Part 3 of Rejuvenation therapy and sulforaphane

Part 1 focused on the study’s clinical biomarkers. Part 2 highlighted its epigenetic clocks. Now we’ll look at rejuvenation of cognitive function.

Charts for this study’s most relevant human aging applications – measured by the new human-rat relative biological age clock – were in supplementary data due to combining the study’s untreated tissue samples into clock training data. Reanalyses showed:

“Using the final version of the epigenetic clocks, we find that the treatment effects become even more significant especially for the hypothalamus.”

Human-rat relative clock percentages of rejuvenation were:

  • “Blood 70.6%
  • Liver 79.4%
  • Heart 61.6%
  • Hypothalamus 20.9%”

The Discussion section addressed hypothalamus rejuvenation:

“Why does plasma fraction treatment not reduce brain epigenetic age by the same magnitude as it does the other organs? We can only begin to address this question after having first understood what epigenetic aging entails.

As it stands, our knowledge in this area remains limited, but it is nevertheless clear that:

  1. Epigenetic aging is distinct from the process of cellular senescence and telomere attrition,
  2. Several types of tissue stem cells are epigenetically younger than non-stem cells of the same tissue,
  3. A considerable number of age-related methylation sites, including some clock CpGs, are proximal to genes whose proteins are involved in the process of development,
  4. Epigenetic clocks are associated with developmental timing, and
  5. Relate to an epigenomic maintenance system.

Collectively, these features indicate that epigenetic aging is intimately associated with the process of development and homeostatic maintenance of the body post-maturity.

  • While most organs of the body turnover during the lifetime of the host, albeit at different rates, the brain appears at best to do this at a very much slower rate.
  • While most tissues harbor stem cells that are necessary for replenishment and turnover, stem cells in adult brain have only been detected in a defined and very limited area of the subventricular zone, olfactory bulb (in rats), hippocampus and hypothalamic proliferative region.

As such, if plasma fraction treatment’s rejuvenating effect is:

  • Mediated through the process of development and
  • Involves tissue stem cells

then its effect on the epigenetic age of the brain would appear to be modest, which indeed it does.

It is to be noted however, that improving brain function does not depend on neurogenesis as much as it does on synapse formation and factors such as NMDA receptors which decline in density with age.

Assessment of plasma fraction treatment on cognitive function (learning and memory). Rats were subjected to Barnes maze test – nine consecutive days of test where the time (in seconds) required by the rats to find the escape hole (latency) was recorded and plotted. The error bars depict 2 standard errors.

Within a month of plasma fraction treatment, the rats exhibited significantly reduced latency to escape, i.e., they learned and remembered better. After the second month, the treated rats began with a slightly reduced latency period compared to the untreated old rats, and once again, they learned much faster than the latter.

By the third month, it was clear that treated rats remembered the maze much better than the untreated ones even from the first day of test as their latency period was significantly reduced and by the end of the test period their latency was similar to that of the young rats. This feature was sustained and repeated in the fourth month.”

Not sure why there’s a 62-day gap between “Second month” and “Third month.” Maybe it had something to do with “First month” starting 10 days after the first treatment and “Third month” similarly starting 13 days after the second treatment?


Regarding cognitive function, a 2019 Italian paper Polyphenol Health Effects on Cardiovascular and Neurodegenerative Disorders: A Review and Meta-Analysis analyzed pathetic results of experiments with polyphenols other than broccoli sprout compounds:

“Current treatments to halt cognitive decline are limited to counteract symptoms and have a positive impact on cognition and behavior only in a transient manner, without affecting the underlying pathology.

Although some polyphenols might improve specific markers of cardiovascular risk and cognitive status, many inconsistent data are present in literature. Therefore, definitive recommendations for the use of these compounds in the prevention of cardiovascular disease and cognitive decline are currently not applicable.”


Many of us know older people who lived well past the time of good cognitive function. We see how they’re helpless and dependent. We see how others take advantage of them as they decline past the end of their healthspan.

We can make personal plans for that day, sure. But let’s also put some urgency into applying this study’s new human-rat relative biological age clock, and make:

“A step change in aging research. Although conservation of aging mechanism could be equally deduced from the existence of multiple individual clocks for other mammals (mouse, dog), the single formula of the human-rat clock that is equally applicable to both species effectively demonstrates this fact.”

