Be a Senescent Cell Killing Machine

Most of us have accepted that weakness comes with old age. But does it have to? Thanks to a new study published in Nature Medicine, we now have a convincing culprit for age-related physical dysfunction: the accumulation of “zombie” senescent cells. We might be able to fight off senescent cells and their inflammatory secretions with interventions including senolytic drugs, exercise and intermittent fasting.

There’s no question that humans are living longer than they used to. But are they living healthier for longer? Based on a 2010 study from the Journal of the American Geriatrics Society, 45% of people over the age of 85 today are frail, often suffering physical dysfunction and decreased mobility that may lead to hospitalization, placement in a nursing home and mortality. But what causes this frailty and physical dysfunction, in the absence diagnosable disease? One culprit is cellular senescence.

Many of the cells in our body are fated to become senescent at some time or another. Radiation exposure, chemotherapy and metabolic stress including high fat intake have been shown to promote senescent cell accumulation.

Senescent cells look very different from healthy cells. They express different genes, they are prohibited from reproducing or dividing naturally, and they pump out inflammatory “SOS” signals in an attempt to recruit other healthy cells to come to their rescue, usually in vain. These signals include proinflammatory cytokines, chemokines, proteases (enzymes that break down proteins) and other factors that together make up what is called the senescence-associated secretory phenotype (SASP). The inflammation created by senescent cells can lead to tissue dysfunction and even turn healthy cells senescent, like a zombie virus that spreads and eventually causes the zombie apocalypse of late-life frailty. This spreading of cellular senescence may even happen at a distance, with senescent cells in one tissue, like fat, spreading senescence-causing inflammatory signals to cells in another tissue, like muscle.

“Senescence can be induced by such stresses as DNA damage, telomere shortening, oncogenic mutations, metabolic and mitochondrial dysfunction, and inflammation. Senescent cell burden increases in multiple tissues with aging, at sites of pathology in multiple chronic diseases, and after radiation or chemotherapy.” — Xu et al., 2018

Zombie cells sound ominous, even if they are a defense mechanism that our bodies use to neutralize damaged cells that could become cancerous — at least they can’t divide. But what if we could selectively remove senescent cells from the body, or reverse the senescent state?

“We all know that we have a growing aging population, and we are seeing aged individuals suffer with a lot of diseases, most of which are age-related,” says Ming Xu, an assistant professor at the UConn Health Center on Aging and first author on the Nature Medicine study on the impacts of senolytics. “The biggest motivation for my research group is to find a way to improve the quality of late life for these individuals and delay the onset of age-related diseases.”

It’s feasible based on current evidence, Xu says, to delay many diseases of aging by targeting fundamental aging processes like cellular senescence. Xu’s lab is exploring the promise of senolytic drugs, or agents that can eliminate senescence cells. The idea of using senolytics to target and potentially reverse the symptoms of aging, such as physical and cognitive dysfunction, is a relatively new one.

Several years ago, Mayo Clinic researchers developed a seemingly anti-aging mouse model by genetically targeting p16-positive cells. The cyclin-dependent kinase inhibitor p16 is a tumor suppressor protein that increases in tissues with age and that is associated with cellular senescence. You can think of it as a policeman that can permanently revoke a cell’s “license to divide.” It is triggered by severe stress or damage that a cell is unable to repair.

The Mayo Clinic researchers found that they could effectively kill off all of the p16-positive senescent cells in their genetically modified mice by administering a single drug. When the researchers treated these mice to clear out their senescent cells, they found that the mice lived healthier for longer, on average. Even late-life drug treatment helped slow the progression of already established age-related disorders. This indicates that cellular senescence can cause age-related phenotypes and that removal of senescent cells can prevent or delay tissue dysfunction and extend healthspan.

Targeting senescent cells has also been found to prevent age-related bone loss in mice.

“These results indicated to us that senescent cells might play a role in aging, and that they could be a therapeutic target,” Xu said.

“Physical function declines in old age, portending disability, increased health expenditures, and mortality. Cellular senescence, leading to tissue dysfunction, may contribute to these consequences of aging, but whether senescence can directly drive age-related pathology and be therapeutically targeted is still unclear.” — Xu et al., 2018

Several years ago, Xu had a thought that he couldn’t shake. If senescent cells were at least partially responsible for the physical and cognitive dysfunction typically observed in aged animals, young mice should experience accelerated aging if injected with already senescent cells. Xu and colleagues eventually tested this idea by transplanting senescent preadipocytes (stem cells from adipose or fat tissue) extracted from transgenic mice into young, healthy mice. The researchers made these fat tissue cells senescent by exposing them to high levels of radiation or a chemotherapy drug after extracting the cells from their transgenic mice.

Xu and his colleagues were surprised to see that when they transplanted these senescent cells into young mice, they observed an onset of physical dysfunction including weakness and frailty. It was as if the young mice were aging before their eyes.

