Decoupling AMPK from fatty acid synthesis allows maintenance of fitness late in life

Reviewer #1 (Public review):

This rigorous and creative study uses an elegant combination of metabolomics, transcriptomics, and budding yeast molecular genetics to discover that (i) activating AMPK to maintain mitochondrial respiration fueled by cytosolic Acetyl CoA and (ii) increasing fatty acid synthesis independent of respiration drive independent pathways that increase the fitness of replicatively-aged budding yeast cells, albeit without increasing their lifespan. This work will be of interest to scientists in the field of aging and metabolism. Some clarifications in the text would address the following concerns, which would increase the impact of the study:

(1) What does activation of AMPK (via PGDP-Sak1 expression) do to the replicative lifespan? How many bud scars, in general, do the subpopulations that are older - yet have less Tom70 (increased mitochondrial fitness) - have, after the 48 hrs time point that they are examining? How many divisions occurred in this 48hr time period - i.e. is it long enough to have all cells reach the end of their replicative lifespan? This information is important to rule out that a subset of the mutant cells just divided faster and hence had more divisions within 48 hrs (growing faster and living longer are different things). Having identical growth curves doesn't indicate per se that they all divide at the same rate, as there may be a subpopulation that divides faster and a subpopulation that doesn't grow so well.

(2) A2A cells do not have an extended replicative lifespan (RLS) but show an increase in the "low senescence" population (Figure 2). If the cells are not becoming senescent, why don't they have longer RLS? Not having a longer lifespan seems inconsistent with the statement that "bud scar counting confirmed that A2A cells reach a higher age than wild type", which comes back to how many times the cells can divide in the 48hr timepoint studied and their rate of cell division? Also, the lifespan curve shown is plotted against time, not cell division number, which does not take into account different division times of cells within the population (described above). It would be much more useful to show standard lifespan curves showing cell division numbers per lifespan per cell.

(3) Increased "fitness" of the old cells is implied from the increased size of the colonies that the old cells can make. However, this is a measure of the fitness of the daughters per se, not the old mother cells. Are the old mothers just passing on healthier mitochondria and more lipids to the daughters, such that they can divide more times? If the aged cells have an "increased fitness", why don't they divide more times themselves (i.e. live longer?).

(4) The statement is made that "these experiments define two classes of aging cells with distinct metabolic needs, coherent with the model of two aging trajectories previously proposed (referencing Nan Hao's work)". However, the big difference here is that in Nan Hao's work, their two aging trajectories influenced the length of lifespan, but that does not appear to be the case here. That distinction should be made clear. Perhaps the authors could also speculate as to why the A2A yeast stops dividing after presumably the same number of cell divisions, even though they have an activated AMPK and activated fatty acid synthesis pathway.

(5) I am a bit confused by the use of the word "senescence" by this lab here and in their previous growth on galactose studies. If yeast don't senesce, which is usually defined as an irreversible arrest of the cell cycle where cells stop dividing, shouldn't the yeast that do not senesce still be dividing and hence have a longer lifespan? Should a different term be used rather than senescence? Such as "fitness late in life". The authors giving their definition of senescence may help reduce this apparent contradiction.

Reviewer #2 (Public review):

Summary:

In this study, the authors investigate how cytosolic acetyl-CoA metabolism influences replicative aging in budding yeast. They propose that acetyl-CoA regulates aging through three major pathways: (1) mitochondrial transport to support mitochondrial function, (2) fatty acid synthesis, and (3) global protein acetylation. The data show that AMPK activation promotes mitochondrial import of acetyl-CoA and partially mitigates mitochondrial decline in a subset of aging cells.

Furthermore, the engineered A2A strain, which enhances mitochondrial acetyl-CoA utilization while relieving inhibition of fatty acid synthesis, increases the proportion of cells exhibiting a "low senescence" phenotype.

Overall, this is a thoughtful and potentially impactful study that advances our understanding of metabolic control of aging. Addressing the points below, particularly by refining interpretations and, where feasible, incorporating additional analyses, will further strengthen the manuscript and its conclusions.

Strengths:

The study has several notable strengths. It addresses an important question by shifting the focus from lifespan to preservation of late-life fitness, which is highly relevant to aging biology. The work integrates metabolic, genetic, and functional analyses to link cytosolic acetyl-CoA flux with distinct aging outcomes, and the engineering of the A2A strain provides a clear and elegant demonstration of how coordinated pathway modulation can improve cellular fitness.

