Aging causes decreased resistance to multiple stresses and a failure to activate specific stress response pathways

Abstract

In this work, we examine the relationship between stress resistance and aging. We find that resistance to multiple types of stress peaks during early adulthood and then declines with age. To dissect the underlying mechanisms, we use C. elegans transcriptional reporter strains that measure the activation of different stress responses including: the heat shock response, mitochondrial unfolded protein response, endoplasmic reticulum unfolded protein response, hypoxia response, SKN-1-mediated oxidative stress response, and the DAF-16-mediated stress response. We find that the decline in stress resistance with age is at least partially due to a decreased ability to activate protective mechanisms in response to stress. In contrast, we find that any baseline increase in stress caused by the advancing age is too mild to detectably upregulate any of the stress response pathways. Further exploration of how worms respond to stress with increasing age revealed that the ability to mount a hormetic response to heat stress is also lost with increasing age. Overall, this work demonstrates that resistance to all types of stress declines with age. Based on our data, we speculate that the decrease in stress resistance with advancing age results from a genetically-programmed inactivation of stress response pathways, not accumulation of damage.

Keywords: C. elegans; aging; heat shock response; hormesis; induced thermotolerance; stress resistance.

Figure 1. Stress resistance declines with age

Synchronized populations of wild-type worms were aged to 1, 3, 5, 8 and 12 days of adulthood on plates containing 25 μM FUdR. All ages were assayed together at the same time. Multiple types of stress resistance were measured including (A) osmotic stress, (B) heat stress, (C) anoxia, (D-F) oxidative stress and (G,H) exposure to pathogenic bacteria. For all types of stress measured, peak resistance to stress occurred during early adulthood and declined with age. Error bars indicate SEM. *p<0.05, **p<0.01, ***p<0.001.

Figure 2. Stress-responsive reporters are not activated with increasing age

To determine whether different stress response pathways are activated with increasing age, we examined fluorescence in stress-responsive reporter strains at day 1, day 6 and day 10. None of unfolded protein response reporter strains showed increased activity with age (A-C). The two reporters for the SKN-1 mediated oxidative stress response both showed decreased fluorescence with age (D,E). The hypoxia reporter also showed no increase with age (F). The DAF-16 reporter exhibited increased expression at day 6 (G) but not increased activation as measured by nuclear localization (H,I). Finally, the SOD-3 reporter showed increased fluorescence at both day 6 and 10 (J). This increase appeared to be due to a change in expression pattern with increased expression around the vulva (K). These results suggest that any increase in internal stress that occurs with increasing age is insufficient to activate these stress-responsive pathways and or that the stress-responsive lose their ability to respond with age. Note that the mild increase in fluorescence in Phsp-16.2::GFP and Pnhr-57::GFP worms is due to autofluorescence. Error bars indicate SEM. ns = not significant. *p<0.05, **p<0.01, ***p<0.001.

Figure 3. Specific stress-responsive pathways fail to respond to stress with advancing age

To examine the effect of aging on the ability of specific stress-responsive pathways to become activated, we used fluorescent reporter strains and exposed worms to stress at increasing ages. The Pgst-4::GFP (D,E), Pgcs-1::GFP (F,G) and Phsp-6::GFP (B) reporter strains showed a decreased ability to respond to stress with increasing age such that by day 10 of adulthood there was no induction in response to stress. The Psod-3::GFP (I) reporter strain also failed to respond to stress on day 10 of adulthood. In contrast, the Phsp-16.2::GFP (A), Phsp-4::GFP (C), and Pnhr-57::GFP (H) reporter strains all maintained their ability to respond to stress until day 10 of adulthood. (J) Nuclear localization of DAF-16 could be induced by heat stress (35°C, 2 hours), anoxia (24 hours) and oxidative stress (4 mM PQ, 2 days) at all ages. Both bacterial pathogen exposure (P. aeruginosa) and starvation (2 days) showed a decreased ability to cause nuclear localization of DAF-16 with increasing age. Error bars indicate SEM. *p<0.05, **p<0.01, ***p<0.001.