“This is the first time, to our knowledge, that researchers have transplanted senescent cells into young, healthy mice and observed an age-related phenotype as a result,” Xu said. “I actually developed the idea for this experiment several years ago. We’ve observed for some time now that removing senescent cells from aged animals has beneficial effects. I thought, maybe we can do the opposite. By transferring senescent cells into healthy, young animals, we could confirm the causal effects of senescent cells on aging and physical dysfunction, if the young animals ended up looking more like aged animals.”

Xu’s lab specializes in investigating physical function, so they focused on this aspect of aging and senescent cell accumulation in mice. While the exact mechanisms whereby senescent cells lead to physical dysfunction are still unclear, Xu suspects that inflammation plays a big role.

“Transplanting senescent cells into young mice increases their senescent cell burden,” Xu said. “In other words, the transplanted cells induce other cells in the host to become senescent. All of these senescent cells together contribute to the physical dysfunction we see.”

“Previously healthy young adult mice transplanted with [one million] senescent cells had significantly lower maximal walking speed, hanging endurance, and grip strength by 1 month after transplantation compared to mice transplanted with control cells. […] Reduced walking speed began as early as 2 weeks following a single implantation of senescent cells and persisted for up to 6 months, yet the transplanted cells survived in vivo for only approximately 40 days, consistent with the possibility that senescent cells might induce senescence in normal host cells.” — Xu et al., 2018

Xu and his colleagues were able to measure how many of the host organism cells in young mice became senescent following transplantation by looking for genetic markers present only in the transgenic mice that they extracted the cells from. Being able to differentiate the senescent cells they found in their young mice by the cells’ origin, they had proof that the transplanted senescent cells created many more senescent cells in the host mice.

While inflammation may be the primary mechanism whereby transplanted senescent cells cause more host cells to become senescent, this turns out to be a difficult idea to prove. Inflammation and levels of cytokines are difficult to measure and compare between individual animals because of their innate variability. It’s best to compare levels of cytokines and inflammation across different time points but within the same individuals, for example before and after events or exposures known to promote cellular senescence.

The transplantation experiments prompted Xu and colleagues to further explore how targeted clearance of senescent cells could improve the healthspan and physical function of aged mice. But instead of targeting inflammation like many other researchers have tried to do with drugs such as rapamycin, Xu and colleagues are looking to kill the source of these Zombie-spreading signals.

Our immune systems normally keep an eye out for mis-folded proteins, damaged and senescent cells, to clear them from our bodies before they wreak havoc. So why couldn’t Xu’s young mice simply rid their bodies of the transplanted senescent cells? Their immune systems should technically have been able to clear these “zombie” cells as soon as they were introduced.

But if the senescent cell burden becomes too great, these “zombie” cells and their inflammatory secretions can compromise a mouse’s (or a human’s) immune system, leaving it prone to an over-accumulation of senescent cells. Aged mice with weakened immune systems are less able to defend themselves against cellular senescence than healthy young mice.

Once they had shown a causal relationship between cellular senescence and aging-related physical dysfunction, Xu and colleagues set their sights on killing of the “zombie” cells responsible for all of this carnage. They hoped that by doing so, they’d be able to improve the physical function and overall health of both young mice treated with senescent cells, and aged mice.

“In my lab, we’ve found that senescent cells have antiapoptotic pathways that help them survive even though they are very proinflammatory and live in inflammatory environments,” Xu said.

Apoptosis is a natural process of programmed cell death that senescent cells, and cancer cells, sneakily surpass. Xu and colleagues used two drugs, dasatinib and quercetin, that in combination have been shown to effectively and selectively eliminate senescent cells. These drugs trigger apoptosis (programmed cell death) in slow-to-die “zombie” cells. Known generally as senolytics, they disable the senescence-associated antiapoptotic pathways that protect senescent cells from inflammatory environments that would normally trigger apoptosis in healthy cells.

Xu and his colleagues found that the senolytic cocktail of dasatinib plus quercetin alleviated physical dysfunction and increased late-life survival in aged mice. The drugs also alleviated and even prevented physical dysfunction in young mice who received senescent cell transplants. Similar effects have been observed in previous studies, Xu said, but only in animal models. Xu and colleagues went a step further by testing their senolytic drug cocktail in human tissue for the first time. They took fat tissue samples from human patients and treated them with dasatinib plus quercetin in vitro.

“We observed naturally occurring human senescent cells being cleared in these tissue samples by our senolytic drug cocktail,” Xu said. “We also observed a reduction in the inflammatory cytokines in these tissues, while key adipokines were not affected. This demonstrates that these senolytic drugs can decrease inflammation without a global killing effect.”

Adipokines are also cytokines, or cell signaling proteins secreted by adipose tissue, but many of these are actually anti-inflammatory and important for a range of tissue functions. For example, the adipokine called adiponectin is a key regulator of insulin sensitivity and tissue inflammation. Higher levels of adiponectin are associated with greater insulin sensitivity and metabolic health. You wouldn’t want senolytic drugs to inhibit adiponectin at the same time as they are having an effect on inflammatory cytokines.

