Abstract Copper neurotoxicity has been implicated in multiple neurological diseases. However, there is a lack of deep understanding on copper neurotoxicity, especially for low-dose copper exposure. In this study, we investigated the effects of chronic, low-dose copper treatment (0.13 ppm copper chloride in drinking water) on hippocampal mitochondrial and nuclear proteome in mice by 2-dimensional fluorescence difference gel electrophoresis coupled with MALDI-TOF-MS/MS. Behavioral tests revealed that low-dose copper caused spatial memory impairment, DNA oxidative damage as well as loss of synaptic proteins. Proteomic analysis revealed modulation of 31 hippocampal mitochondrial proteins (15 increased and 16 decreased), and 46 hippocampal nuclear proteins (18 increased and 28 decreased) in copper-treated versus untreated mice. Bioinformatic analysis indicated that these differentially expressed proteins are mainly involved energy metabolism (NDUV1, COX5B, IDH3A, and PGAM1), synapses (complexin-2, synapsin-2), DNA damage (PDIA3), apoptosis (GRP75), and oxidative stress (SODC, PRDX3). Among these differentially expressed proteins, synapsin-2, a synaptic-related protein, was found to be significantly decreased as confirmed by Western-blot analysis. In addition, we found that superoxide dismutase [Cu-Zn] (SODC), a copper ion target protein, was identified to be decreased in copper-treated mice versus untreated mice. We also found that stathmin (STMN1), a microtubule-destabilizing neuroprotein, was significantly decreased in hippocampal nuclei of copper-treated mice versus untreated mice. Taken together, we conclude that low-dose copper exposure causes spatial memory impairment and perturbs multiple biological/pathogenic processes by dysregulating the mitochondrial and nuclear proteome, particularly the proteins related to respiratory chain, synaptic vesicle fusion, axonal/neurtic integrity, and oxidative stress. The change of STMN1 and SODC may represent early novel biomarkers of copper neurotoxicity. mitochondrial/nuclear proteomics, biomarkers, copper, neurotoxicity Copper, one of the most abundant metals in the human body (Lewińska-Preis et al., 2011), has the highest levels, in the liver and brain (Szerdahelyi and Kása, 1986). Copper acting as a cofactor or enzyme component, is involved in energy metabolism, antioxidant defense, iron metabolism, and other physiological pathways (Scheiber et al., 2014). Copper also plays an important role in such biological processes as angiogenesis, hypoxia, and neuromodulation (Scheiber et al., 2014). Neurological disorders can arise from both copper deficiency and copper excess (Bulcke et al., 2017). Deficiencies arising from copper malabsorption secondary to gastric bypass surgery or excessive zinc intake can trigger myeloneuropathy (Lorincz, 2018). Copper accumulates in the brain, liver, eyes, and other organs in Wilson’s disease, an inherited autosomal recessive mutation in the ATP7B gene that codes for a copper-transporting P-type ATPase located in the trans-Golgi network (Lorincz, 2018). The neurotoxicity of copper has been implicated in neurodegenerative diseases such as Alzheimer disease (AD), Parkinson disease (PD), and Huntington disease (HD) (Lutsenko et al., 2010; Multhaup et al., 1996; Scheiber et al., 2014). Copper toxicity is associated with abnormal energy metabolism via effects on glycolysis, tricarboxylic acid cycle, and respiratory chain enzymes, and mitochondrial membranes (Haywood et al., 2004; Medeiros and Jennings, 2002; Zischka et al., 2011). Excess copper increased reactive oxygen species (ROS) and mitochondrial oxidative damage (Zafar et al., 2016). Copper and copper complexes can also cause DNA damage (Sagripanti and Kraemer, 1989; Tkeshelashvili et al., 1991), chromatin condensation and marginalization, nuclear division, and apoptosis (Babaei et al., 2012). Copper accumulates in synapses and other parts of the brains of AD patients (Lutsenko et al., 2010; Scheiber et al., 2014) and copper overload can also impair synaptic plasticity and accelerate neurodegenerative processes (Lutsenko et al., 2010). These lines of evidence suggest that the neurotoxicity of copper is closely related to the impairment of mitochondria, nuclei, and synapses. However, the effects of excess copper on the protein content of subcellular organelles is not understood. Here, we investigate the proteomic alterations of mitochondrial and nuclear proteins in the hippocampus of mice exposed to low-dose copper using 2-dimensional fluorescence differential gel electrophoresis (2D-DIGE) coupled with Matrix-Assisted Laser Desorption Ionization—Time Of Flight Mass Spectrometry (MALDI-TOF-MS/MS). This approach has the potential of revealing molecular mechanisms and biomarkers of copper neurotoxicity. MATERIALS AND METHODS Animals and treatment protocol The experimental mice (strain: B6129SF2/J) were purchased Jackson Laboratory (Maine, USA). All the mice were provided with freshly either drinking water (0.005 ppm Cu) or drinking water containing 0.13 ppm copper(II) chloride (CuCl2, Sigma, USA) that was freshly prepared each week and administered for 12 months (Supplementary Figure 1). The selected dose of copper in drinking water was referenced to previous studies and the diet is also consistent with that as previously reported (Sappal et al., 2014; Sparks and Schreurs, 2003). The diet is standard diet purchased from Guangdong Medical Laboratory Animal Center, an official organization responsible for providing animals and diets. The quality control of animals and diets are strict. The diet accords with the international standard and actually contains inorganic forms (copper sulfate). Furthermore, the food intake was similar in Cu-treated versus -untreated mice. Mice were housed in groups of 10 animals per cage (470 × 350 × 200 mm) and provided food and water ad libitum. The housing facility was maintained at a stable temperature (20 ± 2°C) and humidity (55 ± 5%) and on a 12-h light-dark cycle with the light on from 6:00 am to 6:00 pm. This study was approved by The Ethics Committee of the Shenzhen Center for Disease Control and Prevention. Animal