Inhibiting NLRP3 inflammasome activation prevents copper-induced neuropathology in a murine model of Wilson's disease

Abstract

Wilson's disease (WD) is an inherited disorder characterized by excessive accumulation of copper in the body, particularly in the liver and brain. In the central nervous system (CNS), extracellular copper accumulation triggers pathological microglial activation and subsequent neurotoxicity. Growing evidence suggests that levels of inflammatory cytokines are elevated in the brain of murine WD models. However, the mechanisms associated with copper deposition to neuroinflammation have not been completely elucidated. In this study, we investigated how the activation of NLR family pyrin domain containing 3 (NLRP3) inflammasome contributes to copper-mediated neuroinflammation in an animal model of WD. Elevated levels of interleukin-1β, interleukin-18, interleukin-6, and tumor necrosis factor-α were observed in the sera of WD patients and toxic milk (TX) mice. The protein levels of inflammasome adaptor molecule apoptosis-associated speck-like protein containing a C-terminal caspase recruitment domain (ASC), cleaved caspase-1, and interleukin-1β were upregulated in the brain regions of the TX mice. The NLRP3 inflammasome was activated in the TX mice brains. Furthermore, the activation of NLRP3 inflammasome was noted in primary microglia treated with CuCl₂, accompanied by the increased levels of cleaved caspase-1, ASC, and interleukin-1β. Blocking NLRP3 inflammasome activation with siNlrp3 or MCC950 reduced interleukin-1β and interleukin-18 production, thereby effectively mitigating cognitive decline, locomotor behavior impairment, and neurodegeneration in TX mice. Overall, our study demonstrates the contribution of copper overload-mediated activation of NLRP3 inflammasome to progressive neuropathology in the CNS of a murine model of WD. Therefore, blockade of the NLRP3 inflammasome activation could be a potential therapeutic strategy for WD.

Fig. 1. Activation of NLRP3 inflammasome in patients with Wilson’s disease (WD) and the WD animal model.

a Levels of circulating interleukin (IL)-1β, IL-18, IL-6, and tumor necrosis factor (TNF)-α in the serum of healthy controls and patients with WD (n = 25). b Serum IL-1β, IL-18, IL-6, and TNF-α levels in toxic milk (TX) mice and wild type (WT) mice (pg/ml) (n = 16). c IL-1β, IL-18, IL-6, and TNF-α levels in the brain of TX mice and WT controls mice (pg/mg) (n = 16). d Levels of cleaved caspase-1 (casp-1), ASC, and IL-1β in the corpus striatum of TX mice and WT mice were quantified using western blotting (n = 6). e Levels of NLRP1, NLRP2, NLRP3, NLRC4, and AIM2 levels in the corpus striatum of TX mice and WT controls were measured using western blotting (n = 6). Data are presented as mean ± SEM; Student’s t test, ^*P < 0.05, ^***P < 0.001 versus the corresponding control or WT mice.

Fig. 2. CuCl₂ induces interleukin (IL-1β) and IL-18 production and NLRP3 inflammasome activation.

a Primary microglia were treated with CuCl₂ (10 µM) and lipopolysaccharide (LPS, 200 ng/ml), supernatants were collected at 8, 12, and 24 h, and levels of IL-1β were quantified with ELISA; ATP (5 mM) treatment served as the positive control. b Primary microglia were treated with CuCl₂ for 8, 12, and 24 h, and the protein levels of NLRP3, cleaved caspase-1 (casp-1), ASC, and IL-1β were quantified using western blotting; ATP (5 mM) treatment served as positive control. c Primary microglia were treated with or without CuCl₂ (10 µM) and LPS (200 ng/ml) for 24 h, and levels of IL-1β were subsequently quantified using ELISA. d Primary microglia were treated with or without CuCl₂ (10 µM) for 8, 12, 24, and 36 h, and the levels of IL-1β were quantified using ELISA. e The levels of IL-18 in microglia treated with CuCl₂ and LPS for 24 h. Data are presented as mean ± SEM; one-way ANOVA, two-way ANOVA, ^**P < 0.01, ^***P < 0.001 versus the corresponding control group.

Fig. 3. Silencing Nlrp3 expression reduced the caspase-1 and interleukin (IL)-1β protein levels in the Wilson’s disease (WD) animal model.

a Immunofluorescence staining of green fluorescent protein (GFP) in the corpus striatum of toxic milk (TX) mice injected with saline or Nlrp3 siRNA (siNlrp3). Scale bars, 50 µm. b Levels of NLRP3 in the brain of negative control siRNA (NC)- or siNlrp3-injected TX mice were measured using western blotting (n = 3). c Levels of NLRP3, cleaved caspase-1 (casp-1), ASC, and IL-1β in the corpus striatum of the NC- or siNlrp3-injected TX mice and WT mice were quantified using western blotting (n = 3). Data are presented as mean ± SEM; Student’s t test, two-way ANOVA, ^*P < 0.05, ^**P < 0.01, ^***P < 0.001 versus the corresponding WT or NC group.

