Mechanisms Underlying Longevity Regulation and Extension by Genetic, Dietary and Pharmacological Interventions in the Yeast Saccharomyces Cerevisiae

This thesis describes studies in which the yeast Saccharomyces cerevisiae was used as a model organism for unveiling the mechanisms underlying longevity regulation and extension by genetic, dietary and pharmacological interventions. We found that a diet known as caloric restriction (CR) modulates oxidation-reduction processes and reactive oxygen species (ROS) production in yeast mitochondria, reduces the frequency of mitochondrial DNA (mtDNA) mutations, and alters the abundance and mtDNA-binding activity of mitochondrial nucleoid-associated proteins. Our findings provide evidence that these mitochondrial processes play essential roles in regulating longevity of chronologically active yeast by defining their viability following cell entry into a quiescent state. Based on these findings, we propose a hypothesis that ROS, which are mostly generated as by-products of mitochondrial respiration, play a dual role in regulating longevity of chronologically aging yeast. On the one hand, if yeast mitochondria are unable (due to a dietary regimen) to maintain ROS concentration below a toxic threshold, ROS promote aging by oxidatively damaging certain mitochondrial proteins and mtDNA. On the other hand, if yeast mitochondria can (due to a dietary regimen) maintain ROS concentration at a certain “optimal” level, ROS delay chronological aging. We propose that this “optimal” level of ROS is insufficient to damage cellular macromolecules but can activate certain signaling networks that extend lifespan by increasing the abundance or activity of stress-protecting and other anti-aging proteins. In addition, studies presented in this thesis imply that mtDNA mutations do not contribute to longevity regulation in yeast grown under non-CR conditions but make important contribution to longevity regulation in yeast placed on a CR diet. The nonreducing disaccharide trehalose has been long considered only as a reserve carbohydrate. However, recent studies in yeast suggested that this osmolyte can protect cells and cellular proteins from oxidative damage elicited by exogenously added ROS. Trehalose has been also shown to affect stability, folding and aggregation of bacterial and firefly proteins heterologously expressed in heat-shocked yeast cells. Our investigation of how a lifespan-extending CR diet alters the metabolic history of chronologically aging yeast suggested that their longevity is programmed by the level of metabolic capacity - including trehalose biosynthesis and degradation - that yeast cells developed prior to entry into quiescence. To investigate whether trehalose homeostasis in chronologically aging yeast may play a role in longevity extension by CR, we examined how single-genedeletion mutations affecting trehalose biosynthesis and degradation impact 1) the agerelated dynamics of changes in trehalose concentration; 2) yeast chronological lifespan under CR conditions; 3) the chronology of oxidative protein damage, intracellular ROS level and protein aggregation; and 4) the timeline of thermal inactivation of a protein in heat-shocked yeast cells and its subsequent reactivation in yeast returned to low temperature. Our data imply that CR extends yeast chronological lifespan by altering a pattern of age-related changes in trehalose concentration

Xenohormetic, hormetic and cytostatic selective forces driving longevity at the ecosystemic level

We recently found that lithocholic acid (LCA), a bile acid, extends yeast longevity. Unlike mammals, yeast do not synthesize bile acids. We therefore propose that bile acids released into the environment by mammals may act as interspecies chemical signals providing longevity benefits to yeast and, perhaps, other species within an ecosystem

A signal from inside the peroxisome initiates its division by promoting the remodeling of the peroxisomal membrane

We define the dynamics of spatial and temporal reorganization of the team of proteins and lipids serving peroxisome division. The peroxisome becomes competent for division only after it acquires the complete set of matrix proteins involved in lipid metabolism. Overloading the peroxisome with matrix proteins promotes the relocation of acyl-CoA oxidase (Aox), an enzyme of fatty acid β-oxidation, from the matrix to the membrane. The binding of Aox to Pex16p, a membrane-associated peroxin required for peroxisome biogenesis, initiates the biosynthesis of phosphatidic acid and diacylglycerol (DAG) in the membrane. The formation of these two lipids and the subsequent transbilayer movement of DAG initiate the assembly of a complex between the peroxins Pex10p and Pex19p, the dynamin-like GTPase Vps1p, and several actin cytoskeletal proteins on the peroxisomal surface. This protein team promotes membrane fission, thereby executing the terminal step of peroxisome division

