New Peer-Reviewed Paper from SENS Research Foundation Reports a Better Method to Study How Immune Cells Seek and Destroy Senescent Cells

Senolytics — drugs that selectively destroy senescent cells (senolytics) — have been shown to powerfully rejuvenate mice and to reverse multiple diseases of aging in animals, from atherosclerosis, to kidney disease, to osteoporosis, to Parkinson’s disease — and beyond. But because these drugs target pathways that are necessary for normal cell function, they can come with side-effects — the most notable being the crash in platelets (and resulting risk of fatal bleeds or strokes) that can accompany the experimental senolytic navitoclax.

But our bodies are actually equipped with an inborn army of immune cells that selectively identify and destroy senescent cells. To date, the most important of these “immunosenolytic” cells soldiers have seemed to be Natural Killer cells (NK cells) — though we will have an announcement to make on this front in the coming months. But if the body already has this defensive force, how do senescent cells still manage to accumulate in our tissues with age and wreak havoc on our health?

As part of SENS Research Foundation’s work to eradicate dysfunctional cells driving aging (ApoptoSENS), Dr. Amit Sharma and his team at SRF are developing ways to either rejuvenate our own NK cells’ ability to detect and destroy senescent cells in aging tissues, or to augment that capacity with NK cells engineered to be more lethal senescent cell hunters. To reach those goals, they realized they needed a more efficient way to enrich NK cells from mixed white blood cells isolated from human blood. In new peer-reviewed scientific paper, they show how they did it.

Their main innovation might seem to be an obvious one: to actually isolate NK cells and activate them to better mimic the situation in vivo. Previous studies have mostly used mixed populations of blood cells, sometimes removing T-cells or other specific cell types but never ensuring that they had isolated NK cells exclusively. Dr. Sharma used an existing kit to make sure that what they were seeing came from the NK cells and not from other cell types.

They also saw that the way that previous researchers had studied NK cells’ ability to destroy senescent cells was quite different from the conditions NK cells actually confront as they patrol for NK cells in the body, potentially leading scientists to chase after interventions that work well in a Petri dish but have no effect or are unsafe when carried out in living, breathing humans. This is probably because most scientists conducting these studies don’t have a specialized background in immunology: instead, they design their experiments based on a general background in the biology of aging or cancer biology, or as specialists in senescence per se, but

The biggest problem the SRF group corrected is that previous studies have cultured low numbers of senescent cells together with very high numbers of NK cells, creating a situation where NK cells are effectively shooting zombie fish in a barrel. Dr. Sharma’s team tested more realistic conditions in which one NK cell encounters one senescent cell, or no more than three, instead of ratios as high as one senescent cell target to 20 or even 80 NK cells.

Conversely, having given NK cells so many senescent cell targets that they can hardly fail to kill some, most previous studies have only set NK cells loose for a few hours, when NK cells have a half-life in the body of ten hours. So the ApoptoSENS team led by Dr. Sharma gave NK cells more time to engage their enemies, which better reflects how long they actually hang out in tissues, with experiments running from 16 hours to four days. This also gives the NK cells the ability to reveal their full range of powers, since some of their cell-killing weapons only engage over extended time periods.

Finally, previous studies on NK cells’ immune surveillance of senescent cells have cultured them in very high levels of interleukin-2 (IL-2), an immune-signaling molecule that triggers NK cells to create more of themselves and makes them more aggressive. At SRF, Dr. Sharma used levels that reflect levels in the body.

The first set of discoveries revealed by this new system: even when the ratio between NK and their potential targets is quite low, introducing more NK cells into the mix tends to kill more senescent cells, but at the expense of killing more and more healthy bystander cells.

All around, the new paper sets up the SRF ApoptoSENS team — and other scientists who can now read the findings for themselves — on a better path to identify ways to rejuvenate or reinforce senescent cell immune surveillance, reinvigorating NK cells to make them battle-ready once again.

Reference

Kim K, Admasu TD, Stolzing A, Sharma A. Enhanced co-culture and enrichment of human natural killer cells for the selective clearance of senescent cells. Aging (Albany NY). 2022 Mar 4;14(5):2131-2147. doi: 10.18632/aging.203931. Epub 2022 Mar 4. PMID: 35245208; PMCID: PMC8954966.

New Peer-Reviewed Paper from SENS Research Foundation Researchers Reveals Tools to Harness Immune Cells to Seek and Destroy Senescent Cells

Senolytics — drugs that selectively destroy senescent cells (senolytics) — have been shown to powerfully rejuvenate mice and to reverse multiple diseases of aging in animals, from atherosclerosis, to kidney disease, to osteoporosis, to Parkinson’s disease — and beyond. But because these drugs target pathways that are necessary for normal cell function, they can come with side-effects — the most notable being the crash in platelets (and resulting risk of fatal bleeds or strokes) that can accompany the experimental senolytic navitoclax.

But our bodies are actually equipped with an inborn army of immune cells that selectively identify and destroy senescent cells. To date, the most important of these “immunosenolytic” soldiers have seemed to be Natural Killer cells (NK cells) — though we will have an announcement to make on this front in the coming months. But if the body already has this defensive force, how do senescent cells still manage to accumulate in our tissues with age and wreak havoc on our health?

SENS Research Foundation scientists are working to harness NK cells to more safely and effectively clear senescent cells out of our tissues. Dr. Amit Sharma and his team at SRF are developing ways to either rejuvenate our own NK cells’ ability to detect and destroy senescent cells in aging tissues, or to augment that capacity with NK cells engineered to be more lethal senescent cell hunters. To reach those goals, they realized they needed a more efficient way to create large numbers of NK cells from mixed white blood cells isolated from human blood. In new peer-reviewed scientific paper, they showed other scientists how they did it. Their main innovation might seem to be an obvious one: to actually isolate NK cells to study them. Previous scientists have mostly used very mixed populations of blood cells, sometimes removing T-cells or other specific cell types but never isolating NK cells exclusively. Dr. Sharma used an existing kit to make sure that what they were seeing came from the NK cells and not from other cell types.

They also saw that the way that previous researchers had studied NK cells’ ability to destroy senescent cells was quite different from the conditions NK cells actually confront as they patrol for NK cells in the body, potentially leading scientists to chase after interventions that work well in a Petri dish but have no effect or are unsafe when carried out in living, breathing humans. This is probably because most scientists conducting these studies don’t have a specialized background in immunology: instead, they design their experiments based on a general background in the biology of aging or cancer biology, or as specialists in senescence per se, but The biggest problem the SRF group corrected is that previous studies have cultured low numbers of senescent cells together with very high numbers of NK cells, creating a situation where NK cells are effectively shooting zombie fish in a barrel. The Sharma lab tested more realistic conditions in which one NK cell encounters one senescent cell, or no more than three, instead of ratios as high as one NK to 20 or even 80 senescent targets.

Conversely, having given NK cells so many senescent cell targets that they can hardly fail to kill some, most previous studies have only set NK cells loose for a few hours, when NK cells have a half-life in the body of ten hours. So the Sharma lab gave NK cells an amount of time to engage their enemies that reflects how long they actually hang out in tissues, with experiments running from 16 hours to four days. This also gives the NK cells the ability to reveal their full range of powers, since some of their cell-killing weapons only engage over extended time periods.

Finally, previous studies on NK cells’ immune surveillance of senescent cells have cultured them in very high levels of interleukin-2 (IL-2), an immune-signaling molecule that triggers NK cells to create more of themselves and makes them more aggressive. At SRF, Dr. Sharma used levels that reflect levels in the body. 

The first set of discoveries revealed by this new system: NK cells are actually most selective for senescent cells when the ratio between NK and their potential targets are quite low. As you introduce more and more targets into the mix, NK cells tend to kill more senescent cells, but also kill more and more healthy bystander cells. Additionally, their preliminary evidence suggests that NK cells from younger male subjects were more effective at killing senescent cells compared to female subjects, and also more effectively than older people of either gender. This is consistent with previous reports that older people’s NK cell armies included more dysfunctional or exhausted NK cells, which release less of the key immune signaling molecule interferon gamma (IFN-γ) and produce less of the chemicals needed to execute their targets. IF their initial finding of reduced senescent cell-killing ability pans out in a larger study, they can look at these and other mechanisms as potential reasons why and then look for ways to reverse or sidestep the effect of aging.

All around, the new paper sets up the SRF immunosenolytic team — and other scientists who can now read the findings for themselves — on a better path to identify ways to rejuvenate or reinforce senescent cell immune surveillance, reinvigorating NK cells to make them battle-ready once again.

Reference

Kim K, Admasu TD, Stolzing A, Sharma A. Enhanced co-culture and enrichment of human natural killer cells for the selective clearance of senescent cells. Aging (Albany NY). 2022 Mar 4;14(5):2131-2147. doi: 10.18632/aging.203931. Epub 2022 Mar 4. PMID: 35245208; PMCID: PMC8954966.

Michael Rae and Dr. Robert Rodgers discuss Parkinson’s and aging

Parkinson’s disease, like all the so-called diseases of aging, is what happens when the particular metabolic demands on specific cell and tissue types inflict enough damage on themselves to render them unable to do their jobs. As more and more of those cells and tissues become dysfunctional over time, the functional reserve of those tissues is slowly drained, until core function can no longer be sustained and the symptoms of that widespread cellular and molecular injury erupt.

In this interview with the Parkinson’s Recovery radio program, SENS Research Foundation’s science writer Michael Rae discusses “The coming rejuvenation biotechnology revolution for Parkinson’s disease.” In it, they discussed how the cellular and molecular damage of aging most closely involved in Parkinson’s can be removed, replaced, or repaired using rejuvenation biotechnologies, and research underway to make it happen. Here is some of what they talked about.

New Cells for Old …

The most prominent kind of aging damage driving Parkinson’s is the loss of specialized neurons producing the molecular messenger dopamine in a defined area of the brain. Signaling from these neurons suppresses the unregulated firing of neurons controlling motion in the brain, so as these neurons are lost to aging and additional insults, the ability to suppress this unwanted firing declines. Eventually, this becomes manifest as the “motor symptoms” of Parkinson’s: the tremors of the hands, shaking of the head, and problems with walking balance.

The rejuvenation biotechnology solution to this problem is repleniSENS: replacing the lost dopamine-producing neurons with fresh young ones. Early human clinical trials using cells derived from aborted fetuses had mixed results, but a small percentage of the patients who received the cells experienced such spectacular results scientists spent the next several decades working out the cell types, transplant sites, and other details to optimize the procedure so that all people suffering PD  (and eventually, all of us) could benefit.

In addition to what we learned through the ensuing decades of painstaking progress, we now have Shinya Yamanaka’s Nobel-prizewinning biotechnological breakthrough: the ability to take common differentiated cells (such as deep-layer skin cells) and reprogram them into  induced pluripotent stem cells (iPSC), which have the full developmental potential of embryonic stem cells and can be nudged to become any cell type we like — including dopamine-producing neurons for cell replacement.  Today, BlueRock Therapeutics, a stem cell company now owned by Bayer (yes, the aspirin people) is running a clinical trial grafting replacement neurons into the brains of patients with PD. Unlike others, BlueRock is not starting the process off with a patient’s own cells, but is using healthy donor cells from the general public, from which it then uses proprietary bioprocessing to create stable master cell banks of what they call “universal iPSCs” that they have found to be compatible with any patient. It is these cells that are then turned into dopaminergic neurons for transplant into the brain of PD patients — or, in the future, heart muscle cells for people with heart failure, and other medical use-cases.

