Delineation of biochemical, molecular, and physiological changes accompanying bile acid pool size restoration in Cyp7a1(-/-) mice fed low levels of cholic acid.

Cholesterol 7α-hydroxylase (CYP7A1) is the initiating and rate-limiting enzyme in the neutral pathway that converts cholesterol to primary bile acids (BA). CYP7A1-deficient (Cyp7a1(-/-)) mice have a depleted BA pool, diminished intestinal cholesterol absorption, accelerated fecal sterol loss, and increased intestinal cholesterol synthesis. To determine the molecular and physiological effects of restoring the BA pool in this model, adult female Cyp7a1(-/-) mice and matching Cyp7a1(+/+) controls were fed diets containing cholic acid (CA) at modest levels [0.015, 0.030, and 0.060% (wt/wt)] for 15-18 days. A level of just 0.03% provided a CA intake of ~12 µmol (4.8 mg) per day per 100 g body wt and was sufficient in the Cyp7a1(-/-) mice to normalize BA pool size, fecal BA excretion, fractional cholesterol absorption, and fecal sterol excretion but caused a significant rise in the cholesterol concentration in the small intestine and liver, as well as a marked inhibition of cholesterol synthesis in these organs. In parallel with these metabolic changes, there were marked shifts in intestinal and hepatic expression levels for many target genes of the BA sensor farnesoid X receptor, as well as genes involved in cholesterol transport, especially ATP-binding cassette (ABC) transporter A1 (ABCA1) and ABCG8. In Cyp7a1(+/+) mice, this level of CA supplementation did not significantly disrupt BA or cholesterol metabolism, except for an increase in fecal BA excretion and marginal changes in mRNA expression for some BA synthetic enzymes. These findings underscore the importance of using moderate dietary BA levels in studies with animal models.

Unesterified cholesterol accumulation in late endosomes/lysosomes causes neurodegeneration and is prevented by driving cholesterol export from this compartment.

While unesterified cholesterol (C) is essential for remodeling neuronal plasma membranes, its role in certain neurodegenerative disorders remains poorly defined. Uptake of sterol from pericellular fluid requires processing that involves two lysosomal proteins, lysosomal acid lipase, which hydrolyzes C esters, and NPC1 (Niemann-Pick type C1). In systemic tissues, inactivation of either protein led to sterol accumulation and cell death, but in the brain, inactivation of only NPC1 caused C sequestration and neurodegeneration. When injected into the CNS of the npc1(-/-) mouse, 2-hydroxypropyl-ß-cyclodextrin (HP-ß-CD), a compound known to prevent this C accumulation, diffused throughout the brain and was excreted with a t(½) of 6.5 h. This agent caused suppression of C synthesis, elevation of C esters, suppression of sterol regulatory-binding protein 2 (SREBP2) target genes, and activation of liver X receptor-controlled genes. These findings indicated that HP-ß-CD promoted movement of the sequestered C from lysosomes to the metabolically active pool of C in the cytosolic compartment of cells in the CNS. The ED(50) for this agent in the brain was ∼0.5 mg/kg, and the therapeutic effect lasted >7 d. Continuous infusion of HP-ß-CD into the ventricular system of npc1(-/-) animals between 3 and 7 weeks of age normalized the biochemical abnormalities and completely prevented the expected neurodegeneration. These studies support the concept that neurons continuously acquire C from interstitial fluid to permit plasma membrane turnover and remodeling. Inactivation of NPC1 leads to lysosomal C sequestration and neurodegeneration, but this is prevented by the continuous, direct administration of HP-ß-CD into the CNS.

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

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.

Multiple mechanisms limit the accumulation of unesterified cholesterol in the small intestine of mice deficient in both ACAT2 and ABCA1.

Cholesterol homeostasis in the enterocyte is regulated by the interplay of multiple genes that ultimately determines the net amount of cholesterol reaching the circulation from the small intestine. The effect of deleting these genes, particularly acyl CoA:cholesterol acyl transferase 2 (ACAT2), on cholesterol absorption and fecal sterol excretion is well documented. We also know that the intestinal mRNA level for adenosine triphosphate-binding cassette transporter A1 (ABCA1) increases in Acat2(-/-) mice. However, none of these studies has specifically addressed how ACAT2 deficiency impacts the relative proportions of esterified and unesterified cholesterol (UC) in the enterocyte and whether the concurrent loss of ABCA1 might result in a marked buildup of UC. Therefore, the present studies measured the expression of numerous genes and related metabolic parameters in the intestine and liver of ACAT2-deficient mice fed diets containing either added cholesterol or ezetimibe, a selective sterol absorption inhibitor. Cholesterol feeding raised the concentration of UC in the small intestine, and this was accompanied by a significant reduction in the relative mRNA level for Niemann-Pick C1-like 1 (NPC1L1) and an increase in the mRNA level for both ABCA1 and ABCG5/8. All these changes were reversed by ezetimibe. When mice deficient in both ACAT2 and ABCA1 were fed a high-cholesterol diet, the increase in intestinal UC levels was no greater than it was in mice lacking only ACAT2. This resulted from a combination of compensatory mechanisms including diminished NPC1L1-mediated cholesterol uptake, increased cholesterol efflux via ABCG5/8, and possibly rapid cell turnover.

