Skip to main page content
U.S. flag
See More

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2017 Jan 10;18(2):454-467.
doi: 10.1016/j.celrep.2016.12.033.

Reduced Insulin/IGF-1 Signaling Restores the Dynamic Properties of Key Stress Granule Proteins during Aging

Marie C Lechler  1 , Emily D Crawford  2 , Nicole Groh  1 , Katja Widmaier  2 , Raimund Jung  2 , Janine Kirstein  3 , Jonathan C Trinidad  4 , Alma L Burlingame  4 , Della C David  5
Affiliations

Reduced Insulin/IGF-1 Signaling Restores the Dynamic Properties of Key Stress Granule Proteins during Aging

Marie C Lechler et al. Cell Rep. .

Abstract

Low-complexity "prion-like" domains in key RNA-binding proteins (RBPs) mediate the reversible assembly of RNA granules. Individual RBPs harboring these domains have been linked to specific neurodegenerative diseases. Although their aggregation in neurodegeneration has been extensively characterized, it remains unknown how the process of aging disturbs RBP dynamics. We show that a wide variety of RNA granule components, including stress granule proteins, become highly insoluble with age in C. elegans and that reduced insulin/insulin-like growth factor 1 (IGF-1) daf-2 receptor signaling efficiently prevents their aggregation. Importantly, stress-granule-related RBP aggregates are associated with reduced fitness. We show that heat shock transcription factor 1 (HSF-1) is a main regulator of stress-granule-related RBP aggregation in both young and aged animals. During aging, increasing DAF-16 activity restores dynamic stress-granule-related RBPs, partly by decreasing the buildup of other misfolded proteins that seed RBP aggregation. Longevity-associated mechanisms found to maintain dynamic RBPs during aging could be relevant for neurodegenerative diseases.

Keywords: Caenorhabditis elegans; DAF-2; HSF-1; RNA-binding proteins; aging; longevity; neurodegenerative diseases; protein aggregation; stress granules.

