Autophagy activator AA-20 improves proteostasis and extends Caenorhabditis elegans lifespan

Abstract

The degradation of cellular components through autophagy is essential for longevity and healthy aging. However, autophagy function decreases with aging, contributing to age-related diseases. In this study, we characterized a small-molecule activator of autophagy called AA-20 that enhances autophagy and lipid droplet clearance in human cells and in the nematode Caenorhabditis elegans. AA-20 reduces polyglutamine aggregation in an autophagy-dependent manner in both human cells and C. elegans, where it also promotes fitness. Consistently, we found that AA-20 extends lifespan in WT C. elegans, but not in autophagy-deficient mutants. Interestingly, our findings suggest that AA-20 acts, at least in part, through a mechanism involving the transcription factor EB, but without inhibiting the protein kinase mammalian target of rapamycin complex 1. Collectively, our results identify an autophagy activator AA-20, which may have potential therapeutic implications for aging-related proteinopathies and lipid storage disorders.

Keywords: C. elegans; autophagy activator; healthspan; lifespan; lipophagy.

Fig. 1.

AA-20 promotes autophagy in mammalian cells. (A) Chemical structure of compound AA-20. Black: 1,3,5-trisubstituted triazine core, blue: 4-methoxyaniline substituents, red: benzothiazole substituent. (B) Representative GFP:RFP ratio plot depicting the impact of AA-20 on general autophagy in RPE-1 cells expressing GFP-LC3-RFP-LC3ΔG. Cells were treated with AA-20 (400 nM to 10 μM) and imaged every 2 h for 72 h. The green/red ratio is normalized at each time point to DMSO (AA-20, n = 3; DMSO, n = 6; refer to Dataset S1 for all data). Error bars indicate SEM. Statistical significance denoted as *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, by two-way ANOVA with Tukey’s multiple comparison test. (C) Confocal micrographs of HeLa cells immunofluorescence stained with anti-LC3B (green) and DAPI (blue). (Scale bar, 10 μm.) White arrowheads indicate LC3B-positive autophagosomes. (D) Bar graph of LC3B-positive puncta per cell quantified using Imaris software’s Spot Detection function (total ~800 cells analyzed, N = 25 images per condition, three biological replicates). HeLa cells were treated with 10 μM AA-20 or DMSO for 20 h, with bafilomycin A1 (BafA) added at the 18th h to block autophagy at the lysosomal acidification step. (E) Confocal micrographs of HeLa cells incubated with Lysotracker Red and costained with DAPI. Cells were treated with or without 10 μM AA-20 and BafA (250 nM; Scale bar, 20 μm). White arrowheads indicate LysoTracker Red-positive lysosomes. (F) Quantification of average LysoTracker intensity per cell using Imaris software. (G) Further quantification of the average number of LysoTracker Red-positive puncta per cell performed using the Spot Detection function in Imaris software. Analysis included ~250 cells across 25 images per condition with three biological replicates. Error bars indicate SEM. Statistical significance denoted as *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, by two-way ANOVA with Tukey’s multiple comparison test. (H) Cathepsin D activity assay in HeLa cells treated with 0.1% DMSO or 10 μM AA-20 for 20 h. Three biological experiments were performed, each with two replicates (error bars represent SEM). *P < 0.05, ***P < 0.001, by one-way ANOVA with Tukey’s multiple comparison test. (I) Relative activity of lysosomal acid lipase (LAL) measured in human fibroblast PCS201–012 primary cells treated with DMSO or 10 μM AA-20 for 72 h (n = 11 to 12, from three independent repeats, error bars represent SEM). ****P < 0.0001, by the unpaired t-test. (J) Relative activity of glucocerebrosidase (GCase) measured in human fibroblast PCS201–012 primary cells treated with AA-20 at 20 μM for 72 h (n = 6, from two independent repeats, error bars represent SEM). n.s. > 0.05, ****P < 0.0001, by ordinary one-way ANOVA.

Fig. 2.

AA-20 enhances autophagy in C. elegans. Autophagy flux was measured in day 4 adult animals expressing rgef-1p::gfp::lgg-1 (A and B) or lgg-1p::gfp::lgg-1 (C–F), treated with vehicle (DMSO) or 5 μM AA-20 from day 1 of adulthood in liquid at 20 °C. Animals were soaked with DMSO or BafA for 2 h before imaging. GFP::LGG-1/Atg8-positive puncta were quantified from three independent experiments in nerve-ring neurons (A and B, N = 18 to 21 animals), proximal intestinal cells (C and D, N = 18 to 28 cells), and body-wall muscle (E and F, N = 15 to 17 animals). (Scale bar, 10 μm.) Error bars represent SEM. **P < 0.01, ***P < 0.001, ****P < 0.0001, by one-way ANOVA with Tukey’s multiple comparison test. White arrowheads indicate GFP::LGG-1-positive autophagosomes. (G and H) Representative images of LysoTracker red positive animals and intensity analysis in day 4 adult WT animals fed with DMSO or 5 μM AA-20 on day 1 of adulthood from three independent repeats (N = 30 to 31 animals). (Scale bar, 500 μm.) (I) Relative activity of cathepsin D measured in day 4 adult WT animals treated with DMSO or 5 μM AA-20 (n = 4, error bars indicate SEM).

