Clonal expansion of mtDNA deletions in post mitotic tissue
E Stephen1, Dr Craig Stamp1, Sir Prof D Turnbull1 and Dr A Reeve1
Increasing COX-deficiency and accumulation of mitochondrial DNA (mtDNA) deletions are common in ageing neurons. Since it is one of the proposed mechanisms behind deletion formation, it is important to understand the site and level of mtDNA replication.
Levels of replication were measured in the cerebellum and cortex of mice using thymidine analogues CldU and IdU. Mice were labelled with CldU for 4 days and IdU for 15 hours before culling. Brain tissue was immunofluorescently stained to detect CldU and IdU in situ. Levels of these analogues were quantified and normalized to mitochondrial levels. Along with WT, POLG mice were also used for comparison, since these mice have error prone replication and exhibit an accelerated ageing phenotype. This study also studied the location of replication within cells.
The results show an increase in CIdU and ldU incorporation into the mitochondria in the Purkinje cells within cerebellum compared to the pyramidal cells in cortex. POLG mice displayed a similar trend with an overall decrease in replication levels compared to WT animals.
Results also showed perinuclear replication, with decreasing levels with increasing distance from the nucleus. This was further examined in Hek293 cells where, with time, mtDNA replication moved further away from the nucleus.
Previous data suggests that the copy number of mtDNA is increased in the cerebellum with a decrease in the level of deletions in comparison to the cortex. This may be related to the detected increase in replication levels in the cerebellum. Alongside this, increased perinuclear labelling suggests that replication may occur in the perinuclear area and move towards cell periphery. Therefore this study detected increased but variable replication in cerebellar neurons, coupled with a perinuclear location which could favor clonal expansion of mtDNA deletions.