The Effect Of ApoE4 Gene Combined With Aluminium On Cognitive Impairment In Mice And Its Mechanism

Objective:In order to investigate combined effect of genetic risk factor ApoE4 and environmental risk factor aluminium on study and memory,and plasticity of neuronal phenotype and synapse,combined effect was stimulated by intervention of transgenic ApoE4 gene and aluminium exposure in mice,and interaction between two factors was analyzed.Moreover,to discuss the potential mechanism of cognitive damages by the combined effect of ApoE4 gene and aluminium through determination of the contents of A?,the gene and protein expression of APP,ApoER2,LRP1,and VLDLRs,and the level of tau phosphorylation.Methods:Three kinds of male mice were used in this experiment:ApoE knock-out mice?KO?,wild type mice?WT?,and transgenic knock-in mice that express the human ApoE4?KI?.The control group and the aluminium-exposed group has been set.The aluminium-exposed group was given aluminium-maltolate?40?mol/kg?by intraperitoneal injection consecutively for 5 days,and pausing for 2 days,and the exposure duration lasted for 60days.The control group was injected with same volume of saline solution.After the end of the experiment,a ligase test was used to evidence whether ApoE4 gene was stably inherited in the transgenic ApoE4 mice.The aluminium content in brain was determined by Inductively Coupled Plasma Mass Spectrometry.Morris water maze and new object recognition tests were used to detect changes in learning and memory ability of animals.Through HE staining,Golgi staining,and electron microscope experiments,the changes of the hippocampal histopathological structure,the neuronal morphological structure,the dendritic spine density,and the synaptic structure were observed.The content of A?₄₀0 and A?₄₂2 was detected by Elisa method,and the expression of APP,ApoER2,LRP1,VLDLRs and other genes and proteins were detected by RT-PCR and western-blot.At the same time,the protein contents of tau-5,pThr181,pThr231,pSer262 and pSer396were detected by western-blot.Results:1.The bases at corresponding site of rs429358,rs7412 in the transgenetic ApoE4mice that express the human ApoE4 used in the experiment were all CC/CC.This has proved that all mice have stably inherited in the ApoE4 gene of human.The contents of aluminium in the hippocampus in the aluminium-exposed mice were 35.34±5.40?g/g?KO?,33.62±4.00?g/g?WT?,35.56±8.72?g/g?KI?,respectively,and higher than the control group,6.52±2.34?g/g?KO?,5.75±1.20?g/g?WT?,5.80±1.83?g/g?KI?.The differences were statistically significant?P<0.01?.Morris water maze experiment results showed that ApoE4 gene and aluminium exposure have no interaction on the escape latency of the animals?P>0.05?,and their main effects were statistically significant?P<0.05?.The pairwise comparison by using LSD method showed that both ApoE4 and aluminium could extend the escape latency.The number of crossing the platform in the aluminium-exposed mice were 1.75±0.71?KO?,1.50±0.53?WT?,1.25±0.89?KI?,respectively,and less than the control group,4.63±0.92?KO?,2.75±1.28?WT?,2.38±0.92?KI?.Statistical analysis showed that there was an interaction between the animal type and the intervention?P<0.05?,and the individual effects were also statistically significant?P<0.05?.The results from pairwise comparison showed that ApoE4 and aluminium could reduce the number of times that animals cross the platform,and the interaction was existed.Results of the new object recognition test showed that the time used in the KI+Al group of mice in the T2 test period were similar for exploring new objects and familiar objects,and there was no statistical significance?P>0.05?,and other remaining groups all had statistical significance?P<0.05?.The discrimination index in the aluminium-exposed mice were 41.9±9.8?KO?,26.8±9.4?WT?,0.8±16?KI?,respectively,and lower than the control group,55.1±10.7?KO?,46.7±8.7?WT?,40.7±5.9?KI?.Statistical analysis showed that there was an interaction between animal types and interventions?P<0.01?,and their individual effects were also statistically significant?P<0.01?.The pairwise comparison results showed that ApoE4 and aluminium could reduce the animal