Small molecules enhance autophagy and reduce toxicity in Huntington's disease models - PubMed (original) (raw)

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

Sovan Sarkar et al. Nat Chem Biol. 2007 Jun.

Abstract

The target of rapamycin proteins regulate various cellular processes including autophagy, which may play a protective role in certain neurodegenerative and infectious diseases. 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 alpha-synuclein, which are associated with Huntington's disease and familial Parkinson's disease, respectively. 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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Figures

Figure 1

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.

(a) Chemical structures of SMERs 10, 18 and 28. (b) A stable inducible PC12 cell line expressing A53T α-synuclein was induced with 1 μg ml-1 doxycycline for 48 h, and expression of the transgene was switched off for 24 h. Cells were treated with DMSO (control), or with 0.94 μM, 4.7 μM, 47 μM of SMER10, 0.86 μM, 4.3 μM, 43 μM of SMER18 or 0.9 μM, 4.7 μM, 47 μM of SMER28 added in the switch-off period as 1:20000, 1:4000 or 1:400 dilutions of 5 mg ml-1 stock solution (in DMSO), respectively. A53T α-synuclein (α-syn) levels were analysed by immunoblotting with antibody against HA (i) and densitometry analysis relative to actin (ii). Error bars denote S.E.M. _p_=0.0917, _p_=0.009, _p_=0.0001 (for increasing concentrations of SMER10); _p_=0.0068, _p_=0.0023, _p_=0.0002 (for increasing concentrations of SMER18); _p_=.0016, p<0.0001, p<0.0001 (for increasing concentrations of SMER28). (c) COS-7 cells transfected with EGFP-HDQ74 construct for 4 h were treated with DMSO (control), 0.2 μM rapamycin (rap), 47 μM SMER10, 43 μM SMER18 or 47 μM SMER28 for 48 h. The effects of treatment on the percentage of EGFP-positive cells with EGFP-HDQ74 aggregates or apoptotic morphology (cell death) were expressed as odds ratios and the control was taken as 1. Error bars: 95 % confidence interval. p<0.0001 (rap and SMER28), _p_=0.013 (SMER10), _p_=0.019 (SMER18) (aggregation); p<0.0001 (rap, SMER18 and SMER28), _p_=0.004 (SMER10) (cell death). Odds ratios were used to determine pooled estimates for the changes in aggregate formation or cell death, resulting from perturbations assessed in multiple independent experiments (see Supplementary Methods online). (d) Wild-type (ATG5+/+) and knock-out (_ATG5_−/−) Atg5 mouse embryonic fibroblasts (MEFs) were transfected with EGFP-HDQ74 for 4 h and treated with DMSO (control), 47 μM SMER10, 43 μM SMER18 or 47 μM SMER28 for 24 h. The effects of treatment on the percentage of EGFP-positive cells with EGFP-HDQ74 aggregates were expressed as odds ratios and the control (DMSO-treated) values were fixed at 1 for both cell lines. Error bars: 95 % confidence interval. _p_=0.039 (SMER10), p<0.0001 (SMER18 and SMER28) (in ATG5+/+ cells); _p_=0.092 (SMER10), _p_=0.271 (SMER18), _p_=0.358 (SMER28) (in _ATG5_−/− cells). Note that EGFP-HDQ74 aggregation was higher in _ATG5_−/− cells compared to ATG5+/+ cells (Supplementary Fig. 3a online). (e) The percentage of EGFP-positive COS-7 cells with EGFP-HDQ74 aggregates as in Fig. 1c, treated with DMSO (control), 10 μM lactacystin (proteasome inhibitor), or both 10 μM lactacystin along with either 47 μM SMER10, 43 μM SMER18 or 47 μM SMER28 for 48 h, were expressed as odds ratios. Error bars: 95 % confidence interval. p<0.0001 (Control vs Lact), _p_=0.014 (SMER10 vs Lact), p<0.0001 (SMER18 vs Lact), _p_=0.001 (SMER28 vs Lact). ***, p<0.001; **, p<0.01; *, p<0.05; NS, Non-significant.

Figure 2

Figure 2. SMERs 10, 18 and 28 induce autophagy in mammalian cells.