Part 2 of Rejuvenation therapy and sulforaphane

A rejuvenation therapy and sulforaphane focused on the study’s clinical biomarkers and not its biological age measurements. This Part 2 curation of the study highlights its epigenetic clocks because:

“While clinical biomarkers have obvious advantages (being indicative of organ dysfunction or disease), they are neither sufficiently mechanistic nor proximal to fundamental mechanisms of aging to serve as indicators of them. It has long been recognized that epigenetic changes are one of several primary hallmarks of aging.

DNA methylation (DNAm) epigenetic clocks capture aspects of biological age. The discrepancy between DNAm age and chronological age (term as ‘epigenetic age acceleration’) is predictive of all-cause mortality. Pathologies and conditions that are associated with epigenetic age acceleration includes, but are not limited to, cognitive and physical functioning, centenarian status, Down syndrome, HIV infection, obesity, and early menopause.

The [new] human-rat clocks apply to both species. The two human-rat pan-tissue clocks are distinct, by way of measurement parameters. One estimates absolute age (in units of years), while the other estimates relative age, which is the ratio of chronological age to maximum lifespan; with values between 0 and 1. This ratio allows alignment and biologically meaningful comparison between species with very different lifespan (rat and human), which is not afforded by mere measurement of absolute age.

Relative age estimation was made using the formula: Relative age = Age / maxLifespan where the maximum lifespan for rats and humans were set to 3.8 years and 122.5 years, respectively.”

From Supplementary Table 3, old control and old treatment subjects were males 109 weeks old, 55% of their maximum lifespan (109 / 197.6). Young control subjects were males 30 weeks old, 15% of their maximum lifespan.

The money charts for this study’s human aging applications – measured by the new human-rat relative biological age clock – were buried in Supplementary Figure 12, bar plots M through P:

“Human-rat clock measure of relative age defined as age/maximum species lifespan. Each bar-plot reports the mean value and one standard error.”

From Supplementary Table 8, the percentages of rejuvenation for the above bar plots, calculated as “(100 * (1 – Old Treated / Old Control)” were:

  • “Blood 70.6%
  • Liver 79.4%
  • Heart 61.6%
  • Hypothalamus 20.9%”

Let’s return to clinical biomarkers for comparison purposes. The current study measured pro-inflammatory cytokine IL-6 blood plasma levels at every time point, but didn’t publish numbers. Bar plots and narrative were:

“Inflammation is an important response that helps protect the body, but excess inflammation especially in terms of duration of this response can have very detrimental effects instead. This occurs when inflammation fails to subside and persists indefinitely; a condition referred to as chronic inflammation, which for reasons not well-understood, increases with age and is associated with a multitude of conditions and pathologies.

The levels of two of the most reliable and common biomarkers of chronic inflammation, interleukin 6 (IL-6) and tumor necrosis factor α (TNF-α), are found to be considerably higher in old rats, and these were very rapidly diminished, within days by plasma fraction treatment, to comparable levels with those of young rats. This was especially stark with IL-6.

In time, the levels of these inflammatory factors began to rise gradually, but they were once again very effectively reduced following the second administration of the plasma fraction on the 95th day.”

Let’s compare the above IL-6 graphic with IL-6 concentration improvements of our 2018 model clinical trial, Effects of long-term consumption of broccoli sprouts on inflammatory markers in overweight subjects, calculated as (100 * (1 – Day _ mean / Day 0 mean):

Mean pg/ml | % improvement | Period | Broccoli sprout consumption

  • 4.594 | 0% | Day 0 | “One week before the beginning of the intervention period, subjects were asked to avoid the consumption of Brassica vegetables (broccoli, radish, cauliflower, Brussel sprouts, mustards, among others) and their derived products.”
  • 1.748 | 62.0% | Day 0 to 70 | Subjects ate 30 g raw broccoli sprouts every day, and stopped eating them after Day 70.
  • 0.896 | 80.5% | Day 0 to 90 | “After the intervention period, a follow-up recovery period for all subjects continued for another 90 days with no ingestion of broccoli sprouts.”
  • 2.170 | 52.8% | Day 0 to 160 | Subjects had not eaten broccoli sprouts after Day 70.

Results between the studies were similar in that:

  1. IL-6 levels improved during early treatments through rat Day 8 and human Day 70, respectively.
  2. IL-6 levels continued decreasing shortly after treatments for 7 days (through rat Day 15) and 20 days (through human Day 90), respectively.
  3. IL-6 levels rose after rat Day 15 and human Day 90, respectively, but were still significantly below Day 0 values at rat Day 95 and human Day 160.