Chemical genetic screen identifies lithocholic acid as an anti-aging compound that extends yeast chronological life span in a TOR-independent manner, by modulating housekeeping longevity assurance processes

In chronologically aging yeast, longevity can be extended by administering a caloric restriction (CR) diet or some small molecules. These life-extending interventions target the adaptable target of rapamycin (TOR) and cAMP/protein kinase A (cAMP/PKA) signaling pathways that are under the stringent control of calorie availability. We designed a chemical genetic screen for small molecules that increase the chronological life span of yeast under CR by targeting lipid metabolism and modulating housekeeping longevity pathways that regulate longevity irrespective of the number of available calories. Our screen identifies lithocholic acid (LCA) as one of such molecules. We reveal two mechanisms underlying the life-extending effect of LCA in chronologically aging yeast. One mechanism operates in a calorie availability-independent fashion and involves the LCA-governed modulation of housekeeping longevity assurance pathways that do not overlap with the adaptable TOR and cAMP/PKA pathways. The other mechanism extends yeast longevity under non-CR conditions and consists in LCA-driven unmasking of the previously unknown anti-aging potential of PKA. We provide evidence that LCA modulates housekeeping longevity assurance pathways by suppressing lipid-induced necrosis, attenuating mitochondrial fragmentation, altering oxidation-reduction processes in mitochondria, enhancing resistance to oxidative and thermal stresses, suppressing mitochondria-controlled apoptosis, and enhancing stability of nuclear and mitochondrial DNA

Chemical genetic screen identifies lithocholic acid as an anti-aging compound that extends yeast chronological life span in a TOR-independent manner, by modulating housekeeping longevity assurance processes

In chronologically aging yeast, longevity can be extended by administering a caloric restriction (CR) diet or some small molecules. These life-extending interventions target the adaptable target of rapamycin (TOR) and cAMP/protein kinase A (cAMP/PKA) signaling pathways that are under the stringent control of calorie availability. We designed a chemical genetic screen for small molecules that increase the chronological life span of yeast under CR by targeting lipid metabolism and modulating housekeeping longevity pathways that regulate longevity irrespective of the number of available calories. Our screen identifies lithocholic acid (LCA) as one of such molecules. We reveal two mechanisms underlying the life-extending effect of LCA in chronologically aging yeast. One mechanism operates in a calorie availability-independent fashion and involves the LCA-governed modulation of housekeeping longevity assurance pathways that do not overlap with the adaptable TOR and cAMP/PKA pathways. The other mechanism extends yeast longevity under non-CR conditions and consists in LCA-driven unmasking of the previously unknown anti-aging potential of PKA. We provide evidence that LCA modulates housekeeping longevity assurance pathways by suppressing lipid-induced necrosis, attenuating mitochondrial fragmentation, altering oxidation-reduction processes in mitochondria, enhancing resistance to oxidative and thermal stresses, suppressing mitochondria-controlled apoptosis, and enhancing stability of nuclear and mitochondrial DNA

Parallel Profiling of Fission Yeast Deletion Mutants for Proliferation and for Lifespan During Long-Term Quiescence

