Uptake by Human Astrocytes of Lipid Vesicles Modeling the Lipid Composition of Myelin

Abstract

The myelin sheath is composed of around 80% lipids including cholesterol, phospholipids, sphingomyelin, cerebroside sulfate and cerebrosides, and in smaller proportions, ceramides and glycerophosphatides. During pathological processes inside the central nervous system (CNS), the damage of this axonal insulation may expose the surrounding glial cells to lipid aggregates that result from this demyelination process. Indeed, microglia, considered to function as the local macrophages, can phagocyte myelin and cell detritus. Human astrocytes are another key example of glial cells where uptake of myelin debris may take place. Astrocytes are key regulators of several neuronal protective mechanisms, but they are also involved in the pathogenesis of certain autoimmune and inflammatory CNS diseases. Aiming to probe the behavior of human astrocytes exposed to different myelin lipids, we monitor the dynamics of lipid vesicle uptake by culture cells, and explore how varying specific myelin lipid components regulate the uptake kinetics and cell viability. A human astrocyte cell line obtained from a glioblastoma is used for all the experiments. Cells are exposed to NBD-PE or calcein labeled 50 nm small unilamellar vesicles (SUVs) of various lipid compositions reflecting various combinations of the myelin lipid components. Vesicle uptake is then monitored through fluorescence spectroscopy at different time points. Significant uptake is observed within 30 minutes, reaching saturation levels around 2 hours. These results are corroborated through flow cytometry, where astrocyte fluorescence stabilizes at around 2 hours. Additionally, we observe the presence of a smaller population of scatter cells thatshowed higher liposome uptake. Finally, by using fluorescence/DIC microscopy, liposomes are found to spread in the astrocyte cytoplasm after 4 hours of incubations. Interestingly, crowding of liposomes around the nucleus is observed after 12 hours of incubation, suggesting a sorting mechanism to be determined.