Scientists have shown that only some of the hallmarks of aging affect methylation clocks that are used to measure biological age .
The big black box
Epigenetic clocks that measure biological age based on aging-related changes in genome methylation have been a great success. They enable researchers to analyze the effects of various interventions on lifespan without waiting for a lab animal to die, and they work even on cells in vitro.
Yet, despite the strong correlation between existing epigenetic clocks and various aspects of aging, we still hardly know how those clocks work – in particular, what specific biological changes drive them. For now, this ignorance does not preclude scientists from using the clocks, but there are several reasons why discovering their biological underpinnings is important, as it can provide a more nuanced understanding of aging itself.
In this new study led by Ken Raj of Cambridge university and Altos Labs, and Steve Horvath, the world’s foremost authority on epigenetic clocks, the researchers investigated the relationship between epigenetic clocks and several hallmarks of aging . The group used Horvath’s second-generation multi-tissue clock , which is known for its accuracy, and several types of human cells, including keratinocytes and fibroblasts, taken from 14 human donors of various ages.
First, the researchers showed that the clock does not correlate well with cellular senescence. Senescence was induced in cells by three different stressors: irradiation, oncogene activation, and replication. Oncogene activation and irradiation led to senescence quickly, after about two weeks, but it did not increase epigenetic age compared to non-senescent cells of the same chronological age. Conversely, it took cells up to six months to reach replicative senescence, which was duly reflected by an increase in epigenetic age.
Replicative stress is accompanied by telomere attrition, another hallmark of aging in which telomeres get shorter as the cells edge towards senescence. When the researchers added telomerase reverse transcriptase (TERT), an enzyme that keeps telomeres from shortening, the cells continued to proliferate without reaching senescence, and their epigenetic age continued to increase. This means that telomere attrition, like senescence, does not contribute to epigenetic aging.
Another important hallmark of aging is DNA instability. The researchers used irradiation protocols known to induce some amount of double-strand DNA breaks without affecting cellular viability. The cells, immortalized with TERT to avoid replicative senescence, lived on and kept proliferating, while their epigenetic age continued to rise on par with non-irradiated controls, showing that genomic instability had no effect on the epigenetic clock.
Deregulation of nutrient sensing
Interestingly, rapamycin administered to immortalized cells after 80 passages effectively stopped further increase in epigenetic age. Since rapamycin mediates nutrient sensing, the researchers concluded that deregulated nutrient sensing, also a hallmark of aging, does contribute to epigenetic age.
To understand how mitochondrial dysfunction, another hallmark of aging, contributes to epigenetic aging, the researchers treated cells with a compound that inhibits mitochondrial activity, which greatly accelerated their rate of epigenetic aging. Treatment with Bezafibrate, a promoter of mitochondrial activity, reversed this increase. The researchers suggest that the two hallmarks (nutrient sensing and mitochondrial activity) that showed relevance to the epigenetic clock might be linked to each other.
Stem cell exhaustion
To investigate this hallmark of aging, the researchers divided tissue samples in two. They increased the ratio of stem cells, which are characterized by a minuscule rate of epigenetic aging, in one part and depleted stem cells from the other part. As a result, the stem cell-enriched fraction aged slower than the stem cell-depleted fraction.
However, can differentiated cells age at different rates? To answer this question, the researchers took cell samples from two donors, both with an epigenetic age of 23. While their cells clonally expanded, their epigenetic age trajectories diverged significantly from the shared baseline. This divergence could not be explained by the much smaller variability of the clock, confirming that an epigenetic age of a tissue is an average of the epigenetic ages of its cells, which could differ substantially from each other.
Aging and longevity
While rapamycin blocked the increase of epigenetic age in immortalized cells, nicotinamide adenine dinucleotide (NAD), nicotinamide riboside (NR), and metformin all extended the cells’ lifespan but without significantly altering their rate of epigenetic aging. The researchers suggest that, in line with previous research, some perturbations can affect both the rate of epigenetic aging and lifespan, while others can affect lifespan without slowing the rate of epigenetic aging, “indicating that aging and longevity, although intimately associated, may nevertheless be distinct”. Interestingly, in the Intervention Testing Program (ITP) trials, rapamycin did substantially increase lifespan in mice, while both metformin and NR failed to do so.
At a higher level of consideration, the innate nature and inevitability of epigenetic aging contrasts with the stochasticity of wear and tear, which is presumed to exert a measurable aging effect only later in life when damage outstrips repair. This, however, does not argue against the relevance of wear and tear and cellular senescence. Instead, these distinct stochastic processes are likely to synergize with epigenetic aging in manifesting the overall phenotypical features of aging. If a successful strategy against aging is to be found, then these distinct and parallel aging mechanisms must be addressed; for example, by the removal of senescent cells, together with the retardation of epigenetic aging.
This important paper is an attempt to look inside the black box that epigenetic clocks have mostly been so far. It raises many intriguing questions that should be addressed in future research. The researchers did not investigate the two other hallmarks of aging, loss of proteostasis and altered cellular communication, though they cite some previous research that links those hallmarks to epigenetic aging.
 Kabacik, S., Lowe, D., Fransen, L., Leonard, M., Ang, S. L., Whiteman, C., … & Raj, K. (2022). The relationship between epigenetic age and the hallmarks of ageing in human cells. Nature Aging, 1-10.
 López-Otín, C., Blasco, M. A., Partridge, L., Serrano, M., & Kroemer, G. (2013). The hallmarks of aging. Cell, 153(6), 1194-1217.
 Horvath, S., Oshima, J., Martin, G. M., Lu, A. T., Quach, A., Cohen, H., … & Raj, K. (2018). Epigenetic clock for skin and blood cells applied to Hutchinson Gilford Progeria Syndrome and ex vivo studies. Aging (Albany NY), 10(7), 1758.