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Combined pharmacological induction of Hsp70 suppresses prion protein neurotoxicity in Drosophila.

Zhang Y, Casas-Tinto S, Rincon-Limas DE, Fernandez-Funez P - PLoS ONE (2014)

Bottom Line: To validate the therapeutic potential of Hsp70, we treated flies with drugs known to induce Hsp70 expression, including the Hsp90 inhibitor 17-DMAG and the glucocorticoid dexamethasone.Although the individual treatment with these compounds produced no significant benefits, their combination significantly increased the level of inducible Hsp70, decreased the level of total PrP, reduced the accumulation of pathogenic PrP conformers, and improved locomotor activity.Thus, the combined action of two pharmacological activators of Hsp70 with distinct targets results in sustained high levels of inducible Hsp70 with improved behavioral output.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurology, McKnight Brain Institute, University of Florida, Gainesville, Florida, United States of America.

ABSTRACT
Prion diseases are rare and aggressive neurodegenerative disorders caused by the accumulation of misfolded, toxic conformations of the prion protein (PrP). Therapeutic strategies directed at reducing the levels of PrP offer the best chance of delaying or halting disease progression. The challenge, though, is to define pharmacologic targets that result in reduced PrP levels. We previously reported that expression of wild type hamster PrP in flies induces progressive locomotor dysfunction and accumulation of pathogenic PrP conformations, while co-expression of human Hsp70 delayed these changes. To validate the therapeutic potential of Hsp70, we treated flies with drugs known to induce Hsp70 expression, including the Hsp90 inhibitor 17-DMAG and the glucocorticoid dexamethasone. Although the individual treatment with these compounds produced no significant benefits, their combination significantly increased the level of inducible Hsp70, decreased the level of total PrP, reduced the accumulation of pathogenic PrP conformers, and improved locomotor activity. Thus, the combined action of two pharmacological activators of Hsp70 with distinct targets results in sustained high levels of inducible Hsp70 with improved behavioral output. These findings can have important therapeutic applications for the devastating prion diseases and other related proteinopathies.

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Co-treatment of 17-DMAG and dexamethasone reduces PrP and induces Hsp70.A and B, Flies expressing PrP ubiquitously (da>PrP-M9) were fed with combinations of 17-DMAG at 0, 48, and 96 µg/ml and dexamethasone at 0, 24, and 48 µg/ml during development. Analysis of PrP levels in 1 day-old flies by western blot showed significant reduction in PrP in two combinations following quantification and analysis by ANOVA, with 17-DMAG 96 µg/ml and dexamethasone 24 µg/ml inducing the strongest response. C, Analysis of PrP and GFP mRNA by qPCR showed no significant changes in flies treated with the optimum drug combination. D and E, Western blot and quantification of Hsp70 in flies treated with different combinations of 17-DMAG and dexamethasone. Quantification of Hsp70 followed by normalization with a non-specific band (control) revealed that all treatments induced significant increases in Hsp70, with 17-DMAG 96 µg/ml and dexamethasone 24 µg/ml inducing the strongest response. F, Expression of Hsp70 mRNAs by qPCR revealed a four-fold increase in flies treated with the optimum 17-DMAG/dexamethasone cocktail in flies expressing either PrP or CD8-GFP. G, Analysis of Hsc70-4 and Hsp27 mRNA by qPCR in flies treated with the optimum 17-DMAG/dexamethasone cocktail showed no significant changes in these two chaperones. Statistical significance was analyzed by ANOVA multiple comparisons for western blots and t-test for qPCR: * p<0.05; ** p<0.01; *** p<0.001.
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pone-0088522-g003: Co-treatment of 17-DMAG and dexamethasone reduces PrP and induces Hsp70.A and B, Flies expressing PrP ubiquitously (da>PrP-M9) were fed with combinations of 17-DMAG at 0, 48, and 96 µg/ml and dexamethasone at 0, 24, and 48 µg/ml during development. Analysis of PrP levels in 1 day-old flies by western blot showed significant reduction in PrP in two combinations following quantification and analysis by ANOVA, with 17-DMAG 96 µg/ml and dexamethasone 24 µg/ml inducing the strongest response. C, Analysis of PrP and GFP mRNA by qPCR showed no significant changes in flies treated with the optimum drug combination. D and E, Western blot and quantification of Hsp70 in flies treated with different combinations of 17-DMAG and dexamethasone. Quantification of Hsp70 followed by normalization with a non-specific band (control) revealed that all treatments induced significant increases in Hsp70, with 17-DMAG 96 µg/ml and dexamethasone 24 µg/ml inducing the strongest response. F, Expression of Hsp70 mRNAs by qPCR revealed a four-fold increase in flies treated with the optimum 17-DMAG/dexamethasone cocktail in flies expressing either PrP or CD8-GFP. G, Analysis of Hsc70-4 and Hsp27 mRNA by qPCR in flies treated with the optimum 17-DMAG/dexamethasone cocktail showed no significant changes in these two chaperones. Statistical significance was analyzed by ANOVA multiple comparisons for western blots and t-test for qPCR: * p<0.05; ** p<0.01; *** p<0.001.