The current study measured Nrf2 but didn’t publish numbers. Bar plots and narrative were:

“The reduction of these inflammation markers is consistent with the profile of the nuclear factor erythroid 2-like 2 protein (Nrf2), which plays a major role in resolving inflammation, in part by inhibiting the expression of IL-6 and TNF-α. Nrf2 also induces the expression of antioxidants that neutralizes ROS [reactive oxygen species], which is also a significant feature in inflammation.”

A PubMed search on “nrf2 sulforaphane human” didn’t turn up relevant 2020 human in vivo studies. I disregarded reviews, cancer studies, disproven hypotheses, and other compounds listed in the below graphic.

I won’t repeat the entire Nrf2 section from the Part 1 curation, just one graphic and paragraph:

It [sulforaphane] is not only a potent Nrf2 inducer but also highly bioavailable [around 80%], so that modest practical doses can produce significant clinical responses. Other Nrf2 activators [shown in the above image] not only lack potency but also lack the bioavailability to be considered as significant intracellular Nrf2 activators.”


As noted in Reviewing clinical trials of broccoli sprouts and their compounds, there are no sulforaphane clinical trials that also use epigenetic clocks. Broccoli sprouts and their compounds’ effects on human aging is an area that hasn’t drawn attention and funding.

What are the effects that broccoli sprouts and their compounds may have on human aging? With this new human-rat relative biological age clock, researchers can get reliable answers from rat studies, with human clinical trials needed only to confirm those findings!

As rejuvenation research continues, what could people do easily, cheaply, and today for our long-term selves? Don’t know about the hypothalamus, but our blood, liver, and heart biological ages may decrease as we reduce inflammation and oxidative stress by eating broccoli sprouts.

I’m at a similar relative percentage of species maximum lifespan as were the study’s old subjects. It’s my choice as to what my healthspan will be.

There isn’t evidence today to definitively say that changing my inflammatory phenotype with broccoli sprouts has had / will have rejuvenation effects on biological ages of my cells, organs, and body. But if eating broccoli sprouts every day not only reduces chronic inflammation and oxidative stress as expected, but also makes me younger, I could probably learn to live with that. 🙂

Continued with Part 3 of Rejuvenation therapy and sulforaphane.

A rejuvenation therapy and sulforaphane

The founder of the epigenetic clock methodology with the coauthor of Aging as an unintended consequence released a 2020 rodent study “Reversing age: dual species measurement of epigenetic age with a single clock” at https://www.biorxiv.org/content/10.1101/2020.05.07.082917v1.full.pdf:

“We employed six clocks to investigate the rejuvenation effects of a plasma fraction treatment in different rat tissues. Two of these epigenetic clocks apply to both humans and rats.

The treatment more than halved the epigenetic ages of blood, heart, and liver tissue. A less pronounced, but statistically significant, rejuvenation effect could be observed in the hypothalamus.

The treatment was accompanied by progressive improvement in the function of these organs as ascertained through numerous biochemical/physiological biomarkers and behavioral responses to assess cognitive functions. Cellular senescence, which is not associated with epigenetic aging, was also considerably reduced in vital organs.

Plasma fraction treatment consists of two series of intravenous injections of plasma fraction. Rats were injected four times on alternate days for 8 days. A second identical series of injections were administered 95 days later. In its entirety, the experiment lasted 155 days.

Overall, this study demonstrates that a plasma-derived treatment markedly reverses aging according to epigenetic clocks and benchmark biomarkers of aging.”

The study hasn’t been peer reviewed, so can’t be viewed yet as conclusive. Given that researchers’ single-most valuable asset is their reputations, though, will the findings have major revisions?


I was alerted to the study by Josh Mitteldorf’s blog post Age Reduction Breakthrough, who did his usual excellent curation:

“Most of the explosion in aging research (and virtually all the venture capital startups) are looking to treat aging at the cellular level. Their paradigm is that aging is an accumulation of molecular damage, and they see their job as engineering of appropriate repair mechanisms.

The truth, as Katcher [the lead lab researcher] understands it, is that, to a large extent, aging is coordinated system-wide via signal molecules in the blood. The problem is that there are thousands of constituents represented in tiny concentrations in blood plasma, but conveying messages that cells read. Which of these are responsible for aging?

