Reversal of defective lysosomal transport in NPC disease ameliorates liver dysfunction and neurodegeneration in the npc1-/- mouse

Abstract

Niemann-Pick type C disease is largely attributable to an inactivating mutation of NPC1 protein, which normally aids movement of unesterified cholesterol (C) from the endosomal/lysosomal (E/L) compartment to the cytosolic compartment of cells throughout the body. This defect results in activation of macrophages in many tissues, progressive liver disease, and neurodegeneration. In the npc1(-/-) mouse, a model of this disease, the whole-animal C pool expands from 2,082 to 4,925 mg/kg body weight (bw) and the hepatic C pool increases from 132 to 1,485 mg/kg bw between birth and 49 days of age. A single dose of 2-hydroxypropyl-beta-cyclodextrin (CYCLO) administered at 7 days of age immediately caused this sequestered C to flow from the lysosomes to the cytosolic pool in many organs, resulting in a marked increase in cholesteryl esters, suppression of C but not fatty acid synthesis, down-regulation of genes controlled by sterol regulatory element 2, and up-regulation of many liver X receptor target genes. There was also decreased expression of proinflammatory proteins in the liver and brain. In the liver, where the rate of C sequestration equaled 79 mg x d(-1) x kg(-1), treatment with CYCLO within 24 h increased C movement out of the E/L compartment from near 0 to 233 mg x d(-1) x kg(-1). By 49 days of age, this single injection of CYCLO resulted in a reduction in whole-body C burden of >900 mg/kg, marked improvement in liver function tests, much less neurodegeneration, and, ultimately, significant prolongation of life. These findings suggest that CYCLO acutely reverses the lysosomal transport defect seen in NPC disease.

Fig. 1.

Cyclo expands lifespan in the npc1^−/− mouse. (A) Npc1^−/− mice continuously fed ezetimibe, which markedly lowers cholesterol flux into the liver and improves liver function, did not live longer than those animals not fed this compound. (B) A single dose of CYCLO 4.5 or 5.6 administered at 7 days of age did, however, significantly and equally prolong life. (C) Addition of Allo to either the CYCLO 4.5 or CYCLO 5.6 did not significantly further prolong life (number indicated for each group). In each experiment, the untreated control animals were littermates of the treated group.

Fig. 2.

A single dose of CYCLO 4.5 administered to 7-day-old pups markedly altered sterol metabolism in the npc1^−/− mice 24 h later but had no effect in the npc1^+/+ animals. The unesterified and esterified cholesterol concentrations are shown in the liver (A) and brain (B), whereas total cholesterol concentrations are given for the remaining tissues of the carcass (C). (D–F) Cholesterol synthesis rates are shown for the same 3 tissue compartments. These values (G) are combined to give whole-animal cholesterol pools (H) and synthesis rates (I) in these same animals. To obtain these latter values, the rates of incorporation of ³H₂O into sterols by the different organs were converted to absolute rates of cholesterol synthesis. Statistically significant differences are indicated by the different letters (n = 6, P < 0.05).

Fig. 3.

A single dose of CYCLO 4.5 was administered to 7-day-old npc1^−/− and npc1^+/+ pups, and tissues were obtained 24 h later. RNA was extracted from the liver and brain for measurement by quantitative real-time PCR. (A) The mRNA level for ACAT2 is shown. The mRNA levels for hepatic target genes regulated by SREBP2 (B–E) and LXR (F–J) are shown in columns 1 and 2, respectively. (K–O) The third column provides relative mRNA levels in liver for various inflammatory proteins. (P–T) The fourth column of data shows several of these same relative mRNA values in the brain. Statistically significant differences are indicated by different letters (n = 6, P < 0.05).

Fig. 4.

Npc1^+/+ and npc1^−/− mice were administered CYCLO 4.5 at 7 days of age and were then studied at 49 days of age. Total cholesterol concentrations were determined in liver (A), brain (B), and various other tissues (C), and these were combined to give whole-animal cholesterol pools (D). Similarly, cholesterol synthesis was measured in these same tissues (E–G), and these data were combined to give whole-animal synthesis rates (H). In these same groups of animals, liver function tests were also measured (I and J), pyramidal and Purkinje cell numbers were quantitated in the brain (K and L), and daily fecal neutral and acidic sterol output was determined (M and N). Statistically significant differences are indicated by different letters (n = 6, P < 0.05).

Fig. 5.

Representative histological sections are shown of anterior-superior cerebellar vermis from 49-day-old npc1^+/+ (A, D, and G), npc1^−/− (B, E, and H), and npc1^−/− mice treated with CYCLO 4.5 at 7 days of age (C, F, and I). Contiguous sections were stained with H&E (A–C) or for calbindin (D–F) or GFAP (G–I) immunoreactivity. There was substantial loss of Purkinje cells (solid arrows) in untreated npc1^−/− animals (B), which was partially prevented by treatment with CYCLO 4.5 (C). (B and C) Increased numbers of Bergman glia (open arrows) and necrotic Purkinje cells (*) were also visible in the untreated npc1^−/− mice. (E) Calbindin immunoreactivity was markedly decreased in a band-like pattern in the molecular layer of untreated npc1^−/− animals (brackets) reflecting loss of Purkinje cell dendrites. (H) In contrast, there was increased GFAP immunoreactivity in the molecular layer in the same regions of calbindin loss (brackets) reflecting activation of astrocytes. Treatment with CYCLO 4.5 markedly reduced this loss of calbindin expression (F) and diminished GFAP expression (I). (Measurement bars: 100 μm.)