Eukaryotic organisms age, yet detrimental age-associated traits are not passed on to progeny. How life span is reset from one generation to the next is not known. We show that in budding yeast resetting of life span occurs during gametogenesis. Gametes (spores) generated by aged cells show the same replicative potential as gametes generated by young cells. Age-associated damage is no longer detectable in mature gametes. Furthermore, transient induction of a transcription factor essential for later stages of gametogenesis extends the replicative life span of aged cells. Our results indicate that gamete formation brings about rejuvenation by eliminating age-induced cellular damage.National Institutes of Health (U.S.) (Grant GM62207

Amon, Angelika B

Kinde, B.

Unal, Elcin

Science

PubMed

When yeast commits to sex, reversal of age-induced cellular damage rejuvenates its life span.</jats:p

Elçin Ünal

Benyam Kinde

Angelika Amon

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Gametogenesis Eliminates Age-Induced Cellular Damage and Resets Life Span in Yeast

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Gametogenesis eliminates age-induced cellular damage and resets life span in yeast

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Gametogenesis eliminates age-induced cellular damage andresets lifespan in yeastElçin Ünal1, Benyam Kinde2,3, and Angelika Amon11David H. Koch Institute for Integrative Cancer Research, Howard Hughes Medical Institute,Massachusetts Institute of Technology, E76-543, 500 Main Street, Cambridge, MA 02139, USA2Howard Hughes Medical Institute Exceptional Research Opportunities Program (HHMI-EXROP)AbstractEukaryotic organisms age, yet detrimental age-associated traits are not passed on to progeny. Howlifespan is reset from one generation to the next is not known. We show that in budding yeastresetting of lifespan occurs during gametogenesis. Gametes (spores) generated by aged cells showthe same replicative potential as gametes generated by young cells. Age-associated damage is nolonger detectable in mature gametes. Furthermore, transient induction of a transcription factoressential for later stages of gametogenesis extends the replicative lifespan of aged cells. Ourresults indicate that gamete formation brings about rejuvenation by eliminating age-inducedcellular damage.Most if not all eukaryotic organisms age, however, the age-induced changes are nottransmitted to the progeny. How lifespan is reset from one generation to the next is notknown. We wished to test the hypothesis that resetting of lifespan occurs duringgametogenesis. In budding yeast, gamete formation (sporulation) requires meiosis andincludes the generation of new membrane compartments, protein and organelle degradation,and synthesis of a resistant spore wall (1). To determine whether gamete formation causesrejuvenation, we asked whether spores derived from aged cells have reset their lifespan andare young or whether they inherit the progenitor’s age and remain old. We isolatedreplicatively aged cells based on biotin labeling of mother cells (2) and induced them tosporulate in the same flask as young cells (Fig.S1). Upon sporulation, tetrads were dissectedand the replicative lifespan (RLS) of each spore was measured. We found that the lifespansof the spores derived from young and aged cells were indistinguishable in twoSaccharomyces cerevisiae strain backgrounds: W303, in which sporulation efficiencydecreases with age ((3,4); Fig.1B) and A364a, in which sporulation efficiency remains highdespite aging ((4);Fig.S2). In contrast, aged cells obtained by the same procedure but notinduced to sporulate die rapidly (Fig.1A). The RLS of the four spores from a tetrad producedfrom young and aged cells is the same; no statistically significant differences are observed((4); Fig.1C). This is in contrast to mitosis, where age is asymmetrically inherited betweenthe mother cell and the bud, culminating in the production of a