Screening And Verification Of The Protein Related To Neuron Apoptosisi In Down Syndrome

BackgroundDown syndrome (DS) is the most common gross chromosomal abnormalities and birth defect, in which the extra chromosome 21 hinders the growth of children, and causes serious neurologic deficit. Intellectual disabilities not only cause the loss of cognitive abilities, but also result in the development of early onset Alzheimer’s disease (AD). Therefore, strengthening the study of the neural defects in DS is significant to delay the brain damage, improve the cognitive ability and enhance the quality of life. Cell apoptosis plays an important role in nervous system diseases. Down syndrome is a kind of neural degenerative diseases which is similar to AD, and now the conclusion how apoptosis influences Down syndrome is not consistent. This study aims to screen the differential proteins in the hippocampal of DS mice and select key protein by bioinformatics analysis. Then explore the relationship between protein and neuronal apoptosis. This study will be helpful to elucidate the molecular mechanism of DS neural defects.Purposes(1) Selecting Ts65Dn mice as the model of Down syndrome. Using ITRAQ technique to screen and identify the differentially expressed proteins in the hippocampus, which laid the foundation for further study on the molecular mechanism of DS neural defects;(2) Using bioinformatics analysis to clear the differences between protein properties and their interactions, reveal the relatedness of differential protein with occurrence and development of neurological defects in Down syndrome;(3) To clear the state of central nervous system cells apoptosis in Down syndrome and preliminaryly confirmed the relationship between the expression of Akt differential protein and the nerve apoptosis of Down syndrome, and further clarifying the results of the proteomics.Methods(1) Identification of subculture and neuronal apoptosis of Ts65Dn mice This study used Ts65Dn mice as a model of Down syndrome, let the male and female in the cage breed offspring, using the first generation of mice (16 to 18 weeks) as the research object. According the protocol of Jackson Laboratory, we strain identified the generation by detecting mutation of GRCm38.p1 chromosome 17 using molecular biology methods.(2) Using iTRAQ to screen the differential proteins of hippocampus in Ts65Dn miceAfter collecting hippocampus tissues of Ts65Dn, we used protein electrophoresis, protein map for preliminary experiments, and confirmed that the protein content of sample met the requirement. We used enzymolysis and quantification of peptides followed by protein electrophoresis using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE); Using the 4-plex iTRAQ reagent to label the peptide; MS analysis was taken after the separation by Capillary high performance liquid chromatography (HPLC); Using Mascot 2.2 software to take database search and identification, and using Proteome Discoverer 1.3 software for quantitative analysis to ion peak intensity value of peptide(3) Bioinformatics analysis of the differential proteinsFor the 374 differential proteins in hippocampus of Ts65Dn mice which were screened and identified by iTraq technology, we respectively used GO function analysis and enrichment analysis, KEGG pathway analysis, Network analysis, Hub protein analysis and so on, to illustrate the protein features of the physical chemical properties and bioinformatics.(4) Research on the influence of Akt pathway to the neural cell apoptosis in Ts65Dn miceWe selected the hippocampus tissue and used the transmission electron microscopy to directly observe the morphological changes of nerve cell apoptosis in Ts65Dn mice; Using TUNEL method to observe the apoptosis rate of hippocampus nerve cell in Ts65Dn mice. Respectively, using immunohistochemical technique and Western blot technique to observe Akt protein of Ts65Dn mice hippocampus and phosphorylated(p-Akt) expression; We isolated and cultured the neural stem cells from brain tissue of newborn Ts65Dn mice and further induced differentiation into neuronal cells; Using LY294002 to inhibit Akt protein phosphoric acid activation, block PI3K/Akt pathway, and using flow cytometry to detect apoptosis of nerve cells, preliminarily confirm the relationship between the expression of Akt protein and neural cell apoptosis of Ts65Dn mice.Results(1) There were 17 survival first generation mice after mating. We identified the genotype according to the protocol of Jackson Laboratory genotyping methods. Used iTRAQ technology, by Mascot database and quantitative analysis of Proteome Discoverer, we identified 2,805 proteins in the hippocampi of Ts65Dn mice. Compared with normal mice and calculated the index of significance (p-value), there were 374 differentially expressed proteins in hippocampal tissues, including 195 proteins that were significantly increased and 179 that were decreased.(2) Using bioinformatics methods to analyze the differential protein. Among them, GO analysis found that binding protein (334,89.3%), catalytic activity of the protein (102,27.3%) and transferase (39,10.4%) were the largest number of proteins in the Molecular functional category; cellular pathways (238,63.6%), biological regulation pathway (149,39.8%) and metabolic pathways,39.6%(148) were the largest number of biological pathway; Cytology components are mostly cytoplasm (cytoplasm) and extracellular region (extracellular region), the cell membrane (be) ingredients. KEGG analysis found that among the 374 proteins in hippocampus of Ts65Dn mice,there were 143 proteins corresponding to 144 different KEGG pathways, corrected by FDR taking P< 0.05 statistical analysis, and only insulin signaling pathway displayed a significant enrichment.(3) Hippocampus nerve cells of Ts65Dn mice appeared different degrees of apoptosis changes. We found apoptosis change of nerve cells by transmission electron microscopy, nuclear membrane thinning and mild subsidence, chromosome distribution, nuclear pyknosis sex change, cell wall sag, etc. According to the results of TUNEL, positive performance of apoptotic neurons was that the cytoplasm and the nuclei were tan. Compared with the normal mice, Ts65Dn mice hippocampus neuron cell apoptosis rate increased significantly.(4) Immunohistochemistry and Western blot results found:Ts65Dn central nervous tissue and normal mice pyramidal cells both had Akt protein expression. Compared with the normal mice, Akt protein of Ts65Dn mice expressed increasing trend, expression of p-Akt protein was also significantly enhanced. We isolated and cultured the neural stem cells from brain tissue of newborn Ts65Dn mice and further induced differentiation into neuronal cells; Adding LY294002 inhibitors to Ts65Dn mice neurons after 48h, Akt phosphorylation was inhibited, the expression of p-Akt protein decreased obviously, and the apoptosis rate of neurons in Ts65Dn mice had a tendency to discrease.Conclusions(1) Ts65Dn mice were successfully bred. Using iTRAQ to screen and find that there were a large number of differentially expressed proteins in the hippocampus of Ts65Dn mice compared with normal mice, which might be correlated to the neuronal apoptosis.(2) Using bioinformatics methods to analyze the characteristics, interaction and involved signal transduction of the differentially expressed proteins in the hippocampus of Ts65Dn mice from several different angles, the correlation between the differential protein and the DS nerve defect was initially revealed.(3) The apoptosis of hippocampal neurons was increased in DS mice. Meanwhile the expression of Akt protein was increased in the hippocampal tissue and cultured neurons of DS mice, which was related to the apoptosis of neurons.