Session MOE. There are 4 abstracts in this session.


Proteomics and metabolomics in studies of the biology of aging: connecting mitochondrial energetics to physiological function

Peter Rabinovitch

For many decades a prominent theory of aging has centered on the damaging effects of reactive oxygen species (ROS). Mitochondria generate the bulk of cellular ROS as a byproduct of oxidative phosphorylation and the generation of ATP. This was the rational for our studies that began with creation of transgenic mice that express catalase in mitochondria to effectively scavenge ROS. These mice were found to be longer lived and protected against multiple health challenges, including cardiac aging, sarcopenia and some cancers. In search of a parallel pharmacologic approach, similar benefits were found to be conferred by treating mice with the mitochondria-targeted tetrapeptide SS-31 (elamipretide), a drug now in phase II and III clinical studies in man. In recent years it has become evident that the free radical theory of aging is over-simplistic, as the role of ROS and mitochondrial energetics is much more multi-faceted. SS-31 was found to not act as an antioxidant, but instead integrates into the mitochondrial inner membrane, improving the efficiency of oxidative phosphorylation in aged and damaged mitochondria. However, how improvements in mitochondrial energetics in old animals results in improvements in a spectrum of physiologic functions, including heart, muscle and vision, remains to be established. We hypothesize that the acute enhancement of mitochondrial function is followed by persistent structural, proteomic and metabolic remodeling to effect these changes. The application of metabolomics and proteomics (including study of protein abundance, turnover, and post-transactional modifications) has provided essential tools to discover and characterize these mechanisms.
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A High Throughput Proteomics Platform to Investigate Aging

Fiona McAllister

Sample preparation is often the rate limiting part of proteomics experiments and hinders the application of proteomics to large cohorts of samples. To address this and allow the processing of thousands of samples we have set up an automated workflow on a Hamilton liquid handler. We evaluated a number of different sample preparation techniques to identify one that does not rely on precipitation and is compatible with automation. Our workflow is based on 16plex TMT. We have investigated various post translational modifications and evaluated how the modifications change with age. We are comparing the magnitude of the post translational modification changes in young and old mice with the aim to determine which modification changes the most with age.
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Senescence-Based Biomarker Signatures of Aging and Senescence Burden

Nathan Basisty1; Abhijit Kale1; Okhee H Jeon1; Chisaka Kuehnemann1; Therese Payne1; Chirag Rao1; Anja Holtz1; Samah Shah1; Vagisha Sharma3; Luigi Ferrucci4; Judith Campisi1, 2; Birgit Schilling1
1The Buck Institute, Novato, CA; 2Lawrence Berkeley Laboratory, Berkley, CA; 3University of Washington, Seattle, WA; 4National Institute on Aging, Baltimore, MD

Introduction: The senescence-associated secretory phenotype (SASP) has emerged as both a driver of, and promising therapeutic target for, a multitude of chronic age-related conditions, ranging from neurodegeneration to cancer.  The complexity of the SASP has been greatly underappreciated and a small set of factors cannot explain the diverse phenotypes it produces in vivo.  Here we present a comprehensive proteomic analysis of SASPs driven by multiple inducers of senescence in different human cell types. We propose that SASP proteins are promising biomarkers to assess senescent cell burden in aging and disease.

Methods: The ‘core’ and inducer-specific secretomes of senescent cells were profiled by comparing the secreted proteomes of senescent fibroblasts to quiescent fibroblasts following three senescence-inducing stimuli: irradiation-, oncogenic RAS-, or HIV drug atazanavir.  Secreted proteins were obtained from the medium of cells cultured for 24 hours in serum-free conditions. Data-independent acquisitions (DIA) were performed on a TripleTOF 6600.

Results: We identified over 1000 unique SASP proteins, including ‘inducer-specific’ and ‘core’ SASP signatures. The ‘core’ SASP was composed of 151 proteins, with GDF15, CXCL1, MMP1, and STC1 consistently among the top increased proteins in response to all inducers. About 25% of the core SASP was enriched among plasma biomarkers of aging in humans and are proposed as senescence-based biomarkers candidates. To develop oncogene-induced senescence (OIS) signature, we performed multi-omic analysis to identify proteins exclusively expressed by RAS-induced senescent cells at both the protein level (based on our secretomes) and RNA-level (based on published RNA-seq data). We developed an OIS-specific signature composed of 26 proteins, a subset of which is proposed as an in vivo signature of OIS cells.

Discussion: This work will aid in identifying the proteins that drive senescence-associated phenotypes and provide comprehensive catalogs of potential biomarkers for assessing the burden and origin of senescent cells in vivo. 

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Identification of Proteins with Reduced Solubility in Aging Mouse Brains

Cristen Molzahn; Mang Zhu; Lorenz Nierves; Aly Karsan; Philipp Lange; Thibault Mayor
University of British Columbia, Vancouver, Canada

Normal cellular function relies on maintaining the collection of proteins, known as the proteome.  The cell depends on the balance between protein production, folding and degradation to maintain the homeostasis of the proteome (proteostasis).  As an organism ages, cellular response to unfolded proteins declines which increases unfolded protein stress; this results in a decline of proteostasis.  The diminished folding capacity can cause an increase in aggregated proteins, which have been associated with neurodegenerative diseases.  Whereas, the age-related decline in proteome maintenance has been relatively well documented in the model organism C. elegans, a lack of information remains around the link between aging and aggregation in mammalian cells. Further investigation into the changes at the protein level is necessary to better characterize the link between aging and loss of proteostasis in mammals.    

This study focuses on the insoluble portion of the proteome defined as those proteins which pellet after high-speed centrifugation.  It is expected that inclusion in higher molecular weight aggregates is will result in certain proteins becoming more enriched in the pellet fraction upon aging.  Using mass spectrometry-based proteomics approaches we analyzed cortex and hippocampus tissue from mice ages 4, 15 and 23 months revealing age-induced changes in protein solubility.  Proteins enriched in the insoluble fraction upon aging show common functions such as chaperone proteins, oxidoreductases and neurofilament proteins (previously associated with amyotrophic lateral sclerosis).  Additionally, such proteins generally have fewer disordered regions as compared to the proteome and have longer half-lives.  These result suggest common features that may predispose proteins to become less soluble upon aging. By identifying proteins that are affected by aging in the absence of pathology, we can observe how proteostatic decline begins and creates conditions favorable to the formation of neurodegenerative diseases.

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