A proteomic atlas of senescence-associated secretomes for aging biomarker development.

The senescence-associated secretory phenotype (SASP) has recently emerged as a driver of and promising therapeutic target for multiple age-related conditions, ranging from neurodegeneration to cancer. The complexity of the SASP, typically assessed by a few dozen secreted proteins, has been greatly underestimated, and a small set of factors cannot explain the diverse phenotypes it produces in vivo. Here, we present the "SASP Atlas," a comprehensive proteomic database of soluble proteins and exosomal cargo SASP factors originating from multiple senescence inducers and cell types. Each profile consists of hundreds of largely distinct proteins but also includes a subset of proteins elevated in all SASPs. Our analyses identify several candidate biomarkers of cellular senescence that overlap with aging markers in human plasma, including Growth/differentiation factor 15 (GDF15), stanniocalcin 1 (STC1), and serine protease inhibitors (SERPINs), which significantly correlated with age in plasma from a human cohort, the Baltimore Longitudinal Study of Aging (BLSA). Our findings will facilitate the identification of proteins characteristic of senescence-associated phenotypes and catalog potential senescence biomarkers to assess the burden, originating stimulus, and tissue of origin of senescent cells in vivo

Composition and Acidification of the Culture Medium Influences Chronological Aging Similarly in Vineyard and Laboratory Yeast

Chronological aging has been studied extensively in laboratory yeast by culturing cells into stationary phase in synthetic complete medium with 2% glucose as the carbon source. During this process, acidification of the culture medium occurs due to secretion of organic acids, including acetic acid, which limits survival of yeast cells. Dietary restriction or buffering the medium to pH 6 prevents acidification and increases chronological life span. Here we set out to determine whether these effects are specific to laboratory-derived yeast by testing the chronological aging properties of the vineyard yeast strain RM11. Similar to the laboratory strain BY4743 and its haploid derivatives, RM11 and its haploid derivatives displayed increased chronological life span from dietary restriction, buffering the pH of the culture medium, or aging in rich medium. RM11 and BY4743 also displayed generally similar aging and growth characteristics when cultured in a variety of different carbon sources. These data support the idea that mechanisms of chronological aging are similar in both the laboratory and vineyard strains

Late-life restoration of mitochondrial function reverses cardiac dysfunction in old mice

Diastolic dysfunction is a prominent feature of cardiac aging in both mice and humans. We show here that 8-week treatment of old mice with the mitochondrial targeted peptide SS-31 (elamipretide) can substantially reverse this deficit. SS-31 normalized the increase in proton leak and reduced mitochondrial ROS in cardiomyocytes from old mice, accompanied by reduced protein oxidation and a shift towards a more reduced protein thiol redox state in old hearts. Improved diastolic function was concordant with increased phosphorylation of cMyBP-C Ser282 but was independent of titin isoform shift. Late-life viral expression of mitochondrial-targeted catalase (mCAT) produced similar functional benefits in old mice and SS-31 did not improve cardiac function of old mCAT mice, implicating normalizing mitochondrial oxidative stress as an overlapping mechanism. These results demonstrate that pre-existing cardiac aging phenotypes can be reversed by targeting mitochondrial dysfunction and implicate mitochondrial energetics and redox signaling as therapeutic targets for cardiac aging

Quantitative Proteomic Analysis of the Senescence-Associated Secretory Phenotype by Data-Independent Acquisition.

