Abstract

ABSTRACT
Using the Experimental Evolution of Long-Lived Yeast Species for Testing Evolutionary Theories of Aging

Anastasia Glebov, M.Sc.

Aging of multicellular and unicellular eukaryotic organisms is a highly complex biological phenomenon that has various causes and affects numerous processes within cells. As a model organism for elucidating the basic biology and molecular mechanisms of cellular aging in multicellular eukaryotes, we use the baker’s yeast Saccharomyces cerevisiae. The employment of this budding yeast as an advantageous model organism in aging research during the past decade has convincingly demonstrated that longevity signaling pathways and mechanisms of their modulation by dietary and pharmacological interventions are conserved across phyla.

Recently, we designed a chemical genetic screen for small molecules that increase the chronological lifespan (CLS) of yeast under caloric restriction (CR) conditions by targeting lipid metabolism and modulating housekeeping longevity pathways that regulate longevity irrespective of the number of available calories. Our high-throughput screen identified a bile acid called lithocholic acid (LCA) as one of such molecules. The results of our pharmacophore modeling of the anti-aging potential of various species of bile acids imply that the maintenance of the minimal polarity of both the hydrophilic (concave) and hydrophobic (convex) sides of the steroid nucleus - by avoiding the presence of polar substituents at the positions 6, 7 and 12 - is mandatory for the extreme life-extending efficacy of LCA under CR conditions. Such stringent structural requirements are consistent with a target specificity of LCA action as an anti-aging small molecule. We found that the life-extending efficacy of LCA under CR exceeds that under non-CR conditions, being inversely proportional to the concentration of glucose in growth medium and thus in correlation with the extent of calorie supply limitation.

Yeast do not synthesize LCA or any other bile acids produced by mammals. Therefore, we 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. We hypothesize that, because bile acids are known to be mildly toxic compounds, they may create selective pressure for the evolution of yeast species that can respond to the bile acids-induced mild cellular damage by developing the most efficient stress protective mechanisms. It is likely that such mechanisms may provide effective protection of yeast against molecular and cellular damage accumulated with age. Thus, we propose that yeast species that have been selected for the most effective mechanisms providing protection against bile acids may evolve the most effective anti-aging mechanisms that are sensitive to regulation by bile acids. We analyzed how small anti-aging molecules other than LCA (including resveratrol, caffeine and rapamycin) synthesized and released into the environment by one species of the organisms composing an ecosystem extend longevity of many other species within this ecosystem. Based on such analysis, we extended our initial hypothesis on how bile acids govern longevity regulation and drive longevity evolution by suggesting a unified hypothesis of the xenohormetic, hormetic and cytostatic selective forces that impel the evolution of longevity regulation mechanisms at the ecosystemic level.

To test the validity of our hypothesis, we carried out the LCA-driven experimental evolution of long-lived yeast species. Our serial-batch-transfer type of selection yielded three laboratory-evolved yeast strains with greatly extended lifespan. All these strains were able to maintain their prolonged lifespan following storage at -80oC and multiple successive passages in medium without LCA. We demonstrated that in the absence of LCA a mutant allele or alleles selected during the LCA-driven multistep selection process of long-lived yeast species extend longevity and alter the age-dependent dynamics of mitochondrial respiration under both CR and non-CR conditions. We therefore concluded that, consistent with its sought-after effect on longevity regulation pathways, our LCA-driven multistep selection process under laboratory conditions yielded long-lived yeast species whose greatly delayed chronological aging was caused by the selection of a mutant allele or alleles that activate housekeeping longevity assurance pathways. As we recently demonstrated, these housekeeping longevity assurance pathways modulate longevity irrespective of the number of available calories and do not overlap with the adaptable longevity pathways that are under the stringent control of calorie availability [167].

We also revealed that under CR conditions in the absence of LCA a mutant allele or alleles selected during the LCA-driven multistep selection process of long-lived yeast species: 1) enhance the resistance of yeast to chronic oxidative, thermal and osmotic stresses; 2) suppress mitochondria-controlled apoptotic cell death triggered by exogenously added hydrogen peroxide; and 3) attenuate lipid-induced necrotic cell death induced by exogenously added palmitoleic acid. All these processes are known to be governed by the housekeeping longevity assurance pathways that modulate longevity irrespective of the number of available calories [167].

Moreover, the experiments described in this Master’s thesis revealed that the addition of LCA to the laboratory-selected long-lived yeast species cultured under CR conditions 1) further extends their CLS, although to a lesser degree than that of wild-type (WT) strain; 2) causes further changes in the age-dependent dynamics of mitochondrial respiration, although not as dramatic as those seen in WT; 3) further enhances the resistance of yeast to chronic oxidative, thermal and osmotic stresses, although to a lesser extent than those observed in WT; 4) further suppresses mitochondria-controlled apoptotic cell death triggered by exogenously added hydrogen peroxide, although to a lesser degree than those seen in WT; and 5) further attenuates lipid-induced necrotic cell death induced by exogenously added palmitoleic acid, although to a lesser extent than those observed in WT. These findings imply that, although a mutant allele or alleles selected during the LCA-driven multistep selection process of long-lived yeast species greatly impact a compendium of the LCA-sensing housekeeping longevity assurance processes, this allele or alleles do not activate such processes sufficiently enough to attain the maximal CLS achievable under life-extending CR conditions in the presence of LCA.

Altogether, these findings provide the comprehensive evidence of our hypothesis in which the evolution of longevity regulation mechanisms within an ecosystem can be driven by the xenohormetic, hormetic and cytostatic selective forces created by a lasting exposure of an organism to an anti-aging natural compound released by other organisms composing this ecosystem.