treatment and housing were carried out in accordance with the Principles of Laboratory Animal Care (NIH publication No. 8–23, revised 1985) and the Regulations of the Animal Care and Use Committee of the Experimental Animal Center at Shenzhen Center for Disease Control and Prevention. Morris water maze test The Morris water maze test was used to assess spatial learning and memory (D'Hooge and De Deyn, 2001). All the mice used in the Morris water maze test were 15-month-old mice exposed to copper for 12 months. The water maze consists of a round tank (170 cm diameter) filled with 30 cm-depth water, made opaque by addition of white milk powder, maintained at 22 ± 1°C. The tank contained a submerged platform to which the animals can navigate. In the daily training period of 5 days, mice were individually placed in the open-swimming area facing the wall of the pool and starting from 1 of the 4 quadrants. Training was ended when the mouse climbed on the platform. If the mouse failed to find the platform within the allowed time, the animal was guided to the platform by the experimenter and left there for 30 s. The 6th day (day 12) after the learning training, the mice were allowed to swim for 120 s when the platform was removed at the probe trial. The number of platform crossing, the swimming paths, the percentage of time spent, and the percentage of distance travelled in the target quadrant were analyzed to assess the spatial memory of the mice. Isolation of mitochondria After the behavioral test was completed, animals were euthanized with diethyl ether, the brain removed from the skull, and the hippocampus excised. Mitochondria were isolated at 4°C using the Mitochondria Isolation Kit for Tissue (Thermo 89801, ThermoFisher Scientific, Waltham, MA, USA). Hippocampal tissue containing the dentate gyrus (DG), CA3, CA2, and CA1 zone was homogenized for 10 min in 300 ml 1× PBS solution with a Motor Driven Tissue Grinder (Sangon Biotech G506003, Shanghai, China). The homogenates were centrifuged and the resulting pellet was suspended in 800 μl of BSA/Reagent A solution and incubated on ice for exactly 2 min. 10 μl of Mitochondria Isolation Reagent B was added to the suspension and the samples were incubated on ice for 5 min. Then, 800 μl of Mitochondria Isolation Reagent C was added to the tube and mixed. Finally, the mitochondrial pellet was isolated after centrifuging, washed twice with 500 μl in Wash Buffer, and stored at −80°C until use. Isolation of nuclei Hippocampal nuclei were isolated with NE-PER Nuclear and Cytoplasmic Extraction Reagents (ThermoFisher 78835). Bilateral hippocampal tissues were homogenized in 300 ml Cytoplasmic Extraction Reagent I (CER I) for 10 min with the aid of a Motor-Driven Tissue Grinder (Sangon Biotech G506003, Shanghai, China). 17 μl of ice-cold Cytoplasmic Extraction Reagent II (CERII) was added to the swollen cells to gently lyse the cell membrane. Then, 150 μl ice-cold Nuclear Extraction Reagent (NER) was used to extract the nuclear proteins from the pellet. The sample was placed on ice and vortexed for 15 s every 10 min, with 4 repeats. The nuclear extract was transferred to a clean pre-chilled tube and stored at −80°C until use. Protein sample preparation for proteomic analysis DIGE-specific lysis buffer (7 M urea, 2 M thiourea, 30 mM Tris–HCl, 4% CHAPS, pH 8.5) was used to treat the mitochondrial pellets and nuclear extracts of whole hippocampus to extract proteins for differential in-gel electrophoresis (DIGE) analysis. The pellets were incubated for 30 min, centrifuged at 20 000 × g for 60 min at 4°C, at 15 000 × g for 30 min at 4°C, respectively, to remove salt and other impurities. Protein concentration was determined by 2-D Quant Kit (GE HealthCare, Milwaukee, USA), and the protein solutions were stored at −80°C until use. DIGE labeling of proteins CyDye (GE Healthcare) powder was dissolved at a concentration of 200 pmol/μl with 25 μl of 99.8% anhydrous N, N-dimethylformamide (DMF, 227056, Sigma-Aldrich, St. Louis, MO, USA). Each protein sample (25 μg, pH 8.0–9.0) was labeled with 200 pmol of either Cy3 dye (GE Healthcare, 25-8008-61), or Cy5 dyes (GE Healthcare, 25-8008-62). In addition, a mixed sample (25 μg each) labeled with Cy2 dyes was used as an internal standard. In the dark, the labeled reactants were incubated on ice for 30 min, and the reaction was terminated with 10 mM lysine (Sigma-Aldrich, L5626) at 4°C for 10 min. After labeling, the Cy2-, Cy3-, and Cy5-labeled samples were mixed together. IPG buffer (2% [v/v] pH 3–11 NL), 100 μl of 2× lysis buffer (7 M urea, 2 M thiourea, 4% CHAPS, 2% DTT) was added and the samples incubated on ice for 10 min. Finally, rehydration buffer was added to adjust the volume to 450 μl. 2D difference gel electrophoresis The first dimension was performed with the Ettan IPGphor Isoelectric Focusing System (GE Healthcare). Mixtures of Cy2-, Cy3-, and Cy5-labeled proteins were put into 24 cm pH 3–11 NL Immobiline Dry Strips (GE Healthcare). Specific steps (300 V 12 h; 500 V 2 h; 1000 V 2 h; gradient 8000 V 8 h; 8000 V 8 h) were carried out at RT. After isoelectric focusing (IEF), each strip was equilibrated in equilibration buffer (6 M urea, 75 mM Tris–HCl, 30% glycerol, 2% SDS), followed by 1% DTT (Sigma-Aldrich) and 4.5% IAA (Sigma-Aldrich) for 15 min. The equilibrated strips were loaded on the top of 12.5% SDS-PAGE gels with 0.5% (w/v) ultra-low-melting-point agarose sealing solution (25 mM Tris, 192 mM glycine, 0.1% SDS, 0.5% [w/v] agarose, 0.02% bromophenol blue). Electrophoresis was performed with an Ettan DALTsix Electrophoresis System (GE Healthcare) under the following conditions: 1 W/gel for 1 h and, subsequently 11 W/gel for 5 h at 12°C in darkness. Gels were immediately scanned with a Typhoon TRIO Variable Mode Imager (GE Healthcare), and the PMT was set to ensure that the maximum pixel intensity of all gel images remained within a range of 40 000–60 000 pixels. Image analysis The DeCyder software package (Version 6.5 GE Healthcare, Milwaukee, USA) was used to analyze the DIGE gels following the manufacturer’s protocol. For further protein differential analysis, we performed a normalized