Fig. 3.

AA-20 reduces neutral lipid levels in an autophagy-dependent manner in C. elegans. (A) C. elegans stained with ORO and (B) bar graph showing neutral lipid levels in day 5 adult WT (N2) animals, and atg-3(bp412) mutant treated with DMSO or 5 μM AA-20 on day 1 of adulthood (WT-DMSO, N = 52; WT-AA-20, N = 54; atg-3(bp412)-DMSO, N = 34; atg-3(bp412)-AA-20, N = 34, from three biological replicates). Error bars indicate SEM. n.s. > 0.05, *P < 0.05, ***P < 0.001, ****P < 0.0001, by two-way ANOVA with Tukey’s multiple comparison test. (Scale bar, 500 μm.) (C) Animals expressing dhs-3p::dhs-3::gfp. (D) DHS-3 intensity was quantified in day 5 adult animals (WT-DMSO, N = 35; WT-5 μM AA-20, N = 40; atg-3(bp412)-DMSO, N = 32; atg-3(bp412)- 5 μM AA-20, N = 32, from three biological replicates). Error bars indicate SEM. n.s. > 0.05, ****P < 0.0001, by two-way ANOVA with Tukey’s multiple comparison test. (Scale bar, 500 μm.)

Fig. 4.

AA-20 ameliorates protein aggregate overload in both mammalian cells and in C. elegans. PolyQ aggregate loads were quantified in HeLa cells (A and B) and in C. elegans (C–H) after DMSO or AA-20 treatment. Representative confocal fluorescence micrographs of HeLa cells PolyQ-74 (green) (A). (Scale bar, 10 μm.) Cells were transfected with GFP-tagged PolyQ-74 constructs and treated with DMSO or 10 μM AA-20 and further cotreated with either vehicle (DMSO) or BafA for 20 h. GFP-positive Q74 puncta were visualized by confocal microscopy and quantified using Imaris software’s Spot Detection function (B) (total ~45 to 109 cells analyzed, N = 4 to 7 images per condition, from three independent experiments). Error bars represent SEM. ***P < 0.001, by one-way ANOVA with Tukey’s multiple comparison test. White arrowheads indicate GFP-Q74-positive aggregates. (C–H) PolyQ aggregate loads were quantified in C. elegans expressing PolyQ stretches in different tissues after DMSO or 5 μM AA-20 treatment. Representative images of PolyQ in neurons (Scale bar, 10 μm.) (C), intestine (E), and body-wall muscle (G) tissues (Scale bar, 500 μm.) White arrowheads indicate Q40::YFP- or Q35::YFP-positive protein aggregates, respectively. Neuronal PolyQ aggregate numbers were quantified on day 7 of adulthood in WT or atg-3(bp412) animals expressing rgef-1p::Q40::YFP (D) (WT-DMSO, N = 32; WT-5 μM AA-20, N = 38; atg-3(bp412)-DMSO, N = 39; atg-3(bp412)-AA-20, N = 38, from four biological repeats). Intestinal PolyQ numbers were quantified in day 4 adult animals expressing vha-6p::Q44::yfp after treatment with either DMSO or AA-20 on day 1 of adulthood (F) (WT-DMSO, N = 40; WT-5 μM AA-20, N = 40; atg-3(bp412)-DMSO, N = 35; atg-3(bp412)-AA-20, N = 40, from four biological repeats), and muscle PolyQ numbers were quantified in day 4 adult animals expressing unc-54p::Q35::YFP (H) (WT-DMSO, N = 40; WT-5 μM AA-20, N = 39; atg-3(bp412)-DMSO, N = 38; atg-3(bp412)-AA-20, N = 35, from four biological repeats). Error bars represent SEM. n.s. > 0.05, *P < 0.05, ****P < 0.0001, by two-way ANOVA with Tukey’s multiple comparison test.

Fig. 5.

