Extreme-longevity mutations orchestrate silencing of multiple signaling pathways

Abstract

Long-lived mutants provide unique insights into the genetic factors that limit lifespan in wild-type animals. Most mutants and RNA interference targets found to extend life, typically by 1.5- to 2.5-fold, were discovered in C. elegans. Several longevity-assurance pathways are conserved across widely divergent taxa, indicating that mechanisms of lifespan regulation evolved several hundred million years ago. Strong mutations to the C. elegans gene encoding AGE-1/PI3KCS achieve unprecedented longevity by orchestrating the modulation (predominantly silencing) of multiple signaling pathways. This is evident in a profound attenuation of total kinase activity, leading to reduced phosphoprotein content. Mutations to the gene encoding the catalytic subunit of PI3K (phosphatidylinositol 3-kinase) have the potential to modulate all enzymes that depend on its product, PIP3, for membrane tethering or activation by other kinases. Remarkably, strong mutants inactivating PI3K also silence multiple signaling pathways at the transcript level, partially but not entirely mediated by the DAF-16/FOXO transcription factor. Mammals have a relatively large proportion of somatic cells, and survival depends on their replication, whereas somatic cell divisions in nematodes are limited to development and reproductive tissues. Thus, translation of longevity gains from nematodes to mammals requires disentangling the downstream consequences of signaling mutations, to avoid their deleterious consequences.

Figure 1. Survivals of F2 age-1(mg44) homozygotes vs. N2drm controls

Survivals are plotted for age-1(mg44) F2 (squares) and N2drm (circles). (Redrawn from [44]).

Figure 2. Structure of PIP₃

R₁ and R₂ are fatty-acid chains that vary among different molecules.

Figure 3. Protein-kinase activity for endogenous substrates are reduced in age-1(mg44) F2 homozygotes

Day-6 adults were harvested, frozen in liquid nitrogen and ground over dry ice. Kinase activity of cleared, sonicated lysates was assessed by γ-³²P-ATP incorporation per 20 μg protein sample, in 1 min at 30°C. Samples were electrophoresed on gels of 10% acrylamide/SDS. (a) Gel stained with SYPRO Ruby (invitrogen) for total protein. (b) ³²P β-image (Molecular Dynamics Storm) from the gel in a, dried under vacuum. (c) Data summary from 2–3 independent expansions each, of strains N2drm, age-1(hx546), F1 age-1(mg44), F2 age-1(mg44) homozygotes, and daf-16 double mutants with each age-1 allele. (d) Additional controls show that in vitro kinase activity is lower in F2 homozygotes of age-1(mg44), even as day-1 adults, than in F1's at adult days 1 or 6; a second mutation, deleting most of the daf-16 gene, restores age-1(mg44) kinase activity to F1 levels; eggs laid by N2DRM or age-1(mg44) F1 adults are intermediate in kinase activity; and dauer larvae have even higher kinase activity than N2DRM adults. Adapted from [30].

Figure 4. Transcriptional suppression of IIS genes in age-1(mg44)

Transcript levels were assayed by real-time polymerase chain reaction (RT-PCR). Expression histograms are shown superimposed on a schematic diagram of IIS. Yellow arrows show protein phosphorylations (circled P's); orange arrows indicate binding of phosphatidylinositol 3,4,5-triphosphate (PIP3, “structural” symbols). Within each histogram, transcript mean ± SEM (steady-state) is shown on a log(2) scale, comparing wild-type to 4 age-1 mutant groups and to dauer larvae. For each group, fold changes are shown (e.g., “3×”), of age-1 (mg44)-F2 relative to N2drm. Post-gravid age-1(mg44) F1 homozygotes were at adult day 8–9; F2 homozygotes at day 10; N2drm, age1(hx546), and daf-16(mu86); age-1(mg44) double mutants, were all post-gravid adults at adult day 6; and N2drm dauer larvae 1 day after reaching 98% SDS-resistance. Transcript levels are means of 3 independent biological replicates, normalized to the mean of three control gene (β-actin, T08G5.3, and Y71D11.3) that did not differ significantly among strains.

Figure 5. Interactions between IIS and other signal transduction pathways in C. elegans

Figure 6. Three system states of a proposed positive-feedback loop

IIS leads through a series of kinase reactions to phosphorylation of DAF-16, sequestering it in the cytoplasm (reproduction mode). If insulinlike antagonists impede IIS, and/or coactivators reinforce DAF-16, the system switches to longevity mode where DAF-16 prevails and transcriptionally represses its own upstream regulatory kinases – promoting dauer formation in development, or life-extension in the adult. This “flip-flop” circuit, with opposing kinase and transcriptional signals, forms a positive-feedback loop. To recover from the dauer state, reproductive kinases must retain partial function, so favorable signals can shift the balance in their direction. Strong age-1 mutations “fuse the switch” in longevity mode, while conferring a distinctive transcript profile and greatly enhanced survival. Adapted from [30].