Effect Of Low-dose Copper Exposure On Nuclear Proteome Of Hippocampus In WT Mice And 3xTg-AD Transgenic Mice

Objective: Alzheimer's disease(AD)is a common neurodegenerative disease.The main clinical manifestations of cognitive dysfunction,such as progressive decline in memory and so on.The neurotoxicity of copper can accelerate the occurrence and development of AD.However,nuclear dysfunction may be one of the important factors that lead to AD.However,it is unclear how copper affects the nuclear function and accelerates the process of AD.The purpose of this paper is to screen key molecules involved in this process by nuclear proteomics and to confirm the effect of low dose copper exposure on the nuclear proteome of hippocampus in WT mice and3xTg-AD mice.Methods: Six-month-old Wild type(WT)mice and triple-transgenic mouse3xTg-AD(3xTg-AD,Tg)mice of the experimental group were fed with drinking water containing 0.13 ppm of cupric chloride while non-exposed with 3xTg-AD and WT mice as control group.Two months later,animals were euthanized with diethyl ether.First,neuronal damage induced by copper neurotoxicity in WT mice and3xTg-AD mice was examined by immunofluorescence,Nissl staining,Tunel staining and other pathological experiments.ATP Assay Kit was used to determine the ATP levels in brain of AD mice.Subsequently,the effect of low-dose copper exposure on the nuclear proteome of hippocampus of WT mice and 3xTg-AD mice was explored using two-dimensional fluorescence difference gel electrophoresis(2D-DIGE)coupled with mass spectrometry.Finally,differential proteins were further verified using Western blot and immunohistochemistry.Results: 8-OHdG(8-hydroxyguanosine)immunofluorescence results showed that low-dose copper exposure led to DNA damage in hippocampal neurons of3xTg-AD mice.Neuron-specific markers Nissei staining and TUNEL staining showed that low dose of copper exposure caused a decrease in the number of neurons and increased apoptosis.Nuclear proteomic analysis revealed that 37 hippocampal nuclear proteins(12 increased and 25 decreased)in 3xTg-AD vs.WT mice;46 hippocampal nuclear proteins(18 increased and 28 decreased)in copper-treated WT vs.untreated mice;43 modulated hippocampal nuclear proteins(25 increased and 18 decreased)in copper-treated 3xTg-AD vs.untreated mice.Protein functional analysis revealed that these differentially expressed proteins mainly involved energy metabolism(NDUV1,COX5 B,IDH3A,PGAM1,GPDM,KPYM),synapses(complexin-2,synapsin-2,dynamin-1),DNA damage and apoptosis(PDIA3,PSPC1,PURA),oxidative stress(SODC,PRDX1,PRDX3,PRDX6).Further Western blot and immunohistochemical results showed that glycoprotein-related protein PGAM1,tricarboxylic acid cycle-related protein IDH3 A,synaptic-related protein synapsin-2,oxidative stress-related protein SODC,and DNA damage-related protein PDIA3 were consistent with 2D-DIGE in copper-treated vs.untreated WT mice;synaptic-related protein synapsin-2,DNA damage and apoptosis-related proteins PURA,PSPC1 were consistent with 2D-DIGE in copper-treated 3xTg-AD vs.untreated mice.Conclusion: Taken together,low-dose copper decreased ATP content,increased the positive staining of 8-hydroxyguanosine(8-OHdG),a marker of DNA oxidative damage,and caused apoptosis and perturbs multiple biological/pathogenic processes by dysregulating the nuclear proteome.Suggesting that changes in STMN1 and SODC may be early biomarkers of neurotoxicity of copper toxicity and that copper exposure may therefore contribute to the evolution of AD.