Parabiosis: the Dilution Solution?

Summary: Scientists have long marveled at the rejuvenating effects of heterochronic parabiosis. When you mix the blood of a young mouse and an old mouse by joining their circulatory systems, the older animal recovers some features of youth, while the young animal becomes functionally older. While many have assumed that these effects were driven by the infusion of pro-youth factors from the young parabiont into the older one, an alternative “Dilution Solution” hypothesis is possible: that the young blood is instead diluting pro-aging factors from the old animal’s blood, as well as allowing the young animal’s livers and kidneys to filter out metabolic toxins through the young animals’ livers and kidneys.

In 2016, a SENS Research Foundation study in the lab of Drs. Irina and Mike Conboy at UC Berkeley gave significant support to the Dilution Solution, and they have now published a pair of new studies showing that literally diluting the aging plasma with injections of saline plus replacement of the relatively inert transport protein albumin promotes even more dramatic rejuvenation effects on body and brain. The studies also suggest a durable re-set of the aging signaling system after breaking pro-aging signaling feedback loops. In the future, repair of the underlying cellular and molecular damage will abrogate those loops at their root, maintaining the youthful signaling system indefinitely.

When researchers surgically conjoin the circulatory systems of a young and an old animal, something remarkable happens: the older animal recovers some features of youth, while the young animal becomes functionally older. This phenomenon of heterochronic parabiosis was first discovered by Clive McCay of Cornell (best known for his work on caloric restriction) in the 1950s, but after a couple of decades of work, the thread lay dormant for the rest of the century. A dramatic revival of heterochronic parabiosis research was triggered by a seminal study published in 2005 by Irina and Mike Conboy at Berkeley, which demonstrated that exposure to the young animal’s circulation dramatically improved the older parabiont’s regenerative response to injury.

The encouraging results of parabiosis — combined with modern tools of which Clive McCay could only dream — have driven a quest to determine the underlying mechanisms of the phenomenon, and how we might capture some of those mechanisms to develop or improve rejuvenation therapies. Broadly, there are two alternative (but not quite mutually exclusive) possible explanations.

One hypothesis (which we will term the “Infusion Solution”) is that one or more factors circulating in young blood is critical to keeping us young, such that the loss of these factors with age progressively robs us of our youthful health, making us biologically older over time.  This is in some ways the more tantalizing explanation, and for a long time it was the most popular, as it suggests an intrinsic fountain of youth. If we could identify the key youth-promoting components of young blood, we could use standard biological techniques to produce more of them (or analogs with advantageous properties, or drugs that stimulate their production) and deliver them up as anti-aging biologics.

The alternative hypothesis (the “Dilution Solution”) posits that instead of there being “youth-promoting” factors in young blood that decline progressively with age, older blood instead becomes progressively saturated with pro-aging factors.

Logically, either hypothesis could explain the most high-level phenomena of parabiosis. So which one was true?

Round One: Seek and Ye Shall Find (or Not)

One way to resolve this question is to look for actual pro-youth or pro-aging factors that:

  • exist at opposite levels in young and old animals’ blood;
  • can mimic some of the effects of parabiosis when injected into an animal with naturally low levels of the substance; and
  • induce those same pro-aging or rejuvenating effects when depleted from an animal with naturally high levels.

Harvard and Stanford researchers published a paper in 2013 announcing that they had identified growth and differentiation factor 11, or GDF-11, as a key youth-promoting factor. The researchers asserted that GDF-11 restored muscle regeneration after injury, increased muscular strength, reduced steady-state levels of genetic damage, reversed dysfunctional age-related growth of the heart, and boosted production of new neurons (neurogenesis), among other benefits, in the aging mouse.

However, a series of subsequent investigations by other researchers showed to most scientists’ satisfaction that the Harvard and Stanford researchers were misidentifying the factor: the substance they were detecting was probably not even GDF-11, and in any case could not plausibly exhibit the effects that they had ascribed to it.

In fact, no signaling molecules have yet met robust criteria for pro-youth factors, with at most weak or incomplete evidence for things like testosterone. By contrast, a series of studies have identified specific pro-aging factors in old blood that cause the young animal to become functionally older, supporting the Dilution Solution hypothesis.

For instance, levels of eotaxin/CCL11 rise with age in the blood of old mice and in the plasma and cerebrospinal fluid (CSF) of otherwise-healthy aging humans. Eotaxin is an inflammatory factor known previously for its role in the allergic response, not brain aging — but Stanford researchers showed that when delivered to young mice (whether as part of parabiosis or through direct injection), it inhibits the formation of new memories and the adaptation of neuronal circuits.

Similarly, the blood of aging humans and mice contains elevated levels of beta-2-microglobulin (B2M), as does the aging mouse hippocampus (a critical region of the brain for new memory formation). B2M is transferred over to young partners during parabiosis, and has a detrimental effect on cognitive functioning and neurogenesis when injected into either the younger animal’s circulation or directly into the hippocampus. There is furthermore a similar, if more complicated, story to be told about transforming growth factor beta (TGF-β) in parabiosis and impaired memory and muscle repair in aging.

Adding to the growing pool of relevant work, Belgian and US researchers recently discovered that cancer cells adopt a more aggressive character when cultured in aged human serum than in serum from young people. The researchers traced this phenomenon back to rising levels of methylmalonic acid (MMA), a byproduct of metabolism of fatty acids produced by gut bacteria that is usually used as a marker of vitamin B12 deficiency.

It’s not clear what’s driving this rise in MMA with age: it didn’t appear to be due to subjects’ B12 levels, for instance, despite the fact that B12 deficiency becomes more common with age. It’s also not clear whether or how it might relate to the classical effects of heterochronic parabiosis, which mostly affect regenerative capacity and have not previously been linked to cancer. But intriguingly, the researchers found that the effects of MMA on cancer cells appear to involve activation of a vicious cycle of TGF-β signaling, which as noted above lurks in the background of many of the classical parabiosis phenomena. Since aging is the main driver of cancer risk, this finding presses the question of whether MMA might be part of the reason — and whether these preliminary effects might be our first glimpse of much wider pro-aging effects of factors in old plasma.

Overall, when we look for evidence of specific molecular culprits for or medicines against the aging process carried through the circulation that might explain the effects of parabiosis, round one goes to the Dilution Solution.

Round Two: Taking the Waste Treatment Plant Offline

Another possible player in the pro-aging/rejuvenating effects of parabiosis that has been largely ignored until recently is the potential role of metabolic toxins and wastes. In addition to the cellular and molecular damage of aging that accumulates in our bodies over time, the body’s normal metabolic processes also produce an enormous amount of more transient metabolic waste every day. In youth, much of what could go to waste is instead reprocessed and reused, and the rest is detoxified and excreted.

As we age, however, the organs responsible for detoxifying and eliminating these wastes — the kidneys, the liver, and to a lesser extent the lungs — age along with the rest of us, and their ability to remove these wastes progressively degrades. As a result, waste levels in blood circulation rise with age. These metabolic toxins are definitely bad for us — just ask a patient waiting for a liver transplant or on haemodialysis. Consistent with this, a biomarker called cystatin C – the most reliable marker of loss of kidney function — is also a powerful predictor of broader age-related decline, including death from cardiovascular disease or any cause, multimorbidity (suffering multiple diseases of aging at once), and declining physical and cognitive function.

It seems almost inescapable that the improved health of the old parabiont must be a function of more than simply the younger animal’s pro-youth factors, or of having proactively-generated pro-aging factors diluted away. These mere byproducts of normal metabolic processes, rather than intentionally-created pro-aging factors, could also potentially induce an aged animal’s blood to blight its parabiotic partner.

At the urging of SENS Research Foundation CSO Aubrey de Grey, and with SRF funding, pioneering parabiotic researchers Michael and Irina Conboy conducted a study to tease out the role of access to a young animal’s organs in the parabiosis effect in 2015. Working in the Berkeley lab, SRF alumnus Justin Rebo and Keith Causey built a machine capable of exchanging volumes of blood at will, replacing them with equal volumes of blood (or plasma, or other substitute fluid) from an opposite-aged animal without passing the blood through the opposite-aged parabiont. This isolates the effects of true bioactive signaling factors intentionally produced and circulating in young and old animals from the effects of metabolic wastes produced in the old animal that are (or are not) filtered out by an aging liver and kidneys.

Sure enough, the benefits of directly trading old blood for young were dramatically less impressive than the effects of full-on parabiosis complete with the filtering and detoxification services provided by young liver and kidney function. Notably, while exposure to old blood alone was sufficient to profoundly inhibit neurogenesis in young animals (implying the presence of one or more strong new-neuron-repressive factors in aging blood), there was little or no effect of transferring young blood alone to an aging animal on its brain’s neurogenesis (implying the absence of any active pro-neurogenic factors in young blood). Similarly, the mere transfer of old blood into young animals weakened the animals’ abilities to hang from the ceiling of their cage, and such animals failed to show improvements in hanging after practice — whereas old animals did not get any stronger when given blood from a young animal but not organ access.

