Niemann-Pick Type C disease: characterizing lipid levels in patients with variant lysosomal cholesterol storage

Abstract

A central feature of Niemann-Pick Type C (NPC) disease is sequestration of cholesterol and glycosphingolipids in lysosomes. A large phenotypic variability, on both a clinical as well as a molecular level, challenges NPC diagnosis. For example, substantial difficulties in identifying or excluding NPC in a patient exist in cases with a "variant" biochemical phenotype, where cholesterol levels in cultured fibroblasts, the primary diagnostic indicator, are only moderately elevated. Here we apply quantitative microscopy as an accurate and objective diagnostic tool to measure cholesterol accumulation at the level of single cells. When employed to characterize cholesterol enrichment in fibroblasts from 20 NPC patients and 11 controls, considerable heterogeneity became evident both within the population of cells cultured from one individual as well as between samples from different probands. An obvious correlation between biochemical phenotype and clinical disease course was not apparent from our dataset. However, plasma levels of HDL-cholesterol (HDL-c) tended to be in the normal range in patients with a "variant" as opposed to a "classic" biochemical phenotype. Attenuated lysosomal cholesterol accumulation in "variant" cells was associated with detectable NPC1 protein and residual capability to upregulate expression of ABCA1 in response to LDL. Taken together, our approach opens perspectives not only to support diagnosis, but also to better characterize mechanisms impacting cholesterol accumulation in NPC patient-derived cells.

Fig. 1.

Automated image analysis reliably quantifies cholesterol levels in Niemann Pick Type C (NPC) patient fibroblasts. (A) Primary human skin fibroblasts from an unaffected individual (neg. ctrl 1; left panel), one NPC patient with moderately elevated cellular cholesterol (“variant” biochemical NPC phenotype; ΔNPC1_v03; middle panel), and one NPC patient with pronounced cholesterol storage (“classic” NPC-phenotype; ΔNPCx_c01; right panel). Cells were cultured under control conditions on glass-bottom slides, fixed, and stained with the cholesterol binding dye filipin. Images were acquired on an automated epifluorescence microscope. Bar = 20 μm. Perinuclear areas encompassing lysosomes (blue/red) were determined from microscopic images with masks generated by the automated image analysis software DetecTiff^©. Green lines delineate extra-perinuclear background areas. (B) Scattergraph showing the correlation of mean perinuclear filipin signal intensity/cell within 11 selected images from four different fibroblast cultures, as quantified by DetecTiff^© (x axis) relative to manual quantification within masks generated with ImageJ software. On average, 71 cells/image were analyzed. (C) Scattergraph showing the correlation of mean perinuclear filipin signal intensity/cell from five different fibroblast cultures as quantified by DetecTiff^© (y axis; means from three to five images/cell line) relative to total cellular levels of free cholesterol as determined biochemically from cell extracts.

Fig. 2.

Analysis of fibroblasts from 20 different NPC patients reveals a continuous spectrum of filipin signal intensities. (A) Comparison of the range of filipin signal intensity distributions (y axis) among fibroblasts from ten NPC patients visually classified as “classic” biochemical phenotype (ΔNPC_c), ten patients classified as “variant” phenotype (ΔNPC_v), four obligate heterozygotes (ΔNPC1_het), two healthy control individuals (neg. ctrl), and five patients in which NPC disease had been considered unlikely (noNPC) (x axis). Signal intensities were quantified by DetecTiff^© from, on average, 39 images/cell line (range: 10–144) with data from up to 2,208 fibroblasts/patient. Box plots show medians (bars), lower and upper quartiles (boxes), 10th and 90th percentiles (whiskers), and outliers (○). (B) Representative images from 10 “variant” patients (ΔNPC_v01-10), 1 “classic” patient (ΔNPCx_c01), and 1 healthy control individual (neg. ctrl 1). Cells cultured under control conditions on glass coverslips were fixed and stained with filipin, and images were acquired on an automated epifluorescence microscope. Bar = 20 μm.

Fig. 3.

Filipin signal intensities in “variant” NPC patient fibroblasts may increase upon exposure to LDL. Fibroblasts from one control individual (neg. ctrl 1), two NPC patients with a “variant” (ΔNPC1_v01; ΔNPC1_v03), and two patients with a “classic” biochemical phenotype (ΔNPC2_c18; ΔNPCx_c01) were cultured either under control culture conditions (10% fetal calf serum; left column) or in lipoprotein-depleted serum (5% LDS) for 48 h before exposure to 50 μg/ml LDL-cholesterol (LDL-c) in 10% FCS for 16 h (middle column). Then cells were fixed and stained with filipin, and images were acquired automatically. Bar = 20 μm. Relative intensity frequency distributions [0..1] (y axis) of mean perinuclear filipin signal intensities as quantified by DetecTiff^© (x axis) from the indicated number of images/cell line (n), and two to three independent replica experiments were quantified. For better comparison, for each cell line, lowest signals were set to 0%, brightest signals to 100%.

Fig. 4.

Patients with a biochemically “variant” phenotype show a disease progression similar to that of other NPC patients. Longitudinal severity scores according to (43) in a cohort of 10 patients with infantile disease onset (<5 years) (A) and 12 patients with juvenile/adult disease onset (>6 years) (B). Scores were assessed by restrospective analysis of clinical data acquired between 2002 and 2010. The progression slope per subgroup (boxes) was determined by linear regression of the depicted curves and averaging for either biochemically “variant” (gray curves) or “other” NPC-patients [i.e., either “classic” patients (n = 6) or patients for which biochemical phenotype was unavailable (n = 10); black curves] separately.

Fig. 5.

The “variant” biochemical NPC phenotype correlates with normalization of plasma HDL levels. Mean plasma levels of HDL-c, LDL-c, total cholesterol, and triacylglycerides (TGs) were determined in 32 NPC patients (“all NPC,” left columns, circles), out of which 7 patients had been diagnosed as “variant” (middle columns; triangles) and 9 as “classic” biochemical phenotype (right columns, squares). Each data point represents mean parameters from 1–14 independent blood samples per patient (with on average approximately three independent measurements/patient). Statistical analysis of source data was performed using the paired 2-tailed Student's t-test (** P < 0.01). The table shows number of patients/parameter in which mean levels fell below the 5th percentile (<5.P.) or above the 95th percentile (>95.P.) of the age- and sex-adjusted reference population.

Fig. 6.

“Variant” NPC fibroblasts show residual NPC1 protein expression and LDL-dependent upregulation of ABCA1. (A) Lysates from fibroblasts of one healthy control individual (neg. ctrl 1), two NPC patients with “classic” (ΔNPC1_c03; ΔNPC2_c18), and two patients with “variant” biochemical phenotype (ΔNPC1_v03; ΔNPC1_v05) were subjected to Western blot and analyzed with antibodies against NPC1, ABCA1, or β-actin, respectively. Cells were cultured either under control conditions (DMEM/10% FCS; middle lane), depleted from sterols by culture in 5% LDS instead of FCS for 72 h (LDS; left lane), or upon sterol depletion for 48 h challenged with 10% FCS/50 μg/ml LDL for 24 h (LDS/LDL; right lane). (B) ABCA1 protein levels under control, sterol-depleted (LDS), and LDL-exposed (LDS/LDL) conditions in (A) were quantified and normalized to β-actin on the identical lane. For each respective patient, relative ABCA1 levels under the indicated culture condition were normalized to control conditions (= 1).