Nöronal Lipid Damlacıklarının İzolasyonu ve Lipido mik Profillemesi

Özet (abstract)

Abstract 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. Key points This protocol describes a robust workflow for pharmacological induction, purification, and characterization of mature lipid droplets from primary cortical neurons without any detectable cytosolic membrane carryover. LC-MS analysis of neuronal lipid droplets can reveal lipids previously uncharacterized by other methods, providing a platform to integrate high-throughput proteomics and other techniques for probing neurodegenerative disease linked metabolic alterations in neurons. Key reference Kumar, M. et al. Triglycerides are an important fuel reserve for synapse function in the brain. Nat Metab 7 , 1392-1403 (2025). https://doi.org:10.1038/s42255-025-01321-x Editorial summary This protocol describes a robust workflow for pharmacological induction, purification, and characterization of mature lipid droplets from primary cortical neurons without any detectable cytosolic membrane carryover. Proposed teaser Isolation and lipidomic profiling of neuronal lipid droplets

Yazarların özeti; kaynağından alınmıştır. Nature Protocols, 2026 · DOI ↗