A Sample of Recent Work on New Means of Detecting and Targeting Senescent Cells

Senescent cells are receiving a great deal more attention from the research community these days, as illustrated by the two papers on methods of senescent cell identification I'll point out today. How things have changed; it wasn't only a few short years ago that scientists struggled to raising funding for animal studies of senescent cell removal, in an environment of little interest in this aspect of cellular biology. That was the state of the field despite the weight of evidence, gathered over decades, for increased cellular senescence in old tissues to be a root cause of aging and age-related disease. Now that studies have demonstrated that targeted clearance of senescent cells improves health and extends healthy life span in mice, and now that the methods of clearance are being used to produce stronger direct evidence for specific age-related disease and loss of function to involve senescent cells, it seems that every other gerontologist is either revising existing views of aging to incorporate cellular senescence or adding studies of cellular senescence to their portfolio.

Most cells fall into a senescent state when they reach the end of their replicative life span, at which point they either self-destruct or are removed by the immune system. Damage from random mutation or a toxic tissue environment can also result in senescence, and should in theory lead to cell death in the same way as for replicative senescence. Complicating the picture somewhat, short-term localized increases in senescent cell presence also appear to be involved in the wound healing process. There may also be numerous multiple distinct forms of senescence with somewhat different behaviors - this is one of many blank spots remaining on the map of cellular biochemistry, presently under active investigation. Regardless, at the end of the day the ideal situation is that all cells that become senescent should self-destruct or be destroyed fairly soon thereafter. Unfortunately that is not the case in practice, and a fraction of these cells linger on, their numbers growing over the years. These cells cause harm primarily through the signals they generate, producing a potent mix of molecules know as the senescence-associated secretory phenotype (SASP) that degrades nearby extracellular matrix structures necessary for tissue function, spurs increased inflammation, and alters the behavior of neighboring cells for the worse. By the time that 1% or more of cells in a tissue have become senescent the SASP and its downstream consequences become a serious threat to health and organ function.

All of this amounts to a very good reason to support research into identification and removal of senescent cells. Therapies capable of clearing senescent cells should produce a form of limited, narrowly focused rejuvenation, improving health at any point in old age. Those therapies will have to be accompanied by improved assays in order to determine exactly how well they remove senescent cells, as well as to definitively establish links between senescence and specific aspects of age-related degeneration. Below find linked a couple of interesting open access papers in which the authors explore potential new approaches to assessing levels of cellular senescence in tissues and tissue samples. The more of this sort of thing the better, to my eyes. Competition tends to result in better solutions at the end of the day.

Detecting senescence: a new method for an old pigment

Senescent cells have been recently shown to contribute causally to the aging process. Elimination of senescent cells by suicide gene-meditated ablation of p16Ink4a-expressing senescent cells in INK-ATTAC mice has led to improvements in healthspan and lifespan suggesting that senescent cells are drivers of aging. This has prompted the scientific community to identify new interventions to target senescence as a therapy against aging and age-related diseases. However, despite remarkable advances, the detection of senescent cells, particularly in tissues, is still a major challenge. There are several reasons, both of a biological and methodological nature, which have hindered the identification of specific markers able to determine whether a cell is senescent or not.

Firstly, while senescence is characterized by numerous changes in gene expression, very few of these differences are exclusive to senescent cells. Secondly, senescence is a kinetic, multifactorial process, with several phenotypic changes occurring at different time points following the initial cell cycle arrest. This could explain why aged tissues are highly heterogeneous, possibly containing cells at different stages of the senescent programme. Thirdly, senescent cells manifest the phenotype differently depending on the type of inducing stimuli or the cell type. Finally, recent data have highlighted that senescence may play different physiological roles in different contexts. For instance, an 'acute' type of senescence has been shown to play a beneficial role during processes such as development or tissue repair, while a 'chronic' type of senescence may contribute to aging and age-related disease. The recent realization that there may be different types of senescent cells in tissues has created an additional obstacle to the identification of a universal marker.

