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Erythromycin Slows Aging of Saccharomyces cerevisiae

^a Department of Biological Sciences, University of Iowa, Iowa City

John R. Menninger, Department of Biological Sciences, University of Iowa, Iowa City, IA 52242-1324 E-mail: john-menninger{at}uiowa.edu.

Decision Editor: John Faulkner, PhD

	Abstract

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Life span was measured by counting budding cycles in cohorts of yeast cells treated with erythromycin, paraquat, or geneticin. Paraquat treatment increases oxidative stress; geneticin treatment increases errors during cytoplasmic protein synthesis. Treating with either or both compounds resulted in shorter life spans. Saccharomyces cerevisiae strain K65-3D grown in 16 µg/ml erythromycin, a treatment that results in more accurate protein synthesis by bacteria, had a mean life span that was significantly longer (27%) than that of untreated yeast cells. The life spans of petite variants with no detectable respiratory activity or extranuclear DNA were not affected by this dose of erythromycin, which appeared, therefore, to exert its effect on aging by means of mitochondria. Fitting the data to Weibull and Gompertz distributions allowed calculation of an accelerated life model that relates life span to dose of erythromycin.

ERYTHROMYCIN, an antibiotic produced by Saccharopolyspora erythraea (Streptomyces erythreus), binds to the large subunit of procaryotic ribosomes and interferes with protein synthesis, most likely by stimulating dissociation from the ribosomes of peptidyl-tRNA ⁽¹⁾. At low doses, erythromycin preferentially inhibits the synthesis of proteins that contain missense errors ⁽²⁾. Erythromycin also perturbs protein synthesis in yeast mitochondria ⁽³⁾, possibly by the same mechanism although this has not been established. Substantial free radical production may occur in mitochondria during aerobic metabolism ⁽⁴⁾. We hypothesized that treating yeast cells with erythromycin might reduce errors in mitochondrial protein synthesis and as a consequence perhaps reduce the generation of free radicals, permitting a longer life span. This hypothesis is thus an attempt to combine the free radical theory of aging ⁽⁵⁾ and the protein error theory of aging ^(6)(7)(8).

As an experimental organism, budding yeast offers the simplicity of a cellular assay to evaluate aging in a eucaryotic organism with mitochondria. A newly formed cell of Saccharomyces cerevisiae grows to a certain size and then puts forth a bud. After a suitable interval the mother cell puts forth another bud. There are a finite number, depending on the strain, of such generations before division ceases ⁽⁹⁾; the total number of generations measures yeast life span ⁽¹⁰⁾. Chronological age appears not to limit life span: yeast cell division can be slowed significantly by experimental manipulation, but the cells still undergo the average number of divisions characteristic of the strain ⁽¹¹⁾.

We used micromanipulation to remove buds from cohorts of yeast cells growing on agar. This allowed us to measure mean and maximum life spans under the influence of different doses of antibiotics. Mean life span was reduced by treating with paraquat, which increases oxidative stress in cells, including yeast ^(12)(13), or geneticin (G418), an aminoglycoside antibiotic that increases missense errors during translation ⁽¹⁴⁾. Both mean and maximum life spans increased in response to erythromycin treatment, in a manner that was dose dependent. The response to erythromycin was not observed in petite strains of yeast that lacked normal mitochondrial function. The slowing of yeast aging elicited by erythromycin was apparently mediated via mitochondria.

	Methods

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Strains and Growth Media
The homothallic diploid strain K65-3D (Mata/Mat lys2-1 tyr1-1 his7-2 can^r ura3-13 ade5 met13d trp5-2 leu1-d ade2-1) was obtained from Robert Malone (University of Iowa). Strain X2180-1A was obtained from S. Michael Jazwinski (Louisiana State University). Growth media included YPD (2% peptone, 1% yeast extract, and 2% D-glucose) and YPG (2% peptone, 1% yeast extract, and 3% glycerol). Solid media contained 2% agar. For bacterial contamination to be prevented, 100 µg/ml ampicillin was added to all media; we know of no interaction between ampicillin and the other antibiotics tested.

