Reversing Aging: Epigenetic Rejuvenation

Scientists have known for a long time that Nature has the power to rejuvenate cells — after all, every new baby is born young despite having started as an oocyte of the same age as its mother (all human oocytes are formed while the mother herself is an embryo).

However, only about a decade ago have we managed to harness that power for ourselves. In 2006 Yamanaka and Takahashi have discovered that using 4 DNA transcription factors (Oct4, Sox2, Klf4 and c‐Myc, abbreviated as OSKM) an adult cell can be turned back into an essentially embryonic stem cell. Of course, in hindsight, early signs that this is possible can be traced as far back as 1962 when Sir John B. Gurdon has shown that he could clone a frog using the nucleus harvested from its adult cell. Both Gurdon and Yamanaka received a Nobel Prize in 2012 for their groundbreaking insights.

The next important discovery came in subsequent work by other researchers. It turned out that cellular “reprogramming” — the process of returning a cell back to its embryonic roots — is not a sudden, stepwise process, but, instead, a gradual one. Moreover, the reprogramming process seems to retrace the steps a cell takes as it usually proceeds down its differentiation path. That is, the cell can be viewed as getting progressively ‘younger’, culminating in the ‘youngest’ state of all — embryonic.

Armed with this knowledge, Belmonte et al. hypothesized as far back as 2013 that this fortuitous fact can be used for partial in vivo rejuvenation:

[T]he fact that reprogramming, or de-differentiation to iPSCs, proceeds in a stepwise manner suggests that the process can be stopped before the acquisition of an embryonic-like signature

It is probably around the same time that Belmonte et al. asked themselves the following question: if we learn to rewind the epigenetic state of all cells in the body just a little, without causing them to lose their phenotypes, might we expect to see positive systemic rejuvenating effects? In 2016 they published a seminal paper which seems to give a cautious affirmative answer. Yes, using in vivo OSKM reprogramming we seem to be able to prolong lifespan in progeric mice by up to 50%.

Let us dive a little deeper into that paper. What exactly have Belmonte et al. done? They employed a special genetic tool widely used by geneticists for many years: a Tet-On gene cassette. Basically, it is a tool that enables experimenters to turn on at will whatever genes they pack into that cassette. Normally those genes would be ‘off’, i.e. silent, not expressed. But when animals would be given a special ‘activator’ (tetracycline or doxycycline), those genes would spring to life. So Belmonte et al. used a special strain of progeric (i.e. fast-aging) LAKI mice, which had a Tet-On cassette with OSKM genes inserted into their DNA. Using doxycycline, they induced periodic epigenetic rejuvenation in these mice and observed that this resulted in increased lifespan. They have also inserted this cassette into regular, wild type mice to study additional aspects of OSKM rejuvenation.

Fast-aging (LAKI) Mice

The first thing Belmonte et al. have set out to do was establish the safest dosing regimen for the OSKM genes. It was known previously that OSKM induction can produce a type of cancer called teratoma. The researchers obviously wanted to avoid this. Thus, the first thing they tested was how long they could keep OSKM genes active before problems start. To do so, they gave mice consecutive daily doses of doxycycline and monitored their weight and survival.

As they observed that some mice started to die after 3 consecutive days of OSKM induction, they settled on a cyclic 2/5-day treatment regimen: 2 days of treatment followed by a 5-day break. They started treating the mice at 8 weeks of age and continued weekly treatments until death. With this protocol Belmonte et al. observed a 33–50% median lifespan increase (depending on which control group to compare to). Even more impressive that the median lifespan increase was the fact that by the time all mice without OSKM genes died (week 22), 75% of mice treated with epigenetic rejuvenation were still alive.

In the survival graph below the blue line represents the treatment group and all other lines represent various controls:

They had 3 control groups: two groups of LAKI mice without the Tet-On cassette (one of which was still getting weekly doxycycline doses to make sure the drug itself does not produce any life increasing effects) and one control group with the OSKM cassette (which they call ‘4F’ for ‘4 factors’) but without weekly activation.

The treated mice not only lived longer but also were biologically younger as measured by several biomarkers:

• lower markers of senescent cells (p16Ink4a and beta-galactosidase)
• lower marker of double-stranded DNA breaks (gamma-H2AX)
• lower metalloprotease levels
• lower interleukin-6 levels
• lower levels of mitochondrial reactive oxygen species (‘free radicals’)
• higher number of hair follicles

Finally, in a separate study, Mario Blasco’s team has demonstrated that OSKM induction increases telomerase levels in LAKI mice and thus lengthens their telomeres. Maria Blasco is famous for studying life-extending effects of telomerase — work that inspired Liz Parrish to inject herself with the hTERT gene therapy.

Normal Mice

Lastly, Belmonte et al. turned to normally aging mice to see if OSKM induction produces rejuvenating effects in them as well. They used two different experiments to do so — inducing beta cell and muscle cell injury in 12-month old wild type mice with the Tet-On OSKM cassette and then measuring differential recovery rates between groups previously treated with a 3-week OSKM induction and those not treated. In both cases they observed significantly better recovery in those mice that have been epigenetically rejuvenated prior to being subjected to above injuries.

Summary

The researchers concluded their paper with the following remarks that seem to indicate that they support both the programmed aging hypothesis and the conjecture that epigenetic regulation is the primary implementation of the aging program:

Although previous studies have indicated that expression of the Yamanaka factors in vivo can lead to cancer development or teratoma formation (Abad et al., 2013; Ohnishi et al., 2014), here, we demonstrate that tumor formation can be avoided by short-term induction of OSKM. Cyclic induction of OSKM in vivo ameliorated hallmarks of aging and extended the lifespan of a mouse model of premature aging. Additionally, short-term induction of OSKM improved the regenerative capacity of pancreas and muscle following injury in physiologically aged mice. Together, these results show that partial in vivo reprogramming might be used to modulate aging hallmarks and significantly benefit organismal health.

Our observations may reinforce the potential role of epigenetic changes as drivers of aging and highlight the plasticity of the aging process, which might be altered by cellular reprogramming in vivo. In addition, our results suggest that aged cells undergo a process of molecular rejuvenation during the initial stages of cellular reprogramming to pluripotency.

…

Reprogramming, currently an experimental tool to study development and cellular differentiation, may provide additional insights into the mechanisms of aging. Proposed drivers of physiological aging include the accumulation of DNA damage, increased ROS production, telomere shortening, cellular senescence, and defects in nuclear envelope architecture (Bernardes de Jesus and Blasco, 2013; Guarente, 2008;Haigis and Sinclair, 2009; Kennedy and Lamming, 2016; Soultoukis and Partridge, 2016; Steffen and Dillin, 2016). Multiple studies using animal models have demonstrated that the manipulation of these aging drivers leads to the manifestation of molecular hallmarks of aging that are shared between premature aging models and physiological aging (Garcia-Prat et al., 2016; Mitchell et al., 2015). We hypothesize that the emergence of these molecular hallmarks during organismal aging results from the translation of aging signals by a unique and universal epigenetic program. Our results suggest that this epigenetic program, which is reset during embryogenesis, can also be experimentally altered by partial cellular reprogramming at later stages of life. Resetting of the aging clock by epigenetic reprogramming, which is also observed during somatic nuclear transfer, might allow for a deeper understanding of the molecular and cellular mechanisms underlying the aging process. Eventually, it may, as well, lead to the development of therapeutic strategies with the goal of ameliorating age-related diseases and thus improving health and longevity.

I could not agree more. The only thing I would change in the above quote is the word ‘eventually’ in the final sentence. I say the time to act is now.