A deep sequencing investigation of mitochondrial DNA damage in cholinergic neurons of the Pedunculopontine Nucleus

In Parkinson’s disease (PD), patients often exhibit higher levels of somatic mitochondrial DNA (mtDNA) damage, particularly large-scale deletions, in their brains. This damage is associated with losing neurons, including nigral dopaminergic and cholinergic neurons in the Pedunculopontine Nucleus (PPN). Unlike dopaminergic neurons, PPN cholinergic neurons tend to maintain wild-type mtDNA levels by increasing mtDNA copy number (mtCN) in response to mtDNA deletions.Our study isolated single cholinergic neurons from post-mortem PPN tissue of PD patients and controls, examining mtDNA deletions' location, size, breakpoints, and heteroplasmy levels using ultra-deep sequencing. Comparisons were made with nigral dopaminergic neurons, aiming to understand compensatory responses to mtDNA damage. Additionally, single-cell qRT-PCR assessed the expression of nuclear genes related to mitochondrial biogenesis/maintenance.Preliminary findings confirm higher levels of large deletions in PD patients compared to controls, with deletions ranging from 5-140bp (small) to >1,400bp (large). The location of large deletions suggests replication errors cause their generation. Thus, data indicate a similar mechanism of deletion generation to POLG/aged controls in this neuronal type. Point mutation levels did not significantly differ between groups. Additionally, low numbers of small deletions suggest oxidative damage is not a significant driver of mtDNA damage in these neurons.