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

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 ß-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.

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

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.

ABCA1 plays no role in the centripetal movement of cholesterol from peripheral tissues to the liver and intestine in the mouse.

This study uses the mouse to explore the role of ABCA1 in the movement of this cholesterol from the peripheral organs to the endocrine glands for hormone synthesis and liver for excretion. The sterol pool in all peripheral organs was constant and equaled 2,218 and 2,269 mg/kg, respectively, in abca1(+/+) and abca1(-/-) mice. Flux of cholesterol from these tissues equaled the rate of synthesis plus the rate of LDL-cholesterol uptake and was 49.9 mg/day/kg in control animals and 62.0 mg/day/kg in abca1(-/-) mice. In the abca1(+/+) animals, this amount of cholesterol moved from HDL into the liver for excretion. In the abca1(-/-) mice, the cholesterol from the periphery also reached the liver but did not use HDL. Fecal excretion of cholesterol was just as high in abac1(-/-) mice (198 mg/day/kg) as in the abac1(+/+) animals (163 mg/day/kg), although the abac1(-/-) mice excreted relatively more neutral than acidic sterols. This study established that ABCA1 plays essentially no role in the turnover of cholesterol in peripheral organs or in the centripetal movement of this sterol to the endocrine glands, liver, and intestinal tract for excretion.

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

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.

Central nervous system: cholesterol turnover, brain development and neurodegeneration.

The average amount of cholesterol in the whole animal equals approximately 2100 mg/kg body weight, and 15% and 23% of this sterol in the mouse and human, respectively, is found in the central nervous system. There is no detectable uptake across the blood-brain barrier of cholesterol carried in lipoproteins in the plasma, even in the newborn. However, high rates of de novo cholesterol synthesis in the glia and neurons provide the sterol necessary for early brain development. Once a stable brain size is achieved in the adult, cholesterol synthesis continues, albeit at a much lower rate, and this synthesis is just balanced by the excretion of an equal amount of sterol, either as 24(S)-hydroxycholesterol or, presumably, as cholesterol itself.

Inhibiting intestinal NPC1L1 activity prevents diet-induced increase in biliary cholesterol in Golden Syrian hamsters.

Niemann-Pick C1-like 1 (NPC1L1) facilitates the uptake of sterols into the enterocyte and is the target of the novel cholesterol absorption inhibitor, ezetimibe. These studies used the Golden Syrian hamster as a model to delineate the changes in the relative mRNA expression of NPC1L1 and other proteins that regulate sterol homeostasis in the enterocyte during and following cessation of ezetimibe treatment and also to address the clinically important question of whether the marked inhibition of cholesterol absorption alters biliary lipid composition. In hamsters fed a low-cholesterol, low-fat basal diet, the abundance of mRNA for NPC1L1 in the small intestine far exceeded that in other regions of the gastrointestinal tract, liver, and gallbladder. In the first study, female hamsters were fed the basal diet containing ezetimibe at doses up to 2.0 mg.day(-1).kg body wt(-1). At this dose, cholesterol absorption fell by 82%, fecal neutral sterol excretion increased by 5.3-fold, and hepatic and intestinal cholesterol synthesis increased more than twofold, but there were no significant changes in either fecal bile acid excretion or biliary lipid composition. The ezetimibe-induced changes in intestinal cholesterol handling were reversed when treatment was withdrawn. In a second study, male hamsters were given a diet enriched in cholesterol and safflower oil without or with ezetimibe. The lipid-rich diet raised the absolute and relative cholesterol levels in bile more than fourfold. This increase was largely prevented by ezetimibe. These data are consistent with the recent finding that ezetimibe treatment significantly reduced biliary cholesterol saturation in patients with gallstones.

GM2/GD2 and GM3 gangliosides have no effect on cellular cholesterol pools or turnover in normal or NPC1 mice.

