Amyloid-binding compounds maintain protein homeostasis during ageing and extend lifespan

Abstract

Genetic studies indicate that protein homeostasis is a major contributor to metazoan longevity. Collapse of protein homeostasis results in protein misfolding cascades and the accumulation of insoluble protein fibrils and aggregates, such as amyloids. A group of small molecules, traditionally used in histopathology to stain amyloid in tissues, bind protein fibrils and slow aggregation in vitro and in cell culture. We proposed that treating animals with such compounds would promote protein homeostasis in vivo and increase longevity. Here we show that exposure of adult Caenorhabditis elegans to the amyloid-binding dye Thioflavin T (ThT) resulted in a profoundly extended lifespan and slowed ageing. ThT also suppressed pathological features of mutant metastable proteins and human β-amyloid-associated toxicity. These beneficial effects of ThT depend on the protein homeostasis network regulator heat shock factor 1 (HSF-1), the stress resistance and longevity transcription factor SKN-1, molecular chaperones, autophagy and proteosomal functions. Our results demonstrate that pharmacological maintenance of the protein homeostatic network has a profound impact on ageing rates, prompting the development of novel therapeutic interventions against ageing and age-related diseases.

Figure 1. Amyloid-binding compounds extend C. elegans lifespan

a, Dose–response Kaplan–Meier survival curves of synchronously ageing hermaphrodite wild-type (N2) populations exposed to 0 μM (control) to 500 μM ThT at 20 °C. b, Per cent change in median lifespan of N2 populations cultured on 0–500 μM ThT and curcumin. c, ln-linear plot of age-specific mortality rate with age for control and 50 μM ThT-treated C. elegans. d, Effect of 50 μM ThT and 100 μM curcumin on motility of N2 worms evaluated as the mean number of body bends in a 20-s period in 15 individual worms throughout life (upper panel) and after 12 days of treatment (lower panel) with ThT and curcumin. Data are presented as bends min⁻¹ and represent the average of three independent experiments. *P < 0.0001. e–g, Dose–response Kaplan–Meier survival curves of synchronously ageing hermaphrodite N2 populations exposed to 0 μM (control) to 1 μM of BM (e), HBT (f) and HBX (g) at 20 °C. Plots are representative of three independent experiments.

Figure 2. ThT and curcumin rescue a paralysis phenotype and slow protein aggregation in vivo

a, b, The paralysis phenotype associated with protein aggregation is suppressed by 25 μM ThT, 50 μM ThT, 100 μM curcumin in CL4176 (*P < 0.001, **P < 0.0001) expressing Aβ(3–42) (a) and AM140 (*P < 0.05, **P < 0.01) expressing polyQ (b) after 1 and 8 days at 25 °C, respectively. Error bars represent the mean ± s.e.m. of four independent experiments. c, Temperature-sensitive strain HE250 [unc-52(e669su250)II] after 36 h at 25 °C showing the typical paralysis phenotype (left upper panel) and the rescue elicited by 50 μM ThT (right upper panel). Arrows indicate the halos of clearance in the bacterial lawn characteristic of paralysed worms. Lower panel shows protection (± s.e.m.) of the HE250 paralysis phenotype by 50 μM ThT, 100 μM curcumin (Cur) and 100 μM rifampicin (Rif). *P < 0.0001, **P < 0.01. n = 4 independent experiments. d, Perlecan immunolocalization showing disruption/aggregation pattern after 24 h at 25 °C, as compared with worms raised at the permissive temperature (upper panel), and the suppression of disruption by 50 μM ThT treatment. Sixteen of twenty worms showed similar perlecan distribution in three independent experiments. Arrows indicate perlecan aggregates. Scale bar, 30 μm. e, Immunolocalization of aggregation-prone soluble oligomeric protein (A11 antibody, red) and Aβ(3–42) (green) in the presence or absence of 50 μM ThT in CL4176. Scale bar, 10 μm. Error bars represent the mean ± s.e.m., 11 worms per group in 3 independent experiments. *P < 0.0001. f, Immunolocalization of aggregation-prone soluble oligomeric protein (A11 antibody) in the presence or absence of 50 μM ThT and under heat shock (HS) in 11 days old wild-type N2 worms. Scale bar, 20 μm. Error bars represent the mean ± s.e.m., 11 worms per group, of 3 independent experiments. *P < 0.0001. Scale bar, 10 μm.

Figure 3. Dependency of ThT suppression of protein aggregation-associated paralysis on protein homeostasis factors

RNAi by feeding was used to knockdown the expression of genes encoding proteostatic factors in HE250 in the presence or absence of 50 μM ThT and the paralysis phenotype was scored after 36 h. Proportion of worms paralysed is plotted (mean ± s.e.m.). *P < 0.01, **P < 0.001, ***P < 0.0001 versus control vector (CV).

Figure 4. ThT enhancement of lifespan depends on HSF-1 and SKN-1 transcription factors but not on DAF-16

a–c, Effect of 50 μM ThT and 100 μM curcumin on Kaplan–Meier survival curves of synchronously ageing PS3551 [hsf-1(sy441)I] (a), CF1038 [daf-16(mu86)I] (b) and DA465 [eat-2(ad465)II] (c) lifespan. d, Effect of reducing SKN-1 by skn-1 RNAi on the increase in lifespan elicited by ThT. Plots are representative of three independent experiments. e, ThT and curcumin (Cur) treatment does not result in DAF-16::GFP relocalization as compared to control strain [daf-16::daf-16-gfp + rol-6] (upper left). Control strain under heat shock (HS) produces a clear relocalization of DAF-16::GFP to the nuclei of intestinal cells.