In preprints: an epigenetic clock that does not tick

Aging is an inevitable process characterised by the gradual decline of molecular and physiological functions. This decline can manifest at multiple levels – from decreased cellular integrity, through impaired tissue homeostasis, to complex functional decay of the organism, decreasing its ability to survive. One of the main factors contributing to preservation of cellular integrity is the maintenance of appropriate gene expression patterns, regulated by epigenetic marks. These marks include histone modifications and DNA methylation. With increasing age, the epigenetic landscape becomes progressively altered, leading to genomic instability and changes in gene expression. Epigenome dysregulation has thus been recognised as one of the hallmarks of aging (López-Otín et al., 2023).

DNA methylation, localised at cytosines within CpG dinucleotides, is among the most studied epigenetic modifications during aging, showing general genome-wide age-dependent hypomethylation (López-Otín et al., 2013). However, some regions, such as the promoters of certain genes, become hypermethylated during aging, leading to transcriptional silencing (Maegawa et al., 2010). While it is believed that most age-related DNA methylation changes occur stochastically across cell populations, some loci appear to gain or lose DNA methylation in a non-stochastic manner (Pal and Tyler, 2016). It is not fully understood whether such changes are somehow programmed, or rather occur at regions prone to DNA methylation alterations. Nevertheless, a set of CpG sites was identified to have the same pattern of age-associated DNA methylation changes across different individuals of the same species; this ‘epigenetic clock’ can be used to predict the chronological age (Field et al., 2018; Horvath, 2013). Approximately 1000 of these CpGs are conserved across more than 100 mammalian species (Lu et al., 2023), as well as between mammals and frogs (Zoller et al., 2024).

Urodele amphibians, which include salamanders and newts, are the only vertebrates capable of substantial regeneration of their body parts, and do not appear to suffer aging-associated physiological decline (Kara, 2009). The most widely studied, but also critically endangered, urodele amphibian is the axolotl (Ambystoma mexicanum), which is well known for reaching sexual maturity without undergoing metamorphosis. Axolotls exhibit multiple characteristics of negligible aging, including resistance to cancer and other aging-associated diseases, and lack of accumulation of senescent cells with increasing age (Ingram, 1971; Yun, 2021; Yun et al., 2015). In their preprint, Haluza and colleagues build the first axolotl epigenetic clock to determine whether these amphibians, despite their apparent lack of physiological aging, exhibit signs of molecular aging (Haluza et al., 2024 preprint). In particular, the authors are interested in aging-associated changes in the DNA methylome, which have been previously demonstrated to occur in mammals and frogs (Lu et al., 2023; Zoller et al., 2024).

Haluza and colleagues measured DNA methylation levels at highly conserved CpG sites by leveraging a commonly used array platform (Arneson et al., 2022). They sampled six different tissues from 30 individuals between 4 weeks and 21 years of age, thus covering the entire lifespan of the axolotl, and used these data to build their axolotl epigenetic clock. Strikingly, while the clock can be used to accurately estimate chronological age in individuals that are under 4 years old, it was not possible to build a clock for the later stages of the axolotl lifespan. This may reflect a stabilisation of DNA methylation patterns in individuals above 4 years old.

To understand why the epigenetic clock does not tick for older axolotls, the authors took two directions. First, they demonstrated that DNA methylation changes in axolotls under 4 years of age are to some extent shared with frogs and humans with the same relative age (determined as chronological age to maximum lifespan ratio). The CpGs with methylation changes that are most highly correlated with chronological age in axolotl during this early period are associated with developmentally relevant genes, such as homeobox transcription factors or their regulators, as well as with genes previously linked to aging – some of which are shared between axolotl, frogs and humans. In contrast, the authors find that CpGs related to genes that are targets of Polycomb Repressive Complex 2 (PRC2, which catalyses a silencing tri-methylation of lysine 27 on histone 3) and have been demonstrated to gain DNA methylation with age in frogs and mammals (Lu et al., 2023; Zoller et al., 2024), do not become considerably hypermethylated in axolotl tissues.

In the second approach, the authors performed Oxford Nanopore sequencing to profile the DNA methylation landscape in the axolotl genome at different ages and confirmed that the axolotl DNA methylome in the promoter regions remains stable during aging, even when only lowly methylated promoters or PRC2 target genes are considered. Importantly, the DNA methylation landscapes of a 3.5-year-old and a 10-year-old individual were more similar than those of 3.5-year-old and younger individuals. This suggests that, although the axolotl DNA methylome undergoes dynamic changes during early life, it stabilises as these organisms age, unlike in other previously tested vertebrates. Overall, despite the general similarity in epigenetic aging between axolotl, frogs and mammals in early life, it seems that older frogs and humans accumulate DNA methylation changes consistent with conventional organismal aging, while aging axolotls deviate from this pattern.

In their preprint, the authors reveal the striking stability of the axolotl epigenome during aging, and demonstrate for the first time that axolotls not only manifest negligible physiological aging, but also negligible aging at the epigenetic level. This is in contrast with long-lived mammals with low aging-associated physiological decline, such as bats and naked mole-rats, which demonstrate conventional changes in CpG methylation with increased age (Horvath et al., 2022; Wilkinson et al., 2021). Although it is not known how this unique DNA methylome stability relates to axolotl neoteny and/or regenerative capacity, it clearly makes the axolotl a unique model to further study anti-aging mechanisms at the molecular level.