Can a Useful Biomarker of Aging be Built from Very Simple Measures?

There is considerable interest in the research community in the construction of a low-cost, reliable biomarker of biological age. The intent is to use such a test immediately before and after the application of a potential rejuvenation therapy to establish how well it worked. It must therefore accurately assess overall health, mortality risk, and remaining life expectancy. Currently DNA methylation assays are a leading approach to the creation of a robust biomarker of aging, as some portions of the changing pattern of DNA methylation are a fairly good reflection of cellular reactions to the damage and decline of aging. Is it possible to produce something far less complicated, however, a biomarker that uses only existing measures of health, but that is nonetheless good enough to evaluate near future rejuvenation therapies? This is an open question, one that can be argued either way.

Mammalian aging is characterized by a gradual decline of numerous health parameters with multiple biochemical, physiological and behavioral manifestations. Several animal models have been successfully used over the last several decades to address mechanistic aspects of aging and development of age-related diseases. In most of these studies the major metric parameter for assessing pro-/anti-aging effect of genetic, nutritional or pharmacological manipulation has been the animals' lifespan. While being informative, longevity by itself however, cannot provide an assessment of the animal's health status, which, like in humans, can significantly decline at older ages and therefore reduce the quality of life. This concern is particularly relevant to research focused on developing the "healthspan"-extending pharmaceuticals, efficacy of which may not be necessarily translated in increased longevity but rather in prolongation of healthy life and require quantitative objective assessment.

Clinical studies in humans measure age-related declines in performance by quantifying the frailty index (FI), which reflects accumulation of health deficits during chronological aging. Since numerous studies have shown that many age-associated changes that occur in humans are also present in aged mice, FI was recently introduced as a measure of mouse aging to pre-clinical models. However, standardized and comprehensive approaches for FI measurements, which will address changes in a broad spectrum of physiological functions, are still missing. Here we describe the development of an alternative scoring system, based on a selected set of non-invasive quantitative and physiological parameters, which could be repetitively used in the same animal over the course of its entire lifespan. We refer to this set of parameters as physiological frailty index (PFI). After measuring 29 diverse parameters including physical (body weight and grip strength), blood cell composition, metabolic, and immune properties, we selected those that show statistically significant change with age. These parameters were used to create PFI of individual mice of different chronological age. The observed gradual increase in mean PFI values with age suggests that our approach can reliably detect the scale of age-dependent health deterioration in a quantitative manner.

We also validated our approach of PFI by testing detrimental (feeding high fat diet, HFD) and beneficial (treatment with mTOR inhibitor rapamycin) factors on animals' longevity. We demonstrated acceleration of growth of PFI in animals placed on a high fat diet, reflecting aging acceleration by obesity. Additionally, we showed that PFI could reveal the anti-aging effect of mTOR inhibitor rapatar (a bioavailable formulation of rapamycin) prior to registration of its effects on longevity. PFI also revealed substantial sex-related differences in normal chronological aging and in the efficacy of detrimental (high fat diet) or beneficial (rapatar) aging modulatory factors.


Calico Partners to Obtain Protein Degradation Technology

For those who like reading the Calico tea leaves, here are a few details on one of their recent partnerships. Calico, the California Life Company, is the aging research venture funded by Google. It launched a few years back, but so far those involved appear to be doing nothing particularly radical, insofar as we know anything about what is going on there. Calico is certainly not supporting the SENS view of damage repair as the best way to treat aging, and may well be turn out to be simply a larger and more secretive version of the Ellison Medical Foundation in the end: an expansion of the largely investigative work already taking place at the NIA, undertaking no projects with the potential to make a large difference to the course of aging in humans. More research is always better than less research, of course, but nonetheless this has grown to have the look of another missed opportunity to add to the recent history of aging research.

Here, Calico is partnering to obtain access to a technology that could be turned to ways to adjust the level of any one or any few of the proteins present in a cell. The approach works by harnessing one of the cell's established recycling mechanisms. This might be intended as an alternative to methods such as RNA interference for use in adjusting cellular operation. The goal is to tinker with the switches and dials of metabolism, all of which are influenced or determined by levels of specific proteins, in order to test approaches that might slightly slow aging by reducing the pace at which damage accumulates. More positively, it might be turned to degrading forms of metabolic waste that cause aging, though beyond amyloid and Alzheimer's disease, there is little sign that Calico researchers are interested in the list of waste compounds outlined in the SENS rejuvenation research proposals, such as cross-links, lipofusin, and so forth.

C4 Therapeutics (C4T) and Calico today announced a five-year collaboration to discover, develop, and commercialize therapies for treating diseases of aging, including cancer. Under the terms of the agreement, the parties will leverage C4T's expertise and capabilities in targeted protein degradation to jointly discover and advance small molecule protein degraders as therapeutic agents to remove certain disease-causing proteins. The partnership will pursue preclinical research and Calico will be responsible for subsequent clinical development and commercialization of resulting products that may emerge from the collaboration.

"We know from decades of translational research that it can be incredibly challenging to find effective pharmacologic inhibitors of many of the biologically well-validated targets, particularly in cancer. Through the alternative strategy of specifically targeting such proteins for degradation, we believe we have the opportunity to identify promising new therapeutics in cancer and in other diseases as well. We're looking forward to collaborating with C4T's scientists and applying their protein degradation technology to the discovery and development of effective new treatments."

C4 Therapeutics is a private biotechnology company developing a new class of drugs based on Targeted Protein Degradation (TPD) to address a broad range of life-threatening and life-impairing diseases. C4T's platform uses small molecule drugs to direct the machinery of the ubiquitin-proteasome system to selectively degrade disease-relevant proteins for therapeutic benefit. This distinctive mechanism provides new opportunities to target traditionally difficult-to-treat diseases and diseases plagued by drug resistance.


What Next for UNITY Biotechnology?

What follows here is an inside baseball discussion relating to the companies working on senolytic therapies, biotechnologies capable of selectively destroying senescent cells. The presence of these cells is one of the causes of aging and age-related disease, and their removal is the first of a number of rejuvenation therapies based on the SENS vision that will emerge over the next few decades. Human trials of the first senolytics will be starting this year and next, and by the mid-2020s most people in the wealthier parts of the world will have the opportunity to remove this part of the burden of aging. This is a wondrous development: based on research to date, removing senescent cells from old individuals is a robust and reliable way to turn back the clock on many measures of aging and markers of age-related disease.

I should preface the rest of this post by noting that competition in the marketplace is a great thing, but only because some investors suffer meaningful losses. The threat of loss is necessary to the alchemy by which self-interest is turned into altruism. Only competition with real penalties for failure can drive the faster progress that benefits everyone. Yet regardless of who wins or loses in terms of the value of their shares, we all win when reasonably priced senolytic therapies become widely accessible. In that sense, investing in credible ventures aiming at the production of rejuvenation therapies is a great opportunity: even failure contributes to progress, and the outcome in the end is that we are all better off. Someone achieves the goal, someone deploys the treatment.

UNITY Biotechnology is presently at the head of the current crop of companies focused on treating aging and age-related disease through the clearance of senescent cells. They hold the leading position by virtue of the involvement of the principal research groups in the field, having big names from the pharmaceutical and biotech field running the show, and having recently raised more than $100 million to push the first of these therapies through the US regulatory progress and into the clinic. Yet I can't say as I think that their position is as enviable and commanding as it might first appear to be. From a competitive point of view, they actually have very little going for them at the moment aside from that war chest and the credibility it took to raise it.

Having made that statement, I should defend my position. The chief problem I see for UNITY is one of technology. They are taking a small molecule drug approach within the current regulatory system, and the currently available stable of senolytic drug candidates with which they entered the picture are chemotherapeutics with significant side-effects. Other drug candidates are emerging quite rapidly in the research community, some of which may have far fewer side-effects, but switching would mean starting fresh, or licensing fresh from current owners. It might also mean moving from a well-characterized drug with excellent pharmacology data, such as navitoclax, to a drug that still needs that data established. The situation is actually worse than this, however. Competitors with far better senescent cell clearance technologies, approaches with essentially zero side-effects, are emerging at the rate of one every year or so. Oisin Biotechnologies was the first, using a programmable gene therapy approach, and just this week another group announced their intent to form a company to develop their FOXO4-p53 interference method.

UNITY didn't emerge from thin air, and their precursor company does have a variety of patents and experience in trying to get immunotherapies and engineered viruses to work as senolytic treatments. They had plenty of time to try to get that to work and did not achieve those goals. With what is now a great deal of funding in comparison to the past, they could go back and try to make one of those approaches work. There is a great deal of uncertainty in that sort of endeavor, however. They should and no doubt will turn some of their funding to longer-term technology development with perhaps a five-year horizon, but when you take as much venture funding as this company has, the clock ticks very aggressively. They have to build a multi-billion-dollar valuation company pretty quickly, within the next couple of years. That has to be done on the basis of human trials starting right about now, since those clinical trials will take a year or so to run from idea to publication of results.

Another threat is that of medical tourism. Not everyone in this global industry is going to care about the opinions of the FDA. Given that existing senolytic drugs, and many of the new ones, can be purchased from established suppliers, there really is little to stop a large industry of medical tourism springing up for the very same drugs that UNITY is trying to put through the system. Or at the very least for drugs that are similar enough in effectiveness and side-effects. That will hamper UNITY's ability to charge regulatory capture prices; it is harder to do that when people can just go to Mexico at a tenth or less of the US price. That in turn will harm their valuation and ability to raise further funding needed to run treatments through the FDA gauntlet.

A final consideration is that everything UNITY spends money on today helps their competitors just as much as it helps them. One might argue that the big UNITY war chest is really largely a charity fund for industry development. Being a competitor to UNITY is one of the greatest places to be in modern for-profit biotechnology. They are doing all the work to prove out the industry, raise its profile, and demonstrate with ever-better clarity that targeted destruction of senescent cells successfully treats aging. They are doing more than their part to set a high initial valuation for any other new company with a credible technology for targeting senescent cells. This is all wonderful from the point of view of anyone waiting for the end result to emerge in clinics, but pretty terrible for UNITY from a competitive point of view. They'll be hip-deep in highly effective competing companies come this time in 2019.

As I see, it the UNITY management has a few options when it comes to strategy. Firstly they can forge ahead in the hope that regulatory barriers are good enough to allow a large valuation based on approval for a chemotherapeutic that is (a) inferior to a range of other treatments only a little behind in time to market, and (b) also available on the open market for medical tourism. Secondly they can couple that approach with significant investment in development of new drugs that can be patented, variants of those already discovered. These two are more or less the standard playbook for a new pharmaceutical entity, so they may well do this and only this. If they do, I think that their competitors, already equipped with far superior products, will eat their lunch over the next few years, however. The third option is to continue to prove out the market, make life easy for competitors, run the first trials, pull in another even larger round of funding at some ridiculous valuation, and then use those funds to buy the best of the crop of competitors, solving the technology problem.

This last option seems plausible, and is not uncommmon. I think it quite likely that UNITY will kick off their chemotherapeutic trials, publish the promising initial results while downplaying the side-effects, raise series C in 2018, and then buy whichever of the young companies in the space with a better senolytic technology wants to sell for a quick turnaround. Despite the enormous size of the target market here, ultimately every adult human much over the age of 30 buying a treatment every few years, not every entrepreneur wants to spend a decade fighting the FDA to make progress towards a narrow, limit use of their therapy. A quick win and sudden wealth is a strong temptation; anyone starting up a senescence-focused biotech company these days will have an acquisition by UNITY somewhere in mind as an option - as will the investors who back those companies.

Considering Uncoupling in Calorie Restriction Mimetics

This open access paper on mitochondrial function considers the mechanism of uncoupling in calorie restriction and in drugs that seek to emulate some of the benefits produced by calorie restriction, known as calorie restriction mimetics. Mitochondria generate energy stores for use in cells, but with greater uncoupling that effort creates heat instead. This is a part of the normal process of body temperature regulation in mammals. However, uncoupling also changes the output of reactive oxygen species (ROS) from the mitochondria, a feature observed in the methods shown to modestly slow aging in short-lived species. Mitochondrially generated ROS are both a signal that spurs greater cellular housekeeping and a source of damage, so either somewhat more or somewhat less than the usual output might be beneficial.