Figure 4. The ability to respond to stress decreases with age

Worms treated with a mild 35°C heat stress exhibit increased resistance to a subsequent more severe heat stress and increased lifespan. To determine how the ability to respond to stress changes with age, worms were exposed to a mild 35°C heat stress for 2 hours at increasing ages (Day 1, Day 3, Day 5, Day 7, Day 9 and Day 11 of adulthood). The following day worms were exposed to a 37°C heat stress while a separate group was used to measure lifespan. Worms were able to respond to heat stress by increasing resistance to heat and by increasing lifespan until day 7 of adulthood inclusive.

Figure 5. Duration of induced thermotolerance persists until day 8 of adulthood

To determine the duration of induced thermotolerance, worms were exposed to a mild 35°C heat stress for 2 hours on day 1 of adulthood. Resistance to a subsequent 37°C heat stress was then tested at increasing ages (Day 2, Day 4, Day 6, Day 8 and Day 10). Worms treated with a mild heat stress maintain increased resistance to heat stress for at least 8 days. Error bars indicate SEM. *p<0.05, **p<0.01, ***p<0.001.

Figure 6. Activation of heat shock response (HSR), mitochondrial unfolded protein response (mitoUPR) and endoplasmic reticulum unfolded protein response (ER-UPR) lasts for multiple days after induction

Fluorescent reporters strains were used to monitor the activation of the HSR, mitoUPR and ER-UPR following exposure to stress on day 1 of adulthood. Fluorescent reporter activity was monitored daily. (A) The HSR, as measured by the Phsp-16.2::GFP reporter strain, remains activated for at least 5 days following a mild 35°C heat stress for 2 hours. (B) The mitoUPR, as measured by the Phsp-6::GFP reporter strain, remains active for at least 7 days following exposure to 4 mM paraquat for 1 day (oxidative stress). (C) The ER-UPR remains active for only 2 days after exposure to 5 μg/ml tunicamycin for 1 day (ER stress). Error bars indicate SEM. *p<0.05, **p<0.01, ***p<0.001.

Figure 7. DAF-16 is required to maintain induced thermotolerance

Survival under 37°C heat stress and induced thermotolerance was equivalent between daf-16 and WT worms (A). Although daf-16 worms exhibit increased survival under heat stress one day after exposure to a mild 35°C heat stress for 2 hours (C), the induced thermotolerance is lost less than 3 days after the heat stress (C-F). daf-16 worms are able to activate the HSR, mitoUPR and ER-UPR in response to heat, oxidative stress and tunicamycin, respectively, to the same extent as WT worms at day 1 of adulthood (G). As in WT worms, the HSR in daf-16 worms, as measured by Phsp-16.2::GFP reporter activity, remains activated for at least 5 days following a mild 35°C heat stress for 2 hours (H). Also, similar to WT worms, the mitoUPR in daf-16 worms, as measured by Phsp-6::GFP reporter activity, remains active for at least 7 days following exposure to 4 mM paraquat for 1 day (oxidative stress). Error bars indicate SEM. *p<0.05, **p<0.01, ***p<0.001.

Figure 8. HSF-1 is required for induced thermotolerance and full activation of heat shock response and mitochondrial unfolded protein response

While hsf-1 mutant worms show equivalent 37°C heat stress survival compared to WT worms (A), hsf-1 mutants failed to exhibit induced thermotolerance under heat stress after exposure to a mild 35°C heat stress for 2 hours (B-F). hsf-1 mutants are able to increase Phsp-16.2::GFP reporter activity in response to heat stress, but the magnitude of increase is markedly less than WT (G). Similarly, hsf-1 mutants show decreased induction of the Phsp-6::GFP mitoUPR reporter in response to oxidative stress compared to WT worms (G). Once activated, the duration of the HSR, as measured by Phsp-16.2::GFP reporter activity, is reduced in hsf-1 worms compared to WT (H). Similarly, the duration of the mitoUPR in hsf-1 worms is also decreased compared to WT worms after induction with 4 mM PQ (I). Error bars indicate SEM. *p<0.05, **p<0.01, ***p<0.001.