The detection of senescence-associated β-galactosidase (SA-β-Gal) activity at pH 6 is probably the most widely utilized method for identification of senescent cells. Nevertheless, there are major limitations to this method. Given the growing realization that senescence is a multifactorial process, a multimarker approach is being favoured by many researchers in the field. Examples of currently used markers are as follows: increased expression of cyclin kinase inhibitors p21 and p16 and absence of proliferation markers; telomere-associated DNA damage foci; senescence-associated heterochromatin foci; loss of lamin B1; senescence-associated distension of satellites (SADS); and expression of components of the SASP amongst several others. Nonetheless, there is also growing realization that many of these markers are not exclusive to all types of senescence and may only occur in specific cell types.

Lipofuscin is a nondegradable aggregate of oxidized lipids, covalently cross-linked proteins, oligosaccharides and transition metals which accumulate within lysosomes. Multiple studies indicate that lipofuscin accumulates in various tissues and species with age, particularly postmitotic tissues such as the brain and cardiac and skeletal muscle. However, lipofuscin has also been shown to accumulate during replicative senescence of human fibroblasts. Lipofuscin is autofluorescent and can be visualized using fluorescent microscopy; however, several other histochemical methods have been described based on lipid detection, such as staining using Sudan Black B (SBB) amongst others. Here, a structurally similar compound to SBB has been designed and coupled to biotin. Commercially available SBB contain numerous impurities which impact on staining quality and justified the need to synthesize a new analogue. The chemical coupling with biotin allows its detection using antibiotin antibodies and thereby increases its detection sensitivity. This method is versatile: it can be used in fresh, frozen cells and tissues, but also in fixed material. Furthermore, it can be identified in cells using both microscopy and flow cytometry.

While the authors have convincingly demonstrated that lipofuscin accumulation correlates with senescent markers in cell culture and that lipofuscin increases in tissues with age, future work should investigate more thoroughly whether and to what extent the lipofuscin signal overlaps with other established senescent markers. A separate question which arises from this work is whether lipofuscin accumulation is a mere consequence of the induction of the senescence programme or whether its accumulation contributes causally to the development of senescence.

Senescent cells expose and secrete an oxidized form of membrane-bound vimentin as revealed by a natural polyreactive antibody

Studying the phenomenon of cellular senescence has been hindered by the lack of senescence-specific markers. As such, detection of proteins informally associated with senescence accompanies the use of senescence-associated β-galactosidase as a collection of semiselective markers to monitor the presence of senescent cells. To identify novel biomarkers of senescence, we immunized BALB/c mice with senescent mouse lung fibroblasts and screened for antibodies that recognized senescence-associated cell-surface antigens by FACS analysis and a newly developed cell-based ELISA. The majority of antibodies that we isolated, cloned, and sequenced belonged to the IgM isotype of the innate immune system.

In-depth characterization of one of these monoclonal, polyreactive natural antibodies, the IgM clone 9H4, revealed its ability to recognize the intermediate filament vimentin. By using 9H4, we observed that senescent primary human fibroblasts express vimentin on their cell surface, and mass spectrometry analysis revealed a posttranslational modification on cysteine 328 (C328) by the oxidative adduct malondialdehyde (MDA). Moreover, elevated levels of secreted MDA-modified vimentin were detected in the plasma of aged senescence-accelerated mouse prone 8 mice, which are known to have deregulated reactive oxygen species metabolism and accelerated aging.

Based on these findings, we hypothesize that humoral innate immunity may recognize senescent cells by the presence of membrane-bound MDA-vimentin, presumably as part of a senescence eradication mechanism that may become impaired with age and result in senescent cell accumulation. Given the growing evidence that oxidized proteins are involved in the development of human disease, the detection and monitoring of secreted proteins like oxidized vimentin is certain to become a vital and noninvasive biomarker for monitoring age-related illnesses.


Why was the technique of inoculating mice with senescent cells to find a surface marker only used now?

Posted by: Jim at February 20th, 2017 9:21 AM

@Jim: I imagine that there is a lot of obvious-in-hindsight stuff that hasn't been done yet in this field because of lack of ability to raise funding. Funding is now much easier to obtain, thanks to the rising profile of the field. It was only six years ago that funding was next to impossible to find, and really that only thawed out in earnest a year or two ago. We should expect a whole lot of funding and papers over the next couple of years, given the events of the past year.

Posted by: Reason at February 20th, 2017 9:37 AM
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