The Nrf2-NLRP3-caspase-1 axis mediates the neuroprotective effects of Celastrol in Parkinson's disease

Abstract

Parkinson's disease (PD) is a chronic neurodegenerative disorder that is characterized by motor symptoms as a result of a loss of dopaminergic neurons in the substantia nigra pars compacta (SNc), accompanied by chronic neuroinflammation, oxidative stress, formation of α-synuclein aggregates. Celastrol, a potent anti-inflammatory and anti-oxidative pentacyclic triterpene, has emerged as a neuroprotective agent. However, the mechanisms by which celastrol is neuroprotective in PD remain elusive. Here we show that celastrol protects against dopamine neuron loss, mitigates neuroinflammation, and relieves motor deficits in MPTP-induced PD mouse model and AAV-mediated human α-synuclein overexpression PD model. Whole-genome deep sequencing analysis revealed that Nrf2, NLRP3 and caspase-1 in SNc may be associated with the neuroprotective actions of celastrol in PD. By using multiple genetically modified mice (Nrf2-KO, NLRP3-KO and Caspase-1-KO), we identified that celastrol inhibits NLRP3 inflammasome activation, relieves motor deficits and nigrostriatal dopaminergic degeneration through Nrf2-NLRP3-caspase-1 pathway. Taken together, these findings suggest that Nrf2-NLRP3-caspase-1 axis may serve as a key target of celastrol in PD treatment, and highlight the favorable properties of celastrol for neuroprotection, making celastrol as a promising disease-modifying agent for PD.

Keywords: Caspase-1; Celastrol; NLRP3; Nrf2; Parkinson's disease; α-synuclein.

Fig. 1

Neuroprotective effects of celastrol in MPTP-induced PD mouse model. a Diagram of the experimental design. MPTP (20 mg/kg）or vehicle (saline) was injected intraperitoneally (i.p.) for 5 consecutive days starting on day −4 after acclimation (3 days). Then, mice were i.p. treated with celastrol (10 μg/kg) or vehicle (DMSO) per day for 7 days. Tissues were harvested for molecular analyses at day 26 after the last behavior test. b Representative photomicrographs and unbiased stereological counts of TH staining in SNc, Scale bars 400 μm. Data are mean ± s.e.m.; n = 10. c Protein levels of TH, DAT and β-actin in SNc. Data are mean ± s.e.m.; n = 10. d Time to traverse beam apparatus, time to descend pole, Hind-limb clasping reflex score, fall latency from an accelerating rotarod and gait analysis (e). Data are mean ± s.e.m.; n = 10. f Relative mRNA expression of the indicated genes in SNc. Data are mean ± s.e.m.; n = 8. The one-way ANOVAs were used for statistical analysis followed by Bonferroni's post hoc test. *P < 0.05, **P < 0.01, and ***P < 0.001. ns, not significant.

Fig. 2

Gene profile analysis of SNc in MPTP-induced PD mice treated with celastrol. a Hierarchical clustered heatmap of gene expression profiles for celastrol or vehicle treatment in SNc under the condition of MPTP injection. b Heatmap of DEGs of MPTP-received mice treated with celastrol or vehicle. c KEGG analysis highlights the DEGs of celastrol treatment compared with vehicle in MPTP-recieved mice, upregulated genes are colored in red, downregulated genes are colored in blue. (P < 0.05 with unpaired two-tailed Student's t-tests). d Gene Ontology enrichment was based on DEGs that have a P value smaller than 0.05. Enrichment analysis for Gene Ontology terms among the genes of a gene–trait correlation module was performed using Metascape. e Volcano plot displays DEGs of celastrol treatment compared with vehicle in MPTP-received mice. Significantly altered genes are colored in blue, insignificantly altered genes are colored in red. f Venn diagram of overlapping significantly changed genes (±1.2 fold, P < 0.05). The top ten overlapping genes are presented in diagram. g Representative immunoblots and quantification of relative protein levels in SNc. Data are mean ± s.e.m.; n = 5 biologically independent animals; *P < 0.05, **P < 0.01, and ***P < 0.001. h Protein–protein interaction network identified among Nrf2, NLRP3 and Caspase-1 using STRING database. i Human SNc mRNA correlations analysis by Gene network.