On the other hand, receipt of young blood did enhance the repair of old animals’ muscles after injury, although the effects were less impressive than what’s seen in parabiosis — and in this case, there was no inhibitory effect on the muscles of young animals exposed to old blood. Similarly, the ability of an old animal’s injured liver to regenerate was enhanced by young blood, and existing age-related fibrosis improved. These experiments show that these health effects are not mediated primarily by removal of metabolic wastes, though certainly they could still be mediated by dilution effects rather than true active-factor transfers.

Round two, then, was a mixed decision, but advantage: Dilution Solution.

Round Three: Clearing the Waters

By this point, a direct test of the dilution hypothesis would seem to be in order — and recently the Conboys ran one. With the blood-replacement machine up and running, they replaced half the blood of old mice — not with young blood, but with saline solution, plus an amount of the albumin protein family equivalent to that in blood, to avoid losing albumin’s important non-signaling functions in transporting different substances around the body.

Like young blood itself, this “neutral blood exchange” (NBE) substitute (as they called it) would lack all of the pro-aging factors that an old mouse’s blood would contain (as well as the metabolic sludge its aging organs would have failed to remove) — but, importantly, would not contain any of the pro-youth molecules that the Infusion Solution hypothesis supposes are responsible for the effects of heterochronic parabiosis.

Remarkably, a single NBE treatment rejuvenated muscle repair capacity of old mice to equivalent levels of quite young control animals, including major improvements in the number of muscle stem cells engaged to regenerate the damaged muscle (the “regenerative index”), the area of muscle where such cells were active, and in the level of fibrosis left behind (Figure 1). NBE also significantly improved liver health in old animals, partially reversing their fibrosis and reducing the pathological fat deposits in the organ.

Figure 1.

After “Dilution” with saline and albumin, aged mouse serum’s effect on muscle repair is rejuvenated. Redrawn from (1).

Looking at the animals’ brains, the Conboys saw something even more intriguing. A single exchange of half the old animals’ blood for saline solution not only matched the effects of young blood on neurogenesis (the birth of new neurons): it boosted neurogenesis by approximately eightfold — substantially more than had been observed in previous heterochronic parabiosis experiments in their own and other researchers’ labs.

Additional direct evidence against the Infusion Solution hypothesis can be found by drilling a bit further into the details of study. First, if there really were strong youth-promoting factors circulating in young blood, then administering saline-albumin to young animals would have diluted them away and impaired such animals’ regenerative powers. Instead, NBE had no effect on muscle repair or liver health in young animals, demonstrating that such factors either do not exist, or play so little a role in maintaining these processes that you can cut their level in half without consequence.

In fact, NBE actually seemed to enhance neurogenesis even in young mice, although to a lesser degree than it did in the old. Anyone trying to explain the effects of heterochronic parabiosis in the old as the effect of such pro-youth factors will have to explain why halving the concentration of such factors not only lacks harmful effects — but actually seems to benefit the brain.

Spurred on by these striking but limited results, the Conboys looked more broadly at the effects of dilution on the aging brain and cognitive function in a followup paper, showing that a single round of NBE was able to reduce the level of overactive immune cell activity and inflammation in the aging brain, and completely wipe out the difference between young and old animals’ ability to discriminate novel objects by appearance or by texture. On that test, dilution made old brains think like young ones.

Might these effects be due to elimination of senescent cells in the brain? It seemed plausible: senescent cells certainly cause inflammation in their environment, and several previous studies had reported that conventional parabiosis lowered the burden of senescent cells in aging animals. In this followup study, the Conboys confirmed that dilution had the same effect on the aging brain, to a degree similar to the effect of the well-established senolytic (senescent cell-killing) drug navitoclax. Despite this, navitoclax had much less effect on brain inflammation and aggravated immune cells than NBE — and it failed entirely to improve age-related neurogenesis impairment in the aging animals’ brains.

An important but intriguing caveat to these results is that navitoclax is not expected to cross the blood-brain barrier (BBB) — the protective system of tight junctions that shields the brain from toxic substances. The drug’s effects on senescent cells in the brain and resulting inflammation and neurogenesis may therefore have been largely limited to indirect effects of eliminating senescent cells elsewhere in the body (which would prevent inflammatory factors from those cells from reaching the brain), plus a small amount of leak-in of navitoclax because of the age-related breakdown of the BBB. Senolytic drugs or immune therapies that can more effectively reach the aging brain might produce much more profound effects.

Round three again goes to the Dilution Solution, marking strike three for youth-promoting factors.

Hints in Humans

Based on the dilution study, and supported by the earlier study involving direct blood exchange between young and old animals (taking the filtering and detoxification machinery of the liver and kidneys out of the picture), it seems clear that most or all of the effect of young blood on older animals is due to alleviation of the suppressive effects of factors in aged blood on the animal’s tissues.

Would these same effects occur in humans? That study has not been performed (yet! See below), but one part of the animal study did hint that NBE might have similar effects on the liquid information superhighway of aging humans. In a tiny pilot study, the Conboys partnered with Dobri Kiprov, who specializes in apheresis (medical removal of patient blood constituents), to test the effects of the equivalent procedure in humans: an established medical procedure known as therapeutic plasma exchange (TPE), which is used to treat a variety of disorders, mostly of the immune system — as well as, just recently, experimental use to treat advanced COVID-19 cases.

Four volunteers aged 65 to 70 had their blood exchanged with physiologic saline and albumin. Blood samples were taken before and after the procedure and tested for their effects on mouse cells. Compared to blood sampled before TPE, aged human serum taken after a round of dilution increased the proliferation of mouse muscle stem cells, irrespective of added albumin, consistent with what was seen in the mice (Figure 2). Here, as in most of their studies, albumin was apparently not a factor in the results.

Figure 2.

After “Dilution” with saline and albumin, aged human serum’s effect on muscle repair is rejuvenated. Redrawn from (1).

AMBAR, Albumin, Ambiguity

Another trial, completed several years ago, is highly suggestive for dilution effects in aging humans, even though that wasn’t what the trial was designed to test.

Early in the last decade, Grifols — a global plasma products company — launched the AMBAR (Alzheimer’s Management by Albumin Replacement) trial to test albumin as a treatment for Alzheimer’s dementia of aging (AD). This was based on the facts that albumin is both a major extracellular antioxidant, and the carrier of about 90% of the beta-amyloid (Abeta) in the circulation — Abeta being one of the two key damaged proteins responsible for driving AD.

Previous research had shown that the pool of soluble Abeta in the brain exists in a kind of dynamic balance with the pool of Abeta in the blood. Therefore, lowering the level of circulating Abeta creates an osmotic force that draws soluble Abeta out of the brain, potentially offering protection against harmful effects. This “peripheral sink” approach had previously been demonstrated in animals using antibodies that trap Abeta in the blood, and was thought to be the mechanism for some of the passive antibodies targeting Abeta in human clinical trials.

In AMBAR, Grifols scientists hypothesized that AD patients’ albumin might be so saturated with Abeta that it would have lost its ability to draw out any more. Replacing that old, saturated albumin with fresh new protein would thus restore the youthful capacity to pull Abeta out of the brain and eliminate it via the liver.

The Grifols researchers also thought that patients would benefit from the fresh albumin’s antioxidant capacity. Albumin is a major antioxidant in blood plasma, but becomes more oxidized with age, and even more so in several diseases of aging — including AD, where albumin is more oxidized not only in plasma, but even more so in the cerebrospinal fluid.

Additionally, some patients in the trial were given Grifols’ intravenous immunoglobulin (IVIG) — a mixture of the antibodies in normal, non-infected people’s plasma, hypothesized to possibly contain natural antibodies targeting Abeta and thus further enhance the effect. IVIG had shown promise in several smaller clinical trials, but was largely abandoned after failing in a large one. Grifols scientists mostly included it to replace the antibodies that would be depleted by TPE, but also hoped that it might have some therapeutic effect of its own if combined with the effects of TPE and albumin itself.  

The researchers also entertained the possibility that TPE itself would remove other toxic substances from AD patients’ plasma — but as a kind of haemodialysis, without imagining the sweeping effects seen in the Dilution Solution experiments (or in heterochronic parabiosis). Their real focus was on the albumin.

There were four groups in the trial. One group got a placebo treatment through every step of the trial, on the same schedule as the other groups: a yellowish fluid was circulated around the equipment, but was never actually connected to the patient’s circulation, giving the appearance of TPE without in any way changing a patient’s blood. All three of the remaining groups underwent six weekly sessions of conventional TPE — considered an intensive regime — and then, after a short break, received one session once a month of TPE ‘fortified’ with extra low- or high-dose albumin, with or without IVIG, for the next year.

The results were finally published this summer — and they look very promising. Overall, Therapeutic Plasma Exchange slowed AD patients’ decline in self-care ability by a remarkable 52% (see Figure (A)), and strongly appeared to slow cognitive decline — by an even larger two-thirds (Figure (G)) — but the pooled cognitive effects did not quite pass the standard test of statistical significance.

When you drill down one layer, both the cognitive effects and the activities of daily living were statistically significant when the moderate AD patients were considered separately (see Figures (B,H)). By contrast, there was no effect on either parameter in mild AD patients considered separately, seemingly for no other reason than the lack of any detectable decline in any of the mild AD groups over the course of the trial (Figures (C, I)).