There are drugs known to reliably produce greater mitochondrial uncoupling, but there has little development of their use as therapeutics for aging, even now that the research community has more enthusiasm for the goal of slowing aging via pharmaceuticals. This is possibly because unbounded increases in uncoupling via drug administration are fairly dangerous: too much is potentially lethal due to raised body temperature and harmful effects on mitochondrial biochemistry. Since this lack of safety at the higher end is an inherent feature of uncoupling in mammals, it may well be the case that direct intervention in the uncoupling process will remain less desirable in comparison to the range of other potential approaches to modestly slow aging in humans. That said, the researchers here point out a family of self-limiting uncouplers that may not exhibit this problem; we shall see how it goes in the years ahead.

Caloric restriction (CR) is the best-studied and most reliable way to increase lifespan. CR affects most of experimental model organisms, from unicellular ones to mammals. Signaling cascades responsible for the effects of CR were studied in detail at the cellular level as well as at the levels of tissues and the whole organisms. Increasing levels of AMP and NAD+, which activates deacetylases, were shown to be the key factors initiating these cascades at the cellular level. One could expect that under the conditions of CR the cells attempt to save energy. Many cellular changes indeed make bioenergetics more economical: CR decreases the rate of protein biosynthesis and activates autophagy. It would be natural to presume that CR also raises the efficiency of mitochondrial energy production, i.e. that it increases the coupling of respiration and oxidative phosphorylation. However the opposite appears to take place.

It has been shown that mice under CR conditions accumulate UCP proteins (uncoupling proteins) in their muscle mitochondria. UCP proteins catalyze an electrogenic process of transporting the dissociated forms of free fatty acids from the inner to the outer layers of mitochondrial inner membrane. In the outer layer the free fatty acids are protonated, and then in the neutral form return to the inner layer. As this decreases the level of the transmembrane potential, the proteins of the UCP family act as natural uncouplers of respiration and oxidative phosphorylation. Indeed, during starvation there is simultaneous accumulation of UCP2 and UCP3, and a decrease a decrease in the efficiency of oxidative phosphorylation. What is the physiological role of uncoupling activation upon CR? On one hand, CR induces mitochondrial biogenesis and respiration. On the other hand, it has been shown that mitochondrial hyperpolarization can induce a strong increase in ROS (reactive oxygen species) generation. Probably, the increased expression of UCP is an "insurance" against the oxidative stress caused by mitochondrial hyperpolarization.

There is probably another advantage of using uncouplers as CR mimetics. As aforementioned, increase in NAD+ levels is one of the best-studied ways of geroprotection. There are many works showing that increasing NAD+ concentration via activation of its biosynthesis leads to lifespan increase in experimental animals. Importantly, in terms of lifespan increase in the sum of NAD+ and NADH concentrations is less relevant than the concentration of the oxidized form. At the same time, reduction of NAD+ to NADH takes place during cellular catabolic reactions (glycolysis and TCA cycle). Therefore, interfering with cell metabolism could be an efficient way of increasing NAD+ concentration. The addition of the uncoupler FCCP at low concentration has been shown to increase ATP level in neurons due to a compensatory response to a temporal depolarization. Earlier, it has been suggested that a slight decrease in the transmembrane potential can prevent the reaction of one-electron reduction of oxygen, which leads to ROS formation. At the same time, such a decrease may not affect the rate of ATP synthesis; thus, such uncoupling was called "mild". In other words, mild uncoupling is aimed at stimulating NAD+-dependent processes rather than at stimulation of AMPK.

What level of uncoupling is most suitable for the purposes of geroprotection? As mentioned, a small decrease in proton resistance of the strongly energized mitochondrial membranes can induce a significant decrease in ROS production and an increase in NAD+/NADH ratio without affecting ATP concentration. According to our line of reasoning, such level of uncoupling combined with AMPK activation is sufficient for efficient interference with the aging process. Theoretically, one could consider a higher level of uncoupling leading to a strong depolarization of the membranes and, as a consequence, a significant increase in ADP/ATP ratio. Apparently, such treatment could lead to a lethal deenergization of cells. Therefore, a relatively weak level of uncoupling seems to be preferential.

Which uncouplers should be used? Probably, the anionic compound dinitrophenol is the best-studied uncoupler in terms of its effects on mammalian physiology. In particular, it has been used on humans as a weight loss treatment. However, it was reported that its use was accompanied by a set of negative side effects. Recently, we reported uncoupling activity of a unique type of chemical compounds - lipophilic penetrating cations. Most of the studies on such compounds were performed on dodecyltriphenylphosphonium (C12TPP). A potential advantage of using such compounds is that their mitochondrial accumulation is proportional to the level of the transmembrane potential. For this reason, penetrating cations affect highly polarized mitochondria to greater extent than mitochondria with relatively low potential levels. In other words, they cause self-limiting (mild) uncoupling.


NAD Precursor NMN Improves DNA Repair in Mice

Sirtuin research, much hyped and in the end producing nothing other than more knowledge of metabolism, has somewhat transitioned into a focus on nicotinamide adenine dinucleotide (NAD) these days. NAD is central in cellular energy production and mitochondrial activity, and appears involved in many of the same processes that sirtuins influence. It is still the case that compelling demonstrations of slowed aging or enhanced longevity in laboratory animals have yet to emerge from this line of research, such as via the use of the NAD precursor nicotinamide mononucleotide (NMN) as a dietary supplement. The research is academically interesting, as here where it is shown to affect DNA repair mechanisms, but from a practical point of view for the treatment of aging this still appears to be another marginal approach, lacking the ability to produce reliable and significant effects on aging and longevity.

DNA repair is essential for cell vitality, cell survival and cancer prevention, yet cells' ability to patch up damaged DNA declines with age for reasons not fully understood. New findings offer an insight into how and why the body's ability to fix DNA dwindles over time and point to a previously unknown role for the signaling molecule NAD as a key regulator of protein-to-protein interactions in DNA repair. If affirmed in further animal studies and in humans, the findings can help pave the way to therapies that prevent DNA damage associated with aging and with cancer treatments that involve radiation exposure and some types of chemotherapy, which along with killing tumors can cause considerable DNA damage in healthy cells. Human trials with NMN are expected to begin within six months, the researchers said.

The investigators started by looking at a cast of proteins and molecules suspected to play a part in the cellular aging process. Some of them were well-known characters, others more enigmatic figures. The researchers already knew that NAD, which declines steadily with age, boosts the activity of the SIRT1 protein, which delays aging and extends life in yeast, flies and mice. Both SIRT1 and PARP1, a protein known to control DNA repair, consume NAD in their work. Another protein DBC1, one of the most abundant proteins in humans and found across life forms from bacteria to plants and animals, was a far murkier presence. Because DBC1 was previously shown to inhibit vitality-boosting SIRT1, the researchers suspected DBC1 may also somehow interact with PARP1, given the similar roles PARP1 and SIRT1 play.

To get a better sense of the chemical relationship among the three proteins, the scientists measured the molecular markers of protein-to-protein interaction inside human kidney cells. DBC1 and PARP1 bound powerfully to each other. However, when NAD levels increased, that bond was disrupted. The more NAD present inside cells, the fewer molecular bonds PARP1 and DBC1 could form. When researchers inhibited NAD, the number of PARP1-DBC1 bonds went up. In other words, when NAD is plentiful, it prevents DBC1 from binding to PARP1 and meddling with its ability to mend damaged DNA. What this suggests is that as NAD declines with age, fewer and fewer NAD molecules are around to stop the harmful interaction between DBC1 and PARP1. The result: DNA breaks go unrepaired and, as these breaks accumulate over time, precipitate cell damage, cell mutations, cell death and loss of organ function.

Next, to understand how exactly NAD prevents DBC1 from binding to PARP1, the team homed in on a region of DBC1 known as a Nudix homology domain (NHD), a pocket-like structure found in some 80,000 proteins across life forms and species whose function has eluded scientists. The team's experiments showed that NHD is an NAD binding site and that in DBC1, NAD blocks this specific region to prevent DBC1 from locking in with PARP1 and interfering with DNA repair. To determine how the proteins interacted beyond the lab dish and in living organisms, the researchers treated young and old mice with the NAD precursor NMN, which makes up half of an NAD molecule. NAD is too large to cross the cell membrane, but NMN can easily slip across it. Once inside the cell, NMN binds to another NMN molecule to form NAD. As expected, old mice had lower levels of NAD in their livers, lower levels of PARP1 and a greater number of PARP1 with DBC1 stuck to their backs.

However, after receiving NMN with their drinking water for a week, old mice showed marked differences both in NAD levels and PARP1 activity. NAD levels in the livers of old mice shot up to levels similar to those seen in younger mice. The cells of mice treated with NMN also showed increased PARP1 activity and fewer PARP1 and DBC1 molecules binding together. The animals also showed a decline in molecular markers that signal DNA damage. In a final step, scientists exposed mice to DNA-damaging radiation. Cells of animals pre-treated with NMN showed lower levels of DNA damage. Such mice also didn't exhibit the typical radiation-induced aberrations in blood counts, such as altered white cell counts and changes in lymphocyte and hemoglobin levels. The protective effect was seen even in mice treated with NMN after radiation exposure.


Selective Destruction of Senescent Cells by Interfering in FOXO4-P53 Crosstalk

Today, another research group announced their entry to the field of senescent cell clearance as a means to treat aging, along with the intent to commercialize their novel method of achieving selective destruction of senescent cells in aged individuals. Senescent cells accumulate with age as a result of the normal operation of living tissues: cells become senescent when damaged or when they reach the Hayflick limit on replication. Near all are destroyed, either through the programmed cell death mechanism of apoptosis, or by immune cells attracted by the signal molecules generated by senescent cells. Unfortunately, some linger, resistant. The number of these cells grows over the years, and the signals they generate start to create harmful outcomes in nearby cells and tissue structures, and in addition spur rising levels of chronic inflammation. The increasing presence of senescent cells is one of the root causes of degenerative aging and directly contributes to many specific age-related diseases.

The best and most direct approach to the phenomenon of cellular senescence is to periodically destroy these cells, reducing their numbers to the greatest extent possible. These numbers are never enormous, perhaps a few percent of most tissues in late old age, depending on the details. Removal can proceed as slowly as needed to be safe in older individuals if there are risks of lysis side-effects due to the amount of cell debris generated by senescent cell destruction. While senescence has short-term roles to play in tumor suppression, by shutting down the ability to replicate in potentially cancerous cells, and in wound healing, these cells have no clear and evident long-term use in the body. So a treatment that gets rid of near all of these cells, undergone once every few years, would in fact be a narrow means of rejuvenation. It would make aged tissues less aged. This has been demonstrated in studies of senescent cell removal showing life extension in mice, as well as those that have demonstrated specific improvements and reversals in the pathology of various age-related diseases and aged tissues.

The methodology developed by the researchers noted here is, at the very high level, analogous to that involved in some of the senolytic drug candidates evaluated to date - though it has the merit of having far fewer side-effects per this report. It involves sabotaging one of the mechanisms that lingering senescent cells use in order to resist the fall into apoptosis, but which in normal cells has no important role to play. Thus drug molecules can be delivered everywhere, and will only produce significant effects in cells that are senescent. In the case of drugs like navitoclax, that mechanism involves inhibiting bcl-2 family proteins. The mechanism here is quite different, involving FOXO4's influence on p53, but I wouldn't be surprised to see it turn out to be a part of the same system of inhibition of apoptosis. Almost all cellular mechanisms can be influenced in many ways, by tinkering with the activities and actions of many directly and indirectly involved proteins, and it is not unusual for research groups initially working on a diverse set of proteins to find that they end up in the same place at the end of the day.

