Is there a master timekeeper, upstream of the methylation clock?

I have promoted the idea that aging is programmed and that the program is epigenetic. Hence epigenetic age is fundamental. But what is it that imprints epigenetic age on the chromosomes and keeps it updated? Is the “methylation clock” responding to a higher authority, a separate clock which coordinates epigenetic age throughout the body? Do epigenetic clocks in different tissues talk to each other? Such questions are important not just for theoretical understanding, but also because they have two practical consequences. (1) Can we rejuvenate the body with system-wide signaling, or do we have to de-age cell-by-cell?  (2) Can we be confident that if we set back the body’s methylation age the person will feel younger and live longer? 

I have been reading and thinking about these questions for several weeks, and I can report no clear answers.


Is aging a cell-by-cell deterioration, or is it orchestrated at the level of the whole body and managed through signal molecules in the blood? If pressed, I think everyone would have to admit there is some of each, and differences within the community of aging biologists are about the relative importance of the former vs the latter.

One thing I think we ought to be able to agree on is that the system level, including signal molecules in the blood, makes a vastly more accessible target for anti-aging interventions. Repairing the body cell-by-cell is a daunting proposition; whereas modifying levels of signal molecules in the blood is a piece of cake, once we identify those molecules and determine their optimal youthful levels. The words “low-hanging fruit” come to mind, as well as “Pascal’s wager”.

If there are multiple, independent aging clocks, it is probable that the one that registers the oldest age is the one that can kill us, independent of the others. To make the big leaps in life extension that we are looking for, we probably will need to reset all the clocks. How much do the cell-level and system-level clocks talk to each other? How much progress can we expect to make by working at the (more accessible) system level without addressing the (more challenging) cell-level aging?

Why is the preponderance of research devoted to aging at the cellular level? A small part of the explanation comes from scientific inertia; aging was understood in terms of increased cellular entropy for many years, whereas the paradigm of central control remained in a Russian backwater until publication of the Stanford parabiosis experiments in 2005. A larger part of the explanation has been the infusion of venture capital into aging research in the last decade. You can’t patent hormones and you can’t make money from rebalancing blood levels of the body’s native signal molecules. I believe that the profit motive has deeply corrupted aging research, as it corrupted medical research through the previous century.

I am passionate about these issues, but I leave them aside to talk about questions of fundamental interest: Differential gene expression—epigenetics, and methylation in particular— seem able to change the body’s age state. It seems clear that gene expression is the primary way in which the age state of the body is transmitted and coordinated system-wide. But is gene expression the end of the line, the ultimate upstream aging clock? Or is there a “higher authority” that keeps track of time and programs the body’s methylation, etc accordingly? Does the epigenetic state of the body constitute an autonomous time-keeping mechanism, or is there a time-keeping reference clock, perhaps in the hypothalamus, which dictates the body’s age through secretions, and distant cells respond to these secretions by adjusting their methylation patterns?

And, if the answer is that methylation constitutes an independent clock, does that clock advance cell-by-cell independently, or does gene expression at the cell level export proteins that coordinate methylation age across the body?

I don’t have answers, but several experiments bear on these questions, and offer a nuanced outlook.

The practical question

We need measures of biological age in order to efficiently tell us when we are on the right track with an anti-aging intervention. Methylation clocks are presently the best technology we have for measuring biological age. So, can we be confident that if an intervention sets back the methylation clock that the intervention really is making a person (or animal) younger?

Reasons to think yes:

  • Methylation clocks track chronological age better than any other biomarker
  • Some of the difference between methylation age and chronological age is meaningful. It correlates with mortality. In other words, each of the major methylation clocks is a better predictor of life expectancy than chronological age. Remarkably, this is true even for the clock algorithms that were trained only on chronological age.
  • There are theoretical reasons for believing that epigenetics is the primary driver of aging, so that methylation changes may actually be close to the causal nexus of biological age. (This conclusion is especially cogent for theorists like me who believe that aging is an evolutionary program; however, there are also prominent scientists in the field who reject programmed aging but embrace epigenetics as a primary driver of aging.)