Weaknesses:

(1) While the manuscript focuses on mitochondrial transport and fatty acid synthesis, cytosolic acetyl-CoA is also a key regulator of histone acetylation and chromatin silencing. It would strengthen the study to consider whether acetyl-CoA depletion contributes to improved fitness through enhanced rDNA silencing. Given the well-established role of rDNA instability in yeast aging, additional experiments examining rDNA silencing and stability would be valuable. For example, monitoring rDNA copy number changes (not necessarily ERCs) under AMPK activation, oleic acid supplementation, and in the A2A strain, similar to approaches used in the authors' prior work, would help clarify whether chromatin regulation contributes to the observed phenotypes.

(2) The current data do not fully distinguish whether AMPK activation and oleic acid supplementation act on distinct subpopulations of aging cells. An alternative explanation is that oleic acid supplementation enhances mitochondrial function and acts additively with AMPK activation, thereby increasing the fraction of cells in the "low senescence" state. Since this distinction is not central to the main conclusions, I suggest softening the language around subpopulation specificity. Emphasizing instead that the A2A strain coordinately modulates multiple branches of acetyl-CoA metabolism to improve late-life fitness would maintain the strength of the central message without overinterpretation.

(3) The manuscript proposes that lipid starvation and excess acetyl-CoA are major drivers of senescence in distinct subpopulations of wild-type aging cells. This conclusion is not yet fully supported by the presented data. Direct measurements of age-dependent divergence in acetyl-CoA and fatty acid levels at the single-cell level would be needed to substantiate this model. Based on the current evidence, a more conservative interpretation would be that aging cells exhibit differential sensitivity to perturbations in acetyl-CoA and lipid metabolism. Accordingly, I recommend revising the statement in the Abstract ("We further implicate lipid starvation and excess acetyl coenzyme A availability as major drivers of senescence...") and the corresponding discussion text to better align with the data.

Reviewer #3 (Public review):

Summary:

These findings suggest that PGPD-SAK1 yeast show a subpopulation with lowered TOM70-GFP expression in high bud scar staining aged cells. Deletion of CAT2 or MLS1 reduces this effect. A PGPD-SAK1 acc1S1157A double mutant (called "A2A" here) shows an even larger effect of lowered tom70 expression in high bud scar staining aged cells. Utilization of various additional mutants involved in acetyl-CoA transport, carnitine shuttle, respiration, etc., leads the authors to conclude that these shifts in TOM70-GFP in aged cells are linked to the AMPK-fatty acid metabolic regulatory system.

Strengths:

These extensive and clearly described experiments reveal interesting changes in TOM70-GFP intensity in subsets of aged yeast in several mutants eventually identified as linked to the AMPK-fatty acid metabolic regulatory system.

Weaknesses:

(1) 3 biological replicates for mRNASeq is low.

(2) While "Traditional conceptions of ageing implicate a progressive accumulation of damage leading to systemic degradation in performance until death, with evolutionary pressures acting to maximise early life fitness and fecundity at the expense of ageing health." is tangential perhaps to the data and conclusions of the study, both claims of this sentence are at best controversial, and the manuscript is no weaker for their omission.

(3) The statement that "Here, we determine the basis of senescence and fitness loss in replicatively ageing yeast" is a bit strong as a summary of the present careful work presented here. If the authors had created yeast mutants that retained fitness indefinitely, this would be a more appropriate strength of claim to summarize the work.

Author response:

Public Reviews:

Reviewer #1 (Public review):

This rigorous and creative study uses an elegant combination of metabolomics, transcriptomics, and budding yeast molecular genetics to discover that (i) activating AMPK to maintain mitochondrial respiration fueled by cytosolic Acetyl CoA and (ii) increasing fatty acid synthesis independent of respiration drive independent pathways that increase the fitness of replicatively-aged budding yeast cells, albeit without increasing their lifespan. This work will be of interest to scientists in the field of aging and metabolism. Some clarifications in the text would address the following concerns, which would increase the impact of the study:

(1) What does activation of AMPK (via PGDP-Sak1 expression) do to the replicative lifespan? How many bud scars, in general, do the subpopulations that are older - yet have less Tom70 (increased mitochondrial fitness) - have, after the 48 hrs time point that they are examining? How many divisions occurred in this 48hr time period - i.e. is it long enough to have all cells reach the end of their replicative lifespan? This information is important to rule out that a subset of the mutant cells just divided faster and hence had more divisions within 48 hrs (growing faster and living longer are different things). Having identical growth curves doesn't indicate per se that they all divide at the same rate, as there may be a subpopulation that divides faster and a subpopulation that doesn't grow so well.

