Functional Effects of a Neuromelanin Analogue on Dopaminergic Neurons in 3D Cell Culture

The substantia nigra pars compacta (SNpc) is a discrete region of the brain that exhibits a dark pigment, neuromelanin (NM), a biomaterial with unique properties and the subject of ongoing research pertaining to neurodegenerative conditions like Parkinson's disease (PD). Obtaining human tissue to isolate this pigment is costly and labor intensive, making it necessary to find alternatives to model the biochemical interaction of NM and its implications on PD. To address this limitation, we modified our established silk 3D brain tissue model to emulate key characteristics of the SNpc by using a structural analogue of NM to examine the effects of the material on dopaminergic neurons using Lund's human mesencephalon (LUHMES) cells. We utilized a sepia-melanin, squid ink, derived NM analogue (NM-sim) to chelate ferric iron, and this iron-neuromelanin precipitate (Fe-NM) was purified and characterized. We then exposed LUHMES dopaminergic cells to the NM-sim, Fe-NM-sim, and control vehicle within 3D silk protein scaffolds. The presence of both NM-sim and Fe-NM-sim in the scaffolds negatively impacted spontaneous electrical activity from the LUMES networks, as evidenced by changes in local field potential (LFP) electrophysiological recordings. Furthermore, the Fe-NM-sim precipitate generated peroxides, depleted nutrients/antioxidants, and increased protein oxidation by carbonylation in sustained (>2 weeks) 3D cultures, thereby contributing to cell dysfunction. The results suggest that this 3D tissue engineered brain-like model may provide useful readouts related to PD neuro-toxicology research.