Epigenetic reprogramming as a key to reverse ageing and increase longevity

Beatriz Pereira a,1, Francisca P. Correia a,1, Inˆes A. Alves a,1, Margarida Costa a,1, Mariana Gameiro a,1, Ana P. Martins b, Jorge A. Saraiva b,* a Department of Chemistry, University of Aveiro, Aveiro, Portugal b LAQV-REQUIMTE, Department of Chemistry, University of Aveiro, Aveiro, Portugal  Ageing Research Reviews Volume 95, March 2024, 102204

Ronald Peters, MD, MPH  – Comments

The epigenome is the collective of chemical modifications to DNA and associated proteins that controls which genes are turned on or off without changing the DNA sequence. Epigenetic changes are triggered by a variety of signals from outside and within the body telling cells how to adjust to any challenging situation.  Diet and nutrients direcctly influence the epigenome similar to excercise which can induce methyltion and demethylation of DNA in muscle affecting genes related to metabolism and inflammation.  Environmental toxins such as chemicals and heavy metals can alter DNA metylation and histone patterns as well in response to the toxic effect.  This article is exploring the complex chemistry of the epigenetics with an eye towards medications to reverse  effects, usually related to both cellular and human aging.  Also, psychological stress can trigger hormones such as cortisol and noreinehrine which leads to a cascade of epigenetic changes.  Epigenetic changes created by consciousness are a powerful force and a bit more mystifying than the molecular world reflected in the article.  Basically, your DNA is the hardware and the epigenome is the software, which responds moment to moment to your thoughts, emotions and how you live your life. 

A B S T R A C T

The pursuit for the fountain of youth has long been a fascination amongst scientists and humanity. Ageing is broadly characterized by a cellular decline with increased susceptibility to age-related diseases, being intimately associated with epigenetic modifications. Recently, reprogramming-induced rejuvenation strategies have begun to greatly alter longevity research not only to tackle age-related defects but also to possibly reverse the cellular ageing process. Hence, in this review, we highlight the major epigenetic changes during ageing and the state-of- art of the current emerging epigenetic reprogramming strategies leveraging on transcription factors. Notably, partial reprogramming enables the resetting of the ageing clock without erasing cellular identity. Promising chemical-based rejuvenation strategies harnessing small molecules, including DNA methyltransferase and his- tone deacetylase inhibitors are also discussed. Moreover, in parallel to longevity interventions, the foundations of epigenetic clocks for accurate ageing assessment and evaluation of reprogramming approaches are briefly pre- sented. Going further, with such scientific breakthroughs, we are witnessing a rise in the longevity biotech in- dustry aiming to extend the health span and ideally achieve human rejuvenation one day. In this context, we overview the main scenarios proposed for the future challenges associated with such an emerging field. Ultimately, this review aims to inspire future research on interventions that promote healthy ageing for all.

 Introduction

 Ageing

 Ageing is a ubiquitous natural process marked by a progressive decline in cellular, tissue, and physiological functions across all organ systems (Fakouri et al., 2019; Guo et al., 2022). This process is accompanied by an exponential increase in mortality following the Gompertz law (da Silva and Schumacher, 2019). As cells divide and proliferate, telomeres progressively shorten until they stop the cell cycle, leading to death and cell senescence, a well-known ageing hallmark. Furthermore, over-proliferation of cells accelerates telomere shortening, potentially contributing to premature ageing and the development of age-related diseases, including cancer. Conversely, insufficient cellular proliferation can also contribute to ageing by leading to tissue atrophy and the inability to repair damage. Therefore, maintaining a balance in cellular proliferation is vital for healthy ageing (Vaiserman and Krasnienkov, 2021).

Moreover, ageing is a complex process that manifests itself through a combination of variable and predictable changes. For instance, thymic involution, presbyopia, and sarcopenia are well-documented and somewhat deterministic phenomena observed in most vertebrates. However, the rate and extent of those can vary greatly among in- dividuals (Crooke et al., 2022; Larsson et al., 2019; Liang et al., 2022; Thomas et al., 2020). Therefore, despite certain predictable aspects, the overall process of ageing occurs in a non-linear or inconsistent manner (Laffon et al., 2021).

Several studies suggest that ageing should be considered a disease, emphasizing the plasticity of the ageing process, having a potential for treatment, rather than an inevitable process (Guo et al., 2022; Stallone et al., 2019). Notably, there have been many efforts to formally classify ageing as a disease, as such a step is fundamental to formally advance appropriate clinical diagnosis and longevity interventions (Calimport et al., 2019; Khaltourina et al., 2020). For instance, organ and tissue senescence-related disease codes have been proposed to be included in ageing classifications in the World Health Organization (WHO) Inter- national Classification of Diseases (ICD) (Calimport et al., 2019).

