Celastrol enhances transcription factor EB (TFEB)-mediated autophagy and mitigates Tau pathology: Implications for Alzheimer's disease therapy

Abstract

Alzheimer's disease (AD), characterized by the accumulation of protein aggregates including phosphorylated Tau aggregates, is the most common neurodegenerative disorder with limited therapeutic agents. Autophagy plays a critical role in the degradation of phosphorylated Tau aggregates, and transcription factor EB (TFEB) is a master regulator of autophagy and lysosomal biogenesis. Thus, small-molecule autophagy enhancers targeting TFEB hold promise for AD therapy. Here, we found that celastrol, an active ingredient isolated from the root extracts of Tripterygium wilfordii (Lei Gong Teng in Chinese) enhanced TFEB-mediated autophagy and lysosomal biogenesis in vitro and in mouse brains. Importantly, celastrol reduced phosphorylated Tau aggregates and attenuated memory dysfunction and cognitive deficits in P301S Tau and 3xTg mice, two commonly used AD animal models. Mechanistical studies suggest that TFEB-mediated autophagy-lysosomal pathway is responsible for phosphorylated Tau degradation in response to celastrol. Overall, our findings indicate that Celastrol is a novel TFEB activator that promotes the degradation of phosphorylated Tau aggregates and improves memory in AD animal models. Therefore, Celastrol shows potential as a novel agent for the treatment and/or prevention of AD and other tauopathies.

Keywords: Alzheimer's disease (AD); Autophagy; Celastrol; Lysosome biogenesis; TFEB; Tau; Therapeutic target; mTOR.

Graphical abstract

Figure 1

Celastrol promotes TFEB-mediated autophagy and lysosome biogenesis. (A) Chemical structure of celastrol (Cel). (B, C) Celastrol promotes TFEB translocation from the cytoplasm into the nucleus in HeLa cells stably expressing 3xFlag-TFEB as reflected by immunostaining and Western blot analysis (at least 250 cells were counted for each group, scale bar = 15 μm). (D) Celastrol increased the levels of LC3B-II and LAMP1. (E) Celastrol further increased LC3B-II levels in the presence of lysosome inhibitor CQ (chloroquine). (F) tf-LC3 staining further confirmed that celastrol promoted autophagy flux (scale bar = 10 μm). (G) Celastrol increased lysosomal numbers as reflected by Lysotracker Red DND-99 staining using flow cytometry assay. The TFEB activator Torin 1 was used as a positive control. (H) Celastrol increased the expression of multiple autophagy–lysosome-related genes as reflected by qPCR assay. (I) Knockout of the expression of TFEB attenuated celastrol-induced autophagy flux. All the values are average ± SEM from at least three independent experiments. ∗P < 0.05, ∗∗P < 0.01 vs. the control group or as indicated analyzed by one-way ANOVA.

Figure 2

Celastrol promotes the nucleus accumulation of TFEB via mTORC1 inhibition. (A, B) Celastrol inhibited the phosphorylation of TFEB (Ser142 and Ser211) in a time- and dose-dependent manner in CF7 cells. (C) Transfection of HEK293 cells with two phosphomimetic TFEB mutants [TFEB(S142D), TFEB(S211D)] inhibited nuclear translocation of TFEB after celastrol treatment (scale bar = 10 μm). (D) After pretreatment of CF7 cells with calcium chelator BAPTA-AM (20 μmol/L), calcineurin inhibitors FK506 (10 μmol/L) and CSA (cyclosporin A, 20 μmol/L), or PP2A inhibitor OA (okadaic acid, 400 nmol/L) for 30 min and then followed with celastrol for another 9 h, the results show that these inhibitors did not attenuate celastrol-induced nuclear accumulation of TFEB. (E) Celastrol inhibited the phosphorylation of RPS6KB1 in HEK293 cells. (F) Knockdown of TSC2 attenuated celastrol-induced dephosphorylation of mTORC1 substrate RPS6KB1. All the values are average ± SEM from at least three independent experiments. ∗P < 0.05, ∗∗P < 0.01 vs. the control group or as indicated analyzed by one-way ANOVA.

Figure 3

Celastrol is a blood–brain barrier permeable and enhances autophagy and lysosomal biogenesis in mouse brains. (A) Celastrol is brain permeable. Celastrol concentration in the brain was determined by LC–MS methods after giving mice 2 mg/kg celastrol by gavage. C_max = 766.67 ng/g (n = 5). C57 mice received 1 and 2 mg/kg/day celastrol for 7 consecutive days by gavage. (B, C) Celastrol promoted TFEB translocation from the cytoplasm into the nucleus in animal brains. (D–H) Celastrol increased autophagy and lysosome biogenesis. The expression of LC3B-II, LAMP1, cathepsin B, and cathepsin D in the frontal cortex was examined by Western blotting after treatment with celastrol. All the values are average ± SEM (n = 4–6). ∗P < 0.05 vs. Vehicle group analyzed by one-way ANOVA.

