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Examining the Role of Upstream Regulators on DAF-16 in Aging 

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Albert B. Grandy III, Theodore M. Nelson, PhD.

Department of Human Science, Georgetown University, Washington D.C.

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BACKGROUND

Lifespan ASSAY

RESULTS

TAKE AWAY:

The insulin signaling pathway likely influences lifespan in C. elegans.

CONCLUSIONS

Study Goal: Do ins-18, ins-1, and daf-2 mutations play a role in the life cycle of C. elegans?

AGING

  • Autophagy is the catabolic process where the cell eliminates unnecessary cellular components to maintain energy homeostasis
  • Autophagic activity has been shown to decline with age.

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MODEL ORGANISM

  • C. elegans are used as model organisms
  • Possess homologs around ⅔ of all human disease genes.
  • Short lifespan (20 days).
  • High fertility rates (250 eggs per worm)

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INSULIN SIGNALING

  • The insulin signaling pathway in C. elegans plays a key role in longevity and autophagic gene expression.
  • There are 37 insulin peptides in C. elegans, most of which bind to the receptor daf-2.
  • daf-2 activation inhibits daf-16 expression
  • daf-16 expression induces autophagic gene expression

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A. daf-2 mutants display greatest average longevity

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B. ins-1 mutants have greater longevity compared to wild type and ins-18 mutants

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C. DAF-16 regulated transcripts, ctl2 and sod3, vary in their expression pattern

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  • Lifespan assay of IK, VC, and N2 indicate that ins-1 mutants display greater longevity, but not as much as daf-2 mutants. ins-18 mutants display lower life cycle than the wildtype.

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  • qPCR was used to analyze expression of known DAF-16 transcripts, ctl2 and sod3 across the mutant strains. Gene expression ratios display the target sample gene expression relative to a reference sample, normalized to reference gene. Surprisingly, ins-18 mutants showed higher expression ratios than either ins-1 or daf-2 mutants. Wildtype expression levels are reported as 1.0.

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  • Average lifespan assay of all strains indicate that daf-2 (DR) mutants display the greatest longevity. Results are reported for 5 life cycles.

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  • daf-2 mutants display an increased longevity over time compared to the other strains.
  • ins-18 mutants show less longevity compared to ins-1 mutants
  • ins-18 mutants show higher expression ratios compared to other strains
  • INS-18 mutant is hypothesized to be an antagonist of DAF-2 and INS-1 mutant is an agonist for DAF-2 [see possible mechanism below]
  • However, DAF-16 regulated transcript expression does not match our hypothesis. This result needs to be confirmed.
    • It is possible that longevity is insulin, but not DAF-16 dependent

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