Fig 1.
Progressive accumulation of cholesterol, GSLs and foam cells in Npc1nih/nih mice liver.
(A) H&E staining of liver sections of Npc1+/+ and Npc1nih/nih at 20, 40, 60 and 80 days of age. CV = central vein, PV = portal vein. Scale bar = 100 μm. (B) Levels of total cholesterol, ceramide and GSLs, glucosylceramide, lactosylceramide and globotriaosylceramide in liver lysates of Npc1+/+ (WT), Npc1+/nih (HET) and Npc1nih/nih (KO) mice at the age of 20–23, 40–43, 60–63 and 84–90 days. Data (n = 6–12, mean ± S.D.) were analysed using a two-way ANOVA: *** P < 0.001 from Npc1+/+.
Fig 2.
Progressive accumulation of cholesterol and GSLs in Npc1nih/nih mice spleen.
(A) H&E staining of spleen sections of Npc1+/+ and Npc1nih/nih at 20, 40, 60 and 80 days of age. FC = follicle centre, PALS = peri-arteriolar lymphocyte sheath, WP = white pulp, RP = red pulp. Scale bar = 100 μm. (B) Levels of total cholesterol, ceramide and GSLs, glucosylceramide, lactosylceramide and globotriaosylceramide in spleen lysates of Npc1+/+ (WT), Npc1+/nih (HET) and Npc1nih/nih (KO) mice at the age of 20–23, 40–43, 60–63 and 84–90 days. Data (n = 3–6, mean ± S.D.) were analysed using a two-way ANOVA: * P < 0.05, ** P < 0.01 and *** P < 0.001 from Npc1+/+.
Fig 3.
Gpnmb gene expression is increased with disease progression in Npc1nih/nih mice viscera.
Gpnmb (top-panel) and the macrophage marker Iba-1 (bottom-panel) gene expression relative to the house-keeping P0 gene in liver and spleen of Npc1+/+ (WT), Npc1+/nih (HET) and Npc1nih/nih (KO) mice at the age of 20–23, 40–43, 60–63 and 84–90 days. Data (n = 3–6, mean ± S.D.) were analysed using a two-way ANOVA: * P < 0.05, ** P < 0.01 and *** P < 0.001 from Npc1+/+.
Fig 4.
Gpnmb positive cells are increased with disease progression in Npc1nih/nih mice liver.
"Composite" panels show immunostaining of Gpnmb in magenta and Iba-1 in yellow and methylgreen nuclear counterstain of liver of Npc1+/+ and Npc1nih/nih at 20, 40, 60 and 80 days of age. Brightfield scans were analysed using spectral imaging, Iba1 and Gpnmb are displayed with a color-coded intensity scale. CV = central vein, PV = portal vein. Scale bar = 100 μm.
Fig 5.
Gpnmb positive cells are increased with disease progression in Npc1nih/nih mice spleen.
"Composite" panels show immunostaining of Gpnmb in magenta and Iba-1 in yellow and methylgreen nuclear counterstain of spleen of Npc1+/+ and Npc1nih/nih at 20, 40, 60 and 80 days of age. Brightfield scans were analysed using spectral imaging, Iba1 and Gpnmb are displayed with a color-coded intensity scale. Red blood cells (rbc) were imaged as well (red colour, “composite” panels). FC = follicle centre, PALS = peri-arteriolar lymphocyte sheath, RP = red pulp. Scale bar = 100 μm.
Fig 6.
Total Cholesterol and GSLs accumulate in macrophages exposed to U18666A.
(A) total cholesterol, (B) ceramide, (C) sphingosine, (D) glucosylceramide (GlcCer), (E) dihydro-ceramide (Dihydro-Cer), and (F) lactosylceramide (LacCer) levels in RAW264.7 murine macrophages exposed to 1, 5 and 10 μM of U18666A for 24 h. Data (n = 3, mean ± S.E.M.) were analysed using an unpaired t-test. * P < 0.05, ** P < 0.01 and *** P < 0.001 from DMSO control.
Fig 7.
Gpnmb gene and protein expression are increased in macrophages exposed to U18666A.
RAW264.7 murine macrophages were exposed to 1, 5 and 10 μM of U18666A for 24 h. (A) Gpnmb mRNA levels and (B) Gpnmb protein levels. (C) Soluble Gpnmb assayed by ELISA of the cell culture medium. Data (n = 3 mean ± S.E.M.) were analysed using an unpaired t-test. * P < 0.05, ** P < 0.01 and *** P < 0.001 from DMSO control.
Fig 8.
Soluble Gpnmb levels are increased in NPC mice and patient plasma.
(A) Plasma sGpnmb levels were assayed by ELISA in plasma of Npc1+/+ (WT), Npc1+/nih (HET) and Npc1nih/nih (KO) mice at the age of 20–23, 40–43, 60–63 and 84–90 days. Data (n = 3–6, mean ± S.E.M.) were analysed using a two-way ANOVA: * P < 0.05, ** P < 0.01 and *** P < 0.001 from Npc1+/+. (B) Plasma levels of sGpnmb in Npc1+/+ (WT) and Npc1nmf164 mice at 110 days of age. Data (n = 3–5, mean ± S.E.M.) were analysed using an unpaired t-test: ** P < 0.01. (C) Plasma levels of sGpnmb in healthy volunteers (n = 9), carriers of one known mutation on the Npc1 gene (n = 8) and confirmed NPC patients (n = 17) detected by ELISA, were analysed using the Matt-Whitney u test: * P < 0.05.
Fig 9.
GSL synthesis inhibition abrogates Gpnmb induction.
(A) Total cholesterol, ceramide, glucosylceramide (GlcCer) and lactosylceramide (LacCer) levels of RAW264.7 murine macrophages exposed to 10 μM U18666A and/or 250 μM NB-DNJ for 20 h. (B) Gpnmb, Ccl3 and Cathepsin D (CtsD) gene expression, and sGpnmb levels of RAW264.7 cells exposed to 10 μM U18666A and/or 250 μM NB-DNJ for 20 h. Data (n = 3 mean ± S.E.M.) were analysed using an unpaired t-test. * P < 0.05, ** P < 0.01 and *** P < 0.001.
Fig 10.
Correlation between glycosphingolipids and sGpnmb levels.
Correlation between cell lysate (A) glucosylceramide (GlcCer) or (B) lactosylceramide (LacCer) and sGpnmb from the medium of RAW264.7 cells exposed to 1.25, 2.5, 5 and 10 μM of U18666A for 20 h. Correlation between tissue lysate (C) hepatic or (D) splenic GlcCer levels and plasma sGpnmb of Npc1nih/nih mice at the age of 20–23, 40–43, 60–63 and 84–90 days. Correlation between plasma (E) GlcCer or (F) chitotriosidase activity and sGpnmb levels from diagnosed NPC patients (n = 17). Pearson correlational analysis was used to evaluate correlation.