Fig. 3

Neuroprotective effects of celastrol in PD are Nrf2 dependent. a Representative TH staining of SNc and STR, unbiased stereological counts of TH⁺ neurons in SNc and the quantity of TH⁺ striatal fiber density in STR of WT and Nrf2-KO mice. Scale bar, 400 μm for SNc, 2 mm for STR. b Representative diagram of gait test of WT and Nrf2-KO mice treated with celastrol or vehicle. c Representative immunoblots and quantification of TH, DAT in SNc or STR levels of WT and Nrf2-KO mice. Data are mean ± s.e.m.; n = 9. d Time to traverse beam apparatus, time to descend pole, hind-limb clasping reflex score, fall latency from an accelerating rotarod. Data are mean ± s.e.m.; n = 9. Two-way ANOVA followed by Tukey's post hoc test. *P < 0.05, **P < 0.01, and ***P < 0.001. ns, not significant.

Fig. 4

Neuroprotective actions of celastrol A in PD are mediated by Nrf2-NLRP3 axis. a Representative TH staining of SNc and STR, unbiased stereological counts of TH⁺ neurons in SNc and the quantity of TH⁺ striatal fiber density in STR of WT and NLRP3-KO mice. Scale bar, 400 μm for SNc, 2 mm for STR. b Representative diagram of gait test of WT and NLRP3-KO mice treated with celastrol or vehicle. c Representative immunoblots and quantification of TH, DAT levels in SNc or STR of NLRP3-KO mice. Data are mean ± s.e.m.; n = 9. d Time to traverse beam apparatus, time to descend pole, hind-limb clasping reflex score, fall latency from an accelerating rotarod. Data are mean ± s.e.m.; n = 9. Two-way ANOVA followed by Tukey's post hoc test. *P < 0.05, **P < 0.01, and ***P < 0.001. ns, not significant.

Fig. 5

Celastrol suppresses caspase-1 to protect dopaminergic neurons from MPTP neurotoxicity. a Representative TH staining of SNc and STR in WT and Caspase-1-KO mice, unbiased stereological counts of TH⁺ neurons in SNc and the quantity of TH⁺ striatal fiber density in STR of WT and Caspase-1-KO mice. Scale bar, 400 μm for SNc, 2 mm for STR. b Representative diagram of gait test of WT and Caspase-1-KO mice treated with celastrol or vehicle. c Representative immunoblots and quantification of TH, DAT levels in SNc or STR of WT and Caspase-1-KO mice. Data are mean ± s.e.m.; n = 9. d Time to traverse beam apparatus, time to descend pole, hind-limb clasping reflex score, fall latency from an accelerating rotarod. Data are mean ± s.e.m.; n = 9. Two-way ANOVA followed by Tukey's post hoc test. *P < 0.05, **P < 0.01, and ***P < 0.001. ns, not significant.

Fig. 6

Celastrol protects against the loss of dopaminergic neurons in h-α-syn overexpressing mice. a Diagram of the experimental design. Briefly, 4 days after stereotactical injection of AAV-α-syn-GFP, mice i.p. received celastrol (10 μg/kg) or vehicle (DMSO) per day for 42 days, tissues were harvested for molecular analyses at day 42 after the last behavior test. b Representational diagram of AAV injection site in mice and the co-labelling of TH with h-α-syn showed that h-α-syn mainly expressed within the SNc dopaminergic neurons. c Representative TH staining and quantification of SNc dopaminergic neurons in h-α-syn overexpressing mice treated with celastrol. Scale bar, 400 μm. Data are mean ± s.e.m.; n = 10. d Protein levels of TH, DAT and β-actin in SNc of mice treated with celastrol or vehicle under the condition of human α-syn overexpression within SNc. Data are mean ± s.e.m.; n = 10. e, f Time to traverse beam apparatus, time to descend pole, hind-limb clasping reflex score, fall latency from an accelerating rotarod and gait analysis. Data are mean ± s.e.m.; n = 10. *P < 0.05, **P < 0.01 and ***P < 0.001 by one-way ANOVA with Bonferroni's post hoc test. g Schematic summary of the mechanism underlying the neuroprotective actions of celastrol in PD.

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