Investigating mtDNA replication in neurons; implications for mtDNA deletion formation

Abstract

Mitochondria contain their own DNA (mtDNA) which can be damaged, leading to the formation of mtDNA deletions (ΔmtDNA). These are particularly prominent in post mitotic tissues and progressively accumulate with advancing age. Within the brain, ΔmtDNA may reach levels of 50% and has been linked to COX-deficiency and neurodegeneration. Although these ΔmtDNA are well characterised, the definitive mechanism for their formation and accumulation to high levels is unknown. One of the suggestions for deletion formation is inadequate replication. This study aimed to further understand how mtDNA replication differs between neurons and with age, and whether such changes could be associated with the formation and accumulation of mtDNA deletions. This was investigated in different ways. Various regions of the brain differ in the levels of deletions. Therefore, the mtDNA replication levels in different regions were investigated via thymidine analogue labelling to identify any alterations. Significant differences were noticed between different regions with cerebellum generally presenting increased signal and the SN presenting the lowest. Since ageing and disease is a risk factor of ΔmtDNA accumulation, aged mice and PolgAmut/mut mice were used, demonstrated a general decrease in thymidine analogue signalling with increased age and with a replication defect. The replication levels obtained from this study were compared to another study from the literature investigating the levels of deletions from different brain regions. No correlation was observed suggesting that although mtDNA replication is altered within different regions in the brain, there could be other mechanisms effecting the formation and accumulation of ΔmtDNA. Additionally, the thymidine analogue labelling was developed and optimised for in vitro use on HeLa cells and was used to label iPSCs and differentiated neurons from a patient with a large scale mtDNA deletion. Two isogenic cell lines of varying heteroplasmy (>10% and 40%) were used for this study to see the effect of altered ΔmtDNA on replication. The methodology was optimised successfully for further use. Along with the total replication levels, the location of replication was also investigated to understand the impact of this on the accumulation of ΔmtDNA. This is due to previous theories that have suggested that since replication and mitophagy happen in close proximity to the nucleus and each other, this increases the chances of a dysfunctional mitochondria, containing ΔmtDNA, replicating. The results suggested that majority of the replication happened in the perinuclear region, and preliminary data on the location of mitophagy also showed a perinuclear increase. To further investigate the health and connectivity of the mitochondria in the perinuclear region I compared them to distal mitochondria. This novel ‘proof-of-concept’ study investigated the morphology and structural connectivity of mitochondria in human brain tissue using electron microscopy techniques. This study was successful and preliminary data from dopaminergic neurons are discussed. In conclusion although mtDNA replication levels and replication location varies between neurons, ageing and genotype suggesting implications for ΔmtDNA formation.