See Supplemental Table 1 for the primary antibodies information. ELISA kits for detection of NO, IL-1β, and TNF-α were purchased from YanTai Science & Biotech and Beijing 4A Biotech Co. The DCFH-DA for the detection of ROS and Nuclear and Cytoplasmic Protein Extraction Kit were purchased from Beyotime Biotechnology China. Alexa Fluor 488-, Alex Fluor 594-conjugated secondary antibodies were purchased from Jackson ImmunoResearch, PA, USA. HRP-conjugated secondary antibodies were obtained from GE Healthcare Life Science, Beijing, China. The reagents for the quantitative real-time PCR (qPCR) were purchased from Promega Biotech Co., Ltd., Beijing, China. All primers used in qPCR were obtained from Sangon Biotech Co., Ltd., Shanghai, China. Tetrathiomolybdate (TM), 99.97% trace metals basis, was purchased from Sigma-Aldrich. Protein G beads were purchased from Thermo Fisher Scientific, China. SOD and GSH were purchased from Nanjing Jiancheng Bioengineering Institute, China.

The APP/PS1 transgenic mice [B6C3-Tg (APPswe,PSEN1dE9)85Dbo/J(#004462)] and the wild-type mice were obtained from the Jackson Laboratory (Bar Harbor, ME, USA). All animal experimental procedures were approved by the Laboratory of Animal Ethical Committee of China Medical University. Six-month-old male mice were randomly divided into two groups (N = 6 per group): control group and TM group. In the control group, mice were given distilled water. In the TM group, mice were treated with TM (2.8 mg/kg/d) in drinking water (12 mg/L) daily for 3 months. Six-month-old wild-type mice were randomly divided into two groups (N = 6 per group): control group and TM group. In the control group, mice were given distilled water. In the TM-treated group, mice were treated with TM (2.8 mg/kg/d) in drinking water (12 mg/L) daily for 3 months.

After 3 months treatment, the mice were sacrificed and the brains were removed quickly on ice. Half of the brains were frozen in −80°C for biochemical analysis. And the other half of brains were fixed into 4% paraformaldehyde.

BV-2 cells were cultured in DMEM (high glucose) with 10% fetal bovine serum and L-glutamine in 5% CO₂ at 37°C. Firstly, the cells pretreated with different doses of TM (6, 12, 24 μM) for 30 min (Wei et al., 2014), and then LPS was added and incubated with cells for 18 h. After 18 h of LPS treatment, the growth medium was collected for the detection of NO, IL-1β, and TNF-α (ELISA Kit) according to the manufacturer's instructions.

To measure the content of copper in BV-2 cells, cells were pretreated with 6 μM TM for 30 min and then added with LPS to incubate for 18 h. After three times of PBS washing, the cells were collected and centrifuged at 800 rpm for 5 min. The supernatant was discarded, cells were digested with 500 μL of 90% HNO₃ at 95°C for 30 min. The samples of cells were diluted, and then the content of copper in the diluted solutions was detected by using the 7500a- ICP-MS (Aglient Technologies InC., USA). Data acquisition mode (spectral analysis) is ⁶³Cu.

Total RNA was extracted from BV-2 cells and tissues using the Total RNA Kit (OMEGA Georgia, USA) according to the manufacturer's instruction and reverse transcribed into cDNA with the GoScript™ Reverse Transcription System (Promega, Madison, USA) on the Bio-rad CFX PCR System. The relative mRNA expression was calculated by Bio-Rad CFX software using ΔΔCt methods. All primers were span an exon-exon junction. All primers and the GenBank accession numbers were listed in Table 1. All the experiments were repeated at least for three times.

BV-2 cells were treated with 6 μM TM for 30 min prior to LPS stimulation. After 18 h of LPS treatment, the cells were washed with PBS three times and then fixed with 4% paraformaldehyde for 15 min. And then were incubated with 0.1% Triton X-100 for 2 min after washing with PBS for three times. The sections were treated with 5% goat serum/PBS for 30 min and then incubated with primary antibodies overnight at 4°C. After washed, sections were incubated with Alex Fluor 594-conjugated secondary antibodies for 1.5 h. Images were taken using the Leica TCS SP8 laser scanning confocal microscope with 40 × objectives.

The extraction of the cytosolic and nuclear protein was performed according to the manufacturer's instructions kit. The concentration of the proteins was measured by Bradford assay (Ernst and Zor, 2010).

Cells and tissues were lysed with RIPA lysis buffer [50 mM Tris-HCL (pH 7.5), 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS, 5 mM EDTA, 25 mM NaF, and 2 mM Na₃VO₄, 1 mM PMSF] containing 1:100 diluted protease inhibitor cocktail (Sigma, St. Louis, MO, USA). The same amount of protein (50 μg) was separated on a 10% SDS-PAGE and transferred onto PVDF membranes, and probed with a panel of primary antibodies (Supplemental Table 1). All the experiments were repeated at least for three times.

