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Query: UNIPROT:P06889 (Mol)
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Resequencing genes in individuals at extremes of the population distribution constitutes a powerful and efficient strategy to identify sequence variants associated with complex traits. An excess of sequence variants at one extreme relative to the other that is not due to chance or to population stratification constitutes evidence for genetic association and implies the presence of functionally significant sequence variants. Recently, we reported that non-synonymous sequence variants in Niemann-Pick type C1-like 1 (NPC1L1), an intestinal cholesterol transporter, were significantly more common among individuals with low cholesterol absorption than in those with high cholesterol absorption. To determine whether sequence variations identified in individuals with low cholesterol absorption affect protein function, we performed studies in cultured cells and in families. Expression of the mutant proteins in Chinese hamster ovarian-K1 cells revealed that a majority (14 of 20) of the variants identified in low absorbers were associated with very low levels of NPC1L1 protein. In two extended families, mean cholesterol absorption levels, as measured using stable isotopes, were significantly lower in family members with the sequence variants than in those without the variant. These data indicate that the excess of sequence variations in individuals with extreme phenotypes reflects an enrichment of functionally significant variants. These findings are consistent with in silico predictions that some sequence variations found in healthy individuals are as deleterious to protein function as mutations that, in other genes, cause monogenic diseases. Such sequence variations may explain a significant fraction of quantitative phenotypic variation in humans.
Hum Mol Genet 2008 Jul 15
PMID:Functional characterization of genetic variants in NPC1L1 supports the sequencing extremes strategy to identify complex trait genes. 1841 23

GFP-tagging is widely used as a molecular tool to localize and visualize the trafficking of proteins in cells but interpretation is frequently limited by the low resolution afforded by fluorescence light microscopy. Although complementary thin-section immunogold electron microscopic techniques go some way in aiding interpretation, major limitations, such as relatively poor structural preservation of membrane systems, low labelling efficiency and the two-dimensional nature of the images, remain. Here we demonstrate that the electron microscopic technique freeze-fracture replica immunogold labelling overcomes these disadvantages and can be used to define, at high resolution, the precise location of GFP-tagged proteins in specific membrane systems and organelles of the cell. Moreover, this technique provides information on the location of the protein within the phospholipid bilayer, potentially providing insight into mis-orientation of tagged proteins compared to their untagged counterparts. Complementary application of the freeze-fracture replica immunogold labelling technique alongside conventional fluorescence microscopy is seen as a novel and valuable approach to verification, clarification and extension of the data obtained using fluorescent-tagged proteins. The application of this approach is illustrated by new findings on PAT-family proteins tagged with GFP transfected into fibroblasts from patients with Niemann-Pick type C disease.
J Cell Mol Med 2009 Jul
PMID:GFP-tagged proteins visualized by freeze-fracture immuno-electron microscopy: a new tool in cellular and molecular medicine. 1862 50

Exposure of cells or animals to stress frequently induces acid sphingomyelinase (ASM)-mediated ceramide production that leads to cell death. Consistent with this, overexpression of ASM in subcutaneous B16-F10 mouse melanomas, in combination with irradiation, resulted in tumors that were up to 12-fold smaller than irradiated control melanomas. Similarly, when irradiated melanomas were pretreated with a single, peritumoral injection of recombinant ASM (rhASM), the tumors were up to threefold smaller. The in vivo effect of ASM was likely due to enhanced cell death of the tumor cells themselves, as well as the surrounding microvascular endothelial cells. In vitro, rhASM had little or no effect on the growth of tumor cells, even in combination with irradiation. However, when the culture media was acidified to mimic the acidic microenvironment of solid tumors, rhASM-mediated cell death was markedly enhanced when combined with irradiation. Microscopic analysis suggested that this was associated with an increase in autophagy. rhASM has been produced for the treatment of the lysosomal storage disorder, type B Niemann-Pick disease, and is currently being evaluated in a phase-1 clinical trial. Based on the data presented in this article, we propose that further investigation of this protein and gene as antineoplastic agents also is warranted.
Mol Ther 2008 Sep
PMID:Acid sphingomyelinase overexpression enhances the antineoplastic effects of irradiation in vitro and in vivo. 1862 57

