| Objective:To explore the role of exogenous iron on psychobehavioral and brain pathological features in Tau-P301 S transgenic mice by an intraperitoneal injection of ferric ammonium citrate(FAC).And to investigate the effect of exogenous iron ions on neuronal activity and Tau hyperphosphorylation and aggregation by treatment human neuroblastoma cells(SH-SY5 Y cells)and polypeptide fragment of the third repeat unit in the microtubule-binding domain of the Tau(Tau R3)with FAC and ferric chloride(Fe Cl3).Methods:1.Psychobehavioral tests in the Tau-P301 S mice after an intraperitoneal injection of FACTau-P301 S mice and wild-type(WT)littermate control mice were used at 5-month age in this study,which randomly divided into WT + Veh,WT + FAC,P301 S + Veh and P301 S + FAC.Mice were injected with FAC(40 mg.kg-1)or equivalent FAC solvent(Vehicle,Veh)every other day.After 8 weeks,behavioral tests were performed,and injection of FAC was continued until the end of all behavioral tests.1.1 Marble burying test: Mice were placed in a cage with fresh corn cob bedding,an array of 15 glass marbles was spaced over the bedding.Mice were allowed to move freely,the number of marbles buried was recorded after 2 h.1.2 Open field test(OFT): Mice were allowed to move freely in the center of the open field with a side length of 40 cm for 5 min.Total distance moved and the percentage of time spent in the center area were recorded and analysed.1.3 Treadmill test:Mice were placed into the track,the speed of 10 m.s-1 was setted,and the movement level was analysed after 5 min.2.The examinations of pathological characteristics after an intraperitoneal injection of FACAfter the behavioral experiment,to avoid the irritating effects of treadmill test on mice,the brain tissues of mice were collected a week after recovery from behavioral tests.Then,the following experiments were performed: iron content in hippocampus of mice was performed by Perl’s iron staining;the expression level of p-tau in hippocampus and cortex was detected by Western blot;neurofibrillary tangles(NFTs)in the hippocampus brain slices was detected by immunohistochemistry.3.FAC and FeCl3 were treated in SH-SY5 Y cells and molecular experimentsSH-SY5 Y cells,as a cell model,were treated with FAC and FeCl3 at different concentrations(10 μg.m L-1,100 μg.m L-1,200 μg.m L-1 and 400 μg.m L-1)of Fe3+.Cell viability was examined by CCK8 assay,and the appropriate intervention concentrations(10 μg.m L-1,200 μg.m L-1)were selected.Intracellular iron content was detected by Perl’s iron staining.After 24 h of FAC and Fe Cl3 intervention in SH-SY5 Y cells transfected with Tau-P301 L plasmid,the expression level of the phosphorylated Tau(p-tau)protein was detected by immunoblotting.The effects of iron on Tau aggregation was detected by the molecular experiments.The changes in fluorescence intensity of the core fragment Tau R3aggregation-bound Thioflavin T(Th T)were detected in real time by a multi-function microplate reader.Meanwhile,after Tau R 3 incubated with Fe Cl3 and FAC for 96 h,the fibers formed by Tau R3 aggregation were observed under a transmission electron microscope(TEM).Results:1.Iron causes psychobehavioral abnormalities and decreased activities of daily living in Tau-P301 S miceThe results of marble burying test showed that,compared with Veh-treated Tau-P301 S mice,the number of marbles buried was decreased in FAC-treated Tau-P301 S mice(P <0.05).The results of OFT showed that,the total distance was decreased in Veh-treated Tau-P301 S mice compared with Veh-treated WT mice(P < 0.05),while the total distance of FAC-treated Tau-P301 S mice displayed shorter than in Veh-treated Tau-P301 S mice,(P< 0.05).In the treadmill test,there was no significant difference in total movement distance between groups during 5 minutes(P > 0.05).2.Iron induces Tau phosphorylation in the brain tissues of miceThe results of Perl’s staining showed that,iron content of FAC-treated Tau-P301 S mice was higher than veh-treated Tau-P301 S mice(P < 0.05).The results of immunoblotting in hippocampus of mice showed that,the t-tau protein expression levels of Veh-treated Tau-P301 S mice was higher than that in Veh-treated WT mice(P < 0.05),while the t-tau protein expression level of FAC-treated Tau-P301 S mice was not significantly different from that of Veh-treated Tau-P301 S mice(P > 0.05).The p-tau protein expression level of Veh-treated Tau-P301 S mice was clearly higher than that Veh-treated WT mice(P < 0.05).The relative expression of p-tau in the P301 S FAC group was higher than that in P301 S + Veh group(P < 0.05).The results of immunoblotting in cortex of mice showed that,the t-tau protein expression level of Veh-treated Tau-P301 S mice was higher than that of Veh-treated WT mice(P < 0.05),while the t-tau protein expression level of FAC-treated Tau-P301 S mice was not significantly different from that of Veh-treated Tau-P301 S mice(P > 0.05).Moreover,the p-tau protein expression level of Veh-treated Tau-P301 S mice was clearly higher than that of Veh-treated WT mice(P <0.05).The relative expression of p-tau in P301 S + FAC group was higher than that in P301 S + Veh group(P < 0.05).Meanwhile,the results of immunohistochemical also displayed that FAC treatment caused an increase in the relative area of NFTs in the hippocampus of Tau-P301 S mice(P < 0.05).3.Iron induced decreased cellular activity in SH-SY5 Y cells,and promoted hyperphosphorylation of Tau and aggregationAfter FAC treatment,the results of CCK8 assay showed that the cell viability in the200 μg.m L-1 and 400 μg.m L-1 group were lower than that in the control group(P < 0.05).Meanwhile,after Fe Cl3 treatment,the cell viability in the 200 μg.m L-1 and 400 μg.m L-1group were lower than in the control group(P < 0.05).Furthermore,after FAC treatment,the results of Perl’s staining showed that the nuclei and cytoplasm in the 200 μg.m L-1group were mainly yellowish-brown,whereas the nuclei and cytoplasm in the control group were mainly hyacinthine,and the positive cell rate in the 200 μg.m L-1 group was higher than that in the control group(P < 0.05);after Fe Cl3 treatment,the results showed that the cytoplasm in the 200 μg.m L-1 group was mainly yellowish-brown compared to the control group,and the positive cell rate was up-regulated(P < 0.05).After FAC treatment,the immunoblotting results showed that the p-tau protein expression levels in the 200 μg.m L-1 group were higher than in the Tau group(P < 0.05).Meanwhile,after Fe Cl3 treatment,the p-tau protein expression level in the Tau + 200 μg.m L-1 group was higher than in the Tau group(P < 0.05).In the Th T experiment,Fe Cl3 increased the fluorescence intensity of Tau R3 in the 84 h and 96 h(P < 0.05).The TEM results also showed the aggravated accumulation of Fe3+ and Tau R3 and formed the deposition of Tau fibers.Conclusion:1.Iron aggravates psychobehavioral abnormalities in Tau-P301 S mice.2.Iron causes Tau hyperphosphorylation in the hippocampus and cortex of Tau-P301 S mice and aggravates NFTs occurrence.3.Iron increases p-tau expression in the SH-SY5 Y cells,and promotes aggregation of Tau and induces neurotoxicity. |
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