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Epigenetic aging measurements can vary by the time of day at which they are taken, according to a study published in Aging Cell.

The circadian rhythm

Living on Earth, organisms have evolved to adapt to our planet’s day/night cycle. This adaptation is known as the circadian rhythm, and it influences many aspects of biology. As these researchers [1] and others have found, this even includes the number of white blood cells circulating in the bloodstream. Because different white blood cells often have different measured epigenetic ages, measuring the total epigenetic age of white blood cells can give different values over the course of a day.

Beginning with a one-person cohort

This research was done using white blood cells taken every three hours, over a period of 72 hours, from one 52-year-old man. Two sets of cells were measured: neutrophils and white blood cells that had been mostly depleted of neutrophils (WBC-Neu).

The researchers found 58,459 epigenetic sites in WBC-Neu that oscillated over 24 hours. These sites had significant overlap with the 2013 Horvath clock, which is touted to work on all tissues: that clock measured this person as being three years older at noon than at midnight. Similar oscillations were also found in the 2013 Hannum clock and the 2016 Lin clock: this particular clock had a variation of 5.5 years over the day.

In total, 8 of the 17 clocks were found to be significantly affected by the circadian rhythm when measuring WBC-Neu cells, and all of them followed the same pattern: older in the day and younger at night. Even GrimAge2, a 2022 clock, was significantly influenced in this way, as were clocks that are made with the principal components (PCs) of other epigenetic clocks.

To confirm their findings, then researchers then moved on to samples taken from a different group of people [2]. While that study had been originally geared to test stress rather than circadian rhythms, the researchers found the same thing: the participants were reported as having younger WBC-Neu values at 4:15 PM than at 12:45 PM.

Number and types of cells matter

Analyzing two other previously collected datasets [3, 4], the researchers found that natural killer (NK) cells were consistently found to be older than B cells and CD4+ T cells. Therefore, blood samples with more NK cells gave higher epigenetic ages. How much this affected the results varied by the particular clock.

While it is possible to partially offset these differences by adjusting for cell types, there are still variances within single cell types. Purified neutrophils taken from the 52-year-old man, along with another cohort of men aged 30 to 54, were reported as having statistically significant differences over the course of a day in 3 of the 17 clocks. Additionally, the number of different types of white blood cells is, itself, a biomarker of aging and disease.

Most critically, these oscillations may have confounded results from previous studies, as they can be stronger than the effect sizes found in those studies. For example, if researchers are testing lifestyle interventions and conclude that these interventions affect epigenetic age by roughly a year, this may be caused entirely by the time of day rather than the actual intervention. Significant work needs to be done to make sure that testing of interventions that affect epigenetic age are not being influenced by this or other confounders.

Literature

[1] Oh, G., Koncevičius, K., Ebrahimi, S., Carlucci, M., Groot, D. E., Nair, A., … & Petronis, A. (2019). Circadian oscillations of cytosine modification in humans contribute to epigenetic variability, aging, and complex disease. Genome biology, 20, 1-14.

[2] Apsley, A. T., Ye, Q., Etzel, L., Wolf, S., Hastings, W. J., Mattern, B. C., … & Shalev, I. (2023). Biological stability of DNA methylation measurements over varying intervals of time and in the presence of acute stress. Epigenetics, 18(1), 2230686.

[3] Reinius, L. E., Acevedo, N., Joerink, M., Pershagen, G., Dahlén, S. E., Greco, D., … & Kere, J. (2012). Differential DNA methylation in purified human blood cells: implications for cell lineage and studies on disease susceptibility. PloS one, 7(7), e41361.

[4] Wang, X., Campbell, M. R., Cho, H. Y., Pittman, G. S., Martos, S. N., & Bell, D. A. (2023). Epigenomic profiling of isolated blood cell types reveals highly specific B cell smoking signatures and links to disease risk. Clinical Epigenetics, 15(1), 90.

Tiny bubbles that cells use to communicate with each other prolonged lifespan and reversed numerous aging phenotypes when taken from young mice and injected into old ones, even though the treatment started late in life [1].

