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. Murphy CT, McCarroll SA, et al Kenyon lab, UCSF
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. Tatar, Kopelman, Epstein
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
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.

51. Weight at birth and during the first three weeks were the same as in normal (wild-type) mice.
After the weaning period (around 20 days) male Igf1r+/- mice grew slightly less than normal mice, being about 8% smaller at 7 weeks.
Female Igf1r+/- mice were within 6% of the weight of normal mice.
The weight differences affected all tissues and persisted throughout life.

52. The Igf1r+/- mice produced half the normal amount of IGF-1R.
Serum levels of IGF-1 were elevated in adult Igf1r+/- mice, possibly as a response to the low levels of the receptor.

53. The following factors were all normal in the Igf1r+/- mice:
Food intake
Resting metabolic rate
Circadian activity
Body temperature (often lower in other long-lived mutants)
Non-fasting insulin levels
Sexual maturation and litter size

54. Resistance to free radicals
Adult normal and Igf1r+/- mice were treated with paraquat to induce ROS.
Igf1r+/- mice lived longer after paraquat treatment than did normal mice. The relative difference was greater in female than in male Igf1r+/- mice.
Treated mouse embryonic fibroblast cells with peroxide (H2O2) to induce ROS, and found that Igf1r+/- cells survived better than cells from normal mice.

55. Conclusions
These experiments show that a decrease in IGF-1 receptor levels can increase lifespan in a mammalian species.
These results indicate that the link between insulin-like signaling and longevity observed among invertebrates appears to operate in higher vertebrates.

56. The magnitude of the change in lifespan is gender-dependent, consistent with gender-dependent effects seen in Drosophila and long-lived mouse mutants.
It is possible that the life-extending effects of calorie restriction are due to reduced levels of circulating IGF-1, mimicking the IGF-1R reduction in this experiment.

57. Extended lifespan with anti-oxidants

58. Extension of Lifespan by Overexpression of Superoxide Dismutase in Drosophila melanogaster
Orr and Sohal

60. Background
Hypothesis: oxygen free radicals/reactive oxygen species (ROS) cause of aging
Main assumption of this theory is that normal antioxidant defense levels are not sufficient, so that some ROS escape elimination.
ROS cause molecular damage, some of which is irreparable, accumulates with age

61. A direct causal link between ROS and aging has not been established.
If ROS cause aging, then enhanced defense against ROS should
Reduce oxidative stress
Decrease the rate of aging
Extend lifespan

62. Orr and Sohal decided to test the theory.
Examine effects of over-expressing Cu-Zn superoxide dismutase (SOD) and catalase in flies
SOD and catalase are the major defenses against ROS in the mitochondria

63. Mitochondria produce energy (ATP) and free radicals

64. SOD converts superoxide anion radical -O2 to peroxide H2O2
catalase breaks down H2O2 into water and oxygen

65. Methods
Created transgenic flies that had extra copies of the SOD and catalase genes
Compared lifespan to controls
Compared metabolism, activity to controls

66. Results Flies that overexpressed SOD and catalase
Lived ~ 30% longer than controls (median and maximum lifespan)
Had lower levels of damage due to ROS
Had higher metabolic rates at older ages
Had delayed loss of motor ability

67. Melov, Ravenscroft, Malik et al.
Extension of Lifespan with Superoxide Dismutase/Catalase Mimetics in Worms
Melov, Ravenscroft, Malik et al.

68. Background
If ROS contributes to aging, then aging can be slowed by reducing the effects of ROS.
This can be done in 2 ways:
reduce the amount of ROS generated
increase the amount of antioxidant repair activities.
Genetic mutations and manipulations that resist oxidation also extend lifespan.

69. Hypothesis Synthetic superoxide dismutase/catalase mimetics can :
Extend lifespan in wildtype worms
Restore lifespan in short-lived worm mutants that lack mitochondrial SOD.

70. Materials 2 mimetics were tested: Adult worms
EUK-8 (has SOD & catalase-like activity)
EUK-134 (an analog of EUK-8 with more catalase activity).
Adult worms

71. Divide worms into several groups:
untreated wildtype control
worms treated with EUK drugs
short-lived mutants (lacking mitochondrial SOD) .
Introduce varying concentrations of mimetics into the medium.
Mimetics entered worms by ingestion

72. Results: wildtype worms
SOD/catalase mimetic increased lifespan of wildtype 54%.
no overall dose response observed
aging worms eat less, mimetic levels decline
fertility unchanged
body size unchanged

75. Results: mutant worms
restored normal lifespan (up 67%)

76. Conclusions
Findings are consistent with amelioration of chronic endogenous oxidative stress.
Mimetics extend lifespan by bolstering natural antioxidant defenses.

77. Does insulin mediate CR effects?
Lambert and Merry tested if CR benefits were reversed by high insulin levels in rats
Showed that CR decreases insulin levels and decreases free-radical levels in the mitochondria
Showed that artificially increasing insulin levels counteract the reduction of free-radical levels in the mitochondria

78. Do caloric restriction, insulin-family receptors, and reactive oxygen species share related mechanisms in aging?
Probably yes, via increased production or reduced scavenging of free radicals, accompanied by damage to DNA (particularly in the mitochondria) and to other important biomolecules.

79. Are anti-oxidants likely effective anti-aging drugs?
Depends on
Species and strain
Genetic background, pharmacogenomics
Dose
Interaction and co-operation with other steps in the affected pathways
Ability of anti-oxidants to reach mitochondria
Rate of elimination of anti-oxidants from the body