The genomic organization and polymorphism analysis of the human Niemann-Pick C1 gene.

Niemann-Pick C (NP-C) is a fatal autosomal recessive storage disorder characterized by progressive neurodegeneration and variable hepatosplenomegaly. At the cellular level, cells derived from an affected individual accumulate unesterified cholesterol in lysosomes when cultured with low-density lipoprotein. The NP-C gene was identified at 18q11. The transcript is 4.9 kb encoding a 1278-amino-acid protein. We have defined the genomic structure of NPC1 along with the 5' flanking sequence. The NPC1 gene spans greater than 47 kb and contains 25 exons. Exons range in size from 74 to 788 bp with introns ranging in size from 0.097 to 7 kb. All intron/exon boundaries follow the GT/AG rule. The 5' flanking sequence has a CpG island containing multiple Sp1 sites indicative of a promoter region. The CpG island is located in the 5' flanking sequence, exon 1 and the 5' end of intron 1. We have also identified multiple single nucleotide polymorphisms in the coding and intronic sequences.

The Niemann-Pick C1 protein resides in a vesicular compartment linked to retrograde transport of multiple lysosomal cargo.

Niemann-Pick C disease (NP-C) is a neurovisceral lysosomal storage disorder. A variety of studies have highlighted defective sterol trafficking from lysosomes in NP-C cells. However, the heterogeneous nature of additional accumulating metabolites suggests that the cellular lesion may involve a more generalized block in retrograde lysosomal trafficking. Immunocytochemical studies in fibroblasts reveal that the NPC1 gene product resides in a novel set of lysosome-associated membrane protein-2 (LAMP2)(+)/mannose 6-phosphate receptor(-) vesicles that can be distinguished from cholesterol-enriched LAMP2(+) lysosomes. Drugs that block sterol transport out of lysosomes also redistribute NPC1 to cholesterol-laden lysosomes. Sterol relocation from lysosomes in cultured human fibroblasts can be blocked at 21 degrees C, consistent with vesicle-mediated transfer. These findings suggest that NPC1(+) vesicles may transiently interact with lysosomes to facilitate sterol relocation. Independent of defective sterol trafficking, NP-C fibroblasts are also deficient in vesicle-mediated clearance of endocytosed [14C]sucrose. Compartmental modeling of the observed [14C]sucrose clearance data targets the trafficking defect caused by mutations in NPC1 to an endocytic compartment proximal to lysosomes. Low density lipoprotein uptake by normal cells retards retrograde transport of [14C]sucrose through this same kinetic compartment, further suggesting that it may contain the sterol-sensing NPC1 protein. We conclude that a distinctive organelle containing NPC1 mediates retrograde lysosomal transport of endocytosed cargo that is not restricted to sterol.

Complementation studies in human and feline Niemann-Pick type C disease.

Complementation studies were performed to determine if the gene responsible for the major form of human Niemann-Pick type C disease (NPC) and a feline model of NPC are orthologous. Cell fusions between human NPC and feline NPC fibroblasts were conducted to assess whether the multinucleated heterokaryons that were formed showed a reversal of the NPC phenotype. Cultured fibroblasts from NPC-affected humans and NPC-affected cats were hybridized and then analyzed for complementation by challenging the cells with low-density lipoprotein (LDL) and subsequently staining with the fluorescent antibiotic filipin to visualize any abnormal accumulation of unesterified cholesterol. All of the multinucleated cells formed from these fusions retained the NPC staining phenotype, indicating an absence of complementation and suggesting that the underlying defect in the major form of human NPC and this feline model of NPC involve orthologous genes.

Niemann-Pick C disease: cholesterol handling gone awry.

Niemann-Pick C disease (NPC) is a debilitating, recessive disorder in humans that causes unrelenting neurological deterioration and is complicated by the presence of lipid-laden foamy cells in the major organs of the body. NPC fibroblasts cultured with an excess of low density lipoprotein (LDL) abnormally sequester cholesterol in their lysosomes. Biochemical analyses of NPC cells suggest an impairment in the intracellular transport of cholesterol to post-lysosomal destinations occurs in NPC. The recent identification of the NPC gene, NPC1, provides a definitive diagnosis of the disease and a means of studying this key component of intracellular cholesterol transport and homeostasis.

