Genetic background modifies phenotypic severity and longevity in a mouse model of Niemann-Pick disease type C1

Abstract

Niemann-Pick disease type C1 (NPC1) is a rare, fatal neurodegenerative disorder characterized by lysosomal accumulation of unesterified cholesterol and glycosphingolipids. These subcellular pathologies lead to phenotypes of hepatosplenomegaly, neurological degeneration and premature death. NPC1 is extremely heterogeneous in the timing of clinical presentation and is associated with a wide spectrum of causative NPC1 mutations. To study the genetic architecture of NPC1, we have generated a new NPC1 mouse model, Npc1^em1PavNpc1^{em1Pav/em1Pav} mutants showed notably reduced NPC1 protein compared to controls and displayed the pathological and biochemical hallmarks of NPC1. Interestingly, Npc1^{em1Pav/em1Pav} mutants on a C57BL/6J genetic background showed more severe visceral pathology and a significantly shorter lifespan compared to Npc1^{em1Pav/em1Pav} mutants on a BALB/cJ background, suggesting that strain-specific modifiers contribute to disease severity and survival. QTL analysis for lifespan of 202 backcross N2 mutants on a mixed C57BL/6J and BALB/cJ background detected significant linkage to markers on chromosomes 1 and 7. The discovery of these modifier regions demonstrates that mouse models are powerful tools for analyzing the genetics underlying rare human diseases, which can be used to improve understanding of the variability in NPC1 phenotypes and advance options for patient diagnosis and therapy.This article has an associated First Person interview with the first author of the paper.

Keywords: Genetic modifiers; Mouse models; NPC1; Niemann-Pick disease type C1.

Fig. 1.

Generation of the Npc1^em allele and analysis of mRNA and protein levels in Npc1^em/em mutant mice. (A) Sanger sequencing results from Npc1^em/em mice. The bottom chromatogram shows the Npc1^em/em mutant genomic DNA sequence (missing the nine base-pair deletion, AGTAACATC) compared to the control Npc1^+/+ sequence on top. (B) RT-qPCR analysis using Taqman assays of Npc1 mRNA normalized to Gapdh. Liver and brain tissues were collected from both Npc1^em/em mutants (n=3) and control Npc1^+/+ littermates (n=3) at P68. Liver showed significantly higher levels of mRNA in the Npc1^em/em mutants compared to controls (P=0.0031). Npc1 mRNA levels in the brain were unchanged between the two groups (P=0.1749). **P≤0.01, unpaired Student's t-test. (C) Western blot analysis shows greatly reduced NPC1 protein levels in Npc1^em/em mutants. Liver and brain tissues were harvested at 8 weeks from Npc1^em/em mutants and control littermates (Npc1^+/+).

Fig. 2.

Abnormal lysosomal function leads to lipid accumulation and storage pathology in Npc1^em/em mice. (A) Brain and liver were collected from Npc1^+/+ controls (n=4) and Npc1^em/em mutants (n=3) between P63 and P68. Npc1^em/em mutants showed significantly elevated levels of total GSLs compared to controls in both the brain (P=0.0037) and liver (P=0.0051). All animals were on a C57BL/6J genetic background. Total GSLs (LacCer+GA2+Gb3+GM3+GM3Gc+Gb4+ GM2Gc levels) were measured in liver homogenates. Total GSLs (LacCer+GA2+Gb3+GM3+GM2+GA1+GM1a+GD1a+GD1b+GT1b+GQ1b) were measured in whole-brain homogenates. **P≤0.01, unpaired Student's t-test. (B) FACS results from primary fibroblasts that were derived from two different Npc1^em/em mutants and two Npc1^+/+ controls (each labeled #1 and #2) were stained with LysoTracker (LTR). Data are mean±s.d. for six technical replicates for each primary fibroblast cell line. These results showed a significantly higher fold change in LTR staining in Npc1^em/em-derived fibroblasts compared to controls (P=0.0117; nested Student's t-test). (C) Histological analysis of liver (left panels), spleen (center panels) and cerebellum (right panels) of Npc1^+/+ controls (top row) and Npc1^em/em mutants (bottom row). In Npc1^em/em liver, H&E staining showed numerous foam cells (indicated by arrowheads). In Npc1^em/em spleen, H&E staining showed a foamy appearance due to lipid-laden macrophages (indicated by arrowheads) that resulted in architectural disruption of both the white pulp (WP) and red pulp (RP). In the cerebellum, calbindin staining (brown) revealed extensive loss of Purkinje neurons in Npc1^em/em mutants compared to control littermates; Nissl (blue) staining of cerebellum. Scale bars: 100 µm for liver; 500 µm for spleen; 25 µm for cerebellum.

