Fig 2: Patient P1 has differentially expressed genes (upregulated) associated with the UPR, but other NPC1 mutants exhibit delayed trafficking to lysosome. (a) List of genes with altered expression (p < 0.05, fold change =1.5) in P1 patient compared with P4, P5, and controls and P4 and P5 versus controls. Those that are upregulated exclusively in P1 are marked red. (b) After cluster analysis, the results showed that the most part of the genes are associated with ER stress and the UPR. (c) Relative expression levels of mRNA for HSPA1A, HSPA5, HSP90B1, and HMOX1 genes (four of the obtained hits). Error bars represent standard deviation (SD) from three independent experiments. ***p < 0.001 P1 versus CTRL by one-way ANOVA with Bonferroni's multiple comparison post hoc test (n = 3 in each group); ns = not significant. (d) CTRL and NPC1 primary human fibroblasts were treated with PNGaseF to deglycosylate the NPC1 protein (all N-linked oligosaccharides) and Endo H, and subjected to Western blotting (WB) using anti-NPC1 antibody. In CTRL the NPC1 protein is mostly Endo H-resistant, but in all patients, the Endo H-sensitive form is more abundant, suggesting that part of the protein was retained in the ER, and did not go for complex sugars formation, a process that only happens in the Golgi. Bands corresponding to complex glycosylated (Endo H-resistant) and mannose rich forms (Endo H-sensitive) were quantified and are displayed as Endo H-sensitive/Endo H-resistant ratio. (e) Immunostaining followed by confocal laser scanning microscopy of CTRL fibroblasts revealed that endogenous NPC-1 protein colocalizes with LAMP1 and partially colocalizes with calnexin. In primary human fibroblasts of patient P1, NPC1 colocalizes much less with LAMP1. Instead, it presents a higher colocalization with calnexin. Scale bar 25 µm

Fig 3: The NGS-targeted gene panel allowed the screening of two NPC patients. One harbors known disease-causing variants and for the second patient, a functional study was conducted. (a) Workflow of the study of the patients. (b) Clinical manifestation timeline for patient P1, with the first symptoms at 6 years old (juvenile form), cognitive impairment, ataxia, dystonia, and vertical supranuclear gaze palsy (VSGP) at 21 years old, compatible with an NPC diagnosis. (c) Unesterified cholesterol was labeled with filipin and staining intensity calculated from three independent experiments for patient P1, compared with a classical (patient P5) and variant (siblings P3/P3'). Data are mean ± SEM, ***p < 0.001, **p < 0.01 by ANOVA with Tukey's post hoc test. ns = not significant

Fig 4: Activity of selected small molecules inhibitors of NPC1 and related compounds against SARS-CoV-2. Infectivity of SARS-CoV-2 in (A) Vero E6 and (D) A549-ACE2 cells at 24 hpi measured by immunofluorescence microscopy. The percentage of infection efficiency was determined by determining the total fluorescence per well, using vehicle-treated cells (DMSO) and mock-infected cells as reference values. Normalized dose-response sigmoidal curve displaying dose values at nM scale on X axis and % of infection inhibition on Y axis using GraphPad 6 in (B) Vero E6 and (E) A549-ACE2. IC50 values (µM) were determined for these compounds for (C) Vero E6 and (F) A549-ACE2. The curves for the determination of CC50 in Vero E6 and A549-ACE2 cells are shown in Fig. S5B and S5C respectively.

Fig 5: Impact of NPC1 mutation on colocalization with StARD9. (A,B) Degree of colocalization of mCherry–StARD9 (red) with wild-type NPC1–EGFP (A, green) or with I1061T mutant NPC1–EGFP (B, green), as indicated by the sample image (left) and calculated Pearson correlation coefficient (right). Cells are outlined in white. n=10. Scale bar: 5 µm. (C) Summary of comparison of cells analyzed (n=10 each) by Pearson coefficient test. Dashed lines in violins represent median values and dotted lines indicate the 25th and 75th percentiles. P-values were obtained from a two-tailed unpaired Student's t-test.