Clearing out the 2019 queue of interesting papers

I’m clearing out the below queue of 27 studies and reviews I’ve partially read this year but haven’t taken the time to curate. I have a pesky full-time job that demands my presence elsewhere during the day. :-\

Should I add any of these back in? Let’s be ready for the next decade!

Early life “Early Behavioral Alterations and Increased Expression of Endogenous Retroviruses Are Inherited Across Generations in Mice Prenatally Exposed to Valproic Acid” (not freely available) “Consolidation of an aversive taste memory requires two rounds of transcriptional and epigenetic regulation in the insular cortex” (not freely available) “Intergenerational transmission of depression: clinical observations and molecular mechanisms” (not freely available)

mother “Epigenomics and Transcriptomics in the Prediction and Diagnosis of Childhood Asthma: Are We There Yet?” epigenetic clocks: estimating gestational age using placental DNA methylation levels” “Mismatched Prenatal and Postnatal Maternal Depressive Symptoms and Child Behaviours: A Sex-Dependent Role for NR3C1 DNA Methylation in the Wirral Child Health and Development Study” “Environmental influences on placental programming and offspring outcomes following maternal immune activation” “5-Hydroxymethylcytosine in cord blood and associations of DNA methylation with sex in newborns” (not freely available) “Paternal diet impairs F1 and F2 offspring vascular function through sperm and seminal plasma specific mechanisms in mice” “Sex differences in the epigenetic regulation of chronic visceral pain following unpredictable early life stress” (not freely available) “Genome-wide DNA methylation data from adult brain following prenatal immune activation and dietary intervention” in depression vulnerability and resilience: novel targets for preventive strategies”

Later life “Effect of Flywheel Resistance Training on Balance Performance in Older Adults. A Randomized Controlled Trial” “Eccentric Overload Flywheel Training in Older Adults” “Epigenetic regulation of the innate immune response to infection” (not freely available) “Hair Cell Regeneration” (not freely available) Modifications as an Intersection Between Diet and Longevity” “Serotonin transporter gene methylation predicts long-term cortisol concentrations in hair” (not freely available) “Frailty biomarkers in humans and rodents: Current approaches and future advances” (not freely available) “Neural mechanisms underlying adaptive and maladaptive consequences of stress: Roles of dopaminergic and inflammatory responses “In Search of Panacea—Review of Recent Studies Concerning Nature-Derived Anticancer Agents” “Reversal of oxycodone conditioned place preference by oxytocin: Promoting global DNA methylation in the hippocampus” (not freely available) “Different epigenetic clocks reflect distinct pathophysiological features of multiple sclerosis” “The Beige Adipocyte as a Therapy for Metabolic Diseases” “Bone adaptation: safety factors and load predictability in shaping skeletal form” (not freely available) “Successful treatment of post-traumatic stress disorder reverses DNA methylation marks” (not freely available) “Editing the Epigenome to Tackle Brain Disorders” (not freely available)

A blood plasma aging clock

This 2019 Stanford human study developed an aging clock using blood plasma proteins:

“We measured 2,925 plasma proteins from 4,331 young adults to nonagenarians [18 – 95] and developed a novel bioinformatics approach which uncovered profound non-linear alterations in the human plasma proteome with age. Waves of changes in the proteome in the fourth, seventh, and eighth decades of life reflected distinct biological pathways, and revealed differential associations with the genome and proteome of age-related diseases and phenotypic traits.

To determine whether the plasma proteome can predict chronological age and serve as a “proteomic clock,” we used 2,858 randomly selected subjects to fine-tune a predictive model that was tested on the remaining 1,473 subjects. We identified a sex-independent plasma proteomic clock consisting of 373 proteins. Subjects that were predicted younger than their chronologic age based on their plasma proteome performed better on cognitive and physical tests.

The 3 age-related crests were comprised of different proteins. Few proteins, such as GDF15, were among the top 10 differentially expressed proteins in each crest, consistent with its strong increase across lifespan. Other proteins, like chordin-like protein 1 (CHRDL1) or pleiotrophin (PTN), were significantly changed only at the last two crests, reflecting their exponential increase with age.