These studies investigated the role of gangliosides in governing the steady-state concentration and turnover of unesterified cholesterol in normal tissues and in those of mice carrying the NPC1 mutation. In animals lacking either GM2/GD2 or GM3 synthase, tissue cholesterol concentrations and synthesis rates were normal in nearly all organs, and whole-animal sterol pools and turnover also were not different from control animals. Mice lacking both synthases, however, had small elevations in cholesterol concentrations in several organs, and the whole-animal cholesterol pool was marginally elevated. None of these three groups, however, had changes in any parameter of cholesterol homeostasis in the major regions of the central nervous system. When either the GM2/GD2 or GM3 synthase activity was deleted in mice lacking NPC1 function, the clinical phenotype was not changed, but lifespan was shortened. However, the abnormal cholesterol accumulation seen in the tissues of the NPC1 mouse was unaffected by loss of either synthase, and clinical and molecular markers of hepatic and cerebellar disease also were unchanged. These studies demonstrate that hydrophobic interactions between cholesterol and various gangliosides do not play an important role in determining cellular cholesterol concentrations in the normal animal or in the mouse with the NPC1 mutation.

Genetic variations and treatments that affect the lifespan of the NPC1 mouse.

Niemann-Pick type C (NPC) disease is a multisystem disorder caused primarily by a mutation in the npc1 gene. These studies evaluated the effect of genetic background, deletion of additional genes, and administration of several agents on the age at death in a murine model of this disorder. Such factors as differing strain background or genetic drift within a given background in the npc1(-/-) mouse significantly altered the age at death and the degree of organ disease. Genetic deletion of Siat9 (GM3 synthetase) or Nr1h2 [liver X receptor (LXR)beta] shortened the life of the npc1(-/-) animals. Daily treatment of the npc1(-/-) mice with an LXR agonist or administration of a single dose of cyclodextrin, with or without the neurosteroid allopregnanolone, significantly slowed neurodegeneration and increased the lifespan of these animals. These data illustrate that the age at death of the npc1(-/-) mouse can be significantly influenced by many factors, including differences in strain background, other inactivating gene mutations (Siat9 and lxrbeta), and administration of agents such as LXR agonists and, particularly, cyclodextrin. It is currently not clear which of these effects is nonspecific or which might relate directly to the molecular defect present in the NPC1 syndrome.

Liver X receptor activation enhances cholesterol loss from the brain, decreases neuroinflammation, and increases survival of the NPC1 mouse.

Although cholesterol is a major component of the CNS, there is little information on how or whether a change in sterol flux across the blood-brain barrier might alter neurodegeneration. In Niemann-Pick type C (NPC) disease, a mutation in NPC1 protein causes unesterified cholesterol to accumulate in the lysosomal compartment of every cell, including neurons and glia. Using the murine model of this disease, we used genetic and pharmacologic approaches in an attempt to alter cholesterol homeostasis across the CNS. Genetic deletion of the sterol transporters ATP-binding cassette transporter A1 (ABCA1) and low-density lipoprotein receptor in the NPC1 mouse did not affect sterol balance or longevity. However, deletion of the nuclear receptor, liver X receptor beta (LXRbeta), had an adverse effect on progression of the disease. We therefore tested the effects of increasing LXR activity by oral administration of a synthetic ligand for this transcription factor. Treatment with this LXR agonist increased cholesterol excretion out of brain from 17 to 49 microg per day, slowed neurodegeneration, and prolonged life. This agonist did not alter synthesis of cholesterol or expression of genes associated with the formation of 24(S)-hydroxycholesterol or neurosteroids such as CYP46A1, 3alphaHSD, and CYP11A1. However, levels of the sterol transporters ABCA1 and ATP-binding cassette transporter G1 were increased. Concomitantly, markers of neuroinflammation, CD14, MAC1, CD11c, and inducible nitric oxide synthase, were reduced, and microglia reverted from their amoeboid, active form to a ramified, resting configuration. Thus, LXR activation resulted in increased cholesterol excretion from the brain, decreased neuroinflammation, and deactivation of microglia to slow neurodegeneration and extend the lifespan of the NPC1 mouse.

Receptor-mediated and bulk-phase endocytosis cause macrophage and cholesterol accumulation in Niemann-Pick C disease.

