A somatic cell defect is associated with the onset of neurological symptoms in a lysosomal storage disease

Abstract

Mutations in individuals with the lysosomal storage disorder Niemann-Pick disease, type C1 (NPC1) are heterogeneous, not localized to specific protein domains, and not correlated to time of onset or disease severity. We demonstrate direct correlation of the time of neurological symptom onset with the severity of lysosomal defects in NPC1 patient-derived fibroblasts. This is a novel assay for NPC1 individuals that may be predictive of NPC1 disease progression and broadly applicable to other lysosomal disorders.

Keywords: Lysosomal storage disorders; NPC1; Neurodegenerative disorders; Niemann–Pick disease, type C1.

Figure 1. Fibroblasts from individuals with NPC1 show quantitative increases in lysosomal storage defects that correlate with age of onset of neurological symptoms

(A) Confocal microscopic images of LysoTracker red staining in fibroblasts from an individual with NPC1 (NPC-25, right) compared to control fibroblasts (CTL1, left). Increased cellular staining in NPC1 is consistent with increased cholesterol storage in late endosomes/lysosomes. Nuclear DAPI staining in blue; scale bar = 20 μm. (B) Scatter dot plot comparing LysoTracker staining (LTR; y-axis) of individual, NPC1 patient-derived fibroblast lines (N=27) with staining of control fibroblast lines (N=4). NPC1 fibroblasts exhibit significantly greater LysoTracker staining than control cells (Student's two-tailed t-test, p<0.0001) with greater variance (NPC1 standard deviation = 8.6). (C) Linear regression of fold change increases in LysoTracker staining intensity of NPC1 cells (y-axis) versus the age of onset of neurological symptoms (x-axis) showed significant correlation. Each blue circle represents results from a single NPC1 patient cell line. Log₁₀-transformed values are presented and were used for statistical analyses of these normally distributed data. (D) Scatter dot plot of NPC1 patients sorted into categories based upon neurological age of onset, as follows: early infantile (2 months – 2years) n=6; late infantile (2 – 6 years), n=8; juvenile (6 – 15 years), n=9; and adolescent/adulthood (≥15 years), n=4. LysoTracker staining intensity for early infantile was significantly different from that of all other categories (1-way ANOVA with Tukey's post-hoc test; p<0.0001). For (B-D), LysoTracker staining measurements are expressed as fold increase over unstained cells derived from the same individual, to remove any effects of autofluorescence). Box and whiskers plots in (B, D) show mean and standard deviation. Statistical analyses performed using Prism (GraphPad).