discrimination index and the interaction was existed.2.The results of HE staining showed that the size and morphology of hippocampal neurons in each group were normal,and no changes such as atrophy and apoptosis were observed.Golgi staining showed that neurons were fractured in the KI+Al group.The number of dendritic intersections at a distance of 20?m from the cell body in the aluminium-exposed mice were 5.29±0.75?KO?,4.60±0.20?WT?,4.15±0.28?KI?,respectively,less than the control group,7.20±0.64?KO?,5.44±0.33?WT?,5.35±0.31?KI?,and the dendritic spine density in the aluminium-exposed mice were 0.53±0.06?KO?,0.34±0.05?WT?,0.26±0.04?KI?,respectively,lower than the control group,0.72±0.07?KO?,0.57±0.06?WT?,0.39±0.05?KI?.Statistical analysis showed that there was an interaction between animal types and interventions?P<0.05?,and their individual effects were also statistically significant?P<0.01?.The pairwise comparison results showed that ApoE4 and aluminium could reduce the number of dendritic intersections and dendritic spine density with an interaction.The electron microscope experiments,the thickness of the postsynaptic density?PSD?in the aluminium-exposed mice were31.80±2.01 nm?KO?,26.07±3.98 nm?WT?,19.67±2.06 nm?KI?,respectively,less than the control group,41.02±2.31 nm?KO?,35.17±2.75 nm?WT?,33.19±3.49 nm?KI?,and the curvature of the synaptic interface in the aluminium-exposed mice were 8.96±1.54?KO?,4.54±1.05?WT?,2.57±0.89?KI?,respectively,less than the control group,26.70±7.95?KO?,17.34±1.96?WT?,6.40±1.16?KI?.Statistical analysis showed that the animal type and the intervention all had an interaction effect?P<0.05?,and the individual effects were also statistically significant?P<0.01?.The comparison of the results in pairs showed that ApoE4 and aluminium could reduce PSD thickness and curvature of synaptic interface with interaction.3.Elisa method detected A?content that animal types and interventions had no interaction on A?₄₀0 content?P>0.05?,and their main effects were not statistically significant?P>0.05?.The content of A?₄₂2 in the aluminium-exposed mice were14.10±7.69 pg/ml?KO?,16.29±6.50 pg/ml?WT?,29.10±15.06 pg/ml?KI?,respectively,and higher than the control group,7.01±3.75 pg/ml?KO?,11.06±4.23 pg/ml?WT?,13.13±6.92 pg/ml?KI?.Statistical analysis showed that there was an interaction in animal types and interventions?P<0.01?,and their individual effects were also statistically significant?P<0.01?.The results of pairwise comparison showed that ApoE4 and aluminium could increase A?₄₂2 content in the brain.The results of gene and protein determination showed that there was no interaction on animal types and interventions with APP gene and protein expression?P>0.05?,but their main effects were statistically significant?P<0.01?.The pairwise comparison results showed that ApoE4 and aluminium could increase the expression of APP genes and proteins.There was no interaction in animal types and interventions with ApoER2 gene?P>0.05?,but their main effects were statistically significant?P<0.01?.The pairwise comparison results showed that ApoE4and aluminium could reduce ApoER2 gene expression.The expression of ApoER2protein in the aluminium-exposed mice were 0.788±0.074?KO?,0.660±0.084?WT?,0.284±0.053?KI?,respectively,and lower than the control group,1.000±0.000?KO?,0.764±0.091?WT?,0.516±0.082?KI?.Statistical analysis showed that there was an interaction in animal types and interventions?P<0.05?,and their individual effects were also statistically significant?P<0.01?.The pairwise comparison results showed that ApoE4 and aluminium could reduce ApoER2 protein expression with interaction effect.For LRP1 gene and protein,there was no interaction between animal types and intervention?P>0.05?,but their main effects were statistically significant?P<0.01?.The pairwise comparison results showed that ApoE4 and aluminium could reduce LRP1 gene and protein expression.For VLDLRs genes and proteins,there was no interaction between animal types and interventions?P>0.05?,but their main effects were statistically significant?P<0.01?.The pairwise comparison showed that both ApoE4 and the aluminium could reduce VLDLRs gene and protein expression.4.The