Peptide targeting senescent cells restores stamina, fur, and kidney function in old mice

Regular infusions of a peptide that can selectively seek out and destroy broken-down cells that hamper proper tissue renewal, called senescent cells, showed evidence of improving healthspan in naturally-aged mice and mice genetically engineered to rapidly age. The peptide took over four years of trial and error to develop and builds on nearly a decade of research investigating vulnerabilities in senescent cells as a therapeutic option to combat some aspects of aging. It works by blocking the ability of a protein implicated in senescence, FOXO4, to tell another protein, p53, not to cause the cell to self-destruct. By interfering with the FOXO4-p53 crosstalk, the peptide causes senescent cells to go through apoptosis, or cell suicide. "Only in senescent cells does this peptide cause cell death. We treated mice for over 10 months, giving them infusions of the peptide three times a week, and we didn't see any obvious side effects. FOXO4 is barely expressed in non-senescent cells, so that makes the peptide interesting as the FOXO4-p53 interaction is especially relevant to senescent cells, but not normal cells."

Results appeared at different times over the course of treatment. Fast-aging mice with patches of missing fur began to recover their coats after 10 days. After about three weeks, fitness benefits began to show, with older mice running double the distance of their counterparts who did not receive the peptide. A month after treatment, aged mice showed an increase in markers indicating healthy kidney function. "The common thread I see for the future of anti-aging research is that there are three fronts in which we can improve: The prevention of cellular damage and senescence, safe therapeutic removal of senescent cells, to stimulate stem cells - no matter the strategy - to improve tissue regeneration once senescence is removed."

Targeted Apoptosis of Senescent Cells Restores Tissue Homeostasis in Response to Chemotoxicity and Aging

To identify potential pivots in senescent cell viability, we initiated this study by investigating whether apoptosis-related pathways are altered in senescent cells. We performed unbiased RNA sequencing on samples of genomically stable primary human IMR90 fibroblasts and IMR90 induced to senesce by ionizing radiation (IR). As senescent cells are reportedly apoptosis-resistant, we expected pro-apoptotic genes to be repressed. Surprisingly, however, senescent IMR90 showed an upregulation of prominent pro-apoptotic "initiators" PUMA and BIM while the anti-apoptotic "guardian" BCL-2 was reduced. This suggested senescent IMR90 are primed to undergo apoptosis but that the execution of the death program is restrained. We reasoned such a brake could potentially be a transcriptional regulator and focused on transcription factors that have previously been linked to apoptosis, including STAT1, 2, and 4; RELB; NFκB; TP53; and FOXO4.

Interference with JAK-STAT signaling is known not to affect the viability of senescent cells, and we have previously observed similar effects for NFκB and p53 inhibition. Our interest was therefore directed to a factor that has not yet been studied as such, FOXO4. FOXO4 belongs to a larger mammalian family, with FOXO1 and 3 being its major siblings. FOXOs are well studied in aging and tissue homeostasis as targets of insulin/IGF signaling and as regulators of reactive oxygen species. Whereas senescence-inducing IR showed only mild effects on the expression of FOXO1 and 3, both FOXO4 mRNA and protein expression progressively increased. We therefore wondered whether FOXO4 could function to balance senescence and apoptosis. We stably inhibited FOXO4 expression using lentiviral shRNA. FOXO4 inhibition prior to senescence-induction resulted in a release of mitochondrial cytochrome C and BAX/BAK-dependent caspase-3 cleavage. In addition, FOXO4 inhibition in cells that were already senescent, but not their control counterparts, reduced viability and cell density. Together, these show that after acute damage FOXO4 favors senescence over apoptosis and maintains viability of senescent cells by repressing their apoptosis response.

Research on peptide chemistry has shown that protein domains containing natural L-peptides can sometimes be mimicked by using D-amino acids in a retro-reversed sequence. Modification of peptides to such a D-retro inverso (DRI)-isoform can render peptides new chemical properties, which may improve their potency. As a cell penetrating peptide the D-retro inverso (DRI)-isoform of FOXO4, FOXO4-DRI, differs from other senolytic compounds by being designed around a specific amino acid sequence in a molecular target only mildly expressed in most normal tissues. Though a more thorough analysis is required, at least as far as tested here FOXO4-DRI appears to be well tolerated, which is an absolutely critical milestone to pass when aiming to treat relatively healthy aged individuals.

Reviewing What is Known of the Aging of Stem Cells

One of the important contributions to the aging process is a progressive reduction in stem cell activity. The majority of tissues in the body are in a constant process of turnover. The somatic cells making up the bulk of all tissues reach the Hayflick limit on replication and self-destruct, and are replaced by new cells generated by tissue-specific stem cell populations. With age, these stem cells spend ever more time quiescent, and thus the supply of new somatic cells declines, causing tissues and organs to deteriorate and ultimately fail. This loss of stem cell support is thought to have evolved as part of a balance between risk of death by cancer versus risk of death through failing tissues. As cells become more damaged with age, the risk of cancer with cell activity increases. Lower cells of stem cell activity dampen that risk somewhat, at the cost of a slower decline into frailty and disease. Still, restoration of youthful stem cell activity is one necessary component of any future toolkit of rejuvenation therapies. To the degree that this raises cancer risk, that is an additional challenge to overcome along the way, not a reason to stand back and do nothing.

Aging is an unavoidable physiological consequence of the living animals. Mammalian aging is mediated by the complex cellular and organismal processes, driven by diverse acquired and genetic factors. Aging is among the greatest known risk factors for most human diseases, and of roughly 150,000 people who die each day across the globe, about two thirds die from age-related causes. In the modern era, one of the emerging fields in medicine is stem cell research, as stem cells have the remarkable potential for use to treat a wide range of diseases. Stem cells are undifferentiated pluripotent cells that can give rise to all tissue types and serve as a sort of internal repair system. Until the recent advance in development of induced pluripotent stem cells (iPSCs), scientists primarily worked with two kinds of pluripotent stem cells from animals and humans: embryonic stem cells, which are isolated from the inner cell mass of blastocysts, and non-embryonic "somatic" or "adult" stem cells, which are found in various tissues.

Although stem cell science promises to offer revolutionary new ways of treating diseases, it is identified that aging affects the ability of stem (and progenitor) cells to function properly, which ultimately can lead to cell death (apoptosis), senescence (loss of a cell's power of division and growth), or loss of regenerative potential. Aging may also shift gene functions, as reported for some genes, such as p53 and mammalian target of rapamycin (mTOR), which are beneficial in early life, but becomes detrimental later in life. In this regard, a novel theory, namely the "stem cell theory of aging", has been formulated, and it assumes that inability of various types of pluripotent stem cells to continue to replenish the tissues of an organism with sufficient numbers of appropriate functional differentiated cell types capable of maintaining that tissue's (or organ's) original function is in large part responsible for the aging process.

In addition, aging also compromises the therapeutic potentials of stem cells, including cells isolated from aged individuals or cells that had been cultured in vitro. Nevertheless, in either case, understanding the molecular mechanism involved in aging and deterioration of stem cell function is crucial in developing effective new therapies for aging- as well as stem cell malfunction-related diseases. In fact, given the importance of the aging-associated diseases, scientists have developed a keen interest in understanding the aging process as well as attempting to define the role of dysfunctional stem cells in the aging process.

From the various advances in stem cell research, it is clear that we grow old partly because our stem cells grow old with us. The functions of aged stem cells become impaired as the result of cell-intrinsic pathways and surrounding environmental changes. With the sharp rise in the aging-associated diseases, the need for effective regenerative medicine strategies for the aged is more important than ever. Fortunately, rapid advances in stem cell and regenerative medicine technologies continue to provide us with a better understanding of the diseases that allows us to develop more effective therapies and diagnostic technologies to better treat aged patients.


Strategies for Cardiovascular Regeneration via Cell Therapies

Researchers here review one slice of the cell therapy field, examining the use of mesenchymal stem cells to provoke greater regeneration of heart tissue than normally takes place. While stem cell therapies are generally at least marginally beneficial, with reduction in inflammation the most reliable outcome to date, the research community has so far struggled to consistently produce larger benefits when it comes to heart damage in older people.

The treatment approach for the majority of cardiovascular disease is to administer drugs, and some cases may require surgery such as coronary angioplasty with stent insertion. The incidence of cardiovascular disease has continued to increase, and aside from transplantation, other therapies, despite recent advances in heart treatments, cannot fundamentally remedy the major etiology of cardiovascular disease; thus, there is a limit to how much treatment outcomes can be improved with the current approaches. Although various studies have been conducted ​​to overcome the limitations of cardiovascular therapies, stem cell therapy using several types of stem cells such as hematopoietic stem cells (HSCs), mesenchymal stem cells (MSCs), cardiac stem cells (CSCs), and endothelial progenitor cells (EPCs) provides an alternative approach, and ​​remarkable advances have been made in clinical and basic research.

Among adult stem cells, MSCs are frequently used to treat the most common cardiovascular diseases. MSCs can be found in the bone marrow (BM), adipose tissue, umbilical cord blood (UCB), and many other tissues. They have self-renewing properties and are multipotent progenitor cells that can differentiate into various lineages such as osteocytes, chondrocytes, adipocytes, and myocytes. MSCs also have immunomodulatory properties, and in addition MSCs are unlikely to lead to immune rejection. The therapeutic benefit of this approach is based on the potency of secretion of beneficial cytokines and growth factors for tissue repair/regeneration, as well as the immunomodulation effect and/or their differentiation for regenerating damaged organs.

MSCs can be applied for cardiovascular regeneration and provide therapeutic benefit for cardiovascular disease. However, MSCs have several disadvantages regarding their therapeutic application, including their very low survival rate in vivo and integration rate into the host cells after transplantation. Another limitation is the low accuracy in delivering the stem cells to the damaged site. Various attempts have been made to improve the poor survival and longevity of engrafted MSCs. The first step in developing therapeutic strategies is the identification of more effective reagents for promoting the ability of stem cells via understanding stem cell niche modulators. An emerging promising therapeutic strategy is the preconditioning of MSCs before transplantation using cytokines and natural compounds that induce intracellular signaling or niche stimulation through paracrine mechanisms. Another is a tissue engineering-based therapeutic strategy involving a cell scaffold, a cell-protein-scaffold architecture made of biomaterials such as extracellular matrix or hydrogel, and cell patch- and 3D printing-based tissue engineering, to enhance cell survival via cell-cell communication or cell-scaffold interactions. Because of its numerous applications, a combined therapeutic strategy that includes cell priming and tissue engineering technology is a promising therapeutic approach for cardiovascular regeneration.


Methuselah Foundation Launches Methuselah Fund

Those in the audience who have been members of this community for a while will know that the Methuselah Foundation has long acted as a form of incubator for biotechnology startups relevant to healthy life extension. The organization has undertaken this role in addition to funding some of the most promising research efforts in the field, such as the SENS rejuvenation biotechnology projects. This incubator work hasn't involved a large number of startups in total, since we are not yet either a vast or a wealthy community when considered in the grand scheme of things, and it is the case that the Methuselah Foundation is powered by our charitable donations. Nonetheless, with our support in past years, the Methuselah Foundation helped to launch the noted bioprinting company Organovo, and provided seed funding for Oisin Biotechnologies, pioneering a programmable gene therapy approach to clearance of senescent cells. Another company presently in the early stages of being shepherded by the Methuselah Foundation is Leucadia Therapeutics, working on a novel approach to clearing metabolic waste from the aged brain. There is a demonstrated track record of success here, moving in parallel to the progress in longevity science that has taken place in the laboratory.

The future isn't just a matter of funding research in non-profit organizations - at some point the leap must be made to a company and for-profit development. Given the growth in interest and funding for the treatment of aging as a medical condition that has taken place in recent years, and given that a number of professional venture funds dedicated to longevity science startups are now emerging, it is time for the Methuselah Foundation to formalize its efforts as an incubator of startups and pull in more funding to help advance the state of the art. Hence David Gobel, Sergio Ruiz, and the other busy folk at the Methuselah Foundation have been working hard these past months to put together the Methuselah Fund. This investment vehicle is tailored to the way that the Methuselah Foundation works with companies, an effective approach that has advanced the state of the field and moved us closer to the era of functional, widely available rejuvenation treatments. This means Methuselah Foundation funding and guidance for the most promising science and scientific groups, to help them reach the point at which they can and should launch a company, followed by Methuselah Fund assistance in the business and venture worlds to help to make that company a success.