PubMed Disclaimer

Figures

None
Graphical abstract
Figure 1
Figure 1
RNA Granule Components No Longer Aggregate in Long-Lived Animals with Reduced daf-2 Signaling (A) Distribution of fold changes with age in insolubility for 260 proteins (n = 3, biological replicates). Fold changes are measured by iTRAQ quantification. (B) Flowchart describing the segregation of insolubility fold changes with age into different groups for analysis. (C) Proteins with reduced aggregation in daf-2 RNAi conditions and U2OS proteins precipitated by b-isox are enriched in aliphatic amino acids. Unequal variance t test: proteins aggregating more with age in long-lived animals (n = 43, >1.2-fold), p = 0.04; proteins aggregating less with age in long-lived animals (n = 81, <0.83-fold), p = 6.1E-15; proteins precipitated by b-isox (n = 126), p = 1.3E-9. (D) Proteins that aggregate in both control and daf-2 RNAi conditions are enriched in extended stretches of β-sheet propensity. Unequal variance t test: proteins aggregating more with age in long-lived animals (n = 43, >1.2-fold), p = 0.002; proteins aggregating less with age in long-lived animals (n = 81, <0.83-fold), p = 0.09; proteins precipitated by b-isox (n = 126), p = 0.6. (E) Large SDS-insoluble aggregates precipitated by 20,000 × g. Immunoblots detecting RBPs PAB-1, FIB-1, HRP-1 (unspecific band noted by asterisk), and CAR-1 (truncated band marked by asterisk). Relevant protein bands are indicated by red arrow. (F) Flowchart showing a high overlap between proteins precipitated by b-isox and aggregation-prone proteins in C. elegans, in particular those no longer aggregating in daf-2(−) conditions. See also Figures S1, S2, and S3 and Tables S1, S2, S3, and S4.
Figure 2
Figure 2
Stress-Granule-Related RBPs PAB-1 and TIAR-2 Aggregate with Age in C. elegans (A) tagRFP::PAB-1 expressed in the pharyngeal muscles forms stress granules upon heat stress (2 hr, 32°C) on day 1 of adulthood. No stress granules are visible after recovery (+24 hr). Scale bars: z stack projection, 15 μm; single-plane insets, 5 μm. (B) tagRFP::PAB-1 distribution changes from a diffuse pattern in young animals to a punctate pattern in aged worms. Scale bars: z stack projection, 15 μm; single-plane insets, 5 μm. (C) Venus::TIAR-2 expressed in the pharyngeal muscles forms stress granules upon heat stress (2 hr, 32°C) on day 1 of adulthood. No stress granules are visible after recovery (+24 hr). Scale bars: z stack projection, 15 μm; single-plane insets, 5 μm. (D) Venus::TIAR-2 accumulates mainly in stress-granule-like puncta with age. Scale bars: z stack projection, 15 μm; single-plane insets, 5 μm. (E) Increased tagRFP::PAB-1 aggregation with age in a population of C. elegans. Day 5 or 8 versus day 2: ∗∗∗∗p < 0.0001. (F) Increased Venus::TIAR-2 aggregation with age in a population of C. elegans. Day 7 versus day 2: ∗∗p < 0.01. (G) tagRFP::PAB-1 co-localizes with stress-granule-like Venus::TIAR-2 puncta in double-transgenic animals. Representative single-plane images. Scale bar, 7 μm. See also Figure S4.
Figure 3
Figure 3
TIAR-2 and PAB-1 Accumulate in Stress-Granule-like Puncta and Large Immobile Puncta in Aged C. elegans (A) Small Venus::TIAR-2 puncta formed with age are similar to stress granules formed during heat stress. Representative single-plane images and masks of puncta for size quantification. Scale bars, 5 μm. (B) Size quantification of Venus::TIAR-2 puncta from masks of representative single-plane images in (A). (C) Representative single-plane images and masks of puncta for size quantification showing large tagRFP::PAB-1 puncta formed with age compared with stress granules assembled during heat stress. Scale bars, 5 μm. (D) Size quantification of tagRFP::PAB-1 puncta from masks of representative single-plane images in (C). Puncta larger than 0.5 μm2 were considered as “large” puncta. (E) Representative immobile tagRFP::PAB-1 puncta at day 11 assayed by FRAP. Bleached area is marked by white box. Scale bar, 4 μm. (F) Representative immobile Venus::TIAR-2 puncta at day 8 assayed by FRAP. Bleached area is marked by white box. Scale bar, 2 μm. (G) FRAP analysis of immobile tagRFP::PAB-1 puncta present in aged worms (days 11–12). Quantification of relative fluorescence intensity (RFI) over time. Number of animals = 6, puncta evaluated = 6, mean ± SD is represented. (H) Venus::TIAR-2 puncta monitored by FRAP were highly immobile both in young (day 2) and in aged (day 8) animals. In both young and aged animals: animals = 5, puncta evaluated = 5, mean ± SD is represented. (I) Quantification of FRAP results shows increased immobility of tagRFP::PAB-1 puncta with age. Twenty percent of tagRFP::PAB-1 puncta present in young worms (day 2) were immobile (number of animals = 18, puncta evaluated = 57) compared with 51% in aged worms (days 11–15) (number of animals = 18, puncta evaluated = 55).
Figure 4
Figure 4
Aggregation of Stress Granule Component PAB-1 with Age Is Associated with Reduced Fitness (A) Animals with tagRFP::PAB-1 aggregation are significantly smaller than animals without aggregation (day 7, p < 0.0001). Data are represented with Tukey-style box plots and mean indicated by + (animals without aggregation n = 99, with aggregation n = 125). See also Figure S5B. (B) High levels of tagRFP::PAB-1 aggregation are associated with reduced survival. Survival curve of Pmyo-2::tagRFP::PAB-1 animals grown at 20°C until day 7, sorted by their aggregation levels at day 7, and then transferred to 25°C (repeat 1: p = 0.029; see Table S5). See also Figure S5, Table S5, and Movie S1.
Figure 5
Figure 5
HSF-1 Activity during Development Protects against PAB-1 Aggregation in Adulthood in daf-2 Mutant and Wild-Type Adults (A) Delayed tagRFP::PAB-1 aggregation with age in daf-2 mutant background. Days 5 and 7, daf-2(−) versus wild-type background: ∗∗∗∗p < 0.0001. (B) Delayed Venus::TIAR-2 aggregation with age in daf-2 mutant background. Days 1, 4 and 7, daf-2(−) versus wild-type background: ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗∗p < 0.0001, respectively. (C) Levels of tagRFP::PAB-1 aggregation are highly increased at all ages examined in hsf-1(−); daf-2(−) animals compared with daf-2(−) animals. DAF-16 moderately protects against tagRFP::PAB-1 aggregation at day 11. daf-2(−) compared with hsf-1(−);daf-2(−): ∗∗∗∗p < 0.0001; daf-2(−) compared with daf-16(−);daf-2(−): ∗p = 0.02. (D) Head regions of representative animals expressing Pmyo-2::tagRFP::PAB-1 in daf-2(−), hsf-1(−); daf-2 (−) and daf-16(−); daf-2(−) mutants at day 11. Scale bars: z stack projection, 15 μm; single-plane zoom, 5 μm. (E) hsf-1 mutation alone increases tagRFP::PAB-1 aggregation dramatically, even at day 2. ∗∗∗∗p < 0.0001. (F) HSF-1 activity during development is essential in order to delay tagRFP::PAB-1 aggregation (control: L4440 empty vector). Days 1 and 2 with RNAi treatment from egg, ∗∗∗∗p < 0.0001. (G) Delayed KIN-19::mEOS (monomeric EOS) aggregation with age in daf-2 mutants is dependent on DAF-16, but not on HSF-1. daf-2(−) compared with hsf-1(−); daf-2(−): day 2, ∗∗p = 0.0049; day 6, ∗p = 0.01. daf-2(−) compared with daf-16(−);daf-2(−): ∗∗∗p = 0.0003, day 10. See also Figure S6 and Table S6.
Figure 6
Figure 6
PAB-1 Aggregation Is Accelerated by KIN-19 (A) tagRFP::PAB-1 co-localizes with KIN-19::Venus in large aggregates in double-transgenic animals. Representative head region displayed in 3D. Scale bars, 10 μm; overlay and zoom scale bar, 5 μm. (B) Representative immobile mixed tagRFP::PAB-1 (magenta) and KIN-19::Venus (yellow) puncta at day 7 assayed by FRAP. Bleached area is marked by white box. Scale bar, 2 μm. (C) Accelerated tagRFP::PAB-1 aggregation in the anterior pharyngeal bulb in double transgenics compared with single transgenics. Days 2, 4, and 7: ∗∗∗∗p < 0.0001. (D) Moderately reduced KIN-19::Venus aggregation in the anterior pharyngeal bulb in double transgenics compared with single transgenics. Day 7: ∗∗p = 0.0083. (E) No significant increase of tagRFP::PAB-1 aggregation in the presence of mEOS2 overexpression. At all ages, p > 0.05. See also Figure S6.
Figure 7
Figure 7
Other Longevity Pathways Prevent PAB-1 Aggregation with Age (A) Dietary restriction delays tagRFP::PAB-1 aggregation with age. Day 8, eat-2(−) versus wild-type background: ∗∗∗p < 0.001. (B) Inhibition of mitochondrial function by cyc-1 RNAi halts tagRFP::PAB-1 aggregation. Days 1 and 8, cyc-1 RNAi versus control: ∗p < 0.05 and ∗∗∗∗p < 0.0001, respectively.
See this image and copyright information in PMC