expression of tau-5 protein in the aluminium-exposed mice were 1.405±0.150?KO?,1.747±0.305?WT?,2.339±0.460?KI?,respectively,and higher than the control group,1.000±0.000?KO?,1.327±0.098?WT?,1.538±0.210?KI?.Statistical analysis showed that there was an interaction in animal types and interventions?P<0.05?,and their individual effects were also statistically significant?P<0.01?.The results of pairwise comparison showed that ApoE4 and aluminium could increase tau-5 protein expression with interactions.The expression of pThr181 protein in the aluminium-exposed mice were 1.638±0.292?KO?,2.22±0.500?WT?,3.438±0.719?KI?,respectively,and higher than the control group,1.000±0.000?KO?,1.434±0.350?WT?,1.905±0.418?KI?.Statistical analysis showed that there was an interaction in animal types and interventions?P<0.01?,and their individual effects were also statistically significant?P<0.01?.The pairwise comparison showed that ApoE4 and aluminium could increase the expression of pThr181 protein with an interaction.The expression of pThr231 protein in the aluminium-exposed mice were 1.722±0.338?KO?,2.549±0.571?WT?,4.176±0.577?KI?,respectively,and higher than the control group,1.000±0.000?KO?,1.543±0.409?WT?,2.215±0.470?KI?.Statistical analysis showed that there was an interaction in animal types and interventions?P<0.01?,and their individual effects were also statistically significant?P<0.01?.The pairwise comparison results showed that ApoE4 and aluminium could increase the expression of pThr231 protein with an interaction.There is no interaction among pSer262 protein,animal types and interventions?P>0.05?,and the main effect of animal types were statistically significant?P<0.01?.The pairwise comparison results showed that ApoE4 could increase the expression of pSer262 protein.The expression of pSer396 protein in the aluminium-exposed mice were 1.325±0.192?KO?,1.687±0.404?WT?,2.423±0.490?KI?,respectively,and higher than the control group,1.000±0.000?KO?,1.305±0.107?WT?,1.564±0.254?KI?.Statistical analysis showed that there was an interaction in animal types and interventions?P<0.05?,and their individual effects were also statistically significant?P<0.01?.The pairwise comparison results showed that ApoE4 and aluminium could increase the expression of pSer396protein with an interaction.Conclusion:1.The single action of ApoE4 could cause rise in A?₄₂2 content in mice brain,rise in APP gene and protein expression,decline in ApoER2,LRP1,VLDLRs gene and protein expression,rise in t-tau and p-tau levels in the brain,and decline in neuronal complexity and synaptic structural plasticity.Finally it would affect the learning and memory ability of mice.This experiment further confirmed that ApoE4 is a risk genetic factor for AD.2.Subchronic aluminium exposure could cause rise in aluminium content in the brain,rise in A?₄₂2 content,rise in APP gene and protein expression,decline in ApoER2,LRP1,VLDLRs gene and protein expression,and rise in levels of brain t-tau and p-tau,decline in neuronal complexity and plasticity of synaptic structures,and decline in learning and memory ability in mice.Also it has further confirmed that aluminium is a dangerous environmental factor for AD.3.Through factorial analysis,the effects of the combined effect of ApoE4 and aluminium on AD animals were mainly as follows:rise the content of A?₄₂2 in the brain,affect the morphology,complexity and structural plasticity of synapses,reduce the content of ApoER2 protein and increase in t-tau and p-tau levels,increase the phosphorylation of tau231 and tau396 sites,and reduce cognitive function in mice.4.From the perspective of animal experiments at the first time,this experiment confirmed that genetic factors?ApoE4?and environmental factors?aluminium?could enhance the development of AD.The mechanism may be the combination of the two factors that block the ApoER2 receptor cycle and make the expression of post-transcriptional of ApoER2 decline to affect the clearance of A?,rise the levels of t-tau and p-tau,thereby damages the morphology and synaptic plasticity of neurons,which in turn affects the learning and memory abilities of animals,and finally enhance the development of AD process.