1. Replicative aging in budding yeast cells Dr. Michael McMurray Dept. Molecular & Cell Biology

2. Outline Intro to yeast replicative aging Senescence factors: molecular determinants of yeast longevity and senescence – longevity: how long an individual survives – senescence: the breakdown of normal physiological function with age What can yeast aging tell us about aging in humans?

3. What about simple eukaryotic cells that do express telomerase? Cells of baker’s yeast, Saccharomyces cerevisiae, always express telomerase, and telomeres do not shorten (in normal strains) Microbial populations are “immortal”: they can be passaged/propagated forever Does this mean that these cells are also immortal? Telomere shortening limits replicative lifespan in mammalian cells that don’t express telomerase

4. ? The symmetry of cell division and replicative aging

5. “virgin” cell daughter 1 1st Generation (cell cycle) dead cell (lysis) nth daughter n 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) The Cell Spiral Model of Yeast Aging

6. 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 “Old” cells are very rare in a large, exponentially growing population (1/2 a+1 )

7. What limits yeast lifespan? 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”…

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

9. What is the yeast senescence factor? or, as it turns out: What are the yeast senescence factors? 1)extrachromosomal rDNA circles (ERCs) 2)dysfunctional mitochondria 3)oxidatively damaged proteins

10. 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 Sir2 Sinclair and Guarente Cell 91:1033 (1997)

11. Dysfunctional mitochondria as a senescence factor Only mitochondria with newly-replicated mtDNA segregate to daughter cells; old ones stay in mother Old mother cells show signs of mitochondrial dysfunction and pass on dysfunctional mitochondria to their daughters dysfunctional mitochondria

12. Oxidatively damaged protein is preferentially segregated to mother cell Damaged protein accumulates in aging mother cells Past some threshold age (>10), it is inherited by the daughters of old mothers Is Damaged Protein a Senescence Factor? Aguilaniu, et al. Science 299:1751 (2003)

13. 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: Express telomerase Divide asymmetrically Undergo senescence No ERCs!

14. Human colon stem cells and their progeny accumulate dysfunctional mitochondria cells with normal mitochondria cells with dysfunctional mitochondria (mtDNA) Schon, J. Clin. Invest. 112: 1312 (2003) Young patientOld patient

15. Hernebring, et al. PNAS 103: 7700 (2006) stem cellprogeny cell DNA stem cell marker damaged proteins Mouse stem cells also accumulate damaged proteins, and generate progeny with little damage

16. Calorie Restriction (CR) Extends Lifespan in Yeast and Mammals Decreasing caloric intake (without starvation) increases longevity Works in yeast, flies, rats, mice, worms, … Many reports claim 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 –How could Sir2 work in organisms that lack ERCs? Recent work has shown that in yeast CR may actually be SIR2- (and ERC-) independent –CR decreases levels of oxidatively damaged protein –CR alters metabolism, promotes mitochondrial respiration

17. 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?

18. Genetic instability in aging yeast cells After about 25 divisions, aging mother cells begin to produce daughters that are genetically unstable High rates of mitotic recombination at multiple chromosomes What is the senescence factor responsible for this aging phenotype? Altering ERC levels alters lifespan, but does not accelerate or delay onset of genetic instability (still 25 generations) CR completely prevents genetic instability

19. Conclusions Budding yeast cells are uniquely tractable for aging research Yeast replicative aging involves longevity regulation as well as senescence phenotypes unlinked from longevity The search continues for the senescence factors responsible for yeast aging phenotypes May be a good model for stem cell aging