The Methuselah Fund is also tailored for the people in our community, those of us who have over the years gained the ability and willingness to invest a few tens of thousands of dollars or more for the long-term, but who cannot undertake the much greater risk of investing in startups directly. The Methuselah Fund is presently open to anyone in our community who can make that level of commitment, not just professional investors. If you are a member of the Methuselah 300, you can complete your pledge of $25,000 by investing in the Methuselah Fund. Like most venture funds, this one has a set lifespan, running until 2030. That is 13 years to achieve great things along the way by investing in companies that are pursuing the SENS vision of clearing out or repairing the molecular damage that causes aging and age-related disease - and not just by investing, but by actively helping these companies to succeed at every step along the way. When it comes to research and development for the treatment of aging and there are few networks as good as those surrounding the Methuselah Foundation and their allies, such as the SENS Research Foundation and other similar organizations.

Is Fight Aging! investing in the Methuselah Fund? Yes, that is underway. I, like most people past the first few decades of life, have savings that become more or less accessible for investments depending on the level of risk involved. Funding startups, as I have done of late, a little, is the most risky activity one can undertake. While it is technically for profit, it is far easier to think of it in the same way as a charitable donation to a research non-profit: it goes to fund a specific line of research, that is the primary goal, and anything more than that is pleasant but not expected. One shouldn't put money into startups that one cannot afford to lose. An investment vehicle like the Methuselah Fund is diversified across a number of companies, however, and therefore less risky. As a fund that invests in startups, excellent track record of the Methuselah Foundation notwithstanding, it still bears considerably more risk than the sort of diversified investments a good portfolio manager will tell you adopt - but no-one lives forever. Yet. If this interests you, I'd suggest reading up on the life-cycle of venture funds, as money put in is locked away for the duration, and ask the Methuselah Foundation folk for a prospectus - contact information can be found in the interview below.

Tell us a little about the motivations here. Why the Methuselah Fund, and why now?

We want to accelerate mission results. As you know, Methuselah Foundation has been working hard during the last 16 years to extend the healthy human lifespan. We have long had the self-imposed goal of making 90 the new 50 by 2030 - it helps to inspire a sense of urgency appropriate to the great level of harm caused by aging. Since we now have only 13 years left to achieve this lofty but attainable goal, we will use an investment fund as a means to add fuel to the fire. Having this financial arm will allow us to exert our influence with companies with ground-breaking technology and approaches to medical issues, turning them towards the same mission as we have. Our ultimate goal is to make new medical treatments readily available to the public, as fast as possible, and thereby extending the healthy human lifespan.

Thus we are constructing a fund that will make it easier for companies to extend the healthy human life span. Unfortunately, history has shown it is often the case that when a venture capital approach is taken to increasing human longevity, the companies involved are required by the investors to pivot away from their original mission of treating aging and instead focus on an "FDA approved" disease such as cancer or type 2 diabetes. We want to avoid those outcomes. We do not believe in covenants that unduly restrict a company from going after therapies to treat the causes of aging. Beyond that, we are guided by Benefit Corporation principles, allowing us to create benefit for all stakeholders, not just the shareholders. This set of principles also gives us an expanded purpose beyond maximizing per-share value; we explicitly include general and specific public benefit. For example, if it makes better sense for a company to be merged in order to achieve our mission by 2030 and give up the potential for a billion dollar unicorn because it might kill the mission, we will prefer the mission. Unlike traditional venture capital, we are not afraid to leave a little money on the table when it benefits the real end goal of achieving more healthy life.

Why now? Because time is pressing. There are only 13 years left until 2030: 13 years to make 90 year-olds feel like they are 50 again. It is time to accelerate progress via the Methuselah Fund. The idea for this type of fund has been around for several years now. However, for it to be successful several things needed to happen, principally 1) emergence of an organization with a proven track record, 2) the development of a more mature view of longevity as a field of investment by the investment community. The Methuselah Foundation now has a significant tract record of positive investments (such as Organovo and Silverstone). These investments as a whole have been both mission-critical and highly profitable. That, compounded with leading the founding rounds of investments in Oisin Biotechnologies and Leucadia Therapeutics, has increased our investment experience and solidified our networks in the venture and scientific communities. Further, the investment community has matured, and events such as the founding of Apollo Ventures and the large investment in UNITY Biotechnology show that it is time to help steer the field to maximize progress towards our goal of far greater health in old age.

What are some of the irons you have in the fire at the moment?

The Methuselah Fund is our main iron in the fire at the moment and we are getting good traction. From a start-up standpoint, we are focusing our attention on two companies with the technology and knowledge to revolutionize their parts of the medical industry in ways relevant to our mission: Oisin Biotechnologies and Leucadia Therapeutics. We are constantly looking through the noise in search of the next shrewd investments, of course.

You've had a ringside seat and often a key role in many of the changes to happen in the aging research community this past fifteen years. What is your take on where things will go as we approach 2020?

Our hope is that as we approach and then pass 2020 we will start to see more products and treatments arriving in clinics via routes that circumvent the traditionally long, expensive, and heavily regulated paths to market, such as via medical tourism. Once this gets underway in any meaningful fashion, medical products of all sorts will have the added pressure of competitors being delivered in a fraction of the time, but with greater efficacy than ever before. Investors will finally see that investing in drugs that take decades to show returns is a bad business, and the whole house of cards will start to be dismantled into favor of something better. Given this process, everyday people will reap the benefits of: 1) cost savings due to a shorter incubation time for new products and 2) treatments arriving soon enough to matter for those who need them. Beyond 2020, we certainly hope to see new treatments that focus on preventing disease rather than merely patching it over: a medical reactionary culture will be overtaken by one of progressive better forms of prevention.

How will Methuselah Foundation change going forward, now that it is paired with Methuselah Fund?

The Methuselah Foundation will continue to organize research prizes and issue research grants as it has always done, with a focus on groups that can be aided in reaching the stage of commercial development. However, with the addition of the Methuselah Fund we will now have a powerful catalytic tool for our long-term mission, one that will bring in new resources and help to expand the breadth of these efforts.

You've often expressed a guiding vision for rejuvenation therapies of "clearing out the junk"; what does this mean in practice?

There are the scientific goals that, when reached as a whole, will make longer healthier human lifespans possible - see the SENS vision. We believe in making our goals easily understandable by all humans, however, since the cause of longer, healthier lives needs the backing of not just the scientific community but every one of us. The goal you mentioned is plain and simple: "Get the Crud Out". We call it "Crud" and not just "Junk" to make a point... no one likes crud! As we age, our bodies are weighed down by inefficiencies that create a slew of side-effects like inflammation and malfunctioning cells: this is easily understood. With "Get the Crud Out" we can cover the safe removal of senescent cells, broken mitochondria, and other destructive biological structures, as well as clearing out the various forms of waste products and byproducts (amyloid, lipofusin, cross-links, etc). That one goal covers a wide range of what we need to accomplish, makes the point, and expresses it simply enough to be a rallying slogan.

Among our other goals, which are equally important are: "New Parts for People," covering technologies such as bioprinting that will create new organs, bones, and vasculature; "Restore the Rivers," working to restore the circulatory system to full youthful competence and thus remove that contribution to conditions such as dementia and heart disease; "Debug the Code," to restore the informational integrity and viability of cells; "Restock the Shelves," investing in processes that replenish or restore stem cell and immune system populations; "Lust for Life," initiatives that restore the capacity for joy and resilience via rejuvenated senses and bodies. After all, what good would it be to live for hundreds of years without happiness, joy, and the fully functioning biology needed for both of those?

What can we in the community do to help make Methuselah Fund a success?

We are presently in the initial funding phase of the Methuselah Fund... the Founders' Round. We would love to talk to those who have the capacity to help back this exciting venture, or who know those who can. Interested parties can let us know and we will reach out with further details: please contact Sergio Ruiz at

Arguing that Selection Pressure Diminishes with Age Even in Immortals

Evolutionary arguments relating to aging are tricky things, typically hinging on details that can be credibly argued either way. You might look at the recent resurrection of group selection in the service of explaining the origins of aging, for example. This is a much debated area of theory, with little in the way of true consensus on exactly why it is that near all species undergo aging. Fortunately, an explanation for aging isn't strictly necessary in order to make inroads into addressing the processes of aging as we observe them today, but evolutionary theories considered in general have in the past proven to be very helpful guides in a variety of medical research. The molecular biochemistry of living beings is vast and enormously complex, and researchers have to start their investigations somewhere.

As old as the evolutionary theory of senescence is its underlying and widespread tenet that senescence evolves because survivorship dwindles with age. Consequently, higher mortality should lead to more senescence. In contrast, several authors have indisputably shown over the last decades that this logic is incorrect. Yet, these results did not suffice to erase the prevailing misconception, which is problematic, because empirical studies keep on testing a theoretical prediction that is, as such, not predicted. What to do, when something repeatedly proven to be wrong is still taken to be right? Here we attempt to advance understanding of the evolutionary theories of aging conditional on survivorship. Clearly, survivorship falls over age, and clearly adding some fixed amount of mortality at every age makes survivorship fall off more steeply. But such a shift in mortality merely reduces fitness; it does not change the selection gradients over the fitness landscape. The selection gradients still decline following the same pattern as in the absence of such mortality; selection does not favor young over old ages more or less strongly than before.

When it comes to understanding why we age, the rarity of survival to old age alone has long served as the explanation for declining selection gradients. This seems curious, because life is driven by birth and death together. Why should one side - survivorship - suffice to explain fundamental patterns of life, such as aging? We have demonstrated that reproduction plays an important role. Births keep on adding new individuals to the population, fueling a population growth factor that reduces the share of old organisms in the population. Even in the absence of death, as we demonstrate, births are enough to achieve declining selection gradients. Mortality is not the all-important driver of selection gradients.

We argue that older organisms have already produced a larger share of their total lifetime reproduction. Therefore a progressively smaller proportion of total production is affected by anything that happens to an organism at higher ages, and the organism will already have passed on its genes. Whether a change at some age affects evolution to a smaller or larger degree hinges not on survivorship per se, but on the relative abundance of individuals and their reproductive values. Provided the population is non-decreasing, the stable age distribution is always dominated by younger individuals over older individuals as a result of reproduction. This is true even in the hypothetical case of zero mortality. Survivorship can be changed by an age independent mortality term without affecting the selection gradients. Similarly, changes in age independent mortality leave optimal strategies unaffected.

The arguments laid out in this paper have theoretical and practical consequences. Empirical research has shown little support for the "central prediction" of the evolutionary theory of senescence, that a higher level of extrinsic mortality (predators, harsh environments, laboratory manipulations) should lead to a higher rate of senescence. A number of authors have called for a more involved theory of senescence, in which mortality is state dependent, and/or in which density effects play a prominent role. The results derived here and elsewhere make clear why there is little support for the central prediction. It is not just that this prediction is not born out in biological reality; life history theory simply makes no such prediction. After decades of theoretical work, we are still challenged to develop theory that provides more than an incidental match with the data. Our results corroborate the need for theory that is more involved; it may include combinations of age- and stage-specific mortality, density effects, and/or interaction mortality. Such a theory should involve mechanisms of senescence, as evolutionary pressures alone are only half the story.


Results from a Human Trial of Stem Cell Therapy Following Stroke

For the past few years, researchers have been running a trial of a first generation stem cell therapy for stroke patients. This used stem cell lines cultured from donors rather than from the patients. As is the case for some other forms of cell therapy, the resulting benefits appear linked to reduced inflammation rather than any other effects of the transplanted cells. Otherwise, the effects on patients were not as large as hoped would be achieved via this type of approach.

A trial looking at whether a single dose of millions of adult, bone-marrow-derived stem cells can aid stroke recovery indicates it's safe and well-tolerated by patients but may not significantly improve their recovery within the first three months. However, the trial does provide evidence that giving the therapy early - within the first 36 hours after stroke symptoms surface - may enhance physical recovery by reducing destructive inflammation as well as the risk for serious infections and that these benefits might continue to surface many months down the road. "There is solid evidence from our basic science work and now some indicators from this phase 2 patient trial that giving these stem cells can safely help dial back the body's immune response to stroke injury that can ultimately further damage the brain and body."

