Temporal requirements of insulin/IGF-1 signaling for proteotoxicity protection

Abstract

Toxic protein aggregation (proteotoxicity) is a unifying feature in the development of late-onset human neurodegenerative disorders. Reduction of insulin/IGF-1 signaling (IIS), a prominent lifespan, developmental and reproductive regulatory pathway, protects worms from proteotoxicity associated with the aggregation of the Alzheimer's disease-linked Abeta peptide. We utilized transgenic nematodes that express human Abeta and found that late life IIS reduction efficiently protects from Abeta toxicity without affecting development, reproduction or lifespan. To alleviate proteotoxic stress in the animal, the IIS requires heat shock factor (HSF)-1 to modulate a protein disaggregase, while DAF-16 regulates a presumptive active aggregase, raising the question of how these opposing activities could be co-regulated. One possibility is that HSF-1 and DAF-16 have distinct temporal requirements for protection from proteotoxicity. Using a conditional RNAi approach, we found an early requirement for HSF-1 that is distinct from the adult functions of DAF-16 for protection from proteotoxicity. Our data also indicate that late life IIS reduction can protect from proteotoxicity when it can no longer promote longevity, strengthening the prospect that IIS reduction might be a promising strategy for the treatment of neurodegenerative disorders caused by proteotoxicity.

Fig 1

Late life insulin/IGF-1 signaling reduction promotes DAF-16 nuclear localization. (A) DAF-16::GFP expressing worms (strain TJ356) were grown on control bacteria (EV) to either day 1 or 9 of adulthood, and transferred onto daf-2 RNAi bacteria. Green fluorescent protein (GFP) signal was visualized 0, 3 or 6 h after the transfer. Six hours after transfer, GFP signal in worms that were treated during early and late adulthood were concentrated in the nuclei. (B) Day 9 DAF-16::GFP worms were placed on daf-2 RNAi for 6 h, fixed and stained with DAPI. Co-localization of the DAPI and GFP signals (arrows) confirmed the nuclear localization of DAF-16 in day 9 old worms that were fed daf-2 RNAi.

Fig 2

Timing requirements for daf-2 RNAi mediated protection from Aβ proteotoxicity. (A) Aβ worms were transferred from empty vector (EV) bacteria onto daf-2 RNAi bacteria at either day 1, 5 or 9 of adulthood. Paralysis rates decreased upon transfer to daf-2 RNAi compared to EV-grown control worms at all tested ages. (B) Three independent repeats of (A) indicate that the reduction of Aβ toxicity observed in worms transferred at day 9 is reproducible and significant. (C) Lifespan of control Aβ worms grown throughout life on EV bacteria and their counterparts which were transferred from EV onto daf-2 RNAi bacteria at day 9 of adulthood are undistinguishable (mean lifespan: 15.48 and 15.35 days respectively, P_value = 0.772). Lifespan of worms that were transferred from EV bacteria onto daf-2 RNAi at day 5 of adulthood were significantly shorter than these of their counterparts which were grown on daf-2 RNAi throughout life (mean lifespan: 17.04 and 23.74 respectively, P_value = 3.55E-09).

Fig 3

(A, B) In-vitro kinetic aggregation assay reveals that Aβ worm homogenates that were treated with daf-2 RNAi either early (days 1–5 of adulthood) (A) or late (days 9–13 of adulthood) (B) in life had higher Aβ seed content compared to their control untreated age-matched counterparts. (C) Western blot analysis using Aβ monoclonal antibody (6E10) indicated that high-MW Aβ aggregates contents in insoluble fractions (debris) of Aβ worm that were treated with daf-2 RNAi either early (lane 2) or late (lane 4) in life were higher compared to their control untreated age-matched counterparts (lanes 1 and 3 respectively).

Fig 4

Timing requirements for daf-16 and hsf-1 RNAi mediated protection from Aβ proteotoxicity (A) Aβ worms were grown on daf-2 RNAi bacteria throughout life or were transferred to daf-16 RNAi bacteria on either day 1, 5 or 9 of adulthood. Development on daf-2 RNAi did not protect the worms from Aβ mediated paralysis compared to EV and daf-16 RNAi controls. Worm transferred from daf-2 onto daf-16 RNAi at either day 5 or 9 of adulthood were protected for 2 days after exposure to daf-16 RNAi. (B) Aβ worms developed on daf-2 RNAi were transferred onto hsf-1 RNAi at either day 1, 5 or 9 of adulthood. Development on daf-2 RNAi protected the worms from paralysis for 8 days while animals transferred at day 5 were protected for one additional day. All panels display one of three independent experiments.

Fig 5

Timing requirement for reduced insulin/IGF-1 signaling (IIS), daf-16 and hsf-1 to counter Aβ proteotoxicity in the worm. IIS reduction during development has no effect on Aβ proteotoxicity if daf-16 is attenuated at day 1 of adulthood. In contrast, IIS attenuation during reproductive adulthood and midlife protect from Aβ. This protection is associated with Aβ hyper-aggregation and dependent in daf-16. hsf-1 is foremost required for protection from Aβ proteotoxicity during larval development but is also required for a lesser extent during early adulthood and midlife.