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Proteomics, "Zombie" Cells and Clinical Biomarkers of Aging

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Proteomics, "Zombie" Cells and Clinical Biomarkers of Aging

Brightfield microscopy images of senescent lung fibroblasts (left) and senescent renal epithelial cells (right). Blue staining of the cells is an indicator of senescence-associated beta-galactosidase activity, a marker present only on senescent cells. Credit: Nathan Basisty, PhD.
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Growing old and aging – some of us revel in the luxury of it whilst others would most likely press a pause button on life if they could.

Over the last few centuries the human life expectancy has continued to rise dramatically, provoking philosophers, historians and scientists alike to explore what it means to grow older and how the human "biological clock" functions.

"Zombie cells" and aging

In such pursuit, scientists have expanded our understanding of the role of senescent cells in biological aging.


Briefly, cellular senescence is a process whereby cells stop dividing and go through phenotypic changes, such as secretome and chromatin changes in addition to tumor-suppressor activation.1 Senescent cells accumulate in several organs as we grow older, and are involved in tissue dysfunction and in numerous pathologies such as cancer. Therefore, they are generally considered to be a "hallmark" of aging and have earned the nickname "zombie" cells.2

In mouse models, genetic ablation of senescent cells and their secretory products (known as the senescence-associated secretory phenotype, or SASP) has been demonstrated to revert tissue dysfunction and improve conditions such as cardiomyocyte hypertrophy, tumorigenesis, atherosclerosis and osteoarthritis.2

Now, whilst there are many uncertainties in life, one (albeit somewhat macabre) certainty is that we will age, and we will die; this is the paradigm of human life. But of course, when have scientists ever drawn within the lines?

“Death is inevitable but aging is not,” said Nir Barzilai, founding director of the Institute for Aging Research at the Albert Einstein College of Medicine, New York.


Credit: Cristian Newman on Unsplash.


In pursuit of the fountain of youth

The increased understanding of senescent cell biology has resulted in an array of start-up companies seemingly springing up from the ground in the pursuit of developing senolytics, molecular drugs that destroy senescent cells, or senomorphics, drugs that suppress the SASP.

Of course, one application of such compounds could be to explore the "reversal" of human aging. A variety of senolytic compounds are available for purchase online, branded as "life activators". *

An alternative, perhaps more clinically relevant application of senolytics or senomorphics is as therapeutics for the treatment of aging-related pathologies.

For this to be achieved, a comprehensive understanding of the abundance of senescent cells and their secretory products in cells, tissues and organs is required: in other words, a simple biomarker of senescence, or "aging".

In a new study published in PLOS Biology, scientists led by postdoc Nathan Basisty at the Buck Institute have harnessed advances in proteomics to extensively profile the SASP of human cells. Their analyses are collated in a database that is now available for researchers in the field.

Proteomics advances our understanding of human aging

Technology Networks
spoke with Judith Campisi, professor at Buck and senior author of the study, and Birgit Schilling, senior co-author and assistant professor and director of the Buck Institute's Proteomics and Mass Spectrometry Center, to learn more about the technological advances that have enabled this research and the projected impact the study will have on the field.

"Judy is a pioneer in the field of senescence and has really brought the biology forward contributing to our understanding of the role of cellular senescence in aging," Schilling says. "We partnered up around 10 years ago to combine Judy's biology with the modern proteomics technologies that I have in my lab. In 2010 Judy published a paper where she used an antibody array to look at the SASP of senescent cells. We thought, 'let's use discovery technologies and take a closer look in a more unbiased fashion'."

Mass spectrometry (MS) was key for the scientists here; specifically, a "data independent approach", or DIA.

If you're unfamiliar with the differences between DIA and data dependent acquisition (DDA), make sure to check out our infographic, DIA vs DDA.

"DIA, what used to be called SWATH, is a MS technology method where, in contrast to DDA, we use windows and take entire peptide ranges from the MS1 data, rather than focusing on a select number of peptides that are eluted. This allows for discovery and quantification at the same time," Schilling comments.