BV-2 cells were lysed with 1% NP40 lysis buffer [20 mM Tris-HCL (pH 7.5), 150 mM NaCl, 1% NP-40, 5 mM EDTA, 20 mM beta-glycerophosphate, 10% glycerol, 0.5 mM DTT, 25 mM NaF, 1 mM Na₃VO₄, 1 mM PMSF] containing 1:100 dilution protease inhibitor cocktail for 30 min on ice. Cell lysates were incubated with 2 μg TRAF6 antibody and protein G beads at 4°C overnight. Followed by washing, the proteins were eluted from beads using SDS sample buffer and analyzed by 10% SDS-PAGE.

All data are expressed as the mean ± standard deviation (SD). Statistical analysis among two-group comparison was performed with 2-tailed Student's t-test with or without Welch's correction. Statistical analysis among three or more groups was performed with One-way ANOVA followed by Bonferroni's post-hoc test. p < 0.05 was considered to be statistically significant.

Inflammatory cytokines, such as IL-1β and TNF-α, are believed to induce neurodegeneration, including AD (Liu and Hong, 2003). Therefore, we detected the expression of iNOS and inflammatory cytokines, such as IL-1β, IL-6, and TNF-α, in the brain of TM-treated APP/PS1 Tg mice. After being treated with TM for 3 months, although the mRNA expression of IL-1β and IL-6 were not reduced by TM-treatment (Supplementary Figure 1), the expression of TNF-α (Figures 1A,B) and iNOS (Figures 1C,D) were significantly reduced in TM-treated APP/PS1 Tg mice as compared with control mice. Meaning while, TM had no effect on the expression of iNOS and TNF-α in wild-type mice (Supplementary Figure 2). These findings suggested that TM could reduce inflammatory responses in the brain of APP/PS1 Tg mice. As a huge amount of ROS are produced among Aβ aggregation, the enzymatic and non-enzymatic antioxidant defenses including superoxide dismutase (SOD), glutathione peroxidase (GPX), catalase (CAT), ascorbic acid (vitamin C), α-tocopherol (vitamin E), glutathione (GSH), β-carotene, and vitamin A protect cells from oxidative-stress injury. In order to determine whether the anti-inflammatory effects of TM is depending on copper associated signaling pathways, we detected the SOD1 activity in TM-treated APP/PS1 mice and control mice. Our results indicated that TM increased the levels of SOD1 (Figure 2A), but had no effect on GSH (Figure 2B), suggesting that TM specifically reduced bioavailability of copper in APP/PS1 Tg mice (Figure 2).

Inflammatory cytokines are predominantly produced by microglia in the brain. Then we determined whether TM inhibited the secretion of inflammatory cytokines in microglial cells. Firstly, we detected the effect of TM on the content of copper. The results showed that copper levels were increased in LPS-induced group, as compared with the control group, and the increased copper in LPS-induced BV-2 cells could be abolished by TM-pretreatment (Figure 3A). As a hippocampal neurotransmitter and a neurotoxic mediator, NO production is positively associated with the impairment of serotonergic transmission in the brain under stressful situations (Joca et al., 2007), and its neurotoxicity has been attributed to the inhibition of mitochondrial respiration (Bolanos et al., 1997). To evaluate the anti-inflammatory responses of TM on the microglial cells, the NO secretion and the iNOS expression in LPS-induced microglial cells were detected in mouse microglial cells (BV-2 cells). After being pretreated with TM (6, 12, 24 μM) for 30 min followed by stimulation with LPS (1 μg/mL) for 18 h, the NO production of BV-2 cells was detected by using Griess agent. The results showed that NO secretion was increased in the LPS-induced group, as compared with the control group (Figure 3B p < 0.001), and the increased-NO secretion in LPS-induced BV-2 cells can be abolished by TM-pretreatment (Figure 3B, p < 0.001). To further understand the mechanism by which TM treatment reduced NO production, we examined iNOS mRNA and protein levels by RT-PCR and western blot, respectively. As showed in Supplementary Figure 3 and Figures 3C,D, the expression of iNOS mRNA (p < 0.001) and protein (p < 0.01) were significantly upregulated in the LPS-induced group, as compared with the control group, and the upregulated expression of iNOS mRNA and protein could be abolished by different dose of TM-pretreatment (Supplementary Figure 3 and Figures 3C,D, p < 0.05), but the intrigue thing was that no dose-dependent effect was observed in this experiment. Taken together, the copper chelator TM decreased inflammatory effects via reducing iNOS expression and NO secretion in LPS-induced microglial cells.

