Isolation and Lipidomic Profiling of Neuronal Lipid Droplets to Unveil the Lipid Landscape of Neurodegenerative Disorders

Recent advances have expanded the role of lipid droplets (LDs) beyond passive lipid storage, implicating their involvement in various metabolic processes across mammalian tissues. Neuronal LDs, whose existence has long been debated, have now been identified in several neural structures, raising questions about their contribution to neurodegenerative disorders. Elucidating the specific chemical makeup of these organelles within neurons is critical for understanding their role in neural pathologies. However, the inherent submicron size and low abundance in neurons have posed challenges in their isolation; and physical and biochemical characterization. Here, we present our protocol which overcomes this obstacle: we blocked neuron-specific triglyceride lipase to enlarge LDs, rendering them amenable to floatation in a sucrose gradient. We further employed an improved methodology of lipidomic analysis on the LDs purified from cultured primary neurons, offering insights into their unique lipid composition. This protocol can be implemented by researchers with basic expertise in neuronal culture, imaging, ultracentrifuge handling, and LC-MS analysis. The complete workflow requires 3-4 weeks, from neuronal culture to lipidomics analysis. Future integration of this method with high-throughput techniques holds promises for uncovering disease-specific alterations in lipid metabolism, providing new avenues for potential therapeutic interventions.