Supposing that the effects in the moderate AD were real: what caused them? Was it the basic TPE that all treatment groups received, or was it the extra albumin added for all groups throughout the last twelve months of the trial? Did the IVIG have any effect at all? If TPE alone was the real driver, are we indeed seeing true dilution effects? There’s unfortunately no way to know for sure: all the treatment groups tend to bunch fairly closely together or to change relative positions throughout the trial ( Figures (D, E, J, and K)).

Overall, AMBAR offers some tantalizing clues about the benefits of the Dilution Solution in Alzheimer’s dementia of aging — but is far from proving or disproving the case.

Plumbing the Depths...

To further investigate the molecular details of what was driving the effects of the Dilution Solution in their study, the Conboys looked to see what circulating proteins had changed in mouse and human serum one month after NBE or TPE (respectively). Proteins that were prominently changed included mediators and regulators of immunity, formation of new blood vessels, and growth factors (including growth factors active in neuronal tissue).

In the brain-centered followup study we discussed above, the researchers took a more specific look at the effects of NBE or TPE on proteins known to play roles in the health of the aging brain. Fifteen such proteins underwent major shifts from the pre-TPE aging human plasma to their levels one month post-dilution, as did eleven such proteins (although a different set) in the rodents after NBE.

Overall, more proteins in both studies actually had their concentrations increase than decrease one month after TPE/NBE, even though the whole basis of the intervention is to dilute the aging blood of such factors. But this isn’t so surprising: just as “dilution” was shown to interrupt the inhibitory influence of old plasma on tissue regeneration, it also released the inhibitory influences on the aging mouse or person’s production of some endogenous proteins, breaking a kind of pathological signaling gridlock enforced by long-set feedback driven by underlying aging changes. The Conboys even presented a theoretical model of how the effect on feedback loops in the system could either have long tails, or shift up and down in waves over time, leading to an extended period of complex changes in blood constituents.

And we should know more before too long. The Conboys have made clear their intent to explore the effects of NBE further, assessing its impact on the fate of newborn neurons, on cognition in treated mice, on metabolic health, and on tissues other than the classical three (liver, brain, and above all muscle) that have been the primary focus of modern heterochronic parabiosis research.

And one step more: Dobri Kiprov, the Conboys’ partner from California Pacific Medical Center in San Francisco, has planned for both near-term individual experiments with TPE and an eventual move to larger and more rigorous studies to test the potential of TPE as a short-term therapy to restore a more youthful signaling environment.

... and Parting the Waters

Remarkable as the heterochronic parabiosis phenomena are — and provocative as the dilution study is — it’s important to ask the next question, which is why the aging blood is so suppressive. The answer, of course, is that as a mouse or a human ages, the cells and tissues that produce, filter, detoxify, respond to, and metabolize signaling factors that circulate in the blood accumulate cellular and molecular damage. Damage, in turn, causes organs to become dysfunctional, which impairs the production, filtration, and metabolism of factors, just as a damaged television satellite can scramble and distort the signal it receives, transmits, and beams down to receivers on the ground. Additionally, damaged tissues proactively send out signals to recruit immune cells and other factors to assist in their repair — but as the burden of unrepaired (and unrepairable) damage accumulates, the net level of these same factors becomes dysfunctional.

A good example of this is, of course, senescent cells. One of the main evolutionary reasons why senescent cells produce the SASP (the “senescence-associated secretory phenotype” cocktail of inflammatory, growth, and other factors) is because the senescence response originally evolved as part of the response to injury and was later adapted as a bulwark against cancer. When senescent cells have served their purpose, they secrete inflammatory factors that attract immune cells to clear them out. But removal is never complete (something we’re working to enhance or rejuvenate at SENS Research Foundation), and as we age, senescent cells accumulate — and with them, exposure to SASP signaling factors increase. Studies have revealed a surprising disconnect between the level of senescent cells in an organism and the level of “classical” SASP factors in the blood, but recently scientists in several different labs have identified rising levels of novel and established SASP products in the aging blood and linked them to age-related frailty and disease.

Time Magazine, February 23, 2004. Copyright 2004 TIME USA, LLC

Another source of aberrant signaling in the aging blood is atherosclerosis, as first became widely known after a 2004 Time magazine cover story. Blood vessels infiltrated by oxidized LDL particles attract macrophages to engulf these particles, and macrophages themselves secrete additional inflammatory factors to recruit other immune cells into the lesion to back them up. Additionally, atherosclerotic lesions are riddled with senescent cells, which of course are a source of SASP. This is why C-reactive protein (CRP) — a general marker of inflammation — is useful as a marker of cardiovascular risk, even though CRP is not itself causally involved in the disease.

An additional source of aberrant signaling specific to the brain is the breakdown of the blood-brain barrier (BBB) — the protective network of tight-wound blood vessels and associated cells that protect the brain from infiltration by both toxins and otherwise-benign circulating factors (including albumin!) that can damage brain neurons if allowed unrestricted access to this critical organ. BBB breakdown has long been linked to brain inflammation and cognitive impairment in aging mice and models of neurological aging diseases, and has more recently been documented in humans using new technology. A recent study modeling BBB breakdown in young, healthy mice showed that the entry of extraneous proteins into the brain leads to brain inflammation and cognitive impairment; notably, the factor that sets this cascade off is none other than TGF-β.

Other sources of pro-aging factors in the aging circulation include the accumulation of cells bearing large deletions in their mitochondrial DNA (which transfer oxidative stress to their neighbors and into the circulation), the accumulation of extracellular aggregates, damage to the heart from decades of hypertension and subclinical heart attacks, and (as discussed previously) accumulating damage to the filtering and detoxification structures of the kidneys and lungs.

From Dilution to the Repair Revolution

Provocative as these results are, the Dilution Solution is at best a stop-gap, comparable to keeping a badly worn-out engine running by means of frequent oil changes and top-ups.

No truly long-term studies have been done on either NBE or parabiosis, though the fact that weekly injections of young plasma starting at middle age and continuing throughout the lifespan failed to extend life in mice, suggesting a possible downside counteracting the benefits seen in short-term studies. One can well imagine that cancer and stem cell depletion might be among these downsides (by promoting the unchecked proliferation of stem cells).

Ultimately, the way to permanently restore the youthful systemic environment and reap the benefits of parabiosis is to repair the underlying damage that deranges it in the first place. This is just common sense: we all know the right way to deal with burning oil is to repair the engine, not constantly change the oil and put buckets under the car.

By ablating senescent cells (via senolytic drugs or immune rejuvenation or enhancement) — including in the brain, where the senolytic drugs used in the Conboys’ brain study couldn’t directly reach; by removing extracellular aggregates; by applying cell therapy and tissue engineering to the damaged kidneys, liver, heart, and other tissues; by repairing the blood-brain barrier and leaky gut; by making mitochondrial mutations harmless via backup copies; and through other damage-repair solutions, rejuvenation biotechnology can return our bodies to the same structural integrity — and therefore, the same signaling environment — that gave us health and vigor in youth.


  1. Mehdipour M, Skinner C, Wong N, Lieb M, Liu C, Etienne J, Kato C, Kiprov D, Conboy MJ, Conboy IM. Rejuvenation of three germ layers tissues by exchanging old blood plasma with saline-albumin. Aging (Albany NY). 2020 May 30;12(10):8790-8819. doi: 10.18632/aging.103418. Epub 2020 May 30. PMID: 32474458; PMCID: PMC7288913.
  2. Boada M, López OL, Olazarán J, Núñez L, Pfeffer M, Paricio M, Lorites J, Piñol-Ripoll G, Gámez JE, Anaya F, Kiprov D, Lima J, Grifols C, Torres M, Costa M, Bozzo J, Szczepiorkowski ZM, Hendrix S, Páez A. A randomized, controlled clinical trial of plasma exchange with albumin replacement for Alzheimer’s disease: Primary results of the AMBAR Study. Alzheimers Dement. 2020 Jul 27. doi: 10.1002/alz.12137. Epub ahead of print. PMID: 32715623.
  3. Mehdipour M, Mehdipour T, Skinner CM, Wong N, Liu C, Chen CC, Jeon OH, Zuo Y, Conboy MJ, Conboy IM. Plasma dilution improves cognition and attenuates neuroinflammation in old mice. Geroscience. 2020 Nov 15. doi: 10.1007/s11357-020-00297-8. Epub ahead of print. PMID: 33191466.

Question of the Month: Senolytics – Solution or Self-Defeating for Senescent Cells?

Q: When senolytic drugs cause senescent cells to die, other (younger) cells need to divide and take the place of the dead cells. This cell division causes telomere shortening, thus possibly creating new senescent cells. How is it that the process of killing senescent cells is not self-defeating if new senescent cells are being created?

There are a couple of ways to come at this question. The first is to just look at the astonishing beneficial effects of senolytic drugs or gene therapies in aging mice and mouse models of age-related disease.1,2 In these studies, senolytic drugs have restored exercise capacity1 and capacity to form new blood and immune precursor cells3 in aging mice to near youthful norms, while preventing age-related lung hypofunction,4 fatty infiltration into the liver,5 weakening or failure of the heart,1,6,7 osteoporosis,8 and hair loss.9 These treatments have also prevented or treated mouse models of diseases of aging like osteoarthritis,10 fibrotic lung disease,11,12 nonalcoholic fatty liver disease (NAFLD),5 atherosclerosis,13,14 cancer15 and the side-effects of conventional chemotherapy,2,16 as well as neurodegenerative diseases of aging like Parkinson’s17 and Alzheimer’s18,19,20 diseases… and on and on! So whatever collateral damage might ensue from ablating senescent cells, it’s pretty clear that senolytic treatments are doing a lot more good than harm.