young daughter and an oldmother cell (5). Thus, sporulation resets RLS.We next asked how sporulation affects age dependent cellular changes such as increasedlevels of protein aggregation (6), aberrant nucleolar structures and increased levels of extraCorrespondence and requests for materials should be addressed to A.A. (angelika@mit.edu).3current address: Harvard Medical School/ Massachusetts Institute of Technology MD-PhD Program, 260 Longwood Avenue,Boston, MA 02115The authors declare no competing financial interests.NIH Public AccessAuthor ManuscriptScience. Author manuscript; available in PMC 2014 February 13.Published in final edited form as:Science. 2011 June 24; 332(6037): 1554–1557. doi:10.1126/science.1204349.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author Manuscriptchromosomal ribosomal DNA circles (ERCs) (7,8). In budding yeast, protein aggregatesassociate with Hsp104 and form foci in replicatively aged cells, which can be visualized byHsp104-eGFP ((6); Fig.S3A–D). During vegetative growth, Hsp104-eGFP foci aredistributed asymmetrically between the mother and daughter cell; less than 10% of youngcells displayed foci, but Hsp104-eGFP foci began to accumulate by generation 8 in mothercells and were present in 85% of aged cells ((4, 6, 9); Fig.S3D). Throughout sporulationmost aged cells contained Hsp104-eGFP foci (mononucleates (95%), binucleates (90%) andtetranucleates (86%)), but the foci were essentially absent in mature tetrads (3%; Fig.2A–B),suggesting that age-associated protein aggregates are cleared during sporulation. Thepolarisome is required for the asymmetric distribution of Hsp104-eGFP foci during mitosis(9). Deleting the genes encoding the polarisome components Bud6 or Spa2 did not interferewith aggregate elimination during sporulation (Fig.S3E–F). Proteasome function alsoappeared dispensable for aggregate clearance. Treatment of cells with the proteasomeinhibitor MG132 after the second division neither prevented sporulation nor the clearance ofHsp104 aggregates ((10); Fig.S3G–H). In contrast, treatment of cells with the autophagyinhibitor chloroquine prevented sporulation and aggregates persisted ((11); Fig.S3G–H),suggesting that an autophagy-dependent process and/or spore formation are required foraggregate clearance.Aged cells are also defective in rDNA metabolism, displaying fragmented nucleoli andforming extra chromosomal rDNA circles (ERCs) (7,8). ERCs decreased considerablyduring sporulation in aged cells, reaching levels similar to young cells (Fig.2C, Fig.S4). Inaged cells, the rDNA structure also underwent dramatic changes as judged by thelocalization of Fob1-GFP, a nucleolar protein that binds to the rDNA (12). In 60% of agedcells, the Fob1-GFP appeared enlarged and discontinuous, likely reflecting rDNAcondensation defects and nucleolar fragmentation, respectively. After aged cells sporulate,more than 90% of the tetrads contained a single Fob1-GFP focus per spore, and displayed amorphology indistinguishable from that of young cells (Fig.2D). Together, our resultsdemonstrate that gamete formation eliminates age-induced protein aggregation and nucleolaraberrations.To determine which aspects of gametogenesis are necessary for RLS resetting, we deletedtwo transcription factors that trigger different stages of sporulation and asked if RLS wasreset. Such studies are possible, because in budding yeast, sporulating cells can resumevegetative growth (return to growth; (4)) provided that the sporulation-inducing cue, nutrientdeprivation, is withdrawn. We first analyzed the RLS of young (1.4±0.6 generations) andaged (16.2±4 generations) cells from a strain that lacks Ime1. Without Ime1, yeast cells areunable to initiate sporulation but still sense nutrient deprivation (13). To ensure that onlycells that responded to the sporulation-inducing cues were included in the analysis, we useda pIME1:mCherry reporter construct (Fig.S5A) (14). Aged ime1Δ cells lost viability rapidly,with a median