How to help your worms live longer

A C. elegans mutant that lives twice as long as wild type.

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

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

• 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

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

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.

Videos of wild-type and daf-2 mutant worms

Daf-2 mutants live longer

Daf-2 mutants are more active

• 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.

Longevity requires daf-16

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.

• 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.

Daf-2, Insulin Receptor-like Gene in Worms

Kimura, Tissenbaum, Liu, Ruvkun

• 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).

• 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.

• 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.

Daf-2 regulates fat accumulation

Genes that act downstream of DAF-16 to influence the lifespan

of Caenorhabditis elegans.Murphy CT, McCarroll SA, et al

Kenyon lab, UCSF

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.

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

• 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).

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.

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.

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%

• 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.

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.

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.

Lifespan extension in the fruit fly, Drosophila

A Mutant Drosophila Insulin Receptor Homolog that

Extends Lifespan and Impairs Neuroendocrine Function

Tatar, Kopelman, Epstein

• 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

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.

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.

Lifespan extension in mice

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

Holzenberger, M. et al.

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.

• 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).

• 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.

• 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.

• 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.

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.

• 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.

• 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).

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.

• 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.

• 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.

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

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.

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.

• 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.

Extended lifespan with anti-oxidants

Extension of Lifespan by Overexpression of Superoxide

Dismutase in Drosophila melanogaster

Orr and Sohal

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

• 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

• 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

Mitochondria produce energy (ATP) and free radicals

• SOD converts superoxide anion radical -O2

to peroxide H2O2

• catalase breaks down H2O2 into water and oxygen

Methods

• Created transgenic flies that had extra copies of the SOD and catalase genes

• Compared lifespan to controls

• Compared metabolism, activity to controls

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

Extension of Lifespan with Superoxide

Dismutase/Catalase Mimetics in Worms

Melov, Ravenscroft, Malik et al.

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.

Hypothesis

• Synthetic superoxide dismutase/catalase mimetics can :– Extend lifespan in wildtype worms– Restore lifespan in short-lived worm mutants

that lack mitochondrial SOD.

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

• 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

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

Results: mutant worms

• restored normal lifespan (up 67%)

Conclusions

• Findings are consistent with amelioration of chronic endogenous oxidative stress.

• Mimetics extend lifespan by bolstering natural antioxidant defenses.

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

• 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.

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