Quantitative role of LAL, NPC2, and NPC1 in lysosomal cholesterol processing defined by genetic and pharmacological manipulations

Abstract

Lipoprotein cholesterol taken up by cells is processed in the endosomal/lysosomal (E/L) compartment by the sequential action of lysosomal acid lipase (LAL), Niemann-Pick C2 (NPC2), and Niemann-Pick C1 (NPC1). Inactivation of NPC2 in mouse caused sequestration of unesterified cholesterol (UC) and expanded the whole animal sterol pool from 2,305 to 4,337 mg/kg. However, this pool increased to 5,408 and 9,480 mg/kg, respectively, when NPC1 or LAL function was absent. The transport defect in mutants lacking NPC2 or NPC1, but not in those lacking LAL, was reversed by cyclodextrin (CD), and the ED₅₀ values for this reversal varied from ~40 mg/kg in kidney to >20,000 mg/kg in brain in both groups. This reversal occurred only with a CD that could interact with UC. Further, a CD that could interact with, but not solubilize, UC still overcame the transport defect. These studies showed that processing and export of sterol from the late E/L compartment was quantitatively different in mice lacking LAL, NPC2, or NPC1 function. In both npc2(-/-) and npc1(-/-) mice, the transport defect was reversed by a CD that interacted with UC, likely at the membrane/bulk-water interface, allowing sterol to move rapidly to the export site of the E/L compartment.

Figures

Fig. 1.

Tissue distribution of NPC1 and NPC2 mRNA in normal mice. RNA was pooled from 3–10 mice for each organ (all males except for uterus, ovary, and placenta) and evaluated using Northern analysis.

Fig. 2.

Alterations in cholesterol metabolism in the npc2−/− mouse compared to those seen in the npc1−/− animal. Groups of npc+/+, npc1−/−, and npc2−/− animals were studied at 49 days of age. The npc+/+ group included combined data derived from both npc1+/+ and npc2+/+ animals because these were virtually identical. Relative organ weight is expressed as a percentage of whole animal weight. Cholesterol content is given as the mg of cholesterol present in each whole organ and rates of sterol synthesis are also expressed per whole tissue. From these data, whole animal weights (G), cholesterol pools (N), and synthesis rates (U) were calculated. The columns represent means ± 1 SEM for 6–14 mice in each group, and significant differences (P < 0.05) among the groups are indicated by different letters.

Fig. 3.

Comparison of various molecular, metabolic, and clinical parameters in the npc2−/− and npc1−/− mice. Groups of npc1+/+, npc2+/+, npc1−/−, and npc2−/− mice were studied at 49 days of age and a variety of measurements were made, both in vivo and in vitro. These include the relative expression of mRNA for several markers of inflammation, plasma lipid levels, and rates of hepatic fatty acid synthesis, and various clinical measures of hepatic and brain disease. The mRNA levels shown for the npc1−/− and npc2−/− mice are expressed relative to the appropriate npc1+/+ and npc2+/+ control animals (set at 1.0). The columns represent means ± 1 SEM of 6 (mRNA measurements) or 6–12 animals in each group. The survival studies (R) utilized 76 npc1−/− and 14 npc2−/− animals. Significant differences (P < 0.05) among groups are indicated by different letters.

Fig. 4.

The effective dose (ED50) of HP-β-CD leading to 50% suppression of cholesterol synthesis in various organs of the npc1−/− and npc2−/− mice. Utilizing animals that were 49 days old, individual mice were given subcutaneously a dose of HP-β-CD that varied from 0 to 8,000 mg/kg. Twenty-four h later, the rate of cholesterol synthesis was measured in all of the major organs. The mean rates of synthesis at each dose are shown in this diagram. However, the sigmoid curves and ED50 values shown for each organ were generated by computer-fitting the individual data points from 68 npc1−/− and 53 npc2−/− animals (GraphPad Prism). These log10 ED50 values were also converted to the dose of HP-β-CD giving 50% inhibition of synthesis. In the case of the lung, the data points would not converge onto a sigmoid curve, i.e., the ED50 values were infinitely high.

Fig. 5.

The ability of HP-β-CD, HP-α-CD, and SBE7-β-CD to overcome the cholesterol export defect in npc1−/− and lal−/− mice. Seven-day-old mice of each genotype along with appropriate controls were administered a single subcutaneous dose of either HP-β-CD, HP-α-CD, or SBE7-β-CD (4,000 mg/kg). Twenty-four h later, rates of cholesterol synthesis were measured in various organs including the liver, spleen, and carcass. In this case, carcass refers to all residual tissues after removal of the liver, spleen, brain, and lungs, and consists mostly of muscle, bone, and adipose tissue. The columns represent means ± 1 SEM for 3–6 animals in each group and significant differences (P < 0.05) among groups are indicated by different letters.

Fig. 6.

Model illustrating cholesterol movement out of the late E/L compartment of cells into the cytosolic compartment. Lipoproteins carrying sterol esters enter the late E/L compartment where the CE normally is hydrolyzed by LAL, and the resulting UC is moved to the export site by the sequential activities of NPC2 and NPC1. In the normal adult mouse (A), the rate of UC movement through this pathway in all organs totals about 140 mg/day/kg. When LAL is nonfunctional (B), the CE is probably trapped in smectic liquid crystals, and UC output from the E/L compartment drops to near zero. However, when either NPC2 or NPC1 is mutated (C), UC continues to be generated and both interacts with the membrane phospholipids and precipitates as crystals of cholesteryl monohydrate. The UC in the wall of the lysosome presumably can still diffuse along the plane of the inner leaflet to the exit site so that, under these conditions, the rate of sterol movement out of the E/L compartment is only modestly reduced to 80–100 mg/day/kg. When cyclodextrin is administered to such animals (D), this rate of diffusion may be markedly increased.