analysis for each protein spot to find significantly different spots (p ≤ .05). Spot picking and in-gel digestion A total of 1.5 mg of protein samples was used to run 2-DE using identical conditions as above. These gels were stained with Coomassie blue solution (0.12% Coomassie Brilliant Blue G-250, 20% ethanol, 10% phosphoric acid, and 10% ammonia sulfate). Spots of interest detected by Decyder software analysis were digested overnight at 37°C with trypsin (Promega Corp., WI, USA) in 1.5 ml Eppendorf centrifuge tube. Mass spectrometry A total of 0.6 μl of peptide extract was used for the MADL-TOF-MS (AB SCIEX, MALDI-TOF/TOF 5800 mass spectrometer) analysis. Samples were crystallized with 0.4 mg/ml α-cyano-4-hydroxycinnamic acid in 30% acetonitrile/0.06% trifluoroacetic acid directly on the target and dried at RT. The spectra were externally calibrated. The SwissProt databases (Matrix Science, UK) for mouse mitochondrial and nuclear proteins were searched in MASCOT database. The search was carried out using the Mus musculus database. The search was conducted with a tolerance on mass measurement of 100 ppm in the MS mode and 0.5 Da in the MS/MS mode. The fixed carbamidomethyl modification was taken into account, and up to 2 missed cleavages per peptide were allowed. Bioinformatics analysis We analyzed proteomic results using a variety of methods and approaches. DAVID version 6.7 (https://david-d.ncifcrf.gov/) was used to elucidate the biological function of the identified protein (Huang et al., 2009a, b). STRING database version 10.0 (https://string-db.org/) (Xu et al., 2016) was used to analyze protein-protein interaction networks. STRING-generated network visualized and edited in Cytoscape version 3.5.1. Venn Diagram Generator (http://www.pangloss.com/seidel/Protocols/venn.cgi) was used to perform a logistic analysis of the mitochondrial proteome and nuclear proteome. Cytoscape 3.5.1 software and plug-ins were used to analyze Wiki paths (Xu et al., 2016). Western-blot analysis Hippocampal mitochondria from copper-treated and untreated mice were extracted with lyse mitochondrial buffer with 2% CHAPS in Tris buffered saline (TBS; eg, 25 mM Tris, 0.15 M NaCl; pH 7.2, Product No. 28379, Thermo Fisher Scientific, USA). Hippocampal nuclear extract from each group was extracted with lysis buffer (Beyotime, Haimen, China) with phosphatase inhibitor cocktail (Thermo Fisher Scientific, USA). The concentration of total proteins was measured with the BCA protein assay kit (Thermo Fisher Scientific, USA). The protein samples were mixed with loading buffer and heated for 8 min at 100°C, then separated on 12% SDS-PAGE and transferred to PVDF membranes. Membranes containing the transferred proteins were blocked with 5% skim milk in TBST (150 mM NaCl, 10 mM Tris, 0.1% Tween-20, pH 8.0). The blocked membranes were incubated with anti-β-actin (1:1000, Santa Cruz, CA, sc-47778), anti-VDAC1 (1: 2000, Abcam, ab154856), anti-synapsin II (1: 5000, Abcam, ab76494), anti-COX5B (1:1000, Abcam, ab180136), anti-QCRI (1: 1000, Abcam, ab131152), anti-IDH3A (1: 1000, Abcam, ab58641), anti-PGAM1 (1: 1000, Abcam, ab184232), anti-PDIA3 (1: 2000, Abcam, ab10287), anti-GRP75 (1: 500, Santa Cruz, sc-133137), and anti-SODC (1: 5000, Abcam, ab13498) in TBST buffer overnight at 4°C. After washing in TBST, the membranes were incubated with a 1: 3000 dilution of anti-rabbit or anti-mouse IgG HRP secondary antibody diluted in TBST for 1 h. Subsequently, the membranes were washed in TBST (4 × 10 min) and developed using chemiluminiscence reagents from an ECL kit (Thermo Scientific Pierce ECL, USA). The blots were detected on a phosphorimager and analyzed using ImageQuant 1D software. TUNEL staining The mouse brain tissues were fixed by immersion in 4% paraformaldehyde for 48 h, dehydrated in ethanol, embedded in paraffin and 5 μM coronal sections prepared with a microtome. For the assay of apoptosis, TUNEL staining was performed using the DeadEnd™ Fluorometric TUNEL System as described in the instructions provided with the kit. The images were taken using an inverted light microscope (Olympus 1X51, Tokyo, Japan). Statistical analysis The data were expressed as mean ± standard error (SEM) and analyzed using GraphPad Prism 6.0 statistical software (GraphPad Software, Inc.). The significance of the differences between the 2 groups of mice was determined by unpaired t-test. The level of significance was set at p < .05. RESULTS Spatial Memory Impairment Caused by Low-Dose Copper Exposure The Morris water maze test was used to investigate the effect of copper exposure on the spatial learning and memory abilities of mice. During the 5-day training period, all the mice showed significant reduction in the time to find the hidden platform, while copper-exposed mice spent longer time than the untreated mice (Figure 1A). Six days after the training period, the probe trial was performed to evaluate the spatial memory of the mice. Compared with the control mice, the percentage of time spent and the percentage of the distance traveled in the correct quadrant were significantly decreased in copper-exposed mice versus control animals (Figs. 1B–D). Additionally, an increased trend in the probe time and decreased platform crossing number were observed for copper-exposed mice relative to the control mice (Figs. 1E and 1F). These data demonstrated that low-dose copper treatment of mice caused spatial memory impairment. Figure 1. View largeDownload slide The effects of low-dose copper treatment on the spatial learning and memory abilities of mice. For the navigation test, the escape latency was measured to determine the effects of oral copper treatment on the spatial learning ability of mice. Three-months-old male mice were treated with low-dose copper in drinking water for 12 months. A, The probe test; B, the probe time; The representative swimming path; C, The percentage of distance traveled in the target quadrant; D, The percentage of time spent in the target quadrant; E, The crossing number; F, The probe time (s). *p < .05 versus the control mice. (Mean ± SEM, n = 9–12 for each group). Figure 1. View largeDownload slide The effects