Fission Yeast Lifespan Research Articles

Factors governing the transcriptome changes and chronological lifespan of fission yeast during phosphate starvation

Starvation of Schizosaccharomyces pombe for inorganic phosphate elicits adaptive transcriptome changes in which mRNAs driving ribosome biogenesis, tRNA biogenesis, and translation are globally downregulated while those for autophagy and phosphate mobilization are upregulated. Here, we interrogated three components of the starvation response: upregulated autophagy; the role of transcription factor Pho7 (an activator of the PHO regulon); and upregulated expression of ecl3, one of three paralogous genes (ecl1, ecl2, ecl3) collectively implicated in cell survival during other nutrient stresses. Ablation of autophagy factor Atg1 resulted in early demise of phosphate-starved fission yeast, as did ablation of Pho7. Transcriptome profiling of phosphate-starved pho7Δ cells highlighted Pho7 as an activator of genes involved in phosphate acquisition and mobilization, not limited to the original three-gene PHO regulon, and additional starvation-induced genes (including ecl3) not connected to phosphate dynamics. Pho7-dependent gene induction during phosphate starvation tracked with the presence of Pho7 DNA-binding elements in the gene promoter regions. Fewer ribosome protein genes were downregulated in phosphate-starved pho7Δ cells versus wild-type, which might contribute to their shortened lifespan. An ecl3Δ mutant elicited no gene expression changes in phosphate-replete cells and had no impact on survival during phosphate starvation. By contrast, pan-ecl deletion (ecl123Δ) curtailed lifespan during chronic phosphate starvation. Phosphate-starved ecl123Δ cells experienced a more widespread downregulation of mRNAs encoding aminoacyl tRNA synthetases vis-à-vis wild-type or pho7Δ cells. Collectively, these results enhance our understanding of fission yeast phosphate homeostasis and survival during nutrient deprivation.

Metformin extends the chronological lifespan of fission yeast by altering energy metabolism and stress resistance capacity.

The antiaging properties of metformin used for the treatment of type-2 diabetes mellitus have been studied extensively, but there is more to discover regarding underlying mechanisms. Here, we show that metformin significantly prolongs the chronological lifespan (CLS) of Schizosaccharomyces pombe through mechanisms similar to those observed in mammalian cells and other model organisms. While the presence of metformin in the medium caused an increase in carbohydrate consumption and ATP production, it reduced reactive oxygen species production and alleviate oxidative damage parameters such as lipid peroxidation and carbonylated proteins. We also tested whether the effect of metformin changed with the time it was added to the medium and observed that the lifespan-prolonging effect of metformin was related to the glucose concentration in the medium and did not prolong lifespan when added after glucose was completely depleted in the medium. On the other hand, cells inoculated in glucose-free medium containing metformin also showed extended lifespan suggesting that mechanisms other than that solely depend on glucose availability may be involved in extending the lifespan. These results suggest that metformin prolongs lifespan especially affecting energy metabolism and stress resistance capacity and that fission yeast can be effectively used when investigating the antiaging mechanisms of metformin.

Absence of the Rpb9 subunit of RNA polymerase II reduces the chronological life span in fission yeast.

Fission yeast RNA polymerase II consists of 12 subunits, Rpb1-Rpb12. Among these subunits, Rpb9 is the only subunit whose absence does not cause lethality under optimum growth conditions in fission yeast. However, an rpb9 null fission yeast mutant exhibits a slow-growth phenotype under optimum growth conditions and a defect in survival under environmental and genotoxic stress conditions. To further gain an understanding of its physiological roles, in the present study we have elucidated the role of the Rpb9 subunit in chronological aging using fission yeast as the model organism. Our results provide evidence that the absence of Rpb9 reduces the chronological life span in fission yeast. Our data further shows that lack of Rpb9 in fission yeast causes oxidative stress sensitivity and accumulation of reactive oxygen species during the stationary phase. Our domain mapping experiments have demonstrated that the Rpb9 region encompassing its amino-terminal zinc finger domain and the central linker region is important for the role of Rpb9 in chronological aging. Finally, we also show that expression of the budding yeast or human Rpb9 ortholog can functionally complement the reduced chronological life span phenotype of the fission yeast rpb9 deletion mutant. Taken together, our study has identified a new role of the Rpb9 subunit in chronological aging.

Expression of Huntingtin and TDP-43 Derivatives in Fission Yeast Can Cause Both Beneficial and Toxic Effects.