Other groups are taking the patient-identical route. These include Dr. Jun Takahashi of Kyoto University in Japan, whose small ongoing trial recruited just 7 subjects and administered cells to its first patient in November of 2018, and Aspen Biotechnologies, which is led by Dr. Jeanne Loring at the Scripps Institute, a good friend of SENS Research Foundation. Additionally, it was only just revealed last year that a dramatic self-funded experiment by a wealthy PD sufferer resulted in millions of his own reprogrammed cells being transplanted into his brain as early as September 2017. And we’re even still learning things about grafting fetal neurons, because of a revamped European trial with curtailed ambitions known as Transeuro.

… And Out with the Old!

Meanwhile, we’ve learned about the evidence supporting a causal role for senescent cells in PD — and the potential of apoptoSENS (destruction of dysfunctional cells) to help keep these “zombie” cells from destroying some of those dopamine-producing neurons in the first place. People with PD have more senescent cells in their brains than do aging people not diagnosed with a specific neurological disease — specifically, senescent astrocytes, which help maintain the integrity of the crucial barrier that protects the brain from toxins in the circulation, and that also act as a kind of support cell for the neurons, providing them with nutrients and maintaining the balance of ionic flows. Scientists led by Julie Anderson and Judith Campisi at the Buck institute showed in animal models and cell culture that when astrocytes turn senescent, betray the very neurons they exist to protect and support, killing them.

But purging senescent astrocytes from the brains of mice with a model of PD prevented the loss of dopamine-producing neurons and enabling the generation of new neurons to begin again. This meant that after senolytic treatment, the PD mice were still able to move like non-PD mice. With ongoing trials of senolytic drugs and a host of startups in the space, the potential to bring this experiment to life in aging humans is breathtaking.

The Roto-Rooter Route to Rejuvenation

They also talked about the many lysoSENS immunotherapies in clinical trials or in the pipeline to remove aggregated alpha-synuclein from aging neurons and prevent the “nonmotor symptoms” of PD, and the related role of “minor” mutations in a lysosomal enzyme that create a vicious circle with alpha-synuclein — and Aspen Biotechnology’s plans to eliminate the problem.

And much more! Hear the full audio here.

Hyperbolic Hyperbaric “Age Reversal”

Lower-quality, clickbait-hungry media outlets love sensationalist claims, but one does expect better from the public relations department of an internationally-respected research university. And it was an easy jump from the already-overstated “In First, Aging Stopped in Humans” and “treatments can reverse two processes associated with aging and its illnesses” to saying that a treatment “can reverse aging process” — and to then land in a mud-pit of self-parody with “Human ageing reversed in ‘Holy Grail’ study, scientists say.”

The actual findings of a recent study on hyperbaric oxygen treatment (HBOT) were much more limited. Despite some intriguing indicators, the actual impact of HBOT on aging based on this study is entirely unclear, quite plausibly negligible, and in any case objectively less impressive than that of (say) regular exercise, which certainly does not “reverse aging.”

Originally developed to treat divers with decompression sickness or dangerous nitrogen bubbles in their blood after surfacing too quickly, HBOT involves administering 100% oxygen to subjects resting in a chamber where the atmospheric pressure is artificially elevated. This enhances the dissolution of oxygen directly into the plasma (normally, it’s almost entirely carried in the red blood cells), and the dissolved oxygen in turn drives out the nitrogen, while delivering more oxygen to the tissues. Later, it was found that repeatedly subjecting people or experimental animals to HBOT can cause an adaptive response that paradoxically resembles being subjected to inadequate oxygen (hypoxia) — a phenomenon referred to as the hyperoxic-hypoxic paradox.

Under the relatively loose 510(k) regulations for medical devices in the United States (standards that were modified over 2019-2020 to still-unclear effect), HBOT devices are also “cleared” (but not approved) for carbon monoxide poisoning and a surprising range of other indications, including treatment of chronic wounds and necrotizing soft tissue infections, burn or crush injuries, and unexplained sudden sensorineural hearing loss. Regulation of medical devices is similarly inadequate internationally. Some clinicians also administer HBOT to patients with post-traumatic stress disorder (PTSD) and traumatic brain injury (TBI), although the evidence base for these uses is weak. Device manufacturers and clinics sometimes push the line even further, advertising their HBOT devices and services for yet more speculative indications, resulting in FDA warnings to consumers and the occasional reprimand to manufacturers, although enforcement is hampered by a trend in the courts to recognize broad commercial freedom of speech rights.

So what about HBOT for aging?

The Study Setup

The study recruited 35 independent-living men in good functional and cognitive condition (granted the effects of aging — they were age 64 and older, with some in their early 80s) and had them complete a baseline assessment. They lost five participants right there — a problem that gets compounded as the study goes along, as we’ll see. The remaining thirty subjects underwent 90-minute HBOT sessions five days a week over the course of three months, for a total of 60 sessions. Whole blood samples were taken at the beginning of the study, at the midway point, and after the last session, followed by one last blood test a week or two after their last HBOT session. When the samples were viable, scientists tested the lengths of the subjects’ blood cell telomeres (the now-famous “shoestring nibs” on the ends of our chromosomes that keep them from unravelling after multiple cell divisions), as well as looking for what they characterize as “senescent” T-cells.

This is where the attrition problems started to get worse. After five of the initial recruits failed to complete their baseline survey, only 30 people remained to contribute samples — and of those, the researchers had to throw out four patients’ telomere analyses and ten patients’ “senescent” T-cell analyses, either because the samples contained too few cells to analyze, or because of lab technician errors. As such, the stated findings are based on very few data points indeed. And aside from reducing the statistical power to attribute (or not) any changes observed to random chance, counting only those data points may also have actively skewed the results. People who don’t complete a study or whose biological samples are not measurable could reflect real differences in the people who finish a trial versus those that began it: for instance, participants providing viable samples may have had unusually robust blood cells for their age, while those who completed baseline surveys may have been more conscientious (thus more inclined toward healthy lifestyle practices) than average.

Because of this potential source of bias, the accepted way to analyze a human clinical study is a so-called intention-to-treat analysis (ITT), in which you use all of the data from all of the subjects whether they actually finished the study (or gave viable samples) or not. Instead, in this case, they just ignored all the people who didn’t complete their initial assessment (that actually is sometimes acceptable, even in ITT), and also performed their analyses only on the people whose samples were all viable. To put it in fewer words, the researchers only compared people who started to the same people if they finished. Yet those who finished are skewed away from the whole group of those who started the study in the first place. Moreover, with no control group of any kind, we don’t know what would have happened anyway to another group of similarly-situated people who spent time under a kind of “placebo HBOT,” such as subjecting them to only very mild increases in atmospheric pressure, breathing something closer to atmospheric air (as).

So, all right: the meal comes with a cartoonishly-large pillar of salt on the side. But with all those caveats, what did they say they found?

After completing the protocol plus a two-week recovery period, telomere lengths in viable samples across several populations of immune cells “increased significantly by over 20% following HBOT,” and “There was a significant decrease in the number of senescent T-helpers by -37.30%±33.04 post-HBOT (P<0.0001)” while “T-cytotoxic senescent cell percentages decreased significantly by -10.96%±12.59 (p=0.0004)”. Is that actually what they found? And suppose that they had shown that (and shown it convincingly): would that be enough to justify a claim of having “stopped” or even “reversed aging”?

Tee-Tottering Telomeres

Let’s first look at the reported change in telomere length. It’s true that, when you look at a large population of people, longer blood cell telomeres and slower blood cell telomere shortening tend to correlate with poorer health outcomes. But that doesn’t make blood cell telomere length even a good proxy for current biological age at the individual level — let alone a causal driver of aging..

First, although blood cell telomere lengths do overall shrink over the course of multi-year periods when you look at aggregated data from an entire population of people, individuals’ telomere lengths swing wildly up and down over the course of mere months — that is, over lengths of time that include the entire duration of the HBOT study. So testing individual subjects’ telomere lengths as a measure of their individual biological age, and then testing again just a couple of months later, guarantees results that are corrupted by lot of sheer noise — and remember, just 24 subjects even had results the lab could read in the first place.

In fact, up to a third of individuals tracked show stable or even increased telomere lengths in their blood cells when re-tested as much as a decade later, due to a mixture of what are presumed to be real changes and lab artifacts. Obviously, degenerative aging is not being arrested or even massively reversed in one person in three across the population every ten years: imagine what an exciting breakthrough it would be to make that happen! So we know from that alone that we can’t use individual people’s blood cell telomere lengths as reliable indicators of age-related change, even over the course of a ten-year period. We therefore can’t possibly take a similar claim for HBOT seriously if it’s based on the same measure taken from 24 people over the course of mere months.

Moreover, although blood cell telomere length does correlate with telomere length in some tissues, the correlation is pretty weak, ranging from explaining 2% of the variation in the testes to a maximum of 14% in one peripheral nerve — and it has no correlation at all to the telomere lengths of about one-third of our tissues! Whatever blood cell telomere length tells us about health, in other words, it’s a pretty lousy proxy for whatever role telomere length plays in aging across the body as a whole.

Worse: calendar age itself — the most important determinant of biological age, which is what advocates want to use blood cell telomere length to measure — explained just 3.3% of the person-to-person variability in telomere length when all tissues were taken into account, and such important contributors to accelerating aging such as body mass index (BMI, as a proxy for obesity) and smoking status explained less than 1% of variation. And African Americans’ telomere lengths are longer than those of Americans of European descent in in nearly all tissues — a result consistent with multiple previous studies looking at blood cell telomere lengths. Yet we know that African Americans suffer with a higher burden of age-related disease and shorter life expectancies than white or Asian Americans. If we’re looking for a measure of biological aging, it makes little sense to use a metric that bears so little relationship to key predictors of future ill-health and death.

The weakness of telomere length as a biomarker of aging becomes clearer when you compare it to more robust markers, such as epigenetic aging clocks or algorithmic scores calculated mostly from common blood blood-test markers. In a comparative study, eleven candidate biomarkers of aging were compared to see how closely they would reflect the impact of aging on a group of older people in the domains of physical functioning (measured on tests of things like balance, grip strength, and motor coordination), rate of cognitive decline, and subjective signs of aging such as having an “old” face. One of the composite scores and one of the early iterations of an epigenetic aging clock consistently correlated with these age-related outcomes (albeit quite modestly in both cases), but telomere length failed to correlate with any of them. Similarly, telomere length was not associated with current health status in a cohort of 50-something New Zealanders, as measured on the  SF-36 evaluation of health status.

And those first-generation epigenetic aging clocks are far inferior as predictors of future age-related morbidity and mortality than more recent iterations such as GrimAge and DNAm PhenoAge, as well as non-epigenetic biomarker composites such as the PhenoAge blood test composite score against which DNAmPhenoAge itself was trained up by machine learning. Indeed, a comparison study of aging Swedes found that eight out of nine different biological age scores predicted risk of death over the next twenty years beyond the predictive power of calendar age itself. Telomere length was the odd man out, being the only one that failed to add value to knowing a subject’s calendar age.

So that’s telomere length. What about the reduction in “senescent” T-cells?

There’s “Senescence” … and then there’s Senescence

Anyone who’s been following research on senescent cells and their roles in diseases of aging and age-related ill-health — and on the sweeping rejuvenation effects of triggering those cells to self-destruct with “senolytic” drugs — will be excited by the authors’ conclusion that their “study indicates that HBOT may induce significant senolytic effects” and “suggests a non-pharmacological method, clinically available with well-established safety profile, for senescent cells populations decrease.” But does the study actually support that claim?

