Transcript, protein, metabolite and cellular studies in skin fibroblasts demonstrate variable pathogenic impacts of NPC1 mutations

Abstract

Background: Niemann-Pick type C (NP-C) is a rare neurovisceral genetic disorder caused by mutations in the NPC1 or the NPC2 gene. NPC1 is a multipass-transmembrane protein essential for egress of cholesterol from late endosomes/lysosomes. To evaluate impacts of NPC1 mutations, we examined fibroblast cultures from 26 NP-C1 patients with clinical phenotypes ranging from infantile to adult neurologic onset forms. The cells were tested with multiple assays including NPC1 mRNA expression levels and allele expression ratios, assessment of NPC1 promoter haplotypes, NPC1 protein levels, cellular cholesterol staining, localization of the mutant NPC1 proteins to lysosomes, and cholesterol/cholesteryl ester ratios. These results were correlated with phenotypes of the individual patients.

Results: Overall we identified 5 variant promoter haplotypes. Three of them showed reporter activity decreased down to 70% of the control sequence. None of the haplotypes were consistently associated with more severe clinical presentation of NP-C. Levels of transcripts carrying null NPC1 alleles were profoundly lower than levels of the missense variants. Low levels of the mutant NPC1 protein were identified in most samples. The protein localised to lysosomes in cultures expressing medium to normal NPC1 levels. Fibroblasts from patients with severe infantile phenotypes had higher cholesterol levels and higher cholesterol/cholesteryl ester ratios. On the contrary, cell lines from patients with juvenile and adolescent/adult phenotypes showed values comparable to controls.

Conclusion: No single assay fully correlated with the disease severity. However, low residual levels of NPC1 protein and high cholesterol/cholesteryl ester ratios associated with severe disease. The results suggest not only low NPC1 expression due to non-sense mediated decay or low mutant protein stability, but also dysfunction of the stable mutant NPC1 as contributors to the intracellular lipid transport defect.

Keywords: Cholesterol transport; Lysosomal storage disease; Mutant protein; Niemann-pick type C; Proteostasis.

Fig. 1

aNPC1 promoter haplotype variants and respective luciferase reporter activities in %. Reporter activity of pGL3 basic vector was 0.1 ± 0.1% of Haplotype 1 construct activity. Haplotype 3 is present in controls only. b Immunoreactive NPC1 protein in skin fibroblast lines (Western blotting). The cell line numbers and phenotypes are indicated on the top. Equal amount of protein (8 μg) was applied per line. Abnormal banding associated with p.Y276H is indicated by arrowheads. CCD data were used for the quantification. c The mutations are depicted using crystal structure of NPC1 protein 3JD8 [40] and the domains are color-coded according to Li [11]. A schematic of primary structure of the mature NPC1 protein is shown below the structure - domain color coding. NTD – N-terminal domain, TM – transmembrane domain, MLD - middle luminal domain, SSD – sterol sensing domain. The most severe mutations are indicated in bold font. Most of the mutations are in lumenal domains I and C (color-coded circles beside the mutation labels). d Representative images of human skin fibroblasts of a control and patients with selected forms of the disease. 1st column: direct filipin stained cultures. 2nd column: confocal microscopy images anti-NPC1 signal. 3rd column: merge of anti-LAMP2 (red) and anti-NPC1 (green) signal, 4th column: co-localization overlay maps. Values 0 - 1 of the pixels are displayed using lookup table LUT 0–1. All images were processed equally

Fig. 2

Overview of NP-C1 patient fibroblasts analyses. Strip plots show results of assays for the four phenotypic subgroups. Solid circles represent individual patient cell lines. Numbers adjacent to the circles represent patient number. The long-dashed and dashed horizontal lines represent the average control levels and standard error of means, respectively. Asterisks above the boxplots (2a, 2b, 2d, 2e) mark groups that differ significantly from controls. The groups were compared by one-way ANOVA test followed by post-hoc Tukey’s HSD test adjusted for unequal sample sizes. aNPC1 mRNA expression level, b NPC1 protein level by Western Blotting, c endosomal / lysosomal localisation of NPC1 protein. NPC1 vs. LAMP2 Object Pearson’s colocalization coefficient, d unesterified cholesterol / cholesterol ester ratio by MS/MS, e direct filipin staining of cellular unesterified cholesterol, f cholesterol esterification rate of LDL-derived cholesterol