of low-dose copper treatment on the spatial learning and memory abilities of mice. For the navigation test, the escape latency was measured to determine the effects of oral copper treatment on the spatial learning ability of mice. Three-months-old male mice were treated with low-dose copper in drinking water for 12 months. A, The probe test; B, the probe time; The representative swimming path; C, The percentage of distance traveled in the target quadrant; D, The percentage of time spent in the target quadrant; E, The crossing number; F, The probe time (s). *p < .05 versus the control mice. (Mean ± SEM, n = 9–12 for each group). Neuronal Cell Apoptosis Caused by Low-Dose Copper We performed TUNEL staining to measure apoptosis in the hippocampus of copper-treated and untreated mice. The data showed significantly increased apoptosis positive staining in hippocampal CA1, CA3, and DG of copper-treated animals relative to that of the control mice (p < .05) (Figs. 2A–D), suggesting that low-dose copper treatment caused neuronal apoptosis in the mouse hippocampus. Figure 2. View largeDownload slide Apoptosis associated with oral copper treatment. A, A representative image of hippocampal neuronal apoptosis; B, The number of apoptotic cells in CA1; C, The number of apoptotic cells in CA3; D, The number of apoptotic cells in DG. *p < .05 versus the control mice. Scale bar = 100 μm. Figure 2. View largeDownload slide Apoptosis associated with oral copper treatment. A, A representative image of hippocampal neuronal apoptosis; B, The number of apoptotic cells in CA1; C, The number of apoptotic cells in CA3; D, The number of apoptotic cells in DG. *p < .05 versus the control mice. Scale bar = 100 μm. Differentially Expressed Hippocampal Mitochondrial and Nuclear Proteins To assess the effects of low-dose copper treatment on protein expression in the hippocampus, we performed comparative proteomic analysis, using 2D-DIGE, image analysis, in-gel digestion, MS identification, and database, on mitochondrial and nuclear fractions prepared from the hippocampus of copper-treated mice and the non-exposed mice. We also made a separate comparison of mitochondrial and nuclear co-dysregulated proteins in copper-treated mice. Representative 2D-DIGE gel images of hippocampal mitochondrial and nuclear proteins are shown in Supplementary Figures 2A–D and 3A–D. Protein spots with at least 1.1-fold and a p-value < .05 were considered differentially expressed and selected for MS/MS identification (Supplementary Figs. 2E and 3E). Based on the data obtained from the SwissProt database, the gI accession number, the percentage of sequence coverage, the theoretical pI, and Mascot scores, differentially expressed proteins in hippocampal tissues are shown in Figure 3. Protein identification relied on at least 2 different peptide sequences and multiple peptide hits corresponding to every MS/MS event. The MS/MS signals for 2 representative proteins, SODC and STMN1, are shown in Supplementary Figures 4A–D. Figure 3. View largeDownload slide Differentially expressed hippocampal mitochondrial and nuclear protein spots between copper-treated versus untreated mice identified by 2D-DIGE/MALDI-TOF-MS/MS. A and B, Low-dose copper treatment resulted in the differential expression of 31 mitochondrial proteins and 46 nucleoproteins that involve energy metabolism, oxidative stress, synaptic dysfunction, DNA damage and apoptosis, and cytoskeletal integrity. Figure 3. View largeDownload slide Differentially expressed hippocampal mitochondrial and nuclear protein spots between copper-treated versus untreated mice identified by 2D-DIGE/MALDI-TOF-MS/MS. A and B, Low-dose copper treatment resulted in the differential expression of 31 mitochondrial proteins and 46 nucleoproteins that involve energy metabolism, oxidative stress, synaptic dysfunction, DNA damage and apoptosis, and cytoskeletal integrity. The Effects of Copper on the Hippocampal Mitochondrial Proteome of Mice Thirty-one mitochondrial protein spots were differentially expressed in the hippocampus of copper-treated versus untreated mice (Figure 3A). Among these proteins, 15 protein spots showed an increased and 16 a decreased abundance. Prominent relatively increased protein abundance was seen for: (1) phosphoglycerate mutase 1 (PGAM1), which catalyzes the interconversion of 3-phosphoglycerate to 2-phosphoglycerate. Oxidative modification of PGAM1 may lead to inhibition of glycolysis and eventually to ATP depletion (Sultana et al., 2010); (2) 2-oxoglutarate dehydrogenase (ODO1), which is associated with familial and sporadic forms of AD and other age-related neurodegenerative diseases (Albers et al., 2000; Gibson et al., 1998). Markedly higher protein abundance in hippocampal mitochondrial fractions of copper-treated mice versus untreated mice was seen for: (1) adenylate kinase isoenzyme 4 (KAD4), which monitors disturbances of cell energy charge and participates in regulation of differentiation and maturation of cells as well as in apoptosis and oncogenesis (Wujak et al., 2015); (2) mitochondrial inner membrane protein (IMMT), which plays an important role in mitochondrial membrane stability and is associated with apoptosis (Thong and Tsoukanova, 2017); (3) heat shock protein 60 (HS60), a molecular chaperone that maintains mitochondrial oxidative phosphorylation and TCA enzyme anti-amyloid stress (Mangione et al., 2016; Veereshwarayya et al., 2006), and mediates the translocation of APP to mitochondria in 3×Tg-AD cells and human AD tissues (Walls et al., 2012). Gene ontology analysis was performed to functionally categorize the differentially expressed mitochondrial proteins in hippocampus of copper-treated mice by biological processes and molecular functions. The results for biological processes revealed strong enrichment for: generation of precursor metabolites and energy process, glycolysis process, oxidation reduction process, electron transport chain process, hexose catabolic process, and glucose catabolic process (Figure 4A). All of these biological processes are related to energy metabolism. Molecular function annotation revealed strong enrichment of NADH