But let’s drill down on the underlying reasoning of the question a little more. Suppose (as the question posits) that every time you destroy a senescent cell, a progenitor cell (one of the partly-specialized tissue-specific cells that repopulate a tissue with mature cells specific to that tissue) replicates to create a new cell to take its place. In fact, studies do show that when senescent cells are killed in a tissue, the progenitor cells begin to multiply and/or to function better as stem cells. This benefit is not due to the progenitor cells automatically replicating themselves and taking the place of the senescent cell, but because the baleful secretions spewed out of senescent cells inhibit the progenitor cells’ regenerative function, such that destroying senescent cells allows the progenitor cells to begin working properly again. This is observed in blood-cell-forming cells,3 cardiac progenitor cells,6 bone-forming cells,8 and the cells that form new fat cells — in both mice21 and now (in a small, short-term clinical trial) even in humans!22

So does this support the worry behind the question? Not really. It just takes a moment’s thought to realize that just one such replication can’t possibly be enough to drive a stem/progenitor cell into senescence: if it did, of course, senolytic therapies would fail to reduce the net burden of senescent cells. But studies clearly show that administering senolytics does lower the overall number of senescent cells in aging and diseased tissues.

Also, if these drugs were not killing more senescent cells than they indirectly produced, you wouldn’t get relief from the harmful effects of having a high burden of senescent cells — and, of course, you do, in multiple tissues and in multiple models of aging and age-related disease.

Going Back to the Well for More

Still, even if a single round of senolytics isn’t enough to drive your stem cells senescent, what if you turn one tissue stem cell senescent for every two times they are triggered to proliferate by senolytic therapy — or every three, or four, or ten? Might a single round of senolytic drugs be a net benefit, whereas repeated treatments over a lifetime would deplete tissue stem cells step by step, eventually riddling the body with senescent cells and leaving the patient (murine or human) worse off over the long term?

Fortunately, we have long-term studies to address that question — and they tell us again that the answer is “no.”

In a study that played a critical role in launching the senolytic drug revolution,15 mice were engineered with a genetic self-destruct mechanism built into all of their cells, which would lie dormant until activated by a two-part command: one, the expression of the gene p16, which is characteristic of senescent cells; and two, an activating drug that scientists could administer to control the pace of senolytic activity. The researchers then waited until the animals were 12 months old (in human terms, this is similar to a person in his or her early 40s) before administering the drug for the first time. They then continued administering the drug every two weeks for the next six months — at which point the animals had received thirteen rounds of senescent cell-clearing therapy, and were roughly similar to humans in their mid-fifties.15

The study clearly showed that the animals benefitted from senolytic therapy, even after undergoing round after round of treatment across the span of their natural middle age through to early natural seniority.15 For instance, the animals whose senescent cell autodestruct mechanism had been triggered subsequently retained more functioning filtering units in their kidneys with age, and fewer of them died in middle age and early seniority.15

More directly on point with our question, the researchers looked to see the effects of multiple rounds of senolytic therapy on total senescent cell burden, and whether they substantially depleted the animals’ reserves of functional progenitor cells by forcing them to divide their way into senescent doom.14 To do this, the investigators looked at the effects of treatment on the preadipocytes (the progenitor cells that form fat cells (adipocytes)) in the animals’ fat tissue — a convenient place to look, because it’s easy to get at, and because it accumulates substantial numbers of senescent cells with age.

After some 13 rounds of senolytic therapy, the treated animals had only one eighth the number of senescent preadipocytes as controls (Figure 1(a)) — a very substantial net reduction. Yet even so, the treated animals still had just as many functional progenitors left — or so close to as many that the difference was indistinguishable from chance (Figure 1(b)).15

Figure 1.

Activating senolytic “self-destruct” genes throughout midlife sustainably slashes senescent cell count (a) with no or very small effects on progenitor numbers (b) in adipose tissue. Bars in (b) with # are differences that were statistically significant. Redrawn from (15).

In another study,23 researchers administered the natural senolytic compound fisetin to mice every single day, starting from the point when they were already in early seniority (and thus already had both a large number of senescent cells, and a dwindling supply of progenitor cells) and continuing on until their death. The extended treatment slashed the number of senescent cells in most tissues in the order of 50%. On top of that, the animals lived substantially longer, and their tissues suffered less severe age-related degenerative lesions than control animals.23

Out with the Old - and In With the New

That said, it is important to have plenty of functional cells around in order to reap the full benefits of senolytic therapy. This was illustrated in a study using senolytic drugs or “self-destruct” genes to treat animal models of osteoarthritis.10 When joint disease was initiated by injury in young animals, the insult led to chronic joint damage and an accumulation of senescent cells in the synovium (the membrane surrounding the joint, where is the cells that maintain the complex fluid that lubricates the joint reside). Eliminating senescent cells reduced the inflammation in the joint, prevented joint erosion, and alleviated signs of pain in the animals.10

But senolytic treatment was much less effective when the scientists repeated the experiment in old animals, and the reasons why shed some light on our original question. Compared to young animals, the old animals accumulated many more senescent cells after their joints were injured, and those cells developed at deeper layers of the tissue, accompanied by more severe osteoarthritis.10 Perhaps this is because in the aging animals, more cells had already suffered significant aging damage, and were thus primed to be tipped over into senescence when injured. And when old animals were then administered senolytic treatment, the remaining healthy cells didn’t respond as well: the burden of zombie cells went down, and the old animals still got some pain relief, but genes that helped the young animals regenerate their damaged joints were not activated, and the cartilage quality score did not improve. The researchers suggest that this could be due to a decline in the number or functional capacity of the non-senescent cartilage-forming cells, driven by degenerative aging processes.10

Similarly, destroying senescent cells in aging mice reduced the excessive numbers of osteoclasts (cells that break down bone) that accumulate in aging bone, but did not restore the dwindling supply of bone-forming osteoblasts, lack of which no doubt constrained the rejuvenating effects of senolytic treatment in restoring the aging bone.8

In both of these cases, the lack of a boost to the number or activity of youthful cells was not the result of damage from the senolytic drug: the failing supply of such cells had already occurred before treatment was initiated. So the problem is not that senolytic therapies stop working or become counterproductive over time: rather, it’s that they only target one kind of aging damage, whereas aging drives disease and disability because of the accumulation of multiple kinds of cellular and molecular damage in our tissues with age. The solution is to pair the killing of senescent cells with the introduction of fresh, new functional cells via cell therapy. (See our analysis of a previous study on senolytic therapy in models of Parkinson’s disease for the clinical path ahead on such combination therapies).

And to return more directly to the original question, this would also be the solution if it ultimately turns out that many decades of of senolytic therapy really did drive too many tissue stem cells into senescence. But as we’ve seen, all the evidence suggests that this won’t be a problem during the decades of our current lifespans.


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Nothin’ Gonna Hold Me Back: Clearance of Senescent Cells for Tissue Rejuvenation

Aging bodies become increasingly burdened over time with dysfunctional cells resistant to apoptotic or other clearance. The most well-known of these are so-called “senescent” cells, originally characterized by Leonard Hayflick as mitotic cells that reached growth arrest after a limited replicative lifespan (later associated with telomere attrition) under unphysiological conditions in culture. Later research has revealed that few cells reach a “senescent” state through sheer replicative exhaustion: instead, senescence has emerged as a programmed response to DNA damage or oncogenic stress, and as part of the resolution of wound healing.(1) Unfortunately, the near-term benefits of these functions — in preventing damaged cells from progressing to cancer, and in preventing fibrosis –are coupled to deleterious long-term consequences, whose effects worsen as the burden of such cells rises with aging. First, the loss of mitotic competence of stem cells denies proliferative tissues of the capacity for renewal. Secondly, the secretory and other phenotypes of such cells progressively derange local and systemic metabolism and tissue function, rendering tissues more vulnerable to metastasis, promoting systemic inflammation, and otherwise impairing tissue function.(1-4)

To bypass the disruptive effects of the age-related accumulation of senescent cells, some investigators are working on possible ways to manipulate the signaling pathways involved in enforcing the senescent phenotype. This approach bears with it great risks, however, because of the very purposes of senescence to which allusion was made above: returning senescent cells to their normal differentiated function and replicative capacity could lead cells bearing oncogenic mutations to progress into metastatic disease, and aberrant resumption of the wound-healing response leading to fibrosis.(1,4) The regenerative engineering solution to this dilemma is therefore the ablation of such cells, to eliminate their contribution to age-related loss of homeostasis without reactivating the more acute risks against which the senescence machinery was activated in the first place.(5)

As widely covered in the mainstream press, a successful proof-of-principle study for this rejuvenation biotechnology has now been performed.(6)

The study was performed using several founder lines, bred onto a background strain of mice hypomorphic for BubR1 (BubR1H/H), a key component of the mitotic checkpoint machinery. Principal investigator Jan van Deursen, Professor of Biochem/Molecular Biology and of Pediatrics at the Mayo Clinic location in Minnesota, had already discovered(7) that BubR1H/H mice “have a markedly shortened lifespan and exhibit a variety of age-related phenotypes, including infertility, lordokyphosis, sarcopenia, cataracts, [subcutaneous] fat loss, cardiac arrhythmias, arterial wall stiffening, impaired wound healing and dermal thinning.”(6) Some, but not all, of these phenotypes were associated with a high age-related incidence in senescent (p16Ink4a-positive) cells.(6-8) and van Deursen and colleagues had already demonstrated that breeding BubR1H/H mice onto a p16Ink4a homozygous-null genetic background attenuated their development of p16Ink4a-senescent cell-associated aging phenotypes and modestly increased their very low survivorship.(8) Imputation of these results specifically to the animals’ age-related, low-BubR1-driven rise in  p16Ink4a-expressing senescent cells was, however, limited: limited by the very nature of so-called “accelerated aging” models such as BubR1H/H,(9) and limited by the lifelong, global absence of p16Ink4a expression in the backcrossed mice.