Murine model of Niemann-Pick C disease: mutation in a cholesterol homeostasis gene.

An integrated human-mouse positional candidate approach was used to identify the gene responsible for the phenotypes observed in a mouse model of Niemann-Pick type C (NP-C) disease. The predicted murine NPC1 protein has sequence homology to the putative transmembrane domains of the Hedgehog signaling molecule Patched, to the cholesterol-sensing regions of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase and SREBP cleavage-activating protein (SCAP), and to the NPC1 orthologs identified in human, the nematode Caenorhabditis elegans, and the yeast Saccharomyces cerevisiae. The mouse model may provide an important resource for studying the role of NPC1 in cholesterol homeostasis and neurodegeneration and for assessing the efficacy of new drugs for NP-C disease.

Niemann-Pick C1 disease gene: homology to mediators of cholesterol homeostasis.

Niemann-Pick type C (NP-C) disease, a fatal neurovisceral disorder, is characterized by lysosomal accumulation of low density lipoprotein (LDL)-derived cholesterol. By positional cloning methods, a gene (NPC1) with insertion, deletion, and missense mutations has been identified in NP-C patients. Transfection of NP-C fibroblasts with wild-type NPC1 cDNA resulted in correction of their excessive lysosomal storage of LDL cholesterol, thereby defining the critical role of NPC1 in regulation of intracellular cholesterol trafficking. The 1278-amino acid NPC1 protein has sequence similarity to the morphogen receptor PATCHED and the putative sterol-sensing regions of SREBP cleavage-activating protein (SCAP) and 3-hydroxy-3-methyl-glutaryl coenzyme A (HMG-CoA) reductase.

Substantial narrowing of the Niemann-Pick C candidate interval by yeast artificial chromosome complementation.

Niemann-Pick disease type C (NP-C) is an autosomal recessive lipidosis linked to chromosome 18q11-12, characterized by lysosomal accumulation of unesterified cholesterol and delayed induction of cholesterol-mediated homeostatic responses. This cellular phenotype is identifiable cytologically by filipin staining and biochemically by measurement of low-density lipoprotein-derived cholesterol esterification. The mutant Chinese hamster ovary cell line (CT60), which displays the NP-C cellular phenotype, was used as the recipient for a complementation assay after somatic cell fusions with normal and NP-C murine cells suggested that this Chinese hamster ovary cell line carries an alteration(s) in the hamster homolog(s) of NP-C. To narrow rapidly the candidate interval for NP-C, three overlapping yeast artificial chromosomes (YACs) spanning the 1 centimorgan human NP-C interval were introduced stably into CT60 cells and analyzed for correction of the cellular phenotype. Only YAC 911D5 complemented the NP-C phenotype, as evidenced by cytological and biochemical analyses, whereas no complementation was obtained from the other two YACs within the interval or from a YAC derived from chromosome 7. Fluorescent in situ hybridization indicated that YAC 911D5 was integrated at a single site per CT60 genome. These data substantially narrow the NP-C critical interval and should greatly simplify the identification of the gene responsible in mouse and man. This is the first demonstration of YAC complementation as a valuable adjunct strategy for positional cloning of a human gene.

Intracellular trafficking of the free cholesterol derived from LDL cholesteryl ester is defective in vivo in Niemann-Pick C disease: insights on normal metabolism of HDL and LDL gained from the NP-C mutation.