Fig. 3.

Progressive weight loss and neurodegenerative phenotypes are accompanied by reduced lifespan in Npc1^em/em mice. (A) Average weight loss onset occurred at 59.8 days, ranging from 58 to 66 days for females (left) and 60.7 days, ranging from 55 to 66 days for males (right). Npc1^+/+ controls (blue), n=8 females, n=9 males; Npc1^em/em mutants (red), n=19 females, n=21 males. (B) Progressive neurodegeneration and motor impairment in Npc1^em/em mutants (Npc1^em/em mutants, n=6; Npc1^+/+ controls, n=7). A combination of six different neurodegenerative phenotypes and behavioral tests were scored weekly starting at weaning (P28) and combined into a composite score (see Materials and Methods). A higher score means greater severity; Npc1^em/em mutant scores were significantly different than Npc1^+/+ controls. **P≤0.01, ****P≤0.0001 (two-way ANOVA with repeated measures and Bonferroni's correction; P<0.0001 for time, genotype and interaction). (C) Survival analysis of Npc1^em and Npc1^nih alleles on a C57BL/6J background. Survival of Npc1^em/em mice was greatly reduced compared to Npc1^+/+, with a median survival of 70 days (n=34). Furthermore, the survival of Npc1^em/em mice was slightly increased in comparison to mice homozygous for the Npc1^nih null allele [67 days, n=12; P=0.028, log-rank (Mantel-Cox) test]. Gender composition for both groups was: Npc1^nih/nih (females, n=4; males, n=8), Npc1^em/em (females, n=14; males, n=20).

Fig. 4.

Visceral pathology in Npc1^em/em mutants varies with genetic background. (A) Liver tissues from age-matched (P21) Npc1^em/em mutants on a C57BL/6J genetic background (left) or a BALB/cJ N6 intercross genetic background (right). Tissues were stained with the macrophage marker CD68 (brown). Npc1^em/em mutants on a C57BL/6J genetic background showed a greater accumulation of foam cells compared to the Npc1^em/em mutants on a BALB/cJ N6 intercross genetic background. A higher magnification view (bottom row) shows that the foam cells appeared to be larger in size owing to lipid storage. (B) Quantification of CD68⁺ signal showed a significant difference between a C57BL/6J genetic background and a BALB/cJ N6 intercross genetic background. ****P<0.0001, unpaired Student's t-test. Each dot represents the average of nine independent fields within an ROI, and nine ROIs were analyzed for each animal. C57BL/6J, n=4; BALB/cJ, n=3. Scale bars: 200 µm, top row; 10 µm, bottom row.

Fig. 5.

Strain-specific lifespan differences in Npc1^em/em mutants map to chromosomes 1 and 7. (A) Lifespan was measured for Npc1^em/em mutants on three different genetic backgrounds: C57BL/6J, BALB/cJ N4 intercross and N2 backcross [B6J(B6JCF1)]. Median survivals were: C57BL/6J, 70 days (n=34); BALB/cJ N4 intercross, 83.5 days (n=10); N2 [B6J(B6JCF1)] backcross, 78 days (n=202). The N2 backcross Npc1^em/em mutants showed a wide range in lifespan, spanning the extremes of both the Npc1^em/em C57BL/6J and Npc1^em/em BALB/cJ N4 intercross mutants. Mean±s.d. values were: Npc1^em/em C57BL/6J, 69.7±4.4 days; Npc1^em/em BALB/cJ N4 intercross mutants, 84.3±7.3 days; Npc1^em/em N2 backcross, 78.3±6.8 days. ****P<0.0001, one-way ANOVA. Each dot represents an individual animal. (B) Frequency distribution of lifespan in Npc1^em/em mutants on a C57BL/6J background (n=34, black) shows distribution for the population is centered at 70 days. In contrast, frequency distribution of N2 [B6J(B6JCF1)] Npc1^em/em mutants (n=202, gray) shows a greater range with a mean at 78 days, which is located at the high end of the C57BL/6J population. (C) Genetic linkage results between lifespan of Npc1^em/em mutants and strain-specific markers for C57BL/6J and BALB/cJ. Genome scan results from the B6J(B6JCF1) N2 backcross generation by individual chromosome locations identified areas with significant LOD scores on chromosome 1 (LOD=5.57) and chromosome 7 (LOD=8.91). The marker with the highest score (peak) at chromosome 1 was UNC99871 at 40.76 cM, and at chromosome 7 was UNC13374196 at 51.5 cM. Analysis was performed with a 5% significant threshold of LOD>3.09.