Two things that could go wrong:

  • There could be a higher authority, a centralized clock that sets up the methylation state. If this is the case, then setting back the body’s methylation age may be temporary, and the methylation state will revert to the age programmed by a central clock. (Cavadas and Cai have adduced evidence that aging signals are transmitted from the hypothalamus.)
  • In the worst case, methylation changes with age could be an adaptive response when the body senses the accumulation of damage. In other words, the body changes its gene expression when damaged because it is working overtime to repair that damage. In this case, resetting methylation state to a younger age just makes the body less able to cope with the consequences of aging and actually shortens lifespan.

Evidence from parabiosis

In parabiosis, a young mouse is surgically joined to an older mouse of the same genotype. Tissues of the old mouse respond by becoming functionally younger. Since this 19th-century finding was brought to the modern scientific community, the search has been on for chemical factors in the blood that either promote aging or promote youth. [read more].

The parabiosis phenomenon and related findings in rejuvenation through blood plasma transfusions has led to a paradigm that says aging is coordinated throughout the body by signals in the blood. To the extent that single cells age, this is happening under central control, and the process can even be reversed if the cell is exposed to the right signals.

BUT

Evidence from bone marrow transplants

Bone marrow transplants are the most powerful available treatment for leukemia, and are also applied for some rarer diseases. The bone marrow comes with the epigenetic age of the donor, and thus the (white) blood cells subsequently generated by the transplanted bone marrow also carry age information. Several different studies [ref, ref, ref, ref] have found consistently that the white blood cells (and presumably the bone marrow from whence they came) retain the age signature of the donor. The donor may be younger or older than the patient. In either case, the methylation age of the patient’s white blood cells—post-op and for years afterward—remains keyed to the donor and does not correlate significantly with the patient’s age.

The lesson of parabiosis experiments was supposed to be that cell aging is not cell-autonomous, but rather a response to signals in the blood that instruct the cells what age to be. Young somatic cells could be aged rapidly in an old blood plasma environment, and — more impressively — old somatic cells could be made younger in a young environment.

Now we have a series of bone marrow transplant studies where the methylation age of the donor is the determining factor, not the patient into which the marrow was transplanted. Bone marrow contains the stem cells from which blood cells grow. Blood cells turn over every few months and they represent an accessible tissue sample which reflects the age state of the bone marrow in approximately real time.

“We found that the DNAm age of the reconstituted blood was not influenced by the recipient’s age, even 17 years after HSCT, in individuals without relapse of their hematologic disorder.” Soraas et al (2019)

This seems on its face to contradict our paradigm from parabiosis that says cell age is not cell-autonomous, but is programmed by the environment. How can we interpret the two results together? Some possibilities…

  • Maybe only differentiated somatic cells are susceptible to age programming by plasma proteins, and not stem cells.
  • Maybe these stem cells are providing the biochemical environment in the plasma. Maybe the stem cells and the white blood cells that they generate are the agents that secrete the plasma proteins responsible for sending age signals.
  • Please think creatively about other possibilities.

Another result from these bone marrow studies

Consistently, the blood cells get older after transplant, whether they are transplanted from young-to-old or from old-to-young. This says two things. First, the point of comparison is the donor age, i.e., the age of the cells pre-transplant, and not the age of the patient who is associated with the systemic environment. Second, the cells seem to age rapidly after transplant, as measured by the methylation age. From there, the age of the cells may (in some studies) revert slowly to their original age trajectory over a period of several years.

Why the rapid methylation aging? It seems like a good guess that the rapid aging initially comes from high rates of reproduction in these transplanted cells that are generating a whole new source of much-needed blood. Could this be a link between telomeres and methylation age (which previously were found to be inversely correlated? Or is there another mechanism by which stem cells keep track of the number of times they have divided asymmetrically?

Am I the only one asking these questions?

Already a decade ago I was thinking about the question How Does the Body Know How Old It Is? Questions about time-keeping mechanisms and coordination of age information through the body go hand-in-hand with conceptions of aging as a programmed phenomenon, and perhaps the prejudice against programmed aging helps to explain the fact that few aging researchers are thinking in this way. A welcome exception is this article by Argentine gerontologists, which I was delighted to discover just yesterday. Lehmann et al:  Hierarchical Model for the Control of Epigenetic Aging.

Although there is evidence suggesting that the cellular epigenetic clock possesses an intrinsic ticking rate [ref, ref, ref] multiple observations at organismal level in humans and other mammals lead to the inference that in vivo, the ticking rate of the clock in tissues is synchronized by a master pacemaker.