It seems a DIA approach provides a more "holistic" view of what's going on at the molecular level, ensuring that even the proteins with low expression levels are captured. "It really overcomes the randomness of sampling that other technologies, such as DDA, suffer with. Applying this technology provided us with comprehensiveness that hasn't been seen before."

Utilizing the DIA approach, the scientists expanded on the proteins known to be secreted by senescent cells to over 1000. Their initial research adopted human lung fibroblasts and epithelial cells from the kidney. The data from these initial analyses were then compared with markers of aging found in the Baltimore Longitudinal Study of Aging that involves more than 3,200 volunteers.

Creating a "whole body" biomarker of aging

Basisty and team discovered a "core" set of senescence factors (secreted by all different types of senescent cells that were studied) were elevated significantly in human plasma as we age. The scientists suggest that these core factors may provide the basis for developing "whole body" biomarkers of aging. Campisi explains this to be a clock of biological age, not chronological age: "The idea is that you could look at samples that are easily obtained, such as plasma, or even better, urine or saliva, and look for these proteins that we know are markers of senescence and that also increase with age. From this data, we'd be able to determine how old an individual is biologically."

Some of the "core" senescence factors uncovered were metalloproteases which are already known to play a role in inflammation. However, several other less-researched factors were discovered in the study, such as growth/differentiation factor 15 (GDF15), a stress response cytokine.

"This is really where the unbiased approach of our technology came in. One of our colleagues, Luigi Ferrucci, MD, the director of the Baltimore Longitudinal Study of Aging, conducted a study in human plasma and he found that GDF15 is one of the best biological aging predictors in his data set. That was one of our most robust senescence markers," Schilling remarks.

Ferrucci notes that "The development of senescence-associated biomarkers will allow us to identify factors that drive aging and disease in specific tissues and will hopefully lead to early detection and interventions that will prevent disease progression. We are pleased to partner with the Buck Institute in this effort."

Senescence – it's not always a bad thing

"The complexity of the SASP, which is typically monitored by a few dozen secreted proteins, has been greatly underappreciated, and a small set of factors cannot explain the diverse phenotypes senescence produces in vivo," said Basisty. He describes the
SASP Atlas as a comprehensive proteomic database of soluble and exosome SASP factors originating from multiple senescence inducers and cell types. Each profile consists of extremely different, distinct proteins in addition to the "core" subset of proteins that are elevated in all SASPs.

"There are now companies [and research institutes] who are using senolytics, the idea being that they can selectively kill senescence. What we've shown over the last 10 years is that, although it's true that senescent cells contribute to many age-related diseases, they're not always bad. The idea is that, by understanding what each cell type secretes as part of their senescence phenotype, we can
help the pharmaceutical industry design more specific drugs."

"Proteomics allowed us to take a totally unbiased approach in this project and is the perfect example of how you can utilize high-end technology to move forward with biological questions," concludes Schilling. "Our hope is that the SASP Atlas will facilitate identification of proteins that drive specific senescence-associated phenotypes and catalog and develop potential senescence biomarkers to assess the burden and origin of senescent cells in vivo."

Judith Campisi and Birgit Schilling, Buck Institute, were speaking with Molly Campbell, Science Writer, Technology Networks.


*Technology Networks does not endorse the purchase of such products.

References:

1.       M. van Deursen. (2014). The role of senescent cells in ageing. Nature. DOI: 10.1038/nature13193.

2.       Paez‐Ribes,González‐Gualda, Doherty and Muñoz‐Espín. (2019). Targeting senescent cells in translational medicine. EMBO Mol Med. DOI: 10.15252/emmm.201810234.

3.       Basisty et al. (2020). A Proteomic Atlas of Senescence-Associated Secretomes for Aging Biomarker Development. Science Advances. DOI: http://dx.doi.org/10.1371/journal.pbio.3000599

Meet The Author
Molly Campbell
Molly Campbell
Science Writer
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