Niemann-Pick C disease (NP-C) is a rare inborn error of metabolism with hepatic involvement and neurological sequelae that usually manifest in childhood. Although in vitro studies have shown that the lysosomal distribution of LDL-derived cholesterol is defective in cultured cells of NP-C subjects, no unusual characteristics mark the plasma lipoprotein profiles. We set out to determine whether anomalies exist in vivo in the cellular distribution of newly synthesized, HDL-derived or LDL-derived cholesterol under physiologic conditions in NP-C subjects. Three affected and three normal male subjects were administered [14C]mevalonate as a tracer of newly synthesized cholesterol and [3H]cholesteryl linoleate in either HDL or LDL to trace the distribution of lipoprotein-derived free cholesterol. The rate of appearance of free [14C]- and free [3H]cholesterol in the plasma membrane was detected indirectly by monitoring their appearance in plasma and bile. The plasma disappearance of [3H]cholesteryl linoleate was slightly faster in NP-C subjects regardless of its lipoprotein origin. Appearance of free [14C] cholesterol ill the plasma (and in bile) was essentially identical in normal and affected individuals as was the initial appearance of free [3H]cholesterol derived from HDL, observed before extensive exchange occurred of the [3H]cholesteryl linoleate among lipoproteins. In contrast, the rate of appearance of LDL-derived free [3H]cholesterol in the plasma membrane of NP-C subjects, as detected in plasma and bile, was retarded to a similar extent that LDL cholesterol metabolism was defective in cultured fibroblasts of these affected subjects. These findings show that intracellular distribution of both newly synthesized and HDL-derived cholesterol are essentially unperturbed by the NP-C mutation, and therefore occur by lysosomal-independent paths. In contrast, in NP-C there is defective trafficking of LDL-derived cholesterol to the plasma membrane in vivo as well as in vitro. The in vivo assay of intracellular cholesterol distribution developed herein should prove useful to quickly evaluate therapeutic interventions for NP-C.

Genetic heterogeneity in Niemann-Pick C disease: a study using somatic cell hybridization and linkage analysis.

The primary molecular defect underlying Niemann-Pick C disease (NPC) is still unknown. A wide spectrum of clinical and biochemical phenotypes has previously been documented. Indication of genetic heterogeneity has recently been provided for one patient. In the present study, somatic cell hybridization experiments were carried out on skin fibroblast cultures from 32 unrelated NPC patients covering the range of known clinical and biochemical phenotypes. The criterion for complementation was the restoration of a normal intracellular fluorescent pattern in polykaryons stained with filipin to document cholesterol distribution. Crosses between the various cell lines revealed a major complementation group comprising 27 unrelated patients and a second minor group comprising 5 patients. Linkage analysis in one multiplex family belonging to the minor complementation group showed that the mutated gene does not map to the 18q11-12 region assigned to the major gene. Patients in the first group spanned the whole spectrum of clinical and cellular phenotypes. No consistent clinical or biochemical phenotypes was associated with the second complementation group. Three of the five group 2 patients, however, presented with a new rare phenotype associated with severe pulmonary involvement leading to death within the first year of life. No biochemical abnormality specific of either group could be demonstrated with regard to tissue lipid storage pattern, intralysosomal cholesterol storage, and regulation of cholesterol homeostasis. Mutations affecting at least two different genes have thus been shown to underlie NPC. The two gene products may function together or sequentially in a common metabolic pathway affecting intracellular cholesterol transport.

Neurofibrillary tangles in Niemann-Pick disease type C.

Niemann-Pick disease type C (NPC) is an autosomal recessive disease, belonging to a clinically heterogeneous group of lipid storage diseases, distinguished by a unique error in cellular trafficking of exogenous cholesterol, associated with lysosomal accumulation of unesterified cholesterol. Unlike Niemann-Pick disease types A and B, there is no primary genetic defect in sphingomyelinase in NPC. During the routine neuropathological study of NPC patients, we found neurofibrillary tangles (NFT) in a series of cases with a slowly progressive chronic course. These were not associated with beta-amyloid deposits. The NFT were most frequent in the orbital gyrus, cingulate gyrus and entorhinal region of the cerebral cortex, but were also frequently found in the basal ganglia, thalamus and hypothalamus. In one of the most severely affected case, the NFT were even found in the neurons in the inferior olivary nucleus and in the spinal cord. The NFT were immunostained with Alz 50, and consisted of paired helical filaments. The distribution of the neurons bearing the NFT was generally similar to that of the swollen storage neurons, and storage neurons often contained NFT in their perikarya and/or in the meganeurites. However, neurons with NFT could be noted without swollen perikarya. The coexistence of neuronal storage and NFT in NPC without amyloid deposits suggests that perturbed cholesterol metabolism and/or lysosomal membrane trafficking may play a role in the formation of NFT, and that amyloid deposits are not necessarily the prerequisite for NFT formation. The results of our study also suggest that NFT formation may be a rather nonspecific cellular reaction of neurons to certain slowly progressive metabolic perturbations of an as yet undefined nature.