Increasing AMPK activity increases replicative lifespan [PMID: 25869125], but given our finding that AMPK activation splits the population, such replicative lifespan assays are hard to interpret. Bud scar counts have a similar issue. Hence we restricted the lifespan and bud scar analyses to wt and A2A which are more homogenous (Figures S2 B and E). A2A cells at 48h have ~25% more bud scars than wt cells. Yes, by 48h most of the cells have lost viability (Figure 2E). The reviewer is correct that you can't properly compare the lifespan curves if the cells divide at different rates, hence our follow-up test of wt at 48h vs A2A at 40h viability after we had confirmed that these timepoints captured cells at equivalent replicative ages (Figure 2D,E). This shows that viability of A2A is slightly lower than wt at matched age, indicating a slightly shorter lifespan.

(2) A2A cells do not have an extended replicative lifespan (RLS) but show an increase in the "low senescence" population (Figure 2). If the cells are not becoming senescent, why don't they have longer RLS? Not having a longer lifespan seems inconsistent with the statement that "bud scar counting confirmed that A2A cells reach a higher age than wild type", which comes back to how many times the cells can divide in the 48hr timepoint studied and their rate of cell division? Also, the lifespan curve shown is plotted against time, not cell division number, which does not take into account different division times of cells within the population (described above). It would be much more useful to show standard lifespan curves showing cell division numbers per lifespan per cell.

Our observation that cells can reach the end of life without senescing is consistent with other studies that have studied the life course of individual cells by microscopy [PMID: 31291577, 32675375]. These studies always highlight some proportion of the cells that reach the end of life with no or minimal senescence, though this fraction varies with the experimental system. The question of why cells lose viability without senescing is a complete unknown in the field, but reflects a wider lack of consensus as to why yeast lose viability with replicative age.

We are wary about making strong statements on lifespan for exactly the reason the reviewer picks out. In liquid culture we can only assess viability over time, and it is clear from the comparison of liquid and solid media lifespans performed by the Gottschling lab [PMID: 19652178] that culture system has a huge effect on lifespan, with cells in classical microdissection-based lifespan assays living far longer than they do in liquid. This of course means that classical microdissection assays are not very useful for A2A so we are left with an unsatisfactory approximation. We have therefore restricted our conclusion on lifespan to simply say that lifespan of A2A cells is not extended which our data in Figures 2D,E,S2B does support (see also answer to Q1), and therefore with the majority of A2A cells showing low senescence marks and high fitness at 48h we can conclude that lifespan and fitness loss must be separable.

We will note these limitations of lifespan measurements in the manuscript.

(3) Increased "fitness" of the old cells is implied from the increased size of the colonies that the old cells can make. However, this is a measure of the fitness of the daughters per se, not the old mother cells. Are the old mothers just passing on healthier mitochondria and more lipids to the daughters, such that they can divide more times? If the aged cells have an "increased fitness", why don't they divide more times themselves (i.e. live longer?).

Yes, colony growth speed is defined by daughter cell replication, and as long as the daughters and subsequent generations divide at the same rate irrespective of whether they come from a young or old mothers then the size of the colony after 24 hours varies based on the time it took the initial mother to produce a daughter. This is what the assay really measures. We note that aged wildtype mothers often do not divide at all in the first 24 hours after being put on an agar plate (hence the tiny reported colony size), even though they do eventually produce a daughter which then forms a colony, whereas A2A cells tend to produce the first daughter rapidly whether young or old. It is known that daughters of aged wildtype mothers also divide slower, which will also contribute to differences in colony size, and this may well result from a lipid and/or mitochondrial contribution, but the primary driver of colony size in 24 hours is the time the mother took to initially divide. We will add this detail to the manuscript.

As noted above, the mechanistic basis of lifespan is unknown, but although senescence can shorten lifespan, our work and that of others shows that lifespan is still limited in the absence of senescence.

