Chronic Copper Exposure Causes Spatial Memory Impairment, Selective Loss Of Hippocampal Synaptic Proteins, And Activation Of PKR/eIF2α Pathway In Mice

Objective: Copper is a necessary metal element required for humangrowth and development, and plays an important role in the development of brain and immune system.However, excessive copper intake could lead to irreversible damage to cells and neurotoxicity.Environmental pollution has become increasingly prominent with industry devoloping, and copper pollution poses agreat threat to human health. However, neurotoxicity of copper exposure and the molecular mechanisms remain poorly understood. Studies have shown that copper exposure led to elevated oxidative stress levels and caused neurotoxicity in vivo. In this study, we exposed C57BL/6 male mice(2-3 months old) to copper for 3 months(drinking water supplemented with 250.0 ppm Cu SO4) and explored the potential neurotoxicity of copper and the molecular mechanisms. The accomplishment of this study would provide experimental evidence for health risk assessment of copper exposure. Methods: ICP-MS was used to measure the content of free copper, protein-bound copper, total copper, calcium,magnesium, zinc, iron in the serum, hippocampus and cortex. Morris water maze test was performed to detect the ability of spatial learning and memory of the mice exposed to copper.The expression levels of copper metabolism-related proteins, oxidative stress-related proteins,double stranded RNA-dependent protein kinase-eukaryotic initiation factor 2α(PKR-e IF2α)pathway-related proteins and synaptic proteins in hippocampus were measured by Western-blot analysis. Immunofluorescence was used to detect the change of oxidative stress marker, 8-hydroxy-deoxyguanine and to analyze the co-localization between activating transcription factor 4(ATF-4) with synaptic proteins(synapsin 1 or PSD-93); TUNEL staining was performed to measure the change of cellular apoptosis in hippocampus. Results:The free copper and protein-bound copper content was significantly elevated in serum,hippocampus and cortex in copper-exposed mice, while the content of calcium,magnesium, zinc, iron level was not significantly changed. Morris water maze test revealed that copper exposure led to impairment of long-term spatial memory, but did not yield a deterious effect on spatial learning ability of the mice. The expression of two copper metabolism-related proteins in hippoampus, ceruloplasmin and superoxide Dismutase 4, wasdownregulated and upregulated respectively. The expression level of nitrotyrosine, a marker of oxidative stress, was significantly elevated after copper exposure. The activity of PKR was significantly increased as evidenced by increased ratio of phospho-PKR at Thr451 and total PKR in hippocampus of copper-exposed mice. The phosphorylation of eukaryotic initiation factor 2α(e IF2α) at Ser51, downstream signaling pathway of PKR, was significantly elevated in hippocampus of copper-exposed mice. The levels of ATF-4 was up-regulated by copper treatment. The phosphorylation of c AMP responsive element-binding protein(CREB) at Ser15 was significantly decreased in hippocampus of copper-exposed mice. The levels of synaptic proteins including synapsin 1, complexin-1/2, PSD-93, PSD-95 were down-regulated after copper exposure, while the levels of NR2 A, NR2 B were not significantly changed. The expression of C/EBP homologous protein(CHOP) was significantly increased after copper exposure. Immunofluorescent analysis showed that the levels of 8-hydroxy-2 deoxyguanosine(8-Ohd G) were elevated in hippocampal CA1, CA3 and DG region after copper treatment.Meanwhile, up-regulation of ATF-4 and down-regulation of synaptic proteins(synapsin 1 and PSD-93) occurred in the same neuronal cells. TUNEL staining showed that copper exposure caused significant cell apoptosis in hippocampus. Conclusion: Chronic copper exposure elevated free copper content in serum, hippocampus and cortex, and caused neurotoxicity in hippocampus of mice. Copper exposure led to abnormal expression of ceruloplasmin and superoxide dismutase, two copper metabolism-related proteins. Copper exposure also caused oxidative stress and activated PKR/e IF2α signaling pathway, as evidenced by increased phosphorylated PKR, increased phosphorylated e IF2α, up-regulated ATF-4, inhibition of CREB, and expression of pro-apoptotic factor-CHOP. Based on the data, we concluded that copper exposure caused spatial memory impairment in mice via PKR/e IF2α mediated down-regulation of synapse-related proteins and enhanced apoptosis in hippocampus.