These studies explored the roles of receptor-mediated and bulk-phase endocytosis as well as macrophage infiltration in the accumulation of cholesterol in the mouse with Niemann-Pick type C (NPC) disease. Uptake of LDL-cholesterol varied from 514 microg/day in the liver to zero in the central nervous system. In animals lacking LDL receptors, liver uptake remained about the same (411 microg/day), but more cholesterol was taken up in extrahepatic organs. This uptake was unaffected by the reductive methylation of LDL and consistent with bulk-phase endocytosis. All tissues accumulated cholesterol in mice lacking NPC1 function, but this accumulation was decreased in adrenal, unchanged in liver, and increased in organs like spleen and lung when LDL receptor function was also deleted. Over 56 days, the spleen and lung accumulated amounts of cholesterol greater than predicted, and these organs were heavily infiltrated with macrophages. This accumulation of both cholesterol and macrophages was increased by deleting LDL receptor function. These observations indicate that both receptor-mediated and bulk-phase endocytosis of lipoproteins, as well as macrophage infiltration, contribute to the cholesterol accumulation seen in NPC disease. These macrophages may also play a role in parenchymal cell death in this syndrome.

Lysosomal unesterified cholesterol content correlates with liver cell death in murine Niemann-Pick type C disease.

Niemann-Pick type C (NPC) disease is a multisystem disorder resulting from mutations in the NPC1 gene that encodes a protein involved in intracellular cholesterol trafficking. Significant liver dysfunction is frequently seen in patients with this disease. The current studies used npc1 mutant mice to investigate the association between liver dysfunction and unesterified cholesterol accumulation, a hallmark of NPC disease. Data from 92 npc1(-/-) mice (age range, 9-56 days) revealed a significant positive correlation between the plasma activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) and whole liver cholesterol content. In 56 day old npc1(-/-) mice that had been fed from 35 days of age a rodent diet or the same diet containing either cholesterol (1.0%, w/w) or ezetimibe (a sterol absorption inhibitor; 0.0125%, w/w), whole liver cholesterol content averaged 33.5 +/- 1.1, 87.9 +/- 1.7, and 20.8 +/- 0.9 mg, respectively. Again, plasma ALT and AST activities were positively correlated with hepatic cholesterol content. In contrast, plasma transaminase levels remained in the normal range in npc1(+/+) mice, in which hepatic esterified cholesterol content had been increased by 72-fold by feeding a high-cholesterol, high-fat diet. These studies suggest that the late endosomal/lysosomal content of unesterified cholesterol correlates with cell damage in NPC disease.

Cholesterol substrate pools and steroid hormone levels are normal in the face of mutational inactivation of NPC1 protein.

Mutational inactivation of NPC1 largely blocks the movement of LDL-derived cholesterol from the lysosome to the metabolically active, cytosolic pool of sterol that is the substrate for steroid hormone production. Such a block might, in theory, lead to deficiencies in circulating levels of testosterone, progesterone, and corticosterone. However, there are at least two other sources for cellular cholesterol, de novo synthesis and scavenger receptor class B type I-mediated uptake of HDL cholesteryl ester (CE). In this study, we measured the rates of net cholesterol acquisition by these three pathways in the adrenal, ovary, and testis. In all three organs, the majority (81-98%) of cholesterol acquisition came from the selective uptake of CE from HDL and de novo synthesis. Furthermore, in the npc1(-/-)mouse, the cytosolic storage pool of CE in a tissue such as the adrenal remained constant (approximately 25 mg/g). As a result of these alternative pathways, the plasma concentrations of testosterone (3.5 vs. 2.5 ng/ml), progesterone (8.5 vs. 6.7 ng/ml), and corticosterone (391 vs. 134 ng/ml) were either the same or elevated in the npc1(-/-)mouse, compared with the control animal. Thus, impairment of cholesterol acquisition through the NPC1-dependent, clathrin-coated pit pathway did not limit the availability of cholesterol substrate for steroid hormone synthesis in the steroidogenic cells.

Cholesterol accumulation and liver cell death in mice with Niemann-Pick type C disease.

Niemann-Pick type C (NPC) disease develops as a result of mutations in the NPC1 gene that encodes a protein involved in the net movement of unesterified cholesterol from the late endosomal/lysosomal compartment to the metabolically active pool of sterol in the cytosol of virtually every cell in the body. Although early publications emphasized the neurodegeneration occurring in children with this mutation, more recent clinical information suggests that serious liver disease also is an important part of this syndrome. These studies, therefore, were undertaken to characterize the liver dysfunction seen in mice with this same mutation. The NPC mouse develops significant hepatomegaly that reaches 8% of body weight at 5 to 6 weeks of age. This increase in liver size is associated with a linear increase in cholesterol content and with accumulation of amorphous cellular inclusions in both hepatocytes and macrophages. During the few weeks after birth, significant elevation of the plasma alkaline phosphatase level occurs, as also is seen in the human infant with this disease. At 4 to 5 weeks of age, plasma aminotransferase levels also rise abruptly. Histologically, at this time there is apoptosis, but no excess deposition of collagen or glycogen. mRNA expression is elevated for caspase 1, caspase 6, and several enzymes associated with sterol biosynthesis and bile acid formation. In conclusion, the NPC mouse has liver disease similar to that seen in the NPC infant and represents a relevant model for exploring the molecular events occurring in this form of liver disease.