We observed a prominent shift in multiple biological pathways with aging:

  • At young age (34 years), we observed a downregulation of proteins involved in structural pathways such as the extracellular matrix. These changes were reversed in middle and old ages (60 and 78 years, respectively).
  • At age 60, we found a predominant role of hormonal activity, binding functions and blood pathways.
  • At age 78, key processes still included blood pathways but also bone morphogenetic protein signaling, which is involved in numerous cellular functions, including inflammation.

These results suggest that aging is a dynamic, non-linear process characterized by waves of changes in plasma proteins that are reflective of a complex shift in the activity of biological processes.” “Undulating changes in human plasma proteome across lifespan are linked to disease”

A non-critical review of the study was published by the Life Extension Advocacy Foundation. Frequent qualifiers like “could,” “may,” and “possible” were consistent with the confirmation biases of their advocacy.

There were several misstatements of what the study did, including the innumerate:

  1. “used around half of the participant data to build a “proteomic clock”
  2. tested it on the other half of the participants
  3. a total of 3000 proteins”

Per the above study quotation, the numbers were actually:

  1. Closer to two thirds (2,858 ÷ 4,331), not “around half”;
  2. The other third (1,473 ÷ 4,331), not “the other half”; and
  3. 2,925 not 3000.

The final paragraph and other parts of the review bordered on woo. Did a review of the findings have to fit LEAF’s perspective?

In contrast, Josh Mitteldorf did his usual excellent job of providing contexts for the study with New Aging Clock based on Proteins in the Blood, emphasizing comparisons with epigenetic clock methodologies:

“For some of the proteins that feature prominently in the clock, we have a good understanding of their metabolic function, and for the most part they vindicate my belief that epigenetic changes are predominantly drivers of senescence rather than protective responses to damage.

Wyss-Coray compared the proteins in the new (human) proteome clock with the proteins that were altered in the (mouse) parabiosis experiments, and found a large overlap [46 proteins change in the same direction and define a conserved aging signature]. This may be the best evidence we have that the proteome changes are predominantly causal factors of senescence.

46 plasma proteins

Almost all the proteins identified as changing rapidly at age 78 are increasing. In contrast, a few of the fastest-changing proteins at age 60 are decreasing (though most are increasing). GDF15 deserves a story of its own.

The implication is that a more accurate clock can be constructed if it incorporates different information at different life stages. None of the Horvath clocks have been derived based on different CpG sites at different ages, and this suggests an opportunity for a potential improvement in accuracy.”

A commentator linked the below study: “GDF15 Is an Inflammation-Induced Central Mediator of Tissue Tolerance” (not freely available)

which prompted his response:

“Thanks, Lee! This is just the kind of specific information that I was asking for. It would seem we should construct our clocks without GDF15, which otherwise might loom large.”

Epigenetic inheritance and microRNAs

This 2019 Canadian rodent study found:

“Folic acid (FA) supplementation mitigates sperm miRNA profiles transgenerationally following in utero paternal exposure to POPs [persistent organic pollutants]. Across the F1 – F4 generations, sperm miRNA profiles were less perturbed with POPs + FA compared to sperm from descendants of dams treated with POPs alone..and only in F1 sperm.

The POPs mixture represents the pollutant composition found in Ringed seal blubber of Northern Quebec which is a traditional food of Inuit people in that region.

F0 founder dams were gavaged with the POPs mixture corresponding to 500 µg PCBs/kg body weight or corn oil (CTRL) thrice weekly and were fed the AIN-93G diet containing either 2 mg/kg (1X) or 6 mg/kg (3X) of FA ad libitum. Treatments were only administered to F0 founder dams for 9 weeks in total; 5 weeks before mating to untreated males at postnatal day 90 and until parturition. Subsequent lineages, F1 through F4, were neither exposed to POPs nor 3X FA – instead they received 1X FA diet ad libitum.”

Folic acid’s mechanisms weren’t clear:

“The protective role of FA supplementation in the F1 sperm may be partly explained by its antioxidant activity if the miRNA changes are caused by oxidative stress induced by POPs exposure. If, however, the miRNA changes in POPs exposed sperm are due to an altered methylation capacity or dysregulated nucleotide synthesis or mutations, then the increased availability of methyl groups provided by FA supplementation may mitigate the POPs effect by supporting DNA repair through nucleotide synthesis. Additional studies of the interaction between POPs and FA are required.”

