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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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Related in: MedlinePlus

17-DMAG has no effect on PrP expression.A, Flies expressing PrP ubiquitously (da>PrP-M9) were fed with 17-DMAG at 0, 12, 24, 48, and 96 µg/ml during development. Analysis of PrP levels in 1 day-old flies by western blot showed no significant changes. Tubulin was used as loading control to normalize protein levels. B, Quantification of the triplicate experiment followed by analysis with ANOVA. No statistical differences were identified in these experiments.
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pone-0088522-g001: 17-DMAG has no effect on PrP expression.A, Flies expressing PrP ubiquitously (da>PrP-M9) were fed with 17-DMAG at 0, 12, 24, 48, and 96 µg/ml during development. Analysis of PrP levels in 1 day-old flies by western blot showed no significant changes. Tubulin was used as loading control to normalize protein levels. B, Quantification of the triplicate experiment followed by analysis with ANOVA. No statistical differences were identified in these experiments.

Mentions: Fortunately, a few derivatives of geldanamycin recently become commercially available. Several of these analogues work at sub-micromolar concentrations, but are poorly soluble in water, like 17-AAG. In contrast, 17-DMAG is water soluble, making this compound the best candidate for in vivo delivery [23]. Taking advantage of its solubility, we incorporated 17-DMAG at concentrations between 12 and 96 µg/ml into standard fly media to make it available throughout all developmental stages to flies expressing PrP. Then, we collected 1 day-old flies and conducted western blot to compare the steady-state levels of total PrP using the 6D11 anti-PrP antibody. For quantification, we performed the experiments in triplicate and detected tubulin as loading control to normalize expression data. After averaging the normalized PrP levels, we found that PrP expression was mostly unchanged in the 17-DMAG treatments (Fig. 1A and B and Table 1).


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)

17-DMAG has no effect on PrP expression.A, Flies expressing PrP ubiquitously (da>PrP-M9) were fed with 17-DMAG at 0, 12, 24, 48, and 96 µg/ml during development. Analysis of PrP levels in 1 day-old flies by western blot showed no significant changes. Tubulin was used as loading control to normalize protein levels. B, Quantification of the triplicate experiment followed by analysis with ANOVA. No statistical differences were identified in these experiments.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3921213&req=5

pone-0088522-g001: 17-DMAG has no effect on PrP expression.A, Flies expressing PrP ubiquitously (da>PrP-M9) were fed with 17-DMAG at 0, 12, 24, 48, and 96 µg/ml during development. Analysis of PrP levels in 1 day-old flies by western blot showed no significant changes. Tubulin was used as loading control to normalize protein levels. B, Quantification of the triplicate experiment followed by analysis with ANOVA. No statistical differences were identified in these experiments.
Mentions: Fortunately, a few derivatives of geldanamycin recently become commercially available. Several of these analogues work at sub-micromolar concentrations, but are poorly soluble in water, like 17-AAG. In contrast, 17-DMAG is water soluble, making this compound the best candidate for in vivo delivery [23]. Taking advantage of its solubility, we incorporated 17-DMAG at concentrations between 12 and 96 µg/ml into standard fly media to make it available throughout all developmental stages to flies expressing PrP. Then, we collected 1 day-old flies and conducted western blot to compare the steady-state levels of total PrP using the 6D11 anti-PrP antibody. For quantification, we performed the experiments in triplicate and detected tubulin as loading control to normalize expression data. After averaging the normalized PrP levels, we found that PrP expression was mostly unchanged in the 17-DMAG treatments (Fig. 1A and B and Table 1).

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