Niemann-Pick C1 expression is not regulated by the amount of cholesterol flowing through cells in the mouse.

The Niemann-Pick C1 (NPC1) protein functions to regulate the transport of cholesterol from late endosomes/lysosomes to other cellular compartments after lipoprotein uptake through the coated-pit pathway. The present study examines the relative expression of NPC1 mRNA and NPC1 protein in different tissues of the mouse in relation to the uptake of total cholesterol carried in chylomicron remnants (CMr-TC), low density lipoproteins (LDL-TC), cholesteryl ester carried in high density lipoproteins (HDL-CE), and cholesterol synthesis. Results from this study demonstrate that the highest relative expression of NPC1 is in the liver, which is also the tissue with the highest uptake of CMr-TC, LDL-TC, HDL-CE, and cholesterol synthesis. However, there was no similar relation in the remaining tissues. To examine the relative expression of NPC1 in relation to the amount of cholesterol that flowed through the coated-pit pathway, mice were fed a diet supplemented with increasing amounts of cholesterol or cholestyramine. The results from this study demonstrated that there was no relation between the relative expression of NPC1 and the amount of cholesterol that flowed through the coated-pit pathway. We conclude that the relative expression of NPC1 is not regulated by the flow of cholesterol through cells in the mouse and is therefore constitutive.

Molecular, anatomical, and biochemical events associated with neurodegeneration in mice with Niemann-Pick type C disease.

In Niemann-Pick type C (NPC) disease, cholesterol associated with either apoE or apoB100 is taken up by cells in all tissues, including the central nervous system, through clathrin-coated pits and becomes trapped in late endosomes and lysosomes. This study defines the functional, biochemical, and molecular events that ensue as nerve cell death occurs. In mice homozygous for a mutation in NPC1, neuromuscular dysfunction begins at 5 weeks and death occurs at 13 weeks of age. Cholesterol accumulates in every tissue in the body. Purkinje cell loss in the cerebellum begins at 3 to 4 weeks of age and is nearly complete by 11 weeks. This neurodegeneration in the cerebellum is associated with increases in the levels of mRNA for caspase 1, caspase 3, NPC2, LipA, apoE, apoD, glial fibrillary acidic protein, and tumor necrosis factor-alpha, but not for most target genes of the LXR nuclear receptors. The level for apoER2 is significantly reduced. These studies show there is a compensatory increase in NPC2 and LipA in an attempt to overcome the physiological defect caused by the mutation. Nevertheless, neurodegeneration proceeds utilizing apoptosis with activation of glial cells, increased apoE and apoD synthesis, and increased cholesterol turnover across the CNS.

Delineation of molecular changes in intrahepatic cholesterol metabolism resulting from diminished cholesterol absorption.

The absorption of cholesterol by the small intestine is a major route for the net entry of cholesterol into the body and can therefore affect the plasma low density lipoprotein-cholesterol (LDL-C) concentration. These studies used ezetimibe, a potent inhibitor of cholesterol absorption, to delineate the biochemical and molecular changes in intrahepatic metabolism and biliary lipid secretion when there is a major reduction in chylomicron cholesterol delivery to the liver. In female LDL receptor (LDLR)-deficient (LDLR-/-) mice fed a basal diet containing ezetimibe (0-10 mg/day/kg body weight), cholesterol absorption was reduced up to 91%, fecal neutral sterol excretion was increased up to 4.7-fold, and plasma total cholesterol concentrations decreased by up to 18%. Blocking cholesterol absorption prevented the accumulation of very low density lipoproteins and LDL in the circulation of LDLR-/- mice fed a lipid-rich diet. In female LDLR+/+ mice fed the lipid-rich diet with ezetimibe, the relative mRNA level for the LDLR in the liver was 2-fold greater than in matching mice given the lipid-rich diet alone. We conclude that in the mouse the reduction in plasma LDL-C levels induced by blocking cholesterol absorption reflects both a diminished rate of LDL-C production and a modest increase in hepatic LDLR expression.