The Case For: Senolytics

In this new series of articles, LEV Foundation’s Chief Science Monitor, Maximus Peto, introduces the interventions used in the first Robust Mouse Rejuvenation study and explores the scientific rationale for their selection.

The second article in the series will be published in early 2024.

Introduction

As of 2023, the flagship research program at LEVF is our Robust Mouse Rejuvenation (RMR) studies, the first of which (RMR-1) was initiated in early 2023. In this program, we seek to investigate the potential lifespan-enhancing effects of combining multiple interventions in middle-aged mice which have been previously shown to extend the lifespans of lab mice.

For RMR-1, we decided to test a senolytic intervention as one of the four interventions administered in combination to our study mice at Ichor Life Sciences (Syracuse, NY). Those four interventions are:

  • Senescent cell ablation via galactose-conjugated Navitoclax (“Nav-Gal”)

  • Rapamycin in food at 42 ppm

  • Enhanced telomerase expression via repeated TERT gene therapy (via nasally administered AAV-mTERT)

  • Hematopoietic stem cell transplantation

In this essay, we hope to answer readers’ questions about the role of cellular senescence in aging and the role of senolytic therapies in rejuvenation. We will further speculate on how reducing the burden of senescent cells might synergize with other rejuvenation therapies.

We will:

  1. Briefly describe senescent cells and their associations with adverse health in lab animals and humans.

  2. Concisely explain senolytics and some information about how they work.

  3. Summarize some benefits that have been associated with senescent cell removal.

  4. Summarize the most well-studied senolytics to date with some accompanying scientific references.

  5. Explain the senolytic method we chose for RMR-1 and why.

  6. Speculate about how senolytic treatment might interact with the other RMR-1 interventions we chose.  

In this particular essay, it is not our intention to be encyclopedic in our review of these topics (there has been a large amount of research done on senescent cells and senolytics over just the past decade), but we do hope that this review will inform and excite our supporters, colleagues, and collaborators.  

Let’s get started by first discussing senescent cells.

What are senescent cells?

The word senescence is related to the Latin word for “old man” — senex. Senescent cells are unusual in part because they don’t display normal proliferation behavior. In general, cells become senescent when they experience a certain level of stress or cellular damage. These factors appear to initiate a kind of stress response in the cell which involves restricting the proliferation of the damaged cell.

Senescent cells chronically secrete a group of inflammatory proteins which is often referred to in the scientific literature as the “senescence-associated secretory phenotype”, abbreviated “SASP”. The SASP proteins appear to play a role in normal wound healing processes but also seem to be associated with adverse effects if they are chronically present at high levels (for a discussion on this point, see Paramos-de-Carvalho et al., 2021. Their illustration on this topic is presented below).

Figure 2 from Paramos-de-Carvalho et al., 2021) illustrating the differing contexts and effects of transient vs. persistent SASP.

Senescent cells arise normally during developmental processes and during wound healing in normal, healthy people (Gal et al., 2022) — they’re not particularly unusual or harmful per se. However, the prevalence of senescent cells clearly increases with age, such that older people can have a much higher baseline prevalence of senescent cells than younger people (e.g., see Ogata et. al, 2023). And this increased prevalence seems to be associated with negative health effects.

Next, let’s explore some of the adverse effects associated with the elevated prevalence of senescent cells.

What adverse effects are senescent cells associated with?

There have been many studies showing an association between the elevated prevalence of senescent cells and adverse health effects. Later in this essay, we will summarize some of the scientific studies that reported that removal of senescent cells was associated with benefits. That the removal of senescent cells results in benefits suggests that their presence in the first place was doing some harm.

However, there is at least one other line of evidence supporting the hypothesis that an elevated burden of senescent cells is associated with adverse effects: transplanting additional senescent cells into lab animals, which has also been reported to induce negative health outcomes and accelerate disease.

Finally, there are also studies which report that elevated senescent cell prevalence is associated with disease states, which suggests the disease might be at least in part caused by the elevated prevalence of senescent cells.

Below are some example studies which reported an association between elevated senescent cell prevalence and adverse health effects:

  1. Xu et al. (2017) measured the effects of transplanting a small number of senescent cells into knee joints of mice. They reported that these senescent cells “caused leg pain, impaired mobility, and radiographic and histological changes suggestive of [osteoarthritis].” These effects were stronger than those observed when non-senescent cells were transplanted, suggesting that senescent cells might contribute to the development of tissue degeneration in the tissues they reside.

  2. In a different study, Xu et al. (2018) reported that transplantation of a relatively small number of senescent cells into young mice was associated with a rapid reduction in physical function. They reported: “Previously healthy young adult mice transplanted with [senescent] cells had significantly lower maximal walking speed, hanging endurance, and grip strength by one month after transplantation compared to mice transplanted with [non-senescent] cells”.