The study at 33 centers in the United States and the United Kingdom from October 2011 to December 2015 included 129 adults with moderately severe strokes. A dose of 400 million cells were given to a handful of patients to establish safety, the dose was then increased to 1,200 million cells for the majority of patients. About half of patients received a single dose of the stem cells while the remainder received placebo. Patients in both arms were able to also have received standard stroke therapies. While the study made several adjustments along the way to enable better enrollment, it was an early adjustment in the timeframe for giving the therapy that may have impacted results. Trial leaders extended the timeframe for therapy from the original 24 to 36 hours - which was suggested by previous animal studies - to 24 to 48 hours. That adjustment was in response to limited hours at some centers to thaw and otherwise prepare the cells for patients as they qualified for the study. Now cell developers have reduced thaw times from 6 hours to 30 minutes and made the process much easier, which should enable tighter timeframes for giving the treatment moving forward.

Although the primary analysis of results was done at 90 days, about 80 percent of study participants were followed for a full year. It was those longer-term results, particularly in the small number of patients who got therapy early, that suggested the cell therapy group might be more likely to continue to recover, with reduced disability and fewer infections one year out than the placebo group. The multipotent cells, dubbed MultiStem, were developed by the international biotechnology company Athersys Inc., which also funded the clinical trial. Doses given in the study were the largest ever given in a human cell therapy trial.

Researchers who have studied the cells believe they primarily work by modulating the body's immune response, which can go a bit haywire following a stroke. An immune response is definitely needed to help the brain heal and to remove debris generated by dead or damaged tissue. But there also may be a secondary response that includes immune organs like the spleen, beginning to shrink in size within the first hours after symptoms of stroke. "Some inflammation is good, but in a big stroke, it almost always overshoots. We think this secondary neuroinflammatory process is preventing the natural healing tendencies of the body. We think cell therapy prevents this early egress of cells from the spleen that go to the brain and, by doing that, they also prevent the later exhaustion of the spleen and immune system."


Predicting Mortality from Ten DNA Methylation Sites

A growing number of researchers are developing and testing various implementations of a DNA methylation biomarker of aging. There is even a US company offering a low-cost test for those who want to give it a try. The quality of resulting data and degree of testing and validation accomplished for these various approaches is quite varied. Some provide only loose correlations with mortality and life expectancy, while others produce estimates of age with a five year margin of error. This depends as much on the intentions of the research team as on the details of construction of the biomarker. Not every team has the funding or time to prove their case very rigorously in large data sets, versus creating an initial proof of concept to show that their approach to the biomarker is worthy of that funding and time.

How do these DNA methylation biomarkers differ form one another? DNA methylation is a form of epigenetic marker, a molecular decoration on DNA that can occur at any CpG site in the genome. This mark determines the pace at which proteins are produced from the related genetic blueprint, which genes are active and which are silent, and is one part of the many regulatory mechanisms that drive changes in cell behavior. All of the switches and dials inside the machinery of a cell can be traced back through chains of cause and consequence to a matter of how much of a particular protein is being produced. A cell's epigenetic configuration is a reaction to the circumstances that cell finds itself in. Some portion of that set of circumstances is due to the age of the tissue within which the cell is situated. Since we all age for the same underlying reasons, we all accumulate the same molecular damage, some of the epigenetic changes that occur with aging are shared, and can in principle indicate the level of damage present in an individual's tissues - a measure of biological age. But which epigenetic marks? That is the question. The choice of CpG sites to evaluate, the weight given in the final score to any one site, and the way in which that score is calibrated against test data: all these are ways in which DNA methylation biomarkers can differ from one another.

The development of at least one reliable, accurate biomarker of aging is an important step in the infrastructure needed for rapid progress in rejuvenation biotechnologies. For approaches based on the SENS vision of damage repair, it is straightforward enough to determine how effective a therapy is within its own paradigm. For example, given the ability to clear senescent cells from aged tissues, researchers can immediately follow such a treatment by measuring how great a percentage of senescent cells have been cleared. A senescent cell clearance therapy that clears half of all such cells is better than one that only clears a quarter of them. That doesn't tell us how great an extension of healthy life span will result from the treatment, however. At the present time the only way to assess that outcome is to wait and see. Waiting to see is, unfortunately, expensive and slow: it is an investment of years and millions of dollars in any earnest study, even in mice. That slows down the pace of progress. An independent biomarker such as DNA methylation might be able to short-cut that waiting game by providing a rapid measure of the degree of rejuvenation achieved immediately following the application of an intervention to treat the causes of aging.

DNA labels predict mortality

What does the methylation status in the DNA reveal about a person's health, his or her susceptibility to disease or, in short, an individual's mortality risk? Researchers investigated the cases of 1,900 participants of two epidemiological studies called ESTHER and KORA. They used DNA from blood cells as the basis of their investigation. All study subjects were older adults and had provided blood samples when they entered the study. This was up to 14 years ago and many of them had died since then. Methyl groups are only attached to a certain combination of DNA building blocks called CpGs. For almost 500,000 of these positions, the researchers analyzed whether their methylation levels revealed a statistical link to survival. After rigorous statistical review, it finally boiled down to 58 CpGs that showed a strong correlation between methylation status and mortality.

These 58 CpGs were all located in genomic regions for which an association with various diseases is well documented. Interestingly, 22 of the 58 CpGs were identical with methylation positions that the researchers had recently found in a study on the epigenetic impacts of smoking. Of all health risk factors, smoking hence appears to leave the strongest tracks in the genome. "The good news is that the level of DNA methylation is not written in stone. Unlike mutations in the DNA building units, it is reversible. That means, for example, that an unfavorable methylation status may change after smoking cessation and the mortality risk may drop again significantly."

Of the 58 CpGs, the scientists selected those ten with the strongest correlation with mortality. This epigenetic risk profile alone enabled them to predict the so-called all-cause mortality (cancer, cardiovascular diseases, and others). Study participants whose genome exhibited an "unfavorable" methylation status at five or more of these sites had a risk of death within the 14-year observation period that was seven times that of study participants whose methylation at these positions showed no abnormalities. "We were surprised that the methylation status of only ten positions of our genome correlates so strongly with all-cause mortality. We found even stronger links to mortality from cardiovascular diseases. Now it is important to find out which prevention measures are most effective to achieve a beneficial impact on the methylation profile and mortality."

DNA methylation signatures in peripheral blood strongly predict all-cause mortality

DNA methylation (DNAm) has been revealed to play a role in various diseases. Here we performed epigenome-wide screening and validation to identify mortality-related DNAm signatures in a general population-based cohort with up to 14 years follow-up. In the discovery panel in a case-cohort approach, 11,063 CpGs reach genome-wide significance. 58 CpGs, mapping to 38 well-known disease-related genes and 14 intergenic regions, are confirmed in a validation panel. A mortality risk score based on ten selected CpGs exhibits strong association with all-cause mortality, showing hazard ratios of 2.16 (1.10-4.24), 3.42 (1.81-6.46) and 7.36 (3.69-14.68), respectively, for participants with scores of 1, 2-5 and 5+ compared with a score of 0. These associations are confirmed in an independent cohort and are independent from the 'epigenetic clock'. In conclusion, DNAm of multiple disease-related genes are strongly linked to mortality outcomes.

The recently established epigenetic clock (DNAm age) has received growing attention as an increasing number of studies have uncovered it to be a proxy of biological ageing and thus potentially providing a measure for assessing health and mortality. Intriguingly, we targeted mortality-related DNAm changes and did not find any overlap with previously established CpGs that are used to determine the DNAm age. Our findings are in line with evidence, suggesting that DNAm involved in ageing or health-related outcomes are mostly regulated by DNAm regions other than the established age-related DNAm. The difference could also plausibly result from the fact that DNAm age was originally trained as precisely as possible to track chronological age and might thus be more indicative of natural ageing beyond the effect of disease, as exemplified by the much stronger association of DNAm age with mortality in oldest population (mean age 86.1 years) to whom common chronic diseases, such as CVD and cancer, might not continue to pose predominant risks.

The Trans-NIH Geroscience Interest Group

One of the active formal networks for scientists interested in treating aging as a medical condition is the Trans-NIH Geroscience Interest Group (GSIG), with a focus on public funded research and research groups. The thrust of their efforts is to achieve a modest slowing of the aging process by adjusting the operation of metabolism so as to slow down the rate at which the molecular damage of aging accumulates. These are generally people who - in public at least - do not support SENS rejuvenation research and the goal of repairing the cell and tissue damage that causes aging in order to reverse the progression of aging. The sort of future they envisage is one of slightly longer human life spans achieved through the use of calorie restriction mimetic drugs and the like, and so the interventions supported include the metformin trial, investigations of rapamycin, and so forth, nothing that is at all likely to produce sizable benefits for older people.

Insofar as useful outcomes result from the GSIG, I think it likely that some will be indirect, in the sense of obtaining greater support for treating aging rather than the effects of aging, that will in turn translate into more funding for projects like the SENS programs that can make a large difference. Secondly, direct benefits may emerge from the GSIG focus on biomarkers of aging, assuming that the DNA methylation approach isn't already good enough for practical purposes, and that other technologies must be explored. Good biomarkers of biological age are necessary for the rapid and cost-effective development of rejuvenation therapies, as when the only viable way to determine effectiveness is to try a treatment in mice and then wait a few years, progress is necessarily slow and expensive. With a biomarker, however, such a trial might be accomplished in a few weeks or months and at a much lower cost, assessing the degree of rejuvenation achieved with a measurement soon after treatment.

During a 2010 workshop organized by the Alliance for Aging Research, a discussion was held about the idea that aging is at the core of all chronic diseases, and one of us mentioned, without much pre-conceptualization, that since aging biology is at the core of all the diseases that concern them, then every institute within the NIH should have a Division of Aging Biology. The idea remained and over discussions in the ensuing months, this concept was further developed as a possible activity to be proposed across the entire NIH. As we refined the ideas and prepared to engage others, it became obvious that geroscience was a proper name for the initiative. Thus was born the Trans-NIH Geroscience Interest Group, GSIG.

Interestingly, the concepts of geroscience have long been understood both by scientists and the general public, as well as literature and the arts. However, the concept was slow in gaining recognition in medical spheres because of the ingrained notion that age is not a modifiable factor. While this is obviously true for chronological age (as the passage of time) it is also well recognized that good health at older ages can be attained by relatively simple interventions (which as behavioral changes, appear difficult for many people). Acceptance of age as the major risk factor for chronic diseases is implicit in the recommendations we receive if we visit a medical doctor for any malady: in addition to disease-specific interventions (statins, metformin, antidepressants), we are often counseled to "eat well, exercise moderately, and refrain from smoking." These are non-specific recommendations aimed at "healthier aging," but physicians seem loath to say so directly.

What has changed the perceptions is the astonishing advances made in the last couple of decades by scientists focused on understanding the basic biological underpinnings of the aging process, independently of disease. This has led to a few publications, including those from the GSIG, that have attempted to classify the main hallmarks or pillars currently believed to be the main drivers of the aging process. These conceptual advances have worked synergistically with reports from the NIA-supported Interventions Testing Program, which aims to test, in a variety of animal models, mostly pharmacological interventions that lead to an increase in both lifespan and healthspan.

Acceptance of the geroscience concept within the NIH proceeded at such a fast pace that an action plan was much less developed than the conceptual arguments used to form the group. An important strategic point was to keep the initial goals simple and attainable. This required a focus primarily on informational activities that would not require significant dollar investments on the part of participating institutes. Also, because the entire concept had been developed as a means to capitalize on the advances in basic aging research, the initial goal statement indicated that the focus was to be on basic biology, although we recognized the translational value of the effort. Current efforts are focused primarily on three areas where the GSIG recognizes an urgent need for further research: development of more appropriate animal models, enhancing the focus of geroscience on health irrespective of disease, and identification of suitable molecular and cellular biomarkers of the aging process. Taken together, these efforts aim at developing a deeper understanding of the basic biology driving all chronic diseases, and harnessing that knowledge for the betterment of health and well-being.