Micromanipulation
Cells from exponentially growing liquid cultures (YPG in most cases; YPD for petite strains) were streaked onto one section of a YPD plate containing ampicillin and other drugs as appropriate. YPD plates were incubated at room temperature for several hours and then examined for growth. From presumptively growing clumps of four or more cells, individual or budded cells (initial cells) were moved to an uninhabited portion of the plate with a fiber-optic needle. Newly arising buds (founder cells) were isolated by micromanipulation and moved to a new portion of the plate. Daughters produced by founder cells were generally removed and discarded after both the founder and the daughter cell each produced another bud, thus ensuring complete cytokinesis. This process continued until budding ceased; the number of buds produced by each founder cell was recorded. Cohorts included all founder cells that produced at least one bud after micromanipulation. Plates were incubated at room temperature (24°C) during the day but shifted to 4°C for 12 hours overnight. Any effects of temperature shifts during life-span assessment were assumed to influence equally cells treated with various doses of antibiotics, and therefore differences between treatments were assumed to be normalized for such effects ⁽¹⁵⁾. A founder cell was considered to have completed its life span if it failed to produce a bud during 24 hours of incubation at 24°C or if it lysed.

Sucrose Gradient Method
This method is modified from one published previously ⁽¹⁶⁾. Cells were grown for 3 to 4 days at 30°C, resuspended in water, and sonicated twice at 150 W (Biosonik IV microprobe) for 30 seconds, with icing between, which produced >90% unbudded cells. Sucrose gradients (0–50%) were prepared by freezing, in dry ice, 50-ml polycarbonate Oakridge tubes containing 41 ml of 25% sucrose, then slowly thawing at 4°C. One milliliter of 10⁸ cells was layered on each gradient, which was spun in a swinging bucket rotor for 4–5 minutes at 400 x g. Buds were recovered from the topmost and mothers from the bottommost of the two or three visible bands in each gradient and were pooled before the cell density was assayed by means of hemacytometer counting. Diluted samples containing 50–200 cells were spread on several YPD plates and incubated at 30°C for 3 days to determine colony-forming units. Viability was defined as the ratio of colony-forming units to microscopically counted cells. When a mother cell reaches the end of its replicative life span, the ability to form a colony, measured by its viability, becomes zero. The viability of buds was similar to reported values ⁽¹⁶⁾. To assay life span, cohorts of buds were resuspended in YPD at a density approximately three doublings less than saturation (the density at which growth ceases), then treated for 24 hours at 30°C with 100 µg/ml of 8-hydroxyquinoline, which synchronizes the cells in early G₁ ⁽¹⁷⁾. The cells were then sedimented and resuspended in an equal volume of YPD. At 30°C, it took 16 hours (longer for the oldest cells) to reach saturation, which occurred after three doublings. These cells were sonicated and separated on sucrose density gradients; bands from the top (buds) and bottom (mothers) were collected and pooled. As a way to assess any pathological effects of treatments, viabilities of both buds and mothers were determined. Synchronized growth, sonication, and separation were repeated until either the concentration of mother cells was too low to continue or the growth in 16 hours was significantly reduced. There was significant loss of yield after each separation.

Statistical Analyses
Separate cohorts of equally treated cells had mean life spans that never varied more than ±10% and often varied less than ±5%. Life spans of pooled sets were also assessed for reproducibility by an analysis of variance (ANOVA). The log likelihood of a distribution used to represent survival data was calculated by summing the natural logarithm of the distribution's probability density function, evaluated at each cell life span. Parameters (maximum likelihood estimates, or MLEs) for distributions were those that maximized the log likelihood and were estimated by the methods of Newton–Raphson, steepest ascent, or other algorithm. Parameters for the Weibull distribution (, r) were fit by MLE; for the gamma distribution (, r) by the formula of Greenwood and Durand ⁽¹⁸⁾ or MLE; and for the Gompertz distribution (q₀, r) by least-squares regression of the natural logarithm of the force of morality (q_i = age-specific death rate = hazard function): ln (q_i) = ln (q₀) + r G_i, where G_i are the life spans (failure times) measured in generations, or by MLE. To derive an accelerated life-span model for cells treated with erythromycin, the Weibull and Gompertz distributions for untreated cells were transformed to depend on the MLE value of ß in the equation (G_ij) = (G_i) x exp(ß dose_j), where G_i is the value of the life span for the ith treated cell and dose_j is the jth dose of erythromycin. Expected values of functions of life spans for the various distributions were calculated from standard formulae or by numerical integration of the function times the probability density function of the distribution.

