Weekly cyclodextrin administration normalizes cholesterol metabolism in nearly every organ of the Niemann-Pick type C1 mouse and markedly prolongs life

Abstract

Niemann-Pick type C1 (NPC1) disease arises from a mutation inactivating NPC1 protein that normally moves unesterified cholesterol from the late endosomal/lysosomal complex of cells to the cytosolic compartment for processing. As a result, cholesterol accumulates in every tissue of the body causing liver, lung, and CNS disease. Treatment of the murine model of this disease, the npc1 mouse, s.c. with β-cyclodextrin (4000 mg/kg) one time each week normalized cellular cholesterol metabolism in the liver and most other organs. At the same time, the hepatic dysfunction seen in the untreated npc1 mouse was prevented. The severity of cerebellar neurodegeneration also was ameliorated, although not entirely prevented, and the median lifespan of the animals was doubled. However, in contrast to these other organs, lung showed progressive macrophage infiltration with development of lipoid pneumonitis. These studies demonstrated that weekly cyclodextrin administration overcomes the lysosomal transport defect associated with the NPC1 mutation, nearly normalizes hepatic and whole animal cholesterol pools, and prevents the development of liver disease. Furthermore, this treatment slows cerebellar neurodegeneration but has little or no effect on the development of progressive pulmonary disease.

Figure 1

Effect of weekly CYCLO administration on organ weights and various parameters of cholesterol metabolism in the npc1^−/− mouse. Both npc1^+/+ and npc1^−/− mice were treated subcutaneously, weekly with either saline or CYCLO (4,000 mg/kg), and then studied at 49 days of age. The relative organ weights were measured in six major tissues (A–F). The content of total cholesterol present in each of these tissues was quantitated (G–L), as was the component of this sterol pool that was esterified (M–R). Both of these latter values are given as the mg of sterol present in the whole organs. Cholesterol synthesis rates in these same tissues are also shown (S–X), and are expressed as the nmol of [³H]water incorporated into sterols per hour per organ. Each column represents the mean ± 1 SEM for six animals. Significant differences (P<0.05) among groups are designated by different symbols in each panel.

Figure 2

Effect of weekly CYCLO administration on the relative mRNA levels of various inflammatory proteins in brain, liver, and lung. Both npc1^+/+ and npc1^−/− mice were treated weekly with either saline or CYCLO, and then studied at 49 days of age. Each column represents the mean ±1 SEM for six mice. Significant differences (P<0.05) among groups are indicated by different symbols in each panel.

Figure 3

Effect of weekly CYCLO administration on whole animal cholesterol metabolism, liver function tests, plasma total cholesterol concentrations and cerebellar neurodegeneration. Both npc1^+/+ and npc1^−/− mice were treated weekly with either saline or CYCLO, and then studied at 49 days of age. Whole animal cholesterol pools are expressed as mg of cholesterol per kg body weight (A). Whole animal synthesis rates (B) are expressed as mg of cholesterol synthesized per day per kg body weight. The liver function tests ALT (C), AST (D), and alkaline phosphatase (E) were quantitated, as were the levels of hepatic triacylglycerol (F). The plasma total cholesterol concentrations were also measured (G). The numbers of Purkinje cells in the cerebellum were counted, and are expressed relative to the number found in the npc1^+/+ mice treated with saline (H). Each column represents the mean ± 1 SEM for six animals in each group. Significant differences (P<0.05) among groups are indicated by different symbols in each panel.

Figure 4

Age at death of npc1^−/− mice treated with CYCLO. This figure shows three groups of npc1^−/− animals that were either untreated, administered a single dose of CYCLO (4,000 mg/kg) once at 7 days of age, or treated weekly with this same dose of CYCLO. There were 76 untreated mice, 32 treated once, and 20 treated weekly. Both the single and weekly doses of CYCLO significantly (*) prolonged the life of these npc1^−/−animals (P<0.001).

Figure 5

Representative histological sections of four tissues in npc1^−/− mice treated with CYCLO. Both npc1^+/+ and npc1^−/− mice were administered saline, while other groups of animals were given CYCLO (4,000 mg/kg) weekly. Histological preparations were then made of the cerebellum, liver, lung, and kidney at either 49 or 160 days of age. The solid arrows point to Purkinje cells in the cerebellum (A–E), to lipid-laden macrophages in the liver and lung (F–O), or to vacuolization in the kidney epithelium (P–T). The open arrows in panels B and C point to pyknotic Purkinje cells. The bar equals 50 μm (P), and all panels are at the same magnification (400X).

Figure 6

Flow of cholesterol from the late E/L compartment into the cytosolic compartment in npc1^−/− mice that were either untreated or were depleted of sequestered sterol pools. Two groups of npc1^−/− mice were used in these studies. One group received no prior treatment until 48 days of age at which time they were given a single dose of either saline (0) or CYCLO (4,000 mg/kg) (+), and then studied 24 hours later (A, C, E, G, I, K). The second group received weekly CYCLO administration beginning at 7 days of age to deplete tissue cholesterol pools. At 48 days of age these animals were then given either saline (0) or CYCLO (+), and studied 24 hours later (B, D, F, H, J, L). The level of cholesteryl ester found in the liver, spleen, and kidney was quantitated (A–F) as was the rate of cholesterol synthesis in these organs (G–L). Each column represents the mean ± 1 SEM for 4–8 animals in each group. Significant differences (P<0.05) among groups are indicated by different symbols in each panel.