Celastrol inhibits polyglutamine aggregation and toxicity though induction of the heat shock response

Abstract

Heat shock proteins (hsps) are protective against the harmful effects of mutant expanded polyglutamine repeat proteins that occur in diseases such as Huntington's, prompting the search for pharmacologic compounds that increase hsp expression in cells as potential treatments for this and related diseases. In this paper, we show that celastrol, a compound recently shown to up-regulate hsp gene expression, significantly decreases killing of cells expressing mutant polyglutamine protein. This effect requires the presence of the transcription factor responsible for mediating inducible hsp gene expression, HSF1, and is correlated with decreased amounts and increased sodium dodecyl sulfate (SDS) solubility of polyglutamine aggregates. These results suggest the potential of celastrol as a therapeutic agent in the treatment of human polyglutamine expansion diseases.

Fig. 1

Celastrol treatment reduces Q57-YFP cytotoxicity. a Hsp70 protein level is increased by celastrol treatment. HeLa cells were treated with the indicated concentrations of celastrol for 24 or 48 h, after which cell extracts were made and subjected to Western blot using antibodies against hsp70 or β-actin (upper panel). These results were quantified using the ImageQuant program, and the values for the two treatment times, grouped by celastrol concentration, were graphed (lower panel). b HeLa cells were transfected with Q57-YFP along with celastrol treatment at the concentrations indicated. After 48 h, the amount of cell death was determined by trypan blue assay. Data are shown as means ± SE (*P < 0.007, **P < 0.004, ***P < 0.0001, for each celastrol concentration treatment vs no celastrol)

Fig. 2

Celastrol treatment reduces number of cells containing Q57-YFP aggregates and increases Q57-YFP solubility. HeLa cells were transfected with Q57-YFP along with celastrol treatment at the concentrations indicated. a After 48 h of transfection, the formation of Q57-YFP aggregates was quantified using fluorescence microscopy. Visual fields which contained similar numbers of cells (based on the density of nuclei stained by Hoechst) were chosen under 20× objective, and then the number of aggregates in each field of vision was counted. Three different visual fields were quantified in each case, and data are shown as means ± SE (*P < 0.004, **P < 0.001, ***P < 0.0003, for each celastrol concentration treatment vs no celastrol). b To determine the amount of Q57-YFP monomer that could be solubilized from aggregates in lysates of the transfected cells by SDS treatment, the protein concentration of the insoluble fraction of the cell lysates was determined, and then 40 μg of protein was subjected to SDS solubilization treatment, followed by Western blot using anti-GFP antibody

Fig. 3

Celastrol effects on Q57-YFP toxicity and aggregates in PC12 cells. PC12 cells were transfected with Q57-YFP along with celastrol treatment at the concentrations indicated. After 48 h, the amount of cell death was determined by trypan blue assay (a), the number of cells containing Q57-YFP aggregates was quantified using fluorescence microscopy (b), and the amount of Q57-YFP monomer solubilized from aggregates by SDS treatment visualized by Western blot using anti-GFP antibody (c). In a and b, data are shown as means ± SE [*P < 0.0001 and **P < 0.0001 (a), *P < 0.001 and **P < 0.0001 (b), in each case for each celastrol concentration treatment vs no celastrol)

Fig. 4

HSF1−/− cells exhibit higher Q57-YFP aggregation and cell death. HSF1−/− and wild-type MEF cells were transfected with Q57-YFP, and after 48 h, cell death was examined by trypan blue assay (a), and the number of cells containing Q57-YFP aggregates was quantified using fluorescence microscopy (b), or filtration assay, in which 30 μg of the insoluble fraction was filtered through 0.2 μm cellulose acetate membrane, and the aggregates retained on the membrane were immunoblotted using anti-GFP antibody (c). In a and b, data are shown as means ± SE. *P < 0.008 (a); *P < 0.003 (b)

Fig. 5

Celastrol decreases Q57-YFP toxicity and aggregation in wild-type but not HSF1−/− MEF cells. a Celastrol treatment does not induce hsp70 expression in HSF1−/− cells. Wild-type and HSF1−/− MEF cells were treated with no celastrol or 0.4 μM celastrol, and after 12 h, cell extracts were made, and 5 μg protein was loaded into each lane for immunoblotting with anti-hsp70 antibodies. b and c Wild-type and HSF1−/− MEF cells were transfected with Q57-YFP, with celastrol at the indicated concentrations added at the same time. After 48 h, cell death was examined by trypan blue assay (b), with data shown as means ± SE (*P < 0.002 for HSF2+/+ 0.4 μM celastrol vs no celastrol; *P < 0.002 for HSF2−/− 0.4 μM celastrol vs no celastrol), and amount of aggregated Q57-YFP was determined by filtration assay followed by anti-GFP Western blot (c)