Lehmann cites as prima facie evidence for this

For a given chronological age, it was found that in DNA samples taken from whole blood, peripheral blood mononuclear cells, buccal epithelium, colon, adipose, liver, lung, saliva, and uterine cervix, Horvath’s algorithm read essentially the same epigenetic age, the only exceptions being some brain regions and very few other organs.

In addition, she cites Katcher’s success in rejuvenating rats (and their diverse organs) using only a set of intravenous signals. The article goes on to propose a model in which there are four time-keepers in the body, coordinated by signals in the circulatory systems. The four are:

  1. Methylation
  2. Light-sensing and neural processing
  3. Neuroendocrine signaling (esp the suprachiasmatic Nucleus of the Hypothalamus)
  4. The Immune system, including thymic involution

Curiously, she does not include the replication counter implicit in telomere shortening, which Fossel, Blasco and other luminaries have adduced as the primary source of aging. I also would add that the hypothalamus is the best candidate we have, not just for one aging clock among several, but as a central, coordinating organ.

Fig. 1. Proposed organismal regulatory network in mammals. The diagram includes the autonomic nervous system (ANS, acting via neurotransmitters), the neuroendocrine system (NES, acting via blood-borne hormones), the immune system (acting via blood-borne cytokines and thymic hormones), the circadian clocks (acting via blood-borne hormones and neurotransmitters) and a putative pathway connecting the neuroendocrine network to the DNAm clock in organs and cells. All networks act on peripheral organs. Inset- Bidirectional interactions among all networks including (in red) the hypothetical DNAm network.

 

In addition to Lehmann, there is a 2021 review by Raj and Horvath, speculation from the horse’s mouth. They note that all the Horvath clocks are based on small differences in % of cells methylated at a given site (conventionally notated as β).

Increase in epigenetic age is contributed by changes of methylation profiles in a very small percent of cells in a population.

One way to interpret this fact (my speculation, not R&H) is that immune sensitivity, (anti-) oxidation, and inflammation are all under tight homeostasis in the body, because these are sensitive functions, balanced on a knife edge between insufficient protection and self-destruction. It is easy to tip the balance over toward self-destruction with small changes in the set point for a few signal molecules in blood plasma.

Another way to interpret this (again, my speculation) is that it is only a handful of cells at the tail end of the distribution that go over an edge into a state where they cause all the damage of aging. This hypothesis is consonant with the story about short telomeres, cell senescence, SASP, and the powerful benefit of senolytics. However, a big hole in the narrative is that it requires a set of CpG’s that would be capable of precipitously tipping the cell over into a toxic state. We know that critically short telomeres can do this, but there is no study yet of methylation-induced cell senescence. R&H speculate about such a mechanism connected with PCR=Polychrome Repressive Complex.

Raj and Horvath also stress the continuity between epigenetic changes that begin in utero, associated with development, and the changes that lead to senescence late in life. Blagosklonny as well has emphasized this point.

“Collectively, these five features of DNA methylation allow one to summarize with some degree of certainty that epigenetic ageing is a measure of change of epigenetic heterogeneity, contributed by a relatively small percentage of cells, seemingly in line with developmental processes that are conserved across species and begins very soon after conception. This seemingly inescapable deduction provides us with a reference point against which models and hypotheses can be measured.”

If I may carry the logic of these two experts one step further, I would emphasize the role that methylation has in determining what hormones and enzymes are secreted into the blood. Therein lies the possibility that intracell methylation clocks are coordinating, both with other cells and with other clocks, via signal molecules in blood plasma.

Other provocative findings that we might hope to integrate into a theory of aging

The methylomes of naked mole rats age at a normal rate, but the phenotypes of the rats themselves show no signs of age [ref]. Males and females age epigenetically in somewhat different ways [ref].  Methyl donor molecules in the diet can lead to a younger methylome, with benefits both for hyper- and hyomethylated regions (validated for MTHFR snps only) [ref]. When human fibroblasts are reprogrammed (with RNA) to turn them into neurons, they remember their Horvath age even after forgetting their identity [ref]. BMI is associated with accelerated methylation aging [ref]. Mice challenged with a high-fat diet can be brought back to normal weight with a normal diet, but accelerated methylation aging persists [ref]. Cessation of smoking decreases Hannum and Horvath DNAmAge [href]. The methylation shadow cast by years of smoking is a better predictor of subsequent morbidity and mortality than the smoking history itself [ref]. Methylation image of telomere length is a better prediction of age and mortality than is telomere length itself [ref]. Pregnancy increases Hannum Age, DNAmAge, and PhenoAge [ref].
(Apologies to Rafil Kroll-Zaiti)

