Abstract
Oxidative stress has been closely linked with ageing. The ‘mitochondrial free radical theory of ageing’ states that mitochondria are both a source of reactive oxidants while also being sensitive to oxidative stress. Peroxiredoxin (Prx) redox proteins are key regulators of oxidative stress and have been associated with pro-longevity. However, little research has been undertaken investigating the role Prxs play in biological ageing. Prxs continually cycle between their reduced and oxidised forms to reduce cellular hydrogen peroxide levels. Preliminary research performed on Dunedin Study participant blood samples at age 45 identified that cytosolic peroxiredoxin 2 (Prx2) in red blood cells was highly reduced whilst mitochondrial peroxiredoxin 3 (Prx3) appeared more oxidised in platelets of participants with a faster pace-of-ageing. These findings led to questions surrounding the relationship between Prx oxidative phenotypes within and between blood cell types and the formation of the hypothesis that the Prx oxidative phenotypes observed in blood cells manifest from mitochondrial dysfunction in haematopoietic stem cells (HSCs).
This project aimed to provide further insight into Prx redox status and its role in biological ageing by addressing three main aims. Firstly, by determining whether the epigenetic regulation of the genes in the peroxiredoxin-thioredoxin network change with age. Secondly, by investigating the relationship between Prx1, Prx2 and Prx3 oxidation state within and between platelets and peripheral blood mononuclear cells (PBMCs). Lastly, by determining the impact promoting mitochondrial stress on haematopoietic progenitors has on Prx oxidative phenotype in differentiated megakaryocytes.
The CpG islands associated with the promoter regions of the genes in the peroxiredoxin-thioredoxin network were found to be predominantly unmethylated in individuals aged 18-25 and 70-80-years-old, indicating the genes are actively expressed during ageing. Prx2 exhibited lineage specific oxidative phenotypes in blood cells. A significant increase in Prx2 oxidation state was observed in the PBMC samples compared to the platelet samples. The platelet samples reflected the Prx2 oxidation state observed in the red blood cells of Dunedin Study participants. Prx1 and Prx3 were found to be highly reduced in both platelets and PBMCs. However, difficulties with the western blotting technique did not allow for accurate quantification of Prx1 and Prx3 oxidation state in platelets. During PMA-induced megakaryocytic differentiation of K-562 cells, an increase in oxidation of Prx3, and Prx1 to a lesser degree, was observed, whilst the oxidation state of Prx2 remained consistent. The targeted mitochondrial stressor MitoParaquat was found to be ineffective at promoting mitochondrial stress in K-562 cells as no change in Prx3 oxidation state was identified.
Overall, this work provides further insight into the redox status of Prxs and their role in biological ageing. The results suggest that the genes of the peroxiredoxin-thioredoxin network are actively expressed throughout life and that the redox state of Prxs is not set in progenitor cells. The observation that Prx3 oxidation increases during megakaryocytic differentiation supports findings of altered mitochondrial functions during megakaryocytic differentiation of K-562 cells. Further research is required to investigate the impact promoting mitochondrial stress in haematopoietic progenitors has on the Prx oxidative phenotype in differentiated megakaryocytes.