(4) The statement is made that "these experiments define two classes of aging cells with distinct metabolic needs, coherent with the model of two aging trajectories previously proposed (referencing Nan Hao's work)". However, the big difference here is that in Nan Hao's work, their two aging trajectories influenced the length of lifespan, but that does not appear to be the case here. That distinction should be made clear. Perhaps the authors could also speculate as to why the A2A yeast stops dividing after presumably the same number of cell divisions, even though they have an activated AMPK and activated fatty acid synthesis pathway.

We will add this distinction. As noted above, we are wary of making strong statements regarding lifespan as the assays we can do in liquid culture are limited. We are therefore similarly wary about speculating about causes for the lack of lifespan difference because in reality all we can do is rule out a big effect. We would love to speculate on why the A2A cells don't have an extended lifespan, but at this point we don't have any good ideas on this point!

(5) I am a bit confused by the use of the word "senescence" by this lab here and in their previous growth on galactose studies. If yeast don't senesce, which is usually defined as an irreversible arrest of the cell cycle where cells stop dividing, shouldn't the yeast that do not senesce still be dividing and hence have a longer lifespan? Should a different term be used rather than senescence? Such as "fitness late in life". The authors giving their definition of senescence may help reduce this apparent contradiction.

We completely agree, this is confusing and noted this distinction in the Introduction. Use of the term senescence to mean a loss of fitness late in life in yeast stems from the classical definition of senescence as applied to whole organisms. However, the term senescence as applied to cells has a more specific meaning in terms of the cell cycle as the reviewer notes. As an individual S. cerevisiae is both a cell and an organism, the terminology clashes. However, the marker we largely employ (Tom70-GFP) which in our hands is a very good proxy for fitness was originally defined as marking the senescence entry point (SEP), so overall we feel we can't avoid the term.

Reviewer #2 (Public review):

Summary:

In this study, the authors investigate how cytosolic acetyl-CoA metabolism influences replicative aging in budding yeast. They propose that acetyl-CoA regulates aging through three major pathways: (1) mitochondrial transport to support mitochondrial function, (2) fatty acid synthesis, and (3) global protein acetylation. The data show that AMPK activation promotes mitochondrial import of acetyl-CoA and partially mitigates mitochondrial decline in a subset of aging cells.

Furthermore, the engineered A2A strain, which enhances mitochondrial acetyl-CoA utilization while relieving inhibition of fatty acid synthesis, increases the proportion of cells exhibiting a "low senescence" phenotype.

Overall, this is a thoughtful and potentially impactful study that advances our understanding of metab to olic control of aging. Addressing the points below, particularly by refining interpretations and, where feasible, incorporating additional analyses, will further strengthen the manuscript and its conclusions.

Strengths:

The study has several notable strengths. It addresses an important question by shifting the focus from lifespan to preservation of late-life fitness, which is highly relevant to aging biology. The work integrates metabolic, genetic, and functional analyses to link cytosolic acetyl-CoA flux with distinct aging outcomes, and the engineering of the A2A strain provides a clear and elegant demonstration of how coordinated pathway modulation can improve cellular fitness.

Weaknesses:

(1) While the manuscript focuses on mitochondrial transport and fatty acid synthesis, cytosolic acetyl-CoA is also a key regulator of histone acetylation and chromatin silencing. It would strengthen the study to consider whether acetyl-CoA depletion contributes to improved fitness through enhanced rDNA silencing. Given the well-established role of rDNA instability in yeast aging, additional experiments examining rDNA silencing and stability would be valuable. For example, monitoring rDNA copy number changes (not necessarily ERCs) under AMPK activation, oleic acid supplementation, and in the A2A strain, similar to approaches used in the authors' prior work, would help clarify whether chromatin regulation contributes to the observed phenotypes.

We have data addressing this point that we will add to the manuscript. In short, we see no difference in gene expression from Sir2-repressed sub-telomeric regions or MAT loci, but the genome-wide gene expression dysregulation associated with age is partially suppressed in PGPD-SAK1. However, A2A does not suppress this further, so it is not critical for the suppression of senescence in A2A though we are following this up. ERC accumulation is higher in A2A at 48h, consistent with the cells being older, meaning that ERCs are unlinked to senescence onset as we have previously reported. There is a strong upregulation of transcripts from Sir2-repressed rDNA intergenic spacers with age in all genotypes, but we attribute this simply to the copy number increase of these regions on ERCs rather than a defect in silencing. We have previously looked for heritable changes in rDNA copy number arising during ageing and found (to our surprise) absolutely nothing, so we don't expect any changes under these conditions.

