The big news: (1) the active ingredient in E5 is exosomes. (2) Young exosomes retain their ability to rejuvenate across mammalian species lines. Work that remains: (1) calibrate a new generation of methylation clocks. (2) Combine removal of old exosomes with addition of young exosomes. Optimize dosage and timing of treatments.
What is in E5, Katcher’s fountain of youth? From the beginning, it was described as a “plasma fraction”, leaving us guessing. Blood plasma contains thousands of chemical species in tiny quantities that nevertheless have powerful, systemic signaling effects. I thought once the patent was applied for, there would be no more need for secrecy, and the patent would reveal the formula; but the patent application was for a process, not a substance. Katcher and Sanghavi claimed rights for a broad array of substances extracted from the blood in different “embodiments” of the patented technique.
Then, in last month’s preprint posted on BioRxiv, the authors casually revealed the secret, almost parenthetically. “the exosome fraction of the plasma, which we term as E5”. So E5 is not large proteins or small proteins, it’s not non-coding RNAs — according to this document, E5 is made of exosomes and exosomes alone. Independently, a group from the Smidt Heart Institute published in July their finding that exosomes from embryonic plasma had a profound rejuvenating effect, while plasma without exosomes had no effect. (Akshay alerted me to that paper when it came out.)
This new preprint reports on experiments using E5 from piglets to rejuvenate a total of 14 rats. Epigenetic markers, cognitive, physical, and metabolic markers all improved impressively. Some of the limitations of Katcher’s experiments remain to be resolved. Because of small numbers and killing of test rats to obtain tissue samples, we still do not have a strong indication to what extent the restoration of all these markers of age translates into increased longevity.
A major finding is that exosomes from a young pig can rejuvenate an old rat. It is remarkable, very lucky, and certainly not to be taken for granted that such specific signals can cross species lines. What a horrible situation we would have been in if it turned out that the best anti-aging treatment available depended on harvesting the blood of babies.
There were only 14 treated rats in this round of experimentation. 6 males in round 1 were sacrificed so that methylation of their organs could be tested. 6 female and 2 male rats in round 2 were kept alive long enough for their blood to be drawn for methylation testing, but not long enough to determine their lifespans.
Methylation clocks: For 6 treated rats, age 109 weeks, these were the average epigenetic ages by tissue:
The preprint does not provide error bars for these average epigenetic ages, but I expect (based on the published minimum and maximum, and the small sample size) that the 95% confidence intervals are broad, corresponding to rejuvenation by anything between 30% and 70%.
The fact that various tissues are rejuvenated by exosomes indicates that exosomes are a medium for transmission of age information through the body, and that young exosomes in the blood are able to induce system-wide changes in gene expression.
|It’s common for medical gerontologists to frame their work in terms of their favorite evolutionary theory of aging, which is always the 70-year-old, conservative theory that won’t raise any hackles. I’m grateful that Dr Katcher is unabashed about framing his medical research in evolutionary terms, and that his evolutionary theory agrees with mine. Aging is a timed program of self-destruction, coordinated through the body by chemical signals, and evolved because ecological communities are more stable when individuals have predictable lifespans.
The hypothalamus is a neuroendocrine region of the the brain that has been associated with circadian timekeeping (day-night) and also with tracking biological age. The fact that gene expression in the hypothalamus was rejuvenated to a far lesser extent than the other three tissues suggests that hypothalamus plays a special time-keeping role, and that perhaps the hypothalamus is a source for these exosomes in vivo. It may be that the hypothalamus gradually erases half the epigenetic gains from the E5 treatment over time. From this and previous tests, we still have limited data on long-term benefits of E5 treatment.
“we measured the levels of the following biomarkers on 30, 60, 90, 120 and 155 days from the start of the experiment: bilirubin, serum glutamic-pyruvic transaminase (SGPT) and serum glutamic-oxaloacetic transaminase (SGOT) to monitor liver function; triglycerides (TG), HDL and cholesterol to monitor risk of atherosclerosis and heart disease, and liver function as well; glucose to monitor the pancreas and diabetes; and creatinine and blood urea nitrogen for kidney function. The levels of all these biomarkers in the treated old rats were altered towards the values of young rats, without exception.”
(Note: Blood sugar and triglycerides in treated old rats was reduced to levels indistinguishable from untreated young rats. In humans, Type 2 Diabetes is a primary means by which the body destroys itself. — JJM)
Cognitive recovery: Treated rats learned to escape from a Barnes maze faster than old rats.
