Cholesterol signaling at the endoplasmic reticulum occurs in npc1(-/-) but not in npc1(-/-), LDLR(-/-) mice

It remains controversial whether deficiency of the Niemann-Pick C1 (npc1) protein results in altered cholesterol signaling at the endoplasmic reticulum (ER). In this report, we have measured the processed, nuclear form of sterol regulatory element binding protein (SREBP)-1 in livers of npc1 wild-type, heterozygous, and homozygous deficient mice, alone, and in combination with deficiencies of the low density lipoprotein receptor (LDLR) or the multiple drug resistant (mdr)1a, P-glycoprotein. Cleavage of SREBPs to activated forms normally occurs when the ER is deficient in cholesterol. A large decrease in processed SREBP-1 was evident in fasted npc1(-/-) mice and npc1(-/-), mdr1a(-/-) mice, with no decrease evident in npc1(-/-), LDLR(-/-) mice. These results suggest that the increase in cellular cholesterol which occurs in npc1(-/-) and in npc1(-/-), mdr1a(-/-) mice includes the sites responsible for cholesterol signaling, while the similar increase in cholesterol found in npc1(-/-), LDLR(-/-) mice does not.     

Decreased hepatic accumulation and enhanced esterification of cholesterol in mice deficient in mdr1a and mdr1b P-glycoproteins

Class I P-glycoproteins [Pgp; MDR1 (ABCB1) in humans, mdr1a and mdr1b in mice] confer resistance to structurally diverse chemotherapeutic drugs in cultured cells and intact animals, but the function of these proteins in normal physiology remains poorly characterized. Based on studies in cell culture, a putative role for class I Pgp in absorption and intracellular trafficking of sterols has been proposed. We examined wild-type and mdr1a(-/)-/1b(-/)- mice to determine whether class I Pgp affects cholesterol absorption and esterification in vivo. Using a dual-isotope protocol, absorption of orally administered radiolabeled cholesterol into serum did not differ between wild-type and mdr1a(-/)-/1b(-/)- mice, demonstrating that class I Pgp is not essential for overall absorption of cholesterol through the intestine. However, the ratio of oral to intravenous labeled cholesterol in liver was decreased significantly in mdr1a(-/)-/1b(-/)- mice. In the liver, but not other tested organs, deletion of class I Pgp enhanced kinetics of esterification of an oral bolus of radiolabeled cholesterol without affecting esterification of cholesterol administered intravenously. Steady-state hepatic content of cholesterol and esterified cholesterol also were unaffected by absence of mdr1a and mdr1b.Thus, in normal animals, class I Pgp functions to kinetically increase hepatic accumulation and decrease esterification of orally administered cholesterol in vivo.     

Defect in fatty acid esterification of dolichol in Niemann-Pick type C1 mouse livers in vivo

Fatty acid esterification of dolichol and cholesterol in Niemann-Pick type C1 mouse (Balb/c NIH npc1(-/-)) livers was investigated in response to treatment with peroxisomal proliferators. These inducers have hypolipidemic properties and influence the mevalonate pathway and the intracellular transport of the final products of this biosynthetic route. Such inducers are consequently interesting to use in a disease model with defective intracellular transport of lipids. In wild-type mice, the levels of dolichol and cholesterol found as free alcohols were not changed to any great extent upon treatment with the peroxisomal inducers dehydroepiandrosterone, clofibrate and diethylhexylphtalate. In contrast, the amounts of dolichyl esters increased whereas cholesteryl esters decreased by the same treatments. The rate of enzymatic esterification of dolichol in isolated microsomes was accordingly elevated after 5- to 7-day treatments with the efficient peroxisomal proliferators DEHP and PFOA, while the corresponding esterification of cholesterol was decreased. Upon peroxisomal induction in npc1(-/-) mice, the enzymatic dolichol esterification in vitro increased whereas the low concentration of dolichyl esters remained unchanged. The results thus demonstrate that the induction of fatty acid esterification of dolichol in vivo is impaired in npc1(-/-) mouse liver. It is therefore proposed that the intracellular lipid transport defect in npc1(-/-) mouse liver disables either dolichol and/or the fatty acid from reaching the site of esterification in vivo. This proposal was strengthened by the finding that the amount of dolichol was decreased in an isolated Golgi fraction from npc1(-/-) mice.     