Mentions: Since 17-DMAG and dexamethasone have different molecular targets, but result in Hsp70 induction, these compounds offer the opportunity to examine their potential synergistic activity. To explore the potential benefit of a combined regimen of these two Hsp70-inducing compounds, we raised flies expressing PrP ubiquitously as above in standard media containing combinations of high concentrations of 17-DMAG and dexamethasone or control media (Fig. 3A). Then, we collected 1 day-old flies, performed western blot, and quantified the bands as described above. All drug combinations tested reduced total PrP compared with non-treated controls, although only two conditions were significantly reduced (Figs. 3A and B). The combinations of 17-DMAG at 48 µg/ml with dexamethasone at 24 µg/ml resulted in 19% reduction in total PrP, but did not reach statistical significance (p = 0.2) (Table 3). The combination of 96 µg/ml of 17-DMAG and 24 µg/ml of dexamethasone produced a 51% reduction in the steady-state levels of PrP that was statistically significant (p = 0.0036). Also, 48 µg/ml of 17-DMAG and 48 µg/ml of dexamethasone reduced PrP by 32% (p = 0.03). Thus, the combined treatment with 17-DMAG and dexamethasone achieved a robust reduction of PrP stead-state levels.


Combined pharmacological induction of Hsp70 suppresses prion protein neurotoxicity in Drosophila.

Zhang Y, Casas-Tinto S, Rincon-Limas DE, Fernandez-Funez P - PLoS ONE (2014)

Co-treatment of 17-DMAG and dexamethasone reduces PrP and induces Hsp70.A and B, Flies expressing PrP ubiquitously (da>PrP-M9) were fed with combinations of 17-DMAG at 0, 48, and 96 µg/ml and dexamethasone at 0, 24, and 48 µg/ml during development. Analysis of PrP levels in 1 day-old flies by western blot showed significant reduction in PrP in two combinations following quantification and analysis by ANOVA, with 17-DMAG 96 µg/ml and dexamethasone 24 µg/ml inducing the strongest response. C, Analysis of PrP and GFP mRNA by qPCR showed no significant changes in flies treated with the optimum drug combination. D and E, Western blot and quantification of Hsp70 in flies treated with different combinations of 17-DMAG and dexamethasone. Quantification of Hsp70 followed by normalization with a non-specific band (control) revealed that all treatments induced significant increases in Hsp70, with 17-DMAG 96 µg/ml and dexamethasone 24 µg/ml inducing the strongest response. F, Expression of Hsp70 mRNAs by qPCR revealed a four-fold increase in flies treated with the optimum 17-DMAG/dexamethasone cocktail in flies expressing either PrP or CD8-GFP. G, Analysis of Hsc70-4 and Hsp27 mRNA by qPCR in flies treated with the optimum 17-DMAG/dexamethasone cocktail showed no significant changes in these two chaperones. Statistical significance was analyzed by ANOVA multiple comparisons for western blots and t-test for qPCR: * p<0.05; ** p<0.01; *** p<0.001.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3921213&req=5

