• Log in with Facebook Log in with Twitter Log In with Google      Sign In    
  • Create Account
  LongeCity
              Advocacy & Research for Unlimited Lifespans

Photo

Rapamycin retards epigenetic ageing of keratinocytes independently of its effects on replicative senescence, prolifer...

rapamycin senescence

  • Please log in to reply
No replies to this topic

#1 Engadin

  • Guest
  • 198 posts
  • 580
  • Location:Madrid
  • NO

Posted 29 May 2019 - 05:31 PM


Rapamycin retards epigenetic ageing of keratinocytes independently of its effects on replicative senescence, proliferation and differentiation

 

 

Published at Rejuvenation Science News (RSN)

 

 

 

Abstract

The advent of epigenetic clocks has prompted questions about the place of epigenetic ageing within the current understanding of ageing biology. It was hitherto unclear whether epigenetic ageing represents a distinct mode of ageing or a manifestation of a known characteristic of ageing. We report here that epigenetic ageing is not affected by replicative senescence, telomere length, somatic cell differentiation, cellular proliferation rate or frequency. It is instead retarded by rapamycin, the potent inhibitor of the mTOR complex which governs many pathways relating to cellular metabolism. Rapamycin, however, is also an effective inhibitor of cellular senescence. Hence cellular metabolism underlies two independent arms of ageing – cellular senescence and epigenetic ageing. The demonstration that a compound that targets metabolism can slow epigenetic ageing provides a long-awaited point-of-entry into elucidating the molecular pathways that underpin the latter. Lastly, we report here an in vitroassay, validated in humans, that recapitulates human epigenetic ageing that can be used to investigate and identify potential interventions that can inhibit or retard it.

 

 

INTRODUCTION

 

One of the biggest challenges in ageing research is the means of measuring age independently of time. This need becomes particularly clear when we wish to evaluate the effects of drugs or compounds on ageing, where the use of time as a measure of age is clearly inappropriate. In recent years, several age-estimators known as epigenetic clocks have been developed, which are based on methylation states of specific CpGs, some of which become increasingly methylated, while others decreasingly so with age [1]. Age estimated by these clocks is referred to as epigenetic age or more precisely, DNA methylation age (DNAm age). The “ticking” of these clocks is constituted by methylation changes that occur at specific CpGs of the genome. Significantly, the increased rate by which these specific methylation changes occur is associated with many age-related health conditions [19], indicating that epigenetic clocks, capture biological ageing (epigenetic ageing) at least to some extent. The numerous epigenetic clocks that have been independently developed [1016] differ in accuracy, biological interpretation and applicability, whereby some epigenetic clocks are compatible only to some tissues such as blood. In this regard, the pan-tissue epigenetic clock [2] stands out because it is applicable to virtually all tissues of the body, with the exception of sperm. It estimates the same epigenetic age for different post-mortem tissues (except the cerebellum and female breast) from the same individual [2,8]. Although the pan-tissue epigenetic clock performs extremely well with in vivo cell samples, its accuracy was not as good with fibroblasts and other in vitro cell samples. We addressed this recently by developing an even more accurate multi-tissue age estimator, which we refer to as skin & blood clock [3], which is applicable for in vivo as well as in vitrosamples of human fibroblasts, keratinocytes, buccal cells, blood cells, saliva and endothelial cells. In vitrohuman cell culture systems offer many advantages including tight control of growth conditions, nutrients, cell proliferation rates, detailed morphological analyses and genetic manipulation, all of which are impractical or inappropriate in human cohort studies. Hence the availability of an in vivo epigenetic clock, such as the skin & blood clock that can also be used for in vitro experiments is an important and significant step towards uncovering the molecular mechanisms that underpin epigenetic ageing.

 

Although the molecular mechanisms of epigenetic ageing remain largely uncharacterised, the cellular aspects however, have been explored to a greater albeit limited degree. The similar epigenetic ages detected amongst different tissue of the same body [2,8] suggests that epigenetic age is not a measure of cellular proliferation since the rate and frequency of proliferation differ greatly between different tissues such as blood, which is highly proliferative and heart cells, which are post-mitotic. It is intuitive to make a connection between epigenetic ageing and senescent cells, which increases in number with age and which mediates phenotypic ageing [8,17]. This attractive link, however, was discounted by previous reports which clearly excluded DNA damage, telomere attrition and cellular senescence as drivers of epigenetic aging [18].

 

A way to further characterise epigenetic ageing is through the evaluation of validated anti-aging interventions on it. Such an intervention is the nutrient response pathway regulated by the mammalian target of rapamycin (mTOR) [1921]. Although originally developed as an immunosuppressant, rapamycin has emerged as one of the most impressive life-extending compounds [22]. It has been repeatedly shown to extend the lives of different animal species including those of yeast [23], flies [24] and mice [25,26]. The structure of rapamycin presents two major sites for potential interactions. The binding of one site to FKBP12 protein, allows its other site to bind and inhibit the mTOR kinase [27]. This kinase is part of a complex that promotes cell growth, proliferation and cell survival [28,29]. This may be why mTOR activity is often elevated in cancer cells; the rationale behind its use as an anti-cancer drug [30]. By inhibiting mTOR activity, rapamycin also recapitulates to some extent, the effect of calorie-restriction, which has also been repeatedly shown to prolong the lives of many different animal species [31]. As such, rapamycin is widely considered to be a promising anti-ageing intervention. Here we characterise epigenetic aging in primary human keratinocytes from multiple donors by testing their sensitivities to rapamycin and we observed that it can indeed mitigate epigenetic ageing independently of cellular senescence, proliferation, differentiation and telomere elongation.

 

 

Results
  Opposing effects of Rapamycin and ROCK inhibitor on keratinocyte proliferation

The availability of an epigenetic clock, such as the skin & blood clock, which is applicable to cultured cells, allows epigenetic ageing to be studied beyond the purely descriptive nature afforded by epidemiological analyses alone. Towards this end, we have established in vitro epigenetic ageing systems using primary human cells. One of this is based on primary keratinocytes that are derived from healthy human skins. As previously reported by others, we observed that the proliferation rate of these cells, which is defined as the number of population doublings per unit of time, can be significantly altered by different compounds. Rapamycin, which is the primary focus of this investigation reduces cellular proliferation rate, while Y-27632, which inhibits Rho kinase (ROCK inhibitor) increases it, and a mixture of both modestly alleviates the repressive effect of rapamycin (Figure 1 and Table 1). The opposing effects of these compounds on keratinocyte proliferation present us with the opportunity to test whether cellular proliferation rate impacts epigenetic ageing while carrying out our primary aim of interrogating the effects of rapamycin on epigenetic ageing.

 

 

Figure 1. Effects of rapamycin and Y-27632 on the proliferation of keratinocytes from Donors A, B and C that were used in the subsequent experiments. Cells from Donors A, B and C were cultured in the continued presence of the indicated compounds. Population doubling at every cell passage was ascertained until replicative senescence, and plotted against time.

 

 

.....

 

 

 

F U L L   T E X T : AgING

Attached Thumbnails

  • img.png






Also tagged with one or more of these keywords: rapamycin, senescence

1 user(s) are reading this topic

0 members, 1 guests, 0 anonymous users