​A STEPWISE 2D-3D SCREENING STRATEGY FOR IDENTIFYING NEUROPROTECTIVE NATURAL COMPOUNDS USING HUMAN NEURONAL MODELS AND MIDBRAIN ORGANOIDS

Neurodegenerative diseases are characterized by progressive neuronal loss preceded by early functional dysfunction, underscoring the need for in vitro models that capture early pathological changes rather than terminal cytotoxicity. Here, we established a stepwise, human-relevant 2D-3D drug screening strategy that integrates a human-derived neuronal cell model with a disease-relevant brain organoid system to identify neuroprotective natural compounds. A neuronal differentiation condition was established for SH-SY5Y cells using retinoic acid, generating a neuron-like 2D model exhibiting neuronal marker expression. Neuronal differentiation was validated by immunocytochemistry and western blotting, confirming mature neuronal features. Mitochondrial reactive oxygen species (ROS)–driven neuronal dysfunction was induced using rotenone, a mitochondrial complex I inhibitor, under conditions that preserved overall cell viability while eliciting oxidative stress responses. Neuronal differentiation and stress-induced dysfunction were evaluated through morphological analysis, neurite quantification, and stress-associated markers expression. Complementing these analyses, a luciferase reporter system was established, enabling quantitative monitoring of oxidative stress– and inflammation-related signaling pathways. Candidate compounds were screened for their ability to modulate ROS-associated neuronal stress responses under established 2D pathological conditions. In parallel, disease-relevant pathological conditions were established in human midbrain organoids, in which neuronal identity was confirmed and rotenone treatment elicited neuronal impairment consistent with neurodegenerative pathology. Compounds demonstrating protective functional profiles in the 2D screening stage were subsequently validated in 3D environment. Collectively, this study presents a scalable and human-relevant 2D–3D screening framework for neuroprotective drug discovery. This work was supported by National Research Foundation of Korea by the Korea Government (NRF-2019R1A5A2026045, RS-2023-00262332, RS-2024-00450091).