Abstract
Ageing is a major risk factor for many human diseases, however there is limited information on the underlying molecular processes. Mitochondrial dysfunction is one of the hallmarks of ageing and involves a loss of ATP production and increased production of reactive oxygen species. My host laboratory has measured an increase in mitochondrial oxidative stress in platelets from faster ageing individuals within The Dunedin Study. Red blood cells from faster agers were also better able to recover after being challenged with hydrogen peroxide. This led to the hypothesis that oxidative stress triggers an adaptive response to produce red blood cells better able to cope with additional oxidants.
I have used Bristol Erythroid Line-Adult (BEL-A) cells, an immortalised erythroid progenitor cell line that can be successfully differentiated into functional reticulocytes, to explore the effects of mitochondrial dysfunction on the differentiation and the phenotype of resultant cells. Conditions for successful BEL-A expansion and differentiation were optimised with differentiation characterised by changes to cell morphology and surface marker expression. The CD36 cell surface marker decreased over 12 days of differentiation, and glycophorin A expression increased. Mitochondrial density and membrane potential decreased in signal over the first four days of differentiation. The morphology of differentiating cells was characterised with live cell microscopy and cytospins.
I attempted to genetically modify the BEL-A cell line using CRISPR to introduce a mutation in the POLG polymerase proofreading domain. This would have led to the accumulation of mutations in mitochondrial DNA and increased oxidative stress. However, this proved unsuccessful as I was unable to isolate the POLG mutant BEL-A cells. As an alternative approach, mitochondrial-targeted paraquat (MitoPQ) was used to increase superoxide production inside mitochondria. When BEL-A cells were treated with a dose of MitoPQ that did not interfere with their proliferation or viability, expression of the mitochondrial antioxidant peroxiredoxin 3 doubled, suggesting an adaptive response in the cell to mitochondrial oxidative stress.
Further work is required to determine the effect of this stress on the phenotype of the differentiated cells. These results will help to understand some of the fundamental processes occurring in haematopoietic cells during human ageing.