Abstract Although aging is the biggest risk factor for human chronic (cancer, diabetic, cardiovascular, and neurodegenerative) diseases, few interventions are known besides caloric restriction and a small number of drugs (with substantial side effects) that directly address aging. Thus, there is an urgent need for new options that can generally delay aging processes and prevent age-related diseases. Cellular aging is the basis of aging processes. The chronological lifespan (CLS) of yeast Saccharomyces cerevisiae is the well-established model system for investigating the interventions of human post-mitotic cellular aging. CLS is defined as the number of days cells remain viable in a stationary phase. We developed a new, cheap, and fast quantitative method for measuring CLS in cell cultures incubated together with various chemical agents and controls on 96-well plates.  Our PICLS protocol with (1) the use of propidium iodide for fluorescent-based cell survival reading in a microplate reader and (2) total cell count measurement via OD600nm absorption from the same plate provides real high-throughput capacity. Depending on logistics, large numbers of plates can be processed in parallel so that the screening of thousands of compounds becomes feasible in a short time. The method was validated by measuring the effect of rapamycin and calorie restriction on yeast CLS. We utilized this approach for chemical agent screening.  We discovered the anti-aging/neuroprotective potential of 2,5-anhydro-D-mannitol (2,5-AM) and suggest its usage individually or in combination with other antiaging interventions.

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【For more info: david.deng@wecistanche.com / WhatApp:86 13632399501】

Keywords Saccharomyces cerevisiae ·  Chronological lifespan · Chemical screening · Antiaging compound · 2,5-anhydro-D-mannitol

Introduction

The growing fraction of the elderly (>65  years)   among the total population (from~ 10% worldwide  (above 20% in first world countries) in 2020 to extrapolated 16% in 2050), as well as their increasing life expectancy (by ~ 10 years since 1960 in first world countries), confronts the world with increasingly difficult problems [1, 2]. Besides generally reducing life activity, aging remains the greatest risk factor for the development of chronic diseases that, subsequently, compromise independent human life and finally lead to death [3–7]. The current medical approaches to prevent age-related pathologies are recommendations for a healthy lifestyle,   including exercise and diet. However, these interventions alone are not sufficient to prevent the onset of age-related diseases.

Aging is characterized by a progressive loss of physiological integrity, efficiency in cellular functions, and metabolic signaling [8]. Increasing efforts are directed to understand cellular aging processes affecting the highly interconnected and functionally redundant gene and protein interactions network [8,  9]. Despite the complexity of aging, recent research in different model systems, including mammals, has demonstrated that delayed aging and increased health span are feasible by anti-aging interventions such as rapamycin drug administration application and calorie (glucose) restriction [10–15]. Therefore,   expanding the repertoire of anti-aging compounds that can be utilized as geroprotecting therapeutics and reducing their unwanted side effects is one of the promising strategies that can delay aging and prolong health span.

Recent research has shown that molecular mechanisms observed in human aging are conserved in different organisms, including single-cell yeast [8, 10,  16, 17]. The budding yeast Saccharomyces cerevisiae is one of the most studied model organisms for uncovering the biological processes involved in cellular aging. The benefits of studying aging in yeast include a short generation time, a tractable lifespan, and its amenability to high-throughput assays. Therefore, this organism became a powerful tool for the identification of anti-aging interventions. Yeast is commonly used to study aging in two distinct ways:   replicative lifespan (RLS) and chronological lifespan  (CLS) [18]. The RLS measures the number of times an individual cell divides, an aging model for mitotic cells such as stem cells. The CLS measures the length of time a non-dividing cell remains viable in the stationary phase, an aging model for post-mitotic cells such as neurons. The viability of aging yeast cells under nutrient-deprived stationary phase conditions decreases, and they eventually die.

