Identifying the mechanism behind mitochondrial changes in the cholinergic neurons of the pedunculopontine nucleus in patients with Parkinson’s disease

Abstract

n 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 neuronal loss and has been seen in multiple neuronal types including nigral dopaminergic of the substantia nigra (SN) 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. This study employed ultra-deep sequencing to analyse single cholinergic neurons isolated in an unbiased fashion from postmortem PPN tissue of PD and controls, examining both the mtDNA deletions, and point mutations across various parameters. Comparative analysis of mtDNA deletions was also conducted with nigral dopaminergic neurons to elucidate compensatory response to mtDNA damage. Additionally, singleneuron qRT-PCR was used to quantify the expression of three nuclear genes involved in mitochondria biogenesis, maintenance and mitophagy. Our findings confirm significantly higher levels of large mtDNA deletions in PD patients compared to aged controls, with deletions ranging from 5-140bp (small) to >1,400bp (large). The distribution of large deletions suggests that replication errors are the primary mechanism underlying their generation, aligning with patterns observed in POLG patients and aged controls. Conversely, the low prevalence of small deletions suggests that oxidative damage is not a major contributor of mtDNA damage in these neurons. Point mutation levels did not differ significantly between groups across various parameters assessed. At the transcriptional level, PINK1 expression was upregulated in PD neurons, whereas no significant changes were observed in TFAM or PGC1α expression. These findings suggest that the mitophagy pathway plays a prominent role in regulatory response to mitochondrial changes in PD.