Dietary restriction mitigates age-related accumulation of DNA damage, but not all changes in mouse corneal epithelium

Significance Statement

It is well known that dietary restriction, the reduction of food intake, is able to extend life span in animal models. In addition, it has been shown to have great health benefits in various animal species from unicellular budding yeast, to nematode worms, flies up to mammals such as rats and mice while still contradictory results are reported in primates. However, the underlying mechanisms for these beneficial effects are still not well understood. One possible mechanism is the decrease of oxidative stress and the resulting DNA damage.

In our study we analysed levels of general DNA damage, measured as g-H2AX foci (a phosphorylated histone) as well as telomere-associated foci (TAFs) that identify DNA damage foci that are specifically localised to telomeres, the protective ends of chromosomes. Although mice have much longer telomeres than humans that don’t shorten significantly like they do in human cells with much shorter telomeres, it had been shown recently, that even long telomeres in mice can accumulate damage and might thus contribute to cellular senescence and the ageing process.

A cell type of particular interest in ageing research are tissue stem cells. While in the human eye there are clearly defined stem cells in the limbus of the cornea, these are less well defined in mouse eyes. Adult stem cells are able to up-regulate telomerase, an enzyme that can maintain telomeres. These stem cells have regenerative properties and differentiate into new, functioning tissue. For example, human limbal stem cells are used for transplantation in cases when the eye of a patient has been damaged which is both painful and diminishes the eyesight.

We found various changes in the mouse cornea that were caused by old age, but could be mitigated in mice that had a reduced food intake due to dietary restriction. We found more changes in the central part of the cornea than in the peripheral part and thus suggest, that the latter might be the location of limbal stem cells in the mouse eye. This result also means that our findings are most likely also true for the human eye.

Although dietary restriction is probably difficult to apply to humans, intensive research is underway to find effective treatments and drugs such as for example rapamycin which activates a process named autophagy that removes damaged cellular components and thus “rejuvenates” tissues. The future will show whether we can apply such compounds to improve our health and delay the ageing process.

Figure Legend

Representative images of g-H2AX DNA damage foci (green) in the central cornea of young, old and dietary restricted mice (same age as the old, ad libitum fed) mice. Blue signals are DAPI for nuclear staining.

Journal Reference

Exp Gerontol. 2015;67:72-9.

Hallam D¹, Wan T¹, Saretzki G².

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Institute for Cell and Molecular Bioscience, Newcastle University Institute for Ageing, Campus for Ageing and Vitality, Newcastle University Newcastle upon Tyne, UK.
Institute for Cell and Molecular Bioscience, Newcastle University Institute for Ageing, Campus for Ageing and Vitality, Newcastle University Newcastle upon Tyne, UK. Electronic address: [email protected].

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Abstract

The cornea protects the anterior eye and accounts for two thirds of the eyes refractive capacity. The homeostasis of corneal epithelium is thought to be maintained by putative stem cells residing in the epithelial basal layer. As a tissue constantly exposed to environmental stress, the cornea is hypothesised to accumulate persistent DNA damage events with time in stem cell populations. Recently, telomere associated DNA damage foci (TAFs) have been suggested as a marker for persistent DNA damage which can be used to detect senescent cells during ageing. Dietary restriction(DR) is the only known non-genetic intervention that is able to increase both life and health span among various animal species. The aim of this study was to analyse changes in corneal properties with age and under 16 months of DR. We employed immunofluorescence staining for ɣH2A.X together with telomere fluorescence in situ hybridisation (immuno-FISH) on mouse corneas from young, old ad libitum (AL) fed as well as dietary restricted (DR) mice. Our data show that the central corneas of old mice had significantly more general and telomere-associated DNA damage compared to young mice while DR treatment was able to reduce the amount of DNA damage significantly. We also found that the thickness of the peripheral region of the cornea, where the putative stem cells may reside, decreased with age regardless of whether the animals underwent DR treatment or not. Number of bullae, which indicates corneal edema, accumulated in old corneas in the central area and DR treatment mitigated the formation of these bullae. Moreover, the protein levels of the stem cell marker TAp63 decreased with age only in the central but not the peripheral region of the cornea. This finding suggests that epithelial progenitors might be better maintained in the peripheral than the central cornea during ageing. Together with the finding that the peripheral corneal showed no increase in DNA damage during age, we speculate that in mice, like humans, the putative stem cells reside in the peripheral cornea.

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About the author

Gabriele Saretzki completed her undergraduate study and master’s at St. Petersburg University (Russia) in genetics of microorganisms in 1982. She did her PhD in Molecular Biology at the Department of Genetics at the Humboldt-University in Berlin in 1990. She then worked as Research Assistant at the Institute of Pathology of the Charite’ (Medical faculty) of the Humboldt University where she became interested in telomeres, telomerase, oxidative stress and ageing research. She was instrumental in identifying telomeres as indicators of oxidative stress and their use as biomarkers of ageing and disease. In 2001 she moved to the Institute for Ageing and Health at Newcastle University. Since 2002 she is a Lecturer in Ageing Research and is now a member of the Institute for Cell and Molecular Biosciences at the Medical Faculty at Newcastle University where she continues to work on telomerase, neurodegenerative diseases and oxidative stress in ageing and cellular senescence.

About the author

Dean Hallam completed his undergraduate study in Genetics at the University of Aberystwyth and acquired his MSc in Molecular Genetics at the University of Leicester in 2010 with a project on the effects of ageing on myosin molecular motors. In 2011 he moved to Newcastle and completed his PhD in 2015 under the supervision of Dr. Gabriele Saretzki in the Institute for Ageing and Health, working on the effects of ageing on the corneal epithelium. Since April 2014 he works as a Research Associate with Professor Majlinda Lako at the Institute of Genetics Medicine on modelling age related macular degeneration using iPSC-RPE (induced pluripotency stem cells-retinal epithelial pigment) cells.

About the author

Tengfei Wan conducted his undergraduate studies at the College of Life Sciences, Northeast Forestry University (China), and finished his Master of Research on Ageing and Health at the Medical School of Newcastle University (UK) in 2013. During his master’s study, Tengfei has investigated the accumulation of DNA damage on mouse corneal epithelium basal cells during ageing, and the effects of interventions such as dietary restriction and rapamycin supplementation on this process. He is now doing his PhD at the Institute for Cell and Molecular Bioscience at the Campus for Ageing and Vitality of Newcastle University in Dr. Gabriele Saretzki’s group, investigating the relationship between neurondegenerative diseases and telomerase on a mouse model.