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.

51. Experiments To discover the molecular mechanisms of aging in yeast. To determine the role of superoxide dismutases in lifespan extension caused by mutations in Sch9 and cAMP/PKA pathway. To investigate the role of proteins that function upstream of PKA to regulate longevity.

52. Materials and Methods Yeast strains lacking RAS2, SOD2 and MSN2/MSN4. SDC medium with 2% glucose supplemented with amino acids

53. Measurements Determine number of viable cells on day 3 measured in colony forming units (CFU). –Note: A viable cell will reproduce and form a colony in 48 hrs. Compare CFU to protein concentration in diet in a time dependent way. –Note: Should correlate with increased cell damage and lysis. Viability also measured by live/dead fluorescent assay. –(for stationary phase cells) Determine the percentage of live cells by fluorescent microscopy. –(count red/green cells after staining with FUN-1 dye)

54. Determine survival rate in the presence of superoxide agents. –(add paraquat or antimycin A to yeast cultures after 24 hrs.) Determine survival rate in the presence of superoxide inhibitors. –(FCCP & NaCN added at time zero) Oxygen consumption measured. Superoxide dismutase assays performed. Catalase activity assays performed.

55. Experiments and Results

56. The role of Sod2 in lifespan extension Transcription factors: Msn2/Mns4 Gis1 (regulated by Rim 15) activate a variety of stress resistance genes through either: STRE PDS element. SOD2 has promoters containing both STRE & PDS element.

57. Hypothesis: –Sod2 functions downstream from Msn2/Mns4 & Gis1 to promote longevity. Material: –Created mutants with deleted SOD2 in two strains of yeast –cyr1::mTn - usually long-lived –sch9  - usually long-lived (3fold longer lifespan compared to wild type)

58. Results Mutants with deleted sod2 and double mutants sch9/sod2 survived similar to wildtype. –(suggests Sod2 required for usual 3fold longer lifespan of sch9 mutants) Deletion of SOD2 decreased lifespan of cyr1::mTn mutants. –(double mutants sod1/sod2 not studied-both suffer early mortality) Found that SOD2 is expressed at high levels in sch9 mutants

59. An unexpected result Deletion of cyr1::mTn caused low levels of SOD2 mRNA The low levels of SOD2 mRNA may be explained by the early decrease in oxygen consumption rates in these mutants, since the expression of the mitochondrial SOD2 should decrease with the decrease in metabolic rate. This may also explain why the deletion of SOD2 did not abolish the lifespan extension in cyr1::mTn mutants.

60. Superoxide Dismutase and Survival Hypothesis: Superoxide dismutase plays a role in life extension observed in mutants: cyr1::mTn sch9 .

61. Method Measure the chronological lifespan of yeast overexpressing antioxidant enzymes. Deliberately overexpress various combinations of: cytosolic Sod1 mitochondrial Sod2 cytosolic catalase T (CTT1).

62. Results Activity in both Sod1 & Sod2 increased 3fold in SOD1/SOD2 over-expressors. Activity of catalase increased 3fold in catalase expressors. Mean chronological lifespan for SOD1/SOD2 double over-expressors increased 33%. Double overexpression of SOD1 and CTT1 resulted in 10% lifespan extension. Over-expression of SOD1 or SOD2 alone resulted in minor increase in mean survival. Over-expression of cytosolic catalase alone slightly decreased survival.

63. In the SP1 background: CuZnDos, MnSod, and catalase T over- expressed. Over-expression of SOD1 and SOD2 was modest with 10% extension of mean survival. Single over-expression of either SOD1 or SOD2 caused no significant survival. So the results depend on the strain of yeast used (depends on genetic background)

64. Treated wildtype yeast with: FCCP or NaCN which reduce mitochondrial superoxide generation in mammals. Treatment with these superoxide inhibitors increased survival of the wildtype yeast.

65. Survival of Ras Mutants In yeast, Ras1 and Ras2 activate Cyr1, which promotes aging and death. Measured the lifespan of Ras1 and Ras2 deletion mutants. –deletion of Ras1 slightly decreased survival –deletion of Ras2 doubled survival.

66. To confirm role of Ras2 in longevity Tested strains carrying temperature sensitive mutations in the Ras pathway. –(lacking RAS1 and with temp sensitive mutation in RAS2) Again, survival was doubled.

67. Conversely: mutants with constitutionally active Ras2 died early mutant with constitutionally active PKA (bcy1) lived 2 instead of 6 days. Suggests a pathway that includes: –Ras2 –Cyr1 –PKA regulates chronological lifespan.

68. Ras2, Msn2/Msn4, and SOD2 Test whether ras2 mutants are resistant to oxidative stress during aging. Treated mutant strains with superoxide generating agent paraquat. After 7 days of treatment: –Ras2 mutants >70% viable –Wildtype 5% viable

69. Test the role of stress-resistance genes in extended longevity of ras2 mutants. Deleted transcription factors Msn2 and Msn4 in ras2 mutants. The life extension of ras2 was abolished. So Msn2 and Msn4 do mediate longevity extension. Suggests Ras2 and Cyr1 function in the same pathway to: –regulate stress resistance –promote senescence.