The first thing to understand is that despite the fact that it’s standard terminology in the immunology world, “senescent” T-cells aren’t actually “senescent cells” in the sense usually used in the geroscience world. (And no, they aren’t anergic T-cells either, though the two do show some overlap). True senescent cells are in a state of total growth arrest, unable to make new copies of themselves due to an interlocking set of pathways of regulation. By contrast, “senescent” T-cells’ ability to proliferate is reduced, but still fundamentally intact. Additionally, the reasons for “senescent” T-cells’ short telomeres and reduced replicative ability are quite different from those of true senescent cells. T-cells are normally very “trigger-happy” with their telomere-lengthening telomerase enzyme (and possibly use another telomere-lengthening mechanism as well), which allows them to quickly replicate when they encounter a known target and flood the zone with new T-cells. “Senescent” T-cells’ telomere-lengthening activity is sluggish compared to normal T-cells because an energy-sensing pathway tunes down its access to the enzyme. By contrast, true senescent cells actively enforce a state of total growth arrest, using an interlocking set of cellular pathways that are just not active in normal cells, in order to prevent the replication of damaged, often cancer-prone cells.

In short, jumping from post-HBOT reductions in the number of these “senescent” T-cells to potential effects on classical senescent cells is really just a misunderstanding of what kinds of cells are involved in each case.

OK, you say, but still, these so-called senescent T-cells are bad, aren’t they? So getting rid of them must be good — right? Well, maybe (though no one has demonstrated that yet). But did HBOT actually get rid of them in the first place?

The first problem here is that we don’t even know for sure that the researchers were measuring “senescent” T-cells to begin with! T-cells are judged “senescent” based on blunted replicative ability, lack of the marker protein CD28 on their surface, and the abnormal presence of the marker CD57 — but the investigators here were only able to measure CD28, and therefore were judging T-cells “senescent” without actually using the full criteria to test for them. So whether there was even a change in “senescent” T-cells in the first place is quite uncertain. (The same goes, in fact, for natural killer (NK) cells, which are another kind of immune cells. Mature NK cells test negative for the marker CD3 but positive for the marker CD56 — but these investigators tested for cells positive for both markers! Maybe this was just a typo that got repeated throughout the manuscript, but as it stands, it can only further fuel uncertainty around the results as a whole).

Add to that the fact that a mere reduction in the numbers of measured cells doesn’t prove that HBOT destroyed them. Maybe HBOT somehow triggered this group of cells to adopt a different functional status. Or maybe the cells (or a subset of them) retreated back to their reservoirs in the lymph nodes, since the researchers were only sampling them in peripheral blood. Who knows?

What we certainly don’t know is that HBOT treatment destroyed these cells in substantial numbers — and again, even if it did, it would be unclear what the researchers had done, since they didn’t actually fully characterize these as “senescent” T-cells in the first place, and they got the result from just twenty samples (after throwing out ten unusable results) — all in a study with no control group.

“Aging Reversed”?

So in short, the actual details of the study show that even the narrow claims of the study abstract aren’t fully justified. It’s not clear that blood-cell telomeres were lengthened any more than they would have been without HBOT; it’s not clear that “senescent” T-cells were reduced in numbers, let alone actually destroyed; and if “senescent” T-cells had been destroyed, it would not demonstrate a senolytic effect of HBOT, because “those aren’t the ‘senescent’ cells you’re looking for.”

And even if the study had robustly demonstrated that every one of the points above really did occur, it would not constitute “reversing aging” — or even justify the more restrained claims that “blood cells actually grow younger as the treatments progress” or “that the aging process can in fact be reversed at the basic cellular-molecular level.”

Aging Reversed!

The mission of SENS Research Foundation is to accelerate the development of rejuvenation biotechnologies: new therapies that remove, repair, replace, and render harmless the real cellular and molecular damage of aging. Scientific studies have rigorously demonstrated that these proposed therapies can remove or repair members of the various categories of cellular and molecular aging damage in animal models — and in an increasing number of cases, in human clinical trials.

When we say that we aim to “reverse aging,” we actually mean reverse aging: not just that damaged cells and molecules will be removed and replaced, but that health and function will be restored — something the HBOT study did not even attempt to demonstrate. Bona fide rejuvenation can be seen (for example) in aging animals treated with senolytic drugs, and in humans with Parkinson’s disease given even the crude early forms of neuronal replacement therapy (and the first glimpses of its next generation).

Going forward, proof-of-concept studies at our Research Center and in expert labs funded by the Foundation will demonstrate more and more rejuvenation biotechnologies. The emerging rejuvenation biotechnology industry will continue to flourish, advance promising breakthroughs into human trials, and eventually license treatments for clinical use (first in the most at-risk, and increasingly in the otherwise-healthy aging). In a future we can see through a glass darkly, our vision will be revealed: a new humanity, open to an indefinite future free of the specter of decline. That day, we will see aging reversed.

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.

References

  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.

References

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  15. Baker DJ, Childs BG, Durik M, Wijers ME, Sieben CJ, Zhong J, Saltness RA, Jeganathan KB, Verzosa GC, Pezeshki A, Khazaie K, Miller JD, van Deursen JM. Naturally occurring p16(Ink4a)-positive cells shorten healthy lifespan. Nature. 2016 Feb 11;530(7589):184-9. doi: 10.1038/nature16932. Epub 2016 Feb 3. PubMed PMID: 26840489; PubMed Central PMCID: PMC4845101.
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  18. Zhang P, Kishimoto Y, Grammatikakis I, Gottimukkala K, Cutler RG, Zhang S, Abdelmohsen K, Bohr VA, Misra Sen J, Gorospe M, Mattson MP. Senolytic therapy alleviates Aβ-associated oligodendrocyte progenitor cell senescence and cognitive deficits in an Alzheimer’s disease model. Nat Neurosci. 2019 May;22(5):719-728. doi: 10.1038/s41593-019-0372-9. Epub 2019 Apr 1. PubMed PMID: 30936558; PubMed Central PMCID: PMC6605052.
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Smart Bombs Against Senescent Cells

When Dr. de Grey and colleagues proposed ablation of senescent cells (ApoptoSENS) as the “damage-repair” strategy of choice for this kind of aging damage in 2001,[1] you’d’ve been hard-pressed to find the idea even mentioned (let alone advocated) in the scientific literature — and certainly no one was actively working to develop such therapies. And this approach remained largely ignored until 2011, when a powerful proof-of-concept study showed that killing these “zombie cells” using a genetically-engineered “suicide switch” substantially rejuvenated a kind of mouse with a mutation that causes them to accumulate an abnormally high level of these cells.

Soon after that, the Mayo Clinic’s James Kirkland and colleagues developed an ingenious drug-discovery strategy that led to the identification of the first two of a new class of “senolytic” drugs — that is, drugs that selectively destroy senescent cells.[2] Because of their stressful internal environment and the effects of their own secretions, such cells are highly dependent on specific biochemical survival pathways to prevent the activation of their inbuilt “cellular suicide” mechanisms. Senolytic drugs inhibit those pathways, tipping the balance in favor of self-destruction.2,[3]

In the three short years since that initial breakthrough, the progress in ApoptoSENS has been astonishing. A torrent of scientific reports have now shown that ablating senescent cells has sweeping rejuvenative effects — wider-ranging, in fact, than we ourselves had predicted. Drugs and gene therapies that destroy senescent cells can restore exercise capacity,2 lung function,[4] and formation of new blood and immune precursor cells[5] of aging mice to nearly their youthful norms. Senolytic drugs and gene therapies have also ameliorated the side-effects of chemotherapy drugs in mice,[6] and prevented or treated mouse models of diseases of aging such as osteoarthritis;[7] fibrotic lung disease;[8],[9] hair loss;[10] primary cancer[11] and its recurrence after chemotherapy;6 atherosclerosis;[12],[13] and age-related diseases of the heart itself2— as well as preventing Parkinson’s disease[14] and (very recently) frontotemporal dementia,[15],[16] a kind of cognitive aging driven by intracellular aggregates of tau protein, which are also an important driver of Alzheimer’s dementia.

The Need for Smart Weapons

Despite the power of these drugs in animal models, there are still risks associated with them. As noted earlier, senolytic drugs kill senescent cells while sparing normal cells because senescent cells are prevented from collapsing under their self-induced stress by leaning heavily on metabolic pathways that prevent the activation of their in-built cellular suicide (apoptosis) machinery, and/or on other growth and survival pathways. So when these drugs inhibit those same pathways, senescent cells self-destruct, while normal cells not so reliant on these pathways survive.

But while normal cells can handle the inhibition of these pathways when not under stress, they still rely on them when damaged or when the local environment turns hostile: they buy the cells time to hunker down, repair themselves, and in return to normal function when the storm has passed. And some normal cell types are also particularly reliant on some of these pathways. Navitoclax, for instance, which has been used as a senolytic in many studies,5,7,9,13,14 targets a cell-survival factor on which platelets (the cells that allow your blood to clot) are heavily dependent. As a result, Navitoclax was shelved as a cancer therapy despite initial promise, due to cases of dramatic collapses of platelets in some patients and a resulting risk of bleeding to death.[17] And while currently-irreplaceable cells like neurons and heart muscle don’t routinely rely on these pathways, no one wants to lose even a small number of these cells until strong cell therapy is available.

The animal studies of senolytic therapy clearly show that the tradeoff is worth it: that the benefits of purging the aging body of senescent cells far outweigh the dangers of a few lost healthy ones. This is true even in the brain (where killing senescent support cells protects neurons in mouse models of diseases of neurodegenerative aging like Parkinson’s disease14 and tau-driven dementias14,15) and the heart2 (where heart function in aging mice is improved, likely by eliminating senescent cells left over from fibrotic responses to damage in the aging heart). Still, it would be better if there were a way of targeting these drugs more specifically to senescent cells, so that healthy cells wouldn’t be dragged through a trial by fire in order to ensure the elimination of their treacherous neighbors.

And now — at least in mice — there is.

The Calling Card of Senescent Cells

Scientists use a range of different cell markers to identify senescent cells: no one marker is infallible, and different senescence markers are more dominant in different senescent cell types. But the best-established and perhaps most universal sign of all is the activity of an enzyme called senescence-associated beta-galactosidase, or SA-beta-gal. All cells produce SA-beta-gal in their “cellular recycling centers” (lysosomes), but because senescent cells contain an abnormally high number of these organelles, they also produce very high levels of SA-beta-gal — so much so that its activity can be detected under conditions under which it can’t be detected in normal cells.[18]

SA-beta-gal degrades the sugar galactose (one half of the milk sugar lactose), so scientists exploit the overproduction of the enzyme to detect senescent cells using chemically modified forms of lactose that change color when cleaved by the enzyme. But a few years ago, a group of scientists began to wonder if there was a way to take advantage of this property not just to detect senescent cells, but to selectively release drugs that would destroy them.

A Mousetrap With “Cheese” for Senescent Cells

To create a system that would release of cell-destroying drugs selectively in cells with senescent-cell levels of SA-beta-gal, chemists and nanotechnologists working with Drs. José Ramón Murguía and Ramón Martínez-Máñez at the Polytechnic University of Valencia turned to an established platform for the selective delivery of drugs: mesoporous silica nanotubes, or MSNs.[19] These devices are like tiny silicone balls that are neither solid nor simply hollow, but are instead comprised of multiple tiny tubes that come together to form a ball structure, their two ends open at the surface of the ball (Figure 1). The size, shape, and electrical charge of MSNs help determine the tissues in the body in which they accumulate and the cell types that will take them up, and how the cells will handle them once they’re inside. As usually configured, MSNs are taken up by a wide range of cells across the body, which first traffic them to the lysosome and then back out again, releasing them harmlessly back out of the cell.[20],[21]

Figure 1. Core features of mesoporous silica nanoparticles (MSN) with “stopper” molecules. Redrawn from ([22]).