Many neurodegenerative disorders display protein aggregation as a hallmark, Huntingtin and TDP-43 aggregates being characteristic of Huntington disease and amyotrophic lateral sclerosis, respectively. However, whether these aggregates cause the diseases, are secondary by-products, or even have protective effects, is a matter of debate. Mutations in both human proteins can modulate the structure, number and type of aggregates, as well as their toxicity. To study the role of protein aggregates in cellular fitness, we have expressed in a highly tractable unicellular model different variants of Huntingtin and TDP-43. They each display specific patterns of aggregation and toxicity, even though in both cases proteins have to be very highly expressed to affect cell fitness. The aggregation properties of Huntingtin, but not of TDP-43, are affected by chaperones such as Hsp104 and the Hsp40 couple Mas5, suggesting that the TDP-43, but not Huntingtin, derivatives have intrinsic aggregation propensity. Importantly, expression of the aggregating form of Huntingtin causes a significant extension of fission yeast lifespan, probably as a consequence of kidnapping chaperones required for maintaining stress responses off. Our study demonstrates that in general these prion-like proteins do not cause toxicity under normal conditions, and in fact they can protect cells through indirect mechanisms which up-regulate cellular defense pathways.

Lithium can mildly increase health during ageing but not lifespan in mice.

Lithium is a nutritional trace element, used clinically as an anti‐depressant. Preclinically, lithium has neuroprotective effects in invertebrates and mice, and it can also extend lifespan in fission yeast, C. elegans and Drosophila. An inverse correlation of human mortality with the concentration of lithium in tap water suggests a possible, evolutionarily conserved mechanism mediating longevity. Here, we assessed the effects of lithium treatment on lifespan and ageing parameters in mice. Lithium has a narrow therapeutic dose range, and overdosing can severely affect organ health. Within the tolerable dosing range, we saw some mildly positive effects of lithium on health span but not on lifespan.

Identification of sur2 mutation affecting the lifespan of fission yeast.

Yeast is a suitable model system to analyze the mechanism of lifespan. In this study, to identify novel factors involved in chronological lifespan, we isolated a mutant with a long chronological lifespan and found a missense mutation in the sur2+ gene, which encodes a homolog of Saccharomyces cerevisiae sphingolipid C4-hydroxylase in fission yeast. Characterization of the mutant revealed that loss of sur2 function resulted in an extended chronological lifespan. The effect of caloric restriction, a well-known signal for extending lifespan, is thought to be dependent on the sur2+ gene.

Magnesium depletion extends fission yeast lifespan via general amino acid control activation.

Nutrients including glucose, nitrogen, sulfur, zinc, and iron are involved in the regulation of chronological lifespan (CLS) of yeast, which serves as a model of the lifespan of differentiated cells of higher organisms. Herein, we show that magnesium (Mg2+) depletion extends CLS of the fission yeast Schizosaccharomyces pombe through a mechanism involving the Ecl1 gene family. We discovered that ecl1 + expression, which extends CLS, responds to Mg2+ depletion. Therefore, we investigated the underlying intracellular responses. In amino acid auxotrophic strains, Mg2+ depletion robustly induces ecl1 + expression through the activation of the general amino acid control (GAAC) pathway—the equivalent of the amino acid response of mammals. Polysome analysis indicated that the expression of Ecl1 family genes was required for regulating ribosome amount when cells were starved, suggesting that Ecl1 family gene products control the abundance of ribosomes, which contributes to longevity through the activation of the evolutionarily conserved GAAC pathway. The present study extends our understanding of the cellular response to Mg2+ depletion and its influence on the mechanism controlling longevity.

Amino Acids Whose Intracellular Levels Change Most During Aging Alter Chronological Life Span of Fission Yeast.

Amino acid deprivation or supplementation can affect cellular and organismal life span, but we know little about the role of concentration changes in free, intracellular amino acids during aging. Here, we determine free amino acid levels during chronological aging of nondividing fission yeast cells. We compare wild-type with long-lived mutant cells that lack the Pka1 protein of the protein kinase A signalling pathway. In wild-type cells, total amino acid levels decrease during aging, but much less so in pka1 mutants. Two amino acids strongly change as a function of age: glutamine decreases, especially in wild-type cells, while aspartate increases, especially in pka1 mutants. Supplementation of glutamine is sufficient to extend the chronological life span of wild-type but not of pka1Δ cells. Supplementation of aspartate, on the other hand, shortens the life span of pka1Δ but not of wild-type cells. Our results raise the possibility that certain amino acids are biomarkers of aging, and their concentrations during aging can promote or limit cellular life span.

Natural extract and its fractions isolated from the marine bacterium Pseudoalteromonas flavipulchra STILL-33 have antioxidant and antiaging activities in Schizosaccharomyces pombe.