Seeds of Destruction and Renewal

To impute aging phenotypes directly to p16Ink4a-expressing senescent cells, van Deursen and colleagues with expertise in the aging and senescence of the relevant tissues developed and tested the effects of a pharmacologically-inducible system for the ablation of p16Ink4a-expressing cells. To create this system, investigators modified an approach used in earlier research, in which mice were bred with a variant on the Gene-Directed Enzyme Prodrug Therapy (or “suicide gene”) paradigm,(11) using a drug (AP20187) that activated fusion protein apoptosis machinery in cells in which the macrophage- and adipocyte-specific minimal Fabp4 promoter was transcriptionally active.(10) To generate mice in which p16Ink4a-expressing cells could be similarly selectively ablated, van Deursen’s team substituted a fragment of the p16Ink4a gene promoter for the Fabp4 promoter, thereby generating BubR1H/H;INK-ATTAC mice.(6) In such mice, then, p16Ink4a would still be under normal physiological regulation, and still be induced in an abnormally high number of cells due to the mitotic checkpoint dysfunction caused by BubR1 hypomorphism, leading to the same abnormally-rapid accumulation of high burdens of  p16Ink4a-expressing senescent cells — but administration of AP20187 would induce apoptosis selectively in such cells, purging the animals’ tissues of senescent cells while leaving non-senescent cells unscathed.

Testing the System

A range of in vitro and in vivo tests was used to rigorously confirm the selectivity and sensitivity of the system’s activation in, and ablation of,  p16Ink4a-positive senescent cells.(6) In early-aging (2-mo old) BubR1H/H;INK-ATTAC mice, but not young (3-wk-old) mice, transcripts of the system and of reporter green fluorescent protein “were significantly elevated in [subcutaneous] adipose tissue, skeletal muscle and eye, but not in tissues in which endogenous p16Ink4a is not induced, including liver and heart.”(6) Moreover, subcutaneous adipose of prematurely-aged transgenic mice exhibited high levels of staining for the senescence marker senescence-associated-β-galactosidase (SAβ-gal) and expressed high levels of several established markers of senescence, including p21, p19, interleukin-6, (insulin-like growth factor binding protein-2 (Igfbp2), and Pai-1; primary BubR1H/H;INK-ATTAC mouse embryonic fibroblasts forced artificially into senescence by oncogenic Ras or serial passage exhibited a subpopulation that was both GFP+ and stained positively SAβ-gal. BubR1H/H;INK-ATTAC  muscle cells and lens did not stain for SAβ-gal, but did exhibit selective induction of the “senescence genes.”(6)

When BubR1H/H;INK-ATTAC mouse bone marrow cells were pushed into senescence in vitro by the  PPAR-γ-activating drug rosiglitazone, a subpopulation of the cells exhibited high levels of  INK-ATTAC expression and GFP, coupled with SAβ-gal staining; subsequent to treatment with the INK-ATTAC activating drug, these cells rapidly entered into apoptosis, and within 48 h were either destroyed or in the cell death process.(6)

Ablation of Senescent Cells Retards Age-Related Tissue Degeneration

As a first approach, the investigators abrogated the premature age-related rise of p16Ink4a-senescent cell burden in the tissues of BubR1H/H;INK-ATTAC mice by initiating a lifelong course of senescent-cell-ablating AP20187 treatment  at weaning. At 9-10 mo of age, such mice were then compared a to age-matched cohorts of untreated BubR1H/H;INK-ATTAC mice, and to BubR1H/H mice lacking the INK-ATTAC system for selective ablation of p16Ink4a-expressing cells. Relative to both control cohorts of the same age, 9-10 mo old treated mice exhibited dramatically more youthful tissues. Consistent with earlier results, their burden of p16Ink4a-positive senescent cells in muscle, eye, and adipose tissues were far lower. Their muscle fibers had larger diameters, their treadmill endurance was greater and they covered more distance on them. Treated mice had fewer cataracts, and less lordykyphosis. And they suffered less lipoatrophy, with larger fat deposits in multiple depots, higher individual adipocyte volumes, and more proliferating cells marked with BrDU (see Figure 1, below).(6) No treatment-related adverse events presented themselves.(6)

Figure 1: Amelioration of “Premature Aging” Phenotypes in Treated and Untreated BubR1H/H;INK-ATTAC Mice. AP=AP20187 treatment. Reproduced from (6). © Nature Publication Group.

Importantly, “premature aging” phenotypes observed in BubR1H/H mice over time, but that are in tissues where p16Ink4a-positive senescent cells do not accumulate with aging, were not alleviated by drug treatment. Thus, these animals exhibit premature cardiac arrhythmias and stiffening of the arterial wall, and cardiac failure appears to be the main cause of death; yet these tissues are not burdened with an abnormally-high burden of p16Ink4a-senescent cells, and accordingly, ablation p16Ink4a-positive senescent cells in these animals had little tissue-specific or survivorship phenotypic impact.(6)

Following this initial test of abrogating the early, age-related rise in p16Ink4a-expressing cell burden, the investigators probed the effects of  leaving BubR1H/H;INK-ATTAC to undergo 5 months of rapid “premature aging” (and thus, to the attendant accumulation of high levels of p16Ink4a-positive cells and onset of “early-aging” phenotypes), and only then  inducing ablation of senescent cells with the INK-ATTAC drug-activated system (see Figure 2 (g) below).(6) At that point, the animals’ cataracts had already reached peak age-related severity, and remained stable after 5 months of further aging irrespective of treatment. But muscle fibers that continued to atrophy over the ensuing 5 months in control animals remained at their more youthful diameters in animals whose p16Ink4a-positive senescent cells had been ablated, and treadmill times, distance traveled, and work outputs were maintained at substantially more youthful levels (Figure 2, (a) and (b) below). Similarly, the degeneration of adipose tissue cells and depots that occurred over the course of the next 5 months in control animals was virtually abrogated, leaving 10 mo-old animals with substantially the same subcutaneous and other fat tissue (in depth and in cell volume) as they had enjoyed in their relative youth, when treatment was first initiated.(6) 

Figure 2: Ablation of Senescent  p16Ink4a-Expressing Cells Maintains Youthful Muscle and Adipose Tissues. Reproduced from (6). © Nature Publication Group.

Rejuvenation Implications

As noted above, studies involving the use of putative “premature aging” models must be interpreted with caution, as the designation inevitably involves an element of petitio principii: from a subset of similar phenotypes are drawn conclusions of similar aetiology, and from this, further conclusions about the “normal” degenerative aging process (and its biomedical amelioration) are too-readily drawn before the thesis itself has first been established.(9) Indeed, the degenerative aging process is by definition one in which the organism progressively accumulates damage to its cellular and molecular components over time, so any genetic or environmental factor that leads to a greater burden of such damage will bear some resemblance to the aging phenotype, irrespective of the causal origin of the defect or its relationship to “normal” aging.

In the case of this new report,(6) however, while caution is still merited, the nature of the intervention used makes the study relatively free of such complications. The investigators did not simply modulate or normalize the very thing that the mutation (in this case, to the mitotic checkpoint component BubR1) itself disrupts, as in other widely-publicized studies involving putative “accelerated aging” (eg. (12,13)). Rather, the defective checkpoint system was left to proceed, and one of its downstream consequences, which was still under normal regulation — and one known to be directly induced by the normal degenerative aging process — was reversed at the structural level, by clearing out the p16Ink4a-positive senescent cells that had accumulated to an abnormal degree in their tissues. This left some aspects of the abnormal “progeroid” phenotype in these organisms (the cardiovascular defects) intact, but illustrated the dysfunctional consequences  of having tissues riddles with such cells. While still of abnormal origin, there is no strong reason to think that the ongoing effects of a rising burden of such cells would not be similar — and thus, that the effects of ablating such cells are uninformative about the effects of a similar intervention in “normally” aging bodies.*

The links to aging phenotypes, and their near-arrest by ablation of p16Ink4a-expressing senescent cells, appear to be dramatic illustrations of the deleterious effects of the age-related rise in the burden of senescent cells in genetically-intact mammals. The fact that it was the removal of such cells from aging tissues that arrested multiple aging phenotypes is of special importance to the rejuvenation biotechnology approach to preventing and reversing age-related disease and disability: it clearly identifies the damage itself, rather than the abnormal function of either p16Ink4a (which was under normal, physiological regulation, rather than being pharmacologically modulated, or knocked out as in their previous report(8)) or BubR1 (mutation of which, and its direct metabolic sequelae, was not affected by the intervention).

