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. 2009 Sep 1;106(35):14914-9.
doi: 10.1073/pnas.0902882106. Epub 2009 Aug 24.

Collapse of proteostasis represents an early molecular event in Caenorhabditis elegans aging

Anat Ben-Zvi  1 , Elizabeth A Miller, Richard I Morimoto
Affiliations

Collapse of proteostasis represents an early molecular event in Caenorhabditis elegans aging

Anat Ben-Zvi et al. Proc Natl Acad Sci U S A. .

Abstract

Protein damage contributes prominently to cellular aging. To address whether this occurs at a specific period during aging or accumulates gradually, we monitored the biochemical, cellular, and physiological properties of folding sensors expressed in different tissues of C. elegans. We observed the age-dependent misfolding and loss of function of diverse proteins harboring temperature-sensitive missense mutations in all somatic tissues at the permissive condition. This widespread failure in proteostasis occurs rapidly at an early stage of adulthood, and coincides with a severely reduced activation of the cytoprotective heat shock response and the unfolded protein response. Enhancing stress responsive factors HSF-1 or DAF-16 suppresses misfolding of these metastable folding sensors and restores the ability of the cell to maintain a functional proteome. This suggests that a compromise in the regulation of proteostatic stress responses occurs early in adulthood and tips the balance between the load of damaged proteins and the proteostasis machinery. We propose that the collapse of proteostasis represents an early molecular event of aging that amplifies protein damage in age-associated diseases of protein conformation.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
The folding and assembly of metastable proteins in muscle cells are perturbed in early adulthood. (A) Percentage of slow-moving, age-synchronized animals expressing WT (squares) or ts (triangles) paramyosin (unc-15) at 15 °C. Data are mean ± SD, >95 animals per data point. (B) Percent of cells with disrupted myofilaments, quantified from confocal images of MYO-3::GFP fluorescence of animals expressing WT (squares) or ts (triangles) paramyosin at 15 °C. Data are mean ± SD, >210 cells per data point. (C) Differential sensitivity of paramyosin to chymotrypsin proteolysis. Total protein extracts of age-synchronized animals were treated for indicated times, and gels were incubated with paramyosin (Top) or dynamin (as an internal control) (Bottom) antibodies. Proteolysis experiments were performed on 3 independent biological samples. Images are from a representative proteolysis experiment. (D) Quantification of proteolyzed paramyosin (fragment 3 + 4) (black) and dynamin (gray) after 1 h of treatment normalized to day 1. (E and F) Confocal images of body-wall muscles. Age-synchronized animals expressing WT or ts paramyosin grown at 15 °C (E) or 25 °C (F) were stained with anti-paramyosin and phalloidin. (Scale bar: 5 μm.) (G) Percentage of slow movement of animals expressing WT (squares) or ts (triangles) myosin (unc-54) as in (A). Data are mean ± SD, >110 animals per data point. (H) Percent of cells with disrupted myofilaments expressing WT (squares) or (triangles) ts myosin as in (B). Data are mean ± SD, >190 cells per data point. (I) Differential sensitivity of myosin(ts) protein to enterokinase digestion as in (C). Gels were exposed to myosin (Top) or dynamin (Bottom) antibodies. (J) Quantification of digested myosin (fragment 1 levels after 7 h digest) (black) or dynamin (gray) from (I) normalized to day 1. (K and L) Confocal images of animals expressing WT or ts myosin grown at 15 °C (K) or 25 °C (L) as in (E). Labels: wt, wild type (WT); ts, time-sensitive.
Fig. 2.
Fig. 2.
Dynamin(ts) function in different tissues declines with age. Percentage of age-synchronized animals, expressing WT (squares) or ts (triangles) dynamin (dyn-1) showing (A) slow movement, (B) SNB-1::GFP mislocalization in neurons, (C) defective GFP uptake in coelomocytes, and (D) dynamin mislocalization. Data are mean ± SD, >45 animals per data point.
Fig. 3.
Fig. 3.
Diverse ts-mutant proteins expressed in multiple tissues lose functionality with age. Percentage of age-synchronized animals expressing WT (squares) or ts (triangles): (A) ras (let-60), (B) gas-1, (C) acetylcholine receptor (unc-63), or (D) perlecan (unc-52) showing (A) osmoregulation (Osm), (B) EtOH sensitivity, (C) levamisole resistance, or (D) paralysis phenotypes. Data are mean ± SD, >45 animals per data point.
Fig. 4.
Fig. 4.
Different stress responses are compromised early in adulthood. mRNA levels of (A) hsp70(C12C8.1), hsp70(F44E5.4), hsp16.11, and hsp16.2, (B) BIP(hsp-4), and (C) hsc70(hsp-1) from age-synchronized WT adults untreated or treated with (A and C) HS (32 °C; 30 min) or (B) UPR (3 mM DTT; 7 h). Data are relative to untreated animals and normalized to day 1 of adulthood-treated animals. Data are mean ± SE, >3 independent biological samples.
Fig. 5.
Fig. 5.
HSF-1 or DAF-16 modulate the age-dependent misfolding of multiple metastable proteins. (A) Age-synchronized (L4) animals expressing paramyosin(ts) [unc-15(e1402)] or dynamin(ts) [dyn-1(ky51)] were transferred to control (L4440), hsf-1, daf-16, or age-1 RNAi-expressing bacteria and scored for slow movement (black) or defective ssGFP uptake (gray) phenotypes respectively. Data are mean ± SD, >92 per RNAi treatment. (B) Age-synchronized (L4) animals expressing ras(ts) [let-60(ga89)] were transferred to control (circles), daf-16 (squares), or hsf-1 (triangles) RNAi-expressing bacteria and scored for percentage of Osm phenotype with age. Data are mean ± SD, >46 per data point. (C) Age-synchronized dynamin(ts) animals overexpressing hsf-1 (triangles) (hsf-1 o/e, strain AM586) or daf-16 (squares) (daf-16 o/e, strain AM707) were scored for percentage of animals showing dynamin mislocalization with age. Dashed line is dynamin(ts) animals for reference. Data are mean ± SD, >45 animals per data point. (D) Age-synchronized ras(ts) animals overexpressing hsf-1 (triangles) (hsf-1 o/e*, strain AM708) or daf-16 (squares) (daf-16 o/e*, strain AM558) were scored for percentage of animals showing Osm phenotype with age. Dashed line is ras(ts) for reference. Data are mean ± SD, >65 animals per data point. The roller phenotype (rol-6) did not affect the behavior scored (we observed 4.5 ± 4.8 compared to 8.9 ± 8.4% Osm phenotype on day 9 of adulthood of rol-6 or WT animals, respectively).
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