Niemann-Pick C disease (NPC) is an autosomal recessive neurodegenerative disorder caused by the abnormal function of NPC1 or NPC2 proteins, leading to an accumulation of unesterified cholesterol and glycosphingolipids (GSLs) in the lysosomes. The mechanisms underlying the pathophysiology in NPC disease are not clear. Oxidative damage is implicated in the pathophysiology of different neurological disorders and the effect of GSL accumulation on the intracellular redox state has been documented. Therefore, we determined whether the intracellular redox state might contribute to the NPC disease pathophysiology. Because the treatment of NPC mice with allopregnanolone (ALLO) increases their lifespan and delays the onset of neurological impairment, we analysed the effect of ALLO on the oxidative damage in human NPC fibroblasts. Concentrations of reactive oxygen species (ROS) and lipid peroxidation were higher in fibroblasts from NPC patients than in fibroblasts from normal subjects. Fibroblasts from NPC patients were more susceptible to cell death through apoptosis after an acute oxidative insult. This process is mediated by activation of the NF-kappaB signalling pathway. Knockdown of NPC1 mRNA both in normal fibroblasts and in human SH-SY5Y neuroblastoma cells caused increased ROS concentrations. ALLO treatment of fibroblasts from NPC patients or NPC1 knockdown cells reduced the levels of ROS and lipid peroxidation and prevented peroxide-induced apoptosis and NF-kB activation. Thus, these findings suggest that oxidative stress might contribute to the NPC disease and ALLO might be beneficial in the treatment of the disease, at least in part, due to its ability to restore the intracellular redox state.
J Cell Mol Med 2009 Sep
PMID:Oxidative stress in NPC1 deficient cells: protective effect of allopregnanolone. 1877 57

Niemann-Pick type C disease (NPC) is a sphingolipid-storage disorder that results from inherited deficiencies of intracellular lipid-trafficking proteins, and is characterised by an accumulation of cholesterol and glycosphingolipids in late endosomes and lysosomes. Patients with this disorder develop progressive neurological impairment that often begins in childhood, is ultimately fatal and is currently untreatable. How impaired lipid trafficking leads to neurodegeneration is largely unknown. Here we review NPC clinical features and biochemical defects, and discuss model systems used to study this disorder. Recent studies have established that NPC is associated with an induction of autophagy, a regulated and evolutionarily conserved process by which cytoplasmic proteins are sequestered within autophagosomes and targeted for degradation. This pathway enables recycling of limited or damaged macromolecules to promote cell survival. However, in other instances, robust activation of autophagy leads to cell stress and programmed cell death. We summarise evidence showing that autophagy induction and flux are increased in NPC by signalling through a complex of the class III phosphoinositide 3-kinase and beclin-1. We propose that an imbalance between induction and flux through the autophagic pathway contributes to cell stress and neuronal loss in NPC and related sphingolipid-storage disorders, and discuss potential therapeutic strategies for modulating activity of this pathway.
Expert Rev Mol Med 2008 Sep 10
PMID:The pathogenesis of Niemann-Pick type C disease: a role for autophagy? 1878 59

The goal of this study was to identify a mechanism regulating cholesterol accumulation in cystic fibrosis (CF) cells. Both CFTR activation and expression are regulated by the cAMP pathway, and it is hypothesized that a feedback response involving this pathway may be involved in the phenotype of cholesterol accumulation. To examine the role of the cAMP pathway in cholesterol accumulation, we treated two CF model cell lines with the Rp diastereomer of adenosine 3',5'-cyclic monophosphorothioate (Rp-cAMPS) and visualized by filipin staining. Rp-cAMPS treatment eliminated cholesterol accumulation in CF cells, whereas 8-bromo-cAMP treatment led to cholesterol accumulation in wild-type cells. To confirm these findings in an independent model system, we also examined the role of cAMP in modulating cholesterol accumulation in Niemann-Pick type C (NPC) fibroblasts. Expression of the protein related to NPC, NPC1, is also directly regulated by cAMP; therefore, it is postulated that NPC cells exhibit the same cAMP-mediated control of cholesterol accumulation. Cholesterol accumulation in NPC cells also was reduced by the presence of Rp-cAMPS. Expression of beta-arrestin-2 (betaarr2), a marker of cellular response to cAMP signaling, was significantly elevated in CF model cells, Cftr(-/-) MNE, primary tissue obtained by nasal scrapes from CF subjects, and in NPC fibroblasts compared with respective controls.
Am J Physiol Lung Cell Mol Physiol 2008 Nov
PMID:cAMP-mediated regulation of cholesterol accumulation in cystic fibrosis and Niemann-Pick type C cells. 1879 Sep 90