dehydrogenase (quinone) activity, metal cluster binding activity, oxidoreductase activity, iron-sulfur cluster binding activity, nucleotide binding activity, and cofactor binding activity (Figure 4B). Figure 4. View largeDownload slide DAVID Gene Ontology enrichment analysis for the dysregulated mitochondrial proteins and nucleoproteins in hippocampus of copper-treated mice. A, Enrichment analysis for mitochondrial differential proteins by biological processes; B, Enrichment analysis for mitochondrial differential proteins by molecular function; C, Enrichment analysis for nuclear differential proteins by biological process; D, Enrichment analysis for nuclear differential proteins by molecular function. Figure 4. View largeDownload slide DAVID Gene Ontology enrichment analysis for the dysregulated mitochondrial proteins and nucleoproteins in hippocampus of copper-treated mice. A, Enrichment analysis for mitochondrial differential proteins by biological processes; B, Enrichment analysis for mitochondrial differential proteins by molecular function; C, Enrichment analysis for nuclear differential proteins by biological process; D, Enrichment analysis for nuclear differential proteins by molecular function. The Effects of Copper on the Hippocampal Nuclear Proteome of Mice Forty-six nuclear protein spots were differentially expressed between copper-treated and untreated mice (Figure 3B). Among these proteins, 18 protein spots showed increased and 28 decreased abundance copper-treated versus untreated mice. Substantially reduced relative abundance in nuclei of copper-treated mice versus untreated mice was seen for: (1) neurofilament light polypeptide (NFL), a nonspecific but sensitive biomarker of neuronal and axonal damage in neurodegenerative disorders, including multiple sclerosis (MS) (Norgren et al., 2004) and Huntington disease (HD) (Vinther-Jensen et al., 2016); (2) isocitrate dehydrogenase 3 (IDH3A), a TCA cycle enzyme responsible for oxidative decarboxylation of isocitrate to 2-oxoglutarate; (3) protein disulfide-isomerase A3 (PDIA3), which is mainly involved in the regulation of endoplasmic reticulum stress (ERS) and its overexpression can protect neurons from ERS-induced apoptosis (Zhang et al., 2015). (4) peroxiredoxin-3 (PRDX3) and superoxide dismutase [Cu-Zn] (SODC), key players in cellular redox function and protection against oxidative injury. Modestly increased relative abundance was found for: (1) ezrin (EZRI), an important component of the ezrin-radixin-moesin complex and is involved in the regulation of FOXP2 on the development, maintenance and function of the nervous system (Oswald et al., 2017); (2) prohibitin (PHB), an olfactory nerve-related protein, which has an important role in olfactory loss in Alzheimer's patients (Lachen-Montes et al., 2017). Gene ontology analysis was performed to functionally categorize the differentially expressed nucleoproteins in hippocampus of copper-treated mice by biological processes and molecular functions. The results for biological processes revealed strong enrichment for: generation of precursor metabolites and energy process, ATP metabolic process, energy coupled proton transport process, ATP synthesis coupled proton transport process, purine ribonucleoside triphosphate metabolic process, and ribonucleoside triphosphate metabolic process (Figure 4C). Molecular function annotation revealed strong enrichment of inorganic cation transmembrane transporter activity, hydrogen ion transmembrane transporter activity, monovalent inorganic cation transmembrane transporter activity, hydrogen ion transporting ATP synthase activity, proton-transporting ATPase activity, and cation-transporting ATPase activity (Figure 4D). STRING Analysis for the Dysregulated Mitochondrial and Nuclear Proteins STRING analysis was performed to reveal the protein-protein interaction networks among the dysregulated proteins in copper-treated mice. Interactions among the proteins related to energy metabolism were evident, such as NADH dehydrogenase [ubiquinone] flavoprotein 3 (NDUV3), NADH dehydrogenase [ubiquinone] flavoprotein 1 (NDUV1), NADH-ubiquinone oxidoreductase 75 kDa subunit (NDUS1), NADH dehydrogenase [ubiquinone] iron-sulfur protein 4 (NDUS4), cytochrome b-c1 complex subunit Rieske (UCRI), cytochrome b-c1 complex subunit 6 (QCR6), cytochrome c oxidase subunit 5B (COX5B), Cytochrome c oxidase subunit 6A1 (CX6A1), ATP synthase subunit beta (ATPB), ATP synthase subunit d (ATP5H), ATP synthase subunit delta (ATPD), and isocitrate dehydrogenase [NAD] subunit alpha (IDH3A) (Figs. 5A and 5B). In addition, WikiPathway analysis revealed that NDUV1, NDUV3, NDUS1, NDUS4, UCRI, QCR6, COX5B, CX6A1, ATPB, ATPD, and ATP5H are located in the electron transport chain complexes I and III-V (Figure 5C). We also found an interaction between the 2 synaptic vesicle-associated proteins complexin-2 and synapsin-2 (Figs. 5A and 5D). Figure 5. View largeDownload slide View largeDownload slide Bioinformatic analysis. A and B, An intricate protein-protein intreaction network among the dysregulated proteins of mitochondria and nuclei in copper-treated mice; C, Dysregulated mitochondrial and nuclear proteins associated with the electron transport chain in the hippocampus of copper-treated mice; D, Synaptic Vesicle Fusion Pathway; E, The Venn logic diagram between the dysregulated proteins of mitochondria and nuclei in copper-treated mice; F, Commonly differentially expressed proteins. Figure 5. View largeDownload slide View largeDownload slide Bioinformatic analysis. A and B, An intricate protein-protein intreaction network among the dysregulated proteins of mitochondria and nuclei in copper-treated mice; C, Dysregulated mitochondrial and nuclear proteins associated with the electron transport chain in the hippocampus of copper-treated mice; D, Synaptic Vesicle Fusion Pathway; E, The Venn logic diagram between the dysregulated proteins of mitochondria and nuclei in copper-treated mice; F, Commonly differentially expressed proteins. Common Proteins Abnormally Expressed in Hippocampal Mitochondria and Nuclei in Copper-Treated Mice A Venn diagram showed that 6 identical proteins (Figure 5E) were abnormally expressed both in mitochondrial and nuclear fractions of the hippocampus of copper-treated mouse hippocampus: (1) Rho GDP-dissociation inhibitor 1 (GDIR1) is a Rho family of proteins involved in the cytoskeletal reorganization of glial and neuronal cells (Chen et al., 2016); (2) Dihydropyrimidinase-related protein 2 (DPYL2), a neuronal development-related protien (Gu and Ihara, 2000); (3) Synapsin-2 is a synaptic vesicle protein that plays an important role in synaptic signaling; (4) Glutamine synthase (GLNA) is responsible for the synthesis of glutamine from excess ammonium and glutamate; (5) Glyceraldehyde-3-phosphate dehydrogenase (G3P) catalyzes the oxidation (dehydrogenation) and phosphorylation of glyceraldehyde 3 monophosphate to produce 1,3-diglycidylglycerol, which is the central part of glucose metabolism; (6) Cytochrome c oxidase subunit 5B (COX5B), an electron transport chain (ETC)-related protein (Figure 5F). These abnormaly expressed proteins may be involved in copper neurotoxicity. Representative Protein Spots Differentially Expressed in Copper-Exposed Mice Based on the results of functional analysis, we selected 6 representative protein spots in hippocampal mitochondria and 11 representative protein spots in hippocampal nuclei for the fluorescent intensity analysis (Figs. 6A and 6B). The representative mitochondrial proteins are electron transport chain-related proteins NDUAA, COX5B, and UCRI; apoptosis-related protein stress-70 protein (GRP75); synaptic-related proteins DPYL2, synapsin-2, respectively (Figure 6A). The representative nuclear proteins are energy metabolism-related proteins ATP5H, IDH3A, UCRI, and COX5B; DNA damage-related protein transcriptional activator protein Pur-alpha (Pura), PDIA3; synaptic-related proteins complexin-2, DPYL2, and synapsin-2; oxidative stress-related proteins SODC and PRDX3, respectively (Figure 6B). Low doses of copper may exert a toxic effect through the aberrant expression of these molecules. Figure 6. View largeDownload slide Protein spots abnormally expressed in both hippocampal mitochondria and nuclei. A, The protein spots that were abnormally expressed in hippocampal mitochondria; B, The protein spots that were abnormally expressed in hippocampal nuclei. All the values were expressed as mean ± SEM. *p < .05, **p < .01, and ***p < .001 versus the control mice. n = 3–6 for each group. Figure 6. View largeDownload slide Protein spots abnormally expressed in both hippocampal mitochondria and nuclei. A, The protein spots that were abnormally expressed in hippocampal mitochondria; B, The protein spots that were abnormally expressed in hippocampal nuclei. All the values were expressed as mean ± SEM. *p < .05, **p < .01, and ***p < .001 versus the control mice. n = 3–6 for each group. Validation of the Differentially Expressed Proteins by Western-Blot Analysis Subcellular organelle proteomic analysis revealed abnormal expression of synaptic-related proteins, energy metabolism-related proteins, apoptosis-related proteins and oxidative stress-related proteins in hippocampal mitochondria and nuclei of copper-treated versus -untreated mice. Synapsin-2, COX5B, QCRI, IDH3A, PGAM1, PDIA3, GRP75, STMN1, and SODC were further validated by Western-blot analysis. Consistent with 2D-DIGE data, synapsin-2, IDH3A, PDIA3, GRP75, STMN1, and SODC were confirmed to be significantly decreased in hippocampus of copper-treated versus -untreated mice, respectively (Figs. 7A–F). Figure 7. View largeDownload slide Validation of differentially expressed proteins by Western-blot analysis. A and C, The relative levels of synapsin-2 in hippocampal mitochondria of copper-treated mice; B and D, The relative levels of synapsin-2 in hippocampus of copper-treated versus control mice; E and F, The relative levels of COX5B, QCRI, IDH3A, PGAM1, PDIA3, GRP75, STMN1, and SODC in hippocampus of copper-treated versus control mice. Data were expressed as mean ± SEM. *p < .05 and **p < .01 versus the control mice. n = 3 for each group. For STMN1 protein, n = 5 for each group. Figure 7. View largeDownload slide Validation of differentially expressed proteins by Western-blot analysis. A and C, The relative levels of synapsin-2 in hippocampal mitochondria of copper-treated mice; B and D, The relative levels of synapsin-2 in hippocampus of copper-treated versus control mice; E and F, The relative levels of COX5B, QCRI, IDH3A, PGAM1, PDIA3, GRP75, STMN1, and SODC in hippocampus of copper-treated versus control mice. Data were expressed as mean ± SEM. *p < .05 and **p < .01 versus the control mice. n = 3 for each group. For STMN1 protein, n = 5 for each group. DISCUSSION These data contribute to the view drawn from various animal studies that the level of copper in drinking water for safe consumption urgently needs reevaluation (Pal and Prasad, 2015; Pal et al., 2014a). The potential neurotoxicity of copper in drinking water has long been a public health concern (Harvey and McArdle, 2008). Very low doses of copper may cause pathological features associated with Alzheimer's disease (Singh et al., 2013). Copper overload leads to oxidative stress, which can cause DNA breakage, protein oxidation, and lipid peroxidation. The central nervous system (CNS) is particularly susceptible to oxidative stress, which is closely related to cognitive deficits (Kucukatay et al., 2007; Perrotta et al., 2008; Rahman et al., 2009). Some studies have suggested an imbalance of copper homeostasis in neurodegenerative diseases, including AD and PD (Bush, 2013; Desai and Kaler, 2008; George et al., 2009). Low levels of copper could contribute to Aβ accumulation in the brain of normal animals by altering its clearance and/or its production (Singh et al., 2013). Copper neurotoxicity can cause mitochondrial dysfunction, accompanied by synaptic plasticity damage, leading to a gradual decline in memory function, evolution of neurodegenerative diseases (Calkins et al., 2011; Krumschnabel et al., 2005). The importance of nuclei, which are also central to genetic regulation, is self-evident. However, little is known about the neurotoxic effects of copper on nuclei, except