How to help your worms live longer

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How to help your worms live longer
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A C. elegans mutant that lives twice as long as
wild type.

• Kenyon C, Chang J, Gensch E, Rudner A, Tabtiang R.

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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

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• 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

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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

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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 worms
  lifespans
• Mutations did not put worms into dauer stage.

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Videos of wild-type and daf-2 mutant worms
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Daf-2 mutants live longer
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Daf-2 mutants are more active
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• 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.

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Longevity requires daf-16
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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.

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• 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.

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Daf-2, Insulin Receptor-like Gene in Worms

• Kimura, Tissenbaum, Liu, Ruvkun

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• 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).

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• Daf-2 is the only member of the insulin receptor
  family in the worms 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.

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• 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.

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Daf-2 regulates fat accumulation
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Genes that act downstream of DAF-16 to influence
the lifespan of Caenorhabditis elegans.

• Murphy CT, McCarroll SA, et al
• Kenyon lab, UCSF

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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.

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

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• 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).

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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.

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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.

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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

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• 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.

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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.

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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.

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Lifespan extension in the fruit fly, Drosophila
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A Mutant Drosophila Insulin Receptor Homolog that
Extends Lifespan and Impairs Neuroendocrine
Function

• Tatar, Kopelman, Epstein

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• 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

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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.

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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.

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Lifespan extension in mice
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Insulin-like growth factor-1 (IGF-1) receptor
regulates lifespan and resistance to oxidative
stress in mice

• Holzenberger, M. et al.

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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.

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• 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).

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• 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.

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• 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.

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• 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.

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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.

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• 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.

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• 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).

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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.

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• 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.

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• 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.

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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

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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.

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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.

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• 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.

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Extended lifespan with anti-oxidants
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Extension of Lifespan by Overexpression of
Superoxide Dismutase in Drosophila melanogaster

• Orr and Sohal

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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

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• 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

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• 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

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Mitochondria produce energy (ATP) and free
radicals
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• SOD converts superoxide anion radical -O2 to
  peroxide H2O2
• catalase breaks down H2O2 into water and oxygen

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Methods

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

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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

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Extension of Lifespan with Superoxide
Dismutase/Catalase Mimetics in Worms

• Melov, Ravenscroft, Malik et al.

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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.

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Hypothesis

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

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Materials

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

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• 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

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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

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Results mutant worms

• restored normal lifespan (up 67)

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Conclusions

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

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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

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• 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.

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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