The two-species clock[s] was [were] a significant innovation, a first bridge for translating results from an animal model into their probable equivalent in humans. Besides the methylation clock[s], the paper presents evidence of rejuvenation by many other measures. For example:

  • IL-6, a marker of inflammation, was restored to low youthful levels;
  • Glutathione (GSH), superoxide dismutase (SOD), and other antioxidants were restored to higher youthful levels;
  • In tests of cognitive function (Barnes maze), treated rats scored better than old rats, but not as well as young rats.;
  • Blood triglycerides were brought down to youthful levels;
  • HDL cholesterol rose to youthful levels; and
  • Blood glucose fell toward youthful levels.

These results bring together three threads that have been gaining credibility over the last decade. Mutually reinforcing, the three have a strength that none of them could offer separately.

  1. The root cause of aging is epigenetic progression = changes in gene expression over a lifetime.
  2. Methylation patterns in nuclear DNA are not merely a marker of aging, but its primary source. Thus aging can be reversed by reprogramming DNA methylation.
  3. Information about the body’s age state is transmitted system-wide via signal molecules in the blood. Locally, tissues respond to these signals and adopt a young or an old cellular phenotype as they are directed.”

Several of these aging measurements are also positively affected by sulforaphane. Using Sulforaphane: Its “Coming of Age” as a Clinically Relevant Nutraceutical in the Prevention and Treatment of Chronic Disease as a reference:

1. “Chronic inflammation”

“Antioxidants in general and glutathione in particular can be depleted rapidly under conditions of oxidative stress, and this can signal inflammatory pathways associated with NF-κB. SFN [sulforaphane] has been shown to inhibit NF-κB in endothelial cells.

Two key inflammatory cytokines were measured at four time points in forty healthy overweight people [our model clinical trial, Effects of long-term consumption of broccoli sprouts on inflammatory markers in overweight subjects]. The levels of both interleukin-6 (Il-6) and C-reactive protein (CRP) declined over the 70 days during which the sprouts were ingested. These biomarkers were measured again at day 90, wherein it was found that Il-6 continued to decline, whereas CRP climbed again. When the final measurement was taken at day 160, CRP, although climbing, had not returned to its baseline value. Il-6 remained significantly below the baseline level at day 160.”

OMCL2019-2716870.010

2. “Oxidative stress”

“As a mediator for amplification of the mammalian defence system against various stressors, Nrf2 [nuclear factor erythroid 2-related factor 2] sits at the interface between our prior understanding of oxidative stress and the endogenous mechanisms cells use to deal with it. Diseases known to be underpinned by oxidative stress are proving to be more responsive to amplification of cellular defences via Nrf2 activation than by administration of direct-acting antioxidant supplements.

SFN, with absolute bioavailability of around 80%, [is] capable of increasing several endogenous antioxidant compounds via the transcription factor, Nrf2.

Nrf2 is ubiquitously expressed with the highest concentrations (in descending order) in the kidney, muscle, lung, heart, liver, and brain. Nrf2 was shown to prevent endothelial cells from exhibiting a proinflammatory state. Nrf2 is required for protection against glucose-induced oxidative stress and cardiomyopathy in the heart.

Well in excess of 500 genes have been identified as being activated by SFN via the Nrf2/ARE [Antioxidant Response Element] pathway, and it is likely that this underestimates the number as others are being discovered. Of the available SFN clinical trials associated with genes induced via Nrf2 activation, many demonstrate a linear dose-response. More recently, it has become apparent that SFN can behave hormetically with different effects responsive to different doses.

It [sulforaphane] is not only a potent Nrf2 inducer but also highly bioavailable so that modest practical doses can produce significant clinical responses. Other Nrf2 activators [shown in the above image] not only lack potency but also lack the bioavailability to be considered as significant intracellular Nrf2 activators.”


The study’s most relentlessly questioned, scrutinized, and criticized findings may be the two new epigenetic clocks that apply to both humans and rats. The researchers invited other researchers to validate these clocks because:

“If validated, this would be a step change in aging research. Although conservation of aging mechanism could be equally deduced from the existence of multiple individual clocks for other mammals (mouse, dog), the single formula of the human-rat clock that is equally applicable to both species effectively demonstrates this fact.”

The commonalities of this study with efforts to change my inflammatory phenotype with broccoli sprouts were summarized in the Discussion section:

“Apart from rejuvenating the vital organs of the treated rats, plasma fraction also impacted two fundamental physiological processes that underlie a great number of pathologies, namely oxidative stress and inflammation. Within a week of treatment, the markers of chronic inflammation (IL-6 and TNF-α) were significantly reduced and remained low throughout the entire experiment.

