Fig. 5: Npc1^−/− microglia display impairment in myelin turnover.

a, b In vitro myelin phagocytosis assay. Cultured primary microglia isolated from P7 mice from three independent experiments (n = 3) were incubated with fluorescently labeled myelin (green) and analyzed at 24, 48, and 72 h. Microglial lysosomes were stained with anti-CD68 antibody (red). Hoechst was used for nuclear staining (blue). a Both WT and Npc1^−/− microglia showed efficient myelin uptake at 6 h. At 24 h (lower left panels) fluorescent labeling could be found within CD68 positive late endosomal/lysosomal compartments in both WT and Npc1^−/− microglia. At 48 h (upper right panels and higher magnification images shown in b), fluorescently labeled lipid vesicles were observed in WT microglia, indicating myelin turnover. In contrast, Npc1^−/− microglia accumulated myelin within the CD68 positive compartments and no fluorescently labeled lipid vesicles were detected. At 72 h (lower right panels), in most of the WT cells myelin was degraded or signal could be detected in lipid vesicles. In Npc1^−/− microglia, myelin signal was still within the CD68 positive compartments, suggesting compromised myelin turnover. Scale bars: 25 μm. c Primary microglia isolated from P7 WT and Npc1^−/− mice from three independent experiments (n = 3) were fed with fluorescently labeled myelin (green) and analyzed after 72 h. In order to confirm the identity of lipid vesicles forming as a result of myelin turnover, we stained microglia with Nile red (red) to visualize lipid droplets. Boxed regions are enlarged in right panels and reveal co-localization between fluorescently labeled myelin vesicles and Nile red, supporting myelin turnover and lipid droplet formation in WT microglia. In Npc1^−/− microglia, Nile red mainly stained myelin deposits accumulating in late endosomes/lysosomes, instead of lipid droplets at the cell periphery, confirming impairment in myelin turnover. Scale bars: 10 μm.