Extended Life-Span and Stress Resistance in Drosophila

1. Extended Life-Span and Stress Resistance in Drosophila Lin, Seroude, Benzer

2. Objective • Find genes in fruit flies that extend lifespan.

3. Method • This study performed a screen for gene mutations that extend lifespan in flies. • Insert P-elements that disrupt gene function • Recover long-lived mutants • Determine which gene the P-element affected.

4. Methuselah gene was isolated • Mutant methuselah (mth) flies outlived parent strain by ~35%. • Mth mutants also have increased resistance to stress: • starvation • high temp • paraquat

5. Mth subjected to stress • Paraquat induces free radicals • mth was resistant to dietary paraquat. • Normal males with a concentration of 20mM are: • sluggish after 12hrs. • 90% dead after 48hrs. • Mth mutant males at same concentration are: • active after 12hrs. • 50% alive after 48hrs. • Longevity and paraquat resistance are associated.

6. Starvation • Mth had a 50% increase in survival time of the parent strain in the starvation test. • Females were more resistant than males. • Larger body weight may help. • In fact, mth mutants outweighed the parent strain by 20-30%.

7. Temperature • 1.Mth survived longer at 36C than parent strain. • mth appear to have higher expression of heat shock proteins and chaperones • Note: daf-2 and age-1 worms had a higher resistance to heat than control worms.

8. What is the Methuselah gene? • mth appears to be a G Protein Coupled Receptors (GPCR) involved in stress response and biological aging. • GPCR’s are involved in an array of activities: • neurotransmission • hormone physiology • drug response • transduction of stimuli (light and odorants)

9. Extended Lifespan Conferred by Cotransporter Gene Mutation in Drosphila Rogina, Reenan, Nilsen, Helfand

10. Methods • Induced mutations in flies • Identified long-lived mutations • Isolated gene responsible: • I’m Not Dead Yet (INDY)

11. Test in other fly strains • Indy mutations were crossed into several other stocks that were isolated 20-30 yrs ago. • Results showed an extension of lifespan of 40-80%. (only 15% in Luckinbill stock) • So Indy extends life directly. • Note: Indy even extended lifespan of selected long lived lines by a small margin

12. Fertility • Need to confirm that lifespan extention not caused by a drop in fertility. • Compared to control flies, Indy flies were normal or superior in fertility

13. Activity • Need to confirm that lifespan extention not caused by a drop in physical activity. • No significant differences were found in: • flight • courtship • feeding behavior • No differences found between Indy and Controls in early life. • Indy maintained behavioral and locomotor activities at high levels for much longer.

14. When Indy is mildly reduced it extends lifespan. • A further reduction leads to less dramatic extension. (additional 10-20% increase) • Created flies with a single copy of Indy and no normal copy: • Indy activity was reduced. • Lifespan was shortened 10-20%.

15. Indy appears to be involved in intermediary metabolism and may represent a new class of longevity gene. • The genetically induced reduction of dicarboxylic acid cotransporter in the Indy mutants may be creating a state similar to CR.

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

17. 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

18. 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.

19. 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.

20. IGF-1 receptor regulates lifespan and resistance to oxidative stress in mice Holzenberger, M. et al.

21. 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.

22. 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).

23. 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.

24. 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.

25. 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.

26. 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.

27. 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.

28. 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).

29. 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.

30. 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.

31. 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.

32. 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

33. 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.

34. 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.

35. 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.

36. SOD2 Functions Downstream of Sch9 to Extend Longevity in Yeast Fabrizio, Liou, Moy

37. Molecular dissection of aging gene pathways • Want to identify all genes that affect aging: • these are potential drug targets • find genes are "drugable" • Want to understand connections between genes (pathways), so that we can understand: • the mode of action of the gene, • potential side effects of drugs that target the gene, • how the desired drug effect may be circumvented or blocked

38. This paper describes the molecular dissection of an aging pathway, and illustrates the methods used to understand molecular pathways. • Illustrates how seemingly negative and contradictory results may arise

39. Introduction • Yeast cells with ample nutrients typically: • divide rapidly • quickly become overcrowded • then spend the rest of their lives in a stationary phase.

40. Lifespan depends on the food source • Yeast fed SDC (synthetic dextrose complete medium): • reach max viability in 48 hrs • reach max population density by 72 hrs • survive for about 6 days. • respiratory rate remains high for most of lifespan.

41. Yeast fed YPD (rich glucose medium): • grow rapidly (by fermentation) • with overcrowding: • continue to grow in size for some time • decrease metabolic rate • decrease macromolecular synthesis by >100 times. • survive for months slowly utilizing reserve nutrients.

42. Measure lifespan in two ways in yeast • chronological lifespan: days of life • budding lifespan: number of buds generated by a mother cell

43. Chronological lifespan in yeast is: • shortened by: • null mutations in either or both superoxide dismutases • extended by: • 1. overexpression of human oncoprotein Bcl-2, (protects against oxidative stress) • 2. mutations that reduce activity of: • adenylate cyclase (Cyr1) • serine threonine kinase (Sch9).

44. Cyr1 and Sch9 are genes that function in pathways that: • mediate glucose dependent signalling • stimulate growth and glycolysis • decrease stress resistance.

45. Longevity in Cyr1 and Sch9 mutants requires stress-resistance transcription factors: • Msn2 • Msn4 • Rim 15 protease kinase. • Suggests that investing in protection and repair slows aging

46. The super-oxide sensitive enzyme aconitase • The age-dependent inactivation of the super-oxide sensitive enzyme aconitase, which is high in wild-type cells, is decreased in mutations that extend longevity. • Stress resistance proteins appear to have no role in replicative longevity (because deletion of stress resistant transcription factors Msn2/Msn4 has no effect)

47. G-proteins Ras1 and Ras2 function upstream of Cyr1. • They have overlapping roles in: • growth • stress resistance.

48. Msn2 & Msn4 are required for longevity in Cyr1 mutants • regulate genes with a stress response element (STRE) in their promoters. • Among the genes they regulate are those encoding: • heat shock proteins • catalase (CTT1) • DNA-damage-inducing gene (DDR2) • genes involved in storage of nutrients.

49. Msn2 & Msn4 may also regulate SOD. • SOD promoters have a stress response (STRE) sequence.

50. Fabrizio and colleagues performed several experiments to elucidate the molecular mechanisms of aging and death in yeast. • In particular, they looked at the role of superoxide dismutases in relation to mutations in known aging-related genes in yeast.