INVESTIGATING LRRK2-LINKED MITOCHONDRIAL DEFECTS IN PARKINSON’S DISEASE USING A HUMAN DOPAMINERGIC CELL MODEL

Abstract

Mutations in the Leucine-Rich Repeat Kinase 2 (LRRK2) gene, particularly the G2019S variant, are among the most prevalent genetic contributors to familial and sporadic Parkinson’s disease (PD), accounting for nearly 40% of familial and 1–2% of idiopathic cases. These mutations are known to impair mitochondrial homeostasis, disrupt oxidative phosphorylation, and compromise. This study investigated the effects of the LRRK2-G2019S mutation on mitochondrial respiration, ATP production, oxidative stress, and neuronal viability using a human dopaminergic LUHMES cell model. LUHMES neurons expressing wild-type or LRRK2-G2019S were analyzed for mitochondrial bioenergetics using the Seahorse XF Analyzer, ATP synthesis via CellTiter-Glo assay, membrane potential with JC-1 dye, and oxidative stress using DCFDA fluorescence. Mitochondrial morphology was quantified by TOM20 immunostaining, and statistical correlations between functional and viability parameters were assessed using Pearson’s correlation coefficient. LRRK2-G2019S neurons exhibited pronounced reductions in basal (125 → 81 pmol O₂/min), ATP-linked (94 → 61 pmol O₂/min), and maximal respiration (187 → 122 pmol O₂/min)—representing ~34–36% losses (p < 0.01–0.001). ATP concentration decreased from 8.7 ± 0.6 to 6.3 ± 0.4 nmol/mg, while the ATP/ADP ratio fell from 6.1 ± 0.4 to 4.2 ± 0.3. Mitochondrial membrane potential dropped by 25.2%, and ROS levels increased 1.6 ± 0.2-fold (p < 0.001), with MDA rising by 67% and GSH decreasing by 31%. Morphometric analysis showed 52 ± 6% fragmented mitochondria (<2 µm) versus 22 ± 4% in wild-type, alongside a 33% reduction in mitochondrial area and 41% shorter branch length. Viability decreased from 96 ± 4% to 73 ± 5%, strongly correlating with OCR (r = 0.91; p < 0.001) and inversely with ROS (r = –0.86; p < 0.001). The LRRK2-G2019S mutation causes 30–40% bioenergetic loss, 60–70% ROS elevation, and 25% neuronal death, underscoring mitochondrial dysfunction as a key driver of Parkinsonian neurodegeneration and a prime therapeutic target for LRRK2-linked PD.