(2) The current data do not fully distinguish whether AMPK activation and oleic acid supplementation act on distinct subpopulations of aging cells. An alternative explanation is that oleic acid supplementation enhances mitochondrial function and acts additively with AMPK activation, thereby increasing the fraction of cells in the "low senescence" state. Since this distinction is not central to the main conclusions, I suggest softening the language around subpopulation specificity. Emphasizing instead that the A2A strain coordinately modulates multiple branches of acetyl-CoA metabolism to improve late-life fitness would maintain the strength of the central message without overinterpretation.

We agree that oleic acid and the lipids produced downstream of Acc1 in A2A may improve late life fitness via enhanced mitochondrial function, and in support of this Oxygen Consumption Rate is marginally (though significantly) higher in A2A than PGPD-SAK1. We will add this data to the manuscript. However, we disagree with the interpretation of an additive effect as we report throughout the study that AMPK activation and lipid biosynthesis/supplementation affect different sub-populations of cells. We do not observe populations of intermediate senescence cells, rather by flow cytometry and fitness assays we observe individual cells in binary low senescence or high senescence states.

(3) The manuscript proposes that lipid starvation and excess acetyl-CoA are major drivers of senescence in distinct subpopulations of wild-type aging cells. This conclusion is not yet fully supported by the presented data. Direct measurements of age-dependent divergence in acetyl-CoA and fatty acid levels at the single-cell level would be needed to substantiate this model. Based on the current evidence, a more conservative interpretation would be that aging cells exhibit differential sensitivity to perturbations in acetyl-CoA and lipid metabolism. Accordingly, I recommend revising the statement in the Abstract ("We further implicate lipid starvation and excess acetyl coenzyme A availability as major drivers of senescence...") and the corresponding discussion text to better align with the data.

We agree and will adjust the abstract to make it clearer that the lipid starvation / excess acetyl coA interpretation is a model.

Reviewer #3 (Public review):

Summary:

These findings suggest that PGPD-SAK1 yeast show a subpopulation with lowered TOM70-GFP expression in high bud scar staining aged cells. Deletion of CAT2 or MLS1 reduces this effect. A PGPD-SAK1 acc1S1157A double mutant (called "A2A" here) shows an even larger effect of lowered tom70 expression in high bud scar staining aged cells. Utilization of various additional mutants involved in acetyl-CoA transport, carnitine shuttle, respiration, etc., leads the authors to conclude that these shifts in TOM70-GFP in aged cells are linked to the AMPK-fatty acid metabolic regulatory system.

Strengths:

These extensive and clearly described experiments reveal interesting changes in TOM70-GFP intensity in subsets of aged yeast in several mutants eventually identified as linked to the AMPK-fatty acid metabolic regulatory system.

Weaknesses:

(1) 3 biological replicates for mRNASeq is low.

Thank you for pointing this out. We performed another replicate after posting the initial preprint but didn’t update the figure in the eLIFe-reviewed version. We will add this to the scatter plots and analysis in Figure 1, the findings have not changed.

(2) While "Traditional conceptions of ageing implicate a progressive accumulation of damage leading to systemic degradation in performance until death, with evolutionary pressures acting to maximise early life fitness and fecundity at the expense of ageing health." is tangential perhaps to the data and conclusions of the study, both claims of this sentence are at best controversial, and the manuscript is no weaker for their omission.

We actually feel that this sentence is very important to the message of the manuscript, which is that ageing does not necessarily have to involve a loss of fitness before death. Ageing is often described as the progressive wearing out of components leading to decline and death (with an old car often used as an analogy); in the ageing field this is certainly controversial, but outside the field this remains the normal understanding. We think it is important to state this widely held viewpoint with which our findings are hard to reconcile.

Our interpretation that yeast are bet-hedging as a population growth strategy and this drives ageing in the long term is a classic antagonistic pleiotropy - we will add this term (from the citation that is already in the manuscript) and clarify in the discussion to make it obvious why we are introducing this concept in the introduction.

(3) The statement that "Here, we determine the basis of senescence and fitness loss in replicatively ageing yeast" is a bit strong as a summary of the present careful work presented here. If the authors had created yeast mutants that retained fitness indefinitely, this would be a more appropriate strength of claim to summarize the work.

Indeed - we will refine this sentence.