How to help your worms live longer

1. How to help your worms live longer

2. A C. elegans mutant that lives twice as long as wild type. Kenyon C, Chang J, Gensch E, Rudner A, Tabtiang R.

4. Dauer • Dauer is an arrested state in young larvae analogous to hibernation or spore formation. • developmentally arrested, sexually immature • restricted to young larvae (no adults) • induced by food limitation and crowding • worms release a pheromone under these conditions; induces dauer • stress resistant • delays reproduction

5. Can survive long time in dauer state • Physiologic effects similar to CR • When food becomes available, the worms leave the dauer stage, become sexually mature, and have offspring. • Clear survival mechanism

6. Daf-2 (Dauer formation 2) gene • The Daf-2 gene regulates entry into dauer stage. • mediates endocrine signaling, metabolism • increased signaling arrests development in worms inducing a dauer state

7. Daf-2 mutations extend lifespan • Kenyon and colleagues at UCSF found three mutations in the Daf-2 gene (sa189, sa193, and e1370) that greatly extended the worm’s lifespans • Mutations did not put worms into dauer stage.

8. Videos of wild-type and daf-2 mutant worms

9. Daf-2 mutants live longer

10. Daf-2 mutants are more active

11. Mean lifespan for wild type worms was 18 days. • Mean lifespan for daf-2 (sa189) mutant was 42 days. • When all the wild-type animals were dead or immobile, 90% of the daf-2 (e1370) mutants still moved actively. • Daf-2 mutants were not stalled in dauer stage; they became full-size adults that behaved normally, except for slightly smaller than normal brood sizes.

12. Longevity requires daf-16

13. How do daf-2 mutations extend lifespan? • The gene daf-16 acts downstream of daf-2 to promote dauer formation. • Mutations in daf-16 completely block the effects of daf-2 mutations, meaning that a functioning daf-16 is required for extended lifespan. • Identification of C elegans genes that act downstream of daf-16 could lead to a general understanding of how lifespan can be extended.

14. Do humans have a gene like daf-2 that may control lifespan? • Kimura and colleagues determined the DNA sequence of the daf-2 gene, and compared it to other known genes to suggest possible function.

15. Daf-2, Insulin Receptor-like Gene in Worms Kimura, Tissenbaum, Liu, Ruvkun

16. Of human genes, the daf-2 protein is most similar to two closely related human receptors, the insulin receptor (IR) and the insulin-life growth factor receptor (IGF-1R).

17. Daf-2 is the only member of the insulin receptor family in the worm’s genome. • Daf-2 is equally distant from human receptors (35% identical to human) and is probably a homolog of their ancestor. So it may subserve any or all of their functions.

18. Daf-2 and the human insulin receptors both regulate metabolism. • A human diabetic insulin-resistant patient has the same amino acid substitution found in a mutant daf-2. (Pro1178  Leu) • This 14 year old was morbidly obese suggesting that effects of decreased insulin signaling were similar to daf-2 mutants.

20. Daf-2 regulates fat accumulation

21. Genes that act downstream of DAF-16 to influence the lifespan of Caenorhabditis elegans. Murphy CT, McCarroll SA, et al Kenyon lab, UCSF

22. Background • C. elegans normally lives a few weeks, but mutations that decrease insulin/IGF-1 signaling, such as daf-2 insulin/IGF-1 mutants, remain youthful and live twice as long as normal.

23. Daf-2 mutations require functioning daf-16 to extend lifespan.

24. Daf-16 is a FOXO-family transcription factor (regulates expression of other genes) • It should be possible to learn how insulin/IGF-1 signaling influences aging by identifying and characterizing the genes regulated by daf-16. • Animals with reduced daf-2 activity are resistant to oxidative stress, suggesting an increased ability to prevent or repair oxidative damage (damage from free radicals).

25. Methods • Kenyon and colleagues studied gene regulated by daf-16 using two methods: • Microarrays measure gene expression. • RNA interference (RNAi) prevents a gene from producing its corresponding protein, similar in effect to a knock-out.

27. Results • Found two classes of genes • Genes repressed in daf-2 mutants but induced in daf-16 RNAi animals. These are candidates for shortening lifespan. • Genes induced in daf-2 mutants but repressed in daf-16 RNAi animals. These are candidates for genes that extend lifespan.

28. stress response genes • lifespan extension: included genes for increased stress response (genes that prevent or repair damage from free radicals). • Kenyon inactivated these genes using RNAi, and found that lifespan was shortened, up to 20%

30. Some genes protect against bacteria, which the worms eat, but bacteria eventually overwhelm and eat the worm. • Kenyon inactivated these genes using RNAi, and found that lifespan was shortened. • These results confirmed that the genes upregulated by daf-16 promote longer lifespan.