lifespan of 4 generations (Fig.3A). Similar results were obtained with wild-type cells that had responded to sporulation cues as judged by pIME1:mCherry expressionbut had not yet entered the sporulation program (Fig.S5B) Thus, the initiation of sporulationdriven by IME1 is required to reset RLS. Furthermore, nutrient deprivation and othersporulation signals are insufficient to promote RLS resetting.In the absence of the transcription factor Ndt80, yeast cells complete pre-meiotic DNAreplication, initiate recombination and arrest in pachytene (15). We used Zip1-GFP toidentify the ndt80Δ cells arrested in pachytene (Fig.S5C); (16) and found that young cells(1.3±0.6 generations) resumed vegetative growth and divided an average of 21.6±7.9 times(median RLS=21). In contrast, almost 50% of the aged cells (16.8±4.4 generations) lostviability within the first mitotic division. The remaining cells underwent significantly fewerdivisions compared to young cells (3.4±4.9 generations; Fig.3B). We conclude that NDT80-Ünal et al. Page 2Science. Author manuscript; available in PMC 2014 February 13.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author Manuscriptinduced processes are necessary for RLS resetting, and that the events prior to NDT80function such as pre-meiotic DNA replication and recombination are insufficient to promoterejuvenation.Progression through sporulation up to pachytene is not sufficient for resetting of RLS. Thus,later stages of sporulation, the meiotic nuclear divisions and/or spore formation must berequired. To determine whether both meiotic divisions are necessary for RLS resetting, wedeleted SPO12. spo12Δ cells undergo a single nuclear division and form two diploid spores(17). We found that young (1.3±0.6 generations) and aged (14±1.8 generations) spo12Δcells had indistinguishable RLS, suggesting that two consecutive meiotic divisions are not aprerequisite for rejuvenation (Fig.3C). The lifespan of spores within individual two-sporedasci is very similar (Fig.S6) (p [young]= 0.15, p [aged]= 0.29, n=30, Wilcoxon signed-ranktest). To test if resetting of RLS can occur in the absence of any nuclear divisions, weinactivated the polo-like kinase Cdc5 during sporulation (cdc5-mn; (18)). Cells lacking Cdc5do not undergo any meiotic divisions and form single spores. Like spo12Δ spores, cdc5-mnspores obtained from aged cells regained their replicative potential (Fig.3D). We concludethat the meiotic divisions per se are dispensable for RLS resetting and note that our findingsexclude a model where halving of the genome or diluting aging factors brings about theresetting of RLS (4).NDT80-regulated genes that mediate spore formation could be required for rejuvenation. AsNDT80 expression is sufficient to induce the expression of mid and late sporulation genes invegetative cells (15, 19) we determined whether Ndt80 could extend the lifespan ofvegetative cells. We expressed NDT80 from the GAL1-10 promoter (GAL-NDT80), whoseexpression can be regulated by a Gal4-estrogen receptor fusion (Gal4.ER) ((20, 21);Fig.S7A–B). Expression of NDT80 significantly extended the lifespan of mitotic cells(Fig.S7C–D; P-value <0.0001, Z-score=4, Mann-Whitney test). To test whether induction ofNDT80 in replicatively aged cells also extends lifespan, we transiently induced Ndt80 withβ-estradiol in young and aged cells and followed their RLS in the absence of β-estradiol.Aged cells that transiently expressed NDT80 lived significantly longer than aged cellstreated in the same manner but lacking the GAL-NDT80 fusion (Fig.4A, P-value < 0.0001,Z-score=8.23, Mann-Whitney test). Transient expression of NDT80 even led to an extensionof lifespan in young cells (Fig.4A, P-value < 0.0001, Z-score=4.85, Mann-Whitney test).Similar results were obtained in experiments comparing cell divisions in liquid culture,excluding the possibility that transient expression of NDT80 extends life span because itcauses cells to become more resistant to the