Although this field is replete with mechanistic theories exploring the underlying molecular and cellular alterations that might contribute to the ageing phenotype, it remains a constant topic of scientific inquiry. So, even though these rather complex processes do not provide a para- digmatic description of the causes of ageing, they are collectively referred to as hallmarks of ageing (Gems and de Magalha˜es, 2021; Lo´pez-Otín et al., 2013; Tsurumi and Li, 2012).

  • Hallmarks of ageing

 The original hallmarks proposed by Lo´pez-Otín et al. (2013) include genomic instability, telomere attrition, epigenetic changes, loss of proteostasis, deregulated nutrient sensing, mitochondrial dysfunction, cellular senescence, stem cell exhaustion, and altered intercellular communication (Laffon et al., 2021; Lidzbarsky et al., 2018; Lo´pez-Otín et al., 2013; Toniolo et al., 2023).

They can be categorized into three groups: primary, antagonistic, and integrative hallmarks. Primary hallmarks consistently have negative effects and initiate damage accumulation; antagonistic hallmarks have dual effects depending on their intensity, so at high levels, they can exacerbate negative effects; and integrative hallmarks arise when tissue homeostasis mechanisms can no longer compensate for accumulated damage (Aunan et al., 2016). Additionally, these can also be subdivided into molecular, cellular, and systemic hallmarks, based on their impact at different levels (Lo´pez-Otín et al., 2023) (Table 1). Furthermore, the ageing field is continuously expanding and proposing new hallmarks, including extracellular vesicles (Manni et al., 2023), compromised autophagy, microbiome disturbance, altered mechanical properties, splicing deregulation, and inflammation (Lo´pez-Otín et al., 2023; Schmauck-Medina et al., 2022).

  • In the pursuit for the fountain of youth

 Humans have long sought ways to prolong life and restore health, as depicted in the myths of the ‘fountain of youth’ through various cultures and times in history. The metaphor has served as an illustration of anything that potentially increases longevity (Aunan et al., 2016). As the average lifespan increases and the elderly population grows, age-related diseases including neurodegenerative, cardiovascular, and metabolic diseases have become more prevalent, resulting in significant social and economic consequences worldwide (Guo et al., 2022; Lidzbarsky et al., 2018; Toniolo et al., 2023). This escalating burden has placed ageing research at the center stage (Chakravarti et al., 2021), creating a huge academic and commercial industry interest (Aunan et al., 2016). The investigation in this field aims to identify interventions and treatments that can promote healthy ageing and prevent, delay, or even reverse age-related diseases (Aunan et al., 2016; Guo et al., 2022). Cutting-edge technologies such as omics and artificial intelligence show promise in the study of ageing mechanisms and treatment options (Guo et al., 2022). Lifestyle modifications such as caloric restriction (Flanagan et al., 2020), a Mediterranean-style diet (Shannon et al., 2021), and exercise (Rebelo-Marques et al., 2018) can reduce the incidence of age-related conditions (Guo et al., 2022). Moreover, stem cell transplantation, senolytics, elimination of senescent cells, and epigenetic reprogramming offer new directions for treating ageing-related diseases. However, identifying safe pharmaceutical targets for ageing improvement remains a challenge (Guo et al., 2022; Lo´pez-Otín et al., 2013). Despite this and the endless unanswered questions, the pursuit of healthy longevity for humankind continues, until the fountain of youth is finally unlocked (Guo et al., 2022).

Considering the vast amount of information surrounding the com- plex multifaceted molecular processes of ageing and its potential ther- apeutic targets, it is impossible to cover all of them comprehensively within the scope of a paper´s length. Hence, the authors have chosen to narrow their focus to strategies that specifically target epigenetic changes in ageing. Specifically, epigenetic reprogramming appears to be the most promising intervention at the moment, not only allowing it to slow down but also potentially reverse cellular ageing.

The recently proposed Information Theory of Aging (ITOA) posits that ageing results from the gradual loss of cellular information, primarily in the form of epigenetic information, leading to the erosion of cellular identity. In contrast to the somatic mutation theory (Morley, 1995), ITOA not only explains similar ageing patterns in individuals with distinct genomes but also justifies why identical mice and human twins can age at different rates (Lu et al., 2023). Notably, the observation that old cells and tissues can be epigenetically reprogrammed to a more youthful state to achieve lifespan extension without apparently reversing mutations reinforces the suspicion of a predominantly epige- netic basis for ageing (Lu et al., 2023). Therefore, by targeting the epigenome it could be possible to go “to the root” of the ageing process and influence multiple hallmarks simultaneously.