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.     

Differential transport properties of two mdr gene products are distinguished by progesterone

P-glycoprotein is an integral membrane protein that is overproduced in multidrug-resistant cells. It is likely to function as an energy-dependent drug efflux pump to maintain intracellular drug concentrations below cytotoxic levels. Individually isolated multidrug-resistant murine cell lines, J7.V1-1 and J7.V3-1, overproduce P-glycoproteins encoded by the mdr1b and mdr1a genes, respectively. The transport properties of these cell lines and the drug binding characteristics of their P-glycoproteins have been compared. It is concluded that 1) the mdr1a gene product is a more efficient efflux pump than the mdr1b gene product, and 2) whereas a single class of vinblastine binding sites is present in J7.V1-1 membrane vesicles, there appears to be two classes of such sites in J7.V3-1 membrane vesicles. The effects of verapamil and progesterone, two compounds that are known to interact with P-glycoprotein, have been analyzed in the two cell lines. Progesterone inhibited drug binding and efflux and increased drug sensitivity to vinblastine with more potency in J7.V1-1 cells than in J7.V3-1 cells. It is concluded that progesterone, but not verapamil, can be used to differentiate the two mdr gene products in the mouse.     

Cyclodextrins in the treatment of a mouse model of Niemann-Pick C disease.

Niemann-Pick type C (NPC) is a neurodegenerative disorder characterized by greatly altered somatic cholesterol metabolism. The NPC1 gene has recently been cloned and shown to have sequence homology to other sterol-sensing proteins. We have used a mouse model with a disrupted npc1 gene to study the effects of the cholesterol-mobilizing compound, 2-hydroxypropyl-beta-cyclodextrins (HPBCD), on the clinical course of this disorder. Treatment with two HPBCDs, with varying levels of 2-hydroxypropyl substitution, had effects in delaying neurological symptoms and in decreasing liver cholesterol storage while a third HPBCD was without effect. The ameliorating effect was not improved by longer exposure times (commencement of exposure in utero), however, it is not known if there is transplacental transfer of HPBCDs. The combination of HPBCD with probucol or nifedipine (which have previously been shown to lower liver cholesterol in this animal model) markedly decreased liver storage of unesterified cholesterol without altering the depressed levels of esterified cholesterol. The slight effects of the HPBCDs on neurological symptoms may be partially due to their apparent non-permeation of the blood-brain barrier (BBB). This non-permeation was assayed with radioactive tracers and was also present in the mdr1a knockout mice which have greatly increased BBB permeability for many drugs. Intrathecal delivery of HPBCD by an Alzet osmotic minipump did not improve its efficacy in ameliorating neurological symptoms.     

Toxicokinetic and genomic analysis of chronic arsenic exposure in multidrug-resistance mdr1a/1b(−/−) double knockout mice

Multidrug-resistance gene knockout mdr1a/1b(-/-) mice, which are deficient in P-glycoproteins, are more sensitive than wild-type (WT) mice to acute arsenic toxicity. This study assessed toxic manifestations of chronic oral arsenic in mdr1a/1b(-/-) mice, including oxidative stress and altered gene expression, and investigated altered toxicokinetics as a potential basis of enhanced arsenic toxicity. Thus, mdr1a/1b(-/-) and WT mice were exposed to sodium arsenite (0-80 ppm as arsenic) in the drinking water for 10 weeks at which time hepatic arsenic accumulation, lipid peroxidation (LPO), redox status and change in gene expression level were assessed. All mice survived the arsenic exposure, but body weight gain in the highest dose group was reduced in both mdr1a/1b(-/-) and WT mice. Arsenic induced pathological changes, elevated LPO levels and enhanced glutathione S-transferase (GST) activity, in the liver to a greater extent in mdr1a/1b(-/-) than in WT mice. Arsenic also decreased Cu/Zn superoxide dismutase activity in both mdr1a/1b(-/-) and WT mice. The expressions of certain genes, such as those encoding cell proliferation, GST, acute-phase proteins and metabolic enzymes, were modestly altered in arsenic-exposed mice. The expression of cyclin D1, a potential hepatic oncogene, was enhanced in arsenic-exposed mdr1a/1b(-/-) mice only. At the highest level of exposure, hepatic arsenic content was higher in mdr1a/1b(-/-) than in WT mice, suggesting that enhanced accumulation due to transport deficiency may, in part, account for the enhanced toxicity in these mice. In summary, this study shows that chronic arsenic toxicity, including liver pathology and oxidative stress, is enhanced in mdr1a/1b(-/-) mice, possibly due to enhanced accumulation of arsenic as a result of transport system deficiency.     