But what makes MSNs so useful as drug-delivery systems is that their constituent tubes can be packed with any number of different drugs, and their openings on the surface of the nano-balls “capped” with molecular stoppers that keep the drug sealed inside until the MSN encounters chemical or other conditions that can break open the seal (see Figure 1). So the trick is to identify a molecular stopper that is sensitive to chemical or physical conditions that are found in the type of cell that you want to target, and not found in innocent cells that you want to leave alone. If you can do that, then the MSNs will pass harmlessly through innocent bystander cells with the drug still sealed up inside them — but target cells will recklessly tear the lids off of these nanotechnological Pandora’s boxes, releasing a world of trouble onto themselves.18

As we’ve said, SA-beta-gal’s actual function in the cell is to breaks down the sugar galactose: senescent cells just produce a whole lot more of it than normal cells. So to target MSNs to senescent cells, the Spanish team used galactooligosaccharide (GOS) as the stopper molecule — that is, a series of galactose molecules strung together in a chain. The researchers predicted that with their overabundance of SA-beta-gal, senescent cells would whittle down the chain of galactose molecules until they uncapped the MSNs and released their payload, while the same MSNs would pass through normal cells with their contents still safely sealed up.

In a preliminary study, the grad students working with Murguía and Martínez-Máñez packed a fluorescent dye into MSNs, and then stoppered them with GOS  (rho-GOS-MSN), and then watched what happened when they exposed cultures varying in their frequency of senescent cells: a human cell line that had been pushed into senescence by being allowed to replicate under glass until their telomeres ran out; cells taken from patients whose cells turn senescent rapidly because of mutations that prevent them from lengthening their telomeres; and telomerase-active cancer cells, which are called “immortal” precisely because they never run down their telomeres and senesce. As they had hoped, rho-GOS-MSN released their dye into the two senescent cell types, but not in telomerase-active cancer cells, demonstrating the selective release they were looking for.18

Next, to show that GOS-MSN could work in living organisms and not just on cells in a dish, Murguía and Martínez-Máñez’s reached out to biogerontologist Dr. Manuel Serrano at the Spanish National Cancer Institute (CNIO), who has worked extensively on telomere biology and senescence with SENS Research Foundation Research Advisory Board member Dr. Maria Blasco. In a second report, the combined team took tumor-bearing mice and exposed them to DNA-damaging chemotherapy — a treatment that, in mice as in humans, creates a surfeit of senescent cells.6 When they then injected the chemo-treated mice with rho-GOS-MSN, the nanoparticles once again selectively released their dye to label senescent cells, while passing through normal cells without leaving a trace.[23]

Safer, More Effective Cancer Chemotherapy

It’s one thing to label senescent cells with a dye; it’s another thing to kill them — and do so without doing harm to their normal neighbors. To test this, Murguía and Serrano’s team first drove several lines of cancer cells senescent using palbociclib (Ibrance®), a cancer drug that works exactly by shutting down genes that cancer cells require for cell division, thereby driving them into senescence. And this time, they loaded up their GOS-MSN with doxorubicin (AKA Adriamycin®) instead of a dye.[24] Doxorubicin is itself a toxic chemotherapy drug, feared for its potential to damage the hearts of cancer patients that receive it. But for this purpose, its sheer toxicity was useful, because unlike senolytic drugs, doxorubicin is lethal to normal, cancerous, and senescent cells alike. An additional useful feature of doxorubicin is that it’s intrinsically fluorescent, allowing the scientists to easily see where it was realeased.23

GOS-MSN loaded with doxorubicin (DOX-GOS-MSN) passed harmlessly through three non-senescent cancer cell lines, and only released their payload in a small percentage of cells of the same lines that were exposed to palbociclib too briefly to induce widespread senescence (Figure 2(A)). The nanoparticles also had minimal drug release in a fourth line that had been exposed to palbociclib, but which lacks a gene necessary for the drug to induce senescence. But when cancer cells were exposed to palbociclib for long enough to force them into senescence en masse, they lit up with doxorubicin fluorescence (Figure 2(A)), and programmed cell death raged through the population (Figure 2(B)). Meanwhile, pure doxorubicin exhibited no such selectivity, hitting every cell type across the board (Figure 2(A)).

Figure 2. (A): Palbociclib exposure drives cancer cells senescent (left column); DOX-GOS-MSN selectively releases toxic drug into senescent cells (middle column), while doxorubicin alone hits all cell types alike (right-hand column). Adopted from (23), Figure 3(A). (B): DOX-GOS-MSN causes extensive cell death selectively in senescent but not non-senescent cells. Redrawn from (23), Figure 3(B).

The scientists then tried the doxorubicin-loaded GOS-MSN in vivo, in mice that had been implanted with human tumors and then treated with palbociclib. Implanted tumors grew rapidly in untreated mice — and because there were few senescent cells in the tumors, DOX-GOS-MSN alone provided no defense against their growth. Palbociclib alone very substantially slowed the tumor’s growth, implying a massive conversion of formerly rapidly-dividing cancer cells into senescent ones (since that’s how palbociclib fights cancer) (Figure 3).23

By creating that huge mass of cellular zombies, palbociclib gave DOX-GOS-MSN a target at which to shoot. Where palbociclib alone caused tumor growth to slow but not stop, supplementing palbociclib with DOX-GOS-MSN to wipe out the chemo-induced senescent cells led the total tumor mass to shrink away to a smaller size than when either therapy began (Figure 3).23 The researchers got similar results using Navitoclax as the primary cancer therapy: while low-dose Navitoclax has been used successfully as a senolytic in its own right, it was originally intended as a cancer drug before its toxicity to platelets was discovered, and it does work for that purpose at higher doses. And just in case, the researchers repeated experiment with empty GOS-MSN: it had no effect on tumor growth, whether or not the animals had been treated with chemotherapy.23

Figure 3. DOX-GOS-MSN further reduces tumor mass by destroying chemotherapy-associated senescent cells. Redrawn from (23), Figure 3(C).

Murguía and Serrano then looked at another key problem with many otherwise-valuable cancer drugs: toxic side-effects from hitting the wrong cells. As we mentioned earlier, doxorubicin is feared for its potential to ravage heart muscle cells, leading to drug-induced congestive heart failure. And Navitoclax was abandoned as a cancer drug because in addition to killing some kinds of cancer cells, it also caused patients’ clot-forming cells to self-destruct, leading to risk of catastrophic bleeds.

But normal heart muscle cells and platelets are not senescent. So could stoppering these drugs up in GOS-MSN allow them to pass harmlessly through normal heart muscle cells and platelets, but still decimate tumor-associated senescent cells?

Indeed it could! Mice with implanted tumors were first treated with palbociclib, and then with either doxorubicin or Navitoclax — and those, either in “naked” form or sealed up inside GOS-MSN. The two drugs were equally effective at further shrinking the mass of implanted tumor whether they were delivered by direct injection or packaged up in GOS-MSN. But as conventionally delivered, the two drugs caused predictable toxicity to heart muscle cells (DOX) and life-threatening die-off of normal platelets (Nav) (Figure 4). But when packaged up in GOS-MSN, doxorubicin was harmless to the heart (left) and Navitoclax spared most platelets (right) (Figure 4)).23

Figure 4. Packaging Doxorubicin (A) and Navitoclax (B) in GOS-MSN shrinks tumor mass by killing tumor-associated senescent cells, but shields healthy cells from deadly drug toxicities. Redrawn from (23), Figure 3(E) and (F).

Breathe Easy

Senescent cells accumulate in the aging lungs, and people suffering with diseases of lung aging suffer an even higher burden of them. Idiopathic pulmonary fibrosis (IPF) is one of these: characterized by scarring of the lung tissue, victims suffer a progressive decline in lung function, making it hard to breathe — both in the sense that it becomes harder to force your lungs to expand to take in air, but also in the sense that your lungs become less and less effective at taking in oxygen and releasing waste CO2. Press a stethoscope up to a patient’s chest, and you can hear a terrible crackling sound inside their lungs when they inhale — like Velcro® being torn apart. There is today only limited therapy: short of lung transplants for a very few, most patients get by on anti-inflammatories, oxygen, and exercises to help them expand their ravaged lungs. Cases often end in death, if nothing else gets them first.

Previous research had already shown that a variety of apoptoSENS strategies can prevent or reverse IPF in mouse models of the disease,8,9 as well as reversing the “normal” loss of lung function with age.4 Murguía and Serrano wanted to see if DOX-GOS-MSN could similarly restore lung function in mice with a model of IPF.23 After first confirming that GOS-MSN distributed evenly across normal and senescent lung tissue but would only release their sequestered dye in tissue with a high senescent cell burden, they treated mice with either straight doxorubicin or DOX-GOS-MSN for two weeks, starting two weeks after inducing model IPF. Lung dysfunction scores remained stubbornly high in animals treated with plain doxorubicin did nothing to help the animals’ lung dysfunction — but DOX-GOS-MSN restored the lung function of the IPF model mice levels equivalent to young mice not subjected to lung damage (Figure 5(A)). DOX-GOS-MSN also reduced the amount of fibrotic tissue in the animals’ lungs, which untargeted doxorubicin was again unable to do  (Figure 5(B)).

Figure 5. Doxorubicin targeted to senescent cells by GOS-MSN, but not untargeted doxorubicin, normalize lung dysfunction (A) and greatly reduce lung fibrosis (B) in a mouse model of idiopathic pulmonary fibrosis. Redrawn from (23), Figure 4 (B) and (C).

Senolytic Synergy

As recently as 2015, an announcement like this — the selective targeting of senescent cells in vivo withy a targeted drug strategy, leading to significant functional benefits — would have been breathtakingly exciting news. But after three years of nonstop announcements of novel ApoptoSENS therapeutics and of proofs-of-concept in animal models of one disease of aging after another, it’s human nature to begin taking each new announcement in stride. Yet while its inventors have not stressed it, there is a potential further tweak to the GOS-MSN technology that makes it particularly promising, and that should cause us to pay attention.

As we have gone over a couple of times, prior to GOS-MSN, all senolytic drugs derived their selectivity from the fact that senescent cells lean heavily on active cell-survival pathways to keep themselves alive. Drugs that suppress the activity of these pathways are therefore able to tip the balance over toward programmed cell death in these cells, while sparing most normal cells, which don’t rely on these pathways for their ordinary day-to-day existence.2

But while this is true of normal cells under unstressed conditions, normal cells also rely on those same pathways to carry them through times when they are under stress, and to give them time to recover afterward. Thus, although the net effect of these drugs is undeniably positive, their mechanism of action will necessarily entail occasionally killing off healthy cells in a moment of vulnerability that they could otherwise have survived. This is a particularly important potential risk when the cells in question are not readily replaceable and have to last a lifetime, as is the case with heart muscle cells and brain neurons today.

But what if senolytic drugs were packaged up in GOS-MSN? Existing senolytics expose all cells to their effects, depending on the different metabolic states of senescent and normal cells for their selective killing power.  But the selectivity of GOS-MSN particles is different: they work by only releasing their payload of drugs in senescent cells, such that the great majority of normal cells are never exposed to the drugs at all.

And what if rejuvenation biotechnologists took advantage of the strengths of both of these approaches, by loading GOS-MSN with senolytic drugs instead of a generically toxic drug like doxorubicin? You’d expect that this would create a therapy “doubly-selective” for senescent cells: normal cells would almost never be exposed to the drug in the first place — and on the rare occasions when they were, most healthy cells would still escape unscathed, because of the drugs’ intrinsic selectivity for senescent cells.

Conceptually, this combination could lead to greater safety of apoptoSENS therapy, allowing for each round of therapy to use higher doses of drug so as to effect a more thorough clearance of senescent cells, with less risk to normal ones. While the potential has hardly been tested, we’ve already seen a hint of this when Navitoclax was delivered in GOS-MSN in a cancer model: it was just as effective in reducing tumor burden as the “naked” drug, but almost eliminated the loss of platelets that the unpackaged drug entailed (Figure 5(B) above).

