Abstract
BackgroundInduction of the heat shock response (HSR) and increased expression of the heat shock proteins (HSPs) provide mechanisms to ensure proper protein folding, trafficking, and disposition. The importance of HSPs is underscored by the understanding that protein mis-folding and aggregation contribute centrally to the pathogenesis of neurodegenerative diseases.Methodology/Principal FindingsWe used a cell-based hsp70-luciferease reporter gene assay system to identify agents that modulate the HSR and show here that clinically relevant concentrations of the FDA-approved ALS drug riluzole significantly increased the heat shock induction of hsp70-luciferse reporter gene. Immuno-Western and -cytochemical analysis of HSF1 show that riluzole increased the amount of cytosolic HSF1 to afford a greater activation of HSF1 upon heat shock. The increased HSF1 contributed centrally to the cytoprotective activity of riluzole as hsf1 gene knockout negated the synergistic activity of riluzole and conditioning heat shock to confer cell survival under oxidative stress. Evidence of a post-transcriptional mechanism for the increase in HSF1 include: quantitation of mRNAhsf1 by RT-PCR showed no effect of either heat shock or riluzole treatment; riluzole also increased the expression of HSF1 from a CMV-promoter; analysis of the turnover of HSF1 by pulse chase and immunoprecipitation show that riluzole slowed the decay of [35S]labeled-HSF1. The effect of riluzole on HSF1 was qualitatively different from that of MG132 and chloroquine, inhibitors of the proteasome and lysosome, respectively, and appeared to involve the chaperone-mediated autophagy pathway as RNAi-mediated knockdown of CMA negated its effect.Conclusion/SignificanceWe show that riluzole increased the amount of HSF1 to amplify the HSR for cytoprotection. Our study provides novel insight into the mechanism that regulates HSF1 turnover, and identifies the degradation of HSF1 as a target for therapeutics intervention.
Highlights
A common feature of many neurodegenerative diseases is the misfolding-due to genetic as well as epigenetic factors-of specific proteins, aggregation and formation of protein fibrillary structures termed amyloid inside and outside of brain cells [1]; terms such as ‘‘protein mis-folding diseases’’ and ‘‘proteinoapthies’’ have been coined to describe such disorders
We show in fig. 1A that expression of the hsp70reporter was induced 36 fold on average by heat shock
A major obstacle in harnessing such cytoprotective activity for therapeutic purposes is that agents/ conditions that induce the heat shock response (HSR) are proteotoxic, and the induction of heat shock proteins (HSPs) under such conditions represents a compensatory mechanism to rectify the perturbation of protein homeostasis
Summary
A common feature of many neurodegenerative diseases is the misfolding-due to genetic as well as epigenetic factors-of specific proteins, aggregation and formation of protein fibrillary structures termed amyloid inside and outside of brain cells [1]; terms such as ‘‘protein mis-folding diseases’’ and ‘‘proteinoapthies’’ have been coined to describe such disorders. Expression of the expanded polyQ protein in a Drosophila line bearing a dominant-negative Hsp augmented the severity and kinetics of neurodegeneration, suggesting that under normal conditions the endogenous Hsp protein may partially mitigate the toxic effects of the expanded polyQ protein [2]. These considerations suggest that agents that upregulate the HSR and HSP chaperones may hold promise in therapeutics development for the prevention, management, and treatment of neurodegeneration [3,4,5,6]. The importance of HSPs is underscored by the understanding that protein mis-folding and aggregation contribute centrally to the pathogenesis of neurodegenerative diseases
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