Abstract

Aging of multicellular and unicellular eukaryotic organisms is a multifactorial biological phenomenon that has various causes and affects a plethora of cellular activities. I employ the yeast Saccharomyces cerevisiae as a model to study the basic biology and molecular mechanisms of cellular aging in multicellular eukaryotes. The use of this budding yeast as an advantageous model organism in aging research greatly contributed to the current understanding of the molecular and cellular mechanisms underlying longevity regulation in evolutionarily distant eukaryotic organisms, thereby convincingly demonstrating that longevity signaling pathways and mechanisms of their modulation by dietary and pharmacological interventions are conserved across phyla.
To address the inherent complexity of aging from a systems perspective and to build an integrative spatiotemporal model of aging process, I investigated the effect of caloric restriction (CR), a low-calorie dietary regimen, on the metabolic history of chronologically aging yeast. CR has been shown not only to exhibit a robust longevity-extending effect in evolutionarily distant organisms ranging from yeast to rhesus monkeys, but also to improve health by attenuating age-related pathologies and delaying the onset of age-related diseases across phyla. I examined how CR influences the age-related dynamics of changes in the intracellular levels of numerous proteins and metabolites, carbohydrate metabolism, interorganellar metabolic flow, concentration of reactive oxygen species (ROS), mitochondrial morphology, essential oxidation-reduction processes in mitochondria, mitochondrial proteome, frequency of mitochondrial DNA mutations, dynamics of mitochondrial nucleoid, susceptibility to mitochondria-controlled apoptosis, and stress resistance.
Based on my comparison of the metabolic histories of long-lived CR yeast and short-lived non-CR yeast, I concluded that yeast define their long-term viability by designing a diet-specific pattern of metabolism and organelle dynamics prior to reproductive maturation. My data imply that longevity in chronologically aging yeast is programmed by the level of metabolic capacity and organelle organization they developed, in a diet-specific fashion, prior to entry into a non-proliferative state. Therefore, chronological aging in yeast is the final step of a developmental program progressing through a series of checkpoints.
I designed a chemical genetic screen for small molecules that increase the chronological life span (CLS) of yeast under CR by targeting lipid metabolism and modulating housekeeping longevity pathways that regulate longevity irrespective of the number of available calories. My screen identifies lithocholic acid (LCA) as one of such molecules. My evaluation of the life-extending efficacy of LCA in wild-type (WT) strain on a high- or low-calorie diet revealed that this compound extends yeast CLS irrespective of the number of available calories. I found that the extent to which LCA extends longevity is highest under CR conditions, when the pro-aging processes modulated by the adaptable target of rapamycin (TOR) and cAMP/protein kinase A (cAMP/PKA) signaling pathways are suppressed and the anti-aging processes are activated. Furthermore, the life-extending efficacy of LCA in CR yeast significantly exceeded that in yeast on a high-calorie diet, in which the adaptable TOR and cAMP/PKA pathways greatly activate the pro-aging processes and suppress the anti-aging processes. Altogether, my findings suggest that, consistent with its sought-after effect on a longevity signaling network, LCA mostly targets certain housekeeping longevity assurance pathways that do not overlap (or only partially overlap) with the adaptable TOR and cAMP/PKA pathways modulated by calorie availability.
Consistent with my assumption that LCA extends longevity not by modulating the adaptable TOR pathway, I found that lack of Tor1p does not impair the life-extending efficacy of LCA under CR. I also revealed that, LCA extends longevity of the tor1 mutant strain to a very similar degree under CR and non-CR conditions. Thus, by eliminating a master regulator of this key adaptable pathway that shortens the CLS of yeast on a high-calorie diet, the tor1 mutation abolished the dependence of the anti-aging efficacy of LCA on the number of available calories.
My findings revealed 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. I provide evidence that LCA modulates housekeeping longevity assurance pathways by 1) attenuating mitochondrial fragmentation, a hallmark event of age-related cell death; 2) altering oxidation-reduction processes in mitochondria, including oxygen consumption, the maintenance of membrane potential, and reactive oxygen species production; 3) enhancing resistance to oxidative and thermal stresses; 4) suppressing mitochondria-controlled apoptosis; and 5) enhancing stability of nuclear and mitochondrial DNA.
Yeast do not synthesize LCA or any other bile acids produced by mammals. Therefore, I 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. I 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, I 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. I extend my hypothesis on longevity regulation by bile acids by suggesting a hypothesis of the xenohormetic, hormetic and cytostatic selective forces driving the evolution of longevity regulation mechanisms at the ecosystemic level.
To verify my hypothesis empirically, I carried out the LCA-driven multistep selection of long-lived yeast species under laboratory conditions. I found that a lasting exposure of wild-type yeast to LCA results in selection of yeast species that live longer in the absence of this bile acid than their ancestor. My data enabled to rank different concentrations of LCA with respect to the efficiency with which they cause the appearance of long-lived yeast species.
Aging is one of the major risk factors in the onset and incidence of cancer, and cancer is considered as one of the numerous age-associated diseases whose onset can be delayed and incidence reduced by anti-aging interventions. The interplay between aging and cancer is intricate as these two complex and dynamic biological phenomena have both convergent and divergent underlying mechanisms. One of the major objectives of my thesis was to examine if LCA, a novel anti-aging compound that I identified in a high-throughput chemical genetic screen, also exhibits an anti-tumor effect in cultured human cancer cells by activating certain anti-cancer processes that may (or may not) play an essential role in cellular aging. As a model system for addressing this important question (and if LCA indeed displays an anti-tumor effect, for establishing the mechanism underlying such effect), I choose several cell lines of the human neuroblastoma (NB) tumor.
My findings provide strong evidence that LCA exhibits a potent anti-tumor effect in cultured human NB cells by: 1) activating both intrinsic (mitochondrial) and extrinsic (death receptor) pathways of apoptotic death in these cells; 2) sensitizing them to hydrogen peroxide-induced apoptotic death; and 3) preventing growth and proliferation of their neighbouring NB cells in the culture. Importantly, my findings also imply that LCA does not display any of these deleterious effects in human neurons and, therefore, is a selective anti-tumor compound. Moreover, my mass spectrometry-based measurement of intracellular and extracellular levels of exogenously added LCA revealed that this bile acid does not enter cultured NB cells. Thus, LCA prevents proliferation of human NB cells and selectively kills these cancer cells by binding to their surface and then initiating intracellular signaling cascades that not only impair their growth and division, but also cause their apoptotic death. The demonstrated inability of LCA to enter cultured human NB cells suggests that this potent and selective anti-cancer compound is unlikely to display undesirable side effects in non-cancerous human neurons. My findings suggest a mechanism underlying a potent and selective anti-tumor effect of LCA in human NB cancer cells.