Mechanism Of APP Palmitoylation Is Involved In The Increase Of Aβ_1-42 Induced By Aluminum

Objective:To explore the mechanism of APP（Amyloid precursor protein）palmitoylation in the increase of Aβ_1-42 induced by aluminum.Methods:In this study,the mechanism of APP palmitoylation in the increase of Aβ_1-42 induced by aluminum was studied from two levels of whole animal experiment and in vitro cell experiment.Part 1:Animal Experiment1.Animal grouping and exposure:40 healthy male adult SD rats weighing 180-220 g were randomly divided into control group,10μmol/kg Al（mal）₃ groups,20μmol/kg Al（mal）₃groups,40μmol/kg Al（mal）₃ group,using the method of intraperitoneal injection of aluminum maltol,the poisoning every other day,lasting 90 days.2.Through the water maze experiment and the new object recognition experiment to detect the changes in the learning and memory abilities of rats by aluminum exposure.3.Analyze the effects of different concentrations of aluminum on the number of cerebralcortical neurons by Nissl staining.4.Detect the effect of aluminum staining on the content of Aβ_1-42 in the cerebral cortex byELISA.5.The immunoprecipitation-acyl-biotin exchange method（IP-ABE）was used to analyze the effect of aluminum staining on APP palmitoylation level in rat brain cortex.6.Western Blotting was used to detect the effect of aluminum staining on the expression of palmitoyltransferase（zDHHC7）and APP andβ-amyloid precursor protein lyase 1（BACE1）in rat cortex.Part 2:Cell Experiment1.Use rat adrenal pheochromocytoma cells（PC12 cells for short）,and use Al（mal）₃to treat the cells for 36 hours.According to the exposure dose,they were divided into the control group,100μmol/L Al（mal）₃ group,200μmol/L Al（mal）₃ group and 400μmol/L Al（mal）₃ group.2.Detect the effect of aluminum staining on the Aβ_1-42 content of PC12 cells by ELISA.3.The immunoprecipitation-acyl-biotin exchange method（IP-ABE）was used to detect the effect of aluminum staining on the palmitoylation level of APP in PC12 cells.4.Western Blotting was used to detect the effect of aluminum staining on the expression of palmitoyltransferase（zDHHC7）and APP and BACE1 on lipid rafts in PC12 cells.5.After inhibiting APP palmitoylation level through two intervention methods of non-specific chemical inhibitor 2-Bromopalmitate（2-BP）and siRNA targeting zDHHC7（siRNAzDHHC7）,observe the effect of aluminum on Aβ_1-42The impact of the production process.Results:Part 1:Animal Experiment1.General situation of rats:During the process of intraperitoneal injection,the hair,diet,mental state and free activities of rats in each dose group were normal,and their body weight increased with the increase of feeding time.At the end of the experiment,there was no significant difference in the body weight of rats and the brain specific gravity of each dose group（P>0.05）.2.Results of impairment of learning and memory in rats:（1）Results of water maze experiment:From the third day of the positioning navigation experiment,the difference in the escape latency of rats in different aluminum-exposing groups was statistically significant（P<0.05）.The difference between the 40μmol/kg Al（mal）₃ group and the control group was significant.There is a significant difference（P<0.05）.There was a statistical difference between the 20μmol/kg Al（mal）₃ group and the control group and the 10μmol/kg Al（mal）₃ group in the escape latency on the 4th day（P<0.05）.There was a statistical difference between the 20μmol/kg Al（mal）₃ and the control group in the escape latency on the 5th day（P<0.05）.40μmol/kg Al（mal）₃ group and control group,10μmol/kg Al（mal）₃ group and 20μmol/kg Al（mal）₃ group had statistical differences on the 4th and 5th day（P<0.05）.There was no statistical difference in the number of times that rats crossed the platform in each group（P>0.05）,and the residence time in the target quadrant was different（P<0.05）,20μmol/kg Al（mal）₃ group,40μmol/kg Al（mal）₃ The stay time in the target quadrant of the group was shorter than that of the control group（P<0.05）.