Fig 2: 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 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: Celastrol protects against the loss of dopaminergic neurons in h-a-syn overexpressing mice. a Diagram of the experimental design. Briefly, 4 days after stereotactical injection of AAV-a-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-a-syn showed that h-a-syn mainly expressed within the SNc dopaminergic neurons. c Representative TH staining and quantification of SNc dopaminergic neurons in h-a-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 ß-actin in SNc of mice treated with celastrol or vehicle under the condition of human a-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.

Fig 5: 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 ß-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.