Immature Pear Extract Constituents Exert Multifaceted Anti-aging Effects

Cellular senescence causes a gradual loss of physiological functions and induces chronic diseases, which negatively affect the quality of human life. Intervention in the cellular senescence process may reduce the incidence of these diseases while delaying the progression of age-related diseases, thereby prolonging human lifespan. In our previous study, we found that extending the chronological lifespan of budding yeast cells, a suitable cellular model for research on mammalian cells, could be achieved by adding immature pear extract (iPE). Moreover, at the 2020 GSA meeting, using a colony-counting method, we reported that both hydrophilic (WiPE) and hydrophobic (OiPE) iPE components exhibited a chronological lifespan prolongation on yeast cells. In this study, the expression of sirtuin-related genes, which regulate cellular senescence, was verified by quantitative real-time reverse-transcription polymerase chain reaction. Interestingly, sirtuin-related gene expression was significantly increased in the WiPE-treated cells only, and OiPE could extend the chronological lifespan of yeast cells through the mechanisms not involved in sirtuin-related gene expression. In general, hydrophobic and hydrophilic components exhibit different degradation and metabolism in cells. Since each component has a different strategy of absorption and excretion in the body, we hypothesize that iPE with multiple active components will have multifaceted effects on anti-aging. Our research on elucidating the mechanism of lifespan extension by OiPE and its application to mammalian cells is ongoing.

The Role of Sch9 and the V-ATPase in the Adaptation Response to Acetic Acid and the Consequences for Growth and Chronological Lifespan

Studies with Saccharomyces cerevisiae indicated that non-physiologically high levels of acetic acid promote cellular acidification, chronological aging, and programmed cell death. In the current study, we compared the cellular lipid composition, acetic acid uptake, intracellular pH, growth, and chronological lifespan of wild-type cells and mutants lacking the protein kinase Sch9 and/or a functional V-ATPase when grown in medium supplemented with different acetic acid concentrations. Our data show that strains lacking the V-ATPase are especially more susceptible to growth arrest in the presence of high acetic acid concentrations, which is due to a slower adaptation to the acid stress. These V-ATPase mutants also displayed changes in lipid homeostasis, including alterations in their membrane lipid composition that influences the acetic acid diffusion rate and changes in sphingolipid metabolism and the sphingolipid rheostat, which is known to regulate stress tolerance and longevity of yeast cells. However, we provide evidence that the supplementation of 20 mM acetic acid has a cytoprotective and presumable hormesis effect that extends the longevity of all strains tested, including the V-ATPase compromised mutants. We also demonstrate that the long-lived sch9Δ strain itself secretes significant amounts of acetic acid during stationary phase, which in addition to its enhanced accumulation of storage lipids may underlie its increased lifespan.

Longevity Regulation by Proline Oxidation in Yeast

Proline is a pivotal and multifunctional amino acid that is used not only as a nitrogen source but also as a stress protectant and energy source. Therefore, proline metabolism is known to be important in maintaining cellular homeostasis. Here, we discovered that proline oxidation, catalyzed by the proline oxidase Put1, a mitochondrial flavin-dependent enzyme converting proline into ∆1-pyrroline-5-carboxylate, controls the chronological lifespan of the yeast Saccharomyces cerevisiae. Intriguingly, the yeast strain with PUT1 deletion showed a reduced chronological lifespan compared with the wild-type strain. The addition of proline to the culture medium significantly increased the longevity of wild-type cells but not that of PUT1-deleted cells. We next found that induction of the transcriptional factor Put3-dependent PUT1 and degradation of proline occur during the aging of yeast cells. Additionally, the lifespan of the PUT3-deleted strain, which is deficient in PUT1 induction, was shorter than that of the wild-type strain. More importantly, the oxidation of proline by Put1 helped maintain the mitochondrial membrane potential and ATP production through the aging period. These results indicate that mitochondrial energy metabolism is maintained through oxidative degradation of proline and that this process is important in regulating the longevity of yeast cells.