  3. Both p16 and p21 are commonly used as markers of cellular senescence. In one study, Zhang et al. (2022) found evidence of higher expression of both p16 and p21 in the muscles of older men and women, suggesting a higher prevalence of senescent cells there. Moreover, higher expression of p16 and p21 were associated with modestly lower cardiorespiratory fitness and leg strength, suggesting that elevated senescence cell prevalence in muscle tissue is associated with lower muscle function.

  4. A study by Tsuji et al. (2006) reported that people with emphysema (a subtype of chronic obstructive pulmonary disease, abbreviated “COPD”) had higher proportions of lung cells expressing markers of senescence, and the prevalence of these senescence markers correlated with the degree of airflow restriction in these patients. These results suggest that cellular senescence in the lungs is associated with COPD/emphysema.

  5. Liton et al. (2005) reported a four-fold increase in the proportion of glaucoma-related cells expressing beta-galactosidase (a marker of senescence) in patients with glaucoma, suggesting that elevated senescence burden might be associated with glaucoma.

  6. Jeon et al. (2022) found that if young mice shared their circulatory system with old mice, a notable increase in senescence markers and an apparent increase in senescent cell prevalence was observed in the young mice. This increase in senescence markers in the young mice was associated with reduced muscle strength, increased liver fibrosis, increased markers of kidney damage, and reduced exercise endurance, suggesting that even exposure to secreted SASP factors from old mice might induce adverse effects in young animals.

Now that we’ve explored what senescent cells are and some adverse effects that they’ve been associated with, what should we do about them?

One thing we can do is induce them to die.

Many types of cells regularly die and are replaced in the body as part of normal cell turnover. Some research suggests that senescent cells avoid being eliminated by interfering with how the immune system functions (explored by Giannoula et al., 2023). If long-term senescent cells can be eliminated, the negative effects associated with their SASP can be eliminated, which might be the main mechanism by which senescent cell elimination induces health benefits in older animals.

We can induce senescent cells to die with interventions specifically targeting senescent cells while leaving normal cells unharmed. These senescent-cell-death-inducing interventions are called senolytics.

What are senolytics?

The word senolytics is a blend of two words — senescence and lysis. We’ve already discussed what cellular senescence is. Lysis means “a process of disintegration or dissolution”. So, the use of senolytics is an effort to selectively and deliberately induce disintegration or elimination of senescent cells.

One could reasonably want to be cautious about purposefully inducing the death of cells in the body, but there have been multiple reports of health benefits associated with administration of senolytics, particularly when done in animals with elevated senescent cell burden such as animals that are older or have been treated with chemotherapy or radiation — both of which are known to elevate the prevalence of senescent cells.

Benefits associated with senolytic administration

Some of the most promising evidence for the use of senolytics in rejuvenation therapies comes from studies conducted in older laboratory mice.

For example, Baker et al., (2016) reported that removal of cells expressing p16Ink4a (a marker of senescence) between 12 and 18 months of age was associated with a noteworthy 23% increase in the median lifespans of male mice and a 30% increase in the median lifespans in female mice. Yousefzadeh et. al., (2018) reported a 16% extension of median lifespans and 8% extension of maximum lifespans in male and female mice when administered fisetin at the age of 20 months. Xu et al., (2021) reported an 11% extension of median lifespans in male and female mice when those mice were administered procyanidin C1 (which was reported to have senolytic activity) starting at age 20 months. Finally, Ming et. al., (2018) reported that the administration of dasatinib and quercetin in mice starting at age 24 months (quite old) was associated with a very modest 4% extension in the median lifespans of male mice.

These are modest extensions of mouse lifespan relative to some other interventions known to extend mouse lifespans. However, in addition to these modest lifespan benefits, there have also been many studies reporting positive effects besides life extension associated with senolytic administration. These studies include (but are not limited to) the following:

  1. In a study using old lab mice, Dungan et al. (2022) reported: “We find that the clearance of senescent cells with dasatinib + quercetin restores muscle growth in old mice”.

  2. Johmura et al. (2021) found that they could induce senolysis by inhibiting the ability of senescent cells to use glutamine for energy. Using this senolytic method was associated with alleviation of age-related organ dysfunction in mice.

  3. Budamagunta et al. (2023) reported that treating middle-aged rats with either a combination of dasatinib and quercetin or with Navitoclax was associated with “…rescued memory [and] preserved the blood–brain barrier (BBB) integrity”.

  4. The combined use of dasatinib and quercetin in old mice was reported by Dungan et al. (2022) to be associated with better muscle regeneration after chemical injury, evidenced by a larger muscle cross-sectional area 28 days after injury.