Petite Isolation and DAPI Staining
Cells were grown overnight in YPD to a density of approximately 10⁷ cells/ml, then diluted to 10⁴ cells/ml with 1 ml of YPD containing 10 µg/ml of ethidium bromide. The cells were incubated at 30°C for 2 days with occasional mixing during the incubation period. Samples of the cells were plated onto YPD plates and incubated at 30°C for 60 hours. Colonies from these plates were then tested for their respiration status by replica plating onto YPG plates. Colonies that grew poorly or not at all on the YPG plates were retested, and reproducibly defective colonies were saved. For DAPI (4,6-diamidino-2-phenylindole; Sigma, St. Louis) staining, aliquots (2 x 10⁷ cells) of cultures grown overnight in YPD at 30°C were spun down in microfuge tubes, washed in 5% formaldehyde, and then washed in water. Cells were fixed in 64% ethanol for 5 minutes, washed in water, moved into a darkroom, exposed to 0.5 µg/ml of DAPI for 10 minutes, and then washed in water to reduce background staining. Stained cells were resuspended in 10 µl of mount medium (90% glycerol, 10% pH 9.0 bicarbonate buffer, and 1 mg/ml of 1,4 phenylenediamine; Sigma); 5 µl was placed on a glass slide, covered with a coverslip, and sealed with clear nail polish. The slides were stored in a dark box until observed with an ultraviolet microscope.

	Results

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Mean life span was reduced by treating with paraquat or geneticin (G418; Fig. 1, Table 1 ). An ANOVA of all cohorts tabulated showed that the various treatments had a significant effect on mean life span. We could not detect a significant effect on mean life span of K65-3D cells by treating with paraquat at a dose of 65 µg/ml, but a dose of 100 µg/ml reduced mean life span by 29%. Geneticin at 4 µg/ml had little effect, but geneticin at 6 µg/ml reduced mean life span by 22%. Combining the doses of paraquat (65 µg/ml) and geneticin (4 µg/ml) that separately had no significant effect reduced mean life span by 16%.

View larger version (19K): [in this window] [in a new window]   Figure 1. Effect of paraquat and geneticin on survival: paraquat 100 µg/ml (), 65 µg/ml (); geneticin 6 µg/ml (), 4 µg/ml (); paraquat 65 µg/ml plus geneticin 4 µg/ml (); no drugs ().

View larger version (18K): [in this window] [in a new window]   Figure 2. Effect of erythromycin on survival: erythromycin 0 (), 8 (), 12 (), 16 (•), and 24 () µg/ml.

View larger version (17K): [in this window] [in a new window]   Figure 3. Effect of erythromycin on life span. Survival data from cohorts treated with 0 () or 16 (•) µg/ml erythromycin were pooled. The variance of the fraction surviving to a particular generation was calculated by assuming that mean survivals were binomially distributed. Results from several cohorts were combined by calculating means (, •) and standard errors of the mean (SEM, vertical error flags, mostly invisible), weighted according to the original number of cells. Also shown on the figure are the life spans of treated and untreated cells calculated on several bases: mean weighted according to the cohort size, unweighted mean, unweighted median, and unweighted maximum (mean survival of longest lived 10% of the pooled cohorts). Each of these last values is accompanied by an estimate of its standard error (horizontal error flags).

View larger version (16K): [in this window] [in a new window]   Figure 4. Parametric models for effect of erythromycin at 0 () and 16 () µg/ml on survival. Weibull (—), Gompertz (-----), and gamma (–––) survival distributions were fit to the observed life spans by using maximum likelihood estimates for the parameters (see Table 4 ).

(1)

where S is the survival at generation G, relative to survival at generation 0. For MLE estimates of , treatment reduced r by 20%. The Gompertz distribution is

(2)

Treatment with erythromycin reduced the exponential aging rate (r) of the treated cells to 63% of the value for the untreated cells. The gamma distribution is

(3)

which for integral values of is identical to the cumulative Poisson distribution. For MLE estimates of , treatment reduced r by 7%. The accelerated life model for survival assumes that a treatment affecting life span is equivalent to speeding up or slowing down the time of the experiment: values of G in a distribution are multiplied by the function exp(ß). In our case of increased life spans, ß should have a negative value (decelerated life). For the Gompertz distribution, ß_G was (MLE) estimated as -0.0116; for the Weibull distribution, ß_W = -0.0096.