The tiny messengers

For millennia, humans credited young blood with rejuvenating qualities. This belief caused legendary and historic rulers to commit atrocities to get enough of this “elixir of youth”. While these ideas were delusional and barbaric, science has confirmed that there is some truth to them.

Research into heterochronic parabiosis, which involves connecting the vasculatures of a young and an old animal, has shown that it rejuvenates the old member of the pair and makes the young one age faster [2], but the mechanisms are still being elucidated. Recently, extracellular vesicles (EVs) carried by blood have been pinpointed as being responsible for many of these effects.

EVs are tiny bubbles made of a lipid bilayer, the same stuff cellular membranes are made of. Emitted by cells, they carry various molecular cargoes, such as proteins and microRNAs (miRNAs), and facilitate intercellular communication. EVs harvested from young blood have been shown to benefit old organisms [3], but since there are many molecules involved, the investigation into how exactly they do it is still very much ongoing.

Small size, big effect

EVs can be of various sizes, which might affect their qualities. In this new study published in Nature Aging by scientists from Nanjing University in China, the researchers focused on small EVs (sEVs) of less than 200 nanometers in diameter. They repeatedly injected old male mice with sEVs obtained from young mice or humans to explore their rejuvenation potential.

The old mice were injected with the sEV cocktail once a week starting from 20 months of age until death. Young and old controls were instead injected with the same dose of phosphate-buffered saline (PBS).

This led to an approximately 1/8th increase in median lifespan (34.4 vs 30.6 months), which is very significant given that the treatment only started when the mice were already quite old. It also improved various healthspan measures, such as frailty and hair retention.

The researchers analyzed various other aspects of age-related decline. For instance, just like humans, old male mice exhibit signs of reproductive aging, with lower levels of testosterone, low sperm count, and reduced sperm motility. However, the sEV treatment brought sperm concentration and motility, as well as litter size, back to levels comparable to those of young mice.

The good news didn’t end there. The treatment significantly improved the old mice’s fitness, as measured by heat production, oxygen consumption, and locomotor activity. It also increased cardiac performance, slowed bone loss, and partially rescued age-related loss of cortical and hippocampal volume.

All of this led to clear cognitive and physical improvements. In the Morris water maze test, which assesses learning and memory, non-treated aged mice performed much worse than young controls, but this deficiency was almost completely reversed by the sEV treatment. The same happened in the treadmill endurance test, in which non-treated aged mice clocked a much shorter time to exhaustion, but the treated old animals kept pace with young controls.

Notably, the researchers also tried injecting aged mice with sEVs taken from other aged mice, but this failed to ameliorate any age-related deficiencies. When young mice were injected with sEVs from old mice, they experienced physical and cognitive decline, which is in line with previous research on heterochronic parabiosis.

Human EVs work in mice, and maybe vice versa

Digging deeper into the effects of the treatment, the researchers found that, even when administered for only two weeks, it lowered senescent cell burden and brought down reactive oxygen species (ROS) in multiple tissues to levels comparable with young controls. Similar reductions were observed in the levels of advanced glycation end products (AGEs) and lipofuscin. Both are harmful compounds, the accumulation of which is associated with aging phenotypes.

Proteomic analysis of several tissues revealed that sEVs exerted a wide-ranging effect, most of which are related to mitochondrial dysfunction, epigenetic alterations, and genomic instability, all of which are known hallmarks of aging. The researchers found that in hippocampus and muscle, the treatment largely restored markers of mitochondrial health, including ATP production, DNA content, quantity, and morphology.

Since, according to the authors, “the biological activity of sEVs exhibits little to no species specificity”, they tried injecting old mice with sEVs derived from the blood of young humans, recapitulating many of the benefits observed in previous experiments. If the reverse is beneficial, this might solve the problem of EV supply for humans.

Literature

[1] Chen, X., Luo, Y., Zhu, Q., Zhang, J., Huang, H., Kan, Y., … & Chen, X. (2024). Small extracellular vesicles from young plasma reverse age-related functional declines by improving mitochondrial energy metabolism. Nature Aging, 1-25.

