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Summary: Background: Idiopathic rapid eye movement (REM) sleep behaviour disorder (IRBD) represents the prodromal stage of Lewy body disorders (Parkinson's disease (PD) and dementia with Lewy bodies (DLB)) which are linked to variations in circulating cell-free mitochondrial DNA (cf-mtDNA). Here, we assessed whether altered cf-mtDNA release and integrity are already present in IRBD. Methods: We used multiplex digital PCR (dPCR) to quantify cf-mtDNA copies and deletion ratio in cerebrospinal fluid (CSF) and serum in a cohort of 71 participants, including 1) 17 patients with IRBD who remained disease-free (non-converters), 2) 34 patients initially diagnosed with IRBD who later developed either PD or DLB (converters), and 3) 20 age-matched controls without IRBD or Parkinsonism. In addition, we investigated whether CD9-positive extracellular vesicles (CD9-EVs) from CSF and serum samples contained cf-mtDNA. Findings: Patients with IRBD, both converters and non-converters, exhibited more cf-mtDNA with deletions in the CSF than controls. This finding was confirmed in CD9-EVs. The high levels of deleted cf-mtDNA in CSF corresponded to a significant decrease in cf-mtDNA copies in CD9-EVs in both IRBD non-converters and converters. Conversely, a significant increase in cf-mtDNA copies was found in serum and CD9-EVs from the serum of patients with IRBD who later converted to a Lewy body disorder. Interpretation: Alterations in cf-mtDNA copy number and deletion ratio known to occur in Lewy body disorders are already present in IRBD and are not a consequence of Lewy body disease conversion. This suggests that mtDNA dysfunction is a primary molecular mechanism of the pathophysiological cascade that precedes the full clinical motor and cognitive manifestation of Lewy body disorders. Funding: Funded by Michael J. Fox Foundation research grant MJFF-001111. Funded by MICIU/AEI/10.13039/501100011033 “ERDF A way of making Europe”, grants PID2020-115091RB-I00 (RT) and PID2022-143279OB-I00 (ACo). Funded by Instituto de Salud Carlos III and European Union NextGenerationEU/PRTR, grant PMP22/00100 (RT and ACo). Funded by AGAUR/Generalitat de Catalunya, grant SGR00490 (RT and ACo). MP has an FPI fellowship, PRE2018-083297, funded by MICIU/AEI/10.13039/501100011033 “ESF Investing in your future”.

Background: Both idiopathic and familial Parkinson's disease are associated with mitochondrial dysfunction. Mitochondria have their own mitochondrial DNA (mtDNA) and previous studies have reported that the release of mtDNA is a biomarker of Parkinson's disease. Methods: We have now investigated the relationship between mtDNA replication, transcription and release in fibroblasts from patients with idiopathic (iPD) and Leucine-rich repeat kinase 2G2019S -associated Parkinson's disease (LRRK2-PD), using Selfie-digital PCR, a method that allows absolute quantification of mtDNA genomes and transcripts. Findings: In comparison with healthy controls, we found that fibroblasts from patients with iPD or LRRK2-PD had a high amount of mitochondrial 7S DNA along with a low mtDNA replication rate that was associated with a reduction of cf-mtDNA release. Accumulation of 7S DNA in iPD and LRRK2-PD fibroblasts was related with an increase in H-strand mtDNA transcription. Interpretation: These results show that 7S DNA accumulation, low mtDNA replication, high H-strand transcription, and low mtDNA release compose a pattern of mtDNA dysfunction shared by both iPD and LRRK2-PD fibroblasts. Moreover, these results suggest that the deregulation of the genetic switch formed by 7SDNA that alternates between mtDNA replication and transcription is a fundamental pathophysiological mechanism in both idiopathic and monogenic Parkinson's disease. Keywords: mtDNA, Digital PCR, 7S DNA, Parkinson's disease, LRRK2, G2019S LRRK2 missense mutation