Objective: To investigate mitochondrial DNA (mtDNA) alterations in single neurons from Parkinson’s disease (PD) brains and determine their relationship with respiratory chain deficiency.

Background: Mitochondrial dysfunction, particularly deficiency of Complex I (CI), is a central feature of PD. mtDNA defects have been implicated in this process, but their contribution to neuronal respiratory chain deficiency remains unclear. Previous studies have been limited by the inability to directly assess mtDNA integrity and respiratory chain function within the same individual neuron.

Method: Postmortem brain tissue from PD patients and controls was analysed by double immunofluorescence to assess CI status and mitochondrial mass, followed by laser microdissection of CI-deficient and CI-intact neurons. mtDNA copy number and deletion levels were quantified in single neurons using qPCR based on the ND4/ND1 assay. In parallel, single-cell mitochondrial genome sequencing and mtDNA single-molecule fluorescence in situ hybridization (smFISH) were performed to further characterize mtDNA alterations. To our knowledge, this is the first application of single-neuron whole mitochondrial genome sequencing and single-molecule mtDNA FISH in postmortem human brain tissue.

Results: Using single-cell qPCR quantification, we identified a complex and heterogeneous relationship between CI deficiency and mtDNA deletion burden in single dopaminergic neurons of the substantia nigra. While CI-intact neurons show minimal mtDNA deletion burden, CI-deficient neurons exhibit a bimodal distribution: a subset of CI-deficient neurons lacks elevated deletion burden, whereas a distinct subset displays very high level.

Conclusion: These findings indicate that CI loss and mtDNA deletion accumulation are interrelated processes that can also occur in the absence of clinical or pathological PD, reflecting an inherent, age-associated vulnerability of nigral dopaminergic neurons to mitochondrial genome instability. Ongoing analyses using single-neuron mitochondrial genome sequencing and mtDNA smFISH aim to further define the spectrum of mtDNA alterations and will be presented at the conference.

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MDS Abstracts - https://www.mdsabstracts.org/abstract/single-neuron-analysis-of-mitochondrial-dna-alterations-and-respiratory-chain-dysfunction-in-parkinsons-disease/