Table 1  

See original article referenced at the end of this post

The nine original hallmarks, accompanied by a short definition of their contribution to ageing, the categories in which they suit in, and references to papers explaining these hallmarks in detail.

  • Epigenetic dynamics in ageing: normal mechanisms and age-related changes

 Epigenetic modifications, including DNA methylation, histone modifications, and non-coding RNA molecules dynamically regulate gene expression patterns and contribute to cellular identity and func- tion. These mechanisms ensure proper development, tissue mainte- nance, and response to environmental cues throughout an organism’s lifespan (Kane and Sinclair, 2019).

However, with advancing age, the epigenome undergoes profound changes at all levels of chromatin and DNA organization. Epigenetic alterations in ageing encompass reduced global heterochromatin and DNA hypomethylation, site-specific DNA hypermethylation, altered histone modifications, and dysregulation of non-coding RNA (ncRNA) expression (Kane and Sinclair, 2019).

As organisms age, there is a reduction in global heterochromatin and DNA hypomethylation, leading to genomic instability and activation of transposable elements, which can disrupt gene regulation and genome integrity (Kane and Sinclair, 2019; Lee et al., 2020). Site-specific DNA hypermethylation affects specific genomic regions, including gene pro- moters, and can result in the silencing of critical genes involved in cellular processes such as DNA repair, immune response, and meta- bolism. Furthermore, alterations in histone modifications, such as changes in histone acetylation and methylation patterns, impact chro- matin structure and gene accessibility. These modifications influence gene expression profiles and can contribute to age-associated pheno- types (Kabacik et al., 2022; Kane and Sinclair, 2019). Additionally, dysregulation of non-coding RNAs, including microRNAs, long non-coding RNAs, and circular RNAs disrupts gene regulatory networks and cellular homeostasis, further contributing to age-related changes (Kabacik et al., 2022; Wang et al., 2022). These epigenetic changes also contribute to various ageing hallmarks including genomic instability, telomere attrition, loss of proteostasis, cellular senescence, and mito- chondrial dysfunction (Lo´pez-Gil et al., 2023).

Understanding the dynamics of epigenetic modifications during ageing is crucial for unraveling the molecular basis of age-related dis- eases and identifying potential therapeutic interventions (Ji et al., 2023a).

  1. Epigenetic reprogramming

 Reprogramming-induced epigenetic rejuvenation is an emerging field of research focused on countering the ageing process through the modification of epigenetic marks and gene expression patterns. Reprogramming can be carried out in different ways, namely complete reprogramming and partial reprogramming (Basu and Tiwari, 2021; Simpson et al., 2021a).

  • Complete reprogramming and partial reprogramming

 Complete reprogramming

Complete reprogramming refers to the process of converting somatic cells into induced pluripotent stem cells (iPSCs), which exhibit distinc- tive characteristics such as self-renewal ability and the potential to differentiate into various cell types. Pluripotency is supported by a complex network of signaling molecules and genes, particularly Oct4, Sox2, and Nanog (Al Abbar et al., 2020; Teshigawara et al., 2017), which are transcription factors that play a crucial role in maintaining this characteristic.  The interplay between external signalingmolecules and internal factors leads to the development of a specific gene expression pattern and the establishment of an epigenetic state characteristic of stem cells (Simpson et al., 2021b).

This method allows for the generation of patient-specific pluripotentstem cells with fewer ethical concerns compared to embryonic stem cells (ESCs), arising from the destruction of embryos during ESC isolation since these cells are derived from either the inner cell mass or epiblast of blastocysts (Al Abbar et al., 2020; Teshigawara et al., 2017; Zakrzewski et al., 2019). iPSCs closely resemble ESCs in terms of morphology, growth behavior, and responsiveness to growth factors and signaling molecules. Like ESCs, iPSCs can differentiate in vitro into cell types from all three primary germ layers (Puri and Wagner, 2023).

In this approach, ageing and cellular differentiation are inter- connected processes that are intricately linked and hence, cannot be separated. In other words, only by achieving a complete state of de- differentiation in cells and erasing their specific lineage identity, cells can suffer from epigenetic resetting and then be differentiated again. Although it has massive potential for regenerative medicine, this phe- nomenon does not seem to be appropriate for anti-ageing strategies, as it requires the loss of cellular identity and re-establishment of self-renewal capabilities (Al Abbar et al., 2020).