Oxidative stress: ROS markers were restored close to a young state. Antioxidant enzymes GSH, SOD, and catalase were increased close to youthful levels.
Inflammation: TNF-α and IL-6 were reduced close to the levels of young rats. Nrf-2 was increased close to the level of young rats.
Sarcopenia and muscular fitness: Grip strength of treated rats rose within a week to levels comparable to young controls, and remained strong for at least 30 days.
A new/old biological clock based on glycation of antibodies
IgG is a blood component, another name for “antibodies”. These bind specifically to viruses and bacteria and tag the cells for attack by phagocytes, another immune component. Antibodies are not cells or globules but individual Y-shaped protein molecules with both arms of the Y able to bind to the (same) antigen, and the stem of the Y modified to attract a phagocyte only when the other end signals that it’s “got one”.
Immune aging is a big deal, as it was identified as a prime cause of aging by Roy Walford already in the 1960s. Greg Fahy pushed the theory into a therapeutic concept four years ago with his TRIIM program. With TRIIM, the idea was validated that a rejuvenated immune system can signal rejuvenation to epigenetic age and, presumably, other aspects of aging.
|Dr Fahy also subscribes to a programmed theory of aging, and he personally offered me my first opportunity for a wide audience in the anti-aging community in 2010. Mitteldorf_Evolutionary-Origins-of-Aging.pdf
Hence, “immune age” is a promising target for measuring success of anti-aging interventions, independent of methylation age. This idea has been realized with a Glycan-Age clock. Antibodies in the blood are coated in complex carbohydrate (sugar) molecules, or glycans, and the structure of the glycans carry information, analogously to the way that DNA or proteins carry information via their sequencing. The particular sugars attached to antibodies change with age, and they become important signals promoting inflammation later in life. Clocks based on glycation of antibodies are as old as methylation clocks, though they have been developed to a lesser extent.
It is surprising, perhaps, that glycan age was never measured in the TRIIM study, but Katcher’s group added glycan age to the tests on his treated rats.
“a significant (p < 0.05) reduction in the relative abundance of the pro-inflammatory agalactosylated IgG2a glycoform (G0) was recorded. This was accompanied by a simultaneous upsurge in the antiinflammatory digalactosylated glycoform (G2)”
Is there such a thing as “biological age”?
The preprint includes the statement,
“However, in the context of aging and rejuvenation, it is crucial to differentiate between improved health or organ function, which could be achieved via medication or surgery, and genuine molecular age reversal.”
Intuitively, we all feel that this must be a legitimate concept. In our experience, we can look at most people and form a pretty good idea how old they are, and this translates into reliable expectations about their stamina, resistance to disease, and ability to cope with a fundamentally new environment.
But this is because we’ve mostly encountered natural humans. We know, for example, that movie stars can have expensive surgeries and skin treatments that make them look decades younger. We don’t expect that people who have had a face lift will live longer or have more youthful vitality.
It is perfectly possible to imagine anti-aging treatments that rejuvenate the liver but not the nervous system, or that slash the risk of heart disease but increase the risk of cancer. Hence, it is advisable to report a variety of functional and metabolic tests rather than relying on any one measure of “biological age”.
What is measured by the methylation clocks?
For more than a decade, I have been committed to the paradigm that the body’s gene expression changes with age, and that changing gene expression is a driver of aging. Methylation, as a convenient surrogate for gene expression, would then seem to be a reliable measure of biological age.
But in the last four years, I’ve entertained questions about this paradigm, and I am no longer absolutely committed. The crucial question is: Does gene expression change as a driver of aging, the body destroying itself with inflammation and by scaling back autophagy and repair? Or does gene expression change in response to damage accumulated with age, scaling up repair functions to mitigate damage?
As recently as four years ago, I wrote confidently that the former greatly predominated. Epigenetic changes drive aging. On this basis, I was confident that if you change the methylation clock, you must be changing the driver of aging, and this was bound to lead to longer lifespan.
I have since become convinced that both kinds of changes in gene expression accrue with age, and that there is no easy way to tell the drivers from the responses. I first blogged about this in response to the GrimAge clock. GrimAge is the best predictor we have of remaining life expectancy, but it is based not just on drivers of aging but also on the body’s distressed response to toxins and cell-level damage.
The bottom line is that epigenetic clocks are the best measure of life expectancy that we have, and that they are improving every year, but while uncertainty remains, it is well to supplement methylation clocks with specific tests of metabolic function, stamina, inflammation, and cognitive performance.
