The Research On Regulation Mechanism Of Ndfip1 To Iron Ion Metabolism In The Brain Of APP/PS1 Transgenic Mice

Objective:Iron is one of the essential metal ions in the body and plays an important role in various biological metabolic processes in the body.Brain iron belongs to non-heme iron,which is widely distributed in the brain,and its homeostasis is strictly regulated.Studies had shown that brain iron content was positively correlated with age,and brain iron content was increased with age.In some patients of neurodegenerative diseases such as Alzheimer's disease,iron overload or abnormal iron ions were also found in the brain.Experiments had shown that excessive intracellular iron content could cause oxidative stress,protein aggregation and even cell death.Among the many causes of AD,metal ions such as iron,zinc and copper were closely related to APP expression,A?secretion and deposition.It can be seen that maintaining the balance of brain iron homeostasis is crucial.But so far,the specific regulation mechanism of brain iron is still unclear.Ndfip1is an evolutionarily conserved transmembrane protein that specifically binds to the Nedd4 family and is involved in the ubiquitination and degradation of certain proteins.Ndfip1 functions as an adaptor protein,which is linked to the WW domain of the E3ubiquitin ligase Nedd4 family via its two PPXY motifs at the amino terminus,and recruits the target protein,ultimately resulting in polyubiquitination of the target protein and proteasome Identification and degradation.Studies had shown that Ndfip1 was involved in the homeostasis of iron ions and could be involved in the regulation of intracellular iron ion transport.DMT1 is a divalent metal ion transporter that mediates the transfer of iron ions into cells.Experiments had confirmed that Ndfip1 was associated with DMT1,and both of them express in cytoplasm and were co-localized in the Golgi complex.Ndfip1 could negatively regulate the expression and transport function of DMT1,and its mechanism of action was achieved by the ubiquitin-proteasome degradation pathway.The proteasome inhibitor MG132 could inhibit the negative regulation of Ndfip1 on DMT1.It is speculated that Ndfip1 regulates the expression and activity of DMT1,and then regulates the intracellular iron ion concentration to affect the toxic effects of iron ions on nerve cells,and finally exerts its neuroprotective effect.In order to study the role and mechanism of Ndfip1,we detected localization and expression of it in the cortex and hippocampus of brain,and effect of age to it.Otherwise,we also detected localization and expression of Ndfip1 in AD animal model and AD cell model.We want to find the evidence that Ndfip1 participates in brain iron metabolism.After that,we used Ndfip1 plasmid to transfect APPsw cells,and detect expression of APP695,A?1-42 level,expression and activity of DMT1,and cell survival rate.These studies will provide mechanism of theory evidences that Ndfip1 participates in brain iron metabolism.Methods:Immunohistochemistry,immunofluorescence,Western Blot and RT-PCR were used to systematically observe the localization and expression of Ndfip1 in the cortex and hippocampus of C57BL/6 mice.Western Blot was used to detect the expression changes of Ndfip1 and ubiquitin-degrading target protein DMT1 in cerebral cortex and hippocampus of mice of different age groups from 1 week after birth to 48weeks after birth.Immunohistochemistry,immunofluorescence three-label confocal laser scanning,Western Blot and RT-PCR techniques were used to observe the localization and expression of Ndfip1 in the cortex and hippocampus of AD mouse model?APP/PS1transgenic mice?and AD cell models?stable SH-SY5Y cells transfected with human APP gene?.The Ndfip1 eukaryotic overexpression plasmid was constructed and transfected into APPsw cells.The expression of Ndfip1 protein was detected by Western Blot at 24 h,48 h and 72 h after transfection to determine the transfection efficiency of the plasmid.The expressions of APP metabolism and DMT1 protein were detected by Western Blot and ELISA after transfection of Ndfip1 plasmid into APPsw cells.The fluorescence intensity of Ndfip1 plasmid transfected APPsw cells under iron overload condition was quantitatively detected by fluorescence microscopy and fluorescence spectrophotometer.The change of DMT1 transportable iron ability was analyzed by Calcian AM method.The effect of 0⁵00?mol/LFeSO₄ and FeCl₃ on the survival rate of APPsw cells and