for DNA damage. High-throughput proteomic approach is an advantage to assess the neurotoxic effects of copper on subcellular organelles, with a potential to reveal reliable biomarkers of copper neurotoxicity. The present study shows that mice given drinking water containing a low concentration of added copper display impaired saptial memory and hippocampal neuronal apoptosis after 1 year of continuous treatment. Proteomic analysis of hippocampal subcellular fractions revealed that the foregoing behavioral and pathological changes were consistent with the significant differential expression of 31 mitochondrial proteins and 46 nucleoproteins. Bioinformatic analysis revealed these proteins were involved in energy metabolism, synaptic dysfunction, DNA damage and apoptosis, oxidative stress, and cytoskeletal integrity. Among these proteins, the copper neurotoxicity target protein copper-zinc superoxide dismutase (SODC) and the axon-associated protein stathmin (STMN1) were significantly decreased, indicating that the copper exposure also led to altered expression of a copper-containing enzyme and an axonal structural damage protein required for normal neuronal function. Energy Metabolism It has been reported that the neurotoxicity of copper damages the mitochondrial membrane, affects the activity of glycolytic and tricarboxylic acid cyclic (TCA) enzymes, and eventually leads to abnormal energy metabolism (Haywood et al., 2004; Medeiros and Jennings, 2002). Our proteomic data show that oral copper treatment resulted in abnormal expression of a large number of molecules associated with brain energy metabolism. Phosphoglycerate mutase 1 (PGAM1) is an glycolytic metabolic enzyme (Fothergill-Gilmore and Watson, 1989). Isocitrate dehydrogenase [NAD] subunit alpha (IDH3A) is an important rate-limiting enzyme in the TCA cycle and closely related to mitochondrial encephalopathy (Fattal-Valevski et al., 2017). Copper treatement significantly down-regulated the expression of both PGAM1 and IDH3A in the mouse hippocampus. Moreover, Wiki pathway analysis showed that several proteins involved in the mitochondrial respiratory chain showed an abnormal abundance, including: Complex I subunits (NDUV1, NDUV3, NDUS1, and NDUS4), Complex III subunits (UCRI, QCR6, and QCRI), Complex IV subunits (COX5B and CX6A1), and Complex V subunits (ATPB, ATPD, and ATP5H) (Koopman et al., 2013; Mitchell, 1961). Collectively, the abnormal expression of energy metabolism-related proteins, including the glycolysis-related protein PGAM1, TCA-related protein IDH3A, and oxidative phosphorylation (OXPHOS)-related proteins (Newmeyer and Ferguson-Miller, 2003; Saraste, 1999), probably disrupts energy metabolism on which the brain depends for a continuous supply of ATP and may thus sbe central to the neurotoxicity of copper. Oxidative Stress Studies have shown that copper undergoes redox cycling, resulting in the production of ROS as superoxide ion, hydrogen peroxide, and hydroxyl radical. As a consequence, enhanced lipid peroxida-tion, DNA damage, and altered calcium and sulfhydryl homeostasis occur (Stohs and Bagchi, 1995). Copper-zinc superoxide dismutase (SODC) not only mediates the neurotoxicity caused by copper exposure (Pal et al., 2014b), but SOD1 was the first identified genetic risk factor for familial amyotrophic lateral sclerosis (ALS) (Hayashi et al., 2016). The expression level of SOD1 has been linked with AD through network biology, and can be used as a potential marker to predict AD (Pal et al., 2014b). In addition, SODC is released by microglial cells and involved in neuroprotection against 6-OHDA neurotoxicity (Polazzi et al., 2013). Specifically, the decrease of SODC protein and inhibition of enzyme activity disrupt pro- and antioxidant balance and increase of oxidative stress, which may be a potential toxicity mechanism (Hayashi et al., 2016; Polazzi et al., 2013). It has been reported that copper chaperone for superoxide dismutase (CCS) is a very important target protein for the metabolism of copper in the body (Bulcke et al., 2017). SOD1 is closely related to copper-induced neurotoxicity (Harvey and McArdle, 2008; Pal et al., 2014a, b). In present study, we found that the expression of SODC was significantly decreased in the hippocampus of copper-treated versus untreated mice, indicating that this enzyme may be involved in copper neurotoxicity due to induction of oxidative stress. This finding is consistent with reported metabolic role of the copper chaperone for superoxide dismutase (CCS) (Bulcke et al., 2017) and the association of SODC with copper-induced neurotoxicity (Harvey and McArdle, 2008; Pal et al., 2014a, b). ER Stress and Apoptosis Oral copper treatment significantly decreased the levels of both GRP75 and PDIA3 in mouse hippocampus. Protein disulfide-isomerase A3 (PDIA3) is a protein related to ER stress, and knockdown of PDIA3 expression results in an increase in shikonin-induced apoptosis (Trivedi et al., 2016). PDIA3 and PRDX2 protect against oxidative stress-induced apoptosis (Kim et al., 2014). Also, PDIA3 knockdown exacerbates free fatty acid-induced hepatocyte steatosis and apoptosis (Zhang et al., 2015). Overexpression of GRP75 (heat shock protein 70) attenuates lipopolysaccharide (LPS)-induced oxidative and metabolic responses and inhibits inflammatory activation (Voloboueva et al., 2013). GRP75 expression is significantly reduced in brain tissue after intracerebral hemorrhage (ICH), and overexpression of GRP75 inhibits inflammation in the ICH rat model and potentially inhibits neuronal apoptosis (Lv et al., 2017). GRP75 and ginsenoside Rg1 have a synergistic protective effect on t-butyl hydroperoxide damage model in vitro (Lu et al., 2015). The present findings suggest that copper neurotoxicity is associated with an abnormal expression of ER-stress proteins. Synaptic Dysfunction Copper treatment of mice also induced changes in the abundance of synaptic vesicle fusion-related proteins synapsin-2 and complexin-2. Synapsin-2, a synaptic vesicle protein, is an important regulator of presynaptic neurotransmitter release (Ho et al., 2001). Decreased synapsin-2 is closely related