Tetrathiomolybdate Treatment Leads to the Suppression of Inflammatory Responses through the TRAF6/NFκB Pathway in LPS-Stimulated BV-2 Microglia

Abstract

Although the positive relationship between copper and Alzheimer's disease (AD) was reported by a lot of epidemiological data, the mechanism is not completely known. Copper is a redox metal and serves as a mediator of inflammation. Because the homeostasis of copper is altered in Aβ precursor protein (APP) and presenilin 1 (PS1) transgenic (Tg) mice, the using of copper chelators is a potential therapeutic strategy for AD. Here we report that a copper chelator, tetrathiomolybdate (TM), is a potential therapeutic drug of AD. We investigated whether TM treatment led to a decrease of pro-inflammatory cytokines in vivo and in vitro, and found that TM treatment reduced the expression of iNOS and TNF-α in APP/PS1 Tg mice through up-regulating superoxide dismutase 1 (SOD1) activity. In vitro, once stimulated, microglia secretes a variety of proinflammatory cytokines, so we utilized LPS-stimulated BV-2 cells as the inflammatory cell model to detect the anti-inflammatory effects of TM. Our results indicated that TM-pretreatment suppressed the ubiquitination of TRAF6 and the activation of NFκB without affecting the expression of TLR4 and Myd88 in vitro. By detecting the activity of SOD1 and the production of reactive oxygen species (ROS), we found that the anti-inflammatory effects of TM could be attributed to its ability to reduce the amount of intracellular bioavailable copper, and the production of ROS which is an activator of the TRAF6 auto-ubiquitination. Hence, our results revealed that TM-treatment could reduce the production of inflammatory cytokines by the suppression of ROS/TRAF6/AKT/NFκB signaling pathway.

Keywords: ROS; copper; inflammation; microglia; tetrathiomolybdate.

Figure 1

TM leads to suppression of pro-inflammatory cytokines in APP/PS1 Tg mouse brains. APP/PS1 Tg mice were pretreated with TM for 3 months and the brain homogenates were used to analysis the effects of TM on the production of pro-inflammatory cytokines. Lanes 1–3 represented different individuals of control APP/PS1 mice. Lanes 4–6 represented different individuals of TM-treated APP/PS1 mice. (A,B) Immunoblot images (A) and quantifications (B) show that TM reduced the expression of TNF-α. (C,D) Immunoblot images (C) and quantifications (D) show that TM reduced the expression of iNOS. Data are represented as means ± SD. N = 6 mice per group. ^*p < 0.05, ^**p < 0.01. The p-values were calculated using 2-tailed Student's t-test.

Figure 2

TM induces the activity of SOD in APP/PS1 Tg mouse brains. The brain homogenates were used to analysis the effects of TM. (A) TM increased the activity of SOD compared with control. (B) TM had no effect on the activity of GSH. Data are represented as means ± SD. N = 6 mice per group. ^*p < 0.05. The p-values were calculated using 2-tailed Student's t-test.

Figure 3

TM-pretreatment reduces the levels of copper and inhibits the production of NO and the expression of pro-inflammatory cytokines in LPS-induced BV-2 cells. BV-2 cells were pretreated with 6, 12, 24 μM of TM followed by treatment with 1 μg/ml LPS for 18 h. (A) The levels of copper were detected by using the ICP-MS. (B) NO production was detected by Griess agent. (C,D) Immunoblot images (C) and quantifications (D) show that TM-pretreatment suppressed iNOS expression in LPS-induced BV2 cells. (E,F) ELISA assay data show that TM-pretreatment decreased the release of TNF-α and IL-1β in LPS-induced BV2 cells. The absolute values ranges of TNF-α and IL-1β were 1000–2000 pg/ml and 102–993 pg/ml. (G,H) Quantitative real-time PCR (qPCR) data show that TM-pretreatment blocked TNF-α and IL-1β mRNA expression in LPS-induced BV2 cells. Data are represented as means ± SD. of at least three independent experiments (N ≥ 3). ^***p < 0.001 ^**p < 0.01, ^*p < 0.05 compared with the LPS; ###p < 0.001, ##p < 0.01 compared with the control. The p-values were calculated by One-way ANOVA followed by Bonferroni's post-hoc test.

Figure 4

TM-pretreatment has no effect on TLR4 and MyD88 in BV-2 cells induced by LPS. BV-2 cells were pretreated with 6, 12 μM of TM, followed by treatment with 1 μg/ml LPS for 18 h. (A,B) The expression of TLR4 were determined by western blot (upper panel: representative pictures; lower panel: quantifications) and immunofluorescence. (C,D) The expression of Myd88 were determined by western blot (upper panel: representative pictures; lower panel: quantifications) and immunofluorescence. The results are expressed as the mean ± SD. of at least three independent experiments (N ≥ 3).

Figure 5

TM-pretreatment reduces the ubiquitination of TRAF6 and decreases nuclear translocation of NFκB by inhibiting the degradation of IκB-α in LPS-induced BV-2 cells. After treated with TM and LPS, cell lysates were collected and immunoprecipitated with TRAF6 antibody, and the IP sample was detected with ubiquitin antibody. (A,B) Immunoblot images (A) and quantifications (B) show that TM-pretreatment lead to the decrease of TRAF6 ubiquitination. (C) ROS was detected by fluorescent enzyme analyzer. (D,E) Immunoblot images (D) and quantifications (E) show that IκB-α expression was inhibited by LPS, but the inhibition was blocked by pre-treatment with TM. (F) Immunoblot images show that TM prohibited the nucleus localization of NFκB induced by LPS. The results are represented as the mean ± SD of at least three independent experiments (N ≥ 3). ^*p < 0.05 compared with the LPS; #p < 0.05 compared with the control. The p-values were calculated by One-way ANOVA followed by Bonferroni's post-hoc test.

Figure 6

TM-pretreatment significantly decreases AKT phosphorylation in LPS-induced BV-2 cells. (A,B) Immunoblot images (A) and quantifications (B) show that both TM-pretreatment and LPS-stimulation did not affect PI3K expression in BV2 cells. (C,D) Immunoblot images (C) and quantifications (D) show that TM-pretreatment lead to a reduction of AKT phosphorylation induced by LPS. The results are represented as the mean ± SD of at least three independent experiments (N ≥ 3). ^*p < 0.05 compared with the LPS; #p < 0.05 compared with the control. The p-values were calculated by One-way ANOVA followed by Bonferroni's post-hoc test.

Figure 7

The schematic diagram shows that TM suppressed the secretion of NO, TNF-α, and IL-1β by reducing the bioavailability of copper and the activity of its downstream ROS/TRAF6/AKT/NFκB signaling pathway.