Characterization of Extracellular Vesicles from LUHMES Cells: A Baseline for Understanding Mitophagy-Related Communication in Dopaminergic Neurons

Abstract

Parkinson’s disease (PD) is a progressive neurodegenerative disorder increasingly understood as a mitochondrial disease. Mitophagy, the selective clearance of damaged mitochondria, is disrupted in PD, leading to mitochondrial accumulation and neuronal death. To study mitochondrial stress responses in neurons, researchers use stem cell models like LUHMES cells, a human dopaminergic neuronal line ideal for PD modeling. We hypothesize that neurons under mitochondrial stress may activate secretory mitophagy, exporting mitochondrial components via extracellular vesicles (EVs). While EVs have gained attention in neurodegeneration, EVs from healthy dopaminergic neuronal cells remain poorly characterized. Understanding how healthy neurons release mitochondrial cargo could reveal new strategies to counteract mitochondrial dysfunction in PD and related diseases. LUHMES cells were cultured using a novel protocol, and EVs were isolated via ultracentrifugation. These were characterized using western blotting for classical EV markers and mitochondrial proteins, nanoparticle tracking analysis, and immunoprecipitation for EV subtypes. Initial challenges included maintaining LUHMES viability during proliferation, prompting optimization. Western blotting suggests that healthy neurons secrete mitochondrial proteins via EVs, supporting the hypothesis of secretory mitophagy under normal conditions. This work establishes a baseline for future comparisons with PD models, and future studies will explore whether inducing secretory mitophagy could offer a therapeutic approach. Limitations include EV yield variability and weak protein signals, which are currently being optimized.