Bulletin

Katcher has been conducting a longevity trial for rats treated with E5 (background story here). Partial results suggest that treated rats are living statistically longer than untreated, but not as much as you would expect if the greatly reduced methylation age indicated full rejuvenation. The results are preliminary, and I will publish a full analysis in this space as soon as I can get the detailed dataset.

The finding, if validated, suggests that multiple clocks in the body are not completely synchronized, and the “fastest clock wins”, meaning that it kills the animal no matter what the other clocks may say.

Conclusions

I am disappointed as you are in not being able to provide fundamental answers, but I hope that (together with Lehmann, Goya, Raj, and Horvath) we have provided a framework and a set of questions that can guide fundamental research. Very few other researchers are addressing these questions, and the answers will be crucial both for devising effective interventions and also for measuring the effectiveness of interventions that we already have.

I recommend this book for every life extensionist

Surviving Death: a journalist investigates evidence for an afterlife
by Leslie Kean

 

Readers of this blog are interested in life extension. We relish the experience of being alive, and we struggle with dread of death, and there is diversity among us how much relish and how much dread we harbor.

We believe in the methodology of science, and we look to biological science for solutions that will preserve our bodies from the ravages of age. Most of us subscribe to the scientific consensus that our bodies support our experience, our brains engender our consciousness, and without our brains, we would be nothing.

How do we respond when we are presented with scientific evidence that the brain is not the source of consciousness; that experience can exist in the absence of neural activity; that death of the body will change but will not necessarily end the experience that we relish?

If we believe in Science with a capital S, if we have faith in the community of scientists and the conclusions in which a great majority of scientists concur, we say, “This is not worth my time. I know it must be wrong. I’m not going to think about it.”

If, on the other hand, we believe in science with a small s—the scientific method, the gathering of evidence and the testing of hypotheses against all the available evidence—then we read Leslie Kean’s book, and our mouths hang agape, and we wonder how we can ever reconcile what she reports with the picture of the world that has served us so well all our lives.

The material in this book is so radical that if we accept that even some portion of it is reliable, and if we are honest and courageous enough to explore the consequences, then we must rethink our relationship to life extension and then begin to overhaul our relationship to life.

Reincarnation

Kean begins with a story that is stunning enough in its own right, but previously well-established by other researchers. Carol Bowman first interviewed the Leininger family and documented the story of their son, who called himself “James the Third” because he had previously lived the life of James Houston, Jr, a World War II fighter pilot who was shot down over Japan in the Battle of Iwo Jima (1945). As a three-year-old (in 2001), James the Third recognized and name parts of the plane that Houston piloted and the aircraft carrier from which he was deployed. From a period photo, he was able to identify by name other members of Houston’s flight crew as well as his two sisters.

The Leininger case is particularly compelling because it is well-confirmed and includes dramatic detail. But in other respects, it is representative of thousands of stories that have been collected at Univ of Virginia. Most of them involve a sudden, violent death in a previous life, leaving a lingering sense of incompleteness. Frequently, children have knowledge of details from their past lives, and occasionally, children will speak in languages that they were not exposed to in their present reincarnation. It’s called xenoglossy

It is natural to take thees stories as support for a traditional (Buddhist or Hindu) account of reincarnation. In that narrative, each of us is an immortal soul, and we evolve through a series of excursions when we assume physical form for the purpose of education via broadened experience. For the most part, we forget our past during each incarnation, but sometimes memories leak through the veil. Leinginer’s story validates part of this, but is subject to other interpretations as well. Memories may be transferred without any continuity of personality across incarnations. Children may spontaneously experience remote viewing or clairvoyance. If reincarnation is a thing, it may be rare or common, and not necessary universal. The story cracks open our dogmatic commitment to a materialistic perspective, but it does not compel a particular alternative.