Figure 4

Celastrol ameliorates memory impairment and reduces phosphorylated Tau aggregates in P301S Tau mice. (A) Schematic models show experimental design for P301S Tau mice. At the end of celastrol treatment, (B) contextual fear conditioning test results show that celastrol improved memory impairments. (C, D) Open field test results show that celastrol improved exploratory and locomotor behavior. (E) Immunofluorescence analysis and quantification data show that celastrol reduced MC1 levels. (F) Immunofluorescence analysis and quantification data show that celastrol reduced CP13 levels. (G) Immunohistochemistry analysis and quantification data show that celastrol reduced AT8 levels (scale bar = 500 μm). All the values are average ± SEM (n = 7–8). ∗P < 0.05, ∗∗P < 0.01 vs. Vehicle group analyzed by one-way ANOVA.

Figure 5

Celastrol reduces phosphorylated Tau aggregates in P301S Tau mice. (A) The brain lysates of P301S Tau mice in Fig. 4 were separated into sarcosyl-insoluble and sarcosyl-soluble fractions. (B–E) Phosphorylated Tau proteins (AT8, PHF1, CP13, and MC1) from sarcosyl-insoluble fractions were quantified. (F, G) Total Tau proteins and autophagy substrate SQSTM1/p62 from sarcosyl-insoluble fractions were quantified. All the values are average ± SEM (n = 7–8). ∗P < 0.05 vs. Vehicle group analyzed by one-way ANOVA.

Figure 6

Celastrol mitigates memory impairments and reduces Tau aggregates in 3xTg mice. (A) Schematic models show experimental design for 3xTg mice. At the end of drug treatment, (B) contextual fear conditioning test results show that celastrol improved memory impairments. (C, D) Open field test results show that celastrol improved exploratory and locomotor behavior. (E–G) Morris water maze results show that celastrol ameliorated spatial learning and memory impairment in 3xTg mice. (H) Immunofluorescence analysis and quantification data show that celastrol reduced MC1 levels. (I) Immunofluorescence analysis and quantification data show that celastrol reduced CP13 levels. (J) Immunohistochemistry analysis and quantification data show that celastrol reduced AT8 levels (scale bar = 500 μm). All the values are average ± SEM (n = 8). ∗P < 0.05, ∗∗P < 0.01 vs. Vehicle group analyzed by one-way ANOVA.

Figure 7

Celastrol reduces phosphorylated Tau aggregates in 3xTg mice. (A) The brain lysates of 3xTg mice in Fig. 6 were separated into sarcosyl-insoluble and sarcosyl-soluble fractions. (B–E) Phosphorylated Tau proteins (AT8, PHF1, CP13, and MC1) from sarcosyl-insoluble fractions were quantified. (F, G) Total Tau proteins and autophagy substrate SQSTM1/p62 from sarcosyl-insoluble fractions were quantified. All the values are average ± SEM (n = 7–8). ∗P < 0.05 vs. Vehicle group analyzed by one-way ANOVA.

Figure 8

TFEB-mediated autophagy and the lysosomal pathway are required for celastrol-induced phosphorylated Tau aggregate degradation. (A) Celastrol promotes TFEB nucleus accumulation in mouse neuroblastoma N2a cells transiently expressing Flag-TFEB (scale bar = 10 μm). (B) Celastrol-induced autophagy in N2a cells; knockdown of Atg5 attenuated celastrol-induced elevation of LC3B-II levels. (C) Celastrol promoted the clearance of phosphorylated Tau (CP13, PHF1, AT8) in NP40-insoluble fraction in N2a cells transiently expressing P301L Tau; starvation (EBSS) was used a positive control. (D) Celastrol increased autophagy flux; lysosome inhibitor Baf A1 attenuated SDS-insoluble phosphorylated Tau degradation in response to celastrol in N2a cells. (E) Lysosome inhibitor CQ attenuated AT8 degradation in response to celastrol in N2a cells. (F) Knockdown of Atg7 compromised phosphorylated Tau AT8 degradation after celastrol treatment. (G) Knockdown of Tfeb compromised AT8 degradation after celastrol treatment. (H) Schematic models show tau-BiFC sensor based on Venus-based BiFC system. N- and C-terminal constituents of Venus protein were fused to full-length P301L mutated Tau. After Tau formed aggregates, the fluorescence increased. (I, J) By using a Tau-BiFC sensor, representative results from fluorescence microscope and flow cytometry analysis show that celastrol reduced Tau aggregates, and this reduction is inhibited by lysosome inhibitor Baf A1 (scale bar = 50 μm). All the values are average ± SEM from at least three independent experiments. ∗P < 0.05, ∗∗P < 0.01 vs. the control group or as indicated analyzed by one-way ANOVA.