Aging is a complex biological process associated with progressive loss of physiological function and susceptibility to several diseases, such as cancer and neurodegeneration. Cellular senescence and mitochondrial dysfunction have been defined as classical hallmarks of aging and neurodegenerative diseases, but non-invasive biomarkers of these processes, such as plasma proteins, are lacking. We hypothesized that senescent cells and their senescence-associated secretory phenotype (SASP) may qualify as biomarkers for aging and age-related diseases. Our studies thus far have demonstrated that selected SASP-derived biomarker candidates (e.g., GDF15, STC1, SERPINs, MMP1) serve as indicators for human aging as demonstrated in multiple human plasma cohort studies, such as The Baltimore Longitudinal Study of Aging (BLSA) and The Invecchiare in Chianti (InCHIANTI) study.
Another largely underexplored part of the SASP are exosomes – extracellular vesicles - that are involved in signaling from cell to cell, and that carry various types of cargo, such as proteins, RNA, DNAs, metabolites and lipids. We have thus started to not only investigate soluble SASP (SASPAtlas.com), but also to comprehensively characterize exosomes from senescent cells to develop senescence-derived exosome biomarkers. The latter also includes investigations of ‘Mitochondrial Dysfunction-Associated Senescence’ (MiDAS) in a tissue culture model to better understand the molecular links between mitochondrial dysfunction and cellular senescence, and likely these mechanisms may be relevant during aging and disease.
In order to efficiently analyze plasma exosomes we developed a high-throughput method to isolate plasma exosomes by sequential size-exclusion chromatography (SEC) and ultrafiltration (UF) to overcome challenges of exosome contamination with abundant soluble plasma proteins. Quality control analysis of the isolated exosomes confirmed high exosome 'purity’. We performed data-dependent acquisitions (DDA) from offline high-pH reversed-phase fractions of exosome lysate to generate a deep spectral library of ~2,300 proteins. Subsequently, in a pilot aging study we used comprehensive data-independent acquisitions (DIA), to compare plasma exosomes from young (n=5; 20–26 yrs) and old (n=5; 60–66 yrs) individuals. We were able to quantify 1,208 total exosome proteins, and 144 proteins significantly changed between young and old plasma groups (Q<0.05; >1.5-fold change). We also analyzed the exosome miRNA cargo and detected 331 miRNA, some of which changed significantly between young and old individuals. These translational workflows aim to develop 'Biomarkers of Aging' for prognostic and diagnostic applications.
In the future, we intend to perform multi-omics analysis of plasma exosomes obtained from human aging cohort studies as well as from dementia patients – specifically analyzing longitudinal plasma samples.