Digging in to the Mechanisms of A2E in Macular Degeneration

An accumulation of the metabolic waste compound A2E in the retina is associated with the progression of degenerative blindness via conditions such as macular degeneration, and there is strong indirect evidence for it to be a cause of the condition. This is one of numerous forms of waste that accumulate to form lipofuscin deposits inside and outside cells in the retina, but most likely the most important form. The easiest way to prove that causation beyond doubt, and hopefully also develop therapies that actually reverse retinal damage, is to selectively break down and remove A2E. An effort based on drug candidates developed at the SENS Research Foundation is currently underway at Ichor Therapeutics, but sadly this class of intervention, addressing root causes, has never been a priority in the research community as a whole. That point is somewhat illustrated in this open access paper, in which researchers investigate the role of A2E, and conclude by deciding that one of the downstream changes caused by A2E should be a target for therapy rather than the A2E accumulation itself.

Age-related macular degeneration (ARMD) is the leading cause of vision loss in developed countries. Hallmarks of the disease are well known; indeed, this pathology is characterized by lipofuscin accumulation, is principally composed of lipid-containing residues of lysosomal digestion. The N-retinyl-N-retinylidene ethanolamine (A2E) retinoid which is thought to be a cytotoxic component for retinal pigment epithelium (RPE) is the best-characterized component of lipofuscin so far. Even if no direct correlation between A2E spatial distribution and lipofuscin fluorescence has been established in aged human RPE, modified forms or metabolites of A2E could be involved in ARMD pathology.

Mitogen-activated protein kinase (MAPK) pathways have been involved in many pathologies, but not in ARMD. Therefore, we wanted to analyze the effects of A2E on MAPKs in polarized ARPE19 and isolated mouse RPE cells. We showed that long-term exposure of polarized ARPE19 cells to low A2E dose induces a strong decrease of the extracellular signal-regulated kinases' (ERK1/2) activity. In addition, we showed that A2E, via ERK1/2 decrease, induces a significant decrease of the retinal pigment epithelium-specific protein 65 kDa (RPE65) expression in ARPE19 cells and isolated mouse RPE. In the meantime, we showed that the decrease of ERK1/2 activity mediates an increase of basic fibroblast growth factor (bFGF) mRNA expression and secretion that induces an increase in phagocytosis via a paracrine effect. We suggest that the accumulation of deposits coming from outer segments (OS) could be explained by both an increase of bFGF-induced phagocytosis and by the decrease of clearance by A2E. The bFGF angiogenic protein may therefore be an attractive target to treat ARMD.


Functional Tooth Regrowth Demonstrated in a Canine Model

There has been considerable progress over the past decade towards the regrowth or tissue engineering of adult teeth via a number of different mechanisms. These include growing a tooth entirely outside the body, starting from a few cells, an approach that has a range of associated challenges regarding how to guide the growing tissues to form the right shape. Some years back researchers demonstrated a fairly brute force method of providing that guidance in tissue engineered mouse teeth, to pick one example. Then there is the alternative approach in which researchers attempt to create the seed of a tooth, the tooth germ, a collection of cells as similar as possible to those that occur naturally when a tooth grows. The idea here is to enlist the existing environment of the jaw and gum to guide growth of a new tooth; if the artificial seed is close enough to the natural equivalent, then the end result will be a correctly formed tooth. The paper quoted below is an example of the state of the art in this latter approach to adult tooth regrowth: researchers have pushed towards larger animal models, and can now fairly reliably induce the growth of functional replacement adult teeth in canines.

If you read the paper closely, the researchers are still relying heavily on natural tissues to source the relevant cells to make up the seed for a new tooth. They report on mining the naturally grown teeth of animal models in order to demonstrate that suitably arranged cell combinations will then go on to grow new teeth in those same animals when implanted into the jaw. Future work will involve establishing reliable methods of creating patient-matched cells to order, such as via reprogramming of a patient cells sample into induced pluripotent stem cells, and then differentiating the needed cell types from that pluripotent lineage. Not all of the required recipes for the cell types of interest have yet been established, however, so there is a significant amount of work left to be accomplished. Once done, however, that will open the doors to further progress.

How long before we humans will benefit from this sort of approach to tooth regeneration? Dentistry is somewhat less oppressively regulated than the rest of medicine in much of the world, the consequence of a long history of somewhat arbitrary separation of disciplines, and so new innovations in dentistry tend to arrive in clinics more rapidly. If researchers are just now growing new teeth in dogs after ten years of work in bioreactors and rodents, then another decade to reach clinical applications is a fair guess. It is an open question as to how well it will work in older individuals, however. Do old people still exhibit enough of the same guiding signals and cellular behavior in gums and jaw bones? It is well known that regeneration in general declines with age, for reasons that include failing stem cell activity and altered cell signaling that occurs in reaction to rising levels of molecular damage in tissues. The fastest way to find out is to try and see, but we can also survey the sort of work on aging and stem cell biochemistry that is currently taking place in relation to the development of stem cell therapies. That will provide some idea of the additional time and cost imposed by trying to make things work well in older people. There are differences between old tissues and young tissues, and in many cases they are significant enough to require a modified or alternative approach.

Practical whole-tooth restoration utilizing autologous bioengineered tooth germ transplantation in a postnatal canine model

In this study, we demonstrated functional tooth restoration after transplanting bioengineered tooth germ in a postnatal large-animal model. The bioengineered tooth, which was reconstructed using canine permanent tooth germ, developed with the correct tooth structure after autologous transplantation into the jawbone. We also determined that the bioengineered tooth erupted into the oral cavity with the features of proper tooth tissue formation and restored physiological tooth function, such as the response to orthodontic mechanical force. This study represents a substantial advancement in organ replacement therapy through the transplantation of bioengineered organ germ as a practical model for future whole-organ regeneration.

Whole-tooth replacement therapy holds great promise for the replacement of lost teeth by reconstructing a fully functional bioengineered tooth using three-dimensional cell manipulation in vitro. It is anticipated that bioengineering technology will ultimately enable the reconstruction of fully functional organs in vitro through the proper arrangement of epithelial and mesenchymal cell components. Many researchers have attempted to generate bioengineered tooth germ using epithelial and mesenchymal cells from embryonic tooth germ or postnatal tooth germ from various species, including mice, rats and swine. With the goal of precisely replicating the developmental processes that occur in organogenesis, the study of an in vitro three-dimensional cell manipulation method called the bioengineered organ germ method has been recently reported. However, additional evidence of the practical application to human medicine is required to demonstrate the generation of bioengineered tooth germ using postnatal cell sources in a large-animal model.

To achieve whole-tooth restoration in humans, it is desirable to autologously transplant bioengineered tooth germ reconstructed using a patient's own stem cells to prevent immunological rejection, and it is necessary to first establish an autologous tooth germ transplantation system in a large-animal model. We therefore investigated whether the canine bioengineered tooth germ reconstructed using epithelial and mesenchymal components isolated from individual tooth germs could develop after autologous transplantation into the jawbone. We demonstrated that a bioengineered tooth reconstructed from canine permanent tooth germ reproduced the correct tooth structure, including calcified components and enamel and dentin microstructure. Furthermore, the erupted bioengineered tooth had a single-root shape with the proper periodontal tissue structure, and it achieved physiological tooth function in terms of biological response to mechanical stress equivalent to the function of a natural tooth.

If a large-scale culture of epithelium/mesenchymal tooth germ cells were to be established in future, this bioengineered tooth technology would be able to treat a large number of missing teeth. Elderly patients, however, do not have a developing tooth germ that can be used for the reconstruction of bioengineered tooth germ in the patient's own jaw. In the dental field, recent stem cell biology studies have led to the identification of dental stem cells based on tooth organogenesis for tooth tissue regeneration and tooth regenerative therapy. Although these stem cells would be valuable cell sources for stem cell transplantation therapy aimed toward dental tissue regeneration, the tooth inductive potential cells, which can replicate an epithelial-mesenchymal interaction for whole-tooth replacement, has not yet been identified.

A Tour of Some of the Molecular Damage Involved in Aging

The intricate molecular machinery found in cells only functions correctly when it is undamaged, meaning formed of the right atoms and bonds, and that often sizable structure correctly arranged into a particular three-dimensional shape. A cell is essentially a liquid bag of molecules that are constantly coming into contact with one another, however. Large numbers of these molecules react in inappropriate ways or become misfolded, and so a cell incorporates layer upon layer of quality control mechanisms, each of which strives to ensure that cellular machinery remains correct in form and structure. Broken parts are aggressively removed and recycled, but nonetheless some damage inevitably slips through. Aging itself is essentially a process of damage accumulation, at root an accumulation of unwanted and malformed molecules, and then the chain of unfortunate consequences that follows from that state of damage. This open access review covers some of the forms of molecular damage involved in the aging process:

The idea that aging results from the gradual accumulation of molecular damage is deeply rooted in the aging research field, although it can appear in verbal disguises so different as to seem conceptually independent. However, damage is implicit to DNA in the somatic mutation theory of aging, to the extracellular matrix proteins in the cross-linking theory, and to phospholipids in the membrane theory. The free-radical theory implies that reactive oxygen species (ROS) are responsible for damage, and the carbonyl-stress theory blames free carbonyls for it. With regard to the last two theories, the former celebrates its 60th anniversary this year and remains the most influential in the "damage field", and the latter is its extension insofar as it attributes the origin of many of the most noxious molecular species to the free-radical oxidation of metabolites initially devoid of highly reactive carbonyl moieties.

In a metabolic system, not only spontaneous decay and degradation reactions, such as hydrolysis, oxidation, and racemization, but also spontaneous multistage synthetic processes take place. Can the products formed in this way be regarded as metabolites in a strict sense? They are not generated by enzymes, are not used purposefully, and are often hazardous. One way to view them is as damaged metabolites. For example, 5-Scysteinyldopamine is a damaged form of cysteine or dopamine. A related way to conceptualize this phenomenon is to view it as a sort of 'underside' of metabolism or 'parametabolism'. A conceptually similar but more general approach is to regard such unwanted products as a manifestation of the imperfectness of metabolism and its components, which together produce deleterious effects at all levels of biological organization. The totality of such effects has been described as the "deleteriome", which expands with age and represents the biological age of an organism. One way to increasing the deleteriome is by the spontaneous polymerization of damaged metabolites, such as catecholamine-derived quinones. In reality, such polymerization occurs in a milieu abundant in proteins, which are included in the resulting agglomerates, wherein they become covalently modified and misfolded and thus made prone to aggregation. Altogether, this leads to the accumulation of polymers of (damaged) metabolites associated with protein aggregates in the form of lipofuscin, neuromelanin, and other forms often referred to as waste.

A good case for applying the ideas discussed above to a specific situation is provided by bisretinyls, the major constituents of lipofuscin accumulated in the pigmented epithelium of the eye. Bisretinyls are byproducts of visual cycle biochemistry. Without delving into important details and conflicting views, it is sufficient in the present context to point out that the functional demands of light perception ensure that the aldehyde retinal is constantly present free in an environment rich in ethanolamine moieties. The result is the formation of retinyl dimer and a host of related compounds accumulating in photoreceptor membranes, which are constantly shed off to be phagocytized by pigmented epithelium cells. The poorly degradable retinal dimer and related products form lipofuscin deposits in pigmented cells and thus increase the risk of macular degeneration, the most common form of age-related vision loss.

Several lessons follow from the above case. First, damage accumulation results from normal functions, and the pathways of damage formation may become clear only after the molecular details of normal functions become known. Second, damage manifests itself in a functionally significant manner at ages rarely achievable in the wild under the conditions in which the species in question evolved. Therefore, there was no selection pressure towards the prevention of accumulation of this sort of damage. However, there was pressure towards preventing any immediate damage, even at the expense of later adverse consequences. In fact, lipofuscin accumulation in pigmented epithelium is a consequence of clearing of photoreceptor cell membranes from damage caused by retinal liberated in the course of light perception. Third, via a series of transitions through rapidly turning-over cell constituents, damage finally accrues as a slowly turning-over material in the nonrenewable component of a functional system where the deposits of damaged metabolites accumulate.