And there are reasons to believe that the resulting arrest of multiple aspects of tissue aging by removal of  p16Ink4a-expressing senescent cells would indeed translate into the tissues of genetically-intact mice — or humans.


The fact  — and disabling and fatal consequences — of age-related decline in muscle quality and quantity is widely known, but the contribution of “senescent” cells to this degenerative process is not. While some studies have reported no decline in satellite cells (muscle progenitor cells) with aging, others (eg. (14,15)) have found age-related satellite cell attrition consistent with the senescence of a subset thereof; moreover, one such study (15) reported that decreases in the number and quality of satellite cells with aging are reliably associated with elevated expression of  p16Ink4a (contrary to (14)), and with secretory and proteomic abnormalities consistent with a rising burden of senescent cells. Consistent with a causal relationship, (16) reports that the prevalence of limited physical functioning in aging varies depending on p16Ink4a allelic variation, consistent with variations in rate of stem cell attrition with senescence.

There is therefore good reason to expect that the profound arrest of sarcopenic phenotypes observed in p16Ink4a-senescent cell ablated BubR1H/H “premature aging” mice would translate into the human case.


While less well-known (masked as it is and placed out of focus by the overall age-related body composition shift from lean mass to adiposity), there is none the less significant age-related subcutaneous lipoatrophy in aging, most visibly in the sunken appearance of the face. Part of this is a pathological redistribution of adipose from the subcutaneous to the visceral depot, but it now emerges that the subcutaneous depot becomes qualitative as well as quantitatively abnormal in the degenerative aging process also suffers genuine age-related lipoatrophy and lipodystrophy — and that p16Ink4a-driven cellular senescence is at the heart of it.

Subcutaneous adipose tissue contributes to maintenance of insulin sensitivity and other aspects of metabolic homeostasis, through the production of adipose-specific endocrine factors such as adiponectin. Surgical removal of subcutaneous fat reduces adiponectin levels and insulin sensitivity, and transplantation of subcutaneous fat increases both.(17) Slow-aging growth hormone receptor knockout (GHRKO) mice are obese, but highly insulin sensitive: in such animals,  surgical removal of visceral adipose tissue impairs insulin secretion and peripheral insulin action, in part by reducing adiponectin production. (21)  Moreover, while  the link between excessive visceral adipose tissue and age-independent diabetes and metabolic syndrome widely known, recent studies suggest instead that it is the accumulation of senescent subcutaneous adipocyte progenitors — and their abnormal metabolic function — that drives similar diabetes-like phenotypes during the “normal” aging process.(3,18; cf. 19,20) Even in visceral fat, it has recently emerged that the obesity-driven rise in inflammation and insulin resistance is associated with an abnormal accumulation of senescent cells, albeit senescent endothelial cells rather than adipocytes.(20) It was this emerging line of research that van Deursen’s collaborator Dr. James Kirkland presented at the fifth annual Strategies for Engineered Negligible Senescence Conference (SENS5) in September of this year,(18) and it was his expertise in the senescence of adipose tissue that he contributed to the new report on the effects of ablating such cells.(6)

Again, then, there is significant evidence consistent with a role of cellular senescence in age-related lipodystrophy and lipoatrophy, and for the benefits observed in treated mice in these studies to translate into aging humans. It is unfortunate that the investigators did not assess insulin secretion, insulin action, or systemic inflammation in early-aging BubR1H/H;INK-ATTAC  mice, with and without ablation of senescent adipose cells, but reasonable to be optimistic that doing so would yield some normalization of age-related metabolic abnormalities.


There is only the most tentative of evidence suggesting a link between cellular senescence and cataract in “normal” aging.(22) Absence of evidence is, however, not evidence of absence, and certainly the inflammatory secretory profile of senescent cells would, if present, likely accelerate the degenerative course of the disease.


A great deal of evidence has now been amassed that stromal cell senescence plays an important role in laying the groundwork for tumor metastasis, promoting cell proliferation with inflammatory cytokines, encouraging angiogenesis, and degrading the tumor-suppressive action of an intact extracellular matrix.(1) One clear disadvantage of using these “early-aging” mice is that they die too early to develop cancer — too early for ablation of p16Ink4a-positive senescent cells to impact the course of the disease. It would indeed be of great interest to see whether ablation of stromal  p16Ink4a-expressing senescent cells, in otherwise genetically intact INK-ATTAC animals without existing tumors, would lower the animals’ risk for cancer, and put any tumors they might develop on a less malevolent trajectory, than untreated mice. It should be noted, however, that while a study on senescent cell ablation in genetically normal mice would provide at least some evidence on the effect of senescent cells (and their ablation) on promoting cancer, even such a study would likely show less effect than could be anticipated in a large mammal model, since even normally-aging mice rarely suffer metastatic disease to the extent of aging humans, as sheer primary tumor volume is generally sufficient to be fatal to mice.

Other Tissues

As the investigators note, the rapid age-related arterial stiffening and cardiac arrhythmias that appear to be at cause for the majority of deaths in BubR1H/H mice were not attenuated by ablating p16Ink4a-expressing senescent cells — but these tissues had little burden of such cells, so this finding reinforces the conclusion that the multiple aging phenotypes arrested in these mice when senescent cells were ablated is attributable specifically to the removal of their baleful influence on local tissues. On the other hand, there are many other tissues — notably, the kidney and articular cartilage — where p16Ink4a-expressing senescent cells appear to be a contributing factor to human and murine degenerative aging, but which were not evaluated in treated or control mice in this study, and it would be of interest to see the effects of ablation of p16Ink4a-positive senescent cells.

Moreover, there are yet other cell types — such as visceral adipose tissue macrophages and cytotoxic CD8+ T-cells — in which the age-related supernumerary accumulation of dysfunctional and apoptosis-resistant cells appears to play a highly deleterious role on tissue function, but where the cells are not “senescent” cells in the classical sense of p16Ink4a expression and the senescence-associated secretory profile observed in senescent fibroblasts. This study (6) cannot provide evidence directly on the effects of ablating such cells, but it does provide an analogous proof-of-concept for the approach. SENS Foundation is funding ongoing work in the lab of Dr. Janko Nikolich-Zugich to investigate the effects of clearance of anergic, “senescent” cytotoxic CD8+ T-cells on immunosenescence,(22) and is interested in the targeting of other such cells.(2)

Arrest vs Reversal

In the new study,  p16Ink4a-expressing senescent cells were ablated either at weaning or some months later, and assessed several months after the initial intervention. Remarkably enough, the removal of such cells arrested tissue degeneration, holding the muscles and adipose tissue (and, when administered before cataract was mature, lens opacification) at approximately the same relatively youthful condition prevalent when the inducing drug was first administered (see eg. Fig 2(a) above).(6) This is consistent with the deleterious effect of such  cells on tissue function, and with the researchers’ conclusion that “the observed improvements in skeletal muscle and fat of late-life treated 10-month-old BubR1H/H;INK-ATTAC-5 mice reflect attenuated progression of age-related declines rather than a reversal of ageing”.(6) However, it would be useful to see a more thorough analysis of the effect of ablating p16Ink4a-expressing senescent cells, and whether there may instead be evidence of a short-term rejuvenation of tissue function that is slowly lost over time to rising levels of other kinds of aging damage that INK-ATTAC activation does not address. Indeed, as illustrated by the lack of effect of p16Ink4a-expressing cell ablation on lifespan, and by the ongoing degeneration of tissues (such as the heart) in which p16Ink4a-postive senescent cells are not a driver of “early aging,” true rejuvenation requires a comprehensive suite of rejuvenation biotechnologies to remove all forms of aging damage from the aging body.

Translation for Human Rejuvenation Biotechnologies

The investigators boldly, but rightly, conclude that

Our proof-of-principle experiments demonstrate that therapeutic interventions to clear senescent cells or block their effects may represent an avenue for treating or delaying age-related diseases and improving healthy human lifespan.(6)

How might the results of this intervention be translated for human rejuvenation therapies?

There is already evidence that senescent cells are targeted by the innate immune system.(24-28) Dr. Judith Campisi, in fact, has found that activating NKG2D receptors on natural killer (NK) cells engage MHC class I chain-related protein A and B (MICA/B) ligands on senescent cells, leading to their NK-induced apopotsis and subsequent clearance.(29) MICA/B ligands are also used to activate tumor cell destruction by NK cells via NKG2G binding, and tumors evolve resistance by several mechanisms to reduce cell-surface MICA abundance;(30) however, the natural selection mechanisms that drive the evolution of such defenses do not apply to growth-arrested cells. Dr. Campisi has found instead that a minority of senescent cells evade destruction by secreting high levels of matrix metalloproteinases (MMPs), which cleave MICA/B ligands and thereby prevent NKG2D binding.(29) This has led to the hypothesis that the great majority of such cells are destroyed over the lifetime by innate immunity, and that the specific senescent cells that do accumulate with aging are precisely those who had variants that allow MMP overexpression, in a kind of “one-off,” very temporally-extended kind of selection process. Potentially, a kind of intervention that could overcome this resistance to endogenous clearance mechanisms would allow for the purgation of senescent cells from aging tissues.

