Key words and terms

Aging; longevity; C. elegans; DAF-2; insulin/IGF-1 receptor; DAF-16/FOXO; caloric restriction; dauer; stress resistance; antioxidants; chaperones; antimicrobials; regulatory pathways

 

*A note on C. elegans nomenclature: genes are written italicized, in lower-case letters, for example, daf-2.  When we are talking about proteins we use capital letters, for example, DAF-2.

Lecture notes

Introduction

For many years, aging was thought to be a random, entropic process.  Biologists hypothesized that damage accumulated in an unregulated manner in the cells of an organism, leading to its eventual death.  But if this is the case, why is it that animals such as mice, bats, and squirrels, which are of similar size and can live in similar environments, have very different lifespans?  While mice rarely live longer than four years even under protected laboratory conditions, the Eastern Grey Squirrel can live up to 24 years and the longest lived bat species, Myotis brandtii can survive up to 41 years in the wild.  Within the past two decades, research from many labs has led to a new understanding of aging.  This research utilized model organisms such as the nematode Caenorhabditis elegans, the yeast Saccharomyces cerevisiae, and the fruit fly Drosophila melanogaster to show that even single gene mutations can dramatically affect lifespan.  Our lab has used C. elegans to study how genes regulate aging.  Many biological mechanisms controlling other aspects of biology are conserved from C. elegans to mammals and thus our discoveries may be extrapolated to other organisms as well.

 

The DAF-2/insulin/IGF-1 receptor

We found that mutation of a single gene, called daf-2, can double the lifespan of C. elegans.  daf-2 mutants appear healthy and normal when young, just like wild-type animals.  However, at day 13 of adulthood (very old for C. elegans), daf-2 mutants are still healthy and still look young!  This is like a 90-year-old human looking like a 45-year-old.

The daf-2 gene was cloned in Gary Ruvkun’s laboratory and found to encode a hormone receptor.  Since a mutation in daf-2 leads to increased longevity, we know that at least one of the normal functions of daf-2 is to promote or speed up aging.  The DAF-2 receptor is similar to two known hormone receptors found in humans and other mammals:

 

  1. Insulin receptor, which binds to insulin and promotes nutrient uptake into tissues after a meal.
  2. IGF-1 receptor, which promotes growth.

 

Now the big question was, do these receptors and their hormones speed up aging in other organisms besides C. elegans?  The short answer: yes!

 

  1. Fruit flies (Drosophila melanogaster):  mutations in the insulin/IGF-1 receptor as well as other proteins in the same molecular pathway can extend lifespan.
  2. Mice (Mus musculus):  IGF-1 receptor heterozygotes (i.e. they have only one good gene copy) live 20% longer than wild-type mice; Mice missing the insulin receptor in their fat tissue are also long-lived (and don’t get fat on a high-fat diet).
  3. Dogs:  small dogs have a mutation in IGF-1 and live longer than large dogs that don’t have this polymorphism.
  4. Humans: Very recently, DNA variants in a human daf-16-like gene, called FOXO3A, have been associated with exceptional longevity in human cohorts all around the world.

 

 

Can longevity and size be uncoupled?  Does an animal have to be small to be long-lived? No, it appears that a slight reduction in insulin/IGF-1 signaling can increase lifespan without decreasing size. C. elegans with mutations in daf-2 are not small and neither are long-lived flies with modest reduction in insulin/IGF-1 signaling activity. Mice with insulin receptor mutations aren’t small and the long-lived IGF-1 heterozygote mice are only slightly smaller than wild-type.

Since its effects on longevity and size can be uncoupled, perhaps the insulin/IGF-1 receptor works at different times during the life of an animal to control or regulate different processes.  We wanted to find out when the daf-2 gene is needed to control aging in C. elegans.  We did this by turning down the activity of the gene with RNAi treatment.  If C. elegans are exposed to RNAi for their whole lives, they live long.  If C. elegans are subjected to RNAi only during adulthood, they still live long.  However, loss of daf-2 function during development only, does not increase lifespan.  The conclusion: daf-2 acts during adulthood to regulate aging.

 

How does insulin/IGF-1 affect aging?

When a hormone is bound to the DAF-2 receptor, it activates a kinase cascade, resulting in the phosphorylation of the DAF-16 transcription factor.  When this happens, DAF-16, which is a transcription factor required for daf-2 mutants to live long, is excluded from the nucleus.  However, if the DAF-2 receptor or any of the components of the kinase cascade are mutated, DAF-16 is no longer phosphorylated and can thus accumulate in the nucleus.  Here, it regulates the transcription of many genes including antioxidant, chaperone, antimicrobial and metabolic genes.

To find out if these genes are important for lifespan, we used RNAi treatment.  In this way, we could turn down the activity of individual genes and assess their impact on lifespan.  We found that inhibiting the activity of genes positively regulated by DAF-16 shortens the lifespan of long-lived daf-2 mutants.  Likewise, turning down activity of negatively regulated genes lengthens lifespan in wild-type animals.  These effects seem to be additive or cumulative.  For example, RNAi of a gene positively regulated by DAF-16 shortens lifespan in daf-2 mutants, but does not shorten it as much as RNAi of daf-16 itself.  This pathway can be likened to an orchestra.  The individual genes represent the instruments while DAF-16 is like the conductor.  DAF-16 orchestrates the activity of all these individual genes, which then work in concert to regulate aging.

 

What does it all mean?

Why should inhibiting genes necessary for growth and food storage extend lifespan?  After all, failure to produce insulin can lead to diabetes in humans while mice (and flies and worms) without any insulin receptors die before birth.  We hypothesize that when you lower the level of insulin/IGF-1 signaling, the metabolism of the organism shifts from growth and food intake to cellular maintenance and stress resistance.  Indeed, long-lived daf-2 mutants are resistant to many environmental stresses like ultra-violet light, heat, oxidative stress, toxins, and pathogens.

Why should this evolve?  Why aren’t all animals long-lived and resistant to environmental stresses?  One hypothesis is that there are advantages to getting old.  For example, aging may have evolved to eliminate parent-progeny competition.  Another possibility is that these pathways evolved to allow the animal to survive harsh environmental conditions.  In C. elegans we now know that the DAF-2/insulin/IGF-1 signaling pathway not only has effects on aging in the adult, but also much earlier, before puberty, on entry into a larval stage called dauer; if daf-2 activity is turned down slightly, C. elegans are long-lived, but if activity is lowered almost completely, C. elegans will enter the dauer stage before it reaches adulthood.  This stage allows animals to survive for long periods of time in harsh environments with little food.  Thus, it seems that daf-2 has two functions: during development it regulates entry into the dauer stage while during adulthood it regulates aging.  This lifespan module may have evolved not to regulate aging per se, but to allow animals to survive harsh conditions, and survive until conditions become better suited for development and reproduction.  Because dauer formation protects an animal from environmental stress before reproduction, its evolution may have been favored by natural selection.


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