Six new methylation clocks
Prof Horvath (presumably it was Horvath) trained four new clocks on rat tissues plus two new clocks that were cross-trained on humans and rats. Since rats have a maximum lifespan of 3.8 years and humans 122.5 years, one logical way to train a clock that works for both species would be to scale the rat ages and human ages so that one rat year is about 32 human years. This is just what Horvath did.
But in addition, he trained a second combination clock (rat and human) to read in absolute years. This presumes there are changes in epigenetics of a very old rat (say, 3 years) that are parallel to epigenetic changes taking place in a very young human (also 3 years). A strange concept, indeed! What kinds of changes would we expect to take place in a 3-year-old rat and simultaneously in a 3-year-young human? My guess is that the training program is selecting those sites that behave very differently in rat and human, and that results derived for rats with this clock will not translate.
But I would suggest that even the time-scaled clock (based on 32 years of human life = 1 year of rat life) is suboptimal. This is because different events in development and aging may occur at different points in the life cycle. A male rat reaches sexual maturity at about 8 weeks. Using a 32x multiplier, this would correspond to a 5-year-old boy. But in fact, boys don’t reach sexual maturity until well into their teens.
Prof Horvath has been developing methylation clocks for more than a decade, and his work has had a deep impact on the science of aging. To my knowledge, all his clocks are based on straight-line changes in methylation over a lifetime, and I have counseled that this is an unnecessary limitation on the clocks’ accuracy. More realistic clocks can be constructed based on parabolic curves, or even the simple expedient of grafting two straight lines together to create a “spline”. He has been creating cross-species clocks for the last few years, and to my knowledge these have all been based on a single scaling factor for each species. My recommendation is that here it is even more important to project the life cycle of one species onto another using a flexible mapping, pegged at several intermediate time points which are determined in a mathematical optimization process. Cross-species clocks will be of increasing importance as we translate mouse studies to develop experimental protocols for human medicine.
A few paragraphs up, I referred to a major issue in methylation clocks: A given intervention affects a set of methylation sites that are all associated with younger age. Which of these are actually beneficial, leading to life extension; and which represent reversion of the body to a younger state when it had less damage to deal with? For the latter, the younger state corresponds to repair mechanisms that are less active (presumably because they are less needed), and “rejuvenation” to this state could actually lead to shorter life expectancy.
To date, the best methylation clocks have been calibrated by how well they predict future life expectancy. This is, of course, very useful information, but it is all based on naturally-aged individuals. Our interventions that reduce methylation age may not increase lifespan.
In two previous rounds of Katcher’s experiments, males experienced more epigenetic rejuvenation, but they were killed before their lifespans could be measured; while females experienced less epigenetic rejuvenation, and their lifespans were measured to be only modestly extended relative to controls. Unexplained is the fact that the controls lived far longer than Sprague-Dawley rats are usually expected to live, and that a single treated rat lived far longer than 7 others that received the same treatment. These are all curious observations that warrant further investigation.
The failure of Katcher’s rats last year to live as long as their methylation age predicted should be a motivator for future research, studying not just the interventions but also the clocks.
We can try to classify different methylation sites based on theory; but if we want to address the question head-on, we need different experimental interventions that affect different CpG sites. Then we can see which CpG sites, when artificially methylated or demethylated, lead to longer lifespan. Hence, Katcher’s experiments provide a unique opportunity to determine in practice: which methylation sites, when reverted to a younger age state, actually extend lifespan? This is an experimental solution to the stickiest problem in aging clocks: which epigenetic changes correspond to drivers of aging and which correspond to responses to damage?
Do we have methylation for Sima, Katcher’s record-setting long-lived rat? If so, we might compare methylation profiles for Sima to his cage-mates that were treated the same way, yet died many months earlier. This is a start, but what we really need is an experiment in which about a hundred rats (M and F) are treated with different E5 protocols, their methylation profiles are monitored over time, and they are also permitted to live until their natural end. Manipulated methylation profiles can then be correlated with life expectancy, and from this a uniquely useful methylation clock can be constructed.
For me, the bottom line is the same thing I’ve been saying since the beginning: Katcher’s research is the most promising line of investigation in the field of anti-aging medicine, and it is a travesty that it is taking place under a single investigator’s direction, and with very limited resources. It will take extensive lab resources to optimize E5 dosage and timing, and to try combining addition of young exosomes with removal of old exosomes. This same research can be the basis for the first methylation clock that is known to predict lifespan for rejuvenated animals.
I would like to see the abundant money that is now in anti-aging research re-directed into rejuvenation via exosomes, and I would like to see Katcher sharing his techniques openly with the research community.