AA-20 promotes healthspan and lifespan in C. elegans. (A) The number of thrashes per minute were measured on day 5, day 7, day 12, and day 14 in WT animals that were fed with DMSO or 5 μM AA-20 on day 1 of adulthood (DMSO, N = 42; 5 μM AA-20, N = 42, from four biological replicates). Error bars indicate SEM. ****P < 0.0001, by two-way ANOVA with Šídák’s multiple comparisons test. (B) Representative DIC images of animals subjected to Smurf assay. (Scale bar, 500 μm.) (C) Quantification of percent animals with blue dye in body cavity during day 10 of WT animals. Day 10 animals fed with DMSO or 5 μM AA-20 on day 1 were soaked in blue food dye for 3 h. Animals containing blue dye leakages from the intestinal lumen into the body cavity were considered Smurf phenotype. Animals that presented no blue dye leakages from intestine to body cavity but contain dye in gonads were censored from the analysis. Data are the mean ± SEM of four biological repeats, each with 34 to 70 animals per condition. *P < 0.05, by the unpaired t-test. (D) Survival percentage of WT (N2, WT) animals subjected to heat shock. Animals were fed with DMSO or 5 μM AA-20 on day 1 of adulthood. On day 5 of adulthood, animals were subjected to a lethal dosage of heat shock at 36 °C for 7 h and survival was calculated for each plate (N = 24 to 25 plates per condition, from five independent experiments). Error bars represent SEM. ****P < 0.0001, by the unpaired t-test. (E) WT animals were bleached and cultured in liquid from L1 onward and DMSO or 5 μM AA-20 were added once during day 1 of adulthood for subsequent lifespan analysis (WT-DMSO, N = 139; WT-AA-20, N = 141 animals). A total of 13 independent repeats were conducted (see Dataset S1 for all data). (F) Lifespan was analyzed for liquid cultured WT animals added once with DMSO or 5 μM AA-20 during day 5 of adulthood (WT-DMSO, N = 158; WT-AA-20, N = 110 animals). Three independent lifespan analyses were conducted (refer to Dataset S2 for data). (G) Lifespan analysis of WT compared with atg-3(bp412) animals at 20 °C subjected to DMSO or 5 μM of AA-20 treatment. Liquid lifespan assay was performed in WT and atg-3(bp412) animals fed once with DMSO or 5 μM AA-20 on day 1 of adulthood (WT-DMSO, N = 35; WT-5 μM AA-20, N = 34; atg-3(bp412)-DMSO, N = 106; atg-3(bp412)- 5 μM AA-20, N = 103 animals). See Dataset S3 for lifespan results of three independent experimental repeats.

Fig. 6.

AA-20 promotes autophagy in C. elegans via a TFEB/HLH-30-dependent mechanism without triggering its transcriptional activation (A) LysoTracker red staining intensity analysis in day 4 adult WT (N2) and hlh-30(tm1978) animals fed with DMSO or 20 μM AA-20 during day 1 of adulthood from three independent repeats (N = 18 to 22 animals). (B) DHS-3 intensity was quantified in day 5 adult animals (WT-DMSO, N = 30; WT-5 μM AA-20, N = 26; hlh-30(tm1978)-DMSO, N = 27; hlh-30(tm1978)- 5 μM AA-20, N = 28, from three biological replicates). (C–E) PolyQ aggregate loads quantification in WT or hlh-30(tm1978) animals expressing PolyQ stretches in different tissues after DMSO or 5 μM AA-20 treatment. Neuronal PolyQ aggregate numbers were quantified on day 7 of adulthood in WT or hlh-30(tm1978) animals expressing rgef-1p::Q40::YFP (C) (WT-DMSO, N = 26; WT-5 μM AA-20, N = 30; hlh-30(tm1978)-DMSO, N = 25; hlh-30(tm1978)-AA-20, N = 25, from four biological repeats). Intestinal PolyQ numbers were quantified in day 4 adult animals expressing vha-6p::Q44::yfp after treatment with either DMSO or AA-20 on day 1 of adulthood (D) (WT-DMSO, N = 31; WT-5 μM AA-20, N = 36; hlh-30(tm1978)-DMSO, N = 41 hlh-30(tm1978)-AA-20, N = 38, from four biological repeats), and muscle PolyQ numbers were quantified in day 4 adult animals expressing unc-54p::Q35::YFP (E) (WT-DMSO, N = 40; WT-5 μM AA-20, N = 35; hlh-30(tm1978)-DMSO, N = 36; hlh-30(tm1978)-AA-20, N = 40, from four biological repeats). Error bars represent SEM. n.s. > 0.05, *P < 0.05, **P < 0.01, ****P < 0.0001, by two-way ANOVA with Tukey’s multiple comparison test. (F) Lifespan was assayed in WT and hlh-30(tm1978) animals treated with 0.05% DMSO or 5 μM AA-20 coated on NGM plates (WT-DMSO, N = 114; WT-AA-20, N = 114; hlh-30(tm1978)-DMSO, N = 105; hlh-30(tm1978)-AA-20, N = 102 animals). P-values by the log-rank test. See Dataset S4 for experimental repeats. (G) Representative images of day 2 adult C. elegans expressing hlh-30p::hlh-30::gfp + rol-6. (Scale bar, 500 μm.) White arrowheads highlight HLH-30::GFP localized in the nuclei. (H) The percentage TFEB/HLH-30 nuclear localization was quantified for the population within each image of C. elegans after 24 h of treatment (DMSO, N = 28; 5 μM AA-20, N = 30 animals) across three independent experiments. Error bars represent SEM. *P < 0.05, ****P < 0.0001, by the unpaired t-test. (I) Expression of TFEB/hlh-30 and putative autophagy-related and lysosomal target genes was measured using RT-qPCR in day 2 adult C. elegans (5 μM AA-20) WT animals raised at 20 °C, from three independent experiments. Error bars represent SEM. *P < 0.05, ****P < 0.0001, by the multiple t-test.