Effect Of Very Long Chain Fatty Acids On Hyperphosphorylation Of Tau Protein In SH-SY5Y Cells And Its Mechanisms

Background Alzheimer’s disease(AD)is a serious neurodegenerative disease with unknown etiology.The main pathological features of AD are extracellular senile plaques and neurofibrillary tangles(NFTs)in neurons.In recent years,the relationship between very long chain fatty acids(VLCFA)and AD has attracted much attention.VLCFA(mainly C22:0,C24:0 and C26:0)increased in brain and blood of AD patients.Our previous studies found that C26:0 promoted the production of beta-amyloid(Aβ,the main component of senile plaque)in SH-SY5Y cells.These data suggest that VLCFA may be involved in the pathogenesis of AD.Tau protein hyperphosphorylation and aggregation in the form of NFTs is an important mechanism of AD pathogenesis.Glycogen synthase kinase-3beta(GSK-3β)catalyzes phosphorylation of Tau protein.Do VLCFA affect the phosphorylation of Tau protein? The relationship between VLCFA and Tau protein has not been reported.Objective In this study,SH-SY5Y cells were used to study the effects of VLCFA(C22:0,C24:0 and C26:0)on Tau protein hyperphosphorylation in SH-SY5Y cells,and to explore the mechanism of VLCFA in the pathogenesis of AD from the aspects of GSK-3β activity,cell membrane fluidity and oxidative stress.Methods SH-SY5Y cells were randomly divided into four groups: control group(vehicle group),C22:0 group,C24:0 group and C26:0 group.Cell growth covered the bottom of cell culture dish to 60%-70% post-treatment cells.CCK-8 assay was used to detect the activity of cells under different fatty acid concentrations and find out the appropriate concentration of administration;fluorescence inversion microscopy was used to observe cell morphology and fluorescence recovery after photobleaching(FRAP)was used to detect cell membrane fluidity;Western blot was used to determine the protein expression of total TAU-5,p-tau(ser396),GSK-3β,p-GSK-3β(Ser9);and TBA microplate method was used to determine MDA content in cells;The activity of total superoxide dismutase(SOD)was measured by WST-1 method.SPSS 19.0 software(SPSS Inc.,Chicago,IL)was used to analyze the data.The experimental data were expressed as(mean ± standard deviation)((?) ± s).One-way ANOVA was used to compare the differences among the groups,and the level of statistical significance was considered at P < 0.05.Results 1.Fatty acid concentration screening: After CCK-8 experiment screening,this experiment selected 10 μmol/L fatty acid concentration to treat cells.2.Cell membrane fluidity: The fluorescence recovery rate and diffusivity of C26:0 group were significantly lower than those of vehicle group(P < 0.05),and the fluorescence recovery rate and diffusivity of C24:0 group were slightly reduced lower than those of Vehicle group(F=3.56,P= 0.078;F=5.08,P=0.11),but there was no significant difference.3.Relative protein expression Tau-5 protein: There was no significant difference in the relative amount of Tau-5 protein between groups.P-tau(ser396)protein: Compared with vehicle group,the phosphorylation level of p-tau(ser396)protein in C22:0,C24:0 and C26:0 groups increased significantly(F=4.57,P =0.041,P=0.005,P=0.014).GSK-3β protein: There was no significant difference in the relative quantity of GSK-3β protein among the groups.P-GSK-3β(Ser9)protein: Compared with vehicle group,the levels of p-GSK-3β(Ser9)protein(inactive form)in C22:0,C24:0 and C26:0 groups were significantly lower(F=6.31,P=0.029,P=0.004,P=0.002),that is,the activity of GSK-3β was significantly higher.4.MDA content: MDA content in C24:0 and C26:0 groups were significantly higher than that in Vehicle group(F=4.94,P=0.015,P=0.024).5.Total SOD activity: There were no significant differences in SOD activity among groups.Conclusion Very long chain fatty acids increase the phosphorylation level of Tau protein and decrease the inactivation of GSK-3β.Very long chain fatty acids may promote the development of AD by increasing the activity of GSK-3β leading to Tau protein hyperphosphorylation.Very long chain fatty acids can cause oxidative damage and reduce cell membrane fluidity.