Startup Lineup

With those exciting results in hand, Drs. Murguía, Martínez-Máñez, and Serrano have launched a biotech startup to turn GOS-MSN into a human rejuvenation biotechnology. Senolytic Therapeutics (Senolytx) projects that “Designed therapies will be efficacious in treating multiple disorders which are caused and driven by the accumulation of damaged cells. For example, …Idiopathic pulmonary fibrosis (IPF). Also, … cancer cells that have been pushed into senescence by approved chemotherapy and radiotherapy” — that is, exactly the conditions that GOS-MSN treated so successfully in their recent proof-of-concept scientific report.23

And Senolytx is just the latest in a rush of new biotech startups in the senescent cell ablation space, including:

  • UNITY Biotechnology: first out of the gate, with the first human patient receiving injections of UBX0101 directly into osteoarthritic knee joints last summer; more senolytics targeted to diseases of the aging eye and lung as well as to systemic sclerosis are expected to follow.
  • Oisín Biotechnologies: launched in 2015, with seed funding and access to intellectual property from SENS Research Foundation and our allies at the Methuselah Foundation; they are developing a variation on the “suicide gene” strategy used in the first breakthrough in the field, but which will not permanently alter people’s genomes, can be controlled with a triggering drug, and which avoids other risks associated with virus-based gene therapy.
  • Cleara Biotech: working to develop a safer and more effective version of scientific founder Peter de Keizer’s FOXO4-DRI peptide, which triggers cellular suicide in senescent cells by preventing the senescence and apoptosis regulator p53 from binding to FOXO4 (which keeps p53 from initiating the cell’s self-destruct sequence).3
  • FoxBio: a joint venture between Foundation alumnus Kelsey Moody’s Ichor Therapeutics and technology financier Jim Mellon’s Juvenescence Limited. FoxBio plans to develop a system for screening for drugs that can prevent FOXO4 or MDM2 from neutralizing the cell-suicide activity of p53. Previous attempts to do such screens have led to unreliable results, because it has been too hard to manufacture full-length, properly folded, bioactive p53 at scale; as a result, companies working to target such interactions have relied on only small fragments of the full protein, or on proteins that do not reflect the complex three-dimensional structure of that p53 adopts our cells. Ichor believes that they can overcome this problem using their proprietary RecombiPure expression technology, and then begin the kind of highly automated screening of large libraries of molecules that could lead to new senoltyics targeting these key interactions.

It must be emphasized that with the exception of FoxBio (who are pursuing a validated target), all of these startups have already proven that their senescent cell ablation therapeutics can selectively clear senescent cells out of the tissues of living, breathing, aging mice — and in so doing, that they rejuvenate tissue function and/or prevent and treat animal models of diseases of aging.

With so many new companies crowding into the space, and with so many potential routes to licensing by the FDA and other regulatory bodies, it now seems not only possible that apoptoSENS therapies will be the first rejuvenation biotechnologies to achieve regulatory approval, but likely that they will be the first to become widely available. Within the decade, people could be taking them as approved drugs for osteoarthritis, IPF, any of several eye diseases — and possibly more common diseases of aging not long afterward, including diabetes, atherosclerosis, lung diseases other than IPF, and as a follow-on therapy for radiation and toxic chemotherapy for cancer.

The real tantalizing outcome here from a SENS perspective is, of course, that when these uses become widely accepted, prophylactic use of senolytics to keep aging people from developing these conditions in the first place could emerge — even as other rejuvenation biotechnologies enter the physician’s armamentarium. In short, the rapid surge of ApoptoSENS type therapies could well be a significant milestone on the road to comprehensive human rejuvenation.

References

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To Save Your Brain, Slay The Zombie (Senescent) Cells!

It was a bit of a mystery to the scientists investigating the phenomenon: a brain disease driven by the death of specialized neurons was strongly linked to exposure to a particular pesticide. Why, then, didn’t exposing those same neurons directly to that same pesticide seem to affect them?

Parkinson’s disease (PD) is a neurodegenerative disease of aging, whose most obvious symptoms — tremors, gait disorders, and a “mask-like” facial appearance — involve the loss of fine motion control. These symptoms are the result of the loss of specialized cells in an area of the brain called the substantia nigra pars compacta (SNc) that specialize in producing the chemical signal-molecule dopamine and are responsible for turning off excess firing of neurons that control muscles. Once a critical number of these “dopaminergic” SNc neurons are lost, the unbalanced firing of those neurons begins to manifest itself in the main motion-related symptoms of the disease.

In all but a few people with rare mutations, degenerative aging processes (such as the accumulation of mitochondrial mutations in SNc neurons) are primarily responsible for the disease. But lifestyle and environmental factors also damage these neurons, and thus increase the risk of a person developing the disease clinically within a currently-normal lifetime. A striking example of this is MPP+, a well-established neurotoxin that specifically attacks the SNc dopaminergic neurons in lab mice, monkeys — and in humans: MPP+’s parent compound, MPTP, has caused numerous cases of Parkinson’s-like syndrome in young people exposed to it in underground drug labs, or via contaminated street drugs.1

So what other exposures might similarly accelerate the degenerative processes that leads to PD? For a long time, scientists have focused on paraquat, a neurotoxic pesticide banned in the EU in 2007, and subject to restricted use in the United States. Paraquat was originally restricted because it can cause lung damage when workers are exposed to high levels of it in the air, but scientists studying it also noted that it has a strong structural resemblance to MPP+. And sure enough, under some conditions it can cause a Parkinson’s-like syndrome in laboratory animals,2 and a strong and consistent relationship has been found between on-farm exposure to paraquat in farm workers (as opposed to exposure to residues in one’s food) and risk of PD.3

Yet, puzzlingly, paraquat doesn’t seem to be particularly toxic to dopaminergic neurons when tested directly; much of the rodent data that seems to show such an effect is ambiguous or unlikely to reflect paraquat exposures actually present in the brain.2 So what might be going on?

As it turned out, the scientists were looking in the wrong place, much like characters in a zombie movie focused intently on the doors when the zombies are about to break through the windows. Paraquat, it turns out, doesn’t directly kill dopaminergic neurons. Instead, it acts by deranging the cells that are supposed to support and nourish them. Meanwhile, the same thing goes wrong in the aging brain, bringing one more insult to the accumulating cellular and molecular damage that slowly erodes the functional capacity of the aging brain, culminating in Parkinson’s and other degenerative syndromes.

The lesson here isn’t just “avoid exposure to dangerous pesticides.” The same study that revealed this surprising indirect mechanism of paraquat’s neurotoxicity also showed how much of the harm can be blocked, and in doing so revealed a new tool in our toolbox for taking the “normal” Parkinson’s disease of aging out of our futures forever.

A Toxin That Makes Zombies Out of Astrocytes

Astrocytes are a kind of support cell for the neurons in the brain. They provide a source of nutrients, maintain the equilibrium in the fluids that surround the neurons, participate in neural repair, and take up and release brain messenger-molecules. Scientists discovered several years ago, however, that rising numbers of astrocytes in the aging brain become senescent. Senescent cells are like cellular zombies: not exactly dead, but not exactly alive any more either, and deadly to all those around them. Senescent cells lose their normal function in the tissue, cease dividing, and begin secreting a deadly mix of inflammatory and tissue-degrading factors collectively known as the senescence-associated secretory phenotype (SASP) that damages and deranges local tissues. Evidence has accumulated to show senescent cells involved in everything from atherosclerosis, to osteoarthritis, to diabetes, cataracts, and on and on.4

It was no surprise, then, when scientists found that the burden of astrocytes with tell-tale signs of senescence rises with age in the brain5 and even faster in those with Alzheimer’s disease.6

This observation got long-time Parkinson’s researcher Dr. Julie Andersen, senescent cell pioneer Dr. Judith Campisi, and their teams at the Buck Institute wondering: could the same be true of Parkinson’s disease? And if so, could it be part of the explanation for the effect of paraquat? And what are the therapeutic implications of such findings in aging people not exposed to this neurotoxin?

Sure enough, when the researchers examined the brains of PD patients, they found more cells exhibiting signs of senescence than in people without the disease — and especially astrocytes, as they had expected.7 This was true even after matching patients for age, meaning that PD subjects had even more senescent astrocytes in their SNcs than is typical for people their age (ranging in this case from 50–92 years at autopsy) — and remember, aging already drives an increase in the burden of these cells as compared with young people, even in those who have yet to develop Parkinson’s disease.7

So, could senescent astrocytes be the link between paraquat and PD? To investigate this possibility, researchers exposed human astrocytes derived from “reprogrammed” human cells to paraquat. In response, the cells underwent changes indicative of senescence, ceasing cell division and churning out key SASP factors.7 Notably, astrocytes were much more vulnerable to paraquat than skin cells: astrocytes went senescent at a lower concentration of the toxin than was required for skin cells, and the doses that turned skin cells senescent killed astrocytes outright.7

So if aging people accumulate senescent astrocytes in their brains, and if an even higher burden of these cells is linked to Parkinson’s, and if even low doses of paraquat turn astrocytes senescent, then might senescent astrocytes be contributing to Parkinson’s disease? To find out, the team looked at the effects of astrocytes on dopamine-producing neurons as mediated by the SASP. When they harvested SASP-laden culture medium from cells rendered senescent by paraquat and transferred it over to separate cultures of neurons, the senescent cell secretions suppressed the ability of mature dopaminergic neurons to survive, and of neural stem-like cells to reproduce and migrate. None of these toxic effects were observed when the same cells were treated with culture medium from non-senescent astrocytes that had not been subjected to paraquat.7

Making “Zombie Cells” Self-Destruct

So far, all of this work on the effects of paraquat on astrocytes — and the havoc those paraquat-exposed astrocytes wreak on neurons — was restricted to cell culture. To see if those effects might explain the link between paraquat, aging, and PD in living organisms, the team turned to genetically altered mice that the Campisi lab had developed for senescent cell studies. Using these mice gives scientists two important abilities: first, to easily track the burden of senescent cells in the mouse’s tissues and how it is affected by aging, environmental exposures, and anti-senescent-cell therapies; and second, to eliminate those cells at will using an inducible “suicide gene.”

Think of this gene construct as a self-destruct device. The gene remains completely dormant and harmless in a mouse’s cell until it is “armed” by the activation of the key senescence gene, p16INK/4a, which rarely occurs except in senescent cells. But once “armed” by p16INK/4a, the device is set to go off as soon as it gets the signal from a remote-controlled trigger. In this case, the trigger is a drug that interacts with the protein produced by the “suicide gene,” which metabolizes it into a form deadly to the cell, causing it to commit programmed cell death (apoptosis).

Consistent with their previous studies in cell culture, mice exposed to paraquat under realistic conditions developed an increased burden of senescent cells in their brains (Figure 1, (a-c)) — and this plague was almost entirely restricted to astrocytes (Figure 1 (d)).

Figure 1. Paraquat induces senescence in astrocytes in mice; ablation by “suicide gene.” Redrawn from (7).

And sure enough, this increased burden of senescent cells caused the same problems in living mice as in the researchers’ cell culture experiments, as well as in previous work in paraquat-exposed mice. These mice developed hallmark signs of PD in their brains and behavior: loss of dopaminergic neurons in the SNc, impaired generation of new neurons in one of the few regions capable of producing them in adult organisms, and impaired muscle coordination similar to human victims of Parkinson’s (as evidenced by difficulty in rearing up on their hind legs) (Figure 2).

Figure 2. Paraquat-induced senescence is associated with signs of Parkinson’s disease in mice. Rescue by ablation of senescent cells. Redrawn from (7).

But remember, scientists have known for decades that paraquat causes PD-like loss of neurons and symptoms in mice (and humans). How might one prove that the newly-discovered induction of senescence in astrocytes was responsible for the damage, and not some other direct or indirect effect of the toxin? The “damage-repair” heuristic of SENS suggested eliminating the senescent cells themselves, and seeing if that was enough to block the downstream mayhem.