CLS has been traditionally measured by estimating colony-forming unit (CFUs) counts on the agar plate  [18]. Yeast cells in a flask with total media (≥20 ml)   are exposed to a single chemical compound. At different time points (e.g., after 2, 5, and 8 days), small aliquots of the chronological aging culture from the fask are serially diluted, plated onto nutrient agar plates,   and incubated for 2–3 days for colony formation and counting. The survival fraction determines from the  CFU for each chronological age point relative to day  1 (considered 100% cell survival). The CFU approach is quantitative but associated with many downsides.  It requires multiple serial dilutions, plating steps, and incubation periods for appearing the colonies. Colony counting is performed either by manual or expensive instruments. Moreover, this method is unsuitable and much too expensive for high-throughput screening  (HTS) of thousands of compounds because it requires substantial amounts of the drugs tested, large-volume flask cultures, and many agar plates. Unfortunately,   the CFU method and all recent variants for measuring CLS are critically dependent on the ability of the post-mitotic cells to re-enter the mitotic phase. In principle, this methodology cannot distinguish yeast in mitotic arrest from dead cells.

Current technical advancements in CFU-derived methods measure cell survival and growth based on the outgrowth of drug-exposed stationary phase yeast in nutrient-rich culture either in a liquid medium or by a spotting assay on an agar plate [17, 19–21]. After  24  h, the outgrowth of aged cells in liquid medium is measured by the absorbance at OD600nm, whereas in the spotting assay, the outgrowth of spotted aged culture onto agar medium is visually identified after  48  h. The logistical improvement includes the usage of  96-well plates that allow limited chemical or genomewide deletion strain screening. For pooled tagged yeast deletion strains, flow cytometry is required to quantify the individual strain viability [22].

Propidium iodide (PI) is a fluorescent dye that only enters the dead cells and, thus, is a powerful marker for direct quantification of cell viability [23–25].  Previously, studies utilized a PI-based approach for measuring CLS and quantifying cell viability by analyzing the images acquired by a fluorescent cell counter, UV transilluminator, or flow cytometry [26,  27]. However, the multiple sample processing, imaging, and software requirements for data analysis are expensive and time-consuming, which hindered these methods in real high-throughput applications.  The introduction of alternative dyes such as SYTOX   green is of limited value in a high-throughput setting because of the prohibitive costs [26].

Therefore, new methods are required to quantify the viability of the aged cell to avoid the limitations associated with existing methodologies. In this work, we describe PICLS, a PI-based CLS measurement method that is outgrowth-independent. Our new approach relies on incubating yeast cells with various test compounds or differing test media conditions in 96-well plates. We use PI to quantify cell survival. Fast and efficient readout (within~15 min)   is provided by a microplate reader, a low-cost device readily available in most laboratories. This cheap methodology is well suited for large-scale screening of the chemical agents to identify anti-aging compounds as many different compounds, controls,   or dilutions can be conveniently placed on a single plate and large numbers of plates can be processed in parallel. Furthermore, we validated our method by determining the extension of CLS of yeast using known anti-aging interventions such as rapamycin administration and calorie restriction. A quick screen of available chemicals in our laboratory unexpectedly revealed 2,5-anhydro-D-mannitol (2,5-AM) extending the lifespan of yeast. We also confirmed the antiaging activity of 2,5-AM with traditional outgrowth methods. Other tested sugar analogs did not deliver any similar effect.

Results

Development of a method for quantification of   cell viability using propidium iodide fluorescence   measurement in the microplate reader

Our new protocol starts with exposing wild-type yeast cells or their mutants placed into nutrient-rich media in 96-well plates together with a specific concentration of chemical compounds of interest. We use PI as a marker for cell death as this fluorescent dye only enters non-surviving cells [23–25]. The cell viability in the stationary phase culture is quantified after a specified number of days by reading the PI fluorescence in the microplate reader that, in turn, provides an electronically readable spreadsheet for data analysis.