References

    1. Alberti S., Halfmann R., King O., Kapila A., Lindquist S. A systematic survey identifies prions and illuminates sequence features of prionogenic proteins. Cell. 2009;137:146–158. - PMC - PubMed
    1. Arai T., Hasegawa M., Akiyama H., Ikeda K., Nonaka T., Mori H., Mann D., Tsuchiya K., Yoshida M., Hashizume Y., Oda T. TDP-43 is a component of ubiquitin-positive tau-negative inclusions in frontotemporal lobar degeneration and amyotrophic lateral sclerosis. Biochem. Biophys. Res. Commun. 2006;351:602–611. - PubMed
    1. Aulas A., Vande Velde C. Alterations in stress granule dynamics driven by TDP-43 and FUS: a link to pathological inclusions in ALS? Front. Cell. Neurosci. 2015;9:423. - PMC - PubMed
    1. Ayyadevara S., Mercanti F., Wang X., Mackintosh S.G., Tackett A.J., Prayaga S.V., Romeo F., Shmookler Reis R.J., Mehta J.L. Age- and hypertension-associated protein aggregates in mouse heart have similar proteomic profiles. Hypertension. 2016;67:1006–1013. - PMC - PubMed
    1. Balch W.E., Morimoto R.I., Dillin A., Kelly J.W. Adapting proteostasis for disease intervention. Science. 2008;319:916–919. - PubMed

Publication types