Epithelial dysfunction associated with the development of colitis in conventionally housed mdr1a-/- mice

P-glycoprotein, the product of the multidrug resistance protein 1 (MDR1) gene, is a xenobiotic transporter that may contribute to the physiology of the intestinal barrier. Twenty-five percent of mdr1a-deficient (mdr1a(-/-)) mice spontaneously develop colitis at variable ages when maintained under specific pathogen-free conditions. We hypothesized that this disease would result from epithelial dysfunction and that conventional housing would increase incidence and severity of the colitis phenotype. Wild-type congenic FVB (+/+) mice were maintained under the same conditions as controls. Knockout and wild-type mice were matched for age and gender and observed for signs of colitis. Colonic tissues from both groups of mice were examined for macroscopic and microscopic injury and for basal ion transport and transepithelial resistance (TER). Translocation of bacteria across the intestine was assessed by culturing the spleen and mesenteric lymph nodes. Protein analysis was performed by Western blot analysis. All mdr1a(-/-) mice developed weight loss and signs of colitis, whereas wild-type mice never showed such signs. Within the mdr1a(-/-) group, males consistently developed severe colitis earlier than females. Knockout mice showed increased basal colonic ion transport (females, 162.7 +/- 4.6 vs. 49.7 +/- 3.8 muA/cm(2); males, 172.6 +/- 5.6 vs. 54.2 +/- 3.1 muA/cm(2); P < 0.01) and decreased TER (females, 25.4 +/- 0.3 vs. 36.4 +/- 0.8 Omega.cm(2); males, 23.1 +/- 1.0 vs. 38.3 +/- 0.2 Omega.cm(2); P < 0.01) compared with wild-type mice. Barrier dysfunction was accompanied by decreased phosphorylation of tight junction proteins. Expression of cyclooxygenase-2 and inducible nitric oxide synthase in intestinal tissues was increased in the mdr1a(-/-) group (P < 0.01) and correlated with disease severity. Bacterial translocation was greater both in incidence (P < 0.01) and severity (P < 0.001) for the knockout group. With respect to all indexes studied, mdr1a(-/-) males performed worse than females. Our data support the hypothesis that alterations in the intestinal barrier alone, in the absence of immune dysfunction, may rapidly lead to colitis in the setting of a normal colonic flora.     

Requirement of Npc1 and availability of cholesterol for early embryonic cell movements in zebrafish

Niemann-Pick disease, type C (NP-C), often associated with Niemann-Pick disease, type C1 (NPC1) mutations, is a cholesterol-storage disorder characterized by cellular lipid accumulation, neurodegeneration, and reduced steroid production. To study NPC1 function in vivo, we cloned zebrafish npc1 and analyzed its gene expression and activity by reducing Npc1 protein with morpholino (MO)-oligonucleotides. Filipin staining in npc1-morphant cells was punctate, suggesting abnormal accumulation of cholesterol. Developmentally, reducing Npc1 did not disrupt early cell fate or survival; however, early morphogenetic movements were delayed, and the actin cytoskeleton network was abnormal. MO-induced defects were rescued with ectopic expression of mouse NPC1, demonstrating functional gene conservation, and by treatments with steroids pregnenolone or dexamethasone, suggesting that reduced steroidogenesis contributed to abnormal cell movements. Cell death was found in anterior tissues of npc1 morphants at later stages, consistent with findings in mammals. Collectively, these studies show that npc1 is required early for proper cell movement and cholesterol localization and later for cell survival.     