to anxiety and depressive disorders (Etholm and Heggelund, 2009). In addition, the low expression of synapsin-2 can cause synaptic dysfunction, eventually leading to memory disorders (Grebb and Greengard, 1990). Recently, we have reported that ginsenoside Rg1 can improve the behavioral deficits of AD transgenic mice by increasing the levels of SYN2 and some other functional proteins (Nie et al., 2017). Complexin-2 (synaphin) plays an important role in the release of neurotransmitters and can be replaced by synaptic binding proteins to promote vesicular trafficking to synaptic membranes (Reim et al., 2001). During synaptic secretion, the neurons lacking CPLX have a reduced sensitivity to Ca2+, and their neurotransmitter release efficiency is significantly reduced (Reim et al., 2001). Complexin-1 and complexin-2 gene silencing can impair long-term potentiation (LTP), a process closely related to learning and memory (Ahmad et al., 2012). In addition, complexin-2 knockout mice show significant cognitive impairment (Glynn et al., 2003). In the present study, we found that the levels of synapsin-2 and complexin-2 were significantly decreased in the hippocampus of copper-treated versus untreated mice. Wiki Pathway analysis shows that synapsin-2 and complexin-2 are involved in synaptic vesicle fusion pathway. Collectively, our data suggest that abnormal expression of synaptic vesicle fusion-related proteins synapsin-2 and complexin-2 might be involved in the toxic effects caused by low-dose copper treatment. Cytoskeletal Proteins Microtubules, neurofilaments, and microfilaments constitute the cytoskeleton of neurons. Stathmin (STMN1), a microtubule-destabilizing neuroprotein controls the transition from neuronal precursors to early postmitotic neurons (Boekhoorn et al., 2014). Stathmins (stathmin 1–4) are involved in all stages of neuronal differentiation and plasticity (Chauvin and Sobel, 2015). In Drosophila, the microtubule regulatory protein stathmin is required to maintain microtubule integrity and axonal transport (Duncan et al., 2013). Stathmin-deficient mice display age-dependent axonal degeneration in both the central and peripheral nervous system (Liedtke et al., 2002). In the present study, we found that the level of STMN1 was significantly decreased in the hippocampus of copper-treated versus -untreated mice, indicating that decreased STMN1 may contribute to copper neurotoxicity through disruption of axonal integrity. In summary, we show that low-dose oral copper treatment impairs spatial memory, and induces neuronal apoptosis and proteomic changes in the mouse hippocampus. Hippocampal mitochondrial and nuclear proteomic and functional analysis revealed that copper treatment resulted in abnormal expression of proteins related to energy metabolism (PGAM1, IDH3A), oxidative stress (SODC), DNA oxidative damage and apoptosis (PDIA3, GRP75), synapse and cytoskeleton (synapsin-2, complexin-2, STMN1). Among these abnormally expressed proteins, synapsin-2, a synaptic-related protein, and SODC, a copper ion target protein, were significantly decreased in copper-treated mice versus -untreated mice as confirmed by Western-blot analysis. STMN1, a microtubule-destabilizing neuroprotein, was also significantly decreased. Taken together, we conclude that prolonged, low-dose copper treatment of mice causes spatial memory impairment and perturbs multiple biological/pathogenic processes by dysregulating the mitochondrial and nuclear proteome, particularly the proteins related to the respiratory chain, oxidative stress, synaptic vesicle fusion, axonal/neuronal integrity. Changes in STMN1 and SODC abundance may represent early novel biomarkers of copper neurotoxicity (Figure 8). However, the relevance of these experimental findings to the regulation of copper in drinking water is uncertain. The World Health Organization has established a guideline value of 2 mg/l of copper per day, but this was set to present undesirable gastrointestinal responses to higher levels of copper in drinking water (WHO, 2004). The present data should be helpful in establishing copper levels in drinking water that both contribute to the physiological need for this element and protect long-term brain function. Figure 8. View largeDownload slide Proposed potential mechanisms underlying copper neurotoxicity. Copper treatment resulted in proteomic alterations of hippocampal subcellular organelles (mitochondria and nuclei), including proteins related to energy metabolism, oxidative stress, apoptosis, synapses, and cytoskeleton. These changes may represent the molecular basis for copper neurotoxicity, including behavioral impairment, neuronal apoptosis, and DNA oxidative stress. Figure 8. View largeDownload slide Proposed potential mechanisms underlying copper neurotoxicity. Copper treatment resulted in proteomic alterations of hippocampal subcellular organelles (mitochondria and nuclei), including proteins related to energy metabolism, oxidative stress, apoptosis, synapses, and cytoskeleton. These changes may represent the molecular basis for copper neurotoxicity, including behavioral impairment, neuronal apoptosis, and DNA oxidative stress. SUPPLEMENTARY DATA Supplementary data are available at Toxicological Sciences online. ACKNOWLEDGMENT The authors would like to thank Dr Xiao Chen for the technical support in data analysis. FUNDING This work was supported by National Natural Science Foundation of China (81673134, 81401570), Guangdong Provincial Natural Science Foundation (2014A030313715, 2016A030313051), Guangdong Provincial Scheme of Science and Technology (To X.F.Y), Shenzhen Special Fund Project on Strategic Emerging Industry Development (JCYJ20160428143433768, JCYJ20150529164656093, JCYJ20150529153646078, JCYJ20160422143433757 and JCYJ20150529112551484) and Sanming Project of Medicine in Shenzhen (SZSM201611090). REFERENCES Ahmad M. , Polepalli J. S. , Goswami D. , Yang X. , Kaeser-Woo Y. J. , Sudhof T. C. , Malenka R. C. ( 2012 ). 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Toxicological Sciences – Oxford University Press
Published: Mar 30, 2018
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