Investigations into the potential for pharmacological inhibition of the aging process and the onset of age-related disease are increasingly garnering attention. Here, we analyzed the antiaging properties of natural compounds derived from several marine bacteria in vitro and in vivo using the fission yeast Schizosaccharomyces pombe. The Pseudoalteromonas flavipulchra STILL-33 extract exhibited high antioxidant and antiglycation activities in vitro. We then characterized two antioxidant active fractions isolated from this extract. In addition, we showed that the P. flavipulchra STILL-33 extract or either of its two active fractions (Fractions 1 and 2) could extend the longevity of fission yeast. Moreover, the particular extract and two active fractions were found to induce mitochondrial activity and to delay the G1 phase of the fission yeast cell cycle, perhaps by improving the aging process. The P. flavipulchra STILL-33 extract and Fraction 1 also increased the expression of the catalase-encoding ctt1+ gene and thereby decreased the reactive oxygen species level. Structural analysis showed that Fraction 1 was dominated by l-arginine and ipriflavone, and we showed indeed that the two corresponding commercial products increase the fission yeast lifespan. As for Fraction 2 was identified as the putative structure of butamben. Together, these results should facilitate the discovery of additional antiaging compounds from P. flavipulchra and ultimately the development of novel antiaging compounds for pharmaceutical use.

Gas1 mutation extends chronological lifespan via Pmk1 and Sty1 MAPKs in Schizosaccharomyces pombe.

In the longevity research by using yeasts, chronological lifespan is defined as the survival time after entry into stationary phase. Previously, screening for long lived mutants of Schizosaccharomyces pombe was performed to identify the novel factors involved in longevity. From this screening, one long lived mutant called as No.36 was obtained. In this study, we identified the mutation caused in gas1+, which encodes glucanosyltransferase (gas1-287 mutation) is responsible for the longevity of No.36 mutant. Through the analysis of this mutant, we found that cell wall perturbing agent micafungin also extends chronological lifespan in fission yeast. This lifespan extension depended on both Pmk1 and Sty1 MAP kinases, and longevity caused by the gas1-287 mutation also depended on these kinases. In summary, we propose that the gas1-287 mutation causes longevity as the similar mechanism as cell wall stress depending on Pmk1 and Sty1 MAPK pathways.

Maf1-dependent transcriptional regulation of tRNAs prevents genomic instability and is associated with extended lifespan.

Maf1 is the master repressor of RNA polymerase III responsible for transcription of tRNAs and 5S rRNAs. Maf1 is negatively regulated via phosphorylation by the mTOR pathway, which governs protein synthesis, growth control, and lifespan regulation in response to nutrient availability. Inhibiting the mTOR pathway extends lifespan in various organisms. However, the downstream effectors for the regulation of cell homeostasis that are critical to lifespan extension remain elusive. Here we show that fission yeast Maf1 is required for lifespan extension. Maf1’s function in tRNA repression is inhibited by mTOR‐dependent phosphorylation, whereas Maf1 is activated via dephosphorylation by protein phosphatase complexes, PP4 and PP2A. Mutational analysis reveals that Maf1 phosphorylation status influences lifespan, which is correlated with elevated tRNA and protein synthesis levels in maf1∆ cells. However, mTOR downregulation, which negates protein synthesis, fails to rescue the short lifespan of maf1∆ cells, suggesting that elevated protein synthesis is not a cause of lifespan shortening in maf1∆ cells. Interestingly, maf1∆ cells accumulate DNA damage represented by formation of Rad52 DNA damage foci and Rad52 recruitment at tRNA genes. Loss of the Rad52 DNA repair protein further exacerbates the shortened lifespan of maf1∆ cells. Strikingly, PP4 deletion alleviates DNA damage and rescues the short lifespan of maf1∆ cells even though tRNA synthesis is increased in this condition, suggesting that elevated DNA damage is the major cause of lifespan shortening in maf1∆ cells. We propose that Maf1‐dependent inhibition of tRNA synthesis controls fission yeast lifespan by preventing genomic instability that arises at tRNA genes.

Ecl1 is a zinc-binding protein involved in the zinc-limitation-dependent extension of chronological life span in fission yeast.