micromanipulations involved in the pedigreeanalysis (Fig.4B–C). Thus, a transient induction of NDT80 is sufficient to extend thelifespan of replicatively aged cells.To address how transient induction of Ndt80 extends RLS, we monitored age-dependentcellular changes after NDT80 induction. Neither ERCs nor Hsp104-eGFP aggregates werereduced after NDT80 induction, although it is possible that they were reduced at later timepoints (Fig.S8A–B). Furthermore, NDT80 expression extended lifespan in the absence of theautophagy gene ATG1 (Fig.S8C). Together these findings suggest that lifespan extensioncan occur in the absence of ERC and Hsp104-aggregate elimination. While ERCs andHsp104-eGFP aggregates were not affected by transient NTD80 expression, nucleolarmorphology was. The percentage of aged cells with enlarged nucleolar morphologydecreased after NDT80 induction (Fig.4D–E, Fig.S8D). Thus, transient induction of NDT80causes a change in nucleolar/rDNA structure reverting it to a state that resembles themorphology in young cells.We do not yet know whether NDT80- and sporulation-induced RLS resetting use the samemechanism(s). The findings that NDT80 is necessary for lifespan extension duringÜnal et al. Page 3Science. Author manuscript; available in PMC 2014 February 13.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author Manuscriptsporulation and sufficient for lifespan extension during vegetative growth and that nucleolarmorphology is altered under both circumstances, suggest that at least some processes areshared. Irrespective of the relationship between NDT80- and sporulation-induced RLSresetting, we note that resetting of RLS provides an opportunity to dissect the molecularcauses of aging. For instance, elimination of Hsp104 aggregates and ERCs seem unlikely tobe required for NDT80-dependent lifespan extension, but changes in nucleolar function and/or structure may be important. Intriguingly, rDNA instability and not ERCs per se appear tocause aging in yeast (22) and budding yeast cells eliminate most of the nucleolar materialduring spore packaging (23).It will be interesting to investigate whether our findings extend to other species. In C.elegans, a number of longevity mutants exhibit a soma-to-germline transformation thatcontributes to their enhanced survival (24). In mice, reintroduction of telomerase rescues theage-related phenotypes of telomerase-deficient mice (25), suggesting that age-dependentcellular damage can be repaired. Our studies suggest that a transient induction of thegametogenesis program in somatic cells removes age-dependent cellular damage andextends lifespan. Determining how gametogenesis causes the resetting of lifespan willprovide insights into the mechanisms of aging and could facilitate the development ofstrategies for longevity.Supplementary MaterialRefer to Web version on PubMed Central for supplementary material.AcknowledgmentsWe thank Barry Alpert, Monica Boselli, Ann Thompson, Jingxun Chen for technical help, Doug Koshland forreagents, Terry Orr-Weaver, Frank Solomon and members of the Amon lab for comments on the manuscript.Research was supported by NIH grant GM62207 to A.A. A.A. is also an HHMI investigator. E.Ü is a fellow of theJane Coffin Childs Memorial Fund.References1. Neiman AM. Microbiol Mol Biol Rev. 2005 Dec.69:565. [PubMed: 16339736]2. Smeal T, Claus J, Kennedy B, Cole F, Guarente L. Cell. 1996 Feb 23.84:633. [PubMed: 8598049]3. 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[PubMed: 3280136]14. Nachman I, Regev A, Ramanathan S. Cell. 2007 Nov 2.131:544. [PubMed: 17981121]15. Chu S, Herskowitz I. Mol Cell. 1998 Apr.1:685. [PubMed: 9660952]16. Scherthan H, et al. Proc Natl Acad Sci U S A. 2007 Oct 23.104:16934. [PubMed: 17939997]17. Klapholz S, Esposito RE. Genetics. 1980 Nov.96:567. [PubMed: 7021311]Ünal et al. Page 4Science. Author manuscript; available in PMC 2014 February 13.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author Manuscript18. Lee BH, Amon A. Science. 