LUHMES-derived 3D organoids as an enhanced platform for modelling dopaminergic neurodegeneration in Parkinson's disease.

BACKGROUND: Parkinson's disease (PD) research traditionally relies on animal models and two-dimensional (2D) culture models. These models fail to recapitulate the complex cellular architecture and network interactions that are possible with three-dimensional (3D) organoid models. While 3D models allow improved cell-cell interactions, spatial organization, and metabolic microenvironments, however, their utility for modelling dopaminergic neurodegeneration remains underexplored. METHODS: We developed and characterized 3D organoids from LUHMES cells which are human embryonic neuronal precursor cells and a well-established human dopaminergic neuronal cell line. We directly compare their responses to the mitochondrial toxin MPP+ against conventional 2D cultures. Functional readouts included cell viability, ATP production, dopaminergic marker expression (tyrosine hydroxylase, MAP2, β-III tubulin), reactive oxygen species (ROS) generation, electrophysiological activity via multi-electrode arrays (MEA), and calcium signalling dynamics. RESULTS: 3D LUHMES organoids had 9-fold higher synapsin expression compared to 2D cultures, indicating enhanced synaptic maturity and network complexity. Following acute MPP+ exposure (0.25 mM, 24 h), 3D organoids were significantly more sensitive than 2D cultures, with greater reductions in cell viability (35% vs. 31%), ATP production (36% vs. 31%), and dopaminergic marker expression (TH: 60% reduction in both; MAP2: 70% vs. 54%; TUJ1: 62% vs. 20%). ROS production increased uniformly in 3D organoids (85% positive cells) compared to heterogeneous accumulation in 2D cultures (78% positive cells). Functional assessments revealed that 3D organoids displayed higher baseline electrophysiological activity that was sensitive to impairment following MPP+ treatment, spike amplitude, and calcium signalling responses to various stimuli (ATP, glutamate, GABA). CONCLUSION: LUHMES-derived 3D organoids demonstrate greater physiological relevance for modelling PD-related dopaminergic neurodegeneration than 2D cultures. The enhanced sensitivity to mitochondrial toxins, combined with more sophisticated network architecture and functional properties, suggests this model is a valuable platform for mechanistic studies of neurodegeneration and preclinical drug screening. These findings also support the broader adoption of 3D culture systems in neurodegenerative disease research.