SENS Foundation is currently funding work by PhD Candidate Kevin Perrott in Campisi’s laboratory, screening compounds for their effectiveness in mitigating the negative impact of cells exhibiting the classical senescence-associated secretory phenotype (SASP)(1) following senescence induced by treatment with 10 gray of ionizing radiation by selective induction of apoptosis or modulating their secretions. To date, a screen of the collection of FDA-approved drugs in the Prestwick Library has identified a handful of potential candidates which have demonstrated effectiveness at lowering secretion of IL-6, a component of SASP whose concentration tends to rise systemically with aging and here used as a preliminary marker of SASP as a phenotype. In particular, Perrott has recently identified some members of a class of compounds that lower the SASP in irradiated-senescent cells, without reversing growth arrest, and he is currently investigating the mechanisms of this phenomenon.

It is clear that there is substantial distance yet to be traveled. Multiple cell types acquire distinctive “senescent” phenotypes on a cell-type-specific basis, and will require ablation to achieve comprehensive rejuvenation. However, this important proof-of-principle from Dr. van Deursen’s laboratory, and the key validation of the scope of the effects of ablation of these particular senescent cells facilitated by his collaboration with the LeBrasseur and Kirkland labs at Mayo, stands as a key landmark in moving toward the removal of their baleful influence on aging tissues. As ever, SENS Foundation is committed to making investments in critical-path research to advance this key but heretofore-neglected line of biomedical research out of the laboratory, into the clinic, and to uniting the multiple strands of rejuvenation biotechnologies into a comprehensive panel for the restoration of the health, vigor, and open futures of aging humanity.

* The principle caveat would be that the interaction of senescence with defective mitotic checkpoint function within such cells, and the effects upon their neighbors of their state and of their senescence-associated secretory phenotype, would very likely cause some phenomena that would not be observed in p16Ink4asenescent cells or in their effects on neighbors.


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7: Baker DJ, Jeganathan KB, Cameron JD, Thompson M, Juneja S, Kopecka A, Kumar R, Jenkins RB, de Groen PC, Roche P, van Deursen JM. BubR1 insufficiency causes early onset of aging-associated phenotypes and infertility in mice. Nat Genet. 2004 Jul;36(7):744-9. Epub 2004 Jun 20. PubMed PMID: 15208629.

8: Baker DJ, Perez-Terzic C, Jin F, Pitel K, Niederländer NJ, Jeganathan K, Yamada S, Reyes S, Rowe L, Hiddinga HJ, Eberhardt NL, Terzic A, van Deursen JM. Opposing roles for p16Ink4a and p19Arf in senescence and ageing caused by BubR1 insufficiency. Nat Cell Biol. 2008 Jul;10(7):825-36. Epub 2008 May 30. PubMed PMID: 18516091; PubMed Central PMCID: PMC2594014.

9: Miller RA. ‘Accelerated aging’: a primrose path to insight? Aging Cell. 2004 Apr;3(2):47-51. Review. PubMed PMID: 15038817.

10: Pajvani UB, Trujillo ME, Combs TP, Iyengar P, Jelicks L, Roth KA, Kitsis RN, Scherer PE. Fat apoptosis through targeted activation of caspase 8: a new mouse model of inducible and reversible lipoatrophy. Nat Med. 2005 Jul;11(7):797-803. Epub 2005 Jun 19. PubMed PMID: 15965483.

11: Both GW. Gene-directed enzyme prodrug therapy for cancer: a glimpse into the future? Discov Med. 2009 Oct;8(42):97-103. Review. PubMed PMID: 19833053.

12: Sahin E, Colla S, Liesa M, Moslehi J, Müller FL, Guo M, Cooper M, Kotton D, Fabian AJ, Walkey C, Maser RS, Tonon G, Foerster F, Xiong R, Wang YA, Shukla SA, Jaskelioff M, Martin ES, Heffernan TP, Protopopov A, Ivanova E, Mahoney JE, Kost-Alimova M, Perry SR, Bronson R, Liao R, Mulligan R, Shirihai OS, Chin L, DePinho RA. Telomere dysfunction induces metabolic and mitochondrial compromise. Nature. 2011 Feb 17;470(7334):359-65. Epub 2011 Feb 9. Erratum in: Nature. 2011 Jul 14;475(7355):254. PubMed PMID: 21307849.

13: Jaskelioff M, Muller FL, Paik JH, Thomas E, Jiang S, Adams AC, Sahin E, Kost-Alimova M, Protopopov A, Cadiñanos J, Horner JW, Maratos-Flier E, Depinho RA. Telomerase reactivation reverses tissue degeneration in aged telomerase-deficient mice. Nature. 2011 Jan 6;469(7328):102-6. Epub 2010 Nov 28. PubMed PMID: 21113150; PubMed Central PMCID: PMC3057569.

14: Carlson ME, Suetta C, Conboy MJ, Aagaard P, Mackey A, Kjaer M, Conboy I. Molecular aging and rejuvenation of human muscle stem cells. EMBO Mol Med. 2009 Nov;1(8-9):381-91. PubMed PMID: 20049743; PubMed Central PMCID: PMC2875071.

15: G. Butler-Browne, M.-C. LeBihan, A. Bigot, D. Furling, F. Svinartchouk, D. Bechet, V. Mouly. Identification of biomarkers of human muscle aging and senescence. Rejuvenation Res. 2007 Sep;10(Suppl1):S22(Abs 14).

16: Melzer D, Frayling TM, Murray A, Hurst AJ, Harries LW, Song H, Khaw K, Luben R, Surtees PG, Bandinelli SS, Corsi AM, Ferrucci L, Guralnik JM, Wallace RB, Hattersley AT, Pharoah PD. A common variant of the p16(INK4a) genetic region is associated with physical function in older people. Mech Ageing Dev. 2007 Mar 27; [Epub ahead of print] PMID: 17459456 [PubMed – as supplied by publisher]

17: Tran TT, Yamamoto Y, Gesta S, Kahn CR. Beneficial effects of subcutaneous fat transplantation on metabolism. Cell Metab. 2008 May;7(5):410-20. PubMed PMID: 18460332; PubMed Central PMCID: PMC3204870.

18: Kirkland JL. Aging, Adipose Tissue, and Cellular Senescence. Abstracts of Strategies for Engineered Negligible Senescence (SENS) Fifth Conference. August 31-September 4, 2011. Cambridge, United Kingdom. Rejuvenation Res. 2011 Aug;14 Suppl 1:S11-45. PubMed PMID: 21847798.

19: Minamino T, Orimo M, Shimizu I, Kunieda T, Yokoyama M, Ito T, Nojima A, Nabetani A, Oike Y, Matsubara H, Ishikawa F, Komuro I. A crucial role for adipose tissue p53 in the regulation of insulin resistance. Nat Med. 2009 Sep;15(9):1082-7. Epub 2009 Aug 30. PubMed PMID: 19718037.

20: Villaret A, Galitzky J, Decaunes P, Estève D, Marques MA, Sengenès C, Chiotasso P, Tchkonia T, Lafontan M, Kirkland JL, Bouloumié A. Adipose tissue endothelial cells from obese human subjects: differences among depots in angiogenic, metabolic, and inflammatory gene expression and cellular senescence. Diabetes. 2010 Nov;59(11):2755-63. Epub 2010 Aug 16. PubMed PMID: 20713685; PubMed Central PMCID: PMC2963533.

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The Pathological Basis of “Normal” Cognitive Aging

Brain Inflammation from Alzheimer's Disease

The often-mooted question of whether “aging itself” is or is not a “disease” has long been mooted in biogerontological circles, with a long-held rhetorical preference for asserting that it is not, but rather, that it is a risk factor for the specific diseases of aging.1 By contrast, the same fundamental semantic dispute was initially resolved in the opposite direction with regard to age-related cognitive decline and dementia, beginning in the early decades after Alois Alzheimer and Emil Kraepelin first identified the pathological basis of the Alzheimer’s disease (AD) until the early 1970s. For most of the twentieth century, it was held that dementia occurring in younger people should be classified as a disease, whereas dementia should be expected and accepted when it occurred in people at more advanced ages, despite the knowledge that the lesions linked to Alzheimer’s dementia accumulated throughout the course of “normal” aging in middle age and onward, and that the pathological basis of the disorder was the same in both cases.2

But beginning in the 1960s, a loose alliance led by social gerontologists but quickly coming to include biogerontologists, geriatricians, and patient advocacy groups successfully campaigned for a new understanding: that while some level of minor cognitive decline was  indeed a “normal” and inevitable part of aging, the newly-rediscovered clinicopathological entity, “Alzheimer’s disease,” was exactly that: a disease, against which the full force of public and private biomedical research should be mobilized in the pursuit of a cure.2

The veneer of coherence to this division has been peeling away for some years now, with the identification of mild cognitive impairment (MCI) as a prodromal or “preclinical” stage of AD, with cerebrospinal, pathological, and neuroimaging evidence linking it clearly to the core pathology of the clinical disease itself. Indeed, it is now well-established that aggregated beta-amyloid protein (Aβ) and neurofibrillary tangles (NFT — cytoplasmic inclusions composed of phosphorylated and abnormally-cleaved species of tau protein) accumulate progressively in the “normally” aging brain and precede the onset of dementia by decades.5,6 Two recent publications 3,4 clearly refute the principle arguments in favor of this dichotomy, firmly rooting the basis of “normal” age-related cognitive decline in the same pathological lesions of the brain that drive the “dementia” of Alzheimer’s.