The products of the mdr1a and mdr1b genes from multidrug resistant murine cells have similar degradation rates

Two vinblastine-resistant sublines of the murine macrophage-like cell line J774.2, J7.V1-1 and J7.V3-1, overproduce unique forms of P-glycoprotein that are encoded by distinct mdr genes, mdr1b and mdr1a, respectively. Degradation rates of the two P-glycoprotein isoforms were measured by immunoprecipitation of P-glycoprotein. The half-life of immunoprecipitable P-glycoprotein from J7.V1-1 cells was 16.8 +/- 0.5 hours and from J7.V3-1 cells, 17.4 +/- 0.5 hours. This rate was not influenced by the presence of vinblastine in the growth medium. The data indicate that P-glycoproteins derived from distinct genes have similar degradation rates.     

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.     

Drosophila Niemann-Pick type C-2 genes control sterol homeostasis and steroid biosynthesis: a model of human neurodegenerative disease

Mutations in either of the two human Niemann-Pick type C (NPC) genes, NPC1 and NPC2, cause a fatal neurodegenerative disease associated with abnormal cholesterol accumulation in cells. npc1a, the Drosophila NPC1 ortholog, regulates sterol homeostasis and is essential for molting hormone (20-hydroxyecdysone; 20E) biosynthesis. While only one npc2 gene is present in yeast, worm, mouse and human genomes, a family of eight npc2 genes (npc2a-h) exists in Drosophila. Among the encoded proteins, Npc2a has the broadest expression pattern and is most similar in sequence to vertebrate Npc2. Mutation of npc2a results in abnormal sterol distribution in many cells, as in Drosophila npc1a or mammalian NPC mutant cells. In contrast to the ecdysteroid-deficient, larval-lethal phenotype of npc1a mutants, npc2a mutants are viable and fertile with relatively normal ecdysteroid level. Mutants in npc2b, another npc2 gene, are also viable and fertile, with no significant sterol distribution abnormality. However, npc2a; npc2b double mutants are not viable but can be rescued by feeding the mutants with 20E or cholesterol, the basic precursor of 20E. We conclude that npc2a functions redundantly with npc2b in regulating sterol homeostasis and ecdysteroid biosynthesis, probably by controlling the availability of sterol substrate. Moreover, npc2a; npc2b double mutants undergo apoptotic neurodegeneration, thus constituting a new fly model of human neurodegenerative disease.     

The multidrug resistance gene mdr1a influences resistance to ectromelia virus infection by mechanisms other than conventional immunity

P-glycoprotein (P-gp), an ATP-dependent membrane pump encoded by mdr, plays, in addition to its ability to efflux toxins, a role in the resistance to pathogens. We employed mdr1a gene knock out (mdr1a-/-) mice and ectromelia virus (EV) to elucidate the role of P-gp in resistance to EV. Mdr1a-/- mice are more susceptible to EV infection than wild type (wt) mice, showing increased mortality and morbidity. Unexpectedly, virus titres in liver, and in vitro in macrophages and splenocytes were significantly lower in the more susceptible mdr1a-/- mice than wt littermates. Analysis of immunological mechanisms known to influence resistance to EV infection, such as NK and cytotoxic T cell responses, EV specific antibody and cytokine levels did not reveal significant differences between the two strains of mice. Only dendritic cells from mdr1a-/- mice showed impaired migration to the draining lymph nodes compared to wt mice. Our data show that P-gp plays an important role in EV infection by as yet undefined mechanisms.     

Cell-autonomous death of cerebellar purkinje neurons with autophagy in Niemann-Pick type C disease

Niemann-Pick type C is a neurodegenerative lysosomal storage disorder caused by mutations in either of two genes, npc1 and npc2. Cells lacking Npc1, which is a transmembrane protein related to the Hedgehog receptor Patched, or Npc2, which is a secreted cholesterol-binding protein, have aberrant organelle trafficking and accumulate large quantities of cholesterol and other lipids. Though the Npc proteins are produced by all cells, cerebellar Purkinje neurons are especially sensitive to loss of Npc function. Since Niemann-Pick type C disease involves circulating molecules such as sterols and steroids and a robust inflammatory response within the brain parenchyma, it is crucial to determine whether external factors affect the survival of Purkinje cells (PCs). We investigated the basis of neurodegeneration in chimeric mice that have functional npc1 in only some cells. Death of mutant npc1 cells was not prevented by neighboring wild-type cells, and wild-type PCs were not poisoned by surrounding mutant npc1 cells. PCs undergoing cell-autonomous degeneration have features consistent with autophagic cell death. Chimeric mice exhibited a remarkable delay and reduction of wasting and ataxia despite their substantial amount of mutant tissue and dying cells, revealing a robust mechanism that partially compensates for massive PC death.     