Spontaneous chemical reactions between metabolites are often labeled with proper names, such as Schiff, Pictet-Spengler, Amadori, Mannich, or Michael, just because they are typical and will take place wherever the respective reactants come together. Thus, from the chemical point of view, a metabolic system cannot but be plagued with numerous short-circuits, leaks and other adverse concomitants of metabolism. Unwanted reactions of this sort give rise to diverse damage products that increase in number and abundance with age and are adjusted (with regard to both composition and rate of increase with age) by interventions that affect lifespan. These reactions in their entirety are sufficient to cause what is generally termed aging.


A High Level View of Senescent Cell Clearance

This is a better than average popular science article on turning back the progression of aging by removing senescent cells from aged tissues; certainly the bar for article quality set by the mainstream press isn't high, but it is always pleasant to see more authors clearing it. One point worth noting in response to this piece is that we really have little idea as to how the life extension observed in mice lacking senescent cells will scale in humans. Near all methods of extending life in mice to date have been based on modestly slowing aging, changing the operation of metabolism to reduce the rate at which molecular damage accrues. Short-lived species like mice have a much greater response to this sort of thing than do humans, demonstrated when we compare the effects of calorie restriction and growth hormone receptor loss of function mutations. In mice these can extend life by as much as half again, but if that was the case in humans, we'd have certainly noticed by now. Clearing senescent cells is a completely different form of therapy, however, a type of damage repair carried out intermittently rather than an ongoing slowing of damage. I know of no such approach that has been tried in both mice and humans, and thus there is no basis for comparison.

Imagine a world where you could take just a single pill for the treatment or prevention of several age-related diseases. Although still in the realms of science fiction, accumulating scientific data now suggests that despite their biological differences a variety of these diseases share a common cause: senescent cells. This has led scientists to find drugs that can destroy these cells. When cells become damaged, they either self-destruct (apoptosis) or they lose their ability to grow and remain stuck within the body. These are the non-growing senescent cells that no longer carry out their tasks properly. They spew out chemicals that cause damage to cells nearby, sometimes turning them into "zombies" - hence why they are sometimes referred to as "zombie cells". Eventually, the damage builds up so much that the function of bodily organs and tissues, such as skin and muscle, becomes impaired. At this point, we identify the changes as disease.

In 2011 and in 2016, researchers showed, through the use of genetically engineered (transgenic) mice, that the removal of senescent cells reduced cancer formation, delayed ageing and protected the mice against age-related diseases. The mice also lived 25% longer, on average. A similar result in humans would mean an increase in life expectancy from 80 years to 100 years. It was proof-of-principle studies like these that laid the groundwork and inspired other researchers to build on these findings. It is not known how many senescent cells need to be present to cause damage to the body, but the harmful effects of the chemicals they release can spread quickly. A few zombie cells may have a huge impact. Drugs for specifically killing senescent cells in order to extinguish their destructive force have recently been revealed and tested on mice. The collective term for these drugs is "senolytics".

In 2016, two research groups independently published findings on the discovery of two new senolytic drugs which target proteins responsible for protecting senescent cells from cell death. Research showed that the drug ABT-263 (Navitoclax) could selectively kill senescent cells in mice, making aged tissues young again. And scientists have also used the drug ABT-737 to kill senescent cells in the lungs and skin of mice. There has also been a lot of interest in the role of senescent cells in pulmonary diseases caused by damage to the lungs. In late 2016, scientists showed that the removal of senescent cells using genetically engineered mice greatly restored lung function in old mice. In light of these accumulating and highly promising findings, a number of start-up biotechnology companies have been created to exploit the health benefits of targeting senescent cells. Probably the most well funded is UNITY Biotechnology in the US which raised US$116M for research and development.


More on PCSK9 Inhibition to Dramatically Reduce Cholesterol Levels, Lowering the Risk of Later Cardiovascular Disease

There is good evidence for at least some methods of achieving dramatic reductions in blood cholesterol in humans to be safe and reduce the risk of age-related cardiovascular issues. To pick one of the underlying mechanisms involved in these benefits, the common age-related condition of atherosclerosis is at root caused by interactions between damaged cholesterol and the cells of blood vessel walls. Cells become irritated by the presence of that cholesterol, and this begins a series of overreactions and unfortunate events that leads to the generation of fatty plaques that narrow blood vessels and weaken blood vessel walls. In conjunction with the raised blood pressure present in older individuals, this eventually leads to the dramatic structural failure of a stroke or heart attack, when a large blood vessel is blocked or ruptures. If there is less cholesterol in the bloodstream, however, the whole chain of cause and effect slows down. That slowing isn't as good as fixing the issue, such as by effectively sabotaging any one of the bad cellular behaviors that combine to lead to the growth of plaques, but it is certainly a lot better than nothing.

Statins are the obvious item to point out when thinking about cholesterol, aging, and cardiovascular disease. The widespread use of this class of drug has done much to reduce the incidence and mortality of cardiovascular disease. These days, however, researchers are looking into more targeted means of reducing cholesterol levels, forms of therapy that suppress specific genes to achieve much larger reductions than is possible with statins. There are a number potential targets for gene therapies, RNA interference approaches, and the like, such as an ASGR1 mutation that occurs in small number of humans, who have less cholesterol and much lower rates of atherosclerosis. The story is much the same for an ANGPTL4 mutation, also present in a small number of people and associated with significantly reduced cholesterol and cardiovascular risk. For today, however, I'll note a brace of publicity materials on recent attempts to target PCSK9 with the aim of permanently lowering blood cholesterol by a large amount. Several distinct teams appear to have timed their press for the same scientific conference, so all the results appeared in public at much the same time.

New 'gene silencer' drug injections reduce cholesterol by 50% in early research

The first in a new class of gene-silencing drugs, known as inclisiran, has halved cholesterol levels in patients at risk of cardiovascular disease. The findings come from the largest trial yet to test the safety and effectiveness of this kind of therapy. The technique, known as RNA interference (RNAi) therapy, essentially 'switches off' one of the genes responsible for elevated cholesterol, PCSK9. The twice-a-year treatment could be safely given with or without statins, depending on individual patient needs. Eventually, inclisiran could help to reduce the risk of heart attacks and stroke related to high cholesterol. "We appear to have found a versatile, easy-to-take, safe, treatment that provides sustained lowering of cholesterol levels and is therefore likely to reduce the risk of cardiovascular disease, heart attacks, and stroke. These reductions are over and above what can be already be achieved with statins alone or statins plus ezetemibe, another class of cholesterol-lowering drug."

In the study, researchers gave 497 patients with high cholesterol and at high risk of cardiovascular disease either inclisiran at varying doses, or placebo. Seventy-three per cent of these patients were already taking statins, and 31 per cent were taking ezetimibe. Participants, who were recruited from Canada, USA, Germany, Netherlands, and the UK, were excluded if they were taking monoclonal antibodies for cholesterol lowering. Patients were given different doses of inclisiran or placebo via subcutaneous injection, either via a single dose, or via a dose on day one and another at three months. They were followed up regularly for a subsequent eight months and tested for blood cholesterol and side effects. The researchers found that just one month after receiving a single treatment of inclisiran, participants' LDL cholesterol levels had reduced by up to 51 per cent.

PCSK9 Inhibitor Evolocumab Significantly Reduces Adverse Cardiovascular Events When Added to Statin Therapy With No Major Safety Concerns

A new class of cholesterol lowering drugs known as PCSK9 inhibitors has emerged as an effective treatment for drastically lowering LDL cholesterol beyond what is possible with statin therapy alone. Previous research demonstrated that evolocumab, a member of this new class of drugs, effectively reduces LDL cholesterol by approximately 60 percent. Evolocumab is a fully human monoclonal antibody that works by blocking proprotein convertase subtilisin-kexin 9 (PCSK9), a protein that reduces the liver's ability to remove LDL cholesterol from the blood. The FOURIER trial (Further Cardiovascular OUtcomes Research with PCSK9 Inhibition in subjects with Elevated Risk) was designed to determine whether evolocumab, when added to statin therapy, would reduce adverse cardiovascular events.

In this randomized, double-blind, placebo-controlled multinational clinical trial, 27,564 patients aged 40-85 were studied. All trial participants had stable atherosclerotic vascular disease, defined as a medical history of heart attack, stroke or symptomatic peripheral artery disease. On a background of high or moderate intensity statin therapy patients had a LDL cholesterol level of at least 70 mg/dl. Patients received either evolocumab (140mg every two weeks or 420mg every month) or placebo. Similar to data from previous lipid lowering trials, researchers report that evolocumab reduced LDL cholesterol by 59 percent, in this case from a median of 92 mg/dL to a median of 30 mg/dL. The LDL cholesterol lowering effect remained constant over the duration of the trial.

Researchers report that patients treated with evolocumab had a 15 percent reduction in the risk of major cardiovascular events, defined as the composite of cardiovascular death, myocardial infarction, stroke, hospitalization for unstable angina, or coronary revascularization (occurring in 9.8 percent of patients treated with evolocumab vs. 11.3 percent of patients treated with placebo). Additionally, evolocumab reduced the more serious key secondary endpoint, which was a composite of heart attack, stroke or cardiovascular death, by 20 percent (occurring in 7.9 percent of patients treated with evolocumab vs. 9.9 percent in the placebo group). This reduction in risk improved over time, increasing from 16 percent in the first year to 25 percent after the first year.

PCSK9 Inhibitior Bococizumab Produces Varying Results

New results from the clinical trial program, SPIRE (Studies of PCSK9 Inhibition and the Reduction of Vascular Events), which sought to determine the effect of bococizumab, a PCSK9 inhibitor, on LDL cholesterol levels and clinical outcomes in high-risk patients already taking statin therapy, have been presented. Researchers report that bococizumab had short-term benefits on lowering cholesterol levels and significantly reduced the risk of cardiovascular events by 21 percent compared to placebo among those who had baseline LDL cholesterol levels of greater than 100 mg/dL. However, the cholesterol lowering effect tended to diminish over time in some patients and bococizumab did not reduce cardiovascular event rates among those with LDL levels lower than 100 mg/dL.

The SPIRE program involved eight double-blind, placebo controlled clinical trials that were conducted simultaneously. Six lipid-lowering trials randomized 4,449 patients, who previously had a heart attack or stroke or had extremely high baseline cholesterol levels and were on statin therapy, to receive either bococizumab (150 mg subcutaneously every 2 weeks) or placebo to determine the effects on LDL levels. Two other multi-national trials randomized 27,438 patients to either bococizumab or placebo and were designed to evaluate the impact of the drug on cardiovascular outcomes, including nonfatal heart attack and stroke, hospitalization for unstable angina requiring urgent revascularization, or cardiovascular death. Almost all the patients were on statin therapy.

On November 1, 2016, the entire SPIRE clinical trials program was stopped when the sponsor, Pfizer, who manufactures bococizumab, discontinued the development of the drug when initial results from the LDL cholesterol lowering trials indicated that some trial participants had developed anti-drug antibodies, an immunologic response to the drug. Further analysis indicated that bococizumab effectively reduced LDL cholesterol levels by an average of 56 percent after 14 weeks; however, the immunologic reaction attenuated the reduction in LDL cholesterol in approximately 15 percent of those who received the drug. Data also show that there was a wide variation in the magnitude of cholesterol reduction that patients achieved with bococizumab, even among patients who did not develop the immunologic response. Bococizumab is a humanized antibody therapy that works by blocking proprotein convertase subtilisin-kexin 9 (PCSK), a protein that reduces the liver's ability to remove LDL cholesterol from the blood. "We believe that the attenuation of LDL lowering over time in the treatment group was likely due to the fact that bococizumab, a humanized antibody, led to an immunologic response in some patients. The alternative PCSK9 inhibitors evolocumab and alirocumab, which have already been approved for use, are fully human antibodies and do not have this adverse effect."

