Gene activities that mediate increased life span of C. elegans insulin-like signaling mutants - PubMed (original) (raw)

Gene activities that mediate increased life span of C. elegans insulin-like signaling mutants

Andrew V Samuelson et al. Genes Dev. 2007.

Abstract

Genetic and RNA interference (RNAi) screens for life span regulatory genes have revealed that the daf-2 insulin-like signaling pathway plays a major role in Caenorhabditis elegans longevity. This pathway converges on the DAF-16 transcription factor and may regulate life span by controlling the expression of a large number of genes, including free-radical detoxifying genes, stress resistance genes, and pathogen resistance genes. We conducted a genome-wide RNAi screen to identify genes necessary for the extended life span of daf-2 mutants and identified approximately 200 gene inactivations that shorten daf-2 life span. Some of these gene inactivations dramatically shorten daf-2 mutant life span but less dramatically shorten daf-2; daf-16 mutant or wild-type life span. Molecular and behavioral markers for normal aging and for extended life span in low insulin/IGF1 (insulin-like growth factor 1) signaling were assayed to distinguish accelerated aging from general sickness and to examine age-related phenotypes. Detailed demographic analysis, molecular markers of aging, and insulin signaling mutant test strains were used to filter progeric gene inactivations for specific acceleration of aging. Highly represented in the genes that mediate life span extension in the daf-2 mutant are components of endocytotic trafficking of membrane proteins to lysosomes. These gene inactivations disrupt the increased expression of the DAF-16 downstream gene superoxide dismutase sod-3 in a daf-2 mutant, suggesting trafficking between the insulin-like receptor and DAF-16. The activities of these genes may normally decline during aging.

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Figures

Figure 1.

Genetic specificity as a classification tool for RNAi clones shortening the life span of daf-2 mutant animals. (A) Loss of daf-16 shortens N2 life span. Using wild type as a reference strain to define daf-2 pathway specificity would classify daf-16 as outside the daf-2 pathway. Thus, wild-type animals cannot be used to categorize whether a particular gene inactivation functions in the daf-2 pathway. (+) N2 (wild type); control RNAi; (X) daf-2(e1370);daf-16 RNAi; (●) daf-2(e1370);daf-16(mgDf47); control RNAi; (*) N2;daf-16 RNAi. (B) Three RNAi clones all shorten the life span of daf-2(e1370) animals to a similar extent. (◦) daf-2(e1370); control RNAi; (◽) daf-2(e1370);C14A4.9 RNAi; (▵) daf-2(e1370);mep-1 RNAi; (◊) daf-2(e1370);F43D2.1 RNAi. (C) However, these same RNAi clones cause distinct changes in the life span of daf-2(e1370);daf-16(mgDf47) animals. A typical daf-2 pathway-specific gene inactivation shortens the life span of daf-2(e1370) animals but not daf-2(e1370);daf-16(mgDf47) double-mutant animals; e.g., daf-2(e1370);daf-16(mgDf47);C14A4.9 RNAi (◼). A typical gene inactivation functioning in a parallel/converging pathway to daf-2 significantly shortens the life span of daf-2(e1370);daf-16(mgDf47) animals, but to a lesser degree than in daf-2(e1370) animals; e.g., daf-2(e1370);daf-16(mgDf47);mep-1 RNAi (▴). A gene inactivation functioning completely independently from the daf-2 pathway shortens daf-2(e1370) and daf-2(e1370);daf-16(mgDf47) life span to the same degree; e.g., daf-2(e1370);daf-16(mgDf47);F43D2.1 RNAi (◆). (X) daf-2(e1370);daf-16 RNAi; (●) daf-2(e1370);daf-16(mgDf47);control RNAi. (D) Summary for each gene inactivation, comparing the induced life span on daf-2(e1370) (X_-axis) versus daf-2(e1370);daf-16_(mgDf47) (_Y_-axis). For each RNAi clone, the life span is relative to control RNAi in each strain. Pathway specificity: (●) Strictly daf-2; (◦) parallel/converging on daf-2; (●) independent of daf-2. RNAi clones in B and C are marked in red. Gray regions delineate the classes of pathway specificity. (Gray arrow) daf-16 RNAi; (black arrow) empty vector control RNAi.

Figure 2.

