Investigating the role of mitochondrial respiratory chain activity and mitochondrial DNA damage in skin ageing

Abstract

Ageing describes the progressive functional decline of an organism over time, leading to an increase in susceptibility to age-related diseases and eventually to death, and it is a phenomenon observed across a wide range of organisms. Despite a vast repertoire of ageing studies performed over the past century, the exact causes of ageing remain unknown. For over 50 years it has been speculated that mitochondria play a key role in the ageing process, due mainly to correlative data showing an increase in mitochondrial dysfunction, mitochondrial DNA (mtDNA) damage, and reactive oxygen species (ROS) with age. Therefore, a major aim of the current project was to assess mitochondrial dysfunction, in the form of complex II activity, in the skin cells of differently aged humans. Mitochondrial complex II of the electron transport chain (ETC) was chosen to be examined, as it has recently been implicated in the generation of ROS, as well as in the ageing process of lower organisms, and is the least studied complex of the ETC. Complex II activity was found in the present study to decline in an age-dependant manner in human skin fibroblast cells, which may have been partially related to an observed decrease in the expression of specific nuclear-encoded complex II subunits with age. Further investigations into the cause of the decrease in complex II activity with age revealed that the decline was specific to senescent cells, and was not present in non-senescent cells, which was determined following sorting into subpopulations via fluorescence-activated cell sorting (FACS). The decrease in activity with age was not reflected in another mitochondrial complex examined, complex IV, for which there was no alteration in activity with age for either unsorted, senescent, or non-senescent cells. This finding could suggest the specific targeting of complex II in senescent cells only for future age-related therapeutics. Interestingly, an age-dependant decrease in complex II activity was not observed for human skin keratinocytes, despite being observed in human skin fibroblasts. In the present study, it was also observed that different cell types undergo differing rates of maximal complex II activity, which could have important consequences in terms of the rate of ageing of specific cell types. In addition to the observed decrease in mitochondrial complex II activity with age, it was demonstrated in the present study that mtDNA damage is increased with age in the skin of both humans and a taxonomic group for which age- 3 related changes have not been previously studied, the whales. It was confirmed that the T414G mutation, which is a general biomarker for mtDNA mutations, was higher in human skin fibroblasts from older individuals when compared to younger individuals. Furthermore, an increase in mtDNA damage with age was also found in multiple whale species, for which mtDNA damage was measured in the form of strand breaks within a large region of the mitochondrial genome, using novel primers designed and optimised through the present study. Whales from three distinct species were chosen to be examined based on their differing levels of UV exposure, as a model for different ages. It was found that the level of mtDNA damage increased with both natural age and increased UV exposure. The three whale species studied appeared to have developed alternative mechanisms of coping with UV-induced damage. MtDNA damage was found to be lowest in those whales with the highest expression of heat shock protein 70 (Hsp70), suggesting that this UV-defensive mechanism may be useful in future studies for the prevention of age-related phenotypes. Overall, the present study provides important new insights into the potential role of mitochondria in ageing.