32. Comments from Kenyon • Longevity must have evolved not just once, but many times. • Evolutionary theory postulates that lifespan is determined by the additive effects of many genes, consistent with our findings.

33. Comments from Kenyon • The beauty of the insulin/IGF-1 system is that it provides a way to regulate all of these genes coordinately. • As a consequence, changes in regulatory genes encoding insulin/IGF-1 pathway members or daf-16 homologs could, in principle, allow changes in longevity to occur rapidly during evolution.

34. Lifespan extension in the fruit fly, Drosophila

36. A Mutant Drosophila Insulin Receptor Homolog that Extends Lifespan and Impairs Neuroendocrine Function Tatar, Kopelman, Epstein

37. The gene InR is an insulin-like receptor in fruit flies. • It is homologous to insulin receptors in mammals and to daf-2 in worms. • Studied InR gene variants (alleles) in flies

38. Various allele combinations produce different results • Some had a reduced survival rate • Females in one type extended life span by 85% • Males followed the female pattern in most cases • Not all the InR alleles extend longevity because the gene is highly variable. • Some alleles produced developmental defects that carry over into adults.

39. Conclusions • Specific mutations in the Insulin Receptor InR in flies extend lifespan up to 85%. • The similarities in phenotype suggest that insulin signaling may be central to a common mechanism in several species. • Certainly insulin signaling has an effect on neuroendocrine regulation of metabolism and the reproductive state and their associated affects on aging.

40. Lifespan extension in mice

41. Insulin-like growth factor-1 (IGF-1) receptor regulates lifespan and resistance to oxidative stress in mice Holzenberger, M. et al.

42. Background • Insulin and insulin-like signaling molecules have been linked to longevity in nematode worms and in fruit flys (Drosophila melanogaster). • These molecules include daf-2 and the insulin receptor InR. Mutations that inactivate the protein Chico, which acts downstream of InR, also extend lifespan.

43. Most long-lived daf-2 and InR mutants are also dwarfs with low fertility, but some long-lived InR mutants have normal size and fertility, indicating that longevity may be regulated independently of body size and fertility. • daf-2 and InR are structural homologs of a family of vertebrate receptors that includes the insulin receptor and the insulin-like growth-factor type-1 receptor (IGF-1R).

44. In vertebrates, the insulin receptor regulates glucose metabolism, while IGF-1R promotes growth. IGF-1R is activated by its ligand IGF-1, which is secreted in response to growth hormone. • While is has been demonstrated that the InR family of proteins regulate lifespan in invertebrates, it is not yet clear if InR, IGF-1R, or both regulate lifespan in vertebrates.

45. In mice, inactivation of the growth hormone receptor decreases circulating IGF-1, impairs growth development, and increases lifespan. • Calorie restriction, the only intervention demonstrated to reliably and consistently increase mammalian lifespan, always reduces circulating IGF-1.

46. Oxidative stress causes aging. Mouse and fly mutants that are resistant to oxidative stress are long-lived. • Based on this evidence, Horzenberger et al decided to test the hypothesis that mammalian lifespan is regulated by IGF-1R, and to test the effects of oxidative stress on mice with altered IGF-1R.

47. Methods • Recall that most organisms have two copies of each gene, one inherited from each parent. • Using genetic engineering methods, it is possible to delete or otherwise alter one or both copies of a gene, so that the animal has either one or no working copy of the gene. • A mouse altered in this way is called a "knock-out" mouse.

48. When both copies are knocked out, it is called a homozygous null mutant, or a double knock-out. • An IGF-1R double knock-out is annotated Igf1r-/- • When one copy of IGF-1R is knocked out, it is called a single knock-out, annotated Igf1r+/-. • Horzenberger created Igf1r-/- and Igf1r+/- mice. The double knock-out Igf1r-/- mice did not survive. The single knock-out Igf1r+/- mice survived.

49. The mice were fed as much as they wished to eat of a standard diet and kept in standard housing until their natural death. • Adult mice were treated by injection of paraquat to induce oxidative stress. Paraquat is a herbicide that induces formation of reactive oxygen species (ROS).

50. Results • The single knock-out Igf1r+/- mice lived an average of 26% longer than wild-type mice. • Female Igf1r+/- mice lived an average of 33% longer than wild-type, • Male Igf1r+/- mice lived an average of 16% longer.