Can such stories be consistent with the “conservative” view that consciousness is generated by the brain, with all knowledge and experience completely dependent on the physical brain? Only if we postulate new physics that transmits information, not attenuated by time or space, and that our nerves are evolved to take advantage of this yet-to-be-discovered effect. In my mind, this is more of a stretch than simply to adopt William James’s view that the brain is a transducer, not a generator of consciousness.

Near death Experiences

For Kean’s book, reincarnation is just an opener, and as her accounts stretch the limits of our reality to the breaking point, her voice becomes increasingly familiar and convincing. In the last chapters, she relates accounts in the first person, and, fantastic though they are, we find it hard to dismiss her because she has used 300 pages to earn our trust.

Near death experiences are another well-plowed regime for anyone who is open to reading the literature. People in extremis have memories of experience that took place while their EEG plots (electrical activity in the brain) were flatlined and they were technically dead. These often include tunnels with a white light at the end, meetings with deceased relatives, and spirit guides. On the one hand, the cases are more specific in what they can tell us about what it’s like to be dead. On the other hand, they are easier to dismiss as illusions or false memories or hallucinations of an oxygen-starved brain. Kean reminds us of the occasional cases where people with brains that are technically dead remember details of their resuscitation, the doctors or nurses in their hospital room. More occasionally, people report visiting distant relatives during this time. And there is just one case where a woman on an operating table experienced floating up from her body and seeing a sneaker on the roof which could not be seen from any point inside the hospital or from the ground. Her description of the sneaker was later verified.

Disciplining herself to remain objective, Kean acknowledges that reports from people who have had NDEs (and actual deathbed experiences) cannot prove that consciousness outlives the body. But she notes a general similarity between what NDEers report and the accounts of children when they talk about the time between incarnations.

Communicating with the dead

To appreciate mediumship, Kean opines, you have to be there. She incorporates a chapter by a credentialed researcher about rigorously controlled studies, but only after she relates in detail the experiences she had contacting her departed brother and another lost friend through three separate mediums. Some 80% to 90% of the details they report are accurate, including names and recall of specific conversations. But (says Kean), this can’t begin to convey the emotional intimacy of feeling a departed person’s personality coming through in the style and language of the communication. For each of the two deceased persons, Kean reports personal details known only to the deceased and herself, which the medium accurately references.

The medium who makes sceptics pant and tremble like nervous horses |  Europaranormal

Mrs. Piper, 1857-1950

Is this evidence that the medium is in touch with a still-existing spirit of the deceased? Kean and her academic expert both admit that this is a difficult question. If the information is known to the sitter, then the medium could have obtained it through telepathy with the living (and if it is not known to the sitter, how can it be verified?) But mediums themselves report that the way information comes to them feels very different from telepathy, and EEGs of the same person doing psychic readings and mediumship seem to corroborate this.

Finally, Kean reports details of the compelling story of a man whose great uncle died on a battlefield of the Great War contacted him through a medium forty years later and related the exact coordinates of the unmarked grave site in which he was buried.

Physical appearance of the dead in seances

For some reason, it is easier for me personally to accept non-physical transfer of information than to believe in the physical incarnation of ghosts or specters. But by this point in the book, Kean has established herself as such a credible witness that these fantastical tales of her personal experience leave me baffled and perplexed.

The science that we understand gives us the technology for transportation and communication, for comfort and convenience. But the science that we don’t understand imparts to us a sense of awe and wonder, and motivation to continue our investigations in new and creative ways.

I am less concerned than Kean and her experts with distinguishing between explanations from telepathy and from post-mortem survival for the phenomena they describe. The big message for me is one of non-local mind. Once it has been established that mind has an existence that cannot be explained by functions of the brain—that, indeed, a part of the mind’s awareness appears to be untethered to any spatial location—for me, there is no longer any reason to suppose that the mind dies with the brain.

I carry with me from early childhood the memory of repeating the phrase, “I am Josh” and savoring an intuitive conviction of its absurdity. A part of me that was deeper than experience seemed to know that “I” am an abstract observer of this physical universe, and not a piece of matter within it. Today, this is just an intellectual curiosity, as I have long ago lost the cosmic expansiveness of the child’s experience.

I plan to continue pursuing life extension as a celebration of life rather than the hopeful forestalling of a dread event. And the sense of mystery and wonder that these anomalous phenomena provide continues to enhance the time I have on earth.

Anne Jeffreys, Glamorous Ghost of '50s TV, Is Dead at 94 - The New York  Times

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