pone-0088522-g003: Co-treatment of 17-DMAG and dexamethasone reduces PrP and induces Hsp70.A and B, Flies expressing PrP ubiquitously (da>PrP-M9) were fed with combinations of 17-DMAG at 0, 48, and 96 µg/ml and dexamethasone at 0, 24, and 48 µg/ml during development. Analysis of PrP levels in 1 day-old flies by western blot showed significant reduction in PrP in two combinations following quantification and analysis by ANOVA, with 17-DMAG 96 µg/ml and dexamethasone 24 µg/ml inducing the strongest response. C, Analysis of PrP and GFP mRNA by qPCR showed no significant changes in flies treated with the optimum drug combination. D and E, Western blot and quantification of Hsp70 in flies treated with different combinations of 17-DMAG and dexamethasone. Quantification of Hsp70 followed by normalization with a non-specific band (control) revealed that all treatments induced significant increases in Hsp70, with 17-DMAG 96 µg/ml and dexamethasone 24 µg/ml inducing the strongest response. F, Expression of Hsp70 mRNAs by qPCR revealed a four-fold increase in flies treated with the optimum 17-DMAG/dexamethasone cocktail in flies expressing either PrP or CD8-GFP. G, Analysis of Hsc70-4 and Hsp27 mRNA by qPCR in flies treated with the optimum 17-DMAG/dexamethasone cocktail showed no significant changes in these two chaperones. Statistical significance was analyzed by ANOVA multiple comparisons for western blots and t-test for qPCR: * p<0.05; ** p<0.01; *** p<0.001.
Mentions: Since 17-DMAG and dexamethasone have different molecular targets, but result in Hsp70 induction, these compounds offer the opportunity to examine their potential synergistic activity. To explore the potential benefit of a combined regimen of these two Hsp70-inducing compounds, we raised flies expressing PrP ubiquitously as above in standard media containing combinations of high concentrations of 17-DMAG and dexamethasone or control media (Fig. 3A). Then, we collected 1 day-old flies, performed western blot, and quantified the bands as described above. All drug combinations tested reduced total PrP compared with non-treated controls, although only two conditions were significantly reduced (Figs. 3A and B). The combinations of 17-DMAG at 48 µg/ml with dexamethasone at 24 µg/ml resulted in 19% reduction in total PrP, but did not reach statistical significance (p = 0.2) (Table 3). The combination of 96 µg/ml of 17-DMAG and 24 µg/ml of dexamethasone produced a 51% reduction in the steady-state levels of PrP that was statistically significant (p = 0.0036). Also, 48 µg/ml of 17-DMAG and 48 µg/ml of dexamethasone reduced PrP by 32% (p = 0.03). Thus, the combined treatment with 17-DMAG and dexamethasone achieved a robust reduction of PrP stead-state levels.

Bottom Line: To validate the therapeutic potential of Hsp70, we treated flies with drugs known to induce Hsp70 expression, including the Hsp90 inhibitor 17-DMAG and the glucocorticoid dexamethasone.Although the individual treatment with these compounds produced no significant benefits, their combination significantly increased the level of inducible Hsp70, decreased the level of total PrP, reduced the accumulation of pathogenic PrP conformers, and improved locomotor activity.Thus, the combined action of two pharmacological activators of Hsp70 with distinct targets results in sustained high levels of inducible Hsp70 with improved behavioral output.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurology, McKnight Brain Institute, University of Florida, Gainesville, Florida, United States of America.

ABSTRACT
Prion diseases are rare and aggressive neurodegenerative disorders caused by the accumulation of misfolded, toxic conformations of the prion protein (PrP). Therapeutic strategies directed at reducing the levels of PrP offer the best chance of delaying or halting disease progression. The challenge, though, is to define pharmacologic targets that result in reduced PrP levels. We previously reported that expression of wild type hamster PrP in flies induces progressive locomotor dysfunction and accumulation of pathogenic PrP conformations, while co-expression of human Hsp70 delayed these changes. To validate the therapeutic potential of Hsp70, we treated flies with drugs known to induce Hsp70 expression, including the Hsp90 inhibitor 17-DMAG and the glucocorticoid dexamethasone. Although the individual treatment with these compounds produced no significant benefits, their combination significantly increased the level of inducible Hsp70, decreased the level of total PrP, reduced the accumulation of pathogenic PrP conformers, and improved locomotor activity. Thus, the combined action of two pharmacological activators of Hsp70 with distinct targets results in sustained high levels of inducible Hsp70 with improved behavioral output. These findings can have important therapeutic applications for the devastating prion diseases and other related proteinopathies.

Show MeSH
Related in: MedlinePlus