins level is not sufficient to explain the impact of oxidative stress on N2a-swe cells, therefore we have carried pathway analysis by useing GSVA pathway enrichment, Through the analysis of all difference proteins showed that high expression of Aβ in N2a-swe cells did not cause much change in the cell pathways, only glycan degradation, type II diabetes and lysosomal were inhibited. After the N2a-swe cells were treated with resveratrol, The fatty acid degradation, Other glycan degradation, Sphingolipid metabolism and Lysosome four kinds pathways were activated. But Mismatch repair, TGF-beta signaling pathway, Hippo signaling pathway and other nine kinds of signal pathways were inhibited. H2O2 had most significant effect to N2a-swe cells, there were 19 kinds of pathways have been activated, 41 kinds of pathways been inhibited, which maybe associated with high concentrations of H2O2 cause N2a-swe cells oxidative damage. Notably, Alzheimer’s disease, Parkinson’s disease and Huntington’s disease pathways were activated, the above results showed that APP695 gene expression in N2a-swe cells could not activate AD pathway, but oxidative stress combine with excessive of Aβ can activate N2a-swe cells AD pathway.In summary, overexpression of Aβ not only improves N2a-swe cells’ oxidative stress level, but also affects the cells’ neuronal function and energy metabolism. These results support the concluction that Aβ is the Alzheimer’s disease risk factors. The study also showed that high level of oxidative stress will accelerate AD pathological process. While N2a-swe cells were treated with resveratrol, N2a-swe cells occurred some positive roles to alleviate AD pathological process, but also resveratrol cause some negative effects, which may be related to the high resveratrol doses used in our study. At the same time, These results showed that the organism keep normal oxidation-reduction levels can delay the onset of AD pathological process.