NEUROMELANIN-PRODUCING NEURONS IN MESENCEPHALIC CULTURES AS A MODEL SYSTEM FOR INVESTIGATING PARKINSON’S DISEASE NEURODEGENERATION

To further investigate the potential role of neuromelanin (NM) in Parkinson’s disease (PD)-associated neurodegeneration, we used cultured mouse mesencephalic neurons in which lentiviral expression of human tyrosinase (hTYR) induces neuronal melanogenesis. Under optimized conditions, discrete, punctate cytosolic pigmentation appeared in some neuronal cell bodies 5–6 days post-transduction, reaching a plateau by days 9–10, with up to 15–20% of the neurons being pigmented. Bright-field imaging showed that hTYR-induced NM had a granular cytosolic distribution like that of NM in human brain tissue. NM synthesis in culture was primarily driven by the enzymatic conversion of L-tyrosine, as melanogenesis was totally absent in low-L-tyrosine medium. It was also enhanced by L-DOPA but not by dopamine under standard conditions. Neither the lipid peroxidation inhibitor Trolox nor the iron chelator deferoxamine affected NM accumulation. NM build-up led to a moderate yet significant reduction in the number of MAP-2+ neurons, including dopaminergic cells, accompanied by a proportional decrease in [³H]-dopamine uptake. Many melanized neurons exhibited lysosomal expansion (LAMP1-positive granules) and large p62-positive inclusions, indicating ongoing autophagy–lysosomal stress and dysregulated proteostasis. α-Synuclein aggregation was, however, not observable in these neurons. Despite some limitations, this model recapitulates key features of NM-related vulnerability in PD and provides a valuable platform for testing neuroprotective strategies against NM-driven neurodegeneration.