Herein we describe detailed characterization of four common mutations (L302P, H421Y, R496L and DeltaR608) within the acid sphingomyelinase (ASM) gene causing types A and B Niemann-Pick disease (NPD). In vitro and in situ enzyme assays revealed marked deficiencies of ASM activity in NPD cell lines homoallelic for each mutation, although Western blotting and fluorescent microscopy showed that the mutant ASM polypeptides were expressed at normal levels and trafficked to lysosomes. Co-immunoprecipitation of the polypeptides with the ER chaperone, BiP, confirmed these findings, as did in vitro expression of the mutant cDNAs in reticulocyte lysates. We further developed a computer assisted, three-dimensional model of human ASM based on homologies to known proteins, and used this model to map each NPD mutation in relation to putative substrate binding, hydrolysis and zinc-binding domains. Lastly, we generated transgenic mice expressing the R496L and DeltaR608 mutations on the complete ASM knock-out background (ASMKO), and established breeding colonies for the future evaluation of enzyme enhancement therapies. Analysis of these mice demonstrated that the mutant ASM transgenes were expressed at high levels in the brain, and in the case of the DeltaR608 mutation, produced residual ASM activity that was significantly above the ASMKO background.
Mol Genet Metab 2008 Nov
PMID:Characterization of common SMPD1 mutations causing types A and B Niemann-Pick disease and generation of mutation-specific mouse models. 1881 62

Duchenne muscular dystrophy (DMD) and other types of muscular dystrophies are caused by the loss or alteration of different members of the dystrophin protein complex. Understanding the molecular mechanisms by which dystrophin-associated protein abnormalities contribute to the onset of muscular dystrophy may identify new therapeutic approaches to these human disorders. By examining gene expression alterations in mouse skeletal muscle lacking alpha-dystrobrevin (Dtna(-/-)), we identified a highly significant reduction of the cholesterol trafficking protein, Niemann-Pick C1 (NPC1). Mutations in NPC1 cause a progressive neurodegenerative, lysosomal storage disorder. Transgenic expression of NPC1 in skeletal muscle ameliorates muscular dystrophy in the Dtna(-/-) mouse (which has a relatively mild dystrophic phenotype) and in the mdx mouse, a model for DMD. These results identify a new compensatory gene for muscular dystrophy and reveal a potential new therapeutic target for DMD.
Mol Biol Cell 2009 Jan
PMID:Amelioration of muscular dystrophy by transgenic expression of Niemann-Pick C1. 1894 78

Niemann-Pick C (NPC) is a fatal progressive neurolipidosis. Miglustat, an inhibitor of glycosphingolipid synthesis, has been proposed to treat patients but questions remain regarding its efficacy. A major problem has been the lack of suitable objective efficacy endpoints. Three adults with NPC were treated with miglustat for 24 months. Efficacy of treatment was assessed clinically and using brain magnetic resonance spectroscopy. All patients reported mild clinical improvement or stabilization. Furthermore, a sustained decrease in the choline/creatine ratio was observed in all three patients over time. Although these preliminary results require confirmation on a larger cohort of patients, they suggest that miglustat has some beneficial effect on brain dysfunction in NPC and that MRS could be used routinely as a non invasive surrogate marker of treatment efficacy.
Mol Genet Metab 2009 Feb
PMID:24 month-treatment with miglustat of three patients with Niemann-Pick disease type C: follow up using brain spectroscopy. 1901 89

Hyperphosphorylation and aggregation of the microtubule-binding protein tau characterize a diverse array of neurodegenerative disorders. Most of these lack mutations in the encoding MAPT gene, and the role of tau in disease pathogenesis remains controversial. Among these tauopathies is Niemann-Pick type C disease (NPC), a lysosomal storage disorder characterized by progressive neurodegeneration and premature death, most often caused by an inherited deficiency in the intracellular lipid trafficking protein NPC1. To determine the extent to which tau affects NPC pathogenesis, we generated Npc1-/- mice deficient in tau. Unexpectedly, NPC1/tau double null mutants are generated in markedly smaller litters, exhibit an enhanced systemic phenotype and die significantly earlier than NPC1 single null mutants. As autophagy is up-regulated in NPC and protein degradation through this pathway depends on movement along microtubules, we knocked down MAPT expression in NPC1-deficient human fibroblasts and examined effects on this pathway. We show that an acute reduction of tau expression in a cellular model of NPC decreases induction and flux through the autophagic pathway. Our data establish that MAPT deletion exacerbates the NPC phenotype through a mechanism independent of tau protein aggregation and identifies a critical role for tau in the regulation of autophagy in NPC1-deficient cells.
Hum Mol Genet 2009 Mar 01
PMID:Tau deletion exacerbates the phenotype of Niemann-Pick type C mice and implicates autophagy in pathogenesis. 1907 61


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