Isonicotinamide extends yeast chronological lifespan through a mechanism that diminishes nucleotides

Chronological lifespan (CLS) of budding yeast, Saccharomyces cerevisiae, is a commonly utilized model for cellular aging of non-dividing cells such as neurons. CLS is strongly extended by isonicotinamide (INAM), a non-metabolized isomer of the NAD+ precursor nicotinamide (NAM), but the underlying mechanisms of lifespan extension remain uncharacterized. To identify potential biochemical INAM targets, we performed a chemical genetic screen with the yeast gene knockout (YKO) strain collection for INAM-hypersensitive mutants. Significantly enriched Gene Ontology terms that emerged included SWR1 and other transcription elongation factors, as well as metabolic pathways converging on one-carbon metabolism and contributing to nucleotide biosynthesis, together suggesting that INAM perturbs nucleotide pools. In line with this model, INAM effects on cell growth were synergistic with mycophenolic acid (MPA), which extends lifespan by reducing guanine nucleotide pools. Direct measurements of nucleotides and precursors by mass spectrometry indicated that INAM reduced nucleotides, including cAMP, at 24- and 96-hour time points post-inoculation. Taken together, we conclude that INAM extends CLS by perturbing nucleotide metabolism, which may be a common functional feature of multiple anti-aging interventions.

Barcode sequencing and a high-throughput assay for chronological lifespan uncover ageing-associated genes in fission yeast

Ageing-related processes are largely conserved, with simple organisms remaining the main platform to discover and dissect new ageing-associated genes. Yeasts provide potent model systems to study cellular ageing owing their amenability to systematic functional assays under controlled conditions. Even with yeast cells, however, ageing assays can be laborious and resource-intensive. Here we present improved experimental and computational methods to study chronological lifespan in Schizosaccharomyces pombe. We decoded the barcodes for 3206 mutants of the latest gene-deletion library, enabling the parallel profiling of ~700 additional mutants compared to previous screens. We then applied a refined method of barcode sequencing (Bar-seq), addressing technical and statistical issues raised by persisting DNA in dead cells and sampling bottlenecks in aged cultures, to screen for mutants showing altered lifespan during stationary phase. This screen identified 341 long-lived mutants and 1246 short-lived mutants which point to many previously unknown ageing-associated genes, including 46 conserved but entirely uncharacterized genes. The ageing-associated genes showed coherent enrichments in processes also associated with human ageing, particularly with respect to ageing in non-proliferative brain cells. We also developed an automated colony-forming unit assay to facilitate medium- to high-throughput chronological-lifespan studies by saving time and resources compared to the traditional assay. Results from the Bar-seq screen showed good agreement with this new assay. This study provides an effective methodological platform and identifies many new ageing-associated genes as a framework for analysing cellular ageing in yeast and beyond.

Overexpression of a single ORF can extend chronological lifespan in yeast if retrograde signaling and stress response are stimulated

Systematic collections of single-gene deletions have been invaluable in uncovering determinants of lifespan in yeast. Overexpression of a single gene does not have such a clear outcome as cancellation of its function but it can lead to a variety of imbalances, deregulations and compensations, and some of them could be important for longevity. We report an experiment in which a genome-wide collection of strains overexpressing a single gene was assayed for chronological lifespan (CLS). Only one group of proteins, those locating to the inner membrane and matrix of mitochondria, tended to extend CLS when abundantly overproduced. We selected two such strains—one overexpressing Qcr7 of the respiratory complex III, the other overexpressing Mrps28 of the small mitoribosomal subunit—and analyzed their transcriptomes. The uncovered shifts in RNA abundance in the two strains were nearly identical and highly suggestive. They implied a distortion in the co-translational assembly of respiratory complexes followed by retrograde signaling to the nucleus. The consequent reprogramming of the entire cellular metabolism towards the resistance to stress resulted in an enhanced ability to persist in a non-proliferating state. Our results show that surveillance of the inner mitochondrial membrane integrity is of outstanding importance for the cell. They also demonstrate that overexpression of single genes could be used effectively to elucidate the mitochondrion-nucleus crosstalk.