（2）New object recognition experiment results:the difference in new object recognition of rats in different aluminum-exposed groups was statistically significant（P<0.001）.The new object recognition index of the rats in the control group was significantly higher than that of the 10,20,and 40μmol/kg Al（mal）₃ groups（P<0.001）,and the discrimination index showed a downward trend with the increase of the aluminum dye dose（P<0.001）.The10μmol/kg Al（mal）₃ group was significantly lower than the control group（P<0.05）.The discriminative index values of the 20μmol/kg Al（mal）₃ and 40μmol/kg Al（mal）₃ groups were significantly lower than those of the control group and the 10μmol/kg Al（mal）₃ group（P<0.05）.3.Pathological results:Nissl staining results:different degrees of neuron loss can be seen in the cerebral cortex of the rats in the aluminum-stained group.The cortical neurons of the rats in the 20μmol/kg Al（mal）₃ group and 40μmol/kg Al（mal）₃ group were unevenly arranged,with many cavities,and most of the cells showed the absence of Nissl bodies.And the difference in the number of neurons in each group was also statistically significant（P<0.05）.Compared with the control group,the loss of neurons in the 10μmol/kg Al（mal）₃ and 20μmol/kg Al（mal）₃ groups was statistically significant（P<0.05）.Compared with the control group and the 10μmol/kg Al（mal）₃ group,the 40μmol/kg Al（mal）₃ group had more obvious neuron loss（P<0.05）.4.The effect of aluminum staining on the content of Aβ_1-42 in rat brain cortex:The content of Aβ_1-42 in rat cortex shows a gradual increase with the increase of the concentration of aluminum staining.Compared with the control group,the Aβ_1-42 content of the 10μmol/kg Al（mal）₃ group was not statistically different（P>0.05）.Compared with the control group and the 10μmol/kg Al（mal）₃ group,20μmol/kg The difference of Aβ_1-42 content in Al（mal）₃group was statistically significant（P<0.05）,compared with the control group,10μmol/kg Al（mal）₃,20μmol/kg Al（mal）₃ group,40μmol/kg Al（mal）₃ The difference in Aβ_1-42 content of the 3 groups was statistically significant（P<0.05）.5.The effect of aluminum staining on APP palmitoylation level of rat brain cortex:APP palmitoylation level of rat cortex showed a gradual increase with the increase of the concentration of aluminum staining.The palmitoylation levels of APP protein in 20 and40μmol/kg Al（mal）₃groups were higher than those in the control group and 10μmol/kg Al（mal）₃ groups,and the difference was statistically significant（P<0.05）;40μmol/kg Al（mal）₃ groups Compared with the 20μmol/kg Al（mal）₃ group and the 20μmol/kg Al（mal）₃group,the difference was also statistically significant（P<0.05）.6.The effect of aluminum on the expression of zDHHC7 protein in rat brain cortex:With the increase of the concentration of stained aluminum,the expression of zDHHC7 protein showed a gradual increase trend.Compared with the 10μmol/kg Al（mal）₃ group,the zDHHC7 protein expression in the 20 and 40μmol/kg Al（mal）₃ groups increased significantly（P<0.05）.The increase in zDHHC7 protein expression in the 40μmol/kg Al（mal）₃ group compared with the 20μmol/kg Al（mal）₃ group was statistically significant（P<0.05）.7.The effect of aluminum staining on the expression of APP protein on lipid rafts of rat brain cortex:With the increase of the concentration of aluminum staining,the expression level of APP protein on lipid rafts showed a gradually increasing trend.Compared with the control group,the increase of APP protein expression on lipid rafts of 20 and 40μmol/kg Al（mal）₃ groups was statistically significant（P<0.05）.The expression levels of APP in the20 and 40μmol/kg Al（mal）₃ groups were significantly higher than those in the 10μmol/kg Al（mal）₃ group（P<0.05）;compared with 20μmol/kg Al（mal）₃ Compared with the two groups,the increase in APP expression in the 40μmol/kg Al（mal）₃ group was also statistically significant（P<0.05）.Part 2:Cell Experiment1.Cell morphology:The cells of the control group showed clear boundaries and uniformsize.With the increase of the aluminum dose,the number of cells gradually decreased,and the synapses decreased,thickened,and broken.This is especially evident in the 400μmol/L Al（mal）₃group.2.The effect of aluminum staining