Low-dose Copper Exposure Changes The Hippocampal Phosphoproteomic Profile And Perturbs Mitochondrial Function In A Mouse Model Of Alzheimer’s Disease

Objective:Alzheimer’s disease（AD）is a progressive neurodegenerative disease.Clinically,it is characterized by cognitive dysfunction,executive dysfunction,personality and behavior changes,etc.Excessive copper can lead to neurotoxicity and the development of certain neurological diseases.However,the neurotoxicity and potential mechanisms of copper remain poorly understood.The purpose of this study was to investigate the effects of low-dose copper exposure on hippocampal phosphoproteomics and mitochondrial function in AD model mice by using phosphoproteomics technology,and to screen out the toxicity related key molecules and analyze their potential links.Methods:Three transgenic 6-month old AD（3xTg-AD）model mice and6-month old wild type（WT）mice were fed with drinking water of 0.13 ppm copper chloride for 2 months,and 3xtg-ad and WT mice without copper exposure were used as the control group.Firstly,Real-time Quantitative PCR Detecting System（QPCR）,immunofluorescence,hydrogen peroxide production（H₂O₂）,lipid oxidation level（MDA）,cytochrome C oxidase activity and ATP content were used to verify the damage of copper exposure on the mitochondrial function of the hippocampal tissues of WT mice and 3xTg-AD mice,silver staining was used to detect the effect of copper exposure on axonal degeneration of hippocampal neurons in 3xTg-AD mice.Finally,Q Exactive mass spectrometry was used to analyze the effects of low-dose copper exposure on hippocampal phosphoproteins in WT mice and 3xTg-AD mice,and Western blot was used to verify mitochondrial production and mitochondrial dynamics,as well as differentially expressed phosphoproteins.Then explore the potential links between them.Results:QPCR,mitochondrial function testing and western blot results show that compared with unexposed WT mice,low-dose copper treatment of WT mice decreased hippocampal mitochondrial copy number,mitochondrial biogenesis and disrupted mitochondrial dynamics;these changes were associated with increased H₂O₂production,reduced cytochrome oxidase activity and decreased ATP content.Phosphoproteomics identified a total of 3960 unique phosphopeptides（5290phosphorylation sites）from 1,406 phosphoproteins.The copper exposed WT mice had41 differentially expressed phosphoproteins compared with copper unexposed WT mice.And 162 differentially expressed phosphproteins in the copper exposed3xTg-AD mice compared with the unexposed 3xTg-AD mice.These differentially expressed phosphoproteins are involved in neuronal and synaptic functions,transcriptional regulation,energy metabolism and mitochondrial function.In addition,silver staining results showed that copper exposure increased axonal degeneration in3xTg-AD mice compared with the copper unexposed 3xTg-AD mice,which was associated with altered phosphorylation of Camk2αat T286 and phosphorylation of mitogen-activated protein kinase（ERK1/2）,which involved long-term enhanced（LTP）signaling.Mitochondrial dysfunction is mainly related to changes in phosphorylation levels of glycogen synthetase kinase-3 beta（GSK3β）and serine/threonine protein phosphatase 2B catalytic subunit isotype（Ppp3ca）,which is involved in biogenic signal transduction of mitochondria.Conclusions:Low-dose copper exposure changes the phosphorylation of key hippocampal proteins involved in mitochondrial,synaptic and axonal integrity.These data showing that excess of copper speeds some early events of AD changes observed suggest that excess circulating copper has the potential to perturb brain function of wild-type mice and exacerbate neurodegenerative changes in a mouse model of AD.