Epigenetic inheritance mechanisms were also unclear:

“It remains puzzling how environmentally perturbed paternal miRNAs can persist across multiple generations. To become heritable, parts of the sperm chromatin must escape reprogramming, leading to the possibility that sperm miRNA profiles are subsequently modified by environmental factors. There are clear examples of sperm DNA methylation that escape reprogramming and histones can be involved.”

The study may have produced more clarity had its design investigated DNA methylation as Epigenetic transgenerational inheritance extends to the great-great-grand offspring did. That study also had an intercross breeding scheme with the populations for the F1 – F3 generations before an outcross for the F4 generation because:

“An intercross within the exposure lineage population (with no sibling or cousin breeding to avoid inbreeding artifacts) provides the optimal phenotypes (i.e. pathology) and germline epigenetic alterations.”

Which breeding scheme do you think would more fairly represent the humans of this study? I’d guess that intercross would – if all Inuits eat Ringed seal blubber and have children with other Inuits. “Folic acid supplementation reduces multigenerational sperm miRNA perturbation induced by in utero environmental contaminant exposure”

Using oxytocin receptor gene methylation to pursue an agenda

A pair of 2019 Virginia studies involved human mother/infant subjects:

“We show that OXTRm [oxytocin receptor gene DNA methylation] in infancy and its change is predicted by maternal engagement and reflective of behavioral temperament.” “Epigenetic dynamics in infancy and the impact of maternal engagement”

“Infants with higher OXTRm show enhanced responses to anger and fear and attenuated responses to happiness in right inferior frontal cortex, a region implicated in emotion processing through action-perception coupling.

Infant fNIRS [functional near-infrared spectroscopy] is limited to measuring responses from cerebral is unknown whether OXTR is expressed in the cerebral cortex during prenatal and early postnatal human brain development.” “Epigenetic modification of the oxytocin receptor gene is associated with emotion processing in the infant brain”

Both studies had weak disclosures of limitations on their findings’ relevance and significance. The largest non-disclosed contrary finding was from the 2015 Early-life epigenetic regulation of the oxytocin receptor gene:

These results suggest that:

  • Blood Oxtr DNA methylation may reflect early experience of maternal care, and
  • Oxtr methylation across tissues is highly concordant for specific CpGs, but
  • Inferences across tissues are not supported for individual variation in Oxtr methylation.

This rat study found that blood OXTR methylation of 25 CpG sites couldn’t accurately predict the same 25 CpG sites’ OXTR methylation in each subject’s hippocampus, hypothalamus, and striatum (which includes the nucleus accumbens) brain areas. Without significant effects in these limbic system structures, there couldn’t be any associated behavioral effects.

But CpG site associations and correlations were deemed good in the two current studies because they cited:

“Recent work in prairie voles has found that both brain- and blood-derived OXTRm levels at these sites are negatively associated with gene expression in the brain and highly correlated with each other.” “Early nurture epigenetically tunes the oxytocin receptor”

The 2018 prairie vole study – which included several of the same researchers as the two current studies – found four nucleus accumbens CpG sites that had high correlations to humans. Discarding one of these CpG sites allowed their statistics package to make a four-decimal place finding:

“The methylation state of the blood was also associated with the level of transcription in the brain at three of the four CpG sites..whole blood was capable of explaining 94.92% of the variance in Oxtr DNA methylation and 18.20% of the variance in Oxtr expression.”

Few limitations on the prairie vole study findings were disclosed. Like the two current studies, there wasn’t a limitation section that placed research findings into suitable contexts. So readers didn’t know researcher viewpoints on items such as:

  • What additional information showed that 3 of the 30+ million human CpGs accurately predicted specific brain OXTR methylation and expression from saliva OXTR methylation?
  • What additional information demonstrated how “measuring responses from cerebral cortex” although “it is unknown whether OXTR is expressed in the cerebral cortex” provided detailed and dependable estimates of limbic system CpG site OXTR methylation and expression?
  • Was the above 25-CpG study evidence considered?

Further contrast these three studies with a typical, four-point, 285-word limitation section of a study like Prenatal stress heightened adult chronic pain. The word “limit” appeared 6 times in that pain study, 3 times in the current fNIRS study, and 0 times in the current maternal engagement and cited prairie vole studies.

Frank interpretations of one’s own study findings to acknowledge limitations is one way researchers can address items upfront that will be questioned anyway. Such analyses also indicate a goal to advance science.