Likewise, markers of oxidative stress in brain, heart, lung and liver, which were very much higher in control old rats, were at the end of the experimental period, indistinguishable between plasma fraction-treated old rats and young ones. Concomitant with this drastic reduction in oxidative stress was the augmented levels of antioxidants (GSH, Catalase and SOD) in these tissues, indicating that modulating the levels of ROS [reactive oxygen species] to that of youthful rats is at least one way by which plasma fraction suppresses oxidative stress. It remains to be ascertained whether the rate of ROS generation is also reduced.

The levels of Nrf2, a transcription factor that impacts on oxidative stress, as well as inflammation, were raised by plasma fraction treatment of old rats to those of the young ones, indicating yet another level by which this treatment modulates these two critical processes. Collectively, these results show that plasma fraction treatment impacts not only the overt performances of organs, but also the underlying physiological processes that are pivotal for optimal organ function and health.”

Great stuff, huh? Are you ready to change your phenotype?

Continued with Part 2 of Rejuvenation therapy and sulforaphane.

Aging as an unintended consequence

The coauthors of 2018’s The epigenetic clock theory of aging reviewed progress that’s been made todate in understanding epigenetic clock mechanisms.

1. Proven DNA methylation features of epigenetic clocks:

  1. “Methylation of cytosines is undoubtedly a binary event.
  2. The increase in epigenetic age is contributed by changes of methylation profiles in a very small percent of cells in a population.
  3. The clock ticks extremely fast in early post-natal years and much slower after puberty.
  4. Clock CpGs have specific locations in the genome.
  5. It applies to prenatal biological samples and embryonic stem cells.

While consistency with all the five attributes does not guarantee veracity of a model, inconsistency with any one will signal the unlikely validity of a hypothesis.”

2. Regarding what epigenetic clocks don’t measure:

“The effects of

  • Telomere maintenance,
  • Cellular senescence,
  • DNA damage signaling,
  • Terminal differentiation and
  • Cellular proliferation

have all been tested and found to be unrelated to epigenetic ageing.”

3. Regarding cyclical features:

Both the epigenetic and circadian clocks are present in all cells of the body, but their ticking rates are regulated. Both these clocks lose synchronicity when cells are isolated from tissues and grown in vitro.

These similarities compel one to ponder potential links between them.”

This was among the points that Linear thinking about biological age clocks missed.

4. The reviewers discussed 3 of the 5 treatment elements in Reversal of aging and immunosenescent trends:

“It is not known at this stage whether the rejuvenating effect is mediated through the regeneration of the thymus or a direct effect of the treatment modality on the body. Also, it is not known if the effect is mediated by all three compounds or one or two of them.

What we know at this stage does not allow the formation of general principles regarding the impact of hormones on epigenetic age, but their involvement in development and maintenance of the body argue that they do indeed have a very significant impact on the epigenetic clock.”

Not sure why they omitted 3000 IU vitamin D and 50 mg zinc, especially since:

“It is not known if the effect is mediated by all three [five] compounds or one or two of them.”

5. They touched on the specialty of Aging as a disease researchers with:

“Muscle stem cells isolated from mice were epigenetically much younger independently of the ages of the tissue / animal from which they were derived.

The proliferation and differentiation of muscle stem cells cease upon physical maturation. These activities are initiated in adult muscles only in response to injury.

6. The reviewers agreed with those researchers in the Conclusion:

“Epigenetic ageing begins from very early moments after the embryonic stem cell stage and continues uninterrupted through the entire lifespan. The significance of this is profound as the question of why we age has been attributed to many different things, most commonly to ‘wear-and-tear.’

The ticking of the epigenetic clock from the embryonic state challenges this perspective and supports the notion that ageing is an unintended consequence of processes that are necessary for

  • The development of the organism and
  • Tissue homeostasis thereafter.”


https://journals.sagepub.com/doi/10.1177/1535370220918329 “Current perspectives on the cellular and molecular features of epigenetic ageing” (not freely available)

An epigenetic clock review by committee

This 2019 worldwide review of epigenetic clocks was a semi-anonymous mishmash of opinions, facts, hypotheses, unwarranted extrapolations, and beliefs. The diversity of viewpoints among the 21 coauthors wasn’t evident.

