Small molecules enhance autophagy and reduce toxicity in Huntington's disease models

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Abstract

The target of rapamycin proteins regulate various cellular processes including autophagy1, which may play a protective role in certain neurodegenerative and infectious diseases2. Here we show that a primary small-molecule screen in yeast yields novel small-molecule modulators of mammalian autophagy. We first identified new small-molecule enhancers (SMER) and inhibitors (SMIR) of the cytostatic effects of rapamycin in Saccharomyces cerevisiae. Three SMERs induced autophagy independently of rapamycin in mammalian cells, enhancing the clearance of autophagy substrates such as mutant huntingtin and A53T α-synuclein, which are associated with Huntington's disease and familial Parkinson's disease, respectively3,4,5. These SMERs, which seem to act either independently or downstream of the target of rapamycin, attenuated mutant huntingtin-fragment toxicity in Huntington's disease cell and Drosophila melanogaster models, which suggests therapeutic potential. We also screened structural analogs of these SMERs and identified additional candidate drugs that enhanced autophagy substrate clearance. Thus, we have demonstrated proof of principle for a new approach for discovery of small-molecule modulators of mammalian autophagy.

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Figure 1: SMERs 10, 18 and 28 enhance the clearance of mutant aggregate-prone proteins by autophagy in mammalian cell models of Huntington's and Parkinson's disease, independent of rapamycin.
Figure 2: SMERs 10, 18 and 28 induce autophagy in mammalian cells.
Figure 3: SMERs 10, 18 and 28 protect against neurodegeneration in D. melanogaster model of Huntington's disease.
Figure 4: Rapamycin and SMERs have additive protective effects on the clearance and toxicity of mutant aggregate-prone proteins.
Figure 5: Screen of chemical analogs of autophagy-inducing SMERs for their protective effects on the clearance and aggregation of mutant proteins.

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Acknowledgements

We thank T. Yoshimori (Japanese National Institute of Genetics) for the EGFP-LC3 construct, N. Mizushima (Tokyo Metropolitan Institute of Medical Science) for Atg5 and HA-Atg12 constructs, and wild-type and Atg5-deficient MEFs, A.M. Tolkovsky (University of Cambridge) for EGFP-LC3 HeLa stable cell line and N.P. Dantuma (Karolinska Institutet) for UbG76V-EGFP degron HeLa stable cell line. We thank the staff of the Broad Institute Chemical Biology Program (formerly the Institute for Chemistry and Chemical Biology), B. Ravikumar, A. Williams, L. Jahreiss and R. Walker (University of Cambridge) for technical assistance; and S. Haggarty for comments and discussion. This work was supported in part with federal funds from the US National Cancer Institute's Initiative for Chemical Genetics, National Institutes of Health, under Contract No. N01-CO-12400. We are grateful for a Wellcome Trust Senior Fellowship in Clinical Science (D.C.R.), an MRC programme grant, an EU Framework VI (EUROSCA) grant (D.C.R.) and the US National Institute of General Medicine Sciences GM38627 (S.L.S.) for additional funding. S.L.S. is an investigator at the Howard Hughes Medical Institute.

Author information

S.S. and E.O.P. designed, performed and analyzed experiments and helped write the paper; S.I., S.P., A.C., R.L.M., J.A.W. and T.A.L. performed and analyzed experiments; C.J.O. designed and analyzed experiments; S.L.S. and D.C.R. designed and analyzed experiments and helped write the paper.

Correspondence to Stuart L Schreiber or David C Rubinsztein.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

Results of a small-molecule screen for suppressors (SMIRs) and enhancers (SMERs) of the cytostatic effects of rapamycin in yeast, and characterization, potency and selectivity of the identified SMIRs and SMERs. (PDF 185 kb)

Supplementary Fig. 2

Screen for the autophagy-inhibitory SMIRs and the autophagy-inducing SMERs in mammalian cell line. (PDF 48 kb)

Supplementary Fig. 3

The effect of SMERs 10, 18 and 28 on mTOR activity, Beclin-1/Atg6, Atg5, Atg7, Atg12, Atg5-Atg12 conjugation and proteasome activity. (PDF 59 kb)

Supplementary Methods (PDF 279 kb)

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Sarkar, S., Perlstein, E., Imarisio, S. et al. Small molecules enhance autophagy and reduce toxicity in Huntington's disease models. Nat Chem Biol 3, 331–338 (2007) doi:10.1038/nchembio883

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