It is encouraging to see positive effects of senolytics reported in different tissues when they are used in older rodents. These and other studies support the hypothesis that the targeted use of senolytics — particularly in older organisms — might provide enhancements to longevity and quality of life.

What do these senolytic compounds do? How do they cause senescent cells to die and leave healthy cells relatively unharmed? Let’s review those questions next.

How senolytics work and some examples

There appear to be multiple mechanisms by which senolysis can be accomplished. The most common mechanism is to inhibit proteins associated with cell survival during stressful situations.

The survival of senescent cells is dependent on proteins and processes that are different than those used for survival by non-senescent cells. We can exploit these differences to specifically target senescent cells while leaving non-senescent cells relatively unaffected.

For example, there is a family of pro-survival proteins called Bcl, which stands for “B-cell lymphoma”. It was apparently named based on the type of cancer in which it was initially studied (Wikipedia). Bcl proteins are apparently upregulated in senescent cells but not in normal cells (Yousef et al., 2016, Figure 1d), which helps them to survive stressful situations (Cory et al., 2003). Therefore, inhibiting Bcl proteins—and thereby reducing the survivability of senescent cells—appears to cause targeted cell death in senescent cells only.

Indeed, many research groups have tested the idea of inducing senolysis by inhibiting proteins or processes that senescent cells rely on to survive. These include:

  1. Navitoclax (a.k.a. ABT-263) appears to inhibit Bcl-2, Bcl-w, and Bcl-XL, but caution must be exercised because it has significant toxicity for some blood cells such as platelets (Tse et al., 2008).

  2. Dasatinib is a chemotherapeutic drug which appears to induce senolysis by inhibiting a protein called Src kinase (Chan et al., 2012).

  3. Quercetin — which is usually combined with dasatinib — appears to inhibit Bcl-w (Priya et al., 2014).

  4. Fisetin appears to be less toxic than dasatinib or navitoclax and seems to induce senescent cell death by induction of several types of cell death proteins called caspases (Yang et al., 2012).

For the first round of LEVF’s Robust Mouse Rejuvenation experiments (“RMR-1”), we decided to use a modified version of Navitoclax, which we’ll explain next.

The senolytic used in the Robust Mouse Rejuvenation-1 (RMR-1) study by LEVF

We found Navitoclax, also called ABT-263, particularly interesting for our first RMR study for several reasons.

First, Navitoclax appears to be effective at inhibiting Bcl-2, Bcl-w, and Bcl-XL (Tse et al., 2008). Because different cell types can overexpress different survival proteins when they become senescent, the ability of Navitoclax to inhibit all three of these proteins means that it might be relatively more effective at reducing the elevated numbers of senescent cells in many different tissues in the body.

Second, Navitoclax also seems to be increasingly well studied. There have been many scientific reports about its effects on both normal and senescent cells, and this gives us confidence about its possible effects in older animals.

However, Navitoclax has a drawback: it has been shown to be toxic to normal, healthy platelets and other immune cells (Wilson et al., 2010). In a review article about senolytic drugs, Kirkland and Tchkonia (2020) wrote:

Navitoclax (N)…[has] substantial off‐target apoptotic effects on nonsenescent cell types, such as platelets and immune cells…, making them potentially ‘panolytic’. 

Fortunately, some researchers have designed a method to substantially reduce the toxicity of Navitoclax to non-senescent cells. To understand how this was accomplished, one needs to know a little more about senescent cells.

As described earlier, cellular senescence is an unusual state, and accordingly, there are some unusual phenomena that occur in senescent cells that enable us to distinguish them from normal cells. One phenomenon that appears to occur widely in senescent cells but not in normal cells is the unusually high prevalence of an enzyme called beta-galactosidase. When cells become senescent, beta-galactosidase appears to accumulate in lysosomes inside cells.

Simply put, beta-galactosidase is an enzyme that cleaves galactose molecules off other molecules they’re attached to.

Interestingly, it was found that attaching galactose to Navitoclax reduced the toxicity of Navitoclax for normal cells but retained its toxicity for senescent cells. This conjoined chemical was called Nav-Gal (González‐Gualda et al., 2020). The clever aspect of this technique is that — as mentioned earlier — there is a lot of beta-galactosidase in the lysosomes of senescent cells but not in normal cells.

The researchers found that if you give galacto-Navitoclax to mice, it ends up being toxic to senescent cells but has significantly reduced toxicity in normal cells. This is because senescent cells have a lot of beta-galactosidase, which will separate galactose from Navitoclax, leaving the free Navitoclax to do its job inhibiting Bcl-2, Bcl-w, and Bcl-XL and thereby inducing cell death in senescent cells. But because normal cells don’t have as much beta-galactosidase, they end up with mostly galacto-Navitoclax, which isn’t as toxic as Navitoclax by itself, and thus, normal cells are spared from the toxicity of galactose-free Navitoclax.