[2] Ashapkin, V. V., Kutueva, L. I., & Vanyushin, B. F. (2020). The effects of parabiosis on aging and age-related diseases. Reviews on New Drug Targets in Age-Related Disorders, 107-122.

[3] Grigorian Shamagian, L., Rogers, R. G., Luther, K., Angert, D., Echavez, A., Liu, W., … & Marbán, E. (2023). Rejuvenating effects of young extracellular vesicles in aged rats and in cellular models of human senescence. Scientific Reports, 13(1), 12240.

A study published in Aging Cell has reported that older people with better regulated autophagy in their skeletal muscles have less age-related frailty.

Taking out the trash

The researchers begin this paper by discussing the various activities and effects of autophagy. Obviously, too much autophagy is not good, as it aggravates tissue degeneration [1], but a lack of it has also been found to lead to degeneration [2]. The energy-sensing AMPK pathway encourages autophagy, and the related nutrient-sensing mTOR pathway discourages it [3].

There are also multiple types of autophagy, depending on the particular organelle being consumed. Like many things in biology, the full biochemical pathways involved in autophagy have not been entirely mapped out. This work focuses principally on mitophagy, the removal of damaged mitochondria, which is chiefly regulated by PGC-1α [4].

Comparing physical abilities to RNA

After screening, a total of 575 participants, with an average age of 75.9 years, were inducted into this study. Most participants were of European descent, slightly over half were female. Over a third had only one chronic condition, while roughly a sixth had more than one.

A total of 260 genes were chosen for RNA sequencing analysis, based on their roles in autophagy, mitophagy, and/or the mTOR pathway. These genes were checked against key functional metrics, such as mitochondrial function as measured by oxidative phosphorylation (OXPHOS), oxygen consumption, and 400-meter walking speed.

The expression of genes that are central to autophagy machinery were uncorrelated with these outcomes. However, some regulatory genes, such as FoxO1, were found to be significantly negatively correlated, to the researchers’ surprise. Other metabolic regulators were found to have positive correlations, as were genes related to mitochondrial fusion and fission. Some genes related to the mTOR pathway were negatively associated, while others were positively associated.

Unsurprisingly, more OXPHOS was associated with more expression of the sirtuin genes SIRT5 and SIRT3. Multiple mitochondria-related genes were also associated with better oxygen consumption. mTOR and its pathways were associated with better walking speeds.

A potential explanation for contradictory results

Some of these findings are entirely expected. However, some of them, particularly the relationship of more FoxO1 to worse outcomes, goes against a consensus that suggests benefits from this autophagy regulator. These researchers suggest that its upregulation could be a consequence, rather than a cause, of autophagic dysregulation. Increased expression of regulatory genes suggests a need for more regulation, with the body engaging in more quality control in an attempt to compensate.

These results also suggest that inhibiting the effects of mTOR, which naturally inhibits autophagy and is itself inhibited by rapamycin and rapalogs, is a potential path to increased muscle performance in older people. Research in this area has been previously conducted [5], and this paper offers more insight into how such an approach might work.

Literature

[1] Mammucari, C., Milan, G., Romanello, V., Masiero, E., Rudolf, R., Del Piccolo, P., … & Sandri, M. (2007). FoxO3 controls autophagy in skeletal muscle in vivo. Cell metabolism, 6(6), 458-471.

[2] Masiero, E., Agatea, L., Mammucari, C., Blaauw, B., Loro, E., Komatsu, M., … & Sandri, M. (2009). Autophagy is required to maintain muscle mass. Cell metabolism, 10(6), 507-515.

[3] Jung, C. H., Ro, S. H., Cao, J., Otto, N. M., & Kim, D. H. (2010). mTOR regulation of autophagy. FEBS letters, 584(7), 1287-1295.

[4] Vainshtein, A., Desjardins, E. M., Armani, A., Sandri, M., & Hood, D. A. (2015). PGC-1α modulates denervation-induced mitophagy in skeletal muscle. Skeletal muscle, 5, 1-17.

[5] Bodine, S. C. (2022). The role of mTORC1 in the regulation of skeletal muscle mass. Faculty Reviews, 11.