Paired helical filament tau (PHFtau) in Niemann-Pick type C disease is similar to PHFtau in Alzheimer's disease.

Niemann-Pick Type C disease (NPC) is a cholesterol storage disease with defects in the intracellular trafficking of exogenous cholesterol derived from low density lipoproteins. In NPC cases with a chronic progressive course, neurofibrillary tangles (NFTs) that consist of paired helical filaments (PHFs) have been reported. To determine if NPC tangles contain abnormal tau proteins (known as PHFtau) similar to those found in Alzheimer's disease (AD) tangles, we examined the brains of five NPC cases by immunohistochemical and Western blot methods using a library of antibodies to defined epitopes of PHFtau. We show here that PHFtau in tangle-rich NPC brains is indistinguishable from PHFtau in AD brains. We speculate, that the generation of PHFtau in NPC may induce a cascade of pathological events that contribute to the widespread degeneration of neurons, and that these events may be similar in NPC and AD.

Analogues of butyric acid that increase the expression of transfected DNAs.

Butyric acid has many strong effects on gene expression in mammalian and viral systems, as well as in increasing the expression of recombinant DNAs artificially introduced into cultured cells. We screened 14 analogues of butyric acid for their ability to upregulate expression from 3 different recombinant chloramphenicol acetyltransferase expression vectors stably integrated into NIH 3T3 cells that had been transformed by calcium phosphate transfection or electroporation. Butyric acid, 2-bromobutyric acid, 3-bromopropionic acid, 3-mercaptopropionic acid, vinylacetic acid, and butyraldehyde were found to upregulate human immunodeficiency viral long terminal repeat-, SV40 early gene promoter-, and glucocerebrosidase promoter-directed expression of heterologous genes in cultured cells. Three other analogues had lesser effects; and 6 additional analogues had very little, if any, effect.

Linkage of Niemann-Pick disease type C to human chromosome 18.

We analyzed the involvement of chromosome 18 in Niemann-Pick disease type C (NPC), an autosomal recessive cholesterol-processing disorder. Within affected offspring, the chromosome 18 parental contributions were identified by using allele-specific microsatellite markers. Significant linkage of NPC to an 18p genomic marker, D18S40, was indicated by a two-point lod score of 3.84. Analysis of meiotic chromosomal breakpoint patterns among the affected individuals indicated that the NPC gene is pericentromerically localized on human chromosome 18.

Molecular and functional characterization of the murine glucocerebrosidase gene.

A genomic clone of glucocerebrosidase (D-glucosyl-N-acyl-sphingosine glucohydrolase; E.C. 3.2.1.45) purified from a genomic library derived from a Balb/c mouse was analyzed by restriction mapping and nucleotide sequencing of its promoter and protein coding regions. Promoter activity was functionally assessed by ligation of a 2 kb glucocerebrosidase fragment to the protein coding segment of a bacterial neomycin resistance gene. Smaller segments of the 5' flanking sequence were then analyzed for their ability to initiate transcription of the chloramphenicol acetyltransferase reporter gene. A 319 bp Eco RI-Bgl II fragment (containing 259 bp upstream of the cDNA 5' limit) ligated to the chloramphenicol acetyltransferase open reading frame produced considerable activity.