Overexpression of Ecl1-family genes (ecl1 +, ecl2 +, and ecl3 +) results in the extension of the chronological life span in Schizosaccharomyces pombe. However, the mechanism for this extension has not been defined clearly. Ecl1-family proteins consist of approximately 80 amino acids, and four cysteine residues are conserved in their N-terminal domains. This study focused on the Ecl1 protein, mutating its cysteine residues sequentially to confirm their importance. As a result, all mutated Ecl1 proteins nearly lost the function to extend the chronological life span, suggesting that these four cysteine residues are essential for the Ecl1 protein. Utilizing ICP-AES (inductively coupled plasma atomic emission spectroscopy) analysis, we found that wild-type Ecl1 proteins contain zinc, while cysteine-mutated Ecl1 proteins do not. We also analyzed the effect of environmental zinc on the chronological life span. We found that zinc limitation extends the chronological life span, and this extension depends on the Ecl1-family proteins.

An aging-independent replicative lifespan in a symmetrically dividing eukaryote.

The replicative lifespan (RLS) of a cell-defined as the number of cell divisions before death-has informed our understanding of the mechanisms of cellular aging. However, little is known about aging and longevity in symmetrically dividing eukaryotic cells because most prior studies have used budding yeast for RLS studies. Here, we describe a multiplexed fission yeast lifespan micro-dissector (multFYLM) and an associated image processing pipeline for performing high-throughput and automated single-cell micro-dissection. Using the multFYLM, we observe continuous replication of hundreds of individual fission yeast cells for over seventy-five generations. Surprisingly, cells die without the classic hallmarks of cellular aging, such as progressive changes in size, doubling time, or sibling health. Genetic perturbations and drugs can extend the RLS via an aging-independent mechanism. Using a quantitative model to analyze these results, we conclude that fission yeast does not age and that cellular aging and replicative lifespan can be uncoupled in a eukaryotic cell.

Identification of a novel protein kinase that affects the chronological lifespan in fission yeast.

Chronological lifespan is defined by how long a cell can survive in a non-dividing state. In yeast, it is measured by viability after entry into the stationary phase. To understand the regulatory mechanisms of chronological lifespan in Schizosaccharomyces pombe, it is necessary to identify and characterize novel factors involved in the regulation of chronological lifespan. To this end, we have screened for a long-lived mutant and identified that novel gene nnk1+ that encodes an essential protein kinase is the determinant of chronological lifespan. We showed that the expression of major glucose transporter gene, ght5+, is decreased in the isolated nnk1-35 mutant, suggesting that Nnk1 protein is involved in the regulation of ght5+ The consumption of glucose in the growth medium after saturated growth was lower in the nnk1-35 mutant than that in wild-type cell. The isolated ght5 deletion mutant showed long-lived phenotype. Based on these results, we propose that Nnk1 regulates chronological lifespan through the regulation of ght5+ Nnk1 might coordinate glucose availability and lifespan in fission yeast.

Drug synergy drives conserved pathways to increase fission yeast lifespan.

Aging occurs over time with gradual and progressive loss of physiological function. Strategies to reduce the rate of functional loss and mitigate the subsequent onset of deadly age-related diseases are being sought. We demonstrated previously that a combination of rapamycin and myriocin reduces age-related functional loss in the Baker’s yeast Saccharomyces cerevisiae and produces a synergistic increase in lifespan. Here we show that the same drug combination also produces a synergistic increase in the lifespan of the fission yeast Schizosaccharomyces pombe and does so by controlling signal transduction pathways conserved across a wide evolutionary time span ranging from yeasts to mammals. Pathways include the target of rapamycin complex 1 (TORC1) protein kinase, the protein kinase A (PKA) and a stress response pathway, which in fission yeasts contains the Sty1 protein kinase, an ortholog of the mammalian p38 MAP kinase, a type of Stress Activated Protein Kinase (SAPK). These results along with previous studies in S. cerevisiae support the premise that the combination of rapamycin and myriocin enhances lifespan by regulating signaling pathways that couple nutrient and environmental conditions to cellular processes that fine-tune growth and stress protection in ways that foster long term survival. The molecular mechanisms for fine-tuning are probably species-specific, but since they are driven by conserved nutrient and stress sensing pathways, the drug combination may enhance survival in other organisms.

3D-printed microfluidic microdissector for high-throughput studies of cellular aging.

Due to their short lifespan, rapid division, and ease of genetic manipulation, yeasts are popular model organisms for studying aging in actively dividing cells. To study replicative aging over many cell divisions, individual cells must be continuously separated from their progeny via a laborious manual microdissection procedure. Microfluidics-based soft-lithography devices have recently been used to automate microdissection of the budding yeast Saccharomyces cerevisiae. However, little is known about replicative aging in Schizosaccharomyces pombe, a rod-shaped yeast that divides by binary fission and shares many conserved biological functions with higher eukaryotes. In this report, we develop a versatile multiphoton lithography method that enables rapid fabrication of three-dimensional master structures for polydimethylsiloxane (PDMS)-based microfluidics. We exploit the rapid prototyping capabilities of multiphoton lithography to create and characterize a cell-capture device that is capable of high-resolution microscopic observation of hundreds of individual S. pombe cells. By continuously removing the progeny cells, we demonstrate that cell growth and protein aggregation can be tracked in individual cells for over ~100 h. Thus, the fission yeast lifespan microdissector (FYLM) provides a powerful on-chip microdissection platform that will enable high-throughput studies of aging in rod-shaped cells.