As a first validation, we show that working with small amounts in microplate wells delivers results similar to cuvette-based approaches and that the microplate reader is sufficiently sensitive to capture cell survival data. Thus, we analyzed the PI staining of yeast cells. Yeast cells were grown in the glass flask and boiled for 15 min at 100 °C. Live and dead cells were washed and incubated in 1×PBS (phosphate-buffered saline) without and with PI (5 µg/ml)   for 15 min. After incubation, cells were washed and resuspended in PBS. Cells were visualized by microscopy and fluorescence intensity was measured by the microplate reader (Supplementary Fig.  S1). After confirmation that PI is a suitable marker for cell death,   a subsequent analysis was performed for developing the method. PI-stained dead cells were resuspended in PBS to the final 48 OD600nm measured in the cuvette using a spectrophotometer. Next, cells were serially diluted in PBS (OD600nm 48 to 0.05) and transferred to a black 96-well plate (costar 3603). To note, black microplates are four times more expensive than clear plates and less easily available, but we used them since they are more suitable for fluorescence readout due to dampening autofluorescence originating from the samples and microplate surfaces.  With a GelDoc imaging system (Fig. 1A), we visualized the degree of incorporation of PI staining in the dead yeast cells at various dilutions. Using the same plate, we measured the PI fluorescence intensity with a microplate reader at excitation 535 nm and emission  617  nm wavelengths. We found a high linear correlation between PI fluorescence intensity and cellular absorbance at OD600nm (Fig. 1B). This result indicates that our variant of the PI fluorescence–based method is effective for quantifying cell viability.

As a next step, we determined the optimal range of cell density (quantified by OD600nm) so that cell density could be directly measured in 96-well   plates instead of in cuvettes. Using the black microplates with dead yeast cells from the previous experiment, we determined the correlation of  OD600nm measurements taken from the cuvettes and from the microplate directly. We found a high linear correlation between the two series of measurements in the OD600nm range of 0.05 to 12 of the cuvette (Fig.  1C). However, the trend is no longer maintained for higher OD600nm values>12 of the cuvette (Fig.  1D). Next, we determined the relationship between PI fluorescence intensity and cell OD600nm of the 96-well plate within the optimal  OD600nm range 0.05–12. We observe an almost perfect linear correlation (Fig.  1E). Thus, our protocol represents a robust method for quantifying cell viability.

Effect of microplate types on quantification of   cell viability using propidium iodide fluorescence   measurement in the microplate reader

Effect of rapamycin on the chronological lifespan of   the yeast

Rapamycin is one of the well-known anti-aging interventions demonstrated to extend the lifespan of several model organisms, including yeast, nematodes,   fruit flies, and mice [10, 14, 15, 28–30]. It inhibits the nutrient-sensing complex TORC1 (target of rapamycin complex 1). TORC1 is a conserved multi-subunit protein complex in eukaryotic cells that couples nutrients in the environment with cell growth and proliferation [19, 29, 31–34].

We have examined the effect of rapamycin on the chronological lifespan (CLS) of the yeast strain to validate our newly developed protocol. Here and below,   we used the prototrophic yeast strain (CEN.PK113- 7D) in our experiments to avoid the strong effects of amino acid auxotrophy on cell survival in stationary phase culture [35, 36]. Cells were aged with different concentrations of rapamycin in the synthetic defined (SD) medium. Cell growth was measured at different time points (24 h, 48 h, and 72 h). We found that cell growth reached saturation approximately 24  h after incubating with 5  nM or less rapamycin (Fig.  4A).  However, the administration of 10  nM rapamycin slowed cell growth, and saturation was reached only after 48 h (Fig. 4A). Previously, a similar trend was observed for cells growing with rapamycin in a CLS   experiment [37].

As a next step, the CLS of cells grown in different concentrations of rapamycin was measured. We determined the cell survival using PI fluorescence at different chronological age time points. The growth time point 72  h was considered as day 1. We can deduce from the survival graph that different concentrations of rapamycin addition extend the CLS of yeast (Fig. 4B). The survival of aged cells supplemented with rapamycin (5 nM and 10 nM) on day 4   was~75%; however, without rapamycin was~50%.  On day 7, survival of rapamycin-supplemented aged cells was 70%; however, without rapamycin was reduced to less than 20%. CLS extension of aged cells can also be observed at lower concentrations  (0.62–2.5 nM) of rapamycin (Fig. 4B).