To test this notion, the researchers put paraquat-exposed mice on a regimen of the drug serving as a trigger for the suicide device now armed in all their senescent cells, rapidly eliminating the excess senescent astrocytes from their brains (Figure 1, (a-c)). Sure enough, taking senescent cells out of the picture also eliminated the other harmful effects of paraquat: the loss of dopaminergic neurons and the suppression of new neuron generation was prevented, and the mice were able to rear up normally (Figure 2).

But all of this is about the effects of an agricultural pesticide on the brain, which immediately presses the question: does it really tell us anything about how senescent cells contribute to “regular” PD, driven by the intrinsic aging processes responsible for the great majority of cases of PD? It seems that the answer is likely ‘yes.’ Remember first the circumstantial evidence: the researchers not only found that the burden of senescent astrocytes rises with age in the human brain, but that there is a further excess burden in the brains of people who died with PD. Beyond that, Dr. Andersen has shown in unpublished work (presented at the 2014 Rejuvenation Biotechnology Conference8) that eliminating senescent astrocytes confers similar benefits to mice with a model of PD that more closely mimics the fundamental processes that drive Parkinson’s in aging people not suffering from either environmental toxicity or rare, genetic causes of PD. These mice accumulated more senescent astrocytes in their brains with age, along with dopaminergic neuron death and loss of motor coordination. And just as in the case of the paraquat model, all of these effects were greatly reduced or prevented entirely when senescent astrocytes were ablated by the suicide-gene activating drug.8

The First Zombie-Cell Slayers

Unfortunately, you and I were not born with a “suicide gene” built into our cells that we can activate with a trigger drug, so we need a different solution to eliminate senescent cells. But fortunately, the first such rejuvenation biotechnologies are coming on fast.

In recent years, researchers have developed so-called “senolytic” drugs that wipe out senescent cells in aging mice and mouse models of age-related disease, exploiting the high dependence of these cells on specific biochemical survival pathways.9,10 In these studies, senolytic drugs have restored exercise capacity9 and formation of new blood and immune precursor cells11 in aging mice to near youthful norms, and prevented or treated mouse models of diseases of aging like osteoarthritis,12 fibrotic lung disease,13 hair loss,14  atherosclerosis,15,16 and age-related diseases of the heart itself.9 UNITY Biotechnology is leading a growing charge toward the clinic, with human clinical trials expected to begin in 2019. Based on their work in animal models and with studies in knee tissue from humans with osteoporosis, the company plans to use osteoarthritis as the first of several labeled disease indications (see Question Of The Month #3: Making SENS Part of Medicine), to be followed by cataracts, atherosclerosis, and fibrotic lung disease.

SENS Research Foundation and the Next Generation of Senescent Cell Ablatives

Senolytic drugs are exciting, and could yet prove to be the first rejuvenation biotechnology that becomes widely available to the public. Still, we must be realistic in acknowledging the limits of these therapies and continuing to pursue additional avenues of research to account for said limits.

As noted earlier, senolytic drugs are only able to effectively kill senescent cells while sparing normal cells because senescent cells are much more reliant than healthy cells on the activity or expression of specific genes involved in cell survival. But while this is true of normal cells in most unstressed conditions, normal cells also rely on those same pathways to carry them through times when the cell is under stress, and to give them time to recover afterward. Thus, although the net effect of these drugs is undeniably positive, their mechanism of action will necessarily entail occasionally killing off healthy cells at a moment of vulnerability that they could otherwise have survived, including difficult-to-replace cells like heart muscle cells and (ironically) neurons. Improved rejuvenation biotechnologies would target senescent cells more selectively, and SENS Research Foundation is helping to advance those next-generation “senoablatives” even as UNITY prepares for human testing.

One such rejuvenation biotechnology is under development by Oisín Biotechnologies, a startup launched in 2015 with seed funding and access to intellectual property from the Foundation and our allies at the Methuselah Foundation. Oisín’s technology is actually a variation on the “suicide gene” strategy used in the senescent astrocyte study7 and similar systems used in previous proofs-of-concept, including the first breakthrough in the field. But in this case, you don’t have to be born with the “suicide gene” already in your cells to benefit from it. Instead, the “suicide gene” is delivered to aging tissues using a kind of gene therapy.

Unlike the case of most gene therapies that are intended to permanently alter their target cells, however, Oisín’s senoablative genetic constructs will not be inserted permanently into the patient’s genome: instead, its genetic payload will be expressed temporarily from the main body of the cell, following which the construct will be passively degraded by normal cellular metabolism.

After initial testing demonstrated that their senoablative constructs could eliminate up to 80% of senescent cells in cell culture, the Oisín team tested them out in living mice, showing that it can purge senescent cells from multiple tissues in middle-aged animals.

Figure 3. Oisín senolytic gene construct clears senescent cells from aging mouse tissues. From the Oisín Biotechnologies website.

In addition to being much more narrowly-targeted to senescent cells than the senolytic drugs, this system also has several advantages over gene therapies that would be permanently inserted into your cells. The most important such advantages relate to safety. The tools currently available to insert new genes permanently into cells do so at a random site inside the cell, which occasionally results in the disruption of the cell’s native genes at the site of insertion, which could potentially turn the cell cancerous or cause other problems. This is why SENS Research Foundation is working on new ways to safely deliver large genetic payloads to our cells. In the meantime, Oisín’s transient gene expression technology essentially eliminates this risk by not requiring insertion into the cell’s own genome.

Additionally, the fact that Oisín’s “suicide gene” is only present in tissues temporarily means that if any side effects were to emerge, the treatment is rapidly self-extinguishing. Its time-limited activity also allows therapy to be be withdrawn at times when a temporary rise of senescent cells in a tissue is needed as part of physiological processes (such as in resolving fibrosis, in wound healing, and even in pregnancy). Afterward, senoablative therapy can be initiated again to mop up any senescent cells not eliminated by physiological processes, and to resume clearance of the backlog of senescent cells that existed before therapy was temporarily suspended.

Breaking Down the Barricades

In addition to drugs and gene constructs, there’s another strategy in the works for eliminating senescent cells: enhancing the innate immune system’s ability to purge them. Studies over the course of the last decade have revealed the somewhat surprising fact that the body’s immune system routinely eliminates senescent cells from our tissues. While several components of the immune system are involved, the key players are natural killer cells (NK cells). But then why do senescent cells accumulate in our tissues with age? Do aging processes lead to a decline in an initially-robust senescent-cell clearing capacity? Or do NK cells never quite catch all the senescent cells that arise at any given time, and the few that remain slowly accumulate over time? Or are a few senescent cells particularly resistant to immunological clearance? And most importantly: can this innate immune clearance of senescent cells be made more effective, so that our bodies can purge themselves of these zombie cells, without requiring an exogenous treatment?

We announced last year that with the support of the Forever Healthy Foundation, we would be funding a new project in Dr. Campisi’s lab to explore these questions and look for solutions. We are also now preparing to expand this project with a complementary intramural project at our Research Center in Mountain View, California.

Each lab will focus on its strong suit. With their deep expertise in the biology of senescent cells, the Campisi lab will be focused on fundamental research into questions like how senescent cells vary in their susceptibility and resistance to immune clearance (depending on factors like their tissues of residence or the pathway that led them into senescence); the targets and mechanisms used by NK cells to clear senescent cells; and why subsets of senescent cells might persist when their similarly-situated neighbors are cleared out (and what might allow us to overcome that resistance).

Meanwhile, our team at the Research Center will be picking up and running with an emerging mechanism of such resistance, testing one of two different platforms to see if we can deny senescent cells the ability to shield themselves from NK clearance. The details on these complementary projects will be announced later this year.

Progress likewise proceeds apace in rejuvenation biotechnologies targeting the other key aging damage driving Parkinson’s disease specifically. There are now multiple immunotherapies targeting clearance of alpha-synuclein from the brain in early-stage clinical trials, and multiple trials underway or in the works on the next generation of cell replacement therapies for dopaminergic neurons, including the TRANSEURO trial; the Summit4StemCell initiative, put together by Jeanne Loring  — a researcher at The Scripps Research Institute who is exceptionally engaged with turning her research into therapies; a Japanese trial to be run by Jun Takahashi of Kyoto University in Japan (cf. here and here); and a trial centered at Memorial Sloan Kettering Cancer Center headed by cell biologist Lorenz Studer.

The benefits of senescent cell clearance to the health and longevity of aging mice have turned out to be more dramatic and sweeping than anyone ever expected. SENS Research Foundation is working to bring those same benefits to aging humans, advancing us to a future where living long is permanently decoupled from age-related debility and disease.