Aberrant intracellular cholesterol transport disrupts pituitary and ovarian function

Cholesterol is imported and processed to provide substrate for ovarian steroidogenesis. The Niemann Pick type C-1 gene codes for a glycoprotein that processes low-density lipoproteinimported cholesterol. Mutation of this gene causes marked impairment of export of low-density lipoprotein-derived cholesterol from endosomes, and consequent lysosomal accumulation of the sterol. The BALB/c npc(nih-/-) mouse line, bearing spontaneous mutation of the NPC-1 gene, provides a model for investigation of aberrant endosomal cholesterol transfer in the ovary. Female homozygote mutant mice are infertile, with underdeveloped ovarian follicles, reduced steroidogenesis, no ovulation, and no corpora lutea. Mutant ovaries transplanted under wild-type kidney capsules display both ovulation and formation of corpora lutea. Gonadotropin treatment induces ovulation and restores expression of steroidogenic proteins. Pituitary glands of mutants are hypoplastic, and prolactin expression is dramatically reduced compared with wild-type mice. Both long and short splice variants of the dopamine-D2 receptors are overexpressed in the pituitary of BALB/c npc(nih-/-) mice. Chronic treatment of mutant mice with 17beta-estradiol restores pituitary volume, prolactin expression, and folliculogenetic capability. We conclude that inactivating mutation of Niemann Pick C-1 perturbs the hypothalamic-pituitary-ovarian feedback loop. Reduced estrogens attenuate prolactin expression and alter gonadotropin secretion patterns and interfere with normal ovarian follicular development and ovulation.     

Identification of a pharmaceutical compound that partially corrects the Niemann-Pick C phenotype in cultured cells

Niemann-Pick C (NPC) is an autosomal recessive lysosomal lipid storage disease characterized by progressive central nervous system degeneration. In cultured human NPC fibroblasts, LDL-derived cholesterol accumulates in lysosomes and endosomes, LDL-cholesterol transport from endocytic compartments to other cellular compartments is delayed, and LDL does not elicit normal homeostatic responses. Currently, there is no therapy that delays the onset of neurological symptoms or prolongs the life span of NPC children. We have developed and implemented an amphotericin B-mediated cytotoxicity assay to screen for potential therapeutic drugs that induce cholesterol movement in cultured NPC cells. NPC cells are relatively resistant to amphotericin B killing due to intracellular sequestration of cellular cholesterol. The screen was carried out using simian virus 40-transformed ovarian granulosa cells from the npc (nih) mouse model of NPC disease. A library of 44240 compounds was screened and 55 compounds were identified that promote amphotericin B-mediated killing of NPC cells. One compound, NP-27, corrected the NPC phenotype by four different measures of cholesterol homeostasis. In addition to making NPC cells more sensitive to amphotericin B, NP-27 stimulated two separate cholesterol transport pathways and restored LDL stimulation of cholesterol esterification to near normal levels.     

Alternate overexpression of two P-glycoprotein [corrected] genes is associated with changes in multidrug resistance in a J774.2 cell line

In multidrug-resistant murine J774.2 cells, the mdr1a and mdr1b genes encode the 120- and 125-kDa P-glycoprotein precursors, respectively (Hsu, S. I., Lothstein, L., and Horwitz, S.B. (1989) J. Biol. Chem. 264, 12053-12062). It is shown here that a J774.2 cell line selected for vinblastine resistance (J7.V3) switched from the 125- to 120-kDa precursor when cells that were maintained in 20 nM vinblastine were grown in 40 nM vinblastine for 20 months. The rate of switching was accelerated by growing cells in higher levels of vinblastine. These findings suggest that cells which express mdr1a have a selective growth advantage compared to cells which express mdr1b. Consistent with this hypothesis, the switching event that occurs in cells maintained at 40 nM vinblastine was correlated with 3.5-5-fold higher levels of resistance to vinblastine, taxol, and doxorubicin in the absence of any detectable increase in the amount of immunoreactive P-glycoprotein. These findings suggest that P-glycoproteins derived from mdr1a and mdr1b are functionally distinct.