A shortened relative life span without changing the proportion of an animal’s life spent active distinguishes gene inactivations that accelerate aging. (A) High proportions of inactivity (white symbols) precede high proportions of mortality (black symbols). Shown are daf-2(e1370) animals treated with dsRNA to an empty vector control (circles) or to daf-16 (squares). Error bars are population-weighted standard deviations. Both Gompertz (solid gray line) and logit (dotted gray line) fits are shown. (B) Data and fits replotted with log(proportion) scale indicates that the rate of transition from active to lethargic movement and the mortality rate are positively correlated. (C) Clones with proportion active significantly <1. A vertical line indicates an arbitrary cutoff (lower 10% by clone rank) to identify sick clones (gray circles); e.g., those with proportion active <0.52. All other clones (black circles) are, by this definition, healthy. While clones are widely distributed by relative life span, they are clustered vertically near the empty vector control clone (black arrow) and daf-16 RNAi clone (gray arrow) in terms of proportion of life spent active. Error bars indicate mean ± SD. Clones with unquantified uncertainty in life span or activity ratio, derived as the quotient of mean active span to mean life span, are also shown (squares).

Figure 3.

Decreased insulin signaling extends life span and decreases the rate of aging. In contrast, loss of daf-16 and many of the progeric gene inactivations described here shortens life span and increases the rate of aging. (A) Pooled mortality proportions for wild type (N2); empty vector control RNAi (◦), N2; daf-16 RNAi (●), and daf-2(e1370);vector control RNAi (◼). Error bars are population-weighted standard deviations. Both Gompertz (dark-gray line) and logit (light-gray line) fits are shown. (B) Data and fits replotted with log(proportion) scale. (C) Eighty-one gene inactivations had MRDT ratios significantly >1 (black circles), implying faster aging than empty vector control (black arrow). An additional 14 gene inactivations cause slower rates of aging (gray circles), implying that they were short-lived due to a higher probability of dying throughout life unrelated to aging (increased IMR). For four gene inactivations the rate of aging showed no significant difference (white circles) relative to empty vector control. Error bars indicate mean ± SD. Histograms show relative count of clones within each bin. Three data sets of daf-16 RNAi (gray arrows) were significant; these clones showed significant variation in MRDT ratio. The increasing variation reflects an increasing steepness in slope (i.e., a very high rate of aging) that is difficult to accurately measure, given that life span is being scored every other day in daf-2(e1370).

Figure 4.

Vesicular trafficking to the lysosome and protein sorting is required for decreased insulin/IGF1 signaling to extend life span. daf-2(e1370) (circles), pdk-1(sa680) (triangles), or daf-2(e1370);daf-16(mgDf47) (squares) animals on dsRNA to empty vector control (white symbols, A_–_R) or to daf-16 (A), CD4.4 (B), C27F2.5 (C), F17C11.8 (D), Y65B4A.3 (E), T27F7.1 (F), rab-7 (G), M57.2 (H), C31H2.1 (I), gdi-1 (J), vps-16 (K), B0303.9 (L), wwp-1 (M), vrk-1 (N), ptr-23 (O), cua-1 (P), Y47G6A.18 (Q), and F30A10.6 (R) (black symbols). (S) Relative life span (to empty vector control) of daf-2(e1370) (_X_-axis) versus pdk-1(sa680) (Y_-axis) for each endocytosis gene inactivation. Empty vector control is indicated by a black arrow, daf-16 RNAi is indicated by a gray circle and gray arrow. (T) Relative life span (to empty vector control) of daf-2(e1370) (X_-axis) versus daf-2(e1370);daf-16(mgDf47) (_Y_-axis) for each endocytosis gene inactivation. Empty vector control is indicated by a black arrow, daf-16 RNAi is indicated by a gray circle and gray arrow.

Figure 5.

Disruption of normal vesicular trafficking to the lysosome blocks sod-3 induction in daf-2 mutant animals. A representative daf- 2(e1370);sod-3_∷_GFP day 7 (adulthood) animal on RNAi to vector (A), daf-16 (B), CD4.4 (C), C27F2.5 (D), F17C11.8 (E), Y65B4A.3 (F), T27F7.1 (G), rab-7 (H), M57.2 (I), C31H2.1 (J), gdi-1 (K), vps-16 (L), B0303.9 (M), wwp-1 (N), vrk-1 (O), ptr-23 (P), and cua-1 (Q). (R) Quantification of average GFP body fluorescence between the nerve ring and vulva. (Light gray) Pooled animals scored between day 2 and 5 of adulthood; (dark gray) pooled animals scored between days 5 and 11.

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Gene activities that mediate increased life span of C. elegans insulin-like signaling mutants - PubMed (original) (raw)