Thankfully, Xu and colleagues found that the senolytic cocktail of dasatinib plus quercetin not only selectively reduced inflammatory cytokine levels in human tissue, but also appeared to improve human adipose tissue function more broadly as measured by gene expression.

“To our knowledge, this is the first time that these senolytics have been shown to kill senescent cells in any human tissues,” Xu said. “We think this is an important step toward a clinical trial for these drugs.”

Could some of the improved tissue function Xu and colleagues observed have been from reversing cellular senescence as opposed to eliminating senescent cells entirely? The answer to this question is still unclear, Xu said. It’s possible, although the current data points to dasatinib plus quercetin promoting apoptosis in treated tissues.

While Xu and colleagues are focusing their efforts on identifying drugs that can selectively eliminate senescent cells, other researchers are exploring whether healthy lifestyles can help people hone their bodies into senescent cell killing machines. For example, results from a study published in Diabetes in 2016 suggest that exercise can prevent diet-induced cellular senescence and metabolic dysfunction in adipose tissue in mice. The researchers fed mice a “fast food” high fat diet (40% of energy from milk fat) and added high fructose corn syrup to their drinking water (think, soda!). The researchers found that exercise reduced the number of p16-positive senescent cells in transgenic mice fed a fast food diet.

“The harmful effects of the [fast food diet] were associated with dramatic increases in several markers of senescence, including p16, EGFP, senescence-associated β-galactosidase, and the senescence-associated secretory phenotype (SASP), specifically in visceral adipose tissue. We show that exercise prevents both the accumulation of senescent cells and the expression of the SASP, while nullifying the damaging effects of the [fast food diet] on parameters of health.” — Schafer et al., 2016

Intermittent fasting is also a promising intervention for the targeting of senescent cells. “Zombie” cells don’t respond well to being starved of sugar(sugar = brains!!) for long periods of time. Intermittent fasting has been shown in animal models to promote autophagy, or cellular “self-eating” that helps clear out damaged cellular components including mis-folded proteins.

Intermittent fasting may also help reduce inflammation and oxidative stress processes associated with cellular senescence. For example, oxidative stress shortens telomeres, the protective DNA caps at the ends of your chromosomes, which can lead to a cell becoming senescent. Intermittent fasting has been shown to reduce markers of oxidative stress and inflammation (for example, in this study of humans with asthma), making it a promising intervention to at least alleviate senescent cell burden in aging individuals.

Xu hopes in the future to discover a reliable, non-invasive biomarker that researchers may be able to use to measure cellular senescence burden in humans across time. Xu and his colleagues identified senescent cells in their study mice via three different invasive markers: p-16 expression, DNA damage in the telomere region and senescence-associated β-galactosidase activity. All of these markers require tissue sampling.

One promising non-invasive biomarker for cellular senescence is activin A, a protein and growth factor that plays a role in cell proliferation. In a previous research study published in eLIFE, Xu found that activin A increases with age in mice. He also found that senescent fat progenitor cells secrete activin A, and that clearing p-16 positive senescent cells from mice is accompanied by reduced serum levels of activin A. These characteristics make activin A a potentially good blood biomarker for senescent cell burden in vivo.

“The experiments revealed that senescent fat cell progenitors release a protein called activin A, which impedes the normal function of stem cells and fat tissue,” Xu said. “Additionally, older mice had higher levels of activin A in both their blood and fat tissue than young mice.”

There are two different ways to go after senescent cells. One is to selectively kill them off. Another is to block the toxic, inflammatory signals that they spew out. Xu and colleagues are working on senolytic drugs that selectively clear senescent cells from the body by targeting anti-apoptotic pathways or p53 signaling. Other drugs exist that inhibit the inflammatory secretions of senescent cells, known as SASP-inhibitors. Rapamycin and metformin, drugs that are already used in humans, and kinase inhibitors are examples of such drugs. Xu and colleagues show in their recent study that these drugs can have some positive effects on physical function in aged mice.

However, SASP-inhibitors also have problematic side effects that are made worse by the fact that these drugs have to be taken on a regular basis. One of the promising aspects of senolytic drugs that eliminate the senescent cells, Xu says, is that these drugs could work while being administered only intermittently. With these drugs you’d only have to kill off your senescent cells every so often, rather than regularly take drugs that simply inhibit the toxins and inflammatory cell signaling pathways that these cells produce.

“One unique aspect of this study is that we treated mice with senolytic drugs at very late-life stage, or at an equivalent of 80 years old in human years. But even at this late stage, we still saw profound benefits,” Xu said. “We extended the lifespan and healthspan of these mice. It indicates that to slow down aging, it might not be necessary to start treatment in middle-age years — late-life treatment might still result in healthspan and lifespan benefits. But we need human clinical trials to tell us whether these senolytic aging interventions can work in humans.”

TAKEAWAY: EXERCISE AND FASTING COUNTER SENESCENCE

by Paige Jarreau, PhD, published in LifeApps.io on July 25, 2018