Fig. 8. Human blood-derived macrophages from NPC patients resemble pathological alterations of murine Npc1^−/− microglia.

a Proteome analysis of macrophages from 7 NPC patients (NPC) and 3 healthy controls (CTR). The negative log10 transformed p-value of each protein is plotted against its average log2 transformed LFQ ratio between NPC and CTR macrophages. The hyperbolic curves indicate the threshold for a permutation-based FDR correction for multiple hypotheses. Selected proteins altered in human NPC macrophages are highlighted in green and non-significantly changed proteins are encircled in gray. b Validation of MS data via western blot analysis and corresponding quantification. Representative immunoblots from 3 NPC patients and 3 healthy controls reveal increased levels of late endosomal/lysosomal proteins in NPC patient-derived macrophages. GAPDH was used as loading control and quantification was performed by densitometry (ImageJ—NIH) from three independent experiments. Values were normalized on CTR and represent mean ± SEM (unpaired two-tailed Student’s t-test). NPC1 (n = 3, p = 0.0003); NPC2 (n = 3, p = 0.02); LAMP1 (n = 3, p = 0.02); CTSD (n = 3, 0.01); CD68 (n = 3, p = 0.01); GRN (n = 3, p = 0.07). c Ex vivo Aβ plaque clearance assay. Representative immunostaining showing macrophages (CD45, green) plated onto APPPS1 brain section that are clustering around and phagocytosing Aβ plaques, visualized with Thiazine Red (ThR, plaque core, red) and 3552 anti-Aβ antibody (Aβ plaque, white). Hoechst was used for nuclear staining (blue). Scale bar: 25 μm. d Quantification of phagocytosed Aβ plaques. Values are expressed as percentages of amyloid plaque clearance normalized to CTR and represent mean ± SEM from 6 NPC patients and 3 healthy controls (p = 0.19, unpaired two-tailed Student’s t-test). e In vitro myelin phagocytosis assay. Human macrophages from 2 NPC patients and 2 healthy controls were fed with fluorescently labeled myelin (green) and analyzed after 48 h. Myeloid cells were visualized using an antibody against CD45 (red). Hoechst was used as nuclear staining (blue). Boxed regions are enlarged in right panels and show that human CTR macrophages efficiently uptake and turnover myelin as demonstrated by fluorescently labeled lipid droplets at the cell periphery. In contrast, we could not detect fluorescently labeled lipid droplets at the cell periphery in NPC macrophages, suggesting trafficking defect that may preclude myelin turnover. Scale bars: 25 μm and 5 μm (enlargements).