An out-of-date review of epigenetic transgenerational inheritance

This December 3, 2019, French review title was “Transgenerational Inheritance of Environmentally Induced Epigenetic Alterations during Mammalian Development”:

“We attempt to summarize our current knowledge about the transgenerational inheritance of environmentally induced epigenetic changes. While the idea that information can be inherited between generations independently of the DNA’s nucleotide sequence is not new, the outcome of recent studies provides a mechanistic foundation for the concept.

The systematic resetting of epigenetic marks between generations represents the largest hurdle to conceptualizing epigenetic inheritance. Our understanding of the rates and causes of epimutations remains rudimentary.

Environmental exposure to toxicants could promote changes in germline cells at any developmental stage, with more dramatic effects being observed during embryonic germ cell reprogramming. Epigenetic factors and their heritability should be considered during disease risk assessment.”

The review showed an inexplicable lack of thorough research. 2017 was its latest citation of epigenetic transgenerational inheritance studies from the Washington State University labs of Dr. Michael Skinner. I’ve curated six of the labs’ 2019 studies!

  1. Transgenerational diseases caused by great-grandmother DDT exposure;
  2. Another important transgenerational epigenetic inheritance study;
  3. The transgenerational impact of Roundup exposure;
  4. Epigenetic transgenerational inheritance mechanisms that lead to prostate disease;
  5. A transgenerational view of the rise in obesity; and
  6. Epigenetic transgenerational inheritance extends to the great-great-grand offspring.

This lack led to – among other items – equivocal statements where current definitive evidence could have been cited. The review was submitted to the publisher on October 31, 2019, and the above studies were available.

The publisher provided insight into the peer review process via

  • Peer reviewer 1: “Taking into account that this is not my main area of expertise..Do the authors really believe in that?”
  • Peer reviewer 2 provided a one-paragraph non-review.
  • Peer reviewer 3: “The authors are missing a large sector of what types of environmental factors can influence methylation and do not acknowledge that other sources exist.”

The authors responded with changes or otherwise addressed peer reviewer comments. “Transgenerational Inheritance of Environmentally Induced Epigenetic Alterations during Mammalian Development”

Prenatal stress heightened adult chronic pain

This 2019 McGill rodent study found:

Prenatal stress exacerbates pain after injury. Analysis of mRNA expression of genes related to epigenetic regulation and stress responses in the frontal cortex and hippocampus, brain structures implicated in chronic pain, showed distinct sex and region-specific patterns of dysregulation.

In general, mRNA expression was most frequently altered in the male hippocampus and effects of prenatal stress were more prevalent than effects of nerve injury. Recent studies investigating chronic pain-related pathology in the hippocampus in humans and in rodent models demonstrate functional abnormalities in the hippocampus, changes in associated behavior, and decreases in adult hippocampal neurogenesis.

The change in expression of epigenetic- and stress-related genes is not a consequence of nerve injury but rather precedes nerve injury, consistent with the hypothesis that it might play a causal role in modulating the phenotypic response to nerve injury. These findings demonstrate the impact of prenatal stress on behavioral sensitivity to a painful injury.

Decreased frontal mRNA expression of BDNF and BDNF IV in male offspring following neuropathic pain or prenatal stress respectively. Relative mRNA expression of other stress-related genes (GR17, FKBP5) and epigenetic-related genes (DNMTs, TETs, HDACs, MBDs, MeCP2) in male offspring.

A drastic decrease in expression of HDAC1 was observed in all groups compared to sham-control animals. CCI: chronic constriction injury.”

The study’s design was similar to the PRS (prenatal restraint stress) model, except that the PRS procedure covered gestational days 11 to 21 (birth):

“Prenatal stress was induced on Embryonic days 13 to 17 by restraining the pregnant dams in transparent cylinder with 5 mm water, under bright light exposure, 3 times per day for 45 min.”

None of the French, Italian, and Swiss PRS studies were cited.

The limitation section included:

  1. “Although our study shows significant changes in expression of epigenetic enzymes, it didn’t examine the impact of these changes on genes that are epigenetically regulated by this machinery or their involvement in intensifying pain responses.
  2. The current study is limited by the focus on changes in gene expression which do not necessarily correlate with changes in protein expression.
  3. Another limitation of this study is the inability to distinguish the direct effects of stress in utero vs. changes in the dam’s maternal behavior due to stress during pregnancy; cross-fostering studies are needed to address this issue.
  4. Functional experiments that involve up and down regulation of epigenetic enzymes in specific brain regions are required to establish a causal role for these processes in chronic pain.”