  • Partial reprogramming

On the other hand, partial reprogramming focuses on achieving epigenetic rejuvenation while retaining the original cell phenotype, rather than inducing pluripotency. To describe this type of rejuvenation accurately, the term “reprogramming-induced rejuvenation” (RIR) is more suitable, highlighting the nature of the process and the ultimate goal of the interventions. RIR holds potential as a safe anti-ageing treatment that can reverse ageing processes while preserving the iden- tity of cells. This approach suggests that there is a safe time window for rejuvenation and full resetting of the epigenetic clock (Chen and Sku- tella, 2022; Chuang et al., 2017; Puri and Wagner, 2023; Simpson et al., 2021a; Talkhabi, 2019).

  1. Reprogramming-induced epigenetic rejuvenation: emerging anti-ageing strategies

 In this section, we briefly review the current emerging anti-ageing strategies, consisting of the well-known transcription factor-mediated reprogramming and other promising approaches, namely pharmaco- logical interventions based on small molecules, in which DNA methyl- transferase inhibitors and histone deacetylase inhibitors are included.

  • Genetically induced reprogramming mediated by Transcription Factors

 One of the most remarkable and booming reprogramming strategies currently leverages on gene therapies mediated by the ectopic expres- sion of transcription factors (TFs) (Fig. 1). The groundbreaking discov- ery of the Yamanaka factors, i.e., a cocktail of four reprogramming factors – Oct4, Sox2, Klf4, and cMyc (OSKM) – has revolutionized ageing research. The so-called partial reprogramming enables to reset of the epigenetic landscape of cells – DNA methylation patterns – rejuvenating cells and regenerating tissues, without reaching a pluripotency state, thus minimizing the risk of tumorigenesis (Galow and Peleg, 2022; Ji et al., 2023a; Puri and Wagner, 2023; Simpson et al., 2021a). Moreover, transient reprogramming influences major hallmarks of ageing at tran- scriptomic and cellular levels, such as autophagy levels and mitochon- drial membrane potential (Sarkar et al., 2020).

This strategy has been able to dramatically reverse age-related phenotypes in many tissues in both cultured mammalian cells and ro- dent models. In one of the most cited studies to date (Lu et al., 2020), researchers showed that mammalian tissues retain a record of youthful epigenetic information that can be easily accessed to improve tissue function and regeneration in vivo. They were able to safely rejuvenate the age of neurons in retinal ganglion cells (RGCs) and, thus, reverse the vision loss in an aged mouse model of glaucoma. To achieve this, by adeno-associated virus (AAV) delivery, they expressed Oct4, Sox2, and Klf4 (OSK) TFs, excluding Myc, as it is an oncogene that reduces the lifespan in mice. Reversal of DNA methylation of RGCs was intricately linked with axon regrowth and restoration of youthful vision, without oncogenicity or loss of identity (Lu et al., 2020). Indeed, the continuous expression of OSK in the RGCs of glaucomatous mice enabled a year-long significant improvement in the visual function without detrimental effects (Karg et al., 2023). In another study, the Yamanaka factors combined with two accessory factors (Lin28 and Nanog) were transiently expressed before the so-called Point of No Return (PNR), in which the cells return to the initiating somatic cell state (Sarkar et al., 2020). This way, the expression of the TFs in a short time (4 consecutive days) was enough to reprogram the cellular age while failing to erase the complete epigenetic signature, and the methylation age was reversed approximately by 5 years in human endothelial cells. This study was additionally extended to human-aged chondrocytes and murine skeletal muscle stem cells, also achieving partial reverse of gene expression, youthful physiological state, and enhanced regenerative potential (Sarkar et al., 2020). To decipher more closely the mechanisms involved in partial

Fig. 1. Visual Representation of Epigenetic Reprogramming mediated by Transcription Factors (TFs) to reverse the epigenetic clock. Created with BioRender.com.

reprogramming, changes in the DNA methylome, tran- scriptome, and serum metabolites were recently studied in aged mice exposed to a single cycle of transient OSKM expression (Chondronasiou et al., 2022). Rejuvenation was achieved in the pancreas, liver, spleen, and blood in a systemic manner in vivo, even with low reprogramming, thus minimizing the risk of teratoma formation. Interestingly, this study showed that reprogrammed cells may influence the rejuvenation of non-reprogrammed cells, through secretion of soluble factors (Chon- dronasiou et al., 2022). However, to rejuvenate the transcriptome of

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