Cellular senescence is a complex stress response that induces an essentially permanent cell cycle arrest and a complex secretory phenotype termed the senescence-associated secretory phenotype (SASP), which drives numerous aging pathologies. Characterization of the SASP can provide insights into aging and disease mechanisms, aging biomarker candidates, and targets for counteracting the deleterious effects of senescent cells. Here we describe a mass spectrometry (MS)-compatible protocol to (1) generate senescent cells using different stimuli, (2) collect conditioned medium containing proteins secreted by senescent cells (i.e., SASP), and (3) prepare the SASP for quantitative proteomic analysis using data-independent acquisition (DIA) MS. © 2021 The Authors. Basic Protocol 1: Generating ionizing radiation-induced senescent and control cells Alternate Protocol 1: Generating doxorubicin-induced senescent and control cells Alternate Protocol 2: Generating oncogenic RAS-induced senescent and control cells Alternate Protocol 3: Generating mitochondrial dysfunction-induced senescent and control cells Alternate Protocol 4: Generating atazanavir/ritonavir-induced senescent and control cells Support Protocol: A multiple-assay approach to confirm the phenotype of senescent cells Basic Protocol 2: Generating conditioned medium from senescent cells cultured in low serum and quiescent control cells Alternate Protocol 5: Generating conditioned medium from senescent cells cultured in complete medium and quiescent control cells Basic Protocol 3: Quantitative proteomic analysis of the SASP

Quantitative Proteomic Analysis of the Senescence-Associated Secretory Phenotype by Data-Independent Acquisition.

Cellular senescence is a complex stress response that induces an essentially permanent cell cycle arrest and a complex secretory phenotype termed the senescence-associated secretory phenotype (SASP), which drives numerous aging pathologies. Characterization of the SASP can provide insights into aging and disease mechanisms, aging biomarker candidates, and targets for counteracting the deleterious effects of senescent cells. Here we describe a mass spectrometry (MS)-compatible protocol to (1) generate senescent cells using different stimuli, (2) collect conditioned medium containing proteins secreted by senescent cells (i.e., SASP), and (3) prepare the SASP for quantitative proteomic analysis using data-independent acquisition (DIA) MS. © 2021 The Authors. Basic Protocol 1: Generating ionizing radiation-induced senescent and control cells Alternate Protocol 1: Generating doxorubicin-induced senescent and control cells Alternate Protocol 2: Generating oncogenic RAS-induced senescent and control cells Alternate Protocol 3: Generating mitochondrial dysfunction-induced senescent and control cells Alternate Protocol 4: Generating atazanavir/ritonavir-induced senescent and control cells Support Protocol: A multiple-assay approach to confirm the phenotype of senescent cells Basic Protocol 2: Generating conditioned medium from senescent cells cultured in low serum and quiescent control cells Alternate Protocol 5: Generating conditioned medium from senescent cells cultured in complete medium and quiescent control cells Basic Protocol 3: Quantitative proteomic analysis of the SASP

Effect of different carbon sources on culture pH, growth rate, and chronological life span.

<p>Cells were aged in synthetic complete medium supplemented with the appropriate carbon source and pH measurements were taken at both day 2 and day 4 of the experiment. Control concentrations for all carbon sources were tested at 2% w/v, except ethanol and glycerol which were tested at 3%. Moderate dietary restriction (DR) concentrations were at 0.5% for all sources except ethanol and glycerol, which were reduced to 1%. Survival integral (SI) values represent the area under the survival curve between day 2 and day 26.</p><p>*Denotes a statistically significant increase SI in response to DR (p<0.05).</p

Survival integral (SI) values for BY and RM haploid and diploid strains aged in different medium compositions with glucose as the carbon source.

<p>The % refers to the amount of glucose present in the culture medium. Survival integral values represent the average of three biological replicates between days 2 to 45 in the experiment. Values in parentheses are standard deviation. P-value is calculated by Student's T-test for each conditions relative to synthetic complete (SC) 2% for that genotype, except for YEP 0.5% and YEP 0.05%, which are calculated relative to YEP 2%.</p

Strains used in this study.

<p>Strains used in this study.</p

Adjusting synthetic complete medium to a pH of 6.0 extends chronological life span.

<p>Life span extension from buffering at pH 6.0 using a citrate phosphate buffer was statistically significant (p<0.05) in (<b>A</b>) diploid, (<b>B</b>) haploid mating type <i>α</i>, and (<b>C</b>) haploid mating type <b>a</b> of BY4743 (BY) and RM11 (RM). Error bars indicate standard deviation across biological replicates. Corresponding survival integral and p-values are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024530#pone-0024530-t002" target="_blank">Table 2</a>.</p