We have validated our protocol by directly comparing it with two traditional outgrowth approaches that measure the CLS of the yeast. Firstly, we conducted the outgrowth assay in the liquid medium to measure the effect of rapamycin on lifespan. The survival of chronological aging cells was monitored at different age points by transferring a 3-µl   culture into 200μL of YPD medium in a fresh  96-well plate. Outgrowth corresponds to the number of viable cells in the inoculum (Fig. 4C). Aged cell outgrowth registered via OD600nm absorbance was determined using a microplate reader. The survival fraction was calculated from the outgrowth  OD600nm absorbance for each age-point relative to day 1 (considered 100% cell survival). The survival graph is shown in Fig. 4D.

Likewise, we conducted the outgrowth by spotting assay on the agar medium. We spotted 3-µl   aging culture on the YPD agar plate and incubated it for 48 h. The outgrowth of aged cells on the  YPD agar plate was visualized by the GelDoc imaging system (Fig. 4E). As expected, we can observe trends of lifespan extension by rapamycin under both YPD liquid and agar outgrowth assays that parallel those determined with our new protocol.

These results show that our HTS protocol reproduces that rapamycin extends the cellular lifespan.  Furthermore, we assess the Z-factor to evaluate the quality of this method [38]. Generally, Z-factors in the range of 0.5–1.0 are indicative of excellent  HTS assays. The Z-factor was determined for different concentrations of rapamycin with PI fluorescence of aged cells (Supplementary Fig.  S2). Our funding, a Z-factor 0.70 for 5  nM rapamycin and  0.74 for 10  nM rapamycin puts this HTS method into the high-quality category. Thus, our protocol is robust enough to identify potential anti-aging compounds.

For exploring whether the method is effective in a different yeast genetic background, we tested the effect of rapamycin on the CLS of the BY4743 strain.  Saccharomyces cerevisiae BY4743 strain is auxotrophic for histidine, leucine, and uracil. The BY4743   strain was grown in different concentrations of rapamycin in the SD medium supplemented with auxotrophic constituents. Survival of aged cells was quantified using PI fluorescence (Supplementary Fig. S3).  We found that rapamycin increased the viability of  BY4743 cells quantitatively in similar ways as that of  CEN.PK113-7D. Thus, these results reveal that our method is effective in different yeast strains.

Effect of Glucose on the chronological lifespan of the Yeast

We also validated our method by examining the effect of calorie restriction on the chronological lifespan of the yeast. Calorie restriction is one of the established interventions to delay aging and prolong the lifespan across the model organism, including yeast and mammalian cells [11, 13, 16]. To determine the effect of calorie restriction on the CLS of the yeast, we reduce the glucose content in the culture medium. We grew the cell in three different SD medium conditions containing 0.25%, 0.5%, and 2% glucose. We measured cell growth at different time points (24 h, 48 h, and  72  h). All cultures reached growth saturation after  24 h (Fig. 5A). Cell growth was lower at 0.25% and  0.5% compared to 2% glucose (Fig.  5A). We measured the survival of chronological aging for cells grown under different glucose concentrations using the PI fluorescence method (Fig. 5B). We infer from the survival graph that glucose restriction extends the  CLS of the yeast. We also concurrently confirmed the extension of the CLS by outgrowth assays (Fig. 5C,  D, and E), further validating our new method for determining the CLS of the yeast. The effect of glucose restriction on the CLS extension of the BY4743   strain was also shown by the PICLS method (Supplementary Fig. S3). Thus, our approach for measuring the CLS of the yeast represents a novel, fast, and low-cost method for screening the chemical agents and culturing conditions to identify the anti-aging interventions.

Screening the chemical agents using the newly   developed method identified 2,5‑anhydrous‑D‑mannitol   as a novel anti‑aging compound