If this were a better world, the next stage in this process would be the development of one-off gene therapies for use by all adults, carried out long before old age rolls around. That is unlikely to happen any time soon within the bounds of the present regulatory environment, however. Waiting until the damage is underway and the disease process in its comparatively late stages before acting is the great failing of modern medicine and regulation of its use. So much more could be done to slow down and prevent aging by intervening sooner with these new technologies, in adults who are still young and healthy. Acting too late is always a poor strategy in comparison.

The Influence of Children on Late Life Mortality in Humans

Researchers here examine mortality data from one of the wealthier parts of the world, where more people have chosen not to have children, and thus there is a large enough data set to make useful comparisons between older people with and without children. The researchers show that having children adds a few years to life expectancy, conforming to similar past results. The demographic data offers little when it comes to support for any one possible mechanism over others, but some of the options are discussed in the paper. The primary focus is on increased support from children, financial and otherwise, once the parents have entered the frail and vulnerable final stage of aging.

It is well established that parents live longer than non-parents, but the underlying mechanisms are unclear and it is not known how the association changes over the life course. In Sweden and the other Nordic countries, there is an overall trend of increasing levels of childlessness across birth cohorts. It may therefore be valuable to improve our understanding of how childlessness is linked to health and survival chances in old age. We hypothesise that support from adult children to their ageing parents may be of importance for parental health and longevity.

However, there are of course several alternative explanations. For example, the timing and number of children could affect the mortality risk of women through biological pathways. Still, a protective effect of parenthood has been found for mothers and fathers, which may suggest that the biological mechanisms that apply to women is not the only explanation to the association, and other factors matter as well. One such factor could be various types of support from adult children to their ageing parents, such as informational, emotional and social support. In addition, parents have on average more healthful behaviours than childless individuals. It is also possible that the survival advantage of parents over non-parents is due to confounding from biological or social factors influencing the chances of having children and the risk of death. Health-related selection may be important at any phase during the life course, but it seems reasonable that the influence would not increase when parents become very old, but rather being more significant before average life expectancy (LE) as frailer individuals tend to die off. The need for social support from family members may, on the other hand, increase when parents age because ill health becomes more common with increasing age and the ability of self-care may decrease.

How the mortality advantage of parents over non-parents changes over the life span is not known. Previous studies have mainly examined associations between parity and subsequent mortality from 40 years of age up to around 60 years of age. This study closely examines the association between parenthood and longevity, with specific focus on the strength of the association in old age, and with absolute and relative measures. More specifically, we investigate; (1) the association between having a child alive in old age and the risks of death among Swedish men and women, (2) whether the association increases with age of the parent and (3) whether the association persists when stratifying for marital status (taking into account the possible confounding effect of having a partner cohabiting).

This study found an inverse association between having a child and death risks in old age, and, importantly, that the death risk differences between parents and non-parents increased with age of the parent, among men and women. Further, the differences in death risks between individuals with and without children were somewhat larger for men than for women. Our finding that the association grew stronger when parents became older is further in agreement with research suggesting that childless people face support deficits only towards the end of life. However, selective elements and alternative explanations, for example, that parents have more healthy behaviours than non-parents, are not ruled out. The association between having children and mortality persisted when stratifying for marital status, taking into account the possible confounding effect of having a partner. Two of our findings may be interpreted as working against the hypothesis about the importance of social support in older ages: the lack of a stronger mortality association for parents whose children lived fairly close, and the insignificant results for the gender of the child.


Stochastic Nuclear DNA Damage in Aging

Nuclear DNA in every cell accumulates random mutational errors over time. DNA repair mechanisms are highly efficient, but nonetheless, some damage slips through. It is still an open question as to whether this damage is important over the course of the present human life span, at least beyond the matter of cancer risk, where it is well proven that more mutational damage means more cancer. Putting aside cancer for the moment, is there enough random DNA damage occurring in normal individuals to produce enough dysregulation of cellular behavior to in turn lead to meaningful levels of other forms of harm and lost function in tissues? The consensus among researchers is that this is the case, and nuclear DNA damage is listed in the noted hallmarks of aging paper, but that consensus has been challenged. This is largely a debate over theory and indirect evidence at the present time, however: is difficult to produce an animal study in which only random DNA damage is altered in degree, so as to definitively establish differences in outcome. There have been a few promising starts in that direction in the past few years, but much more work remains to be accomplished.

The cells in our bodies are constantly churning out proteins and other structures, built according to the blueprints contained in their DNA, which are crucial to supporting those cells' functions. And while in each cell most of the information contained in its DNA will be ignored, if an area of the genome important to the cell's function is damaged or develops mutations, the cell may produce misshapen proteins or simply stop functioning altogether. The effects of misshapen proteins can range from useless to actively harmful, as when neurons in a brain with Alzheimer's disease produce excessive amounts of the neurotoxic protein amyloid beta.

A few dysfunctional cells here and there don't pose much of a problem, but as more and more cells in a tissue accumulate damage over time, the health of the entire tissue or organ may be compromised. The body's normal way of dealing with such cells is to remove them through a type of programmed self-destruction called apoptosis, making way for new cells. Some cells, however, fail to die and enter a state of senescence, where they are incapable of replicating but are still left to take up space in the tissue. But especially dangerous is the case of a cell with damaged DNA that doesn't enter either apoptosis or senescence: damage drives mutation rate up each time the cell replicates, and if a mutation provides a survival advantage or switches off the cell's protective mechanisms against tumor formation, this can eventually lead to cancer. Cells that divide frequently, such as skin or lung cells, are most susceptible to this danger.

Our bodies may have evolved an impressive variety of damage repair mechanisms, but with increasing exposure to damage-causing agents in the environment and the damages caused by our own internal processes, compounded with the declining effectiveness of our protective mechanisms over time, DNA damage and mutations are bound to accumulate as we age. Some evidence suggests that caloric restriction may mitigate these effects. However, since no drugs yet exist that will prevent or repair DNA damage, all we can do at the moment is try our best to avoid harmful agents like excessive sun exposure and smoking.


Can Pregnancy Effects be Used to Argue For Changes in Cell Signaling as an Effective Approach to Treating Aging?

Over the past decade, a few research groups have provided evidence to suggest that pregnancy can enhance regeneration in the mother. The proposed mechanisms have largely involved capable stem cells from the fetus acting as a mild form of stem cell therapy when they find their way into the mother's tissues. Since then, however, parabiosis research has taken off, in which an old and a young animal have their circulatory systems joined. This has a beneficial effect on the older animal, and researchers have spent a great deal of effort investigating signal molecules in the bloodstream that differ between old and young animals. The initial focus was on finding youthful signals that might help turn back some of the detrimental changes that take place in old tissues, such as loss of regenerative capacity and decline in stem cell activity.

Perhaps unfortunately, the latest update in this line of research puts something of a damper on the idea that young signals can rejuvenate old tissues, and proposes that the effect is achieved through dilution of harmful signals or waste in the old tissues and bloodstream. Still, now that everyone is as much focused on signals as they are on stem cells, it is perhaps time to look back at the evidence for pregnancy effects. Is there anything there that might be used to argue in one direction or another when it comes to the signal environment, aging, and potential therapies? From where I stand the existing evidence looks a little sparse to be building anything atop it, but it doesn't seem like an unreasonable angle to pursue further - at least for those researchers who are focused on slowing aging through forcing change on the signaling environment. That said, there is no necessary reason why these two situations, parabiosis and pregnancy, should in the end turn out to have much in common; it could just as easily be the case that the placental barrier rules out all of the interesting exchanges that take place in parabiosis.

Why does signaling change in old tissues? Those who see aging as an accumulation of molecular damage would say it is a consequence of increased levels of damage. Cells react to their environment, and it is unfortunately the case that some of those reactions go on to cause further harm. See, for example, cells becoming senescent. There is another viewpoint, that of programmed aging, which considers signaling changes a primary cause of aging, and altered cellular behavior then in turn leads to damage. After some years of reading around that area of theory, I still think its proponents have a tough hill to climb in order to make that case. Fortunately, the development of working rejuvenation theories will settle the right and wrong of things more rapidly than the slow battles over theory; practical efforts will come to an answer in the next ten to fifteen years, I'd say. Therapies based on damage repair and therapies based on alteration of signaling are both imminent; one approach will work far better than the other, and that will be that.

Molecular and Cellular Interactions between Mother and Fetus - Pregnancy as a Rejuvenating Factor

Prevention or even reversal of aging have become topics of numerous studies. The discovery of rejuvenating factors, if they exist and are of chemical nature, would become a breaking point in solving many problems in gerontology. Scientists periodically report a discovery of such factors; however, careful examination of their data and technical details of these studies raise doubts if the discovered factors indeed possess rejuvenating properties. In particular, when the circulatory systems of two animals of different ages are connected in a heterochronic parabiotic model, the older partner undergoes rejuvenation. Some recent studies aimed to identify the chemical factor that enters the older organism from the younger one suggested that this rejuvenating factor is GDF11, a differentiation growth factor that circulates in the common blood stream of parabiotic partners. However, more detailed studies failed to unambiguously confirm that GDF11 is responsible for the rejuvenating effect.

The rejuvenating effect of pregnancy remains a subject for discussion. We find it promising to view pregnancy as a parabiotic system in which organisms of different age (young fetus and mature mother) are functionally connected and exchange factors that affect both, in either positive or negative manner. Pregnancy is a great burden for the maternal organism and carries a risk of numerous complications. Hence, for many years, both clinical medicine and academic research have concentrated mostly on negative effects of pregnancy on the mother's health. However, recent studies have shown that pregnancy might have positive effects on the physiological state of many organs and on maternal longevity in general, especially in the absence of pregnancy-accompanying complications. Some studies even discussed the "rejuvenating" effect of pregnancy on the maternal organism.

The regenerative capacity of liver (estimated from the rate of liver regeneration after removal of 2/3 of its volume) in 10-12-month-old mice was four times lower than in young (3-month-old) animals. However, when old mice were in the third trimester of pregnancy, their rate of liver regeneration after hepatectomy was like that in young animals: in both young non-pregnant mice and aged pregnant mice, liver regenerated to its initial volume ~2 days after the surgery, while in old non-pregnant animals, liver volume remained less than 50% of the original one. Another common object for studying regeneration and its decline with age is a skeletal muscle. The regeneration index in 20-month-old mice was almost 10 times lower than in 3-month-old mice. The effects of pregnancy on skeletal muscles were like those observed in liver: pregnancy enhanced two-fold the regeneration of muscles in both aged (10-month-old) and young animals. The number of satellite cells (muscle stem cells) did not differ between the groups, which indicates that deterioration of the regenerative functions was not due to the exhaustion of the pool of these cells. The decline in the regenerative capacity of muscle tissue with aging is believed to be related to changes in regulation, in particular, to inactivation of the Notch signaling pathway. Pregnancy reverses these changes in aged animals and restores the activity of this pathway to the levels typical for young individuals.

The rejuvenating effect of pregnancy might be explained by the donation of fetal cells capable to differentiate. The regenerative properties of stem cells are well known, and therapeutic effects of stem cells injection after brain, liver, or kidney damage have been well described. Some fetal cells enter the maternal circulation and tissues - a phenomenon known as microchimerism. Fetal cells could be found in the mother's blood and tissues several decades after pregnancy. At present, there is no consensus on the mechanisms that would explain the effects of microchimeric cells on the maternal organism. Two main possibilities are discussed: the first one is regulatory interactions, and the second is direct differentiation of microchimeric cells into a type of cells required for the mother's regeneration. In the first case, a limited number of fetal cells act as coordinators of the regenerative process. For example, fetal cells are believed to regulate inflammatory response by downregulating TGF-β biosynthesis and by directing maternal regeneration toward scar-less fetal-like wound healing. A direct contribution of microchimeric multipotent cells to regeneration has been demonstrated in a damaged heart model. Fetal cells actively migrated into the damaged area of the mother's heart, where they differentiated into fully functional cardiomyocytes that contracted synchronously with surrounding cells. In the absence of damage, the number of fetal cells in the heart was 20-fold less.

A major question remains: what exactly is the contribution of each of the factors to the rejuvenating effect of pregnancy on the maternal organism? The answers to this question will allow to develop clinical approaches for protection of pregnant women and health improvement in the human population in general.