on the content of Aβ_1-42 in cells:The content of Aβ_1-42also shows a gradually increasing trend with the increase of the aluminum staining dose.Compared with the control group,the content of Aβ_1-42 in the 200 and 400μmol/L Al（mal）₃groups increased statistically（P<0.05）.3.The effect of aluminum staining on cell APP palmitoylation level:The palmitoylation level of APP in PC12 cells showed an increasing trend with the increase of the staining aluminum concentration.Compared with the 100μmol/L Al（mal）₃ group,the control group had no significant difference in APP palmitoylation（P>0.05）;the 200μmol/L Al（mal）₃ group was compared with the control group and 100μmol/L Al（mal）₃ Compared with the group,the difference was statistically significant（P<0.05）;the 400μmol/L Al（mal）₃ group compared with 100μmol/L Al（mal）₃,200μmol/L Al（mal）₃,APP palmitoylation was different There is statistical significance（P<0.05）.4.The effect of aluminum staining on cell zDHHC7 protein expression:zDHHC7 protein expression also showed a gradually increasing trend with the increase of the aluminum staining dose.Compared with the control group,the zDHHC7 protein expression in the 3groups of 100,200,and 400μmol/L Al（mal）₃ increased,and the difference was statistically significant（P<0.05）.Compared with the 100μmol/L Al（mal）₃ group;the zDHHC7 protein expression in the 400μmol/L Al（mal）₃ group was further increased,and there was a statistical difference（P<0.05）;Compared with 200μmol/L Al（mal）₃,400μmol/L Al（mal）₃ group was statistically significant（P<0.05）.5.The effect of aluminum staining on the expression of APP protein on lipid rafts:The expression of APP protein on lipid rafts showed an increasing trend with the increase of aluminum staining dose,and there was a statistical difference（P<0.05）.Compared with the control group,the increase in APP protein expression on lipid rafts in the 3 groups of 100,200,and 400μmol/L Al（mal）₃ was statistically significant（P<0.05）.Compared with the400μmol/L Al（mal）₃ group,the APP protein expression in the 200 and 400μmol/L Al（mal）₃groups increased statistically（P<0.05）;compared with the 200μmol/L group,400μmol/L Al（mal）₃ groups in the increase of APP protein was also statistically significant（P<0.05）.6.The results of two intervention methods targeting palmitoylase zDHHC7 at the cellular level are as follows:1.Research on non-specific chemical inhibition（1）The effect of aluminum on APP palmitoylation level after 2-BP intervention:The APP palmitoylation level in the 200μmol/L Al（mal）₃ group was significantly higher than that in the control group（P<0.05）,and when the aluminum concentration was certain Under the premise,with the increase of 2-BP concentration,APP palmitoylation level showed a downward trend（P<0.05）.APP palmitoylation level of 200μmol/L Al（mal）₃ group,25μg/ml2-BP+200μmol/L Al（mal）₃ and 50μg/ml 2-BP+200μmol/L Al（mal）₃ and control The group difference was significant（P<0.05）.25μg/ml 2-BP+200μmol/L Al（mal）₃ and 100μg/ml 2-BP+200μmol/L Al（mal）₃ compared with 200μmol/L Al（mal）₃ groups,APP palmitoylation levels were significant Decrease（P<0.05）.Compared with 25μg/ml 2-BP+200μmol/L Al（mal）₃ in the 100μg/ml 2-BP+200μmol/L Al（mal）₃ group,there was a statistically significant difference in APP palmitoylation level（P<0.05）.（2）The effect of aluminum on APP on lipid rafts after 2-BP intervention:The palmitoylation level of APP in the 200μmol/L Al（mal）₃ group was significantly higher than that in the control group（P<0.05）,and at a certain aluminum concentration Under the premise of,with the increase of 2-BP concentration,the expression of APP protein on lipid rafts showed a downward trend（P<0.05）.200μmol/L Al（mal）₃,25μg/ml 2-BP+200μmol/L Al（mal）₃ and 100μg/ml 2-BP+200μmol/L Al（mal）₃ The expression of APP protein on lipid rafts was significantly different from that of the control group（P<0.05）.50μg/ml 2-BP+200μmol/L Al（mal）₃ and 100μg/ml 2-BP+200μmol/L Al（mal）₃ and 200μmol/L Al（mal）₃groups,Compared with the 25μg/ml 2-BP+200μmol/L Al（mal）₃ group,the expression of APP protein on lipid rafts was significantly lower（P<0.05）.Compared with 50μg/ml 2-BP+200μmol/L Al（mal）₃,100μg/ml 2-BP+200μmol/L Al（mal）₃ group,the difference between APP on lipid rafts was statistically significant（P<0.05）).