Cyclodextrin overcomes the transport defect in nearly every organ of NPC1 mice leading to excretion of sequestered cholesterol as bile acid

Abstract

A mutation in NPC1 leads to sequestration of unesterified cholesterol in the late endosomal/lysosomal compartment of every cell culminating in the development of pulmonary, hepatic, and neurodegenerative disease. Acute administration of 2-hydroxypropyl-beta-cyclodextrin (CYCLO) rapidly overcomes this transport defect in both the 7-day-old pup and 49-day-old mature npc1(-/-) mouse, even though this compound is cleared from the body and plasma six times faster in the mature mouse than in the neonatal animal. The liberated cholesterol flows into the cytosolic ester pool, suppresses sterol synthesis, down-regulates SREBP2 and its target genes, and reduces expression of macrophage-associated inflammatory genes. These effects are seen in the liver and brain, as well as in peripheral organs like the spleen and kidney. Only the lung appears to be resistant to these effects. Forty-eight h after CYCLO administration to the 49-day-old animals, fecal acidic, but not neutral, sterol output increases, whole-animal cholesterol burden is reduced, and the hepatic and neurological inflammation is ameliorated. However, lifespan is extended only when the CYCLO is administered to the 7-day-old animals. These studies demonstrate that CYCLO administration acutely reverses the cholesterol transport defect seen in the NPC1 mouse at any age, and this reversal allows the sequestered sterol to be excreted from the body as bile acid.

Fig. 1.

Natural history of the changes in sterol metabolism and clinical findings in npc1^+/+ and npc1^−/− mice as a function of age. The first set of panels illustrate the changes in cholesterol content of the liver (A), carcass (B), brain (C) and whole animal (D) at different ages, and the second set shows the rates of cholesterol synthesis measured in vivo in these same tissues (E–H). The data in panels D and H are normalized to a whole-animal weight of 1 kg. The changes in relative mRNA levels of various macrophage-associated proteins in the liver (I) and cerebellum (J) are also shown as a function of age as is the liver function test ALT (K) and relative Purkinje cell number in the cerebellum (L). These various curves were constructed from data obtained in the current study as well as in previously reported investigations. Each value represents the mean ± 1 SEM for 4–8 animals at each age. NPC, Niemann-Pick type C.

Fig. 2.

Time course for the clearance of CYCLO from the whole animal (A) and plasma (B). These data were determined in 7- and 49-day-old npc1^+/+ and npc1^−/− mice injected at time 0 with a subcutaneous bolus of CYCLO (4,000 mg/kg) also containing [¹⁴C]CYCLO. Groups of animals were then killed 0, 0.5, 1, 2, 3, 6, and 24 h after the injection, and the total body pool and plasma concentration of CYCLO were determined. As data from the two genotypes were the same, they have been combined in this figure. Each data point represents the mean ± SEM for 4–6 animals in each group. CYCLO, 2-hydroxypropyl-β-cyclodextrin.

Fig. 3.

Comparison of the acute effects of CYCLO in the liver of 7- and 49-day-old npc1^+/+ and npc1^−/− mice. Each animal was given a single, subcutaneous dose of CYCLO (4,000 mg/kg) and studied 24 h later. The concentrations of hepatic unesterified cholesterol (A, B) and cholesteryl esters (C, D), as well as rates of cholesterol synthesis (E–F) were determined. The relative mRNA levels of SREBP2 and its target genes (G–L) and various macrophage-associated proteins (M–R) were also measured. The liver function test ALT was quantitated in the 49-day-old (T), but not in the 7-day-old (S) animals. Each column represents the mean ± SEM for 6 animals in each group. Significant differences among groups are designated by different letters in each panel. CE, cholesteryl ester; CYCLO, 2-hydroxypropyl-β-cyclodextrin; LDLR, low density lipoprotein receptor; ND, not determined; NPC, Niemann-Pick type C; SREBP, sterol regulatory element-binding protein; UC, unesterified cholesterol.

Fig. 4.

Comparison of the acute effects of CYCLO in the brain of 7- and 49-day-old npc1^+/+ and npc1^−/− mice. Each animal was given a single, subcutaneous dose of CYCLO (4,000 mg/kg) and studied 24 h later. The concentrations of unesterified cholesterol (A, B) and cholesteryl esters (C, D), as well as rates of cholesterol synthesis (E–F) in the whole brain were determined. The relative mRNA levels of SREBP2 (G–H), HMG-CoA SYN (I, J), and the inflammatory protein CD11c (K, L) were also quantified. Each column represents the mean ± SEM for 6 animals in each group. Significant differences among groups are designated by different letters in each panel. CE, cholesteryl ester; CYCLO, 2-hydroxypropyl-β-cyclodextrin ; NPC, Niemann-Pick type C; SREBP, sterol regulatory element-binding protein; UC, unesterified cholesterol.

Fig. 5.

Comparison of the acute effects of CYCLO in the adrenal gland, spleen, kidney, and lung 49-day-old npc1^+/+ and npc1^−/− mice. Each animal was given a single, subcutaneous dose of CYCLO (4,000 mg/kg) and studied 24 h later. The concentration of unesterified cholesterol (A, D, G, J) and cholesteryl esters (B, E, H, K), as well as rates of cholesterol synthesis (C, F, I, L) were measured in each of these organs. Each column represents the mean ± SEM for 6 animals in each group. Significant differences among groups are designated by different letters in each panel. CE, cholesteryl ester; CYCLO, 2-hydroxypropyl-β-cyclodextrin ; NPC, Niemann-Pick type C; UC, unesterified cholesterol.

Fig. 6.

Whole-animal cholesterol pools and synthesis rates in npc1^+/+ and npc1^−/− mice given CYCLO at either 7 or 49 days of age. These animals were administered either saline or CYCLO at 7 or 49 days of age and then studied 24 h later. The whole-animal cholesterol pools were measured in the 8 groups of mice (A, B) as were the rates of whole-animal cholesterol synthesis (C, D). These values represent the mg of cholesterol per kg body weight and the mg of sterol synthesized per day per kg body weight, respectively. Each bar shows the mean ± SEM for 6 animals, and significant differences are indicated by different letters. CYCLO, 2-hydroxypropyl-β-cyclodextrin ; NPC, Niemann-Pick type C.

Fig. 7.

Fecal neutral and acidic sterol output in npc1^+/+ and npc1^−/− mice after administration of CYCLO. The first point in each panel represents the baseline level of sterol output measured over the 3 days (days 46–48) prior to administration of either saline or CYCLO to mice at 49 days of age (indicated by arrows). Feces were collected in daily aliquots over the next 6 days. The amount of neutral (A, C) and acidic (B, D) sterol in each sample was then determined. Each point represents the mean ± SEM for 6 animals. CYCLO, 2-hydroxypropyl-β-cyclodextrin ; NPC, Niemann-Pick type C.

Fig. 8.

Age at death of animals treated with CYCLO at either 7 or 49 days of age. Groups of npc1^−/− mice were administered a single injection of CYCLO (4,000 mg/kg) at either 7 days of age or 49 days of age, and were then allowed to live out their lives. The control animals received only saline injections. The lifespan of only the animals treated at 7 days of age was significantly (P < 0.05) prolonged. CYCLO, 2-hydroxypropyl-β-cyclodextrin.