When Old Mothers Go Bad: Replicative aging in budding yeast cells Dr. Michael McMurray Dept. Molecular & Cell Biology

Outline Intro to yeast aging A molecular cause of yeast aging SIR2: A conserved regulator of longevity? Aging and genetic instability, in yeast and humans

Cellular senescence: finite replicative capacity of mitotically dividing cells Originally observed in human diploid fibroblasts (Hayflick limit, 1965) Represents a limit on the number of population doublings Caused by telomere shortening in cells that do not express telomerase

What about simple eukaryotic cells that do express telomerase? Cells of baker’s yeast, Saccharomyces cerevisiae, express telomerase Microbial populations are “immortal”, can be passaged forever Does this mean these cells are also immortal?

The symmetry of cell division and replicative aging ?

The Cell Spiral Model of Yeast Aging Generation (cell cycle) “virgin” cell • Sterility • increased size • wrinkles • bud scars • increased generation time AGING Lifespan = n (20-40) Adapted from Jazwinski, et al Exp Geront 24:423-48 (1989) nth daughtern daughter1 1st dead cell (lysis)

How does the population remain immortal? In every daughter cell, the lifespan “clock” is reset to zero Each division produces a cell that can divide many more times Senescent cells are very rare in a large, exponentially growing population (1/2a+1)

What is the role of telomere length in yeast cellular senescence? Telomerase is expressed throughout the lifespan Telomere length is maintained throughout the lifespan Mutating telomerase does cause cellular senescence: telomere shortening, limited population doublings, genomic instability, ALT

What causes yeast aging? A clue: exceptions to the rule of the resetting clock Occasionally, daughters of old mothers are born prematurely aged! Their lifespan equals the mother’s remaining lifespan The asymmetry has broken down -- accompanied by loss of size asymmetry (“symmetric buds”) The daughters of symmetric buds have normal lifespan Suggests these symmetric buds have inherited a “senescence factor”…

The Yeast Senescence Factor Model (1989) Preferentially segregated to mother cell each division Accumulates to high concentrations in old mothers Eventually inhibits cell division, causes other aging phenotypes Is occasionally inherited by symmetric buds

What is the yeast senescence factor? Some clues (late 1990s): Aging is accompanied by fragmentation of the nucleolus The nucleolus assembles at the site of rRNA transcription, the rDNA Sir2 localizes to the nucleolus, and sir2 mutants have a short lifespan sir2 mutants have high levels of extrachromosomal rDNA circles (ERCs) ERCs have the characteristics of the senescence factor…

Extrachromosomal rDNA Circles as a cause of yeast aging Excised from the chromosomal array by recombination Recombination is suppressed by Sir2 Replicate nearly every cell cycle Have a strong mother segregation bias at mitosis High levels can inhibit cell division Inherited by the daughters of old mothers

But, no ERCs in humans! (or mice, or worms, or flies…) Why continue to study yeast aging? Overexpressing SIR2 homologs in flies and worms extends lifespan Perhaps the regulation of lifespan is conserved (and SIR2-dependent) while the molecular effectors of aging vary between organisms Example: calorie restriction (CR)

Calorie Restriction (CR) Extends Lifespan Decreasing caloric intake (without starvation) lengthens lifespan Works in yeast, flies, rats, mice, worms, … Many reports claimed that the CR pathway is SIR2-dependent, supporting theory of SIR2 as master aging regulator Heated debate over the mechanism by which SIR2 influences CR pathway Recent work has shown that in some yeast strains CR is actually SIR2-independent

Genetic instability and Aging Frequencies of mutations and chromosomal rearrangements increase with age in various organisms Incidence of cancer increases dramatically with age: Is this due to accumulation of genetic events at a constant rate over the lifetime, or does aging itself alter the rate of new genetic events?

Yeast pedigree analysis Separate daughter from mother Instead of discarding, isolate daughters Let daughters form colonies Assay for Loss of Heterozygosity (LOH) Change in rate during lifespan? LOH wildtype mutant

An Age-induced Hyper-recombinational State humans yeast After about 25 divisions, aging mother cells begin to produce daughters that are genetically unstable High rates of LOH at multiple chromosomes LOH is caused by recombination, not chromosome loss or deletion Behaves as a “switch” to a new, unstable state Hyper-recombinational state is eventually “diluted” in progeny of old cells

This is reminiscent of the Yeast Senescence Factor! Something accumulates with each cell division in mother Reaches a threshold, causes genetic instability Inherited by daughters of old mothers Eventually “reset” in distant progeny

Are ERCs the cause? Mutations that increase ERCs (sir2) do not accelerate onset of switch Mutations that decrease ERCs do not delay onset of switch In fact, onset of switch is unlinked to lifespan! Suggests an important distinction between longevity and functional senescence

How does Yeast Aging relate to Cellular Senescence in Humans? Telomere-independent Asymmetrically dividing cells For what cell type is this a model?

Stem cells in human aging and cancer Evidence that stem cells are important in aging and cancer Immunological senescence “Cancer stem cells” Stem cells often express telomerase Stem cells divide asymmetrically

Conclusions Yeast aging involves longevity regulation as well as senescence phenotypes unlinked from longevity Genetic instability increases with age in yeast, by an epigenetic hyper-recombinational switch May be a good model for stem cell aging