In addition to NO secretion, LPS stimulation also increased the production of inflammatory cytokines, such as TNF-α and IL-1β (Liu et al., 2011). Then we detected the effect of TM on the production of TNF-α and IL-1β in LPS-induced BV-2 cells. The levels of TNF-α and IL-1β in the growth media were detected by ELISA. Consistent with previous studies, our results indicated that LPS stimulation significantly increased the release (Figures 3E,F) and the mRNA expression (Figures 3G,H) of TNF-α (Figures 3E,G) and IL-1β (Figures 3F,H) in BV-2 cells (p < 0.001), and the increased TNF-α and IL-1β can be suppressed by different dose (6, 12, 24 μM) of TM-pretreatment (Figures 3E-H), suggesting that TM decreased the production of IL-1β and the release of TNF-α.

TLR4 signaling pathway plays a pivotal role in the inflammatory response. Studies have shown that TLR4/Myd88/NFκB signaling pathway is involved in LPS-stimulated BV-2 cells (Dai et al., 2015). On the basis of the above results that the effects of TM on LPS-stimulated BV-2 cells have no dose dependence, 6 μM TM and 12 μM TM were used for the following studies. First, We examined the effect of 6 μM and 12 μM TM on the expression of TLR4 and Myd88 in LPS-induced BV-2 cells, and observed that TM had no effect on the expression of TLR4 (Figures 4A,B, p > 0.05) and Myd88 (Figures 4C,D, p > 0.05). In addition, we also examined the effects of TM on the expression of TLR4 and Myd88 in APP/PS1 Tg mouse brains. Consistent with the results of the cell model, no changes were observed on the expression of TLR4 and Myd88 in APP/PS1 Tg mouse brains (Supplementary Figure 4), suggesting that TM regulated the inflammatory process in BV-2 cells without affecting the expression of the two key molecules, which mediated LPS-induced inflammation. It has been suggested that iron generates superoxide anion to induce ubiquitination of TRAF6 (Zhong et al., 2012). Copper, like iron, is a redox metal that produces free radicals (Greenough et al., 2013). In light of these results, we speculated that TM may reduce the ROS production and then regulate the activity of downstream signaling molecules in TLR4 signaling pathway, such as the ubiquitination of TRAF6, the phosphorylation of AKT and NFκB, and finally reduce the secretion of inflammatory cytokines. To confirm whether TM can reduce the production of ROS to reduce the ubiquitination of TRAF6, we detected the ubiquitination of TRAF6 by using the immunoprecipitation, and found that the LPS-induced ubiquitination of TRAF6 can be abolished by only pre-treated with 6 μM TM for 30 min (Figures 5A,B). Next, we detected the production of ROS induced by LPS with or without TM-treatment. The results showed that the production of ROS induced by LPS could be decreased by pre-treated with 6 μM for 30 min (Figure 5C). As IκB-α is the major negative regulator and the binding partner of NFκB, we determined the expression of IκB-α in the presence or absence of TM in LPS-induced BV-2 cells, and found that the expression of IκB-α was significantly induced in 6 μM TM-pretreated group, as compared with the LPS-induced group (Figures 5D,E, p < 0.05). To evaluate the nucleus translocation of phosphor-NFκB p65 subunit (p-NFκB p65) in LPS-induced BV-2 cells with or without TM-pretreatment, we isolated the cytoplasmic and the nucleus fraction of the treated cells, and detected the expression of p-NFκB p65 and NFκB p65 by western blot. The results indicated that TM-pretreatment significantly suppressed the nucleus translocation of p-NFκB p65 in LPS-induced BV-2 cells (Figure 5F), suggesting that TM treatment reduced inflammatory effects of LPS in BV-2 cells by down-regulating TRAF6/NFκB signaling pathway.

It was previously reported that the PI3K/AKT pathway, the upstream activator of NFκB, was involved in the LPS-induced inflammatory responses (Venkatesan et al., 2010; Saponaro et al., 2012). Therefore, we examined the effect of TM on the activation of PI3K and AKT in LPS-induced BV-2 cells. Since, AKT activation requires the phosphorylation of Thr308 in the activation loop and Ser473 within the carboxyl-terminal hydrophobic motif. We detected Ser473 phosphorylation of AKT in LPS-induced BV-2 cells in the presence or absence of TM-pretreatment. Although no changes were detected in the phosphorylation of PI3K and total AKT in TM-treated APP/PS1 Tg mice (Supplementary Figure 5), lower dose of TM-pretreatment significantly decreased the Ser473 phosphorylation of AKT (Figures 6C,D, p < 0.05) induced by LPS in BV-2 cells without affecting PI3K activity (Figures 6A,B), suggesting that TM suppressed the production of inflammatory cytokines via reducing NFκB-nucleus localization which was mediated by TRAF6 auto-ubiquitination and AKT activation.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.