Saccharomyces cerevisiae, the budding yeast was long history as industrial baker’s yeast due to its ability to produce numerous product such as ethanol, acetate, industrial bakers etc. Interestingly, this yeast was also important tools for studying biological mechanism in eukaryotic cells including aging, autophagy, mitochondrial response etc. S. cerevisiae has arisen as a powerful chemical and genetic screening platform, due to a rapid workflow with experimental amenability and the availability of a wide range of genetic mutant libraries. Calorie restriction (CR) as the reduction of nutrients intake could promote yeast longevity through some pathways such as inhibition of nutrient sensing target of rapamycin (TOR), serine–threonine kinase (SCH9), protein adenylate cyclase (AC), protein kinase A (PKA) and ras, reduced ethanol, acetic acid and apoptotic process. In addition, CR also induces the expression of antioxidative proteins, sirtuin2 (Sir2), autophagy and induction of mitochondrial yeast adaptive response. Three methods, spotting test; chronological life span (CLS) and replicative life span (RLS) assays, have been developed to study aging in S. cerevisiae. Here, we present strategies for pharmacological anti-aging screens in yeast, discuss common pitfalls and summarize studies that have used yeast for drug discovery.
- Saccharomyces cerevisiae
- calorie restriction
- spotting test
- chronological life span
- replicative life span
The budding yeast
Scientist stated that
On the other hand, aging is a multifaceted process of accumulation of cellular, molecular and organ damage, leading to loss of function and increased vulnerability to disease following with the death. Indeed, there is a profound overlap between cellular and molecular pathways that influence aging and those linked to neurodegeneration, cancer, metabolic syndrome, and cardiovascular disorders. Therefore, recent efforts have emerged at the identification of compounds that decelerate the aging process and thus may act as a preventive measure that collectively ameliorates age-related diseases . In fact, studies of aging in mammalian cells are limited by the long lifespan of common model organisms. Rats and mice live 3–5 years and primates up to 40. Nevertheless, aging studies, particularly in rodents, have been highly informative, of the prospective understanding of the genetic factors for modulating longevity . Alternatively, a second approach that has dramatically accelerated aging research is the use of invertebrate model organisms, which age more rapidly and are readily amenable to environmental and genetic manipulation. Even though a variety of organisms have been investigated, a majority of studies have employed worms (
The budding yeast
In this brief chapter, we discussed the utilization of
2. Aging interventing mechanisms in
Biological aging in
Ultimately, CR reported could affect in some distinctive pathways in yeast cells. The first pathway is nutrient-sensing which reduced activity of two major nutrient sensing pathways, due to CR condition, could extend yeast life span. Both nutrient sensing pathways are focused on an amino acid-sensing pathway, including the serine–threonine kinase SCH9 and the target of rapamycin (TOR). Notably, deletion or inhibition of SCH9 and TOR causes an increase of up to several fold in yeast life span. Alterations to reduce nutrient and protein synthesis in CR condition are strongly implicated in extension of yeast lifespan by reduced TOR/SCH9. Extension of yeast lifespan by reduced activity of the TOR pathway depends on the transcription factor Gis1, which activates many protective systems including Mn-SOD [6, 20].
Further, the second pathway includes three proteins including adenylate cyclase (AC), protein kinase A (PKA), and Ras which will inhibit by CR conditions. The activation of two transcription factors (Msn2 and Msn4) that control cellular protection systems is required to mediate the effect of reduced Ras-AC-PKA signaling on yeast lifespan extension. Extension of yeast lifespan by these pathways needs the antioxidant enzyme Mn-SOD (superoxide dismutase), which scavenges the superoxide free radical [11, 21]. Intriguingly, superoxide level increases during yeast aging and is reduced in yeast mutants deficient in Tor-SCH9 or Ras-AC-PKA signaling. As for the yeast cells grow in the high glucose medium, could produce ethanol or acetic acid, which also contribute to chronological aging. Interestingly, deletion of SCH9 or TOR1 promotes removal of ethanol and acetic acid and accumulation of glycerol in the medium and further extend chronological life span by mechanisms similar to those of dietary restriction . More importantly, decreased signaling by the Tor-SCH9 and Ras-AC-PKA pathways is important in response to glucose restriction as well as increased transcriptional activity of Msn2 and Msn4, and the consequent affecting the expression of Pnc1 [nicotinamide deaminase that promotes the activity of the nicotinamide adenine dinucleotide (NAD)- dependent deacetylase Sirtuin 2/Sir2]. The Sir2 have been extensively studied for their potential role as conserved modulators of anti-aging in a various of organisms, including mammals . One mechanism by which Sir2 activity promotes yeast longevity is by suppressing homologous recombination in the rDNA that can promote the formation of extrachromosomal rDNA circles (ERCs). In fact, rDNA instability in general suggested the primary defect causing senescence and cell death .