(a) COS-7 cells transfected with EGFP-LC3 construct for 4 h were treated with DMSO (control), 0.2 μM rapamycin (rap) (positive control), 47 μM SMER10, 43 μM SMER18 or 47 μM SMER28 for 16 h, and analysed by fluorescence microscopy. The effects of treatment on the percentage of EGFP-positive cells with >5 EGFP-LC3 vesicles are shown. Error bars denote S.E.M. p<0.0001 (all SMERs). (b) HeLa cells stably expressing EGFP-LC3 were treated with DMSO (control), 47 μM SMER10, 43 μM SMER18 or 47 μM SMER28 for 24 h. Confocal sections show cells containing EGFP-positive autophagic vesicles. Nuclei are stained with DAPI. Bar, 20 μM. (c) HeLa cells stably expressing EGFP-LC3 were treated for 4 h with DMSO (control) or 200 nM bafilomycin A1 (baf), or with 200 nM bafilomycin A1 and 47 μM SMER10, 43 μM SMER18 or 47 μM SMER28. Cells were left untreated or pre-treated with SMERs for 24 h before adding bafilomycin A1. Levels of EGFP-LC3-II were determined by immunoblotting with antibody against EGFP (i) and densitometry analysis relative to actin (ii). Error bars denote S.E.M. _p_=0.0259 (baf), p<0.0001 (SMER10), _p_=0.0003 (SMER18 and SMER28) vs control; _p_=0.0025 (SMER10), _p_=0.0218 (SMER18), _p_=0.0195 (SMER28) vs bafilomycin A1. ***, p<0.001; **, p<0.01; *, p<0.05.

Figure 3

Figure 3. SMERs 10, 18 and 28 protect against neurodegeneration in Drosophila model of Huntington’s disease.

(a-c) Flies treated with 100 μM SMER10 (a), 200 μM SMER18 (b) or 100 μM SMER28 (c) show a shift in the distribution of the number of rhabdomeres compared to flies treated with DMSO (control) alone (2 days after eclosion). Rhabdomere counts from all 3 independent experiments are included. _n_=600 ommatidia (SMER10), _n_=1500 ommatidia (SMER18) and _n_=600 ommatidia (SMER28). Mann-Whitney test values p<0.0001 (all SMERs). Student’s _t_-test (1 tailed) _p_=0.005 (SMER10), _p_=0.004 (SMER18), _p_=0.03 (SMER28) compared distributions of means of independent experiments. These SMER concentrations cause no overt toxicity to flies (see Supplementary Methods online). Distributions of DMSO-treated flies may vary when SMERs were treated in different experiments at different times. For instance, an individual experiment may have lasted slightly longer or shorter and photoreceptor degeneration is progressive and time-dependent.

Figure 4

Figure 4. Rapamycin and SMERs have additive protective effects on the clearance and toxicity of mutant aggregate-prone proteins.

(a-c) Clearance of A53T α-synuclein (α-syn) in stable PC12 cells as in Fig. 1b, treated with DMSO (control), or with 0.2 μM rapamycin alone, SMER alone [140 μM SMER10 (a), 43 μM SMER18 (b) or 47 μM SMER28 (c)] or both for the 8 h switch-off period, was analysed by immunoblotting with antibody against HA (i) and densitometry analysis relative to actin (ii). The concentration of rapamycin is saturating for its effect on the clearance of A53T α-synuclein. Error bars denote S.E.M. _p_=0.0025 (rap), _p_=0.0018 (SMER10), p<0.0001 (SMER10+rap), p<0.0001 (rap or SMER10 vs SMER10+rap) (a); _p_=0.0069 (rap), _p_=0.0498 (SMER18), p<0.0001 (SMER18+rap), _p_=0.0038 (rap vs SMER18+rap), _p_=0.0007 (SMER18 vs SMER18+rap) (b); p<0.0001 (rap, SMER28, rap vs SMER28+rap, SMER28 vs SMER28+rap) (c). (d-f) The percentage of EGFP-positive cells with EGFP-HDQ74 aggregates (i) and cell death (ii) in COS-7 cells as in Fig. 1c, treated with DMSO (control), or with 0.2 μM rapamycin alone, SMER alone [140 μM SMER10 (d), 43 μM SMER18 (e) or 47 μM SMER28 (f)] or both for 24 h, were expressed as odds ratios. Error bars: 95 % confidence interval. (i), For aggregation: _p_=0.248 (rap), _p_=0.217 (SMER10), p<0.0001 (SMER10+rap), p<0.001 (rap or SMER10 vs SMER10+rap) (d); _p_=0.248 (rap), _p_=0.543 (SMER18), p<0.0001 (SMER18+rap), _p_=0.008 (rap vs SMER18+rap), _p_=0.002 (SMER18 vs SMER18+rap) (e); _p_=0.248 (rap), _p_=0.002 (SMER28), p<0.0001 (SMER28+rap), p<0.0001 (rap vs SMER28+rap), _p_=0.012 (SMER28 vs SMER28+rap) (f). (ii), For cell death: _p_=0.002 (rap), p<0.0001 (SMER10, SMER10+rap, rap or SMER10 vs SMER10+rap) (d); _p_=0.002 (rap), _p_=0.948 (SMER18), p<0.0001 (SMER18+rap), _p_=0.015 (rap vs SMER18+rap), p<0.0001 (SMER18 vs SMER18+rap) (e); _p_=0.002 (rap), p<0.0001 (SMER28, SMER28+rap, rap or SMER28 vs SMER28+rap) (f). Note that we have treated cells for a shorter time in this experiment (24 h), compared to Fig. 1c (48 h) - this probably accounts for the failure of the protective trends of rapamycin and some of the SMERs to reach significance for aggregation, on their own in this experiment. ***, p<0.001; **, p<0.01; *, p<0.05; NS, Non-significant.

