Abstract
The single-celled yeast Saccharomyces cerevisiae is one of the most valuable laboratory models that has been used successfully to identify factors and pathways involved in several cellular processes, the counterparts of which are evolutionarily conserved. Furthermore, it is also a powerful tool for analyzing the effects of molecules of nutraceutical interest with the view of leading to human health benefits and improving the quality of aging. In this context, we present some of the methods that have allowed us to assess the beneficial influence of a form of vitamin B3, namely nicotinamide, on mitochondrial functionality during yeast chronological aging. Mitochondrial dysfunctions are considered to be hallmarks of aging, and of several metabolic and neurodegenerative diseases. More specifically, these methods concern the determination of the respiratory parameters in intact cells in order to evaluate the efficiency of mitochondrial respiration in concert with the risk of superoxide anion (O2−) production, which results from inefficient respiration. In addition, we describe fluorescent staining specific for O2− detection and mitochondrial membrane potential, as well as a simple clonogenic assay based on the ability of cells to grow on a carbon source that requires a functional mitochondrial metabolism.
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References
Bogan KL, Brenner C (2008) Nicotinic acid, nicotinamide, and nicotinamide riboside: a molecular evaluation of NAD+ precursor vitamins in human nutrition. Annu Rev Nutr 28:115–130
Houtkooper RH, Pirinen E, Auwerx J (2012) Sirtuins as regulators of metabolism and healthspan. Nat Rev Mol Cell Biol 13(4):225–238
Imai S, Guarente L (2014) NAD+ and sirtuins in aging and disease. Trends Cell Biol 24(8):464–471
Orlandi I, Pellegrino Coppola D, Strippoli M, Ronzulli R, Vai M (2017) Nicotinamide supplementation phenocopies SIR2 inactivation by modulating carbon metabolism and respiration during yeast chronological aging. Mech Ageing Dev 161(Pt B):277–287
Longo VD, Shadel GS, Kaeberlein M, Kennedy B (2012) Replicative and chronological aging in Saccharomyces cerevisiae. Cell Metab 16(1):18–31
Parrella E, Longo VD (2008) The chronological life span of Saccharomyces cerevisiae to study mitochondrial dysfunction and disease. Methods 46(4):256–262
Koning AJ, Lum PY, Williams JM, Wright R (1993) DiOC6 staining reveals organelle structure and dynamics in living yeast cells. Cell Motil Cytoskeleton 25(2):111–128
Madeo F, Fröhlich E, Ligr M, Grey M, Sigrist SJ, Wolf DH et al (1999) Oxygen stress: a regulator of apoptosis in yeast. J Cell Biol 145(4):757–767
Ocampo A, Liu J, Schroeder EA, Shadel GS, Barrientos A (2012) Mitochondrial respiratory thresholds regulate yeast chronological life span and its extension by caloric restriction. Cell Metab 16(1):55–67
Amberg DC, Burke D, Strathern J (2005) Methods in yeast genetics: a Cold Spring Harbor Laboratory course manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. ISBN-10: 0879697288
Gnaiger E (2012) Mitochondrial pathways and respiratory control. An introduction to OXPHOS analysis, 4th edn. OROBOROS MiPNet Publications, Innsbruck. 2014: 4000 prints. ISBN-13: 978-3-9502399-8-0. https://pdfs.semanticscholar.org/1d1c/e797c78fcdab9de8641ca311d457b03c3a87.pdf
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Orlandi, I., Vai, M. (2020). Assays for Monitoring the Effects of Nicotinamide Supplementation on Mitochondrial Activity in Saccharomyces cerevisiae. In: Guest, P. (eds) Clinical and Preclinical Models for Maximizing Healthspan. Methods in Molecular Biology, vol 2138. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0471-7_16
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DOI: https://doi.org/10.1007/978-1-0716-0471-7_16
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Publisher Name: Humana, New York, NY
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