the effect of Ndfip1 overexpression on the decrease of APPsw cell survival rate induced by 200?mol/L FeSO₄ or FeCl₃ were detected by MTT assay.Results:1.The expression of Ndfip1 in cortex and hippocampus of mice,and changes with age of it:Ndfip1 was widely expressed in cortex and restricted expressed in the CA3of hippocampus in mice.There is no positive expression of Ndfip1 in CA1,CA2 and DG of the hippocampus.Ndfip1 was expressed specially in pyramidal neurons,both cell body and processes.Western Blot results indicated that Ndfip1 was detected in the cortex and hippocampus.The protein content of Ndfip1 in cortex was stronger than that in hippocampus.RT-PCR results indicated that Ndfip1 mRNA was expressed in the cortex and hippocampus,and there was not obvious different between them.Ndfip1 was expressed in cortex and hippocampus of mice at postnatal 1,4,12,24,36,and 48 weeks.Ndfip1 expression in the brain of adult mice was lower than that of postnatal 1 week mice.DMT1-IRE and DMT1-nonIRE were expressed in cortex and hippocampus of mice at postnatal 1,4,12,24,36,and 48 weeks.The expression of DMT1-IRE and DMT1-nonIRE in the brain of adult mice were higher than that of postnatal 1 week mice.With the increase of age,the expression of DMT1-IRE and DMT1-nonIRE in cortex and hippocampus of mice were according to Ndfip1.2.Ndfip1 was expressed in the brain of APP/PS1 transgenic mice and APPsw cells:Ndfip1 was expressed in cortex and hippocampus of APP/PS1 transgenic mice.Ndfip1 was widely expressed in cortex and restricted expressed in the CA3 of hippocampus.Ndfip1 was expressed in cytoplasm and processes of pyramidal neurons.It is interesting that Ndfip1 was expressed in the middle area of senile plaque in cortex and hippocampus of APP/PS1 transgenic mice.Ndfip1protein level in cortex and hippocampus of APP/PS1 transgenic mice were lower than that of control group.There was not obvious different of Ndfip1 mRNA between APP/PS1 transgenic mice and control group mice.In APPsw cells,Ndfip1 was expressed in cytoplasm.Ndfip1 protein level in APPsw cells was lower than that of control group,which accorded to animal experiment.3.Ndfip1 affected the metabolic process of APP and weakened the toxic effects of iron ions on cells by regulating expression and activity of DMT1 in neuron:Ndfip1 plasmid was made to transfect APPsw cells.Ndfip1 protein levels,by Western Blot analyses,were increased 40%and 66%after transfected for 24and 48 hours.The APP695 protein level was obviously down regulated in cells that Ndfip1 plasmid transfected for 48 hours by Western Blot analyses.The DMT1-IRE and DMT1-nonIRE protein levels were obviously down regulated in cells that Ndfip1plasmid transfected for 48 hours by Western Blot analyses.The secretion of A?1-42 is reduced in cells that Ndfip1 plasmid transfected for 48 hours by ELISA.Calcein AM fluorescence technology was used to detect the fluorescence intensity in cells transfected by Ndfip1 which stronger than that in control group.The speed of fluorescence quench in Ndfip1 transfected cells was slower than that in control group.The survival rates of cell that dealed with FeSO₄ or FeCl₃ were detected by MTT assay.We found that the cell survival rate was descend to 20%caused by 200?mol/L FeSO₄ or FeCl₃.Ndfip1overexpression can increase the cell survival rates induced by 200?mol/LFeSO₄ or FeCl₃.Conclusions:1.Ndfip1 was widely expressed in cortex and restricted expressed in the CA3 of hippocampus in mice.Ndfip1 was expressed specially in pyramidal neurons,both cell body and processes.2.Ndfip1 protein levels down regulated while DMT1protein levels up regulated in cortex and hippocampus with age,which implied that the age might be one of the effect factors of Ndfip1 and DMT1 protein expression.3.Ndfip1protein level in cortex and hippocampus of APP/PS1 transgenic mice were lower than that of control group.4.Ndfip1 was expressed in the middle area of senile plaque in cortex and hippocampus of APP/PS1 transgenic mice.5.Ndfip1 protein level in APPsw cells was lower than that of control group.6.Ndfip1 protein levels increased in APPsw cells transfected by Ndfip1.It can be the cell model to research the function of Ndfip1.7.Ndfip1 down-regulated APP695 expression and reduced A?1-42 secretion in APPsw cells.8.Ndfip1 down-regulated the expression and iron transfer capacity of DMT1 in APPsw cells.9.Ndfip1 alleviated the decrease of APPsw cell survival rate caused by excessive iron ions,and had protective role for nerve cells.