Fig 2: LIMP-2-WT but not LIMP-2-G379W/V415W overexpression rescues cholesterol efflux from LEs in LIMP-2 deficient cells. a Principle of late endosomal BODIPY-cholesterol (BC) efflux assay in A431 cells. Cells are loaded for 24 h with oleic acid-conjugated BSA to generate lipid droplets (LD). In parallel, cells are labeled with dextran to visualize late endosomal organelles (LE). Cells are then labeled for 2 h with BODIPY cholesteryl linoleate-labeled LDL (BC LN-LDL) that enters LE, and chased in serum-free medium to monitor the transfer of BC from LE to LD (labeled with LipidTox during the last 30 min of BC-LN-LDL labeling). b Overlaid confocal images of BC (green), dextran/LIMP-2 (blue), and LipidTox (red) in live A431 cells transfected with the indicated siRNAs and/or cDNA constructs (LIMP-2-WT or LIMP-2-G379W/V415W overexpressing cells are outlined) at 0 h and 4 h of chase. Scale bar, 10 µm. c Quantification of the fraction of BC fluorescence residing in dextran-positive or LIMP-2-positive late endosomes (LE) vs. LipidTox-positive lipid droplets (LD) after 4 h of chase (n = 21 (siLIMP-2, siCtrl), 22 (siLIMP2 + WT.mCherry) or 23 (siNPC1, siLIMP-2 + G379W/V415W.mCherry) cells). Data (mean ± SEM) from 3 experiments, t-test (**P = 0.01, ****P < 0.0001). d Wide-field fluorescence micrographs of filipin stained NPC1-deficient CHO M12 cells overexpressing LIMP-2-G379W/V415W-mCherry. Arrowheads indicate examples of filipin-positive LIMP-2-containing LEs. Scale bar, 10 µm. For quantification see 1 G. e Analysis of [3H]cholesterol incorporation into cholesteryl esters in LIMP-2-WT and LIMP-2-KO MEFs in the presence and absence of U18666A or PKF (n = 7 (0 h/5 h), n = 3 (9 h), n = 4 (PKF/U18666A) samples). Data (mean ± SEM) from two (9 h chase) to 3 experiments (5 h chase), unpaired two-tailed Student’s t-test (*P = 0.05). Source data are provided as a Source Data file

Fig 3: NPC1 inhibition down-regulates mTOR.(A) 14 DIV cortical neurons were treated with the NPC1 inhibitor U18666A (2 µg/ml) for 24 h or left untreated. Western blot analysis of total lysates show decrease mTOR-dependent phosphorylation of its substrate S6K. Actin was used to normalize for total protein. (B) The plot compares the levels of S6K phosphorylation between untreated (Control) or U18666A-treated (U18) neurons. (A) Quantification of pS6K levels from Western blot experiments as the one shown in panel (A) (n = 3). The graph shows the mean ± SEM and individual data points. Statistical significance was analyzed by two-tailed unpaired t test (*P < 0.05).

Fig 4: Identification of Ncr1 interaction partners on the vacuole membrane.(A) Ncr1 was tagged with one half of the DHFR enzyme and mated to strains carrying fusion proteins to the other half of the enzyme. Interaction strength was a function of colony size of the diploids on methotrexate divided by their abundance. Ncr1 had high interaction strength with Pmc1 and Fth1 relative to the panel of vacuolar proteins. (B) Pmc1 was tagged with one half of the DHFR enzyme and mated to strains carrying fusion proteins to the other half of the enzyme and had high interaction strengths with Fth11, Apc11, and Ncr1 relative to the panel of vacuolar proteins. Means ± SD, N = 8. (C) Graphic of predicted complex of Ncr1, Pmc1, Apc11, and Fth1. (D) Co-immunoprecipitation of NPC1 and PMCA2 (ATP2B2) from rat cerebellum. (E) qRT-PCR for mRNA expression of PMCA2 (ATP2B2) in wild-type, heterozygous, and homozygous cerebellums of 8-wk Npc1nih balb/c mice. N = 5, ***P = 0.0004 as compared with WT, calculated by one-way ANOVA.

Fig 5: Alterations in protein localization caused by loss of Ncr1 lead to cytoskeleton abnormalities in NPC.(A) Representative images from GFP localization screen including Prk1-GFP and She4-GFP in control (BY4741) relative to ?ncr1 yeast. Prk1 is localized to the actin cortical patch in BY4741 yeast, but in the ?ncr1 yeast Prk1 is found to be diffuse and mainly cytoplasmic. She4 is localized primarily to the cytoplasm where in the ?ncr1 yeast, the localization is more punctate. (B) Representative images of NPC1 and control patient fibroblasts stained for acetylated a-tubulin. Scale bar: 10 µm. (C) Graphical representation of the fluorescent intensities of acetylated a-tubulin. Mean ± SD. N = 10–20 **P < 0.01; t test. (D) Protein levels of a-tubulin, acetylated a-tubulin, aTAT1, HDAC6, and SIRT2 were measured in control and NPC1 patient fibroblasts. (D, E) Quantitation of blots from (D) by densitometry. (F) Treatment with 125 µM NAD for 24 h reduced acetylated a-tubulin expression in NPC1 patient cells, fluorescence measured using a flow cytometer and analysed with FloJo software. Mean ± SD, N = 3 *P < 0.05; one-way ANOVA.