References

  1. Langston JW. The MPTP Story. J Parkinsons Dis. 2017;7(s1):S11-S22. doi: 10.3233/JPD-179006. PubMed PMID: 28282815; PubMed Central PMCID: PMC5345642.
  2. Tieu K. A guide to neurotoxic animal models of Parkinson’s disease. Cold Spring Harb Perspect Med. 2011 Sep;1(1):a009316. doi: 10.1101/cshperspect.a009316. Review. PubMed PMID: 22229125; PubMed Central PMCID: PMC3234449.
  3. Pezzoli G, Cereda E. Exposure to pesticides or solvents and risk of Parkinson disease. Neurology. 2013 May 28;80(22):2035-41. doi:10.1212/WNL.0b013e318294b3c8. PubMed PMID: 23713084.
  4. Kirkland JL, Tchkonia T. Cellular senescence: a translational perspective. EBioMedicine. 2017 Jul;21:21-28. doi: 10.1016/j.ebiom.2017.04.013. Epub 2017 Apr 12. Review. PubMed PMID: 28416161; PubMed Central PMCID: PMC5514381.
  5. Kang C, Xu Q, Martin TD, Li MZ, Demaria M, Aron L, Lu T, Yankner BA, Campisi J, Elledge SJ. The DNA damage response induces inflammation and senescence by inhibiting autophagy of GATA4. Science. 2015 Sep 25;349(6255):aaa5612. doi: 10.1126/science.aaa5612. PubMed PMID: 26404840; PubMed Central PMCID: PMC4942138.
  6. Bhat R, Crowe EP, Bitto A, Moh M, Katsetos CD, Garcia FU, Johnson FB, Trojanowski JQ, Sell C, Torres C. Astrocyte senescence as a component of Alzheimer’s disease. PLoS One. 2012;7(9):e45069. doi: 10.1371/journal.pone.0045069. Epub 2012 Sep 12. PubMed PMID: 22984612; PubMed Central PMCID: PMC3440417.
  7. Chinta SJ, Woods G, Demaria M, Rane A, Zou Y, McQuade A, Rajagopalan S, Limbad C, Madden DT, Campisi J, Andersen JK. Cellular Senescence Is Induced by the Environmental Neurotoxin Paraquat and Contributes to Neuropathology Linked to Parkinson’s Disease. Cell Rep. 2018 Jan 23;22(4):930-940. doi: 10.1016/j.celrep.2017.12.092. Epub 2018 Jan 28. PubMed PMID: 29386135; PubMed Central PMCID: PMC5806534.
  8. Andersen JK. Senescence and the aging brain. Presentation at Rejuvenation Biotechnology 2014 (RB2014). August 21-23, 2014 Santa Clara, California. Parkinson’s Disease Session. Program p. 41. Video Presentation.
  9. Zhu Y, Tchkonia T, Pirtskhalava T, Gower AC, Ding H, Giorgadze N, Palmer AK, Ikeno Y, Hubbard GB, Lenburg M, O’Hara SP, LaRusso NF, Miller JD, Roos CM, Verzosa GC, LeBrasseur NK, Wren JD, Farr JN, Khosla S, Stout MB, McGowan SJ, Fuhrmann-Stroissnigg H, Gurkar AU, Zhao J, Colangelo D, Dorronsoro A, Ling YY, Barghouthy AS, Navarro DC, Sano T, Robbins PD, Niedernhofer LJ, Kirkland JL. The Achilles’ heel of senescent cells: from transcriptome to senolytic drugs. Aging Cell. 2015 Aug;14(4):644-58. doi: 10.1111/acel.12344. Epub 2015 Apr 22. PubMed PMID: 25754370; PubMed Central PMCID: PMC4531078.
  10. Baar MP, Brandt RMC, Putavet DA, Klein JDD, Derks KWJ, Bourgeois BRM, Stryeck S, Rijksen Y, van Willigenburg H, Feijtel DA, van der Pluijm I, Essers J, van Cappellen WA, van IJcken WF, Houtsmuller AB, Pothof J, de Bruin RWF, Madl T, Hoeijmakers JHJ, Campisi J, de Keizer PLJ. Targeted Apoptosis of Senescent Cells Restores Tissue Homeostasis in Response to Chemotoxicity and Aging. Cell. 2017 Mar 23;169(1):132-147.e16. doi: 10.1016/j.cell.2017.02.031. PubMed PMID: 28340339; PubMed Central PMCID: PMC5556182.
  11. Chang J, Wang Y, Shao L, Laberge RM, Demaria M, Campisi J, Janakiraman K, Sharpless NE, Ding S, Feng W, Luo Y, Wang X, Aykin-Burns N, Krager K, Ponnappan U, Hauer-Jensen M, Meng A, Zhou D. Clearance of senescent cells by ABT263 rejuvenates aged hematopoietic stem cells in mice. Nat Med. 2016 Jan;22(1):78-83. doi: 10.1038/nm.4010. Epub 2015 Dec 14. PubMed PMID: 26657143; PubMed Central PMCID: PMC4762215.
  12. Jeon OH, Kim C, Laberge RM, Demaria M, Rathod S, Vasserot AP, Chung JW, Kim DH, Poon Y, David N, Baker DJ, van Deursen JM, Campisi J, Elisseeff JH. Local clearance of senescent cells attenuates the development of post-traumatic osteoarthritis and creates a pro-regenerative environment. Nat Med. 2017 Jun;23(6):775-781. doi: 10.1038/nm.4324. Epub 2017 Apr 24. PubMed PMID: 28436958; PubMed Central PMCID: PMC5785239.
  13. Schafer MJ, White TA, Iijima K, Haak AJ, Ligresti G, Atkinson EJ, Oberg AL, Birch J, Salmonowicz H, Zhu Y, Mazula DL, Brooks RW, Fuhrmann-Stroissnigg H, Pirtskhalava T, Prakash YS, Tchkonia T, Robbins PD, Aubry MC, Passos JF, Kirkland JL, Tschumperlin DJ, Kita H, LeBrasseur NK. Cellular senescence mediates fibrotic pulmonary disease. Nat Commun. 2017 Feb 23;8:14532. doi: 10.1038/ncomms14532. PubMed PMID: 28230051; PubMed Central PMCID: PMC5331226.
  14. Yosef R, Pilpel N, Tokarsky-Amiel R, Biran A, Ovadya Y, Cohen S, Vadai E, Dassa L, Shahar E, Condiotti R, Ben-Porath I, Krizhanovsky V. Directed elimination of senescent cells by inhibition of BCL-W and BCL-XL. Nat Commun. 2016 Apr 6;7:11190. doi: 10.1038/ncomms11190. PubMed PMID: 27048913; PubMed Central PMCID: PMC4823827.
  15. Roos CM, Zhang B, Palmer AK, Ogrodnik MB, Pirtskhalava T, Thalji NM, Hagler M, Jurk D, Smith LA, Casaclang-Verzosa G, Zhu Y, Schafer MJ, Tchkonia T, Kirkland JL, Miller JD. Chronic senolytic treatment alleviates established vasomotor dysfunction in aged or atherosclerotic mice. Aging Cell. 2016 Oct;15(5):973-7. doi: 10.1111/acel.12458. Epub 2016 Aug 5. PubMed PMID: 26864908; PubMed Central PMCID: PMC5013022.
  16. Childs BG, Baker DJ, Wijshake T, Conover CA, Campisi J, van Deursen JM. Senescent intimal foam cells are deleterious at all stages of atherosclerosis. Science. 2016 Oct 28;354(6311):472-477. Epub 2016 Oct 27. PubMed PMID: 27789842; PubMed Central PMCID: PMC5112585.

Pathway To New Therapies

FDA Opens Pathway For Testing Rejuvenation Biotechnologies For The Prevention of Alzheimer's Disease

It’s often assumed that the fact that regulators like the FDA don’t recognize aging as a “disease” is a major challenge to the development of medical therapies targeting degenerative aging processes. In fact, as we’ve discussed before (scroll down to “Question of the Month”), this is not a major hurdle for rejuvenation biotechnologies, because of the specifics of the “damage repair” approach to rejuvenation biotechnology that we’re pursuing at SRF. That said, substantial regulatory reform is needed to create a pathway for investors and pharma to put the necessary time and money into researching and developing rejuvenation biotechnologies such that licensable therapies can come out the other end.

The most important regulatory reform would entail acceptance of novel biomarkers of the removal, repair, replacement, or rendering harmless of specific forms of cellular and molecular aging damage as sufficient basis to grant rejuvenation biotechnologies preliminary licensure. This would then be followed up by further monitoring of patients to ensure that the therapy actually does bend the curve on diseases of aging over the longer term. This standard would mark a break with regulators’ usual insistence (which has been getting more entrenched, rather than less, in recent years) that therapies prove an effect on “hard outcomes” to get approval: things like heart attacks, amputations, or blindness. But people should ideally begin to receive rejuvenation biotechnologies well before patients are in near-term danger of such acute threats to life and health, making it extremely expensive and time-consuming to run a trial.

In 2012, it looked as if significant progress had been made on this front and several others during major stakeholder meetings amongst Alzheimer’s disease (AD) patient and caregiver advocates, researchers, and FDA regulators. In particular, there was consensus on the need to work toward the development of new clinical trial designs and regulatory reforms to advance previously-untested combination therapies for AD into clinical testing. Shortly thereafter, FDA issued draft “Guidance for industry: Alzheimer’s disease: developing drugs for the treatment of early stage disease,” which acknowledged that therapies for AD are unlikely to have substantial effects in “full-blown” dementia, because by that point, the brain has suffered either irreversible damage, or too many kinds of damage for any one therapy to be useful anymore.

Recognizing that fact, they furthermore acknowledged that the industry focus should shift to testing therapies in people who, while they may be experiencing measurable cognitive deficits, are not yet even in the early stages of dementia per se. But the tests currently used in clinical trials to measure the progression of existing dementia are unlikely to be useful in assessing the effects of therapies designed to help people in these very early stages of the disease (let alone people who were aging, but still cognitively normal for their age). And therefore, FDA said, alternative outcome metrics will need to be developed instead.

Then, despite having climbed up to the top of the diving tower, with advocates and scientists cheering at every step, FDA and pharma seemed to hesitate. The same FDA leaders who had previously expressed their openness to bold initiatives instead adopted a more conservative stance on using biomarkers as outcomes for early-stage disease in the Guidance and a parallel New England Journal of Medicine article. Despite acknowledging beta-amyloid and aberrant tau as “leading candidates in this respect,” they hesitated: “Until there is widespread evidence-based agreement in the research community that an effect on a particular biomarker is reasonably likely to predict clinical benefit, we will not be in a position to consider an approval based on the use of a biomarker as a surrogate outcome measure in AD (at any stage of the illness).” However, they did, at least, express openness to the inclusion of favorable effects on biomarkers and imaging tests that reflect the pathological processes that set people on the path to dementia as supportive evidence for efficacy if combined with other preliminary evidence.

Meanwhile, at a research summit in 2014, major pharmaceutical companies also seemed to get cold feet, particularly on the key, related subject of cooperating on combination therapies capable of targeting multiple kinds of aging damage at once.

This February, however, FDA finally took the plunge with a revised Draft Guidance, which would represent a tremendous step in the right direction if finalized as official FDA policy. The results of imaging tests or relevant biomarkers could then be considered sufficient to identify so-called “Stage 1” Alzheimer’s patients as eligible candidates for clinical trials or new therapies, or to test existing therapies that had failed in people with frank AD. These “Stage 1” candidates are described in terms that identify people at an even earlier stage along the insidious path to dementia than the 2013 guidance: outwardly healthy but aging people without very-high-risk mutations or apparent cognitive or functional impairments, but who are nonetheless identified as being at higher risk than most. In short, these individuals would now be eligible to participate in trials of new and old therapies that might prevent them from ever tipping over into major cognitive impairments.

Even more boldly, FDA went on to say that since the march toward cognitive impairment in these “Stage 1” patients will be so hard to reliably pick up using any kind of cognitive tests, therapies that successfully move those biomarkers in the right direction would be granted accelerated approval, with a requirement for further followup to confirm the long-term benefit. In the case of “damage-repair” therapies, such movements would reflect the removal or replacement of the underlying cellular and molecular damage that the biomarkers pick up.

That said, the new Guidance is, surprisingly, a bit vague as to what those biomarkers would actually be, speaking instead of “biomarkers reflecting underlying AD pathophysiological changes;” at one point it even says that “there is no consensus as to particular biomarkers that would be appropriate to support clinical findings in trials in early AD.” It is unclear as to how these assertions would be justified; the leading candidates were not only disclosed in the 2013 Guidance, but are widely accepted in the field: levels of beta-amyloid and aberrant tau in the cerebrospinal fluid, imaging of plaques (and, potentially, aberrant tau) in the brain using radiolabeled tracers, markers of neurodegeneration, and impaired metabolism.

Scientific leaders and advocates hailed the new Guidance document, and will continue to push for more progress in this direction toward a more damage-repair-friendly regulatory climate. At SENS Research Foundation, we advocate for an even wider goal: that such new thinking should be extended to other diseases and debilities of aging. The use of biomarkers and imaging directly reflecting the key cellular and molecular damage driving AD and other neurodegenerative diseases of aging should be extended to testing of therapies in people who are in even greater danger, showing early signs of cognitive problems but still not suffering from full-fledged dementia. Analogously, biomarkers of the cellular and molecular damage that accumulates in our tissues as we age and that drives other age-related illness and debility should also be acknowledged as the best targets for new therapies that would prevent, arrest, and reverse those conditions. And because AD and other diseases of aging involve multiple kinds of cellular and molecular damage, it’s critical that regulators allow the testing of combination therapies potentially capable of attacking multiple kinds of cellular and molecular aging damage, without first needing the constituent therapies to be tested individually.

This step could unleash a new wave of testing of one of the most fearsome and stubborn of aging’s plagues, and finally give us a way to banish it into an indefinite future.

Question Of The Month #16: Any Rejuvenation Relevance for Roundworm Reproduction?

Q: Press coverage of a recent study on the ability of the eggs (oocytes) of the roundworm C. elegans suggested that the researchers had discovered a new way that these cells clear out damaged proteins, and thereby “turn back time” and become “young again.” Is there some way that we could take advantage of this mechanism to remove the junk that accumulates inside cells as part of the degenerative aging process in humans?

A: The headlines were certainly provocative, no doubt emboldened by the paper’s opening line (“Although individuals age and die with time, an animal species can continue indefinitely, because of its immortal germ-cell lineage”[1]) and the fact that the senior author has made landmark contributions to understanding the genes that regulate the rate of aging. Some readers got the impression that this study had uncovered a special molecular mechanism that allows these roundworms’ oocytes uniquely to stay “young,” even as the body as a whole grew old. This impression may have been reinforced by a quote from one researcher, contrasting the aging of the human body with the (seeming) “immortality” of the germline (the “line” of sperm and egg genes that actually passes from generation to generation): “You take humans — they age two, three or four decades, and then they have a baby that’s brand new.”

