Researchers have proposed a new epigenetic clock designed to measure subtle epigenetic changes in vitro [1]. They hope that their discovery will help to expedite the creation of new anti-aging drugs.
Measuring biological age
Aging research is all about the difference between chronological and biological age. The former is simply the passage of time, but researchers can try and slow the advance of the latter. One of the necessary instruments is a clock that can reliably measure the biological age of organisms.
In recent years, several such clocks have been devised based on different principles, including age-related changes in blood composition and telomere shortening. However, probably the most reliable to date are epigenetic clocks, which analyze changes in gene expression [2]. The principle was laid out in a seminal paper by Steve Horvath et al. in 2013 [3]. Epigenetic clocks are usually based on the methylation of CpG sites, areas in the genome where a cytosine nucleotide (C) is followed by a guanine nucleotide (G), which can be either a single “CG” or a recurring sequence such as “CGCGCG”. Cytosines in such sequences can have methyl groups added to them (methylation). Each C can be either methylated or not. A strong correlation has been observed between CpG methylation profile and biological age, although the mechanism of this correlation is not fully understood.
Since there are tens of millions of CpG sites across the genome, epigenetic clocks use only a fraction of them. Researchers fine-tune their clocks by carefully choosing the sites that show the strongest correlation with biological age. For instance, Horvath’s original multi-tissue clock and the skin and blood clock that he and his group developed in 2018 [4] use 353 and 391 sites, respectively.
Designing a clock for cultured cells
Epigenetic clocks have rarely been used for identifying changes in the biological age of cultured cells, as epigenetic changes in cultured cells accumulate faster than in the human body. In vitro experiments could greatly expedite aging research and make it more cost-effective, but the unavailability of reliable epigenetic clocks for such experiments is a major hurdle that this research strives to overcome.
The scientists decided to create a clock that would measure minor epigenetic changes in cultured cells in order to use them as a yardstick for the creation of novel anti-aging drugs. They began with obtaining human mammary fibroblasts (HMF) from a healthy 16-year old donor and culturing them from passage (division) 10 to 20, and such cells enter senescence after 29 passages. Methylation was measured after every other passage at 850,000 CpG sites.
The researchers then tested three of the existing epigenetic clocks: Horvath’s original multi-tissue clock from 2013, the skin and blood clock, and the PhenoAge clock developed by Levine at al. in 2018 [5]. All the clocks showed higher numbers at the end of the experiment than at the beginning, reflecting the aging of the cells involved, but the results were too crude. As the researchers concluded, “none of the existing clocks was ideally suited to accurately measure subtle anti-ageing drug potential in human primary cells in vitro“.
To design a clock that would measure minor changes in methylation that occur between passages, the researchers introduced a second set of cells in addition to the aforementioned HMFs: human dermal fibroblasts (HDF) obtained from a different donor, with a different proliferative span and a different rate of methylation changes. After sifting through hundreds of thousands of CpG sites, the researchers had identified 2,543 that showed the strongest correlation with the aging of both samples, and then narrowed the pool down even more to just 42 predictor CpGs that they used to build what they called CellAgeClock.
The novel clock had been tested on 26 additional cell samples and was able to correctly identify the passage number most of the time, which is impressive given the subtlety of epigenetic changes between passages. The researchers then tried to interpret the readings of other clocks in terms of the number of passages but failed to get consistent results.
Since the original aim was to create a tool that would help discover new geroprotective drugs, the researchers wanted to see how well CellAgeClock measures the rejuvenating power of existing ones. They tested the well-researched rapamycin alongside three newer compounds: torin2, Dactolisib/BEZ235, and trametinib. The clock confirmed the anti-aging prowess of rapamycin and revealed that torin2, Dactolisib/BEZ235, and, to a lesser extent, trametinib, have rejuvenating potential as well. The results were also recreated in vivo by demonstrating that the drugs indeed prolong the lifespan of drosophila flies.
Conclusion
The researchers infer from these results that the epigenetic clock they have devised can successfully predict, based on experiments in vitro, the rejuvenating effect of novel drugs in vivo. If their conclusion is correct, CellAgeClock might indeed make the process of discovering new anti-aging drugs faster and cheaper. However, since only two cell types were used both to design and test the clock, and since three out of four drugs that it was tested on inhibit the mTOR pathway, it remains to be seen whether the positive results can be reproduced for different cell and drug types.
Literature
[1] A CellAgeClock for expedited discovery of anti-ageing compounds. Celia Lujan, Eleanor J. Tyler, Simone Ecker, Amy P. Webster, Eleanor R. Stead, Victoria E. Martinez Miguel, Deborah Milligan, James C. Garbe, Martha R. Stampfer, Stephan Beck, Robert Lowe, Cleo L. Bishop, Ivana Bjedov bioRxiv 803676
[2] Horvath, S., & Raj, K. (2018). DNA methylation-based biomarkers and the epigenetic clock theory of ageing. Nature Reviews Genetics, 19(6), 371.
[3] Horvath, S. (2013). DNA methylation age of human tissues and cell types. Genome biology, 14(10), 3156.
[4] Horvath, S., Oshima, J., Martin, G. M., Lu, A. T., Quach, A., Cohen, H., … & Wilson, J. G. (2018). Epigenetic clock for skin and blood cells applied to Hutchinson Gilford Progeria Syndrome and ex vivo studies. Aging (Albany NY), 10(7), 1758.
[5] Levine, M. E., Lu, A. T., Quach, A., Chen, B. H., Assimes, T. L., Bandinelli, S., … & Whitsel, E. A. (2018). An epigenetic biomarker of aging for lifespan and healthspan. Aging (Albany NY), 10(4), 573.
