Amino acid substitution in NPC1 that abolishes cholesterol binding reproduces phenotype of complete NPC1 deficiency in mice

Abstract

Substitution mutations in adjacent amino acids of the N-terminal domain of NPC1, a lysosomal membrane protein, abolish its cholesterol binding activity and impair its ability to export cholesterol from lysosomes of cultured cells lacking npc1 [Kwon HJ, et al. (2009) Cell 137:1213-1224]. Here, we show that the same two mutations (proline-202 and phenylalanine-203, both changed to alanine) reproduce the phenotype of complete NPC1 deficiency when knocked into the mouse npc1 gene by homologous recombination. Homozygous npc1(pf/pf) mice exhibited neurodegeneration beginning at day 49 and died at a median age of 84 d, as previously reported for mice that lack npc1. Liver and other organs of the npc1(pf/pf) mice accumulated excess cholesterol in lysosomes. In liver, mRNAs encoding several lysosomal proteins were elevated, including NPC1 and NPC2 and several digestive enzymes (acid lipase, β-glucuronidase, and cathepsins B and D). Weekly treatment with hydroxypropyl-β-cyclodextrin (HPCD) beginning at 7 wk reduced hepatic cholesterol accumulation and diminished the lysosomal mRNAs. We conclude that the cholesterol binding site in the N-terminal domain of NPC1 is essential for cholesterol export from lysosomes in living animals as it is in cultured cells. The HPCD-mediated reduction of excess lysosomal enzymes may contribute to the ability of this drug to delay the progression of NPC disease in mice.

Fig. 1.

Generation of npc1 knockin mice harboring two point mutations in NPC1 that abolish cholesterol binding activity. (A) Schematic of the mouse WT npc1 allele and the targeting construct used to generate an npc1^pf allele by introducing two point mutations (P202;F203 to A202;A203) into exon 5 of npc1. The targeting vector also contains a loxP-flanked pgkneopA cassette and two copies of the HSV-TK gene as selection markers. The probe used for the Southern blot is denoted by the horizontal filled rectangle labeled “probe.” (B) Representative Southern blot analysis of EcoRI-digested genomic DNA from tails of mice with the indicated genotypes.

Fig. 2.

NPC1 and NPC2 levels in livers of WT and npc1^pf/pf mice. Nine-week-old male WT and npc1^pf/pf littermates were fed an ad libitum chow diet before study. (A) mRNA analysis. Relative amounts of NPC1 and NPC2 mRNAs in livers were determined by quantitative real-time PCR with apoB as the invariant control. Values represent the amount of mRNA relative to that in WT littermates, which is arbitrarily defined as 1. Each bar represents the mean ± SEM of data from 4 mice. Asterisks denote the level of statistical significance (Student t test) between the WT and npc1^pf/pf mice. **P < 0.01. The number of PCR cycles (cycle threshold, C_t) required to reach the threshold line of 0.15 is shown inside the bar graph. (B) Immunoblot analysis. For each genotype, whole-cell lysates were prepared from four mouse livers. Aliquots of the pooled lysates (40 μg protein) were subjected to 8% SDS/PAGE and immunoblot analysis. β-Tubulin was used as a loading control. (C) Glycosidase digestion. For each genotype, membrane fractions from four mouse livers were pooled. Aliquots of the pooled fractions (40 μg and 20 μg for WT and npc1^pf/pf, respectively) were incubated in the absence or presence of the indicated glycosidase and subjected to SDS/PAGE and immunoblot analysis.

Fig. 3.

Colocalization of transfected NPC1(P202A/F203A) with endogenous LAMP1. SV-589 fibroblasts were transfected with TK-driven plasmids expressing Flag-tagged versions of either WT NPC1 (A–C) or mutant NPC1(P202A/F203A) (D–F) as described in Materials and Methods. Twenty-four hours after transfection, the cells were fixed and immunostained for Flag-tagged NPC1 (red) and endogenous LAMP1 (green). Nuclei were stained blue with DAPI. Fluorescence images were viewed and processed by confocal microscopy as described in Materials and Methods. Each image represents a maximum intensity projection of a confocal z-stack of 32 images. (Scale bars, 10 μm; magnification: 190×.)

Fig. 4.

Growth and survival of WT and npc1^pf/pf mice. (A) Littermate WT (n = 30, 18 male and 12 female) and npc1^pf/pf (n = 30, 18 male and 12 female) mice were weighed weekly beginning at 5 wk of age. (B) The same cohort of WT (n = 30) and npc1^pf/pf (n = 30) mice were followed up to 112 d of age, and the percent survival of each group was plotted as a function of time. For comparison, the reported survival curve for npc1^nih/nih mice (14) was replotted in red.

Fig. 5.

Histology of various tissues from WT and npc1^pf/pf mice. Tissues from 9-wk-old female WT (A–C) and npc1^pf/pf (D–F) littermates were fixed, sectioned, and stained with H&E as described in Materials and Methods. Arrows (A) denote Purkinje cells that are present in cerebellum of WT mice but absent from npc1^pf/pf mice. (Scale bars, 50 μm; magnification: 40×.)

Fig. 6.

Accumulation of cholesterol and gangliosides in organs of npc1^pf/pf mice. (A) Total cholesterol content in various organs of 9-wk-old male WT and npc1^pf/pf littermates was determined as described in Materials and Methods. Each bar represents mean ± SEM of data from four mice. Asterisks denote level of statistical significance (Student t test) between the WT and npc1^pf/pf mice. **P < 0.01. (B) Gangliosides from tissues of 9-wk-old male npc1^pf/pf, npc1^nih, and WT mice were subjected to TLC and visualized by orcinol staining as described in Materials and Methods. For each genotype, equal weights of liver or brain tissue from four mice were pooled. Each lane corresponds to the ganglioside fraction isolated from 3 mg of brain or 1.5 mg of liver (wet weight). Position of migration of standards for GM1, -2, and -3 are indicated.

Fig. 7.

Cyclodextrin treatment of WT and npc1^pf/pf mice. Male WT and npc1^pf/pf littermates were treated with three weekly injections of saline or HPCD beginning at 7 wk of age as described in Materials and Methods. At 9.5 wk of age (3 d after the final injection), the mice were killed to obtain plasma and organs for analysis. (A) Content of total and free cholesterol in livers of WT and npc1^pf/pf mice injected with saline or HPCD. Each bar represents the mean ± SEM of data from five mice. (B) Histology of representative livers from WT and npc1^pf/pf mice injected with saline or HPCD. Livers were fixed, sectioned, and stained with H&E. (Scale bars, 40 μM; magnification: 40×.) (C) Relative amounts of mRNAs in livers of the indicated groups were determined by real-time PCR, with apoB as the invariant control. Values represent the amount of mRNA relative to that in WT littermates injected with saline, which is arbitrarily defined as 1 and denoted by the dotted line. Each bar represents the mean ± SEM of data from five mice. Statistical analysis (A and B) was performed with two-tailed Student t test. *P < 0.05; **P < 0.01; ***P < 0.001.