Genetic factors underlying aging are remarkably conserved from yeast to human. The fission yeast Schizosaccharomyces pombe is an emerging genetic model to analyze cellular aging. Chronological lifespan (CLS) has been studied in stationary-phase yeast cells depleted for glucose, which only survive for a few days. Here, we analyzed CLS in quiescent S. pombe cells deprived of nitrogen, which arrest in a differentiated, G0-like state and survive for more than 2 months. We applied parallel mutant phenotyping by barcode sequencing (Bar-seq) to assay pooled haploid deletion mutants as they aged together during longterm quiescence. As expected, mutants with defects in autophagy or quiescence were under-represented or not detected. Lifespan scores could be calculated for 1199 mutants. We focus the discussion on the 48 most long-lived mutants, including both known aging genes in other model systems and genes not previously implicated in aging. Genes encoding membrane proteins were particularly prominent as pro-aging factors. We independently verified the extended CLS in individual assays for 30 selected mutants, showing the efficacy of the screen. We also applied Bar-seq to profile all pooled deletion mutants for proliferation under a standard growth condition. Unlike for stationary-phase cells, no inverse correlation between growth and CLS of quiescent cells was evident. These screens provide a rich resource for further studies, and they suggest that the quiescence model can provide unique, complementary insights into cellular aging

Uncultured Microbial Phyla Suggest Mechanisms for Multi-Thousand-Year Subsistence in Baltic Sea Sediments

Energy-starved microbes in deep marine sediments subsist at near-zero growth for thousands of years, yet the mechanisms for their subsistence are unknown because no model strains have been cultivated from most of these groups. We investigated Baltic Sea sediments with single-cell genomics, metabolomics, metatranscriptomics, and enzyme assays to identify possible subsistence mechanisms employed by uncultured Atribacteria, Aminicenantes, Actinobacteria group OPB41, Aerophobetes, Chloroflexi, Deltaproteobacteria, Desulfatiglans, Bathyarchaeota, and Euryarchaeota marine group II lineages. Some functions appeared to be shared by multiple lineages, such as trehalose production and NAD+-consuming deacetylation, both of which have been shown to increase cellular life spans in other organisms by stabilizing proteins and nucleic acids, respectively. Other possible subsistence mechanisms differed between lineages, possibly providing them different physiological niches. Enzyme assays and transcripts suggested that Atribacteria and Actinobacteria group OPB41 catabolized sugars, whereas Aminicenantes and Atribacteria catabolized peptides. Metabolite and transcript data suggested that Atribacteria utilized allantoin, possibly as an energetic substrate or chemical protectant, and also possessed energy-efficient sodium pumps. Atribacteria single-cell amplified genomes (SAGs) recruited transcripts for full pathways for the production of all 20 canonical amino acids, and the gene for amino acid exporter YddG was one of their most highly transcribed genes, suggesting that they may benefit from metabolic interdependence with other cells. Subsistence of uncultured phyla in deep subsurface sediments may occur through shared strategies of using chemical protectants for biomolecular stabilization, but also by differentiating into physiological niches and metabolic interdependencies

Purification of Mitochondria from Yeast Cells

Mitochondria are the main site of ATP production during aerobic metabolism in eukaryotic non-photosynthetic cells1. These complex organelles also play essential roles in apoptotic cell death2, cell survival3, mammalian development4, neuronal development and function4, intracellular signalling5, and longevity regulation6. Our understanding of these complex biological processes controlled by mitochondria relies on robust methods for assessing their morphology, their protein and lipid composition, the integrity of their DNA, and their numerous vital functions. The budding yeast Saccharomyces cerevisiae, a genetically and biochemically manipulable unicellular eukaryote with annotated genome and well-defined proteome, is a valuable model for studying the molecular and cellular mechanisms underlying essential biological functions of mitochondria. For these types of studies, it is crucial to have highly pure mitochondria. Here we present a detailed description of a rapid and effective method for purification of yeast mitochondria. This method enables the isolation of highly pure mitochondria that are essentially free of contamination by other organelles and retain their structural and functional integrity after their purification. Mitochondria purified by this method are suitable for cell-free reconstitution of essential mitochondrial processes and can be used for the analysis of mitochondrial structure and functions, mitochondrial proteome and lipidome, and mitochondrial DNA