What do you think about possible human applicability of this study’s “effects of prenatal stress were more prevalent than effects of nerve injury” finding?

Are there any professional frameworks that instruct trainees to recognize that if a person’s mother was stressed while pregnant, their prenatal experiences could cause more prevalent biological and behavioral effects than a recent injury? “Prenatal maternal stress is associated with increased sensitivity to neuropathic pain and sex-specific changes in supraspinal mRNA expression of epigenetic- and stress-related genes in adulthood” (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. “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. “Perinatal compromise contributes to programming of GABAergic and Glutamatergic systems leading to long-term effects on offspring behaviour” (not freely available)

A transgenerational view of the rise in obesity

This 2019 Washington State University rodent study found epigenetically inherited transgenerational effects in great-grand offspring due to their great-grandmothers’ toxicant exposures during pregnancy:

“Previous studies found an increased susceptibility to obesity in F3 generation rats ancestrally exposed to the pesticide DDT, and an increase in a lean phenotype in the F3 generation rats ancestrally exposed to the herbicide atrazine. The present study investigated whether there were common DMR [differential DNA methylated region] and associated genes between the control, DDT, and atrazine lineage male and female adipocytes in order to identify potential novel gene pathways modulated by DNA methylation.

Comparison of epigenetic alterations indicated that there were substantial overlaps between the different treatment lineage groups for both the lean and obese phenotypes. Novel correlated genes and gene pathways associated with DNA methylation were identified, and may aid in the discovery of potential therapeutic targets for metabolic diseases such as obesity.

Given that the first widespread [DDT] exposures to gestating human females started in the 1950s, the majority of the subsequent F3 generation are adults today. Ancestral exposures to environmental toxicants like DDT may have had a role in the dramatic rise in obesity rates worldwide.”

This same research group noted in Transgenerational diseases caused by great-grandmother DDT exposure:

“DDT was banned in the USA in 1973, but it is still recommended by the World Health Organization for indoor residual spray. India is by far the largest consumer of DDT worldwide.

India has experienced a 5-fold increase of type II diabetes over the last three decades with a predisposition to obesity already present at birth in much of the population. Although a large number of factors may contribute to this increased incidence of obesity, the potential contribution of ancestral toxicant exposures in the induction of obesity susceptibility requires further investigation.” “Adipocyte epigenetic alterations and potential therapeutic targets in transgenerationally inherited lean and obese phenotypes following ancestral exposures”

A GWAS meta-analysis of two epigenetic clocks

This 2019 UK human study conducted a meta-analysis of genome-wide association studies of two epigenetic clocks using 13,493 European-ancestry individuals aged between ten and 98 years:

“Horvath-EAA, described in previous publications as ‘intrinsic’ epigenetic age acceleration (IEAA), can be interpreted as a measure of cell-intrinsic ageing that exhibits preservation across multiple tissues, appears unrelated to lifestyle factors, and probably indicates a fundamental cell ageing process that is largely conserved across cell types.

In contrast, Hannum-EAA, referred to in previous studies as ‘extrinsic’ epigenetic age acceleration (EEAA), can be considered a biomarker of immune system ageing, explicitly incorporating aspects of immune system decline such as age-related changes in blood cell counts, correlating with lifestyle and health-span related characteristics, and thus yielding a stronger predictor of all-cause mortality.

The meta-analysis of Horvath-EAA identified ten independent associated SNPs [single nucleotide polymorphisms], doubling the number reported to date, and highlighted 21 genes involved in Horvath-based epigenetic ageing. Four of the ten Horvath-EAA-associated SNPs are mQTL [methylation quantitative trait loci] for CpGs used in the Horvath/Hannum epigenetic clocks. A possible interpretation of this is that the functional mechanism by which these SNPs influence the rate of biological ageing is via altering methylation levels.

