Three-dimensional neuroimmune co-culture system for modeling Parkinson's Disease microenvironments in vitro.

Parkinson's disease (PD) is a complex and multifaceted neurodegenerative disorder that results from multiple environmental factors and multicellular interactions. Although several PD neuropathologies have been identified and described, the thorough understanding of PD pathophysiology and research has been largely limited by the absence of reliable in vitro models that truly recapitulate PD microenvironments. Here, we propose a neuroimmune co-culture system that models PD neuropathologies by combining relevant multicellular interactions with environments that mimic the brain. This system is composed of: (i) 3D bioprinted cultures of mature human dopaminergic neurons grown on extracellular matrix (ECM)-derived scaffolds doped with electroconductive nanostructures, and (ii) a direct co-culture of human astrocytes and differentiated monocytes that models neuroinflammatory responses. When co-cultured in a transwell format, these two compartments recreate relevant multicellular environments that model Parkinson's disease pathologies after exposure to the neurotoxin A53T α-synuclein. With immunofluorescent staining and gene expression analyses, we show that functional and mature dopaminergic 3D networks are generated within our ECM-derived scaffolds with superior performance to standard 2D cultures. Moreover, by analyzing cytokine secretion, cell surface markers, and gene expression, we define a human monocyte differentiation scheme that allows the appearance of both monocyte-derived macrophages and dendritic cell phenotypes, as well as their optimal co-culture ratios with human astrocytes to recreate synergistic neuroinflammatory responses. We show that the combined response of both compartments to A53T α-synuclein stimulates the formation of intracellular α-synuclein aggregates, induces progressive mitochondrial dysfunction and ROS production, downregulates the expression of synaptic, dopaminergic, and mitophagy-related genes, and promotes the initiation of apoptotic processes within the dopaminergic networks. Most importantly, these intracellular pathologies were comparable or superior to those generated with a rotenone-stimulated 2D control that represents the current standard for in vitro PD models and showed increased resilience towards these neurotoxic insults, allowing the study of disease progression over longer time periods than current models.