1. Citations of the coauthors’ works seemed excessive, and they apologized for omissions. However:

  • Challenge 5 was titled “Single-cell analysis of aging changes and disease” and
  • Table 1 “Major biological and analytic issues with epigenetic DNA methylation clocks” had single-cell analysis as the Proposed solution to five Significant issues.

Yet studies such as High-Resolution Single-Cell DNA Methylation Measurements Reveal Epigenetically Distinct Hematopoietic Stem Cell Subpopulations were unmentioned.

2. Some coauthors semi-anonymously expressed faith that using current flawed methodologies in the future – only more thoroughly, with newer equipment, etc. – would yield better results. If the 21 coauthors were asked their viewpoints of Proposed solutions to the top three Significant issues of epigenetic clocks, what would they emphasize when quoted?

3. Techniques were praised:

“Given the precision with which DNA methylation clock age can be estimated and evolving measures of biological, phenotype-, and disease-related age (e.g., PhenoAge, GrimAge)..”

Exactly why these techniques have at times produced inexplicable results wasn’t examined, though. Two examples:

  • In Reversal of aging and immunosenescent trends, the Levine PhenoAge methodology estimated that the 51-65 year old subjects’ biological ages at the beginning of the study averaged 17.5 years less than their chronological age. Comparing that to the Horvath average biological age of 3.95 years less raised the question: exactly why did PhenoAge show such a large difference?
  • The paper mentioned the GrimAge methodology findings about “smoking-related changes.” But it didn’t explain why the GrimAge methylation findings most closely associated with smoking history also accurately predicted future disease risk with non-smokers.

Eluding explanations for these types of findings didn’t help build confidence in the methodologies.

4. A more readable approach to review by committee could have coauthors – in at least one section – answer discussion questions, as Reversing epigenetic T cell exhaustion did with 18 experts.

https://genomebiology.biomedcentral.com/articles/10.1186/s13059-019-1824-y “DNA methylation aging clocks: challenges and recommendations”

A review of fetal adverse events

This 2019 Australian review subject was fetal adversities:

“Adversity during the perinatal period is a significant risk factor for the development of neurodevelopmental disorders long after the causative event. Despite stemming from a variety of causes, perinatal compromise appears to have similar effects on the developing brain, thereby resulting in behavioural disorders of a similar nature.

These behavioural disorders occur in a sex‐dependent manner, with males affected more by externalizing behaviours such as attention deficit hyperactivity disorder (ADHD) and females by internalizing behaviours such as anxiety. The term ‘perinatal compromise’ serves as an umbrella term for intrauterine growth restriction, maternal immune activation, prenatal stress, early life stress, premature birth, placental dysfunction, and perinatal hypoxia.

The above conditions are associated with imbalanced excitatory-inhibitory pathways resulting from reduced GABAergic signalling. Methylation of the GAD1/GAD67 gene, which encodes the key glutamate‐to‐GABA synthesizing enzyme Glutamate Decarboxylase 1, resulting in increased levels of glutamate is one epigenetic mechanism that may account for a tendency towards excitation in disorders such as ADHD.

The posterior cerebellum’s role in higher executive functioning is becoming well established due to its connections with the prefrontal cortex, association cortices, and limbic system. It is now suggested that disruptions to cerebellar development, which can occur due to late gestation compromises such as preterm birth, can have a major impact on the region of the brain to which it projects.

Activation of the maternal hypothalamic-pituitary adrenal (HPA) axis and placental protection. Psychological stress is perceived by the maternal HPA axis, which stimulates cortisol release from the maternal adrenal gland.

High levels of maternal cortisol are normally prevented from reaching the fetus by the 11β-hydroxysteroid dehydrogenase 2 (HSD11B2) enzyme, which converts cortisol to the much less active cortisone. Under conditions of high maternal stress, this protective mechanism can be overwhelmed, with the gene encoding the enzyme becoming methylated, which reduces its expression allowing cortisol to cross the placenta and reach the fetus.”


The reviewers extrapolated many animal study findings to humans, although most of their own work was with guinea pigs. The “suggest” and “may” qualifiers were used often – 22 and 37 times, respectively. More frequent use of the “appears,” “hypothesize,” “propose,” and “possible” terms was justified.

As a result, many reviewed items such as the above graphic and caption should be viewed as hypothetical for humans rather than reflecting solid evidence from quality human studies.