（3）The effect of the chemical agent 2-BP intervention on the aluminum content of Aβ_1-42:the Aβ_1-42 content of the 200μmol/L Al（mal）₃ group was significantly higher than that of the control group（P<0.05）,and the aluminum concentration was constant Under the premise of,with the increase of 2-BP concentration,the content of Aβ_1-42showed a downward rend（P<0.05）.25μg/ml 2-BP+200μmol/L Al（mal）₃,50μg/ml 2-BP+200μmol/L Al（mal）₃,100μg/ml 2-BP+200μmol/L Al（mal）₃ Aβ_1-42 content was significantly different from the 200μmol/LAl（mal）₃ group（P<0.05）.50μg/ml 2-BP+200μmol/L Al（mal）₃ and100μg/ml 2-BP+200μmol/L Al（mal）₃ compared with 25μg/ml 2-BP+200μmol/L Al（mal）₃group,Aβ_1-42 protein content was significantly reduced（P<0.05）.Compared with the100μg/ml 2-BP+200μmol/L Al（mal）₃ group,there was a statistically significant difference in Aβ_1-42 content between the 100μg/ml 2-BP+200μmol/L Al（mal）₃ group and 50μg/ml 2-BP+200μmol/L Al（mal）₃（P<0.05）.2.Research on specific siRNA interference（1）The effect of aluminum on the expression of zDHHC7 gene after zDHHC7siRNA interferes with cells:There was no statistically significant difference in zDHHC7gene expression between the control group,the solvent group,and the NC group（P>0.05）.Compared with the control group,solvent group,and NC group,the expression of zDHHC7gene in the 200μmol/L Al（mal）₃ group increased.zDHHC7siRNA group and control group,solvent group,NC group,compared with 200μmol/L Al（mal）₃ the expression of zDHHC7gene was significantly reduced.Compared with the other five groups,the difference of zDHHC7siRNA+200μmol/L Al（mal）₃ was statistically significant（P<0.05）.（2）The effect of aluminum on APP palmitoylation level after siRNAzDHHC7transfection:There was no significant difference in APP palmitoylation level between the control group,solvent group and NC group（P>0.05）.Compared with the control group,solvent group,and NC group,the level of APP palmitoylation in 200μmol/L Al（mal）₃ group increased.Compared with the control group,solvent group,NC group and 200μmol/L Al（mal）₃,APP palmitoylation in zDHHC7siRNA group had a significantly lower level（P<0.05）.Compared with the other five groups,zDHHC7 siRNA+200μmol/L Al（mal）₃showed a statistically significant difference in APP palmitoylation level（P<0.05）.（3）The effect of aluminum on the expression of APP protein on lipid rafts after siRNAzDHHC7 transfection:There was no statistically significant difference in the expression of APP protein on lipid rafts in the control group,solvent group,and NC group.Compared with the control group,solvent group and NC group,the 200μmol/L Al（mal）₃group had higher expression of APP protein on lipid rafts.Compared with the control group,solvent group,NC and 200μmol/L Al（mal）₃ group,APP protein expression on lipid rafts in zDHHC7siRNA group were significantly reduced（P<0.05）.Compared with the other five groups,zDHHC7 siRNA+200μmol/L Al（mal）₃ had statistically significant differences in APP protein expression on lipid rafts（P<0.05）.（4）The influence of aluminum on the content of Aβ_1-42 after zDHHC7siRNA interferes with cellsThere was no statistically significant difference in Aβ_1-42 content between the control group,the solvent group,and the NC group.Compared with the control group,solvent group and NC group,the content of Aβ_1-42 in the 200μmol/L Al（mal）₃ group increased（P<0.05）.Compared with the control group,solvent group,NC and 200μmol/L Al（mal）₃,the content of Aβ_1-42 in the zDHHC7siRNA group was significantly lower（P<0.05）.Compared with the other five groups,the content of Aβ_1-42 in zDHHC7 siRNA+200μmol/L Al（mal）₃ was statistically significant（P<0.05）.Conclusion:1、The increase in APP palmitoylation level may be related to the increase in Aβ_1-42 caused by aluminum,and the mechanism may involve APP palmitoylation to promote the accumulation of APP protein on lipid rafts.2、The increase in the expression level of zDHHC7 may be one of the reasons for the increase in the level of APP palmitoylation caused by aluminum.