Fig. 4. Nlrp3 silencing inhibits interleukin (IL)-1β and IL-18 production, and reverses behavioral deficits in Wilson’s disease (WD) animal model.

a Immunofluorescence staining and quantification of Iba-1-positive cells in the corpus striatum of the negative control siRNA (NC)- or the siNlrp3-injected toxic milk (TX) mice and wild type (WT) mice. Scale bars, 50 µm. b Immunohistochemical staining and quantification of CD11b-positive cells in the corpus striatum of the NC- or the siNlrp3-injected TX mice and WT mice. Scale bars, 200 µm. c Serum IL-1β, IL-18, IL-6, and tumor necrosis factor (TNF-α) levels in the NC- or the siNlrp3-injected TX mice and WT mice (pg/ml) (n = 6). d IL-1β, IL-18, IL-6, and TNF-α levels in the brain of the NC- or the siNlrp3-injected TX mice and WT mice (pg/mg) (n = 6). e TX mice or WT mice were treated with lentivirus-enveloped siNlrp3 or control virus; activities including total distance and climbing behaviors of open-field were measured (n = 15). f TX mice or WT mice were treated with lentivirus-enveloped siNlrp3 or control virus, and after 3 months, motor functions were tested using a rotarod test apparatus (n = 15). g Escape latency was measured using Barnes maze test (n = 15). h TX mice or WT mice were treated with lentivirus-enveloped siNlrp3 or control virus, and time spent in center of open-field was measured. (n = 15). Data are presented as mean ± SEM; two-way ANOVA, ^*P < 0.05, ^**P < 0.01, ^***P < 0.001 versus the corresponding NC group.

Fig. 5. MCC950 inhibits NLRP3 inflammasome activation in CuCl₂-induced microglia.

a Schematic illustration of the experimental set-up. b Primary microglia were treated with or without CuCl₂ (10 µM) and MCC950 (100 nM) for 24 h, and levels of interleukin (IL-1β) and IL-18 were quantified using ELISA (n = 6). c Primary microglia were treated with or without CuCl₂ and MCC950, and the levels of NLRP3, cleaved caspase-1 (casp-1), ASC, and IL-1β were measured using western blotting (n = 4). d Primary neurons separated from the hippocampus were treated with conditioned medium from CuCl₂- or MCC950-administrated microglia, and the apoptotic neuronal cells were quantified by TUNEL (green) immunofluorescence staining. Scale bars, 100 µm. e Cell viability of hippocampal neurons treated with conditioned medium from CuCl₂- or MCC950-administrated microglia. Data are presented as mean ± SEM; two-way ANOVA, ^*P < 0.05, ^**P < 0.01, ^***P < 0.001 versus the corresponding control group.

Fig. 6. MCC950 treatment protects against behavior impairment and neurodegeneration in the Wilson’s disease (WD) animal model.

a Schematic representation of MCC950 treatment experiments. b–e Toxic milk (TX) mice and wild type (WT) mice were treated with MCC950 or saline, (b) total distance and climbing behaviors were quantified with open-field tests (n = 15). c Motor functions were tested using a rotarod test apparatus (n = 15). d Time spent in center were quantified with open-field tests (n = 15). e Escape latency was measured using Barnes maze test (n = 15). f Fluorescence imaging of Fluoro-Jade B (FJB) staining (green) in the corpus striatum of TX mice and WT mice treated with MCC950 or saline. Scale bar, 50 µm. g Immunofluorescence staining and quantification of NeuN (red) in the corpus striatum of MCC950 or saline-treated TX mice and WT mice. Scale bar, 50 µm. Data are presented as mean ± SEM; two-way ANOVA, ^*P < 0.05, ^**P < 0.01, ^***P < 0.001 versus the corresponding saline-treated group.

Fig. 7. MCC950 inhibits NLRP3 inflammasome activation in the Wilson’s disease (WD) animal model.

a Levels of NLRP3, cleaved caspase-1 (casp-1), ASC, and interleukin (IL)-1β in the corpus striatum of MCC950 or saline-treated toxic milk (TX) mice and wild type (WT) mice were quantified using western blotting (n = 3). b Immunofluorescence staining and quantification of Iba-1positive cells in the corpus striatum of MCC950 or saline-treated TX mice and WT mice. Scale bar, 50 µm. c Immunohistochemical staining and quantification of CD11b-positive cells in the corpus striatum of TX mice and WT mice treated with MCC950 or saline. Scale bars, 200 µm. d Serum IL-1β, IL-18, IL-6, and tumor necrosis factor (TNF)-α levels in TX mice and WT mice treated with MCC950 or saline (pg/ml) (n = 6). e IL-1β, IL-18, IL-6, and TNF-α levels in the brain of TX mice and WT mice treated with MCC950 or saline (pg/mg) (n = 6). Data are presented as mean ± SEM; two-way ANOVA, ^*P < 0.05, ^***P < 0.001 versus the corresponding saline-treated group.

Fig. 8. Inhibiting NLRP3 inflammasome activation prevents copper-induced neuropathology in Wilson’s disease (WD) animal model.

NLRP3 inflammasome activation is actively involved in the progression of WD and its inhibition protects against Cu²⁺-induced neuroinflammation and prevents neuronal injury.