The consequences of mutator-driven mutagenesis and analysis of lifespan extending compounds using outgrowth analysis and replicative lifespan in Saccharomyces cerevisiae

Abstract

The hope of dramatically extending our lifespan has captivated humanity for millennia. Over the last two decades, the biology of aging has matured as a field of study and led to greater engagement and investment in aging as a biological problem that can be understood at the molecular level and treated. Translational Geroscience is an interdisciplinary field descended from basic gerontology that seeks to identify, validate, and clinically apply interventions to maximize healthy, disease-free lifespan. To identify and validate interventions, a translational geroscience research pipeline is proposed that begins with identifying and characterizing interventions in wild type model systems (either invertebrate or vertebrate), validating these interventions using models of genetic diversity and disease models, and finally, testing validated interventions in companion animals and humans (Chapter one). A model of mutator-driven mutagenesis, a potent mechanism to induce genetic diversity in cancer and other microbial populations, using budding yeast is then described. Defects in DNA polymerase δ or ε proofreading alone or combined with defects in mismatch repair are used to model mutator phenotypes and mutator-driven mutation burden in haploid and diploid yeast. This model is used to understand the effects of active mutagenesis and accrued mutation burden on cellular aging (Chapter two). Next, a new system to identify chemical inhibitors of mechanistic Target Of Rapamycin (mTOR) is described. This yeast outgrowth based system measures sensitivity of WT and mutant strains sensitized to mTOR inhibition. A set of nutraceutical compounds were screened using this assay. Of these compounds, caffeine was confirmed to be an mTOR inhibitor (Chapter three). Lastly, this set of nutraceuticals were screened for changes in yeast replicative lifespan (RLS). Two treatments, berberine and green tea extract, reduced RLS. Only one treatment, Pterocarpus marsupium extract (PME), extended cellular lifespan. Within this extract are two molecules with reported healthspan and lifespan properties: pterostilbene and (-)-epicatechin. We tested concentrations of these compounds comparable to those found in PME but did not recapitulate extended lifespan (Chapter four).