2003 Apr 18.300:482. [PubMed: 12663816]19. Chu S, et al. Science. 1998 Oct 23.282:699. [PubMed: 9784122]20. Benjamin KR, Zhang C, Shokat KM, Herskowitz I. Genes Dev. 2003 Jun 15.17:1524. [PubMed:12783856]21. Carlile TM, Amon A. Cell. 2008 Apr 18.133:280. [PubMed: 18423199]22. Ganley AR, Ide S, Saka K, Kobayashi T. Mol Cell. 2009 Sep 11.35:683. [PubMed: 19748361]23. Fuchs J, Loidl J. Chromosome Res. 2004; 12:427. [PubMed: 15252239]24. Curran SP, Wu X, Riedel CG, Ruvkun G. Nature. 2009 Jun 25.459:1079. [PubMed: 19506556]25. Jaskelioff M, et al. Nature. 2011 Jan 6.469:7328.Ünal et al. Page 5Science. Author manuscript; available in PMC 2014 February 13.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptFigure 1. Gametogenesis resets RLSThe average number of cell divisions of the starting cell population is indicated in the graphlegend. The median lifespan is written next to each curve. Error bars denote S.D.A) RLS of young and aged wild-type A702 cells directly after sorting.B) Post-sporulation RLS of spores from young and aged A702 cells.C) Age distribution of spores from A702 in individual tetrads from young and agedprogenitors, n=10. The lifespan of spores from each tetrad is compared to the mean lifespanof young spores to obtain a P-value. The average P-value from 10 tetrads is 0.303 for youngand 0.642 for aged spores (t-test), indicating no statistically significant difference inreplicative age among spores from a given tetrad.Ünal et al. Page 6Science. Author manuscript; available in PMC 2014 February 13.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptFigure 2. Sporulation eliminates age-induced cellular damageA) Analysis of Hsp104-eGFP aggregates in aged sporulating A25825 cells.B) Quantification of Hsp104-eGFP foci in young and aged A25825 cells prior to meiosisI(mononuc), after meiosisI (binuc), after meiosisII (tetranuc) and in tetrads.C) rDNA and ERCs in young and aged A26370 cells.D) Nucleolar morphology in young and aged A26271 cells.Ünal et al. Page 7Science. Author manuscript; available in PMC 2014 February 13.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptFigure 3. IME1 and NDT80 but not the meiotic nuclear divisions are required for lifespanresettingA) RLS of young and aged A23998 (ime1Δ) cells.B) RLS of young and aged A24074 (ndt80Δ) cells. The median lifespan of the aged cells is0. Therefore, the average is shown.C) The lifespans of young and aged A27377 (spo12Δ) cells.D) The lifespans of young and aged A24142 (cdc5-mn) spores.Ünal et al. Page 8Science. Author manuscript; available in PMC 2014 February 13.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptFigure 4. Transient NDT80 expression extends the lifespan of vegetatively growing aged cellsA) Lifespan of young and aged cells from A25823 (GAL4.ER, NDT80) and A25824(GAL4.ER, GAL-NDT80).B) Top; description of the experiment, bottom; aged cells from A27507 (GAL4.ER, NDT80)and A27484 (GAL4.ER, GAL-NDT80) labeled before and after β-estradiol.C) The number of cell divisions after β-estradiol treatment was calculated by the differencebetween the green- and red-labeled bud scars. The distribution of n=60 cells is shown forA27507 (GAL4.ER, NDT80) and n=100 cells for A27484 (GAL4.ER, GAL-NDT80).D) Fob1-GFP in young and aged cells from strains A27507 (GAL4.ER, NDT80) and A27484(GAL4.ER, GAL-NDT80) after 6th β-estradiol treatment.E) Percentage of cells with enlarged Fob1-GFP from A27507 (GAL4.ER, NDT80) andA27484 (GAL4.ER, GAL-NDT80) following β-estradiol treatment. The average of twoindependent experiments is shown. 100 – 200 cells were counted for each time point; errorbars display the range.Ünal et al. Page 9Science. Author manuscript; available in PMC 2014 February 13.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author Manuscript

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Gametogenesis Eliminates Age-Induced Cellular Damage and Resets Life Span in Yeast

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