Father’s age at death, a rough proxy for lifespan, was nominally significantly correlated with both EAA measures, and parents’ age at death was additionally correlated with Hannum-EAA. Aside from these, genetic correlations with age-related traits were surprisingly few: it is possible that this could reflect an overly conservative correction for the multiple tests carried out, or low statistical power, rather than a genuine lack of correlations.

Genetic correlation analysis should be restricted to GWAS with a heritability Z-score of 4 or more, on the grounds of interpretability and power, so the Horvath-based results particularly should be interpreted with caution.”

A non-apologetic way to explain the above graphic is that NONE of these 218 “health and behavioral traits” were any more associated with the studied genetic measurements than would be expected by chance!

Fervent believers in the GWAS methodology’s capability to exactly predict individual phenotypes eventually become victims of the scientific method. These GWAS researchers griped about “overly conservative correction, or low statistical power” and other predictable shortfalls, and ended a long limitations statement with:

“While we have identified a number of SNPs and genes significantly associated with EAA, including genes already known to be related to ageing, the analyses presented here fall short of providing a mechanistic explanation for how these variants and genes act to influence biological age.”

Outside of beliefs, it’s hard to understand why research money keeps pouring into the GWAS dead end. If these researchers and their employing institution and sponsors want to make a difference in human lives, they need to get busy in other areas.

These researchers were employed by the same institution that couldn’t be bothered to scrape together six more weeks of funds to study the transgenerational damaging effects of acetaminophen – an analgesic available to billions of people – in Epigenetics research that was designed to fall one step short of wonderful. “A meta-analysis of genome-wide association studies of epigenetic age acceleration”

Organismal aging and cellular senescence

I’ll curate this 2019 German review through its figures:

“With the discovery of beneficial aspects of cellular senescence and evidence of senescence being not limited to replicative cellular states, a redefinition of our comprehension of aging and senescence appears scientifically overdue.

Figure 1. Current determinants and relevant open questions, marking the processes of aging and senescence as discussed in the text. Aspects represented in green are considered as broadly accepted or scientifically consolidated. Novel aspects that are yet unproven, or are under debate, are highlighted in red.

SASP = senescence-associated secretory phenotype. AASP = putative aging-associated secretory phenotype as suggested in the text.

Figure 2. Theories on the causality and purpose of aging. Graphically summarized are four contrasting concepts crystallized from current evidence addressing the inductive driving force of aging. Apart from a stochastic deleteriome, there are arguments for a pseudo-programmed, programmed or at least partially programmed nature of aging.

Figure 3. Comparative representation of the aging and senescence processes highlighting different levels of interaction and putative sites of interventions.

(1) As discussed in the text, causative mechanisms of aging are still not well understood, however, multiple factors including genetic, epigenetic and stress-related effects seem to have an orchestrated role in the progression of aging. Senescence on the other hand, is seen as a programmed response to different kinds of stressors, which proceed in defined stages. Whether, in analogy, aging also follows a defined program or sequential stages is not known.

(2) Senescence involves autocrine and paracrine factors, which are responsible for a ‘seno-infection’ or bystander effect in neighboring cells. There is currently no direct evidence for a similar factor composition propagating the aging process via a kind of ‘gero-infection’.

(3) Accumulation of senescent cells has been described as a hallmark of aging; however, whether they are a causative factor or a consequence of tissue and organismal aging is still unknown. As discussed in the text, it appears possible that aging and senescence mutually influence each other through positive feedback at this level, leading to accelerated tissue damage and aging.

(4,5) Clearance of senescent or aging cells might constitute putative targets for interventional approaches aimed to reduce or reverse the impact of aging and improve cell and tissue homeostasis by inducing a ‘rejuvenation’ process.

Figure 4. Pathological and beneficial functions of aging and senescence, according to current knowledge. In red are represented pathological consequences and in green beneficial functions of aging and senescence.

The impact of aging has mainly been described at the organismal level, since a complete cellular functional profile has not yet been established. Accordingly, whether beneficial consequences of the aging process exist at the cellular level is unclear.”

The assertion of Figure 3 (2) that:

“There is currently no direct evidence for a similar factor composition propagating the aging process via a kind of ‘gero-infection.”

was shown to be false in Reevaluate findings in another paradigm:

“It was demonstrated that increased aging occurred as a result of lack of gonadotropin-releasing hormone and that increased lifespan resulted from its provision during aging.