The reviewers focused on the prenatal (before birth) period more than the perinatal (last trimester of pregnancy to one month after birth) period. There were fewer mentions of birth and early infancy adversities.

https://onlinelibrary.wiley.com/doi/abs/10.1111/jne.12814 “Perinatal compromise contributes to programming of GABAergic and Glutamatergic systems leading to long-term effects on offspring behaviour” (not freely available)

Caloric restriction’s epigenetic effects

This 2019 US review subject was caloric restriction (CR) without malnutrition:

“Cellular adaptation that occurs in response to dietary patterns can be explained by alterations in epigenetic mechanisms such as DNA methylation, histone modifications, and microRNA. Epigenetic reprogramming of the underlying chronic low-grade inflammation by CR can lead to immuno-metabolic adaptations that enhance quality of life, extend lifespan, and delay chronic disease onset.

Short- and long-term CRs produce significant changes in different tissues and across species, in some animal models even with sex-specific effects. Early CR onset may cause a different and even an opposite effect on physiological outcomes in animal models such as body weight.”

 


1. Charts usually don’t have two different values plotted on the same axis. The review didn’t present evidence to equate survival (left axis) with methylation drift (right axis) per the above graphic. Methylation drift should point in the opposite direction of survival, if anything.

2. No mention was made of the epigenetic clock method of measuring age acceleration, although it’s been available since 2013 and recent diet studies have used it. The sole citation of an age acceleration study was from 2001, which was unacceptable for a review published in 2019.

3. The review provided many cellular-level details about the subject. However, organism-level areas weren’t sufficiently evidenced:

A. Arguments for an effect usually include explanations for no effect as well as opposite effects. The reviewers didn’t provide direct evidence for why, if caloric restriction extended lifespan, caloric overabundance produced shorter lifespans.

B. Caloric restriction evidence was presented as if only it was responsible for organism-level effects. Other mechanisms may have been involved.

An example of such a mechanism was demonstrated in a 2007 rodent study Reduced Oxidant Stress and Extended Lifespan in Mice Exposed to a Low Glycotoxin Diet which compared two 40%-calorie-restricted diets.

The calories and composition of both diets were identical. However, advanced glycation end product (AGE) levels were doubled in standard chow because heating temperatures were “sufficiently high to inadvertently cause standard mouse chow to be rich in oxidant AGEs.”

The study found that a diet with lower chow heating temperatures increased lifespan and health span irrespective of caloric restriction!

  • The low-AGE calorie-restricted diet group lived an average of 15% longer (>20 human equivalent years) than the CR group.
  • 40% of the low-AGE calorie-restricted diet group were still alive when the last CR group member died.
  • The CR group also had significantly more: 1) oxidative stress damage; 2) glucose and insulin metabolism problems; and 3) kidney, spleen, and liver injuries.

https://academic.oup.com/advances/article-abstract/10/3/520/5420411 “Epigenetic Regulation of Metabolism and Inflammation by Calorie Restriction” (not freely available)

Non-emotional memories

This 2019 US review covered memory mechanisms:

“With memory encoding reliant on persistent changes in the properties of synapses, a key question is how can memories be maintained from days to months or a lifetime given molecular turnover? It is likely that positive feedback loops are necessary to persistently maintain the strength of synapses that participate in encoding.

These levels are not isolated, but linked by shared components of feedback loops.”


Despite the review’s exhaustive discussion, the reviewers never came to the point. The word cloud I made of the review’s most frequent thirty words had little to do with why memory occurs:

  • Why do some stimuli evoke a memory in response?
  • Why are almost all of the stimuli an organism receives not remembered?

Much of the discussion was baseless because it excluded emotion. Many of the citations’ memory findings relied on emotion, though.

For example, in the subsection Roles of persistent epigenetic modifications for maintaining LTF [long-term facilitation], LTP [long-term potentiation], and LTM [long-term memory]:

  • Histone acetylation is increased after fear conditioning in the hippocampus and amygdala.
  • Correspondingly, inhibition of histone deacetylase enhances fear conditioning and LTP.
  • Following fear conditioning, histone phosphorylation is also increased.
  • DNA methylation is also up-regulated in the hippocampus and amygdala after fear conditioning, and inhibition of DNA methylation blocks fear LTM.”

http://learnmem.cshlp.org/content/26/5/133.full “How can memories last for days, years, or a lifetime? Proposed mechanisms for maintaining synaptic potentiation and memory”

Our brains are shaped by our early environments

This 2019 McGill paper reviewed human and animal studies on brain-shaping influences from the fetal period through childhood:

“In neonates, regions of the methylome that are highly variable across individuals are explained by the genotype alone in 25 percent of cases. The best explanation for 75 percent of variably methylated regions is the interaction of genotype with different in utero environments.