Figure 5

Figure 5. Screen of chemical analogs of autophagy-inducing SMERs for their protective effects on the clearance and aggregation of mutant proteins.

(a-c) Clearance of A53T α-synuclein (α-syn) in stable PC12 cells as in Fig. 1b, treated for 24 h with either DMSO (control), or with 47 μM SMER10 and its analogs (SMER10a-c) (a), 43 μM SMER18 and its analogs (SMER18a-l) (b), or 47 μM SMER28 and its analogs (SMER28a-k) (c), was analysed by immunoblotting with anti-HA antibody (i) and densitometry analysis relative to actin (ii). All the analogs were used in the cell culture media at 1:400 dilution of 5 mg ml-1 stock solution (in DMSO). Error bars denote S.E.M. _p_=0.0058 (SMER10a), _p_=0.6736 (SMER10b), _p_=0.9507 (SMER10c), _p_=0.0481 (SMER10) (a); _p_=0.0006 (SMER18a), _p_=0.0249 (SMER18b), _p_=0.0167 (SMER18c), _p_=0.0117 (SMER18d), _p_=0.0269 (SMER18e), _p_=0.0165 (SMER18f), _p_=0.0148 (SMER18g), _p_=0.0011 (SMER18h), _p_=0.7369 (SMER18i), _p_=0.0012 (SMER18j), _p_=0.1531 (SMER18k), _p_=0.0006 (SMER18l), _p_=0.0001 (SMER18) (b); _p_=0.0014 (SMER28a), _p_=0.0002 (SMER28b), _p_=0.0001 (SMER28d), _p_=0.0048 (SMER28h), _p_=0.0002 (SMER28i), _p_=0.0162 (SMER28j), p<0.0001 (SMER28c, e-g, k, SMER28) (c). (d-f) The percentage of EGFP-positive cells with EGFP-HDQ74 aggregates in COS-7 cells as in Fig. 1c, treated for 48 h with either DMSO (control), or with 47 μM SMER10 and its analog (SMER10a) (d), 43 μM SMER18 and its analogs (SMER18a, c-h, j, l) (e), or 47 μM SMER28 and its analogs (SMER28a-k) (f), were expressed as odds ratios. All the analogs were used in the cell culture media at 1:400 dilution of 5 mg ml-1 stock solution (in DMSO). Error bars: 95 % confidence interval. _p_=0.003 (SMER10a), _p_=0.004 (SMER10) (d); p<0.0001 (SMER18a, c, d, f, h), _p_=0.009 (SMER18e), _p_=0.001 (SMER18g), _p_=0.382 (SMER18j), _p_=0.067 (SMER18l), _p_=0.031 (SMER18) (e); p<0.0001 (SMER28a, c, e-g, i-k, SMER28), _p_=0.574 (SMER28b), _p_=0.041 (SMER28d), _p_=0.002 (SMER28h) (f). ***, p<0.001; **, p<0.01; *, p<0.05; NS, Non-significant.

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References

    1. Klionsky DJ, Emr SD. Autophagy as a regulated pathway of cellular degradation. Science. 2000;290:1717–1721. - PMC - PubMed
    1. Rubinsztein DC, Gestwicki JE, Murphy LO, Klionsky DJ. Potential therapeutic applications of autophagy. Nat. Rev. Drug Discov. 2007;6:304–312. - PubMed
    1. Ravikumar B, Duden R, Rubinsztein DC. Aggregate-prone proteins with polyglutamine and polyalanine expansions are degraded by autophagy. Hum. Mol. Genet. 2002;11:1107–1117. - PubMed
    1. Ravikumar B, et al. Inhibition of mTOR induces autophagy and reduces toxicity of polyglutamine expansions in fly and mouse models of Huntington disease. Nat. Genet. 2004;36:585–595. - PubMed
    1. Webb JL, Ravikumar B, Atkins J, Skepper JN, Rubinsztein DC. Alpha-Synuclein is degraded by both autophagy and the proteasome. J. Biol. Chem. 2003;278:25009–25013. - PubMed

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