Notably, another crucial mechanism that closely related with yeast aging is autophagy. This cellular process is reported as a highly conserved in organisms from yeast to human, which involves degradation of damaged organelles, circulation of amino acids, proteins, and other metabolites. It also regulates the genomic integrity via suppression of cell division in yeast under CR condition. Notably, decreased or dysfunction expression of autophagy genes leads to shorter lifespan in yeast and fruit fly. Conversely, enhanced autophagy promotes the longevity in aging models and suggested could protect against aging and age-related disorders [25, 26]. Another mechanisms which closely related with yeast aging is mitochondrial adaptive response signaling. Mitochondrial organelle is known to have a basic role in aging and age-related diseases. This organelle contributes to the ATP production, cell homeostasis, and imbalanced reactive oxygen species (ROS) creating a basic role of the cells regulation . In common condition, mitochondrial produces toxic ROS as by product of respiration process. However, on the CR condition, mitochondrial would be more active due to the shift metabolic process occurs from fermentation to respiration (CR) resulting ROS at the initial growth phases, as well. Consequently, yeast cells will adapt (pre-adaptation) with the ROS molecules strating from early growth stage and thus might activate defence mechanisms in the late of growth phase ensuring protection against higher doses of ROS. Those defence mechanisms likely activated antioxidative enzyme i.e. superoxide dismutase (SOD), catalase or glutathione peroxidase and therefore increase yeast lifespan (Figure 2) [28, 29].
Other than the above mechanisms in relation with CR condition resulting yeast lifespan extension. CR was also reported as having other substantial effects in yeast cells i.e. apoptotic process that accelerate aging process, repairing protein damage, NAD+ homeostasis, vacuolar function, genome stability, ribosom biogenesis, proteolysis regulation, and cell hyperthrophy . These valuable insights reflect that CR condition in yeast cells could modulate numerous pathways affected in biological aging mechanism. As for forefront anti-aging methods strategy, yeast cells growth on CR oftenly use as for positive control, whereas growing yeast cells on the normal growth medium with 2% glucose utilize as treatment for anti-aging compounds screening. If the yeast cells viability derived from compounds treatment has similar or higher than positive control, it suggested that corresponding compounds have anti-aging activity in yeast cells. Further research usually applied to investigate the precise mechanisms which modulated by those promising anti-aging compounds inside yeast cells.
3. Methods for investigating anti-aging activity in
On the basis of the current literatures, there were established various potential methods for investigating anti-aging activity derived from chemical compound by using
3.1 Spotting test
This particular method is commonly used in order to observe the viability of
Spotting test was reported as having some advantages in relation to assay anti-aging compounds including simple, fast handling and relatively low cost compare than CLS or other methods. However, this method could only represent a qualitative result of yeast cells viability through the yeast cells density, and thus it should be supported by other methods. As long as for the preliminary screening of numerous compounds acting as anti-aging, spotting method will be recommended. Some previous studies reported the usefulness of spot test to examine the particular compound for delaying aging in
3.2 CLS method using TPC analysis
CLS defines to the length of time a non-dividing cell can maintain viability, as refers to the its ability to re-enter the cell cycle process after a prolonged period of quiescence. Thus, CLS has been exhibited as a model of the viability of post-mitotic yeast cells . Traditionally, CLS has been examined by culturing fresh logarithmic yeast cells on a particular flask until reached the stationary phase (20–25 days) in liquid culture with an appropriate initial OD600 of 0.05–0.1. As for compound treatment is applied to the liquid culture medium soon thereafter yeast innoculated. Further, the yeast cell survival is measured as a function of time by dilution and plating onto a nutrient-rich agar medium at the periodic time (i.e each 3 days). Subsequently, viability is then calculated on the basis of the number of colonies (colony forming units: CFUs) on the nutrient plate agar arising [40, 41]. The budding yeast
Ultimately, CLS method requires a relatively large investment of materials, investigator time and belong to laborious, therefore is not suited for high-throughput screening anti-aging compounds. However, through this particular method, it was obtained the quantitatively results and thus could provide deeply insight for representing the cell viability of the yeast cells. Numerous studies has been applied CLS method for assaying anti-aging compounds derived from multi-resources i.e. Nakaya et al.,  assayed Beauveriolide I isolated from mushroom or Sunthonkun
3.3 RLS method
RLS assay is simple conceptually and shows an advantage of the fact that
As for RLS assay, the
RLS method is reported as having some major weakness which it makes less effective for high-throughput approaches. It is including time-consuming, laborious and relatively intricate in technique due to applying microscopic cells observation prior for plating in the plate medium during assays. Nevertheless, this particular methods will devote precisely quntitatively results and thus could represent the number of yeast cells generation between control and anti-aging compound treatments. To date, some previous studies were reported for using
Antiaging study in yeast was popular using CR condition which has numerous response to prolong yeast lifespan. Aging pathway in CR belong to pro-and anti-aging pathways. As for pro-aging are including TOR, SCH9, Ras protein, AC, PKA, ethanol accumulation, and apoptotic process. On the other hand, anti-aging pathways are including induction of antioxidative enzymes, sirtuin2, autophagy and adaptive response thorough mitochondrial adaptive ROS signaling. CR condition usually use for positive control, while treatment conducted in non-CR/high 2% glucose medium. There are numerous methods for anti-aging study, which the most popular is spotting test, CLS and RLS assays.
Recently, anti-aging in
Even the data from studies could be revisited and mined for potential bioactive substances, data obtained in yeast should not be over-interpreted unduly, and when aiming for applications in humans, validation of compounds in multicellular organisms should be done. So far, the potential of yeast to unravel novel pharmacological interventions against aging is far-reaching, however that it will continue to contribute substantially not only to drug discovery but also in other field such as fermented food, biochemical and bioenergy production.
The authors would like to thank Dr. Rika Indri Astuti Department of Biology, IPB University, Indonesia for permitting the pictures.
Conflict of interest
On behalf of all authors, we declare no conflict of interest.