After developing and validating the new protocol, we utilized this method to screen hundreds of chemical agents for the identification of anti-aging compounds (Supplementary Fig. S4 and Supplementary  Table 1). As one of the surprising results, we found indications for the compound 2,5-anhydro-D-mannitol (2,5-AM) to extend the CLS of the yeast. In Fig. 6, we present the outcome of detailed follow-up experiments for this compound. We tested the antiaging activity of 2,5-AM at different concentrations on the 96-well plate. Firstly, we determined the effect of 2,5-AM on cell growth. Yeast cells were incubated with varying concentrations of 2,5-AM in the SD medium. Cell growth was measured at different time points (24 h, 48 h, and 72 h). We found that cell growth reached the same saturation level after 24  h for all tested concentrations of 2,5-AM  (Fig. 6A). Then, we measured the survival of chronologically aged cells supplemented with different concentrations of 2,5-AM using the PI fluorescence method. As previously, the 72h growth culture was considered day 1 for the CLS analysis. The survival graph is plotted for different chronological age time points (Fig.  6B). We find that 2,5-AM extends the  CLS in a concentration-dependent manner. The survival of aged cells supplemented with 2,5-AM  (8 mM) on day 4 was ~ 80%; however, without 2,5- AM was ~ 50%. On day 7, survival of 2,5-AM supplemented aged cells was ~ 75%; however, without  2,5-AM was reduced to less than 20%. The antiaging activity of 2,5-AM was also verified by outgrowth assays (Fig. 6C, D, and E).

2,5-AM is a sugar molecule that can enter the glycolysis pathway. It gets hydrolyzed only at upstream glycolytic steps that cause accumulation of nonmetabolized 2,5-AM-1,6-bisphosphate (2,5-AMBP) in the cells [39–41]. To test whether 2,5-AM at concentrations similar to the CLS experiment has been processed by glycolysis enzymes, we examined the growing sensitivity of the SNF1 gene deletion strain.  SNF1 is a cellular energy sensor and highly conserved AMP-activated protein kinase (AMPK) in eukaryotes [42, 43]. Yeast cells that lack AMPK   activity are associated with a hypersensitive growth phenotype in the presence of non-metabolized glycolytic intermediates [44]. We measured the growth of the snf1Δ deletion strain with different concentrations of 2,5-AM. We found that 2,5-AM inhibits the growth of the snf1Δ deletion strain compared to the wild-type strain  (Supplementary Fig. S5). This observed growth phenotype indicates that 2,5-AM undergoes glycolysis and gets hydrolyzed into non-metabolized glycolytic intermediates.

2,5-AM is an analog of the sugar moiety fructose  [39]. To clarify whether other sugars also influence the CLS, we examined the effect of fructose, mannitol, and maltose on yeast aging. We also included sorbitol which is a non-metabolized sugar reported to increase the CLS of yeast cells by increasing the osmolarity of the culture medium [20, 45]. Firstly, we tested the effect of fructose, mannitol, maltose, and sorbitol on cell growth. Yeast cells were incubated with various concentrations of fructose, mannitol,   maltose, and sorbitol similar to 2,5-AM in the SD   medium. Like 2,5-AM, fructose, mannitol, maltose,   and sorbitol incubated cells reached growth saturation after 24 h (Fig. 7A). Next, we measured the survival of chronologically aged cells on day 1, day 7, day  14, and day 21. Unlike 2,5-AM, fructose, mannitol,   maltose, and sorbitol supplementation did not extend the lifespan of the yeast (Fig. 7B, C, D, E, and Supplementary Fig.  S6). Remarkably, 2,5-AM extends the lifespan even at the late stage of chronological aging. Aged cells supplemented with 2,5-AM (8 mM)   survive (~65%) on day 21. However, the survival of aged cells without 2,5-AM supplementation was less than 10%.

Much to our surprise, we found that sorbitol at low concentrations tested is unable to increase the  CLS (Fig. 7B, C, D, E, and Supplementary Fig. S6).  We hypothesized that higher sorbitol concentrations might be required to increase the CLS. We tested a   range of higher concentrations including 1 M sorbitol, the concentration previously reported to affect the CLS (18% equivalent to 1 M) [20, 45]. Consistent with previous reports, we also find that very high sorbitol concentrations (between 0.5 and 1  M)   increase the CLS of yeast (Supplementary Fig.  S7),   apparently by increasing the medium’s osmolarity.  Interestingly, the CLS increase by 2,5-AM at a very low concentration (2 mM) suggests its mechanism of antiaging activity is distinct from the osmotic one. These results suggest that the anti-aging activity of 2,5-AM   is specific for this sugar compound and not observed for several other, similarly structured chemicals.

【For more info: david.deng@wecistanche.com / WhatApp:86 13632399501】