In one report,3 researchers at the Rush Alzheimer’s Disease Center used data from 354 older clergy from the prospective Religious Orders Study, who had undergone baseline and up to 13 annual followup rounds of medical and psychological testing which included sound composite measures of global cognition and of specific cognitive functions, and culminated in postmortem brain autopsy. In order to distinguish the long course of age-related cognitive decline from the widely-observed rapid phase of antemortem cognitive decline without falling into the petitio principii of using the clinical diagnostic criteria for dementia or Alzheimer’s disease, the investigators tested a series of statistical models that each fit the observed rates of cognitive decline with an hypothesized acceleration in the last n months of life, and selected the best fit model as the analysis with the highest log likelihood value. The inflection point was thereby determined to occur at ~52 mo antemortem.3

Into this model they fit in the interactions of a pathologic index for lesions related to age-related cognitive decline and dementia: density of NFT, the lesion most strongly associated with the level of cognition in AD; Lewy bodies, the characteristic marker of Lewy body dementia (DLB); and gross and microscopic cerebral infarcts occurring at least 6 mo anemortem. as indicators of stroke and transient ischemic attack. With this analysis, they evaluated relationships between each pathological lesion, as well as a final model including all, with “normal” vs. “disease” related (accelerated terminal) decline.

The results (all emphasis mine):

higher tangle density was associated with more rapid age-related and disease-related decline in global cognition. … [V]irtually no age-related change in global cognition occurred at low levels of tangles (25th percentile…) compared to substantial decline at high levels (75th percentile…). By contrast, much disease-related global cognitive decline occurred despite low levels of tangles, suggesting the involvement of other pathologic factors. …

[B]oth gross … and microscopic … infarction were associated with a more than 2-fold increase in rate of age-related global cognitive decline. By contrast, neither gross nor microscopic infarction was associated with disease-related [global] cognitive decline. … The presence of neocortical Lewy bodies … was associated with an approximate doubling of disease-related decline relative to those without Lewy bodies … and [with] a nearly significant effect on age-related decline.  By contrast, nigral/limbic Lewy bodies [characteristic of the movement disorders of Parkinson’s disease] … were not associated with either age-related or disease-related decline in global cognition …

Higher tangle density was associated with more rapid age-related and disease-related decline in all cognitive systems  … By contrast, cerebral infarction  … and Lewy bodies  … had selective effects across time and cognitive systems. … It is noteworthy that Lewy bodies were associated with decline in episodic memory, a defining characteristic of AD, and that all forms of pathology contributed to age-related decline in working memory, a change often attributed to normal aging. …With all pathologic measures in the same model, tangles continued to be associated with age-related and disease-related decline in multiple cognitive systems; gross infarction was associated with age-related working memory decline; and neocortical Lewy bodies were associated with age-related perceptual speed decline and disease-related decline in episodic and semantic memory …

Age-related cognitive decline was associated with neurofibrillary tangles, cerebral infarction, and Lewy bodies, and was not evident in the absence of these lesions. This indicates that the neurodegenerative lesions traditionally associated with dementia are principally responsible for the gradual age-related cognitive decline that precedes dementia and that AD and related disorders have a much greater impact on late-life cognitive functioning than previously recognized. …

Gradual cognitive decline in old age has mainly been thought to reflect normative age-related developmental processes. In this cohort, however, there was no age-related cognitive decline absent postmortem evidence of neurodegenerative disease, and multiple pathologic lesions were associated with rate of age-related cognitive decline. These data challenge the concept of normative cognitive aging and suggest instead that neurodegenerative disease plays a role in virtually all late-life cognitive decline

The results also indicate that factors other than tangles and neocortical Lewy bodies are contributing to variability in disease-related [terminal] cognitive decline. This could include other pathologic features such as the TAR DNA-binding protein 43 [TDP-43]. In addition, neurodegeneration in the form of loss of neurons and synapses may be the most proximate cause of precipitous cognitive decline, leaving less variability to be accounted for by more distal contributors to neurodegeneration such as tangles and Lewy bodies.3

The second paper4 gave context to this latter allusion: the fact that neuron loss is minimal in the “normally” aging brain, but  is prevalent in the late stages of dementia. This relatively recent observation was greeted with surprise, because it stood in curious contrast to the much longer-established fact of extensive volumetric shrinkage of the brain across the course of “normal” cognitive aging, which had previously been assumed to imply a lifelong process of neuronal loss. Once it became clear that little such loss occurred, this fact was often invoked as evidence of a clear distinction between the age-related changes occurring in “normal” cognitive aging and the pathological processes contributing to dementia. But it left the exact structural basis of age-related brain shrinkage largely unexplained.

The new study4 shows that this long process of brain shrinkage during “normal” aging is largely the result, not of neuronal loss, but of premorbid neuronal atrophy:

This paper reviews recent evidence from magnetic resonance imaging (MRI) studies about age-related changes in the brain. The main conclusions are that

(1) the brain shrinks in volume and the ventricular system expands in healthy aging. However, the pattern of changes is highly heterogeneous, with the largest changes seen in the frontal and temporal cortex, and in the putamen, thalamus, and accumbens. With modern approaches to analysis of MRI data, changes in cortical thickness and subcortical volume can be tracked over periods as short as one year, with annual reductions of between 0.5% and 1.0% in most brain areas.

(2) The volumetric brain reductions in healthy aging are likely only to a minor extent related to neuronal loss. Rather, shrinkage of neurons, reductions of synaptic spines, and lower numbers of synapses probably account for the reductions in grey matter. In addition, the length of myelinated axons is greatly reduced, up to almost 50%.

(3) Reductions in specific cognitive abilities–for instance processing speed, executive functions, and episodic memory–are seen in healthy aging. Such reductions are to a substantial degree mediated by neuroanatomical changes, meaning that between 25% and 100% of the differences between young and old participants in selected cognitive functions can be explained by group differences in structural brain characteristics.4

Ultimately, whether we speak of aging as a biological process that can be separated from specific age-related diseases, or as the premorbid structural basis of age-related disease and frailty, or as itself the ultimate age-related disease, should be regarded as a conceptual convenience bordering on a literary conceit, of no ultimate clinical significance. The aging of the body is a process of accumulating cellular and molecular lesions that degrade the fidelity of the structural basis of normal homeostasis; from an heuristic point of view, it can and should be understood to be a pathological process with a biomedical solution. The strategy of rejuvenation biotechnology is to remove, repair, replace, or render harmless such lesions, restoring the structural integrity of the body to the original order seen in youth. In the process, we will restore health and vigor that emerges from youthful biological structures, eliminating age-related disease and disability even as we eliminate the damage that underlies it.


1: de Grey AD. Resistance to debate on how to postpone ageing is delaying progress and costing lives. Open discussions in the biogerontology community would attract public interest and influence funding policy. EMBO Rep. 2005 Jul;6 Spec No:S49-53. PubMed PMID: 15995663; PubMed Central PMCID: PMC1369265.

2: Ballenger JF. Progress in the history of Alzheimer’s disease: the importance of context. J Alzheimers Dis. 2006;9(3 Suppl):5-13. PubMed PMID: 17004361.

3: Wilson RS, Leurgans SE, Boyle PA, Schneider JA, Bennett DA. Neurodegenerative basis of age-related cognitive decline. Neurology. 2010 Sep 21;75(12):1070-8. Epub 2010 Sep 15. PubMed PMID: 20844243; PubMed Central PMCID: PMC2942064.

4: Fjell AM, Walhovd KB. Structural brain changes in aging: courses, causes and cognitive consequences. Rev Neurosci. 2010;21(3):187-221. Review. PubMed PMID: 20879692.

5:Lemere CA, Masliah E. Can Alzheimer disease be prevented by amyloid-beta immunotherapy? Nat Rev Neurol. 2010 Feb;6(2):108-19.  PubMed PMID: 20140000; PubMed Central PMCID: PMC2864089.

6: Braskie MN, Klunder AD, Hayashi KM, Protas H, Kepe V, Miller KJ, Huang SC, Barrio JR, Ercoli LM, Siddarth P, Satyamurthy N, Liu J, Toga AW, Bookheimer SY, Small GW, Thompson PM. Plaque and tangle imaging and cognition in normal aging and Alzheimer’s disease. Neurobiol Aging. 2010 Oct;31(10):1669-78. Epub 2008 Nov 11. PubMed PMID: 19004525; PubMed Central PMCID: PMC2891885.

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