70. Test if superoxide dismutases function downstream of Ras2/PKA/Msn2/Msn4 pathway. –Deleted SOD2 in ras2 mutants. Found: –lifespan of ras2  sod2  mutants was shortened –but they outlived wildtype by 30%. Confirms that the induction of other systems is important in survival extension.

71. Test if increasing superoxide protection extends extra lifespan of ras2  mutants. –Over-expressed (ox) both SOD1 and SOD2 in ras2 mutants. Found: –ras2  SOD1oxSOD2ox mutants marginally outlived ras2  mutants. –Indicates that ras2  mutants have optimized their protection against superoxide toxicity.

72. Again, the results of the experiment depend on the particular genetic background of the yeast strain, and on the particular intervention intended to affect aging.

73. Age-dependent Metabolic Rates Test if survival extension is a result of early metabolic decrease. –Measured oxygen consumption in longlived mutants. In wildtype respiration is: –low when cells actively growing –remained high until day 5 or 6. In sch9  mutants the age-dependent rates were the same as wildtype. In ras2  and cyr1::mTn mutants metabolic rates decreased 48 hours earlier.

74. However, in ras2  mutants in the SP1 background rates were the same as wildtype. No significant effects were found for over- expression of: –SOD1 SOD2 –SOD1 CTT1. Suggests that an early decrease in age-specific metabolic rate is: –associated with certain mutations that extend survival –but not required for longevity extension.

75. Discussion Expression of mitochondrial SOD2 is required for extended longevity in yeast. Expression can be caused by mutations that decrease the activity of: –Ras/Cyr1/PKA pathway –and Sch9 pathway. Superoxide toxicity definitely plays a role in yeast aging and death. Yet, SOD2 over-expression is not sufficient for maximum survival.

76. Studies show that genes regulated by: –stress resistance transcription factors –kinases (Msn2, Msn4, Rim15) also mediate chronological lifespan.

77. Extended longevity achieved in single mutants by inducing SOD2 expression. The double expression of SOD1 SOD2 extends survival by 30%. No other mutation achieved this.

78. In longlived mutants the superoxide inactivation/reactivation of aconitase is lower than wildtype. Suggests superoxide promotes aging by –decreasing activity of an essential enzyme –leads to generation of highly reactive free radicals.

79. Paraquat and antyimycin A: –promote damage to mitochondria –reduced lifespan. They lead to the production of superoxides that are: –highly toxic to sod2 mutants –decrease survival of wildtype.

80. Mutations that cause respiratory deficiency also cause early death. This is consistent with aging and death due to mitochondrial: –superoxide levels – loss of function.

81. Other systems are involved. Sod2 is required for survival extension. It is not the only factor involved.

82. For example: the over-expression of SOD1 SOD2 has a smaller effect on lifespan than ras2 sch9.

83. Another example: ras2 and cyr1::mTn mutants lacking SOD2 survive for shorter time than single mutants but survive longer than wildtype.

84. Yeast vs higher organisms There is a similarity in: genes pathways between yeast and higher eukaryotes

85. In yeast Downregulating glucose signaling by: ras2 cyr1 sch9 leads to: increased longevity resistance to oxidative stress resistance to heat shock.

86. In cyr1 mutants chronological lifespan mediated by stress resistance transcription factors: Msn2 Msn4. These factors induce the expression of genes encoding for: heat shock proteins catalase DNA-damage inducing gene DDR2 SOD2.

87. In worms signal transduction genes age-1 daf-2 work through stress resistance factor DAF- 16 to: extend survival 65-100% increase thermotolerance increase antioxidant defense.

88. In yeast, chronological lifespan linked to mitochondrial: –superoxide generation –Sod2. In worms the daf-2 pathway regulates: –heat shock proteins –mitochondrial SOD.

89. In yeast the Ras/Cyr1/PKA pathway downregulates: –glycogen storage –genes inducing diauxic shift.(hypometabolic) In worms the daf-2 pathways regulate: –storage of reserve nutrients (fat & glycogen) –switch to dauer state. (hypometabolic)

90. So yeast and worms regulate stress resistance longevity by modulating the activity of similar proteins and pathways. Analogous pathways in fruit flies and mice regulate stress resistance aging.

91. Conclusions Evidence exists for prosenescence pathways: –activated by glucose (and other nutrients) –downregulated by starvation.

92. The pathways include: Ras2/Cyr1/PKA Sch9. They downregulate stress resistance transcription factors: Msn2 Msn4 Gis1. This results in the downregulation of many stress resistance genes including SOD2.

93. In old yeast the combination of: high respiration rate low protection from superoxides leads to: inactivation of aconitase mitochondrial damage leads to death.

94. Negative or contradictory results may arise when experiments are performed on animals with different genetic backgrounds.