Taken together, some readers came away with the suggestion that the fact that babies are born young implies the ability of oocytes to “sweep themselves clean” of their adult parents’ lifetime burden of deformed proteins, and excitedly hoped that the tricks that oocytes use to execute this feat could somehow be engineered into aging cells elsewhere in the body to keep our muscle and brain cells young.

Unfortunately, no such tricks emerged from this study, nor are they likely to. This study1 adds substantial insight to a body of work on roundworm (and later frog) oocyte biology sparked by a discovery made by French scientists in 2010[2] and prior work in yeast and in mouse embryos. However, there is nothing here that can be exploited for developing anti-aging therapies.

Off Again, On Again

The real finding of the paper is better captured by its own title than the newspaper headlines: “A lysosomal switch triggers proteostasis renewal in the immortal C. elegans germ lineage.” The key word in there is not “immortal,” but “renewal” — renewal of “proteostasis,” the somewhat equivocal concept of the young cell’s dynamic maintenance of stably low levels of damaged proteins. As it turns out, the “renewal” in question is a reactivation of the normal “proteostatic” activity of the lysosome — the cell’s recycling center, where old and damaged proteins are broken down into raw materials that can then be reused to build new proteins.

While oocytes are held in storage, they adopt a metabolically dormant state to conserve energy and reduce the production of metabolic wastes. This much is just as true in mammals as it is in the roundworms and frogs studied in this new report. What the new study uncovered is a particular energy-conservation strategy these animals’ oocytes use.

When there is no opportunity for fertilization, the oocytes of roundworms and frogs temporarily deactivate the energy-intensive molecular pumps that keep their lysosomes’ interiors acidic. This acidity is needed for lysosomes break down damaged proteins, so when some oocyte proteins become damaged (notably, via an alteration known as carbonylation), there’s no way for them to be degraded. Instead, these damaged proteins aggregate into clumps and slowly accumulate inside the cell.1

But things change suddenly as fertilization approaches. As a sperm draws near to an oocyte, the oocyte senses the sperm cell’s “major sperm proteins” (yes, that’s the term!), and jumps into action in preparation for fertilization. One of its first actions is to rapidly kick its lysosomes’ ion pumps into gear, awakening their ability break down the waste that’s accumulated while the oocyte was in waiting mode. Once acidified and ready to take in wastes again, the lysosomes actively reach out to nearby aggregated proteins, quickly cleaning house so that the oocyte is ready just in time for the big event.1

Being able to put off normal housekeeping in oocytes until they are just about to be fertilized means that no energy is wasted in maintaining oocytes that may not be fertilized for some time, and may never be fertilized at all. The authors also speculate that the rapid breakdown of the accumulated carbonylated proteins may even provide a source of energy for the oocyte, just in time for the energy-intensive processes that follow fertilization.1 But no special rejuvenative power is involved in this process: other cells clean up these same wastes routinely, as a matter of day-to-day housekeeping, instead of letting them build up until it’s absolutely necessary to get rid of them. You doubtless are familiar with similar patterns of housekeeping in your own circles. Both systems can work, especially in a household that doesn’t create much mess in the first place.

Indeed, part of what makes this system workable is that oocytes don’t generate much waste in their metabolically dormant state, so there’s little for them to clean up when the opportunity for fertilization comes around. Such metabolic dormancy also helps maintain the integrity of oocytes in mammals, although there’s no evidence yet that mammalian oocytes put lysosomal acidification on hold until they’re ready to be fertilized. By contrast, deactivation of the lysosome in cells with very high metabolic activity (like neurons and muscle cells) would be disastrous, because such cells produce much higher levels of waste, and are accordingly already vulnerable to the accumulation of much tougher aggregates even with their lysomes working full-time.

Similarly, the very low metabolic activity of oocytes compared to cells in the rest of the body (especially as compared with highly energy-demanding tissues like muscle and brain) reduces the rate at which mitochondria generate free radicals during energy generation, and thus the rate of formation of mitochondrial mutations and other aging damage. Obviously, our lives would not be possible if we spent our days with energy-intensive tissues like brain and muscle in a similar state of dormancy.

Dying that Others May Live

Coming back to the question of the so-called “immortality” of the germ line: an important feature of many of the mechanisms that keep the germ line clear of major mitochondrial mutations and other aging damage is that they come at the expense of the death of many individual oocytes, which are ruthlessly culled in order to avoid passing such damage on to the next generation. When mitochondrial mutations do occur in aging oocytes despite the protective effect of low mitochondrial respiration, their appearance drives both the programmed cell death of individual oocytes and the culling (atresia) of entire follicles that release new oocytes from the ovaries.[3],[4]

This culling process prevents oocytes dominated by mitochondrial mutations from being fertilized and developing into babies with terrible inherited mitochondrial disease (though tragically, even this mechanism sometimes fails). But it also drives the eventual exhaustion of a woman’s reserve of follicles (and thus, oocytes) with age.3 A woman is born with 1-2 million ovarian follicles; by the time she reaches puberty, she is already down to just 300,000 —  and despite the fact that she only loses one (or a few) eggs per month to ovulation, only a few hundred of the healthiest of them remain when a woman is in her fifties or sixties. Once the ovaries are unable to respond to the hormonal signal to ovulate, this lack of response triggers the hormonal chaos of menopause, and all the age-related problems associated with it.

Programmed cell death also occurs throughout the body, just as it does in oocytes, as individual cells that have suffered mutations or other damage that puts them at risk of turning cancerous sacrifice themselves to protect the body from their own deadly predilection. But we can’t afford to “solve” the rising burden of mitochondrial mutations and other aging damage in our brain neurons and muscle cells with age by killing off even more such cells as soon as they suffer such damage.

The Reality of Reproductive Aging

Despite the lengths to which the body goes to maintain only viable, “young” eggs, oocytes do still manage to degenerate with age, which is part of the reason why older parents are less fertile (rising numbers of the sperm and eggs become duds), have more miscarriages (in addition to problems in the ability of older mothers’ bodies to support the developing embryo, the embryo itself is more likely to inherit fatal flaws acquired in the aging sperm and egg), and are more at risk for birth defects. The example you probably know about already is the increasing risk of congenital abnormalities as women reach middle age, most especially Down syndrome. These increased risks are driven by the accumulation of abnormalities in the oocytes, such as aneuploidy — an abnormal number of chromosomes in the cell.

Mitochondrial free radicals also contribute to abnormalities in organelles and other constituents of aging oocytes.2 Calorie restriction (CR) reduces the rate of accumulation of mitochondrial and chromosomal damage in oocytes in laboratory rodents,[5] consistent with its known ability to lower the rate of production of mitochondrial free radicals and to retard the aging process in many species (although with uncertain effects in primates). In fact, CR is so effective at preserving oocytes and other parts of the female rodent reproductive system against aging damage “that ovulated oocytes of aged female mice previously maintained on CR … are comparable to those of young females during prime reproductive life.”5 When mice are started on CR around the time they first become fertile and then returned to a conventional diet just as the last of littermates kept on a lifelong conventional diet are losing fertility, animals previously kept on CR are able to keep delivering viable pups for much of the remaining lifespan, and do so in higher numbers and with greater offspring survival rates than conventionally-fed animals half their age.[6]

And it isn’t just women whose reproductive systems are ravaged by the degenerative aging process. While men don’t suffer the sudden and dramatic loss of fertility that women do with age, and while congenital diseases are less often directly attributable to the aging of sperm than to the aging of eggs, the fact remains that sperm cells from middle-aged and older exhibit a wide range of abnormalities, and that embryos of older fathers are more likely to spontaneously abort or to suffer genetic disorders — many, again, caused by aneuploidy of the sperm, and also by mutations and by abnormalities in the structure of individual chromosomes.[7]

The silver lining in all of this bad news: because the nature of the degenerative aging process is not different from the aging of the rest of the body at the cellular and molecular level, the “damage-repair” heuristic of rejuvenation biotechnology can be applied to rejuvenate the aging reproductive system just as it can to the rejuvenation of the rest of our bodies.

Real Damage Repair

As you can see, we’re not going to solve the degenerative aging process by borrowing any special tricks from the oocyte.[8] The oocyte doesn’t really have any tricks for us to profitably exploit — and more importantly, no cell in the body is naturally able to remove or repair many of the kinds of damage that accumulate in aging bodies and ultimately lead to age-related disease, debility, and death. The oocyte has no way to clear beta-amyloid from aging brains, or TTR amyloid from aging hearts — nor to cleave AGE crosslinks from aging arteries, to none of which damage they are subject. It has no internal means to replace cells that are lost to aging damage,8 and is no more able to degrade the truly stubborn intracellular aggregates that accumulate in aging cells than any other cell type.

For that, we need a new class of medicines that can do what we can’t do on our own: remove, repair, replace, or render harmless the cellular and molecular damage of aging in our tissues. It is when we develop rejuvenation biotechnologies and deploy them comprehensively that we will finally be able to effectively “turn back time” for aging bodies as a whole.

References

  1. Bohnert KA, Kenyon C. A lysosomal switch triggers proteostasis renewal in the immortal C. elegans germ lineage. Nature. 2017 Nov 30;551(7682):629-633. doi: 10.1038/nature24620. Epub 2017 Nov 22. PubMed PMID: 29168500.
  2. Goudeau J, Aguilaniu H. Carbonylated proteins are eliminated during reproduction in C. elegans. Aging Cell. 2010 Dec;9(6):991-1003. doi: 10.1111/j.1474-9726.2010.00625.x. Epub 2010 Oct 29. PubMed PMID: 21040398.
  3. May-Panloup P, Boucret L, Chao de la Barca JM, Desquiret-Dumas V, Ferré-L’Hotellier V, Morinière C, Descamps P, Procaccio V, Reynier P. Ovarian ageing: the role of mitochondria in oocytes and follicles. Hum Reprod Update. 2016 Nov;22(6):725-743. Epub 2016 Aug 25. Review. PubMed PMID: 27562289.
  4. Ramalho-Santos J, Varum S, Amaral S, Mota PC, Sousa AP, Amaral A. Mitochondrial functionality in reproduction: from gonads and gametes to embryos and embryonic stem cells. Hum Reprod Update. 2009 Sep-Oct;15(5):553-72. doi: 10.1093/humupd/dmp016. Epub 2009 May 4. Review. PubMed PMID: 19414527.
  5. Selesniemi K, Lee HJ, Muhlhauser A, Tilly JL. Prevention of maternal aging-associated oocyte aneuploidy and meiotic spindle defects in mice by dietary and genetic strategies. Proc Natl Acad Sci U S A. 2011 Jul 26;108(30):12319-24. doi: 10.1073/pnas.1018793108. Epub 2011 Jul 5. PubMed PMID: 21730149; PubMed Central PMCID: PMC3145697.
  6. Selesniemi K, Lee HJ, Tilly JL. Moderate caloric restriction initiated in rodents during adulthood sustains function of the female reproductive axis into advanced chronological age. Aging Cell. 2008 Oct;7(5):622-9. doi: 10.1111/j.1474-9726.2008.00409.x. Epub 2008 Jul 24. PubMed PMID: 18549458; PubMed Central PMCID: PMC2990913.
  7. Amaral S, Amaral A, Ramalho-Santos J. Aging and male reproductive function: a mitochondrial perspective. Front Biosci (Schol Ed). 2013 Jan 1;5:181-97. Review. PubMed PMID: 23277044.
  8. The one pseudo-exception is the use of oocytes to engineer pluripotent stem cells that are a perfect match for the patient, in the process known as somatic cell nuclear transfer or “therapeutic cloning.”

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