In this manner:

  1. The aging of hypothalamic microglia leads to
  2. The aging of the hypothalamus, which leads to
  3. Aging elsewhere in the body.

So here we have a multi-level interaction:

  1. The activation of NF-κB leads to
  2. Cellular aging, leading to
  3. A diminished production of GnRH, which then
  4. Acts (through the cells with a receptor for it, or indirectly as a result of changes to GnRH-receptor-possessing cells) to decrease lifespan.

So the age state of hypothalamic cells, at least with respect to NF-κB activation, is communicated to other cells via the reduced output of GnRH.”

The reviewers’ position on Figure 2 was:

“In our view, recent evidence that

  • Senescence is based on an unterminated developmental growth program and the finding that
  • The concept of post-mitotic senescence requires the activation of expansion, or ‘growth’ factors as a second hit,

favor the assumption that aging underlies a grating of genetic determination similarly to what is summarized above under the pseudo-programmed causative approach.”

Their position on Figure 4’s beneficial effects of aging began with the sentence:

“If we assume that aging already starts before birth, it can be considered simply a developmental stage, required to complete the evolutionary program associated with species-intrinsic biological functions such as reproduction, survival, and selection.”

Cited studies included: “Dissecting Aging and Senescence-Current Concepts and Open Lessons”

A strawman argument against epigenetic clocks

This 2019 review of epigenetic clocks by Washington cancer researchers ignored the elephant in the room, and repeatedly returned to an argument for randomness as a cause for aging and disease:

“A time-dependent stochastic event process, like epigenetic drift, could lead to cancer formation through the accumulation of random epigenetic alterations that, through chance, eventually alter epigenetic driver gene expression leading to a clone of cells destined to become cancer..

It is plausible that the stochastic process inherent in epigenetic drift can induce aberrant methylation events that accumulate in normal cells and eventually induce cancer formation.

Epigenetic drift relates to a biological process that changes the DNA methylome with age via stochastic gains or losses of DNA methylation. Epigenetic drift can be understood in terms of errors in DNA methylation maintenance during DNA-replication.

The phenomenon of (epi)genetic drift is generally associated with phenotypic neutrality.

For patients who develop cancer around age 80, the most likely initiation time for the founder adenoma cell is predicted to be very early in life, roughly between the ages 15 to 20 years. This unexpected and provocative finding suggests that the optimal age-range for prevention of colorectal cancer may be in adolescence and early adulthood (and ideally through lifelong) dietary and lifestyle interventions.”

The reviewers’ strawman arguments intentionally mischaracterized aspects of the epigenetic clock:

1. The epigenetic clock founder’s actual view on aging was in The epigenetic clock theory of aging:

“The proposed epigenetic clock theory of ageing views biological ageing as an unintended consequence of both developmental programmes and maintenance programmes, the molecular footprints of which give rise to DNAm age estimators.”

The reviewers omitted this intrinsic view of aging, which didn’t fit into their block labeled Extrinsic per the above graphic.

2. Another misrepresentation was:

“In contrast to epigenetic clocks, epigenetic drift refers to a stochastic process that involves both gains and losses of the methylation state of CpG dinucleotides over time.”

A reader of the original 2013 epigenetic clock study would understand that epigenetic clocks measure “both gains and losses of methylation” as in:

“The 193 positively and 160 negatively correlated CpGs get hypermethylated and hypomethylated with age, respectively.”

3. The reviewers omitted recent epigenetic clock significant developments. For example, there was no mention of the GrimAge study, although it was published before the review was submitted.

4. Epigenetic drift as the cause of aging and disease has abundant contrary evidence. The reviewers tossed in a little toward the end of their directed narrative:

“We found only a small number of drift-related CpG island-gene pairs for which drift correlated positively and significantly with gene expression.

The functional consequences of epigenetic drift need to be further elucidated.”

However, they didn’t acknowledge the elephant in the room: The epigenetic drift paradigm is generally inapplicable to humans because the vast majority of our cells don’t divide/proliferate! “Epigenetic aging: more than just a clock when it comes to cancer” (not freely available)

Epigenetic transgenerational inheritance extends to the great-great-grand offspring

This 2019 rodent study by the Washington State University labs of Dr. Michael Skinner continued to F4 generation great-great-grand offspring, and demonstrated that epigenetic inheritance mechanisms are similar to imprinted genes:

“Epigenetic transgenerational inheritance potentially impacts disease etiology, phenotypic variation, and evolution. An increasing number of environmental factors from nutrition to toxicants have been shown to promote the epigenetic transgenerational inheritance of disease.