Ecl1 is activated by the transcription factor Atf1 in response to H2O2 stress in Schizosaccharomyces pombe

The Ecl1 family genes extend the lifespan of fission yeast when overexpressed. They also cause resistance against H(2)O(2) stress. In this study, we found that the bZip transcription factor Atf1 is a direct activator of the induction of extender of chronological lifespan (ecl1 (+)) by H(2)O(2) stress. Based on ChIP analysis, we identified that Atf1 binds to the upstream DNA region of ecl1(+). Previously, we reported that overexpression of ecl1(+) increased the expression of the catalase-encoding ctt1(+). This ecl1(+)-dependent increase of ctt1(+) expression occurred in ∆atf1 mutant. On the other hand, the activation of ctt1 (+) caused by the ∆pyp1 mutation, which enhances Sty1-Atf1 activity, could occur in ∆ecl1 mutant. Based on these results, we propose that Atf1 can regulate ctt1(+) in both an Ecl1-dependent and an Ecl1-independent manner.

A new pma1 mutation identified in a chronologically long-lived fission yeast mutant

We isolated a chronologically long-lived mutant of Schizosaccharomyces pombe and found a new mutation in pma1+ that encoded for an essential P-type proton ATPase. An Asp-138 to Asn mutation resulted in reduced Pma1 activity, concomitant with an increase in the chronological lifespan of this fission yeast. This study corroborates our previous report indicating Pma1 activity is crucial for the determination of life span of fission yeast, and offers information for better understanding of the enzyme, Pma1.

Screening for long-lived genes identifies Oga1, a guanine-quadruplex associated protein that affects the chronological lifespan of the fission yeast Schizosaccharomyces pombe

Schizosaccharomyces pombe and Saccharomyces cerevisiae are excellent model organisms to study lifespan. We conducted screening to identify novel genes that, when overexpressed, extended the chronological lifespan of fission yeast. We identified seven genes, among which we focused on SPBC16A3.08c. The gene product showed similarity to Ylr150w of S. cerevisiae, which has affinity for guanine-quadruplex nucleic acids (G4). The SPBC16A3.08c product associated with G4 in vitro and complemented the phenotype of an S. cerevisiae Ylr150w deletion mutant. From these results, we proposed that SPBC16A3.08c encoded for a functional homolog of Ylr150w, which we designated ortholog of G4-associated protein (oga1 (+)). oga1 (+) overexpression extended the chronological lifespan and also decreased mating efficiency and caused both high and low temperature-sensitive growth. Deleting oga1 (+) resulted in caffeine-sensitive and canavanine-resistant phenotypes. Based on these results, we discuss the function of Oga1 on the chronological lifespan of fission yeast.

Reversal of slow growth and heartbeat through the restoration of mitochondrial function in clk-1-deficient mouse embryos by exogenous administration of coenzyme Q10

The longevity gene clk-1/coq7 encodes an enzyme that is essential for the biosynthesis of coenzyme Q (CoQ) in mitochondria and regulates the lifespan and behavioral timing in Caenorhabditis elegans and the chronological lifespan in fission yeast. However, whether the mammalian clk-1/coq7 ortholog (clk-1) regulates these phenotypes in mammals remains to be fully evaluated due to the embryonic lethality of clk-1-deficient (clk-1−/−) mice. To investigate whether clk-1 regulates biological functions, such as growth and heartbeat, through CoQ in mouse embryos, we cultivated the cells and hearts of clk-1−/− mouse embryos at embryonic day 10.5 (E10.5) for at least 10days in the presence of fetal bovine serum. In embryonic cells, cardiomyocytes, and hearts, the growth and heart rates were significantly slowed in clk-1−/− compared with wild-type or heterozygous mouse tissues. Moreover, frequent apoptosis and a significant reduction in mitochondrial functions, including membrane potential and ATP production, were observed in the clk-1−/− cells and hearts. The slowed growth and heart rates and the reduced mitochondrial function of clk-1−/− embryonic cells and hearts in culture were almost completely rescued by the administration of exogenous CoQ10. The results indicate that clk-1 regulates growth and heart rates through CoQ-mediated mitochondrial functions in mouse embryos.