The life-span-extending responses to erythromycin are consistent with our initial hypothesis that erythromycin might reduce erroneous protein synthesis by mitochondria, but the observed enhancement of life span might have been caused by some effect not mediated by means of mitochondrial protein synthesis. To test this possibility, we studied petite variants derived from strain K65-3D by treatment with ethidium bromide. Strains that could not grow in YPG medium, in which the primary carbon source glycerol cannot be fermented, were stained with DAPI to identify those lacking DNA outside the nucleus. The life spans of two such petite variants were measured in the presence and absence of 16 µg/ml of erythromycin (Fig. 5, Table 5 ). The mean life span of neither petite derivative of strain K65-3D changed significantly when treated with 16 µg/ml of erythromycin. An ANOVA also revealed no significant difference among the means of pooled untreated or treated cohorts. Neither untreated petite derivative had a mean life span that differed significantly from the untreated K65-3D parent. K65-3D cells were also assayed for the frequency of petites at different ages when treated with erythromycin, using the sucrose gradient method. There was no significant difference detected between treated (8 µg/ml) and untreated cells, either mothers or their offspring, through generation 15 (data not shown), a result similar to that of others ⁽¹⁹⁾. No effects of this dose of erythromycin on rates of growth in YPD or YPG media by the yeast, either grandes or petites, could be detected (data not shown).

View larger version (17K): [in this window] [in a new window]   Figure 5. Effect of erythromycin on survival of two petite derivatives of strain K65-3D: petite 1 (, ), petite 2 (, •); erythromycin 0 (, ), and 16 (, •) µg/ml.

	Discussion

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The effect of erythromycin on S. cerevisiae strain K65-3D was to lengthen its mean and maximum life spans significantly (Table 2 ). Significantly higher doses (>300 µg/ml) of this antibiotic are known to inhibit mitochondrial protein synthesis by mitochondria of cells growing on fermentable carbon sources ⁽²²⁾. We are not aware of any report of an effect by erythromycin on processes in yeast other than mitochondrial protein synthesis. Although we were motivated to test erythromycin because of its known ability to improve the accuracy of protein synthesis in Escherichia coli ⁽²⁾, the effects, if any, of erythromycin on enhancing accuracy during protein synthesis in mitochondria are not known. We used a simple test to verify that at least the likely site of action of erythromycin's effect on life span is the mitochondria. The absence of detectable mitochondrial DNA in petite yeast cells should reduce mitochondrial protein synthesis by depriving ribosomes of substrate mRNAs. Fig. 4 and Table 4 show that petite cells, with no detectable aerobic metabolism nor extranuclear DNA, when treated with erythromycin had life spans indistinguishable from untreated petite cells. It thus seems likely that erythromycin exerts its effect on the aging of normal (grande) S. cerevisiae by interacting with mitochondria.

The observation that untreated petite strains had life spans similar to untreated K65-3D may appear inconsistent with the hypothesis that mitochondrion-generated free radicals play a significant role in limiting life span. In considering this issue, it is important to recall that the life spans of petites bear no predictable relation to the life span of their grande parents ⁽¹⁹⁾. This is perhaps the result of the multiple pathways by which petites arise, reflected in the differing amounts of mitochondrial DNA they retain. Moreover, although respiratory chain proteins encoded by mitochondrial DNA may be missing, those encoded by nuclear DNA are not. These latter components can be detected in the residual membrane-bound structures found in petites ⁽²³⁾. Nuclear-encoded proteins are more numerous in the respiratory chain than mitochondrial-encoded proteins, so it seems possible that variously defective respiration can occur in petites, leading to unpredictable rates of free radical formation.

Error theories of aging propose that the finite life span of an organism results from the inevitable appearance and subsequent accumulation of errors. In essence, errors include the failure of any intrinsic process that maintains the homeostatic sexually mature state ⁽²⁴⁾. The Second Law of Thermodynamics guarantees the appearance of errors. Free energy can in principle be used to reduce the frequency of errors, but an infinite amount of energy would be required to reduce the error rate to zero. Whether errors accumulate and limit life span depends on the relative activities of competing processes, those that generate and those that repair errors.

Others have reported that reducing errors during protein synthesis is associated with increased longevity. Silar and Picard ⁽²⁵⁾ showed an increased life span in filamentous fungi (Podospora anserina) bearing a mutation in eucaryotic elongation factor-1 (eEF-1) that enhances the accuracy of translation. In apparent contradiction, several mutants with decreased translational accuracy also have life spans longer than wild type ^(25)(26). Additional expression of eEF-1 has been reported as leading to an increased life span in Drosophila melanogaster ⁽²⁷⁾. Others have suggested that genetic background and the map location of the insertion element bearing the eEF-1 gene may play a larger role than its added expression ⁽²⁸⁾.