A meta-analysis including 45,821 individuals with attention-deficit/hyperactivity disorder and 9,207,363 controls suggests that conditions such as preeclampsia, Apgar score lower than 7 at 5 minutes, breech/transverse presentations, and prolapsed/nuchal cord – all of which involve some sort of poor oxygenation during delivery – are significantly associated with attention-deficit/hyperactivity disorder. The dopaminergic system seems to be one of the brain systems most affected by perinatal hypoxia-ischemia.

Exposure to childhood trauma activates the stress response systems and dysregulates serotonin transmission that can adversely impact brain development. Smaller cerebral, cerebellar, prefrontal cortex, and corpus callosum volumes were reported in maltreated young people as well as reduced hippocampal activity.

Environmental enrichment has a series of beneficial effects associated with neuroplasticity mechanisms, increasing hippocampal volume, and enhancing dorsal dentate gyrus-specific differences in gene expression. Environmental enrichment after prenatal stress decreases depressive-like behaviors and fear, and improves cognitive deficits.”


The reviewers presented strong evidence until the Possible Factors for Reversibility section, which ended with the assertion:

“All these positive environmental experiences mentioned in this section could counterbalance the detrimental effects of early life adversities, making individuals resilient to brain alterations and development of later psychopathology.”

The review’s penultimate sentence recognized that research is seldom done on direct treatments of causes:

“The cross-sectional nature of most epigenetic studies and the tissue specificity of the epigenetic changes are still challenges.”

Cross-sectional studies won’t provide definitive data on cause-and-effect relationships.

The question yet to be examined is: How can humans best address these early-life causes to ameliorate their lifelong effects?

https://onlinelibrary.wiley.com/doi/full/10.1111/dmcn.14182 “Early environmental influences on the development of children’s brain structure and function” (not freely available)

The epigenetic clock now includes skin

The originator of the 2013 epigenetic clock improved its coverage with this 2018 UCLA human study:

“We present a new DNA methylation-based biomarker (based on 391 CpGs) that was developed to accurately measure the age of human fibroblasts, keratinocytes, buccal cells, endothelial cells, skin and blood samples. We also observe strong age correlations in sorted neurons, glia, brain, liver, and bone samples.

The skin & blood clock outperforms widely used existing biomarkers when it comes to accurately measuring the age of an individual based on DNA extracted from skin, dermis, epidermis, blood, saliva, buccal swabs, and endothelial cells. Thus, the biomarker can also be used for forensic and biomedical applications involving human specimens.

The biomarker applies to the entire age span starting from newborns, e.g. DNAm of cord blood samples correlates with gestational week.

Furthermore, the skin & blood clock confirms the effect of lifestyle and demographic variables on epigenetic aging. Essentially it highlights a significant trend of accelerated epigenetic aging with sub-clinical indicators of poor health.

Conversely, reduced aging rate is correlated with known health-improving features such as physical exercise, fish consumption, high carotenoid levels. As with the other age predictors, the skin & blood clock is also able to predict time to death.

Collectively, these features show that while the skin & blood clock is clearly superior in its performance on skin cells, it crucially retained all the other features that are common to other existing age estimators.”

http://www.aging-us.com/article/101508/text “Epigenetic clock for skin and blood cells applied to Hutchinson Gilford Progeria Syndrome and ex vivo studies”


An introduction to the study highlighted several items:

“Although the skin-blood clock was derived from significantly less samples (~900) than Horvath’s clock (~8000 samples), it was found to more accurately predict chronological age, not only across fibroblasts and skin, but also across blood, buccal and saliva tissue. A potential factor driving this improved accuracy in blood could be related to the approximate 18-fold increase in genomic coverage afforded by using Illumina 450k/850k beadarrays.

It serves as a roadmap for future clock studies, pointing towards the importance of constructing tissue or cell-type specific epigenetic clocks, to more accurately measure biological aging in the given tissue/cell-type, and therefore with the potential to be more informative of disease-risk or the success of disease interventions in the tissue or cell-type of interest.”

http://www.aging-us.com/article/101533/text “Epigenetic clocks galore: a new improved clock predicts age-acceleration in Hutchinson Gilford Progeria Syndrome patients”