Analysis of lifespan extending compounds using growth kinetics and replicative lifespan in Saccharomyces cerevisiae

Abstract

Advances in modern medicine have facilitated stunning rises in life expectancy over the last two centuries. This longer life, however, is concomitant with a gradual decline in health and the onset of multiple age-associated pathologies including heart disease, neurodegeneration and cancer. Risk of developing and dying from these age-associated diseases increases dramatically with age suggesting a common underlying cause. Targeting the aging process itself with clinical intervention may allow for amelioration of the onset and progression of multiple age-associated pathologies simultaneously. The mechanistic Target of Rapamycin (mTOR) signaling pathway has emerged as an important clinical target in this regard with its dysregulation being observed in multiple pathologies including many cancers, autoimmune disorders, heart failure, neurodegeneration and type 2 diabetes. Importantly, genetic or pharmacological inhibition of mTOR signaling results in increased life-span across evolutionary distant organisms suggesting a conserved role for this signaling pathway in longevity regulation. A novel yeast-based system to identify putative mTOR inhibitors is described. Utilizing differential growth kinetics of WT and mutant strains sensitized to mTOR perturbations, this system successfully discriminates between allosteric (i.e. rapamycin) and ATP competitive mTOR inhibitors. A number of nutraceutical compounds were screened and of these, caffeine was confirmed to be an mTOR inhibitor (Chapter two). Next a screen of natural products and natural product mixtures were screened for effects on growth rate, mTOR-mediated growth inhibition, and replicative lifespan (RLS). No mTOR inhibitors were identified but two treatments, berberine and green tea extract significantly reduced RLS. Pterocarpus marsupium extract (PME), a plant extract with a long history of use in Ayurvedic medicine, extended cellular lifespan (Chapter three). Constituents of this extract include several compounds with reported health span and life span extending properties: epicatechin, quercetin, berberine, and pterostilbene. Of these PME constituents, quercetin and epicatechin had no effect on cellular life span, while berberine decreased cellular life span. We describe the lifespan and outgrowth kinetics of pterostilbene-treated cells and report on dose-dependent effects on longevity, potent cytotoxicity, and a possible role for mitochondrial function in mediating these phenotypes (Chapter Four).