Imprinted genes are a special class of genes since their DNA methylation patterns are unchanged over the generation and are not affected by the methylation erasure occurring early in development. The transgenerational epigenetic alterations in the germline appear to be permanently reprogrammed like imprinted genes, and appear protected from this DNA methylation erasure and reprogramming at fertilization in the subsequent generations. Similar to imprinted genes, the epigenetic transgenerational germline epimutations appear to have a methylation erasure in the primordial germ cells involving an epigenetic molecular memory.

Comparison of the transgenerational F3 generation, with the outcross to the F4 generation through the paternal or maternal lineages, allows an assessment of parent-of-origin transmission of disease or pathology. Observations provided examples of the following:

  1. Pathology that required combined contribution of both paternal and maternal alleles to promote disease [testis and ovarian disease];
  2. Pathology that is derived from the opposite sex allele such as father to daughter [kidney disease] or mother to son [prostate disease];
  3. Pathology that is derived from either parent-of-origin alleles independently [obesity];
  4. Pathology that is transmitted within the same sex, such as maternal to daughter [mammary tumor development]; and
  5. Pathology that is observed only following a specific parent-of-origin outcross [both F4 male obesity and F4 female kidney disease in the vinclozolin lineage].”

The study showed that epigenetically inherited legacies extend to the fifth generation. Do any of us know our ancestors’ medical histories back to our great-great-grandparents?

Will toxicologists take their jobs seriously, catch up to the current science, and investigate possible effects in at least the F3 generation that had no direct toxicant exposure? “Epigenetic transgenerational inheritance of parent-of-origin allelic transmission of outcross pathology and sperm epimutations”

Restrict information in the name of science?

A Stanford researcher was annoyed that we live in the 21st century, and advocated we return to previous centuries’ information-flow check valves of wise old men. No doubt the publishers of and subscribers to the Journal of the American Medical Association applauded the same old tired prescription.

Ten instances of the word “should” in the final two paragraphs provided ample evidence of the paper’s intent. Mirroring the current political climate – projecting – accusations made of others were items the accusers were guilty of themselves, such as:

“When these scientists act as investigators in the hundreds of observational studies that they publish, or as editors and peer reviewers in evaluating submissions from others, would they tolerate publishing analyses and funding proposals that might contradict their belief system?” “Neglecting Major Health Problems and Broadcasting Minor, Uncertain Issues in Lifestyle Science”

One of the paper’s references included an informative graphic:

“A histogram of the total number of rumor cascades in our data across the seven most frequent topical categories.” “The spread of true and false news online”

I’ll borrow from the curation of another Stanford paper Online dating cuts out the middlemen in conclusion:

“Are there examples where it wouldn’t potentially improve a person’s life to choose their information sources? Friends, family, and other social groups – and religious, educational, and other institutions – have had their middlemen/guarantor time, and have been found lacking.

Make your own choices for your one precious life.”

Maternal obesity causes heart disease in every offspring generation

This 2019 St. Louis rodent study found:

“We hypothesized that maternal obesity induces cardiac mitochondrial dysfunction in the offspring via transgenerational inheritance of abnormal oocyte mitochondria. All F1 to F3 descendants bred via the female in each generation were nonobese and demonstrated cardiac mitochondrial abnormalities.

Contrary to our hypothesis, male F1 also transmitted these effects to their offspring, ruling out maternal mitochondria as the primary mode of transmission. We conclude that transmission of obesity-induced effects in the oocyte nucleus rather than abnormal mitochondria underlie transgenerational inheritance of cardiac mitochondrial defects in descendants of obese females.”

For some reason, the researchers didn’t cite any of Dr. Michael Skinner’s research on epigenetic transgenerational inheritance. Their time, efforts, and resources would have been more productive had they used Dr. Skinner’s studies – such as the 2018 Epigenetic transgenerational inheritance of ovarian disease – as guides.

A podcast with the researchers is available here. “Maternal High-Fat, High-Sucrose Diet Induces Transgenerational Cardiac Mitochondrial Dysfunction Independent of Maternal Mitochondrial Inheritance” (not freely available)