Extended LifeSpan 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.
• GPCRs 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
• Im 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 gt100 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
• cyr1mTn - 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 cyr1mTn
  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 cyr1mTn 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 cyr1mTn mutants.

60
Superoxide Dismutase and Survival

• Hypothesis
• Superoxide dismutase plays a role in life
  extension observed in mutants
• cyr1mTn
• 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 gt70 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 cyr1mTn 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 cyr1mTn 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.