Stress and aging induce distinct polyQ protein aggregation states

Abstract

Many age-related diseases are known to elicit protein misfolding and aggregation. Whereas environmental stressors, such as temperature, oxidative stress, and osmotic stress, can also damage proteins, it is not known whether aging and the environment impact protein folding in the same or different ways. Using polyQ reporters of protein folding in both Caenorhabditis elegans and mammalian cell culture, we show that osmotic stress, but not other proteotoxic stressors, induces rapid (minutes) cytoplasmic polyQ aggregation. Osmotic stress-induced polyQ aggregates could be distinguished from aging-induced polyQ aggregates based on morphological, biophysical, cell biological, and biochemical criteria, suggesting that they are a unique misfolded-protein species. The insulin-like growth factor signaling mutant daf-2, which inhibits age-induced polyQ aggregation and protects C. elegans from stress, did not prevent the formation of stress-induced polyQ aggregates. However, osmotic stress resistance mutants, which genetically activate the osmotic stress response, strongly inhibited the formation of osmotic polyQ aggregates. Our findings show that in vivo, the same protein can adopt distinct aggregation states depending on the initiating stressor and that stress and aging impact the proteome in related but distinct ways.

Fig. 1.

Osmotic stress results in the rapid aggregation of intestinally expressed polyQ proteins at the threshold for activation. (A) Q44-YFP localization in the intestine of young adult animals following a 4-h exposure to 50 mM NaCl (Control), 500 mM NaCl, 1 M sucrose, day 3–4 post-L4 stage adult (Aging), 37 °C (Heat shock), or 1 mM juglone. (Scale bars, 100 μm.) (B) Quantification of the percentage of animals with >10 Q44 aggregates over time following exposure to the indicated stressor. n > 40 animals per condition. Data shown are mean ± SD. (C) (Upper) Collapsed Z stack of images taken through the entire intestine. Dashed lines indicate intestinal boundary. Arrows point to aging aggregates (Left) or osmotic aggregates (Right). (Lower) Single z-plane image from a deconvolved image series from an aging-induced aggregate (Left) or an osmotically induced aggregate (Right). The arrow indicates a fibrillar extension that emanates from the osmotic aggregates. Similar extensions were never observed in aging aggregates. (D) vha-6p::Q44-YFP animals were grown from the L1 stage on either 50 or 200 mM NaCl. Young adults from either group were then transferred to NGM plates with the indicated NaCl concentration, and the percentage of animals with aggregates was quantified after 4 h. n ≥ 40 animals per condition. Data shown are mean ± SD. (E) Representative images of young adult animals expressing the indicated polyQ-length protein exposed to either 50 or 500 mM NaCl for 4 h. (Scale bars, 100 μm.) (F) Quantification of aggregation properties over time following exposure to 500 mM NaCl. n > 40 animals per genotype. Data shown are mean ± SD.

Fig. 2.

Osmotically induced polyQ aggregation is influenced by growth temperature and age. (A) Representative image of worms grown at the indicated temperature and then exposed to 500 mM NaCl for 4 h as first-day adults. (Scale bars, 100 μm.) (B) Percentage of aggregated young adult animals that express intestinal Q44-YFP following exposure to 500 mM NaCl after growth at the indicated temperature. n > 40 animals for each genotype. Data shown are mean ± SD. (C) Percentage of aggregated animals following a 4-h exposure to 500 mM NaCl at the indicated developmental age. Y.A., young adult. n > 40 animals for each genotype. Data shown are mean ± SD. ***P < 0.001.

Fig. 3.

Aging-induced polyQ aggregates can be differentiated from osmotically induced polyQ aggregates. (A) Time-series confocal images of soluble Q44-YFP (Top), aging-induced aggregates (Middle), and osmotically induced aggregates (Bottom). Boxed regions indicate FRAP areas. (Scale bars, 5 μm.) (B) Fluorescence quantification of bleached area in the indicated form of the Q44-YFP protein. Data shown are mean ± SD (soluble, n = 6; aging, n = 8; osmotic, n = 10). (C) Western blot (anti-GFP antibody) of aging and osmotically aggregated Q44-YFP protein extracted with increasing levels of SDS and urea buffers. Aggregates were isolated by lysis of worms in RIPA buffer followed by high-speed ultracentrifugation. The high-speed pellet was then extracted with RIPA buffer containing the indicated SDS levels (for more details, see SI Materials and Methods). The gel is representative of one of four independent experiments. (D) Quantification of the relative fraction of aggregated Q44-YFP protein extracted with low-SDS RIPA buffers (sum of 0.1% and 0.5% fractions), high-SDS RIPA buffers [sum of 1.0%, 1.5%, 2.0% (wt/vol) fractions], and 7 M urea buffer, expressed as a percentage of the total aggregated material (sum of the material in all lanes). Data shown are the mean ± SD from four independent experiments. *P < 0.05, ***P < 0.001, one-way ANOVA with Tukey’s post hoc test. (E) Deconvolved immunofluorescence image of osmotic Q44-YFP aggregate (Upper, 24 h poststress exposure) or aging Q44-YFP aggregate (Lower, 4-d-old animal) stained with a ubiquitin-specific antibody. Arrows indicate the ubiquitin-stained surface of an aging aggregate. Osmotic aggregates failed to stain with ubiquitin antibodies after they were aged for 4 d, suggesting that prolonged incubation times do not result in ubiquitination of osmotic aggregates. (Scale bars, 5 μm.)

Fig. 4.

Osmotic stress-induced Q-length–dependent protein aggregation in HEK293 cells. Nonthreshold Q19 (A and B) or threshold Q72 (C and D) exposed to either isotonic PBS (A and C) or PBS + 200 mM NaCl (B and D) for 3 h. Arrows point to cytoplasmic osmotically induced polyQ aggregates. (Scale bars, 10 μm.) (E) Quantification of the percentage of cells with aggregates (mean ± SD) over 3 h of exposure to 200 mM NaCl. n = 10–12 cell fields per genotype per time point.

Fig. 5.

Genetic activation of the osmotic stress resistance-signaling pathway but not the insulin-like growth factor–signaling pathway prevents the formation of osmotically induced Q44-YFP protein aggregates. (A) Percentage of aggregated young adult animals that express intestinal Q44-YFP following exposure to 500 mM NaCl for wild type and daf-2(e1370). n ≥ 40 animals for each genotype. ***P < 0.001 vs. control. (B) Representative images of wild type and daf-2(e1370) animals expressing intestinal Q44-YFP following 4 h of exposure to either 50 or 500 mM NaCl. (Scale bars, 100 μm.) (C) Percentage of aggregated young adult animals that express intestinal Q44-YFP following exposure to 500 mM NaCl for wild type, osm-7(n1515), and osm-8(n1518). n ≥ 40 animals for each genotype. (D) Representative images of wild type, osm-7(n1515), and osm-8(n1518) animals expressing intestinal Q44-YFP after 4 h of exposure to either 50 or 500 mM NaCl. (Scale bars, 100 μm.)