There is significant experimental support for one or another version of the free radical theory of aging ^{(5)(23)(29)(30)(31)(32)}. Because substantial amounts of free radicals are generated during normal aerobic metabolism ⁽⁴⁾, it seems likely that damage from free radicals will influence life span. Because much of the free radical load in a eucaryotic cell arises in the mitochondria, there is reason to suspect those organelles as important players in aging ⁽³³⁾. That was one reason why it seemed worth seeking an effect on aging by erythromycin, because it is known to inhibit mitochondrial protein synthesis. (It would be equally sensible to test an antibiotic that reduces translational errors in the cytoplasm because most mitochondrial proteins are synthesized there, but unfortunately no antibiotics are known to act on cytoplasmic ribosomes the way erythromycin does on mitochondrial ribosomes.)

Another reason to study the mitochondrion in this context is the dominance of the aging phenotype in cases of cell fusion ^(34)(35)(36). This also occurs during the rare fusion events that accompany mating by young and old haploid yeast cells ⁽³⁷⁾. Budding in yeast might be considered as a kind of reverse of fusion: one cell giving rise to two. In S. cerevisiae, however, buds are generally young. Either the senescence factors that apparently dominate the product of a cell fusion are not segregated into the bud, it receives a disproportionately high amount of senescence repair activity, or both. If the mitochondrion is the site of the senescence factor, it may be possible to detect this segregation by using advanced cell biological assay methods.

One advantage of fitting parametric models to survival data is the possibility that experimentally testable underlying mechanisms may be suggested. Based on their likelihoods, the Gompertz and Weibull distributions gave reasonably good fits to the life spans of untreated cells and the Weibull and gamma distributions to cells treated with 16 µg/ml of erythromycin. The Gompertz and Weibull are two of the three types of distributions that represent the minimum values of a large random sample. In a series of processes, each one of which must operate to ensure continued life, the first one to fail determines the life span. This kind of underlying mechanism would be expected to show a minimum value survival distribution. The observation that Gompertz and Weibull distributions fit the survival of untreated cells can be taken as evidence that there exists a series of critical processes that determine the life span of yeast. The gamma distribution measures the time to complete a set of equivalent Poisson processes. If there are a set of randomly inactivating, parallel processes, any one of which suffices to ensure continued life, then death will occur when the last one fails, and life span should be represented by a gamma distribution. Applied to our untreated yeast cells, this kind of model predicts approximately seven such processes, each one inactivating with a rate of 0.542 per generation. The effect of adding 16 µg/ml of erythromycin is to increase the number of processes by approximately one and reduce the rate by 7%. To the extent that this kind of multiple-hit model fits our data, it suggests searching for the seven to eight equivalent processes within yeast physiology.

Our results show that treatment with erythromycin, which in procaryotic cells reduces the frequency of erroneous proteins, can increase the life span of yeast. There are many possibilities that might explain this counterintuitive result. One alternative explanation is that inhibiting mitochondrial protein synthesis might lengthen life span. We treated yeast with doses of chloramphenicol within the range that inhibits mitochondrial protein synthesis in vitro ⁽³⁾, which is much lower than required in vivo or to inhibit cell growth on nonfermentable carbon sources ⁽³⁸⁾. We detected only a shortening effect on life span (Table 6 ). We conclude that merely inhibiting mitochondrial protein synthesis is not sufficient to extend life span.

Conclusions
Although we were motivated to test erythromycin because of its effects on reducing the frequency of synthesizing complete erroneous proteins in procaryotic cells, our results do not prove such a process occurred in the treated yeast cells. To trace all the links in this putative causal chain would require direct demonstration that erythromycin preferentially inhibits the synthesis of erroneous proteins in yeast mitochondria. This might be attempted by introducing a reporter gene into the organelle and assessing the temperature sensitivity of its enzyme activity, as has been done with E. coli ⁽²⁾. Techniques for introducing reporters into mitochondria are not yet well developed, so these experiments may take some time to accomplish ⁽³⁹⁾. Alternatively, mass spectroscopic methods may be able to detect the effect of treating with erythromycin on aberrant versions of proteins that are normally synthesized in mitochondria. In the meantime, the effect on life span of mutations in nuclear genes whose products play a role in the accuracy of protein synthesis may be revealing. Another test of erythromycin's role in aging might be to use mutants of yeast that are resistant to its effects on protein synthesis ⁽³⁸⁾.

	Acknowledgments

 
This research was supported by National Institutes of Health/National Institute on Aging Grant T32 AG00214, the Interdisciplinary Research Training Program on Aging, and the Department of Biological Sciences, University of Iowa.

We thank S. M. Jazwinski and R. A. Malone for strains and advice about yeast, J. H. Lemke and J. W. Yankey for statistical consultations, and R. Gesteland for consultations on mass spectrometry.

Received November 27, 2000

Accepted August 15, 2001

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