The difference between Navitoclax and galacto-Navitoclax is illustrated in Figure 1b from González‐Gualda et al., 2020:

Further boosting our confidence in the therapeutic prospects for Nav-Gal was a publication in early 2023 by Estepa-Fernández et al. in which the researchers administered Nav-Gal in conjunction with a chemotherapeutic drug to mice with human breast cancer tumors (called a xenograft). They found that this drug combination reduced the growth of the tumors and reported that Nav-Gal had reduced toxicity relative to Navitoclax alone.

We think this engineering of Nav-Gal is a clever technique to target senescent cells while simultaneously reducing the toxicity of Navitoclax. We also like that Navitoclax seems to have such broad senolytic activity. We’d wanted to see how it might affect the lifespans of older lab mice in LEVF’s RMR studies, so we chose it for inclusion in RMR-1 in 2023.

Speculation about how senolytics might interact with other RMR-1 interventions

Here, we’d like to explain some of our speculations about how Nav-Gal might interact with the other three interventions we chose to include in RMR-1. We listed the four RMR-1 interventions at the beginning of this essay, but for convenience, here they are again, with our senolytic of choice, Nav-Gal, at the top:

  • Senescent cell ablation via galactose-conjugated Navitoclax (“Nav-Gal”)

  • Rapamycin in food at 42 ppm

  • Enhanced telomerase expression via repeated TERT gene therapy (via nasally administered AAV-mTERT)

  • Hematopoietic stem cell (“HSC”) transplantation

There is evidence that a persistent, elevated prevalence of senescent cells inhibits wound healing, immune function, tissue maintenance, and possibly stem cell function, and that these effects might limit the lifespans of aged mice (and we suspect, humans). We imagine that the elimination of senescent cells will enable the more anabolic interventions — such as TERT gene therapy and HSC transplantation — to work more effectively. Particularly in the case of HSC transplantation (#4 above), we suspect that ridding the body of excessive senescent cells and SASP signaling might enable those transplanted HSC to function better than they otherwise would.

For example, consider the case of the bone marrow, which houses both mesenchymal stem cells (MSC) and hematopoietic stem cells (HSC). There is some evidence that mesenchymal stem cells become senescent during aging and secrete proteins which alter the bone marrow microenvironment, which in turn impairs HSC function (Gnani et al., 2019). We imagine that a senolytic intervention which reduces the prevalence of senescent MSCs in the bone marrow could enhance HSC function, which includes the generation of red blood cells and immune cells.

In addition, there is some evidence to suggest that rapamycin and senescent cell ablation might be synergistic. This comes from evidence that rapamycin inhibits a protein complex called mTOR and upregulates autophagy — a process by which tissues and cells recycle their molecular materials. A study by Carroll et al. (2017) found that senescent cells seem to upregulate autophagy, but then also upregulate mTOR to survive the upregulated autophagy. Carroll et al. (2017) wrote (bold emphases ours):

Although mTORC1 activity appears to be supported by autophagy upon starvation, evidence suggests that persistent mTORC1 simultaneously prevents senescent cells from realizing their full autophagic potential, which would otherwise lead to cell death. This seemingly contradictory role for autophagy as a prosurvival and cell death mechanism is a phenomenon also shown to contribute to tumorigenesis and neurodegeneration

It may be that inhibiting mTOR and enhancing autophagy (both accomplished by rapamycin) might facilitate greater senolysis by Nav-Gal by making senescent cells more susceptible to cell death — they might experience elevated autophagy and will fail to survive it with pro-survival mTOR being inhibited by rapamycin. So, we’ll be looking for this interaction between rapamycin and Nav-Gal and should be evident by a greater reduction in senescent cell prevalence in the mice administered both Nav-Gal and rapamycin, relative to mice administered Nav-Gal without rapamycin.

Concluding thoughts

We have discussed many different topics in this article, and the material ranged from an introductory to an advanced level. We hope that this detailed information will help scientific beginners and experts alike to understand what senescent cells are, why they might be a problem during aging, what we might do about them, and why we believe senolytics may be an important part of a comprehensive multi-intervention rejuvenation strategy. We’ll continue to provide public updates on the progress of our RMR-1 study, which is currently in-progress at Ichor Life Sciences.

And if what you learned here inspires you to support more of this type of research, we’d be delighted to receive your financial support to keep doing this important work. You can make a tax-deductible donation to LEVF here.

Maximus Peto
Chief Science Monitor, LEV Foundation